Environmental Risk AssessmentSoil Contamination 14ITave

Environmental Risk
Assessment of Soil Contamination
Edited by Maria C. Hernandez-Soriano

Environmental Risk Assessment of Soil Contamination
Edited by Maria C. Hernandez-Soriano
D3pZ4i & bhgvld, Dennixxx & rosea (for softarchive)

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ISBN-10 953511235X
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Contents
Preface
1 Soil Contamination, Risk Assessment and Remediation

2 Sustainability Aspects of In-Situ Bioremediation of
Polluted Soil in Developing Countries and Remote Regions
3 Chronic Toxicity of Weathered Oil-Contaminated Soil
4 Heavy Metal Contamination in Brazilian Agricultural
Soils due to Application of Fertilizers
5 Beneficial and Negative Impacts on Soil by the Reuse
of Treated/Untreated Municipal Wastewater for Agricultural
Irrigation – A Review of the Current Knowledge
and Future Perspectives
6 The Complex Nature of Pollution in the Capping Soils of
Closed Landfills: Case Study in a Mediterranean Setting
7 Assessment of Soil Contamination of a Cattle Market
around River Ogun Basin, Isheri, Nigeria
8 Assessment of Historical Heavy Metal Pollution of Land
in the Proximity of Industrial Area of Targoviste, Romania
9 Assessment of Soil Pollution with Heavy Metals in Romania
10 Eco-Technological Solutions for the Remediation of
Polluted Soil and Heavy Metal Recovery
11 Selected Bioremediation Techniques in Polluted Tropical Soils
12 Thermal Removal of Polycyclic Aromatic Hydrocarbons from
Gasification Biochars
13 Electrochemical Detection of Mercury Removal from
Polluted Bentonite and Quartz using Different Removing Agents
14 Treatment Methods for Radioactive Wastes and Its
Electrochemical Applications
15 Optical Fibers to Detect Heavy Metals in Environment:
Generalities and Case Studies
16 Metabolomics for Soil Contamination Assessment
VI Contents
17 Phytoremediation of Soils Contaminated with Metals and

Metalloids at Mining Areas: Potential of Native Flora
18 Contribution of Vegetation to Alleviate Slope’s Erosion
and Acidity
19 Metal Contamination of Soils and Prospects of Phytoremediation
in and Around River Yamuna: A Case Study from North-Central India
20 Continuous and Induced Phytoextraction — Plant-Based Methods
to Remove Heavy Metals from Contaminated Soil
21 Diagnosis and Management of Field Pollution in the Case of
an Organochlorine Pesticide, the Chlordecone
22 Chemical and Hydraulic Behavior of a Tropical Soil
Compacted Submitted to the Flow of Gasoline Hydrocarbons
23 Exposure and Hazard Identification of Sulphonamides
in the Terrestrial Environment
24 Heavy Metal Deposition in Soils and Plants Impacted by
Anthropogenic Modification of Two Sites in the Sudan Savanna
of North Western Nigeria
25 Geochemical Speciation and Risk Assessment of Heavy Metals
in Soils and Sediments
26 The Effect of Industrial Heavy Metal Pollution on Microbial
Abundance and Diversity in Soils — A Review
27 Pollution of Pb in Soils Affected by Pyrite Tailings:
Influence of Soil Properties
28 Copper Accumulation in Vineyard Soils: Distribution,
Fractionation and Bioavailability Assessment
29 Characterization and Remediation of Soils and Sediments
Polluted with Mercury: Occurrence, Transformations,
Environmental Considerations and San Joaquin’s Sierra
Gorda Case
30 Conducted and Investigate Arsenic (As), Cobalt (Co) Copper (Cu),
Manganese (Mn), Lead (Pb), and Zinc (Zn) Contamination in
Agricultural' Soils and Wheat Crops Projects at Three Regions
in Libya
31 Risks of Heavy Metals Contamination of Soil-Pant System by Land
Application of Sewage Sludge: A Review with Data from Brazil
Preface
Soil is an irreplaceable resource that sustains life on the planet,

challenged by food and energy demands of an increasing population.
Therefore, soil contamination constitutes a critical issue to be addressed
if we are to secure the life quality of present and future generations.
Integrated efforts from researchers and policy makers are required

to develop sound risk assessment procedures, remediation strategies
and sustainable soil management policies.
Environmental Risk Assessment of Soil Contamination provides a wide

depiction of current research in soil contamination and risk assessment,
encompassing reviews and case studies on soil pollution by heavy metals
and organic pollutants.
The book introduces several innovative approaches for soil remediation

and risk assessment, including advances in phytoremediation and
implementation of metabolomics in soil sciences.
chapter 1
Soil Contamination, Risk Assessment and Remediation
Muhammad Aqeel Ashraf, Mohd. Jamil Maah and

Ismail Yusoff
Additional information is available at the end of the chapter
1. Introduction
Environment pollution is a burning topic of the day. Air, water and soil are being polluted
alike. Soil being a "universal sink" bears the greatest burden of environmental pollution. It is
getting polluted in a number of ways. There is urgency in controlling the soil pollution in order
to preserve the soil fertility and increase the productivity. Pollution may be defined as an
undesirable change in the physical, chemical and biological characteristics of air, water and
soil which affect human life, lives of other useful living plants and animals, industrial progress,
living conditions and cultural assets. A pollutant is something which adversely interfere with
health, comfort, property or environment of the people. Generally most pollutants are
introduced in the environment by sewage, waste, accidental discharge or else they are by-
products or residues from the production of something useful. Due to this our precious natural
resources like air, water and soil are getting polluted.
The basis of agriculture is Soil. All crops for human food and animal feed depend upon it. We
are losing this important natural resource by the accelerated erosion 10 some extent. In addition
to this the enormous quantities of man-made waste products, sludge and other product" from
new waste treatment plants even polluted water are also causing or leading to soil pollution.
In order to preserve the fertility and the productivity of the soil, control measures are to be
taken in a herculean manner, thereby improving the health of all living beings.
Assessing the ecological risk of contaminated soil, pesticide application, sewage sludge
amendment, and other human activities leading to exposure of the terrestrial environment to
hazardous substances is a complicated task with numerous associated problems. Not only is
terrestrial ecological risk assessment a relatively new field of science that has developed
rapidly only since the mid-1980s, but it is also complicated by the fact that soil, in contrast to
most aquatic environments, is very often on private lands and traded as real estate. Profes‐
2 Soil Pollution
sional and economic divergence between the interests of scientists, stakeholders, authorities,
engineers, managers, lawyers, nongovernment organizations (NGOs) and regulators is
therefore not unusual. Even neglecting those aspects, a number of unresolved problems exist
in the way we currently assess risk and manage the impact of anthropogenic substances in the
terrestrial environment.
This chapter does not intend to present a comprehensive review of all published data from
ecological studies at contaminated sites. Instead, the observations from all case studies are
used in the discussion and form the basis for the final conclusion. In each case, we try to answer
the following questions:
1. What is soil pollution and how it occurs?
2. How to determine the ecological risk assessment of the soil?
3. To what extent do soil screening levels (over)estimate risk?
4. Do bioassays represent a more realistic risk estimate?
5. Is it possible to make sound field surveys, or do we lack suitable reference situations?
6. What are the possible soil management methods for the polluted soils?
2. Soil pollution
Soil pollution is defined as the build-up in soils of persistent toxic compounds, chemicals, salts,
radioactive materials, or disease causing agents, which have adverse effects on plant growth
and animal health [1].
Soil is the thin layer of organic and inorganic materials that covers the Earth's rocky surface.
The organic portion, which is derived from the decayed remains of plants and animal, is
concentrated in the dark uppermost topsoil. The inorganic portion made up of rock fragments,
was formed over thousands of years by physical and chemical weathering of bedrock.
Productive soils are necessary for agriculture to supply the world with sufficient food [2].
There are many different ways that soil can become polluted, such as:
• Seepage from a landfill
• Discharge of industrial waste into the soil
• Percolation of contaminated water into the soil
• Rupture of underground storage tanks
• Excess application of pesticides, herbicides or fertilizer
• Solid waste seepage

The most common chemicals involved in causing soil pollution are:
Soil Contamination, Risk Assessment and Remediation 3
• Petroleum hydrocarbons
• Heavy metals
• Pesticides
• Solvents
2.1. Inorganic toxic compounds
Inorganic residues in industrial waste cause serious problems as regards their disposal. They
contain metals which have high potential for toxicity. Industrial activity also emits large
amounts of arsenic fluorides and sulphur dioxide (SO2) [3]. Fluorides are found in the
atmosphere from superphosphate, phosphoric acid, aluminium, steel and ceramic industries.
Sulphur dioxide emitted by factories and thermal plants may make soils very acidic. These
metals cause leaf injury and destroy vegetation.
Copper, mercury, cadmium, lead, nickel, arsenic are the elements which can accumulate in
the soil, if they get entry either through sewage, industrial waste or mine washings. Some
of the fungicides containing copper and mercury also add to soil pollution. Smokes from
automobiles contain lead which gets adsorbed by soil particles and is toxic to plants. The
toxicity can be minimized by building up soil organic matter, adding lime to soils and
keeping the soil alkaline [4].
2.2. Organic wastes
Organic wastes of various types cause pollution hazards. Domestic garbage, municipal sewage
and industrial wastes when left in heaps or improperly disposed seriously affect health of
human beings, plants and animals [5-7]. Organic wastes contain borates, phosphates, deter‐
gents in large amounts. If untreated they will affect the vegetative growth of plants. The main
organic contaminants are phenols and coal.
Asbestos, combustible materials, gases like methane, carbon dioxide, hydrogen sulphide,
carbon monoxide, sulphur dioxide, petrol are also contaminants. The radioactive materials
like uranium, thorium, strontium etc. also cause dangerous soil pollution. Fallout of strontium
mostly remains on the soil and is concentrated in the sediments [8]. Decontamination proce‐
dures may include continuous cropping and use of chelate amendments. Other liquids wastes
like sewage, sewage sludge, etc. are also important sources of soil problems.
a. Sewage and sewage sludge
Soil pollution is often caused by the uncontrolled disposal of sewage and other liquid wastes
resulting from domestic uses of water, industrial wastes containing a variety of pollutants,
agricultural effluents from animal husbandry and drainage of irrigation water and urban
runoff [9-10]. Irrigation with sewage water causes profound changes in the irrigated soils.
Amongst various changes that are brought about in the soil as an outlet of sewage irrigation
include physical changes like leaching, changes in humus content, and porosity etc., chemical
changes like soil reaction, base exchange status, salinity, quantity and availability of nutrients
4 Soil Pollution
like nitrogen, potash, phosphorus, etc. Sewage sludges pollute the soil by accumulating the
metals like lead, nickel, zinc, cadmium, etc. This may lead to the phytoxicity of plants.
b. Heavy metal pollutants
Heavy metals are elements having a density greater than five in their elemental form. They
mostly find specific absorption sites in the soil where they are retained very strongly either
on the inorganic or organic colloids. They are widely distributed in the environment, soils,
plants, animals and in their tissues. These are essential for plants and animals in trace
amounts. Mainly urban and industrial aerosols, combustion of fuels, liquid and solid from
animals and human beings, mining wastes, industrial and agricultural chemicals etc. are
contributing heavy metal pollution. Heavy metals are present in all uncontaminated soils
as the result of weathering from their parent materials. Concentration of heavy metals in
soils and plants is given in Table 1.
Sl.No Heavy metal Hithosphere Soil range Plants
1 Cadmium (Cd) 0.2 0.01-0.7 0.2-0.8
2 Cobalt (Co) 40 1-40 0.05-0.5
3 Chromium (Cr) 200 5-3000 0.2-1.0
4 Copper (Cu) 70 2-100 4-15
5 Iron (Fe) 50,000 7000-5,50,000 140
6 Mercury (Hg) 0.5 0.01-0.3 0.015
7 Manganese (Mn) 1000 100-4000 15-100
8 Molybdenum (Mo) 2.3 0.2-5 1-10
9 Nickel (Ni) 100 10-1000 1
10 Lead (Pb) 16 2-200 0.1-10
11 Tin (Sn) 40 2-100 0.3
12 Zinc (Zn) 80 10-300 8-100
Table 1. Heavy metal concentration in the hithosphere, soils and plants (Ug/gm dry matter)
In agricultural soils, however, the concentration of one or more of these elements may be
significantly increased in several ways, like through applications of chemicals, sewage
sludge, farm slurries, etc. Increased doses of fertilizers, pesticides or agricultural chemi‐
cals, over a period, add heavy metals to soils which may contaminate them. Certain
phosphatic fertilizers frequently contain trace amounts of cadmium which may accumu‐
late in these soils. Likewise, some fertilizers when applied to soils, they add certain heavy
metals which are given in Table 2.
The range of heavy metal contents in sludges is given in Table 3.

Sl.No Fertilizer Co Cr Cu Mn Mo Ni Pb Zn
1 Nitrochalk - - 22 24 - 2 - 15
2 Calcium 0.1 Traces Traces Traces - - - 1
3 Nitrate - - To 10 To 5 - - - -
Ammonium <0.05 to Traces to

4 <5 <5 0.800 0.80 <5 0.800
sulphate 0.22 200
Traces to Traces to Traces to Traces to

5 Super phosphate 0.02-13 0-1000 Traces to 35 70-3000
1000 2842 32 92
Potassium
6 001 - 0-10 Traces-8 <0.05 <1 <1 0-3
chloride
Potassium Traces to .
7 <5 <5 0-300 to 80 0.09 <5 <50 <50
sulphate 33
Table 2. Heavy metal content of fertilizers (ug/gm)
Sl.No Heavy metal Range (ppm)
1 Cadmium < 60-1500
2 Cobalt 2-260
3 Chromium 40-8800
4 Copper 200-8000
5 Iron 6000-62,000
6 Manganese 150-2500
7 Molybdenum 2-30
8 Nickel 20-5300
9 Lead 120-3000
10 Zinc 700-49,000
Table 3. Heavy metal contents in sludges (ppm)
The fate of heavy metals in soil will be controlled by physical and biological processes acting
within the soil. Metal ions enter the soil solution from these various forms of combination in
different rates they may either remain in solution or pass into the drainage water or be taken
up by plants growing on the soil or be retained by the soil in sparingly soluble or insoluble
forms. The organic matter of these soil have great affinity to heavy metals cations which form
stable complexes thereby leading to reduced nutrient content [11-12].
2.3. Organic pesticides

Pesticides are quite frequently used to -control several types of pests now-a-days. Pesticides
may exert harmful effects to micro-organisms, as a result of which plant growth may be
6 Soil Pollution
affected. Pesticides which are not rapidly decomposed may create such problems. Accumu‐
lation is residues of pesticides in higher concentrations are toxic. Pesticides persistence in soil
and movement into water streams may also lead to their entry into foods and create health
hazards. Pesticides particularly aromatic organic compounds are not degraded rapidly and
therefore, have a long persistence time which can be seen in Table 4.
Sl.No Pesticide Persistence time
1 BHC 11 yrs
2 DDT 10 yrs
3 2,4-D 2-8 weeks
4 Aldrin 9 yrs
5 Diuron 16 months
6 Atrazine 18 months
7 Siwazine 17 months
8 Chlordane 12 yrs
9 2,3 6-Trichlorobenzene (TBA) 2-5 yrs
Table 4. Persistence time for some selected pesticides
Mercury, cadmium and arsenic are common constituents of pesticides and all these heavy
metals are toxic. At present DDT and a number of organochlorine compounds used as
pesticides have been declared harmful and banned in U.S.A. and England [13-14]. It is due to
the persistence of their residues in soils for considerable time without losing their toxicity. This
has led to higher concentration of these pesticides in vegetation, in animal flesh and milk.
Eventually man has been affected. In view of their demerits, organochlorines have been
replaced by organophosphate pesticides which are more toxic, but do not leave any residue.
They do not pollute the soil. The rodenticides too add to soil pollution. A major method of
checking this pesticidal pollution is to increase the organic matter content of the sol and choose
such pesticides which are non-persistent and leave no harmful residue.
2.4. Types of soil pollution
• Agricultural Soil Pollution
i. pollution of surface soil
ii. pollution of underground soil
• Soil pollution by industrial effluents and solid wastes
ii. disturbances in soil profile

• Pollution due to urban activities
ii. pollution of underground soil
2.5. Sources of soil pollution
The sources which pollute the soil are twofold: Agricultural sources and non-agricultural
sources. Figure 1 shows the different sources for the soil pollution.
a. Agricultural sources
Soil pollution comes from different sources including agriculture and animal husbandry. Some
of the agricultural practices lead to soil pollution. They are animal wastes, use of long lived
pesticides, herbicides, fungicides, nematocides, etc. fertilizers and some agricultural practices.
b. Non-agricultural sources
Soil pollution by non-agricultural sources is usually the direct result of urban sprawl caused
by rapidly increasing population and a rapidly per capita output of waste related to our
modem way of life. Its materials that find their entry into the soil system have long persistence
and accumulate in toxic concentration and thus become sources of pollution. Some of those
most important soil pollutants are inorganic toxic compounds.
Figure 1. Sources of Soil Pollution [5]

8 Soil Pollution
2.6. Causes of soil pollution

Soil pollution is caused by the presence of man-made chemicals or other alteration in the
natural soil environment. This type of contamination typically arises from the rupture of
underground storage links, application of pesticides, and percolation of contaminated surface
water to subsurface strata, oil and fuel dumping, leaching of wastes from landfills or direct
discharge of industrial wastes to the soil. The most common chemicals involved are petroleum
hydrocarbons, solvents, pesticides, lead and other heavy metals. This occurrence of this
phenomenon is correlated with the degree of industrialization and intensities of chemical
usage. A soil pollutant is any factor which deteriorates the quality, texture and mineral content
of the soil or which disturbs the biological balance of the organisms in the soil. Pollution in
soil has adverse effect on plant growth.
Pollution in soil is associated with
• Indiscriminate use of fertilizers

• Indiscriminate use of pesticides, insecticides and herbicides
• Dumping of large quantities of solid waste
• Deforestation and soil erosion
a. Indiscriminate use of fertilizers
Oxygen from air and water but other necessary nutrients like nitrogen, phosphorus, potassi‐
um, calcium, magnesium, sulfur and more must be obtained from the soil. Farmers generally
use fertilizers to correct soil deficiencies. Fertilizers contaminate the soil with impurities, which
come from the raw materials used for their manufacture. Mixed fertilizers often contain
ammonium nitrate (NH4NO3), phosphorus as P2O5, and potassium as K2O. For instance, As,
Pb and Cd present in traces in rock phosphate mineral get transferred to super phosphate
fertilizer. Since the metals are not degradable, their accumulation in the soil above their toxic
levels due to excessive use of phosphate fertilizers becomes an indestructible poison for crops.
The over use of NPK fertilizers reduce quantity of vegetables and crops grown on soil over the
years. It also reduces the protein content of wheat, maize, grams, etc., grown on that soil. The
carbohydrate quality of such crops also gets degraded [15]. Excess potassium content in soil
decreases Vitamin C and carotene content in vegetables and fruits. The vegetables and fruits
grown on over fertilized soil are more prone to attacks by insects and disease.
b. Indiscriminate use of pesticides, insecticides and herbicides
Plants on which we depend for food are under attack from insects, fungi, bacteria, viruses,
rodents and other animals, and must compete with weeds for nutrients. To kill unwanted
populations living in or on their crops, farmers use pesticides. The first widespread insecticide
use began at the end of World War II and included DDT (dichlorodiphenyltrichloroethane)
and gammaxene. Insects soon became resistant to DDT and as the chemical did not decompose
readily, it persisted in the environment. Since it was soluble in fat rather than water, it
biomagnified up the food chain and disrupted calcium metabolism in birds, causing eggshells
to be thin and fragile. As a result, large birds of prey such as the brown pelican, ospreys, falcons
and eagles became endangered. DDT has been now been banned in most western countries.
Ironically many of them including USA, still produce DDT for export to other developing
nations whose needs outweigh the problems caused by it [16].
c. Dumping of solid wastes
In general, solid waste includes garbage, domestic refuse and discarded solid materials such
as those from commercial, industrial and agricultural operations. They contain increasing
amounts of paper, cardboards, plastics, glass, old construction material, packaging material
and toxic or otherwise hazardous substances. Since a significant amount of urban solid waste
tends to be paper and food waste, the majority is recyclable or biodegradable in landfills.
Similarly, most agricultural waste is recycled and mining waste is left on site. The portion of
solid waste that is hazardous such as oils, battery metals, heavy metals from smelting indus‐
tries and organic solvents are the ones we have to pay particular attention to. These can in the
long run, get deposited to the soils of the surrounding area and pollute them by altering their
chemical and biological properties [17].
d. Deforestation
Soil Erosion occurs when the weathered soil particles are dislodged and carried away by wind
or water. Deforestation, agricultural development, temperature extremes, precipitation
including acid rain, and human activities contribute to this erosion. Humans speed up this
process by construction, mining, cutting of timber, over cropping and overgrazing. It results
in floods and cause soil erosion. Forests and grasslands are an excellent binding material that
keeps the soil intact and healthy. They support many habitats and ecosystems, which provide
innumerable feeding pathways or food chains to all species. Their loss would threaten food
chains and the survival of many species. During the past few years quite a lot of vast green
land has been converted into deserts. The precious rain forest habitats of South America,
tropical Asia and Africa are coming under pressure of population growth and development
(especially timber, construction and agriculture). Many scientists believe that a wealth of
medicinal substances including a cure for cancer and aids, lie in these forests. Deforestation is
slowly destroying the most productive flora and fauna areas in the world, which also form
vast tracts of a very valuable sink for CO2 [18].
e. Pollution due to urbanization
Pollution of surface soils materials (like vegetables, animal wastes, papers, wooden pieces,
carcasses, plant twigs, leaves, cloth wastes as well as sweepings) and many non-biodegradable
materials (such as plastic bags, plastic bottles, plastic wastes, glass bottles, glass pieces, stone /
cement pieces) [19-20]. On a rough estimate Indian cities are producing solid city wastes to the
tune of 50,000 - 80,000 metric tons every day. If left uncollected and decomposed, they are a
cause of several problems such as;
• Clogging of drains: Causing serious drainage problems including the burst / leakage of
drainage lines leading to health problems.
10 Soil Pollution
• Barrier to movement of water: Solid wastes have seriously damaged the normal movement
of water thus creating problem of inundation, damage to foundation of buildings as well as
public health hazards.
• Foul smell: Generated by dumping the wastes at a place.
• Increased microbial activities: Microbial decomposition of organic wastes generate large
quantities of methane besides many chemicals to pollute the soil and water flowing on its
surface
• When such solid wastes are hospital wastes they create many health problems: As they may
have dangerous pathogen within them besides dangerous medicines, injections.
f. Pollution of underground soil
Underground soil in cities is likely to be polluted by
• Chemicals released by industrial wastes and industrial wastes

• Decomposed and partially decomposed materials of sanitary wastes
Many dangerous chemicals like cadmium, chromium, lead, arsenic, selenium products are
likely to be deposited in underground soil. Similarly underground soils polluted by sanitary
wastes generate many harmful chemicals. These can damage the normal activities and
ecological balance in the underground soil
2.7. Effects of soil pollution
a. Agricultural
• Reduced soil fertility

• Reduced nitrogen fixation
• Increased erodibility
• Larger loss of soil and nutrients
• Deposition of silt in tanks and reservoirs
• Reduced crop yield
• Imbalance in soil fauna and flora
b. Industrial
• Dangerous chemicals entering underground water

• Ecological imbalance
• Release of pollutant gases
• Release of radioactive rays causing health problems
• Increased salinity
• Reduced vegetation
c. Urban
• Clogging of drains
• Inundation of areas
• Public health problems
• Pollution of drinking water sources
• Foul smell and release of gases
• Waste management problems
d. Environmental
• Soil becomes unavailable to grow food

• If contaminated soil is used to grow food, the land will usually produce lower yields
• Can cause even more harm because a lack of plants on the soil will cause more erosion
• The pollutants will change the makeup of the soil and the types of microorganisms that will
live in it.
• Thus it's possible for soil pollution to change whole ecosystems
2.8. Control of soil pollution
The following steps have been suggested to control soil pollution. To help prevent soil erosion,
we can limit construction in sensitive area. In general we would need less fertilizer and fewer
pesticides if we could all adopt the three R's: Reduce, Reuse, and Recycle. This would give us
less solid waste.
a. Extraction and separation techniques
In solvent extraction, the contaminated & oil is mixed with an extracting agent in general (an
aqueous solution but preferably an organic solvent). Potential applications include the removal
of metals such as cadmium, copper, zinc, nickel, chromium, arsenic, antimony and lead using
a mineral solution, zinc lead, organo-metallic compounds and some cyanides using sodium
hydroxide solution. Hydrocarbons and halogenated hydrocarbons can also be removed [21].
Contamination is often preferentially present in the finer or coarser fraction of the soil or the
organic components (ex. humus), contamination can therefore, be removed in some cases using
a process which separates the soil into fractions on the basis of specific gravity or particle size
or settling velocity.
b. Thermal methods
In thermal methods, there are two ways of heat treatment; removal of contaminants by
evaporation either by direct heat transfer from heated air or an open flame or by indirect heat
transfer, and destruction of the contaminants directly or indirectly at an appropriate temper‐
12 Soil Pollution
ature. The gas leaving the heating appliance must be treated to destroy or remove any
contaminants or unwanted products of combustion. A related process is stream stripping in
which steam is injected into soil to aid evaporation of relatively volatile contaminants which
may be water soluble or insoluble.
c. Chemical methods
Treatment of the soil in suspension in a suitable liquid and without sludging is the two possible
methods. In these, intimate, contact between soil and chemical is essential and should be
frequently done so that the process of detoxification is complete.
d. Microbial treatment methods
The microbial treatment methods appear to be more promising which can deal with whole
range of organic contaminants including phenol, polychlorinated hydrocarbons, oil and oil
products, dioxins, etc. There are two different ways of approaching the problems.
1. A community of microbes already existing on the site is collected and cultured in the
laboratory.
2. Strains of microbes are developed in the laboratory that is capable of metabolizing

particular chemicals.
Excavation of the soil prior to treatment offers the greatest scope for creating optimum
conditions. The excavated soil can be placed on thin layers to various depth using standard
earth moving techniques and microbes and nutrients applied using standard agricultural
techniques such as fertilizing, ploughing, harrowing, etc.
e. Reducing chemical fertilizer and pesticide use
Applying bio-fertilizers and manures can reduce chemical fertilizer and pesticide use.
Biological methods of pest control can also reduce the use of pesticides and thereby minimize
soil pollution.
f. Reusing of materials
Materials such as glass containers, plastic bags, paper, cloth etc. can be reused at domestic
levels rather than being disposed, reducing solid waste pollution.
g. Recycling and recovery of materials
This is a reasonable solution for reducing soil pollution. Materials such as paper, some kinds
of plastics and glass can and are being recycled. This decreases the volume of refuse and helps
in the conservation of natural resources. For example, recovery of one tonne of paper can save
17 trees.
h. Reforesting
Control of land loss and soil erosion can be attempted through restoring forest and grass cover
to check wastelands, soil erosion and floods. Crop rotation or mixed cropping can improve
the fertility of the land.
i. Solid waste treatment
Proper methods should be adopted for management of solid waste disposal. Industrial wastes
can be treated physically, chemically and biologically until they are less hazardous. Acidic and
alkaline wastes should be first neutralized; the insoluble material if biodegradable should be
allowed to degrade under controlled conditions before being disposed. As a last resort, new
areas for storage of hazardous waste should be investigated such as deep well injection and
more secure landfills. Burying the waste in locations situated away from residential areas is
the simplest and most widely used technique of solid waste management.
2.9. Soil monitoring
The main objective of soil monitoring is to prevent and mitigate contamination by substances
with the potential to exert an adverse effect on the soil itself, and on air, water and organisms
that may contact the soil. Soil monitoring, within the approvals program, is directed primarily
to the assessment of contaminants that have been released to the soil surface. Thus, subsurface
facilities are generally not the reason for soil monitoring, but may be the reason for ground‐
water monitoring [22]. However, where soil contamination is known or suspected to originate
from subsurface sources such as underground tanks or pipes, an assessment will be required.
Where the above considerations indicate soil monitoring is required as a condition of an
Approval, the proponent is required to carry out the following, as specified in the Soil
Monitoring Directive: > prepare a soil monitoring proposal; > execute the approved soil
monitoring plan; > interpret and report the results of the soil monitoring; and > prepare and
execute a soil management plan where indicated by the results of soil monitoring. This
guideline provides a background for the soil monitoring program and a description of soil
management program requirements.
a. Legislative background
The soil monitoring program mostly developed under the Environmental Protection in
support of the following principles: > development must be sustainable, meaning that the use
of resources and the environment today must not impair prospects for their use by future
generations; > the environmental impact of development must be prevented or mitigated; >
polluters should bear the responsibility of paying for the costs of their actions; > remediation
costs should be incorporated into financial planning so that adequate funds are available for
site remediation and planners can know the true costs and benefits of source reduction
programs. Recognizing that under the environmental protection is a shared responsibility, it
follows that both the approval holder and the Department must have a means to assess
environmental performance with respect to the above principles and requirements.
b. Soil quality standards
Environmental Protection expects that approval holders will manage their operations to
prevent substance releases to soil. Substance releases to soil do occur, however, and contam‐
inants are often present above background concentrations at industrial facilities. In view of
this, Environmental Protection should have soil quality standards to guide assessment and
14 Soil Pollution
remediation of soil contamination. Facilities that are currently uncontaminated have the
opportunity to maintain conditions that allow unrestricted land-use. For these facilities, the
minimum standards will be determined by the Tier I criteria or equivalent objectives. Older
facilities, however, were often operated under different standards and environmental man‐
agement practices than are currently acceptable.
2.10. Soil pollution risks
There is an increasing use of risk-oriented policies to deal with the local effects of soil pollution.
The risks that such policies deal with are: human health risks and can also include eco-
toxicological risks. These risks are expressed in terms of negative effects and chances between
0 and 1 that such negative effects will occur. Examples of areas where risk-oriented policies
are applied to soil pollution include the United States of America [23], Canada [24] and
countries in the European Union [25]. Historically, these risk oriented policies have followed
the abandonment of policies aimed at restoring soils to their original ‘clean’ state.
Risk-based criteria or standards, developed in the framework of risk oriented policies, are
applied to risks estimated with deterministic methodologies, following the steps of hazard
characterization, appraisal of exposure and risk characterization, while using exposure-risk
relations established beforehand. Risk-based criteria have been applied to decisions about soil
remediation in the form of soil clean-up standards [26], to the use of soils for specific purposes
and in the United States also to sediment management [27]. The risk-oriented policies consid‐
ered here [28], assume that background exposure to pollutants carries no risk and that a
specified level of soil pollution carries a maximum tolerable or maximum acceptable risk for
organisms living locally. The latter is the main basis for standard setting.
In part, risk-oriented soil pollution legislation includes policy goals that are qualitative [29].
For instance, the primary UK legislation on contaminated soil defines land as contaminated
in need of risk management ‘if significant harm is being caused or there is a significant
possibility of such harm being caused’ [29]. Mostly, however policies have resulted in specific
quantitative values for maximum tolerable or acceptable soil pollution. The analysis of such
values used in different industrialized countries has shown that there are very large differen‐
ces, roughly up to a factor [30]. According to Provoost et al. [31], these differences to a large
extent originate in different political choices (e.g. including or excluding ecotoxicity) and in
different assumptions as to the modeling of exposure to soil pollutants, including site related
factors, such as soil type and building constructions [31].
2.10.1. Risks related to one soil pollutant
In practice, there are several matters which are at variance with the proper establishment of
actual risk related to one soil pollutant. These are: the absence of standards for pollutants,
neglect of background exposure, and neglect of routes of exposure to soil pollution, neglect of
available dose- effect studies and neglect of biological availability. These will now be discussed
in more detail.
a. Absence of quality standards
When data regarding soil pollutants are available, they should be compared with quality
standards reflecting maximum tolerable risk of exposure. However, such standards are not
always in place. For instance, of the volatile organic carbon compounds detected in ground‐
water samples by the US Geological Service, were unregulated- with no standards in place [32].
Similarly Patterson et al. [33] found a variety of brominated ethenes in Australian groundwa‐
ter, all lacking standards.
b. Neglect of background exposure
For a proper estimate of soil pollution related risks, exposure to specific soil pollutants should
be evaluated in combination with exposure to the same substance that is not related to local
soil contamination. Several countries, such as Canada, Germany, Spain and Belgium, do
indeed establish soil clean-up standards while considering background dietary and inhalatory
exposure but others, e.g. Sweden, Norway and the Netherlands, do not [34]. Neglecting
background exposure or specific types of background exposure may have implications for risk
estimates.
c. Neglect of routes of exposure to soil pollution
In evaluating exposure to soil pollutants, assumptions regarding exposure routes are impor‐
tant. In this respect difference between countries may be noted. Soil clean-up standards for
lead of Norway and Sweden differ in part because in Sweden the dominant exposure route is
assumed to be by drinking water and in Norway it is thought to be by drinking water and
ingestion of soil [34].
Inhalation of household dust and soil particles is not always taken into account in govern‐
mental decision making about risks of soil pollution. For instance, in the Netherlands inhala‐
tion of soil particles has been neglected as an exposure route, but in e.g. Spain it is not [34].
Neglect of inhalation would seem at variance with existing studies. Nawrot et al. [35] have
studied the effects of cadmium pollution in soil (around former thermal zinc plants) and found
a significant increase in lung cancer risk correlated with cadmium exposure. They plausibly
explain this in terms of exposure of lung tissue to cadmium present in inhaled soil and
household dust particles.
Household dust particles have also been found to be important in the exposure of children to
pesticides in agricultural settings.
d. Neglect of available dose-effect studies
Akesson et al. [37] have analyzed the effects of low environmental cadmium exposure in
an epidemiological study of Swedish women in the Lund area, being 53-64 years of age,
excluding women from areas with soils heavily polluted by cadmium. Akesson et al. [37]
found associations between the internal dose of cadmium and tubular and glomerular
kidney effects, which may represent early signs of adverse effects. Women with diabetes
seemed to be at increased risk of experiencing such early signs. In view of these data it
seems plausible that at a background exposure that is common in Sweden, old women in
the general population may be at risk for adverse cadmium effects [34] and that even a
16 Soil Pollution
modest increase in cadmium exposure due to polluted soil may lead to added risk.
However, when establishing soil clean-up standards in Sweden this background exposure
has been neglected [34]. Nawrot et al. [35] have studied the relation between mortality and
cadmium body burden in Belgium. They obtained evidence that total mortality and non-
cardiovascular mortality may be elevated at cadmium body burdens which can be found
among the population not living on soils that are currently considered to be a health risk.
Similarly there are now strong indications that the negative effects of lead on the neurophy‐
siologic and sexual development may well be found at the level of background exposure
common in Western European and US cities [37], though soil pollution policy, at least in
European counties, assumes that such background exposure is safe [14].
2.10.2. Ecotoxicological risks
Maximum acceptable or maximum tolerable ecotoxicological risks are usually derived from a
limited number of studies concerning single species under laboratory conditions. Laboratory
conditions may be very different from actual conditions in the field, and thus findings in the
field are often at variance with laboratory studies [35]. In field studies it has been found that
several factors which tend to be neglected in laboratory studies may strongly impact toxic
effects of soil pollutants. These include among others: density and adaptability of populations
of affected organisms, the presence of other environmental stress factors and the presence or
absence of specific landscape elements such as buffer strips [35].
2.10.3. Biological availability
Biologically available pollutants determine risk [3]. Biological availability may vary strongly
for different types of organisms [36]. Biological availability of a compound in a specific soil is
also dependent on physical, chemical and biological and spatial factors [35]. Examples of such
factors are pH, the amount and nature of organic and mineral compounds also present and
the presence of organisms that can mobilize soil pollutants [37-39]. In practice, biological
availability may be much at variance with total concentrations [40].
2.10.4. Combination effects
a. Limited accounting of combination effects

As to the overall risk of soil pollutants, cumulative effects of the combination of substances
present in soils should be considered. However actual standard setting practice has largely
focused on criteria relating to one element or compound. In some cases there are criteria for
groups of compounds [34]. Such criteria limit the amount (in g/kg soil) of groups of compounds
but often do not address the possibility that the risk per unit of weight may be different for
different compounds. An exception to this is criteria for the presence of halogenated dioxins
and benzofurans and planar biphenyls. The establishment of risk in case of exposure to these
compounds uses addition on the basis of equivalent toxicity [40]. This is a major improvement,
though it has been pointed out that this approach may still underestimate the risk of neuro‐
development effects [41].
b. Importance of combination effects
Combination effects may be important in two respects. Firstly, coexisting soil contaminants
may impact each others’ biological availability [43]. Secondly, exposure to a combination of
pollutants may be associated with antagonistic, synergistic and additive interactions of these
pollutants, impacting their effect on organisms [44-47]. Some risks of pollutant mixtures can
be predicted on the basis of existing knowledge. For instance there is a fair chance that there
will be dose additivity when effects are receptor mediated [48]. Also in case of narcotic effects,
joint-mixture ecotoxicological effects may be predicted [48]. If responses are dissimilar,
response addition may be used [49]. A methodology to deal with the ecotoxicity of mixtures
giving rise to both dose-additive and response-additive effects has been proposed [48]. This
two step model evaluates mixture toxicity for the same mode of action with concentration
additivity and the toxicity for different modes of action with response additivity. For deter‐
mining the severity of ecotoxicological effects in case of heavily polluted soils (in which legal
maximum tolerable levels for one or more substances are exceeded), a systematic approach to
combination effects based on a mixture of concentration addition and response addition has
been proposed [41].
2.10.5. Remedies for shortcomings
Remedies would seem possible which would allow for a significant improvement in risk
estimates. Unregulated substances can get standards. Standards may be regularly updated on
the basis of new dose-effect studies. Risk estimates can include both background exposure and
all exposure routes for local soil pollution. Estimates of biological availability can be integrated
in risk assessments and improved by better testing of bioavailability or by in-vivo monitoring
[51]. The deficiencies in taking account of combination effects in ecotoxicity, discussed in
section may be addressed by directly testing of ecotoxicity, when the focus is on ecosystem
functioning [52,53]. However it should be noted that small effects on the functioning of
ecosystems may have large effects over time [35]. This necessitates large numbers of replicate
tests that may well be beyond routine practice [35].
In determining combination effects on human health, direct testing on humans is an ‘unethical

option’. However biomarker-based monitoring of some aspects of soil pollution relevant to
humans may be an option. For instance Roos et al. [54] have applied a biomarker based test to
original and remediated soils that were contaminated by a variety of polycyclic aromatic
hydrocarbons (PAH). They tested the expression profile of cytochromes P 450 [54]. Xiao et al.
[55] have measured genotoxic risk of soil contamination using an in-vitro assay with Salmo‐
nella. Though the relation between such biomarker-based data gathered and the in-vivo risks
awaits further elucidation, the application of tests based on biomarkers for soil pollution is an
interesting option in dealing with combination effects on humans.
Also, estimates of risk may be derived from biomarkers which may be monitored in people
exposed to soil pollution. Such biomarkers have emerged from epidemiological studies
considering the combined effect of substances. An illustration thereof is the study by Lee et al.
[56] which found a graded association of the concentration of blood lead and urinary cadmium
18 Soil Pollution
concentrations with oxidative stress related markers in the US population. This suggests that
oxidative stress may be useful as a biomarker for combination effects. It has furthermore been
proposed to evaluate effects of exposure to nitroarenes by measuring haemoglobin adducts
[57], and of mixtures of volatile organochlorines by measuring glutathione conjugative
metabolites [58]. Bioassays based on aryl hydrocarbon (Ah) receptor mediated mechanisms
have been proposed which will allow a better alternative to the measurement of polyhalogen‐
ated aromatic hydrocarbons [41]. Another option is to estimate risks to human health by taking
into account cumulative combination effects in line with established cause-effect relations and
research into the effects of actual combinations. It has been shown that risks of compounds
with the same targets and the same modes of action may be estimated on the basis of concen‐
tration addition, while including toxicity equivalence factors for the compounds involved [59].
This has been shown to apply to receptor-mediated-and reactive mechanisms of toxicity,

provided that no chemical reactions occur between the components of the mixture considered
[60]. Currently this approach is applied to halogenated dioxins, benzofurans and planar
polybiphenyls, though non-linear interactions are not completely absent in this category of
compounds [61], and neurodevelopment effects may be underestimated, as pointed out before
[41]. Extension of this approach is possible to e.g. polycyclic aromatics, including heterocyclic
polycyclic aromatics [18,62] organophosphates that inhibit the enzyme cholinesterase [44,63],
compounds that bind to estrogen receptors [64-66], carcinogens [67], a variety of petroleum
products [68] and compounds that inhibit the MXR efflux pump [69].
3. Ecological risk assessment
Ecological risk assessment (ERA) is a process of collecting, organizing, and analyzing envi‐
ronmental data to estimate the risk or probability of undesired effects on organisms, popula‐
tions, or ecosystems caused by various stressors associated with human activities. The basic
principles of ecological risk assessment are described in numerous papers [70-72]. All varieties
of ERA are associated with uncertainties. The value or usefulness of the different ERA
methodologies depends on the uncertainty, predictability, utility, and costs. There are typically
two major types of ERA. The first is predictive and is often associated with the authorization
and handling of hazardous substances such as pesticides or new and existing chemicals in the
European Union. This kind of ERA is ideally done before environmental release. The second
type of ERA could be described as an impact assessment rather than a risk assessment, as it is
the assessment of changes in populations or ecosystems in sites or areas already polluted. The
predictive method is based on more or less generic extrapolations from laboratory or controlled
and manipulated semi field studies to real-world situations. The descriptive method is more
site specific as it tries to monitor ecosystem changes in historically contaminated soils such as
old dumpsites or gas facilities or in field plots after amendment with pesticides or sewage
sludge, for example.
Often ERA is performed in phases or tiers, which may include predictive as well as descriptive
methods. The successive tiers require, as a rule of thumb, more time, effort, and money. The
paradigm or schemes for ERA may vary considerable from country to country, but often consist
of an initial problem formulation based on a preliminary site characterization, and a screening
assessment, a characterization of exposure, a characterization of effects, and a risk character‐
ization followed by risk management. Although exposure assessment is often just as or even
more important, this chapter primarily considers effect assessment. In most European
countries, ERA of contaminated soils consists of rather simplified approaches including soil
screening levels (SSL) (a.k.a. quality objectives, quality criteria, benchmarks, guideline values)
and simple bioassays for a first screening of risk [73-75]. National research or remediation
programs have led to the development of a large variety of guideline values.
Although hard to categorize, most fall into two categories: generic or site specific. While the
site-specific guidelines require a characterization of pH, organic matter, etc., at the site, generic
guideline values are more independent of modifying factors and hence straightforward to
legislate. Three major classes of tools for assessing ecological effects may be identified:
standardized ecotoxicity experiments with single species exposed under controlled conditions
to single chemicals spiked to soil; ex situ bioassays, here defined as simple laboratory assays
where single species are exposed to historically contaminated soils collected in the field; and
finally monitoring, analyzing, and mapping of population or community structures in the
field. Furthermore, mesocosm, lysometer, or terrestrial model ecosystems (TME) may be
useful; these may be considered as large (multispecies) bioassays or ecotoxicity tests [76-79].
TMEs have the advantage that they operate with the (relatively) undisturbed intrinsic soil
populations that make up a small food web. TME hence allow the assessment of effects of
toxicants that are mediated through changes in food supply or competition and predation.
One of the keystones in deriving environmental quality criteria is the use of standardized
terrestrial test procedures. The emphasis of these prognostic tests is on reproducibility,
standardization, international acceptance, and site independence. Although increasing in
numbers, relatively few terrestrial tests are still approved by the International Standardisation
Organisation (ISO) or Organization for Economic Cooperation & Development (OECD).
However, other tests have shown promising results and are likely to be prepared for stand‐
ardization in the future [80].
However, the major problem in using simple laboratory tests to extrapolate to contaminated
land may not be the limitations of test species and the natural variation in species sensitivity.
The problems associated with extrapolating from one or a few species, exposed under
controlled and typically optimal conditions, to the complex interaction of species and chemi‐
cals found in most contaminated ecosystems should also cause concern. Although single-
species laboratory tests with spiked materials have their obvious benefits, e.g., they measure
direct toxicity of chemicals and interpretation is therefore simple, supplementary tools are
often needed. Bioassays, as defined in this context are one of the more frequently used higher-
tier alternatives. Basically the same test species may be used in bioassays for assessing the risk
of a specific contaminated soil as in standard laboratory tests. However, bioassays have the
advantage, compared to the use of spiked soil samples, that the exact toxicity of a specific soil
may be accessed directly: this includes the combined and site-specific toxicological effect of
the mixture of contaminants and their metabolites. Furthermore, the in situ bioavailability of
20 Soil Pollution
that specific soil is (at least almost) maintained in the laboratory during the exposure period.
Several studies have shown a reduction in bioavailability and/or toxicity of soils with an old
history of contamination [80-85].
Bioassays are therefore often considered a more realistic tool than generic soil screening levels
based on spiked laboratory soils. However, a number of uncertainties or problems may be
associated with the use of bioassays and the interpretation of their results. First, the test species
are still exposed to the contaminants in a relatively short period compared to the permanent
exposure condition found at contaminated sites. Furthermore, they are exposed under more
or less optimal conditions, in that stressors such as predation inter- and interspecies competi‐
tion, drought, frost, and food depletion are eliminated during exposure. Finally, typically only
a few species are tested individually.
To compensate for some of the limitations just described, contaminated soil may be assessed
using multispecies mesocosms, lysometers, or TME. In these, species interactions may be
evaluated by manually introducing several species to the systems or monitoring the intrinsic
populations of the soil. Natural climatic conditions may be included if the test system is kept
outdoors. However, if we want to get a more realistic and large-scale picture of the impact
caused by, for example, pesticide use or sewage sludge application, or to assess the environ‐
mental health at waste sites, industrial areas, or gas works, it is often necessary to conduct
some kind of field observations. Several case studies exist in which field studies have success‐
fully elucidated the ecological risk of specific activities or the ecological impact at specific sites
[85-87]. The small single-species bioassay, large multispecies TME, and field surveys have
some drawbacks in common. First of all, it may be difficult to actually link the observed effect
to a specific toxic component in the soil. Which of the many substances is actually causing the
majority of the observed effects, or is it perhaps a combination of effects? For a hazard
classification of soils or a ranking of soils this may not be so important.
However, to evaluate potential risk-reduction measures or risk management procedures it

may be important to identify the most problematic substances. A comparison of soil screening
values with measured concentrations for each chemical present at a site may be helpful to
identify the most likely group of substances causing the observed effect. Other possible tools
may include a toxicity identification evaluation (TIE) approach [88]. The TIE approach is a
relatively new method, which aims to identify groups of toxicants in soils with mixed pollu‐
tion. Potentially toxic components present in the soil are fractionated and determined, and the
toxicity of each individual fraction is determined by a Lux bacteria-based bioassay or the
Microtox bioassay. Although perhaps promising, TIE is a time-consuming and hence costly
procedure not yet used routinely. Another crucial issue when analyzing the result of bioassays,
TME, and field studies is the presence or absence of a proper reference site or soil. The control
soil should in principle resemble the contaminated soil in all relevant parameters, e.g., texture,
pH, organic matter, waterholding capacity, and nutrient content, a practical problem that very
often is difficult to solve. The lack of adequate control or reference sites may, however, be
conquered at least partially by the use of multivariate techniques [89], which relate the species
composition and abundance to gradients of pollutants. It is not the intention of this chapter to
present a review of statistical tools for ecological risk assessment, and hence a detailed
discussion about the use of these is not given.
Figure 2. The eight steps in the US-EPA framework for risk assessment of contaminated Superfund sites. DQO = data
quality objectives [89]
However, it is obvious that increased computer power and the presence of new easy-to-use
software tools have increased the possibility to move away from more conventional univariate
statistics such as analysis of variance (ANOVA) to more powerful multivariate statistics that
22 Soil Pollution
use all collected data to evaluate effects at a higher level of organization. Statistical methods
such as the power analysis may also be very useful in planning and designing large-scale
ecotoxicity studies such as mesocosms,TME, or field surveys.
The US-EPA has published an Ecological Risk Assessment Guidance, which should be
followed when assessing risks at Superfund sites. As all sites are considered unique this should
always be done in a site-specific manner. The ERA process suggested by the US-EPA for
Superfund sites follows an eight step process, which can be broken down into four categories,
i.e. 1) planning and scoping, 2) problem formulation, 3) stressor response and exposure
analysis and 4) risk characterisation. Essential for all steps are a negotiation and agreement of
the need for further action between the risk assessor, the risk manager and other stakeholders,
the so-called scientific-management decision points (SMDP).
SMDP made at the end of the screening-level assessment will not set an initial cleanup goal.
Instead, hazard quotients, derived in this step, are used to help determine potential risk. Thus,
requiring a cleanup based solely on those values would not be very likely, although it is
technically feasible. There are three possible decisions at the SMDP:
1. There is enough information to conclude that ecological risks are very low or non-existent,
and therefore there is no need to clean up the site on the basis of ecological risk.
2. The information is not adequate to make a decision at this point, and the ecological risk
assessment process will proceed.
3. The information indicates a potential for adverse ecological effects, and a more thorough
study is necessary.
In the Netherlands contaminated sites are first determined using a set of soil screening levels
called target and intervention values, which take both human and ecological risks into account.
At seriously contaminated sites remediation or other soil management decisions are required
if the risks cannot be neglected based on a site-specific ecological and human risk assessment,
and the chance for dispersion of the contaminants. Until now, the ecological risk assessment
has been based on chemical analysis, including a Decision Table harbouring critical dimensions
of the impacted area. The United Kingdom and Canada have also developed framework for
ecological risk assessment of contamination land. A cornerstone in the UK framework of ERA
is the connection to the statutory regime for identification and control of land potentially
affected by contamination. The UK framework is based on schemes found in e.g. USA, Canada
and the Netherlands. Like these it is a based on a tiered approach where the initial Tier 0 aims
to determine whether a site falls under the Part IIA of the legislation. It involves the develop‐
ment of a Conceptual Site Model (CSM), which described what is already (historically) known
about the site, e.g. whether there is a likely source-pathway-receptor linkage. The conceptual
site model is followed by an initial screening phase (Tier 1) and an actual site-specific charac‐
terisation (Tier 2). Tier 1 is a simple deterministic comparison of chemical residue data and the
soil quality guideline values supplemented with simple soil-specific toxicity testing. The final
step (Tier 3) involves more detailed in-situ studies and for example ecological modelling based
on a more advanced ecological theory. Tier 3 is not likely to be conducted at many sites.
3.1. Descision support system for ecological risk assessment
Ecological Risk Assessment is often a complex process with many variables to take into
account. ERA involves many stakeholders and all have to be dealt with in a clear and consistent
way. A stepwise or tiered approach is therefore useful to overcome the complexity of an ERA.
In order to structure all the information collected, a Decision Support System (DSS) can be
used. Each tier will lead to a decision to proceed or to stop. A number of decisions supporting
systems or frameworks have already been developed in other countries, e.g. UK, the Nether‐
lands and the USA. The DSS presented here is based on basic principles also common in the
methodologies used in the USA and UK. However, in the present DSS measures of bioavail‐
ability and the use of the Triad approach may be built into the system more systematically.
This chapter introduces the overall framework of a novel DSS including the Triad approach
and the challenge to weight and scale results used in that process.
3.2. Framework for ecological risk assessment
Rutgers et al. (2000) developed a basic flowchart for Ecological Risk Assessment [90], which is
used as the backbone of the decision support system (DSS) presented in the Figure 5.
The DSS is separated in three different stages, i.e.

• Stage I. Site characterization and description of land-use.
• Stage II. Determination of ecological aspects.
• Stage III. Site-specific tiered assessment (the Triad):

– Tier 1. Simple screening
– Tier 2. Refined screening
– Tier 3. Detailed assessment
– Tier 4. Final assessment.

Each of these four tiers is based on a weight of evidence (WoE) approach combining three lines
of evidence (Chemistry, (eco)Toxicology and Ecology).
3.2.1. Boundaries of the DSS
The DSS in this chapter is not a full and comprehensive document for managing risk of
contaminated land. It focuses strongly on supporting decisions made when considering risk
to the terrestrial environment. Therefore it addresses only indirectly the risk to ground water
and associated (connected) fresh water systems. Nevertheless information about e.g. reduced
bioavailability may be useful when assessing potential risk for leaching of contaminants to
ground water or fresh water. Furthermore, it is important to realise that the management of a
contaminated site is more than assessing ecological risk. Issues like for example risk for
humans, availability and cost of remediation solutions, development plans for the vicinity or
the region are equally important.
24 Soil Pollution
Figure 3. Basic flowchart for ecological risk assessment [90]

3.2.2. Stage I — Site characterisation and land-use definition
The first step in the DSS is to establish what is often referred to as a Conceptual Site Model. It
aims at involving as many stakeholders as possible in order to describe site characteristics and
to review all available information from the site, e.g. historical information about land-use,
investigation of whether the site may be regulated under specific directives, obvious data gaps
and urgency for reaction and data collection. The spatial borders of the site should be defined
and the current and the future landuse have to be defined. Consultation between administra‐
tors, planners and experts therefore has to take place as early as possible in the process.
a. Initial requirements in the DSS
An inquiry among all stakeholders should be conducted as one of the first initiatives. The aim
should be to collect as much information about soil characteristics as possible.
b. Defining land-use
One of the first actions to be taken among all stakeholders is to decide which landuse is required
for the site, as this will determine the required data collection and testing. Many land-uses may
be defined, but generally the four following overall categories of land-use classes are used:
• industrial area (including infrastructure and pavement).
• urban/residential area (including recreational and green areas).
• agricultural area.
• nature area.
c. When is an ecological risk assessment needed?
Most often a site specific ERA will be initiated only when soil concentrations exceed soil
screening levels. However, this may not in itself be a sufficient criterion to go through the entire
ERA procedure. Some boundary conditions, based on the present and future type of land-use,
the level of contamination and various ecological considerations have to be met in order to
rationalize an ERA. The experts and the rest of the stakeholders should answer a number of
simple questions in order to conclude whether the required boundary conditions are fulfilled.
3.2.3. Stage II — Determination of ecological aspects
At stage II, site-specific ecological features and receptors relating to the land-use defined in
Stage I need to be outlined. This includes aspects like key species and life support functions.
The potential ecological receptors should be identified in order to determine whether potential
source-pathway-receptor linkages can be established. This includes not only ecological
receptors directly linked to the site but also those linked indirectly e.g. through leaching of
contaminants to connected fresh water systems or (migrating) birds or mammals feeding in
the area. In Table 9 some examples are given of land-use and related ecological aspects. This
table can be used as a starting point for the selection of ecological aspect. Experts from
ecotoxicology and ecology should be involved in the selection of ecological aspects.
26 Soil Pollution
3.2.4. Stage III — Site specific instruments (the Triad)
If after finalising Stage I and Stage II it is still considered that there is a need for a site specific
evaluation of ecological risk the process continues to Stage III using the weight of evidence
approach described below.
3.2.5. Weight of evidence approaches
In order to deal with conceptual uncertainties in a pragmatic way, it has been proposed to use
weight of evidence (WoE) approaches for ERA [90-93]. The rationale is, like in justice, that
many independent ways to arrive at one conclusion will provide a stronger evidence for
ecological effects, making ERA less uncertain.
In the sediment research area the application of WoE started at an early stage and was called the
Sediment Quality Triad. For terrestrial ecosystems WoE approaches and the Triad are still in a
developing stage. The Triad approach is based on the simultaneous and integrated deploy‐
ment of site-specific chemical, toxicological and ecological information in the risk assessment as
given in Figure 6. The major assumption is that WoE in three independent disciplines will lead
to a more precise answer than an approach, which is solely based on, for example, the concentra‐
tions of pollutants at the site. A multidisciplinary approach will help to minimise the number of
false positive and false negative conclusions in ERA. It also gives acknowledgement to the fact
that ecosystems are too complex to analyse in one-factorial approaches.
Figure 4. Schematic presentation of the integration of three fields of research according to a Triad [92]
• Chemistry: The concentration of contaminants in the environment (totals, bioavailable),

accumulated in biota, or modelled via food-chains is used for calculation of risks on the basis
of toxicity data from the literature.
• Toxicology: Bioassays with species across genera are carried out in order to measure the
actual toxicity present in environmental samples from the site.
• Ecology: Field ecological observations at the contaminated site are compared to the
reference site. Deviations from the reference site, which can be plausibly attributed to the
contamination levels, are funnelled into the Triad.
3.3. Using the TRIAD in site specific assessment of contaminated soil
Triad is a powerful weight of evidence approach originally developed in order to evaluate

sediment quality. In the terrestrial compartment less experience is available on the practical
use of the Triad. This chapter describes the use of Triad in more detail and gives an insight
into some of the important decisions risk assessors have to make when conducting the Triad
in practise, e.g. how to scale, weight and integrate the outcome of the various investigations.
The Triad approach exists of three lines of evidence (LoE), the so-called Triad “legs”, i.e.
chemistry, (eco) toxicology and ecology. The Triad approach includes a tiered system in which
each consecutive tier is increasingly fine-tuned to the site-specific situation. In the first tier the
research is simple, broad and generic. In later tiers more specific and complex tests and
analyses may be used. For each of the LoE in the Triad there are a variety of analyses or tests
that can be chosen. Some examples are:
• Chemistry: Measurement of total concentrations, bioavailable concentrations, bioaccumu‐

lation, etc.
• Toxicology: Bioassays (in field and/or in lab), biomarkers etc.
• Ecology: Field observations of vegetation, soil fauna, micro-organisms, etc. In Chapter 6, a

number of tests or tools that are for suitable for use in each tier are presented for the
chemistry, toxicology and ecology LoE.
3.4. Decision charts in ecological risk assessment of contaminated sites
3.4.1. Flowcharts
This chapter is an attempt to present a decision support system, which can guide risk assessors
in their assessment of site-specific ecological risk. A number of site-specific questions need to
be answered before a final decision on performing an ecological risk assessment can be made.
This chapter introduces a flow chart for ecological risk assessment of contaminated sites. The
flowchart is presented as decision trees as shown in Figure 8 together with a more in-depth
introduction to the relevant questions that needs to be addressed and answered when
performing a site-specific ecological risk assessment.
28 Soil Pollution
3.4.2. Decision making in ERA

The assessment of ecological risk is performed stepwise in tiers. Higher tiers represent
gradually more and more complex studies, but also more expensive and laborious studies. The
full site-specific risk assessment covers four tiers, i.e.
• Simple screening: Tier 1.

• Refined screening: Tier 2.
• Detailed assessment: Tier 3.
• Final assessment: Tier 4.
The main principle in going from a simple screening over a more refined screening to a detailed
assessment of the contaminated site is to minimize time and effort. The actual performance of
the risk assessment and use of the various tiers may be very site-specific.
a. Tier 1 — Simple screening
After deciding in the two first stages of the ERA that ecological concern needs special consid‐
eration, the risk assessment starts typically with a simple evaluation at the screening level. This
is done in order to minimize costs until new information indicates the need for further
assessment and more sophisticated studies. Therefore, the tools used in the first screening need
not only to be reasonably quick and easy, but also relatively cheap. The tools for use in Tier I
are described in more detail in the toolboxes C1, T1 and E1. On the basis of the results of
instruments used in Tier 1 it is decided to either stop further assessment or continue to a higher
tier.
b. Tier 2 — Refined screening
Tier 2, still considered being at the screening level, aims at refining the measurement of
exposure and at the same time to provide further insight into the toxicological and ecological
properties of the contaminated soil. Tier 2 deviate from the conservatism normally associated
with the use of total concentration in the risk assessment by taking (rough) estimations of
bioavailability into consideration in the chemical LoE. A better screening of the toxicological
and ecological properties of the soil compensates for the reduced conservatism in the Chem‐
istry LoE of the Triad. The tools for use in Tier 2 are described in more details in the toolboxes
C2, T2 and E2. On the basis of the results in Tier 2 a decision should be made to either stop
further assessment or continue to a higher Tier.
c. Tier 3 — Detailed assessment
The tools in Tier 3 differ from the ones used in Tier 1 and Tier 2 in that they are more laborious,
costly and may take longer. On the other hand they are (often) more realistic and/or ecological
relevant in order to give a more comprehensive assessment of the ecological risk at the specific
site. The stakeholders should beforehand negotiate a minimum set of tests. Is it for example
necessary to consider all trophic levels in the toxicological and ecological LoE? Or does the
land-use suggest otherwise? Is it necessary (or possible) to estimate the bioavailability of all
the substances exceeding their SSL? If not, how are the non-investigated substances dealt with?
The tools described for use in Tier 3 are described in more details in the toolboxes C3, T3 and
E3. Depending on the results from Tier 3 a decision should be made to either stop further
assessment or continue with an even more detailed assessment in Tier 4
d. Tier 4 — Final assessment
In Tier 4, the aim of the studies is to answer any remaining questions and to decrease existing
uncertainties and this may often require more in-depth research. Tools in Tier 4 can be similar
to tools of Tier 3, but more focus has to be on site-specific circumstances. For example bioassays
should be done with organisms, which normally occur at the site. Furthermore, it may be more
relevant to consider ecological effects outside the contaminated area on e.g. predators or
herbivores feeding in the area or effects in adjacent fresh water systems. This Tier requires
specialised knowledge and experience with ERA, which implies that costs can be high and
only a limited number of people may be able to perform the tests. Generally only on a very
limited number of site evaluations will include investigations at this level. If the results of Tier
4 still indicate risk there are basically two possible solutions. Accept the risk and leave the
contamination or remove (parts of) the contamination.
3.4.3. Screening tools
3.4.3.1. Triad based selection of methods
For each of the three Lines of Evidence (LoE) in the Triad various methods or tools are available.
In order to facilitate the selection of appropriate tools in the right context, the tools have been
compiled in subclasses or toolboxes. Each of these is a collection of tools considered to be
potentially useful in the designated tiers and LoE of the Triad, i.e. chemistry, toxicology and
ecology. Furthermore, the tools are arranged according to their complexity, price and practic‐
ability or in other words depending on whether they are most useful for screening or detailed
assessment, i.e.
• Toolbox C1. Chemistry tools for simple screening.

• Toolbox T1. Toxicology tools for simple screening.
• Toolbox E1. Ecology tools for simple screening.
• Toolbox C2. Chemistry tools for refined screening.
• Toolbox T2. Toxicology tools for refined screening.
• Toolbox E2. Ecology tools for refined screening.
• Toolbox C3. Chemistry tools for detailed assessment.
• Toolbox T3. Toxicology tools for detailed assessment.
• Toolbox E3. Ecology tools for detailed assessment.
• Toolbox IV. Various tools for the final (Tier 4) assessments.
a. Toolbox C1 — Chemistry tools for simple screening

30 Soil Pollution
At the very first stage of the ERA process, total concentrations of all relevant chemicals are
individually compared to soil screening levels (SSL) in order to evaluate whether there is a
need for a site specific assessment of ecological risk. In the current Stage III of the ERA, this
first generic evaluation of risk is followed by a more site-specific screening of risk including
information from all three lines of evidence in the Triad. In the Chemistry part of the Triad
more site-specific information is collected by:
Refining and targeting the comparison of soil concentrations with soil related benchmarks for
site-specific purposes.
Incorporation of the accumulative risk of a mixture of contaminants by calculating the toxic
pressure (TP) of a mixture and by doing so generating more site-specific insight to the potential
ecological impact of a contaminated site. Each of these steps can be done separately or in
combination, e.g. the TP can be calculated using existing SSL or using new developed bench‐
marks based on either NOEC or EC50 values or site-specific benchmarks can be compared to
soil concentrations individually. The approach entirely depends on the strategy taken by the
stakeholder group and the availability of data.
b. Toolbox T1 — Toxicology tools for simple screening
The main objective of the selected toxicity tests or bioassay at Tier 1 should be to screen the
soil for presence of toxic compounds. This includes toxic degradation products or compounds,
which are not routinely included in various national analytical programs for contaminated
sites. This Tier is the first screening level of the ERA and the cost in form of manpower and
money should hence be relatively low.
c. Toolbox E1 — Ecology tools for simple screening
Ecological surveys or monitoring studies are generally considered a time consuming effort
performed by experts. This is in most cases true, wherefore detailed surveys normally take
place in higher tier assessment. However, in order to ensure that also ecological information
is collected and used in the Triad already in the screening phase, it is recommended to perform
a limited examination of the site. A survey of the area with special focus on visible changes in
e.g. plant cover or presence or absence of specific plants, trees or scrubs may indicate ecological
damage, which can be associated to contaminants present at the site.
If any aerial pictures areavailable from the area these may give valuable information about the
plant cover also historically, which may be helpful in identifying parts of the site where the
impact may be highest (hot spots). At this stage the conclusion can in most cases only be
indicative. Therefore if the results from the other line of evidence may cause any doubt or the
survey indicated potential impact, it is recommended to either continue with a more refined
screening in Tier 2 or go directly to the detailed assessment in Tier 3.
d. Toolbox C2 — Chemistry tools for refined screening
Selective solvent extraction

It may be considered useful to adjust the estimate of exposure by taking bioavailability into
consideration and hereby deviating from the conservatism normally associated to the use of
Figure 5. Equipment for measuring luminescence of Vibrio fischeri [94]
Figure 6. Simple survey of the site [95]

32 Soil Pollution
total concentration in the risk assessment. The principle in this refinement of the ecological
risk assessment is to extract a more ecotoxicologically relevant fraction of the contamination
than the total concentration. The latter generally tends to overestimate the risk of historically
contaminated soils. In this screening phase no attempt is made to estimate the freely dissolved
or readily bioavailable concentration of contaminants. Table 10 explains principal studies that
employed chemical extractants to evaluate bioavailability.
Table 5. Outline of principal studies that employed chemical extractants to evaluate bioavailability
Table 5. Outline of principal studies that employed chemical extractants to evaluate bioavailability
Instead the fraction of the contaminants is extracted, which can be directly compared to the existing soil screening levels. This is
considered to be a relatively simple and quick method to screen for potential risk of contaminants in a more realistic way than
Instead
using total the fraction
concentrations. of the contaminants
The extracted concentration (mg is extracted, whichtocan
kg-1) is compared be directly
the SSL compared
and the result used in to thethe
Triad. It is
therefore a prerequisite of this comparison that the extractability in the tests (with spiked soils) used for deriving SSL to
existing soil screening levels. This is considered to be a relatively simple and quick method is close to
100% byscreen for potential
the methods risk short-term
used. In most of contaminants in aweeks)
tests (< four moreitrealistic way thantousing
will be reasonable assume total
that concentrations.
only little “true” ageing or
The extracted
strong sequestering concentration
occurs (mg kg-1)
and hence a majority of the is compared
spiked chemicalstoare
thestill
SSL and thewith
extractable result
mildused in the
organic Triad.
solvents. However,
for most methods this still has to be fully validated.
It is therefore a prerequisite of this comparison that the extractability in the tests (with spiked
Organicsoils) used
solvents mostfor deriving
frequently SSL
used is close
include to 100% byin the
methanol/water methods
different ratios, used. In most
nbutanol, short-term
ethanol, testsacetate
propanpol, ethyl (< and
four weeks)
tetrahydrofuran (THF)itgiven
will be reasonable
in Table 10. The to assume
method that only
establishes little “true”
preferential ageingoforhydrophobic
partitioning strong sequestering
contaminants to the
occurs
extractant and hence
by increasing a majority
their solubility ofaqueous
in the the spiked chemicals
phase whilst removingarepollutant
still extractable
compoundswith mild
from soil organic
surfaces establishing
equilibrium conditions. No standard protocol has been adopted for mild chemical extractions in relation to bioavailability testing.
solvents. However, for most methods this still has to be fully validated.
Common methodology in literature primarily includes a soil sample to which a volume of chemical extractant is added (generally 1
– 10 g soil,15 – 25 ml extractant). This is followed by a period for mixing, e.g. vigorous mixing for10 – 120 seconds or shaking by
Organic solvents most frequently used include methanol/water in different ratios, nbutanol,
orbital shakers for up to 2 hours. The extraction studies have mostly involved PAH and insecticides (including DDT, DDE, DDD
ethanol,
and atrazine) propanpol,
[93-95]. ethyl
Studies that acetateextractability
haverelated and tetrahydrofuran (THF)
with results from givenhave
bioassays in Table 10. focused
generally The method
on uptake and
establishes
accumulation (% takenpreferential partitioning
up by earthworms of and
or plants) hydrophobic contaminants
bacterial degradation to the
(%removed). extractant
Therefore, since by
convincing
increasing
relationships between their solubility
the chemical andinbiological
the aqueous phase
tests were whilst
found removing
it may indicate a pollutant
potential forcompounds from
such extraction methods to
predict bioavailability.
soil surfaces establishing equilibrium conditions. No standard protocol has been adopted for
e. mild
Toolboxchemical extractions
T2 — Toxicology inrefined
tools for relation to bioavailability testing. Common methodology in
screening
literature
In the first primarily
simple screening includes
of Tier I focus a soil
was onsample to which
marine bacteria and aaquatic/sediment
volume of chemical extractant
living species. In Tier is added simple
2 relatively
(generally
tests with 1 –species
soil dwelling 10 g soil,15
are used–for
25aml extractant).
more This of
refined screening is the
followed by ai.e.
soil samples, period for mixing,
the earthworm e.g.tests and
survival
avoidance tests using soil invertebrates.
vigorous mixing for10 – 120 seconds or shaking by orbital shakers for up to 2 hours. The
extraction studies have mostly involved PAH and insecticides (including DDT, DDE, DDD
and atrazine) [93-95]. Studies that haverelated extractability with results from bioassays have
generally focused on uptake and accumulation (% taken up by earthworms or plants) and
bacterial degradation (%removed). Therefore, since convincing relationships between the
chemical and biological tests were found it may indicate a potential for such extraction
methods to predict bioavailability.
e. Toolbox T2 — Toxicology tools for refined screening
In the first simple screening of Tier I focus was on marine bacteria and aquatic/sediment living
species. In Tier 2 relatively simple tests with soil dwelling species are used for a more refined
screening of the soil samples, i.e. the earthworm survival tests and avoidance tests using soil
invertebrates.
Figure 7. Organisms screening in soil [98]
The habitat function of soils is often assessed using the reproduction test with Eisenia fetida.
The avoidance test with Eisenia fetida is a suitable screening test, which is less cost-intensive in
terms of duration and workload than the reproduction test, and at the same time (normally)
more sensitive than the acute test with the same species.
f. Toolbox E2 — Ecology tools for refined screening
In Tier 2 the observations from the survey may be expanded by simple on-site assessment of
the overall soil functioning or biological activity of the soils. Recommended tools include bait-
lamina sticks and simple microbial tests using general endpoints like soil respiration or C/N
mineralization rates.
34 Soil Pollution
Figure 8. Bait-lamina sticks [99]
The main principle for tests at this level is to be relatively simple and cheap but at the same
time to give valuable information whether or not the soil has lost some of its main services.
Bait-lamina sticks for example have been demonstrated useful for describing biological activity
of the soils in a general matter.
g. Toolbox C3 — Chemistry tools for detailed assessment
The objective of the tools found in this toolbox is to assess the bioavailable and freely dissolved
fraction of pollutants found in pore water of soils from contaminated sites. The methods should
(in principle) be able to mimic the fraction of organic pollutants available for uptake in biota.
The collection of methods includes various non-depleting and depleting pore water extrac‐
tions. Very few terrestrial ecotoxicity data are yet expressed as e.g. pore water concentrations.
Instead, the outcome of the methodologies in this toolbox is compared with water quality
standards.
h. Toolbox T3 — Toxicology tools for detailed assessment
The objective of the tools found in this toolbox is to evaluate the potential impact of contami‐
nated soils to fauna and plants and hereby the entire ecosystems. Some of the methods use
introduced, and not intrinsic, species. The benefit of this is a higher degree of standardisation,
as the species used in these bioassays is easy to maintain in laboratory cultures compared to
naturally occurring species. The drawback may be that their ecological relevance is less
obvious. For example the compost worm Eisenia fetida is used as a surrogate to evaluate risk
to soil dwelling earthworms. Two sets of bioassays are presented. One for directly assessing
potential risk for soil dwelling species, including micro-organisms, plants and soil inverte‐
brates, and one for assessing indirectly risk to aquatic species through e.g. leaching of con‐
taminants. It is often anticipated that soil organisms are exposed to pollutants mainly through
uptake from pore water. Therefore it may also be possible to evaluate, or at least to compare
or rank, the risk of contaminated soil samples to soil dwelling organisms on the basis of the
outcome of the aquatic test using elutriate or pore water. The choice of bioassays depends on
a number of variables, e.g.:
• The current and future land-use, i.e. targets of protection.

• The size of the contaminated area.
• The potential for ground water or surface water contamination.
• The need of many simple tests or fewer more complicated tests.
Figure 9. Simple plant tests [91]
i. Toolbox E3 - Ecology tools for detailed assessment
In this late tier of the Triad, the objective of the activities is community or population response
analysis, typically by conducting field surveys. As these studies (most often) are time con‐
suming, costly and dependent on ecologically, taxonomically and statistical expertise they are
36 Soil Pollution
most frequently done on large-scale sites with a long-term- remediation perspective. In fresh
water ecosystem community surveys have been widely used with relative success. The absence
of species from places where they would be expected to occur could be a strong identification
of unacceptable levels of contaminants. However, this type of studies has only seldom been
used for the terrestrial environment. The reasons for this are many. One of the dominants may
be the lack of a concentration gradient and obvious “upstream” reference sites at most
contaminated areas. No world-wide accepted guideline on how to plan and perform a
terrestrial field survey is available and hence no straight-forward and easy-to-follow descrip‐
tion can be given. The decision on when, where and how to conduct field surveys depends on
a number of issues, e.g. the size of the area, the land-use, the type of contaminants present,
time of the year and last but not least the time and money available to perform the study.
Nevertheless, a number of general considerations have to be made in the planning phase of a
successful field survey. These include (but are not limited to):
• Identify the targets of concern and the species to monitor.

• Elucidate the natural temporal and spatial variation before initiating a field study.
• Use statistical (power) analyses to determine the minimum number of samples or replicates
needed to emonstrate the decided difference, e.g. 25% change.
• In order to establish a cause-effect relationship, a number of confounding parameters need
to be characterized both at the reference and the test site, e.g. soil type, pH, salinity,
hydrology, nutrient- and organic matter content and the presence of other contaminants.
As no single description on how to perform ecological surveys for contaminated sites can be
given, some general considerations and useful references for this tier of the ecological risk
assessment are given below for:
• Assessing impact in the overall biological activity and organic matter breakdown.
• Assessing impact on the microbial community.
• Assessing impact on the plant community.
• Assessing impact on the invertebrate community.
Reference data from reference sites, reference samples and literature

A crucial factor in a risk assessment is the quality of reference data, because the results of the
site-specific ecological measurements or calculations are compared against these data. This is
true for as well chemical information (i.e. background levels in that region), toxicological data
from bioassays (i.e. site relevant reference soil and well characterized control soil in order to
verify the test performance) and ecological field surveys.
The reference soil should in principle resemble the contaminated soil in all relevant parameters,
e.g. texture, pH, organic matter, water-holding capacity, nutrient content. In practice, these
ideal spots are difficult to find. If there is no or inadequate reference information, effects can
only be determined in relative terms by comparison with other sites. This is usually adequate
for determining the degree of urgency and/or the need for remediation. Reference data can be
Figure 10. Litter bags [94]
obtained by including reference sites (preferably more than one) in the sampling scheme,
including reference measurements in the experimental set-up, or by obtaining reference data
from the literature or by expert based judgment.
Higher tier assessment of the impact on biological activity and organic matter breakdown
In addition to the general information about biological activity in soils generated in Tier 2 from
the bait-lamina test, other, slightly more laborious, tests may give additional information about
the overall biological activity in soil, e.g. wheat straw decomposition (litter bag test) and cotton
strip degradation. A review paper from Van Gestel et al. (2003) concluded that while the bait-
lamina gave the best reflection of the biological activity of soil animals, e.g. earthworms,
springtails and enchytraeids, the litter bag test and the cotton strip test are more indicative of
the microbial activity in the soil [96]. Knacker et al. (2003) reviewed the usefulness of five
different litter decomposition tests and concluded that the litter-bag test had distinct advan‐
tages over the others [97]. All of these simple tests only give insight into the overall activity in
soils and the breakdown of organic material. They are hence most suitable on their own in
cases of land-use with low sensitivity, e.g. industrial land. For land-uses where structural
endpoints, e.g. biodiversity or specific species, are the target of protection other endpoint(s)
should be monitored as well.
38 Soil Pollution
Higher tier assessment of the impact on the microbial community
The number of microorganisms, especially bacteria, in soil is extremely large. They differ
widely in their function and sensitivity to chemicals. Besides more classical (and simple)
measures of the microbial community like total bacterial biomass, the number of colony
forming units and substance induced respiration rate (SIR), more advanced methods for
assessing the impact of contamination on soil microorganisms have recently been made
available.
Figure 11. Microbial community [96]
This include microbial “fingerprinting” like phospholipid fatty acid analysis (PLFA) and
community-level physiological-profiling (CLPP) based on metabolic response using BIOLOG
plate systems, and the use of pollution induced community tolerance (PICT).
Higher tier assessment of the impact on the plant community
Plants interact dynamically with the physical and chemical characteristics of soils. Soil types
and site characteristics, therefore, greatly influence the occurrence of plants and their total
aboveground biomass (also called, Net Primary Production, i.e. NPP) within given climatic
conditions and human management. Vegetation cover is an important indicator of soil quality
and a diverse plant community is normally a good indication of essential soil functions such
as the decomposition process, the mineralisation rate, and the occurrence of soil dwelling
animals associated to (fresh) organic matter. Vascular plants are easy to sample. They are
immobile and hence associated to soil contamination (and airborne pollution). Plant com‐
munity parameters like plant cover, aboveground plant biomass, plant shoot/ root ratio,
species diversity and the binary occurrence (presence/absence) of specific indicator species like
metal-tolerant species may be used successfully in ERA.
Figure 12. Plant survey [95]
Higher tier assessment of the impact on the soil invertebrate community

Survey of soil biota in order to evaluate the effect of various sources of pollution on soil
communities on historically contaminated sites have not yet been used on a larger scale by e.g.
consultants. However, numerous (monitoring) studies by various research groups can be
found in the open literature. Methods of surveying include:
• Collection of soil samples followed by extraction in the laboratory.

• Extraction or collection of organisms in the field, e.g. by hand-sorting or by the application
of mustard or formalin.
• Trapping (surface dwelling) animals by the use of e.g. pit-falls. Monitoring species includes
earthworms, snails, oribatid mites, nematodes, springtails, ants, ground-living beetles and
spiders. Most of the studies have been done on metal contaminated sites (see references
below). A substantial amount of work has been put into the challenge of developing a soil
invertebrate system for evaluating risk of pollutants. The only soil invertebrate system that
is used on a regularly basis in the context of ecological risk assessment of contaminated soils
is most likely the nematode Maturity Index (MI). The system is based on the evidence, that
rapid colonising species dominate nematode communities in disturbed ecosystems. In the
40 Soil Pollution
Netherlands experience with surveys of soil invertebrates from the monitoring programme
Biological Indicator for Soil Quality (BISQ) has also been used in ERA..
Figure 13. Soil fauna sampling [98]
j. Toolbox for tests in Tier IV
The final assessment in the ERA process is not likely to be initiated for many contaminated
sites. The choice of additional tests or monitoring at this level of the ERA is bound to be very
site-specific and hence an issue for negotiation between stakeholders and experts. Accumula‐
tion in biota is included in this toolbox as the internal concentration in biota is believed, at least
to some extend, to reflect uptake and then bioavailability. An alternative in this final tier could
also be to model uptake in biota provided sufficient data is available [98].
4. Soil management program (Remediation)
4.1. Reporting norms and standards for contaminated land
The aim is to investigate whether the land has been contaminated, and if contamination has
occurred whether the contamination presents a significant risk of harm. The commonly
encountered international practice consists of three distinct reporting phases. It progresses
Figure 14. Detailed field survey [93]
from Phase 1 desktop and site walkover assessments with limited investigation and testing to
a Phase 2 detailed invasive investigation and testing for site characterisation to a comprehen‐
sive Phase 3 report with an evaluation of remediation objectives and a proposed remediation
plan, supported by control and monitoring measures for the activities.
The reporting system requires norms and standards of practice to be strictly applied, but also
must retain flexibility to allow for decisions on the contaminated status of sites to be made in
the most beneficial manner (considering ecological, social and economic aspects) also taking
into account timeframes. In some cases, urgent priority works may require that the phased
approach to reporting has to move forward in a concurrent single report.
4.2. Requirements for preliminary site assessment phase 1
A preliminary site assessment must consider the following elements
1. Site description-location and size
2. Nature and extent of contamination, contaminants of concern and historic activities that
may be sources of contamination. List all present and past activities at the site that involves
42 Soil Pollution
Figure 15. A phase approach for the assessment and remediation of contaminated land
storage and production, use, treatment or disposal of hazardous material that could
contaminate the site.
3. Describe current condition of the site and contents and the results of any previous
assessment report
4. Local topography and geology, drainage, surface cover, vegetation.
5. Status of ground water, approximate depth to water table
6. Proximity to surface water
7. Proximity to drinking water supplies
8. Annual rainfall and flood potential
9. Land and water use for the nearby areas and site
10. Any other regulations as Regulated by the ministry
All data may not be available, or data may vary in terms of uncertainty, it is thus important to
recognize gaps in the knowledge base and to decide whether additional data must be obtained
on the site characterisation. This may trigger the commencement of Phase 2 Investigations.
The Phase 1 report must make clear recommendations on the status of the contamination risk
posed by the site. If a complete site history clearly demonstrates that the site activities do not
pose a contamination threat then no further investigation is warranted and the site should be
recommended as suitable for reuse. In most cases it is likely that some level of preliminary
investigation will be required to provide the level of certainty required to enable property re‐
development or transfer. A limited investigation of certain subsurface activities, for example,
underground storage tanks, would be necessary to obtain a waiver on the contamination status
of a site at a Phase 1 level of reporting.
4.3. Approval requirements
If soil contaminants are found at concentrations that exceed the applicable standards specified
in the approval, the approval holder is required to implement a soil management program.The
program must first address source control to stop on-going contaminant releases. After the
sources of contamination have been stopped, further assessment and delineation of the
contaminated area may be necessary. When the extent of contamination is understood,
remediation objectives, as described below, must be adopted for the area. Once remediation
objectives have been agreed to, appropriate treatment or containment technologies can be
chosen and the management plan finalized.
4.4. Developing remediation objectives
Remediation objectives may be developed in a variety of ways ranging from generic guidelines
to site-specific risk assessment. Generic guidelines are numerical concentration limits that are
applicable under a variety of site conditions. When neither Environmental Protection nor the
agency has a guideline for a particular substance, four options are available. First, the reme‐
diation objective may be based on the ambient background concentration for the site. Second,
the guideline development protocol may be applied. Third, a remediation objective may be
adopted from another jurisdiction if the proponent can show that the remediation objective is
consistent with the environmental protection goals of the approval. Finally, a remediation
objective may be developed by the proponent using site-specific risk assessment procedures.
A risk assessment for an approved facility will focus on human health concerns but funda‐
mental ecological concerns must also be addressed. Site-specific risk assessment is a means of
quantifying the likelihood that soil contamination will have a harmful effect under conditions
found at a specific site. The essential components of human health and ecological risk assess‐
ments are similar; however, ecological risk assessments tend to be more complex than those
for human health because a wide variety of receptors may have to be considered. Very briefly,
a site-specific risk assessment may be described as consisting of the following steps or
components as shown in Figure 20;
Problem formulation involves developing a conceptual model of the possible contaminant effects
on receptors at the site. The conceptual model describes contaminant distribution and
concentration in relation to the receptors and their patterns of activity on the site. The exposure
assessment describes the pathways by which soil contaminants may be taken up by the receptor.
44 Soil Pollution
This information is combined with receptor characteristics in order to estimate the contaminant
uptake. The toxicity assessment describes the adverse effects that the contaminants may cause
and the dose at which these effects occur.
Figure 16. Risk Assessment Framework
The final step, risk characterization, compares this dose with the uptake rate (estimated during
the exposure assessment) and determines whether or not an adverse effect is likely to occur.
Risk assessment procedures can also be used to back calculate a contaminant concentration in
soil at which no adverse effects are expected. At times, it may not be possible to remediate to
a level compatible with industrial land-use objectives. In such cases, the approval holder must
ensure that the contaminants are contained and receptor exposure does not occur. Formal risk
assessment procedures are necessary to meet these requirements. Typically, an engineered
containment system is required, the performance of which must be confirmed through periodic
inspections and monitoring. The approval holder bears responsibility for the design, con‐
struction, assessment and maintenance of the risk management system, and any necessary
emergency response should the system fail.
4.5. Enforcement policy
Enforcement is generally the last step in a regulatory process that aims to first prevent potential
problems before they arise and to resolve existing problems in a cooperative manner. When
discussions between the approval holder and the Department fail to prevent or resolve a
contravention of an approval or the Act, a number of abatement and enforcement tools are
available to the Department including:
• environmental protection orders;
• warning letters;
• tickets;
• administrative penalties;
• enforcement orders;
• prosecutions;
• court orders; and
• cancellation of approvals or certificates.

Due to various factors, including the high cost involved in remediation interventions, it is
essential that a holistic and tiered, risk‐based approach be adopted that is founded on inter‐
national best practice, to address remediation in a uniform manner across the country. This is
relevant irrespective of the sector of occurrence to safeguard both human health and the natural
environment. The Framework is based on a review of international practice in the developed
countries of the world and the emergence of remediation policy from developing countries,
and an assessment of alternative approaches and methodologies that may find application in
the development of a remediation framework.
5. Conclusion
Soil pollution is a result of many activities and experiments done by mankind which end up
contaminating the soil. Industrial wastes such as harmful gases and chemicals, agricultural
pesticides, fertilizers and insecticides are the most common causes of soil pollution. The others
are ignorance towards soil management and related systems, unfavourable and harmful
irrigation practices, improper septic system and management and maintenance of the same,
leakages from sanitary sewage. There is urgent need for a tiered approach in ecological risk
assessment of contaminated soils. Generic soil screening levels are needed as a first tier. Higher
tiers of ecological risk assessment should, however, contain some kind of site-specific assess‐
ment. It is furthermore important to organize the various studies in a framework or decision
support system that is transparent and useful for all stakeholders. A weight of evidence approach
may be an obvious choice to deal with these uncertainties. The TRIAD approach, which
incorporates and categorizes information in a triangle – chemistry, toxicology, and ecology – is
an appropriate tool for handling conceptual uncertainties. Several remedies to these shortcom‐
ings have been proposed. Regarding ecotoxicity direct testing would allow for a major
improvement in risk estimates. As to human health risks: including biological availability in
46 Soil Pollution
risk estimates, more use of up to date knowledge about exposure routes, dose-effect relations
and combination effects, and biomonitoring of effects are options for improvement.
Acknowledgements
Authors greatly acknowledge the University Malaya Research Grant (RG257-13AFR). More‐
over, the comments of two anonymous reviewers are gratefully acknowledged.
Author details
Muhammad Aqeel Ashraf1, Mohd. Jamil Maah2 and Ismail Yusoff1
1 Department of Geology, Faculty of Science, University of Malaya, Kuala Lumpur,

Malaysia
2 Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur,

Malaysia
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chapter 2
Sustainability Aspects of In-Situ Bioremediation of

Polluted Soil in Developing Countries and Remote
Regions
Anders Jonsson and Henrik Haller
1. Introduction
Xenobiotic and other toxic compounds from industrial activities are presently being accumu‐
lated in the ecosystems at an unsustainable rate. From an anthropocentric point of view such
contamination is unsustainable because it threatens basic ecosystem functions and services
that are necessary to maintain food security and provision of potable ground water. Further‐
more natural habitats for plants and animals are threatened. Thousands of chemical com‐
pounds in varying concentrations and compositions have contaminated a wide range of
habitats [1]. These contaminated sites demand diverse strategies and development of new
technologies that are efficient and cheap enough to make remediation feasible even in remote
areas and where financial support is limited.
Soil remediation has been a developing field for several decades and a common assumption
is that it is a sustainable industry but very often clean-ups demand vast amounts of energy
and lead to severe physical damage to the landscape. Traditional remediation technologies
include soil excavation and landfill disposal. Sustainable remediation technology has been
defined as a “remedy or combination of remedies whose net benefit on human health and the environ‐
ment is maximized through the careful use of limited resources” [1]. Such technologies are only
modestly implemented at present but new landfill legislations against “dig and dump”
technologies can favour green solutions over chemical and energy-intensive techniques in the
future [2].
Microorganisms have colonized some of the most extreme environments on the earth and some
of them are efficient degraders of the pollutants that our industrial society produces. Biore‐
mediation is a technology that uses living organisms and their enzymes to degrade, remove
2 Soil Pollution
or immobilize toxic compounds [3]. Bioremediation have been gaining terrain recently [1] and
some authors predict a further increase as technological advances surmount the limitations
that exist today.
In this chapter we briefly discuss how principles of ecological engineering in concurrence with
the application of basic thermodynamic principles and kinetic modelling can provide useful
tools for the development of energy conserving and economically feasible bioremediation
projects. We further discuss the potential of organic waste materials and by-products in locally
adapted soil bioremediation. Finally we present some illustrative cases of novel research on
sustainable bioremediation for tropical developing countries and remote locations and discuss
some promising fields of future research and possible future applications.
2. Bioremediation and ecological engineering
Ecological engineering has been proposed as a theoretical framework to design “sustainable

ecosystems that integrate human society with its natural environment for the benefit of both” [4].
Bioremediation is generally considered an ecological engineering practice but even if it
addresses one of the core goals of ecological engineering, i.e. restoration of damaged ecosystems
[5], bioremediation can be energy-intensive and have a serious impact on the remediated
ecosystems particularly if excavation and ex situ methods are employed.
Mitsch and Jørgensen [5] identify 5 basic concepts that have been developed to distinguish
ecological engineering from other approaches such as industrial ecology, biotechnology or
environmental engineering. Ecological engineering:
1. is based on the self-designing capacity of ecosystems

2. can be the acid test of ecological theories
3. relies on systems approaches
4. conserves non-renewable energy sources
5. supports ecosystem conservation
If used collectively, these 5 concepts can provide guidelines for design of sustainable biore‐
mediation projects. In a sustainable society the ultimate goal is to limit the rate of pollution to
what ecosystems can assimilate and break down without long term negative effects on
ecosystem functions or human health. Meanwhile that goal is attained we will have to give
the ecosystems a hand to boost their intrinsic biodegradation capacity.
2.1. Self design
Self-design is the property of a system to reorganise in an unstable non-homogeneous

environment [5]. The inclusion of self design in a project can facilitate the implementation of
flexible strategies that adapt to new conditions. The powerful capacity of ecosystems to
reorganise after change can be used in bioremediation projects as a means to reduce costs and
Sustainability Aspects of In-Situ Bioremediation of Polluted Soil in Developing Countries and Remote Regions 3
energy use. Consortiums of soil microorganisms naturally restructure to adapt to new

conditions including contamination and are therefore good candidates for self-design strat‐
egies. A typical microbial community in soil consist of less than 1 % hydrocarbon degraders
but that fraction can increase to 10% after exposure to oil pollutants [6]. An example of how
engineers can accelerate the ecosystems self-designing capacity is the introduction of a
microbial strain with capacity to initiate degradation of high molecular-weight pollutants that
later provide food (energy) for other microorganisms that take care of the metabolic by-
products as the introduced strain dies off. In conventional engineering, items are often
designed to behave in an as predictable way as possible since reliability and robustness are
desired criteria of safety and quality. The outcomes of a technology that depend on self-design,
on the contrary, are often a lot more difficult to predict. Bioremediation projects that include
self-designing strategies thus require thorough monitoring to make sure that the degradation
of the pollutants is satisfactory.
2.2. The acid test
Just as strong acids were traditionally used to distinguish gold from base metals, a full scale
bioremediation project can be used to confirm or reject ecological theories. Ecological theories
provide a useful framework that establishes restrictions and opportunities for bioremediation
strategies. The complex nature of full scale in-situ bioremediation however might lead to
outcomes somewhat different from what was expected. Since intense use of monitoring is
required for full scale implementations of unproven technologies, the result from these can be
used to refine the theories on which the project was based.
2.3. A systems approach
Reductionist analysis of the parts of a system in isolation from each other can provide
important data and basic understanding of significant mechanisms but should be combined
with a holistic view of the entire system in order to achieve a sustainable bioremediation
project. The site specific conditions of the ecosystem that is subject to remediation must be
quantified and analysed with appropriate models. That particular ecosystem however is part
of a greater context whose characteristics must also be born in mind. When bioremediation
strategies are chosen, socioeconomic and even cultural aspects must be taken into account.
Questions like “what is the economic impact of the project?”, “can polluted land be reclaimed
for food production or construction?”, “what resources are available in the human environ‐
ment around the site?” might supply useful information. The use of locally available agricul‐
tural waste products, that are potential polluters by themselves, as amendments (see section
about waste recycling) is an example of advantageous design that requires a systems approach.
The source of the pollution must also be addressed to make sure that the remediation project
isn’t an encouragement to further pollution.
2.4. Non-renewable resources conservation
Energy use is one of the major sustainability issues for conventional remediation projects. Ex-
situ remediation is typically too energy-intensive to be considered ecological engineering.
4 Soil Pollution
Furthermore it resembles what Jørgensen & Mitsch [5] call a shell game, in which pollutants are
moved from one location to another. In sustainable bioremediation external energy input is
ideally used only in the initiation phase to start a process that is subsequently powered by
solar energy and the embodied chemical energy of the pollutant itself. The engineer’s role is
to facilitate the proper conditions in which such a process can take place.
2.5. Ecosystem conservation
The ability to cycle nutrients is a fundamental function of all ecosystems. Within the matter-
cycling function lies the catabolic capacity to degrade and mineralise molecules of varying
complexity, including pollutants. This function alone gives a nonmarket value to ecosystems,
not always recognised by society. The role of ecological engineers is to identify ecosystem
functions that are adoptable to human needs and apply them in ways that doesn’t create any
further degradation of nature. Since bioremediation projects aims at eliminating pollution that
exerts stress on the ecosystem, it is by definition an ecosystem conservation approach.
However, if large amounts of soil are physically removed from the site in ex-situ operations
the remediation itself might be a threat to the ecosystem. Bioremediation projects are typically
involved in the restoration of disturbed ecosystems but measures to prevent toxic compounds
from entering sound ecosystems are likewise part of the bioremediation agenda.
3. Thermodynamic and kinetic considerations on bioremediation
The fact that most organic pollutants are composed of molecules with high embodied energies
is an interesting starting point for any consideration about the best practice for sustainable soil
remediation, especially in remote locations and developing countries where energy-intensive
project are cost-prohibitive. Economic incentives are generally small for any kind of remedia‐
tion practice in such regions and, furthermore, time constraints may not be as limiting as in
highly developed urban environments. Technologies that manage to optimize the use of
ecosystem services provided by indigenous organisms, in combination with a minimum input
of extra energy and material resources, are potentially more sustainable in such conditions.
Figure 1 illustrates some basic principles including thermodynamic driving forces (from
molecules with high to low embodied energy) and available transformation pathways in air
and soil. In the atmosphere, abiotic photochemical reactions rapidly oxidize organic vapours.
The rate determining step in these complex photochemical reactions is usually the initial attack
on the primary pollutant by hydroxyl radical. Typical turnover times of i.e. reactive hydro‐
carbons are only a few hours in the sunlit atmosphere while lack of photochemistry in the
subsurface of soils leads to significantly lower reaction rates and hence to much longer
turnover times of pollutants.
In soils where conditions for microbial activity are unfavourable, degradation relies on abiotic
chemical reactions. In such cases organic pollutants may persist for years to decades or longer
and secondary, incompletely degraded products may accumulate. On the other hand if
conditions are favourable (temperature, electron acceptors, nutrients, moisture, available
primary pollutant by hydroxyl radical. Typical turnover times of i.e. reactive hydrocarbons are only a few hours in the sunlit
atmosphere while
Sustainability lack of of
Aspects photochemistry in the subsurface
In-Situ Bioremediation of soils
of Polluted leads
Soil to significantly
in Developing lower reaction
Countries rates and
and Remote hence to 5
Regions
much longer turnover times of pollutants.
Secondary
CO2/H20
substances
(smog)
Photochemical τ = hours-
Air reactions days
Soil/water
Biologically
Organic substance catalysed
reactions
τ = months-
decades
Non photochemical
reactions: Hydrolysis Secondary
substitution, substances
elimination, reduction,
abiotic oxidation
Figure 1. Chemical and biochemical transformations and mineralisation of organic substances in polluted soil and in air. The broad arrow
Figure 1. Chemical and biochemical transformations and mineralisation of organic substances in polluted soil and in
indicates the thermodynamic driving force for the complete degradation of the pollutant in an oxidising environment. Typical average residence
air. The broad
times ofarrow
organicindicates thein thermodynamic
pollutants (τ) driving force for the complete degradation of the pollutant in an
air and soil are indicated.
oxidising environment. Typical average residence times of organic pollutants (τ) in air and soil are indicated.
carbonInsources,
soils wherelow toxicity
conditions to microorganisms,
for microbial pH, to degradation
activity are unfavourable, mention relies
someonof the most
abiotic determinant
chemical reactions. In such cases
organic pollutants may persist for years to decades or longer and secondary, incompletely degraded products may accumulate.
variables), microorganisms such as bacteria and fungi may use the organic pollutant
On the other hand if conditions are favourable (temperature, electron acceptors, nutrients, moisture, available as a carbon
source sources,
of energy and carbon.
low toxicity Enzymatic
to microorganisms, pH, todegradation
mention some ofisthegenerally muchvariables),
most determinant faster than abiotic oxidation
microorganisms such as bacteria and
fungi may use the organic pollutant as a source of energy and carbon. Enzymatic degradation is generally much faster than
reactions and
abiotic the organic
oxidation reactionspollutant that
and the organic entersthat
pollutant the metabolic
enters pathways
the metabolic pathways of soilmicroorganisms
of soil microorganisms may be partly
may beorpartly
completelyor mineralized.
completely Themineralized. Theandriving
driving force for such enzymaticforce for
process suchtheanfree
is again enzymatic process
energy potential that canisbe used
by the organism. For typical hydrocarbon and oxygenate pollutants the free energy that is available by complete oxidation with
again the free
oxygen as energy potential
electron acceptor, is in that canofbe
the range used
3-12 x 103byKJthe
mol-1organism.
or 37-46 MJ For typical
kg-1, as hydrocarbon
can be seen in table 1. Thisand
energy
oxygenate pollutants the free energy that is available by complete oxidation with oxygen as
potential may be used by a wide range of heterotrophic microorganisms.
electron acceptor,
In areas is in the
of hydrocarbon range of the
contamination 3-12
mostx commonly
103 KJ mol foundor
-1
37-46 MJbacteria
heterotrophic kg-1, as are can
of thebe seen
genera in table
Pseudomonas,
1. ThisAchromobacter,
energy potential may be
Arthrobacter, used byVibrio,
Micrococcus, a wide range of Brevibacterium,
Acinetobacter, heterotrophic microorganisms.
Corynebacterium, Flavobacterium,
Mycobacterium and Nocardia [7]. Some soil microorganisms that are incapable of utilizing the full embodied energy potential
of the pollutant may still be able to initiate catabolic processes that ultimately lead to complete mineralisation if oxygen is
In areas of hydrocarbon contamination the most commonly found heterotrophic bacteria are
present at sufficient concentration. Filamentous fungi, such as e.g., white-rot fungi, can oxidize recalcitrant high molecular
of the genera Pseudomonas,
weight polycyclic Achromobacter,
aromatic hydrocarbons Arthrobacter,
(PAH:s) with their extracellularMicrococcus,
enzymes to moreVibrio, Acinetobact‐
water soluble substances. Both
lignolytic and non lignolytic
er, Brevibacterium, fungi has been shown
Corynebacterium, to initiate degradation
Flavobacterium, of high molecular-weight
Mycobacterium and PAH and the oxidation
Nocardia [7].
products formed are thereby made available for further degradation by bacteria [8]. Such interactions between soil
Some soil microorganisms
microorganisms that
is an important aretoincapable
factor of utilizing
consider in sustainable the full embodied
soil bioremediation energy
and remediation potential
practices that favour a
of the pollutant may still
high soil microbial be able
biodiversity are to initiate
likely catabolic
to be more processes that ultimately lead to complete
sustainable.
mineralisation if oxygen is present at sufficient concentration. Filamentous fungi, such as e.g.,

white-rot fungi, can oxidize recalcitrant high molecular weight polycyclic aromatic hydrocar‐
bons (PAH:s) with their extracellular enzymes to more water soluble substances. Both
lignolytic and non lignolytic fungi has been shown to initiate degradation of high molecular-
weight PAH and the oxidation products formed are thereby made available for further
degradation by bacteria [8]. Such interactions between soil microorganisms is an important
factor to consider in sustainable soil bioremediation and remediation practices that favour a
high soil microbial biodiversity are likely to be more sustainable.
3
6 Soil Pollution
∆Go(minerali-
M So ∆Ho f ∆So f ∆Go f
Category Substance Formula Phase b c sation)
g mol -1
J mol K
-1 -1
KJ mol -1
KJ mol -1
KJ mol -1
KJ mol-1 MJ kg-1
Gasoline Benzene C6H6 Liquid 78.11 173.26# 49.0# -253.78 124.66 6 3 -3202 -41.00
Gasoline Toluene C7H8 Liquid 92.14 220.96 #

12.0 #
-342.59 114.14 7 4 -3823 -41.50
Gasoline m-xylene C8H10 Liquid 106.16 253.80# -25.4# -446.26 107.65 8 5 -4448 -41.90
Gasoline
MTBE C5H12O Liquid 88.15 265.3# -315.4# -650.52 -121.45 5 6 -3273 -37.14
additive
Diesel Hexadecane C16H34 Liquid 226.44 586.18# -456.3# -1728.71 59.11 16 17 -10401 -45.93
Diesel Octadecane C18H38 Solid 254.49 480.2 #

-457.4# -2107.71 171.01 18 19 -11776 -46.27
Diesel Phenanthrene C14H10 Solid 178.23 215.06# 110.1# -520.00 265.14 14 5 -6972 -39.12
Diesel Perylene C20H12 Solid 252.31 264.6 #

182.4 #
-636.14 372.07 20 6 -9682 -38.37
Mineralisation Carbon
CO2 Gas 44.01 213.79# -393.5# 2.81 -394.36
product dioxide
Mineralisation
Water H2O Liquid 18.02 69.95# -285.8# -163.30 -237.19
product
Solid
Element Carbon C 12.01 5.83# 0 0 0
(graphite)
Element Hydrogen H2 Gas 2.02 130.68# 0 0 0
Element Oxygen O2 Gas 32.00 205.15 #

0 0 0
#
NIST, National Institute of Standards and Technology website. Available at: http://webbook.nist.gov/chemistry/name-
ser.html
Table 1. Thermodynamic properties and free energy of mineralisation of some representative “high energy” organic
substances commonly found in polluted soil. Standard entropy of formation (∆Sof) was calculated from standard
entropy values (So) of the substances and their elements (phase indicated in table). Standard free energy of formation
(∆Gof) was calculated from standard enthalpy of formation (∆Hof) and standard entropy of formation. Standard free
energy of mineralisation (∆Go) was calculated for the complete oxidative degradation of the organic compounds to
the thermodynamically most stable end products according to the reaction: 1 mol of substance + a O2 = b CO2 + c H2O.
In oxygen depleted soils microorganisms are unable to mineralise organic compounds to

inorganic molecules only. Complete degradation leaves carbon dioxide and methane as end
products. Intermediate fermentation products, that may be toxic to microorganisms and
plants, may accumulate if they are not converted to methane [7]. Since methane and fermen‐
tation products has retained a substantial part of the embodied energy, the energy gain by
microorganisms by anaerobic degradation is less than under aerobic conditions and the
thermodynamic driving force for biodegradation is thus lower. Methane is also a highly potent
greenhouse gas [9] and its unintended production should, from a sustainability point of view,
be avoided. It is therefore in many cases preferable to maintain aerobic conditions in the soil
during in-situ bioremediation projects. There are however some important exceptions.
Xenobiotic and toxic chlorinated organic compounds such as e.g. highly chlorinated PCB:s,
pentachlorophenol, tri- and perchloroethylene, gamma hexachlorocyclohexane (lindane) and
many others are highly persistent to biodegradation under aerobic conditions. The reason for
this may be lack of metabolic pathways available to common soil microorganisms that can
generate energy gain for the organism. Persistence is also caused by high lipophilicity of many
of these substances which reduce their bioavailability.
Chlorinated substances may be efficiently degraded by microorganisms capable of halorespi‐
ration under anaerobic conditions [10]. The degradation mechanism is characterized as
reductive dechlorination. In contrast to aerobic co-metabolism which does not provide any
energetic benefit for the degrading organism, reductive dechlorination may provide a
substantial free energy yield. The thermodynamic driving force of such reactions is demon‐
strated by the fact that hexachloroethane for example, may be a stronger electron acceptor than
oxygen [11].
3.1. Kinetic considerations
Biodegradation kinetics tells us not only about the rate of degradation of target compounds
and of rate dependence on ambient factors, but by studying the shape of the degradation curves
we may also get valuable information about degradation mechanisms. It is preferable to use
models that have a theoretical basis rather than just empirical mathematical modelling that
have limited predictive ability.
Natural attenuation of organic pollutants are typically mediated by slow growing organisms
and if the concentration of pollutants is much lower than the concentration required for
maximum growth of degrading organism (which is often the case in many polluted soils) then
first order kinetics may apply, i.e. degradation rate is directly dependent on substrate concen‐
tration (Equation 1).
-dC/dt = kC (1)
C is the substrate concentration and k is the first order rate constant. First order kinetics is often
applied in degradation studies because of its simplicity but have little predictive power when
conditions for growth change.
In situations with rapid growth of degrading organisms or when other factors than the
substrate concentration controls the rate of degradation, first order kinetics will not describe
the degradation process accurately. According to the earlier mentioned criteria for sustainable
soil bioremediation, efforts should be made to stimulate growth of indigenous soil microor‐
ganisms that have or can achieve the ability to degrade target pollutants. The addition of
organic waste products from the dairy industry that promotes growth of degrading organisms
has been proved to be an efficient means to increase both rate and total degradation of some
representative diesel range hydrocarbons in soil [12-14]. Although the addition of readily
available carbon sources, such as lactose, lactate and amino acids (components of whey) to
contaminated soil would be expected to trigger exponential growth of organisms, a common
phenomenon in liquid culture media, it seems that the soil matrix itself does not readily
promote exponential growth of degrading organisms. Growth of soil microorganisms is
limited by a number of factors caused by the properties of the soil matrix. Among the limiting
8 Soil Pollution
factors is diffusion of substrate and nutrients but also the specific area available for bacterial
sorption on to soil particles. When a large fraction of this area is occupied by bacteria, further
growth might be increasingly restricted by diffusion of substrate and nutrients into multilayers
of bacteria. Kinetic models that take into consideration slow or restricted (linear growth) may
more accurately describe degradation patterns in real life situations. Figure 2 illustrates the
application of a three-half-order kinetic model (II) originally proposed by Brunner and Focht
[15]. The model assumes linear biomass growth rate and simulates the accumulated 14CO2-
production from phenanthrene-9-14C mineralisation in soil microcosms.
( (
P ( t ) = S0 1 - exp - k1t - k2t 2 2 + k0t )) (2)
P[t] is the cumulative 14CO2-production (%) where t is the time (days), S0 is the amount of
substrate (%) that is “highly bioavailable”, k1 is the first-order rate constant (day-1), k2 is a linear
growth term (day-2), and k0 is the zero order rate constant describing the mineralisation of “less
bioavailable” substrate and indigenous mineralisation (% day-1).The lag time was defined as
the initial time until 1% of the added 14C was detected as 14CO2 [16]. From the end of the lag
phase, iterative non-linear least squares regression was performed to fit data to the model by
using the SOLVER function in Microsoft Excel 2000 [17].
The effects of applications of different amendments on phenanthrene biodegradation are

clearly seen in figure 2. Kinetic parameters such as the linear growth term (k2) and S0 was
significantly affected by the addition of the different amendments (Table 2). It should be noted
that the stimulating effect of complex organic amendments such as whey and fermented whey
may not only be an effect of the addition of a readily available carbon source (that promotes
growth) but also of available micronutrients, such as B-vitamins, that is required to maintain
efficient catabolic pathways for degradation of i.e. polycyclic aromatic hydrocarbons.
Mineralisation
Lag time S0 k1 k2 k0
R after 391 days
(days) (%) (day ) -1
(day ) -2
(% day )-1
(%)
Control 21 51.3a 0 4.35a x 10-5 0 0.973 46.3ab
FW low 24 53.8a 0 5.83b x 10-5 0 0.997 52.1a
FW high 87 - - - - - 14.4c
SW low 21 48.6a 0 4.09a x 10-5 0 0.974 43.4b
SW high 18 63.2b 0 20.8c x 10-5 0.0046a 0.983 66.3d
FW multi 15 65.0b 0 63.6d x 10-5 0.014a 0.992 70.8e
Table 2. Tree-half-order kinetic constants for phenanthrene mineralisation in soil, at 25 °C. Lag time is expressed in
units of days, S0 and mineralisation in %, k1 in day-1, k2 in day-2, k0 in % day-1 and R is the correlation coefficient. Values
having the same superscript letters are not significantly different at a 95 % level. (From [18]. Reprinted with
permission from Elsevier).
P[t] is the cumulative 14CO2-production (%) where t is the time (days), S0 is the amount of substrate (%) that is “highly
bioavailable”, k1 is the first-order rate constant (day-1), k2 is a linear growth term (day-2), and k0 is the zero order rate constant
describing the mineralisation of “less bioavailable” substrate and indigenous mineralisation (% day-1).The lag time was defined
14
as the initial Sustainability
time until 1% of the added
Aspects of In-Situ detected as 14COof2 [16].
C wasBioremediation FromSoil
Polluted thein
end of the lag phase,
Developing iterative
Countries andnon-linear least
Remote Regions 9
squares regression was performed to fit data to the model by using the SOLVER function in Microsoft Excel 2000 [17].
Mineralization (% of theor. 14CO2) 80
70
60 Control
50 FW low
FW high
40
SW low
30 SW high
20 FW multi
10
0
0 100 200 300 400
Time (days)
Figure 2. Effect of added fermented whey and sweet whey on 14C-phenanthrene mineralisation to 14CO2 in soil at 25 oC. FW low = Fermented
whey 210 mg dw kg-1 soil dw; FW high = Fermented whey 2100 mg dw kg-1 soil dw; SW low = Sweet whey 210 mg dw kg-1 soil dw; SW high
Figure
= Sweet whey2. Effect
2100 mgofdwadded fermented
kg-1 soil dw, all addedwhey
at theand sweetofwhey
beginning on 14C-phenanthrene
the experiment. mineralisation
FW multi = Fermented whey 210 mg todw
14
CO in soil
kg-12 soil dw at 25
added at thelow
beginning of the experiment andmgthendwevery -1 th day. The curves are fitted to the data by use of the three-half-order
o
C. FW = Fermented whey 210 kg14 soil dw; FW high = Fermented whey 2100 mg dw kg-1 soilkinetic dw; SW model.
low =
Kinetic
Sweet parameters
whey 210 are given
mg dw in table
kg-12soil
(From
dw; [18].
SWReprinted
high = withSweetpermission from Elsevier).
whey 2100 mg dw kg-1 soil dw, all added at the beginning of
the
The experiment.
effects FW multi
of applications = Fermented
of different whey 210
amendments mg dw kg-1 biodegradation
on phenanthrene soil dw addedareat clearly
the beginning of the
seen in figure 2. experiment
Kinetic and
then every 14 th
day. The curves are fitted to the data by use of the
parameters such as the linear growth term (k2) and S0 was significantly three-half-order
affected kinetic
by the addition of themodel.
differentKinetic parameters
amendments
are given in table 2 (From [18]. Reprinted with permission from Elsevier).
5
4. Waste recycling
Organic by-products are produced globally on a massive scale. Economic operations such as
agriculture, livestock, fishing and forestry [19-21] generate a number of by-products that can
be seen in Figure 3. Frequently unexploited organic waste products create serious environ‐
mental problems when disposed of [20]. Agriculture is the economic base of most tropical
developing countries, accounting for 50 % of the employment [21]. Organic by-products from
agriculture, forestry, fishery and livestock operations are thus available in abundance and are
typically of limited economical value [19]. Animal feed has been proposed as an outlet for some
of these by-products but due to low levels of protein, high levels of moisture and some anti-
nutritional factors i.e. presence of tannins and polyphenols, this practice is limited [22].
However, being inexpensive and easily available sources of carbon, nutrients and bioactive
compounds, many of these by-products can potentially be used as sources of readily available
carbon for bioremediation purposes [23].
10 Soil Pollution
Manure
Residues
Abbatoir
from
Waste
fishery
Dairy
Inexpensive Ligno‐
Industry
Waste
organic cellulosic
crop waste
byproducts
Processed
Non‐ligno‐
residues
cellulosic
from wood Un‐ crop waste
industry processed
wood
residues
Figure 3. Some organic by-products that potentially can be used as amendments for bioremediation.
Figure 3. Some organic by-products that potentially can be used as amendments for bioremediation.
A number of organic
A number by-products
of organic have
by-products have the ability
the ability to enhance
to enhance theofdegradation
the degradation of different
different toxic compounds toxic
including
lignocellulosic wastes such as sugarcane, bagasse and sawdust [26], crop residues such as coffee pulp [27] and molasses [28]
compounds including lignocellulosic wastes such as sugarcane, bagasse and sawdust [26],
and whey, a by-product of the dairy industry [12-14]. Three examples of inexpensive by-products with potential or confirmed
crop residues suchcapacity
bioremediation as coffee pulp [27]
are described anddetail
in more molasses [28] and whey,
below: pyroligneous a by-product
acid, compost of the dairy
teas and whey.
industry [12-14]. Three examples of inexpensive by-products with potential or confirmed
4.1 Pyroligneous
bioremediation capacity are acid
described in more detail below: pyroligneous acid, compost teas
Pyroligneous acid (PA) is a by-product of charcoal production [29] with a limited economic value that is also referred to as
and whey.
wood vinegar, pyrolysis oil, smoke extract, bio-oil etc. [30, 31]. PA is typically disposed of as waste [31] but in Japan it has
been used for centuries to increase crop production and to combat agricultural pests [29, 32]. At present, the majority of the
charcoal consumed worldwide is produced in traditional kilns in developing countries [33]. Such charcoal production lacks
4.1. Pyroligneous acid
appropriate control of the gas and vapours produced in the process and causes land and air pollution [34]. Production systems
exist however that recover these condensate co-products or use them as fuel in the pyrolysis process, reducing the emission to a
Pyroligneous
minimumacid (PA) is
[30]. Several lowatech
by-product of charcoal
kilns that recover the PA from production [29] with
the charcoal production havea been
limited economic
developed including the
advanced Brazilian beehive kiln and the Casamance kiln [35]. In recent years the research and technology concerning PA have
value that is also
advanced referred
significantly to and
[30], as wood vinegar,
the current pyrolysis
trend in charcoal oil, smoke
production extract,
is to improve bio-oil etc.performance
the environmental [30, 31]. while
PA is typically
maintainingdisposed
or improving ofcharcoal
as waste yield[31] but inThe
and quality. Japan it has
chemical been used
composition of PAfor centuries
varies dependingto onincrease
factors such as type
of feedstock, pyrolysis temperature and duration etc. [25, 30]. The most frequently identified compounds in PA include
crop production and to combat agricultural pests [29, 32]. At present, the majority of the
methanol, acetic acid, methyl acetone, acetaldehyde, furan, furfural and volatile organic acids. PA contains phenolic
charcoal consumed
compounds worldwide
that are known to haveisantimicrobial
producedproperties
in traditional
[36] and PA kilns in developing
has successfully been usedcountries [33].growth on
to control fungal
Such charcoal
wood [30]production lacks
and bacterial decay appropriate
of foods [36, 37]. At control of the however,
low concentrations gas andPAvapours produced
has been shown in the
to stimulate germination,
growth and yield in a wide range of plants [37]. Although the growth promoting effect of PA is not yet clearly understood it has
processbeen
andattributed
causestoland and air pollution [34]. Production systems exist however that recover
nutritious components such as nitrates of Ca, Mg, K, magnesium sulphate etc. but also to its stimulatory
these condensate co-products
effect on symbiotic or use
fungi and bacteria [31]. them as fuel
The formation in bodies
of fruit the pyrolysis process, reducing
of hydrocarbon-degrading fungi such asthe Lentinus
edodes and Pleurotus ostreatus was significantly increased after applications of PA to the growth medium [38]. Furthermore
emission to a minimum [30]. Several low tech kilns that recover the PA from the
PA has been shown to significantly increase basal respiration and microbial biomass in highly weathered tropical soils charcoal
production havethat
suggesting been developed
microbes used PA forincluding the advanced
their metabolism [29]. Brazilian beehive kiln and the Casamance
kiln [35]. In recent years the research and technology concerning PA have advanced signifi‐
4.2 Compost teas
cantly [30],
Compost and
teasthe
(CT)current trend in
are liquid compost charcoal
extracts obtainedproduction
when compost isissoaked
to improve
in water forthe environmental
a determined number of days.
performance
Optionallywhile
additivesmaintaining or improving
(derived from by-products) charcoal
such as molasses, fish yield androck
hydrolysate, quality.
dust, yeastThe chemical
extract, humic acids etc.
are used to add nutrients and stimulate microbial activity. To date virtually all research on CT has focused on its potential to
control plant diseases in agriculture. However, the fact that CT have proved to significantly increase soil microbial respiration
and dehydrogenase activity [39] makes it an interesting candidate for bioremediation. A number of production parameters (e.g.
aeration, compost source, nutrient additives etc.) have been manipulated in order to optimize plant disease suppression [40]. To
date, consensus on optimum production parameters for disease suppression hasn’t been reached and some studies report
7
composition of PA varies depending on factors such as type of feedstock, pyrolysis tempera‐

ture and duration etc. [25, 30]. The most frequently identified compounds in PA include
methanol, acetic acid, methyl acetone, acetaldehyde, furan, furfural and volatile organic acids.
PA contains phenolic compounds that are known to have antimicrobial properties [36] and PA
has successfully been used to control fungal growth on wood [30] and bacterial decay of foods
[36, 37]. At low concentrations however, PA has been shown to stimulate germination, growth
and yield in a wide range of plants [37]. Although the growth promoting effect of PA is not
yet clearly understood it has been attributed to nutritious components such as nitrates of Ca,
Mg, K, magnesium sulphate etc. but also to its stimulatory effect on symbiotic fungi and
bacteria [31]. The formation of fruit bodies of hydrocarbon-degrading fungi such as Lentinus
edodes and Pleurotus ostreatus was significantly increased after applications of PA to the growth
medium [38]. Furthermore PA has been shown to significantly increase basal respiration and
microbial biomass in highly weathered tropical soils suggesting that microbes used PA for
their metabolism [29].
4.2. Compost teas
Compost teas (CT) are liquid compost extracts obtained when compost is soaked in water for
a determined number of days. Optionally additives (derived from by-products) such as
molasses, fish hydrolysate, rock dust, yeast extract, humic acids etc. are used to add nutrients
and stimulate microbial activity. To date virtually all research on CT has focused on its
potential to control plant diseases in agriculture. However, the fact that CT have proved to
significantly increase soil microbial respiration and dehydrogenase activity [39] makes it an
interesting candidate for bioremediation. A number of production parameters (e.g. aeration,
compost source, nutrient additives etc.) have been manipulated in order to optimize plant
disease suppression [40]. To date, consensus on optimum production parameters for disease
suppression hasn’t been reached and some studies report inconsistency in the performance
[41]. However a number of reports have shown that aeration and additions of nutrients lead
to significant increases of the cell mass of active bacteria [39, 40, 42] and the addition of humic
acids and yeast extracts significantly increased the fungal populations [43]. Plate counts and
microscopic examination studies have revealed that aerated CT has bacterial population
dominated by Bacillus sp., Lactobacillus sp., Micrococcus lutues, Staphylococcus sciuri, Pseudomonas
putida, Burkholderia glumae and Clavibacter agropyri while species of Aspergillus, Penicillium and
Trichoderma dominated the fungal communities [43]. Many of these organisms have the
capacity to degrade a number of toxic substances including PAH:s and organochlorine
compounds [44, 45].
In a recent study, applications of compost teas made from vermicompost caused significantly
greater dehydrogenase activity and microbial respiration [39] which makes it an interesting
candidate for bioremediation. Vermicompost generally has higher concentrations of available
nutrients (NO3-, exchangeable Ca, P and soluble K) and significantly larger and more diverse
microbial populations than common compost. Microorganisms found in worm castings of
Eisenina fetida have caused increased catabolic activity and enhanced degradation of PAHs in
a number of experiments [46, 47].
12 Soil Pollution
4.3. Whey
Whey is produced when milk casein is removed from the milk in dairy operations to make cheese
and other products. This liquid by-product constitutes between 85-95% of the milk volume and
55% of milk nutrients remain in the whey. About half of the yearly global production of 145
million tonnes is utilized for animal feed etc. The remaining large volumes end up as potential
contaminants in the environment [48]. The chemical content of whey is characterised by lactose,
a number of essential and non-essential amino acids in different proportions, vitamin B 1,2,6,7,12,
folic acid and lactic acid [12, 13]. Relatively few studies have been performed on the effect of
whey on biodegradation of organic pollutants [12]. However, research at Mid Sweden Univer‐
sity has showed that the degradation of aliphatic and aromatic hydrocarbons can be significant‐
ly enhanced by the addition of whey and fermented whey [12, 13]. The biodegradation-
enhancing effect of whey was primarily attributed to an increased microbial biomass stimulated
by the readily available carbon source [12]. The degradation studies also showed a more complex
dependence of carbon sources and growth factors, such as B-vitamins, on the degradation of an
aromatic compound (phenanthrene) compared to an aliphatic compound (hexadecane) [18].
These results indicate that the presence of co-factors such as vitamins and micronutrients may
be important to consider when evaluating the suitability of organic amendments for bioremedia‐
tion of soil contaminated by various types of organic pollutants.
5. Case studies
Different technologies and strategies are necessary to remediate the present and future soil
pollution. As our understanding of the degradation mechanisms of different pollutants evolves,
so do our chances to develop alternative and complementary strategies that can take the
concentrations of toxic compounds to a sustainable level. Since 2006, a research group at Mid
Sweden University has undertaken research dedicated to the development of bioremediation
methods for low priority remediation sites including, remote locations and tropical develop‐
ing countries. Ecological engineering provide an appealing theoretical framework in such
locations where energy and material costs are limiting factors. The research has implied
laboratory experiments as well as pilot scale and in-situ projects in tropical and temperate climate.
It includes pilot scale experiments in an experimental station that was developed in Nicaragua
(se figure 4) and field trials in remote parts of northern Sweden where transport distances are
long and the cold climate constitutes an additional challenge to in-situ bioremediation projects.
5.1. Field station in Nicaragua, designed for bioremediation experiments in a tropical

climate
Waste management programs in developing countries are often deficient and the inappropri‐
ate disposal of hazardous waste creates pollution problems. Furthermore the use of pesticides
and other toxic products is minimally restricted in many developing countries which leads to
extensive contamination with persistent substances such as polycyclic aromatic hydrocarbons
and organochlorine pesticides [49, 50]. The experimental station in Nicaragua (figure 5) is
Figure 4. Left picture: Map of Central America and Nicaragua. Right picture: The red marked area is Chontales, the
region where Mid Sweden University’s pilot scale experimental station is located. (Map of Central America by Виктор В
licensed under Creative Commons Attribution-Share Alike 2.0 Generic, Mapa de Nicaragua mostrando al departamen‐
to de Chontales by Vrysxy licensed under Creative Commons Attribution-Share Alike 3.0 Unported.)
Figure 5. Mid Sweden University´s experimental station at Casa Montesano in Nicaragua.
located on the agro ecological farm Casa Montesano, (11˚59’70˝ N 84˚53’06˝ E), where CIPP, a
local NGO, dedicated to sustainable rural development projects has its headquarters. The
region has a humid tropical climate with an average precipitation of 2000 mm per year and an
annual average temperature between 25 and 28 ˚C. The soil on the experimental site is classified
14 Soil Pollution
as ultisol, a common soil in the humid tropics occupying about 8 % of the global ice-free land
area and supporting 18 % of the world's population.
Figure 6 shows the 24 compartments that were built in concrete with a smooth cement plaster
in two sets of twelve compartments each. The compartments were built to fit 150 L each of
agriculture soil. The purpose of the closed compartments is to test sustainable remediation
methods in conditions as similar to in-situ remediation as possible without risks for pollutants
to escape into nature. All technologies assessed in the experimental station are designed to be
low cost/low technology methods, appropriate to economically marginalised people in
developing countries. Notable is the use of locally available organic waste or by-products such
as whey, pyroligneous acid and compost teas that can be achieved at a very low cost. The pilot
scale research aim to find practical solutions to the obstacles that emerge when positive results
from the laboratory are to be reproduced in the field. This implies dealing with issues like
chemotaxis limitations, i.e. the importance of biodegrading microorganisms to be in close
proximity to the pollutant. That can be achieved by the use of surfactants and solvents and by
avoiding clogging of the soil pores.
The first experiment conducted in the station was on soil spiked with diesel at a concentration
of 5.00 g diesel dw-1 kg-1 soil dw. Whey, pyroligneous acid and compost teas were applied at
a high and a low dose on day 3, 18, 32, 46, 60, 74 and 88 after diesel contamination. The doses
were adjusted to reflect the expected ideal dose (high) and an economically more viable dose
(low). Soil samples were taken on day 2, 7, 12, 17, 31, 45, 59, 73, 87 and 101 after diesel
contamination. Leachate water samples were collected by the end of the experiment in order
to detect presence of metabolic by-products.
When this chapter was written the soil samples were not yet analyzed. The soil samples will
be analysed by GC/FID to determine the effect on degradation from the different amendments.
The amendments’ effect on the microbial composition will subsequently be analysed by
molecular methods. Whey has engendered significant enhancement of the degradation of
aliphatic and aromatic hydrocarbons in previous laboratory experiments within our research
group but compost tea and pyroligneous acid have not been previously tested.
In an upcoming project the research group envisages to use the experimental station for novel
research on relevant pesticides. Many of the least biodegradable pesticides are relatively
persistent to aerobic degradation [27] which poses additional challenges for in-situ remedia‐
tion. These challenges will be addressed constructively in trials with redox barriers and other
strategies.
5.2. Experiences from in-situ remediation trials in remote areas of Northern Sweden
5.2.1. Bioremediation field trial using whey treatment of diesel contaminated soil at a former gas station
in Northern Sweden
A field experiment was carried out at a diesel contaminated site in Gäddede, in the county of
Jämtland, north-western Sweden (65°30’N, 14°09’E) [51], see figure 7. The contaminated site
is an old fuel station, established in the 1960:s. The contaminated site is about 10 m2 in area
Figure 6. The compartments filled with soil at the experimental station in Nicaragua.
and about 0.5 m in depth. Previous investigations had pointed out an area with relatively high
loads of aliphatic and aromatic hydrocarbons C5-C35 (approximately 2200 mg kg-1 dw soil)
dominated by diesel range aliphatics.
This site is representative of the many small contaminated areas that are spread out in rural
areas, and are typically considered low priority sites. According to a study by the Swedish
EPA in 2006 excavation and transport was the dominating remediation technology in Sweden
[52] which is also the case in the county of Jämtland. In this case the distance from Gäddede
to Gräfsåsen, the nearest approved treatment plant for contaminated soil, is 230 km. The
alternative of excavation and transport will therefore be expensive and contribute to air
pollution and greenhouse gas emissions. For such areas, a low cost, easy to apply in-situ
remediation technique would be appropriate. In-situ whey treatment might be such a method.
Addition of whey to increase biological activity in soil for remediation purposes has been
previously demonstrated in laboratory studies [12, 13, 18].
5.2.1.1. Life cycle assessment of in-situ bioremediation with whey treatment in Gäddede in comparison
to traditional excavation and composting
In order to compare the two alternative treatment strategies, a screening life cycle assessment
model was set up. The purpose was to investigate the environmental performance of the whey
method, to benchmark the whey method towards the excavation and composting practice and
16 Soil Pollution
Gädded
Sikås
500 km 0,5 km
Figure 7. The test spots for in-situ bioremediation trials with whey in northern Sweden. (Location map of Scandinavia
by NordNordWest licensed under the Creative Commons Attribution-Share Alike 3.0 Unported) Top picture: Diesel
contaminated site at former gasoline station in Gäddede (Village). Bottom picture: Oil spot at lumberyard in Sikås (Vil‐
lage). © Lantmäteriet 2012/1067.
to identify environmental hot spots in the whey treatment life cycle [53]. The functional unit
of this study was the treatment of 5 m3 of contaminated soil reaching a diesel concentration of
500 mg kg-1 dw (upper limit in generic guidelines for “non sensitive areas”). For impact
assessment the results are characterized as global warming potential (GWP), acidification
potential (AP), eutrophication potential (EP) and photo oxidant creation potential (POCP). It
was assumed that whey is added together with commercial fertilizer to avoid macronutrient
deficit. Since the biological activity only takes place when it is warm enough (this mountainous
region has a cold climate with at least 6 months of winter and a yearly average temperature of
around 0oC) the total treatment time was estimated to two summers. The alternative treatment
assumed in this study is to excavate the 5 m3 of contaminated soil at the site in Gäddede,
transport it to the waste treatment site in Gräfsåsen, Östersund, Sweden, and compost the soil
in windrows after mixing the soil with manure. The windrows are turned using an excavator
and when the concentrations of contaminants are low enough the compost is used as top cover
of a landfill. Figure 8 illustrates a simplified model of the life-cycle process flow diagram for
the whey treatment scenario.
Figure 8. The life cycle inventory model of the whey scenario (From [53]).
The hydrocarbon pollution in the 5 m3 soil to be treated (over an area of 10m2) was assumed
to be the same in the whole mass of soil i.e. 2200 mg kg-1 dw. Comparisons with laboratory
results [12] indicate that an addition of 210 mg dw whey kg-1 diesel polluted soil might give a
sufficient reduction in diesel oil contamination to reach the higher target value of 500 mg
kg-1 dw [54]. This gives a calculated need of about 2 kg dw whey, equivalent to 367 L of whey
(5.5 g dw L-1) for the area to be treated. The laboratory tests also indicate that a number of
successive additions of whey will give a better degradation compared to one large addition,
and eight additions (46 L of wet whey per addition) spaced out with 14 days in between was
assumed to be a good practice. The total time needed for the degradation is from the laboratory
tests estimated to 167 days. Since on-site biodegradation will only take place when it is warm
enough [13] the degradation was assumed to take place during two summer months each year
for two years. To ensure that biodegradation is not limited by macronutrient deficiencies
addition of commercial fertilizer was assumed. It was estimated that a one-time addition of
1.5 kg YaraMila 18-4-14 would provide the necessary nutrients. During the remediation
process whey and fertilizer was assumed to be manually added to the contaminated area.
Whey additions of 46 L every 14 day over a period of two subsequent summers were assumed.
For the intended bioremediation a total of 2 kg dw whey is needed. Data on cheese and whey
production was obtained from Milko dairy in Östersund. To produce 0.9 ton of Edam cheese
and 1 ton of dry whey the cheese dairy on average requires 11000 L of milk. Inventory data
was gathered to describe the use of average Swedish electricity, water and auxiliary chemicals
[55]. Inventory data for milk production, including farming activities, transports and dairy
operations was based on a LCI by the Swedish Diary Association.
Since whey is a by-product of cheese production, economic allocation was used giving an
allocation of 80 % on cheese and 20 % on whey. In a sensitivity analysis the results were
18 Soil Pollution
investigated, assuming that all production environmental impact is allocated to the whey. The
fertilizer production is described using emissions data on the production of 1 kg of fertilizer.
Whey and fertilizer will have to be transported to the contaminated site. Whey is produced in
Sweden by Milko AB is transported over a distance of 230 km from Östersund to Gäddede.
Fertilizer is obtained from commercial fertilizer store and transported the same distance.
Environmental impacts from the transports were calculated using emissions factors assuming
a light distribution truck, Euro 2, vehicle manufactured during 1996-2000.
Figure 9 illustrates a simplified model of the life-cycle process flow diagram for the excavation
and composting treatment scenario. Assuming a soil density of 1922 kg m-3 for the 5 m3 soil to
be treated (over an area of 10 m2) the calculated mass of contaminated soil was 9.6 ton.
Figure 9. The life cycle inventory model of the excavation and composting scenario (From [53]).
It was assumed that the Gäddede case would need a 14 ton excavator for 15 minutes and use
2 L of diesel. Production of the diesel oil was described by inventory data from [56]. The
excavated soil will then have to be transported 230 km from Gäddede to Östersund to a
composting plant. Impacts from the transport is described using emission factors for a medium
sized distribution truck with a payload of 8.5-14 ton, Euro class 2, produced between 1996 –
2000.
100
90
80
70
60
Whey 20 % allocated
50
Whey 100 % allocated
40
Composting
30
20
10
0
GWP 100 AP EP POCP
Figure 10.
Figure 10.Results from
Results the LCA
from models
the LCA for the for
models in-situ
thewhey treatment
in-situ wheyscenario – withscenario
treatment two levels–ofwith
allocation of the whey
two levels production of
of allocation – and
the
the excavation and off-site composting scenario. GWP 100, Global Warming Potential, 100 year time horizon; AP, Acidification Potential; EP,
whey production – and the excavation and off-site composting scenario. GWP 100, Global Warming Potential,
Eutrophication Potential; POCP, Photochemical Ozone Creation Potential. The allocation scenarios mean that respectively 20 and 100 % of 100
the
year time horizon;
environmental AP,
effect of theAcidification Potential;
cheese production EP, Eutrophication
was allocated Potential;
to the whey. Normalized data, POCP, Photochemical
the highest OzoneisCreation
result in each category set to 100%Po‐
tential. The allocation scenarios mean that respectively 20 and 100 % of the environmental effect of the cheese pro‐
(From [53]).
duction was allocated to the whey. Normalized data, the highest result in each category is set to 100% (From [53]).
AtWhen comparing the

Gräfsåsen, theallocated
wasteand the unallocated
treatment andwhey treatment
landfill in scenarios
Östersund, the milk-cheese chain has a rather
the contaminated large
soil is impact,
mixed
especially for eutrophication (EP in diagram). It should be noted that the transport of whey and fertilizer for the whey scenario
with horsewith
is described manure
emissionand placed
factors in windrows
of a truck transport, that in a string-like
means it is assumedarrangement.
to have the necessaryOver
wheytheandperiod
fertilizer2005
as part
of the load of the truck and the off-loading will be part of a delivery route where other parts of the load will be off-loaded in
toother
2009 some 2320 tons of contaminated soil and 1560 tons of manure have been received
places. Arranging good logistics is thus important both for the whey treatment and for the excavation and transport to off-
at
the composting plant in Gräfsåsen, giving an average manure use of 0.67 ton/ton. For the
site composting.
manure transport we assume the same emission factors as for the soil transport and an average
transport distance
5.2.1.2 Results of 50 km. Thefield
of bioremediation manure
trialsgenerations
and microcosm areexperiments
totally allocated to thetreatment
using whey horse keeping.
of the
contaminated soil in Gäddede
Every one month during the summer the windrows are mixed using a mixer mounted on a
The result from whey treatment of the polluted site during a 3 ½ months field experiment was not promising. The experiment
wheel loader.
was initiated 10 MayForwhen
thistheprocess
snow hadhalf the
melted andenvironmental
the soil temperatureimpact
at 18 cm from the5 excavation
depth was o
C. The pollutedinarea
Gäddede
was given
an initial dose of 545 g m-2 NPK fertilizer (18 % N; 4 % P; 10 % K) followed by bi-weekly doses of 3.2 L m-2 milk whey [51].
was assumed. Use of the treated compost as cover of the land o fill is not assumed to be connected
During the course of the summer the soil temperature rose to a maximum of 15 C in the beginning of August and dropped
tothereafter.
any environmental
When the experiment impacts.
was endedThein midresults
August no from the whey
detectable reductiontreatment scenario
of soil hydrocarbons was and the com‐
observed.
posting scenario for the four impact categories GWP, AP, EP and POCP is shown
In parallel to the field experiment, microcosm studies were carried out on soil samples, from the vicinity of the contaminated in Figure
10,
site together
that had beenwith the contaminated
artificially results from withthe sensitivity
diesel analysis
fuel. In the microcosms where
the soil wasall environmental
subjected impacts
to the same treatment as
in the field study with the only difference that the soil was kept at 22 oC. During these conditions the diesel range aliphatics
from milkcompletely
was almost production and
degraded the 60
within cheese dairy
days. This is in are allocated
accordance to the studies
with previous whey.on hexadecane that showed
significant difference in degradation rates at 7 and 22 oC respectively [13]. Soil temperature is probably the most limiting factor
For the GWP,
for in-situ AP and
bioremediation POCP
in cold impact
regions and thecategories
short period ofthe whey
warmth scenarios
during 3 summergivemonthslower results
is rarely enough than
to bringthe
the
soil temperature to levels that give sufficient rates of biodegradation. A possible way to circumvent this problem would be to
excavation
heat up the soiland
with off-site composting
heat injection. scenario,carried
Preliminary calculations whereasout at the EP category
our department shows
reveal that higher
the electric results.
power needed
o
to heat
The EPupresult
a 100 x for
100 xthe
2 mwhey
contaminated volumeis
treatment of totally
polluted soil from 8 to 25on
dependent C by
thee.g.whey
a sufficient number of chain
production heat rods,
and
would be 166 kW during a one month period corresponding to an input heating energy of 98 x 103 kWh. The calculation
originates
assumed that atthe the farm was
soil surface (such as nutrient
insulated runoff,
and that there were noruminating cattle, heat
vertical and downwards vaporization of heat-up
losses during the ammonia
period. The energy needed to maintain the temperature at 25 oC for another 11 month period assuming horizontal and vertical
from
(downwards) heat losses would amount to 235 x 10 kWh. Total energy need for a one year period is then 333 x 10The
manure) or by the production of materials
3 used at the farm (such as fertilizers). 3
kWh.milk
The
farm activity
following
-1 -1
is
input datathe
wasmost
used: Acontributing in
linear temperature
-1 -1
the
drop whey
distancescenario
of 9.1 m tofor
-3
all
ambient four impact
temperatures, categories
heat conductivityshown.
1.5 W
m K , specific heat capacity 800 J kg K and soil density 1300 kg m . These are rough calculations based on some
simplified assumptions about heat transfer in soil but may still give an estimate of the magnitude of the input energy needed for
a temperature enhanced in-situ soil bioremediation project in cold climate. Downscaled to the 10 m2 by 0.5 m of the Gäddede
diesel oil plot the corresponding figures would be 27 and 387 kWh respectively for the heating and 11 months maintenance
input energy need respectively giving a total energy need for a 12 month period of roughly 414 kWh. Part of this energy need
14
20 Soil Pollution
When comparing the allocated and the unallocated whey treatment scenarios the milk-cheese
chain has a rather large impact, especially for eutrophication (EP in diagram). It should be
noted that the transport of whey and fertilizer for the whey scenario is described with emission
factors of a truck transport, that means it is assumed to have the necessary whey and fertilizer
as part of the load of the truck and the off-loading will be part of a delivery route where other
parts of the load will be off-loaded in other places. Arranging good logistics is thus important
both for the whey treatment and for the excavation and transport to off-site composting.
5.2.1.2. Results of bioremediation field trials and microcosm experiments using whey treatment of the
contaminated soil in Gäddede
The result from whey treatment of the polluted site during a 3 ½ months field experiment was
not promising. The experiment was initiated 10 May when the snow had melted and the soil
temperature at 18 cm depth was 5 oC. The polluted area was given an initial dose of 545 g
m-2 NPK fertilizer (18 % N; 4 % P; 10 % K) followed by bi-weekly doses of 3.2 L m-2 milk whey
[51]. During the course of the summer the soil temperature rose to a maximum of 15 oC in the
beginning of August and dropped thereafter. When the experiment was ended in mid August
no detectable reduction of soil hydrocarbons was observed.
In parallel to the field experiment, microcosm studies were carried out on soil samples, from
the vicinity of the contaminated site that had been artificially contaminated with diesel fuel.
In the microcosms the soil was subjected to the same treatment as in the field study with the
only difference that the soil was kept at 22 oC. During these conditions the diesel range
aliphatics was almost completely degraded within 60 days. This is in accordance with previous
studies on hexadecane that showed significant difference in degradation rates at 7 and 22 oC
respectively [13]. Soil temperature is probably the most limiting factor for in-situ bioremedia‐
tion in cold regions and the short period of warmth during 3 summer months is rarely enough
to bring the soil temperature to levels that give sufficient rates of biodegradation. A possible
way to circumvent this problem would be to heat up the soil with heat injection. Preliminary
calculations carried out at our department reveal that the electric power needed to heat up a
100 x 100 x 2 m contaminated volume of polluted soil from 8 to 25 oC by e.g. a sufficient number
of heat rods, would be 166 kW during a one month period corresponding to an input heating
energy of 98 x 103 kWh. The calculation assumed that the soil surface was insulated and that
there were no vertical and downwards heat losses during the heat-up period. The energy
needed to maintain the temperature at 25 oC for another 11 month period assuming horizontal
and vertical (downwards) heat losses would amount to 235 x 103 kWh. Total energy need for
a one year period is then 333 x 103 kWh. The following input data was used: A linear temper‐
ature drop distance of 9.1 m to ambient temperatures, heat conductivity 1.5 W m-1 K-1, specific
heat capacity 800 J kg-1 K-1 and soil density 1300 kg m-3. These are rough calculations based on
some simplified assumptions about heat transfer in soil but may still give an estimate of the
magnitude of the input energy needed for a temperature enhanced in-situ soil bioremediation
project in cold climate. Downscaled to the 10 m2 by 0.5 m of the Gäddede diesel oil plot the
corresponding figures would be 27 and 387 kWh respectively for the heating and 11 months
maintenance input energy need respectively giving a total energy need for a 12 month period
of roughly 414 kWh. Part of this energy need could easily be provided by solar panels.
Furthermore, taking into consideration the very rapid increase in degradation rate even at a
moderately elevated temperature such as 22 oC it is possible that a successful temperature
enhanced remediation of the Gäddede plot could even be carried out within 6 months during
a period when solar panels can provide the total need of input energy. Solar panels can easily
be moved from one project to another so the technology described here should have a potential
for application in in-situ soil bioremediation in remote places and in cold regions.
5.2.2. One-time application of whey to oil spot at a lumberyard in Sikås

An experiment was carried out to test a low-cost and low-tech solution for remediation of soil
contaminated by waste motor oil in a cold region. A 10 m2 spot that had been polluted by
leakage from a 200 l barrel with used motor oil was chosen for the experiment. This spot would
certainly come up low in the priority list for remediation projects due to its location in a remote
region in the northern parts of the county of Jämtland and also due to the scale of the pollution
in proportions to the costs of traditional remediation practices. Such spots are however far
from uncommon and the total impact from the highly toxic substances found in waste oil, such
as carcinogenic PAH, may be significant. The polluted area was within a timber terminal and
the soil was covered with highly compacted sand and gravel. The amount of waste oil that had
leaked from the corroding barrel was estimated to 100 L. The permeability of the soil was very
low and the oil had penetrated the soil to a depth of approximately10 mm or less. Samples
from the plot showed that the oil mainly consisted of high molecular weight aliphatics and
aromatics C16-C35.
The oil spot was divided in two parts separated by a shallow cut made by the shovel that can
be seen in the top picture in Figure 11. 200 L of fresh milk whey was applied to the left side of
the spot. The plot had a slight slope to the left and to the front in the picture. Approximately
50 % of the whey was lost by runoff in these directions due to the low permeability of the soil
but some whey crossed the notch made by the shovel and penetrated the right part of the plot.
The dashed line in the figure separates the treated left part of the plot from the untreated, right
part of the plot. To make sure that lack of macronutrients would not limit the biodegradation
a total of 6 kg commercial granulated fertilizer (N/P/K 18/4/10) was evenly applied to the whole
spot (treated and control).
The photo from April 2007 was taken just before treatment. The snow had just melted and the
average air temperature during April is around 2 oC at this latitude and rose to 15 oC during
July, which is also normal for these latitudes. It is plausible that the soil surface could reach
higher temperatures during a few hot summer days and that the dark colour of the oil would
enhance heat absorption in such cases. Nevertheless, one year later, in May 2008, no difference
whatsoever could be observed between treated and untreated spot, see middle picture in
Figure 11 (note snow remaining in May).
The bottom picture in Figure 11 shows the plot two years and two months after application of
whey to the left part. There is a remarkable difference between the darkness of the sectors and
clearly, degradation of the oil in the treated part had commenced. The person in the picture
indicating the divine between the light and the dark side of the plot had no a priori knowledge
22 Soil Pollution
of the treatment strategy. Soil samples taken from the sectors indicate that the TPH concen‐
tration in the left part is half the concentration in the untreated part.
The long term effect of a one-dose of the liquid amendment (whey) is remarkable. It is worth
to note that the summer 2008 was hotter than normal in the region and the monthly average
temperature anomaly in July was + 1.5 oC according to the Swedish Meteorological and
Hydrological Institute, www.smhi.se/klimatdata/meteorologi. Clearly the low soil tempera‐
ture in this, just as in the above presented case, severely limits the rate of biodegradation, but
when conditions becomes thermodynamically more favourable, the microorganisms are able
to benefit from the extra carbon and micronutrients provided by the amendment, even after a
one year incubation time.
6. Opportunities and future predictions of soil remediation
Remediation of contaminated soils is presently a costly venture but as new technologies emerge
to treat soils in-situ, expenses can diminish considerably. Technology optimists like Ray
Kurzweil forecast that pollution will discontinue to be produced in the future and predicts a set
of technologies (including biotechnologies) that will be used to clean up the previously generated
contamination. Kurzweil expresses a strong faith in nanotechnology that he believes will provide
useful features for remediation of contaminants including; improved catalysis, chemical and
atomic bonding, sensing, and mechanical manipulation. Currently, extensive research is
undertaken to develop nanoproduced crystalline materials for catalysts that have the poten‐
tial to improve chemical yields, reduce toxic by-products, and remove contaminants [59].
To overcome some of the difficulties to degrade recalcitrant compounds research on combined

biological/abiological degradation technologies might provide useful technologies. Pre-
treatments by strong oxidizing agents such as ozone, Fenton´s reagent, potassium permanga‐
nate, ferrate etc. have shown promising results and Singh and Ward predict that combined
chemical/biological treatment of pollutants will play an important role in the future [45]. Strong
chemical oxidants may however severely affect soil microbial populations and furthermore
requires input of energy and material resources.
The use of biocatalysts is another interesting method whose practical applications have been
predicted to increase in the future. This kind of enzymatic remediation can potentially
overcome difficulties such as low survival rate of whole cell organisms and generation of toxic
by-products that are associated with bioaugmentation. Research on enzymatic bioremediation
is at its infancy and few field studies exist. The major limitation for large-scale applications is
currently the prohibitive cost for the production of the enzymes. Molecular tools are increas‐
ingly explored to overcome that obstacle which could decrease the costs significantly in the
future [60]. As we have demonstrated in this chapter the use of readily available organic waste
products as amendments and the appropriate application of principles of ecological engineer‐
ing is potentially a way to make bioremediation more sustainable.
Whey application No treatment
April 2007
May 2008
July 2009
Figure 11. One time treatment of left part of oil spot with 200 l fresh milk whey.
24 Soil Pollution
7. Conclusion
It is a common belief that soil remediation is a sustainable industry but many remediation
projects use vast amounts of energy and often physically damage the remediation site by
removing its topsoil. Ecological Engineering can potentially make remediation projects more
sustainable. The large embodied energy in many organic pollutants may be used as a driver
of a self-designing in-situ remediation process by indigenous microorganisms in the soil.
The intrinsic capacity of soil microbial communities to initiate and accelerate degradation of
soil pollutants rely on the fundamental thermodynamic driving force towards complete
degradation of target organic pollutants to low molecular-weight inorganic compounds such
as carbon dioxide and water. The degradation process releases quantities of useful “free”
energy that can be used by soil microorganisms in their metabolism. Figure 1 illustrates this
together with important abiotic and biotic degradation pathways and intermediate storages.
Table 1 gives some numbers to illustrate the theoretical “energy gain” by degrading organisms
as they catabolise representative soil pollutants. Turnover times for organic pollutants are
typically short in the atmosphere, e.g. hours-days [61] but considerably longer in soil, e.g.
months-decades [7, 44] mainly because of lack of photochemistry in the subsurface of the soil.
The slow biodegradation kinetics in soil can however be considerably improved by the use of
locally available waste materials, such as whey and fermented whey that stimulate microbial
activity, figure 2 and table 2. There are a number of other waste materials that have been
successfully tested by us [12-14, 18] or others [26-28] or remain to be tested (figure 3). Reme‐
diation strategies based on amendments from locally available by-products can be tailored to
suit site-specific conditions and be adapted to a wide range of climates and contexts as
illustrated in the case studies presented in this chapter.
Concepts of Ecological engineering, i.e. measures to stimulate the self-organizing capacity of

ecosystems by the use of organic amendments, conservation of non-renewable energy
resources and ecosystem conservation by in-situ treatment with locally available waste
resources have the potential of making soil remediation projects in low priority sites in
developing countries and in remote regions more sustainable than conventional methods that
rely on costly and less environmentally benign technology. Life cycle assessment (LCA) was
used to evaluate the different remediation technologies from a systems perspective. Results of
the screening LCA study of the whey treatment method versus a conventional dig-transport-
compotation scenario at a remote diesel oil polluted site in Northern Sweden (See map,
Gäddede, figure 7) are shown in figure 10. Clearly, the in-situ whey treatment has an overall
lower environmental impact compared to the transport-composting method. The most
contributing activity for the composting scenario in all of the environmental impact categories
studied was the transport of the excavated soil to the composting site. Thus the transport
distance is of large importance. For sites where the transport distance is much shorter, the
composting scenario would come out much better. This outcome is in accordance with what
has been earlier reported by Suèr et al [57] and Toffoletto et al [58] that transportation and
transport distance is a key element when choosing between in-situ and ex-situ remediation
technology.
In this chapter we have demonstrated that Ecological engineering in concurrence with the
application of basic thermodynamic principles and kinetic modelling of data from laboratory
experiments can provide useful guidelines for the development of energy conserving and
economically feasible bioremediation technologies. Such technologies have the potential of
making in-situ bioremediation of polluted soil in low priority sites in developing countries
and in remote regions more sustainable.
Acknowledgements
We gratefully acknowledge the financial support of the European Union Regional Develop‐
ment Fund. We are indebted to Morgan Fröling and Joseph Akambih Tajam for conducting
the LCA screening analysis of the whey method and to Anja Wedermeyer and Sylvia Vesper‐
mann for calculating energy need for temperature-enhanced bioremediation in cold regions.
Author details
Anders Jonsson* and Henrik Haller
*Address all correspondence to: [email protected]
Department of Ecotechnology and Sustainable Building Engineering, Mid Sweden Universi‐

ty, Sweden
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chapter 3
Chronic Toxicity of
Weathered Oil-Contaminated Soil
Dinora Vázquez-Luna
1. Introduction
Currently, the activities derived from the oil industry, such as extraction, transportation and
processing of oil, have affected natural resources [1-3]. For decades, tropical lands have been
contaminated by chronic oil spills, causing significant changes in physical and chemical
characteristics of the soil, affecting plant development and reducing the growth of microor‐
ganisms [4]. Moreover, oil weathering and adaptation of some plants may hide the toxicity of
high molecular weight hydrocarbons to other organisms. For this reason, it is necessary to
incorporate aspects of chronic toxicity of weathered oil-contaminated soil in the study of “Soil
Pollution”. This chapter aims to examine the chronic effects of old spills on soil, plants and
beneficial microbes, in order to support the creation of new remediation technologies, focused
on face the new challenges of soil contamination.
Oil pollution is a global problem of increasing importance [5], is estimated that every year
numerous spills affecting natural resources of Southeast Mexico, in 2011 were contaminat‐
ed more than 2,063 hectares as a result of 217 oil spills that affected soil and sea; of which
85 were caused by uncontrolled illegal connections, number which increased 204% from
2009-2011 [6]. With respect to the damages to the ground, in 2012 reported an increase of
87% of leaks and spills, its main causes were corrosion damage and failure of materials in
pipelines, in that year 30.07 hectares were contaminated in the Southern Region, joined this,
there is lag in the care of cases from previous years. This year, the oil industry closed its
operations with a total of 163.63 hectares waiting to be remedied, of which, 39.5% are located
in the southeast [7].
The environmental problems caused by oil spills, is not limited to visible pollution, because
there are chronic effects that silently endanger ecosystems, biodiversity and environmental
2 Soil Pollution
balance, due to bioaccumulation, leaching and extension of contaminants into groundwater

with potential effects on all living organisms [8]. In Mexico, current environmental regulations
is the NOM-138-SEMARNAT/SS-2003 which establishes the maximum permissible limits of
hydrocarbons in soil and specifications for its characterization and remediation [9], although
in this standard are omitted criteria for assessing chronic effects on soil microorganisms and
plants as a result of weathered oil contamination. However, the contributions of several studies
show that pollution and waste generation caused by oil activities have deteriorated soil quality
[10,11]; ie, the social responsibility of the industry and the government should not only attend
acute environmental emergencies [12].
Crude oil is a complex mixture of thousands of compounds that when released to the envi‐
ronment, is subjected to physical, chemical and biological processes called weatherability [13].
This process includes adsorption, volatilization, dissolution, biodegradation, photolysis,
oxidation, and hydrolysis. The effects of weathering are difficult to predict because it depends
on many biotic and abiotic factors [14]. Therefore, the mobility of hydrocarbons is also
influenced by natural factors [15], which involve: a) chemical processes (hydrolysis, oxidation,
reduction, photolysis), b) transport and physical processes (adsorption, advection, dispersion,
diffusion, volatilization and dissolution), and c) biological processes (biodegradation, metab‐
olism and toxicity) [16, 17].
When an oil spill occurs, it covers the soil surface, but initially at high viscosity prevents
penetration towards the subsoil. The oil is retained in the topsoil, during this phase, the light
fraction is photo-oxidized and volatilized through the soil pore space and transported to the
atmosphere, in this process involves the first n-alkanes (methane, ethane, propane and butane),
which are evaporated in less than 24 hours in tropical climates [18]. The hydrocarbons which
are not evaporated are incorporated into the soil to form a waterproofing layer that prevents
the normal flow of water. This layer or film affects the structure, porosity, absorption and water
penetration into the soil [19].
Subsequently, the oil soluble fraction diffuses into the soil solution through infiltration. The
behavior of this fraction in the soil depends on the type of texture. The presence of fine texture
allows the volatilization of some compounds (C5-C7), but when the texture is coarse, it can
leach out and transport themselves to the groundwater, affecting other organisms [20],
including the human [21-23]. Otherwise, clay soils rich in organic matter immobilize some
compounds, reducing their toxicity and decreasing its spread and leaching through the soil
profile [24, 25].
The most stable fraction of crude oil is composed with more than 18 carbons (Polycyclic
Aromatic Hydrocarbons [PAHs] and polar compounds), which are adhered on the soil matrix
decreasing the solubility and volatility [26], and increasing the capacity of adsorption on the
mineral and organic fractions, owing to the high content of active surface of the soil (clay 2:1)
and to the high molecular weight hydrocarbons [27]. At this stage, the development of bacteria
and fungi have influence on mineralization as part of natural attenuation process, but it is not
sufficient to remove all hydrocarbons weathered [28].
Chronic Toxicity of Weathered Oil-Contaminated Soil 3
Recent studies indicate that the major impediments to the biodegradation of hydrocarbons are
the physical and chemical properties of the soil, the degree of contamination and the molecular
weight of the compounds (C10-C40), but with Enhanced Natural Attenuation (ENA) may be
observed a biodegradation of 26.4% [29] up to 60% with enriched amendments, although the
n-alkanes are not removed completely [30]. Therefore, it is important to study the toxic
potential, because there are reports indicating that can be bioaccumulated PAHs in vegetables
such as C. pepo sp. [31].
2. Materials and methods
In southeastern Mexico, the land has been affected as a result of extraction; handling and
transportation of oil, there are also zones of oil discharges, which are deposited in the open,
without any environmental protection measure [32]. The bad condition of the pipelines and
the dispersion through surface runoff of rainwater, resulting from weather conditions
become more complicated to calculate the chronic effects of soil contamination by hydrocar‐
bons [10]. Accordingly, this study was conducted in three stages, which were analyzed the
chemical and physical properties of the soil, the toxic effects of weathered hydrocarbons
on the growth and development of seedlings and after 150 and 240 days of exposure, and
finally was evaluated the behavior of beneficial soil microorganisms in rhizospheric soil.
2.1. Effect on the physical and chemical properties of the soil
The study was undertaken at the facilities the Colegio de Postgraduados, located in Tabasco,
Mexico. The soil with weathered oil was collected within 2 km of the Petrochemical "La Venta"
(18 ° 04 '54 "N and 94 ° 02' 31" W), Figure 1. The pollution-free soil according to NOM-138-
SEMARNAT/SS-2003 was located in the community of Santa Teresa Arroyo Hondo. The
objective was to identify the types of soils and their level of similarity.
For both soils were determined the content of organic matter (Walkley and Black), pH
(potentiometry), P and K exchangeable (by extraction with 1N ammonium acetate pH 7,
quantification by atomic absorption and emission respectively), CEC (extraction 1N ammoni‐
um acetate pH 7, quantification by distillation and titration) and texture (Bouyoucos). The
analytical methods used were those indicated in NOM-021-2000-RECNAT [33]. Total Petro‐
leum Hydrocarbons (TPH) were determined by the technique reported in the NOM-138-
SEMARNAT/SS-2003 [9].
2.2. Effects on plants
Bioassays to determine the phytotoxicity of weathered oil on two species of legumes

(Crotalaria incana and Leucaena leucocephala) were carried out in the facilities of the Laboratory
of Soil Microbiology, Colegio de Postgraduados, Tabasco, Mexico. Two states were studied
phenological (plant and seedling), in two treatments: a) soil with 150 mg.kg-1 TPH (control
treatment) and b) soil with 79.457 mg.kg-1 TPH weathered (this has been contaminated by over
4 Soil Pollution
25 years). Both soils were characterized as Gleysols, with the same pedogenetic origin, as
described in the previous section.
Bioassays were established under a Completely Randomized Design (CRD) with three
replications and two legumes, these were selected to be species that grow wild in oiled areas,
but the former has tolerance, while the second shows sensitivity to high concentrations of crude
oil [34]. In each bioassay was used 208 Protocol of the Organization for Economic Cooperation
and Development (OECD) modified according to [35], which allows easily identify the
symptoms of stress in the plant.
Seedling bioassays: 50 seeds were sown C. incana and 25 L. leucocephala by repetition, respectively.
Glass containers were used (32 x 22 x 5.5 cm). The number of seeds sown per plant species was
calculated according to the size of the seed [36], the viability of the species [37] and the area of
the container. Seeds were previously scarified to remove impermeable integument, which
constitute a barrier for germination [38]. Scarification consisted of immersing the seeds in
sulfuric acid for 15 minutes and washed with tap water subsequent to remove all acid residues
[39]. Germination tests were performed to determine the initial seed viability, finding viability
standard values in both blocks [40]. The test lasted 30 days and the variables evaluated were
mortality, height, root length and dry matter accumulation aerial and root.
Plant bioassays: Bioassays were established plants seedlings 30 days from uncontaminated soil.
Subsequently, two of these were transplanted into containers, 15 days after a plant was
removed from each container. Exposure of plants to pollutant lasted 150 days to C. incana and
240 days for L. leucocephala, because of its tolerance respective. The physiological variables were
evaluated: height, root length, biomass (leaves and stems), root biomass and number of nodes,
leaves and fruits. During both assays was provided with water to field capacity and were not
supplied nutrients (N, P and K) to avoid interference on the growth of the specimens. The
material was weighed on an analytical balance to obtain the values of DM. The multiple
comparison of means was performed by Tukey test (a = 0.05). The numerical results were
analyzed with SAS software version 9.1, using PROC GLM.
2.3. Effect on soil microorganisms
Quantification of microorganisms was determined by viable count method for serial dilutions
[41] for Rhizobium extracts in nodules and Free Living Nitrogen Fixing Bacteria (FLNFB) in
rhizospheric soil. Culture media were used combined carbon and yeast-mannitol agar [42].
3. Results and discussion
3.1. Effect on the physical and chemical properties of the soil
The soil profile and laboratory results confirmed that both soils are Gleysols, with the same
pedogenetic origin [43] (Table 1), but the contamination had abnormalities in their chemical
characteristics (Table 2), the which have been reported in other studies undertaken in the South
East (Table 3).
Figure 1. Study zone
Sampling pH OM P K CEC Clay Silt Sand Textural TPH

0-30 cm (H2O) (%) mg.kg-1 Cmol(+)kg-1 (%) classification (mg.kg-1)
Soil † 6.3 10.2 23.1 0.35 45.2 61 21 18 Clayey 150
Soil ††
4.2 25.8 3.58 0.4 43.5 48 33 19 Clayey 79,457
* pH 1:2 (potentiometry), organic matter (Walkley and Black), P and K (extraction with 1N ammonium acetate pH 7 and
quantification by atomic absorption and emission), CEC (Cation Exchange Capacity) (extraction with 1N ammonium
acetate and quantification by distillation and titration), texture (Bouyoucos).
†
Control soil
††
Soil with weathered oil (OM without previous removal of TPH)
Table 1. Chemical properties and concentration of TPH in the soils studied.

6 Soil Pollution
Sampling OM P K CEC
pH
0-30cm (%) mg.kg-1 C mol(+) kg-1
Control soil Moderately acid Medium High Medium Very High
Polluted soil †† Strongly acidic Very high Low Medium Very High
††
Soil with weathered oil (OM without previous removal of TPH)
Table 3. Alterations reported in soils contaminated with TPH, in southeastern Mexico.
Table 2. Interpretation of the characteristics of the soils studied.

Alterations The interpretation was based on the ranges indicated
Effects
in NOM-021-2000-RECNAT.
High ratios of C / N and C / P Unfavorable microbial growth [44]
Retaining TPH fractions in organic matter Alter the solubility of phosphorus [45]
Alterations Effects
Increasing
High ratios of C /Na
N and C / P Limitations
Unfavorable in thegrowth
microbial production
[44] of plants [11,
46]
Retaining TPH fractions in organic matter Alter the solubility of phosphorus [45]
Decreasing
Increasing Na pH Decrease
Limitations in microbionas populations
the production of plants [11, 46][47]
Decreasing pH Decrease microbionas populations [47]

The electrical conductivity can be increased Salinity [48]
The electrical conductivity can be increased up to 5.6 times Salinity [48]
up to 5.6 times
Interruption in the interaction between cations Ca and K Reduction in capacity of soil to retain Ca and K [32]
Interruption in the interaction between Reduction in capacity of soil to retain Ca
cations
Table Ca andreported
3. Alterations K and
in soils contaminated with TPH, in K [32] Mexico.
southeastern
Also, many physical properties of soil are altered, such as water retention capacity; this is
Also, many physical properties of soil are altered, such as water retention capacity; this is
because when an oil spill occurs, hydrocarbons bit compete with water filling the pores
because when an oil spill occurs, hydrocarbons bit compete with water filling the pores [49].
[49]. On the other hand, increasing the moisture content in the soil reduces the adsorp‐
On the other hand, increasing the moisture content in the soil reduces the adsorption of
tion
liquid liquid
of hydrocarbons
hydrocarbons in theinorganic
the organic matter
matter and and
clay clay
and and
the the filling
filling of of
thethepores
poresand
and
capillaries (Figure 2) [50].
capillaries (Figure 2) [50].
Figure 2. Alteration in the retaining moisture of soil.

Figure 2. Alteration in the retaining moisture of soil.
The oil can form macroaggregates and macropores that increase water flow (Figure 3), and
The oil can form macroaggregates and macropores that increase water flow (Figure 3), and
these changes inhibit the water retention in the soil, which is moistened only after a long period
these changes inhibit the water retention in the soil, which is moistened only after a long
of contact [51], so that the plants may suffer water stress and die.
period of contact [51], so that the plants may suffer water stress and die.
Figure 3. Formation of macroaggregates in soil with 12,155 mg.kg-1 TPH weathered.
3.2. Effects on plants
The results indicated that the lower height and root length were found in soil with 79,457
mg.kg-1 of weathered oil, this can be explained because of the limited development of these.
Both legumes formed less biomass in soils with higher concentrations of oil, which was
associated with lower production of biomass in leaves and stems, as a result of the presence
of the contaminant in the soil (Table 4 and 5), which limits the entry of water to the plant.
However, the hydrophobic effect also affects the early stages of germination as evidenced with
a delay of up to five days in the emergency [52], even in high concentrations of TPH, germi‐
nation may be inhibited completely [53].
Some authors found that exposure to concentrations of 2 791, 9 025 and 79 457 mg.kg-1 of
petroleum hydrocarbons in soil inhibited the vegetative growth and reduced plant biomass in
seedlings of Echinochloa polystachya, Brachiaria mutica and Cyperus sp [10]. Others establish
increased toxicity on the dry weight of rice seedlings (Oryza sativa) after 25 days of exposure
to 90,000 mg.kg-1 weathered oil [54]. Biomass reduction is possibly due to widespread damage,
which begins in the root system, hindering vegetative growth and therefore the accumulation
of plant biomass. However, the first studies mentions that at low concentrations, oil could
stimulate vegetative growth [55].
Crotalaria incana seedlings did not form nodules in soil contaminated with 79,457 mg.kg-1 of
weathered oil after 30 days of exposure to oil. These results were similar to those obtained
according to [34], who found that nodulation was completely inhibited in Crotalaria sp. and
Mimosa pigra by concentrations above 50,000 mg.kg-1.
8 Soil Pollution
In both plant species, growth decreased with higher concentration of hydrocarbons in soil
(Table 5 and 6). This response may be related to decreased water absorption through the roots
for the presence of the hydrophobic film formed by the oil added to the soil [56]. The lack of
water absorbed decreases cell turgor, reduces or inhibits the processes of incorporation of
nutrients and also affects vegetative growth [57, 58]. Water stress is related to the water
potential gradient (which depends on the conditions present in the soil) and the membrane
permeability to water (which depends on the species [59] therefore, Leucaena leucocephala
shown to be more tolerant to water deficit).
Root Biomass
Concentration Height Mortality
Species length Leaves Stems Aerial Root Total
mg.kg-1
cm (%) gr
Leucaena 150 7.5a 19.0a 0.0a 3.3a 1.3a 4.6a 1.1a 5.7a
79,457 5.1b 5.1b 4.2a 1.5b 0.5b 2.0b 0.5b 2.5b
Values with different letter are statistically different (Tukey, p = 0.05)
Table 4. Response of Crotalaria incana seedlings to 30 days of weathered oil exposure.
Root Biomass
Concentration Height Mortality
Species length Leaves Stems Aerial Root Total
mg.kg-1
cm (%) gr
Crotalaria 150 9.8a 9.8a 2.6a 1.6a 1.0a 2.6a 0.6a 3.2a
79,457 2.9b 2.8b 28.6a 1.0a 0.5b 1.5b 0.4b 1.9b
Table 5. Response of Leucaena leucocephala seedlings to 30 days of weathered oil exposure.
Crotaria incana plants showed a greater effect on biomass in response with exposure to the
weathered oil (Figure 4), which may be related to increased toxicity of recalcitrants com‐
pounds. There are also some soil properties which allow the adsorption of pollutants [60]. Clay
soils with high organic content and low pH may favor the persistence of toxic substances in
the soil for a long time after the oil spill occurred [61, 34], due to the adhesiveness of organic
matter [62].
Furthermore, fine texture allows the oil form a coarse structure on the outside and around the
conglomerate making it waterproof [63], this has effects on root development, growth, and as
a result will cause decrease in the accumulation biomass [59]. On the other hand, soil contam‐
ination by hydrocarbons can also modify some characteristics such as texture, bulk density,
ratio of the particle size of the soil, reducing aeration and affecting the productive development
of plants [64, 65].
Furthermore, fine texture allows the oil form a coarse structure on the outside and around
the conglomerate making it waterproof [63], this has effects on root development, growth,
and as a result will cause decrease in the accumulation biomass [59]. On the other hand, soil
contamination by hydrocarbons can also modify Chronic some characteristics suchOil-Contaminated
Toxicity of Weathered as texture, bulkSoil 9
density, ratio of the particle size of the soil, reducing aeration and affecting the productive
development of plants [64, 65].
a) b)
c) d)
FigureFigure 4. Toxicity
4. Toxicity on Leucaena
on Leucaena leucocephala
leucocephala seedlings
seedlings with: a) 150 with:
mg.kg-1a)y 150 mg.kg
b) 79,457 -1 y -1
mg.kg b)TPH
79,457 mg.kgand
weathered, -1
plantsTPH weathered, and plants with c) 150 mg.kg y d) 79,457 mg.kg TPH weathered.
with c) 150 mg.kg-1
y d) 79,457 mg.kg -1
TPH weathered.-1 -1
Root Biomass
Species Concentration Height Knot Leaves Fruits
length Aerial Root Leaves Stems Inflorescences Seeds Pods
mg.kg-1 cm Number gr
Crotalaria
150 91.7a 17.3a 16.0a 112.7a 54.0a 47.5a 5.6a 11.0a 14.7a 1.5a 9.6a 10.8a
incana
79,457 32.3b 7.0b 15.0a 2.7b 0.0b 0.6b 0.2b 0.1b 0.6b 0.0b 0.0b 0.0b
Table 6. Response of Crotalaria incana to 150 days of weathered oil exposure. 11
Root Biomass
Species Concentration Height Knot Leaves Fruits
length Aerial Root Leaves Stems Inflorescences Seeds Pods
mg.kg-1 cm Number gr
Leucaena
150 149.0a 107.7a 43.7a 19.1a -- 88.6a 34.0a 19.1a 69.5a -- -- --
leucocephala
79,457 66.7b 7.3b 31.0b 4.6b -- 15.9b 6.9b 4.6b 11.3b -- -- --
Table 7. Response of Leucaena leucocephala to 240 days of weathered oil exposure.

10 Soil Pollution
3.3. Effect on soil microorganisms
In plants Leucaena leucocephala high concentration of weathered oil did not affect populations
of Rhizobium into nodules, as happened in the case of C. incana, in which both populations were
significantly lower in a shorter time exposure (Figure 5). This is because the oil alters the
physical and chemical characteristics of the soil, causing the blockage of gas exchange with the
atmosphere and affecting microbial populations. Furthermore, the weathered oil is adsorbed
in the ground, being less accessible and more difficult to degrade by microorganisms [66, 67].
This may bring a direct impact on rhizobia, because they are aerobic bacteria that remain in
the soil as saprophytes, until they infect a radical hair.
Some authors mention that soil conditions have a marked effect on rhizobia, because they can
impact the survival and the infectivity of root hairs [68, 69]. However, there are many other
factors that influence on effectiveness symbiosis such as specificity and virulence of the
bacterium Rhizobium, nutrimental factors, soil temperature and pH [70], the latter is of utmost
importance because has been reported to decrease significantly in contaminated soil [38].
Added to this there are other factors such as the accumulation of heavy metals and salts that
affect soil microbiota [11, 4].
Crotalaria incana Leucaena Leucocephala

4.5E+06 2.5E+07
4.0E+06
Microorganism (CFU g-1)
3.5E+06 2.0E+07
Rhizobium
3.0E+06
1.5E+07
2.5E+06 Free living nitrogen
fixing bacteria
2.0E+06
1.0E+07
1.5E+06
1.0E+06 5.0E+06
5.0E+05
0.0E+00 0.0E+00
150 79,457 150 79,457
Concentration (mg.kg-1) Concentration (mg.kg-1)
Figure 5. Quantification of populations of Rhizobium extracts in nodules and Free Living Nitrogen Fixing Bacteria in
rhizospheric soil.
L. leucocephala almost doubled FLNFB populations with 79,457 mg.kg-1 of weathered oil. This
can be explained because in tolerant species, some microorganisms can increase their popu‐
lations in the presence of hydrocarbons [71], allowing support microbial growth. As well as
[72] argue that tolerant plants are promising tools to accelerate the removal of PAH in long
term polluted soils, due to their ability to thrive in a contaminated site, and its success is
probably influenced by the relative amount of exudates and other compounds within the root,
that stimulate microbial growth [73]. On the other hand, several authors argue that changing
the C: N: P, the water content and the water retention capacity of clay soils are crucial to obtain
the highest rate of degradation of TPH [74, 75].
4. Conclusion
Soil is one of the most valuable resources that humanity has due to the variety of services
offered and which depend for food. Currently, there is tremendous competition for land use,
either for use as human settlements, commercial, industrial or produce raw materials (wood)
and food, so weathered oil pollution is one of the most important challenges for scientists,
because not only it is decontaminated, it comes to restoring the quality and safety of the soil,
its properties and microbial component vital for production and proper plant development.
Acknowledgements
The author wants to especially thank Dr. Macario Vázquez Rivera, Irma Luna Capetillo, Mayra
Vázquez Luna and Daniel Lara Rodríguez for their valuable support and the Technical Council
of COLPOS for their scientific consultancy. Finally, the author wants to thank the Universidad
Veracruzana for financing the publication of the present article.
Author details
Dinora Vázquez-Luna*
Address all correspondence to: [email protected]
Universidad Veracruzana, México
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chapter 4
Heavy Metal Contamination in Brazilian Agricultural

Soils due to Application of Fertilizers
Affonso Celso Gonçalves Jr., Herbert Nacke,

Daniel Schwantes and Gustavo Ferreira Coelho
1. Introduction
The Brazilian grain production reaches record productivities each year and among the major
crops, stand out the maize (Zea mays L.) and soybean (Glycine max L.). For these two cultures,
the obtaining of the maximum yield has a fundamental importance in the current global
scenario [1]. In this relentless pursuit of higher productivities, farmers use technologies related
to various areas, such as new forms of fertilizer and pesticide application, different fertilizers,
crop breeding, equipment and techniques of planting and harvesting. In order to increase the
efficiency in this activity, due to the increased requirement for competitiveness of economic
globalization, one of the tools found by farmers is the use of fertilizers containing micronu‐
trients in their crops [2]. Among the various factors of production, the need of balanced and
sustainable fertilizers occupies a prominent place, which should include not only the primary
and secondary macronutrients, but also micronutrients, which are not considered in routine
fertilization by most farmers. Currently, with the advancement of the industries of fertilizers,
micronutrients are added directly in the formulation of basic fertilizers (N:P2O5:K2O), which
can be found in the market of domestic and imported agricultural inputs [3]. Among the several
micronutrients used in Brazilian and Global agriculture, zinc (Zn) is a major one, having its
need scientifically established over 70 years being its main functions related to photosynthesis,
respiration, protein synthesis and plant membrane permeability [2].
Nutrient uptake by crops is influenced by various factors, including climatic conditions such
as rain and temperature, genetic differences between varieties, type of soil, nutrient content of
soil and various cultural practices.
2 Soil Pollution
In the strategy of reducing costs, both in manufacture of fertilizers, as well in croplands, the
use of industrial waste for the obtaining of micronutrients has been widely used. These wastes,
when used appropriately and rationally present themselves as a good solution for agriculture,
but their improper use can cause serious damage to soil, plants and human beings. Several
researches are touting in scientific works, facts that generate severe criticism to the fertilizer
industry, mainly because the involvement of these industries in operations that endanger the
environment, where the main factor is the use of raw materials containing toxic metals such
as cadmium (Cd ), lead (Pb) and chromium (Cr) in the manufacture of fertilizers [4]. In that
way, the fertilizers used to supply micronutrients have often, in their composition, in addition
to the desirable elements, toxic metals such as Cd, Pb and Cr [5,6].
The purpose of this chapter is to assist in developing a more adequate environmental legisla‐
tion, that ensures a sustainable environment and a production of quality food. In that way,
this chapter highlight the lack of criteria, in the current Brazilian environmental legislation,
regarding the allowed limits of toxic heavy metals in agricultural soils and in the use of
fertilizers contaminated with heavy metals in agriculture, as well, demonstrate that the use of
these contaminated inputs can cause degradation of agricultural soils, making food production
unviable at these locations. Furthermore, it was made the study of three cases about the
existence of fertilizers contaminated with toxic metals in Brazilian agriculture and the impact
caused by its use. The chapter also presents some strategies for recuperation and remediation
of soils contaminated with toxic heavy metals.
The presence of heavy metals in fertilizers is a extremely important subject, since investigations
in Brazil, coordinated by prosecutors in São Paulo, show evidence of entry loads containing
toxic waste through irregular importation and smuggling, several times in Brazilian ports.
These investigations shown that companies and manufacturers of agricultural inputs, are
importing these toxic chemicals from United States, Canada, Mexico, Spain, Holland and
England to be used as raw material in the manufacture of fertilizers to supply micronutrients.
Brazil presents itself as one of most promising nations of contemporary and future world,
however, for the effective consolidation of its presence among the great powers is extremely
necessary maintain the sustainability of its agroecosystem. Therefore, the Brazilian agricultural
activity should be developed without damaging the environment and thus continue to grow
in a rational and appropriate way, avoiding harm to future generations. In the world of
agribusiness, which needs to be highly competitive, concepts such as traceability, environment
and risk assessment are fundamental starting points for those who want to establish and grow.
2. Potential contamination of soil by agricultural activities
Due to its continental dimensions, Brazil has a great diversity of soil types in different regions
of the country. Most of these soils have limitations for agriculture, characteristic of tropical
regions such as acid, deep and highly weathered soils, with low nutrient availability and high
exchangeable acidity (Al3+), resulting in a low cation exchange capacity and low availability of
P [7].
Heavy Metal Contamination in Brazilian Agricultural Soils due to Application of Fertilizers 3
These factors highlight the need for adopting management strategies that aim to increase the
chances of good crop of the culture when grown in soils of low fertility, giving them the ability
to meet the nutritional requirements of these plants. The practices of fertilization and soil
amendment aim to make soil suitable for the development of major crops, providing the
necessary nutrients for the plants and decreasing the acidity of the soil. These practices are
adopted throughout the world, and are based on the prerogative of the need to produce food
to sustain the world population that grows rapidly each year.
According to the Food and Agriculture Organization of the United Nations (FAO) [8], in 1980
there were 4.4 billion people on the planet, 5.7 billion in 1995 and 6.9 billion in 2010. Studies
estimate that by 2050 there will be 9.4 billion people and in 2200 about 11 billion [9]. Based on
this scenario, agricultural activities have expanded greatly since 1945, currently holding
approximately 38% of the land surface. Between 1985 and 2005, the acreage increased about
7%, generating large increases in food production [10].
This pressure on agricultural activities promotes an increasing fertilizer consumption per each
year that goes around the globe (Figure 1). Studies show that in the early 60's used to be about
146 million tons of fertilizer, increasing in 2010 to 176 million of tons, demonstrating a
significant increase in the consumption of these inputs [11]. Also according to the International
Fertilizer Association (IFA) [11], Brazil is the fourth largest consumer of mineral fertilizers
(N:P2O5:K2O) in the world, only behind China, India and the United States. In recent years the
consumption of nutrients by Brazilian agriculture has increased considerably being that in
1961 Brazil was only the 25th world consumer, however in the 1990, the country was already
appearing as the seventh in the rankings. Regarding to micronutrients, there is an increase in
consumption of 13.3 times in the period 1990-2003 [12].
People
-1
World - 120 countries - (ten thousand t m )
-1
Brazil (thousand t m )
9
18000
7x10
16500
9
6x10 15000
Fertilizer consumption
13500
Population growth
9
5x10 12000
9 10500
4x10
9000
9
3x10 7500
6000
9
2x10 4500
9 3000
1x10
1500
0 0
1959 1962 1965 1968 1971 1974 1977 1980 1983 1986 1989 1992 1995 1998 2001 2004 2007 2010 2013
Years
Figure 1. Population growth and consumption of fertilizers in Brazil and in the world [11]
4 Soil Pollution
However the use of these products is often not done correctly. The plan for fertilizer recom‐
mendation involves planning for the collection of soil samples, soil chemical analysis and
recommendation by an appropriate professional, something that many producers do not
perform, often using pre-determined recommendations without professional assistance.
Another problem is the lack of crop rotation and the use of highly productive cultivar which,
according to reference [13], require intense macronutrient application, resulting in micronu‐
trient deficiencies in the soil.
Overuse of fertilizers can also provide soil contamination, it increases the concentration of a
particular element or substance of environmental interest above its naturally occurring in the
soil [7]. This contamination is related to eutrophication (excess nutrients), in this case the major
nutrients to be considered are nitrogen (N) and phosphorus (P). Another concern is the
availability of harmful elements such as toxic metals. These elements can express their
pollutant potential directly on soil organisms, due to the availability to plants in phytotoxic
levels, plus the ability to transfer to the food chain through the own plants, or by contamination
of soil and water resources [14].
The presence of harmful elements in fertilizers is worrying because until the 70's, most
fertilizers were obtained directly from ores found in nature, and although they also contained
toxic metals, the beneficiation process of this material was less offensive from the environ‐
mental standpoint, because the contaminant levels were very low [6]. Recently, the shortage
of mineral resources and modern extraction methods required large investments in equipment
and technology, creating a technical difficulty and the pursuit of lower costs of production,
which eventually encouraged companies to seek a cheaper solution such as using industrial
waste, that may be feasible since it does not contaminate the environment. However, according
to the Environmental Sanitation Technology Company of São Paulo State (CETESB) [15], in
São Paulo, until the end of 2012, 4572 contaminated areas were found, and approximately 4%
of these areas were contaminated with industrial waste, and among the contaminants, toxic
metals are in 4th place, behind Polycyclic aromatic hydro carbons contaminants (PAHs),
aromatic solvents and liquid fuels.
Between the sources of soil contamination from waste, there is the contamination of sewage
sludge, that even though has a variable composition, with macro, micro nutrients and organic
matter, their use may be limited by the presence of pathogens and toxic metals [16,17]. Studies
evaluating the use of sewage sludge in sugarcane found that the doses used increased levels
of chromium, nickel (Ni), Pb and zinc (Zn) in the soil [18].
Reference [19] studying the availability of Cd in dystrophic soil submitted to the application
of sewage sludge, observed that at the first hour of contact between the sewage sludge and
soil occurred transference of Cd to the solution. After this interval, the Cd concentration in the
solution decreases, occurring stabilization in the following hours, due to adsorption and ion
exchange which act providing less metal. According to the authors, in all observed time
intervals, the levels of Cd were higher than reference values for CETESB (<0.5 mg kg-1) [20],
which characterize the contamination of the soil by sewage sludge.
The use of pig manure as fertilizer is also a practical often carried out mainly in south of Brazil,
however, some studies have shown risks as to its overuse. Reference [21] evaluated the
accumulation of metals in the soil under the systematic application of swine manure, and found
an increase in the availability of copper (Cu), zinc and manganese (Mn). The authors also claim
that in these cases there is a need to monitor these elements over time, so that the total contents
do not exceed the critical values. In study conducted in China [22], the application of manure
in soil for 16 years resulted in higher accumulation of Cd. One of the major sources of metals
such as Pb, Ni, Cr and Cd in pig manure is mineral supplied by commercial feed [21].
The metals are also components of many pesticides, mainly Cu, Zn and Pb, which cause high
soil contamination by these elements [23], another case is the use of Cu, that has been used
since 1917 for the Bordeaux mixture [24]. In a study conducted in Caetés watershed in the
county of Paty's Ensign, Rio de Janeiro, Brazil, it was aimed to evaluate the soil contamination
by toxic metals, from the intensive use of agrochemicals, in which the authors found an increase
in the total levels of metals in soil, stream water and dams. The soil metal concentration not
reached critical established levels, however the result was worrying about the water stream
and dam, which showed total contents of Cd, Mn and Pb above maximum standards estab‐
lished by the Brazilian legislation [23].
In England and Wales, reference [25] developed an inventory on the input of metals in
agricultural soils. Forms of contamination are the most diverse, the main sources are generated
by atmospheric deposition, sewage sludge, manure, inorganic fertilizers, lime, agrochemicals,
irrigation water and industrial waste.
All these reports point to industrialization as the main cause for the large amount of metal
dispersed in the environment, and human as the protagonist of all this pollution, because
usually mankind seeks the easiest way supply their needs while generating countless volumes
of waste. These actions put us in the position of predator. And thus there is a need of legislations
that seek to limit the presence of these contaminants in the soil. In Table 1, the international
maximum levels of metals allowed in soils of various places show what would be, in theory
at least, a concern of many countries in the world about metal contamination.
Toxic Metals (mg kg-1)

Countries
As Cd Pb Cr Hg
(*)
European Comunities - 1-3 50-300 - 1-1,5
(**)
United States 14 1.6 50-300 1500 0.5
(**)
Poland (1977 – 1993) 30 1-3 70-150 50-80 5
(**)
Germany (1984 – 1992) 20 3 – 1.5 100 100 2
(**)
United Kingdon 10 3-15 500-2000 - -
(**)
Austria 50 5 100 100 5
Source: * [26], ** [27]
Table 1. Maximum permitted levels of toxic metals in the soil in some countries
6 Soil Pollution
Currently in Brazil, the legislation that deals about soil contamination is Resolution n°. 420 of
2009 of the National Council of Environment (CONAMA) [28], which aims to establish the
criteria and values that guide of soil quality as the presence of chemical substances, among
them the toxic metals. The resolution establishes three categories of guiding values which are:
The Reference Value Quality (RVQ) which is the concentration of a substance that will define
the natural quality of the soil, based on previous studies for each Brazilian state, the Prevention
Value (PV), which refers to the concentration limit of a substance in the soil, and the Investi‐
gation Value (IV) which is the concentration of a substance in the soil of which there are
potential risks, direct or indirect, to human health. Table 2 shows the values set for preventing
and detecting toxic metals in soil.
However, the Resolution n°. 420 is being severely questioned by authorities and researchers,
since it present several misconceptions that violate the Federal Constitution regarding the
"preservation, enhancement and restoration of environmental quality propitious to life" (this
issue will be addressed with greater emphasis on section 5 of this chapter).
IV
Metal PV
Agricultural Area Residential Industrial
As (mg kg )-1
15.0 35.0 55.0 150.0
Cd (mg kg )-1
1.3 3.0 8.0 20.0
Pb (mg kg-1) 72.0 180.0 300.0 900.0
Cr (mg kg-1) 75.0 150.0 300.0 400.0
Hg (mg kg-1) 0.5 12.0 36.0 70.0
PV: Prevention Value; IV: Investigation value
Table 2. Values guiding to the presence of toxic metals in Brazilian soils in accordance with Resolution n° 420 of 2009
of CONAMA [28]
3. Fertilizers versus contaminants
There are three different categories of agricultural fertilizer: macronutrient fertilizer to supply
major nutrients (N, P and K); macronutrient fertilizer to supply secondary nutrients [calcium
(Ca), magnesium (Mg) and sulfur (S)] and micronutrients [Cu, Zn, Ni, Mn, iron (Fe), molyb‐
denum (Mo), boron (B), chlorine (Cl), sodium (Na), cobalt (Co) and silicon (Si)]. All of these
nutrients can be applied through simple fertilizer (only one nutrient) or compound form
(formulations with more than one nutrient).There is a further classification based on the
essentiality of nutrients, where metals may be classified as essentials, such as B, Cu, Fe, Mn,
Mo and Zn; beneficial, such as Co, Ni and V; and toxic, such as Cd, Cr, Hg and Pb. Thus, it
should be noted that the essential and beneficial elements can become toxic at high concen‐
trations [30].
According to reports developed by the United States Environmental Protection Agency

(USEPA) [31] and The Weinberg Group (requested by The Fertilizer Institute) [32], the main
metal contaminants that are found in agricultural fertilizers are Cd, Cr, Co, Cu, Pb, Mo, Ni,
Zn, arsenic (As), mercury (Hg) selenium (Se), vanadium (V) and radio 266 (Ra). It can be
observed in this list, that among the metals commonly found as contaminants in fertilizers,
occur the presence of micronutrients, required for plant development.
According to USEPA [31], among the fertilizers used to supply macronutrients, the phosphate
shown the highest levels of toxic metals (especially Cd), followed by nitrogen and potassium
fertilizers. With respect to micronutrients, the sources used to obtain it are varied, resulting in
the occurrence of fertilizers from low to high levels of metals. In general, micronutrient sources
have higher level of contaminant in comparison to sources of macronutrients, on the other
hand, are applied larger amounts of fertilizer to supply the macronutrients, therefore, the
possibility of soil and environment pollution with the use of any type of contaminated fertilizer
is real and should be seriously studied.
The presence of contaminants in fertilizers occurs most of the time with the use of industrial
waste or low cost sources for raw materials. These sources, when treated and used properly
and rationally, present themselves as a good alternative to agriculture, however its improper
use can cause serious damage to the soil, plants and the human beings.
This fact becomes even more important in the case of fertilizers to supply micronutrients,
which often are obtained from raw materials originated from industrial wastes, which may
show, in addition of the desirable elements, toxic metals. In this situation, the benefits are
highly attractive for the fertilizer industries, once these companies acquire low quality raw
material at low costs, without generating any type of waste, incorporating everything in their
products.
One of the main cases regarding the presence of contaminants in fertilizers happened in the
90s in the United States, where many farmers and social actors reported the possibility of the
presence of toxic metals in these inputs. The impact generated and population pressure led to
the development of several research regarding the contamination of fertilizers and soil
pollution, culminating with the publication by the U.S. government, of the first law regulating
the content of toxic elements in the United States in 1998. From these studies and based on
Canadian law, in 2002, the USEPA published the federal law that regulates the production of
fertilizers to supply Zn from industrial waste [33].
Another case reported in the literature is the presence of contaminants in raw materials from
China. In a publication from the Washington Department of Ecology (WDE) [34] it was
reported the presence of Cd in raw materials for the manufacture of fertilizers, with samples
showing more than 20% of Cd, i.e., more than 200,000 ppm. It is also reported that the final
products produced with the use of these contaminated sources presented 10,600 ppm of Cd.
The report also warns that Cd present in the analyzed material does not naturally occur in
natural mineral sources, leading to the conclusion that the incorporation of Cd or the use of
wastes contaminated with this metal was occurred.
8 Soil Pollution
In Brazil, there are frequent reports of companies selling fertilizer, mineral and organic, outside
the standard quality. The most common cases are related to the presence of nutrients amounts
below the specified labels, since this factor is more investigated. However, there are few studies
evaluating the presence of toxic metals in these inputs, although the few that were conducted
demonstrated the presence of these contaminants in fertilizers [6, 35, 36].
Currently, most grain-producing countries and other plant materials have laws regarding the
limits of metals in fertilizer, which are presented in Table 3, where it can be observed a large
variation between the allowed values. In general, the stipulated limits are for metals in
phosphate fertilizer and micronutrient fertilizers, but there are some normatives that consider
fertilizers in general, regardless of its composition or purpose. The limits of toxic metals can
also be determined based on the amounts of nutrients or total amount in fertilizers.
As Cd Pb Cr
Country Fertilizer type
------------------------- mg kg-1 ------------------------------
Switzerland 1
Phosphate NA 50.0 NA NA
Finland 1
Sweden1 Phosphate NA 100.0 NA NA
Norway1 Phosphate NA 100.0 NA NA
Denmark 1
Belgium1 Phosphate NA 210.0 NA NA
Germany1 Phosphate NA 210.0 NA NA
Austria 1
U.S. (California) 2
Phosphate 2.0* 4.0* 20.0* NA
U.S. (Washington)3 Phosphate 13.0 165.0 61.0 1.0
Australia 1
Japan 1
Phosphate 50.0 343.0 100.0 5.0
New Zealand1 Phosphate NA 280.0 NA NA
U.S. (Texas)4 Anyone 41.0 39.0 300.0 17.0
Canada 1
Anyone 75.0 20.0 500.0 NA
China4 Anyone 50.0 8.0 100.0 NA
U.S. (California) Micronutrient 13.0** 12.0** 140.0** NA
U.S. (Washington) Micronutrient 112.0** 83.0** 463.0** 6.0**
Australia Micronutrient NA 50.0 2000.0 5.0
1 [38], 2 [39], 3 [40], 4 [41]; * - Maximum allowed value per percentage point of P2O5 in the fertilizer; ** - Maximum
allowed value per percentage point of the micronutrients sum in the fertilizer ; NA – not available
Table 3. International maximum limits of contaminants allowable in fertilizers

The Brazilian fertilizers law is based on the Normative Instruction (NI) 27/06 [37], which states
like contaminating toxic metals, the elements As, Cd, Pb, Cr and Hg, determining the maxi‐
mum allowable limits of these metals in four different categories of fertilizers (Table 4).
Maximum permitted quantity of the metal contaminant (mg kg-1)

Fertilizer type
As Cd Pb Cr Hg
Simple mineral fertilizer to

2.0* 4.0* 20.0* 40.0* 0.05*
suply P2O5
Compound mineral
fertilizer with 500.0** 15.0** 750.0** 500.0** 10.0**
micronutrient
Compound mineral
fertilizer with
250.0 57.0 1000.0 NA NA
macronutrient and
micronutrient
Mineral fertilizer to suply

4000.0 450.0 10000.0 NA NA
micronutrient
* - Maximum allowed value per percentage point of P2O5 in the fertilizer; ** - Maximum allowed value per percentage
point of the micronutrients sum in the fertilizer; NA – Not Available.
Table 4. Maximum allowable limits for metal contaminants in Brazilian fertilizers [37]
There are some doubts in relation to NI 27/06, which consider the legislation incipient, since
that it does not determines which raw materials can be used as sources of nutrients in the
fertilizers. Nutrient sources for macronutrients generally present good quality, however, for
micronutrients, the use of low quality materials is frequently observed in Brazil, as mentioned
previously, resulting in a possible contamination of the environment.
The legislation also does not tell how it will be the inspection of fertilizers industries, moreover
tolerating the presence of contaminants in amounts up to 30% higher than the accepted values
(Table 4). Another worth mentioning fact, is that the limits should be revised, since Article 5
affirms the need of a revision of the values predicted by NI 27/06 four years after its publication.
Nowadays, still does not exist an adequate legislation in Brazil regarding the use of toxic waste
in the production of micronutrients. However, there are technical studies which classify these
elements as hazardous materials, an example is the NBR 10004 of the Brazilian Association of
Technical Standards (ABNT) [42], which stablish that the use of this wastes may present a risk
to public health, causing or contributing to an higher incidence of diseases and mortality, also
presenting adverse effects on the environment when handled or disposed inappropriately.
Another issue that is being discussed by the scientific community is that the determination of
contaminants limits in fertilizers provides for the industries the possibility to include, as a
source of nutrients, contaminated materials or with low quality, i.e., by the laws, the govern‐
10 Soil Pollution
ment provided the industries, exactly what they wanted, the possibility to incorporate low-
quality raw materials and contaminated to supply nutrients [43]. It is observed so that there
is a strong influence of the fertilizer industry in the studies and laws that limit the contaminants
in these materials, showing that this problem is not only related to the environmental field,
but also with the political and economic sector.
4. Metals: Toxicity in the foodchain
Due to its mobility in aquatic systems and to its toxicity to life forms, the toxic metals or heavy
metals, once in surface or in groundwater, are mentioned as one of the most problematic
inorganic contaminants in the environment.
The toxic meals are strongly pollutants elements and present many harmful effects to ecosys‐
tems, causing physical-chemical alterations to water, what promote the decrease of water
quality and mortality of the fauna and flora, prejudicing consequently, the human health [44].
The term heavy metal, is many times used with connotations of pollution and toxicity.
However, “heavy” is conventionally used to imply the high density of the metal, referring to
the pure element or to an alloy of metallic elements. The expression heavy metal apply to
elements which have specific mass higher than 5 g cm-3 or which have atomic number higher
than 20 [6].
Some metals, as the case of Cd, Pb, Ni and others, even when its presence is diluted in waters,
many times in undetectable quantities, have recalcitrant effect, i.e., its persistence in the
environment imply in process as biomagnification, which regards to the accumulation of these
harmful substances in the many trophic levels of the ecological chains of ecosystems.
The contamination by toxic metals in animals can occur due to inhalation, skin penetration or
ingestion of contaminated waters and foods, this last caused mainly by the capacity of plants
in bioccumulate these elements in high proportions and later offer them to animals and human
beings [45].
In animals, the toxic metals act in highly specific reactions, enzymatic in the most part and in
alterations in systems that evolves these reactions, resulting in negative effects. One classic
example is the metalloenzymes, in which dimensional structure is changed if one “intruder
metal” binds to its active site, what prevents the enzymatic action [46].
Metal contaminations in the environment results from a sum of natural processes, between
which also make part the natural sources of contamination, however, it is a common sense that
the sources so called anthropogenic are more frequent when compared to the natural sources
of contamination [47].
Significant increases of the metals concentrations are found in areas of intense industrial
activity, where the accumulation can be many times higher than the average levels from
non-contaminated areas. However, areas distant from the industrial centers in many cases
also show metals contamination, due to the atmospheric transportation of these com‐
pounds [48] and in the case of agricultural soils, due to the use of fertilizes contaminated
with these metals [6].
The contamination by metals is arising from the continuous disposal of industrial, domestic
or livestock wastes in soils, rivers, lakes and seas. These pollutants include the toxic metals,
which cause great concern to public health, because they are incorporated very easily in the
tropic chain. These harmful elements cannot be destroyed by biological degradation, as in the
case of the most part of organic pollutants. Incidences of the accumulation of metals in fish,
oysters, mussels, sediment and other components from the aquatic systems have been reported
in many countries around the world [49, 50].
Once the metals are present in cultivated soils, they are likely of radicular absorption by plants.
It is important to point out that some of these elements present biological function whereas
others are much known as toxic even in low concentrations. This way, the plants can be
considered as constituents of the foundation of the trophic chain, causing great concern due
to the possibility of the occurrence of toxic concentrations of certain metals which are trans‐
ported from plants to higher levels of the food chain [51].
The toxic metals are not only subjected to radicular absorption by plants when presents in the
soil solution, because may also occur the fixation of the metals in the soil particles, seepage/
leaching to lower levels of soil or even the surface runoff, reaching the groundwater, rivers,
lakes and seas. In general, there are two types of soils in Brazil, sandy and clayey, so that in
sandy soils, due to the low ion exchange capacity, the metals are easily moved in the environ‐
ment by leaching or surface runoff, reaching easily the hydric resources. In the other hand, in
clayey soils, due to the higher ion exchange capacity, the metals tend to stand immobilized in
the soil particle, becoming available for the plants in more than one cultivation season.
The toxic metals resulting from the anthropogenic activities, when disposed incorrectly in the
environment, directly or indirectly reaches the top of the chain, i.e., humans will have contact
with these elements by the consumption of food from vegetal or animal origin, contaminated
by water, soil or air. Once in the human organism, these metals present cumulative character,
i.e., are accumulated in the live tissues, i.e., hardly are eliminated by the organism. Down below
are related some of the characteristics of some toxic metals and its respective collateral effects
caused to human organisms.
4.1. Arsenic (As) – CAS ID 7440-38-2
In humans, the ingestion of high levels of arsenic can result in death. Exposure to lower levels
can cause nausea and vomiting, decreased production of red and white blood cells, abnormal
heart rhythm, damage to blood vessels, and a sensation of "pins and needles" in hands and
feet. The ingestion of inorganic arsenic can increase the risk of skin cancer and cancer in the
liver, bladder, and lungs. Inhalation of inorganic arsenic can cause increased risk of lung
cancer. Exposure to higher than average levels of arsenic occur mostly in the workplace, near
hazardous waste sites, or in areas with high natural levels. At high levels, inorganic arsenic
can cause death [52].
12 Soil Pollution
Aquatic organisms accumulate arsenic mainly as inorganic forms, and some of the organisms
such as phytoplankton, bacteria, etc. transform them into methylated and organic forms [53].
As is associated with ores containing metals, such as copper and lead. Arsenic may enter the
environment during the mining and smelting of these ores. Small amounts of arsenic also may
be released into the atmosphere from coal-fired power plants and incinerators because coal
and waste products often contain some arsenic. It cannot be destroyed in the environment. It
can only change its form, or become attached to or separated from particles. It may change its
form by reacting with oxygen or other molecules present in air, water, or soil, or by the action
of bacteria that live in soil or sediment [52].
Researching the effects of arsenic accumulation from plants cultivated near pressure-treated
wood, Cao and Ma [54] found out that elevated As concentrations were observed in the soils
adjacent to fences and poles, because As was frequently used for wood treatment, in order to
increase the wood life time. Eating vegetables from these contaminated soils may pose a risk
of As exposure since vegetables would readily take up As from these soils.
As, according to the Agency for Toxic Substances and Disease Registry (ATSDR) [55] is the
first substance on the Priority List of Hazardous Substances.
4.2. Cadmium (Cd) – CAS ID 7440-43-9
At room temperature (25 °C/73.4 °F) the Cd is presented at solid state, with atomic number of
48 and atomic mass of 112.41 g. This element belong to the group of the metal of gray silver
metallic color and it is available in many forms including sheet, granule, pellet, powder, rod,
wire, among other. It is a soft metal, highly toxic which can be easily cut with a knife, and
mostly produced as a byproduct of the extraction of Zn, Cu and Pb [56].
It is considered one of the most toxic metals, presenting important harmful effects for biological
activity on soil, plant metabolism, human and animal health. The abundance of Cd in magmatic
and sedimentary rocks, generally not more than 0.3 mg kg-1, being that this metal tends to be
concentrated in clayey deposits. The Cd is strongly associated to Zn in the geochemistry,
however, have a strong affinity with S, presenting a higher mobility than Zn in acid environ‐
ments [27].
Generally ore (CdS) is associated as a contaminant (0.25 to 0.5%) to ore Zn and Pb. According
to the International Cadmium Association [57], Cd has been mainly used in manufacturing
NI-Cd batteries, though, also has other applications as pigments, coatings, stabilizers for PVC
(polyvinyl chloride) and alloys.
One of the main problems associated with Cd is its final destination, because it can reach the
soil or the air through the burning of urban waste or the burning of fossil fuels, in this way
polluting the environment and causing damage to the ecosystem [58].
In humans, Cd affects several organ systems, as the Cardiovascular (Heart and Blood Vessels),
Developmental (effects during periods when organs are developing), Gastrointestinal
(Digestive), Neurological (Nervous System), Renal (Urinary System or Kidneys), Reproductive
and Respiratory (From the Nose to the Lungs), Cd is also known as a carcinogen agent [52].
According to the Agency for Toxic Substances and Disease Registry [55] Cd is the seventh
substance on the Priority List of Hazardous Substances.
4.3. Lead (Pb) – CAS ID 7439-92-1
The Pb is rarely found in its natural state, but in combination with other elements, being the
most important ores which present this metal are: Galena, cerussite, anglesite, perite and
others.
Among the toxic metals, Pb is present in larger amounts in the earth's crust, with an average
concentration between 10 and 20 mg kg-1 [59], therefore, is among the most used metals
worldwide scale of Fe> Al> Cu> Zn> Pb [60].
To be quite versatile, Pb is much used in industrial products, and can be hardened by the
addition of other metals and metal alloy being used as producing components, welds and other
materials, in addition to being able to form various compounds employed in chemical industry
[61].
According to unofficial information from the National Department of Mineral Production

(DNPM), the main consuming sectors Pb are: accumulators (batteries) 80%, representing
approximately 88 tons of metal, oxides, 12%, equivalent to 13 tons; and electronic (alloys,
solders and various), 8%, with 9000 tons.
The Pb in soil is extremely stable and highly toxic to humans and animals, is currently ranked
as the second most dangerous element in the Priority List of Hazardous Substances [55]. Most
of the lead enters the human body through the respiratory and gastrointestinal routes, and
after absorption, it can be found in the blood, soft and mineralized tissues [62].
In humans, the Pb poisoning may initially cause lack of appetite, metallic taste in mouth,
muscle discomfort, malaise, headache and abdominal cramps strong. However, in childhood,
symptoms often linked to the deposition of this metal in the brain are predominant. In its
inorganic Pb2+ is an inhibitor of enzymes, further affecting the nervous system [63].
The organ systems affected by Pb are the Cardiovascular (Heart and Blood Vessels), Devel‐
opmental (effects during periods when organs are developing), Gastrointestinal (Digestive),
Hematological (Blood Forming), Musculoskeletal (Muscles and Skeleton), Neurological
(Nervous System), Ocular (Eyes), Renal (Urinary System or Kidneys) and Reproductive [52].
4.4. Chromium (Cr) – CAS ID 7440-47-3
With regard to heavy metal Cr, this has turned into a serious pollutant of air, soil and water.
About 70% of the production of Cr is used in alloys, including stainless steel and 15% in
industrial chemical processes, mainly in leather tanning, pigments, electrochemical and
cooling towers [64].
The ions of Cr are found in different oxidation states, however, the trivalent form (Cr3+) is the
most common, while the hexavalent form (Cr6+) depends on the more specific conditions of
pH and redox potential [65].
14 Soil Pollution
The toxicity of Cr depends on its oxidation state. While the Cr3+ is relatively stable and
immobile, the Cr6+ moves easily through the soil and aquatic environments, with a strong
oxidizing agent capable of being absorbed even by the skin in this way, the Cr presents itself
as a carcinogenic species [66, 67].
In humans, the Cr affects the immune, renal (kidney or urinary system) and respiratory tract
(the nose to the lungs), and is known as a powerful carcinogen [52].
According to reference [68], a lethal dose of Cr6+ is between 50 and 100 mg kg-1, a value much
lower when compared to Cr3+, the values are between 1900 and 3300 mg kg-1. Since the Cr6+
occupies the 17th position in the Priority List of Hazardous Substances [55].
4.5. Nickel (Ni) – CAS ID 7440-02-0

The Ni occurs in the earth's crust in the order of 0.01%, mainly as sulfide, oxide and silicate
minerals. Geological activities as natural weathering and volcanoes led the natural distribution
of this metal in natural environments at modest levels. The Ni metal and its compounds are
used in multiple applications as stainless steel and other alloys, castings, catalysts, batteries,
electronics, ceramics, pigments and even coins [69].
Among the main uses of this metal can still cite it use in coating parts, in a process called
electroplating, widely used around the world by Electroplating industries [70].
Although not an element extensively released to the environment, this can present a risk to
human health. Epidemiological investigations and experimental studies have shown that
certain compounds of Ni are extremely carcinogenic after inhalation, however, the same
studies show that these risks are limited by conditions specific occupational exposure to this
metal [71].
Results show that this element can cross the placenta and present embryological and terato‐
genic properties. The main danger of Ni to the man beside the carcinogenicity is related to the
ability to cause sensitivity reactions [71].
The most common harmful health effect in the general population is allergic contact dermatitis
elicited by prolonged skin contact of sensitized individuals with Ni. Therefore, the majority of
studies related to health effects of Ni to humans concern airborne Ni particulate matter.
Strongest evidence for high cancer risk was found for sulfidic nickel species (NiS, NiS2 and
Ni2S3) in Ni refinery dust. On the molecular level the toxic Ni species responsible for severe
health effects, as allergic contact dermatitis and respiratory tract cancer, has been suggested
to be caused by Ni2+ [69].
Ni affects the following organ systems: Cardiovascular (Heart and Blood Vessels), Dermal
(Skin), Immunological (Immune System), Respiratory (From the Nose to the Lungs) [52].
Actually, Ni ranks the 57th position on the Priority List of Hazardous Substances [55].
4.6. Copper (Cu)- CAS ID 7440-50-8

The Cu, despite being a heavy metal, meets the criteria of essentiality for plants and microor‐
ganisms, being classified as micronutrient [72]. This metal is essential to human health, and is
present in all human tissues and many fluids and with amino acids, fatty acids and vitamins,
is required for the metabolic processes [73].
Excess copper may be toxic to humans because of the affinity metal with S-H groups of many
proteins and enzymes associated with diseases such as epilepsy, melanoma and rheumatoid
arthritis, as well as the loss of taste [72]. Toxic levels of Cu occur naturally in some soils, whereas
others may contain high levels of Cu as a result of the anthropogenic release of contaminated
materials into the environment through mining, smelting, manufacturing, agriculture and
waste disposal technologies [72].
In plants, either deficient or in excess, Cu can cause disorders in plant growth and development
by adversely affecting important physiological process. In particular photosynthetic electron
transport is altered under deficiency and excess of Cu. Thus, for healthy plant growth and
development Cu must be acquired from the soil, transported throughout the plant, distributed
and compartmentalized within different tissues and its content carefully regulated within
different cells and organelles. For this purpose, plants, like all other organisms, have homeo‐
static mechanisms to maintain the correct concentrations of essential metal ions. Membrane
transport systems are likely to play a central role in these processes [73].
Copper ranks the 125th position on the Priority List of Hazardous Substances [55].
4.7. Manganese (Mn)- CAS ID 007439-96-5

According to the International Manganese Institute (IMnI) [74], this metal is the fourth most
used in terms of tonnage, being ranked behind Fe, Al and Cu, being the production of Mn
alloys about 17.7 million metric tons at 2011. Mn the second major abundant metal and 12th
most abundant element in the earth’s crust, being its ores deposits usually of sedimentary
origin, with oxide ore layers inter-bedded with iron-rich formations [75].
Steel is basically an alloy of iron and carbon, consisting of an iron phase and iron carbides.
Crude steel produced from iron contains an undesirable amount of oxygen and some sulphur.
Mn plays a key role because of two important properties: its ability to combine with sulphur
and its powerful deoxidation capacity [76].
High grade Mn ores (> 40%) are typically processed into suitable metallic alloy forms by
pyrometallurgical processes. Low grade manganese ores (< 40%) are conventionally processed
by pyrometallurgical reductive roasting or melting followed by hydrometallurgical processing
for production of chemical manganese dioxide (CMD), electrolytic manganese (EM) or
electrolytic manganese dioxide (EMD) [77].
In the human body, Mn displays a somewhat unique behavior with regard to its toxicity. It is
relatively non-toxic to the adult organism except to the brain where it causes Parkinson-like
symptoms when inhaled even at moderate amounts over longer periods of time [78].
This element, when in large amounts, affect fertility in mammals and are toxic to the embryo
and fetus, what implicates that pregnant women should not be exposed to Mn anytime [78].
Due to the substitution of Pb by Mn in gasoline, reference [79], researching the cultivation of
beans and oats in soil next to roads, found not only Mn concentrations significantly higher in
16 Soil Pollution
organic soil but also higher accumulation found in fruits and stems, suggesting that the
addition of MMT to gasoline can be the cause for this increase in exchangeable Mn in organic
soils.
According to the Agency for Toxic Substances and Disease Registry [55], Mn is the 140th
substance on the Priority List of Hazardous Substances.
5. Case studies: Contaminated fertilizers in Brazilian crops
Based on information obtained from seizures made in Brazilian seaports, related to the entry
of loads of industrial waste with the presence of toxic metals for use as source of micronutrients
in fertilizers [6], three studies were conducted to evaluate the possibility of soil and plants
contamination using different sources and Zn levels, since this is main micronutrient applied
in the Brazilian agricultural crops. All studies were conducted at the State University of West
Paraná - Brazil, being one for soybean [2], one for the wheat crop [4] and the last one for the
corn crop [5].
In the work done with the soybean crop [2], an experiment was conducted in 2007, in Palotina-
PR, with coordinates 24° 18' S and 53° 55' W (Figure 2) in a soil classified as Hapludox (RED).
It were used four different fertilizers for the Zn supply sold in Brazil, in three doses (0.0; 0.9
and 1.8 kg ha-1). The results of this study demonstrated the presence of toxic metals (Cd, Pb
and Cr) in the soybean leaf tissue (Table 5) at levels above of the tolerable in agricultural crops
[27]. We also observed the occurrence of these three metals in the soil after harvest (Table 6),
which compared to the Brazilian legislation, that establishes the maximum concentrations of
contaminants in soils [28], classify the obtained values as above the maximum allowed for the
soil be agriculturable (IV - Agricultural area) in the case of Cd. For Pb and Cr the values
obtained in the study were below of the prevention value (PV), which is the maximum
allowable value of the contaminant in soil to that it is able to sustain its biological processes.
The work done with the wheat crop [4] was conducted on the residual fertilization of the work
with the soybean crop, since the concentrations of the metals Cd, Pb and Cr in the soil of the
experiment were significant, especially in the case of Cd. The results of this study showed no
accumulation of Cd in the leaves of wheat at the level of detection by the analytical method
(flame atomic absorption spectrometry - FAAS), however, we found concentrations of Pb and
Cr. For Pb the maximum concentration found was under the tolerable limit, and for the Cr,
the maximum concentration found was above the tolerable level for crops [27]. One of the main
conclusions of this work was the occurrence of availability of Cr and Pb to wheat plants with
fertilization performed in previous crop (soybean), demonstrating the persistence of contam‐
inants in soil and their occurrence in more than one crop agriculture.
The work conducted with maize crop was conducted in Mercedes, Paraná State-Brazil, with
geographic coordinates of 24° 25' S and 54° 15' W (Figure 3) in a Rhodic Eutrudox. The
fertilization of this experiment was performed with eight different sources of Zn in four
different doses (0.0; 2.0; 4.0 and 6.0 kg ha-1). In the chemical analysis performed in the fertilizers
Figure 2. Local where the experiment involving fertilization with Zn in soybean and wheat culture were performed
[80]
used in the work, it was observed that one of the sources presented 11039.46 mg of Pb kg-1,
being the maximum limit permitted by Brazilian law (NI 27/06) [37] 10000.00 mg of Pb kg-1
(Table 3). However, as previously mentioned in the section 3, the NI 27/06 tolerates the
presence of contaminants up to 30% of the maximum permitted value, in this way, the fertilizer
would not be considered as irregular, and can be freely traded.
Figure 3. Local where the experiment involving fertilization with Zn in corn crop was performed [80]
18 Soil Pollution
Regarding the leaf concentration of Cd, Pb and Cr (Table 5), it was observed that only the Pb
concentrations was found at detectable levels by the used method (FAAS), where the leaf
content of Pb in corn plants can be considered inside the tolerable limits [27]. For the soil, it
was observed the occurrence of Cd, Pb and Cr (Table 6), so that the concentrations of Cd were
below the maximum allowable limit so that the soil is agriculturable (IV - Agricultural area),
and the Pb and Cr were below the PV, which is the maximum allowable value of the contam‐
inant in the soil, to that it be able to sustain its biological processes.
Agricultural crop Tolerable foliar

Metal
Soybean* Wheat** Corn*** level****
Cd (mg kg-1) 0.73 ND ND 0.05 a 0.50
Pb (mg kg )-1
25.53 1.95 8.67 0.50 a 10.00
Cr (mg kg )
-1
3.36 9.28 ND 2.00
ND – not detected by the method used (FAAS) with a quantification limit of 0.01 mg L-1; * - results from reference [2]; **
- results from reference [4]; *** - results from reference [5]; **** - Tolerable foliar level according to reference [27].
Table 5. Foliar levels of Cd, Pb and Cr in experiments conducted with mineral fertilizers to supply Zn in different crops
in Brazil
Classification of the metal value in

Agricultural crop
Metal the soil*
Soybean* Corn** PV IV – Agricultural area
Cd (mg dm ) -3
5.52 2.52 1.30 3.00
Pb (mg dm ) -3
24.25 51.48 72.00 180.00
Cr (mg dm ) -3
22.19 18.43 75.00 150.00
* - results from reference [2]; ** - results from reference [5]; *** - Classification of the metal value in the soil according to
reference [28]; PV–Prevention Value; IV – Investigation Value for agricultural area.
Table 6. Levels of toxic metals Cd, Pb and Cr in soil after cultivation of various crops fertilized with Zn in Brazil
Based on the results obtained in the three presented studies it can be inferred that the presence
of metal contaminants in fertilizers is real and should be studied more carefully by regulatory
authorities in Brazil. The foliar and soil levels of the metals Cd, Pb and Cr found cause concern
from the environmental point of view, due the possibility of entry of these contaminants in the
food chain, either by consumption of contaminated materials or pollution of water resources
from contaminants in the soil. It should be noted that the values presented in these works were
obtained with only one fertilization, so that with successive fertilizations may occur a greater
availability of these contaminants to the environment.
It is worth affirm also that the laws related to the presence of contaminants [37], and levels of
toxic heavy metals allowed in Brazilian soils [28], are being severely questioned by Brazilian
researchers and government agencies. Among the key questions are the lack of criteria for
determining and monitoring the presence of contaminants in fertilizers, passivity with the
addition of toxic metals in brazilians soils by the minimum quality and lack of discussion with
researchers and entities responsible for the drafting of legislation.
It should furthermore be highlighted that before approval of Resolution No. 420 of CONAMA
[28], there was serious questioning on the part of Brazilian authorities and researchers in
relation to the criteria used for its formulation by means of Proceeding 02000.002955/2004-69
[81-83], thus confirming the need for more societal discussion (especially among the special‐
ized technical and scientific segments in a multidisciplinary manner) before this is submitted
for approval of possible resolution. The resolution, moreover, violates the Brazilian Federal
Constitution with respect to the ‘‘preservation, improvement and recovery of the environ‐
mental quality favorable to life’’ because it permits the introduction of contaminants in the soil
up to the maximum limit permitted by the resolution, thus allowing the use of contaminated
fertilizers and soil conditioners in agriculture until they reach and exceed these values and
thereby degrade environmental quality. Therefore, the need for more in-depth field studies in
relation to environmental legislation before it is drafted and approved becomes clear. These
studies must be undertaken together with environmental regulatory bodies and research
entities in the country. Based on the results obtained in this study, it is evident that some of
the Zn fertilizer sold by sources in Brazil significantly affect the quantity of Cd, Pb, and Cr in
soil. If their use is continued, there may be contamination of soil, surface and underground
waters, plants, animals, and humans, leading to severe damage to health as well as the
environment. This fact raises concern because according to references [84-86], even low levels
of heavy metals in soil may be available to plants, an effect that is difficult to ascertain because
the symptoms they case occur only over the long term, thus increasing even more the hazards
associated with the use of contaminated materials in agricultural activities.
In a review about the environmental risks of adding phosphate fertilizers contaminated with
trace elements in long-term, reference [87] warn about the possibility of increasing concentra‐
tions of As, Cd and Pb in agricultural soils using contaminated fertilizers. The authors also
emphasize that to prevent the entry of potentially toxic elements in agriculture, it is necessary
a strict control on the sources of macro and micronutrients used in agriculture. This demon‐
strates the importance and need for further studies related to the use of fertilizers contaminated
with toxic metals in crops and the environmental impacts that these sources of nutrients can
cause.
6. Sustainable strategies for soil remediation
The introduction of toxic chemicals by man in various environmental compartments (soil,

water and atmosphere), the of controlled or uncontrolled elimination of wastes (accidental
spills, mining processes and smelting of metallic ores, fertilizer application, sewage sludge and
20 Soil Pollution
pig manure in agricultural soils) puts the ability to self-cleaning of contaminated ecosystems
in doubt. Consequently, the pollutants that accumulate toxic metals are of concern in relation
to human exposure of ecosystems and the potential impact [88].
Today there are several techniques for soil decontamination. It is worth mentioning that the
choice of one or other technique, among other factors, also depends on the characteristics of
the contaminant in question, between the different techniques is possible enumerate:
1. Thermal treatment: Use of heat to remove, stabilize or destroy the contaminants;
2. Physical treatment: Physical processes used to separate toxic substances from the envi‐
ronment;
3. Chemical treatment: Uses chemical reactions to remove, destroy or modify toxic substan‐
ces;
4. Biological treatment: Makes use of natural metabolic means, such a microorganisms and
other biological agents to remove, destroy or modify the contaminants;
5. Stabilization / solidification: This step the contaminants are stabilized chemically and / or
modified to reduce the potential for contamination.
These technologies can in many cases be applied “in situ”, i.e., at the site of origin of the
contamination, or “ex situ” outside the place of contamination, which results in the removal
and transport of the contaminated soil.
Among the physical-chemical treatments, these mostly are based on soil washing. These
methods are based on the technological principle of the transfer of a contaminant from the soil
for a liquid or gaseous phase. The main products are treated soil and the contaminants
concentrated. The specific process of treatment depends on the type of contaminant, in
particular as regards the type of connection established with the soil particles.
It is observed by the description above, that such treatments become process more expensive
the decontamination because are many cases in which the soil treatment requires the removal
of several layers of the same and specific treatment in laboratories.
Thus, many approaches have been developed, evaluated and performed to deal with soil
pollution. Current technologies for decontamination soil are often expensive and of high
energy consumption, and in many cases, the soil cannot be reused after treatment [89]. Thus,
several studies have been developed to promote proper techniques and low cost of remediation
in order to prevent the spread of these contaminants in the food chain [88, 90].
As an alternative, an ecological approach technology that involves the use of plants to clean
up or remediate soils contaminated with toxic metals, called phytoremediation, has been
developed and encouraged by the fact of being a simple and inexpensive option for remedia‐
tion of contaminated soils [90-92].
Phytoremediation can be divided into phytoextraction (hiperacumulation) phytostabilization,

rhizofiltration, fitotransformation and fitovolatilization (Figure 4).
Phytorremediation
Phytoextraction Phytostabilisation Rhizofiltration Phytovolatilization Phytotransformation
Reduction of Removal by Absorption and release Decomposition of

Uptake and mobility, restriction
translocation of adsorption and to the atmosphere of contaminant by
of metals in root precipitation on the volatile materials metabolism or plant
metals from soil tissue and
to the top tissues surfaces or radical (compounds containing enzymes
precipitation in the absorption in the Hg and As)
of the plant. root zone. roots (roots only).
Figure 4. Types and characteristics of phytoremediation [92,93]
However for the phytoremediation to be effective it depends on the selection of plant species,
preferably hyperaccumulators, which are able to grow in soils of low fertility, and degraded
by the presence of contaminants [94].
In studies carried out in Iran using Euphorbia macroclada and Centaurea virgata, demonstrate
that the sampled species not only were able to grow in soils highly contaminated by metals,
as well as were capable of accumulating high concentrations of Zn, Mn, Cu, Pb and Fe, in this
way these plants can be classified as hyperaccumulators, in other words, have adequate
potential for phytoremediation of contaminated soils [95].
However, the metal accumulation by the plants is only efficient after the contaminant be
removed from the soil, for example, by harvesting the plant matter. If most of the toxic metals
are located on the shoots, the harvest can be performed using traditional farming methods. In
general, it is necessary to harvest the plants before the fall of the leaves, or before his death
and decomposition, so that contaminants do not disperse or return to the soil.
After harvesting, the biomass must be processed for extraction and recollection of the majority
of metals. Alternatively, the volume and the weight of the biomass can be reduced by thermal
processes, physical, chemical or microbial. In the case of burning plant, for example, the energy
produced represents an economic value of the process and the ash may be treated as an ore,
which may still be extracted the metallic contaminant (especially if the ashes are enriched in
one or two metals).
Even Brazil showing great potential species for bioremediation and phytoremediation, the
recovery of contaminated areas, due to the great biodiversity and climate, favoring biological
processes in pollution treatment, knowledge about these species of plants and microbial
communities in phytoremediation potential are still scarce [96].
In research carried out by reference [97], it was reported that some plants were able to
remediate soils contaminated with metals such as Brassica juncea for phytoextraction of Cd and
Cr6+, Cu, Ni and Pb, Avena sativa for Zn, Pisum sativum L. and Zea mays L. for removal of Pb.
However, from an economic point of view, the use of these species of accumulator plants
generally do not generate financial returns to the farmers, because these plants only remove
the contaminant from the soil, being later discarded after the remediation. Thus if the farmer
22 Soil Pollution
in question require to decontaminate a particular area, the phytoremediation only would

generate costs, especially with seeds, agricultural machinery, irrigation, etc.
Nevertheless, currently there is a widespread idea that the species used for phytoremediation
of soil can also generate economic gains beyond decontamination. The use of oil crops, for
example, Crambe abyssinica H., Moringa oleifera L. and Jatropha curcas, in which the product
marketing is not consumed as food, but as biofuel (biodiesel), is a possibility to decrease the
cost of recovery of the contaminated area. Thus the fact that the plant has accumulated large
amounts of heavy metal will have little influence on the end product of the culture, generating
profit for the property.
Moreover, oil crops like C. abyssinica, M. oleifera and J. curcas, have been the subject of
research in the removal of toxic metals such as Cd, Pb, Cr and water through the adsorp‐
tion process, in which the pie their seeds are used as adsorbent [98-101]. The results of these
studies demonstrated great potential for removal of these metals, and that these adsorb‐
ents are obtained at minimal charges, because they are wastes generated by the extraction
of vegetable oil.
Thus, the use of cultures for vegetable oil production and recovery of contaminated areas can
be an interesting alternative, which will generate the money from oil production and recovery /
stabilization of the contaminated area with the development of these cultures, preventing them
be lost through leaching or soil surface runoff and reach the groundwater and surface water
and groundwater. However, more researches are still needed In order to evaluate this
possibility, focusing on the development of oil-producing plants and cycles of addition of the
adsorbent material and the biodegradability thereof. Between this and other issues to which
scientific research is driven, many studies still need to be developed so that, in one way or
another, the quest for sustainability will one day be achieved.
7. Conclusion
Based on information existing in the Brazilian and international literature, we can make some
important observations:
The fertilization of plants is one of the major reasons of the increase in food production across
the globe. Consequently, the use of fertilizers in agricultural crops is extremely high and
growing, however, for the maintenance of this production capacity, we should be very careful
with the fertilizers sources that are being used in agriculture.
The presence of toxic metals in fertilizers has been reported in the major food producing
countries, like United States, China and Brazil. This demonstrates that this issue deserves
further detailed studies of long-term about the successive addition and the effects of these
contaminants to crops, soil, supply chain and the wider environment. Thus, the institutions of
research and supervision, have to evaluate the products that are used for the manufacture of
fertilizers on the market, as this is an activity that generate large amounts of money and that
will always be a target of people with questionable character.
The current legislation, not only in Brazil, but internationally, with respect to the use of wastes
as sources of nutrients, presence of contaminants in fertilizers and presence of toxic metals in
soil, need higher accompaniments and answers for some of the community questions, because
the legislation must ensure that the agricultural soils are not being degraded and becoming
unproductive. In Brazil, it is strongly recommended that the laws must be harder and restric‐
tive, until we have reliable and conclusive results related to the addition of contaminated
fertilizer in long-term. In this case, it should be emphasized that these laws should be devel‐
oped based on results from institutional researches and reliable groups, without the influence
of political and industrial groups. Meanwhile, the damage caused by man in air, soil and
waters, must be repaired, by remediation methods (such as phytoremediation) aiming not only
the decontamination, but also ensuring a small profit, as an incentive to the farmer.
Brazil is considered as one of the countries with largest biodiversity and most promising in
food production for the globe, however, for that prediction become a reality, it is very
important that the productive areas are not degraded. The technologies developed by our
civilization provide ways to avoid the degradation of our environment, but often they are not
used because they prevent higher industrial profits, this should not occur, because the balance
of the ecosystem and the quality of human life should be above of any monetary profit.
Author details
Affonso Celso Gonçalves Jr.1, Herbert Nacke2, Daniel Schwantes3 and

Gustavo Ferreira Coelho4
1 Western Paraná State University, Center of Agricultural Sciences, Marechal Cândido Ron‐
don, Brazil
2 University Center Dynamic of Cataracts, Technological Center, Foz do Iguaçu, Brazil
3 Pontifical Catholic University of Paraná, Polytechnic School, Toledo, Brazil
4 Federal University of Paraná, Palotina, Brazil
References
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24 Soil Pollution
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um, lead and chromium toxic heavy metals in soil and soybean leaf tissue fertilized
with different sources of NPK+Zn. Ciência e Agrotecnologia 2011;35(1), 884-892.
[3] Gonçalves Jr AC, Nacke H, Marengoni, NG, Carvalho, EA, Coelho GF. Yield and
production components of soybean fertilized with different doses of phosphorus, po‐
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chapter 5
Beneficial and Negative Impacts on Soil by the Reuse of

Treated/Untreated Municipal Wastewater for
Agricultural Irrigation – A Review of the Current
Knowledge and Future Perspectives
Juan C. Durán–Álvarez and Blanca Jiménez–Cisneros
1. Introduction
The scarcity of water for human use, such as food and energy production, manufacturing,
drinking water and ecosystem conservation is a global problem for which the solution goes
beyond merely the preservation of freshwater sources [1–2]. Although three quarters of the
Earth´s surface is covered by water, most of this water is either contained in oceans or confined
in glaciers [3]. The volume of freshwater available for human activities (less than 1%) is
unequally distributed throughout the globe; in some cases this water is confined to the deep
sub–soil or is polluted [4]. Furthermore, the desertification of large areas caused by climate
change has intensified the lack of water sources in cities and rural areas throughout the world
[5]. Water scarcity results in food scarcity, since 70% of the water withdrawn for human
activities goes to agriculture [6]. In zones where rain–fed agriculture is practiced, decay in crop
yields is observed when droughts occur, which results not only in the scarcity of food but also
the decrease in incomes due to falling crop sales [7]. The use of freshwater for agricultural
irrigation limits the volume of freshwater available for human consumption; therefore,
recycling of water becomes necessary for agricultural irrigation in dry zones. The idea of
reusing wastewater to irrigate is not new; it actually originated around 3000 B.C. People in
these ancient civilizations knew that wastewater contained both water and compounds that
benefited the soil and thus they used it in a planned way to increase crop yields [8].
Commonly, reusing wastewater in agriculture is considered a deleterious practice since it may
introduce pollutants to the environment, spread waterborne diseases, generate odor problems
and result in aversion to the crops. Nevertheless, this kind of reuse may result in some benefits
2 Soil Pollution
for soils, crops and farmers. Nowadays, the reuse of wastewater in agriculture is seen in some
countries as a convenient environmental strategy [9–10]; municipal wastewater is therefore
considered an appropriate option for reuse. This kind of wastewater contains a significant load
of biodegradable organic material (carbon and nitrogen) as well as most of the mineral
macronutrients (e.g. phosphorous, potassium, magnesium and boron) and micronutrients (e.g.
molybdenum, selenium and copper) which are necessary for the growth of crops. Accumula‐
tion of organic matter in soil by irrigation with wastewater can be beneficial as it may result
in the enhancement of the physical structure of the soil, the increase in the soil microbial activity
and the improvement of soil performance as a filter and degrading media for pollutants.
Conversely, a fraction of the organic matter contained in wastewater is due to the occurrence
of organic pollutants (e.g. polyaromatic hydrocarbons and polychlorinated biphenyls) and
pathogenic microbial agents [11–12]. Because of the presence of organic, inorganic and
microbial pollutants in wastewater, a prior step of depuration is necessary before reuse in
irrigation in order to avoid the pollution of soil, crops and the nearby water sources, and thus
the dissemination of waterborne diseases or the degradation of soil. The extent at which
wastewater has to be treated prior to irrigation depends on the restrictions established in local
or international water quality criteria for irrigation [13]. Primary treatment schemes (coagu‐
lation–flocculation with sedimentation or aerobic/anaerobic stabilization pounds) are used for
treating wastewater to irrigate crops that are not intended for human consumption (e.g.
fodder), while secondary treatment of wastewater (biological treatment followed by disinfec‐
tion) is recommended when unrestricted crops are irrigated [14–15]. In developing countries,
most or the whole volume of wastewater produced in cities is treated prior to irrigation, while
in low income countries wastewater treatment is not a priority, and thus untreated or partially
treated wastewater or a mixture of treated and untreated wastewater is commonly used for
agricultural purposes [12, 16]. In Mexico, China, India and Pakistan, for instance, large areas
exist where untreated wastewater has been reused in agricultural irrigation for a considerable
time [17]. The World Health Organization estimates that nearly 20 million hectares throughout
the world are irrigated using untreated wastewater [18]. It is also reported that in some cities
up to 80% of the vegetables locally consumed are produced using wastewater for irrigation
[19]. The application of wastewater to soil, particularly untreated wastewater, followed by its
infiltration poses a significant risk of pollution, not only to soil and crops but also to the surface
and subterranean water sources surrounding the irrigated area [20–21].
Pollution by pathogenic agents is the main cause of concern regarding the application of
treated/untreated wastewater to soil. Due to the variety of microorganisms entering the soil
via the wastewater there is a high risk of enteric disease outbreaks for farmers and consumers
[22–23]. This chapter addresses the contamination of wastewater irrigated soils by helminths
(intestinal worms) and pathogenic bacteria common in developing countries (where untreated
wastewater is used to a greater extent), as well as the risk of outbreaks of parasitic diseases for
both farmers and consumers in agricultural areas where untreated wastewater is reused. The
occurrence of antibiotic resistance in indigenous organisms of soil and pathogens reaching soil
via wastewater is gaining the attention of scientists and health organizations around the world
[24–25], thus a review of what it is known and the research opportunities in this field are
presented in the text. With regard to organic pollution, a current topic of interest is the entry
Beneficial and Negative Impacts on Soil by the Reuse of Treated/Untreated Municipal Wastewater for... 3
to the soil and potential risks within crops of so–called “contaminants of emerging concern”.
These pollutants are substances that have not previously been considered as pollutants since
they are part of everyday products; however, due to the subtle but harmful effects that these
substances may cause in a variety of aquatic and terrestrial organisms, concerns have risen
due to their continuous entry into the environment via wastewater [26]. A review on the
presence of some organic contaminants of emerging concern, such as pharmaceutical sub‐
stances, personal care products and industrial additives, in wastewater–irrigated agricultural
soils is presented in this chapter along with some of the known potential effects caused to soil
organisms, plants and consumers. Such effects have just begun to be elucidated, and only for
some groups of contaminants of emerging concern [27–28], even though it is now known that
up to 7 million commercially available chemicals are routinely disposed of in sewage after use
[29]. In this regard, this chapter makes some suggestions regarding the next steps in the toxicity
studies for this class of pollutants, such as testing the synergistic effects of mixtures of
contaminants of emerging concern in soil organisms.
In spite of the variety and quantity of contaminants that soil regularly receives through
wastewater irrigation, this ecosystem possesses self–purification processes that maintain
homeostasis within the system. Such self–purification processes may either inactivate or
reduce the population of pathogenic microorganisms reaching the soil via wastewater through
predation by the indigenous microbiota within the soil [30–31], the production of antibiotics
by some organisms in the rhizosphere [32] and by retention of microorganisms in the surface
layers of the soil profile through physical and chemical processes. For organic pollutants,
mechanisms such as photolysis and biodegradation promote the dissipation of contaminants
in the soil, while adsorption onto the soil particles lead to the retention –and the potential
confinement– of organics within the solid matrix [33]. In this chapter, current knowledge
concerning the environmental fate of pathogen and organic contaminants of emerging concern
in wastewater irrigated soils is discussed, highlighting the laboratory approaches that show
the best results in simulation of the conditions in the field. Knowledge of the environmental
fate of contaminants in irrigated soils is important in order to perform more accurate risk
assessment studies on contamination of water sources, soil and crops in wastewater irrigated
areas; furthermore, it provides information to policy makers to make proper legislation aimed
at promoting environmentally responsible management of treated/untreated wastewater in
agricultural irrigation.
Depuration of wastewater prior to its reuse is the most plausible option to prevent soil
pollution by wastewater reuse. However, since wastewater represents a cheap source of water
and fertilizer for farmers [34], it is necessary to consider the needs of users before planning
schemes of wastewater treatment. The use of wastewater treatment systems aimed at removing
carbon, nitrogen, phosphorous and minerals in wastewater leads to the reduction in quality
of effluents as fertilizers, impacting crop yields and thus in the livelihood of farmers. In this
sense, the use of advanced primary treatment systems could be a feasible option to: a) remove
suspended solids, pathogens and heavy metals in wastewater without significantly impacting
the content of nutrients in effluent; b) preserve the quality of agricultural soils to properly
perform ecosystem services such as the production of food; and, c) fulfill the needs of farmers
4 Soil Pollution
that use wastewater as a source of water and nutrients. Treating wastewater by these kinds of
systems may be an opportunity to couple sanitation with reuse within a program of compre‐
hensive management of wastewater, the recycling of nutrients and the use of soil as a food
producer and purification system.
This chapter aims to describe what it is known and what it is unknown regarding the positive
and negative impacts of the reuse of treated/untreated wastewater in agricultural irrigation.
It will be shown in detail how this practice can benefit soil and farmers, while at the same time
posing a risk of contamination to the ecosystem. Emphasis is given to the purification processes
occurring in the soil and how soil manages the continuous entrance of pollutants via waste‐
water. Lastly, some perspectives for further studies on the presence and environmental fate of
pollutants in wastewater irrigated soils are proposed.
2. Impacts of wastewater reuse in agriculture
The reuse of wastewater results in both beneficial and negative impacts on soil, some of which
are explained in this section. The aim is to identify both and to understand their origins in
order to assist scientists and policy makers to balance them and even to greater advantage of
the benefits compared to the drawbacks in certain situations.
2.1. Benefits of wastewater reuse in agriculture
Figure 1 summarizes the positive impacts of reusing wastewater in agricultural irrigation in

all of its forms. The extent of the positive impacts depends on local conditions of the specific
project.
2.1.1. Benefits in crops
Since wastewater is produced constantly and thus is always available, it is possible to select a
wider range of crops to be sown year–round, specifically those of high profitability which
normally have higher and more stringent water demands in terms of quantity and timing. The
consistent use of wastewater in irrigation may stabilize the content of nutrients in the soil, even
when growing crops with high nutritional requirements; this is because the continuous
withdrawal of nutrients by plants is compensated by the constant input of organic and mineral
components into the soil via wastewater. Examples of how the reuse of wastewater has led to
increases in crop yields in arid zones can be found worldwide. Studies conducted in Hubli–
Dharwad, India, showed that irrigation with treated and untreated wastewater made it
possible to produce vegetables during the dry season; yields and selling prices increased by
3–5 times compared to the kharif (monsoon) season [35]. In Pakistan, Ghana and Senegal the
reliability and flexibility of wastewater supply allows rural and urban farmers to cultivate
profitable crops in a shorter time, resulting in several harvests per year (3 to 6) [36–37]. Treated/
untreated wastewater is a source of organic matter and the same large diversity of nutrients
contained in any formulated fertilizer. It is estimated that 1,000 m3 of municipal wastewater
applied to one hectare can contribute 16–62 kg of organic nitrogen, 4–24 kg of phosphorus, 269
kg of potassium, 18–208 kg of calcium and 9–110 kg of magnesium each year [16]. Table 1
shows the contribution of water and nutrients that untreated wastewater make to several
crops.
in crops The increase in crop yields
Improvement of the soil

performance as a wastewater
treatment system
Increase of the soil microbial

in soil activity
Improvement of the physical

structure of soil
The reduction of costs

in local economy caused by the use of
Benefits of reusing fertilizers and freshwater
wastewater in for irrigation
agricultural irrigation
Wastewater can be purified

during the irrigation and
in sanitation further the infiltration
through the soil
The contribution to food

in livelihood security of people at local
and regional level
It avoids discharging
pollutants to surface water
It can be a low-cost option to

in sustainability manage wastewater and to
reintegrate water to the
environment
The potential to recharge

certain type of aquifers
through wastewater infiltration
With information of references [14, 18, 21, 36, 37]
Figure 1. Beneficial impacts of reusing wastewater for agricultural irrigation

6 Soil Pollution
Nitrogen is a plant macronutrient which can be found in the form of nitrate ions (N–NO3),
mostly in treated wastewater, or as ammoniacal nitrogen (N–NH4+) and organic nitrogen in
untreated wastewater. The sum of all these forms is known as total nitrogen (TN). Most crops
absorb nitrates to the greatest extent (85% of the nitrate contained in wastewater); whereas
50% of ammoniacal and less than 30% of organic nitrogen contained in wastewater can be
assimilated as it is by plants. The remaining nitrogen is taken up by soil microorganisms and
transformed into nitrates or volatilized as N2. In wastewater irrigated soils, organic nitrogen
is transformed into nitrates by soil microorganisms to a greater extent than that observed in
non–irrigated agricultural soils [38]. Problems related to high inputs of nitrate ions are due to
their high solubility in water, and thus their rapid percolation through the soil to the aquifer.
Water requirements Nutrients and sodium contribution by WW (kg ha/year)

Crop
(mm/year) Ntotal Ptotal K Ca Mg Na
Maize 673 108–418 27–162 13–465 121–1401 61–741 182–1226
Green tomatoes 653 104–405 26–157 13–451 118–1358 59–718 176–1188
Chili 601 96–373 24–144 12–415 108–1250 54–661 162–1094
Beans 370 59–229 7–89 7–255 67–770 33–407 100–673
Wheat 520 83–322 21–125 10–359 94–1082 47–572 140–946
Barley 516 83–320 21–124 10–356 93–1074 46–568 139–939
Alfalfa 1360 218–843 54–326 27–938 245–2829 122–843 367–2475
Marrow 364 58–226 15–87 7–251 66–757 33–400 98–662
Oats 353.6 57–219 14–85 7–244 64–735 32–389 95–644
Source: reference [16]
Table 1. Contribution of nutrients and sodium from untreated wastewater and water requirements of demandant
crops
A significant quantity of nitrate leaching through soil subsequently becomes unavailable

for plants; this does not necessarily represent a problem, as nitrate is continuously supplied
to soil via wastewater. More important, the presence of nitrates in subterranean water is
related to occurrence of methemoglobinemia disease in infants ingesting nitrate at levels
higher than 45 mg/L via drinking water [39]. The quantity of nitrogen washed out from
soil depends on the irrigation rate, the frequency of rain events, the type of crops sown
and the characteristics of the soil [40]. The amount of nitrogen that can be applied to soil
to produce minimal nitrate leaching rates depends on the demand of crops, which usually
varies between 50 and 350 kg of nitrogen per hectare [40]. Such demand is within or slightly
above the amount of nitrate supplied by treated wastewater. In this sense, the limited
removal of nitrogen by wastewater treatment would not significantly affect the input of
this macronutrient to agricultural soils.
Phosphorous is another plant macronutrient, which is very scarce in soil, at the point it needs
to be added through the application of fertilizers. Due to its stability and low solubility, this
nutrient can be accumulated in soil. Wastewater normally contains small amounts of phos‐
phorous, so its use for irrigation is beneficial to plants and it does not impact negatively upon
the environment, even if applied consistently for long periods of time [40–41]. The recycling
of phosphorous and nitrogen in wastewater–irrigated soils is important because it allows
closure the P cycle rather than its breakage. Breakage of the cycle occurs when phosphorous
is removed from wastewater during treatment, becoming trapped in sludge and dumped to
confinement sites or landfills. An advantage of the availability of phosphorus in wastewater
is that it is partly bound to organic components and thus it cannot form complexes with iron
or aluminum ions upon its entry to soils [16]. In contrast to phosphorous, potassium is
contained in soil at high concentrations (around 3% of the lithosphere) but in chemical forms
that impede its bioavailability. As a result it is necessary to add potassium to soils via fertilizers.
Approximately 185 kg of potassium per hectare are required to cultivate some crops [16].
Sewage contains low concentrations of potassium, insufficient to cover the theoretical demand
in most cases. Meeting the demand for potassium in irrigated soils will depend on the amount
of wastewater supplied at each irrigation event, the wastewater quality and the frequency of
irrigation. Fertilization with potassium has not resulted in adverse impacts to the environment
[42]. Recycling nutrients by the reuse of wastewater promotes savings in energy, which would
otherwise be consumed in the production of fertilizers [43]. In particular, the recycling of
phosphorus is important since the world’s phosphorus reserves are becoming scarce [44].
Fertilizing agricultural soils by the reuse of wastewater invariably leads to the increase of crop
yields. An example of this can be found in Mezquital Valley, Mexico [45]; in this respect, Table
2 shows the differences in the agricultural production in croplands of Mezquital Valley when
either untreated wastewater or groundwater is used for agricultural irrigation.
Crop yield (tons/ha)

Crop Increment (%)
Untreated wastewater Groundwater
Corn 5.0 2.0 150
Barley 4.0 2.0 100
Tomato 35.0 18.0 94
Oats for forage 22.0 12.0 83
Alfalfa 120.0 70.0 71
Chili 12.0 7.0 70
Wheat 3.0 1.8 67
Source: references [16, 52]
Table 2. Comparison of crop yields for some vegetables in plots where wastewater and groundwater are used for
agricultural irrigation (Mezquital Valley, central Mexico)
8 Soil Pollution
The use of wastewater in Mezquital Valley has also contributed to changing the land‐
scape of the zone, transforming barren soils into productive and green vibrant soils, as
shown in Figure 2.
Figure 2. Comparison of untreated wastewater irrigated (right side) and rain–fed (left side) croplands from Mezquital
Valley, central Mexico
2.1.2. Benefits in soil quality
In order to define the improvements in soil quality produced by the application of treated/
untreated wastewater it is necessary to establish the use of the irrigated soil. It is known that
soil complies with five ecological functions: a) a medium for plant growth (including agricul‐
ture); b) a biodiversity pool and habitat for plants and (micro and micro) fauna; c) a carbon
sink; d) a storage, filter and transforming medium for nutrients, pollutants and water; and, e)
a landscaping and engineering medium. [46]. This chapter focuses on the functions of soil as
a medium for plant growth as well as in its role as a transforming medium for nutrients and
pollutants.
In addition to the continuous supply of nutrients to the soil, irrigation with treated/untreated
wastewater confers significant improvements in soil quality. Favorable changes reported in
irrigated soils comprise: a) an improvement in the physical structure of soil; b) an increase in
soil microbial activity; and, c) the improvement of the soil performance as a wastewater
treatment system.
Improvement of the physical structure of soil. The physical structure of soil is defined as the
arrangement of the solid particles and the size, shape and interconnection of pores and voids.
Soil structure is closely related to its capacity to store and transport gases and water (and thus
dissolved substances) [47]. Gas exchange between the soil and the atmosphere determines
whether aerobic, anoxic or anaerobic conditions prevail within the soil. This in turn regulates
the metabolism of soil microorganisms and impacts, inter alia, upon the nitrogen fixation, the
transformation of soil organic matter and the degradation of pollutants. Additionally, the
physical structure of soil affects the plant growth by influencing root distribution and thus the
ability to take up water and nutrients [48]. Improvements in the physical structure of soil are
related to the increase in both the stability of the soil aggregates and soil porosity. The
enhancement of the physical structure of soil results in a rise in agronomic productivity, the
augmentation of water infiltration through soil to the aquifer and a decrease in erodibility [49].
The hierarchical theory of aggregation proposes that microaggregates (particle size below 250
µm) in the soil are formed initially by the attachment of organic material to some inorganic
components of soils (e.g. clay and hydroxides); in turn these microaggregates join together to
form macroaggregates (particle size above 250 µm). Alternatively, macroaggregates can form
around the particulate organic matter, while exudates produced by soil microorganisms serve
as cementing agents, making micro and macroaggregates more stable [50]. Microaggregates
can be also formed from bacterial colony clusters which use bacterial polysaccharide exudates
to bind with clay particles. The clay particles act as a protective shell for clusters and macro‐
aggregate formation continues as described above [51].
Since the formation of aggregates in the soil is related to the presence of organic matter, and
in some cases microorganisms, it might be expected that the continuous supply of these two
elements via wastewater would result in the increased formation and stability of soil aggre‐
gates and thus an improvement in the physical structure of soil. For example, the study referred
in [52] establishes that increased soil microbial activity due to the augmentation of organic
carbon content by the application of wastewater impacts positively upon the stability of soil
aggregates. Furthermore, there are substances contained in wastewater other than organic
matter and microorganisms that may contribute to the formation and stability of soil aggre‐
gates. Calcium and magnesium cations, which are abundant in wastewater, increase the
formation of microaggregates through cationic bridging between clay and organic matter,
resulting in aggregation. In arid soils and soils with low organic matter contents, insoluble
calcium and magnesium carbonates can trigger the formation of soil micro and macroaggre‐
gates [51]. Additionally, calcium can inhibit clay dispersion, and thus the breakup of aggre‐
gates, when sodium concentration increases in soil [53]. Dissolved organic matter in
wastewater can form complexes with iron and aluminum in soil forming mobile organo-
metallic compounds which can further precipitate and act as cores for microaggregates
formation. Particulate organic matter (i.e. suspended solids in wastewater) may enhance the
binding of microaggregates to subsequently form macroaggregates; for instance, extracellular
polysaccharides of microorganisms in the surface of suspended solids can act as binding agents
in the formation of macroaggregates [54]. In the case of phosphorous, the formation of
insoluble aluminum and calcium phosphates in the soil can induce the formation of microag‐
gregates and additionally it may act as a macroaggregate binding agent [55]. The entry of
certain chemicals to the soil via wastewater increases the stability of soil aggregates. For
example, hydrophobic substances (e.g. surfactants, lipids and hydrocarbons) decrease the
wettability of aggregates by inducing water repellency, which in turn leads to increased
cohesiveness and low decomposition rates of soil aggregates [51]. Agricultural activities in
wastewater irrigated soils may also contribute to the improvement of the physical structure
of soil. Previous studies have found that some crops (i.e. maize, alfalfa and leguminous plants)
have beneficial effects on the conservation of the physical structure of soil. Aggregation of soil
particles tends to increase when planting crops characterized by high density and long length
of roots; this is because chemicals released by roots (i.e. mucilage) enhance the stability of soil
10 Soil Pollution
aggregates in the rhizosphere by increasing the bond strength and decreasing the wetting rate
[56]. According to the study reported in reference [57], roots of leguminous crops increase the
aggregation of soil particles. Corn (Zea mays) residues (leaves and shoots) also increase
aggregation of soil particles compared with other crops; this is attributable to the liberation of
phenolic compounds from plant tissues, since phenols favor the agglutination of particles and
prevent wetting [57–58]. Municipal and industrial wastewater may also be a source of phenolic
compounds to soil through irrigation, producing similar effects to those of corn wastes [59].
The study referred to in [58] demonstrated that the stability of soil aggregates is high for
continuous cultivation of alfalfa (Medicago sativa), while the opposite effect was observed for
soybean. This is attributable to the low concentration of phenols in the latter [60]. Some studies
have addressed the changes in the physical structure of agricultural soils caused by long–term
irrigation with wastewater. The results of these studies show a decrease in soil porosity caused
either by occlusion of pores by the suspended solids contained in wastewater or by the
augmentation of micropores (radius < 0.01 µm) in the soil matrix [61–62]. Depending on the
method of water application during irrigation, an increase in the compaction of soil may be
observed in the plot after an irrigation event [63]. Soils irrigated by flooding exhibit high
compaction while water dropping effects (erosion) may be observed in soils irrigated by
spraying. In any of both cases, wastewater irrigated soils exhibit large populations of earth‐
worms which may assist in the formation and connection of pores within the soil matrix.
Undoubtedly wastewater contains agents that improve the physical structure of soil. However,
studies performed so far show contrasting results, either an increase in the soil microporosity
or soil compaction. It is therefore necessary to carry out studies aimed at measuring changes
in the physical structure of soil throughout several irrigation cycles and for longer periods
(months or years); additionally, it is of interest to assess changes in the physical structure of
soil at landscape level (piedmont or catena), as it may be useful for evaluating the horizontal
displacement of soil particles and nutrients.
Increase of soil microbial activity. Either due to the extra supply of organic carbon or because
of the addition of microorganisms via wastewater, microbial activity in wastewater irrigated
soils tends to be higher than that found in non–irrigated soils [64–65]. This increase in the
microbial activity of the soil brings benefits to both agriculture and the development of flora
and fauna in the soil ecosystem. According to the study reported in reference [66], the C/N
ratio in soils irrigated with wastewater for long periods tends to decrease by up to 45%, which
implies an improvement in the nutritional conditions for soil microorganisms. The authors
report an increase in the population of copinotrofic and oligotrophic bacteria (234 and 217%,
respectively), as well as in the populations of actinomycetes (234%) and fungi (206%) in soils
irrigated with wastewater for 100 years compared with those populations found in non–
irrigated soils. Rises in the metabolic activity of soil, measured as the production of ATP and
enzymatic activity have been also reported [65-66]. According to reference [66], soil enzymatic
activity remained unchanged 20 years after wastewater irrigation ceased. In contrast, the study
referred to in [67] shows that elevated microbial activity in soils irrigated with treated
wastewater decreases after few days without irrigation. Due to the augmentation of the
populations of bacteria, actinomycetes and fungi in the irrigated soil, a rise in the rhizospheric
activity is experienced, resulting in: a) the increase in the growth and development of plants;
b) high rates in stabilization of organic matter entering the soil through wastewater; c) higher
performance of the depuration of wastewater and degradation of the pollutants fixed in the
soil in comparison with non–irrigated soils; and, d) the improvement in the formation and
stability of soil aggregates. The latter may be explained by the role of polysaccharides exuded
by bacteria as transient binding agents, which initialize aggregation of soil microaggregates
[67]. The transformation of carbon and nitrogen by soil microorganisms supports the prolif‐
eration of soil (micro and macro) fauna which is essential for soil formation as well as for the
development of plants. According to the work referred to in [68], the use of treated wastewater
to irrigate an agricultural soil over 20 years has resulted in the improvement of the metabolic
efficiency of soil microflora to transform carbonaceous and phosphorous substances into
nutrients readily available to plants and macrofauna.
Soil biomass has proven to be capable of adsorbing a certain proportion of heavy metals
contained in the wastewater. For instance, the study referred in [69] found biosorption rates
for cadmium and nickel within the range of 5 to 55 mg/g of biomass in a soil that had been
irrigated with wastewater for two decades. In that soil, the predominant bacteria after
irrigation were Enterobacteriaceae and Pseudomonas.
The effect of wastewater irrigation on soil nitrogen fixing organisms has been little studied.
An increase in soil nitrifying activity accompanied by a low rate of denitrification has been
observed in wastewater irrigated forest soils [70], while in the study referred to in [71] a peak
in N2O production in a soil irrigated with treated wastewater was reported, followed by an
immediate drop in gas production. So far, the metabolic processes performed by different soil
microbial species in wastewater irrigated soils have been little explored. However, it is
important to keep in mind the important role that soil microorganisms play in both the
development of the soil and plants as well as in the purification of wastewater when planning
agricultural systems based on the reuse of wastewater. Even when soil microbial populations
show some kind of resilience to a wide variety of contaminants, some other chemicals can
cause not only toxic effects to soil microorganisms but the proliferation of pathogenic organ‐
isms and the occurrence of antibiotic resistance within the agricultural soils.
Improvement of soil performance as a wastewater treatment system. As it is known, the

application and infiltration of wastewater through soil results in its purification. In practice,
specific wastewater treatment systems are based on soil infiltration, which have been demon‐
strated to improve water quality to levels obtained using tertiary treatment systems [72–73].
Purification of wastewater is one of the ecological functions of soil; through this mechanism,
soil maintains, at least partially, the quality of surface and groundwater bodies. The extent at
which this natural system works is highly variable, from almost nonexistent to very high,
depending on local conditions and types of pollutants. Table 3 shows the extent to which
pollutants in wastewater are removed by infiltration through the soil. The application of
wastewater to soil reduces the content of pathogenic microorganisms by 6–7 log units for
bacteria and 100% for helminths and other protozoa. Total organic carbon can be reduced by
up to 90%, while levels of recalcitrant compounds in wastewater, such as phosphorus (20–
90%), nitrogen (20–70%), and metals (70–95%) are also reduced dramatically. In sewage,
organic phosphorus (5–50 mg/L) is biologically converted to phosphate; subsequently, in
12 Soil Pollution
alkaline or calcareous soils, phosphate precipitates with calcium to form calcium phosphate
and remains available for plants. In contrast, in acidic soils phosphate reacts with iron and
aluminum oxides to form insoluble compounds, which are unavailable to plants. Sometimes
soluble phosphate is initially immobilized by adsorption onto soil particles and then slowly
returns to insoluble forms, allowing for further adsorption of mobile phosphate. This process
is generally known as phosphate aging [72].
Variable Effect
Organic matter Biodegradable material is reduced by more than 90%, while less readily biodegradable
material is adsorbed and later biodegraded or volatilized.
Nitrogen Nitrogen is removed from water at a level similar to tertiary treatment systems by
transformation in soil as well as by assimilation by soil microorganisms and plants.
Phosphorus Phosphorous is reduced to levels of 1 mg/L or less by assimilation by plants.
Microorganisms Helminth eggs and protozoa are easily removed by straining in the soil surface; bacteria and
viruses can also be adsorbed onto the soil particles and then desiccated or killed by
indigenous soil microorganisms. The performance of these processes depends on the texture,
physical structure and organic matter content of soil.
Heavy Metals Heavy metals can be removed by the formation of complexes with soil organic matter,
precipitation or methylation at efficiencies of 70–95%.
Toxic organic Most are retained in soil and then biodegraded at different rates.
compounds
Table 3. Processes in soil that improve the quality of the wastewater, relative to selected parameters
Most of the organic compounds (natural and synthetic) in sewage are rapidly transformed in
soil to stable, and in some cases non–toxic, organic compounds (e.g. humic and fulvic acids).
Actually, soil biodegrades a greater amount and variety of organic pollutants than that
reported for water streams. Wastewater application to soil under controlled conditions (e.g.
limited irrigation rate and intermittent flooding) permits the biodegradation of hundreds of
kilograms of carbonaceous substances per hectare per day, with no impact on the environment
[72]. Total organic carbon levels in wastewater are dramatically reduced from levels of 80–200
mg/L to 1–5 mg/L in the infiltrated water [74]. Heavy metals can be removed from wastewater
during soil infiltration and confined within the organic domain of the soil for several hundred
years. Metals are retained in the surface layer of the soil either by complexation with soil
organic matter or by precipitation at high pH values. Only a small fraction of metals infiltrates
to lower layers of the soil profile and even less can be assimilated by crops. For instance, around
80–94% of cadmium, copper, nickel, and zinc can be removed in the first 5–15 cm of the soil
profile, 5–15% is leached to lower layers and only 1–8% can be absorbed by grass [75]. A similar
process occurs with fluorine [76]. This phytoremediation process is used to treat wastewater
in planned natural treatment systems such as wetlands. However, it is necessary to be aware
that some edible crops are able to take up heavy metals to a greater degree than grasses [77].
The capability of soil to act as a filter and transforming medium for wastewater pollutants can
be observed in both long–term and newly wastewater irrigated soils [72, 78]. The operation of
this natural purification system is closely related to the physical and chemical properties of the
soil and thus modifications in soil characteristics caused by irrigation with wastewater may
either improve or worsen the performance of this natural wastewater treatment system. The
increase in the soil organic matter content is the main factor resulting in an improvement in the
removal of biological, organic and inorganic pollutants as wastewater leaches through the soil.
This is because soil organic matter promotes the immobilization of pollutants either by
adsorption or formation of complexes, while at the same time stimulating the proliferation of
degrading microorganisms [78–79]. Regularly, heavy metals are fixed in the upper layers of the
soil profile by complexation with organic matter [65], thus organic matter enrichment in
wastewater irrigated soils results in greater retention of heavy metals by the solid matrix. Heavy
metals cannot be biodegraded but they may be modified by soil microorganisms. Biological
methylation of metals and metalloids, such as selenium, arsenic and mercury, has been reported
in wastewater irrigated soils. It is expected that this process is elevated in wastewater irrigat‐
ed soils, where microbial biomass occurs at higher levels than in non–irrigated soils. Methyla‐
tion of heavy metals leads either to reduced toxicity or increased loss of metals in soil through
volatilization [80–81]. Another process observed in long–term wastewater irrigated soils, related
to those aforementioned, is the potential of soil microorganisms to develop resistance to the
harmful effects caused by the presence of heavy metals in the solid matrix [69, 82]. Such resistance
is similar to that developed to antibiotics and has been reported for cadmium, chromium, zinc
and nickel in soils irrigated with wastewater over the long term [69, 83]. It is plausible that the
expression of these resistances results in an increase of heavy metal methylation in the soil,
which allows soil microorganisms to survive and to continue with those metabolic functions
that increase agricultural productivity and purify wastewater. With regard to organic contam‐
inants, the increase in the soil organic matter content produces, in most cases, an incremental
boost in the adsorption of solutes onto soil particles. Studies referred to in [84] found in‐
creased adsorption of organic contaminants (i.e. pesticides, pharmaceuticals and estrogenic
hormones) in long–term wastewater irrigated soils compared to rain–fed soils from the same
agricultural area. Organic compounds displaying high hydrophobicity are adsorbed by soil not
only faster and to a greater extent but with greater strength than is observed for semi–polar and
polar compounds [85]. The increase in the hydrophobicity of soil due to the application of
wastewater increases the capacity of such soils to strongly retain non–polar organic contami‐
nants within the solid matrix. The increase in the adsorption of organic pollutants by soil results
in an extended retention time in the solid matrix, encouraging biodegradation processes. Similar
to the results reported for adsorption, higher rates of biodegradation of organic pollutants have
been observed in treated/untreated wastewater irrigated soils compared to non–irrigated ones
[86]. This may be caused, on the one hand, by the continuous supply of organic matter to the
soil via wastewater, which can be used by soil microorganisms as co–substrate in the biodegra‐
dation of target organic pollutants, and on the other hand, by the prolonged exposure of soil
organisms to pollutants. The latter case can be understood as the acclimation of the degrad‐
ing organisms to the occurrence of organic pollutants in the soil followed in the short term by
the acquisition of the capability for using organic contaminants as a carbon source. The increase
in the soil organic matter content caused by wastewater irrigation has a positive impact not only
in the adsorption of organic compounds but also on the retention by soil of wastewater–
14 Soil Pollution
borne pathogens. This is due to the high affinity of the cell membranes to the organic domain
of soil. The study referred to in [87] reports a higher adsorption of enteric bacteria Escherichia
coli and the enteric protozoa Giardia lamblia in long–term wastewater irrigated soils compared
with long–term groundwater irrigated soils from the same agricultural zone.
In general terms, an increase in pH values has been observed in agricultural soils irrigated
with treated/untreated wastewater; although in less cases soil pH tended to decrease following
the application of wastewater [81]. The first phenomenon is attributed to the continuous
addition of salts (carbonates, calcium, magnesium, sodium) in wastewater. The second case is
explained by the high mineralization rate of organic matter in the irrigated soil, which is highly
dependent on the soil type, the climatic conditions of the site, and the quality of wastewater,
among other reasons. The increase in soil pH, in combination with the continuous supply of
organic matter, results in the buffering of soil pH, which prevents the drop of soil pH values
during rain events (including acid rain). Stabilization of soil pH values also contributes to the
retention of heavy metals in the surface layers of the soil by the formation of insoluble basic
salts. Furthermore, basic values of soil pH can facilitate the adsorption of neutral and basic
organic contaminants; as these compounds tends to be better adsorbed to neutral and basic
soils than to acidic ones.
Since wastewater irrigation improves the physical structure of soil (i.e. increased formation
and greater stability of aggregates), aerobic conditions may be maintained within the soil
matrix; which in turns contributes to an increase in the aerobic biodegradation rate of organic
pollutants. Additionally, an increase in the adsorption of pollutants can be achieved in better
structured soils due to the increase in the specific surface area of soil particles. Moreover, higher
biodegradation of the adsorbed contaminants can be expected as long as they remain available
to microorganisms after adsorption. In irrigated soils where occlusion of the pores by the
suspended solids in wastewater occurs, anoxic conditions may be achieved. Under such
conditions, toxic species of heavy metals are chemically reduced into non–toxic species (e.g.
Cr+6 into C+3 and As+5 into As+3), then they may be immobilized by the formation of insoluble
hydroxides. The extent to which wastewater irrigation contributes to the function of the soil
as filter and degradation medium for pollutants is just beginning to be studied. The potential
of soil to act as an efficient wastewater depuration system is a powerful argument to convince
policy makers that agricultural irrigation with treated/untreated wastewater can be an
appropriate strategy to simultaneously solve problems of water stress and low agricultural
productivity with no negative impacts in the quality of water sources surrounding the
irrigation site. This, of course, is achieved when all of the appropriate precautions to avoid
contamination are taken at each site.
2.2. Negative impacts of wastewater reuse in agriculture
The main drawback of reusing treated/untreated wastewater in agriculture is the pollution of

soil, the potential contamination of crops and water sources, and the inherent risk of harmful
effects that contamination poses to the exposed organisms. Even when soil acts as an efficient
living filter to remove, inactivate and transform the pollutants contained in wastewater, it is
not fully effective at eliminating some of them. Moreover, as a result of the increasing industrial
development, wastewater irrigated soils continuously receive newly synthesized substances,

which may negatively impact the effectiveness of soil as a treatment system by poisoning the
degrading microorganisms, destroying the physical structure of soil or damaging the natural
cycles occurring within soil. The pollutants received by soil via wastewater may be different
in developing and developed countries. Examples of this include pathogenic microbial agents.
In developed countries most wastewater is treated prior to reuse and thus pathogens are not
present in irrigation water, while in developing countries untreated wastewater is used in most
of cases. Pathogens vary for different zones; for instance, the enteric protozoa Giardia is
commonly found in wastewater of developing countries (Latin American and African
countries), while the parasitic protozoa Cryptosporidium occurs in developed countries (United
States and western European countries). Similar to microorganisms, some organic pollutants
can be found in wastewater from developing countries and not in developed countries.
Examples include some herbicides (e.g. DDT and atrazine) whose use is restricted in developed
countries; on the other hand, nanomaterials and new–generation antibiotics, all of which are
much more likely to occur in wastewater of developed countries. The determination of
pollutants in soil initially requires specific sampling methods which take into consideration
the heterogeneity of the soil matrix. In addition, specialized extraction techniques able to
efficiently isolate analytes (or microorganisms) from soil are necessary prior to analysis.
Specialized analytical methods have been developed and validated for the determination of
trace contaminants and microorganisms in soil. However, in most cases, these methods are
time–consuming, expensive and require the use of specialized reagents and personnel. It is
therefore necessary to continue research towards the development of simpler and environ‐
mentally–friendly analytical techniques. Determining the occurrence and concentration of
contaminants in soil is a task that requires a significant effort; however, this is only a part of
the job. The study and understanding of the environmental fate of contaminants in soil is also
a priority task to accomplish truly useful environmental risk assessment studies comprising
soil, water sources, crops, farmers and consumers. Knowing the environmental fate of
contaminants in the soil is necessary to understand the potentialities and limitations of each
soil as a natural purification system of wastewater and an effective tool to define the capacity
of each site to support wastewater irrigation in agriculture. Since soil is a complex and
heterogeneous matrix, the fate of contaminants can vary significantly from one site to another.
In this sense, it is worth defining which parameters are determinant in the fate of contaminants
within soil and, on the basis of this knowledge, elucidating the fate of contaminants in other
sites using mathematical tools to achieve such extrapolations. In this section, attention will be
focused on pathogenic microbial agents, heavy metals and organic pollutants contained in
municipal wastewater. The occurrence of such pollutants in wastewater–irrigated soil as well
as their environmental fate in soil is addressed; additionally the most significant effects of these
contaminants will be treated in some detail. Lastly, perspectives for further studies on the
occurrence and fate of the studied pollutants in soil are presented.
2.2.1. Soil pollution by pathogenic microbial agents
Contamination of soil and crops by pathogenic agents is the effect of wastewater reuse in
agriculture that receives most attention from environmentalists and scientists. Municipal
16 Soil Pollution
wastewater contains a huge quantity and variety of bacteria, protozoa and viruses passed from
human and animal feces and urine; therefore this water is a vector for intestinal infections
(although some other diseases can spread from the environment via wastewater). Exposure
may be direct through contact or ingestion of wastewater and soil, or indirect through contact
with sick people or by ingestion of polluted crops, meat or milk. There are four groups at risk:
a) farmers and their families, b) crop handlers, c) product consumers and d) people living
nearby to irrigated fields. For any of these groups children and elderly are the most vulnerable,
especially when they are undernourished. The most affected group is agricultural workers due
to their high exposure to wastewater and contaminated soils [18]. Table 4 shows the risk of
infection of water–borne diseases for vulnerable groups in irrigated areas using treated/
untreated wastewater.
Effects caused by microbial pollution in soil. Several diarrheal outbreaks have been associ‐
ated with the use of wastewater to irrigate [18, 88]; however, since this occurs in places where
sanitation, hygiene practices and drinking water are of low quality it is always difficult to
define their specific contribution to the total diseases burden. Cholera, caused by the bacterium
Vibrio cholera, is one infection closely linked to wastewater irrigation in poor countries. Other
intestinal diseases related to the use of wastewater to irrigate are traveler’s diarrhea caused by
Escherichia coli, shigellosis caused by Shigella spp., gastric ulcers caused by Helicobacter pylori,
giardiasis caused by the parasitic protozoan Giardia intestinalis and amebiasis caused by
Entamoeba histolytica. Additionally, viral enteritis (caused by rotaviruses) and Hepatitis A are
the most reported viral infections caused by consumption of polluted vegetables [89]. Some
studies [90] report skin diseases, such as dermatitis (eczema), in farmers that come into contact
with untreated wastewater and wastewater irrigated soil. Nail problems in farmers, such as
koilonychias (spoon–formed nails), have also been reported as related to the presence of fungi
in wastewater irrigated soils [91]. Health and growth problems have been observed in cattle
that consume forage produced by wastewater irrigation. Furthermore, in low income areas
where water is scarce, cattle are not only fed with fodder grown using wastewater but also
they are allowed to drink the wastewater used for irrigation. Some protozoa can survive in the
surface layers of soil or even in aerial parts of crops; animals can be infected after eating these
crops, although this is a remote way of transmission. There is some evidence indicating that
beef tapeworm (Taenia saginata) can be transmitted from livestock fed with wastewater–
irrigated forage to meat consumers. Furthermore strong evidence indicates that cattle grazing
on fields freshly irrigated with raw wastewater or drinking from raw wastewater canals or
ponds can become heavily infected by Cysticercus bovis, the early stage of the Taenia saginata
life cycle [88].
Microbial agents in wastewater irrigated soils. The study of microbial contamination by the
use of treated/untreated wastewater in agricultural irrigation is focused in the pollution of
crops rather than the soils receiving wastewater. This is because, on the one hand, a greater
number of people are exposed to pathogenic microorganisms through consumption of
contaminated crops, meat and milk than by direct contact with irrigated soils, and on the other
hand, the difficulties in the analysis of microorganisms in soil; for instance, the inherent
problems of extracting microorganisms from such a complex matrix as the soil. Studies in the
Group exposed Helminth infections Bacterial/viral infections Protozoan infections

Consumers Significant risk of Ascaris Cholera, typhoid and Evidence of parasitic protozoa
infection for both adults and shigellosisoutbreaks reported due to found on the surface of
children consuming the consumption of polluted crops. wastewater–irrigated
vegetables contaminated with Seropositive responses for vegetables, but no direct
helminth ova. Helicobacter pylori in crop evidence of disease
consumers. Increase in risks of transmission.
suffering non–specific diarrhea
when concentration of
thermotolerant bacteria in
wastewater used for irrigation
exceeds 104 CFU/100 mL.
Farm workers and Significant risks of Ascaris Increased risk of diarrheal diseases Risk of Giardia intestinalis
their families infection for both adults and for children in contact with infection insignificant for
children in contact with wastewater when it exceeds 104 contact with both treated/
untreated wastewater and CFU/100 mL for thermotolerant untreated wastewater and
irrigated soils. Risk remains, coliforms. Elevated risk of Salmonella soil. Increased risk of
especially for children, when infection in children exposed to amoebiasis observed due to
wastewater presents more untreated wastewater and contact with untreated
than 1 nematode egg per litre. wastewater irrigated soil. Elevated wastewater and wastewater
Increased risk of hookworm seropositive responses to norovirus irrigated soil.
infection in farmers. in adults exposed to partially treated
wastewater and wastewater
irrigated soil.
Nearby High risk of infections when Sprinkler irrigation with untreated No data of protozoan
communities flood and furrow irrigation is wastewater and high aerosol infections transmission during
used. Ascaris transmission not exposure associated with Increased irrigation with wastewater.
studied for sprinkler irrigation. rates of bacterial infections due to
the use of partially treated
wastewater (104–105 CFU/100 mL or
less). No risks of viral infection
associated with sprinkler irrigation.
Table 4. Summary of health risk associated with the use of wastewater in agriculture
Mezquital Valley, Central Mexico, found the occurrence of fecal contamination indicators
(Escherichia coli). Giardia lamblia cysts and helminth eggs (Ascaris lumbricoides) at different
depths of long–term wastewater irrigated soils. Results shown in Figure 3 evidence the
accumulation of the three microorganisms in the first few centimeters of the soil profile,
indicating that infectious agents are removed from wastewater at the beginning of percolation
through soil; such removal can be achieved by several physical and chemical phenomena. In
this study, the content of pathogenic microorganisms in soils with different time under
irrigation was also evaluated. Results showed that the accumulation of microorganisms in the
18 Soil Pollution
tested soils is not related to the time under irrigation, suggesting that soils have mechanisms
to inactivate and/or destroy these microorganisms after irrigation.
As mentioned above, different types of microorganisms can be found in wastewater irrigated

soils depending on the zone where reuse is taking place. For example, the study referred in [92]
showed a higher prevalence of Cryptosparidium spp. compared with Giardia spp. in wastewater
irrigated and manure amended soils of dairy farms in southeastern New York. Cryptosporidi‐
um is a protozoan commonly found in developed countries, while different species of Giardia
are widespread in developing countries. In this respect, the study referred in [93] found the
occurrence of Ascaris lumbricoides, hookworm and Trichiuris trichiura in 69% of the soil samples
taken in an untreated wastewater irrigated area in West Bengal, India.
1.00E+04 E. coli A. lumbricoides eggs G. lamblia cysts 10
8
1.00E+03
ova/g - cysts/g
CFU/g soil
6
1.00E+02
4
1.00E+01
2
1.00E+00 0
0 15 30
Depth (cm)
Figure 3. Abundance of three pathogenic microorganisms in a long–term wastewater irrigated soil at different
depths
The entry of antibiotic–resistant pathogens (ARPs) and antibiotic resistance genes (ARGs) into
the soil via wastewater is an emerging issue. Since municipal wastewater contains both sub–
therapeutic amounts of antibiotics, ARPs and ARGs –which occur to a greater extent when
sewer systems combine municipal and hospital wastewater– [94], these substances can reach
the soil, modifying the dynamic of soil microbial populations. Antibiotic resistance may occur
naturally in the soil, and to a greater extent in the rhizosphere, which functions as a hotspot
for both antibiotic–resistant bacteria and ARGs [32]. Previous studies have found the presence
of opportunistic pathogens (Stenotrophomonas maltophilia, responsible for respiratory tract
infections and endocarditis) in the rhizosphere of Brassicacaea type plants [95]. The transfer of
ARGs from these opportunistic bacteria to human pathogens reaching the soil through
wastewater has not yet been demonstrated. The ARPs reaching the soil through wastewater
may survive on the soil surface and, if conditions are appropriate, reproduce or migrate to
surface and groundwater sources. ARGs may be mobilized into aquifers by infiltration of
wastewater or into surface water sources by runoff. So far a relationship between the presence
of traces of antibiotics in wastewater and the occurrence of antibiotic resistance in the irrigated
soil has not been categorically established. In previous studies the incidence of two sulfona‐
mide resistance genes (sul1 and sul2) was determined in the Mexico City wastewater, agricul‐
tural soils irrigated with such wastewater over different time periods and rain–fed soils [96].
The authors found the presence of ARGs in the three analyzed matrices; the concentration of
resistance genes was 150 to 1500 times higher in irrigated soils than in non–irrigated ones. The
occurrence of ARGs was positively related to the time under irrigation, with a higher content
of resistance genes occurring in Enterococci bacteria living in soils irrigated for longer periods
of time [96]. Such behavior may indicate that prolonged irrigation with wastewater promotes
both the proliferation of indigenous ARPs in soil, due to the high and constant supply of
nutrients via wastewater, and the increase in the assimilation of resistance genes due to the
higher biomass content in old wastewater irrigated soils. Conversely, studies reported in [97]
found that the abundance of isolates resistant to tetracycline, ciprofloxacin, sulfonamides and
erythromycin were identical in wastewater irrigated soils and freshwater irrigated soils
despite the high load of ARGs and ARPs in the wastewater used for irrigation. In this regard,
the study in reference [98] found, by comparing the resistome of soils irrigated either with
wastewater or groundwater, that Entetococci bacteria in freshwater irrigated soil were highly
resistant to a greater number of antibiotics (erythromycin, tylosin, tetracycline, and ciproflox‐
acin) than long–term wastewater irrigated soil, which showed resistance to lincomycin and
daptomycin. Furthermore, no differences were found in the content of ARPs when wastewater
and freshwater irrigated soils were compared, suggesting that ARPs rarely survive after they
enter soil via wastewater. Even though it seems unlikely that development of antibiotic
resistance to human pathogens in wastewater irrigated soil is related to the input of antibiotics
and resistant organisms via wastewater, it is worth, as a next step, studying the exchangeable
genetic material (e.g. plasmids), since such material can be assimilated by soil microorganisms,
inducing antibiotic resistance. Many questions remain about the mechanisms leading to the
transference of this type of genetic material [99].
Microbial pollution in crops. Crops are polluted by direct contact with wastewater during
irrigation. Pollution of the edible parts of plants depends not only on the quality of water, but
also on the quantity applied to soil, the irrigation method and the type of crop. For example,
zucchini when spray–irrigated with wastewater accumulate higher levels of pathogens on
their surface than other crops. Zucchini have a hairy and sticky cover and grows close to the
ground, which favors the attachment of pathogens. Microbial contamination of crops can occur
not only as a result of wastewater irrigation but also during washing, packing, transportation
and marketing. These problems are frequently not addressed, giving the impression that
irrigation is the only source of microbial pollution [100]. In a previous study, referred in [101],
it was found that less microbial pollution of crops is caused if irrigation is performed by
subsurface dripping than through sprinklers, furrows or flooding. Moreover, the study
reported in [102] showed that subsurface irrigation does not pollute crops even when using
wastewater with 6–7 x 105 CFU/100 mL of fecal coliforms and 225 helminth ova/L. Microbial
pollution of crops also depends on the type of crop. Fruits from trees are rarely polluted when
20 Soil Pollution
irrigation is not provided using sprinklers (this is not a common procedure used to apply
wastewater since sprinkler heads tend to become clogged). Fruits grow far from the watering
sites when furrow and flood methods are used. The microbial contamination of crops in
wastewater irrigation systems is closely related to the survival of microorganisms. Table 5
shows the survival times of some pathogens in agricultural soils and crops irrigated with
wastewater.
Survival time (days)

Pathogen
Soil Crops
Ascaris lumbricodes eggs 180 30
Salmonella spp. 80 25
Fecal coliforms <70, but usually <20 <30, but usually <15
Vibrio cholera <20, but usually <10 <5, but usually <2
Entamoeba histolytica <20 <10
Trichuris trichiura eggs >180 <60, but usually <30
Taenia saginata eggs >180 <60, but usually <30
Enterovirus <40 <20
Source: references [105–106]
Table 5. Survival of selected pathogens in soil and crops irrigated with wastewater
Both pathogenic and non–pathogenic microorganisms display differences in their survival in

soil and crops. For instance, the non–pathogenic fecal coliform indicator E. coli can survive in
soil for nearly a month, while the pathogenic strain of E. coli O157:H7 survives at most for 14
days in spinach leaves [103]. It is known in some detail that survival of pathogenic bacteria
can increase by internalization within the plant tissues [104]. Previous studies indicate that E.
coli can translocate from soil to leaves of lettuce through the root system [107]. In contrast, the
results reported in reference [108] indicate that translocation of pathogenic bacteria to the
edible parts of crops via the root system is quite unlikely. It is more likely that pathogens enter
to the edible parts of crops through wounds in vegetal tissues [109]. Wounded tissues have
been demonstrated allow the entrance of Salmonella and E. coli to lettuce and tomato plants
[110–111]. Similarly, it is reported that E. coli can use the stomatal cavities in leaves to enter
the internal structure of lettuce [115]. The pathway of this kind of entry is still unknown. Once
inside the plant tissues, pathogen survival rates improve since they can use cellulose as their
main source of carbon. Protozoa are larger in size than bacteria and thus they cannot access
the internal parts of the plants; however, these pathogenic organisms can adhere to the surface
of edible plants and remain there by the excretion of polymers which facilitate adhesion. Table
6 shows some examples of the occurrence of protozoa in crops irrigated with treated/untreated
wastewater.
Pathogen Crop Occurrence Reference

Giardia lamblia Potatoes 5.1 cysts/kg Crops irrigated using untreated
Coriander 254 cysts/kg wastewater in Marrakesh. [112]
Mint 96 cysts/kg
Carrots 155 cysts/kg
Radish 59.1 cysts/kg
Ascaris lumbricoides Potatoes 0.18 eggs/kg
Turnip 0.27 eggs/kg
Coriander 2.7 eggs/kg
Mint 4.63 eggs/kg
Carrots 0.7 eggs/kg
Radish 1.64 eggs/kg
Enterobius vermicularis Lettuce 10–40 cysts/kg Crops irrigated using treated and
Parsley 10–60 cysts/kg untreated wastewater in
Cress 10–20 cysts/kg Kahramanmaras, Turkey. [113]
Spinach 1–3 cysts/kg
Entamoeba hystolitica Lettuce 10–50 cysts/kg
Parsley 10–50 cysts/kg
Giardia lamblia Lettuce 10–20 cysts/kg
Ascaris lumbricoides Lettuce 10–30 eggs/kg
Parsley 10–30 eggs/kg
Trichuris trichiura Spinach 3.3% of the analyzed samples. Crops grown in soils irrigated with raw
Pudina 3.1% of the analyzed samples. wastewater in West Bengal, India. [93]
Coriander 5% of the analyzed samples.
Hookworm Lettuce 9.4% of the analyzed samples.
Parsley 3.3% of the analyzed samples.
Spinach 6.7% of the analyzed samples.
Pudina 9.4% of the analyzed samples.
Celery 3.6% of the analyzed samples.
Ascaris lumbricoides Lettuce 43.8% of the analyzed samples.
Parsley 23.3% of the analyzed samples.
Spinach 36.7% of the analyzed samples.
Pudina 50% of the analyzed samples.
Celery 25% of the analyzed samples.
Helminth eggs Leafy vegetables 100 eggs/kg Vegetables irrigated with untreated
Cauliflower wastewater in Faisalabad, Pakistan.
[114]
Table 6. Occurrence of some pathogen protozoa on the surface of crops irrigated using treated/untreated
wastewater
22 Soil Pollution
Fate of pathogenic microorganisms in soil. Upon their arrival to irrigated soils, microorgan‐
isms can either survive or be inactivated/killed by the physical and chemical processes
naturally occurring in soil as well as by predation by indigenous soil organisms. Given the
case that these microorganisms can survive in the soil, they may subsequently colonize soil
particles, infiltrate the soil to the aquifer or migrate through across the landscape by runoff.
Processes affecting the environmental fate of the pathogenic microorganisms in soil are shown
in Figure 4. Previous experiments have demonstrated that some microorganisms can vertically
and/or horizontally mobilize through the soil, travelling long distances from the initial point
of contamination [116]. Bacterial migration in soil has been reported up to 830 meters, while
for viruses such displacement is significantly lower, i.e. up to 408 m [117–118]. Survival of
pathogens is related with their environmental fate since the longer the lifetime of the micro‐
organisms the larger the distance they can travel. As indicated in Table 5, bacteria can survive
for long periods compared to viruses, and thus bacteria can be transported farther. Climatic
conditions also impact upon pathogen transportation; for instance, in frozen soils pathogens
can survive longer and thus they can be transported farther than in tropical and desert soils
[119]. Microorganisms can be more easily displaced through coarse textured soils than fine
textured ones. The study referred to in [118] found greater mobilization of coliforms in sand–
gravel soil than in fine sand. In fact, in coarse sandy soils, the vertical movement of microor‐
ganisms can be as rapid as that observed for inorganic tracers. In this regard, the results
reported in reference [120] evidence that infiltration of streptomycin–resistant E. coli can be
compared with that of the chloride tracer in undisturbed soil columns, even when different
soil textures are compared. Since the transportation of microorganisms is similar to that
observed for tracers, the physical structure of soil is the determinant factor in them reaching
the aquifer; therefore, a greater occurrence and interconnection of pores within the solid matrix
may result in efficient infiltration of water and thus bacteria. Studies on the movement of
pathogens in the field confirm the rapid movement of pathogenic bacteria observed in
laboratory tests. These studies also found a high concentration of bacteria and viruses in
groundwater [121]. In addition to the higher quantity and interconnection of pores, the
increased transport of bacteria and viruses through the soil can be explained by the presence
of preferential paths within the soil matrix. Such preferential paths are referred to cracks,
fractures, worm holes and channels formed by plant roots or fauna in the soil. Studies reported
in reference [122] show that larger microorganisms (E. coli) can mobilize deeper into soil than
smaller coliphages. Moreover, the study referred to in [123] confirms that bacterial cells smaller
than 1 µm in diameter are more rapidly transported through soil than larger organisms.
The chemical properties of soil can also impact upon the vertical and horizontal transport of
microorganisms. The mineral composition of soil can favor adhesion of microbial cells, eggs
or cysts onto soil particles. Several types of bacterial cells have been shown to strongly adhere
to the mineral domain of soil and aquifer material [124]; once adhered, bacteria can replicate
and form biofilms on the surface of soil particles. In wastewater irrigated soils, the accumulated
organic matter as well as the continuous input of dissolved organic matter via wastewater may
enhance the proliferation of bacteria. With regard to parasites, the study referenced in [87]
found that Ascairs lumbricoides eggs and Giardia lamblia cysts adhere to the mineral fraction of
wastewater irrigated soils more rapidly and more strongly than to the organic domain. In the
Source: modified from [121]
Figure 4. Factors affecting the environmental fate of pathogenic microorganisms in wastewater irrigated agricultural
soils
case of Ascaris eggs, adhesion occurs with the silica in sand particles. In contrast to protozoan
eggs, studies referred to in [125] suggest that adhesion or adsorption of protozoan cysts may
be related to soil organic matter rather than the mineral fraction of soil. This has been attributed
to the hydrophobic nature of the cysts walls. According to the findings reported in [126]
detachment of bacteria from soil particles is effected by the composition of the irrigation water.
In that study, Pseudomonas sp. showed enhanced transport when distilled water was used for
detachment in column experiments, compared with 0.01 M NaCl. Such results suggest that
clean water can efficiently wash off the polysaccharides excreted by bacterial cells which act
as an adhesive between soil particles and bacteria. The opposite effect has been observed for
Ascaris eggs. When soil is washed with NaOCl, eggs are effectively detached from soil
particles; this is because sodium hypochlorite can destroy the albuminose layer that coats the
surface of helminth eggs and which anchors with the soil particles [127]. According to the
established in [87], the environmental relevance of studying the impact of this salt on the
detachment of eggs from soil relies on the fact that NaOCl can be found in reclaimed water,
as it is commonly used for disinfection of effluents.
Once microorganisms are retained by soil, either by adsorption/adhesion or straining, they can
be inactivated or eliminated by desiccation. This phenomenon is particularly important in arid
areas where high levels of solar radiation are reported. The environmental fate of microor‐
ganisms in soil also depends on the native microorganisms living in the solid matrix. Predators
of wastewater-borne pathogenic bacteria in soil include Streptomycetes, Myxobacteria,
Bdellovibrio and nematodes [121]. The presence of plants may affect the persistence and
movement of microorganisms in soils. On the one hand, pathogen can found favorable
conditions for survival in the rhizosphere due to the high content of nutrients in this zone; and
24 Soil Pollution
on the other hand, native bacteria in rhizosphere can be natural predator of those pathogens,
while roots may excrete antibiotics that inhibit or kill pathogenic microorganisms.
2.2.2. Soil pollution by heavy metals
Given that most agricultural wastewater irrigation is performed using municipal wastewater,
which contains negligible amounts of heavy metals [11], the occurrence of these elements in
wastewater irrigated soils is usually significantly lower than the maximum permissible
concentrations established by international regulations. However, there are some cases where
care should be taken when reusing wastewater in irrigation, e.g. close to tanneries, metal
processing or mining areas [91]. Different levels of risk are perceived for the different heavy
metals. While some of them are nutrients for plants at trace concentrations, others have been
shown to produce harmful effects on exposed organisms, or are absorbed by plants and
accumulated through the food web. Table 7 presents the risks that are incurred by the presence
of some heavy metals in soil.
Risks characteristics Metal
Low risk Mn, Fe, Zn, Cu, Se, Sb
High risk Cr, As, Pb, Hg, Ni, Al, Cd
Essential micronutrient to plants Cu, Fe, Mn, Mo, Zn, Ni
Beneficial for some crops Co, Na, Si
Can accumulate in crops to levels that are toxic for consumers Cd, Cu, Mo
No human toxicological threshold established for wastewater intended for Hg

irrigation
Relatively high threshold for wastewater used in irrigation Cu, Fe, Mn, Zn
Low absorption by plants Co, Cu, Mn, Zn
Source: with information from [75]
Table 7. Heavy metal risk characteristics during irrigation
Cadmium is the metal with the highest associated risk. It is toxic to humans and animals in
doses much lower than those that visibly affect plants; furthermore crop uptake (which is
notably high in acidic soils) can increase the dose consumed by organisms and in turn
accumulation in animal tissue. Absorbed cadmium in animals is stored in kidney and liver,
although meat and milk products have shown to be little affected by cadmium accumulation
[75]. There is a relatively good knowledge to allow the setting of limits regarding the acceptable
amount of heavy metals contained in wastewater used to irrigate. In the study referred to in
[128], numerical calculation of the limits for the maximum tolerable pollutant concentration
in wastewater irrigated soils was carried out (health–based targets). This was based on the
acceptable daily human intake (ADI) for selected heavy metals and the amount that can be
“permitted” to accumulate in soil before harmful effects occur in consumers of crops (Table
8). This analysis assumed: a) only two exposure routes (wastewater → soil → plant → human;
and, wastewater → crop → human); b) a global diet in which the daily intake of grains/cereals,
vegetables, root/tuber crops and fruit accounts for ~75% of daily adult food consumption; c) a
body mass for adults of 60 kg; d) all of the food grain, vegetables, root/tuber crops and fruits
are obtained from land irrigated with wastewater; and, e) a total daily intake of pollutants by
this consumption path of 50% of the ADI (the remaining 50% of the ADI was attributed to
background exposure). Table 8 shows the inputs of heavy metals by wastewater to irrigated
soils, assuming an application of treated wastewater of approximately 1.2 m/year, which is
roughly the amount of water required to produce a crop cycle in an arid zone.
Element Maximum input by wastewater Maximum tolerable concentration

(kg/ha/year) (mg/kg)
Arsenic 0.6–12 9
Cadmium 0.06–0.24 7
Chromium 1.2–60 3200
Lead 1.2–60 150
Mercury 0.12–0.12 5
Nickel 0.24–12 850
Selenium 0.24–0.6 140
Silver 1.2 3
Source: reference [128]
Table 8. Maximum tolerable concentration of heavy metals in wastewater irrigated soils
Health effects associated with the use of water heavily contaminated with industrial discharges
for agricultural irrigation have been reported. In Japan, itai–itai disease, a bone and kidney
disorder associated with chronic cadmium poisoning, occurred in areas where rice paddies
were irrigated with water from the contaminated Jinzu River [129]. In some parts of China, the
use of industrial wastewater for irrigation was associated with a 36% increase in hepatomegaly
(enlarged liver) and 100% increase in both cancer and congenital malformation rates [130].
With regard to the occurrence of heavy metals in agricultural soils irrigated using wastewater,
the study referred in [131] presents an inventory of sources of some heavy metals (zinc, copper,
nickel, lead, chromium and cadmium) in agricultural soils of England and Wales. Results
showed that the greatest contribution of heavy metals in those soils comes from the application
of sludge from wastewater treatment plants, while irrigation appeared to be of little impor‐
tance as a source of heavy metals in soils. According to this investigation, which followed the
rates of deposition of heavy metals in the studied soils, the time required for metal concen‐
trations to reach maximum values permitted by international regulations is 80 years for zinc
26 Soil Pollution
and at least 1256 years for cadmium. In this respect, study referred to in [132] showed that
concentrations of heavy metals in long–term untreated–wastewater irrigated soils in central
Mexico were 10 times lower than the limits set by the Danish regulations; moreover, the authors
estimated that another century of irrigation is necessary to exceed these values. In most cases,
metals have little impact on aquifers. According to the results reported in [133] the most toxic
metals to humans –cadmium, lead, and mercury– were absent in groundwater at five sites in
the United States after 30–40 years of applying secondary and primary effluents at rates
between 0.8 m/year and 8.6 m/year to different crops. The reason given was that the pH values
greater than 6.5 in soil and wastewater resulted in the precipitation of the entire amount of
metals. Metals are normally bonded into the organic matter through the formation of organo–
metallic complexes, which are not bioavailable to plants. The addition of lime and wastewater
to soil assists the precipitation of metals, while the addition of chemical fertilizers has the
opposite effect, since over the long term they tend to lower the soil pH and thus solubilize
metals.
In contrast, agricultural soils have been reported in which the concentration of heavy metals,
such as cadmium and zinc, are close to reaching the maximum levels set out in international
regulations. In these cases, the factors leading to an exacerbated soil contamination and thus
increased risk of groundwater and crop pollution are: a) sandy soil texture; b) acidic to neutral
soil pH; c) low organic matter content; and/or, d) the use of industrial wastewater for agricul‐
tural irrigation [134–135]. In such cases, the cessation of agricultural irrigation with wastewater
is recommended, together with allowing the recovery of soil through remediation techniques
such as phytoremediation.
2.2.3. Soil pollution by organic compounds
Pollution of soil by organic substances has been a matter of concern to scientists and organi‐
zations regulating the quality of soil, water sources and food for several decades. An extensive
body of work exists addressing the degradation of soil by conventional organic pollutants (e.g.
pesticides, polyaromatic hydrocarbons, organochlorides, paraffin, organic solvents, etc.).
However, in sites where treated/untreated wastewater is disposed of by agricultural irrigation
one can find organic substances different to those commonly studied and reported in literature
treating oil spills, mining zones or soil polluted by industrial wastewater. Most of the dissolved
and particulate organic matter contained in municipal wastewater is produced by the degra‐
dation of human and animal excreta, hence organic matter in wastewater is composed mainly
by saccharides, lipids, amino acids and proteins; however, a tiny fraction of the organic
material in wastewater originates from chemicals contained in everyday consumer products
used and disposed of via sewage by people in urban and rural areas. According to [24],
thousands of organic compounds are contained in municipal wastewater at trace levels and
there is a lack of knowledge regarding the effects that such substances may cause to the exposed
organisms, either by themselves or in combination with other compounds or groups of organic
compounds. This group of chemicals is referred as “organic pollutants of emerging concern”
(OPECs) [136]; though they should actually be listed as priority pollutants in cases where
wastewater is used to irrigate crops, since these contaminants are in contact with soils, crops
and water sources near the irrigation area [137]. Over the last three decades, significant work
in the field of analytical chemistry has been carried out in order to extract, isolate and quantify
some of these pollutants in wastewater and soils. Frequently found OPECs in such complex
matrices are pharmaceutically active compounds (PACs) and their metabolites, personal care
products (e.g. disinfectants, fragrances, insect repellents, sunscreens, etc.), sweeteners,
stimulants (e.g. caffeine and psychoactive drugs), detergents and their metabolites, plasticizers
and industrial additives (e.g. additives in gasoline) [137]. Almost all of the studies addressing
the removal of OPECs in wastewater treatment plants report that most of these substances are
partially degraded/removed in primary and secondary treatment systems –and some pollu‐
tants are only partially removed even in tertiary treatment systems– [138]. Because of this,
OPECs occur in irrigated soils if either treated or untreated wastewater is used in irrigation.
Effluents of wastewater treatment plants contain a small fraction of the parent substance as
well as the by–products generated during treatment. However, some of the compounds may
be retained and concentrated in the sludge produced during wastewater treatment and reach
the environment via the use of sludge (or biosolids) as soil amendments in agriculture. Due to
continuous industrial development, the number of organic substances contained in wastewa‐
ter is constantly increasing; in fact, most of these substances are not tested before they are
released onto the market, and therefore their potential risks or the side effects they cause in
non–target organisms in soils or water bodies is yet unknown.
Effects caused by domestic wastewater–related organic pollutants in irrigated soils. As

mentioned above, due to the ever growing pool of organic compounds discharged to the soil
via wastewater, there is a general lack of knowledge regarding the effects that such substances
cause to exposed organisms. In general terms, municipal wastewater is the main vector of
OPECs to reach the environment, so that these substances are ubiquitous at sites where
wastewater streams occur. Pharmaceutically active compounds (PACs) are designed to cause
a defined effect on target organisms; however, when trace amounts of these substances are
transported by wastewater into environment, they can interact with non–target organisms.
One effect that has captured the attention of the scientific community in recent years is the
development of antibiotic resistance by pathogenic microorganisms due to the occurrence of
antibiotics in wastewater, surface water bodies and soils receiving wastewater [139–140].
However, a large number of studies on this subject report that proliferation of antibiotic–
resistant pathogens is quite unlikely in wastewater irrigated soils [97–99]. Conversely, the
study referenced in [96] attempts to relate the occurrence of sulfonamide and fluoroquinolone
antibiotics with the emergence of antibiotic resistances in wastewater and long–term waste‐
water irrigated soils. The authors reported a relationship between time under irrigation and
the frequency of detection of antibiotic resistance genes in soils. In the case of non–antibiotic
PACs, the most studied compounds –because they are the most used worldwide– are the
analgesic and anti–inflammatory drugs [141]. Compounds such as ibuprofen, naproxen,
diclofenac, paracetamol and ketoprofen have been shown to cause systemic damages in
aquatic species; damages in liver, gills and kidney are commonly reported [142]. The non–
steroidal anti–inflammatory drug diclofenac has been demonstrated to cause visceral gout in
vultures; in fact, the presence of diclofenac in livestock was the cause of the mass death of three
species of vulture in India and Africa [143]. Other studies show that chronic exposure to traces
28 Soil Pollution
of anti–inflammatory drugs leads to a lessening in the development of human embryo cells

[144]. The occurrence of psychotropic agents at trace levels in water bodies polluted by
wastewater discharges has been shown to alter the behavior of some fish species, suppressing
their survival instincts against predators [145]. With regard to OPECs that are not pharma‐
ceutically active compounds, there is significant concern that they may alter hormone homeo‐
stasis in organisms. These substances, known as endocrine disruptors, can mimic or compete
with natural hormones by binding with active sites on hormone receptors, causing reduced or
disproportionate hormonal responses in the affected organisms [146]. The most potent
endocrine disruptors found so far in municipal wastewater are the natural and artificial
estrogenic hormones –the latter are used as birth control agents– and the regulators of thyroidal
function, followed by plasticizers (e.g. phthalates and bisphenols), surfactants and their
metabolites and some industrial additives [147]. Endocrine disruptors are suspected of causing
the feminization or masculinization of fish and reptile populations as well as the occurrence
of breast cancer, imbalances in thyroidal function, teratogenic effects (e.g. cryptorchid) in
mammals, and even obesity in mammals (obesogens) [148–150]. There is a serious lack of
knowledge regarding the effects caused by OPECs in soil organisms. Studies on this field have
been little developed compared to those for water bodies. Table 9 shows some examples of
effects caused to soil organism by the occurrence of OPECs.
The effects caused by this class of pollutants are not limited to soil organisms and impacts can
be observed in the soil matrix. For example, surfactants can, on the one hand, decrease the
capillarity and penetrability of soil as well as increase the solid–liquid contact angle, the shape
factor and the sorptivity of soil particles. On the other hand, the input of these substances can
increase the desorption of previously sorbed organic molecules on the soil particles, which in
turn increases the bioavailability and mobility of the desorbed compounds [163]. To evaluate
the toxic effects caused by the occurrence of OPECs to soil organisms two approaches are
commonly used, i.e. acute and chronic toxicity studies. For the former, high concentrations of
target pollutants are supplied to studied organisms under controlled conditions for a short
period; chronic toxicity tests, on the other hand, are based on prolonged exposure of organ‐
isms to low (i.e. environmentally representative) doses of the studied pollutants. So far, most
toxicity studies dealing with OPECs have been carried out using the acute toxicity approach.
Even though these studies do not fully represent the conditions observed in the field, they
provide valuable information on the subject of impacts caused by this kind of contaminants to
soil organisms. Studies evaluating chronic toxic effects of pollutants are more representative of
field conditions, i.e. toxic substances enter to soil in small doses over long periods. In this regard,
conducting long–term toxicity studies that evaluate the chronic effects caused by OPECs in soil
organisms are a priority. Several toxicity studies report that the effects of organic pollutants on
soil organisms (i.e. reduction in soil respiration, enzymatic activity and nitrification/denitrifica‐
tion rates) are observed in the early days of exposition; then, after a short period (4 to 10 days)
soil recovers to its basal conditions [153–154, 159, 161]. The next step in toxicity studies for these
emerging pollutants is to determine the dynamics of the toxic effects on soil organisms after
tens or hundreds of growing cycles in which target contaminants are continuously supplied;
i.e. under conditions similar to what occurs in long–term irrigated areas.
Compound Effect in soil organisms Reference
Estrone, 17β estradiol (hormones) Negative impacts on the vegetative cycle of alfalfa (Medicago [151]
sativa).
Sex hormones Shift in sex ratio of free life nematode communities in soil. [152]
Triclosan (antibacterial agent) Inhibition in plant growth (rice and cucumber). Effect [153]
concentrations 50 (EC50, i.e. 50% of exposed population was
affected) were 57 and 108 mg/kg for rice and cucumber
respectively. Inhibition of soil respiration and phosphatase
activity at concentration levels higher than 10 mg/kg.
Reduction in soil respiration 4 days after supplying the [154]

compound. The observed effects were dependent on the
adsorption of the compound onto the soil.
Bisphenol A (plasticizer) Shift in sex ratio to female individuals in isopod (soil [155]
arthropod) communities.
Abamectin (anthelminthic) Negative impacts on reproduction of Folsomia fimetaria and [156]

Folsomia candida (soil arthropods) at concentrations of 0.25
and 0.5 mg/kg of soil (dry mass), respectively. Negative
impacts on reproduction of soil earthworms at concentration
levels of 0.06 mg/kg of soil (dry mass).
Fenbendazole and cypermethrin Negative impacts on degrading microorganisms of dung. [157]

(antiparasitic)
Sulfonamide and tetracycline Inhibition of the soil microbial activity by 10% (ED10) at [158]
antibiotics concentrations of 0.003–7.35 µg/g of soil (dry mass). Shifts in
fungi:bacteria ratio.
Sulfadiazine (antibiotic) Decrease in denitrification rates when the input of antibiotic [159]
was 100 mg/kg of soil (dry mass).
Significant decrease in the bacteria:fungi ratio [160]
Chlortetracycline, tetracycline, tylosin, Decrease in crop growth (sweet oat, rice and cucumber). [161]
sulfamethoxazole, sulfamethazine and Inhibition of the microbial activity of soil (soil respiration and
trimethoprim (antibiotics) phosphatase enzyme activity).
Human and veterinary Decrease in growth and development of Phaseolus vulgaris L., [162]
pharmaceutically active substances Glycine max, Medicago sativa, Zea mays, and several other
crops.
Table 9. Summary of negative effects on soil organisms caused by the occurrence of pollutants of emerging concern
at trace levels
Occurrence of domestic wastewater–related organic pollutants in irrigated soils. In spite of

the fact that wastewater is the main vehicle allowing OPECs to reach soil, very few studies
reporting the presence of these pollutants in wastewater irrigated soils have been carried out.
30 Soil Pollution
This finds an explanation, on the one hand, in the inherent difficulty of extracting and isolating
organic compounds at trace levels from the soil matrix and, on the other hand, in the fact that
analyzing this type of pollutants is relatively expensive. Figure 5 shows the sites where
monitoring studies aimed at determining the occurrence of OPECs in wastewater irrigated
soils have been performed. In this figure the number of sites monitored is contrasted with the
20 countries with the highest use of untreated wastewater for agricultural irrigation. Most of
the monitoring studies are concentrated in China, the country using the highest volume of
untreated wastewater in agriculture [17], followed by the United States and Mexico –the latter
is the second placed country in terms of reuse of untreated wastewater for irrigation–.
Figure 5. Monitoring studies for pollutants of emerging concern in wastewater irrigated soils throughout the world
and comparison with the 20 countries using the largest volumes of raw wastewater for agricultural irrigation
Efforts in monitoring emerging pollutants in developing countries where the use of raw
wastewater is widespread are of value; this requires cooperation with research centers where
analytical techniques are currently validated to perform soil analyses or by sharing “know
how” and technology with developing countries in order to perform analysis on site. Deter‐
mination of OPECs in soil requires an exhaustive extraction step, which in most cases has to
be carried out at a moderately high temperature, particularly in the case of analysis of
thermolabile compounds (e.g. sulfonamide antibiotics). Extraction methods such as pressur‐
ized fluids extraction, microwave assisted extraction and ultrasonic assisted extraction are
preferred over traditional Soxhlet extraction techniques, since they guarantee greater contact
between the solvent and the soil particles, resulting in higher recoveries of analytes. Analysis
of OPECs is commonly accomplished using either liquid or gas chromatography techniques;
although liquid chromatography is preferred as it is more suitable for the analysis of polar
compounds, i.e. most PACs [164]. Monetary costs of these analyses are relatively high and
analysis entails the use of potentially dangerous chemicals, which is in part the reason why
monitoring studies for OPECs in soils are not carried out in poor countries. So far, the most
reported emerging pollutants in wastewater irrigated soils are the pharmaceutically active
substances (e.g. antibiotics, non–steroidal anti–inflammatory agents, anticonvulsants, antico‐
agulants and sex hormones), followed by plasticizers (e.g. phthalic acid esters and bisphenol
A), metabolites of surfactants (e.g. nonylphenol, octylphenol) and antibacterial and antimy‐
cotic agents (e.g. triclosan and triclocarban). Table 10 shows the concentrations of some OPECs
reported for wastewater irrigated areas.
Overall, higher concentrations of OPECs are found in the first 30 cm of the soil profile. Such
behavior suggests that these compounds are retained by the organic matter accumulated over
time of irrigation; which is consistent with the organic nature of these contaminants, although
several of them display some polarity. Concentration levels reported for the monitored PACs
range from below the detection limits of the analytical techniques to tens of µg/kg of soil (dry
mass). Monitoring studies referred to in [165] report concentration levels of the pharmaceuti‐
cals ibuprofen, naproxen and carbamazepine in the range of 0.25 to 6.48 µg/kg of soil (dry
weight) in Phaeozem and Leptosol soils that have been irrigated using untreated wastewater
for eight decades. Other studies [168] found average concentrations of 1.8 µg/kg for triclosan
and 2.5 µg/kg for estrone. In contrast to PACs, concentrations reported for plasticizers and
surfactants are of the order hundreds of µg/kg of soil (dry mass). For example, in the study
referenced in [170] concentrations of 14–80 µg/kg are reported for nonylphenols, while
concentrations of 140–2610 µg/kg were observed for some plasticizers. Concentrations of up
to 7110 µg/kg of the plasticizer di–2(ethylhexyl)phthalate, have been reported elsewhere [175].
High concentrations of plasticizers in soils are explained by the ubiquity of these compounds
in environment. Phthalic acid esters are contained in almost all plastic products and can easily
leach from the solid matrix (i.e. the plastic articles). Once phthalates are released from the solid
matrix, they can get into environment not only via wastewater but by aerial deposition, using
dust particles as carriers [176]. Nonylphenols, the major by–products of the anaerobic biode‐
gradation of surfactants [177], are commonly found in wastewater irrigated soils due to the
significant presence of detergents in municipal wastewater in combination with the anaerobic
conditions prevailing in sewerage systems. In contrast to PACs, plasticizers and surfactant
metabolites are non–polar in nature and for this reason, higher adsorption can occur for these
compounds in soil, causing not only their build up in the surface layer of soil but the potential
decrease in their bioavailability to soil microorganisms. Most monitoring studies of OPECs in
environmental solid matrices are focused on determining these contaminants in biosolids
amended soils rather than in wastewater irrigated soils. This is necessary since: a) biosolids in
wastewater treatment plants concentrate organic pollutants during water depuration, hence
a greater concentrations of contaminants are expected in biosolids amended soils than in
treated/untreated wastewater irrigated soils; b) the use of biosolids as agricultural soil
amendment is a more socially acceptable practice than reusing wastewater, thus it tends to be
more practiced (or at least more reported) than wastewater irrigation, and it therefore becomes
necessary to determine the pollutant load reaching the soil in this manner; c) since analysis of
32 Soil Pollution
Compound Concentration (µg/kg) Comments Reference

Carbamazepine 0.28–0.94 Concentration range observed in the surface layer (0–10 cm depth) of a [165–167]
treated wastewater irrigated soil during an irrigation cycle (May to October).
The lowest concentration was observed before irrigation started while the
highest concentration was determined in soil at the end of the irrigation cycle.
Irrigation at the site has been occurring for the last 30 years.
5.14 and 6.48 Concentrations found in the surface layer (0–10 cm depth) of Leptosol and
Phaeozem soils, respectively that has been irrigated using untreated
wastewater for 85 years.
4.92, 2.9 and 1.92 Concentrations found in forested, grass–covered and cultivated soil irrigated
with treated wastewater for more than 25 years. Carbamazepine was found
mainly in the first 30 cm of the soil profile.
Ibuprofen <LOD–3; <LOD–3 Concentration ranges observed in loamy sand and sandy loam turf soils (0–30 [168]
Naproxen <LOD–12.5; <LOD–9.5 cm depth) irrigated with treated wastewater at an irrigation rate of 1.1–1.2
Triclosan <LOD–6; <LOD–2.8 and 1.5–1.6–fold the evapotranspiration rate, respectively. Wastewater
Bisphenol A <LOD–1.25; <LOD–1 irrigation has been occurring at the site for almost 20 years.
Estrone <LOD; <LOD–5.3
Ibuprofen <LOD and 0.25 Concentrations found in the surface layer (0–10 cm depth) of Leptosol and [165]
Naproxen 0.73 and 0.55 Phaeozem soils that have been irrigated using untreated wastewater for 85
Nonylphenols 123 and 41 years.
Triclosan 18.6 and 4.4
Bisphenol A 14.8 and <LOD
Di–n–butylphthalate 552 and 244
Butylbenzylphthalate 346 and 171
Di–2–(etylhexyl)phthalate 2079 and 820
Clofibric acid <LOD–9 Concentration range observed in soil from a golf course irrigated with [169]
reclaimed wastewater
Triclocarban <LOD –105 Concentration ranges for pharmaceuticals and endocrine disrupting chemicals [170]
4–nonylphenol 14.2–60.3 in agricultural soils of Hebei province, north China, which have been irrigated
Salicylic acid 1.4–10.7 using treated wastewater for more than 50 years.
Tetracycline <LOQ –19.9
Oxytetracycline 1.1 –16, maximum 212
Trimethoprim <LOQ –2.6
Primidone <LOQ –3.3
Omeprazole 6.5–24.3 Ranges of concentration found in two agricultural soils irrigated with treated [171]
Spironolactone 0.6 wastewater in Spain. Pollutants were found at higher concentrations in the
surface layer of the soils.
Diazepam 4.65 Concentration found in an agricultural soil irrigated with treated wastewater. [172]
Carbamazepine 5.77 Pollutants were accumulated in the surface layer of the studied soil (0–30 cm).
Butylbenzylphthalate 59–1580 Ranges of concentration of phthalate esters in agricultural soils irrigated with [173]
Di–2–(etylhexyl)phthalate 107–29370 untreated wastewater in the peri–urban area of Guangzhou city
Di–n–butylphthalate 9–2740
Di–n–amylphthalate 1–80
Caffeine 14 Concentrations reported for volcanic soils (Vitric, Orthic, Allophanic soils) [174]
Amitriptyline <5 irrigated using treated wastewater for more than 15 years (at rates of 70 mm/
Carbamazepine 217 weak) in Rotorua, New Zealand.
Chlorpromazine <5
17α ethynilestradiol <5
Diltiazem <248
Thioridazine <259
<LOD: concentrations below the limit of detection of the analytical method used for determination.
<LOQ: below the limit of quantification of the analytical technique.
Table 10. Concentrations of organic pollutants of emerging concern in treated/untreated wastewater irrigated soils
OPECs in soil is expensive, these types of monitoring studies are conducted mainly in
developed countries, where the use of biosolids as soil amendment is practiced more inten‐
sively than the reuse of treated/untreated wastewater in agricultural irrigation. At present
there are no regulations that establish maximum permissible concentrations for organic
pollutants of emerging concern in soils. The development of such regulations relies on the
results obtained in both acute/chronic toxicity tests and in health risk assessments.
Environmental fate of organic pollutants of emerging concern (OPECs) in wastewater

irrigated soils. The environmental fate of OPECs in the soil is governed by the physical and
chemical properties of both the compounds and the soil as well as by the climatic conditions
of the site where reuse is taking place. The chemical properties of the organic pollutants
significantly impacting the environmental fate of OPECs are polarity, hydrophobicity and
volatility. Table 11 shows some OPECs found in municipal wastewater which serve as
examples of the differences in the chemical properties affecting the environmental fate of
OPECs in soil. Due to the organic nature of OPECs, soil organic matter, mainly its non–polar
fraction, plays a determinant role in the retention of these pollutants in soil [178]. However,
sorption onto soil organic matter does not occur equally for all contaminants, since polar
molecules tend to remain soluble in water rather than be retained in the soil organic matter;
conversely, non–polar molecules are instantaneously adsorbed by soil organic matter [179].
The polarity of organic compounds is determined by the presence of ionizable radicals within
the molecules; carboxyl, phenol, amine and amide moieties may gain or lose protons, depend‐
ing on soil pH values, acquiring a positive or negative charge, respectively. The compounds
for which functional groups lose protons may be poorly retained by soil due to repulsion forces
between the deprotonated radical and the negatively charged soil particles (i.e. organic matter
and clay); this results in the facilitated leaching of organic pollutants into the aquifer [180].
However, when functional groups within organic molecules gain positive charge, they may
be retained onto the soil particles by cation exchange –as occurs for some tetracycline antibi‐
otics– [181]. In both cases, the organic moiety within OPEC molecules may be held in the soil
organic domain by hydrophobic affinity. In general, the pH of wastewater irrigated soils tends
to be neutral to basic [81], which results in low retention of negatively charged compounds
compared to neutral or positively charged organic compounds [180]. Studies referenced in
[182] found that non–steroidal anti–inflammatory drugs (NSAIDs) such as naproxen, which
can produce negatively charged molecules after the ionization of the carboxyl functional
group, are adsorbed to a lower extent than other compounds displaying higher hydrophobic‐
ity, such as carbamazepine or triclosan, in organic soils with high clay content. Organic
compounds lacking of ionizable functional groups or displaying non–ionizable functional
groups express their hydrophobicity by spontaneously migrating from water to the soil
organic domain [183]. In wastewater irrigation systems, dissolved and particulate organic
matter contained in wastewater tends to accumulate in the surface soil horizons, significantly
favoring the build up of these compounds in topsoil. In studies referred to in [184] a greater
accumulation of hydrophobic compounds, such as carbamazepine and esters of phthalic acid,
was found in surface horizons of the irrigated soils, whereas hydrophilic compounds, namely
ibuprofen, naproxen and diclofenac, were found in subsurface horizons. This behavior is
explained, on the one hand, because hydrophilic compounds remain dissolved in water rather
34 Soil Pollution
Polarity (ionization state at commonly found soil pH values)

Positive Negative Positive/Negative (zwitterions)
Erythromycin (antibiotic) Naproxen (non–steroidal analgesic drug) Ofloxacin (antibiotic)
pKa 8.91 pKa 4.15 pKa 6.27 (COOH); pKa 8.87 ( NH2 +)
Hydrophobicity
Hydrophobic Hydrophilic
Di–2–(ethylhexyl)phthalate (plasticizer) Ciprofloxacin hydrochloride (antibiotic)
pKow 7.5 pKow – 0.82

Solubility in water at 25°C: 4.1x10–2 g/L Solubility in water at 25°C: 30 g/L
Volatility
Volatile Non–volatile
Galaxolide (fragrance used in detergents) Bisphenol A (plasticizer precursor)
Vapor pressure: 7.27x10–2 Pa Vapor pressure: 9.33x10–6 Pa
Table 11. Relevant physical and chemical properties in terms of the environmental fate of emerging pollutants in soil
than being retained in soil and, on the other hand, because of hydrophilic compounds are more
susceptible to desorption from soil either during further irrigation or heavy rain events, and
thus tend to rapidly reach subsoil and the aquifer [180, 182].
The chemical structure also affects the environmental fate of OPECs in soil. Molecules
displaying aromatic moieties, such as carbamazepine and naproxen, have been shown to be
strongly retained by soil organic matter –both to the aliphatic and aromatic fractions of soil
organic matter– compared with compounds that have no resonance structures [182, 185–186].
This behavior is explained by the formation of bonds between aromatic rings within the solute
molecules and the soil organic matter [185]. Nonylphenols and octylphenol compounds have
surfactant properties as they possess an aliphatic chain and a phenol moiety at the edge of the
molecule [177]. Due to this structure, these compounds can promote resolubilization of organic
contaminants retained in soil, although the estimated risk of this occurrence is considerably
low [187]. The presence of heteroatoms in organic molecules can impact upon their environ‐
mental fate in soil. For example, oxygen atoms within the ciprofloxacin molecule can form
covalent bonds with aluminum and iron oxides in soil, resulting in irreversible adsorption of
the compound onto the solid matrix [188]. With respect to volatile OPECs, artificial fragrances
represent the best example of this feature; these compounds are contained both in personal
care products and detergents. Typically, the more volatile compounds are also hydrophobic,
so they can be spontaneously retained in topsoil and then volatilize when temperature
increases [189]. Since irrigation using untreated wastewater, which contains large amounts of
fragrances, is carried out in arid areas, it is expected that a significant fraction or all of the
fragrance molecules are rapidly volatilized upon their input to soil via wastewater. Volatili‐
zation of OPECs in wastewater irrigated soils is still an unexplored issue; studies aimed at
determining the fraction of organic contaminants that can be volatilized in soil enriched with
organic matter via wastewater irrigation are still needed.
Natural attenuation processes leading to the removal and dissipation of OPECs in soil are
shown in Figure 6. Contaminants may either dissipate in soil by photodegradation, biodegra‐
dation or chemical degradation (hydrolysis, oxidation or reduction) mechanisms; they may be
accumulated in soil by adsorption or removed from soil by volatilization. There is a significant
lack of information in the literature with regard to the natural photodegradation (i.e. photolysis
of compounds by sunlight) of emerging pollutants in soil. The information available on the
photodegradation of pesticides in agricultural soils is useful in elucidating, to some extent, the
potential for photodegradation of OPECs in soil. Studies on natural photodegradation of the
pesticide quinalphos showed that photodegradation takes place only in the first 2–5 mm of
soil (photic layer); this photolysis takes place in two stages, each one at a different depth [190].
In the uppermost soil layer (the first 2 mm) direct photodegradation of the organic compounds
(i.e. the transformation of compounds due to the direct incidence of photons) occurs; in this
same layer, the production of free radicals (e.g. hydroxyl radicals and excited dissolved organic
matter) occurs due to the breakdown of the soil organic matter. In the second stage, the free
radicals migrate to the lower photic layer through facilitated transport by soil moisture;
subsequently, photolytic transformation occurs below by the action of free radicals generated
in the upper layer of soil (indirect photodegradation) [191]. As a result of the aforementioned
aspects, soil moisture content as well as organic matter are determinant factors in the photo‐
degradation of organic contaminants retained in the soil surface [191]. The physical structure
of the soil can also significantly impact upon the photodegradation of organic pollutants, as it
defines the depth to which solar radiation can penetrate the soil. In the study referenced in
[192], sunlight photolysis of 4–nonylphenol in biosolids amended soils was studied. Photolysis
resulted in 40% conversion of the compound within 30 days, with photodegradation observed
36 Soil Pollution
in the first 5 mm of the soil. Since natural photodegradation occurs only in the soil surface
layers, the organic compounds retained in the topsoil will be the most exposed to direct
sunlight, although this does not necessarily imply increased rates of photodegradation. An
example of this is the anticonvulsant agent carbamazepine, which is prominently retained in
topsoil but has demonstrated poor photodegradation in water studies. Conversely, the anti–
inflammatory drug diclofenac has been shown to present significant photoactivity [193], but
it is less well retained in the topsoil. Due to this, photodegradation is unlikely to occur in soil
by direct or indirect means. In spite of almost all of the studies evaluating the natural photo‐
degradation of OPECs have been carried out in aqueous matrices [194], the results obtained
in these experiments provide valuable information concerning the photoactivity of such
compounds; which can be useful for studying the photolysis of organic pollutants in soil. For
example, it is known that the NSAID ibuprofen and the anticonvulsants drugs carbamazepine
and primidone are poorly photodegraded in water whereas the antibacterial agent triclosan,
the antibiotic drug sulfamethoxazole and the NSAIDs diclofenac and naproxen are readily
photodegraded [194]. These results can be the basis to establish experiments aimed at deter‐
mining or modeling the photodegradation of organics in soil. In general terms, the natural
sunlight reaching the troposphere (i.e. the surface of earth) does not possess enough energy
to mineralize most photodegradable compounds [195]; therefore a wide variety of by–products
occurs when organic contaminants in water and soil are photodegraded. It is known that, in
some cases, more harmful compounds can be produced by photodegradation of some organic
pollutants. For example, 2,8–dichlorodibenzo–p–dioxin is produced by the natural photode‐
gradation of the antibacterial agent triclosan [196]. Differently to triclosan, its breakdown
product has the potential to cause cancer in mammals. Another example is the antiepileptic
drug carbamazepine, which photodegrades to acridone [197], a compound related to the
occurrence of cancer in aquatic species.
Most photodegradation studies of OPECs (in water matrices) have been carried out in
developed countries at latitudes higher than 30°N [194]. It is therefore necessary to investigate
the intensity of photodegradation processes occurring at lower latitudes, in zones where higher
incidence of sunlight occurs and treated/ untreated wastewater irrigation is more intensively
practiced.
Biodegradation of OPECs in soil has been studied in greater detail than photodegradation.
Laboratory studies have found that biodegradation of emerging pollutants occurs optimally
under aerobic conditions, while negligible transformations have been observed under
anaerobic conditions [198]. This implies that biodegradation of this kind of contaminants is
more likely to occur in well–structured soils, where tillage activities are frequently carried out,
which allows better gas exchange through the soil matrix. The opposite behavior may be
observed in anoxic/anaerobic soils, for instance in paddy fields. The antiepileptic drug
carbamazepine is reported as one of the most refractory organic pollutants in soil, which has
led researchers to consider this antiepileptic agent as a marker for anthropogenic contamina‐
tion of surface and groundwater bodies [199]. In the study referenced in [200], mineralization
of carbamazepine in soil was found to be less than 2%, after 120 days of incubation under
aerobic conditions, while the reported in [201] show half–life times of 472 days in aerobic
Figure 6. Processes involved in the environmental fate of emerging pollutants in soil
biosolids amended soils. Other compounds listed as recalcitrant in soil are the X–ray contrast
media iopaminol, iomeprol and iohexol, whose biodegradation kinetic rate constants range
from 0.29 to 0.46 µM/day [202]. Pharmaceuticals, such as the antiepileptic drug primidone and
the psychoactive diazepam have shown recalcitrance in water [203]; further studies are
necessary in order to elucidate whether such behavior may also occur in soil. Substances
designed to exert an effect on microorganisms have been shown to be rapidly biodegraded in
soil. Examples of these are antibacterial agents such as triclosan, triclocarban and antibiotic
substances [204–205]. Triclosan and triclocarban have been shown to be biodegraded in aerobic
soils after 18 and 108 days, respectively [198], whereas the antibiotic compounds erythromycin,
oleandomycin, tylosin, tiamulin and salinomycin displayed half–lives in aerobic soil of 20, 27,
8, 16 and 5 days, respectively [205]. Endocrine disrupting compounds, such as phthalate esters
have been shown to efficiently biodegrade in agricultural soils, displaying half–lives of 7.8 to
8.3 days for di-butyl phthalate and 26–30 days for di–2–(ethylhexyl)phthalate [206]. Currently,
few soil microorganisms have been identified as degraders of emerging pollutants. For
example, the fungi Trametes versicolor has been demonstrated to degrade naproxen [207], while
Rhodococcus rhodochorus bacteria [208] have been shown to degrade carbamazepine down to
levels of 15% of its initial concentration in soil. In the case of phthalates (plasticizers) bacteria
belonging to groups of Corynebacterium, Mycobacterium and Nocardia were demonstrated to
degrade up to 90% of di–butyl phthalate within 48 hours in biodegradation experiments using
isolated bacteria cultivated in saline solution [209]. Knowing the species of microorganisms
that perform the biodegradation of OPECs in soil is useful in order to design engineered
systems to treat wastewater and polluted soils based on the increased ability of degraders to
degrade specific compounds by acclimatization and bioaugmentation. Such systems were
38 Soil Pollution
tested in [210] using the fungus Trametes versicolor to degrade up to 94% of carbamazepine in
wastewater after 6 days in an air pulsed bed bioreactor. Biodegradation of OPECs in soil is
influenced by the sorption phenomenon, therefore soil characteristics such as the content of
organic matter, soil texture and soil pH are crucial for this process to occur. Adsorption of the
organic contaminants onto the surface of the soil particles may favor biodegradation when the
sorbed compounds are still bioavailable; conversely, when strong adsorption occurs (chemi‐
sorption on soil organic matter, clay or soil micropores) it can result in decreased bioavailability
of the compounds and thus in the confinement of the pollutants within the soil matrix. Other
properties of soil involved in the biodegradation of OPECs are: a) the climatic conditions of
the site; b) the physical structure of soil; c) the soil moisture; and, d) the adaptation of soil
organisms to biodegrade the target pollutants. It is possible that microorganisms in long–term
wastewater irrigated soils more efficiently biodegrade OPECs than those living in non–
irrigated soils or soils irrigated for a shorter time. This is due to the ability of soil microorgan‐
isms to adapt to using emerging pollutants as a carbon source. In this sense, studies comparing
the degradation efficiency of OPECs in long–term wastewater irrigated soils with that
observed in non–irrigated soils or newly irrigated soils are needed in order to establish
appropriate strategies to prevent contamination of groundwater. Very few efforts have been
made to determine the nature and quantity of by–products generated in soil by the biodegra‐
dation of OPECs. As shown in [200], biodegradation of emerging pollutants can generate by–
products that can be more harmful than the original substance [196–197], thus the presence of
these by–products as well as their environmental fate should be priority for further research.
Those emerging pollutants that are not degraded by soil microorganisms may either accumu‐
late in soil, be assimilated by plants (if they are bioavailable) or be degraded by other mecha‐
nisms (e.g. photodegradation or hydrolysis). In the case of carbamazepine, studies referred to
in [184] explain that this compound is one of the most highly accumulated in wastewater
irrigated soils. Moreover, carbamazepine can be assimilated by plants in wastewater irrigation
systems at environmentally relevant concentrations (i.e. within the range 1–3 µg/L). The study
referenced in [211] shows that cucumber (Cucumis sativus L.) can accumulate carbamazepine
in different parts of the plant: 4.5 µg/kg in roots, 1.9 µg/kg in stem, 39.9 µg/kg in leaves and
2.1 µg/kg in fruit. According to the authors, phytotoxic effects were observed when carbama‐
zepine was supplied to soil by irrigation at concentrations as high as 10,000 µg/L. Results of
this study show that consumption of carbamazepine polluted cucumber results in doses of 1
ng of carbamazepine per gram of fruit. Other studies show that soybean (Glycine max L.) can
take up carbamazepine, triclosan and triclocarban in roots, stems and leaves at concentrations
of 1.3–3.4 µg/kg for carbamazepine and 2.4–13.7 µg/kg for the antibacterial agents triclosan
and triclocarban. Concentrations of antibacterial agents in plants at a second harvesting were
found to be higher than those obtained in the first one; this may be due to the accumulation
of contaminants in the soil, as a bioavailable pool, between each irrigation events [212]. To
date, the study of the assimilation of OPECs by plants in wastewater irrigated soils is still
limited; moreover, priority should be given to develop health risk assessment studies related
to the consumption of contaminated crops.
Adsorption (i.e. retention of solutes on the surface of the soil particles) of OPECs in the soil is
a decisive process in their environmental fate, since through this process contaminants may
either be retained or migrate into the aquifer. In cases where organic pollutants are retained
in topsoil, photodegradation or volatilization phenomena can easily take place. The strength
of the bonds that pollutants establish with soil particles determines the bioavailability of
molecules to plants and soil microorganisms. Adsorption of pollutants onto soil is measured
by the distribution coefficient (Kd) which relates the amount of compound retained by soil to
the mass remaining in the liquid phase [213]. Several models to determine the distribution
coefficient of organic compounds have been developed; such models vary in complexity and
the accuracy with which they represent the field conditions; yet simple adsorption models
such as linear, Langmuir and Freundlich are the most used [213]. Due to their organic nature,
OPECs tend to be rapidly and strongly adsorbed by soil organic matter; due to this effect, non–
polar emerging pollutants, such as phthalates, have been shown to instantly adsorb onto
organic soils [214]. On the other hand, OPECs displaying negative charge at the soil pH values,
as occurs for NSAIDs, exhibit less adsorption by soil due to the repulsive forces between the
negatively charged moiety within the molecule and the soil particles displaying negative
charges (i.e. organic matter and clay) [182]. Accumulation of organic matter in wastewater
irrigated soils increases the soil's ability to adsorb organic compounds. The proof of this can
be found in the study referenced in [96], which reports greater adsorption of the antibiotics
sulfamethoxazole and ciprofloxacin in long–term wastewater irrigated soils compared to non–
irrigated ones from the same area. In addition to soil organic matter, OPECs may be retained
by the inorganic domain of soil; for instance, ciprofloxacin showed strong and instantaneous
adsorption by iron oxides and clay in agricultural soils, which was achieved by the formation
of covalent bonds between metals in the soil and the oxygen atoms within ciprofloxacin
molecules [188]. Furthermore, adsorption of carbamazepine by smectite type clays has been
reported by [215]. According to studies referred to in [182, 185, 215], the adsorption of OPECs
with multiple aromatic rings is more efficient in soils displaying a high content of humified
organic matter –which displays higher aromaticity than labile organic matter–. Polyaromatic
compounds can establish π–π bonds between the aromatic rings within the pollutant mole‐
cules and aromatic compounds contained in soil organic matter. The formation of such bonds
should be studied in future research in order to determine the optimum chemical character‐
istics of soil organic matter which enable better retention of contaminants, hence preventing
their mobilization into the aquifer and/or making them available for uptake by plants. OPECs
may be adsorbed by dissolved organic matter to soil via wastewater. Adsorption of organic
pollutants to dissolved organic matter increases the solubility of the compounds and hence
facilitates the lixiviation through soil. Studies referenced in [216–217] report that compounds
such as naproxen, carbamazepine and sex hormones can be adsorbed onto dissolved organic
matter, notably to the hydrophobic and neutral hydrophilic fractions of dissolved organic
matter. The speed of formation and strength of bonds between organic compounds and the
dissolved organic matter varies depending on the quality of both wastewater and dissolved
organic matter in soil [217]. The continuous occurrence of OPECs in wastewater irrigated soils
can impact upon the adsorption of other organic pollutants; this is because at the time emerging
pollutants enter to soil via wastewater, some of the active adsorption sites in soil are still
40 Soil Pollution
occupied by previously adsorbed pollutants. In the study referred to in [182], the distribution
coefficients of three OPECs, namely naproxen, carbamazepine and triclosan, were determined
by an adsorption model which takes into account the previous presence of organic pollutants
in the soil (the initial mass model [218]). The authors found modest differences between the
values obtained in their study and those reported in the literature. However, it was observed
that compounds previously adsorbed onto soil, i.e. naproxen and carbamazepine, were
released from the solid matrix each time wastewater “washes” the soil in each irrigation event,
resulting in a risk of contamination of the aquifer.
The transportation of OPECs through soil is closely related to their adsorption onto the solid
matrix. Transport studies can be performed using different approaches, either packed soil
columns or undisturbed soil columns tests. Transport of OPECs and pathogens is better
described using the undisturbed soil column approach; through this approach, it is possible
to evaluate the impact of both physical and chemical properties of soil on the transport of
pollutants. In transport assays using undisturbed soil columns it is possible to assess the impact
of preferential paths on the transport of solutes and particles, at the same time determining
the effect of chemical properties of soil in the retention of solutes under dynamic flow
conditions. The type of clays in soil significantly impacts on the transport of organic pollutants.
The presence of expansive clays in soil results in the disappearance of preferential paths in the
porous network of soil once clay becomes wet, which in turn provokes the decay in transport
of contaminants contained in water. However, in such cases, soil conditions become anaerobic
and thus organic pollutants are biodegraded with difficulty. The understanding of the
environmental fate of OPECs in wastewater irrigated soil still has many gaps. It is therefore
important to carry out studies on the laboratory scale and then in the field (plot level or
landscape level) in order to determine the fate of these substances under real conditions.
Results of these studies are of great importance, on the one hand, to allow more accurate and
useful risk assessment studies and, on the other hand, to determine the characteristics of the
sites suitable for irrigation with treated/untreated wastewater without posing a risk to the
health of organisms and to the quality of crops and water sources. Lastly, regulations for
OPECs in soil should be established in order to set maximum concentration limits for the
accumulation of these compounds in terms not only of the effects caused to soil organisms,
but also their potential to reach groundwater.
3. Perspectives for further studies
Reuse of wastewater in agricultural irrigation is a complex issue that requires the development
of numerous studies in different disciplines; in this section some perspectives for further
studies are presented.
1. Long–term studies aimed at determining the improvement of soil properties to produce
food. Such studies should compare the rate of entry and conversion of carbon, nitrogen
and phosphorus in irrigated soils in order to obtain a mass balance showing either the
sustainability or the accumulation of organic matter in wastewater irrigated soils.
Moreover, studies demonstrating the long–term increase in the soil's ability to treat
wastewater used for irrigation should be carried out for each of the properties addressed
in this chapter, as well as those considered appropriate in each system.
2. The determination of OPECs and pathogens in soils irrigated with wastewater. Such
monitoring studies can be used to establish an inventory of contaminated sites that reflects
the level of pollution in developed and developing countries. This can help in proposing
ad hoc solutions for each site.
3. Determining feasibility and the mechanisms that can lead to horizontal propagation of
antibiotic resistance genes in soil microorganisms (either innocuous microorganisms or
opportunistic pathogens).
4. Chronic toxicity studies of OPECs in wastewater irrigated soils covering either several
crop cycles, several generations of organisms or several years. Toxicity studies should
address the effects of the presence of mixed contaminants at trace levels (environmentally
relevant concentrations) on soil organisms. Such studies should be conducted including
new emerging contaminants, e.g. nanoparticles.
5. The study of the environmental fate of emerging contaminants using different model
molecules in soil. Such environmental fate studies should be carried out at laboratory and
field scale. In the case of environmental fate studies at laboratory scale, conditions used
should be those that best emulate field conditions, e.g. sunlight lamp intensities similar
to those observed in the field for testing photodegradation or undisturbed soil columns
in transport assays through soil.
6. The determination and quantification of the by–products appearing in soil upon dissipa‐
tion of OPEC. Harmful compounds such as dioxins, chlorophenols and polyaromatics
may be produced in soil from substances such as triclosan and carbamazepine. Discerning
the occurrence and fate of these substances in soil should be addressed in future studies
7. Determination of the environmental fate of organic, inorganic and microbial contaminants

in agricultural soil remaining after irrigation with wastewater has ceased. Worldwide,
notably in developed countries, there are several sites where irrigation with wastewater
has been stopped after a considerable time; in such cases, it is necessary to know the fate
of the pool of pollutants that accumulated in soil during continuous input via wastewater.
Phenomena such as the release of heavy metals confined in soil organic matter can occur
when soil organisms start to mineralize organic carbon accumulated in the soil. In
addition, the soil microorganisms can lose the capacity to treat pollutants in wastewater,
leaving the soil vulnerable in cases where wastewater irrigation is restarted.
8. Studies elucidating the conditioning methods for agricultural soils newly irrigated with
wastewater. Since in arid regions a considerable increase in the area under irrigation is
being observed, it is necessary to use current knowledge to implement regulations
establishing the optimal conditions for soils candidate to receive treated/untreated
wastewater. These are necessary to prevent soil degradation and contamination of water
sources in the irrigated area.
42 Soil Pollution
9. Studies on the migration of contaminants in soil due to extreme events caused by climate
change. Extreme rainfall events can cause an incremental increase in the mobilization of
organic contaminants retained in the surface layers of soil into aquifers or to non–irrigated
soils affected by runoff. However, increases in temperature can decrease the biodegrada‐
tion of organic pollutants in the soil due to excessive drying of the solid matrix.
10. The development and implementation of wastewater treatment systems to remove

organic, inorganic and biological pollution without reducing the content of organic matter
in the water. These systems must be inexpensive for dissemination in developing
countries. Advanced primary treatment systems may represent a plausible strategy in
such cases.
11. The development and validation of environmentally–friendly analytical techniques for

the determination of OPECs in soils..
4. Conclusions
The reuse of treated/untreated municipal wastewater for agricultural irrigation definitely has
positive impacts on soil as a medium for the development of plants and animals; additionally,
this practice results in positive impacts on the welfare of farmers due to the monetary savings
and profits that they obtain by the use of wastewater as a fertilizer and water source for crops.
Similarly, the soil's ability to self–cleanse and treat the wastewater supplied at each irrigation
event increases with the reuse of wastewater. The accumulation of organic matter in the soil
surface results in changes in soil pH to neutral and basic values, an improvement of soil
physical structure and an increase in the soil microbial activity. Together with this, soil
organisms become acclimatized to the presence of contaminants and thus their resilience to
the harmful effects caused by pollutants increase. These phenomena lead to an improvement
in the ability of the soil to act as a filter and transforming medium for contaminants and thereby
to an increase in its capacity to treat wastewater. Such an improvement in soil functions can
be capitalized by the State and the conventional treatment regime can be changed to a cheaper
one driven by natural attenuation mechanisms. This in turn improves the quality of life of
people living in the area by increasing food production and the possibility of obtaining profit
by sales of produce. The responsible reuse of municipal wastewater for agricultural irrigation
can help to mitigate three problems which are a priority in developing countries: a) water stress
in arid areas where rain–fed agriculture makes development uncertain. In such areas fresh‐
water sources are used for agriculture rather than human consumption, and therefore the reuse
of municipal wastewater not only results in savings of freshwater but also in the recharge of
the aquifer in the irrigated area. Recharge is with good quality water produced by infiltration
of wastewater through the soil; b) the food crisis and the lack of jobs in rural and peri–urban
areas in developing countries. Reuse of wastewater represents a way of producing food for
consumption and sale; and, c) the treatment of municipal wastewater generated in urban and
rural areas through a low cost natural treatment systems which in turn generate profits for
population.
In order to reuse wastewater responsibly and exploit its inherent benefits for soil and people
living in the irrigated area, the occurrence of contaminants in wastewater –especially untreated
wastewater– must be kept in mind. The presence of pathogenic microorganisms and the
potential for antibiotic resistance dissipation via wastewater should be priority concerns in
designing wastewater reuse schemes in agricultural areas, notably when using raw wastewa‐
ter. Attention should be paid to the fate of emerging contaminants in wastewater irrigation
schemes including its transportation through irrigation canals, storage in dams and deposition
in agricultural soils and transport to aquifers. Another priority is the elucidation of the chronic
toxic effects caused by the continuous presence of traces of emerging contaminants in irrigated
soils. Since the group of OPECs is quite broad, model compounds should be selected to
determine the rate at which they are dissipated or retained/transported through soil, as well
the risk of these compounds reaching the aquifer or being assimilated by plants. Despite the
spread of antibiotic resistance in the environment it has not been conclusively shown the role
that irrigation with treated/untreated wastewater plays in this. To date, the concentrations of
OPECs found in soil irrigated with wastewater are lower than the toxicity thresholds reported
in literature. The precautionary principle states that wastewater must be minimally treated
before irrigation in order to remove pathogenic microorganisms and trace of heavy metals, as
well as to reduce as much as possible the concentration of emerging pollutants. Other areas of
opportunity to be developed in order to reduce the risk of soil degradation and effects on soil
organisms are: a) the development of environmentally friendly everyday–consumer products,
containing organic compounds that have been proven to have no harmful effects on living
organisms even at trace concentrations. Consumer products must follow strict risk assessments
before release to the market; b) an improvement in health systems in cities in order to reduce
the incidence of infectious diseases that ultimately generate biological contamination of soil,
especially in irrigation systems using raw wastewater; c) the maintenance of wastewater
irrigation schemes fed with municipal wastewater in order to avoid a high input of heavy
metals and refractory organic compounds to soil and crops through irrigation; and, d) the ad
hoc treatment of municipal wastewater to allow its reuse in agricultural activities. Low cost
treatment systems aimed at removing microorganisms, suspended solids and trace heavy
metals are recommended to treat wastewater without affecting its properties as a fertilizer and
source of organic matter to improve physical, chemical and microbiological soil properties.
Such an approach allows soil to fulfill its ecological functions as a generator of food and
livelihoods and as a protective barrier to the aquifer.
Author details
Juan C. Durán–Álvarez1 and Blanca Jiménez–Cisneros2
1 Centre of Applied Science and Technological Development, National Autonomous Uni‐

versity of Mexico, Mexico D.F., Mexico
2 International Hydrological Programme (IHP), UNESCO, Paris, France

44 Soil Pollution
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chapter 6
The Complex Nature of Pollution in the Capping Soils of

Closed Landfills: Case Study in a Mediterranean Setting
Jesús Pastor, María Jesús Gutiérrez-Ginés,

Carmen Bartolomé and Ana Jesús Hernández
1. Introduction
1.1. Waste landfills capped with soil
In most developed countries there is serious concern about the state of waste landfills that were
closed towards the end of the past century. Economic growth and urban development during
this period generated vast amounts of domestic and industrial waste, and this waste was
deposited in landfills without its separation or prior treatment. Today, countries with emerg‐
ing economies or countries in settings of poverty are facing a similar situation, whereby the
uncontrolled disposal of waste has led to regions with worryingly high levels of pollutants
that affect the atmosphere, soil and water resources.
In the Mediterranean setting, most landfills have been sealed simply by capping with soil from
the surroundings. This soil has given rise to a plant cover emerging from the existing seed
bank. Besides recovering the visual impacts on the landscape of mountains of rubbish, a plant
cover will avoid the spread of pollutants to other ecosystems once the landfill has been closed.
However, the implantation of such a cover is conditioned by interactions among several
factors, which are responsible for the complex nature of soil pollution affecting closed landfills.
In the literature, there is a lack of work related to pollution of the capping soils of closed
landfills. Bibliography concerning landfills is mostly focused on leachates and their effect on
water. In case of soil-related works, they were not accomplished in Mediterranean environ‐
ment. Specially, quantitative data of landfill soil pollution and its possible effects on colonizing
plant population is not available.
2 Soil Pollution
For all these reasons, the purposes of this chapter are: i) to describe the profile of solid waste
landfills sealed with soil in the Mediterranean setting; ii) to focus on the study of a given case
in order to present a research methodology that can be used in other scenarios with a similar
problem. In the sections detailing this case study, we describe the methods and techniques
employed for studying landfill’s remediation and discuss the data obtained to give an
overview of the topic examined.
2. Profile of solid waste landfills in the Mediterranean region
Numerous waste tips in the central Iberian Peninsula capped with soil over the 1980s and 90s
have been widely described from an interdisciplinary perspective in [1]. Research efforts have
focused on the pollutants present in the soils used to seal 20 of these landfills and on factors
inducing the spread of pollutants. These studies have been aimed at designing measures to
remediate the visual impacts of solid waste landfills (Figure 1).
Figure 1. Based on [7]
The figure shows the main impacts (not only visual) produced by a landfill sealed with soil.
The most important impact is pollution produced by surface and deep leachates of polluting
The Complex Nature of Pollution in the Capping Soils of Closed Landfills: Case Study in a Mediterranean Setting 3
substances generated by surface run off and rainwater infiltration [2-6]. This type of pollution
especially affects ecosystems in the main areas of leachates discharge i.e., the foots of landfill
slopes grazed by domestic and wild animals [5]. Effects are nevertheless also produced on
crops, particularly cereals such as barley, which are sometimes grown on the landfill itself
(usually on its platforms). We have noted that this cereal accumulates heavy metals (unpub‐
lished data). In addition, soil pollution may spread to nearby rivers, on which these mountains
of waste seem to hang, or to streams, which transport pollutants to areas beyond the landfill.
Table 1 summarizes the main plant communities detected at the 20 landfill sites examined,
along with a summary of their main characteristics according to [8]. Despite the 20 years passed
since the landfills were closed, plant cover generally lacks a bush stratum. Existing commun‐
ities are those classified as ruderals and nitrophiles with a dominance of annual species whose
life cycle is typical of the Mediterranean region. Many of these landfills still show large
expanses of soil unable to sustain plant growth while other areas boast good plant cover,
though with a low diversity. In general, all landfill sites are grazed by itinerant herds of sheep.
Phytosociological class Main characteristics
9. ISOETO-NANOJUNCETEA Br.-Bl. & Tüxen ex Pionner annual and dwarf perennial ephemeral isoetid
Westhoff, Dijk & Passchier 1946 communities on periodically flooded bare soils
12. PHRAGMITO-MAGNOCARICETAE Klika in Klika Swampy, fenny, lacustrine and riverine helophyte communities
& V. Novák 1941 dominated by perennial graminoids, sedges, forbs and herbs of
fresh and brackish waters
20. JUNCETEA MARITIMI Br.-Bl. in Br.-Bl. Roussine & Perennial grasslands growing on coastal and inland temporary
Nègre 1952 wet or inundated salt marshes
34. ARTEMISIETEA VULGARIS Lohmeyer, Preising & Perennial and tall biennial forbs, grasses and thistle pioneer
Tuxen ex von Rochow 1951 ruderal and nitrophilous sunny communities growing on rich soils.
37. PEGANO-SALSOLETEA Br.-Bl. & O. Bolós 1958 Nitrophilous or halo-nitrophilous dwarf scrub communities,
including anthropogenic alloctonous shrubby vegetation
38. POLYGONO-POETEA ANNUAE Rivas Martinez Annual pioneer ephemeral and exceptionally small creeping
1975 perennial nitrophilous anthropozoogenic heavy trodden
communities of urban and rural paths
39. STELLARIETEA MEDIAE Tüxen, Lohmeyer & Annual ephemeral weeds, ruderal, nitrophilous and semi-
Preising ex von Rochow 1951. nitrophilous communities
39A. Stellarienea mediae -Cultivated field weed communities
39B. Chenopodio-Stellarienea Rivas Goday 1956 -Ruderal, nitrophilous and seminitrophilous communities.
39e. Thero-Brometalia (Rivas Goday&Rivas Martinez -Subnitrophilous Mediterranean annual ephemeral grassland-like
ex Esteve 1973) O. Bolós 1975 spring blooming communities.
39f. Sisymbrietalia officinalis J. Tüxen in Lohmeyer & - Nitrophilous and temperate annual ephemeral grassland-like.
al. 1962 em. Rivas-Martinez, Fernándz-González & Path, roadside and rural often trampled communities
Loidi 1991
4 Soil Pollution
Phytosociological class Main characteristics
40. GALIO-URTICETEA Passarge ex Kopecký Perennial hemycriptophyte and climbing tall herbs of nitrified
wood fringes and other semi-shaded anthropogenic biotope
communities.
41. CARDAMINO HIRSUTAE-GERANIETEA PURPUREI Annual spring and summer ephemeral internal and external shrub
(Rivas-Martínez, Fernández-González & Loidi 1999) fringes slightly nitrified semi-shaded communities, growing on
Rivas-Martínez, Fernández-González & Loidi classis rich organic nutrient soils.
nova, stat. Nov.
43. TRIFOLIO-GERANIETEA Müller 1962 Semi-shaded perennial herb communities of scarce moisture
external fringe woodlands. Calcareous or mesoeutrophic rich soils
in temperate submediterranean central Iberian territories.
50. TUBERARIETEA GUTTATAE (Br.-Bl. in Br.-Bl., Therophytic grasslands. Pioneer spring and early summer
Roussine & Nègre 1952) Rivas Goday & Rivas ephemeral plant acidophilous or calcifugous communities,
Martínez 1963 nom. mut. Propos. dominated by non nitrophilous annual short herbs and grasses,
but localized only in dry or initial soils, mostly in
submediterranean or step territories.
51. FESTUCO-BROMETEA Br.-Bl. & Tüxen ex Br.-Bl. Perennial xerophytic and mesophytic grasslands. Anthropogenic
1949 grazed baso-neutrophilous or slightly acidophilous mesophytic or
slightly xerophytic nutrient rich-pastures largely covered by
perennial grasses.
54. POETEA BULBOSAE Rivas Goday & Rivas Western Mediterranean oceanic thermo- to supramediterranean
Martínez in Rivas-Martinez 1978 upper semiarid to humid pastures, grazed and manured,
dominated by dwarf perennial grasses and other nutritious
prostrate chamaephytes…..
56. LYGEO-STIPETEA Rivas-Martinez 1978 nom. Mediterranean perennial basophilous xerophytic tall bunchy
Conserv. Propos. dense or short open grasslands.
57. STIPO GIGANTAE-AGROSTIETEA CASTELLANAE Silicicolous perennial grasslands rich in endemics, serial of Quercus
Rivas-Martínez, Fernández-González & Loidi 1999 rotundifolia and other Quercus natural potential forest
communities.
59. MOLINIO-ARRHENATHERETEA Tüxen 1937 Mesophile to wet often manured meadows and pasture
communities on deep and moist soils, widely spread by grazed
and anthropic activities
Table 1. Phytosociological classes and mean characteristics of the main species found at the landfills.
Most of the capped landfills are mixed dumps containing both domestic and industrial waste.
Besides mitigating the visual impacts of a landfill, the plant cover prevents its collapse and the
pollution of other ecosystems by deposited waste materials.
However, in such scenarios the stability of plant communities that become established from
the seed bank of the capping soil layer is threatened. Among others, the factors that give rise
to this situation are continued waste disposal after the landfill’s initial sealing, the scarce
volume of capping soil present and land use projects implemented without a priori planning.
3. Case study: The Getafe landfill (Madrid)
3.1. Geomorphological characterization
Here we examine the case of a closed landfill in the Madrid Autonomous Community. This
site can be described as one of the most complex scenarios observed among the soil-capped
solid waste landfills of the central Iberian Peninsula despite its many features common to all
the landfills examined in this region [1]. Located in the municipal district of Getafe (Madrid),
this landfill was first described by [9], when it occupied an area of around 70,000 m3. Fifteen
years later (in 2009), the site covered some 95,000 m2 of land.
Continuous waste dumping and subsequent capping with soil from the surroundings has
determined the complex morphology of this landfill. In the photo in Figure 2, the landfill
appears as a flattened hill rising out of a plain.
Figure 2. Picture of the whole landfill in spring of 2009
The landfill site has three main zones: a zone (western) mostly containing solid domestic waste,
and two zones (central and eastern) mainly accommodating industrial waste and some inert
compounds. We have designated these latter zones “rubble tips” to distinguish them from the
landfill proper (Figure 3).
The flatted tops of the landfill correspond to platforms, yet more outstanding are its 12 slopes
showing a high variety of exposures (across their 360º). Slope heights are 10-20 m and gradients
are 50%. Their profiles are straight and many slopes overlap one another. Many slopes show
signs of erosion, especially in troughs, often exposing their waste materials. Leachate surface
runoff may be observed in three main discharge areas. The westernmost discharge area
occupies a wetland. The other two areas, south of the rubble tips form shallow water sheets in
the wettest months and quickly dry when rain ceases at the end of spring. In all these discharge
zones and at the foots of the slopes, sheep herds may be found grazing. What is more, these
and other animals drink any water that accumulates in these areas in 5 to 6 months of the year.
6 Soil Pollution
3.2. Composition of the capping soil layer: Factors linked to fertility, salinity, metal toxicity,
organic compounds and erosion
To identify the soil factors that mainly determine the landfill’s vegetation, mostly arising from
the seed bank of the capping soil, we used a stratified sampling procedure (platforms, adjacent
rubble tips and main surface leachate discharge zones). At each site, samples were collected
using a hoe from the top soil layer (0-10 cm) to give an average soil sample. 57 of such samples
were transported to the laboratory, where they were air-dried and sieved (< 2 mm). These
samples were then subjected to each of the techniques mentioned in the following sections.
Sampling sites 27 to 31 correspond to piles of waste deposited directly in the easternmost
discharge zone with no type of cover at all. Although these samples do not correspond to the
capping soil, they were collected to assess the possible effect of these waste materials on the
soils of the discharge zone in future studies. Corresponding results do not appear in the tables
provided below.
Figure 3. Main areas of the landfill and sites where capping soil samples were collected. 03/08/2009 Google Earth
Image. UTM coordinates: X=442599 Y=4459459, 30T.
a. Soil fertility indicators

In the 57 soil samples, we examined several variables related to soil fertility with consequent
impacts on the vegetation. The procedures described in [10] were used to determine: pH in
water and in a saturated soil paste, percentage organic matter by potassium dichromate
reduction, Kjedahl total nitrogen, pseudototal (by extraction with nitric and perchloric acids
at 4:1) and exchangeable (by extraction with ammonium acetate, pH 7) concentrations of Ca,
K, Na and Mg, and pseudototal and bioavailable P and P2O5 concentrations, analyzed by
plasma emission spectroscopy (ICP-OES).
Sampling point pH OM. N Ca K Na Mg P P2O5
T E T E T E T E T E
Platform
G-23 8.5 2.9 0.09 61566 6 1729 5 923 2 7111 3 205 0 130
G-24 8.0 1.2 0.06 5483 66 8855 4 244 5 4869 6 598 7 240
G-45 8.1 1.3 0.08 7345 30 6892 4 223 4 7085 4 553 3 100
G-46 7.8 1.4 0.08 15253 28 11362 7 276 6 22144 4 542 8 260
G-47 8.3 1.8 0.10 14890 22 10051 3 257 5 11553 4 569 4 110
G-48 7.9 1.5 0.10 9818 34 9400 5 218 7 11036 5 462 7 150
Slope
G-6 7.5 4.0 0.05 6627 27 2495 5 86 16 1316 7 70 0 40
G-8 8.1 0.9 0.05 42871 9 3600 7 355 6 10842 2 32 0 50
G-11 8.2 2.5 0.12 42156 12 6790 12 337 16 7992 4 542 4 300
G-16 7.8 2.2 0.12 46571 14 4478 14 502 12 5795 5 317 0 110
G-17 8.0 2.3 0.06 57028 8 2524 10 604 6 6123 6 132 0 45
G-18 8.2 2.8 0.18 38558 9 3262 9 760 15 6124 7 113 0 15
G-19 7.6 1.2 0.19 11643 27 8270 4 241 15 6617 8 441 0 60
G-20 7.8 1.4 0.09 4051 59 11010 5 243 6 6765 9 547 9 240
G-21 7.9 0.6 0.05 8366 41 7671 4 222 7 5506 8 494 8 200
G-22 8.1 0.9 0.06 7296 53 9209 4 254 5 5810 5 590 6 190
G-39 8.1 1.1 0.05 25150 8 4800 4 350 6 8250 3 50 0 20
Foot of slope
G- 3 A 7.9 0.9 0.06 3969 53 6902 7 221 27 6222 7 258 10 140
G- 3 F 7.6 2.1 0.09 5922 46 7492 6 214 18 10126 5 258 14 200
G-9 8.2 1.2 0.06 42176 10 3206 7 467 5 4391 4 106 0 60
G-10 8.2 0.9 0.03 29893 9 4759 4 339 5 5086 5 137 0 20
G-37 7.8 3.7 0.17 10122 41 8360 8 224 9 4598 7 429 19 320
G-38 7.8 2.3 0.11 14063 35 5774 5 215 32 4861 11 444 9 200
Discharge zone
G- 2 A 7.7 6.2 0.29 13490 39 7245 10 324 52 9071 6 279 9 140
G- 2 B 7.8 7.8 0.37 28560 21 6702 9 252 34 8336 7 366 12 220
G- 4 A 7.5 3.8 0.13 12337 20 3570 12 162 26 3884 5 10 0 1050
G- 4 B 7.8 3.8 0.10 11135 19 3468 8 177 32 3577 8 8 0 70
G-7 7.5 5.6 0.25 37831 33 7442 3 308 32 7953 10 276 0 115
Table 2. pH, organic matter (OM, %), nitrogen (N, %), pseudo-total concentration (T) of nutrient elements (mg kg-1)
and percentage of exchangeable fraction (E) in soil samples collected from landfill proper.
8 Soil Pollution
The results of these determinations in each soil sample are provided in Tables 2 and 3. All
results are provided to highlight the huge variation existing for each factor. pH varied from
7.0 to 8.5, given the alkaline nature of the surrounding soils used to cap the landfill. The
distributions of all variables failed to vary significantly between the landfill proper and rubble
tips.
b. Heavy metals and trace elements toxic for plants
Although some preliminary results regarding soil pollution due to heavy metals, organic
compounds and salinity have been already described [1, 7, 11, 12] here we examine this issue
in detail. All 57 soil samples were subjected to inductively coupled plasma optical emission
spectometry (ICP-OES) to determine pseudototal (after prior extraction with nitric and
perchloric acids, 4:1 [13]) and bioavailable (after prior extraction with ammonium acetate +
EDTA using the method [14]) concentrations of Al, Mn, Zn, Cu, Pb, Cr and Ni. In addition,
total As concentrations were determined by X-ray fluorescence in 48 samples. Total Hg levels
were determined using an Advanced Mercury Analyser (AMA-254, LECO Company, Czeck
Republic) according to the procedure described by [15] in 34 selected samples of the 57 soil
samples collected [16].
Tables 4 and 5 provide the metal and trace element concentrations detected in the capping soil
and discharge area samples. We also examined total Al and Mn levels: Al concentrations in
the landfill ranged from 8123 to 50747 mg kg-1, and Mn concentrations from 205 to 7432 mg
kg-1. In the rubble tips, concentration ranges were higher for Al and lower for Mn. Given the
alkaline nature of the soils, these elements are not considered hazardous for plant populations
and are therefore not included in the tables.
The sites showing the highest levels of all elements occurred on the landfill’s slopes and these
showed an uneven spatial distribution. However, the most contaminated sites were simulta‐
neously polluted by all elements. The percentage of a metal found in its bioavailable form was
also highly variable. Despite being poorly mobile, Pb showed high bioavailability percentages.
Cd was also highly bioavailable. Most variation was shown by Zn and Cu. The metals
appearing in lowest concentrations were Cr and Ni.
Apart from the trace element bioavailability study conducted according to the method of [14],
we performed a more exhaustive analysis of metal bioavailability in the soil samples. To this
end, we undertook sequential extraction by the BCR method optimized by [17]. Sequential
extraction serves to indicate the fractions of each metal that are bioavailable (F1: exchangeable),
reducible (F2: bound to oxyhydroxides), oxidizable (F3: bound to organic matter) and residual
(F4). Given that it is the landfill proper that shows the higher concentrations of these metal
pollutants, 5 sites were selected representing platform, slope and discharge zones showing
variable concentrations of these types of pollutant. The sites were selected according to their
known distributions of metals; we have preserved the numbers assigned to their collection
sites. Table 6 provides total concentrations of each metal in each sample calculated as the sum
of all fractions. The reader may find the percentages of each metal found in each fraction in
Figure 4.
Sampling pH OM. N Ca K Na Mg P P2O5

point
T E T E T E T E T E
Platform
G- 49 8.1 1.7 0.11 13588 24 7139 7 102 15 10111 5 613 3 18
G- 50 7.6 1.6 0.12 10910 38 7464 7 120 11 9279 4 297 11 1171
G- 51 7.8 2.2 0.10 14428 23 9093 5 134 8 10778 2 873 6 66
G- 52 7.8 0.8 0.04 5342 42 6711 3 106 11 6411 4 283 4 12
G- 53 7.7 2.3 0.10 15831 21 7671 8 97 12 7853 3 580 12 54
Slope
G- 13 8.3 1.5 0.08 26245 16 2653 16 187 11 3343 6 106 21 30
G- 15 7.7 1.2 0.09 16589 50 7331 5 198 8 4009 7 249 4 8603
G- 32 7.8 0.7 0.05 63966 14 6989 33 238 12 7583 5 261 3 4008
G- 40 8.1 1.0 0.04 14200 28 7550 3 250 6 10350 2 350 4 264
G- 42 8.2 0.2 0.03 9617 35 3704 4 158 16 4643 4 289 12 26
Foot of slope
G- 12 7.9 1.4 0.07 10790 37 7968 4 214 7 7278 9 423 4 144
G- 14 8.0 1.0 0.06 17651 27 3658 7 127 11 3126 9 68 0 159
G- 33 7.9 1.4 0.09 17519 27 7125 6 190 7 5067 6 536 6 72
G- 34 7.0 2.5 0.13 31469 17 7942 5 248 22 6187 7 469 5 790
G- 35 7.9 1.2 0.08 25913 19 8208 5 173 12 7176 7 428 5 44
G- 41 7.9 1.1 0.10 24281 20 8442 4 246 6 8838 1 593 19 25903
G- 43 8.0 0.8 0.05 72211 6 7503 3 246 6 10099 3 362 8 993
G- 44 7.9 1.3 0.07 17827 19 7959 3 315 13 5894 5 253 4 287
Discharge zone
G- 1 A 7.7 4.2 0.20 72005 34 4234 5 1281 74 8466 22 177 10 5918
G- 1 B 7.7 2.0 0.11 43501 21 7456 4 292 19 7316 3 265 6 3830
G- 5 7.3 5.8 0.24 25041 30 8150 8 412 40 11338 3 449 6 3305
G- 25 8.0 2.6 0.12 15164 28 8552 6 310 27 5753 10 407 5 74
G- 26 7.8 1.1 0.07 64650 9 6624 3 198 15 6081 5 240 4 819
G- 36 7.5 5.6 0.30 34429 25 9961 5 306 27 8024 8 498 3 4245
Table 3. pH, organic matter (OM, %), nitrogen (N, %), pseudo-total concentration (T) of nutrient elements (mg kg-1)
and percentage of exchangeable fraction (E) in soil samples collected from rubble tips.
10 Soil Pollution
Sampling point Zn Cu Pb Cd Cr Ni As Hg
T B T B T B T B T B T B
Platform
G-23 9491 23 4593 3 4421 50 93 35 531 0.9 231 1.1 271 11
G-24 137 20 14 18 19 70 0.0 3.2 2.2 8.7 1.5 n.d 0.0
G-45 148 2.1 8.9 13 28 8.2 0.0 7.3 0.6 5.6 4.1 14 -
G-46 147 2.1 38 17 35 48 0.0 2.2 2.3 12 4.0 22 -
G-47 126 3.8 12 14 11 43 0.0 4.7 1.1 9.9 3.4 n.d -
G-48 175 5.3 43 16 22 40 0.0 18 0.6 21 3.4 n.d -
Slope
G-6 640 22 1916 21 132 29 0.0 256 0.1 35 2.5 119 0.3
G-8 13029 27 1055 8.1 12689 23 185 34 269 1.7 86 1.9 282 4.0
G-11 3247 20 240 11 579 51 10 67 154 0.6 43 2.0 n.d 0.5
G-16 10777 25 748 9.3 5734 38 142 41 150 3.0 42 2.6 n.d 3.0
G-17 17416 26 1313 8.5 12612 28 308 37 242 1.5 60 2.2 n.d 4.9
G-18 22992 28 1804 9.4 18136 30 306 36 298 1.8 80 3.7 685 4.2
G-19 5190 25 125 14 1198 47 29 52 40 3.3 15 2.6 n.d 0.2
G-20 1085 2.6 18 9.4 106 12 0.3 64 9.8 0.5 9.0 0.9 12 0.0
G-21 129 9.0 11 21 18 37 0.0 3.5 1.4 7.0 1.7 13 0.0
G-22 168 9.3 14 20 25 53 0.0 9.1 0.8 8.8 1.8 23 0.0
G-39 17830 76 1445 20 12555 68 190 60 587 2.2 210 2.5 492 -
Foot of slope
G- 3 A 61 2.2 9.1 14 18 59 0.0 2.3 12 5.1 9.2 - -
G- 3 F 69 4.6 31 6.2 8.1 40 0.0 9.2 3 5.2 7.0 - -
G-9 12632 25 1181 10 7179 37 257 48 236 2.4 90 2.1 306 2.4
G-10 15184 33 1493 13 10085 48 155 42 504 2.2 206 2.2 n.d 3.3
G-37 559 66 20 25 28 61 0.0 3.8 4.7 8.5 5.9 15 0.0
G-38 108 18 15 24 23 65 0.0 15 1.0 7.4 6.7 19 0.0
Discharge zones
G- 2 A 528 17 36 27 117 36 0.0 6.6 6.1 8.6 5.1 - -
G- 2 F 366 14 35 18 64 28 0.0 5.7 4.9 7.3 5.5 - -
G- 4 A 577 19 882 27 149 29 0.0 110 0.1 156 1.5 - -
G- 4 F 477 19 1260 26 139 19 0.0 153 0.2 213 1.5 - -
G-7 2417 33 151 27 290 48 2.5 86 9.5 3.4 34 8.9 54 0.3
Ref. (pH"/>7) 450 210 300 3 150 112 29* 1.5
Table 4. Pseudo-total concentration (T) of trace elements (mg kg-1) and percentage of bioavailable fraction (B) in soil
samples collected from landfill proper. nd: not detected; -: not analyzed. Reference levels for alkaline soils according to
Spanish law (RD1310/1990), *As Dutch reference level.
Sampling Zn Cu Pb Cd Cr Ni As Hg
point T B T B T B T B T B T B
Platform
G-49 10 0.0 13 21 0.0 0.0 1.8 0.0 5.4 0.0 n.d -
G-50 17 0.0 21 23 22 54 0.0 3.4 0.0 7.8 0.0 n.d -
G-51 13 0.0 17 19 0.0 0.0 2.4 0.0 6.5 0.0 n.d -
G-52 9 0.0 14 18 0.0 0.0 3.6 0.0 8.0 0.0 12 -
G-53 13 0.0 14 29 0.0 0.0 6.8 0.0 7.7 0.0 15 -
Slope
G-13 1261 18 783 7.0 164 35 0.0 378 0.1 223 1.2 42 0.8
G-15 159 15 34 14 28 48 0.0 17 0.7 12 3.3 29 0.0
G-32 166 11 40 54 24 42 0.0 14 3.8 11 3.4 26 0.1
G-40 120 35 15 14 15 86 0.0 3.5 3.4 5.0 6.6 14 -
G-42 72 12 10 9.2 10 43 0.0 6.7 1.5 4.1 7.1 n.d -
Foot of slope
G-12 1123 2 83 14 129 10 0.0 10 1.2 25 2.6 25 0.0
G-14 341 16 266 11 64 32 0.0 94 0.1 75 1.6 22 0.2
G-33 146 19 29 14 26 56 1.2 90 7 2.5 8.8 3.5 22 0.0
G-34 224 18 28 22 48 40 1.5 3.6 10 1.9 12 4.4 37 0.1
G-35 76 4.8 14 18 11 33 0.9 24 13 1.2 9.1 3.2 31 0.0
G-41 1259 1.3 38 13 148 7.6 1.4 26 9.1 1.2 11 4.1 21 -
G-43 226 2.8 22 5.4 24 12 0.0 1.1 9.1 10 2.2 100 -
G-44 1869 10 73 21 215 54 3.5 67 18 0.9 22 3.6 30 -
Discharge zone
G- 1 A 238 23 18 32 57 43 0.0 2.9 12 4.6 9.0 - -
G- 1 F 74 4.0 13 19 17 22 0.0 1.5 18 6.4 5.2 - -
G-5 252 65 56 31 66 41 0.0 10 0.1 8.3 5.2 31 0.2
G-25 171 13 25 19 44 45 0.0 5.7 3.3 10 4.0 27 0.4
G-26 126 8.1 63 8.5 83 32 0.0 24 1.0 16 2.0 36 0.2
G-36 904 35 68 41 152 62 1.7 11 9.2 2.8 14 7.5 40 0.6
Ref. (pH"/>7) 450 210 300 3 150 112 29* 1.5
Table 5. Pseudo-total concentration (T) of trace elements (mg kg-1) and percentage of bioavailable fraction (B) in soil
samples collected from rubble tips. nd: not detected; -: not analyzed. Reference levels for alkaline soils according to
Spanish law [18], *As Dutch reference level.
12 Soil Pollution
The results of these tests prompt the following conclusions. Cd and Zn showed highest
percentages in the bioavailable fraction. In the case of Cd, this finding is of major concern given
its high concentration in the soils examined and this situation has been also described by [19].
Arsenic shows the highest residual percentage and thus its available levels are low. The
bioavailable fraction of Cu is fairly low, while remaining fractions vary according to the soils.
The behavior of Pb was more irregular among the different soils. In general, the residual
fraction was low. The organic matter fraction was variable being greatest at site 18. Its
bioavailable fraction was very high at site 39, which is worrying given the high concentration
of this heavy metal at this site.
Sample Zn Cu Pb Cd As
G-7 wetland 1425 102 278 3 16
G-18 slope 127529 1441 19500 364 32
G-23 platform 33446 2042 11057 106 10
G-39 slope 76120 1272 20032 223 14
G-46 platform 610 41 182 2 0
Table 6. Total concentration of trace elements (mg kg-1) in soils selected for conducting the sequential fractionation
Figure 4. Percentage of metal content that is in each fraction
Clearly the presence of very high concentrations of trace elements and heavy metals is a
problem for the establishment of plant populations, worsened by the fact that the sites of
highest concentrations coincide with zones of intense slope. In effect, zones corresponding to
samples 17 and 18, along with 10 and 39, are naked slopes with practically no plant cover in
comparison with surrounding zones.
c. Salinity
Salinity has been described as one of the main impacts on the plant populations and animals
of the closed landfills of the Iberian Peninsula’s central region [5, 12, 20, 21] as well as landfills
in other environments [22-25]. This problem is therefore closely examined in the case of the
Getafe landfill. Electrical conductivity was determined in all the soil samples collected, along
with F-, Cl-, NO2-, NO3-, PO43 and SO42- anion concentrations by ion chromatography. These
results may be found in Tables 7 and 8. As for the other chemical properties of the soil, points
showing greatest salinity were unevenly distributed throughout the capping soil.
By comparing soils from the landfill and rubble tips by principal components analysis (PCA)
(Statgraphics 15), we were able to observe that electrical conductivity was related to the Cl-
and SO42- contents of the landfill cap and also to F-, Na, NO3- concentrations in the case of the
rubble tips. The distribution of sites appearing on the new coordinate axes seems to indicate
higher salinity in discharge areas. To confirm this observation, we conducted an analysis of
variance (ANOVA) of the factors electrical conductivity and Cl- level in the different landfill
areas. Our findings indicate that both variables were significantly higher in the soils of the
wetlands where the landfill’s runoff is deposited.
d. Organic compounds
Pollution by organic compounds is also a concern emerging from studies designed to address
the topic of sealed landfills, as many recently banned compounds, dumped in landfills and
numerous affected ecosystems, have been detected [26]. The organic compounds determined
in the soil samples and the techniques used for this purpose were: total hydrocarbons by
infrared spectrometry (UNE 77307); organochlorine insecticides and polychlorinated biphen‐
yls (PCBs) by gas chromatography (ISO 10382); and polycyclic aromatic hydrocarbons (PAHs)
(ISO 18287) and phenols (U.S. E.P.A 3550B, U.S. E.P.A 3650B and U.S. E.P.A 8401) by gas
chromatography. The reader is referred to [1] for descriptions of these techniques and their
modifications for the present purposes.
Table 9 shows the great variety of organic pollutants that may be found in the Getafe landfill.
Those detected at concentrations higher than permitted levels and widely distributed at the
site were total hydrocarbons, PCBs, the PAHs with a greater number of rings and some
organochlorine insecticides. In general, the sites showing most pollution of this type were those
also showing most heavy metal pollution.
Given that total hydrocarbons were detected at all the sites in which these were examined
(N=43), Table 10 presents the differences detected.
14 Soil Pollution
Landfill samples EC F- Cl- NO2 - NO3 - PO4 3- SO4 2-
Platform
G- 23 132 5.5 4.6 1.4 5.4 0.0 26
G- 24 107 1.8 8.1 1.6 52 2.1 11
G- 45 114 2.6 12 1.5 3.3 2.1 8.5
G- 46 161 7.4 8.5 1.1 5.3 2.3 31
G- 47 116 2.4 8.9 1.7 7.6 1.6 10
G- 48 113 2.6 13 1.6 3.7 3.1 8.0
Slope
G- 6 175 2.8 6.4 0.7 3.0 0.0 147
G- 8 317 19 6.0 1.4 17 0.0 398
G- 11 159 7.6 7.7 1.0 3.6 0.0 41
G- 16 1853 10 38 1.3 70 0.0 4063
G- 17 587 14 10 1.4 22 0.0 592
G- 18 985 31 121 2.6 193 0.0 635
G- 19 1329 13 32 2.0 1152 0.0 691
G- 20 154 1.6 5.2 1.3 38 2.2 15
G- 21 171 2.5 6.3 0.9 94 0.9 91
G- 22 157 1.7 4.9 1.1 15 1.1 123
G- 39 366 18 8.6 3.0 41 0.0 214
Foot of slope
G- 3 A 270 3.2 33 1.2 28 0.0 96
G- 3 F 340 1.6 30 1.5 110 5.4 67
G- 9 212 18 8.7 1.3 13 0.0 139
G- 10 237 17 5.3 1.1 12 0.0 194
G- 37 391 1.5 27 1.0 112 0.0 65
G- 38 369 4.2 60 1.3 7.3 0.0 656
Discharge zones
G- 2 A 1490 3.8 135 2.9 93 2.0 1938
G- 2 F 1960 1.6 77 1.5 0.0 0.0 3322
G- 4 A 1490 3.7 125 1.9 148 2.9 1647
G- 4 F 1500 2.9 43 1.0 34 0.0 1729
G- 7 1878 10 85 1.3 20 1.0 4866
Table 7. Electrical conductivity (EC, µS cm-1) and anion concentration (mg kg-1) in soil samples collected from landfill
proper.
Tip samples EC F- Cl- NO2 - NO3 - PO4 3- SO4 2-
Platform
G-49 135 1.5 6.6 0.0 0.0 5.2 18
G-50 826 1.1 3.2 2.4 0.0 0.0 1171
G-51 169 1.7 8.3 0.0 0.0 5.0 66
G-52 60 2.4 2.9 2.6 0.0 2.8 12
G-53 206 1.2 5.1 2.6 0.6 8.0 54
Slope
G- 13 170 7.3 7.2 0.8 18 0.0 30
G- 15 1719 3.7 4.4 0.8 12 0.0 8603
G- 32 2360 9.4 6.9 1.0 67 0.0 4008
G- 40 299 2.8 5.1 1.0 3.8 0.0 264
G- 42 99 3.0 7.9 1.5 4.3 1.8 26
Foot of slope
G- 12 90 0.0 1.1 0.0 0.0 0.0 144
G- 14 193 4.2 15 1.9 13 0.0 159
G- 33 327 2.5 13 1.0 143 0.0 72
G- 34 744 3.8 56 1.5 21 1.5 790
G- 35 198 2.7 13 2.1 37 2.8 44
G- 41 1716 1.9 4.8 1.2 19 0.0 25903
G- 43 877 2.1 3.6 1.0 11 0.0 993
G- 44 384 4.4 46 1.4 5.0 0.0 287
Discharge zones
G- 1 A 8220 8.6 7570 0.0 495 0.0 5918
G- 1 F 2350 2.9 199 2.3 29 0.0 3830
G- 5 2180 3.8 260 2.0 201 0.0 3305
G- 25 280 3.3 35 1.9 24 0.0 74
G- 26 709 6.0 12 1.5 60 0.0 819
G- 36 2500 5.2 69 3.6 43 0.0 4245
Table 8. Electrical conductivity (EC, µS cm-1) and anion concentration (mg kg-1) in soil samples collected from rubble
tips.
16 Soil Pollution
Pollutant Max conc. Ref* Pollutant Max conc. Ref*
PAHs Insecticides
Naphthalene 0.23 1 Alfa-HCH 0.01 0.01
Acenaphthene 0.04 6 Beta-HCH 0.27 0.01
Fluorene 0.09 5 Gamma-HCH 0.48 0.01
Anthracene 0.46 45 Hexachlorobenzene 0.04 0.01
Fluoranthene 2.59 8 Endosulfan 0.07 0.6
Pyrene 2.03 6 p.p'-DDE 0.02 0.6
1,2-benzanthracene 0.99 0.2 Hydrocarbons
Chrysene 1.11 20 Total concentration 3408 50
Benzo(b)fluoranthene 1.85 0.2 Conc. of aromatics 335
Benzo(k)fluoranthene 0.89 2 Conc. of aliphatics 3073
Benzo(a)pyrene 1.56 0.02 Phenols
Indene-1,2,3-(cd)pyrene 1.63 0.3 Phenol 0.05 7
Dibenzo(a,h)anthracene 0.14 0.03 Cresols 0.02 4
2,4,6-trichlorophenol 0.01 0.9
PCBs 3.05 0.01 Pentachlorophenol 0.01 0.01
Table 9. Maximum concentration of organic pollutants found in soils of Getafe landfill (mg kg-1) and maximum
allowed values according to Spanish law (*Ref, [27])
e. Factors linked to soil erosion
Signs of soil erosion observed on the landfill’s slopes prompted us to address this matter, given
the significant effect that soil particle size and the loss of certain fractions can have on the ability
of plant species to take root.
The traditional method of Bouyoucos to determine sand, mud and clay fractions was used on
all 57 soil samples. In addition, the Mastersizer-S was used to assess particle size by the
dispersion and diffraction of a laser light beam as it crosses a suspension of the sample. This
technique and the sample preparation method are described in [1]. Particle size was deter‐
mined in 43 of the samples to establish the type of particle that may be lost through erosion.
Significant differences in this variable were detected in several fractions of fine sand between
soil from the landfill cap and soil from the rubble tips. These results are provided in Figure 5
and table 11. The high standard deviation of the data determined that only differences in the
sand fraction of the rubble tip soil were significant.
Although the results obtained using both granulometric techniques are not comparable since
the first method gives a percentage weight while the second procedure provides percentage
volumes, both revealed that the most marked differences among the higher zones, slopes and
lower zones occur in the rubble tips adjacent to the landfill. Table 11 shows the different
Landfill Landfill Tips
Sampling point HC Sampling point HC Sampling point HC
Platform Foot of slope Slope
G-23 901 G- 3 A 13 G-13 215
G-24 78 G- 3 F 13 G-15 52
Slope G-9 92 G-32 33
G-6 2423 G-10 154 Foot of slope
G-8 93 G-37 33 G-12 174
G-11 230 G-38 26 G-14 169
G-16 62 Discharge zone G-33 52
G-17 63 G- 2 A 5.1 G-34 19
G-18 3408 G- 2 F 5.1 G-35 22
G-19 95 G- 4 A 854 Discharge zone
G-20 33 G- 4 F 854 G- 1 A 7.5
G-21 42 G-7 123 G- 1 F 7.5
G-22 78 G-5 67
G-39 87 G-25 24
G-26 18
G-36 36
Ref. 50 Ref. 50 Ref. 50
Table 10. Total concentration of hydrocarbons (HC, mg kg-1) in points of landfill proper and rubble tips and maximum
allowed values according to Spanish law (Ref, [27])
Figure 5. Mean percentage of each textural fraction determined by Bouyoucos technique in samples from platforms
(P), slopes (S), foots of slopes (FS) and discharge zones (D) in landfill proper and tips. Different letters mean significant
differences between means in the same area (Bonferroni, 95%)
18 Soil Pollution
granulometric fractions analyzed. For the rubble tips, results indicate the dragging of fine
sands from slopes towards the lower zones accompanied by the consequent build-up of coarse
sands. Although with a lack of significance, differences were also observed in the remaining
fractions.
These data do not seem to clearly indicate the signs produced by the in situ transport of
particles from the higher to the lower zones of slopes and discharge areas. No distinguishing
factors were revealed in a discriminatory analysis (figure 6). The findings of such a study also
indicate the heterogeneity of the situations arising on even a single slope and increase the
complexity of understanding the plant colonization pattern, which may vary as small patches
depending on these variations produced on a small scale.
Range of particle size (mm)
Fine sand Fine sand Fine sand Medium Coarse Very coarse
Area Clay Mud
A B C sand sand sand
<0.002 0.002-0.02 0.02-0.05 0.05-0.1 0.1-0.2 0.2-0.5 0.5-1 1-3.2
Landfill
Platform M 0.34 1.93 2.69 7.64 13.7 16.4 23.8 33.4
SD 0.23 0.65 1.39 4.28 6.72 3.10 9.05 14.5
Slope M 0.46 3.12 3.32 8.85 14.0 14.8 16.7 38.6
SD 0.50 2.28 0.64 2.06 3.4 3.51 7.65 13.9
Foot of Slope M 0.76 3.87 3.59 9.15 14.3 16.2 13.8 38.4
SD 0.65 2.81 0.64 1.87 1.81 3.95 1.55 3.84
Discharge zone 0.35 2.30 4.43 11.9 17.6 15.6 13.0 34.8
Tips
Slope M 0.20 1.72 3.10 8.89 a 14.5 a 13.3 14.2 b 44.1
SD 0.25 0.62 1.37 4.14 6.21 3.8 2.92 15.8
Foot of Slope M 0.34 2.30 4.45 12.9 ab 20.1 ab 16.7 12.9 ab 30.4
SD 0.23 0.45 1.02 3.27 5.03 5.4 1.79 14.5
Discharge zone M 0.26 2.76 5.78 16.0 b 22.9 b 17.8 9.73 a 24.8
SD 0.21 1.08 2.30 4.95 3.42 1.30 2.20 10.2
Table 11. Mean (M) and standard deviation (SD) of percentages of each granulometric fraction in different areas of
landfill and tips. Different letters in the same range of particle size mean significant differences between means
(Bonferroni, 95%)
3.3. Heterogeneous distribution of pollutants
Through PCA, we tried to gain insight into the structure of the soil cap used to seal the landfill.
In Figure 7A, it may be seen that the first axis, or component, is closely and positively linked
to heavy metal and organic compound pollution although Na and F also appeared in this group
of variables, and negatively related to soil fertility due to the presence of K and P. The second
component was more related to soil salinity, represented by electrical conductivity, chlorides,
sulfates, nitrates and nitrites. When organic components and the trace elements Hg and As
were excluded, results failed to vary significantly and the first component continued to be
positively and closely linked to the presence of heavy metals and negatively linked to that of
K (Figure 7B). The second component, more related to salinity or electrical conductivity, this
time was linked more to chlorides than the other anions.
Figure 6. Representation of discriminant functions calculated with Mastersizer results of landfill soil samples, grouped
in platforms (P), slopes (S), foots of slopes (FS) and discharge zones (D).
These findings confirm our previous results indicating that despite the uneven distribution of
pollutants, at the most polluted sites all pollutants contribute to this contamination. The PCA
plot of points on the new axes (Figures 7C and 7D) serves to visually identify the sites showing
highest heavy metal pollution as the landfill slopes and those with the greatest salinity as the
rubble tips. The platforms emerged as the least polluted sites both in terms of heavy metals
and salts contents.
The chemical analysis results reveal great heterogeneity in both the distributions and concen‐
trations of pollutants. As an example of the complexity of the problem addressed, Zn concen‐
trations range from 9 mg kg-1 to 23000 mg kg-1; maximal Cd concentrations are 308 mg kg-1 (of
which 85% represents the easily soluble fraction) and the maximal concentration of total
hydrocarbons is 3408 mg kg-1.
The spatial distributions of these factors determined using a Geographical Information System
(ArcMapTM software, v. 9.3.1., ESRI) are depicted in Figure 8.
20 Soil Pollution
4. Understanding the complex nature of landfill soil caps with the view of
restoring the impacts of pollution
The mountains of waste and rubble we have created are new landscape features that most
often emerge in areas around cities. These scenarios can be viewed as laboratories for research
into the environmental impacts of landfills that were capped without prior treatment of the
deposited waste. Even considering that the restoration of degraded ecosystems is a systemic
topic, the functionality of this epistemological approach arises from the fact that ecosystems
are dynamic systems that evolve and co-evolve with human activity.
Figure 7. Principle components analysis of the soil chemical variables showing points appearing on the new coordi‐
nate axes. A) PCA of the whole set of variables, N = 29, B) PCA excluding As, Hg, hydrocarbons and PCBs increasing the
number of cases to N = 52, C) representing the 52 points on the new axes created by the PCA in Figure B), D) expan‐
sion of plot C) from –2.5 to 1 abscissa and -2 to 2.5 ordinate. L, landfill proper; T, rubble tips; Pl, platforms; S, slopes; FS,
foot of slopes; D, discharge zone.
The complexity of the problem faced arises from questions related to the secondary ecological
succession (from the capping soil’s seed bank), which interacts with the primary succession
that is possible in this new ecosystem in the landscape. Besides restoring its impacts, efforts
need to also focus on revegetating the landfill system itself.
Hence, these landfills may be considered a new type of ecosystem in which primary and
secondary successions coincide. They are thus of great interest for ecological science since they
provide a real scenario for investigating the measures we should install to restore a degraded
and polluted ecosystem and help us identify the plant species related to their varied forms of
pollution. This will enable researchers to select the most appropriate plant species for revege‐
tation efforts rather than simply establishing a green cover once a landfill has been capped.
Figure 8. Spatial distribution of Zn, Cd, total hydrocarbons and electrical conductivity
22 Soil Pollution
The ecological theory that is most applicable to the restoration of the environmental impacts
of capped landfills addresses the stress and ecological strategies of herbaceous species.
The classification of plant life cycle strategies described by Grime combines the stress intensity
with the perturbation intensity [7, 28]. Thus, “competing species” are more appropriate for
landfills with a low intensity of perturbation and stress, “ruderal” species adapt better to
conditions of low stress and intense perturbation, and “stress-tolerants” are ideal for settings
of intense stress and scarce perturbation. When both these factors are excessive, this approach
is ineffective.
It should not be forgotten, however, that different types of ecosystem respond differently to a
given perturbation, and vice-versa, that a given ecosystem can respond in many different ways
to different perturbations. We also need to be aware of the vast environmental variability and
randomness that exists along with other associated forms of uncertainty [29].
5. Conclusions
If a sealed landfill needs to be revegetated, it will be necessary to study the fertility of its soil
cover, heavy metals and trace elements that can cause plant toxicity, salinity and organic
compounds in the capping soil layer. The research methodology used in the landfill case study
can be followed in other scenarios with a similar problem.
The analysis of all considered parameters and the heterogeneous distribution of pollutants
indicate that a single-species cover should be avoided. It will be necessary to create a multi‐
species cover that will adapt to the heterogeneous distribution of the organic and inorganic
pollutants present in capping soils and to the morphological features of the landfill’s slopes.
From a scientific viewpoint, the scenario of the closed waste landfill has enabled the in depth
study of what we have called the erosion-pollution binomial. This is the complex situation
found in the capping soils of closed landfills in the Mediterranean setting. The plant species
used for their revegetation should have the capacity to show an adequate response to this
biome. To find such species, there is an urgent need for autecological studies and studies
designed to assess native and commercial plant species that are able to adapt to these particular
conditions. This is the reason why these results should not be extrapolated to other non-
Mediterranean settings.
Acknowledgements
Authors acknowledge program P2009/AMB-1478 Community of Madrid Program (EIADES).

MJGG was funded by the FPU fellowship (AP2008-02934) of Spain’s Ministry of Education.
Author details
Jesús Pastor1, María Jesús Gutiérrez-Ginés1*, Carmen Bartolomé2 and Ana Jesús Hernández2
1 Department of Environmental Biology, MNCN, CSIC, Madrid, Spain
2 Department of Life Sciences, Alcalá University, Alcalá de Henares, Spain
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24 Soil Pollution
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[12] Pastor J, Hernández AJ. La restauración en sistemas con suelos degradados: estudios
de casos en vertederos, escombreras y emplazamientos de minas abandonadas. In:
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[21] Pastor J, Hernández AJ. Heavy metals, salts and organic residues in old solid urban
waste landfills and surface waters in their discharge areas: Determinants for restor‐
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[22] Cheng CY, Chu LM. Phytotoxicity data safeguard the performance of the recipient
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[24] Tatsi AA, Zouboulis AI. A field investigation of the quantity and quality of leachate
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1997.
chapter 7
Assessment of Soil Contamination of a Cattle Market

around River Ogun Basin, Isheri, Nigeria
A.A. Adeyi, O.M. Omidiran and O. Osibanjo
1. Introduction
Continuous increase in environmental pollution caused by the expansion and amplification

of domestic, industrial, and agricultural waste generation has resulted in steadily growing
number of contaminants release into the environment [1]. The uncontrolled discharge of
pollutants into natural sinks such as aquatic ecosystems in developing countries like Nigeria
has prompted a need for periodic assessment of the level of pollutants released into the
environment. Contamination of water resources and soil with toxic metals and priority organic
pollutants represents a major environmental challenge being a source of human exposure to
these toxicants with potential deleterious human health effects. Soils in several parts of Nigeria
have accumulated considerable amount of toxic elements arising largely from unsustainable
consumption and production activities such as illegal mining of solid minerals, uncontrolled
land filling of post-consumer hazardous wastes including electronic waste or e-waste [2, 3],
uncontrolled industrial emissions, and vehicular emissions; as well as environmentally
unsound petroleum exploration, production and exploitation activities, and accidental crude
and petroleum products oil spills etc., [3].
Soil is the foundation of terrestrial ecosystems and a non- renewable fundamental agricultural
resource, inextricably linked to productivity, land development and environmental quality [4].
Soils may contain many organic and inorganic compounds of natural and anthropogenic
origin. The concentrations and toxicity of these compounds present in environmental media
range very widely and depend on possible interactions (synergies) among chemicals. The
continuous introduction of these contaminants into the environment has resulted in their
accumulation. Soil pollution with heavy metals occurs not only near mines and smelters, but
also on agricultural land because of the application of sewage sludge as fertiliser and metal-
based pesticides [5]. Extensive soil contamination with hazardous pollutants including
2 Environmental Risk Assessment of Soil Contamination
inorganic (i.e. Cd, Pb, Cu, Hg, As etc) and organic (i.e. pesticides, dioxins, polyaromatic
hydrocarbons etc) pollutants has increasingly been of global concern over the last decades,
especially in Asian [6] and other developing countries, where urbanization and industrializa‐
tion have occured rapidly and haphazardly.
Heavy metals continue to receive increasing attention due to a better understanding of their
fate as well as toxicological relevance in ecosystems and human health [7]. Pollution of soils
by heavy metals represents one of the most important ecological problems today [8]. Anthro‐
pogenic emission of heavy metals in the environment during the last century has led to
increasing accumulation of metals in soils and natural waters in both urban and rural areas.
Heavy metals are ecologically harmful because they tend to bio-accumulate over time in soils
and plants with ability to have a negative influence on the physiological activities of plants
(e.g. photosynthesis, gaseous exchange and nutrient absorption), influencing reductions in
plant growth, dry matter accumulation and yield [9]. Heavy metal pollution exerts toxic effects
on soil microbial biomass [10] and microbial processes such as soil respiration, nitrogen
mineralization, and enzyme activities [11]. Soil contamination by heavy metals has also been
shown to inhibit soil microbial activities [12], in turn reducing soil fertility and inhibiting the
germination of certain seed plants [13]. Heavy metals contamination may also produce
nutrient imbalance in plants with adverse effects on the synthesis and functioning of many
biologically active compounds [14]. Subsequent accumulation of metals in the food chain [15]
is detrimental to human health.
Heavy metals transport in soil profile is a major environmental concern because even slow
transport through the soil may eventually lead to deterioration of groundwater quality.
Preferential flow can accelerate the movement of water and solutes through soil profile [16].
Soil pollution can lead to water pollution if toxic chemicals leach into groundwater, or if
contaminated runoff reaches streams, lakes, or oceans. The decomposition of organic materials
in soil can release sulphur dioxide and other sulphur compounds, causing acid rain which can
leach heavy metals from soil. Thus, there is need to reduce the introduction of heavy metals
into the environment from anthropogenic sources since metals are persistent, toxic and non-
biodegradable [3]. In addition, chemicals that are not water-soluble contaminate plants that
grow on polluted soils, and they also tend to accumulate increasingly toward the top of the
food chain. Both organic and inorganic contaminants are important in soil management. The
most prominent chemical groups of organic contaminants are petroleum hydrocarbons,
Polycyclic Aromatic Hydrocarbons (PAHs), Polychlorinated Biphenyls (PCBs), chlorinated
aromatic compounds, detergents, and pesticides. Inorganic species include nitrates, phos‐
phates, and heavy metals such as cadmium, chromium and lead.
Organochlorine pesticides not only accumulate in animal tissues; many are extremely stable
and persist in soil and plants, and if soluble enough can reach groundwater or surface waters.
They can therefore enter the food chain not only via their target and non-target organisms, but
also imbibed water and via plants eaten by herbivores, including cattle [17]. Their persistence
in soils depends both on the nature of the soil and their own physico-chemical properties. Light
soils facilitate the water-borne transport of soluble pesticides [18]. Soils with high clay and
organic matter contents tend to retain both the more soluble pesticides (because of their high
Assessment of Soil Contamination of a Cattle Market around River Ogun Basin, Isheri, Nigeria 3
water storage capacity) and the more hydrophobic pesticides (because of their high specific
surface area and other sorption-favouring properties) [19,20]. Because of the dangers they pose
to non-target organisms, DDT and a number of other organochlorine pesticides were banned
in most countries in the 1970s and 1980s, at least for agricultural use. However, because of their
resistance to degradation processes and immobility, many are still found in high concentra‐
tions in soils to which they were formerly applied, where they constitute a reservoir and
secondary source of ongoing contamination of waters, wildlife, and crops [21,22,23].
1.1. Health and environmental effects of heavy metals

Metals are particularly toxic to the sensitive, rapidly developing systems of foetuses, infants,
and young children, otherwise known as ‘’vulnerable group’’. Some metals, such as lead and
mercury, easily cross the placenta of mothers and damage the brain of foetuses. Childhood
exposure to some metals such as lead and mercury can result in learning difficulties, memory
impairment, damage to the nervous system, and behavioural problems such as aggressiveness
and hyperactivity. At higher doses, heavy metals can cause irreversible brain damage.
Children may receive higher doses of metals from food than adults, since they consume more
food for their body weight than adults [24].
Exposure to manganese (Mn) is usually via inhalation, which results in the main cause of
its toxicity. Mn toxicity has been reported through occupational (e.g. welder, miner) and
dietary overexposure and is evidenced primarily in the central nervous system, although
lung, cardiac, liver, reproductive and fetal toxicity have been noted [24]. Mn neurotoxici‐
ty results from an accumulation of the metal in brain tissue [25]. Brain permeability to
manganese is higher than that to iron and zinc. Manganese is easily concentrated in the
brain, especially in the basal ganglia, and can cause an irreversible neurological syn‐
drome similar to Parkinson’s disease [24].
Following long-term exposure to cadmium, the main health concerns are its toxicity to the
kidney and bones, arising via ingestion and inhalation, and its lung carcinogenicity seen in
exposed workers following inhalation [26]. Over a period of time, cadmium accumulates in
the kidney and, if the organ concentration exceeds a critical threshold, the tubule cells become
damaged and renal function impaired. This cadmium build-up also affects vitamin D metab‐
olism, disturbing the calcium balance within the body, which may lead to a decrease in the
mineral content within the bones, resulting in osteoporosis and osteomalacia [26,27]. Its
toxicity is linked with reproduction problem because it affects sperm and reduces birth weight.
It is a potential carcinogen and seems to be a causal factor in cardiovascular diseases and
hypertension. Large concentrations of Cd in the soil are associated with parent material (black
slates) and most are manmade (burning of fossil fuels, application of fertilizers, sewage sludge,
and plastic waste) [28,29]. In humans, long-term exposure is associated with renal dysfunction.
The average daily intake for humans is estimated as 0.15 µg from air and 1 µg from water.
Smoking a packet of cigarettes can lead to the inhalation of around 2-4 µg of cadmium, but
levels may vary widely [27].
Mercury is a toxic substance which has no known function in human biochemistry or physi‐
ology and does not occur naturally in living organisms [27]. Mercury is a toxic heavy metal
and a persistent environmental pollutant. Known sources of Hg in soil e.g. mercury-in-bulb

thermometers used in hospitals; alkyl mercury compounds especially methyl mercury used
as fungicide in agriculture, present in coal and hydrocarbon gas etc. Exposure to mercury is
associated with serious adverse health and developmental effects, especially in pregnant
women, developing foetuses, and young children [24]. This heavy metal is toxic even at low
concentration to a wide range of organisms including humans. The organic form of mercury
can be particularly toxic, and the methyl-and ethyl-forms have been the cause of several major
epidemics of poisoning in humans resulting from the ingestion of contaminated food, e.g. fish.
Two major epidemics in Japan were caused by the release of methyl and other mercury
compounds from an industrial site followed by accumulation of the chemicals in edible fish.
The poisoning became well-known as Minamata disease [28,29]. Inorganic mercury poisoning
is associated with tremors, gingivitis and/or minor psychological changes, together with
spontaneous abortion and congenital malformation. Monomethylmercury causes damage to
the brain and the central nervous system, while foetal and postnatal exposure have given rise
to abortion, congenital malformation and developmental changes in young children [27]. The
negotiations under UNEP of a new Minamata Mercury Convention is under way to underscore
international concerns about the environmental and health impact of mercury and the need
for coordinated global action to address the issue effectively. Average concentrations of
mercury in soil are 6.6 mg/kg, 6.6 mg/kg, 24 mg/kg and 50 mg/kg in agricultural, residential/
parkland, commercial and industrial soils, respectively [30].
Lead has been known to be toxic since the 2nd century BC in Greece. It is a widespread
contaminant in soils. Lead poisoning is one of the most prevalent public health problems in
many parts of the world. It was the first metal to be linked with failure in reproduction. It can
cross the placenta easily. It also affects the brain, causing hyperactivity and deficiency in the
fine motor functions, thus, it results in damage to the brain. The nervous systems of children
are especially sensitive to Pb leading to retardation. It is also cardiotoxic and contributes to
cardiomyopathy (disease of the heart muscle leading to the enlargement of the heart) [28,29].
Lead affects almost every organ system in the human body. The central nervous system is
particularly vulnerable in infants and children under age six. The effects are the same whether
it is breathed or swallowed. Large amounts of lead exposure may lead to blood anaemia, severe
stomach ache, muscle weakness, and brain damage. Lower levels of exposure, may affect a
child's mental and physical growth leading to learning disabilities and seizures [24]. Major
sources of human exposure to lead include old lead water pipes, tetraethyl lead from gasoline,
lead in paint and improper disposal of used lead acid battery (ULAB) in dump sites. In humans,
exposure to lead can result in a wide range of biological effects depending on the level and
duration of exposure. Various effects occur over a broad range of doses, with the developing
foetus and infant being more sensitive than the adult. High levels of exposure may result in
toxic biochemical effects in humans, which in turn cause problems in the synthesis of haemo‐
globin, effects on the kidneys, gastrointestinal tract, joints and reproductive system, and acute
or chronic damage to the nervous system. Lead poisoning, which is so severe as to cause
evident illness, is now less common. This could be due to international efforts at phasing out
lead in gasoline and paints in many parts of the world including developing countries [31]
indeed. At intermediate concentrations, however, there is persuasive evidence that lead can
have small, subtle, subclinical effects, particularly on neuropsychological developments in

children. Some studies suggest that there may be a loss of up to 2 IQ points for a rise in blood
lead levels from 10 to 20 µg/dl in young children [26]. Uncontaminated soil contains lead
concentrations less than 50 ppm but soil lead levels in many urban areas exceed 200 ppm [32].
The EPA's standard for lead in bare soil in play areas is 400 ppm by weight and 1200 ppm for
non-play areas [33].
Arsenic is well-known as a poison and a carcinogen. It has an average concentration in the soil
of 5 to 6 mg/kg [28]. Its amount in the soil is related to rock type and industrial activity [34]. It
is used in combination with other materials in pigments, poison gases and insecticides (such
as Paris green, calcium arsenate and lead arsenate) and is well known from former use as a rat
poison. Arsenic has a long history of medical applications; before penicillin was developed an
arsenic compound was used to treat syphilis and yaws. It is used in ammunition manufactur‐
ing, semi-conductor manufacturing, as a preservative in tanning and taxidermy, as well as on
the exterior of wood such as deck and playground materials as well as by-product of copper
smelting [34]. Acute (short-term) arsenic poisoning may cause nausea, vomiting, diarrhea, and
weakness, loss of appetite, shaking, cough and headache while chronic (long-term) exposure
may lead to a variety of symptoms including skin pigmentation, numbness, cardiovascular
disease, diabetes, and vascular disease. Arsenic is also known to cause a variety of cancers
including skin cancer (non-melanoma type), kidney, bladder, and lung, prostate and liver
cancer.
Chromium is required for carbohydrate and lipid metabolism and the utilization of amino
acids. Its biological function is also closely associated with that of insulin and most Cr-
stimulated reactions depends on insulin. However, excessive amount of the metal can cause
toxicity. Toxic levels are common in soils applied with sewage sludge [28,29]. Chromium is
used in metal alloys and pigments for paints, cement, paper, rubber, and other materials. Low-
level exposure can irritate the skin and cause ulceration. Long-term exposure can cause kidney
and liver damage, and damage to circulatory and nerve tissue. Chromium often accumulates
in aquatic life, adding to the danger of eating fish that may have been exposed to high levels
of chromium [27].
Nickel occurs in the environment only at very low levels. Humans use nickel for many
applications like the use of nickel as an ingredient of steel and other metal products [27], in
the metallurgical, chemical and food processing industries, especially as catalysts and
pigments [35]. Foodstuffs have low natural content of nickel but high amounts can occur in
food crops growing in polluted soils. Humans may also be exposed to nickel by inhalation,
drinking water, smoking, and eating contaminated food [27]. Uptake of high quantities of
nickel can cause death, systemic effects, respiratory effects, cardiovascular effects, gastroin‐
testinal, haematological musculoskeletal, hepatic, renal, endocrine, metabolic, immunological
and lymphoreticular, neurological, reproductive, developmental effects and cancer [36].
Nickel occurs naturally in the Earth's crust with an average concentration of 0.0086% (86 ppm).
The nickel content of soil may vary depending on local geology [37].
Copper is an essential substance to human life, but in high doses it can cause anaemia, liver
and kidney damage, and stomach and intestinal irritation. People with Wilson's disease are at
greater risk for health effects from overexposure to copper. Copper normally occurs in drinking
water from copper pipes, as well as from additives designed to control algal growth [28]. The
major sources of environmental copper releases include the mining, smelting and refining of
copper, industries producing products from copper such as wire, pipes and sheet metal, and
fossil fuel combustion [38].
1.2. Health and environmental effects of some common Persistent Organic Pollutants
(POPs)
Persistent organic Pollutants (POPs) are associated with serious human health problems,
including cancer, neurological damage, birth defects, sterility, and immune system defects. US
Environmental Protection Agency (USEPA) and the Stockholm Convention have classified
certain POPs as probable human carcinogens, including aldrin, dieldrin, chlordane, Dichlor‐
odiphenyltrichloroethane (DDT), heptachlor, Hexachlorobenzene (HBC), toxaphene, and
Polychlorinatedbiphenyls (PCBs) [39,40,41]. Laboratory studies have shown that low doses of
POPs may affect organ systems. Chronic exposure to low doses of certain POPs may affect the
immune and reproductive systems. Exposure to high levels of certain POPs can cause serious
health effects or death. The primary potential human health effects associated with POPs are
cancer; immune system suppression; nervous system disorder; reproductive damage; altered
sex ratio; reduced fertility; birth defects; liver, thyroid, kidney, blood, and immune system
damage; endocrine disruption; developmental disorders; shortened lactation in nursing
women; and chloracne and other skin disorders.
Studies have linked POPs exposure to declines, diseases, or abnormalities in a number of

wildlife species, including certain kinds of fish, birds, and mammals [39,42]. Wildlife also can
act as sentinels for human health: abnormalities or declines detected in wildlife populations
can sound an early warning bell for people. For example, the behavioural abnormalities and
birth defects in fish, birds, and mammals in and around the Great Lakes. People are mainly
exposed to POPs through contaminated foods. Less common exposure routes include drinking
contaminated water and direct contact with the chemicals. In people and other mammals alike,
POPs can be transferred through the placenta and breast milk to developing offspring. In
addition, sensitive populations, such as children, the elderly, and those with suppressed
immune systems, are typically more susceptible to many kinds of pollutants, including POPs.
Because POPs have been linked to reproductive impairments due to their endocrine disrupting
properties, men and women of child-bearing age may also be at risk [43-47].
Organochlorine pesticides are a large class of multipurpose chlorinated hydrocarbon chemi‐

cals, some of which are POPs. They break down slowly in the environment and accumulate in
the fatty tissues of animals. Thus, they stay in the environment and food web long after being
applied [48]. Many organochlorine pesticides are endocrine disrupting chemicals, meaning
they have subtle toxic effects on the body’s hormonal systems [27]. Endocrine disrupting
chemicals often mimic the body’s natural hormones, disrupting normal functions and
contributing to adverse health effects. Organochlorine pesticide is ubiquitous environmental
contaminants because they break down very slowly. The effects that some of the most common
organochlorine pesticides have on humans as well as animal’s health are as follows:
Acute exposure to chlordane can possibly cause neurological effects while long-term exposure
can damage the liver, kidney, and other internal organs. Significant immune system changes
have also been reported in exposed workers. There is some evidence of carcinogenicity [49].
Research has also indicated that chlordane is an endocrine disruptor. The half-life of chlordane
in soil has been reported to be from one to three years. Chlordane binds to aquatic sediments
and bioconcentrates in the fat of organisms.
DDT is a possible human carcinogen; some of its metabolites Dichlorodiphenyldichloroethy‐

lene (DDE) and Dichlorodiphenyldichloroethane (DDD) are probable human carcinogens [49].
DDT and its breakdown products are thought to be endocrine disruptors. DDT is lipophilic
and will both bio-concentrate and bio-magnify. It is present almost everywhere in the envi‐
ronment, and residue has been detected in the arctic. DDT breaks down into the related
compounds; DDE and DDD. In the environment, DDT and its metabolites have a half life of
10 to 15 years in soil. DDT is highly toxic to birds and fish, DDE and DDD are also toxic and
persistent [50]. Some DDT may evaporate from soil and enter the air, and some may be broken
down by the sun or microorganisms.
Heptachlor is metabolised in animals to heptachlor epoxide, whose toxicity is similar to that

of heptachlor [49]. Both binds to animal fat. Heptachlor and heptachlor epoxide are possible
human carcinogens and are thought to be endocrine disruptors. At high levels or from chronic
exposure, they can cause central nervous system and liver damage. The half life of heptachlor
in temperate soil is up to two years. Heptachlor has been found in treated wastewater from
industrial processes including coal mining, foundries, and nonferrous metals manufacturing.
It has been detected in the blood of cattle in the U.S. and heptachlor has been strongly
implicated in the decline of several wild bird populations [50].
Mirex is probably a carcinogen and is also thought to be an endocrine disruptor. It may cause
damage to skin, liver, nervous, and reproductive systems at high levels [49]. Crustaceans may
be the most sensitive organisms to mirex. It can bio-accumulate and bio-magnify and is very
persistent, with a half life of up to 10 years in soil. It has been detected in arctic freshwater.
Lindane, which belongs to the class of new POPs under the Stockholm Convention may be a
carcinogen and is thought to be an endocrine disruptor. It can cause nervous system effects
and pulmonary oedema from short, and acute exposures. Long-term exposure can cause liver
and kidney damage, as well as various blood disorders [49]. Lindane bio-concentrates slightly
in fish and crustaceans, and can remain in the air for up to 17 weeks and travel long distances.
It is broken down quickly in water [50]. Lindane exposure has been associated with recurrent
miscarriage. Lindane also disrupts natural levels of estrogens, androgens and thyroid hor‐
mones in rodents [51]. Lindane produces behavioural and neurochemical changes in devel‐
oping rats at doses that do not produce symptoms in adults. This is significant because it
suggests that lindane may have the greatest effect during development [52]. Both acute and
sub-chronic exposures to lindane were found to reduce dopamine concentrations in the brains
of rats by about 46%. Dopamine is an important neurotransmitter with effects on mood and
behaviour [53]. Lindane exposure during postnatal development in rats was associated with
alterations in levels of detoxification enzymes in the brain that lasted until adulthood. It is not
known how this alteration might affect the outcome of future exposures [54]. Exposure to
lindane decreased both thyroid hormones and progesterone in developing lambs [55S].
Dieldrin can cause poisoning in humans and mammals following exposure via the skin, oral
intake or inhalation. It acts as a stimulant to the central nervous system and accumulates in
fatty tissue causing severe damage to the liver and kidneys. Animal experiments have revealed
a carcinogenic effect, but as yet no teratogenic action. Residue levels of aldrin (mostly present
as dieldrin) do not accumulate indefinitely, as the result of yearly applications at a constant
dosage rate, but will reach a maximum level which is, in practice, of the order of that resulting
from a single annual dose [56,57]. There is virtually no leaching of aldrin into deeper soil layers
and thus no contamination of groundwater, which is eventually used as drinking or irrigation
water [58,59].
Endosulfan is highly toxic to the nervous system and can cause circulatory problems, head‐
ache, vomiting and diarrhea. Thus, it is one of the new POPs under the Stockholm Convention.
It is also a suspected hormone disruptor [49]. Endosulfan has shown no potential to accumulate
over time in animals. It is more water soluble than other organochlorine pesticides, such as
DDT, and is less persistent in the body because it metabolises quickly [60].
1.3. Health and environmental effects of Polycyclic Aromatic Hydrocarbons (PAHs)
Because of combustion of fossil fuels and organic waste, PAHs are ubiquitous in the environ‐
ment. Studies show that certain PAHs metabolites interact with DNA and are genotoxic,
causing malignancies and heritable genetic damage in humans. Many of these compounds
have carcinogenic and mutagenic activities and present a hazard for human health [61]. In
humans, heavy occupational exposure to mixtures of PAHs entails a substantial risk of lung,
skin, or bladder cancer. PAHs generally have a low degree of acute toxicity to humans. The
most significant endpoint of PAHs toxicity is cancer. Increased incidences of lung, skin, and
bladder cancers are associated with occupational exposure to PAHs. It is difficult to ascribe
observed health effects in epidemiological studies to specific PAHs because most exposures
are to PAHs mixtures. Animal studies show that certain PAHs affect the hematopoietic,
immune, reproductive, and neurologic systems and cause developmental effects [62].
1.4. Soil contamination by cattle wastes
Cattle manure is a major waste product of agricultural practice involving the breeding and
rearing of cattle. Inappropriate disposal of manure can create environmental problems such
as odours and leaching of nitrate ion and other pollutants into groundwater. Repeated annual
application of manure with high salt content caused a build up of soluble salts in soils, sufficient
to lower their productivity [63]. During the last three decades, dairy farm facilities have
received attention from the public and regulations due to increased environmental concern.
These facilities concentrate in certain regions of the USA (e.g. California, New York, Minnesota,
and Pennsylvania) and generate considerable amount of manure, which can harm soil and
water quality. Manure varies in mineral composition depending on the type of animal and the
ration fed. It was reported that cattle (Bos Taurus) manure contains 2.7 to 9.5% N, 0.5 to 0.8%
O, 1.7 to 2.9% K, 1.4 to 20% Ca, and 0.61 to 0.76% Mg (% dry waste basis) [64]. In addition,
varying amounts of Al, Fe, Mn, S, Cl, Cu, Zn, and B and trace concentrations of Co, Cd, Cr, Ni,
As and Se are present. Large fractions of these elements are present in a water-soluble form.
With high precipitation, almost all regions where dairy farm facilities concentrate, great
qualities of these elements are lost through surface runoff and leaching into groundwater.
Manure contains a large amount of soluble and insoluble organic substances. Organic matter
content ranges between 80% and 90% (dry weight basis) [64]. Pyrolysis field ionization mass
spectroscopy (Py-FIMS) was also used to analyse cow manure samples [65]. The organic
components that were identified include lignins, dimeric lignins, pesticides, lipids, monoest‐
ers, fatty acid, sterols, and heterocyclic nitrogen compounds.
The aim of this study was to determine the distribution, concentration and profiles of some
ubiquitous environmental pollutants such as heavy metals, polycyclic aromatic hydrocarbons
(PAHs) and organochlorine pesticides in soil of a cattle market around River Ogun Basin,
Isheri, Nigeria so as to ascertain the level of contamination of the soil resulting from the
anthropogenic activities taking place in the area.
2. Experimental
2.1. General description of ogun river basin
The River Ogun basin is situated in south western part of Nigeria covering a total area of 22.44
cubic kilometres. It arises in Oyo state, on the southern side of Yoruba plateau, and runs south
into Ogun state, passing through Abeokuta before entering Lagos state and finally discharging
into the Lagos Lagoon. The elevation of the northern, eastern, southern, and western boun‐
daries are 457 m, 366 m, 426 m, and 230 m, respectively above the sea level [66]. Various
organizations are active in the development of socio-economic conditions of the Ogun river
basin. The Ogun-Osun River Basin Development Authority (OORBDA) is responsible for the
management of the water resources. The Ogun river basin is among others contributing a
diversion at Mokoloki for irrigated farming of the upland. Another is the Ogun State Agricul‐
tural Development Project, which has incorporated a fishery component to increase fish
production and availability. Ogun River is used as a source of water supply for domestic,
industrial, agricultural, and recreational purposes along the states it traverses. This site has
become a place of interest considering its constant and continuous pollution owing to the fact
that it serves as a focal point of some commercial activities in the ever growing cattle market
around the basin [67]. Figure 1 shows the map of the study area.
2.2. Sampling design
The soil samples were collected from the cattle market situated around the Ogun River, Isheri
along Lagos-Ibadan Express Road. The soil samples were collected at two different depths of
0-15 cm and 15-30 cm with non-metallic sampler. The sampling site was divided into three
zones as follows:
Figure 1. Map of river Ogun showing the Sampling Points
1. Samples were collected at five different spots (SA1-SA5) upstream the market across the
river at a distance of about 100 m to the market. The major human activities on this side
of the river were sand mining and fishing. The fishing activity was simple and involved
the use of paddled canoe and boat with fishing net and hooks. The natural physical feature
of these areas was shrub growth. The samples from this area were pooled together to form
a representative sample, A1S1 (representing composite sample of the random samples
collected from 0-15 cm depth), and A2S2 (representing composite sample of the random
samples collected from 15-30 cm depth).
2. Random samples were also collected at five different locations including the market centre
and the areas surrounding it. The activities and the features observed at the different
points were;
i. SB1: Old car scraps, marshy area located about 50 m to the market centre
ii. SB2: Auto-mechanic workshop and residential tents built with planks and polypro‐
pylene materials
iii. SB3: Cattle rearing and selling with other petty trading
iv. SB4: Slaughter houses, wastewater discharge point, animal hair and horn burning
with tyres, animal blood cooking and processing, and solid waste dumpsite
v. SB5: Children playground at the river bank
The samples from this area were pooled together and a representative sample, B1S1 (repre‐
senting composite sample of the random samples collected from 0-15 cm depth), and B2S2
(representing composite sample of the random samples collected from 15-30 cm depth) were
obtained for the analysis.
3. Two samples were collected downstream the market from two points to form a composite
samples C1S1 and C2S2 for the 0-15 cm and 15-30 cm depths, respectively. The activities
around this point are
i. SC1: Petty commercial activities around a police post in a slum with most houses built
with plank, rust iron sheet and polypropylene material;
ii. SC2: A dumpsite that was almost swept off by erosion into the river situated at the
river bank.
2.3. Sample collection, treatment and preservation
Two samples were collected from each area of sampling around the cattle market to form
a composite. Control sample was also collected in a rural community, Lalupon in Ibadan.
The sample portion that is to be analysed for PAHs and organochlorine pesticides were
collected in a glass bottle wrapped with aluminium foil to prevent exposure to sunlight, as
organochlorine pesticides are known to be sensitive to sunlight, while the sample to be
analysed for heavy metals and physico-chemical parameters were collected in a poly‐
thene bag. The samples were preserved in an ice chest for onward transportation to the
laboratory, where they were kept in a refrigerator. The samples collected for heavy metals
and physico-chemical parameters were air-dried at room temperature until they were
properly dried and large objects (sticks, stones, wood, and e.t.c) were manually removed.
The samples were then gently ground with a porcelain mortar and pestle, and sieved
through a 2 mm sieve and stored in polythene bags until sample digestion. The samples
collected for PAHs and organochlorine pesticides analyses were not air-dried to minimise
loss of those components which can vaporise easily [68].
2.4. Analytical procedure
2.4.1. Determination of soil pH
Approximately 20.0 g each of the air-dried and sieved soil samples (< 2 mm) were weighed,
50 ml distilled water was added and then mixed and allowed to stand for 30 minutes. Sus‐
pension was stirred every 10 minutes during this period. The suspension was allowed to settle
for another 30 minutes and a pH meter electrode (Jenway 3510) was placed in the suspension
and the reading was taken after some seconds. The electrode was removed from the suspension
and rinsed thoroughly with distilled water and excess water was carefully dried.
2.4.2. Determination of soil organic matter
About 0.70 g each of the air-dried and sieved soil samples were weighed and 10.0 ml of 1.0 N
K2CrO7 solution and 20.0 ml concentrated H2SO4 were added immediately [69]. The solution
was mixed for 1 minute by swirling until the soil and reagents were thoroughly mixed. The
mixture was allowed to stand for 30 minutes after which 100 ml distilled water was added and
then allowed to cool under running water. 3-4 drops of Ferroin indicator was added and then
titrated with 0.5 N Ferrous Ammonium Sulphate. Near the end-point, the solution took a
greenish colour and later changed to dark green. At this point, the 0.5 N Ferrous Ammonium
Sulphate was added drop by drop until the colour changed from green to reddish-brown at
the end point. The Ferrous Ammonium Sulphate solution was standardized using 1 N
potassium dichromate before use. Blank titration was carried out in the same manner, but
without the soil sample.
The % organic Carbon was determined as follows:

(meq. FAS for blank − meq FAS for sample) (0.003 x 100 f )
% Organic Carbon =
Weight in g of air − dried soil
Where, f = 1.334
% Organic matter = 1.729 x % Total Organic Carbon
meq- Milligram equivalent = normality of solution x ml of solution used.
2.4.3. Determination of soil particle size by the Bouyoucos hydrometer method
This is a measure of the size distribution of individual particles in a soil sample. Particle size
distribution analysis is often used in soil science to evaluate soil texture. The soil texture is
based on different combination of sand, silt, and clay separately, that makes up the particle
size distribution. The size range of sand is 2.0 mm-0.05 mm, silt is 0.05 mm-0.002 mm and clay
is less than 0.002 mm (< 0.002 mm). Particle size distribution analysis result can be used to
predict the water retention capacity and unsaturated hydraulic conductivity of soils [70].
About 50.0 g of air-dried soil sample sieved to < 2 mm was weighed into 250 ml beaker. 20 ml
of 5.0 % Sodium Hexametaphosphate was added along with 350 ml distilled water. The
mixture was stirred mechanically on an end-to-end shaker at 100 revolutions per minutes for
5 minutes. The water-soil slurry was transferred to a sedimentation cylinder and sufficient
water was added to bring the level to the 1000 ml mark. The top of the cylinder was covered
and inverted several times until all the soil particles are in suspension. The cylinder was placed
on the flat surface and the time was recorded. The soil hydrometer was placed on the suspen‐
sion carefully and slided slowly into the suspension until the hydrometer was floating. The
first reading was taken on the hydrometer at 60 s after the cylinder was set down. The
hydrometer was removed and the temperature of the suspension was measured. After the first
hydrometer reading, the suspension was left standing for 3 hours and the second reading was
taken. The temperature of the suspension was also measured at this point. The first reading
measured the percentage of silt and clay in suspension while the second reading indicated the
percentage of clay in the suspension. When taking the hydrometer reading, the hydrometer
was carefully lowered into the cylinder about 25 s before the reading was taken to ensure that
it came to rest before the appointed reading time. The readings were taken at the top of the
meniscus formed by the suspension around the stem of the hydrometer. As soon as the reading
was taken, the hydrometer was carefully removed and rinsed with distilled water. The results
were corrected to a temperature of 20 oC and 2.0 were subtracted to compensate for the added
dispersing agent.
Correction factor for Temperature = 0.3 [Observed Temperature (T oC) – 20 oC]
% Clay + % Silt = [H1 + 0.3 (T1 - 20) – 2.0] 2
% Clay = [H2+ 0.3 (T2 - 20) – 2.0] 2
Sand = 100 – [H1 + 0.3 (T1 - 20) – 2.0] 2
Silt = 100.0 – (% Sand + % Clay)
Where,
H1 = Hydrometer reading at 60 s
T1 = observed Temperature at 60 s (= 28 oC)
H2 = Hydrometer reading at 3 hours
T2 = observed Temperature at 3 hours (= 28 oC)
2.4.4. Metal analysis
Acid digestion procedure was employed for the determination of the concentrations of the
environmentally available metals in the soil samples. Prior to the determination of the metal
concentration in soil, sample digestion is a necessary pre-treatment step. The most common
method for determining the concentrations of metal contained in a soil sample is the total
elemental analysis. The review on total metal concentration determination in soil samples
revealed that heavy metals bound to non-silicates and silicate structures were determined with
concentrated acids such as HCl, HClO4, and HF [71]. However, in recent times, strong acid
extractant such as concentrated nitric acid, aqua regia (a mixture of concentrated HCl and
concentrated HNO3 in ratio 3:1) and dilute form of the acids are used to determine total metals
in contaminated soils [72]. The acids dissolve almost all elements that could become environ‐
mentally available especially metal oxides and carbonates [73].
However, heavy metals bound to silicate structures are not normally dissolved since they are
not usually mobile in the environment [74] but the extractant normally give a reliable measure
of metals added to soils as non-silicates from industrial sources that has potential for natural
leaching and biological processes. The use of perchloric acid and hydrofluoric acid is generally
fading away since complete dissolution of the soil is no longer required for total metal analysis
in environmental work [75].
Most current environmental analysis work that involved total metal analysis has employed
the use of 2 M HNO3 [3,76,77]. The levels of heavy metals extracted with 2 M HNO3 have been
reported to represent maximum contents of potentially available metals for plants [77]. In this
study, approximately 1.0 g each of the composite soil samples were digested with 20 ml of 2
M HNO3 by heating the vessel in a water bath between 90-100oC for 2 hours with shaking every
20 minutes. The vessel was allowed to cool to room temperature. The digestate was filtered
and made up to mark with distilled water. Two replicate samples were digested in the same
way together with a reagent blank and all the samples were analyzed using Buck 200A Atomic
Absorption Spectrophotometer using air-acetylene flame.
2.5. Organochlorine pesticides and PAHs analysis
2.5.1. Soil sample extraction

A solvent mix of pesticide residue (PR) grade acetone and methylene chloride (50:50) was
prepared and 50 ml of the solvent mix was added to about 10 g of the sample, spiked with 1
ml of surrogate standard [78,79]. The sample was placed in the ultrasonic bath (Grant Instru‐
ments) and sonicated for about 10-15 minutes at 70oC. Then 10 g of anhydrous sodium sulphate
(Na2SO4) was added to the sample to remove the water present initially in the sample and
shaken gently until a clear extract was developed. The procedure was repeated once more with
an additional 50 ml of solvent mix. The solvent was then concentrated on a rotary evaporator
(Buchi equipment) and exchanged with 5 ml of n-hexane then re-concentrated to 1 ml. The
concentration of the sample extract was necessary to remove the acetone and methylene
chloride used in the extraction. In the course of the concentration of the solvent on the
evaporator, the acetone and methylene chloride was removed leaving n-hexane with the
extract. This is necessary to prepare the extract for clean-up and fractionation in a column
containing n-hexane-mixed silica slurry. The extracted sample was then fractionated into the
aliphatic and aromatic fractions (PAHs) using silica gel column [80]. The same procedure was
repeated for the extraction of organochlorine pesticide in the sample using acetone and
dichloromethane (DCM) solvent mixture (50:50).
2.5.2. Column packing for fractionation and clean-up of extract

Glass column was packed with 10 g of 100-200 mesh silica gel preconditioned (baked) at 105oC
overnight. The silica was mixed with n-hexane to form slurry. The column was then eluted
with about 15 ml of PR grade n-hexane and the solvent was collected to waste. Caution was
taken not to allow the column to dry up. Using 1 ml pipette, 1 ml of the extract in n-hexane
was added onto the column and then eluted with 60 ml of n-hexane. The aliphatic fraction was
collected and the column was then eluted with 40 ml of PR grade dichloromethane (DCM) and
another fraction was collected, which is the Polycyclic Aromatic Hydrocarbons (PAHs)
fraction. Each of these fractions was concentrated to 1 ml using a rotary evaporator. After
concentrating the PAH fraction to 1 ml, 5 ml of PR grade n-hexane was added and was further
concentrated to remove the DCM using the rotary evaporator. The concentrates were later
transferred into 2 ml sample vials using the graduated 2 ml pipettes. The final volumes of the
extracts were noted and the sample extracts were subjected to GC-MS analysis. The same clean-
up procedure was carried out on the organochlorine pesticide extract by eluting the column
with PR grade n-hexane.
2.5.3. Instrumental analysis
The extracts were analysed for PAHs and organochlorine pesticides using a GC equipped with
MS detector. Separation was achieved by injecting about 1 µL of each extract into the GC system
(in a splitless mode) through a capillary column (3.0 m length, 0.25 mm internal diameter, 0.25
µm film thickness). Helium gas was used as the carrier gas. Table 1 summarize the GC/MS
condition. The quantification limit of the PAHs in the standard and the samples was 0.001
ppm, while that of the organochlorine pesticide residue was 0.002 ppm. The average response
factor for the weight ranges were calculated and used for sample quantification.
GC/MS condition PAHs Organochlorine pesticide

Instrument Shimadzu GC-MS QP2010 Shimadzu GC-MS QP2010
Column HP-1MS (Cross linked PH ME siloxane) HP-1MS (Cross linked PH ME siloxane)
19091S-933 19091S-933
Film thickness: 0.25 µm, Length: 3.0 m, Film thickness: 0.25 µm, Length: 3.0 m,
Column ID: 0.25 mm Column ID: 0.25 mm
Injection method Splitless mode Splitless mode
Injection volume 1.0 μL 1.0 μL
Carrier gas Helium (1.2 ml/min) Helium (1.18 ml/min)
Injection temperature 250 ºC 250 ºC
Oven temperature GC oven temperature was kept GC oven temperature was kept
programme 60 ºC for 1 min 80 ºC for 1 min
First ramp at 5 ºC/ min to 180 ºC First ramp at 5 ºC/ min to 200 ºC
Second ramp at 10 ºC/min to 280 ºC Final temperature at 10 ºC/min to 270 ºC
Final temperature at 10 ºC/min to 300 ºC
MS mode Total ion current (TIC) mode Selected Ion Monitoring Mode (SIM)
Ion source temperature 200 ºC 200 ºC
Interface temperature 250 ºC 250 ºC
Table 1. Operational conditions of GC/MS for PAHs and organochlorine pesticide

The concentration of each analyte was determined by calculating the amount of analyte or
hydrocarbon range injected from the peak response in area ratio. The contribution from the
solvent front and the surrogate compound were excluded from the total area of the sample.
A( p ) x Rf x V f x Df x 1000
Cf =
Wi
Where,
Cf = Final Sample concentration (µg/L)
A(p) = Measured area of peak (peaks)
Wi = Initial weight extracted (g dry weight)
Vf = Final extract volume (ml).
Df = Dilution factor of sample or extract if diluted.
Rf= Response factor from the calibration standard calculation
Concentration (P )
Rf =
Area (P )
Concentration (p) = Total concentration of range
3.1. Soil pH
Soil pH is one of the most important physico-chemical properties which control many other
soil physical, chemical, and biological properties. The pH of a soil is affected by the concen‐
tration of CO2 in the soil air, salt concentration (salt effect), and the presence of colloidal
particles (suspension effect). The higher the CO2 concentration in the soil solution, the lower
the pH, and the pH of a neutral or calcareous soil is very sensitive to small changes in CO2
concentration. pH greatly affects the solubility of minerals in soils. Most minerals are more
soluble in acid soils than in neutral or slightly basic solutions [81]. The pH of a soil is related
to the bioavailability of metals in the soil to plants. At low pH, metals are easily bioavailable
because there is increase in the solubility of metals. However, at high pH i.e. slightly alkaline
to highly alkaline conditions, metals are not easily mobilized, because they are not easily
soluble at high pH, hence not easily bio available [82]. The pH of the composite soils collected
at the five different points (SA1-SA5) opposite the market at a distance of about 100 m to the
market was slightly acidic with the pH of the topsoil (0-15cm) slightly lower than that of the
subsoil (15-30cm) except at the downstream of the river (sampling area C) (Table 2).
3.2. Soil Organic Matter
Soil organic matter is any material produced originally by living organisms (plant or animal)
that is returned to the soil and goes through the decomposition process. At any given time, it
Physical Sampling Area A Sampling Area B Sampling Area C

Parameter A1S1 A2S2 B1S1 B2S2 C1S1 C2S2
(0-15cm) (15-30cm) (0-15cm) (15-30cm) (0-15cm) (15-30cm)
pH 5.43 5.58 6.69 7.19 6.82 6.11
% Organic
0.83 1.05 0.97 1.80 0.88 1.31
matter content
% Clay 24.8 20.8 22.8 26.8 24.8 28.8
% Silt 20 24 18 28 24 22
% Sand 55.2 55.2 59.2 45.2 51.2 49.2
Table 2. Summary of Soil physicochemical parameters
consists of a range of materials from the intact original tissues of plants and animals to the
substantially decomposed mixture of materials known as humus. Most soil organic matter
originates from plant tissue. Plant residues contain 60-90 percent moisture. The remaining dry
matter consists of carbon (C), oxygen, hydrogen (H) and small amounts of sulphur (S), nitrogen
(N), phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg). Although present in
small amounts, these nutrients are very important from the viewpoint of soil fertility man‐
agement [83]. Soil organic matter (SOM) serves as a soil conditioner, nutrient reservoir,
substrate for microbial activity, preserver of the environment, and major determinant for
sustaining and increasing agricultural productivity. The OM content of soils ranges from less
than1% in desert soils to close to 100% in organic soils. A typical agricultural soil may contain
between 1 and 5% OM in the top 15 cm [84]. The percentage organic matter of the soil samples
collected around the site is shown in Table 2. Generally, the subsoil has more organic matter
content than their respective topsoil. The top soil contains less than 1% OM while the subsoil
contains higher values. Soil organic matter performs very important functions in the soil such
as: acts as a binding agent for mineral particles, this is responsible for producing friable (easily
crumbled) surface soils; increases the amount of water that a soil may hold; and provides food
for organisms that inhabit the soil. Humus is an integral component of organic matter because
it is fairly stable and resistant to further decomposition. Humus is brown or black and gives
soils its dark colour. Like clay particles, humus is an important source of plant nutrients.
3.3. Particle size distribution (soil texture)
Particle size is a fundamental property of any sediment, soil or dust deposit that can provide
important clues to nature and provenance and influences a variety of other properties [85].
Soil particle size distribution is of greta importance to soil water movement, soil erosion and
soil solute migration [86]. It is the determination of the proportion of each soil fraction in the
samples and includes the % clay, % silt and % sand. Generally, in all the soil samples collected,
sand has the highest percentage. The topsoil of the site has the highest percentage of sand in
all the sampling areas. Figure 2 shows the variation in the physico-chemical parameters in the
soil.
3.4. Concentration of heavy metals in soil
The results of heavy metals analyzed in the soil samples are shown in Table 3 while Figure 3
shows the distribution around the study site. There is variation in the distribution of the metals
around the market with some of the metals such as lead (23.8 mg/kg) and copper (10.4 mg/kg)
having the highest concentrations in the subsoil (15-30 cm). The concentrations of all the metals
around the market is the highest when compared to the downstream and upstream of the river.
This may be attributed to the anthropogenic activities in this area. In most cases, the subsoil
has higher concentration of the metals than their respective topsoil, except Zn, Cd, and Ni.
However, Cadmium compounds being of very low solubility in water will have little down‐
ward movement and accumulate in the topsoil [87]. This may be due to strong adsorption of
Cd by the surface soils, which have a higher organic matter content and higher pH; factors
which are known to increase Cd adsorption in soils [88]. Also, it was reported that Cd
accumulated in the soil surface layers and had low vertical movement when mobility of heavy
metals contained in the sludge and the wastewater used for the irrigation was studied in the
soil samples collected from the soil profiles from the surface to 100 cm depth of farmland [89].
The mean concentrations and standard deviation of Pb, Zn, Cu, Cr, Cd and Ni in the topsoil
(0 – 15cm) and subsoil (15 – 30cm) around the river basin at Isheri are 14.6 ± 4.5 mg/kg and 18.1
± 8.1 mg/kg; 38.0 ± 27 mg/kg and 44.6 ± 28 mg/kg; 6.41 ± 3.3 mg/kg and 7.77 ± 3.7 mg/kg; 7.35
± 0.1 mg/kg and 10.5 ± 0.4 mg/kg; 0.74 ± 0.3 mg/kg and 0.63 ± 0.03 mg/kg ; and 3.81 ± 0.5
mg/kg and 5.70 ± 0.3 mg/kg, respectively.
Comparing the results of heavy metal concentrations obtained in this study with the control
sample collected at a rural community, Lalupon in Ibadan and some typical values in rural
and urban soil around the world (Table 4). The average metal concentrations in both the top
and subsoil were higher than the values obtained from the control sample. There are no
background and baseline data on toxic metals concentrations in Nigeria. Metal concentrations
in the study site when compared to the control sample suggested that anthropogenic inputs
have occurred over the years. The concentrations of all the metals were below the values
reported [90] except Cd which appeared higher in both the top and the subsoil of the study
area.
Sampling Area A Sampling Area B Sampling Area C Mean Conc.

Metals
A1S1 A2S2 B1S1 B2S2 C1S1 C2S2 Topsoil Subsoil
(0-15cm) (15-30cm) (0-15cm) (15-30cm) (0-15cm) (15-30cm) (mg/kg) (mg/kg)
Pb 12.9 13.9 17.7 23.8 11.4 12.4 14.6±4.5 18.1±8.1
Zn 20.8 20.7 57.0 64.7 19.0 24.5 38.0±4.5 44.6±28
Cu 6.12 6.19 8.73 10.4 4.09 5.14 6.41±3.3 7.77±3.7
Cr 10.4 10.4 7.3 10.7 7.4 10.2 7.35±0.1 10.5±0.4
Cd 0.73 0.55 0.93 0.61 0.55 0.65 0.74±0.3 0.63±0.03
Ni 5.27 4.72 4.19 5.91 3.43 5.48 3.81±0.5 5.70±0.3
Table 3. Heavy metal concentrations (mg/kg) in the soil around the cattle market in Isheri, Nigeria
10.5 ± 0.4 mg/kg; 0.74 ± 0.3 mg/kg and 0.63 ± 0.03 mg/kg ; and 3.81 ± 0.5 mg/kg and 5.70 ± 0.3
mg/kg, respectively.
Comparing the results of heavy

Assessment metal
of Soil concentrations
Contamination of a Cattleobtained in this
Market around study
River Ogun with
Basin, the control
Isheri, Nigeria sample
19
collected at a rural community, Lalupon in Ibadan and some typical values in rural and urban soil
around the world (Table 4). The average metal concentrations in both the top and subsoil were higher
than the
There values obtained
is positive from thecorrelation
and significant control sample.
between There
Zn,arePbno
andbackground
Cu and Niand baseline
with data on toxic
Cr. Atmos‐
metal concentration in Nigeria. Metal concentrations in the study site when compared to the control
pheric
sample fallout of thethat
suggested metals such as leadinputs
anthropogenic from the
havemotor vehicle
occurred overexhaust fromThe
the years. theconcentrations
nearby busy of all
Lagos-Ibadan
the metals were express
belowroad as well
the values as movement
reported [90] exceptof goods around
Cd which the market
appeared higher inmay bothhavethe top and
the subsoil of
contributed the study
to the area.
high concentrations of heavy metals in the soil. Also, there is positive and
There is positive and significant correlation between Zn, Pb and Cu and Ni with Cr. Atmospheric
significant correlation
fallout of the between
metals such organic
as lead matter
from the andvehicle
motor Ni, Pb,exhaust
and Cu.from the nearby busy Lagos-Ibadan
express road as well as movement of goods around the market may have contributed to the high
concentrations of heavy metals in the soil. Also, there is positive and significant correlation between
organic matter and Ni, Pb, and Cu.
70
60
Physicochemical parameters
50
pH
40
Organic m atter content (%)
% Clay
% Silt
30
% Sand
20
10
0
A1S1 A2S2 B1S1 B2S2 C1S1 C2S2
Samples
Figure 1. Variation in the physicochemical parameters in the soil around the cattle market in Isheri,
Nigeria
Figure 2. Variation in the physicochemical parameters in the soil around the cattle market in Isheri, Nigeria
Pb B Zn Cd CuC Cr
Mean Ni
Conc.
Sampling Area A Sampling Area Sampling Area
A1S1(this study) A2S2
Metals Topsoil B1S1 BS
14.6 2 2 38
C1S0.74
1 C2S2
6.41 7.35
Topsoil
3.81
Subsoil
Subsoil (this study) 18.1 44.6 0.63 7.77 10.5 5.70
Control site (0-15cm) 9.12 ND ND 4.57 4.83 0.57
Rural–urban
15
35 90 0.35 30 70 -
Soils average (Bowen, 1979)
Table 4. Comparison of mean concentrations (mg/kg) of metals in this study with the control sample and typical soil
values around the world
Table 4. Comparison of mean concentrations (mg/kg) of metals in this study with the control sample
and typical soil values around the world
250
200
Concentration(mg/kg)
150 Dow nstream (15-30cm)

Dow nstream (0-15cm)
Around the Market (15-30cm)
Around the Market (0-15cm)
100 Upstream (15-30cm)

Upstream (0-15cm)
50
0
Pb Zn Cu Cr Cd Ni
Metals
Figure 3. Heavy metal distribution in the soil around the cattle market in Isheri, Nigeria
Figure 3. Heavy metal distribution in the soil around the cattle market in Isheri, Nigeria
3.5. Concentration of Polynuclear Aromatic Hydrocarbons (PAHs)

3.5. Concentration of Polynuclear Aromatic Hydrocarbons (PAHs)
The results of the PAHs in the soil samples collected along the River Ogun basin at Isheri,
The results of the PAHs in the soil samples collected along the River Ogun basin at Isheri, along
along Lagos-Ibadan Express road are shown in the Table 5. Regular burning of all kinds of
Lagos-Ibadan Express road are shown in the Table 5. Regular burning of all kinds of waste,
waste, tyres etc. was observed in this area and this might be responsible for the presence of
tyres etc. was observed in this area and this might be responsible for the presence of PAHs
PAHs around the study site. Some of the PAHs such as naphthalene, phenanthrene,
around the study
fluoranthene, site. Some
chrysene of the PAHs and
benzo(a)anthracene suchperylene,
as naphthalene,
were foundphenanthrene,
at significantlyfluoranthene,
higher
chrysene benzo(a)anthracene
concentrations around the area. and
Outperylene, werespecies
of the seven foundregarded
at significantly higher
as potential concentrations
carcinogenic
around the area. namely:
compounds, Out of the seven speciesbenzo(a)anthracene,
chrysene, regarded as potential benzo(k)flouranthene,
carcinogenic compounds,
namely: chrysene, benzo(a)anthracene,
benzo(g,h,i)perylene, benzo(k)flouranthene,
dibenzo(a,h)anthracene, indeno(1,2,3-cd)pyrene, benzo(g,h,i)perylene,
and benzo(a)pyrene,diben‐
zo(a,h)anthracene, indeno(1,2,3-cd)pyrene,
chrysene has the highest and benzo(a)pyrene,
concentration in the samples chrysene
collected at all the samplinghas the Its
points. highest
concentration in the
concentration was samples
relatively collected
higher at all the
in the subsoil sampling
(15-30cm) points.
than the topsoilIts(0-15cm)
concentration
except was
at sampling
relatively point
higher in C
the(downstream), wherethan
subsoil (15-30cm) there
theis topsoil
little variation
(0-15cm) inexcept
the concentrations
at sampling inpoint C
both layers. Figure 3 shows the variation in the concentration of PAHs with depth.
(downstream), where there is little variation in the concentrations in both layers. Figure 4
shows the variation in the concentration of PAHs with depth.
21
Sampling area A Sampling area B Sampling area C

PAHs Component A1S1 A2S2 B1S1 B2S2 C1S1 C2S2
(0-15 cm) area(15-30
Sampling A cm) Sampling
(0-15 cm)
area B(15-30 cm) (0- 15area
Sampling cm) C (15-30 cm)
PAHs Component
Naphthalene A1S419
1 A2S2 244 B1S1 159 B2S2 663 C1S1 668 C2S2 609
(0-15 cm) (15-30 cm) (0-15 cm) (15-30 cm (0- 15 cm) (15-30 cm)
2-methyl Naphthalene
Naphthalene 419158 244 103 159 64.3
663 234
668 245
609 227
Acenaphthylene
2-methyl 8.1 4.0 8.4 24.2 20.2 25.8
158 103 64.3 234 245 227
Naphthalene
Acenaphthene 3.7 19.7 3.8 9.3 9.1 69.2
Acenaphthylene
Flourene 8.117.9 4.0 9.3 8.4 18.3 24.2 18.1 20.2 29.6 25.8 18.4
Acenaphthene 3.7 19.7 3.8 9.3 9.1 69.2
Phenanthrene 60.4 47.5 91.7 76.4 129 85.7
Flourene 17.9 9.3 18.3 18.1 29.6 18.4
Anthracene
Phenanthrene 60.414.8 47.5 11.9 91.7 17.2 76.4 8.3 129 22.9 85.7 6.4
Anthracene
Flouranthene 14.841.2 11.9 121 17.2 69.1 8.3 31.8 22.9 69.8 6.4 38.9
Flouranthene
Pyrene 41.214.8 121 69.1 69.1 57.5 31.8 18.7 69.8 43.7 38.9 19.6
Pyrene 14.8 69.1 57.5 18.7 43.7 19.6
Chrysene 93.4 273 81.4 239 193 192
Chrysene 93.4 273 81.4 239 193 192
Benzo(a)anthracene
Benzo(a)anthracene 105105 115 115 ND ND 53.2 53.2 50.3 50.3 ND ND
Benzo(k)flouranthene
Benzo(k)flouranthene 19.819.8 59.3 59.3 25.2 25.2 7.1 7.1 12.0 12.0 9.3 9.3
Benzo(b)flouranthene
Benzo(b)flouranthene 5.8 5.8 18.0 18.0 4.1 4.1 18.5 18.5 51.1 51.1 49.5 49.5
Perylene
Perylene 10.610.6 30.3 30.3 181 181 ND ND 641 641 399 399
Benzo(g,h,i)perylene ND 12.5 8.5 ND ND ND
Benzo(g,h,i)perylene ND 12.5 8.5 ND ND ND
Dibenzo(a,h)anthrace
Dibenzo(a,h)anthracene NDND 12.6 12.6 ND ND ND ND ND ND ND ND
ne
Indeno(1,2,3-
Indeno(1,2,3-cd)pyrene
3.6 3.6 ND ND
ND ND
ND ND
32.0 32.0
ND ND
cd)pyrene
Total PAHs Concentration
Total PAHs 976 1150 790 1400 2220 1750
(µg/kg)
Concentration 976 1150 790 1400 2220 1750
(µg/kg)
Table 5. PAHs concentration (µg/kg) in the soil around the cattle market in Isheri, Nigeria
Table 5. PAHs concentration (μg/kg) in the soil around the cattle market in Isheri, Nigeria
Concentration (m g/kg) with Depth
3
Downstream (15 - 30cm
2.5
Downstream (0 - 15cm)
2
Around the Market (15 - 30cm
1.5
Around the Market (0 - 15cm)
1 Upstream (15 - 30cm)
0.5 Upstream (0 - 15cm)
0
A n nt hr e
en h th n e
Py n e
nz a )a rys e
n o h) e ry e
Fl t hra n e
nz )flo r a e
nz g ,h ery ene
ne
P h F l o e ne
nt e
b ) a ne
an e
, 2 t h ne
ht e
en hy e
d) ne
a en
h en
de a, )p len
B e o(k nth en
ra n
ur e n
ap len
A c pht len
A c ap ale
he
re
e
e
o( ur ce
-c ce
(1 an le
en ur
flo nt h
P th
ou c
h
py
a a
,3 ra
l N th
h y ph
I n o( , i
e t Na
B e o(
b e o(
nz
Di n z
Be
Be
m
2-
Polycyclic Aromatic Hydrocarbons
Figure 4. Variation in the concentration of PAHs with respect to depth in the soil around the cattle market in Isheri,
Nigeria
22
Comparing the values obtained in this study with background soil concentrations of PAHs
(Table 5) [91], the concentrations obtained in this study was higher than the concentrations in
rural and agricultural soils in most cases except benzo(g,h,i) perylene and indeno(1,2,3-
cd)pyrene. There is a significant and positive correlation between phenanthrene versus
flourene and perylene; flouranthene versus pyrene, benzo(k)flouranthene, dibenzo(a,h)an‐
thracene and benzo(g,h,i)perylene; and benzo(k) flouranthene versus benzo(g,h,i)perylene
and dibenzo(a,h)anthracene.
Compounds Concentrations (x 10-3 mg/kg)

Rural Soil Agricultural Soil Urban Soil
Acenaphthene 1.7 6 NE
Acenaphthylene NE 5 NE
Anthracene NE 11-13 NE
Benzo(a)anthracene 5-20 56-110 169-59,000
Benzo(a)pyrene 2-1,300 4.6-900 165-220
Benzo(b)fluoranthene 20-30 58-220 15,000-62,000
Benzo(c)perylene NE 53-130 60-14,000
Benzo(g,h,i)perylene 10-70 66 900-47,000
Benzo(k)fluoranthene 10-110 58-250 300-26,000
Chrysene 38.3 78-120 251-640
Fluoranthene 0.3-40 120-210 200-166,000
Fluorine NE 9.7 NE
Indeno(1,2,3-cd)pyrene 10-15 63-100 8,000-61,000
Phenanthrene 30.0 48-140 NE
Pyrene 1-19.7 99-150 145-147,000
Note: NE-Not established
Table 6. Background soil concentrations of PAHs
3.6. Organochlorine pesticides
There is variation in the concentrations of the organochlorine pesticides (OCP) in the soil
samples collected around the cattle market (Table 6). The major OCP residue identified in the
area with their percentage concentrations were p,p'-DDT, 344 µg/kg (24.5%), δ-BHC, 290
µg/kg (20.7%), Endosulfan sulphate, 186 µg/kg (13.2%), lindane (γ-BHC), 129 µg/kg (9.1%),
Endrin, 110 µg/kg (7.8%), Aldrin, 93.9µg/kg (6.7%), Dieldrin, 93.8 µg/kg (6.7%), o,p-DDE, 45.0
µg/kg (3.2%), β-BHC, 42.1 µg/kg (3.0%), Endosulfan II, 34.5 µg/kg (2.5%), and Heptachlor, 22.8
µg/kg (1.6%). The variation in the concentrations of the organochlorine pesticides with respect
to depth around the cattle market is presented in Figure 4. Dieldrin, a metabolite of aldrin is
present at higher concentration in most of the sampling points when compared to the parent
aldrin. The presence of o,p’ DDE revealed that there is historical use as well as recent us of the
parent DDT in the vicinity of the cattle market. Though, DDT has been banned for over 20
years, it’s presence around the cattle market indicate that it is illegally imported into the
country as it is not produced in Nigeria. Also, lindane, one of the nine new POPs was found
at high concentration around the market. The presence of some of the banned chemical residue
around the cattle market revealed that there is weak regulation and low enforcement on
banned chemicals in the country.
In most cases, the concentrations of organochlorine pesticides is highest at the sampling point
C which could be attributed to the use of pesticides to control household pests and insects in
the area; as the area is a slum with most houses built of planks, rust iron sheets and polypro‐
pylene material and a dumpsite at the bank of the river. There is very strong and significant
correlation at the 0.01 levels (2-tailed) between DDT versus δ-BHC, heptachlor, aldrin and
dieldrin; dieldrin versus δ-BHC, heptachlor and aldrin; aldrin versus heptachlor as well as
endrin versus δ-BHC.
Sampling area A Sampling area B Sampling area C
A1S1 A2S2 B1S1 B2S2 C1S1 C2S2
Organochlorine Pesticide (0-15cm) (15-30cm) (0-15cm) (15-30cm) (0-15cm) (15-30cm)
α-BHC 13.2 ND ND ND ND ND
β-BHC 34.5 7.6 ND ND ND ND
Lindane 108.5 12.0 7.5 ND ND ND
δ-BHC 47.4 ND 53.7 60.8 64.4 63.5
Heptachlor 3.3 ND 3.7 5.3 6.0 4.5
Aldrin 13.6 ND 14.2 16.4 34 14.7
Heptachlor-epoxide ND ND ND ND ND ND
cis-Chlordane ND ND ND ND ND ND
Endosulfan I ND ND ND ND ND ND
trans-Chlordane ND ND ND ND ND ND
DDE ND ND 14.0 14.3 16.7 ND
Dieldrin 15.2 ND 15.1 16.4 22.2 24.9
Endrin 23.4 ND 28.0 23.7 13.5 20.9
Endosulfan II ND ND ND ND 15.8 18.7
Endosulfan sulphate ND ND ND ND 186 ND
p,p' DDT 45.6 ND 59.4 66.0 97.8 75.2
Methoxychlor ND ND ND ND ND ND
Total organochlorine
pesticides concentration 305 19.6 196 203 456 222
(µg/kg)
Table 7. Organochlorine pesticides concentrations (µg/kg) in the soil around the cattle market in Isheri, Nigeria
0.4
0.35
0.3
Concentration (mg/kg) with Depth
0.25 Downstream Area (15-30cm)

Downstream Area (0-15cm)
M arket Area (15-30cm)
0.2
M arket Area (0-15cm)
Upstream Area (15-30cm)
0.15 Upstream Area (0-15cm)
0.1
0.05
0
ne
II
ri n
rin
in
r
ate
T
lo
D
r
da
D
d
ld
d
ch
ph
fa
D
iel
En
D
n
A
ta
ul
Li
l
p'
p'
su
ep
os
o,
p,
n
H
lfa
En
su
do
En
Organochlorine Pesticides
Figure5.
Figure 5. Variation
Variationininorganochlorine
organochlorinepesticides
pesticide residue
residue concentrations
concentrations with respect to
with respect to depth
depth in
in the
the soil
soil around the
around
cattle the cattle
market market
in Isheri, in Isheri, Nigeria
Nigeria
4. Conclusion
4. Conclusion
In this study, the distribution, concentration and profiles of some ubiquitous environmental
In this study,
pollutants suchtheasdistribution,
heavy metals concentration
(Pb, Ni, Cu, andCr, profiles
Zn and of Cd)some ubiquitous
, polycyclic environmental
aromatic
hydrocarbons (PAHs) and organochlorine pesticides in soil of a cattle market around River
pollutants such as heavy metals (Pb, Ni, Cu, Cr, Zn and Cd), polycyclic aromatic hydrocarbons
Ogun Basin, Isheri, Nigeria were assessed as well as some soil physic-chemical
(PAHs) and organochlorine pesticides in soil of a cattle market around River Ogun Basin,
characteristics. The pH of the top soil was observed to be lower than the corresponding
Isheri,
subsoilNigeria were
in most of assessed
the areas as well
sampled as some
except soildownstream
the areas physico-chemical
the market.characteristics.
The pH of theThe pH of
the top soil was observed to be lower than the corresponding subsoil
topsoil being lower than the subsoil may be responsible for the high metal concentrationsin most of in the areas
sampled
the subsoil. Low pH of top soil means more heavy metals will be dissolved in top soil and than the
except the areas downstream of the river. The pH of the topsoil being lower
subsoil mayofbethe
the texture responsible for theand
soil being sandy high metal
loose alsoconcentrations
might favour thein the subsoil. Low pH
migration/leaching ofof top soil
means
heavy more
metalsheavy metals
to lower will beThe
fractions. dissolved in top
solubilising soil metals
heavy and theintexture
the topofsoil
themay
soil being
be sandy
and loose into
migrating alsothe
might favour
subsoil, theinmigration/leaching
except cadmium, which wasof heavy metals
concentrated totop
in the lower
soil.fractions.
The The
soil of the entire
solubilising heavysample
metalsarea was
in the topsandy loamy
soil may soil with low
be migrating intoconcentration of organic
the subsoil, except in cadmium,
matter. The cattle wastes released in the area might have relatively increased the organic
which was concentrated in the top soil. The soil of the entire sample area was sandy loamy soil
matter content of the soil. Though, the topography of the area and the location of the cattle
with low concentration of organic matter. The cattle wastes released in the area might have
market, being sloppy and close to the river basin favoured the washing away of the top soil
relatively increased the organic matter content of the soil. Though, the topography of the area
and the location of the cattle market, being sloppy and close to the river basin favoured the
25
washing away of the top soil by run-off into the river and this might be responsible for the
high organic matter in the subsoil. The pH of the soil of the area was slightly acidic to neutral.
The soil sample with high organic matter content has high metal concentrations in most cases
except Cd, Pb, Cu and Cr. Generally, there is positive and significant correlation between the
organic matter content versus Pb, Zn, Cu, Ni and Cr. There is variation in the concentrations
of PAHs and organochlorine pesticides residues with depth around the river basin. The
detection of banned organochlorine pesticides in the soil of the area could be attributed to both
the historical use as well as recent use of these chemicals in the area. Thus, there is need for
stricter regulations on banned chemicals to safe our environment from ubiquitous and
persistent environmental pollutants.
Author details
A.A. Adeyi, O.M. Omidiran and O. Osibanjo
Analytical and Environmental Chemistry Unit, Department of Chemistry, University of

Ibadan, Ibadan, Nigeria
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Health 1987 2: 140-148.

chapter 8
Assessment of Historical Heavy Metal Pollution of Land

in the Proximity of Industrial Area of Targoviste,
Romania
Carmen Cristina Elekes
1. Introduction
Contamination of soil and water with organic and inorganic pollutants is a subject of interest
to European policy, looking for new ways of preventing pollution and remediating polluted
sites. Source and type of contamination influences the nature of pollution and methods of
remediation. Thus, accidental discharge can be isolated more easily from a small area of soil,
while the contamination of water is more difficult to control. Deposition of particulate matter
can contaminate wide surfaces with low concentrations of contaminants. However, particulate
matter is the main source of contamination of soils with heavy metals.
Many international researches have studied the concentrations of metals in soil, in correlation
with texture, structure and pH of soil. In order to reduce the concentration of heavy metal to
a level allowing development of all crop plants under a secure intake level of heavy metals,
pollution mitigation measures should be applied on those soils. Depending on land use, the
legislation of each country establishes the normal and maximal limits of heavy metals in soil.
The development of metallurgical activities in an irresponsible manner and without taking

into account the environmental damage, lead to a historical accumulation of heavy metals in
soil. The concentration of heavy metals in soil varies significantly depending on the type of
soil and geographic region. This indicates that the parental material, climatic conditions and
human activities have a predominant impact on the chemical forms (speciation) and on the
mobility of metals in soil. In addition to the methods of prevention and mitigation of pollutants
emissions, there are a number of methods of remediation, based on soil stripping and replace‐
ment or methods of bioremediation.
Across Europe, an extensive study was conducted concerning the concentration of heavy
metals in soil [1], involving the collaboration of several organizations: EuroGeo Survey,
Geological Survey of Finland (GTK) and the Forum of European Geological Survey Directors
(FOREGS). The project was conducted between 1996 and 2003, but unfortunately did not cover
the Romanian territory. At the end of the project were drawn distribution maps of all metals
in soils and sediments found along rivers and a Geochemical Atlas of Europe was designed.
Another significant study is Alina Kabata-Pendias book "Trace Elements in Soils and Plants"
published so far in four editions [2]. This book provides a concise but comprehensive overview
of the biogeochemistry of trace elements found in the soil-plant system. Includes over 400
references to recent studies that have been conducted to determine the metal content of the
soil-plant system and highlights the significance of anthropogenic factors leading to the change
of state elements in soil and plants. Subjects are bioindicators behaviour in the environment,
soil remediation, and hyperaccumulation and hyperextraction of heavy metals from soil.
Organizations such as the Food and Agriculture Organization (FAO) and World Health
Organization (WHO) have established very comprehensive reports related to the concentra‐
tion of metals in food and doses considered daily necessary, maximum intake for different age
groups, maximum limits in food and soils (Table 1). In Romania, the reference values for trace
metals in soils are governed by Order 756 / 3rd of November 1997 [3]. It regulates normal
values, alert thresholds and action levels for different trace elements by use of soils.
Heavy Canadian Standard Dutch standard

metal Arable land Inhabited area Industrial area Arable land Inhabited area Industrial area
Cu 150 100 150 36 100 500
Zn 600 500 600 140 500 3000
Cd 3 5 3 0, 8 5 20
Pb 375 500 375 85 150 600
Table 1 Maximum limits of heavy metals in soil, according with Canadian and Dutch standards (ppm) [4]
The present study completes FOREGS project and aims to establish the concentrations of heavy
metals in an industrial area, near the city of Targoviste. Depending on the climatic character‐
istics, on topography and on pollution rose, we have established representative points of soil
sampling for the area. Samples were collected from both the surface of soil to determine the
horizontal distribution of heavy metals in the industrial area, as well as on profile (0-40 cm) to
determine the vertical distribution of the metals and to assess the extent of historical pollution.
2. Heavy metals occurrence in soil and potential impact on life quality
The problem of soil contamination with heavy metals, fuel and other toxic materials is a reality
worldwide. Following the accidents which occurred with discharges of toxic materials on the
Assessment of Historical Heavy Metal Pollution of Land in the Proximity of Industrial Area of Targoviste, Romania 3
ground, the affected area increased by infiltration of substances into groundwater. The
groundwater is carrying the pollutants to residential areas, endangering the health of residents.
Figure 1. Transfer of metallic particulate to human body
Heavy metals are natural components, which occurred in high concentrations under natural
conditions. In the twentieth century, metalliferous uploading of air, water, soil and therefore
of plants and the human body, has become an important concern of international researchers.
Heavy metals are considered a risk for living organisms because they tend to bioaccumulate.
Bioaccumulation is an increase of concentration of a chemical in living organisms, as compared
to the concentration of the element in the environment. Compounds are accumulated in living
organisms by uptake from environment and storage at a higher rate than that of metabolism
or excretion.
Emissions from metallurgical plants are transported by air masses and then deposited on the
ground, leading to an increase of metal concentration in the upper layer of soil. Plants,
perennial grasses especially have a high storage capacity of such metals in their shoots. The
plants loaded with large amounts of heavy metals are consumed by animals that are grazing
of this land (Figure 1).
Compounds are accumulated in living organisms by uptake from environment and storage at
a higher rate than that of metabolism or excretion.
Emissions from metallurgical plants are transported by air masses and then
4 Environmentalon
deposited Riskthe
Assessment
ground, of Soil Contamination
leading to an increase of metal concentration in the upper layer of
soil. Plants, perennial grasses especially have a high storage capacity of such metals in
their shoots. The plants loaded with large amounts of heavy metals are consumed by
Metal concentration
animals that are grazingin soil of
varies
thissignificantly
land (Figure depending
1). on the soil type, but also by region
[5, 6]. This
Metalindicates that the in
concentration parent materialsignificantly
soil varies and climaticdepending
conditions have a predominant
on the soil type, but also by
impact on
region the chemical
[5,6]. state of metals
This indicates that inthe
soil.parent
Kabata-Pendias
materialandand Krakowiak
climatic[7] set a factor have a
conditions
of soil parameters
predominant impact(RDI on- Relative
the chemical explanation
stateindex) basedinonsoil.
of metals the calculation of correlation
Kabata-Pendias and Krakowiak
coefficients
[7] set a factormatrix for metals
of soil parametersand some (RDIsoil parameters:
- Relative pH, clay content,
explanation cation exchange
index) based on the calculation
ofcapacity, substance
correlation the organic
coefficients matrixcontent
forofmetals
the soiland
and some
iron. For approx.
soil 1000 samples,
parameters: the content,
pH, clay
strongest
cation positive linear
exchange correlation
capacity, was obtained
substance for metals
the organic and fine
content of fraction
the soilofand
the soil.
iron.This
For approx.
relationship
1000 samples,varies for strongest
the the metals studied
positiveandlinear
is wellcorrelation
illustrated bywasthe content of heavy
obtained metals and fine
for metals
in soil which
fraction of theincreases
soil. This with increasing clay
relationship varies content.
for the The higheststudied
metals value ofandthe isRDI
wellwhen
illustrated by
correlated
the contentwith clay content
of heavy metals(60-75%) was calculated
in soil which increases on Zn,
withFe, Ni and Cr,clay
increasing while the lower
content. The highest
value of
value (10-30%)
the RDI waswhen
calculated for Cd, Pb,
correlated withCu, andcontent
clay Mn (Figure 2).
(60-75%) was calculated on Zn, Fe, Ni
and Cr, while the lower value (10-30%) was calculated for Cd, Pb, Cu, and Mn (Figure 2).
Cd
Cd a) Pb b)
Pb
Cu Cu
Mn Mn
Zn Zn
Fe Fe
Ni Ni
Cr Cr
0% 20% 40% 60% 80% 0% 10% 20% 30% 40%
Figure 2 Relative explanation index (RDI), of statistically significant relationship at 99%
Figure 2. Relative
confidence explanation
level, between index (RDI), metals
heavy of statistically
in soilsignificant
and clay relationship
contentat< 99%
0.02confidence
mm (a) andlevel, cation
betweenexchange
heavy metals in soil and clay content < 0.02 mm (a) and cation exchange capacity in soil (b) [2]
capacity in soil (b) [2]
Copper (Cu) is an important element for all life forms, but can be toxic in high concentrations.
Copper (Cu) is an important element for all life forms, but can be toxic in high
The average Cu content in the lithosphere is 70 ppm. In natural soils, the average concentration
concentrations. The average Cu content in the lithosphere is 70 ppm. In natural soils, the
is 2-40 ppm. As described for Ni, Cu has no similarities with any other metal regarding his
average concentration is 2-40 ppm. As described for Ni, Cu has no similarities with any
chemical behaviour in soil. Significant quantities of Cu in the soil are connected in the minerals,
other metal regarding his chemical behaviour in soil. Significant quantities of Cu in the soil
therefore, this metal is supplied only by a very slow decay processes. Cu may occur in the form
are connected in the minerals, therefore, this metal is supplied only by a very slow decay
of readily soluble salts (copper nitrate, copper sulphate), and as an oxide and hydroxide. It
processes. Cu may occur in the form of readily soluble salts (copper nitrate, copper
binds to organic matter, ferric oxide and Al. Intake of Cu in plants can be increased by low pH
sulphate), and as an oxide and hydroxide. It binds to organic matter, ferric oxide and Al.
and organic fertilizers. Cu concentration can increase significantly under the effect of anthro‐
Intake of Cu in plants can be increased by low pH and organic fertilizers. Cu concentration
pogenic activities (non-ferrous metal processing, the use of substances for plant protection).
can increase significantly under the effect of anthropogenic activities (non-ferrous metal
In humans, the contamination has not yet been notified by the dietary intake of Cu in the body,
processing, the use of substances for plant protection). In humans, the contamination has
but its high concentration may cause liver damage.
Zinc (Zn) is widespread in nature and the average content of Zn in the lithosphere is about 80
ppm. Unpolluted soil contains an average of 15-100 ppm of Zn. Zinc binds to organic matter
and ferric and Mn oxides. It occurs in large amounts in the layers of the clay minerals. Under
natural conditions, in A horizon of soil from wet areas, with slightly acid pH, more than half
of the Zn is bound to organic material [4]. Because of the extensive use of Zn in industry, the
Zn content in soil surrounding the industrial areas can reach even 5000 ppm [2]. The effect of
Zn is particularly harmful because its accumulation leads to accumulation of other heavy

metals, such as Pb, Cu and cadmium (Cd). Concomitantly, in the lime-rich soils, the plants
show Zn deficiency symptoms. Also, Cd is strongly chemical bond to the Zn, as the proportion
Zn/Cd in soil is constant. The availability to plants can be influenced by the concentration of
cadmium in soil, pH conditions, temperature, amount of organic matter, and presence of other
metals. Cadmium is irreversibly bound by ferric and manganese oxides in soil, and by clay
minerals that influence the Cd mobility.
Lead (Pb) in soil is largely associated with colloidal organic matter, which results in a high
proportion of Pb accumulated in the top 5-15 cm of contaminated soils. The Pb concentration
decreases with depth in a soil profile [2]. In the geosphere, the average Pb concentration is
about 16 ppm. The increasing of Pb concentration may be caused by the accumulation of fuel
combustion residues from the transportation, by application of sewage sludge and by the use
of some pesticides in gardens or orchards. Increasing soil pH may decrease the absorption of
Pb in soil. Plants are able to accumulate significant amounts of Pb (300-400 ppm) in pollution
conditions without noticeable symptoms [2]. High concentration of Pb particularly affects the
neurovegetative functions, hampers blood and cause chronic emphysema in humans.
Tin (Sn) concentrations in soil are generally low, with values of 2-3 mg/kg in unpolluted areas
and can reach 200 – 1000 mg/kg in areas of high tin deposits [8] or in areas influenced by
anthropogenic activities including smelters of ferrous and non-ferrous metals and coal-fired
power plant [9].
Cobalt (Co) is widely distributed in rocks and soils and always occurs in nature in association
with nickel and usually with arsenic [10]. The common Co minerals are smaltite (CoAs2) and
cobaltite (CoAsS) and the most important sources of cobalt are residues from the smelting of
arsenical ores of nickel, cobalt and lead [10]. Cobalt in environment may represent a hazard to
human health and is considered a metal with marked allergenic potential.
Chromium (Cr) is used in alloying metals, in the industry of paints, cement, paper, rubber and
other materials. Exposure to low concentrations of Cr produce skin irritation and ulceration,
and long-term exposure can cause kidney and liver diseases, and diseases of the circulatory
system and nervous tissue. Chromium accumulates especially in aquatic fish and the con‐
sumption increases the risk of a high intake of this metal.
Nickel (Ni) is necessary in small quantities in the human body to produce red blood cells [4],
but greater amounts (>1.0 mg/d) may become toxic. Short-term exposure to Ni does not cause
health problems, but over a long period leads to weight loss, heart and liver diseases, and skin
irritation. Nickel can accumulate in aquatic organisms, but its presence increases for higher
levels on the food chain.
Manganese (Mn) is a metal naturally ubiquitous in the environment, found in many types of
rocks and soil, essential for normal physiologic functioning in humans and animal. Exposure
to low levels of Mn is considered to be nutritional for humans. Long-term exposure to high
levels of Mn by inhalation in humans may result in central nervous system effects. The metallic
Mn is used in steel production to improve hardness, stiffness and strength. Mn is also used in
carbon steel, stainless steel and high-temperature steel, along with cast iron and superalloys
[11]. The average Mn levels in soil range from 40 to 900 ppm [11].
Molybdenum (Mo) is a valuable alloying agent which contributes to the hardness and
toughness of quenched and tempered steels. Mo also improves the strength of steel at high
temperature. In the environment, Mo differs from the other micronutrients in soils because it
is less soluble in acid and more soluble in alkaline soils. Mo availability to plants is sensitive
to pH and drainage conditions. Some plants can have up to 500 ppm of the metal when they
grow on alkaline soils [12].
2.1. Heavy metal pollution of soil

The presence of heavy metals in natural and contaminated soils shows a great variability in
both the horizontal and vertical dimensions [2]. Chemical pollution of soils in Romania is
affecting approx. 0.9 million ha of soil, of which 0.2 million ha are affected by excessive
pollution. Adverse effects are particularly strong to pollution by heavy metals (Cu, Pb, Zn, Cd)
and sulphur dioxide, identified especially in Baia Mare, Zlatna and Copsa Mica. Although in
last years a number of industrial units have been closed and others have reduced activity, the
soil pollution is quite high in some areas: Targu Mures, Turnu Magurele, Tulcea and Slatina.
Oil pollution and salt water from oil wells and transport affects approximately 50, 000 ha. Soil
damaged by excavation comprises 15, 000 ha and constitutes the most serious form of damage
to soil, encountered in the mining industry, for example in the mining basin of Oltenia.
Suitability of land affected by this type of pollution decreased by 1-3 classes, and some of these
areas has become unproductive. Soil cover with solid waste and residues caused sealing of
approximately 18 000 ha of farmland and meadows [13]. Direct economic damage on agricul‐
tural production due to these restrictions is estimated by reducing it by about 20% per year.
The study of Lăcătuşu and Ghelase [14] aimed to assess the abundance of anthropogenic heavy
metals in soils at various distances from Romfosfochim SA Valea Călugărească. The research‐
ers compared the specific data of metal concentrations in polluted soils with those of similar
soils, not subject to pollution effects. The results showed a decrease in the percentage of
geogenic abundance with proximity to the source of pollution, although the concentrations of
Cu, Pb, Zn and Cd are significantly higher at distances between 0-500 m compared to the
distance of 6 km [14]. The depth of penetration into the soil of heavy metals from industrial
emissions is shallow (up to 15 cm) in forest soils and up to 30-40 cm in arable soils [15].
Vrînceanu et al. [16] have published the results of research on polluted soils from Copsa Mica,
showing metal concentrations for Cu, Zn, Pb and Cd in soil, with values between 69-136 mg/
kg, 962-2191 mg/kg, 1182 -1978 mg/kg and 30-42 mg/kg respectively. In 2002 a study showed
that in the soil from Baia Mare the Cu concentration exceeded 9.5 times the maximum limits
and 4.8 times the alert and action limits. The concentration of Pb exceeded 132 times the alert
threshold and 66 times the limit of intervention and the concentration of Zn exceeded 11 and
6 times these limits respectively [17]. Recent researches in Baia Mare showed some decreasing
of heavy metal concentration, but the average values of these concentrations exceed 6 times
the maximum level of lead. In the case of Cu, Zn and Cd the average values exceed the
maximum level by 10, 3 and 7 times. Multiple pollution average index for the four heavy metals
determines the classification of this area as excessive pollution class (values greater than 16)
in the layer of 0-10 cm and as very strong pollution class in the layer of 10-20 cm. The maximum
values of this index reached 78.2 in some excessively polluted areas. In 1994, a land of 21, 875
ha (3, 245 ha of forest and 18, 630 ha of agricultural land) have been severely affected by heavy
metal pollution by exceeding the maximum limits for Pb, Cu, Zn and Cd [16].
Maximum levels of metal pollution were detected in Baia Mare for Cd, in Copşa Mica for Pb
and Zn, and in Valea Călugărească for Cu [15]. Besides these considerations we can add the
ecological accident occurred at Baia Mare in 2000 that led to the contamination of water and
soil with cyanide from extraction plants. Specific conditions of those soils (moderate to severe
acidity) are favouring the translocation of pollutants from soil to plants, animals and humans,
leading to an increase of metal toxicity and a reduction of soil and water quality. Research
conducted in 2000 in several areas in the south of Romania have shown the persistence of
severe soil pollution with heavy metals in the vicinity of industrial plants (S.C. Neferal and
S.C. Acumulatorul-Bucureşti-Pantelimon, S.C. Turnu S.A. – Turnu Măgurele) and pollution
with fluoride near ALRO-Slatina.
All these environmental assessments showed the necessity of soil quality improving policy,
by changing the land use and by replacing the food crops with industrial crops. Also these
areas need measures to reduce the pollution and its toxic effects. Exploit of Romanian peat
deposits for the purpose of complete the organic matter of soil, could be a source for improving
soil quality and reducing pollution [18].
2.2. Availability of toxic metal compounds in the soil for plants
A global statistical evaluation of the substances exchange between soil and plants, led to the
conclusion that the percentage itself is influenced by the following parameters:
• Soil texture;
• Carbon content of organic substances;
• Cation exchange capacity of the soil;
• Calcium carbonate equivalent;
• Oxides and hydroxides, mainly Fe, Mn and Al;
• pH values - dominant factor.

Soil organic fraction plays an extremely important role because they can delay both the
accumulation and transfer of metals and their movement into the soil. Metal toxicity in soil
can be increased or reduced by soil organic fraction. Soil pH directly influences the availability
of metals as soil acidity determines solubility of element and its ability to move in the soil
solution. Regarding the content of phosphorus in soil, the presence of high doses of P2O5 can
increase or decrease metal uptake. In addition, the accumulation of metals is directly influ‐
enced by the plant physiology. For example, Cd uptake in grain has been described to be either
antagonistic or synergistic with high concentrations of Pb in the soil [19].
In discussions about soil protection and remediation, pollutant limits for various elements
have been established only under certain conditions and soil parameters. It was not taken into
account the specific conditions such as the fact that on low-carbon light soils there is strong
influence of rainfall leading to a strong acid mobilization and uptake into plants of toxic heavy
metals. This does not happen on heavier soils rich in limestone.
The solubility of Zn in soil was studied by Herms and Brummer [20], which demonstrated the
extent to which this element is dissolved by increasing acidity of the soil and became available
for plants uptake. A pH value of 5 of low-Zn soil could lead to lasting effect of uptake large
amounts of Zn, with all the negative consequences that result. The balance of Zn in the soil
solution is carried out according to the pH of soil: at 1200 mg/kg of Zn and a pH of 7, at 100
mg/kg of Zn and pH of 6, and at only 40 mg/kg Zn and a pH of 5. This indicates that also the
low-Zn soil can store dangerous amounts of available Zn.
3. Studied area
Metallurgy activities produce gas, wastewater and waste containing pollutants that can be
sources of risk under normal handling, and especially for irresponsible handling of equipment.
The main resulted pollutants are: CO2, CO, NO, SO, VOCs (e.g. BaP, PAHs, dioxins, Freon),
particulate heavy metals (Pb, Cu, Zn, As, Cr), cyanides, phenols, heavy metals and sometimes
toxic organic compounds in waste material.
Annual average concentrations exceed the maximum permitted levels in many localities (Baia
Mare, Copsa Mica, Medias, Targoviste, Arad, Deva, etc.), for both particulate matter and for
sediment, coming mainly from industry of steel.
The main polluter from industrial zone of Targoviste city was SC Mechel Targoviste SA,
located in the south of the city. By the metallurgical activity, emissions were resulting, with
significant concentrations of pollutants, including heavy metals. In the process of obtaining
steels at SC Mechel SA, technological flow was served by Electric steelmaking 2, EBT electric
oven with a capacity of 70 tons/hour and Continuous casting plant (in billets) with a maximum
capacity of production of 2.5 tons/hour. The activity SC Mechel Targoviste SA was assisted by
filtration systems so that the environmental impact to be reduced to a lower limit.
During the elaboration of steel in Electric steelmaking no. 2, total dust are emitted to air, metals
(Cd 0.05 mg/Nm3, Pb 0.3 mg/Nm3 and Cr + Cu + Mn + Ni + Zn 5.0 mg/Nm3), organic compounds,
nitrogen oxides (NOx), carbon monoxide (CO) and sulphur oxides (SOx) [21]. In addition to
these emissions of heavy metal particulates, SC Mechel SA polluted the soil by the waste and
slag dumps generated during the metallurgical processes and stored in an open deposit.
Dispersion of pollutants in the atmosphere largely depends on emission characteristics,
meteorological factors, topography, soil roughness, the height of buildings, especially the stack
height. Among the meteorological factors, wind direction, intensity, and thereof frequency,
are the dominant factors on which we can determine the wind rose indicating the direction of
predominant movement of air masses. Based on the wind rose [22] can be designed the
Figure 3. Dispersion and transport of pollutants particles according to the pollution direction compass, relative to the
most significant pollution source, the furnace of the Electric steelmaking no. 2 belonging to SC Mechel SA
pollution direction compass which indicates the direction of dispersion and transport of
pollutants particles (Figure 3). In the middle of pollution direction compass is the most
significant pollution source, the furnace of the Electric steelmaking no. 2 belonging to SC
Mechel SA. East was one of the predominant directions of pollutants transport and the particles
have deposited on the ground (agricultural fields) during the process of steel elaboration.
In addition to the pollution produced by ground deposition of particles emitted by the SC

Mechel SA chimney, these soils are affected by deposit of slag and skim from the same plant.
The dumps that are within walking distance (less than 10 m) of agricultural land of residents
from Colanu, Romania, are subject to a continuous process of erosion and transport by wind,
smaller particles from the surface of stockpiles being transported up to 10 km. Dispersion of
the slag particles is due to transport activities to and from the place of storage of metal waste.
Characteristic types of soils for studied area are gray luvisol and gray brown luvisol (Figure
4) according SRTS-2003 (reddish brown after SRCS 1980), soils of a reddish hue, quite evident
in the upper horizon and really evident in the middle horizon. Water and air permeability of
the soil was moderate. Humus content was about 3%, nutrient supply was moderate, the soil
reaction was low-acid with pH values in the range of 6.0 to 6.4, and the degree of base saturation
was 80% to 85%.
Figure 4. Gray brown luvisol profile (a) and brown-yellowish-rusty upper horizon (b)
Bioaccumulation in upper and middle horizon was low; plant debris was mostly decomposed
by the action of fungi, and could be observed the formation of small amounts of humus with
predominating fulvic acids. Due to intense alteration of mineral component occurs removal of
clay colloid from the surface, with accumulation in the Bt horizon, where the profile shows a
textural differentiation.
In soils with alluvial B horizon, such as soil in the industrial area of the Targoviste city, colloidal
order mineral fraction of this horizon contains large amounts of colloidal hydroxides (e.g.
Fe(OH)3 and Al(OH)3) and various hydrated iron and aluminium sesquioxides free-form
occurs removal of clay colloid from the su
the profile shows a textural differentiation.
the
orde
amo
Al(O
sesq
amo
coul
soils
The
brow
Figure 5. Iron hydroxides
Figure 5. Iron hydroxides
rust
(mR2O3 nH2O) [23]. Large amounts of iron hydroxides in the humus horizon could be observed
for the brown and reddish-brown soils from the industrial zone of Targoviste (Figure 5). The dep
presence of these hydroxides is manifested by brown-yellowish, brown, reddish-brown,
4. Material and method
yellowish-rusty or rusty lit of the horizon where they are deposited.
4.1 Experimental design

Sampling points were chosen
to reflect a snapshot of the impact of

4.1. Experimental design
Sampling points were chosen to reflect a snapshot of the impact of metallurgical activities in
metallurgical activities in this area by
this area by particles emissions. Based on weather conditions and pollution direction compass
of Dâmboviţa County (Figure 6), were determined that areas found at SW and W towards the
particles emissions. Based on
emission source are the most affected. In that location have been chosen the harvest area Zone
I – industrial fields, with two subzones, for SW and W directions respectively. Land at NE, E
weather conditions and pollution
and SE from the source of pollution, are grouped according with the use category in Zone II –
agricultural fields sensitive to high concentrations of heavy metals.
direction compass of Dâmboviţa

County (Figure 6), were determined
that areas found at SW and W
The presence of these hydroxides is manifested by
brown-yellowish, brown, reddish-brown, yellowish-
12 rusty orRisk Assessment
Environmental rusty of lit of the horizon where they are
Soil Contamination
deposited.
d
n
hosen
pact of
rea by
d on
ollution
boviţa
mined
nd W
are the
n have
Zone I
two
ections
d SE from Figure
the source 6. Sampling
of directions
pollution, are cityfrom
groupedindustrial accordingarea of with
Targoviste city according to the pollution rose
Figure 6. Sampling directions from industrial area of Targoviste according to the pollution rose
agricultural fields sensitive to high concentrations of heavy
Sampling of soil was done at distances between 50 and 1000 meters from the source of
pollution, from five different points, chosen according to triangle method. The results of metal
ne at distances between 50 and 1000 meters from the source
concentration represent the average of these five samples. From each sampling point, samples
were taken from three layers: the upper layer (0-5 cm depth), middle layer (5-20 cm depth) and
points, chosen according to triangle method. The results of
lower layer (20-40 cm depth). These layers were chosen according with the depth to which the
the average of these five samples. From each sampling point,
roots of culture plant normally develop.
layers: the upper

The soil samples layer (0-5incm
were processed depth),
the laboratory middle
for elemental layer
analysis (5-20The
by ICP-AES. cm soil
samples were dried at 40 °C for 24 hours, ground to a fine powder, sieved at 250 µm (according
0 cm depth). These layers were chosen according with the
to SR ISO 11464).
ure plant normally develop.
4.2. Analytical methods
Determination of heavy metal concentration in soil was done on replicates samples by

Inductively Coupled Plasma - Atomic Emission Spectrometry method (ICP-AES). The soils
samples were mineralized in Berghof microwave digester, using a mixture 1:1 with nitric acid
(according with Berghof method) prior to ICP-AES analyses. The advantage of ICP-AES is the
multielemental detection (Cu, Zn, Sn, Pb, Co, Ni, Mn, Cr and Mo) [24]. For this research,
analyses were conducted with Liberty 110 spectrometer of Varian brand. The minimal
detection limits of device range according to the analysed element and is 0.4 mg/kg for Zn, Mn
and Cu; 0.5 mg/kg for Cr and Co; 0.6 mg/kg for Sn, Ni and Pb. The concentrations values for
analysed metals were expressed in milligrams of metal per kilogram of dry soil (mg/kg).
The soil pH was determined with a portable pH-meter, WTW 3110 SET 2, with precision of
0.01 units. For pH analyses, 5 g of each soil sample were mixed with 50 ml KCl 0.1N, F 1000,
Tt 0.0056 g/ml and homogenized for 15 minutes with a magnetic stirrer.
5. Results
5.1. Statistical results of heavy metal concentration
In the industrial area of Targoviste city, the concentration of heavy metals in soil was highly
dependent on the metal species, the position of sampling point towards the source of pollution,
the stack, and the depth of sampling. The general statistical results of heavy metal concentra‐
tion in studied area are presented in Table 2.
The variation of Cu concentration in the 0-5 cm layer of soil was higher than the variation on
the profile. Cu concentration ranged from 578.4 mg/kg in the surface layer, to 170.4 mg/kg (5
-20 cm) and 152.4 mg/kg (20-40 cm). The Cu concentration variation showed that 75% of the
samples had values of concentration up to 135.9 mg/kg, and only 25% of the samples had higher
concentrations than this value. The same pattern of variation of Cu concentration was followed
for the deeper layers. For the layer of 5-20 cm depth, most samples (75%) had concentrations
within a narrow range, from 28.7 to 43.6 mg/kg and for the layer of 20-40 cm depth 75% of
samples range between 22.0 to 55.2 mg/kg.
The variation of Zn concentration was very high at the surface of soil profile (827.7 mg/kg) and
lower in the deeper layers of soil (128.8 mg/kg and 186.9 mg/kg respectively. In all the three
studied layers, the concentration of Zn in most of the samples was placed in a very small range
as compared to the magnitude of total concentration range. In the surface layer, 50% of the
samples showed a concentration of Zn from 42.6 to 86.1 mg/kg and 75% were found between
42.64 to 225.03 mg/kg. The same pattern of distribution of Zn concentration was followed in
the deeper layers, 75% of the samples being in a relatively narrow range of concentration from
44.4 to 72.9 mg/kg for the depth of 5-20 cm and 33.1 to 81.8 mg/kg for the layer of 20-40 cm
depth.
The values of Sn concentration showed a higher homogeneity in soil when compared to Cu

and Zn concentration. Sn concentration ranged from 92.7 mg/kg at the surface of profile to
about 40 mg/kg in deeper layers (5-40 cm). The four quadrants of Sn concentration distribution
showed narrow values, though there was a concentration of Sn values (50%) in the range 32.7
to 53.9 mg/kg for 0-5 cm layer. In the middle layer (5-20 cm), about 75% of the sample ranged
from <LD to 20.9 mg/kg. At 20-40 cm depth, quadrants II and III of the concentration distri‐
bution were distributed on the interval from 15.39 to 22.8 mg/kg, the remaining 50% of the
samples having values lower or higher than this range.
Metal Depth Mean±SD Min-Max Median Q1 Q3 Skewness Kurtosis
Cu 0-5 cm 125.5±162.6 22.0-600.4 36.5 30.9 135.9 2.1185 4.6664
5-20 cm 61.1±55.0 28.7-199.1 35.1 32.5 43.6 1.8284 1.9995
20-40 cm 63.3±53.1 22.0-174.5 38.1 35.4 55.2 1.5680 0.7874
Zn 0-5 cm 223.9±269.0 42.6-870.3 86.1 73.3 225.0 1.6278 1.3552
5-20 cm 74.3±42.9 44.4-173.2 54.1 48.4 72.9 1.5888 1.0601
20-40 cm 75.6±50.61 33.1-220.1 53.1 49.4 81.8 1.9591 4.0515
Sn 0-5 cm 65.1±30.2 32.7-125.4 53.9 41.9 84.8 0.7305 -0.7436
5-20 cm 12.9±12.9 <LD-44.4 14.9 <LD 20.9 0.8094 0.8786
20-40 cm 17.8±10.9 <LD-38.2 19.4 15.4 22.8 -0.3501 0.0826
Pb 0-5 cm 76.4±98.9 0.6-294.3 43.3 0.66 92.1 1.3987 0.8529
5-20 cm 47.9±42.6 <LD-121.0 42.5 20.86 64.1 0.7683 -0.5585
20-40 cm 34.0±43.6 <LD-145.6 26.3 <LD 35.6 1.5800 2.0421
Co 0-5 cm 16.2±4.8 7.1-23.5 17.3 12.9 19.1 -0.5281 -0.4808
5-20 cm 14.7±5.1 6.7-21.5 13.6 10.3 19.4 -0.0702 -1.6268
20-40 cm 13.1±4.0 6.7-19.9 14.9 9.5 15.6 -0.3423 -0.9854
Ni 0-5 cm 65.1±58.5 13.8-185.4 37.6 22.0 84.8 1.0999 -0.1291
5-20 cm 20.2±15.9 8.7-52.7 13.1 12.3 14.8 1.6371 0.8719
20-40 cm 23.2±23.9 4.4-72.4 14.2 8.3 17.7 1.5576 0.7595
Mn 0-5 cm 1579.7±352.3 1159.9-2348.0 1419.1 1373.2 1731.3 0.9420 0.0714
5-20 cm 1348.9±243.6 758.9-1677.3 1384.1 1322.7 1504.3 -1.2771 1.5204
20-40 cm 1367.5±327.9 720.4-1763.1 1486.3 1258.4 1601.1 -1.0239 -0.1494
Cr 0-5 cm 114.6±125.5 13.7-315.6 25.1 21.6 205.7 0.7343 -1.3288
5-20 cm 46.0±51.7 16.2-168.3 22.7 17.81 30.4 1.7522 1.5205
20-40 cm 41.0±52.9 8.2-159.9 18.3 13.8 21.5 1.6981 1.1754
Mo 0-5 cm 7.2±7.8 0.6-23.4 4.5 0.6 10.5 1.0334 -0.1858
5-20 cm 3.0±3.4 <LD-10.8 1.2 0.7 5.1 1.1289 0.2488
20-40 cm 2.4±3.3 <LD-9.7 1.0 0.6 1.6 1.6104 0.9883
LD – limit of detection
Table 2 Statistical results of heavy metal concentration (mg/kg) in soil surrounding the industrial area of Targoviste
city
The variability of Pb concentration was very high, most of the samples showing very low levels,
even below the detection limit of the analytical method, in the entire soil profile studied, while
some samples presented significant concentrations of Pb up to 294.3 mg/kg (0-5 cm), 121.0 mg/
kg (5 to 20 cm deep) and 145.6 mg/kg (20-40 cm). The distribution of the samples in terms of
the concentration was varying, 75% of the samples from the surface of soil profile were in the
range of concentrations from 0 to 92.1 mg/kg, and the remaining 25% were in the range of 92.1
to 294.3 mg/kg. The Pb distribution was less variable in the deeper layers, the four quadrants
of the concentration being distributed at relatively equal intervals, especially for the depth 5-20
cm. Heterogeneity of the sample in terms of the concentration of lead in the ground was
indicated by the high value of the standard deviation, higher than the average concentration
throughout the studied soil profile.
The concentrations of Co in the samples were distributed homogeneously throughout the

range of concentrations for both the upper layer and deeper layers of 5-20 cm and 20-40 cm
respectively. The homogeneity of the samples was indicated by the low values of standard
deviation, between 29% and 34% of the average, and by the median value that was very close
to the average value, 17.3 mg/kg, 13.6 mg/kg, respectively 14.9 mg/kg for the three depths. On
the surface of the soil profile, 50% of the analysed samples were in the range of concentration
from 12.9 to 19.1 mg/kg.
In surface layer of soil, the range of Ni concentration varied widely, up to 185.4 mg/kg. The
soil sample from surface layer presented heterogeneous distribution of Ni, with 75% of the
samples from 0-5 cm depth showing a concentration ranging 13.8 to 84.8 mg/kg, and only 25%
of the samples ranging 84.8 to 185.4 mg/kg. The Ni concentration variation was lower in deeper
layers; most of the samples (75%) were covered by a much narrower range of concentration
from 8.7 to 14.8 mg/kg for 5-20 cm depth, and 4.4 to 17.7 mg/kg for 20-40 cm depth.
Distribution of samples within the range of Mn concentration was uniform and 50% of the
samples were within the range of 1373.2 to 1731.3 mg/kg, while the remaining 50% of the
samples from the surface of the soil profile were higher or lower than this range. The same
pattern of samples distribution was followed in the deeper layers, where 50% of the samples
were concentrated in the middle of the range of Mn concentration. The standard deviation of
the Mn concentration indicates that the samples had similar values of concentration for each
layer, accounting 22%, 18% and 24% respectively for the three layers of 0-5 cm, 5-20 cm and
20-40 cm.
The wide variation of Cr concentration in the samples was reflected by the high value of
standard deviation, greater than the average concentration for all three depths. Half of the
samples from 0-5 cm layer of soil showed Cr concentrations in a very small range between 13.7
and 25.1 mg/kg. The remaining 50% of the samples were distributed in a wider concentration
range between 25.1 and 315.6 mg/kg. At depths greater than 5 cm, 75% of the investigated
samples had concentrations of Cr in a narrow range of 14.2 mg/kg for the depth of 5-20 cm and
13.3 mg/kg for the depth of 20-40 cm, while the remaining 25% had concentrations in the range
of 137.9 mg/kg and 138.4 mg/kg respectively for the two layers of soil.
The range of Mo concentration was higher in the surface of soil profile (22.8 mg/kg)
compared to the range of concentration of deeper layers of soil (10.8 mg/kg and 9.7 mg/kg
respectively). The majority of samples (75%) showed low levels of Mo, up to 10.5 mg/kg
on the surface and 5.1 mg/kg and 1.6 mg/kg in the deeper layers of 5-20 cm and 20-40 cm
respectively. The concentration varied greatly, with values of standard deviation higher
than the mean concentration.
5.2. Horizontal distribution of heavy metals
The horizontal distribution of heavy metals and level of metal pollution in the industrial area
of Targoviste city was established by comparing to the Romanian legislation [3], which
regulates normal values, alert thresholds and action levels for different trace elements by use
of soils, agricultural and industrial land (Table 3).
Limit Cu Zn Sn Pb Co Ni Mn Cr Mo
NV 20 100 20 20 15 20 900 30 2
ATA 100 300 35 50 30 75 1500 100 5
ATI 250 700 100 250 100 200 2000 300 15
NV – normal values; ATA – alert threshold for agricultural soil; ATI – alert threshold for industrial soil
Table 3 Normal values and alert thresholds of heavy metal concentration (mg/kg) for agricultural and industrial soil in
Romania [3]
The concentration of Cu on the surface layer of soil differed greatly between the two studied
zones (I and II) (Table 4). Thus, in Zone I, on the SW and W directions towards the emission
source, the concentration of Cu reached 401.44 mg/kg and 134.58 mg/kg, values of 7 to 20 times
higher than normal levels in this category of soils [25]. In Zone II, the concentrations of Cu on
the NE, E and SE directions were slightly greater than the normal value of concentration,
ranging between 22.37 and 36.61 mg/kg.
In the case of Zn, the maximum values allowed for industrial soil, 700 mg/kg were overcome
on W direction to the source, while the value on SW direction was below that limit, but
exceeded the normal value of Zn in soil. The soils in Zone II showed a deficiency of Zn at
surface layer and in the deeper layers. In the upper horizon, the Zn concentration was between
46.03 and 86.07 mg/kg, values that are below the normal value of this element in soil [26].
Sn concentrations in the analysed soils were above normal values (Table 4) in both the Zone
I and Zone II. A concentration of about 6 times the normal limit was found in soils under the
SW direction. For other samples, the concentration of Sn in soil ranged from 33.77 to 82.90 mg/
kg, the lowest value being on E direction and the highest on SE direction.
Although the normal value of Pb in soil is 20 mg/kg, the concentration of this element in the
studied samples of soils did not exceed the threshold. The soil in Zone II showed the lowest
concentration. In the SE direction, Pb concentration in soil was the highest, 43.35 mg/kg. The
soil in the Zone I, showed values of Pb concentration that exceeded 4 to 12 times the normal
values: 85.38 mg/kg in the SW direction and 252.00 mg/kg on the W direction.
The concentrations of Co in soils were not much higher than the normal values of metal
concentration in soil. Higher values were found in the soil from Zone I, 19.93 mg/kg in the W
Direction towards the emission source

Heavy metal
SV V NE E SE
Cu 401.44 134.58 32.41 22.37 36.61
Zn 194.28 719.13 74.45 46.03 86.07
Sn 112.31 52.79 43.78 33.77 82.90
Pb 85.38 252.00 0.60 0.76 43.25
Co 20.42 19.93 14.11 8.94 17.48
Ni 166.64 83.84 15.01 22.42 37.39
Mn 1264.26 2156.48 1726.44 1362.45 1388.85
Cr 200.31 312.33 13.88 24.98 21.68
Mo 9.79 0.6 20.57 0.65 4.45
Exceed the alert Exceed the alert

Under normal limit Exceed normal limit threshold for agricultural threshold for industrial
soil soil
Table 4 Horizontal distribution of heavy metal concentrations (mg/kg) in the industrial area of Târgovişte city
direction and 20.42 mg/kg in the SW direction. The soils in the Zone II showed values of Co
concentrations that varied between 8.94 and 17.48 mg/kg. The lowest value was on E direction,
and the highest value on SE direction.
The concentration of Ni in soils varied widely in the two studied zones. In Zone I, the con‐
centration of Ni was between 4 and 8 times higher than the normal value of metal concentration
in soil, 83.84 mg/kg in the W direction and 166.64 mg/kg on the SW direction. The soil in Zone
II showed Ni concentration close to normal values, ranging from 15.01 to 37.39 mg/kg. The
highest value was for the Ni concentration on SE direction.
Concentration of Mn in the soil was two times higher than the normal value and range between
1264.26 mg/kg and 2156.48 mg/kg. The lowest value of Mn concentration was on SW direction
and the higher on the W direction.
Concentrations of Cr in soils from the industrial area were very different between the two
studied zones. In Zone I, the concentrations were extremely high compared to the normal limit
(30 mg/kg), reaching values over 10 times higher: 200.33 mg/kg and 312.33 mg/kg on SW and
W directions respectively. In Zone II, Cr concentration showed values below the normal
concentration of Cr in this type of soil, and varied between 13.88 and 24.98 mg/kg [27].
Molybdenum concentration in the surface layer of soil varied within wide limits, irrespective
of the position towards the source of pollution. In Zone I, concentration of Mo has the minimum
value in the W direction (0.6 mg/kg), and the maximum value in the SW direction (9.79 mg/
kg). This value was about 5 times higher than the normal value of Mo concentration in soil. In
Zone II, the concentration of this metal varied also within wide limits, from 0.65 mg/kg in the
E direction to 20.57 mg/kg in the NE direction. The recorded values were more than 10 times
higher than normal values.
Molybdenum
5.3. Vertical concentration
distribution in the surface layer of soil varied within wide limits,
of heavy metals
irrespective of the position towards the source of pollution. In Zone I, concentration of Mo
hasThethe minimum
vertical value inofthe
distribution W direction
heavy metals in (0.6 mg/kg),
the soil profileand
(0 –the maximum
40 cm) is shown value in the7SW
in figures –
direction (9.79 mg/kg). This value was about 5 times higher than the
14, which indicates the level of heavy metals in the three soil layers: 0 – 5 cm, 5 – 20 cm and normal value of20Mo
concentration in soil.
– 40 cm. For each In Zone
layer, linearII, the concentration
regression was calculated of this metal varied
to indicate also within
correlations widemetal
of heavy limits,
from 0.65 mg/kg in the E direction to 20.57 mg/kg in the NE direction. The recorded values
concentrations with the pH.
were more than 10 times higher than normal values.
The high concentrations
5.3 Vertical of Cuofinheavy
distribution the SW direction were maintained at very high level in the
metals
depthThe vertical
of soil distribution
profile (Figure 7).of In heavy metals in Cu
the W direction, theconcentration
soil profile (0 – 40 cm)
decrease is shown
to depth of 40 in
figures 7 – 14, which indicates the level of heavy metals in the three soil layers: 0 – 5 cm, 5
cm to levels of 27 mg/kg. The soil in Zone II showed similar values of Cu concentration on the
– 20 cm and 20 – 40 cm. For each layer, linear regression was calculated to indicate
entire profile of soil. Between the Cu concentration and the pH of soil was observed a moderate
correlations of heavy metal concentrations with the pH.
correlation in the
The high surface layer of
concentrations ofsoil
Cu(0.3) and
in the SWvery low correlations
direction for the two
were maintained atdeeper
very highlayers.
level
All the correlations were positive.
in the depth of soil profile (Figure 7). In the W direction, Cu concentration decrease to depth
of 40 cm to levels of 27 mg/kg. The soil in Zone II showed similar values of Cu concentration
Extremely high Zn concentration from the surface layer of soil did not maintained in depth.
on the entire profile of soil. Between the Cu concentration and the pH of soil was observed a
Below 5 correlation
moderate cm depth the in concentration
the surface layer of Znofdecreased
soil (0.3)toand values
verylower than 35 mg/kg
low correlations for(Figure
the two
8). In the SW direction, the concentration
deeper layers. All the correlations were positive. of Zn in the deeper layers of soil remained at levels
comparable to the
Extremely surface
high layer, ranging between
Zn concentration from the154.38 surfaceandlayer
194.28 of mg/kg.
soil didThenotlowest values in
maintained
wereBelow
depth. in the middle
5 cm depthof thethe
soilconcentration
profile, and the of highest
Zn decreasedvalues on to surface
values layer.
lower The
thandesign
35 mg/kgof
the Zn8).
(Figure distribution
In the SWindirection,
soil was maintained in ZoneofII.Zn
the concentration The
inhighest
the deeper concentrations wereremained
layers of soil on the
at surface,
levels comparable
decreased into thethe surface
middle layer,
of soil ranging
profile, and between
increased 154.38 and part
in the lower 194.28 mg/kg.
of the The
profile.
lowest values were in
The concentrations of the
Zn tomiddle of were
this area the soil profile,
between andand
46.03 the86.07
highest
mg/kg.values on surface
Correlation layer.
between
Thethe design of the Zn
Zn concentrations distribution
in the soil with its in pH
soilis strong
was maintained
in the upper in layerZone II.and
(0.48), The veryhighest
low
concentrations were on the surface, decreased in the middle of soil profile, and increased in
to depth.
the lower part of the profile. The concentrations of Zn to this area were between 46.03 and
86.07 mg/kg. Correlation between the Zn concentrations in the soil with its pH is strong in
the upper layer (0.48), and very low to depth.
450.00
400.00
Cu concentration (mg/kg)
350.00
R² = 0.0824
SW
300.00
R² = 0.0662 W
250.00 R² = 0.3003 6.00 7.00 8.00
200.00 NE
150.00 E
6.00 7.00 8.00
100.00 6.00 7.00 8.00
SE
50.00
0.00
0-5cm 5-20 cm 20-40 cm
Figure 7. Copper vertical distribution in soil and correlation with pH
Figure 7. Copper vertical distribution in soil and correlation with pH
800.00
700.00
Zn concentration (mg/kg)
R² = 0.1782
600.00 SW
500.00 R² = 0.4896
R² = 0.0218 W
400.00
6.00 7.00 8.00 6.00 7.00 8.00 NE
300.00
6.00 7.00 8.00 E
200.00
100.00 SE
0.00
0-5cm 5-20 cm 20-40 cm
Figure 8. Zinc vertical distribution in soil and correlation with pH
Figure 8. Zinc vertical distribution in soil and correlation with pH
In contrast to Cu and Zn, Sn distribution in the soil profile showed a very wide
variance (Figure
In contrast to Cu and 9). TheZn, Snsurface layer showed
distribution Sn
in the soil concentration
profile showed a much higher
very wide than in
variance depth.
(Figure
For all studied direction, was observed that the concentration of Sn decreased towards the
9). The surface layer showed Sn concentration much higher than in depth. For all studied
centre, and grow on the bottom of the profile. Correlation between the Sn concentrations
and the pHwas
direction, observed
of soil that thebut
is positive, concentration
to a different of Sn decreased
extent, towards
depending on the
thecentre,
depth.and Thus,growthe
on the bottom
surface layer had of the profile.
a very lowCorrelation
correlation,between the Sn
in the 5-20 cmconcentrations and the was
layer the correlation pH oflow,soiland
is
positive,
the bottombut of to
soila different
profile theextent, depending
correlation on the depth.
was moderate Thus, the surface layer had a very
(> 0.4).
low correlation,
Distribution in the
of Pb 5-20incm layer
the soilthe
didcorrelation
not followwas low, and but
a pattern, the bottom of soilone
varied from profile theto
area
another. Thus,
correlation wasthe high Pb
moderate concentration from the surface, in the W direction, decreased
(> 0.4).
sharply towards the middle of soil profile, and continues to decrease to the bottom of the
Distribution
profile (Figureof10).
Pb inInthe thesoilSWdidandnot Efollow a pattern,
directions, the but
Pb varied from onewas
concentration areahigher
to another.
in the
Thus, and
middle the high
lowerPb soilconcentration
profile than infrom the surface,
the upper horizon,in far
theexceeding
W direction, the decreased
normal value sharply
of Pb
towards the middle
concentration in soil.ofInsoiltheprofile,
NE and andSEcontinues to decrease
directions, to the bottom
Pb concentrations of the
were profile
similar (Figure
throughout
the
10).entire
In the soil
SW andprofile. The correlation
E directions, between thewas
the Pb concentration Pb higher
concentrations in soil
in the middle andandlower thesoil
pH
decreased R² = 0.0003
profile thanfromin thetheupper surface to the
horizon, depth of the
far exceeding soilnormal
profile.value
The ofcorrelation was moderate
Pb concentration in soil.
positive
In the NEin the
andupper layer (0.47),
SE directions, and low but positive
Pb concentrations to thethroughout
were similar bottom of soil profile.soil profile.
the entire
The correlation
120.00 between the Pb concentrations in soil and the pH decreased from the surface
to the depth of soil profile. The correlation was moderate positive in the upper layer (0.47),
Sn concentration (mg/kg)
and low100.00
but positive 6.00to the7.00bottom8.00 R² = 0.1099
of soil profile.
R² = 0.4283 SW
The distribution
80.00 of Co on the soil profile varied from one point to another (Figure 11). In the
SW, SE and E directions, the Co concentration in the soil increased slightly in the middle W of
60.00and decreased sharply towards the bottom of it. In the W direction, the Co
soil profile
6.00 7.00 8.00 NE
concentration decreased sharply towards the centre of soil profile 6.00 and continues
7.00 8.00 to decrease
40.00
to the bottom thereof. In the NE direction, the Co concentration is higher in the lower part E of
the profile as compared to the upper layers. The correlation between the Co concentrations in
20.00 SE
soil and pH varied on the soil profile as intensity. In the upper layers, the correlation is positive,
but very0.00
low (<0.2), while the lower layers showed weak negative correlation (0.2 - 0.4).
0-5cmalong the profile
Nickel concentration varied 5-20 cm Much higher
of soil. 20-40 cmwere observed in the
values
surface layer of soilFigure
compared
9. Tin to the values
vertical representing
distribution in soil andthe concentration
correlation with pHof Ni in the middle
of the profile. At the bottom of the profile, the concentrations increased slightly (Figure 12).
R² = 0.0003
120.00
Sn concentration (mg/kg)
100.00 6.00 7.00 8.00 R² = 0.1099

R² = 0.4283 SW
80.00
W
60.00
6.00 7.00 8.00 NE
40.00 6.00 7.00 8.00
E
20.00 SE
0.00
0-5cm 5-20 cm 20-40 cm
Figure 9. Tin vertical distribution in soil and correlation with pH
Figure 9. Tin vertical distribution in soil and correlation with pH
300.00
300.00
Pb concentration (mg/kg)
Pb concentration (mg/kg)
250.00
250.00
R²R²==0.3508
0.3508 R² SW
SW
200.00 R²==0.1048
0.1048
200.00 R²R²= =0.4714
0.4714 W
W
150.00
150.00 6.00
6.00 7.00
7.00 8.00
8.00 6.00
6.00 7.00
7.00 8.00
8.00 NE
6.00
6.00 7.00
7.00 8.00
8.00
100.00
100.00 E
50.00
50.00 SE
0.00
0.00
0-5cm
0-5cm 5-20cm
5-20 cm 20-40 cm
20-40 cm
Figure
Figure 10.
10. Leadvertical
Lead verticaldistribution
distributionininsoil
soiland
andcorrelation
correlationwith
with pH
pH
Figure 10. Lead vertical distribution in soil and correlation with pH
The distribution of Co on the soil profile varied from one point to another (Figure 11).
In theTheSW, SE and Eofdirections,
distribution Co on thethe soilCo concentration
profile varied from in one
the point
soil increased
to anotherslightly
(Figurein 11).
the
For soilofsamples
In middle
the SW, soil andcollected
SE profileE and in the SW sharply
decreased
directions, the direction, the metal
towards
Co concentration theinconcentrations
bottom
the soil it. In maintained
of increasedthe Wslightly at high
direction,
in the
the
value
Co
middle ofonsoil
theprofile
concentrationentiredecreased
soil profile
and compared
sharply
decreased to towards
towards
sharply otherthedirections.
centre
the bottom ofInsoil
the W direction,
profile
of it. In theand the surface
continues
W direction, to
the
layer
Codecrease showed
concentration much
to the higher
bottom
decreased concentrations
thereof. In the
sharply NEofdirection,
towards Ni,
theand in the
the
centre of deeper
Co layersand
concentration
soil profile the
is value
higher ofinNi
continues the
to
lower
decrease part
to of
concentration thethe
wereprofile as compared
still thereof.
bottom high. the toNEthe
TheIncorrelation upper layers.
between
direction, the
theNiCo The correlation
concentrations
concentration in between
soil and the
is higher the
inpHCo
the
concentrations
waspart
lower low,of the in
positive soil and
and
profile pH varied
decreased
as compared on the with
in intensity
to the soil profile
upper as intensity.
thelayers.
depth profile. In the upper
The correlation between layers,
the the
Co
correlation is in
concentrations positive,
soil andbut pHvery lowon
varied (<0.2), while
the soil the as
profile lower layers In
intensity. showed
the upperweak negative
layers, the
correlation
correlation (0.2
Except forissamples- 0.4).
collected
positive, but very westlowfrom the pollution
(<0.2), while the source,
lowerdistribution
layers showed of Mnweakwas approxi‐
negative
mately25.00
correlation uniform throughout the soil profile, the concentration ranging between 942.32 mg/kg
(0.2 - 0.4).
and 1578.64 mg/kg at 5-20 cm depth, and between 796.68 and 1631.13 mg/kg at 20-40 cm depth
R² = 0.217
(Figure 13). The surface layer showed a low correlation between the Mn concentrations and
o concentration (mg/kg)
20.00
6.00 7.00 8.00 SW
15.00 W
NE
10.00
R² = 0.0755 R² = 0.2217 E
5.00 SE
25.00
R² = 0.217
Co concentration (mg/kg)
20.00
6.00 7.00 8.00 SW
15.00 W
NE
10.00
R² = 0.0755 R² = 0.2217 E
5.00 SE
0.00 6.00 7.00 8.00 6.00 7.00 8.00

0-5cm 5-20 cm 20-40 cm
Figure 11. Cobalt vertical distribution in soil and correlation with pH
Figure 11. Cobalt vertical distribution in soil and correlation with pH
Except
Nickel for samples collected
concentration varied west
alongfrom
the the pollution
profile source,
of soil. Muchdistribution of Mn were
higher values was
pH, in the
approximately
observed 5-20
in the cm layer
uniform
surface the of
throughout
layer correlation
soilwas
thecompared
soil strong
profile, the(>
tothe 0.6) and
concentration
values in the lowerthe
ranging
representing part of the
between soil
942.32
concentration
of profile
mg/kgNi inandcorrelation
the1578.64 was
middle mg/kg
of moderate.
the at 5-20 At
profile. cmthe depth, and of
bottom between 796.68
the profile, theand 1631.13 mg/kg
concentrations at 20-
increased
40 cm depth
slightly (Figure(Figure
12). For13).
soilThe surface
samples layer in
collected showed
the SWa direction,
low correlation
the metalbetween the Mn
concentrations
concentrations
maintained at high and pH,
value in on
thethe
5-20 cm layer
entire the correlation
soil profile comparedwas strongdirections.
to other (> 0.6) and in the
In the W
lower part of the soil profile correlation was moderate.
direction, the surface layer showed much higher concentrations of Ni, and in the deeper
layers180.00
the value of Ni concentration were still high. The correlation between the Ni
concentrations in soil and the pH was low, positive and decreased in intensity with the depth
profile.160.00
Ni concentration (mg/kg) Ni concentration (mg/kg)
140.00
Except for samples collected west from the pollution R²source,
= 0.1159 distribution of Mn
R²profile,
= 0.1165 SWwas
approximately
120.00 uniform throughout
R² = 0.362 the soil the concentration ranging between 942.32
mg/kg and 1578.64 mg/kg at 5-20 cm depth, and between 796.68 and 1631.13 mg/kg W at 20-
100.00
40 cm depth (Figure 13). The surface layer showed a 6.00 7.00 8.00
low correlation between the Mn
6.00 7.00 8.00
80.00 and pH, in the 5-20 cm layer the correlation was strong (> 0.6) and NE
concentrations in the
lower part 6.00
of the soil
60.00 profile7.00 8.00 was moderate.
correlation
E
40.00
180.00
SE
20.00
160.00
0.00
140.00 R² = 0.1159
R² = 0.1165 SW
120.00 0-5cm
R² = 0.362 5-20 cm 20-40 cm
W
100.00 Figure 12. Nickel vertical distribution in soil and correlation
6.00 7.00 with pH
8.00
6.00 with
12. Nickel vertical distribution in soil and correlation
Figure 80.00 7.00pH 8.00 NE
2500.00
60.00 6.00 7.00 8.00 R² = 0.6073 R² = 0.3893 E
The soils in Zone II showed similar values of Cr concentration on the entire soil profile,
Mn concentration (mg/kg)
2000.00
40.00
observing only a slight increase in the6.00
median7.00
layer (Figure
8.00 14). In the SW and W directions,
SW
SE
6.00 7.00 8.00
Cr distribution
20.00 differed on the soil profile. In the surface layer the concentration had high
1500.00 W
values,0.00
but they decreased with the depth. Only in the surface layer the concentration of Cr in
0-5cm
the soil had a strong correlation with pH of5-20
soilcm
(> 0.6). 20-40 cm NE
1000.00
Figure 12. Nickel vertical distribution in soil and correlation with pH
R² = 0.1357 E
500.00
SE
0.00 6.00 7.00 8.00
0-5cm 5-20 cm 20-40 cm
Figure 13. Manganese vertical distribution in soil and correlation with pH
2500.00
R² = 0.6073 R² = 0.3893
Mn concentration (mg/kg)
2000.00
6.00 7.00 8.00 6.00 7.00 8.00 SW
1500.00 W
NE
1000.00
R² = 0.1357 E
500.00
SE
0.00 6.00 7.00 8.00
0-5cm 5-20 cm 20-40 cm
The soils in Zone II showed similar values of Cr concentration on the entire soil
The Mo
profile, concentration
observing only varied
a slightwidely in depth
increase in theof median
soil profile (Figure
layer 15).14).
(Figure In the
InSW,
the NE
SWand
andSEW
directions the concentrations decreased significantly in the middle of the profile and remain
directions, Cr distribution differed on the soil profile. In the surface layer the concentration
had high
at the samevalues,
value atbutthethey
bottomdecreased with
of it. In the the depth.
W direction, Only concentration
a higher in the surface layer
of Mo the
(4.94
concentration of Cr in the soil had a strong correlation with pH of soil (> 0.6).
mg/kg) was found at a depth of 5-20 cm. The best correlation between the Mo concentration
The Mo concentration varied widely in depth of soil profile (Figure 15). In the SW, NE
in soil
and SE and pH was
directions at concentrations
the depth of 5-20 cm, with values
decreased > 0.4. Thein
significantly upper and lower
the middle layers
of the hadand
profile a
lower at
remain correlation
the samebetween
value atMotheconcentration
bottom of it. InandthepH.W direction, a higher concentration of Mo
(4.94 mg/kg) was found at a depth of 5-20 cm. The best correlation between the Mo
concentration in soil and pH was at depth of 5-20 cm, with values > 0.4. The upper and
lower layers had a lower correlation between Mo concentration and pH.
350.00
350.00
300.00
(mg/kg)
R² = 0.1202
300.00 SW
(mg/kg)
250.00 R² = 0.1202 R² = 0.1557

SW
W
Cr concentration
250.00
200.00 6.00 7.00 8.00 R² = 0.1557
6.00 7.00 8.00 W
Cr concentration
200.00
150.00 6.00 7.00 8.00 NE
R² = 0.7064 6.00 7.00 8.00
150.00
100.00 NE
E
R² = 0.7064
100.00
50.00 6.00 7.00 8.00 ESE
50.00
0.00 6.00 7.00 8.00 SE
0-5cm 5-20 cm 20-40 cm
0.00
0-5cm
Figure 14. 5-20 cm in soil and correlation
Chromium vertical distribution 20-40 cmwith pH
Figure 14.
R²Chromium
= 0.1599 vertical distribution in soil and correlation with pH
Figure 14. Chromium vertical distribution in soil and correlation with pH
25.00
ncentration (mg/kg)
R² = 0.4282
20.00
6.00 7.00 8.00 R² = 0.1598 SW
6.00 7.00 8.00
15.00 W
6.00 7.00 8.00 NE
10.00
E
R² = 0.1599
25.00
Mo concentration (mg/kg)
R² = 0.4282
20.00
6.00 7.00 8.00 R² = 0.1598 SW
6.00 7.00 8.00
15.00 W
6.00 7.00 8.00 NE
10.00
E
5.00
SE
0.00
0-5cm 5-20 cm 20-40 cm
Figure 15. Molybdenum vertical distribution in soil and correlation with pH
Figure 15. Molybdenum vertical distribution in soil and correlation with pH
The statistical analysis indicated that the heavy metal concentration in soil is
The statistical
negatively analysis with
correlated indicated that theasheavy
the depth, metal concentration
the metal concentration indecreased
soil is negatively
with the
correlated
increasingwith the depth,
of depth (Tableas5).
the metal
The concentration
correlation decreased with
was statistically the increasing
significant of depth
at level lower than
(Table 5). The
5%. Heavy correlation
metal was statistically
concentration in soil wassignificant
positively at level lower
correlated withthan 5%.ofHeavy
the pH metal
soil, with low
concentration in soil was
to moderate intensity, positively
except the Mncorrelated with thewhich
concentration pH ofshowed
soil, with low to
a very lowmoderate
negative
concentration.
intensity, except the Mn concentration which showed a very low negative concentration.
Table 5. Pearson coefficient of correlation between heavy metal concentrations in soil and depth and pH
of soil
Cu Zn Sn Pb Co Ni Mn Cr Mo
Cu Zn Sn Pb Co Ni Mn Cr Mo
a a a a a a c a c
Depth
Depth -0.2454
-0.2454 a
a -0.3469 -0.5965 -0.2588 -0.2907 -0.3879
-0.3469 a -0.5965 b -0.2588 a -0.2907 c -0.3879 a -0.2639
a a a a a -0.2639
c -0.3277 -0.3469
-0.3277 a -0.3469
a c
c a
pH 0.3796 0.4171 0.1554 0.5066 0.5675 0.3991 -0.0995 0.5007 0.0932
pH 0.3796
ª - p < 0.05;
b a
0.4171
- p < 0.01; - p <0.1554
c a
0.001b 0.5066 a 0.5675 c 0.3991 a -0.0995 c 0.5007 a 0.0932 a
ª - p < 0.05; b - p < 0.01; c - p < 0.001
5.4 Vertical mobility index of heavy metals in soil

Table 5 Pearson coefficient of correlation between heavy metal concentrations in soil and depth and pH of soil
The vertical mobility index (VMI) represents the relative explanation of heavy metal
concentration between two underlying layer of soil: between 0-5 cm and 5-20 cm; between
5.4.
5-20Vertical
cm and mobility
20-40 index of heavy
cm (Figure 16).metals in soilmobility between layers was statistically
The metal
significant at 95% confidence level. VMI will indicate a very weak mobility of metal at values
The
lowervertical mobility
than 20%, weak index (VMI)
mobility of represents the relative
metal at values between explanation of heavy
20 and 40%, metalmobility
moderate concen‐of
tration
metal at between
values two underlying
between 40 andlayer
60%, of strong
soil: between
mobility0-5
at cm and between
values 5-20 cm; between 5-20 cm
60 and 80%, and
and
very20-40 cmmobility
strong (Figureat 16). The metal
values mobility
higher than 80%.between layers was statistically significant at 95%
confidence level. VMI
Distribution of will
Cu inindicate
the soila very weak
profile wasmobility
given byofthemetal at values
very strong lower than 20%,
correlation of the
concentration
weak mobility ofof this element
metal in the
at values three layers
between 20 andof40%,
soil moderate
profile. High valuesofofmetal
mobility vertical mobility
at values
index were observed between the concentrations of Cu in the two underlying layers, which
between 40 and 60%, strong mobility at values between 60 and 80%, and very strong mobility
demonstrated the very high mobility of Cu in the soil, mobility influenced also by the pH.
at valuesofhigher
Values than 80%.
correlation between concentrations at different depths indicated weak Zn mobility
on the soil profile to depth of 20 cm and a very strong mobility between 20 and 40 cm.
Distribution of Cu in the soil profile was given by the very strong correlation of the concen‐
tration of this element in the three layers of soil profile. High values of vertical mobility index
were observed between the concentrations of Cu in the two underlying layers, which demon‐
metal at values between 40 and 60%, strong mobility at values between 60 and 80%, and
very strong mobility at values higher than 80%.
Distribution of Cu in the soil profile was given by the very strong correlation of the
strated the very high
concentration mobility
of this elementofinCu inthree
the the soil, mobility
layers of soilinfluenced also
profile. High by the
values ofpH. Values
vertical of
mobility
correlation between
index were concentrations
observed between the at different depthsofindicated
concentrations Cu in theweak
two Zn mobilitylayers,
underlying on the soil
which
demonstrated
profile to depth ofthe
20 very high
cm and mobility
a very of mobility
strong Cu in thebetween
soil, mobility
20 andinfluenced
40 cm. also by the pH.
Values of correlation between concentrations at different depths indicated weak Zn mobility
on the soil profile to depth of 20 cm and a very strong mobility between 20 and 40 cm.
Mo 1% 82%
Cr 39% 99%
Mn 64% 88%
Ni 89% 97%
Co 62% 78%
between 1st layer and 2nd layer
Pb 30% 82%
Sn 17% 71% between 2nd layer and 3rd layer
Zn 6% 88%
Cu 94% 98%
0% 50% 100% 150% 200%
Figure 16. Vertical mobility index (VMI) of heavy metals between the layers of the soil profile,
Figure 16. Vertical mobility index (VMI) of heavy metals
statistically between
significant the layers
at 95% of the soil
confidence profile, statistically significant at
level
95% confidence
Thelevel
mobility index of Sn was different depending on the depth. In the surface layer,
this metal had a weak mobility; the correlation between the two layers (0-5cm and 5-20 cm)
Thewasmobility
very index
low. At of greater
Sn was different
depths, depending
Sn mobilityonindex the depth. In the surface
was higher, layer,
indicated bythis
themetal
strong
hadcorrelation
a weak mobility;
of the the correlation between
concentrations at depths the two layersthan
greater (0-5cm
20 and
cm.5-20 cm) was very
Correlation of Pb
low. At greater depths,
concentration between Sn layer
mobility index
of 0-5 cmwas andhigher,
the otherindicated by thewas
two layers strong
verycorrelation
low (<0.3). of A
thestrong
concentrations
correlationat depths greater
exists only than 205-20
between cm. Correlation
cm and 20-40 of Pbcmconcentration
depth whichbetween
indicatelayer
a very
strong
of 0-5 cm andmobility of Pb.
the other twoThe
layerscorrelation
was verybetween
low (<0.3). the concentrations
A strong correlationof exists
Co inonly
the between
soil profile
indicated a strong mobility of metal between the surface layer and underlying layers. The
5-20 cm and 20-40 cm depth which indicate a very strong mobility of Pb. The correlation
correlation between the Ni concentrations of different depths of soil profile is very strong,
between the aconcentrations
indicating of CoofinNithe
very strong mobility soil which
in soil profileincreased
indicatedwitha strong
depth. mobility of metal
between the Thesurface layerbetween
correlation and underlying layers. Theof
the concentrations correlation between
Mn in different the of
depths Nithe
concentra‐
soil profile
tions of different
is very strong, depths of soil profile
which indicated that is
a very strong,increase
significant indicating a very
in the strong mobility
concentration of MnofinNithe
in soil
upperwhich
layerincreased
will lead with
to andepth.
increase of Mn concentration also in the depth due to very strong
mobility of this metal. The correlation between the concentrations of Cr in different depths of
Thesoil
correlation betweenathe
profile indicated weakconcentrations
mobility of Cr ofbetween
Mn in different depths
the surface of the
layer andsoil
theprofile
middleislayer
very of
strong, which indicated
soil profile and a very thatstrong
a significant increase
mobility in thein lower
the concentration
part of theofsoil
Mn profile.
in the upper layer
Correlations
willbetween
lead to the
an increase of Mn concentration
Mo concentrations were differentalso on
in the
the depth due to
soil profile: very
very strong
weak mobility
in its of
upper layer
thisand strong
metal. Theincorrelation
the lower layers
between of soil
the profile.
concentrations of Cr in different depths of soil profile
indicated 6. Discussion
a weak mobility of Cr between the surface layer and the middle layer of soil profile
Because of the metallurgical activities carried out in the vicinity of Targoviste, which
and a very strong mobility in the lower part of the soil profile. Correlations between the Mo
produced in time significant amounts of particulate matter with a high content of Pb, Cr, Cu,
concentrations
Mn, Ni and Zn, were different
the quality on the soil profile:
of agricultural soilsvery weak in itsinfluenced
is negatively upper layer byand
the strong in the
concentrations
lower layersmetals,
of heavy of soil profile.
which represent a risk of toxicity to humans. In order to evaluate the degree
of loading of soils with heavy metals, the present study shows: (i) the horizontal distribution
of heavy metals, depending on the prevailing direction of the air masses movement (wind
6. Discussion
Because of the metallurgical activities carried out in the vicinity of Targoviste, which produced
in time significant amounts of particulate matter with a high content of Pb, Cr, Cu, Mn, Ni and
Zn, the quality of agricultural soils is negatively influenced by the concentrations of heavy
metals, which represent a risk of toxicity to humans. In order to evaluate the degree of loading
of soils with heavy metals, the present study shows: (i) the horizontal distribution of heavy
metals, depending on the prevailing direction of the air masses movement (wind rose) and (ii)
vertical distribution to highlight the mobility of heavy metals into the soil profile, depending
on the depth to which the plant roots can be found [15].
Previous studies have shown Targoviste area as an area of potential risk of heavy metal toxicity,
along with other sources of pollution in Romania, as Copşa Mică, Deva and Baia Mare. This
study presents the results of the concentration of heavy metals in soils near Targoviste, and
are considered the high risk toxic metals such as Cu, Zn, Sn, Pb, Co, Ni, Mn, Cr, Mo. The
analysis of horizontal distribution of heavy metals (Table 4) indicated that the agricultural soil
in the vicinity of metallurgical unit were loaded with heavy metals concentrations exceeding
several times the normal values and even alert threshold. Some values indicated concentra‐
tions of Cu and Zn even higher than in Copşa Mică [16] and Baia Mare [17].
Soils most affected by heavy metal pollution were those located on SV and V directions against
the source of pollution, which are the predominant direction of air masses movement in the
studied area. Also the other studied directions are characterized by concentration of heavy
metals higher than normal limits, constituting a potential risk in the use of those soils in
agriculture.
Analysis of vertical distribution of heavy metals indicated that high levels of heavy metals are
caused primarily by industrial activity and less by the concentration of metals in the parent
material. The concentration of all metals was higher in surface layer of soil (0-5 cm) because
of deposition of particulate matter [21] and lower in the deeper layers of soil, possibly due to
binding of metals to soil organic fractions (in the surface layer) that have slowed the metal
mobility on the profile and decreased the transfer of metals to the underlying layers. Statistical
analysis of correlations between the metal concentrations in soil and depth of the soil layer
showed a negative correlation, which confirmed the hypothesis that the high concentration of
heavy metals in soils from the industrial area of Targoviste, comes from a source of pollution
and not from the parent material.
The metal mobility from the surface layers to the deeper layers can be explained on the basis
of vertical distribution pattern of heavy metal. In order to measure the metal mobility on the
profile of soil, a vertical mobility index (VMI) was calculated. This index indicated the extent
to which that metal from a top layer of soil was transferred to the deeper layer by leaching and
adsorption to soil particles. Depending on the type of soil, the mobility varies, promoting or
inhibiting the rate of metal transfer from surface soil to groundwater, where the toxicity hazard
is more difficult to control and the remediation actions are trickier. The most mobile heavy
metals in investigated soils were Cu and Ni, with a mobility of 94 % and 89 % respectively,
between the two layers from the surface of soil profile and a mobility of 98 % and 97 %
respectively, between the two deeper layers. The hazard risk of these two metals to get into
the groundwater was expressed by the high values of VMI. Also Mn and Co showed quite
high values of VMI, which placed them as potential hazardous metals. Analysis of heavy metal
mobility is important because once they reached the groundwater they present a greater risk
to humans. Long-term exposure to high concentrations of these metals can cause serious
illnesses in humans [4, 10, 11], and the intervention for remediation of soil is urgent, before the
heavy metals will reach the groundwater.
7. Conclusions
Following chemical analysis by ICP-AES, we found the heavy metal concentration levels above
the normal value of an industrial soil, and sometimes even exceeding the alert limit (according
to Order 756/1997).
Distribution of heavy metals on soil profile showed that accumulation of metal in the soil
decreases with the depth. The concentrations were higher on the surface of the soil profile.
This indicates a continuous impact of metallurgical activities on soil by accumulating new
quantities of metals.
The concentration of heavy metals in soil was also considered to be in connection with the pH
and chemical composition of soil: the presence or absence of other metallic elements.
The vertical mobility index indicated moderate to strong mobility of majority of studied metals,
especially in the lower layer of the soil profile, 20-40 cm.
Acknowledgements
The research is part of the PhD thesis “Studies and research concerning the remediation of
heavy metal polluted soil by eco-technological procedure”, in Romanian, realized by the
author in 2011. Is a good opportunity to thank my colleagues Sorin Ciulei for his support in
soil assessment and evaluation, Irina Fierascu and Radu Claudiu Fierascu for their support in
the sample analyses by ICP-AES. Also I want to thank my thesis coordinator, Prof. Georghe
Ionita for guidance in preparing the thesis and to Prof. Dumitru Ion for the financial support
for the publication.
Author details
Carmen Cristina Elekes*
“Valahia” University of Târgovişte, Dâmboviţa, Department of Environmental Engineering,

Romania
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chapter 9
Assessment of Soil Pollution with Heavy Metals in

Romania
Anca Maria Moldoveanu
1. Introduction
Human activities, industry, erosion and the continuous spread of urbanization can induce soil
pollution in various ways. The pollution of soil can be dangerous for human health because
the toxic substances can enter the crops and the ground water. The soil, due to its properties
and structure, plays the role of a filter that can retain and be a deposit for toxic substances. The
most frequent contaminants of soil in Europe are heavy metals and mineral oil. The group
‘heavy metals’ for the purpose of discussing health risks or impacts generally includes: Arsenic
(As), Lead (Pb), Cadmium (Cd), Chromium (Cr) (although only the form Cr(VI) is toxic),
Copper (Cu), Mercury (Hg), Nickel (Ni), Zinc (Zn).
Several of these elements are necessary for human health and are beneficial when taken into
the body in foods or as supplements at appropriate, low levels. Conversely, cadmium, lead
and mercury have no known biological function and are toxic to humans.
The sources of heavy metals that pollute the soil can be human activities like metalliferous
mining (As, Cd, Cu, Ni, Pb, Zn), smelters (As, Cd, Pb), metallurgy, electronic industry (where
metals are used in batteries, semiconductors, circuits), rolling (Ni, Cd, Pb, Hg, Se), dyes and
paints industry (Pb, Cr, As, Se, Mo, Cd, Co, Ba, Zn), plastics industry (Cd, Zn, Pb, Sn are used
as polymer stabilizers), chemical industry (using Pb, Ni, Nb, Hg, Pt, Ru as electrode catalysts),
wood industry (As, Cr and Cu). In the vicinity of furniture factories and wood-processing,
these elements were often identified as soil and water pollutants [1, 2, 3, 4, 5, 6, 7, 8, 9].
Also, by storing municipal waste, special and hazardous waste, the soil may be contaminated
with various heavy metals.
The combustion of fossil fuels leads to the presence of Cd, Zn, As, Se, Cu, Mn, V, in the ashes
and particulates of combustion. Some heavy metals (Se, Te, Pb, Mo, Li) are added to fuels and
lubricants to improve their properties. [1, 2, 3, 4, 5, 6, 7, 8, 9].
Corrosion of metals contained in various materials (Cu, Pb pipes and roofs, Cr, Ni, Co in
stainless steel, Cr, Pb painting) can cause soil pollution. Agro-livestock activities can also cause
soil pollution (As, Cu, Zn can be added to feed pigs and poultry, Cd, As, Pb, Mn, Cu, Zn can
be present in phosphate fertilizers and pesticides). [1, 2, 3, 4, 5, 6, 7, 8, 9].
Pollution by heavy metals and many organic contaminants is practically irreversible (Euro‐
pean Commission, 2012).
Excess heavy metal accumulation in soils is toxic to humans and other animals.
Exposure to heavy metals is normally chronic (exposure over a longer period of time), due to
food chain transfer. Acute (immediate) poisoning from heavy metals is rare, but possible,
through ingestion or dermal contact. Chronic problems associated with long-term heavy metal
exposures are [10, 11, 12, 13, 14, 15, 16, 17, 18, 19] for:
1. Lead [20-60] – is well known to be toxic and its effects have been more extensively
reviewed than the effects of other trace metals. Lead can cause serious injury to the brain,
nervous system, red blood cells, and kidneys. Exposure to lead can result in a wide range
of biological effects depending on the level and duration of exposure. Various effects occur
over a broad range of doses, with the developing young and infants being more sensitive
than adults. Lead poisoning severe enough to cause evident illness is now very rare. Lead
can be harmful after uptake from food, air, water or contaminated dust. Lead is a
particularly dangerous chemical, as it can accumulate in individual organisms, as well as
entire food chains.
2. Cadmium – affects kidney, liver, and GI tract [28, 30, 61, 62, 63].
3. Arsenic – is associated with skin damage, increased risk of cancer and problems with
circulatory circulatory system, affects kidneys and the central nervous system [64, 65, 66,
67, 68, 69].
4. Chromium is associated with allergic dermatitis in humans [70, 71].
5. Mercury is associated with kidney damage [72, 73, 74, 75].
6. The most common problem caused by cationic metals (metallic elements whose forms in
soil are positively charged cations e.g., Pb2+) are mercury, cadmium, lead, nickel, copper,
zinc, chromium and manganese. The most common anionic compounds (elements whose
forms in soil are combined with oxygen and are negatively charged e.g., MoO42-) are
arsenic, molybdenum, selenium, and boron.
1.1. The aim of the study

This study tried to assess the level of soil contamination with heavy metals (lead, cadmium,
mercury, manganese, nickel, total chromium, zinc, cobalt, copper and arsenic) in 34 counties
in Romania. No previous data exists as a reference level of soil pollution.
Assessment of Soil Pollution with Heavy Metals in Romania 3
2. Material and methodology
Sampling was done in 2012, in the fall (September – October). Soil samples were taken from
max 20 cm deep, from areas around children's units like nurseries, kindergartens, schools or
playgrounds for children. The samples were taken in county capital cities. Also, soil samples
were taken from areas of land used for agricultural purposes like farmlands and gardens.
For metal analysis, 0.2 g of ground soil were digested with aqua regia using microwave
digestion (EN 13346 :02) followed by filtration and adjusting the volume in a volumetric flask.
Concentrations of Pb, Cd, Cu, Cr were measured using the electrothermal atomic absorption
spectrometry (ISO 11464:2006, SR ISO 11047/1999, EPA 6020). Concentrations of Zn, Mn, Co,
Ni, Cr were measured using flame AAS (atomic absorption spectrometry) and concentrations
of Hg and As were measured using atomic absorption spectrometry with cold vapour (HVG-
AAS) and SR EN 1485/2007. [76, 77]
Despite the importance of soil pollution for our society, there is no EU legislation specifically
targeting the protection of soil [78, 79]. A regulation regarding the soil pollution exists in
Romania (Order nr. 756, November, the 3th, 1997). In this Order [80], the following definitions
are of importance: environmental impact, types of land uses that require a certain soil quality,
soil characterized by a maximum acceptable level of pollutants - sensitive and less sensitive
soil, remediation targets, potentially significant pollution, significant pollution, threshold alert
– (concentrations of pollutants in air, water, soil or emissions / discharges, which are designed
to alert the competent authorities of a potential impact on the environment and determines
the triggering of additional monitoring and / or reduction of concentrations of pollutants in
emissions / evacuations), intervention threshold, reference sample [80].
Reference Alert thresholds

Alert thresholds Action levels - Action levels – soil
Heavy Metals threshold for - soil less
- sensitive soil soil sensitive less sensitive
sensitive soil sensitive
Arsenic ( As) 5 15 25 25 50
Cadmium (Cd) 1 3 5 5 10
Cobalt (Co) 15 30 100 50 250
Chromium (Cr) : Total

30 100 300 300 600
Chromium total
1 4 10 10 20
Hexavalent Chromium
Copper ( Cu) 20 100 250 200 500
Manganese (Mn) 900 1500 2000 2500 4000
Mercury (Hg) 0.1 1 4 2 10
Nickel (Ni) 20 75 200 150 500
Lead( Pb) 20 50 250 100 1000
Zinc ( Zn) 100 300 700 600 1500
Table 1. Reference values for traces of heavy metals in soil - Heavy metals (mg/kg dry substance). Values are stipulated
in Order 756/1997 for the approval of the Regulation on the assessment of environmental pollution [80].
3. Results and discussions
Cadmium in the soil registered levels that ranged from 0 and 0.86 mg/kg, in the urban areas
and in the rural areas. Compared to the threshold value it can be seen that the levels of cadmium
in soil, in both urban and rural areas, have not exceeded the threshold value (1mg/kg). It can
be seen that the level of cadmium in soil is slightly higher in rural areas compared to urban
areas.
Lead in soil, registered levels that ranged from 0 and 96.11 mg/kg in the urban areas and from
0 to 147.67 mg/kg in rural areas. It can be seen that, both in the urban and rural areas, the lead
levels in soil exceeded the threshold value (20 mg/kg). The alert threshold for sensitive soil (50
mg/kg) was exceeded in both the urban and rural areas.
Figure 1. Lead levels in soil in urban areas
Figure 2. Lead levels in soil in rural areas
Comparing the two charts it can be seen that, in the same area (Constanța county), the level of
lead in soil is over the alert threshold in the urban and in the rural area. In other two counties
(Buzău and Ialomița) the levels of lead in soil are exceeding the threshold limit both in the
urban area and in the rural area. There are other two counties beside Constanta, where the
levels of lead are exceeding the alert threshold in urban areas (Olt and Sibiu). In rural areas it
can be seen that the number of counties where lead levels in soil exceed the threshold is higher
than in urban areas.
Copper in soil registered levels that ranged from 0 and 128.5 mg/kg in the urban areas and
from 0 to 80.43 mg/kg in rural areas. It can be seen that the levels identified in soil exceeded,
both in the urban and rural areas, the threshold value (20 mg/kg). Also, the alert threshold for
sensitive soil (100 mg/kg) was exceeded in a single urban area. In rural areas a slightly higher
level of copper in the soil was observed.
Figure 3. Copper levels in soil in urban areas
Figure 4. Copper levels in soil in rural areas

The alert threshold was exceeded in a single county in urban areas. The number of counties
where was found levels of copper in soil, in rural areas, that exceeded the threshold was double
in comparison with urban areas.
Nickel in soil registered levels that ranged from 0 and 44 mg/kg in the urban areas and from
0 to 41.6 mg/kg in rural areas. The levels of nickel found in soil samples exceeded the threshold
value (20 mg/kg), in the urban and rural areas. Nickel recorded slightly higher levels in the
soil of rural areas, but, also, were found peaks in urban soil. The alert threshold for sensitive
soil (75 mg/kg) was not exceeded in either urban or rural area.
Figure 5. Nickel levels in soil in urban areas
Figure 6. Nickel levels in soil in rural areas
As it can be seen in the charts above, the soil pollution with nickel is similar in the urban and
rural areas, regarding the number of counties affected.
Manganese in soil registered levels that ranged from 0 and 1256.57 mg/kg in the urban areas
and in rural areas the levels ranged from 0 to 1072. 62 mg/kg. Compared to the threshold value,
the levels of manganese found in soil exceeded the threshold, both in the urban and rural areas
(900 mg/kg). Alert threshold (1500 mg/kg) was not exceeded in neither rural areas nor urban
areas.
Figure 7. Manganese levels in soil in urban areas
Figure 8. Manganese levels in soil in rural areas
The threshold was exceeded, both in urban and rural areas, in only one county in the middle
of the country (Mureș).
Concerning the levels of total chromium in soil, values are similar, in urban and rural areas.
The levels found in soil were between 0 and 40.81 mg/kg in urban areas and between 0 and
57.23 mg/kg in rural areas. Only the threshold (30 mg/kg) was exceeded in the urban and rural
areas and not the alert threshold (100 mg/kg).
Figure 9. Total chromium levels in soil in urban areas
Figure 10. Total chromium levels in soil in rural areas
The total chromium levels registered in soil exceeded the threshold in a small number of
counties. Only in one county (Mureș), levels of chromium exceeding the threshold were found
in both the urban and rural areas.
Cobalt levels were higher in rural areas compared to urban areas. The highest value was found
in the soil of a farmland in Covasna county, where the level of cobalt was equal to 20.23
mg/kg, exceeding the threshold value (15 mg/kg) but not the alert threshold (30 mg/kg).
Figure 11. Total cobalt levels in soil in urban areas
Figure 12. Total cobalt levels in soil in rural areas
From the charts, it can be seen that it is only one hot spot where the levels of cobalt in soil
exceeded the threshold.
Zinc levels ranged between 0 and 275.35 mg/kg in the soil of urban areas and between 0 –
131.79 mg/kg in the soil of rural areas. The levels recorded were similar in urban and rural
areas. One single higher value was found in Sibiu county.
Figure 13. Zinc levels in soil in urban areas
Figure 14. Zinc levels in soil in rural areas
A similar situation with zinc pollution of the soil was found in the urban and in the rural areas
of Romania as number of counties affected.
Mercury levels in the soil ranged between 0 and 0.265 mg/kg in the soil of urban areas and
between 0 – 0.305 mg/kg in the soil of rural areas. The threshold value is equal to 0.1 mg/kg
and was exceeded in urban and rural areas, but the alert threshold is equal to 1 mg/kg and was
not exceeded in neither urban nor in rural areas. Special mention must be made for district
Ferneziu of the town Baia Mare where there is a historical pollution of the environment. The
level of mercury found in this area was equal to 0.265 mg/kg.
Figure 15. Mercury levels in soil in urban areas
Figure 16. Mercury levels in soil in rural areas
For the situation of soil pollution with mercury it must be stressed that, in the area of Mara‐
mureș there is a historical pollution due to a non-ferrous industry existing for a long period
of time.
Arsenic levels in soil were between 0 and 8.22 mg/kg in urban areas and 0 and 9.59 mg/kg soil.
Arsenic concentrations were higher in rural areas compared to urban soil. The reference
threshold is equal to 5 mg/kg and the alert threshold is equal to 15 mg/kg. The threshold value
was exceeded but the not the alert threshold.
Figure 17. Arsenic levels in soil in urban areas
Figure 18. Arsenic levels in soil in rural areas
Comparing the two maps, it can be seen that, levels of pollution exceeding the threshold were
found in the Sibiu and Mureș County, in the middle of the country, in both urban and rural
areas.
4. Conclusions
This study is the first study that tried to assess the situation of soil pollution with heavy metals
in Romania.
After assessment of the soil contamination with heavy metals in 34 counties in Romania, it can
be concluded that for all investigated metals except cadmium the threshold values were
exceeded.
Alert thresholds were exceeded for lead and copper, both in urban and rural areas, especially
in two counties: Maramureș county and Sibiu county. Areas like Maramureș county or Sibiu
county have a historic pollution due to the industry present since 1844 in Baia Mare, Mara‐
mures county and 1936 în Copșa Mică, Sibiu county.
A national screening program was performed in the years 2008-2010 in Maramureș county (Baia
Mare city and especially Ferneziu district) to see the impact on human health in connection with
lead pollution. In the years 2008 and 2009 a group of 91 children was investigated [81, 82].
The results showed the following situation:
Lead in blood up to Lead in blood up to 20 Lead in blood up to Lead in blood up

Year of study Lead in blood > 35 μg/dl
10 μg/dl μg/dl 30 μg/dl to 35 μg/dl
2008 -
% of
95.4 97.8 97.8 97.8 2.2
investigated
children
2009 -
% of
58.70 71.74 84.06 89.13 10.87
investigated
children
Table 2. Lead levels in blood in city Baia Mare, Maramureș county in 2008 and 2009.
A special situation had the children living near the factory that processes lead in the district
Ferneziu in Baia Mare county.
During the year 2010, the study group was made of 94 pregnant women and 94 new born
babies. The blood was taken from the new born cord in the case of children [83].
Level of lead in blood

0-10 μg/dl 10-20 μg/dl 20-30 μg/dl > 35 μg/dl
of pregnant women
% of the study group 94.7 1.06 2.12 2.12
Table 3. Lead levels in blood in the group of pregnant women, in city Baia Mare, Maramureș county, in 2010.
Level of lead in blood

of cord of new born 0-10 μg/dl 10-20 μg/dl 20-30 μg/dl > 35 μg/dl
babies
% of the study group 93.6 1.06 - 5.34
Table 4. Lead levels in blood in the cord of new born babies, in city Baia Mare, Maramureș county, in 2010
For copper, alert thresholds was exceeded, both in urban and rural areas. In Suceava county,
copper mining was performed since 1922. In this area, there were taken rehabilitation measures
in the period between1999-2005. Still, some of the effects of the ancient pollution situation can
be seen today.
All the other investigated metals in the soil, both in residential areas and agricultural areas
(which both can be classified as sensitive lands), do not exceed the alert threshold for sensitive
lands.
For cadmium, nickel, manganese, cobalt and arsenic higher levels were recorded in the soil of
rural areas than in the soil of urban areas.
For total chromium, zinc and mercury similar levels were recorded in the soil of urban and
rural areas.
It must be stressed that in the areas where the alert thresholds were exceeded, pollution with
impact on the environment can be present. The situation can be labelled as “potentially
significant pollution”. In this case, the competent authorities can take action to reduce the
pollutants emissions in the air or to take measures to diminish the possibilities to contaminate
the soil directly or through water.
Author details
Anca Maria Moldoveanu1,2*
1 University of Medicine and Pharmacy, “Carol Davila" Bucharest, Romania
2 National Institute of Public Health, Bucharest, Romania
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chapter 10
Eco-Technological Solutions for the Remediation of

Polluted Soil and Heavy Metal Recovery
Carmen Cristina Elekes
1. Introduction
Increasing industrial activities and the lack of appropriate measures to counteract its effects
are causing a progressive pollution of air, water and soil with heavy metal emissions. Studies
have shown that after the downturn of the industrial activity of metallurgical plant, as is in
case of the industrial platform of Targoviste (Romania), heavy metals do not persist in air or
water, but tend to concentrate especially in soil and sediment. The heavy metal concentration
exceeding the threshold in soil can be considered as risk for human health and remediation
technics should be applied to decrease the metal content in soil. The classical methods of soil
remediation are expensive and some of them involve the removal of huge volume of soil. An
alternative of such methods are the bioremediation methods which involve only eco-friendly
materials and procedures, lead to metal recovery with minimal impact on the environment
and are cost-effective.
Phytoremediation is a process which uses green plants to remediate the soil polluted with
heavy metals or other contaminants. The use of different species of plants in the bioremediation
process of polluted soils is an adequate option, with minimal influence over the environment,
without destroying the soil, which also provides the opportunity to recover the heavy metals.
Phytoremediation is a cheaper method, by 50-80% compared to other methods of bioreme‐
diation [1]. The disadvantage of this method is that it can be a much more slowly process of
remediation, requiring several seasons of plant growth. The contaminants may reduce the
growth of plants and the resulted biomass, enriched with heavy metals, is potentially harmful
in the food chain.
Through the application of phytoremediation on heavy metals polluted soils, the resulting
plant biomass will have a high content of toxic metals [2]. This biomass is considered waste
and requires controlled and responsible disposal because of the risk of toxicity for environ‐
2 Soil Pollution
ment, and transfer into the food chain. With the decomposition of plant biomass, metals can
be washed by rain and transported back into the soil. In order that the phytoremediation
process to result in effective outcomes and the level of heavy metals from the environ‐
ment to decrease, not only to move those metals from one area to another, the remedia‐
tion of polluted soils should end with quantitative recovery of metals [3]. The recovery of
heavy metals has the advantage of increasing the economic value of the phytoremediation
process by transforming this method in a financial self-supporting approach of environmen‐
tal remediation.
There have been numerous studies on the phytoremediation process, having examined the
species of plants that have greatest ability to accumulate heavy metals, factors affecting results
of phytoremediation and the areas to be covered with plants for remediation purpose, but
studies on treatment, storage or use of resulting biomass are insufficient. Some studies
presented the possibilities of heavy metal recovery from different waste, even from agricul‐
tural waste. The present research aims to put in one sentence the phytoremediation process
and the recovery of heavy metals from the phytoremediation by-products.
The research focused on identifying methods of heavy metal recovery from ash, resulted from
the incineration of biomass. The phytoremediation process needs to end up with the heavy
metal recovery to obtain (a) de-polluted soil, (b) ash with low content of heavy metals, that
can be used as fertilizer in agriculture and (c) amounts of heavy metals that can be recovered
in the industry to obtain an economic advantage by financially self-supporting of the phytor‐
emediation process. Because of the lack of researches in this domain, this research was
conducted based on the results of those studies that aim to recover metals from different kind
of waste (from agriculture, sewage sludge, or woods).
2. Heavy metals in soil
Heavy metals are harmful for the human health, because they tend to accumulate in the living
organisms. This bioaccumulation is caused by the high rate of absorption comparing to the
rate of metabolism or excretion of the harmful compounds. The emissions from the metallur‐
gical plants are transported by air masses up to 10 km from the pollution source and are
deposited on the ground leading to an increase of heavy metal concentration in the upper layer
of soil.
In soil, the soluble metals go into the soil solution and can be absorbed or immobilized by
plants or can be leached to the deeper layer of soil and to the ground water (Figure 1). Some
metals are chemical or physical adsorbed to soil particles. The fate of metals in soil depends
on the depth of soil layer, on the erosion processes and on the pH. The heavy metals adsorbed
to the soil particles from the upper layer can be subject to the erosion processes and transported
by surface waters or by wind. The metals absorbed to deeper soil particles can be subject to
microbiological and chemical degradation, can be stabilized by the plant root, or can cross
from stable to available forms according with pH. The biological activity influences the speed
Eco-Technological Solutions for the Remediation of Polluted Soil and Heavy Metal Recovery 3
and rate of pollutants degradation and the clay-humus complex represents an efficient buffer
in neuter or alkaline soil reaction [4].
Figure 1. The fate of pollutants in soil [2]
The availability of metallic compounds in soil for plants depends on the soil texture, organic
matter content of soil, cation exchange capacity, calcium carbonate equivalent and pH. Soil
organic substances play an extremely important role because they can delay both the accu‐
mulation and transfer of metals and their movement into the soil. Metal toxicity in soil can be
increased or reduced by the organic substances. Soil pH directly influences the availability of
metals as soil acidity determines the metal solubility and its ability to move in the soil solution.
Concerning the content of phosphorus in soil there are areas where uptake of metals is
accelerated or rather diminished due to the presence of high doses of P2O5. In addition, the
physiology of the plant species influences accumulation of metals. For example, in the case of
cadmium uptake by grain was noted either a competition or a synergism in case of high
concentrations of lead in the soil [5].
For soil protection, the limits for various pollutants have been established only under certain
conditions and soil parameters. It was not taken into account the fact that on light soils, low-
carbon, there is a strong influence of acid rainfall leading to a strong mobilization and uptake
into plants of toxic heavy metals. This does not happen on heavier soils rich in limestone.
The solubility of zinc in soil was studied by Herms and Brummer [6], who demonstrated the
extent to which this element is dissolved by increasing acidity of the soil and became available
to plants absorption. In a low-zinc soil, a pH value of 5 could lead to a lasting effect of uptake
large amounts of zinc, with all the negative consequences that result. Zinc equilibrium in the
soil solution is realized at the level of 1200 mg/kg soil and a pH of 7, at level of 100 mg/kg soil
4 Soil Pollution
and pH of 6 and at a level of only 40 mg/kg at a pH of 5. These levels of equilibrium make the
low-zinc soil to release in the soil solution dangerous amounts of this element.
3. Phytoremediation process
Phytoremediation is defined as the process which uses green plants for the relief, transfer,
stabilization or degradation of pollutants from soil, sediments, surface waters and ground‐
water.
In order to be used in phytoremediation, the selected plant species must be tolerant for the
pollutant to be extracted, to quickly develop high biomass, to accumulate metals in harvestable
parts, to have a well-developed root system and have a high bioaccumulation factor. This factor
must be 20 or more for the phytoremediation to reduce the contamination of soil by 50% over
a period of 10 crops [7]. The level of metal bioaccumulation and recovery is directly propor‐
tional to the quantity of biomass.
Plants that accumulate high levels of metals are known as hyperaccumulators and can
accumulate 50-100 times more metal than a normal plant. There are about 400 hyperaccumu‐
lator species and the level of concentration is 10000 mg/kg for Zn and Mn, 1000 mg/kg for Cu,
Co, Ni and As, and 100 mg/kg for Cd [8].
Thlaspi caerulescens species is the most studied and known as a hyperaccumulator plant with
tolerance capacity for high concentrations of heavy metals in soil (e.g. Cu, Zn, Ni, Cd). Other
tolerant species for heavy metals is Berkheya coddii, which was studied in South Africa, on
ultramafic soils enriched with Ni [9]. The authors found, in leaves, values of Ni concentration
about 18000 mg/kg, exceeding several times the metal content in soil (1300 mg/kg) without
presenting symptoms of toxicity.
Uptake by the root is the most important way to get trace elements in plants, but have been
observed absorption and adsorption processes of metals at level of other tissues. Metal uptake
in plants is influenced by the species-specific ability, pedological factors, of which the most
important are pH, Eh, fluid regime, clay content, cation exchange capacity, nutrient balance
and concentration of other heavy metals. Also, the weather demonstrated some indirect effects
on metals absorption rate in plants, mainly due to the influence on the amount of water in the
environment [10]. In general, a high-temperature environment positively influences the
absorption of micro-nutrients by plants [11].
A disadvantage of phytoremediation is that many hyperaccumulator plants produce a small

quantity of biomass. For example, Thlaspi caerulescens produces only 2-5 tons/ha, but are plants
that produce a larger quantity of biomass 9 t/ha for Alyssum bertolonii, or even 22 t/ha for Berkhya
coddii [12].
Hyperaccumulation involves the absorption, transport and translocation of metals in tissues,

where can be stored large amounts of these elements. One of the most studied mechanisms for
the metal isolation use metallothionein-derived peptides and phytochelatins. Metal binds to
organic sulfite in cysteine, which form the majority of metallothionein-derived peptides. It has
been shown that metallothioneins and phytochelatins are stimulated by exposure to metals
[13].
Phytoremediation of heavy metal polluted soils involves the following processes (Figure 2):
a. Phytoextraction represents the process which uses plants for the absorption, translocation
and accumulation of pollutants from soil, to root systems and shoots. Herbaceous plants
are suitable for phytoextraction because they grow quickly, forming a large amount of
biomass and can remediate different types of soils. Four species of grass, Vetiveria
zizoniodes, Paspalum notatum, Pennisetum glaucum, Stenotaphrum secundatum were used to
decontaminate open mines. The first species is the most effective one, and the addition of
fertilizers is not necessary.
b. Phytodegradation represents the process that uses plants for degradation of organic
compounds. The organic compounds are degraded by plants or isolated in vacuoles for
later degradation. In general, organic compounds can be degraded on several levels:
partial conversion to less toxic compounds, partial degradation and subsequent isolation
and complete degradation.
c. Phytovolatilization represents the process of absorption of pollutants by plants and
volatilization into the atmosphere by the foliar system. Some plants are able to convert
the metal ions in the more volatile forms by the phytovolatilization, which may reduce
the toxicity and can translocate the metals to the stomata. Poplar can volatilize trichlor‐
ethylene, eucalyptus can volatilize methyltertiarybutylether, selenium can be trans‐
formed into Indian mustard and methyl mercury associated with plant roots can be
converted from Hg2+ to volatile Hg.
d. Phytostabilization represents the process that use the plants which showed immobiliza‐
tion capacity for some metals or capacity for create binding condition for metals which
will be adsorbed to soil particles and will be less available. These plants can also transform
some toxic molecules in less toxic forms (e.g. Cr6+ to Cr3+). To stabilize heavy metals in soil
have to be used plants with low capacity of accumulation to reduce the dispersion of
metals by grazing or by plant death. Among the remediation techniques of mining areas,
phytostabilization is a method with good results in prevention of acid mine discharges
and metal stabilization [14].
Heavy metals behave differently and have a different mobility depending on plant species.
Therefore, Pb, Cr and Cu tend to be stabilized and retained in the root, Cd, Ni and Zn are more
easily translocated to aerial tissues, and Cd is transported even to the harvestable tissues of
plants [15].
For Pb there are some plant species, such as Brassica juncea, Vetiveria zizinioides, Cardaminopsis
halleri, Cynodon dactylon and Sorghum halepense presenting hyperaccumulative capacity. To
improve the ability of plants to accumulate heavy metals, the polluted soil can be amended
with chelates that increase the metal bioavailability. This method has disadvantages of the
high risk of metals to be more easily leached to the groundwater, in addition to the higher cost
of remediation.
6 Soil Pollution
Figure 2. Phytoremediation of heavy metals polluted soil [2]
Among the plants that can be used in phytoremediation of mine tailings, studies have focused
on Eriophorum angustifolium, a plant resistant to substrates with a wide range of pH from 10.9
to 2.7. Other species of plants that can be grown in a low pH environment are Carex rostrata,
Eriophorum scheuchzeri, Phragmites australis, Typha angustifolia, Typha latifolia, which grows to a
pH value of 2.1, 4.4, 2.1, 3.0 and 2.5 respectively [14].
The studies made since 1977 by the American biologist Dr. Robert Brooks [16], have shown
that metals can be extracted from plants (e.g. Ni, Zn, Pb and Au), but the facility of this process
depends on the density and solubility of the elements. From the first experiments was obtained
0.01 g of Ni from few kilograms of plant biomass and, more recently, 10 g of Au were obtained
from a two hectares of rape culture, established in the vicinity of abandoned mines in Cali‐
fornia.
4. Methods for heavy metal recovery
So far there have been numerous studies on the phytoremediation process, having examined
the species of plants that have a greater ability to accumulate heavy metals, factors affecting
the results of phytoremediation and areas that should be remediated with plants. In terms of
treatment, storage or heavy metal recovery from the biomass resulted from the phytoreme‐
diation process the reference studies are scant.
Delplanque et al. [17] conducted a study on the behavior of metals during the combustion of
leaves and shoots of Salix (grown for phytoremediation), describing the type of ash obtained
from the use of biomass as an energy source and the level of heavy metal concentration. At the
end of their study, the authors concluded that the combustion of biomass obtained from
phytoremediation reduces the waste volume, but ash cannot be used as fertilizer in agriculture
due to high levels of heavy metals.
The international researches in the last decade deal with developing several techniques and
methods of incineration and recovery of heavy metals from industrial waste [18,19], garbage
[20] and vegetable waste resulting from agriculture [21,22,23]. Unlike industrial waste and
garbage, the biomass obtained from the phytoremediation process has a higher content of
organic matter and low ash content, only 5% of the dry matter [24]. Compared with agricultural
waste, collected from the fields containing heavy metals within normal levels, biomass
resulting from phytoremediation process contains much higher concentrations of heavy
metals. Considering these aspects and morphological differences between the species used in
phytoremediation, heavy metal recovery methods must be adapted for each type of biomass
used (grass species, tree species), according to the concentration and metal mixture.
4.1. Thermal treatment
During the thermal treatment (incineration or pyrolysis), the organic matter from biomass is
destroyed and metals remain in ash as oxides [25] which can be recovered by hydrometallur‐
gical processes [22,23,26] and bio-hydrometallurgical processes [27], ion exchange
[28,29,30,31,32], flotation [19,33], magnetic field [34,35] or electrolysis [3,18,21] and bio-
electrochemical procedures [36].
Due to the high content of oxygen, plant biomass can be easily incinerated, requiring only
small volume of air during the combustion. In addition, relatively low sulfur content of the
biomass is an advantage because there are no harmful gases released in the atmosphere during
the combustion [20]. A negative aspect of incineration at very high temperatures (over 600 °C)
is the fact that some heavy metals, including those with the greatest risk of toxicity, are volatile:
Pb, Zn, Cd, Se [37]. Volatilization of metals can be exploited as an advantage in the recovery
of metals, because these elements condense in the cooler areas of the incineration oven and are
adsorbed on fine particles of ash retained in the cyclone or on filters [37,38]. The metal recovery
is more efficient due to higher metal concentrations by mass of ash [20,37]. The mechanism
which determines the behavior of metals during incineration process is characterized by three
aspects: (a) evaporation in the combustion areas and condensation of metals in the lower
temperature zones of the furnace, (b) physical adsorption on specific surface area of ash
particles, and (c) chemo-absorption [38]. The surface area of ash particle is determined
primarily by unburned carbon and assessed by electron microscopy (SEM) and is a determin‐
ing factor for the adsorption of volatilized metals [18,38].
To minimize the risk of volatile metals reaching the atmosphere, the incineration plant shall
be fitted with ash particle filtering and retrieval (filters and cyclone). Mercury, selenium and
arsenic are metals with the highest percentage of vaporization into incineration system.
8 Soil Pollution
Mercury is highly volatile and can be delivered almost entirely as vapor in the form of HgO
and HgCl2. Oxidized form of mercury is easily collected from air pollution control system [37].
Keller et al. [3] demonstrated by their study that, better results of the thermal treatment of the
biomass are obtained by pyrolysis, under reducing conditions, compared to incineration. The
researchers aimed only the recovery of volatile metals (especially copper and zinc), from shoots
of Salix use in phytoremediation. These statements are subject to the heavy metal content of
ash, which have to lie under the maximum permissible level if they are used as amendment.
4.2. Hydrometallurgical processes
Extraction of heavy metals by ash leaching is a complex chemical process which offers the
possibility of obtaining quantitative precipitated metal. The solutions used for leaching of ash
must be environmentally friendly, efficient, cheap and with a high capacity for regeneration.
In the leaching process, the extraction of heavy metals is subject to such factors as the solubility
and availability of the metal. The solubility can be influenced by pH, the chemical form of
inorganic species, organic matter, and the reducing properties. Most metals from waste
indicated a higher solubility in acid solutions [39]. Singer et al. [40] tested the extraction of
aluminum with citric acid at different temperatures and the results showed that the extraction
of metal was considerably influenced by the concentration of the acid used and by temperature.
Based on this study, Machado et al. [23] have studied the recovery of Ni and Zn in a multi-
component solution by precipitation in the form of alkali metal hydroxide. As a result of the
experiment the researchers obtained a recovery rate of over 99% and a purity of 92% for Ni
and 99.4% for Zn. The main factor which influences the precipitation of metals from NaOH
solution was the pH, which defined the precipitation of Ni at pH 14 and the precipitation of
Zn at pH 10. Recovery of Cd, Cu and Pb by leaching with NaHCO3 was investigated by Lezcano
et al. [22] who obtained different amounts of metal in relation to pH.
To increase the solubility of metals from ores, Hoque & Philip [27] proposed the introduction
of a microbial population, to convert insoluble metal sulfides into soluble metal sulfates. For
example, for the extraction of copper from copper sulfide, it was oxidized by microorganisms
to copper sulfate. The metal ions have been concentrated in the aqueous phase and the solid
residues were removed [27]. A similar technology is used for the conversion of solid metal in
water soluble form, in the presence of microorganisms. The technology is called biooxidation
and is used for the microbiological oxidation of metal-containing minerals to be extracted.
Bio-hydrometallurgical processes are used in copper metallurgy in the presence of the

bacterium Thiobacillus ferooxidans, carrying bivalent iron to trivalent iron oxidation [41]:
CuFeS2+ 4 O2 → CuSO4+ FeSO4

FeSO4+ H2SO4+ ½ O2+ bacteria → Fe2(SO4)3+ H2O
2 Fe2(SO4)3+ CuFeS2+ 3 O2+ 2 H2O → CuSO4+ 5 FeSO4+ H2O
The reaction takes place in aqueous solution, and the last two reactions are cyclic ensuring the
continuous development of the leaching process of the chalcopyrite, while copper passes in
the solution as sulfate. Another type of bacteria is used to oxidize the sulfur (Thiobacillus
sulfooxidans). The solutions obtained by leaching, after purification and concentration, can be
processed to extract metal ions which we are interested in.
4.3. Electrochemical processes

Electrochemical processes for the extraction of heavy metals have the advantage of selective
recovery of metals, depending on the metals reduction potential of the metal to be extracted,
but in order to obtain a high purity for every metal, tests must be performed to optimize the
factors which influence the metal deposition on the electrode (pH and the electrolyte concen‐
tration, the temperature of the electrolytic bath and the metal species). The metal extraction by
electrolysis is common use in the metallurgy of zinc, copper, nickel, etc.
Each metal has a specific ion discharge potential, which corresponds to the minimum potential
at which an ion electrode begins to discharge continuously and visible (substance discharge).
When a substance has more ions, they are discharged successively as they achieve the potential
of each download. On this basis is realized the separation and selectively deposition of a
number of metal ions from the same solution, if their discharge potentials are differing by at
least 0.2 V, otherwise they are deposited at the same time. The ions can be electro-gravimetric
separated with a determination error of less than 0.1%.
Fukuta et al. [42] have obtained the selective recovery of Cu, Ni and Zn with sodium sulfide,
but in 2011, Machado et al. [43] conclude that, because of the difference in deposition potential
of only 0.25 V, the separate recovery of Ni and Zn by electrolysis may be compromised, with
the risk of co-deposition of the two metals. For this reason, the research was continued and the
electrolyte was used to test the removal of the two metals by precipitation. Ni extraction by
electrochemical processes was tested also by Lee [18]. The subject solution of this experiment
was a spent electro-less nickel plating solution, the electrode used as the anode was made of
platinum, and the cathode was made of stainless steel. Just as in the previous studies, pH
played a key role in the extraction of Ni, and, at the end of the process, the metal was obtained
in the form of nickel hydroxide and nickel fine particles.
The study conducted by Kirkelund et al. [21] to remove the Cd from the residual plant material
by electrochemical methods is based on the principle of electro-migration of ions in solution,
in an electric field. The researchers used membranes for the anions and cations exchange to
optimize the process. Optimization of results for metal extraction was pursued also by Modin
et al. [36] who applied a bio-electrochemical process for recovering Cu, Pb, Cd and Zn in dilute
solutions. The anode was inoculated with micro-organisms from the sewage sludge and in the
anode chamber a nutrient solution was circulated. The advantage of this method is the less
energy consumption for the metal discharge and the selectivity of metal extraction [44].
The present study was conducted on seven perennial grass species from Juncaceae and Poaceae
family, to find the best solutions for the phytoremediation of soils in the vicinity of metallur‐
10 Soil Pollution
gical plant of Targoviste (Figure 3). The aim of research was to evaluate the capacity of these
plants species to accumulate heavy metals which were found in high concentrations (above
the normal range in agricultural soils) on the studied site. The best accumulative species, Lolium
perenne was used in an experiment of heavy metal extraction from plant biomass to test the
efficiency of metal recovery methods in case of phytoremediation procedure.
Figure 3. Studied area in the vicinity of metallurgical plant of Targoviste
5.1. Research course
Sampling points of plants and soil were chosen so that the results to reflect a snapshot of the
impact of metallurgical activities in this area by particles emissions. Sampling was done at
distances between 500 and 1000 meters from the source of pollution, from three different
points, chosen according to triangle method. The results of metal concentration represent the
average of these three samples. The depth of sampling was chosen according with the depth
to which the roots of plant culture normally develop. Was formed a mean sample from the
column of soil between 0 – 20 cm depth.
Plants were harvested in two seasons, summer and autumn 2008. Was harvested the entire
plant, including the root system. For each plant sample, the soil underneath was collected,
down to the horizon where the plant developed its root system. The soil was used to establish
the bioaccumulation factor of each plant species by comparing the metal concentration in
plants with the metal concentration in soil.
The harvested plants were wild growing species which already were adapted to high pollution
level of the soil. They were perennial grasses, which usually are used as forage for animals:
Lolium perenne, Festuca pratensis, Stipa capillata, Agrostis alba, Cynodon dactylon, Agrostis tenuis
and Luzula campestris.
The plants and soil samples were processed in the laboratory for elemental analysis by ICP-
AES (see section 5.2). After harvesting, the fresh plants sample were cleaned with deionized
water to remove the soil particles, dried at 60 °C for few hours, ground to a fine powder and
analyzed to establish the metal concentrations. The soil samples were dried at 40 °C for 24
hours, ground to a fine powder, sieved at 250 µm (according to SR ISO 11464).
Based on the results of bioaccumulation for studied plant species, Lolium perenne was chosen
for the subsequent experiment for heavy metal recovery. In the vicinity of metallurgical plant,
a 10x10 m experimental plot was cultured with this species. During the growing period no
amendments were added. After one growing season, the plants were mowed and used for the
experiment of heavy metal extraction by hydrometallurgical and electrochemical processes.
Figure 4. Plant biomass incineration: (a) heat treatment oven with forced rod, (b) incineration process
First the plants were dried for few hours, at 105 °C to remove the water and to decrease the
biomass volume [45]. After drying stage, the plants were incinerated in alumina crucibles using
a heat treatment oven with forced rod (Figure 4), nominal voltage of 220 V, nominal amperage
of 30 A, and maximum temperature 1300 °C. The incineration was conducted at temperature
between 400 °C and 600 °C in two stages. The first stage was the heating up to 500 °C for 30
minutes, followed by a 90 minutes of burning at temperature of 500 - 600 °C. Weighing was
made for the raw biomass, for the biomass after drying and after incineration to establish the
percentage of dry matter and percentage of ash.
Figure 5. Electrolyses cell

12 Soil Pollution
Leaching of ash was made by mixing 10 g of ash with 200 ml of nitric acid (HNO3), concentrated
to 65%. The mixture was heated to boiling point for 60 minutes and then cooled down to room
temperature. After complete cooling, in the mixture were added, on ice, 200 ml of concentrated
sulfuric acid (H2SO4). After half an hour of rest, the mixture was filtered.
For the extraction of heavy metals, an electrolytic cell was designed (Figure 5), with stainless
steel electrodes and 200 ml of the filtrate obtained from the leaching of the ash as electrolyte.
The electrolysis was carried out for 90 minutes with an electric intensity of 1.5 A, and a voltage
of 11.4 V. The microscopic and gravimetric methods were used to assess the metal deposition
on electrodes.
5.2. Analytical methods
Determination of heavy metal concentration, in both plants and soil underneath, was done by
Inductively Coupled Plasma - Atomic Emission Spectrometry method (ICP-AES). For analyze,
the samples were mineralized in Berghof microwave digester, plants by mixture with 10 ml
of nitric acid concentrated 65% and 2 ml of hydrogen peroxide, and soil in mixture 1:1 with
nitric acid (according with Berghof method). The advantage of this method is the multiele‐
mental detection, which give the possibility, in one shot, to read a wide range of elements [46].
For this research, analyzes were conducted with Liberty 110 spectrometer of Varian brand.
The minimal detection limits of device range according to the analyzed element and is 0.4 mg/
kg for Zn, Mn and Cu; 0.5 mg/kg for Cr and Co; 0.6 mg/kg for Sn, Ni and Pb. The concentrations
values for analyzed metals were expressed in milligrams of metal per kilogram of dry soil or
plants (mg/kg).
The soil pH was determined with a portable pH-meter, WTW 3110 SET 2, with precision of
0.01 units. For pH analyzes, 5 g of each soil sample were mixed with 50 ml KCl 0.1N, F 1000,
Tt 0.0056 g/ml and homogenized for 15 minutes with a magnetic stirrer.
The deposition on electrodes was evaluated by microscopy and quantitative assessed by

Energy Dispersive X-Ray Fluorescence method (EDXRF) [47], using a PW4025 – MiniPal –
Panalytical type EDXRF Spectrometer. The XRF determinations were conducted in Helium
atmosphere, excited for 300 s, without any filter, at 16 kV voltage. The current intensity was
automatically adjusted by the use of a 3.6 µm Mylar tissue [48]. The surface of electrodes was
evaluated for heavy metal concentration before and after electrolysis.
5.3. Data analysis
The bioaccumulation factor (BF) for studied plants was calculated as the ratio between metal
concentration in plants and metal concentration in soil:
C plant
BF = (1)
Csoil
where: Cplant represents the metal concentration in plants and Csoil represents the metal
concentration in soil.
To calculate the percentage of metal concentration by drying, the metal content of dry matter
was reported to the element content of the fresh biomass and to calculate the percentage of
metal concentration by incineration, the metal content of ash was reported to the element
content of the dry matter.
Percentage of metal extraction into solution (%) was calculated as ration between the metal
content of 400 ml solution (filtered leachate) and the metal content of 10 g of ash:
solution concentration* 400 1000

Percentage of metal extraction into solution ( % ) = (2)
ash concentration* 10 1000
The quantity of metal recovered by electrolysis was calculated according with the mass of
metal deposition on cathode (0.7 mg), percentage of ash from the fresh biomass (5.1%) and the
mass of fresh plants that can be harvested from one hectare (40 t):
metal concentration in cathode deposit ( mg kg )

Recoverd metal ( m g 5 g ash ) = * 0.7 (3)
1000
From 40 t of fresh biomass of Lolium perenne that can be harvested from one hectare, with a
percentage of ash of 5.1%, can be obtained 2.04 t of ash.
recoverd metal ( mg 5g ash )

Recoverd metal ( g per ha ) = * 2.04 (4)
5
6. Results
6.1. Heavy metal concentration in soil
The soil samples consisted in the upper layer of soil, 0 – 20 cm, where the most of the roots can
be found. The content of soil in macronutrients was about 13 g/kg for Ca, 3 g/kg for Mg, and
1 g/kg for P and K. The soil reaction had the value of 7.30±0.42. Heavy metal concentration in
soil (Table 1) was compared to the normal values of agricultural soils and alert thresholds for
industrial soils according with the Romanian regulations [49]. The average content of Cu, Sn
and Pb in soil exceeded the alert threshold for agricultural soils, 100, 35, 50 mg/kg respectively,
but in some sampling points the concentrations exceeded even the alert threshold for industrial
soils, 250, 100, 250 mg/kg. For Zn, the mean concentration in soil was in normal limit for
agricultural soil (300 mg/kg), but in some sampling points exceeded the alert threshold of 700
mg/kg. The Co concentration in soil had low values with a uniform distribution between the
14 Soil Pollution
sampling points. None of the samples showed values of Co concentration higher than 30 mg/
kg, the alert threshold for agricultural soils. The mean value of Ni concentration did not exceed
the alert threshold for agricultural soils (75 mg/kg), but the concentration was varying in a
wide range from one sampling point to another and some results showed values of concen‐
tration close to the alert threshold for industrial soil (200mg/kg). The mean concentration of
Mn in studied sample of soil was higher than alert threshold for agricultural soil (1500 mg/kg),
and some of results showed values of Mn concentration that exceeded the alert threshold for
industrial soils (2000 mg/kg). The mean concentration of Cr was just under the alert threshold
for agricultural soils (100 mg/kg), but some sampling points showed values for Cr concentra‐
tion higher than the alert threshold for industrial soils (300 mg/kg).
Soil concentration (mg/kg) Soil concentration (mg/kg)

Metal Metal
Mean ±SD Range Mean ±SD Range
Cu 152.4±177.7 21.9-600.4 Co 15.9±4.3 7.1-23.54
Zn 194.3±231.7 42.6-870.3 Ni 53.4±48.6 11.9-185.4
Sn 65.7±30.7 24.6-125.41 Mn 1545.7±334.07 1159.9-2348.76
Pb 65.2±87.2 <LD-294.3 Cr 98.0±112.0 12.9-315.6
<LD – below limit of detection
Table 1. Heavy metal concentration in soil underneath plants (mg/kg dry matter)
The wide variability of metal concentration in soil was according with the orientation and the
distance against the pollution source. Some heavy metals showed a uniformity in metal
distribution (e.g. Co and Sn), which is probably because of the geological origin of these metals,
more than from the pollution source.
6.2. Heavy metal bioaccumulation in plants
Perennial grasses develop a large plant biomass in a relatively short time and are known as
heavy metal tolerant biosystems, accumulating high levels of these elements. Also, perennial
grasses have a high content of dry matter: Lolium perenne 36%, Festuca pratensis 33%, Stipa
capillata 43%, Agrostis alba 42%, Cynodon dactylon 40%, Luzula campestris 50% and Agrostis
tenuis 46%, compared to other species such as Papaver rhoeas, Cirsium arvense or Artemisia
vulgaris which have only 12 – 36 % dry matter.
According to the perennial grasses tolerance for heavy metals and because of environmental
and weather conditions favorable to their development, analyzed plant species have adapted
to the toxic heavy metal concentration in soil and accumulated these elements to high levels
[50]. In addition to these aspects, the concentration of metals in plants was influenced by plant
age, topography and synergistic and antagonistic effects of the elements found in soil.
The heavy metal concentration in perennial grasses was widely different between the species
for all studied metals (Table 2). Copper concentration range between 1.76 and 113.83 mg/kg,
with the highest value for F. pratensis. In the same species was found the highest value for the
tin concentration, 379 mg/kg, while the lower value of tin concentration was for L. campestris,
8 mg/kg. Zinc concentration range for studied species between 62 – 922 mg/kg, and lead
concentration varies between not detectable level of concentration in most of studied species
and 201 mg/kg. The maximum values of zinc and lead concentration were found for L.
perenne species, 922 mg/kg and 201 mg/kg respectively.
The cobalt concentration was below limit of detection for three of studied species. The highest
values of this element concentration were found for species S. capillata and A. tenuis. The
accumulation of Co was influenced by the metal concentration in soil and by the soil moisture
which lead to the leaching of some cobalt compound and increasing of cobalt availability for
plants. The mean values of Ni concentration in studied species of plants varied widely from
one species to another even inside of the same genus. The lowest value of Ni concentration
was 3.88 mg/kg for A. tenuis and the highest was for A. alba, 60.23 mg/kg. The range of Mn
concentration was between 165.9 mg/kg and 703.92, with the highest values of concentration
for L. perenne species. The mean concentration of Cr in studied plants ranged between 10.04
mg/kg for L. campestris and 191.99 mg/kg for S. capillata.
Species Cu Zn Sn Pb
L. perenne 61.95±15.7 921.67±136.2 217.83±40.1 201.23±14.9
F. pratensis 113.83±22.8 130.62±48.2 379.23±103.2 <LD
S. capillata 10.04±3.9 88.22±2.8 265.25±17.4 <LD
A. alba 31.83±5.5 85.34±7.12 250.04±74.2 5.21±3.2
A. tenuis 0.99±0.6 72.33±3.8 235.94±12.1 2.72±0.1
C. dactylon 25.11±2.4 62.09±2.0 186.13±31.4 <LD
L. campestris 1.76±0.4 71.69±2.9 8.38±0.9 7.38±0.5
Species Co Ni Mn Cr
L. perenne <LD 12.55±4.6 703.92±156.2 114.19±35.4
F. pratensis <LD 33.58±11.6 341.90±41.5 173.99±28.6
S. capillata 4.49±0.4 27.72±2.5 342.13±16.6 191.99±12.8
A. alba 3.20±2.0 60.23±20.5 362.07±79.2 56.75±15.1
A. tenuis 4.60±0.2 3.88±0.4 269.39±16.5 21.96±2.53
C. dactylon 2.61±1.1 28.60±10.6 296.33±48.7 28.95±3.9
L. campestris <LD 17.18±1.41 165.90±0.8 10.04±1.4
Table 2. Mean concentration of heavy metals in perennial grasses (mg/kg dry matter)
16 Soil Pollution
For phytoremediation process to be effective it is better to use those biosystems species adapted
to the climatic and soil conditions of the area to be de-polluted. For this reason, the species
used in the studies were chosen from those plants that normally grow in the industrial area of
the city of Targoviste, perennial grass which are effective to mowing and rebuild their
vegetative mass. In addition, the losses caused by death of leaves are greatly reduced.
The bioaccumulation capacity of plants was estimated as the ratio of metal content in soil and
the metal concentration in plant. This ratio is called bioaccumulation factor (BF) [7] and we
evaluated as weak accumulators the species which have a BF value between 0.8 - 1.2, as good
accumulators the species with a value of BF between 1.5 - 5.0 and hyperacumulators those
species with higher BF than 5.0 (Table 3).
Absorption and accumulation of metals in perennial grasses was influenced by both species
and the soil underneath, pH, moisture and metal content in soil. The bioaccumulation of the
studied metals was differently influenced by pH of soil and metal content.
Even F. pratensis and L. perenne showed the highest values of Cu, Zn, Sn concentration, they
did not show the highest accumulation capacity for those metals. The best accumulator for Cu,
Zn and Sn were the plants of C. dactylon species which showed BF values of 1.12, 1.37 and 6.06
respectively for Cu, Zn and Sn. Lead was very well accumulated by L. campestris which showed
a very high level of metal bioaccumulation, 12.3. Tin was the metal with best bioaccumulation
in perennial grasses.
Metal BF Accumulation gradient Metal BF Accumulation gradient
Cu 0.88±0.2 A. alba – weak Pb 1.04±0.1 L. perenne – weak
1.12±0.1 C. dactylon – weak 4.54±0.2 A. tenuis – good
Zn 0.92±0.1 L. campestris – weak 12.3±0.9 L. campestris – hyper
0.98±0.1 A. tenuis – weak Co <0.8 Not accumulative
1.00±0.1 A. alba – weak Ni 1.26±0.05 L. campestris – good
1.31±0.3 L. perenne – good 1.27±0.42 C. dactylon – good
1.37±0.1 C. dactylon – good 1.63±0.63 A. alba – good
Sn 2.43±0.1 S. capillata – good Mn <0.8 Not accumulative
3.00±0.8 A. alba – good Cr 0.83±0.08 F. pratensis – weak
4.10±0.8 F. pratensis – good 1.16±0.23 C. dactylon – weak
4.11±0.6 L. perenne – good 1.51±0.11 A. tenuis – good
5.85±0.1 A. tenuis – hyper 2.11±0.10 S. capillata – good
6.06±0.3 C. dactylon – hyper 2.68±0.75 A. alba – good
Table 3. Bioaccumulation factor (BF) of heavy metals in plant species – metal accumulation capacity of plants (not
accumulative, weak accumulative, good accumulative or hyper accumulative)
None of the studied species of perennial plants showed accumulative capacities for either Co
or Mn. This was probably because of the exclusion mechanism of these plants for the two
elements. For Ni, three of studied species showed accumulative capacity: L. campestris, C.
dactylon and A. alba, while for Mn five studied species showed accumulative capacity. For the
phytoremediation of soils polluted with Ni and Cr, the best species to use is A. alba, because
it showed the highest values of BF, 1.63 and 2.68 respectively.
6.3. Heavy metal extraction from plant biomass
Following the phytoremediation of soil polluted with heavy metals, a crop of 400 kg of plants
was obtained which contain heavy metals, but the concentrations of these elements in the biomass
is very low for the use of these plants for heavy metal extraction process. In order to concen‐
trate the metal, plants were subjected to a drying process in which the mass of substance decreased
by 64%, registering a percentage of metal concentration of 243.9%. The percentage of ash resulted
from the incineration of dry biomass was 14.3% and the percentage of metal concentration in the
plant ash increased by values between 544.5 - 2282.1% (Table 4) The differences of metal
concentration by incineration was because of volatilization of some elements or because of the
adsorption on flying ash which was lost during the incineration. Because of that, future researches
will be needed for the construction of an integrated system of plant biomass incineration,
designed with filters and cyclone for the recovery of all particles of ash.
In this experiment, the ash was used for metal extraction by leaching. The results showed low
effectiveness of this method, because the metals from ash were not fully extracted in the
leachate. The weakest extraction in the leachate was for tin, only 16.3% from the total quantity
of tin in the ash. A fair extraction was only for Ni and Cr which were extracted 92.6% and 87.8%
respectively from the ash (Table 5).
Future research should be conducted to establish methods of metal extraction by leaching of

ash with better results for the majority of heavy metals to be recovered.
Concentration in plants (mg/kg) Percentage of concentration %

Metal
fresh biomass dry matter ash by drying by incineration
Cu 81.35 198.41 1454.08 243.9% 732.8%
Zn 172.87 421.63 2295.92 243.9% 544.5%
Sn 106.77 260.42 3367.35 243.9% 1293.0%
Pb 3.05 7.44 76.53 243.9% 1028.6%
Co < LD < LD 306.12 - -
Ni < LD < LD 2142.86 - -
Mn 179.99 438.99 3214.29 243.9% 732.2%
Cr 65.08 158.73 3622.45 243.9% 2282.1%
Table 4. Metal concentration in plant biomass by drying and incineration (%)

18 Soil Pollution
Ash concentration Percentage of metal extraction into

Metal Solution concentration (mg/l)
(mg/kg) solution* (%)
Cu 1454.08 11.20 30.8%
Zn 2295.92 30.45 53.1%
Sn 3367.35 13.70 16.3%
Pb 76.53 1.25 65.3%
Co 306.12 < LD -
Ni 2142.86 49.60 92.6%
Mn 3214.29 35.5 44.2%
Cr 3622.45 79.55 87.8%
* 10 g of ash were used to prepare 400 ml solution
Table 5. Heavy metal extraction by leaching of ash
In the process of electrolysis, the cathode layout has changed on the surface. At macroscopic
level could be observed the metal deposition after the electrolysis - oxides spots and at the
microscopic level could be observed a smoothing of the surface (Figure 6).
There was a difference in mass of the cathode of 0.7 mg, from 4.6242 g before to 4.6249 g after
electrolysis.
Figure 6. Metal depositions on electrode at 1x, 40x and 80x magnification [2]
The quantitative evaluation of metal deposition on cathode was made by EDXRF and different
metal concentrations were observed. The most effective deposition was for Ni, Mn and Cr,
which showed concentration of 3.07, 2.2 and 7.3 g/kg respectively.
6.4. Balance of phytoremediation and heavy metal extraction

The experiment of phytoremediation of heavy metal polluted soil in the vicinity of Targoviste
city showed the results for one growth season of perennial grass Lolium perenne (Table 6). The
metal concentration in soil as evaluated before and after the plant culture and after mowing
the results showed a decreasing of metal concentration in soil with 0.3 – 5.9%. The concentra‐
tion of cobalt and nickel had the lowest decreasing. The best accumulated heavy metals by
plants were Zn and Pb. The accumulation of Pb was because of the high concentration of this
metal in soil and the Zn accumulation was because of the synergic effect of the Pb concentration
in soil on the accumulation of Zn [2].
Metal concentration in soil

(mg/kg dry matter) Percentage of metal Metal content in plant
Metal BF
extraction by plants (%) (mg/kg dry matter)
before after
Cu 313.54 303.62 3.2% 198.41 0.653
Zn 141.04 132.72 5.9% 421.63 3.177
Sn 107.36 102.78 4.3% 260.42 2.642
Pb 71.99 68.23 5.2% 7.44 0.109
Co 16.77 16.72 0.3% < LD -
Ni 52.93 52.63 0.6% < LD -
Mn 1176.02 1152.63 2.0% 438.99 0.381
Cr 90.66 87.56 3.4% 158.73 1.813
Table 6. Results of the phytoremediation of heavy metal polluted soil near Targoviste
The synergic effect of the Pb concentration in soil can be observed also in the value of Zn
bioaccumulation factor, 3.177. During the experiment of phytoremediation, Lolium perenne
showed good accumulative capacity for chromium also.
By reporting the results to the quantity of ash used in the experiment, small quantities of heavy
metals were obtained (Table 7).
According with the biomass quantity that can be harvested from one hectare cultivated with
Lolium perenne species (40 tons) and ash content of raw material (5.1%), in a growing season of
this species, can be extracted from soil about 0.13 g of Cu, 0.27 g of Zn, 0.07 g of Sn, 0.04 g of
Co, 0.88 g of Ni, 0.63 g of Mn, and 2.09 g of Cr. The efficiency of heavy metals extraction by
electrolysis can be improved by increasing the leaching efficiency.
Even if these amounts are very small and the economic value of process is almost nonexistent,
the immense advantage of metal recovery is the extraction of these elements from soil and the
decreasing of toxicity risk caused by the presence of heavy metals in the environment.
20 Soil Pollution
Metal concentration in leaching Metal concentration in

Recovered metal
Metal solution (mg/l) cathode deposit
before after (mg/kg) µg/5 g ash g per ha
Cu 11.20 7.40 445.92 0.31 0.13
Zn 30.45 22.40 946.39 0.66 0.27
Sn 13.70 11.45 260.91 0.18 0.07
Pb 1.25 0.55 < LD - -
Co < LD < LD 142.31 0.10 0.04
Ni 49.60 23.35 3076.37 2.15 0.88
Mn 35.5 16.935 2222.486 1.56* 0.63
Cr 79.55 17.70 7312.619 5.12** <2.09
* 2% of Mn deposit is probably because of anodic dissolution

** The results were probably contaminated because of high content of Cr in the stainless steel (14%)
Weight of metal deposition – 0.7 mg; fresh biomass per hectare – 40 t; percentage of ash – 5.1%
Table 7. Results of heavy metal extraction from Lolium perenne, by electrolysis
7. Discussion
The present study concerned a novel approach of the phytoremediation process: phytoreme‐
diation followed by heavy metal extraction. The researches concerned first the analysis of soil
to reveal the necessity of remediation, then researches concerned the species used for phytor‐
emediation and methods of processing the resulted biomass. The treatment of biomass
enriched with heavy metals led to the recovery of these elements as alloy, which can be
economically exploited. Also, the resulted ash was characterized as material with low heavy
metal concentration, which can be used in agriculture or can be disposed without the risk of
toxicity.
Analysis of soil samples from the vicinity of metallurgical plant of Targoviste indicated an
exceeding of alert threshold for Cu, Sn, Pb, Mn and Cr, with metal concentration levels that
could be harmful for both the agricultural and industrial soil [49]. According to these aspects
and because the land was used as pasture, the soil needed interventions for decreasing the
heavy metal concentration. The best way to remediate this land was the use of wild growing
perennial plants to extract heavy metals from soil and the resulted biomass to be mowed and
disposed as hazardous material. The wild growing plants have the advantage of ecological
adaptability and low costs for starting and maintaining the crop [2].
Metal concentration was evaluated for seven perennial grasses, regarding the bioaccumulation
capacity of these species, to establish the most effective species to be used in remediation of
studied area. Results indicated heavy metal concentrations for Lolium perenne similar to results
from previous articles: chromium and copper concentration showed values similar to the
results presented by Arienzo et al., 35 and 70 mg/kg for Cr and Cu respectively [50]. Instead,
the results of present study indicated 2 and 6 times higher concentrations for Pb and Zn when
compared with plants from unpolluted sites and similar results when compared with plants
from metallurgical site [50]. The same values of Pb and Zn concentrations were obtained also
by Bidar et al. [51] when have researched the behavior of Trifolium repens and Lolium perenne
growing in a heavy metal contaminated field. This study indicated that the two metals, Pb and
Zn, are accumulated predominantly in roots (269.98 mg/kg for Pb and 1511.18 mg/kg for Zn),
and in lower concentrations in shoots (45.65 mg/kg for Pb and 218.15 mg/kg for Zn) [51]. The
results of soil phytoremediation can be improved by the addition of chelates such as EDGA
which lead to a 2 times increasing of heavy metal uptake by plant [52].
For a correct evaluation of the most efficient species to be used in phytoremediation, the
bioaccumulation factor should be investigated because it indicated the metal accumulation
ability of each species by comparing the metal concentration in plant with the metal content
of the soil [7]. The results of present study indicated that, despite some species showed a high
metal concentration in shoots, the metal uptake was caused by the high metal content of soil,
not by the accumulation capacity of that species [2]. Only some of the studied species accu‐
mulated heavy metals in higher concentration than the metal content of soil (BF > 1) (Table 3).
For effective results of the phytoremediation, the process should be implemented for a long
period of time, with many growing seasons followed by mowing of plants [7]. Therefore, from
a phytoremediation process implemented at commercial scale will result huge quantities of
plant biomass which should be treated as hazardous material because of the contamination
with heavy metals [53]. The leaching test showed that the composting of this biomass is not
efficient, because by composting process are formed soluble organic compounds that enhanced
metal solubility and availability for plants [53]. Based on these requirements, the present study
aimed the development of a novel method of thermal treatment of the biomass from phytor‐
emediation to reduce the initial volume to 5.1%, by drying and incineration. A similar percent
(2 – 5%) of mass reduction was indicated by Ghosh and Singh [53], after biomass combustion.
Thereby, the much smaller quantities of ash can be properly disposed, but still with the risk
of heavy metal toxicity. Also, the resulted ash can be used in phytomining process to recover
the saleable heavy metals [53].
In 1994, the development of novel methods for metal recovery from ash resulted from the plant
incineration have been referred as future research, known as extraction from bio-ore [54]. The
recovered metals could reveal the secondary potential of hyperaccumulating species, the
economic value besides the environmental benefits.
In the incineration process, the organic matter is destroyed, releasing metals as oxides [53]. At
higher temperature, over 600ºC, the most toxic metals became volatile [37], and because of this
reason the thermal treatment should not exceed this temperature [2]. Thus, by the incineration
of plant biomass, the metal concentration is increasing by ash mass and the volatilization
should be avoided. A very important factor that influenced the metal recovery efficiency was
the leaching solution, which determined the metal solubility according with the pH of leachate.
The use of nitric acid and sulfuric acid as leaching reagent had a positive influence on the nickel
and chromium solubility. The lowest solubility showed tin and copper. The experimental
results showed that the methods used for thermal treatment of plant biomass and the leaching
22 Soil Pollution
method were efficient and a quantity of 0.7 mg of alloyed (Ni, Cr and Mn was majority) metals
was obtained. For a selective recovery of heavy metals is necessary that the electrochemical
process should be conducted according to the differences of deposition potential of each metal
[42, 43].
The balance of phytoremediation and heavy metal extraction indicated that the thermal
treatment followed by ash leaching and electrolysis was an efficient method of metal extraction
from the phytoremediation by-products – the plant biomass. By this process could be recov‐
ered saleable heavy metals and the waste resulted from phytoremediation was a heavy metal
low-concentration material, without toxicity risk.
8. Conclusions
For the studied area, in the vicinity of metallurgical plant of Targoviste city, the heavy metal
concentration in soil for Cu, Sn, Pb and Mn exceeded the alert threshold for agricultural soil.
For Zn, Ni and Cr, some of the results showed values that exceeded also the alert threshold
for industrial soils.
The metal concentration in soil was according with the position against the pollution source.
The heavy metal concentration was widely different in the seven studied species of perennial
grasses, and the bioaccumulation capacity was different according with the species and metal
concentration in soil. Even a species accumulate high concentrations of metals, this could be
because of the high content of metal in soil, not because the species showed accumulative
capacity – BF higher than 1 (Table 8).
Metal Maximal concentration in studied species (mg/kg) Maximal value of bioaccumulation factor
Cu 113,83 Festuca pratensis 1,12 Cynodon dactylon
Zn 921,67 Lolium perenne 1,36 Cynodon dactylon
Sn 379,23 Festuca pratensis 6,06 Cynodon dactylon
Pb 66,30 Lolium perenne 12,29 Luzula campestris
Co 4,6 Agrostis tenuis 0,40 Agrostis tenuis
Ni 60,23 Agrostis alba 1,63 Agrostis alba
Mn 703,93 Lolium perenne 0,33 Lolium perenne
Cr 191,99 Stipa capillata 2,67 Agrostis alba
Mo 25,63 Festuca pratensis 15,34 Cynodon dactylon
Table 8. The maximal values of heavy metal concentration and bioaccumulation factor for studied plant species
The thermal treatment of plant biomass was an effective method for metal concentration in
the material which will be used in metal recovery.
The leaching method should be improved for the extraction of a higher percentage of metals
from ash. A quantity of 0.7 mg of metals deposition was obtained after the run of an electro‐
chemical procedure for metal recovery.
By thermal treatment, leaching and electrolysis, small quantities of heavy metals can be
recovered from plants used in phytoremediation of polluted soils. The extraction of these
elements has advantage for environmental protection, by decreasing the risk of toxicity of
heavy metals in soil.
Acknowledgements
The research is part of the PhD thesis “Studies and research concerning the remediation of
heavy metal polluted soil by eco-technological procedure”, in Romanian, realized by the
author in 2011. Is a good opportunity to thank my colleagues Adrian Catangiu for his involve‐
ment in the experiment of metal recovery by electrolysis and Irina Fierascu and Radu Claudiu
Fierascu for their involvement in the sample analyses by ICP-AES and EDXRF. Also I want to
thank my thesis coordinator, Prof. Georghe Ionita for his support.
Author details
Carmen Cristina Elekes*
Department of Environmental Engineering, “Valahia” University of Târgovişte, Dâmboviţa,

Romania
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chapter 11
Selected Bioremediation Techniques

in Polluted Tropical Soils
Emmanuel Uzoma Onweremadu
1. Introduction
Ecosystems are regularly confronted with natural environmental variations and disturbances
over time and geographic space. A disturbance is any process that removes biomass from a
community, such as fire, flood, drought, or predation. Disturbances occur over vastly different
ranges in terms of magnitudes as well as distances and time periods (Levin, 1992) and are both
the cause and product of natural fluctuations in death rates, species assemblages, and biomass
densities within an ecological community. These disturbances create places of renewal where
new directions emerge out of the patchwork of natural experimentation and opportunity
implying a good measure of ecological resilience is a cornerstone theory in ecosystem (Folke,
et al., 2004).One of such disturbances is pollution which alters ecological balance.
Intense industrial activity and urbanization in recent times, especially in developing countries,
have led to serious environmental pollution, resulting in a large number and variety of
contaminated sites which became a threat to the local ecosystems. In all these, natural resources
such as soils, water, air and vegetation are adversely affected.
Industrial revolution gave birth to environmental pollution which continued till today. It was
a revolution that led to the emergence of great factories and consumption of immense
quantities of fossil fuels which was associated with an unprecedented rise in air pollution and
large volume of industrial chemical discharges. This was added to the growing population
with a load of untreated human waste. The Second World War made pollution to become a
popular issue due to radioactive fallout from atomic warfare and testing. Pollution began to
draw major public attention with the emergence of cities and megacities associated with a
stockpile of refuse and characterized by substantial output of sewage and particulate matter.
Pollution defines the introduction of harmful substances often referred to as contaminants into
the natural environment that cause adverse change. The term contamination is in some cases
used interchangeably with pollution in environmental chemistry, where the main interest is
the harm done on a large scale to humans or to organisms or environments that are important
to human beings. Common soil contaminants include chlorinated hydrocarbons, heavy metals
such as chromium, cadmium–found in rechargeable batteries, and lead–found in lead paint,
aviation fuel and still in some countries, gasoline, zinc, arsenic and benzene. Recycling
industrial byproducts into fertilizer may result in the contamination of soils with various
metals. Ordinary municipal landfills are the source of many chemical substances entering the
soil environment and often reaching groundwater, emanating from the wide variety of refuse.
In the case of the term contamination, it is the presence of a minor and unwanted constituent
in a material, in a physical body or in the natural environment. In chemistry, contamination
usually refers to a single constituent, but in specialized fields the term can also mean chemical
mixtures, even up to the level of cellular materials.
Pollution may take various forms including discharge of deleterious chemical substances on
natural substances. Pollution can be point source or nonpoint source pollution.
Sometimes pollution takes the form of harmful energy such as noise, heat or light. Generally
speaking, foreign substances and energies which contaminate natural resources are referred
to as pollutants. Substances contain some level of impurity; and this may become an issue if
the impure chemical is mixed with other chemicals or mixtures and causes additional chemical
reactions. Sometimes, the additional chemical reactions are beneficial, in which case the label
‘contaminant’ is replaced with reactant or catalyst. When additional reactions are detrimental,
other terms such as toxin or poison depending on the chemistry involved are used. However,
if no remedial action is undertaken, the availability of arable land for cultivation will decrease,
because of stricter environmental laws limiting food production on contaminated lands.
Inorganic and organic contaminants typically found in urban areas are heavy metals and
petroleum-derived products. The presence of both types of contaminants on the same site
presents technical and economic challenges for decontamination strategies. There have also
been some unusual releases of polychlorinated dibenzodioxins, commonly called dioxins for
simplicity.
In Nigeria, there is paucity of soil information leading to several forms of soil degradations.
Except in recent times environmental impact assessments (EIAs) are rarely conducted on
natural resources before embarking on major projects. The EIAs are often not backed up with
necessary implementation legislations. Mineral exploration and exploitation as well as various
construction activities are known to have negative impact on surface and subsurface soils,
surface and groundwater, rocks and rocklike minerals, atmospheric resources, vegetation and
wildlife.
Available soil data are not problem-solving (Lal and Ragland,1993).Non-use of soil survey
data and information has led to soil and soil-related environmental problems such as nutrient
depletion, nutrient imbalances, multiple nutrient deficiencies, nutrient toxicity, general decline
in soil quality and yield decline. The situation is often aggravated by socioeconomic pressures
Selected Bioremediation Techniques in Polluted Tropical Soils 3
mainly resulting from poverty and inability to afford relevant inputs of agricultural produc‐
tion. Sound characterization and classification of soils based on quality and proper presenta‐
tion of such information in user-friendly form is a necessary adjunct in sustained use of soils.
Again, soil quality data will go a long way in promoting bio-safety of farm products for both
local consumption and their internationalization.
Primarily, this paper is aimed at reviewing crude oil and non-crude oil polluted soils of tropical
soils with particular emphasis on Nigeria. Specifically, some biotechnological methods are
suggested for the amelioration of contaminated soils. A good knowledge of status and
distribution of polluted soils will go a long way in assisting in the production of land use maps
which will facilitate policy and legislations on soil and soil-related natural resources. Land use
maps derived from soil survey and land evaluation are useful in soil management as well as
in vulnerability and risk assessments. This is true as soil quality problems vary requiring
different remediation strategies to overcome.
Remediation deals with the removal of pollutants or contaminants from natural resources. The
affected natural resources may include soil, groundwater, surface water sediment, vegetation,
rock minerals, wildlife and air. A major aim of remediation is the recovery and general
protection of human health and the environment. Sometimes, remediation is done in places
intended for redevelopment. Remediation goes with an array of regulatory requirements, and
its assessments are based on human health and ecological risks.
Several approaches are used in the remediation of polluted soils, ranging from biological,
chemical and engineering techniques. Sometimes, it may require a combination of organic
and inorganic strategies. For instance the Neapolitan yellow tuff (NYT) was utilized as a
component of an organo-mineral sorbent/exchanger soil conditioner with pellet manure
(NYT/PM) to reduce the mobility of Cd and Pb and recover plant performance in heavily
polluted soils from illegal dumps near Santa Maria La Fossa (Lower Volturno river basin,
Campania Region, southern Italy). Pot experiments were performed by adding the NYT/PM
mixture (1:1, w/w) to polluted soil at the rates of 0%, 25%, 50% or 75% (w/w). Wheat
(Triticum aestivum) was used as the test plant. The addition of organo-zeolite NYT/PM
mixture significantly reduced the DTPA (diethylene-triamine-pentaacetic acid)-extractable
Cd and Pb from 1.01 and 97.5 mg kg−1 in the polluted soil, to 0.14 and 11.6 mg kg−1,
respectively, in the soil amended with 75% NYT/PM. The best plant response was ob‐
served in amended soil systems treated with 25% NYT/PM, whereas larger additions
induced plant toxicities due to increased soil salinity.
When a soil on site is found to be contaminated to a depth of several metres and construction
work needs to get started in a few months’ time, soil replacement is the fastest remedy.
However, some of the contaminated areas can be restored by combining modern and age-old
methods. This is where plants and their microbial partners may enter the picture now and in
the future. This because heavy metals in soils with residence times of thousands of years
present numerous health dangers to higher organisms (Garbisu and Alkorta,2001). They are
also known to decrease plant growth, ground cover and have a negative impact on soil
microflora (McGrath et al.,2001). There is increasing and widespread interest in the mainte‐
nance of soil quality and remediation strategies for management of soils contaminated with
trace metals, metalloids or organic pollutants. Heavy metals are deposited in soils by atmos‐
pheric input and the use of mineral fertilizers or compost, and sewage sludge disposal.
Conventional remediation methods usually involve excavation and removal of contaminated
soil layer, physical stabilization and washing of contaminated soils with strong acids or HM
chelators (Steele and Pichtel,1998). Bioremediation, that is. the use of living organisms to
manage or remediate polluted soils, is an emerging technology. It is defined as the elimination,
attenuation or transformation of polluting or contaminating substances by the use of biological
processes.
It is no new discovery that many plant species can grow in soils contaminated by various
pollutants. Some species can even sequester or decompose contaminants. Soil and plant
microbes help plants survive in harsh conditions.
Bioremediation includes the productive use of biodegradative processes in the elimination or

detoxification of pollutants that have found their way into the environment, especially where
such pollutants are capable of threatening public health. Some of the methods are ex situ while
others are in situ. The ex situ bioremediation techniques involve the excavation or removal of
soil from ground. A good number of in situ bioremediation techniques are generally the most
desirable options due to cheapness and less disturbances since they provide the services in place
avoiding excavation and transport of contaminants. Processes include phytoremediation,
phytostabilization,phytotransformation,phytoextraction,rhizofiltration and phytoscreening.
Phytoremediation involves the treatment of polluted natural resource through the use of plants
that mitigate the problem without the need to excavate the contaminant material and dispose
of it elsewhere. The use of plants in remediation has been growing rapidly in popularity
worldwide for the last twenty years or so. Phytoremediation may be defined as use of
vegetation to contain, sequester, remove, or degrade organic and inorganic contaminants in
soils, sediments, surface water and groundwater. Phytoremediation is a technology that uses
plants to remove contaminants from soil and water (Raskin and Ensley,2000). The basic idea
that plant can be used for environmental remediation is very old and cannot be traced to any
particular source. However, a series of fascinating scientific discoveries combined with an
interdisciplinary research approach have allowed the development of this idea into a prom‐
ising, cost-effective, and environmental friendly technology.
Certain plants and microorganisms are able to precipitate metal compounds in the rhizo‐
sphere. Efficacy was shown by the use of lead pyromorphite (Cotter-Howells et al.,1999), as
phytoremediation may provide an effective means to reduce metal toxicity as well as metal
mobility ( Cotter-Howells and Caporn,1996). This is referred to as phytoimmobilisation.
Although the application of microbial biotechnology has been successful with petroleum-
based constituents, microbial digestion has met limited success for widespread residual
organic and metals pollutants. Vegetation-based remediation shows potential for accumulat‐
ing, immobilizing, and transforming a low level of persistent contaminants. We can find five
types of phytoremediation techniques, classified based on the contaminant fate: phytoextrac‐
tion, phytotransformation, phytostabilization,phytodegradation, rhizofiltration, even if a
combination of these can be found in nature.
Phytoremediation consists of reducing or eliminating pollutant concentrations in contaminat‐

ed soils, water, or air, with plants. Selected plant species are able to contain, degrade, or
eliminate metals, pesticides, solvents, explosives, crude oil and its derivatives, and various
other contaminants from the media that contain them. Boyd and Javre (2001) reported
phytoenrichement of soils by Sebertia acuminata in New Caledonia. In phytoremediation, the
assumption is that certain plants called hyperaccumulators are able to bioaccumulate,
degrade,or render harmless contaminants found in natural resources such as soils, water, and
air. The maize plant (Zea mays) showed high tolerance towards Cr with negligible concentra‐
tion in leaves (Lasat et al.,1998). A plant is said to be a hyperaccumulator if it can concentrate
the pollutants in a minimum percentage which varies according to the pollutant involved.
More than 1000 mg/kg of dry weight for nickel, copper, cobalt, chromium or lead; or more than
10,000 mg/kg for zinc or manganese are recommended (Baker and Brooks,1989). In addition
to this, it is assumed that hyperaccumulating plants can be found thriving under very harsh
conditions or under situations that are not ideal for plant growth.
Some plants are able to translocate and accumulate particular types of contaminants. Plants
can be used as biosensors of subsurface contamination, thereby allowing investigators to
quickly delineate contaminant plumes (Burken et al.,2011). Chlorinated solvents have been
observed in tree trunks at concentrations related to groundwater concentrations (Vroblesky et
al.,1998). Phytoscreening often leads to more optimized site investigations and reduce
contaminated site cleanup costs. Phytoremediation has become increasingly popular and has
been employed at sites with soils contaminated with lead, uranium, and arsenic and it has the
advantage that environmental concerns may be treated in situ.
The technology of phytoremediation has been successfully used in the restoration of aban‐
doned metal-mine sites, reducing the impact of sites where polychlorinated biphenyls have
been dumped during manufacture and mitigation of on-going coal mine discharges.
There are a range of processes mediated by plants which are useful in soil and soil-related
environmental problems. Processes include phytostabilization, phytotransformation,phytoex‐
traction,rhizofiltration and phytoscreening.
Phytostabilization entails the reduction of the mobility of substances in the environment. This
could be done by limiting the leaching of substances from the soil. Its main focus is on long-
term stabilization and containment of the pollutant. Plants can reduce wind erosion; or their
roots can prevent water erosion, immobilize the pollutants by adsorption or accumulation,
and provide a zone around the roots where the pollutant can precipitate and stabilize.
Phytostabilization focuses mainly on sequestering pollutants in soil near the roots but not in
plant tissues. By this, pollutants become less bioavailable to livestock and wildlife, and human
exposure is drastically reduced.
Phytoextraction is the uptake and concentration of substances from the environment into the
plant biomass. The use of plants to mine toxicants is called phytomining. Phytoextraction
employs metal hyperaccumulator plant species to transport high quantities of metals from
soils into the harvestable parts of roots and aboveground shoots (Chaney et al.,1997). Phy‐
toextraction is an innovation using higher plants for in situ decontamination of metal-polluted
soils, sludges and sediments (Wenzel and Jockwer,1999). Large biomass production and high
rates of metal uptake and translocation into the shoot system are critical in achieving reason‐
able metal extraction rates. Effective phytoextraction requires both plant genetic ability and
the development of optimal agronomic management practices (Gupta and Sinha,2007). Hyper
accumulators are defined as plants that contain in their tissue more than 1,000 mg kg-1 dry
weight of Ni, Co, Cu, Cr, Pb, or more than 10,000 mg kg-1 dry weight of Zn, or Mn (Steele and
Pichte,1998). Hyper accumulation is thought to benefit the plant by means of allelopathy,
defence against herbivores, or general pathogen resistance in addition to metal tolerance
(David et al.,2001). In-situ phytoextraction of Ni by a native population of Alyssum murale on
an ultramafic site (Albania) have been reported (Bani et al.,2007). In the case of phytomining,
the use of native flora (including local populations of hyperaccumulators) with limited
agronomic practices (extensive phytoextraction) could be an alternative to intensively
managed crops. The use of plants in remediation has been growing rapidly in popularity
worldwide for the last twenty years or so. In general, this process has been tried more often
for extracting heavy metals than for organics The technique of phytoextraction uses plants to
remove contaminants from soils, sediments or water into harvestable plant biomass. Such
organisms that absorb larger-than-normal amounts of contaminants from the soil are referred
to as hyperaccumulators. Examples of hyperaccumulators are Athyrium yokoscense (Japanese
false spleenwort), Avena strigosa (Brittle oat), Crotalaria juncea (Sunn hemp), Eichhornia cras‐
sipes (water hyacinth), Pistia stratiotes (water lettuce). Helianthus annuus (Sunflower), Salix
viminalis (Basket willow), Lemna minor (Duckweed), Amaranthus retroflexus (Redroot Amar‐
anth), Glomus intradices (Mycorrhizal fungus), Eragrostis bahiensis (Bahia lovegrass), Cynodon
dacvtylon (Bermuda grass), Festuca arundinacea (Tall fescue), Lolium perenne (Perennial
ryegrass), Panicum virgatum, (Switchgrass), Phaseolus acutifolius (Tepary beans), Cocos nuci‐
fera (Coconut tree), Spirodela polyrhiza (Giant duckweed), Tagetes erecta (African-tall) and Zea
mays (Maize)
In phytoremediation, plants absorb contaminants through the root system and store them in
the root biomass and/or transport them up into the stems and/or leaves. A living plant may
continue to absorb contaminants until it is harvested. Thereafter the process, the cleaned soil
can support other vegetation with significant healthfulness.
Some transgenic plants containing genes for bacterial enzymes have been found to be effective
hyperaccumulators (Meagher, 2000). Salt-tolerant plants like sugar beets are commonly used
for the extraction of sodium chloride in reclaiming soils previously flooded by salt water.
Sunflower (Helianthus annuus) is an effective hyperaccumulator in cleaning soils contaminated
with arsenic. In general, plants with non-invasive and moisture-tolerant root systems can be
planted on the embankments. Crops most commonly planted in decontamination systems in
Colombia are plantain (Musa paradisiaca), papaya (Carica papaya), bore (Alocasia macrorrhiza),
sugar cane (Saccharum officinarum) and nacedero tree (Trichanthera gigantea). They are com‐
monly used for forage production in Colombia. Under local conditions it produces about 10
tons of dry matter ha/year with 18 per cent of protein in the foliage dry matter. A good number
of them grow very well in the sub-Saharan Africa, therefore are suggested for phytoremedia‐
tion in that region.
There are two major forms of phytoextraction, namely assisted or natural phytoextraction. In
induced or assisted phytoextraction, hyper-accumulators are cultivated for the purpose of
remediation. It is associated with the use of chelators in soils to increase metal solubility or
mobilization so that the plants can absorb them more easily. In natural phytoextraction, plants
naturally take up the contaminants in soil unassisted. Many natural hyperaccumulators are
metallophyte plants that can tolerate and incorporate high levels of toxic metals.
An advantage of phytoextraction is friendly moderate impact in the soil ecosystem. Most
traditional methods commonly used for cleaning up heavy metal-contaminated soil disrupt
soil structure and reduce soil productivity, but phytoextraction has the ability of cleaning up
the soil without causing any kind of harm to soil quality and soil structural integrity. In
addition to this, phytoextraction is cost-effective when compared with other soil remediation
techniques, although it is frequently argued argued that significant effects are only achieved
in the long term.
Phytotransformation describes chemical modification of environmental substances as a direct
result of plant catabolic and anabolic activities. These activities lead to inactivation, degrada‐
tion or immobilization. The degradation as caused by plants is referred to as phytodegradation,
On the other hand, immobilization is known as phytostabilization which is a process of
reducing the mobility of substances in the environment, for example, by limiting the leaching
of substances from the soil.
Certain plants render organic pollutants, such as pesticides, explosives, solvents, industrial
chemicals, and other xenobiotic substances non-toxic by their metabolism. Sometimes,
microorganisms living in association with plant roots may metabolize these substances in soil
or water. These complex and recalcitrant compounds cannot be broken down to basic mole‐
cules (water, carbon-dioxide, etc.) by plant molecules, and, hence, the term phytotransforma‐
tion represents a change in chemical structure without complete breakdown of the compound.
The term "Green Liver Model" is used to describe phytotransformation, as plants behave
analogously to the human liver when dealing with these xenobiotic compounds or foreign
compounds (Burken et al., 2004). After uptake of the xenobiotics, plant enzymes increase the
polarity of the xenobiotics by adding functional groups such as hydroxyl groups (-OH).
This is known as Phase I metabolism, similar to the way that the human liver increases the
polarity of drugs and foreign compounds. Whereas in the human liver enzymes such as
Cytochrome P450s are responsible for the initial reactions, in plants enzymes such as nitrore‐
ductases carry out the same role.
In the second stage of phytotransformation, known as Phase II metabolism, plant biomolecules
such as glucose and amino acids are added to the polarized xenobiotic to further increase the
polarity (known as conjugation). This is again similar to the processes occurring in the human
liver where glucuronidation (addition of glucose molecules by the UGT (e.g. UGT1A1) class
of enzymes) and glutathione addition reactions occur on reactive centres of the xenobiotic.
Phase I and II reactions serve to increase the polarity and reduce the toxicity of the compounds,
although many exceptions to the rule are seen. The increased polarity also allows for easy
transport of the xenobiotic along aqueous channels.
In the final stage of phytotransformation (Phase III metabolism), a sequestration of the

xenobiotic occurs within the plant. The xenobiotics polymerize in a lignin-like manner and
develop a complex structure that is sequestered in the plant. This ensures that the xenobiotic
is safely stored, and does not affect the functioning of the plant. However, preliminary studies
have shown that these plants can be toxic to small animals (such as snails), and, hence, plants
involved in phytotransformation may need to be maintained in a closed enclosure.Hence, the
plants reduce toxicity (with exceptions) and sequester the xenobiotics in phytotransformation.
Trinitrotoluene phytotransformation has been extensively researched and a transformation
pathway has been proposed (Subramanian et al.,2006).
In the case of organic pollutants, such as pesticides, explosives, solvents, industrial chemicals,
and other xenobiotic substances, certain plants, such as Cannas, render these substances non-
toxic by their metabolism. In other cases, microorganisms living in association with plant roots
may metabolize these substances in soil or water. These complex and recalcitrant compounds
cannot be broken down to basic molecules (water, carbon-dioxide, etc.) by plant molecules,
and, hence, the term phytotransformation represents a change in chemical structure without
complete breakdown of the compound. The mechanism is likened to the Green Liver Model
which is used to describe phytotransformation, as plants behave analogously to the human
liver when dealing with these foreign compound/pollutant (Burken, 2004), After uptake of the
xenobiotics, plant enzymes increase the polarity of the xenobiotics by adding functional groups
such as hydroxyl groups (-OH).
This is known as Phase I metabolism, similar to the way that the human liver increases the
polarity of drugs and foreign compounds. Whereas in the human liver enzymes such as
Cytochrome P450s are responsible for the initial reactions, in plants enzymes such as nitrore‐
ductases carry out the same role.In the Phase II metabolism, plant biomolecules such as glucose
and amino acids are added to the polarized foreign compound pollutants to further increase
the polarity. This is known as conjugation and is again similar to the processes occurring in
the human liver where glucuronidation and glutathione addition reactions occur on reactive
centres of the xenobiotic.
Phase I and II reactions serve to increase the polarity and reduce the toxicity of the compounds,
although many exceptions to the rule are seen. The increased polarity also allows for easy
transport of the xenobiotic along aqueous channels.In the Phase III metabolism, the foreign
pollutant compounds are a sequestered within the plant. The xenobiotics polymerize in a
lignin-like manner and develop a complex structure that is sequestered in the plant where they
are safely stored. However, such plants can be toxic to small animals like snails, and, hence,
plants involved in phytotransformation may need to be maintained in a closed enclosure.
Plants therefore reduce toxicity and sequester the xenobiotics through phytotransformation.
Trinitrotoluene phytotransformation has been extensively researched and a transformation
pathway has been proposed (Subramanian et al.,2006).
A significant number of organic chemicals and many inorganic ones are subject to enzymatic
attack through the activities of living organisms. Efficacy of microbes in decontamination
depends on some edaphic properties such as soil pH soil aeration, soil nutrient status, soil
moisture, soil temperature, soil texture and type of heavy metal (Vidali,2001). According to
Thapa et al. (2012,) most of modern society's environmental pollutants are included among
these chemicals, and the actions of enzymes on them are usually lumped under the term
biodegradation. The productive use of biodegradative processes eliminate or detoxify pollutants
that have found their way into the environment and threaten public health, usually as
contaminants of soil, water, or sediments is bioremediation (Thapa et al.,2012).
Some microbes can reduce activity of different types of heavy metals. Agricultural wastewater
treatment can be effectively undertaken through biological processes involving the activity of
microorganisms such as bacteria, algae, fungi, plants and animals (Chara et al.,1999). This they
can do by their ability to convert active forms of toxic metals to inactive forms. However, choice
of microbes depends on the availability of energy sources of the organisms in question. Other
environmental conditions like temperatures, oxygen, moisture and the presence of hazardous
contaminant contribute immensely in influencing efficacy of microbes in remediation pro‐
grammes. The aerobic bacteria recognized for their degradative abilities are Pseudomonas,
Alcaligenes, Sphingomonas. These microbes have often been reported to degrade pesticides and
hydrocarbons, both alkanes and polyaromatic compounds. Many of these bacteria use the
contaminant as the sole source of carbon and energy. The contact between the bacteria and
contaminant is a precondition for degradation. Some bacteria are mobile and exhibit a
chemotactic response, sensing the contaminant and moving toward it (Burken et al., 2011).
Soil fungi are very helpful in cleaning the pedosphere. The use of fungi in remediation is
mycoremediation. Mycoremediation is a form of bioremediation in which fungi are used to
decontaminate the area. The term mycoremediation refers specifically to the use of fungal
mycelia in bioremediation. One of the primary roles of fungi in the ecosystem is decomposition,
which is performed by the mycelium. The mycelium secretes extracellular enzymes and acids
that break down lignin and cellulose, the two main building blocks of plant fiber. These are
organic compounds composed of long chains of carbon and hydrogen, structurally similar to
many organic pollutants. The key to mycoremediation is determining the right fungal species
to target a specific pollutant. Certain strains have been reported to successfully degrade the
nerve gases VX and sarin.
In one conducted experiment, a plot of soil contaminated with diesel oil was inoculated with
mycelia of oyster mushrooms ; traditional bioremediation techniques (bacteria) were used on
control plots. After four weeks, more than 95% of many of the PAH (polycyclic aromatic
hydrocarbons ) had been reduced to non-toxic components in the mycelial-inoculated plots.
It appears that the natural microbial community participates with the fungi to break down
contaminants, eventually into carbon dioxide and water. Wood-degrading fungi are particu‐
larly effective in breaking down aromatic pollutants (toxic components of petroleum ), as well
as chlorinated compounds.
Rhizofiltration is the uptake of metals into plant roots. Mycofiltration is a similar process, using
fungal mycelia to filter toxic waste and microorganisms from water in soil. Soils Arbuscular
mycorrhizae (AM) are ubiquitous symbiotic associations between higher plants and soil fungi
(Brown and Wilkins,1985) and their extra-radical mycelium form bridges between plant roots
and soil, and mediate the transfer of various elements into plants. There is also a growing body
of evidence that arbuscular mycorrhizal fungi can exert protective effects on host plants under
conditions of soil metal contamination. Binding of metals in mycorrhizal structures and

immobilization of metals in the mycorrhizosphere may contribute to the direct effects. Indirect
effects may include the mycorrhizal contribution to balanced plant mineral nutrition, espe‐
cially P nutrition, leading to increased plant growth and enhanced metal tolerance. It has been
widely reported that ectomycorrhizal and ericoid mycorrhizal fungi can increase the tolerance
of their host plants to heavy metals when the metals are present at toxic levels. The underlying
mechanism is thought to be the binding capacity of fungal hyphae to metals in the roots or in
the rhizosphere which immobilizes the metals in or near the roots and thus depresses their
translocation to the shoots (Smith and Read, 1997). Arbuscular mycorrhizal plants may exhibit
much lower shoot concentrations of Zn and higher plant yields than non-mycorrhizal controls,
indicating a protective effect of mycorrhizas on the host plants against potential Zn toxicity
(Diaz et al.,1996). It has been demonstrated that at high soil heavy metal concentrations,
arbuscular mycorrhizal infection reduced the concentrations of Zn, Cd and Mn in plant leaves
(Heggo et al.,1990). Field investigations have indicated that mycorrhizal fungi can colonize
plant roots extensively even in metal contaminated sites (Sambandan et al.,1992).
Phytodegradation is commonly applied as a phytoremediation measure. Phytodegradation
(also rhizodegradation) is the breakdown of contaminants through the activity existing in the
rhizosphere. Rhizobacteria are effective in nickel extraction (Abou-Shanab et al.,2003). It is
facilitated by the presence of proteins and enzymes produced by the plants or by soil organisms
such as bacteria, yeast, and fungi. Rhizodegradation is a symbiotic relationship where the
plants provide nutrients necessary for the microbes to thrive, while microbes provide a
healthier soil environment.
Rhizofiltration is a water remediation technique that involves the uptake of contaminants by
plant roots. Rhizofiltration is used to reduce contamination in natural wetlands and estuarine
areas.
Phytodegradation or rhizodegradation is the breakdown of contaminants through the activity
existing in the rhizosphere due to the presence of proteins and enzymes produced by the plants
or by soil organisms such as bacteria, yeast, and fungi. Rhizodegradation is a symbiotic
relationship where the plants provide nutrients necessary for the microbes to thrive, while
microbes provide a healthier soil environment.
Soils that have been contaminated for a long time may undergo prolonged remediation (Olson
et al.,2007) and are less responsive to rhizodegradation than their freshly contaminated
counterparts (Gunderson et al.,2007). There is therefore a need for enhancement of bioavaila‐
bility as a key for successful biodegradation. Often times, selection and engineering of plants
and microbial strains that modify solubility and transport of organic pollutants through
exudation of biosurfactants become necessary and promising (Wang et al.,2007). In enhancing
rhizodegradation, gene cloning of plants containing bacterial enzymes for the degradation of
organic pollutants such as PCBs will be helpful in this regard. Other practices include the use
of of root-colonising bacteria like Pseudomonas fluorescens expressing degradative enzymes
such as ortho-monooxygenase for toluene degradation (Yee et al.,1998).In Nigeria, soils and
sediments polluted with crude oil hydrocarbons are of major environmental concern on
various contaminated sites. Hydrocarbon-degrading microorganisms are ubiquitously
distributed in soils and constitute less than 1% of the total microbial communities but may
increase to 10% in the presence of crude oil (Atlas,1995). However, use of fertilizers in
hydrocarbon-contaminated soils act as biostimulants in such conditions. Some microbes are
able to use HC as a carbon and energy source (van Hamme et al.,2003) preferentially in the
absence of a readily available carbon source like labile natural organic matter. Read et al. (2003)
observed increased phosphorus mobilisation due to exudation of biosurfactants by lupine
(Lupinus angustifolius)
Rhizofiltration is a water remediation technique that involves the uptake of contaminants by
plant roots. Rhizofiltration is used to reduce contamination in natural wetlands and estuary
areas.
Bioremediation can be classified as ex situ and in situ bioremediation. The former techniques
involve the excavation or removal of soil from ground. Important ex situ treatments are
composting, biopiles land farming, and bioreactors. In situ is a simple technique in which
contaminated soil is excavated and spread over a prepared bed and periodically tilled until
pollutants are degraded. The goal is to stimulate indigenous biodegradative microorganisms
and facilitate the aerobic degradation of contaminants. The practice is limited to the treat‐
ment of superficial 10–35 cm of soil. Since land farming has the potential to reduce monitor‐
ing and maintenance costs, as well as clean-up abilities, it has received much attention as a
disposal alternative. In land farming, contaminated soils are combined with nonhazardous
organic amendments such as manure or agricultural wastes. Organic materials in land farming
supports the development of a rich microbial population and elevated temperature Compost‐
ing is a process of piling contaminated soil organic substances such as manure or agricultural
wastes. The added organic material supports the development of a rich microbial population
and elevates temperature of the pile. Stimulation of microbial growth by added nutrients results
in effective biodegradation in a relatively short period of time characteristic of composting.
Sometimes, biopiles are used in bioremediation. A biopile is a hybrid of land farming and
composting; and is used for treatment of surfaces contaminated with petroleum hydrocar‐
bons. Biopiles are improved forms of land farming that tend to control physical losses of the
contaminants through leaching and volatilization. Land farming is a method in which contami‐
nated soil is spread over a prepared bed along with some fertilizers and occasionally rotated.
It stimulates the activity of bacteria and enhances the degradation of oil. But, the use of biopiles
provides a favourable environment for autochthonous aerobic and anaerobic microorganisms.
Composting is a process of piling contaminated soil organic substances such as manure or
agricultural wastes. The added organic material supports the development of a rich microbial
population and elevates temperature of the pile. Stimulation of microbial growth by added
nutrients results in effective biodegradation in a relatively short period of time (Thapa et al.,
2012).
Most in situ bioremediation techniques are generally the most desirable options due to
cheapness and less disturbances since they provide the services in place avoiding excavation
and transport of contaminants. This could useful in pro-poor communities common in sub-
Saharan Africa. However, in situ remediation is among other factors governed by depth of
soils for its efficacy. In many soils effective oxygen is also a prerequisite. Examples of important
in situ bioremediation include are biosparging, bioventing, in situ biodegradation, and

bioaugmentation. The Deinococcus radiodurans is used for metal remediation in radioactively
polluted environments (Brim et al.,2000).
Crude oil is a mixture of thousands of varying chemical compounds. Given that composition
of each type of oil is unique, there are different ways to bioremediate them using microbes and
flora. Bioremediation can occur naturally or can be encourage with addition of microbes and
fertilizers.
The microbes present in the soil at early stage recognize the oil and its constituents by
biosurfactants and bio emulsifiers. After this, they attach themselves and use the hydrocarbon
present in the petroleum as a source of energy. However, low solubility and adsorption of high
molecular weight hydrocarbons can pose as a limiting factor to their availability to microor‐
ganisms. But, addition of biosurfactants enhances the solubility and removal of these contam‐
inants. Again, rates of oil biodegradations increases with addition of biosurfactants.
Volatility, volubility, and susceptibility to biodegradation differ distinctly among constituents
of crude oil. Some compounds are easily degraded, some resist degradation and some are non-
biodegradable (Mukred et al.,2008). Yet, biodegradation of different petroleum compounds
occurs simultaneously but at different rates because different species of microbes preferentially
attack different compounds. This scenario leads to progressive and successive disappearance
of constituents of crude oil over time.
Microbes produce enzymes in the presence of carbon sources, and these enzymes are respon‐
sible for the break down of hydrocarbon molecules. Many different enzymes and metabolic
pathways are involved in the degradation of hydrocarbons contained in crude oil polluted
soils. It implies that complete hydrocarbon degradation requires an appropriate enzyme,
unavailability of which either prevents or minimizes its breakdown.
Bioremediation has various benefits of outstanding environmental and agricultural
implications.
People perceive bioremediation as an acceptable strategy for the transformation of a wide
variety of pollutants, often involving recycling (Polprasert, 1989).
Byproducts from bioremediation treatment are usually harmless products. Such residues
include carbon dioxide, water, and cellular biomass, implying that most hazardous contami‐
nants can be transformed to harmless products thereby eliminating the chance of future
liability associated with treatment and disposal of contaminated material.
Processes involved in bioremediation can be conducted on-site without causing a major
disruption of normal activities of the ecosystem. But, this, they need to transport quantities of
waste off site and the potential threats to human health and the environment that can arise
during transportation are eliminated.
Bioremediation is cheap when compared with other technologies that are used for clean-up of
toxic waste. Some of the contaminants are sources of energy to the soil microbes thereby
sustaining microbial biodiversity. Certain bacteria are mobile and exhibit a chemotactic
response, sensing the contaminant and moving toward it.
Bioremediation was described as a strategy for integrated and sustainable development

(Preston and Murgueitio, 1992). More possibilities of recycling wastes within farming
systems become available as wastes from one process become inputs for another (Preston
and Murgueitio 1992).
2. Limitations of bioremediation
1. Bioremediation is limited to those compounds that can be degraded biologically. How‐

ever, not all compounds are susceptible to rapid and complete degradation. Some
substrates such as straw, saw dust and maize cobs can be used to facilitate contact between
soil microbes and toxicants.
2. Biological processes are often highly specific. For instance, anaerobic bacteria used for
bioremediation of polychlorinated biphenyls in river sediments, dechlorination of the
solvent trichloroethylene. The white rot fungus Phanaerochaete chrysosporium have the
ability to degrade an extremely diverse range of persistent or toxic environmental
pollutants
3. Research is needed to develop and engineer bioremediation technologies that are

appropriate for sites with complex mixtures of contaminants that are not evenly dispersed
in the environment.
4. Bioremediation often takes longer than other treatment options, such as excavation and
removal of soil or incineration.
5. Regulatory uncertainty remains regarding acceptable performance criteria for bioreme‐

diation and there are no acceptable endpoints for bioremediation treatments. 6) There are
some concerns that the products of biodegradation may be more persistent or toxic than
the parent compound.
3. Justification
Soil pollution is widespread in Nigeria leading to varying forms of degradation. Soil pollu‐
tion in Nigeria is associated with loss of bioresources especially plant materials. In reaction to
this, it becomes imperative to use biological techniques in restoring and resisting further
degradation.
Nigeria, the most populous country in Africa, is situated on the Gulf of Guinea in West Africa
with a land area of 351,649 sq mi (910,771 sq km); and total area of 356,667 sq mi (923,768 sq
km). Nigerian population is estimated to be152,217,341 with a growth rate: of 1.9%; Its
neighbors are Benin, Niger, Cameroon, and Chad. The lower course of the Niger River flows
south through the western part of the country into the Gulf of Guinea. Swamps and mangrove
forests border the southern coast; while inland areas are hardwood forests.The vegetation of
Nigeria northwards include derived savannah,guinea savannah,sudan savannah and sahel
savannah. Seven major soil groups in Nigeria include Alfisols, Ultisols, Inceptisols, Entisols,
Vertisols, Oxisols and Histosols. Arable farming is a major socioeconomic activity while crude
oil prospecting is a chief source of national revenue. Oil and oil-related activities plus urban
wastes constitute significant mechanism of pollution.
Figure 1. Location map of the study area
Soil samples were collected by random sampling in polluted soils studied. Soil samples were
collected from epipedons (0-15 cm depth) only. The soil samples were sieved using 2-mm sieve
before they were subjected to various laboratory analyses.
Cation exchange capacity was measured by ammonium acetate method at a pH 7 (Soil Survey
Staff, 2003). Soil pH was determined using a 1:1 soil :water ratio (Soil Survey Staff, 2003). Total
carbon content of soils was estimated by loss on ignition using LECO equipment (Leco Corp,
St. Joseph, M. I.). Exchangeable Ca and Mg in ammonium acetate extracted solution (leachate)
were estimated with atomic absorption spectrophotometer (Perkin Elmer Model) while
exchangeable K and Na were measured by flame photometry. Total nitrogen was estimated
by microkjeldahl (Bremner, 1996). Available phosphorus was obtained using Bray P No.2
method (Olsen and Sommers, 1982).
Soil heavy metals were extracted with 1 M ammonium acetate (NH4OAc) [1:4 ratio of soil / 1
M NH4OAc extraction solution : 60 minutes reaction time (Reed and Martens, 1996]. After
extraction, the suspension was centrifuged at 7500 x g relative centrifuge force for 30 minutes
and the supernatant was passed through a Whatman No.42 paper. The filter paper was allowed
to drip dry. The metal concentrations in the supernatant after centrifugation were determined
using Inductively Coupled Plasma Optical Emission Spectroscopy, IRIS N701776 (Thermo
Jarrell Ash Corporation). The total petroleum hydrocarbon (TPH) was determined using
Fourier Transform Infra-red spectrometry (FTIR) (QAL/AM/S 16) at wavelengths ranging from
2800-3200 cm. The digestion and analytical procedures were checked by analysis of DOLT-3
Matrix Certified Reference Material with known concentration for heavy metals (Cantillo and
Calder, 1990).
5. Results
Table 1 shows chemical composition of cassava sludge as opposed to compost derived from
it. The result showed that cassava sludge had higher values of total carbon and total nitrogen
than the compost manure formed from it.Resultantly, the C:N ratio was 17 when compared to
10 got from the compost. Values of exchangeable calcium and potassium were 9.8 and 18.3
cmol/kg, respectively in the compost manure as opposed to 9.3 and 7.4 cmol/kg in cassava
sludge. Composting of cassava sludge reduced total values of heavy metals in it (Table 1). The
pHKCl value of composted cassava sludge increased to 6.3 (Table 1).
Properties Sludge Final Compost
Total carbon(g/kg) 342 138
Total nitrogen(g/kg) 20 13
Carbon-nitrogen ratio 17 10
Exchangeable calcium(cmol/kg) 9.3 9.8
Exchangeable magnesium(cmol/kg) 7.8 5.3
Exchangeable potassium(cmol/kg) 7.4 18.3
Available phosphorus(mg/kg) 2.2 1.5
Total chromium(mg/kg) 2.6 0.9
Total cadmium(mg/kg) 3.3 2.4
Total vanadium(mg/kg) 2.8 1.2
Total nickel(mg/kg) 7. 2,3
Total petroleum hydrocarbon(mg/kg) 58.6 12.4
pHKCl 4.9 6.3
(Source: Onweremadu, 2008)
Table 1. Typical chemical characteristics of the cassava sludge and the produced compost (dry weight basis)
Certain factors influence ability of microbes to cause degradation (Table 2).Soil moisture
requirement for optimum degradation of oil is high to very high ( 30-90 %) while 25- 28 % soil
Environmental conditions affecting Condition required for microbial Optimum value for an oil
degradation. Parameters activity degradation
Soil moisture 25–28% of water holding capacity 30–90%
Soil pH 5.5–8.8 6.5–8.0
Aerobic, minimum air-filled pore space

Oxygen content 10–40%
of 10%
Nutrient content N and P for microbial growth C:N:P = 100:10:1
Temperature (°C) 15–45 20–30
Hydrocarbon 5–10% of dry weight of

Contaminants Not too toxic
soil
Heavy metals Total content 2000 ppm 700 ppm
Type of soil Low clay or silt content
Sources:Vidali,2001; Thapa et al.2012,
Table 2. Factors Influencing Microbial Activities
moisture is required since a good number of these organisms are aerobes. Again, optimal soil
pH range for oil degradation is 6.5 to 8.0 while microorganisms require 5.5 to 8.8.
Pollutant Time in Days
((mg/kg)) 30 60 90 120 180 LSD0.05
Chromium 15 25 35 40 50 1.25
Cadmium 35 60 65 75 90 0.92
Vanadium 20 25 30 35 40 1.08
Nickel 25 40 60 80 85 0.96
TPH 1.8 2.5 6.5 7.5 9.0 0.09
TPH= total petroleum hydrocarbon
(Source: Onweremadu, 2008)
Table 3. Effect of composted sludge on removability of contaminants at room temperature with time (days)
There was significant reduction ( p=0.05) in the concentration of heavy metals and total
petroleum hydrocarbon with time when treated with compost manure derived from cassava
sludge (Table 3).However, removability rate varied among soil pollutants over time. Higher
of values of Nickel were removed between 60 and 120 days while Vanadium was steadily
degraded in the soil. Generally, more values of these soil toxicants were removed in cumulative
terms from soils on a long-term. But, heavy metal concentration differed between rainy and
dry seasons prevalent in the area as well as between land use types (Table 4). There were greater
variations among heavy metals in rainy season in soils affected by automobile services when
compared with values in dry season in the same land use. The variation was highest in
cadmium (CV=79%), followed by Nickel (CV=48 %) and least in mercury (CV=0 %).Similar
trend was observed in arable soils of the area (Table 4).
Automobile wastes
Heavy metal Arable land Automobile
RS DS RS DS
Cd 34 35 79 17
Cr 35 31 37 25
Ni 25 37 48 18
Hg 54 1 0 49
Pb 20 20 36 32
CV=coefficient of variation in percentage, DS=dry season, RS=rainy season, Cd=cadmium, Cr=chromium, Ni=nickel,
Hg=mercury, Pb=lead
(Onweremadu et al., 2007).
Table 4. Seasonal variability (CV) of heavy metal concentration (mg/kg) in soils affected by
6. Discussion
Exchangeable basic cations are significantly deficient in Nigerian soils particularly polluted
soils. Application of composted cassava sludge will enrich these soils with basic cations
thereby reducing predominance of acidic radicals. Composted cassava sludge has high content
of these neutralizing cations plus appreciable values of total nitrogen which is easily leached
in these tropical soils (Table 1).Again, the pH value of the compost (6.3) (Table1) is optimal for
microbial activity in soils (Table 2) as most tropical crops perform optimally at ph range of 5.5
to 6.5 (Ahn,1979). Composted cassava sludge progressively removed heavy metals and total
petroleum hydrocarbons in crude oil polluted soils of Nigeria (Table 3). In a similar study,
Wen et al. (2002) reported a depression in the phytoavailability of copper using sludge compost
but Sims and Kline (1991) observed variations in characteristics of different composts and
extractants, implying compost type influences uptake and availability.in different media..It is
possible sorption ability of organic materials my depend on the specific surface area of each
type. However,the concentration of these toxicants vary with season as indicated in Table 4,
implying that their application strategies will follow the temporal variability. It was observed
that impact of composted sewage waste became significant after thirteen years in an experi‐
ment conducted by Nogueirol et al. (2013) using three rates of sludges with maize and sugar
cane as test crops.
Knowledge on the fate of heavy metals in soil–plant system is of great importance as that helps
in predicting food quality especially in popular crops. Highest concentrations of heavy metals
were recorded in the topmost layer of soils (Yedilar et al.,1994) and they reported that concen‐
trations in rice leaves and grains were lower than values observed in rice roots in China.
Composted sludge has high content of organic carbon which has the capacity of absorb‐
ing organic pollutants in soil thereby reducing their solubility as demonstrated by several
authors (Hernandez-Soriano et al.,2007; Stevens-Garmons et al.,2012). In addition, organic
carbon inputs might enhance the soil buffer capacity. The application of slightly acidic
sewage (6.3), will also affect the mobility and bioavailability of metals present in soils. The
evolution of heavy metal distributions and bioavailability depended not only on total metal
concentration but also on other properties such as pH, organic matter decomposition and
dissolved organic carbon (Miaomiao et al.,2009) and they reported that composting systems
decreased soil pH, soil organic matter and dissolved carbon. Their results varied between
sewage sludge and swine waste. In some studies conducted in China, soil pH, soil organic
matter and clay content were used to predict phytoavailability of Cadmium in paddy
soils,stating that pH negatively correlated with cadmium cont of soils (Krebs et al.,1998).
Martinez and Motto, (2000) reported that solubility availability, mobility and toxicity of
heavy metals to plants increases as pH decreases. In a study at Suwon, Korea. Kim et al.
(2009) remarked that translocation of heavy metals depended more on the variety of crop
rather on soil physicochemical properties.
7. Conclusion
Polluted soils and soil-related natural resources can be decontaminated using living organisms
including plant species classified as hyperaccumulators. Besides several microbial species can
be used in bioremediation process since they can perform and efficient biodegradation
activities. Efficacy in microbial degradation as well as microbial growth and activity are readily
affected by edaphic characteristics like aeration, pH, temperature, and moisture. Bioremedia‐
tion ensures a minimum impact on the ecosystem, minimizing the presence of potentially toxic
by-products after the process.. There are ex situ and in situ methods of bioremediation; but,
most in situ bioremediation techniques are generally the most desirable options due to cost-
effectiveness and limited impact since they can be performed in situ; avoiding excavation and
transport of contaminants. The in situtechnique proves more affordable in rural communities
with higher prevalence of poverty. The main drawback is associated with long-term character
of bioremediation. However, most bioremediation techniques are constrained by high
specificity of operation, long period of degradation and lack of acceptable endpoints of
treatment. Further research is needed to overcome the time constraint of bioremediation
processes as well and to increase the spectrum of activity of microbial species to address a
variety of wastes that are currently generated and entering the soil..
Author details
Emmanuel Uzoma Onweremadu*
Department Of Soil Science and Technology, Federal University Of Technology, Owerri,

Nigeria
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chapter 12
Thermal Removal of Polycyclic Aromatic Hydrocarbons

from Gasification Biochars
Sebastian Meyer, Bruno Glaser, Daniel Fischer,

Peter Quicker, Yves Noel and Georg Kuffer
1. Introduction
The number of small-scale (< 200 kW electrical capacity) wood gasifiers used for electricity and
heat provision in Central Europe is increasing. After the wood gasification process, about 10%
of the dry-mass of the wood feedstock input are left in form of wood gasification residues [1]
consisting of mixture of char and ash. Inspired by the Terra preta phenomenon and the
intention to generate own humus rich black earths [2], especially farmers are interested in the
opportunity to use the carbonaceous and nutrient-rich gasification residues produced by their
own gasifiers for farm fertilizer production, soil amelioration and for carbon sequestration [3].
However, gasification residues are known for their high content in polycyclic aromatic
hydrocarbons (PAH) [4]. PAH are carcinogenic, persistent, accumulate in organisms and
partly inhibit reproduction [5]. Due to that, gasification residues with very high PAH contents
need to be classified as hazardous waste. As a moderate increase in temperature substantially
increases the vapor pressure of PAH, thermal processes can be used to volatize and subse‐
quently remove PAH from background matrixes [6]. To reduce the PAH content in gasification
residues, a thermal process – the so called PAH volatilization unit has been developed by a
German wood gasifier manufacturer. In the following, a critical assessment of the functional
efficiency of this patented process is described.
The research work was carried out with the objective to evaluate the effectiveness of the PAH
volatilization unit in reducing the PAH content in gasification residues. This objective was
chosen to better assess the environmental suitability of using gasification residues as soil
amendment. Our working hypothesis was that it is possible to technically reduce the PAH
content in gasification residues to a level which allows for an agricultural use of the gasification
2 Soil Pollution
residues in line with the soil protection regulations in Germany. As a precondition for this
evaluation, the suitability of the analysis methods DIN 13877:A and DIN 13877:B for the
determination of the PAH content in wood gasification residues had to be checked. Based on
the results of [7], our working hypothesis in this regard was that analysis method DIN 13877:B
would be more suitable for the analysis task as compared to analysis method DIN 13877:A.
2.1. Comparison of PAH analysis methods for gasification residues (1st experiment)
Hilber et al. 2012 [7] demonstrated that the selection of an appropriate solvent is crucial to
determine the PAH content in biochars. They recommend a Soxhlet-extraction with toluene
for the PAH analysis of biochars. To complement and to reassess this work, the PAH content
(sum of the 16 PAH defined by the US Environmental Protection Agency EPA) of three samples
of the same production batch of gasification residues was determined using two different
extraction methods (cold extraction with acetone according to DIN 13877:A and Soxhlet
extraction with toluene for 5 hours according to DIN 13877:B). The gasification residues had
been produced from poplar wood chips in a commercially operated fixed-bed Joos-Spanner
biomass gasifier (30 kW electrical capacity) of the German manufacturer Spanner Re² GmbH.
The PAH analyses of all experiments described on the following pages were carried out by the
commercial laboratory Görtler Analytical Services GmbH in Vaterstetten, Germany.
2.2. Testing the functional efficiency of the PAH volatilization unit (2nd and 3rd experiment)
In a next step, the functional efficiency of a PAH volatilization unit developed by a cooperation
of the RWTH Aachen and the German manufacturer Spanner Re² GmbH was assessed.
Gasification residues produced under identical process conditions as described in the first
experiment were treated in the PAH volatilization unit as illustrated in Figure 1:
This experimental setting was slightly modified for a 3rd experiment: The sealing air stream
was reduced. In addition to that, air from the external source (17) was intermittently (within
the standstill periods of the conveyor screws) blown into the gasification residue stream within
the heating chamber to supply oxygen to the hot gasification residues. In this technical design
version of the process, about 4% of the flue gas from the PAH volatilization unit was recircu‐
lated back into the air stream which supplied both the combustion chamber and the sealing
air stream.
The PAH content of three samples of the processed batches of gasification residues was
analyzed according to DIN 13877:B in both experiments.
2.3. Redesign of the PAH volatilization unit (4th experiment)
In a redesigned version of the PAH volatilization unit, the manufacturer prevented the
recirculation of PAH into the PAH volatilization unit by blowing clean air from an external
Thermal Removal of Polycyclic Aromatic Hydrocarbons from Gasification Biochars 3
Figure 1. PAH volatilization unit (Source: Patent Specification) The gasification residues entered the unit on the left
side (1) and were being transported by a motor- (4) driven long conveyor screw (5) in an upper tube (24) through a
heat exchanger (23) until the heating chamber (16) on the far right side of the unit. From this point, they were trans‐
ported back through the heat exchanger by a motor- (11) driven second long conveyor screw (12) in a second tube
(22) on the bottom side of the unit until the exit (6) on the bottom left site of the unit. Both conveyor screws were
operated according to the following time schedule: 15 seconds operation, 30 seconds stop, 15 seconds operation and
so forth... Within the heating chamber, the gasification residues got in contact with the outer side of a hot (surface
temperature: about 600 °C) tube (26) transporting hot wood gas produced in the wood gasifier (not shown in the
figure). It is technically possible to supply air from an external source (16) to the gasification residue stream inside the
heating chamber. However, this option was not used for the 2nd experiment. A hot external air stream used for the air
supply of the wood gasifier entered (13) the heat exchanger at a temperature of about 300 °C and left (14) the heat
exchanger at a temperature of about 375 °C, thereby effectively cooling the wood gasification residues on their way
through the heat exchanger. Sealing air from the same external source as the heating chamber air stream was blown
into the left side (6) of the bottom tube of the PAH volatilization unit in counter flow principle and left the unit on the
top left side (1) of the unit. This counter current air flow was introduced to prevent volatized PAH from leaving the
PAH volatilization unit through the exit for the processed gasification residues. In the 2nd experiment, about 5 % of the
air leaving the unit was recirculated back into the sealing air stream.
source in the sealing air nozzle and by discharging the airstream with the volatized PAH in
the gas engine where the wood gas of the gasifier was burned. Further modifications of the
redesigned PAH volatilization unit included larger conveyor screw diameters and conveyor
screw tube diameters. Gasification residues from a 45 kWel wood gasifier fed by a mixture of
chips from different sort of woods were treated in this modified PAH volatilization unit in a
4th experiment. The sealing air stream and the heating chamber air stream volumes were
increased as compared to the 2nd and 3rd experiment. The PAH content of one sample of the
processed batch of gasification residues was analyzed according to DIN 13877:B.
2.4. PAH volatilization in a laboratory scale experiment (5th experiment)
To evaluate the general capability of thermal processes to volatize and subsequently remove
PAH from gasification residues, the following experiment was carried out:
20 gram samples of gasification residues from a 45 kWel wood gasifier fed by a mixture of chips
from pine trees and spruce were filled in an open steel container (20mm width x 20mm depth
x 100mm height). This container was placed in an electrical box furnace (Nabertherm, Model
4 Soil Pollution
LH 30/14) which had been heated to specified temperature levels (550° C, 650° C and 700° C
in three consecutive trials). During the experiments, the box furnace was purged by inert gas
of type Argon 4.6. After 30 minutes, the container was removed from the furnace and imme‐
diately cooled down in a water quench. During the cooling process, the container was purged
from above with Argon 4.6. Due to that, the complete heating and cooling procedure took place
in an oxygen-free environment.
The PAH content of the three samples (one for each temperature level) was analyzed according
to DIN 13877:B.
2.5. Statistical analysis
All statistical tests were conducted with the open source software R 3.0.1 (R CORE TEAM
2012). Due to the low number of samples per treatment (n=3), particular care and attention was
paid to the statistical requirements and assumptions. In this respect, our treatments didn’t
show neither a normal distribution nor homogenous variances among groups so that the
Student's t-test couldn’t be applied. Therefore, we applied a permutation version of ANOVA
according to [8] for the statistical analysis of the obtained results from the experiments 1, 2 and
3. For this purpose we used the package “ImPerm” [9].
3. Results
3.1. Comparison of PAH analysis methods for gasification residues (1st experiment)
Table 1 summarizes the Σ EPA16 PAH content in the three gasification residue samples in mg/
kg dry mass (DM) as determined by the two different analytical methods. On average, the
analysis according to DIN 13877:B resulted in PAH contents more than 4 times higher
compared to the analysis according to DIN 13877:A. The standard errors of the mean (SEM)
are indicated after the average values. The difference between the sample means (n=3) of the
two analysis methods is significant (p < 0.01) according to the applied permutation version of
ANOVA.
Figure 2 depicts the results from Table 1 in two box-and-whisker plots.
3.2. Functional efficiency of the PAH volatilization unit (2nd and 3rd experiment)
Table 3 summarizes the Σ EPA16 PAH content in the gasification residue samples of the
production batch after the treatment in the PAH volatilization unit (PAH-VU) in the 2nd
experiment. On average, the PAH content of the processed gasification residues was 58% lower
as compared to the unprocessed residues from the 1st experiment. The difference between the
sample means (n=3) of the two analysis methods is significant (p < 0.01) according to permu‐
tation version of ANOVA applied. The standard errors of the mean are indicated after each
average value.
Figure 3 depicts the results from Table 3 in two box-and-whisker plots.

Extraction with acetone Extraction with toluene

Gasification Residues
according to DIN 13877:A according to DIN 13877:B
(untreated) Σ EPA16 PAH content Σ EPA16 PAH content
mg/kg DM mg/kg DM
sample 1 542 3,056
sample 2 571 1,009
sample 3 504 2,702
Average 539 ± 16 (SEM) 2,255 ± 516 (SEM)
Table 1. Comparison of the two PAH analysis methods using either acetone or toluene for extraction (1st experiment).
SEM indicates standard error of the mean.
3500
3000
2500
2000
1500
1000
500
0
DIN 13877:A DIN 13877:B
Figure 2. Boxplots indicating the median (central black bar), the minimum and maximum (lower and upper whisker) and the lower and upper
Figure
quartile2. Boxplots
(lower indicating
end upper thebox)
end of the median
of the (central
Σ EPA16 black bar), the
PAH contents minimum
in mg/kg DM inand maximum
three gasification(lower
residueand upper
samples whisker)
after application of the
and the method
analysis lower and upper quartile
DIN 13877:A (lower
(left) and DIN end(right).
13877:B upper end of the box) of the Σ EPA16 PAH contents in mg/kg DM in
three gasification residue samples after application of the analysis method DIN 13877:A (left) and DIN 13877:B (right).
Sample 1 Sample 2 Sample 3 Sample 1 Sample 2 Sample 3
mg/kg DM mg/kg DM
Naphthalene 460 480 420 1,200 570 1,200
2-Methylnapthalene 63 66 61 490 130 330
Acenaphtylene 43 47 43 580 120 340
Acenapthene 14 15 14 110 15 54
Flourene 8,5 8,4 8,9 180 38 120
6 Soil Pollution

Sample 1 Sample 2 Sample 3 Sample 1 Sample 2 Sample 3
mg/kg DM mg/kg DM
Naphthalene 460 480 420 1,200 570 1,200

Acenaphtylene 43 47 43 580 120 340
Acenapthene 14 15 14 110 15 54
Flourene 8,5 8,4 8,9 180 38 120
Phenanthrene 12 15 13 690 170 580
Anthracene 1,8 2,4 2 120 32 120
Flouranthen 1,5 1,9 1,7 94 31 140
Pyrene 1,4 1,6 1,5 70 27 110
Benzo(a)anthracene < 0,01 < 0,01 < 0,01 4,5 2,4 12
Chrysene < 0,01 < 0,01 < 0,01 4,8 2,4 18
Benzo(b)flouranthene < 0,01 < 0,01 < 0,01 1,5 0,43 3,4
Benzo(k)flouranthene < 0,01 < 0,01 < 0,01 0,24 0,05 0,64
Benzo(a)pyrene < 0,01 < 0,01 < 0,01 0,65 0,19 1,9
Indeno(1,2,3.cd)pyren < 0,01 < 0,01 < 0,01 0,25 0,11 0,9
Dibenz(a,h)anthracene < 0,01 < 0,01 < 0,01 0,05 < 0,01 0,31
Benzo(g,h,i)perylene < 0,01 < 0,01 < 0,01 0,16 0,16 0,4
Σ EPA16 PAH 542 571 504 3,056 1,009 2,702
Table 2. Comparison of the two PAH analysis methods using either acetone or toluene for extraction (1st experiment).
Detailed results.
Gasification Production batch without treatment from 1st Production batch with treatment in the PAH-VU
Residues experiment from 2nd experiment
Σ EPA16 PAH content (DIN 13877:B, extraction Σ EPA16 PAH content (DIN 13877:B, extraction
with toluene) with toluene)
mg/kg DM mg/kg DM
sample 1 3,056 1,291

sample 2 1,009 731
sample 3 2,702 806
Average 2,255 ± 516 (SEM) 943 ± 143 (SEM)
Table 3. PAH reduction in PAH volatilization unit (2nd experiment). SEM indicates standard error of the mean.
Σ EPA16 PAH content (DIN Σ EPA16 PAH content (DIN 13877:B,
13877:B, extraction with toluene) extraction with toluene)
mg/kg DM mg/kg DM
sample 1 3,056 1,291
sample 2 Thermal Removal of Polycyclic Aromatic Hydrocarbons
1,009 731from Gasification Biochars 7
sample 3 2,702 806
Average 2,255 ± 516 (SEM) 943 ± 143 (SEM)
Table 3. PAH reduction in PAH volatilization unit (2nd experiment). SEM indicates standard error of the mean.
Figure 3 depicts the results from Table 3 in two box-and-whisker plots:
3500
3000
2500
2000
1500
1000
500
0
1st experiment 2nd experiment
Figure
quartile3. Boxplots
(lower indicating
end upper thebox)
end of the median
of the(central
Σ EPA16black bar), theinminimum
PAH contents mg/kg DM and maximum
in three (lower
gasification and
residue upperfrom
samples whisker)
the 1st experiment
and
(left)the
andlower and
from the 2ndupper quartile
experiment (lower end upper end of the box) of the Σ EPA16 PAH contents in mg/kg DM in
(right).
three gasification residue samples from the 1st experiment (left) and from the 2nd experiment (right).
Gasification Residues Σ EPA16 PAH content
(processed) (DIN 13877:B)
Sample 1 Sample 2 Sample 3
mg/kg DM
Naphthalene 850 370 510
Gasification 2-Methylnapthalene
Residues Σ41EPA16 PAH 15content 17
1-Methylnapthalene 32 14 14
Acenaphtylene 14 4.3 7.9
Acenapthene Sample 13.4 Sample 1.42 Sample
1.1 3
Flourene 3.6 4 0.94
Phenanthrene 330 mg/kg250 DM 200
Anthracene 40 37 32
Naphthalene 850 370 510
Flouranthen 26 32 33
2-Methylnapthalene Pyrene 41 19 1526 1817
Benzo(a)anthracene 1.5 2 1.2
1-Methylnapthalene 32 14 14
Chrysene 2.4 3.4 1.9
Acenaphtylene Benzo(b)flouranthene 14 0.27 4.30.54 7.9
0.23
Benzo(k)flouranthene 0.05 0.04 0.05
Acenapthene Benzo(a)pyrene 3.4 0.06 1.40.1 1.1
0.06
Flourene Indeno(1,2,3.cd)pyren 3.6 0.18 <0.01
4 0.03
0.94
Dibenz(a,h)anthracene <0.01 <0.01 <0.01
Phenanthrene 330 250 200
Anthracene 40 37 32
8 Soil Pollution

mg/kg DM
Pyrene 19 26 18
Benzo(a)anthracene 1.5 2 1.2
Chrysene 2.4 3.4 1.9
Benzo(b)flouranthene 0.27 0.54 0.23
Benzo(k)flouranthene 0.05 0.04 0.05
Benzo(a)pyrene 0.06 0.1 0.06
Indeno(1,2,3.cd)pyren 0.18 <0.01 0.03
Benzo(g,h,i)perylene 0.04 0.07 0.05
Σ EPA16 PAH 1,291 731 806
Table 4. PAH reduction in PAH volatilization unit (2nd experiment). Detailed results.
Table 5 summarizes the Σ EPA16 PAH content in the gasification residue samples of the
production batch after the treatment in the PAH volatilization unit in the 3rd experiment. On
average, the PAH content of the processed gasification residues was 36% lower as compared
to the unprocessed residues from the first experiment. The difference between the sample
means (n=3) of the two analysis methods is not significant (p < 0.05) according to permutation
version of ANOVA applied. The standard errors of the mean are indicated after each average
value.
Gasification Production batch without treatment from 1st Production batch with treatment in the PAH-VU
Residues experiment from 3rd experiment
mg/kg DM mg/kg DM
sample 1 3,056 1,713
sample 2 1,009 1,292
sample 3 2,702 1,298
Average 2,255 ± 516 (SEM) 1,434 ± 113 (SEM)
Table 5. PAH reduction in PAH volatilization unit (3rd experiment). SEM indicates standard error of the mean.
13877:B, extraction with toluene) extraction with toluene)
mg/kg DM mg/kg DM
sample 1 3,056 1,713
sample 2 Thermal Removal of Polycyclic Aromatic Hydrocarbons
1,009 from Gasification Biochars
1,292 9
sample 3 2,702 1,298
Average 2,255 ± 516 (SEM) 1,434 ± 113 (SEM)
Figure
Table 5. 4PAH
depicts the
reduction in results from Table
PAH volatilization unit (3rd 5 in two box-and-whisker
experiment). plots:
SEM indicates standard error of the mean.
Figure 4 depicts the results from Table 5 in two box-and-whisker plots:
3500
3000
2500
2000
1500
1000
500
0
1st experiment 3rd experiment
Figure
quartile4. Boxplots
(lower indicating
end upper end of the median
the box) of the(central
Σ EPA16black
PAH bar), theinminimum
contents mg/kg DMand maximum
in three (lower
gasification andsamples
residue upper from
whisker)
the 1st experiment
and
(left)the
andlower and
from the 3rd upper quartile
experiment (lower end upper end of the box) of the Σ EPA16 PAH contents in mg/kg DM in
(right).
three gasification residue samples from the 1st experiment (left) and from the 3rd experiment (right).
mg/kg DM
Naphthalene 1,000 860 980
(processed)
Acenaphtylene 1.1(DIN 13877:B)
0.47 0.52
Acenapthene 0.06 0.05 0.03
Flourene <0.01 <0.01 <0.01
Phenanthrene 480 mg/kg390DM 240
Anthracene 86 23 34
Naphthalene Flouranthen 1,000 87 86012 980
26
Acenaphtylene 1.1 0.47 0.52
Acenapthene 0.06 0.05 0.03
Flourene <0.01 <0.01 <0.01
Phenanthrene 480 390 240
Anthracene 86 23 34
Pyrene 59 6.8 17
Benzo(a)anthracene <0.01 <0.01 <0.01
Chrysene <0.01 <0.01 <0.01

10 Soil Pollution

mg/kg DM
Benzo(b)flouranthene <0.01 <0.01 <0.01
Benzo(k)flouranthene <0.01 <0.01 <0.01
Benzo(a)pyrene <0.01 <0.01 <0.01
Indeno(1,2,3.cd)pyren <0.01 <0.01 <0.01
Benzo(g,h,i)perylene <0.01 <0.01 <0.01
Σ EPA16 PAH 1,713 1,292 1,298
Table 6. PAH reduction in PAH volatilization unit (3rd experiment). Detailed results.
3.3. Redesign of the PAH volatilization unit (4th experiment)
Table 7 summarizes the Σ EPA16 PAH content in the gasification residue sample of the
production batch after the redesign of the PAH volatilization unit (PAH-VU) in the 4th
experiment. The PAH content of the processed gasification residues was 82% lower as
compared to the average PAH content in the unprocessed residues from the 1st experiment.
Gasification Production batch without treatment from Production batch with treatment in the PAH-VU
Residues 1st experiment from 4th experiment
mg/kg DM mg/kg DM
sample 1 3,056 396
sample 2 1,009
sample 3 2,702
Average 2,255 ± 516 (SEM)
Table 7. PAH reduction in PAH volatilization (4th experiment). SEM indicates standard error of the mean.
3.4. PAH volatilization in a laboratory scale experiment (5th experiment)
Table 9 summarizes the Σ EPA16 PAH content in the gasification residue samples treated in
the laboratory scale experiment in an oxygen-free environment. Following a temperature
treatment of at least 650 °C, the PAH contents in the gasification residues decreased drastically.
Gasification Residues Σ EPA16 PAH content (DIN

(processed) 13877:B)
Sample 1
mg/kg DM
Naphthalene 160
Acenaphtylene 58
Acenapthene 2.8
Flourene 12
Phenanthrene 74
Anthracene 14
Flouranthen 35
Pyrene 32
Benzo(a)anthracene 2.5
Chrysene 3.2
Benzo(b)flouranthene 0.96
Benzo(k)flouranthene 0.32
Benzo(a)pyrene 0.59
Indeno(1,2,3.cd)pyren 0.49
Dibenz(a,h)anthracene 0.1
Benzo(g,h,i)perylene 0.5
Σ EPA16 PAH 396
Table 8. PAH reduction in PAH volatilization (4th experiment). Detailed results.
Gasification Σ EPA16 PAH content (DIN 13877:B, extraction

Residues with toluene)
mg/kg DM
550 °C 1,000
650 °C 1.3
700 °C 0.28
Table 9. PAH content in gasification residues from the laboratory scale experiment (5th experiment)
4. Discussion
The comparison of the PAH analysis methods described in section 2.1 confirms the recom‐
mendation of Hilber et al. 2012 [7]: Soxhlet extraction with toluene resulted in a much higher
extraction of PAH from the gasification residues as compared to the cold extraction with
12 Soil Pollution

550 °C 650 °C 700 °C
mg/kg DM
Naphthalene 590 0.77 0.18
Acenaphtylene 0.3 0.19 < 0.01
Acenapthene 1.3 < 0.01 < 0.01
Flourene 0.2 < 0.01 < 0.01
Phenanthrene 230 0.23 < 0.01
Anthracene 43 0.04 < 0.01
Flouranthen 57 0.02 < 0.01
Pyrene 57 0.03 < 0.01
Benzo(a)anthracene 6.6 < 0.01 < 0.01
Chrysene 13 < 0.01 < 0.01
Benzo(b)flouranthene 3,6 < 0.01 < 0.01
Benzo(k)flouranthene 1,3 < 0.01 < 0.01
Benzo(a)pyrene 0.95 < 0.01 < 0.01
Indeno(1,2,3.cd)pyren 2.5 < 0.01 < 0.01
Dibenz(a,h)anthracene 0.1 < 0.01 < 0.01
Benzo(g,h,i)perylene 0.56 < 0.01 < 0.01
Σ EPA16 PAH 1,000 1.3 0.28
Table 10. PAH content in gasification residues from the laboratory scale experiment (5th experiment). Detailed results.
acetone (see Table 1). Thus, analysis method DIN 13877:B was applied for the PAH analysis
in the subsequent experiments 2-5.
[10] observed a positive correlation between the pyrolysis temperature (between 200 °C and
500 °C in the experiment carried out) applied for the production of biochars from lake
sediments and the sorption of Phenanthrene to these biochars. As gasification temperatures
are higher [1] than the pyrolysis temperatures applied in this experiment, we explain our
analysis results by a strong sorption of PAH to the carbonaceous matrix of the gasification
residues.
[11] recently compared recovery rates after reflux extraction with toluene and a 1:1 acetone/
cyclohexane mixture of the three surrogate PAH Acenaphtene-d10, Phenanthrene-d10 and
Chrysene-d10 which had been previously added to a pyrolysis char made form orchard
pruning. The experiment resulted in higher recovery rates with the solvent toluene as com‐
pared to the solvent mixture acetone/cyclohexane for the high molecular weight PAH Phe‐
nanthrene-d10 (68% compared to 41%) and Chrysene-d10 (58% compared to 7%). The recovery
rate with the solvent toluene was though lower as compared to the acetone/cyclohexane
mixture for the low molecular weight PAH Acenaphtene-d10 (68% compared to 80%).
The differences in the recovery rates stated above are yet small if compared to our analysis
results (see Table 1). Reflux extractions are usually carried out at temperatures close to the
boiling point of the solvent applied. We thus presume that the strong difference in our analysis
results after applying the analysis methods DIN 13877:A (low temperature extraction) and
13877:B (high temperature extraction) can be mainly explained by the temperature difference
between the two extraction methods. We recommend to directly compare Soxhlet extractions
with the solvents toluene and a 1:1 acetone/cyclohexane mixture for the determination of the
Σ EPA16 PAH content of gasification residues for future research.
Based on the results described above, the effectiveness of the legal provisions of the German
Federal Soil Protection Act (Bundesbodenschutzgesetz [BBodSchG]) [12] and the German
Federal Soil Protection and Contaminated Sites Ordinance (Bundesbodenschutzverordnung
[BBodSchV]) [13] for the protection of agricultural soils against PAH pollution in Germany
was analyzed. Special attention was paid to the suitability of the PAH analysis methods
specified in these regulations. The German Federal Soil Protection and Contaminated Sites
Ordinance defines precautionary values for the PAH content of soils. These precautionary
values are set at 10 mg/kg DM for the Σ EPA16 PAH content in the finely granulated part of
soils with humus contents larger than 8% and at 3 mg/kg DM for the Σ EPA16 PAH content
of soils with humus contents of less or equal 8%. The German Federal Soil Protection and
Contaminated Sites Ordinance specifies a number of analysis methods which may be used to
determine the PAH content of soils in accordance with this ordinance. Among them are the
analysis methods DIN 13877:A (cold extraction with acetone) and DIN 13877:B (Soxhlet
extraction with toluene). However, our analysis results in section 3.1 have clearly shown that
DIN 13877:A is not suitable to determine the PAH content in gasification residues. Thus, the
regulations of the German Federal Soil Protection Act and the German Federal Soil Protection
and Contaminated Sites Ordinance currently cannot prevent the application of gasification
residues with high PAH contents to agricultural soils, if the cold extraction with acetone is
applied for their characterization. It is recommended to close this legislative loophole. Apart
from that, it should be noted that there is no boundary value for PAH loads added to soil
(however for the PAH content in the soil) in the German soil legislation.
While thermal processes are already used to remove PAH from contaminated soils [6], this
principle was applied for the PAH removal from wood gasification residues for the first time
to our knowledge. The results displayed in Table 3, Table 5 and Table 7 indicate a reduction
of the Σ EPA16 PAH content of the gasification residues by 36% to 82% after the treatment in
the PAH volatilization unit. Still, the residual PAH contents in the gasification residues are too
high to allow for an agricultural use.
The residual PAH content in the gasification residues was higher in the third experiment as
compared to the second experiment. This result might be explained by a difference in the PAH
content of the untreated gasification residues (although these were produced under the same
production conditions) or by a change in the airflow conditions within the PAH volatilization
14 Soil Pollution
unit caused by modifications of the sealing air stream and the heating chamber air stream
volumes. As already mentioned, gasification residues which had already passed through the
hot heating chamber got in contact with the volatilized PAH contained in the sealing air which
was blown into the lower conveyor screw. We assume that this is one of the reasons for the
high residual PAH content of the gasification residues in the 2nd and 3rd experiment.
This hypothesis is supported by the comparably low PAH content of 396 mg/kg DM which
was reached after the redesign of the PAH volatilization unit which prevented the recirculation
of volatized PAH. Independent from the avoided recirculation effect, the increase in the air
supply to the heating chamber in the 4th experiment has with high probability promoted the
oxidation (combustion) of gasification residues in the heating chamber. The resulting temper‐
ature increase might have supported a more complete volatilization of the PAH from the
gasification residues as compared to the 2nd and 3rd experiment.
The 5th experiment proves that thermal volatilization processes are capable in reducing the
PAH content of gasification chars to levels which are acceptable for agricultural applications.
It can be derived from the data presented in Table 9, that minimum process temperatures of
about 650 °C are necessary for an effective removal of PAH from gasification residues.
Based on the laboratory scale experiment, the following additional technical design modifi‐
cations are suggested to further improve the functional efficiency of the PAH volatilization
unit:
• The gasification residues need to be sufficiently hot to allow for a complete volatilization of
the PAH sorbed to the residue surfaces. In a modified heat exchanger, the hot wood gas
should be used to heat the gasification residues in counter-flow principle. It should be noted
in this context that the boiling temperatures of the analyzed PAH are in the range of 218 °C
(naphthalene) to 536 °C (Indeno(1,2,3-cd)pyrene) [6],[14].
• The aim of the PAH volatilization unit is to reduce the PAH content in the gasification
residues. If the gasification residues can be sufficiently heated in the heat exchanger, it is
not necessary anymore to supply oxygen into the PAH volatilization unit to burn a part of
the residues. In this case, cheating chamber air (and perhaps sealing air as well) would not
be needed anymore.
• The volatilized PAH need to be efficiently removed from the gasification residues before
condensing again to the surface of the residues. If the sealing air stream is removed, there
is a need for another transport method to remove the volatized PAH. It is suggested to
vacuum the volatized PAH through a heat-resistant filter installed at the hottest point of the
heat exchanger and thus separate them from the hot gasification residues.
Since the use of biochars in agriculture promises beneficial effects for soil amelioration and
climate change mitigation (see [15] and [16]), it is recommended to continue the technical
development to obtain gasifier residues with low PAH contents which might subsequently be
used as soil amendment. The joint composting of gasification residues and organic feedstock
sources could help to further reduce any remaining PAH in the gasification residues via
biodegradation [14] and to prevent nutrient losses in the composting process. In case further
experiments would affirm the viability of the preparation of gasification residues with very
low PAH contents which also meet all other applicable environmental standards for soil
amendments, a disposal problem could be turned into the valuable resource supply option for
the agricultural sector.
5. Conclusion
The results endorse the suitability of PAH analysis method DIN 13877:B for the determination
of the PAH content in gasification residues. Related to that, our findings give evidence that
DIN 13877:A is not suitable to determine the PAH content in gasification residues. Thus, the
regulations of the German Federal Soil Protection Act and the German Federal Soil Protection
and Contaminated Sites Ordinance currently cannot prevent the application of gasification
residue with high PAH contents to agricultural soils. It is recommended to close this legislative
loophole.
A reduction of the Σ EPA16 PAH content in gasification residues by 36% to 82% was achieved
by the tested PAH volatilization unit. However, the residual Σ EPA16 PAH content in the
gasification residues (between 396 and 1,713 mg/kg DM) is still far too high to allow for an
agricultural use of the residues. There are though promising technical redesign options
available to further improve the effectiveness of the examined thermal process. In a laboratory
scale experiment, the Σ EPA16 PAH content of gasification residues was reduced to values
below 2 mg/kg DM following thermal processing at temperatures of 650 °C and more.
Acknowledgements
We thank Matthias Wanderwitz for his support to the sample preparation and Spanner Re²
GmbH for financial support to the research work.
Author details
Sebastian Meyer1*, Bruno Glaser1, Daniel Fischer1, Peter Quicker2, Yves Noel2 and
Georg Kuffer3
1 Soil Biogeochemistry, Martin-Luther-University Halle-Wittenberg, Germany
2 Unit of Technology of Fuels, RWTH Aachen University, Aachen, Germany
3 Spanner Re² GmbH, Germany

16 Soil Pollution
References
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[2] Glaser, B.; Haumaier, L.; Guggenberger, G.; Zech, W. The‘Terra Preta‘phenomenon: a
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(1), 37‐41.
[3] Kuzyakov, Y.; Subbotina, I.; Chen, H.; Bogomolova, I.; Xu, X. Black carbon decompo‐
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ty 2012, 41, 1001–1013.
[5] Bruno Veyrand; Véronique Sirot; Sophie Durand; Charles Pollono; Philippe March‐
and; Gaud Dervilly-Pinel; Alexandra Tard; Jean-Charles Leblanc; Bruno Le Bizec.
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mental Engineering [Online] 2001, 127 (11), 986.
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tion of PAHs in Biochar: A Prerequisite To Ensure Its Quality and Safe Application.
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Co.: Shelter Island, NY 11964, 2011.
[9] Wheeler, B. lmPerm. Permutation tests for linear models. http://cran.r-

project.org/web/packages/lmPerm/index.html (accessed August 02, 2013).
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content. Chemosphere [Online] 2013, 90 (2), 782–788.
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[12] Bundesministeriums der Justiz. Gesetz zum Schutz vor schädlichen Bodenveränder‐
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chapter 13
Electrochemical Detection of Mercury Removal from

Polluted Bentonite and Quartz using Different
Removing Agents
I. Robles, Luis A. Godínez, J. Manríquez,

F. Rodríguez, A. Rodríguez and E. Bustos
1. Introduction
Heavy metals (also known as the trace metals) are a large group of elements which are
industrially and biologically important; in consequence they are defined as the group of
elements with an atomic density greater than 6 g cm-3. Some of these heavy metals are toxic to
living organisms in high concentrations. Heavy metals of greatest concern in terms of human
health, agriculture and ecotoxicology are arsenic (As), cadmium (Cd), mercury (Hg), lead (Pb),
tallium (Tl), and uranium (U). However, some other heavy metals, such as cobalt (Co), chrome
(Cr), manganese (Mn), and zinc (Zn), are necessary for the healthy growth of plants and / or
animals but only in small concentrations. In addition, deficiencies of "essential" trace elements
or heavy metals (often called "micronutrients") have limited agricultural productivity in many
parts of the world, which include the lack of Zn, Cu and Mn in crops and Co, Mn, Cu and Zn
in livestock (Alloway, 1990).
Mercury (Hg) has a high surface tension, meaning it easily forms small, compact, spherical
drops. Although mercury droplets are stable compared to those of other metals, they can
evaporate at high vapor pressures. High mercury concentration in the environment can be
dangerous and increases as elemental mercury evaporates and enters the atmosphere. In
addition, high concentrations of indoor mercury may pose an inhalation risk.
Mercury, which is commonly found in the earth’s crust as salts such as mercuric sulfide, is
released into the environment by volcanic eruptions. In addition to volcanic eruptions, erosion
of rocks and soils can also release mercury. Anthropogenic sources of mercury include mercury
produced as a by-product of the mining and refinement of others metals like copper, gold, lead
2 Environmental risk assessment of soil contamination
and zinc. Mercury can also be released through recycling operations and is sometimes obtained
from natural gas or other fossil fuels.
Approximately a third of the mercury circulating in the environment is produced naturally,

while the remaining two-thirds are from industrial activity or other human activities. Due to
the increase in industrial activity, the quantity of mercury circulating in the environment
(atmosphere, soils, lakes, streams and oceans) has increased two to fourfold since the beginning
of the industrial era. As a result, mercury levels in our environment are dangerously high.
Most of the mercury in the environment, released from both natural and anthropogenic sources,
is elemental mercury (Hg0), including 99% of total atmospheric mercury. However, biogeochem‐
ical transformations of mercury can create Hg1+ and Hg2+. Most inorganic mercury com‐
pounds (Hg1+) are mildly water soluble and can be found in soils and sediments. In contrast,
inorganic Hg2+ compounds vary depending on the local chemical atmosphere. Hg2+ has a short
atmospheric lifetime (minutes) and is quickly removed through deposition due to its high surface
activity and water solubility. Soil microorganisms oxidize mercury to its 2+ form (Leopold et al,
2010; Godarzi et al, 2012; Slowey et al, 2005; Mikac, 1999, Shi et al, 2005).
For the determination of metal in low concentrations a number of techniques can be applied
(Table 1), in particular colorimetry and atomic absorption spectrometry. Colorimetric deter‐
minations do not need expensive instrumentation, but there are some disadvantages such as
the need for destruction of the organic material and a separation step to avoid metallic
interferences. Atomic absorption is very useful for the sensitive detection and quantitative
determination of many metals, especially when using the flameless technique, but the
instrumental requirements are rather expensive, another disadvantage of this techniques is
that they require pretreatment of the sample differs often from metal to metal so that they do
not lend themselves for systematic analyses in which it is not known which metal (s) may, or
not be presented (Bodle et al, 1980; Bigham, 1990; Sarzaniniet al, 1994).
Techniques for total mercury determination include hydride generation atomic absorption
(Chapple, 1990) and cold vapor atomic absorption (US-EPA Method 7471). For a general
determination of mercury species, high-performance liquid chromatography with inductively
coupled plasma mass spectrometry can be used (Fodör, 2000, 2005, 2007; Franke and Zeeuw,
1976). In consequence, there is a need for the relatively simple and rapid analytical procedure
capable to carry out systematic screening for heavy metals (Franke and Zeeuw, 1976). Some
electrochemical techniques can determine all the different mercury species and oxidation
states.
Electrochemistry provides analytical techniques characterized by instrumental simplicity,

moderate cost and portability. Some as stripping methods use a variety of electrochemical
procedures which all share a characteristic initial stage. First, the analyte is deposited on an
electrode, usually starting from a stirring solution. After an exact period of time, electrolysis
is interrupted, stirring is stopped and the quantity of analyte deposited is measured using
voltammetric procedures. During the second stage of the analysis, the analyte is redissolved
or liberated from electrode (Woolever et al, 2001).
Electrochemical Detection of Mercury Removal from Polluted Bentonite and Quartz using Different Removing Agents 3
Technique Hg / ng mL-1
X – rayFluorescense. 10
Neutron Activation. 2
Gold Film. 0.5
Differential Pulse Voltammetry. 0.04
Cold Vapour Atomic Absorption (CVAAS). 0.01
Cold Vapour Atomic Fluorescense (CVAFS). 0.0001
Inductively Coupled Plasma – Mass Spectrometry (ICP – MS). 0.001
Inductively Coupled Plasma – Atomic Emission Spectrometry (ICP – AES). 50
Table 1. Analytical techniques to determine mercury.
In Anodic Stripping Voltammetry (ASV, Figure 1), the electrode behaves as a cathode during
deposition and as an anode during redissolution, where it is oxidized by the analyte again and
returns to its original form (Skoog, 1997; Dogan-Topal et al, 2010; Pineda et al, 2009; Anguiano
et al, 2012; Bustos, 2012). ASV has been used to detection of trace metals ions in solution at
~10-11 M (or sub-ppb) concentrations have been reported (Hubert, 2001). This technique is
advantageous to other analytical techniques is its simplicity of use, low cost of instrumentation,
and being monodestructive (Wooleveret al, 2001).
Figure 1. Potential sequence used as perturbation in an ASV experiment.
As shown in Figure 1, there are three sequential steps in a typical ASV experiment: deposition
or preconcentration, quiet time and linear stripping. During the deposition step, the electro‐
active species are reduced up to their zero-valence form under stirred electrolytic solutions.
In this sense, Ef must to be a more negative potential than Ei. Later, the stirring is stopped
during the quiet time in order to avoid convection transport of ions through the solutions.
Finally, the species previously reduced are selectively oxidized during an anodic linear sweep
and the current is recorded as function of the potential applied without stirring of the electro‐
lytic solutions (Scholz, 2002).
Figure 2. Potential wave form used as perturbation in a DSPV (A) and NPV (B) experiment.
Differential Pulse Stripping Voltammetry (DSPV, Figure 2A) is comparable to Normal Pulse
Voltammetry (NPV, Figure 2B) in that the potential is also scanned with a series of pulses.
However, it differs from NPV because each potential pulse is fixed, of small amplitude (10 to
100 mV), and is superimposed on a slowly changing base potential. Current is measured at
two points for each pulse, first point just before the application of the pulse and second one at
the end of the pulse (Ari et al, 1990; Brett and Oliveira, 1998).These sampling points are selected
to allow for the decay of the nonfaradaic (charging) current. The difference between current
measurements at these points for each pulse is determined and plotted against the base
potential (Woolever et al, 2001; Franke and Zeeuw, 1976).
Square Wave Voltammetric (SWV) technique is among the most sensitive means, for the direct
evaluation of concentrations; it can be widely used for the trace analysis. The perturbation
consists of a square wave having constant amplitude, superimposed at the same time upon a
staircase wave form. The current is measured at the end of each forward half-cycle (If) and at end
of each reverse half-cycle (Ir). The difference between both current values (If-Ir) is displayed as a
function of the applied potential E as shown in Figure 3. Therefore, the effect of the charging
current is notably decreased in SWV since any residual charging current is completely re‐
moved (Bard and Rubinstein, 1999; Ari et al, 1990; Scholz, 2002; Zbigniew et al, 1994; Brett and
Oliveira, 1998).
SWV has several advantages, has an excellent sensitivity and the rejection of background
currents, this speed coupled with computer control and signal averaging allows for experi‐
Figure 3. Potential wave form used as perturbation in a SWV experiment.
ments to be performed repetitively and increases the signalto- noise ratio. Applications of SWV
include the study of electrode kinetics with regard to preceding, following, or catalytic
homogeneous chemical reactions (Franke and Zeeuw, 1976; Dogan – Topal et al, 2010).
Using ASV, the concentration of mercury in liquid samples can be determined. For the
determination of mercury in soil, ASV is used after an acid digestion which removes mercury
from soil samples (Lamble and Hill, 1998). Some alternative methods for the removal of
mercury in soil samples also exist, such as the use of removing agents as called in Table 2, in
these studies removing agents were used to remove some heavy metals from different samples
(Reddy and Camesselle, 2009; Reddy, 2005; Haheb, 2012; Tandy et al, 2004; Rhazi, 2002; Cox,
1996; Robles et al, 2012). In this paper, we compared the efficiency of eight removing agents
showed in Table 2 in the ability to remove mercury from polluted bentonite and quartz
comparing DSPV, SWV and ASV.
Removing agent Concentration Sample Removed Metal η/%
Ethylenediamine
0.2 M 20
tetraacetic acid (EDTA)
Diethylenetriamin Low permeability soil

0.2 M 50
pentaaceticacid (DTPA) polluted with heavy Hg
Potassium iodide (KI) 0.2 M metals 80
Hydroxypropyl-β-
10% 15
cyclodextrine (HPCD)
Ethyleneediaminetetraac 275 mgL-1,

etic acid + cysteine+ Rawfish Hg 90
1.25%,
sodium chloride
Removing agent Concentration Sample Removed Metal η/%
0.5 %
(EDTA +Cys+NaCl)
Respectively
Ethylenediamine (Cu)- EDTA (84),

tetraacetic acid (EDTA) NTA (66)
Ethylenediamine
EDDS (67)
disuccinicacid (EDDS)
Iminodisuccinicacid Polluted (Pb)- EDTA (94),

0.4 and 4 mM Cu, Pb
(IDSA) soil NTA (65),
Ácidometilglicindi acetic
(MGDA)
EDDS (67)
Nitrilotriacetic acid
(NTA)
0.02 mMg-1 Co, Ca, Cr, Cu (53),

(Co2+ and Ca2+)
Chitosan Polluted water
1.2 mMg-1 (Cr3+) Cu Co (11)
2 mMg (Cr )
-1 3+
Potassium iodide (KI) 0.1 M Polluted soil Hg 99
Potassium iodide (KI) 0.1 M 62

Polluted
Ethylenediamine Hg
0.1 M soil 75
tetraacetic acid (EDTA)
Table 2. Scientific publications about metal extractions facilitated by removing agents.
Table 2 contains references about different removing agents reported to remove different
metals. These studies analyzed the removal of metals from different samples, obtaining the
highest efficiencies using removing and complexing agents by exchanging charges of cations
to remove mercury in the first case, or coordinating cations in the second case (Wypych,
2004; Montuenga, 1979; Přibil, 1982; Buffle, 1990; Spencer et al. 2000; Malone, 1999).
2. Experimental procedure
Stripping voltammetric measurements were carried out using the potensiostat / galvanostat
Epsilon modular electrochemical analysis system. Three-electrode electrochemical cell (Figure
4) was used with glassy carbon electrode as working electrode, a platinum wire as auxiliary
electrode and an Ag/AgCl as reference electrode (Pineda et al, 2009; Anguiano et al, 2012;
Bustos, 2012). Glassy carbon electrode was polished in cloth with an alumina suspension of 1,
0.3 and 0.05 μm. Between each polish it was rinsed with deionized water, and then it was
sonicated during 5 min in deionized water to eliminate any residual alumina.
Figure 4. Scheme of a 3-electrode cell used in voltammetry techniques, where gas inlet is used for bubbling electro‐
lyte solution with an inert gas and with controlled temperature.
2.1. Reagents and solutions
Reagents used in this study were: potassium chloride, potassium iodide, potassium hydroxide,
sodium hydroxide, etilendiamintetracetic acid, and hydrochloric acid obtained from J. T.
Baker; sodium chloride from Sigma Ultra; chitosan from crab shells practical grade, β-
ciclodextrin hydrate, L-cysteine were obtained from Aldrich. For mercury pollution were used
mercury chloride (II) from Merk, and mercury oxide (II) obtained from Hach. Calcium
bentonite from Lodbent Bentonite, and sand white quartz from Sigma Aldrich.
Removing agents tested were 0.1 M potassium iodide (KI), 0.1 M potassium chloride (KCl), 0.1
M potassium hydroxide (KOH), 0.1 M hydrochloric acid (HCl), 0.1 M ethylenediaminetetra‐
acetic acid (EDTA), 10% hydroxypropyl-β-cyclodextrine (HPCD) in deionized water, 0.01 M
chitosan and a mixture of 275 mgL-1 EDTA, 1.15% cysteine and 0.5% sodium chloride (NaCl).
All of the agents, with the exception of chitosan, had previously been tested for the ability to
remove metals in soil samples. Control experiments were carried out with water. Solutions
were prepared using water type I, according to ASTM-D1193-99. Chitosan were dissolved in
acetic acid. Samples of quartz and calcium bentonite were polluted with mercuric chloride
(HgCl2) and mercuric oxide (HgO) at concentrations of 10 and 25 mgL-1. Eight removing agents
were tested to find the most effective. The percentage of mercury removed was quantified by
ASV after extracting the liquid from the bentonite/quartz samples.
2.2. Techniques and procedures
Electrochemical techniques as Anodic Stripping Voltammetry (ASV), Differential Stripping

Pulse Voltammetry (DSPV) and Square Wave Stripping Voltammetry (SWSV) were tested to
select adequate technique to quantify mercury removal efficiency. After select adequate
technique, calibration curves were created for all different removing agents for the addition
of both HgCl2 and HgO.
Experimental conditions for ASV were as follows: pre-concentration potential –0.6 V vs. Ag/
AgCl, deposition time 6 min, quiet time 30 s, scan rate 20 mV s-1. An increase in signal due to
increasing mercury was monitored and recorded along with the increment in current associ‐
ated with the concentration addition. For SWSV were used an initial potential of -0.2 mV, a
deposition potential of -0.6 V for a deposition time of 10 s; a quiet time of 5 s, a SW frequency
of 50 Hz, a potential step of 0.005 V. For DSPV were used an initial potential of -0.2 mV, a
deposition potential of -0.6 V for a deposition time of 10 s; a quiet time of 5 s, a potential step
of 4 mV, a pulse width of 50 ms, a pulse period of 200 ms, pulse amplitude of 50 mV. All
experiments were carried out at room temperature (25±1°C) (Anastasiadou et al, 2010).
Calibration curves for mercuric quantification were done using electrochemical techniques to
select the best.
3.1. Selection of detection technique
To select the technique to quantify mercury was realized calibration curves with both mercury
compounds by ASV, DSPV and SWSV. Figure 5 shows the comparison of mercury detection
using different electrochemical techniques. Mercury chloride was not showed but resembled
same behavior. Table 3 shows comparison of electrochemical techniques of both mercury
compounds, where it shows that any technique can be used to quantify mercury by its low
detection and quantification limits, but the use of ASV shows the best fit with the lowest DL
and QL. In consequence, ASV was selected to quantify mercury in solution, which was
removed from polluted bentonite and quartz.
Mercury m DL QL
Technique Linear Equation R2
Compound (µA mM-1) (pM) (nM)
ASV 2549.30x - 0.0355 0.993 2549.30 112.043 0.373
HgCl2 DPSV 299.43x - 0.5035 0.978 299.43 422.226 1.407
SWSV 467.50x - 1.3763 0.981 467.50 479.046 1.597
ASV 6793.30x - 5.4037 0.998 6793.30 42.046 14.015
HgO DPSV 1313.70x - 1.0952 0.968 1313.70 96.237 32.079
SWSV 2347.90x - 1.6533 0.971 2347.90 95.385 31.795
Table 3. Parameters comparison of stripping voltammetry to quantify mercury.R2 is the correlation coefficient, m
represents the slope of linear regression; DL means Detection Limits, QL represents Quantification Limits.
9 Table 3 mM-1 mM-1
9 12 (Figure 7). (Figure 6).

9 Figure 5 Could you please change this figure by the annexed figure please?
0.000024
0.000022
0.000020
ASV
0.000018
0.000016
Current (A)
0.000014
0.000012 SWSV
0.000010
DSPV
0.000008
0.000006
0.000004
0.000002
0.000000
0.0 0.1 0.2 0.3 0.4 0.5
Potential (V)
Figure 5. ASV-DSPV-SWSV
5. ASV-DSPV-SWSV
Figure comparison tocomparison to select
select technique technique
to quantify mercuryto quantify
removed. mercury removed.
3.2. Electrochemical responses of removing and complexing agents
After obtaining encouraging calibration results for mercury detection using ASV with 3
sequential addition on removing agents proposed. Electrochemical answer of removing and
complexing agents in the presence of mercury compounds was obtained (Figure 6).
Table 4 shows corresponding equations of different removing agents, including fitting (R2),
sensibility (obtained from the slope m), detection and quantification limits (DL and QL
respectively) for both mercury compounds. DL and QL represent fundamental performance
characteristics of measurement processes, where DL or Limit of Detection (LOD) is defined as
an indicator of the minimum detectable analyte net signal, amount or concentration. His term
is widely understood and quoted by most chemists as a measure of the inherent detection
capability. In general, the LOD is taken as the lowest concentration of an analyte in a sample
that can be detected, but not necessarily quantified, under the stated conditions of the test. In
another hand, QL or Limit of Quantification (LOQ) is the lowest concentration of an analyte
in a sample that can be determined with acceptable precision and accuracy under the stated
conditions of test. The general equation to determine detection and quantification limit is:
LOD F (SD )
LOQ = b
Where F is a factor of 33 and 10 for LOD and LOQ, respectively. SD represent the standard
deviation of the ordinate intercept, or residual standard deviation of the linear regression; and
b the slope of the regression line. For a linear calibration curve, it is assumed that the instrument
response y is linearly related to the standard concentration x for a limited range of concentra‐
No. Delete Replace with
10 4 Figure 8 Figure 7
10 Environmental
10 Could
Figure 6 risk you please
assessment of soilchange this figure by the annexed figure increasing its size 50 % please?
contamination
350.0µ
HCl
A 350.0µ
KCl
B 350.0µ
KI
C
HCl + HgO KCl + HgO KI + HgO
300.0µ HCl + HgCl2 300.0µ KCl + HgCl2 KI + HgCl2
300.0µ
250.0µ 250.0µ
250.0µ
200.0µ 200.0µ
Current (uA)
Current (uA)
Current (uA)
200.0µ
150.0µ 150.0µ
150.0µ
100.0µ 100.0µ
100.0µ
50.0µ 50.0µ 50.0µ
0.0 0.0 0.0
-50.0µ -50.0µ -50.0µ

-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
Potential (V) Potential (V) Potential (V)
350.0µ
KOH
KOH + HgO
D 350.0µ
H2O
E 350.0µ
EDTA
EDTA + HgO
F
H2O + HgO
300.0µ 300.0µ 300.0µ
250.0µ
KOH + HgCl2
250.0µ
H2O + HgCl2
250.0µ
EDTA + HgCl2
A
200.0µ 200.0µ 200.0µ
Current (uA)
Current (uA)
Current (uA)
150.0µ 150.0µ 150.0µ
100.0µ 100.0µ 100.0µ
50.0µ 50.0µ 50.0µ
0.0 0.0 0.0
-50.0µ -50.0µ -50.0µ

-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
Potential (V) Potenial (V) Potential (V)
350.0µ
Chitosan
G 350.0µ
HPCD
HPCD + HgO
H 350.0µ
(EDTA+Cys+NaCl)
(EDTA+Cys+NaCl) + HgO
I
Chitosan + HgO
300.0µ 300.0µ HPCD + HgCl2 300.0µ (EDTA+Cys+NaCl) + HgCl2
Chitosan + HgCl2
250.0µ 250.0µ 250.0µ
200.0µ 200.0µ 200.0µ

Current (uA)
Current (uA)
Current (uA)
150.0µ 150.0µ 150.0µ
100.0µ 100.0µ 100.0µ
50.0µ 50.0µ 50.0µ
0.0 0.0 0.0
-50.0µ -50.0µ -50.0µ

-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
Potential (V) Potential (V) Potential (V)
Figure 6. Electrochemical behavior of mercury compounds to removing agents: (A) HCl, (B)KCl, (C)KI, (D) KOH, (E) H2O,
Figure 6. Electrochemical behavior of mercury compounds to removing agents: (A) HCl, (B) KCl, (C) KI, (D) KOH, (E)
(F) EDTA, (G) chitosan, (H) HPCD and (I) EDTA+Cys+NaCl.White the ASV showed before, calibration curves of each of one
H2O, (F) EDTA, (G) chitosan, (H) HPCD and (I) EDTA + Cys + NaCl.
mercury compounds added to removing agents was obtained as Figure 7 shows with the ASV response for chitosan.
Page Line No.

No. 0.00012
A
Delete B Replace with
0.00011
10 Figure 7 Could you please change this figure by the annexed figure please?
0.00010
0.00009
0.00008
Current (A)
0.00007
0.00006
0.00005
0.00004
0.00003
0.00002
0.00001
0.00000
0.0 0.1 0.2 0.3 0.4 0.5

Potential (V)
Figure 7. 7. ASV detection

ASV detection
Figure of HgO of HgO addition
addition to chitosan
to chitosan in 0.1 in
M0.1
HClMusing
HCl using vitreous
vitreous carbon,
carbon, platinum
platinum wirewire
andand
Ag|AgCl as
Ag|AgCl
work, counter asreference
and work, counter and reference
electrode electrode
with a scan with
speed of 70amV
scan
s-1 speed
(A), and 70 mVfits-1
oflinear of(A),
HgO and linear fit
addition toof HgO (B).
chitosan
addition to chitosan (B).
Page Line No.

No. Delete Replace with
11 32 Figures 9 and 10. Figures 8 and 9.
4
11 33 (Figure 11), (Figure 10),
11 44 (Figure 11B). (Figure 10B).
11 45 (Figure 11A). (Figure 10A).

HgO
Removing Agent m DL QL
Equation R2
(mA mg-1) (μg L-1) (μg L-1)
HCl y=0.0077x+0.0980 0.993 0.0077 0.2000 0.6680
KCl y=0.0093x+0.0421 0.999 0.0093 0.0676 0.2250
KI y=0.0141x+0.1194 0.982 0.014 0.0292 0.0974
KOH y=0.0035x+0.0280 0.994 0.0035 0.2750 0.9160
EDTA y=0.0039x+0.0369 0.988 0.0039 1.2400 4.1400
HPCD y=0.0069x+0.0730 0.982 0.0069 0.1100 0.3660
Cys+EDTA+NaCl y=0.0083x+0.0162 0.999 0.0083 0.0454 0.1510
Chitosan y= 0.0054x+0.0401 0.992 0.0054 0.1480 0.4930
Complexing Agent HgCl2
HCl y=0.009x+0.0576 0.995 0.009 0.1710 0.5710
KCl y=0.0092x+0.0538 0.993 0.0092 0.0684 0.2280
KI y=0.0106x+0.1018 0.980 0.0106 0.0386 0.1290
KOH y=0.0005x+0.0484 0.970 0.0005 1.9200 6.4100
EDTA y=0.0033x+0.0421 0.975 0.0033 1.4700 4.8900
HPCD y=0.0056x0.0487 0.995 0.0056 0.1350 0.4510
Cys+EDTA+NaCl y=0.0163x-0.1353 0.987 0.0163 0.0231 0.0770
Chitosan y=0.0046x+0.0406 0.986 0.0046 0.1740 0.5790
Table 4. Calibration curves corresponding to each removing agents.
tion. This model is used to compute the sensitivity b and the LOD and LOQ. Therefore, the
LOD and LOQ can be expressed as
3Sa 10Sa
LOD = b ; LOD = b
Where Sa is the standard deviation of the response and b is the slope of the calibration curve.
The standard deviation of the response can be estimated by the standard deviation of either
y-residuals, or y-intercepts, of regression lines. This method can be applied in all cases, and it
is most applicable when the analysis method does not involve background noise. It uses a
range of low values close to zero for calibration curve, and with a more homogeneous
distribution will result in a more relevant assessment (Currie 1995, 1999; Guidance 2000).
As can be seen in Table 4, we obtained a good QL and DL of mercury compounds in the

presence of removing and complexing agents. So ASV is a good technique to determine
mercury concentration in presence of removing agent too. In this way, removal efficiencies of
mercury were analyzed in the next section.
3.3. Mercury removal efficiencies
Removal percentages were calculated in base of fitting equations for all the different removing
agents, they are show in Figures 8 and 9. Due to structural differences in bentonite and quartz
(Figure 10), mercury removal behaved differently for the two.
Figure 8. Removal percentages of mercury (HgCl2 and HgO) in bentonite using different removing agents with 10 mg
L-1 (A) and 25 mg L-1 (B) mercury concentration.
Figure 9. Removal percentages of mercury (HgCl2 and HgO) in quartz using different removing agents with 10 mg L-1
(A) and 25 mg L-1 (B) mercury concentration.
Figure 10. Representation of the structure of bentonite (A) and quartz (B) (Bruker AXS Advanced X-Ray Solutions Soft‐
ware).
Quartz is ordered as strong matrix of oxygen-silicon bonds, which prevent metal ions from
penetrating its structure (Figure 10B). On the other hand, metal ions can easily slip into
bentonite, which is formed of stacked layers, due to the gaps between the layers (Figure 10A).
Because of the strong organized bonds in quartz, mercury is more easily removed from quartz
than bentonite. Moreover, the particle size of bentonite is lower than quartz, and therefore has
a bigger surface area and as a synthetic colloid, in addition, it has negative charges which
attract positive ions. Once bentonite and quartz samples were contaminated with HgO or
HgCl2, removing agents were added. After 24 h the sample was collected and analyzed by
ASV.
In order of the results obtained, bentonite adsorbed chitosan easily, swelling up dramatically
which made it difficult to remove the supernatant. Therefore, the removal of mercury from
bentonite using chitosan was inefficient (Figure 8, less than 5 %). This effect was consequence
of the affinity of chitosan to the layers of bentonite and it was retained inside of this clay. In
contrast, when the chitosan was used to remove Hg2+ from quartz (Figure 9), this got a removal
close to 30 % when it was 10 mg L-1, and the double of removal was when quartz had 25 mg
L-1 mercury; this increase was proportional to the concentration of mercury ions over quartz,
where chitosan took off the pollutant from particle surface to the solution. Anyway, all the
different extracting agents used to remove mercury remove metals by electrostatic forces,
forming ionic bonds. Thus, magnitude depends on ionic charge.
On the other hand, complexing agents act differently with metallic ions. Metals tend to lose
electrons during chemical reactions, creating metallic ions. The positive charge of these
cations attracts negative ions to form complexes held together by covalent bonds. Donat‐
ing species (ligands) needs to have a lone pair of electrons which can be donated to form
a bond. Water, ammonia and halides are common inorganic ligands (Buffle, 1990; Ma‐
lone, 1999; Montuenga, 1979).
Stronger compounds as EDTA and chitosan tend to remove more mercury contamination
present in the bentonite and quartz samples, than 0.1 M KI, 0.1 M KCl, 0.1 M KOH, 0.1 M HCl,
0.1 M EDTA, 10 % HPCD, 0.01 M chitosan and a mixture of 275 mg L-1 EDTA, 1.15 % cysteine
and 0.5 % NaCl by the different arrangement of mercury ions in these chemical solutions, with
less than 10 % removal from bentonite (Figure 8), and less than 20 % from quartz (Figure 9) in
both concentrations of Hg2+: 10 and 25 mg L-1.
With ASV has been possible study the thermodynamic and kinetic of adsorption of Hg (II) on
Ca-Bentonite, detecting the pollutant in solution to confirm that Ca-Bentonite has a good
adsorption capacity of Hg2+ adjusting the results to Freundlich isotherm as a mathematical
model, with a pseudo second order of reaction. Using the standard potential (E°) obtained after
the ASV, we obtained different thermodynamic parameters as equilibrium constant (Keq), free
standard energy (ΔG°) and entropy (ΔS°) of Hg2+ – Ca-Bentonite. These results indicate us that
the process of adsorption is spontaneous, endothermic and irreversible by a possible inclusion
and interchange of Hg2+ with Ca2+ between the Bentonite slides.
In addition, we have used the ASV to evaluate the electrorremediation of mercury polluted
soil using complexing agents like EDTA removing up to 75 % of metal contaminants in mercury
polluted soil samples by wetting them with 0.1M EDTA, placing them in an experimental cell
equipped with Ti electrodes, and then applying a 5 V electric field for 6 hours in a batch reactor;
Hg2+ was removed around 87 % in a time of 9 hours close to the anode side by the presence
this complexing agent (Robles et al, 2012).
4. Conclusions
ASV was selected as an electrochemical technique to quantify the percentage of mercury

removed in liquid samples, which were derived from bentonite and quartz samples previously
polluted with two mercury compounds (HgO and HgCl2). Eight removing agents were
analyzed: 0.1 M KI, 0.1 M KCl, 0.1 M KOH, 0.1 M HCl, 0.1 M EDTA, 10 % HPCD, 0.01 M chitosan
and a mixture of 275 mg L-1 EDTA, 1.15% cysteine and 0.5 % NaCl, using both extracting (ionic
bond complexes) and complexing agents (covalent bond complexes).
The best result of the removal of Hg2+ from bentonite and quartz were obtained with com‐
plexing agents: EDTA and chitosan. For bentonite, the most effective removing agent was
EDTA which removed 17 % of both Hg compounds. In the case of quartz, chitosan was the
best removing agent, removing 62 % of HgO and 53 % of HgCl2.
This is the first report of the use of chitosan to remove mercury ion from bentonite and quartz,
which is a very good new alternative of complexing agent because it is biodegradable, cheap
and easy to obtain (derived from shrimp exoskeleton), with potential application to quantify
mercury in polluted soil during some remediation strategy to verify its cleaning without a pre-
treatment of sample.
Acknowledgements
The authors would like to thank to Consejo Nacional de Ciencia y Tecnología de los Estados
Unidos Mexicanos (CONACyT) with the project CONACYT 193883-UANL, L’Oreal, United
Nations Educational, Scientific and Cultural Organization (UNESCO), Academia Mexicana de
Ciencias (AMC), and Fundación México – Estados Unidos para la Ciencia (FUMEC) for the
funding of this research. I. Robles is grateful to CONACyT for her scholarship.
Author details
I. Robles, Luis A. Godínez, J. Manríquez, F. Rodríguez, A. Rodríguez and E. Bustos*
Centro de Investigación y Desarrollo Tecnológico en Electroquímica S. C. Parque Tecnológi‐

co Querétaro Sanfandila, Pedro Escobedo, Mexico
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chapter 14
Treatment Methods for Radioactive Wastes and Its

Electrochemical Applicactions
V. Valdovinos, F. Monroy-Guzman and E. Bustos
1. Introduction
There are many uses of radioactive materials which improve or facilitate human activities or
quality of life of people. These uses are given in different fields of technology, ranging from
power generation to supply entire cities or areas, to medical and industrial uses, even the
smoke detectors in buildings. All these applications generate radioactive waste that may
represent risks to the environment or to human beings, but it is necessary to have special
attention to the management of radioactive waste.
In this chapter there are information about the generalities of radioactive wastes, such as its
definition, origin, classification and stages of radioactive waste management. In addition, there
are information about the current state of research and technologies which have been proposed
for the treatment of radioactive waste, with their advantages and disadvantages, in special
case of the electrochemical techniques to treat radioactive waste with theoretical considera‐
tions and cases of study. At the end of this chapter, there is information about the risk
assessment and development of future strategies.
2. Radioactive waste
2.1. Origin of radioactive waste
Radioactive waste are created from all activities that radioactive materials are used, either as
part of the process or the use of such materials as a constituent of equipment or instruments
that allow the realization of a practice. Due to large differences in the characteristics of the
waste generated in the different areas and to a better understanding of these origins, is
convenient to differentiate the activities coming from nuclear fuel cycle of applications coming
from medical, research activities and industrial uses (Radiation Safety General Regulations,
1988; Méndez de Vigo, 2000).
2.2. Classification of radioactive wastes
Classification of radioactive waste is in order of any stage from its origin just to their collection,
segregation, treatment, conditioning, storage, transportation and final disposal. In this case,
in the Figure 1 is the physical classification of radioactive waste (IAEA, 1970 and 1994).
ACTIVITY HALF-LIVE SOURCES FORM
LOW LEVEL VERY SHORT LIVE COMPACTIBLE

WASTE < 100 days SOLID
NON-COMPACTIBLE
SEALED SOURCES
INTERMEDIATE SHORT LIVE AQUEOUS

LEVEL WASTE < 30 years LIQUID
ORGANIC
HIGH LEVEL LONG LIVE OPEN SOURCES

WASTE >30 years BIOLOGICAL
Figure 1. Classification of radioactive waste (own creation).
Quantitative values of allowable activity content for each significant radionuclide will be
specified on the basis of safety assessments for individual disposal sites, and the radiological
classification of radioactive waste is (IAEA, 2009):
• Exempt waste (EW): Waste that meets the criteria for clearance, exemption or exclusion from
regulatory control for radiation protection purposes (IAEA, 2004).
• Very short lived waste (VSLW): waste that can be stored for decay over a limited period of
up to a few years and subsequently cleared from regulatory control according to arrange‐
ments approved by the regulatory body, for uncontrolled disposal, use or discharge. This
class includes waste containing primary radionuclides with very short half-lives often used
for research and medical purposes.
• Very low level waste (VLLW): Waste that does not necessary meet the criteria of EW, but
that does not need a high level of containment and isolation and, therefore, is suitable for
disposal in near surface landfill type facilities with limited regulatory control. Such landfill
type facilities may also contain other hazardous waste. Typical waste in this class includes
Treatment Methods for Radioactive Wastes and Its Electrochemical Applicactions 3
soil and rubble with low levels of activity concentration. Concentrations of longer lived
radionuclides in VLLW are generally very limited.
• Low level waste (LLW): Waste that is above clearance levels, but with limited amounts of
long lived radionuclides. Such waste requires robust isolation and containment for periods
of up to a few hundred years and is suitable for disposal in engineered near surface facilities.
This class covers a very broad range of waste. LLW may include short lived radionuclides
at higher levels of activity concentration, and also long lived radionuclides, but only at
relatively low levels of activity concentration.
• Intermediate level waste (ILW): Waste that because of its content, particularly of long lived
radionuclides, requires a greater degree of containment and isolation that provided by near
surface disposal. However, ILW needs no provision, or only limited provision, for heat
dissipation during its storage and disposal. ILW may contain long lived radionuclides, in
particular, alpha emitting radionuclides that will not decay to a level of activity concentra‐
tion acceptable for near surface disposal during the time for which institutional controls can
be relied upon. Therefore, waste in this class requires disposal at greater depths, of the order
of tens of meters to a few hundred meters.
• High level waste (HLW): Waste with levels of activity concentration high enough to
generate significant quantities of heat by the radioactive decay process or waste with large
amounts of long lived radionuclides that need to be considered in the design of a disposal
facility for such waste. Disposal in deep, stable geological formations usually several
hundred meters or more below the surface is generally recognized option for disposal of
HLW.
2.3. Management of radioactive waste
The ultimate goal of waste management lies in its restraint and seclusion of the human
environment, for a period of time and under conditions such that any release of radionuclides
does not pose unacceptable radiological risk to people or the environment. Management
should ensure that all charges are minimal for future generations.
A responsible management of radioactive waste requires the implementation of measures

aimed at protecting human health and the environment. The basic steps for effective manage‐
ment of radioactive waste are part of a global system, ranging from waste generation to final
disposal are: minimization of radioactive waste, pretreatment, characterization, treatment,
conditioning, transport, storage and disposal (IAEA, 1970; Figure 2).
A management system should to be applied in all steps of radioactive waste management, in

order to ensure that activities, facilities, equipment and waste products in meeting the overall
safety, health, environmental, security, quality and economic requirements, with safety and
environmental protection being of primary importance (IAEA-2008, IAEA 2006a, 2006b).
Management system has to contain a description of the processes and supporting information
that explain how work is to be prepared, reviewed, carried out, recorded, assessed and
improved. In the design of work processes, a detailed sequence of steps in the activities for
WASTE GENERATION
short live PRE-TREATMENT

long live
MINIMIZATION
COLLECTION
SEGREGATION
TREATMENT
VOLUME REDUCTION
CONDITIONING
INMOBILIZATION
PACKAGING
STORAGE STORAGE
FOR DECAY INTERIM
SHIPMENT
SEWER LANDFILL
DISPOSAL DISPOSAL
STACK NEAR SURFACE UNDERGROUND
Figure 2. Steps for managing radioactive wastes (IAEA, 2001).
pretreatment, treatment, conditioning and disposal wastes should be considered, such as: (i)
characterization of waste at each step in the overall waste management program, (i) analytical
methods such as sampling protocols for waste characterization or process control; (iii)
monitoring of discharges; (iv) monitoring for clearance purposes; (v) non-destructive exami‐
nation and testing; (v) heat treatment, (vi) use of special handling tools and techniques,
protective clothing or facilities for radiation protection, etc.
Identification of items should be established and documented on the basis of the importance
to safety and environmental protection or waste isolation. Records shall specify: (a) the origin
of the waste and the processes that generated it; (b) pretreatment of the waste; (c) clearance of
the waste; (d) discharge of the waste; (e) characterization of the waste; (f) treatment of the
waste; (g) design of the containers and/or packages and of equipment, structures, systems and
components for the pretreatment, treatment of the waste.
Additionally, assessments must perform on work processes used in the waste management.
Inspections and measurements have to being performed and the associated records main‐
tained. Consequently, controls to activities, facilities, equipment and waste products will have
to be designed, considering factors such as: (a) the quantities and potential hazards (radiolog‐
ical and non-radiological, for example chemical) of the waste, and the necessary degree of
isolation; (b) the dispersibility and mobility of the waste forms involved and the necessary
degree of containment; (c) the interval before disposal; (d) experience with, and maturity of,
the technology and the potential for future advances; (e) the reliability of equipment and its
function in relation to safety and environmental protection; (f) the complexity and degree of
standardization of the activities; (g) the novelty and maturity of the activities; (h) ease of
operation, maintenance of equipment and eventual decommissioning of the facility, etc.
Quality assurance requirements should be established for all phases of the waste management
process to ensure that each waste is correctly processed and the final waste form has the
required properties. This requires quality assurance parameters to be specified. Performance
data must be documented for each parameter in order to demonstrate compliance. The quality
assurance program should cover the following aspects: (1) waste characterization, (2) waste
management process specification, (3) processing conditions, (4) product specification and (5)
storage or disposal (HSEEA 2007; IAEA 2008, IAEA 2006).
2.4. Safety guide
Safety is a top priority in radioactive waste management, because of this, the purpose of this
section is to present a brief guideline of recommended procedures for working with radioactive
wastes. The safety aspects and environmental protection which need to be considered in the
handling and processing of radioactive wastes are mainly associated with: flammability,
toxicity, explosion and radioactivity.
Some of the most important aspects to consider regarding safety and environmental protection
in the radioactive waste management are (IAEA 2008, IAEA 2006a,2006b):
1. The handling, processing and storage radioactive wastes require a permit which is
authorized by a local regulatory agency.
2. The wastes must be adequately characterized; chemically, physically, and radiological as

a precursor to waste management. The composition of waste should be known with
sufficient accuracy that nuclear and conventional safety and environmental protection are
not compromised. Toxic or hazardous constituents should be characterized by analytical
means or from knowledge of the processes, so that hazards associated with treatment
methods of waste can be identified. The reachability of radionuclides, toxic materials and
the generation rates for volatile organic compounds or powders and other hazardous
gases should be determined. It is important to know the chemical stability of radioactive
waste: flammability, corrosively, reactivity, pyrophoricity, rapid oxidation promotion,
biodegradability and the chemically incompatible waste forms should be carefully
controlled. The amount of mobilizing agents such as chelating compounds, particularly
stable ones, should be kept to a minimum. Waste containing hazardous constituents that
are mobile in the environment, or constituents that enhance the mobility of radionuclides
should avoid.
3. Several possible process options have to be identified for treating radioactive wastes and
before selecting it should include a safety analyses. To prefer processes the cheapest and
simplest to procure and operate. The best practical environmental options should be
selected, with radiation doses and discharges as low as reasonably achievable (ALARA).
4. Radioactive liquid and gaseous discharges should be as low as reasonably achievable

(ALARA) and meet regulatory and environmental restrictions.
5. It is suggested that cheap simple structures will be adequate for installation.
6. Personnel protection should be used in installation: protective clothing, rubber gloves,

face mask, visors and personal dosimeters.
7. It is necessary to assess the radiation exposure of the operators on the site and to estimate
the frequency and consequences of possible accidents related to the facility and the
materials which it handles;
8. The waste processing area must to have suitable fire protection systems, adequate
ventilation, and for the waste contains liquids a collection sump.
9. To ensure that radiation exposure to the workforce is as low as reasonably achievable

(ALARA).
10. It is recommended having a central waste management facility.
11. Details of the safety methodology will be given in a planned technical document.
3. Treatment of radioactive waste
The aim of the radioactive waste treatment is to minimize the volume of waste requiring
management. Treatment process selection for waste depends upon its radiological and
physicochemical properties and the quantity (IAEA, 2001; Adenot et al, 2005; Chang, 2001).
The objective of this section is to provide a brief overview of usually techniques applied for
the treatment of radioactive waste as Figure 3 shows.
3.1. Treatment of aqueous waste
The processes available for treating aqueous radioactive waste are mainly: ion exchange/
sorption, chemical precipitation, evaporation or ultrafiltration/reverse osmosis. However,
liquid containing suspended matter must be treated to remove the particulates before primary
treatment or after it. Sedimentation, decantation, filtration or centrifugation are treatments
used commonly to clear the effluent wastes or to remove miscellaneous debris or insoluble
particles (IAEA, 2001; Adenot et al, 2005; Abdel et al, 2011).
ion exchange
sorption
chemical
precipitation
evaporation
compaction
solid/liquid
separation incineration
aqueous compactible
TREATMENT OF
LIQUIDS SOLIDS
RADIOACTIVE WASTES
organic non-compactible
distillation
Decontamination
incineration Crushing
wet oxidation cutting
acid digestion
electrochemical
oxidation
Figure 3. Classification of the treatment process applied to radioactive wastes (authors own creation).
3.1.1. Chemical precipitation
Chemical precipitation processes are regularly used for removing radioactivity from low and
intermediate level aqueous wastes at fuel reprocessing facilities, research laboratories and
power stations. Precipitation processes are greatly versatile, relatively low investment and
operational costs; and may treat from large volumes of liquid effluents containing relatively
low concentrations of active species to those containing large amounts of particulates or high
concentrations of inactive salts. However, in some cases, a pretreatment stage, such as
oxidization of organic contaminants, decomposition of complexed species, pH adjustment,
change of the valency state or adjust the ionic species, should be applied prior to the formation
of precipitate in order to improve the process. Radionuclides can be removed by precipitation,
co-precipitation with a carrier or sorption on to particulates present in the waste (IAEA, 2001;
Adenot et al, 2005).
3.1.2. Ion exchange/sorption
Ion exchange methods have extensive applications to remove soluble radionuclides from
liquid waste produced in nuclear fuel cycle operations, radioisotope production and research
facilities. It is very effective at transferring the radioactive content of a large volume of liquid
into a small volume of solid.
Ion exchange process involves the replacement of cations or anions between an insoluble solid
matrix containing ionizable polar groups and a liquid solution. When the ionic groups are
negatives the exchange will involve cations and when they are positively charged they involve
anions. The process is selective, stoichiometric and, as a rule, reversible; therefore ion ex‐
changers can be “regenerated” and radioactive liquid waste recovered with high activity
content or if the exchangers become “exhausted” they are removed and treated as radioactive
wastes.
A wide range of materials is available for the ion exchange treatment of radioactive liquids:
(a) natural ion exchangers (clays, zeolites, cellulose, charcoals, collagen) and (b) synthetic
materials such as zeolites, hydrous oxide gels of metals or organic resins formed by highly
polymerized cross-linked hydrocarbons containing ionic groups (sulfonic acid, carboxylic
acid, amino groups, etc.).
Ion exchange processes can be operated in batch or continuous modes and if the wastes contain
high concentrations of salts, suspended solids, organic contaminants or the radionuclide ionic
form not suitable, the liquid wastes will have to be pre-treated before exchange process (IAEA,
2001 and 2002; Adenot et al, 2005).
3.1.3. Evaporation
Evaporation process is effective for concentrating or removing salts, heavy metals and a variety
of hazardous materials from waste effluent, reducing large volumes of liquid wastes with high
factor decontaminations. The process is commonly used for the treatment of high, intermediate
and low level waste effluents; in particular for the treatment of small volumes of highly active
effluents and may be carried out through the use of commercially available evaporation
equipment. However, evaporation has some important limitations: unsuitable for waste
effluents containing large concentrations of inactive salts, expensive because its large energy
requirement and the presence of some organic compounds can produce explosions during
evaporation (IAEA, 2001; Adenot et al, 2005).
3.2. Treatment of radioactive organic liquid
Liquid scintillation, solvents, oils and diverse biological fluids, generated in nuclear research
centers, medical centers or industries are considered as radioactive organic liquid wastes.
These wastes may present radioactive and chemical or biochemical hazards requiring treat‐
ments to remove or destroy chemically or biochemically hazardous components. The objective
is to reduce the volume of radioactive waste which requires storage, transport, conditioning
and land disposal, eliminating the organic components to enhance compatibility of the treated
waste with secondary conditioning processes.
Processes such as incineration, wet oxidation, acid digestion, electrochemical oxidation and
distillation, can be applied for treating radioactive organic liquid (IAEA, 1992 and 2001).
3.2.1. Incineration
Incineration is used for reduction of solid and liquid radioactive waste volume, downscaling
land requirements for disposal. Incineration combusts or oxidizes wastes at high temperatures,
generating as end products of the complete incineration: CO2, H2O, SO2, NO, and HCl gases.
Emission control equipments for particulates, SO2, NOx and products of incomplete oxidation
are needed to control emissions of regulated air pollutants.
The disadvantages of radioactive waste treatment with incineration are: off-gas filtering
system are required to control radioactive discharges, thickening and dewatering wastes
pretreatment may be required, is not economical for small solid waste plants and secondary
waste volumes may be large (Chang, 2001; IAEA, 1992, 2001 and 2006; NEA, 1999).
3.2.2. Wet oxidation
The organic components of radioactive wastes such as ion exchange resins, foams, cellulosic
waste and liquid scintillation can be transformed, degraded, or immobilized using wet
oxidation.
Wet oxidation remedial process involves injecting an oxidizing agent, such as hydrogen
peroxide, activated sodium persulfate, ozone, Fenton’s Reagent (hydrogen peroxide with an
iron catalyst) or other oxidant into the subsurface to destroy organic compounds.
The complete mineralization of carbon-based compound wastes by most chemical oxidizers

produces carbon dioxide, water, and oxygen as well as minor concentrations of nontoxic ions,
salts, and acids. Wet oxidation is thus a process analogous to incineration, with the advantage
of using low temperatures (Twissel and Holt, 1996; IAEA, 1992; Chang, 2001).
3.2.3. Acid digestion
Acid digestion is an oxidative destruction technology for some liquid organic wastes
(hexane, TBP) and organic constituents of mixed waste such as cellulose (paper), polyethy‐
lene, latex rubber, Tyvek™, neoprene, polyvinylchloride, polystyrene ion exchange resins,
filters, plastics, and/or chlorinated cutting oils organic, that may reduce the waste volume
of 20 to 100 times. Acid digestion process uses a mixed of nitric acid in a phosphoric acid
carrier solution at temperatures below 200°C and at atmospheric or moderate pressures (<
20 psig). The principal organic portion of the waste is broken down and mineralized by
the acid solution producing: inorganic constituents in solution, which can be immobilized
easily in a glass or ceramic, and gases (CO2, CO, O2 and NOx) that can be treated in an off-
gas scrubbing system, to convert NOx to reusable nitric acid (Report OST, 1999; IAEA, 1992
and 2001; Adenot et al, 2005).
3.2.4. Distillation
Distillation is a radioactive waste volume reduction technique used for pretreating liquid
scintillation and miscellaneous solvent waste in conventional equipment. The process is
simple, known, and cost effective if the valuable solvent is recycled or reused. The active
residue could be either immobilized or destroyed by incineration (IAEA, 2001).
3.3. Treatment of solid waste
Solid wastes are produced by all applications and uses of radioactive materials, in normal
operations and maintenance activities. Solid, low and intermediate level wastes are generally
segregated into combustible, compactible and non-compactible forms.
Treatments for solid waste are used to reduce the waste volume and/or convert the waste into
a form suitable for handling, storage and disposal (IAEA, 2001 and 2002; Chang, 2001; Adenot
et al, 2005; NEA, 1999).
3.3.1. Decontamination
Decontamination is defined as the removal of contamination from areas or surfaces of facilities

or equipment by washing, heating, chemical or electrochemical action, mechanical cleaning or
by other means. The decontamination objectives are mainly: to reduce the volume of equip‐
ment and materials requiring storage and disposal in licensed disposal facilities, to remove
contamination from components or systems, to reduce dose levels in the installations and to
restore sites and facilities to an unconditional-use condition. Decontamination processes may
divide into chemical, electrochemical and mechanical processes:
• Chemical decontamination. In the chemical decontamination are used concentrated or

dilute chemical reagents in contact with the contaminated item, to dissolve the contamina‐
tion layer, covering the base metal and eventually a part of the base metal.
• Decontamination by melting presents the particular advantage of homogenising a number

of radionuclides in the ingots and concentrating other radionuclides in the slag and filter
dust resulting from the melting process, thus decontaminating the primary material. The
problem with inaccessible surfaces or complex geometries is eliminated and the remaining
radioactivity content is homogenised over the total mass of the ingot.
• Mechanical and manual decontamination included wet or dry abrasive blasting, grinding
of surfaces and removal of concrete by spalling or scarifying, washing, swabbing, foaming
agents, and latex-peelable coatings. These techniques are most applicable to the decontami‐
nation of structural surfaces which may be cleaned by sweeping, wiping, scrubbing or
removed by grit blasting, scarifying, drilling and spalling.
A wet abrasive-blasting system uses a combination of water, abrasive media and compressed
air, and is normally applied in 24 a self-contained, leaktight, stainless steel enclosure. The dry
abrasive-blasting technique, commonly called sandblasting or abrasive jetting, uses abrasive
materials suspended in a medium that is projected onto the surface being treated, resulting in
a uniform removal of surface contamination. The scarification process removes the top layers
of a contaminated surface down to the depth of the sound, uncontaminated surface.
There are two basic disadvantages with the mechanical methods: the surface of the workpiece
has to be accessible and many methods may produce airborne dust (IAEA, 2001 and 2002;
Chang, 2001; Adenot et al, 2005; NEA, 1999).
3.3.2. Compaction
Compaction is performed in order to reduce the waste volume and concentrates the radionu‐
clides. Plastics, paper, absorbent material, and cloth are compatible in conventional compac‐
tors. Metal pipe, valves, conduit, wood, and other like items are compatible in super
compactors. Compactors can range from low-force compaction systems (~5 tons or more)
through to presses with a compaction force over 1000 tons (super compactors). Volume
reduction factors are typically between 3 and 10, depending on the waste material being treated
(IAEA, 2001 and 2002).
3.3.3. Cutting
Cutting and sawing operations are carried out mainly on large items which consist usually of
metals or plastics. This waste has to be reduced in size to make it fit into packaging containers
or to submit it to treatment such as incineration. The cutting is carried out either in the dry
state in cells, using remote control when necessary and with conventional tools, or underwater.
The cutting may also be done with plasma-jets, laser torches, or explosive fuses.
3.3.4. Crushing
Crushing techniques may be used for size reduction of friable solids (glass, concrete, ceramics).
In principle, all types of mill, grinder, and crushing machines of conventional technology can
be used.
3.3.5. Shredding
Shredding reduces void space and is particularly effective when plastics are compacted. Air,
which is trapped between the folds of bulk plastic and in plastic bags and sleeving, takes up
storage space. When the plastic is shredded, better use is made of the waste container space.
3.3.6. Incineration
The size reduction, mixing and blending of the solid wastes is necessary for successful
combustion operation.
4. Electrochemical treatment of radioactive waste
4.1. Electroremediation
The electrochemical treatment, electroremediation, also known as electrokinetic remediation

(EKR) process is classified as a physicochemical technology by the electrochemical transfor‐
mation or destruction of organic and inorganic wastes, which offers many advantages such as
the capacity to remove organic and inorganic pollutants by applying direct electric current
into the soil. The EKR is easy to operate and involves the installation of electrodes into the
organic or inorganic waste and the application of a low voltage gradient or direct current
through them (Vazquez et al, 2007). This process is capable of mineralizing the organics into
carbon dioxide and water completely, without emission of any toxic materials like dioxins.
Several metal oxidizing agents like Ag(II)/Ag(I), Ce(IV)/Ce(III), Co(III)/Co(II), etc., have been
tested with this process in pilot and commercial scale systems (Adenot et al, 2005; Chang,
2001; Prabhakaran et al, 2009; Farmer et al, 1991).
This technology requires having humid waste into which electrodes are inserted, the electro‐
des’ terminals are connected to a power supply, and an electric current or potential gradient
is applied to generate an electrical field. During this process, electrode reactions take place on
its surface, generating protons (H+) and hydroxyl (-OH) at the anode and the cathode, respec‐
tively. The concentration of these ions near the electrodes creates an acid front that moves from
anode to cathode and a basic front that moves from cathode to anode. These species interact
with the soil to carry pollutants out into the pore solution. Some transport phenomena occur
in the liquid phase of soil when direct current or voltage gradient is passed through the
electrodes, such as ion migration (electromigration), electroosmosis and electrophoresis
(Murillo – Rivera et al, 2009; Alcántara et al, 2008), inducing complex and coupled electro‐
chemical and properties of matrix. In this case, the transport phenomenon depends on the
chemistry of the pollutant (Pamuku and White, 1992; Reddy and Chinthamreddy, 2003; Braud
et al, 1998).
If the pollutant is inorganic, as metals, these can be removed by electromigration, because the
compounds are dissociated in ions. If the ions have negative charge (anions) they will move
toward the anode, and if they have positive charge (cations) they will move toward the cathode,
an important characteristic which can determine where the metal, in ionic form, can be
recovered (Virkutytea et al, 2002; Figure 4). Non-polar pollutants or organic pollutants can be
removed by electroosmosis, attributed to the excess charges on the soil surface. There occurs
the net ionic migration that represents the bulk movement along pore fluid through the
electrical double layer of charge at the solid–liquid interface (Al-Shahrani and Roberts, 2005).
And finally, electrophoresis is the movement of charged solid particles, including clay particles
and bacterial cells with size less than 20 m, in response to the electrostatic potential gradient.
As in electromigration, positively charged particles migrate towards the cathode, and nega‐
tively charged particles migrate towards the anode, or they both simply move by a mechanical
transport (Hamed et al, 1991; Méndez et al, 2012).
On other hand, many different soil remediation technologies are available to clean polluted
waste, and most of them remove pollutants from the soil–water complex for further treatment
or disposal in a more concentrated form. However, when the pollutants are persistent, toxic
or simply have low solubility and a strong adsorption to soil surfaces and organic matter, the
traditional remediation technologies are used, such as washing, and land-farming, amongst
others. In these cases, electroremediation, also known as electrokinetic remediation (EKR)
process is classified as a physicochemical technology, which offers many advantages such as
the capacity to remove organic and inorganic pollutants by applying direct electric current
into the soil, even in clays (Virkutytea et al, 2002). The EKR is easy to operate and involves the
installation of electrodes into the soil or waste and the application of a low voltage gradient or
direct current through them. This technology has the capacity to removed heavy metals (Pb°,
Hg°, Cd°, Ni°, Cu°, Zn°, Cr°); toxic anions (NO3−, SO42−); mixtures of organic and ionic
pollutants; cyanide; explosive compounds; hydrocarbons (gasoline, diesel, oil, BTEX: benzene,
toluene, ethyl benzene and xylene); polychlorinated biphenyls (PCBs) and radioactive species
(137Cs, 90Sr, 60Co, 238Ur); from both saturated and unsaturated soils, sludges and sediments
(Virkutytea et al, 2002; Hamed et al, 1991).
Figure 4. Electromigration of ions in an electrochemical arrangement to remove pollutant from the matrix (authors
own creation).
Some researchers have published (Pamuku et al, 1990 and 1992; Reddy et al, 2003; Hamed and
Bhadra, 1997; Reed et al, 1999; Khan and Alam, 1994) that the EKR application was better in
clayed soils that had low concentration of heavy metal than even expensive regular mining
procedures. However, a disadvantage was the time required to achieve over 90 % metal
removal.
In order to improve the EKR process and diminish the removal time, some efforts have been
focused on changing some operational parameters. For example, some researcher have
modified the pH and the current density, introducing chemical compounds on electrolyte
chambers (Yeung et al, 1996; Cox et al, 1996), or have added complexing and chelating agents
to desorb the pollutant from soils (Cundy and Hopkinson, 2005; Ruiz et al, 2011; Colleta et al,
1997), or have modified the system, placing reactive barriers into the soil matrix (Ribeiro et al,
2005; Gómez et al, 2009) or simply they have tested different electrode material.
Thus, the efficiency of pollutant removal using EKR depends of many factors. However, it is
important to select a good electrode material, especially when electrochemical technologies
are used. Their selection can be according the thermodynamic and kinetic reactions carried
out on their surfaces or by their mechanical, thermal and corrosion resistance.
4.2. Electrodes during the electrokinetic remediation of wastes
In that sense, some materials, as the case of titanium (Vázquez et al, 2004), platinum, gold,
silver, stainless steel, among others used in EKR, suffer a kind of passivation, generating an
oxide film on their surfaces which cover the active sites. This behavior occurs during the
experimental conditions, which increases the electrical resistances in the system. For that
reason, it is necessary to pre-treat or pre-activate the material before using, to increase
roughness or surface active sites. Also, carbon electrodes have been used in EKR processes
because of their low cost and accessibility (Saichek and Reddy, 2003; Hu et al, 2002) and because
they are inert. However, these kinds of materials commonly form bonds with the species in
solution or form oxide film. As well, they can adsorb some species on their surface.
In order to increase the active sites, eliminate the passivation phenomenon, increase electrode
life and improve the oxidant activity, it is necessary to modify electrode surfaces to obtain high
overpotentials. Consequently, some electrode materials have been modified with metallic
oxide, forming a thin layer on a base metal (usually titanium), i.e. Ti|SnO2–Sb2O3, Ti|IrO2–
Ta2O5, Ti|IrO2, Ti|RuO2, and carbon|TiO2, among others (Hu et al, 2002; Méndez et al, 2012).
These kinds of electrodes can be used as anodes in order to promote electrochemical oxidation.
For that reason, the name “Dimensionally Stable Anodes” (DSA) was proposed by Comninellis
and Pulgarin (Comninellis and Pulgarin, 1991) who demonstrated the high reactive surface of
iridium DSA. These are able to degrade organic compounds by the presence of hydroxyl
radicals on their surface, while being mechanically resistant to this reactive species and to pH
changes (Comninellis, 1994).
4.3. Configuration of electrodes during an electrokinetic remediation of wastes
Furthermore, most conventional in situ methods have trouble managing the remediation of
fine-grained soils with one or more of the following conditions: high water content, high
organic matter content, high metal concentration; whereas electroremediation is suitable for
wastes (Saichek and Reddy, 2005). Although, there are some aspects of electroremediation that
require attention before the technology can be successfully implemented in the field, such as:
soil characterization, type and concentration of pollutant and electrode material, as well as
electrode array configuration and spacing.
In the last three decades, different research groups have been working in the electrokinetic
treatment at different levels: laboratory, pilot system and directly in the site pursuing to
remove organic pollutants (Gilbon, 2001), inorganic pollutants and radioactive wastes, among
others (Hamed et al, 1991). Also, there are reports about the successful implementation of
electrokinetic treatment in situ; one example is reported by Monsanto, DuPont and General
Electric, who used the LasagnaTM remediation treatment in situ to remove trichloroethylene
with 98 % of efficiency (USEPA, 1997). Another practical example was developed by Sandia
National Laboratories, who removed chromium in situ using Iridium/Titanium electrodes
protected with a porous ceramic coating (Fernández, 2012).
Previous research has been focused on maximize electrokinetic extraction efficiency. Most
laboratory or pilot electroremediation studies have been carried out in one dimension (1D)
array, having only one anode (+) and one cathode (-), separated by the polluted soil. For field
applications, the system must be effective and efficient; therefore it is required an appropriate
electrode configuration, in order to accomplish this goal EKR treatment should be implement‐
ed in a bidimensional system (2D), this means that electrode array must be set up in respect
to the x-y axis; however, there are few reports evaluating the performance of electrode
configurations in the x-y plane, and there is no experimental data about performance of
electrokinetic treatment with different electrode arrays (Méndez et al, 2012; Pérez – Corona et
al, 2013; Reddy et al, 2001).
In addition, several flushing solutions have been investigated, such as water, acids, bases,
chelating agents, alcohol, and other additives (Chaiyaraksa and Sriwiriyanuphap, 2004). In
practice, acid washing and chelator soil washing are the two most prevalent removal methods
(Giannis et al, 2007; Rampley and Ogden, 1988). Also, most of the electrokinetic equipment
has been manufactured as a horizontal type. Laboratory-scale electrokinetic remediation has
performed for TRIGA soil during the past 5 years, but in this time, a study related to pilot-
scale electrokinetic remediation was performed (Kim et al, 2002 and 2003).
4.4. Destruction of radioactive organic wastes
The process developed for the removal of organic contaminants from bulk water using
graphite based adsorbents with electrochemical regeneration at the University of Manchester
(Brown and Roberts, 2007), was adjusted for the destruction of radioactive organic wastes,
specifically oils contaminated with alpha radioactivity produced at Magnox Ltd nuclear
decommissioning site in UK (Wickenden, 2001). This approach comprises four stages:
1. Emulsification: the oils contaminated are emulsified in water using CLAX 200S as organic
emulsifying agent to give a stable emulsion.
2. Adsorption: the emulsion is vigorous mixing with the graphitic adsorbent (NyexTM 1000
supplied by Arvia Technology Ltd) by fluidizing the adsorbent using air sparging. A quick
adsorption is produce by the non-porous nature of the NyexTM.
3. Sedimentation: when the fluidising air is turn off, NyexTM particles precipitate to form a
bed in the anode compartment of the electrochemical cell.
4. Electrochemical destruction: two electrodes are placed either side of the bed and a direct
electric current is passed through the bed which destroys the pollutant through anodic
oxidation of the organic matter. Oxidation of the organic matter may produce soluble
breakdown products or off gases (CO, CO2) and small amounts of H2 and Cl2 at the
electrodes. After electrochemical treatment, the regenerated adsorbent is ready for
immediate reuse and the whole cycle is repeated (Brown et al, 2013).
The treatment of radioactive oils by adsorption and electrochemical regeneration systems has
been achieved at pilot scale 200 L. The latter consisted of three bipolar stacks of six electro‐
chemical cells of each with an electrode area of 2 500 cm2. Graphite plate bipolar electrodes
were used and a micro-porous polyethylene membrane (Daramic, Grace GMBH) separated
the adsorbent bed from the cathode. The catholyte solution, 0.3 wt % NaCl solution acidified
to a pH of less than 2, was stored in a small tank and pumped through the cathode compart‐
ments of the six cells. The distribution of the radioactivity after the process was examined in
the samples of the adsorbent and electrolyte after each regeneration cycle or after each
destruction cycle for 1.5 L of contaminated oil using high-resolution gamma spectrometry for
241
Am, 154Eu, 155Eu, 137Cs and 60Co, liquid beta scintillation counting for 3H,14C, 55Fe, 63Ni, 90Sr,
241
Pu and 36Cl, and alpha spectrometry for plutonium isotopes (239/240Pu, Pu) and 241Am (Brown
et al, 2013).
The process was achieved with a specific treatment rate of 0.63 μL cm−2 h−1 using a current
density of 10 mA cm−2. Regeneration was carried out a current of 1 A (20 mA cm−2) for 25 h L
−1
of oil with a regeneration energy of 48 kWh L−1 of oil. And the oil loading on the adsorbent
was less than 25 wt % on the adsorbent to avoid excessive cell voltage.
The process of adsorption coupled with electrochemical regeneration can remove and destroy
around 95 % radioactive oils in the first cycle, and over 99 % of the emulsified oil. Around 80
– 90 % of the majority of the radioactive species are transferred to the aqueous phase with a
negligible discharges of tritium gaseous; hence no off-gas treatment before direct discharge to
atmosphere is necessary (Brown et al, 2013).
Electrochemical oxidation using a boron-doped diamond (BDD) anode coupled with sonica‐
tion is a promising method to oxidise (Low Level Waste) LLW or Intermediate Level Wastes
(ILW) oils to carbon dioxide and water. The boron-doped diamond (BDD) electrodes contain
non-aggressive and non-corrosive chemicals, are ease of disposal of the spent electrolyte and
allow simple electrochemical cell configuration. Electro-oxidation tests were performed into
an electrochemical cell which comprised a 250 mL beaker fitted with a rubber bung that held
a BDD (DIAFILM PE TM) anode and a stainless steel cathode applied 0.1 A, a cell potential of
5 – 15 V and sonication. The electrolyte contained oil, sodium sulphate and sodium hydroxide
added at the start of the test to maintain an alkaline pH in order to trap the carbon dioxide as
carbonate. The method was effective for unused hydraulic oil, vacuum pump oil and a waste
used machine tool oil (Taylor et al, 2009).
Mediated Electrochemical Oxidation (MEO) process has been used by destroying the organic
components of combustible mixed wastes and for dissolving radioactive materials, such as
transuranic oxides (PuO2). The radioactive components of the wastes dissolved in the electro‐
lyte, can be recovered or immobilized for disposal (Chiba et al, 1995). The destroying organic
process is accomplished via a mediator (oxidizing agent).
Several metal oxidizing agents like Ag(II)/Ag(I), Ce(IV)/Ce(III), Co(III)/Co(II),Fe(IV)/Fe(III)

etc., in nitric or sulfuric acids have been tested (Chiba et al, 1995; Farmer et al, 1995). In
particular, the Ag(II)/Ag(I) based MEO system is capable of destroying cutting oil, cellulose
(paper and cloth), rubber (latex), plastics (Tyvek, polyethylene and polyvinyl chloride),
biomass (bacteria) and ion exchange resins, attaining high destruction efficiencies at reasona‐
ble coulombic efficiencies (Chiba et al, 1995).
Ag(II) in a nitric acid solution produces highly reactive OH and NO3 radicals which attack
organic compounds converting most of them to CO2, water, and inorganic ions. The resulting
Ag(I) is recycled to Ag(II) at the anode of an electrochemical cell to maintain a supply of oxidant
and minimize consumption of Ag. The Ag(II) migrates back into the bulk electrolyte to
continue the oxidation process. A microporous membrane is usually placed between the
electrodes to prevent the oxidizer produced at the anode from being reduced at the cathode.
Ag(II) is a very effective oxidizing agent for the destruction of nonhalogenates organic
compounds. Unfortunately, halide ions liberated during the destruction of halogenated
organics react with Ag(II) to form insoluble precipitates. Therefore systems tolerant to halide
anions such as Ce(IV)/Ce(III), Co(III)/Co(II) and Fe(IV)/Fe(III) have been tested. Tests at Pacific
Northwest Laboratory with the Cerium/HNO3 system has shown that cerium provides the
same or better oxidation rates for cutting oils that silver system (Schwinkendorf and Hart,
1995).
4.5. Examples of electrochemical treatments of radioactive waste
4.5.1. 137Cs and 134Cs removal from radioactive ash
Combustible waste contaminated with 137Cs and 134Cs, generated by the tsunami within
Fukushima, was incinerated, producing a large quantity of radioactive ash. A washing–
electrokinetic decontamination method was developed by Kim and collaborators to decon‐
taminate these radioactive ashes (Kim et al, 2002 y 2003). The process consists in a washing of
contaminated ash with HNO3 in three cycles using pH of 0 and ash (1g) – nitric acid (1 mL)
mixture; the waste solution generated is precipitated with CaO and reused only those
produced in the third washing. If the residual radioactivity of the washed ash is higher than
the clearance concentration level, the washed ash is treated by electrokinetic equipment for
decontamination. The removal efficiency of 137Cs from radioactive ash is inversely proportional
to an increase in the mixing ratio of ash weight (g) / solution volume (mL) and directly
proportional to the increase in the concentration of HNO3.
A pilot-sized washing–electrokinetic equipment was manufactured to remove cesium from

lots of radioactive ash. The equipment consisted of 200 L washing equipment, 50 L
electrokinetic equipment, and 150 L precipitation equipment. The electrokinetic equip‐
ment consisted of a couple of anode rooms, electrokinetic ash cells, cathode rooms and
metal oxide separators. Cesium from radioactive ashes moves to the cathode room through
electro-migration and electro-osmosis. 87 – 89 % of 134Cs and 137Cs in the radioactive ashes
were removed in three washings and around 98 – 100 % from the waste solution after
precipitation with CaO. If the radioactive ashes contain more than 30 – 40 Bq g-1 of 134Cs
and 137Cs the electrokinetic process requires between 8 – 10 days after three washings to
reach below 2.0 Bq g-1 (Kim et al, 2013).
4.5.2. Sodium recovery from alkaline nuclear waste
An electrochemical salt-splitting process has been developed to recover and recycle NaOH
from radioactive wastes containing large amounts of sodium salts. Sodium separation process
can save costs by reducing the disposal volume of wastes and by producing NaOH for recycle
into waste treatment processes such as sludge leaching, regenerating ion exchange resins,
inhibiting corrosion in carbon-steel tanks, or retrieving tank wastes (Fontain et al, 2009). The
process is based on a two-compartment cell separated by a cation selective membrane. The
waste enters into the anolyte electrodes, sodium ions migrate across membrane into the
catholyte, under the influence of an applied electrical potential. Hydroxides form at the
cathode by the reduction of water producing thus a sodium hydroxide solution (Kurath et al,
1997; Hobbs, 1999; Ambashta and Sillanpää, 2012; Fountain et al, 2009).
Platinized titanium (Pt/Ti) anodes and cathodes, and a Ceramatec® NASD membrane were
tested by Hobb, applying a current density of 300 A m-2 and a voltage of 5 - 5.6 V during a run
time of 70 - 104 h. This approach allows retain anionic species such as nitrate, aluminate or
sulfate in the compartment anolyte of the electrochemical cell, and can produce caustic from
radioactive wastes with low levels of gamma radioactivity, which could be released for off-
site use without further treatment (Hobbs, 1999).
Pacific Northwest National Laboratory (PNNL) and Ceramatec Inc. tested a NaSelect®
(sodium super ionic conductor) ceramic membrane to separate sodium from Low Activity
Waste (LAW) streams at Hanford site (Fountain et al, 2009; Pendleton et al, 2011). A pilot scale
system was designed, built and operated to process 7 L h-1 of LAW. The process selectively
removed up to 80 % of sodium hydroxide from LAW and produced up to 50 % concentrated
caustic for reuse in removal aluminum during sludge washing as a pretreatment step in the
vitrification of radioactive waste; reducing about 39 % the waste volume. NaSelect® ceramic
membrane prevents migration of mono valent and multi valent elements and other radionu‐
clides to the sodium hydroxide stream (Pendletonet al, 2011; Figure 5).
Electrolytic decontamination is accomplished by applying a low dc voltage through an

electrolyte to induce a chemical reaction. Contamination is removed at the anode the working
electrode and goes into solution. The cathode (counter electrode) can be constructed from
stainless steel. Electrolyte solutions such as phosphoric or sulfuric acid, used normally in
electropolished, have to be changed after a few articles decontaminated, resulting in large
quantities of radioactive toxic waste that must be treated and/or disposed. Due to this,
electrolytes with a very low toxicity from which the radioactive materials can be easily
separated and recycled, are investigated and applied. Sodium nitrate has been chosen as
electrolyte to decontaminate metals contained Pu and Am (Wedman et al, 1996). In this
medium, both actinides can be precipitated or entrained in the ferric hydroxide formed as
surface metal is removed, resulting a clean surface, free of contamination, and the separation
of the radioactive waste from the solution. Thus, the electrolyte solution can be recycled,
greatly reducing waste.
For this process, the appropriate current densities appear to be in the range of 0.1 to 0.2 A
cm-2. Higher current densities result in higher metal removal rates, but adversely affect the
anode cathode
(+) (-)
NaOH NaOH
depleted concentrated
Na+
O2(g) H2(g)
+ +
2H2O 2OH-
4OH- 2H2O
waste Dilute NaOH

(anolyte) (catholyte)
Ion-selective
membrane
Figure 5. Electrochemical cell for the recovery of sodium hydroxide from alkaline salt solutions (Hobbs, 1999).
surface morphology by causing roughening, pitting, or burning. Optimum situation for the
electrolytic decontamination process is the treatment of metal surfaces that have been electro‐
polished before contamination (Wedman et al, 1996).
NaOH solutions have been used as electrolytes to decontaminate metal surface contaminated
by tritium. In this approach, the metal to be decontaminated is submerse in the electrolyte,
connected to the negative pole, and the anode (polytetrafluoroethylene wax-impregnated
graphite), to positive pole; applying a current densities in the range of 10 - 50 mA cm-2. The
tritium adsorbed on the cathode surface is replaced by the hydrogen and ejected to the
electrolytic solution. This process is proper to small metallic parts of complex geometry or
large pieces with simple geometry; and does not modify the surface metal, thus the objet can
be reused (Bellanger, 1992).
Other decontamination process for contaminated stainless steel equipment is accomplished

by dissolution of the metal surface that is being decontaminated when it is in contact with a
solution composed by an inorganic reduction-oxidation systems such as Ce(IV)/Ce(III), Cr(Vl)-
Cr(III, Ag(II)-Ag(l) or Co(III)-Co(II) in nitric acid. This solution is subsequently reoxidized by
electrolysis (Lerch et al, 1980).
4.5.3. Remotion of 137Cs from soil
The thick-target PIXE analysis was successfully applied at least to the analysis of the electro‐
kinetic behavior of cesium mixed with the soil as CsBr. By using cesium as a test pollutant, we
could clearly observe the effect of the electrolysis without taking into account complicated
interactions between the contaminant and the soil, such as dissolution of soil particles and
adsorption phenomena.
The water supply to the anode well was effective to enhance the removal rate. From the
observed fast migration of the pore water, as well as from the result of a simple calculation on
the electrophoretic flow velocity, it was concluded that the migration of cesium observed was
due mainly to the electroosmotic flow. Remediation performance by increasing the electric
conductivity of the soil by mixing NaCl was possible. This result was consistent with the
reduction of the electroosmotic flow velocity due to the elevated ionic strength. It was found
that the addition of NaCl makes no sense also from the viewpoint of the potential hazard due
to toxic gas emission as well as the cost of electricity. Owing to the interference by major
metallic elements in the soil, the detection sensitivity of the present method based on the simple
LX-ray measurement was not enough to investigate behavior of the trace level Cs contami‐
nants. In order to attenuate only Ti-Kα but to allow high transmission of Cs-Lα radiation, a
thin foil of a single element having an absorption edge at an appropriate energy.
4.5.4. Remotion of 60Co and 137Cs from soil
The pilot-scale electrokinetic remediation equipment suitable for the geological characteristics
of a South Korean nuclear facility site was developed for the remediation of radioactive soil.
The optimal experimental conditions were obtained with 50 L electrokinetic remediation
equipment and the results are as follows: the removal efficiencies Co2+ and Cs+ from the
artificially contaminated soil after 15 days were 98.4 % and 94.9 % respectively, and the
generated effluent volume was 3.4 mL g-1. The removal efficiencies of 60Co and 137Cs by nitric
acid were increased by 3.1 % and 2.0 % more than those by acetic acid. The removal efficiencies
of 60Co and 137Cs from the soil of high concentrations (1 320 Bq kg-1 and 1 720 Bq kg-1) were
increased by 14.4 % and 3.8 % more than those from the soil of low concentrations (110 Bq
kg-1 and 120 Bq kg-1).
The removal efficiency of 137Cs from the soil of average 1.4 mm particle size was increased by
about 2.3 % more than that from the soil of an average 0.7 mm particle size. The removal
efficiencies of 60Co and 137Cs by the application of an electric current of 15 mA cm-2 were
increased by 1.4 % and 4.4 % more than those by the application of 10 mA cm-2. A total removal
efficiency of 60Co and 137Cs from the radioactive soil of about 2 000 Bq kg-1 was 95.8 % during
electrokinetic remediation by the application of an electric current of 15 mA cm-2 for 55 days
(Gye – Nam et al, 2013).
On other hand, a soil washing system with a hydro-cyclone was very effective for
decontaminating the radionuclides in the TRIGA (Training, Research, Isotope, General
Atomic) soil in Korea. The size of the contaminated TRIGA soil ranged from very fine to
more than 1.0 mm. The volume of soil particles larger than 1.0 mm was 28 - 52 %, and the
volume of soil particles smaller than 0.06 mm was less than 4 - 10.5 %, but the volume of
medium-sized soil particle was 43 - 61 %. The radioactive concentration was strongest in

the soil particle smaller than 0.063 mm, as predicted. Oxalic acid was the best chemical
agent for washing, especially for cobalt. A scrubbing time of 4 h was the optimum time to
obtain a removal efficiency of more than 75 % for 137Cs and 60Co. A mixing ratio of the soil
weight to the volume of the oxalic acid solution of 1:10 was the best for washing; two
scrubbing cycles with 1.0 M oxalic acid avoided the generation of a considerable amount
of waste solution. The removal efficiency with a hydro-cyclone was 30 % higher than that
without. Vertical plates and alum had important roles in reducing the sedimentation time.
The waste solution could be reused up to five times after passage through a column of a
strong acid resin (Gye – Nam et al, 2007).
4.5.5. Remotion of 238U, 137Cs and 85Sr from soil
Experimental study seems to demonstrate that the feasibility of electrokinetic soil remediation
for the removal of radionuclide contaminants from soils. The experimental results indicate that
the technique is effective in radionuclide contaminants from soils with a relatively small
amount of energy. Uranium and strontium were efficiently removed from kaolinite by
electrokinetic remediation. In the case of cesium, the removal rate may be significantly slower
than those of uranium and strontium. This is due to the lower ionic mobility and the affinity
of cesium onto kaolinite. Acetic acid was effective as enhancing agent for buffering hydroxide
ions produced by the cathode reaction, and prevented the precipitation of uranium ions in the
cathode region.
Accordingly, the acetic acid increased the removal efficiency and decreased energy consump‐
tion. The use of citric acid was not efficient in removing uranium from kaolinite, because the
direction of electromigration was opposite to that of electroosmosis. Since most metal–citrate
chelates were negatively charged, they were transported toward the anode by electromigration
while electroosmosis flowed toward the cathode. Therefore, removal efficiency significantly
decreased. This result indicates that the selection of enhancement agent should be considered
with respect to contaminant type and site characteristics. The electrokinetic removal of
uranium from the soil weathered from uraniferous black shale was not efficient. This was due
to the low proportion of the mobile fraction, since most uranium exists as residual fractions
derived from enriched uraniferous parent rocks (Kyeong-Hee et al, 2003).
5. Risk assessment and development of future strategies
Nuclear site operations and successful site restoration depend on the availability of suitable
waste management routes and facilities. Effective management of both radioactive and non-
radioactive waste is essential to the delivery is a significant part of the process.
Strategic decisions about waste management are informed by the following key principles:
risk reduction is a priority, centralised and multi-site approaches should be considered where
it may be advantageous, waste should be minimised and the waste hierarchy should be used
as a framework for waste management decision making and enables an effective balance of
priorities including value for money, affordability, technical maturity and the protection of
health, safety, security and the environment.
For Low Level Waste, disposal will be in fit for purpose facilities that reflect the nature of the
wastes to be managed. Within this overall framework our priority is to achieve risk reduction
by dealing with waste in ageing storage facilities and placing it into safer modern storage
conditions. Diverse radioactive waste management and disposal solutions will be pursued
where these offer benefits over previous arrangements. New waste management approaches
will often require different transport arrangements and will be a matter of great interest to
planning authorities and people living close to the sites involved.
Some general ideas about the underpinning strategies are showed in Figure 2, the basic steps
for effective management of radioactive waste are part of a global system, ranging from waste
generation to final disposal are: minimization of radioactive waste, pretreatment, characteri‐
zation, treatment, conditioning, transport, storage and disposal (IAEA, 1970). All of these ideas
with the intention to reduce the volume of radioactive wastes.
6. Conclusions
Radioactive materials are extensively used in industrial and research activities into medical,
agricultural and environmental applications, and in various other areas. During the produc‐
tion and use of these materials, radioactive waste will inevitably arise; this must be managed
with particular care owing to its inherent radiological, biological, chemical and physical
hazards.
Producers and users of radioactive materials must be sure that a waste management strategy
exists prior to the start of waste generation. A well-developed waste management strategy
should consider the entire sequence of waste management operations, from the waste’s
production until its final disposal, including the various regulatory, sociopolitical and
economic issues. The identified goal of radioactive waste management can be met with
reasonable cost and resource use by implementing a carefully planned waste management
strategy using appropriate technologies.
Waste containing long lived radionuclides must be treated, conditioned, stored and disposed
of at a repository specifically designed for this purpose. Sample storage capacity is needed for
the decay of short lived radionuclides and for storing long lived waste prior to, and after, the
treatment and conditioning steps. Decay is the only natural way of reducing radioactivity (the
process of transmutation of some long lived radionuclides is not considered viable at this time).
Since radionuclides have decay rates ranging from days to thousands of years, proper
segregation of wastes depending on their half-lives, and separate treatment and conditioning
of these wastes, is an important factor in the overall scheme of radioactive waste management.
Treatment of waste involves operations intended to benefit safety and/or economy by changing
the characteristics of the waste. There are different procedures to the treatment of radioactive
waste, like:
1. Treatment of aqueous waste: ion exchange/sorption, chemical precipitation, evaporation,

ultrafiltration / reverse osmosis (UF/RO), incineration, solid / liquid separation (sedimen‐
tation, filtration), centrifugation, hydrocyclone techniques, chemical precipitation (co-
precipitation or isomorphous precipitation with the carrier, adsorption on to the floc or
on added absorbers, flocculation, sedimentation, pH adjustment, chemical oxidation and
reduction, ion exchange / sorption, evaporation and reverse osmosis.
2. Treatment of radioactive organic liquid: incineration, wet oxidation, electrochemical oxida‐

tion, acid digestion, distillation, phase separation by adduct formation and biological
digestion.
3. Treatment of solid waste: decontamination, compaction, incineration and electroremedia‐

tion.
In the case of electroremediation treatment of radioactive wastes offers many advantages such
as the capacity to remove organic and inorganic pollutants by applying direct electric current
into the soil, even in clays, it is easy to operate and involves the installation of electrodes into
the soil or waste and the application of a low voltage gradient or direct current through them.
In consequence, the EKR is a very good technological alternative to remove radioactive species
as 22Na, 137Cs, 134Cs, 85Sr, 90Sr, 60Co and 238Ur from both saturated and unsaturated radioactive
soils, sludges, ash and sediments, with less time and high efficiencies of removal in comparison
of the other alternatives to remove radioactive wastes.
Acknowledgements
Unidos Mexicanos (CONACyT) with the project 193883 of projects and thematic networks
strengthening research, L’Oreal, United Nations Educational, Scientific and Cultural Organi‐
zation (UNESCO), Academia Mexicana de Ciencias (AMC), Fundación México – Estados
Unidos para la Ciencia (FUMEC). V. Valdovinos is grateful to CONACyT for her scholarship.
Author details
V. Valdovinos1, F. Monroy-Guzman2 and E. Bustos1*
1 Centro de Investigación y Desarrollo Tecnológico en Electroquímica, S. C., Parque Tecno‐

lógico, Querétaro, Sanfandila, Pedro Escobedo, Querétaro, México
2 Centro Nuclear “Dr. Nabor Carrillo Flores”, La Marquesa, Ocoyoacac, Carretera México-
Toluca, Edo, México
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chapter 15
Optical Fibers to Detect Heavy Metals in Environment:

Generalities and Case Studies
J. A. García, D. Monzón, A. Martínez, S. Pamukcu,

R. García and E. Bustos
1. Introduction
The fiber optic sensors can be used to create a truly distributed chemical sensing capability for
selectively detecting metal compounds by spatial and temporal acquisition over large distan‐
ces in the subsurface. In addition the fiber optic sensors have several advantages such as small
size, light weight, immunity to electromagnetic interference (EMI), high temperature per‐
formance, large bandwidth, high sensitivity, and environmental ruggedness (Krohn, 1988).
Most current technologies capable of detecting contaminants use strategically placed sensing
or monitoring devices. This works reasonably well if plausible event location is known, hence
settle recording vast amounts of benign data over time until the appearance of the suspected
event. This approach remains limited for application in large spatial scales in the geo-envi‐
ronment and subsurface. A simple approach is to suppress all the benign data by triggered
transmittal of the signals only at the spatial and temporal vicinity of the event. This, in essence
the “truly distributed” sensing capable of delivering the event signal “wherever” and
“whenever” it might occur, as opposed to only at strategic places where the sensing devices are
pre-located. The revolutionary advances in flexible sensing and distributed data processing
permits us sensing in this truly distributed manner.
Sensors based on fiber optic cable functions make use of the following important features of
the cable to sense the environment: (1) optical loss: intrinsic and extrinsic energy loss proper‐
ties, (2) refractive index: index profile in radial direction and the reduction of index fluctuation
along the axial direction; (3) shape: cross sectional shape and size, the surface finish and the
fluctuation of the size along axial direction. Present fiber optic sensors mostly use energy loss
principles (i.e., changes in optical power in linearly positioned wave-guides) for chemical
2 Soil Pollution
detection. These can be limited for distributed applications if energy depletes over a short
stretch of the fiber sensor, or frequent sensor points are needed at a prohibitively expensive
cost. Other sensors use the changes in refractive index and/or cross sectional size of the fiber
cable that change the light scattering property in optical fibers, known as Brillouin scattering
(Horiguchi et al, 1995; Kee et al, 2000).Fiber optic sensing based on Brillouin scattering has
been used successfully in civil infrastructure for health monitoring (Bao et al, 2001; Ohno et al,
2001). In this chapter a background on use of optical fibers for chemical sensing and new
developments and proposed advancements are discussed.
2. Background
2.1. Overview of fiber optic sensors
In an optical fiber sensor a physical, chemical or biological variable can interact with the light
and produce a change in one of their parameters. It is desirable to produce an optical signal
related uniquely to the parameter of interest. These sensors use the optical fiber either as the
sensing element (intrinsic sensors), or as a means of relaying signals from remote sensing area
to the signal processor (extrinsic sensor), or both. Optical fiber sensors take advantage of the
inherent fiber optic characteristics which include their lightweight, of very small size, passiv‐
ity, low-power requirement, resistance to electromagnetic interference, environmental impact
and corrosion, their bandwidth, and flexibility. They can be installed in areas normally
inaccessible by conventional sensors, they can be interfaced with data communication systems
and pose no risk of electric shock in live measurements. These attributes have allowed optical
fiber sensors to displace traditional sensors for measurement and monitoring of rotation,
acceleration, electric and magnetic field, temperature, pressure, acoustics, vibration, linear and
angular position, strain, humidity, viscosity, pH, gas and chemical content among many
others.
Use of Fiber Optic Sensors is a viable real-time data gathering approach by surface-adhering
or embedding the fiber to a specimen under evaluation. The concept of embedding fiber-optic
sensors into structures has generated a great deal of interests in aerospace engineering initially
and more recently in civil engineering. There are several types of chemical sensing techniques
based on optical waveguides (Ho et al, 2001). Among those are fiber Bragg gratings (FBG),
which is marking of a fiber with a laser to create a local narrow band pass filter sensitive to
environmental parameters (Guemes et al, 1998; Schulz et al., 1998). Optical time domain
reflectometry (OTDR) consists of sending a powerful light pulse and observe modification in
the reflected light due to local in homogeneties along the fiber. The pulse losses correspond to
specific environmental interaction. The evanescent pulse technique is also based on OTDR, in
which the fiber cladding is modified to interact with the environment and the pulse travels
partially through the cladding. These sensors demand large optical power, due to the cumu‐
lative energy loss at the points of contact with the chemicals.
Optical Fibers to Detect Heavy Metals in Environment: Generalities and Case Studies 3
Over the last decade, there has been rapid development in the area of smart sensor technolo‐
gies, in particular using structurally integrated optical fiber to form the basis for smart structure
technology. A variety of configurations have been developed for measurement of strains and
deformations in structures, including localized-type such as fiber Bragg gratings and multi‐
plexed long gauge interferometric sensors, and distributed sensing schemes including
Stimulated Brillouin Scattering (SBS) or Brillouin Optical Time Domain Analysis (BOTDA)
(Bao et al, 2001) and Brillouin Optical Time Domain Reflectometry (BOTDR) (Pamukcu et al,
2006; Anastasio et al, 2007).
Between different types of optical sensors reported, there are those based on sensitive coatings
onto the fiber surface, Fabry-Perot interferometers, long-period fiber gratings (LPFG), LPFG
with sensitive films, hetero-core devices, fiber Bragg gratings on doped fibers (i.e, Germanium
doped). Fiber gratings are structures consisting of a periodic perturbation of the optical
and/or geometrical properties of an optical fiber. Depending on the pitch of the perturbation,
fiber gratings fall into two distinct categories: short period gratings, known as fiber Bragg
gratings (FBGs) and, long period gratings (LPFGs). Stretching the fiber gratings causes a
change in grating period, hence the wavelength of the reflected light. This makes the FBGs
ideal for localized temperature and strain measurements. Unlike FBGs in which counter
directional coupling occurs in the core, co-directional coupling occur in LPFGs between the
core and cladding. This feature renders LPFGs sensitive not only to temperature and strain,
but also to bending causing a curvature, to hydrostatic pressure, to torsion and to ambient
refractive index changes. The closer the ambient refractive index to that of the cladding the
stronger the sensitivity to refractive index changes. It is this high sensitivity that has piqued
the interest in development of various types of refractive index-based LPFG sensors which
constitute most of the chemical sensing applications (Orellana and Haigh, 2008; Kasik et al,
2010).
Point detection fiber optic sensors have been developed successfully for measurement of liquid
levels, chemical species, drugs, environmental agents (such as pollutants and pesticides),
biochemical reactions, and to monitor a wide variety of chemical processes (Wolfbeis, 2000).
A fiber optic laser induced breakdown spectroscopy method was demonstrated in the field
using a push-cone device, which is a single point, single time measurement technique. The
most common configuration for optical pH sensors, and other environmental parameters,
employs a fluorescence indicator (Lee et al, 2000). Among the different types of optical fiber
devices used in pH sensing are, hetero-core fibers, U-bend fibers, fiber Bragg and long-period
gratings, fibers and fiber tips with active doped cladding, among others (Kocincovaet al, 2007).
Some of the substances that can be detected or identified using optical fiber sensors are volatile
organic compounds (alcohols, formaldehydes, methane, ketones, COx, O2, and H2), some
metallic ions like Ca, Al, Cu, Zn, Hg, V and Pb (Jeronimoet al, 2007; Wolfbeis, 2008).
Wide application of advanced chemical sensing in the environment may suffer from scaling
issues. The real-world conditions often require self-referencing, spatially distributed, tempo‐
rally continuous, and chemically selective sensors for monitoring regions spanning over long
lengths or wide areas. When large area monitoring for chemical agent intrusion is required,
use of currently available point sensors can be cost prohibitive. Other non-point, distributed
4 Soil Pollution
detection methods based on energy loss principles (Buerck et al, 2001) may also be inadequate
when scaled to wide area monitoring due to extensive energy input requirements.
One of the unique features of the optical fiber technology is the possibility to construct
distributed sensors, in which the measuring can be determined along a line of space with a
given spatial resolution (Galindez-Jamioy et al, 2012) by, for example, Brillouin optical time
domain analysis (BOTDA) (Cui et al., 2009, 2010 and 2011); an hetero-core LPFG sensors. In
here, we examine current and proposed application of these techniques to spatially distributed,
temporally continuous, and chemically selective sensing applications in soil and water
environment. The premise of Brillouin technique goes back to 1920 when physicist Leon
Brillouin first studied the diffusion of light by acoustic waves. The phenomenon he observed
was a frequency change of scattered light. The first major papers related to distributed fiber
optic sensor based on Brillouin were generated in mid-nineties (Bao, et al. 1995; Fellay et al,
1997). Current research on Brillouin sensing may be divided in three categories: photonics (the
physics of Brillouin); data processing and post processing to improve signal to noise ratios,
and applications of distributed sensing to civil infrastructure and environment.
2.2. Fundamentals of optical techniques
The interest on optical techniques to measure or detect chemical agents have been continuously
extending and growing over the last forty years. Special attention has been focused on the
development of optical sensor to detect heavy metals, due to the hazardous effects of these
ions on the health of human beings and ecosystems. Optical methods have the advantage of
being fast, simple, compact, portable, low-cost, with sensitivities and resolutions improved to
detect in the picomolar range.
Combined with other technologies, like microfluidics systems, optical waveguides, or MEMs,
optical methods are suitable for application where conventional electrodes cannot be used
because of their large size or because of the risk of electrode shock during in vivo measure‐
ments. Due to their minute size; these optical microsystems are capable of gathering diverse
data with a small amount of analyte. The diversification of optical techniques have made
possible to construct novel sensing platforms to detect heavy metals in air, water or soil, food
and beverages, or biological samples.
Optical sensors to detect heavy metals employ an optical transduction technique, i. e. an

element that “translate” the chemical variable into an optical signal (intensity, wavelength,
polarization or phase), to yield analyte information (McDonagh et al, 2008, Grattan and
Meggitt, 1999). Optical chemical sensors can be categorized, according to the transduction
technique, in direct sensors and reagent-mediated sensing systems. In direct sensors the
element of interest is detected directly via an optical property of the sample such as scattering
or florescence, for example.
However, most heavy metals optical sensor uses an intermediate agent. Most of the optical
chemical techniques to detect heavy metals are based on optical absorption, fluorescence,
Raman spectroscopy, or surface plasmon resonance, whereby the perturbed signal is related
to the reaction of the intermediate agent under the presence of a specific heavy metal. In
general, all these techniques involves the interaction of an incident beam over an analyte or
indicator element yielding transmitted, reflected or fluorescent signal. A schematic represen‐
tation of the spectroscopic principle, the working mechanism of an optical sensor is shown in
Figure 1.
IF
IR
II IT
IF
Figure 1. Representation of the optical signals in an optical chemical sensor. The incident beam II interacts with the
heavy metal sensitive layer and depending on the optical properties of the sample one of the three signals (transmit‐
ted IT, reflected IR, and generated by fluorescence or scattering IF) will be produced and will give information about the
type and concentration of heavy metal present on the sample.
2.2.1. Absorbance-based techniques
Among the optical chemical techniques, the simplest to implement is that based on the
measurement of light absorbed by a sensitive heavy metal layer. Absorption in a gas or liquid,
where it is assumed that each single molecules equally contributes to the total light absorbed,
may be characterized by a Beer-Lambert law, or simply the Beer law,
IT = I I 10-e Cd (1)
where IT and II represents the intensity of the transmitted and incident beam, ε is the molar
absorptivity (Lmol-1 cm), and C is the concentration (mol L-1) of the absorbing species and d is
the absorption path length (cm). In the case of a solid, absorbing and homogeneous medium,
the transmitted signal is calculated using the Lambert Bouguer law, expressed as I T = I I e −αx ,
where x is the thickness of the medium and α is the extinction coefficient. The Beer law can
also be expressed in terms of the absorbance (or optical density) A:
æI ö
A = log10 ç I ÷ = e Cd (2)
è IT ø
There exists a linear relation between the absorbance and the concentration of the element to
be measured. However, in order to observe the linear dependence of absorbance on concen‐
tration, the incident beam should be ideally monochromatic. In the case that a wide broadband
light source is used, the contribution of all wavelengths must be considered, in such cases the
equation (2) becomes:
6 Soil Pollution
æ I I (l ) d l ö
A = log10 ç
ò ÷ (3)
ç ò I T (l ) d l ÷
è ø
Also the presence of highly absorbing or highly scattering media should produce a deviation
from perfect Beer law behavior. In the case that more than one absorbing material is present
the absorbance contribution of each species must be considered. In most of the absorbance-
based heavy metals sensors an intermediate agent, an optical film that changes its absorbance
according to the concentration of a specific heavy metal, is used (Antico et al, 1999;Guo et al,
2006).
A special case of the absorption-based sensors are those schemes where materials that change
their color under the presence of a specific heavy metal are used (Balaji et al, 2006; Prabhakaran
et al, 2007). The reaction of the sensitive components to the concentration of a specific ion
produces a photochromic reaction that can be observed with a naked eye. Such materials are
often in solution, but for sensing the most attractive are those that can be deposited as thin
films over a substrate. The instrumentation of absorption-based sensors is the simpler of the
optical heavy metal techniques, since it can be implemented with a monochromatic light source
and a photodetector. This also makes this technique very susceptible to be implemented in
microscopic opto-fluidic configurations that could diversify the technique.
2.2.2. Reflectance-based techniques
It is well-known that chemical reactions could lead to changes in the complex refractive index
of a substance; this fact has been impulse researchers to design and fabricate materials that
react with heavy metals that can be used as transducers. When these materials, commonly in
the form of a thin layer, are illuminated with an appropriated light the signal will be partially
or totally reflected. However, this reflectance will change when the layer is in contact with a
specific metal that it reacts with. If the refractive index of the layer is purely real, the changes
in the reflected signal can be estimated by using the Fresnel formulae.
However, in most cases the optical response of these materials under the presence of heavy
metals are more complex and involve a change in the real and imaginary parts of the refractive
index, that produce changes in reflectivity and absorbance. Also, there is a contribution of
scattered light. So, the reflected signal is composed of light from different sources, however,
also in this complex response the signal reflected is used to deduce, directly or indirectly, the
concentration C of the heavy metals.
The reflected-based techniques are specially used in optical fiber schemes since the set-up is
very simple to implement (Yusofand Ahmad, 2003, Guillemain et al, 2009). The material
sensitive to the heavy metals are directly deposited over the fiber tip or in a substrate that will
be illuminated by an optical fiber. The reflected signal is usually collected by the same fiber,
but frequently another fiber or fibers are used to collect it. The reflected signal propagates
along the fiber to the detector, where it is analyzed in order to determine the heavy metal
present and their concentration (Figure 2).
Light Isolator
source Optical fiber
Transducer
Coupler
Signal
processing Detector
Analyte
Figure 2. Diagram of a reflectance-based optical chemical sensor based on optical fibers.
2.2.3. Fluorescence-based techniques
Some materials have the property of being fluorescent when they are illuminated with a light
source of appropriated wavelength. The fluorescence is the optical radiation generated when
electrons of an atom or molecule return from the excited to the ground state after absorption
of a photon from an excitation light source. In general the energy of the excited photon is lower
than the absorbed one so the wavelength of the fluorescence signal is longer than that of the
excitation.
The intensity of the fluorescent signal (IF) is proportional to the intensity of light absorbed by
the sample (II-IT), therefore it is possible to establish a direct relation between the intensity of
the fluorescent signal and the concentration of an absorbing material. This feature is very
important for sensing since intensity of the fluorescence increases as the concentration of the
absorbing species augments. Although, we have just made reference to the fluorescence
intensity, for sensing, the decay time of the fluorescence signal is more frequently used because
this parameter is less sensitive to source fluctuations, interference from ambient light or drift
due to aging of detector. It is possible to design and fabricate a fluorescent material sensitive
to a specific heavy metal. Thus, the intensity, wavelength and life time of the fluorescent signal
will change under the presence of this metal. Fluorescence-based techniques are the most used
to detect the presence of heavy metals due to its extraordinary sensibility (Mayra et al.,
2008;Achatz et al, 2011, Aksuner, 2011).
2.2.4. Surface plasmon resonance-based techniques
The most popular label-free refractometric technique is the Surface Plasmon Resonance (SPR),
since it allows the direct observation of chemical reactions in real time without the use of
markers or labels. SPR is a quantum optical-electrical phenomenon produced by the interaction
of light with a metal surface. Actually, the surface plasmon is a charge density oscillation that
exists at a metal-dielectric interface. The plasmon propagates in a direction parallel to the
metal-dielectric interface in the boundary of the metal and the external medium (Figure 3).
These oscillations are very sensitive to any change in the optical refractive index of the material
at the boundary. The optical excitation of plasmon can be achieved in a three-layer system
consisting of a thin metal film sandwiched between two isolators of different dielectric constant
8 Soil Pollution
(Maier, 2007), where the phase-matching condition between the optical and plasmon wave
vector is fulfilled. In the optical domain, the surface plasmon excitation will be observed as an
intensity transmission loss at a specific wavelength. The wavelength of the dip depends on the
refractive index of the two dielectrics and the thin metal film, and the propagation constant of
the optical waveguide. There are three common method to excite surface plasmon, using a
prism coupler and the attenuated total reflection, a periodic grating, and an optical waveguide
planar (Figure 3A) or cylindrical. The prism coupler technique is the most popular since
exhibits a good sensitivity, stability, and reproducibility for the measurement of heavy metals
(Forzani et al, 2007; Lin et al, 2009; Abdi et al, 2011; Fen et al, 2012 and 2013; Fen and Yunus,
2013). For heavy metal detection a sensitive thin film layer is deposited over the thin metal
film, so when the target heavy metal interacts with the layer a refractive index change is
produced. The surface plasmon conditions changes and the peak wavelength shifts as can be
seen in Figure 3B. SPR is the most sensitive refractometric method, with a theoretical resolution
of 1x10-7, so it is possible to detect very small traces of heavy metals.
Plasmon Sample
Thin metal film
Fundamental mode
Optical waveguide
Figure 3. (A) A schematic representation of the surface plasmon excitation in a dielectric planar waveguide coupler
Figure 3. (A) (B)
configuration. A schematic representation
Optical transmission of the
spectra of surface plasmonoptical
an hetero-core excitation
fiberincoated
a dielectric
with aplanar
20 nmwaveguide coupler
thin gold film when
configuration.
the (B) Optical
fiber is immersed in transmission
a liquid withspectra of an hetero-core
a refractive optical1.375,
index of 1.365, fiber coated with a 20
and 1.385, nm thin
black, red gold
and film when
green line
the fiber is
respectively. immersed in a liquid with a refractive index of 1.365, 1.375, and 1.385, black, red and green line respective‐
ly.
2.3. FUNDAMENTALS OF BOTDA AND LPFG BASED SENSING.
Figure 4, 5and 6show the stimulated Brillouin scattering based BOTDA photonics configuration and the principle of
measurement used at Lehigh University Geo-sensing laboratory, respectively (Texier et al, 2005; Pamukcu et al,
2006; Turel and Pamukcu, 2006; Anastasio et al, 2007). Brillouin is a nonlinear effect, in which light is scattered at
well-defined points along the fiber where the acoustic properties of the fiber are locally modified by the environment.
The stimulated Brillouin scattering (SBS) it is an acoustic – optical process which is useful for distributed
measurements of a probe beam by the SBS interaction with a counter-propagating nanosecond pump pulse. In the
SBS technique, as in a null detector, the pump and probe are initially de-tuned by a (frequency) that is slightly
2.3. Fundamentals of BOTDA and LPFG based sensing
Figure 4, 5 and 6 show the stimulated Brillouin scattering based BOTDA photonics configu‐
ration and the principle of measurement used at Lehigh University Geo-sensing laboratory,
respectively (Texier et al, 2005; Pamukcu et al, 2006; Turel and Pamukcu, 2006; Anastasio et al,
2007). Brillouin is a nonlinear effect, in which light is scattered at well-defined points along
the fiber where the acoustic properties of the fiber are locally modified by the environment.
The stimulated Brillouin scattering (SBS) it is an acoustic – optical process which is useful for
distributed measurements of a probe beam by the SBS interaction with a counter-propagating
nanosecond pump pulse. In the SBS technique, as in a null detector, the pump and probe are
initially de-tuned by a (frequency) that is slightly greater than the Brillouin frequency.
Therefore, in unstressed fiber, the base line remains flat resulting in a self-referenced sensor
eliminating the need for duplication with another reference fiber.
The Brillouin line being intrinsically narrow (~20 - 50 MHz), the initial de-tuning can be quite
small so that the amount of strain required to generate a signal is also quite small (0.001 %),
allowing for higher resolution and sensitivity of the sensor compared to other fiber-based
measurement techniques. When SBS based sensors are used for environmental sensing the
fiber is hitched or bonded with selective polymer transducers that are mass detectors in direct
contact with the surrounding medium. The polymer reacts to the surrounding (i.e. moisture,
pH, target chemical) by selectively absorbing the target compound and it swells. Localized
swelling of the bonded or hitched polymer produces tangential, axial or radial stresses on the
fiber depending on the physical coupling. These stresses result in axial straining of the fiber
and a measurable change in its local acoustic properties, hence a Brillouin scatter of the
transmitted light. The location of the generated signal is determined by time domain reflec‐
tometry.
Figure 4. SBS based BOTDR photonics set up at Lehigh Geo-Sensing Laboratory.

10 Soil Pollution
Figure 4. SBS based BOTDRphotonics set up at Lehigh Geo-Sensing Laboratory.
Time t Time t +t1
Time t +t2 @t
Here the probe light originates from the left end and is
reflected back from the right end of the fiber. Both signals
are generated using the same laser source.
@ t +t
The frequency difference between probe and pump wave
correspond to the Brillouin shift of the relaxed fiber.
@t +t2
The energy transfer from the pump to the probe wave
occurs all along the fiber.
CASE I
Figure 5. Measurement principles of SBS based sensing: case I.
Figure 5. Measurement principles of SBS based sensing: case I.
7
Time t +t2 @t
Here the sensed event constrains the fiber locally and
the frequency of the probe is modified
@ t +t
Pump-probe frequency difference matches the local
Brillouin shift at the sensor location
@t +t2
occurs nowhere except at the sensor location
CASE II
Time t +t2 @t
Here the sensed event constrains the fiber locally
@ t +t
The frequency difference between probe and pump wave
correspond to the Brillouin shift of the relaxed fiber
@t +t2
occurs all along the fiber except at the sensor location
CASE III
Figure 6. Measurement principles of SBS based sensing: case II and III.
Figure 6. Measurement principles of SBS based sensing: case II and III.

In the case of LPFG`s the environmental changes produce a spectral shifts, that required a sophisticate or a
complicate equipment. These devices are very sensitive to temperature changes so to measure another different
parameter it is necessary to make the corresponding compensation. One alternative to avoid these difficulties are
hetero-core fibers. These devices are constructed by changing the diameter of the core in a small length (mm) in a
transmission line (Figure 5 and 6), which causes the optical wave to expand within the cladding in the single-mode
region of the hetero-core, thus the evanescent field can easily interact with the external medium.
Owing to the core diameter mismatch, some of the light is guided by the cladding of the SM fiber (Figure 7). This
makes the transmission of the device dependent on the refractive index of the external medium. The sensor exhibits
maximum transmission changes when the index of the sample medium approaches that of the SM fiber cladding. The
device can operate at different wavelengths as well as when coated with thick films made of variable index materials.
Moreover, standard emitters, fibers, detectors, etc., are needed to fabricate the sensor, which makes it attractive for
In the case of LPFG`s the environmental changes produce a spectral shifts, that required a
sophisticate or a complicate equipment. These devices are very sensitive to temperature
changes so to measure another different parameter it is necessary to make the corresponding
compensation. One alternative to avoid these difficulties are hetero-core fibers. These devices
are constructed by changing the diameter of the core in a small length (mm) in a transmission
line (Figure 5 and 6), which causes the optical wave to expand within the cladding in the single-
mode region of the hetero-core, thus the evanescent field can easily interact with the external
medium.
Owing to the core diameter mismatch, some of the light is guided by the cladding of the SM
fiber (Figure 7). This makes the transmission of the device dependent on the refractive index
of the external medium. The sensor exhibits maximum transmission changes when the index
of the sample medium approaches that of the SM fiber cladding. The device can operate at
different wavelengths as well as when coated with thick films made of variable index materials.
Moreover, standard emitters, fibers, detectors, etc., are needed to fabricate the sensor, which
makes it attractive for diverse applications (Villatoro and Monzon-Hernandez, 2006).
Figure 7. Schematic representation of the hetero-core fiber structure. A piece of single-mode fiber (SMF) of length L is
inserted between two multimode fibers (MMF). Cladding diameter of both fibers is 125 μm but the core diameters are
different.
3. Investigation
3.1. BOTDA/BOTDR applications and proposed sensor configurations
The usefulness of SBS for sensing is general: any change in external conditions, that affects the
acoustic properties of the optical fiber, can in principle be detected. This is true of direct
temperature and pressure changes, but can also be true of changes in chemical environment
that can be made to result in temperature and pressure changes. An SBS based BOTDR sensing
system was used to detect water content changes in soil. Water transducers (hydrophilic
polymers) were tested to correlate Brillouin strain response to the water content of the
surrounding soil environment. In these experiments, the optical fiber was wound and secured
about discretely placed discs (2 cm length x 5 cm diameter) of AEP60 hydrophilic polymer
(Figure 8A), stringed along 100-m fiber continuous optical fiber. The diameter of the polymer
3. INVESTIGATION.
3.1. BOTDA/BOTDR APPLICATIONS AND PROPOSED SENSOR CONFIGURATIONS.

12 Soil Pollution
The usefulness of SBS for sensing is general: any change in external conditions, that affects the acoustic properties
of the optical fiber, can in principle be detected. This is true of direct temperature and pressure changes, but can also
be true of changes in chemical environment that can be made to result in temperature and pressure changes. An
SBS based BOTDR sensing system was used to detect water content changes in soil. Water transducers (hydrophilic
disc waswere
polymers) selected
tested to accommodate
to correlate the minimum
Brillouin strain response tocurvature of bending
the water content of the fiber,
of the surrounding as shown
soil environment.
In these experiments, the optical fiber was wound and secured about discretely placed discs (2 cm length x 5cm
in Figure 8B.
diameter) of AEP60 hydrophilic polymer (Figure 8A), stringed along 100-m fiber continuous optical fiber. The
diameter of the polymer disc was selected to accommodate the minimum curvature of bending of the fiber, as shown
in Figure 8B.
A
20
16
Output Power, mW
DBF Laser @ 200 mA/ 10k

12
4
Minimum diameter of curvature = 3.29 cm
0
0 2 4 6 8 10 12
Bending Diameter, cm
Figure 8. A photograph of the AEP60 hydrogel polymer discs used in sensor (A); assembly and test results for
Figure 8. Afiber
minimum photograph of the assessment
bending radius AEP60 hydrogel
(B). polymer discs used in sensor (A); assembly and test results for mini‐
mum fiber bending radius assessment (B).
9
The optical fibre at the inlet and outlet of the string of four transducers were spliced to spools
of fibre on each end, and connected to the photonic set-up. Each water transducer was then
embedded in a wet clay sample of predetermined water content (5, 10, 20 and 30 % by dry
weight of clay), as shown in the inset sketch of Figure 9B.
The clay samples were packed in equal volume, watertight, cylindrical cells of 14-cm diameter
and 28-cm height. The experiments were conducted in a temperature-controlled environment,
at 25°C so that Brillouin scattering measurements were not influenced by thermal expansion
or contraction of the fibre. The Brillouin shift was measured with 5 to 20 minute intervals up
to the maximum observable swelling. When no significant change in Brillouin shift was
recorded for three consecutive measurements, the transducers were removed from the soil
chambers and left for open air-drying.
The Brillouin shift measurements were recorded with 5 to 20 minute intervals until the drying
phase was completed. Since Brillouin readings could be recorded for strains as low as 10, very
small volume change due to water absorption could be detected in a few minutes. Figure 9B
Figure 9A shows the calibration of Brillouin shift versus water content gain of the hydrogel disc coupled to the
optical fiber. The hysteresis between the swelling and drying cycles is attributed to the diffusion trends affected by
the geometry of the hydrogel transducers. The ends of the discs were exposed to diffusion as well as the
shows the time
cylindrical ratewhich
surfaces of Brillouin signal
resulted in changes
uneven during the
strain distribution in thehydrogel
fiber. swelling and drying cycles
of the 4-different water content clay hosts.
10.92
Swelling phase
Drying phase
10.90
Brillouin shift (GHz)
10.88
10.86
10.84
10.82
-5 5 15 25 35 45
Hydrogel water content (%)
A
11.5
Brillouin frequency shift
11.4 30%
11.3 20%
(GHz)
11.2
10%
11.1
11
Clay water content = 5%
10.9
0 240 480 720 960 1200 1440 1680
Time, min
Figure 9. (A) Calibration of Brillouin Shift of fiber versus the water content by dry weight of the
Figure 9. (A)hydrogel
Calibration of Brillouin
disc. Shiftsignal
(B) Brillouin of fibershift
versus
as the water content
a function by dry weight of
of swelling/shrinkage theand
time hydrogel disc. (B) Bril‐
clay water
louin signal shift as a(Inset
content function of swelling/shrinkage
– sketch of experimental set time and clay water content (Inset – sketch of experimental set
up).
up).
The optical fibre at the inlet and outlet of the string of four transducers were spliced to spools of fibre on each end,
Inand
theconnected
experimentsto thedescribed above,Each
photonic set-up. the AEP60 polymerwas
water transducer usedthenwould typically
embedded expand
in a wet from of
clay sample
38predetermined
% to 400 % waterovercontent
dry volume when exposed to water. They are non-toxic and are manufac‐
(5, 10, 20 and 30% by dry weight of clay), as shown in the inset sketch of Figure 9B.
tured in medical
The clay samples weregrades, approved
packed for use
in equal volume, in human
watertight, wound
cylindrical care
cells applications.
of 14-cm This
diameter and 28-cmgroup ofThe
height.
polymers
experimentsdoes
werenot swell ininhydrocarbons
conducted and chlorinated
a temperature-controlled environment,solvents
at 25°Cand so has
that high thermal
Brillouin scattering
measurements were not influenced by thermal expansion or contraction of the fibre. The Brillouin shift was measured
resistance.
with 5 to 20They
minuteare cross-linked
intervals to give them
up to the maximum mechanical
observable stability
swelling. When and accurate
no significant changeexpansion
in Brillouin shift
characteristics.
was recorded for The
threewater absorption
consecutive and expansion
measurements, factorwere
the transducers canremoved
be accurately
from the pre-defined
soil chambers and at the
left for
open air-drying.
formulation stage. Full expansion is reproducible over many wetting and drying cycles and is
consistent over a wide range of pH and dissolved 10solid concentrations. A different integration
of optical fiber and polymer transducer was used to improve the polymer response kinetics as
The Brillouin shift measurements were recorded with 5 to 20 minute intervals until the drying phase was completed.
Since Brillouin readings could be recorded for strains as low as 10, very small volume change due to water
14 Soilabsorption
Pollution could be detected in a few minutes. Figure 9B shows the time rate of Brillouin signal changes during the
hydrogel swelling and drying cycles of the 4-different water content clay hosts.
In the experiments described above, the AEP60 polymer used would typically expand from 38% to 400% over dry
volume when exposed to water. They are non-toxic and are manufactured in medical grades, approved for use in
shown
humaninwound
Figurecare10. In this design,
applications. theofpolymer
This group wasnot
polymers does reduced
swell in to smaller size
hydrocarbons discrete sleeves
and chlorinated solvents and
(1cm length x 0.3 cm diameter) that were bonded over the optical fiber. The bonding
has high thermal resistance. They are cross-linked to give them mechanical stability and adhesive
accurate expansion
characteristics. The water absorption and expansion factor can be accurately pre-defined at the formulation stage.
used
Fullwas Locktite
expansion 414, a super
is reproducible overbonding, quick
many wetting and drying adhesive
drying cycles and iscontaining
consistent overcyanoacrylate and
a wide range of pH and
is intended forconcentrations.
dissolved solid plastics andAvinyl. The
different Locktite
integration 414 was
of optical fiber applied
and polymer to transducer
each endwas of the
usedpolymer
to improve the
polymer response kinetics as shown in Figure 10. In this design, the polymer was reduced to smaller size discrete
sleeve also.
sleeves (1cm length x 0.3 cm diameter) that were bonded over the optical fiber. The bonding adhesive used was
Locktite 414, a super bonding, quick drying adhesive containing cyanoacrylate and is intended for plastics and vinyl.
The Locktite 414 was applied to each end of the polymer sleeve also.
Step 1 Step 2
Step 3
Figure 10. The working principle of the integrated sensor. Step 1 - Solution sorbed by the chemical selective
Figure 10. The
polymer working
sleeve bondedprinciple of the
onto the integrated
fiberoptic cable.sensor.
Step Step 1 - Solution
2 - The selectivesorbed by the
polymer chemical
swell selective polymer
upon encountering the target
sleeve bonded or
compound onto
ionthe fiberoptic
in the cable.
solution. StepStep
3 -2 -The
Theswelling
selectiveof
polymer swell upon
the bonded encountering
polymer the target
sleeve induces compound
a “tangential pull” or
or ion in the
“axial solution.
strain” Stepfiber
on to the 3 - The swelling of the bonded polymer sleeve induces a “tangential pull” or “axial strain”
locally.
on to the fiber locally.
The reduced size was anticipated to improve the swelling kinetics and alleviate the hysteresis affects observed in the
previous configuration. The working principle of the integrated sensor is also depicted in Figure 10, where first the
The reduced
influx size was
of the target anticipated
substance to improve
(e.g. water) the swelling
into the polymer kinetics
transducer causesand alleviate
swelling of the the hysteresis
bonded polymer. The
swelling causes the bonding interface to strain and cause the fiber elongate in tangential pull. The fiber strain can
affects
then be recorded with location and amplitude, as shown in Figure 11, indicating where along the fiber linesensor
observed in the previous configuration. The working principle of the integrated the influx of
is also depicted
the target in Figure
substance 10, where
had occurred, firstthethe
and also influx quantity
calibrated of the target substance
of the substance (e.g.
based onwater) intoofthe
the degree swelling
of the polymer, respectively.
polymer transducer causes swelling of the bonded polymer. The swelling causes the bonding
interface to strain and cause the fiber elongate in11
tangential pull. The fiber strain can then be
recorded with location and amplitude, as shown in Figure 11, indicating where along the fiber
line the influx of the target substance had occurred, and also the calibrated quantity of the
substance based on the degree of swelling of the polymer, respectively.
A B
Figure 11. The operation schematic and the photograph of a spent water sensor: (A) Conceptual schematic of the
integrated
Figure sensor
11. The assembly
operation and its and
schematic working principle as of
the photograph a distributed water
a spent water sensor.
sensor: (A)(B) A laboratory
Conceptual prototype
schematic of
of the
the integrated
integrated water
sensor sensor.and its working principle as a distributed water sensor. (B) A laboratory prototype of the
assembly
integrated water sensor.
Repeated swelling and shrinkage tests of the polymer sleeve component of the integrated water sensor showed
Repeatedof swelling
hysteresis length and and shrinkage
diameter teststhe
change. Both of length
the polymer sleeve
and diameter of thecomponent
of the polymerofsleeve
the integrated
expanded by
water
35% sensor
(~ 0.35 showed
cm and hysteresis
~0.1 cm, of length
respectively) and
after being diameter
soaked change.
in water for threeBoth
days.the
Thelength
majorityand diameter
of this expansion
occurred during the first 12 hours of soaking. Increasing with every cycle, the final dry length of the polymer was
of the of the polymer sleeve expanded by 35 % (~ 0.35 cm and ~0.1 cm, respectively)
greater than the original by ~ 0.025 cm (~2.5%).As more cycles were completed, the diameter increased to its
after being
soaked in
maximum water
faster, but for
the three
value ofdays. The majority
this maximum of this
decreased. Theexpansion
final diameteroccurred during
of the polymer the first
sleeve 12
was fairly
hours of soaking. Increasing with every cycle, the final dry length of the polymer was greater
constant for each cycle, slightly less than the original by ~ 0.0005 cm (~0.2%).
thanmagnitude
The the original by ~of0.025
and rate cmcorrelated
swelling (~2.5 %).directly
As more withcycles were
the initial completed,
water content when thethe
diameter increased
polymer sleeves were
to its maximum
embedded in test clayfaster,
specimens butofthe valuewater
different of this maximum
contents. Once again decreased. The occurred
the full swelling final diameter
between 8oftothe
12 h
when the sleeves were embedded in wet clay. Figure 12A shows that the polymer linear extension and clay water
polymer
content sleeve
relation waswas fairly
fairly constant
linear. for relation
The linear each cycle, slightlyforless
is desirable thancalibration.
robust the original by 12B
Figure ~ 0.0005
showscm the
(~0.2 %). frequency shift response of the integrated water sensor to clay water content increase. The figure plots
dimensionless
two spectrums, the shift spectrum at the location of the expanded polymer and a spectrum near the polymer location
that does not undergo the swelling stress.
The magnitude and rate of swelling correlated directly with the initial water content when the
polymer
The actual sleeves were embedded
Brilloin frequency in test clay
shift was measured specimens
0.0432 of different
GHz corresponding water
to axial contents.
strain of 0.098% Once again
for the fiber.
The actual elongation of the polymer rod was 3 mm, which fully transferred to the fiber would have corresponded to
the full swelling occurred between 8 to 12 h when the sleeves were embedded in wet
an average strain of 0.14% for the fiber over the 12.6 ns pulse. This meant that about 70 percent of the axial tug clay.
Figure 12A
generated shows
by the that
swelling of the polymer
polymer linear extension
was transferred to the fiber.and
Theclay
otherwater content
30 percent can berelation was
attributed fairly
to slippage
across the length of the bond between the fiber and the polymer sleeve or other process related inefficiencies.
linear. The linear relation is desirable for robust calibration. Figure 12B shows the dimension‐
less frequency shift response of the integrated water sensor to clay water content increase. The
figure plots two spectrums, the shift spectrum at the location of the expanded polymer and a
spectrum near the polymer location that does not undergo the swelling stress.
The actual Brilloin frequency shift was measured 0.0432 GHz corresponding to axial strain of
0.098 % for the fiber. The actual elongation of the polymer rod was 3 mm, which fully trans‐
ferred to the fiber would have corresponded to an average strain of 0.14 % for the fiber over
the 12.6 ns pulse. This meant that about 70 percent
12 of the axial tug generated by the swelling
of polymer was transferred to the fiber. The other 30 percent can be attributed to slippage
16 Soil Pollution
Figure 12. (A) Variation of embedded AEP60 polymer sleeve length vs clay w/c in swelling behavior. (B)
Figure 12. (A)
Shifted Variation
frequency of embedded
spectra AEP60sensor
for integrated polymer sleeve
under lengthstresses
swelling vs clay w/c
with in swelling water.
increased behavior. (B) Shifted fre‐
quency spectra for integrated sensor under swelling stresses with increased water.
across the length of the bond between the fiber and the polymer sleeve or other process related
inefficiencies.
These test results demonstrated the viability of integration of optical fiber with reactive
polymer as a BOTDR based sensor. Yet, the slow kinetics of the response (~12 hr for full
swelling in case of water) rendered the prototype assembly limited for quick detection and
measurement purposes. Hence a new polymer and fiber-optic cable configuration is proposed
where a thin layer (e.g. on the order of few hundred μm) of the reactive polymer is brushed
and bonded onto the fiber-optic cable. Figure 13 presents variations of the conceptual sensor
where the reactive polymer coat is continuous. The working principle of this new configuration
is similar to the previous ones described, all based on BOTDR, with the exception that the
continuous coating of a thin layer reactive polymer is anticipated to provide a truly distributed
and fast detection mechanism.
13
Figure 13. Advanced integrated chemical sensors based on BOTDR/BOTDA: (A) Cross-section of integrated
withAdvanced
Figure 13.
sensor integrated
core/shell polymer chemical
layers; sensors based on
(B) Working BOTDR/BOTDA:
principal (A) Cross-section
of an integrated sensor of integrated
with sensor
polymer/jacket
with core/shell polymer layers; (B) Working principal of an integrated sensor with polymer/jacket combination.
combination.
3.2. Reactive polymers principles used as sensors

These test results demonstrated the viability of integration of optical fiber with reactive polymer as a BOTDR based
Point detection fiber optic sensors have been developed for measurement of liquid levels,
sensor. Yet, the slow kinetics of the response (~12 hrs for full swelling in case of water) rendered the prototype
chemicallimited
assembly species for(inorganic and and
quick detection organic), drugs, purposes.
measurement environmental
Hence a agents (such asand
new polymer pollutants
fiber-optic and
cable
configuration
pesticides),isbiochemical
proposed where a thin layer
reactions, and(e.g.
toon the orderaof
monitor few hundred
wide varietyμm) of the reactive
of various polymer
chemical is brushed
processes
and bonded onto the fiber-optic cable. Figure 13 presents variations of the conceptual sensor where the reactive
(Wolfbeis,
polymer coat 2000). One ofThe
is continuous. theworking
majorprinciple
components of aconfiguration
of this new sensor system is the
is similar sensing
to the or ones
previous recognition
described,
all based onPolymers
element. BOTDR, with the often
have exception
beenthatutilized
the continuous coating of sensing
as a chemical a thin layer reactive polymer
material. is anticipated
The interaction ofto
provide a truly distributed and fast detection mechanism.
an analyte with the polymer coating is of prime importance. A signal needs to be generated
once this interaction occurs, and much work has been carried out to understand the interaction
14
18 Soil Pollution
of the polymer coating with the analyte molecules, especially in terms of the diffusion behavior
of the analyte through the polymer coating to the actual sensor itself and its subsequent
enrichment within the polymeric coating layer (Philips, et al, 2003).
The integrated chemical sensor based on BOTDR discussed in here is a single SBS (Stimulated
Brillouin Scattering) sensing optical fiber bonded with such a polymer, which swells selectively
in the presence of a target chemical. The polymer coat is cross-linked to swell in a preferential
direction. During swelling, the bonded polymer coat exerts a radial or tangential force at the
polymer-fiber interface, hence an axial strain on the fiber. The SBS signals are generated along
the fiber at the discrete points of chemical contact triggered by polymer swelling, hence the
local axial strains in the fiber. Consequently, the location of the target chemical can be detected
simply by linear positioning of the SBS signals over the entire length of the cable. Because the
detection is based on local physical changes in the fiber and not on loss of transmitted light,
widely distributed sensing is possible without high power requirements. The specific detection
and measurement components of the integrated sensor described above include, a core/shell
type (multi-layer) polymer coating, and an inner fiber optic cable core, or a rigid woven jacket
conceptualized in Figure 13.
In this conceptual design, the outer cross-linked polymer coating (shell) serves as a rigid, high
permeability filter and confinement to the inner polymer (core). The core is a flexible, chemi‐
cally selective polymer, preferably with rapid mass sorption kinetics. While the highly
networked rigid polymer shell confines and directs the cores welling toward the fiber optic
cable, the selective detection of chemicals is based on the thermodynamics and kinetics of
chemical sorption and swelling of the core polymer layer. A volume change or “swelling”
occurs in the inner flexible polymer layer as a result of mass sorption of the surrounding
analyte. Solute/solvent sorption interrupts the intermolecular forces between individual
chains of a lightly cross-linked or a linear polymer adjacent to the glass fiber, resulting in
swelling forces. The outer, permeable but rigid polymer layer comprised of highly networked
cross-linked chains help confine the volume change tendency and direct part of the swelling
forces inward (radially and/or longitudinally) thus creating a hoop or a tangential stress on
the fiber at the polymer interface. This “pinch” or tug” of the fiber cause changes in the elastic
and refractive properties of the fiber locally, generating a shift in its original Brillouin frequency
at that local. Comparing the new frequency to the original, it is then possible to quantify the
change in terms of the swelling forces and the quantity of the absorbed substance.
A multi-chemical sensor can be developed by bundling polymer-coated fibers of different

functions and sensitivities. The entire sensor assembly can be several tens of kilometers of
optical fiber hosting several 100 measurement locations on the same line, each at a minimum
spatial resolution of 1-meter. The photonics assembly connected to one end of the fiber allows
fast detection of discrete sensing locations rendering the entire assembly a multiplexed
network of many point sensors on the same transmittal line. Hence, the fiber line coupled with
the stringed transducers can be embedded linearly or laced into a host medium (i.e. water
pipeline, paved surfaces, porous media such as soil or concrete) to detect target chemical(s)
online over large distances, areas or spaces by linear positioning of the fiber.
3.2.1. Reactive polymers
The molecularly imprinted polymers are often used to improve selectivity (Wolfbeis, 2000;
Philips et al, 2003). The incorporation of desired functional monomers into the polymer
structure further enhances the selectivity to a given analyte. These polymers are cross-linked
and prepared by free radical polymerization processes such as solution or dispersion poly‐
merization with acrylic or vinyl monomers. Polyelectrolyte gels are charged cross-linked three-
dimensional networks of monomers that possess high swelling capability due to solvent
sorption. The amount of swelling is known to be a string function of pressure, temperature,
ion concentrations and pH changes (Siegel, 1993; Siegel et al, 1998; Matsuo and Tanaka, 1988).
Their swelling and kinetics depend on parameters such as the degree of cross-linking (Skouri
et al, 1995), external salt molarity (Yin et. al, 1992), and the degree of gel ionization rule
(Katchalsky and Michaeli, 1995; Yin et al, 1992).
An emulsion or miniemulsion polymerization approach can be utilized to prepare film-

forming polymer latexes with desired functional moieties to be used to coat optical fibers. The
base latex polymer may be based on acrylic (e.g., n-butyl acrylate, n-butyl methacrylate) or
styrene/acrylic film-forming compositions (i.e., with glass transition temperatures (Tg) of room
temperature or lower). These latexes are prepared by conventional emulsion polymerization
or by a miniemulsion polymerization process in the case where monomers with very low water
solubility are used. In the miniemulsion polymerization process, the monomer would be
emulsified in the presence of an aqueous surfactant (such as sodium lauryl sulfate) / costabil‐
izer (e.g., hexadecane dissolved in the (co)monomer mixture) combination using a high shear
device to form miniemulsion droplets which could then be polymerized in the presence of a
free radical such as potassium persulfate.
The polymer would also be crosslinked to varying extents using crosslinking monomers such
as ethylene glycol dimethacrylate, divinylbenzene, or bisacrylamide. In addition, functional
monomers can be copolymerized along with the base acrylic or styrene/acrylic monomers. One
type of monomer is a carboxylic acid such as methacrylic acid (MAA), which copolymerizes
well with the base monomers. At high concentrations, this latex could function as an alkali-
swellable latex whereby the latex particle size, and coating swellability, would increase
dramatically upon neutralization in aqueous solutions of high pH (e.g. > 10) which would
trigger a sensor response. N-methylol acrylamide (NMA) may also be incorporated into the
base copolymer composition to obtain a crosslinked polymer, which can also act as a hydrogel,
which could also swell when exposed to water. In addition, the monomer, N-(isopropylacry‐
lamide) (NIPAM) will also be utilized for forming hydrogel particles which can swell when
exposed to water. Incorporating NIPAM into a polymer composition would also lead to the
formation of a thermosensitive polymer coating since poly(NIPAM) exhibits a strong phase
transition above 32°C. It is also possible to copolymerize a alkoxysilane-containing monomer
with the acrylic or styrene-acrylic monomers via miniemulsion polymerization to enhance the
compatibility of the polymer coating with the glass optical fiber.
20 Soil Pollution
3.2.2. Bonding/lamination
The extent of bonding of the polymer coating to the glass optical fibers is critical. The optical
fibers can either be coated with the manufacturer’s cladding removed or in place. Preliminary
experiments have shown that it is difficult to coat the uncoated glass fibers. These fibers are
brittle without the manufacturer’s cladding in place; the composition of which is unknown.
The fiber can be passed through a coagulant bath prior to its immersion in the latex bath.
Similar to dip coating, which has been utilized in preliminary coating experiments, the latex
will coagulate onto the glass fiber. The surface of the fiber needs to be made hydrophilic for
this process. This can be achieved by either physical adsorption of nonionic water-soluble
polymer such as poly(vinyl alcohol) (PVOH) or by corona treatment of the fiber surface. In
addition, the polymer processing techniques used in wire coating applications can also be
applied to the case of the optical fibers. Important coating parameters would include the solids
content of the latex (a high solids content is needed to control the rheology of the dispersion
to be coated; a reasonable viscosity is needed for effective coating). Latexes can also be made
self-thickening by the incorporation of carboxyl groups into the latex particles. A thickener can
also be added to a latex composition to adjust the coating viscosity.
In addition, the surface tension of the latex would need to be controlled to give good wetting
onto the glass fiber. Contact angle measurements on glass substrates can be used to determine
the optimum wetting behavior before moving on to the glass fiber itself. The thickness of the
polymer coating would also need to be varied to determine the necessary thickness needed to
give a good, measurable response when exposed to solvent or water containing the heavy
metal ions. If the coating is not thick enough, the response to the analyte may be too weak. If
the coating is not uniform on the fiber, there would be unexposed regions of the fiber which
would affect the detection limit and sensitivity. In addition, there needs to be good adhesion
of the coating to the fiber, otherwise delamination could occur. Silane adhesion promoters can
be explored to enhance adhesion of the polymer coating onto the glass fiber substrate. The
drying temperature and drying conditions (e.g., time and temperature that the latex-coated
fibers are dried in an oven to ensure good film integrity or the use of forced heated air flow
over the fibers) are also critical coating variables to be investigated.
3.2.3. Kinetics
The kinetics of swelling of the polymer coatings when exposed to aqueous or organic media
needs to be evaluated by monitoring the changes in the dimensions of the coating or the
gravimetric uptake of the media by the polymer. The time-dependent changes can be analyzed
to give an idea of the best polymer architecture to obtain an optimum sensor response when
exposed to a given chemical. The time constant for the sensor response needs to be determined
and correlated with the swelling kinetics of the polymer coating to achieve the best sensor
performance.
3.3. Development of a prototype ph sensor with reactive polymer coating
The development of an optical fiber pH sensor based on hetero-core fiber structure coated with
an acrylic polymer doped with Prussian blue is discussed here. In this design, the pH changes
of the surrounding medium affects the Prussian blue present in the layer and produce a change
in the refractive index of the layer. The pH changes are then observed as an increment in the
hetero-core transmission signal.
3.3.1. Building of hetero-core optical fibers with reactive layer
The hetero-core fibers were constructed using two different length and two different types of
optical fiber. In this case two types of single-mode fibers (SMA and SMB) and two of multimode
fibers (MMA and MMB) were used. First, two pieces of MM fiber, stripped of its coating
polymer (3 cm section) were spliced to a stripped SM fiber on each side. The hetero-core fibers
were treated with Prussian Blue 0.1 mM (PB), polyvinyl alcohol(PVOH) at 4 %,acrylic polymer
emulsion (APE) at 50 % plus and their combinations, like PVOH + PB and APE + PB to develop
a reactive coat over the stripped surfaces. A small U-shape container made of a glass capillary
was fixed to a mechanical mount and was filled with the mixture of polymer support and
Prussian blue sensitive material. Then the single-mode section of the hetero-core fiber was
immersed for 5 minutes into the solution after which the fiber was removed and dried at room
temperature. In this manner the sensitive material was adhered to the single-mode section of
the hetero-core fiber.
One end of the hetero-core fiber was connected to a white light source Yokogawa AQ4305 and
the other to the spectrum analyzer Ando AQ6315A (Figure 14A). The set-up was used to
measure the transmission light during the modification process of the fiber and later to
measure the response of the modified fiber to pH changes. In order to test the sensitivity of
the device to changes in pH, a test was designed which consisted of immersing the optical fiber
section modified with PVOH/PB or APE/PB in a Petri dish where the pH was varied by adding
0.1 M NaOH or 0.1 M HCl, recording each transmission spectrum changes in the wavelength
range from 350 nm to 1700 nm (Figure 14B).
The transmission spectra of two hetero-core fibers with PVOH/A (5 mm and 10 mm sections)
were measured in different pH solutions are shown in Figure 14A and 14B, respectively. As
seen in figure 15 the device has good sensitivity (-1.5 dB and -2 dB approximately), however
the signal is erratic and not repeatable for different pH changes. This was attributed to
solubility of PVOH in acidic conditions, and checked visually and with the transmission
spectrum analysis. A new polymer, acrylic polymers emulsion (APE) was selected to replace
PVOH. This polymer has similar characteristics as PVOH. It is water soluble, inexpensive, and
colorless when dried, and has been reported as a good support in manufacturing of modified
electrodes for pH determination.
The transmission spectra of two hetero-core fibers with APE/A (5 mm and 10 mm sections)
were measured in different pH solutions are shown in Figure 16A and 16B, respectively. As
seen in Figure 16A, the device showed very obvious changes when subjected to acidic and
basic conditions. The presence of three peaks in visible region, 400, 500 and 700 nm wave‐
22 Soil Pollution
B
Figure 14. Set up to test prototype hetero-core fibers for chemical detection: (A) Diagram of the set-up to
measure the transmission signal of the hetero-core fibers; (B) Diagram of the test system to determine the
Figure 14. Set up to test prototype hetero-core fibers for chemical detection: (A) Diagram of the set-up to measure
sensitivity of hetero-core fiber to pH changes.
the transmission signal of the hetero-core fibers; (B) Diagram of the test system to determine the sensitivity of hetero-
core fiber to pH changes.
The transmission spectra of two hetero-core fibers with APE/A (5mm and 10mm sections) were measured in different
lengths
pH were
solutions noted
are shown when16A
in Figure optical fiber
and 16B, was in As
respectively. air.seen
These signals
in Figure were
16A, the attributed
device showed veryto obvious
light
absorption
changes when and loss to
subjected ofacidic
lightand
by basic
index refraction
conditions. Thechanges
presence by the composite
of three material.
peaks in visible In acidic
region, 400, 500 and
700 nm wavelengths were noted when optical fiber was in air. These signals were attributed to light absorption and
pH of
loss values
light bythe light
index losses changes
refraction were in by
thetherange of 1.5material.
composite to 3 dB In
while
acidicfor
pHbasic
valuespHthelevels transmission
light losses were in the
near of
range to1.5
0.5todB.
3 dB while for basic pH levels transmission near to 0.5 dB.
In order to identify the origin of signals found, the concentration of Prussian blue was increased
to 2 mM. As we can see in Figure 16B, the intensity of transmission peaks at 400, 500 and 700
nm was increased, suggesting that they are due to increased concentration of PB in the
composite. It also shows that the device sensitivity increased from 1.5 to 3 dB with 1mM
concentration of PB until 4 to 6 dB with 2 mM concentration of PB for acidic pH solutions, but
behaved same as previous in basic solutions. Finally there was a good return to initial
conditions after each change of interface (Figure 17).
Subsequently we performed a sensitivity analysis for pH changes by taking the APE/PB

modified fiber signal in air to use as a normalizing reference. As shown in Figure 18, there is
good sensitivity to pH values lower than 7 with18gains up to 6 dB at 400 nm (absorption or loss
peak), whereas above pH 7 the peak is inverted, turning in a gains peak which may be due to
7 Device MM/SM/MM (62.5/5/62.5) 5mm

PVOH/AP (50:50) (4%/1mM)
Transmission(dB)
5
4
1
2
3
3 4
5
7
8
9
2 10
11
Air
400 600 800 1000 1200 1400 1600

Wavelength(dB)
4.0 Device MM/SM/MM (62.5/5/62.5)10mm

PVOH/AP (50:50) (4%/1mM)
3.5
3.0
Transmission(dB)
2.5 1.6
1.87
2.01
2.45
2.0 2.6
3
4
5
1.5 9
11
12
12.5
1.0 13
1
Air
0.5 Water
400 600 800 1000 1200 1400 1600

Wavelength(nm)
Figure 15.Transmission spectra of hetero-core fiber with PVOH/AP and its sensitivity to pH change with
Figure 15. Transmission spectra of hetero-core fiber with PVOH/PB and its sensitivity to pH change with 5 mm (A) and
5 mm (A) and 10 mm (B) length.
10 mm (B) length.
the hydration process of the polymer and breaking of complex of PB by hydration. To identify
the changes in transmission spectrums, the most characteristic signals (400, 700 and 800 nm)
and the response to 1500 nm (common wavelength in telecommunication systems) were
plotted independently.
19
24 Soil Pollution
Device MM/SM/MM (50/5/50) 10mm

2 APE(50%):AP(1mM) - (1:1)
-2
Transmission(dB)
-4
-6
-8 Air
NaOH (0.1M)
Air
HCl (0.1M)
-10 Air
NaOH (0.1M)
Air
-12
400 600 800 1000 1200 1400 1600
Wavelength (nm)
Device MM/SM/MM (50/5/50) 10mm

APE(50%):AP(2mM) - (1:1)
0
-2
-4
Transmission(dB)
-6
-8
Air
-10 NaOH (0.1 M)
Air
-12 HCl (0.1M)
Air
NaOH (0.1M)
-14 Destilled water
Air
-16
400 600 800 1000 1200 1400 1600
Wavelength (nm)
Figure 16. Transmission spectra of hetero-core fiber with APE/AP and its sensitivity to pH change with
Figure 16. Transmission spectra of hetero-core fiber with APE/PB and its sensitivity to pH change with 1 mM (A) and 2
1 mM (A) and 2 mM (B) AP.
mM (B) PB.
20
Device MM/SM/MM (62.5/5/62.5) 10mm

APE(50%):AP(2mM) (1:1)
6
Transmission(dB)
1
2
4
6
4 7
9
10
11
2 12
0
400 600 800 1000 1200 1400 1600
Wavelength (nm)
Figure 17. Transmission spectrum normalized to air of the modified hetero-core device with APE/PB (2 mM) and its
sensibility to pH changes.
Analyzing the charts of Figure 18 shows that the transmission intervals are decreasing with
increasing wavelength, which demonstrates good sensitivity of the device. As previously
mentioned, the pH changes were more evident at pH values less than 7, due to that pH values
higher than 7, it promotes the process of hydration of Prussian blue complex (Equation 4) and
the signal grows weak on each pH change (García – Jareño et al, 1996).
4-
Fe 4 éë Fe ( CN )6 ùû + 12OH - Û 4Fe ( OH )3 + 3 éë Fe ( CN )6 ùû (4)
3
Based on the results obtained so far, the 10 mm rather than the 5 mm length hetero-core device
is recommended since their sensitivity and the evanescent wave field is bigger than 5 mm
length devices. Also the 10 mm length device provides a gain of about 2 dB at throughout the
analysis spectrum.
Actually, all devices showed in this paper are preparing to their application in real scenarios,
with the intention to quantify physicochemical properties directly to polluted soil without
extraction from the field and pre-treatment of sample, which could reduce time and costs of
analytical determination, increasing the sensibility, detection and quantification limits in
comparison with spectroscopic and spectrometric techniques, to take the best professional
decision to remediate in the better technical conditions the polluted soil.
26 Soil Pollution
8.5
9
@ 700 nm
@ 400 nm 8.0
8
7 7.5
Transmission (dB)
Transmission (dB)
6 7.0
5
6.5
4
6.0
3
5.5
2
1 5.0
0 4.5
0 2 4 6 8 10 12 0 2 4 6 8 10 12
pH pH
8.5 5.5
@ 800 nm
@ 1500 nm
8.0
5.0
7.5
Transmission (dB)
Transmission (dB)
4.5
7.0
6.5 4.0
6.0
3.5
5.5
3.0
5.0
0 2 4 6 8 10 12 0 2 4 6 8 10 12
pH pH
Figure
Figure 18. Transmission spectrum
18.Transmission spectrum of
of hetero-core
hetero-core device
devicemodified
modifiedwith
with APE/PB
APE/APand
anditsitssensibility
sensibilitytotopH
pHchanges at at
changes
400,
400,700,
700,800
800and
and1500
1500nmnmwavelength.
wavelength.
4. Conclusion
Analyzing the charts of Figure 18 shows that the transmission intervals are decreasing with increasing wavelength,
which demonstrates good sensitivity of the device. As previously mentioned, the pH changes were more evident at
pH values less than 7, due to that pH values higher than 7, it promotes the process of hydration of Prussian blue
complex (Equation 4) and the signal grows weak on each pH change (García – Jareño et al, 1996).
For pollution detection and soil remediation purposes it is essential to have relevant and
reliable information on the soil structure,- the hydrogeological circumstances and accumulation
Fe4[Fe(CN)6]3 + 12OH  4Fe(OH)3 + 3[Fe(CN)6]4- (4)
zones of the detected pollutants. Combined application of geological, hydrogeological and
geophysical investigations
Based on the results prior
obtained so the10
far, the placement
mm rather of thetheoptical
than fiber hetero-core
5mm length in the fielddevice
may isincrease the
recommended
efficiency of the monitoring
since their sensitivity technique.
and the evanescent wave field is bigger than 5 mm length devices. Also the 10 mm length
device provides a gain of about 2 dB at throughout the analysis spectrum.
Spatially
Actually, allresolved mapping
devices showed in thisof chemical
paper constituents
are preparing is an important
to their application need in
in real scenarios, witha the
variety of to
intention
environmental and geo-environmental
quantify physicochemical applications.
properties directly to polluted For extraction
soil without example,fromspatially
the fieldresolved analyte of
and pre-treatment
sample, which could reduce time and costs of analytical determination, increasing the sensibility, detection and
monitoring can simultaneously indicate and locate when an accepted level of exposure to toxic
quantification limits in comparison with spectroscopic and spectrometric techniques, to take the best professional
or explosive
decision species
to remediate hasbetter
in the beentechnical
exceeded, and the
conditions canpolluted
track its
soil.source.
The capability of long-range distributed sensing is unique to optical – fiber technology. A

distributed fiber optic sensor returns a value of a target measurement as a function of the linear
position along the fiber length. The only contact between the point to be measured and the
22
observation area is the optical fiber.
Acknowledgements
The authors would like to thank the Consejo Nacional de Ciencia y Tecnología de los Estados
Unidos Mexicanos (CONACyT), L’Oreal, Academia Mexicana de Ciencias (AMC) and
Fundación México – Estados Unidos para la Ciencia (FUMEC).J. A. García is grateful to
CONACyT for his scholarship.
Author details
J. A. García1, D. Monzón2, A. Martínez2, S. Pamukcu3, R. García4 and E. Bustos1*
1 Centro de Investigación y Desarrollo Tecnológico en Electroquímica S.C., Querétaro, Méxi‐

co
2 Centro de Investigaciones en Óptica A. C., León, México
3 Fritz Engineering Laboratory, Lehigh University, Bethlehem, USA
4 Laboratorio de Química Atmosférica, Centro de Ciencias de la Atmósfera, Universidad

Nacional Autónoma de México, Ciudad Universitaria, Coyoacán, Mexico
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chapter 16
Metabolomics for Soil Contamination Assessment
Maria C. Hernandez-Soriano and

Jose C. Jimenez-Lopez
1. Introduction
The evaluation of biological responses to assess and predict the impact of environmental
changes in ecosystems functioning is receiving increasing attention, and current research
focuses on overcoming concerns about the specificity of biomarkers (Amiard-Triquet et al.,
2012). Generally, biomarkers are chemicals, metabolites, susceptibility characteristics, or
physiological changes that relate to the exposure of an organism to a chemical. Accordingly,
a selected biomarker, i.e. biological response, can be linked to a specific environmental
exposure, being representative of the health status of the ecosystem studied. The identification
of biomarker profiles has been possible upon the development of metabolomics. Those profiles
allow the genuine identification of the relevant biological response/s associated to a particular
exposure while the assessment of a single biomarker could only estimate the potential response
of the ecosystem to a particular pollutant.
Metabolomics is the generic name assigned to a scientific field that addresses the characteri‐
zation of low molecular weight organic metabolites released by living organisms in response
to environmental stimuli. Morrison et al. (2007) provided an extended definition ‘‘the appli‐
cation of metabolomics to the investigation of both free-living organisms obtained directly
from the natural environment (whether studied in that environment or transferred to a
laboratory for further experimentation) and of organisms reared under laboratory conditions
(whether studied in the laboratory or transferred to the environment for further experimen‐
tation), where any laboratory experiments specifically serve to mimic scenarios encountered
in the natural environment’’.
The methodological approach of metabolomics relies on a comprehensive analysis of the set
of metabolites or “metabolome” produced in response to particular environmental stimuli.
Accordingly, the metabolome is the pool of metabolites, small molecules, within a cell, tissue,
organ, biological fluid, or entire organism (Miller, 2007). Exposure of an organism to an

external stressor will result in changes at the level of the metabolome (Ankley et al., 2006; van
Ravenzwaay et al., 2007), and such changes may constitute a highly sensitive indicator of an
external stress. Therefore, metabolomics has potential as a sensitive and rapid technique that
can elucidate the relationships between metabolite levels and an external stressor, such as
contaminant exposure, nutritional deficit or a disease.
The main advantage of metabolomics over traditional research is to overcome the bias
associated to the assessment of predefined metabolites (Singh, 2006). Among the diverse
applications of omic profiling methods to the environmental sciences, ecotoxigenomics
addresses the response of organisms to pollutants based on the different sensitivity of species
to toxicants (Spurgeon et al., 2008). Currently, the implementation of metabolomics in
ecological risk assessment is still at an early stage, mostly applied as a screening tool to assess
the potential toxic effects of pollutants or to determine the mode of action (MOA) of a toxicant.
Otherwise, application of metabolomics for environmental monitoring allows the study of a
large variety of species from relatively uncontrolled environments.
Bundy et al. (2009) highlight the challenge of identifying a large number of metabolites and
the necessity of creating metabolite databases specifically dedicated to environmental issues.
Multivariate statistical analysis has proved highly effective for metabolite identification.
Thus, principal component analysis (PCA) is used to identify differences between metabol‐
ic profiles of organisms exposed to organic or inorganic pollutants (Jones et al., 2014; Kwon
et al., 2012; Lankadurai et al., 2011). Besides, association between the metabolic profile and
biological factors evaluated as markers for exposure to pollutants can be modelled by partial
least squares (PLS) regression analysis (Ellis et al., 2012).
The implementation of metabolomics for the assessment of soil contamination is neverthe‐

less at an early stage (Viant, 2009). A basic screening of published research in the web of
science returns circa 100 items for the search “soil pollution-metabolomics”, with a
significant launch in 2011 (Figure 1), reduced to 21 records when the search is narrowed
with the term “biomarkers”, published in the period 2007-2013. However, emerging
regulatory challenges demand the advance of toxicity testing. Toxicogenomics tools have
been presented as an advanced from the current methodologies used for regulatory decision
making in ecotoxicology, which entirely rely on whole animal exposures and adverse effects
on survival, growth, and reproduction (Ankley et al., 2006). From the acquisition of
reproducible metabolic profiles as response to the presence of specific pollutants in soil
(Jones et al., 2008) to the application of metabolomics techniques to the study of the response
of the entire community of a soil to factors such as pollution and climate change (Jones et
al., 2014), the implementation of metabolomics in ecotoxicology is a sound answer to the
current needs of society and the environment (van Ravenzwaay et al., 2012).
During the last decade a number of general revisions about the application of metabolo‐
mics in environmental health assessment have been published (Bundy et al., 2009; Miller,
2007; Snape et al., 2004; van Ravenzwaay et al., 2007; Viant et al., 2003). The present review
specifically summarizes the most significant research concerning implementation of
advanced from the current methodologies used for regulatory decision making in ecotoxicology, which entirely
rely on whole animal exposures and adverse effects on survival, growth, and reproduction (Ankley et al., 2006).
From the acquisition of reproducible metabolic profiles as response to the presence of specific pollutants in soil
(Jones et al., 2008) to the application of metabolomics techniques to the study of the response of the entire
Metabolomics
community of a soil to factors such as pollution and climate change for2014),
(Jones et al., Soil Contamination Assessment
the implementation of 3
metabolomics in ecotoxicology is a sound answer to the current needs of society and the environment (van
Ravenzwaay et al., 2012).
Published items in each year Citations in each year
Figure 1. Citation report for the search “soil pollution-metabolomics” as obtained from Thomson Reuters (January 2014).
Figure 1. Citation report for the search “soil pollution-metabolomics” as obtained from Thomson Reuters (January
2014).
During the last decade a number of general revisions about the application of metabolomics in environmental
health assessment have been published (Bundy et al., 2009; Miller, 2007; Snape et al., 2004; van Ravenzwaay et
al., 2007; Viant et al., 2003). The present review specifically summarizes the most significant research concerning
metabolomics
implementationinofsoil contamination
metabolomics assessment.
in soil contamination The main
assessment. The mainobjectives ofthis
objectives of this revision
revision are i) are
to i)
to provide a systematized
provide a systematized outline outline for the ofapplication
for the application metabolomicsof metabolomics
in risk in contamination,
assessment of soil risk assessmentii) to of
soilprovide
contamination, ii) to provide a rapid guide to the methodological approaches current‐
a rapid guide to the methodological approaches currently optimized and iii) to unify and simplify the
knowledge currently available in the topic to provide an accessible tool for further advance in the
ly optimized
implementation and iii) to unify
of metabolomics andassessment.
in risk simplify the knowledge currently available in the topic
to provide an accessible tool for further advance in the implementation of metabolomics in
risk2.assessment.
Methodological approaches
2.1. Metabolites isolation
2. Methodological
Generally, metabolites are approaches
extracted from intact organisms (occasionally from selected relevant tissues) that have
been exposed to the studied toxicant by moderate chemical extraction (Baylay et al., 2012; Yuk et al., 2010). The
organisms commonly selected for toxicity testing are earthworms (Table 1), particularly the genus Eisenia, which
2.1. are
Metabolites isolation
a classic model organism for toxicity assays (Sanchez-Hernandez, 2006; Van Gestel et al., 1992; van Gestel et
al., 1989) and have been since long included in official guidelines (OECD, 1984, 2004). Earthworms ingest large
Generally, metabolites are extracted from intact organisms (occasionally from selected relevant
amounts of soil and uptake a significant amount of contaminant through the skin. Therefore they are
continuously exposed to contaminants. Extractions performed with methanol-chloroform (Baylay et al., 2012) or
tissues) that have been exposed to the studied toxicant by moderate chemical extraction
(Baylay et al., 2012; Yuk et al., 2010). The organisms commonly selected for toxicity testing are
earthworms (Table 1), particularly the genus Eisenia, which are a classic model organism for
toxicity assays (Sanchez-Hernandez, 2006; Van Gestel et al., 1992; van Gestel et al., 1989) and
have been since long included in official guidelines (OECD, 1984, 2004). Earthworms ingest
large amounts of soil and uptake a significant amount of contaminant through the skin.
Therefore they are continuously exposed to contaminants. Extractions performed with
methanol-chloroform (Baylay et al., 2012) or phosphate buffer solution (Yuk et al., 2010) on
pulverized or lyophilised organisms are described to extract the maximum number of
metabolites while allowing the performance of reliable analyses.
The isolated extracts usually might not require further sample treatment prior to analysis,
which minimizes the introduction of artefacts but also facilitates the development of low cost,
rapid methodologies.
2.2. Metabolites determination: chromatography, spectroscopy and spectrometry
The leading analytical techniques in metabolomics for soil contamination assessment are
proton nuclear magnetic resonance spectroscopy (1H NMR) and gas chromatography–mass
spectrometry (GC-MS), as thoroughly reported in Table 1, both allowing the identification of
compounds at molecular level in the analysed substances. Several authors have also imple‐
mented high pressure liquid chromatography (HPLC) and ultra high pressure liquid chro‐
matography (UPLC) coupled with mass spectrometry detector (MS) for the assessment of
biological responses to soil contamination with heavy metals (Hédiji et al., 2010; Hughes et al.,
2009). Overall, these analytical techniques allow the determination and identification of the
metabolites that foremost represent the metabolic alterations related to the toxic effects of
organic or inorganic contaminants in soil.
Animal model Biomarkers of

Technique Organic toxicant Reference
tested contaminant exposure
D
[2-hexyl-5-ethyl-3-
1
H NMR
2-fluro-4-methylaniline E. veneta furansulfonate, maltose], (Bundy et al., 2002)
GC/MS
I
inosine monophosphate
1
H NMR
3-trifluromethyaniline E. veneta I
lactate (Lenz et al., 2005)
GC/MS
1
H NMR I
[Ala, Gly, Asn, glucose,
3-trifluromethylaniline E. veneta (Warne et al., 2000)
GC/MS citrate, succinate]
D
[acetate, malonate],
1
H NMR
3-fluro-4-nitrophenol E. veneta I
[succinate, trimethylamine- (Bundy et al., 2001)
GC/MS
N-oxide]
D
2-hexyl-5-ethyl-3-
1
H NMR
3,5-difluroaniline E. veneta furansulfonate, Iinosine (Bundy et al., 2002)
GC/MS
monophosphate
1
H NMR D
[Maltose, hexyl-5-ethyl-3-
4-fluroaniline E. veneta (Bundy et al., 2002)
GC/MS furansulfonate]
1
H NMR Aroclor 1254 E. fetida No significant changes (Fitzpatrick et al., 1992)
1
H NMR Caffeine E. fetida I
fumarate (McKelvie et al., 2011)
1
H NMR Carbamazephine E. fetida D
[Fumarate, Glu, Val, DLeu] McKelvie et al. (2011)
(Heimbach, 1988)
1
H NMR Carbaryl E. fetida D
[Phe, Tyr, Lys, Ala, Val, Leu] (Edwards and Bater,
1992)
1
H NMR Chlorpyrifos E. fetida No significant changes (Yu et al., 2006)
1
H NMR Chlorpyrifos L. rubellus I
fumarate (Baylay et al., 2012)
Metabolomics for Soil Contamination Assessment 5

1
H NMR I
[Ala, betaine, ornithine],
spectroscopy, Chlorpyrifos C. elegans D
[choline, Glu, Gly, Ile, (Baylay et al., 2012)
GC–MS lactate, n-buterate, taurine]
D
[Phenylalanine, Alanine, (Drewes and Vining,
1
H NMR Dimethyl phthalate E. fetida
Leucine, Valine] 1984)
1
H NMR
DTT and Endosulfan E. fetida I
[maltose, Ala, Leu] (McKelvie et al., 2009)
GC/MS
Significant fluctuations in
1-D & 2-D 1H
glutamine/GABAeglutamate
NMR Endosulfan E. fetida (Yuk et al., 2013)
cycle metabolites and
spectroscopy
spermidine
Significant fluctuations in
1-D & 2-D 1H
glutamine/GABAeglutamate
NMR Endosulfan Sulfate E. fetida (Yuk et al., 2013)
cycle metabolites and
spectroscopy
spermidine
1
H NMR Estrone E. fetida D
[Adenine, Glu] McKelvie et al. (2011)
D
Toxicological endpoints
1
H NMR Imidacloprid/
L. rubellus (survival, weight loss, and Baylay et al. (2012)
GC-MS analysis Thiacloprid
reproduction)
1
H NMR Naphthalene E. fetida D
[Ala, Leu, Val, Lys] (Brown et al., 2009)
1
H NMR Nonylphenol E. fetida D
[adenine, Glu] McKelvie et al 2011
Polybrominated
1
H NMR diphenyl ethers (PBDE) E. fetida D
Maltose, I[Lys, Glu] McKelvie et al. (2011)
209
I
[succinate, HEFS, Glu], D[leu,
1
H NMR Perfluorooctanoic acid E. fetida Val, LyS, Phe, Arg, maltose, (Lankadurai et al., 2012)
ATP]
I
[succinate, HEFS, Glu], D[leu,
Perfluorooctane
1
H NMR E. fetida Val, Lys, Phe, Arg, maltose, (Lankadurai et al., 2012)
sulfonate
ATP]
Leu, Val, Ala, Lys and maltose

1
H NMR PHA E. fetida changed in response to PAH (Brown et al., 2009)
exposure.
I
[Ala, betaine, Scyllo- and
(Lankadurai et al., 2011)
H NMR
1
Phenanthrene E. fetida myo-inositol, cholesterol,
Brown et al. (2010)
phosphatidylcholine], DGlu

McKelvie et al. (2011)

Polychlorinated
1
H NMR E. fetida I
ATP (Whitfield Åslund et al.,
biphenyl
2011)
Poly brominated
1
H NMR E. fetida I
[lys, Glu], Dmaltose McKelvie et al. (2011)
diphenyl ethers
D
[lactate, tetradecanoic acid,
1
H NMR hexadecanoic acid,
Pyrene L. rubellus (Jones et al., 2008)
GC/MS octadecanoic acid], I[Ala, Leu,
Val, Ile, Lys, Tyr, methionine]
1-D & 2-D 1H

NMR Rifluralin E. fetida I
[Ala, Gly, maltose, ATP] Yuk et al. (2011)
spectroscopy
No significant changes were

1
H NMR Thiacloprid L. rubellus Baylay et al. (2012)
reported
1-D & 2-D 1H

NMR Trifluralin E. fetida I
[Ala, Gly, ATP], Dmaltose Yuk et al. (2011)
spectroscopy
1
H NMR
I
phytochelatins,
spectroscopy andCd C. elegans (Hughes et al., 2009)
D
cystathionine
UPLC-MS
I
choline, D[glucose, citrate,
1
H NMR, HPLC- malate, glutamine,
Cd S. lycopersicum (Hédiji et al., 2010)
PDA asparagine, Phe, Tyr, Val, Ile,
trigonelline]
I
[Phe, glucose, malate,
arachidonic acid, fumarate,
Lys, Tyr, monosaccharides,
1
H NMR, GC-MS monophosphorylated form
Chlorpyrifos + Ni L. rubellus (Baylay et al., 2012)
analysis of inositol, succinate, uracil,
ethanolamine, Pro,
putrescine],
D
myo-inositol,
1
H NMR I
[Ala, creatine, His, lactate,
spectroscopy, Chlorpyrifos + Ni C. elegans betaine, carnosine], (Jones et al., 2012)
GC–MS D
[choline, Gly, Ile, leu, lys, Val]
1
H NMR Cu (II) L. rubellus I
His (Gibb et al., 1997)

1
H NMR
I
[Asn, choline, Gln, lactate,
spectroscopy, Ni C. elegans Jones et al. (2012)
succinate], D[Ala, Ile, Gly, Val]
GC–MS
i) Exposure to Pb - DTyr.
ii) Exposure to Zn - I[Val, Ile,
Leu, Thr, Ala, Asn, Phe,
fosfocoline], D[acetate, Gly,
glucose, fumarate and
1
H NMR Pb, Zn S. salsa ferulate]. (Wu et al., 2013)
iii) Exposure to Pb & Zn -
I
[Ala, Asn, Tyr, Phe], D[acetate,
succinate, Asp, malonate,
fructose, glucose, fumarate
and ferulate]
Mixed metals
1
H NMR (Cadmium, copper, lead L. rubellus I
[maltose, His] (Bundy et al., 2004)
and zinc)
I
[Thr, Leu, Tyr, betaine, Ser,
Lys, Ile, Ala, Arg, Val,
1
H NMR Tellurite P. pseudoalcaligenes (Tremaroli et al., 2009)
glutathione, adenosine],
D
[Lactate, Gly]
I
[Phe, Tyr, Lys, Glu, Ala, (Whitfield Aslund et al.,
H NMR
1
Titanium dioxide E. fetida
lactate, Val, Leu], Dmaltose 2012)
D = decrease; I = increase
Table 1 Metabolic responses of test organism following exposure to selected environmental contaminants in contact
tests.
2.3. Metabolomics data analysis

The general approach to data analysis in metabolomics can be summarized in three main
stages: explorative, supervised and biological interpretation (Smilde et al., 2010). The explor‐
ative phase aims to find groups, clusters and outliers in metabolites and samples studied while
the supervised discriminates two or more groups to make predictive models and to find
biomarkers (Amiard-Triquet et al., 2012; Dallinger-Marianne, 2000; van Ravenzwaay et al.,
2007). Multivariate methods are currently preferred, although univariate and semi-univariate
methods have been commonly used for selecting biomarkers. For instance, the lysosomal
system was identified as a particular target for the toxic effects of pollutants in soil organisms.
However, it is nonspecific as a marker and only included in a suite of biomarkers among
diverse soil invertebrate species can provide the necessary specificity for risk assessment
purposes (Kammenga et al., 2000). Finally, the biological interpretation seeks the links between
metabolome data and underlying metabolic networks through metabolite set enrichment,
pathway analysis and metabolic network inference (Trygg et al., 2006). Thus, finding metab‐
olite relationships is essential to determine comprehensive and meaningful metabolic changes
as biological response to environmental stimuli (Ellis et al., 2012; Morrison et al., 2007).
Accordingly, such extensive evaluation of the impact of pollutants in the metabolism of target
organisms is the approach that can add value to the assessment of soil health and viability of
soil organisms undergoing stress from pollution.
3. Metabolomics bioinformatics
Information processing by bioinformatics tools and computational biology methods has

become essential for solving complex biological problems in genomics, proteomics, and
metabolomics. Understanding “omics” data requires both common statistical and computa‐
tional based methods due to the multi-dimensional and complexity level of the data.
Data-analytical methods for the study of biological systems as developed in the field of
computational biology provide a suit of indispensable tools to survey the outcome of metab‐
olomics studies. First, computational biology allows a fast screening of the large biological and
chemical data sets generated (Shulaev, 2006), and therefore the identification of the most
relevant metabolites, i.e. compounds specifically representative of the metabolic changes in
the model system following exposure to different concentrations of organic and inorganic
toxicants. As a result of the large number of variables (metabolites) studied, metabolomics
studies encompass a significant statistical power for the systematic detection of biological
responses to environmental changes (van Ravenzwaay et al., 2012). Second, the mathematical
models developed in computational biology allow the identification of relationships between
the external stimuli and the metabolic response (Zhang et al., 2010). Third, the implementation
of computational algorithms to structural biology makes possible to discover the structure-
function of new macromolecular compounds, the functional enzymatic conversion and
changes in their activity, as well as their molecular interaction and relationship with others
compounds in the pathways where they are involved (Jimenez-Lopez et al., 2013). Moreover,
it is possible to detect patterns in such biological responses and establish significant dose-
response relationships. Besides, pattern recognition reduces the metabolomics data from
hundreds of variables to two or three components that are orthogonal to each other. Overall,
this advance of computational biology has been possible due to three significant technological
breakthroughs: high-information-content data streams, novel bio-statistical methods, and the
computational power to analyse these data.
Data processing and statistical analyses are commonly performed using multivariate (typically
a principal component analysis (PCA) and (or) partial least squares (PLS) regression analysis)
and univariate (t-test) analyses (Brown et al., 2010; Jones et al., 2014; McKelvie et al., 2011; Yuk
et al., 2013). These analyses are performed in combination with the quantification and
identification of the metabolites. Subsequently, biological interpretation of the data is neces‐
sary for understanding the link between the external stimulus and the metabolic response of
the organisms.
Principal component analysis is the most widely used multivariate statistical approach in
metabolomics, used to explain the overall variability in a data set via a a set of uncorrelated
variables called principal components (PCs), which are linear combinations of the original
variables (Trygg et al., 2006). The organization of samples in PCA scores plots is based on the
similarities between their metabolic profiles. Thus, PCA allows for dimensional reduction of
the data into a low dimensional plane, such as PC1 versus PC2. The scores plot (e.g., PC1 versus
PC2) allows for a visual examination of the relationship between the samples based on their
metabolic profiles. In a 1-D PCA loadings plot, the contribution (or weight) of each metabolite
to the discrimination of the sample classes along one component is represented by the intensity
of the metabolite peak. In the 2-D PCA loadings plot discrimination is performed by selecting
the points that are scattered further away from the tight cluster of points found near the origin.
Other widely used multivariate statistical tools in metabolomics are PLS regression analysis
and PLS discriminant analysis (PLS-DA). Both PLS-regression and PLS-DA are methods for
samples classification, with pre-defined variables added to maximize the separation between
the sample classes and to construct predictive models. The predefined variables for PLS-
regression are measurable quantities such as the contaminant exposure concentration.
Validation methods such as the leave-one-out cross validation are used to test the robustness
of the models generated by PLS-regression, PLS-DA, OPLS, and OPLS-DA (Whitfield Åslund
et al., 2011).
Although metabolomics studies mostly use multivariate statistics, univariate statistical
analyses can contribute to the information gained from a study. Thus, t-tests can be used to
assess the significance of the separation between the controls and stressed organisms in PCA
and PLS-DA scores plots. Also, t tests can be used to determine which metabolites in the 1H
NMR spectra of the treatment class increased or decreased significantly relative to the controls.
4. Biomarkers
The somewhat secondary significance of biological responses for soil contamination assess‐
ment was customarily associated to the limitation of biomarkers as measurable responses to
contaminants, which classically could only provide an indication of exposure to contaminants
in soil (Sanchez-Hernandez, 2006). The development of metabolomics, considered an “emerg‐
ing field” as late as mid-2010, has provided the tools for the determination of multiple
biomarkers across different levels of biological organization, and therefore a better assessment
of the ecological consequences of contamination. Since the creation of the first metabolomics
web database, METLIN (Smith et al., 2005), 60,000 metabolites has been incorporated, a rapid
development closely related to the evolution of mass spectrometry instrumentation and data
analysis tools. Currently, the number of databases and metabolites registered is continuously
increasing. Table 2 summarizes some of the most relevant databases operative and the
corresponding website is also indicated. Further information on metabolomics databases can
be obtained from the metabolomics society (http://www.metabolomicssociety.org). For

instance, ChemSpider is an aggregated database of organic molecules containing more than
20 million compounds from many different providers. At present the database contains
information from such diverse sources as a marine natural products database, ACD-Labs
chemical databases, the EPA's DSSTox databases and from a series of chemical vendors. It has
extensive search utilities and most compounds have a large number of calculated physico-
chemical property values.
One of the goals in bioinformatics is to establish automated and efficient ways to integrate
large, biological datasets from multiple sources. This objective is challenging because data
sources are heterogeneous in terms of their functions, structures, data access methods and
dissemination formats. In addition, the enormous quantity of information produced by
“omics” is handled via computers that systematically analyze and store the accumulating
sequence, structure and function data. Databases are essential in metabolomics because they
provide a rapid and specific tool to identify the compounds isolated from an organism exposed
to a particular environmental challenge. Thus, the KNApSAcK package provides tool for
analysing datasets of mass spectra as well as for retrieving information on metabolites by
entering the name of a metabolite, the name of an organism, molecular weight or molecular
formula. A list of metabolites that are associated to a taxonomic class can be obtained by search
with the taxonomic name, from which information of individual metabolites can be retrieved.
The NIST Chemistry WebBook provides access to chemical and physical property data for
chemical species. The data provided in the site are from collections maintained by the NIST
Standard Reference Data Program and outside contributors. Data in the NIST Chemistry
WebBook can be found by direct searches for chemical species or indirect searches based on
related data. Specific databases are also being developed, such as LIPID MAPS, currently the
largest database of lipid molecular structures. Otherwise, SetupX combines mass spectrometric
and biological metadata, which is a step forward in the organization of information generated
by metabolomics analysis.
METLIN http://metlin.scripps.edu/index.php
LIPID MAPS http://www.lipidmaps.org/
KEGG http://www.genome.jp/kegg/pathway.html
ChemSpider http://www.chemspider.com/
SetupX http://fiehnlab.ucdavis.edu/projects/binbase_setupx
KNApSAcK http://kanaya.naist.jp/KNApSAcK/
NIST http://webbook.nist.gov/chemistry/
MassBank http://www.massbank.jp/
HMP http://www.hmdb.ca/
IIMDB http://metabolomics.pharm.uconn.edu/iimdb/
Table 2 Selected metabolomic databases.

Metabolomic databases are thus accompanied by accurate description of the biological study
design and accompanying metadata reporting on the laboratory workflow from sample
preparation to data processing.
Currently, standard analyses focus on the determination of amino acids, mono- and disac‐
charides, lipids/fatty acids, short chain fatty acids and small phenolics. Accordingly, it is
possible to already launch the standardization of metabolomics analysis. For instance, the
Northwest Metabolomics Research Center (University of Washington) has established a
relevant list of target compounds to evaluate biological responses to changes in the environ‐
ment. The list of compounds is summarized in Table 3.
Metabolic Pathways Number of Metabolites

Alanine, aspartate and glutamate metabolism 15
Arginine and proline metabolism 23
Butanoate metabolism 18
Citrate cycle (TCA cycle) 11
Cysteine and methionine metabolism 14
Fatty acid metabolism 3
Glutathione metabolism 14
Glycine, serine and threonine metabolism 21
Glycolysis / Gluconeogenesis 16
Histidine metabolism 13
Lysine biosynthesis 7
Lysine degradation 6
Nitrogen metabolism 9
Oxidative phosphorylation 6
Pentose phosphate pathway 10
Phenylalanine metabolism 10
Phenylalanine, tyrosine and tryptophan biosynthesis 8
Purine metabolism 30
Pyrimidine metabolism 30
Pyruvate metabolism 10
Synthesis and degradation of ketone bodies 4
Tryptophan metabolism 15
Tyrosine metabolism 18
Valine, leucine and isoleucine biosynthesis 11
Valine, leucine and isoleucine degradation 5
Table 3 Summary of metabolites and metabolic pathways representative of biological responses to environmental
stimuli.
The information of metabolites and metabolic pathways has been obtained from the website
of Kyoto Encyclopedia of Genes and Genomes (Kegg, http://www.genome.jp/kegg/). Accord‐
ing to the research results summarized in Table 1, the implementation of metabolomics in the
assessment of soil contamination indicates that contaminants in soil affect several of the major
metabolic pathways in living organisms (Table 3), including glycolysis, trycarboxylic acids
cycle and amino acids metabolism. Moreover, data analysis indicates an overall reduction in
the production of the associated metabolites. For instance, the interference in amino acids
specialized pathways results in a decreased synthesis of purine and pyrimidine nucleotides
(Brown et al., 2010; McKelvie et al., 2011). These nucleotides are essential for the production
of the energy (ATP molecules) that drive most of the enzymatic reactions in living organisms,
but also protein synthesis is consequently hampered, which explain the negative effect in
processes such as antioxidant activity.
Another emerging group of biomarkers, as highlighted in several studies, are lipids (Rochfort
et al., 2009; Sanchez-Hernandez, 2006). Rochfort et al., (2009) indicate that lipophilic extracts
can be used in field based metabolomics experiments to investigate different treatment effects
on earthworms. Lipid metabolism is highly sensitive to environmental contaminants (Vega-
López et al., 2013), with increasing production of lipoprotein vesicle and lipid peroxidation
rate during early stages of the biological response to the presence of a toxicant (Lankadurai et
al., 2011). Relatedly, earthworm esterases has been proposed as biomarkers for pesticide
contamination in soil (Sanchez-Hernandez, 2010). Esterases are directly involved in the natural
tolerance of earthworms to pesticides, and can therefore be used as specific biomarkers, but
furthermore, their characterization by metabolomics approach might help to select the
appropriate earthworm species for regulatory toxicity testing. Overall, the increasing specif‐
icity of the research performed in ecotoxigenomics will allow a realistic and meaningful
incorporation of biological responses in ecological risk assessment.
5. Oxidative stress in contaminated soil
The induction of the oxidative stress response by the presence of toxic compounds in the
environment is a primary mechanisms of defence, although prolonged exposure to contami‐
nants is likely to overwhelm this short-term defence (Regoli et al., 2002).
Metabolites such as proline possibly detoxify the ROS under stress in vivo (Smirnoff, 1993).
Exposure of plants to both redox active, for example, Cu and Hg, and other metals, for example,
Cd and Zn, induces the generation of free radicals that leads to oxidative stress. This represents
one of the major causes of toxicity particularly due to redox metals. The cells are equipped
with an elaborate network of antioxidative enzymes and low molecular weight metabolites
which mitigate the oxidative stress. Proline scavenges different free radicals in certain in vitro
generation and detection systems.
Proline quenches ROS and reactive nitrogen species (RNS), which relieves the oxidative
burden from the glutathione system. Moreover, polyamines also have an antioxidative role by
quenching the accumulation of O2.- probably through inhibition of NADPH oxidase (Pascha‐
lidis and Roubelakis-Angelakis, 2005). This may facilitate phytochelatin synthesis and enhance
metal tolerance (Siripornadulsil et al., 2002).
Overall, oxidative defence response to toxicity or other environmental stress involves the
generation of oxygenated metabolites from exposed organisms and activation/inhibition of the
production of antioxidants enzymes and metabolites such as glutathione. The depletion of
antioxidants for prolonged exposures might result in the decrease of the response effectiveness
and eventual imbalance between generation and elimination of reactive oxygen species.
Depletion of glutathione appears to be a major mechanism in short-term heavy metal toxicity
(Schutzendubel and Polle, 2002). In accordance with this hypothesis, a good correlation
between glutathione contents and tolerance index was observed with 10 pea genotypes
differing in Cd sensitivity (Metwally et al., 2005). High GSH concentrations in hyperaccumu‐
lator T. Goesingense coincided with high constitutive activity of serine acetyl transferase (SAT);
SAT catalyses the acetylation of L-Ser to OAS which in turn provides the carbon skeleton for
Cys biosynthesis. Elevated GSH levels in T. Goesingense also coincided with the ability both to
hyperaccumulate Ni and to resist its damaging oxidation effects.
The significance of glutathione and the metal-induced phytochelatins (PCs) in heavy metal
tolerance has been studied intensely (Rauser, 1995). However, PCs are important for detoxi‐
fication of only a limited set of metals such as Cd2+, Cu2+ and AsO22- while Zn2+ and Ni2+ are
poor inducers of PCs and exhibit low binding affinity. Most other metals lack significant
binding.
Evaluation of metabolites related to oxidative response constitutes a relevant group of target

compounds for risk assessment. Although oxidative response to soil contamination has been
classically addressed in plants, the study of this response in soil microorganisms is already
being introduced in ecotoxicology as a fundamental part of the biological response of soil
microorganisms to soil contamination (Boer et al., 2013; Tremaroli et al., 2009). Accordingly,
Boer et al. (2013) describe the attenuation of the oxidative response for springtails in laboratory
tests, which constitutes and early detection of soil pollution, and standardized test have been
developed.
6. Metabolites related to soil contamination with organic compounds
The importance of the identification of biomarkers and metabolic pathways specifically related
to soils contamination with a particular pollutant or group of pollutants has been already
highlighted through this chapter. From the information summarized in Table 1 and Table 3 it
is possible to infer that soil contamination with organic compounds, namely pesticides o
polycyclic aromatic hydrocarbons, abates essential metabolic pathways such as the trycarbox‐
ylic acid cycle and the oxidative stress response, while lipid metabolism appears to be
enhanced. However, the advance in the application of bioinformatics is providing further
progress in terms of identification of specific biomarkers for risk assessment of individual
target compounds. Thus, toxicity of endosulfan has been directly related with alterations of
the GABA-glutamine cycle (Yuk et al., 2013), while chlorpyrifos depresses the Cori cycle and
reduces the production of phospholipids, as indicated by lower levels of choline (Jones et al.,
2012). Baylay et al. (2012) specifically relates chlorpyrifos toxicity to increased levels of
fumarate, an intermediate of the trycarboxylic acid cycle. Research conducted with the same
earthworm (E. fetida) and other families of organic compounds revealed a different metabolic
response (Brown et al., 2010; Lankadurai et al., 2012), confirming the capability of metabolo‐
mics to discriminate the metabolic pathways involved in the response to a particular toxic
compound. Moreover, the results strongly suggest that sets of biomarkers might be soon
sufficiently reliable as for their implantation in in toxicity standardized test.
The relevance of these and future studies on the development of risk assessment strategies is
aggravated by the inherent risk of soil contamination for human health. Soil contaminants may
be responsible for health effects costing millions of euros. Health problems range from cancer
(arsenic, asbestos, dioxins), to neurological damage and lower IQ (lead, arsenic), kidney
disease (lead, mercury, cadmium), and skeletal and bone diseases (lead, fluoride, cadmium).
Overall, few studies have been conducted on the toxicity of complex chemical mixtures in soils.
The effects of the soil and organisms within it upon organic pollutants are unknown. The data
currently available correspond mostly to short-term studies and high level exposure of these
chemicals, which is less relevant to the potential low-level, long term health impacts on living
organisms near to contaminated soil.
7. Metabolites related to soil contamination with heavy metals
The uptake of excess metal ions is toxic to most organisms, and the biochemical impact of metal
ions on the cells varies with the chemistry of the element as their chemical nature. In plants,
phytotoxicity of heavy metals in most parts can be attributed to symplastic accumulation of
heavy metals, such as the cytosol and chloroplast stroma. Metal-induced changes in develop‐
ment are the result of either a direct and immediate impairment of metabolism or signaling
processes that initiate adaptive or toxicity responses that need to be considered as active
processes of the organism. Transport processes have been recognized as a central mechanism
of metal detoxification and tolerance (Hall, 2002; Hall and Williams, 2003).
Some metals, for example, Zn and Cu, are essential for normal plant growth and development
as they serve as structural and functional components of specific proteins. Other metals, for
example, Cd and Pb, have no known function in plants although a Cd requirement for carbonic
anhydrase from marine diatoms has been reported (Lane and Morel, 2000).
Upon exposure to metals, organisms often synthesize a set of diverse metabolites that accu‐
mulate to concentrations in the millimolar range, particularly specific amino acids, such as
proline and histidine, peptides such as glutathione and phytochelatins (PC), and the amines
spermine, spermidine, putrescine, nicotianamine, and mugineic acids that can be detected as
response to these metals exposure. The advance of toxicogenomics in relation to organic
contaminants is significantly ahead of the equivalent research in metal contaminated soil
(Table 1). Nevertheless, research conducted up to date has yielded a number of biomarkers
representative of the biological response of soil microorganisms to metals toxicity. Thus, soil
contamination with Pb has been related with an enhancement of lipid metabolism (Sanchez-
Hernandez, 2006) and more directly with reduction of tyrosine levels (Wu et al., 2013).
Otherwise, Cd toxicity promotes the secretion of phytochelatins in C. elegans, likely at the

expenses of the sulphur metabolism, as suggested by the reduction in cystathionine (Hughes
et al., 2009), while the response of tomato plants to Cd involves several biochemical pathways
(Hédiji et al., 2010). These examples illustrate the genuine specificity of biological reactions to
different metals but also the variation in representative biomarkers among different organ‐
isms. Accordingly, exposure of C. elegans to Ni (Jones et al., 2012) yields a different metabolome
than Cd since different biochemical pathways are affected.
In plants, data currently available demonstrate the significance of nitrogen-containing
metabolites beyond phytochelatins and glutathione in plant response and acclimation to heavy
metals. The various metal ions have specific chemical properties and induce distinct responses
of adaptation and damage development. Thus, accumulating N-metabolites display a variety
of functions, i.e. metal ion chelation, antioxidant defence, protection of macromolecules, and
possibly signalling.
Proline is an extensively studied molecule in the context of plant responses to abiotic stresses.
Up-regulation of proline is often encountered in plants under heavy metal stress, comparable
to what occur under other abiotic stresses. When compared at equal toxic strength, proline
accumulation decreased in the order Cd > Zn > Cu (Schat et al., 1997). In addition, it has been
suggested different functions of proline under metal-stress, being involved in osmoregulation,
metal chelation, antioxidant, and regulator of specific functions in plant morphogenesis.
Furthermore, Ni-hyperaccumulation has been specifically linked to histidine production
(Krämer, 2005), particularly for Saccharomyces cerevisiae (Pearce and Sherman, 1999). The
beneficial role of high histidine levels has been shown in transgenic Arabidopsis thaliana which
accumulated about 2-fold higher histidine levels than wild- type plants and showed more than
10-fold increased biomass production in the presence of toxic Ni in the growth medium
(Wycisk et al., 2004).. Moreover, cell surface-engineered yeast displaying a histidine oligo‐
peptide (hexa-His) has been shown to adsorb 3–8 times more copper ions than the parent strain,
being more resistant to Cu than the parent (Kuroda et al., 2002).
Otherwise, polyamine contents are altered in response to the exposure to heavy metals.
Weinstein et al. (1986) showed an increment in putrescine content in Cd-treated oat seedlings
and detached oat leaves with a marginal rise in spermidine and spermine content. They
influence a variety of growth and development processes in plants and have been suggested
to be a class of plant growth regulators and to act as second messengers (Kakkar and Sawhney,
2002). It has been suggested that they could stabilize and protect the membrane systems against
the toxic effects of metal ions, particularly the redox active metals.
Overall, the number of studies remains rather scarce, and the preliminary results available in
the literature merely constitute a launching platform for this promising research field.
8. Future perspectives
The main objective of metabolomics implementation in soil risk assessment is to meet the
continuously increasing demand of safety data from human and ecological risk assessments.
Accordingly, regulatory programs worldwide are currently incorporating tests with end-
points that involve the effects of chemicals and the impact in specific metabolic pathways
(Ankley et al., 2006). Toxicological end-points can be general biological responses such as
survival or weight loss (Baylay et al., 2012), but specific biomarkers provide the accuracy that
was classically elusive for test with living organisms
Several issues immediately arise from the summary here presented, such as the need to
perform field toxicological test, with natural soils rather than use artificial soils, as was the case
with some of the studies listed in Table 1. Ecotoxigenomics can also benefit from the incorpo‐
ration of further analytical techniques. Techniques based on mass spectrometry are certainly
required to understand the mechanisms involved in the alteration of metabolic pathways as
response to toxicants. However, for screenings which merely require the detection of differ‐
ences between metabolic phenotypes, optical methods such as FT-IR would be suitable,
particularly if extremely high sample throughput is required (Bundy et al., 2009). Although
no data was available in the existing literature, Figure 2 illustrates the change in the fingerprint
of organic compounds in a soil amended with different sources of carbon collected 10 after the
application. While some of the groups of compounds might be merely related to the sources
of carbon added, the variations in the signal associated to polysaccharides (600-1000 cm-1) can
be associated to changes in the metabolic fingerprint of the soil system and therefore linked to
microbiological activity in soil. Overall, the introduction of these results seeks to encourage
further characterization of families of compounds in intact soil (or functional pools such as
aggregates) in relation with soil processes, an approach that can find immediate application
in the assessment of biological responses to toxic compounds in soil.
(a) (b)
3.0 3.0
S S
SL SBL
2.5 2.5
SP SBP
SM
SBM
2.0
Absorbance
Absorbance
2.0
1.5
1.5
1.0
1.0
0.5
0.0 0.5
4000 3000 2000 1000 4000 3000 2000 1000
Wavenumber (cm-1) Wavenumber (cm-1)
Figure 2. Absorption spectra obtained by Fourier transform infrared spectroscopy (FTIR) for an agricultural soil (S), soil
amended
Figurewith fresh residues
2. Absorption spectra(a): dry leaf
obtained bylitter (SL),transform
Fourier peanut infrared
shell (SP), maize residue
spectroscopy (FTIR)(SM), and
for an soil amended
agricultural with
soil (S), soilbio‐
char (b) derived
amended from
with freshthose feedstocks
residues (BL,
(a): dry leaf BP(SL),
litter or BM). Spectra
peanut presented
shell (SP), (after(SM),
maize residue 10 dandincubation)
soil amendedarewith
the biochar
average (b)of 5
derived
spectra from those
obtained feedstocks
for different (BL, BP of
samples or each
BM). Spectra presented
treatment. (after 10 d incubation)
Hernandez-Soriano et al.,are the average of
unpublished 5 spectra obtained
data.
for different samples of each treatment. Hernandez-Soriano et al., unpublished data.
The The
variability
variabilityofofbiological responses
biological responses hashas been
been one one of the
of the mainmain obstacles
obstacles for theirfor their implemen‐
implementation in
tation in standardized
standardized riskHowever,
risk assessment. assessment. However,
the examination the examination
of changes of changes
in biological processes in analytical
by accurate biological
techniques and powerful statistical tools has launched a new era in our understanding of the soil processes. The
possibility of identifying the most sensitive metabolites for a certain toxicant and develop a tailored standardized
test is the ultimate goal pursued.
Acknowledgments
MCH-S thanks The University of Queensland for a postdoctoral research fellowship. JCJ-L thanks the European
research program Marie Curie (FP7-PEOPLE-2011-IOF) for his PIOF-GA-2011-301550 grant.
processes by accurate analytical techniques and powerful statistical tools has launched a new
era in our understanding of the soil processes. The possibility of identifying the most sensitive
metabolites for a certain toxicant and develop a tailored standardized test is the ultimate goal
pursued.
Acknowledgements
MCH-S thanks The University of Queensland for a postdoctoral research fellowship. JCJ-L
thanks the European research program Marie Curie (FP7-PEOPLE-2011-IOF) for his PIOF-
GA-2011-301550 grant.
The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Author details
Maria C. Hernandez-Soriano1 and Jose C. Jimenez-Lopez2
1 School of Agriculture and Food Sciences, The University of Queensland, St Lucia QLD,
Australia
2 The UWA Institute of Agriculture, The University of Western Australia, Crawley, Perth
WA, Australia
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chapter 17
Phytoremediation of Soils Contaminated

with Metals and Metalloids at Mining Areas:
Potential of Native Flora
Paulo J.C. Favas, João Pratas, Mayank Varun,

Rohan D’Souza and Manoj S. Paul
1. Introduction
Contaminated soils and residues can be remediated by various methods, such as: removal,
isolation, incineration, solidification/stabilization, vitrification, thermal treatment, solvent
extraction, chemical oxidation, etc. These methods have the disadvantage of being very
expensive and in some cases, they involve the movement of contaminated materials to
treatment sites thus, adding risks of secondary contamination [1-3]. Therefore, currently
preference is being given to in situ methods that are less environmentally disruptive and more
economical. In this context, biotechnology offers phytoremediation techniques as a suitable
alternative.
Phytoremediation can be understood as the use of plants (trees, shrubs, grasses and aquatic
plants) and their associated microorganisms in order to remove, degrade or isolate toxic
substances from the environment [3-8]. The word “phytoremediation” derives from the Greek
«phyton», meaning “plant”, and Latin «remedium», which means “to remedy” or “to correct”.
Substances that may be subjected to phytoremediation include metals (Pb, Zn, Cd, Cu, Ni, Hg),
metalloids (As, Sb), inorganic compounds (NO3- NH4+, PO43-), radioactive chemical elements
(U, Cs, Sr), petroleum hydrocarbons (BTEX), pesticides and herbicides (atrazine, bentazone,
chlorinated and nitroaromatic compounds), explosives (TNT, DNT), chlorinated solvents
(TCE, PCE) and industrial organic wastes (PCPs, PAHs), and others [5].
2. Phytoremediation strategies
Phytoremediation techniques include different modalities, depending on the chemical nature

and properties of the contaminant (if it is inert, volatile or subject to degradation in the plant
or in the soil) and the plant characteristics (Figure 1). Thus, phytoremediation essentially
comprise six different strategies, though more than one may be used by the plant simultane‐
ously.
Figure 1. Schematic representation of phytoremediation strategies.

Phytoremediation of Soils Contaminated with Metals and... 3
1. Phytodegradation (Phytotransformation): organic contaminants are degraded (metabo‐

lized) or mineralized inside plant cells by specific enzymes that include nitroreductases
(degradation of nitroaromatic compounds), dehalogenases (degradation of chlorinated
solvents and pesticides) and laccases (degradation of anilines). Populus species and
Myriophyllium spicatum are examples of plants that have these enzymatic systems [9,10].
2. Phytostabilization (Phytoimmobilization): contaminants, organic or inorganic, are

incorporated into the lignin of the cell wall of roots cells or into humus. Metals are
precipitated as insoluble forms by direct action of root exudates and subsequently trapped
in the soil matrix. The main objective is to avoid mobilization of contaminants and limit
their diffusion in the soil [3,11-13]. Species of genera Haumaniastrum, Eragrostis, Ascole‐
pis, Gladiolus and Alyssum are examples of plants cultivated for this purpose.
3. Phytovolatilization: this technique relies on the ability of some plants to absorb and
volatilize certain metals/metalloids. Some element ions of the groups IIB, VA and VIA of
the periodic table (specifically Hg, Se and As) are absorbed by the roots, converted into
non-toxic forms, and then released into the atmosphere. As examples the species
Astragalus bisulcatus and Stanleya pinnata for Se or transgenic plants (with bacterial genes)
of Arabidopsis thaliana, Nicotiana tabacum, Liriodendron tulipifera or Brassica napus for Hg can
be mentioned [13-18]. This technique can also be used for organic compounds.
4. Phytoextraction (Phytoaccumulation, Phytoabsorption or Phytosequestration): this

involves the absorption of contaminants by roots followed by translocation and accumu‐
lation in the aerial parts. It is mainly applied to metals (Cd, Ni, Cu, Zn, Pb) but can also
be used for other elements (Se, As) and organic compounds. This technique preferentially
uses hyperaccumulator plants, that have the ability to store high concentrations of specific
metals in their aerial parts (0.01% to 1% dry weight, depending on the metal). Elsholtzia
splendens, Alyssum bertolonii, Thlaspi caerulescens and Pteris vittata are known examples of
hyperaccumulator plants for Cu, Ni, Zn/Cd and As, respectively [3,19-26].
5. Phytofiltration: this uses plants to absorb, concentrate and/or precipitate contaminants,

particularly heavy metals or radioactive elements, from an aqueous medium through their
root system or other submerged organs. The plants are kept in a hydroponic system,
whereby the effluents pass and are “filtered” by the roots (Rhizofiltration), or other organs
that absorb and concentrate contaminants [13,27,28]. Plants with high root biomass, or
high absorption surface, with more accumulation capacity (aquatic hyperaccumulators)
and tolerance to contaminants achieve the best results. Promising examples include
Helianthus annus, Brassica juncea, Phragmites australis, Fontinalis antipyretica and several
species of Salix, Populus, Lemna and Callitriche [3,16,29-31].
6. Rhizodegradation (Phytostimulation): growing roots promote the proliferation of

degrading rhizosphere microorganisms which utilize exudates and metabolites of plants
as a source of carbon and energy. In addition, plants may exude biodegrading enzymes
themselves. The application of phytostimulation is limited to organic contaminants [3,27].
The microbial community in the rhizosphere is heterogeneous due to variable spatial
distribution of nutrients, however species of the genus Pseudomonas are the predominant
organisms associated with roots [13,32,33].
There are other strategies, which are considered categories of phytoremediation by some
authors, but actually, they are mixed techniques or variations of the above mentioned strat‐
egies. These include:
a. Hydraulic barriers: some large trees, particularly those with deep roots (e.g., Populus sp.),
remove large quantities of groundwater during transpiration. Contaminants in this water
are metabolized by plant enzymes, and vaporized together with water or simply seques‐
tered in plant tissues [3,34].
b. Vegetation covers: Herbs (usually grasses), eventually shrubs or trees, establish on
landfills or tailings, are used to minimize the infiltration of rain water, and contain the
spread of pollutants. The roots increase soil aeration thus, promoting biodegradation,
evaporation and transpiration [7,35-37]. The difficulty of this technique is that tailings
generally are not suitable for the development of plant roots. However, various investi‐
gations have been undertaken with the aim of developing processes of cultivation in
tailings. For example a technique in which an organic soil composed of sawdust, plant
remains, and some NPK-fertilizers is deposited on the surface was utilized by Hungarian
agronomists (Biological Reclamation Process, BRP), [38]. The workers were able to obtain,
at the end of a single biological cycle, 76 different plant species including cereals, shrubs,
fruit trees and even large trees like oaks and pines.
c. Constructed wetlands: these are ecosystems consisting of organic soils, microorganisms,
algae and vascular aquatic plants in areas where the water level is at/near the surface, at
least part of the year. All the components work together in the treatment of effluents,
through the combined actions of filtration, ion exchange, adsorption and precipitation
[27,39,40]. It is the oldest method of wastewater treatment and is not regarded as proper
phytoremediation, since it is based on the contributions of the entire system [3,41]. Good
cleaning efficiency, low cost of construction along with easy operation and maintenance
are the main advantages. It is widely applied in the treatment of domestic, agricultural
and industrial waste water, but has proved to be suitable also for treating acid mine
drainages [42-45].
d. Phytodesalination: it is a recently reported [13,46] emerging technique that utilizes
halophytes to remove excess salts from saline soils. The potential of Suaeda maritima and
Sesuvium portulacastrum in removal and accumulation of NaCl, from highly saline soils,
has been demonstrated [47]. Although it has its peculiarities, this technique is a modality
of phytoextraction.
3. Advantages and limitations of phytoremediation
Phytoremediation offers several advantages, but also some disadvantages, which should be
considered when seeking to apply this technology (Table 1). If low cost is an advantage, the
time necessary to observe the results can be long. The pollutant concentration and the presence
of other toxins should be within the tolerance limits of the plant to be used. Selecting plants
with the efficiency for remediating varied contaminants simultaneously is not easy. These
limitations and the possibility of these plants entering in the food chains, should be taken into
account when applying this technology.
Advantages Limitations
Limited to shallow soils or where contamination is

In situ and passive technique
localized to the surface (< 5 m)
Still under development and therefore not accepted by

Uses solar energy and is low cost
many regulatory agencies
Has reduced environmental impact and contributes to There is little knowledge of farming, genetics,
the landscape improvement reproduction and diseases of phytoremediating plants
Metal concentrations in the soil can be toxic and lethal to

High acceptance by the public
plants
Provides habitat for animal life Generally, plants are selective in metal remediation
Treatment slower than the traditional physico-chemical

Reduction in dispersal of dust and contaminants by wind
techniques
Contamination may spread through the food chain if

Reduction of surface runoff
accumulator plants are ingested by animals
Efficient phytoremediating plants may not adapt to

Reduction of leaching and mobilization of contaminants
climatic and environmental conditions at contaminated
in soil
sites
Harvesting of the plants or organs that have

If the plants release compounds to increase the mobility
accumulated metals is easy to accomplish with existing
of the metals, these can be leached into groundwater
technology
The area to be decontaminated must be large enough to

The harvested biomass can be economically valuable
allow application of cultivation techniques
Plant process more easily controlled than those of Toxicity and bioavailability of degradation products
microorganisms remain largely unknown
Table 1. Advantages and limitations of phytoremediation [3,7,8,48,49].
4. Phytoextraction and phytostabilization
Phytoextraction and phytostabilization are the two techniques most useful for phytoremedia‐
tion of metal and metalloid contaminated soils. Phytoextraction has been widely studied,
mainly due to the potential for high efficiency and possible economic value (in metal recovery,
energy production) [3,23,24,48,50,51]. Preferably, plants used in phytoextraction should
present, among others, the following characteristics [13,23,52,53]:
1. tolerance to high concentrations of metals;

2. accumulate high concentrations in their aerial tissues;
3. rapid growth;
4. high biomass production;
5. profuse root system;
6. easy to cultivate and harvest.
Phytoextraction can only be considered effective if the accumulated contaminant is subse‐

quently removed through harvesting (Figure 2). If most of the captured heavy metals are
translocated to shoots, traditional farming methods can be used for harvesting. It is important
to harvest the plants before leaf-fall or death and decomposition to ensure that contaminants
do not disperse or return to the soil [20].
Figure 2. Schematic representation of phytoextraction of metals from soil.
After harvesting, biomass may be processed for extraction and recovery of metals (phytomin‐
ing). The commercial value of metals such as Ni, Zn, Cu or Co may encourage the phytore‐
mediation process. Alternatively, thermal, physical, chemical or microbiological processes can
be used to reduce the volume/ weight of biomass. In the case of incineration of plants the energy
produced represents an economic opportunity, and the ash can be further processed for
extraction of metals. However, this process must be very careful, given the possible chemical
elements accumulated, to prevent any dispersion mechanisms of contaminants.
According to McGrath and Zhao [22], phytoextraction efficiency is determined by two key
factors: the ability to hyperaccumulate metals and the biomass production. Therefore, if these
factors influence the phytoextraction, they can be optimized to improve the phytoremedia‐
tion process. One possibility is the addition of chemical agents into the soil in order to increase
the bioavailability of metals and their root uptake [54,55]. This form of assisted phytoreme‐
diation (or induced phytoremediation) has shown great potential and has been widely
studied (Figure 3).
Figure 3. Schematic representation of the processes of natural (A) and assisted (B) phytoextraction.
Although hyperaccumulators are phytoextractors par excellence, usually they are low biomass
producers. Thus, it is generally accepted that plants with a significant biomass production
capacity can compensate their relatively lower metal accumulation capacity, to an extent where
the amount of metal removed can be higher [51].
Phytoextraction potential can be estimated by calculation of bioconcentration factor (or

biological absorption coefficient) and translocation factor [51,56]. The bioconcentration factor
(BCF), which is defined as the ratio of the total concentration of element in the harvested plant
tissue (Cplant) to its concentration in the soil in which the plant was growing (Csoil), is calculated
as follows:
C plant
BCF = Csoil
(1)
Translocation factor (TF), defined as the ratio of the total concentration of elements in the aerial
parts of the plant (Cshoot) to the concentration in the root (Croot), is calculated as follows:
Cshoot
TF = Croot
(2)
The commercial efficiency of phytoextraction can be estimated by the rate of metal accumu‐
lation and biomass production. Multiplying the rate of accumulation (metal (g)/plant tissue
(kg)) by the growth rate (plant tissue (kg)/hectare/year), gives the metal removal value (g/kg
of metal per hectare and per year) [3,19,54,57]. This rate of removal or extraction should reach
several hundred, or at least 1 kg/ha/year, for the species to be commercially useful, and even
then, the remediation process may take from 15 to 20 years [3].
Some soils are so heavily contaminated that removal of metals using plants would take an
unrealistic amount of time. The normal practice is to choose drought-resistant fast-growing
crops or fodder which can grow in metal-contaminated and nutrient-deficient soils.
In contrast to phytoextraction, phytostabilization aims at reducing the mobility of contami‐
nants in the soil. In this technique, contaminated soil is covered by vegetation tolerant to high
concentrations of toxic elements, limiting the soil erosion and leaching of contaminants in to
groundwater. Mobility of contaminants can be reduced by surface adsorption/accumulation
in roots as well as their precipitation in rhizosphere by induced changes in pH or by oxidation
of the root environment [3,12,58]. For example, the immobilization of arsenic in iron plates in
the rhizosphere of salt marsh plants [58,59]. Phytostabilization can also be promoted by plant
species with the capacity to exude high amounts of chelating substances. These substances
lead to immobilization of contaminants by preventing their absorption, while simultaneously
reducing their mobility in soil. Thus, plants with phytostabilization potential can be of great
value for the revegetation of mine tailings and contaminated areas [58,60].
5. Phytoremediation potential of native flora of contaminated soils
It is possible to find a wide variety of plant species that can colonize areas highly pollut‐
ed with heavy metals and metalloids, such as mine tailings or soils degraded and contami‐
nated by mining/industrial activities. These are referred to as metallophyte and
pseudometallophyte species.
Metallophytes are endemic plant species of natural mineralized soils and, therefore, have
developed physiological mechanisms of resistance and tolerance to survive on substrates
with high metal levels [61,62]. Since metallophytes, in general, and hyperaccumulators, in
particular, are relatively rare and usually produce reduced biomass, the study of pseudome‐
tallophytes, indigenous species of contaminated soils, is of great value. Pseudometallo‐
phyte species (or facultative metallophytes) aren’t specialized in metalliferous soils and
have a more extensive distribution, but, due to selective pressure, are capable to survive
in metalliferous soils [63-65]. Thus, the high pressure of metalliferous soils (natural or
contaminated by human action) allows the selection of populations of common species,
with higher tolerance than other populations of the same species. Therefore, their capaci‐
ty of adaptation to these environments and, eventually, of accumulation of metals and
metalloids, can be very interesting with a view to their use, for example, in ecological
restoration, phytoremediation and bioindication actions.
In recent decades many studies have been conducted in contaminated mining and industrial
areas and in natural metalliferous soils [35,51,58,66-78] in order to inventory and screen the
indigenous species and evaluate their potential for phytoremediation of contaminated soils.
5.1. Phytoremediation potencial of native flora of contaminated soils in Portugal
Several studies to survey the indigenous plant species of diverse contaminated areas and
evaluate their potential for phytoremediation have been performed in Portugal [79-93]. The
authors have undertaken studies to evaluate the phytotechnological potential (phytoreme‐
diation, phytomining, bioindication, biogeochemical prospecting) of native flora of soils
enriched with metals and metalloids, in distinct abandoned mining areas of tin/tungsten
(Sn/W), copper (Cu), lead (Pb), uranium (U), and chromium (Cr) and the results are
presented in this chapter.
5.1.1. Native flora of old mining areas
In the old mining areas studied, several line transects were made in mineralized and non-
mineralized zones as well as tailings. Soils and plants were collected at 20 m intervals along
the line transects (0, 20, 40 m, etc.) in circle of ≅2 m radius. At each location four random partial
soil samples weighing 0.5 kg each were collected from 0 to 20 cm depth and mixed to obtain
one composite sample to save time and costs. These were oven-dried at a constant temperature,
manually homogenized and quartered. Two equivalent fractions were obtained from each
quartered sample. One was used for the determination of pH, and the other for chemical
analysis. The samples for chemical analysis were sieved using a 2 mm mesh sieve to remove
plant matter and subsequently screened to pass through a 250 µm screen. Samples were also
obtained from all species of plants whenever found growing within the 2 m radius of each
sampling point. The plant sample focused on the aerial parts, taking into consideration similar
maturity of the plants and the proportionality of the different types of tissues, or the separation
of different types of tissues (leaves and stems) in some species. In the laboratory, the vegetal
material was washed thoroughly, first in running water followed by distilled water, and then
dried in a glasshouse. When dry, the material was milled into a homogenous powder. Soil pH
was determined in water extract (1:2.5 v/v). The soil and plant samples were acid-digested for
elemental analysis. Analytical methods included colorimetry for W, atomic absorption
spectrophotometry (AAS, Perkin-Elmer, 2380) for Ag, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn and
hydride generation system (HGS) for As and Sb. Fluorometry (Fluorat-02-2M analyzer,
Lumex) was the methodology that was adopted for the determination of the U content in the
plant and soil samples. Data quality control was performed by inserting triplicate samples into
each batch. Certified references materials were also used.
5.1.1.1. Tin/tungsten mines
The studied areas included several abandoned Sn/W mines (Sarzedas mine, Fragas do Cavalo
mine, Tarouca mine, Vale das Gatas mine, Adoria mine, Ervedosa mine, Regoufe mine, and
Rio de Frades mine). Results obtained from Sarzedas (Central Portugal) and Vale das Gatas
mines (Northern Portugal) are presented.
A summary of trace element data in soil from the Sarzedas mine is shown in Table 2. Among
the elements present in the soils, Ag, As, Pb, Sb and W show the most relevant anomalies. Soil
pH was negatively correlated to mineralization. Low pH values observed near the mineralized
area can be explained by the presence of sulfides in the mineralization [84]. High levels of
sulfides, in particular pyrite and arsenopyrite that are easily weathered, favors the dissolution
of toxic elements, allowing higher dispersion and bioavailability.
Range Mean Median Standard deviation
pH 3.3 – 5.2 4.7 4.8 0.5
Ag 0.69 – 1.91 0.98 0.92 0.32
As 11.1 – 651 76.3 19.9 181
Co 5.40 – 14.9 8.80 8.41 2.60
Cr 50.7 – 129 96.5 100 26.3
Cu 15.5 – 78.2 40.7 35.1 21.1
Fe 21,881 – 58,644 39,981 37,356 12,883
Mn 22.0 – 92.0 50.0 47.0 22.0
Ni 11.2 – 52.5 21.8 19.7 10.2
Pb 35.7 – 417 85.4 53.9 106
Sb 30.5 – 5,986 663 87.8 1,689
W 0.80 – 684 663 2.90 52.3
Zn 29.0 – 127 58.8 53.3 24.3
Table 2. Trace elements content (mg/kg) and pH of soil samples (N=24, Sarzedas mine).
In the flora of Sarzedas mine area, As was accumulated in aerial tissues of Pinus pinaster and
Digitalis purpurea. Therefore, these species are suited for recognizing the anomaly. High
accumulation of As was present in leaves (Figure 4), and it increased in the older tissues. This
translocation is a common mechanism in plants to avoid toxicity in young leaves as their
metabolic activity is higher [84]. Digitalis purpurea also accumulated substantial amount of Sb
(Figure 5), indicating its tolerance to this element, although the assimilation occured at low
concentrations in the soil. Species that are capable of accumulating W are D. purpurea, Cistus
ladanifer, P. pinaster, Calluna vulgaris and Helichrysum stoechas (Figure 6).
Figure 4. Accumulation of As (mg/kg DW) in plant species of the Sarzedas mining area.
Figure 5. Accumulation of Sb (mg/kg DW) in plant species of the Sarzedas mining area.
Figure 6. Accumulation of W (mg/kg DW) in plant species of the Sarzedas mining area.
It was concluded that the species and organs best suited for biogeochemical prospecting and/
or with potential for mine restoration in the Sarzedas mine area are by order of importance: 1)
As: old needles of P. pinaster, aerial tissues of C. vulgaris, Chamaespartium tridentatum, leaves of
C. ladanifer, Erica umbellata and Quercus ilex subsp. ballota; 2) Sb: D. purpurea, E. umbellata, stems
of C. ladanifer, C. vulgaris, C. tridentatum and stems of P. pinaster; 3) W: D. purpurea, C. triden‐
tatum, old stems and needles of P. pinaster, stem and leaves of C. ladanifer, E. umbellata and
stems and leaves of Q. ilex [84].
pH 3.5 – 6.3 5.0 5.0 0.8
As 26.7 – 5,770 446 56.7 1,178
Cu 11.7 – 352 88.0 29.0 101
Fe 18,482 – 60,100 33,039 29,443 12,463
Mn 103 – 898 336 167 248
Ni 11.6 – 61.2 30.6 23.6 15.1
Pb 55.4 – 6,299 499 102 1,285
Zn 63.1 – 469 180 125 112
W 2.00 – 636 73.8 10.6 162
Table 3. Trace elements content (mg/kg) and pH of soil samples (N=69, V. Gatas mine).
Very high maximum values for Pb (6,299 mg/kg), As (5,770 mg/kg) and W (636 mg/kg) were
observed at the Vale das Gatas mine (Table 3). The Cu-Mn-W-As-Pb-Zn association, which
reflects the presence of mineralised veins in the area, is inversely correlated with pH [93]. In
general, the content variations in plant materials were strongly related to the content variations
in soils. It has also been verified that in contaminated locations or tailings, the concentration
of metals in plant tissues is high due to the high metal concentrations in the soil.
The leaves of Agrostis castellana and Holcus lanatus reflect the Cu, Pb and Ni pedogeochemical
anomalies. The aerial parts of Pteridium aquilinum and Juncus effusus seem to be indicative of
Zn anomalies in the soil [94]. Holcus lanatus and A. castellana were the main accumulators of
As (Figure 7), Cu (Figure 8), Fe (Figure 9) and Pb (Figure 10) and good accumulators of Zn
(Figure 11). Pteridium aquilinum was a good accumulator of As, Pb and Zn (Figures 7, 10, 11).
Juncus effusus appeared to be a Zn accumulator (Figure 11).
Figure 7. Accumulation of As (mg/kg DW) in plant species of the V. Gatas mining area.
Figure 8. Accumulation of Cu (mg/kg DW) in plant species of the V. Gatas mining area.
Figure 9. Accumulation of Fe (mg/kg DW) in plant species of the V. Gatas mining area.
Figure 10. Accumulation of Pb (mg/kg DW) in plant species of the V. Gatas mining area.
Figure 11. Accumulation of Zn (mg/kg DW) in plant species of the V. Gatas mining area.
The P. pinaster trees growing on the tailings and contaminated soils of Vale das Gatas mine
accumulated the studied elements in quantities greater than observed in plants of the areas
representative of the local geochemical background. These values were also higher than those
typically observed in this species.
In the P. pinaster samples from tailings and contaminated soil locations, the older needles (2-
and 3-years-old) show a tendency to accumulate higher concentrations of As, Fe, Zn, Pb and
W while Ni and Cu were preferentially accumulated in young needles and stems (1-year-old)
[93]. This allowed the authors to conclude that the metal/metalloid concentrations of elements
in plants depend as much on the plant organ as on its age and in biogeochemical studies, it is
important not to mix foliar and woody material in the same sample. The species showed a
great variability in the accumulation behaviour of As, Fe, Mn, Cu, Zn, Pb, Ni, and W with the
age of the organ. Thus, the 1-year-old needles and stems accumulated higher levels of Cu
(Figure 8) and Ni (Figure 12). While the older needles accumulated higher levels of As, Fe, Pb,
Zn and W (Figures 7, 9, 10, 11 and 13). The 2-years-old stems may also be appropriate samples
to detect higher levels of Fe, Zn and Pb.
Figure 12. Accumulation of Ni (mg/kg DW) in plant species of the V. Gatas mining area.
Figure 13. Accumulation of W (mg/kg DW) in plant species of the V. Gatas mining area.
5.1.1.2. Copper mines
The São Domingos mine (abandoned in 1966) located in south-east Portugal is also included
in this study. This is one of the historical mining centres, known for its activity since pre-Roman
times, with extraction of gold, silver and copper [95] though copper production was the
highlight.
A summary of soil trace element data is presented in Table 4. High levels of As, Cu, Pb and
Zn were recorded in the soils. Copper concentration in soils reached up to 1,829 mg/kg as a
result of the former activities at the site (copper smelter). Maximum concentration of As in
soils was very high, reaching 1,291 mg/kg. The concentration of Pb in the soil was also very
high, 2,694 mg/kg as the average value registered. The average Zn concentration in soils was
of 218 mg/kg but it could reach 714 mg/kg, a level that can be extremely toxic for plants. Cobalt
and Cr concentrations in soils were normally low, ranging from 20.1 to 54.3 mg/kg for Co and
5.1 to 84.6 mg/kg for Cr. Nickel and Ag were also low, varying from 27.2–52.9 mg/kg and 2.5–
16.6 mg/kg, respectively.
pH 4.0 – 6.7 5.1 5.1 0.6
Ag 2.50 – 16.6 7.50 7.00 3.60
As 37.2 – 1291 393 353 324
Co 20.1 – 54.3 31.0 29.4 8.40
Cr 5.10 – 84.6 26.5 8.30 31.7
Cu 87.3 – 1,829 553 444 443
Ni 27.2 – 52.9 42.2 43.9 6.60
Pb 234 – 12,218 2,694 2,355 2,345
Zn 104 – 714 218 163 145
Table 4. Trace elements content (mg/kg) and pH of soil samples (N=21, S. Domingos mine).
In plants, Pb concentration was rather high for some species, varying from 2.9 to 84.9 mg/kg
dry weight (DW) (Figure 14). Semi-aquatic species sampled in the mining area, Juncus
conglomeratus and Scirpus holoschoenus, showed high accumulation of Pb in plant tissues. Lead
above 20 mg/kg DW was found in leaves of two species of Cistus, typical Mediterranean shrubs
known for their tolerance to drought and low nutrients availability. Arsenic concentration in
plant tissues ranged from 0.3 to 23.5 mg/kg DW. Maximum As was recorded in J. conglomera‐
tus, Thymus mastichina, J. effusus and S. holoschoenus [82]. Semi-aquatic species from the
Juncaceae family showed the highest content of both metals. Copper concentration in plant
tissues ranged from 3.60 to 28.9 mg/kg DW (Figure 15). These Cu values are within the range
considered normal for plants [96]. The species Cistus monspeliensis and Daphne gnidium showed
the highest Zn concentrations [82]. A few trees, Eucalyptus, Quercus and Pinus species, were
found in the contaminated area showing accumulation of different metals in the aboveground
tissues. Due to their high biomass, they can be very effective for metals phytoextraction and
phytostabilization especially when established in the less contaminated soils on the peripheral
zone of the study area [82].
Figure 14. Accumulation of Pb (mg/kg DW) in plant species of the S. Domingos mining area.
Figure 15. Accumulation of Cu (mg/kg DW) in plant species of the S. Domingos mining area.
5.1.1.3. Lead mines
The Barbadalhos mine is an abandoned Pb mine in Central Portugal. It was exploited for Pb
by underground mining from 1887 till the 1940s. The concentrated ore was smelted on site. As
per the usual practice at the time, tailings were deposited on the ground.
Metal concentrations in soil are shown in Table 5. Lead concentration in soils reached 9,331
mg/kg while the average value was 928 mg/kg; obviously due to mining of galena at the
site. In soils from mineralized zone, the mean Pb concentration (2,380 mg/kg) was nearly
9 times the threshold for industrial soils suggested by Canadian Environmental Quality
Guidelines [97].
pH 3,6 – 6.4 4.7 4.6 0.5
Ag 0.71 – 13.0 1.71 1.06 2.03
As 2.77 – 208 16.9 8.07 31.7
Co 3.74 – 50.5 20.1 16.7 12.4
Cr 61.3 – 196 89.0 85.7 22.1
Cu 21.4 – 193 41.7 34.5 27.9
Fe 24,145 – 98,510 40,283 38,497 13,751
Mn 44.4 – 2,224 596 381 588
Ni 7.68 – 87.0 30.5 28.1 12.2
Pb 24.4 – 9,331 928 68.8 2,119
Zn 30.4 – 517 134 90.3 109
Table 5. Trace elements content (mg/kg) and pH of soil samples (N=45, Barbadalhos mine).
Samples from 49 species of the native flora were investigated at this site. Individual elements
and species displayed different trends of accumulation. All plants collected along mineralized
zone accumulated eight metals (Ag, Co, Cr, Cu, Fe, Ni, Pb, and Zn) but many plants from non-
mineralized zone accumulated only five metals (Ag, Cu, Fe, Pb, and Zn). A few however did
accumulate the remaining three (Co, Cr, and Ni); bringing the count of metals accumulated at
par with those of mineralized zone [92].
Most plants were seen to be tolerant of soil Pb concentrations. In mineralized zone, Pb

concentrations in plants ranged from 1.11 to 548 mg/kg DW. This is far above the 100 – 400 mg
Pb/kg content considered toxic for most plants [98]. Significant accumulation of Pb was seen
in Cistus salvifolius (548 mg/kg), Lonicera periclymenum (318 mg/kg), Anarrhinum bellidifolium,
Phytolacca americana, Digitalis purpurea, Mentha suaveolens (255 – 217 mg/kg) [listed in decreas‐
ing order] (Figure 16). Pteridophytes like Polystichum setiferum, Pteridium aquilinum, and
Asplenium onopteris also showed 117 – 251 mg/kg Pb in aerial parts. In plants from non-
mineralized zone, Pb content was not significant ranging from 0.94 to 11.6 mg/kg.
Though at first glance maximum Pb content observed in trees like Acacia dealbata (84 mg/kg:
leaves), Olea europaea (62 mg/kg: twigs), and Quercus suber (58 mg/kg: twigs) from mineralized
zone is not very impressive compared to that of smaller plants mentioned above, nevertheless
these trees can be very effective due to their higher biomass. When combined with the hardy
nature, biomass and abundance of this species, the moderate accumulation indicates immense
potential for phytoextraction of Pb in the area [92].
In mineralized zone, Zn concentrations in plants reached 1,020 mg/kg in D. purpurea. And

ranged from 262 to 887 mg/kg in L. periclymenum, P. americana, Solanum nigrum, P. setiferum,
M. suaveolens, Viola riviniana, and A. bellidifolium [listed in decreasing order] [92].
Figure 16. Accumulation of Pb (mg/kg DW) in plant species of the Barbadalhos mining area.
5.1.1.4. Uranium mines
The old U mine of Sevilha (Central Portugal) is one of several small mines exploited by ENU
(Portuguese Uranium Company). After the removal of the main ore body, the site was filled
with the mine wastes and a reclamation process was initiated. This action was somewhat
unsuccessful because the selected allochthonous plant species (Lupinus sp.) did not survive.
Current U soil contamination on the Sevilha mine ranges from 8 to 560 mg/kg [99]. Species of
Compositae and Ericaceae (among the most abundant families of terrestrial plants) accumu‐
lated highest U concentrations (Figure 17). Among Compositae members, an average of 4.91
mg/kg DW and a maximum of 13.1 mg/kg DW was found in Helichrysum stoechas and an
average of 4.07 mg/kg DW and a maximum of 10.5 mg/kg DW was recorded in Hypochaeris
radicata (Figure 17). In Erica umbellata an average of 1.70 mg/kg DW and a maximum of 7.50
mg/kg DW were obtained (Figure 17). Even though the concentrations obtained in the latter
are not high, it is particularly interesting because it has a high bio-productivity. This accumu‐
lation potential might be intensified if uptake enhancement strategies, such as addition of citric
acid, are adopted. A restoration program can be applied to the soils of Sevilha mine by adopting
revegetation with endemic species allied to a process of continuous phytoremediation that
avoids dispersion of U into the streamlets.
Even though the soils in this mine are not highly contaminated, the lixiviation of refilling
materials has been responsible for the dispersion of U into ground and superficial water bodies.
These waters are being used for subsistence agriculture and, therefore, the risk of contamina‐
tion spreading to humans can be acute, due to food chain accumulation [99]. The plant survey
revealed that some of the native plant species are well adapted to U contamination in soils,
therefore, they are metallotolerants. Their phytoremediation potential has to be evaluated.
Dispersion of U into the streamlets can be minimized by a strategic combination of terrestrial
and aquatic plant phyto-systems. Revegetation with Helichrysum stoechas, Hypochaeris radica‐
ta and Erica umbellata will allow fixation of U in the plants and a consequent reduction in its
dispersion. This site can be an excellent prototype for the restoration of other mines in Portugal
where levels of contamination are a matter of concern.
5.1.1.5. Chromium mines (Serpentine soils)
The abandoned mining area of Pingarela in North-east Portugal has serpentine soils and
associated flora. These soils are disproportionately rich in trace elements like Ni, Cr, Co and
poor in Ca. Serpentine outcrops have been referred to as barrens because they are often sparsely
vegetated and extremely poor in essential nutrients, hence not of much agricultural value.
Serpentine ecosystems can generally be distinguished by their grey-green or reddish rocky
soils (soils are very thin), and shrubby or stunted vegetation with plants having small leathery
leaves.
Plant species found on serpentine soils can be divided into two groups: (a) serpentine-tolerant
or serpentine-facultative plants, which are able to survive on serpentine but grow better
elsewhere; (b) serpentinicolous, serpentine-endemic or serpentine-obligate plants, which grow
exclusively on serpentine soils and are not found on other substrates [83,100]. Both these
Figure 17. Accumulation of U (mg/kg DW) in plant species of the Sevilha mining area.
groups include species with different efficiencies to uptake or exclude a variety of metals.
Serpentinophytes often experience drought, nutrient stress and excessive exposure to heavy
metal and high light intensity. This means there is less substrate in which nutrients and water
can be held and made available to plants.
An area of ~8,000 ha in north-east Portugal is serpentinized with characteristic geology and

flora. The serpentine plant community and respective soils were analyzed [83] to examine the
trace metal budget in different tissues of the plants exhibiting resistance to trace metals. 135
plant species belonging to 39 families and respective soils were analyzed for total Co, Cr, Cu,
Fe, Mn, Ni, Pb and Zn. Heavy metal concentrations recorded in sampled soils are shown in
Table 6. The high contents of Ni and Cr obtained were to be expected, considering the
geochemistry of the sampling site. However, the high variability of Cr, Ni, Fe and Mn in the
soils is noteworthy.
pH 4.9 – 9.3 6.7 6.7 0.6
Co 56.0 – 151 82.3 81.1 18.5
Cr 200 – 6,822 1,622 1,410 1,064
Cu 30.8 – 221 101 99.3 34.7
Fe 95.0 – 110,418 82,950 84,711 14,502
Mn 1,007 – 1,835 1,389 1,363 175
Ni 102 – 2,295 918 883 464
Pb 18.5 – 46.6 29.2 29.1 5.78
Zn 63.0 – 242 110 112 24.8
Table 6. Trace element content (mg/kg) and pH of serpentine soil samples (N=74, Pingarela mine).
The Ni hyperaccumulating endemic of this region is Alyssum serpyllifolium subsp. lusitani‐

cum, which concentrated 38,105 mg Ni/kg DW in the aboveground tissues (Figure 18).
Bromus hordeaceus with 1,467 mg Ni/kg DW and Linaria spartea with 492 mg Ni/kg DW in
the aerial parts, also showed high concentration of Ni. Four other taxa viz.—Plantago radicata,
Ulmus procera, Lavandula stoechas and Cistus salvifolius showed more than 100 mg Ni/kg DW
(Figure 18).
Chromium has low solubility in the serpentine soil solution due to the relatively high pH
values of these soils [83]. This is reflected in the low uptake of this element by plants, which
in general did not exceed 40 mg/kg. However, concentrations of 707 mg Cr/kg DW were
reported in the above ground parts of L. spartea (Figure 19). Alyssum serpyllifolium also
presented high content of Cr, reaching a maximum of 130 mg Cr/kg DW. Ulmus procera showed
a content of 173 mg Cr/kg DW in the twigs.
Figure 18. Accumulation of Ni (mg/kg DW) in serpentine plant species of the Pingarela mining area.
Figure 19. Accumulation of Cr (mg/kg DW) in serpentine plant species of the Pingarela mining area.
Although leaves of serpentine plants show 10 times higher Co levels than those of plants
growing on non-serpentine soils, the absolute concentrations are only about 10 mg/kg DW in
most species. In this study highest accumulation of Co was found in the aboveground tissues
of A. serpyllifolium with 145 mg Co/kg DW and L. spartea with 63.2 mg Co/kg DW [83].
6. Conclusion
The physico-chemical properties of the metalliferous or metal-contaminated soils tend to

inhibit soil-forming processes and plant growth. In addition to elevated metal/metalloid
concentrations, other adverse factors included absence of topsoil, erosion, drought, compac‐
tion, wide temperature fluctuations, absence of soil-forming fine materials and shortage of
essential nutrients [84,101]. Degraded soils of mines usually have low concentrations of
important nutrients, like N, P and K [102]. Toxic metals can also adversely affect the number,
diversity and activity of soil organisms, inhibiting soil organic matter decomposition and N
mineralization processes. The chemical form of the potential toxic metal, the presence of other
chemicals which may aggravate or attenuate metal toxicity, the prevailing pH and the poor
nutrient status of contaminated soil affects the way in which plants respond to it. Substrate
pH affects plant growth mainly through its effect on the solubility of chemicals, including toxic
metals and nutrients.
Metal toxicity issues do not generally arise in the case of native flora, considering that native
plants become adapted over time to the locally elevated metal levels [76,103]. Native plants
may be better phytoremediators for contaminated lands than the known metal hyperaccumu‐
lators because these are generally slow growing with shallow root systems and low biomass.
Plants tolerant to toxic metals and low nutrient status with a high rate of growth and biomass
are the ideal species to remediate degraded soils and habitats like those around mines. The
native flora displayed its ability to withstand high concentrations of heavy metals in the soil.
Some species also displayed variable accumulation patterns for metals at different soil
concentrations. This variation was also observed in different parts of the same plant suggesting
that full consideration of plant–soil interactions should be taken into account when choosing
plant species for developing and utilizing methods such as phytoremediation.
Indigenous plant species growing on tailings and contaminated soils show tolerance to
imposed stress conditions (metal-contamination and nutrient deficiency) and can fulfill the
objectives of stabilization, pollution attenuation and visual improvement. Besides, these
species are drought-resistant and some even exhibit high biomass and bioproductivity. In fact,
the constraints related to plant establishment and amendment of the physical–chemical
properties of the metalliferous soils depends upon the choice of appropriate plant species.
Hence, the plant community tolerant to toxic trace elements plays a major role in remediation
of degraded mine soils.
The existing natural plant cover at abandoned mining sites can be increased manifold by wide-
scale planting and maintenance of native species with higher metal accumulation potential for
some years. Even dispersal of seeds obtained from plants on site is to be encouraged. Adding
organic amendment is essential to facilitate the establishment and colonization of these
“pioneer plants”. They can eventually modify the man-made habitat and render it more
suitable for subsequent plant communities. Allowing native species to remediate soils is an
attractive proposition since native wild species do not require frequent irrigation, fertilization,
and pesticide treatments, while simultaneously a plant community comparable to that existing
in the vicinity can be established.
Therefore, mine restoration could benefit from a broader perspective including different
groups of plant species as they can perform distinct functional roles in the remediation process.
The use of leguminous plants, for example, may enrich the nutrient content and the combined
used of perennials and annuals can provide substantial inputs in terms of organic matter and
nutrient recycling, thus contributing in distinct ways to the development of the soil [82,104].
This approach requires more information about plant communities growing on metal-
contaminated soils in order to accurately determine their potential for remediation of polluted
soils at abandoned mines. Ideal phytoremedial candidates can be screened out from the native
flora and after assessing their individual requirements, suitable conditions/amendments can
be created to develop them as good competitors with enhanced growth and proliferation than
their counterparts growing on the same metal contaminated nutrient depleted soils.
Significant accumulation of heavy metals and metalloids in both soils and native wild flora
suggests that metal contamination is a matter of great concern in the studied mining areas.
The native flora displayed its ability to withstand high concentrations of heavy metals/
metalloids in the soil. However, accumulation patterns of metals/metalloids in the plants
tested differed. As metal concentrations in above ground parts were maintained at low
levels, metal tolerance in most cases may mainly depend on their metal excluding ability.
However, metal/metalloid concentrations higher than toxic level in some species like
Agrostis castellana (for As, and Fe), Cistus ladanifer subsp. ladanifer (for Cr, and W), Cistus
salvifolius (for Ni, and Pb), Digitalis purpurea subsp. purpurea (for Sb, W, and Zn), Helichry‐
sum stoechas and Hypochaeris radicata (for U), Holcus lanatus (for As, Cu, and Fe), Lonicera
periclymenum, Mentha suaveolens and Phytolacca americana (for Pb, and Zn), Pinus pinaster (for
As, W, and Zn), Polystichum setiferum and Solanum nigrum subsp nigrum (for Zn), Pteridi‐
um aquilinum (for As), as well as the serpentine plant species Alyssum serpyllifolium subsp.
lusitanicum, Lavandula stoechas subsp. sampaiana, Linaria spartea subsp. virgatula and Ulmus
procera (for Cr, and Ni) and Bromus hordeaceus and Plantago radicata subsp. radicata (for Ni)
indicate that internal detoxification metal tolerance mechanisms might also exist; there‐
fore, their utility for phytoremediation is possible. Furthermore, the plants could grow and
propagate in substrata with low nutrient conditions which would be a great advantage in
the revegetation of mine tailings. It was also observed that despite lower accumulation,
trees of the studied regions can be very effective due to their higher biomass.
Some of the studied species also showed variable accumulation patterns for metals at different
soil concentrations. This difference was also noted between parts of the same plant suggesting
that full consideration of plant–soil interactions should be taken into account when choosing
plant species for developing and utilizing methods such as phytoremediation.
Acknowledgements
This study was partially supported by the European Fund for Economic and Regional
Development (FEDER) through the Program Operational Factors of Competitiveness (COM‐
PETE) and National Funds through the Portuguese Foundation for Science and Technology
(PEST-C/MAR/UI 0284/2011, FCOMP 01 0124 FEDER 022689).
Author details
Paulo J.C. Favas1,3*, João Pratas2,3, Mayank Varun4, Rohan D’Souza4 and Manoj S. Paul4
1 University of Trás-os-Montes e Alto Douro, UTAD, School of Life Sciences and the
Environment, Vila Real, Portugal
2 Department of Earth Sciences, Faculty of Sciences and Technology, University of Coimbra,

Coimbra, Portugal
3 IMAR-CMA Marine and Environmental Research Centre, Faculty of Sciences and

Technology, University of Coimbra, , Portugal
4 Department of Botany, St. John’s College, Agra, India

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chapter 18
Contribution of Vegetation to Alleviate Slope’s Erosion

and Acidity
Normaniza Osman, Mohammed Saifuddin and

Aimee Halim
1. Introduction
Most of the slope soils in the tropical region such as in Malaysia is arid and infertile due to lack
of buffering capacity and low clay activities which resulted in soil acidity. In addition, high
intensity rainfall and extreme conditions of slope such as transient drought and lack of
nutrients have reduced the survival and growth of potential plants. Rain water percolation
which leaches basic elements such as calcium, magnesium, potassium and sodium from the
soil profile is also another factor which contributes to soil acidity.
Soil acidity has a huge negative impact on fertility, biological activities and plant produc‐
tivity. Fortunately, the use of vegetation and their association with microbes have great
potential to alleviate soil acidity and erosion problems. Moreover, heavy metal-tolerant
plants also play an important role in phytoremediation. Therefore, proper plantation and
management of plants in polluted and acidic soil may significantly contribute to restoring
the natural environment.
The practice of using vegetation, so-called “Bio-engineering” technique, combines an ecolog‐

ical, mechanical and hydrological concept which has been successfully applied to alleviate soil
erosion in Malaysia [1]. The combined right choices of plants and planting technique confer
numerous advantages such as producing a high biodiversity, low cost-maintenance, self-
sustainability as well as environmental friendly slope [1]. Nonetheless, the interaction of
vegetation and soil are complex as it is involved with, inter alia, varying soil properties, soil
pollutants and different type of plant coverage. Therefore, in light of fast developmental land
transformation and climate change, it is of a great challenge to integrate all plant-soil properties
and its interaction with the whole ecosystem and environment.
2 Soil Pollution
1.1. Objectives of the chapter
This book chapter will explore the attributions of vegetation to alleviate the most common
slope problems in Malaysia; acidity and erosion. This chapter will also provide potential
characteristics of plants to reinforce soil and the mechanism of Aluminium (Al) accumulator
plant to alleviate soil acidity. The specific aims of this book chapter are to:
a. Describe the ecological approach towards slope stabilization;
b. Outline the vegetation effects on erosion rate and carbon sequestration;
c. Elucidate the mechanism of Al accumulator plant to alleviate soil acidity and
d. Exhibit some potential slope plants.
2. Ecological approaches to stabilize slope
There are many types of natural and manmade slopes exist in Malaysia. Due to the nature of
topography e.g. slope angle, length, aspect, gradient and curvature, and the weather condi‐
tions, these slopes become unstable and make serious geologic hazards. Around the world,
landslide depends on the geological characteristics, hydrological condition and rainfall
distribution [2,3]. In Malaysia, significant numbers of slope failure are reported on manmade
and residual soil slopes especially at the time of high intensity rainfall due to rapid change of
soil properties, particularly physical properties, such as bulk density, cohesiveness and shear
strength [4]. However, there are three common triggering factors for slope failure in respect
to Malaysia which are intense rainfall, water level change and change of slope loading (Figure
1), indicating hydrological condition gives infavorable impact on the slope stability. Apart
from that, landslides also occurred due to the human activities such as cultivation, excavation
for housing, foot paths and deforestation or construction works in hillsides and lack of
plantation in hilly area [5,6]. In addition, improper slope design might cause manmade slope
failure which has been acknowledged as one of the most reasons for frequent disaster around
the world [7]. To overcome this problem, the slopes need to be proper designed, considering
the geological characteristics, structural model, local weather and soil characteristics [8]. From
eco-physiological point of view, the plant availability, so-called bioengineering systems, has
become an alternative solution to minimize the impacts and stabilize the slope [1,9].
Bioengineering systems are extremely beneficial as it incorporates with geotechnical engi‐

neering, ecological and biological aspects [7,11]. In bioengineering techniques, less heavy
equipments and live plants are usually used which are cost effective and provided long-term
soil stability [12]. Generally, newly cut and manmade slope soils are unstable and expose to
soil loss. Hence, it is needed to be made more resistant to erosion. In our study, Nordin et. al.
[13], have discovered that different composition of root distribution and branching between
two potential slope plants, namely; Acacia mangium and Leucaena leucocephala (Table 1) caused
the differences in pull-out values. In tandem with L. leucocephala’s ability to establish on slope’s
harsh condition and reinforced the soil [14], it exhibited the higher force of pull-out, thus,
Contribution of Vegetation to Alleviate Slope’s Erosion and Acidity 3
Figure 1. Common triggering factors which is related to slopes erosions [10].
showing its capability to resist the uprooting forces. This feature helps the species anchorage
and resists the slope against forces such as wind. Interestingly, the study also found that the
pull-out force is much affected by the tensile strength (R=0.88; Figure 2). The tensile strength
decreased with increasing root diameter. Thus, this property of higher tensile strength in the
finer roots would ultimately yield an increase in shear strength of the root-soil composite in
the field and provide better ductility to the root-soil composite with a higher capacity to
withstand surface erosion and runoff [15].
Species Pull-out Force (KN) Tensile strength (N/mm2)
Acacia mangium 1.69 ± 0.34 54.37 ± 10.80
Leucaena leucocephala 2.25 ± 0.45 104.83 ± 18.72
Significant difference between species at LSDp<0.05 = 0.73 (pull-out) and 18.9 (tensile strength)
Table 1. Pull-out test and tensile strength of Acacia mangium and Leucaena leucocephala (data are means ± standard
error)
Knowing the importance of vegetation, however, the problem of vegetation establishment on

freshly cut slope is a succession problem, in which naturally, the process takes a longer time.
Fortunately, with the human helps and input, we can accelerate this process. Normaniza and
Barakbah [1] proposed a concept for establishing vegetation cover on slopes which enhanced
the process of natural succession (Figure 3). The introduction of pioneer species on slope is to
initiate the succession process and later to accelerate the ecological restoration as a whole.
Initially, the proposed pioneer species is to improve the quality of soil, be it acidic or infertile
soil.
4 Soil Pollution
Figure 2. The relationship between pull-out force and root tensile strength [13].
In relation to the proposed concept, the selected pioneer must exhibit prominent characteristics
which include high growth rate, good root profiles, water relations and high tolerance to a
wide range of adverse factors with regard to soil quality, microclimate and mechanical stress
[9,16,17]. It is anticipated that once the pioneer is established, the succession process would be
enhanced through the changes of abiotic and biotic factors. Consequently, influx of other
species will enrich the plant biodiversity of slopes. This plant community changes would not
only hasten the process of natural succession but would also attract small animals such as
insects and birds to the ecosystem. This fauna association would assist in promoting "flora-
fauna" interaction via being the agents of seed dispersal, which would ultimately enhance the
natural plant succession process.
This ecological approach towards slope stabilization has been proven on slope projects
conducted by our team at several highways cut slopes in Malaysia [1,18] (Figure 4). Our study
showed that legume trees which act as pioneers had increased the species diversity and slope
stability parameters of the slope (Table 2). The legume tree plots exhibited higher root length
density and resulted in a lower saturation level of the soil. In addition, penetration resistance
was observed to increase with presence of legume trees. The increment of shear strength at 30
cm soil depth was prominent in legume tree plot. Overall increment of slope stability param‐
eters indicated the positive effect of legume trees in reducing the probability slope failures,
since most surface failures occur at a depth of 20-50 cm [1]. In contrast, the grasses plots had
almost saturated, 83.9-93.2%, a characteristic of failing slope.
In relation to the proposed concept, the selected pioneer must exhibit prominent characteristics which include high growth rate, good root
profiles, water relations and high tolerance to a wide range of adverse factors with regard to soil quality, microclimate and mechanical
stress [9,16,17]. It is anticipated that once the pioneer is established, the succession process would be enhanced through the changes of
abiotic and biotic factors. Consequently, influx of other species will enrich the plant biodiversity of slopes. This plant community changes
would not only hasten the process of natural succession but would also attract small animals such as insects and birds to the ecosystem.
This fauna association would assist in promoting "flora-fauna" interaction via being the agents of seed dispersal, which would ultimately
enhance the natural plant succession process.
SOIL
Nitrogen fixation
PLANT AMENDMENT biotic/abiotic
SPECIES (Low fertility and
Acidity) SPECIES
INFLUX
initial
succession
+ succession
+ succession BIODIVERSITY
FLORA-FAUNA
INTERACTION
+ succession
NATURAL SLOPE
SUCCESSION STABILITY
+ mechanical and
hydrological factors
Figure 3. A proposed concept for establishing vegetation cover on slopes and enhancing the process of natural succession [1].
This ecological approach towards slope stabilization has been proven on slope projects conducted by our team at several highways cut
slopes in Malaysia [1,18] (Figure 4). Our study showed that legume trees which act as pioneers had increased the species diversity and
slope stabilityFigure 3. A proposed
parameters of the slopeconcept forThe
(Table 2). establishing vegetation
legume tree coverhigher
plots exhibited on slopes and enhancing
root length density andthe process
lower of natural
saturation suc‐
level. In
cession [1].
addition, penetration resistance was observed to increase with presence of legume trees. The increment of shear strength at 30 cm soil
depth was prominent in legume tree plot. Overall increment of slope stability parameters indicated the positive effect of legume trees in
reducing the probability slope failures, since most slope failures occur at a depth of 20-50 cm [1]. In contrast, the grasses plots had almost
saturated, 83.9-93.2%, a characteristic of failing slope.
Parameters Grasses Legume trees
Table 2. Slope stability parameters
Saturation level (%) between the plots (grasses and legume trees) 83.9-93.2 73.6-81.2
Parameters Grasses Legume trees
Root
Saturation level (%)length density (Km m-3) 83.9-93.2 0.8673.6-81.2 9.4
Root length density (Km m-3) 0.86 9.4
Penetrability Penetrability (24 months) (MPa)
(24 months) (MPa) 0.97-1.5 0.97-1.5
1.44-2.01 1.44-2.01
Shear strength (at 30 cm soil depth at 24 months) (KPa) 100.5±5.5 104.6±2.4
Shear strength (at 30 cm soil depth at 24 months) (KPa) 100.5±5.5 104.6±2.4
3
Table 2. Slope stability parameters between the plots (grasses and legume trees)
6 Soil Pollution
Figure 4. Topographic map of surveyed slopes in Peninsular Malaysia; a) Faculty of Science, University of Malaya; lati‐
tude 030 07’ 28.5’’ N, longitude 1010 39’ 14.6’’ E and b) Batu 38, Pusat Pengajian Luar, University of Malaya, Ulu Gom‐
bak; latitude 030 20’ 45.27’’ N, longitude 1010 46’ 26.52’’ E.
3. Impacts of vegetation on soil erosion and carbon sequestration
3.1. Soil erosion
The use of vegetation to control soil erosion has been practiced for many centuries, firstly
introduced in China in 16th century to stabilize dam [19]. Nowadays, this practice of vegetation
has been successfully applied to stabilize slope throughout the world. The vegetation and
erosion process are interrelated by the ability of the plant life growing on soil and the inter‐
action of root and soil [20]. But the interaction of vegetation and soil are complex as it involved
with, inter alia, the combination of soil types, plant coverage and the steepness of slope. There
are many factors also responsible for controlling soil erosion such as soil elements, soil density,
slope length, existing plant species and plant position on slope, plant age, plant coverage and
plant root distribution. Moreover, the revegetation process also influenced by the plant-soil
interaction such as soil acidity, nutrient content, and drought conditions. Toriman and Shukor
[21] found that in a forest area of Malaysia, interception reduces 23.9% of the total rainfall and
it is varying subjected to plant canopy, density and types of plants.
In our research findings, the plant density treatment (i.e. low, medium and high densities) of
the potential slope plant species, Melastoma malabathricum provided the significant findings on
the interception process at the sloping areas. A higher plant density increased the leaf area
index (LAI) (Figure 5) as well as contributed to a higher plant growth. In addition, the highest
plant density in the studied plots recorded the lowest erosion rate, indicating that soil erosion
was lower at the area with a higher vegetation density by intercepting rainfall by plant canopy
(LAI) (Figure 6).
Figure 5. The Leaf Area Index (LAI) of M. malabathricum of three different density treatments.
Furthermore, the higher the plant density, the higher the soil carbon content is. This result also
indicates that the increased of species density influenced in carbon cycle via storing the large
amount of carbon in soil through photosynthesis and respiration. It can be explained by the
large amount of litter fall on the soil surface, thus, enhanced the decomposition process which
in turns, increased the organic matter and mineral content at the top layer of soil. Apart from
that, the amount of soil carbon was directly related to the root length density (Figure 7). Higher
soil carbon content was observed at the greater root length density (RLD) area, indicating the
distribution of soil carbon was induced by the root distribution. The root system supplies
decomposable organic matter in soil and supports a large microbial community in the
rhizosphere [22,23], thus, help in distribution of soil carbon. In addition, a higher plant density
produced more litter on the top layer of the soil surface, which in turns increased the decom‐
position process via carbon and nitrogen cycles [24], hence, increased the soil pH value as well.
In aftermath, the soil pH was enhanced.
8 Soil Pollution
Figure 6. The erosion rate on slope at different plant density of M. malabathricum.
Figure 7. Soil carbon content and root length density (RLD) with depth of the species studied at different plant density.
3.2. Carbon sequestration and carbon sink potentiality
Carbon sequestration, a natural processes in ecosystems where CO2 is absorbed from the
atmosphere and stored it in plants and soil. During the photosynthesis process, plants
absorb CO2 and converted into carbohydrate or starch [25]. In this way, atmospheric carbon
is stored in the leaves, stems, and roots for a long period of time (Figure 8). When a tree
is utilized for wood, its ability to sequester carbon is extended, and the carbon is not
released until the product burns or decomposes. Vegetation plays an important role in
sequestrating carbon, as one way to alleviate global warming, a global issue discussed
nowadays. Whereas, carbon sink potential is defined as a natural entity, process, activity
or mechanism such as plants which can alleviate greenhouse gases from the atmosphere.
It has been reported that forest, for example the tropical rainforest, is one of the largest
carbon sinks in the world [26]. The higher the potential of the plant to absorb CO2, the
greater is the capacity of the plant to be a carbon sink potential. The quantitative meas‐
ure on the CO2 absorption by individual plant will assist to assess the carbon sink
potentiality of plants. The plants which exhibited high photosynthetic components i.e. Amax,
A400, light and CO2 saturation levels, are the good carbon sink plants.
Amax is an indicator of acclimatisation towards elevated CO2 and can be used to determine the
plant growth capacity in a future climatic situation. In our study, Leucaena leucocephala,
Peltophorum pterocarpum and Justicia betonica exhibited higher Maximum Assimilation Rate
(Amax) and Quantum Efficiency (QE) than Lantana camara and Thunbergia erecta (Table 3). From
the simulated CO2 experiments, L. leucocephala, P. pterocarpum and J. betonica seem to show the
ability to utilize high concentrations of CO2 in order to enhance photosynthetic rate. Further‐
more, in the simulated light experiments, no photo-oxidation occurs. It has been reported that
plants which can maintain the use of captured light energy for NADPH and ATP synthesis,
may provide more sink capacity. This ability, then, would diminish the accumulation of
excitation energy in the photosynthetic pigments, which is a major cause of photo-oxidative
damage. Thus, the capacity to resist photo-oxidation is an indication that L. leucocephala, P.
pterocarpum and J. betonica are the good potential carbon sink species. In addition, L. leucoce‐
phala and P. pterocarpum remain photosynthetically comparatively active at lower CO2
concentrations, indicating low CO2 is required to initiate the photosynthesis process of L.
leucocephala and P. pterocarpum. Changes in CO2 concentrations affected the photosynthesis of
both species similarly; L. leucocephala and P. pterocarpum seemed to show higher ability to utilize
high concentration of CO2 in order to enhance photosynthetic rate as compared to other species
studied. As inferred from these findings, L. leucocephala is a good carbon sink plant. Other
species also showed considerably higher carbon sequestration capacity in which they can be
regarded as supportive plants enhancing the carbon sink source when combine-grown on
slope.
4. Rehabilitation of acidic slope
Malaysia’s climate is described as typical tropical with warm, high rainfall intensity as well as
humidity throughout the year. As its experiences high precipitation ranging from 2000 mm to
2500 mm per annum, the highland areas especially slopes are prone to erosion and landslides
[10]. Due to the high rainfall and humid climate throughout the year in Malaysia, most of the
slopes are facing the acidity problem. The acidic and infertile condition of slope does not
encourage the vegetation establishment and consequently the slope turns barren. The barren
slope is prone to erode when rainfall dislodge soil particles and carries them off the acidic
slope. It would form rills and gullies which can trigger landslides. Rainfall hitting the soil
surface can also seal the soil particles and make a crust that prevents infiltration and creates
runoff.
10 Soil Pollution
Figure 8. The process of carbon sequestration [27].
Light response curves Carbon response curves

Plant
Amax Г1 QE Amax A400 gm ГCO2
species
(µmol m−2 s−1) (µE m−2 s−1) (µE m−2 s−1) (µmol m−2 s−1) (µmol m−2 s−1) (µmol m−2 s−1) (ppm)
LL 62 27 0.13 124 58 3 2
PP 36 85.5 0.1 80 34 2 9
JB 12.6 42.8 0.06 37.6 16.2 0.11 70
LC 9.3 55.9 0.06 16.7 10 0.08 48.5
TE 4.3 31.0 0.04 17.7 2.4 0.08 96.5
Maximum Assimilation Rate (Amax), Light Compensation point (Г1), Quantum Efficiency (QE), Photosynthesis at ambient
CO2 concentration (A400), Mesophyll conductance (gm), CO2 compensation point (ГCO2), LL: Leucaena leucocephala, PP;
Peltophorum pterocarpum, JB: Justicia betonica, LC: Lantana camara, TE: Thunbergia erecta
Table 3. Photosynthetic components of plants grown on slope [18]
Acid soil rehabilitation is an essential process of minimizing the acidity level of the soil and
providing a better environment for plant growth. This process also will help offset greenhouse
gas emissions, guarantee more food for an increasing population and contribute to the
economic progress of future generations. There are several methods used in rehabilitating
acidic soil. However, only three methods, which are liming, planting legumes and acid tolerant
plants will be discussed in this chapter.
4.1. Liming
Lime is a material that contains calcium (Ca) or magnesium (Mg) and will neutralize soil
acidity. Carbonates are the most available and widely used liming materials. Lime decreases
the acidity level of soil (increases pH) by changing the hydrogen ions of soil into water and
carbon dioxide (CO2) molecule. One calcium ion (Ca++) from the lime replaces two hydrogen
ions (H+) of soil complex. In addition, one carbonate ion (CO32-) reacts with water molecule
(H2O) to form bicarbonate ion (HCO3-). These react with hydrogen ion (H+) to form H2O and
CO2. Thus, the pH of soil increases due to the concentration of hydrogen ions (H+) has been
reduced (Figure 9).
Figure 9. Reduction of soil acidity (or H+ ions) by lime [28].
Regarding the mechanism of acid soil rehabilitation, liming changes the biological, structural
and chemical components of soils. Organic matter decay is slow in acid soils due to the low
activity levels of soil organisms. However, excessive liming rate would pollute the soil and
cause environmental hazards.
4.2. Planting legumes species
Legumes foster production of a greater total biomass in the soil by providing additional
nitrogen. Soil microbes use the increased nitrogen to break down carbon-rich residues of crops
like wheat or corn. Normaniza and Barakbah [1] introduced a planting technique and docu‐
mented that legumes plants showed high tolerance to acidic condition when planted with lime,
rock phosphate and sphagnum moss (Figure 10). This planting technique known as “Micro‐
climate Plant Propagation Technique” and the supplied chemicals (CaCO3, K2SO4 and MgSO4,
and rock phosphate) assist as a suitable plant supplement to enhance the plant growth.
Another advantage of tree legumes is their deep root systems, a characteristic which confers
persistence even on infertile soils [18]. Several legumes have aggressive taproots reaching six
to eight feet deep and half inch in diameter that open pathways deep into the soil. Legumes
contribute to an increased diversity of soil flora and fauna lending a greater stability to the
total life of the soil [18]. In conclusion, nitrogen-fixing abilities of legumes plants are important
for alleviating soil acidity, maintaining ecosystem fertility and long-term slope stabilization.
4.3. Planting Al-tolerant plants
Aluminium was the major factor for slope acidity and the presence of Aluminium was
unavoidable because it was a part of most clay particles. The mechanism of Al accumulator
12 Soil Pollution
plant to alleviate soil acidity has attracted the interest of plant ecologist and physiologist as
well as evolutionary biologist. In the presence of Al, the tolerant cultivars have efficiently
uptake and utilized Ca and P. The susceptible (Al-sensitive) and intermediate cultivars
exhibited less Ca and P uptake and utilization. The effect of Al on roots indicated that the
nutrient solution having Al at a concentration below 40 mM has stimulated root growth,
increasing the size and number of central cap cells. Beyond 60 mM, root growth was strongly
inhibited with cellular damage in peripheral root cap cells.
Figure 10. Plantation of legume seedling on slope [1].
Revegetation with Al-tolerant plants can be a valuable rehabilitation tool. Al-tolerant plants
can tolerate and accumulate high concentrations of Al in the shoot whereby the growth of the
plants was not affected by Al toxicity. Plants can deal with Al toxicity by setting up several
aluminium tolerance mechanisms. Therefore, on such Al-contaminated soil, planting Al-
tolerant plants plays increasingly important phytoremediation role. Proper management of
these kind of plants in acidic soil may significantly contribute to restoring the natural envi‐
ronment. On the other hand, most of Al-tolerant plants are shrubs for example, M. malabathri‐
cum. These kind of shrubs have woody root systems (M type) that give mechanical support to
slopes stability. Moreover, when M. malabathricum are planted with grass, they can help to
prevent sloughing of the shallow sod layer. The woody top growth also helps to stabilize
rehabilitated areas by reducing surface wind velocity. These shrubs also improve soil and
forest floors by drying them out, adding organic matter, and fix some nitrogen.
4.4. Tolerance mechanism of plants in acidic slope
Some tolerance mechanisms to ensure its survival and growth vary amongst the species. In
some cases, the plant produces small leaves as a response to acidic condition of the soil, for
example smaller and thinner leaves in L. leucocephala as observed in our research (Figure 11).
Figure 11. Leaf necrosis to reduce leaf area - a visual symptom of Aluminium toxicity, a mechanism to resist acidic
condition [18].
Additionally, this visual observation indicated that plants reduced leaf number by leaf wilting
and necrosis. This necrosis symptom is the sign of acid tolerance mechanism of plants, possibly
as a mechanism to reduce leaf area [18]. However, high leaf chlorophyll content was observed
in adapted plants in acidic soil and the plant seems to have recovered from the toxicity effect
by increasing the nodulation activity. In addition, leaf analysis of acidic and non-acidic treated
L. leucocephala showed that Aluminium concentration was higher by 36% in acidic treated than
those in non-acidic treated L. leucocephala, implying a high Al uptake of L. leucocephala in acidic
slope. The excessive accumulation of Al in leaf may indicate the mechanism of acidity tolerance
of this species. Moreover, the plant which exhibits Al concentration in leaf more than 1000 ppm
is reportedly called an Al accumulator, a mechanism of Al adaptation of a plant to acidity
(Figure 12). M. malabathricum accumulated the highest concentration of Aluminium, which
was almost 1850 ppm, which makes both species an Al accumulator (more than 1000 ppm)
and A. mangium is non-accumulator.
As similarly reported by Watanabe and Osaki [29], most of the plant samples (Evodia latifolia
and Justicia betonica) exhibited general symptoms of Al toxicity which includes curly young
leaves, reduced leaf number and necrosis, as mechanisms to reduce leaf area.
In relation to the Al concentration of the leaf, the soil pH grown with M. malabathricum
increased up to 6.0, meanwhile 5.5 and 5.1 for L. leucocephala and A. mangium, respectively
(Figure 13). The results imply a positive relationship between the concentration of Al in the
leaf and soil pH; as the Aluminium accumulation in leaf increased, the value of soil pH
increased as well. The experiment showed the importance role of plant as an Aluminium
accumulator in rehabilitating the acidic slope.
14 Soil Pollution
Figure 12. Al concentration of leaves of three species studied
Other possible tolerance mechanisms that could be identified in this project were increasing
root length, stomatal conductance and LAI (Figure 14-17). The interaction and compilation of
all tolerance mechanism contribute to the rehabilitation of the acidic soil. The photosynthetic
rate and chlorophyll content of Al-tolerant plants increased with the increasing of Al concen‐
tration. Therefore, as the Al concentration increased, the tolerance mechanism has also
enhanced by increasing the transpiration rate of plant.
Figure 13. Soil pH changes grown by treated plants of the three species studied.
Figure 14. Root length in sandy loam and acidic soil of three species studied
Figure 15. Leaf Area Index in sandy loam and acidic soil of three species studied
16 Soil Pollution
Figure 16. Stomatal conductance in (a) sandy loam and (b) acidic soil of three species studied
Figure 17. Tolerance mechanism of plants [30]

5. Potential slope plants
Numerous studies have been conducted to determine the plant species which are suitable as
slope plants. Normaniza and Barakbah [1] and Stokes et al. [31] referred that native plants
usually increased the success rate of the planting program and reduced the long-term main‐
tenance requirements. Karim and Mallik [32] suggested that the selected plants should be
adapted to local climate and be able to prevent landslides or erosion. Mafian et al. [12] showed
that the reinforcement of soil by vegetation is highly promising solution and this approach
would be more beneficial if the species acutely possessed the mechanical (through reinforce‐
ment of soils by plant root), hydrological (through reduction in runoff and by keeping the slope
relatively dry) and environmental (through the increase in carbon sequestration to counter the
rising carbon dioxide level in atmosphere) aspects.
Different plant species has different hydro-mechanical characteristics and can perform
different roles on slope but certain types of plants are better than others in terms of soil
reinforcement and surface protection [33]. Many problems may occur after planting any type
of plants which does not fulfill the slope plant characteristics. Therefore, selection of plant
species by observing the potential slope plant characteristics is crucial. A set of criteria was
formulated to select potential species for plantation on slope [1,9,16,31]. Physiological charac‐
teristics such as the high photosynthetic rate, transpiration rate and growth rate and root
profiles, such as high cellulose content in roots, fine roots, root biomass, root volume and root
length are considered as major criteria [14,16]. Additionally, the selected plant should exhibit
other prominent characteristics such as good plant-water relations and tolerance of wide range
of adverse condition with regard to soil acidity and water stress [9].
A list of potential tree and shrubs species was presented in Table 4. Based on the observations,
L. leucocephala and P. pterocorpum showed the higher bioengineering characteristics than A.
mangium and M. malabathricum. It was discovered that root architecture of L. leucocephala and
P. pterocorpum was VH and R type, respectively. The VH- and R type root architecture was
considered to be the most effective root system for slope stabilization and soil reinforcement
[34]. The H-types were found to be beneficial for wind resistance. The M-types are regarded
to be beneficial for controlling soil erosion. The extensive root growth and tensile strength L.
leucocephala and P. pterocorpum were claimed to be the cause of tremendous enhancement of
mechanical impacts on soil. Thus, in terms of root properties, L. leucocephala and P. pterocor‐
pum were more prominent to play a major mechanical role on soil and their high root tensile
strength would ultimately improved soil shear strength as well. These introduced tropical
plants will indeed assist eco-engineer to establish bioengineering technique on slope and
provide long-term soil reinforcement.
Many legumes, especially woody trees are particularly planted for controlling soil erosion,
slope stabilization and restoration in tropical countries [13]. Leucaena leucoephala and Peltopho‐
rum pterocorpum, have a potential to be slope plants. L. leucoephala is one of the most productive
fast growing, semi ever green and nitrogen fixing tropical legume trees. In Malaysia, L.
18 Soil Pollution
leucocephala is used as a potential slope pioneer and wind protection. It has aggressive taproots
reaching six to eight feet deep and half inch in diameter that open pathways deep into the soil
(Figure 18). Nodules on the root of plant can fix atmospheric nitrogen and this is perhaps the
most notable aspect that sets them apart from other plants. Additionally, P. pterocarpum is a
woody ornamental plant and has a R type root system [16] (Figure 19). This tree usually is
planted along roadsides, parks and slope. It has high atmospheric nitrogen-fixing potentiality.
Figure 18. Root profile of a potential slope plant- Leucaena leucocephala [18].
M. malabathricum produced the M type root system that makes it suitable to grow at slope area
(Figure 20). Acidic treated M. malabathricum showed a higher root length than non-acidic
treated, implying high water absorption to perform a basic metabolic process such as photo‐
synthesis. Plant released the absorbed water to the atmosphere by transpiring through pores
on the leaves. As a result, the excessive water were removed and resulted in a drier and more
stable slope. Moreover the flowering feature of M. malabathricum can help to enhance the flora-
fauna interaction of the slopes by increasing the biodiversity. Different species have different
mechanical characteristics and ranges acidic soil rehabilitation capacity. Potential slope plants
and their mechanical characteristics were shown in Table 4. Additionally, a list of potential
tree and shrubs species for planting in acidic slope was shown in Table 5.
Figure 19. Potential slope plant- Peltophorum pterocarpum [16].
Figure 20. (a) Plant profile, (b & c) root profile and (d) flowering feature of M. malabathricum.
20 Soil Pollution
Species Root pattern Type of root system Root tensile strength (MPa)
Leucaena
104
leucocephala
VH type VH type
Peltophorum
61
pterocarpum
R type R type
Acacia mangium 54
H type H type
Dillenia suffruticosa -
Heart
Melastoma
29
malabathricum
M type
M type
Table 4. Potential slope plants and their mechanical characteristics [13,16,35].

Species Genus Family Site (soil pH)
Bixa orellana Baxia Bixaceae >5
Leucaena leucocephala Leucaena Mimosoideae > 4, Moderate acidic
Acacia mangium Acacia Fabaceae > 4, Moderate acidic
Bauhinia purpurea Bauhinia Fabaceae > 4, Moderate acidic
Melastoma malabathricum Melastoma Melastomataceae < 3, Severe acidic
Thunbergia erecta Thunbergia Acanthaceae >5
Justicia betonica Justicia Acanthaceae >5
Lantana camara Lantana Verbenaceae >5
Hibiscus mutabilis Hibiscus Malvaceae >5
Vetiver zizanioides Chrysopogon Poaceae >5
Table 5. List of species for planting in acidic slope and classified by slope characteristic [13, 18].
6. General discussion
The observation of this study provides the key findings and contribution of tropical plants to
alleviate soil acidity and soil erosion. The bioengineering characteristics of selected tropical
plants have been intensively assessed to identify their potentiality towards slope stabilization.
This observation also revealed the contribution of pioneer species to enhance the process of
natural succession on slope. Amongst the tropical plants, few were suitable for reinforcing
slope and rehabilitation of acidic slope, exhibiting tolerance mechanism in soil acidic condition.
Based on the field studies, significant morphological and physiological changes were observed
in M. malabathricum in response to severe acidic (< 3) condition. These changes include the
improved photosynthetic rate, transpiration rate, LAI and root system. In addition, the highest
concentration of Al (> 1200 ppm) in the M. malabathricum leaves, either Al-treated or not, has
made this species the most suitable plant for severe acidic slope. This study also reveals an
alternative approach to alleviate the acidity. Besides liming, legume trees were recommended
for acidic slope rehabilitation due to their nitrogen-fixing abilities and extensive root systems
that can penetrate a deeper soil depth. The deeper the soil depth, the higher the soil pH (less
acidic), which makes this condition more conducive for the root establishment as well as the
plant growth as a whole [18].
In terms of the alleviation of soil erosion, the soil reinforcement of slope stability is mainly
depending on the properties of root systems of plant species. Root biomass, root architecture
and tensile strength were remarked as prominent engineering properties of plants to assess its
potentiality for soil reinforcement [13]. The extensive root growth and development of the
taproot (VH-type) of L. leucocephala has resulted in a tremendous enhancement of mechanical
effects on soil. As inferred from these findings, M-type root was suitable in protecting ground
22 Soil Pollution
cover from surficial erosion. In relations to this mechanical aspect, plant density also showed
an important contribution for controlling soil erosion. Research findings exhibited that higher
plant density would reduce the soil erosion rate, indicating a high soil-root interaction and
canopy interception. Apart from that, carbon sink potentiality of plants was also measured via
photosynthetic components of plants grown on slope. It can be envisaged that the outstanding
carbon sink potential of the slope plant community would confer numerous advantages, not
only to improve the global warming and sustain the ecosystem but also to produce more stable
and safe slopes.
7. Conclusion
The new information and findings presented in this chapter have mostly been achieved
through the bioengineering research, carried out in Malaysia. Aluminium accumulating plants
and new potential tropical slope plants are introduced and recommended for the revegetation
programme of the slope to alleviate soil acidity and erosion. These potential tropical plants’
attributions will be beneficial for enlisting database which can be further applied for ground
bio-engineering techniques in other tropical countries in the South-East Asia. It is our earnest
hope that the new discoveries and the outcomes of this knowledge would benefit the soil
science as well as other related disciplines.
Author details
Normaniza Osman*, Mohammed Saifuddin and Aimee Halim
Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur,

Malaysia
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chapter 19
Metal Contamination of Soils and Prospects of

Phytoremediation in and Around River Yamuna: A Case
Study from North-Central India
Manoj S. Paul, Mayank Varun, Rohan D’Souza,

Paulo J.C. Favas and João Pratas
1. Introduction
The rapid industrialization and intensive agricultural activities over the last few decades have
resulted in accumulation of various pollutants in the environment, which are distributed over
wide areas by means of air and water. This has caused visible detrimental effects to the
ecosystem and consequences to human health. Today, many soils throughout the world have
undesirably high concentrations of heavy metals. These include lead (Pb), cadmium (Cd), zinc
(Zn), mercury (Hg), arsenic (As), silver (Ag), chromium (Cr), copper (Cu), iron (Fe), and the
platinum group elements. At low or background concentrations, heavy metals are not
pollutants. They occur naturally in the environment due to their presence in bedrocks. Some
heavy metals such as Zn and Cu are also essential micronutrients for living organisms.
Therefore, the term heavy metal pollution refers to heavy metal levels that are abnormally high
relative to normal background levels. All heavy metals at high concentration have strong toxic
effects and are regarded as environmental pollutants.
Some heavy metals (like Fe, Zn, Ca and Mg) have been reported to be of bio-importance
to man and their daily medicinal and dietary allowances have been recommended.
However, some others (like As, Cd, Pb, and methylated forms of Hg) have been reported
to have no known bio-importance in human biochemistry and physiology and consump‐
tion even at very low concentrations can be toxic [1]. Even for those that have bio-impor‐
tance, dietary intakes have to be maintained at regulatory limits, as excesses result in
poisoning or toxicity [2]. Although individual metals exhibit specific signs of their toxici‐
ty, the following have been reported as general signs associated with Cd, Pb, As, Hg, Zn,
2 Soil Pollution
Cu and Al poisoning: gastrointestinal disorders, diarrhoea, stomatitis, tremor, ataxia,

paralysis, vomiting and convulsion, depression, and pneumonia when volatile vapours and
fumes are inhaled [3]. The nature of effects could be toxic (acute, chronic or sub-chronic),
neurotoxic, carcinogenic, mutagenic or teratogenic.
Pb, Zn, Cu, Co, Mn, Fe, Cr and Cd have been found in the streams and rivers of the Americas,
Europe, Asia, Africa and Australia [4-9]. In India, presence of heavy metals has been reported
in the Brahmaputra [10]; the Kali and Hindon [11]; and more recently, in the Gomti [12]; the
Cauvery [13]; and the Ganga [14].
The Yamuna (also Jamuna or Jumna) is the largest tributary of the Ganga in northern India,
having the total length of about 1376 km. The source of Yamuna is Yamunotri in the Uttarak‐
hand Himalaya, which is north of Haridwar in the Himalayan mountains. Yamuna river flows
through the states of Uttarakhand, Delhi, Haryana and Uttar Pradesh and finally merges with
river Ganges at a sacred spot known as Triveni Sangam in Allahabad. A number of prominent
cities such as Delhi, Mathura and Agra lie on the bank of river Yamuna. Over 57 million people
depend on the Yamuna waters. Just like the Ganges, the Yamuna too is highly venerated in
Hinduism and worshipped as goddess Yamuna, throughout its course.
Due to high density population growth, rapid industrialization, today Yamuna is one of the
most polluted rivers in the world, especially around New Delhi, where 15 drains discharge
waste water into the river. The city dumps ~58% of its waste into it. When the river enters the
city, it is already contaminated with 7500 coliform content per 100 ml. when it leaves the city,
it carries with a dangerously high coliform content of 24 million per 100 ml. Even the ground
water has been affected by leachates that pass down from the dumping sites. According to the
Central Pollution Control Board (CPCB), 70% of the pollution in river is from untreated sewage
and the remaining 30% is from industrial sources, agricultural run-off, garbage etc. The water
quality of Yamuna River falls under the category "E" which makes it fit only for recreation and
industrial cooling, completely ruling out the possibility for underwater life. Almost every year
mass death of fishes is reported. Biological Oxygen Demand (BOD) load increased by 2.5 times
between 1980 and 2005: from 117 tonnes per day in 1980 to 276 in 2005.
Although the government of India has spent nearly $500 million to clean up the river, the
river continues to be polluted with garbage while most sewage treatment facilities are
underfunded or malfunctioning. The Ministry of Environment and Forests (MoEF) of the
Government of India (GOI) took measures to curb pollution in 12 towns of Haryana, 8
towns of Uttar Pradesh, and Delhi under an action plan (Yamuna Action Plan-YAP) which
is being implemented since 1993 [15]. However in 2009, the Union government admitted
the failure of the Ganga Action Plan (GAP) and the Yamuna Action Plan (YAP), saying
that "rivers Ganga and Yamuna are no cleaner now than two decades ago" despite spending
over Rs 1, 700 crore to control pollution [16]. In August 2009, Delhi Jal Board (DJB) initiated
its plan for resuscitating a 22 km stretch of the Yamuna in Delhi by constructing intercep‐
tor sewers, at the cost of about Rs 1, 800 crore [17].
There are three main sources of pollution in the river, namely household and municipal
disposal sites, agricultural run-off, and industrial effluents and run-off. Urban runoff and
Metal Contamination of Soils and Prospects of Phytoremediation in and around River Yamuna: A Case Study... 3
agricultural runoff are mainly non–point sources. The major sources of pollution from
agriculture are fertilizers containing superabundant nutrients such as nitrogen and phospho‐
rus, and heavy metals such as Cd, Cu, Pb and Zn. Water quality may also be altered by other
factors, such as livestock manure, human waste, and atmospheric deposition. Atmospheric
pollutants are often the largest source of waterborne metals. It is estimated that 70% of lead in
water and over 50% of many of the other trace metals in the Great Lakes (USA) are derived
from atmospheric transfer. In general, freshwater ecosystems have low natural background
metal levels and therefore tend to be sensitive to even small additions of most trace metals.
Heavy metal contamination of soils and water from industrial and traffic sources in urban
environments has been studied in North America and Europe [18-22]. Agencies like the World
Health Organization (WHO) and the United states Environment Protection Agency (USEPA)
have set stringent standards for maximum permissible limits of heavy metals, but there is a
paucity of detailed studies on heavy metal pollution and its remediation within industrial
zones in developing countries. Yamuna outnumbers any other river in the number of indus‐
tries on its bank. This is because it passes through many major industrial cities. About 22, 42,
and 17 large and medium industrial units in the states of Haryana, Delhi, and Uttar Pradesh
have been identified as polluting the river in the action plan area. In addition, the water in this
river remains stagnant for almost 9 months in a year aggravating the situation.
According to the Agra District Industrial Centre officials, there were 226 iron foundries and
about 340 metal casting units functioning in Agra in the decade of 1990-2000. Before the revised
pollution control directives put the Agra diesel generator manufacturing industry off its track,
the foundry industry of this town ranked among the country’s largest assemblies of metal
casting industrial units, generating business of over Rs 6, 000 crores. The ban on coking coal
in the blast furnaces utilized by the foundry and metal-casting industry was a serious setback
and the number of industrial units reduced drastically. In August 1999, the Supreme Court
ordered the closure of 53 iron foundries and 107 other factories in Agra. In September 2010, it
again ordered the closure of 212 of the 1, 715 small industries that had failed to disclose their
toxic emission levels to the Uttar Pradesh Pollution Control Board (UPPCB). Another 299 were
required to install pollution controlling devices, failing which they too would face closure.
However, the ground realities are still nowhere near the reduced pollution levels targeted in
Yamuna and its adjacent areas whether Agra or elsewhere, after it leaves the Himalayan
foothills. The status quo, thus, ultimately leaves much to be desired.
Phytoremediation is an emerging technology that employs the use of green plants for the clean
up of contaminated environment. It takes the advantage of the fact that a living plant acts as
a solar-driven pump, which can extract and concentrate certain metals from the environment
[23]. This remediation method maintains the biological properties and physical structure of
the soil. The technique is environmentally friendly, cost-effective, visually unobtrusive, and
offers the possibility of bio-recovery of the metals. In the case of heavy metal contamination
in soil, phytoremediation techniques are narrowed down to Phytoextraction, where plants
remove metals from the soil by concentrating them in their harvestable parts [24], and
Phytostabilization, where plants reduce the mobility and bioavailability of pollutants by
immobilization [25].
4 Soil Pollution
Phytoremediation is becoming possible because of the successful basic and applied research
much of it conducted with the productive interdisciplinary cooperation of plant biologists, soil
chemists, microbiologists and environmental engineers. Extensive progress has been made in
characterizing and modifying the soil chemistry of the contaminated site to accelerate phy‐
toremediation. The greatest progress in phytoremediation has been made with metals [26,
27]. Phytoremediation leaves the topsoil in usable condition and it is aesthetically pleasing. It
requires minimal equipment and less energy inputs as plants do most of the work using solar
energy. Thus, it is an eco-friendly process. The plants used can later be harvested, processed
and disposed off in an environmentally sound manner. This technology has been receiving
attention lately as an innovative, cost-effective alternative to the otherwise tedious and
expensive methods in use which are not only a burden on the exchequer but also require efforts
on recurring basis.
Phytoremediation employing indigenous species can be an ecologically viable option for

sustainable and cost-effective management. Native plants often become adapted to locally
elevated levels of metals in soil at contaminated sites, e.g. mines and industrial zones [28-30]
and metal toxicity issues do not generally arise. Many native, well adapted plants have been
investigated and even used for heavy metal bioindicatoring and phytoremedial purposes
including lemongrass and other wild grasses, vetiver, Sesbania, Avena, Crotalaria, Crinum
asiaticum, Typha latifolia and Calotropis procera etc. [31-35, 28]. Native wild species are also
important to remediate soils in context of the studied area due to a remark (April, 2006) of the
Supreme Court prohibiting the cultivation of plants requiring fertilizers and pesticides along
the Yamuna. In the light of this limitation, native wild species are a viable option since these
do not require agronomic inputs.
Since the river Yamuna is the life line of Mathura and Agra, the existing pollution level has
posed a serious threat not only to the environment but also to the human population. Adjacent
areas are highly polluted and are a sink for a variety of chemicals including heavy metals. The
present study was undertaken: (i) to get a comprehensive profile of eight metals in water and
adjacent soils of the river Yamuna within Mathura, Agra and Bateshwar; (ii) to get a qualitative
and quantitative estimate of the species present at test sites through phyto-sociological
surveys; and (iii) to inventorize species with potential for phytoremediation present on sites
by comparing with those previously reported by the authors as suitable in this context.
2. Case study
Agra (27°10’N, 78°05’E, 169 msl), on the banks of the river Yamuna, is located in Uttar Pradesh
in the north central part of India. It is roughly 200 km south-east of the national capital, New
Delhi. Bounded by the Thar desert of Rajasthan on its south-east, west and north-west
peripheries, it is a semi-arid area. The world renowned Mughal monument, the Taj Mahal is
situated here. It is world renowned for its leather industry and marble handicrafts but it also
boasts a cast iron and engineering goods industry. Mathura (27.28°N 77.41°E) is located
approximately 60 km north of Agra and 145 km south-east of Delhi. According to Hindu
scriptures Mathura is the birthplace of Lord Krishna. It is a fast expanding city with about half
a million residents. Mathura oil refinery is one of the biggest oil refineries of Asia. Textile
printing, dyeing and silver ornament manufacturing are major industries. Apart from these
there are units manufacturing taps, household items, and cotton materials. Bateshwar (26.93°N
78.54°E) is a village on the banks of Yamuna about 120 km downstream from Agra. It is an
important spiritual and cultural centre for Hindus.
Figure 1. Map of the study area
The study area was divided into three zones (Figure 1); all three along the course of Yamuna
and covering two cities viz. Mathura (zone 1) and Agra (zone 2) and a large village i.e.
Bateshwar (zone 3). The distance between zones 1 and 2 is 80 km and zones 2 and 3 is 120 km
downstream. In all, a total distance of 200 km was covered along the course of river. In each
zone, 5 sites were selected ~1 km apart. Five random soil samples were taken from 0-15 cm
depth at each site. A total of 75 soil samples (25 from each zone) were analyzed in order to
obtain a complete profile. The same number of river water samples was collected from
midstream at a depth of about 0.3 m. Soil from the botanical garden of St. John’s college, Agra,
was utilized as control.
The statistical significance of differences among mean metal content in water and soil was
independently determined by one-way analysis of variance (ANOVA) followed by Fisher’s
LSD test. Pearson’s coefficient for correlation of water and soil data was analyzed at a signif‐
icance level of P < 0.05 and P < 0.01 with SPSS 16.0 statistics software.
6 Soil Pollution
3. Physico-chemical profile
3.1. Water
Physico-chemical properties of the water samples collected from the study zones are men‐
tioned in Table 1. The pH values indicate neutral nature of river water acceptable as per BIS
[36] and WHO [37] guidelines. A reading of 6.5 to 7.5 is considered neutral, suitable for general
plant growth [38]. Conductance which reflects the status of major ions/inorganic pollution and
is a measure of total dissolved solids and ionized species in the water, varies between 434 –
503 µmho/cm. Total dissolved solids were highest in zone 2. The hardness of water body is
regulated largely by the levels of Ca and Mg salts. Other metals if present such as Fe, Al and
Mn may also contribute to hardness. Most parameters were within their respective acceptable
limits [36, 37]. Electrical conductivity was low. High COD, BOD and low DO in zones 1 and 2
are due to the discharge of huge amount of the untreated urban and industrial wastewater/
effluents indiscriminately. All three zones were faecally contaminated. Bacterial contamina‐
tion ranged from19000—93000 coliform/100ml; the values are much higher than recommended
values of 1coliform/100ml. Most of these coliforms were of faecal type due to gravity discharge
of faecal wastes in adjacent areas along the river.
Acceptable
Parameters Zone 1 Zone 2 Zone 3
Limits [36, 37]
pH (1:2.5) 7.31 7.23 7.61 6.5-8.5
Total Dissolved Solids (mg/l) 266 314 245 500
Conductivity (µmho/cm) 462 503 434
N-NO3 (mg/l) 3.54 4.37 4.11 10
N-NH3 (mg/l) 2.34 2.36 1.63 10
Total hardness (mg/l) 227 223 210 250
Total alkalinity (mg/l) 203 188 209
Chemical Oxygen Demand (mg/l) 23.5 24.3 11.3
Chloride (mg/l) 8.5 8 9 250
Fluoride (mg/l) 0.37 0.37 0.32
Dissolved Oxygen (mg/l) 5.63 - 8.67 >5
Biological Oxygen Demand (mg/l) 9.63 10.7 5.34
Sodium (mg/l) 6.81 6.62 7.15
Potassium (mg/l) 0.38 0.4 0.38
Ca hardness (mg/l) 124 131 115
Mg hardness (mg/l) 83 96.4 91.6
Faecal coliforms (MPN/100ml) 86000 93000 19000
Streptococcus (MPN/100ml) 64000 71000 17000
Table 1. Physico-chemical profile of water

3.2. Soil
The soil of the study area is characterized by alluvium, which is an admixture of gravel, sand,
silt and clay in various proportions deposited during the quaternary period. The area is a part
of Indo-Gangetic alluvium of quaternary age and is made up of recent unconsolidated
fluviatile formations comprising sand, silt, clay and kankar with occasional beds of gravel. The
topsoil is coarse and angular sand with small clay fraction. The sub-soil is sandy throughout.
The stabilized topsoil is reddish brown with sand and clay mixed. The minimum depth of
topsoil layer is 60 cm.
Physico-chemical properties of soil samples are given in Table 2. The topsoil in the study area
is sandy loam (sand 60-80%, silt 10-24%, clay 8-16%). It has high exchangeable sodium
percentage (ESP) values and moderate water retaining capacity. The sub-soil is sandy through‐
out. Soil pH ranged from neutral to alkaline. Zones 3, 2 and 1 were classified as very low, low
and medium in organic matter, respectively.
Electrical Organic Avail. Avail. Avail.

pH
Zone Conductivity Matter Phosphate Potash Nitrogen
(1:2.5)
(dS/m) (1:2.5) (%) (kg/ha) (kg/ha) (kg/ha)
1 7.06-7.12 0.46-.50 1.44-1.53 108-115 236-298 53.4-60.8
2 6.24-6.81 0.33 0.38 0.8-1.2 131-140 65-94 50.1-55.2
3 7.43-7.6 0.44-0.47 0.5-0.72 50-65 143-178 75.2-87.8
Control 7.21 0.54 1.68 70.5 393 112.9
Table 2. Physico-chemical profile of soils
The electrical conductivity (EC) of soils ranged from 0.33-0.54 dS/m. Zone 1 and 2 soils fall in
very high (>100 kg/ha), soils from zone 3 and control site in the high (50-100 kg/ha) phosphate
availability bracket. Soils from zones 1, 3 and control displayed medium (130-330 kg/ha) potash
levels while zone 2 was low (<130 kg/ha) in available potash. Nitrogen content in the soil
samples ranged from 50.1 – 112.9 kg/ha.
4. Heavy metal profile
4.1. Water
Concentrations of heavy metals in the water samples collected from different location have
been summarized in Table 3. It is clearly evident from the table that heavy metals were
consistently higher in zone 2 compared to zones 1 and 3. Cr content was markedly higher
among the metals in zone 2 followed by zone 1. The concentration of heavy metals in water
samples ranged from 0.018 – 0.095 mg Pb l-1, 0.025 – 0.341 mg Cd l-1, 0.47 – 1.76 mg Zn l-1, 0.27
8 Soil Pollution
– 1.58 mg Cu l-1, 0.001 – 0.005 mg Co l-1, 0.80 – 9.37 mg Cr l-1 and 0.078 – 0.32 mg Ni l-1. As was
not detected in any sample.
Zone Pb Cd Zn Cu Co Cr Ni
Range 0.025-0.041 0.05-0.136 0.7-1.02 0.86-0.98 0.002-0.004 2.87-4.23 0.078-0.12
1 Avg. 0.036a 0.088a 0.868a 0.916a 0.003a 3.55a 0.097a
SD 0.007 0.037 0.128 0.046 0.0005 0.540 0.016
Range 0.066-0.095 0.159-0.341 1.37-1.76 1.27-1.58 0.004-0.005 6.42-9.37 0.17-0.32
2 Avg. 0.082b 0.243b 1.56b 1.40b 0.005b 7.914b 0.256b
SD 0.013 0.072 0.166 0.112 0.001 1.138 0.071
Range 0.018-0.028 0.025-0.03 0.47-0.61 0.27-0.33 0.001-0.002 0.8-0.97 0.009-0.015
3 Avg. 0.023c 0.028c 0.54c 0.3c 0.001c 0.864c 0.01c
SD 0.004 0.004 0.058 0.042 0.0003 0.066 0.003
F value * * * * * * *
Permissible WHO [37] 0.01 0.003 5 2 - - -
limits USEPA [39] 0.015 0.005 5 1.3 - - -
World Average 0.004 0.001 0.2 1.4 - - -
#
As content below detection limit.
F value :“∗” statistically significant. Different letters in the same column denote significant statistical difference
(P≤0.001) in mean metal contents in water samples from different zones
SD- Standard deviation.
WHO – World Health Organization.
USEPA – United States Environment Protection Agency.
Table 3. Heavy metal content of water samples (mg L-1)
All the metals in water samples were positively (P<0.01) correlated with each other (Table 4).
In other words, metal concentration trends were identical and increased simultaneously for
Pb, Cd, Zn, Cu, Co, Cr, and Ni.
Cd Zn Cu Co Cr Ni
Pb 0.970** 0.985** 0.875** 0.924** 0.960** 0.962**
Cd 0.977** 0.902** 0.944** 0.954** 0.963**
Zn 0.925** 0.953** 0.976** 0.966**
Cu 0.963** 0.940** 0.899**
Co 0.961** 0.930**
Cr 0.947**
** Correlation is significant at the 0.01 level (two-tailed) (two-tailed; n=75)
Table 4. Correlation coefficients: water heavy metal concentrations

Higher concentrations of metals in zone 2 (Figure 2) may be attributed to the discharge of

industrial effluents from various sources including untreated sewage, municipal waste and
agrochemical runoff from the nearby villages directly into the river. The concentrations of Co
and Ni were found to be negligible at all sites. Due to the neutral to alkaline nature of river
water, most of the heavy metals have precipitated and settled as carbonates, oxides, and
hydroxide bearing sediments and elevated levels indicates higher exposure risks to the benthic
biota of the river. Based on the WHO [37] and USEPA [39] drinking water standards (Table
3) the results in the present investigation show that Pb, Cd and Cr at all sites and Ni at most
sites far exceeded the prescribed limits. Cu values from zone 2 were above the USEPA [39]
threshold. One Way ANOVA and Fisher’s LSD test indicate the difference in mean content of
each metal among zones was highly significant statistically (P ≤ 0.001).
Figure 2. Average heavy metal content in water samples
When compared with the metal profile of the rivers around the world (Table 5) the situation
does not seem that desperate here, at least as far as heavy metal contamination is concerned.
The picture, however, is quite different when we consider the WHO guidelines for drinking
water and World average of trace elements in unpolluted rivers [56, 57], the concentration
ranges of Pb and Cd were well above the international guidelines and acceptable concentra‐
tions for drinking water (Table 3). When compared to the world average of trace elements for
unpolluted rivers, the river was considered polluted by Pb, Cd, Zn and Cu.
10 Soil Pollution
Rivers Pb Cd Zn Cu Co Cr Ni References
Yamuna river (present 0.018-0.09 0.001-0.00

0.05-0.341 0.47-1.76 0.27-1.58 0.8-9.37 0.009-0.32
study) 5 5
Cauvery river, India 13.35 - 47.51 4.57 8.25 1.01 4.53 [13]
Brahmaputra river, India - - 916 108 168 222 179 [10]
Ganga river, India 76.36 11.5 332.5 48.39 - 5.36 4.88 [14]
Gomti river, India 3.058 - 63.022 - - 0.064 0.013 [12]
Challawa river, Nigeria 0.44 - 1.2 0.22 - 0.47 - [40]
Mghogha river, Morocco 48.25 0.36 299.5 56.7 - 86.4 46.83 [41]
Sava river, Croatia 34 0.5 91 24 - - - [42]
Pasig river, Philippines 70 - 530 - 160 - 21.2 [43]
Rhine river, Netherland 188.2 7.1 684.3 62.5 6.4 33.7 [44]
Zhujiang, China 75.2 - 212 51 17.8 70.6 61.8 [45]
Almendares river, Cuba 93 2.5 262 158 - 90 - [46]
Montevideo, Uruguay 44-128 1-1.6 174-491 58-135 - 79-253 - [47]
Ribeira river, Brazil 767102 0.2-5.5 15-5090 60 - - [48]
Amazon river, Brazil 83 - 110 37.5 - 65 26.7 [49]
Danube river, Serbia

28.65 3.12 253.74 36.29 - 76.26 70.1 [50]
and Montenegro
Msimbazi river, Tanzania - 0.9 79 14 - 12 8.7 [51]
Brisbane River, Australia 20.1-81.9 1.9 40.8-144.0 31.1-30.2 - 14.2-54.3 - [52]
Siahroud river, Iran 9.7 0.05 14.9 - - 1.03 - [53]
Gediz River, Turkey 1.3 - 2.6 - 1.6 - 4 [54]
Avg. shale value/ world

20 0.3 95 45 90 68 [55]
avg.
Table 5. Average heavy metal concentrations of rivers around the world (mg L-1)
4.2. Soil
Concentrations of heavy metals in the soil samples have been summarized in Table 6. Quan‐
titatively the metals were observed in the sequence Pb > Zn > Cr > Ni > Cu > As > Cd > Co
(Figure 3), though their thresholds for concern, mobility in soil and toxicity are different so
this trend does not necessarily reflect the threat of individual metals. Pb and Zn were found
in fairly higher concentrations at all the sampling locations. Generally, an overall linear
increasing trend of metal contamination was noted from site 1, before the Yamuna enters the
city of Mathura, to site 10 where the river leaves Agra. Thus, maximum values for all metals
were observed in the samples pertaining to Agra. In the third zone metal concentrations were
seen to decrease gradually. One-way ANOVA and Fisher’s LSD test indicate that mean Pb and
Co content was different at all sites (P ≤ 0.001); while mean Cr, Cd, Cu, Ni, and As in zone 2
differed significantly from zone 1 and 3 (P ≤ 0.001). The latter did not differ significantly among
themselves. Mean Zn content in zone 1 differed significantly from zone 2 and 3 (P ≤ 0.05). The
difference between the latter was not significant statistically.
Zone Pb Cd Zn Cu Co Cr Ni As
Range 157-230 8.6-20.6 87.3-136 22.4-41.5 1.84-4.8 26.3-53.2 23.1-41.2 14.2-20.4
1 Avg. 200a 13.4a 116a 30.5a 3.71a 40.7a 33.6a 17.2a
SD 29.6 5.36 21.4 8.14 1.25 9.77 6.78 2.97
Range 241-285 17.8-25.2 129-222 50.4-64.2 5.91-15.2 76.3-104 57.8-71.3 22.2-28.6
2 Avg. 261b 20.3b 173b 57b 9.6b 86.7b 63.5b 25.2b
SD 16.1 3.13 37.6 5.93 3.80 10.5 5.44 3.10
Range 111-136 6.02-14.6 115-167 22.4-30.1 2.91-6.4 14.7-45.6 17.8-32.4 7.9-17.6
3 Avg. 125c 10.2a 144b 25.7a 4.95c 28.1a 23.3a 13.8a
SD 10.4 3.71 21.9 3.43 1.29 11.8 5.57 4.01
Range 13.6-18.4 1.23-1.87 39.6-54.3 12.8-24.3 1.68-2.53 10.2-14.3 7.3-9.7 3.03-5.7
Control Avg. 15.6 1.6 47.4 18 2.1 12 8.6 4.2
SD 1.88 0.25 5.3 4.53 0.31 1.55 1 0.99
F value * * * * * * * *
Suggested thresholds
Industrial 600 22 360 91 - 87 50 12
in soil [58]
Residential 140 10 200 63 - 64 50 12
Suggested thresholds
Background - - 140 - - 100 35 29
in soil [59]
Intervention - - 720 - - 380 210 55
Class I 35 0.2 100 35 - 90 40 15
Threshold values [60] Class II 250 0.3 200 50 - 150 60 30
Class III 500 1.0 500 400 - 300 200 40
F value :‘*’ statistically significant. Different letters in the same column denote significant statistical difference (P≤0.05)
in mean metal contents in soil samples from different zones.
SD- Standard deviation.
Table 6. Heavy metal content of soil samples (mg kg-1)

12 Soil Pollution
Figure 3. Average heavy metal content in soil samples
All the metals in soils were positively (P<0.01) correlated with each other (Table 7). Significant
negative correlation was observed between metal concentrations and soil pH (P<0.01). The
same was observed in the case of Zn and Co with Organic matter. Phosphate is able to increase
water-soluble lead forms from contaminated soils by 56.8– 100% [61]. This is clearly shown by
the phosphate values (Table 2) obtained for different samples with maximum in zone 2
followed by zone 1 which probably led to higher Pb values in zones 1 and 2 (Table 6). Fertilizers
contain from trace to several ppm of Pb, Zn, Cu, Mg [62, 63]. High P2O5-blended fertilizers and
the pure phosphates, contain significant concentrations of several elements of potential
environmental or agronomic concern [62, 64].
Cd Zn Cu Co Cr Ni As OM pH
Pb 0.821** 0.479** 0.889** 0.629** 0.909** 0.933** 0.894** 0.426** -0.802**
Cd 0.701** 0.886** 0.724** 0.862** 0.848** 0.906** 0.0876 -0.621**
Zn 0.723** 0.915** 0.689** 0.653** 0.744** -0.493** -0.371**
Cu 0.805** 0.966** 0.972** 0.932** 0.0260 -0.773**
Co 0.809** 0.784** 0.806** -0.304** -0.529**
Cr 0.991** 0.944** 0.0816 -0.834**
Ni 0.936** 0.138 -0.837**
As 0.106 -0.739**
OM -0.242*
* Correlation is significant at the 0.05 level (two-tailed; n=75)
** Correlation is significant at the 0.01 level (two-tailed)
Table 7. Correlation coefficients: soil heavy metal concentrations

Agra is the fourth most populated city in Uttar Pradesh, India. With a population of 1.7 million
(2011 census) it generates about 700 tonnes of solid wastes every day. It is also a major cause
for adding contamination to soil and groundwater. Solid waste is also discharged from 200
hospitals and nursing homes along with 168 foundries, 52 tanneries, 300 shoe industries, 200
petha (a local sweet) manufacturing units, 50 dairies, 56 electroplating units, 15 silver vibrators
and 15 galvanizing units. Significantly higher amount of metal pollution in the samples from
the city (sites 6-10) is obviously due to untreated domestic/wastewater, sewage and industrial
effluent discharged at these sites throughout the year. The increasing contamination as one
proceeds downstream mirrors the extent of damage caused to the pedosphere.
Mean concentrations of heavy metals in soils at the sites studied were compared with threshold
values of soil suggested by the Canadian Environmental Quality Guidelines [58]. It was
observed that As (sites 1-13) and Ni (sites 6-10) crossed their respective industrial thresholds
while the other metals (Pb, Zn and Cu) are well within it. Mean concentrations of As at sites
4-10 were approximately twice the thresholds suggested. Cd and Cr levels were above their
thresholds only at site 10. However, the situation is drastically different in the perspective of
the residential limits where in addition to these, the thresholds are exceeded even by Pb, Cd
(10 sites each), Cr (5 sites) and also Zn and Cu at one site.
On comparing metal concentrations with the values suggested for soil remediation by VROM,
Netherlands [59], values of Zn (sites 7-13), Ni (sites 4-10) and Cr (site 10) were above the
background values but below the intervention level. It is significant to note that in studies
similar to the present one, the degree of contamination and the resulting ‘hazard indices’ for
soils may vary when different thresholds, existing in only a few countries, are considered [65].
To increase the reliability of risk estimation due to contaminants, global consensus on such
thresholds is urgently needed.
The concentrations of As are usually low, less than 6 ppm, for geological and soil environment
[64]. It is estimated that about 60% As in the environment is from anthropogenic sources
including As-based pesticides, fertilizers, and wastes from mines, smelter and tannery
industries [66]. The relatively high values of As in the samples seem to be directly related to
the discharge of domestic and industrial effluent as well as use of phosphate fertilizers,
pesticides used in the agricultural activities in the region.
Highly significant positive correlation (P<0.01) was observed between soil and water content
of Pb, Cd, Cu, Co, Cr and Ni. The results also indicate that metal concentrations in soil were
higher than those in the water. This distribution pattern of heavy metals between the water
phase and soil is expected as most heavy metal speciation studies have reported a similar
pattern of distribution both in sea water as well as in lakes [67-69].
Several authors have pointed out the need for a better knowledge of urban soils [18, 70]. In the
past few years, studies on urban soils in many cities have been carried out around the world.
Some examples are Spanish [19, 71] and Italian cities [21, 72]. Other examples for European
cities are Aberdeen [73], Athens [74], Oslo [22] and Belgrade [18]. The mean heavy metal
contents for all zones are compared in Table 8 to those of some cities around the world. The
differences concerning population, living habits, industrial activities, etc., cause significant
14 Soil Pollution
differences in the metal contamination profile. Compared to average concentrations in urban

soils in the world, the mean concentrations of Pb and Cu are up to 2—4 times higher in some
cases but still less than London, Naples and Palermo. In the case of Cd, it is many times higher
than Kattedan (India). Zn and Cr contents do not differ much; still they are less than those of
Naples and Madrid. Ni content is more than almost all European cities, but less than Kattedan
and Firozabad in India. Co values are less than those reported from other industrial regions
of India. As content is less than that of Firozabad.
City Pb Cd Zn Cu Co Cr Ni As Reference
London 294 - 183 73 - - - - [75]
Madrid 161 - 210 72 - 75 14 - [76]
Rostock 83 - 100 35 - 48 30 - [77]
Sevilla 161 - 107 64.6 - 42.8 23.5 - [19]
Belgrade 53.2 - 129.1 29 - 33.2 67.4 - [18]
Palermo 253 - 151 77 - 39 19.1 - [72]
Naples 262 - 251 11 - 74 - - [21]
Nanjing 107.3 - 162.6 66.1 - 84.7 - - [78]
Hong Kong 93.4 - 168 24.8 - n.a. - - [79]
Kattedan 195-6241 0.08-0.16 130-3191 72-1450 12-36 77-586 63-494 0.10-0.21 [80]
Firozabad 35.5-781 3.64-107 76.4-1247 22.4-300 10.9-63.7 19.1-158 23-218 9.25-204 [29]
Zone 1 200 13.4 116 30.4 3.70 40.7 33.6 17.2
Present
Zone 2 261 20.3 173 57.0 9.60 86.7 63.5 25.2
Study
Zone 3 125 10.2 144 25.7 4.96 28.1 23.3 13.8
Table 8. Average heavy metal concentrations in urban soils from different cities across the world (mg kg−1)
It is encouraging to note that the mean concentrations of individual metals are below those
reported from other industrial hubs within India i.e. Kattedan (Andhra Pradesh) [except Cd
and As] and Firozabad (Uttar Pradesh). Kattedan Industrial Development Area (KIDA) is a
major industrial area of Andhra Pradesh and houses 400–500 industries, including 150 large
scale industries and 300 small-scale industries. Major sources of metals pollution are battery,
electrode, oil refining, metal plating, textile, pharmaceutical, chemical paints, rubber, petro‐
chemicals, glass, therapeutics, and Pb extraction facilities [81]. This is also one of the contami‐
nated areas identified by the Central Pollution Control Board (CPCB) in New Delhi, and
referred to as an ecological disaster area [81]. Firozabad is the hub of the Indian Glass industry.
5. Assessment of heavy metal contamination in soil
Assessment of soil contamination was performed by the contamination index (Pi) and inte‐
grated contamination index (Pc) as expressed by fuzzy functions [82, 29, 28]. Class I criteria [60]
could be used as no-polluted threshold; Class II as lowly polluted threshold value; and while
Class III as highly polluted threshold value. Pi values ≤ 1 indicate no contamination; 1 ≤ Pi ≤ 2
indicates low contamination; 2 ≤ Pi ≤ 3 indicates moderate contamination; while Pi > 3 indicates

high contamination.
Individual elements displayed remarkably different patterns of accumulation in soils.

Furthermore, observed differences in the magnitude of accumulation suggest that the relative
contribution of the individual elements to total heavy metal contamination varies. Figure 4
shows the proportions of contamination levels (from Pi values) in the soil samples from all the
sites studied. Except for 76% samples from zone 2, which showed moderate Pb contamination,
the rest exhibited low contamination zone as did all samples from zones 1 and 3. In case of Cd,
all samples were in the high contamination zone. For Zn, 24% samples from zone 2 were
moderately contaminated while 72%, 76%, and 100% samples from zones 1, 2, and 3, respec‐
tively were in the low contamination range. For Cu, 88% samples from zone 2 were moderately
contaminated while 36% and 12% samples, from zones 1 and 2, respectively were in the low
contamination range. All samples from zone 3 indicated no contamination. Except for zone 2
(20% samples) in the low contamination zone, the remaining samples did not indicate Cr
contamination. For Ni, 12% and 72% samples from zones 1 and 2, respectively were moderately
contaminated while 16% and 22% samples from, respectively were in the low contamination
range. All samples from zone 3 indicated no contamination. In the case of As, 64%, 100%, and
60% samples from zones 1, 2, and 3 were in the low contamination range.
Figure 4. Contamination indices (Pi) of heavy metals in soil samples
Thus, zone 1 was found to be lowly contaminated with Pb, Zn, Cu, Ni and As but highly
contaminated with Cd. Zone 2 exhibited low to moderate contamination of Pb, Zn, Cu, Ni; low
Cr and As contamination; and high Cd contamination. Zone 3 was lowly polluted with Pb and
Zn. As contamination ranged from none to low. No Cu, Cr and Ni contamination was observed.
These results agree with the findings regarding metal contamination of soil due to the glass
industry at Firozabad, India [29]. Of the nine elements studied, Zn, Cd, and As showed a
16 Soil Pollution
greater accumulation in all soils, whereas, accumulation of Ni and Cu was high in limited
samples.
Integrated Contamination Indices (Pc) were calculated for all soils to assess the extent of heavy
metal contamination at the sites. Pc is defined as the summation of the difference between the
contamination index for each metal and 1 (one). It is categorized under the following heads:
Pc ≤ 0 no contamination; 0 ≤ Pc ≤ 7 low contamination; 7 ≤ Pc ≤ 21 moderate contamination; Pc
> 21 high contamination. Threshold values for Co could not be obtained hence this metals was
excluded in the calculation. A clear ascending trend is visible in the Pc values for all sites (Figure
5). Pc values generally show a moderate to high contamination at studied sites. The Pc indices
indicate that 45% sampling locations fall in the moderate contamination while 55% of the
samples fall in the high contamination range. All the samples from zone 2 fell under the high
contamination category. While in zone 1, 60% samples come under moderate and 40% under
high contamination level category. In zone 3, 76% and 24% samples were in the moderate and
high contamination range, respectively.
50
40
30
Pc
20
10
0
Zone 1 Zone 2 Zone 3
Figure 5. Integrated contamination indices (Pc) of soil samples. Black and gray lines are the upper threshold values of
moderate and low contamination, respectively
The effect of the glass industry on urban soil metal characterization was assessed at 25 test
sites at Firozabad, India [29]. The area is characterized by little or no monitoring of industrial
processes, usage and disposal of hazardous chemicals. A comprehensive profile of Zn, Mn,
Co, Cd, Pb, Cr, Ni, Cu and As contamination was obtained. Zn, Cd, and As showed a greater
accumulation in all soils, whereas, accumulation of Ni and Cu was high in limited samples.
Integrated contamination indices (Pc) indicate that 60% of the sites were in the high contami‐
nation range and 28% were in the moderate contamination range with just 12% sites on the
border of the moderate to low contamination range. [83] assessed the impact of both landuse
and soil textures on Cd, Zn, Pb and Cu based on samples collected from the major landuse/
landcover pattern of Dutch forests and aerable soils drawn from six different sites. Metal
content in agricultural and industrial soil is found to be higher than the forest soil.
The fact that no Pc value in the present investigation fell within the low contamination range
was not surprising, given the fact that the study was being carried out in an area which has
already been contaminated with metals, but moderate to high indices in zone 1 and 2 are
alarming because these include heavily populated areas. The local populace is, thus, exposed
to wide range of historically well established toxins and even carcinogens. The situation is
surely compounded by vehicular pollution at urban sites (1-10). Vehicular emissions are a
significant source of many pollutants [21, 84].
6. Phytosociological studies
Plants show differing morpho-physiological responses to soil metal contamination. Most are
sensitive to very low concentrations; others have developed tolerance, and a reduced number
accumulate metals. The latter capacity has practically opened up the way to phytoextraction.
Hyperaccumulation is an unusual occurrence, seen in a narrow range of species which often
grow in metal-rich soils. The following thresholds for metal hyperaccumulation in shoots,
without evident symptoms of toxicity, have been suggested [85]: 100 mg kg-1 for Cd, 1, 000 mg
kg-1 for Ni, Cu, Co and Pb, and 10, 000 mg kg-1 for Zn and Mn. Known hyperaccumulators are
generally minor vegetation components in most European and North American habitats.
Currently, more than 400 hyperaccumulator species are known, belonging to 45 different
botanical families, among which the most frequent are Brassicaceae and Fabaceae [86].
Lack of information on the agricultural management of hyperaccumulators, together with
slow-growing and poor shoot and root growth, increase the difficulties in the practical
application of these species in remediation projects [87]. Hence, the potential for any plant
species to remediate successfully heavy metal contaminated sites depends on all of the
following prerequisite factors: a) the amount of metals that can be accumulated by the
candidate plant, b) the growth rate of the plant in question, and c) the planting density [88].
The growth rate of a plant in a chemically contaminated soil is important from the perspective
of biomass. Parameters like basal area, canopy, abundance, dominance of species can help
obtain a more rounded picture in the case of mixed planting or natural flora at a contaminated
site. The rate of metal removal from the soils can be calculated if information on the above
mentioned parameters is available. In addition, versatility of the candidate plant to tolerate
and at the same time accumulate multiple metal contaminants and/or metal-organic mixtures
would be an asset for any phytoremediation system.
The choice of plant species is thus, an important task in any phytoremediation based technique.
Decontaminating a site in a reasonable number of harvests requires plants that are both high
18 Soil Pollution
yielders of biomass and good metal accumulators by dry weight. It has been demonstrated [89,
90] that, wild native plants may be better phytoremediators for waste lands than the known
metal bioaccumulators like Thlaspi caerulescens and Alyssum bertolonii because the latter are
slow growing with shallow root systems and low biomass. Also, the technology for their large-
scale cultivation is not fully developed; therefore, their use is rather limited [91].
If soil at contaminated sites, e.g. mines, industrial zones is naturally high in a particular metal,
native plants will often become adapted over time to the locally elevated levels [28-30] and
metal toxicity issues do not generally arise. Successful establishment and colonization of
several pioneer plant species tolerant to Pb/Zn mine spoils has also been demonstrated with
tolerant plants including Phragmites australis, Vetiveria zizanioides, and Sesbania rostrata [31, 92].
Many native, well adapted plants have been investigated and even used for heavy metal
bioindicatoring and phytoremedial purposes including lemongrass and other wild grasses,
vetiver, Sesbania, Avena, Crotalaria, Crinum asiaticum, Typha latifolia and Calotropis procera etc.
[31-35]. Phytoremediation employing indigenous species can be an ecologically viable option
for sustainable and cost-effective management.
An important component of any ecosystem is the species it contains. Species also serve as good
indicators of the ecological condition of a system [93]. Ecological surveys are necessary for an
adequate characterization of a plant community and also to know the diversity and dispersion
status of species in the area. Phytosociology aims to characterize and classify plant commun‐
ities in terms of composition and structure.
At all sampling sites within a zone, ecological indices [relative frequency, relative density,
relative dominance and importance value index (IVI)] were estimated, by using a 1m2 quadrat.
Sampling was done randomly at 10 spots at each site within a zone. The data were compiled
and analysed according to some workers [94-96].
Relative density is the proportion of density of a species (plants/unit area) to that of the stand
as a whole. The dispersion of species in relation to that of all the species is termed as relative
frequency of a species. Relative dominance is the proportion of the basal area of a species to
the sum of the basal area of all species present. Basal area refers to area covered by the plant’s
stem and leaves one inch above the ground surface. The overall picture of ecological impor‐
tance of a species in relation to the community structure can be obtained by adding the values
of the above three parameters [97].
A total of 22 weed species were recorded from the sites (Table 9). Most of the weeds recorded
are herbs except Calotropis procera and Datura stramonium which are shrubby in nature. Two
grasses i.e. P. annua and C. dactylon were observed. The phytosociological parameters obtained
from the sites clearly indicate that there are naturally occurring plant species which have the
capacity to tolerate the heavy metal content of the soils. The floral composition of the three
zones varied qualitatively and quantitatively. Most species were seen to grow vigorously.
Relative frequency, relative density, relative dominace and IVI indicate that Calotropis procera,
Parthenium hysterophorus, Chenopodium murale, Croton bonplandianum, Rumex dentatus, Amar‐
anthus spinosus, Datura stramonium and Withania somnifera were the most abundant weeds. All
of these species have been reported as potential phytoremediators in earlier studies. It is
important to note that floral diversity decreased with increasing contamination profile of the
sites. Maximum species (20) were observed in zone 3, followed by zones 1 and 2.
Relative Relative Relative

Zone Plants IVI
frequency density dominance
Amaranthus spinosus 8.42 10.55 0.15 19.12
Rumex dentatus 6.32 6.00 0.07 12.38
Calotropis procera 8.42 10.31 0.18 18.92
Croton bonplandianum 6.32 6.00 0.23 12.54
Chenopodium murale 6.32 6.24 0.01 12.56
Datura stramonium 7.37 9.83 0.16 17.37
Stellaria media 5.26 4.32 0.00 9.58
Withania somnifera 8.42 9.59 0.13 18.15

1
Heliotropium ellipticum 5.26 3.12 0.00 8.38
Achyranthes aspera 6.32 5.76 0.01 12.08
Parthenium hysterophorus 7.37 6.95 0.01 14.33
Amaranthus alba 5.26 4.56 0.01 9.83
Boerhaavia diffusa 3.16 2.64 0.01 5.80
Euphorbia hirta 6.32 6.00 0.00 12.31
Sida longifolia 4.21 4.56 0.00 8.77
Gnaphalium luteo-album 5.26 3.60 0.02 8.88
Abutilon indicum 7.25 4.75 0.06 12.06
Rumex dentatus 11.59 13.65 0.15 25.39
2 Withania somnifera 8.70 8.61 0.10 17.40
Cynodon dactylon 7.25 3.56 0.00 10.81
Sida longifolia 7.25 2.97 0.00 10.21
Boerhaavia diffusa 4.35 5.64 0.00 9.99

20 Soil Pollution
Relative Relative Relative

Zone Plants IVI
frequency density dominance
Abutilon indicum 4.76 3.46 0.01 8.23
Cynodon dactylon 4.76 2.91 0.06 7.74
Poa annua 4.76 3.28 0.01 8.05
Rumex dentatus 3.97 1.64 0.01 5.62
Barleria diffusa 4.76 4.55 0.00 9.32

3
Sida longifolia 4.76 3.64 0.00 8.41
Withania somnifera 4.76 3.83 0.09 8.68
Boerahvia diffusa 4.76 2.55 0.00 7.31
Sida cordifolia 4.76 3.28 0.00 8.04
Euphorbia hirta 3.17 3.46 0.00 6.64
Ageratum conyzoides 3.17 2.37 0.00 5.55
Datura stramonium 4.76 4.19 0.00 8.95
Tridex procumbens 3.17 4.01 0.00 7.18
IVI- Importance Value Index
Table 9. Phytosociological parameters of flora at test sites
C. procera has been demonstrated as a potential phytoremediator species. The shrub showed
good accumulation of metals and is a potential phytoextractor for As and Zn as well as a
promising phytostabiliser for Pb, Cd, Cu and Mn [28, 29, 35]. C. procera was observed to have
a high degree of sociability i.e. relative frequency, relative density, relative dominance and IVI.
P. hysterophorus was also important in this context and was most dominant in zone 2. This
species has been identified for As phytoextraction along with A. spinosus, C. bonplandianum,
and D. stramonium [28]. The latter two have also been indicated for phytoextraction—C.
bonplandianum for Mn and D. stramonium for Mn, Cr, and Cu—together with R. dentatus for
Pb. Another species with high IVI, C. murale has been suggested for Zn, Cd, Pb and Cu
phytoextraction [28, 29]. Among the less dominant species, Tridex procumbens and Euphorbia
hirrta have also been reported as promising tools for phytoextraction of Mn and As, respec‐
tively [28]. E. hirrta and D. stramonium were not found in zone 2.
Poa annua has been identified as a phytostabilizer for Mn, Cd, and As and phytoextractor
for Cu and Pb. Cu concentrations up to 742.06 mg kg-1 dry weight have been reported in
P. annua shoots [28, 29]. Poa annua was observed only at sites in zone 1.
Other species found at the sites have also been indicated for further studies following initial
field surveys. Gnaphalium luteo-album (Mn and As); Withania somnifera (Cu); and Heliotropium
ellipticum (As) have shown promise as phytostabilisers for these metals and metal combina‐
tions [28, 29].
7. Discussion
The occurrence as well as concentrations of heavy metals like Pb, Zn, Cu, Co, Mn, Fe, Cr and
Cd in streams and rivers all over the world is increasing. In the present case study, heavy metal
contamination was consistently higher in city of Agra, which may be attributed to the heavy
industrialization combined with agricultural and urban runoff. The situation is made worse
by atmospheric deposition, again attributable to industrial and vehicular pollution. In general,
freshwater ecosystems have low natural background metal levels and therefore tend to be
sensitive to even small additions of most trace metals. The river water far exceeded the limits
of metals prescribed by WHO and USEPA for drinking water standards and Pb, Cd, and Cr
content at all sites and Ni at most sites exceeded the prescribed limits. In a heavily populated
country like India where a sizeable portion of the population is illiterate and resides in slums/
poorly planned neighbourhoods without proper sanitation and drainage, day-to-day activities
also contribute to the overall pollution load. Provision of suitable alternatives along with
proper education and awareness is integral to the minimization of this problem at the source.
Apart from taking measures like effluent treatment before it enters the river and subsequent
treatment of river water at the most polluted sites, a steady flow of water is to be ensured
throughout the year, by way of channelizing the river with canals at crucial points. Such
measures can address this problem to a substantial extent. Expenditure of more than US$ 500
million without much success appears to be an unjustified proposition.
Phytoremediation has been receiving attention lately as an innovative, cost-effective alterna‐
tive to the otherwise tedious and expensive methods in use, which are not only a burden on
the exchequer but also, require efforts on a recurring basis. Lack of information on the
agricultural management of hyperaccumulator species, together with their poor biomass and
root proliferation, increases the difficulties in their practical application. It has been amply
demonstrated that wild native plants may be better phytoremediating tools. These species can
be an ecologically viable option for sustainable and cost-effective management especially in
scenarios where expertise, technical expertise and/or funding is a limiting factor. Ecological
surveys are necessary for adequate characterization of a plant community and subsequent
identification of prospective candidates for phytoremedial strategies since metal toxicity issues
generally do not arise in plants already established on contaminated soils. Allowing native
22 Soil Pollution
species to remediate site is an attractive proposition since these species do not require frequent
irrigation, fertilizers, and pesticide treatments, while simultaneously a plant community
comparable to that existing in the vicinity can be established. The outcome is, thus site
remediation, ecological restoration and addition in aesthetic value. This is also in concurrence
with the ruling (2006) of the Hon’ble Supreme Court of India prohibiting cultivation of plants
requiring fertilizers and pesticides along the river Yamuna. Using these perennial phytore‐
medial candidates without any special needs holds much promise in this context. In addition,
versatility of the candidate plant to tolerate and at the same time accumulate multiple metal
contaminants and/or metal-organic mixtures would be an asset for any phytoremediation
strategy.
Acknowledgements
Financial support from University Grants Commission [F. no. 35-47/2008(SR)] is gratefully
acknowledged. This study was partially supported by the European Fund for Economic and
Regional Development (FEDER) through the Program Operational Factors of Competitiveness
(COMPETE) and National Funds through the Portuguese Foundation for Science and Tech‐
nology (PEST-C/MAR/UI 0284/2011, FCOMP 01 0124 FEDER 022689.
Author details
Manoj S. Paul1*, Mayank Varun1, Rohan D’Souza1, Paulo J.C. Favas2,4 and João Pratas3,4
1 Department of Botany, St. John’s College, Agra, India
2 Department of Geology, School of Life Sciences and the Environment, University of Trás-os-
Montes e Alto Douro, Vila Real, Portugal
3 Department of Earth Sciences, Faculty of Sciences and Technology, University of Coimbra,

Coimbra, Portugal
4 IMAR-CMA Marine and Environmental Research Centre, Faculty of Sciences and

Technology, University of Coimbra, Coimbra, Portugal
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chapter 20
Continuous and Induced

Phytoextraction — Plant-Based Methods to
Remove Heavy Metals from Contaminated Soil
Maciej Bosiacki, Tomasz Kleiber and

Bartosz Markiewicz
1. Introduction
As a result of the continuous civilization progress and the increasing human population we
have been observing an accelerating process of environmental pollution, frequently leading
to its complete degradation. The primary cause of environmental pollution is associated with
the rapid development of motorization and industry (particularly power industry and
mining), progressing urbanization, improved standards of living worldwide, intensive
farming (application of high amounts of mineral fertilizers and herbicides), along with
numerous other anthropogenic factors. These sources contribute to increased concentrations
of many chemical elements and compounds in the atmosphere, soil, water and plants (includ‐
ing crops with edible parts for human consumption).
The cycle of chemical elements and compounds in nature is influenced not only by human
activity, but also nature itself, in which progressing geological processes occur such as volcanic
eruptions, shifts of tectonic plates or natural disasters.
In recent years the ecological awareness of the general public has increased, with decontami‐
nation of the polluted environment being perceived as an essential requirement. The aim of
reclamation of polluted areas is to restore ecosystems polluted by human activity to the
condition resembling their former natural state.
There are many methods applied in environment decontamination worldwide, including

const-intensive, conventional physico-chemical methods. Scientists continue to search for
novel, more effective and economically viable methods of pollutant inactivation. In recent
2 Soil Pollution
years an increasing body of research has focused on engineering bioremediation, such as e.g.
an in situ technology of phytoremediation, utilizing the capacity of plants to accumulate high
amounts of pollutants from the environment. Research conducted for many years now has
identified a relatively numerous group of plants potentially applicable in this new environ‐
ment decontamination technology.
At present a major ecological problem is connected with the penetration to the environment
of heavy metals, which at higher concentrations are strongly toxic to humans and animals
(resulting in e.g. increased disease incidence), and have a negative effect on soil properties as
well as quality and physiological activity of plants.
Literature sources present various definitions of an element to be considered a heavy metal.
One of these hypotheses says that they are elements with specific gravity greater than 4.5, 5, 6
and 7 g cm-3. It is a physical term, which is understood and used differently in various contexts.
There are also many definitions based on atomic number. Some of them are considered to be
nutrients for living organisms (essential nutrients e.g. Fe, Cu, Zn, Mn, Ni) and others are
redundant or toxic (e.g. Cd, Pb, Hg, Al, As). Their common characteristic is connected with
the fact that at excessive concentrations in the environment they have an adverse effect on
plant growth and development, and when incorporated in the food chain may also pose a
hazard for animals and humans.
2. Soil factors influencing availability of heavy metals to plants
Physico-chemical properties of soil influencing contents of phytotoxic forms of heavy metals

include the type of soil, its grain size distribution, reaction, organic matter content, sorption
properties and redox potential [43, 67, 68, 73].
Mother rock is a natural source of heavy metals in soils. The amounts of elements coming
from mother rock constitute the geochemical background posing no threat to soil fertility
[67]. Other sources of heavy metals include geochemical processes and anthropogenic
factors. In soil heavy metals are subjected to processes affecting their concentrations and
chemical forms [4]. In individual soil horizons their content depends on anthropogenic and
climatic factors [67, 114].
Soil reaction is a major factor influencing the form and availability of heavy metals to plants
[67]. Soil acidity may lead to their increased concentrations in soil [3, 13, 30, 48, 93, 112] and
their excessive uptake by plants [42, 93]. According to Tyler and Olsson [118], concentrations
of Cu and Pb increase also at pH 7.5 – 7.8 as a result of formation of stable complexes with
ligands, which solubility is connected with solubility of organic substance. Liming results in
a reduced content of available forms of heavy metals in soil [39]. Soil reaction is a factor
determining the force with which heavy metals are bound by organic substance and mineral
compounds [2, 60].
Organic substance found in soil contributes to the limitation of the amounts of heavy metals
available to plants [38, 47], since it binds very strongly Cr3+, Fe3+, Pb2+, Ni2+ and Co2+ ions and
Continuous and Induced Phytoextraction — Plant-Based Methods to Remove Heavy Metals from Contaminated Soil 3
to a lesser extent also Mn2+ and Zn2+ [68]. Organic matter binds heavy metals into water
insoluble forms or forms sparingly soluble in water [79], thus reducing the share of plant-
available forms of heavy metals [19], and in this way it limits their toxicity to plants [48, 87].
Heavy metals differ in the force, with which they are bound by the sorption complex and they
are connected mainly with the silt fraction [67]. In soil they undergo exchange and biological
sorption. They may also be precipitated in the form of insoluble compounds [83]. Availability
of heavy metals in soil is influenced by the cation sorption capacity. Introduction of compounds
enhancing sorption capacity to soil causes a reduction of amounts of available metal forms in
soil [106].
Availability of heavy metals is also dependent on the redox reactions taking place in soil [83].
Oxide forms of heavy metals become readily available to plants at a low redox potential [33].
The application of mineral fertilizers changes amounts of phytoavailable forms of heavy metals
in soil. The effect of used fertilizers on physico-chemical and biological properties of soil causes
a reduction or an increase of amounts of phytotoxic forms in the soil environment [105]. When
applying mineral fertilizers we introduce heavy metals to soil, which contents in fertilizers are
connected both with the raw material from which they were produced and the technological
process of fertilizer production. The greatest contamination with heavy metals, particularly
cadmium, is found in phosphorus fertilizers [52] and calcium fertilizers, mainly being by-
products of various branches of industry [27, 69]. Heavy metal contents in phosphorus
fertilizers depend on the fertilizer type [52] and solubility [57]. The application of phosphorus
fertilizers leads to the transition of soluble phosphate forms into sparingly soluble zinc, copper,
cadmium and lead phosphates [30], leading to the reduction of heavy metal contents in plants.
3. Admissible heavy metal contents in soil binding in Poland
In Poland respective boundary heavy metal contents are specified in the Ordinance of the
Minister of the Environment of 9 September 2002 on soil quality standards and land quality
standards. These standards were established taking into consideration the current and
forecasted functions for the following categories of land types (Table 1):
1. Category A:
a. Landed property incorporated into an area legally protected on the power of the
regulations of the Water Act,
b. Areas protected on the power of regulations on nature protection if maintenance of

the current soil pollution levels does not pose a threat for human health or the
environment – for these areas the concentrations meet the standards resulting from
the actual status, subject to points 2 and 3;
2. Category B – land classified as agriculturally utilized area except for land covered by
ponds and ditches, forested areas as well as areas covered by trees and shrubs, barren
4 Soil Pollution
land, as well as developed and urbanized areas except for industrial areas, surface mining
land in use and municipal areas;
3. Category C – industrial areas, surface mining land in use, transportation areas.
Attachment to the Ordinance of the Minister of the Environment of 9 September 2002 (item
1359)
Category B Category C
Depth [m below ground level]
0-0.3 0.3-15.0 >15 0-2 2-15

Pollutant Category A
Water permeability of soil [m/s]
Up to below Up to below Up to below
1∙10-7 1∙10-7 1∙10-7
Arsenic 20 20 20 25 25 55 60 25 100
Barium 200 200 250 320 300 650 1000 300 3000
Chromium 50 150 150 190 150 380 500 150 800
Tin 20 20 30 50 40 300 350 40 300
Zinc 100 300 350 300 300 720 1000 300 3000
Cadmium 1 4 5 6 4 10 15 6 20
Cobalt 20 20 30 60 50 120 200 50 300
Copper 30 150 100 100 100 200 600 200 1000
Molibdenum 10 10 10 40 30 210 250 30 200
Nickel 35 100 50 100 70 210 300 70 500
Lead 50 100 100 200 100 200 600 200 1000
Mercury 0,5 2 3 5 4 10 30 4 50
Table 1. Admissible concentrations in soil (mg kg-1 dry matter)
Based on the multiannual analyses the Institute of Soil Science and Plant Cultivation in Puławy,
Poland (IUNG) specified boundary heavy metal contents in soils to be utilized agriculturally
(Table 2). As it was reported by Ociepa et al. [94], the mean content of heavy metals in
agriculturally utilized soils in Poland is lower than in countries of Western Europe or the USA,
which results mainly from a lesser share of farms with intensive agricultural production
systems and a lesser intensity of industrial processes. The same authors reported that approx.
90% agriculturally utilized area have natural levels of toxic metals. Several percent of this area
have elevated contents (I0) of mainly cadmium and zinc, while approx. 3% are polluted (II-V0)
with metals. The greatest percentages of polluted soils are found in the Śląskie, MałoPolande
and Wrocławskie provinces of Poland.
Soil pollution rates

Metal Soil class*
0 I II III IV V
a 30 70 100 500 2500 >2500

lead
b 50 100 250 1000 5000 >5000
(Pb)
c 70 200 500 2000 7000 >7000
a 50 100 300 700 3000 >3000

zinc
b 70 200 500 1500 5000 >5000
(Zn)
c 100 300 1000 3000 8000 >8000
a 15 30 50 150 750 >750

copper
b 25 50 80 100 500 >500
(Cu)
c 40 70 100 150 750 >750
a 10 30 50 100 400 >400

nickel
b 25 50 75 150 600 >600
(Ni)
c 50 75 100 300 1000 >1000
a 0, 3 1, 0 2 3 5 >5
cadmium
b 0, 5 1, 5 3 5 10 >10
(Cd)
c 1, 0 3, 0 5 10 20 >20
Pollution rate: 0 – natural content, I – elevated content, II – weak pollution, III – medium pollution, IV – strong pollution,
V – very strong pollution
*soil classes:
a. Very light soils with low silt and clay contents ( < 10%), irrespective of pH (in KCl)
- light soils (10 - 20% silt and clay), highly acid (pH < 4.5) and acid (pH 4.5 – 5.5)
b. Light soils (10 - 20% silt and clay), neutral reaction (pH > 6.5)
- medium soils (20 - 35% silt and clay), highly acid (pH < 4.5) and acid (pH 4.5 – 5.5)
c. heavy soils (>35% silt and clay), highly acid (pH < 4.5)
- mineral organic soils (organic substance at 6 - 10%)
d. Medium heavy soils (20-30% silt and clay) and heavy soils (> 35%).
Table 2. Boundary values in mg kg-1 heavy metals in the surface layer (0 – 20 cm) of soils with different degrees of
contamination (IUNG 1992)
4. Contents of heavy metal soluble forms and reaction of surface horizon

(0 - 20 cm) of soils in green belts adjacent to selected transportation routes
in the city of Poznań (western Poland)
Anthropopressure affects physical, biological and chemical properties of soil. Soils in urban
areas, located along transportation routes, are exposed to heavy metal pollution, originating
from substances produced during combustion of fuels, abrasion of road surfaces and tires,
6 Soil Pollution
granular materials falling onto the ground during transport, etc. [24, 36]. Platinum metals,
which have been employed in production of car catalysts, reach environment and cause
contamination of soil, plants and water [84]. Moreover, chemical substances used in winter to
eliminate black ice (e.g. sodium or calcium chlorides) as well as deposition of dusts and water
migration of elements contribute to soil degradation and deterioration of plant growth
conditions. In urban areas strong alkalization of soil is frequently observed, which significantly
reduces contents of soluble forms of metallic components [12, 27, 74].
According to the Ordinance of the Minister of the Environment of 9 September 2002 on soil
quality standards and land quality standards currently binding in Poland (the Journal of Law
Dziennik Ustaw no. 165, item 1359), soil category B comprises soils in urbanized areas, for
which the admissible heavy metal level (mg kg-1 dry matter) in the upper 0 - 30 cm layer is Ni
100, Cd 4, Pb 100 and Cr 150, respectively.
Legal regulations pertain to total heavy metal contents in soils; however, many authors claim
that it is not always a direct indicator of their bioavailability [44]. In the opinion of Gorlach
and Gambuś [53], the most appropriate measure is to assess soil contents of soluble forms of
trace elements, as they may be absorbed by plants.
The aim of the studies conducted by the authors was to determine what amounts of soluble
forms of heavy metals are available for plants in the 0 - 20 cm layer of soil in green areas located
in the vicinity of selected transportation routes in the city of Poznań, Poland. Collected soil
samples were tested for soil reaction and contents of selected heavy metals (cadmium, lead,
chromium and nickel, classified as metallic micronutrients, essential elements at the same time
having a negative effect on plants when found in greater amounts).
Material and methods
Soil samples from green areas were collected in October 2012 in the vicinity of transportation routes in the city of
Poznań, Poland. Forty five streets were selected for analyses and soil samples were collected using an Egner sampling
stick from a 0 - 20 cm layer at a distance of 0.5 - 2.0 m from the roadway. Along each analyzed street 4 bulk samples
were collected, comprising 15 individual samples (4 x 15 = 60 individual samples). Heavy metals (Cd, Pb, Cr and Ni)
were extracted from soil using Lindsay’s solution containing in 1 dm3: 5 g EDTA (ethylenediaminetetraacetic acid), 9
cm3 25% NH4OH solution, 4 g citric acid and 2 g Ca(CH3COO)2·2H2O. Next they were assayed by flame atomic
absorption spectroscopy (FAAS) with an AAS 3 Zeiss apparatus. Active acidity expressed in pH (H2O) was determined
by potentiometry (soil : water = 1:2), [50].
Results were analyzed determining their minimum, maximum and mean values (in the case of pH its logarithmic value
was considered), standard deviation, coefficients of variation and empirical distribution for individual chemical
parameters. Mean results of chemical analyses are presented in Table 3, while stem-and-leaf displays are given in Figs.
1 - 2.
In these analyses soil pH fell within the range of 4.32 – 8.26, while its coefficient of variation
was as low as 9% (Table 3). For most plants optimal soil reaction is pHH2O 6.0 – 6.5. Only 2.2%
soils had a highly acid reaction (at pH 4.3 – 5.0), whereas a vast majority had an alkaline reaction
pH>7.4 (46.7% soil samples) and a neutral reaction (37.8%), comprising jointly 84.5% samples
(Figure 1). Elevated soil pH is connected with an excessive content of alkaline ions, i.e. calcium,
magnesium, sodium and potassium. Alkalization of urban soils may result e.g. from the use
of salts such as sodium chloride to remove black ice from roadways, as well as strongly
alkalizing dusts (containing e.g. CaO, MgO, Na2O, K2O), being by-products of carbon com‐
bustion to heat houses [27, 74]. In turn, Kleiber [74] to a much greater degree suggests strong
alkalization of soil (pH in H2O up to 10.95) as a factor having a potentially negative effect on
plant growth and development.
In the opinion of Klimowicz and Melke [75], in urban areas traffic-related pollution is more
dangerous than industrial pollution, since it is spread in relatively large amounts, at low
heights, in the respiration zone of humans, animals and plants. Relief features, distance from
a roadway and intensity of vehicle traffic have a decisive effect on the contents of heavy metals
in soils adjacent to transportation routes in urban green areas. In the soil found at the car market
in Słomczyn near Warsaw (one of the biggest car markets in Poland) contents of zinc, lead and
copper are as follows (in mg kg-1 d.m.): the car sale point Zn at 612.3, Pb at 397.8 and Ni at 94.5,
while at the spare part sale point and warehouse they are: Zn at 679.1, Pb at 420.4 and Ni at
114.0, respectively [115].
Soil pollution with heavy metals is typically assessed on the basis of total contents of elements
[44]. Those authors claimed that this assessment should be supplemented with an analysis of
heavy metal contents directly available to living organisms.
This study consisted in the determination of contents of soluble heavy metal forms (Table 3).
Cadmium content in soils fell within the range of 0.16 – 0.42 mg Cd dm-3 and it was of relatively
limited variability (CV=17.50%). As many as 55.1 % samples contained this heavy metal at
<0.21 - 0.26 mg Cd dm-3 (Figure 2). Bach [9] in soils of green areas adjacent to transportation
routes in the city of Krakow, Poland found contents of soluble cadmium forms to range from
0.21 to 1.54 mg kg-1 d.m. soil. In most tested soils Bach [9] recorded cadmium content from 0.4
to 0.8 mg kg-1 d.m. soil. According to Kabata-Pendias and Pendias [67], natural total cadmium
content (the so-called background) in Polish soils is 0.3 mg Cd kg-1 d.m.
Lead content ranged from 0.79 to 42.96 mg Pb dm-3, at the same time being highly variable
depending on the location (CV=110.78%). As many as 84.4% tested soils had low contents of
this heavy metal (up to 9.22 mg Pb dm-3), while samples with 42.96 mg Pb dm-3 accounted for
2.2.% (Figure 2). A low lead content (max. up to 4.3 mg Pb dm-3) was reported in his study by
Kleiber [74]. Lead content (soluble forms) in soils adjacent to transportation routes in the city
of Krakow ranged from 11.1 up to 142.8 mg kg-1 d.m. soil, of which the highest proportion
(53%) comprised soils with its contents from 40 to 80 mg Pb kg-1 d.m. soil. Mean content of
bioavailable Pb forms in soils of allotment gardens located in the right-bank Warsaw was 332.7
mg kg-1 d.m. [41].
The primary source of lead pollution in soils (adjacent to transportation routes) up to 2005 was
connected with tetraethyl lead, commonly added to gasoline as an antiknock agent [62]. In
turn, the authors of this chapter in their analyses recorded a low content of soluble lead content
in the surface soil layer in areas adjacent to transportation routes in the city of Poznań, Poland.
8 Soil Pollution
No lead pollution in soils adjacent to exit routes leading from Poznań was detected by Hofman
and Wachowski [62].
In turn, chromium content ranged from 0.26 to 0.67 mg Cr dm-3. The coefficient of variation
for chromium was similar to that calculated for nickel, amounting to CV 22.34%. The greatest
proportion (44.4%) of soils was found within the range of chromium contents from < 0.36 to
0.46 mg Cr dm-3 (Figure 2). Results of analyses conducted by Bach [9] indicate that the content
of chromium (soluble forms) in soils in areas adjacent to transportation routes in the city of
Krakow ranged from trace values to 10.23 mg Cr kg-1 d.m.
The recorded nickel content ranged from 0.43 to 1.25 mg Ni dm-3. The variability of levels for
this metal was relatively medium-ranged (CV=23.19%). Most of soil samples (84.4%) were
characterized by nickel contents falling within the range of values from < 0.43 to 0.63 mg Ni
dm-3 (Figure 2). In soils found in green areas adjacent to transportation routes in Krakow,
Poland the content of nickel soluble forms ranged from 1.07 to 6.38 mg kg-1 d.m. soil [9]. In
contrast, in soils collected from allotment gardens located in right-bank Warsaw the mean
content of bioavailable nickel forms was 28.4 mg kg-1 d.m. [41].
Environmental pollution with heavy metals constitutes a serious problem in some regions of
Poland. According to Dmochowski et al. [41], high emissions of heavy metals originating from
a dense network of transportation routes with high intensity vehicle traffic causes their
accumulation in soils and crops produced in allotment gardens located in Praga Południe, the
right-bank district of Warsaw, Poland.
In the opinion of Heck et al. [59], introduction of advanced catalytic systems, containing
platinum, rhodium and palladium and constituting a source of environmental pollution also
for soils in areas adjacent to transportation routes makes their monitoring a necessary practice.
In the nearest future it will be required to create an effective system of environmental pollution
monitoring.
pH Contents (mg kg-1 d.m.)

No. Street
(H2O) Ni Cd Pb Cr
1 28 czerwca 56 r. 7.67 0, 49 0, 23 14, 81 0, 31
2 Aleje Niepodległości 7.58 0, 51 0, 28 10, 74 0, 33
3 Aleje Solidarności 7.37 0, 58 0, 27 3, 36 0, 49
4 Arciszewskiego 7.30 0, 48 0, 20 6, 55 0, 41
5 Armii Poznań 7.72 0, 59 0, 21 42, 96 0, 55
6 Biskupińska 7.76 0, 52 0, 23 2, 18 0, 46
7 Dolna Wilda 8.26 0, 44 0, 18 4, 00 0, 38
8 Droga Dębińska 7.37 0, 48 0, 23 6, 76 0, 34


No. Street
(H2O) Ni Cd Pb Cr
9 Fredry 7.30 0, 62 0, 33 6, 83 0, 60
10 Głogowska 6.86 0, 62 0, 31 3, 56 0, 57
11 Hercena 7.00 0, 58 0, 21 1, 91 0, 46
12 Hetmańska 7.45 0, 43 0, 19 3, 92 0, 40
13 Jarochowskiego 7.60 0, 55 0, 24 3, 24 0, 46
14 Kopanina 6.64 0, 73 0, 26 7, 45 0, 39
15 Królowej Jadwigi 6.55 0, 86 0, 22 8, 70 0, 35
16 Krzywoustego 6.98 0, 58 0, 28 4, 31 0, 57
17 Księcia Mieszka I 7.17 0, 50 0, 23 18, 89 0, 39
18 Kuźnicza 6.29 0, 70 0, 28 6, 17 0, 43
19 Lechicka 8.18 0, 55 0, 25 2, 89 0, 52
20 Leszczyńska 7.63 0, 57 0, 28 3, 46 0, 67
21 Lodowa 7.72 0, 51 0, 21 3, 10 0, 48
22 Maczka 7.48 0, 60 0, 30 6, 07 0, 56
23 Małopolska 7.47 0, 62 0, 29 2, 97 0, 64
24 Marcelińska 7.89 0, 55 0, 26 2, 78 0, 59
25 Niestachowska 5.84 0, 71 0, 25 10, 97 0, 38
26 Nowowiejskiego 7.14 0, 49 0, 19 3, 02 0, 36
27 Opieńskiego 7.32 0, 47 0, 22 0, 79 0, 39
28 Opolska 7.76 0, 62 0, 28 3, 65 0, 64
29 Ożarowska 7.50 0, 51 0, 24 3, 76 0, 41
30 Piątkowska 7.40 0, 50 0, 26 2, 64 0, 46
31 Piłsudskiego 7.50 0, 60 0, 26 2, 10 0, 57
32 Polanka 6.45 0, 60 0, 24 5, 73 0, 41
33 Poznańska 6.99 0, 44 0, 16 2, 49 0, 26
34 Przemysłowa 7.36 0, 60 0, 28 2, 54 0, 60
35 Serbska 7.79 0, 60 0, 24 2, 34 0, 63
10 Soil Pollution

No. Street
(H2O) Ni Cd Pb Cr
36 Słowiańska 6.87 0, 63 0, 23 5, 31 0, 29
37 Stablewskiego 7.55 0, 57 0, 23 3, 28 0, 46
38 Starołęcka 7.12 0, 63 0, 29 6, 01 0, 53
39 Stróżyńskiego 7.53 0, 49 0, 23 2, 71 0, 46
36 Słowiańska 6.87 0,63 0,23 5,31 0,29
40 Szpitalna 4.32 0, 82 0, 23 11, 84 0, 50
37 Stablewskiego 7.55 0,57 0,23 3,28 0,46
41 Umultowska 6.79 1, 25 0, 42 3, 03 0, 65
38 Starołęcka 7.12 0,63 0,29 6,01 0,53
3942 Widłakowa
Stróżyńskiego 7.41
7.53 0, 0,49
50 0, 22
0,23 2, 07 2,71 0, 45 0,46
4043 Witosa Szpitalna 7.09

4.32 0, 0,82
48 0, 21
0,23 6, 0711,84 0, 43 0,50
4144 Umultowska
Wojska Polskiego 6.79
6.53 0, 1,25
69 0,42
0, 26 9, 48 3,03 0, 39 0,65
42 Widłakowa 7.41 0,50 0,22 2,07 0,45
45 Zwierzyniecka 7.64 0, 52 0, 23 2, 49 0, 40
43 Witosa 7.09 0,48 0,21 6,07 0,43
mean 7.22 0.59 0.25 6.00 0, 47
44 Wojska Polskiego 6.53 0,69 0,26 9,48 0,39
45Content minimum
Zwierzyniecka 4.32
7.64 0.43
0,52 0.160,23 0.79 2,49 0, 26 0,40
maximum
mean 8.26
7.22 1.25
0.59 0.420.25 42.966.00 0, 67 0,47
Contentdeviation
Standard minimum 4.32
0, 65 0.43
0.14 0.040.16 6.64 0.79 0.10 0,26
Coefficient of variation (%) maximum 8.26

9, 0 1.25
23.19 0.42
17.50 42.96
110.78 22.32 0,67
Standard deviation 0,65 0.14 0.04 6.64 0.10
Table 3. Contents of soluble forms of heavy metals and9,0 soil reaction in the surface soil
23.19 layer (0 - 20 cm)
17.50 in green areas
110.78 22.32
Coefficient
adjacent oftransportation
to selected variation (%) routes in the city of Poznań, Poland
pH
The number of cases
30
46,7%
20 37,8%
10 11,1%
2,2% 2,2%
0
4,3‐5,0 5,0‐6,0 6,0‐6,7 6,7‐7,4 7,4‐8,5
Class left half‐open ranges
Figure 1. The stemplot for pH (H2O) of soil in green areas located near to routes in Poznan
Figure 1. The stemplot for pH (H2O) of soil in green areas located near to routes in Poznan
Cd
30 51,1%
The number of cases
20
20,0% 24,4%
10
2,2% 2,2%
0
0,16‐0,21 0,21‐0,26 0,26‐0,31 0,31‐0,34 0,34‐0,42
Pb
84,4%
40
The number of cases
30
20
10 11,1%
2,2% 0 2,2%
0
0,79‐9,22 9,22‐17,65 17,65‐26,08 26,08‐34,51 34,51‐42,96
Cr
30
The number of cases
44,4%
20
22,2%
10 15,6% 15,6%
2,2%
0
0,26‐036 0,36‐0,46 0,46‐0,56 0,56‐0,66 0,66‐0,76
Ni
84,4%
40
The number of cases
30
20
10 11,1%
2,2% 0 2,2%
0
0,43‐0,63 0,63‐0,83 0,83‐1,03 1,03‐1,23 1,23‐1,43
Figure 2. The stemplot for content of heavy metals in soil of green areas located near to routes in Poznan
Figure 2. The stemplot for content of heavy metals in soil of green areas located near to routes in Poznan
12 Soil Pollution
4.1. Concluding remarks
Based on analyses of soils collected from green areas located in the vicinity of selected
transportation routes in the city of Poznań, Poland in most examined locations an alkaline
reaction pH >7.4 (46.7% soil samples) and a neutral reaction (37.8%) were found, which may
significantly affect habitat conditions for plants.
Moreover, most tested soil samples contained low amounts of soluble forms of cadmium, lead,
chromium and nickel.
5. Heavy metal contents in leaves of selected ornamental plant species

growing in green areas adjacent to selected transportation routes in the city
of Poznań (western Poland)
Effects of soil pollution with heavy metals may be identified by analyzing their content in
plants. Plants may be considered good bioindicators of environmental pollution with heavy
metals. Most frequently healthy leaves are indicator parts of plants; however, it depends on
the species and occasionally even on the cultivar.
The aim of the analyses conducted by the authors in September 2012 was to determine contents
of selected heavy metals in leaves of trees growing in the vicinity of transportation routes in
Poznań (western Poland).

It was decided to conduct analyses on healthy plants with no symptoms of damage. Leaves of trees were collected
from the central parts of long shoots, distributed at various sides around the circumference of the crown. A total of 15
- 20 leaves were collected from each tree. Leaves were collected from 10 trees of a given genus (1 bulk sample
comprised approx. 150 - 200 leaves). The collected material was dried at a temperature of 45 - 50°C and then
homogenized. In order to determine total forms of heavy metals they were mineralized in a mixture of nitric and
perchloric acids (v:v=3:1; Σ 30 cm3). Following mineralization Cd, Cr, Pb and Ni contents were determined by atomic
absorption spectrometry (AAS) in a Carl Zeiss Jena apparatus.
High variation was observed in the contents of heavy metals in leaves of tested trees (Acer sp.,
Betula sp., Robinia sp., Salix sp., Tilia sp.), (Table 4). Mean content of cadmium amounted to 1.18
- 1.74 mg Cd kg-1 (for Tilia sp. and Betula sp.), for chromium it was 0.46 - 0.82 mg Cr kg-1 for
Tilia sp. and Acer sp. (except for Betula sp. – trace content), that of lead ranged from 1.55 to 3.28
mg Pb kg-1 (for Betula sp. and Salix sp., respectively). Nickel content ranged from 1.63 mg Ni
kg-1 (for Tilia sp.) up to 2.23 mg Ni∙kg-1 (for Betula sp.)
Table (5) present contents of heavy metals in leaves of selected genera of trees reported by
Tomašević et al. [117], Lawal et al. [81] and Knezevic et al.[76]. In this study cadmium and
nickel contents in leaves did not exceed the levels detected by those authors. In contrast, they
found markedly lower chromium contents in leaves of Tilia sp., Aesculus hippocastanum and
Azadirachta indica. In this study the content of lead in leaves was markedly lower than that
recorded by those researchers for Tilia sp. and Aesculus hippocastanum, while it was comparable
to that in Azadirachta indica and Paulownia elongata (Tables 4 and 5).
Heavy metals
Sample Content
Cd Cr Pb Ni
Acer sp. Minimum 0.25 Tr. 0.83 0.80
Maximum 1.85 1.79 3.19 2.40
Average 1.31 0.82 2.10 1.81
Betula sp. Minimum 1.16 Tr. 0.55 1.90
Maximum 2.87 Tr. 3.49 2.40
Average 1.74 Tr. 1.55 2.23
Robinia sp. Minimum 0.47 Tr. 1.06 0.50
Maximum 2.54 2.13 5.73 2.90
Average 1.37 0.48 2.78 2.07
Salix sp. Minimum 1.12 Tr. 2.43 1.60
Maximum 1.71 1.69 4.61 2.50
Average 1.39 0.48 3.28 2.10
Tilia sp. Minimum 0.29 Tr. 2.10 0.80
Maximum 2.09 1.16 4.28 2.50
Average 1.18 0.46 3.19 1.63
Table 4. Contents of heavy metals (mg kg-1 d.m.) in leaves of selected genera of trees growing in Poznań (tr. – trace
amounts)
Heavy metals
Sample Content
Cd Cr Pb Ni
Tilia sp.1 Min dl dl 1.88 -
Max 1.4 0.02 11.4 -
Aesculus hippocastanum1 Min 0.4 dl 5.35 -
Max 4.9 0.33 20.3 -
Azadirachta indica2 Min - 0.17 0.77 1.14
Max - 0.37 2.25 1.92
Paulownia elongata S.Y. Hu 3

Min - - 0.94 3.23
Max - - 3.16 6.63
dl detection limit
1
Tomašević et al. 2004; 2 Lawal et al. 2011, 3 Knezevic et al. 2009
Table 5. Contents of heavy metals (mg kg-1 d.m.) in leaves of selected genera of trees
14 Soil Pollution
6. The principle of phytoremediation
Among soil purification methods biological methods are increasingly often focused on as
particularly promising [1, 99, 108]. One of these is phytoremediation, based on the activity of
living organisms [40]. It is an alternative method, competitive in relation to other technologies
extensively applied in pollutant removal from soil. There are methods facilitating deactivation
or removal of toxic substances from the substrate. In most cases they are based on methods of
physico-chemical extraction, but their application is connected with excessive costs and
complete elimination of soil microorganisms. Reconstruction of semi-natural ecosystems in
such cases is a lengthy process.
Obvious advantages of this biological method include its applicability at the contamination
site, as well as relatively low investment outlays and low operating costs at the simultaneous
high effectiveness of the process [95]. In the opinion of Salt et al. [107], other factors promoting
its more common use are connected with the fact that it is an environmentally friendly process,
which does not disturb soil structure, and that it may use many plant species.
The term phytoremediation originates from Greek phyton – a plant and Latin remediare – to
purify. Salt et al. [108], Blaylock and Huang [14], Schnoor [109], Schwitzguebel et al. [111],
McGrath and Zhao [86], Vassilev et al. [122], Vangronsveld et al. [121] and Larcher et al. [80]
stated that phytoremediation is a technology using higher plants to stabilize and either remove
or reduce amounts of soil pollutants, bottom deposits or surface and underground waters.
Cunningham et al. [37] and Salt et al. [107] define a plant as a system of filters and pumps
powered by solar energy and extracting from its environment and accumulating specific
elements.
As it was reported by Pandolfini et al. [96], this biological method is based on the practical use
of three types of physiological response to substances found in the environment, i.e. exclusion,
accumulation and hyperaccumulation.
The term phytoremediation refers to the following methods using higher plants to purify
environmental matrices [49, 98, 108, 119]:
• phytodegradation – the use of plants and microorganisms to degrade organic pollutants,

• phytostabilization – the use of plants to reduce bioavailability of pollutants in the environ‐
ment,
• phytoextraction – the use of plants absorbing pollutants and accumulating them in organs
removed from fields together with crops in order to purify soil from heavy metals and
organic substances,
• phytovolatilization – the use of plants to volatilize pollutants and release them to the
atmosphere,
• rhizofiltration – the use of plant roots to absorb pollutants from water and sewage,
• rhizodegradation – the use of plants to supplement the bioremediation process performed
by microorganisms colonizing the rhizosphere.
In the opinion of Negri et al. [91], the above mentioned phytoremediation technologies act at
three different detoxication levels on pollutants accumulated in the environment, i.e. the
pollution-loaded soil matrix (phytostabilization, rhizofiltration and rhizodegradation), plants
(phytodegradation, phytoextraction, rhizofiltration) and the atmosphere (phytovolatiliza‐
tion). Studies are also being conducted on the application of plants in the technology to
excavate heavy metals, referred to as phytomining [5, 90, 97]. Soils with high heavy metal
contents are planted with plants capable of growing under such adverse conditions and
accumulating selected elements in their biomass. Such biomass is next treated as the so-called
bio-ore. The biomining technlogy to extract heavy metals from soil, created by R. L. Chaney,
J.S. Angle, A.J. Baker and J.M. Li, was patented in 1989 [45].
7. Continuous and induced phytoextraction
Phytoextraction, a phytoremediation method observed in 1885 by Bauman [11], is an

excellent concept of soil purification, which is not yet commonly applied. In many centers
worldwide research is being conducted on phytoextraction in the search for plant species
capable of accumulating heavy metals in their aboveground parts. Within phytoextraction
of heavy metals from soil we may distinguish the so-called continuous and induced
phytoextraction.
Plants used in phytoextraction of heavy metals from soil should exhibit:

• good tolerance to high concentrations of heavy metals
• capacity to absorb and accumulate heavy metals in their aboveground parts
• rapid growth
• high increase in biomass
• resistance to diseases, pesticides and adverse atmospheric conditions
• low cultivation requirements
• easy harvesting and processing.

In continuous phytoextraction heavy metals are absorbed by plants and accumulated
continuously with plant growth [14, 20, 46, 85, 110].
Apart from it being economically attractive, continuous phytoextraction is also environmen‐

tally friendly, leaving the site suitable for cultivation of other plants [37, 63].
In urbanized areas continuous phytoextraction may be used in two types of sites. One
comprises degraded soils in post-industrial areas, while the other, highly promising as a
future application of phytoextraction, is connected with soils in the vicinity of transporta‐
tion routes and in urban green areas. The potential of ornamental plant species most
frequently planted in urban locations is investigated in many research centers worldwide.
16 Soil Pollution
Such species include also Tagetes erecta L. It results from studies conducted by Bosiacki [16,
17] that it is a species exhibiting tolerance to high concentrations of cadmium and lead.
When comparing several cultivars of this species the author found that cv. ‘Hawaii’ has
the greatest contents of cadmium and lead in individual aboveground organs. The highest
cadmium content (96.06 mg kg-1 d.m.) was found in leaves of Tagetes erecta L. cv. ‘Ha‐
waii’, while that of lead (145.00 mg kg-1 d.m.) was recorded in stems. Accumulation of
heavy metals in individual plant organs is not uniform. Bosiacki [15] observed variation in
cadmium contents in individual organs of selected ornamental plant species. The highest
content of this metal was detected in Tagetes erecta ‘Inca Yellow’ in its roots, in Salvia
splendens ‘Fuego’ in leaves and stems, while in Helianthus annuus ‘Pacino’ it was in
inflorescences. Liu et al. [82] stated that Calendula officinalis L. accumulated more Cd and
Pb in roots than in shoots, while Althaea rosea L. collected greater amounts of these elements
in shoots than in roots.
Zhou and Wang [124] when investigating the effect of cadmium on growth in three
ornamental plant species stated that Salvia splendens L. is most sensitive to Cd, while
Abelmoschus manihot Medik and Tagetes erecta L were most resistant.
Phytoremediation of heavy metals from contaminated areas, including urbanized areas,

according to Porębska and Gworek [101] may be conducted using many species of ornamental
plants and vegetables, e.g. Salix viminalis L. (accumuating Zn and Cd), Alyssum bertolonii Desv.
(Ni), Brassica pekinensis Rupr.(Pb), Thlaspi caerulescens J. Presl & C. Presl. (Zn). The same authors
stated that wild-growing plants, commonly considered weeds in cultivation of agricultural
and horticultural crops, may also be used in bioremediation, e.g. Atriplex nitens Schkuhr,
Artemisia vulgaris L., Chenopodium album L., (accumulating greatest amounts of Pb among the
tested plants) and Lacuca serriola L. (accumulating greatest amounts of Zn).
Antonkiewicz and Jasiewicz [6] assessed suitability of Helianthus tuberosus L., Zea mays L., Sida
hermaphrodita Rusby, Amaranthus sp. L. and Cannabis sativa L. in phytoextraction of heavy
metals from soil. In their study the highest contents of cadmium and zinc were detected in
amaranth, while that of lead and nickel in Virginia fanpetals. Jerusalem artichoke had the
greatest content of copper.
Antonkiewicz et al. [7] when investigating phytoextraction of heavy metals (Cd, Pb, Ni, Zn
and Cu) from soil using Virginia fanpetals (Sida hermaphrodita Rusby) found that this species
absorbed the highest rates of nickel from soil and the lowest of copper.
Many authors for phytoextraction of soils polluted with heavy metals recommended sun‐
flower, corn, rape, amaranth, willows, Miscanthus and strong growing cereals, while for
phytoextraction in urban soils he recommended plants with high tolerance to pollution, e.g.
London plane, northern red oak, Japanese larch, poplars, field maple, ashes and dogwoods,
desert false indigo, false Spirea and forsythia.
Larcher et al. [80] conducted pilot-scale studies in the industrial area of Turin (Italy) using two
plant species in phytoremediation of soil to remove heavy metals. They found Helianthus
annuus L. "Holeko HO” and Brassica juncea Czern. "Red Giant" to be highly suitable for
phytoremediation. Those authors stated that further studies in this respect are needed.
In urban green areas the predominant forms are lawns and turf-covered areas in escarpments,
embankments, spoil tips, belts separating roadways, parking lots, gas stations, landfills and
industrial waste dumps. Depending on the use of turf areas it is highly important to select
appropriate species and cultivars of lawn grasses. It results from experiments conducted by
Bosiacki and Zieleziński [23] on the potential of three grass species (Poa pratensis L. ‘Evora’,
Festuca arundinacea Schleb. ‘Asterix’, Festuca rubra L. sensu lato ‘Jasper’) in phytoextraction of
nickel that Poa pratensis L. ‘Evora’ and Festuca arundinacea Schleb. ‘Asterix’ are species showing
the greatest capacity to accumulate this metal. In turn, Festuca rubra L. sensu lato ‘Jasper’ turned
out to be the species showing the greatest capacity to accumulate cadmium and lead [21, 22].
Studies are also conducted on induced phytoextraction using plants producing large amounts
of biomass, but additionally such substances as e.g. chelating carriers affecting mobility of
individual elements and enhancing pollutant accumulation in plant organs (particularly
aboveground parts) are introduced to the environment [35, 92, 123].
Chelating substances are to transform forms of heavy metals sparingly soluble or insoluble in
water into forms available to plants. Studies are being conducted using different chelators,
both natural and synthetic (e.g. humus substances, low molecular organic acids, citric acid,
tartaric acid, amino acids as well as EDTA, EGTA, EDDS, HEDTA, CDTA, EDDHA, DTPA,
NTA). Some of them are biodegraded in soil, while others in combination with a heavy metal
may be leached to ground waters contaminating them. For this reason it is essential in this
technology to determine an appropriate dose and date for the application of a given chelating
agent.
It results from preliminary studies (unpublished data) conducted in 2012 by Bosiacki at the
Department of Plant Nutrition, the Poznań University of Life Sciences, Poland that EDTA
introduced to mineral soil contaminated individually with cadmium at 1.5 mg dm-3 in the form
of cadmium sulfate (3CdSO4 8H2O), lead 100 mg dm-3 as lead acetate [(CH3COO)2Pb 3H2O]
and nickel 50 mg dm-3 as nickel sulfate (NiSO4 6H2O) caused an increase in the contents of
these metals in leaves of Tagetes erecta ‘Taishan Orange’. Doses of EDTA were introduced to
soil in the solution form 35 days after plants had been planted to mineral soil contaminated
with these metals. Samples of plant material (leaves) were collected 25 days after the applica‐
tion of EDTA to soil. Contents of individual heavy metals in leaves of Tagetes erecta ‘Taishan
Orange’ are presented in Figs. 3, 4 and 5. The research found that acid (EDTA) has improved
the efficiency of phytoextraction of cadmium, lead and nickel in soil by Tagetes erecta L.
'Taishan Orange'.
Identification of compounds complexing toxic heavy metals and at the same time biodegrad‐
able in the soil medium is crucial for induced phytoremediation.
At present studies are being conducted at the Department of Plant Nutrition, the Poznań
University of Life Sciences, Poland on the application of a biodegradable compound in
phytoextraction of heavy metals from contaminated soils.
18 Soil Pollution
40 34,21c
(mg kg‐1 d.m.)

30
40
20 16,89b 17,03b 17,67b 34,21c
d.m.)
kg‐1Cd 30
10
40
9,14a
34,21c
16,89b 17,03b 17,67b
d.m.)
20
0
30
kg‐1(mg
9,14a
0 25 50 75 100
10 16,89b 17,03b 17,67b
Cd (mg Cd
20 Doses EDTA (mg dm‐3)

0 9,14a
10
0
*homogeneous groups were identified 25 test, p = 0.0550(values denoted with
using the Duncan 75 identical letters
100do not differ signific
*homogeneous groups were 0 identified using the Duncan test,
EDTAp(mg
antly)
Doses = 0.05
dm‐3)(values denoted with identical letters do not
-3
differ significantly)
Figure 3. The effect of increasing EDTA doses introduced to soil (contaminated with 1.5 mg Cd dm ) on cadmium content in leaves
0 25 50 75 100
of Tagetes erecta ‘Taishan Orange’
‐3)
*homogeneous groups were identified using the DuncanDoses
test, p =EDTA (mg dm
0.05 (values denoted with identical letters do not differ signific
Figure 3. The effect of increasing EDTA doses introduced to soil (contaminated with 1.5 mg Cd dm-3) on cadmium
antly)
80 70,21c
content in leaves of Tagetes erecta ‘Taishan Orange’
Figure 3. The effect of increasing EDTA doses introduced to soil (contaminated with 1.5 mg Cd dm-3) on cadmium content in leaves
(mg kg‐1 d.m.)
*homogeneous groups were identified using of

theTagetes
Duncan erecta
test, p =‘Taishan Orange’
0.05 (values denoted with identical letters do not differ signific
60 antly)
1.5 mg Cd dm-3) on cadmium content in leaves
Figure 3. The effect of increasing EDTA doses introduced to soil (contaminated with39,78b
80 of33,23b 35,11b Orange’
Tagetes erecta ‘Taishan 70,21c
40
d.m.)
60
80 15,31a
20 70,21c
kg‐1Pb
39,78b
35,11b
d.m.)
40 33,23b
60
0
kg‐1(mg
0
15,31a 25 50 39,78b
75 100
20
40 33,23b 35,11b
Pb (mgPb
Doses EDTA (mg dm‐3)

0
20 15,31a
0 25 50 75 100
0
*homogeneous groups were identified
0 using the Duncan
25 test, p = 0.05 (values
50 denoted with75identical letters-3100
do not differ significantly)
Figure 4. The effect of increasing EDTA doses introduced to soil (contaminated with 100 mg Pb dm ) on lead content in leaves of
Doses‘Taishan
Tagetes erecta EDTA (mg dm‐3)
Orange’
*homogeneous groups were identified using the Duncan test, p = 0.05 (values denoted with identical letters do not
*homogeneous groups were identified using the Duncan test, p = 0.05 (values denoted with identical letters do not differ significantly)
300 268,34e
Figure 4. The effect of increasing EDTA doses introduced to soil (contaminated with 100 mg Pb dm-3) on lead content in leaves of
250 Tagetes erecta ‘Taishan Orange’
(mg kg‐1 d.m.)
Figure 4. The*homogeneous
effect of increasing EDTAusing
groups were identified doses introduced
the Duncan test, p = to
0.05soil (contaminated
(values with
denoted with identical 100
letters do mg Pb dm
not differ
-3
) on lead content
significantly)
of200 178,76d
100 mg Pb dm-3) on lead content in leaves of
in leavesFigure
of Tagetes erecta
4. The effect ‘TaishanEDTA
increasing Orange’doses introduced to soil (contaminated with
300 Tagetes erecta ‘Taishan Orange’ 268,34e
150 111,25c
250
kg‐1Nid.m.)
100
300 57,21b 268,34e
200 178,76d
50
250 18,78a
d.m.)
150 111,25c
0
200 178,76d
(mg
100 0 57,21b
25 50 75 100
kg‐1
150
Ni (mg Ni
50 18,78a 111,25c
1000 57,21b
50 18,78a
0 25 50 75 100
0 identified using the Duncan test,Doses
*homogeneous groups were EDTA
p = 0.05 (mg
(values dm‐3with
denoted ) identical letters do not differ significantly)
Figure 5. The effect of increasing EDTA 0 doses introduced
25 to soil (contaminated
50 75mg Ni dm-3) on
with 50 100nickel content in leaves of
Tagetes erecta ‘Taishan Orange’‐3
Doses EDTA (mg dm )
Identification
*homogeneousof compounds complexing
groups were identified usingtoxic heavy test,
the Duncan metals
p = and
0.05 at the same
(values time
denoted biodegradable
with identical lettersindothe
notsoil medium
differ is crucial for
significantly)
5. The effect of increasing EDTA doses introduced to soil (contaminated with 50 mg Ni dm-3) on nickel content in leaves of
Figurephytoremediation.
induced
*homogeneous groupsgroups
*homogeneous werewere
identified using
identified using
Tagetes
the Duncan
erecta
the Duncan ‘Taishan
test,
test, p = 0.05
Orange’
p =denoted
(values 0.05 with
(values denoted
identical with
letters do not identical
differ letters do not
significantly)
Figure 5. The effect
Identification of increasingcomplexing
of compounds EDTA doses introduced
toxic to soiland
heavy metals (contaminated
at the same with
time 50 mg Ni dm-3)inonthe
biodegradable nickel content in
soil medium is leaves
crucialoffor
induced phytoremediation. Tagetes erecta ‘Taishan Orange’
Figure 5. The effect ofofincreasing

Identification EDTA doses
compounds complexing toxicintroduced
heavy metals to
andsoil (contaminated
at the with 50
same time biodegradable in mg Ni medium
the soil dm-3) on nickelforcontent
is crucial
in leaves of Tagetes
induced erecta ‘Taishan Orange’
phytoremediation.
8. The phenomenon of hyperaccumulation
As it was stated by Brooks [28] the discovery and description of a phenomenon termed
hyperaccumulation has contributed to the practical use of plants to remove metallic pollutants
from soil. According to Boyd and Martens [25] and Brown et al. [29], plant species referred to
as hyperaccumulators are genetically and physiologically capable of accumulating large
amounts of heavy metals with no symptoms of toxicity. Threshold values of metal concentra‐
tions have been used to define metal hyperaccumulation, including 100 mg kg-1 dry weight of
shoots for Cd, 1000 mg kg-1 for Cu, Ni, Pb and 10 000 mg kg-1 for Zn [10, 28, 86]. Cline et al. [34]
stated that concentrations of heavy metals in tissues of hyperaccumulator plants should be 1
- 2%. Van der Ent et al. [120] recommend the following concentration criteria for different
metals and metalloids in dried foliage:100 µg g-1 for Cd, Se and Ti; 300 µg g-1 for Co, Cu and
Cr; 1000 µg g-1 for Ni, Pb and As; 3000 µg g-1 for Zn; 10000 µg g-1 for Mn, with plants growing
in their natural habitats. There are over 400 known plant species from 45 families classified as
hyperaccumulators. Most species belong to the families Brasicaceae and Fabaceae [103]. Ap‐
proximate numbers for various elements are as follows: Ni (450), Cu (32), Co (30), Se (20), Pb
(14), Zn (12), Mn (12), As (5), Cd (2), Tl (2), [120]. Moreover, hyperaccumulators typically
accumulate most efficiently one heavy metal and to date no plant has been identified which
would be capable of accumulating in its tissues all toxic metals [37]. According to Vangrons‐
veld et al. [121] successful phytoremediation depends first of all on the selection of an appro‐
priate plant species. For this reason in the opinion of Baker et al. [10] it would be a practical
solution to grow many different species at the same time.
Most natural hyperaccumulators are plants characterized by slow growth and production of
low amounts of biomass. These traits result in a limited applicability of these plant species in
phytoextraction of heavy metals from soil [37]. An example in this respect may be provided
by Thalspi caerulescens L., producing only 5 ton dry matter per hectare [32], whereas it is cost-
effective to use plants, which yield 20 ton per hectare and the accumulation of metal in
aboveground parts is over 1% [63].
Studies are being conducted worldwide on the use of plants producing large amounts of
biomass (for energy generation purposes) in the phytoextraction of heavy metals from polluted
soils. They include annual plants such as e.g. cereals and rape, and perennials e.g. willows,
which capacity for phytoextraction of heavy metals was confirmed by Greger and Landberg
[56] and Boyter et al. [26]. Other perennial species of energy crops include Spartina pectinata,
Sida Hermaphrodita, Rosa multiflora, Helianthus tuberosus, Populus spp., and Robinia pseudoaca‐
cia. The most significant parameters of these plants include high annual increment of biomass
and its high calorific value [116].
Ociepa et al. [94] stated that plants grown for energy purposes may play a considerable role
in view of the assumptions of the Common Agricultural Policy and the environmental
protection policy of the European Union. One of such plant species is Miscanthus × giganteus
Greef and Deu.
In the years 2008 – 2011 at the Department of Plant Nutrition, the Poznan University of Life
Sciences, Poland, studies were conducted to assess applicability of Miscanthus × giganteus Greef
20 Soil Pollution
and Deu. in phytoextraction of heavy metals from soil. The aim of the conducted analyses was
to determine what amounts of cadmium and lead are transported to aboveground parts of
Miscanthus × giganteus and whether Miscanthus × giganteus would be suitable for rapid
phytoextraction of cadmium, lead from polluted soils.
The vegetation experiment was conducted in an unheated plastic tunnel with suspended sides, of 6 x 30 m in size. at
the Marcelin Experimental Station of the Poznan University of Life Sciences.
Seedlings of Miscanthus × giganteus were produced at a tissue culture laboratory of Vitroflora. Plants were planted in
the beginning of May in drainless containers (of 7 dm3) filled with previously prepared substrate. The experiment
comprised 16 combinations (in each year of the study) and each combination consisted of six replications. A
replication comprised one plant growing in pots.
Phytoremediation of cadmium and lead by Miscanthus × giganteus was investigated in two years of growth with the
plants being grown in two substrates. at four levels of metal contents. Since light soils with low contents of organic
matter predominate in Poland. such soil was selected for the experiment. Another substrate was a mixture of this
mineral soil with highmoor peat. Highmoor peat was added to increase the amount of organic matter in the mineral
soil.
Substrates: mineral soil (sand) and mineral soil with highmoor peat (1:1 v/v)
Doses of cadmium: control (native contents of cadmium), 3, 5 and 10 mg dm-3)
Doses of lead: control (native contents of cadmium), 250, 1000 and 5000 mg dm-3).
In mineral soil the method according to Mocek and Drzymała [88] was used to determine particle density (which
amounted to 2.65 g cm-3) and bulk density (1.62 g cm-3). Total porosity of mineral soil was 38.9%. Moreover. grain size
distribution of mineral soil was determined by the densimetric method according to Prószyński [88]. On the basis of
the percentages of fractions the grain size class of soil was identified (according to the guidelines of the Polish Society
of Soil Science) - sand.
Prior to the establishment of the experiment, Corg content in mineral soil was determined according to the Tiurin
method [51]. Content of organic carbon in sand (the Tiurin method) was 0.55% (0.95% humus). while the percentage
of organic matter in the mixture of sand and highmoor peat (from loss on ignition) was 10.05%. In the substrate
composed of a mixture of mineral soil with highmoor peat (1:1 v/v) the percentage of organic substance was
determined by loss on ignition the substrate by the direct method at high temperature in the presence of oxygen,
under the influence of which organic substance is decomposed (carbon is released in the form of CO2. hydrogen in the
form of H2O and nitrogen as N2, while the other elements remain in ash).
Experiments were conducted using highmoor peat by Hartmann (sphagnum peat. ground. fractional with acid
reaction (pH 4.50). This peat has a high water capacity, at the same time retaining an elastic structure. The weight of 1
dm3 peat was 490 grams.
In order to obtain an appropriate pH for growing of Miscanthus × giganteus a neutralization curve was plotted for the
analyzed substrates. On its basis the dose of CaCO3 required for the maintenance of pH within the range of 6.5-7.0
was established. The reaction of the substrate (mineral soil + highmoor peat) was regulated using 3 g dm-3 CaCO3
(chemically pure reagent). The substrate composed of mineral soil did not require reaction control. Despite that fact 1
g dm-3 CaCO3 was applied in order to maintain pH at 6.5-7.0. An adequate amount of calcium carbonate was
introduced to each experimental container with the substrate. Cadmium and lead contents in analyzed substrates
after liming amounted: in mineral soil Cd 0.27, Pb 27.32 mg dm-3 and mineral soil with highmoor peat Cd 0.18, Pb
12.03 mg dm-3. Two weeks after liming nutrients cadmium and lead were introduced to the substrate. Cadmium and
lead was introduced only in the first year of the experiment in the form of chemically pure reagents (C.P.): cadmium
sulfate (3CdSO4 8H2O), lead acetate [(CH3COO)2Pb∙3H2O]. Pre-vegetation fertilization (in the first year) with macro-
and micronutrients was determined taking into consideration initial nutrient contents in substrates. after liming
reaching the following levels (in mg dm-3): N 200, P 120, K 250, Mg 100, Fe 50, Mn 20, B 1.5 and Mo 1.5. All macro-
and micronutrients were introduced in the form of solutions using chemically pure reagents (potassium
monophosphate, potassium nitrate, ammonium nitrate, magnesium saltpeter, magnesium sulfate, iron sulfate, copper
sulfate, zinc sulfate, manganese sulfate, ammonium molybdate, borax). In the second year of the experiment an
identical experimental design was used as in the first year. After plant cutting in the first year of the experiment
containers with polluted substrates were stored in an unheated tunnel to the next vegetation year (the second year of
the study). In the second year of the study in March - prior to the beginning of vegetation substrate samples were
collected and chemical analyses were performed to determine nutrient contents. On this basis nutrient fertilization
was established (leading to nutrient contents at the same levels. which were applied in the first year of the
experiment). Nutrients in substrates were determined using the “Universal” method [78] in CH3COOH solution at a
concentration of 0.03 mol dm-3, pH in water was determined by potentiometry (the substrate to water ratio of 1:2),
while conductometry was applied to determine EC (mS cm-1), (the substrate to water ratio of 1:2), [50].
The following nutrient determination techniques were applied: N – NH4 and N – NO3 by microdistillation (Bremner
modified by Starck), P by colorimetry using the vanadium-molybdenum method, K, Ca and Na by flame photometry,
Mg by atomic absorption (AAS), Cl and S – SO4 by nephelometry [78].
In October in each year of the study prior to harvesting plant height was measured. Dry weight of plants was recorded
and samples of plant material were collected for analyses.
Harvested plant material (entire aboveground mass) was dried in an extraction drier at a temperature of 105ºC for 48
h. Next the material was ground and at 2.5 g from each sample it was digested in a mixture of concentrated HNO3
(ultra pure) and HClO4 (analytically pure) at a 3:1 ratio [18]. Content of cadmium and lead in the plant material were
determined by flame atomic absorption spectrophotometry (FAAS), AAS-3 spectrophotometer by Zeiss. Moreover,
content of metals in the reference material (Pseudevernia furfuracea BCR®-482/2009) was determined.
In the first and second year of the study samples of substrate were collected after harvest, from which metals were
extracted using the Lindsey’s solution containing in 1 dm3: 5 g EDTA (ethylenediaminetetraacetic acid), 9 cm3 25%
NH4OH solution, 4 g citric acid and 2 g Ca(CH3COO)2 2H2O. Next this metal was assayed by flame atomic absorption
spectrophotometry (FAAS), AAS-3 spectrophotometer by Zeiss.
Results of content of cadmium and lead in substrates and aboveground parts of Miscanthus × giganteus were
elaborated statistically in the Statobl program applying a one-way analysis of variance for orthogonal factorial
experiments, with differences between means determined at a significance level p=0.05.
Miscanthus × giganteus Greef and Deu. (Figure 6) is an interspecies hybrid of a diploid Chinese
silver grass [Miscanthus sinensis (Thunb.) Anderss.] and a tetraploid Amus silver grass
[Miscanthus sacchariflorus (Maxim.) Benth], belonging to the family Poaceae [54, 55, 61]. After
three years of culture the yield of dry matter is 20 to 35 t and plant height is 3 - 4 m [102].
Miscanthus × giganteus originates from South-Eastern Asia and it has been grown in Europe,
initially as an ornamental plant, for over 50 years. In Poland it still is not a popular crop. In the
nearest future interest in growing of this plant in Poland will be increasing. This abundant
grass forms new shoots stretching outwards from rhizomes (underground runners), forming
an increasingly bigger rounded cluster [102].
22 Soil Pollution
Analyses conducted by physiologists classify Miscanthus to the group of plants of the C-4
pathway, which are characterised by a highly efficient photosynthesis process, ensuring a
rapid and high increase in biomass, at a simultaneous lower transpiration coefficient, i.e. lower
water consumption [58, 89].
Proposed methods to manage Miscanthus after phytoextraction of heavy metals from soil
include combustion (ashes – hazardous waste), bio-ore, paper and pulping industry, produc‐
tion of particleboards as well as chemical industry (packaging plastics).
Figure 6. Miscanthus × giganteus Greef and Deu. in the third year of growth
The aim of the conducted analyses was to determine the effect of increasing doses of cadmium,
lead introduced to mineral soil (sand) and to mineral soil with an addition of highmoor peat
(at a 1:1 ratio, v/v), on the tolerance index of Miscanthus x giganteus. The tolerance index (T )
i
was calculated, i.e. the ratio of the yield (dry biomass) obtained in metal-polluted soil to the
yield (dry biomass) produced in unpolluted soil. This index is considered as the most reliable
indicator of the toxic effect of heavy metals contained in soils and substrates exercised on
plants.
Ti < 1 value lower than one - inhibition of growth or plant death
Ti = 1 value equal one - no effect of increased metal contents on yielding
Ti > 1 value greater than one - positive effect of metal on yielding.
In the first year of growth of Miscanthus × giganteus the tolerance index greater than one was
recorded in plants in mineral soil polluted with 3 mg Cd dm and 1000 mg Pb dm (Table 6).
-3 -3
In the second year of growth the tolerance index greater than one was found in plants in
mineral soil contaminated with all the used cadmium pollution rates and in soil, to which 250
and 1000 mg Pb dm were introduced. The tolerance index greater than one was also recorded
-3
in the substrate constituting a mixture of mineral soil with highmoor peat contaminated with
3 mg Cd dm (in the first year of growth) and 5 mg Cd dm (in the second year of growth).
-3 -3
Substrates
Dose of metal Mineral soil Mineral soil + highmoor peat

Metal
(mg dm-3)
The first year of The second year The first year of The second year
growth of growth growth of growth
3 1.02 1.20 1.02 0.88
Cd 5 0.99 1.34 0.83 1.01
10 0.70 1.24 0.95 0.85
250 0.80 1.30 0.78 0.99
Pb 1000 1.01 1.28 0.99 0.90
5000 1.00 0.98 0.93 0.58
Table 6. The tolerance index (Ti) in Miscanthus x giganteus
In the opinion of Arduini et al. [8] and Kozak et al. [77], Miscanthus is a less tolerant species to
high concentrations of heavy metals in comparison to osier and it is necessary to conduct
further tests on the applicability of varieties of this species in the phytoextraction of heavy
metals.
Kalembasa and Malinowska [72], when testing different clones of Miscanthus found differences
in their capacity to accumulate individual heavy metals, particularly cadmium. Iżewska [65]
in the third year of Miscanthus culture recorded a greater content of cadmium and a lower
content of lead in its biomass. In the opinion of Kalembasa and Malinowska [71], contents of
cadmium and lead in biomass are also dependent on the harvest date of Miscanthus. The
highest cadmium concentration was recorded in Miscanthus biomass at the begining of June,
lead at the beginning of September and October. Those authors stated that the triploid
Miscanthus species was characterized by higher contents of cadmium and lead in its biomass
in comparison to the diploid species. According to those authors, nitrogen, phosphorus and
potassium fertilization has an effect on cadmium content in biomass of diploid Miscanthus
genotypes.
Based on studies conducted by the authors of this chapter it was found that cadmium applied
at 3 and 5 mg dm mineral soil in the first year of growth of Miscanthus × giganteus had no
-3
significant effect on the content of this metal in aboveground parts of plants in comparison to
those growing in soil with no addition of this metal (Table 7). In the second year of growth in
24 Soil Pollution
mineral soil to which 5 mg Cd dm were introduced a significantly greater content of this metal
-3
was detected in aboveground parts of plants in comparison to plants growing in unpolluted

soil. In plants growing in mineral soil the greatest Cd content was found in the second years
of growth of Miscanthus × giganteus growing in soil polluted with 10 mg Cd dm .
-3
In a mixture of mineral soil with highmoor peat, to which 3 mg Cd dm were introduced both
-3
in the first and second year of growth of Miscanthus × giganteus no significant differences were
observed in the contents of this metal in aboveground parts. The greatest cadmium content
was recorded in plants growing in the substrate polluted with 10 mg Cd dm in the first and
-3
second years of growth.
Year of culture
Dose of metal 1st year 2nd year

Substrate
(mg dm-3)
min.-max. range min.-max. range
SD mean SD mean
range R range R
Control 0.98-1.29 0.31 0.12 1.17 ab 1.29-1.52 0.23 0.10 1.39 ab
Cd 3 1.31-1.99 0.68 0.24 1.70 bc 1.14-2.59 1.45 0.51 2.06 a-d

Mineral soil
Cd 5 1.33-3.69 2.36 0.78 2.61 bcd 1.78-4.69 2.91 0.97 3.52 d
Cd 10 3.56-8.34 4.78 1.62 5.51 e 6.16-12.62 6.46 2.81 9.30 g
Control 0.64-1.33 0.69 0.25 0.97 a 0.95-1.46 0.51 0.19 1.20 ab
Mineral soil + Cd 3 1.34-1.93 0.59 0.22 1.60 abc 1.38-1.90 0.52 0.20 1.66 abc
highmoor
peat Cd 5 2.39-3.96 1.57 0.67 2.97 cd 2.68-4.78 2.10 0.97 3.41 d
Cd 10 5.23-9.34 4.11 1.55 7.46 f 5.54-11.17 5.63 2.48 8.66 fg
*homogeneous groups were identified using the Duncan test, p=0.05 (values denoted with identical letters do not
Table 7. Contents of Cd (mg kg dry weight) in aboveground parts of Miscanthus × giganteus growing in substrates
-1
polluted with cadmium
A significantly greater lead content in aboveground parts of Miscanthus × giganteus in com‐

parison to the control was found in plants growing in all tested substrates contaminated with
this metal (Table 8). In plants growing in mineral soil contaminated with 10 mg Pb dm a higher
-3
content of this metal was detected in the first year of growth and it was the highest content
recorded in the analyses. No differences were observed in lead contents in aboveground parts
of Miscanthus × giganteus in the first and second year of growth in a mixture of mineral soil
with highmoor peat.
Year of culture
Dose of
1st year 2nd year
Substrate metal
(mg dm ) -3
min.-max. range min.-max. range
SD mean SD mean
range R range R
Control 1.23-1.77 0.54 0.21 1.54 a 1.76-2.74 0.98 0.38 2.31 a
Pb 250 29.87-48.25 18.38 7.44 39.29 b 27.37-46.78 19.41 7.71 36.83 b

Mineral soil
Pb 1000 56.89-75.43 18.54 6.92 62.17 b 53.45-73.56 20.11 7.58 64.88 bc
Pb 5000 71.46-280.62 209.16 85.24 210.20 e 58.56-131.34 72.78 29.19 104.49 d
Control 1.14-1.62 0.48 0.18 1.47 a 1.12-1.67 0.55 0.21 1.38 a
Mineral soil + Pb 250 27.89-40.99 13.10 5.65 34.29 b 21.67-45.62 23.95 8.00 31.92 b
highmoor
peat Pb 1000 48.67-61.78 13.11 4.74 53.48 b 40.56-62.38 21.82 7.80 49.30 b
Pb 5000 73.56-110.34 36.78 12.56 92.98 cd 53.56-137.89 84.33 31.89 96.46 d
*homogeneous groups were identified using the Duncan test, p=0.05 (values denoted with identical letters do not
Table 8. Contents of Pb (mg kg dry weight) in aboveground parts of Miscanthus × giganteus growing in lead-polluted
-1
substrates
In the conducted analyses the cadmium and lead concentration indexes were calculated for
aboveground parts of Miscanthus × giganteus. The metal concentration index was caluclated
from the formula
C=a:b
a - content in a plant growing in polluted substrate
b - content in a plant growing in unpolluted substrate.
The greatest concentration index was recorded for lead in the first year of growth in the case
of plants growing in mineral soil (Table 9). Plants growing in mineral soil were characterized
by a greater concentration index for cadmium and lead in the first year of growth. An identical
dependence was found in plants growing in a mixture of soil and peat, except for plants
growing in a substrate contaminated with 5000 mg Pb dm-3, in which a higher lead concen‐
tration index was found in the second year of growth.
Miscanthus sp. was tested on heavy metal contaminated arable soil in Southern Poland [110].
The authors concluded that this species accumulates high amounts of metals what may cause
high emission of contaminants during biomass combustion.
26 Soil Pollution
According to Kalembas [70] in ash of Miscanthus sinensis Thumb. the content of individual
heavy metals ranks in the following decreasing levels: Zn>Cd>Pb>Ni>Cu>Cr.
Substrates
Dose of
Mineral soil Mineral soil + highmoor peat
Metal metal
The first year of The second year of The first year of The second year of
(mg dm-3)
growth growth growth growth
3 1.45 1.48 1.65 1.38
Cd 5 3.06 2.53 3.06 2.84
10 7.69 6.69 7.69 7.22
250 25.51 15.94 23.33 23.13
Pb 1000 40.37 28.09 36.38 35.72
5000 136.49 45.23 63.25 69.90
Table 9. Metal concentration indexes in aboveground parts of Miscanthus x giganteus
Both in soil and in a mixture of soil and peat a lower cadmium content was recorded after the
second year of culture except for substrates contaminated with 10 mg Cd dm-3, in which this
dependence was not observed (Table 10).
When analyzing lead content in tested substrates after the completion of growth a lower Pb
content was found also in the second year except for a mixture of mineral soil with peat, to
which lead was not introduced (table 11).
In the substrate being a mixture of soil with peat lower contents of cadmium and lead were
observed in comparison to those recoded in mineral soil in all the experimental variants (Tables
10 and 11).
Year of growth
Type of pollution Substrate
1st year 2nd year
Control mineral soil 0.09 c 0.07 b
(native content of Cd mg dm-3) soil + peat 0.07 b 0.05 a
Weak pollution mineral soil 2.23 d 1.09 b
(Cd 3 mg dm-3) soil + peat 1.79 c 0.77 a
Medium pollution mineral soil 4.12 d 2.65 c
(Cd 5 mg dm-3) soil + peat 2.51 b 1.32 a
Strong pollution mineral soil 6.37 b 6.39 b
(Cd 10 mg dm ) -3
soil + peat 5.43 a 5.22 a
*homogeneous categories were identified with the Duncan test, p=0.05 (values denoted with identical letters do not
Table 10. Cadmium contents (extracted with Lindsey’s solution) in substrates (in mg dm-3) after the completion of
plant growth in the first and the second year of analyses
Year of growth
Type of pollution Substrate
1st year 2nd year
Control mineral soil 24.84 c 15.18 b
(native content of Pb mg dm-3) soil + peat 6.95 a 4.79 a
Weak pollution mineral soil 227.71 d 192.68 c
(Pb 250 mg dm-3) soil + peat 130.31 b 104.74 a
Medium pollution mineral soil 837.70 d 575.34 b
(Pb 1000 mg dm-3) soil + peat 665.30 c 236.88 a
Strong pollution mineral soil 3755.06 d 3304.71 c
(Pb 5000 mg dm-3). soil + peat 2431.14 b 1401.27 a
*homogeneous groups were identified with the Duncan test, p=0.05 (values denoted with identical letters do not dif‐
fer significantly)
Table 11. Contents of lead (extracted with Lindsey’s solution) in substrates (in mg dm-3) after the completion of plant
growth in the first and the second years of analyses
9. The amount of ash (%) after incineration of aboveground parts of

Miscanthus × giganteus
Aboveground parts of Miscanthus × giganteus were incinerated at a temperature of 450ºC in a

Linn Elektro Therm furnace. Incineration was performed in two stages:
• stage I - preliminary carbonization at a temperature of 100°C for 1 h
• stage II – combustion at 450°C for 5 h.
Incineration was performed on 20 randomly selected samples of aboveground dry matter of

Miscanthus × giganteus. Assuming dry matter used in the incineration process as 100%. after
combustion a mean 8.20% ash was obtained (SD 0.55), (Table 12).
The amount of ash left after combustion of aboveground parts of Miscanthus × giganteus was
analyzed in terms of empirical probability distribution. Mean observations of the amount of
ash were ordered in the so-called stemplot. in which separate intervals called classes were
identified. Five classes were determined for the amount of ash left after incineration and next
the number of observations (the so-called class frequencies) belonging to them were specified
for each class. In order to ensure uniqueness of classification of observations it was assumed
that classes are left side open intervals and right side closed intervals. Moreover, frequencies
were added which when multiplied by 100 determine the percentage of observations of the
amount of ash left after combustion of plants as classified to individual classes.
Among all observations after the combustion process the highest percentage (40%) was found
for the amount of ash within the range of 8.44 to 8.88%. The amount of ash within the range
of 8.00 to 8.44% ranked second constituting 30% observations.
28 Soil Pollution
In a study conducted by Kalembasa [70] largest content of raw ash was obtained from the Sida
hermaphrodyta Rusby. (59.5 kg t ), less Miscanthus sinensis Thumb. (52.5 kg t ) and at less Salix
-1 -1
sp. (31.5 kg t ).
-1
Number Frequencies Cumulative

Miscanthus × giganteus Classes
fi fi/n frequencies
V = (6.68-7.12] 1 1/20 1/20

IV = (7.12-7.56] 1 1/20 2/20
Amount of ash after combustion in
III = (7.56-8.00] 4 4/20 6/20
(%)
II = (8.00-8.44] 6 6/20 12/20
I = (8.44-8.88] 8 8/20 20/20
Total 20 1
Table 12. The stemplot for the amount of ash (%) obtained after combustion of aboveground pars of Miscanthus ×
giganteus
Concluding remarks
Miscanthus × giganteus may be used in phytoextraction of cadmium and lead from polluted
soils; however, when assessing its potential for phytoextraction of these metals from soil it was
found not to be considerable. This plant need to be further tested in terms of cadmium and
lead phytoextraction capacity from contaminated soil in natural environment.
The study was financed from funds for science in the years 2008 - 2011 as a research project
no. N N305 085535
Acknowledgements
The authors express their gratitude to the Management Board of the Municipal Green Areas
in Poznań and the Management Board of the Municipal Road Network in Poznań for facili‐
tating the soil monitoring project.
Author details
Maciej Bosiacki*, Tomasz Kleiber and Bartosz Markiewicz
Department of Plant Nutrition, University of Life Sciences in Poznan, Poland

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chapter 21
Diagnosis and Management of Field Pollution in the

Case of an Organochlorine Pesticide, the Chlordecone
T. Woignier, F. Clostre, P. Cattan, J. Levillain,

Y.M. Cabidoche and M. Lesueur-Jannoyer
1. Introduction
1.1. Background
The consequences of pesticide pollution of soil and water can be extremely damaging for
both the environment and human health. Chlordecone (CLD), an organochlorine insecticide
(C10Cl10O), was used to control the banana black weevil more than twenty years ago in the
French West Indies but continues to contaminate the environment today [1, 2]. CLD pollu‐
tion occurs in many parts of the world [3-5] and CLD is very persistent, sorptive and highly
lipophilic [6]. Its persistence in soils is due to its low solubility in water [7], high affinity for
organic matter with a Koc (soil organic carbon content/water partitioning coefficient) of be‐
tween 2.5 and 20 m3kg-1 [1, 8], and to its chemical structure (Figure1), which makes it poorly
biodegradable [9]. Similar characteristics are shared by all organochlorine pesticides and
persistent organic pollutants.
CLD causes diffuse pollution in agricultural soils [10], which in turn become a continuous
source of contamination for water resources, crops and animals [1, 3, 11, 12]. In addition, its
long-term effects on human health and child development linked to the consumption of pol‐
luted food and water are now a serious concern [13-15].
1.2. Objective
This chapter explores the multifactorial nature of soil pollution and its evolution at field and
regional scale. We focus on two key factors that determine CLD contamination and disper‐
sion in ecosystems. One is physical and the other anthropogenic. The first is the influence of
clay microstructure on (1) the concentration of CLD in the soil, (2) the bioavailability of the
2 Soil Pollution
pollutant for crops and water resources, and (3) the efficiency of the remediation process.
The second factor is past and present farming practices through their effects on (1) the stock
of pollutant and its components in the soil and its distribution at the plot and regional
scales, and (2) the diffusion of the pollutant in the different compartments of the environ‐
ment. These two factors determine the contributing areas and their pollutant stock, the po‐
tential availability and fluxes of the pollutant, and hence its potential transfer to different
environmental compartments.
Figure 1. The chlordecone molecule (C10Cl10O)
2. Influence of clay microstructure
The studied soils come from Guadeloupe (16°15 N, 61°35 W) and Martinique (14°40 N, 61°00
W) in the French West Indies. These volcanic islands rise to 1467 m and 1388 m elevation
respectively. Rainfall is high and ranges from 1000 to 10 000 mm/year depending on the ele‐
vation and geographical area. All primary minerals of andesitic rocks are weathered, so that
soils have a high content of secondary minerals (clays): halloysite for nitisol, halloysite and
Fe-oxihydroxides for ferralsol, and allophane for andosol, the three main soil types contami‐
nated in French West Indies [1, 10, 16].
These volcanic soils have a high infiltration capacity (saturated hydraulic conductivity
greater than 60 mm h−1) [1, 10]. However in the ‘‘clay’’ matrix of andosols, there are pores
smaller than 1 micron, where water and solute transfers are slow. All these soil types are
acidic (pH = 4.5 -6), which prevents clay dispersion and sheet erosion [1]. Among these soils,
the carbon content of andosol is particularly high, which may influence the retention of the
compound (stock) and its availability.
Indeed all soils are not equivalent in terms of pesticide contamination and in their ability to
transfer the pollutant to water and to plants [17, 18]. For example, although andosols are
highly polluted [1, 10, 16], data show they release less pesticide to percolating water and
crops than other soils [1, 19]. In the case of CLD, one explanation for the retention effect re‐
ported in the literature is the high organic content of these soils and the high affinity of the
pesticide for soil organic carbon [1, 8].
Diagnosis and Management of Field Pollution in the Case of an Organochlorine Pesticide, the Chlordecone 3
However, these volcanic soils contain amorphous clay, allophane, whose structure and
physical properties differ from those of the crystalline clays found in nitisols and ferralsols.
Allophane clay is amorphous and has physical features that closely resemble those of nano
porous materials: large pore volume and water content, a broad pore size distribution, a
high specific surface area and a fractal structure [20-22]. One objective of this chapter is to
show the influence of the clay microstructure on the accumulation and retention of chlorde‐
cone in soils.
2.1. Allophane microstructure
Figure 2 shows the pore volume and specific surface area as a function of the allophane con‐
tent of a set of andosols. There was a clear increase in these two textural features with an
increase in allophane content, showing that allophane clay favors larger porous features.
The pore volume and the specific surface area were well correlated with allophane content
(respectively P<0.0001 r2=0.87and P <0.0001, r2=0.80). The specific surface area was as high as
180 m2.g-1 and pore volume close to 2.5 cm3.g-1. This combination of high specific surface
area and large pore volume suggests that the porous structure is made up of both micro-
and mesopores.
Figure 2. Pore volume (empty circles, ○) and specific surface area (black squares, ∎) versus allophane content
Figure 3 shows the structure of amorphous clay in comparison with classical phyllosilicate
clay: kaolinite or halloysite. These micrographs confirm the spongy structure of allophane
clay.
The morphology of the allophane aggregates is peculiar [21]. Allophane has a very open
structure made up of aggregated small particles (3-5 nm) that form clusters of around 10-20
nm. The clusters can stick together and form larger and larger aggregates up to ~ 100 nm in
size. In comparison, the plate-like particles of phyllosilicate clay are 300-1000 nm in size
(Figure 3). This aggregation mechanism is in agreement with results in the literature [22, 24].
4 Soil Pollution
Figure 3. Scanning electronic micrographs showing the structure of phyllosilicate clay and amorphous clay from [23]
Wada [22] describes allophane particles as nearly spherical, with diameters ranging from 3-5
nm. The aggregation mechanism corresponds to a fractal morphology. Several authors as‐
sumed that allophane could have a fractal structure [20, 22]. The structure of allophane can
be studied at nanoscale using scattering experiments to quantify the fractal features of the
soil samples. Small angle X-ray scattering (SAXS) experiments make it possible to calculate
the fractal dimension Df (which expresses the compactness and tortuosity of the clusters)
and the extent of the fractal aggregates (ξ) [25]. The fractal extent can be considered as the
size of the tortuous “nano-labyrinth”. SAXS curves [26] show that the fractal dimension Df is
constant (2.5-2.7). Table 1 lists changes in ξ versus allophane content. Our results showed
that the size of the fractal labyrinth increased with an increase in allophane content (P=0.001
and r2=0.71).
Allophane (%) 0 3 5 8 10 12 13 15 18 22
ξ ( nm) 0 12 23 22-32 18 23-35 23 35 34-45 42-60
Table 1. Fractal range (ξ) versus allophane content
These data (high specific surface area and pore volume, and fractal features) describe a high‐
ly tortuous microstructure and small mesopores, suggesting that accessibility inside the clay
microstructure is reduced.
2.2. Pesticide sequestration in allophane
Allophane clay has a spongy structure comprising aggregated small particles that form a
tortuous network with small pores. This peculiar structure influences the concentration of
pesticide in the soil.
Figure 4 shows the marked increase in CLD concentration in soils with increasing allophane
content (P<0.0001 and r2=0.807). This finding confirms previous data in the literature [1, 16,
27] stating that allophanic soils are more contaminated than other kinds of tropical soils,
18
Chlordecone contamination in soils

16
14
12
(mg.kg-1)
10
8
6
4
2
0
0 5 10 15 20
allophane (%)
Figure 4. Soil CLD contamination versus allophane content. CLD data have a confidence interval of 30%
which contain the usual crystalline clays (like halloysite and kaolinite). Figure 4 also clearly
shows the link between CLD contamination of the soil and the allophane content of the soil
and one can thus assume that the pesticide retention properties of the soil are partially de‐
pendent on the features of allophane.
Another interesting result of our previous study was that for similar soil CLD contents,
crops cultivated on allophanic soils were much less contaminated than the same crops culti‐
vated on soils containing classical clays, [26]. We calculated the mean soil to plant transfer
(expressed in µg.kg-1 of fresh matter / µg.kg-1 of dry soil), for different crops. Table 2 con‐
firms that, whatever the crop concerned [26], CLD soil to plant transfer was always higher in
halloysite soils than in allophanic soils. For the three crops studied (lettuce, yam, and das‐
heen) the ratio was close to 3.
Soil to crop transfer Soil to crop transfer Ratio

Crops
(halloysite soils) (allophanic soils) (halloysite: allophanic)
Lettuce 0.64 (0.14) 0.23 ( 0.15) 2.83
Yam 3.2 (2.1) 1.04 ( 0.75) 3.13
Dasheen 12.2 (5.8) 3.26 (2.56) 3.73
Table 2. Mean CLD transfers from halloysite and allophanic soils to lettuce, yam and dasheen, expressed in µg.kg-1 of
fresh matter / µg.kg-1 of dry soil (standard deviation in brackets) [26]
2.3. Trapping mechanism in allophane
The results shown in Figure 4 and Table 2 may appear contradictory because one would ex‐
pect allophanic soils, which are more contaminated, to strongly pollute cultivated vegeta‐
6 Soil Pollution
bles. One explanation for the observed effect is that CLD is trapped in the microstructure of
the allophane clay, thus reducing its transfer from the soil to the plant. The influence of soil
allophane content on CLD retention is the signature of the peculiar microstructure of the al‐
lophane aggregate. The spongy structure influences transport inside the allophane aggre‐
gates. The SAXS data made it possible for us to propose a mechanism for the retention of
pesticides in allophanic soils and also for the limited release of CLD to crops and water re‐
sources [1, 19]. At the scale of the allophane, accessibility is difficult because of the fractal
structure and small pore size of allophane clay. CLD transfers within the soil depend on hy‐
draulic conductivity (K) and diffusion processes (Di) in the porous microstructure. The frac‐
tal structure allows an approximation of K and Di at the aggregates scale l [26] through the
following equations:
û û (
K ( l ) ¥ éë1 - éë l a ù Df -3 ù l 2 and Di ¥ 1 - ( l a )
Df - 3
) [1 - 2 3 (2 - (l / a) ) (l a)
Df - 3 Df - 3 3 2
) ]. (1)
Figure 5. Relative K (○) and Di (▲) versus the scale length, l (nm)
We calculated the transport properties (hydraulic conductivity and diffusion, inside the allo‐
phane fractal aggregate, i.e. between 3 and 60 nm (Table 2). Figure 5 shows changes in K and
Di normalized to K and Di at l = 60 nm. Hydraulic conductivity decreased by 4 orders of
magnitude and Di decreased by 20 orders of magnitude when l decreased from 60 to 4 nm.
The very low calculated Di and K suggest that CLD is trapped in the porosity. The trapping
mechanism is favored by the large size of the fractal labyrinth. The higher the allophane
content, the bigger the labyrinth and the higher the retention. Like nanoporous materials
[28], the paradox of allophane clay is that it has large porosity but poor transport properties.
In these fractal structures, possible reactions with chemical or biological species that could
extract the pesticides are thus hindered; the pesticide remains trapped inside allophane clay
and cannot be extracted.
2.4. Influence of allophane on the decontamination process
Soil features influence pollutant availability and hence natural decontamination of the soil.
In the case of CLD and other organochlorine pesticides, natural decontamination is very
slow, and pollution management and remediation have to take into account any soil proper‐
ties that could influence the efficiency of the treatment considered [1].
Although different strategies are proposed in the literature to remediate diffuse pollution of
CLD including phytoextraction [29] and microbial degradation [30, 31], to date, these ap‐
proaches have not been very successful. Recently “In situ chemical reduction” was tested for
the reduction of the pesticide in the soil [32], but the technique was clearly less efficient in
soils containing allophane clay. Six months of in situ chemical reduction resulted in an 88%
decrease in CLD content in halloysite soils but only in a 47% decrease in allophanic soils.
The efficiency of the in situ chemical reduction technique was probably affected by poor ac‐
cessibility to the allophane clusters. Trapping thus likely reduces the degradation of pesti‐
cides in andosols. Whatever the soil decontamination process used to desorb or degrade the
CLD, the confining structure of the clay has to be taken into account to ensure the process is
effective.
3. Key farming practices
Soil type determines the potential for CLD sequestration but, in the analysis of risk and in
the diagnosis of soil pollution, farming practices are key factors that determine the level of
the pollutant stock, its potential availability, and its potential transfer to different environ‐
mental compartments [1, 10, 33]. In this section, we examine the effect of three main types of
practices characteristic of past and present farming practices. The first is the level of intensi‐
fication of the cropping system, which, in the case of CLD, determines the initial input [1, 10,
33]. The second is soil tillage, which determines the depth (and hence the volume) of soil
affected by the pollution [1, 10, 33]. The third is organic matter amendment, which affects
the availability of CLD [34].
3.1. Level of intensification of the cropping system
It is a truism to say that soils are polluted because they have received pollutants. Farmers
use a wide range of strategies based on pesticides to protect their crops. These strategies
mainly depend on the intensification of the cropping system [35-37]. Intensive systems pro‐
duce a higher cash flow and more profit than small diversified systems [38, 39] and farmers
tend to minimize the risk of yield loss by intensifying chemical pest control [10]. These farm‐
ers have to deal with higher pest pressure than systems that include crop rotation and diver‐
sification, and consequently require more frequent treatments [40, 41]. In agro-industrial
banana plantations in the French West Indies, agronomic and economic conditions led to the
intensive use of agrochemicals [39] i.e. frequent applications and/or high doses of the persis‐
tent molecule CLD over large areas. In 2013, twenty years after the treatments ended, the
8 Soil Pollution
ubiquitous presence of the CLD in different environmental compartments raises the ques‐
tion of the impact of these past applications on soil contamination and its true extent.
To answer this question, the link between CLD supply and soil contamination was first ex‐
amined using a simple CLD leaching model called WISORCH [1]. This model predicts soil
CLD content based on the history of CLD applications under different farming systems, dif‐
ferent soil types, and different average annual rainfalls. The model accounts for andosol, ni‐
tisol and ferralsol through their main characteristics, notably the soil-water partition
coefficient relative to organic C content (Koc in m 3 kg-1), soil organic carbon content, depth of
tillage and soil bulk density.
Simulation results first showed that the schedule of CLD applications, i.e. CLD loads, had
the most impact. Long after the application of CLD, the effect of CLD loads on soil CLD con‐
tent meant that soil decontamination was extremely slow. By exploring different assump‐
tions, the WISORCH model provided two main explanations for the slowness of
decontamination: 1) the absence of degradation; 2) only lixiviation by percolation water can
slowly reduce soil contamination. These assumptions are consistent with the lack of evi‐
dence for natural degradation of CLD reported in the literature [9]; given the very low vola‐
tility of CLD, water is the only vector of CLD dispersion.
In addition to the main effect of CLD applications, WISORCH simulations identified tillage
depth as the second factor that influences soil CLD content. This factor depends on cropping
systems and is discussed below. Here we simply note that physical factors like soil gravi‐
metric carbon content, bulk density and average annual rainfall have less impact. Conse‐
quently, human activity was the first determinant of soil contamination.
The WISORCH model explained why large areas are still contaminated even though treat‐
ments were halted long ago. As the model first identified the schedule of pesticide applica‐
tions, this suggested that tracing the history of these applications would help assess the level
and distribution of soil contamination at a regional scale. A historical analysis was per‐
formed in Guadeloupe using maps of banana plantations at different dates. The results re‐
vealed three gradients of land use for banana in 1 145 plots (1 376 ha) that were analyzed for
the presence of CLD [10].
Overall, plot contamination increased with the duration of land use in andosols and ferral‐
sols. Table 3 shows that on average, plots with short term banana land use were less conta‐
minated than those with medium and long term use. The lack of a significant difference
between medium and long term banana land use in CLD stocks and concentrations was con‐
sistent with the widespread use of CLD in the 1980s and the 1990s, but this was not the case
in the 1970s, when CLD was used frequently but not systematically. The effect of banana
land use duration was significant for andosols and ferralsols but not for nitisols. This differ‐
ence in behavior between soils could be explained by their ability to retain CLD. In this case,
unlike andosols and ferralsols, the lower retention capacity of nitisols could mask the impact
of variations in CLD inputs with respect to the length of time the land was used for banana
cultivation.
Land use duration
log([CLD]) Short Medium Long Mean soil
Andosol 0.87 a 1.14 b 1.16 b 1.11 c
(1.39) (2.13) (2.19) (2.05)
Ferralsol 0.25 a 0.52 b 0.38 ab 0.45 a
(0.28) (0.69) (0.46) (0.57)
Nitisol 0.62 a 0.66 a 0.64 a 0.65 b
(0.86) (0.94) (0.89) (0.91)
Mean duration 0.64 a 0.91 b 0.91 b
(0.9) (1.48) (1.48)
Table 3. Effect of soil type and banana land use duration (short, medium and long term) on the log of mean soil CLD
concentration (corresponding values in mg kg-1 in brackets). Differences in means were assessed at two levels:
globally, for the “mean soil” column and “mean duration” rows (in italics); for each soil, i.e. each row (in normal font).
For each case, the letters a, b and c indicate significant differences between factor levels at p <0.05 (Kruskal-Wallis
test). [10]
The soil sorption capacity was first assessed by inverting the WISORCH model [1] to deter‐
mine Koc. Results showed that andosols had a higher sorption capacity (Koc of 12-25) than fer‐
ralsols (Koc of 7.5-12) and nitisols (Koc of 2-3).
This is consistent with our previous results concerning CLD sorption. Indeed, these differen‐
ces in Koc do not stem from the chemical composition of the clays, since the allophane in an‐
dosols does not significantly differ from phyllosilicate clays (nitisols and ferralsols) [42]. The
higher apparent Koc in andosols could be the result of the allophane microstructure, leading
to lower CLD availability.
Moreover, large farms were more contaminated than small farms [10]. We selected farms
with more than 10 plots to assess the intra-farm variation in CLD concentrations. Figure 6
shows that inter-farm variation in CLD concentration was far higher than the intra-farm var‐
iation whatever the soil type, meaning the farm factor was decisive in explaining the distri‐
bution of CLD concentration. Regardless of the type of soil, the treatment strategy used on a
given farm was an essential component of soil contamination.
Concerning the type of soil and other physical factors of CLD retention in soils, at regional
scale, the effect of the type of soil was clear. Analysis of CLD concentrations (Table 2) re‐
vealed significant differences between soils (p<0.05 Kruskal-Wallis test) showing that ando‐
sols were the most contaminated. Values of CLD concentrations in andosols were 2.3-fold
higher than in nitisols, and 3.6-fold higher than in ferralsols. Organic carbon (OC) content
was only calculated for andosols to avoid possible interactions with other physical variables
(notably, allophane). Results showed that andosols with high OC content tended to retain
CLD better. However, the relationship was weak. A probable explanation is that OC had
less impact due to the variability of inputs. Finally, although carbon is considered to deter‐
10 Soil Pollution
Figure 6. Distribution of CLD concentration on farms located on andosols and nitisols with more than 10 banana
plots. Each box represents a farm. The bottoms and tops of the boxes represent the 25th and 75th percentile; the
band inside the box is the median; the whiskers extend to the most extreme data point, which is no more than 1.5
times the interquartile range of the box [10].
mine sorption of CLD, at the regional scale, organic carbon was not a prime factor in ex‐
plaining variations in CLD concentrations.
To summarize, findings at the regional scale were consistent with the findings of the WI‐
SORCH model at the plot scale. Farming systems mainly explained soil CLD contamination,
and physical factors like soil carbon content had less influence. Concerning the type of soil,
although the findings were the result of observations at different scales - region, plot, micro‐
structure – they all highlighted the specific sorption capacity of andosols for CLD.
These important observations made it possible to draw maps of areas with a risk of CLD
contamination in Guadeloupe and Martinique based on soil type and on an historical analy‐
sis of the supply of CLD [43, 44]. They also identified a pollution system characterized by
remarkable inertia comprising (i) a persistent molecule – CLD; (ii) intensive large scale ap‐
plications; (iii) andosols with high organic matter content and an allophanic microstructure
that can trap pollutants. This inertia was assessed by simulating changes in soil CLD con‐
tamination using the WISORCH model. Figure 7 shows that one hundred years will be
needed to clean up nitisols, and six hundred years to clean up andosols (Cabidoche et al.,
2009).
Figure 7. Simulation of changes in soil CLD contamination in Guadeloupe
Figure 7 highlights the need to use different temporal scales to assess pollution: a long term
scale when soil retention properties are probably the main explanation for the spatial varia‐
bility of soil contamination; short and medium term scales when applications of the pollu‐
tant explain most spatial variability. From a management point of view, authorities are thus
justified in focusing on reducing pollutant loads (the frequency of application and the quan‐
tities of pollutant used) on cropped areas, even in the case of less persistent molecules. In the
case of CLD, the persistence of pollution calls for further research on soil decontamination.
However, in the meantime, different agricultural practices can help manage the risk of con‐
tamination, this being the case of soil tillage and organic matter amendment.
3.2. Tillage practices
A second step of the diagnosis is the analyses of soil tillage The heterogeneity of CLD con‐
tent at field scale and the effect of tillage were investigated [33].
12 Soil Pollution
In the French West Indies, different tillage practices are used on banana plantations. These
range from no tillage, especially on sloping plots, to regular deep tillage to a depth of 60 cm
or more, every four years [10, 45]. The mode of application of the pesticide (powder spread
on the ground around the foot of the banana tree), and the semi-perennial arrangement of
trees [46] account for the high heterogeneity observed at field scale. Indeed in our study, in
the same plot, CLD contamination of the upper soil layer (0-30 cm) could range from 0.2 to
2.7 mg kg-1 and, in plots of less than 1 ha, from 2.9 to 17.6 mg kg-1.
Figure 8. Ratio of CLD contents in the 0-30 cm and 30-60 cm soil layers as a function of the depth of tillage, adapted
from [33].
Figure 8 shows that, whatever the soil type, the upper soil layer (0-30 cm) was generally
more contaminated than the lower layer (30-60 cm). With no tillage, the 0-30 cm layer was
four times more contaminated than the 30-60 cm layer (21 mg kg-1 and 5 mg kg-1). We also
showed that tillage had a significant effect (P <0.0001) on the horizontal distribution of the
contaminant, leading to pesticide dilution in the soil profile. With deep tillage (60 cm and
deeper), whatever the type of soil and mean CLD content, CLD content was similar at the
two sampling depths, with mean values of 11 vs. 12 mg kg-1, 12 vs. 11 mg kg-1 and 2 vs. 2 mg
kg-1. Thus CLD content was homogenized in the 0-60 cm layer. In plots where tillage was
shallower, the upper layer was still significantly more contaminated than the lower layer al‐
though the proportion depended on the tillage depth. This result is in accordance with re‐
sults observed for DDT [47].
Likewise, tillage tended to reduce CLD horizontal heterogeneity. For this reason, when sam‐
pling soil, it is important to take such heterogeneity into account at intra-field scale for accu‐
rate assessment of CLD content. For sampling, it is recommended to subdivide all plots of
more than 2 ha and, more generally, to use an appropriate sampling procedure that takes
into account landscape (slope and resulting erosion), field history (tillage, cropping system,
former inter-row distance, etc.) but also the reasons for sampling: cropping system manage‐
ment or analysis of overall risk.
Tillage is of major concern because it modifies the vertical distribution of the pollutant with‐
in the soil profile, and hence the volume of soil that is contaminated and the level of contam‐
ination. The risk of the pollutant being transferred to the crop depends on the type of soil
and soil CLD content [19, 26]. Thus, in some cases (mainly low soil CLD content), this will
determine the range of crops that can be cultivated while respecting regulatory thresholds,
in particular the maximum residue limit of 20 µg kg-1 fresh matter for food products [23].
It is also important to keep in mind that tillage practices may negatively affect CLD seques‐
tration and distribution. Indeed tillage during the dry season can cause surface desiccation
and reduce pore volume, which will irreversibly alter the micro-structure of allophane [45,
48], thus possibly modifying soil CLD sequestration potential. Moreover, inappropriate till‐
age practices increase the risk of erosion, driving the top soil layer downslope [49] thereby
modifying CLD distribution at plot scale, with higher CLD content at the bottom of the
slope than at the top [33].
In conclusion, analyzing past and present farming practices provides insight into CLD con‐
tent and distribution at intra-field scale. These practices can also affect CLD availability by
modifying soil retention properties.
3.3. Pesticide sequestration by compost addition
The use of soil organic matter to control the environmental mobility and fate of pesticides
has already been reported in the literature [50-53]. Here, we propose an alternative strategy
which is quite the opposite of total soil decontamination: CLD sequestration enhanced by
soil organic amendment. Because CLD is tightly trapped in the soil, an alternative solution
to decontamination may be to further increase its sequestration in the soil thereby reducing
pesticide diffusion into the environment. This could be a way to reduce further release of
CLD from contaminated soils towards other environmental compartments until efficient re‐
mediation techniques become available. We now examine the hypothesis that adding organ‐
ic matter to contaminated soils improves their CLD sequestration ability with the objective
not of removing the pesticide from the soil but rather of controlling its release into the envi‐
ronment.
As detailed above, combined with high organic matter content, the microstructure and the
large specific surface area of clay favor the accumulation of pollutants in the soil. In allo‐
phanic soil, the high CLD content is the result of the combination of CLD’s high affinity for
the soil organic content and the poor accessibility of CLD into the mesopore structure.
With the aim of preventing consumer exposure, we tested the incorporation of compost in
soils as a possible way to reduce plant contamination [34], based on the hypothesis that add‐
ing organic matter would improve CLD trapping and thus reduce its bioavailability for
crops. We characterized the transfer of CLD from soil to radish, a crop belonging to roots
and tubers, a CLD sensitive group. Two months after incorporation of the compost, the con‐
tamination of the different plants organs was 3, 15 and 5 times lower in small roots, tubers
and leaves, respectively than without added compost (Figure 9.).
14 Soil Pollution
Figure 9. CLD transfer in radish organs (leaves, tubers and fine roots) with added compost (in grey) and without (in
white).
These experiments also showed that adding compost closed the microstructure of allophane
clays, thus favouring CLD retention in allophanic soils. Adding compost altered the porosi‐
ty of the allophane clay in the size range 10 to 60 nm, while the intensity of this effect varied
with the allophane content. We suggest that these pore changes are the consequence of ca‐
pillary stress and of the low mechanical properties of the fractal structure [54].
Organic matter (OM) amendments aim to modify chemical conditions within the soil pro‐
file. As mentioned above, the OM soil status influences the availability of the pollutant, a
factor of primary importance for both pollutant transfer and degradation [55]. At field scale,
OM amendments modify the potential sequestration of persistent organic pollutants in the
soil by enhancing the soil’s sorption capacity for CLD [56]. This sorption capacity depends
on the quantity of OM supplied, the type of OM (stable vs. labile) and the frequency of the
application. This practice needs to be studied over time, as OM degrades and could modify
the OM – pollutant relationships.
Finally, all these practices depend on farm strategies. When the aim is to modify these prac‐
tices, both the scale and the type of farm need to be taken into account. In the case of CLD
pollution, analyses revealed a strong “farm effect” [10]. A typology should be built includ‐
ing the farms’ overall strategy and objectives, the types of crops grown, practices (more or
less intensive) and the farms’ specific field orientation (how often the land is used for each
crop).
4. Discussion
When dealing with agricultural soil pollution, two major tasks must be included in the risk
and management analysis: the characterization and analysis of soil physical properties and
farmers’ past and present practices (Figure 10). These two domains provide information on
the level of soil pollution, on the behavior and fate of the pollutant in the soil, and on its
remediation potential.
Figure 10. Key determinants of CLD soil pollution.
For soil physical properties, porosity can range from micro-, to meso- and macro-porous in
volcanic soils. Fluids containing inorganic and organic solutes and gaseous species can occu‐
py the pores and several factors (size, shape, distribution and connectivity of the pore geo‐
metries) determine how fluids migrate into and through the porosity and ultimately adsorb
and react with the solid surfaces. The low hydraulic conductivity calculated for fractal allo‐
phane aggregates thus explains the high pesticide content of these materials. Because of the
resulting low hydraulic conductivity, fluid exchange is slow and pesticide bioavailability is
consequently reduced. This leads to the accumulation of pesticides that are not easily chemi‐
cally or biologically transformed. Future studies on pesticide degrading microorganisms
[57] and other bioremediation tools to clean up polluted soils should take the high soil or‐
ganic carbon content and CLD accessibility in volcanic soils into account.
At field scale, past pesticide application practices account for the potential stock of pollutant
in the soil, and an historical analysis will improve the initial diagnosis in terms of the quan‐
tity (doses) of pollutant and its horizontal distribution. Modeling and mapping are appro‐
priate tools to roughly simulate current levels of pollution in the field and to identify the
parts of a field that contribute to environmental pollution (water, food, animals). Current
16 Soil Pollution
practices explain the diffusion of pollution from the soil reservoir: tillage accounts for the
vertical distribution of the pollutant, i.e. dilution of the pollutant in the soil profile, and or‐
ganic matter amendments account partially for the availability of the pollutant. In this way,
current practices modify the level and volume of polluted soil and the environmental state
of the field (surface conditions, OM content, soil profile, etc.), which in turn, influence the
fate of the pollutant in the environment. Variability among and within farms also needs to
be taken into account by building a farm typology including the general orientations and
strategies of the farm and farm practices.
Finally, at the regional scale, all these factors combine to determine soil pollution. In the case
of CLD, in agreement with the results of physical analyses, andosols, which contain allo‐
phanic clay and have high organic matter content, are the most polluted. In addition, the ef‐
fect of agricultural practices was evidenced by a strong farm effect and an effect of how long
the land was used for banana cultivation.
These two aspects (agricultural practices and soil physical properties) proved to be effective
in the diagnosis of CLD pollution and in management analysis and could make sense for
other forms of agricultural pollution, especially in the case of persistent pesticides. To illus‐
trate this point, the WISORCH model, which accounts for agricultural loads as well as soil
and climate characteristics, can simulate changes in soil pollution over time. In the case of
CLD contamination, simulation results showed that soils will remain contaminated for six
hundred years, with andosols decontaminating at a slower rate than other soils.
5. Conclusion
Wide use of chlorinated pesticides such as chlordecone has led to severe contamination of
the environment in the French West Indies. Chlordecone is an organochlorine insecticide
that was regularly applied to banana crops more than 20 years ago and is now on the list of
POPs prohibited by the Stockholm Convention. Chlordecone is a hydrophobic, non-biode‐
gradable pesticide that is strongly bound to the organic soil matrix which is why soils are
still contaminated today. The level of soil pollution and the fate of the pollutant in the eco‐
systems depend on two main determinants at two different scales: agricultural practices and
the soil physical properties, i.e. the clay microstructure.
Obviously, the history of agricultural practices (land-use changes and the intensity of pesti‐
cide use) partly explains soil contamination. The stocks of pesticide still present in soil are
important indicators, which calls for the reinforcement of databases on agricultural practi‐
ces, especially on pesticide use. However, soil type mainly explains the concentration of pol‐
lutant in the soil. Our experimental data showed that clay microstructure controls the
accumulation of pollutants in the soil and their release into the environment. In the case of
andosols, this study revealed the importance of the fractal microstructure and the associated
tortuous porosity in trapping the pollutant. Moreover, as chlordecone tends to bind with
soil organic matter, the high organic content of tropical volcanic soils also favors its reten‐
tion.
More generally, it is worth noting that since pesticide retention depends on soil type, the
concentration of pesticide in the soil cannot provide reliable information about the actual
risk of contamination from the soil to the ecosystem. This makes mapping contamination
hazards problematic because there is no univocal relation between soil pollution and its ca‐
pacity to contaminate crops. Additional studies are thus necessary to characterize the behav‐
ior of pesticides in soils and to identify the link with plant absorption. This will increase the
relevance of mapping pesticide contamination and availability
Concerning possible soil decontamination procedures, the efficiency of chemical or biologi‐

cal species able to degrade the pesticide will be controlled by the microstructure of the clays.
Future research into pesticide degrading microorganisms and other remediation tools that
could be used to clean up polluted soils should take the accessibility of the pesticide in the
soil microstructure into account.
Acknowledgements
Funding was provided by the French Chlordecone National Plan (“JAFA” project), the French
National Research Agency (“Chlordexco” project) and the French Ministry for Overseas
development (MOM).
Author details
T. Woignier1,2*, F. Clostre3,4, P. Cattan5, J. Levillain5, Y.M. Cabidoche6 and

M. Lesueur-Jannoyer3,4
1 CNRS UMR 7263- Institut Méditerranéen de Biodiversité et d’Ecologie marine et continen‐

tale (IMBE), Aix-Marseille Université, Faculté de St-Jérôme, Marseille, France
2 IRD UMR 237- Campus Agro Environnemental Caraïbes, Le Lamentin, Martinique
3 Cirad UR HortSys, TA B-103/PS4, Montpellier, France
4 Cirad/PRAM, UPR HortSys Agroecological Functioning and Performances of Horticultur‐

al Cropping Systems, Le Lamentin, Martinique
5 Cirad UPR Banana, Plantain and Pineapple Cropping Systems, Guadeloupe, France
6 Inra ASTRO (TROpicalAgroSystems) Research Unit, Domaine Duclos, Guadeloupe,

France
18 Soil Pollution
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cides by Clay Minerals and Humic Acid-Clay Complexes. Soil Sci Soc Am J.
2003;67(1):122-31.
[54] Fernandes P, Jannoyer-Lesueur M, Soler A, Achard R, Woignier T. Effects of clay mi‐

crostructure and compost quality on chlordecone retention in volcanic tropical soils:
consequences on pesticide lability and plant contamination. 19th World Congress of
Soil Science, Soil Solutions for a Changing World2010. p. 50-3.
[55] AFNOR. Amendements organiques et supports de culture - Caractérisation de la

matière organique par fractionnement biochimique et estimation de sa stabilité biolo‐
gique - Soil improvers and growing media - Characterization of organic matter by bi‐
ochemical fractioning and estimation of its biological stability. Saint Denis la Plaine,
France: AFNOR; 2009. p. 19.
[56] Dolfing J, Novak I, Archelas A, Macarie H. Gibbs free energy of formation of chlor‐
decone and potential degradation products: implications for remediation strategies
and environmental fate. Environ Sci Technol. 2012;46(15):8131-9. Epub 2012/07/12.
[57] Dolfing J, Novak I, Archelas A, Macarie H. Gibbs free energy of formation of chlor‐
decone and potential degradation products: implications for remediation strategies
and environmental fate. Environ Sci Technol. 2012;46(15):8131-9. Epub 2012/07/12.
chapter 22
Chemical and Hydraulic Behavior of a Tropical Soil

Compacted Submitted to the Flow of Gasoline
Hydrocarbons
Wisley Moreira Farias, Geraldo Resende Boaventura,

Éder de Souza Martins,
Fabrício Bueno da Fonseca Cardoso,
José Camapum de Carvalho and
Edi Mendes Guimarães
1. Introduction
Gasoline is a fuel comprised basically of hydrocarbons such as aromatic, olefinic and saturated
compounds of a carbon chain comprised of 4 to 12 atoms. The aromatic compounds such as
benzene, toluene, ethylbenzene, o-, m-, p-xylene (BTEX) are harmful to human health (Cairney
et. al., 2002). As these compounds are harmful to health, the legislation becomes restrictive.
The U.S. Environmental Protection Agency for drinking water (US EPA) establishes the
maximum concentration of benzene in 5μg.L-1. In Brazil the Ordinance of the Ministry of health
number 2,914 in 12th December 2011, stipulates that the maximum allowable concentration of
benzene is 5 μg.L-1 regulation of drinking water contaminant. Soil in contaminated residential
areas, Brazil has been adopting as intervention guide value, the concentration of benzene 0.08
mg.kg -1 set by the State of São Paulo in 2001. This value indicates the intervention limit of
contamination where there is potential risk to human health.
Brazil produces type-C gasoline which is different than other types due to its anhydrous
alcohol content (ethanol), in the proportion of 25% (Farias, 2003). The alcohols are soluble in
water, and have a significant mobility potential to percolate through the soil until reaching
underground water (Ulrich, 1999; Corseuil and Fernandes, 1999). The alcohol in gasoline in an
2 Soil Pollution
aqueous medium promotes co-solvency which is the increase in the solubility of the hydro‐
carbons in the gasoline in an aqueous solution (Banerjee and Yalkowsky, 1988; Cline et al.,
1991).
Solubility is generally controlled by the polarity effect, which decreases in size for molecules
with the same organic function. Non-polar or weakly polar substances dissolve in similar
solvents. Thus, highly polar compounds dissolve in polar solvents such as water. The polarity
or dipolar moment is proportional to the dielectric constant, and therefore high dielectric
constant compounds (values of 80 for water and 34 for methanol) dissolve ions through
hydration of the disassociated types (Fernandez and Quigley, 1985).
On the surface of clay-minerals, the absorbed water forms a double layer, which reduces the
strength of interaction between the negatively charged clay particles and the cations in the
colloidal solution. The hydrophobic hydrocarbons in the gasoline have low dielectric constant
values, thus provoking the collapse of the double layer. This collapse is due to the contraction
of the double layer through the attraction of the contra-ions which are closer to the superficial
charge of the clay-minerals, favoring flocculation, and consequently the increase in permea‐
bility due to the increase in pore space (Mesri and Olson, 1971; Fernandez and Quigley, 1985
and 1988).
The co-solvêncy is responsible for the partition of BTXs to the aqueous phase, promoting the
reduction of density of colloidal solution of soil, providing increased viscosity and a reduction
of the surface tension (Mcdowell and Powers, 2003). This reduction in surface tension and
generated by the collapse of the electrical double layer that there was between the soil and
water (Farias, 2003).
1.1. Aspects of the transport of pollutants in soils

The transport of pollutants in the soil can occur through the porous medium and saturated or
unsaturated fractured media. This transportation occurs through physical or chemical
processes, or through both processes. The chemical process becomes evident when the velocity
of the fluid is not sufficiently high (i.e., less than 10-6 cm/s), generating a gradient due to the
flow of the solute (contaminating agent) from the more concentrated medium to the less
concentrated one. This process is called molecular diffusion (Rowe, 1988; Pastore and Mioto,
2000). This type of flow has been widely studied with metals and organic compounds in solid
waste landfill leachate contaminants, for application in compacted soil layer, also called liners
(Shackelford and Daniel, 1991; Rowe, 1988; Barone et al., 1988).
Fernandez and Quigley (1985) developed an experimental research program to evaluate the
hydraulic behavior of clayey-like soil (Sarnia, Ontario), permeated with liquid substances such
as benzene, xylene, cyclohexane, aniline, propanol, acetone, alcohol and water. The results
have shown that Hydraulic conductivity increased from 5 x 10-9 to 1 x 10-4 cm.s-1 along with a
decrease in the dielectric constant from 80 (water) to 2 (benzene).
When there is a hydraulic gradient, the velocity of the solvent is relatively high and the
transportation of the solute is practically managed by the velocity of the solvent, a mechanism
Chemical and Hydraulic Behavior of a Tropical Soil Compacted Submitted to the Flow of Gasoline Hydrocarbons 3
which is known as an advection process. In this process, the velocity of the fluid is governed
by Darcy’s Law, which considers not only the characteristics of the soil, but also those of the
fluid (Fernandez and Quigley, 1988).
In order to have good performance, the compacted clay liners must have a hydraulic conduc‐
tivity less than 10-8 cm/s. However Daniel and Koerner (1995) defined that the hydraulic
conductivity of clay liners must be less than or equal to 10-7 cm/s. This low flow is normally
associated with the presence of clay-minerals, and at least 15 to 20% of particles with sizes
under 2 mm, as well as a minimum plasticity greater than 7%, activity greater than 0.3, and
cation exchange capacity (CEC) greater than 100 mmolc/dm3 of soil (Rowe et al.,1995).
The natural organic material of the soils have proven to be efficient in the retarding process
through the sorption of hydrophobic hydrocarbons, which are also found in gasoline (Chiou
et al., 1983; Karickhoff et al., 1979; Schwarzenbach et al., 1993).
1.2. Importance of research
The aim of this study is to evaluate the behavior of a tropical soil, and their performance as
liner against the flow of hydrocarbons from gasoline, by interpreting transportation according
to physical and chemical parameters, as well as micromorphological aspects. For this charac‐
terized the mineralogy of the soil and the influence of his organic matter (OM), considering
the adsorption processes of hydrocarbons from gasoline and hydraulic behavior in the
laboratory by variation of the hydraulic gradient in front of the gasoline flow through
compacted soil. This study also aims to contribute to the understanding of the dynamics of the
flow through the soil of specific groups of compounds: aromatic, olephine, saturated hydro‐
carbons and the ethanol found in Brazilian type-C gasoline (a complex mixture of organic
compounds).
2. Location and Soil classification
The soil sample was collected indisturbed block in depth of 4 m in the experimental field of
foundations and test field of the Civil Engineering Department of the University of Brasília,
located on the University campus in the city of Brasília, Brazil with coordinates 15° 56 ' 45 "S,
47° 52 ' 20" W (Fig. 1).
The sample of lateritic soil typical of the Brazilian cerrado region was studied. According to the
Brazilian Soil Classification System (Embrapa, 1999), the soil was classified as Red Latossoil,
considered as Ustic Rhodic Oxisol according to the U.S. Soil Taxonomy and Geric Ferralsol
Ferric (FAO, IUSS Working Group WRB, 2007). It possesses a silt-clay-like texture, a large
quantity of granular aggregates, and small pores. Visually, it is homogeneous and isotropic,
without the presence of discontinuities.
4 Soil Pollution
Figure 1. Map of location of the soil collection.
3. Methodology
The characterization of the soil involved the use of physical, chemical and mineralogical
analysis.
3.1. Physical tests

Geotechnical tests of physical properties of soils were performed following the Brazilian
Association of Technical Standards (ABNT) procedures: test of limits of consistency called
Atterberg limits following the ABNT NBR 7180/84 plastic limit; 6459/84 liquid limit following
the Casagrande method. Before the grain-size determination, the real density was determined
according the ABNT NBR 6508/84 method. The grain-size distribution curve was determined
using a grain-size digital meter Malvern Mastersizer with lens de 300Rf for grain size range of
0.05 μm to 900μm at 25 ° C. For this analysis, the sample was previously passed through a No.
40 sieve. The analyses of the samples were done either with or without ultrasound dispersion.
Ultrasonic condition was 5 minutes of dispersion in distilled water with ultrasonic level set at
5. The grain size fractions were classified following the Brazilian standard NBR 6502/93.
The degree of flocculation and dispersion of soil particles was determined comparing the
results of grain size determinations before and after ultrasonic dispersion.
3.1.1. Hydraulic conductivity

The test of hydraulic conductivity in compacted soil in standard Proctor energy were per‐
formed in a conventional manner with water using the variable charge and special form for
gasoline (Fig. 2 and Fig. 3).
Figure 2. Hydraulic Conductivity cell.
Figure 3. Schematic of permeameter Cell.

6 Soil Pollution
The gasoline hydrocarbons, for possessing volatile and low-density compounds require a
special sealed cell to avoid losses due to evaporation and leakage and to support the applied
tensions. The material selected for the construction of the special cells was stainless steel, to
avoid reaction and adsorption problems in the walls, which is the case of plastics and acrylics
(Doanhue et al., 1999).
The system used to perform the gasoline’s hydraulic conductivity was similar to that applied
by Fernandes (1989). The special cell may be disassembled, and is made up of three parts. The
first part is a cylinder, where the test material and reservoir are found. This part is 5 mm thick,
110 mm long and has an internal diameter of 77.2 mm. The other two parts are the upper and
lower lids. Both have cavities filled with rubber rings which are able to prevent the reaction
of the hydrocarbons in the gasoline and act as a seal when the cylinder is assembled. The upper
lid has two openings, one for the entry of fluid and the other for the application of vertical
tension with compressed air. The lower lid is made up of an outgoing flow register which is
connected to a collecting container. The two lids are 120 x 120 mm2 square, 10 mm thick. The
connections were made out of aluminum, due to its low cost and flexibility; the connecting
joints were sealed with 3M automotive glue and winding sealing thread, in order to prevent
leaks and to make the system more secure.
The conductivity test was performed with test material 5 cm long, compacted at normal Proctor
energy at optimal water content condition, in the cylinder of the hydraulic conductivity cell.
Then, a thin disk of porous ceramic was placed top of the sample. The small space between
the disk and the cylinder wall was filled with 3M glue to prevent preferential flows along the
wall, and to ensure that the gasoline only passed through the porous ceramic disk. The cell
was then assembled, and the upper and lower lids were connected to the cylinder. The cylinder
is 11 cm high, of which the remaining 6 cm were filled with type C gasoline. After the cell was
totally sealed and connected to the compressed air system, with pressure controlled by a
manometer, it was connected with plastic tubes able to support high pressure. The conduc‐
tivity tests were performed for various applied vertical pressures. For each pressure applied,
the hydraulic conductivity was measured. The pressures were varied to see how the soil
sample behaved with an increase in hydraulic gradient upon the flow of gasoline. The
hydraulic conductivity was measured in the laboratory at static tensions σ of 50, 100, 150, 200,
v
and 300 kPa, with respective hydraulic gradients of 75, 150, 225, 300, and 450.
The residual water of the soil pores mixed with gasoline collected in the test was previously
run through a separating funnel to remove the aqueous phase to later take a reading of
hydrocarbons of the gasoline through infrared technique.
The test material of the lateritic soil sample, before and after the hydraulic conductivity test
conducted with water, and the other with the flow of gasoline, were dried at room temperature.
Micromorphological analyses were performed on Thin Lamina (TL) in vertical sections,
prepared by impregnating the sample with plastic resin (Cardoso, 1995; Martins, 2002). The
instrumental technique used for the microscopic views of the TL was Optical Microscopy.
3.2. Mineralogical characterization
The identification and quantification of minerals in the sample were carried out by the method
developed by Martins (2000). This method involves the use of X-ray diffraction (XRD)
technique for identifying the minerals, chemical analysis for the determination of major
elements (Al, Fe, Si, Ca, Mg and Ti), thermogravimetric analysis (TGA), and the use of Munsell
color code (Munsell color company Inc., 1975). The determination of major chemical elements
was performed by ICP-AES after the fusion of samples with alkaline NaOH as fondant at a
temperature of 450 ° C for 40 minutes using the nickel crucible. Determinations of elements by
ICP / AES (atomic emission spectrometry of Plasma Induced Coupling) were performed with
Thermo Jarrell ASH equipment, model Iris / AP.
The thermogravimetric analysis were applied to quantify the kaolinite and gibbsite. For this
used the TGA Shimadzu equipment with temperature ramp of 20 °C to 1500 °C, with speeds
ranging from 0.2 to 60 ° C / min, using the software TAS60WS for the treatment of data. The
Munsell code was used for determining the ratio of hematite and goethite in the soil samples.
The CEC of soil was determined using the principle of the simple as the sum total of the
exchangeable cations that a soil can adsorb. The determination of the organic matter content
was done prior to extraction using wet oxidation method with potassium dichromate in
sulfuric medium. The excess of dichromate after oxidation was titrated with standard solution
of ferrous ammonium sulfate (Mohr salt).
3.3. Chemical characterization
The pH was measured in the soils samples in distilled water medium using a combined glass
electrode Ag/AgCl (potentiometric method).
In order to study the influence of OM and mineralogy in the gasoline sorption process, an
experiment was performed with samples treated with H2O2 and another without treatment.
The extraction of the OM used 15 g of soil in a porcelain capsule, with 10 mL of H2O2 volume
30% and with agitation in a 50 mL Becker cup. After agitation, there was an effervescent
reaction, when the capsule was covered with clock glass for one night. The process was
repeated until the complete disappearance of the reaction. It was then washed 3 to 5 times in
distilled water, using a Büchner funnel with filtering under reduced pressure. Then, for the
gasoline sorption test, the sample was allowed to dry at room temperature.
The sorption test procedure used 2 g of soil with 25 mL of gasoline placed in an amber glass
jar under agitation for 24 hours at a temperature of 22oC. After this, the samples were centri‐
fuged as in the processes described above, with the removal of a 15 mL portion for analysis.
The hydrocarbons content of the gasoline samples was determined at the National Petroleum
Agency (ANP) laboratory, in Brasilia, with a (FTIR = Fourier Transform Infrared), manufac‐
tured by Grabner Instruments, model IROX 2000. This instrument qualified and quantified the
compounds, generating the mass and volume percentages of the ethanol, aromatic, olephine
and saturated compounds.
8 Soil Pollution
4. Results
Tab. 1 and 2 present data of the physical, chemical and mineralogical Brazilian soil and
constituents of the gasoline type C studied.
Test Lateritic
Atterberg Limits
Liquid limit-WL (%) 41
Plastic limit-WP (%) 29
Plastic Index-IP (%) 12
Activity 0,18
Grain size distribution*
Clay (%) 65
Silt (%) 34
Sand(%) 1
Degree of flocculation (%) 92
Degree of dispersion (%) 8
Chemical Parameters
pH 5,70
Organic Matter content (%) 0,41
CEC (mmolc/dm3) 6,4
Mineralogy
Quartz (%) 30,2
Anatase (%) 1,57
Kaolinite(%) 24,6
Gibbsite (%) 25,5
Goethite (%) 4,6
Hematite (%) 7,5
Illite (%) 2,2
Vermiculite (%) 3,7
Hydraulic Conductivity in water (cm/s) 3,7.E-07
*Grain size data obtained by ultra-sound waves using a laser beam grain size analyser.
Table 1. Characteristics of the soil (Farias, 2003).
Tab. 2 presents the composition of the Brazilian type-C gasoline, according to Farias (2003).
Compounds Mass (%)

Aromatics 20,8
Olefins 22,4
Saturated 31,4
Ethanol 25,4
Table 2. Brazilian Type C gasoline data.
Fig. 4 presents the increase in hydraulic conductivity with an increase in the hydraulic gradient.
At a gradient of approximately 210, conductivity becomes practically constant. Fig. 5 presents
the intrinsic permeability, which considers the characteristics of the soil, but does not consider
the chemical and physical properties of the fluid. Intrinsic permeability reaches values close
to 10-13m2. However, as the hydraulic gradient increases, stability reaches approximately 4.5
10-11m2. 4
Conductivity Hydraulic
Conductivity Hydraulic K.10-8 (cm /s)

3.5
K.10-8 (cm /s)

Hydraulic conductivity (cm/s)
2.5
10 110 210 310 410 510
1.E-05 10 110 210 310 410 510 2
Conductivity Hydraulic (cm/s)
1.E-05
1.5
1
1.E-06
1.E-06
0.5
0
1.E-07 0.00 0.2
1.E-07
1.E-08
1.E-08 Hidraulic Gradient
Hidraulic Gradient 1.80

Figure 4. Behavior of hydraulic conductivity and hydraulic gradient of laterite soil on the gasoline flow.
1.60
0 50 100 150 200 250 300 350 400 450 500 1.40
1,E-10
1.20
Intrinsic permeability
1,E-11 1.00
C/Co
( cm 2)
0.80
1,E-12
0.60
1,E-13
0.40
1,E-14 0.20
Hydraulic Gradient
0.00
0.13
Figure 5. Behavior of the intrinsic permeability and hydraulic gradient of laterite soil on the gasoline flow.
A
10 Soil Pollution
Fig. 6 depicts the behavior of the hydraulic conductivity relative to the volume of pores while
undergoing saturation in the test material with gasoline at a tension of σ of 50 kPa. The v
saturation process takes place with the expulsion of the interstitial water accumulated in the
pores due to optimal compacting moisture content (w = 26%) is the test material at normal opt
Proctor energy. It may be observed that as the volume of pores in the gasoline flow increases,
conductivity decreases from 4 to 2 x 10 cm.s . This suggests that the behavior of the reduction
-8 -1
may be represented by a second-order equation.
4.5
Hydraulic Conductivity
3.5
K.10-8 (cm /s)
2.5
2 y = -3.3548x2 + 2.762x + 3.5475

1.5 R² = 0.9576
0.5
0
0.00 0.50 1.00 1.50
Pore Volume
Figure 6. Behavior of the lateritic soil saturated with gasoline at 50 kPa.
Fig. 7 presents the saturation process at a σ tension of 50 kPa, based on the ratio between the
v
concentration (C) of the gasoline hydrocarbons passing through the soil sample, and the initial
concentration (C ) added to the reservoir, in relative to the volume of pores. The hydrocarbons
o
concentration data are from the Light Non-aqueous Liquid Phase (LNALP), after the flow
through the soil sample in the hydraulic conductivity test.
w γ γdmax γs Sr Vv
Sample e n
(%) (kN.m-3) (kN.m-3) (kN.m-3) (%) cm3
lateritic* 1,7 17,7 17,4 27,5 0,58 0,4 8,1 134,3
lateritic** 1,7 15,8 15,6 27,5 0,77 0,4 6,2 178,5
lateritic*** 1,8 14,7 14,5 27,5 0,90 0,5 5,3 210,0
*Dry soil sample before the hydraulic conductivity test
**Dry soil sample after the hydraulic conductivity test with the water flow
*** Dry Soil sample after the hydraulic conductivity test with the gasoline flow
Table 3. Result of the physical parameters of the test material.

Hydraulic conductivit
1.E-06
1.E-07
The results in Tab. 3 present

1.E-08 the physical parameters of the compacted test materials dried at
room temperature before and after the hydraulic conductivity

Hidraulic test. Highlights the volume of
Gradient
voids (Vv), which changes substantially when there is a flow of gasoline. The degree of
saturation also decreases after the flow of gasoline.
1.80
1.60
1.40
1.20
1.00
C/Co
0.80
0.60
0.40
0.20
0.00
0.13 0.36 0.65 0.90 1.14
Pore volume
Aromatics olefins Saturated Ethanol
Figure 7. Light non-aqueous liquid phase ratio of the gasoline relative to the volume of pores of the lateritic soil in a
saturation process at 50 kPa.
The micromorphology of the three compacted soil samples was important in order to visualize
the behavior of the test material before the hydraulic flow (Fig. 8), after the hydraulic flow with
water, and after the flow with gasoline. It must be noted that the grains of quartz make up
approximately 40% of the total solid material; variable in size, 0.12 mm on average; and overall,
are sub-rounded to angular. They are highly fractured, without orientation and their contours
present corrosion. In spite of the compacting, the structure of this soil is not totally dispersed,
for microaggregations of oxyhydroxides of Fe and Al remain, forming micropores. The
compacted soil sample submitted to percolation in water showed a single micro-structural
difference relative to the one performed on the LT of the compacted soil sample. Actually,
there was an increase in small canal-type voids, generated by the flow of water (Fig. 9). The
micromorphology regarding the LT of the compacted soil submitted to the flow of gasoline
also showed only a quantitative increase in canal-type voids (Fig. 10). However, this variation
was greater than that registered in the previous sample with the water flow.
12 Soil Pollution
Figure 8. Photomicrography of the porfirosquelic APE, aggregates, and quartz grains of the compacted lateritic soil.
Parallel nichols (N//).
Figure 9. Photomicrography showing the nodules and canal- and chamber-type voids of the compacted lateritic soil
submitted to percolation with water. Parallel nichols (N//).
Figure 10. Photomicrography showing the canal-type voids of the compacted lateritic soil submitted to percolation
with gasoline. Parallel nichols (N//).
Fig. 11 presents the results of the adsorption of the ethanol and aromatic substances in the
samples with and without the extraction of organic matter with the use of hydrogen peroxide.
Note that the samples treated with extractor presented low adsorption. Aromatic compounds
showed no adsorption after extraction of organic matter contained in the soil.
200,00
Concentration ( g/g)
150,00
Ethanol
100,00
Aromatics
50,00
0,00
Without With extraction
extraction
Figure 11. Results of the adsorption of the gasoline hydrocarbons in the soils with and without the extraction of the
soil organic matter.
Gasoline ethanol can be adsorbed on the sites of hydroxyls of the octahedron of Al, exposed
by fractures, Scrubs or crystalline lattice imperfections, or by interactions with the Fe oxides
and hydroxides and Al amorphous. This occurs from adsorption of hydrogen bonds, which
can also occur with water strongly adsorbed on the surface of the clay minerals (Fig. 12).
14 Soil Pollution
Figure 12. Coordination of interaction of hydrogen and hydroxyl ethanol exposed in the clay mineral (1:1).
5. Discussion of the results
The discussion of the results is focused on three main aspects. The first considers mineralogical,
chemical and physical characteristics of the material with potential for liners. The second aspect
is assessed the performance of Laterite soil on gasoline hydrocarbon flow subjected to high
hydraulic gradients, causing an acceleration of the process of formation of flow channels for
compressed soil in power of Proctor. The last aspect to be evaluated is the power of gasoline
hydrocarbon adsorption by soil with OM and no OM.
5.1. The delimiters criteria material with potential for liners

Evaluating the criteria prescribed by Rowe et al., (1995) the soil presents considerable levels
of Fe oxides and hydroxides and Al (hematite, goethite and gibbsite) and kaolinite with only
30.2% of quartz. As the mineral vermiculite is low soil activity levels was 0.18, less than the 0.3
suggested by the literature. However this value of activity indicates that the material is not
expandable, being a good quality for liners. The cationic exchange capacity (CEC) also
presented low value (6.4 mmolc/dm3) comparing with value defined in literature. Tropical soils
lateritic in general are highly weathered with low or no mineral content of 2:1, which are typical
of temperate climate. Therefore, the activity and CEC are low. The granulometry performed
65% clay fraction indicated more than 20% of particles less than 2 mm confirming the material
rich in clay fraction indicating low permeability material when compressed. Thus, the
hydraulic conductivity parameter value introduced into water in the order 10-7 cm/sec
subjected to a pressure of 20 kPa (Tab. 1). These results of the hydraulic conductivity charac‐
terize the material with great potential for liner according to predefined values in the literature.
5.2. Lateritic soil performance as Liner

The hydraulic conductivity of gasoline type C brazilian obtained values between 10-8 to 10-7
cm/s to a gradient of 75 with a pressure of 50 kPa, which corresponds to 5 m of the water
column (Fig. 4). Such a result of hydraulic conductivity defines the material as great for barrier
on gasoline hydrocarbon flow according to predefined values in literature (Rowe et al., 1995;
Daniel and Koerner, 1995). With the increase of the hydraulic gradient there was an increase
in hydraulic conductivity until it reaches a level of stabilization in gradient greater than 210
(Fig. 4). Although it does not occur to the destruction of the liner to the 210 to 450 gradients
suggests avoid gradients greater than 100 in the projects of protection of underground fuel
tanks, ensuring in this way a hydraulic conductivity around 10-7 cm/s for liners according to
the literature. The intrinsic permeability or specific considers simply the porous medium, not
considering the characteristics of fluid. The values found for intrinsic permeability compacted
laterite soil is similar to that found in the literature to clay (Freeze and Cherry, 1979).
The compacted soil voids indexes suffered increased 0.58 before tests to 0.77 with water flow
and 0.90 with gasoline flow in hydraulic gradient of 75. The empty volume also increased from
134.3 to 210.0 (Tab. 3). The soils studied presented a high degree of flocculation due to the
aggregates of the oxyhydroxides of Fe and Al. Even when compacted, they contain micro-
aggregates which are not destroyed. When a flow is established through the soil, the micro-
aggregates may interconnect, forming flow channels. The physical behavior provoked by the
flow may be visualized in the micromorphology of the samples in Fig. 8, 9 and 10. However
even with these micros channels formed in the compacted soil hydraulic conductivity limit of
10-7 cm/s (Daniel and Koerner, 1995) is not affected considering a gradient of 100.
5.2.1. Adsorption performance for hydrocarbons of gasoline
The performance of laterite soil to a gasoline hydrocarbon flow subjected to a pressure of 50

kPa with 75 gradient was evaluated for pore volume and the ratio C/Co in the process of
saturation of the compacted clay liner for gasoline, in other words, there was the expulsion of
the water contained in the soil by the process of compression to achieve the optimum water
content of compaction. The reason indicates that values above 1 there is an LNAPL phase
concentration of groups of substances evaluated. The groups were evaluated for aromatic,
olefins, saturated and ethanol.
In Fig. 7, the aromatic compounds appear as constants in the saturation process. Since they are
hydrophobic, their polarity is low, and are more easily transported in the soil. The olefines and
saturates have a greater C/Co ratio in the LNAPL due to their low solubility in water, being
lower than the aromatic compounds, which are more affected by ethanol through co-solvency.
In the 0.13 a 0.36 pore volume range, the ethanol is partitioned to the aqueous phase and, as
the saturation of the pores with gasoline increases, the C/Co ratio for ethanol in the LNAPL
also increases. The partitioning of the ethanol for the aqueous phase is natural and is due its
polarity, which makes it mixable in water. Thus, the ethanol, along with the other hydrophobic
compounds in the gasoline, favors the collapse of the double layer, as well as the increase in
micropores (Rowe et al., 1995)
The results in Fig. 11 show that the soil organic matter, although in low quantities, has an
influence of almost 0.41 %, in the sorption process. The soil studied was collected at a depth
of 4 meters, thus contained evolved organic matter, possibly fulvic acid. The removal of the
16 Soil Pollution
organic material with hydrogen peroxide showed low ethanol sorption. The aromatic com‐
pounds, which are hydrophobic, were not absorbed.
Evaluating the transportation of gasoline compounds by soil with application of 50 kPa of

pressure, indicates low retention and greater mobility, because mostly they are hydrophobic
compounds that do not bind the soil particles. Another aspect of this experiment is that has
not been evaluated by diffusion flux, which occurs at speeds equal to or less than 10-10 cm/s.
The test of sorption to organic matter proved to be important in the retention process of ethanol.
In view of the low adsorption of gasoline compounds by soil suggests considering projects of
liners gradients below 75 and pressures less than 50 kPa ensuring a hydraulic conductivity
greater than 10-8 cm/s and use clayey material rich in organic matter to promote greater
retention of ethanol and avoid or reduce the effect of co-solvency.
6. Conclusion
Since the lateritic soil studied possesses a high aggregation capacity, even when compacted at
normal Proctor energy, micropores remain which, in high hydraulic gradient situations, are
interconnected, forming flow channels. However, even under higher hydraulic gradients in
the gasoline percolation tests, this soil presents good material of liners. This is due to the
stabilizing of the flow channels formed, favoring also the stabilizing hydraulic conductivity.
The measure that gasoline occupies the pores in the process of saturation the concentration of
ethanol increases. This is due to the polarity of the ethanol. The aromatic compounds maintain
a C/Co ration close to 1, as the volume of pores increases, indicating that these are tracers due
to their low dielectric constant and polarity. Due to their low solubility in water, the olephines
and saturates are more present in the LNAPL phase. These hydrocarbons may form emulsions,
favoring transportation through the soil in the aqueous phase.
Regarding the retarding potential of the lateritic soil, evaluated by the sorption parameter, it
is not directly correlated with the mineralogy, because the aromatic compounds are not
absorbed when the organic material is extracted. Actually, this sorption may be correlated with
a certain type of humic substance, which may be interacting with the poly-amorphs of the
oxyhydroxides of Fe and Al in the soil, favoring interaction with the aromatic compounds.
Finally, a low hydraulic gradient context (< 75), hydraulic conductivity < 10-8 and organic
matter, in lateritic soil can improve the performance of liner.
Acknowledgements
The authors are gratefully to the Conselho Nacional de Desenvolvimento Científico e Tecno‐
lógico – CNPq, CAPES and ANP for the fellowships and financial support granted to the
accomplishment of this research.
Author details
Wisley Moreira Farias1, Geraldo Resende Boaventura1, Éder de Souza Martins2,

Fabrício Bueno da Fonseca Cardoso3, José Camapum de Carvalho1 and
Edi Mendes Guimarães1
1 Universidade de Brasília, Brasília-DF, Brazil
2 Embrapa/ Cerrados, Planaltina –DF, Brazil
3 ANA-Agência Nacional de Águas –Brasília-DF, Brazil
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chapter 23
Exposure and Hazard Identification of Sulphonamides in

the Terrestrial Environment
Anna Białk-Bielińska, Joanna Maszkowska,

Alan Puckowski and Piotr Stepnowski
1. Introduction
For ten years now, interest has been increasing in research focused on pharmaceutical residues
in the environment. Special attention has been given to the residues of antimicrobials, since it
has been demonstrated, that due to the formation of the dangerous phenomenon of bacterial
resistance, these substances could pose a real threat not just to ecosystems, but also to human
health. Most antimicrobial agents are used in large quantities for different purposes in
veterinary medicine.
Various antibiotics are commonly used in this field, but we shall concentrate on sulphonamides
(SAs). The physicochemical properties and chemical structures of selected SAs are presented
in Table 1.
Having been used for more than fifty years, SAs are among the oldest groups of veterinary
chemotherapeutic substances. They are inexpensive and offer a broad spectrum of activity for
the prevention and treatment of bacterial infections. After tetracyclines, they are the most
commonly consumed veterinary antibiotics in the European Union [1,10]. However, as animals
do not completely metabolize these compounds, a large fraction of them is being excreted
unchanged in faeces and urine. Therefore, both the unmetabolized drugs and their metabolites
are discharged to the environment, mainly to the soil component, either directly by grazing
animals or indirectly during the application of manure [11]. Once in the soil, they and their
transformation products are distributed among its different compartments and can be
transported to the surface and ground waters. The physicochemical properties of these
compounds, the applied dosage and the nature of the environmental compartment where they
are released and further interact, comply the whole process.
2 Soil Pollution
Substance [CAS]
Chemical structure Selected physicochemical properties
Abbreviation
N N
M = 284.7 g mol-1
Sulphachloropyridazine Cl
HN pKa2 = 1.72
[80-32-0]
pKa3 = 6.39
SCP H 2N S O
logP = 0.71
O
N
M = 250.3 g mol-1
Sulphadiazine
N pKa2 = 1.98
[68-35-9] HN
pKa3 = 6.01
SDZ H 2N S O logP = -0.09
O
OMe M = 310.3 g mol-1

Sulphadimethoxine O
pKa2 = 2.5
[122-11-2] H2N S NH N
N pKa3 = 6.0
SDM O
OMe logP = 1.63
Sulphadimidine N M = 278.3 g mol-1

(sulphamethazine) pKa2 = 2.46
HN N
[57-68-1] pKa3 = 7.45
SDMD (SMZ) H 2N S O logP = 0.27
M = 214.2 g mol-1
Sulphaguanidine O NH
pKa2 = 2.8
[57-67-0] H2N S NH C
NH2 pKa3 = 12.0
SGD O
logP = -1.22
N
M = 264.3 g mol-1
Sulphamerazine
N pKa2 = 2.16
[127-79-7] HN
pKa3 = 6.80
SMR H 2N S O logP = 0.11
O
N O
M = 253.3 g mol-1
Sulphamethoxazole
HN pKa2 = 1.81
[723-46-6]
pKa3 = 5.46
SMX H 2N S O
logP = 0.89
O
Exposure and Hazard Identification of Sulphonamides in the Terrestrial Environment 3
Substance [CAS]
Chemical structure Selected physicochemical properties
Abbreviation
O N M = 280.3 g mol-1
Sulphamonomethoxine
H2N S N N pKa2 = 1.73
[1220-83-3] H
O pKa3 = 6.22
SMM
OMe logP = -0.04
N
M = 249.2 g mol-1
Sulphapyridine
HN pKa2 = 2.37
[144-83-2]
H2N S O pKa3 = 7.48
SPY
O logP = 0.03
S
M = 255.3 g mol-1
Sulphathiazole
N pKa2 = 2.06
[72-14-0] HN
pKa3= 7.07
STZ H 2N S O
logP = -0.04
O
O N
M = 267.3 g mol-1
Sulphisoxazole
HN pKa2 = 2.15
[127-69-5]
pKa3= 5.00
SSX H 2N S O
logP = 1.01
O
Table 1. The structures and physicochemical properties of the sulphonamides discussed in this chapter (according to
[1-9])
SAs are fairly water-soluble, polar compounds, however quite persistent - resistant to biode‐
gradation [10,12-13] and hydrolysis [3]. This goes a long way to explaining why they have been
regularly detected in both aquatic and terrestrial environments in the last ten years [1,10-11].
Although SA concentrations in environmental samples are relatively low (at the ppb or ppt
level), they are continuously being released, so ultimately they may pose an elevated risk. SAs
are designed to specifically target the biosynthetic pathway of folate (an essential molecule
required by all living organisms) by competitively inhibiting the conversion of p-aminobenzoic
acid (pABA); hence, they not only target bacteria but can also elicit hitherto unknown effects
in environmentally relevant, non-target organisms like invertebrates and plants [14-16]. As
they belong to different trophic levels, these taxonomic groups may be exposed to SAs to
various extents.
So far only a small number of studies concerning the presence and effects of SAs in the soil
environment have been carried out. Hence, there are a number of very pertinent questions that
4 Soil Pollution
need to be addressed, for example: (i) What is the fate of these compounds in the terrestrial
environment? (ii) What are the effects of their presence in the soil environment? (iii) Do they
pose a risk to different soil organisms and also to human health?
For these reasons, the aim of this chapter is to review and summarize existing knowledge of
the fate and effects of SA residues in the terrestrial environment.
Conventionally, the environmental fate of antimicrobials in the soil ecosystem is assessed with
respect to their persistence and sorption onto soil. In the case of SAs, as they are very stable,
only photodegradation process could have recognizable influence on their elimination from
the environment [1,10]. However this process in the soil ecosystem is of lesser importance.
Therefore, sorption processes influence the environmental behaviour of SAs to the greatest
extent, so it is these that we shall be discussing in detail.
Although a few review papers have been published summarizing the available ecotoxicity
data of pharmaceuticals, including some SAs [14-15,17-18], they focus on aquatic organisms
rather than soil. In this chapter, therefore, we shall discuss the available data on SA toxicity
towards different soil organisms on various trophic levels like bacteria, invertebrates and
plants. These results will be discussed with respect to the existing requirements for the
environmental risk assessment of veterinary pharmaceuticals (VICH, 2000 [19]; VICH, 2004
[20]; EMEA, 2007 [21]; EMEA, 2008a [22]; EMEA, 2008b [23]). In addition, we shall identify
some areas where future work is warranted as well as the needs for further investigations.
2. Fate of SAs in the terrestrial environment
2.1. Basic concept of sorption modelling studies
Sorption is defined as a phenomenon during which chemicals become associated with solid
phases. Immensely important, this process affects the fate of chemicals in the environ‐
ment [24-25]. Adsorption/desorption experiments are useful for generating essential
information on the mobility of various contaminants and their distribution in the soil, water
and air. Assessing the distribution of a chemical between the soil and aqueous phases is
not a straightforward process, however. It depends on various factors, such as the chemi‐
cal nature of the substance and the characteristics of the soil (e.g. pH, organic matter (OM)
content, clay fraction content). Furthermore, climatic factors such as rainfall, temperature,
sunlight intensity and wind, which can affect the strength of sorption, also have to be taken
into consideration. Thus, the numerous phenomena and mechanisms involved in the
adsorption of a chemical by soil cannot be completely defined by simple laboratory models.
Nevertheless, such investigations can provide valuable information on the environmental
relevance of the adsorption of chemicals [26].
Laboratory sorption experiments can be carried out under static (batch) and dynamic
(column) conditions. Static tests are commonly used when the aim of the study is to calculate
the distribution coefficient (Eq. 1), in equilibrium time, which is specific to every chemi‐
cal. Column tests, on the other hand, enable time-dependent monitoring of contaminant
leaching from soil and waste materials; in addition, the flow-through pattern of such tests
resembles actual environmental conditions. Therefore, the release of a contaminant
depending on local equilibrium time and advection conditions can be evaluated based on
column testing systems [27].
Both column and batch tests can be used to assess the possible leaching/release potential of
contaminated materials on the soil – ground water pathway [28-30]. The release potential of
water-soluble contaminants can be assessed as an expression of the source term, which gives
an indication of their bioavailability. In this case a batch test provides a snapshot of a particular
liquid-to-solid ratio. This type of test focuses on constituent solubility and release over a range
of conditions by varying a single parameter (e.g. final extract pH, liquid-to-solid (LS) ratio) [27].
The common approach for modelling sorption results involves using only the initial linear part
of the isotherm, plotted as cw and cs:
c s = Kd × c w (1)
where: Kd is the partition coefficient; cs is the content of test substance adsorbed on the soil at
adsorption equilibrium (mg kg-1); cw is the mass concentration of test substance in the aqueous
phase at adsorption equilibrium (mg L-1).
The sorption isotherm can be also mathematically described by the Freundlich or Langmuir
model.
The Langmuir equation is written as:
c max × K L × c w
cs = (2)
1 + K L × cw
where: cs is the content of test substance adsorbed on the soil at adsorption equilibrium (mg
kg-1); cw is the mass concentration of test substance in the aqueous phase at adsorption
equilibrium (mg L-1); cmax is the maximum sorption capacity of the sorbent; KL is the Langmuir
constant.
The linear form of this equation is:
1 1 1
= + (3)
c s K L c max c w c max
In the Langmuir model the mass of solute sorbed per unit mass of sorbent (cs) increases linearly
as the solute concentration increases. The model is based on three main assumptions:
• the sorption energy is the same for all sites and is independent of the degree of surface
coverage;
6 Soil Pollution
• sorption occurs only at localized sites, with no interaction between adjoining sorbed
molecules;
• the sorption maximum (cmax) represents a monolayer coverage.

Based on these findings, it is justified that the Langmuir isotherms are observed only very
rarely in case of sorption of when organic compounds are sorbed in such a complex and
heterogeneous sorbent as soils [31].
The Freundlich model takes the following form:
c s = K F × c 1/n
w (4)
where: cs is the content of test substance adsorbed on the soil at adsorption equilibrium (mg
kg-1); cw is the mass concentration of test substance in the aqueous phase at adsorption
equilibrium (mg L-1); KF is the Freundlich adsorption coefficient; n is the regression constant
Based on the value of 1/n, which describes the relative magnitude and diversity of energies
associated with sorption, the mechanism of this process can be defined more accurately [31-36].
If 1/n = 1, adsorption is linear, which indicates the occurrence of homogeneous energy sites on
the sorbent. This type of adsorption is generally typical at very low solute concentrations with
many sites on the sorbent still available for possible interaction with the adsorbate. A value of
1/n > 1 indicates a concave, upward-curving isotherm, which is sometimes referred to as a
solvent-affinity type isotherm (S-type), where the sorption energy increases with increasing
surface concentration. However, phenomena unrelated to the sorption of the analyte to the
sorbate surface, such as strong adsorption of the solvent, strong intermolecular attraction
within the adsorbent layers, penetration of the solute in the adsorbent, and the monofunctional
nature of the adsorbate, can also affect the shape of the isotherm [31]. When 1/n < 1, the isotherm
is of the convex, downward-curving Langmuir-type (L-type), where the marginal sorption
energy decreases with increasing surface concentration. This may occur when the competition
of solvent for sites is minimal or the absorbate is a planar molecule [36].
In order to calculate the Freundlich factors as 1/n or KF, Eq. 4 can be linearized by a logarithmic
transformation:
log c s = 1 / n log c w + log K F (5)
It has been shown that the sorption of pharmaceutical compounds (PCs) to soils is influenced
by solution chemistry and the type of mineral and organic sorbents [24,25,37]. Pharmaceuticals
can exist as either neutral or charged species (e.g. cations, anions, zwitterions) [24], depending
on the pH. Various adsorptive forces may therefore be acting. Whereas neutral molecules
partition to solid matrices via relatively weak van der Waals and electron donor-acceptor
interactions, charged species can interact via stronger electrostatic mechanisms, such as cation
exchange, cation-bridging and complexation. The acid-base equilibrium of sulphonamides
resulting from either the loss or the gain of a proton is similar for all sulphonamides apart from
sulphaguanidine (see Białk-Bielińska et al. [3]). The basic properties are due to the aniline
group present in all SAs and specific to each SA heterocyclic base. SAs can thus be described
by three dissociation constants. Nevertheless, since the presence of the protonated form of the
heterocyclic functional group is extremely unlikely, only two of the possible ionized functional
groups existing in the structure of SA molecules are taken into consideration – during sorption
experiments [38-45]. Hence, Ka2 is the dissociation constant for the equilibrium between the
positively charged, protonated amino group of a SA and its electrically neutral conjugate base,
whereas Ka3 refers to the equilibrium involving the loss of the SA proton to yield its negatively
charged conjugate (Figure 1) [7].
O Ka2 O Ka3 O
+
H3N S N R H2N S N R + H+ H2N S N R
H H
O O O
Figure 1. Dissociation equilibrium of sulphonamides [7]
Assessing the sorption of veterinary pharmaceuticals in soils is extremely important for

estimating the risk of the large-scale usage of veterinary medicines to human health and
environmental matrices, because this affects the fate and transport of chemicals in ground
water.
2.2. Sorption potential of SAs to soils
Although SA sorption is quite a common topic of investigation, authors generally focus on just
a few SAs, so that knowledge of the sorption behaviour of some of them (e.g. sulphaguanidine,
sulphisoxazole) is still limited. So far, only a few review papers have been published describing
the sorption of SAs to soils [16,46-49]. However, they cover a wide range of pharmaceuticals,
so SAs are inadequately reviewed. Furthermore, since 2011 (when the last review on SA
sorption was published), new data have been published, which are included in the present
review. The available information on the sorption of the most commonly investigated com‐
pounds will therefore be discussed in depth.
The most widely investigated SA is sulphamethazine (SMZ). The level to which its undergoing
sorption onto soils was investigated already more than thirty years ago by Langhammer [50].
That author calculated adsorption coefficients for four different soils, differing in pH and OM
content. Based on the values of the distribution coefficients (from 1.0 to 3.1 L kg -1), this drug
can be considered as a very mobile chemical. These results are in accordance with the inves‐
tigations of other researchers, such as Thurman et al. [51] and Tolls et al. [52], who reported
low sorption coefficients for SMZ (0.6 L kg -1 and 3.0 L kg-1 respectively) or Thiele-Bruhn et al.
[53], who gave a Kd value of 2.4 L kg-1 for humus-rich soil. This was also supported by
Lertpaitoonpan et al. [54], who examined this SA in terms of the distribution in soils varying
in OM content (Kd lies between 0.2 and 3.9 L kg-1 depending on the physicochemical parameters
of soils). However, Fan et al. [55] reported a higher sorption potential of the polar metabolite
8 Soil Pollution
of SMZ (N4-acetyl-SMZ) during a miscible-displacement experiment (column test). The Kd

values obtained by these authors range from 7.5 andto 206.2 L kg-1 and are much higher than
previous data for the native compound. However, this may well be due to the polar functional
group present in N4-acetyl-SMZ, which could enhance the association of this compound to the
negatively charged soil surfaces via cation bridging or complexes. The high mobility of SMZ
was also reported by Kurwadkar et al. [41], who observed a 50-90% release of SMZ from a soil
column system. The most recent studies presented by Leal et al. [56] underscore the concern
regarding the possible occurrence of this compound in the environment. These authors
investigated a number of different Brazilian soils, finding a tendency for SMZ to leach from
soil matrices.
Much attention has also been given to calculating the sorption potential of sulphadiazine
(SDZ). Just recently, Doretto et al. [45] reviewed the available literature data through Freund‐
lich sorption coefficient (KF) for SDZ. On this basis they concluded that the potential of SDZ
to interact with soil particles is relatively low and depends on the type of soil, thus on the
physicochemical properties of the sorbent. In another work, these authors demonstrated the
weak interaction of SDZ with binding sites on the soil surface (KF values from 0.4 to 2.6 µg 1-1/
n
(cm3)1-1/ng-1). The column studies of Wehrhan et al. [57] showed that the amount of leached
SDZ depends strongly on the duration of the process. The eluted mass fraction was consider‐
ably higher in long-pulse experiments (83 and 61% respectively) than in short-pulse ones,
during which only 18% was leached. Furthermore, these authors recorded the highest
concentrations at the top of the column, with concentrations steadily decreasing towards the
bottom. In the column with the short pulse application, solute concentrations were relatively
uniformly distributed. Environmental conditions like rainfall can therefore affect the distri‐
bution of contaminants in soil.
It was also observed that SDZ exhibits, for example, a lower tendency to be retained in solid
matrices than SMZ [53,56], with respective Kd values for SDZ and SMZ varying from 2.0 to 2.4
L kg-1 as reported by Thiele-Bruhn et al. [53], and from 5.2 to 10.5 L kg -1, as obtained by Leal
et al. [56].
Although sulphachloropyridazine (SCP) is not as widely studied a sulphonamide as SMZ or
SDZ, this drug has been extensively examined using various tests besides batch or laboratory
column tests. The data available in the literature show a sorption potential in soil similar to
that of other SAs. For example, Boxall et al. [58] reported low sorption coefficients for SCP in
soil and a soil/slurry mixture ranging from 0.9 to 1.8 L kg-1. They also confirmed the high
mobility of SCP in field studies, demonstrating the rapid transport of SCP to surface waters
for concentrations as high as 590 µg L-1. Other studies are also consistent with this statement
[38,44,52,56], giving Kd values from 0.7 to 70.1 L kg-1.
Blackwell et al. [59], who also examined the leaching of SCP under field conditions, detected
this compound in surface run-off samples even at a concentration of 25.9 µg L-1 following
application at a rate of 1.18 kg ha-1. These authors reported the occurrence of SCP in soil water
samples at a concentration of 0.8 µg L-1 at 40 cm depth as long as 20 days after treatment. On
the basis of their results the authors concluded that SCP poses a moderate risk of contaminating
ground or surface water but that its potential to accumulate in soils is low. Further lysimeter-
based studies by Blackwell et al. [60] sporadically detected SCP in leachate at levels from 0.7
to 8.5 µg L-1, depending on the irrigation conditions. SCP was applied in slurry (197 mL per
lysimeter), which corresponds to a SCP application rate of 5.2 mg (1.18 kg ha-1). The authors
concluded that this compound has the potential to reach ground and surface waters. On the
basis of a lysimeter study, field investigations and laboratory column tests, Kay et al. [61,62,63]
pointed out that soil tillage prior to slurry application can significantly reduce losses of SCP
to tile drainage systems, thereby reducing the risk of surface water contamination by SCP
residues in the slurry. The observed losses of SCP in a soil column with a soil surface broken
as a result of tillage fell from 54.6 % of the applied amount to zero [62].
Studies of sulphamethoxazole (SMX) have reported a similar sorption potential to that of SMZ
[40]. With some exceptions, Leal et al. [56] recorded similar values of the distribution coefficient
Kd for both SMX and SMZ in an examination of thirteen soils. The results obtained by Yu et al.
[64] are in agreement with that. These authors calculated a Kd of 18.9 L kg-1 for one of three
investigated soils. Their aim was to assess the suitability, inter alia, of SMX as a wastewater
indicator. However, owing to the formation of non-extractable residues, such an application
of SMX was regarded as limited. On the basis of a few investigations into the sorption of SMX
to activated sludge, we can state that the sorption potential of sulphonamides to this sorbent
is much greater than to soils [65-69]. Hrsing et al. [65] presented Kd values for SMX ranging
from 280 to 370 L kg-1, depending on the type of activated sludge. The results are consistent
with the investigations of Hyland et al. [66], who studied the sorption of 75 pharmaceuticals
onto activated sludge, obtaining a Kd value of 269 L kg-1, or with those of Göbel et al. [67], who
obtained a similar value of Kd for SMX. In contrast, Yang et al. [68] reported a lower sorption
of SMX to activated sludge (Kd = 28.6 L kg-1). However, these differences may have arisen, for
example, from the different methodologies used in the tests. Nevertheless, Yang et al. [68, 69]
concluded that sorption of SAs to activated sludge is highly reversible (the amount retained
after desorption is 0.9% of the original dose of 100 µg L-1). Therefore, the use of sewage sludge
as fertilizer may constitute an additional source of SAs in ground and surface waters. More‐
over, since SAs may be taken up by farmland crops, as demonstrated by Li et al. [70], the use
of sewage sludge as fertilizer poses a serious risk to human health as well.
Knowledge of the sorption potential of other SAs like sulphathiazole (STZ), sulphapyridine
(SPY), sulphamerazine (SMR), sulphadimethoxine (SDM), sulphamonomethoxine (SMM),
sulphaguanidine (SGD) or sulphisoxazole (SSX) is very limited. In the literature there are only
a few reports dealing with the sorption of these pharmaceuticals. Kd for STZ adsorption onto
soil particles ranges from 1.0 to 62.5 L kg-1 depending on soil properties [56]. In the case of
SDM, Sanders et al. [71] pointed out that the linear sorption coefficient for SDM differs
somewhat, ranging from 0.4 to 25.8 L kg-1 as a single solute and from 2.5 to 22.1 L kg-1 as a co-
solute with ormetoprim, another antimicrobial. Moreover, the sorption of SDM was less linear
in combination with ormetoprim. In turn, Maszkowska et al. [72] did not determine the
influence of the co-solute on sulphonamide release. SDM exhibited a similar leaching behav‐
iour from the soil when it was tested alone or in a mixture with SGD. Nevertheless, these
authors also reported the considerable mobility of three SAs (SDM, SGD, SSX) in three different
soils; SDM was released the slowest from the soil column. These results are consistent with
10 Soil Pollution
those published previously by Białk-Bielińska et al. [43], who showed that SDM had a greater
sorption potential than SGD (Kd = 0.3 – 107.5 L kg-1 for SDM, 1.0 – 31.0L kg-1 for SGD). SDM
was also found to have the strongest tendency of all the SAs investigated to interact with
activated sludge [68,69]. In addition, these authors investigated the sorption strength of
sulphamonomethoxine (SMM) on activated sludge, finding a lower affinity of SMM than of
SDM for activated sludge particles. Jin et al. [73] demonstrated the relatively high mobility of
SDM in soil, obtaining a Kd of 18.9 L kg-1. These authors also highlighted the influence of
different co-contaminants on adsorption. They concluded that anionic surfactants and urea
could adversely affect the sorption potential of SDM, whereas cationic surfactants could
improve the retention of SDM on soil particles. Figueroa-Diva et al. [40] found that SMR
exhibited the lowest level of sorption of the four SAs (SDM, SMX, SMZ and SMR) that they
examined. According to Thiele-Bruhn et al. [53], SPY was the most strongly retained SA in the
soil matrix, with Kd higher than that of SMZ, SDZ and SDM.
Summing up, the available data indicate that determining the environmental fate of SAs in
soils is not an easy task, as this depends largely on the physicochemical properties of soils and
the chemical structures of the SAs. Nevertheless, one can infer from these results that these
pharmaceuticals will tend to leach into ground or surface water rather than persist in soils.
These data also show, however, that a certain amount of the SAs entering the soil can be
retained there for quite a long time. Furthermore, their sorption to soils can increase or decrease
depending on a number of different factors, which are discussed below.
2.3. Factors influencing sorption of SAs to soils
2.3.1. Influence of the organic/mineral composition of soil
Soils can be regarded as mixtures of mineral and organic fractions. The differences in their
texture, structure, consistency, colour, chemical, biological and other characteristics arise from
the type of parent material. Soils are therefore diverse matrices in which different sorption
mechanisms can occur. The organic matter (OM) content undoubtedly plays a critical role in
the sorption capacity of soils [74]. Overall, in accordance with the available literature data, it
has been shown that OM positively affects sorption strength of organic compounds. Figueroa-
Diva et al. [40] reported values of Kd for all examined SAs increasing in the same sequence as
the organic carbon content (ƒOC) in the soils they investigated. Białk-Bielińska et al. [43] pointed
out that SAs predominantly interact with soil OM by nonbonding van der Waals interactions
and hydrogen bonding. Furthermore, such weak bonding forces are susceptible to desorption,
resulting in the free release of SAs following their prior surface adsorption, an observation
previously made by Thiele-Bruhn et al. [53]. The authors indicated that the influence of soil
OM on adsorption depends not only on the organic carbon content, but also on its composition.
Sukul et al. [39] demonstrated increased sorption of SDZ in soils in the presence of manure
compared to soil without manure, which greatly emphasizes the role of dissolved OM and
organomineral soil particles in SDZ sorption. On the basis of original research and literature
available data, Hou et al. [75] demonstrated a positive relationship between Kd and the organic
carbon content (ƒOC) for SMX sorption on soils/sediments with ƒOC > 2%. However, for
adsorbents with ƒOC < 2%, a lower ƒOC could result in increased sorption, suggesting competi‐
tion between SMX and organic matter on mineral particles. Hyland et al. [66] confirmed the
positive influence of organic matter on SA sorption. The high values of Kd for SMX sorption
onto activated sludge are fully justified, due in great part to the organic carbon (average ƒOC =
44.1%) in the sorbent. Leal et al. [56] analysed the influence of the OM and clay content on the
sorption of several SAs, concluding that hydrophobic partition was important in SA sorption.
Nevertheless, they also found that non-hydrophobic interactions with organic and/or mineral
surfaces, mainly with Al and Fe oxides and hydroxides (abundant in the investigated soils)
were also important in SA retention in soils. Boxall et al. [58] determined the influence of the
type of mineral fraction in soil (clay or sand) on Kd. Their results showed that clay had a greater
sorption capacity for SCP than sand. The same was reported by Ter Laak et al. [38], whose Kd
value for soil with greater amount of clay was twice as high as that for sandy soil. During field
studies, however, Boxall et al. [58] reported faster leaching of SCP to ground water from a clay
site than from a sandy site, an observation corroborated by Fan et al. [55]. The Kd values for
SMZ were positively related to the OM content in case of sorbents without sand. However,
the latter authors’ Kd value was higher for sand (%OM=0) than for soil containing OM. They
explained this as having resulted from the transport of SAs on mobile colloids (< 2 µm,
dissolved organic matter and clay-sized materials) in accordance with EPA [76], which resulted
in faster elution from a soil column with OM content than from sand.
2.3.2. Influence of pH
In the context of the acid-base equilibrium of SAs, pH can strongly affect sorption. This has
been confirmed in many investigations. The overall trend presented in the literature indicates
decreasing sorption of SAs with increasing pH. This is explained by the amphoteric nature of
SAs, which consequently can occur in cationic, anionic or neutral form. The strongest possible
interactions (ion-exchange mechanism) arise from competition for negatively charged sites on
the soil surface between a cationic analyte and other cations present in the solute. Nevertheless,
the existence of cationic SAs is limited due to the relatively low pKa2 value. Cation exchange is
therefore not regarded as a favourable mechanism for SA sorption to soil matrices [39].
Although decreasing SA sorption is observed at high pH, Sukul et al. [39] achieved relatively
strong adsorption in the case of one soil at a pH where the anionic form of SDZ was dominant,
claiming that this was due to possible partition to the positively charged surfaces of pedogenic
oxides, very abundant in the clay fraction [39,53]. Kim et al. [42] and Białk-Bielińska et al. [43]
also observed a negative correlation between Kd and pH. The former authors considered that
the changes were better evident for soil with a greater OM content. Pinna et al. [77], in turn,
did not observe such a strong dependence on OM content. The addition of cow manure (ƒOC
= 30.58) did not significantly affect antibiotic sorption to one of the investigated soils, but did
increase the extent of sorption to another soil about three times, even though larger amounts
of manure had been added to the first soil than to the second one. These authors concluded
that the greater sorption to the second soil prior to the addition of cow manure was most
probably due to the low pH of the soil suspension rather than to its high organic carbon content.
On the other hand, the high pH of the first soil suspension (7.8) could have been responsible
for the reduced sorption, despite the considerable amount of OM in this amended soil.
12 Soil Pollution
2.3.3. Influence of ionic strength
Another environmentally important factor that can affect SA sorption is ionic strength. But
this has not been examined extensively. Ter Laak et al. [38] carried out sorption studies of SCP,
among other compounds. Generally speaking, they did not observe any significant influence
of ionic strength, except in the case of one soil (clay loam), in which sorption doubled when
the CaCl2 concentration was raised from 0.006 to 0.2 M. The authors concluded that this
increase in sorption was probably due to the neutral form of SCP increasing from 3.3 to 8.3%
because of the decreasing pH. Protons are displaced from the cation-exchange sites by the
addition of Ca2+ cations, which are ultimately responsible for the decrease in pH. Elevated
cation concentrations near negatively charged soil surfaces, resulting in a decrease in the
electrostatic repulsion of negatively charged sorbate molecules and soil particles, is another
explanation considered by those authors. Srinivasan et al. [44] reported the different behaviour
of SMX under conditions of increasing ionic strength. They explained the increasing Kd for
SMX in the case of one soil as being due to cation bridging. Since positively charged calcium
ions are present in the solution, bonding of anionic SMX to calcium is possible. In addition,
the occurrence of a salting out effect, reducing the solubility of SMX in the salt solution so that
it precipitates in the soil, was taken into consideration as a possible reason for the increase in
sorption. The positive influence of ionic strength on sorption can also be attributed to the
replacement of protons from the soil surface as the ionic strength increases, causing a slight
reduction in pH, and shifting acidic SMX towards neutral forms that are more strongly sorbed
than the anionic forms. Two other soils examined by Srinivasan et al. [44] exhibited an opposite
and irregular trend in sorptive affinity of SMX, with elevated ionic strengths resulting in
decreased sorption coefficients of SMX in the case of both soils. A slight decrease in sorption
with increasing ionic strength of solute was also observed by Białk-Bielińska et al. [43] in the
case of SDM and SGD and three soils. Srinivasan et al. [44] concluded that the ionic composition
plays an important role in the sorption of ionizable organic compounds. Nevertheless, they,
too, highlighted the necessity for further research in view of the conflicting results published
in the literature.
2.4. Available data on the presence of SAs in soils
Although many methods have been developed in the past decade for the analysis of SAs in
aqueous matrices, only a few are described in the literature for the determination of these
contaminants in soil matrices. This is because the chemical analysis of pharmaceuticals from
soil matrices is complicated by the need for extraction. Hence, our knowledge about the
quantity of SAs in solid matrices is still limited. Nevertheless, the available literature data
indicate their occurrence in agricultural soils after conventional fertilization. In a two-year
monitoring study Höper et al. [78] determined SMZ at a concentration of 11 µg kg-1. Pawelzick
et al. [79] reported a maximum concentration of 4.5 µg kg-1 for SMZ; these results are in
agreement with Hu et al. [80], who demonstrated the occurrence of SMX (0.03 – 0.9 µg kg-1)
and SCP (0.18 – 2.5 µg kg-1). Karcı et al. [81] found three SAs in agricultural soils in Turkey at
concentrations even two orders of magnitude higher than those reported in previous studies:
STZ (0.05 – 0.4 mg kg-1), SCP (0.05 – 0.1 mg kg-1) and SMX (0.05 – 0.1 mg kg-1). There are some
discrepancies in the available literature data, which may be due to differences in the physico‐
chemical properties of the solid samples examined. They may also stem from the intensity of
fertilization and the initial quantities of SAs applied in animal husbandry. Nevertheless, even
low concentrations of SAs reported in soil samples may contaminate other environmental
compartments as a result of release via desorption.
In general, concentration limits of antibiotics in the environment are not regulated, even
though growing public concern has been taken into account in the prescription of environ‐
mental risk assessments of veterinary pharmaceuticals in the USA and Europe [19-23]. For
these reasons, it is still necessary to develop analytical methods for the quantitation of the most
important SAs in soil samples: this will help to estimate the exposure potential as well as the
concentration of these substances in the terrestrial environment. Nevertheless, for a full risk
assessment of these compounds not only is an exposure assessment necessary but also a hazard
characterization, which addresses the question whether a substance has the potential to cause
harmful effects. This will be discussed below.
3. Effects of sulphonamides in the soil environment
3.1. Introduction to soil ecotoxicology

SAs are commonly present in agricultural soils, though in fairly low concentrations (ppb and
ppt levels), and are continuously being released into the environment via several routes (e.g.
grazing animal faeces, manure spreading, WTP effluents). Such a state of affairs requires an
Environmental Risk Assessment investigation, which should answer the basic question of
whether the presence of SAs in soils poses a hazard. Ecotoxicology is the discipline that
addresses this issue. It encompasses the study of organisms, populations, communities and
ecosystems in terms of exposure to chemical agents, i.e. their transfer from the environment
to organisms and their toxic effects. Simply put, it is the science of assessing the effects of toxic
substances on ecosystems in order to protect these as a whole, rather than particular compart‐
ments, such as populations or organisms. In a practical manner, besides toxic effects, ecotox‐
icology explores the occurrence, distribution, accumulation and dissipation of anthropogenic
toxic substances in ecosystems. The fundamental tools for this kind of research are ecotoxico‐
logical tests [82-83].
3.1.1. Ecotoxicological tests

These tests are a special group of quantitative research methods based on a thorough assess‐
ment of the impact of toxic substances (single or mixtures) on living organisms. Quantification
of the results enables us to estimate the cumulative toxicity and the interactions between the
introduced substances [82]. Obtaining such data enables scientists to extrapolate the results
and define safe concentration levels in the ERA process (which is described in greater detail
in Section 4 of this chapter).
In order to prepare valid ecotoxicity tests numerous factors need to be considered, the main
ones being exposure time (acute or chronic), type of medium used, target species, toxic
14 Soil Pollution
substance concentration range and choice of endpoint (e.g. mortality, growth inhibition,
respiration).
Some 60 years ago scientists realized the need to establish uniform, standard test procedures
in order to increase the repeatability and comparability of data obtained from tests. Researchers
publish their own designs for tests together with results, enabling others to mimic the
conditions for further experiments and allowing a better comparison of the results. Until now
many standardized test procedures have been established by various environmental and
governmental organizations/institutions. The best-known of these are the Organization for
Economic Cooperation and Development (OECD), International Standards Organization
(ISO), American Public Health Association (APHA), Environmental Protection Agency (EPA),
American Society for Testing and Materials (ASTM) and International Seed Testing Associa‐
tion (ISTA), as well as many others from non-English speaking countries, like the the German
Institute for Standardization (Deutsches Institut für Normung – DIN) and Polish Norms
(Polskie Normy – PN). With this in mind it is common practice to perform tests strictly
according to a chosen norm, or to modify just some aspects of a method as and when the
conditions require this [82,84].
3.1.2. Soil organisms in ecotoxicological studies
The guidelines for ecotoxicological tests recommend using the best suited organisms. If the
species stipulated in a guideline is unavailable, a similar one can be chosen, but it is important
to select species that are extensively described in the literature. As species usually differ
between ecosystems, their choice should take account of specific local conditions [82]. It is very
important to realize that no single species is representative of all ecosystems; several single-
species and multispecies tests have to be carried out in order to evaluate the behaviour of a
toxic substance in an ecosystem.
Three main groups of organisms are evaluated in soil ecotoxicology: plants, microorganisms
(microfauna) and animals (pedofauna). In the case of pedofauna, most ecotoxicological studies
of soils are based on invertebrates and focus on worms, collembolans or enchytraeids because
of their rapid reproduction times and easy maintenance. The most often examined endpoints
here are weight change, survival, reproduction and behaviour (e.g. avoidance). Spermato‐
phytes are the most popular plants, in which the measured effects usually relate to physio‐
logical disorders, growth inhibition and seed germination. Microorganisms constitute a very
sensitive indicator of chemical stress as there are many parameters that can be evaluated: the
usual ones are respiration, nitrification and growth. However (as in the case of SAs), micro‐
organisms are also regularly evaluated for the occurrence and magnitude of increasing
resistance towards pharmaceutical compounds [82-84].
3.2. Available data on sulphonamide soil ecotoxicology
An extensive literature review of data on SA soil toxicity has shown that there is a considerable
gap in knowledge concerning the effects of these substances towards soil organisms. The vast
majority of publications are dedicated to the analysis of microorganisms, followed by a small
number of works on plants and just a few on pedofauna. Investigations involving the deter‐
mination of quantified dose-response data (such as EC50 – the median effective concentration)
are rare. However, rather more experiments have been done to detect the ecotoxicity of SAs
(e.g. effects observed at a single concentration). Several investigations into the accumulation
of these drugs in plants and the problem of bacterial resistance have also been done and are
summarized below.
3.2.1. Toxic effects of sulphonamides towards soil organisms
Literature results in the form of a dose-response relationship concerning soil organisms

affected by SAs are relatively scant in comparison to the numbers available for aquatic
organisms. Nonetheless, such material as has been gathered does allow us to establish some
basic trends and consider the potential risk posed by SAs in the soil environment. Table 2 lists
effective concentrations of SAs towards soil organisms determined for different taxonomic
groups. Some of the endpoints deemed less relevant have not been included in Table 2, but
they will nevertheless be mentioned in the following section.
To make the results presented in Section 3.2. more transparent, all cited concentrations have
been recalculated into ppm units. However, it is of paramount importance to bear in mind that
each individual study was designed separately; this implies, for example, differences between
the media (soil or liquid) used. Careful thought is therefore advisable in this respect as one
could grossly over- or underestimate the inferences drawn from the results. On that account,
for a more detailed inquiry, we recommend that the reader refer to the original versions of the
cited papers.
The pedofauna is the most understudied group of soil organisms mentioned in the literature:
there have been just a handful of studies. Very interesting experiments using SMX, SDZ, SPY
and SMZ were conducted on the nematode Caenorhabditis elegans, where several kinds of effects
were evaluated (behavioural – movement, and growth – body length) during 24 – 96 hours.
The second generation, not exposed to SAs, was examined in the same manner. The results
showed that SAs affected growth and behaviour in all the exposed nematodes in a time- and
concentration-dependent way. Also, as one might expect, behavioural effects were more
sensitive than growth in all cases. Interestingly though, transgenerational effects were
observed: the unexposed progeny of the examined nematodes exhibited significant toxic
effects. This was speculated to correspond to the ability of SMX, SDZ, SPY to penetrate the
placenta and the secretion of SDZ, SPY and SMZ in maternal milk [85-86]. A different study
group evaluated the effects of SCP in a multispecies soil system, where one of the examined
endpoints was the mortality of earthworms Eisenia fetida. In this case no effect was observed
for up to 21 days of exposure to SCP at concentrations reaching 100 ppm [87].
Two independent groups performed studies of the potential impact of SMX and SMZ towards
plant growth and soil quality. The plants investigated were rice Oryza sativa L., cucumber
Cucumis sativus L., endive Cichorium endivia [88], lettuce Lactuca sativa, alfalfa Medicago sativa
L. and carrot Daucus carota [89]. There are several differences between the approaches of the
two groups, such as the time of exposure, range of concentrations or the types of tests used.
Despite this, the results are comparable and some conclusions are shared. In all the investiga‐
tions SMX and SMZ were deemed to have the potential to affect soil organisms in environ‐
mentally relevant concentrations. Seed germination was found to be an insensitive endpoint.
One of the groups [88] evaluated seed germination using the root length of seedlings in order
16 Soil Pollution
to obtain better results. In nearly all cases SMX was found to be more effective than SMZ. Rice
and carrot were found to be the most sensitive organisms with respective EC10 values of 0.1
ppm and 0.011 ppm [88-89]. Additionally, one group explored soil respiration and soil
phosphatase activity: in these cases the respective EC10s for SMX were 7 ppm and 1 ppm [88].
The inference to be drawn here is that antibiotic residues in manure and soils may affect soil
microbial and enzyme activities.
A relatively original investigation was performed to assess the impacts of anthropogenic
stressors (i.a. SAs) on symbiotic plant-microbe relationships [90]. The authors studied the
effects of SMX on the arbuscular mycorrhizal fungus Glomus intraradices grown on carrot D.
carota root-organ cultures. The assay endpoints were root length (carrot), hyphae growth and
spore production (fungus). The exposure period lasted up to 28 days and the highest concen‐
tration tested was 1 ppm. SMX was found to be effective at low concentrations towards both
organisms: the respective EC50s for carrot and fungus (hyphae growth) were 0.0454 and 0.0451
ppm. Assessment of the endpoints was as follows: root lengths responded quickly to the
presence of phytotoxic pharmaceuticals in the culture medium; hyphae length was a sensitive
endpoint after 21 days’ exposure; spore production required 28 days’ exposure before
significant differences could be detected [90].
The toxicity of STZ towards soybean (Glycine max (L.) Merr.) was evaluated as a potential
nitrification inhibitor [91]. The effects were measured according to the growth of these plants.
Fresh weight and dry weight of roots and plant tops were measured. The concentration range
for STZ reached 200 ppm. STZ drastically reduced both main root elongation and lateral root
development, the suppression increasing concentration-wise. Effects on soybean plants were
detectable but statistically non-significant at a concentration of 10 ppm. STZ EC50 for dry root
yield was equal to 29.5 ppm. It is worth noting that STZ inhibited root growth more than top
growth.
The effects of sulphamonomethoxine sodium (SMM-Na) and sulphadiazine sodium (SDZ-Na)
were investigated in three plant species: wheat Triticum aestivum L., Chinese cabbage Brassica
campestris L. and tomato Cyphomandra betacea [92]. All of the plants exhibited linear correlations
between the effects (root and shoot elongation) and SA concentrations. Seed germination was
also considered, but was not sensitive to toxicity within the chosen range of SA concentrations.
The tests were conducted over 2-5 days. The data acquired showed that wheat was the plant
most sensitive to the toxicity of SDZ-Na with an IC50 = 28.1 ppm and that cabbage was the most
sensitive to SMM-Na with an IC50 = 27.1 ppm. Worthy of note is the fact that in this study root
and shoot elongation of the three crops exhibited different sensitivities, depending on the
particular drug and plant species [92].
A study was developed specifically for SAs; it attempted to assess different susceptibility
patterns of soil bacteria Pantoea agglomerans and standard antibiotic test bacteria Pseudomonas
aeruginosa, depending on intercellular and environmental pH [93]. The results of the study
revealed the effects at low concentrations (max. 20 ppm) of 8 SAs (SMX, STZ, SDZ, SDM, SMZ,
SCP, SPY and SGD) at different pH values (from 5 to 8). The effects corresponding to the most
sensitive pH values are listed in Table 2. The brief conclusion of this work is that the effects of
SAs on microbial soil populations may depend closely on the ability of the bacteria to regulate
their intercellular pH [93].
Substance Type of test Species Critical effect Time Toxicity [ppm] Ref.
24 h EC10 > 100
nematode 48 h EC10 = 1.02

tailored design body length * [85]
Caenorhabditis elegans 72 h EC10 = 0.0302
96 h EC10 = 0.00131
EC50 = 8
rice Oryza sativa L.
NOEC = 1
cucumber Cucumis root length (seed EC50 > 300

ISTA 1985 4-5 d
sativus L. germination) NOEC = 1
EC50 = 69
endive Cichorium endivia
NOEC = 0.1 [88]
EC50 = 13
OECD 1984 root length (plant NOEC = 1
20 d
(modified) cucumber Cucumis growth)* EC50 > 300
sativus L. NOEC = 100
tailored design soil microbe soil respiration 2d EC50 = 7
EC50 > 10
lettuce Lactuca sativa
EC10 = 1.367
5d
SMX EC50 > 10
ASTM 2003 alfalfa Medicago sativa L. root length* [89]
EC10 > 10
EC50 = 0.06
carrot Daucus carota 7d
EC10 = 0.011
bacteria Pseudomonas
EC50 = 2.98
aeruginosa
DIN 58959-7 growth inhibition 24 h [93]
bacteria Pantoea
EC50 = 0.34
agglomerans
EC50 = 0.0627
14 d
LOEC = 0.01
EC50 = 0.0612
carrot Daucus carota root length 21 d
LOEC = 0.03
EC50 = 0.0454
28 d
tailored design LOEC = 0.01 [90]
14 d EC50 = 0.5029
EC50 = 0.0749
mycorrhizal fungus 21 d
hyphae length* LOEC = 0.3
Glomus intraradices
EC50 = 0.0451
28 d
LOEC = 0.3
soybean Glycine max (L.) growth reduction – fresh

STZ tailored design 61 d ED50 = 42 [91]
Merr. weight
18 Soil Pollution
growth reduction – dry

ED50 = 33.5
yield top
growth reduction – dry

ED50 = 29.5
yield root
EC50 = 5.47
aeruginosa
bacteria Pantoea
EC50 = 0.77
agglomerans
EC50 = 2.85
aeruginosa
SDZ DIN 58959-7 growth inhibition 24 h [12]
bacteria Pantoea
EC50 = 0.5
agglomerans
wheat Triticum aestivum

2d IC50 = 28.1
L.
cabbage Brassica root elongation

SDZ-Na OECD 1984 3d IC50 = 31.3 [92]
campestris L. inhibition *
tomato Cyphomandra
5d IC50 = 92.9
betacea
wheat Triticum aestivum

2d IC50 = 120.7
L.
cabbage Brassica root elongation

SMM-Na OECD 1984 3d IC50 = 27.1 [92]
campestris L. inhibition *
tomato Cyphomandra
5d IC50 = 88.0
betacea
EC50 > 20
aeruginosa
SDM DIN 58959-7 growth inhibition 24 h [93]
bacteria Pantoea
EC50 = 2.05
agglomerans
EC50 = 45
NOEC = 1
cucumber Cucumis root length (seed EC50 > 300

ISTA 1985 4-5 d
sativus L. germination) NOEC = 1
EC50 = 37
endive Cichorium endivia
SDMD (SMZ) NOEC = 0.1 [88]
EC50 = 43
OECD 1984 root length (plant NOEC = 1
20 d
(modified) cucumber Cucumis growth)* EC50 > 300
sativus L. NOEC = 100
tailored design soil microbe soil respiration 2d EC50 = 13

EC50 >10
lettuce Lactuca sativa
EC10 = 0.851
5d
EC50 > 10
ASTM 2003 alfalfa Medicago sativa L. root length* [98]
EC10 = 5.336
EC50 >10
carrot Daucus carota 7d
EC10 = 0.065
EC50 > 20
aeruginosa
bacteria Pantoea
EC50 = 1.14
agglomerans
EC50 = 7.08
aeruginosa
SCP DIN 58959-7 growth inhibition 24 h [93]
bacteria Pantoea
EC50 = 0.48
agglomerans
EC50 > 20
aeruginosa
SPY DIN 58959-7 growth inhibition 24 h [93]
bacteria Pantoea
EC50 = 2.22
agglomerans
EC50 > 20
aeruginosa
SGD DIN 58959-7 growth inhibition 24 h [93]
bacteria Pantoea
EC50 > 20
agglomerans
* this author also examined the results for several other endpoints – see Section 3.2.1.
Table 2. Sulphonamide soil ecotoxicology – literature review
3.2.2. Other relevant soil ecotoxicology data

Several publications by Migliore et al. have shed much light on the toxicity and bioaccumu‐
lation of SDM for different terrestrial plants [94-98]. The species included in the research
belonged to two groups:
• crop plants Panicum miliaceum L., Pisum sativum L., Zea mays L. and Hordeum distichum L.
• weeds Amaranthus retroflexus L., Plantago major L., Rumex acetosella L and Lythrum salicaria
L.
All the plants exhibited bioaccumulation and toxicity during post-germinative development
at concentrations of 300 ppm, though of course to different extents. Lythrum salicaria L., exposed
to lower concentrations, demonstrated a hormetic response. Crop plants accumulated SDM at
dissimilar rates but always higher in roots than in foliage. In order to present the versatility of
these results, those of additional research using other SAs and terrestrial plants are listed in
Table 3.
20 Soil Pollution
Substance Organism Observed effects Ref.
Panicum miliaceum L., Pisum sativum L., Zea mays L.,

Hordeum distichum L., Amaranthus retroflexus L., accumulation, growth inhibition,
SDM [94-98]
Plantago major L., Rumex acetosella L. and Lythrum hormesis
salicaria L.
SDZ
accumulation, toxic effects (root
SDM Salix fragilis L., Zea mays L., Hordeum vulgare L. [101-103]
physiology impairment)
SMZ
SDZ Brassica rapa L. and Lumbricus terrestris (p), activated non-extractable residues – low uptake
[104]
SMX sludge (sm) by organisms
SDZ soil microorganisms respiration inhibition, adaptation [105]
SDZ Eisenia fetida ( )

p
no accumulation (uptake detected) [106]
SDM Rhizobium etli ( )–Phaseolus vulgaris L. symbiosis

sm
growth inhibition of both organisms [107]
SDZ accumulation, soil microbial biomass

SMZ Brassica chinensis L. and soil microorganisms inhibition - higher effects for [70]
SMX combined pollution
12 SAs Arthrobacter globiformis (sm) no effect in t = 4 h [100]
SMX Salmonella typhimurium ( ) sm

mutagenic activity [108]
SDZ Zea mays L. and soil microorganisms molecular-chemical pattern changes [109]
soil respiration and bacterial

SDZ soil microorganisms community structure were influenced [110]
only after the addition of glucose
SDZ soil microorganisms soil community structure shift [111]
SDZ Triticum aestivum L. accumulation (mainly in roots) [112]
Lupinus luteus, Pisum sativum L., Lens esculenta root growth inhibition, necrotic
SMZ Medik., Glycine max (L.) Merr., Vigna angularis, changes, cytochrome c oxidase activity [113]
Medicago sativa L. shifts
SDM
Pisum sativum L., Cucumis sativus L. accumulation (mainly in roots) [114]
SMX
SMX Brassica campestris L. minimal accumulation [115]
Lolium perenne L., Poa pratensis L., Poa trivialis L.,

SMZ accumulation [116]
Nasturtium officinale R. Br.
SDM Solanum tuberosum L., Daucus carota, Zea mays L.,

accumulation [117]
SMR Lycopersicon esculentum Mill.
SDZ Lactuca sativa, Daucus carota not detected in subject plants [118]
All organisms in Table 3 belong to one of three groups: soil microorganisms (sm), pedofauna (p) or terrestrial plants.
Table 3. Simplified list of published SA soil ecotoxicology research

As stated before soil microbiota is sensitive and easy to evaluate; hence, it is often exam‐
ined for several target effects. In some cases, however, the results are closely dependent
on incubation time – OECD 209 guidelines recommend relatively short exposure times –
which can lead to underestimated results. This issue has been mentioned by authors
working with SAs [99-100]. Moreover, microorganisms are often not the primarily evalu‐
ated body in a test design. In fact effects on microbe communities are sought as addition‐
al results, helpful in monitoring the conditions in the test environment. Some research
papers covering the soil ecotoxicity of SAs (not mentioned hitherto) are very briefly sum‐
marized in Table 3.
3.2.3. Development of resistance of soil microorganisms to sulphonamides
The current increasing interest in research into pharmaceutical residues in the environment
has drawn the attention of scientists to the causation of bacterial resistance by antimicrobial
residues. As the case of SA residues in terrestrial compartments is no exception, relevant
research results have been published by several authors.
The effect of SCP together with pig slurry was examined during a three-week exposure, using
Biolog® multiwall plates to determine pollution-induced community tolerance (PICT). Several
physiological processes were monitored as well as community-level physiological profiling
(CLPP) [119]. As a result of the tests it was established that the soil microbial community’s
tolerance increased as soon as 7 days following exposure. Indeed, a SCP concentration of 7
ppm was sufficient to trigger the first effects. An increase in tolerance has been reported for a
procedure comparable to normal agricultural practice [119].
Several investigations have been conducted with SDZ in manure. It was established that
such a combination synergistically increased antibiotic resistance in bacteria. Some of the
explored variations of the tests included the use of a multispecies system (microcosm)
with soil bacteria, the preparation of tests on different kinds of soil and multiple amend‐
ment with pig manure. SA resistance genes were detected using hybridization and the
polymerase chain reaction (PCR). In all cases there was a significant increase in resistance,
though differing in extent depending on the test design. Following amendment, the bacte‐
rial populations carrying the SA resistance genes introduced to the soil declined strongly
in the first weeks; nonetheless, they have the potential to be present for several months
[120-122].
Sulphamethoxazole (SMX) was also examined with respect to its effect on soil bacteria. Two
methods were used to assess PICT: leucine incorporation and Biolog® plates. Community
structure was assessed using phospholipid fatty acid (PLFA) analysis, CLPP and bacterial
growth. No effect was seen at 1 ppm SMX. At higher concentrations (20 – 500 ppm) effects
were significant but relatively small (a ca twofold increase in community tolerance). None‐
theless, the impact of SMX on soil reflected both the direct inhibition of bacterial growth rates
and changes in community structure [123].
22 Soil Pollution
4. Environmental risk assessment of veterinary pharmaceuticals (VPCs) in

soil ecosystems
Guidelines describing how the environmental risks of veterinary products should be assessed
for a range of countries have been published [22]. The approach used in Europe is based on
the recommendations of the International Co-operation on Harmonization of Technical
Requirements for Registration of Veterinary Products (VICH), which has attempted to
harmonize the environmental risk assessment requirements of veterinary products in the USA,
Europe and Japan. The approach is a two-phase process [19-20,22]. According to these
guidelines for the environmental risk assessment (ERA) of VPCs, the ERA process starts with
an initial exposure assessment (Phase I). With some exceptions, a fate and effects analysis
(Phase II) is only required when exposure-based thresholds, the so-called action limits, are
exceeded in different environmental compartments. Thus, risk assessment, described by the
Risk Quotient (RQ), is carried out by calculating the ratio of the predicted (or measured)
environmental concentration (PEC or MEC respectively) and the predicted biological non-
effective concentrations (PNEC) on non-target organisms. The PNEC is ultimately derived
from the toxicity data by applying an assessment factor (AF), usually calculated as the ratio of
EC50 or NOEC to AF. The assessment factor (in the range from 10 to 1000) takes into account
interspecies variation, acute/subchronic to chronic extrapolation and laboratory data to field
impact extrapolation. For example, for acute toxicity tests its value is 1000. Nevertheless, if RQ
is less than one, no further testing is recommended. Calculations of environmental concentra‐
tions rely on information on treatment dosage and intensity along with default values for
standard husbandry practices and are based on a total residue approach reflecting worst-case
assumptions. No fate and effects analysis is required for VPCs if the predicted environmental
concentration in soil (PECsoil) is < 100 µg/kg dry weight of soil. In this case the ERA is brought
to a close. However, if PECsoil is higher than the action limit, then Phase II, divided into two
parts, comes into play: Tier A, in which the possible fate of the pharmaceutical or its metabolites
and its effects on earthworms (mortality) and plants (germination and growth) as well as the
effects of the test compound on the rate of nitrate mineralization in soil are determined; and
Tier B, in which only effect studies are recommended for affected taxonomic levels (when RQ
> 1 or in the case of soil microorganisms an effect > 25 %); no further guidance on Tier B testing
is provided. There are no requirements regarding the species of organisms that should be used
at this stage apart from the statement that the study using terrestrial plants should be repeated
on two additional species from the most sensitive species category in the Tier A study, in
addition to repeating the study on the most sensitive species. However, if after Tier B testing
RQ is still > 1, more studies may be needed in order to further elucidate the effects on terrestrial
ecosystems [19-23].
The main problem associated with this approach is the fact that the no actual sales figures or
measured environmental concentrations are at hand when a risk assessment is conducted.
Therefore, only crude PEC calculations are performed [124]. Moreover, the (eco)toxicity tests
in Phase II are carried out only for single compounds and on a limited range of species. As
these compounds occur in natural media not as single, isolated drugs but usually together with
other compounds of the same family or the same type, accumulated concentrations or
synergistic-antagonistic effects need to be considered. For these reasons, Schmitt et al.
suggested checking whether the current action limits are sufficiently protective for aquatic and
terrestrial organisms by at least performing limit tests to confirm the absence of effects due to
pharmaceuticals with PECs below the action limits [125]. Therefore, it seems to very important
to check whether other pharmaceuticals do not also pose a threat to such organisms. In
addition, it must be highlighted that contaminants in ecosystems can cause adverse effects,
and that the severity of such effects is not dependent on the total substance concentration but
on the bioavailable concentration of the xenobiotics. Therefore, the central concept for an
assessment of soils should be the bioavailability representing the link between exposure and
effect of contaminants. As a consequence a “bioavailability concept” was developed, which
links environmental chemistry and ecotoxicology. According to Frische et al. [126] “bioavail‐
ability describes the complex processes of mass transfer and uptake of contaminants into soil-
living organisms which are determined by substance properties, soil properties, the biology
of organisms and climatic influence. The bioavailable contaminant fraction in soil represents
the relevant exposure concentration for soil organisms”. For these reasons, risk assessments
and subsequent regulatory measures (permission to use chemicals, threshold values and
redemption goals in soils) are particularly subject to considerable uncertainties as long as
nominal and measured total concentrations are the basis of exposure assessment [126]. Owing
to necessary extrapolations, the present non-consideration of bioavailability in practice can
result in both over- and underestimating the risks posed by chemicals to soils, which is why
it should be included in such an ecotoxicological test. To date, only a few authors have
investigated the ecotoxicity potential of pharmaceuticals with respect to their bioavailability
in soil samples (e.g. [127]).
5. Risk assessment of sulphonamide residues in soil ecosystems
The approach to risk evaluation of sulphonamides in the terrestrial environment, based on the
discussed issues, is presented in Figure 2.
Although, as stated in Section 2, available studies have demonstrated that the sorption
potential of SAs is low, this feature, along with their mobility, is strongly influenced by the
physicochemical properties of soils, the ionic strength of soil solutions as well as the physico‐
chemical properties of the drug itself. Hence, even a slight change in these parameters can
greatly increase the immobilization of SAs in soil. Furthermore, it must be emphasized that
SAs are continuously being released into terrestrial ecosystems; therefore, the kind of exposure
of soil organisms may be subjected to will resemble that of traditional pollutants (e.g. pesti‐
cides, detergents), even those of limited persistence. Consequently, SAs (like other pharma‐
ceuticals) may be considered pseudo-persistent, which explains why they are detected in soils,
even at such high concentrations. For all the above reasons, the ERA of SAs is a very complex
and difficult process. The simple approach defined in the available guidelines, while undoubt‐
edly useful, may not be sufficient. In accordance with existing requirements [19-23] we present
a calculation of RQ for three SAs, assuming the worst case scenario; maximum reported
24 Soil Pollution
Figure 2. Risk evaluation of sulphonamides in the terrestrial environment
concentrations (Section 2.4) were used for the MEC value, PNEC was calculated on the basis
of the data presented in Table 2, and AF was set at 1000 to ensure reliability (see Table 4).
MECsoil PNEC=EC50/AF
Substance Organism RQ=MEC/PNEC
[(µg kg-1] (AF=1000) [µg kg-1]
rice Oryza sativa L. 13 7.7

100
soil microbe 7 14.3
SMX
0.9
soil microbe 7 0.13
STZ 400 soybean Glycine max (L.) Merr. 42 9.5

11
soil microbe 13 0.84
SMZ
4.5
soil microbe 13 0.35
Table 4. RQ calculations for three SAs (RQs >1 are presented in bold)
The data presented in Table 4 support our above statement, as different RQs were obtained,
depending on the data used in this evaluation. It seems that a single environmental concen‐
tration, which can differ in time and place, as well as the PEC values, which may also differ in
different countries, can lead to the over- or underestimation of the risk posed by these
compounds. Hence, in our opinion, more realistic (reliable) approaches should be incorporated
that are based on data obtained from longer term monitoring studies in each country.
6. Conclusions
Our knowledge of the presence of SAs in soils is increasing, but information in the peer-
reviewed literature regarding the fate and ecotoxicological effects is still limited.
As sorption to the soil matrix governs the transport, persistence and (bio)availability of these
chemicals in the environment, it can be assumed that low Kd values, together with the physi‐
cochemical properties of these compounds, indicate that they are highly mobile, readily
bioavailable and easily transported from soil surfaces to aquifers, causing surface- and
groundwater contamination. Being readily bioavailable to micro-organisms, plants and
animals, SAs can affect these directly; indeed, they have the potential to affect entire terrestrial
ecosystems. The literature records the effects of many SAs on soil organisms, although these
are mainly microorganisms and plants; as there are few data on pedofauna, it is impossible to
form any clear judgment in this respect. SAs have been detected in soils, and the evidence
points to possible effects on soil organisms at environmentally relevant concentrations.
Furthermore, SAs can be accumulated by several terrestrial plants, such as the willow Salix
fragilis L., which could be employed for the phytoremediation of SA-contaminated soils.
However, some vegetables are also reported to accumulate SAs, which could lead to adverse
effects along the food chain, ultimately affecting human health. Nevertheless, research into
bioaccumulation as well as the phytoremediation of these compounds is still needed.
The most and least sensitive endpoints in plant studies are root length and seed germination
respectively. The effects of SAs on microorganisms have been studied in many ways, e.g. with
single species and multispecies designs, and different endpoints. Most of the available data
show a strongly dose-dependent relationship for the explored endpoints. Moreover, their
toxicity can be strongly influenced by the pH in the environment and organisms. Furthermore
the issue of microorganisms developing antibiotic resistance is related to SAs. Especially when
SA-contaminated manure is used, there is a noticeable increase in resistance genes.
Hardly any information has been found concerning the toxicity of SA mixtures in soils. Since
these compounds are almost always present in the form of mixtures in the environment, this
issue is one to be addressed in the future. Furthermore, there is a lack of data relating to the
long-term exposure of non-target organisms, and especially how continuous exposure for
several generations may affect a whole population.
In conclusion, the presented data on the fate and potential effects of SAs in the terrestrial
environment appear to indicate a possible negative impact on soil ecosystems and imply a
threat to public health. However, further studies are necessary to characterize the risk
completely.
26 Soil Pollution
Abbreviations
Abbreviation Full name Abbreviation Full name
AF Assessment Factor PICT Pollution-induced Community Tolerance
APHA American Public Health Association PN Polish Norms (Polskie Normy)
American Society for Testing and

ASTM PNEC Predicted Non-Effective Concentrations
Materials
Community-level Physiological
CLPP RQ Risk Quotient
Profiling
German Institute for

DIN Standardization (Deutsches Institut SA(s) Sulphonamide(s)
für Normung)
EC50 Effective Concentration SCP Sulphachloropyridazine
ED50 Effective Dose SDM Sulphadimethoxine
European Medicines Evaluation

EMEA SDMD (SMZ) Sulphadimidine (Sulphamethazine)
Agency
EPA Environmental Protection Agency SDZ Sulphadiazine
ERA Environmental Risk Assessment SGD Sulphaguanidine
IC50 Inhibitory Concentration SMM Sulphamonomethoxine
International Organization for

ISO SMR Sulphamerazine
Standardization
International Seed Testing

ISTA SMX Sulphamethoxazole
Association
Lowest Observed Effect

LOEC SPY Sulphapyridine
Concentration
Measured Environmental
MEC SSX Sulphisoxazole
Concentration
No Observable Effect
NOEC STZ Sulphathiazole
Concentrations
Organization for Economic Veterinary International Conference on

OECD VICH
Cooperation and Development Harmonization
OM Organic Matter VPCs Veterinary pharmaceuticals
pABA p-aminobenzoic acid WTP Wastewater Treatment Plant
Predicted Environmental
PEC ƒOC Organic Carbon Content
Concentration
Table 5. List of abbreviations used in the text

Acknowledgements
Financial support was provided by the Polish National Science Centre under grants
DEC-2011/03/B/NZ8/03009 and DEC-2011/03/B/NZ8/03010 and DS 30-8110-D195-13.
Author details
Anna Białk-Bielińska, Joanna Maszkowska, Alan Puckowski and Piotr Stepnowski
Department of Environmental Analysis, Faculty of Chemistry, University of Gdańsk,

Gdańsk, Poland
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chapter 24
Heavy Metal Deposition in Soils and Plants Impacted by

Anthropogenic Modification of Two Sites in the Sudan
Savanna of North Western Nigeria
Sunday Paul Bako,

Augustine Uwanekwu Ezealor and Yahuza Tanimu
1. Introduction
Heavy metals occur naturally in the ecosystem, most of them in trace quantities [1]. Depending
on their concentration, some of them like Zn, Fe, Mn and Cu are essential plant nutrients [2,
3, 4], others like Pb, Hg and Cr do not have any known use to plants.
Deposition of heavy metals in soils from anthropogenic activities have been implicated for an
increase in heavy metal concentration above background and recommended levels [5, 6, 7].
Heavy metals are important components of agro-allied products such as pesticides, herbicides,
fertilizers; manufacturing and other synthetic products such as paints and batteries [8]. Mining
activities, industrial, municipal and domestic wastes have been reported to be important
sources of heavy metal pollution to the environment [9].
Combustion of fuel from petroleum, abrasion of tyres, brake lining, corrosion of the body work
of vehicles and engine wear have been associated with elevated concentrations of Cd, Cu, Mn,
Ni, V and Zn [10, 11, 12, 13].
Excessive concentration of heavy metals in the environment is of great concern because of their
non-biodegradability. Therefore, their persistence in the environment portends health hazard
to plants and animals and consequently trigger ecological imbalance in the ecosystem [14].
Another concern that high concentrations of heavy metals raise is their ability to bioaccumulate
across the food chain, with members that are high up the food chain having concentration of
such metals several times higher than what is obtainable in the environment [15, 16, 17].
2 Soil Pollution
The effect of metals in living organisms could be chronic, due to exposures over a long period
of time as a result of food chain transfer or acute poisoning due to ingestion or dermal contact
[18]. The concentration of heavy metals in the shoot of plants may vary with season as a result
of inherent growth dynamics of the plant; metal concentration and its bio-availability in the
environment. Data on the response of plants to anthropogenic modification of the environ‐
ment, particularly in relation to soil and air pollution by heavy metals in northern Nigeria is
limited and includes earlier assessments [3, 6, 15, 16] and more recently [19], in which an
attempt was made to document the visual symptoms expressed by some ruderal plant species
in relation to air pollution as a step towards developing a reference for field identification of
pollution events [19].
This study was carried out to determine the spatial and seasonal variations of heavy metal
deposition in soils and plants in Nigeria’s Sudan Savanna in order to assess the extent of
pollution and to identify indigenous plant species that may be pollution tolerant and thus have
potential for use in phytorememediation of heavy metal polluted sites.
2.1. Study area
The two sampling sites were located in Katsina State, a state that falls within the Sudan Savanna
eco-region of northern Nigeria (See Fig 1).
The Katsina Steel Rolling Mill (See Fig 2) was established in the mid 1970s with an installed
capacity of 207, 000 mts per annum [20]
Zobe dam was constructed in 1977 to provide Portable water and for irrigation. It has a storage
capacity of 170 Million cubic meters, a surface area of 39.6 km2 and supports 8137 ha of irrigated
land (See Fig 3).
Sampling
Location/Site Longitude Latitude Altitude
Stations
1 7 37 06. 46 E 12 57 24.72 N 557m
Katsina Steel Rolling Mill environs 2 7 37 10. 23 E 12 57 10.23 N 553m
3 7 37 23.06E 12 57 23.07 N 556m
1 7 27 56.40 E 12 21 42. 18 N 510 m
Zobe Dam catchment 2 7 27 4.8 E 12 21 19. 12 N 520 m
3 7 27 52.31 E 12 22 28. 19 N 520 m
Table 1. Geographical Positioning Coordinates of sampling stations at Katsina Steel Rolling Mill environs and Zobe
Dam catchment, Katsina state, north Western Nigeria.
Heavy Metal Deposition in Soils and Plants Impacted by Anthropogenic Modification of Two Sites in the Sudan… 3
Figure 1. Katsina State, Nigeria showing approximate location of the Steel Rolling Mill and Zobe dam.
4 Soil Pollution
Figure 2. Environs of the Katsina Steel Rolling Mill Showing approximate sampling points.
Figure 3. Environs of Zobe Dam showing approximate sampling points
2.2. Sample collection
Soil and plant samples were collected in both dry and wet seasons from three sampling stations
per location in 5m by 5m quadrats. Soil samples were collected at 0-15cm depth using a soil
auger. The shoot of herbaceous plants with relative abundance greater than five per quadrat,
were clipped with a pair of secateurs for heavy metal analysis. Identification of plant samples
were confirmed in the herbarium of the Department of Biological Sciences, Ahmadu Bello
University, Zaria.
6 Soil Pollution
2.3. Sample preparation
Plant samples were washed with tap water and then with distilled water to remove debris and
surface contamination. Samples were then bulked and air dried to remove excess moisture.
Similarly, samples of the soils collected were bulked into composite samples and air dried for
three days (72 hours).
Dried plant and soil samples were ground using a porcelain mortar and pestle and sieved to
attain a uniform particle size. Each sample was put in a small transparent polythene bag and
labeled.
2.4. Metal analysis
Analysis of the elemental content of the samples was done using the Energy dispersive X-ray
fluorescence Spectroscopy (EDXRF) method [21].
The samples were ground manually to powder with an agate mortar and pestle to grain size
of less than 125µm. Pellets of 19mm diameter were prepared from 0.3-0.5kg powder mixed
with three drops of organic liquid binder and pressed at 10 tons of pressure in a hydraulic
press.
Measurements were performed using an annular 25mCi 109Cd as the excitation source, that
emits Ag – K rays (22.1 KeV) in which case all elements with lower characteristic excitation
energies are accessible for detection in the samples. The system consists furthermore of Si (Li)
detector, with a resolution of 170eV for the 5.90KeV line, coupled to a computer controlled
ADC – card.
Quantitative analysis of the sample was done using the Emission Transmission (E-T) method
and that involves the use of pure target material (Mo) to measure the absorption factors in the
sample.
The Mo target served as a source of monochromatic X-rays, which are excited through the
sample by primary radiation and then penetrate the samples on the way to the detector. In this
way, the absorption factor is experimentally determined which the program uses in the
quantification of concentration of the elements. In addition, the contribution to the Mo-K peak
intensity by the Zr-K is subtracted for each sample.
Sensitivity calibration of the system was performed using thick pure metal foils (Ti, Fe, Co, N,
Zn, Nb, Zr, Mo, Sn, Ta and Pb) and stable chemical compounds (K2CO3), CaCO3, Ce2O3), WO3,
ThO2, U3O3). The spectra for the samples were collected for 3000s with the 109Cd source and
the spectra were then evaluated using the AXIL-QXAS program [22]. 109Cd source was used
for the analysis of K, Th, Y, Zr, Nb and Mo.
The accuracy and precision of the measurements was confirmed though an analysis of IAE –
V10 (hay powder) and IAEA – 259 (cabbage) certified reference material, distributed by
International Atomic Energy Agency (IAEA).
2.5. Bioconcentration factor (Enrichment Coefficient) [23]
This estimates the capacity of plants to accumulate metals, and was computed for each species
as:
Mean concentration of metal in the plant

BCF =
Mean concentration of metal in the soil
3. Results
3.1. Metal concentration in soils and plant samples
In the soils around the Zobe dam catchment, the mean concentration of metals generally
followed the order Fe>Mn>Zn>Cr>Cu>Ni>Cd and Fe>Mn>Zn>Ni>Cr>Cu>Cd in dry and wet
seasons respectively. In soils in the environs of the Katsina Steel Rolling Mill (KTSRM), the
mean wet season concentration trend followed the order Fe>Mn>Zn>Cr>Ni>Cu>Cd and
Fe>Mn>Zn>Cu>Cr>Ni>Cd in the dry season (Table 2; Fig 4). Differences in soil concentrations
of all the metals between seasons were not statistically significant (P=0.05)
3.2. Chromium
The soil around the Zobe dam catchment had a mean Cr concentration of 66.00 mg/kg and
70.53 mg/kg, in dry and wet seasons respectively (Table 2; Fig 4). The mean Cr concentration
in the environs of the Katsina Steel Rolling Mill was 46.67 mg/kg in the dry season and 70.53mg/
kg in the wet season. The wet season concentrations were observed to be higher than acceptable
limits for soils in Canada and the Netherlands (Table 3).
Metals
Sampling Locations Seasons and P Values
Cr Cu Ni Cd Fe Mn Zn
DRY 66.00 29.66 26.87 0.15 22700.00 360.58 263.33
Zobe Dam WET 70.53 7.92 184.57 1.05 55516.93 891.66 428.45
P value 0.13ns 0.33ns 0.64ns 0.29ns 0.25ns 0.33ns 0.34ns
DRY 46.67 56.91 46.31 0.33 18266.67 277.22 693.67
KTSRM WET 69.58 12.05 84.50 0.00 58867.00 745.75 385.00
P value 0.23ns 0.60ns 0.12ns 0.32ns 0.17ns 0.94ns
Differences in soil heavy metal content were not significant (ns) between seasons in both locations.
Table 2. Mean Seasonal Heavy Metal Concentration (mg/kg) and T-test P-values of Soils collected from Zobe dam
catchment and the environs of Katsina Steel Rolling Mill (KTSRM)
8 Soil Pollution
Recommending Agency/Country Cr Cu Ni Cd Fe Mn Zn
WHO [24] 100 30 80 NA NA NA 200
Mexico (Agricultural soils) [25] NA NA 1600.00 37.00 NA NA NA
USA [26] 3, 000 4, 300 75.00 NA NA 7500
Japanese MOE [27] 250 NA NA 150 NA NA NA
Agriculture 64 63 50 14 NA NA NA
Canadian Residential/Park 64 63 50 10 NA NA NA
CCME [28] Commercial 87 91 50 22 NA NA NA
Industrial 87 91 50 22 NA NA NA
Austria [29] 100 60 to 100 50 t0 70 1 to 2 NA NA NA
Germany [29] 60 40 50 1 NA NA NA
France [29] 150 100 50 2 NA NA NA
Luxembourg [29] 100-200 50 to 140 30 to 75 1 to3 NA NA NA
Netherlands [29] 30 40 15 0.5 NA NA NA
Sweden [29] 60 40 30 0.4 NA NA NA
United Kingdom [29] 400 135 75 3 NA NA NA
NA — Not available
Table 3. International Recommended Levels of Heavy Metals in Soils (mg/kg)
In the plant samples, the highest concentration of Cr was recorded in Senna siberiana (2.64 mg/
kg) in the dry season and Englerina gralicinus and Terminalia mollis (3.30 mg/kg) in the wet
season (Fig 5). “Normal” concentration of Cr in plants is described as being from 0.1 to 0.5 mg/
kg, while concentrations between 5 to 30 mg/kg as “Excessive” and 75 to 100 mg/kg, “Phyto‐
toxic” (Table 4).
3.3. Copper
Mean concentrations of Cu in the soil samples collected around the Zobe dam catchment
was 29.66mg/kg in the dry season and 7.92 mg/kg in the wet season. Around the environs
of KTSRM, the mean Cu concentration was observed to be 56.91 mg/kg in the dry season
and 12.05 mg/kg in the wet season (Table 2; Fig 4). Only soil samples collected from the en‐
virons of KTSRM in the dry season were observed to have mean Cu concentration above
the acceptable limits of the WHO (30 mg/kg), Germany, the Netherlands and Sweden (40
mg/kg) Table 3).
In plant samples collected, the highest concentration of Cu was observed in members of the
genus Diospyros, D. piscatoria, 10.41 mg/kg and D. mespiliformis 10.40 mg/kg in the dry season.
In the wet season, the concentration of Cu was generally higher in the plants, with the highest
concentration recorded in Combretum mucronatum (34.58 mg/kg) (Fig 5). Concentrations
between 20 to 100 mg/kg in plants have been described as being excessive (Table 4).
250.00
a
200.00
150.00
Cr
100.00
Cu
50.00
0.00 Ni
‐50.00 Dry Wet Dry Wet
Zobe KTSRM
1200.00
1000.00 b
800.00
600.00 Mn
400.00
Zn
200.00
0.00
Dry Wet Dry Wet
Zobe KTSRM
80000.00
60000.00
c
40000.00
20000.00 Fe
0.00
Dry Wet Dry Wet
Zobe KTSRM
2.50
2.00
1.50 d
1.00
0.50 Cd
0.00
‐0.50 Dry Wet Dry Wet
‐1.00 Zobe KTSRM
‐1.50
Fig. 4. Seasonal variation in heavy metal concentration (mg/kg) in soils of Zobe dam
Figure 4. Seasonal variation in heavy metal concentration (mg/kg) in soils of Zobe dam catchment and the environs of
catchment and the environs of Katsina Steel Rolling Mill (KTSRM)(a, Cu, Cr and Ni; b, Mn
Katsina Steel Rolling Mill (KTSRM)(a, Cu, Cr and Ni; b, Mn and Zn; c, Fe; and d, Cd).
and Zn; c, Fe; and d, Cd).
Table 4: Range Values of Heavy Metals in Vegetation (mg/kg)

10 Soil Pollution
NORMAL DEFICIENCY EXCESSIVE PHYTOTOXIC
Cd NA NA NA 5 to 30
Zn 27-150 10 to20 100 to 400 70 to 400
Cr 0.1- 0.5 NA 5 to 30 75-100
Cu 5.1-30 2 to 5 20-100 60-125
Ni 0.1 -5 NA 10-100 100
Table 4. Range Values of Heavy Metals in Vegetation (mg/kg)
3.4. Nickel
In soils around the Zobe dam catchment, the mean Ni concentration was 184.57 mg/kg in the
wet season and 26.87 mg/kg in the dry season. Whereas mean concentrations of Ni of 46.31
mg/kg and 84.50. mg/kg were observed in dry and wet seasons respectively, in soils around
the environs of the KTSRM (Table 2; Fig 4). The mean concentration of Ni in both sites were
observed to be higher than the acceptable limits of WHO (80 mg/kg), USA (75 mg/kg), Canada,
France and Germany (50 mg/kg), Austria (50 to 70 mg/kg), Luxembourg (30-75 mg/kg); and
Netherlands, Sweden and the UK (Table 3).
In plant samples, Senna siberiana had the highest Ni concentrations of 3.69 mg/kg and 13.77
mg/kg in dry and wet seasons respectively in the environs of KSTRM. Diospyros piscatoria was
observed to have the highest concentration of 2.20 mg/kg for Ni in the dry season while Ipomoea
ascarafolia had the highest concentration of 3.63 mg/kg among plants collected in the wet season
around the Zobe dam catchment (Fig 5). Normal concentration of Ni in plants has been
described to be between 0.1 to 5 mg/kg, whereas concentrations between 10 to 100 mg/kg as
excessive (Table 4).
3.5. Cadmium
Soils around the Zobe dam catchment had a mean Cd concentration of 0.15 mg/kg in the dry
season and 0.33 mg/kg in the wet season. In the environs of KTSRM, a mean concentration of
1.05 mg/kg was observed in the dry season. In the wet season Cd concentrations were below
detectable limits in the wet season (Table 2; Fig 4). The above mean concentrations were found
to be within the acceptable limits of the WHO and the Countries listed in Table 3.
Ipomoea ascarafolia had the highest concentration of cadmium in dry (3.90 mg/kg) and wet (1.03
mg/kg) seasons among plants sampled around the Zobe dam catchment. In the environs of
KTSRM, Ziziphus abyssinica was observed to have the highest concentration of 4.5 mg/kg of Cd
in the dry season, while in the wet season Cadaba farinosa was observed to have the highest
concentration of 0.54 mg/kg (Fig 5).
16 a
14
12
10
8
6
4 Cr
2 Cu
0
Ni
Ipomea ascarafolia
Senna sieberiana
Cyperus rotondus
Guiera senegalensis
Senna sieberiana
Diospyros mespiliformis
Leptadenia hastata
Leptadenia hastata
Diospyros piscatoria
Boscia senegalensis
Ziziphus abyssinica
Senna occidentalis
Cd
Zobe 1 Zobe 2 Zobe 3 KSRM1 KTSRM2
40
35
b
30
25
20
15
10 Cr
5 Cu
Ni
0
Cadaba farinosa
Cadaba farinosa
Senna sieberiana
Dicrostachys cinerea
Ipomea ascarafolia
Guiera senegalensis
Crateva religiosa
Guiera senegalensis
Gymnema sylvestris
Combretum micrumtues
Senna occidentalis
Combretum nigricans
Combretum nigricans
Boscia senegalensis
Leptadenia hastata
Gardenia rubescens
Capparis polymopha
Terminalia mollis
Combretum nigricans
Senna sinquena
Capparis polymopha
Coldenia procumbens
Englerina gralicinus
Cd
Stylosanthes arecta
Zobe 1 Zobe 2 Zobe 3 KTSRM 1 KTSRM 2 KTSRM 3
Fig 5: Concentration (mg/kg) of Cr, Cu, Ni and Cd in plants collected from Zobe Dam
catchment and the environs of Katsina Steel Rolling Mill (KSRM) during the Dry (a)
and Wet (b) Seasons
Figure 5. Concentration (mg/kg) of Cr, Cu, Ni and Cd in plants collected from Zobe Dam catchment and the environs
of Katsina Steel Rolling Mill (KTSRM) during the Dry (a) and Wet (b) Seasons
Soil samples collected in the wet season were observed to have a higher concentration of Fe
(891.66 and 745.75 mg/kg in Zobe dam catchment and the environs of KTSRM, respectively)
than the dry season (360.58 and 277.22 mg/kg in Zobe dam catchment and environs of
KTSRM, respectively (Table 2; Fig 4).
Plant species with the highest concentration of Fe in the wet season were Ipomoea
ascarafolia (115.50 mg/kg) and Guiera senegalensis (264.10 mg/kg) for Zobe dam
catchment and environs of KTSRM, respectively. In the dry season, Coldenia procumbens
(184.00 mg/kg) and Guiera senegalensis (176.84 mg/kg) were observed to have the highest
12 Soil Pollution
3.6. Iron
Soils in the two study sites were observed to have a higher concentration of Fe in the dry season
than in the wet season. Soils from Zobe dam catchment and the environs of KTSRM had means
of 22, 700 and 555, 16.93 mg/kg; and 18, 266.67 and 58867 mg/kg in dry and wet seasons,
respectively (Table 2; Fig 4).
Soil samples collected in the wet season were observed to have a higher concentration of Fe
(891.66 and 745.75 mg/kg in Zobe dam catchment and the environs of KTSRM, respectively)
than the dry season (360.58 and 277.22 mg/kg in Zobe dam catchment and environs of KTSRM,
respectively (Table 2; Fig 4).
Plant species with the highest concentration of Fe in the wet season were Ipomoea ascarafolia
(115.50 mg/kg) and Guiera senegalensis (264.10 mg/kg) for Zobe dam catchment and environs
of KTSRM, respectively. In the dry season, Coldenia procumbens (184.00 mg/kg) and Guiera
senegalensis (176.84 mg/kg) were observed to have the highest concentration of Fe Zobe dam
catchment and the environs of KTSRM, respectively (Fig 6).
3.7. Manganese
The mean concentration of Manganese in soils was observed to be higher in the wet season
(891.66 and 745.75mg/kg) than in the dry season (360.66 and 277mg/kg) for Zobe dam catch‐
ment and KTSRM respectively.
In plants, Diospyros piscatoria (36.5 mg/kg) and Boscia senegalensis (102.87 mg/kg) were observed
to have the highest Mn concentration during the dry season in Zobe dam catchment and
environs of KTSRM, respectively. In the wet season, Capparis polymopha (syn. C. tomentosa)
(80.72 mg/kg) and Senna siberiana (190.60 mg/kg) were observed to have the highest concen‐
tration of Mn in Zobe reservoir and KTSRM, respectively (Fig 6).
3.8. Zinc
Zn concentrations in soils showed a similar pattern with Mn and Fe in Zobe dam catchment
during the dry season, being higher in the wet (428.45 mg/kg) than the dry season (263.67 mg/
kg). A reversed trend was observed in the environs of KTSRM, with the concentration being
higher in the dry season (693.67 mg/kg) than the wet season (385.00 mg/kg) (Table 2; Fig 4).
These observed mean concentrations of Zn were higher than the 200 mg/kg acceptable limit
of the WHO.
Leptadenia hastata (38.5 mg/kg), Senna siberiana (54.84 mg/kg) were observed to have the highest
concentration of Zn in the dry season for Zobe dam catchment and the environs of KTSRM,
respectively. In the wet season, the highest concentrations of Zn in plant samples were
presented by Capparis polymopha (syn. C. tomentosa) (158.50 mg/kg) and Senna siberiana (132.60
mg/kg) (Fig 6). Normal concentrations of Zinc in plants fall between 27 to 150 mg/kg. However,
concentrations between 100 to 400 mg/kg may be considered Excessive, while from 70 to 400
mg/kg, as Phytotoxic (Table 4). This is dependent on the plant species in question.
Figure
0
100
150
200
250
300
350
400
450
0
100
150
200
250
300
50
50
Senna occidentalis
catchment
Senna occidentalis
Leptadenia hastata
Zobe 1
Ipomea ascarafolia
6. Concentration
Wet (b) Seasons
Leptadenia hastata
Zobe 1
Gardenia rubescens
and the
Senna sinquena
Ipomea ascarafolia
(mg/kg)
Gymnema sylvestris
Zobe 2
Combretum micrumtues
environs
Leptadenia hastata
Capparis polymopha
of Fe, of
Coldenia procumbens
Zobe 3
Zobe 2
Guiera senegalensis
MnKatsina
Senna sieberiana
Cadaba farinosa
and Zn inSteel
13
Senna sieberiana
Cyperus rotundus
KTSRM 1
Zobe 3
Combretum nigricans
Capparis polymopha
plantsRolling
Guiera senegalensis
Katsina Steel Rolling Mill (KTSRM) and the Dry (a) and Wet (b) Seasons
collectedMill
Crateva religiosa KTSRM1
Stylosanthes arecta
KTSRM 2
Combretum nigricans
Boscia senegalensis
Boscia senegalensis
from(KTSRM)
Cadaba farinosa
Senna sieberiana
Terminalia mollis
KTSRM2
KTSRM 3
Guiera senegalensis
Zobe dam catchment

Ziziphus abyssinica
Combretum nigricans
and the Dry

and the
Fe
Fe
Zn
Zn
Mn
Mn
Fig 6: Concentration (mg/kg) of Fe, Mn and Zn in plants collected from Zobe dam
(a)environs
Heavy Metal Deposition in Soils and Plants Impacted by Anthropogenic Modification of Two Sites in the Sudan…
and of
13
14 Soil Pollution
4. Bioconcentration Factor (BCF) of heavy metals in plants
Chromium: BCF for Cr was generally low across seasons and site. In the two study areas, the
BCF of Cr ranged between 0.00 to 0.03 in the dry season, while in the wet season it varied
between 0.00 and 0.07. Combretum mucronatum was observed to have the highest BF value (Fig.
7and 8).
Copper: BCF for Cu (Fig. 7 and 8) was generally higher in the wet season than in the dry season,
with Combretum mucronatum having the highest value of 6.16. Ipomoea ascarafolia was observed
to have the highest BF in the dry season. Across seasons and sites, other plant species that had
BCF >1.0, were: Gardenia rubescens, Senna singuena, Gymnema sylvestris, Capparis polymopha,
Guiera senegalensis, Englerina gracilinus, Senna siberiana, Combretum nigricans, Dicrostachys
cinerea, Crateva religiosa, Stylosanthes arectalea and Terminalia mollis (Fig. 7 and 8).
Nickel: Highest BCF of Ni was observed in Combretum nigricans (1.61) and Capparis polymo‐
pha (syn. C. tomentosa) (0.61). Most plants show a higher BF during the wet season in compar‐
ison to the dry season (Fig. 7 and 8).
Iron: BCF of Fe was generally low, with the highest value of 0.07 observed in Senna siberiana,
across seasons and sites (Fig. 7 and 8 ).
Manganese: For Manganese, BCF was generally low (<1.0) (Fig. 9) across seasons and sites. In
the wet season, Combretum nigricans was observed to show a high BF of 0.53 and 0.41 of Mn in
two different replicate plots. Guiera senegalensis was observed to accumulate Mn to a BF of 0.38.
Boscia senegalensis was observed to have a BF of 0.62 during the dry season (Fig. 7 and 8).
Zinc: For Zinc, BCF was also generally low (<1.0) (Fig. 9) across seasons and sites.The highest
BCF of Zn were observed in Ipomoea ascarafolia (0.70), Capparis polymopha (syn. C. tomen‐
tosa) (0.59), Guiera senegalensis (0.41) and Combretum nigricans (0.50) (Fig. 7 and 8).
Cadmium: Senna occidentalis and Ipomoea ascarafolia were observed to have considerably high
BCF of 20.69 and 13.45 respectively. Most of the other plants were observed to have recorded
higher BF in the wet season than the dry season. Across sites and seasons, other species that
had BCF >1.0, were Leptadenia hastata, Capparis polymopha, Senna siberiana and Ziziphus abyssinica
(Fig. 9)
Table 5 presents a summary of the most frequently accumulated metal in all species encoun‐
tered at the sampling locations. Eighteen plant species were found to bioaccumulate metals
(BCF >1) in their above ground part to varying degrees. Cu was the most frequently accumu‐
lated, being found to occur in all of the 18 species (100%) across sites and seasons. This was
followed by Cd in 6 species (33.33%) and Ni in only one (05.6%) species.
0.25
a
0.20
0.15
0.10
0.05
Cr
0.00 Cu
Leptadenia hastata
Ipomea ascarafolia
Leptadenia hastata
Boscia senegalensis
Combretum nigricans
Senna sieberiana
Cyperus rotundus
Senna sieberiana
Ziziphus abyssinica
Guiera senegalensis
Senna occidentalis
Ni
Zobe 1 Zobe 3 Zobe 3 KTSRM 1 KTSRM 2 KTSRM

3
0.70
b
0.60
0.50
0.40
0.30
0.20
Fe
0.10 Mn
Zn
0.00
Ipomea ascarafolia
Guiera senegalensis
Boscia senegalensis
Senna occidentalis
Leptadenia hastata
Leptadenia hastata
Cyperus rotundus
Combretum nigricans
Senna sieberiana
Senna sieberiana
Ziziphus abyssinica
Zobe 1 Zobe 3 Zobe 3 KTSRM 1 KTSRM 2 KTSRM 3
Fig 7: Bioconcentration of (a) Cr, Cu and Ni, (b) Fe, Mn and Zn in plants collected from
Figure 7. Bioconcentration of (a) Cr, Cu and Ni, (b) Fe, Mn and Zn in plants collected from Zobe dam catchment and
Zobe dam catchment and the environs of Katsina Steel Rolling Mill during the Dry
the environs of Katsina Steel Rolling Mill during the Dry Season
Season
15
16
ment and
Soil Pollution
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
during
Figure 8.collected
Senna occidentalis Senna occidentalis Senna occidentalis
Leptadenia hastata
the environs
Leptadenia hastata Leptadenia hastata
Zobe 1
Zobe 1
Zobe 1
the Wet
Ipomea ascarafolia Ipomea ascarafolia Ipomea ascarafolia
Bioconcentration
Gardenia rubescens Gardenia rubescens Gardenia rubescens
from Zobe
Senna sinquena Senna sinquena
of Katsina
Senna sinquena
Gymnema sylvestris Gymnema sylvestris Gymnema sylvestris
Zobe 2
of (a) dam
Zobe 2
Zobe 2
Combretum… Combretum… Combretum…
Cu and
Capparis polymopha Capparis polymopha Capparis polymopha
Coldenia procumbens Coldenia procumbens Coldenia procumbens
Zobe 3
Zobe 3
Zobe 3
Guiera senegalensis Guiera senegalensis Guiera senegalensis
catchment
Englerina gralicinus Englerina gralicinus Englerina gralicinus
Ni, (b) Fe and

and
Cadaba farinosa Cadaba farinosa Cadaba farinosa
c
a
16
Senna sieberiana Senna sieberiana Senna sieberiana
Cr, the
KTSRM 1
KTSRM 1
KTSRM 1
Combretum nigricans Combretum nigricans Combretum nigricans
Capparis polymopha Capparis polymopha Capparis polymopha
Dicrostachys cinerea Dicrostachys cinerea Dicrostachys cinerea
SeasonSteel Rolling Mill during the Wet Season

and (c)environs
Creteva religiosa Creteva religiosa Creteva religiosa
Mn and Zn
Stylosanthes… Stylosanthes… Stylosanthes…
KTSRM2
KTSRM2
KTSRM2
Boscia senegalensis Boscia senegalensis Boscia senegalensis
ofinKatsina
Cadaba farinosa Cadaba farinosa Cadaba farinosa
Terminalia mollis Terminalia mollis Terminalia mollis
KTSRM3
KTSRM3
KTSRM3
Guiera senegalensis
plants collected
Guiera senegalensis Guiera senegalensis
Steel Rolling
from Zobe
Zn
Ni
Cr
Cu
Fe
Mn
Fig 8: Bioconcentration of (a) Cu and Ni, (b) Fe and Cr, and (c) Mn and Zn in plants
Mill
dam catch‐
ing Mill
0.00
5.00
10.00
15.00
20.00
25.00
Senna occidentalis
Figduring
Leptadenia hastata
Zobe 1
Ipomea ascarafolia
Gardenia rubescens
Senna sinquena
Gymnema sylvestris
Combretum mucronatum
Capparis polymopha
Zobe 2
Leptadenia hastata
9: Bioconcentration
Senna sieberiana
the Wet and Dry Seasons

Cyperus rotundus
3
Zobe
Coldenia procumbens
Guiera senegalensis
Cadaba farinosa
Senna sieberiana
Combretum nigricans
KTSMM 1
Capparis polymopha
17
Boscia senegalensis
Senna sieberiana
Ziziphus abyssinica
Creteva religiosa
KTSRM 2
Stylosanthes arrectalea
Combretum nigricans
Boscia senegalensis
Cadaba farinosa
Terminalia mollis
Guiera senegalensis
KTSRM 3
Combretum nigricans
environs of Katsina Steel Rolling Mill during the Wet and Dry Seasons
DRY SEASON Cd
WET SEASON Cd
of Cd in plants collected from Zobe dam catchment and the

Heavy Metal Deposition in Soils and Plants Impacted by Anthropogenic Modification of Two Sites in the Sudan…
Figure 9. Bioconcentration of Cd in plants collected from Zobe dam catchment and the environs of Katsina Steel Roll‐
17
18 Soil Pollution
S/No. Family Plant Species Frequency of Occurrence Phtoextractive potential (as function of
(%) BCF)
Zobe Dam KTSRM Cu Ni Cd
Wet Dry Wet Dry Wet Dry Wet Dry Wet Dry
1 Caesalpiniaceae Senna
0 33 0 0 - - - - - +
occidentalis
2 Asclepiadaceae Leptadenia
33 67 0 0 + - - - - +
hastata
3 Convolvulaceae Ipomea
33 33 0 0 + - - - + +
ascarafolia
4 Rubiaceae Gardenia
33 0 0 0 + - - - - -
rubescens
0 33 0 0 + - - - - -
singuena
6 Asclepiadaceae Gymnema
33 0 0 0 + - - - - -
sylvestre
7 Combretaceae Combretum
33 0 0 0 + - - - - -
mucronatum
8 Capparidaceae Capparis
33 0 0 0 + - - - + -
polymopha
0 33 0 0 + - - - - -
sieberiana
10 Combretaceae Guiera
0 0 66 33 + - - - - -
senegalensis
11 Loranthaceae Englerina
0 0 33 0 + - - - - -
gralicinus
12 Combretaceae Combretum
0 0 100 0 + - + - - -
nigricans
13 Mimosaceae Dicrostachys
0 0 33 0 + - - - - -
cinerea
0 0 33 33 + - - - - +
sieberiana
15 Rhamnaceae Ziziphus
0 0 0 33 - - - - - +
abyssinica
16 Capparidaceae Crateva
0 0 33 0 + - - - - -
religiosa
17 Papilionaceae Stylosanthes
0 0 33 0 + - - - - -
arectalea
18 Combretaceae Terminalia
0 0 33 0 + - - - - -
mollis
(+) = BCF > 1; (-) = BCF < 1
Table 5. Seasonal occurrence of plant species with potential for phytoextraction (BCF) of Cu; Ni and Cd in the
catchment of Zobe Dam and the environs of Katsina Steel Rolling Mill.
5. Discussion
Concentrations of heavy metals in soils were generally observed to be higher during the wet
season in both locations although the differences were not statistically significant (P=0.05). This
differs somewhat with the findings of [30], who observed a higher concentration of these metals
in the dry season than the wet season. Seasonal variations in patterns of metal deposition could
be related to the intensity and duration of climatic variables such as precipitation, temperature
etc., that interface with topography, drainage, soil structure/texture etc., to determine the
physicochemical properties of the soil in a particular location. Soil physicochemical properties
have complex, interdependent effects on metal solubility, with the most important of these
including solution composition (inorganic and organic solubles), Eh, and pH; type and density
of charge on soil colloids; and reactive surface area, that interact with factors like metal
concentration and form, particle size distribution, quantity and reactivity of hydrous oxides,
mineralogy, degree of aeration and microbial activity [31]. The aggregate effects of these
complex interactions determine the bioavailabilty of metals to plants.
The above acceptable limits of the observed concentrations of Cr, Cd, Cu, Ni and Zn may be
attributed to some of the human activities (mainly agriculture and industrial) going on around
the sites. This presents health risks to humans and other animals as the metals contaminate
both aquatic and terrestrial ecosystems. Above background values of these metals may have
resulted from metal smelting and electroplating activities, burning of fossil fuels, application
of phosphate fertilizers, disposal of solid wastes, and quarry activities [32, 33, 34, 35]. High
levels of heavy metals in terrestrial or aquatic ecosystems ultimately end up being transmitted
and accumulated in the food chain. Health risks to humans arise when metal polluted water
is used as drinking water or when animals that have consumed vegetative materials in which
metals have accumulated, are used for food. Furthermore, cultivation of crops on metal
polluted soils indicates a possibility of consuming crops in which metals have accumulated.
Although, specific effects of the various metals on human health have been discussed by
several authors, the biotoxic effects of metals to humans have generally been outlined as
ranging from gastrointestinal disorders, diarrhoea, stomatitis, tremor, hemoglobinuria, ataxia,
paralysis, vomiting and convulsion, depression, coughing and wheezing, respiratory inflam‐
mation, dermatitis, leukocytis, low blood pressure, jaundice haemolytic anemia pneumonia
and coma to death (Cd, Pb, As, Hg, Zn, Cu, Cr, Ni and Al). The nature of these effects could
be toxic (acute, chronic or sub-chronic), neurotoxic, carcinogenic, mutagenic or teratogenic.
For example, see [15, 16, 40, 41, 42].
Concentration of Cr in all the plants observed was found to be below the 5 to 30 mg/kg
described as phytotoxic to plants [35, 36]. The excessive to phytotoxic concentrations of Zn in
Capparis polymopha (syn. C. tomentosa) and Guiera senegalensis; Cd in Senna occidentalis; Ni in
Senna siberiana and Cu in Combretum mucronatum, may be a consequence of the high values
observed in the soils or direct deposition from the atmosphere. In addition to soil concentra‐
tions of metals, other factors that determine the uptake, translocation and accumulation of
metals in plants include soil pH, cation exchange capacity, organic matter content, soil texture
and interaction with other metals, as well as translocation factor (rate of movement of metals
20 Soil Pollution
between root and shoot tissues) for the particular metal [43, 44]. Heavy metals in toxic
concentrations within the plant have inhibitory effects on enzymatic activity, stomatal
function, photosynthesis and nutrient uptake, which may be expressed visually as chlorosis,
reduced/stunted growth and yield depression. Plants vary widely in their ability to tolerate
high concentrations of metals in their tissues. This variation is usually natural and dependent
on inherent genetic factors. The genetic disposition confers the ability to employ a range of
avoidance/exclusion or detoxification mechanisms that enable the plants cope with high metal
loads. These may include the binding of metals (e.g. Ni and Cr) with amino acids, peptides
and organic acids to form low molecular weight compounds, formation of phytochelatins, by
binding (e.g. Cu and Pb) with sulphur-rich proteins and cellular adaptations. Other strategies
may involve roles for mychorrhizas, the cell wall, extra-cellular exudates, efflux pumping
mechanisms in the plasma membrane and formation of stress proteins etc [3, 45, 46, 47].
Plants with BCF of metals >1.0, have been described as suitable for phytoextraction [37, 38].
Some of the plants observed in this study with this potential include; Combretum mucronatum,
Ipomoea ascarafolia, Gardenia rubescens, Senna singuena, Gymnema sylvestre, Capparis polymopha
(syn. C. tomentosa), Guiera senegalensis, Englerina gracilinus, Senna siberiana, Combretum nigri‐
cans, Dicrostachys cinerea, Crateva religiosa, Stylosanthes arectalea and Terminalia mollis for Cu;
Senna occidentalis, Ipomoea ascarafolia, Leptadenia hastata, Capparis polymopha (syn. C. tomentosa),
Senna siberiana and Ziziphus abyssinica for Cd; Combretum nigricans for Ni. The ability of these
plants to concentrate high levels of these metals suggests that they may have a good potential
for phytoremediation.
No hyper-accumulator was observed in this study. Hyper-accumulators are plants that can
accumulate at least 0.1% wt of Cu, Cd, Cr, Pb, Ni and Co or 1% wt of Zn and Mn [39].
There are possibilities for genetic modification of plants to enhance their capacity for metal
tolerance [48].
6. Recommendations for further research
There is a great need to establish environmentally safe limits for metals in plant and soils of
the various eco-regions in Nigeria. This need is emphasized by the observed variations in
published background values from one country to another and even within the same country.
These background values are often dependent on the geological history of the area. A com‐
parison of observed field values with national recommended levels for heavy metals, devel‐
oped from the background values will give a more realistic assessment of the pollution status.
Furthermore, the search for alternative green technology that can be employed in remediation
of pollution events must necessarily be a continuous one, due to the relative low cost and
environmental friendliness of this option as compared to others. In this regard, ruderal species
rather than those with agricultural value must be the candidates of choice for avoidance of
obvious conflicts. The species that have indicated potentials for phytoextraction of Cu, Cd and
Ni in this research may therefore be further evaluated.
7. Conclusion
The concentrations of Cr, Cu, Ni and Zn in soils around the Zobe dam catchment and the
environs of Katsina Steel Rolling Mill were found to be above the acceptable limits. This
presents health risks to humans and other animals as the metals contaminate both aquatic and
terrestrial ecosystems.
Although no hyper accumulator plant species was encountered in this study, eighteen (18)
plant species were identified to have high bioconcentration of metals, which indicated
tolerance to excessive or phytotoxic metal concentrations. In addition, they generally produce
high above ground biomass, due to rapid vegetative growth. These plants include: Combre‐
tum mucronatum, Ipomoea ascarafolia, Gardenia rubescens, Senna singuena, Gymnema sylvestre,
Capparis polymopha (syn. C. tomentosa), Guiera senegalensis, Englerina gracilinus, Senna
siberiana, Combretum nigricans, Dicrostachys cinerea, Crateva religiosa, Stylosanthes
arectalea, Terminalia mollis, Senna occidentalis, Leptadenia hastata, and Ziziphus abyssinica.
These present further possibilities for evaluating metal tolerance in relation to their potential
use in phytoremediation programmes in metal polluted sites.
Acknowledgements
We wish to appreciate the University Board of Research of the Ahmadu Bello University, Zaria,
Nigeria for providing the funds for this research. We also thank Mallam Gallah S. Umar who
helped with the field identification of plant species and collection of plant/soil samples.
Author details
Sunday Paul Bako1, Augustine Uwanekwu Ezealor2 and Yahuza Tanimu1
1 Department of Biological Sciences, Ahmadu Bello University, Zaria, Nigeria
2 Department of Forestry and Environmental Management, Michael Okpara University of

Agriculture, Umudike, Nigeria
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chapter 25
Geochemical Speciation and Risk Assessment of Heavy

Metals in Soils and Sediments
Santosh Kumar Sarkar, Paulo J.C. Favas,

Dibyendu Rakshit and K.K. Satpathy
1. Introduction
Heavy metal pollution is a serious and widely environmental problem due to the persistent
and non-biodegradable properties of these contaminants. Sediments serve as the ultimate sink
of heavy metals in the marine environment and they play an important role in the transport
and storage of potentially hazardous metals. They are introduced into the aquatic system as a
result of weathering of soil and rocks, from volcanic eruptions and from a variety of human
activities involving mining, dredging, processing and use of metals and/or substances
containing metal contaminants. Heavy metals entering natural water become part of the water-
sediment system and their distribution processes are controlled by a dynamic set of physico‐
chemical interactions and equilibria. The properties of metals in soils and sediments depend
on the physiochemical form in which they occur [1]. Heavy metals are distributed throughout
soil and sediment components and associated with them in various ways, including adsorp‐
tion, ion exchange, precipitation and complexation and so on [2]. Changes in environmental
conditions, such as temperature, pH, redox potential and organic ligand concentrations, can
cause metals to be released from solid to liquid phase and sometimes cause contamination of
surrounding waters in aquatic systems [3]. They are not permanently fixed by soil or sediment.
Therefore, it cannot provide sufficient information about mobility, bioavailability and toxicity
of metals if their total contents are studied alone.
Natural and anthropogenic activities have the capacity to cause changes in environment
conditions, such as acidification, redox potential, or organic ligand concentrations, which can
remobilize contaminated soils and sediments releasing the elements from soils and sediments
and pore water to the water column resulting contamination of surrounding waters. Daily
tidal currents, wind energies, and storms in coastal and estuarine systems can cause periodical
2 Soil Pollution
remobilization of surface sediments [4]. More turbulent conditions, such as seasonal flooding
or storms, or bioturbation, due to feeding and movement of benthic organisms, can expose
anoxic sediments to oxidant conditions. In addition, activities such as dredging result in major
sediment disturbances, leading to changes in chemical properties of sediment [5].
The remediation of heavy metal pollution is often problematic due to their persistence and
non-degradability in the environment. As a sink and source, soils and sediments consti‐
tute a reservoir of bioavailable heavy metals and play a significant role in the remobiliza‐
tion of contaminants in the aquatic systems under favorable conditions. Such potential of
sediment for being a sink as well as a source of contaminant can make sediment chemis‐
try and toxicity key components of the quality of aquatic system. Much concern has been
focused on the investigation of the total element contents in soils and sediments. Howev‐
er, it cannot provide sufficient information about mobility, bioavailability and toxicity of
elements and thus may not be able to provide information about the exact dimension of
pollution. The data on total contents of metals are quite insufficient to estimate the possible
risk of remobilization of total metals under changing environmental conditions and potential
uptake of liberated metals by biota and thus the determination of different fractions assume
great importance. This has been described as “speciation” [6]. Since each form have different
bioavailability and toxicity, the environmentalists are rightly concerned about the exact
forms of metal present in the aquatic environment.
The concept of speciation dates back to 1954 when Goldberg introduced the concept of
speciation to improve the understanding of the biogeochemical cycling of trace elements in
seawater. Kinetic and thermodynamic information together with the analytical data made
it possible to differentiate between oxidized versus reduced, complexed or chelated versus
free metal ions in solution and dissolved between particulate species. Florence [7] has
defined the term speciation analysis as the determination of the individual physicochemi‐
cal forms of the element, which together make up its total concentration in a sample.
According to Lung [8], speciation analysis involves the use of analytical methods that can
provide information about the physicochemical forms of the elements. Schroeder [9]
distinguishes physical speciation, which involves differentiation of the physical size or the
physical properties of the metal, and chemical speciation, which entails differentiation
among the various chemical forms. The main objective of measuring metal species relates
to their relative toxicities to aquatic biota. The second and long term aim of speciation
studies is to advance an understanding of metal interactions between water and bed
sediments in an aquatic ecosystem. In the last decade researchers have followed different
sequential extraction techniques for the fractionation of metals in sediments of different
river systems. Rauret et al. [10] studied the speciation of copper and lead in the sedi‐
ments of River Tenes (Spain) while Pardo et al. [11] studies the speciation of zinc, cadmi‐
um, lead, copper, nickel and cobalt in the sediments of Pisuerga River, Spain, in order to
establish the extent to which these are polluted and their capacity to remobilization. Jardo
and Nickless [12] investigated the chemical association of zinc, cadmium, lead and copper
in soils and sediments of England and Wales. In most samples, these four metals were
Geochemical Speciation and Risk Assessment of Heavy Metals in Soils and Sediments 3
associated with all the chemical fractions. Tessier et al. [13] studied speciation of cadmi‐
um, cobalt, copper, nickel, lead, zinc, iron and manganese in water and sediments of St.
Fransois River, Quebec, Canada. Elsokkary and Muller [14] studied speciation of chromi‐
um, nickel, lead and cadmium in the sediments of Nile River, Egypt, reporting that a high
proportion of chromium, nickel and lead is bound to organic material and sulphides, while
cadmium is bound to carbonate fraction. Ure [15] and Rauret [16] have reviewed the
chemical extraction procedures used for heavy metal determinations in contaminated soils
and sediments. Owing to the need for validation of extraction schemes, the EC Measure‐
ment and Testing Programme (formerly BCR) has organized a project for improving the
quality of determinations of extractable heavy metals, where development and validation
of extraction procedures has been discussed [17, 18].
The present article aims to summarize the potentials of sequential extraction technique
adopting different analytical protocols for gaining information on the mobility and dynamics
of operationally determined chemical forms of heavy metals in soils and sediments. The BCR
(Community Bureau of Reference, now superseded by the Standards, Measurement and
Testing Programme of the European Community) procedure has been illustrated considering
the case study of Ganges (Hugli) River Estuary and adjacent Indian Sundarban mangrove
wetland (a UNESCO World Heritage Site), northeastern part of the Bay of Bengal. In addition,
the authors also evaluate the modified BCR sequential extraction technique as devised by
various scientists, the risk assessment code (RAC) as well as assessment of toxicity comparing
with sediment quality guidelines. The RAC classification is based on the strength of the bond
between the metals and the different geochemical fractions in sediments or soils and the ability
of metals to be released and enter into the food chain.
2. Sequential extraction: Merits and demerits
The sequential extraction provides more or less detailed information concerning the origin,
mode of occurrence, biological and physicochemical availabilities, mobilization and transport
of heavy metals. The procedure stimulates the mobilization and retention of these species in
the natural environment using changes in environmental condition such as pH, redox potential
and degradation of organic matter [16]. A series of reagents is applied to the sample, increasing
the strength of the extraction at each step, in order to dissolve the trace metal present in
different sediment phases. The extractants are inert electrolytes, weak acids, reducing agent,
oxidizing agents and strong mineral acids [19].
The 3-stage sequential extraction procedure proposed by the European Community Bureau of
Reference (BCR) was developed in an attempt to standardize the various schemes described
in the literature [2, 20, 21], since the use of different procedures, varying in the number of steps,
types of reagents and extraction condition. Hindered comparison of results obtained in the
many studies of heavy metals chemical fractionation in environmental samples [22].
The BCR methods has been widely adapted by various authors, and applied to a range of type
of solid sample including fresh water sediment [23-25], salt water sediment [26-28], sewage
4 Soil Pollution
sludge and particulate matter [29-31]. This scheme enables us to associate the meals with one
of the following four geochemical phases:
I. Acid-soluble phase: This phase is made up of exchangeable metals and others bound to
carbonates that are able to pass easily into the water column, for example, when the pH drops.
It is the fraction with the most labile bond to the soil/sediment and, therefore, the most
dangerous for the environment.
II. Reducible phase: This phase consists of metals bound to iron and manganese oxides that
can be released if the sediment changes from the oxic to the anoxic state, which could be caused,
for example, by the activity of microorganisms present in the soils/sediments.
III. Oxidisable phase: This shows the amount of metal bound to organic matter and sulphides,
which can be released under oxidizing conditions. Such conditions can occur, for example, if
the sediment is resuspended (by dredging, currents, flooding, tides, etc.) and the sediment
participles come into contact with oxygen-rich water.
IV. Residual phase : Lithogenous and inert (Non-bioavailable).
The heavy metals in the soils and sediments are bound to different fractions with different
strengths, the value can, therefore, give a clear indication of soil and sediment reactivity, which
in turn assess the risk connected with the presence of heavy metals in a terrestrial or aquatic
environment. The rationale of the sequential extraction procedure is that each successive
reagent dissolves a different component, which can content heavy metals within their
crystalline structures. Under natural conditions, metals in minerals are unlikely to experience
significant release over the time frames of interest [32, 33].
3. Analytical protocols for sequential extraction
In recent years a great number of papers have been published on various analytical techniques
proposed for the fractionation analysis of trace elements in various environmental samples
(soils, sediments, etc.). An approach that has been found to be preferable is the fractionation
of heavy metal into operationally defined forms under the sequential action of different
extractants [2]. Selective extractants, used in sequential extraction procedures, are aimed at the
simulation of natural conditions whereby metals associated with certain soil (sediment)
components can be released. For example, changes in the ionic composition affecting adsorp‐
tion–desorption reactions or a decrease in pH may lead to the release of metals, retained on a
matrix by weak electrostatic interactions or co-precipitated with carbonates (“exchangeable”
and “acid soluble” forms). Decreasing the redox potential can result in dissolution of oxides,
unstable under reducing conditions, and liberation of scavenged metals (“reducible” forms).
Changes in oxidizing conditions may cause the degradation of organic matter and release of
complexed metals (“oxidizable” forms). Finally, the destruction of primary and secondary
mineral lattice releases heavy metal retained within the crystal structure, e.g., due to isomor‐
phous substitution (“residual” forms) [2]. The nominal “forms” determined by operational
fractionation can help to estimate the amounts of total metals in different reservoirs which
could be mobilized under changes in the chemical properties of the soil [34]. Since the 1970s a
considerable number of extraction procedures have been proposed for determining the forms
of heavy metal [2, 35-39]. Most of these procedures are based on the scheme of Tessier et al. [2].
Although most of the extracting reagents were originally used in the chemical analysis of soils,
the procedures proposed have been tested on a wide variety of contaminated environmental
samples—sediments, road dust, sewage sludge, etc.
Sequential extraction can be useful to have an operational classification of metals in different

geochemical fractions [2] which is the most reliable criteria to quantify the potential effect of
soil/sediment contamination by heavy metals. This can provide information about the
identification of the main binding sites, the strength of element binding to the particulates and
the phase associations of trace elements in soil/sediment. Following this basic scheme, some
modified procedures with different sequences of reagents or operational conditions have been
developed [40-43]. Considering the diversity of procedures and the lack of uniformity in
different protocols, a European Community Bureau of Reference (BCR, now the European
Community Standards Measurement and Testing Program) method was proposed [6] and was
applied by a large group of researchers [31, 44-47]. In this study, we followed the sequential
extraction procedure proposed by the European Union's Standards, Measurements and
Testing program [3].
4. Modified BCR Sequential extraction process
As discussed above it is evident that sequential extraction provides valuable information

regarding identification of main binding site, the strength of the element binding to the
particulates and the phase associations of heavy metals in sediments. However, various
complicated sequential extraction procedures were experimented to provide more detailed
information regarding different metal phase associations [2, 48, 49]. A wide range of techniques
is available whereby various extraction reagents and experimental conditions are used. These
techniques involve a 5-step [2], 4-step (BCR, Bureau Commune de Reference of the European
Commission), 6-step [50] and 7-step [51, 52] extraction, and are thus becoming popular
methods to be used for sequential extraction [53, 54]. Following this basic scheme, some
modified procedures with different sequences of reagents or operational conditions have been
developed [40-43].
Several sophisticated instruments have been used for the determination of heavy metals
contents in marine environments. These include; flame AAS [55, 56], atomic fluorescence
spectrometry [57], anodic stripping voltametry [58, 59], ICP-AES [60] and ICP-MS [61, 62].
Heavy metal mobility and bioavailability depend strongly on their chemical and mineralogical
forms in which they occur [63]. Several speciation studies have been conducted to determine
study different forms of heavy metals rather their total metal content. These studies reveal the
level of bioavailability of metals in harbour sediments and also confirm that sediments are
indicators of heavy metal pollution in marine environment [64-67].
6 Soil Pollution
Since the early 1980s and 1990s sequential extraction methodology has been developed to
determine speciation of metals in sediments [2, 68] due to the fact that the total concentra‐
tion of metals often does not accurately represent their characteristics and toxicity. In order
to overcome the above mentioned obstacles it is helpful to evaluate the individual fractions
of the metals to fully understand their actual and potential environmental effects [2]. To
date, strong acid digestion is used often for the determination of total heavy metals in the
sediments. However, this method can be misleading when assessing environmental effects
due to the potential for an overestimation of exposure risk. Moreover, in order to deter‐
mine the mobility of heavy metals in sediments, various sequential extraction procedures
have been developed [69-71].
Among a range of available techniques using various extraction reagents and experimen‐
tal conditions to investigate the distribution of heavy metals in sediments and soils, the 5-
step Tessier et al. [2] and the 6-step extraction method, Kersten and Fronstier [50] were
mostly widely used. Following these two basic schemes, some modified procedures with
different sequences of reagents or experimental conditions have been developed [40-43].
Considering the diversity of procedures and lack of uniformity in different protocols, a
BCR, Bureau Commun de Recherche (now called the European Community (EC) Stand‐
ards Measurement and Testing Programme) method was proposed [6]. It harmonized
differential extraction schemes for sediment analysis. The method has been validated using
a sediment certified reference material BCR-701 with certified and indicative extractable
concentration of Cd, Cr, Cu, Ni, Pb and Zn [72]. This method was applied and accepted
by a large group of specialists [31, 44, 45, 47, 73, 74] despite some shortcoming in the
sequential extraction steps [75, 76].
Wang et al. [77] used a modified Tessier sequential extraction method to investigate the
distribution and speciation of Cd, Cu, Pb, Fe, and Mn in the shallow sediments of Jinzhou Bay,
Northeast China. This site was heavily contaminated by nonferrous smelting activities. They
found out that the concentrations of Cd, Cu and Pb in sediments was to be 100, 73, 13 and 7
times, respectively, higher than the National guidelines (GB 18668-2002). The sequential
extraction tests revealed that 39%-61% of Cd was found in exchangeable fractions. This shows
that Cd in the sediments posed a high risk to the local environment. Copper and Pb were found
to be at moderate risk levels. According to the relationships between percentage of metal
speciation and total metal concentration, it was concluded that the distributions of Cd, Cu and
Pb in some geochemical fractions were dynamic in the process of pollutants migration and
stability of metals in marine sediments from Jinzhor Bay decrease in the order Pb>Cu>Cd.
Yuan et al. [78] applied BCR-sequential extraction protocol to obtain metal distribution
patterns in marine sediments from the East China Sea. The results showed that both the total
contents and the most dangerous non-residual fractions of Cd and Pb were extremely high.
More than 90% of the total concentration of V, Cr, Mo and Sn existed in the residual fraction
while more than 60% of Fe, Co, Ni, Cu, and Zn were mainly present in the residual fraction.
Manganese, Pb, and Cd were dominantly present in the non-residual fractions in the top
sediments.
Jones and Turki [79] worked on distribution and speciation of heavy metals in surface
sediments from the Tees estuary, North East England. Tessier et al. [2] metal speciation scheme
modified by Ajay and van Loon [80] was used for the study. They observed out that the
sediments were largely organic-rich clayey silts in which metal concentrations exceed back‐
ground levels, and which attain peak values in the upper and middle reaches of the estuary.
Chromium, Pb and Zn were associated with the reducible, residual, and oxidizable fractions.
Cobalt and Ni were not highly enriched while Cu is associated with the oxidizable and residual
fractions. Cadmium is associated with the exchangeable fractions.
Pempkowlak et al. [81] investigated the speciation of heavy metals in sediments and their
bioaccumulation by mussels. They used a 4-step sequential extraction procedure adapted from
Forstner and Watmann [82]. Their investigation which was characterized by varying metal
bioavailability was aimed at revealing differences in the accumulation pattern of heavy metals
in mussel inhabiting that inhabit in sediments. The bioavailabilities of metals were measured
using the contents of metals adsorbed to sediments and associated with Fe and Mn hydroxides.
The biovailable fraction of heavy metals contents in sediments collected from Spitsbergen
represented a small proportion (0.37% adsorbed metals and 0.11%, are associated with metals
hydroxides). It was also revealed that the percentages of metals adsorbed and bound to
hydroxides of the sediments ranged from 1 to 46% and 1 to 13%, respectively.
Wepener and Vermeulen [66] investigated on the concentration and bioavailability of selected
metals in sediments of Richards Bay harbor, South Africa. Sequential extraction of sediments
was carried out according to Tessier et al. [2] method. The following metals were investigated:
Al, Cr, Fe, Mn, and Zn. Their studies revealed that metals concentrations in sediments samples
varied only slightly between seasons, but showed significant spatial variation, which was
significantly correlated to sediment particle size composition. Highest metal concentration was
recorded in sites with substrates dominated by fine mud. Manganese and Zn had more than
50% of this concentration in reducible fraction while more than 70% of the Cr was associated
with the inert fractions and the concentration recorded at some sites were still above action
levels when considering only the bioavailable fractions. They also concluded that the concen‐
tration of Zn recorded was not elevated their results were compared with the historic data.
Coung and Obbard [54] used a modified 3-step sequential extraction procedure to investigate
metal speciation in coastal marine sediments from Singapore as described by the European
Community Bureau of Reference (ECBR). Highest percentages of Cr, Ni, and Pb were found
in residual fractions in both Kranji (78.9%, 54.7% and 55.9% respectively) and Pulang Tokong
(82.8%, 77.3% and 62.2% respectively). This means that these metals were strongly bound to
sediments. In sediments from Kranji, the mobility order of heavy metals studied were
Cd>Ni>Zn>Cu>Pb>Cr while sediments from Pulan Tekong showed the same order for Cd, Ni,
Pb and Cr, but had a reverse order for Cu and Zn (Cu>Zn). The sum of the 4-steps (acid soluble
+ reducible + oxidizable + residual) was in good agreement with the total metal content, which
confirmed the accuracy of the microwave extraction procedure in conjunction with the GFASS
analytical method.
Fedotov et al. [83] applied a modified technique for accelerated fractionation of heavy metals
in contaminated soils and sediments using rotating coiled columns. Rotating coiled columns
8 Soil Pollution
(RCC) is valuable for the continuous-flow sequential extraction and can be successfully applied
to the dynamic leaching of heavy metals from soil and sediments. This is a fluoroplastic or
steel coil wound around a rigid cylindrical drum, which revolves about its axis and, at the
same time, revolves around the central axis of the device called planet centrifuge. The
stationary (liquid, solid, or heterogeneous) phase is retained in the column because of the
centrifugal force field, and the mobile liquid phase is continuously pumped through the
column. A solid sample was retrieved in the rotating column as the stationary phase under
the action of centrifugal forces while different elements (aqueous solution of complexing
reagents, mineral salts and acids) were continuously pumped through. This procedure
developed is time saving and requires only 4-5 hr instead of the several days needed for
individual sequential extraction. Losses of solid sample are minimal. Further studies are
needed to better estimate the reproducibility of the technique.
Nemati et al. [84] used a modified BCR sequential extraction procedure (SEP) in combination
with ICP-MS to obtain the metal distribution patterns in different depths of sediments from
Sungai Buloh, Selangor, Malaysia. The results showed that heavy metal contaminations at
Sungai Buloh River sediments were more severe than at other sampling sites, especially for
Zn, Cu, Ni and Pb. Nevertheless, the element concentrations from top to bottom layers
decreased predominantly.
Mossop et al. [85] compared of original and modified BCR sequential extraction procedures
for the fractionation of Cu, Fe, Pb, Mn and Zn in soils and sediments. The procedures were
applied to five soil and sediment substrates: a sewage sludge-amended soil, two different
industrially contaminated soils, river sediment and intertidal sediment. Extractable Fe and Mn
concentrations were measured to assess the effects of the procedural modifications on
dissolution of the reducible matrix components. Statistical analyses (two-tailed t-tests at 95%
confidence interval) indicated that recovery of Fe in step 2 was not markedly enhanced when
the intermediate protocol was used. However, significantly greater amounts were isolated
with the revised BCR scheme than with the original procedure. Copper behaved similarly to
Fe. Lead recoveries were increased by use of both modified protocols, with the greatest effect
occurring for the revised BCR extraction. In contrast, Mn and Zn extraction did not vary
markedly between procedures. The work indicates that the revised BCR sequential extraction
proves better attack on the Fe-based components of the reducible matrix for a wide range of
soils and sediments.
5. Sequential extraction of metals in sediments of the Hugli River Estuary

and Indian Sundarban wetland: A case study
5.1. Materials and methods
5.1.1. Sample collection and sediment quality analysis

The delta region formed by Hugli (Ganges) River Estuary (HRE) and is famous for its luxuriant
mangrove vegetation, known as Sundarban wetland, acclaimed as UNESCO World Heritage
Site for its capacity of sustaining an excellent biodiversity. The wetland is characterized by a
complex network of tidal creeks, which surrounds hundreds of tidal islands exposed to
different elevations at high and low semi-diurnal tides. This is one of the most sensitive and
vulnerable ecosystems in the world and suffers from environmental degradation due to rapid
human settlement, tourism and port activities, operation of mechanized boats, deforestation,
and increasing agricultural and aquaculture practices. The ongoing degradation is also related
to huge siltation, flooding, storm runoff, atmospheric deposition, and other stresses resulting
in changes in water quality, depletion of fishery resources, choking of river mouth and inlets,
and overall loss of biodiversity. Moreover, the rapid economic development in this deltaic
region has caused highly dense areas of human activity and led to serious contamination
including heavy metals and persistent organic pollutants (POPs).
Nine sampling sites, namely Barrackpur (S1), Dakshineswar (S2), Babughat (S3), Budge budge
(S4), Ulubaria (S5), Diamond Harbor (S6), Frezergunge (S7), Gangasagar (S8), and Haribhanga
(S9) were selected considering the existence of typical sediment dispersal patterns along the
drainage network systems (as shown in Figure 1) and their position was fixed by a global
positioning system (GPS). The stations are representative of the variable environmental and
energy regimes that cover a wide range of substrate behavior, wave–tide climate, and intensity
of bioturbation (animal–sediment interaction), geomorphological–hydrodynamic regimes and
distances from the sea (Bay of Bengal). The sites are exposed to a variable level of heavy metal
contamination mainly from anthropogenic sources as mentioned earlier. Six sampling sites
(S1 to S6) have been chosen along the lower stretch of Hugli River Estuary, while residual three
sites (S7 to S9) were taken into account in the coastal regions of Sundarban wetland. All
sampling sites together with the main stresses to which they are subjected are presented in
Table 1.
During winter months (January–March 2009) surface sediment samples weighting 10 g were
randomly collected in triplicate from the top 3–5 cm of the surface at each sampling site during
low tide using a grab sampler, pooled and thoroughly mixed. Immediately after collection, the
samples were placed in sterilized plastic bags in the ice box and transported to the laboratory.
Samples were oven dried at 50°C, most gently disaggregated, transferred into precleaned inert
polypropylene bags and stored in deep freeze prior to analyses. Each sample was divided into
two aliquots: one unsieved (for the determination of sediment quality parameters) and the
other sieved through 63 μm metallic sieves (for elemental analyses). Organic carbon content
was determined following a rapid titration method [86] and pH with the help of a deluxe pH
meter (model no. 101E) using combination glass electrode manufactured by M.S. Electronics
Pvt. Ltd. (India). Mechanical analyses of sediment were done by sieving in a Ro-Tap Shaker
manufactured by W.S. Tyler Company, Cleveland, Ohio.
5.1.2. Analytical procedure
To determinate the total element concentration, sediment samples were digested in polyte‐
trafluoroethylene vessels with aqua regia (HCl/HNO3, 3:1) and HF neutralized with H3BO3 in
a 650 W microwave oven (CEM MDS 2000) with a program consisting of a 20-min ramp and
a 30-min hold at 100% power in pressure and temperature controlled conditions (150 psi and
10 Soil Pollution
Figure 1. Map showing the location of the monitoring sites (S1 to S9) covering Hugli River Estuary and Sundarban
mangrove wetland along with the location of the major industries.
180°C). The digested samples were filtered, transferred to polyethylene containers and stored
at +4°C until analysis. All reagents were Suprapur® grade (Merck). Reagent blank was
processed with the samples and did not show any significant contamination. Accuracy of the
procedure was checked using two different certified reference materials (CRM): MESS-2 and
PACS-2, which are both marine sediments certified by the National Research Council of
Canada for the element content. The MESS-2 recovery ranged between 91% and 116% for all
the elements (Table 2). Precision, calculated as relative standard deviation (RSD%), resulted
always lower than 5%.
Station number Site Main stresses

S1 Barrackpur Industrial effluents, domestic sewage disposal, boating, bathing.
S2 Dakshineswar Industrial and domestic effluents, boating, bathing, idol immersion site.
S3 Babughat Power plant discharges, domestic sewage, boating, idol immersion site.
S4 Budge budge Domestic and industrial effluents, bathing, boating.
S5 Ulubaria Domestic and industrial effluents
Boating, recreational activities, bathing, fishing, jetties for fishing
S6 Diamond Harbour
trawlers
S7 Frezergunge Tourist activities, ferry services, fishing
Boating, tourist activities, dredging, fishing, agricultural, domestic and
S8 Gangasagar
aquaculture practices
S9 Haribhanga Boating, fishing and ferrying
Table 1. Details of the nine sampling sites and the main stresses to which they are subjected.
Al As Cd Co Cr Cu Fe Mn Ni Pb Zn
Found 86,613± 24.0± 0.230± 15.6± 112± 38.4± 47,385± 372± 54.1± 20.0±
170±12
MESS-2 17,773 2.4 0.010 1.5 12 6.1 3,668 42 3.8 1.4
Found 70,190± 29.2± 1.59± 12.8± 94.9± 307± 46,630± 465± 44.3± 184±
398±16
PACS-2 3,784 1.3 0.80 0.6 4.4 22 1,411 23 2.5 10
Certified 85,698± 20.7± 0.240± 13.8± 106± 39.3± 43,504± 365± 49.3± 21.9±
172±16
MESS-2 2,600 0.8 0.010 1.4 8 2.0 2,266 21 1.8 1.2
Certified 66,125± 26.2± 2.11± 11.5± 90.7± 310± 43,738± 440± 39.5±
183±8 364±23
PACS-2 3,184 1.5 0.15 0.3 4.6 12 585 19 2.3
Recovery
101% 116% 95.8% 113% 106% 97.8% 109% 102% 110% 91.2% 99.0%
MESS-2
Recovery
106% 111% 75.4% 111% 105% 99.1% 107% 106% 112% 100% 109%
PACS-2
Table 2. Results of certified reference materials MESS-2 and PACS-2 as well as the observed values. All the values are
expressed in μg/g of dry weight. MESS-2 and PACS-2 recovery rates are also reported.
In this study, we followed the sequential extraction procedure proposed by the European
Union's Standards, Measurements and Testing program [3]. Selective extraction is based on
the procedure used by Tessier et al. [2] with improvements made according to the BCR, which
examined and finally eliminated irreproducibility sources. It is made up of three steps, which
dissolve the following phases, respectively: exchangeable and bound to carbonate, bound to
Fe and Mn oxides and hydroxides, bound to organic matter and sulphides. Exchangeable and
bound to carbonate phase (phase 1) is extracted with 0.11 M acetic acid, while the fraction
bound to Fe–Mn oxides (phase 2) with 0.5 M hydroxylamine hydrochloride, adjusted to pH 2
with nitric acid (65%). The phase bound to organic and sulphides (phase 3) is extracted with
8.8 M hydrogen peroxide (stabilized at a pH included between 2 and 3), treated at 80°C in a
microwave oven using a program consisting of a 30-min ramp and a 60-min hold at 50% power
12 Soil Pollution
in pressure and temperature controlled conditions (80 psi and 85°C), and 2 M ammonium
acetate adjusted to pH 2 with nitric acid (65%). Each extraction was carried out overnight (16
h) at room temperature. All the reagents employed were Tracepur® grade (Merck Eurolab,
Italy). After each extraction, the samples were separated from the aqueous phase by centri‐
fuging at 4, 000 rpm for 15 min. The sediments were washed with Milli-Q water and centrifuged
again. The wash water was added to supernatants. The element content of the residual phase
was obtained from the difference between the total content and the sum of phases 1, 2 and 3,
according to Ianni et al. [37, 38], Ramirez et al. [39], and Mester et al. [27]. Sequential extraction
reagent blanks showed no detectable contamination. Accuracy of the procedure was checked
with BCR-701 (SM&T). The recovery rates for trace elements in the standard reference material
ranged between 77% and 118% (Table 3). Precision, calculated as RSD%, resulted generally
lower than 5%, except As and Cr in the phase 1 (~20%).
Al As Cd Co Cr Cu Fe Mn Ni Pb Zn
Found
2.57± 6.09± 2.06± 2.41± 47.7± 43.8± 14.5± 3.38±
BCR-701 198±1 180±1 185±4
0.28 0.09 0.08 0.51 1.7 5.8 0.3 0.35
step 1
Found
3,451± 16.5± 3.37± 3.22± 39.2± 7,042± 24.5±
BCR-701 100±2 128±3 111±2 102±1
46 0.3 0.08 0.03 0.4 106 0.4
step 2
Found
1,912± 3.09± 0.28± 1.86± 64.8± 1,147± 31.9± 17.4± 7.15± 58.4±
BCR-701 169±4
74 0.20 0.01 0.17 1.5 56 2.6 1.7 0.12 5.0
step 3
Certified
7.34± 2.26± 49.3± 15.4± 3.18±
BCR-701 n.a. n.a. n.a. n.a. n.a. 205±6
0.35 0.16 1.7 0.9 0.21
step 1
Certified
3.77± 26.6±
BCR-701 n.a. n.a. n.a. 45.7±2 124±3 n.a. n.a. 126±3 114±5
0.28 1.3
step 2
Certified
0.27± 55.2± 15.3± 54.2±
BCR-701 n.a. n.a. n.a. 143±7 n.a. n.a. 9.3±2.0
0.06 4.0 0.9 2.0
step 3
Recovery
n.a. n.a. 83.0% n.a. 107% 96.8% n.a. n.a. 94.4% 106% 90.3%
step 1
Recovery
n.a. n.a. 89.4% n.a. 85.9% 80.6% n.a. n.a. 92.3% 87.9% 89.4%
step 2
Recovery
n.a. n.a. 104% n.a. 118% 117% n.a. n.a. 114% 76.9% 108%
step 3
Table 3. Results of certified reference materials BCR-701 as well as the observed values (expressed in μg/g of dry
weight) together with recovery rates for each step. n.a.= not available.
The elemental concentrations were determined with an inductively coupled plasma atomic
emission spectrometer Vista Pro (Varian), with the internal standard method. Cadmium was
determined by electrothermal atomization atomic absorption spectrometry. A Varian Spectra
A300 spectrometer with Zeeman effect background correction and autosampler Varian Model
96 was used employing the standard addition method for calibration. All the metal analyses
were performed at the Department of Chemistry and Industrial Chemistry of the University
of Genoa (Genoa, Italy).
5.1.3. Statistical analyses
Principal component analysis (PCA) was used to characterize the metal composition in
sediments, and cluster analysis was used for grouping the sampling stations. Principal
component analysis (PCA) is a multivariate statistical technique used for data reduction and
for deciphering patterns within large sets of data. With PCA, a large data matrix is reduced to
two smaller ones that consist of principal component (PC) scores and loadings. PC loadings
are eigenvectors of the correlation or covariance matrix depending on which is used for the
analysis. The PC scores contain information on all of the variables combined into a single
number, with the loadings indicating the relative contribution of each variable to that score
[87]. Hierarchical cluster analysis (HCA) characterizes similarities among samples by exam‐
ining interpoint distances representing all possible sample pairs in high-dimensional space.
The sample similarities are represented on two dimensional diagrams call dendrograms [88].
All statistical analyses were performed using the computer software STATISTICA (StatSoft,
Inc. 2001).
5.2. Results and discussion
5.2.1. Sediment geochemistry
Table 4 shows values of pH; organic carbon (%); and percentage of sand, silt, and clay in
sediments of the nine sampling sites. Organic carbon values, ranging from 0.22% (in station
S8) to 1.02% (in station S2), are low in comparison with values found in sediments from other
Indian coastal areas, such as Gulf of Mannar [89], Cochin [90], and Muthupet mangroves [91].
The low organic carbon values might be related with the poor absorbability of organics on
negatively charged quartz grains, which predominate in sediments in this estuarine environ‐
ment [92]. In addition, the constant flushing activity by tides along with the impact of waves
can support the low percentage of organic carbon in the sediments. The sediments of the
studied stations are characterized by slightly basic pH (7.50–8.36) with maximum values
recorded in the stations closest to the sea (stations S6, S8, and S9) and minimum in station S7.
These were different from the low pH values in most of the mangrove swamps in Hong Kong
[93], where sediments were not frequently flooded by the tide and become acidic in reducible
conditions. With respect to texture, the sediment samples show a variable admixture of sand,
silt, and clay. Clay fractions dominate in low-energy areas of suspensional deposits. On the
contrary, silt, and sand dominates where the energy level is high. Sediments from station S7
14 Soil Pollution
Latitude and Organic

Stations Salinity pH Sand (%) Silt (%) Clay (%)
Longitude carbon (%)
22º43′ 16″ N
S1 0 7.86 0.35 4 87.1 8.9
88º21′ 20″ E
22º39′ 17″ N
S2 0 7.80 1.02 1 76.5 22.5
88º12′ 25″ E
22º33′ 53″ N
S3 0 7.90 0.52 2.24 41.97 55.79
88º20′ 19″ E
22º30′ 10″ N
S4 0–2.5 7.60 0.74 18.25 47.42 34.33
88º11′ 48″ E
22º28′ 06″ N
S5 0–1 7.90 0.91 16.7 69.6 13.7
88º06′ 54″ E
22º11′ 14″ N
S6 0–5.6 8.36 0.56 3.15 41.13 55.71
88º11′ 15″ E
21º34′ 44″ N
S7 30–34.3 7.50 0.36 98.02 0.18 0
88º15′ 03″ E
21º38′ 15″ N
S8 32–35 8.14 0.22 32.85 58.45 8.7
88º03′ 53″ E
21º34′ 20″ N
S9 35 8.10 0.46 39.3 44.25 16.45
88º01′ 25″ E
Table 4. Geographical position, physicochemical and textural properties of sediment samples of 9 sampling sites.
contain higher percentage of sand (98%) compared to the others, while sediments from S1, S2,
S5, and S8 contains higher percentage of silt (more than 50%) compared to the others. A variable
mixture of sand, silt, and clay is present in the other stations and reflect a variable amount of
erosion and deposition.
5.2.2. Total element concentrations
Total element concentrations in the investigated stations varied in a narrow range of values
(Table 5) and were comparable with data obtained for other Indian coastal areas [94, 95]. Datta
and Subramanian [96] found very similar trace element concentrations throughout the Bengal
Basin, where anthropogenic perturbation is low and river channel may receive a several
centimeter-thick sediment layer in a single event during peak flow, preventing to bear the
signature of an accumulation of trace elements. The highest concentrations for As, Cu, Fe, Mn,
and Ni were measured at station S9 while for Cd and Pb at station S3, close to Calcutta city
(about 4.5 million residents, but about 14.2 million including suburbs). An anthropogenic input
from vehicular traffic and in-dustrial activities may cause high Cd and Pb con-centrations
measured in samples collected in the Calcutta urban area. The lowest element concentrations
were found at station S5. Very low (close to the detection limit) Cd concentration was found
in the coastal stations (S7, S8, and S9).
Stations Al As Cd Co Cr Cu Fe Mn Ni Pb Zn
S1 70,289 8.81 0.165 13.0 67.6 27.8 37,737 591 31.9 20.4 86.6
S2 70,879 8.44 0.452 14.0 74.8 36.8 39,405 625 34.2 22.3 90.7
S3 72,134 8.65 1.79 14.5 73.5 32.3 40,070 712 35.0 33.2 83.1
S4 72,613 8.49 0.492 14.9 76.8 27.9 40,303 726 35.0 19.6 80.4
S5 62,044 6.41 0.220 12.1 58.2 21.1 33,428 597 27.5 17.0 64.1
S6 64,325 6.79 0.106 12.0 64.8 32.0 34,273 613 31.3 17.9 69.6
S7 77,529 7.77 0.044 14.0 75.1 28.4 40,084 389 38.1 20.5 74.4
S8 68,146 8.08 0.027 12.7 62.5 22.2 36,786 511 34.3 19.7 61.4
S9 72,666 9.40 0.044 14.1 74.2 36.6 40,838 785 40.1 22.9 74.9
Table 5. Total element concentrations (μg/g) in sediments of 9 sampling sites (instrumental precision, calculated as
RSD%, resulted lower than 5% for each element in all samples).
The geoaccumulation index (Igeo) of Muller [97] has been calculated for the analyzed elements,
by comparing current concentrations with pre-industrial levels, in order to estimate the metal
contamination in sediments. The equation used for the calculation of Igeo is: log2 (Cn/1.5 Bn),
where Cn is the measured content of element “n” and Bn the element’s content in “average
shale” [98]. Factor 1.5 is used because of possible variations in background values for a given
element in the environment, as well as very small anthropogenic influences [99]. As shown in
Figure 2, all sediments fall in class 0 for Al, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn, therefore the
area is not contaminated for these elements. Unlike the Hugli river, in other rivers of the Bengal
Basin, such as Meghna and Brahmaputra, Cr exhibits higher Igeo values respect to the other
elements [96]. For Cd, two stations fall in class 1 and three in class 2 exhibiting a moderate
contamination for this element. In all stations, As falls in class 2 (moderate pollution). In this
area, As contamination was already observed in previous studies and it is probably due to
groundwater contamination [100]. This contamination can have natural origin, such as coal
seams in Rajmahal basin and arsenic mineral in mineral rocks in the upper reaches of the
Ganges river system. The highly reducing nature of groundwater would reduce As, causing
the possible desorption of As [101].
5.2.3. Speciation patterns
The potential environmental risk of trace elements in sediments is associated with both
their total content and their speciation. The chemical partitioning of the considered elements
(Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) from each extraction step has been
described. Aluminum, Cr, and Fe are present mainly in the residual phase, representing
95.8–96.8%, 88.9–91%, and 83.0–94.7% of the total concentration, respectively, which implies
that these elements are strongly linked to the inert fraction of the sediments. This result
was in good agreement with data reported by several studies carried out worldwide in
marine coastal areas [45, 46, 78, 102]. The high percentage of Fe in the residual phase
16 Soil Pollution
indicates that most of the Fe exists as crystalline Fe peroxides (goethite, limonite, magnet‐
ite, etc.). The remaining Fe is associated with the reducible phase (mean, 11.25%). Large
amounts of Fe accumulate in the residual phase probably because it is basically of natural
origin (it is the most common element in the earth’s crust).
Concentrations of Al, Fe, and Cr are very low in exchangeable phase (0.08%, 0.26%, and 1.72%
as mean values, respectively), limiting their potential toxicity as pollutants. It should be noted
that sediments always act as reservoir for elements; therefore, their potential risk of pollution
to environment has always to be considered.
Arsenic, Co, Ni, and to some extent Zn, are found mainly in the residue (∼50% of the total
concentration). Nickel and Co are associated to the residue respectively for 56% and 74% of
the total concentration, with a speciation similar in all the samples. A mean of 23% of Co is
present in the phase 2. The highest percentage of labile Co (~13%) was measured in S6 (Diamond
Harbour) and S8 (Gangasagar) and can be due to a recent input of this element. The dominant
proportion of Ni in the residual phase is in agreement with the results of other studies [27,
46]. Nickel is present, apart from the residue, in phases 2 and 3 (about 10% in each phase).
Arsenic is distributed mainly between the residual (mean 47%), the reducible, and the
oxidizable phases (mean 19% and 22%, respectively). Acharyya et al. [101] observed that As is
adsorbed to iron-hydroxide-coated sand grains and to clay minerals in the sediments of the
Ganges delta from West Bengal. Among the studied elements, As is found with the greatest
proportion in the oxidizable phase coinciding with organic and sulfur compounds. Arsenic is
present in the phase 1 for about 10% of the total content, in station S7 phase 1 percentage rises
up to 16%. The lower land alluvial basin of the Ganges River is recognized as an arsenic-
affected area. Arsenic in solution probably is easily entrapped in the fine grained organic-rich
sediments deposited in the Ganges delta [101]. The percentage of silt (lower than 70% except
in S1 and S2) may have contributed to a low retention of dissolved As since coarse sediments
are less efficient at retaining As.
Cadmium was mainly present in the labile phase (more than 60%) in all the stations with the
exception of station S7, where the Cd labile percentage represents only 25% of the total
concentration. Cadmium concentrations were negligible in phases 2 and 3. The highest labile
Cd concentration was measured at station S3, the closest to the city of Calcutta. Datta and
Subramanian [96] found that the concentrations of elements in the non-detrital phases were
higher in stations sampled in the Hugli river around Calcutta than in samples collected along
Brahmaputra and Meghna rivers. The petroleum refinery, industrial, and mining effluents
carried by the Hugli river may be responsible for this higher concentrations of non detrital
fractions.
About 40% of the total Cu concentration is associated to the residue, while 33% of Cu is bound
to Fe-Mn oxide and hydroxide (phase 2). The high percentage of Cu in the residue is likely due
to the fact that Cu is easily chemisorbed on or incorporated in clay minerals [103]. All the
samples showed lower Cu concentrations in exchangeable phase, with percentage ranging
from 7% (S7) to 22% (S5), with a mean of 15%. Copper is characterized by high complex constant
with organic matter thus it can be hypothesized that Cu is bound to labile organic matter such
as lipids, proteins, and carbohydrates. On the other hand, high-element concentration in labile
phase could be related to recent coastal input [39].
Manganese was found in all the four sediment phases, as observed by other researchers [45,
104]. Manganese is the most mobile element since it is present with the highest percentage (a
mean of 42%) in the labile phase. This is probably because of the known close association of
Mn with carbonates [105] as endorsed by other workers [69, 106]. In this phase, weakly sorbed
Mn retained on sediment surface by relatively weak electrostatic interactions may be released
by ion exchange processes and dissociation of Mn-carbonate phase [2]. The result indicates
that considerable amount of Mn may be released into environment if conditions become more
acidic [107]. The highest Mn labile percentage was measured in S6 (57%). Differently, in S7, Mn
in the residue represents 65% of the total concentration, while the labile Mn is only 15%. A
substantial Mn percentage was also found in the residue (mean 37.8%), followed by the
reducible phase (14.7%), in which Mn exists as oxides and may be released if the sediment is
subjected to more reducing conditions [108].
The major geochemical phase for Pb in these sediments was the Fe-Mn oxides phase (mean
55.7%) followed by the residual phase (mean 30.2%) while lower percentage of the total Pb are
bound to exchangeable-labile (mean 5.3%) and oxidizable phases (mean 6.8%). At S3 (Babugh‐
at), the reducible part is as high as 65% and only 19.9% of the total is associated with the residue.
Atmospheric input as fallout from vehicular emission can be probably the major input of Pb
for this station. The relatively high percentage of Pb in reducible phase is in agreement with
the known ability of amorphous Fe–Mn oxides to scavenge Pb from solution [109, 110]. Caille
et al. [111] observed that resuspension of anoxic sediment results in a rapid desorption of Pb
and Cu adsorbed to sulphides. Thus, a high element percentage in the reducible fraction is a
hazard for the aquatic environment because Fe and Mn species can be reduced into the
porewaters during early diagenesis by microbially mediated redox reactions [112]. Dissolution
will also release Pb associated with oxide phases to the porewater possibly to the overlying
water column [113] and to benthic biota [79]. The major sources of Pb are from intensive human
activities, including agriculture in the drainage basin [114], auto exhaust emission together
with atmospheric deposition [115]. In addition, a substantial contribution from the factories
located in the upstream of the Hugli river dealing with Pb producing lead ingots and lead
alloys play a vital role as referred by Sarkar et al. [ 116].
The percentage of Zn in residue is highly variable (38.5–70%) and the distribution pattern in
each fraction showed the following order: residual>reducible>oxidizable> exchangeable and
bound to carbonates. There was some difference in Zn speciation among the sampling sites:
in stations S1, S2, and S3 about 40% of Zn is present in the residue, while in the other stations
this percentage increases to more than 60%. In station S1, the exchangeable and oxidisable
phases shared over 22% of the total Zn, whereas labile Zn was as low as 4.6% at S7. A major
part of Zn (16.3%) is associated with Fe–Mn oxide phase, because of the high stability constants
of Zn oxides. Iron oxides adsorb considerable quantities of Zn and these oxides may also
occlude Zn in the lattice structures [117].
The BCR procedure as discussed above showed satisfactory recoveries, detection limits, and
standard deviations for determinations of heavy metals/metalloid in the sediments. It is
18 Soil Pollution
evident from the present results of the fractionation studies that the metals/metalloids in the
sediments are bound to different fractions with different strengths leading to variations in
mobility and availability and some of them show significant spatial variations subject to
diverse environmental stresses. This type of association between metals and the sediments can
be understood in detail by sequential extraction techniques. Hence the application of sequen‐
tial extraction is fully justified as the quantification of different forms of metal is more
meaningful than the estimation of its total metal concentrations. The strength values can,
therefore, give a clear indication of sediment reactivity, which in turn assess the risk connected
with the presence of metals in this wetland environment. The results obtained suggest the need
for corrective remediation measures due to the higher accumulation of potentially dangerous
metals/metalloids, which in most cases exceed the limits established by certain legislation.
5.2.4. Comparison with Sediment Quality Guidelines
Results obtained after total and sequential extraction are compared with Sediment Quality
Guidelines (SQGs). Table 6 reports consensus-based values, such as TEC (concentration below
which harmful effects on sediment-dwelling organisms were not expected) and PEC (concen‐
tration above which harmful effects on sediment-dwelling organisms were expected to occur
frequently), and effect range-low and range-medium, such as ERL (concentrations below
which adverse biological effects were observed in less than 10% of studies) and ERM (con‐
centrations above which effects were more frequently observed in more than 75% of studies).
Element Phase Si<TEC TEC Si<TEC<PEC PEC Si<ERL ERL ERL<Si<ERM ERM
Total All 9.79 None 33 S5,S6,S7,S8 8.2 S1,S2,S3,S4,S9 70
As
Labile All None All None
S1,S2,S4,S5, S1,S2,S4,S5,
Total 0.99 S3 4.98 1.2 S3 9.6
S6,S7,S8,S9 S6,S7,S8,S9
Cd
S1,S2,S4,S5, S1,S2,S4,S5,
Labile S3 S3
S6,S7,S8,S9 S6,S7,S8,S9
Total None 43.4 All 111 All 81 None 370
Cr
S1,S3,S4,S5,
Total S1,S4,S5,S7,S8 31.6 S2,S3,S6,S9 149 34 S2,S9 270
Cu S6,S7,S8
Total None 22.7 All 48.6 None 20.9 All 51.6
Ni
Total All 35.8 None 128 All 46.7 None 218
Pb
Total All 121 None 459 All 150 None 410
Zn
Table 6. Sediment Quality Guidelines concentrations with respect to total and labile element concentrations found in
the analyzed samples (expressed as μg/g of dry weight).
Comparing our results with the SQGs, it is revealed that for Pb and Zn in all the stations the
measured concentrations are lower than both TEC and ERL. As regards Cd, concentration
measured in station S3 is higher than TEC and ERL but lower than PEC and ERM both in term
of total and labile concentration. For this station, some possible toxic effect on benthic organism
can be hypothesized, in particular because of the large amount of element bound to the most
labile phase of the sediment. Considering Cu, some stations (S2, S3, S6, and S9) exhibit total
concentrations higher than TEC but lower than PEC. Concentrations of Cu are higher than
ERL but lower than ERM only in stations S2 and S9. Since only 7–22% of total Cu is bound to
the labile phase, in all stations Cu labile concentrations are lower than TEC and ERL. Total As
concentrations in stations S1, S2, S3, S4, and S9 are higher than ERL value but lower than TEC
value. Since more than 50% of total As is not found in the residue, attention should be paid to
a change in the environment conditions which could induce a release of As from the sediments.
Total Ni and Cr concentrations are higher than TEC (Ni is also higher than ERL) but lower
than PEC (and ERM in the case of Ni) in all the stations. Nevertheless, more than 70% of Ni as
well as 90% of Cr are present in the residual fractions, therefore adverse impacts on organisms
is very much negligible.
Mean sediment quality guidelines quotients (mSQGQ) have been developed for assessing the
potential effects of contaminant mixtures in sediments [118]: they are determined by calculat‐
ing the arithmetic mean of the quotients derived by dividing the concentrations of chemicals
in sediments by their respective SQGs. The probability of observing sediment toxicity can be
estimated by comparing the mSQGQ in a sample to previously published probability tables.
It is important to keep in mind that mSQGQs cannot be used to accurately predict the uptake
and bioaccumulation of sediment-bound chemicals by fish, wildlife, and humans, even if there
is considerable evidence that this assessment tool can be predictive of the presence or absence
of toxic effects [118].
SQGQs are calculated for seven elements considering ERM as sediment quality guidelines
(Table 7). The mean quotient values ranges from 0.16 in station S5 to 0.24 in station S3. Using
PEC values instead of ERM, the mean SQGQ ranges from 0.25 in station S5 to 0.38 in station
S3 (Table 7).
Stations SQGQPEC SQGQERM

S1 0.30 0.19
S2 0.33 0.21
S3 0.38 0.24
S4 0.33 0.21
S5 0.25 0.16
S6 0.28 0.18
S7 0.32 0.21
S8 0.28 0.18
S9 0.34 0.22
Table 7. Mean Sediment Quality Guidelines Quotients calculated for the nine stations using PEC and ERM as SQGs.
20 Soil Pollution
Compiled data from multiple data sets reporting 10-day toxicity test conducted on amphipod
species in saltwater showed that the incidence of toxicity for a range of SQGQ of 0.25–0.5 is
∼35%, while for a mean SQGQ range from 0.1 to 0.25, the incidence of toxicity lowers to ∼20%.
Measures recorded in a survey of Biscayne Bay (port of Miami and the adjoining saltwater
reaches of the lower Miami River, FL, USA) showed that the average amphipod survival
(Ampelisca abdita) decreased slightly from the least contaminated (ERMQ <0.03) to the inter‐
mediate category, (ERMQ included in 0.03–0.2 range) then decreased greatly in the most
contaminated sediments (ERMQ included in 0.2–2 range). Therefore, we can presume a low
toxicity of sediments sampled in the nine stations for benthic organisms. It is important to note
that the benthic response to contaminants covaried among stations with both the mean ERM
quotients and the effect of natural factors, such as the sediment texture, TOC, and salinity [118].
5.2.5. Statistical analyses
The relationships between variables and the differences between stations were evaluated by
PCA. The analysis was performed on 36 objects (four sediment phases for nine stations) and
11 variables (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Zn). Two significant components were
identified explaining 68.3% and 14.5% of the total variance, respectively. By studying the
loadings of the variables (Figure 2a) on the components it can be seen that all the elements
except Cd, Mn, and Pb are significantly correlated.
Unlike the other elements, most of Cd and Mn is present in the first phase: labile Cd and Mn
represent more than 60% and 40% of the total concentration, respectively, except in station S7.
Cadmium and Mn speciation can be ascribed to their considerable affinity for carbonates. Lead
is the only element which is bound to the reducible phase for more than 50%. Lead is a very
reactive element in water column and, having scavenging type behavior, is easily bound to
hydroxy- and oxyligands. Copper is positively and significantly correlated with all elements
except Cd and Mn, but with lower correlation coefficients (0.66–0.81).
In the score plot (Figure 2b) phases 1, 2, 3, and 4 (corresponding to labile, reducible, oxidizable,
and residual phases respectively) are identifiable by 1, 2, 3, and 4 suffix, respectively. In all
stations, residue concentrations were characterized by negative values of PC1 and conse‐
quently by high concentrations of Al, As, Co, Cr, Fe, Ni, and Zn. Conversely, in the positive
PC1 semi-axis labile and oxidizable metal concentrations, which represent a small percentage
of the total elements, are distributed. For all stations, reducible concentrations are distributed
along the positive PC2 semi-axis, i.e., high Pb concentrations, with a maximum for station S3
and a minimum for S5. The group formed by elements bound to organic matter and sulphides
(phase 3) is characterized by low values of both PC1 and PC2. Therefore, a low percentage of
elements (higher than 20% exclusively for all As data and for Zn in stations S1 and S3) is bound
to the oxidizable phase, suggesting the presence of an oxidant environment. High Mn and Cd
concentrations are associated with negative values of PC2, therefore a relatively high concen‐
tration of labile Mn and Cd is present in all samples (in particular in S3), except station S7.
Samples are prevalently grouped in relation to the sediment geochemical phase, suggesting a
similar element speciation among the stations. Station S7 represents an exception, in fact the
labile fraction is closely associated to the oxidizable phase group.
Figure 2. Principal component analysis: a) variable plot; b) score plot (phase 1, 2, 3 and 4, corresponding to labile,
reducible, oxidizable and residue, are identifiable by 1, 2, 3 and 4 suffix and different colors in the score plot).
A HCA was carried out by applying Euclidean distances to quantitatively identify specific
groups of similar stations. In the dendrogram of the sampling stations (Figure 3), we can note
two main clusters: the first represented by station S7, characterized by the highest element
percentage bound to residue, and the second constituted by all the remainder stations. In the
second group, a subgroup formed by station S5 and S6 can be individuated.
Station S7 was sampled in a marine coastal environment; it is characterized by a peculiar grain

size percentage respect to the other stations, being the sand percentage as high as 98.6%. In
general, the concentrations of elements are much higher in fine than in coarse fraction because
the fine fraction larger specific surface facilitates absorption processes. As previously noted
22 Soil Pollution
Figure 3. Dendrogram indicating linkage of sites on the basis of element concentrations.
by Ramirez et al. [39], this pattern is particularly evident for Cd. It is interesting to note that
the marine coastal stations S8 and S9 are more similar to river stations than to station S7. Both
the stations S8 and S9 are, in fact, located in front of the Hugli river runoff, while station S7 is
located easternmost and probably is less influenced by the Hugli river discharge.
5.3. Conclusion and recommendation
The study provides valuable information on the potential mobility of trace elements in
sediments collected along the stretch of Hugli River and in the Sundarban mangrove wetland
(northeastern part of the Bay of Bengal). The results obtained adopting BCR sequential
extraction method provided the following important information: (i) Al, Cr, and Fe were found
mostly in the residual phase while the other elements were found in the four phases sharing
different proportions; (ii) the dominant Cd, Mn, and Pb proportion was found in the non-
residual fractions and (iii) Mn had the highest percentage in the labile phase. This is worthwhile
to mention that coastal environment of West Bengal is considerably constrained due to
implementation of dredging, construction of port/ harbor and other industrial activities. The
authors strongly recommend for periodical monitoring on the bioavailability and mobility of
trace elements, control the mixing of effluent of the concentration of heavy metals in the region,
environmental remediation, treatment of industrial effluent and municipal wastewater for
effective management of this estuarine system. It is wisely suggested that an environmental
recovery framework should be urgently implemented to avoid extension of heavy metal
contamination (especially As).
6. Case studies from other coastal regions in India
Although the importance of metal speciation and fractionation has been realized in developed
countries, the subject has not really taken off in India and only few references are available on
the speciation of metals in Indian rivers. Speciation of selected heavy metals geochemistry in
surface sediments (n=10 was studied by Venkatramanan et. al. [119] from Tirumalairajan river
estuary, east coast of India. The results obtained from sequential extraction showed that a
larger portion of the metals were associated with the residual phase, although they are
available in other fractions too.
Trace metal fractionation in the Pichavaram mangrove–estuarine sediments in southeast coast

of India was studied by Ranjan et. al. [120] considering the pronounced changes due to
occurrence of tsunami (2004). A 5-step sequential extraction procedure was applied to assess
the effects of tsunami on mobility and redistribution of selected elements (Cd, Cr, Cu, Fe, Mn,
Ni, Pb, and Zn) in coastal sediments revealed that metals in the residual fraction (lattice bound)
had the highest concentration suggesting their non-availability and limited biological uptake
in the system. Majority of the metals (except Mn) do not constitute a risk based on the different
geochemical indices.
Fractionation of selected metals in the sediments of Cochin estuary and Periyar River (south‐
west coast of India) was studied by Mohan et. al. [121]. The results reveal that remobilization
potential of metals bound is in the range of low to medium risk to various sedimentary phases
is different and is based on bond strength. Therefore, the strength values can give a clear
indication of sediment reactivity that can be used to assess the risk related with metals to the
aquatic organisms.
7. Risk Assessment Code (RAC)
The risk assessment code (RAC) mainly applies the sum of the exchangeable and carbonate
bound fractions for assessing the availability of metals in sediments. These fractions are
considered to be weakly bonded metals which may equilibrate with the aqueous phase and
thus become more rapidly bioavailable [11, 33]. This is important because the fractions
introduced by anthropogenic activities, such as agricultural runoff and tourism, are typified
by the adsorptive, exchangeable, and bound to carbonate fractions, which are weakly bonded
metals that could equilibrate with the aqueous phase and thus become more rapidly bioavail‐
able [122]. According to RAC guideline (Table 8), for any metal, soil/sediment which can release
in exchangeable and carbonate fractions, less than 1% of the total metal will be considered safe
for the environment and soil/sediment with 11-30% carbonate and exchangeable fractions will
be at medium risk to the environment. On the contrary, soil/sediment releasing in the above
fractions more than 50% of the total metal has considered being highly dangerous, which can
be easily enter the food chain [123].
24 Soil Pollution
Grade Exchangeable and bounded to carbonate metal (%) Risk
I <1 No risk
II 1 – 10 Low risk
III 11 – 30 Medium risk
IV 31 – 50 High risk
V >50 Very high risk
Table 8. Criteria of Risk Assessment Code [123].
Heavy-Metal Fractionation in surface sediments was studied by Dhanakumar et. al. [124] in
the Cauvery river estuarine region, southeastern coast of India. The results revealed that most
of the samples fall under the category from low- to high-risk class and from low-risk to very
high-risk class in terms of labile fractions of Pb as well as Zn and Cu, respectively.
8. Conclusion
From the above discussion it is revealed that geochemical fractionation approach to the
chemical speciation has provided a useful tool and opens a new dimension in assessing the
potential mobility/bioavailability of heavy metals and metalloids in soils/sediments and opens
a new dimension in the field of ecology and environmental chemistry. More efficient, non-
laborious and time saving processes techniques in this field of chemical speciation are also
coming up to get valid information regarding geochemical behavior of soils/sediments. Besides
geochemical fractionation, Dezileau et al. [125] opined that total Fe or Fe/Al may be used to
infer millennial-scales climate changes in the south eastern pacific while performing sequential
extraction of Fe in marine sediments from the Chileau continental margin. However, the
chemical partitioning should be carefully used in the assessment of environmental pollution
as large amount of metals may naturally occur as anthropogenic fractions (including loosely
bonded ions, sulfide ions and metals associated with sediments).
Acknowledgements
The authors gratefully acknowledge full support and cooperation of the Springer press, UK
for extending permission in publishing the research paper of the journal Environmental
Monitoring & Assessment, vol. 184(12), pp:7561-77, 2012.
This study was partially supported by the European Fund for Economic and Regional
Development (FEDER) through the Program Operational Factors of Competitiveness (COM‐
PETE) and National Funds through the Portuguese Foundation for Science and Technology
(PEST-C/MAR/UI 0284/2011, FCOMP 01 0124 FEDER 022689).
Author details
Santosh Kumar Sarkar1, Paulo J.C. Favas2,3*, Dibyendu Rakshit1 and K.K. Satpathy4
1 Department of Marine Science, University of Calcutta, Calcutta, West Bengal, India
2 Department of Geology, School of Life Sciences and the Environment, University of Trás-
os-Montes e Alto Douro, Vila Real, Portugal
3 IMAR-CMA Marine and Environmental Research Centre, Faculty of Sciences and Technol‐
ogy, University of Coimbra, Coimbra, Portugal
4 Indira Gandhi Centre for Atomic Research, Environment and Safety Division, Kalpakkam,
Tamil Nadu, India
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chapter 26
The Effect of Industrial Heavy Metal Pollution on

Microbial Abundance and Diversity in Soils — A Review
Anna Lenart-Boroń and Piotr Boroń
1. Introduction
Metals are essential components of the ecosystem, whose biologically available concentrations
depend mainly on geological and biological processes [1]. There are several definitions of
heavy metals, and some of them are based on the mass density of these elements. Authors of
numerous publications use different limits to define the threshold density for a “heavy metal”,
ranging from 3.5 to 7 g×cm-3, however, the majority of authors suggests that the mass density
of heavy metals should be greater than 4.5 g×cm-3 [2]. Within the group of heavy metals one
can distinguish both elements that are essential for living organisms (microelements) and the
elements whose physiological role is unknown and thus they are “inactive” towards organ‐
isms. The metals that serve as microelements in living organisms usually occur in trace
amounts, precisely defined for each species and both their deficiency and excess badly affect
living organisms [3]. The term “heavy metal” is linked in many people’s minds to metals that
are toxic. However, this is not always the truth. The effect of any substance on a living system
is always dependent on its available concentration to cells. Also, several heavy metal ions are
crucial in metabolic processes at low concentrations but are toxic at high concentrations [2].
Nevertheless, locally elevated levels of these elements can create significant environmental
and health problems when the release of metals through various biological, geological and
anthropogenic processes far exceeds its natural content resulting from processes of metal
cycling. Heavy metal pollution of terrestrial environments is of great concern, due to the
persistence of metals in the ecosystem and their threat to all living organisms [4].
Given the importance of the subject of soil heavy metal pollution and its effect on soil micro‐
organisms, this chapter gives an overview of the severity of the problem when it comes to the
reaction of soil microbial community to the environmental pollution. The first part of this
chapter deals with the abundance of microorganisms in soils and their role in this environment.
The next part concerns major sources of heavy metals in soils with particular emphasis on the
most important source of soil pollution, i.e. human activity (and more precisely – industry and
mining). The following part discusses the effects that toxic levels of heavy metals may have
on the microbial population in soils. The last two parts of this chapter describe the ways of
dealing with heavy metal pollution – one introduces the term of phytoremediation (soil
remediation with the use of plants) and the other one focuses on the use of microorganisms
resistant to heavy metals in the process of soil remediation.
2. The complexity of microbial community in soils
Except for occasional insects or earthworms, once visible traces of plant biomass are removed,
soil appears as a lifeless mass, that is composed of mineral particles and organic residues.
However, even desert soils are abundant source of living microorganisms. This seemingly
lifeless matter contains complex microbial community, including bacteria, fungi, protozoa and
viruses. The integrity of the aboveground and belowground ecosystems depends on the
stability, resilience and function of the soil microbial community [5].
Soil is an interesting medium for growing microorganisms, as it contains various nutrients that
the microbes need for their metabolism. Unfortunately, nutrients are not always readily
available [6]. However, it is one of the richest reservoirs of microorganisms, i.e. 1 gram of
agricultural soil may contain even several billion colony forming units (CFUs) of microorgan‐
isms belonging to thousands of different species [7], and even though microorganisms
constitute less than 0.5% of the soil mass, they have a major impact on soil properties and
processes [5]. Table 1 presents the average numbers of soil microorganisms in a “typical”
temperate soil. Destruction of the soil microbiota through mismanagement or environmental
pollution causes decline or even death of the aboveground plant and animal populations.
Numbers
Organisms Biomass [wet kg×ha-1]
Per m2 Per g
Bacteria 1013-1014 108-109 300-3000
Actinomycetes 1012-1013 107-108 300-3000
Fungi 10 -10
10 11
10 -10
5 6
500-5000
Microalgae 10 -10
9 10
10 -10
3 6
10-1500
Table 1. Relative numbers and approximate biomass of the soil microbiota in a fertile soil [8].
The most characteristic feature of microbial habitats is the great micro-spatial variability of
environmental parameters, like temperature or nutrient availability. Many basic requirements
of heterogeneous microorganisms are satisfied by various soil microhabitats. This is the reason
why, in ecological terms, a number of varying microbial niches can be described. Therefore,
the microbial community is composed of diverse taxa with different nutritional demands
The Effect of Industrial Heavy Metal Pollution on Microbial Abundance and Diversity in Soils — A Review 3
within small microenvironments [9]. Analysis of the spatial distribution of bacteria at micro‐
habitat levels showed that in soils subjected to different fertilization treatments, the majority
of bacteria were located in micropores of stable soil micro-aggregates (2 – 20 µm), as they
contained over 80% of cells [10]. Such microhabitats offer the most favorable conditions for
microbial growth in terms of water and substrate availability, gas diffusion and protection
against predation. The microhabitat-adapted groups of microorganisms form so-called
consortia which are held together by mutually facilitating metabolic processes. The consortia
are characterized by more or less sharp boundaries, and variable level of interaction with each
other and with other parts of the soil biota. Numerous investigations emphasize the impact of
soil structure and spatial isolation on microbial diversity and community structure [11]. Some
studies indicate that the soil particle size affects the diversity of microorganisms and com‐
munity structure to a greater extent than other factors such as bulk pH and the type or amount
of available organic compounds [12]. Other investigations show that the type and amount of
available organic substrates strongly affect the abundance of microbial groups and their
functional diversity in soils [13]. Fierer and Jackson [14] claim that the structure of soil bacterial
communities is not random also at continental scale and that the diversity and composition of
soil bacterial communities at large spatial scales can be predicted to a large extent by a single
variable, that is soil pH. The diversity of soil microorganisms comprises different levels of
biological organization. It includes genetic variability among taxa (species), number (richness),
relative abundance (evenness) of taxa and functional groups within communities [11]. The
overall biodiversity of soil microflora comprises bacteria, fungi, actinomycetes and photosyn‐
thetic microorganisms [6].
Bacteria constitute the most numerous group of soil microbes – a teaspoon of productive soil
contains between 100 million and 1 billion bacterial cells. As soil environment changes rather
drastically, spore-forming bacteria tend to be the most common. When environmental
conditions become too difficult for normal growth, the bacteria form spores and remain
dormant until the environment returns to proper conditions [6]. They facilitate various
processes in soils, e.g. those related to water dynamics, nutrient cycling or disease suppression
[15]. Soil-dwelling bacteria may be divided into different groups based on:
• Shapes: rods (also called bacilli), sphere (also called cocci) and spiral (also called spirilla)
• Their reaction to oxygen: aerobic (bacteria that need oxygen for their survival) and anaerobic
(the ones that do not require oxygen and in most cases cannot bear oxygen that is deadly
for them)
• Result of Gram staining: Gram negative (stain pink and have thinner cell walls, they are the
smallest ones and tend to be more sensitive to water stress) or Gram positive (stain violet,
have thicker cell walls, are larger in size and tend to resist water stress)
• Source of carbon they use: autotrophs (obtain carbon from carbon dioxide – some autotro‐
phic bacteria directly use sunlight in order to produce sugar from carbon dioxide, while
others depend on various chemical reactions) or heterotrophs (they obtain carbohydrates
from their environment)
• Classification based on phyla: based on morphology, barcode DNA sequences, physiolog‐

ical requirements and biochemical characteristics, bacteria have been classified into 12
phyla. Each phylum corresponds to a number of bacterial species and genera [15].
Tate [5] lists the most commonly encountered soil bacterial genera as: Acinetobacter, Agrobac‐
terium, Alcaligenes, Arthrobacter, Bacillus, Brevibacterium, Caulobacter, Cellulomonas, Clostridium,
Corynebacterium, Flavobacterium, Hyphomicrobium, Metallogenium, Micrococcus, Mycobacterium,
Pseudomonas, Sarcina, Streptococcus and Xanthomonas. These are the heterotrophic bacteria that
are augmented in soil by autotrophic and mixotrophic representatives, including nitrifiers,
Thiobacillus species and iron bacteria.
Bacteria facilitate a number of physical and biochemical alterations or reactions in soils and
thereby directly or indirectly support the development of higher plants. Their performance is
vital for a variety of processes that include: decomposition of cellulose or other carbohydrates
(e.g. Bacillus, Achromobacter, Cellulomonas, Clostridium, Methanococcus), ammonification
(Bacillus, Pseudomonas), nitrification (Nitrosomonas, Nitrobacter), denitrification (Achromobacter,
Pseudomonas, Bacillus, Micrococcus) and nitrogen fixation (symbiotic Rhizobium, Bradyrhizobi‐
um etc., non-symbiotic Azotobacter, Beijerinckia) [16].
On the other hand, soil fungi form three functional groups: decomposers, mutualists and
pathogens. Fungi, along with bacteria, are important decomposers of hard to digest organic
matter and they increase nutrient uptake of phosphorus. Mycorrhizal fungi support plants by
promoting root branching and increasing nitrogen, phosphorus and water uptake. They
improve plant resilience to pests, diseases or drought and improve soil structure, as fungal
hyphae binds soil particles together to create water-stable aggregates. They in turn create the
pore spaces in the soil that enhance water retention and drainage [17]. The most common fungi
found in soil belong to the Penicillium and Aspergillus genera together with the representatives
of the Zygomycetes and the mycorrhizae-associated Ascomycetes and Basidiomycetes [5].
Actinomycetes are a large group of microorganisms, systematically identified as bacteria, that

grow as hyphae. They decompose a wide range of substances, but they are particularly
important in degrading recalcitrant (difficult to degrade) compounds such as chitin, lignin,
keratin and cellulose. Moreover, they produce a number of secondary metabolites such as
antibiotics i.e. streptomycin [18] or geosmine which is responsible for “earthy” smell after soil
plowing [15]. Actinomycetes are important in forming stable humus, which enhances soil
structure, improves soil nutrient storage and increases water retention in soils. According to
Tate [5], the most commonly encountered soil actinomycetes belong to Nocardia and Strepto‐
myces genera.
Algae are the most common among photosynthetic microorganisms found in soil. They are
found only near soil surface, where light is readily available [6]. The most common genera of
green algae found in soil are: Chlorella, Chlamydomonas, Chlorococcum, Protosiphon etc. and that
of diatoms are Navicula, Pinnularia. Synedra, Frangilaria. Their functions include the mainte‐
nance of soil fertility, increasing water retention capacity of soil, prevention of soil erosion due
to the fact that they act as cementing agents in binding soil particles. They add organic matter
to soil after the cell death and thus increase the amount of organic carbon, while their photo‐
synthetic activity release large quantity of oxygen that facilitate the aeration in submerged soils
or oxygenate the soil environment. They also take part in weathering rocks, thus building up
the soil structure [19]
Although biomass of all microorganisms living in soil constitutes only several percent of
organic matter content, they play an important role in the functioning of entire ecosystems [20].
They take part in soil formation, mineralize organic substances, provide plants with bioavail‐
able compounds, cooperate with plants or may be used as a source of insecticidal substances
[21]. One of the most important and most widely studied microbial groups in terms of
beneficial effects to soil and plants is the group of Plant Growth Promoting Rhizobacteria
(PGPR) [22]. This group includes bacterial species from genera such as Azotobacter, Azospiril‐
lum, Bacillus, Burkholderia, Enterobacter, Erwinia, Flavobacterium, Pseudomonas and Rhizobium
[23]. Activity of these bacteria significantly increases plant growth and yield due to a variety
of mechanisms, such as phytohormone production, symbiotic and asymbiotic N2 fixation,
production of siderophores, activity against phytopathogenic microorganisms, synthesis of
antibiotics, enzymes and/or fungicidal compounds, as well as solubilization of mineral
phosphates and other nutrients [24]. PGPR may improve plant growth, salinity and metal
toxicity stress tolerance, as well as they are able to produce phytohormones such as indole-3-
acetic acid (IAA) [25]. Some PGPR produce the enzyme 1-aminocyclopropane-1-carboxylate
(ACC) deaminase, which hydrolyses ACC, the immediate precursor of ethylene in plants. By
decreasing its concentration in seedlings and thus its inhibitory effect, these PGPR stimulate
seedlings’ root length [26]. Figure 1. shows the ways how Plant Growth Promoting Rhizobac‐
teria can stimulate plants. Bacteria from the genus Rhizobium form symbiotic associations with
roots of leguminous plants like clovers, peas or alfalfa. These Gram-negative, rod-shaped
bacteria infect growing root hairs, forming visible nodules. In this form of symbiosis, plants
supply simple carbohydrates to bacteria while bacteria convert nitrogen (N2) from air into the
forms (NO3- or NH4+) that plant can use. When leaves or roots from the plant decompose,
nitrogen content increases in soil [15]. Some microbial species are capable of detergent
decomposition, taking part in self-purification process of soils. Decomposers are particularly
important in immobilizing or retaining nutrients in their cells, thus preventing the loss of
nutrients, such as nitrogen, from the rooting zone.
Despite beneficial effects of numerous soil microbes on plant growth or development, soil
structure and functioning, some soil-dwelling microorganisms may cause plant, animal and
human diseases. Similarly to the beneficial soil microflora, soil pathogens include bacteria,
fungi and viruses. One of the example of the most important or best known plant pathogens
include Agrobacterium tumefaciens (whose updated scientific name is now Rhizobium radiobact‐
er) [27] which is the causal agent of crown gall disease of walnuts, grape vines, stone fruits and
many others. These bacteria infect plant roots and induce cells to divide (due to overproduction
of auxin and cytokinin), causing a tumor-like swellings that contain infected cells [28]. Erwinia
carotovora (or now called Pectobacterium carotovorum) and Erwinia amylovora, the Gram-negative
plant pathogens with a diverse host range cause infections of numerous agriculturally and
scientifically important plant species, such as potato, apple, pear and some members of the
family Rosaceae [29]. Soil is also an abundant source of fungal pathogens. Among them we may
Figure 1. Summary of mechanisms employed by Plant Growth Promoting Rhizobacteria to stimulate plant develop‐
ment.
distinguish Rhizoctonia solani, a plant pathogenic fungus with a wide host range and worldwide
distribution. It causes collar rot, crown rot, root rot, damping off and wire stem [30]. It mainly
attacks plant seeds below the soil surface, but may also infect leaves and stems. Due to a variety
of hosts that this pathogen attacks, it is of great importance and is detrimental to a variety of
crops. The Armillaria root rot, caused by several species of basidoimycete genus Armillaria –
the honey fungus is, on the other hand, one of the greatest threat for woody plants [31]. Another
example of soil-borne plant pathogens is an important genus of fungi – Fusarium, which
contains a number of, worldwide distributed, phytopathogenic species [32]. Moreover,
Fusarium has also been more recently reported as an emerging human pathogen for immuno‐
compromised patients [33]. Clostridium tetani is an example of one of the most dangerous soil-
borne human pathogens. It is a tetanus-causing Gram-positive bacterium, whose transmission
occurs through the contamination of wounds with soil carrying its spores [34]. Generally, soil
is a typical carrier of human bacterial and fungal pathogens. Another example of them is
Bacillus anthracis, the causative agent of anthrax, which is found worldwide in a variety of soil
environments. Inhalation of B. anthracis spores can be fatal. Nevertheless, the incidence of both
of these fatal diseases has been largely controlled in developed countries due to the develop‐
ment of vaccines [35].
Undoubtedly, soil is an inexhaustible reservoir of microorganisms, both beneficial and

pathogenic ones. Causing the imbalance between groups of soil macro- and microorganisms
may be irreversible and result in a variety of effects, sometimes unpredictable. Such imbalance
may be caused by soil pollution resulting from developing industry, therefore understanding
the sources and effects of industrial soil pollution is an important element in preventing the
environmental degradation.
3. Sources of soil heavy metal pollution
Chemical compounds, entering the ecosystem as a result of different human activities, may
accumulate in soil and water environments. Therefore, soil may be regarded as a long-
term reservoir of pollutants, from which these compounds may be introduced to food chains
or groundwater [36]. Inappropriate and careless disposal of industrial waste often results
in environmental pollution. The pollution includes point sources such as emission, efflu‐
ents and solid discharge from industry, vehicle exhaustion and metal smelting or mining,
as well as nonpoint sources (e.g. the use of pesticides or excessive use of fertilizers) [37].
Each of the sources have their own damaging effects on plant, animal and human health,
but those that add heavy metals to soils are of serious concern due to the persistence of
these elements in the environment. They cannot be destroyed, but are only transformed
from one state to another [38].
Soil pollution may be defined as presence of xenobiotics (e.g. chemical compounds, radioactive
elements) that alters the soil properties – both chemical, physical and biological. Soil pollution,
including heavy metals, may be of natural origin, like volcanic eruptions, animal excrements
or ore leaching. Nevertheless, human activity and mostly chemical industry, mining and
metallurgy, as well as municipal management and traffic emissions are the main source of
environmental pollution. Some authors also mention that waste disposal, waste incineration,
fertilizer application and long-term application of wastewater in agricultural lands may result
in heavy metal pollution of soils [39].
Heavy metals occur naturally in soils due to pedogenetic processes of weathering parent
materials, however concentrations of these metals are regarded as trace (<1000 mg×kg-1) and
rarely toxic [40]. Due to the disturbance and acceleration of the natural slow geochemical cycles
of metals by man, most soils of rural and urban environments accumulate one or more heavy
metals above the defined background levels, high enough to cause risks to ecosystems [41].
Nevertheless, heavy metals occurring in soils from anthropogenic sources tend to be more
mobile, therefore more bioavailable than pedogenic or lithogenic ones [42].
Communication routes, such as roads, railways etc., are an important source of soil pollution,
especially in the case of lead and zinc. Despite restricted use of leaded gasoline adopted in
most countries, lead remains one of the most serious automotive-originating metal pollutant.
The areas located nearby roads, particularly in urban sites, are the most vulnerable to auto‐
motive pollution. Apart from lead and zinc, chromium, cadmium, nickel and platinum are
among the pollutants emitted by combustion engine-powered vehicles [43]. Heavy metals
enter the environment as a result of tire wear and damage of vehicle parts. Moreover, grease
used in vehicles may also be the source of cadmium pollution along roads [44]. Nickel emission
results from this metal being added in gasoline and atmospheric abrasion of nickel-containing
parts of automobiles [45]. The changes in the concentrations of lead, nickel, cadmium, copper
and zinc in roadside soils are frequently attributed to traffic density [46].
Standard agricultural practices are also a significant source of heavy metals in soils, as
application of fertilizers and pesticides has contributed to a continuous accumulation of these
elements. Heavy metals can accumulate in soils due to the application of liquid and solid
manure, as well as inorganic fertilizers [47]. The application of numerous biosolids, such as
livestock manures, composts and municipal sewage sludge on agricultural soils leads to the
accumulation of various heavy metals, such as, Cd, Cr, Cu, Hg, Mo, Ni, and Zn [48]. Lime and
superphosphate fertilizers contain not only major elements necessary for plant nutrition and
growth but also trace metal impurities such as cadmium. The presence of high concentrations
of Cd in some fertilizers (particularly in phosphatic fertilizers) is of most concern due to the
toxicity of this metal and its ability to accumulate in soils as well as due to its bioaccumulation
in plant and consequently in animal tissues [49, 50]. Additionally, copper-containing com‐
pounds have been widely used in agricultural practice as pesticides. Copper oxychloride is
annually applied on vineyards as a fungicide to control a significant number of plant diseases.
Inevitably, this Cu ends up in the agricultural soil and adjacent pristine natural vegetation [51].
Lead arsenate was used in fruit orchards for many years to control some of the parasitic insects.
Arsenic-containing compounds were also extensively used to control pests in banana planta‐
tions in new Zealand and Australia [52]. High fertilizer applications and acid atmospheric
deposition, combined with insufficient liming, may also cause a decrease in pH and thus
increase heavy metal bioavailability, aggravating the problem of deteriorating food quality,
metal leaching and impact on soil organisms [53]. The application of municipal wastewater or
industrial waste as fertilizers and liming agents in agriculture is a separate issue. Application
of this type of waste requires constant monitoring of the amount and proportion of harmful
factors, including heavy metals. The high risk of soil pollution with Cd, Zn, Ni and Pb as a
result of industrial waste application as fertilizers was also evidenced [50].
Airborne sources of heavy metals include stack emissions or fugitive emissions such as dust
from storage areas or waste heaps. Stack emissions can be distributed over a wide area by
natural air currents, while fugitive emissions are often distributed over much smaller areas.
In general, concentrations of pollutants are much lower in fugitive emissions compared to
stack emissions. The type and concentration of metals emitted from both types of sources
depend on site-specific conditions. All solid particles in smoke from fires and other
emissions from factory chimneys are deposited on land or sea. Most forms of fossil fuels
contain some heavy metals and this form of environmental pollution has been increasing
since the industrial revolution began. For instance, very high concentrations of Cd, Pb and
Zn have been found in plants and soils adjacent to smelting plants. Another major source
of soil pollution is the aerial emission of lead from combustion of petrol containing tetraethyl
lead; this contributes substantially to the content of Pb in soils in urban areas and in those
adjacent to major roads [52].
Another, and one of the most significant sources of heavy metal pollution of soils, includes
heavy industry, e.g. mining and metallurgy. Industrial airborne heavy metal contamination
of the nonferrous smelters surrounding landscapes is a well-known and widely occurring
phenomenon. Emissions of metallurgical dust are spread according to the wind direction and
particle size while soil is the main receiver of heavy metals in dry land. Dust emissions from
smelters using sulfide copper-nickel ores are similar, regardless of their location, owing to the
fact that the same raw materials are used in metallurgical processes. The following major metal-
containing compounds are deposited onto the landscape in the form of dust emissions from
smelters: pentlandite (Ni,Fe)9S8, pyrrotite Fe7Sg(Nix), chalcopyrite CuFeS2, chalcosite Cu2S,
covellite CuS, cuprite Cu20, tenorite CuO, and metal copper and nickel [54]. Surface soil layers
in the mining or metallurgy areas are often heavily polluted with copper. In the vicinities of
steel plants the concentration of this element exceeds several thousand ppm and the pollution
remains for a long time, even after the operation of mines or steel plants had been stopped [50].
The fine fractions of dust are enriched with lead, arsenic, and zinc. The quantity and compo‐
sition of dust derived from different sources (metallurgical processes) varies according to the
raw materials and the condition of the gas cleaning systems [54]. The cause for the frequently
widely dispersed metal pollution in habitats of mining areas was found in the formation of
acid mine drainage (AMD). The runoff from mining heaps of active and abandoned mines can
be extremely acidic, with pH values reaching as low as pH 2 [9]. Chemical and biological
oxidation of the abundant mineral pyrite (FeS2) occurs after the unearthing of pyrite-containing
rock formations and results in an acidification of the dump material [55]. Under acidic
conditions, the majority of heavy metals is leached from the waste dump and they are
transported as AMD in streamwaters [9]. Galvanization industry may cause soil pollution with
silver as well as other industrial facilities that use silver salts. Additionally, the increased
amount of silver may by introduced to soils with municipal sewage. Municipal sewage
contains also large amounts of highly soluble forms of zinc, which may then easily contaminate
soil environment [50]. Zinc is also extensively used in metallurgical industry, as an anti-
corrosion agent in alloys and in galvanization. It is frequently used in paint industry [50]. The
concentration of cadmium highly increases in soils polluted with emissions from nonferrous
metal plants, which constitute over 60% of all anthropogenic sources of this element in soils.
Municipal sewage contains on average 10 – 40 ppm of cadmium, while industrial sewage may
contain over 1000 ppm. This is also a case of large amounts of lead that may be introduced into
soils from municipal sewage and waste, as they contain mobile forms of this element. This may
result in large increase in the concentration of lead in soils that may exceed several times the
admissible limits. Additionally, dust emissions from landfills of nonferrous metal plants may
become dangerous sources of lead in soils [50]. Table 2 shortly summarizes the major sources
of different heavy metals in soil.
Heavy metals Sources
As Semiconductors, petroleum refining, wood preservatives, animal feed additives, coal power
plants, herbicides, volcanoes, mining and smelting
Cu Electroplating industry, smelting and refining, mining, biosolids
Cd Geogenic sources, anthropogenic activities, metal smelting and refining, fossil fuel burning,
application of phosphate fertilizers, sewage sludge
Cr Electroplating industry, sludge, solid waste, tanneries
Pb Mining and smelting of metalliferous ores, burning of leaded gasoline, municipal sewage,
industrial wastes enriched in Pb, paints
Hg Volcano eruptions, forest fire, emissions from industries producing caustic soda, coal, peat and
wood burning
Se Coal mining, oil refining, combustion of fossil fuels, glass manufacturing industry, chemical
synthesis (e.g., varnish, pigment formulation)
Ni Volcanic eruptions, land fill, forest fire, bubble bursting and gas exchange in ocean, weathering of
soils and geological materials
Zn Electroplating industry, smelting and refining, mining, biosolids
Table 2. Different sources of heavy metals in soils [56]
4. The effects of heavy metals on soil microorganisms
Metals without biological function are generally tolerated only in minute concentrations,
whereas essential metals with biological functions, are usually tolerated in higher concentra‐
tions [9]. They have either metabolic functions as constituents of enzymes or meet structural
demands, e.g. by supporting the cell envelope. Frequently the concentration and the speciation
of metal determine whether it is useful or harmful to microbial cells [9].
Microorganisms are the first biota that undergoes direct and indirect impacts of heavy metals.
Some metals (e.g. Fe, Zn, Cu, Ni, Co) are of vital importance for many microbial activities when
occur at low concentrations. These metals are often involved in the metabolism and redox
processes. Metals facilitate secondary metabolism in bacteria, actinomycetes and fungi [9; 57].
E.g. chromium is known to have stimulatory effect on both actinorhodin production and
growth yield of the model actinomycete S. coelicolor [58]. However, high concentrations of
heavy metals may have inhibitory or even toxic effects on living organisms [59]. Adverse effects
of metals on soil microbes result in decreased decomposition of organic matter, reduced soil
respiration, decreased diversity and declined activity of several soil enzymes [60]. Some of the
general changes in morphology, the disruption of the life cycle and the increase or decrease of
pigmentation are easy to observe and evaluate [9]. Rajapaksha et al. [61] compared the
reactions of bacteria and fungi to toxic metals in soils (Zn and Cu). They concluded, that
bacterial community is more sensitive to increased concentrations of heavy metals in soils than
the fungal community. The relative fungal/bacterial ratio increased with increasing metal
levels. Those authors also noticed the varying effect of soil pH on the microbial reaction to soil
pollution, i.e. that lower pH in contaminated soils enhanced the negative effect on bacteria,
but not on fungi.
The toxic concentration of heavy metals may cause enzyme damage and consequently their
inactivation, as the enzymes-associated metals can be displaced by toxic metals with similar
structure [59]. Moreover, heavy metals alter the conformational structures of nucleic acids
and proteins, and consequently form complexes with protein molecules which render them
inactive. Those effects result in disruption of microbial cell membrane integrity or destruc‐
tion of entire cell [62]. Heavy metals also form precipitates or chelates with essential
metabolites [63].
Various metals may affect different microbial populations and the resulting impact may vary
depending on the metal whose limit concentrations in soils were exceeded. For instance, the
pollution of soils with copper affects microorganisms that take part in nitrification and
mineralization of protein compounds [50]. Silver is one of the most toxic metals to heterotro‐
phic bacteria. This effect is used for the production of antiseptic preparations. However, there
are some silver-resistant bacteria, both in clinical and natural conditions. Some strains of
Thiobacillus ferrooxidans are able to accumulate particularly large amounts of silver [50]. About
100 ppm of zinc in soils may inhibit nitrification processes and about 1000 ppm inhibits the
majority of microbiological processes in soils [64]. Microorganisms play vital role in circulation
and transformation of mercury compounds in the environment. Numerous bacteria and fungi
show high tolerance (also acquired) to increased concentrations of mercury in soils. However,
some microorganisms are sensitive to excess mercury, e.g. the concentration of <10 ppm Hg
may have toxic effects on nitrifiers in soils [50]. Increased concentrations of lead in surface soil
layers negatively affect soil microflora. Processes of organic matter decomposition, particularly
cellulose, are inhibited as a result of decreased enzymatic activity of microorganisms. This
results in soil degradation. Biosorption of lead by soil microorganisms reaches on average 0.2%
of this metal, but in some cases it may reach even 40% of biomass and may be used for biological
remediation [50]. Some studies indicate that long-term contamination of soils with heavy
metals has adverse effects on soil microbial activity. For instance, Juwarkar et al. [65] while
researching the remediation strategies for cadmium and lead contaminated soils, compared
the numbers of the selected groups of microorganisms in natural and heavy metal spiked soils.
The results that they obtained indicated that the examined microbial groups were much less
abundant in contaminated soils than in natural ones [Table 3]. On the other hand, Lenart and
Wolny-Koładka [66] recorded significantly variable numbers of the selected microbial groups
while analyzing the uncontaminated and heavy metal contaminated soils of ArcelorMittal
steelworks in Cracow. Similarly, their results indicated that except for fungi, the soil-dwelling
microorganisms were much less abundant in heavy metal polluted soils than in uncontami‐
nated soils (Table 4). Heavy metal contamination results in reduction of microbial biomass and
even if they do not cause the reduction in their number – they reduce biodiversity or disturb
the community structure [64].
Analyzed microorganisms [CFU×g-1] Natural soil Heavy metal contaminated soil
Total number of bacteria 17×10 5

58×104
Total number of fungi 26×103 42×102
Actinomycetes 43×103 18×101
Azotobacter spp. 23×103 17×101
Rhizobium spp. 21×10 3

16×102
Table 3. Microbiological characteristics of natural and heavy metal spiked spoil samples in Nagpur (India) [65]
Analyzed microorganisms [CFU×g-1] Uncontaminated soil Heavy metal contaminated soil
Total number of mesophilic bacteria 22.50×10 – 10.44×10

2 6
0 – 13.15×105
Total number of fungi 84.00×101 – 21.03×103 0 – 57.90×103
Actinomycetes 62 – 99.50×103 0 – 20.26×103
Azotobacter spp. 0 – 28.90×102 0 – 57.00×101
Table 4. Ranges of the selected microbial groups in heavy metal contaminated and uncontaminated soils of
ArcelorMittal steelworks in Cracow, Poland [66].
However, one of the reasons of decreasing biodiversity of microorganisms in heavy metal

polluted soils is the selection for tolerant species or strains. Metal exposure may lead to the
establishment of tolerant microbial populations, that are often represented by several Gram-
positive genera such as Bacillus, Arthrobacter and Corynebacterium or Gram-negatives, e.g.
Pseudomonas, Alcaligenes, Ralstonia or Burkholderia [68]. It was shown that the impact of heavy
metals on the bacterial metabolism depends on the growth form. The resistance towards metals
seems higher in consortia than in pure cultures [69]. A great number of heavy metal-resistant
bacteria, such as e.g. Cupriavidus metallidurans possess efflux transporters that excrete toxic or
overconcentrated metals outside the cell [70]. Efflux transporters have high substrate affinity
and can therefore maintain low cytosolic concentration of metals [9]. Alternatively, microbial
cells may prevent the intoxication by the release of metal-binding compounds into the
extracellular surroundings. In that case, metals are chelated outside the cell and thus blocked
from entering the cell through the membrane transporters that otherwise facilitate the influx
[9]. Some fungal and bacterial species are able to keep metals outside their cells by the
extracellularly active melanin [71]. It is a secondary metabolite that has strong cation chelating
properties through the anionic function such as carboxyl and deprotonated hydroxyl groups
[9]. A substantial number of soil microorganisms, such as widespread fungus Aspergillus
niger, solubilize metals by the release of organic acids or by the immobilization of metals
through excretion of different compounds, such as oxalates [72]. Some microorganisms possess
the abilities to protect their cells by a cytosolic sequestration mechanisms. These mechanisms
are activated once the metal enters the cell and cannot be excreted. In this case internal inclusion
bodies, e.g. polyphosphate granules (volutin) bind large amounts of metal cations [73].
Investigation and understanding of microbial resistance mechanisms towards heavy metals

are crucial for the potential application of microorganisms for remediation of polluted soils.
5. General outline of soil remediation strategies
The overall objective of any soil remediation approach is to create a final solution that is
protective both for human health and the environment [74]. For heavy metal-polluted soils,
the physical and chemical form of the heavy metal contaminant in soil strongly influences the
selection of the appropriate remediation treatment approach. Details on the physical charac‐
teristics of polluted soils, type and level of the pollution at the site must be known to enable
accurate assessment of the problem severity and adjustment of remedial measures [52].
Remediation of heavy metal-polluted sites is very expensive and difficult, therefore the best
method to protect the environment from contamination is to prevent it. Nevertheless, it is not
always possible and once metals are introduced and pollute the soil, they will remain there.
Unlike carbon-based organic pollutants, heavy metals cannot be degraded or eliminated
completely, therefore the traditional treatments for heavy metal pollution of soils are compli‐
cated and cost-intensive.
There are several technologies for remediation of heavy metal-polluted soils. One of the
classifications divides the methods into in situ and ex situ treatment technologies. In situ (in
place) means that the polluted soil is treated in its original location, i.e. it remains at the site or
in the subsurface. Such technologies remove the pollutant from soil without excavation or
removal of the soil. In this case fixing agents are applied on the unexcavated soil. This
technique’s advantages may include low invasiveness, simplicity and rapidity. Moreover, it
is fairly inexpensive and generates relatively low amount of waste. However, it is only a
temporary solution. This is due to the fact that when physicochemical properties of soil change,
the pollutants may again become active. Moreover, the reclamation process is applied only to
the surface layer of soil [75]. Ex situ means that the treated soil is removed or excavated from
the site [52]. It is applied in areas where heavily polluted soil must be removed from its place
of origin and its storage is associated with high ecological risk. Fast and easy applicability,
relatively low costs of investment and operation are the advantages of this method. On the
other hand, it is highly invasive to the environment, generates a significant amount of solid
wastes, and it is necessary to control the stored waste permanently. Evanko and Dzombak [76]
divide in situ remediation strategies into solidification/stabilization, vitrification, soil flushing,
electrokinetic extraction and biological treatment. Ex situ treatment technologies are divided
by these authors into: solidification/stabilization, soil washing, vitrification and pyrometal‐
lurgical separation. Another classification of remedial strategies divides the technologies
under five categories of general approaches to remediation: isolation, immobilization, toxicity
reduction, physical separation, and extraction. There are several physicochemical techniques
that include excavation and burial of soil at a hazardous waste site, chemical processing of soil
to immobilize metals, leaching by using acid solutions or appropriate leachants to desorb
metals from soil followed by the return of clean soil to the site [77], precipitation or flocculation
followed by sedimentation, ion exchange, reverse osmosis and microfiltration [78]. Neverthe‐
less, physicochemical techniques for heavy metal remediation are generally costly and have
side effects [37]. Therefore, continuous efforts have been made to develop techniques that are
easy to use, sustainable and economically feasible.
6. The use of plants for biological remediation of heavy metal polluted soils
Phytoremediation is one of the best techniques for treatment of heavy metal-polluted sites. It
is an in situ strategy that uses vegetation and associated microbiota together with agronomic
practices to aid in metal remediation [79]. It is based on the use of special type of plants to
decontaminate soil by inactivating metals in the rhizosphere or translocating them in the aerial
parts [56]. Some plants developed mechanisms to remove ions selectively from the soil to
regulate the uptake and distribution of metals. Potentially useful phytoremediation technol‐
ogies for heavy metal-polluted sites include phytoextraction, phytostabilization and rhizofil‐
tration [75].
Phytoextraction uses hyperaccumulating plants to remove metals from soil by absorption into
the roots and shoots of the plant. The aboveground shoots can be then harvested to remove
metals from the site and subsequently stored as hazardous waste or employed for the recovery
of metals. The ideal plant for phytoextraction should grow rapidly, produce high amount of
biomass and be able to tolerate and accumulate high metal concentrations in shoots [80].
Hyperaccumulating plants belong to the families of Brassicaceae, Fabaceae, Euphorbiaceae,
Asterraceae, Lamiaceae, and Scrophulariaceae [77]. Studies indicate that many Brassica species,
such as B. juncea, B. napus or B. rapa exhibit enhanced accumulation of Zn and Cd [81] In
comparison to conventional methods like e.g. soil excavation (ex situ remediation), phytoex‐
traction is time consuming, but on the other hand it is cost-effective and less labor-intensive [9].
Phytostabilization is based on the use of plants to limit the mobility and bioavailability of
metals in soil. Plants used in this method are characterized by high tolerance of metals in
surrounding soils together with their low accumulation. Phytostabilization can be carried out
through the process of sorption, precipitation, complexation, or metal valence reduction. This
technique is useful for the removal of Pb, As, Cd, Cr, Cu, and Zn [82]. This process is advan‐
tageous because in this case disposal of hazardous material/biomass is not required, and it is
very effective when rapid immobilization is needed to preserve soils or ground and surface
waters [82].
Rhizofiltration (or phytofiltration) removes metals from contaminated soil via absorption,
concentration and precipitation by plant roots. This technique is used to remove pollutants
from groundwater and aqueous-waste streams rather than for the remediation of polluted soils
[76]. Apart from the above described phytoremediation methods, some authors [83] include
also phytovolatization and phytodegradation.
Phytovolatization involves the use of plants to volatilize pollutants from their foliage such as
Se and Hg, while phytodegradation uses plants and associated microorganisms to degrade
organic pollutants. Even though phytoremediation strategies are inexpensive, effective,

environmentally friendly and can be implemented in situ, a substantial proportion of metal
pollutants are unavailable for root uptake by field grown plants [84]. Therefore, methods of
increasing phytoavailability of heavy metal pollutants in soil and their transport to plant roots
are vital to the success of in situ phytoremediation. In this case it is useful to apply microbial
populations that are able to affect trace metal mobility and availability to plants, through the
release of chelators, acidification and redox changes [85]. It was proved that the presence of
rhizosphere bacteria increases the available concentrations of various heavy metals to hyper‐
accumulative plants [80]. Microbial populations may be used not only for increasing metal
bioavailability to plants, but also for the promotion of hyperaccumulative plant growth
through N2 fixation, production of phytohormones and siderophores, and transformation of
nutrients [26]. Figure 2 summarizes the mechanisms of plant-mediated remediation of
contaminated soils.
Figure 2. Mechanisms of phytoremediation involved in purifying contaminated soils and physiological processes that
occur in plants during phytoremediation.
7. Application of microorganisms to remediate heavy metal-polluted soils
Another approach for biological remediation of heavy metal-polluted soils includes the use of
microorganisms to detoxify metals by valence transformation, extracellular chemical precipi‐
tation or volatilization etc. [56]. Bioleaching is the method that uses microorganisms to
solubilize heavy metal pollutants either by direct bacterial processes, or as a result of interac‐
tions with metabolic products, or both [76]. It can be used in situ or ex situ to help to remove
the pollutants from soils. This process is based on mobilization of metal cations from insoluble
ores by biological oxidation and complexation. This process was adapted from mining
industry for the use in soil remediation and a general term covering both bioleaching and
biooxidation techniques could be “biomining”. This technique is mainly employed for copper,
cobalt, nickel, zinc and uranium, which are extracted either from insoluble sulfides or (in the
case of uranium) from oxides [86]. The classical bioleaching bacteria belong to the genus
Acidithiobacillus (A. thiooxidans and A. ferrooxidans), Acidiphilium, Acidimicrobium, Ferromicrobi‐
um or Sulfobacillus [86].
Another solution for soil bioremediation using microorganisms is to apply microbially-
mediated biochemical processes, such as oxidation/reduction or methylation reactions [87].
Often, biostimulation and bioaugmentation are the components of bioremediation strategies.
Biostimulation is a form of in situ bioremediation which uses growth rate stimulation nutrients,
electron donors or acceptors to encourage the growth of site-specific indigenous microorgan‐
isms capable of degrading environmental pollutants. Common electron donors and acceptors
used in biostimulation include: acetate, sulfate, nitrate and ethanol [88]. Bioaugmentation is
the introduction of specific competent microorganisms to the local microbial population in
order to increase the metabolic capacities needed for remediation [89]. Biosorption is a
physicochemical process that occurs naturally and allows to passively concentrate and bind
contaminants onto the microbial cell structure [90]. Metal biosorption by living organisms is
a complicated process that consists of two steps. In the first step, metal ions are adsorbed on
the cell surface by interactions between metals and cell surface functional groups. Biosorption
of metal ions occurs primarily on the outer surface of microbial cells and is the first step in the
interactions between metals and microbial cell walls [4]. The cell wall consists of a variety of
polysaccharides and proteins, and hence offers a number of active sites capable of binding
metal ions [91]. Differences in the cell wall composition among various microbial groups, i.e.
algae, bacteria, cyanobacteria and fungi, cause significant differences in the type and amount
of metal ions binding to them [91]. Physical adsorption via electrostatic or van der Waals forces
allow to retain metal ions on the outer surfaces of bacterial cells. In addition to physical
adsorption, ion exchange and complexation are believed to be the dominant mechanisms
involved in metal biosorption [4]. The first step, passive biosorption, is metabolism-independ‐
ent and proceeds rapidly by any one or a combination of metal binding mechanisms. In the
second step, due to active biosorption, metal ions penetrate the cell membrane and enter into
the cells. This is, however, a slowly occurring process. Active mode is metabolism-dependent
and related to metal transport and deposition [91]. There are several microbial genera and
species capable of metal biosorption. Fungi were found to be efficient biosorbent organisms,
as their cells are characterized by a high percentage of cell wall material, which shows excellent
metal binding properties [92]. Aureobasidium pullulans, Cladosporium resinae, Aspergillus niger,
Aspergillus versicolor or Rhizopus nigricans are the fungal species proved to be effective in heavy
metal biosorption [91]. Numerous studies also identified several species of bacteria as efficient
metal accumulating microorganisms. For instance, Bacillus spp. has been reported to have a
high potential of metal sequestration and has been used in commercial biosorbent preparation
[91]. Other bacterial species capable of metal transformation include, among others: Escherichia
coli, Pseudomonas maltophilia, Shewanella putrefaciens, Pseudomonas aeruginosa, Enterobacter
cloacae [4].
Mechanisms involved in biochemical interactions between bacteria and metal ions involve
specific enzymes that catalyze the oxidation, reduction, methylation, dealkylation and
precipitation reactions. Microorganisms transform a substantial number of metals and
metalloids by reducing or oxidizing them directly to a lower or higher redox state. Addition‐
ally, indirect oxidation or reduction is an alternative for immobilization of toxic metals in the
environment. Methylation is an important process involved in geochemical cycling of metals
and the removal of metal pollutants from soils. Methylation processes derive the methyl group
from methylocarbolamine (CH3B12) which is implicated in the methylation of multiple metals
and metalloids, such as Pb, Sn, Pd, Pt, Au, Ti, As, Se and Te [93]. Methylation of Hg, Sn and
Pb can be mediated by a range of microbes, including Clostridium spp., methanogens and
sulfate-reducing bacteria under anaerobic conditions and principally by fungi (e.g. Penicilli‐
um spp. and Alternaria spp.) under aerobic conditions. Methyl groups are enzymatically
transferred to metals and a given species may transform a number of different metals [94].
Methyl-metal compounds are generally highly volatile and available to plants [50]. Another
mechanism that has the potential for the application in heavy metal-polluted sites is the
production of siderophores by different microbial genera. Siderophores are the largest class
of compounds that can bind and transport Fe. They are highly specific Fe(III) ligands and are
excreted by a wide variety of fungi and bacteria to aid Fe assimilation [94].
Microorganisms play an important role in the environmental biogeochemical cycle of metals

and their properties are of significant interest in the remediation of contaminated sites. The
microbial ability to absorb and transform metals is a promising aspect in respect of solving the
pollution problems [4]. The potential of numerous microbial metal transformations in treat‐
ment of environmental pollution may be employed and some processes are already in
commercial operation. However, many processes are still at the laboratory scale and yet to be
tested in a rigorous applied and/or commercial context [94]. Another interesting aspect of the
microbial community is their ability to multiply even under undesirable environmental
conditions. These microorganisms sometimes affect soil environment more quickly than
abiotic processes can. Therefore, the structure of soil microbial populations may be useful as
a highly sensitive bioindicator of soil disturbance and progress of remediation [95].
Facing the increasing heavy metal pollution severity accompanied by rising land prices the
communities around the world need to struggle for available investment grounds. This is
mostly the problem of big cities, especially those with limited opportunities for development
due to geographical barriers such as seashores, mountain ranges or desert areas. In such
situations the polluted industrial areas cannot be left unused for long time to recover naturally.
This creates a need for the development of various remedial procedures adjusted to changing
contamination level, environmental conditions, available time and funding. Thus, remedial
measures need to be almost always modified in order to meet those criteria. This makes that
the continuous effort should be made to increase the effectiveness, flexibility and decrease the
cost and side effects of the procedures available today. Although a number of measures was
developed to remove the even toxic level of contamination, there are many degenerated areas
that still cannot be successfully treated now. Those cases involve sites where remediation
would be too expensive, time consuming or even technically disputable with currently
available treatment procedures.
8. Conclusion
Heavy metals pose a significant threat towards the soil environment and the rapid industri‐
alization will result in increasing problems of environmental pollution. Therefore, it is
necessary to carry out the continuous monitoring of both industrial areas and their vicinities
for possible transgressions of the limits given by the authorities. When necessary, the remedial
measures should be applied as soon as possible by all available means. On the other hand,
research should be promoted to understand the mechanisms of microbial response to heavy
metal pollution and to enable screening for possible resistant microorganisms that could be
used for both remediation and restoration of soil environment fertility.
Author details
Anna Lenart-Boroń1 and Piotr Boroń2
1 Department of Microbiology, Faculty of Agriculture and Economics, University of

Agriculture in Cracow, Cracow, Poland
2 Department of Forest Pathology, Faculty of Forestry, University of Agriculture, Cracow,

Poland
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chapter 27
Pollution of Pb in Soils Affected by Pyrite Tailings:

Influence of Soil Properties
F. Martín, M. Simón, I. García, A. Romero and

V. González
1. Introduction
Pb is considered one of the most common contaminant in the environment [1] and it is
considered neither essential nor beneficial for plants or animals but highly toxic. Pb is one of
the less mobile heavy metals so its compounds tend to accumulate in soils and sediments, and
it is slightly affected by microbial degradation [2-4]. It has a strong affinity by the sulphur, so
is frequently associated to sulphides, being the galena (PbS) the most frequent ore mineral. Pb
has two stable oxidation states: Pb(II) and Pb(IV), but the most dominant species in nature is
the plumbous one. Geochemically, Pb2+ can replace isomorphously K+ in silicate lattices or
Ca2+ in carbonates and apatites, due to the similar ionic radius with these both elements [2].
In crustal rocks, the average Pb content is approximately 16 mg kg-1 [5]. In igneous rocks trend
to increase from ultrabasic (gabbro 1.9) to acid (granite 22.7), meanwhile shales and mudstones
have an average Pb content of 23 mg kg-1 and sandstones 10 mg kg-1, being limestones and
dolomites the rocks with higher average Pb content (71 mg kg-1). The average concentration
in uncontaminated soils is estimated to be 40 mg kg-1, ranging from 2 to 200 mg kg-1 [6, 7].
On 25 April 1998, a breach in the dam of the holding pond of tailings from a pyrite mine in
Aznalcóllar (S Spain) spilled around 45 x 105 m3 of acidic waters and toxic tailings containing
high concentrations of Zn, Cu, Cd, As, Pb, Sb, Bi and Tl into the Agrio and Guadiamar river
basins, affecting around 43 km2 [8]. The soils were polluted in two stages [9]. First, the toxic
water (liquid phase) and tailings (solid phase) covered the soils and penetrated in depth
through macro and micropores; the acidic water had far lower heavy-metal content (Pb
concentration ranging from 2.05 to 2.60 mg dm-3) than the tailings had (Pb concentration
ranging from 4352.3 to 9635.9 mg kg-1). In the second stage, the oxidation of the pyrite tailings
2 Soil Pollution
causes sulphides to oxidize to sulphates, a marked fall in pH and a solubilization of heavy

metals [10]. The lowest pH values were reached 25 days after the spill, and the total Pb
concentration at 88 days doubled or tripled (depending the soil type) the initial value of
contamination in the upper 10 cm of the soils [11].
The present study provides an evaluation of the contamination processes occurring just after
the removal of the tailings and part of the upper soil, to assess the behaviour of the residual
Pb concentrations in the affected area at this early stage. The aim of the present work is to
analyse the mobility and availability of Pb in the initial steps of the contamination process, to
obtain useful information in the planning of different strategies for future remediation
activities in other affected areas based on a detailed study of the soil properties.
2. Material and methods
After the removal of the tailings, the basin was gridded into 91 sectors of 400 x 400 m, and in
the centre of each sector a plot 10 x 10 m was laid out. In every plot, soils samples were taken
at the four corners and centre of this plot, at depths of 0-10, 10-30 and 30-50 cm. For each depth,
250 g of soil from the five sampling points per plot were mixed and homogenized, providing
three samples per plot, for a total of 273 soil samples for the entire basin. All the samples were
air dried and screened to 2 mm, and the percentages of gravels (>2mm) and fine earth (<2mm)
were determined. The laboratory analyses were made with the fine-earth fraction.
Particle-size distribution was measured by the pipette method after eliminating organic matter
with H2O2 and dispersion with sodium hexametaphosphate [12]. The pH was measured
potentiometrically in a 1:2.5 soil:water suspension, as recommended by the International
Society of Soil Science, in a CRISON Digit 501 instrument. The CaCO3 equivalent was deter‐
mined manometrically by the Barahona method [13]. Total carbon and total sulphur were
measured by dry combustion with a LECO mod. SC-144DR instrument. Organic carbon was
calculated as the difference between total carbon and inorganic carbon from CaCO3. The cation-
exchange capacity (CEC) was determined with 1N Na-acetate at pH 8.2 [14], measuring the
sodium in a METEOR NAK-II flame-photometer. The total concentration of iron (Fet) was
measured by X-ray fluorescence in a Philips PW-1404 instrument, from a disc of soil and
lithium tetraborate in a ratio of 0.6:5.5. Amorphous and poorly crystallized iron (Feo) and
aluminium (Alo) oxides were extracted with oxalic-oxalate [15] and measured by atomic-
absorption spectroscopy.
Samples of air-dried soils, ground to < 0.05 mm, were digested in open flasks by strong acids
(HNO3:HF in a ratio 2:3) at 160 ºC till dry, and 100 ml HNO3 at 4% were added. In the digested
samples, total Pb (Pbt) was measured by ICP-MS with a PE SCIEX-ELAN 5000A spectrometer.
A multi-element calibration standard 4 (Perkin-Elmer) was used with Rh as the internal
standard. The detection limit for Pb was 0.001 μg L-1, and the accuracy of the method was
corroborated by analyses (six replicates) of a standard reference material: SRM 2711 (soil with
moderately elevated trace-element concentrations [16]). For Pb, the mean certified value was
Pollution of Pb in Soils Affected by Pyrite Tailings: Influence of Soil Properties 3
1162.0 mg kg-1 with a standard deviation of 31.0; the mean experimental value was 1138.1 mg
kg-1 with a standard deviation of 11.0.
Soluble Pb (Pblw) was measured in the extract from a saturated soil paste. Pb extracted by
calcium chloride 0.01 M (Pbc) using the method [17], Pb extractable by acetic acid 0.43 M (Pba)
following [18], and Pb extractable by oxalic-oxalate pH 3 (Pbo) as in [15]. For the EDTA 0.05 M
extractable fraction (Pbe) method [19] were followed. The Pb of all the extracts was measured
by ICP-MS.
For the statistical analysis, the SPSS 15.0 program was used. The areal distribution of the
contamination was established by the algorithm of the least Euclidean, using the software
Surfer 7.0.
The climate of this area is typically Mediterranean (hot, dry summers; cold, wet winters;
temperate autumns and springs with variable rainfall). The mean annual rainfall of 613 mm,
the mean temperature of 17.7 °C, and the potential evapotranspiration of 900 mm (taken from
7 weather stations near the spill, over 27 years). The affected soils were Typic Xerofluvents and
Typic Xerothents [20] developed on alluvial deposits coming from the erosion of sedimentary
and metamorphic materials of the Agrio and Guadiamar River basin, mainly gravels, sands,
silts and clays from the Miocene and Plio-Quaternary.
We assigned five different soil types, according to the soil properties between 0 and 50 cm in
depth, with significant differences (p < 0.05) by a cluster analysis via the k-mean method (Table
1). Soil types 1 and 2 (hereafter Group A), located in the upper part of the basin, were acidic,
with little or no carbonate; type 1 had a loam texture while type 2 was dominated by gravel
and sand. Soil types 3, 4 and 5 (hereafter Group B) were predominantly neutral or slightly
alkaline and carbonated, the main differences between them being texture (type 3, clay loam;
type 4, loam; and type 5, silty clay).
Soil Gravel Sand Silt Clay CaCO3 OC CEC

n pH
type (%) (%) (%) (%) (%) (%) (cmolc kg-1)
1 36 5.2 (3.5) 50.7 (18.4) 30.8 (14.1) 18.5 (6.9) 5.8 (1.9) 1.7 (3.5) 1.0 (0.4) 15.5 (5.8)
2 33 37.2 (24.5) 76.2 (17.1) 13.6 (9.3) 10.2 (8.6) 5.9 (1.8) 0.8 (1.7) 0.4 (0.3) 7.7 (3.7)
3 63 6.9 (9.4) 19.6 (11.2) 49.4 (9.7) 31.0 (11.2) 7.7 (0.5) 10.0 (5.0) 1.1 (0.5) 18.1 (5.2)
4 87 4.8 (8.5) 48.8 (12.6) 32.5 (9.4) 18.7 (6.0) 7.8 (0.7) 6.9 (6.3) 0.9 (0.4) 12.1 (4.2)
5 54 8.2 (9.6) 4.9 (7.1) 47.7 (11.2) 47.4 (14.0) 8.0 (0.3) 13.6 (4.9) 1.3 (0.5) 22.9 (4.9)
Table 1. Mean and (standard deviation) of the particle size, pH, CaCO3, organic-carbon (OC) content, and cation
exchange capacity (CEC) in the different soil types (n = number of samples in each soil type).
4 Soil Pollution
Figure 1. Location of the study area, including the distribution of the different soil types analysed in Table 1. Group A
include non-carbonate soils and Group B consist of carbonate soils.
As mentioned above, these soils were quite homogeneous in depth, so the variation in the soil
properties with the depth were not significant in the cases of texture, calcium-carbonate
content, organic-carbon content and cation-exchange capacity. Nevertheless, total sulphur
content (St), related to the contamination, registered its highest values in the upper 10 cm in
all soil types, with values declining progressively and significantly (p < 0.001) in depth (Fig.
1). The difference in St between the first 10 cm and the interval 10-30 cm was greater in Group
A soils than in Group B. In addition, the contamination also affected the pH increasing the
acidity towards the soil surface, and with significant differences in depth. In the Group B soils,
where differences between depths were less pronounced for the presence of CaCO3, the pH
remained higher, rarely falling below 6.0 (Fig. 1).
St (%) pH
0 2 4 6 8 10 12 0 1 2 3 4 5 6 7 8 9
0 - 10 0 - 10
Group A soils Group A soils

10 - 30 10 - 30
30 - 50
Depth (cm)
30 - 50
0 2 4 6 8 10 12 0 1 2 3 4 5 6 7 8 9
0 - 10 0 - 10
Group B soils Group B soils

10 - 30 10 - 30
30 - 50 30 - 50
Figurecontent
Figure 2. Total sulphur 2. Total(Ssulphur content (St) and pH in the two soil groups (horizontal lines
t) and pH in the two soil groups (horizontal lines range from minimum to maximum
range from minimum to maximum values; boxes show the confidence interval at
values; boxes show the confidence interval at 95% for the mean; vertical lines mark the mean values).
95% for the mean; vertical lines mark the mean values).
Total Pb content
Total Pb content
In uncontaminated soils next to the affected area the concentrations of the total Pb (Pb t) were
In uncontaminated soils next to the affected area the concentrations of the total Pb
between 22.0
(Pbt) and
were74.6 mg kg
between 22.0, with
-1
a mean
and 74.6 mg value of 41.8
kg-1, with a meanmg value
kg-1 [9]. Meanwhile,
of 41.8 in contami‐
mg kg-1 [9].
nated soils, the-1mean
Meanwhile, Pb content soils,
in contaminated in thetheuppermost 10
mean Pb content cmin was 385.8 mg
the uppermost 10kg
cm , ranging
-1 was 385.8
between
mg kg , ranging-1between 35.8 and 3231.0 mg kg-1. This mean value significantly declined
35.8 and 3231.0 mg kg . This mean value significantly declined with depth
with depth in all soil types (Fig. 2), with a distribution similar to that of St (Fig.1).
in all soil types
(Fig. 2), with a distribution
Consequently, similar
in all soils, to that
a highly of St linear
significant (Fig.1). Consequently,
relationship in was
(p < 0.001) all soils,
found a highly
significantbetween
linearPb t and St (Table 2).
relationship (p < 0.001) was found between Pbt and St (Table 2).
Pbt
variable value Pbt
std. Error P value
2
r = 0.832 < 0.001
variable value std. Error P value
Intercept 27.58 10.95 0.012
St 22.45
r2 = 0.832 0.65 < 0.001 < 0.001
Intercept 27.58 model of the Pbt (mg kg ) and St (g kg ) in all soils.

Table 2. Multivariable regression 10.95
-1 0.012
-1
St 22.45 0.65 < 0.001
Table 2. Multivariable regression model of the Pbt (mg kg-1) and St (g kg-1) in all soils.
In addition, the Pbt concentration in the 10-30 cm layer did not significantly differ from that of
the 30-50 cm layer, and had values higher than unaffected soils (doubled or tripled the mean
value for uncontaminated soils), which could be associated with a previous contamination in
the area [21].
6 Soil Pollution
In addition, the Pbt concentration in the 10-30 cm layer did not significantly differ
from that of the 30-50 cm layer, and had values higher than unaffected soils (doubled or
tripled the mean value for uncontaminated soils), which could be associated with a previous
contamination in the area [21].
Pbt (mg kg-1)

0 500 1000 1500 2000 2500 3000 3500
0 - 10
Group A soils
10 - 30
Depth (cm)
30 - 50
0 500 1000 1500 2000 2500 3000 3500
0 - 10
Group B soils
10 - 30
30 - 50
Figure 3. Total Pb content (Pbt) in the two soil (horizontal lines range from
Figure 3. Total Pbminimum
content (Pbto maximum values; boxes show the confidence interval at 95% for
t) in the two soil (horizontal lines range from minimum to maximum values; boxes show
the mean;
the confidence interval at 95%vertical lines vertical
for the mean; mark the mean
lines mark values).
the mean values).
In any case, the pH range as well as the St and Pbt values in the upper 10 cm of the soils were
In any case,
very broad, indicating the pH
a highly heterogeneous as the St and Pbas
range as well contamination, t values in the upper 10 cm of
previously reported in [9].
the soils were very broad, indicating a highly heterogeneous contamination, as previously
To quantify the degree of Pb
reported in [9]. To quantifyt pollution,
the degree of Pbt pollution, we usedlevels
we used the intervention proposed by
the intervention the
levels
Andalusian Regional
proposed Government
by the Andalusian (350
Regionaland 500 mg kg-1 for
Government (350agricultural
and 500 mg soils
kg-1with pH < 7 and
for agricultural
-1
> 7, respectively;
soils with pHand< 1000
7 andmg> 7,kg-1
for natural
respectively; parks).
and 1000 mgThekgsoilforuse in theparks).
natural area was
The mainly
soil use for
in
the area was mainly for agriculture and grazing and, according to these levels, 15.0% of the
agriculture and grazing and, according to these levels, 15.0% of the area proved to be uncon‐
area proved to be uncontaminated, with values below the reference level for -1 the area (< 75
taminated,
mg with
kg ), values
-1
23.7% below the referenceforlevel
was contaminated for the area
agricultural use, (<
and75 10.6%
mg kgwas ), 23.7% was
seriously
contaminated for agricultural
contaminated (> 1000 use,
mg kgand -1
). 10.6% was seriously
The remaining 50.7%contaminated
had anomalous (> values
1000 mg kg-1). the
(above The
remaining 50.7% level
reference had anomalous
and below thevalues (abovelevels).
intervention the reference level and below the intervention
levels). Pb extractions
Pb extractions
To assess the speciation of Pb in the polluted soils, selective extractions using reagents with
different strengths were used, although most of them are not specific to a given soil phase [22],
they provide useful information in relation to mobility and availability of the pollutant
element. The average concentration and standard deviation of the different extracted forms of
Pb are shown in Table 3.
In general, water extracted forms constitute the soluble fraction [23, 24]; forms extracted with
CaCl2 is considered the exchangeable fraction [25, 26]; forms extracted with acetic acid is
Soil Depth Pbe Pba Pbo Pbc Pbw

type (cm) Mean SD Mean SD Mean SD Mean SD Mean SD
1 0-10 149.29 155.50 41.16 49.52 17.92 28.14 4.00 5.94 0.32 0.24
10-30 29.74 54.56 21.30 48.65 7.21 8.83 0.86 2.72 0.30 0.57
30-50 13.39 17.94 7.80 10.59 4.12 5.47 0.70 1.92 0.40 0.63
2 0-10 117.96 137.29 43.48 63.77 5.61 12.60 7.74 15.42 0.39 0.35
10-30 25.61 22.38 9.21 9.89 4.35 6.12 1.31 1.52 0.29 0.33
30-50 19.58 28.56 18.79 32.71 4.61 4.13 1.47 3.69 0.21 0.20
3 0-10 83.00 68.52 31.59 28.93 5.36 23.32 0.49 0.49 0.58 0.98
10-30 31.79 22.31 9.88 6.66 0.79 1.63 0.20 0.36 0.45 0.78
30-50 29.53 20.67 8.66 9.79 1.49 5.36 0.73 1.27 0.69 1.49
4 0-10 55.21 68.16 20.29 38.82 2.11 3.23 1.61 5.46 0.74 1.32
10-30 37.39 69.96 8.87 14.87 4.55 9.14 1.18 3.46 0.65 1.45
30-50 26.55 27.98 7.11 9.91 4.01 8.41 0.48 0.60 1.10 2.05
5 0-10 37.86 35.56 11.56 13.09 5.65 13.49 0.80 0.76 0.17 0.50
10-30 20.58 13.32 4.61 2.35 3.41 5.80 0.89 0.99 0.11 0.11
30-50 15.94 14.61 4.20 3.21 2.21 4.47 0.83 0.80 0.14 0.20
Table 3. Mean content (mg kg-1) and standard deviation (SD) in the five soil types for Pb extracted with EDTA (Pbe),
acetic acid (Pba), oxalic-oxalate (Pbo), calcium chloride (Pbc) and water (Pbw).
used as the bio-available fraction associated with carbonates and also specifically adsorbed
by oxides as well as organic matter [25, 27-29]; forms extracted with EDTA is also consid‐
ered to be the bio-available fraction associated with carbonates, inorganic precipitates,
amorphous oxides and organic matter [24, 27-30]; and the forms extracted with oxalic oxalate
is held on, or occluded in, amorphous or poorly crystallized iron, aluminium or manga‐
nese oxides [22, 31].
The reagent that extracted the highest concentrations of Pb was EDTA (around 20 - 25 % of the
total Pb), indicating that a very high proportion of the Pb forms inorganic precipitates easily
complexed by this reagent, but not dissolved by acetic acid or oxalic-oxalate. When all soils
were pooled, the Pb extracted with EDTA (Pbe) was related to total Pb (Pbt), sulphates (SO42-),
and amorphous iron oxides (Feo) with highly significant (p < 0.001) relationship (Table 4).
Therefore, apart from the relation with the total contamination (Pbt), we can suppose that the
main forms in which Pb is retained in the soils could be iron oxy-hydroxysulphates. The
presence of iron compounds promote the sorption of Pb in the soil [32]; and these results are
consistent with the obtained by [33] in alluvium contaminated by metal in an adjacent mining
area (Tinto river basin). These authors pointed that the highest amounts of Pb of all contami‐
nant metal-bearing minerals in the area were characterised by Fe oxy-hydroxysulphate
minerals.
8 Soil Pollution
Pbe
value std. Error P value VIFa
All soils r2 = 0.726 < 0.001
Pbt 0.121 0.008 < 0.001 1.689
SO4 2-
0.014 0.002 < 0.001 1.552
Feo 0.003 0.001 0.019 1.736
Group A soils Pbe
r2 = 0.867 < 0.001
Pbt 0.207 0.017 < 0.001 1.966
SO4 2-
0.008 0.003 0.004 1.966
Group B soils Pbe
r2 = 0.651 < 0.001
Pbt 0.100 0.008 < 0.001 1.416
Feo 0.006 0.001 < 0.001 1.416
a
The variance inflation factor (VIF) is the reciprocal of tolerance. Values next to 1 indicate the independent variables have
no redundant information.
Table 4. Multivariable Regression Model of the Pbe (mg kg-1), Pbt (mg kg-1), SO42- (mg kg-1) and Feo (g kg-1) in the
different soil groups.
Nevertheless, when the soils were separated into acidic (Group A) and neutral alkaline (Group
B), the former correlated significantly Pbe with Pbt and SO42, meanwhile the latter correlated
Pbe with Pbt and Feo (Table 4), indicating that the soil properties of each group are essential in
the forms of Pb retention. In the case of acid soils, the relationship between Pbe and SO42- is
significant, but the one with the Feo disappear. In these soils, the pH is maintained in acidic
values, so the iron released from the pyrite oxidation (Fe2+) is mobile, being able to rise and
accumulate in the soil surface by the effect of the semi-arid climate of the area. This process
occurs under Mediterranean climatic conditions, where the precipitation are lower than the
evapotranspiration and the predominant upward movement of the soil solutions generate the
formation of hard surface crusts, of scarce mm thickness and high iron concentration, observed
by us in the acid soils of the affected area [34]. According to this behaviour, the Pb retention
in acid soils seems to be related mainly to the precipitation as Pb sulphate (PbSO4); this
retention has been already reported as the main process in the Pb immobilization from acid
mine waters [35].
In the case of basic soils, the relationship between Pbe and Feo is statistically significant, but
the one with the SO42- disappears. In these soils, the presence of calcium carbonate pro‐
duce the releasing of Ca2+ by the acidification caused by the oxidation of sulphides, and the
precipitation of gypsum (CaSO4 2H2O) is dominant. These reactions could be responsible in
the disappearance of the statistical relation between Pbe and sulphates, but solubility of
gypsum allows to maintain certain amounts of soluble sulphates (1.51 10-2 M, at 25 ºC), so
the formation of other types of sulphates is possible, being the presence of jarosite [KFe3(SO4)2
(OH)6] common in this type of contamination [36]. In addition, the basic values of the pH
encourage the precipitation of iron oxy-hydroxydes, but the Pb retention by poorly crystal‐
lized iron oxides in our basic soils, seems to be less important than the one produced by iron
oxy-hydroxysulphates [37]. In fact, the concentration of Pb extracted by oxalic-oxalate (Pbo)
is very low (between 1 and 3% of the total Pb). According to these results, we suppose that
the main Pb-bearing mineral in basic soils should be plumbojarosite [PbFe6(SO4)4 (OH)12].
The formation of this mineral has also been described in others sulphide mine contamina‐
tion of the area [33, 38].
The Pb extracted with calcium chloride (Pbc) in Group A soils was significantly related only
to the percentage of clay fraction (Table 5). This implies that, in acid soils, a little fraction of Pb
is retained by the clay fraction as easily exchangeable forms. On the contrary, in Group B soils,
the Pbc was nor significantly related to any of the properties studied, but only to the Pb soluble
in water (Pbw). In addition, if we compare the amounts of Pbc to the amounts of Pbw in basic
soils, we note that there are no statistical differences between the two forms.
Pbc
Group A soils value std. Error P value
r2 = 0.836 0.011
Intercept 0.807 0.066 0.029
clay 0.058 0.013 0.011
Pbw
Group B soils
r2 = 0.871 < 0.001
Intercept 1.304 0.122 < 0.001
clay - 0.024 0.004 < 0.001
Table 5. Multivariable Regression Model of the Pbc (mg kg-1) and clay (%) in acid soils (Group A); and Pbw (mg kg-1)
and clay (%) basic soils (Group B).
Soluble in water Pb (Pbw) was not significantly related to any of the soil properties in Group
A soils, while in Group B soils, Pbw proved to be negatively and significantly related to the
clay fraction (Table 5), implying the retention of the soluble Pb forms by this soil fraction. This
decrease in availability of Pb in relation to clay content is in agreement with the described by
[39], although the type of clay should be studied in more detail [40]; in this way, high illite
content has been described as an efficient mineral in the adsorption of Pb in soils [41]. Finally,
we found that the concentrations of Pbw was generally higher in basic soils in relation to the
acid ones (Fig. 3), being this difference between groups statistically significant (p < 0.05). This
finding contradicts the usual increase in Pb solubility in acidic conditions [42, 43]. Therefore,
10 Soil Pollution
this variation could be related to the apparent solubility and stability of the iron oxy-hydrox‐
ides precipitated at different values of pH [44, 45], but more detailed studies are carrying out
to verify this point.
Pbw (mg kg-1)

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8
Group A
soils
Group B
soils
Figure 4. Soluble Pb content (Pbw) in the two Group of soils (A: acid, B: basic). (boxes show
the confidence interval at 95% for the mean; vertical lines mark the mean values).
Figure 4. Soluble Pb content (Pbw) in the two Group of soils (A: acid, B: basic). (boxes show the confidence interval at
95% for the mean; vertical lines mark the mean values).
CONCLUSIONS
The mobility and solubility of Pb in the contaminated soils of this study is mainly
controlled by the soil properties and ageing (oxidation) of the pyrite tailings. Regardless of
the soil type, the Pb accumulates mainly in the top of the soils without contaminating the
4. Conclusionssubsoil or groundwater. The climatic conditions (semi-arid Mediterranean climate) produce
the upward movement of the soil solutions, which cause the formation of surface crusts but
only in acidic soils (where the iron and contaminants remain soluble in higher proportion).
The mobility andThesolubility of Pb in the contaminated soils of this study is mainly controlled
main Pb-bearing mineral in acid soils should be Pb-sulphates, and in basic ones should
be iron hydroxysulphates (mainly plumbojarosite). The amounts of Pb retained by iron
by the soil properties and ageing
oxyhydroxides (oxidation)
are very little in allof
soilthe pyrite
types. The tailings.
soluble PbRegardless ofhigher
is significantly the soil type,
in basic
the Pb accumulates mainly in the top of the soils without contaminating the subsoil
soils than in acid ones, which contradict the normal behaviour of this contaminant in soils.
According to the obtained results, more detailed studies in relation to the solubility and
or
groundwater. Thestability
climatic conditions
of the (semi-arid
different phases Mediterranean
precipitated at the earlyclimate)
stages ofproduce the upward
the contamination are
movement of the required,
soil solutions, which cause the formation of surface crusts but only in acidic
to improve the applications in the future of adequate remediation measurements in
similar affected areas.
soils (where the iron and contaminants remain soluble in higher proportion). The main Pb-
bearing mineral in acid soils should be Pb-sulphates, and in basic ones should be iron hydrox‐
ysulphates (mainly plumbojarosite). The amounts of Pb retained by iron oxyhydroxides are
very little in all soil types. The soluble Pb is significantly higher in basic soils than in acid ones,
which contradict the normal behaviour of this contaminant in soils. According to the obtained
results, more detailed studies in relation to the solubility and stability of the different phases
precipitated at the early stages of the contamination are required, to improve the applications
in the future of adequate remediation measurements in similar affected areas.
Author details
F. Martín1, M. Simón2, I. García2, A. Romero1 and V. González2
1 Departamento de Edafología y Química Agrícola, Facultad de Ciencias, Universidad de

Granada, Granada, Spain
2 Departamento de Agronomía, Área de Edafología y Química Agrícola, Universidad de Al‐

mería, Almería, Spain
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minated soil depends on the nature of bioreceptor Ecotoxicology and Environmental
Safety 2012;78 344–350.
[43] Cheyns K., Peeters S., Delcourt D., Smolders E. Lead phytotoxicity in soils and nu‐
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[44] Bigham J M., Schwertmann U., Traina SJ., Winland RL., Woolf M. Schwertmannite
and the chemical modelling of iron in acid sulfate waters. Geochim. Cosmochim. Ac‐
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[45] Yu J-Y., Heo B., Cho J-P., Chang H-W. Apparent solubilities of schwertmannite and
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chapter 28
Copper Accumulation in Vineyard Soils: Distribution,

Fractionation and Bioavailability Assessment
Marija Romić, Lana Matijević, Helena Bakić and

Davor Romić
1. Introduction
Metals are chemically very reactive in the environment, which results in their mobility and
bioavailability to living organisms. Metals can be present in all environmental compart‐
ments as different species, with the TMs associated with different ligands, but never being
irreversibly transformed or metabolized, and in those meaning metals are different from or‐
ganic compounds. People can be exposed to high levels of toxic metal by breathing air,
drinking water, or eating food that contains it. As a consequence, metals get into the human
body by different routes - by inhaling, over skin, and ingestion of contaminated food. The
issue of toxicity is usually merely a matter of quantity, with the range varying for each ele‐
ment.
1.1. Why we need to study trace metals in soils?
Soil is an important compartment of the environment in which anthropogenic loading of

trace metals puts ecosystems and their inhabitants at a health risk. Repeated use of metal-
enriched chemicals, fertilizers, and organic amendments such as sewage sludge as well as
wastewater may cause contamination at a large scale. So far, it is believed that most soils in
Europe have not been significantly enriched in trace metals by anthropogenic activity. This
is changing as livestock production expands, fertilizer application increases, and biosolids
and effluent applications to agricultural soils become more common. Accumulation of trace
metals in soil has potential to restrain the soil functions, cause toxicity to plants, and enter
the food chain.
2 Soil Pollution
Many chemical reactions are responsible for the behaviour of TMs in soils, but the most im‐
portant processes that control their bioavailability and mobility are precipitation-dissolu‐
tion, adsorption-desorption, and complexation. The ability of soils to adsorb metal ions from
aqueous solution is of special interest and has consequences for both agricultural issues such
as soil fertility and environmental questions such as remediation of polluted soils and waste
deposition [1].
Metal-soil interaction is such that when metals are introduced at the soil surface, their mobi‐
lisation does not occur to any great extent unless the metal retention capacity of the soil is
overloaded, or metal interaction with the associated waste matrix enhances mobility [2]. The
most important interfaces involved in TMs transformation in soils are mineral groups com‐
monly found in soil: aluminosilicates, oxides and organic matter. Through their surface elec‐
trochemical properties, these soil minerals control adsorption, transformation, and release
behaviour of chemical constituents (e.g. nutrients and contaminants) to water and soil solu‐
tion [3]. Furthermore, soil-surface electrochemical properties vary between soil types and
depend on factors such as parent material, climate, and vegetation.
So that, full understanding and prediction of chemical behaviour of an element in the terres‐
trial environment is possible only by identification of all forms in which that element can be
found in soil under different environmental conditions. Copper is one of the major toxic
metals, and a highly reactive one, as well. Elevated levels of Cu in agricultural soils result
from the use of Cu-containing compounds to control plant diseases and from application of
manure or sewage sludge. These applications may lead to gradual accumulation of Cu in
the soil and thereby increase Cu toxicity toward crop and beneficial microorganisms. In this
article, the actual risk of high concentrations of copper and its mobility in vineyard soils is
reviewed considering sources, chemical processes in soil and biogeochemical behaviour of
copper as well as impact on agroecosystem and environment in general.
1.2. Sources and behaviour of copper in soils

Copper occurs in the Earth's crust at concentrations between 25-75 mg kg-1, with the aboun‐
dance pattern that shows the tendency for the concentration in mafic igneous rocks (60-120
mg kg-1) and argillaceous sediments (40-60 mg kg-1), but it is rather excluded from the carbo‐
nate rocks (2-10 mg kg-1) [4]. Values for soil contents generally range worldwide from 1 to
140 mg kg-1 depending on the nature of the soil parent material.
In soil solids and solution copper occurs almost exclusively as the divalent cation Cu2+, and
the reduction of Cu2+ to Cu+ and Cuo is possible under reducing conditions. As a chacophile,
copper associates with with sulfide in the very soluble minerals, Cu2S and CuS. Being very
reactive in soil, copper is found in all matrix components. Most of the colloidal soil material
(clay minerals, oxides of Mn, Al i Fe, and organic matter) adsorb copper strongly, and in‐
creasingly so as the pH is raised [5]. For copper, specific adsorption, which is not significant
for the most of metal ions, seems to play a more important then nonspecific adsorption.
Amorphous and cristalline oxides of Fe and Al easy adsorb Cu2+, regardless the excess of al‐
kali metals in the solution [6]. However, the most important sink for Cu is soil organic mat‐
ter, and its complexation with organic matter is one of the most efficient mechanisms of Cu2+
Copper Accumulation in Vineyard Soils: Distribution, Fractionation and Bioavailability Assessment 3
retention in soil [7]. This restricts Cu bioavailability, but also considerably reduces the risks
of phytotoxicity of the accumulated anthropogenic input and its vertical migration. Organi‐
cally complexed Cu2+ is bound more tightly than any other divalent transition metal and of
low lability these complexes results in limiting copper bioavailability. This prevents copper
mobility and its transport through soil to underground, and reduces substantially the risk of
the groundwater contamination.
1.3. Anthropogenic inputs and copper contamination of cultivated soils
Trace elements in general enter an agroecosystem through both natural and anthropogenic
processes. The latest includes TEs inputs through use of agrochemicals, farm manure, bio‐
solids and composts, industrial and municipal waste, irrigation, and wet and/or dry depos‐
its. Being widely used, copper is a common metal pollutant released to environment as a
result of man's activities. Copper is an essential nutrient, but in excess in soils it becomes
toxic to plants and some micro-organisms, disrupting nutrient-cycling and inhibiting the
mineralisation of essential nutrients such as nitrogen and phosphorus. Some species accu‐
mulate copper. Toxic effects on fish and other aquatic organisms have also been observed.
For humans, excess amount of this trace metal can have serious health effects.
Elevated levels of copper in agricultural soils result from the use of Cu-containing com‐
pounds to control plant diseases and from application of manure or sewage sludge. In‐
creased concentration of Cu in soils under long-term production of grapevine, citrus and
other fruit crops have been recorded in numerous studies. The Bordeaux mixture, an effi‐
cient agent for prevention of vine «Downy Mildew», Plasmopara viticola, has been routinely
used in Europe since the end of the 19th century with its concentrations and the number of
treatments depending on weather conditions, infection intensity and vineyard management.
The century-old practice of using Cu-sulphates and other copper containing fungicides to
protect grapevine, but also other agricultural crops, in temperate and tropic climatic regions,
resulted in significant Cu accumulation in soils [8]. Most of the copper accumulated in
leaves and soil by spraying will be retained in topsoil through the biological cycle and till‐
age [9-11]. Comparison of copper contents of 110 to 1500 mg kg-1 with its usual content in
agricultural soils (20 – 30 mg kg-1) points to their connection with such practice [12]. Copper
can be either a micro nutrient or a toxic element which depends on the copper concentra‐
tion. Determination of the total content of metals in soils is an important step in estimating
the hazards to the vital roles of soil in the ecosystem, and also in comparison with the quali‐
ty standards in terms of the effects of pollution and sustainability of the system.
From the ecotoxicological point of view, it is equally important to determine the bioavaila‐
bility of Cu accumulated in vineyards, i.e., the fraction of the total metal content in soil that
can be utilized by biota [13]. It depends on the soils properties, temperature, water content
and aeration, and also on plant species. The toxicity of copper is essentially observed in acid
soils, but not in calcareous soils and for copper contents as high as those reported in vine‐
yard soils. A number of authors have found positive correlation between copper retention
and pH [14, 15] and sum of bases or exchangeable calcium [16]. The bioavailability of copper
has also been reported to decrease when the cation exchange capacity or the level of organic
4 Soil Pollution
matter increases [5, 15]. Various electrolytes such as water, buffered or unbuffered salt solu‐
tions, chelating agents, diluted acids or a mix of these reagents can be used to estimate the
biavailability of copper in soils (reviews given by [17, 18]).
1.4. Fractionation and bioavailability of copper in soils
The concepts of «bio-availability» and «bio-accessibility» were introduced to express wheth‐

er the actual concentration of a toxic element would have effects on organisms [19]. The
main challenge that comes out from the assessment of loads of trace and toxic metals is the
methodology of determination or prediction of the trace element content in a soil that results
in toxicity [20]. Trace element mobility and bioavailability is determined by their transfer be‐
tween the soil solid phase and the soil solution [21], and trace element in soils can be divid‐
ed into inert and the potentially toxic labile fraction [22]. Thus, the impact of trace metals on
soil and the surrounding environment in most cases cannot be predicted simply by measur‐
ing their total concentration. This is because only soluble and mobile fractions have the po‐
tential to leach or to be taken up by plants, and enter the food chain. Furthermore, Cu
content in the plants usually does not well correspond to the total soil copper content [23].
Water-soluble and exchangeable copper fractions are considered to be bioavailable; copper

complexed with oxide, carbonate and organic matter are potentially bioavailable fractions;
and mineral fraction is considered to be non-bioavailable [24]. However, fractionation does
not provide information about species of metals in soil. Metal speciation is one of the most
important properties that determine the behavior and toxicity of metals in the environment.
Chemical speciation of an element refers to its specific form characterized by a different iso‐
topic composition, molecular structure, and electronic or oxidation state [25]. Speciation is
the process of identification and determination of different chemical and physical forms of
elements present in a sample [26]. TMs speciation is determined by their reactivity and
physical and chemical properties of the soil. Metals are very reactive in the environment and
can relatively easy change form in soil, making their speciation non stable. Among many
chemical processes that are involved in the transformation of TEs in soils, precipitation-dis‐
solution, adsorption-desorption and complexation are the most important in controlling
their bioavailability and mobility. Metals that occur in cationic forms have a higher ability of
binding to negatively charged soil colloids, and are thus less bioavailable, but more easily
accumulate in soil, unlike the anionic forms that are mainly present in soil solution and are
more bioavailable, but are more readily leached from the soil. Formation of complexes with
soil organic matter, adsorption onto the surfaces of clays and Fe and Mn oxides regulate the
behavior of copper in the soil [23]. Thus, copper bioavailability in soil depends on soil pH,
redox potential, CEC, amount and nature of organic matter and soil minerals [27].
Chemical extractions are used for soil copper bioavailability predictions, often in compari‐
son with its content in plants [23]. Copper is not readily mobile in plants and root concentra‐
tion is considered to be a good indicator of the plant copper content. It has been assumed
that the factors affecting metal fractionation and bioavailability in soil include root-induced
pH changes, metal binding by root exudates, root-induced microbial activities and root de‐
pletion as a consequence of plant uptake [28]. In the root developing zone, rhizosphere,
processes that control the mobility, transformation and toxicity of metals in soil are under
direct influence of plant roots and may differ from those in bulk soil. Thus, root activities
can considerably modify TM speciation in the rhizosphere. Metal plant root adsorption is
determined by ionization of negatively charged binding sites for metal on root surfaces.
Cupric ions bind to a specific carrier on the root cell plasmalemma surface [29] and plant
uptake depends on the available copper in the soil and the nutritional status of a plant [30].
Soil pH determines aqueous metal speciation, affecting metal sorption and desorption on
the solid phase [31], but plant roots as well. As the degree of biotic ligands ionization in‐
creases with pH, the metal ion root adsorption increases. Vice versa, the adsorption capacity
of the plant root for cupric ion decreases with decreasing solution pH. Rhizosphere pH
modifications by plant are a known occurrence and root zone alkalization may decrease the
exposure of plant roots to copper by promoting formation of organic complexes and reduc‐
ing copper solubility throughout rhizosphere area [32]. Furthermore, plant may directly de‐
crease copper bioavailability near roots by excretion of metal-binding compounds that can
complex the free cupric ions [33].
In this article, the actual risk of high concentrations of copper and its mobility in vineyard
soils is reviewed considering sources, chemical processes in soil and biogeochemical behav‐
iour of copper as well as impact on agroecosystem and environment in general.
2. Case study: Spatial distribution of copper concentrations in vineyard

soils of Croatia: Wine–growing subregion of Plesivica
Elements inherited from the bedrock are partitioned within the soil through specific process‐
es. Besides the parent material characteristics, geomorphology and landscape features con‐
tribute greatly to the variability of elements distribution. The topography is especially
important, since it affects water infiltration and drainage on the one hand and soil erosion
on the other. This variability is additionally enhanced in cultivated soils, especially in moun‐
tain regions and on sloping terrains where erosion processes are more expressed [34].
Steep southern slopes of the mountains in north-western Croatia have been used for centu‐
ries to grow vine and produce wine (Figure 1). Wine quality is almost always associated
with the location, which means with the specific natural viticultural environment where soil
is one of the major factors: firstly in terms of soil physical properties, and secondly in terms
of soil inorganic chemistry [35, 36].
Although the systems of vineyard planting and maintenance in north-western Croatia have
been changing with time and modern plantations prevail nowadays, parcels under tradi‐
tional cultivation and old, almost forgotten, cultivars can still be found. Therefore, the aim of
this study was to explore the distribution and retention pattern of copper concentrations in
vineyard soil and, and to study the copper speciation and its distribution within five opera‐
tionally defined fractions to assess its bioavailability and possible downward movement.
6 Soil Pollution
Figure 1. Grapevine plantation in the study region (Site Lokosin dol, Plesivica wine-producing subregion, Croatia, pho‐
to M. Romic)
2.1. Study area
Research was carried out on different wine-producing subregions in Croatia, in the wider
Zagreb region: the wine-growing subregion of Plesivica (approximate coordinates: latitude,
45° 42’ and longitude, 15° 37’). The area of Žumberak, which is a mountainous territory lo‐
cated to the west of Zagreb, is bordered to the north and west by Slovenia, and to the south
by Kupa River (Figure 3). Soils are developed on Pannonian sediments. This sediments con‐
sist of limy marls, sands, sandstones, conglomerates and breccias [37-39]. Week consistency
of those sediments, solid Triassic dolomite as bedrock and periodic streams led to fast ero‐
sion and filling in valleys and formation of amphitheater-shaped valleys. Namely, those
landscape shapes were formed by the distinct climatic oscillations and pulsation between
glacial and stadial periods, along with constant tectonic activity and elevation of terrain [39].
The landscape reflects the features of the Dinaric, so numerous formations characteristic of
the karst are found on highly dissected limestone terrains. As the Zumberacka Mt. piedmont
spreads perpendicularly to the mountains, some slopes are firmly interlinked by ridges,
forming well protected, amphitheatre shaped, vineyard areas in the wine-growing subre‐
gion of Plesivica.
Anthropogenic vineyard soils, classified as Aric Anthrosols [40], have been developed on
Tertiary sediments and Pleistocene loams. Owing to exceptional geomorphological and
agro-ecological conditions, these locations have been occupied almost exclusively by vine‐
yards for many decades.
The climate of the wider area is humid and the average annual rainfall is 836 mm. The mean
annual temperature is 10.3 °C, ranging from –0.6 °C (January) to 20.4 °C (July).
To investigate the spatial variability of surface soils, 67 soil samples were taken at the nodes
of a square grid at intervals of 1 km (Figure 3). The samples were defined as composite sam‐
ples made up of 10 increments collected from the soil upper 10 cm in a cross pattern, with a
5 m distance between increments (Eijkelkamp soil sampling kit used). Site descriptions were
registered at the time of sampling to record the sample location in relation to vineyard char‐
acteristics and major environmental features.
Three soil profiles were then located according to the landscape feature: profile 1 (Aric An‐
throsols) was dug in the vineyard plot down to the parent material (80 cm depth) at the 258
m asl, profile 2 (Aric Anthrosols) was located in vineyard plot at 231 m asl, and profile 3
(Colluvic soil) was located in the meadow at the foot of the hill 200 m asl, with the altitude
difference of 28 % between P1 and P2, and 13 % between P-2 and P-3. Table 1 reports the
selected physical and analytical features of soil profiles, and Figure 2 shows a sequence of
horizons.
Exchangeable cations
Profile / Depth CaCO3 Corg CEC Sand Silt Clay
pHH2O (cmol(+)kg-1)
Horizon cm % % cmol+kg-1
Caex Kex Mgex Naex %
P 0 - 45 8.2 48.8 2.6 24.1 0.63 2.14 0.09 24.0 11 64 26

P1
C 45 - 100 8.5 52.1 0.9 15.5 0.16 1.32 0.03 13.2 14 65 21
PI 0 - 35 8.2 47.7 3.1 25.9 0.72 2.22 1.02 26.1 11 63 27
P2 P II 35 - 90 8.4 47.3 1.8 26.9 0.31 2.30 0.11 26.6 8 59 33
C 90 - 130 8.5 51.0 0.5
Aa 0 - 10 7.9 41.0 3.6 32.6 0.18 2.30 0.05 31.1 15 68 17
Ap 10 - 30 8.2 46.2 2.4 29.1 0.16 1.64 0.03 28.6 13 64 23
P3 I 30 - 50 8.2 33.9 1.8 28.2 0.19 1.64 0.05 30.5 9 59 32
II 50 - 80 8.3 34.2 0.8 25.2 0.17 1.64 0.09 26.6 9 56 35
Cg 80 - 150 8.4 44.2 22.0 0.20 1.64 0.25 20.9 11 58 31
Table 1. Selected physical and chemical features of soil profiles
2.2. Chemical analysis
Soil samples were air-dried, sieved at 2-mm, and subjected to the following analyses: pH in
a 1:5 soil/water ratio (MettlerToledo MPC 227 pH- meter), soil organic carbon (SOC) by sul‐
fochromic oxidation [41], calcium carbonate (CaCO3) by the volumetric calcimeter method
after HCl attack, and effective cation exchange capacity (CEC) using BaCl2 solution. Particle
8 Soil Pollution
Figure 2. Soil profiles description (Site Lokosin dol, Plesivica wine-producing subregion, Croatia, photo M. Romic)
size distribution was determined by the pipette method after disaggregation in sodium py‐
rophosphate (HRN ISO 11277:2004). Soil samples were also digested in aqua regia [42] with
the microwave technique on a MARSXpress system (CEM).
Copper concentrations in soil digests were determined by inductivey coupled plasma opti‐
cal emission spectroscopy (ICP-OES) on a Vista MPX AX (Varian). All concentrations were
calculated on the basis of dry weight of samples (105 °C, 24 h). Quality control procedure
consisted of reagent blanks, duplicate samples and several referenced soil and sediment
samples with similar matrix from the inter-laboratory calibration program [43]. Maximum
allowable relative standard deviation between replicates was set to 10 %.
Two other methods were used for evaluating soil available copper: DTPA extraction [44]
and calcium chloride extraction [45].
DTPA extraction: 10 g of soil were extracted with 20 ml DTPA 0.005 M + TEA 0.1 M + CaCl2
0.01 M for 2 h at 20 °C under stirring (Heidolph PROMAX 2200 used), prior to being filtered.
Calcium chloride extraction: 0.5 g of soil were extracted with 50 ml of CaCl2 0.01 M for 2 h at
20 °C under stirring, prior to being filtered.
2.3. Metal fractionation
The selective sequential dissolution procedure was employed to divide metals into five sol‐
id-phase fractions [46, 47]. Chemical reagents and the experimental conditions applied are
summarized in Table 2. In this procedure, 0.5 g of each sample was weighted into 50 ml
polyethylene centrifuge tube and the extractions were carried out directly in the tubes, ex‐
cept in the last step where Teflon tubes for microwave digestion were used. At each extrac‐
tion step, after shaking and equilibration, solid-liquid separation was achieved by
centrifugation at 3500 rpm (2000 x g) for 10 min (centrifuge Sigma 3-15 used). The superna‐
tant was filtered through S&S 583 filter paper and placed to an acid-washed polyethylene
tube. The solid residue was washed three times successively with 5 ml of ethanol and the
liquid was discarded leaving the residue soil for the next step. The supernatant obtained at
each step was analysed for metals using inductively coupled plasma optical emission spec‐
troscopy (ICP-OES) on a Vista MPX AX (Varian). Single element standards were prepared
for each extraction in the same solution as the extracting agent to minimise matrix effects.
Blanks were used for background correction and other sources of error.
Fraction Abbreviation Procedure
Exchangeable EXCH (F1) 0.1 M Mg(NO3)2 pH=7, 10 ml, room temperature,

shake for 2 h
Associated with carbonates CARB (F2) 1M CH3COONa + CH3COOH (pH = 5.0), 10 ml, room
temperature, shake for 6 h
Associated with Fe/Mn oxides Fe/Mn OX (F3) 0.04 M NH2OH.HCl in 25 % HOAc, 25 ml, water-bath
at 90o for 3 h
Associated with organic matter ORG (F4) 0.1 M K4P2O7 25 ml, shake for 24 h
Residual RESID (F5) 4 M HNO3, 50 ml, microwave digestion
Table 2. The selective sequential dissolution procedure; chemical reagents and the experimental conditions.
2.4. Metal statistical analysis and data management
Linear regression: Relations between extractible copper in soils and soil properties were ana‐
lyzed by simple and multiple linear regression [48]. General conditions for model applica‐
tion were verified after parameter determination. Thus, residuals should follow normal
distribution, the assumption of homoscedasticity of variable variances should be proven,
and they should be independent of one another. Distribution normality was tested using the
Shapiro-Wilk test, while the other hypotheses were checked visually after the graphs were
drawn. Details given by [49]. All soil data were incorporated into the GIS database.
3. Copper concentrations in surface vineyard soil
Total copper concentrations in vineyard soils ranged from 30 to 700 mg kg-1, while total cop‐
per in 88 % samples exceeded the maximum tolerant concentration under the Croatian regu‐
lation of 100 mg kg-1 [50] (Figure 3).
10 Soil Pollution
Figure 3. Study area, surface soil sampling scheme and interpolated map of total surface soil copper concentrations
[51]
The amount of copper built up in the surface vineyard soil was estimated on a per hectare
basis taking into account the total copper concentration and the weight of 10 cm thick sur‐
face layer of soil, assuming a bulk density of 1.5 (Table 3). Approximate plantation age (with
within-decade precision) was estimated for most plots, and a detailed history for some plots:
time of the first and possibly second deep ploughing, time of vineyard restoration or supple‐
mentary planting, common growing practice in the past, and thereby also approximate fre‐
quency of copper fungicide applications. More than 64 % vineyards are more than 40 years
old and it is assumed that the same percent of all plots received similar annual amounts of
copper, ranging from 2 to 5 kg ha-1. This certainly allows only an estimation of the overall
copper input into soil throughout the vineyard history. The upper layer of plots planted
with about a century old vines exhibited high copper contents.
Location1 CuTOT(mg kg-1) CuDTPA (mg kg-1) CuCaCl2 (mg kg-1) CuTOT/ha (kg ha-1)2
351 363 136 1.15 491

352 154 48 0.63 208
353 380 142 1.17 514
354 389 121 0.74 525
355 181 56 0.73 244
356 166 54 0.55 224
357 655 368 1.45 884
358 586 296 1.96 790
359 369 135 0.94 498
360 565 296 1.35 763
361 633 340 2.08 855
362 641 228 1.68 866
Location1 CuTOT(mg kg-1) CuDTPA (mg kg-1) CuCaCl2 (mg kg-1) CuTOT/ha (kg ha-1)2
363 336 196 1.24 454

364 385 152 1.15 519
365 263 101 0.82 355
366 667 316 1.36 901
367 615 332 1.56 830
368 419 143 1.19 565
369 494 166 1.47 667
370 244 95 0.93 330
371 519 282 1.15 701
372 134 47 0.42 181
373 199 71 0.52 268
374 623 348 1.84 841
375 35 4.32 0.10 47
376 389 140 1.26 525
377 442 138 0.96 597
379 252 104 0.92 340
380 30 808 0.93 40
381 531 198 1.17 717
382 305 117 1.04 412
383 453 199 1.35 612
384 360 131 1.2 486
385 79 21.2 0.21 107
386 398 146 1.36 537
387 324 120 0.94 437
390 51 8.28 0.21 69
391 74 11.8 0.11 100
392 228 89 0.83 307
395 533 170 1.46 720
396 627 254 1.66 847
397 32 12.4 0.10 43
398 43 10.6 0.21 58
399 610 180 1.98 823
401 691 278 1.93 933
402 427 154 1.35 576
1
Location: All the data available from the GIS database
2
Cu/ha (kg ha-1) Calculated from the data on total Cu (mg kg-1) in the top 10 cm assuming a bulk density of 1.5
Table 3. Total Cu, extractable Cu and total amount of Cu per ha in the upper layer (10 cm) of vineyard soils (n=67)
(Romic et al., 2004)
12 Soil Pollution
Determination of the total metal content in soils is an important step in assessing the haz‐
ards to the vital roles of soils in the ecosystem, but also in comparing them with quality
standards referring to the effects of contamination and system sustainability. However, the
mobility and availability of soil copper are governed by the processes of dynamic equilibri‐
um, and not only by its total concentration [52]. This study shows that the mobile (CaCl2-
extractable) fraction of copper in vineyard soils amounted only to 0.2 – 3.1 % of its total
content. As Cu accumulation in the studied soils is restricted to surface layer the risk of Cu
phytotoxicity for grapevine is small, since grapevine develops most of its roots at a depth
>30 cm, depending on the soil type and the profile depth. Material suspended by erosion,
however, carries away also a part of the applied copper, and redistribution of this material
depends on a number of factors (relief, size and shape of the drainage basin, etc.). Halamic
et al. [53] applied factor analysis in geochemical investigations of stream sediments in drain‐
age basins in the Mt. Medvednica region, which also includes part of the studied area, with‐
out determining correlation of copper concentrations with any lithological unit, so they
assumed anthropogenic influence, mostly grapevine production. Ribolzi [54] carried out the
research in the Mediterranean drainage basins of wine-growing regions in France with the
aim to characterize copper forms in suspended material and recorded an average total con‐
centration as high as 245 mg Cu kg-1, but Brun et al. [55] reported the maximum of 250 mg
kg-1 of copper in vineyard soils of the Mediterranean part of France.
3.1. Correlation between total, extractable Cu and soil properties
In the total copper concentration extracted with aqua regia, DTPA-extractable copper
amounted to 12-81 % and CaCl2- extractable to 0.2-3.1 %. Both DTPA- and CaCl2-extractable
copper were largely explained by the total copper concentrations, as confirmed by their high
correlation coefficient (R = 0.899 and R = 0.896). They were also highly correlated to each
other (R = 0.763) (Table 4).
Cutotal CuDTPA CuCaCl2 pHH2O Org. C CEC CaCO3

Cutotal 1 0.899*** 0.896*** 0.388** 0.601*** 0.277* 0.314**
CuDTPA 1 0.763*** 0.321** 0.395*** 0.084ns 0.236ns
CuCaCl2 1 0.392 ***
0.734 ***
0.257 *
0.341**
pHH2O 1 0.121 ns
0.315 **
0.587***
Org. C 1 0.339 **
0.173ns
CEC 1 0.130ns
CaCO3 1
ns - not significant
*
Correlation significant at p < 0.05
**
***
Table 4. Correlation matrix, upper triangle

Highly significant positive correlation was also determined between total copper and organ‐
ic matter in soil, and a weaker correlation, but still significant, between total copper and cati‐
on exchange capacity and carbonate content (Table 4). In the case of extractable copper, a
significant correlation was determined between CuDTPA and organic carbon content and pH,
but not between cation exchange capacity and carbonate content. Significant correlation was
recorded between all the analyzed soil properties and CuCaCl2.
3.2. Linear regression of total and extractable copper and selected soil properties
To establish the relation between copper fractions after particular extractions (aqua regia,
DTPA and CaCl2) and soil properties that may affect their behavior in soil and availability to
plants, the multiple linear regression analysis was done. The model included those variables
for which correlation probability p<0.05 was determined [48]. For the regression model of
aqua regia extracted copper, this condition was met by the following properties: CuDTPA,
CuCaCl2 and cation exchange capacity (CEC).
For total copper, the regression model explains 92 % of total variance (Table 5). The largest
contribution to the variance in regression was that of CuDTPA.
Variable explained: CuTOT
Source of variation Degree of freedom Sum of squares F Pr > F
CuDTPA 1 301395 101,8 0.0000

CuCaCl2 1 192403 65,0 0.0000
CEC 1 30384 10,3 0.0021
Total 63 2319596
Regression equation: CuTOT = -63.42 + 0.956* CuDTPA + 170.9*CuCaCl2 + 3.761*CEC

R2 = 0.92
Table 5. Linear regression of total copper (CuTOT) as a function of DTPA-extractable (CuDTPA), CaCl2-extractable (CuCaCl2)
and cation exchange capacity (CEC).
Concentration of total copper in vineyard topsoil went up with an increase in the cation ex‐
change capacity. Square root transformation (SQRT) was applied tothe regression model of
DTPA-extractable copper, whereby the model conditions were satisfied, and the trans‐
formed variables SQRT(CuDTPA) were predominantly dependent on Cutot. As this relation
was already determined, it was omitted from the model. Two other properties met the con‐
dition of correlation probability p<0.05: organic matter content (Org-C) and CaCl2-extracta‐
ble copper (CuCaCl2), and they were included into the model. For CuDTPA, the regression
model explains 85 % of total variance (Table 6).
14 Soil Pollution
Variable explained: SQRT (CuDTPA)
CuCaCl2 1 685.7 240.0 0.0000

Org-C 1 44.54 15.59 0.0002
Total 63 1224
Regression equation: SQRT(CuDTPA) = 5.534 + 9.400*CuCaCl2 – 1.326* Org-C

R2 = 0.853
Table 6. Linear regression of DTPA-extractable (CuDTPA), as a function of CaCl2-extractable (CuCaCl2) and soil organic
matter (Org. C)
Two parameters were included into the regression model of CaCl2-extractable copper: or‐
ganic matter content (Org-C) and pH, and the model explains 62 % of total variance (Table
7). Concentrations of CaCl2- extractable copper mainly depend on pH, which relation was
also confirmed by this investigation. However, since these are predominantly alkaline soils,
this relation is not as strong as in the case of soils with a more varying pH [55].
Variable explained: CuCaCl2
Org-C 1 8.30 83.37 0.0000

pH 1 1.62 16.23 0.0002
Total 64 17.34
Regression equation: CuCaCl2 = -2.673 + 0.379* Org-C + 0.337* pH

R2 = 0.621
Table 7. Linear regression of CaCl2-extractable copper as a function of soil organic matter (Org. C) and pH
Over 65 % of vineyard plots under study were more than 40 years old, and some have been
continuously cultivated for more than 100 years. It is assumed that the same percent of par‐
cels received a similar annual amount of copper, ranging from 2 to 5 kg ha-1. This, naturally,
does not allow an exact estimate of the overall copper input into soil throughout the vine‐
yard history. Vineyard age parameter was not therefore included in the multiple linear re‐
gression model. There is, however, strong statistical evidence that an increase in vineyard
age is related to the increase of expected total copper content (Figure 4). According to histor‐
ical documents, the vine downy mildew infection started spreading in the vineyards of
northwestern Croatian in 1882, and the Bordeaux mixture application became indispensable
during the wine boom period at the end of the 19th century. Recognizing the benefits, wine-
growers often did not observe the recommended concentrations and application times, and
a large number of treatments, as many as 8 to 14, were often applied at positions exposed to
disease attacks. However, numerous other factors, such as scattering during applications,
washing off the leaves by rain, input of treated plant residues into soil, tillage and erosion,
make it difficult to establish the relation between vineyard age and accumulated copper. In
France, for example, the Bordeaux mixture has been used since 1855, and it was found that
after several decades of its continuous application the soil total copper reached a concentra‐
tion of as much as 1.0 g kg-1 [56]. Research done by Deluisa et al. [11] on 43 plots in a humid
region of northern Italy revealed an average copper accumulation in topsoil of 297 mg kg-1.
Moolenaar and Beltrami [57] have calculated that organic protection of grapevine, which im‐
plies exclusive use of the Bordeaux mixture, can result in an increase of soil copper concen‐
tration up to 600 mg kg-1 after 100 years.
Figure 4. Vine age as a function of total soil copper
Diethylenetriaminepentaacetic acid (DTPA) is a potent synthetic chelating agent, and the

method of extraction with DTPA was developed for the purpose of determining zinc, iron,
manganese or copper deficiency in neutral and carbonate soils [58]. Haq and Miller [59] re‐
ported negative results of the DTPA test, which they explained by their failure to determine
sufficiently significant relations between concentrations of metals (copper and manganese)
extracted from soil and those found in the tested plants. Mention should be also made of the
research done by O’Connor [60], who gave a number of comments on the DTPA test, based
also on non-significant correlation between DTPA-extractable metals in soil and their con‐
centrations in plants. Regardless of the above considerations, DTPA is the most widely used
agent for extraction of “available” cadmium, copper, nickel and zinc, and thereby also the
most standardized one [44, 61, 62]. Starting from the fact that the data on total copper con‐
tent reveals very little about its bio-availability, such strong correlation between copper ex‐
tracted with aqua regia and DTPA actually indicates that neither the latter extraction
method is suitable for assessing copper availability to plants.
Merry et al. [63] in vineyard soils recorded 25-35 times higher contents of copper, lead and
arsenic, originating from plant protection agents, than their common values in uncontami‐
16 Soil Pollution
nated soils; they also determined a strong correlation between total and DTPA-extractable
copper (0.93 < r < 0.96). Also Brun et al. [55] found that the regression model CuDTPA ex‐
plained 90 % of total variance in vineyard soils, its largest part referring to total copper con‐
tent.
When cation exchange capacity was included into the model, it was found that the DTPA-
extractable copper decreased with increasing cation exchange capacity.
Soil extraction 0.01 M CaCl2 is the method that was increasingly used in the last decade for
soil testing to determine soil fertility and the behavior of nutrients and contaminants in the
soil. The capabilities of instrumental chemical analysis have improved to such an extent,
even in the last few years, that it become possible to determine very low concentrations of
nutrients and pollutants in soil extracts [45]. The advantage of this method for determining
metal concentrations in soil is that the concentration of electrolytes stays practically constant
and metal concentrations reflect the difference in binding strength or solubility between
soils. The extractant is an unbuffered solution and therefore the measured metals reflect
their availability at the pH of the soil.
The best criterion of the efficiency of the method for determining the soil bioavailable frac‐
tion is the high correlation between the Cu content observed in plants grown in situ, at least
for neutral to acid soils [55].
3.3. Vertical distribution of total copper in soil profiles

High copper concentrations were found in vineyard soils down to 20 cm depth (to 800 mg
kg-1 in profile 1, and to 500 mg kg-1 in profile 2) (Figure 5). Profile 1 is situated at a higher
altitude, erosion is more pronounced, and the anthropogenic horizon is less thick. Marl ap‐
pears already at 45 cm depth, so that the root zone extends into horizon C as well, thus
opening the transport routes of water, dissolved substances and solid particles deeper into
the profile. In profile 2, the anthropogenic horizon is much thicker, while total copper con‐
centrations are lower down to 30 cm depth. Erosion material was deposited at the base of
the slope, so that as much as 100 mg kg-1 of copper was found in the topsoil of colluvial soil,
to which no copper agents for plant protection had ever been directly applied. Accumula‐
tion of copper in colluvial soil (profile 3) was recorded down to 30 cm depth, that is, over
the entire depth of the humus-accumulative horizon. Uniform copper concentrations of <25
mg kg-1 were found at greater depths.
Land use for agriculture causes great changes in the natural properties of soil. Translocation
of soil by tillage may be the key reason for redistribution of soil particles within the profile
and over the entire site, while erosion due to tillage is especially present in hilly landscapes
[64]. Tillage and homogenization of several natural horizons alter both morphological and
physicochemical characteristics of soil.
3.4. Sequential extraction

Results of the five-step sequential extraction are presented in Figure 6, which shows the dis‐
tribution of exchangeable (Exch), carbonate-associated (Carb), Fe-Mn oxides-bound (Fe-Mn
Figure 5. Verical distribution of total copper within the soil profiles
ox), organic-bound (Org) and residual (Resid) copper found in the soil samples. These five
metal fractions were separated in soil samples from 10-cm profile layers of Aric Anthrosols
and master horizons of Calcaric Regosols. The data were used to calculate the relative error
(RE %), which for most metals amounted to ±10 %.
In the surface 10 cm of profile 1, 877 mg kg-1 of copper was determined by extraction in aqua
regia. The sum of copper fractions separated by selective sequential extraction is slightly
lower and amounts to 816 kg-1 (RE = 7 %). As much as 47 % of total copper was bound in the
organic fraction, 20 % in the residual fraction, and 18 % and 16 % in the reductive and carbo‐
nate fractions, respectively. Down to 30 cm depth, the share of copper in exchangeable frac‐
tion was <1 %, whereas it was not detected in deeper layers (Figure 6). However, copper
distribution per fractions changes with the profile depth. As the total concentration decreas‐
es to background values, the share of copper in residual fractions increases, since complex‐
ing ability decreases with the reduced amount of organic matter.
Considering the high content of the organic fraction (54 % in the 0–10 cm layer and 27 % at
60–80 cm depth), a very probable mechanism is translocation through complexation with
soil organic matter. However, copper is certainly not translocated in the same way with sur‐
face eroded material as vertically through the profile depth. Eroded material deposited at
the base of hillsides under vineyards is richer in silt, and copper is mostly strongly bound in
the residual fraction. In the surface 10 cm of soil of the profile 1, 20 % of total copper content
is bound in the residual fraction. In the profile 2, copper content in the residual fraction was
increased to 21 %, while it amounted to 50.3 % in colluvial soil (profile 3).
18 Soil Pollution
Figure 6. The distribution of exchangeable (Exch), carbonate-associated (Carb), Fe-Mn oxides-bound (Fe-Mn ox), or‐
ganic-bound (Org) and residual (Resid) copper in soils
Copper distribution per fractions in profile 2 resembled that in the preceding profile. High
copper concentrations decrease with depth, the profile being deeper as well. Copper content
of <1 % was found in the exchangeable fraction, and that of 3 % at 60–80 cm depth. It is obvi‐
ous that copper translocation occurs within the profile, but this is also the zone where most
of the vine roots develop.
Although no copper fungicides had ever been directly applied to the area on which profile 3
was dug, as much as 100 mg Cu kg-1 was determined in topsoil by extraction in aqua regia.
Background concentrations were reached at 30 cm depth, and copper was predominantly
bound in the residual fraction (47 %-63 %). There was a significant share of the organic frac‐
tion, but it decreased with depth from 33 % to 15 %. The content of exchangeable copper
was higher than in vineyard soils – 2 % to 5 %.
Research has shown that soil type is the main factor of accumulation and distribution of
both natural and anthropogenic concentrations of heavy metals. In natural profiles, the in‐
digenous element distribution is generated by long-term pedogenesis [65]. In the case of cul‐
tivated soils, their characteristics in time and space change in dependence on the ecological
environment and land use and management, whereby also their production capacity and
environmental impact get changed [66, 67]. When growing woody crops, and thus also
grapevine, soil is homogenized to a greater depth, first by deep ploughing to xx cm depth
before the setting up of the plantation, and then by regular tillage. This somewhat disrupts
the morphogenetic soil properties, changes the sequence of genetic horizons, and often
deepens the active part of the profile. Such changes are naturally reflected in the distribution
of elements, changes in their mobility and bioavailability. Selective sequential extraction was
used in this research to determine the way and strength of binding and retention of heavy
metals in soil, which under certain conditions enables the estimation of potential mobility
and bioavailability.
The spatial variability of trace metals in agricultural surface soils of the wider Zagreb area
has shown that the application of agrochemicals has caused high accumulation of copper
and zinc [51, 68]. This especially applies to vineyard soils, but also to orchard and vegetable
garden soils. The presence of a buffer material, such as carbonate, can be particularly impor‐
tant in the retention of heavy metals. The trace metal retention capacity of silty soils with
high carbonate content can be as high as, or higher than, the retention capacity of certain
clayey soils [69].
In soils and sediments that were receiving high concentrations of copper (along with other
metals) for at least 6 years, Hickey and Kittrick [70] established that about 28 % copper was
bound in the organic fraction. It is also in the soils treated with waste sludge or stable man‐
ure rich in copper that most of this metal is bound in the organic fraction [71, 72]. Affinity of
humic and fulvo acids to copper sorption was, among others, reported by Senesi et al. [73]
while McLaren et al. [74], pointed to the importance of soluble copper chelates in soil solu‐
tion. In soils of lighter texture, poorer in organic matter, the added copper is initially re‐
tained in the exchangeable fraction, whereafter it is translocated into the carbonate fraction
[75]. Incubation in the laboratory experiment revealed that translocation of copper to more
stabile fractions was much slower in texturally light, but acid, vineyard soil, also poor in or‐
ganic matter [76]. However, Flores-Velez et al. [10] report that in the case of sandy acid vine‐
yard soils the selective sequential extraction procedure was not selective enough to specify
the form of copper. The same authors report that copper in anthropogenic vineyard soil,
originating from Cu-fungicides, was concentrated in the coarse organic fraction (plant resi‐
dues) and in the mineral colloid fraction. In soils or sediments deficient in organic matter, a
larger part of copper was bound to Fe and Mn oxides that it was found in this research.
Thus, Szarek–Gwiazda and Mazurkiewicz–Boron [77] found that 40.2-54.1 % of total copper
in fluvial sediment was bound to Mn oxides and amorphous Fe hydroxides, and only about
10 % to the organic fraction. These authors maintain that Fe(III) and Mn(IV)-oxides can oc‐
cur either as coats on detritus particles, as cement between them or as pure concretions.
20 Soil Pollution
Their ability to adsorb and control heavy metal distribution between the solution and the
matrix has been thoroughly explained in scientific literature (e.g., [78]).
Romic et al. [49] did not establish, either by individual correlations or by factorial analysis in
the vineyard soils of NW Croatia, the importance of the contents of clay, Fe and Mn oxides
or cation exchange capacity for copper sorption in soil, though some authors stress the im‐
portance of these fractions [74]. In their investigations, the relation between the content of
metals and soil properties was assessed on the basis of their total contents. In this research,
correlations were determined between copper fractions and the selected soil properties: sig‐
nificant correlation was recorded between the contents of organic C and ORG and RESID
fractions, or its total content, whereas no significant correlation was found between copper
in the said fractions and cation exchange capacity, or total carbonates. This corroborates the
reports that the distribution of copper of anthropogenic origin among fractions depends pre‐
vailingly on soil organic matter.
Finally, the use of copper containing fungicides is allowed in the organic agriculture by the
European Union regulation, and the official guidelines for soil copper content are usually
derived from the total soil copper content. However, these guidelines should be modified
according to the soil properties, such as pH and organic matter content, which will affect the
Cu solubility, and consequently its bioavailability. Furthermore, soil copper thresholds
should be confirmed with toxicological data obtained for biota (e.g. plants, microorganisms,
invertebrates). Above mentioned implies that site-specific guidelines should developed for
the risk assessment of soil copper toxicity.
4. Summary
Increased anthropogenic inputs of trace metals in soils have received considerable attention
since they can enter the food chain by different ways. Soils receiving repeated applications
of fungicides, pesticides or manure exhibit high concentrations of extractable metals, espe‐
cially copper. From the commercial aspect, wine-growers are now showing increasing inter‐
est in the effects of soil composition, its fertility and texture upon wine quality. Special
importance is laid on the influence of soil geochemical characteristics, including accumula‐
tion of certain toxic elements, on grape and wine quality. A variety of factors, both spatial
and temporal, affect the grape quality, many of them being specific exactly to the given
wine-growing site. Grape growing conditions and enological potential have been created,
among other factors, also by landscape characteristics: soil, climate and topography. These
factors are much less changeable than biological (cultivar, stock) or human (ampelotechnics,
vinification) factors and for this reason the concept of viticultural terroir is based on the sim‐
ple relationship between soil and wine. Since terroir is defined as an interactive ecosystem, it
is very difficult to evaluate scientifically its contribution to plant capacity to accumulate bio‐
active phytochemicals good for human health.
Soil is a factor of the natural environment and its effect on wine quality and grape composi‐
tion is highly complex, since it affects mineral nutrition of grapevine, water uptake, as well
as rooting depth and rhisosphere temperature. Land use for agriculture causes great
changes in the natural properties of soil. Translocation of soil by tillage may be the key rea‐
son for redistribution of soil particles within the profile and over the entire site, while ero‐
sion due to tillage is especially present in hilly landscapes. Tillage and homogenization of
several natural horizons alter both morphological and physicochemical characteristics of
soil, which affect metal behaviour in soil – plant system.
The mobility and bioavailability of metals depends not only on the its total concentration in
soil but also on soil properties, metal characteristics and environmental conditions. Trace
metals are present in soil in various forms: water soluble, exchangeable, carbonate associat‐
ed, oxide associated, bound on organic matter and residual forms. Obviously, various metal
forms have different mobility. Water soluble and exchangeable fractions are readily released
to the environment, whereas the residual fractions are immobile under natural conditions.
The most common observation in majority of studies dealing with sequential or partial ex‐
traction of soil trace elements, including copper as well, is that they are not completely spe‐
cific to metals or chemical phases. The complexity of bioavailability phenomenon comes out
from an arry of matrix-related, species-related and metal-related issues.
Plant copper uptake occurs predominantly from the soil solution, indicating that soil charac‐
teristics that determine copper solubility, will also determine the copper phytoavailability.
However, roots can alter the chemical mobility and thus the bioavailability of copper in the
rhizosphere. Remediation of contaminated vineyard soils is an issue of debate and it should
be focused on growing practice and vineyard management more than application of the re‐
mediation techniques per se (i.e. phytoextraction). Periodical monitoring of soil quality indi‐
cators is a required wine-growing practice. If systematically collected information is
associated to a location, then it can be useful to all participants in the complex process of
grape and wine production, wine marketing and consumption. Use of advanced analytical
and information technologies may significantly improve the production and contribute to
rational utilization of resources.
Author details
Marija Romić, Lana Matijević, Helena Bakić and Davor Romić
University of Zagreb, Faculty of Agriculture, Croatia
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chapter 29
Characterization and Remediation of Soils and

Sediments Polluted with Mercury: Occurrence,
Transformations, Environmental Considerations and
San Joaquin’s Sierra Gorda Case
I. Robles, J. Lakatos, P. Scharek, Z. Planck,

G. Hernández, S. Solís and E. Bustos
1. Introduction
Soil as important part of the ecosystems which must be protected in the environment
context, and it is necessary be studied the possible overall impact of measures for protec‐
tion, with a very special attention from mining activities. The soil resource occupies a
fundamental part of the ecosystems; when a soil is degraded, the others components of the
ecosystems are degraded too.
The fate of the heavy metal in soils depends upon many soil processes that are governed by
several soils properties of which soil pH and redox potential are known to be the most
important parameters. Thus, the solubility of trace elements is often shown as a function of
pH affected by amount and kind of organic matter. Trace elements are known to be accumu‐
lated in surface soils as a result of contamination from point sources as mining activities. An
appreciable amount of the soils has been made unusable because of pollution. Highly conta‐
minated soils belong to a high healthy risk to human being and their environmentally harmful
effects. That is why soil should be correctly understood and underestimated long range lethal
effects that can have irreversible consequences. The improvement of soils damaged and
contaminated by pollutants need of the particular soils, requires a full understanding of soil
properties and of the deteriorating factors.
Mercury is one of the most toxic elements to human health and ecosystem; because of all
mercury species are toxic. A wide variety of mercury species exist in the environment and its
2 Soil Pollution
various chemical forms can differ in bioavailability, transport, persistence, and toxicity. Still,
every mercury species is toxic with methyl mercury being the most toxic species. The World
Health Organization (WHO) recommends a maximum methyl mercury intake of 1.6 μg Kg-1
per week, while the Environmental Protection Agency (EPA) lists a maximum recommended
intake of 0.1 μg Kg-1 of body weight per day for adults. Due to high bioaccumulation, mercury
is found on many levels of the food chain (Hinton and Veiga, 2001; Bengtsson, 2008). Any form
of mercury in the environment may evolve into a more toxic species (methyl mercury) under
biogeochemical transformation processes (Figure 1). Due to these processes and the high
mobility of mercury species, a good understanding of how mercury species transform and
accurate monitoring are essential for assessing the risk of mercury in the environment.
The impact of mercury depends strongly on its chemical species; understanding mercury
transformations and the impact of its various chemical forms are vital to preventing harmful
effects on humans and the environment. Nevertheless, the physicochemical characteristics of
mercury are either useful or necessary for many industrial and agricultural applications, and
mercury may be scattered over large area, depending on the source (Leopold et al, 2010).
Figure 1. Biogeochemical transformation processes of mercury (Leopold et al, 2010).
Mercury concentrations in ground water indicate that the highest concentration of mercury in
groundwater comes from the soil and from aquifers. While simulating mercury predictions
can often be difficult, mercury can be estimated in experiments conducted in batch mode or
in columns. The percentage of Hg that can potentially leach from the soil was previously
estimated in batch experiments. Distribution analyses of species in leachate confirmed the
Characterization and Remediation of Soils and Sediments Polluted with... 3
presence of inorganic species (Hg2+ and Hg0) ranging from 90 – 100 % (Bollen, 2008; Harvey,
2002).
The most commonly used techniques for the remediation of mercury contaminated soils have
been classified as either excavation techniques or containment techniques, and are grouped as
follows (Hinton and Veiga, 2001): (a) ex situ treatments: physical separation, thermal treat‐
ments, hydrometallurgical treatments; (b) In situ recuperation: vapor extraction coupled with
evaporation (soil), permeable reactive barriers; (c) In situ leaching and extraction: electrokinetic
separation, interceptor systems, phytoremediation, passive remediation; (d) containment:
pump and treat impermeable barriers, sealed surfaces and drainage, stabilization and solidi‐
fication, sediment covering.
2. Physicochemical properties of mercury
Mercury (Hg) is a chemical element with an atomic number of 80. Mercury is a silver plated
heavy metal, liquid and odorless at normal conditions. It easily alloys with many other metals
like gold or silver producing amalgams, is insoluble in water and soluble in nitric acid. The
main source of Hg is cinnabar or mercury sulfide (HgS), a stable compound and insoluble
usually recovered as a byproduct of ore processing. Mercury in this form is found in the earth's
crust average concentrations of 0.5 ppm (Hinton and Veiga, 2001).
Mercury is one of the most toxic elements to human health and ecosystem. At temperatures
above 40 °C mercury produces toxic and corrosive fumes. It is harmful by inhalation, ingestion
and contact, is a very irritating to skin, eyes and respiratory tract, even to nervous system, its
gaseous form is absorbed by lung tissues (Hinton and Veiga, 2001, Bengtsson, 2008).
A wide variety of mercury species exist in the environment and its various chemical forms can
differ in bioavailability, transport, persistence, and toxicity. Still, every mercury species is toxic
with methyl mercury being the most toxic species. This element can exist in the environment
as elemental (Hg0), oxidized inorganic (Hg2+ -mercuric, Hg22+-mercurous) or oxidized organic
(methyl/ethyl mercury) forms. Mercuric and mercurous forms are more stable under oxidizing
conditions. In moderately reducing conditions, the organic or inorganic mercury can be
reduced to its elemental form and be converted to forms leased by biotic or abiotic processes:
these are the most toxic forms of mercury, as well as being soluble and volatile. Hg (II) forms
strong soluble complexes with a variety of organic and inorganic ligands oxidized in aqueous
systems. Hg sorption in soil, sediment and humic materials is an important mechanism for the
removal of mercury from solutions, another mechanism, a high pH is their co-precipitation
sulfide (HgS) (Leopold et al, 2010).
Any form of mercury in the environment may evolve into a more toxic species (methyl
mercury) under biogeochemical transformation processes. Due to these processes and the high
mobility of mercury species, a good understanding of how mercury species transform and
accurate monitoring are essential for assessing the risk of mercury in the environment. The
impact of mercury depends strongly on its chemical species; understanding mercury trans‐
4 Soil Pollution
formations and the impact of its various chemical forms are vital to preventing harmful effects
on humans and the environment. Nevertheless, physicochemical characteristics of mercury
are either useful or necessary for many industrial and agricultural applications, and mercury
may be scattered over large area, depending on the source (Leopold et al, 2010; Nick, 2012).
The metal mercury (Hg0) is mainly used to produce chlorine gas and caustic soda, and is part
of some types of alkaline batteries, fluorescent lamps, electrical contacts, and instruments such
as pressure gauges and thermometers, among others. Hg salts are used in antiseptic ointments
and creams and skin lightening. Among the activities that generate the most pollution by Hg,
is the burning of coal and chlor-alkali plants: other important sources are mining and metal‐
lurgy and the burning of municipal solid waste, which may contain instruments such as
pressure gauges, thermometers, alkaline batteries and fluorescent lamps. The mercury
released into the air tends to settle and adhere to soil organic matter (Hinton and Veiga,
2001; Nick, 2012).
Natural and anthropogenic mercury emissions are mainly in the form of elemental mercury
(Hg0), which makes up about 99 % of total atmospheric mercury. However, biogeochemical
transformations can oxidize it, forming Hg+ and Hg2+. Most inorganic Hg-compounds are water
soluble in small doses, and can be found in soil and sediments. In contrast, the presence of
inorganic forms of Hg2+ bonded to organic and/or inorganic species ([HgClx]2-x; [HgII-DOC];
[HgS]) depends on the local chemical environment. The life time of these compounds in air is
very short (on the scale of minutes) and they are rapidly removed by deposition processes
because of high water solubility and surface activity. Figure 2 shows the main mercury species
in the atmosphere, hydrosphere and sediment (Leopold et al, 2010; Nik, 2012; Slowey et al,
2005; Wartel et al, 1999; Shi et al, 2005).
Over 90 % of surface water mercury is from atmospheric deposition. Hg2+ usually undergoes
a biomethylation process that forms methylmercury (MeHg, CH3Hg+) and dimethyl mercury
(DMeHg, (CH3)2Hg), though these reactions can be reversed using microorganisms and/or
photolytic decomposition. All these species are highly mobile.
Three main forms of mercury are found in natural waters: elemental mercury (Hg0), inorganic
Hg2+ (Hg2+and its complexes) and organic mercury (MeHg, MeHg complexes and DMeHg).
With solubility (at 25°C) of 0.08 mg L-1, Hg0 can be found at all depths. Inorganic mercury
(Hg2+) and MeHg forms complexes with other dissolved compounds in fresh water, but for the
most part, only forms complexes with chlorine in sea water. DMeHg is found in the deep sea.
(Wartel et al, 1999, Shi et al, 2005, Slowey et al, 2005).
3. Edaphology properties of soil related with mercury
Soil is a collection of natural bodies on the Earth’ surface, in places that most of them have been
modified by man in its quality and containing living matter and supporting or capable of
supporting plants. Soil grades at its lower margin to hard rock or to earthy materials virtually
devoid of roots, animals or marks of other biologic activity.
Figure 2. Distribution of mercury species in atmosphere, hydrosphere and sediment (Leopold et al, 2010).
In general, physical, chemical and biological soil characteristics are highly correlated param‐
eters that are necessary to understand. The specific elemental composition of each particular
soil reflects, to a degree modified over time by weathering and the chemical composition of
the parent material from which the soil is formed. For instance, the extractability of the different
elements depends on the soil properties.
Soil as important part of the ecosystems which must be protected in the environment context,
and it is necessary be studied the possible overall impact of measures for protection, with a very
special attention from mining activities. The soil resource occupies a fundamental part of the
ecosystems; when a soil is degraded, the others components of the ecosystems are degraded too.
The fate of the heavy metal in soils depends upon many soil processes that are governed by
several soils properties of which soil pH and redox potential are known to be the most
important parameters. Thus, the solubility of trace elements is often shown as a function of
pH affected by amount and kind of organic matter. Trace elements are known to be accumu‐
lated in surface soils as a result of contamination from point sources as mining activities.
An appreciable amount of the soils has been made unusable because of pollution. Highly
contaminated soils belong to a high healthy risk to human being and their environmentally
harmful effects. That is why soil should be correctly understood and underestimated long
range lethal effects that can have irreversible consequences. The improvement of soils
damaged and contaminated by pollutants need of the particular soils, requires a full under‐
standing of soil properties and of the deteriorating factors.
Mercury is a microelement: its Clark value in the Earth’s crust is 56 μg Kg-1 (Fügedi et al,
2011). It is characterized by a dual geochemical behavior: it is liable to extreme concentration
6 Soil Pollution
and to dispersion, the latter resulting in an approximately entirely even concentration. It is

found either as a native metal (near to 80 % in hydrothermal and vapors) or in cinnabar,
corderoite, livingstonite and other minerals. Cinnabar (HgS) is the most common ore. Mercury
ores usually occur in very young orogenic belts where rock of high density on upper mantle
is forced to the crust of the Earth (Ozerova, 1996).
Given that mercury is enriched by an extremely wide variety of geological processes

(Fergusson, 1990) from the formation of hydrocarbon to hydrothermal mineral occurrences, it
can be regarded as an universal geochemical indicator of young geological effects; its disper‐
sion halos are more extensive than that of any other element (Fügedi et al, 2011).
Historically there were two main registered Mercury mines: Almaden (Spain) and Idrija
(Slovenia) in Europe. Later new occurrences were found in California and worldwide. It was
used in gold separation. In Mexico we know mercury mines from the Pre-Hispanic era (Scharek
et al., 2010) and a usage in cultic fests (Figure 3). In 2005, China was the top producer of mercury
with almost two-thirds global share followed by Kyrgyzstan. Several other countries are
believed to have unrecorded production of mercury from copper electro winning processes
and by recovery from effluents.
Figure 3. Typical cinnabar occurrences in a limestone system (Formation Las Trancas, San Joaquin, Querétaro, Mexico,
photo by P. Scharek)
4. Mobilization and loadings the mobilized mercury
The mobilization of the different mercury forms can due to by evaporation and dissolution.
Here we do not deal with the erosion which able to mobilize all form of mercury if it has
attached to the solid particle, however in case of soils and fly as particles in the flue gas this
particle associated mobilization mechanism can play important role in the mercury transport.
4.1. Evaporation
The evaporation governed by the vapor pressure depends on the volatility of the compound
and the temperature. Concerning the volatility of the different mercury compounds evapora‐
tion at ambient temperature can be significant in case of elemental and the organic mercury
cases, however the volatilization of the other mercury forms (the inorganic mercury com‐
pounds) can become considerable if the temperature reaches a couple hundred degree
centigrade. All of the mercury compounds have relatively low boiling points (Table 1), some
of them decompose before melting, others can sublimate. Based on these data it is obvious the
vaporization can play important role of the mercury compound transport and mobilization.
Melting T, ˚C Boiling T, ˚C
Hg
-38.9 356.5
HgS (cinnabar, α) 580 sublimate
HgS (metacinnabar, β) 446 sublimate
Hg2O 100 decompose
HgO 500 decompose
Hg SO4 450 decompose
Hg2Cl2 302 384
HgCl2 277 304
HgBr2 237 322
HgI2 259 354
CH3HgCl - 92
(CH3)2Hg -43 94
Table 1. Melting and boiling points of the mercury and mercury compounds
The partial pressure of the elemental mercury (Hg0) reach 1 Pa at 42 ˚C and enhances expo‐
nentially till the boiling point (Tb = 356.5 ˚C). At 20 ˚C the Hg vapor pressure is 0.18 Pa the Hg
concentration in the air saturated with the mercury is 7.64 10-8mol dm-3 = 15.3 μg m-3. Due to
this high volatility the elemental mercury evaporate if stored and processed an open container.
Elemental mercury can escape from solution if the oxidized mercury is able to reduce. The
8 Soil Pollution
analytical data will be inaccurate if the sample is not preserved agents the elemental mercury
formation.
Concerning the global mercury contamination till the middle of the past century evapora‐
tion of elemental mercury used to extract silver and gold was the main source of the
mercury emission, (Nriahu, 1994). All the once produced and recently available elemental
mercury stock (in the past five century one million tons was produced from cinnabar and
from other ores) if in used either evaporation or after transformation can contribute to the
mercury contamination worldwide (Hylanderand Meili, 2003). This is the reason why the
elemental mercury use is banned. Recently one of the most significant sources of mercu‐
ry emission by evaporation is the coal firing. During the coal burning the mercury associated
with pyrite and be organically bonded to the coal minerals are released in the combus‐
tion flame as elemental mercury, which is partially oxidized to Hg(II) in homogeneous and
heterogeneous catalytic reaction governed by the chlorine and the ash content of the
combustion gases (Sondreal et al, 2004).
Generally accepted view is that the evaporated oxidized forms of mercury contaminate the
environment locally, close to the emission source. However they can transform to elemental
mercury and depend on this transformation rate it can become part of the global mercury cycle.
The transformation of the oxidized to reduce the reduced to oxidized forms can happen both
in gas and aquatic environment according to the circumstances. There is similar transformation
between the inorganic and organic forms.
Different species of macro algae from the dissolved mercury can produce different methylated
mercury compounds in the ocean. Because of these methylated mercury compounds have high
volatility and at the dimethylated form has low solubility in ocean water they are easily emitted
into the atmosphere and can contribute significantly to the global atmospheric mercury
(Pongratz and Heuman, 1998). Beside this different bacteria (e.g. sulfate reducing) and in case
of abiotic route the tin- alkyls and the humic acids also can transform the dissolved mercury
(II) to methyl mercury form (Weber, 1993). A quite detailed set of possible transformation in
gas and aquatic media and the Henry constants which inform about the dissolved compound
volatility are collected by Shon at al, 2005.
During heating mercury compounds can transforms directly or via oxides to elemental
mercury. Beside the elemental mercury only the halogenides and the sulfides since last have
a tendency to sublimate can occur in evaporated forms. The sulfides at presence of oxygen at
600 °C transforms to Hg and SO2, however in presence of Fe and CaO the HgS also will
decompose to Hg and Fe- or Ca- sulfides. Using the temperature programmed evaporation
technique based on the volatility difference of mercury compounds the compound forms can
be distinguished and can use for mercury speciation in solids (Lopez-Anton et al, 2010, 2011).
In a high temperature process since the mercury compounds decompose the original specia‐
tion of mercury does not preserve a new speciation can formed which determined by the gas
composition. In the high temperature gases high portion of mercury exists in elemental and
just a small portion in oxidized form. This is the reason why these technologies such as coal
fired energy production, the cement kiln, the incineration has difficulty in the mercury capture.
Focusing to the soil, the heating comes from sunlight can mobilize only the weakly sorbed
elemental and organic mercury but the fire on the soil surface, for example the forest fire can
evaporate the less volatile mercury forms as well. This case the contamination level of the fired
soil decreases but, due to the transport, at other places the contamination becomes higher
(Caldwel et al, 2000).
The volatilization can be the cause of contamination but can use for decontamination as well.
Based on the volatilization of mercury compounds, mercury removal process was established
from coal cleaning by mild pyrolysis (Wang et al., 2000) and for the soil cleaning by thermal
treatment. In case of coals the speciation of mercury determines the maximum efficiency of
the mercury removal. The efficiency of the process generally remains below 100 %, (bituminous
coal case at 500 ˚C it was aprox. 75 %). Since the efficiency remains below 100 % the rest of
mercury still remain in the process and pass to the flue gas after the coal burning. The speciation
of the mercury in the contaminated soil also has influence on the efficiency of the thermal
remediation, see more details later.
Concerning that the different mercury forms exhibit different volatility the actual distribution
of the mercury species in a medium the rate of the transformation process which able to modify
it together govern the mercury mobilization by evaporation.
It is well known, if elemental mercury forms in the water this elemental mercury can easily
escape to the gas phase. It is quite intensive if gas bubbling through the water or the water
surface is disturbed (Okouchi and Saaski, 1984). Sunlight induced H2O2 formation in alkaline
condition can result reduction of the oxidized mercury forms to elemental mercury. This can
explains that the Hg concentration above the lake water surface can be higher day time than
night. The fulvic and humic compounds are able to complex the mercury (II) ion in aquatic
media but these compounds can take part in the mercury alkylations, further at a suitable pH
can work as a reducing agent. The redox potential at 0 pH for Hg (II) reduction is 0.85 V(Allard
and Arsenie, 1991).
This type of mercury transformation between oxidized and reduced forms together with the
alkylation will generate not only a modification between the concentrations of the mercury
species in the aquatic phase but will modify the mercury transport between the phases. The
mercury transformation processes are important in the technological processes used for the
mercury removal since can effect they efficiency (Somoano et al., 2007).
4.2. Dissolution
The mobilization by dissolution can arrange two groups: (a) dissolutions ways exist in the
nature (b) dissolution way can be applied in the laboratory and in the remediation technology
to determine the loading forms or remove the mercury from the contaminated media.
4.2.1. Dissolutions ways exist in the nature
The solubility of elemental mercury and the ore of mercury can find in the nature (cinnabar
etc.) are very low in water. This low solubility result low mercury concentration level in aquatic
10 Soil Pollution
phase and restricts the transport between phases by dissolution. However the oxidation both
cases enhances these mercury forms solubility. The elemental mercury can be oxidized by
ozone, halogens, some components of acid rains, or by oxy-acids in laboratory (HNO3 and the
hot H2SO4) resulting a soluble form. The ozone in air if does not consumed by the other more
reactive air contaminants can oxidize Hg to Hg(II) (Iverfeld and Linquist, 1986; Shonet al, 2005).
In aquatic media oxidation can occur at acidic conditions if the sunlight produces oxidative
radicals OH, or peroxides. This process can play role in the trap of the physically dissolved
elemental mercury in water, and also can hinder the transformation of the oxidized mercury
forms towards the reduced elemental mercury direction. The oxidative transformation of
elemental mercury is essential in case of many mercury capture process since the oxidized
forms of mercury has higher tendency to sorb and dissolve, therefore different oxidation
procedures are available and applied in the demercurysation technologies (Ko et al, 2008;
Lakatos et al, 2009; Sondreal et al, 2004).
However the mercury in the natural minerals is in the oxidized forms these minerals luckily
due to the very low solubility can be considered not a mobile occurrence of the mercury. The
environmental risk improves if the natural processes can transform the minerals a more soluble
form. One of the most significant ore transformations which effect the mercury mobilization
is the sulfide ore oxidation in the air. The oxidations of sulfides to sulfate a considerable
enhancement ensue in mercury solubility (Holley et al, 2007). This process, the oxidation of
the tailings, can accused for the mercury contamination all around the abandoned mercury
ore mines.
Solubility in Water
Compound
c, ppm
Hg 0.049*
HgS (cinnabar, α) 0.01
HgS (metacinnabar, β) -
HgSO4 -
Hg(NO3)2 soluble
Hg2O 51
HgO 51
Hg2Cl2 10
HgCl2 66 000*
HgBr2 5 100
HgI2 51
CH3HgCl 5 780
(CH3)2Hg -
*Solubility from paper of Ko et al, 2008.
Table 2. Solubility of different mercury compounds.

Among the mercury compounds (Table 2) the mercury-chloride and nitrates are those which
have the highest solubility in water. The simple cationic form of Hg(II) is not the common form
in the aquatic media, it exist only in acidic solutions, at less acidic condition the dissolved
mercury appears as HgOH+, HgOHCl, Hg(OH)2 and HgCl2 molecules and complex anions
HgCl42- at high chloride concentration. It means that the sea water contains the oxidized
mercury mainly in this chlor- complex form. Beside the chlor- complex the mercury -fulvo and
-humic complexes also exists in aquatic environment. The speciation in the solution, the
molecular forms govern the mercury loadings and play important role at the way and
efficiency of the removal.
4.2.2. Dissolution for leaching mercury from different medium

Beside the thermal way and the application of the species sensitive analytical methods for
mercury analysis (XPS, EXAFS etc.) the sequential extraction is often applied technique to
specify the mercury chemical form and associations in solids. The thermal methods and the
species sensitive elemental analysis can distinguish the elemental Hg, the HgS forms and the
organically bonded mercury forms which generally exist in the soil. However these techniques
do not allow doing any estimation about mobility and bioavailability of mercury. To get the
loading specific information in soil for mercury, beside the classical Tessier six step extraction
used generally, different modified procedure are available for Hg which able to distinguish
better the mercury forms than the Tesssier method can do (Orecchio and Polizzotto, 2013; Han
et al,2006). For example it can determine mercury bounded to amorphous iron oxides (by
NH4 oxalate-oxalic acid extraction), mercury bonded to crystalline iron oxides (by
NH2OH•HCl- 25 % acetic acid extraction), non-cinnabar mercury (elemental mercury, organic
bounded, humine bounded (by 4 M HNO3 extraction), cinnabar mercury (by extracted with
saturated Na2S,Han et al, 2006). The advantage of this protocol is the ability of the separation
of humic and sulfide bounded mercury which important in the soil case. Two set of sequential
extraction regime can compared at Table 3 and 4.
Mobilization of mercury can occur through complex formation, ligand exchange reactions with
chloride and sulfur-containing ligands which leading to enhanced Hg solubility in soil
solutions. The sulfur containing ligands: tiosulfates (S2O32), thyocyanites (SCN-) can mobilize
the mercury efficiently and could improve the phytoextraction efficiency (Moreno et al, 2004).
Removal mercury by phytoextraction from soils and others soil like materials eg. waste water
plants biosolids often need additives which improve the solubility of the mercury. These
mobilizations agents are used in accelerate phytoextraction. One type is the chelating agents:
citrate, oxalate, malate, succinate, tartarate, salicilate, acetate, and amino-poly-carboxylic acids:
EDTA (Lomonte et al, 2011). Since the EDTA is persistent compound, recently the biodegrad‐
able ethylendiamine-disuccinate (EDDS) or nitrilotriacetic acid (NTA) suggested as alternative
chelator instead of EDTA (Evangelu et al, 2007).
Specific compounds used the mercury extraction from tissues: they can pay role in case of
poisoning for detoxification : EDTA was tested for detoxification by Aposhian (Aposhian et
al,1995), 2,3- dimercapto- 1- propansulfonate was used to extract mercury from tissues of rats
exposed to different mercury compounds (Buchet and Lauwerys, 1989). The EDTA was not
12 Soil Pollution
Mobilization Protocol Determination of Association of Mercury to Soil Component
Loading form of mercury Extraction procedure
Water soluble mercury NH4-acetate

Exchangable mercury (1 M NH4-acetate pH 7 set with NH4OH: solid:liquid 1:25, 30 min 25 ºC)
Carbonate bounded mercury

Hidroxylamine – HCl
Easily reducible oxides bounded mercury
(0.1 M NH2OH.HCl +0.01 M HCl solid:liquid1:25, 30 min 25 ºC)
( Mn-oxides)
H2O2
Elemental and organic bounded mercury (3 mL 0.1 M HNO3+5 mL 30 % H2O2 80 ºC 2 h; 2 mL H2O2 80 ºC 1 h; 50
mL 1M NH4- acetate)
NH4-oxalate –oxalic acid

Amorphous iron- oxide bounded mercury
(0.2 M oxalate buffer 1:1, pH 3.25 solid:liquid1:25)
Hydroxilamine – HCl- acetic acid (hot)

Crystalline iron oxide bounded mercury
(0.04 M NH2OH.HCl in 25 % acetic acid 97-100 ºC 3 h solid:liquid 1:25)
Non cinnabar bounded mercury

4 M HNO3
( Hg, organically bounded, humin bounded)
(4 M HNO3 80 ºC16 h solid:liquid1:25)
TOT (non cinnabar mercury)
Na2S
Cinnabar bounded mercury
(4 mL saturated Na2S 12 h repeated twice)
Table 3. Mobilization protocol for determination of association of mercury to soil component (Han et al, 2006).
Mobilization Protocol Determination of Association of Mercury to Soil Component
Loading form of mercury Extraction procedure
Water soluble mercury H2O100ºC, 1h stirred, solid:liquid 1:8
Na-acetate
Exchangable mercury
(1M Na-acetate, 1 h stirred, solid:liquid1:8
Na-acetate –aceticacidpH 5
Carbonate bounded mercury
(1M Na-acetate –acetic acid pH 5, 4 h stirred, solid:liquid1:8)
Fe, Mn-oxide bounded Hydroxylamin HCl - acetic acid

mercury 0,04 M NH2OH•HCl in 25 % Acetic acid, 96 °C, 6 h, Solid:liquid 1:8
Mineralisation by HNO3-H2O2
(a)heated previously 180 ºC - organic bounded - microwave digestion
Elemental and organic bounded mercury
in cc HNO3-H2O2 mixture; (b) no heat - elemental + organic bounded -
microwave digestion in cc HNO3-H2O2 mixture)
Aqua regia
Sulfid bounded mercury
(HCl:HNO3 3:1)
Table 4. Mobilization protocol for determination of association of mercury to soil component (Orecchio and
Polizzotto, 2013).
found the best for mercury removal in human application since does not the best chelator for
Hg and has side effects, however there are two other compounds which suggested to keep in
stock in any poison control center as mercury chelator DMPS (2,3-dimercapto-1-propane
sulfonic acid, (unithiol)) and DMSA (meso-2,3 dimercapto succinic acid, succimer, Guzzi et al,
2010).
4.3. Loadings of mercury

The mercury contaminations are in the environment can exist different phases: as vapor in the
air (Hg, and compounds, particle associated), dissolved in aquatic media (Hg2+, Hg(OH)2,
HgCl2, HgCl42-, different complexes, particle associated) and solid as precipitates or minerals
and in associated forms bonded to different manner to the component of different solids (soil,
fly ash, waste water sludge, etc). In the previous section it was demonstrated that how the
associations can be identified.
The loadings of mercury to solid can be considered as positive or negative phenomenon. It can
restrict the dispersion of the contamination one side it is positive, the negative this way it can
preserve the contamination. Since the loadings depends on the character of the collector and
the speciation of the mercury, difficult to establish general rules for this process. However it
can state that the elemental mercury has low sorption ability, the cationic sorbs better than the
anionic forms on clays, and negatively charged carbon surfaces (coals, activated carbons,
humic materials) the loading is more effective to that surfaces which have contain sulfides. It
was interesting findings after the cinnabar oxidation a part of liberated mercury could load to
the cinnabar surface this way it can be not just the source but the collector of mercury ions.
Unfortunately the most toxic forms (alkyls) have the highest ability for bioaccumulation.
The nature works against the mercury contamination. Except the alkylation it transforms the
mercury toward the most stable less soluble form. Near the chlor-alkali plant the total mercury
sometimes reach the four order of magnitude higher level, than the background concentration,
luckily it found a non-volatile and non-soluble associations since transforms to sulfides
(Bernaus et al, 2006).
The history of mercury contamination is recorded by loadings. The dept profile of mercury
concentration on peat can provide a clear picture how the mercury contamination changed
during the mankind history (Barraclough et al, 2002).
The loadings play important role in the environmental technologies used for decrease the
mercury emission or clean the contaminated medium. Sulfur and halogen containing carbons,
oxidative inorganic sorbents (Lakatos et al, 2009) were developed for elemental mercury
removal from flue gas. Beside a range of, classical, functionalized sorbents, sulphur containing
carbon nanotubes widen the collection one can chose among for eliminate the mercury
contamination in aquatic media (Pillay et al, 2013).
The coals especially the low rank and the oxidized coals are very good mercury ion collectors.
Due to this feature we must face that the coal-firing are the main source of the anthropogenic
mercury contamination nowadays. However this material offers us an application for cure a
slice of the mercury problem: remove the mercury from aquatic media. It can use in batch mode
14 Soil Pollution
or dynamic systems as the reactive barrier material, by the high mercury capture coal are able
to retard or remove the aquatic mercury contamination (Lakatos et al,1999).
5. Remediation of polluted soil with mercury
The most commonly used techniques for the remediation of mercury contaminated soils have
been classified as either excavation techniques or containment techniques, and are grouped as
follows (Hinton and Veiga, 2001): excavation and ex-situ treatments, containment and in situ
chemical treatment (Figure 4).
Figure 4. Representation of the techniques for the remediation of mercury contaminated soils: (A) excavation and ex-
situ treatments, (B) containment and (C) in situ chemical treatment.
5.1. Excavation and ex situ treatments
They will be treated off-site soil contaminated when removing soil or contaminated soil-like
materials to a place outside the place in which they are located, for submission to authorized
treatment fixtures. (Hinton and Veiga, 2001):
1. Physical separation. Mercury has affinity for the smallest particles of soil.
2. Thermal treatment. The volatility of mercury increases with increasing temperature;

therefore, a heat treatment technique of excavated soil is a potentially effective technique
for the removal of mercury in soils.
3. Hydrometallugical treatment. Chemical extraction of mercury in contaminated soils can be

induced by four mechanisms: desorption of adsorbed species, oxidation of metallic
mercury, use of strong complexing agents, and dissolution of Hg precipitate. The
efficiency and mechanism employed decrease with respect to time due to recomplexation,
readsorption and removal of the soluble fraction. Two of the two most promising
hydrometallurgical techniques are electrokinetics and electroleaching / methods of
leaching.
4. In situ recuperation. In situ techniques have not been studied as much as ex situ techniques
due to surface heterogeneity and longer treatment times. However, in situ techniques may
be promising due to better cost-effectiveness and practicality.
5. Vapor extraction coupled with evaporation. Vacuums are used in unsaturated zone to remove
volatile and semi-volatile contaminants.
6. Permeable reactive barriers. Dissolved compounds react with compounds found on the walls
and then precipitate out. This technique has been employed for the treatment of sites
contaminated with organic compounds and metals. These barriers are placed perpendic‐
ularly to the flow of contaminants.
7. In situ leaching and extraction. Used together with pumps in treatment, this method uses
chemicals injections to improve the solubility of mercury in groundwater.
8. Electrokinetic separation. This process involves the generation of an electric field by

applying a potential difference or current into a soil matrix. Metals such as mercury
migrate towards electrodes placed in the soil where they accumulate and can be removed
at a lower cost by excavating the affected area.
9. Interceptor systems. Interceptor systems such as ditches and drains are simple and effective
for the recovery of mercury as free product, but these treatments are limited by site
topography and stratigraphy.
10. Phytoremediation. Some plants have the ability to assimilate and concentrate metals in soil.
The recovery of these metals occurs after collecting and incinerating the plants.
11. Passive remediation of the wetlands. Using wetlands to immobilize mercury is a controversial
topic as some wetlands contain microorganisms that can convert mercury into even more
toxic species.
5.2. Containment
In the containment treatment the soils are treated on one side of the contaminated site, where
the processing is performed on an area adjacent to the contaminated site or an area within the
contaminated site upon removal of soil or soil-like materials. In this classification are (Hinton
and Veiga, 2001):
16 Soil Pollution
1. Pump and treat. With certain contaminants or systems, pollution removal is not possible
and it is necessary to protect hydraulic content. When the contaminant mass remains in
the subsurface, pump and treat systems can prevent site contamination.
2. Impermeable barriers (sealed surfaces and drainage). Mud barriers are slightly permeable
barriers made of bentonite or cement-bentonite mixtures. Generally, these barriers are
between 0.5 and 2 m thick and have a maximum depth of 50 m. There are other types of
barriers that are constructed by injection molding or by vibratory forces. On the other
hand, surface seals and drainage are used to controlling filtration and limit pollutant
movement towards groundwater.
3. Stabilization and solidification. Stabilization and solidification techniques use both in situ or
ex situ conditions by mixing impacted sites. Stabilization attaches contaminants to the soil
structure, which usually decreases soil permeability. Moreover, solidification improves
the physical characteristics of materials such as mudor sediments; they can be excavated
and transported more easily.
4. Sediment covering. In situ covering involves placing an insulating layer over the contami‐
nated material.
5.3. In situ chemical treatment
Another option is the in situ chemical treatment option, which is the name of all treatments
that involve the injection of a chemical reagent into an aquifer source upstream of the conta‐
minated site. This chemical agent reacts with the contaminant, transforming it into an innoc‐
uous form; eventually, it can pump through a given volume of water which can later be
recycled for injection. The following actions must be considered:
1. Increase the output rate of the ground water through the contaminated zone by increasing
the hydraulic gradient through injection and extraction.
2. Transform the contaminant using chemical reaction within the aquifer.
5.4. Electroremediation of polluted soil with mercury
Electroremediation has been successfully applied in a variety of soil restoration studies, this
methodology having the advantage of exhibiting simultaneous chemical, hydraulic and
electrical gradients. Indeed, for efficient mercury removal from a saturated soil with electro‐
remediation, application of either an electric field or direct current through two electrodes
(anode and cathode) is required. These are usually inserted in wells containing a supporting
electrolyte made from inert salts, leading to improved electric field conductive properties
(Rajeshwar et al, 1994; Huang et al, 2001; Acar and Alshawabkeh, 1993).
Furthermore, since electroremediation is a physicochemical technique based on ion transport,

it is an excellent tool for the removal of inorganic species, such as Hg+2 (Rajeshwar et al, 1994;
Bustos, 2013). The main advantages of electroremediation, as compared with other soil
treatment procedures, are (Huang et al, 2001; Acar and Alshawabkeh, 1993; Ibañez et al, 1998;
Segall and Bruell, 1992; Cabrera – Guzmán et al, 1990): (1) electroosmotic flow is not dependent
on either pore or particle size, (2) hydraulic gradient is enhanced by electromigration, (3)
treatment can be applied in situ, (4) it can be applied to low permeability soils, (5) there is
minimal disruption of normal activities at the site, (6) the required investment is usually lower
than that for other conventional treatments, and (7) it can be applied in conjunction with
techniques such as pumping, vacuum extraction or bioremediation.
The processes taking place during electroremediation can be classified into two main catego‐
ries: (a) processes occurring as a consequence of the applied electric potential. These processes
include electromigration (ion transport), electroosmosis (mass transport), and electrophoresis
(charged particle transport); (b) processes occurring in the absence of an electric potential. This
includes concentration induced processes like diffusion, sorption, complexation, precipitation
and acid - base reactions (Reed et al, 1995; Bustos, 2013).
Specifically, for mercury polluted soil electroremediation, the use of complexing agents like
ethylendiaminetetraacetic acid (EDTA), KI, and NaCl under a constant potential gradient has
been reported (Reddy et al, 2003). Based on the above precedents, the electroremediation was
developed aided by extracting agents for mercury removal from San Joaquin’s Sierra Gorda
soil samples (Figure 5, Robles et al, 2012).
Electroremediation of mercury polluted soil, facilitated by the use of complexing agents,

proved to be an attractive alternative treatment for the removal of mercury from polluted soil
in mining areas located at Sierra Gorda in Queretaro, Mexico (Figure 5A and 5B). Implemen‐
tation of this remediation protocol is expected to improve the living conditions and general
health of the population in the Mine “El Rincón” in San Joaquin (Figure 5C). Experimental
observations suggest that it is possible to remove up to 75 % of metal contaminants in mercury
polluted soil samples by wetting them with 0.1 M EDTA, placing them in an experimental cell
equipped with Ti electrodes, and then applying a 5 V electric field for 6 hours (Figure 5D,
Robles et al, 2012). When we followed the electrochemical removal of mercury in a batch reactor
(Figure 6A), it was removed around 87 % of Hg2+ in a time of 9 hours close to the anode side
by the presence of EDTA (Figure 6B). The pH remains nearly constant at 4 and conductivity
showed values close to 10 mS cm-1 by the ionic species.
The efficient removal of mercury contaminants observed under these conditions is attributed
to electromigration of the coordination complexes that form between the terminal hydroxyl
groups in EDTA and divalent mercury (Hg+2), which is probably strengthened by supramo‐
lecular interactions between unshared electrons at EDTA’s tertiary amino nitrogens and Hg
+2
. These interactions are particularly effective with the presence of potassium ions. This
observation is supported by molecular modeling of several possible interactions in the
proposed complex using the Density Functional Theory method (B3LYP LANL2DZ, Robles et
al, 2012, Figure 7).
18 Soil Pollution
B C
A D
Figure 5. Localization of Queretaro in Mexico (A) with satellite image from San Joaquin’s Sierra Gorda, Queretaro (B)
where there is the Mine “El Rincón” (C) with high concentration of Hg2+, which was removed with electroremediation
process in continues flow in presence of EDTA (D).
Figure 6. Electroremediation process in batch reactor assisted by EDTA (A), and its corresponding removal percentage
of Hg2+ followed during 13 h of treatment, close to anode and cathode.
Figure 7. Optimized conformation and molecular structure of the proposed 2 Hg+2 / 2 EDTA / 4 Na+ complexes (B3LYP
LANL2DZ, Robles et al, 2012).
6. Conclusions
Mercury is a non-essential metal that can bioacumulated in living organisms, causing toxic
effects of various kinds. Therefore, it is vital to understand how this metal is transmitted
through the environment and the changes that occur due to contact with living organisms, or
environmental conditions such as high temperatures or strong winds. A study of the reactions
that form organic or inorganic compounds, which are even more toxic, is also necessary to
limit mercury toxicity. This research gives a brief overview of the techniques commonly used
for treatment of sediment and soil contaminated with mercury, mobilization and loadings the
mobilized mercury across different matrixes of environment, in specially electroremediation
of mercury polluted soil, facilitated by the use of complexing agents as EDTA, proved to be
an attractive alternative treatment for the removal of mercury from polluted soil in mining
areas.
Acknowledgements
Unidos Mexicanos (CONACyT), L’Oreal, Academia Mexicana de Ciencias (AMC), Fundación
México – Estados Unidos para la Ciencia (FUMEC) and the International Cooperation Program
across Bilateral Cooperation Mexico – Hungary for the funding of this research. The contri‐
bution of J. L to this research was (partially) carried out in the framework of the Center of
Excellence of Sustainable Resource Management / Applied Materials Science and Nano-
Technology / Mechatronics and Logistics / Innovative Engineering Design and Technologies
at the University of Miskolc. I. Robles is grateful to CONACyT for her scholarship.
20 Soil Pollution
Author details
I. Robles1, J. Lakatos2, P. Scharek3, Z. Planck3, G. Hernández4, S. Solís4 and E. Bustos1*
1 Centro de Investigación y Desarrollo Tecnológico en Electroquímica, S. C. Parque Tecnoló‐

gico Querétaro S/N, Sanfandila, Pedro Escobedo, Querétaro, Mexico
2 University of Miskolc, Faculty of Material Sciences and Engineering, Institute of Chemis‐

try, Miskolc, Hungary
3 Geological and Geophysical Institute of Hungary, Budapest, Hungary
4 Centro de Geociencias, Campus UNAM-Juriquilla, Querétaro, Qro., Mexico
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ments of the Seine estuary (France). Applied Organometallic Chemistry, 13 (10), 715 –
725.
[55] Weber J. H. (1993). Review of possible path for abiotic methylation of mercury(II) in
the aquatic environment. Chemosphere, 26, 2063-2077.
chapter 30
Conducted and Investigate Arsenic (As), Cobalt (Co)

Copper (Cu), Manganese (Mn), Lead (Pb), and Zinc (Zn)
Contamination in Agricultural' Soils and Wheat Crops
Projects at Three Regions in Libya
Youssef F. Lawgali, Andy A. Meharg and

Yousef M.T. Elgimati
1. Introduction
Libya is one of the driest countries in the world [2]. The rainfall in the northern part of the
country varies between 100–500 mm/year, while the southern region receives only 10 mm/year
and some parts receive no precipitation at all [17]. Average evaporation rates are also high,
ranging from 1.7 mm/day in the north to 6.9 mm/day in the south, much higher than the
average annual rainfall. Irrigation water use has increased as part of the green revolution
program to make a Libya self-sufficient in terms of food production, e.g. for wheat and barley.
Agriculture in Libya developed remarkably during the last two decades, as can be seen from
the large increase in the irrigated areas, which has doubled during the period 1975–2005
[11,15]. The cultivated area has also increased, from 282,250 ha in 1975 to 470,000 ha in 2000
[15]. Increased agricultural activities have resulted in higher demands for groundwater,
irrigation water and applications of fertilizer and have also resulted in higher salinity (nitrate,
chloride, Na, Ca, Mg, ammonia) and increases in some trace elements in soil [3]. The devel‐
opment in agriculture has occurred at great cost to the environment, and concern is now being
expressed about the impact of agricultural chemicals. Whereas agricultural production
increased from 1137 thousand tons in 1975 to 1779 thousand tons in 2000, the agrarian
productivity per hectare decreased from 4 tons/hectare in 1975 to 3.5 tons/hectare in 1980 and
1990, and then showed a slight increase to 3.7 tons/hectare in 2000. Libya’s annual import of
fertilizer is about 2 × 108 metric tons, including fertilizer nutrients N, P205, and K20.1. [5,6-7].
[6] Observed that application of fertilizers to dry land not only increases plant shoot and stem
growth, but also increases evapotranspiration through development of a larger root system
and greater extraction of stored water. [4.14] found that the amount of N and P fertilizer used
in the Montanan North African soils ranged from 20–120 kg/ha and 0–30 kg/ha, respectively.
However, under irrigated conditions, critical P levels are high (i.e. 12–15 mg/kg). The high pH
affects the availability of many crop nutrients in the southern Libyan soil [13].
The objective of the present study was to establish levels of As and other trace elements in the
grains and soils of the Libyan arable belt. Soil and wheat samples were collected from 3 Libyan
agricultural regions, East (ESR), West (WSR) and South (SSR). Each region included several
sampling districts (Fig.1).
2.1. Sample preparation
Seventy-nine samples each of soils, shoots and wheat grains from arable land were collected
for the study, representing major soil types and climatic regions of agronomic cultures in the
Eastern, Western and Southern soil regions of Libya (ESR, WSR and SSR, respectively). Each
region included several sampling districts (52, 10 and 17, respectively). Soil samples were taken
from the root zone of each sample, and within the soil horizon (0–100 mm), using a spiral auger
of 20–50 mm diameter. Three samples were taken around the plant to make composite soil
samples and stored in airtight bags. The sampling was carried out between May 26th and June
30th in 2007. The sampling points are shown in Fig. 1.
M SF
BEN SLQ DRN
WSR TH
ESR
MAK ARL
TAS
Figure 1. Soil survey sampling Figure

locations1Soil survey sampling locations in Libya.
in Libya.
All samples were air-dried and homogenized by grinding and sieving through a 2 mm mesh. A soil sample of 0.2 g
was placed in a digestion tube, 2 ml concentrated HNO3 added and the sample pre-digested (i.e. left to stand
overnight). 2 ml of H2O2 was added immediately before digestion on a digest block at 100 °C for 1 hour, 120
°Cfor 1 hour and then at 140 °C until samples were clear (c. 2 hours). Samples were then heated to 160 °C until the
sample digest appeared clear or a pale soil color.
Quality control and assurance

Each batch of 40 samples digested included a blank, a spike and a soil reference material (NCS ZC 73007) (Table
1). The samples were allowed to cool, decanted into well-labelled 15 ml centrifuge tubes and made up to 10 ml
with ultra pure decant deionised water (18.2 M) obtained from a Milli-Q system (Millipore) and analysed by ICP-
MS. If the outcome of the SRM NCS ZC 73007 was above 25 % of the certified values, the analysis of the batch
Conducted and Investigate Arsenic (As), Cobalt (Co) Copper (Cu), Manganese (Mn), Lead (Pb), and Zinc (Zn)... 3
All samples were air-dried and homogenized by grinding and sieving through a 2 mm mesh.
A soil sample of 0.2 g was placed in a digestion tube, 2 ml concentrated HNO3 added and the
sample pre-digested (i.e. left to stand overnight). 2 ml of H2O2 was added immediately before
digestion on a digest block at 100 °C for 1 hour, 120 °Cfor 1 hour and then at 140 °C until
samples were clear (c. 2 hours). Samples were then heated to 160 °C until the sample digest
appeared clear or a pale soil color.
Quality control and assurance
Each batch of 40 samples digested included a blank, a spike and a soil reference material (NCS
ZC 73007) (Table 1). The samples were allowed to cool, decanted into well-labelled 15 ml
centrifuge tubes and made up to 10 ml with ultra pure decant deionised water (18.2 M)
obtained from a Milli-Q system (Millipore) and analysed by ICP-MS. If the outcome of the
SRM NCS ZC 73007 was above 25 % of the certified values, the analysis of the batch was
rejected.
Soil sample analysis by ICP-MS
Cd, Pb, Cu, P, Zn, Co, Mn and As concentrations were measured in all soil sample digestions
by An ICP-MS Agilent™ 7500c (Agilent Technologies, Tokyo, Japan) octopole reaction system
(ORS)-ICP-MS with a Meinhard nebulizer was used to measure the elements Ti (m/z 49), Mn
(m/z 55), Co (m/z 59), Cu (m/z 63), Zn (m/z 64, 66, 67, 68), As (m/z 75), Se (m/z 77, 78, 82) and
In (m/z 115). Hydrogen (H) was employed as the reaction gas for the ORS, set at a flow rate of
1.8 ml/min. With this method, low detection thresholds can be obtained for elements such as
metalloids that have high ionization energies (e.g. As).
Soil As extraction
Soil extraction to determine As fractionation was performed. Sample material was oven-dried
at 90 °C for 48 hrs. Extracts were obtained by shaking 1 g of soil with 10 ml of 0.1 % HNO3, as
described by [10]. The soil was mixed with 10 ml of 0.1 % HNO3 in 15 ml plastic centrifuge
tubes shaken at a speed of 60 rpm for 24 hrs. After shaking, soil solids were separated from
the solution by centrifugation at 2958 g for 15 min. The concentrations of As were determined
as described above for the soil digestion. Quality controls (spikes and blanks) were run with
each digest set of 40 samples. Concentrations were determined using five point calibrations.
Soil As extraction and analysis by hydride generation (HG)

The 0.1 % NHNO3 water-soluble As fraction was determined by hydride generation (HG,
Perkin-Elmer A. Analyst 300) [10]; in order to achieve this, the system must first be calibrated
using a standard curve. AsV was reduced to AsIII by a mixture of hydrochloric acid (HCL) (30
% v/w), potassium iodide (KI) (10 % w/v) and ascorbic acid (5 % w/v). The solution was
pumped into a gas-liquid separator and reacted with sodium borohydride and sodium
hydroxide (NaBH4) (0.2 % w/v solution in 0.1 M NaOH) to from arsenic hydride generation
[10]. Sample concentrations were calibrated using a standard curve of 0.5, 1, 5, 10, 15 and 20
μg/l As, in 10 % HCl 1 % KI and further diluted where necessary. All As concentrations were
below the limit of detection (LOD).
Soil As and other element extraction and analysis by ICP-MS
Since the analysis by hydride generation of all Asconcentrations were below the LOD, As and
other elements (Cd, Pb, Ni, Cu, P, Zn, Cu, Co and Mn) were analysed with ICP-MS.
The analytical procedures used ICP-MS to extract As and other elements extraction from the
soil are identical to those outlined in section 3.2.4 (Soil As extraction), as are the aristar grade
reagents used.
Minitab v. 15 was used for all statistical analyses.
3. Results
The certified reference material (CRM) as well as the LOD and spike recoveries for each of the
trace elements analysed are presented in Table 1.
Co Zn Cu As Cd Pb Mn
CRM (%) n=4 87 ±0.1 66 ±1.7 87 ±1.3 103 ±0.2 101 ± 0.01 90 ±1.2 88 ±5.1
Spike (%) n=4 97 ±0.7 96 ±7.4 112 ± 3.1 123 ±2.5 81 ±0.1 97 ±1.2 104 ± 5.4
LOD (mg/kg) 0.01 0.30 0.20 0.01 0.01 0.03 0.14
Table 1. Quality control measurements for analysis of Co, Zn, Cu, As, Cd, Pb and Mn concentration in Libyan
agricultural soils. (CRM = NCS ZC 73007).
It should be noted that the concentration of Cd in all samples was below the LOD. The
descriptive statistics for trace element concentrations in agricultural soil investigated in this
study. They are provided for each region, with a summary of the As levels and other trace
element concentrations in soils sampled from ESR, WSR and SSR. A one-way analysis of
variance (ANOVA) showed that the differences in As levels between location means are highly
significant (P<0.001,). ESR recorded the highest mean As the level in Libyan soil (8.10 ± 0.48
mg/kg), followed by WSR (1.97± 0.41mg/kg) and SSR (1.70 ± 0.58 mg/kg), i.e. As mean
concentrations in ESR are nearly 4.8 and 4.1 times higher than in SSR and WSR, respectively.
Conversely, the average As levels from the three regions were below 10 mg/g dry weight (dwt).
The concentration of As in the agricultural soil, therefore, ranged from 0.01–18.94 mg/kg in
ESR, 1.19–2.48 mg/kg in WSR and 0.73–2.92 mg/kg in SSR.The underlying rock structure in
ESR is composed of Lower Palaeozoic rocks. The clay agricultural soils of ESR recorded the
highest mean As the level in Libyan soil (8.10 mg/kg); this may have some relation to the parent
material.
10
n=52
4
n=10
n=17
2
Mean As concentration (mg /Kg)
0
ESR SSR WSR
Sampling sites
Figure 2. Mean As concentrations in agricultural soils from Libya (Error bars represent ± std. error).
The descriptive statistics for trace element concentrations in the Libyan agricultural soil
investigated in this study are shown in Appendix.1, together with a summary of the levels of
As and other trace elements.
3.1. Southern soil regions (SSR)
In SSR, the sites used for sampling were ARL, JAF and TAS (sites are shown in Fig.1). The
mean As levels in sites in SSR decreased from TAS > JAF >ARL. In general, As levels were low
in SSR, ranging from 1–2.9 mg/g, 0.7–1.8 mg/g and 1.1–1.7 mg/g for the TAS, JAF and ARL
sites, respectively.
3.2. Eastern soil regions (ESR)
The sampling sites in ESR were BEN, DRN, HEN, MR, SF and SLQ (sites are shown in Fig.1).
The mean As levels in these sites decreased from SF > DRN > BAY > MAR > HEN > BEN >SLQ.
In general, As levels in ESR were high, ranging from 0.01 to 18.9 mg/kg.
Western soil regions (WSR)
Samples in WSR were taken from two sites, THR and SIRT (Fig.1). WSR recorded the second
highest mean total As level compared with ESR and SSR (2 ± 0.1 mg/kg vs.8.1± 0.50 and 1.7±
0.14, respectively). The mean As levels in sites in WSR decreased from THR > SIRT ( range 2–
2.5 mg/g and 1.2–2.1mg/g for THR and SIRT, respectively).
Results of analysis of variance (ANOVA)

A one-way analysis of variance (ANOVA) was performed on each of the three regions studied.
For SSR, the ANOVA showed that that the mean As levels between sites were not significantly
different (SSR: P=0.398), but were for ESR and WSR (P<0.0016, P<0.015, respectively).
Multivariate analysis results
Principal component analysis (PCA)
PCA was applied in each region to assist in identifying sources of elements. 3-D plots of the
PCA loadings are presented in Fig.3, where the relationships among the six trace elements are
readily seen. As expected, four factors were obtained, accounting for 97.4, 98.0 and 99.4 % of
the total variance in ESR, SSR and WSR, respectively. In ESR, Factor 1 is dominated by Co, Cu,
Pb and Mn, while in SSR it was dominated by Co, Zn, As and Pb and in WSR by Co, Zn, As
and Mn (accounting for 67.4, 69.9 and 90.7 % of the total variance, respectively). In each area,
the loadings of certain minerals were lower than the others, which may, therefore, imply a
quasi-independent behaviour within the group. These elements were Cu and Mn (0.42 and
0.4, respectively) for ESR, Co and Zn (0.5 and 0.4, respectively) in SSR and Zn and As (0.4 and
0.4, respectively) in WSR.
Factor 2 is dominated by Zn and As in ESR, by Mn in SSR and by Cu and Pb in WSR (accounting
for 23.1, 15.3 and 6.0 % of the total variance, respectively). Factor 3 is dominated in ESR by As
and Pb, by Zn As and Cu Pb in SSR and by Zn and As in WSR (accounting for 4.1, 7.7 and 1.4
% of the total variance, respectively). Zn is negatively correlated with Cu in SSR, and with As
in WSR. In each region, certain elements (As and Zn in ESR, Mn in SSR and Cu in WSR) were
separated from the others by a large distance in the 3-D PCA loading plot, suggesting that
these elements are poorly correlated and may have different sources (Fig.3). Factor 4 is
dominated by Cu, As and Mn (accounting for 2.7 % of the total variance; Mn is negatively
correlated with Cu and As) in ESR, Zn, As and Pb (accounting for 5.4 % of the total variance;
Zn and Mn are negatively correlated with As and Pb) in SSR and Pb and Mn (accounting for
1.3 % of the total variance; Mn is negatively correlated with Pb) in WSR.
Pearson's correlation matrix for the trace element concentrations in agricultural soils from
Libya.
The correlations between trace element concentrations in agricultural soils showed a high,
positive, linear relationship (Table 2). To determine whether geochemical associations exist
between the different trace elements analysed in this study, the datasets for agricultural soils
from each region and soils from ESR, WSR and SSR were subjected to simple correlation
analysis. A scatter plot for the trace element correlations that were found to be significant
(P<0.05) in some of the soils studied are presented in Fig.4. The correlation analysis was also
performed on the composite data sets of all agricultural soils for this study. The Pearson
correlation coefficients and P-values for Co, Zn, Cu, As, and Pb were significant and all the
analyses are shown in Table 2.
Manganese
A summary of trace element Pearson correlation coefficients of soils from different regions of
Libya are shown in Table 2, Fig.4 and Appendices 3.11, 3.12 and 3.13.Mn concentration showed
1.0 Cu
0.8
0.6 Mn Pb
0.4 As
PCA 3
0.2
0.0 Co
Zn
-0.2 1.0
-0.4 0.8
0.6
-0.6 0.4
1
0.2
A
0.0
C
1.0 0.8 -0.2
P
0.6 0.4 -0.4
0.2 0.0 -0.2 -0.6
-0.4 -0.6
PCA
2
1.0
As
0.8
Cu
0.6
0.4 Mn Pb
PCA 3
0.2 Co
0.0
-0.2 Zn
1.0
0.8
-0.4 0.6
0.4
-0.6
1
0.2
A
0.0
1.0 0.8
PC
-0.2
0.6 0.4 -0.4
0.2 0.0 -0.6
-0.2 -0.4
-0.6
PCA
2
1.0 Pb
0.8 Zn
0.6 Co
0.4
PCA 3
Cu
0.2
0.0 Mn
As
-0.2 1.0
-0.4 0.8
0.6
-0.6 0.4
1
0.2
A
1.0 0.8 0.0

C
0.6 0.4 -0.2

P
0.2 0.0 -0.4

-0.2 -0.4 -0.6
-0.6
PCA
2
Figure 3. PCA loading plots for the three rotated components for a) SSR, b)WSR and c) ESR.
Figure 3. PCA loading plots for the three rotated components for a) SSR, b)WSR and c) ESR.
Pearson's correlation matrix for the trace element concentrations in agricultural soils from Libya.
a significant correlation with Co, Zn, Cu and Pb in WSR and ESR. However, Mn is not
correlated with AsTheand Pb in ESR
correlations or SSR,
between tracerespectively, or with in
element concentrations theagricultural
other metals (Co, Zna and
soils showed high, positive,
Cu) in SSR, reflecting
linear different
relationshipsources
(Table 2).for Mn and the
To determine other
whether elements.
geochemical Moreover,
associations existMn is not
between the different
trace elements analysed in this study, the datasets for agricultural soils from each region and soils from
correlated with As and not significant in ESR and SSR, which may suggest a common origin
ESR, WSR and SSR were subjected to simple correlation analysis. A scatter plot for the trace element
(Table 2 and Fig.4).
correlations that were found to be significant (P<0.05) in some of the soils studied are presented in Fig.
Lead .4. The correlation analysis was also performed on the composite data sets of all agricultural soils for
this study. The Pearson correlation coefficients and P-values for Co, Zn, Cu, As, and Pb were significant
The Pb concentration showed
and all a significant
the analyses correlation
are shown in Table 2. with Co, Zn, Cu and As concentrations
in all three of the agricultural soil regions (Fig.5a, b & c and Table 2).
Manganese
Arsenic
As is not correlated with Co and Cu in ESR; however, As concentration showed a significant

correlation with Zn concentrations in all three of the soil regions. Trace element correlations
with As were slightly varied across regions. As is also positively correlated with Co, Cu and
Zn in SSR and WSR (Table 2 and Fig.6a, b & c).
Zinc
Zn concentration correlated well with Co and Cu in soils from WSR and SSR sites but not with
those from ESR (Table 2). The Cu-Zn and Zn-Co correlations, however, remained significant
(P<0.01) when the analysis was performed on the composite data sets (Figs 3.7 and 3.8,
respectively). [6]Reported that in Libya, depending on the crop and soil, farmers use an average
of 100 kg/hectare of phosphate fertilizer annually.
ESR WSR SSR
Mn-Co (+ C * ) (+ C * ) (+ 0 NS)
Mn-Zn (+ C * ) (+ C * ) (+ 0 NS )
Mn-Cu (+ C * ) (+ C * ) (+ 0 NS )
Mn-As (+ 0 NS ) (+ C * ) (+ C NS )
Mn-Pb (+ C * ) (+ C * ) (+ 0 NS )
Pb-Co (+ C * ) (+ C * ) (+ C * )
Pb-Zn (+ C * ) (+ C * ) (+ C * )
Pb-Cu (+ C * ) (+ C * ) (+ C * )
Pb-As (+ C * ) (+ C * ) (+ C * )
As-Co (+ 0 * ) (+ C * ) (+ C * )
As-Zn (+ C * ) (+ C * ) (+ C * )
As-Cu (+ 0 * ) (+ C * ) (+ C * )
Cu-Co (+ C * ) (+ C * ) (+ C * )
Cu-Zn (+ 0 * ) (+ C * ) (+ C * )
Zn-Co (+ 0 * ) (+ C * ) (+ C * )
+: positive correlation (significant at P< 0.05)

–: negative correlation (significant at P< 0.05)
NS: not significant at P< 0.05
*:significant at P< 0.05
0: no correlation
C: correlation (r> 0.50)
Table 2. Summary of trace element correlation in soils from different regions of Libyan soils (see for correlation
coefficients and P-values).
20 80
18 a b
70
16
60
14
50
12
Zn
40
Co
10
8 30
6
20
4
10
2
0 0
0 200 400 600 800 1000 0 200 400 600 800 1000
Mn Mn
30
20
c d
25
15
20
10
Cu
15
As
10 5
5 0
0
0 200 400 600 800 1000
0 200 400 600 800 1000
Mn
Mn
30
e
25
20
Pb
15
10
5 ESR
SSR
0 WSR
0 200 400 600 800 1000
Mn
Figure 4. Mn correlations against other trace elements in agricultural soils from ESR, SSR and WSR sites.
20 80
18 a b
70
16 60
14
50
12
Zn
40
Co
10
8 30
6 20
4
10
2
0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Pb Pb
30 20
c d
25
15
20
10
Cu
15
As
5
10
5 0
0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Pb Pb
1000
e
800
600
Mn
400
ESR
200 SSR
WSR
0
0 5 10 15 20 25 30
Pb
Figure 5. Pb correlations against other trace elements in agricultural soils from ESR, SSR and WSR sites.
20 80
18 a b
70
16
14 60
12 50
Co
10
Zn
40
8
30
6
4 20
2 10
0 0
0 5 10 15 20 0 5 10 15 20
As As
1000
30
c e
25 800
20
600
Mn
Cu
15
400
10
200
5
0
0 0 5 10 15 20
0 5 10 15 20
As
As
30
d
25
20
Pb
15
10
ESR
SSR
5
WSR
0
0 5 10 15 20
As
Figure 6. As correlations against other trace elements in agricultural soils from ESR, SSR and WSR sites.
20 80
18 a b
70
16
60
14
12 50
Co
10
Zn
40
8
30
6
20
4
2 10
0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Cu Cu
20 30
c
d
25
15
20
10
Pb
15
As
5
10
0 5
0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Cu Cu
1000
e
800
600
Mn
400
200 ESR
SSR
WSR
0
0 5 10 15 20 25 30
Cu
Figure 7. Cu correlations against other trace elements in agricultural soils from ESR, SSR and WSR sites.
20 30
18 a b
25
16
14
20
12
Cu
Co
10 15
8
10
6
4
5
2
0 0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80
Zn Zn
30
20 d
c 25
15
20
10
Pb
15
As
5 10
5
0
0
0 10 20 30 40 50 60 70 80
0 10 20 30 40 50 60 70 80
Zn
Zn
1000
e
800
600
Mn
400
ESR
SSR
200 WSR
0
0 10 20 30 40 50 60 70 80
Zn
Figure 8. Zn correlations against other trace elements in agricultural soils from ESR, SSR and WSR sites.
Overview of total shoot, soil and grain As for ESR, SSR and WSR
A one-way analysis of variance (ANOVA) was performed on each of the three regions studied.
The ANOVA showed that that the mean As levels for grain between sites were not significantly
different (P=0.662 ), but that those for soil and shoot were (soil: P<0.001; shoot: P<0.002). Fig.9
shows a comparison of the distribution of As concentrations in the shoot, soil and grain from
ESR, WSR and SSR.
100
80
Cumulative distribution (%)
60
40
20
0
0 5 10 15 20
Soil A s (mg/kg)
100
80
60
40
20
0.0 0.1 0.2 0.3 0.4

Shoot As (mg/g)
95
85
75
65
55
45
35
25
15
5
0.0 0.1 0.2 0.3 0.4 0.5
Grain As (mg/kg)
Figure 8 Cumulative ranked distribution of As concentrations in Libyan agricultural soil, shoot and grain
Figure 9. Cumulative ranked distribution of As concentrations in Libyan agricultural soil, shoot and grain from ESR
from ESR (solid), WSR (dash) and SSR (square dot).
(solid), WSR (dash) and SSR (square dot).
13
The mean shoot As content for SSR (range 0.07–0.35 mg/kg; mean: 0.19 ± 0.02 mg/kg) was higher
than for ESR (range 0.04–0.34 mg/kg; mean: 0.13 ± < 0.01 mg/kg) and WSR (range 0.01–0.19 mg/
kg; mean: 0.12 ± < 0.01mg/kg). At the 50th percentile, shoot As content for SSR (0.17 mg/kg) was
more than 1.5 times that recorded for ESR (0.12 mg/kg) and WSR (0.11 mg/kg; Fig.9).
ESR recorded the highest mean As level in Libyan soil (8.10 ± 0.48 mg/kg, range 0.01–18.94 mg/
kg), followed by WSR (1.97± 0.41 mg/kg, range 1.19–2.48 mg/kg) and SSR (1.70 ± 0.58 mg/kg,
range 0.73–2.92 mg/kg), i.e. mean As concentrations in ESR are nearly 4.8 and 4.1 times higher
than in SSR and WSR, respectively.
The mean grain As content for SSR (range 0.02–0.2 mg/kg; mean: 0.07±0.01 mg/kg) was also
significantly higher (P< 0.01) than for ESR (range 0.02–1.1 mg/kg; mean: 0.05 ± 0.02 mg/kg) and
WSR (range 0.02–0.04 mg/kg; mean: 0.02 ± < 0.01mg/kg). At the 50th percentile, grain As content
for SSR (0.05 mg/kg) was over twice that recorded for ESR (0.02 mg/kg) and WSR (0.02 mg/kg;
Fig.9).
The ranges of total grain, shoot and soil As concentrations over the three regions studied were
relatively wide; however, the distribution was markedly skewed, with 65 % in ESR having
grain, shoot and soil As concentrations of < 0.03, 0.17 and 9.9 μg, respectively, 21 % in SSR <
0.09, 0.28 and 1.8, respectively and 12 % in WSR < 0.02, 0.16 and 2.4, respectively (Fig.9).
As transfer from soil to shoot and grain
The range of As shoot/soil transfer factors (shoot/soil TFs) was considerable. Minimum and
maximum shoot/soil TFs were 0.06 for ESR, 0.04–0.29 for SSR and 0.04 for WSR, while means
and medians were 0.1 and 0.02, respectively, for ESR; 0.13 and 0.12 for SSR and 0.06 and 0.06
for WSR. In SSR, the median shoot/soil TF was nearly twice as high as that in ESR and WSR.
In this study, transfer of As was an order of magnitude greater from shoot to grain, despite
lower rates of soil to grain transfer. The differences in these transfer ratios may be due to
differences in As speciation. [18],reported that, in general, higher shoot As levels were
consistent with low grain/shoot transfer factors.
Estimation of bioavailable As concentrations by nitric acid extraction
Nitric acid extraction is a widely used method of estimating the bioavailable concentration of
elements. The precision of the results in the present work was controlled by analysis of CRM,
blank and spike samples. The correlation factors showed good precision, with the exception
of two analytical bioavailable and total results (Table 3). A summary of the bioavailable As in
soils sampled from ESR, WSR and SSR are shown in Table 3. A one-way analysis of variance
(ANOVA) showed that the differences in As levels between location means are highly
significant (P<0.001, Table 3). SSR recorded the highest mean levels of bioavailable soil As (0.05
± 0.01 mg/kg) (Fig.10). In contrast, the highest total mean As level was recorded in ESR (8.10
± 0.48 mg/kg;.Table 3 and Fig.11b). The mean bioavailable As concentrations in the three
regions studied decreased from SSR > ESR > WSR (Table 3) and was in general low, ranging
from 0.02–0.07 mg/g, 0.02–0.03 mg/g and 0.01–0.14 mg/g for SSR, ESR and WSR, respectively.
Bioavailable concentrations of As are 1–5 % of the total concentrations. In this study, the highest
median bioavailable / median total (B/T) As was recorded in SSR (Table 3, Fig.10 and 3.11b).
Regions
T T T B B B (B/T)*100
Elements
mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg ra B/Tb
Mean Median Range Mean Median Range %
As ESR 7.81 8.45 24.36 0.02 0.01 0.13 0.1 0.2
SSR 4.05 2.48 6.50 0.05 0.05 0.05 –0.4 2.0
WSR 4.42 3.96 7.16 0.02 0.02 0.02 –0.1 0.5
Co ESR 11.49 11.63 15.11 0.76 0.02 5.59 0.4 0.2
SSR 2.27 2.27 2.26 0.10 0.02 0.33 –0.4 0.9
WSR 2.28 2.33 2.26 0.02 0.02 0.01 0.9* 0.7
Cu ESR 15.39 15.32 23.12 0.29 0.06 2.30 0.3* 0.4
SSR 4.28 4.22 7.21 0.08 0.05 0.33 –0.1 1.2
WSR 4.46 3.07 7.57 0.04 0.05 0.05 0.6 1.5
Pb ESR 14.36 14.06 24.11 0.22 0.01 2.98 0.2 0.1
SSR 3.05 3.04 3.36 0.01 0.01 0.06 –0.2 0.3
WSR 3.39 3.46 1.69 0.00 0.01 0.01 –0.6 0.3
Mn ESR 492.30 504.60 780.60 58.50 8.50 300.50 0.5* 1.7
SSR 117.80 111.40 241.50 14.17 1.34 72.57 0.7* 1.2
WSR 95.66 96.89 82.96 1.15 0.98 2.50 –0.3 1.0
Ni ESR 0.72 0.10 4.32
SSR 0.19 0.15 0.29
WSR 0.09 0.10 0.03
P ESR 7.66 1.95 105.61
SSR 52.80 33.40 154.40
WSR 8.92 9.96 12.27
a
* Correlation is significant at the 0.05 level
b
B/T= Median bioavailable/ Median total %
Table 3. The descriptive statistics for total (T) and bioavailable (B) trace element concentrations in Libyan soils (mg/kg).
There was no correlation between the mean total and the bioavailable As concentrations in
ESR and WSR. However, there was a negative correlation between the mean total and the
bioavailable As in SSR. [16]. reported that appreciable As can move with leaching water,
especially in coarse-textured soils. The mean and median bioavailable As concentration values
for SSR were close to the those for WSR and ESR (Table 3). The correlation factors between the
bioavailable and the total element concentrations were: 0.073, –0.419 and –0.097 (As); 0.371, –
0.349 and 0.892 (Co); 0.318, –0.124 and 0.614 (Cu); 0.24, –0.161 and –0.545 (Pb); and 0.466, 0.659
and –0.309 (Mn) for ESR, SSR and WSR, respectively (Table 3, Fig.10). Adriano (1986) reported
that As mobility and phytotoxicity is usually greater in sandy than in clayey soils. The SSR
samples had high B/T bioavailable As, Co, Pb and Mn contents, compared to B/T total elements
(Table 3 &Fig.10). In these samples, the levels of bioavailable elements were derived from P
fertilization ) [12]. The low correlation value for total Cd is because the concentrations were
lower than the ICP-MS detection limit. Weathering will increase bioavailability of trace
elements in SSR. [1],found that at P:As ratios of 4:1 or greater, phytotoxicity on wheat was
markedly reduced and deduced phytotoxicity to be a function of P concentration.
0.16 6
a b
0.14 5
0.12
4
0.10
Co "total"
As "total"
3
0.08
0.06 2
0.04
1
0.02
0
0.00
0 5 10 15 20 25 30 0 2 4 6 8 10 12 14 16 18 20
As "bioavailable" Co "bioavailable"
2.5 3.5
c d
3.0
2.0
2.5
1.5
2.0
Pb "total"
Cu "total"
1.0 1.5
1.0
0.5
0.5
0.0
0.0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Cu "bioavailable" Pb "bioavailable"
350
e
300
250
Mn "total"
200
150
100
50
ESR
0 SSR
WSR
0 200 400 600 800 1000
Mn "bioavailable"
Figure 10. Scatter diagram of total vs. bioavailable trace element concentrations in agricultural soils from ESR, SSR
and WSR.
10
a
As mg/kg
6
0
East South West
0.06
b
0.05
0.04
As mg/kg
0.03
0.02
0.01
0.00
East South West
Figure 11. Comparison of (b) soil extractable of As with (a) total soil concentrations in Libya
4. Discussion
Total As mean concentrations in ESR are nearly 4.8 and 4.1 times higher than in SSR and WSR,
respectively. Total As loads in ESR may be related to the soil parent material, which is
comprised of subsequently covered Lower Palaeozoic rocks. This may be the reason why the
clay agricultural soils of ESR have the highest background levels of As. This is in agreement
with [8], who reported higher concentrations of trace elements in Eastern Libyan soil than in
other areas. In the present study, the trace elements present in Eastern Libyan soil (Co, Zn, Cu,
As, Cd, Pb and Mn) were nearly five times higher than in WSR and SSR. Trace element
concentrations in agricultural soils showed positive linear correlations (Table 2).
In this study, transfer of As was an order of magnitude greater from shoot to grain, despite
lower rates of soil to grain transfer. In addition, this study considered samples of soil, shoot
and wheat grains from arable land with a sampling design that allowed for the consideration
of intra-field variations. The differences in these transfer ratios may be due to differences in
As speciation. [18]reported that, in general, higher shoot As levels were consistent with low
grain/shoot transfer factors. Although this similarity in trends for plants from varying
geographical locations strongly suggests the involvement of plant physiological regulation,
the actual mechanisms involved are still far from clear. However, the findings from the present
study indicate that As transfer to grain isgoverned by multiple factors (Fig. 7.1). The bioavail‐
ability of trace elements in soils is influenced by a wide array of biophysical factors (Fig. 7.1),
including pH, redox potential, organic matter content and soil texture. These factors were,
however, not considered in the experimental/sampling design since the main thrust of the field
studies was to establish the contribution of elevated soil As to grain As levels.
In this study, the highest median bioavailable / median total (B/T) As was recorded in SSR
(Table 3, Fig.10 and 3.11b).[15]reported that appreciable amounts of As can move with leaching
water, especially in coarse-textured soils. This is in agreement with the [9], which reported that
As mobility and phytotoxicity is usually greater in sandy than in clayey soils. The SSR samples
had high B/T bioavailable As, Co, Pb and Mn contents, compared to B/T total elements (Table
3 &Fig.10). In these samples, the levels of bioavailable elements derived from P fertilization
(Hurd-Karrer 1939) and the high pH affects the availability of many crop nutrients in the
southern Libyan soil [12]. Weathering will increase bioavailability of trace elements in SSR.
[10]found that at P:As ratios of 4:1 or greater, phytotoxicity on wheat was markedly reduced
and deduced phytotoxicity to be a function of P concentration.
The present study agrees with [16], who reported that the available soil moisture holding
capacity for silty clay loams is three times higher than for loamy sands. Therefore, in the present
study, total As mean concentrations in ESR are nearly 4.8 times higher than in SSR. Total As
loads in Libyan agricultural soil may be related to the soil moisture holding capacity, with
different types of soil having varying capacities and also different levels of As.
5. Future research
• To investigate the differential efficiency with which the main species of As found in
Mediterranean conditions wheat are transferred to the grain, resulting either from remobi‐
lised shoot As, or coming direct from the soil.
• Effects of As on As uptake, speciation, and nutrient uptake by winter wheat (Triticum
aestivum L.) under hydroponic conditions.
• Determination and comparison of total As concentrations and speciation in different parts
of the wheat plant at various stages of growth and in different genotypes.
• Future research needs to focus on the balance of trace elements in an agro-ecosystem,

elaboration of soil chemical and biochemical parameters that can be used to diagnose soil
contamination with or deficiency in trace elements, and quantification of trace metal
transport from an agro ecosystem to the environment.
• Investigate the relationship between mobility and leaching of As and soil profile depth.
• Set up baseline concentrations of As in Libya soil and wheat. This is of great importance
since Libya, as a nation, consumes an enormous quantity of rice.
• The determination of the trace elements iron, zinc, phosphors, cobalt and cadmium in
Libyan arable soils is essential for the management of fertilizers and pest side in that soils
projects.
Acknowledgements
I wish to express my deepest gratitude to:Friends: Dr Youssef Gamtey Benghazi University,for

his assistance and guidance in statistical.To Mrs Susan McCourt of the Queen Mother Library,
University of Aberdeen, for her assistance and guidance in the use of the Refworks software
and to Mrs Alison Sage for her copy editing. Libyan Agricultural Projects (Aril, Maknowsa,
Tasswat, Bengzh, Darna Srit and Sluq) for soil and plant sampling.
Author details
Youssef F. Lawgali1*, Andy A. Meharg1 and Yousef M.T. Elgimati1
1 Department of plant production, University of Sirte, Libya
2 School of Biological,Queen's University Belfast, UK
3 Benghazi University, Libya
References
[1] Adriano, D.C., Bolan, N.S., Vangronsveld, J. and Wenzel, W.W. (2005). Heavy metals.
In: Daniel Hillel, ed, Encyclopedia of Soils in the Environment. Oxford: Elsevier, pp.
175-182
[2] Atkinson K, Waugh B. Morphology and mineralogy of red desert soils in the Libyan
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[3] Beke GJ, Entz T, Graham DP. Long-term quality of shallow ground water at irrigated
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116-128.DOI.org/10.1061/(ASCE)0733-9437(1993)119:1(116)
[4] Cooper JR, Gilliam JW. Phosphorus redistribution from cultivated fields into riparian
areas. Soil Science Society of America Journal. 1987;51(6):1600-4. DOI.10.2136/
sssaj1987.03615995005100060035x
[5] Cooper PJM, Gregory PJ. Soil water management in the rain-fed farming systems of
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10.1111/j.1475-2743.1987.tb00711.x
[6] El-Ghawi U, Pátzay G, Vajda N, Bódizs D. Analysis of selected fertilizers imported to

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Radioanalytical and Nuclear Chemistry. 1999;242(3):693-701.DOI:10.1007/BF02347381
[7] El-Ghawi, U., Vajda, N. and Pátzay, G. (1999). Determination of some trace elements
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[8] El-Ghawi UM, Bejey MM, Al-Fakhri SM, Al-Sadeq AA, Doubali KK. Analysis of Lib‐
yan arable soils by means of thermal and epithermal naa. Arabian Journal for Science
and Engineering. 2005;30(1 A):147-53..
[9] Environment CCoMot. Arsenic. Canadian Water Quality Guidelines for the Protec‐
tion of Agricultural Water Uses. 1999.
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sessment of bioavailable arsenic and copper in soils and sediments from the Antofa‐
gasta region of northern Chile. Science of the Total Environment. 2002;286(1-3):
51-9.DOI.org/10.1016/S0048-9697(01)00962-7
[11] Huda FS. Development of Rural Areas and Societies in Libya. Szent Istvan Universi‐
ty [Online]. Available: http://phd.okm.gov.hu/disszertaciok/tezisek/2005/tz_hu1937.
pdf [9/2009, 2009]. 2004.
[12] Hurd-Karrer A. Antagonism of certain elements essential to plants toward chemical‐

ly related toxic elements. Plant Physiology. 1939;14:9-29Doi: 10.1104/pp.14.1.9
[13] Phosyn. Wadi Aril Prodction Project. May 2005. York - UK: Phosyn.. 2005.
[14] Ryan JaM, A.E. Fertilizer use efficiency under rainfed agriculture in West Asia and
North Africa, ICARDA, ed. In: The Fourth Regional Workshop, Aleppo, Syria: ICAR‐
DA.. 1992.
[15] Shaki AA, Adeloye AJ. Mathematical modelling of effects of Irawan irrigation project
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chapter 31
Risks of Heavy Metals Contamination of Soil-Pant

System by Land Application of Sewage Sludge: A Review
with Data from Brazil
Alysson Roberto Baizi e Silva and Fábio Camilotti
1. Introduction
The discharge of urban sewage directly to rivers and lakes is among the principal causes of
surface water contamination. Contaminated water resources threaten the water supply of cities
and the ecological equilibrium of aquatic ecosystems. To minimize or avoid these negative
impacts, the sewage needs to be treated.
Sewage treatment is increasing to the extent that is necessary to maintain the water quality.
However, despite purifying municipal wastewater, making it suitable for discharge in
receiving water bodies such as rivers and lakes, the sewage treatment process generates a large
volume of sludge that needs to be destined appropriately and quickly to avoid its accumulation
and consequently its transformation into environmental liabilities in wastewater treatment
plants (WTPs).
The options for disposal of sewage sludge are varied. In general, they include: (i) land
application, (ii) industrial reuse, (iii) disposal in landfills, (iv) incineration and (v) discharge
to oceans [1]. Land application and disposal in landfills are the most widely adopted disposal
methods in various parts of the world [2, 3]. The application to land is considered the most
attractive option, because the sewage sludge can improve soil conditions for agricultural
production, since it is rich in organic matter and plant nutrients [4, 5]. However, there are
undesirable constituents in its composition.
Heavy metals, such as cadmium (Cd), lead (Pb), among others, are the most concern undesir‐
able constituents [5-7], since they may be toxic to microorganisms, plants, animals and humans
in not very high concentrations [8-10]. Therefore, soils amended with sewage sludge should
be evaluated for heavy metal contamination in order to prevent its excessive entry into the
food chain, thus reducing the risk of toxicity in living organisms posed by the method of
disposal of sludge on land.
The evaluation of heavy metals in soil can be made in several ways. The basic and most
common is to quantify the total concentration [11]. The great advantage of this measure is its
robustness, that is, it does not easily change with environmental conditions, but it may be
altered by the addition of metals from external sources, such as sludge, which allows its use
as an indicator of soil contamination by heavy metals. However, the total concentration not
satisfactorily represents the amount of metal that would be available for uptake by plants, that
is, the fraction of contaminant which could cause phytotoxicity and enter the food chain,
affecting animals and humans. Due to its direct relationship with potential toxic effects, the
available concentration should be determined in addition to total concentration.
The availability of heavy metals to plants has been characterized using chemical extractants,
some of them already employed in routine soil analysis, as Mehlich 1, Mehlich 3 and DTPA.
In general, extractants are (i) acid, (ii) chelating agents, (iii) acid-chelating or (iv) saline
solutions [11]. The chemical nature of the extracting solution interferes with the ability of
extracting metals and, ultimately, the efficiency of the extractant to represent the available
fraction. Therefore, the extractants should be systematically tested before being used in
monitoring heavy metals in soils amended with sewage sludge.
Fractionation is also interesting technique to evaluate heavy metals in soils. Its principle is
to separate the metals in soil fractions in which they have variable solubility [12]. With this
procedure, it is possible to determine the contribution of each fraction in the availability of
metals to plants [13, 14]. It also allows selecting the best chemical extractant, based on its
relationship with the fractions that most contribute to uptake of metals by plants. More‐
over, the redistribution of metals among fractions in response to changes in soil condi‐
tions can be studied [15]. Thus, the fractionation can indicate whether the metals added to
the soil by the sludge are to be redistributed in fractions in which they are either more or
less available, that is, whether they have either greater or lesser potential to cause toxici‐
ty problems respectively [7, 16].
The study of speciation is another interesting strategy to evaluate heavy metals in soils, since
it enables to distinguish different chemical species in the soil solution. Each species has a
particular chemical behavior in terms of availability and mobility in soil. Free ions are more
relevant to the availability of metals, because they are the preferred forms of plant uptake [11].
In contrast, organo-metal complexes are more related to the mobility of metals and, conse‐
quently, to their leaching [17]. Thus, the speciation can indicate if the risk of phytotoxicity and
contamination of the food chain is higher or lower than the risk of groundwater contamination,
based on the proportion of the chemical species formed in response to application of sewage
sludge.
Besides the contamination of soil, it is also necessary to evaluate whether the sewage sludge
applied to land can contaminate crops with heavy metals. The assessment of crop contamina‐
tion must include studies of differential capacity of uptake, translocation, accumulation and
allocation of metals in different plant species. In the case of allocation, it is essential to evaluate
Risks of Heavy Metals Contamination of Soil-Pant System by Land Application of… 3
the metals of interest in the harvested parts, especially those that are edible [18]. As a result of
these investigations, plants can be separated by their susceptibility to contamination with
heavy metals and thus the indication of using less susceptible plants in areas that receive
sewage sludge can be made.
In Brazil, the sewage treatment has grown considerably in recent years. In 2001, 25.6 % of the
sewage generated were treated [19]. Ten years later, in 2011, this index had risen to 37.5 % [20].
Growth should remain strong, since there is a significant amount of resources to be invested
in sanitation in the country [21]. The expansion in sewage treatment causes inevitable increase
in the generation of sewage sludge.
The Brazilian production of sewage sludge is estimated at 150-220 thousand tons per year (dry
basis) [22] with a perspective to increase. As in other parts of the world, land application has
been one of the preferred forms of sludge disposal [1]. As seen above, this option requires a
careful evaluation of heavy metals in soils amended. In recent decades, many experiments
have been conducted in Brazil to evaluate the effects of sewage sludge on heavy metals in soils
and crops. The results of these works can help technicians to manage more safely sludge
application to land.
Thus, our objective was to review the scientific literature on the impacts of sewage sludge on
heavy metals in soils and plants in conditions of Brazil, attempting to evaluate the risks of
contamination of the soil-plant system. We focused on studies involving field experiments
because they represent better real situations of management of the sludge.
2. Heavy metals: An overview
Heavy metals comprise a class of not very well defined chemical elements. They have been
commonly characterized as metals and metalloids (semimetals) with density higher than 5 g
cm–3 associated with problems of environmental contamination and toxicity, although some
of them are required in low concentrations for some organisms such as higher plants, animals
and humans [23, 24]. However, this definition (attempt) has been criticized for its chemical
and toxicological inadequacy and it has had no institutional support of IUPAC (International
Union of Pure and Applied Chemistry) [25]. Despite the criticism, there is no more appropriate
term to refer collectively to the elements known as heavy metals. Therefore, we use this
terminology in this text and support its use in the definition introduced by Hawkes [26],
according to which heavy metals comprise a block in the Periodic Table with all metals and
metalloids in Groups 3 to 16 that are in periods 4 and greater. As some authors consider
selenium (Se) it as a metalloid and therefore heavy metal [10, 27], we also consider it as
metalloid in this chapter, even though it is a non-metal. Thus, we expand the definition of
Hawkes [26] to include Se as a heavy metal, keeping in mind which this redefinition is simply
operating, just to join in the same class specific chemical elements commonly associated to
problems of environmental contamination and toxicity.
Heavy metals can be divided according to their need for different organisms. There are those
which are doubtless essential and those which are not recognized as essential (Table 1). Copper
(Cu), iron (Fe), manganese (Mn), molybdenum (Mo) and zinc (Zn) are essential to plants,
animals and humans. Cobalt (Co) and Se are essential only to animals and humans, while
chromium (Cr) and nickel (Ni) are essential to humans and plants, respectively. In contrast,
arsenic (As), Cd, Pb and mercury (Hg) are not essential to any of these organisms. However,
essential or not essential metals may be toxic. For example, manganese (Mn) is an essential
element (i.e., micronutrient) to plants, but its excessive uptake can cause toxicity in crops [28,
29]. On the other hand, As, Cd, Pb and mercury (Hg) are not essential to humans but in excess
can also cause toxicity. People exposed to Pb or Hg develop neurological disorders, while
exposure to Cd is associated with kidney damage and fragile bones, and various forms of
cancer can occur due to the ingestion of food or water contaminated with As [30]. A summary
of the toxicity of heavy metals commonly associated with environmental contamination is
given in Table 1.
Heavy metal
Organism† Essentiality‡ Toxicity§ Reference
(Symbol)
Increased oxidative stress and reduced plant
Plants No [31-33]
growth.
Arsenic (As)
Animals No Blindness and reduced weight gain. [9]
Humans No Increased cancer risk. [30]
Plants No [34, 35]
growth.
Cadmium (Cd)
Animals No Reduced weight gain. [36]
Humans No Kidney damage and fragile bones. [30]
Plants No [9, 37]
growth. Cr(VI) is more toxic than Cr(III).
Chromium (Cr) Animals No ? [38]
Allergy and increased cancer risk. Cr(VI) is more toxic
Humans Yes [39-41]
than Cr(III) or Cr(V).
Chlorosis in younger leaves (restricted Fe
Plants No [42, 43]
translocation) and reduced plant growth.
Cobalt (Co)
Animals Yes Body weight loss and muscular incoordination. [9, 44]
Humans Yes Cardiomyopathy and Increased cancer risk. [45]
Intervenial chlorosis in younger leaves, reduced
Plants Yes branching, thickening, darkening of rootlets and [9, 43, 46]
reduced plant growth.
Copper (Cu)
Animals Yes Gastroenterits, liver damage and death. [9, 47]
Wilson’s disease, hemolysis, hepatic necrosis and
Humans Yes [9, 48]
kidney damage.
Leaf bronzing, roots with black coating and reduced
Plants Yes [9, 49]
plant growth. Common in flooded rice.
Iron (Fe)
Anorexia, diarrhea, metabolic acidosis, reduced
Animals Yes [9, 44]
body growth rate and death.
Heavy metal
(Symbol)
Vomiting, diarrhea, metabolic acidosis and
Humans Yes increased risk of atherosclerosis and Alzheimer’s [9, 50]
disease.
Chlorosis, root system darkening, stunted plant
Plants No [51]
growth and increased oxidative stress.
Appetite loss, diarrhea, anemia and body weight
Lead (Pb) Animals No [9]
loss.
Neurological problems (from headache to
Humans No [9, 30]
psychosis) and kidney damage.
General chlorosis, necrotic leaf spots and stunted
Plants Yes [9]
plant growth.
Anemia, gastrointestinal lesions and growth
Manganese (Mn) Animals Yes [9]
retardation.
Psychiatric disturbance and neurodegenerative
Humans Yes [9, 52]
disorder, including Parkinson’s disease.
Hypertrophic root, retarded plant growth and
Plants No [53, 54]
increased oxidative stress.
Vomiting, bloody diarrhea and necrosis of the
Mercury (Hg) Animals No [9]
alimentary mucosa.
Neurological disturbances, kidney damage and
Humans No [30]
decreased fertility.
Yellow or orange-yellow chlorosis, seedling injury,
Plants Yes [9, 55]
delayed maturity and reduced plant growth.
Molybdenum (Mo) Diarrhea, anorexia, depigmentation of hair and
Animals Yes [9]
wool and neurological disturbance.
Humans Yes Possibly acting joints, hyperuricosuria and gout. [9, 56]
Increased oxidative stress, retarded germination,
Plants Yes stunted root growth, chlorosis, inhibited plant [9, 57]
growth and reduced yield.
Nickel (Ni) Animals No ? [37]
Skin allergies, lung fibrosis, kidney and
Humans No cardiovascular system damage and stimulation of [58]
neoplastic transformation.
Chlorosis, wilted and dried leaves, reduced plant
Plants No [59]
growth and plant premature death.
Selenium (Se) Impaired vision, anemia, loss of hair, ataxia, stiffness
Animals Yes [9, 60]
of joints, paralysis, atrophy of heart and death.
Humans Yes Hypochromic anemia, damaged nails and hair loss. [9, 61]
Chlorosis (Fe-deficiency-induced), stunted plant
Zinc (Zn) Plants Yes [9, 62]
growth and reduced yield.
Heavy metal
(Symbol)
Diarrhea, anorexia, jaundice, kidney and abomasum
Animals Yes [9, 63]
damage, arthritis and weight loss.
Diarrhea, nausea, vomiting, epigastric pain, lethargy,
Humans Yes anemia, neutropenia, impaired immune function [9, 64]
and decreased HDL cholesterol.
†In case of animals, they are livestock animals.
‡Yes: essentiality recognized. No: essentiality unrecognized.
§Most common manifestations, symptoms and consequences of heavy metal toxic effects.
Table 1. Essentiality and toxicity of heavy metals for different organisms.
The relative importance of heavy metals toxicity was addressed by McLaughlin et al. [10] in
terms of food chain contamination. According to these authors, Cd is the metal with greatest
potential to contaminate plants and subsequently to be transferred to animals and humans
that eat these contaminated plants or part of them. This statement is based on the fact that (i)
Cd poses animal and human health risks in plant tissue concentrations that are not generally
phytotoxic and (ii) Cd concentrations in agricultural soils are increasing in many parts of world
due to Cd inadvertent additions through the use of fertilizers, sewage sludge and soil amend‐
ments. Due to the high risk of contaminating the food chain, the risk of Cd to cause toxicity is
considered to be high as well. Despite increased concern with Cd, the toxicity risk of other
heavy metals should not be neglected.
The toxicity of heavy metals in living organisms is a phenomenon somewhat complex. Toxic
effects of a metal depend on a number of factors that often include (i) rate, (ii) exposure time,
(iii) tolerance of the organism and (iv) environmental conditions. In recent years, the effect of
the interaction between heavy metals on the expression of toxicity has been considered very
intensely. As a result of the interaction, a given metal may increase or decrease the negative
effects of other metal in the organism [65].
Despite the complexity, the toxicity of heavy metals in plants and in animals and humans that
eat contaminated plants is primarily associated with previous environmental contamination.
Soils may be contaminated with such hazardous elements by the use of sewage sludge. High
concentrations of metals in the sludge increase the risks of contamination and therefore
toxicity. Thus, it is important to know the chemical composition of sewage sludge.
3. Sewage sludge
Sewage sludge is generally defined as a semi-solid or liquid organic residue generated during
the treatment of domestic sewage. He has also been called biosolids, but this term is more
appropriate for the sludge that was conveniently selected and treated to allow its application
to land (i.e., agricultural recycling) [6, 66]. Although the use of the term biosolids is becoming
common, sewage sludge was used in this text because it encompasses sludges with different
types and degrees of treatment and is the term adopted by the Brazilian legislation [67].
The chemical composition of sewage sludge is highly variable, particularly in terms of heavy
metals. Such variability can be seen in Table 2 for sludge generated at WTPs in Brazil. The
coefficients of variation (CVs) of heavy metal concentrations are somewhat high. Cd, Co,
Cr and Ni concentrations had the highest CVs, above 80 %, and Zn the lowest, 43 %.
Intermediate variability was observed for Cu, Fe, Mn, Mo and Pb. It was not possible to
calculate CV for As, Hg and Se because of the paucity of data. The high variability is
explained primarily by the origin of the sludges, since they were obtained in WTP from
different cities, each with particular characteristics of generation and wastewater treat‐
ment as well as treatment of generated sludge. However, there was also considerable
variation for a same city, which indicates the occurrence of temporal variability (e.g.,
seasonal, annual etc.) for concentration.
Despite the high variability, the concentrations of heavy metals in sewage sludge for land
application may not exceed limits established by environmental institutions. In Table 2 two
limits for each metal are shown, one from the Environmental Agency of the State of São Paulo
(CETESB) and other from the Brazilian National Environment Council (CONAMA). There is
agreement for the limits between the institutions only for the Se. For all elements, CONAMA
limits are lower. As CONAMA rule is federal and more restrictive, it must prevail over the
rule of CETESB, which is state and less restrictive.
Considering Resolution # 375/2006 of CONAMA, some sewage sludges presented in Table 2
had heavy metal concentrations above the established limits. Seven, six, four and three sludges
were with concentrations of Zn, Ni, Cr and Pb, respectively, higher than the maximum
allowable concentrations. Generally, more than one metal was in excess, but not necessarily.
Improper sludges were generated in the cities of Barueri, Franca and São Paulo, however,
Franca produced sludges less contaminated with heavy metals, which probably reflects its
lower level of industrialization in relation to the other two cities.
The concentrations of Cd and Cu in sewage sludges were below the limits of CONAMA (Table
2). Similarly, the concentrations of As, Hg, Mo and Se were also below, but there were few
values determined, making this finding unrepresentative. However, the high number of cases
in which these elements were below the limit of detection (LOD) of the analytical method
suggests low concentrations, probably far from reaching the maximum allowable concentra‐
tions. Although they are not legally restrictive, Co, Fe and Mn were in relatively high concen‐
trations in the sludges analyzed.
The finding that the Cd did not exceed the limit established by CONAMA is the most positive
result of this survey of sewage sludge used in experiments conducted in Brazil. This result
indicates that the sludge has low potential for soil contamination with this heavy metal. Soil
contaminated with Cd is a constant concern throughout the world, because this metal can
easily enter the food chain and impose risks to human health [10]. On the other hand, the sludge
seems to be a considerable source of other heavy metals such as Cr and Pb. Considering that
8
State City As Cd Co Cr Cu Fe Hg Mn Mo Ni Pb Se Zn Reference

mg kg–1
SP Barueri 28.4 385.0 784.3 238.6 152.8 1568.5 [68]
SP Barueri 16.0 386.0 534.0 286.0 171.0 1649.0 [68]
SP Barueri 664.0 228.0 268.0 152.0 1800.0 [69]
SP Barueri 551.0 294.0 595.0 371.0 3810.0 [69]
SP Barueri 660.0 257.0 360.0 180.0 2328.0 [69]
SP Barueri 719.0 263.0 354.0 171.0 1745.0 [69]
SP Barueri 627.0 287.0 350.0 155.0 2354.0 [69]
SP Barueri <1.0 12.8 12.2 823.8 1058.0 54181 429.0 <0.01 518.4 364.4 2821.0 [70]
SP Barueri <1.0 9.5 5.0 1071.0 1046.0 32500 335.0 <0.10 483.0 233.0 3335.0 [70]
SP Barueri <1.0 9.4 9.3 1297.2 953.0 37990 418.9 <0.01 605.8 348.9 3372.0 [70]
SP Barueri 11.0 808.0 722.0 222.0 231.0 186.0 2159.0 [71]
SP Barueri 10.0 736.0 690.0 194.0 297.0 173.0 2930.0 [71]
SP Barueri 8.0 798.0 998.0 206.0 299.0 169.0 2474.0 [71]
SP Barueri 8.0 798.0 998.0 206.0 299.0 169.0 2474.0 [71]
SP Barueri 20.3 477.7 754.1 355.3 155.8 1926.9 [72]
SP Franca <1.0 3.3 5.0 633.8 239.8 33793 349.3 <0.01 54.7 199.6 1230.0 [71]
SP Franca <1.0 2.0 4.9 1325.0 259.0 31700 267.0 <0.10 74.0 118.0 1590.0 [71]
SP Franca <1.0 2.1 4.8 1230.0 240.9 24176 232.5 <0.01 72.4 140.5 1198.0 [71]
SP Franca 3.3 29.0 284.5 572.6 184 730.0 2.8 56.6 77.3 1028.3 [73]
SP Franca 2.0 102.0 204.0 18883 243.0 69.0 100.0 1279.0 [74]
SP Franca 1.5 41.3 34.8 43.8 [75]
SP Franca 1.5 41.3 34.8 43.8 [75]
SP Franca 1.5 41.0 34.3 42.4 [75]
SP Franca 1.6 41.7 35.2 45.3 [75]
SP Franca 98.0 42224 242.0 9.8 127.0 1868.0 [76]
SP Jundiaí <0.10 5.8 149.3 284.1 <0.10 676.8 <0.10 41.8 283.1 <0.10 1364.8 [77]
SP Jundiaí <0.01 6.6 188.5 864.8 <0.01 693.3 <0.01 35.4 206.6 <0.01 1738.1 [77]
SP Jundiaí 4.2 14.0 277.7 304.1 1.1 9.8 65.6 201.6 1.8 1869.9 [78]
SP Piracicaba <0.02 207.2 192.9 30295 173.0 <0.01 107.6 943.4 [79]
RJ Rio de Janeiro 0.8 47.0 60.0 82.0 217.0 [80]
SP São Paulo 791.0 38000 315.0 322.0 1888.0 [81]
SP São Paulo 975.0 44000 423.0 401.0 2904.0 [81]
SP São Paulo 1148.0 41000 479.0 459.0 3326.0 [81]
SP São Paulo 989.0 84000 223.0 1240.0 4035.0 [81]
Table 2. Heavy metals in sewage sludge samples used in experiments conducted in Brazil.
Statistics
n 1 23 7 23 30 14 1 26 3 31 30 1 30
Minimum (mg kg–1) 4.2 0.8 4.8 41.0 47.0 184 1.1 60.0 2.8 34.3 42.4 1.8 217.0
–1
Maximum (mg kg ) 4.2 28.4 29.0 1325.0 1148.0 84000 1.1 730.0 9.8 1240.0 371.0 1.8 4035.0
Coefficient of variation (%) 91 79 81 51 51 51 54 92 53 43
Limits (mg kg–1)
By CETESB† 75 85 4300 57 75 430 840 100 7500 [82]
By CONAMA‡ 41 39 1000 1500 17 50 420 300 100 2800 [67]
†Technical Norm # P4.230/1999 of CETESB (Environmental Agency of the State of São Paulo).
‡Resolution # 375/2006 of CONAMA (Brazilian National Environment Council).
the sludge can introduce these metals in excess, soils amended with sewage sludge should be
carefully evaluated for accumulation and availability of Cu, Cr, Mn, Pb and Zn. Additionally,
the Cd should also be monitored because it is highly dangerous.
4. Heavy metals in soils amended with sewage sludge
4.1. Total concentration
The determination of the total concentration of heavy metals in soils has been performed
with previous extraction of metals from samples using different mixtures of acids. In Brazil,
the methods of extraction most widely used are presented in Table 3. Because of the
difference in the chemical composition, some of them may produce quite contrasting results.
Comparison between the methods HNO3–H2O2–HCl and HClO4–HF shows this contrast. It
is observed in Table 4 that concentrations of Cr, Pb and Zn extracted with HNO3–H2O2–
HCl were 72 %, 31 % and 62 % lower than concentrations of these metals extracted with
HClO4–HF. For Cd, however, the results were not different between the two methods,
suggesting that the difference in values between them may depend on the type of metal in
question. The contrasting results between these methods are due to extraction differential
capacity of the reagent mixtures employed in each of them. The HNO3–H2O2–HCl mix‐
ture does not normally extract metals bound to silicates. On the other hand, hydrofluoric
acid contained in the HClO4–HF mixture is able to dissolve silicates and extract metals
eventually present in this mineral class. Therefore, concentrations of metals extracted with
HNO3–H2O2–HCl tend to be smaller in relation to extraction with HClO4–HF. Thus, it is
necessary to standardize the method of extraction of heavy metals to assess the contamina‐
tion of soil amended with sewage sludge.
Extraction method Common name External heating Denomination by USEPA† Reference

HNO3–H2O2–HCl Method 3050B [83]
HNO3–HCl 1+3 Aqua regia Microwave oven Method 3051A [84]
HNO3–HClO4 5+1 Nitric-perchloric [85]
HClO4–HF [86]
†United States Environmental Protection Agency. The composition of the extraction methods 3050B and 3051A may
vary slightly but it always has HNO3.
Table 3. Methods commonly used in Brazil for extraction of heavy metals from soils.
Brazilian official institutions of environment, as CETESB [82] and CONAMA [67], recommend
the use of the methods 3050B and 3051A of United States Environmental Protection Agency
(USEPA) [83, 84] for extraction of heavy metals in soils amended with sewage sludge. Such
methods are not designed to extract fully the metals from the soil, since they normally do not
dissolve elements bound to silicates, which are not generally available in the environment,
Heavy metal†
Extraction method Cd Cr Pb Zn
mg kg–1
HNO3–H2O2–HCl 1.93 a 28.72 b 15.72 b 64.82 b
HClO4–HF 1.99 a 104.17 a 23.05 a 171.97 a
†Means within a column followed by the same letter are not significantly different according to Tukey test (p < 0.05).
Values are means of four sewage sludge rates.
Source: Adapted from Nogueira et al. [71].
Table 4. Cd, Cr, Pb and Zn extracted by two different methods from a Brazilian Oxisol amended with sewage sludge.
thus without implications for environmental contamination. They extract only elements which
could become environmentally available, consequently harmful to living organisms. As the
extraction is not fully, the terms pseudototal concentration and total recoverable concentration
have recently been employed to designate the concentrations of metals extracted by these
official methods, although the use of the term total concentration is still very common. In recent
years, many field experiments with sewage sludge were performed in Brazil, particularly in
São Paulo state, therefore there are several data on total concentrations of heavy metals in soil
which allow studying the risks of contamination pose by agricultural use of the waste. A
summary of these experiments is presented below.
Oliveira and Mattiazzo [68] conducted an experiment with sugarcane to test varying rates of
sewage sludge applied to an Oxisol for two consecutive years, in which heavy metals were
added in different amounts, including rates above the annual maximum rates allowed in São
Paulo state by CETESB. The data in Tables 5 and 6 show that there was at least one rate above
the annual rate allowed for Cd, Cu, Ni, Pb and Zn, but there was no rate higher than the
maximum cumulative rate. In soil, the total concentrations of Cu and Zn (extraction with HCl–
HNO3 3+1 in microwave) in layer 0-0.20 m depth increased with increasing sewage sludge rate
in two years especially for the higher rates in the second year, indicating a cumulative effect.
Cd and Pb were below the LOD of the analytical method (Atomic Absorption Spectrometry–
AAS). Ni was detected only in the second year and at the two highest rates of sludge. The
addition of metals above the allowed maximum rates increased total concentrations of Cu, Ni
and Zn in the second year above the natural concentrations established for São Paulo state
(Tables 3 and 7), suggesting that soils receiving high loads of heavy metals by the application
of excessive rates of sewage sludge could be contaminated in a short time.
In addition to the possibility of rapidly contaminating the soil with heavy metals, high rates
of sewage sludge cause prolonged effect on contamination. Martins et al. [81] observed linear
increases in total concentrations of Cu and Zn (extraction with HNO3–HClO 5+1) in a clayey
Oxisol in the year of application of sewage sludge single rates which reached maximum of 80
Mg ha–1, being in this rate added quantities of these metals that exceeded the maximum annual
limits established for São Paulo state. Four years later, increases were still linear and the
concentrations were similar to the first year, indicating that these elements persist in the soil
for a long time. In fact, high persistence is a characteristic of heavy metals added to soils [87].
On the other hand, low rates of sewage sludge applied to land do not increase excessively the
total concentrations of heavy metals in soils. Oliveira et al. [69] found that the total concentra‐
tions of Cu, Ni, Pb and Zn (extraction with HNO3–H2O2–HCl) in 0-0.20 m layer of an Oxisol
cultivated with maize increased after five annual applications of sewage sludge rates up to 10
Mg ha–1 year–1, but the increases did not exceed the limits of these heavy metals allowed in São
Paulo state. In the study conducted by Silva et al. [88], the total concentrations of Cu, Ni and
Zn (extraction with HCl–HNO3 3+1) in 0-0.20 m layer of a clayey Oxisol cultivated with maize
increased in response to rates of sewage sludge from the Barueri and Franca municipalities,
but they increased less with Franca sewage sludge, which had lower concentrations of these
metals and it was applied at lower rates. Thus, application of low rates of sludge with low
concentrations of heavy metals in their composition seems to be a strategy to minimize the
excessive accumulation of heavy metals in soils.
However, this strategy may be insufficient for Cd. Nogueira et al. [78] observed that application
of 10.8 Mg ha–1 of sewage sludge, rate defined to supply 100 % of N required by sugarcane,
increased total concentrations of As, Cd, Cu, Ni, Pb and Zn (extraction by Method 3051A – see
Table 3) in 0-0.20 m layer of an Ultisol in assessments performed 360 and 720 days after
application of the sludge. While the concentrations of As, Cu, Ni, Pb and Zn were well below
the limits established by CETESB and CONAMA, the concentration of Cd (0.2 mg kg–1) was
relatively close to the limit of CETESB (< 0.5 mg kg–1, Table 7). Although the concentration of
Cd has more than double to reach this limit, the fact that the limit is too low cause concern in
relation to any increase in Cd concentration due to new applications of sewage sludge. In the
work of these authors has been reported for the first time in Brazil the effect of sewage sludge
on the accumulation of Se in soil. Se concentration increased from 0.068 to 0.092 mg kg–1 with
application of 10.8 Mg ha–1 of sludge, but this increase was below the limit of CETESB (0.25
mg kg–1) and far below the limit of CONAMA (5 mg kg–1).
The concentrations of heavy metals presented above refer only to the topsoil (0-0.20 m depth).
However, deeper and stratified sampling can give an idea of how these metals are distributed
among soil layers. Merlino et al. [73] evaluated the concentrations of Cd, Cr and Pb (extraction
with HNO3–H2O2–HCl) in the layers 0-0.10, 0.10-0.20 and 0.20-0.40 m of an Oxisol cultivated
with maize after 11 years of annual application of sewage sludge rates up to 20 Mg ha–1. The
concentrations of Cr and Pb increased only in the 0-0.10 m layer, suggesting accumulation in
the superficial layer. There was no effect on the concentration of Cd. In another study, there
was also no effect on total concentrations of Cd, Cr, Ni and Pb (extraction with HNO3–H2O2–
HCl) in the layers 0-0.10, 0.10-0.20, 0.20-0.30, 0.30-0.40 e 0.40-0.50 m depth of a clayey Oxisol
after four annual applications up to 15 Mg ha–1 year–1 to supply nitrogen (N) for sugarcane [75].
In contrast, Oliveira and Mattiazzo [89] observed significant increases in total concentrations
of Zn (extraction with HCl–HNO3 3+1 in microwave) until the layer of 0.40-0.60 m of an Oxisol
amended with relatively high rates of sewage sludge (> 30 Mg ha–1 year–1) for two consecutive
years and cultivated with sugarcane, suggesting that Zn was leached to layers below the
incorporation layer of sludge (0-0.20 m depth). For Cu and Cr, there was no evidence of
leaching. It was not possible to assess the mobility of Cd, Ni and Pb in soil, because their
concentrations were below the LOD of the analytical method (AAS).
12
cultivated with sugarcane

Sewage sludge rate Cd Cr Cu Ni Pb Zn
1996/97 1997/98 1996/97 1997/98 1996/97 1997/98 1996/97 1997/98 1996/97 1997/98 1996/97 1997/98 1996/97 1997/98
_________ –1
Mg ha–1 _________ Rate applied to soil (kg ha )
0 0 0 0 0 0 0 0 0 0 0 0 0 0
33 37 0.9 0.6 13 14 26 20 8 11 5 6 52 61
66 74 1.9 1.2 25 29 52 40 16 21 10 13 104 122

99 110 2.8 1.8 38 42 78 59 24 31 15 19 155 181
–1
Total concentration in soil† (mg kg )
0 0 nd‡ nd 16.57 15.55 17.86 16.87 nd nd nd nd 21.14 20.19
33 37 nd nd 21.56 18.14 19.47 26.17 nd nd nd nd 39.22 50.03
66 74 nd nd 25.30 25.21 27.90 36.39 nd 10.65 nd nd 40.41 77.11
99 110 nd nd 25.59 27.19 30.97 42.85 nd 14.63 nd nd 41.27 97.21
†In the 0–0.20 m soil layer. Digestion of soil samples was performed with HCl + HNO3 (3:1) in a microwave oven.
‡Not detected. Concentration of the metal was below the limit of detection of the analytical method (AAS).
Source: Adapted from Oliveira and Mattiazzo [68].
Table 5. Rate and total concentration of heavy metals in a Brazilian Oxisol amended with sewage sludge and
Norm of Abrangency As Cd Cr Cu Hg Mo Ni Pb Se Zn
Annual loading rate (kg ha–1 365 days–1)
CETESB† São Paulo State 2.0 1.9 75 0.85 21 15 5.0 140
Cumulative loading rate (kg ha–1)
CETESB† São Paulo State 41 39 1500 17 420 300 100 2800
CONAMA‡ Brazil 30 4 154 137 1.2 13 74 41 13 445
†Technical Norm # P4.230/1999 of CETESB (Environmental Agency of the State of São Paulo) [82].
‡Resolution # 375/2006 of CONAMA (Brazilian National Environment Council) [67].
Table 6. Loading rates for heavy metals applied by sewage sludge in São Paulo State and Brazil
Risks of Heavy Metals Contamination of Soil-Pant System by Land Application of…
13
14
Norm of Abrangency As Cd Co Cr Cu Hg Mo Ni Pb Se Zn
Limits (mg kg–1)
CETESB† São Paulo State 3.5 <0.5 13 40 35 0.05 <4 13 17 0.25 60
CONAMA‡ Brazil 15.0 1.3 25 75 60 0.50 30 30 72 5 300
†CETESB [90].
‡Resolution # 420/2009 of CONAMA [91].
Table 7. Limits for heavy metals in soils from São Paulo State and Brazil
Besides increasing or not be changed, the concentrations of heavy metals can also decrease in
response to the application of sewage sludge, as shown in the work of Macedo et al. [92].
Concentrations of Cd, Cr and Pb (extraction with HNO3–H2O2–HCl) were evaluated in the
layers 0-0.10, 0.10-0.20 and 0.20-0.40 m of a clayey Oxisol cultivated with maize after 11 years
of application of sludge sewage rates up to 20 Mg ha–1 year–1. In general, the application of
sewage sludge reduced the concentrations of heavy metals in the surface layers, with decreas‐
ing quite evident for Cd in layer of 0-0.10 m depth. This reduction was not expected since
metals were added to the soil. Probably, the heavy metals added and part of those natives may
have combined with components of sewage sludge forming highly stable compounds resistant
to the attack of the extractant used.
4.2. Availability
Availability refers to the amount of a chemical (e.g., heavy metal) in the soil that would be
available to be absorbed by plants or other biological receptors (e.g. microorganisms). The
availability to plants is called phytoavailability, which is a specific term for this class of
organisms. In recent years, the availability has also been called bioavailability. Although
bioavailability not yet has a clear and accepted definition [93], it has been used to characterize
the availability of heavy metals in the environment [94]. However, the uncertainty about the
meaning of bioavailability restricts the expansion of its use and thus contributes to the
maintenance of the term availability.
The availability of a heavy metal is given by its available concentration. Quantification of the
available concentration is performed by extracting the fraction of the metal that is sufficiently
soluble to be absorbed by plants. The extraction of this fraction is performed using chemical
extractants that try to simulate the potential for uptake of an element of the soil by the roots
of plants. There are several extractants which can be used to extract heavy metals from the soil.
The extractants used to assess the availability of heavy metals added to Brazilian soils by the
application of sewage sludge to land are usually the same ones used in the evaluation of
cationic micronutrients, especially Mehlich 1 and DTPA as the most common. Other extrac‐
tants, as 0.1 mol L–1 HCl and Mehlich 3, have also been used in this assessing. The concentration
of a metal in the soil can only be considered available if it is closely and positively correlated
with the concentration of the metal in plant tissues. In terms of assessing the contamination,
the available concentration can be correlated to the concentration of the metal in raw or
processed agricultural product. When there is no correlation or the correlation is poor, it is
more appropriate to use the term extractable concentration.
Knowledge of available concentrations is essential for assessing the environmental impact of
sewage sludge application to land, since such concentrations represent the amount of heavy
metals that could be leached to reach groundwater or could be absorbed by plants and
transmitted to the food chain levels until reaching the man. Assessment of the availability of
heavy metals in soils amended with sewage sludge was carried out in some field experiments
conducted in Brazil. The main results are summarized below.
Oliveira and Mattiazzo [68] found different effects of sewage sludge rates (Table 5) on the
availability of some heavy metals evaluated. The sludge was applied for two consecutive years
to an Oxisol cultivated with sugarcane, and concentrations of available Cd, Cr, Cu, Ni, Pb and
Zn by extractants 0.1 mol L–1 HCl, Mehlich 3 and DTPA were determined in soil samples
collected in 0-0.20 m depth one year after each application of the waste. Increase in rates of
sewage sludge consistently increased concentrations of Cu and Zn extracted with the three
extractants in both evaluations. There was increase in concentration of available Ni only at the
second assessment and for sewage sludge treatments. The concentrations of Cd, Cr and Pb
were below the LOD of the analytical method (AAS). Variations in concentrations of DTPA-
Cu and DTPA-Zn between the control (without application of sewage sludge) and the
maximum rate of the waste (110 Mg ha–1) were from 0.70 to 10.70 mg kg–1 and 0.62 to 19.12 mg
kg–1, respectively. The higher values of Cu and Zn are considered high and very high, respec‐
tively, for soils of São Paulo state (Table 8), assuming a similarity between the values expressed
in mg kg–1 and mg dm–3. However, the authors did not report any negative consequence of
these high concentrations to the crop. Positive correlations between the availability of Cu and
Zn in soil assessed with three extractants and the concentrations of these metals in different
plant components (leaf+1, stalk and juice) were significant when included data from treatments
with sewage sludge (three rates), treatment with mineral fertilization and control. However,
when included only the sludge treatments, there were few significant correlations, indicating
generally low efficiency of extractants to assess the availability of Cu and Zn in soil amended
with sewage sludge. The exceptions were significant correlations between 0.1 mol L–1 HCl-Zn
and juice-Zn in the two years of evaluation and between Zn extracted by the three extractants
and stalk-Zn and juice-Zn only in the second year.
Limits
Interpretation
Cu Fe Mn Zn
_______________________________________________________
mg dm –3 ___________________________________________________________
Low 0.0-0.2 0-4 0.0-1.2 0.0-0.5

Medium 0.3-0.8 5-12 1.3-5.0 0.6-1.2
High 0.9-1.5 13-24 5.1-9.0 1.3-2.3
Very high 1.6-15 25-60 10-50 2.4-15
Toxicity >130
Source: Adapted from Abreu et al. [93].
Table 8. Interpretation limits for concentrations of cationic micronutrients extracted by DTPA pH 7.3 in soils from São
Paulo State, Brazil
In another study with sugarcane, Nogueira et al. [78] applied sewage sludge rates up to 10.8
Mg ha–1 to an Ultisol to supply 100 % of N required by the crop, and after 360 and 720 days
they assessed the availability of heavy metals in the layer of 0-0.20 m depth using DTPA. The
concentrations of Cd, Cu, Ni, Pb and Zn available increased with increasing sludge rates in
the two evaluation periods, reaching maximum values of 0.112, 2.64, 0.47, 2.09 and 7.61,
respectively. Concentrations of Cu and Zn are considered very high (Table 8), while the
concentrations of Cd, Ni and Pb are above the normal range for soils of São Paulo state (Table
9), indicating contamination. Nevertheless, there was no toxicity in the plant. The extractant
used was efficient only for Cd and Zn, since their “… available concentrations in the soil were
highly correlated with the concentrations in leaf with top visible dewlap, stalk and juice. For
concentrations of As, Cr and Se, there was no effect of sewage sludge application. All metals
intentionally evaluated in this study were detected, probably by the use of inductively coupled
plasma mass spectrometry (ICP-MS), which had LODs very low.
Limits
Abragency
Cd Cr Ni Pb
_________________________________________________________
mg dm–3 _________________________________________________________
São Paulo state 0.020 0.030 0.180 0.85
Other states 0.019 0.007 0.147 0.76
†Values above the limits are indicative of contamination.
Source: Adapted from Abreu et al. [93].
Table 9. Limits for heavy metals extracted by DTPA pH 7.3 in soils from São Paulo State and other states in Brazil†.
The availability of heavy metals in soils amended with sewage sludge has also been evaluated
for maize crop. Martins et al. [81] found that sewage sludge rates up to 80 Mg ha–1 increased
the concentrations of extractable Cu, Ni and Zn by DTPA and Mehlich 3 in a clayey Oxisol
with or without lime after a maize cultivation. However, the effect of liming on extractability
of these metals was different between the two extractants. Liming reduced the concentrations
of extractable Ni and Zn by DTPA, but did not change concentrations of Cu. In the case of
Mehlich 3, concentrations of extractable Cu and Zn increased with liming, which was unex‐
pected, because it is well known the fact that the increase in soil pH due to liming generally
decreases the availability of cationic micronutrients. Positive correlations between soil Cu or
Zn extracted by DTPA and Mehlich 3 and concentrations of Cu and Zn in leaf+4 and shoots of
maize were observed, suggesting that extractants were effective in assessing the availability
of these micronutrients. In the case of Ni, it has not been possible to establish these relationships
because its concentration in plant tissues was below LOD of the analytical method (AAS).
Silva et al. [88] applied sewage sludges from Franca and Barueri municipalities (São Paulo
state) in three consecutive crops of maize to a clayey Oxisol. For both sludges, rates were to
supply up to eight times the amount of N required by the crop, reaching maximum values of
30 and 64 Mg ha–1 for sludges of Franca and Barueri, respectively. Soil samples were taken
from the layer 0-0.20 m depth after each waste application and before each maize sowing for
assessing availability of Cu, Mn, Ni, Pb and Zn by DTPA and Mehlich 1. The concentrations
of Cu, Ni and Zn extracted by both extractants increased in response to the application of the
two sludges in the three maize crops. For Mn, the extractability varied with sludge, crop and
extractant. In extraction with Mehlich 1, the concentration of Mn increased in the three crops
for Franca sewage sludge and only in the first crop for Barueri sludge. The concentration of
Mn extracted by DTPA increased for both sludges, but only in first and second crops. There
were no significant changes in the extractability of Pb measured by Mehlich 1 for both sludges
in all crops. In contrast, the concentration of Pb extracted by DTPA increased in all crops for
Barueri sludge and in the second crop for Franca sludge. In all cases in which concentrations
of extractable metals increased, increases were higher for Barueri sludge, probably because it
had the highest concentrations of metals and was applied in higher rates. Regardless of sewage
sludge, increases in concentrations of extractable metals by both extractants were lower in the
third than in the second crop, suggesting that successive applications of sewage sludge
reduced the solubility of such elements. The significance of the correlations between the
concentrations of Cu, Mn, Ni, Pb and Zn in soil and leaf below the ear and grains varied with
sewage sludge and crop for both extractants, indicating that they were not consistently
effective in assessing the availability of these metals to maize. The element that had more cases
of significance was Zn.
In long term experiments conducted by Oliveira et al. [69], sewage sludge rates up to 10 Mg
ha–1 year–1 were applied to two Oxisols (Typic Haplorthox and Typic Eutrorthox) for five years
and in each year the soils were cultivated with maize. The concentrations of Cu, Ni and Zn
extractable by Mehlich 1 in layer of 0-0.20 m depth increased in both soils after the fifth year
of sludge application. The concentration of extractable Pb in this layer increased only in Typic
Haplorthox. In layer of 0.20-0.40 m depth, there was an increase in extractability of Ni in both
soils, whereas concentrations of extractable Cu, Pb and Zn were increased only in Typic
Haplorthox. There was no change in concentration of extractable Mn in any of the layers of
both soils. Although the concentrations of extractable Cu, Ni, Pb and Zn increased with sludge
application, these increases were not adequately correlated with the accumulation of these
metals in the shoot and grains of maize plant, indicating that the Mehlich 1 was ineffective in
assessing the availability of such heavy metals in both soils. Although the extractant has been
inefficient to evaluate the availability, increased extractability in layer of 0.20-0.40 m depth of
Typic Haplorthox suggests that Cu, Ni, Pb and Zn were leached due application of sewage
sludge.
The results presented in Galdos et al. [77] also suggest the occurrence of leaching, but not for
all metals evaluated. The authors applied sewage sludge to a clayey Oxisol to supply up to
double of N requirement of maize by two successive crops. The maximum rates were 21.6 Mg
ha–1 in first crop and 20.5 Mg ha–1 in second crop. The sludge was incorporated into soil layer
of 0-0.10 m depth. The concentrations of extractable Cu, Ni and Zn by DTPA in soil layers of
0-0.05, 0.05-0.10 e 0.10-0.20 m depth were evaluated 267 days after first application and 179
days after second application. The applications of sewage sludge increased the concentrations
of Cu and Ni in the layers 0-0.05, 0.05-0.10 m only in the second evaluation, but did not change
concentrations in 0.10-0.20 m layer in any of the evaluations, indicating no leaching, probably
because the concentrations of these metals in sludge were low (284.1 and 864.8 mg kg–1 Cu and
41.8 and 35.5 mg kg–1 Ni). In contrast, Zn concentrations increased in all layers, including the
layer of 0.10-0.20 m in both evaluations, suggesting that Zn was leached to the layer below the
incorporation layer of sewage sludge. The leaching may have occurred because of high
concentrations of Zn in sludge (11,364.8 and 1,738.1 mg kg–1). Increased mobility of heavy
metals in soils amended with sewage sludge is very concerning because the metals leached
can reach groundwater, contaminating it.
4.3. Chemical fractions

In Brazil, field studies about fractionation of heavy metals in soils amended with sewage sludge
are scarce. Among the few studies that exist, one of Nogueira et al. [94] can be considered
comprehensive because they evaluated Cd, Pb and Zn in different soil fractions [exchangeable
(Exch), organic matter (OM), amorphous Fe oxide (AFeO), crystalline Fe oxide (CFeO ), and
residual (Res)] and in soil organic matter (SOM) chemical fractions [fulvic acid (FA), humic
acid (HA) and humin (Hum)] after nine annual applications of sewage sludge rates up to 20
Mg ha–1 to a clayey Oxisol cultivated with maize. The Cd concentration in the fraction Res was
not altered by application of sewage sludge, and concentrations in other fractions were below
the LOD of the analytical method (AAS). Similarly, there was no effect of sludge on concen‐
tration of Pb in fractions AFeO, CFeO and Res, and the concentrations in other fractions were
below the LOD. In contrast, the sludge application increased the concentration of Zn in all
fractions, mainly in AFeO and CFeO fractions, indicating that a considerable part of the added
Zn was adsorbed on these oxides. In the fractions of SOM, Cd and Pb concentrations were not
changed in response to sewage sludge application. While the concentration of Zn in Hum
fraction was not changed, the concentrations in FA and HA fractions increased by sludge
application, indicating that, in SOM, the added Zn is bound to fractions less stable and thus
can become more easily available in the environment.
In another study, the redistribution of Ni among humic fractions of a medium-textured Oxisol
cultivated with maize and amended with sewage sludge rates up to 20 Mg ha–1 year–1 for six
years was assessed by Melo et al. [95]. The authors observed that the application of sewage
sludge increased more the proportion of Ni in the humin fraction (Figure 1). This means that
the sewage sludge redistributed added Ni preferably to more stable fraction of SOM. As the
humin fraction is insoluble in acid and alkaline medium, Ni associated with it can be consid‐
ered unavailable to plants and not directly subject to leaching, which favors its accumulation
in soil layer where sewage sludge is incorporated.
4.4. Speciation
In this work, speciation refers to the separation of heavy metals in different chemical forms
and possible oxidation states (e.g., chemical species) in the soil solution. Such metals can be
separated basically into free ions, complexes and ion pairs [11, 96]. The participation of each
form in the total concentration of a metal in the soil solution can be estimated by using the
following general procedures: (i) extraction of soil solution, (ii) determining total concentration
of cations, anions and organic compounds in solution and (iii) calculation of the activity of the
metals of interest, based on determined concentrations, using specific software for this
purpose. Speciation is the basis of the free ion activity model (FIAM) and also other models,
which relate the responses of plants to different chemical species of metals present in the soil
solution [96].
Plants uptake heavy metals preferably as free ions. Thus, if these ions are increased in the soil
solution, so they can be absorbed in excess, and excessive uptake of these metals can cause
phytotoxicity and also contamination of the food chain. Indeed, uptake and toxicity of metals
in plants generally correlate better with the activity of free ions [11, 96]. On the other hand,
7
Page 34, Line 20 – Replace “heavy metal” by “heavy metals”.
15
Ni in organic matter fractions (mg kg-1)

Fulvic acid a
Humic acid
12 Humin
9
c
3 a
bc bc
c
ab b ab a
0
0 5 10 2.5 + 20†
Annual sewage sludge rate (Mg ha-1)
Source: Adapted from Melo et al. [95].
Figure 1. Nickel associated to soil organic matter chemical fractions in medium-textured Oxisol amended with sewage
Figure applied
sludge 1 annually during six years and cultivated with maize. Means within each fraction followed by the same
letter are not significantly different according to Tukey test (p < 0.05). †2.5 Mg ha–1 in 1st, 2nd, and 3rd years and 20
Mg ha–1 in 4th, 5th and 6th years.
other chemical species that are not immediately relevant to the uptake of plants can also cause
environmental impact. Heavy metals bound to dissolved organic matter, forming organo-
metal complexes in the soil solution, may have increased their mobility in the soil profile, as
demonstrated in experiments with undisturbed soil columns [17], favoring the leaching of
these potentially toxic metals, which increases the risk of groundwater contamination
Addition of organic materials to the soil is able to change the availability and mobility of heavy
metals in the soil solution, which can minimize some environmental impacts and maximize
others. Organic materials added to the soil can decrease the activity of free ions [97] and,
consequently, reduce the availability of metals to plants, minimizing the risk of phytotoxicity
and contamination of the food chain. In contrast, the addition of organic materials can increase
the activity of organo-metal complexes [97] and thus increasing mobility of heavy metals,
which become more readily leachable, maximizing the risk of groundwater contamination.
Sewage sludge, as an organic material, can also generate these contrasting environmental
effects.
As occurs for fractionation, field studies about speciation of heavy metals in soils amended
with sewage sludge are also rare in Brazil. The only study found with these characteristics was
the Silva’s MS thesis [98]. In this work, sewage sludge rates up to 20 Mg ha–1 year–1 were applied
for seven years to a clayey Oxisol cultivated with maize. The author evaluated chemical species
of heavy metals in the soil solution from the layer 0-0.20 m depth at 22, 26, 37 and 43 months
after the seventh application of sludge. Considering all evaluation times, Cd, Cr, Mo, Ni and
Pb concentrations in the soil solution were generally below the LOD of the analytical method
used (Inductively Coupled Plasma-Optical Emission Spectroscopy–ICP-OES). On the other
hand, Cu and Zn were consistently detected. The data of speciation for Cu and Zn show that
application of sewage sludge generally decreased the proportion of these metals as free ions
(Cu2+ and Zn2+) and increased their proportion as organo-metal complexes [dissolved organic
carbon-Cu (DOC-Cu) and DOC-Zn]. The observed decrease in the proportion of Cu and Zn
free ions in the soil solution indicates that sewage sludge can restrict the availability and
consequently uptake of theses metals by plants, reducing the risk of phytotoxicity and
contamination of the food chain. In contrast, increased proportions of COD-Cu and COD-Zn
in the soil solution indicate that the sludge can intensify the leaching of Cu and Zn, increasing
the risk of groundwater contamination. These results suggest that the risk of environmental
contamination by application of sewage sludge on land should be better assessed considering
the intensity these two contrasting effects.
4.5. Risks of contamination
Sewage sludge is an organic material containing heavy metals. Therefore, its application to
land generates risks of soil contamination by heavy metals. Although it is difficult to quantify
this risk, due to the complex interaction among the factors that determine it, it is possible to
mention some steps that can minimize it. Knowledge of these measures is relevant, since they
allow the adoption of a more appropriate management for the disposal of sewage sludge on
land.
As noted above, research about the impact of sewage sludge agricultural use on soil contam‐
ination by heavy metals has advanced considerably in recent years in Brazil. Field experiments
using the total concentration as an indicator of heavy metal contamination have shown
interesting results. Sludge rates above 65 Mg ha–1 year–1 can quickly contaminate (approxi‐
mately two years) the soil by Cu, Ni and Zn. Once contaminated with Cu and Zn, soil may
remain in this condition for several years, even without additional application of sewage
sludge. Zn may be leached in soils amended with relatively high rates of sludge (> 30 Mg ha–
1
year–1). In contrast, rates as low as 10 Mg ha–1 year–1, applied for five years, have low potential
for soil contamination by Cu, Ni, Pb and Zn. Single application of sludge rate of this magnitude
is unlikely to contaminate soil for As and may be insufficient to cause contamination by Cd.
But as the limits for Cd in soils are very low (Table 7), any increase in its total concentration is
concerning. Therefore, maximum rates for annual increase of Cd total concentration in soil
could be adopted as an optional security criterion to restrict application of sewage sludge on
land even though there is no legal restriction to its application.
Studies reviewed in this work also show the usefulness of available concentration to assess
soils contaminated by heavy metals. Sewage sludge rates relatively low (~ 10 Mg ha–1 year–1)
can contaminate soil by Cd and Zn due to the excessive increase in available concentrations of
these metals by DTPA. Similarly, soil contamination by Cu and Zn, indicated by increase in
available concentrations by DTPA and Mehlich 3, may occur by applying high sludge rates.
Liming reduces the availability of Zn and hence the potential for contamination of the soil, but
only DTPA is able of detecting this reduction. Mehlich 1 extractant, which is widely used in
routine soil analysis (soil testing) in Brazil, seems to be inefficient to assess whether a soil is
contaminated by heavy metals. Likewise, Mehlich 3 and DTPA are not always efficient. The
alternative extractant 0.1 mol L–1 HCl has also shown poor performance. As the factors
associated to inefficiency of the extractants are variables (e.g., type of heavy metal, soil class,
type of crop, cropping sequence and origin of sewage sludge) and they may act in combination,
the available concentration can not yet be considered a consistent and relatively safe indicator
for assessing the risk of heavy metals contamination in soils amended with sewage sludge.
Nevertheless, extractable concentrations by DTPA and Mehlich 1 may be useful to monitor the
leaching of heavy metals in soils amended with sewage sludge. Relatively low sludge rates (~
10 Mg ha–1 year–1) applied for a long time (5 years) stimulate the leaching of Cu, Ni and Pb, as
indicated by increase in extractable concentrations by Mehlich 1 in depth. High sludge rates
(~ 20 Mg ha–1 year–1) with high concentration of Zn in its composition (> 1,300 mg kg–1) have
the potential to leach Zn from the superficial layers of the soil in a short time (2 years), as
suggested by the increase in extractable concentrations by DTPA in depth. Thus, the extractable
concentration, used in the monitoring of leaching, can help in assessing the risk of groundwater
contamination by these heavy metals added to the soil by sewage sludge application to land.
The results of fractionation of heavy metals, albeit very limited, have shown opposite trends
for Zn and Ni. Zn added to the soil by the sludge is preferably bound to Fe and Al oxides. In
this form, its solubility is limited and thus the risk of being excessively absorbed by plants or
leached is reduced. On the other hand, added Zn which binds to SOM is most often associated
with fulvic and humic acids. As these humic substances are poorly stable, its transformation
tends to release Zn retained in soil organic matrix, thereby increasing the risk of contamination.
In contrast, added Ni which binds to SOM is mainly in humin fraction. Since this fraction is
very stable, it restricts the solubility of Ni, minimizing the risk of contamination of crops and
groundwater.
Speciation of heavy metals in soils amended with sewage sludge is still incipient. We found
only one work on this topic, which is a MS thesis. The results of this thesis, however, have
shown significant trends. Applications of sewage sludge rates as low as 20 Mg ha–1 year–1 for
seven years declined forms of Cu and Zn (Cu2+ and Zn2+) that are preferentially absorbed by
plants, but increased forms of these metals (Cu-DOC and DOC -Zn) that are easily leached.
This means that the sewage sludge, when applied in low rates over a long period, decreases
the risk of phytotoxicity and contamination of the food chain, but increases the risk of
groundwater contamination.
Simultaneous use of different measures to evaluate heavy metals in soils can increase security
in the risk assessment of environmental contamination due to the application of sewage sludge
to land. From this perspective, total concentration, available concentration, chemical fractions
and chemical species of metals should be assessed together to better characterize the dynamics
of possible contamination. These different measures represent a gradient of solubility with
immediate and potential impact on plant uptake and leaching of heavy metals in soils. Thus,
the environmental risk posed by the application of sewage sludge could be predicted for short
and long term. In addition, these integrated measures could also be useful for the development
of mathematical models to predict the availability of heavy metals as free ions using easily
measurable input variables (e.g., total metal concentration, pH and SOM) [96]. Such models
would be particularly advantageous in predicting the activity of free ions in response to
changes in soil properties by the application of sewage sludge [96]. We did not find studies in
Brazil using this integrated approach. The studies summarized in this work enable only
superficial considerations, because the data are from independent experiments, and only for
Zn, since it was the only metal with results for all measures. Total concentration is shown to
be a good measure of soil contamination by Zn, but only at high rates of sewage sludge. Under
these conditions, the leaching of metal can be found by the total concentration. For relatively
low rates of sludge, however, this measure suggests only low potential for contamination. In
both cases, it is not possible to establish a close and reliable relationship between total
concentration and toxic effects. Nevertheless, availability and chemical fractions indicate
environmental contamination even with application of relatively low rates of sludge. Specia‐
tion, in turn, suggests that the leaching of Zn may be more relevant than its excessive uptake
by plants, which increases the risk of groundwater contamination to the detriment of phyto‐
toxicity and contamination of the food chain.
5. Heavy metals in crops grown on soils amended with sewage sludge
5.1. Concentration in plants
Land application of sewage sludge can successively increase heavy metals availability in soil,
uptake by plants and accumulation in plant tissues. Thus, concentration in plant tissue can be
used as an indicator of heavy metals transfer from soils amended with sludge to plants and of
the entry of hazardous elements in the food chain. It can also be used to evaluate the phyto‐
toxicity and contamination of harvested products by heavy metals added to the soil by sewage
sludge application, thus presenting important role in determining crop performance and
quality of food originated in field. The concentrations of heavy metals in plants vary depending
on several factors, including rate of sewage sludge, type of heavy metal, type of plant and
analyzed plant part. These factors were studied in field experiments conducted in Brazil,
particularly in São Paulo state. Their main results are summarized below.
Galdos et al. [77] evaluated the concentrations of Cu, Ni and Zn in the middle third of the leaf
below the ear of maize grown for two years on a clayey Oxisol amended with sewage sludge
to supply up to twice the N requirement of the crop. The maximum rates were 21.6 Mg ha–1 in
the first year and 20.5 Mg ha–1 in the second year. Cu concentration in the leaf was changed
only in the first year, increasing with application of the maximum rate of sludge. The waste
had no effect on the concentration of Ni, which was measured only in the second year. For Zn,
the concentration in the leaf increased progressively with sewage sludge rates in the two years.
The concentrations of Cu and Zn were within the range of concentrations suitable for the crop
[99], indicating no phytotoxicity, which is consistent with the absence of adverse effects of the
waste on productivity of grains.
Oliveira et al. [69] applied sewage sludge rates up to 10 Mg ha–1 year–1 to two Oxisols (Typic
Haplorthox and Typic Eutrorthox) for five years and measured the concentrations of Cu, Mn,
Ni, Pb and Zn in shoot and grains of maize in the 5th year. There was no effect of the sludge
on concentration of Cu in any case. The Mn concentration varied only in the shoot, decreasing
in Typic Haplorthox and not having clear effect on Typic Eutrorthox. The sludge did not
change the concentration of Ni in shoot and grains, except for concentration in grains in the
Typic Eutrorthox which was below the LOD of the analytical method (AAS). The Pb concen‐
tration was not changed in the shoot and it was below the LOD in grains. The concentration
of Zn increased in shoot and grains, with the exception of the concentration in the grains in
Typic Eutrorthox, which has not changed. Increased concentrations of Zn were below the
tolerance limit in foods as corn (50 mg kg–1) established by Brazilian Health Surveillance
Agency [100].
Silva et al. [88] applied sewage sludge from Franca and Barueri municipalities to a clayey
Oxisol in three successive annual crops of maize and measured the concentrations of Cu, Mn,
Ni, Pb and Zn in middle third of leaf opposite and below the ear and in its grains. Sludge rates
were defined to supply until eight times the amount of N required by the crop, reaching
maximum values of 30 and 64 Mg ha–1 for ludges from Franca and Barueri, respectively. The
concentration of Cu in leaf increased only in the first crop with Barueri sludge application and
in third crop with Franca sludge application. Despite the increase, the values are below the
toxic level (> 50 mg kg–1) for maize leaf presented in Barbosa Filho et al. [99]. The concentration
of Mn in the leaf increased with increasing sludge rates, except for the first crop when was
used Franca sewage sludge. Suitable concentrations of Mn in maize leaf can be as high as 214
mg kg–1 [99]. No concentration exceeded this value suggesting that there was no toxicity of
Mn. The concentration of Ni increased only in second crop by Franca sludge application. On
the other hand, the Zn concentration increased with increasing rates of both sludges and in all
crops. The only situation in which the concentration of Zn in leaf was slightly above the
appropriate range of concentrations (15-100 mg kg–1) presented in Barbosa Filho et al. [99] was
in third crop with application of higher Barueri sludge rate. However, this excess Zn in leaf
must not have been toxic to the plant, since no symptoms of toxicity were reported. In grains,
there was much less cases of elevated concentrations of heavy metals when compared with
the leaf, suggesting some limitation in the redistribution of these metals to the harvested plant
part. Consistent increases were observed for Mn in second crop with sludge application, Ni
also in second crop, but with Barueri sludge application, and Zn in the second and third crops
with application of the sludges. In the first crop, metals concentrations were below the LOD
of the analytical method used (ICP-OES). The concentrations of Cu, Ni, Pb and Zn in grains
were below the maximum limits for cereals in general (30, 5.0, 8.0 and 50 mg kg–1, respectively)
by Brazilian Food Industry Association [101] and Mn concentrations were below the critical
range presented in Kabata-Pendias and Pendias [102] for grains of plants grown in contami‐
nated soils with Mn.
Oliveira et al. [71] evaluated the concentrations of Cd, Cr, Pb and Zn in stem, leaves, straw,
ear husk, cobs and grains of maize grown on a clayey Oxisol after nine years of annual
applications of sewage sludge rates (Table 10). The Cd concentrations were below the LOD of
the analytical method (AAS) (0.06 mg kg–1 in grains and 0.2 mg kg–1 in other plant tissues). Cr
concentration increased in stem and leaves in response to sludge application and was below
the LOD in ear husk, cob and grains. Pb concentration increased in stem, leaves and ear husk,
did not change in cob and was below the LOD in grains. Except for grains, all plant parts had
increases in Zn concentration. These results show that stem and leaves were the most sensible
plant tissues for expressing the effect of sewage sludge rates on Cr, Pb and Zn concentrations
in maize.
In other long term experiment, Cd, Cr and Pb concentrations were evaluated in leaf apposite
and below the ear, whole plant and grains of maize after 11 annual applications of sewage
sludge rates up to 20 Mg ha–1 to an Oxisol [73]. There was no effect of sludge on Cd and Pb
concentrations in leaf. For Cr in this plant part, its concentrations were below the LOD (< 0.19
mg kg–1) of the analytical method (AAS). Similarly, concentrations of these heavy metals were
below the LOD in grains (0.03, 0.15 and 0.19 mg kg–1 for Cd, Cr and Zn, respectively). When
considered the whole plant, Cd, Cr and Pb concentrations were not significantly changed by
sewage sludge applications.
There are also results for sugarcane. Camilotti et al. evaluated the concentrations of Cd, Cr, Ni
and Pb in stem and leaves of sugarcane cultivated in a clayey Oxisol after three [103] and four
[75] annual applications of sewage sludge rates, which reached a maximum of 15 Mg ha–1, with
the goal of supplying 100 % and 200 % of N required by the crop. There was no effect of three
applications of sewage sludge in concentrations of Cr in stalk and leaves, Ni in leaves and Pb
in stalk and leaves, as well as Cd concentrations in all plant parts analyzed. Cr in leaves and
Ni in stalk were below the LOD of the analytical method used (AAS). Similarly, the fourth
sludge application did not affect the concentrations of Pb in leaves, but all other cases the
concentrations of evaluated heavy metals were below the LOD.
In an experiment conducted on Ultisol, Nogueira et al. [78] applied sewage sludge rates up to
10.8 Mg ha–1 at sugarcane planting for supplying N required by the crop and evaluated As,
Cd, Cr, Cu, Ni, Pb, Se and Zn concentrations in leaf (with top visible dewlap), stalk and juice
at the harvest time of plant cane (first year) and ratoon cane (second year). In these two
evaluations, As concentration increased in leaf and was not affected in stalk and juice by sludge
application. Cd concentration increased in leaf, stalk and juice in both crops. On the other hand,
Pb concentration decreased in stalk and juice of plant cane and was not affected in other
situations. Cu concentration decreased in stalk of plant cane and leaf of ratoon cane and was
not affected in other cases. Ni concentration also was not generally affected, but it increased
in stalk of plant cane and decreased in stalk of ratoon cane. For Pb concentration, there was no
effect of sludge rates. Se concentration was affected only in leaf of plant cane, increasing with
sludge application. Zn concentration increased in all situations. There were no cases of
concentration below the LOD probably because the authors used ICP-MS.
Sewage sludge rate Heavy metal in plant tissue
Annual Cumulative Cr Pb Zn
Mg ha –1 ______________________________
mg kg –1 _____________________________
Stem
0 0 0.13 c† 0.68 b 15.76 c
5 45 0.33 b 0.61 b 19.11 c
10 90 0.45 a 0.83 a 26.62 b
2.5 + 20‡ 127.5 0.19 c 0.47 c 44.35 a
Leaves
0 0 0.74 b 1.10 c 29.35 b
5 45 1.20 a 1.43 b 28.54 b
10 90 1.48 a 1.72 a 39.34 ab
2.5 + 20‡ 127.5 0.67 b 1.64 ab 50.77 a
Ear husk
0 0 < 0.3 4.28 ab 13.03 b
5 45 < 0.3 3.95 b 12.91 b
10 90 < 0.3 4.86 a 17.71 ab
2.5 + 20‡ 127.5 < 0.3 4.70 ab 20.52 a
Cob
0 0 < 0.3 0.88 a 13.49 b
5 45 < 0.3 0.98 a 15.93 b
10 90 < 0.3 1.04 a 27.47 a
2.5 + 20‡ 127.5 < 0.3 0.98 a 32.35 a
Grains
0 0 < 0.09 < 0.4 32.36 a
5 45 < 0.09 < 0.4 29.32 a
10 90 < 0.09 < 0.4 35.77 a
2.5 + 20‡ 127.5 < 0.09 < 0.4 36.70 a
†Means within a column in each plant tissue followed by the same letter are not significantly different according to
Tukey test (p < 0.05).
‡2.5 Mg ha–1 in 1st, 2nd, and 3rd years and 20 Mg ha–1 in 4th, 5th, 6th, 7th, 8th and 9th years.
Source: Nogueira et al. [71].
Table 10. Cr, Pb and Zn concentrations in stem, leaves, ear husk, cob and grains of maize after nine annual
applications of sewage sludge to a clayey Oxisol from São Paulo State, Brazil.
5.2. Risks of contamination
The results reviewed above have shown contrasting effects of sewage sludge on the concen‐
trations of heavy metals in maize and sugarcane. Sludge effects on Cu, Pb and Ni concentra‐
tions ranged greatly. On the other hand, its effects were more consistent for Zn. Sludge
application generally increased Zn concentrations in plants, but not in phytotoxic levels.
Increases occurred even in edible parts of crops, such as grains of corn, but concentrations did
not exceed the limit established by Brazilian legislation. For sugarcane, in addition to Zn
accumulation, Cd concentrations may also increase with sludge application. However, even
increasing, Cd concentrations remained very low.
These findings support the view that the sewage sludge applied to land has low potential to
contaminate maize and sugarcane with heavy metals. However, this does not mean that risks
of contamination of other crops are also low. Vegetable crops, for example, can easily be
contaminated by heavy metals since they have contact with sewage sludge applied to soil or
bed. Because of this high risk of contamination, Brazilian law prohibits the agricultural use of
sewage sludge for production of vegetable crops and also other crops [67].
6. Summary and conclusions
The expected increase in the generation of sewage sludge in Brazil should intensify its disposal
on land. Although the sludge is rich in organic matter and plant nutrients, it has heavy metals
in its composition. Due to the presence of potentially toxic metals, application of sludge to land
requires a risk assessment of soil-plant system contamination. Knowledge of the sludge
composition in terms of heavy metals is the first step in assessing risks of contamination.
We have shown in this work that concentrations of heavy metals in the sludge generated in
Brazilian WTPs can vary widely depending on the metal. Cd concentration is extremely
variable, while Zn concentration varies relatively little. High variability of Cd concentration
may hamper the planning sludge application by the need for additional adjustment in order
to avoid excessive application of Cd in soil. Zn concentrations found in thesludges analyzed
were very high. Thus, the risk of contamination by Zn can be considered high as well.
Soil contamination by Cu, Ni and Zn, as determined by the total concentration, can be rapid,
since high rates of sludge are applied to land. In contrast, low rates of sludge have shown low
potential to contaminate soil with Cu, Ni, Pb and Zn. However, available concentrations can
indicate contamination by Cu and Zn even with application of low rates of sludge. Further‐
more, they can detect changes in the solubility of metals due to liming.
Nevertheless, the available concentrations depend on the efficiency of the extractant. DTPA,
0.1 mol L–1 HCl, Mehlich 1 and Mehlich 3 have not been as efficient as needed. DTPA has shown
best performance among these extractants. DTPA and Mehlich 1 extractants can also be used
to monitor the leaching of heavy metals such as Cu, Ni, Pb and Zn in soils amended with
sewage sludge, whose importance is to assess the soil contamination at depth and the potential
for contamination of groundwater. The fractionation and speciation data have confirmed that
Zn has a potential to be leached in soil amended with sludge, which increases the risk of
groundwater contamination. Regard to contamination of the plant, the results have shown that
the application of sewage sludge has low potential to contaminate maize and sugarcane with
Cu, Ni, Pb and Zn.
Despite the apparent low risk of contamination of the soil-plant system by heavy metalsdue
to the application of sewage sludge to land, this review has shown that (i) the results from the
field experiments are limited to a restricted number of potentially toxic metals, (ii) the chemical
extractantsaregenerally inefficient to estimate the available concentrations for these metals in
a range of situations and (iii) the studies on fractionation and speciation of these hazardous
elements in soils are still incipient. Furthermore, studies have not used an integrated approach
involving total concentration, available concentration, chemical fractions and chemical species
of heavy metals to assess more reliably the risk of phytotoxicity and contamination of the food
chain and groundwater. Thus, this scenario suggests that the application of sewage sludge to
land should be as restrictive as possible in Brazil.
Author details
Alysson Roberto Baizi e Silva1* and Fábio Camilotti1
1 Embrapa Eastern Amazon, Belém, PA, Brazil
2 Jaboticabal College of Technology, Jaboticabal, SP, Brazil
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Environmental Risk

Assessment of Soil Contamination

Edited by Maria C. Hernandez-Soriano

Edited by Maria C. Hernandez-Soriano

D3pZ4i & bhgvld, Dennixxx & rosea (for softarchive)

Publishing Process Manager

Published 26 March, 2014

1 Soil Contamination, Risk Assessment and Remediation

17 Phytoremediation of Soils Contaminated with Metals and

Soil is an irreplaceable resource that sustains life on the planet,

Integrated efforts from researchers and policy makers are required

Environmental Risk Assessment of Soil Contamination provides a wide

The book introduces several innovative approaches for soil remediation

Soil Contamination, Risk Assessment and Remediation

Muhammad Aqeel Ashraf, Mohd. Jamil Maah and

Additional information is available at the end of the chapter

2. How to determine the ecological risk assessment of the soil?

3. To what extent do soil screening levels (over)estimate risk?

4. Do bioassays represent a more realistic risk estimate?

5. Is it possible to make sound field surveys, or do we lack suitable reference situations?

• Discharge of industrial waste into the soil

• Percolation of contaminated water into the soil

• Rupture of underground storage tanks

• Excess application of pesticides, herbicides or fertilizer

• Solid waste seepage

2.1. Inorganic toxic compounds

2.2. Organic wastes

b. Heavy metal pollutants

Sl.No Heavy metal Hithosphere Soil range Plants

1 Cadmium (Cd) 0.2 0.01-0.7 0.2-0.8

2 Cobalt (Co) 40 1-40 0.05-0.5

3 Chromium (Cr) 200 5-3000 0.2-1.0

4 Copper (Cu) 70 2-100 4-15

5 Iron (Fe) 50,000 7000-5,50,000 140

6 Mercury (Hg) 0.5 0.01-0.3 0.015

7 Manganese (Mn) 1000 100-4000 15-100

8 Molybdenum (Mo) 2.3 0.2-5 1-10

9 Nickel (Ni) 100 10-1000 1

10 Lead (Pb) 16 2-200 0.1-10

11 Tin (Sn) 40 2-100 0.3

12 Zinc (Zn) 80 10-300 8-100

The range of heavy metal contents in sludges is given in Table 3.

2 Calcium 0.1 Traces Traces Traces - - - 1

Ammonium <0.05 to Traces to

Traces to Traces to Traces to Traces to

Table 2. Heavy metal content of fertilizers (ug/gm)

Sl.No Heavy metal Range (ppm)

1 Cadmium < 60-1500

Table 3. Heavy metal contents in sludges (ppm)

2.3. Organic pesticides

Sl.No Pesticide Persistence time

3 2,4-D 2-8 weeks

9 2,3 6-Trichlorobenzene (TBA) 2-5 yrs

Table 4. Persistence time for some selected pesticides

2.4. Types of soil pollution

• Agricultural Soil Pollution

i. pollution of surface soil

ii. pollution of underground soil

• Soil pollution by industrial effluents and solid wastes

i. pollution of surface soil