Wwtbugs PDF

WASTEWATER TREATMENT
BUGS
CONTINUING EDUCATION COURSE
PROFESSIONAL DEVELOPMENT COURSE
WWTBUGS©8/1/2018 www.abctlc.com 2 Toll Free (866) 557-1746
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United States Library of Congress Number TX 6-600-029
ISBN 978-0-9799928-5-8
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Water Chemistry Courses. Most unaccredited photographs have been taken by TLC
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following address: TLC, P.O. Box 3060, Chino Valley, AZ 86323
Information in this document is subject to change without notice. TLC is not liable for errors
or omissions appearing in this document.
Contributing Editors
James L. Six Received a Bachelor of Science Degree in Civil Engineering from the
University of Akron in June of 1976, Registered Professional Engineer in the State of Ohio,
Number 45031 (Retired), Class IV Water Supply Operator issued by Ohio EPA, Number
WS4-1012914-08, Class II Wastewater Collection System Operator issued by Ohio EPA,
Number WC2-1012914-94
Joseph Camerata has a BS in Management with honors (magna cum laude). He retired
as a Chemist in 2006 having worked in the field of chemical, environmental, and industrial
hygiene sampling and analysis for 40 years.
James Bevan, Water Quality Inspector S.M.E. Twenty years of experience in the
environmental field dealing with all aspects of water regulations on the federal, state, and
local levels. Teacher and Proctor in Charge for Backflow Certification Testing at the ASETT
Center in Tucson for the past 15 years and possess an Arizona Community College,
Special Teaching Certificate in Environmental Studies.
Dr. Pete Greer S.M.E., Retired biology instructor, chemistry and biological review.
Jack White, Environmental, Health, Safety expert, City of Phoenix. Art Credits.

Technical Learning College’s Scope and Function
Welcome to the Program,
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Course Structure
TLC's online courses combine the best of online delivery and traditional university
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Contact Numbers
Fax (928) 468-0675
Email [email protected]
Telephone (866) 557-1746

Course Description
Wastewater Treatment Bugs CEU Training Course

This is a review of various and complex wastewater treatment methods, water quality,
sampling techniques, bug identification, disinfection, sludge disposal and related WWT
subjects. This course is general in nature and not state specific but will contain various
wastewater treatment microorganisms, including activated sludge methods and
wastewater quality procedures. You will not need any other materials for this course.
Intended Audience
Wastewater Treatment Operators; Pretreatment and Industrial Waste Inspectors. The
target audience for this course is the person interested in working in a wastewater
treatment or pretreatment/industrial wastewater facility and/or wishing to maintain CEUs
for certification license or to learn how to do the job safely and effectively, and/or to meet
education needs for promotion.
Prerequisites: None
Course Procedures for Registration and Support

All of TLC’s correspondence courses have complete registration and support services
offered. Delivery of services will include e-mail, web site, telephone, fax and mail support.
TLC provides immediate and prompt service.
When a student registers for a correspondence course, he or she is assigned a start date
and an end date. It is the student's responsibility to note dates for assignments and keep
up with the course work. If a student falls behind, he or she must contact TLC and request
an end date extension in order to complete the course. It is the prerogative of TLC to
decide whether to grant the request.
All students will be tracked by a unique number assigned to the student.
Instructions for Written Assignments

The Wastewater Treatment Bugs CEU training distance learning course uses a fill-in-the-
blank style answer key. You can write your answers in the assignment or type out your
own answer key. TLC would prefer that you type out and e-mail final assignment to TLC,
but it is not required.
Feedback Mechanism (examination procedures)

Each student will receive a feedback form as part of the study packet. You will be able to
find this form in the front of the course assignment or lesson.
Security and Integrity

All students are required to do their own work. All lesson sheets and final exams are not
returned to the student to discourage and sharing of answers. Any fraud or deceit and the
student will result in forfeiture of all fees, and the appropriate agency will be notified.
Grading Criteria
TLC will offer the student either pass/fail or a standard letter grading assignment. If TLC
is not notified, you will only receive a pass/fail notice.

Required Texts
The Wastewater Treatment Bugs CEU training course will not require any other materials.
This course comes complete. No other materials are needed.
Environmental Terms, Abbreviations, and Acronyms

TLC provides a glossary that defines, in non-technical language, commonly used
environmental terms appearing in publications and materials. It also explains
abbreviations and acronyms used throughout EPA and other agencies. You can find the
glossary in the rear of the manual.
Recordkeeping and Reporting Practices

TLC will keep all student records for a minimum of five years. It is your responsibility to
give the completion certificate to the appropriate agencies. TLC will mail a copy to Indiana,
Pennsylvania, Texas, or any other state that requires a copy from the Training Provider.
ADA Compliance
TLC will make reasonable accommodations for persons with documented disabilities.
Students should notify TLC and their instructors of any special needs. Course content may
vary from this outline to meet the needs of this particular group. There is an alternative
assignment available.
The final grade options are as follows:

Letter grade (A, B, C, D, F) - These grades are awarded based on the course grading
scale. Withdrawn (W or Y) - Students who enroll but do not participate in the class may
withdraw themselves by calling Admissions and Records, or their instructor may withdraw
them. Either case will result in a grade of "W." Note that participation means the completion
of a single homework assignment or an exam. Completion of the pretest and/or syllabus
receipt does not imply course participation.
Credit/no credit option (P/Z) - None Available
Note to students: Keep a copy of everything you submit. That way if your work is lost
you can submit your copy for grading. If you do not receive your graded assignment or
quiz results within two or three weeks after submitting it, please contact your instructor.
We expect every student to produce his/her original, independent work. Any student
whose work indicates a violation of the Academic Misconduct Policy (cheating, plagiarism)
can expect penalties as specified in the Student Handbook, which is available through
Student Services; contact them at (928) 468-0665.
A student who registers for a Distance Learning course is assigned a "start date" and an
"end date." It is the student's responsibility to note due dates for assignments and to keep
up with the course work.
If a student falls behind, she or he must contact the instructor and request an extension of
her/his end date in order to complete the course. It is the prerogative of the instructor to
decide whether or not to grant the request. You will have 90 days from receipt of this
manual to complete it in order to receive your Continuing Education Units (CEUs) or
Professional Development Hours (PDHs). A score of 70% or better is necessary to pass
this course.

If you need any assistance, please email all concerns to [email protected].
Educational Mission
The educational mission of TLC is:
To provide TLC students with comprehensive and ongoing training in the theory and
skills needed for the environmental education field,
To provide TLC students opportunities to apply and understand the theory and skills
needed for operator certification,
To provide opportunities for TLC students to learn and practice environmental

educational skills with members of the community for the purpose of sharing diverse
perspectives and experience,
To provide a forum in which students can exchange experiences and ideas related to
environmental education,
To provide a forum for the collection and dissemination of current information related to
environmental education, and to maintain an environment that nurtures academic and
personal growth.
Course Objective
To provide a detailed understanding wastewater treatment microorganisms, activated
sludge, wastewater treatment sampling techniques and biological monitoring, bug
identification and microorganism control methods.

Important Information about this Manual
This manual has been prepared to educate employees in the general awareness of dealing with
complex wastewater treatment procedures and regulatory requirements for safely handling
hazardous and toxic materials. The scope of the problem is quite large, requiring a major effort to
bring it under control. Employee health and safety, as well as that of the public, depend upon
careful application of safe treatment procedures. The manner in which we deal with such hazards
will affect the earth and its inhabitants for many generations to come.
This manual will cover general laws, regulations, required procedures and generally accepted
policies relating to wastewater treatment and wastewater sampling. It should be noted, however,
that the regulation of wastewater treatment, sampling and other hazardous materials is an ongoing
process and subject to change over time. For this reason, a list of resources is provided to assist
in obtaining the most up-to-date information on various subjects.
This manual is not a guidance document for employees who are involved with pollution control or
wastewater treatment. It is not designed to meet the requirements of the United States
Environmental Protection Agency (EPA) or Department of Labor-Occupational Safety and Health
Administration (OSHA) or state environmental or health departments.
This course manual will provide general educational awareness guidance of activated sludge. This
document is not a detailed wastewater treatment textbook or a comprehensive source book on
occupational safety and health.
Technical Learning College or Technical Learning Consultants, Inc. makes no warranty, guarantee
or representation as to the absolute correctness or appropriateness of the information in this
manual and assumes no responsibility in connection with the implementation of this information. It
cannot be assumed that this manual contains all measures and concepts required for specific
conditions or circumstances. This document should be used for educational guidance and is not
considered a legal document.
Individuals who are responsible for the treatment of wastewater, wastewater sampling or the health
and safety of workers at wastewater sites should obtain and comply with the most recent federal,
state, and local regulations relevant to these sites and are urged to consult with OSHA, EPA and
other appropriate federal, state, health and local agencies.

TABLE OF CONTENTS
Acronyms................................................................................ 15
Clean Water Act Section......................................................... 21
Effects of WWT Pollutants...................................................... 27
Preliminary Treatment............................................................ 29
Secondary Clarification.......................................................... 37
Flow........................................................................................ 47
Processed Solids.................................................................... 49
Water Quality Criteria............................................................. 59
Nitrogen Control...................................................................... 61
Nitrification.............................................................................. 69
Nitrogen Removal.................................................................. 71
Phosphorus............................................................................ 79
Phosphorus Removal............................................................. 81
Chemical Feeding................................................................... 85
Advanced Solids.................................................................... 87
Water Quality Trading............................................................. 93
Wastewater Sampling............................................................. 95
Pretreatment........................................................................... 99
Proper Sample Handling........................................................ 121
Field Blanks............................................................................. 125
Laboratory Analysis Section................................................ 137
pH............................................................................................ 141
Dissolved Oxygen……………………….....................………... 155
Dissolved Oxygen Procedure……………......................….…. 157
TDS………………..................……………......................…...... 159
Sludge Volume Indexing………………….....................……... 167
Mixed Liquor……………………………......................……...... 169
Microlife .................................................................................. 175
Microorganisms in Lagoons.................................................... 179
Advanced Methods................................................................. 183
Algae ...................................................................................... 193
Bacteria Section..................................................................... 195
Filamentous............................................................................ 209
Microthrix................................................................................ 211
PAX........................................................................................ 215
Sphaerotilus natas.................................................................. 217
Nostocoida limicola................................................................ 218
Thiothrix................................................................................. 219
Activated Sludge Section....................................................... 223
Activated Sludge Methods...................................................... 229
Complete Mix Process............................................................ 233
Contact Stabilization............................................................... 235
Extended Aeration.................................................................. 237
Aeration................................................................................... 245

Blowers.................................................................................. 249
Secondary Clarifiers................................................................ 253
Scum Removal...................................................................... 257
Review Process Goals........................................................... 259
Nitrification/Denitrification........................................................ 261
Key Design Issues.................................................................. 265
Biological Phosphorus Removal.............................................. 273
RAS/WAS Systems................................................................ 275
RBC........................................................................................ 279
Emerging Technologies.......................................................... 283
Types of Filters........................................................................ 291
Chlorine.................................................................................. 295
Chlorine Introduction............................................................... 299
Chlorine Exposure.................................................................. 301
Chlorine Basics...................................................................... 303
Chemical Equations................................................................ 307
Alternative Disinfectants.......................................................... 341
Ultraviolet Radiation................................................................ 343
Respiratory Protection............................................................. 373
Confined Space...................................................................... 375
Entry Permit............................................................................ 383
Responsibilities....................................................................... 385
Charge of Entry....................................................................... 387
Glossary.................................................................................. 407
Microorganism Appenidix....................................................... 445
Chlorine Charts…………………………………........................ 507
Conversion Factors................................................................ 519
References............................................................................. 525
This course contains general EPA’s CWA federal rule

requirements. Please be aware that each state implements
wastewater/safety/environment regulations that may be more stringent
than EPA’s regulations. Check with your state environmental agency
for more information.

Common Wastewater Acronyms and Terms
Acronyms and Abbreviations
A/E Contract: Architectural and Engineering Contracts
A/O: Pho-redox
AMSA: Association of Metropolitan Sewerage Agencies
AOB: Ammonia Oxidizing Bacteria
ASM: Activated Sludge Model
AT3: Aeration Tank 3
BABE: Bio-Augmentation Batch Enhanced
BAF: Biological Aerated Filter
BAR: Bio-Augmentation Regeneration/Reaeration
BCFS: Biological Chemical Phosphorus and Nitrogen Removal
bDON: Biodegradable Fraction of Dissolved Organic Nitrogen
BHRC: Ballasted High Rate Clarification Processes
BNR: Biological Nutrient Removal
BOD: Biochemical Oxygen Demand
BOD5: Biochemical Oxygen Demand (5-day)
BPR: Biological Phosphorus Removal
COD: Chemical Oxygen Demand
CSO: Combined Sewer Overflow
CWA: Clean Water Act
CWSRF: Clean Water State Revolving Fund
D&D: Drying and Dewatering Facility
DAF: Dissolved Air Flotation
DNR: Department of Natural Resources
DO: Dissolved Oxygen
DON: Dissolved Organic Nitrogen
E1: Estrone
E2:17 ß-estradiol
EBPR: Enhanced Biological Phosphorus Removal
EDC: Endocrine Disrupting Chemicals
EDTA: Ethylene Diamine Tetraacetic Acid
EE2: 17α-ethynylestradiol
EPA: U.S. Environmental Protection Agency
EPA or USEPA: United States Environmental Protection Agency
FFS: Fixed-film Systems
FWPCA: Federal Water Pollution Control Act
FWS: Free Water Surface
GAO: Glycogen Accumulating Organism
GIS: Geographic Information System
HHWP: Household Hazardous Waste Collection Program
HRT: Hydraulic Retention Time
I&C: Instrumentation and Control System
I/I: Infiltration and Inflow
iDON: Inert Dissolved Organic Nitrogen
ISF: Intermittent Sand Filter
ISS: Inline Storage System
IWA: International Water Association
IWPP: Industrial Waste Pretreatment Program

LIMS: Laboratory Information Management Systems
MAUREEN: Mainstream Autotrophic Recycle Enhanced N-removal
MBBR: Moving-Bed Biofilm Reactor
MBDT: Minority Business Development and Training
MBE: Minority Business Enterprise
MBR: Membrane Bioreactor
MGD: Million Gallons per Day
MLE: Modified Ludzack Ettinger
N: Nitrogen
NOAA: National Oceanic and Atmospheric Administration
NOB: Nitrite Oxidizing Bacteria
NPDES: National Pollutant Discharge Elimination System
NTT: Nitrogen Trading Tool
ORD: EPA Office of Research and Development
ORP: Oxidation Reduction Potential
OWM: EPA Office of Wastewater Management
P: Phosphorus
P2: Pollution Prevention Initiative
PAH: Polycyclic Aromatic Hydrocarbons
PAO: Phosphate Accumulating Organism
PHA: Polyhydroxyalkanoates
PHB: Poly-B-hydroxy-butyrate
PHV: Poly-hydroxy valerate
POTW: Publicly Owned Treatment Works
PPCPs: Pharmaceuticals and Personal Care Products
QA/QC: Quality Assurance and Quality Control
RAS: Return Activated Sludge
RBC: Rotating Biological Contactor
rbCOD: Readily Biodegradable Chemical Oxygen Demand
rDON: Recalcitrant Dissolved Organic Nitrogen
RO: Reverse Osmosis
RSF: Recirculating Sand Filters
S/W/MBE: Small, Women's, Minority Business Enterprise
SAV: Submerged Aquatic Vegetation
SBR: Sequencing Batch Reactors
SHARON: Single Reactor High-activity Ammonia Removal over Nitrite
SND: Simultaneous Nitrification-Denitrification
SRT: Solids Retention Time
SSES: Sewer System Evaluation Survey
SSO: Sanitary Sewer Overflow
SWIS: Subsurface Wastewater Infiltration System
TAT: Technical Advisory Team
TDS: Total Dissolved Solids
TKN: Total Kjeldahl Nitrogen
TMDL: Total Maximum Daily Loads
TN: Total Nitrogen
TP: Total Phosphorus
TSS: Total Suspended Solids
TUDP: Bio-P Model of the Delft University of Technology
USDA: U.S. Department of Agriculture
USGS: U.S. Geological Survey

VFA: Volatile Fatty Acids
VIP: Virginia Initiative Plant
VSS: Volatile Suspended Solids
WAS: Waste Activated Sludge
WEF: Water Environment Federation
WERF: Water Environment Research Foundation
WPAP: Water Pollution Abatement Program
WQS: Water Quality Standard
WWTP: Wastewater Treatment Plant
When sludge is not removed or is settling improperly, the sludge will denitrify and rise to
the top. The sludge will flow over the weir as seen in this photograph and clog filters.

Clean Water Act Section
What is Wastewater Treatment?
Wastewater treatment is the process of cleaning used water and sewage so it can be returned
safely to our environment. Wastewater treatment is the last line of defense against water pollution.
If you envision the water cycle as a whole, you can clean water produced by wastewater treatment
is the same water that eventually ends up back in our lakes and rivers, where we get our drinking
water.
Why Are Wastewater Treatment Plants Important?

Wastewater treatment plants are vital to our communities. They protect public health by eliminating
disease-causing bacteria from water. By protecting water quality, wastewater treatment plants
make it possible for us to safely enjoy the recreational use of clean oceans, lakes, streams and
rivers.
33 U.S.C. s/s 1251 et seq. (1977)

The Clean Water Act is a 1977 amendment to the Federal Water Pollution Control Act of 1972,
which set the basic structure for regulating discharges of pollutants to waters of the United States.
The law gave the EPA the authority to set

effluent standards on an industry basis
(technology-based) and continued the
requirements to set water quality standards for
all contaminants in surface waters. The CWA
makes it unlawful for any person to discharge
any pollutant from a point source into navigable
waters unless a permit (NPDES) is obtained
under the act.
The 1977 amendments focused on toxic

pollutants. In 1987, the PCA was reauthorized
and again focused on toxic substances,
authorized citizen suit provisions, and funded sewage treatment plants (POTW's) under the
Construction Grants Program.
The CWA provides for the delegation by the EPA of many permitting, administrative, and
enforcement aspects of the law to state governments. In states with the authority to implement
CWA programs, the EPA still retains oversight responsibilities. In 1972, Congress enacted the first
comprehensive national clean water legislation in response to growing public concern for serious
and widespread water pollution. The Clean Water Act is the primary federal law that protects our
nation’s waters, including lakes, rivers, aquifers, and coastal areas.
Lake Erie was dying. The Potomac River was clogged with blue-green algae blooms that were a
nuisance and a threat to public health. Many of the nation's rivers were little more than open sewers
and sewage frequently washed up on shore. Fish kills were a common sight. Wetlands were
disappearing at a rapid rate.
Today, the quality of our waters has improved dramatically as a result of a cooperative effort by
federal, state, tribal and local governments to implement the pollution control programs established
in 1972 by the Clean Water Act. The Clean Water Act's primary objective is to restore and maintain
the integrity of the nation's waters. This objective translates into two fundamental national goals:
 eliminate the discharge of pollutants into the nation's waters, and

 achieve water quality levels that are fishable and swimmable.

The Clean Water Act focuses on improving the quality of the nation’s waters. It provides a
comprehensive framework of standards, technical tools and financial assistance to address the
many causes of pollution and poor water quality. This includes municipal and industrial wastewater
discharges, polluted runoff from urban and rural areas, and habitat destruction.
For example, the Clean Water Act requires major industries to meet performance standards to
ensure pollution control; charges states, and tribes with setting specific water quality criteria
appropriate for their waters and developing pollution control programs to meet them; provides
funding to states and communities to help them meet their clean water infrastructure needs;
protects valuable wetlands and other aquatic habitats through a permitting process that ensures
development, and other activities are conducted in an environmentally sound manner. After 25
years, the act continues to provide a clear path for clean water and a solid foundation for an
effective national water program.
In 1972
Only a third of the nation's waters were safe for fishing and swimming. Wetlands losses were
estimated at about 460,000 acres annually. Agricultural runoff resulted in the erosion of 2.25 billion
tons of soil and the deposit of large amounts of phosphorus and nitrogen into many waters. Sewage
treatment plants served only 85 million people.
Today
Two-thirds of the nation's waters are safe for fishing and swimming. The rate of annual wetlands
losses is estimated at about 70,000-90,000 acres according to recent studies. The amount of soil
lost due to agricultural runoff has been cut by one billion tons annually, and phosphorus and
nitrogen levels in water sources are down. Modern wastewater treatment facilities serve 173 million
people.
The Future
All Americans will enjoy clean water that is safe for fishing and swimming. We will achieve a net
gain of wetlands by preventing additional losses and restoring hundreds of thousands of acres of
wetlands. Soil erosion and runoff of phosphorus and nitrogen into watersheds will be minimized,
helping to sustain the nation's farming economy and aquatic systems. The nation's waters will be
free of effects of sewage discharges.
Rotifer, an excellent MO or Microorganism or Indicator Organism.

Wastewater Treatment WWT Introduction
During the early days of our nation’s history, people living in both the cities and the countryside
used cesspools and privies to dispose of domestic wastewater. Cities began to install wastewater
collection systems in the late nineteenth century because of an increasing awareness of
waterborne disease and the popularity of indoor plumbing and flush toilets.
The use of sewage collection systems brought dramatic improvements to public health, further
encouraging the growth of metropolitan areas. In the year 2000 approximately 208 million people
in the U.S. were served by centralized collection systems.
Wastewater Treatment
In 1892, only 27 American cities provided wastewater treatment. Today, more than 16,000 publicly-
owned wastewater treatment plants operate in the United States and its territories. The
constructions of wastewater treatment facilities blossomed in the 1920s and again after the
passage of the CWA in 1972 with the availability of grant funding and new requirements calling for
minimum levels of treatment. Adequate treatment of wastewater, along with the ability to provide a
sufficient supply of clean water, has become a major concern for many communities.
What is in Wastewater?
Wastewater is mostly water by weight. Other materials make up only a small portion of wastewater,
but can be present in large enough quantities to endanger public health and the environment.
Because practically anything that can be flushed down a toilet, drain, or sewer can be found in
wastewater, even household sewage contains many potential pollutants. The wastewater
components that should be of most concern to homeowners and communities are those that have
the potential to cause disease or detrimental environmental effects.
Basic Wastewater Treatment Processes

Physical
Physical processes were some of the earliest methods to remove solids from wastewater, usually
by passing wastewater through screens to remove debris and solids. In addition, solids that are
heavier than water will settle out from wastewater by gravity. Particles with entrapped air float to
the top of water and can also be removed. These physical processes are employed in many modern
wastewater treatment facilities today.
Biological
In nature, bacteria and other small organisms in water
consume organic matter in sewage, turning it into new
bacterial cells, carbon dioxide, and other by-products.
The bacteria normally present in water must have oxygen

to do their part in breaking down the sewage. In the
1920s, scientists observed that these natural processes
could be contained and accelerated in systems to remove
organic material from wastewater. With the addition of
oxygen to wastewater, masses of microorganisms grew
and rapidly metabolized organic pollutants.
Any excess microbiological growth could be removed

from the wastewater by physical processes.
Activated Sludge is a suspended growth process for removing organic matter from sewage by
saturating it with air and microorganisms that can break down the organic matter. Advanced
Treatment involves treatment levels beyond secondary treatment.

Chemical
Chemicals can be used to create changes in pollutants that increase the removal of these new
forms by physical processes. Simple chemicals such as alum, lime or iron salts can be added to
wastewater to cause certain pollutants, such as phosphorus, to floc or bunch together into large,
heavier masses which can be removed faster through physical processes. Over the past 30 years,
the chemical industry has developed synthetic inert chemicals known as polymers to further
improve the physical separation step in wastewater treatment. Polymers are often used at the later
stages of treatment to improve the settling of excess microbiological growth or biosolids.
Organisms
Many different types of organisms live in wastewater and some are essential contributors to
treatment. A variety of bacteria, protozoa, and worms work to break down certain carbon-based
(organic) pollutants in wastewater by consuming them. Through this process, organisms turn
wastes into carbon dioxide, water, or new cell growth.
Bacteria and other microorganisms are particularly plentiful in wastewater and accomplish most of
the treatment. Most wastewater treatment systems are designed to rely in large part on biological
processes.
Pathogens
Many disease-causing viruses, parasites, and bacteria also are present in wastewater and enter
from almost anywhere in the community. These pathogens often originate from people and animals
are infected with or are carriers of a disease. Graywater and blackwater from typical homes contain
enough pathogens to pose a risk to public health. Other likely sources in communities include
hospitals, schools, farms, and food processing plants.
Some illnesses from wastewater-related sources are relatively common. Gastroenteritis can result
from a variety of pathogens in wastewater, and cases of illnesses caused by the parasitic protozoa
Giardia lambia and Cryptosporidium are not unusual in the U.S. Other important wastewater-related
diseases include hepatitis A, typhoid, polio, cholera, and dysentery. Outbreaks of these diseases
can occur as a result of drinking water from wells polluted by wastewater, eating contaminated fish,
or recreational activities in polluted waters. Some illnesses can be spread by animals and insects
that come in contact with wastewater.
Even municipal drinking water sources are not completely immune to health risks from wastewater
pathogens. Drinking water treatment efforts can become overwhelmed when water resources are
heavily polluted by wastewater. For this reason, wastewater treatment is as important to public
health as drinking water treatment.
Ciliate
Vorticella is a stalked ciliate. There are at least a dozen species found in activated sludge
ranging in length from about 30 to 150 μm. These organisms are oval to round shaped,
have a contractile stalk, a domed feeding zone, and a water vacuole located near the
terminal end of the feeding cavity.

Domestic Wastewater Characteristics
Typical major pollutant characteristics of domestic wastewater
Type Pollutant Conc. (mg/L)
Physical Total Suspended Solids 300
Volatile Suspended Solids 240
Fixed Suspended Solids 60
Total Dissolved Solids 440
Volatile Suspended Solids 175
Fixed Suspended Solids 265
Temperature 10 - 25 oC
Color Grey - Black
Chemical BOD5 250

COD 500
TOC 160
Total N 40
Organic N 15
Free ammonia N 25
Nitrite N 0
Nitrates N 0
Total P 9
Organic P 4
Inorganic P 5
Alkalinity 100
Fats, oil and grease (FOG) 100
Microbiological Total coliform 108 - 109 MPN/L

Fecal coliform 107 - 108 MPN/L
Non-fecal coliform 9x107 - 9x108 MPN/L
Total viruses 1,000-10,0000 infectious units/L
This course contains general EPA’s CWA federal rule requirements. Please be
aware that each state implements wastewater/safety/environment regulations that
may be more stringent than EPA’s regulations. Check with your state
environmental agency for more information.

Typical Flow Rate of Domestic Wastewater
Typical BOD5 Variation of Domestic Wastewater

Effects of Wastewater Pollutants
Effect of BOD
o Depletes dissolved oxygen from streams, lakes and oceans.
o May cause death of aerobic organisms (fish kills, etc.).
o Increases anaerobic properties of water.
Effect of TSS
o Increases turbidity
 Less light - reduced photosynthesis.
 Causes fish's gills to get plugged up.
o Increases silting
 Reduces lifetime of lakes.
 Changes benthic (i.e., bottom) ecology.
Effects of Phosphorous and Nitrogen

o Increases algal photosynthesis (eutrophication)
 Increased plant life on surface.
 Reduces light in lower levels.
Additional Effects of Nitrogen

o Organic nitrogen and ammonia are converted to nitrates in water.
o Nitrates are converted to nitrites in digestive system.
o Nitrites are assimilated into blood stream where they are converted by respired
oxygen to nitrates.
o May cause suffocation (blue baby syndrome).
Effect of pH
o Organisms are very susceptible to acids and bases.
o Recommended to have near neutral conditions (6.5 - 8.5).
Effect of Pathogens
May infect:
o Humans
o Animals
Domestic waste overflow at the headworks. Yes, incredibly headworks do over-flow,

usually due to rags, grease and debris or operator error. We do not like to see this
happening and are very careful about letting the public and state regulatory agencies
see this activity. One activity the State does not want to see but will happen during a
rain storm is bypassing untreated waste to the outfall.

Preliminary Treatment
Primary Treatment
The initial stage in the treatment of domestic wastewater is known as primary treatment. Coarse
solids are removed from the wastewater in the primary stage of treatment. In some treatment plants,
primary and secondary stages may be combined into one basic operation. At many wastewater
treatment facilities, influent passes through preliminary treatment units before primary and
secondary treatment begins. One of the most common forms of pollution control in the United
States is wastewater treatment.
The country has a vast system of collection sewers, pumping stations, and treatment plants.
Sewers collect the wastewater from homes, businesses, and many industries, and deliver it to
plants for treatment. Most treatment plants were built to clean wastewater for discharge into
streams or other receiving waters, or for reuse.
Years ago, when sewage was dumped into waterways, a natural process of purification began.
First, the sheer volume of clean water in the stream diluted wastes. Bacteria and other small
organisms in the water consumed the sewage and other organic matter, turning it into new bacterial
cells; carbon dioxide and other products. Today’s higher populations and greater volume of
domestic and industrial wastewater require that communities give nature a helping hand. The basic
function of wastewater treatment is to speed up the natural processes by which water is purified.
There are two basic stages in the treatment of wastes, primary and secondary.
In the primary stage, solids are allowed to settle and removed from wastewater. The secondary
stage uses biological processes to further purify wastewater. Sometimes, these stages are
combined into one operation.
The Preliminary Treatment is purely physical stage consisting of Coarse Screening, Raw Influent
Pumping, Static Fine Screening, Grit Removal, and Selector Tanks. The raw wastewater enters
from the collection system into the Coarse Screening process. After the wastewater has been
screened, it may flow into a grit chamber where sand, grit, cinders, and small stones settle to the
bottom.
Removing the grit and gravel that washes off streets or

land during storms is very important, especially in cities
with combined sewer systems. Large amounts of grit and
sand entering a treatment plant can cause serious
operating problems, such as excessive wear of pumps
and other equipment, clogging of aeration devices, or
taking up capacity in tanks that is needed for treatment.
In some plants, another finer screen is placed after the

grit chamber to remove any additional material that might
damage equipment or interfere with later processes.
The grit and screenings removed by these processes

must be periodically collected and trucked to a landfill for
disposal or are incinerated.
Collected Grit►

The Coarse Screening consists of a basket shaped bar
screen which collects larger debris (several inches in
diameter) prior to the Raw Influent Pumping. This debris is
removed and placed into a dumpster for disposal into the
landfill.
The wastewater then passes into the Raw Influent

Pumping process that consists of submersible centrifugal
pumps. These influent pumps operate under a principal
termed prerotation, which allows them to vary their pump
rate hydraulically without the use of complex and
expensive electronics.
Manual and Mechanical Bar Screens

The flow then passes into the Static Fine Screening
process which consists of two stationary (or static)
screens which remove finer debris not captured by the
coarse screens. This screened debris is then dewatered
and collected in hoppers for disposal into a landfill.
The wastewater then passes into the Grit Removal

process which consists of two vortex grit separators
which produce a whirlpool action to force the finest debris
to the outside perimeter for subsequent collection. This
debris is then collected in hoppers, dewatered, and
disposed into a landfill. The screened and de-gritted
wastewater then enters into Primary Sedimentation.
Fine Screening►

Primary Sedimentation
With the screening completed and the grit removed, wastewater still contains dissolved organic
and inorganic constituents along with suspended solids. The suspended solids consist of minute
particles of matter that can be removed from the wastewater with further treatment such as
sedimentation or gravity settling, chemical coagulation, or filtration.
Primary Clarifier
Pollutants that are dissolved or are very fine and remain suspended in the wastewater are not
removed effectively by gravity settling. When the wastewater enters a sedimentation tank, it slows
down and the suspended solids gradually sink to the bottom. This mass of solids is called primary
sludge. Various methods have been devised to remove solids, newer plants have some type of
mechanical equipment to remove the settled solids and some plants remove solids continuously
while others do so at intervals.
Secondary Treatment
After the wastewater has been through Primary Treatment processes, it flows into the next stage
of treatment called secondary. Secondary treatment processes can remove up to 90 percent of the
organic matter in wastewater by using biological treatment processes. The two most common
conventional methods used to achieve secondary treatment are attached growth processes and
suspended growth processes.
The Secondary Treatment stage consists of a biological

process such as Oxidation Ditches and a physical process,
Secondary Clarification. The Preliminary Treatment stage
removes as much solids as possible using physical
processes, however, very fine solids are still present that
cannot be removed physically.
The wastewater enters from Preliminary Treatment into the

Oxidation Ditches process which is a biological process
consisting of large oval shaped basins which are capable of
removing these finer solids. This is accomplished by
maintaining a population of microorganisms within the
oxidation basins which consume the very fine solids (which
are primarily organic) and also adhere to the solids
themselves. By consuming and adhering to these finer solids
they form larger and heavier aggregates that can by
physically separated.
Thus, after this process has taken place within the Oxidation Ditches the wastewater then enters
Secondary Clarification process which can provide this physical separation.

Mechanical Bar Screens. Operators are necessary to pick up trash that is blown off the
rakes.
Here is a grinder pump that is installed after the bar screens. This debris is sent to the
landfill.

Caked grease stuck on weir.
Floating scum in primary clarifier.

Maintenance on a circular clarifier should be performed annually.
Scum Box

Scraping mechanism inside a clarifier.
Scum rake collecting oil and other floating particles.

Secondary Clarification Process
The Secondary Clarification process consists of four rectangular tanks which provide quiescent
(or calm) conditions which allow the larger aggregates of solids and microorganisms to settle out
for collection. The clear overflow (or upper layer) is collected at
the end of the tank and passed onto the Tertiary process for
additional treatment if available.
The majority of microorganism-rich underflow (or lower layer) is

re-circulated to Tanks as Return Sludge to help sustain the
microorganism population in the Oxidation Ditches process.
However, if all the underflow was returned the plant would soon
become overloaded with solids, therefore, a small portion of this
mixture termed Waste Sludge is removed from the system for
disposal. The Waste Sludge is transported into the Solids
Handing process for disposal.
Fixed Film Systems

Fixed film systems grow microorganisms on substrates such as rocks, sand or plastic. The
wastewater is spread over the substrate, allowing the wastewater to flow past the film of
microorganisms fixed to the substrate. As organic matter and nutrients are absorbed from the
wastewater, the film of microorganisms grows and thickens. Trickling filters, rotating biological
contactors, and sand filters are examples of fixed film systems.
Suspended Film Systems

Suspended film systems stir and suspend microorganisms in wastewater. As the microorganisms
absorb organic matter and nutrients from the wastewater, they grow in size and number. After the
microorganisms have been suspended in the wastewater for several hours, they are settled out as
sludge. Some of the sludge is pumped back into the incoming wastewater to provide "seed"
microorganisms. The remainder is wasted and sent on to a sludge treatment process. Activated
sludge, extended aeration, oxidation ditch, and sequential batch reactor systems are all examples
of suspended film systems.
Lagoon Systems
Lagoon systems are shallow basins that hold the wastewater for several months to allow for the
natural degradation of sewage. These systems take advantage of natural aeration and
microorganisms in the wastewater to renovate sewage.

Other Important Wastewater Characteristics
In addition to the many substances found in wastewater, there are other characteristics system
designers and operators use to evaluate wastewater. For example, the color, odor, and turbidity of
wastewater give clues about the amount and type of pollutants present and treatment necessary.
The following are some other important wastewater characteristics that can affect public health and
the environment, as well as the design, cost, and effectiveness of treatment.
Sampling Industrial Waste, in this photograph, the Inspector or Sampler is shaking the
sample to make sure that the sample is mixed-up before pouring off a smaller sample into
the smaller sample bottles on the ground. Normally, these Inspectors or Samplers will
work in pairs. Get used to having wastewater and/or industrial waste/odors all over your
clothes. But other than that, spiders, grease, confined spaces, irate customers, the
interesting odors and dangerous Hydrogen Sulfide gas; this is a good job to have, a secure
and well-paying job.
Temperature
The best temperatures for wastewater treatment probably range from 77 to 95 degrees Fahrenheit.
In general, biological treatment activity accelerates in warm temperatures and slows in cool
temperatures, but extreme hot or cold can stop treatment processes altogether. Therefore, some
systems are less effective during cold weather and some may not be appropriate for very cold
climates.
Wastewater temperature also affects receiving waters. Hot water, for example, which is a byproduct
of many manufacturing processes, can be a pollutant. When discharged in large quantities, it can
raise the temperature of receiving streams locally and disrupt the natural balance of aquatic life.
pH
The acidity or alkalinity of wastewater affects both treatment and the environment. Low pH indicates
increasing acidity while a high pH indicates increasing alkalinity (a pH of 7 is neutral). The pH of
wastewater needs to remain between 6 and 9 to protect organisms. Acids and other substances
that alter pH can inactivate treatment processes when they enter wastewater from industrial or
commercial sources.

Wastewater Components
Hydrogen Sulfide and Ammonia
The gases hydrogen sulfide and ammonia can be toxic and pose asphyxiation hazards. Ammonia
as a dissolved gas in wastewater also is dangerous to fish. Both gases emit odors, which can be a
serious nuisance. Unless effectively contained or minimized by design and location, wastewater
odors can affect the mental well-being and quality of life of residents. In some cases, odors can
even lower property values and affect the local economy.
Hydrogen sulfide or H2S problems are very common in the collection and wastewater system. There
are many chemicals used to help or treat this problem. Here are a few used in the treatment of
hydrogen sulfide problems: Salts of zinc, lime, hydrogen peroxide, chlorine and magnesium
hydroxide. Hydrogen sulfide production in collection systems can cause a number of problems such
as corrosion of the pipes, manholes, and creation of hazardous atmospheres and foul odors. The
best method of controlling hydrogen sulfide is to eliminate its habitat or growth area by keeping
sewers cleaner, this will harbor fewer slime bacteria.
Here are some important statements regarding the reduction of hydrogen sulfide: Salts of zinc and
iron may precipitate sulfides, lime treatments can also kill bacteria which produce hydrogen sulfide,
but this creates a sludge disposal problem and chlorination is effective at reducing the bacteria
which produce hydrogen sulfide. Hydrogen sulfide conditions occur in the sewer system because
of the lack of oxygen.

Pollutants, Oxygen-Demanding Substances
Dissolved oxygen is a key element in water quality that is necessary to support aquatic life. A
demand is placed on the natural supply of dissolved oxygen by many pollutants in wastewater. This
is called biochemical oxygen demand, or BOD, and is used to measure how well a sewage
treatment plant is working. If the effluent, the treated wastewater produced by a treatment plant,
has a high content of organic pollutants or ammonia, it will demand more oxygen from the water
and leave the water with less oxygen to support fish and other aquatic life.
Organic matter and ammonia are “oxygen-demanding” substances. Oxygen-demanding

substances are contributed by domestic sewage and agricultural and industrial wastes of both plant
and animal origin, such as those from food processing, paper mills, tanning, and other
manufacturing processes. These substances are usually destroyed or converted to other
compounds by bacteria if there is sufficient oxygen present in the water, but the dissolved oxygen
needed to sustain fish life is used up in this break down process.
Pathogens
Disinfection of wastewater and chlorination of drinking water supplies has reduced the occurrence
of waterborne diseases such as typhoid fever, cholera, and dysentery, which remain problems in
underdeveloped countries while they have been virtually eliminated in the infectious micro-
organisms, or pathogens, may be carried into surface and groundwater by sewage from cities and
institutions, by certain kinds of industrial wastes, such as tanning and meat packing plants, and by
the contamination of storm runoff with animal wastes from pets, livestock and wild animals, such
as geese or deer. Humans may come in contact with these pathogens either by drinking
contaminated water or through swimming, fishing, or other contact activities. Modern disinfection
techniques have greatly reduced the danger of waterborne disease.
Nutrients
Carbon, nitrogen, and phosphorus are essential to living organisms and are the chief nutrients
present in natural water. Large amounts of these nutrients are also present in sewage, certain
industrial wastes, and drainage from fertilized land. Conventional secondary biological treatment
processes do not remove the phosphorus and nitrogen to any substantial extent. They may convert
the organic forms of these substances into mineral form, making them more usable by plant life.
When an excess of these nutrients over-stimulates the growth of water plants, the result causes
unsightly conditions, interferes with drinking water treatment processes, and causes unpleasant
and disagreeable tastes and odors in drinking water.
The release of large amounts of nutrients, primarily phosphorus but occasionally nitrogen, causes
nutrient enrichment which results in excessive growth of algae. Uncontrolled algae growth blocks
out sunlight and chokes aquatic plants and animals by depleting dissolved oxygen in the water at
night. The release of nutrients in quantities that exceed the affected waterbody’s ability to assimilate
them results in a condition called eutrophication or cultural enrichment.
Inorganic and Synthetic Organic Chemicals

A vast array of chemicals is included in this category. Examples include detergents, household
cleaning aids, heavy metals, pharmaceuticals, synthetic organic pesticides and herbicides,
industrial chemicals, and the wastes from their manufacture. Many of these substances are toxic
to fish and aquatic life and many are harmful to humans. Some are known to be highly poisonous
at very low concentrations. Others can cause taste and odor problems, and many are not effectively
removed by conventional wastewater treatment.
Thermal
Heat reduces the capacity of water to retain oxygen. In some areas, water used for cooling is
discharged to streams at elevated temperatures from power plants and industries. Even discharges
from wastewater treatment plants and storm water retention ponds affected by summer heat can
be released at temperatures above that of the receiving water, and elevate the stream temperature.
Unchecked discharges of waste heat can seriously alter the ecology of a lake, a stream, or estuary.

Organic Matter
Organic materials are found everywhere in the environment. They are composed of the carbon-
based chemicals that are the building blocks of most living things. Organic materials in wastewater
originate from plants, animals, or synthetic organic compounds, and enter wastewater in human
wastes, paper products, detergents, cosmetics, foods, and from agricultural, commercial, and
industrial sources.
Organic compounds normally are some combination of carbon, hydrogen, oxygen, nitrogen, and
other elements. Many organics are proteins, carbohydrates, or fats and are biodegradable, which
means they can be consumed and broken down by organisms. However, even biodegradable
materials can cause pollution. In fact, too much organic matter in wastewater can be devastating
to receiving waters.
Large amounts of biodegradable materials are dangerous to lakes, streams, and oceans, because
organisms use dissolved oxygen in the water to break down the wastes. This can reduce or deplete
the supply of oxygen in the water needed by aquatic life, resulting in fish kills, odors, and overall
degradation of water quality.
The amount of oxygen organisms need to break down wastes in wastewater is referred to as the
biochemical oxygen demand (BOD) and is one of the measurements used to assess overall
wastewater strength.
Some organic compounds are more stable than others and cannot be quickly broken down by
organisms, posing an additional challenge for treatment. This is true of many synthetic organic
compounds developed for agriculture and industry.
In addition, certain synthetic organics are highly toxic. Pesticides and herbicides are toxic to
humans, fish, and aquatic plants and often are disposed of improperly in drains or carried in
stormwater. In receiving waters, they kill or contaminate fish, making them unfit to eat. They also
can damage processes in treatment plants. Benzene and toluene are two toxic organic compounds
found in some solvents, pesticides, and other products. New synthetic organic compounds are
being developed all the time, which can complicate treatment efforts.

Oil and Grease
Fatty organic materials from animals, vegetables, and petroleum also are not quickly broken down
by bacteria and can cause pollution in receiving environments. When large amounts of oils and
greases are discharged to receiving waters from community systems, they increase BOD and they
may float to the surface and harden, causing aesthetically unpleasing conditions.
They also can trap trash, plants, and other materials, causing foul odors, attracting flies and
mosquitoes and other disease vectors. In some cases, too much oil and grease causes septic
conditions in ponds and lakes by preventing oxygen from the atmosphere from reaching the water.
Onsite systems also can be harmed by too much oil and grease, which can clog onsite system
drainfield pipes and soils, adding to the risk of system failure. Excessive grease also adds to the
septic tank scum layer, causing more frequent tank pumping to be required. Both possibilities can
result in significant costs to homeowners.
Petroleum-based waste oils used for motors and industry are considered hazardous waste and
should be collected and disposed of separately from wastewater.

Inorganics
Inorganic minerals, metals, and compounds, such as sodium, potassium, calcium, magnesium,
cadmium, copper, lead, nickel, and zinc are common in wastewater from both residential and
nonresidential sources. They can originate from a variety of sources in the community including
industrial and commercial sources, stormwater, and inflow and infiltration from cracked pipes and
leaky manhole covers. Most inorganic substances are relatively stable, and cannot be broken down
easily by organisms in wastewater.
Large amounts of many inorganic substances can contaminate soil and water. Some are toxic to
animals and humans and may accumulate in the environment. For this reason, extra treatment
steps are often required to remove inorganic materials from industrial wastewater sources. For
example, heavy metals which are discharged with many types of industrial wastewaters are difficult
to remove by conventional treatment methods. Although acute poisonings from heavy metals in
drinking water are rare in the U.S., potential long-term health effects of ingesting small amounts of
some inorganic substances over an extended period of time are possible.
Nutrients
Wastewater often contains large amounts of the nutrients nitrogen and phosphorus in the form of
nitrate and phosphate, which promote plant growth.
Organisms only require small amounts of nutrients in biological treatment, so there normally is an
excess available in treated wastewater. In severe cases, excessive nutrients in receiving waters
cause algae and other plants to grow quickly depleting oxygen in the water, deprived of oxygen,
fish and other aquatic life die, emitting foul odors.
Nutrients from wastewater have also been linked to ocean "red tides" that poison fish and cause
illness in humans. Nitrogen in drinking water may contribute to miscarriages and is the cause of a
serious illness in infants called methemoglobinemia or "blue baby syndrome."
Solids
Solid materials in wastewater can consist of organic and/or inorganic materials and organisms. The
solids must be significantly reduced by treatment or they can increase BOD when discharged to
receiving waters and provide places for microorganisms to escape disinfection. They also can clog
soil absorption fields in onsite systems.
Settleable solids: Certain substances, such as sand, grit, and heavier organic and inorganic
materials settle out from the rest of the wastewater stream during the preliminary stages of
treatment. On the bottom of settling tanks and ponds, organic material makes up a biologically
active layer of sludge that aids in treatment.
Suspended solids: Materials that resist settling may remain suspended in wastewater. Suspended
solids in wastewater must be treated, or they will clog soil absorption systems or reduce the
effectiveness of disinfection systems.
Dissolved solids: Small particles of certain wastewater materials can dissolve, like salt in water.
Some dissolved materials are consumed by microorganisms in wastewater, but others, such as
heavy metals, are difficult to remove by conventional treatment. Excessive amounts of dissolved
solids in wastewater can have adverse effects on the environment.
Gases
Certain gases in wastewater can cause odors, affect treatment, or are potentially dangerous.
Methane gas, for example, is a byproduct of anaerobic biological treatment and is highly
combustible. Special precautions need to be taken near septic tanks, manholes, treatment plants,
and other areas where wastewater gases can collect.

Other Wastewater Treatment Components
Biochemical Oxygen Demand
Biochemical Oxygen Demand (BOD or BOD5) is an indirect measure of biodegradable organic
compounds in water, and is determined by measuring the dissolved oxygen decrease in a
controlled water sample over a five-day period.
During this five-day period, aerobic (oxygen-consuming) bacteria decompose organic matter in the
sample and consume dissolved oxygen in proportion to the amount of organic material that is
present. In general, a high BOD reflects high concentrations of substances that can be biologically
degraded, thereby consuming oxygen and potentially resulting in low dissolved oxygen in the
receiving water.
The BOD test was developed for samples dominated by oxygen-demanding pollutants like sewage.
While its merit as a pollution parameter continues to be debated, BOD has the advantage of a long
period of record.
Organic Carbon
Most organic carbon in water occurs as partly degraded plant and animal materials, some of which
are resistant to microbial degradation. Organic carbon is important in the estuarine food web and
is incorporated into the ecosystem by photosynthesis of green plants, then consumed as
carbohydrates and other organic compounds by higher animals. In another process, formerly living
tissue containing carbon is decomposed as detritus by bacteria and other microbes.
Total Organic Carbon

(TOC) bears a direct relationship with biological and chemical oxygen demand; high levels of TOC
can result from human sources, the high oxygen demand being the main concern.

Flow
Whether a system serves a single home or an entire community, it
must be able to handle fluctuations in the quantity and quality of
wastewater it receives to ensure proper treatment is provided at all
times. Systems that are inadequately designed or hydraulically
overloaded may fail to provide treatment and allow the release of
pollutants to the environment.
To design systems that are both as safe and as cost-effective as

possible, engineers must estimate the average and maximum
(peak) amount of flows generated by various sources. Because
extreme fluctuations in flow can occur during different times of the
day and on different days of the week, estimates are based on
observations of the minimum and maximum amounts of water
used on an hourly, daily, weekly, and seasonal basis. The
possibility of instantaneous peak flow events that result from
several or all water-using appliances or fixtures being used at once
also is taken into account.
The number, type, and efficiency of all water-using fixtures and

appliances at the source is factored into the estimate (for example, the number and amount of
water normally used by faucets, toilets, and washing machines), as is the number of possible
users or units that can affect the amount of water used (for example, the number of residents,
bedrooms, customers, students, patients, seats, or meals served).
Waterless urinals are reducing water use but are concentrating the wastestream. Water
conservation education is taught at schools and this too is affecting our flow dynamics and
microorganisms or MO’s. Anything new always affects the bugs and no one cares but us.
According to studies, water use in many homes is lowest from about midnight to 5 a.m.,
averaging less than one gallon per person per hour, but then rises sharply in the morning
around 6 am to a little over 3 gallons per person per hour. During the day, water use drops
off moderately and rises again in the early evening hours. Weekly peak flows may occur
in some homes on weekends, especially when all adults work during the week. In U.S.
homes, average water use is approximately 45 gallons per person per day, but may range
from 35 to 60 gallons or more.

Peak flows at stores and other businesses typically occur during business hours and during meal
times at restaurants. Rental properties, resorts, and commercial establishments in tourist areas
may have extreme flow variations seasonally. Estimating flow volumes for centralized treatment
systems is a complicated task, especially when designing a new treatment plant in a community
where one has never existed previously.
Engineers must allow for additional flows during wet weather due to inflow and infiltration of extra
water into sewers. Excess water can enter sewers through leaky manhole covers and cracked
pipes and pipe joints, diluting wastewater, which affects its overall characteristics. This can increase
flows to treatment plants sometimes by as much as three or four times the original design load.
Grout is used to prevent infiltration into manholes.
The main focus of wastewater treatment plants is to reduce the BOD and COD in the effluent
discharged to natural waters, meeting state and federal discharge criteria. Wastewater treatment
plants are designed to function as "microbiology farms," where bacteria and other microorganisms
are fed oxygen and organic waste. Treatment of wastewater usually involves biological processes
such as the activated sludge system in the secondary stage after preliminary screening to remove
coarse particles and primary sedimentation that settles out suspended solids. These secondary
treatment steps are generally considered environmental biotechnologies that harness natural self-
purification processes contained in bioreactors for the biodegradation of organic matter and
bioconversion of soluble nutrients in the wastewater.
Application Specific Microbiology

Each wastewater stream is unique, and so too are the community of
microorganisms that process it. This "application-specific
microbiology" is the preferred methodology in wastewater treatment
affecting the efficiency of biological nutrient removal. The right
laboratory prepared bugs are more efficient in organics removal if
they have the right growth environment. This efficiency is multiplied
if microorganisms are allowed to grow as a layer of biofilm on
specifically designed support media. In this way, optimized
biological processing of a waste stream can occur. To reduce the
start-up phase for growing a mature biofilm one can also purchase
"application specific bacterial cultures" from appropriate
microbiology vendors.
Draining Biofilm

Processed Wastewater Solids
Biosolids are processed wastewater solids (“sewage sludge”) that meet rigorous standards
allowing safe reuse for beneficial purposes. Currently, more than half of the biosolids produced by
municipal wastewater treatment systems are applied to land as a soil conditioner or fertilizer and
the remaining solids are incinerated or landfilled.
Large solids treatment facility

Ocean Dumping
Ocean dumping of these solids is no longer allowed.
Biosolids Stabilization
Prior to utilization or disposal, biosolids are stabilized to control odors and reduce the number of
disease-causing organisms. Sewage solids, or sludge, when separated from the wastewater, still
contain around 98 percent water. They are usually thickened and may be dewatered to reduce the
volume to be transported for final processing, disposal, or beneficial use.
Dewatering Processes
Dewatering processes include drying beds, belt filter presses, plate and frame presses, and
centrifuges. To improve dewatering effectiveness, the solids can be pretreated with chemicals such
as lime, ferric chloride, or polymers to produce larger particles which are easier to remove.
Centrifuge Filter Press

Digestion
Digestion is a form of stabilization where the volatile material in the wastewater solids can
decompose naturally and the potential for odor production is reduced. Digestion without air in an
enclosed tank (anaerobic solids digestion) has the added benefit of producing methane gas which
can be recovered and used as a source of energy. Stabilization of solids may also be accomplished
by composting, heat treatments, drying or the addition of lime or other alkaline materials. After
stabilization, the biosolids can be safely spread on land.
Land Application
In many areas, biosolids are marketed to farmers as fertilizer. Federal regulation (40 CFR Part 503)
defines minimum requirements for such land application practices, including contaminant limits,
field management practices, treatment requirements, monitoring, recordkeeping, and reporting
requirements. Properly treated and applied biosolids are a good source of organic matter for
improving soil structure and help supply nitrogen, phosphorus, and micronutrients that are required
by plants.
Biosolids have also been used successfully for many years as a soil conditioner and fertilizer, and
for restoring and re-vegetating areas with poor soils due to construction activities, strip mining or
other practices. Under this biosolids management approach, treated solids in semi liquid or
dewatered form are transported to the soil treatment areas. The slurry or dewatered biosolids,
containing nutrients and stabilized organic matter, is spread over the land to give nature a hand in
returning grass, trees, and flowers to barren land.
Restoration of the countryside also helps control the flow of acid drainage from mines that
endangers fish and other aquatic life and contaminates the water with acid, salts, and excessive
quantities of metals.
Incineration
Incineration consists of burning the dried solids to reduce the organic residuals to an ash that can
be disposed of or reused. Incinerators often include heat recovery features. Undigested sludge
solids have significant fuel value as a result of their high organic content.
However, the water content must be greatly reduced by dewatering or drying to take advantage of
the fuel potential of the biosolids. For this reason, pressure filtration dewatering equipment is used
to obtain biosolids which are sufficiently dry to burn without continual reliance on auxiliary fuels. In
some cities, biosolids are mixed with refuse or refuse derived fuel prior to burning. Generally, waste
heat is recovered to provide the greatest amount of energy efficiency.
Beneficial Use Products from Biosolids

Heat dried biosolids pellets have been produced and used extensively as a fertilizer product for
lawn care, turf production, citrus groves, and vegetable production for many years. Composting of
biosolids is also a well-established approach to solids management that has been adopted by a
number of communities. The composted peat-like product has shown particular promise for use in
the production of soil additives for re-vegetation of topsoil depleted areas, and as a potting soil
amendment.
Effective pretreatment of industrial wastes prevents excessive levels of unwanted constituents,

such as heavy metals (i.e. cadmium, mercury, and lead) and persistent organic compounds from
contaminating the residuals of wastewater treatment and limiting the potential for beneficial use.
Effective stabilization of wastewater residuals and their conversion to biosolid products can be
costly. Some cities have produced fertilizers from biosolids which are sold to help pay part of the
cost of treating wastewater. Some municipalities use composted, heat dried, or lime stabilized
biosolid products on parks and other public areas.

Decentralized (Onsite and Cluster) Systems
A decentralized wastewater system treats sewage from homes and businesses that are not
connected to a centralized wastewater treatment plant. Decentralized treatment systems include
onsite systems and cluster systems. An onsite system is a wastewater system relying on natural
processes, although sometimes containing mechanical components, to collect, treat, disperse or
reclaim wastewater from a single dwelling or building. A septic tank and soil adsorption field is an
example of an onsite system.
A wastewater collection and treatment system under some form of common ownership that collects
wastewater from two or more dwellings or buildings and conveys it to a treatment and dispersal
system located on a suitable site near the dwellings or buildings is a cluster system.
Decentralized systems include those using alternative treatment technologies like media filters,
constructed wetland systems, aerobic treatment units, and a variety of soil dispersal systems. Soil
dispersal systems include pressure systems such as low pressure pipe and drip dispersal systems.
These systems treat and disperse relatively small volumes of wastewater, and are generally are
found in rural and suburban areas.
While septic tanks and soil absorption systems have significant limitations, decentralized systems
can effectively protect water quality and public health from groundwater and surface water
contamination if managed properly (i.e. properly sited, sized, designed, installed, operated, and
maintained). Nitrate concentrations in groundwater that exceed the drinking water standards can
cause health problems.
Onsite Treatment
Onsite wastewater systems contain three components: a treatment unit which treats water prior to
dispersal into the environment; a soil dispersal component which assures that treated water is
released into the environment at a rate which can be assimilated; and a management system which
assures proper long term operation of the complete system.
Disinfection of the treated effluent may be provided prior to dispersal. A typical onsite system
consists of a septic tank followed by an effluent distribution system. Alternative treatment systems
include aerobic treatment and sand filtration systems. We will cover this in much more detail in a
few more pages.
Conventional Septic Tanks

A septic tank is a tank buried in the ground used to treat sewage without the presence of oxygen
(anaerobic). The sewage flows from the plumbing in a home or small business establishment into
the first of two chambers, where solids settle out. The liquid then flows into the second chamber.
Anaerobic bacteria in the sewage break down the organic matter, allowing cleaner water to flow
out of the second chamber. The liquid typically discharges through a subsurface distribution
system. Periodically, the solid matter in the bottom of the tank, referred to as septage, must be
removed and disposed of properly.
Aerobic Treatment Units

Aerobic treatment units are also used to provide onsite wastewater treatment. They are similar to
septic tanks, except that air is introduced and mixed with the wastewater inside the tank. Aerobic
(requiring oxygen) bacteria consume the organic matter in the sewage.
As with the typical septic system, the effluent discharge from an aerobic system is typically released
through a sub-surface distribution system or may be disinfected and discharged directly to surface
water. Aerobic treatment units also require the removal and proper disposal of solids that
accumulate in the tank.

Media Filters
Media filters are used to provide further treatment of septic tank effluent, and provide high levels of
nitrification. They can be designed to pass the effluent once or multiple times through the media
bed. Media, such as sand, acts as a filter. The media is placed two to three feet deep above a liner
of impermeable material such as plastic or concrete. Septic tank effluent is applied to the filter
surface in intermittent doses and is further treated as it slowly trickles through the media. In most
media filters, wastewater is collected in an underdrain then either pumped back to the filter bed or
to other types of treatment. We will cover this in much more detail in a few more pages.
Dispersal Approaches
Traditional onsite systems include treatment units followed by a drainfield or absorption field.
Wastewater from the treatment unit is dispersed through a suitable soil layer where it receives
additional treatment by the soil microorganisms and filtering properties of the soil. If the soil is
unsuitable for the installation of a soil absorption field, alternative methods can be used to further
treat or distribute the treated effluent.
The most common alternative dispersal systems include low-pressure pipe, mounds, drip disposal,
and evapotranspiration beds.
Absorption Field
When soil conditions permit, the most common method to disperse septic tank or aerobic system
effluent is an absorption field consisting of a series of perforated parallel pipes laid in trenches on
gravel or crushed stone or as a direct discharge to the soil through trenches.
Typically, effluent flows into the absorption field from a distribution box which maintains an even
flow of effluent to the absorption field. From there, the effluent drains through the stone and into
the soil which provides further treatment.
Mound System
When the soil is not conducive to percolation or when the groundwater level is high, a mound
system is commonly used. A mound system is a distribution system constructed above the original
ground level by using granular material such as sand and gravel to receive the septic tank effluent
before it flows to the native soil below.
The effluent flows to a dosing tank that is equipped with a pump. Here the effluent is stored until
there is sufficient liquid. Once the liquid is pumped out, it moves evenly throughout the mound
before reaching less permeable soil or groundwater. The granular material acts as a treatment
medium and improves the removal of pollutants in ways that may not be provided by substandard
native soils.
Drip Dispersal System

Where soils are very thin or have reduced permeability, drip dispersal systems can be utilized. The
typical drip system operates like drip irrigation at a moderately high pressure. The components of
a drip system include filters to remove solids, a network of drip tubes to disperse liquid into soil,
tanks to hold liquid, and controllers to regulate the flow to the drip system.
Evapotranspiration Beds
Evapotranspiration (ET) bed is an onsite dispersal system where pretreated wastewater
evaporates from the soil surface or is transpired by plants into the atmosphere. Usually, ET beds
are used in arid climates and there is no discharge either to surface or groundwater. Vegetation is
planted on the surface of the sand bed to improve the transpiration process and landscaping
enhances the aesthetics of the bed.

Both photographs show the aeration sequence for an SBR.
Sequence Batch Reactor.

Anoxic zone, denitrification area.
The same area from above photo, but clean and dry, these are porous air diffusers. A
rare photo.

This clarifier is used to thicken sludge prior to the digester, this is not a digester but looks
like one. Notice the light on the top for Operators to look inside.
Two massive anaerobic digesters.

Management of Decentralized Systems
Ensuring performance of decentralized wastewater treatment systems is an issue of national
concern because these systems are a permanent component of our nation’s wastewater
infrastructure. Twenty-five percent of households nationwide and one-third of the new homes being
constructed are served by onsite systems. Many of the existing systems do not perform adequately
due to a lack of management. Therefore, the EPA promotes the sustained management of
decentralized wastewater systems to enhance their performance and reliability. The EPA strongly
encourages communities to establish management programs for the maintenance of onsite
systems in addition to improving local requirements for onsite system siting and system design.
Communities benefit from effective onsite system management programs by enjoying improved
protection of public health and local surface water and groundwater resources, preserving rural
areas, protecting property owners’ investments through increased system service life, and avoiding
the need to finance costly central wastewater collection and treatment systems.
Dispose of Household Hazardous Wastes Safely

Many household products are potentially hazardous to people and the environment and never
should be flushed down drains, toilets, or storm sewers. Treatment plant workers can be injured
and wastewater systems can be damaged as a result of improper disposal of hazardous materials.
Other hazardous chemicals cannot be treated effectively by municipal wastewater systems and
may reach local drinking water sources. When flushed into septic systems and other onsite
systems, they can temporarily disrupt the biological processes in the tank and soil absorption field,
allowing hazardous chemicals and untreated wastewater to reach groundwater.
Some examples of hazardous household materials include motor oil, transmission fluid, antifreeze,
paint, paint thinner, varnish, polish, wax, solvents, pesticides, rat poison, oven cleaner, and battery
fluid. Many of these materials can be recycled or safely disposed of at community recycling centers.
A drive-thru household hazardous waste collection site, trying to keep the toxic material
out of the sewer system. These workers usually get to keep lots of goodies and take the
materials home while at the same time keeping the bad stuff from upsetting the plant. I
worked a day at one of these facilities and I was amazed with the chemicals that people
keep around the home, example, 1 pound of liquid Mercury and another had a bottle of
Sodium Cyanide.

Water Quality Criteria
Many types of microscopic plants and animals, such as plankton, water beetles, and insects that
live in or on the water, serve as food for small fish. Small fish are eaten by larger fish which, in turn,
are consumed by even larger fish. These large fish may ultimately be consumed by humans. All
life along the food chain is dependent on the water environment and it is for this reason that the
quality of the nation's surface waters must be protected.
The Clean Water Act directs the EPA to develop criteria for water quality that accurately reflect the
latest scientific knowledge about the effects of pollutants on aquatic life and human health. In
developing these criteria, the EPA examines the effects of specific pollutants on plankton, fish,
shellfish, wildlife, plant life, aesthetics, and recreation in any body of water. This includes specific
information on the concentration and dispersal of pollutants through biological, physical, and
chemical processes as well as the effects of pollutants on biological communities as a whole.
States may use the criteria that are developed by the EPA to help set water quality standards that
protect the uses of their waters or they may develop their own water quality criteria. The EPA
publishes human health and aquatic life criteria and is currently developing sediment and biological
criteria. These criteria are complementary; each is designed to protect specific types of living
organisms or ecological systems from the adverse effects of pollution.
Human Health Criteria

People can potentially be exposed to water pollutants when they drink untreated surface water or
eat fish, shellfish, or wildlife that have been contaminated by pollutants in surface waters. To reduce
the risk to humans from these sources, the EPA scientists research information to determine the
levels at which specific chemicals are not likely to adversely affect human health.
The EPA publishes these levels as human health criteria that the states use, along with other
information, to set allowable concentrations of pollutants in their water quality standards. In this
way, the EPA and the states work together to protect people from exposure to harmful pollutants
in surface waters.
Aquatic Life Criteria

Aquatic life criteria provide protection for plants and animals that are found in surface waters. The
EPA develops these criteria as numeric limits on the amounts of chemicals that can be present in
river, lake, or stream water without harm to aquatic life. Aquatic life criteria are designed to provide
protection for both freshwater and saltwater aquatic organisms from the effects of acute (short term)
and chronic (long term) exposure to potentially harmful chemicals. Aquatic life criteria are based
on toxicity information and are developed to protect aquatic organisms from death, slower growth,
reduced reproduction, and the accumulation of harmful levels of toxic chemicals in their tissues that
may adversely affect consumers of such organisms.
Sediment Quality Criteria Guidance

In a healthy aquatic community, sediments provide a habitat for many living organisms. Worms,
plants, and tiny microorganisms living in or on the sediment sustain the fish and shellfish that, in
turn, nourish larger fish, wildlife, and man.
Pollutants in the Sediment

Controlling the concentration of pollutants in the sediment helps to protect bottom dwelling species
and prevents harmful toxins from moving up the food chain and accumulating in the tissue of
animals at progressively higher levels. This is particularly important at the lower levels of the food
chain because the concentration of many pollutants may increase at each link in the food chain. A
pollutant level in the sediment that does not harm snails of small fish may bioaccumulate in the
food chain and become very harmful to larger fish, birds, mammals, wildlife, and people.

The EPA develops sediment quality criteria guidance on the concentrations or amounts of individual
chemicals that can be present in river, lake, or stream sediments and still protect sediment-dwelling
organisms and ultimately animals higher in the food chain from the harmful effects of toxic
pollutants.
Biological Criteria
A water body in its natural condition is free from the harmful effects of pollution, habitat loss, and
other negative stressors. It is characterized by a particular biological diversity and abundance of
organisms. This biological integrity--or natural structure and function of aquatic life--can be
dramatically different in various types of water bodies in different parts of the country. Because of
this, the EPA is developing methodologies that states can use to assess the biological integrity of
their waters and, in so doing, set protective water quality standards. These methodologies will
describe scientific methods for determining a particular aquatic community's health and for
maintaining optimal conditions in various bodies of water.
Summary
The goal of all biological wastewater treatment systems is to remove the non-settling solids and
the dissolved organic load from the effluents by using microbial populations. Biological treatments
are generally part of secondary treatment systems. The microorganisms used are responsible for
the degradation of the organic matter and the stabilization of organic wastes. With regard to the
way in which they utilize oxygen, they can be classified into aerobic (require oxygen for their
metabolism), anaerobic (grow in absence of oxygen) and facultative (can proliferate either in
absence or presence of oxygen although using different metabolic processes). Most of the micro-
organisms present in wastewater treatment systems use the organic content of the wastewater as
an energy source to grow, and are thus classified as heterotrophes from a nutritional point of view.
The population active in a biological wastewater treatment is mixed, complex and interrelated.
Genera
By example, in a single aerobic system, members of the genera Pseudomonas, Nocardia,
Flavobacterium, Achromobacter and Zooglea may be present, together with filamentous organisms
(Beggioata and Spaerotilus among others). In a well-functioning system, protozoas and rotifers are
usually present and are useful in consuming dispersed bacteria or non-settling particles. More
extensive description and treatment of the microbiology of wastewater treatment systems are given
elsewhere (Stanier, 1976). The organic load present is incorporated in part as biomass by the
microbial populations, and almost all the rest is liberated as gas (carbon dioxide (CO2) if the
treatment is aerobic, or carbon dioxide plus methane (CH4) if the process is anaerobic) and water.
In fisheries wastewaters the non- biodegradable portion is very low.
Unless the cell mass formed during the biological treatment is removed from the wastewater (e.g.,
by sedimentation or other treatment described in the previous section), the treatment is largely
incomplete, because the biomass itself will appear as organic load in the effluent and the only
pollution reduction accomplished is that fraction liberated as gases. The biological treatment
processes used for wastewater treatment are broadly classified as aerobic in which aerobic and
facultative micro-organisms predominate or anaerobic which use anaerobic micro-organism.
If the microorganisms or Bugs are suspended in the wastewater during biological operation, the
operations are called "suspended growth processes", while the micro-organisms that are attached
to a surface over which they grow are called "attached growth processes". This section explains
the principles and main characteristics of the most common processes in each case.
Aerobic Processes
In these, the reactions occurring can be summarized as:
Organic load + Oxygen + more cells + CO2 + H2O

Nitrogen Control
Nitrogen in one form or another is present in municipal wastewater and is usually not removed by
secondary treatment. If discharged into lakes and streams or estuary waters, nitrogen in the form
of ammonia can exert a direct demand on oxygen or stimulate the excessive growth of algae.
Ammonia in wastewater effluent can be toxic to aquatic life in certain instances. By providing
additional biological treatment beyond the secondary stage, nitrifying bacteria present in
wastewater treatment can biologically convert ammonia to the non-toxic nitrate through a process
known as nitrification. The nitrification process is normally sufficient to remove the toxicity
associated with ammonia in the effluent. Since nitrate is also a nutrient, excess amounts can
contribute to the uncontrolled growth of algae. In situations where nitrogen must be completely
removed from effluent, additional biological process can be added to the system to convert the
nitrate to nitrogen gas. We will cover this in much more detail in a few more pages.
Conversion of Nitrate to Nitrogen Gas

The conversion of nitrate to nitrogen gas is accomplished by bacteria in a process known as
denitrification. Effluent with nitrogen in the form of nitrate is placed into a tank devoid of oxygen,
where carbon-containing chemicals, such as methanol, are added or a small stream of raw
wastewater is mixed in with the nitrified effluent. In this oxygen free environment, bacteria use the
oxygen attached to the nitrogen in the nitrate form, releasing nitrogen gas. Because nitrogen
comprises almost 80 percent of the air in the earth’s atmosphere, the release of nitrogen into the
atmosphere does not cause any environmental harm.
Biological Phosphorus Control

Like nitrogen, phosphorus is also a necessary nutrient for the growth of algae. Phosphorus
reduction is often needed to prevent excessive algal growth before discharging effluent into lakes,
reservoirs and estuaries. Phosphorus removal can be achieved through chemical addition and a
coagulation-sedimentation process discussed in the following section. Some biological treatment
processes called biological nutrient removal (BNR) can also achieve nutrient reduction, removing
both nitrogen and phosphorus.
Most of the BNR processes involve modifications of suspended growth treatment systems so that
the bacteria in these systems also convert nitrate nitrogen to inert nitrogen gas and trap phosphorus
in the solids that are removed from the effluent.

Coagulation-Sedimentation Process
A process known as chemical coagulation-
sedimentation is used to increase the removal of solids
from effluent after primary and secondary treatment.
Solids heavier than water settle out of wastewater by
gravity. With the addition of specific chemicals, solids
can become heavier than water and will settle.
Alum, lime, or iron salts are chemicals added to the

wastewater to remove phosphorus. With these
chemicals, the smaller particles ‘floc’ or clump together
into large masses. The larger masses of particles will
settle faster when the effluent reaches the next step the
sedimentation tank. This process can reduce the
concentration of phosphate by more than 95 percent.
Although used for years in the treatment of industrial

wastes and in water treatment, coagulation-
sedimentation is considered an advanced process
because it is not routinely applied to the treatment of municipal wastewater. In some cases, the
process is used as a necessary pretreatment step for other advanced techniques. This process
produces a chemical sludge, and the cost of disposing of this material can be significant.
Carbon Adsorption
Carbon adsorption technology can remove organic materials from wastewater that resist removal
by biological treatment. These resistant, trace organic substances can contribute to taste and odor
problems in water, taint fish flesh, and cause foaming and fish kills.
Carbon adsorption consists of passing the wastewater effluent through a bed or canister of
activated carbon granules or powder which remove more than 98 percent of the trace organic
substances. The substances adhere to the carbon surface and are removed from the water. To
help reduce the cost of the procedure, the carbon granules can be cleaned by heating and used
again.
Granular Carbon
The Use or Disposal of Wastewater Residuals and Biosolids

When pollutants are removed from water, there is always something left over. It may be rags and
sticks caught on the screens at the beginning of primary treatment. It may be the solids that settle
to the bottom of sedimentation tanks. Whatever it is, there are always residuals that must be reused,
burned, buried, or disposed of in some manner that does not harm the environment.
The utilization and disposal of the residual process solids is addressed by the CWA, Resource
Conservation and Recovery Act (RCRA), and other federal laws. These Federal laws re-enforce
the need to employ environmentally sound residuals management techniques and to beneficially
use biosolids whenever possible. We will cover this in much more detail in a few more pages.

Nitrogen and Phosphorus Removal at Wastewater Treatment
Plants
Nutrients
Nutrients are chemical elements or compounds essential for plant and animal growth. Nutrient
parameters include ammonia, organic nitrogen, Kjeldahl nitrogen, nitrate nitrogen (for water only)
and total phosphorus. High amounts of nutrients have been associated with eutrophication, or over-
fertilization of a water body, while low levels of nutrients can reduce plant growth and (for example)
starve higher level organisms that consume phytoplankton.
The purpose of this section is to provide an overview of the major factors driving decisions to
enhance nutrient removal at WWTPs. This section characterizes the industry based on U.S.
Environmental Protection Agency (EPA) survey information. This section describes the negative
impacts of nutrient enrichment, highlighting the history of water quality changes in key regions of
the country. EPA and State initiatives to reduce nutrient pollution from wastewater treatment
discharges are summarized in this training course. Lastly, we will highlight several barriers to
enhancing nutrient removal at wastewater plants.
Status of Wastewater Treatment in the U.S.

The 1972 Amendments to the Federal Water Pollution Control Act (FWPCA) (Public Law 92-500),
also known as the Clean Water Act (CWA), established the foundation for wastewater discharge
control in the U.S. The CWA’s primary objective is to “restore and maintain the chemical, physical,
and biological integrity of the Nation’s waters.” The CWA established a program to ensure clean
water by requiring permits that limit the amount of pollutants discharged by all municipal and
industrial dischargers into receiving waters. Discharges are regulated under the National Pollutant
Discharge Elimination System (NPDES) permit program. As of 2004, there were 16,583 municipal
wastewater utilities [also known as Publicly Owned Treatment Works (POTWs)] regulated under
the CWA, serving approximately 75 percent of the Nation’s population (U.S. Public Health Service
and USEPA, 2008) with the remaining population served by septic or other onsite systems.
Wastewater treatment has generally been defined as containing one or more of the following four
processes: (1) preliminary, (2) primary, (3) secondary, and (4) advanced - also known as tertiary
treatment.
Preliminary treatment consists of grit removal, which removes dense inert particles and screening
to remove rags and other large debris. Primary treatment involves gravity settling tanks to remove
settleable solids, including settleable organic solids. The performance of primary settling tanks can
be enhanced by adding chemicals to capture and flocculate smaller solid particles for removal and
to precipitate phosphorus. Secondary treatment follows primary treatment in most plants and
employs biological processes to remove colloidal and soluble organic matter. Effluent disinfection
is usually included in the definition of secondary treatment.
EPA classifies advanced treatment as “a level of treatment that is more stringent than secondary
or produces a significant reduction in conventional, non-conventional, or toxic pollutants present in
the wastewater” (U.S. Public Health Service and USEPA, 2008). Other technical references
subdivide advanced treatment, using the terms “secondary with nutrient removal” when nitrogen,
phosphorus, or both are removed and “tertiary removal” to refer to additional reduction in solids by
filters or microfilters (Tchobanoglous et al, 2003).
Effluent filtration and nutrient removal are the most common advanced treatment processes. The
CWA requires that all municipal wastewater treatment plant discharges meet a minimum of
secondary treatment. Based on data from the 2004 Clean Watersheds Needs Survey, 16,543
municipal WWTPs (99.8 percent of plants in the country) meet the minimum secondary wastewater
treatment requirements.

Of those that provide at least secondary treatment, approximately 44 percent provide some kind of
advanced treatment (U.S. Public Health Service and USEPA, 2008).
Nutrient Impairment of U.S. Waterways

The harmful effects of eutrophication due to excessive nitrogen and phosphorus concentrations in
the aquatic environment have been well documented. Algae and phytoplankton growth can be
accelerated by higher concentrations of nutrients as they can obtain sufficient carbon for growth
from carbon dioxide. In addition to stimulating eutrophication, nitrogen in the form of ammonia can
exert a direct demand on dissolved oxygen (DO) and can be toxic to aquatic life. Even if a treatment
plant converts ammonia to nitrate by a biological nitrification process, the resultant nitrate can
stimulate algae and phytoplankton growth. Phosphorus also contributes to the growth of algae.
Either nitrogen or phosphorus can be the limiting nutrient depending on the characteristics of the
receiving water.
Nitrogen is typically limiting in estuarine and marine systems and phosphorus in fresh water
systems. According to the 2007 report Effects of Nutrient Enrichment in the Nation’s Estuaries: A
Decade of Change, increased nutrient loadings promote a progression of symptoms beginning with
excessive growth of phytoplankton and macroalgae to the point where grazers cannot control
growth (Bricker et al., 2007). These blooms may be problematic, potentially lasting for months at a
time and blocking sunlight to light-dependent submerged aquatic vegetation (SAV). In addition to
increased growth, changes in naturally occurring ratios of nutrients may also affect which species
dominate, potentially leading to nuisance/toxic algal blooms. These blooms may also lead to other
more serious symptoms that affect biota, such as low DO and loss of SAV. Once water column
nutrients have been depleted by phytoplankton and macroalgae and these blooms die, the bacteria
decomposing the algae then consume oxygen, making it less available to surrounding aerobic
aquatic life.
Consequently, fish and invertebrate kills may occur due to hypoxia and anoxia, conditions of low to
no DO. Eutrophic conditions may also cause risks to human health, resulting from consumption of
shellfish contaminated with algal toxins or direct exposure to waterborne toxins. Eutrophication can
also create problems if the water is used as a source of drinking water. Chemicals used to disinfect
drinking water will react with organic compounds in source water to form disinfection byproducts,
which are potential carcinogens and are regulated by EPA.
Advanced eutrophic conditions can lead to “dead zones” with limited aquatic life, which describes
the hypoxia condition that exists in the Northern Gulf of Mexico. A recent U.S. Geological Survey
(USGS) report titled Differences in Phosphorus and Nitrogen Delivery to the Gulf of Mexico from
the Mississippi River Basin documents the contribution of nitrogen and phosphorus from
agricultural and non-agricultural sources in the Mississippi River basin (Alexander et al., 2008).
On June 16, 2008 the joint federal-state Mississippi River/Gulf of Mexico Watershed Nutrient Task
Force released its 2008 Action Plan for Reducing, Mitigating, and Controlling Hypoxia in the
Northern Gulf of Mexico and Improving Water Quality in the Mississippi River Basin, which builds
upon its 2001 plan by incorporating emerging issues, innovative approaches, and the latest
science, including findings from EPA’s Science Advisory Board.
Improvements include more accountability through an Annual Operating Plan, better tracking of
progress, state and federal nutrient reduction strategies, and a plan to increase awareness of the
problem and implementation of solutions (USEPA, 2008b).
Nutrient pollution has also caused significant problems in the Chesapeake Bay. Elevated levels of
both nitrogen and phosphorus are the main cause of poor water quality and loss of aquatic habitats
in the Bay. Significant algae blooms on the water surface block the sun’s rays from reaching
underwater bay grasses. Without sunlight, bay grasses cannot grow and provide critical food and
habitat for blue crabs, waterfowl, and juvenile fish.

The Chesapeake Bay Program estimates that 22 percent of the phosphorus loading and 19 percent
of the nitrogen loading in the Bay comes from municipal and industrial wastewater facilities
(Chesapeake Bay Program, 2008). The first national attention to nutrient contamination occurred
in the Great Lakes. In the 1960s Lake Erie was declared “dead” when excessive nutrients in the
Lake fostered excessive algae blooms that covered beaches and killed off native aquatic species
due to oxygen depletion. At that time, phosphorus was the primary nutrient of concern due to the
advent of phosphate detergents and inorganic fertilizers. With the enactment of the CWA and the
Great Lakes Water Quality Agreement in 1972, a concerted effort was undertaken to reduce
pollutant loadings, including phosphorus in the Lake.
Although the health of the Lake improved dramatically, in recent years, there has been renewed
attention to the re-emergence of a “dead” zone in Lake Erie, again due to nutrient loadings. Recent
studies by scientists and the National Oceanic and Atmospheric Administration (NOAA) have also
hypothesized a relationship between excessive nutrients in the Lake and the presence of two
aquatic invasive species – the zebra mussel and the quagga mussel (Vanderploeg et al., 2008).
Development and population increases in the Long Island Sound Watershed have resulted in a
significant increase in nitrogen loading to the Sound. The increased nitrogen loads have stimulated
plant growth, increased the amount of organic matter settling to the benthic zone, lowered DO
levels, and changed habitats.
The primary concerns in the Sound include hypoxia, the loss of sea grass, and alterations in the
food web. Management efforts are currently underway to reduce nitrogen pollution by more than
half with a focus on upgrading WWTPs with new technologies and removing nitrogen by reducing
polluted run-off through best management practices on farms and suburban areas (Long Island
Sound Study, 2004). The above represent four examples of impaired large water bodies impacted
by nutrient loadings. There are more than 80 additional estuaries and bays, and thousands of rivers,
streams, and lakes that are also impacted by nutrients in the U.S. In fact, all but one state and two
territories have CWA section 303(d) listed1 water body impairments for nutrient pollution.
Collectively, states have listed over 10,000 nutrient and nutrient–related impairments.
Climate change may also be a significant influence on the development of future eutrophic
symptoms. According to the report Effects of Nutrient Enrichment in the Nation’s Estuaries: A
Decade of Change, the factors associated with climate change that are expected to have the
greatest impacts on coastal eutrophication are:
• Increased temperatures
• Sea level rise
• Changes in precipitation and freshwater runoff
Increased temperatures will have several effects on coastal eutrophication. Most coastal species
are adapted to a specific range of temperatures. Increases in water temperatures may lead to
expanded ranges of undesirable species. Higher temperatures may also lead to increased algal
growth and longer growing seasons, potentially increasing problems associated with excessive
algal growth and nuisance/toxic blooms.
Additionally, warmer waters hold less DO, therefore potentially exacerbating hypoxia.
Temperature-related stratification of the water column may also worsen, having a further negative
effect on DO levels.
Climate change models predict increased melting of polar icecaps and changes in precipitation
patterns, leading to sea level rise and changes in water balance and circulation patterns in coastal
systems. Sea level rise will gradually inundate coastal lands, causing increased erosion and
sediment delivery to water bodies, and potentially flooding wetlands. The increased sediment load
and subsequent turbidity increase may cause SAV loss. The positive feedback between increased
erosion and algal growth (as erosion increases, sediment associated nutrients also increase,
stimulating growth) may also increase turbidity. The loss of wetlands, which act as nutrient sinks,
will further increase nutrient delivery to estuaries.

Another report titled Aquatic Ecosystems and Global Climate Change – Potential Impacts on Inland
Freshwater and Coastal Wetland Ecosystems in the United States notes that climate change of the
magnitude projected for the U.S. over the next 100 years will cause significant changes to
temperature regimes and precipitation patterns across the U.S. (Poff et al., 2002). Such alterations
in climate pose serious risks for inland freshwater ecosystems (lakes, streams, rivers, wetlands)
and coastal wetlands, and may adversely affect numerous critical services provided to human
populations.
These conclusions indicate climate change is a significant threat to the species composition and
function of aquatic ecosystems in the U.S. However, critical uncertainties exist regarding the
manner in which specific species and whole ecosystems will respond to climate change. These
arise both from uncertainties about how regional climate will change and how complex ecological
systems will respond.
Indeed, as climate change alters ecosystem productivity and species composition, many
unforeseen ecological changes are expected that may threaten the goods and services that these
systems provide to humans. Required by Section 303(d) of the CWA, the 303(d) list is a list of
state’s water bodies that do not meet or are not expected to meet applicable Water Quality
Standards with technology-based controls alone.
Federal and State Initiatives to Reduce Nutrient Pollution

NPDES Permitting
Established by the FWPCA Amendment of 1972, EPA’s NPDES permit program has been the
primary mechanism for controlling pollution from point sources. Point sources are discrete
conveyances such as pipes or man-made ditches. Individual homes that are connected to a
municipal system, use a septic system, or do not have a surface discharge do not need an NPDES
permit; however, POTWs and other facilities must obtain permits if they discharge directly to
surface waters.
NPDES permits for wastewater discharges contain, among other information, effluent limits for
“conventional” pollutants such as biochemical oxygen demand (BOD), total suspended solids
(TSS), and pH as well as limits for specific toxicants including various organic and inorganic
chemicals. Permits may also include effluent limits for “non-conventional” pollutants such as
nitrogen and phosphorus.
Effluent limits can be technology-based and/or water-quality based. EPA has established
technology-based, secondary treatment effluent limits for BOD as 5-day biochemical oxygen
demand (BOD5), TSS, and pH.
Water-quality based effluent limits are set if the technology-based limits are not sufficient to
maintain the water quality standards (WQS) of the receiving water. Federal and State regulations
related to WQSs and Total Maximum Daily Loads (TMDLs) are expected to drive down NPDES
effluent limits for nitrogen and phosphorus. WQS define the goals for a water body by designating
its uses, setting criteria to protect those uses, and establishing provisions to protect water bodies
from pollutants. Criteria can be narrative or numeric.
Regulatory agencies can adopt nutrient criteria to protect a water body against nutrient over-
enrichment and eutrophication caused by nitrogen and phosphorus. In June 1998, EPA issued a
National Strategy for the Development of Regional Nutrient Criteria. This was followed by
publication of recommended nutrient criteria for most streams and lakes in 2001. In a January 9,
2001 Federal Register notice, EPA recommended that states and other regulatory agencies
develop a nutrient criteria plan to outline their process for adopting such nutrient criteria (Federal
Register, 2001).
As of May 2007, only a handful of States and Territories had adopted nutrient criteria for nitrogen
and phosphorus (USEPA, 2007a), although many have made progress in criteria development.

In a memo dated May 25, 2007, EPA encouraged all regulatory agencies to “…accelerate their
efforts and give priority to adopting numeric nutrient standards or numeric translators for narrative
standards for all waters in States and Territories that contribute nutrient loadings to our waterways”
(USEPA, 2007b).
CWA Section 303(d) requires states to develop TMDLs for water bodies on the 303(d) list of
impaired waters. A TMDL is a calculation of the maximum amount of a pollutant a water body can
receive and still meet WQS. TMDLs serve as a tool for implementing WQS. The TMDL targets or
endpoints represent a number where the applicable WQS and designated uses (e.g., such as public
water supply, contact recreation, and the propagation and growth of aquatic life) are achieved and
maintained in the water body of concern.
TMDLs identify the level of pollutant control necessary to meet WQS and support the designated
uses of a water body. Once a TMDL is set, the total load is allocated among all existing sources.
The allocation is divided into two portions - a load allocation representing natural and non-point
sources and a waste load allocation representing NPDES permitted point source discharges.
In many regions, water bodies have a poor ability to assimilate nutrients or water bodies are already
impaired from past pollution and the water body cannot handle large loads of additional nutrients.
In these cases, TMDLs may require nutrient permit levels to be even lower than what might be
allowed otherwise by nutrient criteria.
The spinning reel for this oxidation ditch is mixing or aerating properly.

Nitrification
It was once thought that only two bacteria were involved in nitrification: Nitrosomonas europaea,
which oxidizes ammonia to nitrite, and Nitrobacter winogradskyi, which oxidizes nitrite to nitrate. It
is now known that at least 5 genera of bacteria oxidize ammonia and at least three genera of
bacteria oxidize nitrite (Holt et al., 1994). Besides oxygen, these nitrifying bacteria require a neutral
pH (7-8) and substantial alkalinity (these autotrophs use CO2 as a carbon source for growth). This
indicates that complete nitrification would be expected at pond pH values between pH 7.0 and 8.5.
Nitrification ceases at pH values above pH 9 and declines markedly at pH values below 7. This
results from the growth inhibition of the nitrifying bacteria. Nitrification, however, is not a major
pathway for nitrogen removal in lagoons. Nitrifying bacteria exists in low numbers in lagoons. They
prefer attached growth systems and/or high MLSS sludge systems.
Anaerobic Bacteria
Anaerobic, heterotrophic bacteria that commonly occur in lagoons are involved in methane
formation (acid-forming and methane bacteria) and in sulfate reduction (sulfate reducing bacteria).
Anaerobic methane formation involves three different groups of anaerobic bacteria that function
together to convert organic materials to methane via a three-step process. General anaerobic
degraders - many genera of anaerobic bacteria hydrolyze proteins, fats, and poly saccharides
present in wastewater to amino acids, short-chain peptides, fatty acids, glycerol, and mono- and
di-saccharides. These have a wide environmental tolerance in pH and temperature.
Photosynthetic Organisms
Acid-forming bacteria - this diverse group of bacteria converts products from above under
anaerobic conditions to simple alcohols and organic acids such as acetic, propionic, and butyric.
These bacteria are hardy and occur over a wide pH and temperature range.
Methane forming bacteria - these bacteria convert formic acid, methanol, methylamine, and acetic
acid under anaerobic conditions to methane. Methane is derived in part from these compounds
and in part from CO2 reduction. Methane bacteria are environmentally sensitive and have a
narrow pH range of 6.5-7.5 and require temperatures > 14o C.
Note that the products of the acid formers (principally acetic acid) become the substrate for the
methane producers. A problem exists at times where the acid formers overproduce organic acids,
lowering the pH below where the methane bacteria can function (a pH < 6.5). This can stop
methane formation and lead to a buildup of sludge in a lagoon with a low pH. In an anaerobic
fermenter, this is called a "stuck digester". Also, methane fermentation ceases at cold temperature,
probably not occurring in most lagoons in the wintertime in cold climates. A number of anaerobic
bacteria (14 genera reported to date (Bolt et al., 1994)) called sulfate reducing bacteria can use
sulfate as an electron acceptor, reducing sulfate to hydrogen sulfide.
This occurs when BOD and sulfate are present and oxygen is absent. Sulfate reduction is a major
cause of odors in ponds. Anaerobic, photosynthetic bacteria occur in all lagoons and are the
predominant photo-synthetic organisms in anaerobic lagoons. The anaerobic sulfur bacteria,
generally grouped into the red and green sulfur bacteria and represented by about 28 genera
(Ehrlich, 1990), oxidize reduced sulfur compounds (H2S) using light energy to produce sulfur and
sulfate. Here, H2S is used in place of H2O as used by algae and green plants, producing S04-
instead of O2. All are either strict anaerobes or microaerophilic. Most common are Chromatium,
Thiocystis, and Thiopedia, which can grow in profusion and give a lagoon a pink or red color.
Finding them is most often an indication of organic overloading and anaerobic conditions in an
intended aerobic system.
Conversion of odorous sulfides to sulfur and sulfate by these sulfur bacteria is a significant odor
control mechanism in facultative and anaerobic lagoons, and can be desirable.

Nitrogen and Phosphorus Removal Technologies
Introduction
This section provides information on a number of different technologies that can reduce nitrogen
and phosphorus levels. The actual technology selected will be site-specific and dependent on many
factors including soil conditions, influent water quality, required effluent levels, disposal options,
availability of land, cost, etc. In some cases, a combination of technologies may be necessary to
effectively remove all the contaminants of concern. Small system owners and operators should
work closely with their state onsite and decentralized program staff as well as engineers to ensure
that the technologies selected will work effectively in combination to achieve the effluent goals.
Nutrient Removal Technologies

Fixed-film Systems - Aerobic/anaerobic trickling filter package plant
Fixed-film systems (FFSs) are biological treatment processes that employ a medium such as rock,
plastic, wood, or other natural or synthetic solid material that will support biomass on its surface
and within its porous structure (USEPA, 2008c). Trickling filter FFSs are typically constructed as
beds of media through which wastewater flows.
Oxygen is normally provided by natural or forced ventilation. Commercial on-site systems use
synthetic media and receive wastewater from overlying sprayheads for aerobic treatment and
nitrification. Nitrified effluent returns to the anoxic zone to mix with either septic tank contents or
incoming septic tank effluent for denitrification. A portion of the denitrified effluent is discharged for
disposal or further treatment. Aerobic tanks are available in residential or small community sizes.
Typical trickling filters systems currently available are capable of producing effluent BOD and TSS
concentrations of 5 to 40 mg/L. Nitrogen removal typically varies from 0 to 35 percent although
removal percentages as high as 65% have been demonstrated through USEPA’s Environmental
Technology Verification (ETV) program. Phosphorus removal is typically 10 to 15 percent.

Higher removal occurs at low loading rates in warm climates. Systems can be configured for single-
pass use where the treated water is applied to the trickling filter once before being disposed of, or
for multi-pass use where a portion of the treated water is cycled back to the septic tank and re-
treated via a closed loop.
Multi-pass systems result in higher treatment quality and assist in removing Total Nitrogen (TN)
levels by promoting nitrification in the aerobic media bed and denitrification in the anaerobic septic
tank. Factors affecting performance include influent wastewater characteristics, hydraulic and
organic loading, medium type, maintenance of optimal DO levels, and recirculation rates.
Sequencing Batch Reactor (SBR)

The SBR process is a sequential suspended growth (activated sludge) process in which all major
steps occur in the same tank in sequential order (USEPA, 2008d). The SBR system is typically
found in packaged configurations for onsite and small community or cluster applications. The major
components of the package include the batch tank, aerator, mixer, decanter device, process control
system (including timers), pumps, piping, and appurtenances. Aeration may be provided by diffused
air or mechanical devices. SBRs are often sized to provide mixing as well and are operated by the
process control timers.
Mechanical aerators have the added value of potential operation as mixers or aerators. The
decanter is a critical element in the process. Several decanter configurations are available,
including fixed and floating units. At least one commercial package employs a thermal processing
step for the excess sludge produced and wasted during the “idle” step. The key to the SBR process
is the control system, which consists of a combination of level sensors, timers, and microprocessors
which can be configured to meet the needs of the system.

SBRs can be designed and operated to enhance removal of nitrogen, phosphorus, and ammonia,
in addition to removing TSS and BOD. Package plant SBRs are suitable for areas with little land,
stringent treatment requirements, and small wastewater flows such as RV parks or mobile homes,
campgrounds, construction sites, rural schools, hotels, and other small applications. These
systems are also useful for treating pharmaceutical, brewery, dairy, pulp and paper, and chemical
wastes (USEPA, 2000d).
Intermittent Sand Filters (ISF)

ISF is used to describe a variety of packed-bed filters of sand or other granular materials available
on the market (USEPA, 2008g). Sand filters provide advanced secondary treatment of settled
wastewater or septic tank effluent. They consist of a lined (e.g., impervious PVC liner on sand
bedding) excavation or structure filled with uniform washed sand that is placed over an underdrain
system. The wastewater is directed onto the surface of the sand through a distribution network and
allowed to percolate through the sand to the underdrain system. The underdrain system collects
the filter effluent for further processing or discharge.
Sand filters are aerobic, fixed-film bioreactors. Bioslimes from the growth of microorganisms
develop as films on the sand particle surfaces. The microorganisms in the slimes capture soluble
and colloidal waste materials in the wastewater as it percolates over the sand surfaces. The
captured materials are metabolized into new cell mass or degraded under aerobic conditions to
carbon dioxide and water. Most biochemical treatment occurs within approximately 6 inches of the
filter surface. Other treatment mechanisms that occur in sand filters include physical processes,
such as straining and sedimentation, to remove suspended solids within the pores of the media.
Most suspended solids are strained out at the filter surface.
Chemical adsorption can occur throughout the media bed. Adsorption sites in the media are usually
limited, however. The capacity of the media to retain ions depends on the target constituent, the
pH, and the mineralogy of the media. Phosphorous is one element of concern in wastewater that
can be removed in this manner, but the number of available adsorption sites is limited by the
characteristics of the media.
Sand filters can be used for a broad range of applications, including single-family residences, large
commercial establishments, and small communities. Sand filters are frequently used to pretreat
septic tank effluent prior to subsurface infiltration onsite where the soil has insufficient unsaturated
depth above ground water or bedrock to achieve adequate treatment. They are also used to meet
water quality requirements (with the possible exception of fecal coliform removal) before direct
discharge to surface water.
Sand filters are used primarily to treat domestic wastewater, but they have been used successfully
in treatment trains to treat wastewaters high in organic materials such as those from restaurants
and supermarkets. Single-pass ISF filters are most frequently used for smaller applications and
sites where nitrogen removal is not required. However, they can be combined with anoxic
processes to significantly increase nitrogen removal.
Recirculating Sand Filters (RSF)

Recirculating filters using sand, gravel, or other media provide advanced secondary treatment of
settled wastewater or septic tank effluent (USEPA, 2008h). They consist of a lined (e.g., impervious
PVC liner on sand bedding) excavation or structure filled with uniform washed sand that is placed
over an underdrain system. The wastewater is directed onto the surface of the sand through a
distribution network and allowed to percolate through the sand to the underdrain system. The
underdrain system collects and recycles the filter effluent to the recirculation tank for further
processing or discharge.
The basic components of recirculating filters include a recirculation/dosing tank, pump and controls,
distribution network, filter bed with an underdrain system, and a return line. The return line or the
underdrain must split the flow to recycle a portion of the filtrate to the recirculation/dosing tank.

A small volume of wastewater and filtrate is dosed to the filter surface on a timed cycle 1 to 3 times
per hour.
Recirculation ratios are typically between 3:1 and 5:1. In the recirculation tank, the returned aerobic
filtrate mixes with the anaerobic septic tank effluent before being reapplied to the filter. RSFs can
be used for a broad range of applications, including single-family residences, large commercial
establishments, and small communities. They produce a high quality effluent with approximately
85 to 95 percent BOD and TSS removal. In addition, almost complete nitrification is achieved.
Denitrification also has been shown to occur in RSFs. Depending on modifications in design and
operation, 50 percent or more of applied nitrogen can be removed (USEPA, 1999). To enhance
this capability, they can be combined with a greater supply of biodegradable organic carbon, time,
and mixing than is normally available from the conventional recirculation tank.
Natural Systems
The natural systems described here include constructed wetlands and floating aquatic plant
treatment systems. Wetland systems are typically described in terms of the position of the water
surface and/or the type of vegetation grown. Most natural wetlands are free water surface (FWS)
systems where the water surface is exposed to the atmosphere; these include bogs (primary
vegetation mosses), swamps (primary vegetation trees), and marshes (primary vegetation grasses
and emergent macrophytes) (USEPA, 2000e). subsurface flow (SF) wetlands are specifically
designed to treat or polish wastewater and are typically constructed as a bed or channel containing
appropriate media.
Constructed wetlands treat wastewater by bacterial decomposition, settling, and filtering. As in tank
designs, bacteria break down organic matter in the wastewater, aerobically, anoxically and
anaerobically. Oxygen for aerobic decomposition is supplied by the plants growing in the wetland.
Solids are filtered and finally settle out of the wastewater within the wetland. After about two weeks
in the wetland, effluent is usually discharged by gravity to an unlined wetland bed. If these systems
discharge effluent to surface ditches, they require a NPDES permit.
The submerged plant roots do provide substrate for microbial processes. However, the amount of
oxygen that emergent macrophytes can transmit from the leaves to their roots is negligible
compared to the oxygen demand of wastewater. Therefore, subsurface flow wetlands are devoid
of oxygen. The lack of oxygen in these subsurface flow systems means that ammonia oxidation via
biological nitrification will not occur without the use of an additional unit process, such as a gravel
trickling filter for nitrification of the wastewater ammonia. Vertical flow wetland beds are a
modification of subsurface flow wetlands which contain gravel or coarse sand and are loaded
intermittently at the top surface. Unlike ammonia oxidation, nitrate removal in a subsurface flow
wetland can be rapid and effective because the anoxic conditions and carbon sources necessary
to support the treatment reactions occur naturally in these systems.
FWS wetlands with long detention times can remove minor amounts of phosphorus through plant
uptake, adsorption, complexation, and precipitation. However, removal via plant uptake is limited
to phosphorus retained in plant litter that is buried by sediments before plant decomposition occurs
(i.e. peat building process). Phosphorus removal is typically greater in the first year or two because
of soil absorption and rapidly expanding vegetation but decreases when the system reaches
equilibrium, and unburied plant litter releases phosphorus back into the water as it decomposes.
Phosphorus removal is also possible with the use of an addition process, such as chemical addition
and mixing prior to a final deep settling pond.
Aquatic systems using duckweed have been used for a number of years to treat wastewater for
various purposes (WEF, 2001). Duckweed (Lemna spp.) are floating macrophytes. Duckweed
fronds can double their mass in two days under ideal conditions of nutrient availability, sunlight,
and temperature. Although duckweed can be found in most regions, the rate of growth is optimal
at 20 to 30o C and they grow best in a pH range of 3.5 to 8.5.

Duckweed can grow about six months per year in most U.S. climates. High levels of BOD and TSS
removal have been observed from duckweed systems. To achieve secondary treatment most
duckweed systems are coupled with either facultative or aerated ponds. Nitrogen is removed by
plant uptake and harvesting, by denitrification, or a combination of the two. Typically, less than 1
mg/L of phosphorus can be removed by plant uptake and harvest. If significant phosphorus removal
is required, chemical precipitation with alum, ferric chloride, or other chemicals used in a separate
treatment step is necessary. The major disadvantage of duckweed systems is the large amount of
biomass produced by the rapidly growing plants, which creates a solids handling requirement
similar to handling sludge at an aerobic wastewater treatment facility.
Proprietary Filters/Improved and Emerging Technologies

A number of companies have developed proprietary nitrogen and phosphorus removal
technologies that can be used at centralized wastewater treatment facilities as well as at onsite,
decentralized systems. This section provides a general description of some of these technologies
without mentioning specific trade names.
Sustainable Nutrient Recovery

While the U.S. is primarily addressing nutrient removal concerns through development of WQSs
and treatment at centralized wastewater facilities, a number of European countries including
Switzerland, Sweden, and the Netherlands are conducting research on innovative sustainable
nutrient recovery systems. The concept behind these new technologies is to separate and treat
toilet waste before it leaves the home or building and mixes with the larger waste stream to be
carried to WWTPs.
Recent studies have shown that about 80 percent of the nitrogen and 50 percent of the phosphorus
in wastewater are derived from urine although urine makes up only 1 percent of the volume of
wastewater (Larsen and Leinert, 2007). Separating the urine from wastewater could offer various
advantages: WWTPs could be built on a smaller scale, water bodies will be better protected from
nitrogen and phosphorus pollution, nutrients could be recycled for agricultural use, and various
constituents of concern including hormones and pharmaceutical compounds could be removed
before being mixed with wastewater and released to the environment.
A major benefit would be reduced energy consumption at WWTPs as a result of reduced treatment
requirements for nitrogen. Also, separating 50 to 60 percent of urine could reduce in-plant nitrogen
gas discharges and result in fewer impurities in methane captured from sludge digestion.
Organizations such as the Swiss Federal Institute of Aquatic Science and Technology (Eawag) are
currently experimenting with the development and application of “NoMix technology” to separate
urine from solid waste at the toilet bowl. While similar in size and shape to current toilets, this new
technology has two waste pipes – a small front one that collects and diverts urine into a storage
tank, and a larger rear waste pipe that operates like a standard toilet. The first of these toilets were
installed in two “eco-villages” in Sweden in 1994 and since then have spread to other locations
throughout the country and to Denmark, the Netherlands, and Switzerland. The concept is now
taking hold in Austria and Germany. While the pollutant-free urine, or “urevit,” can be spray-applied
directly onto agricultural fields; in the Netherlands, a company called Grontmij trucks stored urine
to a special treatment plant where the phosphate is precipitated out as a mineral called struvite and
used as a fertilizer.
Novaquatis, a branch of Eawag is experimenting with extracting nitrogen and potassium from urine
that can be sprayed directly onto crops. Eawag is also experimenting with a pilot decentralized
basement sewage plant where domestic wastewater is treated in a MBR so it can be reused for
flushing the toilets or watering the garden and the sewage sludge is composted. While still
experimental, some of these technologies may have practical future applications if widely
applicable low-cost solutions can be found for urine transport, or stable and cost-effective
technologies can be developed for decentralized treatment.

While studies of consumer attitudes and acceptance appear to be positive, technological
improvements are still needed to prevent clogging in pipes, to identify best treatment options that
can be applied in practice; and to identify how and where to convert urine to fertilizer.
Sustainability concerns are also driving the wastewater treatment industry to start looking at sludge
as a renewable resource. Historically, agricultural use has been the traditional approach for
disposal of municipal sludge due to its high nutrient content for fertilizing crops, and its low cost
approach. As scientific advances detect smaller and smaller quantities of contaminants (i.e., heavy
metals, pathogenic microorganisms, pharmaceuticals, and personal care products), the public,
farming organizations, and the food industry are raising concerns about continuing this practice. As
noted above, researchers are discovering that valuable products can be generated from sewage
treatment byproducts such as energy extracted from anaerobic digestion, construction materials
such as bricks, and nutrients such as phosphorus that can be extracted from sludge and used as
fertilizer.
In February 2008, the non-profit Global Water Research Coalition, an international water research
alliance formed by 12 world-leading research organizations, released a report titled, State of
Science Report: Energy and Resource Recovery from Sludge (Kalogo and Monteith, 2008). The
report focuses on:
• The international situation of energy and resource recovery from sludge
• How the use of different sludge treatment processes affects the possibility of recovering energy
and/or materials from the residual sludge
• The influence of market and regulatory drivers on the fate of the sludge end-product
• The feasibility of energy and resource recovery from sludge
• The social, economic, and environmental performance (triple bottom line or TBL assessment) of
current alternatives technologies
Four market drivers are identified and discussed including:

- Sustainability and environmental concerns, such as the threat of soil pollution, global warming
and resource depletion
- Rising energy costs and the need of more electricity and heat to operate the plants
- Requirements for high quality of resources for industrial applications, such as calcium phosphate
for the phosphate industry
- Regulation as a factor stimulating the development of new technologies
In the report, energy recovery technologies are classified into sludge-to-biogas processes, sludge-
to-syngas processes, sludge-to-oil processes, and sludge-to-liquid processes. The technologies
available for resource recovery discussed in the report include those to recover phosphorus,
building materials, nitrogen, and volatile acids.
The report, which covers both established as well as emerging technologies, will be used as the
basis for development of the coalition’s future strategic research plan on energy and recovery from
sludge. As a technical resource, it provides a valuable overview of sludge disposal practices in
various countries such as the U.S., the Netherlands, the United Kingdom, Germany, Sweden,
Japan, and China; and presents a number of treatment processes for resource recovery.
Other groups have looked at recovering phosphorus from the supernatant from anaerobic
digestion. Several different processes have been proposed that rely on precipitation of the
phosphorus as either struvite or calcium phosphate. Work is underway on projects in Italy,
Germany, the Netherlands, and Canada (SCOPE, 2004).

Nutrient Constituents in Wastewater and Measurement Methods
This section provides an overview of the sources, forms, and measurement methods for nitrogen
and phosphorus in wastewater.
Nitrogen
Nitrogen is an essential nutrient for plants and animals. Approximately 80 percent of the earth’s
atmosphere is composed of nitrogen and it is a key element of proteins and cells. The major
contributors of nitrogen to wastewater are human activities such as food preparation, showering,
and waste excretion. The per capita contribution of nitrogen in domestic wastewater is about 1/5th
of that for BOD. Total nitrogen in domestic wastewater typically ranges from 20 to 70 mg/L for low
to high strength wastewater (Tchobanoglous et al., 2003). Factors affecting concentration include
the extent of infiltration and the presence of industries. Influent concentration varies during the day
and can vary significantly during rainfall events, as a result of inflow and infiltration to the collection
system.
The most common forms of nitrogen in wastewater are:

• Ammonia (NH3)
• Ammonium ion (NH4+)
• Nitrite (NO2-)
• Nitrate (NO3-)
• Organic nitrogen
Nitrogen in domestic wastewater consists of approximately 60 to 70 percent ammonia-nitrogen and

30 to 40 percent organic nitrogen (Tchobanoglous et al., 2003; Crites and Tchobanoglous, 1998).
Most of the ammonia-nitrogen is derived from urea, which breaks down rapidly to ammonia in
wastewater influent. EPA approved methods for measuring ammonia, nitrate, and nitrite
concentration use colorimetric techniques. Organic nitrogen is approximated using the standard
method for Total Kjeldahl Nitrogen (TKN) (APHA, AWWA, and WEF, 1998).
The TKN method has three major steps:

(1) digestion to convert organic nitrogen to ammonium sulfate;
(2) conversion of ammonium sulfate into condensed ammonia gas through addition of a strong
base and boiling; and
(3) measurement using colorimetric or titration methods. Because the measured concentration
includes ammonia, the ammonia-nitrogen concentration is subtracted from the TKN to determine
organic nitrogen.
Nitrogen components in wastewater are typically reported on an “as nitrogen” basis so that the total
nitrogen concentration can be accounted for as the influent nitrogen components are converted to
other nitrogen compounds in wastewater treatment.
WWTPs designed for nitrification and denitrification can remove 80 to 95 percent of inorganic
nitrogen, but the removal of organic nitrogen is typically much less efficient (Pehlivanoglu-Mantas
and Sedlak, 2006). Domestic wastewater organic nitrogen may be present in particulate, colloidal
or dissolved forms and consist of proteins, amino acids, aliphatic N compounds, refractory natural
compounds in drinking water (e.g. Humic substances), or synthetic compounds (e.g. ethylene
Diamine tetraacetic acid (EDTA)).
Organic nitrogen may be released in secondary treatment by microorganisms either through

metabolism or upon death and lysis. Some nitrogen may be contained in recondensation products.
Hydrolysis of particulate and colloidal material by microorganisms releases some organic nitrogen
as dissolved, biodegradable compounds.

Amino acids are readily degraded during secondary biological treatment, with 90 to 98 percent
removal in activated sludge systems and 76 to 96 percent removal in trickling filters. However, other
forms of organic nitrogen may be more persistent in wastewater treatment processes.
The importance of organic nitrogen has increased as effluent limits on nitrogen have become more
stringent. With more impaired waterways from nutrient loads, effluent limits for total nitrogen (TN)
concentrations of 3.0 mg/L or less are becoming more common. The dissolved organic nitrogen
(DON) concentration in the effluent from biological nutrient removal treatment facilities was found
to range from 0.50 to 1.50 mg/L in 80 percent of 188 plants reported by Pagilla (STAC-WERF,
2007) and values as high as 2.5 mg/L were observed.
Thus, for systems without effluent filtration or membrane bioreactors (MBRs) that are trying to meet
a TN treatment goal of 3.0 mg/L, the effluent DON contribution can easily be 20 to 50 percent of
the total effluent nitrogen concentration, compared to only about 10 percent for conventional
treatment (Pehlivanoglu-Mantas and Sedlak, 2004).
The chemical composition of DON in wastewater effluents is not completely understood. Sedlak
(2007) has suggested that only about 20 percent of the DON has been identified as free and
combined amino acids, EDTA, and other trace nitrogen compounds. About 45 percent may be
unidentified low molecular weight compounds and the other 35 percent as unidentified high
molecular weight compounds containing Humic acids and amides. Similar results were found by
Khan (2007). Early work by Parkin and McCarty (1981) suggested that 40 to 60 percent of effluent
DON is non-bioavailable. The non-bioavailable portion is also referred to as recalcitrant DON
(rDON).
Primary clarifier

Phosphorus
Total phosphorus (TP) in domestic wastewater typically ranges between 4 and 8 mg/L but can be
higher depending on industrial sources, water conservation, or whether a detergent ban is in place.
Sources of phosphorus are varied. Some phosphorus is present in all biological material, as it is
an essential nutrient and part of a cell’s energy cycle. Phosphorus is used in fertilizers, detergents,
and cleaning agents and is present in human and animal waste.
Phosphorus in wastewater is in one of three forms:

• Phosphate (also called Orthophosphate)
• Polyphosphate, or
• Organically bound phosphorus.
The orthophosphate fraction is soluble and can be in one of several forms (e.g., phosphoric acid,
phosphate ion) depending on the solution pH. Polyphosphates are high-energy, condensed
phosphates such as pyrophosphate and trimetaphosphate. They are also soluble but will not be
precipitated out of wastewater by metal salts or lime. They can be converted to phosphate through
hydrolysis, which is very slow, or by biological activity.
Organically bound phosphorus can either be in the form of soluble colloids or particulate. It can
also be divided into biodegradable and non-biodegradable fractions. Particulate organically bound
phosphorus is generally precipitated out and removed with the sludge. Soluble organically bound
biodegradable phosphorus can be hydrolyzed into orthophosphate during the treatment process.
Soluble organically bound non-biodegradable phosphorus will pass through a wastewater

treatment plant. A typical wastewater contains 3 to 4 mg/L phosphorus as phosphate, 2 to 3 mg/L
as polyphosphate, and 1 mg/L as organically bound phosphorus (WEF and ASCE, 2006).
Phosphorus content in wastewater can be measured as

• Orthophosphate
• Dissolved orthophosphate
• Total phosphorus
• Total dissolved phosphorus (i.e., all forms except particulate organic phosphorus)

EPA approved laboratory methods rely on colorimetric analysis. Colorimetric analysis measures
orthophosphate only, so a digestion step is needed to convert polyphosphate and organic
phosphorus to orthophosphate to measure TP. The persulfate method is reported to be the most
common and easiest method (WEF and ASCE, 2006). To determine dissolved phosphorus (either
total dissolved phosphorus or total dissolved orthophosphate), the sample is first filtered through a
0.45-micron filter.
USEPA approved colorimetric methods are routinely used to measure phosphorus levels as low as
0.01 mg/L. On-line analyzers that use the colorimetric method are available from venders (e.g., the
Hach PhosphaxTM SC phosphate analyzer).
Ion chromatography is a second common technique used to measure orthophosphate in

wastewater. As with colorimetric methods, digestion is required for TP measurement, with
persulfate digestion recommended (WEF and ASCE, 2006).

Phosphorus Removal by Chemical Addition
The purpose of this section is to describe techniques for phosphorus removal by chemical addition.
It summarizes issues associated with chemical feed location, mixing, and sludge production. An
overview of advanced solids separation processes is also provided.
Principles
Chemical precipitation for phosphorus removal is a reliable, time-tested, wastewater treatment
method that has not drastically changed over the years. To achieve removal, various coagulant
aids are added to wastewater where they react with soluble phosphates to form precipitates. The
precipitates are removed using a solids separation process, most commonly settling (clarification).
Chemical precipitation is typically accomplished using either lime or a metal salt such as aluminum
sulfate (alum) or ferric chloride. The addition of polymers and other substances can further enhance
floc formation and solids settling. Operators can use existing secondary clarifiers or retrofit primary
clarifiers for their specific purposes.
Aluminum and Iron Salts

Alum and ferric or ferrous salts are commonly used as coagulant and settling aids in both the water
and wastewater industry. They are less corrosive, create less sludge, and are more popular with
operators compared to lime. Alum is available in liquid or dry form, can be stored on site in steel or
mild concrete, and has a near unlimited shelf life. Ferric chloride is similar although care is needed
during handling because of corrosivity. If an industrial source is available such as waste pickle
liquor, ferrous chloride or ferrous sulfate have been used for phosphorus removal. Ferrous forms
should be added directly to aerobic reactors rather than to anaerobic reactors such as primary
settling basins because the ferrous iron needs to oxidize to ferric iron for best results.
The molar ratio of aluminum to phosphorus required for phosphorus removal ranges from about
1.38:1 for 75 percent removal, 1.72:1 for 85 percent removal, and 2.3:1 for 95 percent removal.
For iron compounds, a ratio of about 1:1 is required, with a supplemental amount of iron (10 mg/L)
added to satisfy the formation of hydroxide (WEF and ASCE, 1998).

For additional removal of phosphorus with aluminum and iron salts, a ratio of between 2 and 6 parts
metal salt to 1-part phosphorus may be required for adequate phosphorus removal.
To supplement stoichiometry calculations, designers should consider jar tests and, in some cases,
full-scale pilot tests to gauge the effects on the required dose of competing reactions; the influence
of pH and alkalinity, adsorption, and co-precipitation reactions; and the interaction with polymers
that are added to increase coagulation and flocculation (WEF and ASCE, 1998; Bott et al. 2007).
Aluminum or ferric iron salts can be added to the primary clarifier, secondary clarifier, tertiary
clarifier, or directly into the activated sludge aeration tank. Multiple additions can increase
phosphorus removal efficiency. Ferrous salts can only be added to the aeration basin since it needs
to be oxidized to ferric to precipitate the phosphorus.
The solubility of aluminum and iron salts is a function of pH. The optimum solubility for alum was
previously reported to occur at a pH range of 5.5 to 6.5, significantly lower than most influent
wastewater. Recent studies (Szabo et al., 2008) showed that the range for both iron and alum is
between 3.5 and 7.5 with the highest efficiency between pH 5.5 and 7.
Chemicals such as lime compounds, caustic soda, and soda ash can be used to raise the pH of
the waste stream prior to biological treatment processes or discharge. It is important to understand
that alkalinity is consumed during the precipitation reactions, and precipitation will be incomplete if
insufficient alkalinity is present.

Lime
Although lime had lost favor due to issues associated with chemical handling, sludge production,
and re-carbonation, it has recently been considered more often because of its ability to reduce
phosphorus to very low levels when combined with effluent filtration and the microbial control
properties associated with its high pH.
When lime is added to wastewater, it first reacts with the bicarbonate alkalinity to form calcium
carbonate (CaCO3). As the pH increases to more than 10, excess calcium ions will react with
phosphate to precipitate hydroxylapatite [CA5(OH)(PO4)3].
Because it reacts first with alkalinity, the lime dose is essentially independent of the influent
phosphorus concentration. Tchobanoglous et al. (2003) estimates the lime dose to typically be 1.4
to 1.6 times the total alkalinity expressed as CaCO3.
The typical reaction between calcium compounds and phosphorus is represented below:
5Ca2+ + 4OH- + 3HPO4- Ca5OH(PO4)3 + 3H2O (4-3)
The molar ratio required for phosphorus precipitation with lime is approximately 5:3, but can vary
from between 1.3 to 2, depending on the composition of the wastewater. As with iron and aluminum
salts, jar tests can be used to determine correct doses for a specific wastewater stream
(WEF,1998).
Lime addition can raise the pH to greater than 11. Because activated sludge processes require pH
levels below 9, lime cannot be added directly to biological treatment processes or it will cause
process upsets. Lime can be added to primary sedimentation tanks and removed with the primary
sludge or it can be added as a tertiary treatment process after biological treatment. When added to
primary tanks, it will also result in the removal of colloidal material through coagulation and settling,
with a concomitant removal of TSS up to 80 percent and chemical oxygen demand (COD) up to 60
percent.
In either case, pH adjustment is needed and typically accomplished by adding CO2 or a liquid acid
such as sulfuric acid, nitric acid, or hypochlorite (Tchobanoglous et al., 2003; USEPA, 1999a).
Hortskotte et al. (1974) showed that when the primary effluent is discharged directly to a nitrifying
activated sludge plant, the hydrogen ions produced may neutralize the high pH. However, when
denitrification is practiced and the operator wishes to make use of the soluble COD in the primary
effluent, the effluent must be neutralized before discharging it to the anoxic zone.
Lime requires special handling and operations practices that further set it apart from chemical
precipitation by metal salts. Although the formation of carbonate scaling on equipment and pipes
is a drawback of lime treatment, lime slaking, where quicklime (CaO) is reacted with water to form
calcium hydroxide (Ca(OH)2), is the biggest operational disadvantage.

Location of Chemical Feed and Mixing
Lime or metal salts can be added at several locations throughout the treatment plant to remove
phosphorus.
“Pre-precipitation” is when chemicals are added to raw water to precipitate phosphorus in the
primary sedimentation basins.
“Co-precipitation” involves adding chemicals to form precipitates that can be removed with
biological sludge.
“Post-precipitation” is when chemicals are added after secondary sedimentation and precipitants
are removed in a tertiary process such as sedimentation or filtration (Tchobanoglous et al., 2003).
Because it requires a high pH to achieve a low phosphorus concentration, lime cannot be added
directly to biological reactors or to the secondary clarifiers.
Multipoint additions of iron or aluminum salts have been very effective and can typically remove
more phosphorus than single-point applications. There are several advantages to post-precipitating
phosphorous using a tertiary treatment technique (after biological processes in a separate reactor):
• Microorganisms rely on phosphorus as a food source. If too much phosphorus is removed prior
to biological treatment, biological processes may suffer. For activated sludge, the minimum ratio of
phosphorus to BOD5 for a rapidly growing (low solids retention time (SRT)) system is typically
about 1:100 (WEF and ASCE, 1998).
• Competing chemicals in the primary sedimentation basins can increase the required dose.
• Phosphorus enters the treatment plant as soluble orthophosphate, soluble polyphosphates, and
organically bound phosphorus. Most of the polyphosphates and much of the organically bound
phosphorus are converted to more simple orthophosphates during biological treatment. If the
influent contains significant polyphosphates and/or organically bound phosphorus, locating
chemical treatment after biological processes would be more efficient and achieve lower effluent
levels.
• The removal of carbonate alkalinity and phosphorus by lime prior to biological treatment can have
a negative impact on nitrification processes (WEF and ASCE, 1998). Also, removing phosphorus
to very low concentrations upstream of denitrification filters can negatively affect the denitrification
process. Previous studies showed that the hydroxide alkalinity can be balanced by the hydrogen
ions produced during nitrification.
• Sludge recalcification can be used to achieve high removal efficiencies using lime in tertiary
treatment. One potential advantage to adding chemicals during primary treatment instead of tertiary
treatment is reduced capital costs and space requirements as a result of removing additional BOD
and TSS and reducing the load to downstream processes, thereby reducing the size of the
subsequent activated sludge basins and the amount of oxygen transfer needed.
Chemicals should be well mixed with the wastewater to ensure reaction with soluble phosphates
and formation of precipitates. Chemicals may either be mixed in separate tanks or can be added
at a point in the process where mixing already occurs. Bench-scale and pilot scale tests are often
used to determine the correct mixing rate for a given composition of wastewater and chemicals
used, including polymer (USEPA, 1999a).

Advanced Solids Separation Processes
The effectiveness of phosphorus removal by chemical addition is highly dependent on the solids
separation process following chemical precipitation. Direct addition of metal salts to activated
sludge processes followed by conventional clarification can typically remove TP to effluent levels
between 0.5 and 1.0 mg/L (Bott et al., 2007). Tertiary processes (post-secondary treatment) can
be used to remove phosphorus to very low (< 0.1 mg/L) concentrations. For example, Reardon
(2005) reports that four WWTP with tertiary clarifiers achieved TP levels of between 0.032 and 0.62
mg/L. Two common tertiary processes are clarification and effluent filtration. These approaches
can be used separately or in combination. The next section presents a detailed discussion of
effluent filtration. Advances in tertiary clarification processes are discussed below.
The types of clarifiers used for tertiary processes include conventional, one or two-stage lime,
solids-contact, high-rate, and ballasted high-rate (BHRC). Several patented BHRC using different
types of ballast such as recycled sludge, microsand, and magnetic ballast (USEPA, 2008a) have
been developed in recent years. The advantages of high-rate clarification are that the clarifiers
have a smaller footprint and are able to treat larger quantities of wastewater in a shorter period of
time. In addition, as an add-on during wet weather, they can help prevent sanitary sewer overflows
(SSOs) and combined sewer overflows (CSOs).
The following patented processes are examples of high rate clarification including
performance estimates:
• DensaDeg® uses a coagulant in a rapid mix basin to destabilize suspended solids. The water
flows into a second tank where polymer (for aiding flocculation) and sludge are added. The sludge
acts as the “seed” for formation of high density floc. This floc is removed in settling tubes (USEPA,
2008). The main advantages of this process are a smaller footprint and denser sludge which is
easier to dewater. Pilot testing for City of Fort Worth, Texas found a phosphorus removal rate of
88-95% for DensaDeg® (USEPA, 2003).
• Actiflo® uses a coagulant in a rapid mix basin to destabilize suspended solids. The water flows
to a second tank where polymer (for aiding flocculation) and microsand are added. Microsand
provides a large surface onto which suspended solids attach, creating a dense floc that settles out
quickly. Clarification is assisted by lamella settling. Product pilot testing in Fort Worth, Texas
showed a phosphorus removal efficiency of 92-96% for Actiflo® (USEPA, 2003).
• The CoMag process uses the addition of magnetic ballast with metal salts to promote floc
formation. Settling is followed by high gradient magnetic separation for effluent polishing and
recovery of the magnetic ballast (USEPA, 2008a). CoMag is currently in operation at a 4.0 million
gallons per day (MGD) wastewater treatment plant in Concord, Massachusetts. The vendor has
guaranteed an effluent phosphorus concentration not to exceed 0.05 mg/L (EPA Region 10, 2007).
Other Design and Operational Issues

Phosphorus removal by chemical addition is limited to the soluble phosphates in the waste stream.
Organically bound phosphorus and polyphosphates will not be removed by chemical treatment
unless they are coagulated with the chemicals and removed in the sludge. Chemicals can be added
after biological treatment to capitalize on the conversion of polyphosphates and organically bound
phosphorus to phosphates by microorganisms in activated sludge.
The success of phosphorus removal by chemical addition depends on proper instrumentation and
control. Dosage control typically takes the form of manual operation (for small systems),
adjustments based on automatic flow measurements, or the more advanced on-line analyzers with
computer-assisted dosage control.

Chemical properties of any water used for making solutions should be considered – tap water high
in suspended solids could cause sludge to form when mixed with coagulants (WEF and ASCE,
1998) and could lead to clogging of chemical feed lines. Smith et al. (2008) found that factors such
as pH, complexation, mixing, and the coagulant used can limit the removal of phosphorus,
especially in the range of <0.1 mg/L.
Impacts on Sludge Handling and Production

Sludge handling and production is generally considered to be one of the main downsides of
chemical addition. Chemical precipitation methods always produce additional solids due to
generation of metal- or calcium- phosphate precipitates and additional suspended solids (WEF and
ASCE, 1998). Chemically treated sludge has a higher inorganic content compared to primary and
activated sludge and will increase the required size of aerobic and anaerobic digesters. Additional
sludge production can be estimated using reaction equations. The use of metal salts can result in
increased inorganic salts (salinity) in the sludge and in the effluent.
Salinity can create problems when biosolids are land applied or when the effluent is returned to
existing water supply reservoirs. Biological phosphorus removal was developed in South Africa due
to the high rate of indirect recycling of wastewater effluent which led to excessive total dissolved
solids (TDS) during dry periods. High total salts can reduce germination rates for crops and
negatively affect the soil structure.
Lime traditionally produces a higher sludge volume compared to metal salts because of its reaction
with natural alkalinity. An advantage of lime sludge is that some stabilization can occur due to the
high pH levels required. One disadvantage is that lime can cause scaling in mechanical thickening
and dewatering systems. There are also differences in the amount and characteristics of sludge
generated by alum versus ferric salts. Although alum tends to produce less sludge than do ferric
salts, alum sludge can be more difficult to concentrate and dewater compared to ferric sludge.
Biological Nitrogen Removal

This section provides an overview of the principles behind biological nitrogen removal and
describes the common design configurations in use today. It identifies key operational and design
issues (including impacts on sludge handling and production), provides general guidelines on
process selection, and summarizes ongoing research efforts in this area. Process configurations
that are designed to remove both nitrogen and phosphorus are described latter.
Principles
In wastewater treatment, nitrogen removal occurs in two sequential processes: nitrification and
denitrification. An overview of each process is provided below.
Nitrification
Nitrification is an aerobic process in which autotrophic bacteria oxidize ammonia or nitrite for energy
production. Nitrification is normally a two-step aerobic biological process for the oxidation of
ammonia to nitrate. Ammonia-nitrogen (NH3-N) is first converted to nitrite (NO2 -) by ammonia
oxidizing bacteria (AOB). The nitrite produced is then converted to nitrate (NO3-) by nitrite oxidizing
bacteria (NOB). Both reactions usually occur in the same process unit at a wastewater treatment
plant (e.g. activated sludge mixed liquor or fixed film biofilm).
The group of AOB most associated with nitrification is the Nitrosomonas genus, although other
AOB such as Nitrosococcus and Nitrosospira can contribute to the process. Nitrobacter are the
NOB most associated with the second step, although other bacteria including Nitrospina,
Nitrococcus, and Nitrospira have been found to also oxidize nitrite (Tchobanoglous et al., 2003;
USEPA, 2007c).

AOB and NOB are classified as autotrophic bacteria because they derive energy from the oxidation
of reduced inorganic compounds (in this case, nitrogenous compounds) and use inorganic carbon
(CO2) as a food source. Nitrifying bacteria require a significant amount of oxygen to complete the
reactions, produce a small amount of biomass, and cause destruction of alkalinity through the
consumption of carbon dioxide and production of hydrogen ions. For each gram (g) of NH3-N
converted to nitrate, 4.57 g of oxygen are used, 0.16 g of new cells are formed, 7.14 g of alkalinity
are removed, and 0.08 g of inorganic carbon are utilized in formation of new cells (Tchobanoglous
et al., 2003).
Nitrifying bacteria grow slower and have much lower yields as a function of substrate consumed,
compared to the heterotrophic bacteria in biological treatment processes. The maximum specific
growth rate of the nitrifying bacteria is 10 to 20 times less than the maximum specific growth rate
of heterotrophic bacteria responsible for oxidation of carbonaceous organic compounds in
wastewater treatment. Thus, the nitrification process needs a significantly higher SRT to work
compared to conventional activated sludge processes. The SRT needed for nitrification in an
activated sludge process is a function of the maximum specific growth rate (which is related to
temperature), the reactor dissolved oxygen concentration, and pH. Nitrification rates decline as the
DO concentration decreases below 3.0 mg/L and the pH decreases below 7.0 mg/L. With sufficient
DO and adequate pH, typical nitrification design SRTs range from 10 to 20 days at 10oC and 4 to
7 days at 20oC (Randall et al.,1992).
Denitrification
In municipal and industrial wastewater treatment processes, denitrification is the biological
reduction of nitrate or nitrite to nitrogen gas (N2) as indicated by equation below.
NO → NO → NO → N O → N (5-1)
It is accomplished by a variety of common heterotrophic microorganisms that are normally present

in aerobic biological processes. Most are facultative aerobic bacteria with the ability to use
elemental oxygen, nitrate, or nitrite as their terminal electron acceptors for the oxidation of organic
material.
Heterotrophic bacteria capable of denitrification include the following genera: Achromobacter,

Acinetobacter, Agrobacterium, Alcaligenes, Arthrobacter, Bacillus, Chromobacterium,
Corynebacterium, Flavobacterium, Hypomicrobium, Moraxella, Nesseria, Paracoccus,
Propionibacteria, Pseudomonas, Rhizobium, Rhodopseudmonas, Spirillum and Vibrio
(Tchobanoglous et al., 2003).
Recent research has shown that nitrite reduction is accomplished by a much more specialized
group of heterotrophic bacteria than those performing the conversion of nitrate to nitrite (Katehis,
2007).
Denitrification by heterotrophic nitrifying bacteria and by autotrophic bacteria has also been
observed. An example of a heterotrophic nitrifying bacteria that can denitrify is Parococcus
pantotropha, which obtains energy by nitrate or nitrite reduction while oxidizing ammonia under
aerobic conditions. A readily available carbon source, such as acetate, is needed (Robertson and
Kuenen, 1990). The conditions required for this form of denitrification are not practical in biological
wastewater treatment.
An autotrophic denitrifying bacteria of practical significance in wastewater treatment is that in the

Anammox process used to remove nitrogen in return streams from anaerobic digestion sludge
dewatering filtrate or centrate. These bacteria have been identified as a member of bacteria in the
order Planctomycetales (Strous et al, 1999). Under anaerobic conditions, ammonia is oxidized with
the reduction of nitrite with the final product as nitrogen gas. The reaction is best accomplished at
temperatures above 25°C and they are slow growing organisms.

Facultative denitrifying bacteria will preferentially use oxygen instead of nitrate. In the absence of
oxygen, however, they will carry out nitrite and/or nitrate reduction. Microbiologists generally use
the term anaerobic to describe biological reactions in the absence of oxygen. To distinguish
anaerobic conditions for which the biological activity occurs mainly with nitrate or nitrite as the
electron acceptor, the term “anoxic” has been applied.
Although oxygen is known to inhibit denitrification, denitrification has been observed in activated
sludge and fixed film systems in which the bulk liquid DO concentration is positive. This is due to
the establishment of an anoxic zone within the floc or biofilm depth. Hence, a single system can
carry out simultaneous nitrification and denitrification. The DO concentration that is possible for
simultaneous nitrification and denitrification depends on a number of factors including the mixed
liquor concentration, temperature, and substrate loading. The DO concentration above which
denitrification is inhibited may vary from 0.10 to 0.50 mg/L (WEF and ASCE, 2006; Tchobanoglous
et al., 2003; Barker and Dold, 1997).
The organic carbon source for denitrifying bacteria can be in the form of:
• Soluble degradable organics in the influent wastewater
• Soluble organic material produced by hydrolysis of influent particulate material
• Organic matter released during biomass endogenous decay
A general rule of thumb is that 4 g of wastewater influent BOD is needed per g of NO3-N to be
removed through biological treatment (Tchobanoglous et al., 2003). When denitrification occurs
after secondary treatment, there is little BOD remaining so a supplemental carbon source is often
needed.
The most common exogenous carbon source in use is methanol; however, due to issues regarding
its safety, cost, and availability, some wastewater systems are using alternative carbon sources
such as acetic acid, ethanol, sugar, glycerol, and proprietary solutions depending on the needs of
their particular facility (deBarbadillo et al., 2008).
Biological denitrification reactions produce alkalinity and heterotrophic biomass. Based on the
stoichiometry of the reactions, denitrification will produce a 3.57 mg/L of alkalinity as CaCO3 for
each mg/L of NO3ˉ−N consumed. Heterotrophic biomass produced can be estimated as 0.4 g
volatile suspended solids (VSS) produced for every gram of COD consumed. Growth kinetics for
denitrifiers are dependent on a number of factors including carbon substrate type and
concentration, DO concentration, alkalinity, pH, and temperature, with carbon source being the
most important.
Current Configurations
Biological nitrogen removal can be accomplished by a variety of treatment configurations using
suspended growth, attached growth, or combined systems. In the past, some WWTPs were
required to only remove ammonia-nitrogen in wastewater to reduce toxicity to aquatic organisms
with no limits on nitrate or total nitrogen.
However, most treatment plants are now required to remove nitrogen because both ammonia-
nitrogen and nitrate-nitrogen can stimulate algae and phytoplankton growth and lead to
eutrophication of U.S. waterways. For biological nitrogen removal, it is essential that nitrification
occur first followed by denitrification.
Biological Nitrogen Removal Process Configurations

Biological nitrogen removal systems achieve nitrification and denitrification along with BOD
reduction in bioreactors followed by secondary clarification. Processes can be either suspended
growth or hybrid systems that use a combination of attached growth (biofilms) and suspended
growth technologies. Configurations within each of these classifications are discussed below. Note
that biological processes that removal both nitrogen and phosphorus are discussed later in this
manual.

Suspended Growth Systems Modified Ludzck Ettinger (MLE) process
The most common nitrogen removal process used at WWTPs is the Modified Ludzck Ettinger
(MLE) process, which is considered a pre-denitrification, single sludge system. The process
includes an initial anoxic zone, followed by an aerobic zone. In the anoxic zone, nitrate produced
in the aerobic zone is reduced to nitrogen gas. This process uses some of the BOD in the incoming
waste. Nitrification occurs in the aerobic zone along with the removal of most of the remaining BOD.
At the end of the aerobic zone, pumps recycle the nitrate-rich mixed liquor to the anoxic zone for
denitrification.
Total nitrogen removal for the MLE process is typically 80 percent, and the process achieves total
effluent nitrogen concentrations ranging from approximately 5 to 8 mg/L with internal nitrate recycle
ratios of 2 to 4 based on the influent flowrate (2-4Q).
Four-Stage Bardenpho Process

The four-stage Bardenpho process builds on the MLE process, with the first two stages being
identical to the MLE system (anoxic zone followed by an aeration zone with a nitrate-rich recycle
from the aeration to the anoxic zone). The third stage is a secondary anoxic zone to provide
denitrification to the portion of the flow that is not recycled to the primary anoxic zone. Methanol or
another carbon source can be added to this zone to enhance denitrification. The fourth and final
zone is a re-aeration zone that serves to strip any nitrogen gas and increase the DO concentration
before clarification. Some configurations have used an oxidation ditch instead of the first two
stages. This process can achieve effluent TN levels of 3 to 5 mg/L.
Sequencing Batch Reactors

Sequencing batch reactors (SBRs) are fill and draw batch systems in which all treatment steps are
performed in sequence for a discreet volume of water in a single or set of reactor basins.
SBRs use four basic phases for most systems:

Fill: water is added to the basin and is aerated and mixed
React: Biological processes are performed
Settle: All aeration and mixing is turned off and the biomass is allowed to settle
Decant: Clarified effluent is removed and biomass is wasted as necessary
The SBR control system allows it to mimic most other suspended growth processes such as the
MLE or Four-Stage Bardenpho system. It typically completes 4 to 6 cycles per day per tank for
domestic wastewater. If properly designed and operated, SBRs can achieve about 90 percent
removal of nitrogen (WEF and ASCE, 2006).
Oxidation Ditches
Oxidation ditches are looped channels that provide continuous circulation of wastewater and
biomass. A number of operating methods and designs have been developed to achieve nitrogen
removal, all of which work by cycling the flow within the ditch between aerobic and anoxic
conditions. DO can be added to the aerated zone using horizontal brush aerators, diffused aerators
with submersible mixers, or vertical shaft aerators (WEF and ASCE, 2006). Patented designs
include the NITROX process, Carrousel, and BioDenitro (WERF, 2000a). Many oxidation ditch
configurations can achieve simultaneous nitrogen and phosphorus removal.
Step Feed
The step feed biological nitrogen removal process splits the influent flow and directs a portion of it
to each of several anoxic zones, with the highest proportion of influent flow going to the first zone
and steadily decreasing until the last anoxic zone prior to clarification. The biomass in the later
stages are not just treating influent flow but are also used to reduce nitrate from the upstream
zones.

The step feed system provides flexibility for systems to handle wet-weather events. It can also be
compatible with existing conventional “plug flow” activated sludge processes and it does not require
the installation of recycle pumps and piping.
Disadvantages include the need to control the DO concentration of aeration zones preceding the
downstream anoxic zones and the need to control the flow splitting to the step feed points.
Attached Growth and Hybrid Systems Integrated Fixed-Film Activated Sludge (IFAS)
Integrated fixed-film activated sludge (IFAS) is any suspended growth system (e.g., MLE, Four-
Stage Bardenpho) that incorporates an attached growth media within the suspended growth reactor
in order to increase the amount of biomass in the basin.
IFAS systems have higher treatment rates than suspended growth systems and generate sludge
with better settling characteristics. Many types of fixed and floating media are available, including:
• Rope: also called looped-cord or strand media. Consists of a polyvinyl chloride-based material
woven into rope with loops along the length to provide surface area for the biomass (WERF, 2000b).
Proprietary designs include Ringlace, Bioweb, and Biomatrix (USEPA, 2008a).
• Moving Bed with Sponge: proprietary products include Captor and Linpor (USEPA, 2008a).
• Plastic Media: several types of free-floating plastic media are available from Kaldness. Other
media types include fabric mesh (e.g., AccuWeb) and textile material (Cleartec).
Moving-Bed Biofilm Reactor (MBBR)

The moving-bed biofilm reactor (MBBR) is similar to the IFAS system in that it uses plastic media
with a large surface area to increase biomass within the biological reactor. The MBBR media is
submerged in a completely mixed anoxic or aerobic zone. The plastic media are typically shaped
like small cylinders to maximize surface area for biomass growth. The difference between MBBR
and IFAS is that MBBR does not incorporate return sludge (WERF, 2000b).
Membrane Bioreactor (MBR)

MBRs are commonly designed for nitrogen removal, using membranes for liquid-solids separation
following the anoxic and aerobic zones instead of conventional clarification. Membranes can be
submersed in the biological reactor or located in a separate stage or compartment. Low-pressure
membranes (ultrafiltration or microfiltration) are commonly used. Systems can be pressure driven
or vacuum. All systems use an air scour technique to reduce buildup on the membranes (USEPA,
2007d; USEPA, 2008a).
Membrane materials are either organic polymers or inorganic materials such as ceramics. They
are designed in modular units and are typically configured as either hollow fiber bundles or plate
membranes (USEPA, 2007d) For biological nutrient removal applications, the design SRTs and
design principals for MBR systems are similar to those used for systems with secondary clarifiers.
One of the main differences is that the MBR systems operate at a higher MLSS concentration which
results in smaller tanks and smaller space requirements. In addition, membrane separation
provides for greatly reduced TSS in the effluent, typically below 1.0 mg/L, and hence slightly greater
removal of nitrogen and phosphorus. Operational issues include potential for membrane biofouling
and increase pumping costs (USEPA, 2007d; WEF,2005).

Nutrient Removal for Small Communities and Decentralized
Wastewater Treatment Systems
Approximately 25 percent of the U.S. population is served by onsite septic or decentralized
systems. Onsite septic systems treat and dispose of effluent on the same property that produces
the wastewater, whereas decentralized treatment refers to onsite or cluster systems that are used
to treat and dispose of relatively small volumes of wastewater, generally from dwellings and
businesses that are located relatively close together. In many cases, wastewater from several
homes is pretreated onsite by individual septic tanks before being transported through alternative
sewers to an offsite decentralized treatment unit that is relatively simple to operate and maintain.
The remaining 75 percent of the population is served by centralized wastewater treatment facilities,
which collect and treat large volumes of wastewater. There is, in fact, a growing movement toward
decentralized or clustered wastewater treatment systems to reduce cost, to provide groundwater
recharge near the source, and for speed and ease in siting since they are generally located
underground. The use of residential cluster development is gaining in popularity across the U.S. as
a means to permanently protect open space, preserve agricultural land, and protect wildlife habitat
(Mega et al., 1998).
As part of these developments, wastewater systems such as community drainfields, irrigation

systems, and package plants are being installed to reduce infrastructure investment and minimize
adverse environmental impacts. Additional alternatives that include aerobic tanks, sand filters, and
constructed wetlands can be used to reduce nutrient pollution; particularly in sensitive coastal areas
or over sensitive, unconfined aquifers used for drinking water (Anderson and Gustafson, 1998).
Phosphorus Removal
Few phosphorus removal processes are well developed for onsite wastewater systems application
(USEPA, 2008e). The controlled addition of chemicals such as aluminum, iron, and calcium
compounds with subsequent flocculation and sedimentation has had only limited success because
of inadequate operation and maintenance of mechanical equipment and excessive sludge
production. Most notable successes have come with special filter materials that are naturally high
in their concentration of the above chemicals, but their service lives are finite. Studies of high-iron
sands and high-aluminum muds indicate that 50 to 95 percent of the phosphorus can be removed.
However, the life of these systems has yet to be determined, after which the filter media will have
to be removed and replaced. Use of supplemental iron powder mixed with natural sands is also
being researched. Aside from specialized filter media, the most likely phosphorus-reduction
systems are iron-rich intermittent sand filter (ISF) media and SBRs. These are discussed in more
detail below.
Nitrogen Removal
Processes that remove 25 to 50 percent of total nitrogen include aerobic biological systems and
media filters, especially recirculating filters (USEPA, 2008f). The vast majority of on-site and cluster
nitrogen-removal systems employ nitrification and denitrification biological reactions. Most notable
of these are recirculating sand filters (RSFs) with enhanced anoxic modifications, SBRs, and an
array of aerobic nitrification processes combined with an anoxic/anaerobic process to perform
denitrification. Some of the combinations are proprietary.
A few recently developed highly instrumented systems that utilize membrane solids separation
following biological nitrification and denitrification are capable of removing total nitrogen down to
very low concentrations (i.e. 3 – 4 mg/L TN). Nitrogen removal systems generally are located last
in the treatment train prior to subsurface wastewater infiltration system (SWIS) disposal or surface
water disposal, in which case a disinfection step is typically required. Usually, the minimum total
nitrogen standard that can be regularly met is about 10 mg/L. These technologies can be either
above ground or below ground.

Water Quality Trading
Water quality trading is a market-based approach to improve and preserve water quality. Trading
can provide greater efficiency in achieving water quality goals by allowing one source to meet its
regulatory obligations by using pollutant reductions created by another source that has lower
pollution control costs. For example, under a water quality trading program, a POTW could comply
with discharge requirements by paying distributed sources to reduce their discharges by a certain
amount. The use of geographically-based trading ratios provides an economic incentive,
encouraging action toward the most cost effective and environmentally beneficial projects.
EPA issued a Water Quality Trading Policy in 2003 to provide guidance to States and Tribes on
how trading can occur under the CWA and its implementing regulations. The policy discusses CWA
requirements that are relevant to water quality trading including: requirements to obtain permits,
antibacksliding provisions, development of WQSs including an antidegradation policy, NPDES
permit regulations, TMDLs and water quality management plans. EPA also developed a number of
tools and guidance documents to assist states, permitted facilities, non-point sources, and
stakeholders involved in the development of trading programs. Recently, the U.S. Department of
Agriculture (USDA) National Resources Conservation Service released a Nitrogen Trading Tool
(NTT) prototype for calculating nitrogen credits based on the Nitrogen Loss and Environmental
Assessment Package Model (Gross et al., 2008).
Water quality trading programs have been successfully implemented in several states and
individual watersheds across the county. For example, nitrogen pollution from point sources into
the Long Island Sound was reduced by nearly 25 percent using an innovative Nitrogen Credit
Trading Program. In Connecticut, the program was implemented among 79 sewage treatment
plants in the state. Through the Nitrogen Credit Exchange, established in 2002, the Connecticut
program has a goal of reducing nitrogen discharges by 58.5 percent by 2014. A recent American
Society of Civil Engineers journal article points out, however, that regulatory frameworks for water
quality trading programs have yet to be adopted by the majority of States. Barriers to adopting such
programs include uncertainty in: (1) the mechanisms for determining appropriate credits and ratios
between point sources and distributed sources; and (2) approaches to ensure that promised
reductions actually occur (Landers, 2008).
Aeration is often used to refresh the wastewater flow at the influent channel.

Wastewater Sampling Information Section
Required Containers, Preservation Techniques, and Holding Times
40 CFR 136.3
Parameter No./name Container Preservation Maximum holding time
Table IA--Bacteria Tests:

1-4 Coliform, fecal and total. P,G.............. Cool, 4C, 0.008% Na2S2O3...... 6 hours.
5 Fecal streptococci.......... P,G.............. Cool, 4C, 0.008% Na2S2O3 ...... 6 hours.
Table IA--Aquatic Toxicity
Tests:
6-10 Toxicity, acute and P,G............ Cool, 4C ............... 36 hours.
chronic.
Table IB--Inorganic Tests:

1. Acidity.................... P, G............. Cool, 4C................... 14 days.
2. Alkalinity................. P, G............. Cool, 4C................... 14 days.
4. Ammonia.................... P, G............. Cool, 4C, H2SO4 to pH< 2..... 28 days.
9. Biochemical oxygen demand.. P, G............. Cool, 4C................... 48 hours.
10. Boron..................... P, PFTE, or HNO3 TO pH2..................... 6 months. Quartz.
11. Bromide................... P, G............. None required................... 28 days.

14. Biochemical oxygen demand, P, G............. Cool, 4C............ 48 hours. carbonaceous.
15. Chemical oxygen demand.... P, G............. Cool, 4C, H2SO4 to pH<2..... 28 days.
16. Chloride.................. P, G............. None required................... 28 days.
17. Chlorine, total residual.. P, G............. None required ........................ Analyze immediately.
21. Color..................... P, G............. Cool, 4C................... 48 hours.
23-24. Cyanide, total and P, G............. Cool, 4 deg.C, NaOH to pH>12, 14 days.
amenable to chlorination. 0.6g Ascorbic acid
25. Fluoride.................. P................ None required................... 28 days.
27. Hardness.................. P, G............. HNO3 to pH<2, H2SO4 to pH <2....... 6 months.
28. Hydrogen ion (pH)......... P, G............. None required................... Analyze immediately.
31, 43. Kjeldahl and organic P, G............. Cool, 4 deg.C, H2SO4 to pH <2..... 28 days.
nitrogen.
Metals:
18. Chromium VI............... P, G............. Cool, 4C................... 24 hours.
35. Mercury................... P, G............. HNO3 to pH<2.................. 28 days.
3, 5-8, 12, 13, 19, 20, 22, P, G............. ......do........................ 6 months.
26, 29, 30, 32-34, 36, 37, 45, 47, 51, 52, 58-60, 62,63, 70-72, 74, 75. Metals, except boron,
chromium VI and mercury.
38. Nitrate................... P, G............. Cool, 4 deg.C................... 48 hours.

39. Nitrate-nitrite........... P, G............. Cool, 4 deg.C, H2SO4 to pH <2..... 28 days.
40. Nitrite................... P, G............. Cool, 4 deg.C................... 48 hours.
41. Oil and grease............ G................ Cool to 4 deg.C, HCl or H2SO4 to pH <2 to 28 days.
42. Organic Carbon............ P, G............. Cool to 4 deg.C HC1 or H2SO4 to pH <2 or 28 days.
44. Orthophosphate............ P, G............. Filter immediately, Cool, 4 deg.C. ............. 48 hours.
46. Oxygen, Dissolved Probe... G Bottle and top. None required......... Analyze immediately.
47. Winkler................... ........ G Bottle and top. Fix on site and store in dark... 8 hours.
48. Phenols................... G only........... Cool, 4 deg.C,..... H2SO4 to pH <2 28 days.
49. Phosphorus (elemental).... G................ Cool, 4 deg.C................... 48 hours.
50. Phosphorus, total......... P, G............. Cool, 4 deg.C, H2SO4 to pH <2…………..28 days.
53. Residue, total............ P, G............. Cool, 4 deg.C................... 7 days.
54. Residue, Filterable....... P, G............. Cool, 4 deg.C.................... 7 days.
55. Residue, Nonfilterable P, G............. Cool, 4 deg.C........................ 7 days.

56. Residue, Settleable (TSS)........ P, G............. Cool, 4 deg. C........................ 48 hours.
57. Residue, volatile......... P, G............. Cool, 4 deg. C....................... 7 days.
61. Silica.................... P, PFTE, or Quartz…….. Cool, 4 deg. C.................. 28 days.
64. Specific conductance...... P, G.......... . Cool, 4 deg. C....................... 28 days.
65. Sulfate................... P, G............. ...... Cool, 4 deg. C .................... 28 days.
66. Sulfide.... P, G...... Cool, 4 deg. C add zinc acetate plus sodium hydroxide to pH>9. 7 days.
67. Sulfite................... P, G............. None required................... Analyze immediately.
68. Surfactants............... P ,G............. Cool, 4 deg. C................... 48 hours.
69. Temperature............... P, G............. None required................... Analyze.
73. Turbidity................. P, G............. Cool, 4 deg. C................... 48 hours.
Table IC--Organic Tests

13, 18-20, 22, 24-28, 34-37, G, Teflon-lined septum Cool, 4 deg. C, 0.008% NA2S2O3
14 days.
39-43, 45-47, 56, 76, 104, 105, 108-111, 113.
Purgeable Halocarbons. 6, 57, 106.
Purgeable aromatic hydrocarbons G, Teflon-lined septum Cool, 4 deg.C, 0.008% NA2S2O3
14 days.
3, 4. Acrolein and acrylonitrile G, Teflon-lined septum Cool, 4 deg.C, 0.008% NA2S2O3 pH 4-5
14 days.
23, 30, 44, 49, 53, 77, 80, 81, 98, 100, 112. G, Teflon-lined Cool, 4 deg.C, 0.008% NA2S2O3
14 days.
Phenols G, Teflon-lined septum Cool, 4 deg.C, 0.008% NA2S2O3 pH 4-5 7 days until extraction;
40 days after extraction.
7, 38. Benzidines G, Teflon-lined septum………… Cool, 4 deg.C, 0.008% NA2S2O3 7 days
until extraction.
14, 17, 48, 50-52. Phthalate G, Teflon-lined septum Cool, 4 deg.C.................. 7 days until
extraction; esters 40 days after extraction.
82-84. Nitrosamines G, Teflon-lined septum ……Cool, 4 deg. C, 0.008% NA2S2O3…Store in dark
88-94. PCBs G, Teflon-lined septum Cool, 4 deg.C ............. 7 days until extraction; 40 days
after extraction.
54, 55, 75, 79. Nitroaromatics ...... G, Teflon-lined septum………….Cool, 4 deg.C, 0.008%
NA2S2O3 and isophorone
1, 2, 5, 8-12, 32, 33, 58, 59, 74, 78, 99, 101. Polynuclear aromatic hydrocarbons. Cool, 4
deg.C, 0.008% NA2S2O3……Store in dark
15, 16, 21, 31, 87. Haloethers ........ G, Teflon-lined septum....... Cool, 4 deg.C, 0.008%
NA2S2O3 7 days until extraction; 40 days after extraction.
29, 35-37, 63-65, 73, 107. Chlorinated hydrocarbons G, Teflon-lined septum………….Cool, 4
deg.C, 7 days until extraction; 40 days after extraction.
60-62, 66-72, 85, 86, 95-97, 102, 103. CDDs/CDFs aqueous: field and lab G........ Cool, 0-
4 deg.C, pH9, 0.008% NA2S2O3 1 year preservation.
Solids, mixed phase, and ......do......... Cool, 4 deg.C.................. 7 days. Tissue: field
preservation.
Solids, mixed phase, and ......do......... Freeze, -10 deg.C.............. 1 year. Tissue: lab
preservation.
Table ID--Pesticides Tests:

1-70. Pesticides \11\......... ......do......... Cool, 4 deg.C, pH 5-9 ........ Do.
Table IE--Radiological Tests:
1-5. Alpha, beta and radium... P, G............. HNO3 to pH2..................... 6 months.

Polyethylene (P) or glass (G). For microbiology, plastic sample containers must be made of
sterilizable materials (polypropylene or other autoclavable plastic).
Sample preservation should be performed immediately upon sample collection. For composite
chemical samples each aliquot should be preserved at the time of collection. When use of an
automated sampler makes it impossible to preserve each aliquot, then chemical samples may be
preserved by maintaining at 4 degrees C until compositing and sample splitting is completed.
When any sample is to be shipped by common carrier or sent through the United States Mails, it
must comply with the Department of Transportation Hazardous Materials Regulations (49 CFR part
172). The person offering such material for transportation is responsible for ensuring such
compliance. For the preservation requirements of Table II, the Office of Hazardous Materials,
Materials Transportation Bureau, Department of Transportation has determined that the
Hazardous Materials Regulations do not apply to the following materials: Hydrochloric acid (HCl)
in water solutions at concentrations of 0.04% by weight or less (pH about 1.96 or greater); Nitric
acid (HNO3in water solutions at concentrations of 0.15% by weight or less (pH about 1.62 or
greater); Sulfuric acid (H2SO4) in water solutions at concentrations of 0.35% by weight or less (pH
about 1.15 or greater); and Sodium hydroxide (NaOH) in water solutions at concentrations of
0.080% by weight or less (pH about 12.30 or less).
Samples should be analyzed as soon as possible after collection. The times listed are the maximum
times that samples may be held before analysis and still be considered valid. Samples may be held
for longer periods only if the permittee, or monitoring laboratory, has data on file to show that for
the specific types of samples under study, the analytes are stable for the longer time, and has
received a variance from the Regional Administrator under Sec. 136.3(e). Some samples may not
be stable for the maximum time period given in the table. A permittee, or monitoring laboratory, is
obligated to hold the sample for a shorter time if knowledge exists to show that this is necessary to
maintain sample stability. See Sec. 136.3(e) for details. The term ``analyze immediately'' usually
means within 15 minutes or less of sample collection. Should only be used in the presence of
residual chlorine.
Maximum holding time is 24 hours when sulfide is present. Optionally all samples may be tested
with lead acetate paper before pH adjustments in order to determine if sulfide is present. If sulfide
is present, it can be removed by the addition of cadmium nitrate powder until a negative spot test
is obtained. The sample is filtered and then NaOH is added to pH 12.
Samples should be filtered immediately on-site before adding preservative for dissolved metals.
Guidance applies to samples to be analyzed by GC, LC, or GC/MS for specific compounds.
Sample receiving no pH adjustment must be analyzed within seven days of sampling.
The pH adjustment is not required if acrolein will not be measured. Samples for acrolein receiving
no pH adjustment must be analyzed within 3 days of sampling.
When the extractable analytes of concern fall within a single chemical category, the specified
preservative and maximum holding times should be observed for optimum safeguard of sample
integrity. When the analytes of concern fall within two or more chemical categories, the sample
may be preserved by cooling to 4 deg. C, reducing residual chlorine with 0.008% sodium
thiosulfate, storing in the dark, and adjusting the pH to 6-9; Samples preserved in this manner may
be held for seven days before extraction and for forty days after extraction. Exceptions to this
optional preservation and holding time procedure are noted in footnote 5 (re the requirement for
thiosulfate reduction of residual chlorine), and footnotes 12, 13 (re the analysis of benzidine).
If 1,2-diphenylhydrazine is likely to be present, adjust the pH of the sample to 4.0<plus-minus>0.2

to prevent rearrangement to benzidine.

Extracts may be stored up to 7 days before analysis if storage is conducted under an inert (oxidant-
free) atmosphere.
For the analysis of diphenylnitrosamine, add 0.008% NA2S2O3 and adjust pH to 7-10 with NaOH
within 24 hours of sampling.
The pH adjustment may be performed upon receipt at the laboratory and may be omitted if the
samples are extracted within 72 hours of collection. For the analysis of aldrin, add 0.008% NA2S2O3.
Sufficient ice should be placed with the samples in the shipping container to ensure that ice is still
present when the samples arrive at the laboratory. However, even if ice is present when the
samples arrive, it is necessary to immediately measure the temperature of the samples and confirm
that the 4OC temperature maximum has not been exceeded.
In the isolated cases where it can be documented that this holding temperature cannot be met, the
permittee can be given the option of on-site testing or can request a variance. The request for a
variance should include supportive data which show that the toxicity of the effluent samples is not
reduced because of the increased holding temperature.
Common Wastewater Sample Collection Bottles
Above, 625/608, 1657, TTO/Organics, TPH/Oil/Grease

Smaller bottles-TOCs, VOCs, 601/602 and 502.2.

Wastewater/Pretreatment Sampling General Information
In accordance with the Clean Water Act and General Pretreatment Program Regulations, the
POTW conducts a variety of sampling activities which must be closely coordinated.
Each of these activities is briefly described below.
Permit Application Policy - Example

All industrial users that require a permit must be sampled to determine the characteristics of
the wastes to be discharged into the POTW’s sewer system. Prior to the issuance of a permit
for existing industrial users, the POTW samples the user's effluent, and performs the analyses
required by the applicable discharge standards (i.e., Categorical standards or local limits).
For new industrial users,

estimates of the wastes to be
discharged into the POTW’s
sewer system must be
submitted along with the
permit application. No
sampling would be performed
at these new facilities, since
they do not presently
discharge wastes into the
sewer system. A four-day
sampling program is usually
conducted at each site to
collect both composite and
grab (for pollutants not
amenable to composite
sampling) samples as
needed.
Sewer System Evaluation Policy - Example

On a regular basis, selected locations in the sewer system are sampled to develop
background data for purposes of updating the local limits, and to screen areas for higher than
"background" pollutant levels. In addition, problem areas are sampled on an as needed basis
to determine potential sources of POTW Code violations that either occur on a frequent basis,
or are the result of a slug load to the sewer system. To monitor sewers for background
information, the sampling program would typically be conducted over a four-day period. In
instances where the intent is to determine sources of pollutants and/or slug loads, the length
of the program would vary.
Multi-City Users (Metering Stations) Policy - Example

All wastewater, which is transported to the POTW Treatment Plant from the Multi-City or other
domestic users, is analyzed for pollutants of concern to the Industrial Pretreatment Program.
The sampling program is conducted over a five-day period to obtain four days of sampling
data at each sewer location (i.e., a metering station) on a quarterly basis. Once the sampling
dates have been determined, the Water Quality Inspector will notify, in writing, the Sub-
regional Organizational Group (SROG) or equivalent representative for that City of the dates
when the sampling will be conducted.

Sampling Safety Policy - Example
Upon arrival at the site, safety is the priority. A visual inspection must be completed prior to
any entry. The site must be free of any obstructions or hazards which may cause injury when
entering the sampling area. If there are any problems detected, the SROG or equivalent
representative and the Water Quality Inspector should be notified, and no entry should be
attempted until the problem has been corrected.
Metering and Sampling Stations Qualify As Confined Spaces

If all safety criteria have been met, prepare equipment for the site. Check the assignment
sheet to determine what parameters are required to be sampled, which in turn determines the
type of tubing to be used, (i.e. Tygon or Teflon).
The sampler must be completely assembled before performing QA/QC procedures. After
QA/QC is complete, a sufficient amount of weight must be attached to the tubing to keep the
strainer submerged in the effluent for proper siphoning of the sample, without allowing the
strainer to hit the bottom of the flume. Make sure the intake tubing does not kink.
If the metering station has a

flow meter, you may connect
either their cable or a POTW
cable to the sampler from the
flow meter. Occasionally, you
will set up a flow meter to have
a comparison reading.
Determine the pulse rate and

proper setting from the flow,
and program the sampler.
After entering the data into the
sampler, wait to make sure the
equipment is pulling samples.
After the initial set-up of the
sampling equipment, samples
will be collected during the remainder of the sampling period. Split samples may be
requested by the SROG or equivalent representative. If the volume of the sample is
adequate, these may be given, provided the representative supplies the containers and
allows the POTW Inspector to pour off the samples.
Upon exiting the confined space, continue to follow the confined space entry procedures as
outlined by OSHA Standards. When you return to the sampling vehicle, you must immediately
perform field tests and preserve the samples according to the techniques set forth by Standard
Methods or the State/Federal Rule.
All paper work must be filled out completely before the sampling crew's departure. This
paperwork includes the chain of custody which is turned in to the laboratory with the samples,
"Metering Station Field Observation Form" or equivalent form that remains with the sampling
site file, and the Multi-City Metering Station Sample Record of which the original is given to
the Water Quality Inspector or representative and the copy is given to the SROG or equivalent
representative. If there is not a representative at the site, these copies will be turned over to
the Water Quality Inspector with the originals at the end of the week.
Remember, all paperwork should be completed prior to leaving site or

sampling location.

Wastewater and Pretreatment Compliance Monitoring
There are two types of sampling activities that are performed as part of compliance monitoring
for permitted industries: unscheduled and demand.
Unscheduled sampling is used to determine the compliance status of the user. Instances
of noncompliance are often identified during unannounced monitoring visits. No notice is
given for this type of sampling. This type of sampling is performed two to four times a year,
at each industrial user site, over a two to five-day period to obtain sampling data
Demand sampling is usually initiated in

response to a known or suspected violation,
discovered as a result of a self-monitoring
report, routine sampling visit, public complaint,
unusual influent condition at the wastewater
treatment plant, or emergency situations (e.g.,
plant upsets, sewer line blockages, fires,
explosions, etc.).
Most often, this type of sampling is conducted

to support enforcement actions against an
industrial user. This type of sampling activity
is performed on an as needed basis. The
length of the sampling program depends on
the flow, nature of the wastes, and type of
samples (i.e., grab or composite) to be
collected. Typically, composite and grab
samples are collected at each user site.
Nonpermitted Industrial Users (User Rate Charge Program) Policy - Example

On a periodic basis (i.e., once every two to three years), commercial and minor industrial
users are sampled to determine discharge concentrations of various pollutants. Typical types
of users which may be sampled include: restaurants, photo processing laboratories, laundries,
car washes, and printing shops.
A three- to four-day sampling program is usually conducted at each assigned site.

Commercial establishments are sampled to establish BOD and SS levels for various groups
of users for the POTW’s Finance or Utilities Division.
This activity is also helpful in identifying industrial or commercial users which may discharge
pollutants of concern.
Wastewater Treatment Plant Sampling - Example

POTW samples are collected in accordance with the National Pollutant Discharge Elimination
System (NPDES) permit which sets discharge limits for certain pollutants and specifies
sampling frequencies and sample types.
The POTW is responsible for coordinating the plant sampling activity with laboratory personnel
who prepare any special sampling bottles and laboratory appurtenances necessary (i.e. trip
blanks, etc.) to complete the sampling objectives.

Pre-Treatment Monitoring Locations Should:
 be appropriate for waste stream conditions;
 be representative of the discharge;
 have no bypass capabilities; and
 allow for unrestricted access at all times.
Control Authorities should measure flow to allow for collection of flow-proportioned composite
samples, which are required, unless flow-proportional sampling is not feasible. Flow-
proportional composite samples are preferred over time composite samples particularly where
the monitored discharge is intermittent or variable.
Desired analyses dictate the preparation protocols, equipment, and collection bottles to use
to avoid contamination of samples or loss of pollutants through improper collection. Sampling
for such pollutants as pH, cyanide, oil and grease, flashpoint, and volatile organic compounds
require manual collection of grab samples. Similar to composite samples, grab samples must
be representative of the monitored discharge and are to be collected from actively flowing
wastestreams. Fluctuations in flow or the nature of the discharge may require collection of
and hand-compositing of more than one grab sample to accurately access compliance.
To ensure defensibility of data, Control

Authorities should develop and implement
standard operating procedures and
policies detailing sample collection and
handling protocols in accordance with 40
CFR Part 136.
Adherence to proper sample collection

and handling protocols, 40 CFR Part 136
approved analytical methodologies, and
record keeping requirements [40 CFR
§403.12(o)(1)] can be verified through
review of field measurement records,
chain of custodies, and lab reports.
Field measurement records may require

information regarding sample location,
condition of and programmed settings for
sampling equipment, wastewater meter
readings, and information for such
parameters as pH and temperature which
require analysis in the field.
Chain of custody forms serve as a link between field personnel and the laboratory and
contain information regarding the sample matrix, type, and handling. Lab reports should
contain the minimum information specified in 40 CFR §403.12(o)(1)(ii-iv) as well as any
additional information necessary to demonstrate compliance with 40 CFR Part 136
requirements (e.g., analytical methodology, sample preparation date and time, time of
analysis).
Use of standardized forms which prompt recording of information necessary for demonstrating
compliance with applicable requirements, will aid in ensuring it can be used as admissible
evidence in enforcement proceedings or in judicial actions.

Wastewater Plant Sampling Procedure - Example
Set up two samplers or equivalent at the plant influent channel and two samplers at the plant
effluent channel. Two samplers are used to provide sufficient sample quantity and to minimize
sampler failure. All sampling equipment must be prepared and cleaned as established in your
POTW’s procedures. Teflon hose or equivalent is required. Sampling sites are specified in
each plants NPDES permit.
Collect the following composite samples at all sites.
(1) Metals Sample - (one 2-liter plastic bottle)
Preserve with 1:1 nitric acid to a pH < 2. Store sample on ice to four degrees
Centigrade.
(2) Cyanide Sample - one (2-liter plastic bottle)
Collect the cyanide sample as a composite in accordance with NPDES

permit. Check the sample for chlorine. If Cl2 is present, use ascorbic acid to
eliminate chlorine. Add NaOH to a pH > 12. Store samples on ice to four
degrees Centigrade.
(3) EPA Test Method 608 and 625i samples are informational samples only.
These results are used for local limits data.
608 and 625 samples are collected as composite samples. At the influent
channel: Collect one 1-liter amber glass bottle of each sample (608, 625).
Check samples for chlorine. At the effluent channel: Collect one 4-liter amber
glass bottle of each sample (608, 625). Check samples for chlorine. If Cl2 is
present in the samples, use sodium thiosulfate (Na2S203) to eliminate
chlorine. Store samples on ice to four degrees Centigrade.
(4) 625/Phenols are collected as a grab sample. Collect one 4-liter amber glass
bottle at the effluent channel only. Check the sample for chlorine. If Cl2 is
present, use sodium thiosulfate (Na2S203) to eliminate chlorine. Store sample
on ice to four degrees Centigrade.
Bio-Solids Sampling - Example

Bio-solids (dried sludge) samples are
collected at POTWs. Normally, bio-solid
samples will be collected from the final
storage area for dry sludge. The location of
the dried bio-solids may vary based on the
individual plants. Sampling frequency will be
determined on an as needed basis and to
comply with the EPA requirements.
All samples collected are grabs. All samples

are collected using a sterile plastic scoop or
equivalent in order to avoid any
contamination.

The following is a list of samples that are normally collected:
PARAMETER CONTAINER
Helminth Ova & Enteric Virus 1 Qt Plastic Bag (Ziploc)
Metals + 500 ml Plastic Bottle
Nitrogen (total) 4 oz Glass Bottle
TOC (Total Organic Carbon) 4 oz Glass Bottle
Fecal Coliform (autoclaved from lab)
6 hr hold time 500 ml Plastic Bottle
Sample Scheduling - Example

An active file is maintained on each sampling location which contains historical data,
including past process discharge flow readings, water meter readings, sampling dates, and
conditions of sampling site.
Treated Wastewater Effluent River Sampling Activities - Example

When developing a sampling plan for river sampling, the following considerations must be
observed:
(1) Sampling sites must meet the objectives of the program or study.
(2) At the sampling sites the river must be flowing freely and the sample must be
as representative as possible of river flow at that site. Consideration of all
safety factors must be observed.
(3) Samples must be collected at midstream of the main channel at
approximately two-thirds of the depth unless specific depths have been
requested.
(4) All safety precautions must be observed during sampling which includes the
use of harnesses, waterproof boots and other equipment.
Samples from Sewers - Example

Sewer system and user rate sampling are conducted in manholes. General guidelines for
selection of sampling locations include the following:
(1) Samples should be taken
at points of high turbulent
flow to ensure good
mixing and prevent the
deposition of solids.
(2) The sample location
should be easily
accessible and free of any
major safety hazards.
(3) Sample lines should not
be located where there is
surface scum.
(4) If a flow study or a
flow/proportional sampling
event is required, make
sure that the sewer pipe
does not have a curve, a drop in the line or any obstructions. These would
cause false readings.

Co-Removal of Emerging Contaminants
This section provides a brief background on emerging contaminants and key findings from studies
on the co-removal of emerging contaminants by nutrient removal technologies.
Background on Emerging Contaminants

The term “emerging contaminants” refers broadly to those synthetic or naturally occurring
chemicals, or to any microbiological organisms, that have not been commonly monitored in the
environment but which are of increasing concern because of their known or suspected adverse
ecological or human health effects. Emerging contaminants can fall into a wide range of groups
defined by their effects, uses, or by their key chemical or microbiological characteristics. Two
groups of emerging contaminants that are of particular interest and concern at present are
endocrine disrupting chemicals (EDCs) and pharmaceutical and personal care products (PPCPs).
These compounds are found in the environment, often as a result of human activities.
EDCs may interfere with the endocrine systems by damaging hormone-producing tissues,
changing the processes by which hormones are made or metabolized, or mimicking hormones. In
addition to natural and synthetic forms of human hormones that are released into the environment,
there are a multitude of synthetic organic compounds that are able to disrupt the endocrine system.
Public concern about EDCs in the environment has been rapidly increasing since the 1990s when
researchers reported unusual sexual characteristics in wildlife. A report by the USGS, found that
fish in many streams had atypical ratios of male and female sex hormones (Goodbred et al., 1997).
In England, researchers found that male trout kept in cages near WWTP outfalls were developing
eggs on their testes and had increased levels of the protein that is responsible for egg production
(vitellogenin) (Sumpter, 1995; Kaiser, 1996). Follow-up laboratory studies showed that synthetic
forms of estrogen (17α-ethynylestradiol (EE2)) could increase vitellogenin production in fish at
levels as low as 1-10 ng/L, with positive responses seen down to the 0.1-0.5 ng/L level (Purdom et
al., 1994).
Human estrogens have the ability to alter sexual characteristics of aquatic species at trace
concentrations as low as 1 ng/L (Purdom et al., 1994). WWTP effluents have been identified as a
primary source for EDCs in the environment, with the bulk of their endocrine disrupting activity
resulting from human estrogen compounds (Desbrow et al., 1998, Snyder et al., 2001). The
synthetic estrogen, EE2, and the natural estrogens, estrone (E1) and 17β-estradiol (E2), are the
greatest contributors to endocrine disrupting activity in WWTP effluent (Johnson et al., 2001) with
EE2 showing the greatest recalcitrance in WWTPs (Joss et al., 2004). Influent concentrations range
from below detection to 70 ng/L for EE2, 670 ng/L for E1 and 150 ng/L for E2 (Vethaak et al., 2005,
Clara et al., 2005b). Other EDCs include tributyl tin, which was previously used in paints to prevent
marine organisms from sticking to ships, nonylphenol (a surfactant), and bisphenol A (platicizer
and preservative).
PPCPs encompass a wide variety of products that are used by individuals for personal health or
cosmetic reasons, and also include certain agricultural and veterinary medicine products. PPCPs
comprise a diverse collection of thousands of chemical substances, including prescription and over-
the counter therapeutic drugs, veterinary drugs, fragrances, sun-screen products, vitamins, and
cosmetics. Many of these products, notably the pharmaceuticals for human or animal use, are
specifically designed to be biologically active, and some PPCPs may also fall into the category of
EDCs described previously.
Estrogens of Concern
Name Chemical Structure Name Chemical Structure
E1 Estrone C18H22O2
E2 17β-estradiol C18H24O2
E3 Estriol C18H24O3
EE2 17α-ethynylestradiol C20H24O2

Currently, municipal sewage treatment plants are engineered to remove conventional pollutants
such as solids and biodegradable organic material but are not specifically designed for PPCP
removal or for other unregulated contaminants. Wastewater treatment commonly consists of
primary settling followed by biological treatment, secondary settling, and disinfection. This
treatment can remove more than 90 percent of many of the most commonly known or suspected
EDCs found in wastewater influent; however, low concentrations of some suspected EDCs may
remain in the wastewater treatment sludge or effluent (WERF, 2005). As discussed in the next
section, studies have shown enhanced nutrient removal technologies to be effective in removing
low concentrations of some emerging contaminants.
Removal of Emerging Contaminants by Nutrient Removal Technologies

Several studies have examined the effectiveness of current wastewater treatment technologies in
the removal of emerging contaminants. Some of these studies are discussed below and their major
findings are organized under three subsections: role of activated sludge SRT in removal efficiency,
role of nitrifying bacteria in biodegradation, and use of RO to improve removal efficiencies. Details
regarding the study design, such as evaluated treatments and contaminants, and a summary of
major study findings are provided at the end of this section.
The significant findings are also presented as follows:

• Removal efficiencies were enhanced for several investigated contaminants at longer SRTs, with
critical SRTs for some beyond which removal rates did not improve.
• Longer SRTs allow for the establishment of slower growing bacteria (e.g., nitrifying bacteria in
activated sludge), which in turn provide a more diverse community of microorganisms with broader
physiological capabilities.
• Nitrifying bacteria may play a key role in biodegradation but the role of heterotrophic bacteria may
also play a significant role.
• Reverse osmosis has been found to effectively remove PPCPs below detection limits including
those that that were not consistently removed at longer SRTs.
One caveat regarding studies on emerging contaminants is that their concentrations in wastewater
influent are often quite low (e.g., concentrations of ng/L to μg/L range) and may be close to method
detection limits. Therefore, small variations between measured influent and effluent concentrations
may show large variations in apparent removal efficiencies, possibly even producing negative
calculated removals.
Role of Solids Retention Time in Removal Efficiency

The focus of several studies has been the relationship of the SRT to the removal of emerging
contaminants. In particular, many investigated whether longer SRTs would result in increased
removal efficiencies for estrogens and other categories of PPCPs. Longer activated sludge SRTs
allow for the establishment of slower growing bacteria (e.g., nitrifying bacteria in activated sludge),
which in turn provide a more diverse community of microorganisms with broader physiological
capabilities.
Clara et al. (2005a), Kreuzinger et al. (2004), and Oppenheimer et al. (2007) observed enhanced
removal with increasing SRTs for most of the EDCs and pharmaceuticals tested and found no
significant differences in removal performances between conventional activated sludge systems
and MBR when operated at similar SRT10 °C. This is likely due to the molecular weight of the study
compounds, which was smaller than the molecular weight cut-off of the ultrafiltration membranes
in the MBR.
Researchers have observed similar findings for natural estrogens with higher removal percentages
at longer SRTs. Effluent concentrations for three natural estrogens were measured near their
detection limits at SRTs10° C higher than 10 days, with their critical SRTs10° C estimated between
5 and 10 days (Clara et al., 2005a).

High removal rates of > 90 percent were also observed by Joss et al. (2004) in a study in which
they evaluated the removal of E1, E2, and EE2 under aerobic and anaerobic conditions in WWTPs
designed for nutrient removal. Joss et al. (2004) also reported that the maximum efficiency is
dependent on redox conditions, with the highest removal rate occurring during the reduction of E1
to E2 under aerobic conditions. Clara et al. (2005a) cited examples where conflicting results were
obtained for EE2.
Ternes et al. (1999) found no significant elimination of this compound during batch experiments;
however, Baronti et al. (2000) and Joss et al. (2004) report greater than 85 percent removal in full-
scale WWTPs. For the pharmaceuticals ibuprofen and bezafibrate, Clara et al. (2005a) reported
more than 95 percent removal during treatment and calculated the critical value for SRT10° C at 5
days for ibuprofen and about 10 days for bezafibrate. Analogous removal results were obtained in
several other studies (Stumpf et al., 1998; Buser et al., 1999; Zwiener et al., 2001, as cited in Clara
et al., 2005a; Oppenheimer et al., 2007). Clara et al. (2005b) noted no or slight removal of these
two pharmaceuticals and two musk fragrances (tonalide and galaxolide) at a WWTP with a low
SRT of 1 to 2 days.
Clara et al. (2005a, 2005b) also found that the pharmaceutical carbamazepine was not removed
during wastewater treatment. In addition, these studies found contradictory results for diclofenac
(e.g., removal rates ranged from no removal to > 70 percent at SRTs of > 10 days (Clara et al.,
2005b)). Clara et al. (2005a) also cited several examples where conflicting results were obtained
for diclofenac. No significant removal was reported by Buser et al. (1999) and Heberer (2002a);
whereas, Ternes et al. (1998) observed elimination rates of up to 70 percent.
Clara et al. (2005a, 2005b) concluded that the removal potential for conventional WWTPs and
MBRs depends on the SRT. They further concluded that high removal rates can be achieved at
SRTs10° C of more than 10 days. These parameters correspond to the design criteria for nitrogen
removal in the German Association for Water, Wastewater and Waste (ATV-DVWK, 2000) and the
urban wastewater directive of the European Community (91/271/EEC) for WWTPs in sensitive
areas. In its 2005, technical brief, “Endocrine Disrupting Compounds and Implications for
Wastewater Treatment,” WERF summarized information from several studies that examined the
effectiveness of current wastewater treatment technologies in the removal of EDCs.
The classes of EDCs included:

steroids/sterols (naturally occurring, synthetic, and phytoestrogens), organohalides, metals/
organometals, alkyl phenols, polycyclic aromatic hydrocarbons (PAHs)/crude oil, and plasticizers.
Although the WERF 2005 technical brief states that in general, EDC treatment effectiveness is
improved with increased SRT, it does not provide the specific SRTs that are associated with the
cited removal rates.
Oppenheimer et al. (2007) examined the relationship of SRT to treatment removal efficiencies for
20 PPCPs that are commonly found in the influent of U.S. treatment facilities. Many of the studies
already discussed here have been conducted primarily in Europe, were conducted at small-scale
WWTPs and bench/pilot plants under controlled conditions, and focused on estrogens and
prescription pharmaceuticals rather than PPCPs. The Oppenheimer et al. (2007) study also noted
trends regarding the effect of HRT and pure oxygen systems compared to conventional aeration
systems on PPCP removal.
Oppenheimer et al. (2007) defined a minimum critical SRT as the minimum time needed to
consistently demonstrate greater than 80 percent removal. The results of the study showed that
this critical SRT was compound dependent but that the majority of the 20 PPCPs were consistently
removed in those treatment plants operating at SRTs of 5 to 15 days. Specifically, 9 of 12 frequently
occurring PPCPs were effectively removed through secondary treatment (e.g., ibuprofen).

Conversely, six compounds that are routinely detected in influent (i.e., detected in at least 20
percent of the influent samples) were not well removed by secondary treatment (BHA, DEET, musk
ketone, triclosan, benzophenone, galaxolide).
The results for galaxolide conflicted with those reported by Clara et al. (2005b) who generally found
high removal rates with SRTs > 10 days and Kreuzinger et al. (2004) who reported removal at SRT
between 25 to 40 days. Oppenheimer et al. (2007) found that some compounds such as
octylphenol, tri-(chloroethyl) phosphate, and triphenylphosphate were not well removed by
secondary treatment; however, these were seldom detected in the influent samples. Based on
these results, Oppenheimer et al. (2007) concluded that secondary treatment provides an “effective
first barrier” for the 20 PPCPs in the study.
Oppenheimer et al. (2007) also noted trends regarding the effect of HRT and pure oxygen systems
compared to conventional aeration systems on PPCP removal but determined that insufficient data
existed to make any definitive conclusions. When the PPCP removal performance of a high-purity
oxygenated activated sludge plant was compared to a conventional aeration system, the pure
oxygen system showed higher removal rates although its SRT was shorter than the conventional
aeration plant (i.e., 1 day versus 3 days). In addition, different HRTs operating at similar SRTs had
similar removal rates, and therefore suggested that HRT does not significantly affect removal
effectiveness in the investigated PPCPs.
Role of Nitrifying Bacteria in Biodegradation

As discussed above, longer SRTs allow for the establishment of slow-growing nitrifying bacteria
(i.e., ammonia oxidizing bacteria and nitrite-oxidizing bacteria). Several studies evaluated whether
nitrifying bacteria improve the biodegradation of certain emerging contaminants. Major findings
from some of these studies are discussed in this section.
The WERF (2005) technical brief indicated that secondary biological treatment that includes
nitrification, nutrient removal, and disinfection may remove more than 90 percent of certain steroids,
and >95 percent of alkyl phenols; whereas, secondary biological treatment without nitrification and
disinfection may decrease removal of these by more than 15 percent. Batt et al. (2006) investigated
the role of nitrifying bacteria in activated sludge in the biodegradation of two pharmaceuticals,
iopromide and trimethoprim.
The biodegradation of these compounds was conducted in two lab-scale bioreactors using biomass
from a stage-2 activated sludge WWTP (operated at an SRT of 49 days). In one of the bioreactors,
nitrification was not inhibited (Batch-1 reactor); in the other, nitrification was inhibited with
allylthiourea (Batch-2 reactor). Monitoring was also conducted in the WWTP and compared to
results obtained from the batch reactors. Both reactors exhibited high removal rates for iopromide;
however for trimethoprim, Batch-1 showed a high removal rate of 70 percent, contrasted to the
Batch-2 reactor removal rate of approximately 25 percent with nitrification inhibited. Removal rates
within the treatment plant, however, were consistent for both pharmaceuticals, showing significantly
higher removal rate after nitrification (approx. 60 percent for iopromide and 50 percent for
trimethoprim) compared to activated sludge treatment only ( <1 percent for both).
Based on these results, Batt et al. (2006) concluded that nitrifying bacteria have a key role in the
biodegradation of pharmaceuticals in WWTP that are operated at higher SRTs. This conclusion is
supported by Marttinen et al. (2003), who investigated the fate of phthalates in a WWTP with
nitrogen removal and observed that about one third of the removal occurred in the
nitrification/denitrification treatment phase.
Studies by Yi and Harper (2007), Khunjar et al. (2007), and others have focused on the
mechanisms of estrogen removal during nitrification. Possible mechanisms include sorption of
estrogens to solids and biotransformation within the treatment facility, especially in the presence of
nitrifying activated sludges (Khunjar et al., 2007).

Ammonia oxidizing bacteria have monoxygenase enzymes for ammonia oxidation and these
enzymes have been shown previously to be nonspecific and able to accomplish cometabolic
degradation of recalcitrant organics.
Cometabolic degradation is a reasonable hypothesis for estrogen degradation because this

compound is present at low ng/L concentrations that are below those expected to support microbial
growth on that compound alone. One goal of the Yi and Harper (2007) study was to establish
whether biotransformation of EE2 is due to cometabolic activity. They conducted batch experiments
using enriched cultures of autotrophic ammonia oxiders. Their study and others (Vader et al., 2000,
Shi et al., 2004, as reported in Yi and Harper, 2007) showed a strong relationship between
nitrification and EE2 removal in enriched nitrifying cultures. Based on batch tests with and without
a nitrifying bacteria inhibitor, they concluded that EE2 biotransformation can be cometabolically
mediated in bioreactors that are enriched for autotrophic nitrifiers. However, Yi and Harper (2007)
noted that the heterotrophic microorganisms, if present in activated sludge processes, may also be
responsible for some micropollutant biotransformations.
Further work is needed in this area as these tests did not identify the EE2 degradation product to
confirm cometabolic degradation and the role of heterotrophs was not accounted for in some tests.
The focus of a Khunjar et al. (2007) study was to identify the role of ammonia oxidizing bacteria
compared to heterotrophic bacteria in the biotransformation of EE2. They used pure cultures of
ammonia oxidizing Nitrosomonas europaea and heterotrophic cultures that were enriched with
monooxygenase and dioxygenase enzyme systems. Nitrifying activated sludge mixed liquors were
taken from two WWTPs to seed the cultures. EE2 concentrations were 10-15 μg/L. The results of
their study showed significant sorption of EE2 to the predominantly heterotrophic culture but none
to the N. europaea culture.
In addition, biotransformation of EE2 was significant in the N. europaea culture. They observed
three major EE2 metabolites at different phases of N. europaea culture growth that suggest
differential action on each byproduct by the nitrifying bacteria; however, additional work is needed
to identify these byproducts. The authors also noted that additional research is needed with
continuous flow cultivated N. europaea to determine whether these metabolites are likely to be
present in nitrifying activated sludge. Also, N. europaea was not significantly inhibited at EE2
concentrations at or below 10 μg EE2/L, suggesting that ammonia oxidation may not be significantly
impacted by concentrations of EE2 that may be typical of those found in the environment.
Use of Reverse Osmosis to Improve Removal Efficiencies

Several studies describe the effectiveness of RO in the removal of PPCP and EDCs from
secondary wastewater effluent. Braghetta et al. (2002) calculated the removals rates that could be
achieved with a RO step following tertiary treatment for 17 PPCPs. They estimated removals to be
> 90 percent for most of the selected compounds. Lower removal rates were estimated for
diclofenac (55.2 to 62 percent), ketoprofen (64.3 percent), and paraxanthine (73.7 percent).
As previously discussed, the WERF (2005) technical brief evaluated RO removal rates for several
compounds. Specifically, the WERF brief cites numerous studies in which RO achieved removal
rates of 90 percent or better for naturally occurring and synthetic steroids, organohalides, metals
/organometals, and alkyl phenols.
In addition, Oppenheimer et al. (2007) found that RO was effective in removing all 20 investigated
PPCPs below the detection limit including those that were not consistently removed at SRTs of 30
days (i.e., galaxolide) using conventional activated sludge treatment or media filtration.

Hand Compositing - Example
Hand compositing is a series of time proportional grab samples which are collected and
composited by hand. Provided the sample volumes are equal and are collected at even
intervals, the results should be the same as if done by an automatic sampler (i.e., flow
proportional composite sampling). A specific instance where this sampling method may be
used is in metal plating shops which have batch discharges from the treatment tank. Provided
the tank contains a homogeneous mixture, a minimum of four grab samples are taken of equal
amounts and at evenly spaced intervals of time during discharge, to accurately represent the
entire tank.
This should represent the waste characteristics of the entire batch discharge to the sewer.
One hand composite per batch discharge would be equivalent to a 24-hour composite sample
taken at other types of facilities. The sampling data would be compared with the average daily
categorical standards or local limits where applicable.
Parshall Fume and Ultrasonic Flow Meter. Here is a great trick if you do not
have a stand for your ultrasonic probe, simply use a reflective street cone to hold
your probe. Notice the debris and most POTW’s will write a NOV for not
maintaining the flume and/or uncleanness.

POTW’s Wastewater Samples- Example
General
There are four types of samples that are collected by the POTW’s Sampling Section: grab,
time proportional composites, flow proportional composites, and hand composites. The
sampling method used depends largely on the types of analyses to be run, and the nature of
the wastestream being sampled. Each sampling method is described in this section.
Most POTW’s will define the sampling methods which must be used by industrial users (IUs)
to obtain representative samples to show compliance with their permits: Example
(1) A grab sample is an individual sample collected in less than 15 minutes

without regard for flow or time of day. pH, cyanide, oil and grease, sulfide,
and volatile organics must be collected as grab samples.
(2) 24-hour flow proportional composite samples where feasible. The POTW
may waive this requirement if the IU demonstrates that this method is not
feasible. Samples would then be taken by means of time proportional
composite sampling methods or by hand composite where the IU can
demonstrate that this will provide a representative sample of the effluent
being discharged.
The volume of sample to be collected by any of these methods is dependent on the number
and types of analyses that must be performed.
Wastewater Grab Samples

Grab samples are individual
samples collected in less
than 15 minutes without
regard to flow or time of day.
Grab samples are normally
taken manually, but can be
pumped. Oil and grease
samples and purgeable
organics are exceptions and
must be taken manually.
A grab sample is usually

taken when a sample is
needed to:
(1) Provide
information
about an
instantaneous concentration of pollutants at a specific time.

(2) Quantify the pollutants in a non-continuous discharge (e.g., batch
discharge).
(3) Corroborate composite samples if the waste is not highly variable.
(4) Monitor parameters not amenable to compositing such as pH, temperature,
dissolved oxygen, chlorine, purgeable organics and sulfides, oil and grease,
coliform bacteria, and sulfites.

Collecting Procedure for Water/Wastewater Grab Samples
Policy Example
Lower dipper or mouth of the bottle into water just below surface. In some cases, you will
need to rinse the bottle or dipper three times in the sample before obtaining the sample.
Retrieve collected sample to clean processing area.
Rinse the outside of the bottle 3 times to remove contamination.

Pour the sample into the required laboratory bottle.
Filtering (for ortho-P and NOx samples)

You may need to filter the sample; this is true with some water and wastewater samples.
Some surface water virus samples need to be filtered.
 Secure caps tightly.
 Bottle preservation is performed in the truck or lab before sampling.
 Secure sample container caps tightly.
 Label the sample containers and place them in an iced cooler before storage.
Timed Composites
Timed samples are usually taken in instances where the intention is to characterize the wastes
over a period of time without regard to flow, or where the flow is fairly constant. Timed
composite samples consist of a series of equal volume grab samples taken at regular
intervals. Usually the interval is 15 minutes with a maximum sampling duration of 24 hours.
However, other intervals can be used and may be more appropriate under some
circumstances. Samplers are available which can take up to 10 discreet samples per bottle,
for a total of 240 discreet samples. The sampler may be programmed to take any number of
samples into one composite bottle which has a 2.5-gallon capacity.
Flow Proportional Composites

Flow proportional composite samples consist of: a series of grab samples whose volumes
are equal in size and proportion to the flow at the time of sampling. Samples are taken at
varying time intervals, or continuous samples taken over a period of time based on the flow.
Wherever possible, flow proportional sampling is recommended because it most accurately
reflects the nature of the wastestream.
Equal volume samples taken at varying time intervals are most often collected by the sampling
inspectors. A flow measuring device should be used in conjunction with the automatic
sampler.
This sampling method is used for all sampling activities except for instances where grab
samples are required or time proportional sampling is more expedient and can provide the
same accuracy as flow proportional sampling (i.e., constant flow levels).

Pre-Sampling Procedures - Example
To ensure acceptable analytical results, numerous steps must be followed before a sampling
program can be initiated:
(1) Sampling equipment must be clean and in good working order.
(2) Sampling site must be selected.
(3) Types of analyses must be determined.
(4) Proper sample containers must be selected and prepared.
Wastewater Sampling Equipment

The POTW may use one or more of the following portable samplers, ISCO Ultra-Sonic flow
meters, SIGMA Depth Sensor samplers, and SIGMA pH Probe samplers. Safety equipment
and other necessary equipment are also used.
The equipment that is kept in the sampling vehicle is dependent on the types of sampling
activities planned each week, while the equipment stored in the storeroom is for back-up
needs and future sampling demands.
Each sampling vehicle should be equipped with at least one sampler and one flow meter more
than is needed for the particular sampling period. For example, three scheduled flow
proportionate sampling sites would require a vehicle to be equipped with four samplers and
four flow meters. At least one spare battery for each type of equipment taken into the field
should also be placed in the sampling vehicle.
Ancillary equipment, such as supports, harnesses, blowers, etc., that must be carried in each
vehicle will depend on the nature of the sampling location.
In order to keep the equipment in good working order, it should be maintained and cleaned
on a regular basis. Routine maintenance and cleaning procedures should be written into the
procedures.
Sampling Equipment Maintenance Policy - Example

Basic maintenance for samplers includes: periodic calibration, general equipment checking,
and replacement of the internal desiccant and fuses. Routine cleaning should be done as
covered in SOP or equivalence.
Basic maintenance of the flow meters includes: periodic replacement of the internal desiccant,
plotter paper, ribbon, fuses, and any broken re-roll spool assemblies. Note: on this assembly
there are two tabs on the sides of this piece which are extremely thin and easily broken.
The NiCad and Gel Cell or equivalent batteries need to be recharged on a regular basis.
Any battery that reads less than 12.50 when checked should not be installed or left on any of
the sampling equipment. At the battery charging station, areas are set aside for batteries that
need to be charged and batteries already charged.
To prolong battery life, batteries should be charged for a maximum of 24 hours, in accordance
with the procedures described in the manufacturer's operations and maintenance manuals.
It is important to note that charged NiCad batteries, if left unused for a long time, are
nevertheless slowly discharging.

Gel cell batteries are generally more stable. Voltage readings should be taken before the
charged batteries are taken into the field to be sure that they still have a full charge.
When a sampler, flow meter, or ancillary equipment needs more specific repairs, the
manufacturer representative should be contacted and arrangements made for repair or
replacement of the equipment.
Cleaning Automatic Samplers Policy - Example

Samplers, sample jars, grab beakers, and all other equipment used in collecting samples must
be cleaned between their uses at each site, to avoid the possibility of cross contamination.
Latex or nitrile gloves or equivalent should be worn to protect against infections and acid
burns. The following steps should be taken to ensure the proper cleaning of the sampling
equipment.
(1) Break down the sampler and lay the three components in a row.
(2) Place the strainers and weights in a plastic bucket.
(3) Set the glass composite jars and Teflon caps off to the side, to be cleaned
separately from the samplers.
(4) Pour a small amount of diluted (1:128) O-Syl disinfectant and MICRO soap
into each sampler component, the bucket containing the strainers and
weights, and the composite jars.
(5) To clean the sampler components:
(a) Partially fill the sampler bases and cover with water.
(b) Use a brush to scrub the inside and outside of each sampling
component. Using a small bottle brush, thoroughly scrub the inside
of the intake tube and the float housing of the sampler head (these
are critical areas since they come in contact with the sample).
(c) Rinse off the soap with fresh water.
(d) Stack each component so that it will dry quickly and thoroughly.
(e) Reassemble the sampler after the components are dry, and store it
in the proper compartment of the sampling van. Leave the sampler
lid loose so moisture won't be trapped.
(f) Clean the strainers and weights in the bucket. Empty the contents of
the bucket and rinse the bucket, strainers, and weights. After they
have dried, place them in the proper storage areas of the sampling
van.
(g) Drain the wastewater tank of the sampling van into the sewer drain.
(h) Refill the fresh-water tank on the sampling van with potable water.

Sampler Bottle Cleaning and Preparation Policy - Example
(1) Fill each jar with O-Syl (same dilution as used in the sampler disinfection),
MICRO soap, and fresh water.
(2) Thoroughly scrub the inside and outside of the jars until they are sparkling
clean. Make sure that all oil and grease are removed.
(3) Rinse the jars with fresh water.
(4) Pour a small amount of 1:1 nitric acid into one jar, and securely place the
proper Teflon cap on the jar. Swirl the nitric acid throughout the jar, remove
the lid, and pour the nitric acid into the next jar. Repeat this procedure until
all the bottles have been treated. Rinse bottles with water after the acid wash.
NOTE: Wear safety glasses or a full-face shield to protect your eyes.
(5) Place jars in the drying oven. If jars are to air dry use Acetone to clean the
bottles the same way as stated in (4) above. Let the jars and caps dry
completely.
(6) Place the jars, with their caps on loosely, in their respective places on the
sampling van.
Selection of Pretreatment Sampling Site

In order to ensure the collection of valid samples, a representative sampling site must be
selected. For industrial sampling, the sites are designated in the permit.
Industrial Users - Permitted/Non-permitted - Example

The sampling points within an industry vary with each industry depending on the nature of the
process and location of pretreatment facilities. Therefore, exact locations must be identified
on a case by case basis. However, the following general principles apply in all cases:
(1) A permanent sampling location(s) must be identified for use by the POTW
and the IU.
All permitted industries are required to install a sampling vault. The location of the vault is
designated by the enforcement inspector. The enforcement inspector responsible for an
individual company or site is responsible for providing directions (maps) of the specific
sampling points, as well as current copies of permits and the name of the contact person and
phone number. This information needs to be kept current in the sampling file. Locations of
sampling points need to be compared to what is listed on the currant permit. If sampling points
that the POTW is using do not agree with permit location, do not sample -refer to Chief
Inspector or Supervisor.
(2) The sampling location should be easily accessible and relatively free of safety
hazards.
(3) For categorical industries, there should be, if possible, no discharge present
other than that from the regulated process.

If other wastestreams are combined with the regulated wastestream prior to
the sampling location, the combined wastestream formula will need to be
utilized. The sampling crew must be aware of lower limits to correctly show
analysis on chain of custody.
(4) If the rate of industrial process discharge flow is needed (i.e., where mass
limitations are applied), the sampling location will need to be located where
the flow of the wastestream is known or can be measured or estimated and
flow rates for the other wastestreams obtained.
(5) In instances where sampling must be performed in the sewer outside of the
building, the IU must install a sampling vault in accordance with Code.
Sample Type and Analyses

Different sample volumes are required for various analyses. In addition, the laboratory has
developed standard volumes for routine analyses performed on industrial waste samples as
follows:
(1) BOD/COD/TSS (1000-2000 ml, plastic) or equivalent

(2) Heavy metals (500-2000 ml, plastic) or equivalent
(3) Cyanide (2000 ml, plastic) or equivalent
(4) Oil and grease (1000 ml, level-one glass) or equivalent
Selection and Preparation of Sample Containers

The selection of a sample container is based on the parameter to be measured. The inspector
should be familiar with the type of sampling containers and preservatives that are needed.
It is essential that the sample containers be made of chemically resistant material, and do not
affect the concentrations of the pollutants to be measured.
In addition, sample containers should have a closure (i.e., leak proof/resistant, Teflon lined)
that protects the sample from contamination and be properly labeled before leaving the
sampling site.
Wastewater Sample Preservation

Wastewater usually contains one or more unstable pollutants that require immediate analysis
or preservation until an analysis can be made.
Sample preservation is needed for composite samples, for example, which may be stored for
as long as 24 hours prior to transferring them to the laboratory.
Recommended preservatives and holding times that should be used for specific pollutants are
presented in the front of this section.

Quality Assurance/Quality Control Policy - Example
Quality Assurance/Quality Control (QA/QC) measures taken by the sampling crew include
equipment blanks, trip blanks, split samples and duplicate samples. Equipment blanks and
trip blanks are routine QA/QC measures.
Split samples are taken for Local Limits (pretreatment) sampling and when requested by an
industry or laboratory. Split samples requested by an industry are analyzed by their lab at
their expense.
Duplicate samples should be run when requested by a Supervisor or Project Leader.
The laboratory prepares all trip blanks/travel blanks used by the sampling crews. This is
performed in the laboratory rather than in the field in order to assure that there is no field
contamination in the blanks.
Any contamination detected in the blanks would result from field exposure which could in turn
affect collected samples.
Chain-of-Custody
Documentation of all pertinent data concerning the collection, preservation and transportation
of samples is critical to the overall success of the Wastewater Sampling Program. If sampling
is performed for the Pretreatment program, any sampling data may be used as evidence in
court proceedings against a noncompliant industrial user. In this case documentation
becomes critical. This form is a legal document and is of major importance in a court hearing.
Specific procedures with regard to chain of custody are outlined below:
(1) The sampling crew takes a sufficient supply of pre-numbered Industrial Waste
Lab Reports, (custody forms) and sample containers into the field.
(2) The sampling crew fills in the sampling form at the time of sample collection,
and returns the form to the lab along with the collected sample. Specific
information to be completed on the form includes:
(a) CODE: The company ID number assigned by supervisor.
(b) SITE No.: The sampling point ID number assigned by supervisor.
(c) DATE SAMPLED: From - Date sampling began To - Date sample

is pulled. If it is a grab sample, only the date the sample was taken
will be entered with the other line crossed out.
(d) SUBMITTED BY: This will have a preprinted truck number. The
sampling crew will write in their initials on the blank line which
follows.
(e) LABEL: A letter is checked and the type of analysis to be

performed.
(f) PRESERVATIVE: The method of preservation used. See

preservation section to see which preservatives to use.
(g) TYPE OF SAMPLE: Check off whether proportional, timed

composite, hand composite, flow or grab sample.

(h) TIME: The time frame needed for collection of the sample. A
starting time for sample collection, an ending time, and a total time
in hours and quarter hours is recorded, such as 23.25 hours. On a
grab sample only, the end time, which is the time the sample was
taken, will be entered and the other two lines will be crossed out.
(i) RELINQUISHED BY: This is the signature of person that

relinquishes sample to lab personnel, or to any other person taking
custody of the sample.
(j) DATE: Date sample is submitted to the laboratory or relinquished

to another person.
(k) NOTES TO LAB: Includes any special notes to the lab, such as
special analysis required of the sample, a letter code which is
assigned to the entity being tested the amount of flow if sample is
flow proportional, grab sample pH and temperature, and/or actual
sample temperature.
(l) FIELD TEST: Results of any field tests including sample pH,
hexavalent chromium, dissolved sulfides, copper, and residual
chlorine.
(m) RESULTS: The appropriate box(es) need to be checked to

correspond to the label designation chosen above.
(3) When the sampling is completed at a site, the sampling crew labels the bottles
with the label letter designation. The samples are sealed with chain of
custody seals and placed in an ice chest for transportation to the lab.
(4) The sampling crew submits the samples and the chain of custody form to the
laboratory.
(5) The laboratory logs the samples and assigns a Lab Reference Number to the
sample. The sample is tracked by means of this number.
(6) Laboratory personnel sign and date the form, and return it to the sampling
crew who makes two copies of the form. One copy is for the sampling crew
files and the other is for data entry. The original form is returned to the
laboratory. It is also important to note that the sampling vehicle should be
kept locked at all times when the sampling crew is not in the vehicle, or in full
view of the vehicle.

Equipment Maintenance Cleaning Techniques - Example
It is important to keep all equipment used for sampling clean to reduce the risk of cross
contamination.
Is your automatic sampler and equipment clean?
All components of an automatic sampler need to be clean prior to setup, since

contamination can occur.
Automatic sampler - Each section should be clean, especially the inlet tube.
Intake tubing - Cleaned or new Tygon or Teflon should be used according to the samples
you are collecting.
 Intake line strainer - Clean or stainless steel or Teflon.
 Pump tubing - Clean or new medical grade silicone tubing.
Composite bottle - Generally glass is used for this and it’s extremely important that it is
clean.
Automatic sampling equipment - all components of an automatic sampler should be cleaned

using phosphate free soap and rinsed thoroughly prior to setup.
Follow safety procedures (goggles, gloves, etc...) When cleaning equipment!

Composite bottles used in the automatic sampler need to be cleaned prior to setup.
1. Rinse bottle and cap with tap water to remove any residual contaminants.
2. Wash bottle and cap with a phosphate free soap and tap water.
3. Rinse bottle and cap with tap water to flush off any soap.
4. Add 1:1 nitric acid to bottle, cap and shake to cover entire inside of bottle and cap.
Pour out 1:1 nitric acid.
5. Triple rinse bottle and cap with analyte free water.
6. Allow bottle and cap to dry.
7. Store Bottle with cap loosely screwed onto bottle to keep contaminants out.
Tubing - clean or new

Using clean tubing (intake and pump) for each sampling event will eliminate the need to
clean the tubing. If tubing is not going to be changed before a sampling event, then the
tubing will need to be washed.
1. Pump clean tap water through tubing, using the automatic sampler pump to
transfer water from one container to another.
2. Place tubing into another container that has a phosphate free soap in it and pump the
soapy water through the tubing 3 or 4 times while catching the discharge in a separate
container.
3. Pump clean tap water through tubing to rinse out soap.
4. Add 1:1 nitric acid to clean tap water and pump this solution through tubing 3 or 4 times.
5. Pump analyte-free water through tubing until all residual acid has been removed.
6. Keep tubing in a clean place until next sampling event.

Regular Maintenance Includes the Following
Automatic Sampler
 Washing
 Drying
 Change desiccant
 Check full bottle shut-off float
 Recharge battery
Flowmeter
 Washing
 Drying
 Change plotter paper
 Change printer ribbon
 Change desiccant
 Recharge battery
Record keeping - keep a record of cleaning dates. What was cleaned, and when.
A modern wastewater treatment facility may have up to 10 different sampling sites some
are necessary for compliance purposes, some sites are to please certain agendas or for
political purposes. These sampling sites may include Local Limits, QA/QC, Influent,
Outfall, Chlorine residual, and many more sites to maintain compliance and to ensure
that the plant is running efficiently.

Proper Sample Handling- Example
The proper handling of water quality samples also includes wearing gloves. Gloves not only
protect field personnel, but also prevent potential contamination to the water sample. Always
wear powderless, disposable gloves. When sampling for inorganics, wear latex gloves. Nitrile
gloves are appropriate for organics.
The following sections provide a field

reference for chain of custody procedures,
sampling surface water and ground water,
and further provides procedures for
measuring field parameters and handling
water-quality samples.
Use chain-of-custody procedures when

coolers and containers are prepared, sealed
and shipped. They will remain sealed until
used in the field.
When making arrangements with the

laboratory, make sure you request enough
containers, including those for blank and
duplicate samples. Order extra sample
bottles to allow for breakage or
contamination in the field.
Some samples require low-temperature storage and/or preservation with chemicals to

maintain their integrity during shipment and before analysis in the laboratory. The most
common preservatives are hydrochloric, nitric, sulfuric and ascorbic acids, sodium hydroxide,
sodium thiosulfate, and biocides.
Many laboratories provide pre-preserved bottles filled with measured amounts of

preservatives. Although most federal and state agencies allow the use of pre-preserved
sample containers, some may require either cool temperatures or added preservatives in the
field.
When the containers and preservatives are received from the laboratory, check to see that
none have leaked. Be aware that many preservatives can burn eyes and skin, and must be
handled carefully. Sampling bottles should be labeled with type of preservative used, type of
analysis to be done and be accompanied by a Safety Data Sheet (SDS).
Make sure you can tell which containers are pre-preserved, because extra care must be
taken not to overfill them when collecting samples in the field. Check with the laboratory about
quality control procedures when using pre-preserved bottles. Coolers used for sample
shipment must be large enough to store containers, packing materials and ice. Obtain extra
coolers, if necessary. Never store coolers and containers near solvents, fuels or other
sources of contamination or combustion. In warm weather, keep coolers and samples in the
shade.
Field Parameters
Measure and record the field parameters of temperature, electrical conductivity, pH and
dissolved oxygen in an undisturbed section of stream flow. Other parameters may be
measured, if desired.

Sample Station commonly found at most wastewater treatment plants. This tap will
allow the operator to obtain Grab Samples for pH, Temperature, COD, Bacterial,
ORP, OUP, Organics and Inorganic field parameters.

QA/QC Field Procedures for Plant Sampling Example
Duplicate Sampling Procedure
The purpose of Duplicate Samples is to check the laboratory's ability to reproduce
analytical results. Duplicate Samples are to be collected using these steps:
1. Determine amount of sample needed. If a flow proportion sample is required, then

base the amount of sample needed on the current flow reading. If a flow-
proportion sample is not required, then use the predetermined amount for the
sampling site.
2. Collect sample using a grab type sampler or a sampling head.
3. Measure the amount determined in Step 1 using a graduated cylinder or other
accurate measuring device.
4. Pour measured sample into sample container that is not marked as the Duplicate
Sample.
5. Measure same amount as in Step 1.
6. Pour second measured quantity into sample container marked for Duplicate
Sample.
7. Process both samples using standard procedures and submit both samples to
laboratory.
Split Sampling Procedure

The purpose of Split Samples is to check analytical procedures by having the samples
analyzed by two different laboratories. Split Samples are to be collected using these steps:
1. Determine amount of sample needed. If a flow proportion sample is required, then

base the amount of sample needed on the current flow reading. If a flow-proportion
sample is not required, then use the predetermined amount for the sampling site.
2. Collect sample using a grab type sampler or a sampling head.
3. Measure the amount determined in Step 1 using a graduated cylinder or other
accurate measuring device.
4. Pour measured sample into sample container that is not marked as the Split Sample.
5. Measure same amount as in Step 1
6. Pour second measured quantity into sample container marked for Split Sample.
7. Process both samples using standard procedures and submit both samples to the
laboratory. The laboratory will be responsible for submitting the samples to the
outside laboratory that will be analyzing the Split Sample.
Trip Blank Procedure

The purpose of Trip Blanks is to determine if the sample bottles have been adequately
cleaned, and if sample contamination occurs between the time sample bottles leave the
laboratory to the time that samples are returned to the lab.
Trip blanks are prepared by the laboratory using bottles supplied by the sampler. They are
picked up by the person who begins the sampling day. Trip blanks are placed in the cooler
which contains the other samples and remain there until the samples are turned into the
laboratory.

A normal day for a WWT sampler: Here she is looking up a flow rate to determine the
volume of wastewater that is flowing. Many samplers do not know simple math formulas
to determine flow and rely solely upon the electric measuring devices. These measuring
devices can fail or be programmed incorrectly, or batteries can die. Be prepared for the
worst case scenario. Pickle bottle is shown in the middle photograph.

Field Equipment Blank Procedure- Example
The purpose of Field Equipment Blanks are to test the procedure for cleaning the sample
measuring container to determine if cross contamination between sample sites has occurred.
These Blanks are needed only at sites where flow-proportion samples are taken. Follow
these steps when collecting a Field Equipment Blank:
1. Collect Field Equipment Blank AFTER collecting a sample and BEFORE moving to
the next sampling location.
2. After collecting sample, triple rinse sample measuring container, usually a
graduated cylinder, using High Purity water.
3. Open a sealed bottle of High Purity Water.
4. Pour the High Purity Water into the sample measuring container that was just
rinsed.
5. Pour the High Purity water from sample measuring device into sample bottles
labeled for the Field Equipment Blanks.
6. Repeat Steps 3 through 5 until all Field Equipment Blank sample bottles have been
filled.
7. Process samples using standard procedures and submit to laboratory.
An equipment blank is high purity water which has been collected in a composite sample
bottle or a series of discrete bottles from an automatic sampler. Equipment blanks are used
to evaluate the reliability of composite samples collected in the field.
The data produced from the equipment blank indicates the performance of the sample
collection system, which involves the cleaning of sampling equipment, and accessories,
preservation techniques, and handling of samples.
The objective is to demonstrate that the samples are not contaminated by inadequate
cleaning of equipment, contaminated preservation additives or sample collection techniques,
and to provide documented records on Quality Assurance Practices.
Procedures to be followed in collecting the equipment blanks are outlined below.

(Also see QA/QC check list, example).
(1) The sampler is to be assembled completely in the manner determined by

the parameters the crew will be sampling (i.e. if sampling for organics, Teflon
suction tubing must be used at that site). The composite jar inside the
sampler must always be rinsed out thoroughly with high purity water.
(2) Program the sampler to collect the proper amount of high purity water that
is representative of the sample parameters that will be collected at that site.
Grab samples are excluded. Pump high purity water through the strainer
and intake tubing prior to filling the sampler bottle. Then, place the strainer
into as many fresh, uncontaminated bottles of high purity water as needed
to collect the necessary volume of sample.
(3) If the sampler is set up in the discrete mode, the crew must then transfer the
collected samples into the field composite bottle and shake to mix
thoroughly.
(4) Transfer the sample from the field composite bottle into its respective lab
sample bottles. Test and preserve the samples as appropriate for the
parameters being analyzed.

(5) Follow the chain of custody procedures outlined in SOP for turning the
samples in to the laboratory. All paperwork must be completed at this time,
and all bottles must be marked accordingly. Custody seals must be used.
The crew must note the sampling activity in a logbook that is kept specifically
for documenting preparation of equipment blanks and/or any other QA
activities.
Wastewater Sampling Procedures/Techniques

General Guidelines
In general, the following guidelines should be observed in conducting sampling activities:
(1) Samples being collected must be representative of the wastestream being

tested.
(2) Samples shall be collected in

uncontaminated containers and preserved
properly.
(3) Samples should be of sufficient volume for

the required analyses.
(4) Samples should be stored in a manner

which does not alter the properties of the
sample prior to chain of custody transfer.
(5) Samples should be properly and completely identified by marking them

with the proper information.
(6) Sample lines should be as short as possible and the smallest practical
diameter to facilitate purging, reduce lag time, and give adequate
consideration to maximum transport velocity. Also, they should have
sufficient strength to prevent structural failure.
(7) Sample lines should be pitched downward at least 10 percent to prevent

settling or separation of solids contained by the sample.
(8) Samples should be delivered as quickly as possible to the

laboratory.
Specific Techniques
Sampling techniques in addition to the above general guidelines must also recognize
differences in sampling methodology, preservation, and analytical methods.
The following sections specify techniques that differ by pollutant group and discuss such
factors as sampling methodology (e.g., composite, grab, etc.), type of container,
preservation and holding time.

Sampling Techniques for Volatile Organics- Example
Volatile organics are analyzed in accordance with EPA methods 601, 602, and 603.
Due to the volatility of these compounds, only grab samples can be taken. If a composite
sample is needed, individual grab samples must be collected and composited in the
laboratory prior to analysis.
The procedures that must be followed in taking these samples are outlined below.
NOTE: Gloves, clothing, face, and eye protection must be worn when handling volatile
organics.
In addition, the sampling crew must thoroughly clean those parts of the body that have been
exposed to these materials.
(1) For each sampling date, the lab will also provide two additional
bottles to be used as a backup in case of breakage. These sampling
vials are only good for one week. If any are unused, they must be
returned to the lab for disposal.
(2) The lab will provide one sample trip blank per sampling date. This
bottle is to be kept on ice until the samples are submitted to the lab.
At least one day prior to sampling, go to the lab and request the
sample bottles (40 ml vials) for the specific sampling site, as
indicated by the sampling plan. The laboratory will arrange to have
the appropriate number of sample bottles prepared, based on the
number of analyses to be performed. The sampling crew should
make sure that all bottles are provided for these samples by the lab
technicians.
(3) Collect the sample in a clean glass beaker. Test for chlorine with
the Hach test kit. If there is any chlorine residual, neutralize the
chlorine with sodium thiosulfate (Na2S2O3) and retest for chlorine.
Repeat until there is no chlorine residual. Make notes on chain of
custody sheet if extra amounts of sodium thiosulfate are required for
neutralization.
(4) Remove the vials from the ice. There will be two empty vials for the
601 sample and two vials with HCl for the 602. The HCl will already
have been measured into the vials by the lab personnel.
(5) Fill the vial to just overflowing in such a manner that no air bubbles
pass through the sample as the vial is being filled. This is
accomplished by pouring the sample from the beaker into the vial
along the side of the vial to minimize the possibility of entrapping air
in the sample. Do not rinse out or overfill the vials, this will wash out
the preservative in the vial.
(6) Seal the vial so that no air bubbles are entrapped in it. Remember
to put the Teflon side of the cap facing down onto the vial.
(7) To be sure there are no air bubbles, turn the vial upside down and
tap it against the palm of the hand. Check to see if there are air
bubbles along the sides or bottom of the vial. If there are bubbles,

unseal the vial, top off the vial, and reseal. Check the vial again for
the presence of bubbles.
(8) All samples must be maintained at four degrees centigrade from the
time of collection until the time of extraction. Custody seals must be
placed on all samples, and all paper work must be filled out properly.
(9) Return the sample bottles and QA/QC bottles to the laboratory the
same day the sample is collected.
Acid/Base/Neutral Extractable Organics and Pesticides Example

Acid extractable organics are analyzed in accordance with EPA methods 604 and 625.
Base/neutral extractable organics are analyzed in accordance with EPA method 625, or
individual methods for various groups of compounds including EPA methods 605, 606, 607,
609, 611, and 612. Pesticides are analyzed in accordance with EPA method 608.
The procedures that must be followed in taking these samples are outlined below.
(1) Samples must be collected in certified clean one-gallon amber glass bottles
with Teflon lids.
(2) Travel blanks or QA/QC bottles may not be required with the samples.
(3) Grab samples must be collected in amber glass bottles. They do not have
to be completely filled, but must be a minimum of 1/3 to 1/2 full. Bottles
should not be pre-washed with samples prior to filling.
(4) For composite sampling, glass composite bottles must be used and pre-
cleaned. Teflon tubing must be used for the suction piping. The pump tubing
must be medium grade silicone rubber.
(5) The composite bottle in the sampler must be kept refrigerated (putting ice in
the sampler) at 4 degrees Celsius. If amber glass is not used, (i.e. 2 1/2-
gallon clear composite sampler bottle,) the sample must be protected from
the light during collection and compositing. The compositing must be done
in the field (i.e. when discrete sampling has been used).
(6) All samples must be iced at four degrees Celsius from the time of collection
until extraction.
(7) The sample should be checked for the presence of chlorine using field test
kits that provide results in accordance with EPA methods 330.4 and 330.5.
If chlorine is determined to be present, 80 mg of sodium thiosulfate should
be added to each bottle. The sample must be retested for chlorine. This
procedure must be repeated until there is no residual of chlorine shown. The
amount of sodium thiosulfate added must be noted on the chain of custody
if in excess of 80 mg.
(8) All necessary paperwork must be completed at sampling site. All bottles
must be properly labeled, and have custody seal.

Sampling Techniques for Heavy Metals- Example
(1) Generally, all metal samples collected are to be composite samples, i.e.,
flow/composite, time/composite, or hand composite.
(2) For composite sampling, place the lid on the bottle and agitate the bottle to
completely mix the composite sample.
(3) Transfer the required amount from the composite container to either a 500
ml or 2000 ml clean plastic bottle. Check the pH of the sample as
described in Section 8.7.2.5.
Note: For inductively coupled plasma (ICP) metal analysis, a 500 ml

clean plastic bottle is required. For extra metals or metals by
furnace, a 2000 ml clean plastic bottle is required.
(4) Add nitric acid (1:1 solution) to the sample to reduce the pH to below 2.0.
Usually, 2 ml/500 ml is sufficient. Recheck the pH to be sure it is below 2.0.
Make a note on the lab sheet if more than two ml of acid is required to bring
the pH below 2.0.
(5) Label the sample bottle with the corresponding IW number and proper
analysis code letter. Attach the custody seal to the sample, then store in the
ice chest until transferred to the laboratory. Fill out the IW lab sheet with all
the pertinent information, being careful to include all required parameters
and the type of analysis required, e.g., ICP/furnace.
(6) When a grab sample is necessary, rinse out the receiving sample bottle with
an aliquot of the sample stream at least three times. Then fill the sample
bottle and proceed with steps two through four described above.
(7) When a split sample is

requested (i.e., one for
the samplers and one
for the user), the
composite sample is
prepared as described
in item one. Providing
there is sufficient
sample, a portion is
transferred into the
bottle provided by the
user.
(8) If more than one site is sampled per day, a clean composite container (i.e.,
two and one half-gallon glass jar), must be used at each site.
(9) If a discreet sampler is being used, at the time of collection combine all the
samples that have been collected into a single clean composite bottle.
Then follow the preceding steps one through four, and refer to step six if a
split is requested.

Cyanide- Example
To assure that the sample can be analyzed for cyanide, no chlorine can be present in the
sample. Procedures for taking cyanide samples are as follows:
(1) This sample is normally a grab sample. The cyanide sample is a

composite sample when collected as part of Priority Pollutants or Plant
Sampling at the waste treatment plants.
(a) In the sampling file, check the industries' wastewater discharge

permit and locate all cyanide (CN) sampling sites. If the sampling
sites are located in a confined space, follow Confined Space
procedures before collecting the sample or samples.
(b) Collect 2000 ml (maximum), 1000 ml (minimum), of CN sample
into a type C plastic bottle.
NOTE: 2000 ml is the standard, but for batch dischargers 1000 ml is adequate.
(c) Test the cyanide sample for pH and temperature with the pH
meter. Record the results on the custody sheet (Industrial Waste
(IW) lab sheet).
(d) Test for chlorine with Hach Total Chlorine Test Kit (the
instructions are located in the kit)
(e) If chlorine is present in the CN sample, neutralize it with Ascorbic
Acid (C6H8O6). For ascorbic acid neutralization, add C6H8O6, a few
crystals at a time, until five mls of sample in the test tube produces
no color. Then add an additional 0.06 g of C6H8O6 for each liter of
sample volume.
(f) Once all Cl2 has been neutralized, preserve the sample with
Sodium Hydroxide (NaOH) and raise the pH to >12. Verify the >12
pH with a pH meter or pH test strips.
(g) Mark on the side of the CN sample bottle the Lab sheet number
(using a water proof marker), and place a corresponding custody
seal across the sample bottle tightened cap. Place a Cyanide label
on the bottle if cyanide is suspected of being present in the sample.
(h) Store the CN sample in the ice at four degrees Celsius and transport
it to the laboratory.
Total Sulfides
(1) The Total Sulfide sample is collected as a grab sample only. Use a clean
500 ml plastic bottle to collect the sample. This sample may be pumped
into the sample container or collected directly from the discharge side of
the sampling device.
(2) Preserve the sample with 1 ml of 2N Zinc Acetate (C4H6O4Zn) and then
add Sodium Hydroxide (NaOH) to raise the pH > 9.
(3) Label and seal the sample with a custody seal. Cool to 4c.

Oil and Grease/TPH- Example
Oil and grease samples are collected as two separate samples:
METHOD 413.1 (Oil and Grease). Non-volatile hydrocarbons: vegetable oils,

animal fats, waxes, soaps, and related matters.
METHOD 418.1 (TPH). Extractable petroleum hydrocarbons: light fuels and

mineral oils.
(1) This is a grab sample only. The bottle used to take the sample must be the same
bottle given to the laboratory for analysis. Do not pump or transfer the wastewater
sample into the bottle. Obtain a level one clean 1000 ml glass bottle, do not use a
pre-preserved bottle because you will lose the preservative when collecting the
sample.
(2) Collect the sample by placing the bottle neck down (up-side down) into the effluent
stream below the surface. This should be as close to the discharge pipe or point as
physically possible. Turn the bottle, allowing the bottle to fill, while keeping the bottle
below the surface. Remove the filled bottle and cap it. Never skim the surface of
the effluent stream.
(3) Preserve the sample using five ml of sulfuric acid (H2SO4) for method 413.1 or
hydrochloric acid (HCL) for method 418.1 (6:1 Ratio) to a pH of less than two.
Reference 42 of methods 418.1 and 41 of methods 413.1. When more than five ml
of HCL is used to lower the pH to less than two, make note of how much additional
acid is used, and record this on the lab sheet. Also indicate required analyses
method on lab sheet.
(4) After making sure the sample is well mixed and

preserved, seal and attach the proper
identification (custody) label to the bottle. Then
attach a custody seal across the lid. Store all
samples at four degrees centigrade.
(5) Under no circumstances are Inspectors to collect

an oil and grease sample or any other grab
sample for IUs.
(6) All samples must be taken from a good

representative flow. If there is any question as to
whether there is sufficient flow for a representative
sample, do not collect any sample. Make the
necessary notes in the file report as to why no
sample was obtained.

BOD/COD/SS- Example
(1) 24-hour composite sampling is always used for this test. Agitate the bottle
to completely mix the composite sample. Do not allow the solids to settle
out before you pour off the sample.
(2) When more than one sample is being taken from a composite bottle, the
BOD/COD/SS is taken first. The lab needs 1000 ml if the sample is cloudy
or has solids. If the sample is clear, you must collect 2000 ml. Transfer the
appropriate volume to the sample bottle.
(3) Take the pH/temperature of the sample with either pH paper and a
thermometer, or the pH meter carried on the sampling trucks.
(4) Label the sample bottle and place a custody seal over the lid. Store on ice
at four degrees centigrade.
(5) Should split samples be requested, they are given when it is sure there is
enough sample for POTW’s requirements. Users must provide their own
sample containers and allow POTW’s staff to pour off samples.
Rotating Bar Screens

The wastewater headworks is a key sampling location both for compliance and for
process control.

Virus Sampling - Example
Viruses are microbiological organisms which can cause infectious diseases. Wastewater recharge
and sewage disposal into the environment may contribute to the occurrence of viruses in surface
water and groundwater. Viruses are the most mobile and infectious of the waterborne pathogens.
Large volumes of water must be filtered to detect viruses. This involves passing the water samples
through a cartridge filter by use of a gasoline driven pump.
(1) Equipment Needed
Most of the equipment required for virus sampling should be available on the
sampling trucks. However, some equipment is virus sampling specific. The
needed equipment is as follows:
(a) Gasoline/oil powered water pump or equivalent

(b) Hoses - intake (supplied with pump) and discharge (garden type, with
female connectors at both ends)
(c) Two 55-gallon plastic containers or equivalent
(d) Filter apparatus
(e) Cartridge filters
(f) Sodium thiosulfate (two 500 gram bottles/site)
(g) Gasoline can with gas/oil mixture
(h) Hach total chlorine test kit
(i) Large plastic Zip-lock bags (supplied with cartridges)
(j) Chain of custody sheets
(k) Thermometer
(l) Water-proof marker
(m) Latex gloves
(n) Liquid bleach
(o) Cooler with blue ice
(p) pH meter
(2) Sampling Procedure

Check the pump for gas/oil prior to starting (Note: do not fill while it is running).
Make sure the gas/oil mixture is correct by checking the mixing instructions on the
side of the two-cycle pump oil can. Latex gloves should be worn for protection,
and to prevent contamination of the filters.
Connect the hoses and filter housing (with no filter) to the pump, and run the
effluent through it for one to two minutes to flush the system. Next, pump effluent
into the two 55-gallon drums and rinse them out. (Note: If disinfection was not
possible after the last sampling, then 50-100 gallons of effluent should be pumped
through the entire equipment set up prior to placing the filter in the housing.)
Pump effluent almost to the top (just above the handles) of both containers. While
the drums are filling, check the water in the drums for chlorine using the Hach test
kit and record the results and the temperature on the custody sheet.
If chlorine is present and needs to be eliminated, add 500 grams of sodium

thiosulfate to each container to eliminate it.
After visual observation has determined that all the sodium thiosulfate has
dissolved, retest to make sure there is a <0.1 ppm chlorine residual. If chlorine
was removed, take the hose from the channel, allow it to drain, and re-prime the
pump with the de-chlorinated water.

Pump this water through the system to flush it, and adjust the flow to fill a one-
gallon jug in about 15-20 seconds. Don't waste too much water, as the flow can
be adjusted after the filter is inserted. Install the filter into the blue holder, being
very careful not to touch it with your hands (wear clean latex gloves). There are
two black washers that go with the filter, one on the bottom and the other on the
top. Make sure these are aligned with the filter housing to prevent leaking. Screw
the holder and filter onto the apparatus.
Refuel the pump, restart it, and adjust the water flow so that it is close to 15-20
seconds per gallon. Make sure the housing doesn't leak. Try to keep this amount
of flow, since too great a flow will cause pass-through in the filter. Pump the water
from both containers until they are empty.
Stop the pump, remove the filter (wear clean latex gloves), and place it in its original
zip-lock bag. The washers do not need to go with the filter, but if they fall into the bag
it is better to leave them than take the chance of contaminating the filter trying to
remove them.
Fill in the information area on the zip-lock bag with a marker, indicating the plant
being sampled and the date, and put it in the cooler with the blue ice provided. The
blue ice keeps the temperature at 4 degrees Celsius to prevent significant die-off of
the viruses.
While at the site, or later

at the plant, mix a half-
gallon of bleach to 10
gallons of clean water.
Pump it through the flow
system and the
containers. Rinse
everything with fresh
water and drain it so it is
ready for the next time.
Let the pump cool before
storing it. Store the
gas/oil mixture in the
warehouse flammable
storage cabinet.
Parasitological Sampling
Parasitological sampling utilizes the same equipment and techniques as in the virus sampling
described above. However, a different type of filter, which is provided by the Lab, is used.

Field Tests - Example
Sampling Procedures for Hexavalent Chromium (Hach Kit)
(1) Rinse out the two color viewing tubes with a portion of the sample to be tested.
(2) Refill one of the color viewing tubes to the 5 ml mark with a sample (this is the test
sample). Using the clippers provided in the test kit, open one ChromaVer three
chromium reagent powder pillow. Add the contents of the pillow to the sample.
Stopper and shake to mix and put the tube in the color comparator.
(3) Fill the other viewing tube with a sample and put it in the left side of the color
comparator (this is the blank).
(4) Let the viewing tubes sit in the color comparator for approximately 5 minutes. The
samples should not be exposed to direct sunlight.
(5) Hold the color comparator up to a light source and view the two samples through the
two openings in the front. Rotate the dial on the holder until the color appears the
same in both samples. Record the results from the dial (which is read in mg/l Cr +6)
onto the chain of custody form.
Sampling Techniques for Dissolved Sulfides (Chemetrics, Inc. Kit)

(1) Collect a 25 ml grab sample in the container provided.
(2) Add three drops of activator (amber colored liquid) and mix well.
(3) Break a sulfide chemet Type S glass ampule and add the contents to the 25 ml
container.
(4) Let stand five minutes.
(5) Take a reading and record the results on the chain of custody form. If the reading is
0.0 then show the results less than 0.1 mg/l.
Sampling Techniques for Free and Total Chlorine (older colorwheel Hach Kit)
Procedures for determining free chlorine are as follows.
sample). Using the clippers provided in the test kit, open one DPD free chlorine
reagent powder pillow. Add the contents of the pillow to the sample. Stopper and
shake to mix and put the tube in the color comparator. All of the powder does not
have to dissolve to obtain correct readings.
(3) Fill the other viewing tube with the original sample and put it in the left side of the
color comparator (this is the blank).
(4) Let the viewing tubes sit in the color comparator for approximately 1 minute. The
(5) Hold the color comparator up to a light source and view the two samples through the
two openings in the front. Rotate the dial on the holder until the color appears the
same in both samples. Record the results from the dial (which is read in mg/l free
chlorine) onto the chain of custody form.

Procedures for determining total chlorine are as follows.
sample). Using the clippers provided in the test kit, open one DPD total chlorine
reagent powder pillow. Add the contents of the pillow to the sample. Stopper and
shake to mix and put the tube in the color comparator. All of the powder does not
have to dissolve to obtain correct readings.
(3) Fill the other viewing tube with a sample and put it in the left side of the color
comparator (this is the blank).
(4) Let the viewing tubes sit in the color comparator for approximately 3 minutes. The
(5) Hold the color comparator up to a light source and view the two samples through
the two openings in the front. Rotate the dial on the holder until the color appears
the same in both samples. Record the results from the dial (which is read in mg/l
total chlorine) onto the chain of custody form.
COD Reactor

Laboratory Analysis Section
Wastewater treatment operators run laboratory tests and analysis to monitor the treatment
plant operation. These analyses are for testing the process control and indicate how well
a particular process is working. Operators will analyze the results and if needed, will make
operational adjustments.
In a typical wastewater treatment plant, there are several locations to sample. As

wastewater flows through the treatment plant, including the collection system, its
characteristics frequently change. By taking samples at different locations throughout the
process, the operator has a better understanding of how to treat the flow.
Laboratory duties include some of the following:
 Collect and preserve samples

 Prepare samples for analysis
 Analyze samples and interpret results
 Operate and maintain equipment and instruments
 Handle chemicals and wastes (PPE)
 Quality assurance/quality control (Engineering and Administrative controls)
 Manage laboratory
 Laboratory safety (OSHA)

Grab Samples (Snapshot)
A grab sample consists of a single container or large bucket of wastewater analyzed at
one specific time. Grab samples indicate the condition of the wastewater at that specific
time and may or may not represent the normal conditions. Grab samples are required
when the analysis change rapidly. For instance, grab samples are required for certain
tests such as temperature, pH, D.O. (dissolved oxygen), and bacteriological analysis.
Composite Samples
A composite sample consists of several grab samples collected from the same spot over
a specific period of time and merged into a single sample. A composite sample is more
arduous, complicated and usually inconvenient than a simple grab sample. Collecting a
sample every few minutes and adding it to a single bottle is tedious, boring, and costly. To
help solve this problem, a 24-hour automatic sampler is often used. The automatic
sampler consists of a battery pack, a programmable timer, a pump, and as many as 24
bottles.
The automatic sampler has the capability to be programmed to draw a certain volume of
sample every few minutes and deposit each sample into one bottles that are preserved or
refrigerated. At the end of the sampling period, the operator can retrieve the bottles, bring
them back to the lab and create a single composite sample. Analysis can now be
performed on a single composite sample that is more representative of the wastewater
quality than a grab sample.
Unweighted Composite
An unweighted composite collects the same sample volume at a constant time interval.
For example, the operator collects 100 ml every hour for 6 hours. At the end of the time
period, there will be 12 individual bottles representing the wastewater quality over the 6
hour time period. The operator now composites the samples by pouring from each bottle
into a large bottle and mixes the composite.
Flow Weighted Composite

A flow meter is connected to the composite sampler and the sampler is programed to draw
at different flow intervals. As the flow increases so does the number of samples.

The refrigerated automatic WWT sampler will have a Data programmer that will
allow you to set the time to collect the sample or samples. This machine can also
measure the amount of the sample. These can devices also be used for the
collection of composite samples. Sometimes you will see a pH probe with real-time
readings sent to the Operator’s Command Center. These are a common sight at
most wastewater plants and SIUs.

pH Testing Section
In water and wastewater processes, pH is a measure of the acidity or basicity of an

aqueous solution. Solutions with a pH greater than 7 are basic or alkaline and solution or
samples with a pH less than 7 are said to be acidic. Pure water has a pH very close to 7.
Primary pH standard values are determined using a concentration cell with transference,
by measuring the potential difference between a hydrogen electrode and a standard
electrode such as the silver chloride electrode. The pH scale is traceable to a set of
standard solutions whose pH is established by international agreement.
Measurement of pH for aqueous solutions can be done with a glass electrode and a pH
meter, or using indicators like strip test paper.
pH measurements are important in water and wastewater processes (sampling) but also
in medicine, biology, chemistry, agriculture, forestry, food science, environmental science,
oceanography, civil engineering, chemical engineering, nutrition, water treatment & water
purification, and many other applications.

Mathematically, pH is the measurement of hydroxyl ion activity and expressed as the
negative logarithm of the activity of the (solvated) hydronium ion, more often expressed
as the measure of the hydronium ion concentration.
Contents
History
The scientific discovery of the p[H] concept of was first introduced by Danish chemist
Søren Peder Lauritz Sørensen at the Carlsberg Laboratory back in 1909 and revised to
the modern pH in 1924 to accommodate definitions and measurements in terms of
electrochemical cells. In the first papers, the notation had the "H" as a subscript to the
lowercase "p", as so: pH.
Alkalinity
Alkalinity is the quantitative capacity of an aqueous solution to neutralize an acid.
Measuring alkalinity is important in determining a stream's ability to neutralize acidic
pollution from rainfall or wastewater. It is one of the best measures of the sensitivity of the
stream to acid inputs. There can be long-term changes in the alkalinity of rivers and
streams in response to human disturbances.
Reference. Bates, Roger G. Determination of pH: theory and practice. Wiley, 1973.

pH Definition and Measurement
Technical Definition of pH
In technical terms, pH is defined as the decimal logarithm of the reciprocal of the
hydrogen ion activity, aH+, in a solution.
Ion-selective electrodes are often used to measure pH, respond to activity.
In this calculation of electrode potential, E, follows the Nernst equation, which, for the
hydrogen ion can be written as
where E is a measured potential, E0 is the standard electrode potential, R is the gas

constant, T is the temperature in kelvin, F is the Faraday constant. For H+ number of
electrons transferred is one. It follows that electrode potential is proportional to pH when
pH is defined in terms of activity.

International Standard ISO 31-8 is the standard for the precise measurement of pH as
follows: A galvanic cell is set up to measure the electromotive force (EMF) between a
reference electrode and an electrode sensitive to the hydrogen ion activity when they are
both immersed in the same aqueous solution.
The reference electrode may be a silver chloride electrode or a calomel electrode. The
hydrogen-ion selective electrode is a standard hydrogen electrode.
Reference electrode | concentrated solution of KCl || test solution | H2 | Pt
Firstly, the cell is filled with a solution of known hydrogen ion activity and the emf, ES, is
measured. Then the emf, EX, of the same cell containing the solution of unknown pH is
measured.
The difference between the two measured emf values is proportional to pH. This method
of calibration avoids the need to know the standard electrode potential. The
proportionality
constant, 1/z is ideally equal to the "Nernstian slope".
If you were to apply this practice the above calculation, a glass electrode is used rather
than the cumbersome hydrogen electrode. A combined glass electrode has an in-built
reference electrode. It is calibrated against buffer solutions of known hydrogen ion activity.
IUPAC has proposed the use of a set of buffer solutions of known H+ activity.
Two or more buffer solutions should be used in order to accommodate the fact that the
"slope" may differ slightly from ideal.
The electrode is first immersed in a standard solution and the reading on a pH meter is
adjusted to be equal to the standard buffer's value, to implement the proper calibration.
The reading from a second standard buffer solution is then adjusted, using the "slope"
control, to be equal to the pH for that solution. Further details, are given in the IUPAC
recommendations.
When more than two buffer solutions are used the electrode is calibrated by fitting
observed pH values to a straight line with respect to standard buffer values. Commercial
standard buffer solutions usually come with information on the value at 25 °C and a

correction factor to be applied for other temperatures. The pH scale is logarithmic and pH
is a dimensionless quantity.
pH Indicators
Visual comparison of the color of a test solution with a standard color chart provides a
means to measure pH accurate to the nearest whole number. Indicators may be used to
measure pH, by making use of the fact that their color changes with pH. More precise
measurements are possible if the color is measured spectrophotometrically, using a
colorimeter of spectrophotometer. Universal indicator consists of a mixture of indicators
such that there is a continuous color change from about pH 2 to pH 10. Universal indicator
paper is made from absorbent paper that has been impregnated with universal indicator.
pOH
pOH is sometimes used as a measure of the concentration of hydroxide ions, OH−, or
alkalinity. pOH values are derived from pH measurements. The concentration of
hydroxide ions in water is related to the concentration of hydrogen ions by
where KW is the self-ionization constant of water. Taking logarithms
So, at room temperature pOH ≈ 14 − pH. However this relationship is not strictly valid in
other circumstances, such as in measurements of soil alkalinity.
Extremes of pH
Measurement of pH below about 2.5 (ca. 0.003 mol dm−3 acid) and above about 10.5 (ca.
0.0003 mol dm−3 alkali) requires special procedures because, when using the glass
electrode, the Nernst law breaks down under those conditions.
Extreme pH measurements imply that the solution may be concentrated, so electrode

potentials are affected by ionic strength variation. At high pH the glass electrode may be
affected by "alkaline error", because the electrode becomes sensitive to the concentration
of cations such as Na+ and K+ in the solution. Specially constructed electrodes are
available which partly overcome these problems. Runoff from industrial outfalls, restaurant
grease, mines or mine tailings can produce some very low pH values.
Non-aqueous Solutions
Hydrogen ion concentrations (activities) can be measured in non-aqueous solvents. pH
values based on these measurements belong to a different scale from aqueous pH values,
because activities relate to different standard states. Hydrogen ion activity, aH+, can be
defined as:
where μH+ is the chemical potential of the hydrogen ion, μoH+ is its chemical potential in the
chosen standard state, R is the gas constant and T is the thermodynamic temperature.
Therefore pH values on the different scales cannot be compared directly, requiring an
intersolvent scale which involves the transfer activity coefficient of hydrolyonium ion.

pH is an example of an acidity function. Other acidity functions can be defined. For
example, the Hammett acidity function, H0, has been developed in connection with
superacids.
The concept of "Unified pH scale" has been developed on the basis of the absolute
chemical potential of the proton. This scale applies to liquids, gases and even solids.
Applications
Water has a pH of pKw/2, so the pH of pure water is about 7 at 25 °C; this value varies
with temperature. When an acid is dissolved in water, the pH will be less than that of pure
water. When a base, or alkali, is dissolved in water, the pH will be greater than that of pure
water.
A solution of a strong acid, such as hydrochloric acid, at concentration 1 mol dm−3 has a
pH of 0. A solution of a strong alkali, such as sodium hydroxide, at concentration 1 mol
dm−3, has a pH of 14. Thus, measured pH values will lie mostly in the range 0 to 14, though
negative pH values and values above 14 are entirely possible.
Since pH is a logarithmic scale, a difference of one pH unit is equivalent to a tenfold

difference in hydrogen ion concentration.
The pH of an aqueous solution of pure water is slightly different from that of, a salt such
as sodium chloride even though the salt is neither acidic nor basic. In this case, the
hydrogen and hydroxide ions' activity is dependent on ionic strength, so Kw varies with
ionic strength. The pH of pure water decreases with increasing temperatures. One
example is the pH of pure water at 50 °C is 6.55.
Seawater
The pH of seawater plays an important role in the ocean's carbon cycle, and there is
evidence of ongoing ocean acidification caused by carbon dioxide emissions. pH
measurement can be complicated by the chemical properties of seawater, and several
distinct pH scales exist in chemical oceanography.
As part of its operational definition of the pH scale, the IUPAC defines a series of buffer
solutions across a range of pH values (often denoted with NBS or NIST designation).
These solutions have a relatively low ionic strength (~0.1) compared to that of seawater
(~0.7), and, as a consequence, are not recommended for use in characterizing the pH of
seawater, since the ionic strength differences cause changes in electrode potential.
To resolve this problem, an alternative series of buffers based on artificial seawater was
developed. This new series resolves the problem of ionic strength differences between
samples and the buffers. The newest pH scale is referred to as the total scale, often
denoted as pHT.

Calculations of pH
The calculation of the pH of a solution containing acids and/or bases is an example of a
chemical speciation calculation, that is, a mathematical procedure for calculating the
concentrations of all chemical species that are present in the solution.
The complexity of the procedure depends on the nature of the solution.
If the pH of a solution contains a weak acid requires the solution of a quadratic equation.
If the pH of a solution contains a weak base may require the solution of a cubic equation.
For strong acids and bases no calculations are necessary except in extreme situations.
The general case requires the solution of a set of non-linear simultaneous equations.
A complicating factor is that water itself is a weak acid and a weak base. It dissociates
according to the equilibrium
with a dissociation constant, Kw defined as
where [H+] represents for the concentration of the aquated hydronium ion and [OH-]
stands for the concentration of the hydroxide ion. Kw has a value of about 10−14 at 25 °C,
so pure water has a pH of approximately 7.
This equilibrium needs to be considered at high pH and when the solute concentration is
extremely low.

Strong Acids and Bases
Strong Acids and Bases

Strong acids and bases are compounds that, for practical purposes, are completely
dissociated in water. Under normal circumstances this means that the concentration of
hydrogen ions in acidic solution can be taken to be equal to the concentration of the acid.
The pH is then equal to minus the logarithm of the concentration value.
Hydrochloric acid (HCl) is an example of a strong acid. The pH of a 0.01M solution of HCl
is equal to −log10(0.01), that is, pH = 2.
Sodium hydroxide, NaOH, is an example of a strong base. The p[OH] value of a 0.01M
solution of NaOH is equal to −log10(0.01), that is, p[OH] = 2.
From the definition of p[OH] above, this means that the pH is equal to about 12. For
solutions of sodium hydroxide at higher concentrations the self-ionization equilibrium must
be taken into account.
Weak Acids and Bases

A weak acid or the conjugate acid of a weak base can be treated using the same
formalism.
Acid:
Base:
First, an acid dissociation constant is defined as follows. Electrical charges are omitted
from subsequent equations for the sake of generality
and its value is assumed to have been determined by experiment. This being so, there
are three unknown concentrations, [HA], [H+] and [A-] to determine by calculation. Two
additional equations are needed.

One way to provide them is to apply the law of mass conservation in terms of the two
"reagents" H and A.
C stands for analytical concentration. In some texts one mass balance equation is
replaced by an equation of charge balance. This is satisfactory for simple cases like this
one, but is more difficult to apply to more complicated cases as those below.
Together with the equation defining Ka, there are now three equations in three unknowns.
When an acid is dissolved in water CA = CH = Ca, the concentration of the acid, so [A] =
[H]. After some further algebraic manipulation an equation in the hydrogen ion
concentration may be obtained.

Alkalinity
Introduction
Alkalinity of water is its acid-neutralizing capacity. It is the sum of all the titratable bases.
The measured value may vary significantly with the end-point pH used. Alkalinity is a
measure of an aggregate property of water and can be interpreted in terms of specific
substances only when the chemical composition of the sample is known.
Alkalinity is significant in many uses and treatments of natural waters and

wastewaters. Because the alkalinity of many surface waters is primarily a function of
carbonate, bicarbonate, and hydroxide content, it is taken as an indication of the
concentration of these constituents. The measured values also may include contributions
from borates, phosphates, silicates or other bases if these are present. Alkalinity in
excess of alkaline earth metal concentrations is significant in determining the suitability of
water for irrigation. Alkalinity measurements are used in the interpretation and control of
water and wastewater treatment processes.
Titration Method
a. Principle
Hydroxyl ions present in a sample, because of dissociation or hydrolysis of solutes react
with additions of standard acid. Alkalinity thus depends on the end-point pH used.
b. Reagents
i) Standard Hydrochloric Acid – 0.02 N.
ii) Methyl Orange Indicator – Dissolve 0.1 g of methyl orange in distilled water and dilute
to 1 liter.
iii) Sodium carbonate solution, 0.02 N : Dry 3 to 5 g primary standard Na2CO3 at 250oC
for 4 h and cool in a desiccator. Weigh 1.03 gm.
(to the nearest mg), transfer to a 1-L volumetric flask, fill flask to the mark with distilled
water, dissolve and mix reagent. Do no keep longer than 1 week.
c. Procedure
Titrate over a white surface 100 ml of the sample contained in a 250-ml conical flask
with standard hydrochloric acid using two or three drops of methyl orange Indicator.
(NOTE – If more than 30 ml of acid is required for the titration, a smaller suitable aliquot
of the sample shall be taken.)
d. Calculation
Total alkalinity (as CaCO3), mg/l = 10 V or NxVx50x1000
T.A. (as CaCO3) = ----------------------

Sample Amount
Where N = Normality of HCl used
V = volume in ml of standard hydrochloric acid used in the titration.
Alkalinity to Phenolphthalein
The sample is titrated against standard acid using phenolphthalein indicator.
a. Reagents
i) Phenolphthalein Indicator Solution :

Dissolve 0.1 g of phenolphthalein in 60 ml of ETHANOL and dilute with Distilled water to
100 ml.
ii) Standard hydrochloric Acid – 0.02 N.
b. Procedure
Add 2 drops of phenolphthalein indicator solution to a sample of suitable size, 50 or 100
ml, in a conical flask and titrate over a while surface with standard hydrochloric acid.
c. Calculation
1000 V1
Alkalinity to phenolphthalein (as CaCO3), mg/l = --------------------
V2
Where
V1 = volume in ml of standard hydrochloric acid used in the titration, and
V2 = Volume in ml of the sample taken for the test.
Caustic Alkalinity
a. General
Caustic alkalinity is the alkalinity corresponding to the hydroxides present in water and is
calculated from total alkalinity (T) and alkalinity to phenolphthalein (P).
b. Procedure
Determine total alkalinity and
alkalinity to phenolphthalein and
calculate caustic alkalinity as Caustic
shown in Table below. Alkalinity or Carbonate Bicarbonate
Result of Titration Caustic Hydroxide Alkalinity as Concentration as
Alkalinity or Hydroxide Alkalinity Alkalinity as CaCO3 CaCO3
as CaCO3 Carbonate Alkalinity CaCO3
as CaCO3 Bicarbonate
Concentration as CaCO3
Result of Titration
P=0 0 0 0
P<1/2T 0 2P T-2P
P=1/2T 0 2P 0
P>1/2T 2P-T 2(T-P) 0
P=T T 0 0
The alkalinity of water is a measure of its capacity to neutralize acids. The alkalinity of
natural water is due to the salts of carbonate, bicarbonate, borates, silicates and
phosphates along with the hydroxyl ions in free state.
However, the major portion of the alkalinity in natural waters is caused by hydroxide,
carbonate, and bicarbonates which may be ranked in order of their association with high
pH values.
Alkalinity values provide guidance in applying proper doses of chemicals in water and
waste water treatment processes, particularly in coagulation and softening.
Alkalinity (Total)
References: ASTM D 1067-92, Acidity or Alkalinity of Water.

APHA Standard Methods, 19th ed., p. 2-26, method 2320B (1995).
EPA Methods for Chemical Analysis of Water and Wastes, method 310.1 (1983).
The alkalinity of water is a measurement of its buffering capacity or ability to react with
strong acids to a designated pH. Alkalinity of natural waters is typically a combination of
bicarbonate, carbonate, and hydroxide ions. Sewage and wastewaters usually exhibit
higher alkalinities either due to the presence of silicates and phosphates or to a
concentration of the ions from natural waters.
Alkalinity inhibits corrosion in boiler and cooling waters and is therefore a desired quality
that must be maintained. Alkalinity is also measured as a means of controlling water and
wastewater treatment processes or the quality of various process waters. In natural
waters, excessive alkalinity can render water unsuitable for irrigation purposes and may
indicate the presence of industrial effluents.
The Titrimetric Method

CHEMetrics' tests determine total or "M" alkalinity using an acid titrant and a pH indicator.
The end point of the titration occurs at pH 4.5. Results are expressed as ppm (mg/L)
CaCO3.
Hardness (calcium)
Reference: West, T. S., DSC, Ph.D., Complexometry with EDTA and Related Reagents,
3rd ed., p. 46, 164 (1969).
Originally described as water's capacity to precipitate soap, hardness is one of the most
frequently determined qualities of water. It is a composite of the calcium, magnesium,
strontium, and barium concentrations in a sample. The current practice is to assume total
hardness refers to the calcium and magnesium concentrations only.
Completely de-hardened water, resulting from sodium zeolite or other suitable ion
exchange treatment, is required for various processes-including power generation,
printing and photo finishing, pulp and paper manufacturing, and food and beverage
processing. Hard water can cause scale formation on heat exchange surfaces, resulting
in decreased heat transfer and equipment damage.
The Titrimetric Method. This method is specific for calcium hardness. The EGTA titrant in
alkaline solution is employed with zincon indicator. Results are expressed as ppm (mg/L)
CaCO3.
Shelf-life. 8 months. Although the reagent itself is stable, the endpoint indicator has a
limited shelf-life. We recommend stocking quantities that will be used within 7 months.

Dissolved Oxygen
Dissolved oxygen (DO) in water is not considered a contaminant. However, the (DO) level is
important because too much or not enough dissolved oxygen can create unfavorable conditions.
Generally, a lack of (DO) in natural waters creates anaerobic conditions. Anaerobic means without
air. Certain bacteria thrive under these conditions and utilize the nutrients and chemicals available
to exist. Under anaerobic conditions the reaction is:
Anaerobic:
Organics  intermediates + CO2 + H2O + energy
Where the intermediates are butyric acid, mercaptans and hydrogen sulfide gas. At least two
general forms of bacteria act in balance in a wastewater digester: Saprophytic organisms and
Methane Fermenters. The saprophytes exist on dead or decaying materials. The methane
fermenters live on the volatile acids produced by these saprophytes. The methane fermenting
bacteria require a pH range of 6.6 to 7.6 to be able to live and reproduce. Aerobic conditions
indicate that dissolved oxygen is present. Aerobic bacteria require oxygen to live and thrive. When
aerobes decompose organics in the water, the result is carbon dioxide and water.
Aerobic:
Organics + Oxygen  CO2 + H2O + energy
Dissolved Oxygen in a water sample can be detrimental to metal pipes in high concentrations
because oxygen helps accelerate corrosion. Oxygen is an important component in water plant
operations. Its primary value is to oxidize iron and manganese into forms that will precipitate out
of the water. It also removes excess carbon dioxide. The amount of dissolved oxygen in a water
sample will affect the taste of drinking water also.
Methods of Determination
There are two methods that we will be using in the
lab. The membrane electrode method procedure is
based on the rate of diffusion of molecular oxygen
across a membrane. The other is a titrimetric
procedure (Winkler Method) based on the oxidizing
property of the (DO). Many factors determine the
solubility of oxygen in a water sample. Temperature,
atmospheric pressure, salinity, biological activity and
pH all have an effect on the (DO) content.
Iodometric Test
The iodometric (titration) test is very precise and reliable for (DO) analysis of samples free from
particulate matter, color and chemical interferences. Reactions take place with the addition of
certain chemicals that liberate iodine equivalent to the original (DO) content. The iodine is then
measured to the starch iodine endpoint. We then calculate the dissolved oxygen from how much
titrate we use. Certain oxidizing agents can liberate iodine from iodides (positive interference), and
some reducing agents reduce iodine to iodide (negative interferences). The alkaline Iodide-Azide
reagent effectively removes interference caused by nitrates in the water sample, so a more
accurate determination of (DO) can be made.
Methods of analysis are highly dependent on the source and characteristics of the sample. The
membrane electrode method involves an oxygen permeable plastic membrane that serves as a
diffusion barrier against impurities. Only molecular oxygen passes through the membrane and is
measured by the meter. This method is excellent for field testing and continuous monitoring.
Membrane electrodes provide an excellent method for (DO) analysis in polluted, highly colored
turbid waters and strong waste effluents.

These interferences could cause serious errors in other procedures. Prolonged usage in waters
containing such gases as H2S tends to lower cell sensitivity. Frequent changing and calibrating of
the electrode will eliminate this interference.
Samples are taken in BOD bottles where agitation or contact with air is at a minimum. Either
condition can cause a change in the gaseous content. Samples must be determined immediately
for accurate results.
The dissolved oxygen test is the one of the most important analyses in determining the quality of
natural waters. The effect of oxidation wastes on streams, the suitability of water for fish and other
organisms and the progress of self-purification can all be measured or estimated from the dissolved
oxygen content. In aerobic sewage treatment units, the minimum objectionable odor potential,
maximum treatment efficiency and stabilization of wastewater are dependent on maintenance of
adequate dissolved oxygen. Frequent dissolved oxygen measurement is essential for adequate
process control.
Terms Review
Aerobic (AIR-O-bick) - a condition in which free or dissolved oxygen is present in the aquatic
environment.
Aerobic Bacteria (aerobes) – bacteria which will live and reproduce only in an environment
containing oxygen. Oxygen combined chemically, such as in water molecules (H2O), cannot be
used for respiration by aerobes.
Anaerobic (AN-air O-bick) - a condition in which “free” or dissolved oxygen is not present in the
aquatic environment.
Anaerobic Bacteria (anaerobes) – bacteria that thrive without the presence of oxygen.
Saprophytic Bacteria – bacteria that break down complex solids to volatile acids.
Methane Fermenters – bacteria that break down the volatile acids to methane (CH4) carbon
dioxide (CO2) and water (H2O).
Oxidation – the addition of oxygen to an element or compound, or removal of hydrogen or an

electron from an element or compound in a chemical reaction. The opposite of reduction.
Reduction – the addition of hydrogen, removal of oxygen or addition of electrons to an element

or compound. Under anaerobic conditions in wastewater, sulfur or compounds elemental sulfur
are reduced to H2S or sulfide ions.

Procedure for Dissolved Oxygen Determination
METER-PROBE METHOD
1. Collect a water sample in the clean 300-ml glass stoppered

BOD bottle for two or three minutes to make sure there
are no air bubbles trapped in the bottle. Do one Tap water
sample and one DI water sample. Mark the BOD bottles.
2. Insert the DO probe from the meter into your BOD bottles.
Record the DO for Tap and DI water. Now continue with
the Winkler Burette method.
PROCEDURES FOR WINKLER BURET METHOD
1. Add the contents of one MANGANESE SULFATE powder pillow and one ALKALINE
IODIDE-AZIDE reagent powder pillow to each of your BOD bottles (TAP and DI)
2. Immediately insert the stoppers so that no air is trapped in the bottles and invert several
times to mix. A flocculent precipitate will form. It will be brownish-orange if dissolved
oxygen is present or white if oxygen is absent.
3. Allow the samples to stand until the floc has settled and leaves the solution clear (about
10 minutes). Again invert the bottles several times to mix and let stand until the solution
is clear.
4. Remove the stoppers and add the contents of one SULFAMIC ACID powder pillow to
each bottle. Replace the stoppers, being careful not to trap any air bubbles in the bottles,
and invert several times to mix. The floc will dissolve and leave a yellow color if
dissolved oxygen is present.
5. Measure 200 ml of the prepared solution by filling a clean 250-ml graduated cylinder to
the 200-ml mark. Pour the solutions into clean 250-ml Erlenmeyer flasks. Save the last
100 mls for a duplicate.
6. Titrate the prepared solutions with PAO Titrant, 0.025N, to a pale yellow color. Use a
white paper under the flask.
7. Add two droppers full of Starch Indicator Solution and swirl to mix. A dark blue color will
develop.
8. Continue the titration until the solution changes from dark blue to colorless (end point).
Go Slow- drop by drop. Record the burette reading to the nearest 0.01mls.
9. The total number of ml of PAO Titrant used is equal to the mg/L dissolved oxygen.

Dissolved Oxygen Results
Meter Results
1. De-ionized water ___________________________________mg/L
2. Tap water ___________________________________mg/L
3. What is the meter procedure measuring?
4. What factors would determine which the best method to use is?
5. What are two forms of bacteria present in a wastewater digester?
Wrinkler Method Results

1. De-ionized Water
200ml final Burette reading-
Sample initial Burette reading- -_______________ = ______________mg/L

duplicate initial Burette reading- -______________dup=_____________mg/L
mls x 2
2. Tap water
Sample initial Burette reading- -______________=________________mg/L
mls
100ml final Burette reading
Sample initial Burette reading- -______________=_________________mg/L
mls x 2
3. What are some factors that can alter the (DO) content prior to testing?
4. Were your samples anaerobic or aerobic?
5. Why is it important to monitor the (DO) content of water and wastewater?
Be specific and give a detailed explanation.

Total Dissolved Solids Section
Water is a good solvent and picks up impurities easily. Pure water is tasteless, colorless, and
odorless and is often called the universal solvent. Dissolved solids refer to any minerals, salts,
metals, cations or anions dissolved in water. Total dissolved solids (TDS) comprise inorganic salts
(principally calcium, magnesium, potassium, sodium, bicarbonates, chlorides and sulfates) and
some small amounts of organic matter that are dissolved in water.
TDS in drinking-water originate from natural sources, sewage, urban run-off, industrial wastewater,
and chemicals used in the water treatment process, and the nature of the piping or hardware used
to convey the water, i.e., the plumbing. In the United States, elevated TDS has been due to natural
environmental features such as: mineral springs, carbonate deposits, salt deposits, and sea water
intrusion, but other sources may include: salts used for road de-icing, anti-skid materials, drinking
water treatment chemicals, stormwater and agricultural runoff, and point/non-point wastewater
discharges.
In general, the total dissolved solids concentration is the sum of the cations (positively charged)
and anions (negatively charged) ions in the water. Therefore, the total dissolved solids test
provides a qualitative measure of the amount of dissolved ions, but does not tell us the nature or
ion relationships.
In addition, the test does not provide us insight into the specific water quality issues, such as:
Elevated Hardness, Salty Taste, or Corrosiveness. Therefore, the total dissolved solids test is
used as an indicator test to determine the general quality of the water.
Total Solids
The term "total solids" refers to matter suspended
or dissolved in water or wastewater, and is
related to both specific conductance and turbidity.
Total solids (also referred to as total residue) are

the term used for material left in a container after
evaporation and drying of a water sample.
Total Solids includes both total suspended solids,

the portion of total solids retained by a filter and
total dissolved solids, the portion that passes
through a filter (American Public Health
Association, 1998).
Total solids can be measured by evaporating a

water sample in a weighed dish, and then drying
the residue in an oven at 103 to 105° C.
The increase in weight of the dish represents the

total solids. Instead of total solids, laboratories
often measure total suspended solids and/or total
dissolved solids.

Lab tech removing filter for TSS analysis.

Total Suspended Solids (TSS)
Total Suspended Solids (TSS) are solids in water that can be trapped by a filter. TSS can
include a wide variety of material, such as silt, decaying plant and animal matter, industrial
wastes, and sewage. High concentrations of suspended solids can cause many problems
for stream health and aquatic life.
High TSS can block light from reaching submerged vegetation. As the amount of light
passing through the water is reduced, photosynthesis slows down. Reduced rates of
photosynthesis causes less dissolved oxygen to be released into the water by plants. If
light is completely blocked from bottom dwelling plants, the plants will stop producing
oxygen and will die. As the plants are decomposed, bacteria will use up even more oxygen
from the water. Low dissolved oxygen can lead to fish kills.
Sampling downstream from a wastewater plant’s discharge point.
High TSS can also cause an increase in surface water temperature, because the
suspended particles absorb heat from sunlight. This can cause dissolved oxygen levels to
fall even further (because warmer waters can hold less DO), and can harm aquatic life in
many other ways, as discussed in the temperature section. (The decrease in water clarity
caused by TSS can affect the ability of fish to see and catch food.
Suspended sediment can also clog fish gills,

reduce growth rates, decrease resistance to
disease, and prevent egg and larval
development. When suspended solids settle
to the bottom of a water body, they can
smother the eggs of fish and aquatic insects,
as well as suffocate newly hatched insect
larvae. Settling sediments can fill in spaces
between rocks which could have been used
by aquatic organisms for homes.
Dead fish in lake using reclaimed water.►
High TSS in a water body can often mean higher concentrations of bacteria, nutrients,
pesticides, and metals in the water. These pollutants may attach to sediment particles on
the land and be carried into water bodies with storm water. In the water, the pollutants
may be released from the sediment or travel farther downstream. High TSS can cause
problems for industrial use, because the solids may clog or scour pipes and machinery.

Measurement of Total Suspended Solids
To measure TSS, the water sample is filtered through a
pre-weighed filter. The residue retained on the filter is
dried in an oven at 103 to 105° C until the weight of the
filter no longer changes. The increase in weight of the
filter represents the total suspended solids. TSS can
also be measured by analyzing for total solids and
subtracting total dissolved solids.
Total Dissolved Solids (TDS) are solids in water that

can pass through a filter (usually with a pore size of 0.45
micrometers). TDS is a measure of the amount of
material dissolved in water.
This material can include carbonate, bicarbonate,

chloride, sulfate, phosphate, nitrate, calcium,
magnesium, sodium, organic ions, and other ions. A
certain level of these ions in water is necessary for
aquatic life. Changes in TDS concentrations can be
harmful because the density of the water determines the
flow of water into and out of an organism's cells (Mitchell
and Stapp, 1992). However, if TDS concentrations are
too high or too low, the growth of many aquatic lives can
be limited, and death may occur.
Similar to TSS, high concentrations of TDS may also reduce water clarity, contribute to a
decrease in photosynthesis, combine with toxic compounds and heavy metals, and lead
to an increase in water temperature. TDS is used to estimate the quality of drinking water,
because it represents the amount of ions in the water. Water with high TDS often has a
bad taste and/or high water hardness, and could result in a laxative effect.
The TDS concentration of a water sample can be estimated from specific conductance if
a linear correlation between the two parameters is first established. Depending on the
chemistry of the water, TDS (mg/l) can be estimated by multiplying specific conductance
(micromhos/cm) by a factor between 0.55 and 0.75. TDS can also be determined by
measuring individual ions and adding them up.
Conductivity Meter

Suspended Matter for Mixed Liquor and Return Sludge (MLSS)
Suspended matter in mixed liquor and return sludge can be used to determine process
status, estimate the quantity of biomass, and evaluate the results of process
adjustments.
Apparatus
- Buchner funnel and adaptor
- Filter flask
- Filter paper 110 mm diam., Whatman 1-4
- 1030 drying oven
- Desiccator
- Balance
- Graduated Cylinder
Procedure
1. Dry the filter papers in oven at 1030 c to remove all
traces of moisture.
2. Remove papers from oven and desiccate to cool for

approximately 5 minutes.
3. Weigh to the nearest 0.01g and record the mass (W1)
4. Place the paper in the bottom of the Buchner funnel and carefully arrange so
that the outer edges lay snugly along the side. Careful not to touch it with
your finger. Use a glass rod. Wet the paper, turn on the vacuum and make a
good seal, make a pocket covering the bottom of the funnel.
5. Add 20 to 100 mls of sample at a sufficient rate to keep the bottom of the
funnel covered, but not fast enough to overflow the pocket made by the filter
paper. Record the Volume used.
6. Remove the filter paper with tweezers. Dry in a 1030 c oven for 30 minutes.
Remove and desiccate. Reweigh the filter paper (W2) to the nearest 0.01g.
Calculation:
mg/L Suspended Matter
(W2 ) - (W1) x 1000 ML/L

ML Sample
Where:
(W1) and (W2) are expressed in mg.
(W1) = mass of the prepared filter
(W2) = mass of the filter and sample after the filtration step.

Total dissolved solids - The weight per unit volume of all volatile and non-volatile solids
dissolved in a water or wastewater after a sample has been filtered to remove colloidal
and suspended solids.
Top left, filters being baked at 105oC. Right photograph, filters in desiccant.

Sludge Volume Index (SVI)
1. Pour sample of mixed liquor from the process
into a 2 liter settlometer.
2. Allow it to settle for 30 minutes
3. After the time period, read the marking to

determine the volume occupied by the settled
sludge and the reading is expressed in terms of
mL/L and this is figure is known as the sludge
volume SV value.
4. Next, for MLSS, there are actually two

approaches to get the value. A conventional
standard approach is by filtering the sludge,
drying it and then weigh the second portion of
the mixed liquid. However, this can be time
consuming and a faster way is by using MLSS
meter.
.
Calculation:
The results obtained from the suspended matter test
and settleability test on aerated mixed liquor are used to obtain the SVI.
Calculation:
SVI = sludge volume SV x 1000

MLSS

The settleometer is a great tool for operators. It indicates how the solids will settle in the
clarifier and the density of the sludge.
During the settleometer test, operators not only check how the solids settle out they can
also determine the rate of denitrification in the clarifier.

Sludge Volume Index Lab Report Worksheet
Suspended Mater Calculations:
(W1) = mg Duplicate (W1) = mg
(W2) = mg (W2) = mg
mls Sample = mls Sample = .
mg/L suspended matter = dup. .
Settleability Calculations:
% settled sludge = ________________________
(ml of sludge in settled mixed liquor or returned sludge x 100)

1000
Sludge Volume Index Calculations:
(ml of sludge in settled mixed liquor in 30 minutes x 1000 mg/g)

mg/L of suspended matter in mixed liquor

Activated sludge process control calculations may include determination of the thirty and
sixty minute settled sludge volume (SSV30 and SSV60), sludge volume index (SVI) and
pounds of waste activated sludge removed from the process. The sample jars used are
1,000 milliliters or 2,000 milliliters.
Some plants are designed in steps for nitrification/denitrification as shown in the picture
above.
Nitrification is a microbial process by which ammonia is sequentially oxidized to nitrite

and then to nitrate. The nitrification process is accomplished primarily by two groups of
autotrophic nitrifying bacteria that can build organic molecules by using energy obtained
from inorganic sources––in this case, ammonia or nitrite.
Denitrification is the process by which nitrates are reduced to gaseous nitrogen by

facultative anaerobes. Facultative anaerobes, such as fungi, can flourish in anoxic
conditions because they break down oxygen containing compounds to obtain oxygen.

Fecal Coliform Analysis
Sample Collection
Fecal coliform must be collected in a clean, sterile borosilicate glass or plastic bottle
containing sodium thiosulfate. Pre-sterilized bags or bottles containing sodium thiosulfate
can also be used. Sodium thiosulfate is added to remove residual chlorine which will kill
fecal coliforms during transit. 0.1 ml of 10% sodium thiosulfate is added to a 120 ml sample
bottle prior to sterilization. The minimum bottle size should be 120 ml to allow enough
head space (1") for proper sample mixing.
Collection Procedure
Select a site that will provide a representative sample. Fecal
coliform samples are always grab samples and should be drawn
directly from the flow stream without using collection other
devices. We do not want to cross contaminate the sample. Keep
the sample bottle lid closed tightly until it is to be filled.
Remove the cap and do not contaminate the inner surface of the
bottle, neck, threads or cap. Fill the container without rinsing,
being sure to leave ample air space to allow mixing. Rinsing will
remove the dechlorinating agent. All samples should be labeled
properly with date and time of collection, sampler's name, and
sample collection location. Leaking sample bottles allow for
contamination of the sample and should be discarded and the
sampling repeated.
Preservation
Fecal coliform samples should be analyzed as soon as possible after collection to prevent
changes to the microorganism population. Fecal coliforms must be transported on ice, if
they cannot be analyzed within 1 hour of collection. Fecal coliforms transported at ambient
temperature may reproduce and higher bias to the numbers than desired or they may be
killed off resulting in lower numbers, if handled poorly such as transport in sunlight. Fecal
coliform samples should be stored by the laboratory in a refrigerator until time of analysis.
The maximum holding time for state or federal permit reporting purposes is 6 hours.

An incubator for the coliform test. The operator will place the sample in this device for 24
to 48 hours depending on the desired results. There are several different methods to
calculate coliform bacteria. This is an older true and tested method.
This glass bottle is used for quality control (QA/QC) for bacteria samples tubes.

This operator is wearing the proper safety measures for preparation of the fecal test.
This operator is splitting the sample for bacteriological analysis. Always wear gloves for
your and others’ safety. We have all seen the operator holds a sandwich in one hand
while working in the lab, or the operator does not wear gloves at all.

Phase microscopes are used to see indicator bugs and other MO’s microorganisms.
This examination is used so that the operator knows how well the process is working.
This is a filter used for the coliform test.

Microlife - Microorganism Section
We talked about the basic components and designs of wastewater treatment now let’s look at the
main “Team Players”. Your process will respond to whatever direction you give it. You can run
your plant (the team) to always try for the better or be content with the way it is. To get the best, it
takes work!
Most activated sludge processes are used to degrade carbonaceous BOD. It is also possible to
design and/or operate the basic system to oxidize ammonia (nitrification).
Many plants are now designed to achieve nitrification. Other system modifications include
phosphorus removal and biological denitrification. Activated sludge plants are usually designed
from pilot plant and laboratory studies.
From this approach, it is possible to design a process based on the amount of time the sludge
spends in the system, generally termed mean cell residence time (MCRT), or on the amount of
food provided to the bacteria in the aeration tank (the food-to-microorganism ratio, F/M). What does
this mean?
Suppose a person ate 10 pounds of hot dogs (BOD) and weighed 200 pounds (MLSS).
What is the ratio of food to weight?
It would be 10 lbs. to 200 lbs. If we divide 200 into 10, the ratio is .05 or 5%.
Activated Sludge Aeration Basin, you can tell by the bubbles.

Ciliate
Amoeba

F/M and MCRT
The following are some general statements about F/M and MCRT assuming that the
environmental conditions are properly controlled.
a. The optimum operating point of either helps obtain the desired effluent concentration.
b. Both provide a means for maintaining the best effluent and sludge quality.
c. Both techniques attempt to regulate rate of growth, metabolism, and stabilization of food
matter.
d. Both techniques indicate the solids level needed to stabilize the food and attain sludge
quality.
e. The desired solids level is controlled by wasting.
1. To maintain – waste amount of net daily
2. To increase – decrease waste rate
3. To decrease – increase waste rate
f. They are interrelated so changing one control changes the other.
g. Once the control point is set, it should remain constant until change in effluent or sludge
quality requires a change.
The operating control point is that point when the best effluent and sludge quality is obtained for
the existing conditions.

Microorganisms in Lagoons
Before we look at the bugs themselves, let’s look at eating habits. Have you ever met a person who
was a picky eater?
You have people who will put their noses up at some things and others who would eat anything.
Predators typically eat from a narrow set of prey, while omnivores and scavengers eat from a
broader food selection.
 Swimming and gliding ciliates engulf bacteria or other prey.

 Stalked ciliates attach to the biomass and vortex suspended bacteria into their gullets,
while crawlers break bacteria loose from the floc surface.
 Predators feed mostly on stalked and swimming ciliates. The omnivores, such as most
rotifers, eat whatever is readily available, while the worms feed on the floc or prey on larger
organisms. Microorganisms are directly affected by their treatment environment.
 Changes in food, dissolved oxygen, temperature, pH, total dissolved solids, sludge age,
presence of toxins, and other factors create a dynamic environment for the treatment
organisms.
Food (organic loading) regulates microorganism numbers, diversity, and species when other
factors are not limiting. The relative abundance and occurrence of organisms at different loadings
can reveal why some organisms are present in large numbers while others are absent.
Aerobic Bacteria
The aerobic bacteria that occur are similar to those found in other treatment processes such as
activated sludge. Three functional groups occur: freely dispersed, single bacteria; floc-forming
bacteria; and filamentous bacteria. All function similarly to oxidize organic carbon (BOD) to
produce CO2 and new bacteria (new sludge).
Many bacterial species that degrade wastes grow as single

bacteria dispersed in the wastewater. Although these readily
oxidize BOD, they do not settle and hence often leave the system
in the effluent as solids (TSS). These tend to grow in lagoons at
high organic loading and low oxygen conditions. More important
are the floc-forming bacteria, those that grow in a large aggregate
(floc) due to exocellular polymer production (the glycocalyx).
This growth form is important as these flocs degrade BOD and

settle at the end of the process, producing a low TSS effluent.
A number of filamentous bacteria occur in lagoons, usually at specific growth environments.

These generally do not cause any operational problems in lagoons, in contrast to activated
sludge where filamentous bulking and poor sludge settling is a common problem.
Most heterotrophic bacteria have a wide range in environmental tolerance and can function
effectively in BOD removal over a wide range in pH and temperature. Aerobic BOD removal
generally proceeds well from pH 6.5 to 9.0 and at temperatures from 3-4oC to 60-70°C
(mesophilic bacteria are replaced by thermophilic bacteria at temperatures above 35°C). BOD
removal generally declines rapidly below 3-4°C and ceases at 1-2°C.
A very specialized group of bacteria occurs to some extent in lagoons (and other wastewater
treatment systems) that can oxidize ammonia via nitrite to nitrate, termed nitrifying bacteria.
These bacteria are strict aerobes and require a redox potential of at least +200 m V (Holt et al.,
1994).

Aerated Lagoons
The aerated lagoons are basins, normally excavated in earth and operated without solids recycling
into the system. This is the major difference with respect to activated sludge systems. Two types
are the most common: the completely mixed lagoon (also called completely suspended) in which
the concentration of solids and dissolved oxygen are maintained fairly uniform and neither the
incoming solids nor the biomass of microorganisms’ settle, and the facultative (aerobic-anaerobic
or partially suspended) lagoons. In the facultative lagoons, the power input is reduced causing
accumulation of solids in the bottom which undergo anaerobic decomposition, while the upper
portions are maintained aerobic. The main operational difference between these lagoons is the
power input, which is in the order of 2.5-6 Watts per cubic meter (W/m3) for aerobic lagoons while
the requirements for facultative lagoons are of 0.8-1 W/m3. Being open to the atmosphere, the
lagoons are exposed to low temperatures which can cause reduced biological activity and
eventually the formation of ice. This can be partially alleviated by increasing the depth of the basin.
These units require a secondary sedimentation unit, which in some cases can be a shallow basin
excavated in earth, or conventional settling tanks can be used.
Diagram of aerated lagoons.

If excavated basins are used for settling, care should be taken to provide a residence time long
enough for the solids to settle, and there should also be provision for the accumulation of sludge.
There is a very high possibility of offensive odor development due to the decomposition of the
settled sludge, and algae might develop in the upper layers contributing to an increased content of
suspended solids in the effluent.
Odors can be minimized by using minimum depths of up to 2 m, while algae production is reduced
with liquid retention time of less than two days. The solids will also accumulate, all along the
aeration basins in the facultative lagoons and even in comers, or between aeration units in the
completely mixed lagoon. These accumulated solids will, on the whole, decompose in the bottom,
but since there is always a non-biodegradable fraction, a permanent deposit will build up.
Therefore, periodic removal of these accumulated solids becomes necessary. We will cover this
in much more detail in a few more pages.

Advanced Methods of Wastewater Treatment
As our country and the demand for clean water have grown, it has become more important to
produce cleaner wastewater effluents, yet some contaminants are more difficult to remove than
others. The demand for cleaner discharges has been met through better and more complete
methods of removing pollutants at wastewater treatment plants, in addition to pretreatment and
pollution prevention which helps limit types of wastes discharged to the sanitary sewer system.
Currently, nearly all WWTPs provide a minimum of secondary treatment. In some receiving waters,
the discharge of secondary treatment effluent would still degrade water quality and inhibit aquatic
life. Further treatment is needed.
Discharge point from a wastewater plant into a wetlands project.
Advanced Treatment Technologies

Treatment levels beyond secondary are called advanced treatment. Advanced treatment
technologies can be extensions of conventional secondary biological treatment to further stabilize
oxygen-demanding substances in the wastewater, or to remove nitrogen and phosphorus.
Advanced treatment may also involve physical-chemical separation techniques such as adsorption,
flocculation/precipitation, membranes for advanced filtration, ion exchange, and reverse osmosis.
In various combinations, these processes can achieve any degree of pollution control desired. As
wastewater is purified to higher and higher degrees by such advanced treatment processes, the
treated effluents can be reused for urban, landscape, and agricultural irrigation, industrial cooling
and processing, recreational uses and water recharge, and even indirect augmentation of drinking
water supplies.

The rotating biological contactor (RBC) is a fixed film biological secondary treatment
device. The basic process is similar to that occurring in the trickling filter. In operation, a
media, consisting of a series of circular disks mounted side by side on a common shaft is
rotated through the wastewater flow.
RBC’s with integral units treat unsettled sewage and has the capability of providing
primary and secondary settling in the unit.

The most often used ponds in domestic wastewater treatment are the stabilization pond
and facultative lagoon. The stabilization pond is designed to be aerobic throughout its
depth and the facultative lagoon will be anaerobic at the bottom and aerobic at the top.
Stabilization ponds provide secondary biological treatment and are the most commonly
used wastewater pond. Stabilization ponds must be preceded by some form of primary
treatment to reduce the solids entering the pond.
Respiration in lakes recycles organic carbon arising from photosynthesis back to

inorganic carbon. Prior to this transformation, the organic carbon is potentially available
to support secondary production.

Flow to the oxidation ditch is aerated and mixed with return sludge from a secondary
clarifier. A typical process flow diagram for an activated sludge plant using an oxidation
ditch is shown above.
An oxidation ditch is a modified activated sludge biological treatment process that utilizes
long solids retention times (SRTs) to remove biodegradable organics. Oxidation ditches
are typically complete mix systems, but they can be modified to approach plug flow
conditions.
An oxidation ditch is a modified activated sludge biological treatment process that utilizes
long solids retention times (SRTs) to remove biodegradable organics.
Oxidation ditches are typically complete mix systems, but they can be modified to
approach plug flow conditions. (Note: as conditions approach plug flow, diffused air must
be used to provide enough mixing. The system will also no longer operate as an oxidation
ditch).
Typical oxidation ditch treatment systems consist of a single or multichannel configuration

within a ring, oval, or horseshoe-shaped basin. As a result, oxidation ditches are called
“racetrack type” reactors. Horizontally or vertically mounted aerators provide circulation,
oxygen transfer, and aeration in the ditch.
Preliminary treatment, such as bar screens and grit removal, normally precedes the
oxidation ditch. Primary settling prior to an oxidation ditch is sometimes practiced, but is
not typical in this design. Tertiary filters may be required after clarification, depending on
the effluent requirements. Disinfection is required and re-aeration may be necessary prior
to final discharge.

Advantages
The main advantage of the oxidation ditch is the ability to achieve removal performance
objectives with low operational requirements and operation and maintenance costs.
Some specific advantages of oxidation ditches include:

 An added measure of reliability and performance over other biological processes owing
to a constant water level and continuous discharge that lowers the weir overflow rate and
eliminates the periodic effluent surge common to other biological processes, such as
SBRs.
 Long hydraulic retention time and complete mixing minimize the impact of a shock load or
hydraulic surge.
 Produces less sludge than other biological treatment processes owing to extended
biological activity during the activated sludge process.
 Energy efficient operations result in reduced energy costs compared with other biological
treatment processes.
Disadvantages
 Effluent suspended solids concentrations are relatively high compared to other
modifications of the activated sludge process.
 Requires a larger land area than other activated sludge treatment options. This can prove
costly, limiting the feasibility of oxidation ditches in urban, suburban, or other areas where
land acquisition costs are relatively high.
Surface aerators, such as brush rotors, disc aerators, draft tube aerators, or fine bubble diffusers
are used to circulate the mixed liquor.
The mixing process entrains oxygen into the mixed liquor to foster microbial growth and the motive
velocity ensures contact of microorganisms with the incoming wastewater. The aeration sharply
increases the dissolved oxygen (DO) concentration but decreases as biomass uptake oxygen as
the mixed liquor travels through the ditch.
Solids are maintained in suspension as the mixed liquor circulates around the ditch. If design SRTs
are selected for nitrification, a high degree of nitrification will occur.
Oxidation ditch effluent is usually settled in a separate secondary clarifier. An anaerobic tank may
be added prior to the ditch to enhance biological phosphorus removal.
An oxidation ditch may also be operated to achieve partial denitrification. One of the most common
design modifications for enhanced nitrogen removal is known as the Modified Ludzack-Ettinger
(MLE) process.

In this process, an anoxic tank is added upstream of the ditch along with mixed liquor
recirculation from the aerobic zone to the tank to achieve higher levels of denitrification.
In the aerobic basin, autotrophic bacteria (nitrifiers) convert ammonia-nitrogen to nitrite-
nitrogen and then to nitrate-nitrogen. In the anoxic zone, heterotrophic bacteria convert
nitrate-nitrogen to nitrogen gas which is released to the atmosphere. Some mixed liquor
from the aerobic basin is recirculated to the anoxic zone to provide a mixed liquor with a
high- concentration of nitrate-nitrogen to the anoxic zone.
Several manufacturers have developed modifications to the oxidation ditch design to

remove nutrients in conditions cycled or phased between the anoxic and aerobic states.
While the mechanics of operation differ by manufacturer, in general, the process consists
of two separate aeration basins, the first anoxic and the second aerobic.
Wastewater and return activated sludge (RAS) are introduced into the first reactor which
operates under anoxic conditions. Mixed liquor then flows into the second reactor
operating under aerobic conditions. The process is then reversed and the second reactor
begins to operate under anoxic conditions.
The oxidation ditch process is a fully demonstrated secondary wastewater treatment

technology, applicable in any situation where activated sludge treatment (conventional or
extended aeration) is appropriate. Oxidation ditches are applicable in plants that require
nitrification because the basins can be sized using an appropriate SRT to achieve
nitrification at the mixed liquor minimum temperature. This technology is very effective in
small installations, small communities, and isolated institutions, because it requires more
land than conventional treatment plants.

The principle role microorganisms have in the activated sludge process is to convert
dissolved and particulate organic matter, measured as biochemical oxygen demand
(BOD), into cell mass. In a conventional activated sludge process, microorganisms use
oxygen to break down organic matter (food) for their growth and survival. Over time and
as wastewater moves through the aeration basin, food (BOD) decreases with a resultant
increase in cell mass (MLSS concentration). The end product is shown above in the
diagram.
Ammonia to nitrite to nitrate process in fresh water.

Algae
Algae are aerobic organisms that are photosynthetic and grow with simple inorganic compounds
CO2, NH3, NO3, and PO4 using light as an energy source. (**Note that algae produce oxygen during
the daylight hours and consume oxygen at night.)
Algae are desirable in lagoons as they generate oxygen needed by bacteria for waste stabili-zation.
Three major groups occur in lagoons, based on their chlorophyll type: brown algae (diatoms), green
algae, and red algae. The predominant algal species at any given time is dependent on growth
conditions, particularly temperature, organic loading, oxygen status, nutrient availability, and
predation pressures. A fourth type of "algae" common in lagoons is the cyano-bacteria or blue-
green bacteria.
These organisms grow much as the true algae, with the exception that most species can fix
atmospheric nitrogen. Blue-green bacteria often bloom in lagoons and some species produce
odorous and toxic by-products.
Blue-Green Bacteria
Blue-green bacteria appear to be favored by poor growth conditions including high temperature,
low light, low nutrient availability (many fix nitrogen) and high predation pressure. Common blue-
green bacteria in waste treatment systems include Aphanothece, Microcystis, Oscillatoria and
Anabaena.
Algae can bloom in lagoons at any time of the year (even under the ice); however, a succession of
algae types occurs over the season. There is also a shift in the algal species present in a lagoon
through the season, caused by temperature and rotifer and Daphnia predation. Diatoms usually
predominate in the wintertime at temperatures <60°F. In the early spring, when predation is low
and lagoon temperatures increase above 60°F, green algae such as Chlorella, Chlamydomonas,
and Euglena often predominate in waste treatment lagoons. The predominant green algae change
to species with spikes or horns such as Scenesdesmus, Micractinium, and Ankistrodesmus later in
the season when Rotifers and Daphnia are active
(these species survive predation better).
Algae grow at warmer temperatures, longer detention time, and when inorganic minerals needed
for growth are in excess. Alkalinity (inorganic carbon) is the only nutrient likely to be limiting for
algal growth in lagoons. Substantial sludge accumulation in a lagoon may become soluble upon
warming in the spring, releasing algal growth nutrients and causing an algal bloom. Sludge
resolution of nutrients is a major cause of high algal growth in a lagoon, requiring
sludge removal from the lagoon for correction.
Algae on the Secondary clarifier, this is not a good sign.

Treatment Lagoon
The pH at a treatment lagoon is determined by the various chemical species of alkalinity that are
present. The main species present are carbon dioxide (CO2, bicarbonate ion (HCO3), and
carbonate ion (CO23). Alkalinity and pH can affect which species will be present. High amounts of
CO2 yield a low lagoon pH, while high amounts of CO23 yield a high lagoon pH.
Bacterial growth on BOD releases CO2 which subsequently dissolves in water to yield carbonic
acid (H2CO3). This rapidly dissociates to bicarbonate ion, increasing the lagoon alkalinity. Bacterial
oxidation of BOD causes a decrease in lagoon pH due to CO2 release.
Algal growth in lagoons has the opposite effect on lagoon pH, raising the pH due to algal use for
growth of inorganic carbon (CO2 and HCO3). Algal growth reduces the lagoon alkalinity which may
cause the pH to increase if the lagoon alkalinity (pH buffer capacity) is low. Algae can grow to such
an extent in lagoons (a bloom) that they consume all of the CO2 and HCO3 present for
photosynthesis, leaving only carbonate (CO23) as the pH buffering species. This causes the pH of
the lagoon to become alkaline. pH values of 9.5 or greater are common in lagoons during algal
blooms, which can lead to lagoon effluent pH violations (in most states this is pH = 9). It should be
noted that an increase in the lagoon pH caused by algal growth can be beneficial. Natural
disinfection of pathogens is enhanced at higher pH.
Phosphorus removal by natural chemical precipitation is greatly enhanced at pH values greater

than pH = 8.5. In addition, ammonia stripping to the atmosphere is enhanced at higher pH values
(NH3 is strippable, not NH4+).
Protozoans and Microinvertebrates

Many higher life forms (animals)
develop in lagoons. These include
protozoans and microinvertebrates
such as rotifers, daphnia, annelids,
chironomids (midge larvae), and
mosquito larvae (often termed the
zooplankton). These organisms play a
role in waste purification by feeding on
bacteria and algae and promoting
flocculation and settling of particulate
material.
Protozoans are the most common

higher life forms in lagoons with about
250 species identified in lagoons to
date (Curds, 1992).
Rotifers and daphnia are particularly important in controlling algal overgrowth and these often
"bloom" when algal concentrations are high.
These microinvertebrates are relatively slow growing and generally only occur in systems with a
detention time of >10 days. Mosquitoes grow in lagoons where shoreline vegetation is not removed,
possibly causing a nuisance and public health problem.
Culex tarsalis, the vector of Western Equine Encephalitis in the western U.S., grows well in
wastewater lagoons (USEPA, 1983). The requirement for a minimum lagoon bank slope and
removal of shoreline vegetation by most regulatory agencies is based on the public health need to
reduce mosquito vectors.

Bacteria Section More information in the Appendix
Bacteria come in a variety of shapes. The simplest shape is a round sphere or ball. Bacteria formed
like this are called cocci (singular coccus). The next simplest shape is cylindrical. Cylindrical
bacteria are called rods (singular rod). Some bacteria are basically rods but instead of being straight
they are twisted, bent or curved, sometimes in a spiral. These bacteria are called spirilla (singular
spirillum). Spirochaetes are tightly coiled up bacteria.
Bacteria are friendly creatures; you never find one bacteria on its own. They tend to live together
in clumps, chains or planes. When they live in chains, one after the other, they are called
filamentous bacteria - these often have long thin cells. When they tend to collect in a plane or a
thin layer over the surface of an object, they are called a biofilm. Many bacteria exist as a biofilm
and the study of biofilms is very important. Biofilm bacteria secrete sticky substances that form a
sort of gel in which they live. The plaque on your teeth that causes tooth decay is a biofilm.

Filamentous Bacteria
Filamentous Bacteria are a type of bacteria that can
be found in a wastewater treatment system.
They function similar to floc forming bacteria since

they degrade BOD quite well. In small amounts, they
are quite good to a biomass. They can add stability
and a backbone to the floc structure that keeps the
floc from breaking up or shearing due to turbulence
from pumps, aeration or transfer of the water. In large
amounts they can cause many problems. Filaments
are bacteria and fungi that grow in long thread-like
strands or colonies.
Site Specific Bacteria

Aeration and biofilm building are the key operational parameters that contribute to the efficient
degradation of organic matter (BOD/COD removal). Over time, the application-specific bacteria
become site-specific as the biofilm develops and matures and is even more efficient in treating the
site-specific waste stream.
Facultative Bacteria
Most of the bacteria absorbing the organic material in a wastewater treatment system are
facultative in nature. This means they are adaptable to survive and multiply in either anaerobic or
aerobic conditions. The nature of individual bacteria is dependent upon the environment in which
they live. Usually, facultative bacteria will be anaerobic unless there is some type of mechanical or
biochemical process used to add oxygen to the wastewater. When bacteria are in the process of
being transferred from one environment to another, the metamorphosis from anaerobic to aerobic
state (and vice versa) takes place within a couple of hours.
Anaerobic Bacteria
Anaerobic bacteria live and reproduce in the absence of free oxygen. They utilize compounds such
as sulfates and nitrates for energy and their metabolism is substantially reduced. In order to remove
a given amount of organic material in an anaerobic treatment system, the organic material must be
exposed to a significantly higher quantity of bacteria and/or detained for a much longer period of
time. A typical use for anaerobic bacteria would be in a septic tank. The slower metabolism of the
anaerobic bacteria dictates that the wastewater be held several days in order to achieve even a
nominal 50% reduction in organic material. That is why septic tanks are always followed by some
type of effluent treatment and disposal process. The advantage of using the anaerobic process is
that electromechanical equipment is not required. Anaerobic bacteria release hydrogen sulfide as
well as methane gas, both of which can create hazardous conditions. Even as the anaerobic action
begins in the collection lines of a sewer system, deadly hydrogen sulfide or explosive methane gas
can accumulate and be life threatening.
Aerobic Bacteria
Aerobic bacteria live and multiply in the presence of free oxygen. Facultative bacteria always
achieve an aerobic state when oxygen is present. While the name "aerobic" implies breathing air,
dissolved oxygen is the primary source of energy for aerobic bacteria. The metabolism of aerobes
is much higher than for anaerobes. This increase means that 90% fewer organisms are needed
compared to the anaerobic process, or that treatment is accomplished in 90% less time. This
provides a number of advantages including a higher percentage of organic removal. The by-
products of aerobic bacteria are carbon dioxide and water. Aerobic bacteria live in colonial
structures called floc and are kept in suspension by the mechanical action used to introduce oxygen
into the wastewater. This mechanical action exposes the floc to the organic material while
treatment takes place. Following digestion, a gravity clarifier separates and settles out the floc.
Because of the mechanical nature of the aerobic digestion process, maintenance and operator
oversight are required.

Protozoans and Metazoans More information in the Appendix
In a wastewater treatment system, the next higher life form above bacteria is protozoans. These
single-celled animals perform three significant roles in the activated sludge process. These include
floc formation, cropping of bacteria and the removal of suspended material. Protozoans are also
indicators of biomass health and effluent quality. Because protozoans are much larger in size than
individual bacteria, identification and characterization is readily performed. Metazoans are very
similar to protozoans except that they are usually multi-celled animals. Macroinvertebrates, such
as nematodes and rotifers, are typically found only in a well developed biomass.
The presence of protozoans and metazoans and the relative abundance of certain species can be
a predictor of operational changes within a treatment plant. In this way, an operator is able to make
adjustments and minimize negative operational effects simply by observing changes in the
protozoan and metazoan population.
Aspidisca
Nematode

Dispersed Growth
Dispersed growth is material suspended within the activated sludge process that has not been
adsorbed into the floc particles. This material consists of very small quantities of colloidal (too small
to settle out) bacteria as well as organic and inorganic particulate material. While a small amount
of dispersed growth between the floc particles is normal, excessive amounts can be carried through
a secondary clarifier. When discharged from the treatment plant, dispersed growth results in higher
effluent solids.
Taxonomy
Taxonomy is the science of categorizing life forms according to their characteristics. Eighteen
different categories are used to define life forms from the broadest down to the most specific. They
are: Kingdom, Phylum, Subphylum, Superclass, Class, Subclass, Cohort, Superorder, Order,
Suborder, Superfamily, Family, Subfamily, Tribe, Genus, Subgenus, Species and Subspecies.
Identifying the genus is usually specific enough to determine the role of the organisms found in a
wastewater treatment system.
Process Indicators
Following taxonomic identification, enumeration and evaluation of the characteristics of the various
organisms and structures present in a wastewater sample, the information can be used to draw
conclusions regarding the treatment process.
Numerous industry references, such as WASTEWATER BIOLOGY: THE MICROLIFE by the

Water Environment Federation, can be used to provide a comprehensive indication of the
conditions within a treatment process. As an example, within most activated sludge processes, the
shape of the floc particles can indicate certain environmental or operational conditions.
A spherical floc particle indicates immature floc, as would be found during start-up or a process
recovery. A mature floc particle of irregular shape indicates the presence of a beneficial quantity of
filamentous organisms and good quality effluent. An excess of dispersed growth could indicate a
very young sludge, the presence of toxic material, excess mechanical aeration or an extended
period of time at low dissolved oxygen levels.
Certain protozoans, such as amoebae and flagellates dominate during a system start-up. Free
swimming ciliates are indicative of a sludge of intermediate health and an effluent of acceptable or
satisfactory quality. A predominance of crawling ciliates, stalked ciliates and metazoans is an
indicator of sludge with excellent health and an effluent of high quality.

Filamentous Bacteria have Positive aspects:
 They are very good BOD removers.
 They add a backbone or rigid support network to the floc structure.
 Helps the floc structure filter out fine particulate matter that will improve clarifier
efficiency.
 They help the floc settle if in small amounts.
 They reduce the amount of "pin" floc.
Filamentous Bacteria have Negative aspects:

They can interfere with separation and compaction of activated sludge and cause bulking when
predominant.
 They can affect the sludge volume index (SVI).
 They can cause poor settling if dominant.
 They can fill up a clarifier and make it hard to settle, causing TSS carryover.
 They can increase polymer consumption.
 They can increase solids production and cause solids handling costs to increase
significantly.
Filamentous bacteria floc (SEM) or Pin Floc.

Bugs or MOs More information in the Appendix
Four groups of bugs do most of the “eating” in the activated sludge process. The first group is the
bacteria which eat the dissolved organic compounds. The second and third groups of bugs are
microorganisms known as the free-swimming and stalked ciliates. These larger bugs eat the bacteria
and are heavy enough to settle by gravity. The fourth
group is a microorganism, known as Suctoria, which
feeds on the larger bugs and assists with settling.
The interesting thing about the bacteria that eat the

dissolved organics is they have no mouths. The
bacteria have an interesting property, their “fat reserves”
are stored on the outside of their bodies. This fat layer
is sticky and is what the organics adhere to.
Once the bacteria have “contacted” their food, they start

the digestion process. A chemical enzyme is sent out
through the cell wall to break up the organic
compounds. This enzyme, known as hydrolytic enzyme,
breaks the organic molecules into small units which are
able to pass through the cell wall of the bacteria.
In wastewater treatment, this process of using bacteria-

eating bugs in the presence of oxygen to reduce the
organics in water is called activated sludge. The first
step in the process, the contact of the bacteria with the
organic compounds, takes about 20 minutes. The
second step is the breaking up, ingestion and digestion
processes, which takes four to 24 hours.
The fat storage property of the bacteria is also an asset in settling. As the bugs “bump” into each
other, the fat on each of them sticks together and causes flocculation of the non-organic solids and
biomass. From the aeration tank, the wastewater, now called mixed liquor, flows to a secondary
clarification basin to allow the flocculated biomass of solids to settle out of the water. The solids
biomass, which is the activated sludge, contains millions of bacteria and other microorganisms, is
used again by returning it to the influent of the aeration tank for mixing with the primary effluent and
ample amounts of air.

Paramecium sp.
Paramecium is a medium to large size (100-300 μm)
swimming ciliate, commonly observed in activated
sludge, sometimes in abundant numbers. The body is
either foot-shaped or cigar-shaped, and somewhat
flexible.
Paramecium is uniformly ciliated over the entire body

surface with longer cilia tufts at the rear of the cell.
Paramecium swims with a smooth gliding motion. It
may also be seen paired up with another
Paramecium which makes a good diagnostic key.
The cell has either one or two large water cavities
which are also identification tools.
This swimmer moves freely in the water column as it

engulfs suspended bacteria. It has a large feeding
groove used to trap bacteria and form the food
cavities that move throughout the body as digestion
occurs.
Paramecium is described as a filter-feeding ciliate because its cilia move and filter bacteria from
the water.
Vorticella sp.
Vorticella is a stalked ciliate. There
are at least a dozen species found
in activated sludge ranging in
length from about 30 to 150 μm.
These organisms are oval to round
shaped, have a contractile stalk, a
domed feeding zone, and a water
vacuole located near the terminal
end of the feeding cavity.
One organism is found on each

stalk except during cell division.
After reproducing, the offspring
develops a band of swimming cilia
and goes off to form its own stalk.
The evicted organism is called a
"swarmer."
Vorticella feeds by producing a

vortex with its feeding cilia. The
vortex draws bacteria into its gullet.
Vorticella's principal food source is suspended bacteria. The contracting stalk provides some
mobility to help the organism capture bacteria and avoid predators.
The stalk resembles a coiled spring after its rapid contraction. Indicator: If treatment conditions are
bad, for example low DO or toxicity, Vorticella will leave their stalks. Therefore, a bunch of empty
stalks indicates poor conditions in an activated sludge system. Vorticella sp. are present when the
plant effluent quality is high.

Euglypha sp.
Euglypha (70-100 μm) is a shelled (testate)
amoeba. Amoebas have jelly-like bodies.
Motion occurs by extending a portion of the
body (pseudopodia) outward. Shelled amoebas
have a rigid covering which is either secreted or
built from sand grains or other extraneous
materials.
The secreted shell of this Euglypha sp. consists

of about 150 oval plates. Its spines project
backward from the lower half of the shell.
Euglypha spines may be single or in groups of
two or three. The shell has an opening
surrounded by 8-11 plates that resemble shark
teeth under very high magnification.
The shell of Euglypha is often transparent,

allowing the hyaline (watery) body to be seen
inside the shell. The pseudopodia extend
outward in long, thin, rays when feeding or
moving. Euglypha primarily eats bacteria.
Indicator: Shelled amoebas are common in

soil, treatment plants, and stream bottoms
where decaying organic matter is present. They
adapt to a wide range of conditions and
therefore are not good indicator organisms.
Euchlanis sp.
This microscopic animal is a typical rotifer.
Euchlanis is a swimmer, using its foot and
cilia for locomotion. In common with other
rotifers, it has a head rimmed with cilia, a
transparent body, and a foot with two strong
swimming toes.
The head area, called the "corona," has cilia

that beat rhythmically, producing a strong
current for feeding or swimming.
Euchlanis is an omnivore, meaning that its

varied diet includes detritus, bacteria, and
small protozoa. Euchlanis has a glassy
shell secreted by its outer skin. The
transparent body reveals the brain,
stomach, intestines, bladder, and
reproductive organs.
A characteristic of rotifers is their mastax,

which is a jaw-like device that grinds food as it enters the stomach. At times the action of the
mastax resembles the pulsing action of a heart. Rotifers, however, have no circulatory system.
Indicator: Euchlanis is commonly found in activated sludge when effluent quality is good. It
requires a continual supply of dissolved oxygen, evidence that aerobic conditions have been
sustained.

Ciliate above, Amoeba, below.

Nitrobacter winogradskyi
Nitrospira gracilis
Of all biological waste treatment methods, aerobic digestion is the

most widespread process used throughout the world (more than 95%).
Nature gives, takes and does everything in-between. Nowhere is this better exemplified than the
biological solution it offers to mankind's waste problems. An illustration of nature's work is its
influence on the constant cycle of biological waste treatment. Microorganisms, like all living things,
require food for growth.
Biological sewage treatment consists of many different microorganisms, mostly bacteria, carrying
out a stepwise, continuous, sequential attack on the organic compounds found in wastewater and
upon which the microbes feed.
Aerobic digestion of waste is the natural biological degradation and purification process in which
bacteria that thrive in oxygen-rich environments break down and digest the waste. During this
oxidation process, pollutants are broken down into carbon dioxide (CO2), water (H2O), nitrates,
sulfates and biomass (micro-organisms). By optimizing the oxygen supply with so called aerators
the process can be significantly accelerated.

Activated Sludge Aerobic Flocs
Aerobic flocs in a healthy state are referred to as activated sludge. While aerobic floc has a
metabolic rate approximately 10 times higher than anaerobic sludge, it can be increased even
further by exposing the bacteria to an abundance of oxygen. Compared to a septic tank, which
takes several days to reduce the organic material, an activated sludge tank can reduce the same
amount of organic material in approximately 4-6 hours. This allows a much higher degree of overall
process efficiency. In most cases, treatment efficiencies and removal levels are so much improved
that additional downstream treatment components are dramatically reduced or totally eliminated.
Problems may appear during the operation of activated sludge systems, including:
 High solids content in clarified effluent, which may be due to too high or too low solids
retention time and to growth of filamentous microorganisms.
 Rising sludge, occurring when sludge that normally settles rises back to the surface after
having settled. In most cases, this is caused by the denitrification process, where nitrate
present in the effluent is reduced to nitrogen gas, which then becomes trapped in the
sludge causing this to float. This problem can be reduced by decreasing the flow from the
aeration basin to the settling tank or reducing the sludge resident time in the settler, either
by increasing the rate of recycle to the aeration basin, increasing the rate of sludge
collection from the bottom, or increasing the sludge wasting rate from the system.
 Bulking sludge, that which settles too slowly and is not compactable, caused by the
predominance of filamentous organisms. This problem can be due to several factors of
which the most common are nutrient balance, wide fluctuations in organic load, oxygen
limitation (too low levels), and an improper sludge recycle rate.
 Insufficient reduction of organic load, probably caused by a low solids retention time,
insufficient amount of nutrients such as P or N (rare in fisheries wastewaters), short-
circuiting in the settling tank, poor mixing in the reactor and insufficient aeration or presence
of toxic substances.
 Odors, caused by anaerobic conditions in the settling tanks or insufficient aeration in the
reactor.
Filamentous Organisms
The majority of filamentous organisms are bacteria, although some of them are classified as algae,
fungi or other life forms. There are a number of types of filamentous bacteria which proliferate in
the activated sludge process. Filamentous organisms perform several different roles in the process,
some of which are beneficial and some of which are detrimental. When filamentous organisms are
in low concentrations in the process, they serve to strengthen the floc particles. This effect reduces
the amount of shearing in the mechanical action of the aeration tank and allows the floc particles
to increase in size.
Larger floc particles are more readily settled in a clarifier. Larger floc particles settling in the clarifier
also tend to accumulate smaller particulates (surface adsorption) as they settle producing an even
higher quality effluent. Conversely, if the filamentous organisms reach too high a concentration,
they can extend dramatically from the floc particles and tie one floc particle to another (interfloc
bridging) or even form a filamentous mat of extra-large size.
Due to the increased surface area without a corresponding increase in mass, the activated sludge
will not settle well. This results in less solids separation and may cause a washout of solid material
from the system. In addition, air bubbles can become trapped in the mat and cause it to float,
resulting in a floating scum mat.
Due to the high surface area of the filamentous bacteria, once they reach an excess concentration,
they can absorb a higher percentage of the organic material and inhibit the growth of more desirable
organisms.

Filamentous Bacteria Identification
Filamentous Identification should be used as a tool to monitor the health of the biomass when a
filament problem is suspected. Filamentous Identification is used to determine the type of filaments
present so that a cause can be found and corrections can be made to the system to alleviate future
problems. All filamentous bacteria usually have a process control variation associated with the type
of filament present that can be implemented to change the environment present and select out for
floc forming bacteria instead. Killing the filaments with chlorine or peroxide will temporarily remove
the filaments, but technically it is a band-aid. A process change must be made or the filaments will
return with time eventually. Find out what filaments are present, find out the cause associated with
them and make a process change for a lasting fix to the problems.
Filaments, their causes and suggested controls

Low DO Filaments Control
Type 1701 Adjust the aeration rates or F/M (based on aeration solids)
S. natans
H. hydrossis Long RAS lines or sludge held too long in the clarifier can
sometimes cause the growth of low DO filaments even if the
aeration
Waste with limited Nutrients Control
Thiothrix I & II Nutrient addition BOD ratio of 100:5:1
021N and N. Limicola III
Low F/M ratios Control
0041, Nocardia Use of selector, increase RAS
Type 1851, 0961, 0803, 0675 Increase WAS
Some filaments have more than one version of the filament species, with slightly different
characteristics for identification.
 N. Limicola I
 N. Limicola II
 N. Limicola III
 Thiothrix I
 Thiothrix II
Filamentous Identification
Filaments can be internal or external, and they can be free of the floc structures or found intertwined
in the floc. Most labs think that filaments need to be extending from the floc in order to be a problem.
This is not true. Internal filaments can cause more problems than external filaments. Think of
internal filaments causing a structure like a sponge. It will retain water easily and be harder to
dewater, will be hard to compress and will take up more space, thereby increasing solids handling
costs.
Filaments present in the system do not always mean there is a problem. Some filaments are good
if they form a strong backbone and add a rigid network to the floc. They help give the floc more
structure and settle faster. Filaments are good BOD degraders also. They are only a problem when
they become dominant. If filament abundance is in the abundant or excessive range, having a
Filamentous Identification performed is recommended.
When Gram and Neisser stains are performed for filamentous Identification, the types of filaments
found present will be noted on the Floc Characterization sheet to the right of the filament section
and will be noted on the Cover Sheet. A Filament Causes sheet, Filamentous Predominance sheet
and corrective actions will be given and included with the report. A Filamentous Worksheet will be
included. Individual sheets on the actual filaments present in the sample will be included with more
information on that particular filament.

The activated sludge process was invented around 1914 and is today still the most commonly used
biological wastewater treatment process. This widespread use is due to the fact that activated
sludge can be a rather easy process to implement and one that can attain high treatment efficiency.
That is to say, if it works! Activated sludge is susceptible to process disturbances making it a very
problematic technology for many of its users. Problems arise most when the wastewater to be
treated varies significantly in composition and/or flow.
Let’s do a quick review of the Bugs…. We will go much more into detail later…
Nocardia amarae
Nocardia amarae, a common cause of disruptive foaming in waste treatment plants, is a slow
growing, usually gram-positive, chemoautotrophic, filamentous, strict aerobe that produces the
biosurfactant trehalose. Colonies can be brown, pink, orange, red, purple, gray or white, so color
alone is not a key to identifying this species. N. amarae, member of the Actinomycetes family, is
not motile, so it relies on movement of the water to carry it through the system. It produces catalase,
urease and nitrate reductase enzymes, but not casease. The foam from Nocardia amarae is usually
a viscous brown color unless algae are entrapped in it, in which case it appears green and brown.
Nostocoida limicola
Nostocoida limicola is yet another common cause of disruptive foaming in waste treatment plants,
motile in its Hormogonia and sometimes Trichome phases. This oxygenic phototrophic species
often forms a confluent gel encasing flattened discs or large sheets of cells, forming symbiotic
relationships with other species. Staining gram-positive, Nostocoida produces round cells within
tight coil formations. Nostocoida can also be dentified by their starburst effect formations using
phase contrast microscopy at 400 to 1000x magnification. After chlorination, a few dead cells
sticking out identify stress to this species.
Thiothrix
Thiothrix spp., the second most common cause of disruptive foaming in wastewater treatment
plants appears as straight to slightly curved cells with rectangular shape form filaments up to 500
microns in length, in multicellular rigid filaments, staining gram-negative, with obligately aerobic
respiration. Thiothrix are mixotrophic, using several small organic carbons and reduced inorganic
sulfur sources for growth and energy. Thiothrix I is one of the largest filament found using phase
contrast microscopy at 400 to 1000x magnification. Thiothrix II produces rectangular filaments up
to 200 microns in length and is easily identified by their starburst effect formations using phase
contrast microscopy at 400 to 1000x magnification.
Microthrix parvicella
Microthrix parvicella is another common cause of disruptive foaming in waste treatment plants,
producing filaments up to 400 microns in length, easily visualized by phase contrast microscopy at
400x magnification. This species is usually found outside floc, tangling with structures in the
system, but can also be found hanging out of the floc.
Sphaeroliticus natans
Sphaeroliticus natans is another filamentous species, and yet it is reputed to increase settleability
by branching between flocs, increasing surface area. Cells are straight to slightly curved, up to
1000 microns in length and stain gram-negative. These large cells can be easily visualized by
phase contrast microscopy at 100x magnification.
Certain conditions favor the proliferation of filamentous species. A low F/M (food to mass) ratio
favors filamentous organisms, because their higher ratio of surface area to volume provides them
with a selective advantage for securing nutrients in nutrient limited environments. When a plant
runs an extremely long sludge age, the slower growing filaments have a better chance to establish
a strong colony. As a strict aerobe, high levels of oxygen are necessary to sustain this species.
Mesophilic, Nocardia amarae thrives in temperatures from 17 to 37 deg. C.

The presence of high levels of fats, oils and greases or hydrocarbons and phenols, can encourage
this species, particularly when insufficient levels of nitrogen and phosphorus are present to balance
these carbon sources.
A problem that often frustrates the performance of activated sludge is bulking sludge due to the
growth of filamentous bacteria. Sludge bulking can often be solved by careful process
modifications.
However, different filamentous bacteria such as Microthrix, Sphaerotilus, Nostocoida, Thiothrix or

“Type 021N” and others cause bulking for very different reasons. Many filamentous species have
not even been given a scientific name yet. Consequently, in order to make the right kind of process
modification, knowledge to identify them and experience with the process ecology are
required. The potential for instability with activated sludge is an acute problem when strict demands
on treatment performance are in place.
PAX - finally, a Fix for Microthrix

If you ever experienced an overgrowth of Microthrix
parvicella in your activated sludge plant, you will be
aware that it can be very difficult to either eradicate or
control. Microthrix is the most common cause of bulking
and foaming in activated sludge plants (Rosetti et al.
2002), and it appears either essentially alone or in the
company of other filaments.
Microthrix foams appear in many of the photographs of

aeration basins and clarifiers I have collected all over the
world, and many of the plant tours on the Internet show
the same brown stable scums associated with this
organism. Let's face it, Microthrix is just about
everywhere.
Figure 1.
A micrograph of Microthrix parvicella, gram stain x 1000
Microthrix is your enemy - Get to know it!

Microthrix fits into the filamentous bacterial classification of low F/M, which means that it tends to
appear in plants with long sludge ages. Lackay et al. (1999) suggested that M. parvicella and its
low F/M compatriots Haliscomenobacter hydrossis, and types 0092, 0041, 1851, 0803 were also
encouraged to the point of maximum proliferation by alternating anoxic-aerobic conditions
(particularly 30-40% aerobic and 60-70% anoxic) but any alternation of anoxic-aerobic conditions
may cause a problem in single reactor, two reactor, or multireactor systems in which nitrate and/or
nitrite are present throughout the anoxic period, or in the anoxic reactor just prior to the aerobic
reactor. Modern plants incorporating denitrification and/or phosphorus removal are obvious
candidates for bulking and foaming due to Microthrix.
Figures 1 and 2 show typical views of Microthrix by using light microscopy and scanning electron
microscopy respectively. It is not difficult to recognize using standard staining and microscopy,
giving a positive response to Gram stain and being of fairly easily recognized morphology (Seviour
et al. 1999). Of all the filaments creating difficulties in activated sludge plants, it is one of the most
easily recognized, but there is a commercial test kit available which uses fluorescent situ
hybridization (or "FISH") to permit visual identification should one feel the need.

The design of plants can play a significant part in the proliferation of scums and foams and there
are many common mistakes in plant design which assist organisms like Microthrix by retaining
floating masses in dead areas of the plant which have very high MCRT values and continuously
reseed the biomass, (Pitman 1996). These should obviously be avoided (Figs 3, 5 and 6).
Similarly, poor mixing, poorly designed and inadequate aeration systems, cyclic overloading and
low process D.O. levels can contribute to the creation of anoxic and anaerobic zones in what are
supposed to be aeration basins.
Figure 2. A scanning electron micrograph of Microthrix parvicella
Current Remedial Techniques

Jenkins et al. (1993) presented sludge chlorination as a method of choice in the United States to
combat filamentous bulking due to any organism. The success of treatment of Microthrix in mixed
liquor or foams is poor, due it is believed to resistant filamentous bacteria with hydrophobic cell
walls such as M. parvicella and Nostocoida limicola.
Lakay et al. (1988) obtained only a partial elimination of

Microthrix parvicella bacteria at a high chlorine dose. Hwang and
Tanaka found in batch tests that M. parvicella remained intact at
very high chlorine doses, while the microbial flocs were
completely destroyed. Saayman et al. (1996) examined the use
of non-specific chemical treatment in a BNR plant and assessed
the effects of biomass settling characteristics and other
operational parameters.
While chlorine use was the most effective, it was reported to

damage the biomass and cause difficulties in the P removal
process when dosed at high levels, while ozone and peroxide
were less effective in treating settling problems but less of a
problem to the biomass.
Figure 3.
Dry Microthrix parvicella foam
trapped in an anoxic zone of a
BNR plant. aeration basin.

In recent times the introduction of selectors has
been hailed as a major initiative in the control and
elimination of filamentous bacteria (bulking and
foaming) and the maintenance of moderate
biomass SVIs. Evidence on the performance of
selectors in controlling low F/M filaments has been
described as both controversial and ambiguous
and, in the Netherlands, despite incorporating over
80 selectors in full-scale plants, the percentage of
plants with bulking associated with Microthrix
parvicella was unchanged. Other experiences with
the aerobic selector showed only little success in
controlling the growth of M. parvicella in the
presence of long chain fatty acids (LCFA), (Lebek
and Rosenwinkel, 2002) and a comparison of
anoxic selectors at five plants in the US has
demonstrated that performance and effectiveness
varied significantly (Marten and Daigger, 1997).
Figure 4. Typical dark brown Microthrix

parvicella foam on an
More on Microthrix
Mamais et al. 1998 examined the effect of factors such as temperature, substrate type (easily
biodegradable in the form of acetate and slowly biodegradable in the form of oleic acid) on
Microthrix parvicella growth using complete mix with and without selectors (anoxic and anaerobic)
and plug flow reactors. The results indicate that low temperatures and substrates in the form of
long chain fatty acids favor the growth of M. parvicella. The plug flow configuration was shown to
be quite effective in controlling the growth of M. parvicella and producing a sludge with good settling
characteristics, while the presence of a selector, either anoxic or anaerobic, had no significant effect
on the growth of M. parvicella. Maintenance of low sludge ages (5) days has also been reported to
eliminate M. parvicella because it is a slow growing organism, but this is not always operationally
possible.
While it is often convenient to group filaments together, it does appear the Microthrix has received
special attention because of its ability to proliferate. More selective investigation of Microthrix has
indicated that it has quite well defined requirements. The nature of Microthrix is such that it has
the capability of using long chain fatty acids (oleic acid) and their esters (triglycerides of palmitic
and stearic acid) (fats and oils) as sources of carbon and energy.
Lipids and LCFA are present in all domestic wastewater streams and often constitute a significant
part of it. Values of 25-35% of the incoming COD have been reported, and it can support a
substantial biomass production in a treatment plant. LCFA are generally easily consumed in
activated sludge, and the consumption rate of LCFA under aerobic or anoxic conditions has been
found to be rapid.
Studies indicate that M.parvicella consumes exclusively long chain fatty acids (LCFA), and that it
is able to take up LCFA not only under aerobic, but also under anaerobic and anoxic conditions
(Andreasen, K. and Nielsen, P.H. (2000)). It has been reported that M. parvicella is able to out-
compete other bacteria particularly well in alternating anaerobic-aerobic and anoxic activated
sludge systems. This ability is based on a high uptake and storage capacity for LCFA under
anaerobic conditions and a subsequent use of the stored substrate for growth with oxygen (or
nitrate) as electron acceptor.

Rosetti et al. (2002) carried out an extensive
examination of M. parvicella and found that it was a
very versatile organism which could store organic
carbon under anaerobic conditions using stored
polyphosphate for energy (like the organisms
responsible for phosphorus removal). Once exposed to
aerobic conditions it would recover rapidly and resume
growing. Microthrix has a high storage capacity under
all operating conditions (anaerobic, anoxic and
anaerobic). It has a high "substrate affinity" or low Ks,
which means it competes well at low substrate
concentration.
Figure 5.
Microthrix parvicella foam trapped
near a mechanical aerator.
Most interestingly, M. parvicella has a maximum growth rate near 22° C, zero growth rate at 30° C
and is capable of quite reasonably large growth rates at as low as 7° C which gives it a significant
advantage in the competition with floc formers during winter in cold climates.
PAX vs. Microthrix parvicella

Microthrix parvicella is well-equipped to survive, compete and dominate in all kinds of activated
sludge systems. With all of the above in mind, it is pleasing to find that Microthrix does have a
weakness. That weakness is its apparent sensitivity to poly aluminum chloride (PAX) dosing, which
seems to attack the ability of Microthrix parvicella to use lipids by reducing the activity of
extracellular enzymes (lipases) on the surface of the organism rendering the organism relatively
uncompetitive (Nielsen et al. 2003).
Roels et al. (2002) reported a loss of surface scum following

PAX-14 dosing which was probably due to a loss of
hydrophobicity. Full-scale dosages of PAX-14 range from 1.5 to
4.5 g Al3+/kg MLSS/day depending on the sludge retention time
(SRT); the lower the SRT, the higher the dosage and certainly
lower than 7 g Al3+/kg MLSS. Roels et al. (2002) offered the
following empirical formula to establish the dose:
60/SRT = #g of Al3+/kg MLSS
They also recommended the removal of the scum layer before

dosing to allow the concentration and time of dosage to be kept
at a minimum. Removal of the floating sludge layer from the
surface before starting PAX application was necessary to
ensure specific and rapid impact of Al-salts on M. parvicella.
In fact, the stable floating sludge represents an independent

microbial system, into which aluminum can penetrate only at a
limited extent. Dosage should be combined with high oxygen
concentration in the aeration (i.e. above 2.5 mg/L) and the
MLSS concentration low (i.e. under 2.5 g/L) since M. parvicella
competes well at low oxygen levels.
Figure 6. A heavy build-up of

trapped Microthrix parvicella foam
during winter.

Of note was that the morphological properties
of only Microthrix parvicella changed,
apparently leaving the other filaments
remaining unaffected.
Paris et al. (2003) came to a similar

conclusion; by dosing AICl3 (3.5 mg mgAl3+
gMLSS/d), a general improvement of the
settling properties of the activated sludge was
achieved. As the filamentous population of
activated sludge and the occurrence
frequency of M. parvicella dropped, a
decrease of hydrophobicity and floating
tendency of activated sludge was observed.
With low hydrophobicity the sludge does not
tend to float. This has significant relevance for
any measure to prevent floating foams.
Figure 7. An typical view of
Microthrix parvicella (gram stain x 1000)
after extended PAX treatment.
It was observed that by adding PAX a morphological modification of the filamentous bacterium M.
parvicella occurs. The morphological modification is probably the reason why the hydrophobic
property of the filaments decreases. Paris et al. (2003) included micrographs which indicated that
the Microthrix parvicella appeared to shorten in length after dosing (Figure 7) and no longer inhabit
the zones between flocs.
PAX
PAX (or PAX-14 or polyaluminium chloride) used for
Microthrix control is a flocculant or coagulant
commonly used in water and wastewater treatment.
The 14 or other number associated with the name
refers to the particular grade of the chemical. Nielsen
et al. (2003) report that PAX-14 is Al13O4(OH)24
(H2O)127+ and it is produced from Al(OH)3 at high
temperature and high pressure. PAX-14 and 18 are
being used in several countries with good success for
controlling M. parvicella - in particular Denmark where
PAX-14 has been applied successfully in treatment
plants with biological N and/or P removal for 91 out of
500 plants in 2002. Figure 8.
Foam build-up in a secondary
clarifier resulting in solids loss and
turbid effluent.
Proposed Treatment Regime
In the fall, to prevent the normal appearance of M. parvicella during the coming winter and to control
problems with M. parvicella (winter, spring). Dosage: 0.5-1.5g Al/kgSS/day usually added to return
sludge. PAX should be dosed continuously over the treatment period at the chosen level. Removal
of floating sludge before and during dosing is recommended. Microscopic examination of the
biomass and regular testing of biomass settling is also a very good idea and the dosing at the
chosen remedial rate until a target SVI or preferably DSVI is reached should be the rule. It is not
yet fully clear why PAX has the effect that it does, but the research continues. It is known that other
Al salts have little effect on surface associated enzymes after 15 min, and no effect on surface
hydrophobicity and surface associated enzymes.

Sphaerotilus natans
Description and Significance
Sphaerotilus natans is a filamentous bacterium that is covered in a tubular sheath and can be
found in flowing water and in sewage and wastewater treatment plants. While this bacterium
sometimes clogs pipes and causes other similar problems, it does not cause major threat to
wastewater treatment plants nor is it known to be pathogenic.
Long unbranched and ensheathed filaments produced by Sphaerotilus natans IF4.
Relatively long, non-motile filaments (100-1000 µm). Straight or smoothly curved with tree-like false
branching. The cells are round-ended and rod shaped (1.0-1.8 x 1.5-3.0) and are contained in a
clear, tightly fitting sheath. Note: They can be rectangular when the cells are tightly packed within
the sheath. The cell septa are clear and easily observable with indentations. Filaments radiate
outward from the floc surface into the bulk solution and can cause sludge settling interference by
inter-floc bridging. The filament is usually Gram negative and Neisser negative. There are no sulfur
granules. Poly-ß-hydroxybutric acid (PHB) is frequently observed as dark intracellular granules. In
wastewater that is nutrient deficient, an exocellular slime coat may be present. Attached growth is
usually uncommon, but may occur when at low growth rate.
This filament is usually found in environments where there is low

DO or low nutrients (Nor P).
Control
RAS chlorination can be used to get rid of the filaments but
process changes should also be made. Cell lysis occurs readily
on this type of filament, although the empty sheaths still remain.
Sludge wasting is necessary to remove them entirely from the
system.

Manipulation of F/M and DO concentration can be used to control the filaments. Nutrient deficient
wastes can be checked by effluent values of residual NH3 and o-PO4 and should be supplemented
if necessary.
Rank
Sphaerotilus natans ranks 6th in number of predominance. Typically, not found in pulp-mills with
activated sludge.
Nostocoida limicola I and II

Nostocoida limicola I is a bent and highly coiled filament. N. limicola has cells that are oval (0.6-
0.8 µm wide) but are found to be closer to each other and the cell septa are almost
indiscernible. The length of the filament can range from 100 to 200 µm and the majority of the time
the trichome is found within the floc. N. limicola has no sheath and attached growth is rare. It
stains Gram positive and Neisser positive.
Nostocoida limicola II Identification

Medium length, non-motile filaments (100-200
µm). Bent and irregularly coiled filaments with
incidental true branching. Knots sometimes seen.
Cell septa are clear with indentations. Cells are
oval or disc shaped (1.2-1.4 µm). Filaments are
found within the floc structure but may occur in
the bulk solution. The filament staining is variable,
it is usually Gram negative but sometimes positive
and Neisser positive. Usually easy to identify due
to its Neisser staining properties.
Stains entirely purple and looks like stacked discs

(or hockey pucks). In industrial wastes, an
organism that is Gram negative and Neisser
negative occurs. There is no sheath and there are
no sulfur granules. Poly-ß-hydroxybutric acid
(PHB) granules are frequently observed as dark
intracellular granules. Attached growth is usually
uncommon. Three subtypes are known.
Resembles M. parvicella except in its Neisser
staining properties.
Environment
This filament is usually found in environments
where there is low DO or low F/M and the
presence of organic wastes. Wastes containing
starch seem more selective to this filament.
Bulking is more common in industrial wastes. The
filament appears to be facultative fermentative,
which is unique for most filaments.
Control
Manipulation of F/M (usually an increase) and DO concentration can be used to control the
filaments. A selector may be used and chlorination. System changes include changing from a
complete mix to plug flow aeration basin configuration.
N. limicola ranks 12th in number of predominance in industry. Typically, not found in kraft mills.
Common in municipalities.

Thiothrix I & II
Thiothrix species consist of two types of Thiothrix and
they are Thiothrix I and Thiothrix II. Thiothrix filaments
are straight or slightly curved with Thiothrix I having an
overall length of 100-500 µm and individual cells having a
rectangular shape (1.4-2.5 x 3-5 µm). Thiothrix II has
total length varying form 50-200 µm and its cells are
rectangular (0.8-1.4 x 1-2 µm).
Both types of Thiothrix are found stretching from the floc

surface, there is a noticeable septa between cells. Both
species are Gram negative and Neisser negative with
cells that on occasions have sulfur granules.
There are additional structures on Thiothrix trichomes

and they include apical gonidia as well as rosettes and a
sheath is present, incidental attached growth may be
observed. A holdfast may add to the characteristic of
radiating out from a common center, the "starburst
effect".
Relatively large, non-motile filaments (100-500 µm). Straight or smoothly curved filaments with no
branching. Cells are rectangular (1.4 x 2.5 µm) and a clear cell septa is present without
indentations at the septa. Filaments are found radiating outwards from the floc structure causing
inter-floc bridging.
The filament staining is Gram negative or Gram variable

when sulfur granules are present and Neisser negative
with Neisser positive granules observed frequently.
Exhibits bright sulfur granules in the presence of sulfides

under phase contrast (use the S-test). Poly-ß-
hydroxybutric acid (PHB) is frequently observed as dark
intracellular granules.
No attached growth when extending into the bulk

solution. Can form rosettes and the filaments can have
gonidia on the tips. Rosettes are when many filaments
radiate outward from a common origin. Prominent heavy
sheath. Easy to identify due to its large size.
Similar Organisms
Type 021N is similar when in the bulk solution and with no attached growth, although Type 021N
has no sheath.
Environment
This filament is usually found in environments where there are limited nutrients (N or P). It can
also be found in wastes containing specific compounds with sulfides and/or organic acids or
environments with low DO. Sometimes found in plants with high pH in the aeration system.

Thiothrix II

Indicatior Organisms
"Amoeboid" and "amoeba" are used interchangeably. Amoeboids move using

pseudopodia, which are bulges of cytoplasm. Amoebas breathe using their entire cell
membrane that is constantly immersed in water. Excess water can cross into the cytosol.
Amoebas have a contractile vacuole to expel excess water.
Chlamydomonas reinhardtii is known to remove nitrogen and phosphorus from

wastewater.
Euchlanis is commonly found in activated sludge when effluent quality is good. It requires
a continual supply of dissolved oxygen, evidence that aerobic conditions have been
sustained.
This particular Rotifer is a great indicator bug for fresh and brackish water. With the
increasing use of antibiotics, wastewater facilities are noticing passage through the
treatment process. The toxicity effects the reproduction of the organisms in the water.
This ciliate is heavy metal resistant, Stylonychia mytilus, isolated from industrial
wastewater has been shown to be potential bioremediator of contaminated
wastewater. The ability of Stylonychia to take up variety of heavy metals from the medium
could be exploited for metal detoxification and environmental clean-up operations.

Activated Sludge Process Section
Key Terms
Aerobic (AIR-O-bick) - A condition in which free or dissolved oxygen is present in the aquatic
environment.
Aerobic Bacteria – Bacteria which will live and reproduce only in an environment containing
oxygen.
Aerobes -Oxygen combined chemically, such as in water molecules (H2O), cannot be used for
respiration by aerobes
Anaerobic (AN-air O-bick) - A condition in which “free” or dissolved oxygen is not present in the
aquatic environment.
Anaerobic Bacteria – Bacteria that thrive without the presence of oxygen. (Anaerobes)
Saprophytic bacteria – Bacteria that break down complex solids to volatile acids.
Methane Fermenters – Bacteria that break down the volatile acids to methane (CH4) carbon
dioxide (CO2) and water (H2O).
Oxidation – The addition of oxygen to an element or compound, or removal of hydrogen or an

electron from an element or compound in a chemical reaction. The opposite of reduction.
Reduction – the addition of hydrogen, removal of oxygen or addition of electrons to an element

or compound. Under anaerobic conditions in wastewater, sulfur compounds or elemental sulfur
are reduced to H2S or sulfide ions.

Water Bear Tardigrade
This creepy "water bear" has forced scientists to reconsider their definition of what's "alive." When
unable to find water, this insect like critter (which is the size of a grain of sand) stops moving,
breathing, and eating. Even its cells shut down. Dead as a doornail, right? Wrong!
Add water and the critter springs back to life. Scientists have exposed dried-up water bears to
extreme heat, bitter cold, and even massive doses of radiation -- and the teeny animals still revived.
The key to the critters' survival may be a sugar they produce as they dry out. By coating structures
inside and between the critters' cells, the sugar keeps the cells from sticking together and breaking.
When water is added, the sugar dissolves -- and the creepy crawlies burst back into action.

Activated Sludge Words
Amine: A functional group consisting of "-NH2."
Amino acid: A functional group consisting of a carbon with a carboxylic acid, "-COOH" and an amine,
"-NH2." These compounds are the building blocks for proteins.
Anabolism: Biosynthesis, the production of new cellular materials from other organic or inorganic
chemicals.
Anaerobes: A group of organisms that do not require molecular oxygen. These organisms, as well as
all known life forms, require oxygen. These organisms obtain their oxygen from inorganic ions such as
nitrate or sulfate or from protein.
Anaerobic process: A process that only occurs in the absence of molecular oxygen.
Anoxic process: A process that occurs only at very low levels of molecular oxygen or in the absence
of molecular oxygen.
Biochemical oxygen demand (BOD): The amount of oxygen required to oxidize any organic matter
present in a water during a specified period of time, usually 5 days. It is an indirect measure of the
amount of organic matter present in a water.
Carbonaceous biochemical oxygen demand (CBOD): The amount of oxygen required to oxidize
any carbon containing matter present in a water.
Chemical oxygen demand (COD): The amount of oxygen required to oxidize any organic matter in
the water using harsh chemical conditions.
Decomposers: Organisms that utilize energy from wastes or dead organisms. Decomposers
complete the cycle by returning nutrients to the soil or water and carbon dioxide to the air or water.
Denitrification: The anoxic biological conversion of nitrate to nitrogen gas. It occurs naturally in
surface waters low in oxygen, and it can be engineered in wastewater treatment systems.
Deoxygenation: The consumption of oxygen by the different aquatic organisms as they oxidize
materials in the aquatic environment.
Facultative: A group of microorganisms which prefer or preferentially use molecular oxygen when
available, but are capable of using other pathways for energy and synthesis if molecular oxygen is not
available.
F/M Ratio: Another method for control wasting is to maintain a constant food-to-microorganism (F:M
or F/M) ratio. With this method, the operator will try to increase or decrease the MLVSS to match an
increase or decrease in the BOD entering the plant. Most plants will operate best at a specific F/M
ratio between 0.05 - 0.1. If the optimum F/M has been determined from experience and can be
maintained, a good effluent may be produced with consistent plant operation. The F/M ratio is to be
calculated at least weekly and related to the efficiency of treatment plant operation. An F/M ratio
between 0.05 - 0.15 BOD/lb MLSS is usually considered acceptable for an extended aeration process.

Nitrification: The biological oxidation of ammonia and ammonium sequentially to nitrite and then
nitrate. It occurs naturally in surface waters, and can be engineered in wastewater treatment systems.
The purpose of nitrification in wastewater treatment systems is a reduction in the oxygen demand
resulting from the ammonia.
Nitrogen fixation: The conversion of atmospheric (or dissolved) nitrogen gas into nitrate by
microorganisms.
Nitrogenous oxygen demand (NOD): The amount of oxygen required to oxidize any ammonia
present in a water.
NPDES: The National Pollutant Discharge Elimination System. The discharge criteria and permitting
system established by the U.S. EPA as a result of the Clean Water Act and its subsequent
amendments or the permit required by each discharger as a result of the Clean Water Act.
MCRT - Mean Cell Residence Time: The average time a given unit of cell mass stays in the
activated sludge biological reactor. It is typically calculated as the total mixed liquor suspended solids
in the biological reactor divided by the combination of solids in the effluent and solids wasted.
Mixed liquor suspended solids (MLSS): The total suspended solids concentration in the activated
sludge tank.
Mixed liquor volatile suspended solids (MLVSS): The volatile suspended solids concentration in
the activated sludge tank.
Organic compound: Any compound containing carbon except for the carbonates (carbon dioxide,
the carbonates and bicarbonates), the cyanides, and cyanates.
Organic nitrogen: Nitrogen contained as amines in organic compounds such as amino acids and
proteins.
Oxidative phosphorylation: The synthesis of the energy storage compound adenosine triphosphate
(ATP) from adenosine diphosphate (ADP) using a chemical substrate and molecular oxygen.
Secondary treatment: In wastewater treatment, the conversion of the suspended, colloidal and
dissolved organics remaining after primary treatment into a microbial mass which is then removed in
a second sedimentation process. Secondary treatment includes both the biological process and the
associated sedimentation process.
Sludge: A mixture of solid waste material and water. Sludges result from the concentration of
contaminants in water and wastewater treatment processes. Typical wastewater sludges contain from
0.5 to 10 percent solid matter. Typical water treatment sludges contain 8 to 10 percent solids.
Thiols: Organic compounds which contain the "-SH" functional group. Also called mercaptans.
Total dissolved solids: (TDS) Is the amount of dissolved matter in a water.
Total solids: (TS) Is the amount of organic and inorganic matter that is contained in a water.
Total suspended solids: (TSS) Is the amount of suspended (filterable) matter in a water.
Ultimate biochemical oxygen demand (BODu): The total amount of oxygen required to oxidize any
organic matter present in a water, i.e. after an extended period, such as 20 or 30 days.

Virus: A submicroscopic genetic constituent that can alternate between two distinct phases. As a
virus particle, or virion, it is DNA or RNA enveloped in an organic capsule. As an intracellular virus, it
is viral DNA or RNA inserted into the host organisms’ DNA or RNA.
Volatile: A material that will vaporize easily.
Volatile solids (VS) is the amount of matter which volatilizes (or burns) when a water sample is
heated to 550oC.

Activated Sludge Methods
We have some wastewater treatment plants that grow the microorganisms (Bugs) in large tanks.
To have enough oxygen in the tanks we add oxygen by blowing air into the tank full of wastewater
and microorganisms. The air is bubbled in the water and mixes “the bugs,” food and oxygen
together. When we treat wastewater this way, we call it the activated sludge method. With all of
this food and air, the microbes grow and multiply very rapidly.
Pretty soon the population of bugs gets too large and some of them need to be removed to make
room for new bugs to grow. We remove the excess bugs by sedimentation in the same kind of
tanks used for primary treatment. In the tank, the bugs sink to the bottom and we remove them.
The settled bugs are also called waste activated sludge. The waste sludge is treated separately,
and the remaining wastewater is now much cleaner. In fact, after primary and secondary treatment,
about 85% or more of all pollutants in the wastewater has been removed and it goes on to
Disinfection. These systems originated in England in the early 1900's and earned their name
because a sludge (mass of microbes) is produced which aerobically degrades and stabilizes the
organic load of a wastewater. Below diagram shows the layout of a typical activated sludge system.
Diagram of a simple activated sludge system.

For larger systems, especially when high variability is expected, the design involves the use of
multiple aeration tanks and multiple settling tanks. The number of units employed depends on the
flow of wastewater being generated.
Organic Load
The organic load (generally coming from primary treatment operations such as settling, screening
or flotation) enters the reactor where the active microbial population (activated sludge) is present.
The reactor must be continuously aerated. The mixture then passes to a secondary settling tank
where the cells are settled. The treated wastewater is generally discharged after disinfection while
the settled biomass is recycled in part to the aeration basin.
The cells must be recycled in order to maintain sufficient biomass to degrade the organic load as
quickly as possible. The amount that is recirculated depends on the need to obtain a high
degradation rate and on the need for the bacteria to flocculate properly so that the secondary
settling separates the cells satisfactorily. As the cells are retained longer in the system, the
flocculating characteristics of the cells improve since they start to produce extra cellular slime which
favors flocculating.

Common Types
The most common types of activated sludge are the conventional and the continuous flow stiffed
tank, in which the contents are completely mixed. In the conventional process, the wastewater is
circulated along the aeration tank, with the flow being arranged by baffles in plug flow mode. The
oxygen demand for this arrangement is maximum at the inlet as is the organic load concentration.
Diagram of a conventional activated sludge process.

In the completely mixed process the inflow streams are usually introduced at several points to
facilitate the homogeneity of the mixing; if the mixing is complete, the properties are constant
throughout the reactor. This configuration is inherently more stable to perturbations because mixing
causes the dilution of the incoming stream into the tank. In fisheries wastewaters the perturbations
that may appear are peaks of concentration of organic load or flow peaks. The flow peaks can be
damped in the primary treatment tanks. The conventional configurations would require less reactor
volume if smooth plug flow could be assured, which usually does not occur.
Other versions of activated sludge systems (e.g., extended aeration, contact stabilization, step
aeration and pure oxygen processes) are used in other kinds of wastewaters but are not known to be
applied to treat fisheries wastewaters. They are discussed elsewhere (Metcalf and Eddy Inc., 1979;
Eckenfelder, 1980). In all activated sludge systems, the cells are separated from the liquid and
partially returned to the system to have a relatively high concentration of cells that degrade the organic
load in a relatively short time.
Therefore two different resident times are characteristic:

the hydraulic residence time (θH) given by the ratio of reactor volume (V) to flow of wastewater (Q):
θH = V/Q
and the cell residence time (θc) given by the ratio of cells present in the reactor to the mass of cells
wasted per day. Typical θH values are in the order of 3-6 hours, while θc fluctuates between 3 and 15
days. Such difference in resident times is obtained by discharging the clarified effluent but wasting
only a small fraction of the sludge. This in turn can be accomplished by discarding a portion of the
sludge from the settling tank or by wasting a fraction of the outlet of the reactor before entering the
settling tank. In activated sludge systems, organic load removals of 85-95% are the most common.
A key factor in the success of these systems is its proper operation, which requires trained manpower.
Although used by some large fisheries which operate on a year-round basis, activated sludge may
not prove to be economical or feasible for small seafood processors who operate seasonally because
of the need to have a fairly constant supply of wastewater to maintain the micro-organisms.

Basic System Components of Activated Sludge
In the basic “activated” sludge process, emphasis on activated, the wastewater enters an aerated
tank (the dome) where previously developed biological floc particles are brought into contact with
the organic matter (foot-long hot dogs) of the wastewater.
The organic matter is a carbon and an energy source for the bug’s cell growth and is converted
into cell tissue. The oxidized end product is mainly carbon dioxide, CO2. The substance in the
sports dome is referred to as mixed liquor. The stuff in the mixed liquor is suspended solids and
consists mostly of microorganisms, suspended matter, and non-biodegradable suspended matter
(MLVSS).
The make-up of the microorganisms are around 70 to 90% organic and 10 to 30% inorganic matter.
The makeup of cells varies depending on the chemical composition of the wastewater and the
specific characteristics of the organisms in the biological mass. The picture below shows the basic
outline of an aeration tank. Just remember that pretreatment is crucial prior to the activated sludge
process.
Before we dive into the tank, in the space provided, list three key components of
pretreatment (headworks) and how each benefits the process.
1.
2.
3.

Mixed Liquor
Back to the mixed liquor, as it leaves the aeration tank, it usually goes to a clarifier to separate the
suspended solids (SS) from the treated wastewater. The concentrated biological solids are then
recycled back to the aeration tank, as returned activated sludge (RAS), to maintain a concentrated
population of bugs (the team players) to treat the wastewater.
Before we start the game, we need to make sure we have a stadium and all components are in
place and operating properly. In the space provided, define the following terms:
See Glossary in Rear.
Let’s first look at the different aeration tank designs and how they function. We will focus
on the following:
Anaerobic:
Aerobic:
DO:
BOD:
COD:
Process Design:

Complete Mix Activated Sludge Process
In a complete mix activated sludge process, the mixed liquor is similar throughout the aeration tank.
The operating characteristics measured in terms of solids, oxygen uptake rate (OUR), MLSS, and
soluble BOD 5 concentration are identical throughout the tank.
Because the entire tank contents are the same quality as the tank effluent, there is a very low level
of food available at any time to a large mass of microorganisms.
This is the major reason why the complete mix modification can handle surges in the organic
loading without producing a change in effluent quality. The type of air supply used could be either
diffused air or a mechanical aerator. Complete mix process may be resistant to shock loads but is
susceptible to filamentous growths.

Plug Flow Activated Sludge Process
Plug flow tanks are the oldest and most common form of aeration tank. They were designed to
meet the mixing and gas transfer requirements of diffused aeration systems. One characteristic of
the plug flow configuration is a very high organic loading on the MLSS in the initial part of the tank.
The loading is then reduced and the organic material in the raw wastewater is oxidized.
At the end of the tank, depending on detention time, the oxygen consumption may primarily be the
result of endogenous respiration or nitrification, which we will talk more about later on. The same
characteristics are present when the aeration tank is partitioned into a series of compartments.
Each compartment must have the oxygen supply and design to meet the individual compartment
needs. Plug flow configurations have the ability to avoid “bleed through,” the passage of untreated
organics during peak flow. These configurations are often preferred when high effluent DO’s are
sought because only a small section of the tank will operate at a high DO. In a complete mix
configuration, the entire tank must operate at the elevated DO.

Contact Stabilization Activated Sludge Process
Contact stabilization activated sludge is both a process and a specific tank configuration. The
contact stabilization encompasses a short-term contact tank, secondary clarifier, and a sludge
stabilization tank with about six times the detention time used in the contact tank.
Contact stabilization is best for smaller flows in which the MCRT desired is quite long.
Therefore, aerating return sludge can reduce tank requirements by as much as 30 to 40 % versus
that required in an extended aeration system. The volumes for the contact and stabilization tanks
are often equal in size and secondary influent arrangements.
What does this all mean?
They can be operated either in parallel as an extended aeration facility or as a contact stabilization
unit. This flexibility makes them suitable for future expansion to conventional activated sludge,
without increasing the aeration tank, by merely adding more clarification capacity.

Step Feed Activated Sludge Process
Step feed is a modification of the plug flow configuration in which the secondary influent is fed at
two or more points along the length of the aeration tank.
With this arrangement, oxygen uptake requirements are relatively even and the need for tapered
aeration is eliminated.
Step feed configurations generally use diffused aeration equipment. The step feed tank may be
either the long rectangular or the folded design. Secondary influent flow is added at two or more
points to the aeration tank, usually in the first 50 to 75% of the length.
It is also possible to use the same process approach by compartmentalizing the tank and directing
flow lengthwise through the compartments. Usually, the last compartment does not receive any raw
waste.

Extended Aeration Activated Sludge Process
The extended aeration process uses the same flow scheme as the complete mix or plug flow
processes but retains the wastewater in the aeration tank for 18 hours or more.
This process operates at a high MCRT (low F/M), resulting in a condition where there is not enough
food in the system to support all of the microorganisms present. The microorganisms therefore
compete very actively for the remaining food and even use their own cell structure for food.
This highly competitive situation results in a highly treated effluent with low sludge production.
(Many extended aeration systems do not have primary clarifiers and they are package plants used
by small communities.)
The main disadvantages of this system are the large oxygen requirements per unit of waste
entering the plant and the large tank volume needed to hold the wastes for the extended period.
Oxidation Ditch Activated Sludge Process

The oxidation ditch is a variation of the extended aeration process. The wastewater is pumped
around a circular or oval pathway by a mechanical aerator/pumping device at one or more points
along the flow pathway. In the aeration tank, the mixed liquor velocity is maintained between 0.8
and 1.2 fps in the channel to prevent solids from settling.
Oxidation ditches use mechanical brush disk aerators, surface aerators, and jet aerator devices to
aerate and pump the liquid flow. Combination diffused aeration and pumping devices are
commonly used in Europe.

High Purity Oxygen Activated Sludge Process
The most common high purity oxygen activated sludge process uses a covered and staged aeration
tank configuration. The wastewater, return sludge, and oxygen feed gas enter the first stage of this
system and flow concurrently through the tank.
The tanks in this system are covered to retain the oxygen gas and permit a high degree of oxygen
use. A prime advantage of the staged reactor configuration of the oxygenation system is the
system’s ability to match the biological uptake rate with the available oxygen gas purity.
The dissolution of oxygen and the mixing of the biological solids within each stage of the system
are accomplished with either surface aeration devices or with submerged turbine-aeration systems.
The selection of either of these two types of dissolution systems largely depends on the aeration
tank geometry selected.
The particular configuration of oxygenation tank selected for a given system, that is, size of each
stage, number of stages per aeration tank, and number of parallel aeration tanks, is determined by
several parameters including waste characteristics, plant size, land availability, and treatment
requirements.
Aside from the aeration tank, the other key factor in an oxygen activated sludge system is the
oxygen gas source. There are three sources of oxygen supply: liquid oxygen storage, cryogenic
oxygen generation, and pressure-swing adsorption generation.
The first of these requires no mechanical equipment other than a storage tank that is replenished
by trucked-in liquid oxygen. This method is economically feasible for small (less than 4 mgd) or
temporary installations.

Sludge Problems and Solutions
Sludge Age
Activated sludge is recycled back through the aeration basins by returning settled sludge
in the final clarifiers and thus remains in the activated sludge system for a number of days.
For effective treatment, a certain sludge age is desired for the type of activated sludge
system.
For conventional activated sludge, a sludge age of 3-10 days is typical. For extended
aeration activated sludge, older sludge ages of 15-30 days are common. F/M ratio and
sludge age is inversely related (1 divided by the sludge age approximates the F/M ratio).
The older the sludge, the lower the F/M ratio; conversely, the younger the sludge, the
higher the F/M ratio. All three process control methods are regulated by wasting sludge.
It is the key to controlling the activated sludge process. The operator should monitor
MLSS, F/M ratio and sludge age to waste accordingly and thus ensure optimal operations
and process stability.
Excess Solids
Solids are generated by microorganism growth and reproduction. The influent BOD
supplies the food for the growth and reproduction. As microorganisms’ populations
multiply, excess solids (microorganisms) must be removed (wasted). If excess solids are
not removed, the mixed liquor suspended solids (MLSS) and sludge age will increase and
process efficiency will be lowered. Sludge settling rates are affected. Eventually, if excess
solids do not get wasted, they can overflow the clarifier weirs and into the receiving water.
Wasting Sludge
Wasting sludge is the most important operational process control of the activated sludge
process. By wasting sludge on a consistent basis, preferably daily, the biomass within the
aeration tank will remain healthy and at a consistent MLSS level.
Wasting Rates
The concentration of WAS has a direct bearing on how much to waste and the volume
wasted. On a volume basis, a thicker waste activated sludge (high WAS concentration)
will require less amount of wasting than a thinner waste activated sludge (low WAS
concentration).
Clarifier Sludge Blanket

Solids settle and concentrate in the final clarifiers forming a sludge blanket. The sludge
blanket can increase or decrease depending on the RAS flow rate. The proper RAS flow
rate allows for a desired sludge blanket.
Young Sludge
Young sludge consists of sludge which has not yet reached a high enough sludge age to
be most effective in a particular activated sludge process. Billowing whitish foam is an
indicator that the sludge age is too low. Young sludge will often have poor settling
characteristics in the clarifier, and can leave straggler floc in the clarifier effluent. Young
sludge is often associated with a high F/M. To correct for young sludge, it is necessary to
decrease wasting rates. This will increase the amount of solids under aeration, reduce the
F/M ratio, and increase the sludge age.

Old Sludge
Old sludge consists of sludge in which the sludge age is too high to be most effective in a
particular activated sludge process. Dark brown foam and a somewhat greasy or scummy
appearance is an indicator of old sludge. Settling in the clarifier is rapid, but pin floc can
be present in the effluent and the effluent is hazy. Old sludge is often associated with a
low F/M ratio. To correct for old sludge, it is necessary to increase wasting rates and return
less sludge to the aeration basin. This will reduce the amount of solids under aeration,
increase the F/M ratio and decrease the sludge age.
RAS Concentration
Varying the RAS flow rate will affect the concentration and detention time of clarified solids.
Adjusting the RAS pumping rate allows the return of more or less concentrated solids
while also increasing or decreasing the depth of the sludge blanket. RAS flow rates can
be paced off influent flow rates.
Final Clarifier Solids Loading Rate (SLR)

The rate at which the activated sludge is returned from the final clarifiers to the aeration
basins, along with the influent flow, effects the flow of solids into the clarifiers. Aeration
basin mixed liquor suspended solids must have sufficient time to settle and be returned or
wasted in the activated sludge system. Clarifiers are designed for certain solids loading
rates that should not be exceeded.
Hydraulic Overloads
Solids washouts
If the flow is too high through the final clarifier, the solids will not have enough time to settle
and can wash out over the weirs. This can result in a loss of solids from the system and
effluent permit violations.
Reduced treatment efficiency

High flows can reduce the detention time in the aeration basins and thus reduce treatment
efficiency. If too many solids also flow out of the clarifier, there may not be enough biomass
to effectively treat the incoming organic load.
Denitrification
When RAS flow rates are too low, thick sludge blankets in the final clarifier can result. The
operator will see gas bubbles (from nitrogen gas) and rising/floating sludge clumps on the
clarifier surface.
Controlling dissolved oxygen levels in diffused air systems

1. By controlling air valves
2. By controlling the blower output such as using VFDs
3. By increasing or decreasing the number of blowers in operation
4. Cleaning or replacing diffusers
5. Changing the number of diffusers
6. Process control (ex. MLSS levels)
Controlling dissolved oxygen levels in mechanical aeration systems

1. By increasing or decreasing the aerator speed by using VFDs
2. By increasing or decreasing the aerator submergence by adjusting the tank water level
3. By increasing or decreasing the number of aerators in operation
4. Process control (ex. MLSS levels)

[Note: Throttling air valves with a positive displacement blower will not reduce air flow
output but will raise operating pressure of the blower with high electric cost as the result.
Throttling an inlet air valve on a centrifugal blower will reduce air discharge flow.]
Filamentous Bulking Sludge

The sludge blanket in the final clarifier will be near the surface, often with solids going over
the weirs. Confirm by microscopic examination.
Nocardia Filaments Present

Thick, greasy, dark tan foam on aeration basins and possibly on final clarifiers. Confirm
by microscopic examination.
Return Rates Too Low

Thin mixed liquor suspended solids and a sludge blanket build-up of solids. Rising clumps
of sludge or gas bubbles may occur in the final clarifier.
Return Rates Too High

No sludge blanket in the final clarifier and a thin return activated sludge.
Denitrification in Final Clarifier

In the absence of oxygen, a sludge blanket that is too thick and remains in the clarifier too
long can denitrify. Nitrates in the sludge will be converted to nitrogen gas. The release of
nitrogen gas will cause small gas bubbles that will be observed at the clarifier surface.
Clumps of sludge may also rise to the surface.
Low DO in an Aeration Basin

Problem: Dissolved Oxygen Meter/probes
Solution: Check the calibration of DO monitoring equipment. Clean probes and monitoring
equipment regularly to ensure accurate DO measurements
or
Problem: Inadequate air supply
Solution: Increase air supply.
or
Problem: Excessive Organic Loading
Solution: Reduce influent loading through enforcement of the sewer use ordinance; a
pretreatment program; equalization basins or bringing additional aeration basins on-line if
available.
Clarifier Settling Problems and Solutions

Problem: Excessive filamentous organisms
Solution: Adjust the environmental conditions to support a healthier biomass.
or
Problem: Sludge age. Too young or too old a sludge can result in a poor settling sludge.
Solution: Adjust wasting to achieve the proper sludge age.
or
Problem: Clarifier washouts due to high flows
Solution: Develop and implement a collection system CMOM Program to reduce
infiltration/inflow (I/I)
or

Problem: Too many solids in the system
Solution: Waste regularly to maintain proper MLSS, F/M ratio and sludge age for influent
organic loads
Foaming Problems
Problem: Young sludge (white billowing foam)
Solution: Increase sludge age
or
Problem: Filamentous foaming organisms (Nocardia, Microthrix)
Solution: Adjust environmental conditions. Adjust F/M ratio, sludge age and dissolved
oxygen. Reducing incoming grease is one of the most important factors to control surface
filamentous forming organisms.
or
Problem: Industrial/chemical discharges (surfactants, phosphates, etc.)
Solution: Enforce sewer use ordinance
Lack of Nitrification
Problem: Improper environmental conditions
Solution: Nitrifying bacteria are very sensitive to environmental factors, such as very low
dissolved oxygen, alkalinity, and temperatures. An older sludge (> 8 days) is usually
needed for their growth. Adjust these environmental conditions, as you can, to support the
growth of nitrifying bacteria.
Course Bubble Aeration Systems

1. Aeration basins should be drained annually
2. Remove excess settled solids that have accumulated
3. Clean diffusers and piping assemblies as needed
4. Inspect all hardware and components
5. Repair, replace, and tighten components as needed
6. Refill aeration tank following startup procedures
Fine Bubble Aeration Systems

1. Aeration basins should be drained annually
2. Drain aeration basin and leave air on
3. Remove excess settled solids that have accumulated
4. With air on, hose off and wash each diffuser with clean water
5. With air off, if needed scrub each diffuser with either a soft bristle brush or rag.
6. Turn air back on and repeat hosing procedure for each diffuser
7. Inspect all hardware and components
8. Repair, replace, and tighten components as needed
9. Refill aeration tank following startup procedures

Aeration Section
There are several designs and applications for aerators:
 Diffused Aerators
 Mechanical Surface Aerators
 Submerged Turbine Aerators
The two most common types of aeration systems are subsurface diffusion and mechanical aeration.
Diffused air systems have been around longer than you.
Opened tubes were used or perforated pipes located at the bottom of aeration tanks. But a more
efficient process was desired, born to the process, porous plate diffusers. In the diffused air system,
compressed air is introduced near the bottom of the tank. Let’s look at the definition for diffused
aeration:
“The injection of a gas, air or oxygen, below a liquid surface.”
There are a variety of hybrid air diffusion systems used in the process; we will focus on the basic
components.
The following diagram highlights the main parts of the diffused aeration system.

Here is a rare and up-close view of non-porous diffuser heads. Notice the heads that
are missing in the bottom photograph.

All about Aeration
The aerated systems described above need an oxygen supply. Depending on the characteristics
of the process, different designs may be used. The oxygen can be supplied to the activated sludge
by either diffused aeration, by turbine agitation, by static aerators, or by surface coarse or large
bubble diffusers. The last two are also used in lagoon systems.
The diffused aeration systems are also divided into fine bubble, medium and coarse or large bubble
diffusers. The fine bubble diffusers are built of porous materials (grains of pure silica or aluminum
oxide are bonded ceramically or by resins) which provide very small bubbles of high surface area
that favor the oxygen transfer from the air to the wastewater. The medium bubble diffusers are
perforated pipes or tubes wrapped with plastic or woven fabric. The coarse or large bubble diffusers
can be orifice devices of various types, some of which are designed to be non-clogging.
Coarse or Large Bubble Diffusers

With the small or fine bubble diffusers, it is important to use air free of particles that would otherwise
clog them. Although somewhat less efficient for oxygen transfer, the coarse bubble diffusers are
sometimes preferred because the presence of particles in the air is not a critical problem, and also
for their lower cost and maintenance requirements. The diffusers are placed along air manifolds,
close to the bottom of the aeration tanks.
The static aerators are vertical tubes placed at the bottom of the aeration tank, with packing material
along its length. The compressed air is supplied from the bottom of the tubes, forcing a mixture of
air and water through the packing, where most of the oxygen transfer to the wastewater takes
place. They have been used mainly in aerated lagoons.

Oxidation ditches are used for nutrient removal.

Blowers
In the diffused aeration system, blowers are used to circulate the tank’s contents by the air-lift effect.
The air filter on the blower removes dirt from the air. Therefore, helps prevent diffuser clogging.
Before all this begins, we need a power source to drive the blower. Usually, electric motors are
used but in remote locations, gas or diesel engines can be used as well. In some states, solar
energy is available to provide the power.
As illustrated in the photograph below, the rotation of the motor shaft is transferred to the blower
shaft by means of a flexible coupling or through drive belts. The blowers that we will refer to are
centrifugal blowers.
The centrifugal blower works like a centrifugal pump or a fan. Rotating impellers or fans cause
movement of the air through the blowers. You have an intake side that takes in the air and the
discharge side the forces the air out. The number of impellers you have will determine if it is a multi-
stage or single-stage blower. The photograph below illustrates the major components of a
centrifugal blower.
A lobe blower utilizes positive displacement; it also

has an intake and a discharge side. The lobes turn
in opposite directions in the casing. As they turn,
the air is drawn in through the blower inlet and is
trapped. The lobes keep turning, opening the
blower discharge, and forcing the trapped air
through the outlet. Usually, an electric motor
drives the blower with belt pulleys or flexible
couplings.

Before we continue let’s review what you just read about the blowers and motors.
1. What are two ways that the motor and the blowers can be attached?
2. When using flexible couplings, what are some maintenance concerns to consider?
Blowers may be provided with additional equipment. For example, safeguards can be installed to
protect equipment and operators. Temperature sensors can be used for bearing housing, vibration
sensors protect the unit by shutting it down if limits are exceeded. Condensation drains should be
provided on the bottom of blowers to drain off any accumulated moisture.
The compressed air from the blowers moves into a system of pipes and valves. The amount of air
supplied from the blower is controlled by regulating valves mounted on the intake and/or discharge
side of the blower. Usually butterfly valves are used and depending on your budget, you could have
manually operated or use automation.
Blowers usually discharge to a common manifold, so check valves are installed at the discharge of
each blower. The intake and discharge pipes are called the air mains. They are connected by a
flexible connection to allow for vibration and heat expansion in the piping. In the winter months, the
best place to be is in the blower room. There is a pressure relief valve on the discharge manifold
to protect the blower from excessive back pressure overload. When this occurs the operator will be
awakened on the midnight shift. Pressure gauges are used in several areas on the discharge side
of the blowers. In some cases, you may see them on the intake side for use in calculations of pump
efficiency.
On the intake side, where air is supplied, you would have some type of filtering to remove dirt
particles that could clog the diffusers. It also protects the blowers from excessive wear. Replaceable
filter units are the simplest for operations. Bag house dust collectors are bulky and expensive,
though maintenance may be less. In some cases, electrostatic precipitators may be an advantage,
shocking if operators are not careful, in areas of poor air quality. Most systems have utilized
pressure drop measuring to indicate when it is time to replace or clean the units.
The above photograph shows air being unevenly distributed.

Diffusers
There are many different design layouts and patterns of diffuser placement. Systems that allow
longer and more complete contact between the air and the liquid are preferred. We will focus on
fine bubble (porous) diffusers and coarse bubble (nonporous).
Coarse bubble diffusion devices, or large-hole diffusers, produce larger bubbles than porous plates,
porous tubes, or synthetic socks. The larger bubbles provide less surface area for air-liquid contact
and will result in less oxygen transfer efficiency than that obtained with fine bubble diffusers.
Answer this question:
An air stone like those used in aquariums is a good example of a?

A. Porous material
B. Nonporous material
Mechanical Aeration
There are several main types of mechanical
aeration devices. The floating and fixed
bridge aerators are quite common. Some
use a blade to agitate the tank’s surface
and disperse air bubbles into the aeration
liquor. Others circulate the mixed liquor by
an updraft or downdraft pump or turbine.
This action produces surface and
subsurface turbulence, while diffusing air
through the mixed liquor.
The motor speeds are usually in the 1800 rpm range. This speed is reduced to the 30 to 70 rpm
range with gear reducers.

Most vertical motors are mounted on a gear reduction unit as seen in the
photograph on the right. The impeller drive shaft can be enclosed in a
housing connected directly to the gear box. There is a bearing at the
bottom of the shaft that steadies and aligns this shaft. This bearing
needs lubrication, always check your manufactures recommendations.
Some plants use an oxidation ditch in which rotating brushes, blades, or

disks are rotated partially submerged in the mixed liquor. The
turbulence produced traps the air bubbles and keeps the mixed liquor in
motion.
Other systems use both compressed air and a mechanical device to trap
the bubbles. In one such system, submerged turbine aeration, air is
injected below a rotating turbine blade that shears and disperses the air.
Submerged turbine applications have also used a draft tube operating in a downdraft-pumping
mode.
Jet and Aspirator

Aerators provide oxygen transfer by mixing pressurized air and water within a nozzle and then
discharging the mixture into the aeration tank. The velocity of the discharged liquid and the rising
air plume provide the necessary mixing action.

Secondary Clarifiers
Because microorganisms are continually produced, a way must be provided for wasting some of
the generated biological solids produced. This is generally done from the round or rectangular
shaped clarifiers.
Let’s first look at the components of a rectangular clarifier. Most are designed with scrapers on the
bottom to move the settled activated sludge to one or more hoppers at the influent end of the tank.
It could have a screw conveyor or a traveling bridge used to collect the sludge. The most common
is a chain and flight collector. Most designs will have baffles to prevent short-circuiting and scum
from entering the effluent.
The activated sludge is removed from the hopper(s) and returned by a sludge pump to the aeration
tank or wasted. Since we mentioned return and waste what do the following terms represent?
RAS:
WAS:

A settling tank used to remove suspended solids by gravity settling. Commonly referred
to as sedimentation or settling basins, they are usually equipped with a motor driven chain
and flight or rake mechanism to collect settled sludge and move it to a final removal point.

Scum Removal
Scum removal equipment is desirable on secondary clarifiers. Skimmers are either of the type that
rotates automatically or manually. The most important thing to consider is the sludge and scum
collection mechanism. We will talk about “flights and chains”. They move the settled sludge to the
hopper in the clarifier for return and they also remove the scum from the surface of the clarifier.
The flights are usually wood or nonmetallic flights mounted on parallel chains. The motor shaft is
connected through a gear reducer to a shaft which turns the drive chain. The drive chain turns the
drive sprockets and the head shafts. The shafts can be located overhead or below.
Some clarifiers may not have scum removal equipment so the configuration of the shaft may vary.
As the flights travel across the bottom of the clarifier, wearing shoes are used to protect the flights.
The shoes are usually metal and travel across a metal track.
To prevent damage due to overloads, a shear pin is used. The

shear pin holds the gear solidly on the shaft so that no slippage
occurs.
Remember, the gear moves the drive chain. If a heavy load is put
on the sludge collector system, the shear pin should break. This
means the gear would simply slide around the shaft and
movement of the drive chain would stop.

Top photograph, a clarifier’s raking mechanism. Bottom, scum armature equipment.

Scum Removal Equipment
In some circular or square tanks rotating scrapers are used. The diagram below shows an typical scum
removal equipment.
The most common type has a

center pier or column. The major
mechanical parts of the clarifier
are the drive unit; the sludge
collector mechanism; and the
scum removal system. There is
also some related equipment that
we will consider briefly.
Let’s look at the drive unit first.

There are three main parts to the
drive unit; the motor (or gear
motor); the gear reducer; and the
turntable.
The motor is connected to a gear reduction unit which is commonly connected to additional gearing. The
drive cage is rotated around a center column by the motor and gear reduction unit. Although the drive motor
runs at about 1800 rpm, the gear reducer lowers the output speed so that the sludge collector mechanism
goes through one revolution every 20 to 30 minutes. Usually, the motors used on clarifier mechanisms are
totally enclosed, fan cooled motors, suitable for outside operation.
The horsepower of the motor is dependent on the size of the

clarifier. The motor drives the chain and sprocket which drives the
worm gear. The worm gear drives the gear that is mounted on a
shaft that drives the turntable. The motor shaft speed is reduced by
a series of gear reducers.
We looked at the main parts of the drive unit. Now let’s take a look
at the sludge collector and the scum removal system mechanism.
The main parts of the unit are: the rake arm; the scraper blades;
the adjustable squeegees; the surface skimmer; the scum baffles;
and the scum box.
The surface skimmer rotates at the same speed as the collector

mechanism and is usually supported by the collector rake arm. The scum baffle prevents scum from flowing
over the effluent weir. The surface skimmer collects the scum and deposits it in the scum box. The stilling
well or influent baffle projects above the liquid and directs the influent downwards to assist in settling
suspected solids and reduce short circuiting. Another important part of the secondary clarifier is the effluent
weir, launder and pipe.
An effluent weir goes around the circumference of the tank and allows clarified liquid to flow evenly from the
tank. The effluent launder collects the tank overflow and takes it to a low point in the launder where a pipe is
used to take the effluent to the chlorine contact basin or other means of treatment.
Some clarifiers may have a scum trough heater. The scum removal system rotates around the clarifier at a
very slow rate. In subfreezing temperatures, the scum box and pipe could freeze. This problem can be
overcome by using immersion heaters, or putting infrared lamps over the scum box. Some clarifiers are
covered.

As you have previously read, depending on the design and operation of the process, activated sludge
has several interrelated components:
1. Single aeration tank or multiple aeration tanks designed for completely mixed or plug flow.
2. An aeration source to provide adequate oxygen and mixing: sources can be compressed air,
mechanical aeration, or pure oxygen.
3. A clarifier to separate the biological solids (activated sludge) from the treated wastewater.
4. A means of collecting the biological solids in the clarifier and recycling most of them (return activated
sludge, RAS) to the aeration tank.
5. A means of removing or wasting excess biological solids (waste activated sludge, WAS) from the
system.

Review Process Goals
As previously noted, the activated sludge process can be used to remove carbonaceous BOD and also
ammonia (nitrification). We can take the wastewater oxygen demand separated into two categories:
carbonaceous and nitrogenous.
Carbonaceous BOD Removal

The carbonaceous demand should be expressed as a function of the number of days that the demand will
be measured; 3-day, 5-day (most common), 7-day, and 20-day time periods are commonly used. To obtain
only carbonaceous oxygen demand, it may be necessary to inhibit nitrification by adding chemicals.
The rate and extent of BOD5 (5-day BOD) removal in primary treated (settled) or untreated wastewater
depends on the relative quantities of soluble, colloidal, and suspended BOD5, and a soluble BOD5 content
of approximately 20 to 40% of the total. These proportions may vary, particularly in warmer climates where
long collection system residence times and the higher wastewater temperatures may result in a higher
proportion of soluble BOD5. This is caused by the bacterial degradation of a portion of the colloidal and
settleable fractions.
With typical municipal wastewater, a well-designed activated sludge process should achieve a
carbonaceous, soluble BOD5 effluent quality of 5mg/L or less. Similarly, with clarifiers designed to maximize
solids removal at peak flows and adequate process control, the average SS in the effluent should not exceed
15 mg/L. On a practical basis, an effluent with 20/20 mg/L BOD5 and SS should be attained, assuming
proper operation. Potential capabilities of the process are 10/15 mg/L Bod5 and SS. To consistently achieve
values lower than 10/15 mg/L, some type of tertiary treatment is required.
Nitrification
Of the total oxygen demand exerted by the wastewater, there is often a sizeable fraction associated with the
oxidation of ammonia to nitrate. The autotrophic bacteria Nitrosomonas and Nitrobacter are responsible for
this two-state conversion. Being autotrophic, these nitrifying organisms must reduce oxidized carbon
compounds in the wastewater, such as CO2 and its related ionic species, for cell growth. As a result, this
characteristic markedly affects the ability of the nitrifying organisms to compete in a mixed culture.
The nitrifying bacteria obtain their energy by oxidizing ammonia nitrogen to nitrite nitrogen and then to nitrate
nitrogen. Because very little energy is obtained from these oxidation reactions, and because energy is
needed to change CO2 to cellular carbon, the population of nitrifiers in activated sludge is relatively small.
When compared to the normal bacteria in activated sludge, the nitrifying bacteria have a slower reproduction
rate.
Nitrifying organisms are present to some extent in all domestic wastewaters. However, some wastewaters
are not nitrified in existing plants because they are designed for the higher growth rate of bacteria responsible
for carbonaceous removal. As the MCRT is increased, nitrification generally takes place. The longer MCRT
prevents nitrifying organisms from being lost from the system when carbonaceous wasting occurs or, more
accurately, the longer MCRT permits the build-up of an adequate population of nitrifiers.
Because of the longer MCRT required for nitrification, some systems are designed to achieve nitrification in
the second stage of a two-stage activated sludge system.
The oxygen demand for complete nitrification is high. For most domestic wastewaters, it will increase the
oxygen supply and power requirements by 30 to 40% because complete nitrification requires from 4.3 to 4.6
lb. of oxygen for each lb. of ammonia nitrogen (4.3 to 4.6 mg/mg) converted into nitrate, and wastewaters
generally contain 10 to 30 mg/L of reduced nitrogen. Nitrification systems generally are not operated at
intermediate (40 to 80%) removals; stable operation is achieved when essentially complete nitrification
(greater than 90%) occurs.

Minimum acceptable dissolved oxygen (DO) concentrations of 2 to 3 mg/L have been reported, but
nitrification appears to be inhibited when the oxygen concentration is lower than 1 mg/L.
Optimum growth of nitrifying bacteria has been observed in the pH range of 8 to 9 although other ranges
have been reported. A substantial reduction in nitrification activity usually occurs at pH levels below 7,
although nitrification can occur at low pH.
While nitrification occurs over a wide temperature range, temperature reduction results in a slower reaction
rate.
The temperature effect is made less severe by increasing the MCRT. During the conversion of ammonia to
nitrate, mineral acidity is produced. If insufficient alkalinity is present, the system’s pH will drop and
nitrification may be inhibited.
Bacteria Highlights
A change in the numbers or predominance of microorganisms in activated sludge is usually gradual. The
time required for a complete shift from one species to another will normally be seen in: 2 to 3 MCRT's.
A large amount of long filamentous bacteria will prevent good settling. The liquid above this mass is called
the supernatant.
Endogenous respiration of microorganisms in an extended aeration plant will complete the oxidation process
of an organic material.
The bug Nocardia causes frothing.
Saprophytic type bacteria produces the most acid in an anaerobic digester.
The best location for microscopic examination of activated sludge in a conventional system is at the effluent
end of the aeration system. The examination can reveal a predominant number of rotifers and nematodes,
this condition may indicate that the F/M ratio is too low and this would be normal in an extended aeration
process.
Food to microorganism ratio. A measure of food provided to bacteria in an aeration tank.
Food = BOD, lbs/Day

Microorganism MLVSS, lbs
= Flow, MGD x BOD, mg/L x 8.34 lbs/gal

Volume, MG x MLVSS, mg/L x 8.34 lbs/gal
or = BOD, kg/day
MLVSS, kg

Separate Stage Nitrification and Denitrification Systems
Suspended Growth Nitrification
Single-sludge systems for BOD removal and nitrification require that the biomass inventory be retained long
enough to establish a stable population of nitrifiers and that the HRT be such that the biomass can react with
the ammonia-nitrogen entering the system.
The overall approach for designing such systems is to determine the target SRT for the system based on
influent characteristics (i.e., BOD, ammonia-nitrogen, organic nitrogen), environmental conditions such as
temperature and flow characteristics (i.e., average daily, maximum monthly, diurnal peak).
Most activated sludge treatment plants will readily nitrify if they have sufficient aerobic SRT and can deliver
sufficient oxygen maintaining 2 mg/L DO or greater. For plants having difficulty in nitrifying due to insufficient
tank volume, there are some emerging technologies which can improve the process.
One of these is bioaugmentation. Bioaugmentation is accomplished by seeding the activated sludge process
with an external source of nitrifying bacteria (also known as external bioaugmen-tation) or making process
improvements to increase the activity of or enrich the nitrifier population (also known as in situ
bioaugmentation).
External bioaugmentation uses either commercial sources of nitrifiers or sidestream processes to grow
nitrifiers onsite. Early experiences with commercial sources were not consistent, so most work to date has
been with sidestream production onsite (USEPA, 2008a). Two patented sidestream configurations for
external bioaugmentation are the Single reactor High-activity Ammonia Removal Over Nitrite (SHARON)
process and the In-Nitri® process. Both provide high temperature sidestream nitrification using ammonia
from the anaerobically digested sludge dewatering liquid or digested supernatant. The nitrifiers grown in the
sidestream reactor are fed to the main liquid treatment stream.
Both use flow through reactors with hydraulic retention times (HRT) in the 2 to 3-day range. In the SHARON
process, nitrification is stopped mainly at nitrite by such process control methods as low DO concentration,
low pH and/or low SRT. Full-scale operating systems for the SHARON process include installations at
Utrecht, Rotterdam, Zwolle, Beverwijk, Groningen, The Hague in the Netherlands, and a system in New York
City. Seeding from a diffused air biological nutrient removal process to stimulate nitrification in a parallel
oxygen process has proved successful at a number of locations (Bott et al., 2007). Emerging in situ
bioaugmentation technologies used to enhance nitrifier growth and shown to be successful in bench, pilot,
and/or full-scale trials are described briefly below (USEPA, 2008a):
• The Bio-Augmentation Regeneration/Reaeration (BAR) process was developed in the U.S. and is identical
to the Regeneration-DeNitrification (R-DN) process developed independently in the Czech Republic. It works
by recycling ammonia-laden filtrate or centrate from dewatering of aerobically digested sludge to the head of
the aeration tank. The sidestream is fully nitrified, seeding the aeration tank with additional nitrifying bacteria
which allows for reduced SRT.
• Aeration Tank 3 (AT3) is similar to the BAR process except that it sends a smaller fraction of the return
activated sludge (RAS) to the aeration tank in order to stop the nitrification process at the nitrite stage.
• Bio-Augmentation Batch Enhanced (BABE) process uses a SBR to grow nitrifiers by feeding it RAS and
reject water from the sludge dewatering process. After treatment, concentrated nitrifiers are recycled to the
head of the aeration tank.

• The Mainstream Autotrophic Recycle Enhanced N-removal (MAUREEN) Process was developed for the
two-sludge treatment configuration at the Blue Plains Advanced Wastewater Treatment Plant in Washington,
DC. The process involves sidestream treatment of WAS from the second stage to preferentially select AOB
for bioaugmentation to the first sludge stage.
Attached Growth Nitrification

Attached growth processes will also nitrify. Trickling filters and rotating biological contactors (RBCs) have
historically been used for biological treatment of wastewater and can achieve nitrification with a low organic
loading and a relatively high media volume. Typically, nitrification is achieved on the media after most of the
BOD is removed since the heterotrophic population competes with the nitrifying organisms for oxygen and
space on the media.
A major disadvantage of these technologies compared to suspended growth systems is that denitrification is
fully dependent on addition of a supplemental carbon source. Suspended growth processes, on the other
hand, can be designed to denitrify 80 percent or more of nitrate using the incoming BOD as the carbon
source, which is a lower cost solution.
Consequently, trickling filters and RBCs have fallen out of favor for nutrient removal applications. In recent
years, manufacturers have developed new technologies called biological aerated filters (BAF) to achieve
BOD removal and nitrification. USEPA (2008a) identifies two existing BAF designs as established
technologies: the Biofor® system and the Biostyr® system. The Biofor® filtration system is a fixed bed, upflow
system with a dense granular media that is designed to expand during filtration. Air is sprayed into the filter
to maintain an aerobic environment. The Biostyr® system is similar but uses a media that is less dense than
water and held in place during operation by a screen at the top of the cell.
BAF can be configured in series to remove BOD in one unit and ammonia-nitrogen in the next or it can be
designed for BOD removal and nitrification in a single unit depending on process goals. Advantages of BAF
include its smaller footprint, higher hydraulic loading rates, and less susceptibility to washout than suspended
sludge systems (Verma et al., 2006).
Another fixed film process that has gained popularity lately is moving bed biofilm reactors (MBBR). These
reactors involve biofilm attached to a plastic media in a series of fluidized bed reactors. The plastic media
help promote specialization of the biofilm within each reactor for either nitrification or denitrification (WEF and
ASCE, 2006). Mixers or medium bubble diffuse aeration are used to keep the media suspended, depending
on whether the system is anaerobic or aerobic. MBBR has a shorter SRT and smaller footprint than activated
sludge processes. It has also proven to be effective in cold temperatures (Bott et al., 2007).
Separate-Stage Denitrification
A separate-stage denitrification system may be appropriate for plants that are regularly achieving nitrification
and need to add denitrification capabilities. Attached growth systems (denitrifying filters) are more common
than suspended growth systems, although suspended growth systems have been used for some treatment
plants. Suspended growth reactors typically have short SRTs (2 to 3 hrs.) and a small aerated zone following
the denitrification zone to oxidize excess methanol and release contained nitrogen gas bubbles (WEF and
ASCE, 2006).
Denitrification filters typically have a small footprint compared to suspended growth systems and have the
added advantage of achieving denitrification and solids removal simultaneously. They were first installed in
the 1970s and have evolved into two main process configurations (USEPA, 2007c):
• Downflow denitrification filters are deep bed filters consisting of media, support gravel, and a block
underdrain system. Wastewater flow is directed over weirs onto the top of the filter where a supplemental
carbon source, typically methanol, is added. Backwashing (typically air scouring and backwashing with air
and water) is conducted at regular intervals to remove entrapped solids from the filter.

During operation, nitrate is converted to nitrogen gas and becomes entrained in the filter media, increasing
head loss through the filter. To release entrained nitrogen, most denitrification systems have a nitrogen-
release cycle operation that essentially “bumps” the filter by turning on the backwash pump(s) for a short
period of time.
• Upflow continuous backflow filters do not have to be taken off-line for backwashing, as it is an integral part
of the filtering process. Wastewater enters the bottom of the filter where a carbon source, typically methanol,
is added. Water flows up through an influent pipe and is dispersed into the filter media through distributors.
Filtered water discharges at the top of the filter. Filter media continuously travels downward, is drawn into an
airlift pipe at the center of the filter, and is scoured before being returned to the filter bed.
Performance of denitrifying filters depends on many factors including:

• Influent weir configuration
• Filter media
• Underdrain system
• Backwash system
• Flow and methanol feed control
One wastewater system in Connecticut reported that key design issues for them were influent piping design
to minimize aeration, maintaining a consistent flow to the filters, and control of methanol feed based on
influent COD (Pearson et al., 2008).
The tubes held in this photograph should be placed in an autoclave. One tube is a standard or
QA and the other would indicate contamination.

Key Design and Operational Issues
Temperature
In general, as temperature of the wastewater increases, the rate of nitrification and denitrification increases.
For the typical range of liquid temperatures between 10 and 25o C, the nitrification rate will approximately
double for every 8 to 10o C increase in temperature (WEF and ASCE, 2006). Rapid decreases in temperature
without acclimation time will, however, cause even slower nitrification rates than predicted, strictly by the
temperature change. Denitrification rates will also increase with increasing temperature, although not at the
same magnitude as nitrification rates.
Dissolved Oxygen
Nitrifying bacteria are also more sensitive to DO levels as compared to aerobic heterotrophic bacteria, with
growth rates starting to decline below 3 to 4 mg/L with significant reduction below 2 mg/L. The nitrification
rate at a DO concentration of 0.50 mg/L is only about 60 percent of that at a 2.0 mg/L DO concentration.
Studies have shown that the amount of oxygen available to nitrifying bacteria can be limited by floc size,
requiring higher bulk DO concentrations under higher organic loading conditions (Stenstrom and Song,
1991). At DO concentrations less than 0.5 mg/L, the effect is greater for Nitrobacter than for Nitrosomonas.
This can result in higher NO2-N in the effluent and have a negative impact on chlorine disinfection as 1 g of
NO2-N consumes 5 g chlorine for oxidation. DO must normally be less than 0.2 to 0.5 mg/L, otherwise there
will be inhibition of the denitrification process.
pH and Alkalinity
Nitrification generally operates well within a pH range of 6.8 to 8.0 (WEF and ASCE, 2006). At lower pH
values the nitrification rate is much slower and at pH values near 6.0 the nitrification rate may only be about
20 percent of that with a pH of 7.0 (Tchobanoglous et al., 2003). Alkalinity is consumed during the nitrification
process but partially replenished (up to 62.5 percent) during the denitrification process. Depending on the
influent wastewater alkalinity, there may be a sufficient alkalinity reduction due to nitrification to decrease to
unacceptable levels. Addition of chemicals such as lime, sodium hydroxide, or soda ash can be used to
replace the alkalinity consumed by nitrification to maintain acceptable pH levels.
Carbon Sources for Denitrification

Denitrifying bacteria need a readily available carbon food source, such as soluble BOD, to ultimately convert
nitrate to nitrogen gas. WWTPs that meet very low total nitrogen limits typically use a secondary anoxic zone
in which supplemental carbon is added. Supplemental sources can be “internal” such as fermented
wastewater or sludge, or “external” sources such as purchased chemicals.
Methanol is currently the most common external carbon source used in denitrification because of it
low cost. It has several drawbacks, however, namely:
• It is highly flammable and implicated in some storage tank explosions and fires (Dolan, 2007); however,
with proper design and operation problems can be minimized.
• It is not the most efficient source for most treatment configurations.
• Costs have begun to fluctuate widely (deBarbadillo et al., 2008).
• Availability is a problem in some areas (Neethling et al. 2008).
• Reported low growth rates under cold temperatures (Dold et al. 2008).
Other sources of carbon include ethanol, acetic acid, corn syrup, molasses, glucose, glycerol, and industrial
waste products. The WEF Nutrient Challenge Research Plan (2007) identified research on alternative carbon
sources as priority for operators, owners, and engineers of wastewater systems. In December of 2007, the
2nd External Carbon Workshop was held in Washington, DC to discuss the state of the technology and
research needs. WERF is also currently formulating a standard protocol for evaluation of external carbon
alternatives.

Nitrification Inhibition from Toxic Chemicals
Nitrifying bacteria are very sensitive to heavy metals and other inorganic compounds in wastewater. The
Local Limits Development Guidance Manual (USEPA 2004) has been the main source of information on
inhibitory effects for a variety of wastewater treatment processes including nitrification. Appendix G of the
2004 version provides a summary table with the reported range of nitrification inhibition threshold levels for
a number of metals, non-metal inorganics, and organic compounds. Actual inhibitory effects are site-specific
and depend on many factors including the nature of biodegradable organic material, microorganism
speciation, acclimation effects, temperature, and water quality conditions.
Wet Weather Events

Wet weather events can increase inflow and infiltration into the collection system and subsequently increase
the hydraulic load to the wastewater treatment plant. This can in turn reduce the SRT leading to reduced
performance of nitrification process units. In addition, wet weather flows have different characteristics than
typical wastewater influent flow and can be less favorable for nitrification and denitrification. Conditions that
are less favorable for nitrification include decreased alkalinity and sudden temperature drops. Lower
biodegradable COD concentrations and increased DO make wet weather flows less amenable to
denitrification.
Flow equalization basins can be used to handle wet weather events; however, this requires available space
and capital investment. USEPA (2008a) identifies a number of innovative storage and treatment technologies
used to manage influent flows during wet weather events.
Guidance for Selecting Process Modifications

Nitrogen removal requires first that a biological nitrification process be present or that the facility be modified
to accomplish nitrification. Considerably more volume is needed for activated sludge nitrification compared
to designs for BOD removal only. If there is insufficient space to accommodate the increased volume,
suspended growth or hybrid process options that require less space such as the MBR process or IFAS
systems with suspended media in the activated sludge process should be considered. Another option is to
use a fixed film nitrification process after the suspended growth process clarification step. This could be a
BAF or a plastic media trickling filter. However, if nitrogen removal is required, an exogenous carbon source
is needed, which has higher operating costs than using the influent BOD for denitrification.
Nitrification systems need sufficient oxygen transfer for ammonia oxidation in addition to BOD removal. Such
systems should consider the impact to diurnal loadings and ammonia addition in recycle streams. The influent
TN concentration may have daily peak values that are 1.5 to 2.0 times the daily average loading. Higher peak
loadings require longer SRTs to assure that sufficient nitrifying bacteria are present to remove ammonia at a
greater rate, while maintaining a low effluent ammonia concentration. Often anaerobic digester sludge
dewatering operations occur during the day and produce return recycle streams high in ammonia
concentration (500-1000 mg/L) at times that coincide with the high influent diurnal ammonia loads. Recycle
equalization or treatment helps to provide a more stable nitrification system and lower effluent NH3-N
concentrations.
In many cases, it is advantageous to incorporate a denitrification pre-anoxic step with nitrification

(MLE process) due to the many benefits and improved operational stability. The advantages include
1) less aeration energy as the nitrate produced can be used for BOD removal,
2) the production of alkalinity to offset the alkalinity used by nitrification, which in some cases eliminates the
need to purchase alkalinity, and
3) a more stable, better settling activated sludge process as the anoxic-aerobic processes favor good settling
floc-forming bacteria over filamentous growth.

The effluent nitrogen goals greatly affect the process design choices and system operation. For an effluent
goal of 10 mg/L TN, an MLE process is often sufficient for activated sludge treatment with secondary clarifiers
or membrane separation. However, with water conservation leading to more concentrated wastewater, these
processes alone may not be sufficient due to the fact that they are limited to 80-85% removal of the influent
TN.
For TN effluent goals of 3 to 5 mg/L or lower, some form of post anoxic treatment is generally needed. One
option is to convert an MLE process to a Bardenpho process by adding another anoxic aerobic set of tanks.
Although the endogenous respiration rate of the bacteria can be used to consume nitrate in the post anoxic
tanks, it is often necessary to add an exogenous carbon source. Other alternatives to using exogenous
carbon sources include denitrification filters instead of adding more activated sludge tank volume, step feed
with carbon addition in the last pass, and IFAS processes.
Denitrification processes require sufficient carbon to drive the nitrate/nitrite reduction reactions.
Characterization of the influent wastewater with regard to its organic strength and soluble fraction and the
TN and ammonia concentrations is needed to fully understand a system’s carbon needs. In addition, design
and operating methods that eliminate or minimize DO feeding to anoxic zones can reduce the amount of
exogenous carbon needed and provide a more stable operation. Low DO zones prior to downstream anoxic
tanks or for withdrawal of recycle to preanoxic zones should be considered.
Impacts on Sludge Production and Handling

It has been documented by both research and full scale experiments that BOD removal by activated sludge
using nitrate as the electron acceptor instead of DO will result in a 20% or more reduction in waste activated
sludge (WAS) production for the same operating conditions. Full-scale investigations near Melbourne,
Australia achieved as high as a 40% reduction in WAS, and implementation of nitrogen removal at the York
River, VA, plant resulted in a reduction of more than 50% in WAS production.
The impact this will have on total sludge production by a treatment plant will depend upon how much waste
sludge is produced by other treatment units such as primary clarifiers and chemical treatment with
precipitating chemicals.
Additionally, implementation of nitrogen removal at

conventional activated sludge plants can improve
the thickening characteristics due to decreasing
the amounts of filamentous bacteria in the
activated sludge.
If an external carbon source is added to improve

the rate of denitrification, there will be an increase
in WAS production compared to when no external
carbon source is added. If an external carbon
source is used to supplement denitrification, it is
likely that the small increase in solids production
will be offset by endogenous respiration due to
longer SRTs. Solids produced from nitrogen
removal processes generally thicken and dewater
well and show no negative impact on any solids
processing system.

What is in Municipal Wastewater?
Municipal wastewater consists primarily of domestic wastes from households and industrial
wastewater from manufacturing and commercial activities. Both types of wastewater are collected
in sanitary sewers, and are usually treated at a municipal wastewater treatment plant. After
treatment, the wastewater is discharged to its receiving water (i.e., a river, an estuary, or an ocean).
Wastewater entering a treatment plant

may contain organic pollutants (including
raw sewage), metals, nutrients, sediment,
bacteria, and viruses.
Toxic substances used in the home –

motor oil, paint, household cleaners, and
pesticides - or substances released by
industries, also make their way into
sanitary sewers.
Industrial processes, such as steel or chemical manufacturing, produce billions of gallons of

wastewater daily. Some industrial pollutants are similar to those in municipal sewage, but often are
more concentrated. Other industrial pollutants are more exotic and include a variety of heavy metals
and synthetic organic compounds. In sufficient dosages, they may present serious hazards to
human health and aquatic organisms. Unlike municipal or industrial sources of pollution, which
come from a single discrete facility, other sources are usually more diffuse. For example, rainwater
or snowmelt washing over farmlands may carry topsoil and fertilizer residues into nearby streams.
Stormwater
This type of runoff, called stormwater, may carry oil and gasoline, agricultural chemicals, nutrients,
heavy metals, and other toxic substances, as well as bacteria, viruses, and oxygen-demanding
compounds. A recent EPA study indicated roughly one-third of identified cases of water quality
impairment nationwide are attributable to stormwater, whether from farmland, streets, parking lots,
construction sites, or other sources.
Animal Feeding Operations (AFOS)

Animal Feeding Operations are livestock-raising operations, such as hog, cattle and poultry farms,
that confine and concentrate animal populations and their waste. Animal waste, if not managed
properly, can run off to nearby water bodies and cause serious water pollution and public health
risks. There are approximately 450,000 AFOs in the United States.
Acid Mine Drainage

Acid Mine Drainage is one of the most significant environmental impacts resulting from past and
current mining activities. It has been cited as a major cause of stream pollution in northern
Appalachia (PA, W. VA, VA, MD); more than 50 percent of stream miles in PA and WV do not meet
water quality standards because of acid mine drainage impacts. In addition, there are an estimated
200,000 abandoned hardrock mines nationwide and somewhere between 2,000 and 10,000 active
ones. Some of these mining operations produce waste material and other conditions that result in
acid mine drainage as well as discharges of heavy metals which affect aquatic life and drinking
water sources.

Biological Phosphorus Removal and Combination Processes
This section provides an overview of the principles behind biological phosphorus removal (BPR). It describes
existing configurations that can achieve phosphorus removal and in many cases, simultaneous nitrogen
removal. Key operational issues, impacts on sludge handling, and a summary of ongoing research related to
BPR removal are also provided.
Principles
Biological phosphorus removal is achieved by contacting phosphorus accumulating organisms (PAOs) in the
RAS with feed, containing volatile fatty acids (VFA), in a zone free of nitrates and DO (anaerobic zone).
Phosphorus is released in this zone providing energy for uptake of VFAs that are polymerized and stored
inside the PAO cells. The anaerobic zone is followed by an aeration zone where the polymerized VFAs are
metabolized and phosphorus is taken up again to store excess energy from the metabolism.
The phosphorus content of the mixed liquor suspended solids (MLSS) would be similar to that of the waste
activated sludge (WAS). When nitrification occurs in the aeration basin, nitrates will be present in the RAS,
resulting in some metabolism of the VFA before storage, thereby reducing subsequent phosphorus uptake.
Some form of denitrification (anoxic zones) must be used to reduce/remove the nitrates from the RAS. The
process flow sheets now known as Pho-redox (A/O) and 3 Stage Pho-redox (A2/O) as well as the modified
Bardenpho process were first published by Barnard (1975) as the Pho-redox flow sheets for the removal of
phosphorus. The theory for the functioning of the PAO was first suggested by Fuhs & Chen (1975).
Fuhs & Chen Theory

PAOs have the ability to store a large mass of phosphorus in their cells in the form of polypho-sphates.
Polyphosphates are formed by a series of high-energy bonds. The organisms can subsequently get energy
from breaking these bonds. The polyphosphate globules within the cells function just like energy storage
batteries. The storage of polyphosphates (energy), takes place in the aeration zone. In the anaerobic zone,
these obligate aerobic bacteria can take up short chain VFA such as acetate and propionate and store them
in the form of intermediate products such as poly-β-hydroxybutyrate (PHB). The energy for transferring the
food across the cell membranes in the anaerobic zone is derived from breaking phosphorus bonds. Excess
phosphates are released to the liquid in the anaerobic zone.
Some magnesium and potassium ions are co-transported across the cell walls with phosphates. PAOs can
only get energy from the food they have taken up in the anaerobic zone when they pass to the aerobic zone
where oxygen is available. They use oxygen to metabolize the stored products, deriving enough energy to
take up all the released phosphates as well as those in the influent, and store them in the cells. The WAS
will contain sufficient phosphate-enriched PAOs to remove most of the phosphorus from the waste steam
once enhanced BPR is established.
The right carbon source, in this case a combination of acetates and propionates, is essential for BPR. The
wastewater characteristics are thus important. In general, it can be said that you need at least 40:1 COD:TP
or about 18:1 BOD5:TP in the process influent wastewater to reduce effluent phosphorus to less than 1.0
mg/L. In addition, some of the COD should consist of short chain VFAs. More COD may be required if nitrates
must also be denitrified.
Biological phosphorus removal can work in with or without nitrification. When nitrification occurs,
denitrification within the process is important to reduce the nitrates that may be returned with the RAS. While
the anaerobic zone serves mostly as a contact zone for VFAs and PAOs, some fermentation of easily
biodegradable carbon compounds (rbCOD) to acetate and propionate may take place. In most plants the
readily biodegradable material is in short supply and must be reserved for the PAOs.

When nitrate or oxygen is discharged to the anaerobic zone, two things may happen, both
undesirable:
• They will prevent fermentation of rbCOD to acetic and propionic acid.
• Nitrates or DO could serve as electron acceptors for PAOs and other organisms that will metab-olize the
VFA and so deprive the PAOs of the substance that they need to store for growth and phosphorus removal.
In the absence of electron acceptors such as DO and nitrates in the anaerobic zone, PAOs are favored to
grow since they can take up and store the VFA under anaerobic conditions, thereby making it unavailable for
other aerobic and facultative heterotrophs in the aerobic zone.
Biological removal of both nitrogen and phosphorus at the same WWTP is common. Both funct-ions require
a carbon source. While denitrification organisms can feed on quite a number of easily degradable materials
such as methanol, sugar, glucose, acetate and propionate, PAOs are restricted to the latter two for
polymerization and storage (e.g. adding methanol to the anaerobic zone will reduce nitrates but not assist in
the removal of phosphorus).
Current Configurations
The basic design of anaerobic, anoxic, and aerobic zones, in that order, has been achieved in many different
configurations. The configurations vary in the number of stages, the nature and location of recycles, and the
operation of the process. Each process was modified from the standard biological activated sludge design in
order to accomplish various design goals (e.g., protection of the anaerobic zone from excess nitrate recycle).
The primary processes are listed below.
Of these, all will also biologically remove nitrogen except for the Pho-redox process.
• Pho-redox (A/O)
• 3 Stage Pho-redox (A2/O)
• Modified Bardenpho
• University of Capetown (UCT) and Modified UCT (MUCT)
• Johannesburg (JHB), Modified Johannesburg, and Westbank
• Orange Water and Sewer Authority (OWASA)
• Oxidation ditches with anaerobic zones or phases added
• SBR operated with an anaerobic phase
• Hybrid chemical/biological processes
The performance of these technologies depends on many site specific factors, including but not limited to
temperature, hydraulic and organic loading, recycle rates, and return streams. The technologies described
in this section are generally capable of phosphorus removal to effluent levels between 0.5 and 1.0 mg/L.
Operating strategies that can be used to enhance biological treatment and achieve these and, in some cases,
even lower effluent levels.
Biological phosphorus removal can be combined with other technologies to achieve very low effluent
concentrations (< 0.2 mg/L). Chemical addition combined with biological removal of phosphorus has been
used to consistently achieve low levels. WEF and ASCE (1998) recommend that WWTPs have chemical
addition capabilities even for well operating BPR plants to provide backup phosphorus removal in the event
of power outages, pipe breaks, or other unforeseen events.
Solids removal can also be a limiting factor in achieving phosphorus removal below 0.2 mg/L. Very low
phosphorus levels generally require a TSS level of less than 5 mg/L. Tertiary filtration (see membrane
bioreactors), and advanced clarification processes can achieve TSS levels less than 5 mg/L.

Pho-redox (A/O) and 3 Stage Pho-redox (A2/O)
The Pho-redox (A/O) process is a conventional activated sludge system with an anaerobic zone at the head
of the aeration basin. The RAS is pumped from the clarifier to the anaerobic zone. It is a low SRT process,
operated to avoid nitrification. With no nitrates in the RAS the process is reliable and easy to operate except
at temperatures in excess of 25°C when nitrification is difficult to avoid. The 3 Stage Pho-redox (A2/O)
process adds an anoxic zone after the anaerobic zone to achieve de-nitrification.
In addition, a nitrate rich liquor is recycled from the end of the aerobic zone to the head of the anoxic zone to
enhance de-nitrification. A shortcoming of the 3 Stage Pho-redox process is that there will be nitrates present
in the RAS, potentially making the process unreliable.
Modified Bardenpho
The Bardenpho process removes nitrogen to low concentrations. The addition of an anaerobic zone at the
head of the process enables phosphorus removal as well. The process consists of 5 stages: an anaerobic
stage followed by alternating anoxic and aerobic stages. A nitrate-rich liquor is recycled from the first aerobic
stage, designed for complete nitrification, to the first anoxic stage. The RAS is recycled from the clarifier to
the beginning of the anaerobic zone. Since the nitrates in the RAS ranges from 1 to 3 mg/L, it does not
seriously interfere with the mechanism for phosphorus removal as can happen in the 3 Stage Pho-redox
process.
University of Cape Town (UCT) and Modified UCT (MUCT)

The UCT process was designed to reduce nitrates to the anaerobic zone when high removal of nitrates in
the effluent is not required. It consists of three stages: an anaerobic stage, an anoxic stage, and an aerobic
stage. The RAS is returned from the clarifier to the anoxic zone instead of the anaerobic zone to allow for
denitrification and to avoid interference from nitrate with the activation of the PAOs in the anaerobic stage. A
nitrate rich stream is recycled from the aerobic zone to the anoxic zone. Denitrified mixed liquor is recycled
from the anoxic zone to the anaerobic zone.
Several modifications of the process exist. Sometimes it can be difficult to achieve the level of denitrification
in the anoxic zone required to protect the anaerobic zone from nitrates when the zone is receiving both RAS
and high internal nitrate recycle flows. This problem led to the development of the modified UCT process,
which splits the anoxic zone into two stages.
The nitrate rich recycle from the aerobic zone is recycled to the head of the second anoxic stage. The nitrate
containing RAS is recycled to the first anoxic stage where it is denitrified. Next, the denitrified RAS is recycled
from the end of the first anoxic stage back to the head of the anaerobic stage and mixed with the incoming
wastewater.
Johannesburg (JHB), Modified Johannesburg and Westbank

The JHB process is similar to the 3 Stage Pho-redox process, but has a pre-anoxic tank ahead of the
anaerobic zone to protect the zone from nitrates when low effluent nitrates are not required. The low COD of
the wastewater limited the de-nitrification capacity in the original plant (Nothern Works), resulting in nitrates
in the RAS. This reduced BPR so much that a pre-anoxic tank was included on the RAS line to remove the
nitrates from the RAS flow using endogenous respiration, before the flow entered the anaerobic zone.
The modified JHB process adds a recycle from the end of the anaerobic zone to the head of the pre-anoxic
zone to provide residual, readily biodegradable compounds for denitrification.
The Westbank process is similar to the JHB process but adds some primary effluent to the anaerobic zone
to assist in denitrification with the remainder of the primary effluent being discharged to the anaerobic zone.
During storm flows, excess flow is passed directly to the main anoxic zone. VFA obtained from acid
fermentation of the primary sludge is passed to the anaerobic zone.

Orange Water and Sewer Authority (OWASA)
The OWASA process was developed by adding activated sludge from a biological nitrogen removal process
to a trickling filter plant. Then, nitrified effluent from the trickling filter is fed to the aerobic zone of the activated
sludge system. Because the VFAs have been destroyed by the trickling filter, it is necessary to ferment the
settled organic solids from the primary clarifier to produce sufficient VFAs for BPR.
Next, the fermented supernatant is passed to an anaerobic (nutrition) zone and mixed with the RAS to initiate
BPR. Mixed liquor then flows from the nutrition zone to an anoxic zone and then to an aerobic zone.
Alternatively, simultaneous nitrification and denitrification takes place in the aeration zone.
Oxidation Ditches
There are several oxidation ditch designs that can remove phosphorus. They normally consist of an
anaerobic zone ahead of the oxidation ditch whereas simultaneous nitrification and denitrification takes place
within the ditches. Oxidation ditches typically operate as racetrack configurations around a central barrier,
with forward mixed liquor flows of approximately 1 foot per second or more. It is possible, by manipulating
the DO transferred to the mixed liquor, to establish both anoxic, aerobic and near anaerobic zones within the
racetrack configuration, even though the high flow velocities accomplish complete mixing of the wastewater
with the RAS.
There are many forms of oxidation ditches, such as the Carousel, the Pasveer Ditch and the Orbal process.
The Orbal process creates anaerobic and anoxic zones in the outer of three concentric oval shaped ditches
with the RAS recycled from the clarifier to the anoxic zone. It is also possible to introduce an anaerobic tank
before the ditch to accomplish BPR in the combined system. The Pasveer Ditch and the Carousel system
also can be used in conjunction with an anaerobic zone to accomplish BPR, in addition to simultaneous
nitrification and denitrification within the ditches. Because of the very high internal recycle within the ditches,
very low nitrate concentrations can be achieved in the mixed liquor before settling, and anaerobic conditions
are easy to maintain in the anaerobic zone, thereby resulting in efficient BPR. The layout would resemble a
Pho-redox process with simultaneous nitrification-denitrification (SND) in the aeration basin. Alternatively,
the Carousel or Pasveer Ditch could be used as the aeration stage in either the 3 Stage Pho-redox or the
Modified Bardenpho process.
The VT2 process at Bowie, MD, operates two Pasveer ditches in series with dedicated anoxic, near anaerobic
and aerobic zones. It also has a side stream anaerobic zone that receives only 30 percent of the influent flow
to enhance BPR. Denitrified MLSS for the anaerobic zone are obtained from the end of the near anaerobic
zone of the adjacent ditch. Operated without primary sedimentation, the system consistently obtains very low
(<0.25 mg/L) effluent TP without chemicals or effluent filtration. The ditches are operated in series because
the plant has limited clarification capacity, and series operation results in lower MLSS concentrations to the
clarifiers. The biodenipho process also uses pairs of ditches.
The ditches in the biodenipho process operate in alternating anoxic-aerobic modes. An anaerobic tank is
placed before the ditches for BPR and the ditches are alternated between nitrification and denitrification.
Sequencing Batch Reactors (SBR)

SBRs are fill-and-draw reactors that operate sequentially through the various phases by means of adjusting
the mixing and aeration. The reactor phases can be set and automated to allow the mixed liquor to go through
an anaerobic/anoxic/aerobic progression as is necessary for removal of phosphorus and nitrates. Because
of the fill-and-draw nature of SBRs, it actually is necessary to remove the nitrates remaining from the previous
cycle before anaerobic conditions can be established, thus the typical treatment progression becomes
anoxic/anaerobic/aerobic.
However, SBRs are almost always operated without primary sedimentation, so they still usually have a
favorable BOD5:TP ratio for effluent TP of somewhat less than 1.0 mg/L during the settling phase.

Hybrid Chemical / Biological Processes
The PhoStrip configuration, used mainly in non-nitrifying plants, pulls a side stream off the RAS in a
conventional activated sludge plant. The side stream is concentrated and retained for a day or more in a
thickening tank where the solids blanket is deep enough to produce anaerobic conditions and fermentation,
resulting in the release of phosphates by the microorganisms. Lime is then added to the supernatant stream
to precipitate and remove phosphate. The thickened, fermented sludge is passed back to the main aeration
basin. Existing plants include Seneca Falls, NY; Lansdale, PA; Adrian, MI; Savage, MD; Southtowns, NY;
Amherst, NY; and Reno-Sparks, NV.
The Biological Chemical Phosphorus and Nitrogen Removal (BCFS) configuration is similar to the modified
UCT process. In this process, a sludge stream is removed from the anaerobic zone. Ferric chloride is added
to the sludge thickener to remove phosphate. This provides an advantage over chemical addition to the
secondary clarifier because it does not require the chemical sludge to be recycled. There is an existing plant
at Holten in the Netherlands (WEF and ASCE, 2006), but no performance data are available.
Microscope being utilized to view activated sludge MO’s. Thiothrix is a type of filament that can
grow in the aeration basin of an activated sludge plant. Low DO levels are a possible cause to the
growth of this long filament.

A large shallow basin used for wastewater treatment by natural processes involving the use of
algae and bacteria to accomplish biological oxidation of organic matter.

Last Word on Return and Waste Activated Sludge Systems
The RAS system pumps the settled sludge from the secondary clarifier back to the aeration tank. It is
important that this system returns the RAS to the aeration tank before the microorganisms deplete the entire
DO. The RAS must also be as concentrated as possible and the flow must be accurately measured and
controlled.
To accomplish this, the RAS pumping system must have a

positive variable flow control device and the RAS flow must
be adjustable between the minimum and maximum range for
proper process control. The desired return flow to the
aeration tank could also be automatically paced to
secondary influent flow.
All activated sludge processes must have a WAS system to

remove excess microorganisms. This is necessary to control
the F/M and MCRT. If the process is to reliably meet
discharge requirements, this system must provide a positive,
flexible, and reliable means of removing excess
microorganisms.
It is essential for the system to have flow-metering and

pumping equipment that function completely independent of
other activated sludge control devices. The most positive
and flexible system will include an independent pumping system with flow adjustability (for example,
variable speed drive) and a flow meter that provides feedback into a flow-control device.
Such a system can be set for a given wasting rate with complete assurance that variable system head or
concentration conditions will not affect its ability to remove the microorganisms required. WAS systems
must have sufficient capacity to deal with both the hydraulic and/or organic load changes and process
changes.
Aeration and DO Control

The purpose of aeration is two-fold: oxygen must be dissolved in the liquid in sufficient quantities to maintain
the organisms and the contents of the tank must be sufficiently mixed to keep the sludge in suspension.
Mixing energy and oxygen transfer are provided through mechanical or diffused aeration. The amount of
oxygen that has to be transferred by the aeration system is theoretically equal to the amount of oxygen
required by the organisms in the system to oxidize the organic material.
The DO concentration in the aeration tank must be sufficient to sustain at all times the desirable
microorganisms in the aeration tank, clarifier, and return sludge line back to the aeration tank. When oxygen
limits the growth of microorganisms, filamentous organisms may predominate and the settleability and quality
of the activated sludge may be poor.
On the other hand, over aeration can create excess turbulence and may result in the breakup of the biological
floc and waste energy. Poor settling and high effluent solids will result. For these reasons, it is very important
to periodically monitor and adjust the aeration tank DO levels and, for diffused air systems, the air flow rates.

In practice, the DO concentration in the aeration tank should normally be maintained at about 1.5 to 4 mg/L
in all areas of the aeration tank at all times for adequate microorganism activity. Poor sludge settling as a
result of filamentous organisms has been associated with mixed liquor DO concentrations below 0.5 mg/L.
Above 4 mg/L, treatment usually does not significantly improve but power usage increases aeration costs
considerably.
RAS Control
To properly operate the activated sludge process, a good settling mixed liquor must be achieved and
maintained. The MLSS are settled in a clarifier and then returned to the aeration tank as the RAS. This
keeps a sufficient concentration of activated sludge in the aeration tanks so that the required degree of
treatment can be obtained in the allotted time period. The return of activated sludge from the secondary
clarifier to the aeration tank is a key control parameter of the process.
The secondary clarifiers have two basic functions:
 to clarify the secondary effluent through solids/liquid separation; and
 to rapidly collect and thicken the settled solids for return to the aeration tanks or wasting to the sludge
processing facilities.
Example of a Sludge Press.

Constant Rate Versus Constant Percentage Return
There are two basic ways for returning sludge to the aeration tank:
 at a constant rate, independent of the secondary influent flow rate, and

 at a constant percentage of the varying secondary influent flow.
Clarifier size and hydraulics may limit the range of practical return adjustments. Regardless of calculated
values, return rates should not be reduced to the level where slowly moving, thick clarifier sludge will plug
the sludge withdrawal pipes.
Also, low return rates during the night should be increased to approach the anticipated higher return rates
during the day before, rather than after, the increased wastewater flows actually reach the plant. Increasing
the return sludge flow after the flow increase may cause a hydraulic overload condition resulting in a
carryover of solids into the clarifiers (washout).
Constant Rate Control

Returning activated sludge at a constant flow rate that is independent of the secondary influent wastewater
flow rate results in a continuously varying MLSS concentration that will be at a minimum during peak
secondary influent flows and a maximum during minimum secondary influent flows.
The aeration tank and the secondary clarifier must be looked at as a system where the MLSS are stored in
the aeration tank during minimum wastewater flow and then transferred to the clarifier as the wastewater
flow and then transferred to the clarifier as the wastewater flows initially increase.
The clarifier acts as a storage reservoir for the MLSS during periods of high flow. The clarifier has a
constantly changing depth of sludge blanket as the MLSS moves from the aeration tank to the clarifier and
vice versa.
Constant Percentage Control

The second approach is to pace the return flow at a fixed percentage of the influent wastewater flow rate
(Q), at a constant rate (R) R/Q. This may be done automatically with instruments, or manually with frequent
adjustments.
This approach keeps the MLSS and sludge blanket depths more constant throughout high and low flow
periods and also tends to maintain a more constant F/M and MCRT.
Settleability
The settleability test can be used to estimate the desirable sludge return rate. This method uses the sludge
volume in a 2-L settleometer at the end of a 30-minute settling period to represent the underflow and the
supernatant volume to represent the overflow.

Above photograph, Flagella.
Lagoons in series.

Rotating Biological Contactors RBC
Rotating Biological Contactors is a remediation technology used in the secondary treatment of wastewater.
This technology involves allowing wastewater to come in contact
with a biological medium in order to facilitate the removal of
contaminants.
In its simplest form, a rotating biological contactor consists of a

series of discs or media blocks mounted on a shaft which is
driven, so the media rotates at right angles to the flow of
sewage. The discs or media blocks are normally made of plastic
(polythene, PVC, expanded polystyrene) and are contained in a
trough or tank so that about 40% of their area is immersed.
The biological growth that becomes attached to the media

assimilates the organic materials in the wastewater. Aeration is
provided by the rotating action, which exposes the media to the
air after contacting them with the wastewater. The degree of wastewater treatment is related to the amount
of media surface area and the quality and volume of the inflowing wastewater.
Rotating Biological Contactors can be supplied as part of an integral package plant to treat sewage from
various communities. Integral units are provided in sizes of up to a 500-population equivalent. A smaller
version is also available for small private installations.
Modular systems can also be adapted to cater to populations of any number.
Multiple units have been used for populations in excess of 5000.
Each plant is designed to meet the specific requirements of the site and the effluent quality required.
Key Advantages
 Short contact periods are required because of the large active surface.
 Capable of handling a wide range of flows.
 Sloughed biomass generally has good settling characteristics and can easily be separated from the
waste stream.
 Operating costs are low, as little skill is required in plant operation.
 Retention times are short.
 Low power requirements.
 Low sludge production and excellent process control.
Problems
White biomass over most of a RBC disc can be resolved by increasing the age of the sludge.
RBC Principles
The principles of the rotating biological contactor originated in the early 1900's but its application to sewage
treatment did not occur until the 1960's when the present system was developed. The process employed
relies on the well-established principle of biological oxidation using naturally occurring organisms to ensure
even the most stringent effluent standards can be achieved.
Primary Settlement Zone

Incoming flows of crude sewage enter the RBC primary settlement zone, which is designed to have a
buffering capacity of balancing flows up to 6 mgd (million gallons a day).
Settlement solids are retained in the tank's lower region while the partially clarified liquor passes forward to
the biozone where it makes contact with the slowly rotating disks.

Contactors
Installation of Rotating Biological Contactors
Rotating Biological Contactors are available in sizes from 1100mm diameter up to 3800mm in diameter. The
media packs that form the rotors are manufactured from vacuum formed black polyethylene sheets supported
on the central shaft with a galvanized steel framework. The central shaft is manufactured from mild steel
tube, protected internally against corrosion and fitted with end stub shafts, which are supported on split
bearings.
Gearbox and Drive mechanism

Rotation is provided by a shaft mounted gearbox and motor fitted at one end.

Biozone
The rotor assembly is suspended within the biozone with 40% of the diameter submerged in the liquor at any
one time. The disks slowly rotate and the continuous alternate exposure to air and sewage results in a growth
of organisms known as biomass which adheres to the disks. These organisms occur naturally in the sewage
and carry out the purification process by feeding off the impurities present in the sewage. As they have a
short life cycle, these organisms are continually shearing off the rotating disks and pass from the biozone to
the final zone.
The biozone is fitted with a series of baffles between each bank of media to prevent short circuiting and to
ensure maximum performance.
Final Settlement Zone
Culbokie, Scotland Water.
The biomass passes from the biozone into the final settlement zone where it settles to form humus sludge.
This is then regularly pumped out using either an air lift system or submersible pumps and returned to the
primary zone.
The clarified liquid decants from the top of the tank as effluent that can be discharged to a reed bed for further
clarification or direct to a watercourse.

Emerging Technologies
Many plants that are not specifically configured for BPR nevertheless achieve phosphorus removal to less
than 1 mg/L. The first such observation in a nitrifying plant was in a four-stage Bardenpho plant where mixed
liquor was recycled from the second anoxic zone to an unstirred fermenter, then returned to the anoxic zone.
The CATABOL™ and Cannibal Processes claim to reduce excess secondary sludge production by passing
mixed liquor or RAS through an anaerobic (fermenting) stage and then back to the main stream aeration
system. In addition, both processes pass the mixed liquor through a process for removal of some of the inert
solids. Both processes claim to get similar phosphorus removal to that for the Bardenpho plant described
above.
All of these processes rely on the fermentation of some of the mixed liquor for producing VFA that assists in
the biological removal of phosphorus. The Town of Cary, NC, has been using a system by which some of the
sludge in the return streams of a biological nitrogen removal plant is subjected to anaerobic conditions similar
to that of the other processes described above resulting in an effluent phosphorus concentration of less than
0.5mg/L.
There is a similarity between these processes and ad hoc processes for switching off aeration in plug-flow
plants for promoting phosphorus removal. These ad hoc processes take various forms. The Piney Water,
CO, plant is a 5-stage Bardenpho plant with no primary sedimentation and little VFA in the influent, which
resulted in little phosphorus removal. By switching off a mixer in one of the anaerobic zones, sludge settled
to the bottom and fermented, which supplied the VFAs for reducing the orthophosphorus to less than 0.2
mg/L.
A similar operation at the Henderson, NV, plant in a JHB type process had the same effect. Some plug-flow
aeration plants succeeded in reducing phosphorus to below 1 mg/L by turning off aeration at the feed end of
the plant, such as the Blue Lakes and Seneca plants operated by the Metropolitan Council Environmental
Service in Minnesota and the St. Cloud, MN, plant.
The Joppatowne plant operated by Harford County, MD, consists of an MLE plant with some sludge
accumulation in the anoxic zone while reducing the phosphorus from 7 mg/L in the influent to around 1 mg/L
in the effluent. All of these plants use the same principle of fermenting some of the mixed liquor sludge or
underflow from the final clarifiers, either inside the main stream tanks or in a side stream basin. There are
many instances where enterprising operators can achieve 80 percent or more phosphorus removal by turning
off air or mixers in conventional treatment plants. There is a Catabol plant in Cartersville, GA (USEPA,
2008a); however, there are no published data for this plant.
Operational and Design Considerations

Important factors that affect BPR include:
• Bioavailable COD:P ratio in the anaerobic zone influent, including adjustments by VFA addition and sludge
fermentation
• SRT and HRT
• Presence of oxygen or nitrate in the anaerobic zone
• Backmixing of oxygen
• Temperature
• pH
• Secondary release under anaerobic conditions
• Sufficient oxygen in the aerobic zone
• Inhibition
• Flow and load balancing

COD:P Ratio
The PAOs need VFAs in the form of acetic and propionic acid. These acids may be in the feed or can be
produced through fermentation of soluble rbCOD such as sugar, ethanol, etc., in the anaerobic zone. As a
rough estimate of the propensity for phosphorus removal to an effluent concentration less than 1.0 mg/L, the
COD:P ratio typically should be about 40 or more. VFA is produced through fermentation of municipal
wastewater or it can be added as a commercial or waste product. Some wastewater collection systems that
are relatively flat and have long collection times may have sufficient fermentation in the collection system to
provide the necessary VFAs, but it will vary monthly depending upon the temperature and flow conditions in
the collection system. Force mains are excellent fermenters for the production of VFA. Systems that do not
have a COD/P ratio of at least 40 will most likely need to supplement VFAs to achieve effluent phosphorus
concentrations below 1.0 mg/L. However, they will still achieve substantial BPR with lower ratios if
appropriately operated. See below for a more detailed discussion of VFAs.
Recent studies suggest that the instantaneous COD:P ratio is more important than the overall average
(Neethling et al., 2005). Short term drops in the BOD:P ratio in the primary effluent to below that required for
sustainable phosphorus removal correlated well with rises in effluent phosphorus. Intermittent recycles of
phosphorus rich return streams may cause short term variability in the BOD:P ratio. Controlling or eliminating
these recycles can improve plant performance. Weekend changes in the BOD:P ratio also can affect
performance.
Another group of organisms, glycogen accumulating organisms (GAOs), also has the ability to take up
acetate in the anaerobic zone, not by using energy in phosphate bonds but by using stored glycogen as the
energy source. Under certain conditions, such as high temperatures or low phosphorus concentrations
relative to the influent bioavailable COD, they may out-compete PAOs for the VFAs, which would result in
less or no release of phosphorus in the anaerobic zone. This in turn will result in less or no overall phosphorus
removal. GAOs use the stored energy in the form of glycogen to take up VFAs and store them as a complex
carbohydrate containing poly-hydroxy valerate (PHV), instead of PHB formed with poly-phosphorus as the
energy source. When this begins to happen, there is a slow decline of phosphorus removal by the biological
system.
There is still a debate amongst researchers about the conditions likely to favor GAOs over PAOs.
Summarizing a number of publications, it would appear that the following conditions favor the growth of GAOs
over that of PAOs:
• High SRT
• High temperature over 28 °C
• Longer non-aerated zones
• Stronger wastes with low TKN content
• Periods of intermittent low BOD loads
• If the VFA consists mostly of either acetate or propionate
• Polysaccharides such as glucose are fed to the anaerobic zone.
• Low pH in the aerobic zone
Further confirmation is needed for some of these factors.
Volatile Fatty Acid Addition

Only VFAs such as acetic and propionic are taken up by PAOs. Reported doses of VFA for successful
phosphorus removal range from 3 to 20 mg/L VFA per gram of phosphorus removed. These numbers,
however, do not take into account the rbCOD that is fermented in the anaerobic zone.
It is more accurate to look at the rbCOD/P ratio for good phosphorus removal, which ranges from 10 to 16.
(Barnard, 2006). Surveys show that it is rare for a WWTP treating municipal sewage to achieve more than
95 percent removal of phosphorus by biological processes without adding VFAs (Neethling et al., 2005).

An Australian study shows that while both PAOs and GAOs could use acetate, PAOs will have a competitive
advantage when the VFAs consist of roughly equal parts of acetic and propionic acid as a growth medium.
PAOs that are fed on acetate are able to switch to propionate much more quickly and effectively than GAOs
(Oehmen et al., 2005). This finding led to a strategy to feed equal amounts of acetic acid and propionic acid
as the optimal for stimulating PAO growth (Oehmen et al., 2006, Bott et al., 2007). One study shows that
isovaleric acid drives BPR even better than acetic acid (Bott et al.,2007).
Isovaleric acid, however, is much more expensive than acetic acid and is more odorous. It also is not
significantly generated in the primary sludge fermentation process. Addition of rbCOD such as sugars and
alcohols containing two carbons or more can increase phosphorus uptake by PAOs when added to the
anaerobic zone but may cause sludge bulking if dosed in excess (Jenkins and Harper, 2003).
Sludge Fermentation
Anaerobic fermentation produces VFA consisting mainly of acetic and propionic acid. Some configurations,
such as the Westbank and OWASA configurations, make use of anaerobic fermentation of the primary sludge
to provide VFAs to the nutrient removal process. A fermentation process, however, can be added to any
configuration to provide VFAs, especially in areas where little fermentation takes place in the collection
system. Fermentation of the primary sludge or the RAS will produce VFA. Primary sludge fermentation is
used more frequently.
There are several primary sludge fermenter designs that can accomplish this. The simplest configuration
involves allowing the formation of a thicker sludge blanket in the primary clarifier itself and returning some of
the thickened sludge to either the primary clarifier or to a mixing tank ahead of the primary clarifier to allow
elutriation of the VFA to the primary effluent. This is referred to as an activated primary sedimentation tank
(Barnard, 1984). Another variation is to pump some sludge to a complete-mix tank ahead of the primary
clarifier, to accomplish fermentation.
The sludge is then passed to the primary clarifier for elutriation of the VFA. Both of these processes lead to
an increased load on the primary clarifier and some VFA may be lost due to aeration between the primary
clarifier and the anaerobic zone. Sludge age should also be controlled to prevent methanogenic bacteria from
growing and converting the VFA to methane. Usually, a SRT less than 4 days is sufficient for this.
Alternative methods accomplish fermentation in a gravity sludge thickener by holding the sludge under
anaerobic conditions for 4 to 8 days. The supernatant can then be fed directly to the anaerobic zone and a
high load on the primary clarifier can be avoided. Thickening can either be accomplished with a single
thickener or in two stages. The two-stage process can either be a complete mix tank, followed by a thickener
or two thickeners in series. It has been shown that adding molasses or other sources of readily biodegrable
COD can improve the performance of fermenters (Bott et al., 2007).
RAS can also be fermented in a side stream process. The fermentation zone is similar to the anaerobic or
anoxic zone of many biological processes. RAS fermentation could be used in any BPR process, but is most
common in processes without primary clarifiers. Research and experience have revealed some key design
considerations for primary fermenters (WEF and ASCE, 2006).
These processes can have high solids content and may need a positive displacement pump to operate
properly. Because fermentation can lower the pH and produce carbon dioxide and hydrogen sulfide,
corrosion resistant materials should be used. Odor control may also be necessary if hydrogen sulfide is
produced. Monitoring of pH and oxidation reduction potential (ORP) may be desirable to control the process.

Retention Time
The concentration of phosphorus in the sludge typically increases as the SRT increases, although the impact
is very small over the SRT range of 4 to 30 days. Efficient phosphorus uptake typically requires a minimum
SRT of 3 to 4 days depending on temperature. Higher SRTs will not increase phosphorus uptake; given there
is sufficient VFAs available. If SRT becomes too great, however, effluent quality can degrade. This can be
due to release of phosphorus as biomass degrades (WEF and ASCE, 2006). Both anaerobic and aerobic
HRT can affect the amount of phosphorus stored by PAOs. Sufficient time should be allowed for the formation
of VFAs and storage of the Polyhydroxyalkanoates (PHAs) in the anaerobic zone, although the reactions are
relatively fast. If the time is too short, phosphorus uptake in the aerobic zone will be lower than achievable
because insufficient PHAs were stored in the anaerobic zone. It has been reported that the ratio of HRT in
the anaerobic zone to the HRT in the aerobic zone is important. One study found that a ratio of between 3
and 4 for aerobic HRT to anaerobic HRT led to optimal plant operation (Neethling et al., 2005).
Temperature
High temperatures can have an adverse effect on phosphorus removal. At temperatures greater than 28° C,
phosphorus removal will generally be impaired, apparently by the predominance of the GAOs (Bott et al.,
2007). At the low end of the temperature scale, Erdal et al. (2002) found that PAOs outcompeted GAOs at
5° C even though the PAO metabolism was slower at 5° C than at 20° C. The GAOs virtually disappeared in
the 5° C reactor. Modeling studies have shown that GAOs can predominate at higher temperatures because
of their increased ability to uptake acetate at those temperatures compared to PAOs (Whang et al., 2007).
Low temperatures can also lower phosphorus uptake but have been shown to not be an issue in well operated
and properly acclimatized plants (WEF and ASCE, 2006).
Presence of Oxygen or Nitrate in the Aerobic Zone

If oxygen or nitrate is present in the anaerobic zone, organisms that use oxygen or nitrates as electron
acceptors will preferentially grow by fully oxidizing the organics to CO2 and H2O, thereby reducing the VFAs
available for polymerization and storage by the PAOs. Nitrate can also inhibit fermentation of rbCOD because
most of the fermenters are facultative and can use the nitrate as an electron acceptor to fully oxidize the
rbCOD instead of producing VFAs as an end product of fermentation, thus depriving the PAOs of organics
they can polymerize and store. Therefore, recycle of streams containing high DO and nitrate concentrations
to the anaerobic zone should be avoided. Introduction of oxygen through pumps and other devices should
also be avoided.
Avoiding Backmixing of Oxygen

Another potential source of oxygen and nitrates to the anaerobic zone is backmixing from downstream zones.
In configurations where the anaerobic zone is followed immediately by an anoxic or aerobic zone, backmixing
can cause elevated concentrations of nitrates and/or DO in the anaerobic zone leading to favoring of
organisms other than PAOs.
The problem can be avoided by increased baffling or changing the mixing rates. This problem is more likely
to occur when the downstream zone is aerated, because aeration of mixed liquor increases the liquid depth,
making the liquid level in the aerobic zone higher than in the non-aerated zone.
pH
Low pH can reduce and even prevent BPR. Below pH 6.9 the process has been shown to decline in efficiency
(WEF and ASCE, 2006). This is possibly due to competition with GAOs. Filipe, et al. (2001), found that GAOs
grow faster than PAOs at a pH of less than 7.25.
Because many wastewater processes such as chemical addition and nitrification can lower pH, this should
be monitored and adjusted if necessary. It also has been shown that it is not possible to establish enhanced
biological phosphorus removal (EBPR) when the pH is less than 5.5, even though an abundant amount of
acetic acid is present in the anaerobic zone (Tracy and Flammino, 1987; Randall and Chapin, 1997).

Anaerobic Release
Secondary release of phosphorus occurs when the PAOs are under anaerobic conditions in the absence of
a source of VFA. The energy stored as polyphosphate is used for cell maintenance and phosphorus is
released to the liquid phase (Barnard, 1984). There will then be no stored food to supply energy for the uptake
of phosphorus upon subsequent aeration.
This may occur in the following process stages:

• In the anaerobic zone if the retention time is too high and the VFA is depleted well within the required
retention time.
• In the main anoxic zone when that runs out of nitrates.
• In the second anoxic zone there are no nitrates to be removed.
• In the sludge blankets of final clarifiers when the RAS rate is too low and sludge is not removed fast enough.
Additionally, release may happen in aerobic zones that are too large, resulting in stored substrate depletion
and destruction of PAO cells by endogenous metabolism. Since there was no food storage associated with
the phosphorus release, additional carbon is then required to take up the phosphorus released, but the
amount in the influent may be insufficient.
Therefore, chemicals must be added to remove the excess phosphorus. Over-design of biological nutrient
removal systems could thus lead to a higher demand for an external source of VFA. Phosphorus will be
released in sludge treatment processes that are anaerobic. Gravity thickening of BPR sludge can lead to
phosphorus release if long retention times are used. Using mechanical dewatering instead of gravity
dewatering allows less retention time and less phosphorus release (Bott et al., 2007). It is usually
recommended that dissolved air flotation (DAF) be used to thicken BPR sludge to reduce the amount of
phosphorus release. DAF thickening can be quite successful for the reduction of release, but if the thickened
sludge is left on the DAF beach too long before removal, excessive release will occur, just as it will when the
sludge is left too long in a gravity thickener.
Anaerobic digestion will also lead to phosphorus release although some phosphorus will be precipitated as
either a metal salt (e.g. calcium phosphate) or as struvite (magnesium ammonium phosphate, MgNH4PO4).
BPR sludge takes up and releases magnesium along with phosphates, and these two ions combine with
ammonium, also present in abundance in anaerobic digesters, to form struvite.
Struvite formation is very fast, and will continue until one of the three ions is reduced to that ion’s solubility
level. Magnesium is usually present in the lowest concentration, and its depletion typically limits struvite
formation within the anaerobic digester.
Calcium phosphate precipitates also tend to form in anaerobic digesters, but they form much more slowly
than struvite and the formation tends to be non-stoichiometric. If substantial amounts of phosphates are
precipitated by calcium along with the struvite formation, there will be little if any propensity for struvite to
form when the sludge exits the anaerobic digesters. Also, if the digested sludge is composted after
dewatering, the resulting Class A sludge will be enriched in magnesium, phosphorus, nitrogen, and, to a
lesser extent, potassium, which also is taken up and released with phosphorus by PAOs.
Thirty percent of the phosphorus entering the anaerobic digesters at the York River plant during BPR
experimentation was recycled back to the headworks from belt filter press dewatering (Randall et al., 1992).
Alternatives to anaerobic digestion such as composting, drying, or alkaline treatment can be used to reduce
phosphorus release. There have been several studies which have examined using struvite precipitation as a
way of recovering phosphorus from supernatant from digesters. When anaerobic release of phosphorus
occurs, recycling these streams can overload phosphorus removal processes. The effect can be worsened
when the waste handling process is only operated intermittently.

In some instances, there is a high degree of phosphorus precipitation in the anaerobic digesters and with
sufficient VFA in the influent the returned phosphorus may be removed. However, in most circumstances,
some chemicals need to be added to the return streams or to the anaerobic digester itself so that the metal
precipitate will be removed with the dewatered sludge.
Sufficient Oxygen in the Aerobic Zone

It is necessary for oxygen to be present in the aerobic zone for phosphorus to be taken up and retained in
the activated sludge. Maintaining a sufficiently high DO transfer in the aerobic zone enhances process
stability and has been found to be a key factor in phosphorus removal. (Bott et al., 2007)
Inhibition
EBPR, like any biological process, can be inhibited by chemicals toxic to the organisms. Although not as
sensitive to inhibition as nitrification and rare in practice, the BPR process can be inhibited by toxic chemicals,
including high concentrations of acetate (Randall and Chapin, 1997).
Flow and Load Balancing

Flows and loads to wastewater treatment plants can vary widely because of regular diurnal use patterns and
because of larger, more irregular disturbances such as storm events. Peaks in either flow, or nutrient load
can stress the system and cause poor performance. Peaks can be evened out using equalization tanks to
balance the flow. Equalization tanks in combination with nutrient sensors can also be used to balance nutrient
loads. In this case, recycle streams high in nutrient concentrations such as digester supernatant can be
stored during peak nutrient loads and recycled during times when concentrations are lower.
Impacts on Sludge Handling and Removal

Stored phosphorus adds dry weight to the sludge; however, the actual PAO VSS production will be less
because the reaction is less efficient than heterotrophic metabolism using DO as the electron acceptor.
Sludge from BPR will be similar to sludge from conventional activated sludge plants, although it will have a
higher phosphorus content. Varying results have been found with some plants reporting little or no change
in settling and dewatering (Knocke et al., 1992) and others reporting enhanced settling and dewatering
properties (Bott et al., 2007). The sludge produced from the process will also have higher magnesium and
potassium concentrations due to co-uptake of these elements with phosphorus.
Struvite can precipitate in anaerobic processes. With abundant phosphorus and ammonia, it is usually only
the magnesium that is in short supply. Some magnesium is released from the digested cells with the
phosphorus and may increase struvite precipitation. Some processes have proposed precipitating out struvite
or other phosphate solids to avoid phosphorus return in recycle streams (Bott et al., 2007). The struvite
crystals, however, depending upon where they form, can plug centrifuge ports, and pumps and pipes used
to convey the sludge, if not controlled. Plugged lines are very difficult to clean.
Guidance for Selecting Process Modifications

If an existing activated sludge WWTP needs to lower phosphorus levels in its effluent, a number of options
are available. Some key considerations are summarized below. For systems that do not have BPR, an
anaerobic zone can be added at the head of the plant. This may be achieved by switching off aerators at the
head of the reactor or by adding a separate reactor. Mixing in the anaerobic zone should be sufficient to
retain biological solids in suspension without introducing oxygen. If baffling is not already present, it could be
added to achieve separation of the anaerobic and aerobic zones.
Note that baffling is essential to prevent backmixing because the liquid level in the aerated zone will always
be higher than that in the non-aerated zone. Therefore, an overflow baffle should be used between zones.
Considerations should also be made for additional pumping needed for any recycle streams. Proper sizing
of the anaerobic zone is important to ensure sufficient VFA is formed and taken up in the aeration basin. If
an aerobic zone is converted to an anaerobic zone, care should be taken to ensure that the remaining aerobic
zone is sufficiently sized to achieve treatment objectives.

This usually is not a problem because the anaerobic zone seldom needs to be more than 15 percent of the
total volume, and can be considerably less if fermentation is practiced or VFA are added. Note that much of
the BOD in typical municipal sewage will be removed from solution in the anaerobic zone, and this reduces
the required size of the aerobic zone, even though most of the stored BOD will be stabilized in either the
anoxic or aerobic zone, or both.
For plants that already have BPR but need additional phosphorus removal, the designers should start by
identifying areas that may be limiting the current process. For example, if recycle streams are intermittent,
overloading of the process may occur during recycle and the process performance may suffer.
Flow equalization to enable constant recycle flows may be an option in these cases. RAS when returned to
the anaerobic zone may introduce nitrates or oxygen that will interfere with PAO performance. The
phosphorus content of the return streams could be reduced by adding some chemicals to precipitate some
of the phosphorus. Reducing oxygen introduction to the anaerobic zone from upstream processes may be
needed to optimize phosphorus removal.
Plants looking to improve phosphorus removal performance should also closely examine the plant for
secondary release of phosphorus. If sludge blankets in clarifiers are too deep, anaerobic conditions can
develop and cause secondary phosphorus release. This can be minimized by using deeper clarifiers,
maintaining low sludge blankets, and increasing the RAS rate, so that the released phosphorus is pumped
from the bottom of the clarifier rather than flowing over the effluent weir.
Sludge handling can also cause excessive phosphorus release such as in gravity thickeners, DAFs and
anaerobic digesters. If supernatant from these processes when poorly managed is recycled, it can overload
the process. Options in this case would be to eliminate the recycle, improve operation of the process, change
the process, or treat the recycle stream to remove phosphorus before it is returned to the plant.
Another area to examine in seeking improved phosphorus removal is the COD:P ratio. If the ratio is low,
supplementing the current process with VFAs may provide additional removal. VFAs can either be added as
a chemical addition process or produced through fermentation of primary or secondary sludge.
Other ways of improving TP removal include filtration and chemical addition. Phosphorus is often attached
to colloidal particles and very low phosphorus levels usually require removal of TSS. Membrane bioreactors
(MBR) in combination with biological and/or chemical phosphorus removal can result in very low effluent
levels due to enhanced solids removal. Chemical addition with or without filtration can also achieve low
phosphorus levels.
Effluent Filtration
Effluent filtration in combination with chemical precipitation can be used to remove phosphorous down to
very low levels (< 0.1 mg/L). USEPA Region 10 (2007) found that 2-stage filtration through use of a first and
second stage filter or by providing tertiary clarification prior to filtration, resulted in the lowest effluent
phosphorus concentrations of 23 WWTPs evaluated. Effluent filtration can also be used to remove soluble
organic nitrogen that is not removed through biological treatment or settling.

A wide variety of filter types have been used for wastewater treatment, including:
• Conventional down-flow filters

• Deep-bed down-flow filters
• Continuous backwashing upflow sand filters
• Pulsed bed filters
• Traveling bridge filters
• Fuzzy filters
• Discfilter
• Cloth media disk filters
• Membranes
• Blue PROTM process
• Pressure filters
NO2/NO3, Fluoride, Sulfide, Metals, BOD-TDS-TSS

Wide-mouth Sludge/Metals bottle.

Types of Filters
This section describes the various filters listed on above page, presents key design and operating principles,
and summarizes ongoing research and emerging technologies in this area.
Conventional Down-flow Filters

These filters consist of fixed-media beds typically up to 3 feet in depth and are similar to filters used to treat
drinking water. Media can be single media, dual media, or multi media. Single media is typically sand or
anthracite. Dual media combines anthracite and sand. Multi-media filters include a layer of garnet or limonite.
Flow in these filters is by gravity from the top down. Most of the removal occurs in the top few inches of the
media. The filter must be taken off-line periodically to backwash the filter to prevent clogging and too high of
a pressure loss.
Deep-bed Down-flow Filters

These filters are similar to conventional down-flow filters but have deeper beds and larger media size. This
gives the advantage of longer run times between backwashes. The size of the media is limited by the ability
to backwash the filter. Because these filters are more difficult to backwash, air scour is necessary to fully
clean the filter bed.
Continuous Backwashing Upflow Sand Filters

During operation of the continuous backwashing upflow filter, water is introduced through risers at the bottom
of a deep sand bed. Water flows upward through the sand bed and over an overflow weir. Sand and trapped
solids flow downward through the filter and are drawn into the suction of an airlift pipe in the center of the
filter. As the sand travels up the airlift pipe, energy from the air scours the particles and separates the sand
from filtered solids. At the top of the airlift pipe, the clean sand settles back onto the top of the filter and the
solids are carried away into a reject line.
These filters have the advantage of having no moving parts other than the air compressor and requiring less
energy and maintenance than traditionally backwashed filters. They are sometimes referred to by the trade
name Dynasand.
Pulsed Bed Filters

Pulsed bed filters are shallow filters with an unstratified fine sand media. An air pulse disturbs the media and
allows penetration of solids into media bed, allowing the entire filter bed to be used for removal of solids. The
pulse is designed to expand the filter operation and reduce the number of backwash cycles, although the
filter must still be periodically backwashed to remove the solids.
Traveling-Bridge Filters
Traveling-bridge filters consist of long shallow beds of granular media. Wastewater is applied to the top of
the media and flows downward. Each cell is individually backwashed by a traveling-bridge while the other
cells continue to operate. The bridge uses filtered water to backwash the filters and includes surface wash
to breakup matted solids or clumps of solids.
Fuzzy Filters
The fuzzy filter uses a proprietary synthetic filter media that is highly porous. Water flows not only around the
media but also through it, allowing much higher filtration rates. The media is held in place by a metal plate
and flow is from the bottom of the bed upwards. The filter is backwashed by raising the plate and introducing
a horizontal air stream from alternating sides causing the media to roll back and forth. The effluent is returned
to the plant.
Discfilters
Discfilters are a series of parallel mounted disks used to support a cloth filter media. Water enters a central
tube and flows out between the two layers of cloth in each disk. The disks rotate and are normally 60 to 70
percent submerged. The portion above the water is backwashed using spray nozzles.

Cloth Media Disk Filters
The cloth media disk filter is similar to the discfilter listed above. In this case the water flows from the outside
of the partially submerged cloth disks and into a center pipe. Disks continue to rotate
during backwash and water is sucked into the disc using suction heads.
Membranes
Membrane systems use a pressure head to drive water through a permeable membrane. Membrane filters
are typically classified by their pore size which in turn determines the size of the particles they exclude.
Microfiltration, ultrafiltration, nanofiltration, and reverse osmosis (RO) remove increasingly smaller particles.
Microfiltration and ultrafiltration remove 3 to 6 logs of bacteria, 95 percent or more BOD, along with most
particles (WEF, 2006). Nanofiltration removes nearly all particles including some viruses. RO removes all
particles as well as most large dissolved constituents. The energy cost for applying the pressure head and
the need to replace membranes make membrane filtration a more expensive technology. It can achieve very
low concentrations of nutrients and other contaminants, however, and is common in water re-use projects.
Membranes can be configured a number of ways including hollow fiber, spiral wound, plate and frame,
cartridge, or in pressure vessels. Membranes can foul from organics, biological activity, or metals in the
wastewater. Typically the water must be pre-treated before using these membranes. Pretreatment could be
conventional filters, cartridge filters, or larger membrane filters. Disinfection may also be required to prevent
biological fouling.
Blue PROTM Process

The Blue PROTM process uses a continuous backwashing filter that is designed remove phosphorus. Filters
can be run in series for even greater removal. The filter media (sand) is coated with a hydrous ferric oxide
coating, which enhances phosphorus removal through adsorption. A ferric salt is added prior to the filter to
aid in coagulation and to replace the ferric coating which is abraded from the sand. Water flows up through
the filter while the sand travels down. An airlift tube at the bottom of the filter carries the sand upward.
Turbulence from the compressed air knocks accumulated iron and phosphorus along with any solids off the
particle as it travels upward. The iron, phosphorus, and particles are wasted, while the clean sand is
deposited on the top of the bed. The filters can be run biologically active to achieve denitrification.
The Blu-CAT process combines the Blu-Pro process with addition of advanced oxidants. Early pilot tests
show that this process is capable of removing other emerging contaminants along with phosphorus and
microorganisms (USEPA, 2008a).
Pressure Filters
Pressure filters are similar to conventional media filters except they are contained in closed containers and
are filtered under pressure. The increased pressure creates a greater head loss and allows longer times
between backwashes.
Design and Operating Principles

Filtration is mainly affected by the concentration and size distribution of particles entering the filter. Turbidity
is often used as a surrogate for particle concentration. The concentration of particles will affect run-time in
filters and will also affect the required surface area to achieve the desired filtration. The size distribution of
the particles and its relevance to pore size of the granular or membrane filters will affect the removal
mechanisms. Filtration rate is also an important design parameter.
Too fast of a filtration rate can cause floc to break up and pass through the filter. The optimal filtration rate
depends on floc strength, which in turn depends on the biological treatment processes prior to filtration (e.g.,
Higher SRTs lead to weaker flocs). The filtration rate, along with the loading rate will determine the area of
the filter required.
The higher the loading rate, the more frequent backwashes will be required and the greater the head loss
across the filters. Typical filtration rates are 5 to 15 meters per hour for gravity filters and up to 20 meters per
hour for pressure filters (WEF and ASCE, 1998).

Addition of polymers or other coagulant aids can greatly aid filtration. Typical doses for filter influent are 0.05
to 0.15 mg/L of organic polyelectrolyte (WEF and ASCE, 1998), although jar tests are conducted to determine
the proper dose. Too low a dose can allow uncoagulated particles through the filter and too high a dose can
lead to mudballs and filter clogging.
There are several ways the flow rate can be controlled in filters. Constant-rate fixed head filtration maintains
a constant flow through the filter. This will lead to an increased head above the filter as the filter run
progresses. In constant-rate variable head filtration the rate is kept the same and the filter is backwashed
when the head reaches a certain value.
In variable-rate filtration, the rate of filtration decreases throughout the filter run until it reaches a minimum
value and is backwashed. Variable-rate filtration is less common than constant-rate filtration.
Proper backwashing is also important to filter operation. Without proper backwashing there can be
breakthrough of particles and turbidity. Lack of a proper backwash can also lead to accumulation of materials
on the surface of the filter that can form mudballs and cracks, which can allow solids to pass through the
filter. A surface wash or air scour may also be helpful to prevent accumulation of mudballs or grease. Surface
wash or air scour is also helpful for traveling bridge filters. Without surface wash traveling bridge filters are
limited to an influent TSS concentration of 40 to 50 mg/L (WEF and ASCE, 1998).
If membrane filters are used, fouling can be an important consideration. Cellulose acetate membranes can
be damaged by biological activity. Disinfection is often used to prevent biological fouling of the membranes.
Some membrane materials such as polyacramides, however, can be damaged by chlorine. This can be
avoided by using an alternative disinfectant, a different membrane material, or by de-chlorination.
Lowering the pH can help to prevent mineral fouling of nanofiltration or reverse osmosis membranes. Besides
pre-treatment, chemical cleaning of the membranes may also be required periodically. Monitoring of effluent
quality and pressure differential can be important to help identify membrane fouling or failure.
Ongoing Research and Emerging Technologies

The use of membranes as tertiary filtration is an area that has recently expanded. Research continues on
various membrane configurations along with topics such as pre-treatment, membrane cleaning, and removal
of emerging contaminants. Fuzzy filters are also an innovative technology that is beginning to be established
in the wastewater community with several full scale projects.
Other research has focused on enhancements to existing technology. For example, the Blue-Pro system
combines continuous backwashing filters, a well-known technology, with a hydrous ferric oxide coating and
ferric salt addition to remove phosphorus by adsorption as well as filtration.
Mathematical Modeling
8.1 The Need for Models
WWTPs are complex systems that depend on numerous biological, chemical, and physical processes to
achieve effluent goals. Because of the complex behavior of the processes and the variability in wastewater
characteristics, biological populations, and plant design, it is not always possible to predict how changing any
one variable will affect the effluent quality.
Plant designs that work for one influent wastewater and climate may not perform well in different conditions.
Pilot scale or full scale trials can help to determine the effect of various parameters, but costs and time to
cover all possibilities may be prohibitive. Therefore, models fill an important need by enabling simulation of
a process and estimating the impact that changing parameters will have on the treatment effectiveness.

Models can be used for a number of purposes including the design of new WWTPs, the design of retrofits or
upgrades to existing plants, determining how changes in operations may affect effluent concentrations of
permitted contaminants, determining how plants will respond to changes in influent quality or flow, and for
training operators. Not all models can achieve all of these purposes, so models should be selected with the
desired use in mind. There is some disagreement in the literature in the use of the term model.
Some references use the term to refer to sets of mathematical equations that characterize a process, other
references use model to refer to the computer program used to solve these equations. This section will use
the former and will use the term “simulator” to describe the computer program.
Overview of Available Models

Models are sets of equations, generally based on theory and grounded in empirical data that represent a
wastewater treatment process. Each unit process is represented by its own model.
Model equations for processes such as clarification and settling are well known and fairly simple. Modeling
biological wastewater processes such as activated sludge, however, is much more complicated. The primary
set of models for activated sludge processes has been compiled by the International Water Association
(IWA). The first model was developed in 1986 and was called the activated sludge model (ASM). Later known
as ASM1, this model was able to model the biological oxidation of carbon, nitrification, and de-nitrification.
Although the ASM model gained widespread use among both academia and industry, it had limitations. For
example, the model assumed constant temperature and pH, did not include EBPR, and the biological
reactions did not depend on the carbon source.
In order to improve the model, IWA developed four other ASM models; ASM2, ASM2d, ASM3, and ASM3
with BioP. ASM2 and ASM2d were intended to add EBPR. The ASM3 models were intended to deal with
limitations such as the independence of the ASM1 model of temperature and carbon source. In addition,
other models were developed to seek to improve upon the ASM model.
The metabolic biological phosphorus model of the Delft University of Technology (TUDP) was developed to
fully account for the metabolism occurring in PAOs during EBPR. Barker and Dold (1997) developed a model
(B&D) to include different rates of growth depending on the carbon source.
When selecting models, the processes required and the range of normal operating parameters for the plant
should be considered and compared to the available models. For example, if chemical phosphorus removal
is to be used in a plant, the plant is limited to using either the ASM2 or ASM2d models. Each model also has
a range of temperatures and pH over which it is valid.

Chlorine Section

Chlorine Exposure Limits and Related Information
This information is necessary to pass your pre-test.
* OSHA PEL 1 PPM - IDLH 10 PPM and Fatal Exposure Limit 1,000 PPM
The current Occupational Safety and Health Administration (OSHA) permissible exposure limit
(PEL) for chlorine is 1 ppm (3 milligrams per cubic meter (mg/m(3))) as a ceiling limit. A worker's
exposure to chlorine shall at no time exceed this ceiling level. * IDLH 10 PPM
Physical and chemical properties of chlorine: A yellowish green, nonflammable and liquefied gas
with an unpleasant and irritating smell. Can be readily compressed into a clear, amber-colored
liquid, a noncombustible gas, and a strong oxidizer. Solid chlorine is about 1.5 times heavier than
water and gaseous chlorine is about 2.5 times heavier than air. Atomic number of chlorine is 17.
Cl is the elemental symbol and Cl2 is the chemical formula.
Monochloramine, dichloramine, and trichloramine are also known as Combined Available Chlorine.
Cl2 + NH4.
HOCl and OCl-; The OCL- is the hypochlorite ion and both of these species are known as free
available chlorine. These are the two main chemical species formed by chlorine in water and they
are known collectively as hypochlorous acid and the hypochlorite ion. When chlorine gas is added
to water, it rapidly hydrolyzes. The chemical equation that best describes this reaction is Cl2 + H2O
--> H+ + Cl- + HOCl. Hypochlorous acid is the most germicidal of the chlorine compounds with the
possible exception of chlorine dioxide.
Yoke-type connectors should be used on a chlorine cylinder's valve, assuming that the threads on
the valve may be worn.
The connection from a chlorine cylinder to a chlorinator should be replaced by using a new,
approved gasket on the connector. Always follow your manufacturer’s instructions.
On 1-ton Chlorine gas containers, the chlorine pressure reducing valve should be located
downstream of the evaporator when using an evaporator. This is the liquid chlorine supply line and
it is going to be made into Chlorine gas.
In water treatment, chlorine is added to the effluent before the contact chamber (before the clear
well) for complete mixing. One reason for not adding it directly to the chamber is that the chamber
has very little mixing due to low velocities.
Here are several safety precautions when using chlorine gas. In addition to protective clothing and
goggles, chlorine gas should be used only in a well-ventilated area so that any leaking gas cannot
concentrate. Emergency procedures in the case of a large uncontrolled chlorine leak are as follows:
Notify local emergency response team, warn and evacuate people in adjacent areas, and be sure
that no one enters the leak area without adequate self-contained breathing equipment.
Here are several symptoms of chlorine exposure. Burning of eyes, nose, and mouth, coughing,
sneezing, choking, nausea and vomiting, headaches and dizziness, fatal pulmonary edema,
pneumonia, and skin blisters. A little Cl2 will corrode the teeth and then progress to throat cancer.
Approved method for storing a 150 - 200-pound chlorine cylinder: Secure each cylinder in an
upright position, attach the protective bonnet over the valve and firmly secure each cylinder. Never
store near heat. Always store the empty in an upright, secure position with proper signage.

The design of gas chlorine facilities should consider operator and public safety as well as
maintaining long-term plant reliability and operation. Chlorination facilities are designed
such that chlorine gas can be contained in the chlorine storage room. Doors and windows
should be gas-tight to minimize escape of gaseous chlorine to the exterior atmosphere or
building interior.
Leak detectors should be located 1 foot above the floor of the chlorine storage room and
should activate an alarm when a chlorine leak occurs. It is preferable that the detector be
capable of differentiating between two or more chlorine concentrations to alert personnel
of the severity of the release. This would help determine the appropriate procedure for
entrance to the room, ventilation, or other solutions. Self contained breathing apparatus
(SCBA) should not be located within the chlorine storage room. It is preferable that this
equipment be located in a convenient location where personnel can easily access it in the
event of an emergency.
The length of the chlorine gas and liquid chlorine pipelines should be as short as possible.
All the safety equipments should be readily available and handy. The Plant should have
provisions for exhausting chlorine gas, if a leak develops. Ideally a chlorine gas leak
absorption system can be provided for gas leak evacuation and neutralization. An
automatic or manual Shut - Off Valve and Pressure Relief Valve is also included for safe
operation.

Chlorine Introduction
Name: Chlorine
Symbol: Cl
Atomic Number: 17
Atomic Mass: 35.4527 amu
Melting Point: -100.98 °C (172.17 K, -149.764 °F)
Boiling Point: -34.6 °C (238.55 K, -30.279997 °F)
Number of Protons/Electrons: 17
Number of Neutrons: 18
Classification: Halogen
Crystal Structure: Orthorhombic
Density @ 293 K: 3.214 g/cm3
Color: Green
Uses: Water purification, bleaches
Obtained From: Salt
Date of Discovery: 1774
Discoverer: Carl Wilhelm Scheele
Name Origin: From the Greek word khlôros (green)
Chlorine Gas Information

Identifiers
1. CAS No.: 7782-50-5
2. RTECS No.: FO2100000
3. DOT UN: 1017 20
4. DOT label: Poison gas
Safety Data
NIOSH IDHL: 25 ppm
NIOSH Ceiling: 0.5ppm/15 minutes
PEL/TWA: 1 ppm
TLV/TWA: 1 ppm
TLV/STEL: 3 ppm
TLV/IDLH: 25 ppm
Physical Data Chlorinators

1. Molecular weight: 70.9
2. Boiling point (at 760 mm Hg): -34.6 degrees C (-30.28 degrees F)
3. Specific gravity (liquid): 1.41 at 20 degrees C (68 degrees F) and a pressure of 6.86
atm
4. Vapor density: 2.5
5. Melting point: -101 degrees C (-149.8 degrees F)
6. Vapor pressure at 20 degrees C (68 degrees F): 4,800 mm Hg
7. Solubility: Slightly soluble in water; soluble in alkalis, alcohols, and chlorides.
8. Evaporation rate: Data not available.

Chlorine’s Appearance and Odor
Chlorine is a greenish-yellow gas with a characteristic pungent odor. It condenses to an
amber liquid at approximately -34 degrees C (-29.2 degrees F) or at high pressures. Odor
thresholds ranging from 0.08 to part per million (ppm) parts of air have been reported.
Prolonged exposures may result in olfactory fatigue.
Reactivity
1. Conditions Contributing to Instability: Cylinders of chlorine may burst when exposed
to elevated temperatures. Chlorine in solution forms a corrosive material.
2. Incompatibilities: Flammable gases and vapors form explosive mixtures with chlorine.
Contact between chlorine and many combustible substances (such as gasoline and
petroleum products, hydrocarbons, turpentine, alcohols, acetylene, hydrogen, ammonia,
and sulfur), reducing agents, and finely divided metals may cause fires and explosions.
Contact between chlorine and arsenic, bismuth, boron, calcium, activated carbon, carbon
disulfide, glycerol, hydrazine, iodine, methane, oxomonosilane, potassium, propylene, and
silicon should be avoided. Chlorine reacts with hydrogen sulfide and water to form
hydrochloric acid, and it reacts with carbon monoxide and sulfur dioxide to form phosgene
and sulfuryl chloride. Chlorine is also incompatible with moisture, steam, and water.
3. Hazardous Decomposition Products: None reported.
4. Special Precautions: Chlorine will attack some forms of plastics, rubber, and coatings.
Flammability
Chlorine is a non-combustible gas.
The National Fire Protection Association has assigned a flammability rating of 0 (no fire
hazard) to chlorine; however, most combustible materials will burn in chlorine.
1. Flash point: Not applicable.
2. Autoignition temperature: Not applicable.
3. Flammable limits in air: Not applicable.
4. Extinguishant: For small fires use water only; do not use dry chemical or carbon
dioxide. Contain and let large fires involving chlorine burn. If fire must be fought, use water
spray or fog.
Fires involving chlorine should be fought upwind from the maximum distance
possible.
Keep unnecessary people away; isolate the hazard area and deny entry. For a massive
fire in a cargo area, use unmanned hose holders or monitor nozzles; if this is impossible,
withdraw from the area and let the fire burn. Emergency personnel should stay out of low
areas and ventilate closed spaces before entering.
Containers of chlorine may explode in the heat of the fire and should be moved from the
fire area if it is possible to do so safely. If this is not possible, cool fire exposed containers
from the sides with water until well after the fire is out. Stay away from the ends of
containers. Firefighters should wear a full set of protective clothing and self- contained
breathing apparatus when fighting fires involving chlorine.

Chlorine Exposure Limits
* OSHA PEL
The current OSHA permissible exposure limit (PEL) for chlorine is 1 ppm (3 milligrams
per cubic meter (mg/m(3))) as a ceiling limit. A worker's exposure to chlorine shall at no
time exceed this ceiling level [29 CFR 1910.1000, Table Z-1].
* NIOSH REL
The National Institute for Occupational Safety and Health (NIOSH) has established a
recommended exposure limit (REL) for chlorine of 0.5 ppm mg/m(3)) as a TWA for up to a
10-hour workday and a 40-hour workweek and a short-term exposure limit (STEL) of 1
ppm (3 mg/m(3))[NIOSH 1992].
* ACGIH TLV
The American Conference of Governmental Industrial Hygienists (ACGIH) has assigned
chlorine a threshold limit value (TLV) of 0.5 ppm (1.5 mg/m(3)) as a TWA for a normal 8-
hour workday and a 40-hour workweek and a STEL of 1 ppm (2.9 mg/m(3)) for periods not
to exceed 15 minutes. Exposures at the STEL concentration should not be repeated more
than four times a day and should be separated by intervals of at least 60 minutes [ACGIH
1994, p. 15].
* Rationale for Limits

The NIOSH limits are based on the risk of severe eye, mucous membrane and skin
irritation [NIOSH 1992]. The ACGIH limits are based on the risk of eye and mucous
membrane irritation [ACGIH 1991, p. 254].
Chlorine’s Atomic Structure
Isotopes
Isotope Half Life Cl-37 Stable
Cl-35 Stable Cl-38 37.2 minutes
Cl-36 301000.0 years

Top photograph, this blue device prevents the liquid from being pulled and freezing the
lines. Bottom photograph, the application of an ammonia mist to detect a chlorine gas
leak.

Chlorine Basics
Chlorine is one of 90 natural elements, the basic building blocks of our planet. To be
useful, an element must be relatively abundant or have extremely desirable properties.
Chlorine has both characteristics. As a result -- over the course of many decades of careful
research and development -- scientists have learned to use chlorine and the products of
chlorine chemistry to make drinking water safe, destroy life-threatening germs, produce
life-saving drugs and medical equipment, shield police and fire fighters in the line of duty,
and ensure a plentiful food supply.
In 1774, in his small experimental laboratory, Swedish pharmacist Carl Wilhem Scheele
released a few drops of hydrochloric acid onto a piece of manganese dioxide. Within
seconds, a greenish-yellow gas arose. Although he had no idea at the time, he had just
discovered chlorine.
The fact that the greenish-yellow gas was actually an element was only recognized several
decades later by English chemist Sir Humphrey Davy. Until that time, people were
convinced that the gas was a compound of oxygen. Davy gave the element its name on
the basis of the Greek word khloros, for greenish-yellow. In 1810 he suggested the name
"chloric gas" or "chlorine."
One of the most effective and economical germ-killers, chlorine also destroys and
deactivates a wide range of dangerous germs in homes, hospitals, swimming pools,
hotels, restaurants, and other public places.
Chlorine's powerful disinfectant qualities come from its ability to bond with and destroy the
outer surfaces of bacteria and viruses. First used as a germicide to prevent the spread of
"child bed fever" in the maternity wards of Vienna General Hospital in Austria in 1846,
chlorine has been one of society's most potent weapons against a wide array of life-
threatening infections, viruses, and bacteria for 150 years.
When the first men to set foot on the moon returned to earth (Apollo 11 mission: 24.7.69)
a hypochlorite solution was chosen as one of the disinfectants for destroying any
possible moon germs.
What Happens to Chlorine When it Enters the Environment?

 When released to air, chlorine will react with water to form hypochlorous acid and
hydrochloric acid, which are removed from the atmosphere by rainfall.
 Chlorine is slightly soluble in water. It reacts with water to form hypochlorous acid
and hydrochloric acid. The hypochlorous acid breaks down rapidly. The
hydrochloric acid also breaks down; its breakdown products will lower the pH of
the water (makes it more acidic).
 Since chlorine is a gas it is rarely found in soil. If released to soil, chlorine will
react with moisture forming hypochlorous acid and hydrochloric acid. These
compounds can react with other substances found in soil.
 Chlorine does not accumulate in the food chain.

Disinfectant Qualities
Restaurants and meat and poultry processing plants rely on chlorine bleach and other chlorine-
based products to kill harmful levels of bacteria such as Salmonella and E. coli on food
preparation surfaces and during food processing. Chlorine is so important in poultry processing
that the US Department of Agriculture requires an almost constant chlorine rinse for much of
the cutting equipment. In fact, no proven economical alternative to chlorine disinfection exists
for use in meat and poultry processing facilities.
Properties
Because it is highly reactive, chlorine is usually found in nature bound with other elements like
sodium, potassium, and magnesium. When chlorine is isolated as a free element, chlorine is a
greenish yellow gas, which is 2.5 times heavier than air. It turns to a liquid state at -34°C (-
29°F), and it becomes a yellowish crystalline solid at -103°C (-153°F). Chemists began
experimenting with chlorine and chlorine compounds in the 18th century. They learned that
chlorine has an extraordinary ability to extend a chemical bridge between various elements and
compounds that would not otherwise react with each other. Chlorine has been especially useful
in studying and synthesizing organic compounds -- compounds that have at least one atom of
the element carbon in their molecular structure. All living organisms, including humans, are
composed of organic compounds.
Chlorine is one of the most abundant chemical elements on Earth. It is ubiquitous in soils,
minerals, plants and animals. Seawater is a huge reservoir of dissolved chlorine weathered
from the continents and transported to the oceans by Earth's rivers.
Chlorine is also one of the most useful chemical elements. Each chemical element has its own
set of unique properties and chlorine is known as a very reactive element--so reactive, in fact,
that it is usually found combined with other elements in the form of compounds. More than
3,500 naturally occurring chlorinated organic (associated with living organisms) compounds
alone have been identified.
Chlorine's chemical properties have been harnessed innovatively for good use. For example,
this element plays a huge role in public health. Chlorine-based disinfectants are capable of
removing a wide variety of disease-causing germs from drinking water and wastewater as well
as from hospital and food production surfaces. Additionally, chlorine plays an important role in
the manufacture of thousands of products we depend upon every day, including such diverse
items as cars, computers, pharmaceuticals and military flak jackets. As the ninth largest
chemical produced in the U.S. by volume, chlorine is truly a "workhorse chemical."
Released from the Salt of the Earth

Chlorine is produced industrially from the compound sodium chloride, one of the many salts
found in geologic deposits formed from the slow evaporation of ancient seawater. When
electricity is applied to a brine solution of sodium chloride, chlorine gas (Cl2), caustic soda
(NaOH) and hydrogen gas (H2) are generated according to the following reaction:

Co-Products
As the reaction demonstrates, chlorine gas cannot be produced without producing caustic soda,
so chlorine and caustic soda are known as "co-products," and their economics are inextricably
linked. Caustic soda, also called "alkali," is used to produce a wide range of organic and
inorganic chemicals and soaps. In addition, the pulp and paper, alumina and textiles industries
use caustic soda in their manufacturing processes. Thus, the "chlor-alkali" industry obtains two
very useful chemicals by applying electrical energy to sea salt.
Definitions
Chlorine Gas Feed Room
A chlorine gas feed room, for the purposes of this document, is a room that contains the
chlorinator(s) and active cylinder(s) used to apply chlorine gas at a water or wastewater
facility.
Chlorine Gas Storage Room

A chlorine gas storage room, for the purposes of this document, is a room other than a chlorine
gas feed room, in which full, partial, or empty chlorine gas cylinders or ton containers are stored
at a water or wastewater facility.
Gas Chlorinator
A gas chlorinator is a device used to meter and control the application rate of chlorine gas into
a liquid. There is the danger of the gas escaping at a water or wastewater treatment facility.
The gas chlorinator should be isolated from a water or wastewater treatment plant.
Chlorine Cabinet
A chlorine cabinet is a pre-assembled or factory built unit that contains the equipment used to
apply chlorine gas at a water or wastewater treatment facility. It is isolated from a water or
wastewater treatment plant.

Top photograph, a view of the top of a 150 gas cylinder. Bottom, always work in pairs when
working around Chlorine. Here the hoist is being used to move the container.

Chemical Equations, Oxidation States, and Balancing of Equations
Before we breakdown chlorine and other chemicals, let’s start with this review of basic
chemical equations.
Beginning
The common chemical equation could be A + B --> C + D. This is chemical A + chemical B,
the two reacting chemicals will go to products C + D, etc.
Oxidation
The term “oxidation” originally meant a reaction in which oxygen combines chemically with
another substance, but its usage has long been broadened to include any reaction in which
electrons are transferred.
Oxidation and reduction always occur simultaneously (redox reactions), and the substance
which gains electrons is termed the oxidizing agent. For example, cupric ion is the oxidizing
agent in the reaction: Fe (metal) + Cu++ --> Fe++ + Cu (metal); here, two electrons (negative
charges) are transferred from the iron atom to the copper atom; thus the iron becomes positively
charged (is oxidized) by loss of two electrons, while the copper receives the two electrons and
becomes neutral (is reduced).
Electrons may also be displaced within the molecule without being completely transferred away
from it. Such partial loss of electrons likewise constitutes oxidation in its broader sense and
leads to the application of the term to a large number of processes, which at first sight might
not be considered to be oxidation. Reaction of a hydrocarbon with a halogen, for example, CH4
+ 2 Cl --> CH3Cl + HCl, involves partial oxidation of the methane; halogen addition to a double
bond is regarded as an oxidation.
Dehydrogenation is also a form of oxidation; when two hydrogen atoms, each having one
electron, are removed from a hydrogen-containing organic compound by a catalytic reaction
with air or oxygen, as in oxidation of alcohol to aldehyde.
Oxidation Number
The number of electrons that must be added to or subtracted from an atom in a combined
state to convert it to the elemental form; i.e., in barium chloride (BaCl2) the oxidation number
of barium is +2 and of chlorine is -1. Many elements can exist in more than one oxidation
state.
Now, let us look at some common ions. An ion is the reactive state of the chemical, and is
dependent on its place within the periodic table.
Have a look at the “periodic table of the elements”. It is arranged in columns of elements,
there are 18 columns. You can see column one, H, Li, Na, K, etc. These all become ions as
H+, Li+, K+, etc. The next column, column 2, Be, Mg, Ca etc. become ions Be2+, Mg2+, Ca2+,
etc. Column 18, He, Ne, Ar, Kr are inert gases. Column 17, F, Cl, Br, I, ionize to a negative F-,
Cl-, Br-, I-, etc.
What you now need to do is memorize the table of common ions, both positive ions and
negative ions.

Table of Common Ions
Positive Ions
Valency 1 Valency 2 Valency 3
lithium Li+ magnesium Mg2+ aluminum Al3+
sodium Na+ calcium Ca2+ iron III Fe3+
potassium K+ strontium Sr2+ chromium Cr3+
silver Ag+ barium Ba2+
hydronium H3O+ copper II Cu2+
(or hydrogen) H+ lead II Pb2+
ammonium NH4+ zinc Zn2+
copper I Cu+ manganese II Mn2+
mercury I Hg+ iron II Fe2+
tin II Sn2+
Negative Ions
Valency 1 Valency 2 Valency 3
fluoride F- oxide O2- phosphate PO43-
chloride Cl- sulfide S2-
bromide Br - carbonate CO32-
iodide I- sulfate SO42-
hydroxide OH- sulfite SO32-
nitrate NO3- dichromate Cr2O7-
bicarbonate HCO3- chromate CrO42-
bisulphate HSO4- oxalate C2O42-
nitrite NO2- thiosulfate S2O32-
chlorate ClO3- tetrathionate S4O62-
monohydrogen
permanganate MnO4- HPO42-
phosphate
hypochlorite OCl-
dihydrogen
H2PO4-
phosphate
Positive ions will react with negative ions, and vice versa. This is the start of our
chemical reactions. For example:
Na+ + OH- --> NaOH (sodium hydroxide)
Na+ + Cl- --> NaCl (salt)
3H+ + PO43- --> H3PO4 (phosphoric acid)
2Na+ + S2O32- --> Na2S2O3

You will see from these examples, that if an ion of one (+), reacts with an ion of one (-)
then the equation is balanced. However, an ion like PO43- (phosphate) will require an ion of
3+ or an ion of one (+) (but needs three of these) to neutralize the 3- charge on the phosphate.
So, what you are doing is balancing the charges (+) or (-) to make them zero, or cancel each
other out.
For example, since aluminum exists in its ionic state as Al3+, it will react with many negatively
charged ions; for example: Cl-, OH-, SO42-, PO43-.
Let us do these examples and balance them.

Al3+ + Cl- --> AlCl (incorrect)
Al3+ + 3Cl- --> AlCl3 (correct)
How did we work this out?

Al3+ has three positives (3+)
Cl- has one negative (-)
It will require 3 negative charges to cancel out the 3 positive charges on the aluminum
(Al3+).
When the left hand side of the equation is written, to balance the number of chlorine’s (Cl-)
required, the number 3 is placed in front of the ion concerned, in this case Cl-, becomes 3Cl-.
On the right hand side of the equation, where the ions have become a compound
(a chemical compound), the number is transferred to after the relevant ion, Cl3.
Another example:
Al3+ + SO42- --> AlSO4 (incorrect)
2Al3+ + 3SO42- --> Al2(SO4)3 (correct)
Let me give you an easy way of balancing:

Al is 3+
SO4 is 2-
Simply transpose the number of positives (or negatives) for each ion, to the other ion, by placing
this value of one ion, in front of the other ion. That is, Al3+ the 3 goes in front of the SO42- as
3SO42-, and SO42-, the 2 goes in front of the Al3+ to become 2Al3+. Then on the right hand side
of the equation, this same number (now in front of each ion on the left side of the equation), is
placed after each “ion” entity.
Let us again look at:

Al3+ + SO42- --> AlSO4 (incorrect)
Al3+ + SO42- --> Al2(SO4)3 (correct)
Put the three from the Al in front of the SO42- and the 2 from the SO42- in front of the Al3+.
Equation becomes:
2Al3+ + 3SO42- --> Al2(SO4)3. You simply place the valency of one ion, as a whole number, in
front of the other ion, and vice versa.
Remember to encase the SO4 in brackets. Why? Because we are dealing with the sulfate
ion, SO42-, and it is this ion that is 2- charged (not just the O4), so we have to ensure that the
“ion” is bracketed. Now to check, the 2 times 3+ = 6+, and 3 times 2- = 6-. We have equal
amounts of positive ions, and equal amounts of negative ions.

Another example:
NaOH + HCl --> ?
Na is Na+, OH is OH-, so this gave us NaOH. Originally, the one positive canceled the one
negative.
HCl is H+ + Cl -, this gave us HCl.
Reaction is going to be the Na+ reacting with a negatively charged ion. This will have to be the
chlorine, Cl-, because at the moment the Na+ is tied to the OH-. So: Na+ + Cl- --> NaCl
The H+ from the HCl will react with a negative (-) ion this will be the OH- from the NaOH.
So: H+ + OH- --> H2O (water).
The complete reaction can be written:

NaOH + HCl --> NaCl + H2O. We have equal amounts of all atoms each side of the
equation, so the equation is balanced.
or
Na+OH- + H+Cl- --> Na+Cl- + H+OH-
Something More Difficult:

Mg(OH)2 + H3PO4 --> ? (equation on left not balanced)
Mg2+ 2OH- + 3H+PO43- --> ? (equation on left not balanced), so let us rewrite the equation in
ionic form.
The Mg2+ needs to react with a negatively charged ion, this will be the PO43-,
so: 3Mg2+ + 2PO43- --> Mg3(PO4)2
(Remember the swapping of the positive or negative charges on the ions in the left side of
the equation, and placing it in front of each ion, and then placing this number after each ion
on the right side of the equation)
What is left is the H+ from the H3PO4 and this will react with a negative ion, we only have the
OH- from the Mg(OH)2 left for it to react with.
6H+ + 6OH- --> 6H2O
Where did I get the 6 from? When I balanced the Mg2+ with the PO43-, the equation became
3Mg2+ + 2PO43- --> Mg3(PO4)2
Therefore, I must have required 3Mg(OH)2 to begin with, and 2H3PO4, ( because we originally
had (OH)2 attached to the Mg, and H3 attached to the PO4. I therefore have 2H3 reacting with
3(OH)2. We have to write this, on the left side of the equation, as 6H+ + 6OH- because we
need it in ionic form.
The equation becomes:
6H+ + 6OH- --> 6H2O
The full equation is now balanced and is:

3Mg(OH)2 + 2H3PO4 --> Mg3(PO4)2 + 6H2O
I have purposely split the equation into segments of reactions. This is showing you which ions
are reacting with each other. Once you get the idea of equations you will not need this step.
The balancing of equations is simple. You need to learn the valency of the common ions (see
tables). The rest is pure mathematics; you are balancing valency charges, positives versus

negatives. You have to have the same number of negatives, or positives, each side of the
equation, and the same number of ions or atoms each side of the equation.
If one ion, example Al3+, (3 positive charges) reacts with another ion, example OH- (one
negative ion) then we require 2 more negatively charged ions (in this case OH-) to counteract
the 3 positive charges the Al3+ contains.
Take my earlier hint, place the 3 from the Al3+ in front of the OH-, now reads 3OH-, place the 1
from the hydroxyl OH- in front of the Al3+, now stays the same, Al3+ (the 1 is never written in
chemistry equations).
Al3+ + 3OH- --> Al(OH)3
The 3 is simply written in front of the OH-, a recognized ion, there are no brackets placed
around the OH-. On the right hand side of the equation, all numbers in front of each ion on the
left hand side of the equation are placed after each same ion on the right side of the equation.
Brackets are used in the right side of the equation because the result is a compound.
Brackets are also used for compounds (reactants) in the left side of equations, as in
3Mg(OH)2 + 2H3PO4 --> ?

Hard to tell, but these are one-ton chlorine gas containers. Notice the five-gallon bucket of
motor oil in the bottom photograph. Also notice that this photograph is the only eye wash
station that we found during our inspection of 10 different facilities. Do you have an eye wash
and emergency shower?

Chemistry of Chlorination
Chlorine can be added as sodium hypochlorite, calcium hypochlorite or chlorine gas. When any
of these is added to water, chemical reactions occur as these equations show:
Cl 2 + H 2 O → HOCI + HCI
(chlorine gas) (water) (hypochlorous acid) (hydrochloric acid)
CaOCI + H 2 O → 2HOCI + Ca(OH)

(calcium hypochlorite) (water) (hypochlorous acid) (calcium hydroxide)
NaOCI + H 2 O → HOCI + Na(OH)

(sodium hypochlorite) (water) (hypochlorous acid) (sodium hydroxide)
All three forms of chlorine produce hypochlorous acid (HOCl) when added to water.
Hypochlorous acid is a weak acid but a strong disinfecting agent. The amount of hypochlorous
acid depends on the pH and temperature of the water. Under normal water conditions,
hypochlorous acid will also chemically react and break down into a hypochlorite. ion
(OCl - ): HOCI H + + OCI – Also expressed HOCI → H + + OCI –

(hypochlorous acid) (hydrogen) (hypochlorite ion)
The hypochlorite ion is a much weaker disinfecting agent than hypochlorous acid, about 100
times less effective.
Let’s now look at how pH and temperature affect the ratio of hypochlorous acid to hypochlorite
ions. As the temperature is decreased, the ratio of hypochlorous acid increases. Temperature
plays a small part in the acid ratio. Although the ratio of hypochlorous acid is greater at lower
temperatures, pathogenic organisms are actually harder to kill. All other things being equal,
higher water temperatures and a lower pH are more conducive to chlorine disinfection.
Types of Residual
If water were pure, the measured amount of chlorine in the water should be the same as the
amount added. But water is not 100% pure. There are always other substances (interfering
agents) such as iron, manganese, turbidity, etc., which will combine chemically with the
chlorine.
This is called the chlorine demand. Naturally, once chlorine molecules are combined with
these interfering agents, they are not capable of disinfection. It is free chlorine that is much
more effective as a disinfecting agent.
So let’s look now at how free, total and combined chlorine are related. When a chlorine residual
test is taken, either a total or a free chlorine residual can be read.
Total residual is all chlorine that is available for disinfection.
Total chlorine residual = free + combined chlorine residual.

Free chlorine residual is a much stronger disinfecting agent. Therefore, most water regulating
agencies will require that your daily chlorine residual readings be of free chlorine residual.
Break-point chlorination is where the chlorine demand has been satisfied, and any additional
chlorine will be considered free chlorine.
Residual Concentration/Contact Time (CT) Requirements

Disinfection to eliminate fecal and coliform bacteria may not be sufficient to adequately reduce
pathogens such as Giardia or viruses to desired levels. Use of the "CT" disinfection concept is
recommended to demonstrate satisfactory treatment, since monitoring for very low levels of
pathogens in treated water is analytically very difficult.
The CT concept, as developed by the United States Environmental Protection Agency (Federal
Register, 40 CFR, Parts 141 and 142, June 29, 1989), uses the combination of disinfectant
residual concentration (mg/L) and the effective disinfection contact time (in minutes) to measure
effective pathogen reduction. The residual is measured at the end of the process, and the
contact time used is the T10 of the process unit (time for 10% of the water to pass).
CT = Concentration (mg/L) x Time (minutes)
The effective reduction in pathogens can be calculated by reference to standard tables of

required CTs.
Required Giardia/Virus Reduction

All surface water treatment systems shall ensure a minimum reduction in pathogen levels:
3-log reduction in Giardia; and 4-log reduction in viruses.
These requirements are based on unpolluted raw water sources with Giardia levels of = 1
cyst/100 L, and a finished water goal of 1 cyst/100,000 L (equivalent to 1 in 10,000 risk of
infection per person per year). Higher raw water contamination levels may require greater
removals as shown on Table 4.1.
TABLE 4.1
Level of Giardia Reduction
Raw Water Giardia Levels*
Recommended Giardia Log
Reduction
< 1 cyst/100 L 3-log
1 cyst/100 L - 10 cysts/100 L 3-log - 4-log
10 cysts/100 L - 100 cysts/100 L 4-log - 5-log
> 100 cysts/100 L > 5-log
*Use geometric means of data to determine raw water Giardia levels for compliance.
Required CT Value
Required CT values are dependent on pH, residual concentration, temperature, and the
disinfectant used. The tables attached to Appendices A and B shall be used to determine the
required CT.

Calculation and Reporting of CT Data
Disinfection CT values shall be calculated daily, using either the maximum hourly flow and the
disinfectant residual at the same time, or by using the lowest CT value if it is calculated more
frequently. Actual CT values are then compared to required CT values.
Results shall be reported as a reduction Ratio, along with the appropriate pH, temperature, and
disinfectant residual. The reduction Ratio must be greater than 1.0 to be acceptable.
Users may also calculate and record actual log reductions.

Reduction Ratio = CT actual divide by CT required.

Chlorinator Components
A. Ejector
B. Check Valve Assembly
C. Rate Valve
D. Diaphragm Assembly
E. Interconnection Manifold
F. Rotometer Tube and Float
G. Pressure Gauge
H. Gas Supply
Chlorine measurement devices or Rotometers.
Chlorine Safety Information

There is a fusible plug on every chlorine tank. This metal plug will melt at 158 to 165o F. This
is to prevent a build-up of excessive pressure and the possibility of cylinder rupture due to fire
or high temperatures.

Chlorine Review
Chlorine Demand: The minimum amount of chlorine needed to react in a water purification
system; used as a monitoring measurement by system operators.
Chlorine Residual: The concentration of chlorine in the water after the chlorine demand has
been satisfied. The concentration is normally expressed in terms of total chlorine residual,
which includes both the free and combined or chemically bound chlorine residuals.
Combined Chlorine Residual: The amount of chlorine used up in a water purification system;
used as a monitoring measurement by system operators. Combined chlorine is defined as the
residual chlorine existing in water in chemical combination with ammonia or organic amines
which can be found in natural or polluted waters. Ammonia is sometimes deliberately added to
chlorinated public water supplies to provide inorganic chloramines.
Free Chlorine: Free chlorine is defined as the concentration of residual chlorine in water
present as dissolved gas (Cl2), hypochlorous acid (HOCl), and/or hypochlorite ion (OCl-). The
three forms of free chlorine exist together in equilibrium.
Cl2 + H2O HOCl + H+ + Cl-
HOCl H+ + OCl-
Their relative proportions are determined by the pH value and temperature. Regardless of
whether pre-chloration is practiced or not, a free chlorine residual of at least 1.0 mg/L should
be maintained in the clear well or distribution reservoir immediately downstream from the point
of post-chlorination and .2 mg/L in the distribution system to guard against backflow.
Total Chlorine Residual: The total of free residual and combined residual chlorine in a water
purification system; used as a monitoring measurement by system operators. Total chlorine is
the sum of free and combined chlorine. When chlorinating most potable water supplies, total
chlorine is essentially equal to free chlorine since the concentration of ammonia or organic
nitrogen compounds (needed to form combined chlorine) will be very low. When chloramines
are present in the municipal water supply, then total chlorine will be higher than free chlorine.
Pre-chlorination: The addition of chlorine at the plant headworks or prior to other water
treatment or groundwater production processes and mainly used for disinfection and control of
tastes, odors, and aquatic growths.
Post-chlorination: The addition of chlorine after a process or adding chlorine downstream to

meet a demand in the system.
Breakpoint chlorination: Breakpoint chlorination means adding Cl2 to the water until the Cl2
demand is satisfied. Until all the microorganisms are killed.

What is the process of chlorination called as a treatment process and how does it
differ from sterilization?
Chlorination: A method of water disinfection where gaseous, liquid, or dissolved chlorine is
added to a water supply system. Water which has been treated with chlorine is effective in
preventing the spread of disease. The chlorination of public drinking supplies was originally
met with resistance, as people were concerned about the health effects of the practice. The
use of chlorine has greatly reduced the prevalence of waterborne disease as it is effective
against almost all bacteria and viruses, as well as amoeba. Sterilization kills everything.
What are the physical properties of chlorine, what hazards does it present, what
advantages does it have over most other disinfectants, and how does it react with
bacteria?
Physical and chemical properties of chlorine: A yellowish green, nonflammable and liquefied
gas with an unpleasant and irritating smell. Can be readily compressed into a clear, amber-
colored liquid, a noncombustible gas, and a strong oxidizer. Solid chlorine is about 1.5 times
heavier than water and gaseous chlorine is about 2.5 times heavier than air. Atomic number of
chlorine is 17. Cl is the elemental symbol and Cl2 is the chemical formula.
Chlorine reacts with bacteria as if it was very corrosive and burns the skin or covering killing
the bacteria.
What is the purpose of a fusible plug, at what temperature does it melt, and where is it
located on 150-lb. and 1-ton cylinders?
Fusible plug is a safety device that melts. If the temperature of a full Cl2 cylinder is increased
by 50o F or 30o C, a rupture may occur. It will melt at 158 to 165 degrees F. It is found on the
side of a 1-ton container and on top of the 150-pound cylinder and is located in the valve below
the valve seat.
What is the correct procedure to follow in changing a chlorine cylinder and what item
should always be replaced with a new one in doing so?
Hook up the chlorinator to the container or cylinder with the chlorine valve turned off. Use the
gas side not the liquid if using a 1-ton container. Remove the cylinder valve outlet cap and
check the valve face or damage. Clean with wire brush if necessary. If the valve face is smooth,
clean proceed with hooking up the cylinder. Check the inlet face of the chlorinator and clean if
necessary. Place a new lead gasket on the chlorinator inlet, place the chlorinator on the
cylinder valve, install the yoke clamp and slowly tighten the yoke clamp until the two faces are
against the lead gasket. Tighten the yoke, compressing the gasket one half to three quarters
turn, do not over tighten. Replace the lead gasket with every change out.
How, when and where should chlorine residuals be taken and what information do they
provide? The sample must be taken within the distribution system of your PWS. If you take it
before the distribution system you will not get an accurate reading. The sample must be taken
at the same tap that you take the Bac-t sample.

Chlor-Alkali Membrane Process
The electrolysis occurs in a cell containing electrodes submerged in solutions called
electrolytes. One electrode is referred to as the anode and is submerged in a salt water
solution. The second electrode is the cathode and is submerged in a sodium hydroxide (caustic
soda) solution. A membrane is used to keep the two different solutions from mixing. This
particular method of producing chlorine is called the chlor-alkali membrane process.
When a low voltage direct current (DC) power supply is applied to the electrodes in the cell, the
sodium and chlorine ions in the brine are attracted in opposite directions to the polarized
electrodes. The sodium ion passes across an ion selective membrane leaving the chlorine ion
to combine with a second chlorine ion, which makes a chlorine gas bubble at the anode
(electrode).
When the sodium crosses the membrane, it combines with a hydroxyl ion at the cathode
(electrode) making sodium hydroxide, or caustic soda (NaOH). The hydroxyl ion originates from
the dissolution of water at the cathode where hydrogen gas also develops. The membrane in
the cell keeps the two solutions separate; otherwise, the chlorine gas bubble would immediately
combine with the caustic soda forming sodium hypochlorite, or bleach. This process, which
uses a membrane to separate the two solutions, is called the chlor-alkali process. The chemical
equation for the chlor-alkali process is illustrated in the following equation:
2NaCl + 2H2O + electric current (2NaOH + Cl2- +- H2)

Chlorine’s Effectiveness
The effectiveness of chlorination depends on the chlorine demand of the water, the
concentration of the chlorine solution added, the time that chlorine is in contact with the
organism, and water quality. These effects can be summarized in the following manner:
 As the concentration of the chlorine increases, the required contact time to disinfect
decreases.
 Chlorination is more effective as water temperature increases.
 Chlorination is less effective as the water's pH increases (becomes more alkaline).
 Chlorination is less effective in cloudy (turbid) water.
 When chlorine is added to the water supply, part of it combines with other chemicals in
water (like iron, manganese, hydrogen sulfide, and ammonia) and is not available for
disinfection. The amount of chlorine that reacts with the other chemicals plus the amount
required to achieve disinfection is the chlorine demand of the water.
The safest way to be sure that the amount of chlorine added is sufficient is to add a little more
than is required. This will result in a free chlorine residual that can be measured easily. This
chlorine residual must be maintained for several minutes depending on chlorine level and water
quality. Table 4 lists the free chlorine residual level needed for different contact times, water
temperatures and pH levels.
Kits are available for measuring the chlorine residual by looking for a color change after the test
chemical is added. The test is simple and easy for a homeowner to perform. If chlorination is
required for the water supply, the chlorine residual should be tested regularly to make sure the
system is working properly. The kit should specify that it measures the free chlorine residual
and not the total chlorine. Once chlorine has combined with other chemicals it is not effective
as a disinfectant. If a test kit does not distinguish between free chlorine and chlorine combined
with other chemicals, the test may result in an overestimation of the chlorine residual.

Chlorine will kill bacteria in water, but it takes some time (Table 4) . The time needed depends
on the concentration of chlorine. Two methods of chlorination are used to disinfect water:
simple chlorination and superchlorination.
Table 4. Necessary chlorine residual to disinfect water for various contact times, water
temperatures and pH
Water Temp. 50 degrees F
Necessary chlorine residual (mg/l)
Contact time (minutes)
pH 7 pH 7.5 pH 8
40 0.2 0.3 0.4
30 0.3 0.4 0.5
20 0.4 0.6 0.8
10 0.8 1.2 1.6
5 1.6 2.4 3.2
2 4.0 6.0 8.0
1 8.0 12.0 16.0
Water Temp. 32 - 40 degrees F
Necessary chlorine residual (mg/l)
Contact time (minutes)
pH 7 pH 7.5 pH 8
40 0.3 0.5 0.6
30 0.4 0.6 0.8
20 0.6 0.9 1.2
10 1.2 1.8 2.4
5 2.4 3.6 4.8
2 6.0 9.0 12.0
1 12.0 18.0 24.0
Example: What is the necessary chlorine residual for well water with pH 7.5?
The well water is 38 degrees F when it enters the house. The pump delivers 7 gallons per
minute and after the chlorine is added it is held in a 100 gallon holding tank.
1. Contact time (from Table 5) - gallons per minute for 50-gallon tank = 5 minutes
2. Multiply by 2 for a 100-gallon tank = 10 minutes.
3. Necessary chlorine residual (from Table 4)- for water at 38 degrees F and pH 7.5 = 1.8
mg/l.
Simple chlorination involves maintaining a low level of free residual chlorine at a concentration
between 0.30 to .5 mg/l for at least 30 minutes. The residual is measured at the faucet most
distant from the where chlorine is added to the water supply.
To ensure the proper contact time of at least 30 minutes, a holding tank can be installed (Table
5). Pressure tanks, while often thought to be sufficient, are usually too small to always provide
30 minutes of contact time.

Table 5. Available contact time from a 50-gallon holding tank
Water flow rate (gallons per minute) Holding time (minutes)
5 7
7 5
10 3.5
Another way to maintain necessary contact time is to run the chlorinated water through a coil
of pipe (Table 6).
Table 6. Available contact time from 1000 feet of 1-1/4 inch pipe
Water flow rate (gallons per minute) Holding time (minutes)
5 9.2
7 6.6
10 4.6
When the water cannot be held for at least 30 minutes before it is used, super chlorination is
an alternative. For superchlorination, a chlorine solution is added to the water to produce a
chlorine residual of between 3.0 and 5.0 mg/l, which is about ten times stronger than for simple
chlorination.
The necessary contact time for this concentration is reduced to less than five minutes (Table
4). The water will have a very strong chlorine smell. If this is not desirable, the chlorine can be
removed just before it is used with a carbon filter (Note: may not be currently allowed under
your Department of Health for private water supplies).
Oxidation Chemistry
Oxidation chemistry has long been an accepted and effective part of many water treatment
programs. Oxidizing chemicals used in today's water treatment programs include: chlorine,
chlorine dioxide, bromine, bromine/chlorine releasing compounds, ozone and hydrogen
peroxide.
Oxidizing microbiocides are often found at the forefront of many cooling water treatment
programs. In large volume or once-through cooling systems they are usually the primary biocide
and often are the most cost-effective programs available to a plant. When selecting these
economical and versatile chemicals, several factors should be considered before a technically
sound program is implemented. Environmental and regulatory impact, system pH, process
contamination, and equipment capital and maintenance expense all play a role in the decision-
making process.
The primary killing mechanism these types of microbiocides use is oxidizing protein groups
within a microorganism. Proteins are the basic components of essential cellular enzymes that
are necessary for life-sustaining cellular processes such as respiration. The destruction of these
proteins deprives the cell of its ability to carry out fundamental life functions and quickly kills it.
One oxidant is chlorine dioxide, which appears to provide an additional killing mechanism.
Chlorine dioxide is able to diffuse readily through hydrophobic lipid layers of an organism,
allowing it to react with cellular amino acids, which directly inhibits protein synthesis. Since
amino acids are the basic building blocks of all cellular proteins, destruction of these molecules
has a devastating effect on the microorganism.

Staff shall be familiar with the locations of the chemical feed building as indicated by a posted
site plan. Self-contained breathing apparatus (SCBA) and personal protective equipment
should be facing the chemical feed building. Emergency repair kits “B” and “C” should be stored
on site close to the chemical feed building.
Chlorine scrubber

Chlorine Gas Section
Chlorine Gas
Background: Chlorine gas is a pulmonary irritant with intermediate water
solubility that causes acute damage in the upper and lower respiratory
tract. Chlorine gas was first used as a chemical weapon at Ypres, France
in 1915. Of the 70,552 American soldiers poisoned with various gasses in
World War I, 1843 were exposed to chlorine gas. Approximately 10.5
million tons and over 1 million containers of chlorine are shipped in the
U.S. each year.
Chlorine is a yellowish-green gas at standard temperature

and pressure. It is extremely reactive with most elements.
Because its density is greater than that of air, the gas
settles low to the ground. It is a respiratory irritant, and it
burns the skin. Just a few breaths of it are fatal. Cl2 gas
does not occur naturally, although Chlorine can be found in
a number of compounds.
Chlorine gas is likely the most widely used oxidizing

microbiocide. It has traditionally been the biocide of choice
in many cooling water treatment systems. It is a strong
oxidizer that is relatively easy to feed and is quite
inexpensive. Upon introduction into the water stream, chlorine hydrolyzes into hypochlorous
acid (HOCl) and hydrochloric acid (HCl).
This hydrolization provides the active toxicant, HOCl, which is pH-dependent. In alkaline cooling
systems, it readily dissociates to form the hypochlorite ion (OCl-). This dissociation
phenomenon is important to remember when working with systems that will operate at a higher
pH. In alkaline conditions, OCl- becomes the predominant species and lacks the biocidal
efficacy of the non-dissociated form. Considerably more HOCl is present at a pH of 7.0 than at
pH 8.5.
It is also widely known that chlorine is non-selective, making it very sensitive to contamination
from either cooling water makeup or from in-plant process leaks. Ammonia, organic acids and
organic compounds, sulfides, iron and manganese all easily react with HOCl. The amount of
chlorine needed to react with these contamination species is referred to as chlorine demand
and it must be satisfied before active HOCl is available to provide a free chlorine residual.
The combination of high chlorine demand in process-contaminated systems and the

dissociation process in alkaline systems creates the need for greater chlorine feed to obtain the
same microbial efficacy. This results in a higher concentration of HCl in the cooling system.
Since HCl removes alkalinity, pH depression and system corrosion could occur. In low pH water
the passive metal oxide layers protecting the metal may resolubulize, exposing the surface to
corrosion. At free mineral acidity (pH <4.3), many passivating inhibitors become ineffective, and
corrosion will proceed rapidly. Increased chloride may also have a negative impact on system
corrosion. The chloride ion (Cl-) can damage or penetrate the passive oxide layer, leading to
localized damage of the metal surface.

High chlorine concentrations have also been shown to directly attack traditional organic-
based corrosion inhibitors. When these inhibitors are "deactivated," the metal surface would
then be susceptible to corrosion. Process Safety Management (PSM) guidelines dictated by
the U.S. Occupational Safety and Health Administration (OSHA), discharge problems related
to chlorinated organic compounds such as trihalomethane (THM), dezincification of admiralty
brass and delignification of cooling tower wood are other significant concerns associated with
the use of chlorine.
Pathophysiology
Chlorine is a greenish-yellow, noncombustible gas at room temperature and atmospheric
pressure. The intermediate water solubility of chlorine accounts for its effect on the upper airway
and the lower respiratory tract.
Exposure to chlorine gas may be prolonged because its moderate water solubility may not
cause upper airway symptoms for several minutes. In addition, the density of the gas is greater
than that of air, causing it to remain near ground level and increasing
exposure time.
The odor threshold for chlorine is approximately 0.3-0.5 parts per

million (ppm); however, distinguishing toxic air levels from permissible
air levels may be difficult until irritative symptoms are present.
Mechanism of Activity
The mechanisms of the above biological activity are poorly understood
and the predominant anatomic site of injury may vary, depending on
the chemical species produced. Cellular injury is believed to result
from the oxidation of functional groups in cell components, from
reactions with tissue water to form hypochlorous and hydrochloric acid,
and from the generation of free oxygen radicals.
Although the idea that chlorine causes direct tissue damage by

generating free oxygen radicals was once accepted, this idea is now
controversial. The cylinders on the right contain chlorine gas.
The gas comes out of the cylinder through a gas regulator.
The cylinders are on a scale that operators use to measure the amount used each day. The
chains are used to prevent the tanks from falling over. Chlorine gas is stored in vented rooms
that have panic bar equipped doors. Operators have the equipment necessary to reduce the
impact of a gas leak, but rely on trained emergency response teams to contain leaks.
Solubility Effects
Hydrochloric acid is highly soluble in water. The predominant targets of the acid are the epithelia
of the ocular conjunctivae and upper respiratory mucus membranes. Hypochlorous acid is also
highly water soluble with an injury pattern similar to hydrochloric acid. Hypochlorous acid may
account for the toxicity of elemental chlorine and hydrochloric acid to the human body.
Early Response to Chlorine Gas

Chlorine gas, when mixed with ammonia, reacts to form chloramine gas. In the presence of
water, chloramines decompose to ammonia and hypochlorous acid or hydrochloric acid. The
early response to chlorine exposure depends on the (1) concentration of chlorine gas, (2)
duration of exposure, (3) water content of the tissues exposed, and (4) individual susceptibility.

Immediate Effects
The immediate effects of chlorine gas toxicity include acute inflammation of the conjunctivae,
nose, pharynx, larynx, trachea, and bronchi. Irritation of the airway mucosa leads to local edema
secondary to active arterial and capillary hyperemia. Plasma exudation results in filling the
alveoli with edema fluid, resulting in pulmonary congestion.
Pathological Findings
Pathologic findings are nonspecific. They include severe pulmonary edema, pneumonia,
hyaline membrane formation, multiple pulmonary thromboses, and ulcerative tracheobronchitis.
The hallmark of pulmonary injury associated with chlorine toxicity is pulmonary edema,
manifested as hypoxia. Noncardiogenic pulmonary edema is thought to occur when there is a
loss of pulmonary capillary integrity.

Using DPD Method for Chlorine Residuals
N, N – diethyl-p-phenylenediame
Small portable chlorine measuring kit. The redder the mixture the “hotter” or
stronger the chlorine in solution.
Measuring Chlorine Residual

Chlorine residual is the amount of chlorine remaining in water that can be used for
disinfection. A convenient, simple and inexpensive way to measure chlorine residual is to
use a small portable kit with pre-measured packets of chemicals that are added to water.
(Make sure you buy a test kit using the DPD method, and not the outdated orthotolodine
method.)
Chlorine test kits are very useful in adjusting the chlorine dose you apply. You can
measure what chlorine levels are being found in your system (especially at the far ends).
Free chlorine residuals need to be checked and recorded daily. These results should be
kept on file for a health or regulatory agency inspection during a regular field visit.
The most accurate method for determining chlorine residuals to use the laboratory
amperometric titration method.

Amperometric Titration
The chlorination of water supplies and polluted waters serves primarily to destroy or
deactivate disease-producing microorganisms. A secondary benefit, particularly in treating
drinking water, is the overall improvement in water quality resulting from the reaction of
chlorine with ammonia, iron, manganese, sulfide, and some organic substances.
Chlorination may produce adverse effects. Taste and odor characteristics of phenols and
other organic compounds present in a water supply may be intensified. Potentially
carcinogenic chloro-organic compounds such as chloroform may be formed.
Combined chlorine formed on chlorination of ammonia- or amine-bearing waters

adversely affects some aquatic life. To fulfill the primary purpose of chlorination and to
minimize any adverse effects, it is essential that proper testing procedures be used with
a foreknowledge of the limitations of the analytical determination.
Chlorine applied to water in its molecular or hypochlorite form initially undergoes

hydrolysis to form free chlorine consisting of aqueous molecular chlorine, hypochlorous
acid, and hypochlorite ion. The relative proportion of these free chlorine forms is pH- and
temperature-dependent. At the pH of most waters, hypochlorous acid and hypochlorite ion
will predominate.
Free chlorine reacts readily with ammonia and certain nitrogenous compounds to form
combined chlorine. With ammonia, chlorine reacts to form the chloramines:
monochloramine, dichloramine, and nitrogen trichloride.
The presence and concentrations of these combined forms depend chiefly on pH,
temperature, initial chlorine-to-nitrogen ratio, absolute chlorine demand, and reaction time.
Both free and combined chlorine may be present simultaneously. Combined chlorine in
water supplies may be formed in the treatment of raw waters containing ammonia or by
the addition of ammonia or ammonium salts.
Chlorinated wastewater effluents, as well as certain chlorinated industrial effluents,

normally contain only combined chlorine. Historically the principal analytical problem has
been to distinguish between free and combined forms of chlorine.
Hach’s AutoCAT 9000™ Automatic Titrator is the newest solution to hit the disinfection
industry – a comprehensive, benchtop chlorine-measurement system that does it all:
calibration, titration, calculation, real-time graphs, graphic print output, even electrode
cleaning. More a laboratory assistant than an instrument, the AutoCAT 9000 gives you:
 High throughput, performs the titration and calculates concentration, all
automatically.
 Forward titration, USEPA-accepted methods for free and total chlorine and
chlorine dioxide with chlorite.
 Back titration, USEPA-accepted method for total chlorine in wastewater.
 Accurate, yet convenient: the easiest way to complete ppb-level amperometric
titration.

Health Hazard Information
Routes of Exposure
Exposure to chlorine can occur through inhalation, ingestion, and eye or skin contact [Genium
1992].
Summary of toxicology
1. Effects on Animals: Chlorine is a severe irritant of the eyes, mucous membranes, skin, and lungs
in experimental animals. The 1 hour LC(50) is 239 ppm in rats and 137 ppm in mice ()[Sax and
Lewis 1989]. Animals surviving sub-lethal inhalation exposures for 15 to 193 days showed marked
emphysema, which was associated with bronchiolitis and pneumonia [Clayton and Clayton 1982].
Chlorine injected into the anterior chamber of rabbits' eyes resulted in severe damage with
inflammation, opacification of the cornea, atrophy of the iris, and injury to the lens [Grant 1986].
2. Effects on Humans: Severe acute effects of chlorine exposure in humans have been well
documented since World War I when chlorine gas was used as a chemical warfare agent. Other
severe exposures have resulted from the accidental rupture of chlorine tanks. These exposures
have caused death, lung congestion, and pulmonary edema, pneumonia, pleurisy, and bronchitis
[Hathaway et al. 1991]. The lowest lethal concentration reported is 430 ppm for 30 minutes [Clayton
and Clayton 1982].
Exposure to 15 ppm causes throat irritation, exposures to 50 ppm are dangerous, and exposures
to 1000 ppm can be fatal, even if exposure is brief [Sax and Lewis 1989; Clayton and Clayton
1982]. Earlier literature reported that exposure to a concentration of about 5 ppm caused respiratory
complaints, corrosion of the teeth, inflammation of the mucous membranes of the nose and
susceptibility to tuberculosis among chronically-exposed workers.
However, many of these effects are not confirmed in recent studies and are of very dubious
significance [ACGIH 1991]. A study of workers exposed to chlorine for an average of 10.9 years
was published in 1970. All but six workers had exposures below 1 ppm; 21 had TWAs above 0.52
ppm. No evidence of permanent lung damage was found, but 9.4 percent had abnormal EKGs
compared to 8.2 percent in the control group.
The incidence of fatigue was greater among those exposed above 0.5 ppm [ACGIH 1991]. In 1981,
a study was published involving 29 subjects exposed to chlorine concentrations up to 2.0 ppm for
4- and 8-hour periods. Exposures of 1.0 ppm for 8 hours produced statistically significant changes
in pulmonary function that were not observed at a 0.5 ppm exposure concentration. Six of 14
subjects exposed to 1.0 ppm for 8 hours showed increased mucous secretions from the nose and
in the hypopharynx.
Responses for sensations of itching or burning of the nose and eyes, and general discomfort were
not severe, but were perceptible, especially at the 1.0 ppm exposure level [ACGIH 1991]. A 1983
study of pulmonary function at low concentrations of chlorine exposure also found transient
decreases in pulmonary function at the 1.0 ppm exposure level, but not at the 0.5 ppm level [ACGIH
1991].
Acne (chloracne) is not unusual among persons exposed to low concentrations of chlorine for long
periods of time. Tooth enamel damage may also occur [Parmeggiani 1983]. There has been one
confirmed case of myasthenia gravis associated with chlorine exposure [NLM 1995].

Signs and Symptoms of Exposure
1. Acute exposure: Acute exposure to low levels of chlorine results in eye, nose, and throat irritation,
sneezing, excessive salivation, general excitement, and restlessness. Higher con-centrations
causes difficulty in breathing, violent coughing, nausea, vomiting, cyanosis, dizziness, headache,
choking, laryngeal edema, acute tracheobronchitis, chemical pneumonia. Contact with the liquid
can result in frostbite burns of the skin and eyes [Genium 1992].
2. Chronic exposure: Chronic exposure to low levels of chlorine gas can result in a dermatitis known
as chloracne, tooth enamel corrosion, coughing, severe chest pain, sore throat, hemoptysis and
increased susceptibility to tuberculosis [Genium 1992].
Emergency Medical Procedures: [NIOSH to supply]

1. Rescue: Remove an incapacitated worker from further exposure and implement
appropriate emergency procedures (e.g., those listed on the Material Safety Data
Sheet required by OSHA's Hazard Communication Standard [29 CFR 1910.1200]).
2. All workers should be familiar with emergency procedures, the location and proper
use of emergency equipment, and methods of protecting themselves during rescue
operations.
Exposure Sources and Control Methods

The following operations may involve chlorine and lead to worker exposures to this substance:
The Manufacture and Transportation of Chlorine

 Use as a chlorinating and oxidizing agent in organic and inorganic synthesis; in the
manufacture of chlorinated solvents, automotive antifreeze and antiknock compounds,
polymers (synthetic rubber and plastics), resins, elastomers, pesticides, refrigerants, and
in the manufacture of rocket fuel.
 Use as a fluxing, purification, and extraction agent in metallurgy.
 Use as a bacteriostat, disinfectant, odor control, and demulsifier in treatment of drinking
water, swimming pools, and in sewage.
 Use in the paper and pulp, and textile industries for bleaching cellulose for artificial fibers;
use in the manufacture of chlorinated lime; use in de-tinning and de-zincing iron; use to
shrink-proof wool.
 Use in the manufacture of pharmaceuticals, cosmetics, lubricants, flame-proofing,
adhesives, in special batteries containing lithium or zinc, and in hydraulic fluids; use in the
processing of meat, fish, vegetables, and fruit.
 Use as bleaching and cleaning agents, and as a disinfectant in laundries, dishwashers,
cleaning powders, cleaning dairy equipment, and bleaching cellulose.
Methods that are effective in controlling worker exposures to chlorine, depending on the feasibility
of implementation, are as follows: Process enclosure Local exhaust ventilation General dilution
ventilation Personal protective equipment.
Workers responding to a release or potential release of a hazardous substance must be protected

as required by paragraph (q) of OSHA's Hazardous Waste Operations and Emergency Response
Standard 29 CFR.

Good Sources of Information about Control Methods are as Follows:
1. ACGIH [1992]. Industrial ventilation--a manual of recommended practice. 21st ed. Cincinnati,
OH: American Conference of Governmental Industrial Hygienists.
2. Burton DJ [1986]. Industrial ventilation--a self-study companion. Cincinnati, OH: American
Conference of Governmental Industrial Hygienists.
3. Alden JL, Kane JM [1982]. Design of industrial ventilation systems. New York, NY: Industrial
Press, Inc.
4. Wadden RA, Scheff PA [1987]. Engineering design for control of workplace hazards. New
York, NY: McGraw-Hill.
5. Plog BA [1988]. Fundamentals of industrial hygiene. Chicago, IL: National Safety Council.
Chlorine Storage
Chlorine should be stored in a cool, dry, well-ventilated area in tightly sealed containers that are
labeled in accordance with OSHA's Hazard Communication Standard [29 CFR 1910.1200].
Containers of chlorine should be protected from exposure to weather, extreme temperatures
changes, and physical damage, and they should be stored separately from flammable gases and
vapors, combustible substances (such as gasoline and petroleum products, hydrocarbons,
turpentine, alcohols, acetylene, hydrogen, ammonia, and sulfur), reducing agents, finely divided
metals, arsenic, bismuth, boron, calcium, activated carbon, carbon disulfide, glycerol, hydrazine,
iodine, methane, oxomonosilane, potassium, propylene, silicon, hydrogen sulfide and water,
carbon monoxide and sulfur dioxide, moisture, steam, and water. (Sulfur dioxide is used for de-
chlorination).
Workers handling and operating chlorine containers, cylinders,

and tank wagons should receive special training in standard
safety procedures for handling compressed corrosive gases.
All pipes and containment used for chlorine service should be
regularly inspected and tested. Empty containers of chlorine
should have secured protective covers on their valves and
should be handled appropriately.
Spills and Leaks

In the event of a spill or leak involving chlorine, persons not
wearing protective equipment and fully-encapsulating, vapor-
protective clothing should be restricted from contaminated areas until cleanup has been completed.
The following steps should be undertaken following a spill or leak:
1. Notify safety personnel.
2. Remove all sources of heat and ignition.
3. Keep all combustibles (wood, paper, oil, etc.) away from the
leak.
4. Ventilate potentially explosive atmospheres.
5. Evacuate the spill area for at least 50 feet in all directions.
6. Find and stop the leak if this can be done without risk; if not,
move the leaking container to an isolated area until gas has
dispersed. The cylinder may be allowed to empty through a
reducing agent such as sodium bisulfide and sodium bicarbonate.
7. Use water spray to reduce vapors; do not put water directly on
the leak or spill area.

Special Requirements
The U.S. Environmental Protection Agency (EPA) requirements for emergency planning,
reportable quantities of hazardous releases, community right-to-know, and hazardous waste
management may change over time. Users are therefore advised to determine periodically whether
new information is available.
Emergency Planning Requirements

Employers owning or operating a facility at which there are 100 pounds or more of chlorine must
comply with the EPA's emergency planning requirements [40 CFR Part 355.30].
Reportable Quantity Requirements for Hazardous Releases

A hazardous substance release is defined by the EPA as any spilling, leaking, pumping, pouring,
emitting, emptying, discharging, injecting, escaping, leaching, dumping, or disposing into the
environment including the abandonment or discarding of contaminated containers) of hazardous
substances. In the event of a release that is above the reportable quantity for that chemical,
employers are required to notify the proper Federal, State, and local authorities [40 CFR
The Reportable Quantity of Chlorine is 10 Pounds.

If an amount equal to or greater than this quantity is released within a 24-hour period in a manner
that will expose persons outside the facility, employers are required to do the following: Notify the
National Response Center immediately at (800) or at (202) 426-2675 in Washington, D.C. [40 CFR
302.6]. Notify the emergency response commission of the State likely to be affected by the release
[40 CFR 355.40]. Notify the community emergency coordinator of the local emergency planning
committee (or relevant local emergency response personnel) of any area likely to be affected by
the release [40 CFR 355.40].
Community Right-to-Know Requirements

Employers who own or operate facilities in SIC
codes 20 to 39 that employ 10 or more workers
and that manufacture 25,000 pounds or more of
chlorine per calendar year or otherwise use 10,000
pounds or more of chlorine per calendar year are
required by EPA [40 CFR Part 372.30] to submit a
Toxic Chemical Release Inventory form (Form R)
to the EPA reporting the amount of chlorine emitted
or released from their facility annually.
Hazardous Waste Management Requirements

EPA considers a waste to be hazardous if it
exhibits any of the following characteristics:
ignitability, corrosivity, reactivity, or toxicity as
defined in 40 CFR 261.21-261.24. Under the
Resource Conservation and Recovery Act (RCRA) [40 USC 6901 et seq.], the EPA has
specifically listed many chemical wastes as hazardous. Although chlorine is not specifically listed
as a hazardous waste under RCRA, the EPA requires employers to treat waste as hazardous if it
exhibits any of the characteristics discussed above.
Providing detailed information about the removal and disposal of specific chemicals is beyond the
scope of this guideline. The U.S. Department of Transportation, the EPA, and State and local
regulations should be followed to ensure that removal, transport, and disposal of this substance
are conducted in accordance with existing regulations.

Chlorination Equipment Requirements
For all wastewater treatment facilities, chlorine gas under pressure shall not be permitted outside
the chlorine room. A chlorine room is where chlorine gas cylinders and/or ton containers are stored.
Vacuum regulators shall also be located inside the chlorine room. The chlorinator, which is the
mechanical gas proportioning equipment, may or may not be located inside the chlorine room.
For new and upgraded facilities, from the chlorine room, chlorine gas vacuum lines should be run
as close to the point of solution application as possible. Injectors should be located to minimize the
length of pressurized chlorine solution lines. A gas pressure relief system shall be included in the
gas vacuum line between the vacuum regulator(s) and the chlorinator(s) to ensure that pressurized
chlorine gas does not enter the gas vacuum lines leaving the chlorine room.
The gas pressure relief system shall vent pressurized gas to the atmosphere at a location that is
not hazardous to plant personnel; vent line should be run in such a manner that moisture collecting
traps are avoided. The vacuum regulating valve(s) shall have positive shutdown in the event of a
break in the downstream vacuum lines.
As an alternative to chlorine gas, it is permissible to use hypochlorite with positive displacement

pumping. Anti-siphon valves shall be incorporated in the pump heads or in the discharge piping.
Capacity
The chlorinator shall have the capacity to dose enough chlorine to overcome the demand and
maintain the required concentration of the "free" or "combined" chlorine.
Methods of Control
Chlorine feed system shall be automatic proportional controlled, automatic residual controlled, or
compound loop controlled. In the automatic proportional controlled system, the equipment adjusts
the chlorine feed rate automatically in accordance with the flow changes to provide a constant pre-
established dosage for all rates of flow. In the automatic residual controlled system, the chlorine
feeder is used in conjunction with a chlorine residual analyzer which controls the feed rate of the
chlorine feeders to maintain a particular residual in the treated water.
In the compound loop control system, the feed rate of the chlorinator is controlled by a flow
proportional signal and a residual analyzer signal to maintain particular chlorine residual in the
water.
A manual chlorine feed system may be installed for groundwater systems with constant flow rates.
Standby Provision
As a safeguard against malfunction and/or shut-down,
standby chlorination equipment having the capacity to
replace the largest unit shall be provided. For uninterrupted
chlorination, gas chlorinators shall be equipped with an
automatic changeover system. In addition, spare parts shall
be available for all chlorinators.
Weigh Scales
Scales for weighing cylinders shall be provided at all plants
using chlorine gas to permit an accurate reading of total daily weight of chlorine used. At large
plants, scales of the recording and indicating type are recommended. As a minimum, a platform
scale shall be provided. Scales shall be of corrosion-resistant material.

Securing Cylinders
All chlorine cylinders shall be securely positioned to safeguard against movement. Tag the cylinder
“empty” and store upright and chained.
Ton containers may not be stacked.
Chlorine Leak Detection

Automatic chlorine leak detection and related alarm equipment shall be installed at all water
treatment plants using chlorine gas. Leak detection shall be provided for the chlorine rooms.
Chlorine leak detection equipment should be connected to a remote audible and visual alarm
system and checked on a regular basis to verify proper operation.
Leak detection equipment shall not

automatically activate the chlorine
room ventilation system in such a
manner as to discharge chlorine gas.
During an emergency, if the chlorine
room is unoccupied, the chlorine gas
leakage shall be contained within the
chlorine room itself in order to facilitate
a proper method of clean-up.
Consideration should also be given to

the provision of caustic soda solution
reaction tanks for absorbing the
contents of leaking one-ton cylinders
where such cylinders are in use.
Chlorine leak detection equipment

may not be required for very small
chlorine rooms with an exterior door (e.g., floor area less than 3m2).
You can use a spray solution of Ammonia or a rag soaked with Ammonia to detect a small Cl2
leak. If there is a leak, the ammonia will create a white colored smoke, Ammonium Chloride.
Safety Equipment
The facility shall be provided with personnel safety equipment including the following: Respiratory
equipment; safety shower, eyewash; gloves; eye protection; protective clothing; cylinder and/or
ton repair kits.
Respiratory equipment shall be provided which has been approved under the Occupational Health
and Safety Act, General Safety Regulation - Selection of Respiratory Protective Equipment.
Equipment shall be in close proximity to the access door(s) of the chlorine room.
Chlorine Room Design Requirements

Where gas chlorination is practiced, the gas cylinders and/or the ton containers up to the vacuum
regulators shall be housed in a gas-tight, well illuminated, corrosion resistant and mechanically
ventilated enclosure. The chlorinator may or may not be located inside the chlorine room. The
chlorine room shall be located at the ground floor level.
Ventilation
Gas chlorine rooms shall have entirely separate exhaust ventilation systems capable of delivering
one (1) complete air change per minute during periods of chlorine room occupancy only. The air
outlet from the room shall be 150 mm above the floor and the point of discharge located to preclude
contamination of air inlets to buildings or areas used by people. The vents to the outside shall have
insect screens.

Air inlets should be louvered near the ceiling, the air being of such temperature as to not adversely
affect the chlorination equipment. Separate switches for fans and lights shall be outside the room
at all entrance or viewing points, and a clear wire-reinforced glass window shall be installed in such
a manner as to allow the operator to inspect from the outside of the room.
Heating
Chlorine rooms shall have separate heating systems, if a forced air system is used to heat the
building. The hot water heating system for the building will negate the need for a separate heating
system for the chlorine room. The heat should be controlled at approximately 15oC.
Cylinders or containers shall be protected to ensure that the chlorine maintains its gaseous state
when entering the chlorinator.
Access
All access to the chlorine room shall only be from the exterior of the building. Visual inspection of
the chlorination equipment from inside may be provided by the installation of glass window(s) in
the walls of the chlorine room. Windows should be at least 0.20 m2 in area, and be made of clear
wire reinforced glass. There should also be a 'panic bar' on the inside of the chlorine room door
for emergency exit.
Storage of Chlorine Cylinders

If necessary, a separate storage room may be provided to simply store the chlorine gas cylinders,
with no connection to the line. The chlorine cylinder storage room shall have access either to the
chlorine room or from the plant exterior, and arranged to prevent the uncontrolled release of spilled
gas.
The chlorine gas storage room shall have provision for ventilation at thirty air changes per hour.
Viewing glass windows and panic button on the inside of door should also be provided. In very
large facilities, entry into the chlorine rooms may be through a vestibule from outside.
Scrubbers
For facilities located within residential or densely populated areas, consideration shall be given to
provide scrubbers for the chlorine room.
Some WWT plants transfer and store chlorine from tankers to holding tanks as shown in
the above photograph.

Chlorine Demand
Chlorine combines with a wide variety of materials. These side reactions complicate the use of
chlorine for disinfecting purposes. Their demand for chlorine must be satisfied before chlorine
becomes available to accomplish disinfection. Amount of chlorine required to react on various
water impurities before a residual is obtained. Also, means the amount of chlorine required to
produce a free chlorine residual of 0.1 mg/l after a contact time of fifteen minutes as measured by
iodmetic method of a sample at a temperature of twenty degrees in conformance with Standard
methods.
Chlorine Questions and Answer Review
True or False. Even brief exposure to 1,000 ppm of Cl2 can be fatal. True
How does one determine the ambient temperature in a chlorine room? Use a regular thermometer
because ambient temperature is simply the air temperature of the room.
How is the effectiveness of disinfection determined? From the results of coliform testing.
How often should chlorine storage ventilation equipment be checked? Daily.

Alternate Disinfectants
Chloramine
Chloramine is a very weak disinfectant for Giardia and virus reduction; it is recommended that it be
used in conjunction with a stronger disinfectant. It is best utilized as a stable distribution system
disinfectant.
In the production of chloramines, the ammonia residuals in the finished water, when fed in excess
of stoichiometric amount needed, should be limited to inhibit growth of nitrifying bacteria.
Chlorine Dioxide
Chlorine dioxide may be used for taste and odor control, or as a pre-disinfectant. Total residual
oxidants (including chlorine dioxide and chlorite, but excluding chlorate) shall not exceed 0.30 mg/L
during normal operation or 0.50 mg/L (including chlorine dioxide, chlorite and chlorate) during
periods of extreme variations in the raw water supply.
Chlorine dioxide provides good Giardia and virus protection but its use is limited by the restriction
on the maximum residual of 0.5 mg/L ClO2/chlorite/chlorate allowed in finished water. This limits
usable residuals of chlorine dioxide at the end of a process unit to less than 0.5 mg/L.
Where chlorine dioxide is approved for use as an oxidant, the preferred method of generation is to
entrain chlorine gas into a packed reaction chamber with a 25% aqueous solution of sodium chlorite
(NaClO2).
Warning: Dry sodium chlorite is explosive and can cause fires in feed equipment if leaking solutions
or spills are allowed to dry out.
Ozone
Ozone is a very effective disinfectant for both Giardia and viruses. Ozone CT (Contact Time) values
must be determined for the ozone basin alone; an accurate T10 value must be obtained for the
contact chamber, residual levels measured through the chamber and an average ozone residual
calculated.
Ozone does not provide a system residual and should be used as a primary disinfectant only in
conjunction with free and/or combined chlorine.
Ozone does not produce chlorinated byproducts (such as trihalomethanes) but it may cause an
increase in such byproduct formation if it is fed ahead of free chlorine; ozone may also produce its
own oxygenated byproducts such as aldehydes, ketones, or carboxylic acids. Any installed
ozonation system must include adequate ozone leak detection alarm systems, and an ozone off-
gas destruction system.
Ozone may also be used as an oxidant for removal of taste and odor, or may be applied as a pre-
disinfectant.

Ozone
Ozone (O3) is probably the strongest oxidizing agent available for water treatment. Although
it is widely used throughout the world, it has not found much application in the United States.
Ozone is obtained by passing a flow of air or oxygen between two electrodes that are
subjected to an alternating current in the order of 10,000 to 20,000 volts.
3O2 + electrical discharge → 2O3
Liquid ozone is very unstable and can readily explode. As a result, it is not shipped and must
be manufactured on-site. Ozone is a light blue gas at room temperature.
It has a self-policing pungent odor similar to that sometimes noticed during and after heavy
electrical storms. In use, ozone breaks down into oxygen and nascent oxygen.
O 3 → O2 + O
It is the nascent oxygen that produces the high oxidation and disinfections, and even
sterilization. Each water has its own ozone demand, in the order of 0.5 ppm to 5.0 ppm.
Contact time, temperature, and pH of the water are factors to be determined.
Ozone acts as a complete disinfectant. It is an excellent aid to the flocculation and

coagulation process, and will remove practically all color, taste, odor, iron, and manganese.
It does not form chloramines or THMs, and while it may destroy some THMs, it may produce
others when followed by chlorination. Ozone is not practical for complete removal of chlorine
or chloramines, or of THM and other inorganics. Further, because of the possibility of
formation of other carcinogens (such as aldehydes or phthalates) it falls into the same
category as other disinfectants in that it can produce DBPs.
Ozone generator

Ultraviolet Radiation
The enormous temperatures on the sun create ultraviolet (UV) rays in great amounts, and
this radiation is so powerful that all life on earth would be destroyed if these ray were not
scattered by the atmosphere and filtered out by the layers of ozone gas that float some 20
miles above the earth.
This radiation can be artificially produced by

sending strong electric currents through various
substances. A sun lamp, for example, sends out
UV rays that, when properly controlled, result in a
suntan. Of course, too much UV will cause
sunburn.
Open Channel UV Lamp

The UV lamp that can be used for the disinfection of water depends upon the low-pressure
mercury vapor lamp to produce the ultraviolet energy. A mercury vapor lamp is one in
which an electric arc is passed through an inert gas. This in turn will vaporize the mercury
contained in the lamp; and it is a result of this vaporization that UV rays are produced.
Enclosed UV lamp assembly. Assemblies will often need frequent cleaning and
bulb replacements, there are facilities with 1,000’s of bulbs.
The lamp itself does not come into with contact water, the lamp is placed inside a quartz
tube, and the water is in contact with the outside of the quartz tube. Quartz is used in this
case since practically none of the UV rays are absorbed by the quartz, allowing all of the rays
to reach the water. Ordinary glass cannot be used since it will absorb the UV rays, leaving
little for disinfection.

The water flows around the quartz tube. The UV sterilizer will consist of a various number of lamps
and tubes, depending upon the quantity of water to be treated. As water enters the sterilizer, it is
given a tangential flow pattern so that the water spins over and around the quartz sleeves. In this
way the microorganisms spend maximum time and contact with the outside of the quartz tube and
the source of the UV rays.
The basic design flow of water of certain UV units is in the order of 2.0 gpm for each inch of the
lamp. Further, the units are designed so that the contact or retention time of the water in the unit is
not less than 15 seconds. Most manufacturers claim that the UV lamps have a life of about 7,500
hours, which is about 1 year’s time. The lamp must be replaced when it loses about 40% to 50%
of its UV output; in any installation this is determined by means of a photoelectric cell and a meter
that shows the output of the lamp. Each lamp is outfitted with its own photoelectric cell, and with its
own alarm that will be activated when the penetration drops to a present level.
Ultraviolet radiation is an excellent disinfectant that is highly effective against viruses, molds, and
yeasts; and it is safe to use. It adds no chemicals to the water, it leaves no residual, and it does not
form THMs. It is used to remove traces of ozone and chloramines from the finished water. Alone,
UV radiation will not remove precursors, but in combination with ozone, it is said to be effective in
the removal of THM precursors and THMs.
The germicidal effect of UV is thought to be associated with its absorption by various organic
components essential to the cell’s functioning. For effective use of ultraviolet, the water to be
disinfected must be clean, and free of any suspended solids. The water must also be colorless and
must be free of any colloids, iron, manganese, taste, and odor.

These are conditions that must be met. Also, although a water may appear to be clear, such
substances as excesses of chlorides, bicarbonates, and sulfates affect absorption of the ultraviolet
ray.
These parameters will probably require at least filtration of one type or another. The UV
manufacturer will of course stipulate which pretreatment may be necessary.
Removal of Disinfection By-products
Disinfectant By- Disinfectant By-product

Disinfectant
product Removal
Chlorine (HOCl) Trihalomethane Granular Activated Carbon (GAC),
(THM) resins, controlled coagulation,
aeration.
Chloramines GAC-UV
Chloroprene GAC
Chloramines (Chicly) Probably no THM GAC
Others? UV?
Chlorine dioxide Chlorites Use of Fe2+ in coagulation, RO,
(ClO2) Chlorates ion-exchange
Permanganate No THMs
(KMnO4)
Ozone (O3) Aldehydes, GAC
Carboxylics,
Phthalates
Ultraviolet (UV) None known GAC
The table indicates that most of the disinfectants will leave a by-product that is or would possibly be
inimical to health. This may aid with a decision as to whether or not precursors should be removed
before these disinfectants are added to water.
If it is decided that removal of precursors is needed, research to date indicates that this removal can
be attained through the application of controlled chlorination plus coagulation and filtration, aeration,
reverse osmosis, nanofiltration, GAC (Granular Activated Charcoal) or combinations of others
processes.

A true on-line, amperometric, chlorine residual analyzer requires a pH buffer to bring the
sample pH down to a range where optimum free chlorine residuals can be accurately
measured, ideally 4.0 to 4.5 pH. Any amperometric chlorine residual analyzer that claims
buffers are not required uses either a pH buffered electrolyte in the probe, or makes an
electronically simulated pH compensation (which is not a true chlorine residual reading).
The vinegar reduces the pH in the sampling cell, which provides the current potential
needed to measure chlorine residuals accurately.
According to the journal Nature, chlorine atoms can affect nitrogen oxides and ozone
production, reducing the life cycle of methane gas. When exposed to the atmosphere,
chlorine atoms can deplete the ozone. This reduces the ozone's ability to block ultraviolet
rays, which can contribute to skin cancer in humans. It can also contribute to the
greenhouse effect.

CT and Log Inactivation Calculation Overview
This reference takes you step by step through the CT and log inactivation calculation procedure,
through an example calculation, and presents the disinfection segment concept.
“CT” (minutes•mg/L) in the context of water treatment is defined as the product of: C, for “residual
disinfectant concentration” in mg/L (determined before or at the first customer) and T, for the
corresponding “disinfectant contact time” in minutes. CT is a measure of the disinfection process
reaction time, but CT is only one of several variables that control the effectiveness of the disinfection
process.
CTCALC = Concentration Time, Calculated Value (minutes•mg/L)
C = Residual disinfectant concentration measured during peak flow (mg/L)
T = Actual Detention Time (minutes)
CTCALC = C × T
TDT = Theoretical Detention Time (minutes)
V = Volume, based on low water level (gallons)
Q = Peak hourly flow (gpm)
TDT = V/Q
Volume Equations:
Cylindrical: π x r2 x d Pipeline: π x r2 x l
Rectangular: l x w x d
d = minimum water depth
π = 3.1416
Disinfection Segments
Total inactivation = Σ log inactivation from each disinfection segment
Disinfection Profile
Almost all community and non-transient, non-community public water systems that use Surface
Water or Ground Water Under the Direct Influence of Surface Water sources are required to
develop a disinfection profile. Systems are required to retain the disinfection profile in graphic form
and it must be available for review by the state as part of a sanitary survey.
Disinfection Profile and Benchmark

• A disinfection profile is a graphical representation of a system’s level of Giardia lamblia or virus
inactivation measured, at least weekly, during the course of a year.
• A benchmark is the lowest monthly average microbial inactivation during the disinfection profile
time period.
The EPA has developed a disinfection profile spreadsheet calculator that calculates and graphs the
disinfection profile for Giardia and viruses. The spreadsheet can be downloaded from:
http://www.epa.gov/safewater/mdbp/lt1eswtr.html.

Understanding Chlorine Demand
The amount of chlorine used by reactions with substances that oxidize in the water before chlorine
residual can be measured. It is the difference between the amount of chlorine added to wastewater
and the amount of chlorine residual remaining after a given contact time. Chlorine demand may
change with dosage, time, temperature, pH, and the type and amount of pollutants in the water.
The presence of chlorine residual in drinking water indicates that: 1) a sufficient amount of chlorine
was added initially to the water to inactivate the bacteria and some viruses that cause diarrheal
disease; and, 2) the water is protected from recontamination during storage. The presence of free
residual chlorine in drinking water is correlated with the absence of disease-causing organisms, and
thus is a measure of the potability of water.
While chlorine's most important attributes are its broad-spectrum germicidal potency and persistence
in water distribution systems, its ability to efficiently and economically address many other water
treatment concerns has also supported its wide use. Chlorine-based compounds are the only major
disinfectants exhibiting lasting residual properties. Residual protection guards against microbial
regrowth and prevents contamination of the water as it moves from the treatment plant to household
taps.
Definitions
When chlorine is added to water, some of the chlorine reacts first with organic materials and metals
in the water and is not available for disinfection (this is called the chlorine demand of the water). The
remaining chlorine concentration after the chlorine demand is accounted for is called total chlorine.
Total chlorine is further divided into: 1) the amount of chlorine that has reacted with nitrates and is
unavailable for disinfection which is called combined chlorine and, 2) the free chlorine, which is the
chlorine available to inactivate disease-causing organisms, and thus a measure to determine the
potability of water.
For example, if using completing clean water, the chlorine demand will be zero, and there will be no
nitrates present, so no combined chlorine will be present. Thus, the free chlorine concentration will
be equal to the concentration of chlorine initially added. In natural waters, especially surface water
supplies such as rivers, organic material will exert a chlorine demand, and nitrates will form combined
chlorine. Thus, the free chlorine concentration will be less than the concentration of chlorine initially
added.
Chlorine Dose, Demand, and Residual

Most water treatment plants are required to disinfect the water, a process used to kill harmful
bacteria. The most frequently used method of disinfection is the addition of chlorine. Here, we will
briefly introduce three terms used during chlorination - chlorine dose, chlorine demand, and chlorine
residual. These three characteristics are related to each other using the following equation:
(Chlorine demand) = (Chlorine dose) - (Chlorine residual)
The amount of chlorine added to the water is known as the chlorine dose. This is a measured quantity
chosen by the operator and introduced into the water using a chlorinator or hypochlorinator.
As the chlorine reacts with bacteria and chemicals in the water, some of the chlorine is used up. The
amount of chlorine used up by reacting with substances in the water is known as the chlorine
demand. If nothing reacts with the chlorine (as would be the case in distilled water), then the chlorine
demand is zero. However, in most cases the operator should count on some of the chlorine dose
being used up when it reacts with substances in the water.
The amount of chlorine remaining in the water after some of the chlorine reacts with substances in
the water is known as the chlorine residual. This lab introduces a test which can be used to calculate
the chlorine residual. The chlorine residual is the most important of these three values - dose,
demand, and residual - because it represents the actual amount of chlorine remaining in the water
to act as a disinfectant.
The test for chlorine residual is performed frequently at most water treatment plants. Since
regulations require a certain level of chlorine in water at the far ends of the distribution system,
operators should be sure to test the chlorine residual in the distribution system as well as in the clear
well.
Understanding Combined Chlorine Residual

The residual consisting of chlorine that is combined with ammonia, nitrogen, or nitrogenous
compounds (chloramines).
Understanding Free Available Chlorine

The residual consisting of hypochlorite ions (OCl-), hypochlorous acid (HOCl) or a combination of
the two. These are the most effective in killing bacteria.
Total Combined Chlorine Residual

The total amount of chlorine present in a sample. This is the sum of the free chlorine residual and
the combined available chlorine residual.
Understanding Pre-Chlorination
Chlorination is the application of chlorine to water to accomplish some definite purpose. In this
lesson, we will be concerned with the application of chlorine for the purpose of disinfection, but you
should be aware that chlorination can also be used for taste and odor control, iron and manganese
removal, and to remove some gases such as ammonia and hydrogen sulfide. Chlorination is currently
the most frequently used form of disinfection in the water treatment field. However, other disinfection
processes have been developed. These alternatives will be discussed at the end of this lesson.
Pre-Chlorination and Post-Chlorination

Like several other water treatment processes, chlorination can be used as a pretreatment process
(prechlorination) or as part of the primary treatment of water (postchlorination). Treatment usually
involves either postchlorination only or a combination of prechlorination and postchlorination.
Pre-chlorination is the act of adding chlorine to the raw water. The residual chlorine is useful in
several stages of the treatment process - aiding in coagulation, controlling algae problems in basins,
reducing odor problems, and controlling mudball formation. In addition, the chlorine has a much
longer contact time when added at the beginning of the treatment process, so prechlorination
increases safety in disinfecting heavily contaminated water.
Post-chlorination is the application of chlorine after water has been treated but before the water
reaches the distribution system. At this stage, chlorination is meant to kill pathogens and to provide
a chlorine residual in the distribution system. Post-chlorination is nearly always part of the treatment
process, either used in combination with prechlorination or used as the sole disinfection process.
Until the middle of the 1970s, water treatment plants typically used both prechlorination and post-
chlorination. However, the longer contact time provided by prechlorination allows the chlorine to react
with the organics in the water and produce carcinogenic substances known as trihalomethanes. As
a result of concerns over trihalomethanes, prechlorination has become much less common in the
United States. Currently, prechlorination is only used in plants where trihalomethane formation is not
a problem.
Understanding Breakpoint Chlorination

Addition of chlorine to water until the chlorine demand has been satisfied. Since ammonia is present
in all domestic wastewaters, the reaction of ammonia with chlorine is a great significance. When
chlorine is added to waters containing ammonia, the ammonia reacts with hypochlorous acid (HOCl)
to form monochloramine, dichloramine and trichloramine. The formation of these chloramines
depends on the pH of the solution and the initial chlorine-ammonia ratio.

Chlor-Alkali Membrane Process
The chloralkali process (also chlor-alkali and chlor alkali) is an industrial process for the electrolysis
of sodium chloride solution (brine). Depending on the method, several products besides hydrogen
can be produced. If the products are separated, chlorine and sodium hydroxide (caustic soda) are
the products; by mixing, sodium hypochlorite or sodium chlorate are produced, depending on the
temperature. Higher temperatures are needed for the production of sodium chlorate instead of
sodium hypochlorite. Industrial scale production began in 1892. When using calcium chloride or
potassium chloride, the products contain calcium or potassium instead of sodium.
The process has a high energy consumption, for example over 4 billion kWh per year in West
Germany in 1985, and produces equal (molar) amounts of chlorine and sodium hydroxide, which
makes it necessary to find a use for the product for which there is less demand, usually the chlorine.
There are three production methods in use. While the mercury cell method produces chlorine-free
sodium hydroxide, the use of several tons of mercury leads to serious environmental problems. In a
normal production cycle a few hundred pounds of mercury per year are emitted, which accumulate
in the environment. Additionally, the chlorine and sodium hydroxide produced via the mercury-cell
chloralkali process are themselves contaminated with trace amounts of mercury. The membrane and
diaphragm method use no mercury, but the sodium hydroxide contains chlorine, which must be
removed.
Understanding Chlorine’s Effectiveness

In 1881, German bacteriologist Robert Koch demonstrated under controlled laboratory conditions
that pure cultures of bacteria could be destroyed by hypochlorite (bleach). The bulk of chlorine
disinfection research, which was conducted from the 1940s to the 1970s with a focus on bacteria,
provided observations as to how chlorine kills the microorganism. The observations that (1) bacterial
cells dosed with chlorine release nucleic acids, proteins and potassium and (2) membrane functions
such as respiration and active transport are affected more by chlorine than are cytoplasmic
processes, directed researchers’ attention to the surface of the bacterial cell. The hypothesis was
that the bacterial cell wall, under environmental stress, could interact with chlorine.
Chlorine exposure appears to cause physical, chemical, and biochemical alterations to the cell wall,
thus destroying the cell’s protective barrier, terminating vital functions, resulting in death of the
microorganism. A possible sequence of events during chlorination would be: (1) disruption of the cell
wall barrier by reactions of chlorine with target sites at the cell surface, (2) release of vital cellular
constituents from the cell, (3) termination of membrane-associated functions, and (4) termination of
cellular functions within the cell. During the course of this sequence of events, the microorganism
dies, meaning it is no longer capable of growing or causing disease.
Understanding Chlorine Solubility Effects

Chlorine is only slightly soluble in water; its maximum solubility is approximately one percent at 49°
C. At temperatures below this point it combines with water to form chlorine ice, a crystalline
substance. When the water supply to a gas chlorinator is below normal room temperature, it may
cool the chlorine gas to the point at which chlorine ice is formed and accumulates on the needle
valve and gas outlet tube, resulting in erratic feed results. Because the vapor pressure of chlorine
increases with rising temperatures, its solubility also decreases. At 212° F. chlorine is insoluble in
water.
Chlorine dissolved in water forms a weak corrosive mixture of hydrochloric and hypochlorous acid.
The corrosivity of chlorine solutions in water creates problems in handling chlorine spills and chlorine
containers.
Chlorine reacts with many compounds. Because of its great affinity for hydrogen, it removes
hydrogen from some compounds, such as hydrogen sulfide. It also reacts with ammonia or other
nitrogen-containing compounds to form various mixtures of chloramines. It reacts with organic
materials, sometimes with explosive violence.

Chemicals like chlorine, bromine, and ozone are examples of oxidizers. It is their ability to oxidize or
steal electrons from other substances that makes them good water sanitizers. As soon as the
oxidizing agent is added to the water, it begins to combine with microorganisms like bacteria, algae,
and whatever else the water may contain.
Now the free and available oxidizer is combining with contaminants and its effectiveness is reduced
according to how much combining took place. Although the hydrogen ion does not play a direct
reduction role on copper surfaces, pH can influence copper corrosion by altering the equilibrium
potential of the oxygen reduction half-reaction and by changing the speciation of copper in solution
(Reiber, 1989). Copper corrosion increases rapidly as the pH drops below 6; in addition, uniform
corrosion rates can be high at low pH values (below about pH 7), causing metal thinning. At higher
pH values (above about pH 8), copper corrosion problems are almost always associated with non-
uniform or pitting corrosion processes (Edwards et al., 1994a; Ferguson et al., 1996). Edwards et al.
(1994b) found that for new copper surfaces exposed to simple solutions that contained bicarbonate,
chloride, nitrate, perchlorate or sulphate, increasing the pH from 5.5 to 7.0 roughly halved corrosion
rates, but further increases in pH yielded only subtle changes.
The prediction of copper levels in drinking water relies on the solubility and physical properties of the
cupric oxide, hydroxide and basic carbonate solids that comprise most scales in copper water
systems (Schock et al., 1995). In the cupric hydroxide model of Schock et al. (1995), a decrease in
copper solubility with higher pH is evident. Above a pH of approximately 9.5, an upturn in solubility
is predicted, caused by carbonate and hydroxide complexes increasing the solubility of cupric
hydroxide. Examination of experience from 361 utilities reporting copper levels under the U.S. EPA
Lead and Copper Rule revealed that the average 90th-percentile copper levels were highest in
waters with pH below 7.4 and that no utilities with pH above 7.8 exceeded the U.S. EPA's action
level for copper of 1.3 mg/L (Dodrill and Edwards, 1995). However, problems associated with copper
solubility were also found to persist up to about pH 7.9 in cold, high-alkalinity and high-sulphate
groundwater (Edwards et al., 1994a).
In the pH range of 7-9, both the corrosion rate and the degree of tuberculation of iron distribution
systems generally increase with increasing pH (Larson and Skold, 1958; Stumm, 1960; Hatch, 1969;
Pisigan and Singley, 1987). Iron levels, however, were usually reported to decrease with increasing
pH (Karalekas et al., 1983; Kashinkunti et al., 1999; Broo et al., 2001; Sarin et al., 2003). In a pipe
loop system constructed from 90- to100-year-old unlined cast iron pipes taken from a Boston
distribution system, iron concentrations were found to steadily decrease when the pH was raised
from 7.6 to 9.5 (Sarin et al., 2003). Similarly, when iron was measured in the distribution system
following a pH increase from 6.7 to 8.5, a consistent downward trend in iron concentrations was
found over 2 years (Karalekas et al., 1983). These observations are consistent with the fact that the
solubility of iron-based corrosion by-products decreases with increasing pH.
Water with low pH, low alkalinity and low calcium is particularly aggressive towards cement materials.
The water quality problems that may occur are linked to the chemistry of the cement. Lime from the
cement releases calcium ions and hydroxyl ions into the drinking water. This, in turn, may result in a
substantial pH increase, depending on the buffering capacity of the water (Leroy et al., 1996). Pilot-
scale tests were conducted to simulate low-flow conditions of newly lined cement mortar pipes
carrying low-alkalinity water (Douglas et al., 1996). In the water with an initial pH of 7.2, alkalinity of
14 mg/L as calcium carbonate and calcium at 13 mg/L as calcium carbonate, measures of pH as
high as 12.5 were found.
Similarly, in the water with an initial pH of 7.8, alkalinity of 71 mg/L as calcium carbonate and calcium
at 39 mg/L as calcium carbonate, measures of pH as high as 12 were found.

Understanding Amperometric Titration
It appears that DPD colorimetric determination and amperometric titration as described in Standard
Methods are the procedures most commonly used for routine measurement of total chlorine. Few
studies have been conducted to evaluate these or other total residual chlorine measurement
techniques. Bender5 studied approximately 10 test procedures and found that results using the DPD
colorimetric procedure were consistently higher than those using amperometric titration. Brooks and
Seegert6 described an amperometric titration procedure employing a recording polargraph and
microburette, which was reported to be accurate and free from interference. The reliability of the DPD
colorimetric method for free chlorine has been increasingly questioned in recent years. The suitability
of that procedure for accurate total chlorine determinations appears to the authors to be
questionable, as well. Amperometric titration as described in Standard Methods cannot be used to
measure total chlorine concentrations less than about 0.05 mg/L, which is at least an order of
magnitude greater than levels of concern in natural waters for potential toxicity to aquatic organisms.
A reliable, simple procedure for low-level total chlorine determinations is clearly needed.
Analytical Procedure
Section 409C of Standard Methods includes a General Discussion section on amperometric titration
for the determination of chlorine in aqueous solutions. That discussion is applicable to the procedure
used by the authors. Also included in Standard Methods is a section concerning the titration
apparatus. Basically, the titration equipment consists of a buret capable of accurately delivering 0.01
mL of titrant, a sample cup, and a stirring device in which is housed a platinum electrode and a KCl
reference electrode. Several companies manufacture amperometric titrators that fit this general
description. The experience of the senior author is that some of the commercial titrators are less
suitable than others, primarily because of the small surface area of some of the electrodes employed.
A Wallace and Tiernan amperometric titrator was used by the authors in developing and applying
the procedure described below.
Reagents
a. Chlorine-free water. Only distilled or demineralized water that is free of chlorine should be used in
preparing reagents. Chlorine-free water may be prepared by passing distilled or demineralized water
through a suitable activated carbon filter adsorption column. The water may be tested for the
presence of chlorine by titrating a sample as described in the Procedure section. Any deflection in
the meter upon the addition of PAO titrant indicates the presence of chlorine or other oxidants that
would interfere in the titration procedure.
b. Standard phenylarsine oxide (PAO), 0.00564 N. See Standard Methods Section 409B, paragraph
3a.
Standardization – Dilute 50.00 mL of freshly prepared 0.0002256 N potassium biniodate to 200 mL
in chlorine-free water. Add approximately 1.5 g KI and stir to dissolve. Add 1 mL acetate buffer and
allow to stand in the dark for 6 minutes. Titrate using the amperometric titrator and determine the
equivalence point as detailed in the Procedure section. If the standard PAO is 0.00564 N, exactly
2.00 mL of PAO will be required to reach the equivalence point.
c. Phenylarsine oxide titrant, 0.000564 N. Dilute 10.00 mL of 0.00564 N PAO to 100.0 mL in chlorine-
free water.
Standardization – Dilute 5.00 mL of 0.0002256 N potassium biniodate to 200 mL with chlorine-free
water. Add approximately 1.5 g KI and stir to dissolve. Add 1 mL acetate buffer and allow to stand in
the dark for 6 minutes. Titrate using the amperometric titrator and determine the equivalence point
as detailed in the Procedure section below. If the PAO titrant is 0.000564 N, exactly 2.00 mL of PAO
will be required to reach the equivalence point.
d. Potassium biniodate, 0.0002256 N. Dissolve 0.7332 g reagent grade KH(IO3)2 in 500 mL chlorine-
free water and dilute to 1.00 L. Dilute 10.00 mL of that solution to 100.0 mL with chlorine-free water.
That solution is used for the standardization of the PAO and should be freshly prepared.
e. Acetate buffer solution, pH 4. See Standard Methods1 Section 409B, paragraph 3e.
f. Potassium iodide, (KI), reagent grade crystals.

Procedure
a. Titrant selection. Normally a 200-mL sample is used in titration. Each 0.1 mL of 0.000564 N PAO
corresponds to 0.01 mg/L in a 200-mL sample. The titrant normality should be selected such that no
more than about 4 mL of titrant will be required to reach the equivalence point. Thus, if the chlorine
concentration in the majority of the samples to be titrated is less than about 0.4 mg/L, use 0.000564
N PAO as the titrant. If only samples containing chlorine concentrations in excess of 0.4 mg/L are to
be analyzed, use 0.00564 N PAO as the titrant. If samples containing concentrations of chlorine in
excess of about 0.4 mg/L are to be titrated only occasionally and the volume of 0.000564 N PAO
required for titration is found to be excessive, a suitable subsample may be used and diluted to 200
mL with chlorine-free water.
b. Titration procedure (total residual chlorine). Prior to beginning the titration, rinse the buret with
PAO titrant by filling it completely and allowing the titrant to run into an empty sample cup. Repeating
this operation three or four times will ensure that the correct titrant concentration reaches the sample
cup. Remove the sample cup and rinse with distilled water and with the sample to be titrated. Add
200 mL of the sample to the sample cup. Add approximately 1.5 g (± 0.2 g) crystalline KI and allow
to dissolve, using the agitator on the titrator for mixing.
The exact amount of KI added is not critical, but the analyst should weigh 1.5 g of this reagent
periodically to become familiar with the approximate amount required. Add 1 mL of acetate buffer
and allow the microammeter on the titrator to reach a stable reading; the titration should be started
within about 30 seconds following the addition of the KI to the sample.
Full-scale deflection on the microammeter is 100 units. The meter should be initially adjusted to read
between 90 and 100 units. Record the initial reading prior to the addition of titrant. Titrate by adding
suitable volumes of titrant and recording the titrant volume added and the resultant current reading.
At least three (and preferably five to ten) readings of current and titrant volume added should be
obtained prior to passing the equivalence point; then add excess titrant to ensure that there is no
further meter deflection. Record the final meter reading. If, during the titration, the meter reading falls
to near or below 10 units, record the low reading, re-adjust the meter to read between 90 and 100
units, record the high reading, and continue the titration. This approach allows calculation of the total
meter deflection, which is used in determining the equivalence point.
The equivalence point is determined by plotting the total meter deflection as a function of titrant
volume added. It is important that the total meter deflection be used in preparing this plot. A straight
line is drawn through the first few points in the plot and a second straight line is drawn parallel to the
abscissa and corresponding to the final total deflection in the meter reading.
The equivalence point is determined by the intersection of those two lines. When 0.000564 N PAO
is used as the titrant, the chlorine concentration is 0.1-times the titrant volume at the equivalence
point. This plotting procedure is also outlined in the ASTM Water Manual8 under procedures ASTM
D1253 (Tests for Residual Chlorine in Water) and ASTM D1427 (Tests for Residual Chlorine in
Waste Water).
c. Sample storage and handling. Chlorine measurements should be made as soon after sample
collection as possible. Samples to be analyzed for chlorine should be stored in the dark and packed
on ice if they must be held for more than a few minutes before analysis. Chlorine compounds are
highly reactive and may be rapidly lost from samples due to the effects of volatilization,
phototransformation, and chlorine demand. Storage of samples on ice and in the dark between
sampling and analysis will help minimize the rate of dissipation. It is important to estimate the
changes that occur in chlorine content in the subject water between sample collection and analysis.

This can be accomplished by performing a “time-lag” test. To perform a time-lag test, a single large
(approximately 2-L) sample of the water being analyzed is collected. The chlorine concentration in
that sample is determined six to ten times over a period of one to three hours, depending on the
normal sample holding time. The measured concentrations are then plotted as a function of time,
normally on semilog paper. In most cases, the decrease in chlorine concentration over time can be
described by first-order reaction kinetics.
The original chlorine content in any sample can be computed given the measured concentration and
the holding time. A time-lag study should be performed on a regular basis for each type of water
being analyzed because of variability in water compositions. The sample set used for the study
should be handled in the same way as other samples (i.e., the samples should be kept cold and in
the dark). Even when time-lag studies are made a part of the routine analytical procedure, it is
important that the delay between sample collection and chlorine analysis be held to a minimum.
Sodium Hypochlorite
Sodium Hypochlorite, or bleach, is produced by adding elemental chlorine to sodium hydroxide.
Typically, hypochlorite solutions contain from 5 to 15% chlorine, and are shipped by truck in one- to
5,000- gallon containers.
Advantages
 Solution is less hazardous and easier to handle than elemental chlorine
 Fewer training requirements and regulations than elemental chlorine
Limitations
 Limited shelf-life
 Potential to add inorganic byproducts (chlorate, chlorite and bromate) to water
 Corrosive to some materials and more difficult to store than most solution chemicals
 Higher chemical costs than elemental chlorine
Calcium Hypochlorite
 Calcium hypochlorite is another chlorinating chemical used primarily in smaller applications.
It is a white, dry solid containing approximately 65% chlorine, and is commercially available
in granular and tablet forms.
Advantages
 More stable than sodium hypochlorite, allowing longer storage
 Fewer training requirements and regulations than elemental chlorine
Limitations
 Dry chemical requires more handling than sodium hypochlorite
 Precipitated solids formed in solution complicate chemical feeding
 Higher chemical costs than elemental chlorine
 Fire or explosive hazard if handled improperly
 Potential to add inorganic byproducts (chlorate, chlorite and bromate) to water

Onsite Hypochlorite Generation
In recent years some municipalities have installed on-site hypochlorite generators that produce weak
hypochlorite solutions (~0.8%) using an electrolytic cell and a solution of salt water.
Advantages
 Minimal chemical storage and transport
Limitations
 More complex and requires a higher level of maintenance and technical expertise
 High capital cost
 Operating costs are often higher than for commercial hypochlorite
 Requires careful control of salt quality
 Weak solution requires high volume chemical feed and control
 Byproducts in generated hypochlorite may be difficult to monitor and control
 System backup may be more difficult and costly

Ozone
Ozone (O3) is generated on-site at water treatment facilities by passing dry oxygen or air through a
system of high voltage electrodes. Ozone is one of the strongest oxidants and disinfectants available.
Its high reactivity and low solubility, however, make it difficult to apply and control. Contact chambers
are fully contained and non-absorbed ozone must be destroyed prior to release to avoid corrosive
and toxic conditions. Ozone is more often applied for oxidation rather than disinfection purposes.
Advantages
 Strongest oxidant/disinfectant available
 Produces no chlorinated THMs, HAAs
 Effective against Cryptosporidium at higher concentrations
 Used with Advanced Oxidation processes to oxidize refractory organic compounds
Limitations
 Process operation and maintenance requires a high level of technical competence
 Provides no protective residual
 Forms brominated byproducts (bromate, brominated organics)
 Forms nonhalogenated byproducts (ketenes, organic acids, aldehydes)
 Breaks down more complex organic matter; smaller compounds can enhance microbial re-
growth in distribution systems and increase DBP formation during secondary disinfection
processes.
 Higher operating and capital costs than chlorination
 Difficult to control and monitor particularly under variable load conditions
Ultraviolet Radiation
Ultraviolet (UV) radiation, generated by mercury arc lamps, is a non-chemical disinfectant. When UV
radiation penetrates the cell wall of an organism, it damages genetic material, and prevents the cell
from reproducing. Although it has a limited track record in drinking water applications, UV has been
shown to effectively inactivate many pathogens while forming limited disinfection byproducts.
Advantages
 Effective at inactivating most viruses, spores and cysts
 No chemical generation, storage, or handling
 Effective against Cryptosporidium
 No known byproducts at levels of concern
Limitations
 No residual protection
 Low inactivation of some viruses (reoviruses and rotaviruses)
 Difficult to monitor efficacy

 Irradiated organisms can sometimes repair and reverse the destructive effects of UV through
a process known as photo-reactivation
 May require additional treatment steps to maintain high-clarity water
 Does not provide oxidation, or taste and odor control
 High cost of adding backup/emergency capacity
 Mercury lamps may pose a potable water and environmental toxicity risk
Alternative Disinfectants
Up until the late 1970s, chlorine was virtually the only disinfectant used to treat drinking water.
Chlorine was considered an almost ideal disinfectant, based on its proven characteristics:
 Effective against most known pathogens
 Provides a residual to prevent microbial re-growth and protect treated water throughout the
distribution system
 Suitable for a broad range of water quality conditions
 Easily monitored and controlled

Understanding Commonly Used Water Disinfectants
Almost all U.S. systems that disinfect their water use some type of chlorine-based process, either
alone or in combination with other disinfectants. In addition to controlling disease-causing organisms,
chlorination offers a number of benefits including:
 Reduces many disagreeable tastes and odors;
 Eliminates slime bacteria, molds and algae that commonly grow in water supply reservoirs,
on the walls of water mains and in storage tanks;
 Removes chemical compounds that have unpleasant tastes and hinder disinfection; and
 Helps remove iron and manganese from raw water.
As importantly, only chlorine-based chemicals provide “residual disinfectant” levels that prevent
microbial re-growth and help protect treated water throughout the distribution system.
The Risks of Waterborne Disease

Where adequate water treatment is not readily available, the impact on public health can be
devastating. Worldwide, about 1.2 billion people lack access to safe drinking water, and twice that
many lack adequate sanitation. As a result, the World Health Organization estimates that 3.4 million
people, mostly children, die every year from water-related diseases.
Even where water treatment is widely practiced, constant vigilance is required to guard against
waterborne disease outbreaks. Well-known pathogens such as E. coli are easily controlled with
chlorination, but can cause deadly outbreaks given conditions of inadequate or no disinfection. A
striking example occurred in May 2000 in the Canadian town of Walkerton, Ontario. Seven people
died and more than 2,300 became ill after E. coli and other bacteria infected the town’s water supply.
A report published by the Ontario Ministry of the Attorney General concludes that, even after the well
was contaminated, the Walkerton disaster could have been prevented if the required chlorine
residuals had been maintained.
Some emerging pathogens such as Cryptosporidium are resistant to chlorination and can appear
even in high quality water supplies. Cryptosporidium was the cause of the largest reported drinking
water outbreak in U.S. history, affecting over 400,000 people in Milwaukee in April 1993. More than
100 deaths are attributed to this outbreak. New regulations from the U.S. Environmental Protection
Agency (EPA) will require water systems to monitor Cryptosporidium and adopt a range of treatment
options based on source water Cryptosporidium concentrations. Most water systems are expected
to meet EPA requirements while continuing to use chlorination.
The Benefits of Chlorine

Potent Germicide
Chlorine disinfectants can reduce the level of many disease-causing microorganisms in drinking
water to almost immeasurable levels. Chlorine is added to drinking water to destroy pathogenic
(disease-causing) organisms. It can be applied in several forms: elemental chlorine (chlorine gas),
sodium hypochlorite solution (bleach) and dry calcium hypochlorite.
When applied to water, each of these forms “free chlorine” (see Sidebar: How Chlorine Kills
Pathogens). One pound of elemental chlorine provides approximately as much free available chlorine
as one gallon of sodium hypochlorite (12.5% solution) or approximately 1.5 pounds of calcium
hypochlorite (65% strength). While any of these forms of chlorine can effectively disinfect drinking
water, each has distinct advantages and limitations for particular applications. Almost all water
systems that disinfect their water use some type of chlorine-based process, either alone or in
combination with other disinfectants.

Taste and Odor Control
Chlorine disinfectants reduce many disagreeable tastes and odors. Chlorine oxidizes many naturally
occurring substances such as foul-smelling algae secretions, sulfides and odors from decaying
vegetation.
Biological Growth Control

Chlorine disinfectants eliminate slime bacteria, molds and algae that commonly grow in water supply
reservoirs, on the walls of water mains and in storage tanks.
Chemical Control
Chlorine disinfectants destroy hydrogen sulfide (which has a rotten egg odor) and remove ammonia
and other nitrogenous compounds that have unpleasant tastes and hinder disinfection. They also
help to remove iron and manganese from raw water.
Water Treatment
Every day, approximately 170,000 (U.S. EPA, 2002) public water systems treat and convey billions
of gallons of water through approximately 880,000 miles (Kirmeyer, 1994) of distribution system
piping to U.S. homes, farms and businesses. Broadly speaking, water is treated to render it suitable
for human use and consumption. While the primary goal is to produce a biologically (disinfected) and
chemically safe product, other objectives also must be met, including: no objectionable taste or odor;
low levels of color and turbidity (cloudiness); and chemical stability (non-corrosive and non-scaling).
Individual facilities customize treatment to address the particular natural and manmade
contamination characteristic of their raw water.
Surface water usually presents a greater treatment challenge than groundwater, which is naturally
filtered as it percolates through sediments. Surface water is laden with organic and mineral
particulate matter, and may harbor protozoan parasites such as Cryptosporidium parvum and Giardia
lamblia.
Water Distribution
In storage and distribution, drinking water must be kept safe from microbial contamination.
Frequently, slippery films of bacteria, known as biofilms, develop on the inside walls of pipes and
storage containers. Among disinfection techniques, chlorination is unique in that a pre-determined
chlorine concentration may be designed to remain in treated water as a measure of protection against
harmful microbes encountered after leaving the treatment facility. In the event of a significant
intrusion of pathogens resulting, for example, from a broken water main, the level of the average
“chlorine residual” will be insufficient to disinfect contaminated water. In such cases, it is the
monitoring of the sudden drop in the chlorine residual that provides the critical indication to water
system operators that there is a source of contamination in the system.
The Challenge of Disinfection Byproducts

While protecting against microbial contamination is the top priority, water systems must also control
disinfection byproducts (DBPs), chemical compounds formed unintentionally when chlorine and
other disinfectants react with natural organic matter in water. In the early 1970s, EPA scientists first
determined that drinking water chlorination could form a group of byproducts known as
trihalomethanes (THMs), including chloroform. EPA set the first regulatory limits for THMs in 1979.
While the available evidence does not prove that DBPs in drinking water cause adverse health effects
in humans, high levels of these chemicals are certainly undesirable. Cost-effective methods to
reduce DBP formation are available and should be adopted where possible.
Chemical Safety (IPCS 2000) Strongly Cautions:

The health risks from these byproducts at the levels at which they occur in drinking water are
extremely small in comparison with the risks associated with inadequate disinfection. Thus, it is
important that disinfection not be compromised in attempting to control such byproducts. Recent EPA
regulations have further limited THMs and other DBPs in drinking water. Most water systems are

meeting these new standards by controlling the amount of natural organic material prior to
disinfection.
Chlorine and Water System Security

The prospect of a terrorist attack has forced all water systems, large and small, to re-evaluate and
upgrade existing security measures. Since September 11th, 2001, water system managers have
taken unprecedented steps to protect against possible attacks such as chemical or biological
contamination of the water supply, disruption of water treatment or distribution, and intentional
release of treatment chemicals.
With passage of the Public Health Security and Bioterrorism Response Act of 2002, Congress
required community water systems to assess their vulnerability to a terrorist attack and other
intentional acts. As part of these vulnerability assessments, systems assess the transportation,
storage and use of treatment chemicals. These chemicals are both critical assets (necessary for
delivering safe water) and potential vulnerabilities (may pose significant hazards, if released). Water
systems using elemental chlorine, in particular, must determine whether existing protection systems
are adequate. If not, they must consider additional measures to reduce the likelihood of an attack or
to mitigate the potential consequences.
Disinfection is crucial to water system security, providing the “front line” of defense against biological
contamination. However, conventional treatment barriers in no way guarantee safety from biological
attacks. Additional research and funding are needed to improve prevention, detection and responses
to potential threats.
The Future of Chlorine Disinfection

Despite a range of new challenges, drinking water chlorination will remain a cornerstone of
waterborne disease prevention. Chlorine’s wide array of benefits cannot be provided by any other
single disinfectant. While alternative disinfectants (including chlorine dioxide, ozone, and ultraviolet
radiation) are available, all disinfection methods have unique benefits, limitations, and costs. Water
system managers must consider these factors, and design a disinfection approach to match each
system’s characteristics and source water quality.

Understanding Giardia lamblia
Giardia lamblia, discovered approximately 20 years ago, is another emerging waterborne pathogen.
This parasitic microorganism can be transmitted to humans through drinking water that might
otherwise be considered pristine. In the past, remote water sources that were not affected by human
activity were thought to be pure, warranting minimal treatment. However, it is known now that all
warm-blooded animals may carry Giardia and that beaver are prime vectors for its transmission to
water supplies.
There is a distinct pattern to the emergence of new pathogens. First, there is a general recognition
of the effects of the pathogen in highly susceptible populations such as children, cancer patients and
the immunocompromised. Next, practitioners begin to recognize the disease and its causative agent
in their own patients, with varied accuracy. At this point, some may doubt the proposed agent is the
causative agent, or insist that the disease is restricted to certain types of patients. Finally, a single or
series of large outbreaks result in improved attention to preventive efforts. From the 1960’s to the
1980’s this sequence of events culminated in the recognition of Giardia lamblia as a cause of
gastroenteritis (Lindquist, 1999).
Understanding Waterborne Diseases

Detection and investigation of waterborne disease outbreaks is the primary responsibility of local,
state and territorial public health departments, with voluntary reporting to the CDC. The CDC and the
U.S. Environmental Protection Agency (EPA) collaborate to track waterborne disease outbreaks of
both microbial and chemical origins. Data on drinking water and recreational water outbreaks and
contamination events have been collected and summarized since 1971.
While useful, statistics derived from surveillance systems do not reflect the true incidence of
waterborne disease outbreaks because many people who fall ill from such diseases do not consult
medical professionals. For those who do seek medical attention, attending physicians and laboratory
and hospital personnel are required to report diagnosed cases of waterborne illness to state health
departments. Further reporting of these illness cases by state health departments to the CDC is
voluntary, and statistically more likely to occur for large outbreaks than small ones.
Despite these limitations, surveillance data may be used to evaluate the relative degrees of risk
associated with different types of source water and systems, problems in current technologies and
operating conditions, and the adequacy of current regulations. (Craun, Nwachuku, Calderon, and
Craun, 2002).

Laboratory Analysis
Samples need to be kept on ice and shipped to a central laboratory for analysis of coliphage, C.
perfringens, Cryptosporidium, Giardia, and enteric viruses by the current analytical methods. The
single-agar layer (SAL), direct plating method with induction of β-galactosidase (Ijzerman and
Hagedorn, 1992) is recommended for detection of somatic and F-specific coliphage in streamwater
samples. In this method, 100-mL sample volumes are mixed with an agar medium, E. coli host
culture, chemicals that induce the β-galactosidase enzyme, and appropriate antibiotics. The mixtures
are poured into four 150- x 15-mm plates and incubated at 35°C.
Upon infection by coliphage in the water sample, the E. coli host cells are lysed and stable indolyl
product that is dark blue is visible within each plaque. Viral plaques are easily identified and
enumerated by the distinct blue circle. Because of contamination by naturally occurring bacteria in
streamwater samples, antibiotic- resistant host-culture strains, E. coli CN-13 (resistant to nalidixic
acid) and E. coli F-amp (resistant to streptomycin and ampicillin) are used as hosts for somatic and
F-specific coliphage, respectively. Large sample volumes, such as 1-L volumes or greater, are
recommended for detection of coliphage in ground water. Because the SAL method is impractical for
sample volumes above 100 mL, an alternative method should be used for ground-water sample
analysis.
One example, currently being tested by USEPA, is a two-step enrichment presence-absence method
(U.S. Environmental Protection Agency, 1999e). Samples for enumeration of C. perfringens are
analyzed by use of the mCP agar method (U.S. Environmental Protection Agency, 1996c). Standard
MF techniques are used, and the plates are incubated anaerobically for 24 hours at 44.5°C. After
incubation, the plates are exposed to ammonium hydroxide, and all straw-colored colonies that turn
dark pink to magenta are counted as C. perfringens. In the laboratory, C. perfringens analyses are
done on 100-, 30-,and 10-mL volumes of streamwater. In the case of a high-flow or high-turbidity
streamwater sample, lower sample volumes may be plated.
Method 1623 (U.S. Environmental Protection Agency, 1999c) is recommended for detection of
Cryptosporidium oocysts and Giardia cysts in water. The oocysts are concentrated on a capsule filter
from a 10-L water sample, eluted from the capsule filter with buffer, and concentrated by
centrifugation. Immunomagnetic separation (IMS) is used to separate the oocysts from other
particulates in the sample. In IMS, the oocysts are magnetized by attachment of magnetic beads
conjugated to an antibody and then are separated from sediment and debris by means of a magnet.
Fluorescently labeled antibodies and vital dye are used to make the final microscopic identification
of oocysts and cysts. The reverse-transcriptase, polymerase chain reaction (RT-PCR) and cell-
culture methods are recommended for detection of enteric viruses in water samples (G. Shay Fout,
U.S. Environmental Protection Agency, written commun., 1997; U.S. Environmental Protection
Agency, 1996c). To prepare samples for RT-PCR and cell culture, attached viruses are eluted from
a 1MDS filter with beef extract (pH 9.5), concentrated using celite (pH 4.0), and eluted with sodium
phosphate (pH 9.5).
For RT-PCR analysis, viruses are isolated from the eluate by ultracentrifugation through a sucrose
gradient, and trace contaminants are removed by extraction with a solvent mixture. During these
steps, the 10-L streamwater sample (or 2,000-L ground-water sample) is concentrated down to 40
μL. An aliquot of the concentrate is used for RT-PCR, wherein any target viral RNA is converted to
DNA and amplified by use of an enzymatic process. The RT-PCR products are analyzed by agarose
gel electrophoresis and confirmed by hybridization. The enteric viruses detected by use of this
method include enterovirus, hepatitis-A, rotavirus, reovirus, and calicivirus.
For cell-culture analysis, the sample eluate is added to a monlayer of a continuous cell line derived
from African green monkey kidney cells (U.S. Environmental Protection Agency, 1996c). Each cell
culture is examined microscopically for the appearance of cytopathic effects (CPE) for a total of 14
days; if CPE

is not observed in 14 days, a second passage is done. Results are reported as most probable number
of infectious units per volume of water.
QA/QC Activities and Measures

QA/QC activities and measures to take to reduce contamination.
• Use a sterilization indicator, such as autoclave tape, in preparing sample bottles and other
equipment
for collection of microbiological samples to determine whether adequate temperatures and pressures
have been attained during autoclaving.
• Prepare a separate set of sterile equipment for microbiological sampling at each site.
• Before processing samples in the field vehicle, wipe down the area with a disinfectant (such as
isopropyl alcohol) to ensure a sterile working surface.
• Monitor the incubators daily to ensure temperatures are appropriate for the methods used.
For bacteria samples, membrane-filtration (MF) equipment and MF procedure blanks are used to
estimate analytical bias.
Field personnel should do the following:

• Prepare an MF equipment blank, a 50- to 100-mL aliquot of sterile buffered water plated before the
sample—for every sample by field personnel for total coliform, E. coli, and enterococci analyses to
determine the sterility of equipment and supplies.
• Prepare a MF procedure blank, a 50- to 100-mL aliquot of sterile buffered water plated after the
sample— for every fourth sample to measure the effectiveness of the analyst’s rinsing technique or
presence of incidental contamination of the buffered water.
If contamination from a MF equipment or procedure blank is found, results are suspect and are
qualified or not reported. Proper and consistent procedures for counting and identifying target
colonies will be followed, as described in Myers and Sylvester (1997).
• After counting, turn the plate 180° and ensure the second count is within 5 percent of the first count.
Have a second analyst check calculations of bacterial concentrations in water for errors.
For coliphage, Cryptosporidium, Giardia, and enteric virus samples, equipment and field blanks are
used to determine sampling and analytical bias. Equipment blanks for these analyses are different
from the MF equipment blanks for bacterial analysis. An equipment blank is a blank solution (sterile
buffered water) subjected to the same aspects of sample collection, processing, storage,
transportation, and laboratory handling as an environmental sample, but it is processed in an office
or laboratory. Field blanks are the same as equipment blanks except that they are generated under
actual field conditions.
• For enteric virus analysis, collect one equipment blank after collection of the first sample to ensure
that equipment cleaning and sterilization techniques are adequate.
• For coliphage, Cryptosporidium, Giardia, and enteric virus analyses, collect field blanks periodically.
At a minimum, the number of field blanks should equal 5 percent of the total number of samples
collected. Five percent of samples collected for bacterial and viral indicators (total coliforms, E. coli,
enterococci, C. perfringens, and coliphage) should be nested replicate samples to estimate sampling
and analytical variability. For streamwater samples, concurrent replicates to estimate sampling
variability are collected by alternating subsamples in each vertical between two collection bottles.
For ground-water samples, sequential replicates are collected one after another into separate sterile
bottles. Concurrent and sequential replicates are then analyzed in duplicate (split replicates) to
estimate analytical variability.
• Because of the expense associated with collection and analysis of samples for pathogens
(Cryptosporidium and enteric viruses), collect only one replicate sample per year at a site wherein
detection of pathogens was found in an earlier sample.
To assess analytical bias of the sampling and analytical method, 2 to 5 percent of the samples
collected for enteric virus should be field matrix spikes.
• Run all but 10 L of ground water through the 1 MDS filter and collect the remaining 10 L in a carboy.
In the laboratory, the poliovirus vaccine will be added to the 10 L and then passed through the same
1MDS filter. Analysis will be done by use of the cell-culture and RT-PCR methods. • All cell-culture
positive samples are serotyped to identify or discount laboratory contamination. Because of the
variability in the performance of Method 1623 for recovery of Cryptosporidium and Giardia, each
sample will be collected in duplicate—one will be a regular sample and the other a matrix spike. The
laboratory will add a known quantity of cysts and oocysts to the matrix spike to determine recovery
efficiency, as described in USEPA (1999c).
Quality Assurance and Quality Control in the Laboratory

The following criteria may be used to evaluate each production analytical laboratory: (1) appropriate,
approved, and published methods, (2) documented standard operating procedures, (3) approved
quality-assurance plan, (4) types and amount of quality-control data fully documented and technical
defensible, (5) participation in the standard reference sample project (6) scientific capability of
personnel, and (7) appropriate laboratory equipment.
The microbiology laboratories must follow good laboratory practices—cleanliness, safety practices,
procedures for media preparation, specifications for reagent water quality—as set forth by American
Public Health Association (1998) and Britton and Greeson (1989). Some specific guidelines are listed
in the following paragraphs.
Reference cultures are used by the central laboratory to evaluate the performance of the test
procedures, including media and reagents. Pure cultures of E. coli, Enterobacter aerogenes, and
Streptococcus faecalis (American Type Culture Collection, Rockville, Md.) are used to ensure that
MF culture media and buffered water are performing adequately. A pure culture of C. perfringens,
isolated from a sewage sample and verified by standard procedures, is used to evaluate the test
procedure and each lot of media and reagents.
Because contamination of samples from coliphage during the analytical procedure is highly probable
(Francy and others, 2000), a negative control of host and sterile buffered water is run concurrently
with each batch of samples. In addition, to ensure that the method is being executed properly, a
positive-control sewage sample is run with each batch of samples. A laminar flow safety hood is
recommended for processing the samples for coliphage analysis. Alternatively, a separate coliphage
room may be established to discourage laboratory contamination during the analytical process. An
ultraviolet light is installed and operated for 8 hours every night in the safety hood or coliphage room
to reduce contamination.
The laboratory should follow the QA/QC guidelines in Method 1623 (U.S. Environmental Protection
Agency, 1999c) for Cryptosporidium and Giardia and in the cell-culture and RT-PCR analysis for
enteric viruses (G. Shay Fout, U.S. Environmental Protection Agency, written commun., 1997; U.S.
Environmental Protection Agency, 1996c).

In chemistry and common usage, a filter is a device (usually a membrane or layer) that is
designed to physically block certain objects or substances while letting others through,
depending on their size. Filters are often used to remove solid substances suspended in
fluids, for example to remove air pollution, to make water drinkable, and to prepare coffee.
Some devices that are called filters may also carry out other processes, such as waste
treatment, (e.g. biofilter). Several types of filters are used in chemistry in order to facilitate
separation, thereby purifying a liquid (or gas). Many filters use gravity, or gravity enhanced
by vacuum (suction) in order to create this separation, often through a funnel-shaped
device.
Filter efficiency can be improved in a number of ways, such as with the use of fluted filter
paper. Other types of materials may be used to effect separations based on size, similar to
filters, such as molecular sieves.
The process of passing a mixture through a filter is called filtration. The liquid produced
after filtering a suspension of a solid in a liquid is called filtrate, while the solid remaining in
the filter is called retentate, residue, or filtrand.

Mycobacterium
The mycobacteria are a group of slow-growing organisms. The most important is Mycobacterium
tuberculosis, the causative organism of tuberculosis, which takes about 4–6 weeks to grow in the
diagnostic laboratory. M. tuberculosis is not a waterborne pathogen; there are, however, a number
of Mycobacterium species that occur in the environment and can cause disease in humans.
Mycobacterium avium and its related species cause an infection of cervical lymph nodes; it occurs
in the environment and is most probably accompanied by ingestion or inhalation. M. avium can grow
in water to which no additional nutrients have been added; although water treatment processes of
coagulation and filtration appear to reduce the numbers, it is not affected by chlorine levels of 1
mg/ml. It is therefore not surprising that these organisms can regrow and colonize domestic water
systems. Once ingested, M. avium can colonize the pharynx without causing any disease. The
number of cases reported was very low, but patients with HIV/AIDS are very susceptible.
Another species, Mycobacterium xenopi, has been reported as the waterborne cause of spinal
infections following a look-back exercise on over 3000 patients who had undergone discectomy
operations some years beforehand (Astagneau et al. 2001).
Mycobacterium paratuberculosis causes Johne’s disease in cattle. It is a chronic wasting disease

with considerable economic consequences. The organism is extremely difficult to culture; when it
does grow, it is very slow and dependent on an exogenous source of mycobactin, which is an iron
chelating agent produced by all other mycobacteria. Transmission is by either direct or indirect
contact with infected animals and occurs mainly through the faecal–oral route. Organisms are
ingested in large numbers by young animals when they feed in troughs that have been contaminated
by faeces of shedding animals
(Chiodini et al. 1984).
M. paratuberculosis has recently been suggested as a cause of Crohn’s disease, a non-specific

chronic transmural inflammatory disease of humans that affects the intestinal tract, commonly the
ileum. The disease is chronic, debilitating and of a relapsing nature; the symptoms experienced
include diarrhea with blood in the stools and abdominal pain. Complications include obstruction,
fistulation and abscesses. There have been many bacteria implicated over the years, but no definite
etiological agent has been found. It is thought that immunological mechanisms may play an important
role.
Molecular techniques have been developed for the diagnosis of M. paratuberculosis infections and
applied to human surgically resected tissues. M. paratuberculosis was detected in approximately
30% of samples, but the sets of results from different laboratories have been conflicting. Some
studies were unable to detect the organism; in other studies, the organism was detected in a smaller
percentage of healthy subjects. In addition, a few Crohn’s disease patients have shown clinical
remission when treated with anti-tuberculosis drugs. There is therefore much more work to be done
to acquire a better understanding. M. paratuberculosis may be present in surface water contaminated
by cattle faeces. Routine testing for indicator organisms would detect faecal pollution, and normal
water treatment processes of coagulation and filtration are likely to remove mycobacteria. It is
unlikely that drinking-water is a major source of M. paratuberculosis, and its association with Crohn’s
disease is still under investigation.
Burkholderia Pseudomallei
Burkholderia pseudomallei is the cause of melioidosis, an acute pneumonia often followed by
systemic infection with later presentations of abscesses. The organism is widespread in the
environment and was originally described in Rangoon in patients compromised by severe poverty
who had presumably inhaled the organism in dust when sleeping on the ground. It occurs commonly
in southeast Asia and has been detected in service personnel repatriated from those areas in the
past. It was also investigated as a biological weapon by several nations, to be released as an aerosol
and cause pneumonia infection in those exposed. A recent study in Bologna, Italy, detected B.
pseudomallei in 7% of 85 samples of drinking-water collected from public and private buildings.

The mean count was 578 cfu/100 ml. The occurrence of the organism was found to correlate with
the HPC at 22 and 36 °C (Zanetti et al. 2000).
Francisella Tularensis
Tularaemia is a zoonosis caused by a highly infective and virulent organism Francisella tularensis,
which occurs throughout the northern hemisphere but has never been isolated within the United
Kingdom. It occurs in a wide range of animal reservoir posts and can be isolated from the
environment in water and mud. It is transmitted to humans who come in close contact with the animal
reservoir, arthropods that feed on them or debris and dust associated with them. It can also be
transmitted through the ingestion of contaminated water. Human epidemics sometimes occur and
are associated with epizootics in the animal
Bacteria of potential health concern 75 populations, evidenced by die-offs. There are several
presentations of tularaemia in humans, depending on the route of exposure. Ingestion usually results
in
oropharyngeal tularaemia, with fever, pharyngitis and cervical lymphadenitis. Other forms include
ulcero-glandular, pleuropneumonic and typhoidal. Following the recent war in Kosovo, over 900
suspected cases of tularaemia were identified and 327 cases confirmed serologically. The
epidemiological investigation pointed to rodent-contaminated wells, and rodent carcasses found in
some wells tested positive for F. tularensis (Reintjes et al. 2002). In a waterborne outbreak reported
from Spain, 19 cases who had contact with river-caught crayfish were identified (Anda et al. 2001).
Attempts to isolate F. tularensis from water were unsuccessful. Drinking-water was not involved. F.
tularensis is notoriously difficult to culture, requiring a source of cysteine. F. tularensis was
investigated and developed as a biological weapon; the infectious dose was found to be extremely
low — 10 organisms.
Examples of classical biological warfare agents

Agent Disease
Bacillus anthracis Anthrax
Brucella species Brucellosis
Burkholderia mallei Glanders
Burkholderia pseudomallei Melioidosis
Francisella tularensis Tularaemia
Yersinia pestis Plague
Rickettsia species Typhus
Coxiella burnetii Q fever
Clostridium botulinum toxin Botulism
Staphylococcus aureus enterotoxin B Staphylococcal food poisoning
Smallpox virus Smallpox
The remaining bacteria of concern are either heterotrophs that might have a role in disease or
emerging pathogens that do have a role in disease and could possibly be waterborne. It is important
that these organisms and diseases are kept under surveillance in order to confirm or refute the
suggested associations. Many of the organisms are difficult to grow, and there is no validated trigger
of when to look for them.

The HPC does not measure all organisms present, of which many will be non-culturable but viable,
and indeed several of the organisms of concern described above would not grow on HPC media.
The HPC, however, does give an indication of change in the flora of drinking-water, and the HPC
should be evaluated as a trigger for further investigation.
Many new molecular techniques for the detection of pathogens and putative pathogens in water are
being described (Waage et al. 1999a,b,c; Lightfoot et al. 2001). DNA chips that have the capacity to
detect up to 44 pathogens on one single chip are being developed.
These tests are very expensive when compared with the routine monitoring tests carried out in the
water industry and in public health monitoring. The HPC should be evaluated as the signal of
changing events in a drinking-water supply to trigger the utilization of these new molecular tests to
detect the new bacteria of concern and any associated virulence genes.
Understanding Heterotrophic Plate Count

The analytical methods promulgated under the authority of Section 304(h) of the Clean Water Act
are sometimes referred to as the "304(h)" or "Part 136" methods. The methods measure chemical
and biological pollutants in media, such as wastewater, ambient water, sediment, and biosolids
(sewage sludge). These various CWA methods are tested in a variety of labs and matrices. In
addition to Part 136 methods, some approved methods are published or incorporated by reference
at 40 CFR Parts 401-503, approved industry-specific methods.
In addition to general purposes methods, EPA has approved special purpose analytical methods.
These methods were developed to work in samples or for pollutants specific to certain industrial
categories. For example, methods specific to the Pharmaceutical Manufacturing and Pesticide
Chemicals industrial categories are listed in Tables IF and IG, respectively, at 40 CFR Part 136.
Methods specific to other industrial categories are listed or incorporated by reference into the
regulations at 40 CFR Parts 401-503. These include methods specific to the Pulp, Paper, and
Paperboard category (40 CFR Part 430), and specific to Use or Disposal of Sewage Sludge
(biosolids) (at 40 CFR Part 503.) Industry-specific methods that are approved for compliance
monitoring in the industry for which they are designated may be used for general use, if the same
method is listed in Tables IA to IE, or IH at 40 CFR 136.3.
Understanding Total Coliforms

The Total Coliform Rule was published in 1989 and became effective in 1990. The rule set both
health goals (MCLGs) and legal limits (MCLs) for the presence of total coliform in drinking water. The
rule also detailed the type and frequency of testing that water systems must undertake. In 2003, EPA
announced its intent to revise the Total Coliform Rule.

What’s in Water?
Water is the chemical substance with chemical formula H2O: one molecule of water has two
hydrogen atoms covalently bonded to a single oxygen atom. Water is a tasteless, odorless
liquid at ambient temperature and pressure, and appears colorless in small quantities,
although it has its own intrinsic very light blue hue. Ice also appears colorless, and water
vapor is essentially invisible as a gas.
Water is primarily a liquid under standard conditions, which is not predicted from its
relationship to other analogous hydrides of the oxygen family in the periodic table, which
are gases such as hydrogen sulfide. The elements surrounding oxygen in the periodic table,
nitrogen, fluorine, phosphorus, sulfur and chlorine, all combine with hydrogen to produce
gases under standard conditions.
The reason that water forms a liquid is that oxygen is more electronegative than all of these
elements with the exception of fluorine. Oxygen attracts electrons much more strongly than
hydrogen, resulting in a net positive charge on the hydrogen atoms, and a net negative
charge on the oxygen atom.
The presence of a charge on each of these atoms gives each water molecule a net dipole
moment. Electrical attraction between water molecules due to this dipole pulls individual
molecules closer together, making it more difficult to separate the molecules and therefore
raising the boiling point.

Respiratory Protection Section
Conditions for Respirator Use
Good industrial hygiene practice requires that engineering controls be used where feasible to reduce
workplace concentrations of hazardous materials to the prescribed exposure limit.
However, some situations may require the use of respirators to control exposure. Respirators must
be worn if the ambient concentration of chlorine exceeds prescribed exposure limits. Respirators
may be used before engineering controls have been installed, during work operations such as
maintenance or repair activities that involve unknown exposures, during operations that require entry
into tanks or closed vessels, and during emergencies. Workers should only use respirators that have
been approved by NIOSH and the Mine Safety and Health Administration (MSHA).
Respiratory Protection Program

Employers should institute a complete respiratory protection program that, at a minimum, complies
with the requirements of OSHA's Respiratory Protection Standard [29 CFR 1910.134]. Such a
program must include respirator selection, an evaluation of the worker's ability to perform the work
while wearing a respirator, the regular training of personnel; respirator fit testing, periodic workplace
monitoring, and regular respirator maintenance, inspection, and cleaning. The implementation of an
adequate respiratory protection program (including selection of the correct respirator) requires that
a knowledgeable person be in charge of the program and that the program be evaluated regularly.
For additional information on the selection and use of respirators and on the medical screening of
respirator users, consult the latest edition of the NIOSH Respirator Decision Logic [NIOSH 1987b]
and the NIOSH Guide to Industrial Respiratory Protection [NIOSH 1987a].
Personal Protective Equipment

Workers should use appropriate personal protective clothing and equipment that must be carefully
selected, used, and maintained to be effective in preventing skin contact with chlorine. The selection
of the appropriate personal protective equipment (PPE) (i.e., gloves, sleeves, encapsulating suits)
should be based on the extent of the worker's potential exposure to chlorine.
The resistance of various materials to permeation by chlorine liquid and chlorine gas is shown below:
Material Breakthrough Time (hr.) Chlorine Liquid Responder

To evaluate the use of PPE materials with chlorine, users should consult the best available
performance data and manufacturers' recommendations. Significant differences have been
demonstrated in the chemical resistance of generically similar PPE materials (e.g., butyl) produced
by different manufacturers. In addition, the chemical resistance of a mixture may be significantly
different from that of any of its neat components.
Any chemical-resistant clothing that is used should be periodically evaluated to determine its
effectiveness in preventing dermal contact. Safety showers and eye wash stations should be located
close to operations that involve chlorine.
Splash-proof chemical safety goggles or face shields (20 to 30 cm long, minimum) should be worn
during any operation in which a solvent, caustic, or other toxic substance may be splashed into the
eyes.
In addition to the possible need for wearing protective outer apparel e.g., aprons, encapsulating
suits), workers should wear work uniforms, coveralls, or similar full-body coverings that are laundered
each day. Employers should provide lockers or other closed areas to store work and street clothing
separately.

Employers should collect work clothing at the end of each work shift and provide for its laundering.
Laundry personnel should be informed about the potential hazards of handling contaminated clothing
and instructed about measures to minimize their health risk.
Protective clothing should be kept free of oil and grease and should be inspected and maintained
regularly to preserve its effectiveness. Protective clothing may interfere with the body's heat
dissipation, especially during hot weather or during work in hot or poorly ventilated work
environments.

Confined Space Entry Program
Purpose
The Confined Space Entry Program is provided to protect authorized employees that will enter
confined spaces and may be exposed to hazardous atmospheres, engulfment in materials,
conditions which may trap or asphyxiate due to converging or sloping walls, or contains any other
safety or health hazards.
Reference: OSHA-Permit-Required Confined Spaces (29 CFR 1910.146).
Scope
You are required to recognize the dangers and hazards associated with confined spaces, and
this program is designed to assist you in the safety of and compliance with the OSHA standards
associated with such.
Most WWTP’s will utilize the Fire Department for all rescues and additional assistance dealing
with confined spaces, understanding that most Fire Department operations utilize additional in
house SOG's/SOP’s pertaining to such operations.
Definitions
Confined space:
 Is large enough or so configured that an employee can bodily enter and perform
work.
 Has limited or restricted means for entry or exit (i.e. tanks, vessels, silos, storage
bins, hoppers, vaults, and pits are spaces that may have limited means of entry).
 Is not designed for continuous employee occupancy.
Permit required confined space (permit space), is a confined space that has one or more of the
following characteristics:
1. Contains or has a
potential to contain a
hazardous atmosphere.
2. Contains a material that

has the potential for
engulfing an entrant.
3. Has an internal
configuration such that an
entrant could be trapped or
asphyxiated by inwardly
covering walls or by a floor
which slopes downward and
tapers to a smaller cross-
section.
4. Contains any other

recognized serious safety or
health hazard.
Each Permit-Required Confined Space will be marked

"Confined Space - Entry Permit Required".

Confined Space Hazards
Fatalities and injuries constantly occur among construction workers who, during the course of their
jobs, are required to enter confined spaces. In some circumstances, these workers are exposed to
multiple hazards, any of which may cause bodily injury, illness, or death.
Newspaper and magazine articles abound with stories of workers injured and killed from a variety of
atmospheric factors and physical agents. Throughout the construction jobsite, contractors and
workers encounter both inherent and induced hazards within confined workspaces.
Inherent Hazards
Inherent hazards, such as electrical, thermal, chemical, mechanical, etc., are associated with specific
types of equipment and the interactions among them.
Examples include high voltage (shock or corona discharge and the resulting burns), radiation
generated by equipment, defective design, omission of protective features (no provision for
grounding non-current-carrying conductive parts), high or low temperatures, high noise levels, and
high-pressure vessels and lines (rupturing with resultant release of fragments, fluids, gases, etc.).
Inherent hazards usually cannot be eliminated without degrading the system or equipment, or without
making them inoperative. Therefore, emphasis must be placed on hazard control methods.
Induced Hazards
Induced hazards arise, and are induced from, a multitude of incorrect decisions and actions that
occur during the actual construction process. Some examples are: omission of protective features,
physical arrangements that may cause unintentional worker contact with electrical energy sources,
oxygen-deficient atmospheres created at the bottom of pits or shafts, lack of safety factors in
structural strength, and flammable atmospheres.
Typical Examples of Confined Workspaces

Following are typical examples of confined workspaces in
construction which contain both inherent and induced hazards.
Vaults
A variety of vaults are found on the construction jobsite. On various
occasions, workers must enter these vaults to perform a number of
functions.
The restricted nature of vaults and their frequently below-grade

location can create an assortment of safety and health problems.
Oxygen-Deficient Atmosphere
One of the major problems confronting construction workers while
working in vaults is the ever-present possibility of an oxygen-
deficient atmosphere.
Explosive or Toxic Gases, Vapors, or Fumes

While working in an electrical vault, workers may be exposed to the
build-up of explosive gases such as those used for heating
(propane). Welding and soldering produce toxic fumes which are confined in the limited
atmosphere.
Electrical Shock
Electrical shock is often encountered from power tools, line cords, etc. In many instances, such
electrical shock results from the fact that the contractor has not provided an approved grounding
system or the protection afforded by ground-fault circuit interrupters or low-voltage systems.

Purging
In some instances, purging agents such as nitrogen and argon may enter the vault from areas
adjacent to it. These agents may displace the oxygen in the vault to the extent that it will asphyxiate
workers almost immediately.
Materials Falling In and On

A hazard normally considered a problem associated with confined spaces is material or equipment
which may fall into the vault or onto workers as they enter and leave the vault.
Vibration could cause the materials on top of the vault to roll off and strike workers. If the manhole
covers were removed, or if they were not installed in the first place, materials could fall into the vault,
causing injury to the workers inside.
Condenser Pits
A common confined space found in the construction of nuclear power plants is the condenser pit.
Because of their large size, they are often overlooked as potentially hazardous confined spaces.
These below-grade areas create large containment areas for the accumulation of toxic fumes, gases,
and so forth, or for the creation of oxygen-deficient atmospheres when purging with argon, Freon,
and other inert gases. Other hazards will be created by workers above dropping equipment, tools,
and materials into the pit.
Manholes
Throughout the construction site, manholes are commonplace. As means of entry into and exit from
vaults, tanks, pits, and so forth, manholes perform a necessary function. However, these confined
spaces may present serious hazards which could cause injuries and fatalities. A variety of hazards
are associated with manholes. To begin with, the manhole could be a dangerous trap into which the
worker could fall. Often covers are removed and not replaced, or else they are not provided in the
first place.
Pipe Assemblies
One of the most frequently unrecognized types of confined spaces encountered throughout the
construction site is the pipe assembly. Piping of sixteen to thirty-six inches in diameter is commonly
used for a variety of purposes.
For any number of reasons, workers will enter the pipe. Once inside, they are faced with potential
oxygen-deficient atmospheres, often caused by purging with argon or another inert gas. Welding
fumes generated by the worker in the pipe, or by other workers operating outside the pipe at either
end, subject the worker to toxic atmospheres.
The generally restricted dimensions of the pipe provide little room for the workers to move about and
gain any degree of comfort while performing their tasks. Once inside the pipe, communication is
extremely difficult. In situations where the pipe bends, communication and extrication become even
more difficult. Electrical shock is another problem to which the worker is exposed.
Ungrounded tools and equipment or inadequate line cords are some of the causes. As well, heat
within the pipe run may cause the worker to suffer heat prostration.
Ventilation Ducts
Ventilation ducts, like pipe runs, are very common at the construction site. These sheet metal
enclosures create a complex network which moves heated and cooled air and exhaust fumes to
desired locations in the plant.
Ventilation ducts may require that workers enter them to cut out access holes, install essential parts
of the duct, etc. Depending on where these ducts are located, oxygen deficiency could exist. They
usually possess many bends, which create difficult entry and exit and which also make it difficult for
workers inside the duct to communicate with those outside it. Electrical shock hazards and heat
stress are other problems associated with work inside ventilation ducts.
Tanks
Tanks are another type of confined workspace commonly found in construction. They are used for a
variety of purposes, including the storage of water, chemicals, etc.
Tanks require entry for cleaning and repairs. Ventilation is always a problem. Oxygen-deficient
atmospheres, along with toxic and explosive atmospheres created by the substances stored in the
tanks, present hazards to workers. Heat, another problem in tanks, may cause heat prostration,
particularly on a hot day.
Since electrical line cords are often taken into the tank, the hazard of electrical shock is always
present. The nature of the tank's structure often dictates that workers must climb ladders to reach
high places on the walls of the tank.
Sumps
Sumps are commonplace. They are used as collection places for water and other liquids. Workers
entering sumps may encounter an oxygen-deficient atmosphere.
Also, because of the wet nature of the sump, electrical shock hazards are present when power tools
are used inside. Sumps are often poorly illuminated. Inadequate lighting may create an accident
situation.
Containment Cavities
These large below-grade areas are characterized by little or no air movement. Ventilation is always
a problem. In addition, the possibility of oxygen deficiency exists. As well, welding and other gases
may easily collect in these areas, creating toxic atmospheres. As these structures near completion,
more confined spaces will exist as rooms are built off the existing structure.
Electrical Transformers
Electrical transformers are located on the jobsite.
They often contain a nitrogen purge or dry air.
Before they are opened, they must be well vented
by having air pumped in.
Workers, particularly electricians and power plant

operators, will enter these transformers through
hatches on top for various work-related reasons.
Testing for oxygen deficiency and for toxic
atmospheres is mandatory.
Heat Sinks
These larger pit areas hold cooling water in the
event that there is a problem with the pumps
located at the water supply to the plant--normally
a river or lake--which would prevent cooling
water from reaching the reactor core.
When in the pits, workers are exposed to

welding fumes and electrical hazards,
particularly because water accumulates in the
bottom of the sink.
Generally, it is difficult to communicate with

workers in the heat sink, because the rebar in
the walls of the structure deaden radio signals.
Unusual Conditions
Confined Space within a Confined Space
By the very nature of construction, situations are created which illustrate one of the most hazardous
confined spaces of all--a confined space within a confined space.
This situation appears as tanks within pits, pipe assemblies or vessels within pits, etc. In this situation,
not only do the potential hazards associated with the outer confined space require testing,
monitoring, and control, but those of the inner space also require similar procedures.
Often, only the outer space is evaluated. When workers enter the inner space, they are faced with
potentially hazardous conditions. A good example of a confined space within a confined space is a
vessel with a nitrogen purge inside a filtering water access pit. Workers entering the pit and/or the
vessel should do so only after both spaces have been evaluated and proper control measures
established.
Hazards in One Space Entering another Space

During an examination of confined spaces in construction,
one often encounters situations which are not always easy
to evaluate or control. For instance, a room or area which
classifies as a confined space may be relatively safe for
work.
However, access passages from other areas outside or

adjacent to the room could, at some point, allow the transfer
of hazardous agents into the "safe" one. One such instance
would be a pipe coming through a wall into a containment
room.
Welding fumes and other toxic materials generated in one

room may easily travel through the pipe into another area,
causing it to change from a safe to an unsafe workplace. A
serious problem with a situation such as this is that workers
working in the "safe" area are not aware of the hazards
leaking into their area. Thus, they are not prepared to take
action to avoid or control it.
Session Conclusion
In this discussion, we have defined inherent and induced
hazards in confined spaces. We have examined typical
confined spaces on construction sites and we have
described representative hazards within these confined spaces.
Permitted Confined Space Entry Program

Definition of Confined Spaces Requiring an Entry Permit
Confined space:
 Is large enough or so configured that an employee can bodily enter and perform work.
 Has limited or restricted means for entry or exit (i.e. tanks, vessels, silos, storage bins, hoppers,
vaults, and pits are spaces that may have limited means of entry).
 Is not designed for continuous employee occupancy.
Purpose
The Permit Required Space (PRCS) Program is provided to protect authorized employees that will
enter confined spaces and may be exposed to hazardous atmospheres, engulfment in materials,
conditions which may trap or asphyxiate due to converging or sloping walls, or contains any other
safety or health hazards.
Many workplaces contain confined spaces not designed for human occupancy which due to their
configuration hinder employee activities including entry, work and exit. Asphyxiation is the leading
cause of death in confined spaces.
Subpart P applies to all open excavations in the earth's surface.
 All trenches are excavations.
 All excavations are not trenches.
Permit Required Confined Space Entry General Rules

During all confined space entries, the following safety rules must be strictly enforced:
1. Only authorized and trained employees may enter a confined space or act as safety
watchmen/attendants.
2. No smoking is permitted in a confined space or near entrance/exit area.
3. During confined space entries, a watchmen or attendant must be present at all times.
4. Constant visual or voice communication will be maintained between the safety watchmen and
employees entering a confined space.
5. No bottom or side entry will be made or work conducted below the level any hanging material or
material which could cause engulfment.
6. Air and oxygen monitoring is required before entering any permit-required confined space. Oxygen
levels in a confined space must be between 19.5 and 23.5 percent. Levels above or below will require
the use of an SCBA or other approved air supplied respirator. Additional ventilation and oxygen level
monitoring is required when welding is performed. The monitoring will check oxygen levels, explosive
gas levels and carbon monoxide levels. Entry will not be permitted if explosive gas is detected above
one-half the Lower Explosive Limit (LEL).
7. To prevent injuries to others, all openings to confined spaces will be protected by a barricade when
covers are removed.

Appendix A to §1910.146
Permit-Required Confined Space Decision Flow Chart
Note: Appendices A through F serve to provide information and non-mandatory guidelines to assist
employers and employees in complying with the appropriate requirements of this section.
[58 FR 4549, Jan. 14, 1993; 58 FR 34846, June 29, 1993; 63 FR 66039, Dec. 1, 1998]

Confined Space Entry Permit Example
Date & Time Issued Date & time Expires
Space I.D. Supervisor
Equipment Affected Task
Standby Team
Time (am - pm)
Pre-Entry
Atmospheric Checks
Oxygen
Explosive ( % LEL)
Toxic (PPM)
Testers Signature
Pre-entry Fluid System Isolation Yes No
Pumps /lines blinded, blocked, disconnected
Ventilation Source Established
Mechanical Forced Air
Natural Ventilation
Post Ventilation Pre-Entry Atmospheric Checks
Time
Oxygen (%)
Explosive ( % LEL
Toxic (PPM)
Tester Signature
Communication Procedures Established per specific Confined Space SOP
Rescue Procedures established per specific Confined Space SOP
Training Verification - for the following persons & space to be entered YES NO
All persons entering Confined Space
All persons acting as Supervisor for the Entry
All persons assigned backup positions
All persons assigned to monitor access and interior activities
All persons assigned to emergency rescue team
Equipment on Scene YES NO NA YES NO NA
Gas Monitor Life Line
Safety Harness Hoisting
Equipment
Fall Arrest Gear Powered Comm
Eq.
SCBAs Air Line
Respirators
Protective Clothing Elect Gear
Properly Rated
Periodic Atmospheric Checks
Time (am - pm)
Oxygen
Explosive ( % LEL)
Toxic (PPM)
Testers Signature

A review of the work authorized by this permit and the information contained on this Entry Permit.
Written instructions and safety procedures have been received and are understood. Entry cannot
be approved if any squares are marked in the "No" column. This permit is not valid unless all
appropriate items are completed.
Permit Prepared By: (Supervisor) _______________________________
Approved By: (Unit Supervisor) _________________________________
This permit to be kept at job site.
Return job site copy to Safety Office following job completion.
Copies: Safety Office, Unit Supervisor, Job site

Confined Space Duties & Responsibilities
Examples of Assignments
Employees
 Follow program requirements.
 Report any previously un-identified hazards
associated with confined spaces.
 Do not enter any confined spaces that have
not been evaluated for safety concerns.
Management
 Provide annual Confined Space training to all
employees that may need confined space training.
 Ensure confined space assessments have been
conducted.
 Annually review this program and all Entry
Permits.
Rescue or Training Department
 Ensure proper training for entry & rescue teams.
 Provide proper equipment for entry & rescue
teams.
 Ensure all permit required confined spaces are
posted.
 Evaluate rescue teams and service to ensure they
are adequately trained and prepared.
 Ensure rescue team at access during entry into
spaces with Immediately Dangerous to Life or
Health (IDLH) atmospheres.
 Provide annual confined space awareness training to all employees
that may need confined space awareness training.
Entry Supervisor
Entry supervisors are responsible for the overall permit space entry and
must coordinate all entry procedures, tests, permits, equipment and other
relevant activities.
The following entry supervisor duties are required:

Know the hazards that may be faced during entry, including information on
the mode, signs or symptoms, and consequences of the exposure.
Verify by checking that the appropriate entries have been made on the
permit, all tests specified by the permit have been conducted, and that all procedures and
equipment specified by the permit are in place before endorsing the permit and allowing
entry to begin.
Terminate the entry and cancel the permit when the entry is complete or there is a need for
terminating the permit.
Verify that rescue services are available and that the means for summoning them are
operable.
Remove unauthorized persons who enter or attempt to enter the space during entry
operations.

Determine whenever responsibility for a permit space entry operation is transferred and at
intervals dictated by the hazards and operations performed within the space that entry
operations remain consistent with the permit terms and that acceptable entry conditions are
maintained.
Entry Attendants
At least one attendant is required outside the permit space into which entry is authorized for
the duration of the entry operation. Responsibilities include:
 To know the hazards that may be faced during entry, including information on the mode,
signs or symptoms, and consequences of the exposure.
 To be aware of possible behavioral effects of hazard exposure on entrants.
 To continuously maintain an accurate count of entrants in the permit space and ensures a
means to accurately identify authorized entrants.
 To remain outside the permit space during entry operations until relieved by another
attendant (once properly relieved, they may participate in other permit space activities,
including rescue if they are properly trained and equipped).
 To communicate with entrants as necessary to monitor entrant status and alert entrants of
the need to evacuate.
 To monitor activities inside and outside the space to determine if it is safe for entrants to
remain in the space; orders the entrants to immediately evacuate if: the attendant detects a
prohibited condition, detects entrant behavioral effects of hazard exposure, detects a
situation outside the space that could endanger the entrants; or if the attendant cannot
effectively and safely perform all the attendant duties.
 To summon rescue and other emergency services as soon as the attendant determines the
entrants need assistance to escape the permit space hazards.
 To perform non-entry rescues as specified by that rescue procedure and entry supervisor
and not to perform duties that might interfere with the attendants' primary duty to monitor
and protect the entrants.

Duties of the Person Authorizing or in Charge of the Entry
The person who authorizes or is in charge of the permit entry confined space must comply
with the following:
1. Make certain that all pre-entry requirements as outlined on the permit have been completed
before any worker is allowed to enter the confined space.
2. Make certain that any required pre-entry conditions are present.
3. If an in-plant/facility rescue team is to be used in the event of an emergency, make sure they
would be available. If your Employer does not maintain an in-plant rescue team, dial 911 on any
telephone for the Rescue Squad.
4. Make sure that any communication equipment which would be used to summon either the in-
plant rescue team or other emergency assistance is operating correctly.
5. Terminate the entry upon becoming aware of a condition or set of
conditions whose hazard potential exceeds the limits authorized by the entry
permit.
If the person who would otherwise issue an entry permit is in charge of the entry
and present during the entire entry, then a written permit is not required if that
person uses a checklist as provided in the section on "Permits".
This person may also serve as the attendant at the site.
Special Considerations During A Permit Required Entry

Certain work being performed in a permit entry confined space could cause the
atmosphere in the space to change. Examples of this are welding, drilling, or
sludge removal. In these situations, air monitoring of the confined space should
be conducted on a continuous basis throughout the time of the entry.
If the workers leave the confined space for any significant period of time, such
as for a lunch or other break, the atmosphere of the confined space must be
retested before the workers reenter the confined space.
Unauthorized Persons
Take the following actions when unauthorized persons approach or enter
a permit space while entry is under way:
1. Warn the unauthorized persons that they must stay away from the permit space,
2. Advise unauthorized persons that they must exit immediately if they have entered the
space, and
3. Inform the authorized entrants and the entry supervisor if unauthorized persons have
entered the permit space.
Entrants
All entrants must be authorized by the entry supervisor to enter permit spaces, have received
the required training, have used the proper equipment, and observed the entry procedures
and permit requirements.
The following entrant duties are required:

Know the hazards that may be faced during entry, including information on the mode, signs
or symptoms, and consequences of the exposure;
Properly use the equipment required for safe entry;

Communicate with the attendant as necessary to enable the attendant to monitor the status
of the entrants and to enable the attendant to alert the entrants of the need to evacuate the
space if necessary;

Alert the attendant whenever; the entrant recognizes any warning signs or symptoms of
exposure to a dangerous situation, or any prohibited condition is detected; and Exit the
permit space as quickly as possible whenever the attendant or entry supervisor gives an
order to evacuate the permit space, the entrant recognizes any warning signs or symptoms
of exposure to a dangerous situation, the entrant detects a prohibited condition, or an
evacuation alarm is activated.

Hazards
 Explosive / Flammable Atmospheres
 Toxic Atmospheres
 Engulfment
 Asphyxiation
 Entrapment
 Slips & falls
 Chemical Exposure
 Electric Shock
 Thermal / Chemical Burns
 Noise & Vibration
Hazard Control
Engineering Controls
 Locked entry points
 Temporary ventilation
 Temporary Lighting
Administrative Controls
 Signs
 Employee training
 Entry procedures
 Atmospheric Monitoring
 Rescue procedures
 Use of prescribed Personal Protective Equipment
Entry Standard Operating Procedures

This program outlines:
 Hazards
 Hazard Control & Abatement
 Acceptable Entry Conditions
 Means of Entry
 Entry Equipment Required
 Emergency Procedures

Permit Required Confined Space Entry General Rules
During all confined space entries, the following safety rules must be strictly
enforced:
1. Only authorized and trained employees may enter a confined space or act as safety
watchman/attendant.
2. No smoking is permitted in a confined space or near entrance/exit area.
3. During confined space entries, a watchman must be present at all times.
4. Constant visual or voice communication will be maintained between the safety

watchman/attendant and employees entering a confined space.
5. No bottom or side entry will be made or work conducted below the level of any hanging
material or material which could cause engulfment.
6. Air and oxygen monitoring is required before entering any permit-required confined
space. Oxygen levels in a confined space must be between 19.5 and 23.5 percent.
Levels above or below will require the use of an SCBA or other approved air supplied
respirator. Additional ventilation and oxygen level monitoring is required when welding
is performed.
The monitoring will check oxygen levels, explosive gas levels and carbon monoxide
levels. Entry will not be permitted if explosive gas is detected above one-half the Lower
Explosive Limit (LEL), or 10% of a specific gas explosive limit.
7. To prevent injuries to others, all openings to confined spaces will be protected by a

barricade when covers are removed.
Confined Space Entry Procedures

Each employee who enters or is involved in the entry must:
1. Understand the procedures for confined space entry
2. Know the Hazards of the specific space
3. Review the specific procedures for each entry
4. Understand how to use entry and rescue equipment
Confined Space Entry Permits

Confined Space Entry Permits must be completed before any employee enters a permit-
required confined space. The permit must be completed and signed by an authorized
member of management before entry.
Permits will expire before the completion of the shift or if any pre-entry conditions change.
Permits will be maintained on file for 12 months.

Contractor Entry
All work by non-company employees that involves the entry into confined spaces will follow
the procedures of this program. The information of this program and specific hazards of the
confined spaces to be entered will be provided to contractor management prior to
commencing entry or work.
Important Rescue Service Questions

What is the availability of the rescue service?
Is it unavailable at certain times of the day or in certain situations?
What is the likelihood that key personnel of the rescue service might be unavailable at
times?
If the rescue service becomes unavailable while an entry is underway, does it have the
capability of notifying the employer so that the employer can instruct the attendant to abort
the entry immediately?

Confined Space Training
Training for Confined Space Entry includes:
1. Duties of entry supervisor, entrant and attendants
2. Confined space entry permits
3. Hazards of confined spaces
4. Use of air monitoring equipment
5. First aid and CPR training
6. Emergency action & rescue procedures
7. Confined space entry & rescue equipment
8. Rescue training, including entry and removal from representative spaces
Confined Space Training and Education

OSHA's General Industry Regulation, §1910.146 Permit-required confined spaces, contains
requirements for practices and procedures to protect employees in general industry from the hazards
of entry into permit-required confined spaces. This regulation does not apply to construction.
OSHA's Construction Safety and Health Regulations Part 1926 do not contain a permit-required
confined space regulation. Subpart C, §1926.21 Safety training and education specifies training for
personnel who are required to enter confined spaces and defines a "confined or enclosed space."
These requirements are shown below.
§1926.21 Safety training and education. (Partial)

(b)(6)(i) All employees required to enter into confined or enclosed spaces shall be instructed as to
the nature of the hazards involved, the necessary precautions to be taken, and in the use of protective
and emergency equipment required. The employer shall comply with any specific regulations that
apply to work in dangerous or potentially dangerous areas.
(ii) For purposes of paragraph (b)(6)(i) of this section, "confined or enclosed space" means any
space having a limited means of egress, which is subject to the accumulation of toxic or flammable
contaminants or has an oxygen deficient atmosphere. Confined or enclosed spaces include, but are
not limited to, storage tanks, process vessels, bins, boilers, ventilation or exhaust ducts, sewers,
underground utility vaults, tunnels pipelines, and open top spaces more than 4 feet in depth such as
pits, tubs, vaults, and vessels.
OSHA's Construction Regulations also contain requirements dealing with confined space hazards in
underground construction (Subpart S), underground electric transmission and distribution work
(§1926.956), excavations (Subpart P), and welding and cutting (Subpart J).
Further guidance may be obtained from American National Standard ANSI Z117.1-1989, Safety
Requirements for Confined Spaces. This standard provides minimum safety requirements to be
followed while entering, exiting and working in confined spaces at normal atmospheric pressure. This
standard does not pertain to underground mining, tunneling, caisson work or other similar tasks that
have established national consensus standards.
Your Employer is Responsible for Certain Training Requirements
These are as follows:

1. GENERAL As an employer, your employer must ensure that all workers who must enter a permit
entry confined space in the course of their work are informed of appropriate procedures and controls
for entry into such spaces. These workers must be made aware of the fact that an unauthorized entry
could be fatal, and that their senses are unable to detect and evaluate the severity of atmospheric
hazards.
2. TRAINING FOR AUTHORIZED ENTRANTS Your employer must ensure that all authorized
entrants know the emergency action plan and have received training covering the following subjects
prior to entering any permit entry confined space:
a. Hazard Recognition: Each worker must understand the nature of the hazard before entering and
the need to perform appropriate testing to determine if it is safe to enter.
b. Use of Personal Protective Equipment: Each employee must be taught the proper use of all
personal protective equipment required for entry or rescue, and the proper use of protective barriers
and shields.
c. Self-Rescue: Each worker must be trained to get out of the confined space as rapidly as possible
without help whenever an order to evacuate is given by the attendant, whenever an automatic
evacuation alarm is activated, or whenever workers recognize the warning signs of exposure to
substances that could be found in the confined space. They must also be made aware of the toxic
effects or symptoms of exposure to hazardous materials he could encounter in the confined space.
This includes anything that could be absorbed through the skin or which could be carried through the
skin by any solvents that are used. They must be trained to relay an alarm to the attendant and to
attempt self- rescue immediately upon becoming aware of these effects.
d. Special Work Practices or Procedures: Each worker must be trained in any modifications of
normal work practices that are necessary for permit entry confined space work.
3. TRAINING FOR PERSONS AUTHORIZING OR IN CHARGE OF ENTRY In addition to other

requirements already covered, the person authorizing or in charge of entry shall be trained to
recognize the effects of exposure to hazards that could be in the confined space. They must also
carry out all duties that the permit assigns to them.
Rescue practice training. In the photo above, the sand bag represents a fallen victim.

4. TRAINING FOR ATTENDANT Any worker functioning as an attendant at a permit entry confined
space must be trained in the company's emergency action plan, the duties of the attendant, and in;
a. Proper use of the communications equipment furnished for communicating with authorized
workers entering the confined space or for summoning emergency or rescue services.
b. Authorized procedures for summoning rescue or other emergency services.
c. Recognition of the unusual actions of a worker which could indicate that they could be
experiencing a toxic reaction to contaminants that could be present in the space.
d. Any training for rescuers, if the attendant will function as a rescuer also.
e. Any training for workers who enter the confined space, if the permit specifies that the duty of the
attendant will rotate among the workers authorized to enter the confined space.

Confined Space Entry Procedure
Space _________________ Date Last Modified _____________

Place check mark in all applicable areas
Hazards Personal Protective Equipment
Explosive / Combustion Hazard Air supplied Respirator
Exposed Electrical Circuits Air Purifying Respirator
Unguarded Machine Parts Welding Protection
Atmospheric Hazard Gloves
Potential Atmospheric Hazard Hard Hat
Thermal Hazard Ventilation Requirements
Chemical Hazard Continuous ___cuft/min
Note: See Ventilation Guidelines for Confined Spaces for
typical ventilation configurations and formulas.
Fall Hazard
Engulfment hazard Note: Additional ventilation may be required for hot work, grinding or
other operations that would produce airborne fumes, mist or dust.
Entry Supervisor must assess additional ventilation requirements
base on tasks to be performed in the space
Converging Walls
Floors slope-small cross-
section
Slip Hazard
Entry Path Vent Exhaust Point:
Side entry Vent Supply Point:
Bottom entry Space Volume
Door Initial Purge Time= 7.5 X _____ (space volume)
Effective Blower Capacity
Top open entry
Top manhole entry 20 Air Changes per Hour (ACH) for duration of entry
Hinged hatch Minimum initial Purge Time= 20 Minutes

Entry & Rescue Equipment Adequate Blower Capacity (ABC) = ________
ABC = Space Volume x 20 ACH
60 minutes
Life Line
Floor level opening barrier Acceptable Entry Conditions
Body Harness Confined Space Entry permit posted
Tripod Oxygen 19.5 23.5%
Man Winch Lower Explosive Level %
Fall Arrest Unit Toxic fumes/vapors Less than PEL
Emerg Retrieval Line No engulfing material in space
Atmospheric Monitor No hazardous chemicals or material
Blower /Saddle / Trunks Drained - Flushed
Drop Light Rescue Team Available on Site
Communication Gear Ventilation Established & Maintained
Ladder LOTO Electrical components in space
Hand held radios LOTO Mechanical Components in space
Portable Lighting LOTO All pipes to and from space

Other Hazards
Flammable Atmospheres
A flammable atmosphere generally arises from enriched oxygen atmospheres, vaporization of
flammable liquids, byproducts of work, chemical reactions, concentrations of combustible dusts, and
desorption of chemical from inner surfaces of the confined space.
An atmosphere becomes flammable when the ratio of oxygen to combustible material in the air is
neither too rich nor too lean for combustion to occur. Combustible gases or vapors will accumulate
when there is inadequate ventilation in areas such as a confined space.
Flammable gases such as acetylene, butane, propane, hydrogen, methane, natural or manufactured
gases or vapors from liquid hydrocarbons can be trapped in confined spaces, and since many gases
are heavier than air, they will seek lower levels as in pits, sewers, and various types of storage tanks
and vessels. In a closed top tank, it should also be noted that lighter than air gases may rise and
develop a flammable concentration if trapped above the opening.
The byproducts of work procedures can generate flammable or explosive conditions within a
confined space. Specific kinds of work such as spray painting can result in the release of explosive
gases or vapors. Welding in a confined space is a major cause of explosions in areas that contain
combustible gas.
Chemical reactions forming flammable atmospheres occur when surfaces are initially exposed to
the atmosphere, or when chemicals combine to form flammable gases. This condition arises when
dilute sulfuric acid reacts with iron to form hydrogen or when calcium carbide makes contact with
water to form acetylene.
Other examples of spontaneous chemical reactions that may produce explosions from small
amounts of unstable compounds are acetylene-metal compounds, peroxides, and nitrates. In a dry
state, these compounds have the potential to explode upon percussion or exposure to increased
temperature.
Another class of chemical reactions that form flammable atmospheres arise from deposits of
pyrophoric substances (carbon, ferrous oxide, ferrous sulfate, iron, etc.) that can be found in tanks
used by the chemical and petroleum industry. These tanks containing flammable deposits will
spontaneously ignite upon exposure to air.
Combustible dust concentrations are usually found during the process of loading, unloading, and
conveying grain products, nitrated fertilizers, finely ground chemical products, and any other
combustible material.
High charges of static electricity, which rapidly accumulate during periods of relatively low humidity
(below 50%) can cause certain substances to accumulate electrostatic charges of sufficient energy
to produce sparks and ignite a flammable atmosphere. These sparks may also cause explosions
when the right air or oxygen to dust or gas mixture is present.
Toxic Atmospheres
The substances to be regarded as toxic in a confined space can cover the entire spectrum of gases,
vapors, and finely-divided airborne dust in industry. The sources of toxic atmospheres encountered
may arise from the following:
1. The manufacturing process (for example, in producing polyvinyl chloride, hydrogen chloride is
used as well as vinyl chloride monomer, which is carcinogenic).
2. The product stored [removing decomposed organic material from a tank can liberate toxic
substances, such as hydrogen sulfide (H2S)].
3. The operation performed in the confined space (for example, welding or brazing with metals
capable of producing toxic fumes).

During loading, unloading, formulation, and production, mechanical and/or human error may also
produce toxic gases which are not part of the planned operation.
Carbon monoxide (CO) is a hazardous gas that may build up in a confined space. This odorless,
colorless gas that has approximately the same density as air is formed from incomplete combustion
of organic materials such as wood, coal, gas, oil, and gasoline; it can be formed from microbial
decomposition of organic matter in sewers, silos, and fermentation tanks.
CO is an insidious toxic gas because of its poor warning properties. Early stages of CO intoxication
are nausea and headache. CO may be fatal at as little as 1000 ppm or 10% in air, and is considered
dangerous at 200 ppm or 2%, because it forms Carboxyhemoglobin in the blood which prevents the
distribution of oxygen in the body.
CO is a relatively abundant colorless, odorless gas. Therefore, any untested atmosphere must be
suspect. It must also be noted that a safe reading on a combustible gas indicator does not ensure
that CO is not present. CO must be tested for specifically. The formation of CO may result from
chemical reactions or work activities, therefore fatalities due to CO poisoning are not confined to any
particular industry. There have been fatal accidents in sewage treatment plants due to
decomposition products and lack of ventilation in confined spaces.
Another area where CO results as a product of decomposition is in the formation of silo gas in grain
storage elevators. In another area, the paint industry, varnish is manufactured by introducing the
various ingredients into a kettle, and heating them in an inert atmosphere, usually town gas, which
is a mixture of carbon dioxide and nitrogen.
In welding operations, oxides of nitrogen and ozone are gases of major toxicologic importance, and
incomplete oxidation may occur and carbon monoxide can form as a byproduct. Another poor work
practice, which has led to fatalities, is the recirculation of diesel exhaust emissions. Increased CO
levels can be prevented by strict control of the ventilation and the use of catalytic converters.

Irritant (Corrosive) Atmospheres
Irritant or corrosive atmospheres can be divided into primary and secondary groups. The primary
irritants exert no systemic toxic effects (effects on the entire body).
Examples of primary irritants are chlorine, ozone, hydrochloric acid, hydrofluoric acid, sulfuric
acid, nitrogen dioxide, ammonia, and sulfur dioxide. A secondary irritant is one that may produce
systemic toxic effects in addition to surface irritation. Examples of secondary irritants include
benzene, carbon tetrachloride, ethyl chloride, trichloroethane, trichloroethylene, and
chloropropene.
Irritant gases vary widely among all areas of industrial activity. They can be found in plastics
plants, chemical plants, the petroleum industry, tanneries, refrigeration industries, paint
manufacturing, and mining operations. Prolonged exposure at irritant or corrosive concentrations
in a confined space may produce little or no evidence of irritation. This may result in a general
weakening of the defense reflexes from changes in sensitivity. The danger in this situation is that
the worker is usually not aware of any increase in his/her exposure to toxic substances.
Asphyxiating Atmospheres
The normal atmosphere is composed approximately of 20.9% oxygen and 78.1% nitrogen, and
1% argon with small amounts of various other gases. Reduction of oxygen in a confined space
may be the result of either consumption or displacement.
The consumption of oxygen takes place during combustion of flammable substances, as in

welding, heating, cutting, and brazing. A more subtle consumption of oxygen occurs during
bacterial action, as in the fermentation process.
Oxygen may also be consumed during chemical reactions as in the formation of rust on the
exposed surface of the confined space (iron oxide). The number of people working in a confined
space and the amount of their physical activity will also influence the oxygen consumption rate.
A second factor in oxygen deficiency is displacement by another gas. Examples of gases that
are used to displace air, and therefore reduce the oxygen level are helium, argon, and nitrogen.
Carbon dioxide may also be used to displace air and can occur naturally in sewers, storage bins,
wells, tunnels, wine vats, and grain elevators. Aside from the natural development of these
gases, or their use in the chemical process, certain gases are also used as inerting agents to
displace flammable substances and retard pyrophoric reactions.
Gases such as nitrogen, argon, helium, and carbon dioxide, are frequently referred to as non-
toxic inert gases but have claimed many lives. The use of nitrogen to inert a confined space has
claimed more lives than carbon dioxide. The total displacement of oxygen by nitrogen will cause
immediate collapse and death.
Carbon Dioxide
Carbon dioxide and argon, with specific gravities greater than air, may lie in a tank or manhole
for hours or days after opening. Since these gases are colorless and odorless, they pose an
immediate hazard to health unless appropriate oxygen measurements and ventilation are
adequately carried out.
Oxygen Deprivation
Oxygen deprivation is one form of asphyxiation. While it is desirable to maintain the atmospheric
oxygen level at 21% by volume, the body can tolerate deviation from this ideal. When the oxygen
level falls to 17%, the first sign of hypoxia is deterioration to night vision, which is not noticeable
until a normal oxygen concentration is restored. Physiologic effects are increased breathing
volume and accelerated heartbeat.

Between 14-16% physiologic effects are increased breathing volume, accelerated heartbeat,
very poor muscular coordination, rapid fatigue, and intermittent respiration. Between 6-10% the
effects are nausea, vomiting, inability to perform, and unconsciousness. Less than 6%, the
effects are spasmodic breathing, convulsive movements, and death in minutes.
Mechanical Hazards
If activation of electrical or mechanical equipment would cause injury, each piece of equipment
should be manually isolated to prevent inadvertent activation before workers enter or while they
work in a confined space. The interplay of hazards associated with a confined space, such as
the potential of flammable vapors or gases being present, and the build-up of static charge due
to mechanical cleaning, such as abrasive blasting, all influence the precautions which must be
taken.
To prevent vapor leaks, flashbacks, and other hazards, workers should completely isolate the
space. To completely isolate a confined space, the closing of valves is not sufficient. All pipes
must be physically disconnected or isolation blanks bolted in place. Other special precautions
must be taken in cases where flammable liquids or vapors may re-contaminate the confined
space.
The pipes blanked or disconnected should be inspected and tested for leakage to check the
effectiveness of the procedure. Other areas of concern are steam valves, pressure lines, and
chemical transfer pipes. A less apparent hazard is the space referred to as a void, such as double
walled vessels, which must be given special consideration in blanking off and inerting.
Thermal Effects
Four factors influence the interchange of heat between people and their environment. They are:
(1) air temperature, (2) air velocity, (3) moisture contained in the air, and (4) radiant heat.
Because of the nature and design of most confined spaces, moisture content and radiant heat
are difficult to control.
As the body temperature rises progressively, workers will continue to function until the body
temperature reaches approximately 102oF.
When this body temperature is exceeded, the workers are less efficient, and are prone to heat
exhaustion, heat cramps, or heat stroke. In a cold environment, certain physiologic mechanisms
come into play, which tend to limit heat loss and increase heat production. The most severe
strain in cold conditions is chilling of the extremities so that activity is restricted. Special
precautions must be taken in cold environments to prevent frostbite, trench foot, and general
hypothermia.
Protective Insulated Clothing

Protective insulated clothing for both hot and cold environments will add additional bulk to the
worker and must be considered in allowing for movement in the confined space and exit time.
Therefore, air temperature of the environment becomes an important consideration when
evaluating working conditions in confined spaces.
Noise
Noise problems are usually intensified in confined spaces because the interior tends to cause
sound to reverberate and thus expose the worker to higher sound levels than those found in an
open environment.
This intensified noise increases the risk of hearing damage to workers, which could result in
temporary or permanent loss of hearing. Noise in a confined space which may not be intense
enough to cause hearing damage may still disrupt verbal communication with the emergency
standby person on the exterior of the confined space. If the workers inside are not able to hear
commands or danger signals due to excessive noise, the probability of severe accidents can
increase.
Vibration
Whole body vibration may affect multiple body parts and organs, depending upon the vibration
characteristics. Segmental vibration, unlike whole body vibration, appears to be more localized
in creating injury to the fingers and hands of workers using tools, such as pneumatic hammers,
rotary grinders or other hand tools which cause vibration.
Other Hazards
Some physical hazards cannot be eliminated
because of the nature of the confined space
or the work to be performed. These hazards
include such items as scaffolding, surface
residues, and structural hazards. The use of
scaffolding in confined spaces has
contributed too many accidents caused by
workers or materials falling, improper use of
guard rails, and lack of maintenance to insure
worker safety.
The choice of material used for scaffolding

depends upon the type of work to be
performed, the calculated weight to be
supported, and the surface on which the
scaffolding is placed, as well as the
substance previously stored in the confined
space.
Surface residues in confined spaces can

increase the already hazardous conditions of
electrical shock, reaction of incompatible
materials, liberation of toxic substances, and
bodily injury due to slips and falls. Without
protective clothing, additional hazards to
health may arise due to surface residues.
Structural hazards within a confined space

such as baffles in horizontal tanks, trays in
vertical towers, bends in tunnels, overhead
structural members, or scaffolding installed for maintenance constitute physical hazards, which
are exacerbated by the physical surroundings. In dealing with structural hazards, workers must
review and enforce safety precautions to assure safety.
Abbreviations:
PEL - permissible exposure limit: Average concentration that must not be exceeded during 8-
hour work shift of a 40-hour workweek.
STEL - Short-term exposure limit: 15-minute exposure limit that must not be exceeded during
the workday.
REL - Recommended exposure limit: Average concentration limit recommended for up to a
10-hour workday during a 40-hour workweek.
IDLH - Immediately dangerous to life or health: Maximum concentration from which person
could escape (in event of respirator failure) without permanent or escape-impairing effects
within 30 minutes.

Required Confined Space Equipment Policy - Example
Air Testing Equipment
All air-testing equipment should be calibrated in accordance with the manufacturer's instruction.
Oxygen Meters and Monitors

The oxygen content of the air in a confined space is the first and most important constituent to
measure before entry is made. The acceptable range of oxygen is between 19.5 and 23.5 percent.
This content is measured before flammability is tested because rich mixtures of flammable gases
or vapors give erroneous measurement results.
For example, a mixture of 90 percent methane and 10 percent air will test nonflammable because
there is not enough oxygen to support the combustion process in the flammability meters. This
mixture will not support life and will soon become explosive if ventilation is provided to the space.
Before entry, spaces must be ventilated until both oxygen content and flammability are acceptable.
Flammability Meters
Flammability meters are used to measure the amount of flammable vapors or gases in the
atmosphere as a percent of the LEL/LFL. The oxygen content must be near 21 percent for results
to be meaningful.
Toxic Air Contamination Testers

Tests for toxic contaminants must be specific for the target toxin. The instrument manufacturer
should be consulted for interferences. Therefore, it is important to know the history of the confined
space so proper tests can be performed. Part of hazard assessment is to identify all possible
contaminants that could be in the confined space.
Protective Devices
Fall-Protection Equipment
Fall-protection equipment for confined spaces should be the chest-waist harness type to minimize
injuries from uncontrolled movements when it arrests a worker's fall. This type of harness also
permits easier retrieval from a confined space than a waist belt. Adjustable lanyards should be
used to limit free fall to two feet before arrest.
Respirators
An industrial hygienist should select respirators on the basis of his or her evaluation of possible
confined-space hazards. NIOSH-approved respirators should be identified in the approved
procedure required by the confined-space entry permit. It is important to note that air-purifying
respirators cannot be used in an oxygen deficient atmosphere.
Lockout/Tagout Devices
Lockout/tagout devices permit employees to work safely on de-energized equipment without fear
that the devices will be accidentally removed. Lock and tag devices are required to withstand a 50-
pound pull without failure. Devices used to block or restrain stored mechanical energy devices must
be engineered for safety.
Safety Barriers
Safety barriers separate workers from hazards that cannot reasonably be eliminated by other
engineering controls. Required barriers will be identified in the approved confined-space entry
procedure.
Ground Fault Circuit Interrupters

Ground fault circuit interrupter must be used for all portable electrical tools and equipment in
confined spaces because most workers will be in contact with grounded surroundings.

Emergency Response Equipment
Fire Extinguishers
"Hot work" inside a confined space requires that an approved fire extinguisher and a person trained
in its use be stationed in the confined space or in a suitable vantage point where he or she could
effectively suppress any fire that might result from the work.
First Aid Equipment

Blankets, first-aid kit, Stokes stretchers, and any other equipment that may be needed for first-
response treatment must be available just outside the confined space. Medical and safety
professionals should select equipment on the basis of their evaluations of the potential hazards in
the confined space.
Retrieval Equipment
A tripod or another suitable anchorage, hoisting device, harnesses, wristlets, ropes, and any other
equipment that may be needed to make a rescue must be identified in the confined-space safe-
entry procedures.
It is important that this equipment be available for immediate use. Harnesses and
retrieval ropes must be worn by entrants unless they would increase hazards to the
entrants or impede their rescue.

Glossary
2,4-D: A chlorinated phenoxy compound, functions as a systemic herbicide and is used to control many types
of broadleaf weeds. There are many forms or derivatives (esters, amines, salts) of 2,4-D and these vary in
solubility and volatility. Unless otherwise specified, this document will refer to the acid form of 2,4-D. This
compound is used in cultivated agriculture and in pasture and rangeland applications, forest management,
home and garden situations and for the control of aquatic vegetation. 2,4-D was a major component (about
50%) of the product Agent Orange used extensively throughout Vietnam. However most of the problems
associated with the use of Agent Orange were associated with a contaminant (dioxin) in the 2,4,5-T component
of the defoliant. The association of 2,4-D with Agent Orange has prompted a vast amount of study on the
herbicide.
ABIOGENESIS: The concept of spontaneous generation (that life can come from non-life). This idea was
refuted by Pasteur.
ABIOTIC: The non-living components of an organism's environment. The term abiotic is also used to denote
a process which is not facilitated by living organisms.
ABORAL: Pertaining to the region of the body opposite that of the mouth. Normally used to describe radially
symmetrical animals.
ABSCISIC ACID (ABA): A plant hormone that generally acts to inhibit growth, promote dormancy, and help
the plant withstand stressful conditions.
ABSENCE OF OXYGEN: The complete absence of oxygen in water described as Anaerobic.
ABSORPTION SPECTRUM: The range of a material's ability to absorb various wavelengths of light. The
absorption spectrum is studied to evaluate the function of photosynthetic pigments.
ACCURACY: How closely an instrument measures the true or actual value.
ACID ADDITION: Slowly add the acid to water while stirring. An operator should not mix acid and water or
acid to a strong base.
ACID AND BASE ARE MIXED: When an acid and a base are mixed, an explosive reaction occurs and
decomposition products are created under certain conditions.
ACID RAIN: Rain that is excessively acidic due to the presence of acid: causing pollutants in the atmosphere.
Pollutants include nitrogen and sulfur oxides due to burning of coal and oil.
ACID: n acid is a molecule or ion capable of donating a hydron (proton or hydrogen ion H+), or, alternatively,
capable of forming a covalent bond with an electron pair (a Lewis acid). The first category of acids is the
proton donors or Brønsted acids. In the special case of aqueous solutions, proton donors form the
hydronium ion H3O+ and are known as Arrhenius acids. Brønsted and Lowry generalized the Arrhenius
theory to include non-aqueous solvents. A Brønsted or Arrhenius acid usually contains a hydrogen atom
bonded to a chemical structure that is still energetically favorable after loss of H+.
ACIDOSIS: A condition whereby the hydrogen ion concentration of the tissues is increased (and pH
decreased). Respiratory acidosis is due to the retention of CO2; metabolic acidosis by retention of acids due
either to kidney failure or diarrhea.
ACTIVATED SLUDGE PROCESS: A biological wastewater treatment process in which a mixture of
wastewater and biologically enriched sludge is mixed and aerated to facilitate aerobic decomposition by
microbes.
ACTIVATED SLUDGE: The biologically active solids in an activated sludge process wastewater treatment
plant.
ACTIVATING ENZYME: An enzyme that couples a low-energy compound with ATP to yield a high-energy
derivative.
ACTIVATION ENERGY: In a chemical reaction, the initial investment required to energize the bonds of the
reactants to an unstable transition state that precedes the formation of the products.
ACTIVE SITE: That specific portion of an enzyme that attaches to the substrate by means of weak chemical
bonds.
ACTIVE TRANSPORT: The movement of a substance across a biological membrane against its
concentration or electrochemical gradient with the help of energy input and specific transport proteins.
ADAPTATION: Any genetically controlled characteristic that increases an organism's fitness, usually by
helping the organism to survive and reproduce in the environment it inhabits.
ADAPTIVE RADIATION: This refers to the rapid evolution of one or a few forms into many different species
that occupy different habitats within a new geographical area.
ADHESION: In chemistry, the phenomenon whereby one substance tends to cling to another substance.
Water molecules exhibit adhesion, especially toward charged surfaces.
ADP (Adenosine diphosphate): A doubly phosphorylated organic compound that can be further
phosphorylated to form ATP.
ADRENAL GLAND: An endocrine gland located adjacent to the kidney in mammals. It is composed of an
outer cortex, and a central medulla, each involved in different hormone: mediated phenomena.

ADRENALIN: A hormone produced by the pituitary that stimulates the adrenal cortex.
ADSORB: Hold on a surface.
ADSORPTION: Not to be confused with absorption. Adsorption is a process that occurs when a gas or liquid
solute accumulates on the surface of a solid or a liquid (adsorbent), forming a film of molecules or atoms (the
adsorbate). It is different from absorption, in which a substance diffuses into a liquid or solid to form a solution.
The term sorption encompasses both processes, while desorption is the reverse process. Adsorption is
present in many natural physical, biological, and chemical systems, and is widely used in industrial
applications such as activated charcoal, synthetic resins, and water purification. Adsorption, ion exchange,
and chromatography are sorption processes in which certain adsorbates are selectively transferred from the
fluid phase to the surface of insoluble, rigid particles suspended in a vessel or packed in a column. Similar to
surface tension, adsorption is a consequence of surface energy. In a bulk material, all the bonding
requirements (be they ionic, covalent, or metallic) of the constituent atoms of the material are filled by other
atoms in the material. However, atoms on the surface of the adsorbent are not wholly surrounded by other
adsorbent atoms, and therefore can attract adsorbates. The exact nature of the bonding depends on the
details of the species involved, but the adsorption process is generally classified as physisorption
(characteristic of weak van der Waals forces) or chemisorption (characteristic of covalent bonding).
AERATION: The addition of air or oxygen to water or wastewater, usually by mechanical means, to increase
dissolved oxygen levels and maintains aerobic conditions.
AEROBIC DIGESTION: Sludge stabilization process involving direct oxidation of biodegradable matter and
oxidation of microbial cellular material.
AEROBIC: The condition of requiring oxygen; an aerobe is an organism which can live and grow only in the
presence of oxygen.
AIR ENTRAINMENT: The dissolution or inclusion of air bubbles into water.
AIR GAP SEPARATION: A physical separation space that is present between the discharge vessel and the
receiving vessel; for an example, a kitchen faucet.
ALCOHOL: Any of a class of organic compounds in which one or more - OH groups are attached to a carbon
compound.
ALDEHYDE: An organic molecule with a carbonyl group located at the end of the carbon skeleton.
ALGAE: Microscopic plants that are free-living and usually live in water. They occur as single cells floating in
water, or as multicellular plants like seaweed or strands of algae that attach to rocks.
ALKALINE: Having a pH of more than 7. Alkaline solutions are also said to be basic.
ALKALINITY: Alkalinity or AT is a measure of the ability of a solution to neutralize acids to the equivalence
point of carbonate or bicarbonate. Alkalinity is closely related to the acid neutralizing capacity (ANC) of a
solution and ANC is often incorrectly used to refer to alkalinity. However, the acid neutralizing capacity refers
to the combination of the solution and solids present (e.g., suspended matter, or aquifer solids), and the
contribution of solids can dominate the ANC (see carbonate minerals below). The alkalinity is equal to the
stoichiometric sum of the bases in solution. In the natural environment carbonate alkalinity tends to make up
most of the total alkalinity due to the common occurrence and dissolution of carbonate rocks and presence of
carbon dioxide in the atmosphere. Other common natural components that can contribute to alkalinity include
borate, hydroxide, phosphate, silicate, nitrate, dissolved ammonia, the conjugate bases of some organic acids
and sulfide. Solutions produced in a laboratory may contain a virtually limitless number of bases that contribute
to alkalinity. Alkalinity is usually given in the unit mEq/L (milliequivalent per liter). Commercially, as in the pool
industry, alkalinity might also be given in the unit ppm or parts per million. Alkalinity is sometimes incorrectly
used interchangeably with basicity. For example, the pH of a solution can be lowered by the addition of CO2.
This will reduce the basicity; however, the alkalinity will remain unchanged.
ALLANTOIS: One of the four extraembryonic membranes found associated with developing vertebrates; it
serves in gas exchange and as a repository for the embryo's nitrogenous waste. In humans, the allantois is
involved in early blood formation and development of the urinary bladder.
ALLELE: Alternate forms of a gene which may be found at a given location (locus) on members of a
homologous set of chromosomes. Structural variations between alleles may lead to different phenotypes for
a given trait.
ALLOMETRIC: The variation in the relative rates of growth of various parts of the body, which helps shape
the organism.
ALLOSTERIC ENZYME: An enzyme that can exist in two or more conformations.
ALPHA AND BETA RADIOACTIVITY: Represent two common forms of radioactive decay. Radioactive
elements have atomic nuclei so heavy that the nucleus will break apart, or disintegrate spontaneously. When
decay occurs, high-energy particles are released. These high-energy particles are called radioactivity.
Although radioactivity from refined radioactive elements can be dangerous, it is rare to find dangerous levels
of radioactivity in natural waters. An alpha particle is a doubly-charged helium nucleus comprised of two
protons, two neutrons, and no electrons. A beta particle is a high-speed electron. Alpha particles do not
penetrate matter easily, and are stopped by a piece of paper. Beta particles are much more penetrating and
can pass through a millimeter of lead.

ALPHA EMITTERS: Certain minerals are radioactive and may emit a form of radiation known as alpha
radiation. Some people who drink water containing alpha emitters in excess of the EPA standard over many
years may have an increased risk of getting cancer.
ALPHA HELIX: A spiral shape constituting one form of the secondary structure of proteins, arising from a
specific hydrogen: bonding structure.
ALTERNATION OF GENERATIONS: Occurrences of a multicellular diploid form, the sporophyte, with a
multicellular haploid form, the gametophyte.
ALTERNATIVE DISINFECTANTS: Disinfectants - other than chlorination (halogens) - used to treat water,
e.g. ozone, ultraviolet radiation, chlorine dioxide, and chloramine. There is limited experience and scientific
knowledge about the by-products and risks associated with the use of alternatives.
ALTRUISM: The willingness of an individual to sacrifice its fitness for the benefit of another.
ALUMINUM SULFATE: The chemical name for Alum. The molecular formula of Alum is Al2(SO4)3~14H2O. It
is a cationic polymer.
ALVEOLUS: One of the dead-end, multilobed air sacs that constitute the gas exchange surface of the lungs.
AMINO ACID: An organic molecule possessing a carboxyl (COOH) and amino group. Amino acids serve as
the monomers of polypeptides and proteins.
AMINO GROUP: A functional group consisting of a nitrogen atom bonded to two hydrogens; can act as a
base in solution, accepting a hydrogen ion and acquiring a charge of +1.
AMINOACYL: tRNA synthetases- A family of enzymes, at least one for each amino acid, that catalyze the
attachment of an amino acid to its specific tRNA molecule.
AMMONIA: A chemical made with Nitrogen and Hydrogen and used with chlorine to disinfect water. Most
ammonia in water is present as the ammonium ion rather than as ammonia.
AMOEBA: Amoeba (sometimes amœba or ameba, plural amoebae) is a genus of protozoa that moves by
means of pseudopods, and is well-known as a representative unicellular organism. The word amoeba or
ameba is variously used to refer to it and its close relatives, now grouped as the Amoebozoa, or to all
protozoa that move using pseudopods, otherwise termed amoeboids.
AMOEBOID: (cell) A cell which has the tendency to change shape by protoplasmic flow. (movement) A
streaming locomotion characteristic of Amoeba and other protists, as well as some individual cells, such as
white blood cells, in animals.
AMP (Adenosine monophosphate): A singly phosphorylated organic compound that can be further
phosphorylated to form ADP.
AMYLASE: A starch-digesting enzyme.
ANABOLISM: A metabolic pathway of biosynthesis that consumes energy to build a large molecule from
simpler ones.
ANAEROBIC CONDITIONS: When anaerobic conditions exist in either the metalimnion or hypolimnion of a
stratified lake or reservoir, water quality problems may make the water unappealing for domestic use without
costly water treatment procedures. Most of these problems are associated with Reduction in the stratified
waters.
ANAEROBIC DIGESTION: Sludge stabilization process where the organic material in biological sludges are
converted to methane and carbon dioxide in an airtight reactor.
ANAEROBIC: Without oxygen. An organism that lives in the absence of oxygen is called an anaerobe. An
abnormal condition in which color and odor problems are most likely to occur.
ANAGENESIS: A pattern of evolutionary change involving the transformation of an entire population,
sometimes to a state different enough from the ancestral population to justify renaming it as a separate
species; also called phyletic.
ANALOGOUS: Characteristics of organisms that are similar in function (and often in structure) but different
in embryological and/or evolutionary origins.
ANALYST: The analyst must have at least 2 years of college lecture and laboratory course work in
microbiology or a closely related field. The analyst also must have at least 6 months of continuous bench
experience with environmental protozoa detection techniques and IFA microscopy, and must have
successfully analyzed at least 50 water and/or wastewater samples for Cryptosporidium and Giardia. Six
months of additional experience in the above areas may be substituted for two years of college.
ANCESTRAL TRAIT: Trait shared by a group of organisms as a result of descent from a common ancestor.
ANEUPLOIDY: A chromosomal aberration in which certain chromosomes are present in extra copies or are
deficient in number.
ANION: A negatively charged ion.
ANISOGAMOUS: Reproducing by the fusion of gametes that differ only in size, as opposed to gametes that
are produced by oogamous species. Gametes of oogamous species, such as egg cells and sperm, are highly
differentiated.
ANOXIC: A biological environment that is deficient in molecular oxygen, but may contain chemically bound
oxygen, such as nitrates and nitrites.
ANTERIOR: Referring to the head end of a bilaterally symmetrical animal.

ANTHROPOMORPHISM: Attributing a human characteristic to an inanimate object or a species other than
a human.
ANTIBIOTIC: A chemical that kills or inhibits the growth of bacteria, often via transcriptional or translational
regulation.
ANTIDIURETIC HORMONE: A hormone important in osmoregulation (it acts to reduce the elimination of
water from the body.
ANTIGEN: A foreign macromolecule that does not belong to the host organism and that elicits an immune
response.
ANTIMONY: A chemical element with the symbol Sb (Latin: stibium, meaning "mark") and atomic number 51.
A metalloid, antimony has four allotropic forms. The stable form of antimony is a blue-white metalloid. Yellow
and black antimony are unstable non-metals. Antimony is used in flame-proofing, paints, ceramics, enamels,
a wide variety of alloys, electronics, and rubber.
APOMORPHIC CHARACTER: A derived phenotypic character, or homology, that evolved after a branch
diverged from a phylogenetic tree.
APOSEMATIC COLORATION: Serving as a warning, with reference particularly to colors and structures that
signal possession of defensive device.
AQUEOUS SOLUTION: A solution in which water is the solvent.
ARCHAEBACTERIA: A lineage of prokaryotes, represented today by a few groups of bacteria inhabiting
extreme environments. Some taxonomists place archaebacteria in their own kingdom, separate from the other
bacteria.
ARCHENTERON: The endoderm-lined cavity formed during the gastrulation process that develops into the
digestive tract of the animal.
ARISTOTLE: A Greek philosopher often credited as the first to use empirical and deductive methods in logic.
ARTIFICIAL SELECTION: The selective breeding of domesticated plants and animals to encourage the
occurrence of desirable traits.
AS NITROGEN: An expression that tells how the concentration of a chemical is expressed mathematically.
The chemical formula for the nitrate ion is NO3 , with a mass of 62. The concentration of nitrate can be
expressed either in terms of the nitrate ion or in terms of the principal element, nitrogen. The mass of the
nitrogen atom is 14. The ratio of the nitrate ion mass to the nitrogen atom mass is 4.43. Thus a concentration
of 10 mg/L nitrate expressed as nitrogen would be equivalent to a concentration of 44.3 mg/L nitrate expressed
as nitrate ion. When dealing with nitrate numbers it is very important to know how numeric values are
expressed.
AS: The chemical symbol of Arsenic.
ASCUS: The elongate spore sac of a fungus of the Ascomycota group.
ASEXUAL: A type of reproduction involving only one parent that produces genetically identical offspring by
budding or division of a single cell or the entire organism into two or more parts.
ASSORTATIVE MATING: A type of nonrandom mating in which mating partners resemble each other in
certain phenotypic characters.
ASYMMETRIC CARBON: A carbon atom covalently bonded to four different atoms or groups of atoms.
ATOM: The general definition of an ion is an atom with a positive or negative charge. Electron is the name of
a negatively charged atomic particle.
ATOMIC NUMBER: The number of protons in the nucleus of an atom, unique for each element.
ATOMIC THEORY: The physical theory of the structure, properties and behavior of the atom.
ATOMIC WEIGHT: The total atomic mass, which is the mass in grams of one mole of the atom (relative to
that of 12C, which is designated as 12).
ATP (Adenosine triphosphate): A triply phosphorylated organic compound that functions as "energy
currency" for organisms, thus allowing life forms to do work; it can be hydrolyzed in two steps (first to ADP
and then to AMP) to liberate 7.3 Kcal of energy per mole during each hydrolysis.
ATPASE: An enzyme that functions in producing or using ATP.
AUTOGENOUS MODEL: A hypothesis which suggests that the first eukaryotic cells evolved by the
specialization of internal membranes originally derived from prokaryotic plasma membranes.
AUTOIMMUNE DISEASE: An immunological disorder in which the immune system goes awry and turns
against itself.
AUTOPOLYPLOID: A type of polyploid species resulting from one species doubling its chromosome number
to become tetraploids, which may self-fertilize or mate with other tetraploids.
AUTOSOME: Chromosomes that are not directly involved in determining sex.
AUTOTROPH: An organism which is able to make organic molecules from inorganic ones either by using
energy from the sun or by oxidizing inorganic substances.
AUXIN: One of several hormone compounds in plants that have a variety of effects, such as phototropic
response through stimulation of cell elongation, stimulation of secondary growth, and development of leaf
traces and fruit.

AUXOTROPH: A nutritional mutant that is unable to synthesize and that cannot grow on media lacking certain
essential molecules normally synthesized by wild-type strains of the same species.
AXON: A typically long outgrowth, or process, from a neuron that carries nerve impulses away from the cell
body toward target cells.
AXONEME: An internal flagellar structure that occurs in some protozoa, such as Giardia, Spironucleous, and
Trichonmonas.
B
BACKFLOW PREVENTION: To stop or prevent the occurrence of, the unnatural act of reversing the normal
direction of the flow of liquid, gases, or solid substances back in to the public potable (drinking) water supply.
See Cross-connection control.
BACKFLOW: To reverse the natural and normal directional flow of a liquid, gases, or solid substances back
in to the public potable (drinking) water supply. This is normally an undesirable effect.
BACKSIPHONAGE: A liquid substance that is carried over a higher point. It is the method by which the liquid
substance may be forced by excess pressure over or into a higher point.
BACTERIA: Small, one-celled animals too small to be seen by the naked eye. Bacteria are found everywhere,
including on and in the human body. Humans would be unable to live without the bacteria that inhabit the
intestines and assist in digesting food. Only a small percentage of bacteria cause disease in normal, healthy
humans. Other bacteria can cause infections if they get into a cut or wound. Bacteria are the principal concern
in evaluating the microbiological quality of drinking water, because some of the bacteria-caused diseases that
can be transmitted by drinking water are potentially life-threatening.
BACTERIOPHAGE: A bacteriophage (from 'bacteria' and Greek phagein, 'to eat') is any one of a number of
viruses that infect bacteria. The term is commonly used in its shortened form, phage. Typically, bacteriophages
consist of an outer protein hull enclosing genetic material. The genetic material can be ssRNA (single stranded
RNA), dsRNA, ssDNA, or dsDNA between 5 and 500 kilo base pairs long with either circular or linear
arrangement. Bacteriophages are much smaller than the bacteria they destroy - usually between 20 and 200
nm in size.
BACTERIUM: A unicellular microorganism of the Kingdom Monera. Bacteria are prokaryotes; their cells have
no true nucleus. Bacteria are classified into two groups based on a difference in cell walls, as determined by
Gram staining.
BALANCED POLYMORPHISM: A type of polymorphism in which the frequencies of the coexisting forms do
not change noticeably over many generations.
BARIUM: A chemical element. It has the symbol Ba, and atomic number 56. Barium is a soft silvery metallic
alkaline earth metal. It is never found in nature in its pure form due to its reactivity with air. Its oxide is
historically known as baryta but it reacts with water and carbon dioxide and is not found as a mineral. The
most common naturally occurring minerals are the very insoluble barium sulfate, BaSO4 (barite), and barium
carbonate, BaCO3 (witherite). Benitoite is a rare gem containing barium.
BARR BODY: The dense object that lies along the inside of the nuclear envelope in cells of female mammals,
representing the one inactivated X chromosome.
BASAL BODY: A cell structure identical to a centriole that organizes and anchors the microtubule assembly
of a cilium or flagellum.
BASE PAIRING: Complementary base pairing refers to the chemical affinities between specific base pairs in
a nucleic acid: adenine always pairs with thymine, and guanine always pairs with cytosine. In pairing between
DNA and RNA, the uracil of RNA always pairs with adenine. Complementary base pairing is not only
responsible for the DNA double helix, but it is also essential for various in vitro techniques such as PCR
(polymerase chain reaction). Complementary base pairing is also known as Watson-Crick pairing.
BASE: A substance that reduces the hydrogen ion concentration in a solution.
BASEMENT MEMBRANE: The floor of an epithelial membrane on which the basal cells rest.
B-CELL LYMPHOCYTE: A type of lymphocyte that develops in the bone marrow and later produces
antibodies, which mediate humoral immunity.
BELT PRESS: A dewatering device utilizing two opposing synthetic fabric belts, revolving over a series of
rollers to “squeeze” water from the sludge.
BENCH TEST: A small-scale test or study used to determine whether a technology is suitable for a particular
application.
BENIGN TUMOR: A noncancerous abnormal growth composed of cells that multiply excessively but remain
at their place of origin in the body.
BENTHIC: Pertaining to the bottom region of an aquatic environment.
BERYLLIUM: A chemical element with the symbol Be and atomic number 4. A bivalent element, beryllium is
a steel grey, strong, light-weight yet brittle alkaline earth metal. It is primarily used as a hardening agent in
alloys, most notably beryllium copper. Commercial use of beryllium metal presents technical challenges due
to the toxicity (especially by inhalation) of beryllium-containing dusts.

BEST AVAILABLE TECHNOLOGY ECONOMICALLY ACHIEVABLE (BAT): A level of technology based on
the best existing control and treatment measures that are economically achievable within the given industrial
category or subcategory.
BEST MANAGEMENT PRACTICES (BMPs): Schedules of activities, prohibitions of practices, maintenance
procedures, and other management practices to prevent or reduce the pollution of waters of the U.S. BMPs
also include treatment requirements, operating procedures and practices to control plant site runoff, spillage
or leaks, sludge or waste disposal, or drainage from raw material storage.
BEST PRACTICABLE CONTROL TECHNOLOGY CURRENTLY AVAILABLE (BPT): A level of technology
represented by the average of the best existing wastewater treatment performance levels within an industrial
category or subcategory.
BEST PROFESSIONAL JUDGMENT (BPJ): The method used by a permit writer to develop technology-
based limitations on a case-by-case basis using all reasonably available and relevant data.
BETA PLEATED SHEET: A zigzag shape, constituting one form of the secondary structure of proteins formed
of hydrogen bonds between polypeptide segments running in opposite directions.
BETA/PHOTON EMITTER: Certain minerals are radioactive and may emit forms of radiation known as
photons and beta radiation. Some people who drink water containing beta and photon emitters in excess of
the EPA standard over many years may have an increased risk of getting cancer.
BILATERAL SYMMETRY: The property of having two similar sides, with definite upper and lower surfaces
and anterior and posterior ends. The Bilateria are members of the branch of Eumetazoa (Kingdom Animalia)
which possess bilateral symmetry.
BILE: A mixture of substances containing bile salts, which emulsify fats and aid in their digestion and
absorption.
BINARY FISSION: The kind of cell division found in prokaryotes, in which dividing daughter cells each receive
a copy of the single parental chromosome.
BINOMIAL NOMENCLATURE: Consisting of two names. In biology, each organism is given a genus name
and a species name (i.e., the human is Homo sapiens.
BIOCHEMICAL OXYGEN DEMAND (BOD): The BOD test is used to measure the strength of wastewater.
The BOD of wastewater determines the milligrams per liter of oxygen required during stabilization of
decomposable organic matter by aerobic bacteria action. Also, the total milligrams of oxygen required over a
five-day test period to biologically assimilate the organic contaminants in one liter of wastewater maintained
at 20 degrees Centigrade.
BIOGENESIS: A central concept of biology, that living organisms are derived from other living organisms
(contrasts to the concept of abiogenesis, or spontaneous generation, which held that life could be derived
from inanimate material).
BIOLOGICAL MAGNIFICATION: Increasing concentration of relatively stable chemicals as they are passed
up a food chain from initial consumers to top predators.
BIOMASS: The total weight of all the organisms, or of a designated group of organisms, in a given area
BIOME: A large climatic region with characteristic sorts of plants and animals.
BIOSOLIDS: Solid organic matter recovered from municipal wastewater treatment that can be beneficially
used, especially as a fertilizer. “Biosolids” are solids that have been stabilized within the treatment process,
whereas “sludge” has not.
BIOSPHERE: The region on and surrounding the earth which is capable of supporting life. Theoretically, the
concept may be ultimately expanded to include other regions of the universe.
BMR: The basal metabolic rate is the minimal energy (in kcal) required by a homeotherm to fuel itself for a
given time. Measured within the thermoneutral zone for a postabsorptive animal at rest.
BODY FEED: Coating or bulking material added to the influent of material to be treated. This adds “body” to
the material during filtration cycle.
Both measurements (mg/L or KH) are usually expressed "as CaCO3" – meaning the amount of hardness
expressed as if calcium carbonate was the sole source of hardness. Every bicarbonate ion only counts for half
as much carbonate hardness as a carbonate ion does. If a solution contained 1 liter of water and 50 mg
NaHCO3 (baking soda), it would have a carbonate hardness of about 18 mg/L as CaCO3. If you had a liter of
water containing 50 mg of Na2CO3, it would have a carbonate hardness of about 29 mg/L as CaCO3.
Carbonate hardness supplements non-carbonate (a.k.a. "permanent") hardness where hard ions are
associated with anions such as Chloride that do not precipitate out of solution when heated. Carbonate
hardness is removed from water through the process of softening. Softening can be achieved by adding lime
in the form of Ca(OH)2, which reacts first with CO2 to form calcium carbonate precipitate, reacts next with
multi-valent cations to remove carbonate hardness, then reacts with anions to replace the non-carbonate
hardness due to multi-valent cations with non-carbonate hardness due to calcium. The process requires
recarbonation through the addition of carbon-dioxide to lower the pH which is raised during the initial softening
process.
BREAK POINT CHLORINATION: The process of chlorinating the water with significant quantities of chlorine
to oxidize all contaminants and organic wastes and leave all remaining chlorine as free chlorine.

BROMATE: An inorganic anion, bromate is tasteless and colorless, with a low volatility. As a moderately
strong oxidant, bromate is reactive. BrO3- is a bromine-based oxoanion. A bromate is a chemical compound
that contains this ion. Examples of bromates include sodium bromate, (NaBrO3), and potassium bromate,
(KBrO3).
BROMINE: Chemical disinfectant (HALOGEN) that kills bacteria and algae. This chemical disinfectant has
been used only on a very limited scale for water treatment because of its handling difficulties. This chemical
causes skin burns on contact, and a residual is difficult to obtain.
BUFFER: Chemical that resists pH change, e.g. sodium bicarbonate
BULKING SLUDGE: A poor or slow settling activated sludge that results from the prevalence of
filamentous organisms. A phenomenon that occurs in activated sludge plants whereby the sludge occupies
excessive volumes and will not concentrate readily. This condition refers to a decrease in the ability of the
sludge to settle and consequent loss over the settling tank weir. Bulking in activated sludge aeration tanks is
caused mainly by excess suspended solids (SS) content. Sludge bulking in the final settling tank of an
activated sludge plant may be caused by improper balance of the BOD load, SS concentration in the mixed
liquor, or the amount of air used in aeration.
C
Ca: The chemical symbol for calcium.
CADMIUM: A chemical element with the symbol Cd and atomic number 48. A relatively abundant, soft, bluish-
white, transition metal, cadmium is known to cause cancer and occurs with zinc ores. Cadmium is used largely
in batteries and pigments, for example in plastic products.
CAKE: Dewatered sludge material with a satisfactory solids concentration to allow handling as a solid
material.
CALCIUM HARDNESS: A measure of the calcium salts dissolved in water.
CALCIUM ION: Is divalent because it has a valence of +2.
CALCIUM, MAGNESIUM AND IRON: The three elements that cause hardness in water.
CaOCl2.4H2O: The molecular formula of Calcium hypochlorite.
CARBON DIOXIDE GAS: The pH will decrease and alkalinity will change as measured by the Langelier index
after pumping carbon dioxide gas into water.
CARBONATE HARDNESS: Carbonate hardness is the measure of Calcium and Magnesium and other hard
ions associated with carbonate (CO32-) and bicarbonate (HCO3-) ions contained in a solution, usually water.
It is usually expressed either as parts per million (ppm or mg/L), or in degrees (KH - from the German
"Karbonathärte"). One German degree of carbonate hardness is equivalent to about 17.8575 mg/L.
CARBONATE, BICARBONATE AND HYDROXIDE: Chemicals that are responsible for the alkalinity of
water.
CATHODIC PROTECTION: An operator should protect against corrosion of the anode and/or the cathode
by painting the copper cathode. Cathodic protection interrupts corrosion by supplying an electrical current to
overcome the corrosion-producing mechanism. Guards against stray current corrosion.
CAUSTIC SODA: Also known as sodium hydroxide and is used to raise pH.
CAUSTIC: NaOH (also called Sodium Hydroxide) is a strong chemical used in the treatment process to
neutralize acidity, increase alkalinity or raise the pH value.
CENTRATE: The liquid remaining after solids have been removed in a centrifuge.
CENTRIFUGAL FORCE: That force when a ball is whirled on a string that pulls the ball outward. On a
centrifugal pump, that force throws water from a spinning impeller.
CENTRIFUGAL PUMP: A pump consisting of an impeller fixed on a rotating shaft and enclosed in a casing,
having an inlet and a discharge connection. The rotating impeller creates pressure in the liquid by the velocity
derived from centrifugal force.
CENTRIFUGE: A dewatering device relying on centrifugal force to separate particles of varying density such
as water and solids.
CHAIN OF CUSTODY (COC): A record of each person involved in the possession of a sample from the
person who collects the sample to the person who analyzes the sample in the laboratory.
CHECK VALVE: Allows water to flow in only one direction.
CHELATION: A chemical process used to control scale formation in which a chelating agent "captures" scale-
causing ions and holds them in solution.
CHEMICAL FEED RATE: Chemicals are added to the water in order to improve the subsequent treatment
processes. These may include pH adjusters and coagulants. Coagulants are chemicals, such as alum, that
neutralize positive or negative charges on small particles, allowing them to stick together and form larger
particles that are more easily removed by sedimentation (settling) or filtration. A variety of devices, such as
baffles, static mixers, impellers and in-line sprays, can be used to mix the water and distribute the chemicals
evenly.
CHEMICAL OXIDIZER: KMnO4 is used for taste and odor control because it is a strong oxidizer that
eliminates many organic compounds.

CHEMICAL OXYGEN DEMAND (COD): The milligrams of oxygen required to chemically oxidize the
organic contaminants in one liter of wastewater.
CHEMICAL REACTION RATE: In general, when the temperature decreases, the chemical reaction rate
also decreases. The opposite is true for when the temperature increases.
CHEMICAL SLUDGE: Sludge resulting from chemical treatment processes of inorganic wastes that are
not biologically active.
CHLORAMINATION: Treating drinking water by applying chlorine before or after ammonia. This creates a
persistent disinfectant residual called chloramines.
CHLORAMINES: A group of chlorine ammonia compounds formed when chlorine combines with organic
wastes in the water. Chloramines are not effective as disinfectants and are responsible for eye and skin
irritation as well as strong chlorine odors (also known as Combined Chlorine).
CHLORINATION: The process in water treatment of adding chlorine (gas or solid hypochlorite) for purposes
of disinfection.
CHLORINE DEMAND: Amount of chlorine required to react on various water impurities before a residual is
obtained. Also, means the amount of chlorine required to produce a free chlorine residual of 0.1 mg/l after a
contact time of fifteen minutes as measured by Iodometric method of a sample at a temperature of twenty
degrees in conformance with Standard methods.
CHLORINE FEED: Chlorine may be delivered by vacuum-controlled solution feed chlorinators. The
chlorine gas is controlled, metered, introduced into a stream of injector water and then conducted as a
solution to the point of application.
CHLORINE, FREE: Chlorine available to kill bacteria or algae. The amount of chlorine available for
sanitization after the chlorine demand has been met. Also known as chlorine residual.
CHLORINE: A chemical used to disinfect water. Chlorine is extremely reactive, and when it comes in contact
with microorganisms in water it kills them. Chlorine is added to swimming pools to keep the water safe for
swimming. Chlorine is available as solid tablets for swimming pools. Some public water system’s drinking
water treatment plants use chlorine in a gas form because of the large volumes required. Chlorine is very
effective against algae, bacteria and viruses. Protozoa are resistant to chlorine because they have thick coats;
protozoa are removed from drinking water by filtration.
CHLORITE: The chlorite ion is ClO2−. A chlorite (compound) is a compound that contains this group, with
chlorine in oxidation state +3. Chlorites are also known as salts of chlorous acid.
CHROMIUM: A chemical element which has the symbol Cr and atomic number 24. It is a steel-gray, lustrous,
hard metal that takes a high polish and has a high melting point. It is also odorless, tasteless, and malleable.
CHRONIC: A stimulus that lingers or continues for a relatively long period of time, often one-tenth of the life
span or more. Chronic should be considered a relative term depending on the life span of an organism. The
measurement of chronic effect can be reduced growth, reduced reproduction, etc., in addition to lethality.
CIRCULATION: The continual flow of drilling fluid from injection to recovery and recirculation at the surface.
CLARIFIER: A settling tank used to remove suspended solids by gravity settling. Commonly referred to as
sedimentation or settling basins, they are usually equipped with a motor driven chain and flight or rake
mechanism to collect settled sludge and move it to a final removal point.
ClO2: The molecular formula of Chlorine dioxide.
COAGULATION: The best pH range for coagulation is between 5 and 7. Mixing is an important part of the
coagulation process you want to complete the coagulation process as quickly as possible. A chemical added
to initially destabilize, aggregate, and bind together colloids and emulsions to improve settleability, filterability,
or drainability.
COLIFORM TESTING: The effectiveness of disinfection is usually determined by Coliform bacteria testing.
A positive sample is a bad thing and indicates that you have bacteria contamination.
COLIFORM: Bacteria normally found in the intestines of warm-blooded animals. Coliform bacteria are present
in high numbers in animal feces. They are an indicator of potential contamination of water. Adequate and
appropriate disinfection effectively destroys coliform bacteria. Public water systems are required to deliver
safe and reliable drinking water to their customers 24 hours a day, 365 days a year. If the water supply
becomes contaminated, consumers can become seriously ill. Fortunately, public water systems take many
steps to ensure that the public has safe, reliable drinking water. One of the most important steps is to regularly
test the water for coliform bacteria. Coliform bacteria are organisms that are present in the environment and
in the feces of all warm-blooded animals and humans. Coliform bacteria will not likely cause illness. However,
their presence in drinking water indicates that disease-causing organisms (pathogens) could be in the water
system. Most pathogens that can contaminate water supplies come from the feces of humans or animals.
Testing drinking water for all possible pathogens is complex, time-consuming, and expensive. It is relatively
easy and inexpensive to test for coliform bacteria. If coliform bacteria are found in a water sample, water
system operators work to find the source of contamination and restore safe drinking water. There are three
different groups of coliform bacteria; each has a different level of risk.

COLLOIDAL SUSPENSIONS: Because both iron and manganese react with dissolved oxygen to form
insoluble compounds, they are not found in high concentrations in waters containing dissolved oxygen
except as colloidal suspensions of the oxide.
COLORIMETRIC MEASUREMENT: A means of measuring an unknown chemical concentration in water
by measuring a sample's color intensity.
COMBINED CHLORINE: The reaction product of chlorine with ammonia or other pollutants, also known as
chloramines.
COMBINED RADIUM 226/228: Some people who drink water containing radium 226 or 228 in excess of EPA
standard over many years may have an increased risk of getting cancer.
COMPOSITE SAMPLE: A water sample that is a combination of a group of samples collected at various
intervals during the day. A combination of individual samples of water or wastewater taken at predetermined
intervals to minimize the effect of variability of individual samples. To have significant meaning, samples for
laboratory tests on wastewater should be representative of the wastewater. The best method of sampling is
proportional composite sampling over several hours during the day. Composite samples are collected because
the flow and characteristics of the wastewater are continually changing. A composite sample will give a
representative analysis of the wastewater conditions.
COMPOSTING: Stabilization process relying on the aerobic decomposition of organic matter in sludge by
bacteria and fungi.
CONDENSATION: The process that changes water vapor to tiny droplets or ice crystals.
CONTACT STABILIZATION PROCESS: Modification of the activated sludge process where raw
wastewater is aerated with activated sludge for a short time prior to solids removal and continued aeration in
a stabilization tank.
CONTACT TIME (CT): To inactivate viruses and bacteria, the minimum disinfection contact time measured
before the first customer should be six milligrams per minute per liter (6 mg-min/L). This value is called
“Chlorine Contact Time” or CT. To calculate CT, multiply the free chlorine residual concentration (C) times the
contact time (T). To get the required CT value of 6, adjust the free chlorine residual concentration or the
contact time.
CONTACT TIME: If the water temperature decreases from 70°F (21°C) to 40°F (4°C). The operator needs
to increase the detention time to maintain good disinfection of the water.
CONTAMINANT: Any natural or man-made physical, chemical, biological, or radiological substance or
matter in water, which is at a level that may have an adverse effect on public health, and which is known or
anticipated to occur in public water systems.
CONTAMINATION: A degradation in the quality of groundwater in result of the it’s becoming polluted with
unnatural or previously non-existent constituents.
COPPER: The chemical name for the symbol Cu.
CORROSION: The removal of metal from copper, other metal surfaces and concrete surfaces in a
destructive manner. Corrosion is caused by improperly balanced water or excessive water velocity
through piping or heat exchangers.
CORROSIVITY: The Langelier Index measures corrosivity.
CROSS-CONNECTION: A physical connection between a public water system and any source of water or
other substance that may lead to contamination of the water provided by the public water system through
backflow. Might be the source of an organic substance causing taste and odor problems in a water
distribution system.
CROSS-CONTAMINATION: The mixing of two unlike qualities of water. For example, the mixing of good
water with a polluting substance like a chemical.
CRYPTOSPORIDIUM: A disease-causing parasite, resistant to chlorine disinfection. It may be found in fecal
matter or contaminated drinking water. Cryptosporidium is a protozoan pathogen of the Phylum Apicomplexa
and causes a diarrheal illness called cryptosporidiosis. Other apicomplexan pathogens include the malaria
parasite Plasmodium, and Toxoplasma, the causative agent of toxoplasmosis. Unlike Plasmodium, which
transmits via a mosquito vector, Cryptosporidium does not utilize an insect vector and is capable of completing
its life cycle within a single host, resulting in cyst stages that are excreted in feces and are capable of
transmission to a new host.
CRYPTOSPORIDIUM: A parasite that enters lakes and rivers through sewage and animal waste. It causes
cryptosporidiosis, a mild gastrointestinal disease. However, the disease can be severe or fatal for people with
severely weakened immune systems. The EPA and the CDC have prepared advice for those with severely
compromised immune systems who are concerned about Cryptosporidium.
CYANOBACTERIA: Cyanobacteria, also known as blue-green algae, blue-green bacteria or Cyanophyta, is
a phylum of bacteria that obtain their energy through photosynthesis. The name "cyanobacteria" comes from
the color of the bacteria (Greek: kyanós = blue). They are a significant component of the marine nitrogen cycle
and an important primary producer in many areas of the ocean, but are also found on land.
CYANURIC ACID: Chemical used to prevent the decomposition of chlorine by ultraviolet (UV) light.

CYST: A phase or a form of an organism produced either in response to environmental conditions or as a
normal part of the life cycle of the organism. It is characterized by a thick and environmentally resistant cell
wall.
D
DAILY MAXIMUM LIMITATIONS: The maximum allowable discharge of pollutants during a 24 hour period.
Where daily maximum limitations are expressed in units of mass, the daily discharge is the total mass
discharged over the course of the day. Where daily maximum limitations are expressed in terms of a
concentration, the daily discharge is the arithmetic average measurement of the pollutant concentration
derived from all measurements taken that day.
DANGEROUS CHEMICALS: The most suitable protection when working with a chemical that produces
dangerous fumes is to work under an air hood.
DECANT: Separation of a liquid from settled solids by removing the upper layer of liquid after the solids
have settled.
DECOMPOSE: To decay or rot.
DECOMPOSITION OF ORGANIC MATERIAL: The decomposition of organic material in water produces
taste and odors.
DEMINERALIZATION PROCESS: Mineral concentration of the feed water is the most important consideration
in the selection of a demineralization process. Acid feed is the most common method of scale control in a
membrane demineralization treatment system.
DENITRIFICATION: A biological process by which nitrate is converted to nitrogen gas.
DEPOLARIZATION: The removal of hydrogen from a cathode.
DESICCANT: When shutting down equipment that may be damaged by moisture, the unit may be protected
by sealing it in a tight container. This container should contain a desiccant.
DESORPTION: Desorption is a phenomenon whereby a substance is released from or through a surface.
The process is the opposite of sorption (that is, adsorption and absorption). This occurs in a system being in
the state of sorption equilibrium between bulk phase (fluid, i.e. gas or liquid solution) and an adsorbing surface
(solid or boundary separating two fluids). When the concentration (or pressure) of substance in the bulk phase
is lowered, some of the sorbed substance changes to the bulk state. In chemistry, especially chromatography,
desorption is the ability for a chemical to move with the mobile phase. The more a chemical desorbs, the less
likely it will adsorb, thus instead of sticking to the stationary phase, the chemical moves up with the solvent
front. In chemical separation processes, stripping is also referred to as desorption as one component of a
liquid stream moves by mass transfer into a vapor phase through the liquid-vapor interface.
DIATOMACEOUS EARTH: A fine silica material containing the skeletal remains of algae.
DIGESTER: A tank or vessel used for sludge digestion.
DIGESTION: The biological decomposition of organic matter in sludge resulting in partial gasification,
liquefaction, and mineralization of putrescible and offensive solids.
DIRECT CURRENT: A source of direct current (DC) may be used for standby lighting in a water treatment
facility. The electrical current used in a DC system may come from a battery.
DISINFECT: The application of a chemical to kill most, but not all, microorganisms that may be present.
Chlorine is added to public water drinking systems drinking water for disinfection. Depending on your state
rule, drinking water must contain a minimum of 0.2 mg/L free chlorine. Disinfection makes drinking water safe
to consume from the standpoint of killing pathogenic microorganisms including bacteria and viruses.
Disinfection does not remove all bacteria from drinking water, but the bacteria that can survive disinfection
with chlorine are not pathogenic bacteria that can cause disease in normal healthy humans.
DISINFECTION BYPRODUCTS: Disinfection byproducts are chemical, organic and inorganic substances that
can form during a reaction of a disinfectant with naturally present organic matter in the water.
DISINFECTION: The treatment of water to inactivate, destroy, and/or remove pathogenic bacteria, viruses,
protozoa, and other parasites.
DISSOLVED OXYGEN: Can be added to zones within a lake or reservoir that would normally become
anaerobic during periods of thermal stratification.
DISSOLVED SOLIDS: Solids in solution that cannot be removed by filtration with a 0.45 micron filter.
DISTILLATION, REVERSE OSMOSIS AND FREEZING: Processes that can be used to remove minerals
from the water.
DPD METHOD: Presence of free chlorine in the distribution network is indication of correct disinfection.
Chlorine in water is determined according to ISO 7393-2 by colorimetric HACH method on the basis of DPD
(N, N-diethyl - p – phenylendiamine). The photometric detection uses the wave lengths of 490 – 555 nm. Hach
elected, for most of his DPD colorimetric systems, the wavelength of 530 nm.
DRY ACID: A granular chemical used to lower pH and or total alkalinity.

E
E. COLI, Escherichia coli: A bacterium commonly found in the human intestine. For water quality analyses
purposes, it is considered an indicator organism. These are considered evidence of water contamination.
Indicator organisms may be accompanied by pathogens, but do not necessarily cause disease themselves.
ECOLOGY: The study of how organisms interact with their environments.
ECOSYSTEM: The sum of physical features and organisms occurring in a given area.
ECTODERM: The outermost tissue layer of an animal embryo. Also, tissue derived from an embryonic
ectoderm.
EFFECTIVENESS OF CHLORINE: The factors which influence the effectiveness of chlorination the most
are pH, turbidity and temperature. Effectiveness of Chlorine decreases occurs during disinfection in source
water with excessive turbidity.
EFFECTOR: The part of an organism that produces a response to a stimulus.
EFFLUENT: Partially or completely treated water or wastewater flowing out of a basin or treatment plant.
ELECTRON MICROSCOPE: A microscope that focuses an electron beam through a specimen, resulting in
resolving power a thousandfold greater than that of a light microscope. A transmission EM is used to study
the internal structure of thin sections of cells; a scanning EM is used to study the ultrastructure of surfaces.
ELECTRON TRANSPORT CHAIN: A series of enzymes found in the inner membranes of mitochondria and
chloroplasts. These are involved in transport of protons and electrons either across the membrane during
ATP synthesis.
ELECTRON: The name of a negatively charged atomic particle. A negatively charged subatomic particle of
an atom or ion. In atoms, the number of electrons present is equal to the number of positively charged
protons present. Hence, atoms are electrically neutral.
ELECTRONEGATIVITY: A property exhibited by some atoms whereby the nucleus has a tendency to pull
electrons toward itself.
ELECTRONIC CHARGE UNIT: The charge of one electron (1.6021 x 10e - 19 coulomb).
ELECTROSTATIC FORCE: The attraction between particles with opposite charges.
ELECTROSTATIC GRADIENT: The free-energy gradient created by a difference in charge between two
points, generally the two sides of a membrane.
ELEMENT: Any substance that cannot be broken down into another substance by ordinary chemical
means.
ELIMINATION: The release of unabsorbed wastes from the digestive tract.
EMULSION: A suspension, usually as fine droplets of one liquid in another. A mixture made up of dissimilar
elements, usually of two or more mutually insoluble liquids that would normally separate into layers based on
the specific gravity of each liquid.
ENDERGONIC: A phenomenon that involves uptake of energy.
ENDOCRINE: A phenomenon that relates to the presence of ductless glands of the type typically found in
vertebrates. The endocrine system involves hormones, the glands that secrete them, the molecular hormone
receptors of target cells, and interactions between hormones and the nervous system.
ENDONUCLEASE: An enzyme that breaks bonds within nucleic acids. A restriction endonuclease is an
enzyme that breaks bonds only within a specific sequence of bases.
ENDOPLASMIC RETICULUM: A system of membrane-bounded tubes and flattened sacs, often continuous
with the nuclear envelope, found in the cytoplasm of eukaryotes. Exists as rough ER, studded with ribosomes,
and smooth ER, lacking ribosomes.
ENDORPHIN: A hormone produced in the brain and anterior pituitary that inhibits pain perception.
ENDOSKELETON: An internal skeleton.
ENDOSPERM: A nutritive material in plant seeds which is triploid (3n) and results from the fusion of three
nuclei during double fertilization.
ENDOSYMBIOTIC: 1) An association in which the symbiont lives within the host 2) A widely accepted
hypothesis concerning the evolution of the eukaryotic cell: the idea that eukaryotes evolved as a result of
symbiotic associations between prokaryote cells. Aerobic symbionts ultimately evolved into mitochondria;
photosynthetic symbionts became chloroplasts.
ENERGY: The capacity to do work by moving matter against an opposing force.
ENTAMOEBA HISTOLYTICA: Entamoeba histolytica, another water-borne pathogen, can cause diarrhea or
a more serious invasive liver abscess. When in contact with human cells, these amoebae are cytotoxic. There
is a rapid influx of calcium into the contacted cell, it quickly stops all membrane movement save for some
surface blebbing. Internal organization is disrupted, organelles lyse, and the cell dies. The ameba may eat the
dead cell or just absorb nutrients released from the cell.
ENTERIC: Rod-shaped, gram-negative, aerobic but can live in certain anaerobic conditions; produce nitrite
from nitrate, acids from glucose; include Escherichia coli, Salmonella (over 1000 types), and Shigella.
ENTEROVIRUS: A virus whose presence may indicate contaminated water; a virus that may infect the
gastrointestinal tract of humans.
ENTROPY: A type of energy that is not biologically useful to do work (in contrast to free energy).

ENVELOPE: 1) (nuclear) The surface, consisting of two layers of membrane, that encloses the nucleus of
eukaryotic cells. 2) (virus) A structure which is present on the outside of some viruses (exterior to the capsid).
ENVIRONMENT: Water, air, and land, and the interrelationship that exists among and between water, air and
land and all living things. The total living and nonliving aspects of an organism's internal and external
surroundings.
ENZYME: A protein, on the surface of which are chemical groups so arranged as to make the enzyme a
catalyst for a chemical reaction.
EPIDERMIS: The outermost portion of the skin or body wall of an animal.
EPISOME: Genetic element at times free in the cytoplasm, at other times integrated into a chromosome.
EPISTASIS: A phenomenon in which one gene alters the expression of another gene that is independently
inherited.
EPITHELIUM: An animal tissue that forms the covering or lining of all free body surfaces, both external and
internal.
EQUATION: A precise representation of the outcome of a chemical reaction, showing the reactants and
products, as well as the proportions of each.
EQUILIBRIUM: In a reversible reaction, the point at which the rate of the forward reaction equals that of the
reverse reaction. (constant) At equilibrium, the ratio of products to reactants. (potential) The membrane
potential for a given ion at which the voltage exactly balances the chemical diffusion gradient for that ion.
ESSENTIAL: 1) An amino or fatty acid which is required in the diet of an animal because it cannot be
synthesized. 2) A chemical element required for a plant to grow from a seed and complete the life cycle.
ESTIVATION: A physiological state characterized by slow metabolism and inactivity, which permits survival
during long periods of elevated temperature and diminished water supplies.
EUBACTERIA: The lineage of prokaryotes that includes the cyanobacteria and all other contemporary
bacteria except archaebacteria.
EUCHROMATIN: The more open, unraveled form of eukaryotic chromatin, which is available for
transcription.
EUCOELOMATE: An animal whose body cavity is completely lined by mesoderm, the layers of which
connect dorsally and ventrally to form mesenteries.
EUGLENA: Euglena are common protists, of the class Euglenoidea of the phylum Euglenophyta. Currently,
over 1000 species of Euglena have been described. Marin et al. (2003) revised the genus so and including
several species without chloroplasts, formerly classified as Astasia and Khawkinea. Euglena sometimes can
be considered to have both plant and animal features. Euglena gracilis has a long hair-like thing that stretches
from its body. You need a very powerful microscope to see it. This is called a flagellum, and the euglena uses
it to swim. It also has a red eyespot. Euglena gracilis uses its eyespot to locate light. Without light, it cannot
use its chloroplasts to make itself food.
EUKARYOTE: A life form comprised of one or more cells containing a nucleus and membrane - bound
organelles. Included are members of the Kingdoms Protista, Fungi, Plantae and Animalia.
EUMETAZOA: Members of the subkingdom that includes all animals except sponges.
EUTROPHIC: A highly productive condition in aquatic environments which owes to excessive
concentrations of nutrients which support the growth of primary producers.
EVAGINATED: Folded or protruding outward.
EVAPORATIVE COOLING: The property of a liquid whereby the surface becomes cooler during evaporation,
owing to the loss of highly kinetic molecules to the gaseous state.
EVERSIBLE: Capable of being turned inside out.
EXCITABLE CELLS: A cell, such as a neuron or a muscle cell that can use changes in its membrane potential
to conduct signals.
EXCRETION: Release of materials which arise in the body due to metabolism (e.g., CO2, NH3, H20).
EXERGONIC: A phenomenon which involves the release of energy.
EXOCYTOSIS: A process by which a vesicle within a cell fuses with the plasma membrane and releases its
contents to the outside.
EXON: A part of a primary transcript (and the corresponding part of a gene) that is ultimately either
translated (in the case of mRNA) or utilized in a final product, such as tRNA.
EXOSKELETON: An external skeleton, characteristic of members of the phylum, Arthropoda.
EXOTHERMIC: A process or reaction that is accompanied by the creation of heat.
EXOTOXIN: A toxic protein secreted by a bacterial cell that produces specific symptoms even in the
absence of the bacterium.
EXTRINSIC: External to, not a basic part of; as in extrinsic isolating mechanism.
F
F PLASMID: The fertility factor in bacteria, a plasmid that confers the ability to form pili for conjugation and
associated functions required for transfer of DNA from donor to recipient.
F: The chemical symbol of Fluorine.

FACILITATED DIFFUSION: Passive movement through a membrane involving a specific carrier protein;
does not proceed against a concentration gradient.
FACULTATIVE: An organism which exhibits the capability of changing from one habit or metabolic pathway
to another, when conditions warrant. (anaerobe) An organism that makes ATP by aerobic respiration if oxygen
is present but that switches to fermentation under anaerobic conditions.
FAT: A biological compound consisting of three fatty acids linked to one glycerol molecule.
FATTY ACID: A long carbon chain carboxylic acid. Fatty acids vary in length and in the number and location
of double bonds; three fatty acids linked to a glycerol molecule form fat.
FAUNA: The animals of a given area or period.
FECAL COLIFORM: A group of bacteria that may indicate the presence of human or animal fecal matter in
water. Total coliform, fecal coliform, and E. coli are all indicators of drinking water quality. The total coliform
group is a large collection of different kinds of bacteria. Fecal coliforms are types of total coliform that mostly
exist in feces. E. coli is a sub-group of fecal coliform. When a water sample is sent to a lab, it is tested for total
coliform. If total coliform is present, the sample will also be tested for either fecal coliform or E. coli, depending
on the lab testing method.
FECAL COLIFORM: Fecal Coliform and E. coli are bacteria whose presence indicates that the water may be
contaminated with human or animal wastes. Microbes in these wastes can cause short-term effects, such as
diarrhea, cramps, nausea, headaches, or other symptoms.
FECES: Indigestible wastes discharged from the digestive tract.
FEEDBACK: The process by which a control mechanism is regulated through the very effects it brings about.
Positive feedback is when the effect is amplified; negative feedback is when the effect tends toward restoration
of the original condition. Feedback inhibition is a method of metabolic control in which the end-product of a
metabolic pathway acts as an inhibitor of an enzyme within that pathway.
FERMENTATION: Anaerobic production of alcohol, lactic acid or similar compounds from carbohydrate
resulting from glycolysis.
FERRIC CHLORIDE: An iron salt commonly used as a coagulant. Chemical formula is FeCl3.
FILTER AID: A polymer or other material added to improve the effectiveness of the filtration process.
FILTER CAKE: The layer of solids that is retained on the surface of a filter.
FILTER CLOGGING: An inability to meet demand may occur when filters are clogging.
FILTER PRESS: A dewatering device where sludge is pumped onto a filtering medium and water is forced
out of the sludge, resulting in a “cake”.
FILTER: A device utilizing a granular material, woven cloth or other medium to remove pollutants from water,
wastewater or air.
FILTRATE: Liquid remaining after removal of solids with filtration.
FILTRATION RATE: A measurement of the volume of water applied to a filter per unit of surface area in a
given period of time.
FITNESS: The extent to which an individual passes on its genes to the next generation. Relative fitness is the
number of offspring of an individual compared to the mean.
FIXATION: 1) Conversion of a substance into a biologically more usable form, for example, CO2 fixation
during photosynthesis and N2 fixation. 2) Process of treating living tissue for microscopic examination.
FIXED ACTION PATTERN (FAP): A highly: stereotyped behavior that is innate and must be carried to
completion once initiated.
FLACCID: Limp; walled cells are flaccid in isotonic surroundings, where there is no tendency for water to
enter.
FLAGELLIN: The protein from which prokaryotic flagella are constructed.
FLAGELLUM: A long whip-like appendage that propels cells during locomotion in liquid solutions. The
prokaryote flagellum is comprised of a protein, flagellin. The eukaryote flagellum is longer than a cilium, but
as a similar internal structure of microtubules in a"9 + 2" arrangement.
FLAME CELL: A flagellated cell associated with the simplest tubular excretory system, present in
flatworms: it acts to directly regulate the contents of the extracellular fluid.
FLOC SHEARING: Likely to happen to large floc particles when they reach the flocculation process.
FLOCCULANTS: Flocculants, or flocculating agents, are chemicals that promote flocculation by causing
colloids and other suspended particles in liquids to aggregate, forming a floc. Flocculants are used in water
treatment processes to improve the sedimentation or filterability of small particles. For example, a flocculant
may be used in swimming pool or drinking water filtration to aid removal of microscopic particles which would
otherwise cause the water to be cloudy and which would be difficult or impossible to remove by filtration alone.
Many flocculants are multivalent cations such as aluminum, iron, calcium or magnesium. These positively
charged molecules interact with negatively charged particles and molecules to reduce the barriers to
aggregation. In addition, many of these chemicals, under appropriate pH and other conditions such as
temperature and salinity, react with water to form insoluble hydroxides which, upon precipitating, link together
to form long chains or meshes, physically trapping small particles into the larger floc. Long-chain polymer
flocculants, such as modified polyacrylamides, are manufactured and sold by the flocculant producing

business. These can be supplied in dry or liquid form for use in the flocculation process. The most common
liquid polyacrylamide is supplied as an emulsion with 10-40 % actives and the rest is a carrier fluid, surfactants
and latex. Emulsion polymers require activation to invert the emulsion and allow the electrolyte groups to be
exposed.
FLOCCULATION BASIN: A compartmentalized basin with a reduction of speed in each compartment. This
set-up or basin will give the best overall results.
FLOCCULATION: The process of bringing together destabilized or coagulated particles to form larger masses
that can be settled and/or filtered out of the water being treated. Conventional coagulation–flocculation-
sedimentation practices are essential pretreatments for many water purification systems—especially filtration
treatments. These processes agglomerate suspended solids together into larger bodies so that physical
filtration processes can more easily remove them. Particulate removal by these methods makes later filtering
processes far more effective. The process is often followed by gravity separation (sedimentation or flotation)
and is always followed by filtration. A chemical coagulant, such as iron salts, aluminum salts, or polymers, is
added to source water to facilitate bonding among particulates. Coagulants work by creating a chemical
reaction and eliminating the negative charges that cause particles to repel each other. The coagulant-source
water mixture is then slowly stirred in a process known as flocculation. This water churning induces particles
to collide and clump together into larger and more easily removable clots, or “flocs.” The process requires
chemical knowledge of source water characteristics to ensure that an effective coagulant mix is employed.
Improper coagulants make these treatment methods ineffective. The ultimate effectiveness of
coagulation/flocculation is also determined by the efficiency of the filtering process with which it is paired.
FLOOD RIM: The point of an object where the water would run over the edge of something and begin to
cause a flood.
FLORA: The plants of a given area or period.
FLOW CYTOMETER: A particle-sorting instrument capable of counting protozoa.
FLUID FEEDER: An animal that lives by sucking nutrient-rich fluids from another living organism.
FLUID MOSAIC MODEL: The currently accepted model of cell membrane structure, which envisions the
membrane as a mosaic of individually inserted protein molecules drifting laterally in a fluid bilayer of
phospholipids.
FLUX: The term flux describes the rate of water flow through a semipermeable membrane. When the water
flux decreases through a semipermeable membrane, it means that the mineral concentration of the water is
increasing.
FLY ASH: The noncombustible particles in flue gas. Often used as a body feed or solidification chemical.
FOLLICLE STIMULATING HORMONE (FSH): A gonadotropic hormone of the anterior pituitary that
stimulates growth of follicles in the ovaries of females and function of the seminiferous tubules in males.
FOLLICLE: A jacket of cells around an egg cell in an ovary.
FOOD CHAIN: Sequence of organisms, including producers, consumers, and decomposers, through which
energy and materials may move in a community.
FOOD WEB: The elaborate, interconnected feeding relationships in an ecosystem.
FORMAZIN TURBIDITY UNIT (FTU): A unit used to measure the clarity of water. The ISO refers to the units
as FNU (Formazin Nephelometric Units). The technique is the same as that for the NTU, but the calibration
uses microspheres of the polymer formazin.
FORMULA: A precise representation of the structure of a molecule or ion, showing the proportion of atoms
which comprise the material.
FOUNDER EFFECT: The difference between the gene pool of a population as a whole and that of a newly
isolated population of the same species.
FRACTIONATION: An experimental technique that involves separation of parts of living tissue from one
another using centrifugation.
FRAGMENTATION: A mechanism of asexual reproduction in which the parent plant or animal separates into
parts that reform whole organisms.
FREE CHLORINE RESIDUAL: Regardless of whether pre-chloration is practiced or not, a free chlorine
residual of at least 1.0 mg/L should be maintained in the clear well or distribution reservoir immediately
downstream from the point of post-chlorination. The reason for chlorinating past the breakpoint is to provide
protection in case of backflow.
FREE CHLORINE: In disinfection, chlorine is used in the form of free chlorine or as hypochlorite ion.
FREE OIL: Non-emulsified oil that separates from water, in a given period of time.
FREQUENCY DEPENDENT SELECTION: A decline in the reproductive success of a morph resulting from
the morph's phenotype becoming too common in a population; a cause of balanced polymorphism in
populations.
FUNCTIONAL GROUP: One of several groups of atoms commonly found in organic molecules. A functional
group contributes somewhat predictable properties to the molecules that possess them.
FUNDAMENTAL NICHE: The total resources an organism is theoretically capable of utilizing.

G
G: (protein) A membrane protein that serves as an intermediary between hormone receptors and the enzyme
adenylate cyclase, which converts ATP to cAMP in the second messenger system in non-steroid hormone
action. Depending on the system, G proteins either increase or decrease cAMP production.
G1 PHASE: The first growth phase of the cell cycle, consisting of the portion of interphase before DNA
synthesis is initiated.
G2 PHASE: The second growth phase of the cell cycle, consisting of the portion of interphase after DNA
synthesis but before mitosis.
GAMETANGIUM: The reproductive organ of bryophytes, consisting of the male antheridium and female
archegonium; a multi-chambered jacket of sterile cells in which gametes are formed.
GAMETE: A sexual reproductive cell that must usually fuse with another such cell before development
begins; an egg or sperm.
GAMETOPHYTE: A haploid plant that can produce gametes.
GANGLION: A structure containing a group of cell bodies of neurons.
GAP JUNCTION: A narrow gap between plasma membranes of two animal cells, spanned by protein
channels. They allow chemical substances or electrical signals to pass from cell to cell.
GASTRULATION: The process by which a blastula develops into a gastrula, usually by an involution of cells.
GATED ION CHANNEL: A membrane channel that can open or close in response to a signal, generally a
change in the electrostatic gradient or the binding of a hormone, transmitter, or other molecular signal.
GEL ELECTROPHORESIS: In general, electrophoresis is a laboratory technique used to separate
macromolecules on the basis of electric charge and size; the technique involves application of an electric field
to a population of macromolecules which disperse according to their electric mobilities. In gel electrophoresis,
the porous medium through which the macromolecules move is a gel.
GEL: Colloid in which the suspended particles form a relatively orderly arrangement.
GENE AMPLIFICATION: Any of the strategies that give rise to multiple copies of certain genes, thus
facilitating the rapid synthesis of a product (such as rRna for ribosomes) for which the demand is great.
GENE CLONING: Formation by a bacterium, carrying foreign genes in a recombinant plasmid, of a clone of
identical cells containing the replicated foreign genes.
GENE DELIVERY: This is a general term for the introduction of new genetic elements into the genomes of
living cells. The delivery problem is essentially conditioned by the fact that the new genetic elements are
usually large, and by the presence of the outer cell membrane and the nuclear membrane acting as barriers
to incorporation of the new DNA into the genome already present in the nucleus. Viruses possess various
natural biochemical methods for achieving gene delivery; artificial gene delivery is one of the essential
problems of "genetic engineering". The most important barrier is apparently the outer cell membrane, which
is essentially a lipid barrier, and introduction of any large complex into the cell requires a fusion of one kind or
another with this membrane. Liposomes, which consist of lipid membranes themselves, and which can fuse
with outer cell membranes, are thus potential vehicles for delivery of many substances, including DNA.
GENE FLOW: The movement of genes from one part of a population to another, or from one population to
another, via gametes.
GENE POOL: The sum total of all the genes of all the individuals in a population.
GENE REGULATION: Any of the strategies by which the rate of expression of a gene can be regulated, as
by controlling the rate of transcription.
GENE: The hereditary determinant of a specified characteristic of an individual; specific sequences of
nucleotides in DNA.
GENETIC DRIFT: Change in the gene pool as a result of chance and not as a result of selection, mutation,
or migration.
GENETIC RECOMBINATION: The general term for the production of offspring that combine traits of the two
parents.
GENETICS: The science of heredity; the study of heritable information.
GENOME: The cell's total complement of DNA.
GENOMIC EQUIVALENCE: The presence of all of an organism's genes in all of its cells.
GENOMIC IMPRINTING: The parental effect on gene expression. Identical alleles may have different effects
on offspring depending on whether they arrive in the zygote via the ovum or via the sperm.
GENOMIC LIBRARY: A set of thousands of DNA segments from a genome, each carried by a plasmid or
phage.
GENOTYPE: The particular combination of genes present in the cells of an individual.
GENUS: A taxonomic category above the species level, designated by the first word of a species' binomial
Latin name.
GIARDIA LAMBLIA: Giardia lamblia (synonymous with Lamblia intestinalis and Giardia duodenalis) is a
flagellated protozoan parasite that colonizes and reproduces in the small intestine, causing giardiasis. The
giardia parasite attaches to the epithelium by a ventral adhesive disc, and reproduces via binary fission.
Giardiasis does not spread via the bloodstream, nor does it spread to other parts of the gastro-intestinal tract,

but remains confined to the lumen of the small intestine. Giardia trophozoites absorb their nutrients from the
lumen of the small intestine, and are anaerobes.
GIARDIA LAMBLIA: A parasite that enters lakes and rivers through sewage and animal waste. It causes
gastrointestinal illness (e.g. diarrhea, vomiting, cramps).
GIS – GRAPHIC INFORMATION SYSTEM: Detailed information about the physical locations of structures
such as pipes, valves, and manholes within geographic areas with the use of satellites.
GLIAL CELL: A non-conducting cell of the nervous system that provides support, insulation, and protection
for the neurons.
GLIDING: Rod-shaped, gram-negative, mostly aerobic; glide on secreted slimy substances; form colonies,
frequently with complex fruiting structures.
GLOMERULUS: A capillary bed within Bowman's capsule of the nephron; the site of ultrafiltration.
GLUCOSE: A six-carbon sugar which plays a central role in cellular metabolism.
GLYCOCALYX: The layer of protein and carbohydrates just outside the plasma membrane of an animal cell;
in general, the proteins are anchored in the membrane, and the carbohydrates are bound to the proteins.
GLYCOGEN: A long, branched polymer of glucose subunits that is stored in the muscles and liver of animals
and is metabolized as a source of energy.
GLYCOLYSIS: A metabolic pathway which occurs in the cytoplasm of cells and during which glucose is
oxidized anaerobically to form pyruvic acid.
GLYCOPROTEIN: A protein with covalently linked sugar residues. The sugars may be bound to OH side
chains of the polypeptide (O: linked) or the amide nitrogen of asparagine side chains (N: linked).
GLYCOSIDIC: A type of bond which links monosaccharide subunits together in di- or polysaccharides.
GLYOXYSOME: A type of microbody found in plants, in which stored lipids are converted to carbohydrates.
GOLGI APPARATUS: A system of concentrically folded membranes found in the cytoplasm of eukaryotic
cells. Plays a role in the production and release of secretory materials such as the digestive enzymes
manufactured in the pancreas.
GONADOTROPIN: Refers to a member of a group of hormones capable of promoting growth and function of
the gonads. Includes hormones such as follicle stimulating hormone (FSH) and luteinizing hormone (LH) which
are stimulatory to the gonads.
GOOD CONTACT TIME, pH and LOW TURBIDITY: These are factors that are important in providing good
disinfection when using chlorine.
GPM: Gallons per minute.
GRAB SAMPLE: A sample that is taken from a water or wastestream on a one-time basis with no
regard to the flow of the water or wastestream and without consideration of time. A single grab sample
should be taken over a period of time not to exceed 15 minutes. A single water or wastewater sample
taken at a time and place representative of total discharge.
GRADED POTENTIAL: A local voltage change in a neuron membrane induced by stimulation of a neuron,
with strength proportional to the strength of the stimulus and lasting about a millisecond.
GRANUM: A stack-like grouping of photosynthetic membranes in a chloroplast
GRAVITY BELT THICKENER: A sludge dewatering device utilizing a filter belt to promote gravity drainage
of water. Usually precedes additional dewatering treatment.
GRAVITY FILTER: A filter that operates at atmospheric pressure.
GRAVITY THICKENING: A sedimentation basin designed to operate at high solids loading rates.
GROWTH FACTOR: A protein that must be present in a cell's environment for its normal growth and
development.
GT: Represents (Detention time) x (mixing intensity) in flocculation.
GYMNOSPERM: A vascular plant that bears naked seeds not enclosed in any specialized chambers.
H
H2SO4: The molecular formula of Sulfuric acid.
HABIT: In biology, the characteristic form or mode of growth of an organism.
HABITAT: The kind of place where a given organism normally lives.
HABITUATION: The process that results in a long-lasting decline in the receptiveness of interneurons to the
input from sensory neurons or other interneurons (sensitization, adaptation).
HALIDES: A halide is a binary compound, of which one part is a halogen atom and the other part is an
element or radical that is less electronegative than the halogen, to make a fluoride, chloride, bromide, iodide,
or astatide compound. Many salts are halides. All Group 1 metals form halides with the halogens and they are
white solids. A halide ion is a halogen atom bearing a negative charge. The halide anions are fluoride (F),
chloride (Cl), bromide (Br), iodide (I) and astatide (At). Such ions are present in all ionic halide salts.
HALOACETIC ACIDS: Haloacetic acids are carboxylic acids in which a halogen atom takes the place of a
hydrogen atom in acetic acid. Thus, in a monohaloacetic acid, a single halogen would replace a hydrogen
atom. For example, chloroacetic acid would have the structural formula CH2ClCO2H. In the same manner, in
dichloroacetic acid two chlorine atoms would take the place of two hydrogen atoms (CHCl2CO2H).

HALOACETIC ACIDS: Haloacetic acids are carboxylic acids in which a halogen atom takes the place of a
hydrogen atom in acetic acid. Thus, in a monohaloacetic acid, a single halogen would replace a hydrogen
atom. For example, chloroacetic acid would have the structural formula CH2ClCO2H. In the same manner, in
dichloroacetic acid two chlorine atoms would take the place of two hydrogen atoms (CHCl2CO2H).
HAPLOID: The condition of having only one kind of a given type of chromosome.
HARD WATER: Hard water causes a buildup of scale in household hot water heaters. Hard water is a type of
water that has high mineral content (in contrast with soft water). Hard water primarily consists of calcium
(Ca2+), and magnesium (Mg2+) metal cations, and sometimes other dissolved compounds such as
bicarbonates and sulfates. Calcium usually enters the water as either calcium carbonate (CaCO3), in the form
of limestone and chalk, or calcium sulfate (CaSO4), in the form of other mineral deposits. The predominant
source of magnesium is dolomite (CaMg(CO3)2). Hard water is generally not harmful. The simplest way to
determine the hardness of water is the lather/froth test: soap or toothpaste, when agitated, lathers easily in
soft water but not in hard water. More exact measurements of hardness can be obtained through a wet titration.
The total water 'hardness' (including both Ca2+ and Mg2+ ions) is read as parts per million or weight/volume
(mg/L) of calcium carbonate (CaCO3) in the water. Although water hardness usually only measures the total
concentrations of calcium and magnesium (the two most prevalent, divalent metal ions), iron, aluminum, and
manganese may also be present at elevated levels in some geographical locations.
HARDNESS: A measure of the amount of calcium and magnesium salts in water. More calcium and
magnesium lead to greater hardness. The term "hardness" comes from the fact that it is hard to get soapsuds
from soap or detergents in hard water. This happens because calcium and magnesium react strongly with
negatively charged chemicals like soap to form insoluble compounds.
HAZARDS OF POLYMERS: Slippery and difficult to clean-up are the most common hazards associated with
the use of polymers in a water treatment plant.
HEAD: The measure of the pressure of water expressed in feet of height of water. 1 PSI = 2.31 feet of water
or 1 foot of head equals about a half a pound of pressure or .433 PSI. There are various types of heads of
water depending upon what is being measured. Static (water at rest) and Residual (water at flow conditions).
HEADWORKS: The facility at the "head" of the water source where water is first treated and routed into the
HEALTH ADVISORY: An EPA document that provides guidance and information on contaminants that can
affect human health and that may occur in drinking water, but which the EPA does not currently regulate in
drinking water.
HEAT OF VAPORIZATION: The amount of energy absorbed by a substance when it changes state to a gas.
Water absorbs approximately 580 calories per gram when it changes from liquid water-to-water vapor.
HEAT: The total amount of kinetic energy due to molecular motion in a body of matter. Heat is energy in its
most random form.
HELPER T CELL: A type of T cell that is required by some B cells to help them make antibodies or that helps
other T cells respond to antigens or secrete lymphokines or interleukins.
HEMAGGLUTININ: A surface antigen on influenza viruses that controls infectivity by associating with
receptors on host erythrocytes or other cells.
HEMATOPOIETIC STEM CELLS: Cells found in the bone marrow of adult mammals which give rise to
erythroid stem cells, lymphoid stem cells, and myeloid stem cells. Such cells give rise to erythrocytes and a
variety of types of lymphocytes and leucocytes.
HEMOGLOBIN: An iron-containing respiratory pigment found in many organisms.
HEMOLYMPH: In invertebrates with open circulatory systems, the body fluid that bathes tissues.
HEMOPHILIA: A genetic disease resulting from an abnormal sex-linked recessive gene, characterized by
excessive bleeding following injury.
HEPATIC: Pertaining to the liver.
HEREDITY: A biological phenomenon whereby characteristics are transmitted from one generation to another
by virtue of chemicals (i.e. DNA) transferred during sexual or asexual reproduction.
HERPESVIRUS: A double stranded DNA virus with an enveloped, icosahedral capsid.
HERTZ: The term used to describe the frequency of cycles in an alternating current (AC) circuit. A unit of
frequency equal to one cycle per second.
HETEROCHROMATIN: Non-transcribed eukaryotic chromatin that is so highly compacted that it is visible
with a light microscope during interphase.
HETEROCHRONY: Evolutionary changes in the timing or rate of development.
HETEROCYST: A specialized cell that engages in nitrogen fixation on some filamentous cyanobacteria.
HETEROGAMY: The condition of producing gametes of two different types (contrast with isogamy).
HETEROMORPHIC: A condition in the life cycle of all modern plants in which the sporophyte and
gametophyte generations differ in morphology.
HETEROSPOROUS: Referring to plants in which the sporophyte produces two kinds of spores that develop
into unisexual gametophytes, either male or female.

HETEROTROPH: An organism dependent on external sources of organic compounds as a means of
obtaining energy and/or materials. Such an organism requires carbon ("food") from its environment in an
organic form. (synonym-organotroph).
HETEROTROPHIC PLATE COUNT: A test performed on drinking water to determine the total number of all
types of bacteria in the water.
HETEROZYGOTE ADVANTAGE: A mechanism that preserves variation in eukaryotic gene pools by
conferring greater reproductive success on heterozygotes over individuals homozygous for any one of the
associated alleles.
HETEROZYGOUS: The condition whereby two different alleles of the gene are present within the same cell.
HF: The molecular formula of Hydrofluoric acid.
HIGH TURBIDITY CAUSING INCREASED CHLORINE DEMAND: May occur or be caused by the inadequate
disinfection of water.
HIGH-TEST HYPOCHLORITE: A composition composed mainly of calcium hypochlorite is commonly called
high test hypochlorite. High-Test Hypochlorite contains not less than 60.0% of available chlorine.
HISTAMINE: A substance released by injured cells that causes blood vessels to dilate during an inflammatory
response.
HISTOLOGY: The study of tissues.
HISTONE: A type of protein characteristically associated with the chromosomes of eukaryotes.
HIV-1: Acute human immunodeficiency virus type 1 is the subtype of HIV (human immune deficiency virus)
that causes most cases of AIDS in the Western Hemisphere, Europe, and Central, South, and East Africa.
HIV is a retrovirus (subclass lentivirus), and retroviruses are single: stranded RNA viruses that have an
enzyme called reverse transcriptase. With this enzyme the viral RNA is used as a template to produce viral
DNA from cellular material. This DNA is then incorporated into the host cell's genome, where it codes for the
synthesis of viral components. An HIV-1 infection should be distinguished from AIDS. Acquired
immunodeficiency syndrome (AIDS) is a secondary immunodeficiency syndrome resulting from HIV infection
and characterized by opportunistic infections, malignancies, neurologic dysfunction, and a variety of other
syndromes.
HOLOBLASTIC: A type of cleavage in which there is complete division of the egg, as in eggs having little
yolk (sea urchin) or a moderate amount of yolk (frog).
HOME RANGE: An area within which an animal tends to confine all or nearly all its activities for a long period
of time.
HOMEOBOX: Specific sequences of DNA that regulate patterns of differentiation during development of an
organism.
HOMEOSTASIS: A phenomenon whereby a state or process (for example, within an organism) is regulated
automatically despite the tendency for fluctuations to occur.
HOMEOTHEMIC: Capable of regulation of constancy with respect to temperature.
HOMEOTIC GENES: Genes that control the overall body plan of animals by controlling the developmental
fate of groups of cells.
HOMEOTIC: (mutation) A mutation in genes regulated by positional information that results in the abnormal
substitution of one type of body part in place of another.
HOMOLOGOUS CHROMOSOMES: Chromosomes bearing genes for the same characters.
HOMOLOGOUS STRUCTURES: Characters in different species that were inherited from a common ancestor
and thus share a similar ontogenetic pattern.
HOMOLOGY: Similarity in characteristics resulting from a shared ancestry.
HOMOPLASY: The presence in several species of a trait not present in their most common ancestor. Can
result from convergent evolution, reverse evolution, or parallel evolution.
HOMOSPOROUS: Referring to plants in which a single type of spore develops into a bisexual gametophyte
having both male and female sex organs.
HOMOZYGOUS: Having two copies of the same allele of a given gene.
HORMONE: A control chemical secreted in one part of the body that affects other parts of the body.
HOST RANGE: The limited number of host species, tissues, or cells that a parasite (including viruses and
bacteria) can infect.
HUMORAL IMMUNITY: The type of immunity that fights bacteria and viruses in body fluids with antibodies
that circulate in blood plasma and lymph, fluids formerly called humors.
HYBIRD VIGOR: Increased vitality (compared to that of either parent stock) in the hybrid offspring of two
different, inbred parents.
HYBIRD: In evolutionary biology, a cross between two species. In genetics, a cross between two genetic
types.
HYBIRDIZATION: The process whereby a hybrid results from interbreeding two species; 2) DNA hybridization
is the comparison of whole genomes of two species by estimating the extent of hydrogen bonding that occurs
between single-stranded DNA obtained from the two species.

HYBRIDOMA: A hybrid cell that produces monoclonal antibodies in culture, formed by the fusion of a
myeloma cell with a normal antibody-producing lymphocyte.
HYDRATED LIME: The calcium hydroxide product that results from mixing quicklime with water. Chemical
formula is CaOH2.
HYDRATION SHELL: A "covering" of water molecules which surrounds polar or charged substances in
aqueous solutions. The association is due to the charged regions of the polar water molecules themselves.
HYDRIDES: Hydride is the name given to the negative ion of hydrogen, H. Although this ion does not exist
except in extraordinary conditions, the term hydride is widely applied to describe compounds of hydrogen with
other elements, particularly those of groups 1–16. The variety of compounds formed by hydrogen is vast,
arguably greater than that of any other element. Various metal hydrides are currently being studied for use as
a means of hydrogen storage in fuel cell-powered electric cars and batteries. They also have important uses
in organic chemistry as powerful reducing agents, and many promising uses in hydrogen economy.
HYDROCARBON: Any compound made of only carbon and hydrogen.
HYDROCHLORIC ACID: It is the aqueous solution of hydrogen chloride gas (HCl). It is a strong acid, and the
major component of gastric acid, and of wide industrial use. Hydrochloric acid must be handled with
appropriate safety precautions because it is a highly corrosive liquid.
HYDROCHLORIC AND HYPOCHLOROUS ACIDS: The compounds that are formed in water when chlorine
gas is introduced.
HYDROFLUOSILICIC ACID: (H2SiF6) a clear, fuming corrosive liquid with a pH ranging from 1 to 1.5. Used
in water treatment to fluoridate drinking water.
HYDROGEN BOND: A type of bond formed when the partially positive hydrogen atom of a polar covalent
bond in one molecule is attracted to the partially negative atom of a polar covalent bond in another.
HYDROGEN ION: A single proton with a charge of +1. The dissociation of a water molecule (H2O) leads to
the generation of a hydroxide ion (OH-) and a hydrogen ion (H+).
HYDROGEN SULFIDE: A toxic gas formed by the anaerobic decomposition of organic matter. Chemical
formula is H2S.
HYDROLYSIS: The chemical reaction that breaks a covalent bond through the addition of hydrogen (from a
water molecule) to the atom forming one side of the original bond, and a hydroxyl group to the atom on the
other side.
HYDROPHILIC: Having an affinity for water.
HYDROPHOBIC INTERACTION: A type of weak chemical bond formed when molecules that do not mix with
water coalesce to exclude the water.
HYDROPHOBIC: The physicochemical property whereby a substance or region of a molecule resists
association with water molecules.
HYDROSTATIC: Pertaining to the pressure and equilibrium of fluids. A hydrostatic skeleton is a skeletal
system composed of fluid held under pressure in a closed body compartment; the main skeleton of most
cnidarians, flatworms, nematodes, and annelids.
HYDROXYL GROUP: A functional group consisting of a hydrogen atom joined to an oxygen atom by a polar
covalent bond. Molecules possessing this group are soluble in water and are called alcohols.
HYDROXYL ION: The OH- ion.
HYPEROSMOTIC: A solution with a greater solute concentration than another, a hypoosmotic solution. If the
two solutions are separated from one another by a membrane permeable to water, water would tend to move
from the hypo- to the hyperosmotic side.
HYPERPOLARIZATION: An electrical state whereby the inside of the cell is made more negative relative to
the outside than was the case at resting potential. A neuron membrane is hyperpolarized if the voltage is
increased from the resting potential of about -70 mV, reducing the chance that a nerve impulse will be
transmitted.
HYPERTROPHY: Abnormal enlargement, excessive growth.
HYPHA: A fungal filament.
HYPOCHLORITE AND ORGANIC MATERIALS: Heat and possibly fire may occur when hypochlorite is
brought into contact with an organic material.
HYPOCOTYL: The portion of the axis of a plant embryo below the point of attachment of the cotyledons;
forms the base of the shoot and the root.
HYPOOSMOTIC SOLUTION: A solution with a lesser solute concentration than another, a hyperosmotic
solution. If the two solutions are separated from one another by a membrane permeable to water, water would
tend to move from the hypo- to the hyperosmotic side.
HYPOTHESIS: A formal statement of supposition offered to explain observations. Note that a hypothesis is
only useful if it can be tested. Even if correct, it is not scientifically useful if untestable.
HYPOTHETICO-DEDUCTIVE: A method used to test hypotheses. If deductions formulated from the
hypothesis are tested and proven false, the hypothesis is rejected.
If the actual pH of the water is below the calculated saturation pH, the LSI is negative and the water has a
very limited scaling potential. If the actual pH exceeds pHs, the LSI is positive, and being supersaturated with

CaCO3, the water has a tendency to form scale. At increasing positive index values, the scaling potential
increases.
I
IMAGINAL DISK: An island of undifferentiated cells in an insect larva, which are committed (determined) to
form a particular organ during metamorphosis to the adult.
IMBIBITION: The soaking of water into a porous material that is hydrophilic.
IMMUNE RESPONSE: 1) A primary immune response is the initial response to an antigen, which appears
after a lag of a few days. 2) A secondary immune response is the response elicited when the animal
encounters the same antigen at a later time. The secondary response is normally more rapid, of greater
magnitude and of longer duration than the primary response.
IMMUNOGLOBULINE: The class of proteins comprising the antibodies.
IMMUNOLOGICAL: 1) Immunological distance is the amount of difference between two proteins as
measured by the strength of the antigen: antibody reaction between them. 2) Immunological tolerance is a
mechanism by which an animal does not mount an immune response to the antigenic determinants of its own
macromolecules.
IMMUNOMAGNETIC SEPARATION (IMS): A purification procedure that uses microscopic, magnetically
responsive particles coated with an antibodies targeted to react with a specific pathogen in a fluid stream.
Pathogens are selectively removed from other debris using a magnetic field.
IMPELLERS: The semi-open or closed props or blades of a turbine pump that when rotated generate the
pumping force.
IMPERVIOUS: Not allowing, or allowing only with great difficulty, the movement of water.
IMPRINTING: A type of learned behavior with a significant innate component, acquired during a limited critical
period.
IN SERIES: Several components being connected one to the other without a bypass, requiring each
component to work dependent on the one before it.
IN SITU: Treatment or disposal methods that do not require movement of contaminated material.
INCINERATION: The process of reducing the volume of a material by burning and reducing to ash if possible.
INCLINED PLATE SEPARATOR: A series of parallel inclined plates that can be used to increase the
efficiency of clarifiers and gravity thickeners.
INCOMPLETE DOMINANCE: A type of inheritance in which F1 hybrids have an appearance that is
intermediate between the phenotypes of the parental varieties.
INDETERMINATE: 1) A type of cleavage exhibited during the embryonic development in deuterostomes, in
which each cell produced by early cleavage divisions retains the capacity to develop into a complete embryo;
2) A type of growth exhibited by plants: they continue to grow as long as they live, because they always retain
meristematic cells capable of undergoing mitosis.
INDIRECT REUSE: The beneficial use of reclaimed water into natural surface waters or groundwater.
INDUCED FIT: The change in shape of the active site of an enzyme so that it binds more snugly to the
substrate, induced by entry of the substrate.
INDUCTION: 1) The ability of one group of embryonic cells to influence the development of another. 2) A
method in logic that proceeds from the specific to general and develops a general statement which explains
all of the observations. Commonly used to formulate scientific hypotheses.
INDUSTRIAL WASTEWATER: Liquid wastes resulting from industrial processes.
INFECTIOUS PATHOGENS/MICROBES/GERMS: Are considered disease-producing bacteria, viruses and
other microorganisms.
INFECTIOUS: 1) An infectious disease is a disease caused by an infectious microbial or parasitic agent. 2)
Infectious hepatitis is the former name for hepatitis A. 3) Infectious mononucleosis is an acute disease that
affects many systems, caused by the Epstein: Barr virus.
INFLAMMATORY RESPONSE: A line of defense triggered by penetration of the skin or mucous membranes,
in which small blood vessels in the vicinity of an injury dilate and become leakier, enhancing infiltration of
leukocytes; may also be widespread in the body.
INFLUENT: Water or wastewater flowing into a basin or treatment plant.
INFORMATION COLLECTION RULE (ICR): EPA collected data required by the Information Collection Rule
(May 14, 1996) to support future regulation of microbial contaminants, disinfectants, and disinfection
byproducts. The rule was intended to provide EPA with information on chemical byproducts that form when
disinfectants used for microbial control react with chemicals already present in source water (disinfection
byproducts (DBPs)); disease-causing microorganisms (pathogens), including Cryptosporidium; and
engineering data to control these contaminants.
INGESTION: A heterotrophic mode of nutrition in which other organisms or detritus are eaten whole or in
pieces.
INHIBITORY POSTSYNAPTIC POTENTIAL: An electrical charge (hyperpolarization) in the membrane of a
postsynaptic neuron caused by the binding of an inhibitory neurotransmitter from a presynaptic cell to a
postsynaptic receptor.

INITIAL PRECISION AND RECOVERY (IPR): Four aliquots of spiking suspension analyzed to establish
the ability to generate acceptable precision and accuracy. An IPR is performed prior to the first time this
method is used and any time the method or instrumentation is modified.
INNER CELL MASS: A cluster of cells in a mammalian blastocyst that protrudes into one end of the cavity
and subsequently develops into the embryo proper and some of the extraembryonic membranes.
INORGANIC COMPOUND: Compounds that contain no carbon or contain only carbon bound to elements
other than hydrogen.
INORGANIC CONTAMINANTS: Mineral-based compounds such as metals, nitrates, and asbestos. These
contaminants are naturally occurring in some water, but can also get into water through farming, chemical
manufacturing, and other human activities. EPA has set legal limits on 15 inorganic contaminants.
INORGANIC IONS: Present in all waters. Inorganic ions are essential for human health in small quantities,
but in larger quantities they can cause unpleasant taste and odor or even illness. Most community water
systems will commonly test for the concentrations of seven inorganic ions: nitrate, nitrite, fluoride, phosphate,
sulfate, chloride, and bromide. Nitrate and nitrite can cause an illness in infants called methemoglobinemia.
Fluoride is actually added to the drinking water in some public water systems to promote dental health.
Phosphate, sulfate, chloride, and bromide have little direct effect on health, but high concentrations of
inorganic ions can give water a salty or briny taste.
INSERTION: A mutation involving the addition of one or more nucleotide pairs to a gene.
INSOLUBLE COMPOUNDS: Are types of compounds cannot be dissolved. When iron or manganese reacts
with dissolved oxygen (DO) insoluble compound are formed.
INSULIN: The vertebrate hormone that lowers blood sugar levels by promoting the uptake of glucose by most
body cells and promoting the synthesis and storage of glycogen in the liver; also stimulates protein and fat
synthesis; secreted by endocrine cells of the pancreas called islets of Langerhans.
INTAKE FACILITIES: One of the more important considerations in the construction of intake facilities is the
ease of operation and maintenance over the expected lifetime of the facility. Every intake structure must be
constructed with consideration for operator
INTEGRAL PROTEIN: A protein of biological membranes that penetrates into or spans the membrane.
INTERBREED: To breed with another kind or species; hybridize.
INTERFERON: A chemical messenger of the immune system, produced by virus: infected cells and capable
of helping other cells resist the virus.
INTERLEUKIN: 1: A chemical regulator (cytokine) secreted by macrophages that have ingested a pathogen
or foreign molecule and have bound with a helper T cell; stimulates T cells to grow and divide and elevates
body temperature. Interleukin: 2, secreted by activated T cells, stimulates helper T cells to proliferate more
rapidly.
INTERTIDAL ZONE: The shallow zone of the ocean where land meets water.
INTRON: The noncoding, intervening sequence of coding region (exon) in eukaryotic genes.
INVAGINATION: The buckling inward of a cell layer, caused by rearrangements of microfilaments and
microtubules; an important phenomenon in embryonic development.
INVERSION: 1) An aberration in chromosome structure resulting from an error in meiosis or from mutagens;
reattachment in a reverse orientation of a chromosomal fragment to the chromosome from which the fragment
originated. 2) A phenomenon that occurs during early development of sponges at which time the external
ciliated cells become inward-directed.
INVERTEBRATE: An animal without a backbone; invertebrates make up about 95% of animal species.
ION EXCHANGE: An effective treatment process used to remove iron and manganese in a water supply. The
hardness of the source water affects the amount of water an ion exchange softener may treat before the bed
requires regeneration.
ION: A charged chemical formed when an atom or group of atoms has more or less electrons than protons
(rather than an equal number).
IONIC BOND: A chemical bond due to attraction between oppositely charged ions.
IRON AND MANGANESE: In water, they can usually be detected by observing the color of the inside walls
of filters and the filter media. If the raw water is pre-chlorinated, there will be black stains on the walls below
the water level and a black coating over the top portion of the sand filter bed. When significant levels of
dissolved oxygen are present, iron and manganese exist in an oxidized state and normally precipitate into the
reservoir bottom sediments. The presence of iron and manganese in water promote the growth of Iron
bacteria. Only when a water sample has been acidified then you can perform the analysis beyond the 48-hour
holding time. Iron and Manganese in water may be detected by observing the color of the of the filter media.
Maintaining a free chlorine residual and regular flushing of water mains may control the growth of iron bacteria
in a water distribution system.
IRON BACTERIA: In the management of water-supply wells, iron bacteria are bacteria that derive the energy
they need to live and multiply by oxidizing dissolved ferrous iron (or the less frequently available manganese
and aluminum). The resulting ferric oxide is insoluble, and appears as brown gelatinous slime that will stain
plumbing fixtures, and clothing or utensils washed with the water carrying it, and may contribute to internal

corrosion of the pipes and fixtures the water flows through. They are known to grow and proliferate in waters
containing as low as 0.1mg/l of iron. However, at least 0.3 ppm of dissolved oxygen is needed to carry out
oxidation. The proliferation of iron bacteria, in some way, increases the chance of sulfur bacteria infestation.
IRON: The elements iron and manganese are undesirable in water because they cause stains and promote
the growth of iron bacteria.
ISOMER: Molecules consisting of the same numbers and kinds of atoms, but differing in the way in which the
atoms are combined.
ISOSMOTIC: Solutions of equal concentration with respect to osmotic pressure.
ISOTOPE: An atomic form of an element, containing a different number of neutrons than another isotope.
Isotopes vary from one another with respect to atomic mass.
K
K- SELECTION: The concept that life history of the population is centered upon producing relatively few
offspring that have a good chance of survival.
KARYOGAMY: The fusion of nuclei of two cells, as part of syngamy.
KARYOTYPE: A method of classifying the chromosomes of a cell in relation to number, size and type.
KEYSTONE PREDATOR: A species that maintains species richness in a community through predation of
the best competitors in the community, thereby maintaining populations of less competitive species.
KILL = C X T: Where other factors are constant, the disinfecting action may be represented by: Kill=C x T.
KILOCALORIE: A thousand calories; the amount of heat energy required to raise the temperature of 1
kilogram of water by primary C.
KINGDOM: A taxonomic category, the second broadest after domain.
L
L.O.T.O.: If a piece of equipment is locked out, the key to the lock-out device the key should be held by the
person who is working on the equipment. The tag is an identification device and the lock is a physical restraint.
LABORATORY BLANK: See Method blank
LABORATORY CONTROL SAMPLE (LCS): See Ongoing precision and recovery (OPR) standard
LAND APPLICATION: The disposal of wastewater or municipal solids onto land under controlled conditions.
LAND DISPOSAL: Application of municipal wastewater solids to the soil without production of usable
agricultural products.
LANDFILL: A land disposal site that employs an engineering method of solid waste disposal to minimize
environmental hazards and protect the quality of surface and subsurface waters.
LANGELIER INDEX: A measurement of Corrosivity. The water is becoming corrosive in the distribution
system causing rusty water if the Langelier index indicates that the pH has decreased from the equilibrium
point. Mathematically derived factor obtained from the values of calcium hardness, total alkalinity, and pH at
a given temperature. A Langelier index of zero indicates perfect water balance (i.e., neither corroding nor
scaling). The Langelier Saturation Index (sometimes Langelier Stability Index) is a calculated number used to
predict the calcium carbonate stability of water. It indicates whether the water will precipitate, dissolve, or be
in equilibrium with calcium carbonate. Langelier developed a method for predicting the pH at which water is
saturated in calcium carbonate (called pHs). The LSI is expressed as the difference between the actual system
pH and the saturation pH.
LARVA (pl. larvae): A free-living, sexually immature form in some animal life cycles that may differ from the
adult in morphology, nutrition, and habitat.
LEACHATE: Fluid that trickles through solid materials or wastes and contains suspended or dissolved
materials or products of the solids.
LEACHING: A chemical reaction between water and metals that allows for removal of soluble materials.
LEADING STRAND: The new continuously complementary DNA strand synthesized along the template
strand in the 5' --- > 3' direction.
LETHAL CONCENTRATION 50: Also referred to as LC50, a concentration of a pollutant or effluent at which
50 percent of the test organisms die; a common measure of acute toxicity.
LEUKOCYTE: A white blood cell; typically functions in immunity, such as phagocytosis or antibody
production.
LEVELS OF ORGANIZATION: A basic concept in biology is that organization is based on a hierarchy of
structural levels, with each level building on the levels below it.
LICHEN: An organism formed by the symbiotic association between a fungus and a photosynthetic alga.
LIFE: A table of data summarizing mortality in a population.
LIGAMENT: A type of fibrous connective tissue that joins bones together at joints.
LIGAND: A ligand is a molecule that binds specifically to a receptor site of another molecule. A ligase is an
enzyme that catalyzes such a reaction. For example, a DNA ligase is an enzyme that catalyzes the covalent
bonding of the 3' end of a new DNA fragment to the 5' end of a growing chain.
LIGASE: Ligases are enzymes that catalyze the "stitching together" of polymer fragments. DNA ligase, for
example, catalyzes phosphodiester bond formation between two DNA fragments, and this enzyme is involved

in normal DNA replication, repair of damaged chromosomes, and various in vitro techniques in genetic
engineering that involve linking DNA fragments.
LIGNIN: A hard material embedded in the cellulose matrix of vascular plant cell walls that functions as an
important adaptation for support in terrestrial species.
LIMBIC SYSTEM: A group of nuclei (clusters of nerve cell bodies) in the lower part of the mammalian
forebrain that interact with the cerebral cortex in determining emotions; includes the hippocampus and the
amygdala.
LIME SOFTENING: Lime softening is primarily used to “soften” water—that is to remove calcium and
magnesium mineral salts. But it also removes harmful toxins like radon and arsenic. Though there is no
consensus, some studies have even suggested that lime softening is effective at removal of Giardia. Hard
water is a common condition responsible for numerous problems. Users often recognize hard water because
it prevents their soap from lathering properly. However, it can also cause buildup (“scale”) in hot water heaters,
boilers, and hot water pipes. Because of these inconveniences, many treatment facilities use lime softening
to soften hard water for consumer use. Before lime softening can be used, managers must determine the
softening chemistry required. This is a relatively easy task for groundwater sources, which remain more
constant in their composition. Surface waters, however, fluctuate widely in quality and may require frequent
changes to the softening chemical mix. In lime softening, lime and sometimes sodium carbonate are added to
the water as it enters a combination solids contact clarifier. This raises the pH (i.e., increases alkalinity) and
leads to the precipitation of calcium carbonate. Later, the pH of the effluent from the clarifier is reduced again,
and the water is then filtered through a granular media filter. The water chemistry requirements of these
systems require knowledgeable operators, which may make lime softening an economic challenge for some
very small systems.
LIME STABILIZATION: The addition of lime to untreated sludge to raise the pH to 12 for a minimum of 2
hours to chemically inactivate microorganisms.
LIME: The term generally used to describe ground limestone (calcium carbonate), hydrated lime (calcium
hydroxide), or burned lime (calcium oxide).
LINKED GENES: Genes that are located on the same chromosomes.
LIPID: One of a family of compounds, including fats, phospholipids, and steroids, that are insoluble in water.
LIPOSOME: Liposomes are vesicles (spherules) in which the lipid molecules are spontaneously arranged
into bilayers with hydrophilic groups exposed to water molecules both outside the vesicle and in the core.
LISTED HAZARDOUS WASTE: The designation for a waste material that appears on an EPA list of specific
hazardous wastes or hazardous waste categories.
LOCUS: A particular place along the length of a certain chromosome where a specified allele is located.
LOGISTIC POPULATION GROWTH: A model describing population growth that levels off as population size
approaches carrying capacity.
LSI = pH - pHs
LYSOGENIC CYCLE: A type of viral replication cycle in which the viral genome becomes incorporated into
the bacterial host chromosome as a prophage.
LYTIC CYCLE: A type of viral replication cycle resulting in the release of new phages by death or lysis of the
host cell.
M
M PHASE: The mitotic phase of the cell cycle, which includes mitosis and cytokinesis.
M.S.D.S.: Now S.D.S. (Safety Data Sheet) .A safety document must an employer provide to an operator upon
request.
MACROMOLECULE: A giant molecule of living matter formed by the joining of smaller molecules, usually by
condensation synthesis. Polysaccharides, proteins, and nucleic acids are macromolecules.
MACROPHAGE: An amoeboid cell that moves through tissue fibers, engulfing bacteria and dead cells by
phagocytosis.
MAGNESIUM HARDNESS: Measure of the magnesium salts dissolved in water – it is not a factor in water
balance.
MAGNETIC STARTER: Is a type of motor starter should be used in an integrated circuit to control flow
automatically.
MAJOR HISTOCOMPATIBILITY COMPLEX: A large set of cell surface antigens encoded by a family of
genes. Foreign MHC markers trigger T-cell responses that may lead to rejection of transplanted tissues and
organs.
MAKEUP WATER: Fluid introduced in a recirculating stream to maintain an equilibrium of temperature, solids
concentration or other parameters. Also refers to the quantity of water required to make a solution.
MALPHIGHIAN TUBULE: A unique excretory organ of insects that empties into the digestive tract, removes
nitrogenous wastes from the blood, and functions in osmoregulation.
MANGANESE (IV) OXIDE: The chemical compound MnO2, commonly called manganese dioxide. This
blackish or brown solid occurs naturally as the mineral pyrolusite, which is the main ore of manganese. It is
also present in manganese nodules. The principal use for MnO2 is for dry-cell batteries, such as the alkaline

battery and the zinc-carbon battery. In 1976 this application accounted for 500,000 tons of pyrolusite. MnO2
is also used for production of MnO4–. It is used extensively as an oxidizing agent in organic synthesis, for
example, for the oxidation of allylic alcohols.
MANTLE: A heavy fold of tissue in mollusks that drapes over the visceral mass and may secrete a shell.
MARBLE AND LANGELIER TESTS: Are used to measure or determine the corrosiveness of a water source.
MASS NUMBER: The sum of the number of protons plus the number of neutrons in the nucleus of an atom;
unique for each element and designated by a superscript to the left of the elemental symbol.
MATRIX SPIKE (MS): A sample prepared by adding a known quantity of organisms to a specified amount
of sample matrix for which an independent estimate of target analyte concentration is available. A matrix
spike is used to determine the effect of the matrix on a method’s recovery efficiency.
MATRIX: The nonliving component of connective tissue, consisting of a web of fibers embedded in
homogeneous ground substance that may be liquid, jellylike, or solid.
MATTER: Anything that takes up space and has mass.
MAXIMUM CONTAMINANT LEVEL (MCL): The maximum concentration of a chemical that is allowed in
public drinking water systems.
MAXIMUM CONTAMINANT LEVEL (MCLs): The maximum allowable level of a contaminant
MAXIMUM CONTAMINANT LEVEL GOAL (MCLG): The maximum level at which a contaminant can exist in
drinking water without having an adverse effect on human health.
MECHANICAL SEAL: A mechanical device used to control leakage from the stuffing box of a pump. Usually
made of two flat surfaces, one of which rotates on the shaft. The two flat surfaces are of such tolerances as
to prevent the passage of water between them. Held in place with spring pressure.
MECHANORECEPTOR: A sensory receptor that detects physical deformations in the body environment
associated with pressure, touch, stretch, motion, and sound.
MEDIAN BODIES: Prominent, dark-staining, paired organelles consisting of microtubules and found in
the posterior half of Giardia. In G. intestinalis (from humans), these structures often have a claw-hammer
shape, while in G. muris (from mice), the median bodies are round.
MEDIUM WATER SYSTEM: More than 3,300 persons and 50,000 or fewer persons.
MEDULLA OBLONGATA: The lowest part of the vertebrate brain; a swelling of the hindbrain dorsal to the
anterior spinal cord that controls autonomic, homeostatic functions, including breathing, heart and blood
vessel activity, swallowing, digestion, and vomiting.
MEDUSA: The floating, flattened, mouth-down version of the cnidarian body plan. The alternate form is the
polyp.
MEGAPASCAL: A unit of pressure equivalent to 10 atmospheres of pressure.
MEGGER: Used to test the insulation resistance on a motor.
MEIOSIS: A two-stage type of cell division in sexually reproducing organisms that results in gametes with
half the chromosome number of the original cell.
MEMBRANE POTENTIAL: The charge difference between the cytoplasm and extracellular fluid in all cells,
due to the differential distribution of ions. Membrane potential affects the activity of excitable cells and the
transmembrane movement of all charged substances.
MEMBRANE: A thin barrier that permits passage of particles of a certain size or of particular physical or
chemical properties.
M-ENDO BROTH: The coliform group are used as indicators of fecal pollution in water, for assessing the
effectiveness of water treatment and disinfection, and for monitoring water quality. m-Endo Broth is used for
selectively isolating coliform bacteria from water and other specimens using the membrane filtration technique.
m-Endo Broth is prepared according to the formula of Fifield and Schaufus.1 It is recommended by the
American Public Health Association in standard total coliform membrane filtration procedure for testing water,
wastewater, and foods.2,3 The US EPA specifies using m-Endo Broth in the total coliform methods for testing
water using single-step, two-step, and delayed incubation membrane filtration methods.
MESENTERIES: Membranes that suspend many of the organs of vertebrates inside fluid- filled body cavities.
MESODERM: The middle primary germ layer of an early embryo that develops into the notochord, the lining
of the coelom, muscles, skeleton, gonads, kidneys and most of the circulatory system.
MESOSOME: A localized infolding of the plasma membrane of a bacterium.
MESSENGER: (RNA) A type of RNA synthesized from DNA in the genetic material that attaches to ribosomes
in the cytoplasm and specifies the primary structure of a protein.
METABOLISM: The sum total of the chemical and physical changes constantly taking place in living
substances.
METALLOID: Metalloid is a term used in chemistry when classifying the chemical elements. On the basis of
their general physical and chemical properties, nearly every element in the periodic table can be termed either
a metal or a nonmetal. A few elements with intermediate properties are, however, referred to as metalloids.
(In Greek metallon = metal and eidos = sort)
METAMORPHOSIS: The resurgence of development in an animal larva that transforms it into a sexually
mature adult.

METANEPHRIDIUM: A type of excretory tubule in annelid worms that has internal openings called
nephrostomes that collect body fluids and external openings called nephridiopores.
METASTASIS: The spread of cancer cells beyond their original site.
METAZOAN: A multicellular animal. Among important distinguishing characteristics of metazoa are cell
differentiation and intercellular communication. For certain multicellular colonial entities such as sponges,
some biologists prefer the term "parazoa".
METHANE: Methane is a chemical compound with the molecular formula CH4. It is the simplest alkane, and
the principal component of natural gas. Methane's bond angles are 109.5 degrees. Burning methane in the
presence of oxygen produces carbon dioxide and water. The relative abundance of methane and its clean
burning process makes it a very attractive fuel. However, because it is a gas at normal temperature and
pressure, methane is difficult to transport from its source.
METHOD BLANK: An aliquot of reagent water that is treated exactly as a sample, including exposure to
all glassware, equipment, solvents, and procedures that are used with samples. The method blank is used
to determine if analytes or interferences are present in the laboratory environment, the reagents, or the
apparatus.
Mg/L: Stands for "milligrams per liter." A common unit of chemical concentration. It expresses the mass of
a chemical that is present in a given volume of water. A milligram (one one-thousandth of a gram) is equivalent
to about 18 grains of table salt. A liter is equivalent to about one quart.
MICROBE OR MICROBIAL: Any minute, simple, single-celled form of life, especially one that causes
disease.
MICROBIAL CONTAMINANTS: Microscopic organisms present in untreated water that can cause
waterborne diseases.
MICROBIOLOGICAL: Is a type of analysis in which a composite sample unacceptable.
MICROBODY: A small organelle, bounded by a single membrane and possessing a granular interior.
Peroxisomes and glyoxysomes are types of microbodies.
MICROFILAMENT: Minute fibrous structure generally composed of actin found in the cytoplasm of eukaryotic
cells. They play a role in motion within cells.
MICROFILTRATION: A low-pressure membrane filtration process that removes suspended solids and
colloids generally larger than 0.1 micron diameter.
MICROORGANISMS: Very small animals and plants that are too small to be seen by the naked eye and must
be observed using a microscope. Microorganisms in water include algae, bacteria, viruses, and protozoa.
Algae growing in surface waters can cause off-taste and odor by producing the chemicals MIB and geosmin.
Certain types of bacteria, viruses, and protozoa can cause disease in humans. Bacteria are the most common
microorganisms found in treated drinking water. The great majority of bacteria are not harmful. In fact, humans
would not be able to live without the bacteria that inhabit the intestines. However, certain types of bacteria
called coliform bacteria can signal the presence of possible drinking water contamination.
MICROSCOPE: An instrument that magnifies images either by using lenses in an optical system to bend light
(light microscope) or electromagnets to direct the movement of electrons (electron microscope).
MICROTUBULE: A minute tubular structure found in centrioles, spindle apparati, cilia, flagella, and other
places in the cytoplasm of eukaryotic cells. Microtubules play a role in movement and maintenance of shape.
MICROVILLUS: Collectively, fine, fingerlike projections of the epithelial cells in the lumen of the small intestine
that increase its surface area.
MILLIGRAMS PER LITER: (mg/L) A common unit of measurement of the concentration of a material in
solution.
MILLILITER: One one-thousandth of a liter; A liter is a little more than a quart. A milliliter is about two
drops from an eyedropper.
MIMICRY: A phenomenon in which one species benefits by a superficial resemblance to an unrelated
species. A predator or species of prey may gain a significant advantage through mimicry.
MISCIBLE: Capable of being mixed together.
MISSENSE: (mutation) The most common type of mutation involving a base- pair substitution within a gene
that changes a codon, but the new codon makes sense, in that it still codes for an amino acid.
MITOCHONDRIAL MATRIX: The compartment of the mitochondrion enclosed by the inner membrane and
containing enzymes and substrates for the Krebs cycle.
MITOCHONDRION: An organelle that occurs in eukaryotic cells and contains the enzymes of the citric acid
cycle, the respiratory chain, and oxidative phosphorylation. A mitochondrion is bounded by a double
membrane.
MITOSIS: A process of cell division in eukaryotic cells conventionally divided into the growth period
(interphase) and four stages: prophase, metaphase, anaphase, and telophase. The stages conserve
chromosome number by equally allocating replicated chromosomes to each of the daughter cells.
MIXED LIQUOR SUSPENDED SOLIDS: Suspended solids in the mixture of wastewater and activated sludge
undergoing aeration in the aeration basin.

MODEM SYNTHESIS: A comprehensive theory of evolution emphasizing natural selection, gradualism, and
populations as the fundamental units of evolutionary change; also called Neo-Darwinism.
MOISTURE AND POTASSIUM PERMANGANATE: The combination of moisture and potassium
permanganate produces heat.
MOISTURE: If a material is hygroscopic, it must it be protected from water.
MOLARITY: A common measure of solute concentration, referring to the number of moles of solute in 1 L of
solution.
MOLD: A rapidly growing, asexually reproducing fungus.
MOLE: The number of grams of a substance that equals its molecular weight in daltons and contains
Avogadro's number of molecules.
MOLECULAR FORMULA: A type of molecular notation indicating only the quantity of the constituent atoms.
MOLECULAR WEIGHT: The molecular mass (abbreviated Mr) of a substance, formerly also called molecular
weight and abbreviated as MW, is the mass of one molecule of that substance, relative to the unified atomic
mass unit u (equal to 1/12 the mass of one atom of carbon-12). This is distinct from the relative molecular
mass of a molecule, which is the ratio of the mass of that molecule to 1/12 of the mass of carbon 12 and is a
dimensionless number. Relative molecular mass is abbreviated to Mr.
MOLECULE: Two or more atoms of one or more elements held together by ionic or covalent chemical bonds.
MOLTING: A process in arthropods in which the exoskeleton is shed at intervals to allow growth by secretion
of a larger exoskeleton.
MONERA: The kingdom of life forms that includes all of the bacteria.
MONOMER: A small molecule, two or more of which can be combined to form oligomers (consisting of a few
monomers) or polymers (consisting of many monomers).
MONOPHYLETIC: A term used to describe any taxon derived from a single ancestral form that gave rise to
no species in other taxa.
MONOSACCHARIDE: A simple sugar; a monomer.
MORPHOGENESIS: The development of body shape and organization during ontogeny.
MORPHOSPECIES: Species defined by their anatomical features.
MOSAIC: A pattern of development, such as that of a mollusk, in which the early blastomeres each give rise
to a specific part of the embryo. In some animals, the fate of the blastomeres is established in the zygote.
MOTOR NERVOUS SYSTEM: In vertebrates, the component of the peripheral nervous system that transmits
signals from the central nervous system to effector cells.
MPF: M: phase promoting factor: A protein complex required for a cell to progress from late interphase to
mitosis; the active form consists of cyclin and cdc2, a protein kinase.
MUD BALLS IN FILTER MEDIA: Is a possible result of an ineffective or inadequate filter backwash.
MULLERIAN MIMICRY: A mutual mimicry by two unpalatable species.
MULTIGENE FAMILY: A collection of genes with similar or identical sequences, presumably of common
origin.
MUNICIPAL WASTE: The combined solid and liquid waste from residential, commercial and industrial
sources.
MUNICIPAL WASTEWATER TREATMENT PLANT (MWTP): Treatment works designed to treat municipal
wastewater.
MURIATIC ACID: An acid used to reduce pH and alkalinity. Also used to remove stain and scale.
MUST: This action, activity, or procedural step is required.
MUTAGEN: A chemical or physical agent that interacts with DNA and causes a mutation.
MUTAGENESIS: The creation of mutations.
MUTATION: A spontaneous or induced change in a gene's or chromosome's structure or number. The
resulting individual is termed a mutant.
MUTUALISM: A symbiotic relationship in which both the host and the symbiont benefit.
MYCELIUM: The densely branched network of hyphae in a fungus.
MYCOBACTERIUM: Pleomorphic spherical or rod-shaped, frequently branching, no gram stain, aerobic;
commonly form yellow pigments; include Mycobacterium tuberculosis, cause of tuberculosis.
MYCOPLASMA: Spherical, commonly forming branching chains, no gram stain, aerobic but can live in certain
anaerobic conditions; without cell walls yet structurally resistant to lysis; among smallest of bacteria; named
for superficial resemblance to fungal hyphae (myco-means “fungus’).
MYELIN SHEATH: An insulating coat of cell membrane from Schwann cells that is interrupted by nodes of
Ranvier where saltatory conduction occurs.
MYOFIBRILS: Fibrils arranged in longitudinal bundles in muscle cells (fibers); composed of thin filaments of
actin and a regulatory protein and thick filaments of myosin.
MYOGLOBIN: An oxygen-storing, pigmented protein in muscle cells.
MYOSIN: A type of protein filament that interacts with actin filaments to cause cell movement, such as
contraction in muscle cells.

N
NAD+: Nicatinamide adenine dinucleotide (oxidized); a coenzyme present in all cells that assists enzymes in
transferring electrons during the redox reactions of metabolism.
NANO-FILTRATION: A specialty membrane filtration process that rejects solutes larger than approximately
one nanometer (10 angstroms) in size.
NANOMETER: A unit of measure (length). 1 nm is equal to 1 x 10: 9 m, or 1/1,000,000 mm.
NaOCl: Is the molecular formula of Sodium hypochlorite.
NaOH: Is the molecular formula of Sodium hydroxide.
NATURAL ORGANIC MATTER: Organic matter present in natural waters.
NEGATIVE CONTROL: See Method blank.
NEGATIVE FEEDBACK: A primary mechanism of homeostasis, whereby a change in a physiological variable
that is being monitored triggers a response that counteracts the initial fluctuation.
NEPHELOMETRIC TURBIDITY UNIT (NTU): The unit used to describe turbidity. Nephelometric refers to the
way the instrument, a nephelometer, measures how much light is scattered by suspended particles in the
water. The greater the scattering, the higher the turbidity. Therefore, low NTU values indicate high water
clarity, while high NTU values indicate low water clarity.
NEURON: A nerve cell; the fundamental unit of the nervous system, having structure and properties that
allow it to conduct signals by taking advantage of the electrical charge across its cell membrane.
NEUROSECRETORY CELLS: Cells that receive signals from other nerve cells, but instead of signaling to an
adjacent nerve cell or muscle, release hormones into the blood stream.
NEUROTRANSMITTER: The chemical messenger released from the synaptic terminals of a neuron at a
chemical synapse that diffuses across the synaptic cleft and binds to and stimulates the postsynaptic cell.
NEUTRAL VARIATION: Genetic diversity that confers no apparent selective advantage.
NEUTRALIZATION REACTIONS: Chemical reactions between acids and bases where water is an end
product.
NEUTRALIZATION: The chemical process that produces a solution that is neither acidic nor alkaline. Usually
with a pH between 6 and 8.
NEUTRON: An uncharged subatomic particle of about the same size and mass as a proton.
NH3: The molecular formula of Ammonia.
NH4+: The molecular formula of the Ammonium ion.
NITRATES: A dissolved form of nitrogen found in fertilizers and sewage by-products that may leach into
groundwater and other water sources. Nitrates may also occur naturally in some waters. Over time, nitrates
can accumulate in aquifers and contaminate groundwater.
NITROGEN AND PHOSPHORUS: Pairs of elements and major plant nutrients that cause algae to grow.
NITROGEN: Nitrogen is a nonmetal, with an electronegativity of 3.0. It has five electrons in its outer shell and
is therefore trivalent in most compounds. The triple bond in molecular nitrogen (N2) is one of the strongest in
nature. The resulting difficulty of converting (N2) into other compounds, and the ease (and associated high-
energy release) of converting nitrogen compounds into elemental N2, have dominated the role of nitrogen in
both nature and human economic activities. At atmospheric pressure molecular nitrogen condenses (liquefies)
at 77 K (-195.8 °C) and freezes at 63 K (-210.0 °C) into the beta hexagonal close-packed crystal allotropic
form. Below 35.4 K (-237.6 °C) nitrogen assumes the alpha cubic crystal allotropic form. Liquid nitrogen, a
fluid resembling water, but with 80.8% of the density, is a common cryogen. Unstable allotropes of nitrogen
consisting of more than two nitrogen atoms have been produced in the laboratory, like N3 and N4.[1] Under
extremely high pressures (1.1 million atm) and high temperatures (2000 K), as produced under diamond anvil
conditions, nitrogen polymerizes into the single bonded diamond crystal structure, an allotrope nicknamed
"nitrogen diamond."
NITROGEN-FIXING: Rod-shaped, gram-negative, aerobic; convert atmospheric nitrogen gas to ammonium
in soil; include Azotobacter, a common genus.
NO3-: The molecular formula of the Nitrate ion.
NOMENCLATURE: The method of assigning names in the classification of organisms.
NON-CARBONATE HARDNESS: The portion of the total hardness in excess of the alkalinity.
NON-CARBONATE IONS: Water contains non-carbonate ions if it cannot be softened to a desired level
through the use of lime only.
NONCOMPETITIVE INHIBITOR: A substance that reduces the activity of an enzyme by binding to a location
remote from the active site, changing its conformation so that it no longer binds to the substrate.
NON-POINT SOURCE POLLUTION: Air pollution may leave contaminants on highway surfaces. This non-
point source pollution adversely impacts reservoir water and groundwater quality.
NONPOLAR: Electrically symmetrical. For example, in many molecules with covalent bonds, the electrons
are shared equally; the poles are electrically neutral.
NONSENSE MUTATION: A mutation that changes an amino acid codon to one of the three stop codons,
resulting in a shorter and usually nonfunctional protein.

NORM OF REACTION: The range of phenotypic possibilities for a single genotpe, as influenced by the
environment.
NORMALITY: It is the number of equivalent weights of solute per liter of solution. Normality highlights the
chemical nature of salts: in solution, salts dissociate into distinct reactive species (ions such as H+, Fe3+, or
Cl-). Normality accounts for any discrepancy between the concentrations of the various ionic species in a
solution. For example, in a salt such as MgCl2, there are two moles of Cl- for every mole of Mg2+, so the
concentration of Cl- as well as of Mg2+ is said to be 2 N (read: "two normal"). Further examples are given
below. A normal is one gram equivalent of a solute per liter of solution. The definition of a gram equivalent
varies depending on the type of chemical reaction that is discussed - it can refer to acids, bases, redox species,
and ions that will precipitate. It is critical to note that normality measures a single ion which takes part in an
overall solute.
NTU: (Nephelometric turbidity unit): A measure of the clarity or cloudiness of water.
NUCLEAR: 1) (envelope) The surface, consisting of two layers of membrane, that encloses the nucleus of
eukaryotic cells. 2) (pore) An opening of the nuclear envelope which allows for the movement of materials
between the nucleus and surrounding cytoplasm.
NUCLEIC: (acid) A polymer composed of nucleotides that are joined by covalent bonds (phosphodiester
linkages) between the phosphate of one nucleotide and the sugar of the next nucleotide.
NUCLELUS: A small, generally spherical body found within the nucleus of eukaryotic cells. The site of
ribosomal RNA synthesis.
NUCLEOID: The region that harbors the chromosome of a prokaryotic cell. Unlike the eukaryotic nucleus, it
is not bounded by a membrane.
NUCLEOLUS (pl. nucleoli): A specialized structure in the nucleus, formed from various chromosomes and
active in the synthesis of ribosomes.
NUCLEOSIDE: An organic molecule consisting of a nitrogenous base joined to a five- carbon sugar.
NUCLEOSOME: The basic, beadlike unit of DNA packaging in eukaryotes, consisting of a segment of DNA
wound around a protein core composed of two copies of each of four types of histone.
NUCLEOTIDE: The basic chemical unit (monomer) of a nucleic acid. A nucleotide in RNA consists of one of
four nitrogenous bases linked to ribose, which in turn is linked to phosphate. In DNA, deoxyribose is present
instead of ribose.
NUCLEUS: A membrane-bound organelle containing genetic material. Nuclei are a prominent internal
structure seen both in Cryptosporidium oocysts and Giardia cysts. In Cryptosporidium oocysts, there is one
nucleus per sporozoite. One to four nuclei can be seen in Giardia cysts.
NUCLEUS: The membrane bound organelle of eukaryotic cells that contains the cell's genetic material. Also
the central region of an atom composed of protons and neutrons.
NULL: In the scientific method, the hypothesis which one attempts to falsify.
O
O3: The molecular formula of ozone.
OLIGOTROPHIC: A reservoir that is nutrient-poor and contains little plant or animal life. An oligotrophic
ecosystem or environment is one that offers little to sustain life. The term is commonly utilized to describe
bodies of water or soils with very low nutrient levels. It derives etymologically from the Greek oligo (small, little,
few) and trophe (nutrients, food). Oligotrophic environments are of special interest for the alternative energy
sources and survival strategies upon which life could rely.
ONGOING PRECISION AND RECOVERY (OPR) STANDARD: A method blank spiked with known
quantities of analytes. The OPR is analyzed exactly like a sample. Its purpose is to assure that the results
produced by the laboratory remain within the limits specified in this method for precision and recovery.
OOCYST: The encysted zygote of some sporozoa; e.g., Cryptosporidium. The oocyst is a phase or form of
the organism produced as a normal part of the life cycle of the organism. It is characterized by a thick and
environmentally resistant outer wall.
ORGANIC MATTER: Substances containing carbon compounds, usually of animal or vegetable origin.
ORGANIC PRECURSORS: Natural or man-made compounds with chemical structures based upon carbon
that, upon combination with chlorine, leading to trihalomethane formation.
ORGANIC: Relating to, or derived from, a living thing. A description of a substance that contains carbon
atoms linked together by carbon-carbon bonds.
OSMOSIS: Osmosis is the process by which water moves across a semi permeable membrane from a low
concentration solute to a high concentration solute to satisfy the pressure differences caused by the solute.
OXIDE: An oxide is a chemical compound containing at least one oxygen atom as well as at least one other
element. Most of the Earth's crust consists of oxides. Oxides result when elements are oxidized by oxygen in
air. Combustion of hydrocarbons affords the two principal oxides of carbon, carbon monoxide and carbon
dioxide. Even materials that are considered to be pure elements often contain a coating of oxides. For
example, aluminum foil has a thin skin of Al2O3 that protects the foil from further corrosion. Virtually all
elements burn in an atmosphere of oxygen. In the presence of water and oxygen (or simply air), some
elements - lithium, sodium, potassium, rubidium, caesium, strontium and barium - react rapidly, even

dangerously to give the hydroxides. In part for this reason, alkali and alkaline earth metals are not found in
nature in their metallic, i.e., native, form. Caesium is so reactive with oxygen that it is used as a getter in
vacuum tubes, and solutions of potassium and sodium, so called NaK are used to deoxygenate and dehydrate
some organic solvents. The surface of most metals consists of oxides and hydroxides in the presence of air.
A well-known example is aluminum foil, which is coated with a thin film of aluminum oxide that passivates the
metal, slowing further corrosion. The aluminum oxide layer can be built to greater thickness by the process of
electrolytic anodizing. Although solid magnesium and aluminum react slowly with oxygen at STP, they, like
most metals, will burn in air, generating very high temperatures. As a consequence, finely divided powders of
most metals can be dangerously explosive in air.
OXIDIZING: The process of breaking down organic wastes into simpler elemental forms or by products. Also
used to separate combined chlorine and convert it into free chlorine.
OXYGEN DEFICIENT ENVIRONMENT: One of the most dangerous threats to an operator upon entering a
manhole.
OZONE: Ozone or trioxygen (O3) is a triatomic molecule, consisting of three oxygen atoms. It is an allotrope
of oxygen that is much less stable than the diatomic O2. Ground-level ozone is an air pollutant with harmful
effects on the respiratory systems of animals. Ozone in the upper atmosphere filters potentially damaging
ultraviolet light from reaching the Earth's surface. It is present in low concentrations throughout the Earth's
atmosphere. It has many industrial and consumer applications. Ozone, the first allotrope of a chemical element
to be recognized by science, was proposed as a distinct chemical compound by Christian Friedrich Schönbein
in 1840, who named it after the Greek word for smell (ozein), from the peculiar odor in lightning storms. The
formula for ozone, O3, was not determined until 1865 by Jacques-Louis Soret and confirmed by Schönbein in
1867.
P
PACKING: Material, usually of woven fiber, placed in rings around the shaft of a pump and used to control
the leakage from the stuffing box.
PARAMECIUM: Paramecia are a group of unicellular ciliate protozoa formerly known as slipper animalcules
from their slipper shape. They are commonly studied as a representative of the ciliate group. Simple cilia
cover the body which allows the cell to move with a synchronous motion (like a caterpilla). There is also a
deep oral groove containing inconspicuous compound oral cilia (as found in other peniculids) that is used to
draw food inside. They generally feed upon bacteria and other small cells. Osmoregulation is carried out by a
pair of contractile vacuoles, which actively expel water absorbed by osmosis from their surroundings.
Paramecia are widespread in freshwater environments, and are especially common in scums. Paramecia are
attracted by acidic conditions. Certain single-celled eukaryotes, such as Paramecium, are examples for
exceptions to the universality of the genetic code (translation systems where a few codons differ from the
standard ones).
PARTS PER MILLION (PPM): A common unit of measure used to express the number of parts of a
substance contained within a million parts of a liquid, solid, or gas.
PASTEURIZATION: A process for killing pathogenic organisms by applying heat for a specific period of
time.
PATHOGENS: Disease-causing pathogens; waterborne pathogens A pathogen may contaminate water and
cause waterborne disease.
Pb: The chemical symbol of Lead.
PCE: abbr. perchloroethylene. Known also as perc or tetrachloroethylene, perchloroethylene is a clear,
colorless liquid with a distinctive, somewhat ether-like odor. It is non-flammable, having no measurable
flashpoint or flammable limits in air. Effective over a wide range of applications, perchloroethylene is supported
by closed loop transfer systems, stabilizers and employee exposure monitoring.
pCi/L: Picocuries per liter A curie is the amount of radiation released by a set amount of a certain
compound. A picocurie is one quadrillionth of a curie.
PEAK DEMAND: The maximum momentary load placed on a water treatment plant, pumping station or
PERKINESIS: The aggregation resulting from random thermal motion of fluid molecules.
PERMEATE: The term for water which has passed through the membrane of a reverse osmosis unit. The
liquid that passes through a membrane.
PERMISSIBLE EXPOSURE LIMIT (PEL or OSHA PEL): A legal limit in the United States for exposure of an
employee to a substance or physical agent. For substances it is usually expressed in parts per million (ppm),
or sometimes in milligrams per cubic meter (mg/m3). Units of measure for physical agents such as noise are
specific to the agent. Permissible Exposure Limits are established by the Occupational Safety and Health
Administration (OSHA).
pH OF SATURATION: The ideal pH for perfect water balance in relation to a particular total alkalinity level
and a particular calcium hardness level, at a particular temperature. The pH where the Langelier Index equals
zero.

pH: A unit of measure which describes the degree of acidity or alkalinity of a solution. The pH scale runs from
0 to 14 with 7 being the mid-point or neutral. A pH of less than 7 is on the acid side of the scale with 0 as the
point of greatest acid activity. A pH of more than 7 is on the basic (alkaline) side of the scale with 14 as the
point of greatest basic activity. The term pH is derived from “p”, the mathematical symbol of the negative
logarithm, and “H”, the chemical symbol of Hydrogen. The definition of pH is the negative logarithm of the
Hydrogen ion activity. pH=-log[H+].
PHENOL RED: Chemical reagent used for testing pH in the range of 6.8 - 8.4.
PHENOLPHTHALEIN/TOTAL ALKALINITY: The relationship between the alkalinity constituent’s
bicarbonate, carbonate, and hydroxide can be based on the P and T alkalinity measurement.
PHOSPHATE, NITRATE AND ORGANIC NITROGEN: Nutrients in a domestic water supply reservoir may
cause water quality problems if they occur in moderate or large quantities.
PHYSICAL CHEMICAL TREATMENT: Treatment processes that are non-biological in nature.
PICOCURIE: A unit of radioactivity. "Pico" is a metric prefix that means one one-millionth of one one-millionth.
A picocurie is one one-millionth of one one-millionth of a Curie. A Curie is that quantity of any radioactive
substance that undergoes 37 billion nuclear disintegrations per second. Thus a picocurie is that quantity of
any radioactive substance that undergoes 0.037 nuclear disintegrations per second.
PIEZOMETRIC SURFACE: See potentiometric surface.
PIN FLOC: Small flocculated particle size.
PLATE AND FRAME PRESS: A batch process dewatering device in which sludge is pumped under high
pressure through a series of parallel plates, in which a chamber is created between the plates. Each plate is
fitted with filter cloth and the solids are collected in the chambers and the water is filtered from the sludge.
POINT SOURCE DISCHARGE: A pipe, ditch, channel or other container from which pollutants may be
discharged.
POLLUTANT: A substance, organism or energy form present in amounts that impair or threaten an
ecosystem to the extent that its current or future uses are prevented.
POLLUTION: To make something unclean or impure. See Contaminated.
POLYMER: A type of chemical when combined with other types of coagulants aid in binding small suspended
particles to larger particles to help in the settling and filtering processes. Chemical used for flocculation in
dewatering. Also known as a "polyelectrolyte" which is a substance made of giant molecules formed by the
union of simple smaller molecules.
POLYPHOSPHATES: Chemicals that may be added to remove low levels of iron and manganese.
PORE SPACE: The interstitial space between sediments and fractures that is capable of storing and
transmitting water.
POROSITY: A factor representing a rock, soil, or formations percentage of open space available for the
percolation and storage of groundwater.
POSITIVE CONTROL: See Ongoing precision and recovery standard.
POST TREATMENT: Treatment of finished water or wastewater to further enhance its quality.
POST-CHLORINE: Where the water is chlorinated to make sure it holds a residual in the distribution
system.
POTABLE: Good water which is safe for drinking or cooking purposes. Non-Potable: A liquid or water that is
not approved for drinking.
POTENTIAL ENERGY: The energy that a body has by virtue of its position or state enabling it to do work.
POWDERED ACTIVATED CARDON TREATMENT (PACT): A wastewater technology in which powdered
activated carbon is added to an anaerobic or aerobic treatment system. The carbon in the biological treatment
process acts as a "buffer" against the effects of toxic organics in the wastewater.
PPM: Abbreviation for parts per million.
PRE-CHLORINATION: The addition of chlorine before the filtration process will help:
PRE-CHLORINE: Where the raw water is dosed with a large concentration of chlorine.
PRECIPITATE: A solid that separates from a solution.
PRECIPTATION: The phenomenon that occurs when a substance held in solution passes out of solution
into a solid form.
PRELIMINARY TREATMENT: Treatment steps including comminution, screening, grit removal, pre-
aeration, and/or flow equalization that prepares wastewater influent for further treatment.
PRESSURE FILTER: Filter unit enclosed in a vessel that may be operated under pressure.
PRESSURE HEAD: The height of a column of water capable of being maintained by pressure. See also
Total Head, Total Dynamic Head.
PRESSURE MEASUREMENT: Bourdon tube, Bellows gauge and Diaphragm are commonly used to
measure pressure in waterworks systems. A Bellows-type sensor reacts to a change in pressure.
PRESSURE: Pressure is defined as force per unit area. It is usually more convenient to use pressure rather
than force to describe the influences upon fluid behavior. The standard unit for pressure is the Pascal, which
is a Newton per square meter. For an object sitting on a surface, the force pressing on the surface is the

weight of the object, but in different orientations it might have a different area in contact with the surface and
therefore exert a different pressure.
PREVENTION: To take action. Stop something before it happens.
PRIMARY CLARIFIER: Sedimentation basin that precedes secondary wastewater treatment.
PRIMARY SLUDGE: Sludge produced in a primary waste treatment unit.
PRIMARY TREATMENT: Treatment steps including sedimentation and/or fine screening to produce an
effluent suitable for biological treatment.
PROCESS WASTEWATER: Wastewater generated during manufacture or production processes.
PROCESS WATER: Water that is used for, or comes in contact with an end product or the materials used
in an end product.
PROPIONIC ACID: Rod-shaped, pleomorphic, gram-positive, anaerobic; ferment lactic acid; fermentation
produces holes in Swiss cheese from the production of carbon dioxide.
PROTON, NEUTRON AND ELECTRON: Are the 3 fundamental particles of an atom.
PROTOZOA: Microscopic animals that occur as single cells. Some protozoa can cause disease in humans.
Protozoa form cysts, which are specialized cells like eggs that are very resistant to chlorine. Cysts can survive
the disinfection process, then "hatch" into normal cells that can cause disease. Protozoa must be removed
from drinking water by filtration, because they cannot be effectively killed by chlorine.
PSEUDOMONAD: Rod-shaped (straight or curved ) with polar flagella, gram-negative, aerobic; can use up
to 100 different compounds for carbon and energy.
PTFE: Polytetrafluoroethylene.
PUMPING LIFT: The height to which water must be pumped or lifted to, feet of head.
Q
QUANTITATIVE TRANSFER: The process of transferring a solution from one container to another using a
pipette in which as much solution as possible is transferred, followed by rinsing of the walls of the source
container with a small volume of rinsing solution (e.g., reagent water, buffer, etc.), followed by transfer of the
rinsing solution, followed by a second rinse and transfer.
QUICKLIME: A calcium oxide material produced by calcining limestone to liberate carbon dioxide, also
called “calcined lime” or “pebble lime”, commonly used for pH adjustment. Chemical formula is CaO.
QUICKLIME: A calcium oxide material produced by calcining limestone to liberate carbon dioxide, also
called “calcined lime” or “pebble lime”, commonly used for pH adjustment. Chemical formula is CaO.
R
RADON: A gas that can dissolve and accumulate in underground water sources, such as wells, and in the
air in your home. Breathing radon can cause lung cancer. Drinking water containing radon presents a risk of
developing cancer. Radon in air is more dangerous than radon in water.
RAW SEWAGE: Untreated wastewater and its contents.
RAW SLUDGE: Undigested sludge recently removed from a sedimentation basin.
RAW TURBIDITY: The turbidity of the water coming to the treatment plant from the raw water source.
RAW WATER: Untreated surface or groundwater.
REAGENT WATER BLANK: see Method blank.
REAGENT WATER: Water demonstrated to be free from the analytes of interest and potentially
interfering substances at the method detection limit for the analyte.
REAGENT: A substance used in a chemical reaction to measure, detect, examine, or produce other
substances.
RECLAIMED WATER: Wastewater that has been treated to a level that allows for its reuse for a beneficial
purpose.
RECLAMATION: The process of improving or restoring the condition of land or other material to a better or
more useful state.
RECOMMENDED EXPOSURE LIMIT (REL): An occupational exposure limit that has been recommended by
the U.S. National Institute for Occupational Safety and Health to OSHA for adoption as a Permissible Exposure
Limit. The REL is a level that NIOSH believes would be protective of worker safety and health over a working
lifetime if used in combination with engineering and work practice controls, exposure and medical monitoring,
posting and labeling of hazards, worker training and personal protective equipment. No REL has ever been
adopted by OSHA, but they have been used as guides by some industry and advocacy organizations.
RECYCLING: The process by which recovered materials are transformed into new products.
REDOX POTENTIAL: Reduction potential (also known as redox potential, oxidation / reduction potential or
ORP) is the tendency of a chemical species to acquire electrons and thereby be reduced. Each species has
its own intrinsic reduction potential; the more positive the potential, the greater the species' affinity for electrons
and tendency to be reduced. In aqueous solutions, the reduction potential is the tendency of the solution to
either gain or lose electrons when it is subject to change by introduction of a new species. A solution with a
higher (more positive) reduction potential than the new species will have a tendency to gain electrons from
the new species (i.e. to be reduced by oxidizing the new species) and a solution with a lower (more negative)
reduction potential will have a tendency to lose electrons to the new species (i.e. to be oxidized by reducing

the new species). Just as the transfer of hydrogen ions between chemical species determines the pH of an
aqueous solution, the transfer of electrons between chemical species determines the reduction potential of an
aqueous solution. Like pH, the reduction potential represents an intensity factor. It does not characterize the
capacity of the system for oxidation or reduction, in much the same way that pH does not characterize the
buffering capacity.
RELATIVE STANDARD DEVIATION (RSD): The standard deviation divided by the mean times 100.
RELAY LOGIC: The name of a popular method of automatically controlling a pump, valve, chemical feeder,
and other devices.
RESERVOIR: An impoundment used to store water.
RESIDENCE TIME: The period of time that a volume of liquid remains in a tank or system.
RESPIRATION: Intake of oxygen and discharge of carbon dioxide as a result of biological oxidation.
RETURN ACTIVATED SLUDGE: Settled activated sludge that is returned to mix with raw or primary settled
wastewater.
RICKETTSIA: Spherical or rod-shaped, gram-negative, aerobic; cause Rocky Mountain spotted fever and
typhus; closely related to Agrobacterium, a common gall-causing plant bacterium.
ROBERT HOOKE: Coined the term "cell" to describe the structures he saw while examining a piece of cork
using a microscope.
ROTARY DRUM SCREEN: Cylindrical screen used to remove floatable and suspended solids.
ROTIFER: Rotifers get their name (derived from Greek and meaning "wheel-bearer"; they have also been
called wheel animalcules) from the corona, which is composed of several ciliated tufts around the mouth that
in motion resemble a wheel. These create a current that sweeps food into the mouth, where it is chewed up
by a characteristic pharynx (called the mastax) containing a tiny, calcified, jaw-like structure called the trophi.
The cilia also pull the animal, when unattached, through the water. Most free-living forms have pairs of
posterior toes to anchor themselves while feeding. Rotifers have bilateral symmetry and a variety of different
shapes. There is a well-developed cuticle which may be thick and rigid, giving the animal a box-like shape, or
flexible, giving the animal a worm-like shape; such rotifers are respectively called loricate and illoricate.
S
SANITARY SURVEY: Persons trained in public health engineering and the epidemiology of waterborne
diseases should conduct the sanitary survey. The importance of a detailed sanitary survey of a new water
source cannot be overemphasized. An on-site review of the water sources, facilities, equipment, operation,
and maintenance of a public water systems for the purpose of evaluating the adequacy of the facilities for
producing and distributing safe drinking water. The purpose of a non-regulatory sanitary survey is to identify
possible biological and chemical pollutants which might affect a water supply.
SANITIZER: A disinfectant or chemical which disinfects (kills bacteria), kills algae and oxidizes organic
matter.
SATURATED ZONE: Where an unconfined aquifer becomes saturated beneath the capillary fringe.
SATURATION INDEX: See Langelier's Index.
SATURATOR: A device which produces a fluoride solution for the fluoride process. Crystal-grade types of
sodium fluoride should be fed with a saturator. Overfeeding must be prevented to protect public health when
using a fluoridation system.
SCADA: A remote method of monitoring pumps and equipment. 130 degrees F is the maximum
temperature that transmitting equipment is able to with stand. If the level controller may be set with too close
a tolerance 45 could be the cause of a control system that is frequently turning a pump on and off.
SCALE: Crust of calcium carbonate, the result of unbalanced water. Hard insoluble minerals deposited
(usually calcium bicarbonate) which forms on pool and spa surfaces and clog filters, heaters and pumps. Scale
is caused by high calcium hardness and/or high pH. The regular use of stain prevention chemicals can prevent
scale.
SCREENINGS PRESS: A mechanical press used to compact and/or dewater material removed from
mechanical screening equipment.
SCROLL AND BASKET: The two basic types of centrifuges used in water treatment.
SCRUBBER: A device used to removal particulates or pollutant gases from combustion or chemical
process exhaust streams.
SCUM: Floatable materials found on the surface of primary and secondary settling tanks consisting of food
wastes, grease, fats, paper, foam, and similar matter.
SECONDARY CLARIFIER: A clarifier following a secondary treatment process, designed for gravity
removal of suspended matter.
SECONDARY SLUDGE: The sludge from the secondary clarifier in a wastewater treatment plant.
SECONDARY TREATMENT: The treatment of wastewater through biological oxidation after primary
treatment.
SEDIMENT: Grains of soil, sand, gravel, or rock deposited by and generated by water movement.

SEDIMENTATION BASIN: A quiescent tank used to remove suspended solids by gravity settling. Also called
clarifiers or settling tanks, they are usually equipped with a motor driven rake mechanism to collect settled
sludge and move it to a central discharge point.
SEDIMENTATION BASIN: Where the thickest and greatest concentration of sludge will be found. Twice a
year sedimentation tanks should be drained and cleaned if the sludge buildup interferes with the treatment
process.
SEDIMENTATION: The process of suspended solid particles settling out (going to the bottom of the vessel)
in water.
SEDIMENTATION: The removal of settleable suspended solids from water or wastewater by gravity in a
quiescent basin or clarifier.
SENSOR: A float and cable system are commonly found instruments that may be used as a sensor to
control the level of liquid in a tank or basin.
SEPTIC: Condition characterized by bacterial decomposition under anaerobic conditions.
SETTLEABILITY: The tendency of suspended solids to settle.
SETTLEABLE SOLIDS: That portion of suspended solids which are of a sufficient size and weight to settle
to the bottom of an Imhoff cone in one hour.
SETTLED SLUDGE VOLUME: Volume of settled sludge measured at predetermined time increments for
use in process control calculations.
SETTLED SOLIDS: Solids that have been removed from the raw water by the coagulation and settling
processes.
SEWAGE: Liquid or waterborne wastes polluted or fouled from households, commercial or industrial
operations, along with any surface water, storm water or groundwater infiltration.
SEWER GAS: A gas mixture produced by anaerobic decomposition of organic matter usually containing
high percentages of methane and hydrogen sulfide.
SHEATHED: Filamentous, gram-negative, aerobic; “swarmer” (colonizing) cells form and break out of a
sheath; sometimes coated with metals from environment.
SHOCK LOAD: A sudden hydraulic or organic load to a treatment plant, also descriptive of a change in the
material being treated.
SHOCK: Also known as superchlorination or break point chlorination. Ridding a water of organic waste
through oxidization by the addition of significant quantities of a halogen.
SHORT-CIRCUITING: Short Circuiting is a condition that occurs in tanks or basins when some of the water
travels faster than the rest of the flowing water. This is usually undesirable since it may result in shorter
contact, reaction or settling times in comparison with the presumed detention times.
SHOULD: This action, activity, or procedural step is suggested but not required.
SINGLE PHASE POWER: The type of power used for lighting systems, small motors, appliances, portable
power tools and in homes.
SLOP OIL: Separator skimmings and tramp oil generated during refinery startup, shutdown or abnormal
operation.
SLUDGE BASINS: After cleaning sludge basins and before returning the tanks into service the tanks should
be inspected, repaired if necessary, and disinfected.
SLUDGE BLANKET: The accumulated sludge suspended in a clarifier or other enclosed body of water.
SLUDGE DEWATERING: The removal of a portion or majority of the water contained in sludge by means of
a filter press, centrifuge or other mechanism.
SLUDGE DRYING BED: A closed area consisting of sand or other porous material upon which sludge is
dewatered by gravity drainage and evaporation.
SLUDGE REDUCTION: Organic polymers are used to reduce the quantity of sludge. If a plant produces a
large volume of sludge, the sludge could be dewatered, thickened, or conditioned to decrease the volume of
sludge. Turbidity of source water, dosage, and type of coagulant used are the most important factors which
determine the amount of sludge produced in a treatment of water.
SLUDGE: Accumulated and concentrated solids generated within a treatment process that have not
undergone a stabilization process.
SLURRY: A mixture of a solid and a liquid that facilitates the transfer of the solid into a treatment solution.
SOC: A common way for a synthetic organic chemical such as dioxin to be introduced to a surface water
supply is from an industrial discharge, agricultural drainage, or a spill.
SODA ASH: Chemical used to raise pH and total alkalinity (sodium carbonate)
SODIUM BICARBONATE: Commonly used to increase alkalinity of water and stabilize pH.
SODIUM BISULFATE: Chemical used to lower pH and total alkalinity (dry acid).
SODIUM HYDROXIDE: Also known as caustic soda, a by-product chlorine generation and often used to
raise pH.
SOFTENING WATER: When the water has a low alkalinity, it is advantageous to use soda ash instead of
caustic soda for softening water.
SOFTENING: The process that removes the ions which cause hardness in water.

SOLID WASTE: Garbage, refuse, sludge and other discarded material resulting from community activities
or commercial or industrial operations.
SOLID, LIQUID AND VAPOR: 3 forms of matter.
SOLUBILITY: The amount of a substance that can dissolve in a solution under a given set of conditions.
SPADNS: The lab reagent called SPADNS solution is used in performing the Fluoride test.
SPIKING SUSPENSION: Diluted stock suspension containing the organism(s) of interest at a
concentration appropriate for spiking samples.
SPIRILLUM: Spiral-shaped, gram-negative, aerobic; include Bdellovibrio, predatory on other
bacteria.
SPIROCHETE: Spiral-shaped, gram-negative, mostly anaerobic; common in moist environments,
from mammalian gums to coastal mudflats; complex internal structures convey rapid movement;
include Treponemapallidum, cause of syphilis.
SPOROZOITE: A motile, infective stage of certain protozoans; e.g., Cryptosporidium. There are four
sporozoites in each Cryptosporidium oocyst, and they are generally banana-shaped.
SPRAY BOTTLE OF AMMONIA: An operator should use ammonia to test for a chlorine leak around a
valve or pipe. You will see white smoke if there is a leak.
SPRING PRESSURE: Is what maintains contact between the two surfaces of a mechanical seal.
STABILIZATION POND: A large shallow basin used for wastewater treatment by natural processes
involving the use of algae and bacteria to accomplish biological oxidation of organic matter.
STERILIZED GLASSWARE: The only type of glassware that should be used in testing for coliform bacteria.
STOCK SUSPENSION: A concentrated suspension containing the organism(s) of interest that is obtained
from a source that will attest to the host source, purity, authenticity, and viability of the organism(s).
STUFFING BOX: That portion of the pump that houses the packing or mechanical seal.
SUBNATANT: Liquid remaining beneath the surface of floating solids.
SUCCESSION: Transition in the species composition of a biological community, often following ecological
disturbance of the community; the establishment of a biological community in an area virtually barren of life.
SULFATE- AND SULFUR- REDUCING: Commonly rod-shaped, mostly gram-negative, anaerobic; include
Desulfovibrio, ecologically important in marshes.
SULFIDE: The term sulfide refers to several types of chemical compounds containing sulfur in its lowest
oxidation number of -2. Formally, "sulfide" is the dianion, S2-, which exists in strongly alkaline aqueous
solutions formed from H2S or alkali metal salts such as Li2S, Na2S, and K2S. Sulfide is exceptionally basic
and, with a pKa > 14, it does not exist in appreciable concentrations even in highly alkaline water, being
undetectable at pH < ~15 (8 M NaOH). Instead, sulfide combines with electrons in hydrogen to form HS, which
is variously called hydrogen sulfide ion, hydrosulfide ion, sulfhydryl ion, or bisulfide ion. At still lower pH's (<7),
HS- converts to H2S, hydrogen sulfide. Thus, the exact sulfur species obtained upon dissolving sulfide salts
depends on the pH of the final solution. Aqueous solutions of transition metals cations react with sulfide
sources (H2S, NaSH, Na2S) to precipitate solid sulfides. Such inorganic sulfides typically have very low
solubility in water and many are related to minerals. One famous example is the bright yellow species CdS or
"cadmium yellow". The black tarnish formed on sterling silver is Ag2S. Such species are sometimes referred
to as salts. In fact, the bonding in transition metal sulfides is highly covalent, which gives rise to their
semiconductor properties, which in turn is related to the practical applications of many sulfide materials.
SULFUR- AND IRON- OXIDIZING: Commonly rod-shaped, frequently with polar flagella, gram-negative,
mostly anaerobic; most live in neutral (nonacidic) environment.
SUPERNATANT: The liquid layer which forms above the sludge in a settling basin.
SURFACE SEAL: The upper portion of a wells construction where surface contaminants are adequately
prevented from entering the well, normally consisting of surface casing and neat cement grout.
SURFACTANT: Surfactants reduce the surface tension of water by adsorbing at the liquid-gas interface.
They also reduce the interfacial tension between oil and water by adsorbing at the liquid-liquid interface. Many
surfactants can also assemble in the bulk solution into aggregates. Examples of such aggregates are vesicles
and micelles. The concentration at which surfactants begin to form micelles is known as the critical micelle
concentration or CMC. When micelles form in water, their tails form a core that can encapsulate an oil droplet,
and their (ionic/polar) heads form an outer shell that maintains favorable contact with water. When surfactants
assemble in oil, the aggregate is referred to as a reverse micelle. In a reverse micelle, the heads are in the
core and the tails maintain favorable contact with oil. Surfactants are also often classified into four primary
groups; anionic, cationic, non-ionic, and zwitterionic (dual charge).
SUSPENDED SOLIDS: Solids captured by filtration through a 0.45 micron filter membrane.
T
TCE, trichloroethylene: A solvent and degreaser used for many purposes; for example dry cleaning, it is a
common groundwater contaminant. Trichloroethylene is a colorless liquid which is used as a solvent for
cleaning metal parts. Drinking or breathing high levels of trichloroethylene may cause nervous system effects,
liver and lung damage, abnormal heartbeat, coma, and possibly death. Trichloroethylene has been found in
at least 852 of the 1,430 National Priorities List sites identified by the Environmental Protection Agency (EPA).

TDS-TOTAL DISSOLVED SOLIDS: An expression for the combined content of all inorganic and organic
substances contained in a liquid which are present in a molecular, ionized or micro-granular (colloidal sol)
suspended form. Generally, the operational definition is that the solids (often abbreviated TDS) must be small
enough to survive filtration through a sieve size of two micrometers. Total dissolved solids are normally only
discussed for freshwater systems, since salinity comprises some of the ions constituting the definition of TDS.
The principal application of TDS is in the study of water quality for streams, rivers and lakes, although TDS is
generally considered not as a primary pollutant (e.g. it is not deemed to be associated with health effects), but
it is rather used as an indication of aesthetic characteristics of drinking water and as an aggregate indicator of
presence of a broad array of chemical contaminants.
TELEMETERING: The use of a transmission line with remote signaling to monitor a pumping station or
motors. Can be used to accomplish accurate and reliable remote monitoring and control over a long
TEMPERATURE SAMPLE: This test should be performed immediately in the field, a grab sample.
TERTIARY TREATMENT: The use of physical, chemical, or biological means to improve secondary
wastewater effluent quality.
The addition of chlorine to the water prior to any other plant treatment processes.
THE RATE DECREASES: In general, when the temperature decreases, the chemical reaction rate
decreases also.
THICKENING, CONDITIONING AND DEWATERING: Common processes that are utilized to reduce the
volume of sludge.
THICKENING: A procedure used to increase the solids content of sludge by removing a portion of the
liquid.
TIME FOR TURBIDITY BREAKTHROUGH AND MAXIMUM HEADLOSS: Are the two factors which
determine whether or not a change in filter media size should be made.
TITRATION: A method of testing by adding a reagent of known strength to a water sample until a specific
color change indicates the completion of the reaction.
TOTAL ALKALINITY: A measure of the acid-neutralizing capacity of water which indicates its buffering ability,
i.e. measure of its resistance to a change in pH. Generally, the higher the total alkalinity, the greater the
resistance to pH change.
TOTAL COLIFORM: Total coliform, fecal coliform, and E. coli are all indicators of drinking water quality. The
total coliform group is a large collection of different kinds of bacteria. Fecal coliforms are types of total coliform
that mostly exist in feces. E. coli is a sub-group of fecal coliform. When a water sample is sent to a lab, it is
tested for total coliform. If total coliform is present, the sample will also be tested for either fecal coliform or E.
coli, depending on the lab testing method.
TOTAL DISSOLVED SOLIDS (TDS): The accumulated total of all solids that might be dissolved in water.
The weight per unit volume of all volatile and non-volatile solids dissolved in a water or wastewater after a
sample has been filtered to remove colloidal and suspended solids.
TOTAL DYNAMIC HEAD: The pressure (psi) or equivalent feet of water, required for a pump to lift water to
its point of storage overcoming elevation head, friction loss, line pressure, drawdown and pumping lift.
TOTAL SOLIDS: The sum of dissolved and suspended solids in a water or wastewater.
TOTAL SUSPENDED SOLIDS: The measure of particulate matter suspended in a sample of water or
wastewater.
TOXIC: Capable of causing an adverse effect on biological tissue following physical contact or absorption.
TRANSIENT, NON-COMMUNITY WATER SYSTEM: TNCWS A water system which provides water in a place
such as a gas station or campground where people do not remain for long periods of time. These systems do
not have to test or treat their water for contaminants which pose long-term health risks because fewer than 25
people drink the water over a long period. They still must test their water for microbes and several
chemicals. A Transient Non-community Water System: Is not required to sample for VOC’s.
TREATABILITY STUDY: A study in which a waste is subjected to a treatment process to determine treatment
and/or to determine the treatment efficiency or optimal process conditions for treatment.
TRIHALOMETHANES (THM): Four separate compounds including chloroform, dichlorobromomethane,
dibromochloromethane, and bromoform. The most common class of disinfection by-products created when
chemical disinfectants react with organic matter in water during the disinfection process. See Disinfectant
Byproducts.
TUBE SETTLERS: This modification of the conventional process contains many metal tubes that are placed
in the sedimentation basin, or clarifier. These tubes are approximately 1 inch deep and 36 inches long, split-
hexagonal shape and installed at an angle of 60 degrees or less. These tubes provide for a very large surface
area upon which particles may settle as the water flows upward. The slope of the tubes facilitates gravity
settling of the solids to the bottom of the basin, where they can be collected and removed. The large surface
settling area also means that adequate clarification can be obtained with detention times of 15 minutes or less.
As with conventional treatment, this sedimentation step is followed by filtration through mixed media.

TUBERCLES: The creation of this condition is of the most concern regarding corrosive water effects on a
water system. Tubercles are formed due to joining dissimilar metals, causing electro-chemical reactions. Like
iron to copper pipe. We have all seen these little rust mounds inside cast iron pipe.
TURBIDIMETER: Monitoring the filter effluent turbidity on a continuous basis with an in-line instrument is a
recommended practice. Turbidimeter is best suited to perform this measurement.
TURBIDITY: A measure of the cloudiness of water caused by suspended particles. A qualitative measurement
of water clarity which results from suspended matter that scatters or otherwise interferes with the passage of
light through the water.
TURBIDITY: Turbidity can interfere with disinfection and provide a medium for microbial growth. Turbidity may
indicate the presence of disease causing organisms. These organisms include bacteria, viruses, and parasites
that can cause symptoms such as nausea, cramps, diarrhea, and associated headaches.
U
U.S. ENVIRONMENTAL PROTECTION AGENCY: In the United States, this agency responsible for setting
drinking water standards and for ensuring their enforcement. This agency sets federal regulations which all
state and local agencies must enforce.
U.S. ENVIRONMENTAL PROTECTION AGENCY: In the United States, this agency responsible for setting
drinking water standards and for ensuring their enforcement. This agency sets federal regulations which all
state and local agencies must enforce.
ULTRAFILTRATION: A low pressure membrane filtration process which separates solutes up to 0.1 micron
size range.
UNDER PRESSURE IN STEEL CONTAINERS: After chlorine gas is manufactured, it is primarily transported
in steel containers.
UP FLOW CLARIFIER: Clarifier where flocculated water flows upward through a sludge blanket to obtain floc
removal by contact with flocculated solids in the blanket.
V
VANE: That portion of an impeller that throws the water toward the volute.
VAPOR: The gaseous phase of a material that is in the solid or liquid state at standard temperature and
pressure.
VARIABLE DISPLACEMENT PUMP: A pump that will produce different volumes of water dependent on
the pressure head against it.
VELOCITY HEAD: The vertical distance a liquid must fall to acquire the velocity with which it flows through
the piping system. For a given quantity of flow, the velocity head will vary indirectly as the pipe diameter
varies.
VENTURI: If water flows through a pipeline at a high velocity, the pressure in the pipeline is reduced.
Velocities can be increased to a point that a partial vacuum is created.
VERTICAL TURBINE: A type of variable displacement pump in which the motor or drive head is mounted on
the wellhead and rotates a drive shaft connected to the pump impellers.
VIBRIO: Rod- or comma-shaped, gram-negative, aerobic; commonly with a single flagellum; include Vibrio
cholerae, cause of cholera, and luminescent forms symbiotic with deep-water fishes and squids.
VIRUSES: Very small disease-causing microorganisms that are too small to be seen even with microscopes.
Viruses cannot multiply or produce disease outside of a living cell.
VIRUSES: are very small disease-causing microorganisms that are too small to be seen even with
microscopes. Viruses cannot multiply or produce disease outside of a living cell.
VITRIFICATION: Vitrification is a process of converting a material into a glass-like amorphous solid that is
free from any crystalline structure, either by the quick removal or addition of heat, or by mixing with an additive.
Solidification of a vitreous solid occurs at the glass transition temperature (which is lower than melting
temperature, Tm, due to supercooling). When the starting material is solid, vitrification usually involves heating
the substances to very high temperatures. Many ceramics are produced in such a manner. Vitrification may
also occur naturally when lightning strikes sand, where the extreme and immediate heat can create hollow,
branching rootlike structures of glass, called fulgurite. When applied to whiteware ceramics, vitreous means
the material has an extremely low permeability to liquids, often but not always water, when determined by a
specified test regime. The microstructure of whiteware ceramics frequently contain both amorphous and
crystalline phases.
VOID: An opening, gap, or space within rock or sedimentary formations formed at the time of origin or
deposition.
VOLATILE ORGANIC COMPOUNDS (VOCs): Solvents used as degreasers or cleaning agents. Improper
disposal of VOCs can lead to contamination of natural waters. VOCs tend to evaporate very easily. This
characteristic gives VOCs very distinct chemical odors like gasoline, kerosene, lighter fluid, or dry cleaning
fluid. Some VOCs are suspected cancer-causing agents. Volatile organic compounds (VOCs) are organic
chemical compounds that have high enough vapor pressures under normal conditions to significantly vaporize
and enter the atmosphere. A wide range of carbon-based molecules, such as aldehydes, ketones, and other
light hydrocarbons are VOCs. The term often is used in a legal or regulatory context and in such cases the

precise definition is a matter of law. These definitions can be contradictory and may contain "loopholes"; e.g.
exceptions, exemptions, and exclusions. The United States Environmental Protection Agency defines a VOC
as any organic compound that participates in a photoreaction; others believe this definition is very broad and
vague as organics that are not volatile in the sense that they vaporize under normal conditions can be
considered volatile by this EPA definition. The term may refer both to well characterized organic compounds
and to mixtures of variable composition.
VOLATILE ORGANIC COMPOUNDS (VOCs): Solvents used as degreasers or cleaning agents. Improper
disposal of VOCs can lead to contamination of natural waters. VOCs tend to evaporate very easily. This
characteristic gives VOCs very distinct chemical odors like gasoline, kerosene, lighter fluid, or dry cleaning
fluid. Some VOCs are suspected cancer-causing agents. Volatile organic compounds (VOCs) are organic
chemical compounds that have high enough vapor pressures under normal conditions to significantly vaporize
and enter the atmosphere. A wide range of carbon-based molecules, such as aldehydes, ketones, and other
light hydrocarbons are VOCs. The term often is used in a legal or regulatory context and in such cases the
precise definition is a matter of law. These definitions can be contradictory and may contain "loopholes"; e.g.
exceptions, exemptions, and exclusions. The United States Environmental Protection Agency defines a VOC
as any organic compound that participates in a photoreaction; others believe this definition is very broad and
vague as organics that are not volatile in the sense that they vaporize under normal conditions can be
considered volatile by this EPA definition. The term may refer both to well characterized organic compounds
and to mixtures of variable composition.
VOLATILE: A substance that evaporates or vaporizes at a relatively low temperature.
VOLTAGE: Voltage (sometimes also called electric or electrical tension) is the difference of electrical potential
between two points of an electrical or electronic circuit, expressed in volts.[1] It measures the potential energy
of an electric field to cause an electric current in an electrical conductor. Depending on the difference of
electrical potential it is called extra low voltage, low voltage, high voltage or extra high voltage. Specifically
Voltage is equal to energy per unit charge.
VOLUTE: The spiral-shaped casing surrounding a pump impeller that collects the liquid discharge by the
impeller.
VORTEX: The helical swirling of water moving towards a pump.
VORTICELLA: Vorticella is a genus of protozoa, with over 100 known species. They are stalked inverted
bell-shaped ciliates, placed among the peritrichs. Each cell has a separate stalk anchored onto the substrate,
which contains a contracile fibril called a myoneme. When stimulated this shortens, causing the stalk to coil
like a spring. Reproduction is by budding, where the cell undergoes longitudinal fission and only one daughter
keeps the stalk. Vorticella mainly lives in freshwater ponds and streams - generally, anywhere protists are
plentiful. Other genera such as Carchesium resemble Vorticella but are branched or colonial.
VULNERABILITY ASSESSMENT: An evaluation of drinking water source quality and its vulnerability to
contamination by pathogens and toxic chemicals.
W
WAIVERS: Monitoring waivers for nitrate and nitrite are prohibited.
WASTE ACTIVATED SLUDGE: Excess activated sludge that is discharged from an activated sludge
treatment process.
WASTEWATER: Liquid or waterborne wastes polluted or fouled from households, commercial or industrial
operations, along with any surface water, storm water or groundwater infiltration.
WATER HAMMER: A surge in a pipeline resulting from the rapid increase or decrease in water flow. Water
hammer exerts tremendous force on a system and can be highly destructive.
WATER PURVEYOR: The individuals or organization responsible to help provide, supply, and furnish
quality water to a community.
WATER QUALITY CRITERIA: Comprised of both numeric and narrative criteria. Numeric criteria are
scientifically derived ambient concentrations developed by EPA or States for various pollutants of concern to
protect human health and aquatic life. Narrative criteria are statements that describe the desired water quality
goal.
WATER QUALITY STANDARD: A statute or regulation that consists of the beneficial designated use or uses
of a waterbody, the numeric and narrative water quality criteria that are necessary to protect the use or uses
of that particular waterbody, and an antidegradation statement.
WATER QUALITY: The 4 broad categories of water quality are: Physical, chemical, biological, radiological.
Pathogens are disease causing organisms such as bacteria and viruses. A positive bacteriological sample
indicates the presence of bacteriological contamination. Source water monitoring for lead and copper be
performed when a public water system exceeds an action level for lead of copper.
WATER RECLAMATION: The restoration of wastewater to a state that will allow its beneficial reuse.
WATER VAPOR: A characteristic that is unique to water vapor in the atmosphere is that water does not
contain any salts.
WATERBORNE DISEASE: A disease, caused by a virus, bacterium, protozoan, or other microorganism,
capable of being transmitted by water (e.g., typhoid fever, cholera, amoebic dysentery, gastroenteritis).

WATERSHED: An area that drains all of its water to a particular water course or body of water. The land
area from which water drains into a stream, river, or reservoir.
WAVE FUNCTION: A function describing the electron's position in a three-dimensional space.
WHOLE EFFLUENT TOXICITY: The total toxic effect of an effluent measured directly with a toxicity test.
WPCF: Water Pollution Control Facility
WTP: Water Treatment Plant
WWTP: Wastewater Treatment Plant
X
X-RAY DIFFRACTION: A method for establishing structures of crystalline solids using singe wavelength X-
rays and looking at diffraction pattern.
X-RAY PHOTOELECTRON SPECTROSCOPY: A spectroscopic technique to measure composition of a
material.
X-RAY: Form of ionizing, electromagnetic radiation, between gamma and UV rays.
Y
YIELD: The amount of product produced during a chemical reaction.
Z
ZERO DISCHARGE: A facility that discharges no liquid effluent to the environment.
ZONE MELTING: A way to remove impurities from an element by melting it and slowly travel down an ingot
(cast).
ZWITTERION: Is a chemical compound whose net charge is zero and hence is electrically neutral. But there
are some positive and negative charges in it, due to the formal charge, owing to the partial charges of its
constituent atoms.

Waterborne Microorganisms and Bacteria Appendix
This section will give a close-up and short explanation of the major microorganisms
found in wastewater.

Protozoa Section
The diverse assemblage of organisms that carry out all of their life functions within the
confines of a single, complex eukaryotic cell are called protozoa.
Paramecium, Euglena, and Amoeba are well-known examples of these major groups of
organisms. Some protozoa are more closely related to animals, others to plants, and still
others are relatively unique. Although it is not appropriate to group them together into a
single taxonomic category, the research tools used to study any unicellular organism are
usually the same, and the field of protozoology has been created to carry out this research.
The unicellular photosynthetic protozoa are sometimes also called algae and are
addressed elsewhere. This report considers the status of our knowledge of heterotrophic
protozoa (protozoa that cannot produce their own food).
Free-living Protozoa
Protozoans are found in all moist habitats within the United States, but we know little about
their specific geographic distribution. Because of their small size, production of resistant
cysts, and ease of distribution from one place to another, many species appear to be
cosmopolitan and may be collected in similar microhabitats worldwide (Cairns and
Ruthven 1972). Other species may have relatively narrow limits to their distribution.
Marine ciliates inhabit interstices of sediment and beach sands, surfaces, deep sea and
cold Antarctic environments, planktonic habitats, and the algal mats and detritus of
estuaries and wetlands.

Protozoa
Protozoa are around 10–50 micrometer, but can grow up to 1 mm and can easily be seen
under a microscope. Protozoa exist throughout aqueous environments and soil. Protozoa
occupy a range of trophic levels. As predators, they prey upon unicellular or filamentous
algae, bacteria, and microfungi.
Protozoa play a role both as herbivores and as consumers in the decomposer link of the
food chain. Protozoa also play a vital role in controlling bacteria populations and biomass.
As components of the micro- and meiofauna, protozoa are an important food source for
microinvertebrates. Thus, the ecological role of protozoa in the transfer of bacterial and
algal production to successive trophic levels is important. Protozoa such as the malaria
parasites (Plasmodium spp.), trypanosomes and leishmania are also important as
parasites and symbionts of multicellular animals.
Most protozoa exist in 5 stages of life which are in the form of trophozoites and cysts. As
cysts, protozoa can survive harsh conditions, such as exposure to extreme temperatures
and harmful chemicals, or long periods without access to nutrients, water, or oxygen for a
period of time.

Being a cyst enables parasitic species to survive outside of the host, and allows their
transmission from one host to another. When protozoa are in the form of trophozoites
(Greek, tropho=to nourish), they actively feed and grow.
The process by which the protozoa takes its cyst form is called encystation, while the
process of transforming back into trophozoite is called excystation.
Protozoa can reproduce by binary fission or multiple fission. Some protozoa reproduce
sexually, some asexually, and some both (e.g. Coccidia). An individual protozoan is
hermaphroditic.
Classification
Protozoa were commonly grouped in the kingdom of Protista together with the plant-like
algae and fungus-like water molds and slime molds. In the 21st-century systematics,
protozoans, along with ciliates, mastigophorans, and apicomplexans, are arranged as
animal-like protists. However, protozoans are neither Animalia nor Metazoa (with the
possible exception of the enigmatic, moldy Myxozoa).
Sub-groups
Protozoa have traditionally been divided on the basis of their means of locomotion,
although this is no longer believed to represent genuine relationships:
* Flagellates (e.g. Giardia lambia)
* Amoeboids (e.g. Entamoeba histolytica)
* Sporozoans (e.g. Plasmodium knowlesi)
* Apicomplexa
* Myxozoa
* Microsporidia
* Ciliates (e.g. Balantidium coli)
There are many ways that infectious diseases can spread. Pathogens usually have
specific routes by which they are transmitted, and these routes may depend on the type
of cells and tissue that a particular agent targets. For example, because cold viruses infect
the respiratory tract, they are dispersed into the air via coughing and sneezing.
Once in the air, the viruses can infect another person who is unlucky enough to inhale air
containing the virus particles.
Agents vary greatly in their stability in the environment. Some viruses may survive for only
a few minutes outside of a host, while some spore-forming bacteria are extremely durable
and may survive in a dormant state for a decade or more.

Eukaryote
Eukaryotes are organisms with complex cells, in which the genetic material is organized
into membrane-bound nuclei. They include the animals, plants, and fungi, which are
mostly multicellular, as well as various other groups called protists, many of which are
unicellular. In contrast, other organisms such as bacteria lack nuclei and other complex
cell structures, and are called prokaryotes. The eukaryotes share a common origin, and
are often treated formally as a super kingdom, empire, or domain. The name comes from
the Greek eus or true and karyon or nut, referring to the nucleus.
What are Protists?

 They are eukaryotes because they all have a nucleus.
 Most have mitochondria although some have later lost theirs. Mitochondria were
derived from aerobic alpha-proteobacteria (prokaryotes) that once lived within their
cells.
 Many have chloroplasts with which they carry on photosynthesis. Chloroplasts

were derived from photosynthetic cyanobacteria (also prokaryotes) living within
their cells.
Eukaryotic Cells
Eukaryotic cells are generally much larger than prokaryotes, typically with a thousand
times their volumes. They have a variety of internal membranes and structures, called
organelles, and a cytoskeleton composed of microtubules and microfilaments, which plays
an important role in defining the cell's organization.

Eukaryotic DNA is divided into several bundles called chromosomes, which are separated
by a microtubular spindle during nuclear division. In addition to asexual cell division, most
eukaryotes have some process of sexual reproduction via cell fusion, which is not found
among prokaryotes.
Eukaryotic cells include a variety of membrane-bound structures, collectively referred to

as the endomembrane system. Simple compartments, called vesicles or vacuoles, can
form by budding off of other membranes. Many cells ingest food and other materials
through a process of endocytosis, where the outer membrane invaginates and then
pinches off to form a vesicle. It is probable that most other membrane-bound organelles
are ultimately derived from such vesicles.
The nucleus is surrounded by a double membrane, with pores that allow material to move
in and out. Various tube- and sheet-like extensions of the nuclear membrane form what is
called the endoplasmic reticulum or ER, which is involved in protein transport. It includes
rough sections where ribosomes are attached, and the proteins they synthesize enter the
interior space or lumen. Subsequently, they generally enter vesicles, which bud off from
the smooth section. In most eukaryotes, the proteins may be further modified in stacks of
flattened vesicles, called Golgi bodies or dictyosomes.
Vesicles may be specialized for various purposes. For instance, lysosomes contain
enzymes that break down the contents of food vacuoles, and peroxisomes are used to
break down peroxide which is toxic otherwise.
Contractile Vacuoles
Many protozoa have contractile vacuoles, which collect and expel excess water, and
extrusomes, which expel material used to deflect predators or capture prey. In multicellular
organisms, hormones are often produced in vesicles. In higher plants, most of a cell's
volume is taken up by a central vacuole or tonoplast, which maintains its osmotic pressure.
Many eukaryotes have slender motile projections, usually called flagella when long and
cilia when short. These are variously involved in movement, feeding, and sensation. These
are entirely distinct from prokaryotic flagella. They are supported by a bundle of
microtubules arising from a basal body, also called a kinetosome or centriole,
characteristically arranged as nine doublets surrounding two singlets. Flagella also may
have hairs or mastigonemes, scales, connecting membranes, and internal rods. Their
interior is continuous with the cell's cytoplasm.
Centrioles
Centrioles are often present even in cells and groups that do not have flagella. They
generally occur in groups of one or two, called kinetids that give rise to various
microtubular roots. These form a primary component of the cytoskeletal structure, and are
often assembled over the course of several cell divisions, with one flagellum retained from
the parent and the other derived from it.
Centrioles may also be associated in the formation of a spindle during nuclear division.
Some protists have various other microtubule-supported organelles. These include the
radiolaria and heliozoa, which produce axopodia used in flotation or to capture prey, and
the haptophytes, which have a peculiar flagellum-like organelle called the haptonema.

Amoebas
Amoebas (Phylum Rhizopoda) are unicellular protists that are able to change their shape
constantly. Each species has its own distinct repertoire of shapes.
How does an amoeba locomote?

Amoebas locomote by way of cytoplasmic movement. (cytoplasm is the cell content
around the nucleus of the cell) The amoeba forms pseudopods (false feet) with which they
'flow' over a surface. The cytoplasma not only flows, it also changes from a fluid into a
solid state.
These pseudopods are also used to capture prey; they simply engulf the food. They can
detect the kind of prey and use different 'engulfing tactics'.
The image from the last page shows several cell organelles. Left from the center we can
see aspherical water expelling vesicle and just right of it, the single nucleus of this species
can be seen. Other species may have many nuclei. The cell is full of brown food vacuoles
and also contains small crystals.
Protozoa Information
Our actual knowledge of salinity, temperature, and oxygen requirements of marine
protozoa is poor (although some groups, such as the foraminifera, are better studied than
others), and even the broadest outlines of their biogeographic ranges are usually a
mystery.
In general, freshwater protozoan communities are similar to marine communities except

the specialized interstitial fauna of the sand is largely missing. In freshwater habitats, the
foraminifera and radiolaria common in marine environments are absent or low in numbers
while testate amoebae exist in greater numbers. Relative abundance of species in the
marine versus freshwater habitat is unknown.
Soil-dwelling protozoa have been documented from almost every type of soil and in every
kind of environment, from the peat-rich soil of bogs to the dry sands of deserts. In general,
protozoa are found in greatest abundance near the soil surface, especially in the upper 15
cm (6 in), but occasional isolates can be obtained at depths of a meter (yard) or more.
Protozoa do not constitute a major part of soil biomass, but in some highly productive
regions such as forest litter, the protozoa are a significant food source for the
microinvertebrates, with a biomass that may reach 20 g/m2 of soil surface area there.
Environmental Quality Indicators

Polluted waters often have a rich and characteristic protozoan fauna. The relative
abundance and diversity of protozoa are used as indicators of organic and toxic pollution
(Cairns et al. 1972; Foissner 1987; Niederlehner et al. 1990; Curds 1992). Bick (1972), for
example, provided a guide to ciliates that are useful as indicators of environmental quality
of European freshwater systems, along with their ecological distribution with respect to
parameters such as amount of organic material and oxygen levels. Foissner (1988)
clarified the taxonomy of European ciliates as part of a system for classifying the state of
aquatic habitats according to their faunas.

Amoeba
Amoeba (sometimes amœba or ameba, plural amoebae) is a genus of protozoa that

moves by means of pseudopods, and is well-known as a representative unicellular
organism.
The word amoeba or ameba is variously used to refer to it and its close relatives, now
grouped as the Amoebozoa, or to all protozoa that move using pseudopods, otherwise
termed amoeboids.

Paramecia
Paramecia are a group of unicellular ciliate protozoa formerly known as slipper

animalcules from their slipper shape. They are commonly studied as a representative of
the ciliate group. Simple cilia cover the body which allows the cell to move with a
synchronous motion (like a caterpilla).
There is also a deep oral groove containing inconspicuous compound oral cilia (as found
in other peniculids) that is used to draw food inside. They generally feed upon bacteria
and other small cells. Osmoregulation is carried out by a pair of contractile vacuoles, which
actively expel water absorbed by osmosis from their surroundings.
Paramecia are widespread in freshwater environments, and are especially common in

scums. Paramecia are attracted by acidic conditions. Certain single-celled eukaryotes,
such as Paramecium, are examples for exceptions to the universality of the genetic code
(translation systems where a few codons differ from the standard ones).

Symbiotic Protozoa
Parasites
Protozoa are infamous for their role in causing disease, and parasitic species are among
the best-known protozoa. Nevertheless, our knowledge has large gaps, especially of
normally free-living protozoa that may become pathogenic in immunocompromised
individuals. For example, microsporidia comprise a unique group of obligate, intracellular
parasitic protozoa. Microsporidia are amazingly diverse organisms with more than 700
species and 80 genera that are capable of infecting a variety of plant, animal, and even
other protist hosts.
They are found worldwide and have the ability to thrive in many ecological conditions. Until
the past few years, their ubiquity did not cause a threat to human health, and few
systematists worked to describe and classify the species.
Since 1985, however, physicians have documented an unusual rise in worldwide

infections in AIDS patients caused by four different genera of microsporidia
(Encephalitozoon, Nosema, Pleistophora, and Enterocytozoon). According to the Centers
for Disease Control in the United States, difficulties in identifying microsporidian species
are impeding diagnosis and effective treatment of AIDS patients.

Protozoan Reservoirs of Disease
The presence of bacteria in the cytoplasm of protozoa is well known, whereas that of
viruses is less frequently reported. Most of these reports simply record the presence of
bacteria or viruses and assume some sort of symbiotic relationship between them and the
protozoa.
Recently, however, certain human pathogens were shown to not only survive but also to
multiply in the cytoplasm of free-living, nonpathogenic protozoa. Indeed, it is now believed
that protozoa are the natural habitat for certain pathogenic bacteria. To date, the main
focus of attention has been on the bacterium Legionella pneumophila, the causative
organism of Legionnaires' disease; these bacteria live and reproduce in the cytoplasm of
some free-living amoebae (Curds 1992). More on this subject in the following pages.
Symbionts
Some protozoa are harmless or even beneficial symbionts. A bewildering array of ciliates,
for example, inhabit the rumen and reticulum of ruminates and the cecum and colon of
equids. Little is known about the relationship of the ciliates to their host, but a few may aid
the animal in digesting cellulose.
Data on Protozoa
While our knowledge of recent and fossil foraminifera in the U.S. coastal waterways is
systematically growing, other free-living protozoa are poorly known. There are some
regional guides and, while some are excellent, many are limited in scope, vague on
specifics, or difficult to use. Largely because of these problems, most ecologists who
include protozoa in their studies of aquatic habitats do not identify them, even if they do
count and measure them for biomass estimates (Taylor and Sanders 1991).
Parasitic protozoa of humans, domestic animals, and wildlife are better known although
no attempt has been made to compile this information into a single source. Large gaps in
our knowledge exist, especially for haemogregarines, microsporidians, and
myxosporidians (see Kreier and Baker 1987).
Museum Specimens
For many plant and animal taxa, museums represent a massive information resource. This
is not true for protozoa. In the United States, only the National Natural History Museum
(Smithsonian Institution) has a reference collection preserved on microscope slides, but it
does not have a protozoologist curator and cannot provide species' identification or
verification services. The American Type Culture Collection has some protozoa in culture,
but its collection includes relatively few kinds of protozoa.
Ecological Role of Protozoa

Although protozoa are frequently overlooked, they play an important role in many
communities where they occupy a range of trophic levels. As predators upon unicellular
or filamentous algae, bacteria, and microfungi, protozoa play a role both as herbivores
and as consumers in the decomposer link of the food chain.
As components of the micro- and meiofauna, protozoa are an important food source for
microinvertebrates. Thus, the ecological role of protozoa in the transfer of bacterial and
algal production to successive trophic levels is important.

Factors Affecting Growth and Distribution
Most free-living protozoa reproduce by cell division (exchange of genetic material is a
separate process and is not involved in reproduction in protozoa). The relative importance
for population growth of biotic versus chemical-physical components of the environment
is difficult to ascertain from the existing survey data. Protozoa are found living actively in
nutrient-poor to organically rich waters and in fresh water varying between 0°C (32°F) and
50°C (122°F). Nonetheless, it appears that rates of population growth increase when food
is not constrained and temperature is increased (Lee and Fenchel 1972; Fenchel 1974;
Montagnes et al. 1988).
Comparisons of oxygen consumption in various taxonomic groups show wide variation

(Laybourn and Finlay 1976), with some aerobic forms able to function at extremely low
oxygen tensions and to thereby avoid competition and predation. Many parasitic and a
few free-living species are obligatory anaerobes (grow without atmospheric oxygen). Of
the free-living forms, the best known are the plagiopylid ciliates that live in the anaerobic
sulfide-rich sediments of marine wetlands (Fenchel et al. 1977). The importance of
plagiopylids in recycling nutrients to aerobic zones of wetlands is potentially great.
Because of the small size of protozoa, their short generation time, and (for some species)
ease of maintaining them in the laboratory, ecologists have used protozoan populations
and communities to investigate competition and predation.
The result has been an extensive literature on a few species studied primarily under
laboratory conditions. Few studies have been extended to natural habitats with the result
that we know relatively little about most protozoa and their roles in natural communities.
Intraspecific competition for common resources often results in cannibalism, sometimes
with dramatic changes in morphology of the cannibals (Giese 1973). Field studies of
interspecific competition are few and most evidence for such species interactions is
indirect (Cairns and Yongue 1977).
Contractile Vacuoles
Many protozoa have contractile vacuoles, which collect and expel excess water, and
extrusomes, which expel material used to deflect predators or capture prey. In multicellular
organisms, hormones are often produced in vesicles. In higher plants, most of a cell's
volume is taken up by a central vacuole or tonoplast, which maintains its osmotic pressure.
Many eukaryotes have slender motile projections, usually called flagella when long and
cilia when short. These are variously involved in movement, feeding, and sensation. These
are entirely distinct from prokaryotic flagella. They are supported by a bundle of
microtubules arising from a basal body, also called a kinetosome or centriole,
characteristically arranged as nine doublets surrounding two singlets. Flagella also may
have hairs or mastigonemes, scales, connecting membranes, and internal rods. Their
interior is continuous with the cell's cytoplasm.
Centrioles
Centrioles are often present even in cells and groups that do not have flagella. They
generally occur in groups of one or two, called kinetids that give rise to various
microtubular roots. These form a primary component of the cytoskeletal structure, and are
often assembled over the course of several cell divisions, with one flagellum retained from
the parent and the other derived from it.

Centrioles may also be associated in the formation of a spindle during nuclear division.
Some protists have various other microtubule-supported organelles. These include the
radiolaria and heliozoa, which produce axopodia used in flotation or to capture prey, and
the haptophytes, which have a peculiar flagellum-like organelle called the haptonema.
Paramecium
Members of the genus Paramecium are single-celled, freshwater organisms in the
kingdom Protista. They exist in an environment in which the osmotic concentration in their
external environment is much lower than that in their cytoplasm. More specifically, the
habitat in which they live is hypotonic to their cytoplasm. As a result of this, Paramecium
is subjected to a continuous influx of water, as water diffuses inward to a region of higher
osmotic concentration.
If Paramecium is to maintain homeostasis, water must be continually pumped out of the

cell (against the osmotic gradient) at the same rate at which it moves in. This process,
known as osmoregulation, is carried out by two organelles in Paramecium known as
contractile vacuoles.

Protozoan Diseases
Protozoan pathogens are larger than bacteria and viruses, but still microscopic. They
invade and inhabit the gastrointestinal tract. Some parasites enter the environment in
a dormant form, with a protective cell wall called a “cyst.” The cyst can survive in the
environment for long periods of time and be extremely resistant to conventional
disinfectants such as chlorine. Effective filtration treatment is therefore critical to
removing these organisms from water sources.
Giardiasis
Giardiasis is a commonly reported protozoan-caused disease. It has also been
referred to as “backpacker’s disease” and “beaver fever” because of the many cases
reported among hikers and others who consume untreated surface water. Symptoms
include chronic diarrhea, abdominal cramps, bloating, frequent loose and pale greasy
stools, fatigue and weight loss.
The incubation period is 5-25 days or longer, with an average of 7-10 days. Many
infections are asymptomatic (no symptoms).
Giardiasis occurs worldwide. Waterborne outbreaks in the United States occur most
often in communities receiving their drinking water from streams or rivers without
adequate disinfection or a filtration system. The organism, Giardia lamblia, has been
responsible for more community-wide outbreaks of disease in the U.S. than any other
pathogen. Drugs are available for treatment but are not 100% effective.

Cryptosporidiosis
Cryptosporidiosis is an example of a protozoan disease that is common worldwide,
but was only recently recognized as causing human disease. The major symptom in
humans is diarrhea, which may be profuse and watery. The diarrhea is associated with
cramping abdominal pain. General malaise, fever, anorexia, nausea, and vomiting
occur less often. Symptoms usually come and go, and end in fewer than 30 days in
most cases. The incubation period is 1-12 days, with an average of about seven days.
Cryptosporidium organisms have been identified in human fecal specimens from more
than 50 countries on six continents. The mode of transmission is fecal-oral, either by
person-to-person or animal-to-person. There is no specific treatment for
Cryptosporidium infections.
All of these diseases, with the exception of hepatitis A, have one symptom in common:
diarrhea. They also have the same mode of transmission, fecal-oral, whether through
person-to-person or animal-to-person contact, and the same routes of transmission,
being either foodborne or waterborne. Although most pathogens cause mild, self-
limiting disease, on occasion, they can cause serious, even life threatening illness.
Particularly vulnerable are persons with weak immune systems such as those with HIV
infections or cancer. By understanding the nature of waterborne diseases, the
importance of properly constructed, operated and maintained public water systems
becomes obvious.
While water treatment cannot achieve sterile water (no microorganisms), the goal of
treatment must clearly be to produce drinking water that is as pathogen-free as
possible at all times. For those who operate water systems with inadequate source
protection or treatment facilities, the potential risk of a waterborne disease outbreak is
real. For those operating systems that currently provide adequate source protection
and treatment, operating and maintaining the system at a high level on a continuing
basis is critical to prevent disease.

Cryptosporidium
Cryptosporidium is a protozoan pathogen of the Phylum Apicomplexa and causes a

diarrheal illness called cryptosporidiosis. Other apicomplexan pathogens include the
malaria parasite Plasmodium, and Toxoplasma, the causative agent of toxoplasmosis.
Unlike Plasmodium, which transmits via a mosquito vector, Cryptosporidium does not
utilize an insect vector and is capable of completing its life cycle within a single host,
resulting in cyst stages which are excreted in feces and are capable of transmission to a
new host.
A number of species of Cryptosporidium infect mammals. In humans, the main causes of

disease are C. parvum and C. hominis (previously C. parvum genotype 1). C. canis, C.
felis, C. meleagridis, and C. muris can also cause disease in humans. In recent years,
cryptosporidiosis has plagued many commercial Leopard gecko breeders. Several
species of the Cryptosporidium family (C. serpentes and others) are involved, and outside
of geckos it has been found in monitor lizards, iguanas, tortoises as well as several snake
species.
Cryptosporidiosis is typically an acute short-term infection but can become severe and
non-resolving in children and immunocompromised individuals. The parasite is
transmitted by environmentally hardy cysts (oocysts) that, once ingested, excyst in the
small intestine and result in an infection of intestinal epithelial tissue. The genome of
Cryptosporidium parvum was sequenced in 2004 and was found to be unusual amongst
Eukaryotes in that the mitochondria seem not to contain DNA. A closely-related species,
C. hominis, also has its genome sequence available. CryptoDB.org is a NIH-funded
database that provides access to the Cryptosporidium genomics data sets.
When C. parvum was first identified as a human pathogen, diagnosis was made by a
biopsy of intestinal tissue (Keusch, et al., 1995).

However, this method of testing can give false negatives due the "patchy" nature of the
intestinal parasitic infection (Flanigan and Soave, 1993). Staining methods were then
developed to detect and identify the oocysts directly from stool samples. The modified
acid-fast stain is traditionally used to most reliably and specifically detect the presence of
cryptosporidial oocysts.
There have been six major outbreaks of cryptosporidiosis in the United States as a result
of contamination of drinking water (Juranek, 1995). One major outbreak in Milwaukee in
1993 affected over 400,000 persons.
Outbreaks such as these usually result from drinking water taken from surface water
sources such as lakes and rivers (Juranek, 1995). Swimming pools and water park wave
pools have also been associated with outbreaks of cryptosporidiosis. Also, untreated
groundwater or well water public drinking water supplies can be sources of contamination.
The highly environmentally resistant cyst of C. parvum allows the pathogen to survive
various drinking water filtrations and chemical treatments such as chlorination. Although
municipal drinking water utilities may meet federal standards for safety and quality of
drinking water, complete protection from cryptosporidial infection is not guaranteed. In
fact, all waterborne outbreaks of cryptosporidiosis have occurred in communities where
the local utilities met all state and federal drinking water standards (Juranek, 1995).

Giardia Lamblia
Giardia lamblia (synonymous with Lamblia intestinalis and Giardia duodenalis) is a

flagellated protozoan parasite that colonizes and reproduces in the small intestine,
causing giardiasis. The giardia parasite attaches to the epithelium by a ventral adhesive
disc, and reproduces via binary fission. Giardiasis does not spread via the bloodstream,
nor does it spread to other parts of the gastro-intestinal tract, but remains confined to the
lumen of the small intestine. Giardia trophozoites absorb their nutrients from the lumen of
the small intestine, and are anaerobes.
Giardia infection can occur through ingestion of dormant cysts in contaminated water, or
by the fecal-oral route (through poor hygiene practices). The Giardia cyst can survive for
weeks to months in cold water and therefore can be present in contaminated wells and
water systems, and even clean-looking mountain streams, as well as city reservoirs, as
the Giardia cysts are resistant to conventional water treatment methods, such as
chlorination and ozonolysis.
Zoonotic transmission is also possible, and therefore Giardia infection is a concern for
people camping in the wilderness or swimming in contaminated streams or lakes,
especially the artificial lakes formed by beaver dams (hence the popular name for
giardiasis, "Beaver Fever"). As well as water-borne sources, fecal-oral transmission can
also occur, for example in day care centers, where children may have poorer hygiene
practices.

Those who work with children are also at risk of being infected, as are family members of
infected individuals. Not all Giardia infections are symptomatic, so some people can
unknowingly serve as carriers of the parasite.
The life cycle begins with a non-infective cyst being excreted with feces of an infected
individual. Once out in the environment, the cyst becomes infective. A distinguishing
characteristic of the cyst is 4 nuclei and a retracted cytoplasm. Once ingested by a host,
the trophozoite emerges to an active state of feeding and motility. After the feeding stage,
the trophozoite undergoes asexual replication through longitudinal binary fission. The
resulting trophozoites and cysts then pass through the digestive system in the feces. While
the trophozoites may be found in the feces, only the cysts are capable of surviving outside
of the host.
Distinguishing features of the trophozoites are large karyosomes and lack of peripheral
chromatin, giving the two nuclei a halo appearance. Cysts are distinguished by a retracted
cytoplasm. This protozoa lacks mitochondria, although the discovery of the presence of
mitochondrial remnant organelles in one recent study "indicate that Giardia is not
primitively amitochondrial and that it has retained a functional organelle derived from the
original mitochondrial endosymbiont"

Entamoeba histolytica
Entamoeba histolytica, another water-borne pathogen, can cause diarrhea or a more

serious invasive liver abscess. When in contact with human cells, these amoebae are
cytotoxic. There is a rapid influx of calcium into the contacted cell, it quickly stops all
membrane movement save for some surface blebbing. Internal organization is disrupted,
organelles lyse, and the cell dies. The ameba may eat the dead cell or just absorb nutrients
released from the cell.
On average, about one in 10 people who are infected with E. histolytica becomes sick
from the infection. The symptoms often are quite mild and can include loose stools,
stomach pain, and stomach cramping.
Amebic dysentery is a severe form of amebiasis associated with stomach pain, bloody
stools, and fever. Rarely, E. histolytica invades the liver and forms an abscess. Even less
commonly, it spreads to other parts of the body, such as the lungs or brain.
Scientific Classification
Domain: Eukaryota
Phylum: Amoebozoa
Class: Archamoebae
Genus: Entamoeba
Species: E. histolytica

Vorticella
Vorticella is a genus of protozoa, with over 100 known species. They are stalked inverted
bell-shaped ciliates, placed among the peritrichs. Each cell has a separate stalk anchored
onto the substrate, which contains a contracile fibril called a myoneme. When stimulated
this shortens, causing the stalk to coil like a spring. Reproduction is by budding, where the
cell undergoes longitudinal fission and only one daughter keeps the stalk.
Vorticella mainly lives in freshwater ponds and streams - generally anywhere protists are
plentiful. Other genera such as Carchesium resemble Vorticella but are branched or
colonial.
Domain: Eukaryota
Phylum: Ciliophora
Class: Oligohymenophorea
Subclass: Peritrichia
Order: Sessilida
Family: Vorticellidae
Genus: Vorticella

Rotifer
The rotifers make up a phylum of microscopic and near-microscopic pseudocoelomate

animals. They were first described by John Harris in 1696 (Hudson and Gosse, 1886).
Leeuwenhoek is mistakenly given credit for being the first to describe rotifers but Harris
had produced sketches in 1703. Most rotifers are around 0.1-0.5 mm long, and are
common in freshwater throughout the world with a few saltwater species. Rotifers may be
free swimming and truly planktonic, others move by inch worming along the substrate,
whilst some are sessile, living inside tubes or gelatinous holdfasts. About 25 species are
colonial (e.g. Sinantherina semibullata), either sessile or planktonic.
Rotifers get their name (derived from Greek and meaning "wheel-bearer"; they have also
been called wheel animalcules) from the corona, which is composed of several ciliated
tufts around the mouth that in motion resemble a wheel. These create a current that
sweeps food into the mouth, where it is chewed up by a characteristic pharynx (called the
mastax) containing a tiny, calcified, jaw-like structure called the trophi. The cilia also pull
the animal, when unattached, through the water. Most free-living forms have pairs of
posterior toes to anchor themselves while feeding.
Rotifers have bilateral symmetry and a variety of different shapes. There is a well-
developed cuticle which may be thick and rigid, giving the animal a box-like shape, or
flexible, giving the animal a worm-like shape; such rotifers are respectively called loricate
and illoricate.
Like many other microscopic animals, adult rotifers frequently exhibit eutely - they have a
fixed number of cells within a species, usually on the order of one thousand.

Males in the class Monogononta may be either present or absent depending on the
species and environmental conditions. In the absence of males, reproduction is by
parthenogenesis and results in clonal offspring that are genetically identical to the parent.
Individuals of some species form two distinct types of parthenogenetic eggs; one type
develops into a normal parthenogenetic female, while the other occurs in response to a
changed environment and develops into a degenerate male that lacks a digestive system,
but does have a complete male reproductive system that is used to inseminate females
thereby producing fertilized 'resting eggs'.
Resting eggs develop into zygotes that are able to survive extreme environmental
conditions such as may occur during winter or when the pond dries up. These eggs
resume development and produce a new female generation when conditions improve
again. The life span of monogonont females varies from a couple of days to about three
weeks.
Bdelloid rotifers are unable to produce resting eggs, but many can survive prolonged
periods of adverse conditions after desiccation. This facility is termed anhydrobiosis, and
organisms with these capabilities are termed anhydrobionts. Under drought conditions,
bdelloid rotifers contract into an inert form and lose almost all body water; when
rehydrated, however, they resume activity within a few hours.
Bdelloids can survive the dry state for prolonged periods, with the longest well-
documented dormancy being nine years. While in other anhydrobionts, such as the brine
shrimp, this desiccation tolerance is thought to be linked to the production of trehalose, a
non-reducing disaccharide (sugar), bdelloids apparently lack the ability to synthesize
trehalose.
Bdelloid rotifer genomes contain two or more divergent copies of each gene. Four copies
of hsp82 are, for example, found. Each is different and found on a different chromosome,
excluding the possibility of homozygous sexual reproduction.

Various and Commonly found Wastewater Bugs




Bacteria Section


Peritrichous Bacteria
Microbiologists broadly classify bacteria according to their shape: spherical, rod-shaped,
and spiral-shaped. Pleomorphic bacteria can assume a variety of shapes. Bacteria may
be further classified according to whether they require oxygen (aerobic or anaerobic) and
how they react to a test with Gram’s stain. Bacteria in which alcohol washes away Gram’s
stain are called gram-negative, while bacteria in which alcohol causes the bacteria’s walls
to absorb the stain are called gram-positive.

Bacterial Cell
Mitochondria
The bacterial cell is surrounded by a lipid membrane, or cell membrane, which encloses
the contents of the cell and acts as a barrier to hold nutrients, proteins and other essential
components of the cytoplasm within the cell.
As they are prokaryotes, bacteria do not tend to have membrane-bound organelles in their
cytoplasm and thus contain few large intracellular structures. They consequently lack a
nucleus, mitochondria, chloroplasts and the other organelles present in eukaryotic cells,
such as the Golgi apparatus and endoplasmic reticulum.

Bacteria Glossary
Type Characteristics
Rod-shaped, gram-negative, aerobic; highly tolerant of acidic
Acetic acid
conditions; generate organic acids
Rod-shaped or filamentous, gram-positive, aerobic; common in soils;
Actinomycete essential to growth of many plants; source of much of original
antibiotic production in pharmaceutical industry
Spherical, sometimes in clusters or strings, gram-positive, aerobic and
anaerobic; resistant to drying and high-salt conditions; Staphylococcus
Coccoid
species common on human skin, certain strains associated with toxic
shock syndrome
Rod-shaped, form club or V shapes, gram-positive, aerobic; found in
Coryneform wide variety of habitats, particularly soils; highly resistant to drying;
include Arthrobacter, among most common forms of life on earth
Usually rod-shaped, can be gram-positive or gram-negative; have
Endospore- highly adaptable, heat-resistant spores that can go dormant for long
forming periods, possibly thousands of years; include Clostridium (anaerobic)
and Bacillus (aerobic)
Rod-shaped, gram-negative, aerobic but can live in certain anaerobic
Enteric conditions; produce nitrite from nitrate, acids from glucose; include
Escherichia coli, Salmonella (over 1000 types), and Shigella
Rod-shaped, gram-negative, mostly aerobic; glide on secreted slimy
Gliding
substances; form colonies, frequently with complex fruiting structures
Gram-positive, anaerobic; produce lactic acid through fermentation;
Lactic acid include Lactobacillus, essential in dairy product formation, and
Streptococcus, common in humans
Pleomorphic, spherical or rod-shaped, frequently branching, no gram
Mycobacterium stain, aerobic; commonly form yellow pigments; include
Mycobacterium tuberculosis, cause of tuberculosis
Spherical, commonly forming branching chains, no gram stain, aerobic
but can live in certain anaerobic conditions; without cell walls yet
Mycoplasma
structurally resistant to lysis; among smallest of bacteria; named for
superficial resemblance to fungal hyphae (myco- means 'fungus')
Rod-shaped, gram-negative, aerobic; convert atmospheric nitrogen
Nitrogen-fixing
gas to ammonium in soil; include Azotobacter, a common genus
Rod-shaped, pleomorphic, gram-positive, anaerobic; ferment lactic
Propionic acid acid; fermentation produces holes in Swiss cheese from the
production of carbon dioxide
Rod-shaped (straight or curved) with polar flagella, gram-negative,
Pseudomonad
aerobic; can use up to 100 different compounds for carbon and energy
Spherical or rod-shaped, gram-negative, aerobic; cause Rocky
Rickettsia Mountain spotted fever and typhus; closely related to Agrobacterium,
a common gall-causing plant bacterium

Filamentous, gram-negative, aerobic; 'swarmer' (colonizing) cells form
Sheathed and break out of a sheath; sometimes coated with metals from
environment
Spiral-shaped, gram-negative, aerobic; include Bdellovibrio, predatory

Spirillum
on other bacteria
Spiral-shaped, gram-negative, mostly anaerobic; common in moist
environments, from mammalian gums to coastal mudflats; complex
Spirochete
internal structures convey rapid movement; include
Treponemapallidum, cause of syphilis
Sulfate- and
Commonly rod-shaped, mostly gram-negative, anaerobic; include
Sulfur-
Desulfovibrio, ecologically important in marshes
reducing
Sulfur- and Commonly rod-shaped, frequently with polar flagella, gram-negative,
iron-oxidizing mostly anaerobic; most live in neutral (nonacidic) environment
Rod- or comma-shaped, gram-negative, aerobic; commonly with a
Vibrio single flagellum; include Vibrio cholerae, cause of cholera, and
luminescent forms symbiotic with deep-water fishes and squids

Bacteriophage
A bacteriophage (from 'bacteria' and Greek phagein, 'to eat') is any one of a number of
viruses that infect bacteria. The term is commonly used in its shortened form, phage.
Typically, bacteriophages consist of an outer protein hull enclosing genetic material. The
genetic material can be ssRNA (single stranded RNA), dsRNA, ssDNA, or dsDNA
between 5 and 500 kilo base pairs long with either circular or linear arrangement.
Bacteriophages are much smaller than the bacteria they destroy - usually between 20 and
200 nm in size.
Phages are estimated to be the most widely distributed and diverse entities in the
biosphere. Phages are ubiquitous and can be found in all reservoirs populated by bacterial
hosts, such as soil or the intestine of animals.
One of the densest natural sources for phages and other viruses is sea water, where up
to 9×108 virions per milliliter have been found in microbial mats at the surface, and up to
70% of marine bacteria may be infected by phages.

Release of Virions
Phages may be released via cell lysis or by host cell secretion. In the case of the T4 phage,
in just over twenty minutes after injection upwards of three hundred phages will be
released via lysis within a certain timescale. This is achieved by an enzyme called
endolysin which attacks and breaks down the peptidoglycan.
In contrast, "lysogenic" phages do not kill the host but rather become long-term parasites
and make the host cell continually secrete more new virus particles. The new virions bud
off the plasma membrane, taking a portion of it with them to become enveloped viruses
possessing a viral envelope. All released virions are capable of infecting a new bacterium.

Salmonella
Salmonella is a Gram-negative bacterium. It is found in many turtles and other reptiles. In

clinical laboratories, it is usually isolated on MacConkey agar, XLD agar, XLT agar, DCA
agar, or Önöz agar. Because they cause intestinal infections and are greatly outnumbered
by the bacteria normally found in the healthy bowel, primary isolation requires the use of
a selective medium, so use of a relatively non-selective medium such as CLED agar is not
often practiced.
Numbers of salmonella may be so low in clinical samples that stools are routinely also
subjected to "enrichment culture", where a small volume of stool is incubated in a selective
broth medium, such as selenite broth or Rappaport Vassiliadis soya peptone broth,
overnight.
These media are inhibitory to the growth of the microbes normally found in the healthy
human bowel, while allowing salmonellae to become enriched in numbers. Salmonellae
may then be recovered by inoculating the enrichment broth on one or more of the primary
selective media. On blood agar, they form moist colonies about 2 to 3 mm in diameter.

When the cells are grown for a prolonged time at a range of 25—28°C, some strains
produce a biofilm, which is a matrix of complex carbohydrates, cellulose and proteins.
The ability to produce biofilm (a.k.a. "rugose", "lacy", or "wrinkled") can be an indicator of
dimorphism, which is the ability of a single genome to produce multiple phenotypes in
response to environmental conditions. Salmonellae usually do not ferment lactose; most
of them produce hydrogen sulfide which, in media containing ferric ammonium citrate,
reacts to form a black spot in the center of the creamy colonies.
Classification
Salmonella taxonomy is complicated. As of December 7, 2005, there are two species
within the genus: S. bongori (previously subspecies V) and S. enterica (formerly called
S. choleraesuis), which is divided into six subspecies:
* I—enterica
* II—salamae
* IIIa—arizonae
* IIIb—diarizonae
* IV—houtenae
* V—obsolete (now designated
S. bongori)
* VI—indica
There are also numerous (over 2500)

serovars within both species, which are
found in a disparate variety of
environments and which are associated
with many different diseases.
The vast majority of human isolates

(>99.5%) are subspecies S. enterica. For
the sake of simplicity, the CDC
recommends that Salmonella species be
referred to only by their genus and
serovar, e.g.
Salmonella Typhi instead of the more

technically correct designation,
Salmonella enterica subspecies enterica
serovar Typhi.

Shigella dysenteriae
Shigella dysenteriae is a species of the rod-shaped bacterial genus Shigella. Shigella can
cause shigellosis (bacillary dysentery). Shigellae are Gram-negative, non-spore-forming,
facultatively anaerobic, non-motile bacteria.
S. dysenteriae, spread by contaminated water and food, causes the most severe
dysentery because of its potent and deadly Shiga toxin, but other species may also be
dysentery agents. Shigella infection is typically via ingestion (fecal–oral contamination);
depending on age and condition of the host as few as ten bacterial cells can be enough
to cause an infection. Shigella causes dysentery that result in the destruction of the
epithelial cells of the intestinal mucosa in the cecum and rectum. Some strains produce
enterotoxin and Shiga toxin, similar to the verotoxin of E. coli O157:H7. Both Shiga toxin
and verotoxin are associated with causing hemolytic uremic syndrome.
Shigella invades the host through epithelial cells of the large intestine. Using a Type III
secretion system acting as a biological syringe, the bacterium injects IpaD protein into cell,
triggering bacterial invasion and the subsequent lysis of vacuolar membranes using IpaB
and IpaC proteins. It utilizes a mechanism for its motility by which its IcsA protein triggers
actin polymerization in the host cell (via N-WASP recruitment of Arp2/3 complexes) in a
"rocket" propulsion fashion for cell-to-cell spread.
The most common symptoms are diarrhea, fever, nausea, vomiting, stomach cramps, and
straining to have a bowel movement. The stool may contain blood, mucus, or pus (e.g.
dysentery). In rare cases, young children may have seizures. Symptoms can take as long
as a week to show up, but most often begin two to four days after ingestion. Symptoms
usually last for several days, but can last for weeks. Shigella is implicated as one of the
pathogenic causes of reactive arthritis worldwide.

Top Photo: This technician is using Colilert which is a commercially available enzyme-
substrate liquid-broth medium (IDEXX Laboratories, Inc.) that allows the simultaneous
detection of total coliforms and Escherichia coli (E. coli). It is available in the most-
probable number (MPN) or the presence/absence (PA) format. The MPN method is
facilitated by use of a specially designed disposable incubation tray called the Quanti-
Tray®.
Bottom Photo: Another method is using a petri dish with a filter membrane. The broth and
membrane used vary depending on the sample type for water or wastewater.

Escherichia Coli Section
Fecal Coliform Bacteria
Fecal coliform bacteria are microscopic organisms that live in the intestines of warm-
blooded animals. They also live in the waste material, or feces, excreted from the intestinal
tract. When fecal coliform bacteria are present in high numbers in a water sample, it means
that the water has received fecal matter from one source or another. Although not
necessarily agents of disease, fecal coliform bacteria may indicate the presence of
disease-carrying organisms, which live in the same environment as the fecal coliform
bacteria.
Reasons for Natural Variation

Unlike the other conventional water quality parameters, fecal coliform bacteria are living
organisms. They do not simply mix with the water and float straight downstream. Instead
they multiply quickly when conditions are favorable for growth, or die in large numbers
when conditions are not. Because bacterial concentrations are dependent on specific
conditions for growth, and these conditions change quickly, fecal coliform bacteria counts
are not easy to predict. For example, although winter rains may wash more fecal matter
from urban areas into a stream, cool water temperatures may cause a major die-off.
Exposure to sunlight (with its ultraviolet disinfection properties) may have the same effect,
even in the warmer water of summertime.
Expected Impact of Pollution

The primary sources of fecal coliform bacteria to fresh water are wastewater treatment
plant discharges, failing septic systems, and animal waste. Bacteria levels do not
necessarily decrease as a watershed develops from rural to urban. Instead, urbanization
usually generates new sources of bacteria. Farm animal manure and septic systems are
replaced by domestic pets and leaking sanitary sewers.
In fact, stormwater runoff in urbanized areas has been found to be surprisingly high in
fecal coliform bacteria concentrations. General coliforms, E. Coli, and Enterococcus
bacteria are the "indicator" organisms generally measured to assess microbiological
quality of water.

However, these aren't generally what get people sick. Other bacteria, viruses, and
parasites are what we are actually worried about because it is so much more expensive
and tedious to do so; actual pathogens are virtually never tested for.
Coliform Standards (in colonies/100ml)

Drinking water..................................................................1FC
Total body contact (swimming).............................................200FC
Partial body contact (boating)..............................................1000FC
Threatened sewage effluent ................................not to exceed 200 FC
*Total coliform (TC) includes bacteria from cold-blooded animals and various soil
organisms. According to recent literature, total coliform counts are normally about 10
times higher than fecal coliform (FC) counts.
Indicator Connection Varies

Over the course of a professional lifetime pouring over indicator tests, in a context where
all standards are based on indicators, water workers tend to forget that the indicators are
not the things we actually care about. Infection rates are around 5% in the US, and
approach 100% in areas with poor hygiene and contaminated water supplies.
Keep in the back of your mind that the ratio of indicators to actual pathogens is not
fixed. It will always be different, sometimes very different. Whenever you are trying to form
a mental map of reality based on water tests, you should include in the application of your
water intuition an adjustment factor for your best guess of the ratio between indicators and
actual pathogens.
What are these indicators? More information in the Laboratory section.

 General coliforms indicate that the water has come in contact with plant or animal
life. General coliforms are universally present, including in pristine spring water.
They are of little concern at low levels, except to indicate the effectiveness of
disinfection. Chlorinated water and water from perfectly sealed tube wells is the
only water I've tested which had zero general coliforms. At very high levels they
indicate there is what amounts to a lot of compost in the water, which could easily
include pathogens (Ten thousand general coliform bacteria will get you a beach
closure, compared to two or four hundred fecal coliforms, or fifty enterococcus).
 Fecal coliforms, particularly E. coli, indicate that there are mammal or bird feces
in the water.
 Enterococcus bacteria also indicate that there are feces from warm blooded
animals in the water. Enterococcus are a type of fecal streptococci. They are
another valuable indicator for determining the amount of fecal contamination of
water. According to studies conducted by the EPA, enterococci have a greater
correlation with swimming-associated gastrointestinal illness in both marine and
fresh waters than other bacterial indicator organisms, and are less likely to "die off"
in saltwater.

Membrane Filter Total Coliform Technique
The membrane filter total Coliform technique is used at Medina County for drinking water
quality testing. The following is a summary of this test. A sampling procedure sheet is
given to all sample takers by Medina County.
The samples are taken in sterile 100 mL containers. These containers, when used for
chlorinated water samples, have a sodium thiosulfate pill or solution to dechlorinate the
sample.
The sample is placed in cold storage after proper sample taking procedures are
followed. (See sample
procedures below)
The samples are taken to the

laboratory with a chain of
custody to assure no
tampering of samples can
occur.
These samples are logged in

at the laboratory.
No longer than 30 hours can

lapse between the time of
sampling and time of test
incubation. (8 hours for
heterotrophic, non-potable 6
hours, others not longer than
24 hours)
All equipment is sterilized by oven and autoclave.

Glassware in oven at 170oC + 10oC with foil (or other suitable wrap) loosely fitting and
secured immediately after sterilization.
Filtration units in autoclave at 121oC for 30 minutes.
Use sterile petri dishes, grid, and pads bought from a

reliable company – certified, quality assured - test for
satisfactory known positive amounts.
Incubators – 35oC + .5oC (60% relative humidity)
M-endo medium is prepared and heated to near

boiling removed from heat cooled to 45oC pH
adjusted to 7.2 + .2 and immediately dispensed 8ml to
plates. Keep refrigerated and discard after 2 weeks.

Plates can be stored in a dated box with expiration date and discarded if not used. No
denatured alcohol should be used. Everclear or 95% proof alcohol or absolute methyl
may be used for sterilizing forceps by flame.
Procedure:
1. Counters are alcohol wiped.
2. Bench sheets are filled out.
3. Samples are removed from refrigeration.
4. Sterile wrapped utensils are placed on counters.
5. Filtration units are placed onto sterile membrane filters by aseptic technique using
sterile forceps.
6. Sterile petri dishes are labeled.
7. The samples closures are clipped.
8. The sample is shaken 25 times 1 foot in length within 7 seconds.
9. 100 mL is filtered and rinsed with sterile distilled water 3 times.
10. The membrane filter is aseptically removed from filter holder.
11. A sterile padded petri dish is used and the membrane filter is rolled onto the pad
making sure no air bubbles form.
12. The sterile labeled lid is placed on the petri dish.
13. 2 blanks and a known is run with each series of samples.
14. The samples are placed in the 35oC + .5oC incubator stacked no higher than 3 for
22 – 24 hours (Humidity can be maintained by saturated paper towels placed under
containers holding petri dishes.)
15. After 22- 24 hours view the petri dishes under a 10 –15 power magnification with
cool white fluorescent light.
16. Count all colonies that appear pink to dark red with a metallic surface sheen – the
sheen may vary in size from a pin head to complete coverage.
17. Report as Total Coliform per 100 mL.
18. If no colonies are present report as <1 coliform/100mL.
Anything greater than 1 is over the limit for drinking water for 2 samples taken 24 hours
apart. Further investigation may be necessary – follow Standard Methods accordingly.
Photograph and Credits to Mary McPherson

AranTM Aqua Analytical Laboratory Director.

Viruses
Viruses are acellular microorganisms. They are made up of only genetic material and a
protein coat. Viruses depend on the energy and metabolic machinery of the host cell to
reproduce. A virus is an infectious agent found in virtually all life forms, including humans,
animals, plants, fungi, and bacteria. Viruses consist of genetic material—either
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)—surrounded by a protective
coating of protein, called a capsid, with or without an outer lipid envelope. Viruses are
between 20 and 100 times smaller than bacteria and hence are too small to be seen by
light microscopy.
Viruses vary in size from the largest poxviruses of about 450 nanometers (about 0.000014
in) in length to the smallest polioviruses of about 30 nanometers (about 0.000001 in).
Viruses are not considered free-living, since they cannot reproduce outside of a living cell;
they have evolved to transmit their genetic information from one cell to another for the
purpose of replication. Viruses often damage or kill the cells that they infect, causing
disease in infected organisms.
A few viruses stimulate cells to grow uncontrollably and produce cancers. Although many
infectious diseases, such as the common cold, are caused by viruses, there are no cures
for these illnesses.
The difficulty in developing antiviral therapies stems from the large number of variant
viruses that can cause the same disease, as well as the inability of drugs to disable a virus
without disabling healthy cells. However, the development of antiviral agents is a major
focus of current research, and the study of viruses has led to many discoveries important
to human health.

Virions
Individual viruses, or virus particles, also called virions, contain genetic material, or
genomes, in one of several forms. Unlike cellular organisms, in which the genes always
are made up of DNA, viral genes may consist of either DNA or RNA. Like cell DNA, almost
all viral DNA is double-stranded, and it can have either a circular or a linear arrangement.
Almost all viral RNA is single-stranded; it is usually linear, and it may be either segmented
(with different genes on different RNA molecules) or non-segmented (with all genes on a
single piece of RNA).
Capsids
The viral protective shell, or capsid, can be either helical (spiral-shaped) or icosahedral
(having 20 triangular sides). Capsids are composed of repeating units of one or a few
different proteins. These units are called protomers or capsomers. The proteins that make
up the virus particle are called structural proteins. Viruses also carry genes for making
proteins that are never incorporated into the virus particle and are found only in infected
cells. These viral proteins are called nonstructural proteins; they include factors required
for the replication of the viral genome and the production of the virus particle.
Capsids and the genetic material (DNA or RNA) they contain are together referred to as
nucleocapsids. Some virus particles consist only of nucleocapsids, while others contain
additional structures.
Some icosahedral and helical animal viruses are enclosed in a lipid envelope acquired
when the virus buds through host-cell membranes. Inserted into this envelope are
glycoproteins that the viral genome directs the cell to make; these molecules bind virus
particles to susceptible host cells.
Bacteriophages
The most elaborate viruses are the bacteriophages, which use bacteria as their hosts.
Some bacteriophages resemble an insect with an icosahedral head attached to a tubular
sheath. From the base of the sheath extend several long tail fibers that help the virus
attach to the bacterium and inject its DNA to be replicated, direct capsid production, and
virus particle assembly inside the cell.
Viroids and Prions

Viroids and prions are smaller than viruses, but they are similarly associated with disease.
Viroids are plant pathogens that consist only of a circular, independently replicating RNA
molecule. The single-stranded RNA circle collapses on itself to form a rod-like structure.
The only known mammalian pathogen that resembles plant viroids is the deltavirus
(hepatitis D), which requires hepatitis B virus proteins to package its RNA into virus
particles. Co-infection with hepatitis B and D can produce more severe disease than can
infection with hepatitis B alone. Prions are mutated forms of a normal protein found on the
surface of certain animal cells.
Virus Classification
Viruses are classified according to their type of genetic material, their strategy of
replication, and their structure. The ICNV report published in 1995 assigned more than
4000 viruses into 71 virus families. Hundreds of other viruses remain unclassified because
of the lack of sufficient information.

Hepatitis
There are five types of hepatitis -- A through E -- all of which cause inflammation of the
liver. Type D affects only those who also have hepatitis B, and hepatitis E is extremely
rare in the United States.
 Type A hepatitis is contracted through anal-oral contact, by coming in contact

with the feces of someone with hepatitis A, or by eating or drinking hepatitis A
contaminated food or water.
 Type B hepatitis can be contracted from infected blood, seminal fluid, vaginal
secretions, or contaminated drug needles, including tattoo or body-piercing
equipment. It can also be spread from a mother to her newborn.
 Type C hepatitis is not easily spread through sex. You're more likely to get it
through contact with infected blood, contaminated razors, needles, tattoo and
body-piercing equipment, or manicure or pedicure tools that haven't been
properly sanitized, and a mother can pass it to her baby during delivery.
 Type D hepatitis can be passed through contact with infected blood,

contaminated needles, or by sexual contact with an HIV-infected person.
 Type E hepatitis is most likely to be transmitted in feces, through oral contact, or

in water that's been contaminated.

Peptidoglycan
Peptidoglycan, also known as murein, is a polymer consisting of sugars and amino acids
that forms a mesh-like layer outside the plasma membrane of eubacteria. The sugar
component consists of alternating residues of β-(1,4) linked N-acetylglucosamine and N-
acetylmuramic acid residues. Attached to the N-acetylmuramic acid is a peptide chain of
three to five amino acids. The peptide chain can be cross-linked to the peptide chain of
another strand forming the 3D mesh-like layer.

Cyanobacteria
Cyanobacteria
Cyanobacteria, also known as blue-green algae, blue-green bacteria or Cyanophyta, is a

phylum of bacteria that obtain their energy through photosynthesis. The name
"cyanobacteria" comes from the color of the bacteria (Greek: kyanós = blue). They are a
significant component of the marine nitrogen cycle and an important primary producer in
many areas of the ocean, but are also found on land.
Cyanobacteria include unicellular and colonial species. Colonies may form filaments,
sheets or even hollow balls. Some filamentous colonies show the ability to differentiate
into several different cell types: vegetative cells, the normal, photosynthetic cells that are
formed under favorable growing conditions; akinetes, the climate-resistant spores that
may form when environmental conditions become harsh; and thick-walled heterocysts,
which contain the enzyme nitrogenase, vital for nitrogen fixation. Heterocysts may also
form under the appropriate environmental conditions (anoxic) wherever nitrogen is
necessary.
Heterocyst-forming species are specialized for nitrogen fixation and are able to fix nitrogen
gas, which cannot be used by plants, into ammonia (NH3), nitrites (NO2) or nitrates (NO3),
which can be absorbed by plants and converted to protein and nucleic acids.

The rice paddies of Asia, which produce about 75% of the world's rice, could not do so
were it not for healthy populations of nitrogen-fixing cyanobacteria in the rice paddy
fertilizer too.
Many cyanobacteria also form motile filaments, called hormogonia, that travel away from
the main biomass to bud and form new colonies elsewhere. The cells in a hormogonium
are often thinner than in the vegetative state, and the cells on either end of the motile chain
may be tapered. In order to break away from the parent colony, a hormogonium often
must tear apart a weaker cell in a filament, called a necridium.
Each individual cell of a cyanobacterium typically has a thick, gelatinous cell wall. They
differ from other gram-negative bacteria in that the quorum sensing molecules
autoinducer-2[4] and acyl-homoserine lactones are absent. They lack flagella, but
hormogonia and some unicellular species may move about by gliding along surfaces. In
water columns some cyanobacteria float by forming gas vesicles, like in archaea.


Chlorine Charts



MATH CONVERSION FACTORS
1 PSI = 2.31 Feet of Water LENGTH
1 Foot of Water = .433 PSI 12 Inches = 1 Foot
1.13 Feet of Water = 1 Inch of Mercury 3 Feet = 1 Yard
454 Grams = 1 Pound 5280 Feet = 1 Mile
1 Gallon of Water = 8.34 pounds/gal AREA
1 mg/L = 1 PPM 144 Square Inches = 1 Square Foot
17.1 mg/L = 1 Grain/Gallon 43,560 Square Feet = 1 Acre
1% = 10,000 mg/L VOLUME
694 Gallons per Minute = MGD 1000 Milliliters = 1 Liter
1.55 Cubic Feet per Second = 1 MGD 3.785 Liters = 1Gallon
60 Seconds = 1 Minute 231 Cubic Inches = 1 Gallon
1440 Minutes = 1 Day 7.48 Gallons = 1 Cubic Foot
.746 kW = 1 Horsepower 62.38 Pounds = 1 Cubic Foot
DIMENSIONS
SQUARE: Area (sq. ft.) = Length X Width
Volume (cu. ft.) = Length (ft) X Width (ft) X Height (ft)
CIRCLE: Area (sq. ft.) = 3.14 X Radius (ft) X Radius (ft)
CYLINDER: Volume (Cu. ft) = 3.14 X Radius (ft) X Radius (ft) X Depth (ft)
SPHERE: (3.14) (Diameter)3 Circumference = 3.14 X Diameter

(6)

GENERAL
POUNDS PER DAY AKA SOLIDS APPLIED = Concentration (mg/L) X Flow (MG) X 8.34
PERCENT EFFICIENCY = In – Out X 100

In
0
TEMPERATURE: F = (0C X 9/5) + 32
0
C = (0F - 32) X 5/9
CONCENTRATION: Conc. (A) X Volume (A) = Conc. (B) X Volume (B)
FLOW RATE (Q): Q = A X V (Quantity = Area X Velocity)
FLOW RATE (gpm): Flow Rate (gpm) = 2.83 (Diameter, in)2 (Distance, in)
Height, in
% SLOPE = Rise (feet) X 100

Run (feet)
ACTUAL LEAKAGE = Leak Rate (GPD)

Length (mi.) X Diameter (in)
VELOCITY = Distance (ft)

Time (Sec)
N = Manning’s Coefficient of Roughness

R = Hydraulic Radius (ft.)
S = Slope of Sewer (ft/ft.)
HYDRAULIC RADIUS (ft) = Cross Sectional Area of Flow (ft)

Wetted pipe Perimeter (ft)
WATER HORSEPOWER = Flow (gpm) X Head (ft)

3960
BRAKE HORSEPOWER = Flow (gpm) X Head (ft)

3960 X Pump Efficiency
MOTOR HORSEPOWER = Flow (gpm) X Head (ft)

3960 X Pump Eff. X Motor Eff.
MEAN OR AVERAGE = Sum of the Values

Number of Values
TOTAL HEAD (ft) = Suction Lift (ft) X Discharge Head (ft)
SURFACE LOADING RATE = Flow Rate (gpm)

(gal/min/sq. ft.) Surface Area (sq. ft)

MIXTURE = (Volume 1, gal) (Strength 1, %) + (Volume 2, gal) (Strength 2,%)
STRENGTH (%) (Volume 1, gal) + (Volume 2, gal)
INJURY FREQUENCY RATE = (Number of Injuries) 1,000,000

Number of hours worked per year
DETENTION TIME (hrs) = Volume of Basin (gals) X 24 hrs

Flow (GPD)
Slope = Rise (ft) Slope(%) = Rise (ft) X 100

Run (ft) Run (ft)
POPULATION EQUIVENT (PE):

1 PE = .17 Pounds of BOD per Day
1 PE = .20 Pounds of Solids per Day
1 PE = 100 Gallons per Day
LEAKAGE (GPD/inch) = Leakage of Water per Day (GPD)

Sewer Diameter (inch)
CHLORINE DEMAND (mg/L) = Chlorine Dose (mg/L) – Chlorine Residual (mg/L)
MANNING FORMULA
Q = Allowable time for decrease in pressure from 3.5 PSI to 2.5 PSI
q = As below
Q = (0.022) (d12L1)/Q q = [ 0.085] [(d12L1)/(d1L1)]

q
Q = 2.0 cfm air loss

 = .0030 cfm air loss per square foot of internal pipe surface
 = Pipe diameter (inches)
L = Pipe Length (feet)
V = 1.486 R 2/3 S 1/2


V = Velocity (ft./sec.)
 = Pipe Roughness
R = Hydraulic Radius (ft)
S= Slope (ft/ft)
HYDRAULIC RADIUS (ft) = Flow Area (ft. 2)

Wetted Perimeter (ft.)
WIDTH OF TRENCH (ft) = Base (ft) + (2 Sides) X Depth (ft 2)

Slope
AMPERAGE = Voltage
Ohms
VOLTAGE IMBALANCE = Maximum Voltage Deviation (Volts) X 100

Average Voltage (Volts)

LABORATORY
TSS (mg/L) = Paper Wt. And Dried Solids (g) – Paper Wt. (g) X 1,000,000
Milliliters of Sample
BOD (mg/L = (Initial DO – Final DO) X 300

(unseeded) Milliliters of Sample
LANGELIER INDEX = pH - pHs
STABILIZATION PONDS
DETENTION TIME (Days) = Volume of Ponds (gals)

Flow Rate (gals/day)
ORGANIC LOADING (Lbs. Of BOD/Acre/Day) = Pounds of BOD Applied per Day

Surface Areas (Acres)
FIXED MEDIA
HYDRAULIC LOADING (gals/1000 cu. ft./day) = Flow Rate (gals./day)

1000’s Cubic Feet of Media
ORGANIC LOADING (lbs BOD/day/1000 cu. ft.) = Pounds of BOD applied per Day
1000’S OF Cubic Feet of Media
ACTIVATED SLUDGE
DETENTION TIME (hrs.) = Volume of the tank (gals)

Flow Rate (gals/hour)
SVI (mg/L) = Settled Sludge Volume (mls) X 1000

MLSS (mg/L)
SDI (g/ml) = 1 X 100

SVI
F/M = BOD (applied to aerator) X Flow (MGD) X 8.34

Pounds of Solids under Aeration
MCRT (Days) = Pounds of Solids under Aeration  Pounds of Solids in Clarifier

Pounds of Solids Wasted  Pounds of Solids over the Weirs

DIGESTER AND SOLIDS HANDLING
OXYGEN UPTAKE RATE (OUR) = mg of O2 used
Minute
ORGANIC LOADING (lbs./day/cu. ft.) = Pounds of Volatile Solids applied per Day
Volume of Digester (cu. ft.)
VOLATILE SOLIDS REDUCTION = (In – Out) (100%)

In – (In – Out)
DRY POLYMER (lbs) = (Gal. Of solution) X (8.34 lbs./gal.) X (% polymer solution)
SLUDGE APPLICATION (lbs)= (Gal. Of Sludge) X (8.34 lbs./gal.) X (% Solids in sludge)
1 TON = 2,000 lbs I METER = 3.28 Feet

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WASTEWATER TREATMENT

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Hyperlink to the assignment

State Approval Listing URL…

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A second certificate of completion for a second State Agency $50 processing

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Precept-Based Training Course

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Requests for acknowledgements or permission to make copies shall be made to the

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Wastewater Treatment Bugs CEU Training Course

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Instructions for Written Assignments

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Environmental Terms, Abbreviations, and Acronyms

Recordkeeping and Reporting Practices

The final grade options are as follows:

Credit/no credit option (P/Z) - None Available

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To provide opportunities for TLC students to learn and practice environmental

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This course contains general EPA’s CWA federal rule

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Why Are Wastewater Treatment Plants Important?

33 U.S.C. s/s 1251 et seq. (1977)

The law gave the EPA the authority to set

The 1977 amendments focused on toxic

 eliminate the discharge of pollutants into the nation's waters, and

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Rotifer, an excellent MO or Microorganism or Indicator Organism.

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Basic Wastewater Treatment Processes

The bacteria normally present in water must have oxygen

Any excess microbiological growth could be removed

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Chemical BOD5 250

Microbiological Total coliform 108 - 109 MPN/L

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Typical BOD5 Variation of Domestic Wastewater

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Effects of Phosphorous and Nitrogen

Additional Effects of Nitrogen

Domestic waste overflow at the headworks. Yes, incredibly headworks do over-flow,

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Removing the grit and gravel that washes off streets or