SOIL

ORGANIC
MATTER
PROCEEDINGS OF A
SYMPOSIUM
BRAUNSCHWEIG, 6-10 SEPTEMBER 1976
JOINTLY ORGANIZED BY THE
IAEA AND FAO
IN CO-OPERATION WITH AGROCHIMICA

ENERGY A G E N C Y , V IE N N A , 1 9 7 7
SOIL ORGANIC MATTER STUDIES

VOL. II
 PROCEEDINGS SERIES

SOIL ORGANIC MATTER STUDIES

PROCEEDINGS OF A SYMPOSIUM
ON SOIL ORGANIC MATTER STUDIES
JOINTLY ORGANIZED BY
THE INTERNATIONAL ATOMIC ENERGY AGENCY
AND
THE FOOD AND AGRICULTURE ORGANIZATION
OF THE UNITED NATIONS
IN CO-OPERATION WITH AGROCHIMICA
AND HELD IN BRAUNSCHWEIG, 6 -1 0 SEPTEMBER 1976

In tw o volum es

VOL.II

INTERNATIONAL ATOMIC ENERGY AGENCY

VIENNA, 1977
SOIL ORGANIC MATTER STUDIES
IAEA, VIENNA, 1977
STI/PUB/438
ISBN 92-0-010177-1

Printed by the IAEA in Austria

April 1977
 FOREWORD

The nature, content and behaviour of the organic matter, or humus, in soil are factors of
fundamental importance for soil productivity and the development of optimum conditions for
growth of crops under diverse temperate, tropical and arid climatic conditions. Unfortunately,
the study of soil organic matter presents some of the most complex problems with which the
soil specialist and his collaborators in soil biochemistry and soil microbiology have to deal. How­
ever, through the co-operation of scientists of various disciplines and the use of tracer and other
modem techniques in research, valuable contributions have been and are being made to a better
understanding of the behaviour and functions of organic matter in soil; this will, in turn, lead to
the increase in crop production that is sorely needed.
The Food and Agriculture Organization of the United Nations and the International Atomic
Energy Agency had jointly convened two international meetings, the first in 1963 and the Second
in 1968, in co-operation with the International Soil Science Society, to review the progress that
had been achieved through the use of tracer techniques in studies of soil organic matter, and the
ways in which these methods could contribute to the growth of better crops. In the present,
third symposium, as in the two preceding ones, due consideration was given to studies involving
the use of radioactive and stable isotopes, but this third symposium departed from previous
practice in that it was organized in co-operation with AGROCHIMICA and that non-isotopic
approaches to research on soil organic matter were included. The meeting was the first symposium
sponsored by the IAEA and the FAO to deal with the subject matter without any restriction
regarding specific techniques.
The symposium, held at Braunschweig from 6 to 10 September 1976, was attended by 146
participants from 33 countries and two international organizations. A total of 77 papers were
presented and discussed during nine sessions. To make this possible within the limited time of the
meeting some of the papers were presented by rapporteurs. A number of papers, dealing with the
behaviour and functions of organic matter, make a contribution to increasing agricultural pro­
duction by proposing improved management practices. Other papers discussed turnover o f plant
residues, release of plant nutrients through the biodegradation of organic compounds, and nitrogen
economy and the dynamics of transformation of organic forms of nitrogen. Among other topics
raised and discussed were: the biochemical transformation of organic matter, the characterization
of humic acids, carbon dating and the impact of modern techniques on soil organic matter
research.
It is hoped that these Proceedings, which include the papers presented and the discussions,
will lead to further knowledge of the ways in which soil organic matter, with appropriate manage­
ment practices, contributes to increased production of crops for food and industrial purposes.
The organizers of the symposium wish to express their gratitude to the Federal Ministry of
Research and Technology, the Federal Ministry of Food, Agriculture and Forestry, and the
Forschungsanstalt für Landwirtschaft. The staff of the latter organization’s Institut für Bio­
chemie des Bodens in Braunschweig contributed greatly to the success of the symposium.
 E D IT O R IA L N O T E

The papers an d discussions have been e d ite d b y th e editorial s ta f f o f th e In tern ational

A to m ic Energy A g en cy to th e e x te n t con sidered necessary f o r th e rea d er’s assistance. The view s
expressed an d th e general s ty le a d o p te d rem ain, h ow ever, th e respon sibility o f th e n am ed authors
o r participan ts. In a ddition , th e view s are n o t necessarily th ose o f th e g overn m en ts o f the
n om in atin g M em ber S ta tes or o f th e n om in atin g organizations.
Where papers have been in corporated in to these P roceedings w ith o u t resettin g b y th e A gency,
this has been d o n e w ith th e kn ow ledge o f th e au th ors and th eir govern m en t au th orities, an d their
co o p era tio n is gratefu lly ackn ow ledged. The Proceedings have been p rin te d b y com p o sitio n
typ in g a n d p h o to -o ffse t lith ograph y. Within th e lim itation s im posed b y this m eth od, every effo rt
has been m ade to m aintain a high editorial standard, in particular to achieve, w h erever practicable,
co n sisten cy o f u n its an d sy m b o ls an d c o n fo rm ity to th e standards re co m m en d ed b y c o m p e te n t
in tern ation al bodies.
The use in th ese Proceedings o f particu lar designations o f cou n tries o r territories does n o t
im p ly an y ju d g em en t b y th e publisher, th e IA E A , as to th e legal statu s o f such cou n tries or
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The m en tio n o f sp ecific com pan ies o r o f th eir p ro d u c ts or brand nam es does n o t im p ly an y
en d o rsem en t o r recom m en dation on th e p a rt o f th e IAEA.
A u th o rs are th em selves respon sible f o r obtain ing th e necessary perm ission to reprodu ce
co p yrig h t m aterial fro m o th e r sources.
 CONTENTS OF VOL. II

BIOCHEMICAL TRANSFORMATION OF ORGANIC MATTER (Session 6)

Factors affecting the biostability of metabolic materials in soil (IAEA-SM-211 /20)............ 3

L.H. Sorensen
Discussion.............................................................................................................................. 14
Stability constants of some complexes of Argentine humic acids and
micronutrients (IAEA-SM-211/51)..................................................................................... 15
R .A . R esell, A.M . Miglierina, L.Q. d e N ovilla
Discussion............................................................................................................................ 21
Decomposition in soil of specifically carbon-14-labelled DHP and corn stalk lignins,
model humic acid-type polymers and coniferyl alcohols (IAEA-SM-211/4).................... 23
J.P. M artin, K. H aider
Discussion.............................................................................................................................. 32
Transformation d’acides phenoliques simples en substances para-humiques par des
micro-organismes du sol (IAEA-SM-211 /41) ...................................................................... 33
J.-R. Bailly, V. N kundikije-D esseaux, K. A g b ek o
Discussion.............................................................................................................................. 41
Caracterisation et transformations en milieu mull d’un modele humique issu de
l’autoxydation du Systeme catechol-glycine et marque selectivement au carbone-14
(IAEA-SM-211/42)............................................................................................................... 43
F. A n dreu x, D o ro ta G olebiow ska, T herese Chone, F. Jacquin, M. M etch e
Discussion.............................................................................................................................. 57
Effect of nitrogen on the formation of pyrocatechin-humic acid and the nitrogen
linkage characteristic of this acid (IAEA-SM-211/4 3 )........................................................ 59
H. Ö zb ek
Chemical alterations of natural lignin by interactions with humic-like autoxidation
products of pyrogallol(1,2, 3-trihydroxybenzene)(IAEA-SM-211/44)............................ 67
T. W eichelt
Discussion ............................................................................................................................. 83
Possibilities of an economic and non-polluting utilization of lignin
(IAEA-SM-211/45)............................................................................................................... 85
W.H.M. Schweers, W. Vorher
Discussion.............................................................................................................................. 89
Oxidation of some phenolic substances as influenced by clay minerals
(IAEA-SM-211/46).................................................................................................................. 91
Z. Filip, W. Flaig, E. R ie tz

BITUMENS IN SOIL ORGANIC MATTER (Session 7a)

Lipids of microbial origin in soilorganic matter (IAEA-SM-211/4 7 ) .................................... 99

G. H. Wagner, E.I. M u zorew a
Analyse et röle des bitumes dans les sols sableux acides (IAEA-SM-211 /48)....................... 105
E. F ustec-M athon, P. Jam bu, G. Joly, R. Jacqu esy
Discussion................................................................................................................................ 114
CHARACTERIZATION OF HUMIC ACIDS (Session 7b)

Review paper:

Recent findings on the characterization of humic substances extracted from soils from
widely differing climaticzones (IAEA-SM-211/7)............................................................... 117
M. S ch n itzer
Discussion................................................................................. 131
Structure et genese des acides fulviques des sols des Landes du Medoc (France)
(IAEA-SM-211/3 9 )............................................................................................................... 133
D. R ight, P. Jam bu, T. D u pu is
Discussion.............................................................................................................................. 141
Aggregation-dispersion phenomena in humicsubstances (IAEA-SM-21T/72) ....................... 143
N. Senesi, Y. Chen, M. S ch n itzer
Structure chimique des acides humiques et fulviques du sol (IAEA-SM-211 /73)................. 157
J.-A. N eyro u d , M. S ch n itzer
Applications of computer techniques in humus research (IAEA-SM-211/49)...................... 171
B.R. Nagar

CARBON DATING (Session 7c)

Mesures d’activite specifique de fractions de matiere organique appliquees ä l’etude

de revolution des sols de Guyane (IAEA-SM-211 /69)....................................................... 179
J. L. Rapaire, J.F. Turenne
Datations par le carbone-14 naturel de la matiere organique d’horizons spodiques de
podzols des Landes du Medoc (France) (IAEA-SM-211 /7 0 )............................................. 187
D. Right, B. G uillet
The search for biologically inert and lithogenic carbon in recent soil organic matter
(IAEA-SM-211/71).............................................................................................................. 193
H.W. Scharpenseel
Discussion.................................................................................................................................. 201

MODERN TECHNIQUES AND TH EIR IMPACT ON SOIL ORGANIC M ATTER

RESEARCH (Session 8a)

Review paper:

Advantages and limitations of mass- and photo-spectrometry in nitrogen-15-aided

studies (IAEA-SM-211 /8 ).................................................................................................... 205
V. M id d elb o e
Discussion ............................................................................................................................. 210
Studies on soil humic compounds, fungal melanins and model polymers by pyrolysis
mass-spectrometry (IAEA-SM-211/53)............................................................................... 213
K. H aider, B.R. Nagar, C. Saiz, H .L.C. Meuzelaar, J.P. M artin
Discussion.............................................................................................................................. 219
Chromatography of humic substances on controlled pore glass (IAEA-SM-211 /54)............ 221
O. H. D anneberg
Discussion.............................................................................................................................. 228
Review paper:

Role of organic matter in volatilization of sulphur and nitrogen in soils

(IAEA-SM-211/9 )................................................................................................................ 229
J.M. B rem ner
Discussion ............................................................................................................................. 240
Application of high-pressure liquid chromatography to studies of extractable soil
organic matter:Porous silica packings (IAEA-SM-211/5 5 ) ................................................. 241
R.H . L o ep p ert, B.G. V olk
Discussion ............................................................................................................................. 249

SLUDGE (Session 8b)

Municipal sludge as organic fertilizer with special reference to the heavy metals
constituents (IAEA-SM-211/74)......................................................................................... 253
N. el-Bassam, C. Tietjen
Epandage de boues residuaires: repercussions sur l’azote du sol et le prelevement
par du ray-grass de Cd, Cr, Hg et Zn (IAEA-SM-211/7 5 ) .................................................. 259
J. C. Fardeau, G. Guiraud, J o celyn e Jappe, Gisele L lim ous
Determination de l’aptitude ä la biodegradation des boues residuaires d’origines
diverses —Action sur les proprietes physico-chimiques du sol (IAEA-SM-211/76).......... 265
J.L. M orel, F. Jacquin
Evaluation of municipal refuse from Dahomey (Benin) as an organic manure
(IAEA-SM-211/77)......................................................... 277
F.X. K o m a A lim u , I E . S oe A gnie, B.H. Janssen
Discussion.................................................................................................................................. 289
Variation in content of polycyclic aromatic hydrocarbons in soil and plants by using
municipal waste composts in agriculture (IAEA-SM-211/31) ........................................... 291
P.-Chr. E llw ardt

SOIL ORGANIC MATTER COMPONENTS AND PLANT METABOLISM

(Session 9a)

Metabolism of soil-related phenolic compounds in plants and cell suspension cultures

(IAEA-SM-211/56)............................................................................................................... 301
H. Harms
Estudio de la action ejercida sobre la planta de maiz por dos tipos de äcido humico
(IAEA-SM-211/57)............................................................................................................... 307
V. H ernando, В. C. Ortega, C. F ortun
Discussion.............................................................................................................................. 317
Effect of organic matter and salts on the activity of some soil enzymes
(IAEA-SM-211/58)............................................................................................................... 319
AS. A bdel-G haffar, M .H .A. el-Shakw eer, M .A. Barakat
Discussion.............................................................................................................................. 324
INTERACTION BETWEEN AGROCHEMICALS AND ORGANIC MATTER
(Session 9b)

Blocage de molecules s-triaziniques par la matiere organique (IAEA-SM-211/7 8 )............... 327

M. Schiavon, F. Jacquin, C. Goussault
Bound or adsorbed methabenzthiazuron residues in soil (IAEA-SM-211/7 9 )...................... 333
F. Führ, W. M ittelsta ed t, K. H aider
Discussion ................................................................................................................................ 339

PEAT (Session 9c)

Composition and content of amino acids in peat-forming plants and in peats

(IAEA-SM-211/81)............................................................................................................... 343
F. Maciak, H. Süchtig, W. Flaig
Stabilization of organic matter in sand mixed cultures (IAEA-SM-211/82)......................... 359
B. S ch effer
Organic nitrogen content of peat soils combined with different fractions of humic
substances (IAEA-SM-211/83)............................................................................................ 365
W. R ochu s

List of Chairmen of Sessions and Secretariat......................................................................... 371

List of participants.................................................................................................................... 373
Author ind ex ............................................................................................................................ 387
B IO C H E M IC A L T R A N S F O R M A T IO N
O F O R G A N IC M A T T E R
( S e s s io n 6 )
 IAEA-SM-211/20

FACTORS AFFECTING THE BIOSTABILITY

OF METABOLIC MATERIALS IN SOIL
L.H. S0RENSEN
Agricultural Research Department,
Research Establishment Risф,
Roskilde,
Denmark

Abstract

FACTORS A FFECTING THE BIOSTABILITY OF METABOLIC M ATERIALS IN SOIL.

A loam and a sandy soil, incubated w ith 14C-labelled straw for 3 and 12 years respectively, were used for
an investigation o f th e effec ts o f th e addition o f energy material (unlabelled glu cose) and air-drying and grinding
on th e ev olution o f labelled and native C 0 2-C, and on the biom ass. The flush o f CO 2 caused by fum igation with
chloroform was taken as a measure o f the biom ass. The addition o f 5 0 and 2 0 0 mg glucose-С /100 g soil respectively,
resulted in an evolution o f labelled C 0 2-C that was 1.6 and 3 .2 tim es the evolution from unam ended soil ( ‘prim ing’).
The priming was largest after th e sm all addition o f glucose-C when calculated per unit o f glucose-C added. One
addition o f glucose resulted in a decrease in the labelled biom ass in com parison w ith unam ended soil, whereas
after repeated additions it was approxim ately o f th e same size as in th e unam ended soil. Air-drying and grinding
caused an evolution o f labelled C 0 2-C that ranged from 1.2 to 5.2 tim es the evolution from untreated soil.
Grinding had the strongest effect. Air-drying had a m inor influence on the biom ass, whereas grinding had a'
strong influence. The results suggest th at th e increase in evolution o f C 0 2-C caused b y the treatm ents originated
partly from killed biom ass and partly from non-biom ass material. Air-drying and grinding influenced the
evolution o f native CO--C, w hich, on th e w h ole, was parallel to the in flu en ce on the labelled C 0 2-C.

INTRODUCTION

Metabolites, which are synthesized in soils during decomposition of labelled cellulose and
other decomposable materials added to the soils, decay slowly [1, 2]. Conditions such as irregular
chemical structure and interaction with other soil constituents might be the cause of this biostability.
The rate of decomposition of soil organic matter can be increased by certain treatments as,
for example, addition of fresh decomposable materials, drying of the soil, and disruption of the
soil structure [3—5].
The investigation described here is of a preliminary nature; it was performed with the aim
of exploring to what extent the rate of decay of a fraction of labelled materials in soils was
influenced by the above-mentioned treatments, and to what extent the biomass in the soils was
influenced. The flush of C02 caused by fumigation with the vapour of CHC13 was taken as a
measure of the biomass according to the method introduced by Jenkinson [6]. A series of five
papers was recently published by Jenkinson and collaborators [7—11 ]. Evidence is presented in
these papers that the flush of C02 caused by fumigation with CHC13 can be used as a measure of
the soil biomass. It was thus shown that there was a close agreement between biomass-C measured
by fumigation and soil biovolume measured by optical microscopy [9].
A loam and a sandy soil were used for the present investigation, each containing a fraction
of 14C-labelled materials originating from 14C-labelled straw incubated in the soils for 3 and 12 years
respectively.

3
4 S0RENSEN

MATERIALS AND METHODS

Sandy soil: pH 5.8; organic C, 1.9%; N,0.18%; particles <0.02 mm, 15%.
Loam soil: pH 8.1; organic C, 1.0%; N,0.11%; particles <0.02 mm, 32%.
The sandy soil was incubated in the field for 12 years after the addition of 14CTabelled
straw; this was added in 1964 in an amount corresponding to 350 mg C/100g soil [12]. Three
lots of the soil were used for the present investigation: lots 1 and 2 were removed from the
field plot after 8 years, lot 1 was incubated in the laboratory for 4 years moistened to 60% of
the water-holding capacity; lot 2 was air-dried and stored in this condition for 3 years when it
was remoistened; lot 3 was removed from the field plot in April 1976. At the beginning of the
experiments described here lots 1—3 contained 53, 55 and 50 mg labelled C/100 g soil respectively.
About 21% of the labelled C in the soil was in the form of amino acids.
The loam soil was incubated in the laboratory for 3 years after the addition of the labelled
straw, which was added in amounts corresponding to 880 mg C/100 g soil. At the beginning of
the present experiment the soil contained 223 mg labelled C/100 g soil. About 17% of the labelled
C was in the form of amino acids.
Analytical methods and the preparation of the labelled straw have been described in previous
publications [2].

Treatments

Air-drying was performed by placing thin layers of the moist soil on aluminium foil at about
25°C in a slow air stream for 24 h. Oven-drying was performed by placing the air-dried soil in an
oven at 80°C for 24 h. The dried soils were remoistened by adding the required amount of water
followed by gentle stirring with a spatula. Gradual moistening was carried out by placing the air-
dried sample over a layer of water in a closed jar for 16 h; the weight of the soil increased 2%
during this time. The soil sample was then placed on moistened filter paper for 6 h; during this
time the soil absorbed half the required amount of water, the remainder being added as described
above.
Grinding of air-dried soil was performed by means of an electrically powered mortar and
pestle until 95% of the material was < 0.125 mm.
Unlabelled glucose was added to soil samples dissolved in 3 ml of water. The moist soil
samples were left in open jars in the air until they had lost about 3 g in weight, the glucose was
then added and the sample was gently stirred.

Incubation and C02 determination

Samples corresponding to 40—50 g oven-dried soil were placed in wide-mouth 100-ml

Erlenmeyer flasks. Water was added in amounts corresponding to 60% of the water-holding
capacity. To the ground samples was added an amount of water corresponding to 60% of the
water-holding capacity of the same weight of normal soil. The untreated control samples consisted
of soil which had been incubated moist for at least 3 months before the beginning of the present
experiment. The soil was weighed out moist, and dry matter determinations were performed
at the same time.
The flasks were fitted to an aeration system yielding a slow stream of moist C02-free air.
The C02 evolved from the samples was trapped in absorption towers containing 0.2N KOH and
determined by titration. Radioactivity was determined by scintillation counting. The incubation
temperature was 20°C. The flasks were weighed every month and evaporated water was restored;
the loss in weight rarely exceeded 1 g. For further details, see Ref. [12]. All determinations were
performed in duplicate. All results are expressed on an oven-dried soil basis (105°C for 24 h) unless
otherwise stated.
 IAEA-SM-2II/20 5

Biomass determination

At the end of the incubation period that followed the treatments, the flasks containing the
soil samples were removed from the aeration system and placed in a desiccator containing a;beaker
with ethanol-free chloroform. The desiccator was evacuated until the chloroform began to boil,
and then it was closed and left in the dark for 40 h. When opened, the beaker was removed from
the desiccator, which was then evacuated and opened several times to remove traces of chloroform.
The fumigated soil samples were inoculated by the addition of 1 ml of a soil suspension prepared
by shaking 1 g of fresh unlabelled soil with 100 ml of water. The flasks were then connected to
the aeration system, and the amount of C02-C evolved during 10 days of incubation was determined.
The increase in C02-C evolution caused by the fumigation was taken as the amount calculated by
deducting the amount of C02-C evolved from the sample during 10 days of incubation preceding
the fumigation (these values are not shown in the tables) from the value observed after fumigation.
Or it was taken as the difference between the amount evolved from the fumigated soil and the
amount evolved from non-fumigated soil during the second incubation period ranging from the
10th to the 20th day. The first-mentioned procedure was used only when the samples had been
incubated uninterruptedly for 2 months or more. Jenkinson [10] reported that on an average 50%
of the carbon in killed biomass was mineralized to C02 by the 10th day of incubation after
fumigation; this value was used for the calculation of biomass-C in the present investigation.

RESULTS AND DISCUSSION

Rate of decomposition and biomass in untreated soil

The untreated samples of the sandy soil evolved during 285 days of incubation 1.057 mg
labelled C02-C and 37.69 mg native CO2-C/100 g soil (Tables I and II, Fig.l). This represented

TABLE I. EVOLUTION OF LABELLED C02-C FROM THE SANDY SOIL (Lot 1) AFTER
ADDITION OF UNLABELLED GLUCOSE, AND AFTER FUMIGATION WITH CHC13
F um igation was p e rfo rm e d a fter in cu bation f o r 2 8 5 days; biom ass-C was calcu lated fro m these
values

Labelled

COj-C evolved after

T reatm ent3
Biomass-C
Treatment Fum igation
(2 8 5 days) (1 0 days)

m g /100 g %b m g /100 g m g /1 0 0 g %c

N one 1.057 2.0 0 .3 1 2 0 .5 4 2 1.0

135 mg non-labelled
glucose-C added 1.651 3.1 0 .2 8 3 0 .4 7 2 0.9
6 7 5 mg non-labelled
glucose-C added 3.3 5 5 6.3 0 .4 4 6 0 .6 4 8 1.3

a T he glucose was added in 3 portions each corresponding to 45 and 225 mg g lu cose-C /100 g soil respectively;
th e first p ortion was added at th e beginning, th e second and third after incubation periods o f 9 0 and 180 days
respectively.
b Percentage o f labelled C in soil at th e start.
c Percentage o f labelled C remaining in soil after treatm ent and incubation.
6 S0RENSEN

TABLE II. EVOLUTION OF NATIVE AND NON-LABELLED C02-C AFTER REPEATED

ADDITIONS OF NON-LABELLED GLUCOSE TO THE SANDY SOIL (Lot 1)
For fu r th e r explan ation s see Table 1

N ative and non-labelled

C 0 2-C evolved after

Treatment
Biomass-C
Treatment Fum igation
(2 8 5 days) (1 0 days)

m g /1 0 0 g %a m g /100 g m g /1 0 0 g % b

N one 3 7 .69 2.1 2 .98 5 .0 2 2 0.3

135 mg non-labelled
glucose-C added 1 4 0.54 76.2 8.55 1 4 .4 2 0 29.2
675 mg non-labelled
glucose-C added 509.87 7 0 .0 2 1 .0 9 3 0 .0 9 0 12.4

a Percentage o f native C in soil at the beginning, or o f non-labelled glucose-C added (see tex t).
^ Percentage o f native soil-C or non-labelled glucose-C remaining in soil after treatm ent and incubation (see tex t).

PERI OD OF I NCUBA T I ON ( DAYS)

F IG .l. C u m u la tive e v o lu tio n o f labelled CO y C fr o m th e sa n d y so il ( lo t 1} a fte r rep ea ted a d d itio n s o f n o n -

labelled glucose-C. T he g lu c o se was a d d ed in three p o r tio n s each co rresp o n d in g to 4 5 ( V ) a n d 2 2 5 m g (£s)
g lu c o se -C jl 00 g so il respectively; th e fir s t p o r tio n was a d d ed a t th e beginning, th e s e c o n d a n d th ird as in d ica te d
b y arrow s; (O ) u n a m e n d e d soil. C om pare w ith Table /.
 IAEA-SM-211/20 7

TABLE III. EVOLUTION OF LABELLED C02-C FROM THE SANDY SOIL (Lot 2) AFTER
VARIOUS TREATMENTS, AND AFTER FUMIGATION WITH CHC13 ;
F um igation w as p e rfo rm e d a fter in cu bation fo r 77 d a ys fo llo w in g th e treatm en ts; biom assrC
was calcu la ted fr o m th ese values

Labelled '

CO 2 -C evolved after
T reatm ent B iom ass-C ,
Treatment Fum igation
( 1 1 days) (1 0 days)

m g /1 0 0 g %a m g /100 g m g /1 0 0 g % b

N one 0 .9 4 0 1.7 0.4 9 2 0 .7 6 4 . 1.4

Air-drying
norm al m oistening 1.064 1.9 0.4 0 7 0 .6 3 3 ! 1 .2

Air-drying
gradual m oistening 1.085 2 .0 0.3 7 5 0 .5 7 8 ; 1.1

Grinding 2 .2 3 4 4.1 0.251 0 .2 1 6 ; 0.4

a Percentage o f labelled C in soil at the beginning.

k Percentage o f labelled C remaining in soil after treatm ent and incubation.

TABLE IV. NATIVE C02-C EVOLVED FROM THE SANDY SOIL (Lot 2) AFTER VARIOUS
TREATMENTS, AND AFTER FUMIGATION WITH CHC13
F um igation was p e rfo rm e d a fte r in cu bation f o r 77 d a ys fo llo w in g th e treatm en ts; biom ass-C
was calcu la ted fr o m th ese values

Native

CO 2 -C evolved after

Treatm ent Biomass-C

Treatment Fum igation
(7 7 days) (1 0 days)

mg / 1 0 0 g %a mg / 1 0 0 g mg / 1 0 0 g % b

N one 16.69 0.9 5.62 7.36 0.4

Air-drying
norm al m oistening 17.62 1 .0 5.05 6.11 0 .3

Air-drying
gradual m oistening 18.57 1 .0 4 .9 8 6 .4 4 : 0 .4

Grinding 3 0 .3 7 1.7 4 .5 0 4 .4 5 ; 0 .3

a Percentage o f native C in soil at the beginning.

^ Percentage o f native C remaining in soil after treatm ent and incubation.
 S0RENSEN

TABLE V. EVOLUTION OF LABELLED C02-C FROM THE LOAM SOIL AFTER VARIOUS
TREATMENTS, AND AFTER FUMIGATION WITH CHC13
F um igation was p e rfo rm e d a fter incubation fo r 5 8 days fo llo w in g treatm en ts; biom ass-C was
calcu la ted fr o m these values

Labelled

C 0 2-C evolved after

Treatment Biomass-C
Treatment Fum igation
(5 8 days) ( 1 0 days)

m g /100 g %a m g /100 g m g /100 g %b

N one 4 .2 9 0 1.9 5 .737 1 0 .270 4.7

Air-drying 9.061 4.1 5 .6 9 4 9 .9 7 8 4.7
Oven-drying 14.744 6.6 3 .1 1 9 3.971 1.9
Grinding 2 2 .4 3 0 10.1 1.874 1.166 0.6
50 mg non-labelled
glucose-C added 7 .077 3.2 5.275 8 .8 7 6 4.1
200 mg non-labelled
glucose-C added 13.400 6.0 4.951 6 .9 7 6 3.3

3 Percentage o f labelled C present in the soil at the beginning,

k Percentage o f labelled C remaining in the soil after treatm ent and incubation.

TABLE VI. NATIVE AND NON-LABELLED COr C EVOLVED FROM THE LOAM SOIL
AFTER VARIOUS TREATMENTS, AND AFTER FUMIGATION WITH CHCI3
F or fu rth er explan ation see Table V

Native and non-labelled

C 0 2-C evolved after

Treatment Biomass-C
Treatment Fum igation
(5 8 days) (1 0 days)

m g /100 g %a m g /100 g m g /1 0 0 g %b

N one 8.80 0.9 6 .9 4 11.04 1.1

Air-drying 10.22 1.0 6 .97 11.54 1.2

Oven-drying 18.70 1.9 5 .38 6 .9 0 0.7

Grinding 36.43 3.6 5.16 3 .8 0 0.4

50 mg non-labelled
glucose-C added 4 3 .1 4 68.7 10.52 16.23 33.1

200 mg non-labelled
glucose-C added 162.73 7 7 .0 13.96 19.32 18.0

a Percentage o f native C in soil at th e beginning, or o f non-labelled glucose-C added (see tex t).
^ Percentage o f native soil-C or non-labelled glucose-C remaining in soil after treatm ent and incubation (see tex t).
 IAEA-SM-211/20 9

an average of two replicas, the deviation between the two replicas being 4.2 and 6.8% of the
mean for the labelled and the native C respectively. This rate of decomposition corresponds
to a half-life of 25—27 years for both the labelled and the native carbon in the soil. The values
were calculated by means of the formula Ti = О.ЗТ/log (A0/A), where A0 represents the amount
of substance at zero time, and A the amount after time T [13]. The values are considered only
to indicate the order of the size, a longer period of incubation being necessary to establish a
more exact value. The decomposition in lot 2 of the sandy soil proceeds more rapidly (Tables III
and IV). The half-life of the labelled and the native carbon, calculated as above, corresponded to
9 and 17 years respectively. The reason for this difference is that, after removal from the field,
lot 2 of the sandy soil was stored air-dried for 3 years and only remoistened 3 months before the
initiation of the present experiment. The rate of decomposition in the loam soil (Tables Vand VI)
corresponded to a half-life of 6 years for the labelled carbon and 13 years for the native carbon.
At the end of the incubation periods described above, the biomass in the soils was determined.
It accounted for 1.0 and 1.4% of the labelled carbon remaining in lots 1 and 2 of the sandy soil
respectively; of the native carbon, 0.3 and 0.4% were in the biomass. The biomass in a sample
of the sandy soil removed from the field plot in April 1976 (lot 3), and thus incubated uninter­
ruptedly in the field for 12 years, accounted for 2.9 and 0.7% of the labelled and native carbon
respectively. The biomass in the loam soil accounted for 4.7 and 1.1% of the labelled and native
carbon respectively (Tables I—VI).

The priming effect

The addition of unlabelled glucose increased the rate of decomposition of labelled materials
both in the sandy and in the loam soils; the larger the addition, the larger the increase —the
‘priming effect’ (Tables I and V, Fig. 1). The evolution of labelled C02-C ranged from 1.6 to
3.2 times the evolution from unamended samples for both soils. The priming effect, however,
decreased per unit added glucose. The addition of 135 and 675 mg glucose-C, respectively;
to 100 g of the sandy soil resulted thus in a priming of 0.44 and 0.34 mg labelled C02-C per 100 g
of glucose-C added. The corresponding figures for the addition to the loam soil of 50 and 200 mg
glucose-C, respectively, were 5.6 and 4.6 mg labelled C02-C (Table VII). The repeated addition
of glucose to the sandy soil showed that the first addition had the strongest effect, whereas the
third addition did not increase the rate of decomposition of labelled materials initiated by the
second addition. The rate was, however, roughly maintained, indicating that the labelled materials
susceptible to priming were not exhausted. !
The addition of glucose, and the conditions in the soil during the period of incubation
resulting from this, diminished the size of the labelled biomass in the loam soil in comparison
with the unamended soil, and the largest addition caused the largest reduction (Table V). Per
unit of added glucose, however, the reduction caused by the small addition was the largest,
0.028 and 0.016 mg, respectively, per mg of glucose-C after the addition of 50 and 200 mg
glucose C/l 00 g soil (Table VII). Stated as a percentage of the previous increase in evolution of
labelled C02-C, the small addition also had the largest effect on the biomass. The decrease caused
by the addition of 50 mg glucose-C corresponded to 50% of the increase in labelled C02-C,
whereas the decrease caused by the addition of 200 mg glucose-C corresponded to only 36%
of the increase.
The labelled biomass in the sandy soil after repeated additions of glucose was approximately
of the same size as in the unamended soil; in the sample that had received the large addition it
was even slightly larger (Table I). This indicates either that the biomass was not diminished in
this case, or, what is more likely, that repeated additions resulted in such conditions in the soil
that the labelled biomass was able to regain its original size. A low addition of energy material
such as glucose might primarily result in an increased death rate of the biomass, whereas larger
additions might also result in an abundant production of enzymes which render non-biomass
10 S0RENSEN

TABLE VII. INCREASE IN C02 EVOLUTION AND THE CORRESPONDING DECREASE

IN BIOMASS CAUSED BY THE TREATMENTS

Increase in Decrease in
Decrease in
evolution in biomass-C
biomass-C^
c o 2- c a as % o f
Soil Treatment U161Cd5C ill
m g /100 g soil CO 2 evolution

Labelled Native Labelled Native Labelled Native

Air-drying
norm al m oistening 0 .1 2 4 0 .9 3 0 0 .1 3 2 1.250 106 134
Sandy soil Air-drying
(lo t 2) gradual m oistening 0.145 1.880 0 .1 8 6 0 .9 2 0 128 49

Grinding 1.294 13.680 0 .5 4 8 2 .9 1 0 42 21

Air-drying 4.771 1.420 0 .2 9 2 - 0 .5 0 0 6 ND

Oven-drying 10.454 9 .9 0 0 6 .3 0 0 4 .1 4 0 60 42
Grinding 18.140 2 7 .6 3 0 9 .1 0 4 7 .2 4 0 50 26
Loam soil
50 mg non-labelled
glucose-C added 2.787 ND 1.3 9 4 ND 50 ND
2 0 0 mg non-labelled
glucose-C added 9 .1 1 0 ND 3 .2 9 4 ND 36 ND'

135 mg non-labelled
Sandy soil glucose-C added 0 .5 9 4 ND 0 .0 7 0 ND 12 ND
(lo t 1) 675 mg non-labelled
glucose-C added 2.2 9 8 ND - 0 .1 0 6 ND ND ND

a The evolution from untreated soils deducted from the evolution o f C 0 2-C during the period o f incubation
follow ing th e treatm ents.
k Biomass-C in treated soils deducted from biomass-C in untreated soils.

material more susceptible to microbial attack. These aspects have been discussed in greater
detail elsewhere [14]. After repeated additions, the labelled biomass might therefore eventually
regain its original size, or even become larger than in the unamended soil.
A tentative calculation was made of the evolution of non-labelled glucose-C in C02 and the
percentage of glucose-C remaining in the soil and present in the biomass. For this calculation it
was assumed that the evolution of native C02-C and the native biomass in the samples amended
with non-labelled glucose were of the same size as in the unamended soil; these values were
deducted from the total values. These assumptions are not entirely correct since the decomposition
of the native soil organic matter was presumably increased by the added glucose (priming), and the
biomass reduced. The results are consequently only considered to indicate the order of the
size. Of the 135 and 675 mg glucose-C added to 100 g of the sandy soil, 76 and 70% respectively
were evolved as C02, and of the glucose-C remaining in the soil, 29 and 12% respectively were in
the biomass (Table II). The loam soil was amended with 50 and 200 mg glucose-C/100 g; 69 and
77% of this were evolved as C02, and of the amount remaining in the soil, 33 and 18% respectively,
were in the biomass (Table VI).
 IAEA-SM-211/20 11

TREATMENTS

0 U N T R E A T E D V 50 mg
о -------„ -------- Д 200 N O N -L A B E L L E D
+ AIR DRYIN G
▼ 135 G LU C O SE - C

▲ 675_ A D D E D / 100 g SO IL
• OVEN DR YIN G
□ G RINDING

о
(л LA BELLED С NATIVE С
o> 12 12
LOAM 0+ LOAM
о
о |-° +
V
SOIL SOIL
8 8
д
Ol А |-
А -
Е
О О 0 _i
10 20 30 10 20 30 АО
со
со
<
s: 1. 2 12Г
SA N D Y SA N D Y
о
- SO IL - SO IL
m
0.8 8 -
0 О
Н*
t ▼
-
0. A A -
о -

0 ............................ 0 -
1. 0 2.0 3.0 10 20 30 АО

С 0 2-С mg / Ю О д S OI L

F IG .2. T he relation b e tw e e n C 0 2-C ev o lved during th e in c u b a tio n fo llo w in g th e tr e a tm e n ts a n d th e biom ass

p re se n t in th e soils a t th e te rm in a tio n o f in cu b a tio n . T he s y m b o l f o r u n tr e a te d so il ( Q j refers to th e sa n d y
so il flo t 1} th a t received rep ea ted a d d itio n s o f п о п -labelled glucose (T a b le I). T h e s y m b o ls (+ X J refe r fojair­
d ryin g fo llo w e d b y n o rm a l an d gradual m o iste n in g respectively. 1

The effect of drying and grinding

The evolution of labelled C02-C after air-drying was 1.2 and 2.1 times as large as the evolution
from the untreated soil in the case of the sandy and the loam soils, respectively; grinding increased
the evolution 2.4 and 5.2 times. Only the loam soil was subjected to oven-drying, and this
increased the evolution of labelled C02-C 3.4 times (Tables V and VI).
The increase in C02 evolution during the incubations subsequent to the treatments was
followed by a reduction in biomass in comparison with untreated soils (Fig.2). In the sandy soil
this decrease was slightly larger than the previous increase in C02 evolution; it corresponded to
106 and 128% of the increase for air-drying followed by normal and gradual moistening respectively
(Table VII). In the loam soil the decrease in biomass caused by air-drying was insignificant.
It corresponded to only 6% of the previous increase in C02 evolution, whereas oven-drying was
followed by a reduction corresponding to 60%. The decrease in biomass that followed after
grinding corresponded to 42 and 50% of the previous increase in C02 evolution (Table VII).
12 S0RENSEN

T A B L E V III. T H E D IR E C T IN F L U E N C E O F A IR - D R Y IN G A N D G R IN D IN G O N T H E
B IO M A S S

Labelled Native

CO 2 "Ca CO 2 -Ca
Soil Treatm ent *5 Bio- Bio-
mass-C mass-C
A lone + CHC13 A lone + CHCI3

mg / 1 0 0 g soil

No N one 0 .1 2 4 0 .8 5 0 1.452 4 .4 6 0 11.1 3 0 13.3 4 0

Sandy soil
Air-drying 0.3 2 7 0 .972 1.2 9 0 6 .8 1 0 1 1 .5 9 0 9 .5 6 0
(lo t 3)

Grinding 1.231 1.176 ND 17.4 8 0 1 6 .1 3 0 ND

N one 0 .6 1 0 5.821 1 0 .420 0 .7 2 7 5 .8 7 0 10.2 9 0

Loam soil Air-drying 3 .7 1 4 8.921 1 0 .410 6 .4 0 0 9 .3 7 0 5 .9 4 0

Grinding 11.541 11.636 0 .1 9 0 16.4 8 0 1 4 .5 8 0 ND

The evolution o f C 0 2 was determ ined during 10 days o f incubation fo llow ing treatm ent alone or treatm ent plus
fum igation.
^ Fum igation w ith CHC13 was performed im m ediately after treatm ent and rem oistening.

G r a d u a l m o is t e n in g o f t h e s a n d y s o il r e s u lte d i n a s l i g h t l y la r g e r e v o l u t io n o f la b e lle d C 0 2-C

t h a n n o r m a l m o is t e n in g . T h e o p p o s it e r e s u lt h a d b e e n e x p e c t e d s in c e i t w a s b e lie v e d t h a t n o r m a l ,
r a p i d m o is t e n in g m ig h t c a u s e s o m e r u p t u r e o f s o il c r u m b s , a n d t h u s r e n d e r m o r e o f t h e m a t e r ia l
s u s c e p t ib le t o d e c o m p o s it i o n . T h e d if f e r e n c e b e t w e e n t h e t w o t r e a t m e n t s w a s o b s e r v e d a f t e r
t h e f i r s t 1 2 d a y s o f in c u b a t io n .
T h e in f l u e n c e o f d r y in g a n d g r in d in g o n t h e e v o l u t i o n o f n a t iv e C 0 2-C a n d o n n a t iv e b io m a s s
w a s o n t h e w h o l e p a r a lle l t o t h e e f f e c t o n t h e la b e lle d m a t e r ia ls ( T a b le s I V , V I , V I I a n d F ig . 2 ) .

T h e d i r e c t e f f e c t o f a ir - d r y in g a n d g r in d in g o n t h e b io m a s s

T h e v a r ia t i o n s i n s iz e o f t h e b io m a s s a f t e r t h e in c u b a t i o n t h a t f o l l o w e d t h e t r e a t m e n t s a re
n o t n e c e s s a r ily a t r u e m e a s u r e o f t h e in f l u e n c e o f t h e t r e a t m e n t s o n t h e b io m a s s , s in c e n e w
g e n e r a t io n s o f m ic r o - o r g a n is m s m u s t h a v e d e v e lo p e d d u r i n g t h e p e r i o d o f i n c u b a t io n w h e n th e
m a t e r ia l e x p o s e d b y t h e t r e a t m e n t s w a s d e c o m p o s e d . T h e d ir e c t e f f e c t o f a ir - d r y in g a n d g r in d in g
o n t h e b io m a s s i n s o ils w a s t h e r e f o r e in v e s tig a te d b y f u m i g a t i o n im m e d ia t e ly a f t e r t h e t r e a t m e n t s
a n d r e m o is t e n in g . I t w a s a s s u m e d t h a t t h e e f f e c t o f f u m i g a t i o n w o u l d b e t h e s a m e i n r e c e n t ly
t r e a t e d s o ils as i n s o ils w h e r e t h e b io lo g ic a l c o n d i t io n s w e r e s ta b iliz e d d u r i n g a p e r io d o f u n i n t e r ­
r u p t e d i n c u b a t io n . A n in c r e a s e i n t h e e v o l u t io n o f C 0 2 a f t e r f u m ig a t i o n , i n c o m p a r is o n w i t h th e
e v o l u t i o n f r o m t r e a t e d b u t n o t f u m ig a t e d s o ils , s h o u ld t h u s b e a m e a s u r e o f t h e b io m a s s t h a t h a d
s u r v iv e d t h e t r e a t m e n t .
T h e r e s u lt s in d ic a t e d t h a t a ir - d r y in g h a d s o m e e f f e c t o n t h e la b e lle d b io m a s s i n t h e s a n d y
s o il; i t w a s r e d u c e d t o 8 9 % o f t h a t i n u n t r e a t e d s o il, w h e r e a s t h e la b e lle d b io m a s s i n t h e lo a m
s o il w a s u n a f f e c t e d ( T a b le V I I I ) . T h e s e f i n d i n g s a re i n a c c o r d a n c e w i t h t h e p r e v io u s o b s e r v a t io n s
f o r t h e lo a m s o il, w h e r e , a f t e r a ir - d r y in g a n d s u b s e q u e n t in c u b a t i o n , t h e la b e lle d b io m a s s w a s
p r a c t ic a ll y o f t h e s a m e s iz e as t h a t i n u n t r e a t e d s o il ( T a b le V I I ) . T h e in c r e a s e i n C 0 2 e v o l u t io n
c a u s e d b y a ir - d r y in g o f t h e s a n d y s o il w a s f o ll o w e d b y a d e c re a s e in b io m a s s o f a p p r o x im a t e ly
 IAEA-SM-211/20 13

t h e s a m e s iz e ( T a b le V I I ) . T h e o b s e r v a t io n o f t h e d ir e c t e f f e c t is s o m e w h a t c o n t r a r y t o t h a t ,
h o w e v e r ; b o t h o b s e r v a t io n s s u g g e s t t h a t t h e la b e lle d b io m a s s i n t h e s a n d y s o il w a s m o r e s u s c e p t ib le
t o t h e t r e a t m e n t t h a n t h e b io m a s s i n t h e lo a m s o il.
T h e d ir e c t e f f e c t o f a ir - d r y in g o n t h e n a t iv e b io m a s s w a s s t r o n g e r t h a n o n t h e la b e lle d ; th e
n a t iv e b io m a s s w a s r e d u c e d t o 7 2 a n d 5 8 % o f t h a t i n t h e u n t r e a t e d s a m p le s f o r t h e s a n d y s o il
a n d t h e lo a m s o il r e s p e c t iv e ly .
O n t h e w h o le , t h e r e s u lt s s u g g e s t t h a t t h e m a t e r ia l i n t h e s o ils r e n d e r e d d e c o m p o s a b le b y
a ir - d r y in g w a s o n l y p a r t l y d e r iv e d f r o m k i l l e d b io m a s s , a n d le a s t f o r t h e la b e lle d b io m a s s i n th e
lo a m s o il. T h is is i n a g r e e m e n t w i t h o b s e r v a t io n s b y T u c k w e l l a n d J e n k in s o n [ 1 5 ] , w h o f o u n d t h a t
t h e in c r e a s e i n e v o l u t io n o f C 0 2 a f t e r a ir - d r y in g o r ig in a t e d p a r t l y f r o m k i l l e d b io m a s s a n d p a r t l y
f r o m a n o n - b io m a s s c o m p o n e n t . S h ie ld s , P a u l a n d L o w e [ 1 6 ] c o n lu d e d t h a t t h e in c r e a s e d e v o l u t io n
o f la b e lle d C 0 2- C a f t e r p h y s ic a l t r e a t m e n t s c o u l d n o t b e a s c r ib e d s t r i c t l y t o b io m a s s .
T h e n u m b e r s o f v ia b le o r g a n is m s , d e t e r m in e d b y p la t e c o u n t s , h a v e b e e n f o u n d b y s o m e
in v e s t ig a t o r s [ 1 7 , 1 8 ] t o b e l o w e r i n t h e a ir - d r ie d a n d r e m o is t e n e d s o il t h a n i n fr e s h s o il, w h e r e a s
o t h e r w o r k e r s s ta te t h a t a ir - d r y in g h a s n o m a r k e d e f f e c t o n t h e n u m b e r s o f e it h e r f u n g i o r
b a c t e r ia [ 1 9 ] .
G r in d in g h a d a s tr o n g e ffe c t; t h e e v o l u t i o n o f C 0 2 a f t e r f u m ig a t i o n w a s i n t h r e e cases
o u t o f f o u r l o w e r t h a n f r o m t h e g r o u n d b u t u n f u m ig a t e d s o il ( T a b le V I I I ) . T h is s u g g e s ts t h a t
t h e b io m a s s w a s a lm o s t c o m p l e t e ly k i l l e d b y t h e t r e a t m e n t . T h e b io m a s s o b s e r v e d i n t h e g r o u n d
s a m p le s a f t e r t h e s u b s e q u e n t in c u b a t io n , a n d s h o w n i n T a b le s I I I , I V , V a n d V I , s h o u ld c o n s e q u e n t ly
b e n e w b io m a s s d e v e lo p e d d u r i n g t h e d e c o m p o s it i o n o f k i l l e d b io m a s s a n d n o n - b io m a s s m a t e r ia l
e x p o s e d b y th e tr e a tm e n t.
T h e la b e lle d b io m a s s i n t h e u n t r e a t e d s a n d y s o il ( 0 . 7 6 4 m g , T a b le I I I ) c o r r e s p o n d s t o 5 9 %
o f t h e in c r e a s e i n C 0 2 e v o l u t io n c a u s e d b y t h e g r in d in g ( 1 . 2 9 4 m g , T a b le V I I ) . T h e b io m a s s in
t h e u n t r e a t e d lo a m s o il ( 1 0 . 2 7 0 m g , T a b le V ) c o r r e s p o n d s t o 5 7 % o f t h e in c r e a s e c a u s e d b y
g r in d in g ( 1 8 . 1 4 0 m g , T a b le V I I ) . T h is s u g g e s ts t h a t a b o u t 4 0 % o f t h e C 0 2-C re le a s e d as a r e s u lt
o f t h e g r in d in g o r ig in a t e d f r o m n o n - b io m a s s m a t e r ia l.
T h e c o r r e s p o n d in g fig u r e s f o r t h e n a t iv e s o il- C w e r e 5 4 % f o r t h e s a n d y s o il ( in c r e a s e 1 3 . 6 8 0 m g ,
b io m a s s 7 . 3 6 0 m g ) a n d 4 0 % f o r t h e lo a m s o il ( in c r e a s e 2 7 . 6 3 0 m g , b io m a s s 1 1 . 0 4 0 m g ) .
J e n k in s o n [ 2 0 ] c o n c lu d e d t h a t t h e f l u s h o f d e c o m p o s it i o n c a u s e d b y u l t r a s o n ic d is p e r s io n
w a s t o o la r g e t o d e r iv e s o le ly f r o m k i l l e d o r g a n is m s , a n d t h a t n o n - l iv in g p a r t s o f s o il o r g a n ic
m a t t e r m u s t a ls o h a v e b e e n e x p o s e d t o a t t a c k .

REFERENCES

[ 1 ] JEN K INSO N , D.J., Soil Sei. 111 (1 9 7 1 ) 64.

[2 ] S 0R E N S E N , L.H., Soil Biol. Biochem . 4 (1 9 7 2 ) 245.
[3 ] JEN K INSO N , D .S., “ The priming action ” , The Use o f Isotop es in Soil Organic Matter Studies, Pergamon
Press, Oxford (1 9 6 6 ) 199.
[4 ] BIRCH, H.P., Plant Soil 10 (1 9 5 8 ) 9.
[5 ] CRASWELL, E.T., W ARING, S.A ., Soil Biol. B iochem . 4 (1 9 7 2 ) 4 2 7 .
[ 6 ] JENKINSON, D .S., J. Soil Sei. 17 (1 9 6 6 ) 280.
[7 ] JENKINSON, D .S., POWLSON, D .S., Soil Biol. B iochem . 8 (1 9 7 6 ) 167.
[ 8 ] POWLSON, D .S., JEN K INSO N , D .S., Soil Biol. B iochem . 8 ( 1 9 7 6 ) 179.
[9 ] JENKINSON, D .S., POWLSON, D .S., W ED DER BU RN , R.W.M., Soil Biol. B iochem . 8 (1 9 7 6 ) 189.
[1 0 ] JENKINSON, D .S., Soil Biol. B iochem . 8 (1 9 7 6 ) 203.
[1 1 ] JEN K INSO N , D .S., POWLSON, D .S., Sod Biol. B iochem . 8 (1 9 7 6 ) 2 09.
[1 2 ] S 0 R E N S E N , L.H., Soil Biol. B iochem . 6 (1 9 7 4 ) 287.
[1 3 ] COMAR, C.L., R adioisotopes in B iology and Agriculture, McGraw-Hill, N ew York (1 9 5 5 ) 19.
[1 4 ] S 0R E N S E N , L.H., Soil Sei. Soc. A m ., Proc. 39 (1 9 7 5 ) 809.
[1 5 ] TUCKWELL, S.B., JENKINSON, D .S., “ E ffects o f air-drying on th e release o f organically-bound nutrients
by soils” , R otham sted Experim ental Station R eport for 1 9 74, Part I (1 9 7 5 ) 197.
14 S0RENSEN

[1 6 ] SHIELDS, J.A ., PAUL, E.A ., LOWE, W.E., Soil Biol. B iochem . 6 (1 9 7 4 ) 31.
[1 7 ] STEVENSO N , I.L., Plant Soil 8 (1 9 5 6 ) 170.
[1 8 ] SOULIDES, D .A ., ALLISO N, F.E., Soil Sei. 91 (1 9 6 1 ) 291.
[1 9 ] MACK, A .R ., Can. J. Soil. Sei. 4 3 (1 9 6 3 ) 316.
[2 0 ] JENKINSON, D .S., “ The effects o f m echanical disturbance o n th e decom p osition o f soil organic m atter” ,
R otham sted Experim ental Station R eport for 19 7 4 , Part I (1 9 7 5 ) 197.

D IS C U S S IO N

D .R . S A U E R B E C K : Y o u s p e a k o f p r im in g e f f e c t s m e a s u r e d f o r t w o m o n t h s a f t e r t h e
p a r t ic u la r t r e a t m e n t . I n o u r e x p e r ie n c e t h i s a c c e le r a t e d m in e r a liz a t i o n la s ts f o r o n l y a f e w d a y s ,
so t h a t o n p l o t t i n g t h e ra te s o f C 0 2 e v o l u t io n w e g e t a s te p - s h a p e d c u r v e w h i c h s h o w s a b r i e f
in c r e a s e im m e d ia t e ly a f t e r t h e t r e a t m e n t b u t q u i c k l y r e t u r n s t o it s f o r m e r s h a p e .
L .H . S O R E N S E N : W e , t o o , h a v e o b s e r v e d t h a t m o s t o f t h e a c c e le r a t e d e v o l u t io n o f la b e lle d
C 0 2 o c c u r r e d d u r i n g t h e f i r s t f iv e d a y s o f i n c u b a t io n a f t e r t h e a d d i t i o n o f t h e u n la b e lle d g lu c o s e .
T h e r e a f t e r t h e a c c e le r a t e d p r o d u c t i o n o f la b e lle d C 0 2 le v e lle d o f f , a n d a f t e r 3 0 d a y s o f in c u b a t io n
t h e c u r v e w a s a lm o s t p a r a lle l w i t h t h e c u r v e f o r t h e u n a m e n d e d s o il.
 IAEA-SM-211/51

STABILITY CONSTANTS OF SOME COMPLEXES OF

ARGENTINE HUMIC ACIDS AND MICRONUTRIENTS
R . A . R O S E L L * , A . M . M I G L I E R I N A , L .Q . D E N O V I L L A
U n iv e r s id a d N a t i o n a l d e l S u r ,
B a h ia B la n c a ,
A r g e n t in a

A b s tr a c t

STABILITY C O N STA N TS OF SOME COMPLEXES O F ARG ENTIN E HUMIC ACIDS A N D M ICRONUTRIENTS.

One o f th e m ain aims in th e stud y o f th e com p lexes o f hum ic substances and plant mineral nutrients
in th e soil is to ob tain therm odynam ic data w hich, com pared w ith know n system s, can give th e necessary inform ation
to calculate th e elem en t activity in solu tion . This inform ation is essential for predicting th e dynam ics o f such
nutrients in th e soil solu tion , sin ce th e latter is o n e o f th e main location s o f fundam ental reactions related to soil
genesis and fertility. The distribution o f radioactive 6 5 Zn and non-radioactive Cu b etw een a cation exchange resin
and th e hum ic system s w as used to calculate th e stability constants o f th ese elem en ts and the hum ic acids (H A )
extracted from various A rgentine soils. O n th e basis o f th ese stability con stants, th e fo llow ing con clusion s were
obtained: (1 ) C opper w as m ore strongly com p lexed than zinc b y th e hum ic acid o f a S olod soil. Copper had
also alm ost th e sam e value o f th e logarithm o f the stability constant for several soils (8.1 t o 9 .1 ); (2 ) T he value
o f the stability con stant o f th e Zn-HA co m p le x was higher for a C hestnut so il (Petrocalcic Paleustoll) than fo r the
o th er soils studied; (3 ) Since HA is an im portant in solu b le fraction in norm al soil con d ition s, it is suggested that
th e m o b ility o f Cu is lo w in th e soil solu tion o f th ese soils. Therefore, a Cu d eficien cy m ay be predicted or exp ected.

IN T R O D U C T IO N

T h e c a p a c i t y o f h u m i c s u b s ta n c e s t o f o r m s ta b le c o m p o u n d s w i t h m e t a ll ic c a t io n s i n t h e s o il
s o l u t i o n h a s a h ig h p e d o lo g ic a l a n d a g r o n o m ic v a lu e , b u t i t is n e i t h e r w e l l k n o w n n o r u n d e r s t o o d .
O f m o s t i m p o r t a n c e is t h e r e la t i o n s h ip b e t w e e n t h e m a in e s s e n t ia l p l a n t m i c r o n u t r i e n t s a n d t h e
f u l v i c a n d h u m i c a c id s . T h e s e h u m i c f r a c t i o n s a re p o l y e l e c t r o l i t i c m a c r o m o le c u le s , m a in ly a c id ic ,
w h i c h f o r m m e t a ll ic s e q u e s tr a te s a n d c h e la te s t h a t p a r t ic i p a t e i n i m p o r t a n t r e a c t io n s d u r i n g th e
g e n e s is a n d d e v e l o p m e n t o f s o ils . F u r t h e r m o r e , th e s e c o m p le x e s r e g u la t e t h e m o v e m e n t o f s o il
n u t r i e n t s a n d t h e i r a v a i l a b i l i t y t o p la n t s .
T a b le I p r e s e n ts t h e l o g a r it h m o f t h e s t a b i l i t y c o n s t a n t o f s o m e c o m p le x e s o f s o il h u m i c
m a t e r ia ls a n d t h e m i c r o n u t r i e n t s Z n a n d C u . T h e v a lu e o f t h e s t a b i l i t y c o n s t a n t d e p e n d s o n th e
t y p e o f h u m i c s u b s ta n c e a n d t h e p H o f t h e d e t e r m i n a t io n .
T h e o b j e c t o f t h is r e s e a r c h w a s t o e s ta b lis h t h e s t a b i l i t y c o n s t a n t o f c o m p le x e s o f Z n a n d C u
w i t h h u m i c a c id s e x t r a c t e d f r o m s e v e ra l A r g e n t in e s o ils . T h e d i s t r i b u t i o n o f r a d io a c t iv e 65 Z n
a n d n o n - r a d io a c t iv e C u b e t w e e n a c a t io n e x c h a n g e r e s in a n d t h e h u m i c s y s te m w a s u s e d t o c a lc u la t e
th e s t a b ilit y c o n s ta n t. T h is i n f o r m a t i o n is u s e f u l i n c o m p a r in g t h e r e la t iv e s o il c o m p l e x in g p o w e r ,
a n d it s i n f l u e n c e o n t h e d y n a m ic s o f m i c r o n u t r i e n t s in t h e s o il s o lu t i o n .

M A T E R IA L S A N D M E T H O D S

S o ils a n d h u m i c a c id s

T h e h u m i c a c id s w e r e e x t r a c t e d w i t h O .S N N a O H f r o m t h e A 1 h o r i z o n s o f B r u n i z e m , S o lo d
a n d S o lo n e t z s o ils o f B a lc a r c e , A r g e n t in a [ 1 0 ] , a n d f r o m a C h e s t n u t s o il o f B a h ia B la n c a , A r g e n t in a
[11, 12].

* A t present A. von H um boldt Stiftu ng fello w and guest-professor at the Institu t für B iochem ie des Bodens
der Forschungsanstalt für Landwirtschaft, Braunschw eig-Völkenrode, Federal R epublic o f Germ any.

15
16 ROSELL et al.

T A B L E I. L O G A R IT H M O F T H E S T A B IL IT Y C O N S T A N T O F
V A R IO U S O R G A N IC M A T E R IA L S A N D C O P P E R A N D Z IN C

Type o f com p lex Log К (pH ) Reference

Cu-peat 6.5 tu
Cu-fulvic acid 3 .2 3 (3 .5 ) [2]
Cu-humic acid 7 .0 0 (5 .0 ) [2]
Cu-non-dialysable hum us
in th e soil solu tion (N D H SS) 5.5 [3]
Cu-fulvic acid 5.78 (3 .5 ), 8 .6 9 (5 .0 ) [4]
Zn-organic m atter 3 .4 (4 .5 ), 5 .6 (7 .0 ) [5]
Zn-manure (aq ueous extr.) 7.8 (7 .0 ) [6]
Zn-hum ic acid 4 .4 (3 .6 ), 6 .2 (5 .6 ), 6 .8 (7 .0 ) [7]
Zn-fulvic acid 2.8 3 (3 .5 ) [2]
Zn-hum ic acid 2.8 7 (5 .0 ) [2]
Zn-NDHSS 4.3 [3]
Zn-fulvic acid 1 .7 3 (3 .5 ), 2 .3 4 (5 .0 ) [4]

Zn-fulvic acid (in 32 soils) 3.9 to 9 .3 , average 6 .2 (7 .0 ) [8]

Zn-hum ic acid (in 29 soils) 4 .2 to 10.8, average 7.3 (7 .0 ) [8]

Zn-hum ic acid (in 5 soils) 3 .1 3 to 5 .1 3 (6 .5 ) [9]

C a t io n - e x c h a n g e m e t h o d

T h e c a tio n - e x c h a n g e m e t h o d , f i r s t d e v e lo p e d b y S c h u b e r t [ 1 3 ] , a n d a p p lie d b y m o s t o f th e
a u t h o r s in d ic a t e d i n T a b le I , is b a s e d o n t h e r e s in s e le c t i v it y t o a d s o r b fr e e m e t a ll ic c a t io n s ( M e )
f r o m e q u i l i b r i u m s o lu t io n s . I n t h e p r e s e n c e o f a c o m p l e x in g a g e n t ( R ) , t h e m e t a ll ic c a t io n is
d i s t r i b u t e d b e t w e e n t h e r e s in a n d t h e e q u i l i b r i u m s o lu t i o n , d e p e n d in g o n t h e c o n c e n t r a t i o n a n d
c o m p l e x in g p o w e r o f ( R ) .
T h e e q u i l i b r i u m r e a c t io n a n d t h e f o r m a t i o n o f s t a b i l i t y c o n s t a n t ( K ) c a n b e f o r m u la t e d
as f o l l o w s [ 2 ] :

Me + xR = M eRx (1 )

(M e R x )
K. U )
(M e )(R )x "

w h e re (M e ) is t h e a c t i v i t y o f t h e fr e e m e t a ll ic c a t io n a t e q u i l i b r i u m ,

(R ) is t h e a c t i v i t y o f t h e f r e e c o m p l e x in g a g e n t a t e q u i l i b r i u m ,
(M e R x ) is t h e a c t i v i t y o f t h e c o m p l e x , a n d
X is t h e n u m b e r o f m o le c u le s o f t h e c o m p l e x in g a g e n t c o m b in e d w i t h o n e m e t a ll ic
c a t io n .
W o r k in g a t a c o n s t a n t i o n i c s t r e n g t h , b y a d d in g a n e le c t r o li t e t o t h e e q u i l i b r i u m s o lu t i o n , i t is
p o s s ib le t o u s e m o la r c o n c e n t r a t i o n s in s t e a d o f a c t iv i t i e s w i t h o u t a lt e r i n g t h e t h e r m o d y n a m ic
v a lu e a n d m e a n in g o f E q . ( 2 ) .
 IAEA-SM-211/51 17

T h e v a lu e s o f t h e u n k n o w n p a r a m e te r s x a n d ( R ) m u s t b e e s t im a t e d . T h e d is tr ib u t io n
c o n s t a n t A is d e f in e d b y

(M e ) + (M e R x )

w h e re m is t h e a m o u n t o f m e t a ll ic c a t io n b o u n d t o t h e r e s in , a n d
(M e ) + (M e R x ) is t h e t o t a l m e t a ll ic c a t io n in t h e e q u i l i b r i u m s o l u t i o n , M .
I n t h e a b s e n c e o f a c o m p l e x in g a g e n t t h e d i s t r i b u t i o n c o n s t a n t is A 0 :

m
Aq - (4 )
(M e )

E x p r e s s in g E q s ( 2 ) , ( 3 ) a n d ( 4 ) i n l o g a r i t h m i c f o r m a n d e lim i n a t in g te r m s , t h e r e la t i o n o b t a in e d
b y M a r t e l l a n d C a lv in [ 1 4 ] is :

lo g ] = lo g К + x l o g ( R ) (5 )

T h e p l o t lo g v e r s u s lo g ( R ) g iv e s t h e v a lu e s o f x a n d lo g K .

S in c e ( R ) is u n k n o w n a n d v e r y d i f f i c u l t t o o b t a in , th e v a lu e o f lo g ( R ) w a s p l o t t e d u s in g
r e la t iv e c o n c e n t r a t i o n s f o l l o w i n g R a n d h a w a a n d B r o a d b e n t [ 7 ] . T h e f u n c t i o n t h u s o b t a in e d
h a s t h e s a m e s lo p e as E q . ( 5 ) , a n d a llo w s x ( F i g . 1 ) t o b e c a lc u la t e d .

F I G .l. D e te r m in a tio n o f th e n u m b e r o f m o le c u le s (X ) o fa S o lo d h u m ic a c id c o m b in e d w ith o n e z in c c a tio n .

 T A B L E II. D E T E R M IN A T IO N O F T H E T A B L E III. M O L A R C O N C E N T R A T IO N O R
M O L A R C O N C E N T R A T IO N O R A C T IV IT Y (R ) A C T IV I T Y ( R ) O F H U M IC A C ID S O L U T IO N S
O F T H E A l- S O L O D H U M IC A C ID
HA fro m M olar c o n c e n tra tio n , M
C o u n ts/m in
T re a tm e n t m g Z n in s o lu tio n A l-B runizem 4 .81 X 1 0 '4
A ctu a l A verage A l-Solod 9 .0 6 X 1 0“4

A I-Solonetz 9 .5 4 X 1 0 '4
B lank 6581
6668 10
B lank 6756 A l-C h estn u t 12.3 6 X 10~4
W ith HA 5089
5031 7 .5 4
W ith H A 4973

R O SE L L e t al.
T A B L E IV . S T A B IL IT Y C O N S T A N T O F T H E Z n -H A C O M P L E X A T p H 5
Soil HA, A l-Solod

HA
Z n in
0.5 m g /m l (H A ) ^•0 Xo
log (H A ) so lu tio n C o u n ts /m in <*0 *0 X — 1 log — - 1 X log К
s o lu tio n M X 1 0 's X X
(m b )
(m l)

B lank - 8743
0 0 - 29 .8 879 90Л 455 - - - - -
5 9.1 0 .9 5 9 0 4 4 1 .0 1207 8 6 .4 3 1 7 .5 0.43 1 .6 3 3 4 7 1.21 4.5

10 18.0 1 .2 5527 51.1 1504 8 3 .0 2 4 4 .0 0 .8 6 1 .9 3 4 5 0 4.5

15 27.1 1 .4 3297 68 .0 2000 7 7 .4 171.0 1.66 0 .2 2 0 1 1 4.5

20 ' 36 .2 1.55871 69.7 2050 76.8 165.5 1.74 0 .2 4 0 5 5 4 .4

In itia l ac tiv ity o f th e m e tallic so lu tio n : 8 7 4 3 c o u n ts/m in = 3 0 0 p g Z n /flask 4 .4 7

 IAEA-SM-211/51 19

( R ) is c a lc u la t e d b y d e t e r m i n in g t h e m a x im u m c o m p l e x in g c a p a c it y o f t h e H A . T h is is
o b t a in e d b y e s t a b lis h in g t h e a m o u n t o f t h e m e t a ll ic c a t io n t h a t c o m b in e s w i t h a H A s o lu t i o n
o f k n o w n c o n c e n tr a tio n [2 ]. S in c e i t is a ls o k n o w n t h a t x m o le c u le s o f H A a re c o m b in e d w i t h a
m e t a ll ic a t o m ( E q . ( l ) ) , i t is p o s s ib le t o c a lc u la t e t h e n u m b e r o f m o le c u le s o f H A c o n t a in e d in
th e s o lu tio n a n d , th e r e fo r e , it s a c tiv ity (R ) .

Determination o f x

T o 5 0 - m l v o l u m e t r i c f la s k s t h e f o l l o w i n g s o lu t io n s a re a d d e d :
V a r ia b le a m o u n t o f H A s o l u t i o n ( 0 . 5 m g H A / m l )
D i s t i lle d w a t e r ( D W ) u p t o a b o u t 3 5 m l
5 m l 0 .1 N N a C l
3 m l o f m e t a ll ic c h lo r id e s o l u t i o n ( 1 0 0 p g m e t a l / m l ) , w h i c h is ta g g e d w i t h a r a d i o t r a c e r w h e n p o s s ib le
A d j u s t p H t o 5 w i t h d i l u t e H C 1 o r N a O H a n d c o m p le t e t o 5 0 m l v o lu m e w i t h D W .
T h e s o lu t i o n s a re t r a n s f e r r e d t o 1 2 5 - m l e r le n m e y e r fla s k s c o n t a in in g 0 . 5 g D o w e x 5 0 W
( 2 0 — 5 0 m e s h ) r e s in i n t h e N a - f o r m , a n d a re t h e n s h a k e n f o r 2 h i n a c o n s t a n t t e m p e r a t u r e ( 2 5 ° C )
b a th . I n t h e s u p e r n a t a n t t h e c o n c e n t r a t i o n o f f r e e a n d c o m p le x e d m e t a ll ic c a t io n s is d e t e r m i n e d b y
a t o m i c a b s o r p t i o n f o r C u a n d r a d i o a c t i v i t y c o u n t in g f o r 65 Z n .

Distribution constants

T h e f o l l o w i n g p r o c e d u r e is f o l l o w e d :

«о V
(6)
0 _ 1 0 0 -a o X P

w h e re a0 is t h e p e r c e n ta g e o f t h e m e t a ll ic c a t io n c o m b in e d w i t h t h e r e s in
100 - a0 is t h e p e r c e n ta g e o f t h e m e t a ll ic c a t io n r e m a i n in g i n s o lu t i o n
V is t h e v o lu m e o f t h e e q u i l i b r i u m s o lu t i o n , 5 0 m l
P is t h e r e s in w e i g h t , 0 . 5 g.
X is c a lc u la t e d as X 0 , i n t h e p r e s e n c e o f v a r ia b le a m o u n t s o f H A .

Molar concentration or activity (R ) o fH A s

D u p l ic a t e s o lu t i o n s a re p r e p a r e d as f o ll o w s :
5 m l o f c o n e . H A s o lu tio n (5 m g H A / m l)
5 m l O . lN N a C l
1 0 m l o f m e t a ll ic c h lo r id e s o l u t i o n (1 m g m e t a l / m l )
A d ju s t p H to 5
A d u p l ic a t e b la n k ( w i t h o u t H A ) is r u n t o o b t a in t h e i n i t i a l m e t a ll ic c a t io n c o n c e n t r a t i o n .
T h e s o lu t i o n s a re s h a k e n f o r 2 h a t 2 5 ° C , c e n t r if u g e d t o s e p a r a te t h e m e t a ll ic h u m a t e , a n d
a n a ly s e d t o o b t a in t h e r e m a i n in g m e t a ll ic c a t io n i n s o lu t i o n . I n T a b le I I t h e r e s u lt s o b t a in e d w i t h
t h e c o m p l e x b e t w e e n A l - S o l o d H A a n d Z n a re p r e s e n te d ( 2 . 4 6 m g Z n w e r e c o m b i n e d o r c o m p le x e d
b y 2 5 m g H A ).
T a b le I I I p r e s e n ts t h e m o l a r c o n c e n t r a t i o n o r a c t i v i t y ( R ) o f t h e v a r io u s H A s .

Calculation o f stability constants

T a b le I V s h o w s t h e p r o c e d u r e u s e d t o c a lc u la t e th e s t a b i l i t y c o n s t a n t o f t h e c o m p l e x b e t w e e n
t h e A l - S o l o d H A a n d t h e m e t a ll ic c a t io n Z n , a c c o r d in g t o E q . ( 5 ) .
20 ROSELL et al.

TABLE V. S T A B IL IT Y C O N S T A N T S O F
V A R IO U S C O M P L E X E S O F M IC R O N U T R IE N T S
A N D H U M IC A C ID S F R O M A R G E N T IN E S O IL S

L og К (p H 5)
H um ic acid
from soil
Cu Zn

Al-Brunizem 8.4 6.1

A l-Solod 9.1 4.5
Al-Solonetz 9.0 9.5

Al-Chestnut 8.1 9.7

R E S U L T S A N D D IS C U S S IO N

T a b le V p r e s e n ts t h e l o g a r it h m o f t h e s t a b i l i t y c o n s t a n t s o f t h e c o m p le x e s o f m i c r o n u t r ie n t s
( C u a n d Z n ) a n d t h e h u m i c a c id s e x t r a c t e d f r o m s e v e r a l A r g e n t in e s o ils . T h e f o l l o w i n g c o m m e n t s
can be m ade:
1. T h e lo g o f t h e s t a b i l i t y c o n s t a n t s o f t h e C u - H A c o m p le x e s a r e h ig h a n d o f t h e s a m e
o r d e r o f m a g n it u d e ( f r o m 8 .1 t o 9 . 1 ) . T h e S o lo d s o il s h o w s t h e h ig h e s t v a lu e a n d t h e C h e s t n u t
s o il t h e lo w e s t . S in c e H A is a n i m p o r t a n t in s o lu b le f r a c t i o n i n n o r m a l s o il c o n d i t io n s , i t is
s u g g e s te d t h a t t h e a v a i l a b i l i t y o f C u is l o w i n t h e s o il s o l u t i o n o f th e s e s o ils . T h e r e f o r e a C u
d e f ic i e n c y m a y b e e x p e c t e d o r p r e d i c t e d . G o o d m a n a n d C h e s h ir e [ 1 5 ] h a v e r e c e n t ly r e p o r t e d
C u - f i x a t i o n i n o r g a n ic m a t e r ia ls f r o m s t u d y in g t h e e le c t r o n p a r a m a g n e t ic r e s o n a n c e o f t h e c o ­
o r d i n a t i o n c o m p le x e s b e t w e e n C u a n d t h e n i t r o g e n i n p o r p h y r i n d e r iv a t iv e s o f p e a t h u m i c a c id s .
2 . T h e s t a b i l i t y c o n s t a n t s o f t h e Z n - H A c o m p le x e s o f A r g e n t in e s o ils a re h ig h e r t h a n t h e
d a t a r e p o r t e d i n t h e l i t e r a t u r e ( T a b le I ) . T h e v a lu e s a re h ig h e r f o r t h e C h e s t n u t s o il a n d lo w e r f o r
t h e S o lo d s o il, i n in v e r s e o r d e r t o t h a t o f t h e C u c a t io n .
3 . T h e p H d e p e n d e n c y o f t h e s t a b i l i t y c o n s t a n t v a lu e s ( T a b le I ) s u g g e s ts t h e c o n v e n ie n c e
o f s t u d y in g t h e b e h a v io u r o f t h e m a in m ic r o n u t r i e n t s i n s im i la r n a t u r a l c o n d i t io n s t o t h o s e
( c o n c e n t r a t i o n , a c i d i t y , s o lu b le a n d in s o lu b le o r g a n ic m a t t e r , e t c . ) o f t h e s o il s o lu t i o n . I t w o u ld
b e o f s p e c ia l in t e r e s t t o e s ta b lis h t h e s t a b i l i t y c o n s t a n t s o f t h e p l a n t m i c r o n u t r i e n t s a n d t h e
v a r io u s c o m p l e x in g s y s te m s , s u c h as t h e s o lu b le a n d in s o lu b le o r g a n ic m a t t e r , i n t h e s o il s o lu t i o n .
T h is t h e r m o d y n a m ic i n f o r m a t i o n c o u l d h e lp t o o b t a in a b e t t e r a n d c le a r e r p i c t u r e o f t h e g e n e r a l
d y n a m ic s o f m in e r a l c o m p o n e n t s w h i c h p la y a f u n d a m e n t a l r o le i n t h e g e n e s is , d e v e lo p m e n t
a n d f e r t i l i t y o f s o ils .

ACKNO W LEDG EM ENT

T h e C o n s e jo N a c io n a l d e In v e s tig a c io n e s C ie n t if ic a s у T e c n ic a s ( C O N I C E T ) , A r g e n t in a ,
is t h a n k e d f o r f i n a n c ia l h e lp .

REFERENCES

[ 1] C O L E M A N , N.T., M c C L U N G , A.C., M O O R E , D.P., Science 123 (1 9 5 6 ) 330.

[2 ] C O U R P R O N , C., A nn. Agron. 18 (1 9 6 7 ) 623.
[3 ] G E E R IN G , H.R., H O D G S O N , J.F., Soil Sei. Soc. Am., Proc. 33 (1 9 6 9 ) 54.
[4 ] S C H N IT Z E R , M., Soil Sei. Soc. Am., Proc. 33 (1 9 6 9 ) 75.
 IAEA-SM-211/5I 21

[ 5 ] H IM E S , F.L., B A R B E R , S.A., Soil Sei. Soc. Am., Proc. 21 (1 9 5 7 ) 368.

[6 ] M I L L E R , M.H., O H L R O G G E , A.J., Soil Sei. Soc. Am., Proc. 2 2 ( 1 9 5 8 ) 225.
[ 7 ] R A N D H A W A , N.S., B R O A D B E N T , F.E., Soil Sei. 99 (1 9 6 5 ) 362.
[8 ] M A T S U D A , K., IT O , S., Soil Sei. Plant. Nutr. 16 (1 9 7 0 ) 1.
[9 ] A R D A K A N I , M.S., S T E V E N S O N , F.J., Soil Sei. Soc. Am., Proc. 36 (1 9 7 2 ) 884.
[ 1 0 ] R O S E L L , R.A., A L L A N , A.L., A G U L L O , E „ G E L O S , B., L A Z Z A R I , M.A., Turrialba 22 3 (1 9 7 2 ) 327.
[ 11 ] C O N IG L IO , R., R O S E L L , R.A., Ciencia e Investigaeiön (Argentina) 28 (1 9 7 2 ) 271.
[ 12] R O S E L L , R.A., A L L A N , A.L., A G U L L O , E „ G E L O S , B., L A Z Z A R I , M.A., Turrialba 22 3 (1 9 7 2 ) 333.
[ 1 3 ] S C H U B E R T , J., J. Phys. Coll. Chem. 52 (1 9 4 8 ) 340.
[ 14] M A R T E L L , E.A., C A L V IN , M., Chem istry of Metal Chelates C om pounds, Prentice Hall, N ew Y o r k (1952).
[ 15] G O O D M A N , B.A., C H E S H IR E , M.V., Nature (L o n d o n ) N ew Biol. 2 4 4 (1 9 7 3 ) 158.

D IS C U S S IO N

C h. JU S TE : E q u a t i o n ( 1 ) , w h i c h y o u u s e f o r d e t e r m i n in g t h e a p p a r e n t s t a b i l i t y c o n s t a n t s ,
s u g g e s ts t h a t y o u c o n s id e r t h e c o m p le x e s f o r m e d t o b e m o n o n u c le a r . A r e y o u c e r t a in o f t h is ,
f o r i f p o l y n u c l e a r c o m p le x e s a re f o r m e d , r e l a t i o n ( 2 ) is n o l o n g e r a p p lic a b le ?
R .A . R O S E L L : N o , I d o n o t k n o w th e ty p e s o f c o m p le x fo r m e d . I a m a w a re o f th e lim ita tio n s
o f t h e m e t h o d , w h i c h h a v e b e e n p o i n t e d o u t b y s e v e ra l a u t h o r s (e .g . R e f . [ 9 ] ) , b u t o u r in t e r e s t w a s
i n o b t a in in g i n f o r m a t i o n o n t h e r e la t iv e c o m p l e x in g p o w e r o f t h e h u m i c a c id s f r o m d if f e r e n t
s o ils a n d o f t h e v a r io u s e s s e n tia l m ic r o n u t r ie n t s .
C h. J U S T E : D o y o u h a v e a n y h y p o t h e s e s f o r e x p l a in in g t h e d if f e r e n c e s o b s e r v e d i n th e
d e t e r m i n a t io n o f s t a b i l i t y c o n s t a n t s w h e n t h e h u m i c a c id s d i f f e r i n o r ig in ?
R .A . R O S E L L : W e h a v e m a d e n o h y p o t h e s e s a b o u t th e s e d if f e r e n c e s . W e t h i n k t h e y a re
d u e t o d i f f e r e n t f u n c t i o n a l g r o u p s a n d s t r u c t u r a l c o m p o s i t io n s o f t h e h u m i c a c id s . W e p la n t o
s tu d y th is p r o b le m in th e n e a r fu tu r e .
M. S C H N IT Z E R : T h e r e is a t p r e s e n t n o s a t is f a c t o r y m e t h o d f o r d e t e r m i n in g m e t a l- o r g a n ic
m a t t e r c o m p le x e s . M r . R o s e ll h a s d o n e t h e b e s t h e c o u l d c o n s id e r in g t h e k n o w le d g e t h a t is c u r r e n t l y
a v a ila b le . M o r e r e s e a r c h i n t h is f i e l d is n e e d e d t o d e v e lo p b e t t e r m e t h o d s .
\
 IAEA-SM-211/4

DECOMPOSITION IN SOIL OF SPECIFICALLY

CARBON-14-LABELLED DHP AND CORN STALK LIGNINS,
MODEL HUMIC ACID-TYPE POLYMERS
AND CONIFERYL ALCOHOLS
J. P. M A R T I N
D e p a r t m e n t o f S o il a n d E n v ir o n m e n t a l S c ie n c e s ,
U n i v e r s i t y o f C a l if o r n ia ,
R iv e r s id e ,
U n i t e d S ta te s o f A m e r ic a

K . H A ID E R
I n s t i t u t f ü r B io c h e m ie d e s B o d e n s d e r
F o r s c h u n g s a n s t a lt f ü r L a n d w ir t s c h a f t ,
B r a u n s c h w e ig - V ö lk e n r o d e ,
F e d e r a l R e p u b lic o f G e r m a n y

Abstract

DECOMPOSITION IN SOIL OF SPECIFICALLY CARBON-14-LABELLED DHP A ND CORN STALK LIGNINS,

MODEL HUMIC ACID-TYPE POLYM ERS A N D CON IFERY L ALCOHOLS.
Side chain 2-I4C, ring-14C and 0 14CH3-labelled coniferyl alcohols were synthesized. T hese were linked into
m odel DHP lignin using peroxidase and hydrogen peroxide, and in to m odel hum ic acid-type polym ers using m ush­
room phenolase and m ixtures o f h yd roxytolu en es, hydroxyph en ols and h y droxybenzoic and cinnam ic acids.
Labelled corn stalk lignins w ere prepared by injecting side-chain 1 ,2 and 3 14C, ring 14C and 0 14CH3 feruhc acids
as lignin precursors in to th e growing stalks. The labelled con iferyl alcohols, polym ers and lignins were incubated in
G reenfield sandy loam (pH 7 .0 ) adjusted to 60% m oisture capacity. The to ta l C 0 2 and th e 14C 0 2 evolved were
measured over a 28-w eek incubation period. Compared w ith other com m on p henolic substances tested , the coniferyl
alcohol carbons w ere relatively resistant to m icrobial utilization in soil. A fter 28 w eeks, about 4 9 , 4 6 and 66%
respectively o f th e ring-14C, 2-14C (side chain) and 0 14CH3 carbons had evolved as 14C 0 2 . Linkage o f coniferyl
alcohol in to m odel lignins (D H P), plant lignins, and especially in to m od el hum ic polym ers, reduced avaüability. The
carbons in th e m od el hum ic acids were m ost resistant. A fter 28 w eeks, about 5, 6 and 14% respectively o f th e ring,
2- side chain and OCH3 carbons had evolved as C 0 2. T he sam e carbons in the m odel lignins were decom posed to a
greater ex ten t, nam ely, 2 0 , 22 and 45% for th e ring, 2-side chain and OCH3 carbons respectively. The related carbons
in the corn stalk lignin were utilized to a slightly greater ex ten t than th o se in the DHP lignins. Loss o f ring, 1 ,2 and
3- side chain and OCH3 carbons after 28 w eek s w ere about 2 8 , 4 2 , 2 8 , 32 and 52% respectively.

IN T R O D U C T IO N

L ig n in , a r e la t i v e l y s ta b le p h e n o lic p o l y m e r , is t h e s e c o n d m o s t a b u n d a n t p o l y m e r s y n th e s iz e d
b y p la n t s [ 1 — 3 ] . F r o m t h e e c o lo g ic a l p o i n t o f v ie w , i t is i m p o r t a n t t h a t t h e c a r b o n p r e s e n t in
l i g n i n b e re le a s e d as C 0 2 f o r u s e b y n e w g e n e r a t io n s o f li v in g p la n t s [ 4 , 5 ] . A l t h o u g h r e la t i v e l y
r e s is t a n t t o b io d e g r a d a t io n , li g n i n is d e c o m p o s e d i n n a t u r e [ 2 , 6 , 7 ] . A g r o u p o f b a s id io m y c e t e s , th e
w h i t e r o t f u n g i, a re p a r t i c u l a r l y a c t iv e i n li g n i n u t i l i z a t i o n , a n d h a v e r e c e iv e d m o s t a t t e n t i o n in
l i g n i n d e c o m p o s it i o n s tu d ie s [ 2 , 5 , 7 — 9 ] . S o m e o f th e s e f u n g i c a n d e g r a d e u p t o 8 0 % o f a n a v a ila b le
lig n in s o u rc e . O t h e r f u n g i, in c lu d in g Fungi imperfecti and Ascomycetes, a n d b a c t e r ia a re a ls o a b le
t o d e c o m p o s e li g n i n [ 2 , 9 ] . V e r y l i t t l e is k n o w n , h o w e v e r , a b o u t t h e b io c h e m i c a l m e c h a n is m s
in v o lv e d i n t h e d e g r a d a t io n p ro c e s s e s .
L i g n i n is c o n s id e r e d a n i m p o r t a n t s o u r c e o f s t r u c t u r a l u n i t s f o r f o r m a t i o n o f s o il h u m u s
[1 0 — 1 3 ]. I t c o u l d c o n t r i b u t e t o h u m u s f o r m a t i o n i n o n e o r m o r e o f a t le a s t t h r e e w a y s : ( 1 ) th e
li g n i n m o le c u le s c o u l d b e a lt e r e d o r p a r t i a l l y d e g r a d e d t h r o u g h m i c r o b i a l a c t i v i t y , a n d o t h e r o r g a n ic

23
24 MARTIN and HAIDER

c o n s t it u e n t s s u c h as p r o t e in s , p e p t id e s , a m in o a c id s a n d p o ly s a c c h a r id e s w i t h a m in o s u g a r u n it s
c o u l d b e l i n k e d t o t h e a lt e r e d r e s id u e s t h r o u g h e n z y m a t ic o r a u t o x id a t iv e p o l y m e r iz a t io n r e a c t io n s ;
(2 ) p h e n o lic d e g r a d a t io n p r o d u c t s o f l i g n i n t o g e t h e r w i t h m i c r o b i a l l y s y n th e s iz e d a r o m a t i c a n d
o t h e r r e a c t iv e c o m p o u n d s c o u l d u n d e r g o a u t o x id a t iv e o r e n z y m a t ic p o l y m e r i z a t i o n r e a c t io n s e it h e r
e x t r a c e llu la r ly o r w i t h i n m ic r o b i a l c e ll w a lls t o f o r m h u m i c m o le c u le s ; a n d ( 3 ) t h e li g n i n m o le c u le s
in c lu d in g t h e b e n z e n e r in g s c o u ld b e d e g r a d e d t o s im p le a l ip h a t ic c o m p o u n d s , s o m e o f w h i c h
c o u l d b e u t i l i z e d f o r t h e m ic r o b i a l s y n th e s is o f r e a c t iv e p h e n o ls a n d o t h e r s u b s ta n c e s w h i c h c o u ld
b e p o l y m e r iz e d t o f o r m h u m i c m o le c u le s [ 1 0 — 1 8 ] . S e v e ra l in v e s t ig a t o r s b e lie v e t h a t t h e s e c o n d
p a t h w a y m a y b e r e la t i v e l y m o r e i m p o r t a n t t h a n t h e o t h e r t w o [ 1 0 , 1 2 , 1 5 ] .
D H P ( d e h y d r o p o l y m e r ) li g n i n s s y n th e s iz e d b y t h e a c t io n o f p e r o x id a s e a n d li g n i n a lc o h o ls
a p p e a r t o b e s im i la r t o i f n o t i d e n t ic a l w i t h t r u e p la n t lig n in s [ 1 , 5 , 1 9 — 2 1 ]. B y u s in g s p e c i f i c a l ly
14C - la b e lle d li g n i n a lc o h o ls i t is p o s s ib le t o s y n th e s iz e s p e c i f i c a l ly 14C 4 a b e lle d D H P lig n in s . T h e s e
lig n in s , h o w e v e r , a re n o t a s s o c ia te d w i t h c e llu lo s e a n d o t h e r p o ly m e r s as a re t h e li g n i n s i n p la n t s .
B y in je c t in g s p e c i f i c a l ly 14C 4 a b e lle d f e r u l i c a c id s as li g n i n p r e c u r s o r s [ 1 , 2 0 ] i n t o p la n t s te m s , i t is
p o s s ib le t o o b t a in p la n t m a t e r ia l w i t h s p e c i f i c a l ly 14C 4 a b e lle d li g n i n [ 8 ] .
U s e o f s p e c i f i c a l ly 14C 4 a b e lle d li g n i n s f o r b io d e g r a d a t io n s tu d ie s m a k e s i t p o s s ib le t o
d e t e r m in e w h i c h o f th e c o n s t it u e n t u n i t c a r b o n s a re u t i l i z e d m o s t r e a d i ly b y t h e s o il m ic r o b e s , a n d
w h i c h a r e r e la t i v e l y m o r e i m p o r t a n t i n s o il h u m u s f o r m a t i o n . P r e v io u s s tu d ie s w i t h U n k a g e o f
14C 4 a b e lle d p h e n o lic a n d c in n a m ic a c id d e r iv a tiv e s , a m in o a c id s , p e p t id e s , a m in o s u g a rs a n d
p o ly s a c c h a r id e s w i t h a m in o s u g a r c o n s t it u e n t u n i t s i n t o m o d e l h u m i c a c id p o ly m e r s h a v e s h o w n
t h a t a ll t h e c a r b o n s a re s t a b iliz e d t o a c o n s id e r a b le d e g re e [ 1 4 , 1 6 — 1 8 ] . H o w e v e r , s id e c h a in a n d
C O O H g r o u p s , a n d li n k e d p e p t id e s , p r o t e in s , a m in o a c id s , a m in o s u g a rs a n d p o ly s a c c h a r id e c a r b o n s
a re a l i t t l e m o r e s u s c e p t ib le t o d e g r a d a t io n t h a n a re t h e a r o m a t i c r in g c a r b o n s .
T h e p u r p o s e o f t h e s tu d ie s r e p o r t e d w a s t o c o m p a r e t h e d e c o m p o s it i o n i n s o il o f s p e c i f i c a l ly
14C 4 a b e lle d c o n i f e r y l a lc o h o ls i n t h e fr e e s ta te a n d a f t e r lin k a g e i n t o D H P lig n in s , c o r n s t a lk lig n in s
a n d m o d e l h u m i c a c id - t y p e p o ly m e r s i n o r d e r t o b e t t e r u n d e r s t a n d t h e p ro c e s s e s in v o lv e d i n th e
t r a n s f o r m a t io n o f l i g n i n t o s o il h u m i c p o ly m e r s .

M ETHODS

T h e m e t h o d s f o r t h e s y n th e s is o f I4 C - la b e lle d f e r u l i c a c id s h a v e b e e n d e s c r ib e d [ 8 , 2 2 — 2 5 ] .
T h e f e r u l i c a c id s w e r e a c e t y la t e d , c o n v e r t e d t o t h e a c id c h lo r id e s a n d r e d u c e d w i t h L i A l H 4 t o th e
c o n i f e r y l a lc o h o ls . U n la b e lle d c o n i f e r y l a lc o h o l w a s p u r c h a s e d f r o m F l u k a A G , S w it z e r la n d . T h e
n u m b e r in g o f t h e s id e c h a in c a r b o n s w a s b e g u n w i t h t h e a lc o h o l g r o u p [ 3 ] .
F o r p r e p a r a t io n o f m o d e l h u m i c a c id s , 1 .5 m m o l o f 2 ,3 - , 2 , 6 - a n d 3 , 4 - d i h y d r o x y t o lu e n e s ,
c a t e c h o l, p - c r e s o l, o r c i n o l , p h o r o g lu c in o l, p y r o g a ll o l , r e s o r c i n o l , a n d c a f f e i c , 2 , 4 - d i h y d r o x y b e n z o ic ,
3 , 5 - d i h y d r o x y b e n z o ic , g a l li c , p - h y d r o x y b e n z o ic , p - h y d r o x y c in n a m i c , p r o t o c a t e c h u ic , s a lic y l ic a n d
2 , 4 , 6 - t r i h y d r o x y b e n z o i c a c id s w e r e d is s o lv e d i n 1 5 0 0 m l o f 0 . 0 5 M K H 2P 0 4 s o lu t i o n , a n d t h e
m i x t u r e a d ju s te d t o p H 6 . 5 w i t h N a O H . T h r e e m m o l o f t h e d e s ir e d 14C - la b e lle d c o n i f e r y l a lc o h o l
a n d 2 0 0 m g ( 1 0 0 0 0 0 u n i t s ) o f m u s h r o o m p h e n o la s e , o b t a in e d f r o m I C N , I n c . , w e r e a d d e d , a n d
th e m ix t u r e a e ra te d f o r fiv e d a y s .
T h e d a r k s o lu t i o n s w e r e a c id if ie d t o p H 1 .5 w i t h 6 N H C 1 a n d a llo w e d t o s ta n d o v e r n ig h t . T h e
h u m i c a c id p r e c i p it a t e s w e r e r e c o v e r e d b y c e n t r if u g a t io n , w a s h in g w i t h d i s t i ll e d w a t e r a d ju s te d
t o p H 2 . 0 w i t h H C 1 , a n d f r e e z e - d r y in g . T o o b t a i n t h e f u l v i c a c id f r a c t i o n s t h e s u p e r n a t a n t s w e r e
a d ju s t e d t o p H 7 . 0 , c o n c e n t r a t e d t o 4 0 0 m l, a d ju s te d t o p H 2 , d ia ly s e d i n t u b in g s w i t h a 6 0 0 0 m o le ­
c u la r w e i g h t c u t - o f f a g a in s t f r e q u e n t c h a n g e s o f d i s t i ll e d w a t e r , a n d f r e e z e - d r ie d .
T h e m o d e l lig n in s w e r e p r e p a r e d a c c o r d in g t o t h e ‘ Z u t r o p f m e t h o d o f F r e u d e n b e r g a n d
N im z [ 1 9 ] , u s in g h i g h l y p u r i f i e d h o r s e - r a d is h p e r o x id a s e o b t a in e d f r o m N u t r i t i o n a l B io c h e m ic a ls ,
In c . F o r la b e lli n g t h e l i g n i n o f c o r n p la n t s , 1 m l o f s o lu t i o n s c o n t a in in g 1 .5 m g o f la b e lle d f e r u l i c
a c id s w a s in je c t e d i n t o t h e b a s e o f y o u n g c o m s ta lk s b y m e a n s o f a s y r in g e w i t h t h e p is t o n r e m o v e d .
 IAEA-SM-211/4 25

T h e l i q u i d w a s t a k e n u p b y t h e p la n t w i t h i n t w o t o t h r e e d a y s . T h is p r o c e d u r e w a s r e p e a te d th r e e
t im e s o v e r a t w o - w e e k p e r i o d . T h e p la n t s w e r e a llo w e d t o g r o w f o r a n o t h e r th r e e w e e k s . T h e t o p s
w e r e g r o u n d i n l i q u i d N 2 , f r e e z e - d r ie d , e x t r a c t e d th r e e t im e s w i t h 8 0 % b o i l i n g e t h a n o l, a n d d r ie d [ 8 ] .
T h e m a t e r ia l h a d a li g n i n c o n t e n t o f 7 - 8 % w h i c h c o n t a in e d m o r e t h a n 8 0 % o f t h e t o t a l r a d i o a c t i v i t y .
F o r d e c o m p o s it i o n te s ts , G r e e n f ie ld s a n d y lo a m t o p s o il w a s a ir - d r ie d , p a s s e d t h r o u g h a 2 - m m
s c re e n a n d w e ig h e d i n t o 2 5 0 - m l e r le n m e y e r fla s k s i n 1 0 0 g p o r t io n s . T h is s o il h a d a p H o f 7 . 0 ,
a n e x c h a n g e c a p a c i t y o f 11 m e q 1 0 0 g a t p H 7 , a n d c o n t a in e d 1 .2 % o r g a n ic m a t t e r . T h e la b e lle d
c o n i f e r y l a lc o h o l a t c o n c e n t r a t i o n s o f 1 0 0 a n d 1 0 0 0 p p m , a n d t h e la b e lle d lig n in s , m o d e l h u m i c
a c id s a n d c o r n s t a lk s a t c o n c e n t r a t i o n s o f 1 0 0 0 o r 2 0 0 0 p p m , w e r e t h o r o u g h l y m ix e d w i t h t h e d r y
s o ils . T h e m o is t u r e c o n t e n t w a s a d ju s t e d t o 6 0 % o f t h e H ilg a r d c u p m o is t u r e c a p a c i t y ( 1 / 3 b a r ) , t h e
fla s k s a t t a c h e d t o a c lo s e d s y s te m , a n d s l o w l y a e r a t e d w i t h a c o n s t a n t s t r e a m o f C 0 2- fr e e a n d
h u m i d i f i e d a ir . W i t h t h is p r o c e d u r e 0 2 d o e s n o t b e c o m e a l i m i t i n g f a c t o r , a n d t h e m o is t u r e c o n t e n t
o f t h e s o il r e m a in s v i r t u a l l y c o n s t a n t f o r lo n g p e r io d s o f t i m e . M o s t tre a tm e n ts w e re r u n in
d u p l ic a t e . T h e a c t i v i t y o f t h e c o n i f e r y l a lc o h o ls a p p lie d r a n g e d f r o m a b o u t 1 0 0 0 0 t o
5 0 0 0 0 d is / m i n p e r m g . T h e a c t i v i t y o f t h e p o ly m e r s , t h e D H P li g n i n s , a n d t h e c o m s t a lk p r e p a ­
r a tio n ra n g e d f r o m a b o u t 5 0 0 t o 2 0 0 0 d is /m in p e r m g .
T h e C 0 2 e v o lv e d f r o m t h e in c u b a t in g s o ils w a s c o lle c t e d i n K O H s o lu t i o n , a n d t h e t o t a l C 0 2
d e t e r m in e d a t in t e r v a ls b y t i t r a t i o n o f a n a li q u o t w i t h s ta n d a r d H C 1 a f t e r a d d i t io n s o f B a C l2
s o lu t i o n . T h e 14C 0 2 w a s d e t e r m i n e d b y a c i d i f y i n g a n a li q u o t a n d c o lle c t in g t h e I4 C 0 2 r e le a s e d in
2 m l o f N C S r e a g e n t i n a s c i n t i l l a t i o n v ia l f i t t e d w i t h a n a d s o r p t i o n t o w e r . T h e to w e r c o n te n ts
w e r e w a s h e d i n t o t h e v ia l w i t h P P O t o lu e n e c o c k t a i l ( 0 . 5 % w t / w t , 2 , 5 - d ip h e n y lo x a z o l e i n t o l u e n e ) ,
a n d t h e a c t i v i t y d e t e r m in e d i n a B e c k m a n l i q u i d s c i n t i l l a t i o n s y s te m . T h e a c t i v i t y o f t h e c o n i f e r y l
a lc o h o ls , D H P lig n in s , c o m s t a lk s , m o d e l h u m i c a c id s , r e s id u a l s o ils a n d s o il f r a c t i o n s w a s d e t e r m in e d
b y c o m b u s t io n a t 9 7 0 C i n t h e p r e s e n c e o f a m e t a l c a t a ly s t a n d a s tr e a m o f p u r e 0 2 . T h e 14C 0 2
r e le a s e d w a s c o lle c t e d i n N C S r e a g e n t a n d t h e a c t i v i t y d e t e r m i n e d as in d ic a t e d a b o v e .
P o r t io n s o f t h e in c u b a t e d s o ils w e r e e x t r a c t e d w i t h 0 . 5 N N a O H , a n d t h e h u m i c a n d f u l v i c
a c id f r a c t i o n s o b t a in e d b y t h e c la s s ic a l p r o c e d u r e [ 2 6 ] . B e f o r e e x t r a c t i o n a n d c o m b u s t io n t h e
e n t ir e 1 0 0 g p o r t i o n s o f s o il w e r e f i n e l y g r o u n d i n a m e c h a n ic a l g r in d e r . P o r t io n s o f s o il w e r e a ls o
h y d r o l y s e d u n d e r r e f l u x w i t h 6 N H C 1 f o r 1 8 h , a n d t h e a c t i v i t y i n t h e h y d r o l y s e d s o ils a n d i n t h e
h y d r o ly s a t e s d e t e r m in e d .

R ESULTS

D e c o m p o s it i o n p e r c e n ta g e s f o r t h e 14C - la b e lle d c o n i f e r y l a lc o h o ls a re g iv e n i n T a b le I . B ased

o n t o t a l C 0 2 e v o lv e d , a b o u t 5 6 % o f t h e a d d e d c o n i f e r y l a lc o h o l C w a s lo s t d u r i n g t h e 2 8 w e e k s ’
in c u b a t i o n p e r i o d . B a s e d o n e v o l u t i o n o f 14C 0 2 , f r o m 4 6 t o 7 3 % o f t h e s p e c i f i c a l ly 14C - la b e lle d
c a r b o n s w a s lo s t d u r i n g t h is p e r i o d . I n a l l t e s ts a g r e a t e r lo s s o c c u r r e d f r o m t h e 1 0 0 0 p p m t h a n
f r o m t h e 1 0 0 p p m a d d i t io n s . T h e 2 - 14C ( s id e c h a in ) a n d r i n g - 14C w e r e d e g r a d e d a t a b o u t t h e s a m e
r a t e ( 4 6 t o 5 5 % ) w h e r e a s t h e 0 ,4 C H 3 c a r b o n s w e r e u t i l i z e d t o a g r e a t e r e x t e n t ( 6 0 t o 7 3 % ) .
A c c o r d i n g t o T a b le I I , t h e c o n i f e r y l a lc o h o l c a r b o n s w e r e h i g h l y s t a b iliz e d a g a in s t m i c r o b i a l
d e g r a d a t io n t h r o u g h lin k a g e i n t o m o d e l h u m i c - t y p e p o ly m e r s . O n l y 5 t o 7 % o f t h e r in g a n d 2 - s id e
c h a in c a r b o n s w e r e e v o lv e d as 14C 0 2 o v e r t h e 2 8 - w e e k in c u b a t i o n p e r i o d . T h e O C H 3 c a r b o n s w e r e
s l i g h t l y m o r e s u s c e p t ib le t o m i c r o b i a l u t i l i z a t i o n . A b o u t 1 4 % o f th e s e c a r b o n s w a s lo s t as 14C 0 2 .
T h e p e r c e n ta g e r e c o v e r y o f t h e a p p lie d 14C a c t i v i t y r a n g e d f r o m 9 8 t o 1 0 8 % .
T a b le I I I r e c o r d s t h e p e r c e n ta g e lo s s o f t h e s p e c i f i c a l ly 14C - la b e lle d c o n i f e r y l a lc o h o ls a f t e r
lin k a g e i n t o D H P lig n in s . A b o u t t h r e e t o f o u r t im e s as m u c h o f t h e a p p lie d C w a s e v o lv e d as w a s
n o t e d f o r t h e s a m e c a r b o n s l i n k e d i n t o t h e m o d e l h u m i c a c id s . L o s s e s o f t h e 2 - ( s id e c h a i n ) a n d
r in g - c a r b o n s r a n g e d f r o m 2 0 t o 2 3 % , a n d lo s s e s o f t h e 0 14C H 3 c a r b o n f r o m 4 4 t o 5 1 % . R e c o v e r ie s
o f t h e a p p lie d a c t i v i t y v a r ie d f r o m 9 9 t o 1 0 8 % .
26 MARTIN and HAIDER

T A B L E I. D E C O M P O S IT IO N O F S P E C IF IC A L L Y C A R B O N - 1 4 - L A B E L L E D C O N IF E R Y L
A L C O H O L S IN G R E E N F IE L D S A N D Y L O A M

C oncentration D ecom p osition after (w eeks)

14C label
(ppm ) 'l 2 4 8 12 20 28

Percentage o f 14C evolved as 14C 0 2 a

0 14CH3 100 38 43 47 53 56 59 60
1000 54 60 63 67 70 73 73
2-14C 100 17 21 25 32 38 42 43
(side chain) 1000 i6 31 37 43 47 49 50
Ring-14C 100 18 23 28 32 36 44 46
1000 20 25 32 36 40 48 52

Percentage o f total added C evolved as CC>2 b

0 14CH3 1000 26 44 48 49 52 54 56
2-14C 1000 25 43 47 49 50 52 55
(side chain)

Ring-14C 1000 40 45 50 53 55 57 58

a Percentage o f recovered 14C ( 14C 0 2 + residual 14C in soil) evolved as 14C 0 2.

b Based on total C 0 2 evolved minus C 0 2 evolved from controls.

T A B L E II. D E C O M P O S IT IO N I N G R E E N F IE L D S A N D Y L O A M O F S P E C IF IC A L L Y
C A R B O N - 14 - L A B E L L E D C O N I F E R Y L A L C O H O L S L I N K E D I N T O M O D E L P H E N O L A S E
H U M IC P O L Y M E R S 3

D ecom p osition after (w eek s)b T otal 14C activity

I4C label
1 12 recovered0
2 4 8 20 28

‘H um ic acid’ fraction

0 14CH3 4 6 8 10 12 14 15 102
3 5 7 9 11 13 14 100
to

1 2 2 3 4 5 6 102
(sid e chain) 1 1 2 3 4 5 6 108

Ring-14C 1 2 3 4 5 6 7 106
1 2 2 3 4 5 6 99

‘Fulvic acid’ fraction

o 14c h 3 4 6 8 9 10 12 13 104
3 5 7 10 11 13 14 100
2-I4C 1 2 3 4 5 6 6 104
(side chain) 1 2 3 3 4 5 6 110
Ring-14C 1 2 3 4 5 6 6 105
1 2 2 3 4 5 5 98

a Polym ers from a m ixture o f 18 h ydroxytolu en es, p henols and ben zoic acid derivatives; soil addition
1 0 0 0 ppm .
b Percentage o f to ta l recovered activity evolved as 14C 0 2.
c 14C 0 2 evolved + residual 14C in soil
X 1 00 .
to ta l 14C activity added
 IAEA-SM-211/4 27

T A B L E III. D E C O M P O S IT IO N I N G R E E N F IE L D S A N D Y L O A M O F S P E C IF IC A L L Y
C A R B O N - 1 4 - L A B E L L E D C O N I F E R Y L A L C O H O L S L I N K E D I N T O D H P L I G N I N S 3*

Percentage decom p osition after (w eek s)b T otal 14C activity

14C label
1 2 4 8 12 20 28 recovered6*

0 14CH3 (a) 10 16 21 29 34 39 44 99
(a) 8 14 20 29 34 38 44 99
(b) 10 21 30 38 42 47 51 108
(b) 10 21 30 36 39 44 47 104
2-j4C 2 4 7 11 14 19 22 106
2 4 7 12 15 20 23 106
Ring-14C 2 4 7 12 15 18 21 110
2 5 7 12 14 17 20 109

a Soil addition 1 0 0 0 ppm.

b Percentage o f to ta l 14C recovered evolved as l4C 0 2.
c 14CO evolved + 14C in soil
---------- TJ--------------------------- X 1 0 0 0 .
to ta l C activity added

T A B L E IV . D E C O M P O S IT IO N I N G R E E N F IE L D S A N D Y L O A M O F S P E C IF IC A L L Y
C A R B O N - 14 - L A B E L L E D C O N I F E R Y L A L C O H O L S L I N K E D I N T O C O R N S T A L K L I G N I N S ,
L A B E L L E D B Y A B S O R P T I O N O F 0 14C H 3 , S I D E C H A I N O R R I N G L A B E L L E D F E R U L I C
A C ID R E S P E C T IV E L Y 3

Percentage decom p osition after (w eek s)3 T otal 14C activity

1 2 4 8 12 20 28 recovered6

o 14c h 3 7 18 30 40 46 51 54 98
7 17 28 38 43 48 50 97
l- l4C (CH2OH) 7 12 22 27 32 37 40 102
(side chain) 12 17 26 31 36 41 44 101
10 15 23 32 36 37 40 112
10 15 23 33 37 38 41 112
2 -i4C 4 7 9 12 15 23 25 108
(sid e chain) 4 6 9 13 18 24 28 108
i 3 4 5 15 26 28 94
i 4 11 17 21 27 30 96
3-14C 6 8 12 i6 19 24 27 102
(sid e chain) 6 9 13 17 20 25 28 111
7 12 17 23 27 33 35 104
7 12 17 22 26 32 34 98
Ring-14C 3 5 11 19 22 26 29 99
3 5 10 18 21 25 28 101

3 S o il addition 2 0 0 0 ppm .
b Percentage o f to ta l 14C recovered evolved as I4C 0 2.
c 14C 0 2 evolved + 14C in soil
----------- 7 2 -------------:-------------- X 100.
to ta l C activity added
28 MARTIN and HAIDER

TA B LE V. D E C O M P O S IT IO N O F M O D E L P H E N O L A S E P O L Y M E R S ,
D H P L IG N IN S A N D C O R N S T A L K S B A S E D O N T O T A L A D D E D
C A R B O N E V O L V E D A S C 0 2a

Percentage d ecom p osition after (w eek s)b Range at 28 weeks

1 2 4 8 12 20 28 (%>

DHP lignins

5 13 18 23 27 29 30 2 7 -3 4

M odel phenolase polym ers — hum ic acid fractions

1 2 3 3 4 4 5 3 -7

M odel phenolase polym ers — fulvic acid fractions

1 2 3 4 4 5 5 2 -7

Corn stalks

25 36 45 57 57 63 64 6 0 -6 6

a T otal C evolved as C 0 2 m inus C 0 2 evolved from controls.

b Averages o f all polym er and corn stalk treatm ents.

T h e r e la t e d c a r b o n s i n t h e c o r n s t a lk lig n in s w e r e s l i g h t l y m o r e a v a ila b le t o t h e s o il m ic r o b e s
( T a b le I V ) t h a n w e r e t h e s a m e c a r b o n s i n t h e D H P lig n in s . L o s s e s o f r in g - a n d 2 - c a r b o n s ra n g e d
fro m 25 to 30% . A b o u t 4 2 , 3 0 a n d 5 2 % o f t h e 1 - 14C ( C H 2O H ) , 3 - 14C a n d 0 14C H 3 c a r b o n s ,
r e s p e c t iv e ly , w e r e e v o lv e d as 14C 0 2 . T h e r e s id u a l 14C i n t h e in c u b a t e d s o ils p lu s t h a t e v o lv e d as
14C 0 2 c o n s t it u t e d f r o m 9 4 t o 1 1 2 % o f t h e a c t i v i t y a p p lie d . D e c o m p o s it i o n p e r c e n ta g e s b a s e d o n
t o t a l C 0 2 e v o lv e d s u p p o r t e d t h e 14C 0 2 e v o l u t io n d a t a ( T a b le V ) , o r w e r e c o m p a r a b le w i t h
p r e v io u s te s ts .
T h e d i s t r i b u t i o n o f r e s id u a l a c t i v i t y i n t h e s o il h u m i c a n d f u l v i c a c id s , a n d e x t r a c t e d s o il
f r a c t i o n s , a f t e r in c u b a t i o n o f t h e la b e lle d fr e e a lc o h o ls w a s e v e n ly d is t r i b u t e d i n t h e h u m i c a c id ,
f u l v i c a c id a n d e x t r a c t e d s o il f r a c t i o n s ( T a b le V I ) . W i t h a ll t h e p o ly m e r s a n d lig n in s t h e b u l k o f t h e
r e s id u a l a c t i v i t y w a s r e c o v e r e d i n t h e h u m i c a c id f r a c t i o n s . F r o m 3 6 t o 5 8 % o f t h e a p p lie d a c t i v i t y
w a s p r e s e n t i n t h e h u m i c a c id f r a c t i o n a f t e r in c u b a t i o n w i t h t h e D H P lig n in s . F o r t h e c o r n s t a lk
li g n i n a n d m o d e l h u m i c p o l y m e r s , t h e h u m i c a c id f r a c t i o n c o n t a in e d 2 5 t o 5 6 % o f t h e a p p lie d
a c tiv ity . T h e lo w e s t p e r c e n ta g e s w e r e r e p r e s e n te d b y t h e 0 14C H 3 c a r b o n s f o r t h e D H P lig n in s a n d
m o d e l h u m i c p o l y m e r s , a n d 1 - 14C a n d 0 14C H 3 c a r b o n s f o r t h e c o m s t a lk lig n in s .
T h e lo s s o f a c t i v i t y u p o n 6 N H C 1 h y d r o l y s is o f s e le c te d s o ils a f t e r i n c u b a t io n w i t h th e
s p e c i f i c a l ly 14C - la b e lle d p r o d u c t s is g iv e n i n T a b le V I I . T h e g r e a t e s t lo s s e s , 3 8 t o 4 7 % o f th e
r e s id u a l a c t i v i t y , w e r e f r o m t h e s o ils in c u b a t e d w i t h t h e fr e e a lc o h o ls . L o s s e s f r o m t h e s o ils
a m e n d e d w i t h D H P a n d c o m s t a lk li g n i n v a r ie d f r o m 1 8 t o 3 5 % a n d 2 7 t o 3 7 % r e s p e c t iv e ly . In each
g r o u p t h e le a s t lo s s o c c u r r e d f r o m t h e s o ils r e c e iv in g t h e r i n g - 14C - la b e lle d m a t e r ia l.

D IS C U S S I O N

I n c o m p a r is o n w i t h m a n y o t h e r p h e n o lic s u b s ta n c e s te s te d [ 1 6 , 1 7 ] , c o n i f e r y l a lc o h o l is
r e la t i v e l y r e s is t a n t t o m i c r o b i a l d e g r a d a t io n i n s o il. I n a 2 8 - w e e k i n c u b a t io n p e r i o d o n l y a b o u t
5 0 % o f t h e 2 - 14C ( s id e c h a in ) a n d r i n g - 14C , a n d 6 6 % o f t h e 0 I4 C H 3 c a r b o n s , w e r e e v o lv e d as 14C 0 2 .
T h is c o m p a r e s w i t h lo s s e s o f a b o u t 5 3 , 6 9 a n d 7 5 % r e s p e c t iv e ly o f c o m p a r a b le c a r b o n s i n f e r u l i c
a c id o v e r o n l y a 1 2 - w e e k i n c u b a t io n [ 1 7 ] , a n d w i t h g r e a t e r C lo s s e s f r o m b e n z o ic a n d v e r a t r ic
a c id s [ 1 6 ] .
 IAEA-SM-211/4 29

T A B L E V I. D IS T R IB U T IO N O F C A R B O N -1 4 A C T IV IT Y A F T E R S O IL IN C U B A T IO N W IT H
S P E C I F I C A L L Y C A R B O N - 14 - L A B E L L E D C O N I F E R Y L A L C O H O L S , F R E E A N D L I N K E D
IN T O D H P L IG N IN S , C O R N S T A L K L IG N IN S A N D M O D E L H U M IC P O L Y M E R S 3

D istribution o f activity15
Label l4C 0 2 Humic acid Fulvic acid E xtracted soil

Free alcohols
0 14CH3 73 9 9 9
2-14C (side chain) 50 17 16 17
Ring-I4C 52 18 14 16

M odel phenolase hum ic polym ers — hum ic acid fractions

s

15 46 14 25
o
о
X

2-14C (side chain) 6 50 21 23

Ring-14C 6 52 21 21

M odel phenolase hum ic polym ers — fulvic acid fractions

0 14CH3 14 47 13 26
2-14C (side chain) 6 55 20 19
Ring-I4C 5 56 19 20

DHP lignins
0 ,4 CH3 47 36 8 9
2-14C (sid e chain) 22 53 11 14
Ring-14C 21 58 11 10

Corn stalk lignins

0 14CH3 52 24 9 17
1-14C (side chain) 42 29 8 21
2-14C (sid e chain) 28 38 13 21
3-14C (side chain) 32 40 14 14
Ring-14C 28 41 13 18

a 1 0 0 0 ppm additions.
b Based on total activity recovered = 100%.

S in c e c o n i f e r y l a lc o h o l is a la b il e a n d v e r y e a s ily o x id iz e d c o m p o u n d , i t is p r o b a b le t h a t a
p o r t i o n o f t h e w h o l e c o n i f e r y l a lc o h o l m o le c u le s o r s o m e o f t h e e a r l y t r a n s f o r m a t io n p r o d u c t s a re
s t a b iliz e d i n s o il h u m i c f r a c t i o n s , o r i n m ic r o b ia l m e la n in s [ 1 2 , 1 7 , 2 7 , 2 8 ] . M ic r o b ia l m e t a b o lis m
w o u l d in v o lv e t h e f o r m a t i o n o f s im p le a l i p h a t ic c o m p o u n d s s u c h as a c e t ic a n d s u c c in ic a c id s . I f
a ll t h e a p p lie d c o n i f e r y l a lc o h o l w e r e m e t a b o liz e d t h r o u g h o x i d a t i o n t o f e r u l i c a n d v a n i ll ic a c id s ,
d e m e t h o x y l a t i o n t o c a f f e i c a n d p r o t o c a t e c h u ic a c id s a n d r in g c le a v a g e , m o r e t h a n 8 0 % o f t h e
a d d e d c a r b o n w o u l d h a v e e v o lv e d as 14C 0 2 f r o m t h e s o il [ 1 7 ] .
C o n i f e r y l a lc o h o l li n k e d i n t o m o d e l h u m i c a c id p o ly m e r s o r D H P a n d c o m s t a lk li g n i n s w e r e
m u c h le ss a v a ila b le t o t h e s o il o r g a n is m s . C e r t a in c a r b o n s , h o w e v e r , w e r e s t i l l m o r e r e a d i ly u t i l i z e d
th a n o th e rs . T h e c o n i f e r y l a lc o h o l , l i n k e d i n t o t h e m o d e l h u m i c a c id s m a d e f r o m a r e la t i v e l y
30 MARTIN and HAIDER

T A B L E V II. L O S S O F C A R B O N -1 4 A C T IV I T Y U P O N 6 N HC1
H Y D R O L Y S IS O F S O IL S A F T E R IN C U B A T IO N W IT H
S P E C I F I C A L L Y C A R B O N - 14 - L A B E L L E D C O N I F E R Y L A L C O H O L S
A N D L IG N IN S
The figures give percentage values o f the total activity remaining in
the soil after incubation

A ctivity lost upon 6N HC1 hydrolysis

Label
(%)

Free alcohols

0 14CH3 45

2-14C (sid e chain) 47

Ring-14C 38

DHP lignins

0 14CH3 35

2-14C (side chain) 26

Ring-14C 18

Corn stalk lignins

0 14CH3 37

2-i4C (sid e chain) 35

Ring-14C 27

c o m p l e x m i x t u r e o f p h e n o lic s u b s ta n c e s , w a s m o s t r e s is t a n t . A ft e r 2 8 w e e ks, o n ly a b o u t 6% o f
t h e r in g - a n d 2 - ( s id e c h a in ) c a r b o n s a n d 1 4 % o f t h e 0 14C H 3 c a r b o n s h a d e v o lv e d as l4 C 0 2 , a n d th e
b u l k o f t h e e v o lv e d C w a s lo s t d u r i n g t h e f i r s t 1 2 w e e k s . C o n i f e r y l a lc o h o l c a r b o n s li n k e d i n t o
lig n in s w e r e m o r e r e a d i ly a v a ila b le . A f t e r 2 8 w e e k s , 2 0 t o 3 0 % o f t h e r in g a n d 2 - s id e c h a in D H P
li g n i n c a r b o n s , a n d a b o u t 4 5 t o 5 0 % o f t h e O C H 3 c a r b o n s , h a d b e e n lo s t as C 0 2 , a n d d e g r a d a t io n
w a s c o n t i n u i n g a t a s te a d y b u t s lo w r a te . T h e p la n t l i g n i n d e c o m p o s e d a t a s l i g h t l y f a s t e r r a te
t h a n t h e D H P l i g n i n , w h i c h m a y b e e i t h e r r e la t e d t o t h e c lo s e a s s o c ia tio n o f t h e p la n t li g n i n w i t h
c e llu lo s e a n d o t h e r p l a n t p o l y m e r s o r t o a g r e a t e r a v a i la b ili t y o f t h e g ra s s li g n i n t y p e c o m p a r e d w i t h
t h a t f r o m c o n if e r s . I n t h e l i g n i n o f g ra s s e s , a ll t h r e e l i g n i n a lc o h o ls a re r e p r e s e n te d , w h e r e a s t h e
li g n i n o f c o n if e r s is p r i m a r i l y a p o l y m e r o f c o n i f e r y l a lc o h o l . T h e s e o b s e r v a t io n s s u g g e s t t h a t t h e
li g n i n , a lt h o u g h r e la t i v e l y r e s is t a n t t o m i c r o b i a l d e g r a d a t io n , w i l l c o n t in u e t o d e c o m p o s e s lo w ly ,
a n d t h a t g r e a t e r s t a b il iz a t io n o f t h e c o n s t it u e n t u n i t s o c c u r s w h e n t h e y a re l i n k e d i n t o t h e s o il
h u m i c m o le c u le s t h a t c o n t a in a m u c h g r e a t e r v a r ie t y o f c o n s t it u e n t u n i t s .
T h e b u l k o f t h e r e s id u a l c o n i f e r y l a lc o h o l c a r b o n s i n t h e lig n in s w a s r e c o v e r e d i n t h e h u m i c
a c id f r a c t i o n s . T h e s e o b s e r v a t io n s in d ic a t e t h a t b e f o r e l i g n i n h a s b e e n f u l l y d e c o m p o s e d o r
c o n v e r t e d t o h u m i c a c id i t w i l l b e s o lu b le i n d i l u t e N a O H s o lu t i o n s , a n d w i l l b e p r e c i p it a t e d w i t h
a c id s o t h a t t h e p r o c e d u r e , e s p e c ia lly f r o m s o ils r e c e n t ly a m e n d e d w i t h lig n a c e o u s r e s id u e s , w i l l
y i e l d h u m i c a c id t o g e t h e r w i t h p a r t l y d e c o m p o s e d li g n i n . T h e g r e a t e r u t i l i z a t i o n o f t h e O C H 3
g r o u p s b y t h e s o il o r g a n is m w a s r e f le c t e d i n a s m a lle r a m o u n t o f th e s e c a r b o n s i n t h e h u m i c a c id
a n d o t h e r s o il f r a c t i o n s w h e r e a s r in g c a r b o n s w e r e s o m e w h a t e n r ic h e d .
S o m e w h a t s m a lle r a m o u n t s o f t h e r e s id u a l r in g c a r b o n s o f t h e c o n i f e r y l a lc o h o ls w e r e lo s t
u p o n 6 N H C 1 h y d r o l y s is o f t h e i n c u b a t e d s o ils t h a n w e r e lo s t f r o m t h e O C H 3 a n d 2 - ( s id e c h a in )
c a rb o n s . T h is s u g g e s ts t h a t m o r e o f t h e l a t t e r l i g n i n fr a g m e n t s w e r e p r e s e n t i n m i c r o b i a l p r o d u c t s
s u c h as p r o t e in s a n d p o ly s a c c h a r id e s w h e r e a s m o r e o f t h e r in g c a r b o n s w e r e p r e s e n t i n t h e n o n -
 IAEA-SM-211/4 31

h y d r o l is a b l e p a r t . M a y a u d o n a n d B a t is t ic [ 2 9 ] r e p o r t e d f r o m s tu d ie s o n t h e b io lo g ic a l d e g r a d a t io n
o f u n i f o r m l y la b e lle d K la s o n ’s li g n i n t h a t t h is li g n i n d o e s n o t u n d e r g o a s e le c tiv e d e m e t h o x y la t i o n
d u r in g th e d e c a y p ro c e s s . H o w e v e r , K la s o n li g n i n is a h i g h l y d e n a t u r e d li g n i n w h i c h d o e s h o t
n e c e s s a r ily u n d e r g o t h e s a m e d e g r a d a t io n p ro c e s s e s as n a t u r a l l i g n i n p r e p a r a t io n s . G e n e r a l ly , s o il
h u m i c a c id s a re l o w i n m e t h o x y l g r o u p s [ 2 9 ] , a n d a ls o o n l y s m a ll a m o u n t s o f c o m p o u n d s a re
p r e s e n t w h i c h c a n b e d i r e c t l y tr a c e d b a c k t o li g n i n [ 3 0 , 3 1 ]. T h e p r e s e n t s t u d y a ls o s u g g e s ts t h a t
l i g n i n as s u c h , o r e v e n c h u n k s o f s t i l l h i g h l y m e t h o x y la t e d f r a g m e n t s , a re n o t s t a b iliz e d i n t h e
h u m ic c o m p o u n d s .
P u r e c u lt u r e s o f l i g n o l y t i c f u n g i , e s p e c ia lly w h i t e r o t f u n g i, c o m p l e t e ly m e t a b o liz e l i g n i n a n d
u s e i t as a s o u r c e o f c a r b o n a n d e n e r g y [ 9 , 3 2 ] . S im i la r o b s e r v a t io n s w e r e r e p o r t e d r e c e n t ly f o r
c e r t a in l i g n o l y t i c Fungi imperfecti is o la t e d f r o m s o il [ 6 , 8 ] . B r o w n r o t f u n g i, s e v e r a l f u n g i f r o m
a r a b le s o ils a n d s o m e b a c t e r ia [ 9 , T r o ja n o w s k i a n d H a id e r , u n p u b lis h e d ] e f f e c t i v e l y d e m e t h y la t e
l i g n i n , i n t r o d u c e a d d i t io n a l h y d r o x y l g r o u p s a n d in c re a s e t h e c a r b o n y l a n d c a r b o x y l c o n t e n t s
w h i c h le a d s t o a g r e a t e r s o l u b i l i t y [ 9 , 3 3 ] w i t h o u t a r a d ic a l d e g r a d a t io n o f t h e a r o m a t i c f r a m e w o r k .
O u r r e s u lt s i n d ic a t e t h a t s im i la r t y p e s o f r e a c t io n s m a y o c c u r a ls o d u r i n g t h e li g n i n d e g r a d a t io n i n
s o il. K i r k a n d c o - w o r k e r s [ 5 ] a ls o h a v e r e c e n t ly p r e p a r e d s p e c i f i c a l ly 14C T a b e lle d D H P lig n in s ,
a n d f o l l o w e d t h e re le a s e o f th e s e li g n i n c a r b o n s f r o m a f o r e s t s o il. D u r i n g a 2 5 - d a y i n c u b a t io n
p e r i o d , o n l y 4 t o 5 % o f t h e a p p lie d 14C w a s r e c o v e r e d as 14C 0 2 . I n o u r s tu d ie s , 7 % o f t h e 2 - s id e
c h a in a n d r i n g c a r b o n s a n d a b o u t 2 5 % o f t h e G C H 3- c a r b o n s w e r e r e le a s e d d u r i n g t h e f i r s t f o u r
w e e k s o f t h e 2 8 w e e k s ’ in c u b a t i o n p e r i o d . T h e s e d if f e r e n c e s c o u ld b e r e la t e d t o s o il p r o p e r t ie s
in c lu d in g t h e n a t u r e o f t h e m i c r o b i a l p o p u la t i o n . F u r t h e r te s ts a re n e e d e d u s in g a v a r ie t y o f s o ils
a n d w i t h v a r ia t i o n s i n t h e in c u b a t i o n c o n d i t io n s .

AC KNO W LEDG EM ENTS

T h e a u t h o r s t h a n k J .O . E r v in , H . L e m k e a n d E . P le is s f o r t e c h n ic a l a s s is ta n c e .

REFERENCES

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[3] SA R K A N EN , K .V ., LUDWIG, C.H. (E ds), Lignins, W iley-Interscience, N ew York (1 9 7 1 ) 1.
[4] D A G LEY , S „ Am . Sei. 6 3 ( 1 9 7 5 ) 68 1 .
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[6 ] H A ID ER , K ., DOMSCH, K .H ., Arch. M ikrobiol. 6 4 (1 9 6 9 ) 338.
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[9] KIRK, T .K ., “Chem istry o f lignin degradation b y w ood destroying fungi” , B iological T ransform ation o f
Wood b y Microorganisms (LIESE , W., Ed.), Springer-Verlag, Berlin, Heidelberg, N ew Y ork (1 9 7 5 ) 153.
[1 0 ] FELBECK, G .T ., Adv. Agron. 17 ( 1 9 6 5 ) 3 2 7 .
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substances” , Soil C om pon en ts 1: Organic C om ponents (GIESEKING, J.E ., E d .), Springer-Verlag, N ew York,
Heidelberg, Berlin (1 9 7 5 ) 1.
[1 2 ] H A ID ER , K., M ARTIN, J.P., FILIP, Z., “Hum us biochem istry” , Soil B iochem istry 4 (PA U L , E .A .,
M cLAREN, A .D ., Eds), Marcel D ekker, N ew York (1 9 7 5 ) 195. '
[1 3 ] M ARTIN, J.P., H A ID ER , K„ S oil Sei. 111 (1 9 7 1 ) 54.
[1 4 ] BO NDIETTI, E „ M ARTIN, J.P., H A ID ER , K ., S oil Sei. Soc. A m ., Proc. 3 6 (1 9 7 2 ) 5 97.
[1 5 ] KO NO NO VA , M.M., Sov. Soil S ei., N o. 7 (1 9 7 2 ) 27. j
[1 6 ] H A IDER , K., M ARTIN, J.P., S oil Sei. Soc. A m ., Proc. 39 (1 9 7 5 ) 6 5 7 .
[1 7 ] M ARTIN, J.P., H A ID ER , K„ Soil Sei. Soc. A m ., Proc. 4 0 ( 1 9 7 6 ) 3 7 7 . '
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[18] VERM A, L„ M ARTIN, J.P., H A ID ER , K ., Soil Sei. Soc. A m ., Proc. 39 (1 9 7 5 ) 2 79.

[19] FR EU D EN BER G , K., NIMZ, H„ Chem. C om m un. 1966 132.
[2 0 ] FR EU D EN BER G , K ., “T he con stitu tion and biosynthesis o f lignin” , C onstitution and B iosynthesis o f Lignin
(FR E U D E N B E R G , К ., NEISH, A .C ., Eds), Springer-Verlag, N ew Y ork, Heidelberg, Berlin (1 9 6 8 ) 1.
[21] NIMZ, H„ M OGHARAB, I., LÜDEM AN N , H .D ., M akromol. Chem. 175 (1 9 7 4 ) 2 5 6 3 .
[2 2 ] H A IDER , K .,J . Labelled Compd. 2 (1 9 6 6 ) 174.
[2 3 ] H A ID ER , K „ LIM, S., J. LabeUed Compd. 1 (1 9 6 5 ) 294.
[24] KR A TZL, K „ BILLEK, G., H olzforschung 7 (1 9 5 3 ) 66.
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[26] STEVENSO N , F.J., “ Gross chem ical fractionation of-organic m atter” , M ethods o f Soil A nalysis 2
(BLACK, C .A ., Ed.), Am. Soc. A gronom y, Madison (1 9 6 5 ) 1404.
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[2 8 ] ELLIS, D .H ., G R IFFITH S, D .A ., Can. J. M icrobiol. 2 0 ( 1 9 7 4 ) 1 379.
[2 9 ] M A Y AU D O N , J., BATISTIC, L., Ann. Inst. Pasteur 1 1 8 ( 1 9 7 0 ) 191.
[3 0 ] SCHNITZER, M., K H AN, S.U ., H um ic Substances in th e Environm ent, M. D ekker, N ew York (1 9 7 2 ).
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[3 2 ] W ILDUNG, R .E., CHESTER, G., BREM NER, D .E ., Plant Soil 3 2 (1 9 7 0 ) 2 2 1 .
[33] KIRK, T .K ., CHANG, H„ H olzforschung 28 (1 9 7 4 ) 217; ibid. 2 9 (1 9 7 5 ) 56.

D IS C U S S IO N

F. A N D R E U X : W h e n y o u c o m p a r e t h e d e c o m p o s it i o n o f c o n i f e r y l a lc o h o ls i n t r o d u c e d i n t o
t h e s o il e it h e r i n t h e f r e e s ta te o r i n c o m b in e d f o r m , i n s y n t h e t ic o r n a t u r a l p o ly m e r s , th e r e a re
s t r i k i n g d if f e r e n c e s i n t h e k in e t i c s o f C 0 2 re le a s e b e t w e e n t h e f i r s t case a n d t h e s e c o n d .
I w o u l d l i k e t o k n o w w h e t h e r t h e d e c re a s e i n t h e i n i t i a l r a te o f d e c o m p o s it i o n o f c o m b in e d
p r o d u c t s is d u e s o le ly t o t h e i r s t a b il iz a t io n b y r e s is t a n t c h e m ic a l b o n d s , o r w h e t h e r y o u h a v e
a ls o o b s e r v e d a d e p r e s s iv e o r t o x i c e f f e c t o f th e s e p o l y m e r s o n m i c r o b i a l d e v e lo p m e n t ,
e s p e c ia lly o n t o t a l s o il r e s p ir a t io n .
J .P . M A R T I N : N u m e r o u s te s ts h a v e in d ic a t e d t h a t i n t h e q u a n t i t ie s u s e d t h e p h e n o ls a n d
m o d e l p o l y m e r s d o n o t e x e r t a t o x i c e f f e c t o n t h e s o il m ic r o b io lo g ic a l a c t i v i t y . W h e n s u b s ta n c e s
s u c h as g lu c o s e a n d v a r io u s p o ly s a c c h a r id e s a re i n t i m a t e l y a s s o c ia te d w i t h t h e p o l y m e r s t h e y
d e c o m p o s e j u s t as f a s t i n t h e s o il as w h e n a d d e d a lo n e . T h e fig u r e s a t t h e b o t t o m o f T a b le I
r e p r e s e n t t h e a v e ra g e c a r b o n lo s t , a n d a ll th r e e c a n b e r e g a r d e d as r e p lic a t e s . T h e 4 0 % f ig u r e
f o r o n e w e e k a p p e a rs t o b e o u t o f lin e .
 IAEA-SM-211/41

TRANSFORMATION D’ACIDES PHENOLIQUES SIMPLES

EN SUBSTANCES PARA-HUMIQUES
PAR DES MICRO-ORGANISMES DU SOL
J .- R . B A I L L Y , V . N K U N D I K I J E - D E S S E A U X , K . A G B E K O
L a b o r a t o ir e d e p e d o lo g ie ,
U n i v e r s i t e P a u l S a b a tie r ,
T o u lo u s e ,
F ra n c e

Abstract-Rdsumd

TRAN SFO R M A TIO N OF SIMPLE PHENOLIC ACIDS INTO PARAHUM IC SUBSTANCES BY SOIL
MICRO-ORGANISMS.
During the cu ltivation o f soil m icroflora in selective culture media based on sim ple p henolic acids, the
occurrence o f brow n substances resem bling hum ic acids was observed. The in flu en ce o f certain culture conditions
was studied, such as th e nature and concentration o f the phenolic com p ou nd , aeration and so forth , on this effect.
S om e o f the data obtained from biochem ical analysis (hydrolysis and infra-red spectroscop y) confirm the
relationship betw een these brown substances and som e o f the natural hum ic acids. More especially, th e results
obtained by infra-red spectroscop y show that the brown substances obtained by cu ltivation d iffer from natural
hum ic acids on ly by the fact that th e bands for the -C H 2 - , -C H 3 groups substituted in the Ф rings are o f low er
in ten sity and th e fact that -O H alcohol I and alcohol II are present in addition to -O H alcohol III and/or phenol.
Lastly, the m icro-organisms responsible for the reaction were studied. T w en ty-tw o strains producing black
substances were isolated. A high proportion belonged to the genus P s e u d o m o n a s , probably because o f its m obility
and fast reproduction rate, but oth er m icrobial groups were present. On the w hole, th e soil micro-organisms can
produce from sim ple p henolic com pounds, under q uite norm al con d ition s, substances that can be termed
‘parahum ic’.

T R AN SFO R M A TIO N D’ACIDES PHENOLIQUES SIMPLES EN SUBSTANCES PARA-HUM IQUES PAR

DES MICRO-ORGANISMES DU SOL.
Au cours de cultures de m icroflore de sol sur des m ilieu x selectifs ä base d’acides phenoliques sim ples, on
a observt l’apparition de substances brunes ressem blant ä des acides hum iques. On a exam ine l’in flu en ce de
certaines con d ition s de culture (nature e t concentration du co m p o st p h tn o liq u e, aeration, e tc .) sur ce p htn om en e.
Certains resultats d’analyse biochim ique (h ydrolyses, spectroscopie i.r.) confirm ent la parente entre ces substances
brunes e t certains acides hum iques naturels. En particulier, les resultats ob ten us par spectroscopie i.r. m ontrent
que les substances brunes ob ten ues par culture ne different de ces acides hum iques naturels que par une m oindre
m ten site des bandes correspondant aux groupem ents -C H 2“ , “ CH 3 sub stitu te sur les cycles Ф et par la presence
de -O H alcool I e t II en plus des -O H alcool III et/o u p henol. Enfin, on a aborde l’etu d e des m icro-organism es
responsables de la reaction. V ingt-deux souches productrices de substances noires on t e t t isolees. U ne forte
proportion appartient au genre P s e u d o m o n a s , probablem ent ä cause de la m o b ilitt et de la rapidite de m ultiplication
de ces germes, mais d’autres groupes m icrobiens sont reprtsen tts. Au tota l, des m icro-organism es du sol peuvent,
dans des con d ition s qui n’on t rien d’excep tion n el, produire ä partir de com p oses phenoliques sim ples des
substances que Гоп peut appeler para-humiques.

IN T R O D U C T IO N

L ’ h y d r o l y s e d e s a c id e s h u m iq u e s lib e r e c e r te s d e p e t it e s q u a n t i t e s d ’ a c id e s a m in e s e t d e
g lu c id e s , m a is e ile lib e r e p r in c ip a le m e n t d e s c o m p o s e s p h e n o liq u e s [ 1 ] . L e s a c id e s h u m iq u e s
a p p a r a is s e n t d e p lu s e n p lu s c o n s t it u e s d e c o m p o s e s p h e n o liq u e s d iv e r s e m e n t c o n d e n s e s , p o ly m e r is e s
et oxydes.

33
34 BAILLY et al.

C ’ e s t p o u r q u o i o n a e x a m in e i c i la p o s s i b ili t y d e n e o - f o r m a t i o n d e c o m p o s e s h u m iq u e s ä
p a r t i r d ’ a c id e s p h e n o liq u e s s im p le s ( m o n o m e r e s ) p a r d e s m ic r o - o r g a n is m e s d u s o l.

L e s p r e m ie r e s c u lt u r e s a y a n t a b o u t i ä la f o r m a t i o n d e s u b s ta n c e s b r u n e s , o n a s u c c e s s iv e m e n t:
- p r e c is e c e r ta in e s c o n d i t io n s p h y s ic o - c h im iq u e s d u b r u n is s e m e n t ,
- a b o r d c l ’ e t u d e b io c h im iq u e d e s s u b s ta n c e s b r u n e s o b t e n u e s ,
- is o le e t c t u d ie q u e lq u e s m ic r o - o r g a n is m e s c a p a b le s d e p r o d u ir e ces r e a c t io n s .

1. F O R M A T IO N D E S U B S T A N C E S B R U N E S P A R L E S M IC R O - O R G A N IS M E S D U S O L

O n a e x a m in e le s d e u x s e rie s d ’ a c id e s p h e n o liq u e s s im p le s s u iv a n te s :

S e rie C 6- C , S e rie C 6- C 3

A c id e c in n a m iq u e
A c id e o - O H - b e n z o lq u e A c id e o - O H - c in n a m iq u e
A c id e m - O H - b e n z o 'iq u e A c id e m - O H - c in n a m iq u e
A c id e p - O H - b e n z o i'q u e A c id e p - O H - c in n a m iq u e
A c id e 3 ,5 d i- O H - b e n z o lq u e

O n r e a lis e le m il ie u d e c u lt u r e c i-d e s s o u s , d e p H = 7 ,0 , q u i , e x c e p t e le c o m p o s e p h e n o liq u e ,

e s f u n iq u e m e n t m in e r a l:

S o lu t i o n s a lin e s ta n d a r d d e W in o g r a d s k y [ 2 ] 50 m l
S o lu t i o n d ’ o lig o - e le m e n t s 2 m l
N itr a te d ’ a m m o n iu m 2 g
C o m p o s e p h e n o liq u e en g e n e ra l 3 g
E a u d is t i ll d e — q u a n t i t e s ü f f is a n t e p o u r 1000 m l

L e m il ie u s a n s l ’ a c id e p h e n o liq u e e s t n e u t r a lis e p u is s t e r ilis e ä l ’ a u t o c la v e . L ’ a c id e p h e n o liq u e

m is e n s o l u t i o n e t n e u t r a lis e e s t s t e r ilis e p a r f i l t r a t i o n e t a jo u t e a s e p t iq u e m e n t . C e m il ie u p e u t ,
e v e n t u e lle m e n t , e t r e g e lo s e ä 1 5 g/1.
L e c o m p o s e p h e n o liq u e c o n s t it u e la s e u le s o u r c e ä la f o is d e c a r b o n e e t d ’ e n e r g ie ( c u lt u r e s
s e le c tiv e s s e lo n W i n o g r a d s k y [ 3 ] e t P o c h o n [ 4 ] ) .
C om m e inoculum, o n a u t il is e d ’ u n e p a r t l ’ h o r i z o n A , d ’ u n s o l f o r e s t ie r a c id e d e v e lo p p e
s u r te r r a s s e a llu v ia le e t la l i t i e r e v e g e ta le c o r r e s p o n d a n t e ( f o r e t ä d o m in a n c e d e Quercus pubescens ) ,
d ’ a u t r e p a r t u n s o l n e u t r e c u lt i v e s u r m o la s s e e t u n e l i t ie r e f o r e s t ie r e p r o c h e ( d o m in a n c eQuercus
pubescens) t o u j o u r s s u r m o la s s e , s o i t e n t o u t q u a t r e e c h a n t illo n s .
L e s m i l i e u x r e p a r t is e n f i o l e s d e R o u x o u d ’ E r le n m e y e r ( 3 0 0 m l / f i o l e ) s o n t e n s e m e n c e s a
p a r t i r d e 1 g d e s o l o u d e l i t i e r e f o r e s t ie r e ; c e la c o n s t it u e la c u lt u r e I. A p r e s d e v e lo p p e m e n t
m i c r o b ie n e t d is p a r it i o n d e la p lu s g r a n d e p a r t d u c o m p o s e p h e n o liq u e ( d o s a g e s e lo n F o li n -
C io c u lt e u [ 5 ] m o d i f i e p a r M o n t r e a u [ 6 ] , o n r e p i q u e s u r le m e m e m il ie u e t d a n s le s m e m e s c o n d i t io n s
u n e p r e m ie r e f o is ( c u l t u r e I I ) , p u is u n e s e c o n d e f o is ( c u lt u r e I I I ) a f i n d ’ f i lim i n e r le s s u b s ta n c e s
a p p o r te e s p a r Г inoculum i n i t i a l .
S a u f ca s p a r t ic u li e r , t o u t e s le s c u lt u r e s s o n t re a lis e e s ä 3 0 ° C . T o u s le s r e s u lt a t s e t d is c u s s io n s
u lt e r i e u r s p o r t e r o n t u n i q u e m e n t s u r le s c u lt u r e s I I I p r e c e d e m m e n t d e f in ie s .
A p r e s u n te m p s d e c u lt u r e d e 2 ä 5 jo u r s , o n c o n s t a t e :
— u n e m u l t i p l i c a t i o n m ic r o b ie n n e ,
— u n e c o l o r a t i o n m a r r o n p lu s o u m o in s in te n s e , m a is t o u jo u r s b e a u c o u p p lu s f o r t e e t p lu s
r a p i d e m e n t a p p a r u e q u e c e lle q u e Г о п p e u t o b s e r v e r d a n s le s t 6 m o in s n o n e n s e m e n c 6 s ,
— la d is p a r it i o n , o u d u m o in s la f o r t e d i m i n u t i o n , d e la t e n e u r e n c o m p o s e s p h e n o liq u e s [ 5 , 6 ] .
 IAEA-SM-211/41 35

On doit done admettre que l’acide phenolique sert a la fois au developpement microbien
et a la formation des substances brunes.
Un certain nombre de parametres peuvent influer sur les resultats:
— La nature du compose phenolique: l’acide o-OH-benzoique et l’acide o-OH-cinnamique
sont, parmi les composes examines, ceux qui donnent generalement la coloration la plus intense;
e’est pourquoi on les a beaucoup utilises par la suite; des essais paralleles ont montre que la
tyrosine donne aussi des resultats interessante.
— La concentration du compose phenolique: dans l’intervalle 1—6 g/1 d’acide o-OH-benzoique
ou d’acide o-OH-cinnamique, plus la concentration initiale est elevde, plus la diminution de cette
concentration est lente.
— L’apport d’eau de levure (10 ml/1) accelere le brunissement qui se produit 2 ä 3 jours
plus tot, mais ne modifie pas la coloration finalement obtenue.
— Dans l’intervalle 20—50°C, la temperature la plus favorable au developpement microbien
et au brunissement est d’environ 30°C, ce qui confirme bien l’importance du phenomene biologique,
puisqu’une oxydation chimique serait plus intense ä 50°C.

Mais un p h en o m en e sem b le p a rticu lierem en t interessant: Pour etudier l’influence de l’aeration,

au moins d’une maniere qualitative sommaire, on a realise des cultures dans trois conditions
differentes, toutes autres choses etant identiques:
Condition 1: 6paisseur du milieu = 7 cm, culture non agit6e
Condition 2: epaisseur du milieu = 2 cm, culture non agitee
Condition 3: epaisseur du milieu = 2 cm, culture agitee (80 mouvements/min).
On constate que la condition 2, correspondant ä une aeration moderee, permet d’obtenir la
teinte la plus intense.
II semble que cela doive etre mis en relation avec le fait [7] que dans l’oxydation des composes
phenoliques, Poxygene intervient non seulement comme accepteur d’electrons, mais aussi comme
substrat enzymatique. Et, en presence d’une grande quantite d’oxygene, les composes phenoliques
sont en grande partie oxydes jusqu’au terme ultime.
Par ailleurs, des numerations microbiennes, realisees aussi bien par nephelometrie que par
culture sur agar-agar, ont montre que le developpement microbien 6tait plus intense dans les
cultures agitees, oh le brunissement est faible.
Le brunissement maximal semble done lie a une aeration moderee, en correlation avec un
developpement microbien egalement modere.

2. ETUDE BIOCHIMIQUE DES SUBSTANCES BRUNES PRODUITES

2.1. Purification

Le schema de purification est presente ä la figure 1; on peut voir que les substances brunes
ne dialysent pas: ce sont done de grosses molecules; on a constate par ailleurs qu’elles ргё-
cipitent en milieu acide.

2.2. Hydrolyses

Le tableau I indique les echantillons trait es.

On a effectue des hydrolyses acides selon Bohm et Towers [8] (HCl 2N, 1 h, 100°C) qui
attaquent les liaisons ether-oxydes, et des hydrolyses alcalines [8, 9] (OHNa 2N, 12 h, temperature
ambiante, atmosphere d’azote) qui attaquent la liaison ester.
Les composes phenoliques liberes sont extraits par Tether ethylique en milieu acide [10],
separes par Chromatographie en couche mince [ 11 ] et identifies ä la fois par leur Rf (position
36 BAILLY et al.

CULTURE BRUNE

F I G .l. P u r ific a tio n d e s s u b s ta n c e s b r u n e s fo r m e e s d a n s d e s c u ltu r e s m ic r o b ie n n e s .

relative sur le Chromatogramme) et diverses reactions colorees [12]. Le tableau II indique les
resultats obtenus.
Alors que les micro-organismes n’ont initialement ä leur disposition qu’un seul compose
phenolique, on retrouve dans Thydrolysat plusieurs monomeres differents. Sous Taction des micro-
organismes, se produisent done des reactions qui ne sont pas une simple polymerisation du
compose initial:
— Des echantillons, pourtant assez proches par leurs conditions de formation, ont donne par
hydrolyse des listes differentes de monomeres; les conditions experimentales imposees laissent
done encore aux micro-organismes quelques degres de liberte; plus exactement, la selection des
micro-organismes par ces milieux pourtant assez rigoureux doit permettre la survie de micro-
organismes divers, capables done de produire des substances brunes quelque peu differentes.
— Les composes phenoliques liberes par hydrolyse des matieres brunes sont assez semblables
ä ceux que Ton obtient par hydrolyse d’acides humiques naturels [ 1]. Les micro-organismes du
sol sont done capables ä partir d’un acide phenolique de produire des matieres brunes qui, par ce
caractere, sont apparentees aux acides humiques naturels.
 IAEA-SM-211/41 37

TABLEAU I. CONDITIONS D’OBTENTION DES SUBSTANCES BRÜNES ET

ANALYSES EFFECTUEES

A nalyses effec tu ee s
S u b stan ce
E c h an tillo n ca rb o n e e In o cu lu m Eau de levure H y d ro ly se H y d ro ly se S p ectro sco p ie
initiale acide alcaline i.r.

c A cide A i, sol avec + +

o-O H -benzoique fo restier
2 s/1 acide

D A cide A b sol avec + + +

o-O H -benzo'ique fo re s tie r
2 g/1 acide
E A cide A b sol sans + + +
o-O H -benzoique fo restier
2 g/1 acide

F A cide L itie re A q, avec + + +

o-O H -cinnam ique sol fo restier
2 g/1 acide

TABLEAU II. COMPOSES PHENOLIQUES MIS EN EVIDENCE

DANS LES HYDROLYSATS DES SUBSTANCES BRUNES

E chan tillo n
C om pose p h en o liq u e c D E F

A cide o-O H -benzoique X

A cide p -O H -benzoique + + +

A cide 3 ,4 di-O H -benzoique X +

A cide 2,5 di-O H -benzoique +
H y d ro q u in o n e X X

P yrogallol + +
G uaiacol +
D im e th y lp h e n o l n o n precise + X X + +
C orps n o n identifi6s 1 3 2 2

x: H y drolyse alcaline ä fro id

• : H y d ro ly se acide ä ch a u d

2.3. Spectroscopie infrarouge

On a utilise la technique des pastilles de KBr et un spectrophotometre UNICAM SP 200 G

ä rdseau.
La figure 2 presente les spectres des substances brunes obtenues par des cultures microbiennes
et les spectres de la fraction «molecules moyennes» d’acides humiques naturels fractionnes sur
gel de dextrane [13, 14].
38 BAILLY et al.

ГУ.

F I G .2 . S p e c t r e s I r . d 'a c id e s h u m i q u e s n a tu r e ls d e d im e n s io n s m o le c u la ir e s «m o y e n n e s » [1 3 , 1 4 ] e t d e s u b s ta n c e s

b r u n e s o b te n u e s p a r c u ltu r e s s u r a c id e p h e n o liq u e . 1: a c id e s h u m iq u e s e x tr a i ts d 'u n s o l g r is f o r e s tie r 1 6 - 2 9 cm ,

2: a c id e s h u m iq u e s e x tr a i ts d 'u n tc h e r n o z io m e 2 0 - 3 5 cm , D , E : c u ltu r e s s u r a c id e o -O H -b e n z o iq u e (v o ir

ta b le a u I ), F : c u l t u r e s u r a c i d e o - O H - c i n n a m i q u e f v o i r ta b l e a u 1 ). L e s s p e c tr e s s o n t e n r e g is tr e s e n p o u r c e n ta g e

d e lu m ie r e tr a n s m is e .

R essem blan ces [ 14]

— La bande 3400 cm"1correspond aux -OH lies constituant de longues series.

— Le pic 1640 cm"1 correspond aux doubles liaisons C = C (probablement benzeniques)et C= О
(cetonique et -COO"), avec l’epaulement П40 cm“1 des -COOH.

D ifferen ces

— La bande 1000—1100 cm"1 oil se trouve, vers 1035 cm“1, la bande C-O-C, est moins intense
chez les substances brunes provenant de cultures, et ой agissent les groupements substitues sur les
cycles. Mais il faut rappeier ä ce propos que les echantillons D et E sont formes ä partir d’acide
o-OH-benzolque, dcpourvu de tels groupements.
— Les acides humiques naturels absorbent vers 1400 cm“1, et non vers 1300 cm"1, ce qui
indique des - OH alcool III et/ou phenol. Par contre, les substances brunes absorbent aussi a
1300 cm"1, ce qui indique des - OH alcool I et II en plus des - OH alcool III et/ou phenol.
— Par la spectroscopie i.r., les substances brunes extraites des cultures ne different des acides
humiques naturels de «dimensions moyennes» que par une moindre teneur en -CH, -CH2- , -CH3,
 IAEA-SM-211/41 39

TABLEAU III. SOUCHES MICROBIENNES PRODUCTRICES DE SUBSTANCES

PARA-HUMIQUES

C om pos6 p h 6 n o liq u e su r
S o u ch e G enre
le q u el la so u ch e a 6te isolee

'B S 2 P seu d o m o n a s A cide o -O H -benzoique

■ BS02 P seudom onas A cide o -O H -benzoique

,G D 5 P seudom onas A cide o -O H -benzoique

BS4 P seu d o m o n a s A cide o -O H -benzoique

BS5 A c h r o m o b a c te r A cide o -O H -cinnam ique

JB S6 P seu d o m o n a s A cide o -O H -cinnam ique

1 b S06 P seu d o m o n a s A cide o -O H -cinnam ique

BS8 P seu d o m o n a s A cide 3,5-d i-O H -b en zo iq u e =

A cide a-r£ so rcy liq u e
JB S 1 0 P seudom onas T y ro sin e

I bsh P seudom onas A cide o -O H -cinnam ique

rG B 6 P seudom onas A cide o -O H -cinnam ique

GSB5 P seu d o m o n a s A cide o -O H -cinnam ique

G SB 05 P seu d o m o n a s A cide o -O H -cinnam ique

GC5 P seu d o m o n a s A cide o-O H -cin n am iq u e

G L3 P seudom onas A cide o -O H -cinnam ique
^G SC5 P seudom onas A cide o -O H -benzoique
G SA 5 P seu d o m o n a s A cide o -O H -cinnam ique
BS3 A c h r o m o b a c te r A cide o-O H -cinnam ique
■ BA4 A c h r o m o b a c te r A cide o -O H -benzoique
.BB5 A c h r o m o b a c te r A cide o -O H -benzoique
BSOl N o c a r d ia A cide o -O H -benzoique
BS7 F u n g i im p e r fe c ti A cide o -O H -benzoique

peut-etre due au choix des substances initiales, d’une part, et la presence des - OH alcooU et II
en plus des - OH alcool III et/ou phenol d’autre part.
Compte tenu des resultats de la spectroscopie i.r. et des hydrolyses et aussi d’autres resultats
en spectroscopie u.v. et en electrophorese [15], les substances brunes obtenues par culture paraissent
bien meriter l’appellation de substances para-hum iques.

3. ETUDE DES MICRO-ORGANISMES PRODUCTEURS DE SUBSTANCES PARA-HUMIQUES

A partir des cultures en milieu liquide produisant des substances para-humiques, on a isole, par
les techniques classiques (dilution, epuisement, etc.) un certain nombre de souches microbiennes
que Ton a cherche ä situer dans la classification [16].
Le tableau III presente les resultats obtenus. Les accolades regroupent les souches qui, quoique
provenant de travaux d’isolement distincts, ont paru semblables pour les caracteres examines.
Bien entendu, il s’agit lä uniquement de souches qui ont conserve, apres isolement, la
propriete de former des substances brunes ä partir de l’acide phenolique sur lequel elles s’etaient
d£velopp6es.
40 BAILLY et al.

De plus, on n’a ici que des souches pures, ce qui climine d’eventuelles associations micro-
biennes. Enfin, ce travail a ete mene uniquement sur agar-agar, et Гоп peut penser que 1’emploi
de silico-gels aurait permis l’isolement d’autres micro-organismes.
Un grand nombre de souches isolees peuvent etre attribuees aux genres P seudom onas et
A c h ro m o b a c te r; ces micro-organismes sont mobiles par des flagelles et ont un «temps de generation»
assez bref. II est done probable que Гоп a isole preferentiellement les germes les plus mobiles et
qui proliferaient le plus dans les milieux de culture. II reste cependant que de tels germes, presents
dans le sol, ä multiplication rapide, ä forte mobilitc, sont capables, ä partir de composes phenoliques
simples, de synthetiser des substances para-humiques.
Par ailleurs, le tableau III montre qu’un compose phenolique peut donner des substances
brunes sous Taction de souches nettement differentes. A l’inverse, des souches ayant les memes
caracteres (par exemple GL3 et GSC5) ont ete isolees sur deux composes differents. De plus les
souches BS5 et BS6, isolees sur acide o-OH-cinnamique et repiquees sur acide o-OH-benzoique,
continuent, quoique plus lentement, ä у produire des substances brunes.
Bien qu’on ne puisse encore generalise^ il semble done qu’il n’y a pas une grande specificite
des souches microbiennes pour les divers acides phenoliques.
Enfin, la presence dans la liste de Nocardia, actinomycetine, et de Fungi im p erfecti montre
que les eubacteries ne sont pas les seuls germes susceptibles de produire ces reactions. Des germes
appartenant ä d’autres groupes de la classification, peut-etre moins faciles ä mettre en evidence,
peuvent donner un resultat semblable.

CONCLUSION

Lorsqu’on cultive une microflore de sol avec comme seule source de carbone un acide
phenolique, dans des conditions ad equates et qui n’ont den d’exceptionnel, il у a, sous faction
des micro-organismes, formation de substances brunes meritant l’appellation de substances
para-humiques. Parmi les germes responsables il у a une certaine diversity, et on a mis particuliere-
ment en evidence des P seudom onas et des A ch rom obacter, germes particulierement mobiles et
qui se multiplient rapidement.
Cela nous parait etre un resultat important qui s’accorde, entre autres, avec le point de vue
de Mangenot [17] qui ecrit: «N ou s pen son s qu e la p o lym erisa tio n b iologiqu e e st un even em en t
p ro b a b le dans un so l a c tif ».

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P lan t S oil, sous presse.
[1 5 ] B A IL L Y , J.-R ., N K U N D IK IJE -D E S SE A U X , V ., S u r la fo rm a tio n de su b stan ce s n o ire s ä p a rtir de p h en o ls
sim ples p a r des m icro-organism es du sol, P la n t Soil 4 3 (1 9 7 5 ) 2 3 5 —57.
[1 6 ] B R E E D , S .R ., M U R R A Y , E .G .D ., SM ITH , R .N ., e t al., Bergey’s M anual o f D ete rm in ativ e B acterio lo g y ,
7 th E d ., W illiam s an d W ilkins C o m p a n y , B altim o re (1 9 5 7 ).
[1 7 ] M A N G EN O T , F ., «O n th e o x id a tio n o f p h en o lic co m p o u n d s by b ac teria and sy n th esis o f p a ra h u m ic
c o m p o u n d s» , T rans. In t. S ym p. H u m u s e t P lan ta V , S tudies a b o u t h u m u s, P rague, 13—17 S ep t. 1 9 7 1 , 3 7 —42.

DISCUSSION

F. ANDREUX: You have used spectra of salified compounds, which on the one hand causes
you to lose information on the intensity of the bands for the -COOH group, and on the other
obscures the peak at 1610-1640 cm"1 which is characteristic of the conjugate double bonds
C=C or C=0. Have you tried to make these spectroscopic comparisons more accurate by
decationizing beforehand, for instance, with an IF resin?
J.-R. BAILLY: I have been able to remove the ions from these brown substances only very
imperfectly by dialysis. Decationization on resin would require larger amounts of material. We
are working on the production of the brown substances with a fermentor. If we obtain 10 litres
of culture in that way instead of 200 ml I think we could try the treatment you suggested.
H.W. SCHARPENSEEL: We usually correlate the hue (spectral regions) of the Munsell colour
charts primarily with the presence of iron oxides (10 YR, 7.5 YR with goethite, 2.5 YR, 10 R
with haematite, etc.). The effect of the organic matter is more manifest in the categories of value
and chroma. Can your characterization of the humic compounds by spectral region be considered
sufficiently reproducible?
J.-R. BAILLY: Such a correlation would be significant only for a given concentration of
brown substances and for a known liquid thickness. That is impossible at present, as I do not
have enough brown substances. The Munsell colour plates shown on the slides beside the microbial
cultures were only intended to correct any distortion of the colours by the photograph.
W. FLAIG ( S cien tific S ecretary): You indicate in your paper that you isolated 22 strains
which form dark-coloured substances. Is it known whether any of these strains synthesize
phenolic compounds by metabolism?
J.-R. BAILLY: I was referring to 22 isolates, some of which may belong to the same strain.
I do not know whether brown substances are synthesized within the microbe cell or outside it
under the influence of exo-enzymes, but the browning is connected with the microbes’ development,
and phenolic acid is the only organic compound present.
 IAEA-SM-211/42

CARACTERISATION ET TRANSFORMATIONS EN
MILIEU MULL D’UN MODELE HUMIQUE ISSU DE
L’AUTOXYDATION DU SYSTEME CATECHOL-GLYCINE
ET MARQUE SELECTIVEMENT AU CARBONE-14

F. ANDREUX*, Dorota GOLEBIOWSKA**, Therese CHONE*,

F. JACQUIN+, M. METCHE*

* CNRS, Centre de pedologic biologique,

Vandoeuvre-les-Nancy,
France

** Akademia Rolnicza,
Zaklad Fizyki,
Szczecin,
Pologne

+ Institut national polytechnique de Lorraine,

Ecole nationale superieure d’agronomie
et des industries alimentaires,
Nancy,
France

Abstract-Е ёзи тё

D E T E R M IN A T IO N O F T H E C H A R A C T E R IST IC S A N D T R A N SF O R M A T IO N S IN A M U L L M ED IU M O F A
H U M IC M O D E L D E R IV E D F R O M A U T O -O X ID A T IO N O F T H E C A T E C H O L -G L Y C IN E SY STEM A N D
S E L E C T IV E L Y L A B E L L E D W ITH C A R B O N -14.
B ro w n n itro g e n -c o n ta in in g p o ly m ers are p rep are d b y a u to -o x id a tio n a t p H 7 .9 fro m a n eq u im o la r m ix tu re o f
c a te c h o l a n d glycine. S tric t c o n tro l o f th e e x p e rim e n ta l c o n d itio n s y ie ld s p ro d u c ts o f c o n s ta n t c o m p o sitio n , an d
a ffo rd s a basis fo r th e selective p re p a ra tio n o f th re e p o ly m ers labelled w ith 14C o n th e a to m s C , a n d C ; o f th e g ly cin e
a n d o n th e c a te c h o l ring. T h e se p o ly m e rs have a m e a n m olecu lar size sm aller th a n th a t o f h u m ic acid s e x tra c te d
fro m n e u tra l soils b u t th e ir s tru c tu re resem b les m o re th a t o f h y d ro ly tic resid u e s fro m e u tro p h ic h u m ic acid s an d
m elan in s. T h e ir s to ic h io m e tric ra tio is 2 m oles o f gly cin e t o 7 m o les o f ca te c h o l. T h e y are n o t c o m p le te ly h o m o ­
g en e o u s a n d c o n ta in tw o g ro u p s o f su b stan ce s: th e first a n d m o re a b u n d a n t g ro u p is larger an d p e rm its o p tim u m
sta b iliz a tio n o f th e g ly cin e in h ig h ly re s o n a n t s tru c tu re s ; th e seco n d g ro u p rem ain s m o re sensitive to h y d ro ly tic
tr e a tm e n t an d b io d e g ra d a tio n ; th e y are ch a ra cte rized b y p a rtia l fission o f th e a ro m a tic rin g s d u rin g sy n th esis, w h ich
is re sp o n sib le f o r th e fo rm a tio n o f th e ring-deriving -C O O H gro u p s.T h e b io d e g ra d a tio n o f th e se p o ly m ers in a n e u tra l m u ll
reveals tw o successive stages: (a) a n in itia l stage lasting five days, d u rin g w h ic h th e ring-deriving -C O O H g ro u p s are
a c tiv ely d e c a rb o x y la te d , w h ereas th e g ly cin e b o u n d to th e less stab le fra c tio n is d eg rad ed p re fe re n tia lly a n d th e n
m e tab o lized in th e u n e x tra c ta b le m icro b ia l su b stan ce s (m icro b ial h u m in ). T h e n o n -am in o resid u e s o f th e p o ly m ers
th e n h av e a d ep ressan t e ffe c t o n soil re s p ira tio n , w h ic h disappears o n ly w h en th e ir tra n s fo rm a tio n b y d e g ra d a tio n o r
re a c tio n w ith th e soil o rg an ic c o m p o u n d s o cc u rs; (b ) A second stage, ch a ra cte rized b y v ery slow m in eralizatio n and
affe c tin g a lm o s t exclusively th e m o re s tab le p o ly m ers in c o rp o ra te d in to th e h u m in b y p h y sico -c h em ic al in s o lu b iliz atio n .
T h e re is th e n n o f u r th e r p re fe re n tia l d e g ra d a tio n o f th e glycine, th e s ta b ility o f w h ich eq u als th a t o f th e ring-deriving
c a rb o n c o m p o u n d s o f a ro m a tic origin. T h e s tu d y show s th a t th e in c o rp o ra tio n o f am in o -acid resid u e s in to p o ly m ers
o rig in atin g fro m q u in o n e stru c tu re s re p re s e n ts a n im p o rta n t fo rm o f storag e f o r org an ic soil n itro g en .

C A R A C T E R IS A T IO N E T T R A N S F O R M A T IO N S EN M ILIEU M U L L D ’U N M O D E L E H U M IQ U E ISSU D E
L ’A U T O X Y D A T IO N D U S Y STE M E C A T E C H O L -G L Y C IN E E T M A R Q U E SE L E C T IV E M E N T A U C A R B O N E-14.
D es p o ly m eres b ru n s az o tes s o n t p re p a re s p a r a u to x y d a tio n ä p H 7 ,9 d ’u n m elange eq u im o le cu laire de ca tech o l
e t de g lycine. U n c o n trö le rig o u re u x des c o n d itio n s ex p e rim en tale s c o n d u it ä des p ro d u its d e c o m p o sitio n c o n s ta n te ,

43
44 ANDREUX et а].

e t au to rise la p re p a ra tio n de tro is p o ly m eres s£lectivem ent m a rq u es au 14C su r les ato m es d e ca rb o n e C i e t C 2 d e la

glycine e t su r le cycle d u ca tech o l. Ces p o ly m eres p re s e n te rs u n en c o m b re m e n t m o lecu laire m o y e n in fe rieu r ä celui
des acides h u m iq u e s e x tra its des sols e u tro p h e s , e t le u r s tru c tu re se rap p ro c h e davantage d e celle des residus
d’h y d ro ly se des acides h u m iq u es n a tu re ls e t des m elanines. L eur ra p p o rt sto ec h io m e triq u e est d e 2 m oles de glycine
p o u r 7 m oles de ca tech o l. Ils n e s o n t pas to ta le m e n t h o m o g e n es e t c o m p o rte n t d eu x g ro u p es de su b stan ces; les
p rem ieres, p lu s a b o n d a n te s , s o n t de plus g rande dim ension e t p e r m e tte n t u n e stab ilisatio n o p tim ale de la glycine dans
des s tru c tu re s h a u te m e n t re so n n an tes; les secondes d em e u re n t plu s sensibles au x tra ite m e n ts h y d ro ly tiq u e s e t ä la
b io d e g ra d a tio n ; elles se ca ra c te rise n t p a r u n e o u v e rtu re p artielle des n o y a u x a ro m atiq u es au co u rs de la s y n th ese, q ui
est resp o n sab le de la fo rm a tio n de g ro u p e m e n ts -C O O H m atriciels. L a b io d e g rad atio n d e ces p o ly m eres d an s u n
m u ll e u tro p h e m e t e n evidence d e u x S tapes successives: a) U ne e tap e p rim aire de 5 jo u rs , au co u rs de laq u elle les
g ro u p e m e n ts -C O O H m a tric iels s o n t a c tiv em en t d ec arb o x y les, ta n d is q ue la glycine lice ä la fra c tio n la m o in s stab le
est dcgradee p re fe re n tic lle m e n t, p u is m etabolisS e dans les co rp s m icro b ie n s in e x tra c tib le s (h u m in e m icro b ie n n e). Les
residue n o n am ines des p o ly m eres e x e rc e n t alors u n e ffe t d ep ressif su r la re sp ira tio n d u sol, q ui ne d isp ara ft q u ’avec
le u r tra n sfo rm a tio n p a r d eg rad a tio n o u re a c tio n avec les com poses o rg an iq u es d u sol. b ) U ne e ta p e seco n d aire,
caractcrisee p a r u n e m in eralisatio n tre s le n te q u i affe c te p resq u e ex clu siv em en t les p o ly m eres les p lu s stables
in c o rp o res dans l’h u m in e p a r in so lu b ilisatio n phy sico -ch im iq u e. II n ’y a p lu s alors de d eg rad a tio n p refere n tie lle de
la glycine, d o n t la sta b ilite est dgale a celle des com poses c a rb o n e s m atriciels d ’origine a ro m a tiq u e . C ette e tu d e
m o n tre q u e l’in c o rp o ra tio n des rdsidus am ino-acides dans des p o ly m eres d eriv an t de s tru c tu re s q u in o n iq u es c o n s titu e
u n e fo rm e im p o rta n te de sto ck ag e de l’az o te org an iq u e des sols.

1. INTRODUCTION

La stabilisation de l’azote organique dans les composes humiques des sols atteint des degres
divers en fonction du type de structure chimique dans lequel il est engage. Les composes
hydrolysables en milieu chlorhydrique ä chaud, les plus abondants, correspondent ä Tammoniaque,
aux amino-acides, aux amino-sucres, ainsi qu’ä des formes combinces solubles difficilement identi-
fiables [1—6]. L’azote non hydrolysable, abusivement appele parfois «azote heterocycle» correspond
le plus souvent ä des structures complexes provenant de l’interaction entre ces molecules simples et
d’autres composes. Maillard [7] fut l’un des premiers ä montrer que les amino-acides donnaient lieu
ä des condensations avec les sucres pour former des composes noirs, les mclanoi'dines, qu’il comparait
d£ja aux extraits de terreau et de furnier; de faqon generale, tous les aldehydes et les cetones peuvent
participer ä des reactions analogues [8]. Dans les sols la plus grande partie des polycondensats azotes
trouvent leur origine dans la polymerisation oxydative des polyphenols liberes par degradation de la
lignine, des tanins, ou des pigments vegetaux ou microbiens. Sur les sites electrophiles des noyaux
Ortho- ou Paraquinoniques ainsi form6s peuvent alors se fixer les groupements amines, en particulier
ceux des amino-acides N-terminaux des polypeptides [6, 9-11]. Ces reactions, apparentees aux
ph6nomenes de tanage, sont extremement gen6rales et sont egalement avancees pour expliquer la
biosynthese des melanines [12—18]. Lorsque de tels composes sont soumis ä une hydrolyse energique,
seule leur partie «peripherique» se trouve scindee au niveau des liaisons peptidiques, tandis que sub­
sisted les residus amino-acides directement lies ä la matrice polycondensee. La structure chimique
de ces combinaisons est souvent difficile ä elucider, en raison du nombre eleve de leurs precurseurs
phenoliques et des rearrangements profonds qu’elles subissent au cours de la pedogencse. De plus,
leur isolement est souvent malaise car elles peuvent etre modifiees par les reactifs d’extraction. C’est
pourquoi on fait souvent appel ä des systemes modeles: les modeles naturels, en particulier les
melanines fongiques ou les phytomelanines, possedent souvent une structure encore trop complexe,
et il semble qu’on puisse avantageusement leur substituer des modeles synthetiques obtenus en
conditions controlees. Le modele le plus courant correspond au Systeme catechol-glycine; les
mecanismes qui regissent sa preparation sont ä l’heure actuelle bien connus, tant en ce qui conceme
l’oxydation du catechol en quinone [19, 20], que la formation de produits d’addition glycine plus
catechol [21, 22]. Les reactions connexes de degradation oxydative de la glycine, avec formation
d’ammoniaque et d’acide glyoxylique, ont egalement ete etudiees et sont encore sujettes ä differentes
interpretations [21, 22]. Enfin, l’etude des polymeres noirs azotes, qui proviennent de la stabilisation
 IAEA-SM-211/42 45

finale des radicaux semi-quinoniques intermediaries, a fait l’objet de nombreux travaux mettant
indifferemment en jeu des techniques d’oxydation chimique ou enzymatique [23—28]. Les trans­
formations des polyphenols dans les sols ont ete etudiees, souvent par des methodes radioisotopiques,
et portent soit sur des composes libres (29, 30], soit sur leurs polycondensats syntltetiques [31,32].
Toutefois, peu d’auteurs ont jusqu’ä present etudie simultanement les relations structurales existant
entre les conditions de formation de tels polymeres et leur stabilite chimique et biologique. Nous
avons tente d’atteindre un tel resultat en recherchant des conditions moderement autoxydantes et
rigoureusement contrölees permettant une synthese reproductible de polymeres azotes ä partir du
Systeme catechol-glycine. Ces conditions etant fixees, il a ete possible de synthetiser des polymeres
marques selectivement au 14C et de les soumettre apres caracterisation ä differentes conditions de
degradation, soit par voie chimique, soit au contact de la microflore d’un sol ä mull.

2. METHODES EXPERIMENTALES

Un melange equimoldculaire 0,03M de catechol et de glycine dans un tampon phosphate

NaH2P04/Na2HP04 de pH 7,9 est soumis ä agitation pendant 5 jours dans un reacteur traverse par
un courant d’oxygene pur, a 20°C et ä l’obscurite. En cours de reaction, on effectue des preleve-
ments dont on enregistre le spectre d’absorption u.v.-visible (spectrophotometre «Beckman Acta 1»),
Une experience analogue est realisee avec 3 ml de melange, dans des fioles de Warburg de
15 ml montees sur un microrespirometre Gilson permettant de determiner la cinetique de
consommation d’oxygene par le milieu ä 20°C et sous pression normale.
Afin d’effectuer des mesures de chimiluminescence, 3 ml de melange sont preleves ä intervalles
reguliere et introduits dans une cuve en quartz placee contre la fenetre d’un photomultiplicateur
RCA 6655 A, dont la sensibilite maximale est atteinte pour 440 nm, et Ton enregistre le nombre
d’impulsions emises par unite de temps [33].
Trois polymeres sont prepares en reacteur avec 100 ml de solution equimoleculaire:0,03M,
en utilisant des precurseurs marques selectivement au 14C et de radioactivites specifiques connues:

0 catechol + glycine 14Ci

u) catechol + glycine 14C2
iii) catechol l4Cu‘ + glycine

A la sortie de chaque reacteur, le courant gazeux traverse deux pieges successifs contenant
15 ml de NaOH2N, destines ä mettre en Evidence les degagements eventuels de C02. En fin de
reaction, chaque solution est dialysee ä 5°C contre eau distilrie; les dialysats sont stockes au froid,
et les polymeres selectionnes par dialyse sont decationises sur resine Dowex 50 W X 8, 50—100 mesh
(H+), lyophilises et conserves sous vide.
La distribution des sites marques entre effluents gazeux, dialysats et polymeres est determinee
au moyen du spectrometre ä scintillation liquide Packard 3003 equipe d’un dispositif de standardisa­
tion externe [34]. Un bilan de l’azote est effectue par dosage dans les dialysats de l’azote ammoniacal
distillable [35] et de l’azote a-amine libre [36]. La presence de produits carbonyles de degradation
oxydative de la glycine est recherchee par preparation de dinitro-2,4-phenylhydrazones, Chromato­
graphie sur papier avec differents solvants [37] et autoradiographie des chromatogrammes.
Les polymeres sont ensuite soumis ä une etude physico-chimique: filtration sur gels de dextrane
Sephadex G 100 et G 50 superfine, en presence d’un tampon tris/HCl de pH 7,5; analyse elementaire
par combustion en four ferme (analyseur Carlo Erba 1104); spectre d’absorption infrarouge, au
moyen du spectrophotometre Beckman IR 10, apres pastillage dans KBr anhydre; determination des

M arquage u n ifo rm e d u n o y a u aro m atiq u e.

46 ANDREUX et al.

acidites COOH et OH par titration en continu dans l’eau distillee par NaOH 0,01N (Urectron 5,
Tacussel).
Vingt mg de polymeres sont soumis ä une hydrolyse acide par ebullition ä reflux dans 40 ml de
HCl 3N durant 3 heures. Les hydrolysats sont filtres sur membrane Millipore, concentres sous vide
jusqu’a elimination de l’acide, puis redisperses dans l’eau. La solution obtenue est alors percolee sur
une colonne de resine Dowex 50 W X 8, 50—100 mesh (H+), et les produits fixes sont elues ä l’aide
d'une solution de NH4OH 1,0N. Les dosages de C et N hydrolysables sont effectues, et l’on mesure
la distribution des differents sites marques 14C entre les fractions cationiques et anioniques.
Les polymeres selectivement marques 14C sont soumis ä une incubation d’un mois dans un mull
de sol brun lessive forestier dont les principales caractdristiques sont les suivantes:

pH dans l’eau = 5,6 Ferlibre = 2,3%

CEC = 16,2meq/100g C = 3,5%
Saturation en bases = 74,0% N = 0,25%
Argiles =22,6% C/N =14,0%

Des comparaisons sont realisees avec les precurseurs marqufis, glycine 14C!, glycine 14C2,
catechol 14CU; introduits ä l’etat libre dans le sol en quantites äquivalentes ä celles introduites sous
forme combinee dans les polymeres. Le sol seche ä l’air et tamise a 2 mm est reparti en lots de 20 g
dans des erlenmeyers de 500 ml. Chaque lot regoit respectivement 27 g de polymeres (1350 ppm),
3,4 mg de glycine (170 ppm) et 21,4 mg de catechol (1070 ppm), en solution dans un volume d’eau
calcule pour porter le sol ä Thumidite Äquivalente. Les erlenmeyers sont ensuite places dans un
bain thermostate ä 28°C et relies ä un Systeme clos muni d’un dispositif de circulation d’air [34].
Le C 02 dägage par les echantillons est regulierement entrafne par un courant d’air humide exempt
de C02, et piege ä la sortie des incubateurs dans une solution de NaOH 0,5N dont on determine la
radioactivity et que Ton peut titrer en retour par HC1 0,2N.
Apres incubation, les sols sont seches ä 40°C et leur radioactivity est determinee par combustion
dans un analyseur Wösthoff-Carmhograph 8 modifiy. Les essais de redissolution des produits
residuels sont effectuÄs en agitant 1 g de sol successivement avec 25 ml des reactifs suivants: H20,
tampon NaOH/tytraborate de sodium ä pH 9,7, pyrophosphate de sodium ä 1%, NaOH 0,1N [38].

3. RESULTATS ET DISCUSSION

3.1. CinÄtique de la rdaction d’oxydation

3 .1 .1 . S p ectres d ’ab so rp tio n u. v.-visible

Une pigmentation rouge se developpe des le depart de la reaction, se traduisant par une bande
d’absorption centräe ä 480 nm, dont l’intensite maximale est atteinte apres 25 min. Cette coloration
est indicatrice de la formation d’une combinaison entre glycine et catechol [21, 27]. On n’observe
par contre ä ce stade aucune modification des absorptions dans l’ultra-violet, caracterisees par les
maxima ä 215 nm (e max = 6300) et 275 nm (e max = 2300) propres au catechol.
Apres 5 heures, la preparation prend une teinte brun-verdätre coi'ncidant avec l’apparition d’un
maximum spectral ä 610— 620 nm, dü probablement ä la transition n -*• -n* du groupement 1C = 0 de
1'0. benzoquinone, tandis qu’on observe une augmentation sensible de l’extinction dans l’ultra-violet
(fig. 1). Un nouvel epaulement apparaft a 320— 330 nm, que Ton peut attribuer ä une combinaison
glycine-catychol, car on ne l’obtient pas avec le catechol seul. Apres 24 heures, la coloration brun-
verdätre s’accentue, puis la preparation prend une teinte brun-fonce, accompagnee d’un accroissement
general de Tabsorption, avec effacement parallele des maxima. L’extinction continue ainsi ä croftre,
parallelement au brunissement, jusqu’a l’obtention d’une valeur stable ä partir du quatrieme jour.
 IAEA-SM-211/42 47

F1G.1. V ariations au corns d u te m p s d u sp ectre u.v.-visible d e la p reparatio n ( C = 0 ,2 '1 0 ~ ъ m o le /l); P = p o ly m e re s

apres dialyse.

Le spectre est alors pratiquement monotone, avec decroissance progressive de l’extinction vers le
visible, comme e’est le cas pour les composes humiques stables extraits des sols [39, 40].

3 .1 .2 . O x yd a tio n du ca tech o l e t chim ilum in escence

La formation du pigment rouge apres 25 min n’affecte encore qu’une faible proportion de
molecules, comme en temoignent l’absence de modification du spectre u.v. et la faible intensity des
dchanges gazeux. La consommation d’oxygene s’etale sur 4 jours et sa cn^tique presente deux
etapes distinctes (fig. 2). La premiere etape, plus rapide, correspond aux 40 premieres heures de
reaction, et conduit ä la fixation d’environ 0,75 mole d’oxygene par mole de catechol introduit au
depart; la phase plus lente qui lui succede se termine par un palier correspondant ä la consommation
de 1,1 mole d’oxygene par mole de catechol.
La chimiluminescence, qui traduit le taux de desactivation des radicaux semi-quinoniques issus
de l’oxydation du catechol, montre d’abord une croissance pratiquement constante durant les
30 premieres heures, puis atteint un equilibre stationnaire durant 48 heures, avant de decroftre au
cours du quatrieme jour. II semble done que la desactivation des semi-quinones par polymerisation
n’atteint sa valeur optimale que lorsque la plus grande partie des molecules polyphdnoliques sont
oxyddes. Cette observation est parfaitement en accord avec les modifications du spectre u.v.-visible
apres 40 heures de reaction.
48 ANDREUX et al.

F IG .2. C in e tiq u e s com parees des echanges g a ze u x e t d e la c h im ilu m in escen c e au cours d e la syn th e se . A - C 0 2
p ro ven a n t d e la g ly c in e e t d u catechol; В = o x y g e n e c o n so m m e ; C = ch im ilu m in escen ce.

3 .1 .3 . D egradation e t p ro d u ctio n de СОг

La liberation de C02 par le milieu reactionnel interesse en premier lieu le groupement

carboxylique de la glycine, dont plus de 16% se trouvent decarboxyles (fig. 2). Elle est particuliere-
ment rapide en debut de synthese, puisque 13,5% des COOH sont d6ja degrades en 40 heures. Le
C2 de la glycine ne contribue pratiquement pas au degagement de C02, tandis que 3,8% de la radio­
activity du catechol у participent. Un tel degagement implique l’ouverture d’une proportion
 IAEA-SM-211/42 49

importante de noyaux aromatiques, qui peut atteindre 22,8% si Гоп admet qu’une molecule de
catechol libere une molecule de C02.
La desamination des amino-acides au cours de l’oxydation des polyphenols est liee ä l’oxydation
des combinaisons amino-phenoliques et au rearrangement des quinones ainsi formees [21, 22] ; eile
conduit ä la production d’un a-cetoacide, dont la decarboxylation peut etre spontanee en milieu
alcalin. La decarboxylation active de la glycine durant la premiere phase de reaction confirme que
les produits d’addition de la glycine sur le catechol se forment surtout au cours de cette phase et
qu’ils participent aux processus de polymerisation. L’ouverture des noyaux aromatiques se poursuit
durant la phase de polymerisation, et il n’est done pas exclu que des cycles ouverts participent ä la
structure des polymeres [41].

3.2. Identification des produits de reaction

3 .2 .1 . C o m p o sitio n des d ialysats

Dans les dialysats sont prdsents pres de 50% de la glycine initiale, et 17,2 ä 22,7% d’ions NH4+
(tableau I); l’acide glyoxylique provenant de la desamination de la glycine I4C2 est mis en evidence
par Chromatographie sur papier des DNP-hydrazones qui en derivent, suivie d’une autoradiographie.
On deduit du dosage des DNP hydrazones 14C2 que la quantite d’acide glyoxylique presente dans
le milieu correspondant ä moins de 2% des molecules de glycine de depart. L’acide glyoxylique a
done et€ decarboxyle ä mesure de sa formation en liberant du formaldehyde. Toutefois, il n’a pas
dte possible de mettre en evidence la DNP-hydrazone du formaldehyde, ce qui suggere que ce
compose s’est combine ä la glycine en donnant des bases de Schiff et des melanoidines [7, 8].

3.2.2. Caracterisation des p o ly m e re s .

Les polymeres lyophilises ont l’aspect d’une poudre brun-fonce, dont le poids atteint 16,4 ä
25,4% du poids total des composes de depart. Les bilans presentes au tableau II permettent d’observer
que ces polymeres incorporent 27,1% de la radioactivite du catechol, et moins de 10% de celle de
chacun des atomes Ci et C2 de la glycine.
Gräce au marquage selectif, on peut estimer que sur 23 atomes de carbone, 21 proviennent du
catechol et les deux autres respectivement des atomes Ct et C2 de la glycine. La coincidence entre
les nombres d’atomes d’azote et de carbone C! et C2 derivant de la glycine tend ä confirmer que des
moldcules entieres de cet amino-acide ont €t€ incorporees. En moyenne, 1 g de polymeres correspond
ä 1,5 millimole de glycine pour 5,4 millimoles de catechol, soit un rapport stoechiometrique de 1/3,5.
La comparaison entre la composition elementaire calculde et les resultats analytiques (tableau III)
montre une coincidence satisfaisante entre les teneurs en C, N et H des composes. Par contre, on
trouve par le calcul deux fois moins d’oxygene que par analyse. On en ddduit que les composes ont
incorpore de l’oxygene lors de la Synthese, soit par ouverture d’une partie des cycles, soit par hydroxy-
lation des quinones intermediates [20, 40—43].
La titration des acidites -COOH et -OH des polymeres, par comparaison avec celle des
equivalents glycine et catechol engages dans la structure des polycondensats, montre une diminution
significative des -OH initiaux, de 10,9 ä 2,9 meq/g, que Гоп peut imputer ä l’oxydation et ä la forma­
tion de structures hydroxyquinoniques [40, 42]; au contraire, l-’acidite carboxylique s’est elevee de
1,6 ä 4,8 meq/g par suite des ruptures de cycles. En admettant qu’un cycle est ouvert avec formation
de deux groupements -COOH [41 ], on peut estimer que l’acidite matricielle mesuree (3,2 meq/g)
est due ä l’ouverture de 29,6% des noyaux incorpores. Cette acidite est mise en evidence en
spectroscopie infrarouge par l’apparition d’un pic intense ä 1710 cm"1, deplace ä 1590 cm"1 par
salification [24].
En Chromatographie sur gel de dextrane Sephadex G 100, les polymeres montrent un pic
unique dont le maximum correspond а Кду = 0,55 et dont la branche descendante presente un
50 ANDREUX et al.

TABLEAU I. DISTRIBUTION DES FORMES DE L’AZOTE AU

TERME DE LA SYNTHESE (en % de N introduit au depart)

F o rm es de N D ialysats P o ly m eres P ertes

Exces

n n h Y 22,7
17,2 - -
N glycine libre 45,5 _
47 ,8 - -
N com bing 15,5 5,2
14,1 4,5
N to ta l 83,7 5,2 - 11,1
79,1 4,5 - 16,4

TABLEAU II. DISTRIBUTION DES DIFFERENTS SITES MARQUES

AU 14C AU TERME DE LA SYNTHESE (en % de la radioactivity de depart)

Sites co2 D ialysats P o ly m eres P ertes

m arqu6s Exces

G lycine 14C, 15,0 75,2 4,8 - 5,0

16,9 65,8 5,9 - 11,4
G lycine 14C2 0,1 72,8 4 ,7 - 2 2 ,4
0,2 9 6 ,4 7,3 + 3 ,9
0,2 9 3 ,9 7,7 + 1,8

C a te c h o l14Си 3,8 6 8 ,0 27,1 " 1.1

TABLEAU III. COMPARAISON DES COMPOSITIONS ELEMENTAIRES

CALCULEES ET MESUREES DES POLYMERES Resultats en %)

c H N 0

C atech o l 14C y 3 9 ,1 2 3,26 17,38

—COOH 1,86 0 ,1 6 - 4 ,9 6

D c h 2- 2 ,1 0 0,35 - -
nh 2- - 0,3 3 2,31 -

T o tal calcule 4 3 ,0 8 4 ,1 0 2,31 2 2 ,3 4

A nalyse 4 5 ,6 0 3,63 2 ,47 4 8 ,3 0

epaulement de Kav = 0,75. Par comparaison avec des protönes globulaires etalons [44], on peut
estimer que leurs masses molaires se situent entre 25 000 et 15 000 pour le constituant majeur, et
7000 et 4000 pour le second constituant. Ces valeurs, faibles par comparaison avec celles trouvees
pour les acides humiques des sols ä mull [45-48], peuvent s’expliquer par le taux 61eve des o v e r­
tures de cycles au cours de la synthese. En effet, dans les polycondensats naturels on observe souvent
une relation inverse entre le degrd de polycondensation et l’acidite matricielle [47, 49].
 IAEA-SM-211/42 51

TABLEAU IV. STABILITES COMPAREES DES SITES MARQUES

AU 14C ET DE L’AZOTE LORS DE L’HYDROLYSE ACIDE DES
POLYMERES (resultats en %)

Sites H ydrolysables C ations N on catio n s P ertes

14c , 56,7 30,6 22,9 3,2

,4C2 68,1 30,2 26,1 11,8

N -a am ine 30,0 - - -
N N H ,* 19,0 - - -
C atech o l 14Си 28 ,9 3,3 22,3 3,3

3 .2 .3 . D egradation a cid e d es p o lym eres

Lors de l’hydrolyse acide des composes humiques naturels et des modeles incluant des poly­
peptides, les amino-acides N-terminaux demeurent fix6s sur la matrice polycondensee [5, 10, 28, 47];
les polymeres obtenus ä partir du Systeme catechol-glycine devraient done opposer une resistance
61evde au traitement hydrolytique. Ainsi, leur spectre d’absorption presente les caracteristiques
habituelles des residus d’hydrolyse des acides humiques et des melanines [6], avec en particulier un
maximum tres net ä 1610 cm-1 attribue aux liaisons )C = О a - ß conjuguees [50].
L’hydrolyse acide ne modifie pas le spectre du residu solide; 28,9% de la radioactivite provenant
du catechol, 56,7% et 68,1% des radioactivites issues respectivement du C2 et du C2 de la glycine
sont retrouves dans les hydrolysats (tableau IV); 30% de la radioactivite du C2 et du C2 correspondent
ä de la glycine libre retenue sur resine cationique, la fraction non fix6e etant imputable ä des fragments
de polymeres et, ainsi que le suggerent Cranwell et Haworth [51], ä de l’acide glyoxylique. En effet,
la desamination de la glycine est probable, car 19% de l’azote sont liberes sous forme ammoniacale.
La rupture de la moitie de la glycine incorporee dans les polymeres suggere qu’il existe des liaisons de
stabilites differentes, en relation sans doute avec l’aromaticite des cycles matriciels.

3.3. Degradation des polymeres dans un sol ä mull

3 .3 .1 . D egagem en t d e C 0 2

Les courbes de mineralisation en C02 des diffdrents Substrats dans le mull actif sont presentees
a la figure 3. L’oxydation de la glycine libre est la plus active; le rapport i des atomes 14C! et 14C2
restant dans le sol apres 48 heures d’incubation laisse prevoir que la glycine non transformee emprunte
les voies metaboliques classiques de synthese proteique [52]. A l’dquilibre, apres quatre semaines,
78,5% du C2 et 48,6% du C2 sont oxydes en C02.
Dans le cas du catechol 14Cu libre, la vitesse d’oxydation, faible au depart, passe par un
maximum entre 48 et 72 heures et decroft ensuite; 9% seulement des atomes de carbone sont convertis
en C02. La resistance de ce compose ä la biodegradation peut etre attribute soit ä sa toxicite, soit
ä sa rapide humification par oxydation en quinones intermediaires, en accord avec les observations
faites sur des precurseurs polyphenoliques autoxydables, tels que le trihydroxy-1,4,5-naphtalene [6]
ou l’hydroquinone [29].
La mineralisation des polymeres est active et atteint sa vitesse maximale au cours des premieres
24 heures d’incubation, puis decroit et devient pratiquement constante ä partir du cinquieme jour.
Les taux de mineralisation en C02 s’elevent ä 39,5% et 25,9% respectivement pour le Q et le C2 de
la glycine, et 17,7% pour le carbone du catechol. L’incorporation de la glycine et du catechol dans
les polymeres se traduit done par une stabilisation de l’amino-acide, et par une fragilisation du
catechol en raison de l’existence des groupements -COOH matriciels.
52 ANDREUX et al.

F I G .3 . C o u r b e s c u m u l a tiv e s d e la m in e r a lis a ti o n e n C 0 2 d e s S u b s tr a t s m a r q u e s i n c u b e s d a n s le s o l. 1 = g ly c in e 14C 1;

2 = g l y c i n e 14C2; 3 = c a te c h o l 14C u ; 4 - p o l y m e r e s g l y c i n e 14C j; 5 ~ p o ly m e r e s g ly c in e l i C 2 ; 6 = p o ly m e r e s c a te c h o l

14C u,‘ 7 = p o l y m e r e s g ly c in e 14C u + c a t e c h o l 14C u .

3 .3 .2 . M o d ifica tion s de la stru ctu re des p o ly m ir e s

Les essais de reextraction des residus d’incubation avec l’eau et les solvants alcalins [38]
montrent qu’une faible proportion seulement de la radioactivite initiale est remise en solution
(tableau V). Dans le cas des polymeres, cette insolubilisation suggere l’existence de modifications
structurales profondes. Les rapports atomiques C1/C2/Ccatechol sont determines dans le C02 degage
et dans les differents extraits (tableau VI). Par comparaison avec les rapports atomiques de depart,
et en choisissant C2 constant et egal ä 1, on observe qu’au debut de la mineralisation, l’oxydation en
C02 affecte preferentiellement le C, de la glycine. Apres 4 jours d’incubation, les atomes Cj et C2
sont degrades ä la т ё т е vitesse, tandis que l’oxydation des atomes matriciels provenant du catechol
commence ä croftre, jusqu’ä un rapport final proche de celui des produits de depart. A l’equilibre,
les polymeres se trouvent done degrades selon un processus congruent tres lent et continu. La parti­
cipation de processus physico-chimiques ä ces degradations est tres possible dans ce sol neutre
particulierement riche en hydroxydes de fer amorphes.
Les produits de degradation les plus appauvris en radioactivite due au catechol et au Ci sont
reextraits respectivement ä l’eau et dans NaOH ä pH 9,7. Les rapports atomiques des autres extraits
alcalins et des residus se rapprochent davantage de celui des polymeres initiaux avec toutefois une
augmentation sensible du carbone matriciel (tableau VI). Comme dans le cas de l’hydrolyse acide,
l’action de la microflore semble s’etre exercee preferentiellement sur des structures presentant un
moindre degre de stabilisation de la glycine, ainsi que sur des groupements -COOH matriciels
 IAEA-SM-211/42 53

TABLEAU V. DISTRIBUTION DE LA RADIOACTIVITE RESIDUELLE DANS LES

EXTRAITS AQUEUX ET ALCALINS DES SOLS INCUBES

Substrats H2Oa NaOH Pyrophosphate NaOH Total extrait

pH 9 ,7 a l%a 0 ,lN a a b

G lycine 14Ci 2,5 0,6 0,4 0,6 4,1 19,1

G lycine 14 C2 5,5 1,8 1,2 1,9 10,4 20,2

Catechol 14Си 2,7 5,3 6,3 8 ,0 22,3 2 4,5

Polym eres G lycine 14Ci 6 ,i 3,7 3,7 2,5 16,0 2 5 ,6
Polym eres G lycine 14C2 7,7 7,4 4,1 3,4 2 2 ,6 , 29,4
Polym eres C atechol 14Q j 5,5 7,3 4,8 3,8 2 1 ,4 '2 5 ,8

a En % de la radioactivity introduite.
b En % de la radioactivite residuelle apres incubation.

TABLEAU VI. COMPARAISON DE LA DISTRIBUTION DES TROIS SITES MARQUES ET

DE LEURS RAPPORTS ATOMIQUES ENTRE LES DEGAGEMENTS GAZEUX ET LES
COMPOSES RESIDUELS

C 0 2 degage dans M icroatom es C /20 g de sol Rapports atom iques

les periodes
(jours) C, C2 0-catechol C, C2 ^catechol

0 -1 7,2 2,8 2 2 ,6 2,6 1 8,1

1 -2 2,3 1,5 22,6 1,5 1 15,0

2 -3 0,8 1,0 13,2 0,8 1 13,2

3 -4 0,7 0,7 9,6 1 1 13,7
4 -7 1,2 1,0 17,0 1,2 1 17,0
7 -9 0,6 0,6 9,7 1 1 i6 ,2
9 -1 1 0,6 0,6 9,1 1 1 15,2
1 1 -1 5 0,9 0,9 14,5 1 1 16,1
1 5 -1 8 0,4 0,45 8,0 0,9 1 17,8
1 8 -2 2 0,5 0,55 9,6 0,9 1 17,5
2 2 -2 7 0,7 0,7 13,0 1 18,6
1
C 0 2 total 16,1 10,8 148,9 1,5 1 13,8
Extrait H2 0 2,5 3,2 4 6 ,6 0,8 1 14,6
Extrait NaOH pH 9,7 1,5 3,1 61,5 0,5 1 19,8
Extrait Na4P20 7 1,5 1,7 40 ,3 0,9 1 2 3,7
Extrait NaOH 0,1 N 1,0 1,4 3 2 ,0 0,7 1 2 2,8
N on extrait 18,2 21,5 51 1 ,9 0,8 1 23,8

Produit de depart - - - 1 1 2 1 ,0
54 ANDREUX et al.

provenant de noyaux ddjä ouverts. II est probable qu’en cours d’incubation, d’autres noyaux se
trouvent rompus par la microflore, conduisant ä des structures aliphatiques riches en groupements
-COOH, analogues ä l’acide muconique et ä ses derives [53-55]. De tels composes se trouveraient
stabilises sous forme de complexes pseudosolubles avec les cations polyvalents du sol [38, 56, 57].
Les composes les moins degrades correspondraient au contraire ä des macromolecules ä structure
resonnante assurant la stabilisation de la glycine, et susceptibles d’etre insolubilisees par voie physico-
chimique.

3 .3 .3 . In flu en ce su r la m ineralisation des com poses organiques p reex ista n ts

Le dcgagement de C02 total par les sols enrichis est compare avec celui d’un sol non enrichi
incube dans les mcmes conditions. L’apport de glycine exerce une action stim u la n te [58] pratique-
ment immediate: apres 4 jours d’incubation, le degagement de C02 a augmente d’environ 250% par
rapport au temoin. Cette action est au contraire depressive dans le cas du catechol libre et des poly­
meres, au moins durant les premiers jours de l’incubation. Ces composes provoquent done une
perturbation dans l’equilibre biologique du sol, qui ne disparait qu’apres adaptation de la microflore.
Dans le cas des polymeres, il ne semble pas que cette perturbation soit düe ä une toxicitd directe car,
contrairement au catechol seul, leur vitesse initiale de mineralisation est tres elevee; il semble plutot
que leur degradation conduise ä la formation de composes intermediaires presentant une toxicite
temporaire. On a cherche ä confirmer cette hypothese en etudiant revolution au cours de l’incubation
du rapport C2/C] des atomes de carbone degage sous forme de C02. Dans le cas des polymeres, се
rapport passe de 0,38 ä 0,46 entre le premier et le second jour, puis ä une valeur constante comprise
entre 0,50 et 0,60. Dans le cas de la glycine libre, cette valeur est atteinte des le premier jour. On
peut done conclure que durant les premiers jours, les amino-acides presentant les liaisons les plus
fragiles sont d’abord decarboxyles avant d’etre detaches de la matrice des polymeres et de participer
au metabolisme microbien [52, 59]. La fraction matricielle residuelle manifesterait alors un effet
toxique, avant d’etre transformee ä son tour.

4. CONCLUSION

L’autoxydation d’une solution equimoleculaire de catechol et de glycine ä pH 7,9 et 20°C

conduit en 4 jours ä un etat d’equilibre caracteris6 par la formation de polymeres azotes de couleur
foncee, saliflables grace ä une teneur elevee en groupements carboxyliques. Leur elaboration met
en jeu l’oxydation du catechol avec formation intermediaire de radicaux semi-quinoniques et de
quinones, qui reagissent avec la glycine [21,22]. Les produits d’addition ainsi formes s’oxydent ä
leur tour en quinones actives qui subissent des rearrangements provoquant la desamination d’une
Partie de la glycine libre, avec formation d’aeide glyoxylique CHOCOOH, dont la decarboxylation
est tres rapide.
La premiere phase —qui dure entre 30 et 40 heures —de la reaction est caracterisee par
l’oxydation du catechol, et la production de C02 par degradation de la glycine (15 ä 18% du C,)
et des noyaux aromatiques (3,8% du carbone du catechol). A la fin de cette phase, les molecules
oxydees predominent et la polymerisation des radicaux semi-quinoniques peut alors se d6velopper.
A l’equilibre, presque tous les radicaux libres ont ete desactives en conduisant ä des polymeres dont
les quantites et les dimensions dependent etroitement des conditions chimiques de l’equilibre.
Les produits ainsi stabilises contiennent peu de radicaux libres [60], mais leur traitement
alcalin conduit comme dans le cas des acides humiques naturels ä une nouvelle activation des groupe­
ments quinoniques et ä l’accroissement des ouvertures de cycles avec production de C02 [49]. Les
polymeres isol6s presentent des caracteristiques analytiques comparables ä celles des acides humiques
extraits des sols eutrophes; nöanmoins, leur encombrement moleculaire est moindre, en raison du
maintien des conditions d’autoxydation durant toute la duree de la Synthese. Celles-ci, contrairement
 IAEA-SM-211/42 55

ä l’oxydation enzymatique ä pH faiblement acide [19, 26], tendent en effet ä ralentir la polymerisa­
tion au profit de la degradation des noyaux aromatiques [49, 61]. Toutefois, il est bon de preciser
que dans la nature l’autoxydation accompagne toujours l’oxydation enzymatique des precurseurs
polyphenoliques.
Parmi les polymeres isotes, peuvent etre distinguds deux types de composes; les uns, les plus
abondants, se caracterisent par des structures hydroxyquinoniques hautement resonnantes qui
permettent une stabilisation dlevee des substituants glycine; les seconds, de plus petite taille,
contiennent davantage de cycles ouverts porteurs de groupements -COOH [61 ] et presentent un
etat de conjugaison plus faible, qui explique la stabilisation moindre de la glycine. En effet,
l’hydrolyse chlorhydrique ä chaud parvient ä detacher la moitie des molecules de glycine tandis que
l’autre moitie demeure incorporee ä un ptecipite noir tres stable representant environ 70% du poids
total des polymeres. Lors de l’incubation dans le sol, 17,5% du carbone des polymeres sont degages
sous forme de C02. Par rapport ä la glycine et au catechol libres, la combinaison dans les polymeres
se traduit par une stabilisation de la glycine, dont la biodegradation diminue de 50%, et au contraire
par une fragilisation du catechol, dont l’oxydation en C02 augmente de 100%. La biodegradation
parait affecter pteferentiellement les motecules les plus petites, en assurant ä la fois la rupture des
amino-acides et la decarboxylation des groupements -COOH matriciels. Contrairement ä la glycine
libre, les polymeres exercent, vis-ä-vis de la biodegradation des composes organiques preexistants
dans le sol, un effet depressif qui ne prend fin qu’apres 4 jours, avec la fin de la phase primaire de
biodegradation de la glycine liee. Cet effet est etroitement lie ä l’dlimination de l’amino-acide, avec
liberation de composes toxiques d’origine matricielle qui pourraient se fixer aux parois cellulaires
des micro-organismes ou bien etre partiellement biodegrades.
La «phase secondaire» de l’incubation, ainsi que la qualifient de nombreux auteurs [62—64],
est caracterisee par une mineralisation tres lente et stoechiontetrique des polymeres, ce qui suggere
leur incorporation aux composes humiques les plus stables. Les composes reextractibles ä l’eau ou
par NaOH ä pH 9,7 correspondent ä des structures aliphatiques riches en groupements -COOH issus
de la biodegradation des noyaux aromatiques et susceptibles de donner lieu ä la formation de
complexes pseudo-solubles. Les extraits alcalins et l’humine sont au contraire enrichis en noyaux
aromatiques et ne mettent pas en evidence de decarboxylation preferentielle de la glycine. L’activite
biologique tend done ä epargner une fraction homogene et hautement polymerisfe des macromole-
cules introduites, dont la stabilisation dans 1’humine semble faire intervenir des processus physico-
chimiques. Cette stabilisation est ä rapprocher des insolubilisations obtenues dans les sols apres
degradation microbienne des chafnes polypeptidiques rattacltees aux phytomelanines [56]; eile
s’avere etre une forme importante de stockage de l’azote organique dans l’humine des sols.

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[2 7 ] SCH ANEL, L., “ Studies about hum u s” , Trans. Int. Sym p. Hum us et Planta IV , Prague (1 9 6 7 ) 73.
[28] L A D D , J.N ., BU TLER, J.H .R ., A ust. J. SoU Res. 4 (1 9 6 6 ) 41.
[2 9 ] SCHVARTZ, C., «E volution des hydrosolubles de litieres de calluna e t de hetre au cours du processus
d’hum ification», These, N ancy (1 9 7 5 ) 81 p.
[3 0 ] WANG, T.S.C ., CHANG, S.Y ., YEH, K .H ., J. Agric. Ass. China 7 2 (1 9 7 0 ) 30.
[3 1 ] V ERM A, L„ M ARTIN, J.P., H A ID ER , K., SoU Sei. Soc. A m ., Proc. 3 9 2 (1 9 7 5 ) 2 7 9 .
[3 2 ] H A IDER , K „ M ARTIN, J.P., SoU Sei. Soc. A m ., Proc. 39 4 (1 9 7 5 ) 657.
[3 3 ] GOLEBIOWSKA, D ., “ Studies about hum us”, Trans. Int. Sym p. Humus et Planta V , Prague (1 9 7 1 ) 3 69.
[3 4 ] GUCKERT, A „ ROGER, P„ JA CQ UIN, F ., Bull. E N SA N 10 2 (1 9 6 8 ) 69.
[3 5 ] STEVENSO N , F.J., K ID DER , G., TILO, S.N ., SoU Sei. Soc. A m ., Proc. 31 1 (1 9 6 7 ) 71.
[3 6 ] MOORE, S„ STEIN, W .N., J. Biol. Chem. 211 (1 9 5 4 ) 907.
[3 7 ] SMITH, I., in C hromatographie and E lectrophoretic T echniques, V ol. I, William Heinem ann Medical B ooks,
Interscience Publishers, London (1 9 6 0 ) 261.
[3 8 ] BRUCKERT, S„ HETIER, J.M ., GU TIERR EZ, F „ Sei. Sol - Bull. A FES 4 (1 9 7 4 ) 2 25.
[3 9 ] FLA IG , W„ Sei. Sol - BuU. A FES 2 (1 9 7 0 ) 39.
[4 0 ] FLA IG , W., Proc. Int. M eet. H um ic Substances, N ieuw ersluis, Pudoc, W ageningen (1 9 7 2 ) 19.
[4 1 ] CZUCHAJOWSKI, L„ KRECZEK, J., R ocz. G leboz. 16 1 (1 9 6 6 ) 3.
[4 2 ] M ARTIN, J.P., H A IDER , K., BO NDIETTI, E., Proc. Int. Meet. Hum ic Substances, N ieuw ersluis, Pudoc,
W ageningen (1 9 7 2 ) 171.
[4 3 ] PIERPOINT, W .S., B iochem . J. 9 8 (1 9 6 6 ) 567.
[4 4 ] ANDREW S, P„ B iochem . J. 91 (1 9 6 4 ) 22 2 .
[45] BA ILLY , J.R ., M ARGULIS, H., Plant SoU 2 4 (1 9 6 8 ) 34 3 .
[46] BA ILLY , J.R ., Plant Soil 4 4 1 (1 9 7 6 ) 4 9 .
[4 7 ] A N D R E U X , F ., METCHE, M., Rapp. 1er Coli. Int. Biodegradation e t H um ification, N ancy, 1 9 7 4 , PIERRON Ed.
(1 9 7 4 ) 47 9 .
[4 8 ] POSPISIL, F „ R ostl. V yroba 2 0 8 (1 9 7 4 ) 795.
[4 9 ] SWIFT, R .S., PO SNER, A.M ., J. SoU Sei. 23 4 (1 9 7 2 ) 381.
[5 0 ] THENG, B.K .G ., PO SN ER , A .M ., SoU Sei. 104 (1 9 6 7 ) 191.
[5 1 ]
CRANWELL, P .A ., HAWORTH, R .D ., Proc. Int. Meet. H um ic Substances, N ieuw ersluis, Pudoc, Wageningen
(1 9 7 2 ) 13.
[52] M A Y AU D O N , J„ SIM ONARD, P., Ann. Inst. Pasteur 109 (1 9 6 5 ) 2 24.
[5 3 ] H A Y AISH I, O., K A TA G IRI, M„ ROTHBERG, S ., J. Am . Chem. Soc. 7 7 (1 9 5 5 ) 5 4 5 0 .
[54] R U N G , F ., Folia M icrobiol. 16 1 (1 9 7 1 ) 41.
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[5 6 ] C A R BA LLAS, T „ A N D R E U X , F „ JA CQ UIN, F „ Sei. S ol - Bull. A FES 3 (1 9 7 1 ) 29.
[5 7 ] LESPINAT, P .A ., H ETIER, J.M., THOM ANN, C., CHONE, T ., Sei. Sol - BuU. A FES 1 (1 9 7 6 ) 53.
[5 8 ] JENKINSON, D .S ., “ The turnover o f organic m atter in soU” , Rep. FA O /IA E A Technical M eeting on the U se o f
Isotop es in SoU Organic Matter Studies, Pergamon Press, Oxford (1 9 6 6 ) 187.
[5 9 ] THIM ANN, V ., in The Life o f Bacteria, 2nd Ed., McMUlan C om pany, N ew York (1 9 6 3 ) 9 0 9 p.
[6 0 ] A TH ERTON, N .M ., CRANW ELL, P .A ., FLO YD , A .J., HAWORTH, R .D ., Tetrahedron 2 3 (1 9 6 7 ) 1653.
[61] N G U Y EN , Q .H ., METCHE, M„ U RIO N , E„ Bull. Soc. Chim. Fr. 7 (1 9 6 6 ) 2 2 3 2 .
[62] D A N N EBER G , O., B odenkultur 2 2 3 (1 9 7 1 ) 264.
[6 3 ] H A N R IO N , M., TOUT A IN , F ., BRUCKERT, S„ JA CQ UIN, F ., O ecologia Plantarum, 1 0 2 (1 9 7 5 ) 169.
[6 4 ] McGILL, W .B., SHIELDS, J.A ., PA U L, E.A ., SoU B iol. B iochem . 7 (1 9 7 5 ) 57.
 IAEA-SM-211/42 57

DISCUSSION

R.W. ALDAG: In your model experiments, when you observed loss of I4C from the carboxyl
group of glycine, did you also notice an increase in ammonia in the reaction solution or was the
ammonia that may have evolved simultaneously bonded to the newly formed polymer?
F. ANDREUX: Analysis of the dialysable compounds reveals a quantity of free ammonia
corresponding almost exactly to the amount of C02 released. This proves that almost all the
glyoxylic acid formed by de-amination of the glycine is decarboxylated. It seems, therefore, that
all the nitrogen incorporated in the polymers comes from the glycine and not from the ammonia.
 IAEA-SM-211/43

EFFECT OF NITROGEN ON THE FORMATION OF

PYROCATECHIN-HUMIC ACID AND THE
NITROGEN LINKAGE CHARACTERISTIC OF THIS ACID

H. ÖZBEK
Faculty of Agriculture,
University of (Jukurova,
Adana,
Turkey

Abstract

EFFECT O F NITROGEN ON THE FORM ATION OF PYROCATECHIN-HUMIC ACID A N D THE NITROGEN

LINK AG E CHARACTERISTIC O F THIS ACID.
An evaluation o f syn th etic hum ic acids as fertilizers has been undertaken. T he effects o f nitrogen on
h um ification, and th e w ay nitrogen is linked to th e main structure, were studied. T he use o f sy n th etic procedures
to resolve the com p lex problem s o f hum ic acid form ation is n ot a particularly new m ethod but researchers believe
that m od el com p ou nd investigations can m ake valuable contributions to this subject. In th e present stud y,
pyrocatechin was polym erized b y oxid ative coupling reaction w ith nitrogenous com pounds arranged in five
groups including th e con trol group. The polym erization was achieved using air o x y g en as th e oxidizing agent
in alkaline m edium at pH 8.6. To ascertain the h um ification rate, aliquot sam ples were taken after 13, 2 8 , 6 3 ,
8 4 and 102 days o f th e reaction. The samples were precipitated w ith 1N NCI. T h e am ounts o f to ta l hum ic
acids form ed in th e reaction and the nitrogen linked to hum ic acids, as w ell as hydrolysable nitrogen, were
determ ined. As a result, it is suggested that th e form o f nitrogen affects the rate o f hum ification as w ell as the
am ount o f nitrogen linkage to hum ic acids. The longer the h um ification period, th e greater th e am ount o f
nitrogen linked to hum ic acids in the h ydrolysable form . When the am ounts o f hum ic acids, nitrogen con ten t
and th e hydrolysable nitrogen w ere taken in to consideration, th e m ost appropriate form o f nitrogen for
hum ification was found to be nitrogen in th e form o f ammonia.

INTRODUCTION

It is well known that humic acids which do not contain nitrogen do not occur in the nature [1 ].
Humic acids are not only a degradation product, but are also formed by polymerizations in the soil.
Thus, nitrogen is linked to the structure during polymerization [1,2]. It is evident that the linkage
of nitrogen to humic acids can be explained by theoretical chemical principles; however, the
amount of nitrogen linkage to humic acids is particularly important from the point of view of the
soil scientist. Nitrogen also plays an important role in humification. It is almost impossible in
nature to observe how this reaction occurs. All researchers agree that model compound studies
can make a valuable contribution to interpretation of the results.
In recent years, research workers have been united in their efforts to find a source that will
supply the essential need of nitrogen when the plant requires it [3]. This is only possible when
nitrogen is given as organic fertilizer to the soil. Synthetic humic acids containing nitrogen may
serve as a source. On this basis, this study was carried out to elucidate the effects of nitrogen
on humification and the manner in which nitrogen is linked to the main structure.

MATERIALS AND METHODS

Lignin has the most important role in the humification process in the soil [1,4]. Lignin
molecules or degradation products of lignin may directly polymerize to yield humic acids. It is

59
60 ÖZBEK

widely accepted that the basic monomeric units of humic acids are oxidation products of lignin [5].
It is known that oxidative degradation of lignin yields mostly polyphenols. For this reason,
pyrocatechin has been taken as a model compound in the present communication [6]. To provide
nitrogen, several nitrogen-containing compounds were studied, and five different groups were
arranged [7].
The groups are as follows:

Group I Pyrocatechin
” II ” +(NH4)2S04
” III ” + KN03
” IV ” + H2N-CO-NH2
” V ” + H2N-CH2-COOH

A reaction mixture of 5 g pyrocatechin and the nitrogenous compound corresponding

to 1 g nitrogen was placed in a reaction flask equipped with an oxygen inlet tube. The reaction
was established in an alkaline medium at pH 8.6. The alkaline medium is necessary in the oxidative
polymerization to enable the hydrogen in the phenolic hydroxide group to dissociate for coupling
reactions [8]. The pH values lower than 8.6 are not a good medium for the reaction, since only
a few pyrocatechin molecules take place in the polymerization. The pH values over 10 or 11 are
also not suitable, because humic acids formed may have been degraded to yield degradation
products under very alkaline conditions [9].
Oxygen that was used as the oxidizing agent was passed through the stirred solution, and the
reaction mixture was left for humification with continuous bubbling of oxygen [ 10, 11 ]. In order

Sample
( reaction mixture)

centrifuge

I i
centrifugate precipitate
(discarded) washed w ith

acidified H 0
4; ^
centrifuge

l
precipitate centrifugate
(discarded)
drTe^under vacuum
at 105° C
determination of humic
acid formed
___ ±_____

K jeldahl Hydrolysis
Analysis 6 N HCl
I . . Kjeldahl Analysis
Total N determination

Hydrolysable N
determination

F I G .l. S e p a r a tio n o f th e p o ly m e r iz a tio n m ix tu r e .

 IAEA-SM-211/43 61

to follow the humification rate, aliquot samples were taken after 13, 28, 63, 84 and 102 days
of the reaction. They were precipitated with 1N HC1 and were separated according to the scheme
shown in Fig. 1. The amounts of total humic acids formed and the nitrogen linked to humic
acids (total N) were determined.
Determination of hydrolysable nitrogen was carried out, after hydrolysing the samples
with 6N HC1 [9]. The difference between the total and the hydrolysable nitrogen gives,us the
amount of humic acid nitrogen linked as heterocyclic units [2]. The hydrolysable nitrogen is
the nitrogen that can be mineralized during a vegetation period to provide the essential require­
ment of the plant [9].

DISCUSSION

In the five groups, the humification rate and the amount of humic acids formed increased with
time (Table I). Nitrogen speeds up humification, and the rate of this reaction also changes
according to the form of nitrogen used (Fig.2).
As seen in Table 11, in the five groups, at the end of 102 days, the amount of humic acid formed
in the reaction varies from one to group to another.
In the group without nitrogen, about 20% of pyrocatechin is polymerized by 102 days.
However, this ratio is greater in percentage in the other groups.

TABLE I. AMOUNT OF HUMIC ACIDS FORMED DURING

102 DAYS OF HUMIFICATION »

A m ount o f hum ic acids (m g)

Days 13 28 63 84 102

Group I 3 4 0 .0 503.5 65 0 .0 8 5 0 .2 1 0 00.0

Group II 9 9 8 .0 1 0 29.0 1438.0 1 7 6 8 .0 2 0 2 9 .8

Group III 6 2 0 .0 6 2 0 .0 81 0 .0 1 3 5 4 .0 1 3 1 8 .0
Group IV 6 9 4 .0 1 0 24.0 1400.0 2 0 2 8 .0 1 9 9 8 .0
Group V 9 0 0 .0 9 2 0 .0 1106.0 1 1 6 8 .0 1 3 5 5 .0

F IG . 2. F o r m a tio n o f h u m ic a c id s r e la te d to tim e a n d fo r m o f n itr o g e n u se d .

62 ÖZBEK

TABLE II. RATIO OF HUMIC ACIDS FORMED TO THE

PYROCATECHIN USED DURING 102 DAYS (%)

R atio o f hum ic acids to th e p yrocatechin used (% )

Days 13 28 63 84 102

Group I 6 .8 10.07 13.0 17.05 2 0 .0

Group II 19.9 2 0 .4 28.8 3 5 .4 4 0 .6 .
Group III 12.4 12.4 16.1 2 7 .0 2 6 .3 7
Group IV 15.3 20.2 28 .0 40.5 4 0 .0
Group V 18.0 18.4 22.3 22.3 27.1

As humification proceeds, the linkage capability of nitrogen in the reaction mixture to the
humic acid structure has increased, but there are still noticeable differences among them (Fig.3).
These differences are not parallel with the amount of humic acids formed in the reaction. In other
words, the ratio of increase in the amount of humification to the increase in the amount of
nitrogen linked to humic acids is not similar. If this ratio had been equal, the curves in Fig.4 would
have been parallel to the x-axis. Therefore, it is evident that the form of nitrogen used in humifi­
cation mostly affects the linkage capability of nitrogen to humic acid structure.
The amounts of total and hydrolysable nitrogen linked to humic acids during humification
reactions can be seen in Table III. The linkage ratio of nitrogen is indicated in Table IV. The
total nitrogen contents of the humic acids formed and the hydrolysable and unhydrolysable
nitrogen are given in Table V.
 IAEA-SM-211/43 63

F I G .4 . C h a n g e in n itr o g e n c o n t e n t o f h u m ic a c id s d u r in g h u m ific a tio n p e r io d .

TABLE III. AMOUNT OF TOTAL (tot-N) AND HYDROLYSABLE NITROGEN (hyd-N)

LINKED TO HUMIC ACIDS

N itrogen con ten t (m g)

Days 13 28 63 84 102

T ot-N Hyd-N Tot-N Hyd-N Tot-N Hyd-N Tot-N Hyd-N Tot-N Hyd-N

Group II 15.8 5.8 16.0 7.7 25.2 11.5 3 6 .2 15.4 4 4 .8 2 4 .7

Group III 1.1 0.6 1.4 0.7 2.4 1.0 2.3 1.0 12.5 5.2
Group IV 1.2 0 .8 3.5 2 .2 5.6 3.4 2 6 .6 15.9 4 3 .4 2 3 .4
Group V 2 7 .0 10.2 3 3 .0 10.0 42 .2 10.5 4 1 .4 10.4 51.3 12.0

In group II, the humic acids formed in the reaction contain 1.5, 1.74, 2.2% nitrogen after
13, 63, 102 days respectively (Table V). In the other groups, a similar increase can also be
observed. Although humic acid yields at the end of 102 days in group IV are more than in group V,
the amount of nitrogen linkage is higher in group V. In groups III and V, equal amounts of humic
acid are formed. However the amounts of nitrogen linked to the structure vary.
It can be concluded that the longer the humification period, the greater is the amount
of nitrogen linked to humic acids in the hydrolysable form. As seen in Fig. 5, an increase in the
humic acid rate is almost parallel with nitrogen linkage, but the longer the humification, the
smaller is the percentage of hydrolysable nitrogen in the humic acid structure.
64 ÖZBEK

TABLE IV. LINKAGE RATIO OF NITROGEN USED IN THE EXPERIMENT TO THE

HUMIC ACIDS AS TOTAL AND HYDROLYSABLE NITROGEN

Linkage ratio (%)

Days 13 28 63 84 102

Tot-N Hyd-N Tot-N Hyd-N Tot-N Hyd-N Tot-N Hyd-N Tot-N Hyd-N

Group II 1.58 0.58 1.6 0.77 2.52 1.15 3 .6 2 1.54 4 .4 8 2.47

Group III 0.11 0.0 6 0 .1 4 0 .0 1 0 .2 4 0 .1 0 0 .2 3 0 .1 0 1.25 0 .5 2
Group IV 0 .1 2 0.0 8 0.35 0.2 2 0.56 0 .3 4 2 .66 1.59 4 .3 4 2.34
Group V 2 .7 0 1.02 3.3 0 1.03 4 .2 2 1.05 4 .1 4 1.04 5.13 1.20

TABLE V. AMOUNT OF TOTAL, HUMIC AND HYDROLYSABLE NITROGEN (AS

PERCENTAGE OF HUMIC ACID) IN THE HUMIC ACIDS FORMED IN GROUPS II, III, IV
AND V (AS PERCENTAGE OF TOTAL NITROGEN)

Group II Group III

(A s percentage o f total nitrogen) (A s percentage o f to ta l nitrogen)

Total-N
Days D ays
(%) H um ic—N Hydrolysable-N
<.%)
Humic-N Hydrolysable-N

13 1.5 3 1 .0 69 .0 13 0 .17 54.7 54.3

28 1.58 31.3 6 8 .0 28 0.22 5 0.0 5 0.0

63 1.74 34.2 6 5 .8 63 0.29 58.3 4 1.7

84 2.0 0 37.5 62.5 84 0 .1 7 58.3 4 1 .7

102 2 .2 0 4 4 .7 55.3 102 0 .9 4 58.5 4 1.5

Group IV Group V

(A s percentage o f total nitrogen) (A s percentage o f to ta l nitrogen)

Total-N Total-N
Days Days
(%) (%)
Humic-N Hydrolysable-N Humic-N Hydrolysable-N

13 0.1 8 3 3 .4 66.6 13 3.00 6 2.2 37.7

28 0 .3 4 35.7 64.3 28 3.59 6 8.6 3 1 .4

63 0.4 0 3 8 .8 6 1 .2 63 3.81 7 5.2 2 4 .8

84 1.31 4 0 .0 6 0 .0 84 3 .5 4 74.8 2 5.2

102 2.17 46 .2 53.8 102 3 .78 76.7 2 3.3

In group II, the amount of hydrolysable nitrogen in humic acids is 69% after 13 days, and
it decreases to 55.3% at the end of 102 days. Similar variations are also observed in other groups
(Table V). It is obvious that when the humification rate increases, nitrogen is linked to the humic
acids in the form of heterocyclic units. The form of nitrogen here plays the most important role.
For example, the humic acid that links higher amounts of nitrogen is formed in group V which
contains the nitrogen as NH2 group (Fig.4). However, there is less hydrolysable nitrogen in
group V in comparison with other groups (Fig.6).
 IAEA-SM-211/43 65

x---------- G ro u p П
3 * ---------- Group Ш
h----------- Group ЕГ
0.......... G rou p 21

0 5

13 28 63 84 102 T im e
(d a ys)

F IG . 5. L in k a g e o f n itr o g e n to h u m ic a c id s in h y d r o ly s a b le fo r m a c c o r d in g to th e d iffe r e n t n itr o g e n s a m p le s

u s e d in th e r e a c tio n .

100 х--------- Group П

•--------- Group Щ
+ ---------- Group Ш"
0..... Group 2
z
Ъ 70 *-------- --------------
о ----------------- к-

50' ч-
О
er)
о

c 30
.о
о
СП
>»
о Ю
тэ
>»
X
I3 28 63 84 I0 2 ^ т е ,
(d a y s )

F IG . 6. C h a n g e in a m o u n t o f h y d r o ly s a b le n itr o g e n r e la te d to h u m ific a tio n r a te .

CONCLUSION

Pyrocatechin has been successfully polymerized with certain nitrogen-containing compounds

by oxidative coupling. After calculating the amount of humic acids formed, the total and
hydrolysable nitrogen was determined according to the Kjeldahl technique. The form of nitrogen
that affects the rate of humification has been investigated.
According to the results evaluated in this work, it is suggested that the form of nitrogen
affects the rate of humification as well as the amount of nitrogen linked to humic acid structures.
When the following three factors —amount of humic acids, nitrogen content and the
hydrolysable nitrogen —were taken into consideration, the most appropriate form of nitrogen
for humification was found to be nitrogen in the form of ammonia.
66 ÖZBEK

REFERENCES

[1] FLAIG, W. SALFELD, J. Chr., H A IDER , K., Z w ischenstufen bei der Bildung von natürlichen Huminsäuren
und synth etischen V ergleichssubstanzen, Landwirtsch. Forsch. 16 2 (1 9 6 3 ) 85.
[2] FLAIG, W., C hem ische Untersuchungen an H um instoffen, Z. Chem. 4 7 (1 9 6 4 ) 262.
[3 ] FLAIG, W., SÖCHT1G, H., Grundlagenforschung zur Strohdüngung, Kali-Briefe Fachgebiet 8 2 (1 9 6 6 ) 5.
[4] FLAIG, W., BR EYH AN , Th., Über das Vorkom m en von Indolverbindungen in Schwarzerde-Hum insäuren,
Z. Pflanzenernaehr. Bodenkd. 7 5 ( 1 9 5 6 ) 132.
[5 ] K U BIENA, W.L., Entw icklungslehre des Bodens, Springer Verlag, Wien (1 9 4 8 ).
[6] SCHARPENSEEL, H.W., K R A USE, R., Am inosäureuntersuchungen an verschiedenen organischen Sedim enten,
besonders Grau- u. Braunhuminsäurefraktionen verschiedener B od en typen (einsch ließ lich 14C-markierter
Huminsäuren; Z. Pflanzenernaehr. Bodenkd. 9 6 ( 1 9 6 2 ) 11.
[7] IWASCHKEWITSCH, T.M., KUPREWITSCH, W .F., TSCHERBAKOW A, T ., D ie freien Am inosäuren im
B oden, Ber. Akad. WissBelouruss. S.S.R . V I (1 9 6 2 ) 523; Z. Pflanzenernaehr. Bodenkd. 1 0 5 ( 1 9 6 4 ) 56.
[8] ZIECHMANN, W,, KICKUTH, R., D ie Struktur der Huminsäuren, Kolloid Z. 1 7 4 1 (1 9 6 1 ) 39.
[9 ] SCH EFFER , F ., ULRICH, B ., Humus und Humusdüngung I , Stuttgart (1 9 6 0 ) 1 —4 9 , 7 1 —214.
[1 0 ] HOLLEM AN, A .F ., WIEBERG, E., Lehrbuch der anorganischen Chem ie, Walter de Gruyter, Berlin (1 9 6 4 ) 35.
[1 1 ] KICKUTH, R., C hem ische A bbauversuche an einer natürlichen Hum insäure, D issertation, U niversity o f
G öttingen (1 9 5 9 ) 24.
 IA E A -SM -211 /4 4

C H E M IC A L A L T E R A T IO N S O F N A T U R A L L IG N IN B Y j
IN T E R A C T IO N S W IT H H U M IC -L IK E A U T O X ID A T IO N
P R O D U C T S O F P Y R O G A L L O L ( 1 ,2 ,3 -T R IH Y D R O X Y B E N Z E N E )
I
T. WEICHELT j
Interfakultatives Lehrgebiet Chemie, |
University of Göttingen, J
Göttingen, !
Federal Republic of Germany |

Abstraa I
I
CHEMICAL ALTERATIONS OF N A T U R A L LIGNIN BY INTERACTIONS WITH HUMIC-LIKE A UTO XID ATIO N
PRODUCTS OF PYROGALLOL (1 ,2 ,3-TR IH YD R O X YBEN ZEN E). j
Investigations on chem ical interactions b etw een natural lignin and au toxid ation products o f pyrogallol are
described. Pyrogallol after au toxid ation catalysed by Si0 2 under con d ition s similar to nature (soil, p eat) was used
as a m od el substance for a group o f phenols reacting in alm ost th e same w ay. T he m easurem ents w ere carried ou t
b y m eans o f absorption spectroscop y in the regions o f electron excitation as w ell as m olecular vibration and
rotation, and later b y a Warburg apparatus to register th e gas dynam ics, and b y electrical con d uctivity, thin-layer
chrom atography, special colour reactions and various oth er analytical m ethods. Apart from th e fact that interactions
b etw een lignin and autoxidizing pyrogallol could be tested exactly, other im portant results o f the investigations were
th e adsorption o f O 2 and th e release o f CO 2 in the reaction m ixture, and th e structure o f lignin was altered in m any
respects. T he electron rr-orbitals decreased, m ainly because o f a m ore or less drastic dim inution o f fu nction al groups
such as aldehyde and a - and 0 -k eto groups and structures such as stilb en oid e and phenylcoum arone elem ents in th e
aliphatic parts o f lignin, w here also alcoholic and p henolic h yd roxy groups were lost. On the other hand, carboxyl
or ester lactone groups increased as w ell as m eth oxyl. B y chrom atography m eth od s th e form ation o f a com plex
b etw een lignin and parts o f th e hum ic-like au toxid ation products o f pyrogallol could be identified. In general,
lignin was degraded by the indicated chem ical interactions m ainly follow ing o xid ative reaction m echanism s. The
release o f CO 2 even show s th e total d ecom p osition o f som e o f th e material.

1. INTRODUCTION ;

As no reaction between natural lignin and already system-stabilized humic substances by

the methods described here could be proved, the lignin was added to phenols, which during
their autoxidation reacted with lignin to form humic-like substances. This means that the humic
substances can only react with lignin when they are still nascent.
The following experiments, for which pyrogallol was chosen as the model substance to form
humic-like substances, are divided into two groups of special interest: first the experiments to show
the chemical effects of the reaction mixture on the whole, and secondly the group of analytical
measurements that investigate in detail the altered lignin.

2. EXPERIMENTS

2.1. Test conditions

Pyrogallol was the basic material used to produce humic-like autoxidation products catalysed
by finely divided Si02, whereas lignin itself was not altered by Si02.
The experiments were so arranged that all single components (lignin, phenol, catalyst, solvent)
as well as their combinations (reaction mixtures) could be measured and interpreted separately.

67
68 WEICHELT

TABLE I. INTERPRETATION OF ULTRA-VIOLET SPECTRA TO INDICATE CHEMICAL

INTERACTIONS BETWEEN NATURAL LIGNIN AND AUTOXIDIZING PYROGALLOL
0.1 cm cells, E = absorbance (log I/log I0)

N o. C om ponents Meas ured Interpretation (E )

cone. me X.
W ithout Sum o f lignin D iff. betw een
(see Section 2 .2 )
nm E solvents and pyrogallol sum and 4
and S i 0 2 (2 + 3) (effec t o f reaction)
Де

Solvent m ixture 283 0.36

1 plus catalyst 277.5 0.395 - - -
( S i 0 2) 279 0 .3 9

2 Lignin in 1 283 1.24 0 .8 8

2 .27 -
3 Pyrogallol in 1 277.5 1.785 1.39

C om bination o f -0 .2 9
4 279 2.37 1.98 -
2 and 3 in 1 (dim inution)

2.2. Reaction mixture

The reaction mixture consisted of 500 mg lignin, 63 gpyrogallol and 26 g Si02' in 1 litre
7-butyrolacton/H20 (1:1 vol.%). In relation to the whole solution the concentration of pyrogallol
was 0.5M and that of Si02 0.4M. The single components measured in parallel had the same
concentration. Continuous shaking guaranteed a supply of oxygen. Care was taken to ensure that
the concentration was not altered and no evaporation occurred during the reaction procedures so
that exact measurements of all components investigated in parallel could be obtained.

2.3. Investigation of the reaction mixture

2.3.1. E le ctro n spectroscopy

To indicate the chemical interactions between lignin and autoxidizing pyrogallol, first the
absorption spectra in the ultra-violet and visible region were measured after a reaction time of
four weeks. The spectra registered were interpreted at their maxima or at any other suitable
wavelength.

2.3.1.1. Absorption in the ultra-violet region

The registered and interpreted measured values of the ultra-violet spectra from 210—340 nm
are summarized in Table I.

2.3.1.2. Absorption in the visible region

The interpretation of the spectra measured from 340—700 nm show again a diminution of
absorption in the reaction mixture (Table II).
In Tables I and II the absorption was interpreted only at a certain wavelength. But whereas
the spectra in the visible region of all single components (lignin, autoxidizing pyrogallol and solvent)

Merck.
 IA E A -SM -211/44 69

TABLE II. INDICATION OF CHEMICAL INTERACTIONS BETWEEN NATURAL LIGNIN

AND AUTOXIDIZING PYROGALLOL BY THE ABSORPTION OF LIGHT IN THE
VISIBLE REGION
0.1 cm cells, E = absorbance (log I/log I0)

N o. C om ponents Measure d values Interpretation (E )

cone.: ma X. W ithout Sum o f lignin D iff. betw een
(see S ection 2 .2 ) E
nm solvents and pyrogallol sum and 4
and S i0 2 (2 + 3) (effec t o f reaction)
Дб !

Solvent m ixture
1 plus catalyst 400 0 .1

( S i0 2)

2 Lignin in 1 400 0 .375 0.2 7 5

1.575 -
A utoxidizing
3 400 1.4 1.3
pyrogallol in 1

C om bination - 0 .3 5 5
4 400 1.32 1 .2 2
o f 2 + 3 in 1 (dim inution)

TABLE III. EXPERIMENTAL ARRANGEMENTS TO MEASURE THE GAS DYNAMICS

DURING THE REACTIONS BETWEEN LIGNIN AND AUTOXIDIZING PYROGALLOL

N o. Variants Main chamber Side arm*

1 Therm obarom eter 2 .0 ml 7 -butyrolacton: —

H20 ( 1 : 1 vol.%)
2 Solvent m ixture SM = 1.8 ml 7 -butyrolacton: 0 .2 ml 0 .4 S i0 2-
(SM) plus S i0 2 H 2 0 ( 1 :1 vol.%) suspension
3 Lignin in 2 1 . 8 ml lignin solu tion in Suspension
SM, cone.: as above
5 m g/m l = 9 g lignin
4 Pyrogallol in 2 1 . 8 ml pyrogallol solu tion in Suspension
SM, cone.: as above
1/2 m = 63 mg pyrogallol
5 C om bination o f 1 . 8 ml solu tion o f the Suspension
lignin and com bination in SM, cone.: as above
pyrogallol in 2 5 mg lignin in 63 mg pyrogallol

a Start o f the reaction w hen th e S i0 2 in th e side arms has been converted to th e m ain chambers.

show a continual trend of diminishing absorption without special maxima, minima and shoulders,
the ultra-violet spectra (see Section 2.5.1) had rather different shapes depending on the substance
and wavelength.

2.3.2. Gas dyn a m ic in a Warburg apparatus

The chemical interactions between lignin and autoxydation products of pyrogallol were also
investigated by measuring the gas dynamics (Table III).
70 WEICHELT

O j- uptake

F I G .l. 0 2 - u p ta k e o f lig n in ( 1 ), a u to x id iz i n g p y r o g a llo l ( 2 ) a n d th e c o m b i n a tio n (3 ) o f b o th , w ith o u t th e a b ility

o f a b s o r b in g C 0 2 b y N a O H a n d a fte r th e e f f e c t o f th e c h o s e n s o lv e n t m ix tu r e w i th S i 0 2 is s u b tr a c te d . C u rve 4

is t h e c a l c u l a t e d s u m o f o x yg e n u p ta k e o f t h e s e p a r a te ly r e g is te r e d s in g le c o m p o n e n ts lig n in a n d a u to x id iz i n g

p y r o g a llo l (1 + 2 ). T h e a r r o w s in d ic a te th e d iff e r e n c e in 0 2 ~ u p ta k e b e tw e e n th e s u m (4 ) a n d th e c o m b in a tio n (3 ).

D im in u tio n o f 0 2 - u p ta k e in d ic a te s r e le a s e o f g a s.

0 2- uptake

F I G .2 . 0 2 - u p ta k e o f lig n in (1 ), a u to x id iz in g p y r o g a llo l (2 ) a n d th e c o m b in a tio n (4 ) o f b o th w ith th e p o s s ib ility

o f a b s o r b in g C 0 2 b y N a O H . T h e e ffe c t o f th e m e d iu m (s o lv e n t, S i 0 2 ) w a s s u b tr a c te d . T h e a r r o w s in d ic a te th e

0 2-u p ta k e a s th e d iffe r e n c e b e tw e e n c u r v e s 4 a n d 3 , th e la s t b e in g th e c a lc u la te d s u m o f th e s e p a r a te ly m e a s u r e d

s in g le c o m p o n e n ts 1 + 2 .

2.3.2.1. Warburg experiments without C02-adsorption

The gas dynamics were measured at first without NaOH (no C 02-absorption). Some of these
results are shown in Fig.l.
As a result of the reaction between lignin and autoxidizing pyrogallol, the combination of
these two components took up less oxygen than the separately measured single components.
This effect can be explained by a release of gas. A further Warburg experiment was undertaken to
investigate the kind of gas released during the reaction process.
 IAEA -SM -211/44 71

TABLE IV. CHROMATOGRAPHICAL INVESTIGATIONS ON THE REACTION

PRODUCTS OF LIGNIN AND PYROGALLOL

Experim ental D escription o f the chromatogram Remarks

arrangements
Lignin A utoxidizing C om bination
pyrogallol o f lignin
and pyrogallol

Thin layer plates: Rf = 0.89 Rf = 0.75 R f = 0 .6 9 The reaction

cellu lose ( 0 - 0 .2 5 mm) Red spot Brow n spot Very weak red m ixture and
M obile solvent : parts o f it had
n-butanol/dioxan ( 2 : 1 ) Red trace the smallest
Tem p.: 23°C m obility
E xposure tim e: 3 1 /2 h Red coloured Red sp o t at
Colour test: spot at th e the beginning
phloroglucin/H C l beginning

2.3.2.2. Warburg experiment with C02-absorption

It was believed that C02 was the gas released. In order to absorb it, NaOH was placed in the
central chambers of each Warburg vessel. The results are summarized in Fig.2.

2.3.3. Chrom atography

Chromatographic investigations should give further information on the interactions between

lignin and autoxidizing pyrogallol after a reaction time of two months. The summarized results in
Table IV clearly demonstrate a bond between parts of lignin and some of the humic-like aut-
oxidation products of pyrogallol.
The reduced mobility of the new complex between lignin and humic-like autoxidation
products of pyrogallol may be caused by the formation of greater particles (larger molecular mass)
or by a reduction of functional groups because of chemical bonds.

2.3.4. Electrical c o n du ctivity

By measuring the electrical conductivity in the fluid medium of dioxan/water, the interactions
between lignin and autoxidizing pyrogallol could be tested also. Whereas lignin did not change its
conductivity over the whole reaction time, pyrogallol increased its conductivity during its humi­
fication. However, this increase in electrical conductivity was significantly higher when lignin
was also added.

2.3.5. pH-values

The concentration of hydrogen ions in the solutions used in these experiments was very
similar to those in soil. This is shown in Table V.

2.4. Separation of the reaction mixture

To investigate the reacted lignin separately it had to be isolated from the reaction mixture
without further alterations, which was possible by the method of separation outlined in Fig.3.
72 WEICHELT

TABLE V. pH-VALUES DURING THE INTERACTIONS BETWEEN LIGNIN AND

AUTOXIDIZING PYROGALLOL

No. C om ponents pH-v dues

A t the beginning After 2 \ m onths

o f experim ents

7 -b u tyrolacton /H 2 0
1 ( 1 : 1 vol.% ) 5.2 5.2
plus S i0 2

2 Lignin in 1 5.1 5.1

A utoxidizing
3 4.5 3.5
pyrogallol in 1

C om bination
4 (lignin + pyrogallol) 4.5 3.5
in 1

Reaction mixture

(filtration of SiO^)

Coagulation of the lignin in the thirtyfold

amount of Na2S04-solution (1%)

Separation of lignin by filtration Filtrate encases the autoxidation

(membrane ~ 2 jum) or products of pyrogallol
centrifugation. The coagulate
is cleaned by repeated washing
with dist. H20 and n-butanol

The complex between lignin The above-mentioned products

and humic-like autoxidation coagulate with HCI (pH < 3) and
products of pyrogallol is are separated by filtration or
probably present in the centrifugation
butanol fraction

F I G .3 . S e p a r a tio n o f r e a c tio n m ix tu r e .

2.5. Investigations on the reacted lignin

2.5.1. Ultra-violet spectroscopy

The comparison of the u.v. spectra between unaltered lignin and lignin already reacted and
isolated from the reaction mixture shows significant differences (Fig.4). With the help of the so-
called Де-method [1—3], where one spectrum is subtracted from'the other, the difference is made
more evident (Fig.5).
The diminution of absorption of the u.v. light by the reacted lignin can be caused by the loss
of carbonyl groups of various kinds, as well as by the transformation of phenylcoumarone in
phenylcoumaran elements. Further possibilities are the decay of the aliphatic cis- and trans-double
bonds or of such bonds in stilbenoide structures. Some more details are revealed by i.r. spectroscopy.
 IA E A -SM -211/44 73

F I G .4 . U ltr a -v io le t s p e c tr a : ( 1 ) u n r e a c te d lig n in ; ( 2 ) r e a c te d lig n in . C o n c e n tr a tio n : 0 .5 m g fm l; 0 .1 cm c e lls ;

s o lv e n t « n -d io x a n -H 2 0 f9 :1 v o l.% ) , d o u b le - b e a m s p e c tr o p h o to m e te r .

FJG.5. R e s u lt o f th e tie - m e th o d ( th e r e d u c e d a b s o r p tio n o f th e r e a c te d lig n in is d e m o n s t r a te d ) .

F I G .6 . In fr a -r e d s p e c tr a : ( 1 ) u n r e a c te d lig n in , ( 2 ) r e a c te d lig n in . C o n c e n tr a tio n : 3 m g lig n in p lu s 3 0 0 m g K B r.

 -J

TABLE VI. INTERPRETATION OF SOME IMPORTANT INFRA-RED ABSORPTION BANDS (COMPARISON BETWEEN UNREACTED
LIGNIN AND LIGNIN REACTED WITH AUTOXIDIZED PYROGALLOL
D = Density in %; E = Extinction

D ifferen ce b etw een I + II

Reason for absorption + = increase o f absorpt.
Sarkanen (1 9 7 1 ) Unreacted lignin I R eacted lignin II - = decrease o f absorpt.
cm 1 o f th e reacted lignin

2935 OH-stretching, m eth yl, D 7 0 -1 9 6 .9 8 - 14 -

m ethylene E 0 .1 5 5 - 0 .721 = 0 .5 6 6 0 .1 5 5 - 0 .8 5 4 = 0 .6 9 9 - 0 .1 3 3

1920- Carbonyl valence = U nconjugated D 6 2 - 3 5 .9 59 - 37.1 -

10 k eto and carboxyl groups E 0 .2 0 8 - 0 .4 4 5 = 0 .2 3 7 0 .2 2 4 - 0 . 4 3 1 = 0 .2 0 7 - 0 .0 3 0

WEICHELT
1660- Carbonyl valence = para- D 5 7 .2 - 25 5 3 .0 - 2 4 .2 -
55 substituted aryl k eton e E 0 .2 4 2 - 0 .6 0 2 = 0 .3 6 0 .2 7 6 - 0 . 6 1 6 = 0 .3 4 - 0 .0 2 0

1595 A rom atic skeletal vibrations D 5 1 - 9 .3 4 5 .6 - 9 .8

E 0 .2 9 2 - 1 .032 = 0 .7 4 0 .3 4 1 - 1 .0 0 9 = 0 .6 6 8 - 0 .0 7 2

1270 Guaiacyl ring breathing D 4 3 .2 - 0 .1 3 6 .5 - 0.5 -

w ith CO-valence E 0 .3 6 5 - 3 .0 0 0 = 2 .6 3 5 0 .4 3 8 - 2 .3 0 0 = 1 .8 6 2 - 0 .7 7 3

1220 Syringyl ring breathing D 4 2 .7 - 2.2 3 5 .7 - 2 -

w ith CO-valence E 0 .3 7 - 1 .6 5 8 = 1.288 0 .4 4 7 - 1 .6 9 9 = 1 .252 - 0 .0 3 6

1085 0 = 0 deform ation sec. D 41 - 6.2 3 3 .3 - 4 -

alcohol and aliphatic ether E 0 .3 8 7 - 1 .2 0 8 = 0.821 0 .4 7 9 - 1 .3 9 8 = 0 .9 1 9 + 0 .0 9 8

1035- A rom atic C-H in plane D 4 0 .3 -1 .9 3 2 .4 - 1.5 -

30 deform ation, Guaiacyl typ e and E 0 .3 9 5 - 1 .7 2 2 = 1.32 0 .4 9 0 - 1 .8 2 4 = 1 .3 3 4 + 0 .0 0 7
C = 0 d eform ation , prim, alcohol

975 > CH o u t o f plane deform ation D 3 9 .5 - 2 5 .8 3 0 .3 - 2 0 .9 -

(trans) F. 0 .4 0 3 - 0 . 5 8 8 = 0 .1 8 5 0 . 5 1 9 - 0 . 6 8 = 0 .161 -0 .0 2 4
TABLE VII. COMPARISON OF THE EFFECT O F THE PHLOROGLUCIN/HC1 COLOUR TEST ON THE UNREACTED LIGNIN
1 cm cells in th e reference beam : n-dioxan/HCl

N o. Variants Lignin Phloroglucin/H Cl- HClc End cone, E 555 E ^555 E rel. D ecrease o f
solu tion 3 solu tion b o f lignin max. max. aldehyde
colour groups
(m l) (m l) (m l) (m g/m l) effect (%) (%)

IA E A -SM -211 /4 4
a C ontrol
Unreacted solution 2.5 0 .2 5 0 .2 4
1
b lignin Colour
solution 2.5 0.25 - 1.2 0 .9 6 100 -

a C ontrol
solution 2.5 0 .2 5 0 .2 3 _ _
R eacted
b lignin C olour
solu tion 2.5 0.25 - 0 .9 0 .6 7 6 9 .8 3 0.2

Cone. = 5 .0 m g/m l solvent.

b
Cone. = 8 .3 3 m g/m l; H C l:d = 1.125.
D ensity o f HC1= 1.125.
TABLE VIII. INTERPRETATION O F u.v. SPECTRA A FTE R REDUCTION O F THE UNREACTED AND REACTED LIGNIN BY SODIUM On
BOROHYDRIDE.
Solvent: dioxan/H 20 (9 :1 vol. %); 0.1 cm cells; reaction tim e: 11 h; for concentrations see A ppendix.

Measured values in E = ex tin c tio n parts Interpretation

(A ) Unreacted lignin (B ) R eacted lignin

и II j he iv 4 Vs V l6 V II 7 V III 8 IX 9 X,o
X lignin lignin in lignin in Reacted Reacted Reacted D iff. betw een D iff. b etw een D iff. betw een D iff. b etw een V III 8 + IX 9
nm alone NaOH NaOH + lignin alone lignin in lignin in E + 1V 4 n 2 + H i3 v 5 + V I6 (decreased absorption o f
NaBH„ NaOH NaOH + NaBH4 (decreased (e ffe c t o f (e ffe c t o f reacted lignin = higher
absorption o f redu ction o f redu ction o f co n ten ts o f carbonyl groups)
the reacted the unreacted the reacted
lignin) lignin) lignin)

275 1.27 1.38 1.36 1 .1 6 1.135 1.21 - 0 .1 1 - 0 .0 2 - 0 .0 2 5 - 0 .0 0 5

WEICHELT
280 1.49 1.52 1.5 1.31 1.335 1.31 - 0 .1 8 - 0 .0 2 - 0 .0 2 5 - 0 .0 0 5
285 1.585 1.61 1.577 1.37 1.38 1 .3 5 - 0 .2 1 5 - 0 .3 3 -0 .0 3 0 - 0 .0 0 3
2 8 5 .5 1.55 1.613 1.573 1 .3 3 1.376 1.345 -0 .2 2 -0 .0 4 0 - 0 .0 3 1 - 0 .0 0 9
290 1.43 1.57 1.52 1.24 1.335 1.300 - 0 .1 4 - 0 .0 5 - 0 .0 3 5 - 0 .0 1 5
295 1.13 1.41 1.365 0 .9 9 1.2 1 .162 - 0 .1 4 - 0 .0 4 5 - 0 .0 3 8 - 0 .0 0 7
300 0.95 1.29 1.24 0 .8 3 1.1 1.051 - 0 .1 2 - 0 .0 5 - 0 .0 4 9 - 0 .0 0 1
305 0.8 8 5 1.18 1.13 0 .7 6 0 .9 9 2 0 .9 4 5 - 0 .1 2 5 - 0 .0 5 - 0 .0 4 7 - 0 .0 0 3
310 0 .8 4 6 1.050 0 .985 0 .7 2 0 .8 9 0 .8 4 4 - 0 .1 2 6 - 0 .0 6 5 - 0 .0 4 6 -0 .0 1 9
315 0.8 0.93 0 .878 0.6 8 7 0 .8 0 0.75 - 0 .1 1 3 - 0 .0 5 2 -0 .0 5 -0 .0 0 2
320 0 .778 0 .856 0 .8 0.65 0 .7 3 5 0 .6 8 7 - 0 .1 2 8 -0 .0 5 6 - 0 .0 4 0 -0 .0 1 6
325 0.7.4 0 .8 0 4 0 .7 5 0.61 0 .6 9 5 0 .6 3 8 - 0 .1 3 -0 .0 5 4 - 0 .0 5 7 + 0 .0 0 3
330 0.71 0 .778 0 .7 2 0 .5 7 0 .6 6 7 0 .6 0 7 - 0 .1 4 -0 .0 5 8 -0 .0 6 0 + 0 .0 0 2
335 0.6 7 5 0 .755 0 .6 9 7 0 .5 2 8 0 .6 4 0 .5 8 - 0 .1 4 7 -0 .0 5 8 -0 .0 6 0 + 0 .0 0 2
340 0 .6 2 0 .7 3 4 0 .6 7 2 0 .4 8 0 .6 1 5 0 .5 5 - 0 .1 4 -0 .0 6 2 -0 .0 6 0 -0 .0 0 2
345 0.5 4 3 0.71 0.6 4 2 0 .4 0 .5 8 7 0 .5 2 3 - 0 .1 4 3 -0 .0 6 8 -0 .0 6 4 -0 .0 0 4
350 0.4 7 7 0 .687 0.6 1 8 0 .3 7 4 0 .5 5 7 0 .4 9 8 - 0 .1 0 3 -0 .0 7 0 - 0 .0 5 9 - 0 .0 1 1
355 0 .4 0 8 0 .6 5 0 0.4 8 3 0 .3 2 0 .5 2 3 0 .4 7 0 - 0 .0 8 8 -0 .0 6 7 - 0 .0 5 3 -0 .0 1 4
360 0 .3 2 0 .6 0 2 0 .5 4 0 .2 6 5 0 .4 8 3 0 .4 3 4 - 0 .0 5 5 -0 .0 6 2 - 0 .0 4 9 - 0 .0 1 3
 IA E A -SM -211/44 77

2 .5 .2 . In fra -r e d s p e c tr o s c o p y

Com parison o f the i.r. spectra o f the unreacted and th at o f the reacted lignin shows also m any
differences (Fig.6). An exact interpretation o f the spectra was carried o u t in Table VI.

2 .5 .3 . P h lo r o g lu c in /H Q c o lo u r r e a c tio n

The determ ination o f aldehyde groups, which are conjugated w ith the arom atic system o f
lignin, chiefly coniferyl, but also p-coum ar and sinapin aldehyde structures, was carried out by
quantitatively measured colour reactions w ith phloroglucin and HC1 on the unreacted lignin as
well as on th a t reacted w ith autoxidizing pyrogallol. In Table VII the experim ental arrangements,
the measured values and the in terpretation are summarized.
The difference betw een the content in aldehyde structures o f the unreacted lignin and o f
the (reacted) lignin isolated from the reaction m ixture, as show n in Table VII, is very great.

2 .5 .4 . R e d u c tio n w ith s o d iu m b o r o h y d r id e

The purpose o f the NaBH4-reduction was to eliminate all keto groups in the a - and /3-position
o f th e aliphatic side chains as well as all aldehyde groups, in order to show w hether the chemical
interactions betw een lignin and autoxidizing pyrogallol inducted new carbonyl groups as in
carboxyl, lacton o r ester groups. The effect o f the reduction was investigated by u.v. and i.r.
spectroscopy.

2.5.4.1. NaBH4-reduction followed by ultra-violet spectroscopy

The m ethod o f interpretation sum m arized in Table VIII was partly carried out as described
in Refs [1 -3 ].
The results indicate th a t during the interactions betw een lignin and autoxidizing pyrogallol,
except for a decrease in carbonyl groups (as described in Section 2.5.1), new groups were created,
such as carboxyl, ester and lacton groups.

2 .5 A .2 . NaBH4-reduction follow ed by infra-red spectroscopy

The interpretation o f the NaBH4-reduction by i.r. spectroscopy (Table IX) gave some m ore
detailed results on the newly form ed carbonyl groups in the reacted lignin.

2 .5 .5 . P h e n o lic h y d r o x y l g r o u p s

D eterm ination o f the free phenolic hydroxyl groups (Table X) was carried o u t by a
spectroscopically measured colour reaction using l-nitroso-2-naphthol. The experim ents were
controlled by eugenol (1-propane, 2-hydroxy-, 3-m ethoxy-benzene) as a m odel substance for lignin.
More details are given in th e A ppendix.

2 .5 .6 . M e th o x y l g r o u p s

The m ethoxyl groups o f lignin were determ ined as described in Ref. [4]. The measured values
and their in terpretation are sum m arized in Table XI. As show n by th e control substance o-m ethoxy-
benzoic acid, the m easurem ent error was about 3% , so th a t the relatively high increase in m ethoxyl
caused by the interactions betw een lignin and autoxidation products o f pyrogallol is trustw orthy.
 -J
00

TABLE IX. INTERPRETATION O F SOME IMPORTANT FREQUENCIES IN i.r. SPECTRA O F UNREACTED AND REACTED LIGNIN A FTER
ITS REDUCTION WITH NaBH4
For m ore details see Appendix

cm ”1 Reason for Unreacted lignin R eacted lignin D iff. b etw een I + II Remarks
absorption reduced w ith NaBH 4 reduced w ith NaBHU (values unrelated to
Ia IIa reacted lignin)b

OH-valence, 1) 6 1 . 4 - 3 5 4 .7 - 3 .6 —
3420 H bonded 2) 0 .2 1 1 - 1 .5 2 3 = 1 .312 0 .2 6 2 - 1 .0 1 8 = 0 .7 5 6 - 0 .5 5 6
3) 5 8 .4 51.1 - 7 .5

OH-stretching 1) 5 8 . 6 - 9 5 0 .8 - 1 3 _
2935 m ethyl and 2) 0 . 2 3 2 - 1 .0 4 6 = 0 .8 1 4 0 .2 9 - 0 .8 8 6 = 0 .5 9 6 -0 .2 1 8

WEICHELT
m ethylene 3) 4 9 .6 3 7.8 - 1 1 .8
T he reacted lignin
Carbonyl valence 1) 3 8 .7 - 3 6 .5 3 2 .2 - 2 9 .6 - show s an increase in
1 7 5 0 -1 0 unconjugated carbonyl 2) 0 . 4 1 2 - 0 .4 3 8 = 0 .0 2 6 0 .4 9 2 - 0 .5 2 9 = 0 .0 3 7 + 0.0 1 1 carboxyl, ester or
carboxyl groups 3) 2.2 2.6 + 0 .4 lactone groups

1) 2 9 .4 - 1 1 2 4 .6 - 9 .2 _
Carbonyl valence
1 6 6 0 -5 5 substituted 2) 0 . 5 3 2 - 0 .9 5 9 = 0 .4 2 7 0 . 6 0 9 - 1 .0 3 6 = 0 .4 2 7 ±0
aryl k eton e 3) 1 8.4 1 5.4 - 3 .0

Arom atic 1) 1 9 .7 - 3 .3 1 6 .7 - 3 .4 _
1595 skeletal 2) 0 . 7 0 6 - 1 .4 8 2 = 0 .7 7 6 0 . 7 7 7 - 1 .4 6 9 = 0 .6 9 2 -0 .0 8 4
swinging 3) 16.4 13.3 - 3 .1

a 1 = O ptical d en sity in percentage; 2 = E xtinction (log I/log I0); 3 = Scale parts.

b + = increased absorption; — = decreased absorption.
TABLE X. COMPARISON O F THE FREE PHENOLIC HYDROXYL GROUPS IN UNREACTED LIGNIN AND IN LIGNIN REACTED WITH
AUTOXIDATION PRODUCTS O F PYROGALLOL

I = control Lignin 1- Nitrose-

Dioxan H N 0 3b HC1 End. D ilution d = cm E % 385 R eal colour Proportion o f th e C ontent o f Decrease
Substances

sam ple solu tion 3 2- naphthol- cone. (cells) max. effec t OH -groups per phenolic in
II = colour solu tion b (effec t o f w eight unit and OH-groups phenolic
reaction colour fin al absorbance OH-groups
(m l) (m l) (m l) (m l) (m g/m l) reaction) valueb

о I 5 - 1 0.2 5 1.5 3.7 - 0.1 - - „ - -

IA E A -SM -211/44
.00 и 5 1 - 0.25 1.5 3.7 1/3 0.1 0 .3 9 3 1 0 0 .3 9 1.1 100% -
3
= 11.7 = 10.6 4

тз
о I 5 - 1 0.25 1.5 3.7 - 2 - - - - -
Unrea
lignin

II 5 I 0.25 1.5 3 .7 2 1.035 3 - 0 . 5 - 1.035 1 14.6%

= 1.55 1.55 -

•a I 5 - 1 0.2 5 1.5 3.7 - 2 - 8 - -

React
lignin

II 5 1 0.25 1.5 3.7 2 0 .9 7 3 - 0 .5 - 0 .9 7 1 13.6% -6 .8 %

= 1.45 1.45 (reacted
lignin)

3 C oncentration: 0 .5 m g/m l.
b See A ppendix.

-j
vo
80 WEICHELT

TABLE XI. COMPARISON OF THE METHOXYL GROUPS IN THE UNREACTED LIGNIN

AND IN LIGNIN REACTED WITH AUTOXIDATION PRODUCTS OF PYROGALLOL

N o. Sample Weight o f th e V olum e o f OCH3-con ten ta Increase o f

investigated thiosulphate m eth oxy groups
substances
(mg) (m l) (m g) (%) (%)

o-m eth oxy-b en zoic 4 .1 5 -

1 21 6.8
acid 4 .2 8 b —

2 Lignin 22 .0 6 5.23 3 .1 9 14.5 -

Lignin reacted w ith

3 22 .4 7 7 .1 2 4 .3 4 19.3 4.8 ± 0 .1 4 4
au toxy. pyrogallol

a C ontent o f m eth oxyl: volum e o f thiosulphate X 0 .5 1 7 0 6 X factor o f th e thiosulphate solution = mg OCH3 ;

1 ml thiosulp hate = 0 .5 1 7 0 6 m gO C H 3. T h e factor o f th e thiosulphate solution was 1 .1 7 9 , so that the
factor necessary to calculate th e con ten t o f m eth oxy is 0 .6 0 9 6 .
b Calculated value.

F IG . 7. R e la tio n s b e tw e e n lig n in a n d a u to x id a tio n p r o d u c ts o f p h e n o ls a s w e ll a s f u r t h e r fa c to r s c a u s in g th e

d e c o m p o s itio n o f lig n i n .

3. INVESTIGATION ON THE AUTOXIDATION PRODUCTS OF PYROGALLOL

The formation of humic-like substances during the autoxidation process of pyrogallol, which
is parallel with the creation of other substances under the chosen conditions, was demonstrated by
electrical conductivity, absorption spectra in the u.v., visible and i.r. region, also by the solution
and coagulation in various solvents and at different pH-values. However, a detailed description
is not given here.
 IA E A -SM -211/44 81

4. DISCUSSION

With the help of a variety of methods including electron spectroscopy in the u.v. and visible
region, i.r. spectroscopy, and the Warburg technique, and also thin-layer chromatography, the use of
electrical conductivity and the application of special colour reaction, as well as several other
analytical methods, it was not only possible to indicate chemical interactions between natural
lignin and autoxidation products of pyrogallol under test conditions very similar to nature (soil,
peat, etc.), but the many alterations that occurred in the structure of lignin could be measured
analytically also. So apart from the uptake of oxygen, a release of C02 and a diminution of
light absorption in the u.v. and visible region, registered on the reaction mixture of lignin and
pyrogallol, dissolved in 7-butyrolacton/H20 (1:1 vol.%) in the presence of Si02 as catalyst, the in­
vestigations indicated that the тг-orbitals of the lignin had decreased, particularly in cis- and trans­
double bonds and a- and /З-keto as well as aldehyde groups in the aliphatic side chains of lignin and
in phenylcoumarone and stilbenoide structure elements. On the other hand, some ^-electronic
systems were increased by the reaction procedures (carboxyl, ester and/or lacton groups). The
esterification of carboxyl with alcoholic hydroxyl groups, besides radical mechanisms, possibly
forms complexes between lignin and humic-like substances of autoxidized pyrogallol. The
reduction in phenolic hydroxyl groups by the interactions seemed to proceed at least partly parallel
with the formation of oxygen bridges, as could be shown by i.r. spectra, and the increasing content
of methoxyl groups indicated a relative diminution of aliphatic structures.
The reactions and structural alterations of lignin by the interactions with autoxidation
products of pyrogallol are typical for the decay of lignin, where condensation and, above all,
oxidative processes play a large part. The total decay is chiefly related to the aliphatic structures
of lignin.
Because of the fact that lignin in soil can produce phenols by the activity of microbes,
autoxidation of at least part of these phenols leads to the (abiological) decay of lignin, as described
here. These aspects and some further relations are expressed in the scheme shown in Fig.7.
In order to guarantee a better decomposition of straw in soil, where lignin is the limiting
factor of the turn-over in this material, the supply of lignin in soils should be as high as possible,
and burning of straw or other methods of total removal should be kept to a minimum.

5. SUMMARY

Chemical interactions between natural lignin and humic-like autoxidation products of

pyrogallol (1,2,3-tri-hydroxybenzene) produced many effects on the reaction mixture
( 0 2-uptake, C02-release, decrease of absorption in the u.v. and visible region) and altered the
structure of lignin to a large extent (decrease of rr-electron orbitals, loss of phenolic hydroxyl,
alcoholic, aldehyde and keto groups, whereas the methoxyl, carboxyl, ester and/or lacton groups
increased). On the whole the lignin was degraded abiologically, mainly in its aliphatic structure
elements. The results of the experiments described are in many respects similar to the effects which
were obtained in consequence of the chemical interactions between lignin and hydrochinon
(p-di-hydroxybenzene) under soil conditions, although there were also some remarkable
differences [5].

Appendix
DETAILS OF EXPERIMENTS

Apparatus Perkin-Elmer, E T S—3T (u.v.; visible region; near i.r.)

Perkin-Elmer, Grating 4 5 7 (i.r. 4 0 0 0 — 4 0 0 cm -1 ).
82 WEICHELT

M ethods

R ed uction w ith NaBH4 :

(a) 4 m l lignin solu tion (unreacted or from the reaction-m ixture-separated lignin), concentration: 1.0 m g/m l
in n-dioxan; 2 m l distilled H20 w ere added.

(b ) 4 ml lignin solu tion (see above) plus 2 m l 0 .0 3 N NaOH.

(c) 4 m l lignin solu tion (see above) plus 2 ml NaBH4-solution in 0 .0 3 N NaOH, w hich had a concentration o f
0.1 mg NaBH4 per m l 0 .0 3 N NaOH.

T he u.v. spectra was measured on th e dissolved substances, and th e i.r. spectra were measured after
evaporation and preparation o f KBr-pellets.

Phenolic h yd roxy groups

C oncentrations: lignin = 0 .5 m g/m l dioxan

l-nitrose-2-naphthol: 0.01%
H N 0 3 = 2.5N ; H C l:d = 1.125.

The solu tion s were added as described in Table X; reaction tim e: 6 hours; m easurem ent at Xmax 3 8 5 nm.

To calculate the ratios o f th e w hole con ten t o f phenolic OH groups per unit o f w eight, th e m olecular mass
o f a phenylpropane unit in lignin w as assumed as 180. Therefore o n e gram o f lignin has
6.02
-------X 1023 = 3 3 .3 X Ю20 phenylpropane units, whereas eu gen ol has 3 6 .7 X Ю20 m olecules, so that th e ratio
180
o f phenol-bearing units is 1 :1 .1 per unit o f w eight.

Lignin preparation

The lignin was isolated from P i c e a e x c e l s a b y th e slightly m od ified m ethods o f Björkman [6 ] and Pepper,
Baylis and Adler [7]. T hese m eth od s gave lignins very like th ose described in R efs [ 8 ,9 ], and w hich were tested
b y further experim ents [1 0 —12].

REFERENCES

[1] A ULIN -ERD TM A N , G ., U ltraviolet spectroscopy o f lignins and lignin derivates, T echn. A ssoc. Pulp. Paper
Ind. 3 2 ( 1 9 4 9 ) 260.
[2] A ULIN -ERD TM A N , G., Sven. Papperstidn. 56 (1 9 6 8 ) 1187 .
[3] A ULIN -ERD TM A N , G., S A N D E N , R ., A cta Chem. Scand. 2 2 (1 9 6 8 ) 1187.
[4] GATTERM ANN-W IELAND, D ie Praxis des organischen Chem ikers, 41st E dn, Walter DeGruyter, Berlin,
(1 9 6 2 )7 4 .
[5] WEICHELT, T h ., “C hem ical interactions betw een lignin and autoxidizing h ydroch in on ” , Int. Peat Sym posium ,
D anzig, 1 9 74, 88.
[6] BJÖRKM AN, A ., Studies o n fin ely divided w ood: I. Extraction o f lignin w ith neutral solvents, Sven.
Papperstidn. 59 (1 9 5 6 ) 4 7 7 .
[7] PEPPER, J.M ., et al., The isolation and properties o f lignin obtained b y th e acidolysis o f spruce and aspen
w ood in dioxan-water m edium , Can. J. Chem. 37 (1 9 5 9 ) 1241.
[8] ZIECHMANN, W., WEICHELT, Th., Chem ische Veränderungen bei der Isolierung von Lignin,
T eil I: A usbeuten, M ethoxylgehalte, U V-Spektren, Z. Pflanzenernaehr. Bodenkd. (in press).
[9] ZIECHMANN, W., WEICHELT, T h ., Chem ische Veränderungen bei der Isolierung von Lignin,
Teil II: IR-Spektren, Phloroglucin-HCl-Nachweis, Z. Pflanzenernaehr. Bodenkd. (in press).
[10] WEICHELT, Th., M ÜLLER-WEGENER, U ., Fluorescence o f lignin (unpublished).
[11] KRESS, B.M., WEICHELT, Th., ZIECHMANN, W., Phosphorescence o f lignin (unpublished).
[12] WEICHELT, Th., T h e use o f fluorescence and p hosphorescence to investigate biological material,
Int. Peat S ym posiu m , Posen, Sept. 1976 (in press).
 IAE A-SM -211 /4 4 83

DISCUSSION

M. METCHE: In view of your results one question arises whether the autoxidation catalyst
of the pyrogallol'was not copper. Several observations mentioned in the literature seem toindicate
that this autoxidation does not occur when the reaction takes place in a silica vessel and in a
solution of triple-distilled water.
Your paper seems to me very interesting because the model you studied includes processes
involving radicals and it is therefore conceivable that the autoxidation of lignin is promoted by
coupling with a reaction leading to oxidation of a simple compound (pyrogallol). We now have
some results which tend to confirm your observations.
Moreover, such models are made considerably more interesting by observations made on the
organic matter of soils. T. Carballas and F. Andreux have recently demonstrated the primary role
of the process of walnut leaf a-hydrojuglone oxidation in the development of the organic matter
of a soil covered by mixed vegetation (Graminaceae and walnut). 1
T. WEICHEÜT: From the literature and from my own investigations I know that several
copper and various silicium oxide compounds can initiate the so-called autoxidation of a variety
of phenols. Although the research I described was not primarily directed towards studying the
effects of various catalysts that cause the autoxidation of phenols, I did study the effect of Si02,
Si02 •2H20 , Si02 •nH20 , Cun S04, KOH and NaOH in parallel experiments using a variety of
phenols, such as pyrogallol, hydroquinone, p-quinone and pyrocatechin. I first took as a medium
distilled H20 alone and then added one of the catalysts mentioned above. All these gave a clearly
positive effect; I selected Si02 because it had a rather long reaction time, and because it presents
conditions very similar to those of soil (pH, clay minerals). The effect of the various catalysts I
measured by using electron spin resonance (decrease after the starting period), u.v. spectra
(decrease ~ 270—295 nm), absorption spectra in the visible region (increase) and also gravimetrical
determination of the humic substances.
In my investigations, pyrogallol was used as a model substance to form humic substances
(autoxidation products) that altered the lignin by chemical interactions in many ways. But during
these interactions new phenols such as pyrogallol can be produced by decomposition of lignin,
too, as I indicated in Fig.7.
Many thanks for your interest and the reference to additional literature.
W. FLAIG (S cien tific S ecreta ry): How do you explain the increase in ester groups?
T. WEICHELT: The increase in ester groups is due to oxidative processes on various
functional groups of lignin, such as coniferyl and unconjugated and conjugated aldehyde groups,
the latter can be of a syringyl, coniferyl and a p-coumaric nature. All these groups form carboxyl
groups that react with aromatic as well as aliphatic hydroxyl groups of the humic substances
produced by pyrogallol, thus producing the corresponding ester groups. Some of the humic
substances were bound by such ester linkages, whereas another part reacted with lignin by radical
mechanisms.
W. FLAIG ( S cien tific S ecreta ry): Did you consider the possibility of tropolone systems being
formed during oxidation of pyrogallol in your explanation of reactions? The formation of this
type of compound is very rapid.
T. WEICHELT: I regarded the aromatic tropolone systems that have many electron я-orbitals
as very important in the chemical interactions between lignin and autoxidizing pyrogallol, mainly
because of their ability to form bi-radicals and the corresponding mesomeric formations. As the
investigation of the chemical alteration of lignin was the main purpose, the tropolones (or very
similar systems) were estimated only by using u.v. spectra, as was the case for purpurogallols,
gallic acid, etc. Other measurements using fluorescence and phosphorescence (emission) spectro­
scopy also indicated the importance of tropolone systems but the results are not published here.
 IAEA -SM -211 /4 5

P O S S IB IL IT IE S O F A N E C O N O M IC A N D
N O N -P O L L U T IN G U T IL IZ A T IO N O F L IG N IN

W.H.M. SCHWEERS,
Bundesforschungsanstalt für
Forst- und Holzwirtschaft,
Hamburg

W. VORHER
Ordinariat für Holztechnologie der
• Universität Hamburg,
Hamburg,
Federal Republic of Germany

Abstract

POSSIBILITIES OF A N ECONOMIC A N D NON-POLLUTING UTILIZATION OF LIGNIN.

The problem o f lignin u tilization , in spite o f m any years o f effo rt, has n o t been solved satisfactorily. One
o f the m any reasons for th e difficulties encountered in the prod uction o f useful products, either polym eric or
m onom eric, is th e fact that in technical processes, such as w ood pulping or w ood hydrolysis in w hich lignin is
obtained as a by-product, the natural lignin undergoes condensation reactions and/or is transform ed to sulphur-
containing derivatives. These alterations o f the natural lignin m olecu le make it less reactive, and influence
strongly its degradability to m on om eric products. If lignin is isolated from w o o d y plants or oth er lignocellulose-
containing m aterials b y phenolysis, lignin is obtained in a less condensed state and h en ce can easüy be degraded
w ith good yield to m onom eric phenols. A lso its chem ical reactivity, particularly because o f its high con ten t in
phenolic OH-groups, is m uch higher than the reactivity o f th e usual technical lignins. For exam p le, it can be
reacted easüy w ith dnsocyanates to obtain polyurethanes. It could be show n that the phenolysis o f lignin also
is an alternative to existing pulping procedures. H ence it should be possible to obtain also the by-product lignin
from these processes w hich n ow am ount to approxim ately 4 0 m illion ton s annually in a less condensed and higher
reactive state.

Lignocellulosic material, such as wood and straw, is one of the largest renewable resources of
carbon-containing raw material in the world. The annual net primary production of carbon fixed
by various major land ecosystems amounts to approximately 54 X 109 t [1 ].
Since the carbon content of lignocellulosic material is approximately 50%, the production of
total dry mass amounts to 108 X 109 t annually. If one works on an average lignin content of
20% of this material, the annual production of lignin in nature is 21.6 X 109 t, representing
12 X 109 t of organic bound carbon.
So far this vast amount of organic bound carbon has been almost unused as a source of the
production of chemicals or chemical products, since we do not have at our disposal technically
and economically feasible processes to isolate the lignin in such a structural state that allows easy
chemical degradation to useful products.
Technical processes by which lignin is separated from the carbohydrate portion of the ligno­
cellulosic material, such as the different pulping processes and processes for wood hydrolysis,
yield lignin in a drastically altered state. It is more condensed than in its natural form, and further­
more in many of these processes such as the sulphate- and sulphite pulping it is obtained as a
sulphur-containing derivative. The amount of lignin which at present is obtained in such an altered
state as a by-product of these pulping processes is approximately 40 X 106 t annually. Hence
the ‘technical lignins’, äs they are obtained at present, are difficult to degrade and less reactive than
natural lignin, and because of the sulphur content are not suitable for a series of further chemical

85
86 SCHWEERS and VORHER

сн, он сн,он
I 2 I 2
н-с н-с-
H'-С-0— / ~ Л — С-С- СН20Н Н-С+ + НО— S— С-С- СН2ОН

а осн, а осн,
ь 5
I
он

СН2ОН
о
Н-с-
H.-?—О - он
осн,
0 i
1 F I G .l. R e a c tio n o f p h e n o l w ith lig n in .

TABLE I. PULPING OF PINE WOOD WITH PHENOL; PULPING

CONDITIONS

Tim e Tem perature

(h) (°C )

0.5 2 0 to 110

1 110

2 110 to 170

3 170
0-25 170 to 50

Hydrom odul: 1:10

A cid catalyst: HC1 or HOOC-COOH 0.05 — 2%

TABLE II. PULPING OF PINE WOOD WITH PHENOL;

MATERIAL BALANCE

Wood 2 0 0 0 g (o .d .)

Phenol 12364g

Lignin in w ood 28% = 5 6 0 g

Screened pulp 5 3 .3 % = 1066 g

Kappa N o. 6 3 .6
Lignin in pulp 9 .5 4 % = 101.7 g

Screenings 3.5% = 7 0 g

Lignin in screenings 2 8 % = 19.6 g

Lignin extracted 4 3 8 .7 g

Phenol recovered by distillation 12.2 5 6 g

Phenol condensed to lignin 90 g

Crude phenol lignin obtained 1024 g

 IA E A -SM -211/45 87

TABLE III. PULPING OF PINE WOOD WITH PHENOL; PULP QUALITY

Pulp yield

U nscreened 56.8%

Screened 53.3%

Screenings 4.5%

Kappa N o. 6 3.3

T ensile strength 9 .2 0 0 m

Tear factor 53

Burst factor 60

Folding resistance (all at 50° SR) 1.300

Behaviour in bleaching C /E /H /D /E /D 99% GE

TABLE IV. PULPING OF PINE WOOD WITH PHENOL; PYROLYSIS OF PHENOL LIGNIN

P y r o ly s is c o n d itio n s

Temperature 550°C

Sw eeping gas (con tin u ou s) Water vapour

P r o d u c ts o b ta in e d

% based on % based on
crude lignin lignin removed from w ood

Phenol 2 0 .0 4 6 .7 !

o-Cresol 3.9 6.7

p-Cresol 2.0 4.7

E thyl phenol 1.2 2.8

T otal phenols 26.1 6 0 .9

Surplus o f
Phenol 26.2

Phenol m ixture 4 0 .4

Charcoal 29.8

Gases (CO, CH4 , C 0 2) 16.0

Neutrals 14.0

A cids 0 .4

L osses (w ater) 13.7

reactions unless they are desulphurized before use. If one takes into account that the chemical
industry today uses approximately 90 X 106 t annually of organic bound carbon for the production
of its organic products, and is mainly based on crude oil as raw material — a limited and
unrenewable resource which will be available only for the next 30—50 years —it is obvious that
in future it will not be tolerated to waste such large amounts of lignin which contain 22 X 106 t
of organic bound carbon, and hence represent 24% of the amount of carbon that is consumed by
the organic chemical industry.
88 SCHWEERS and VORHER

For an economic use of lignin as a raw material for the production of chemicals it is necessary
to develop processes that allow the separation of lignin from the lignocellulosic raw material in a
less altered form. On the other hand, such a process should also be suitable to obtain the carbo­
hydrate portion of the raw material as pulp in good quality and good yield.
In this connection we have studied the possibilities of pulping wood, and other ligno­
cellulosic matter such as straw, using phenol in the presence of small amounts of an acid catalyst
as pulping agent [2-8]. Phenol reacts with lignin in the presence of acids, as shown schematically
in Fig.l.
The lignin macromolecule is degraded by splitting the ether-bonds, and phenol is condensed
to reactive centres in the а-position of the propane side chains of the phenylpropane units, avoiding
internal condensation of the lignin molecule [9—12].
The phenol lignin formed in this reaction dissolves in the phenol and can be finally extracted
from the crude pulp by isopropanol and 1% NaOH.
The crude phenol lignin is isolated together with the dissolved portion of carbohydrates as
a brown powder by distilling off unreacted phenol and the solvent. This crude material can easily
be utilized for the production of simple monomeric phenols by pyrolysis. The analytical
characteristics of the purified phenol lignin show that it is high in OH-content, and has a low
molecular weight compared with other technical lignins. Recent experiments have shown that
it can be easily reacted with diisocyanates to form polyurethanes.
The pulp obtained by the phenolysis of lignocellulosic materials has also been investigated.
It could be obtained in good yield and it is easily bleachable. Tensile strength, burst factor and
folding resistance are comparable with results obtained by the classical pulping processes. Only
the tear factor is lower, but it is hoped that by changing the pulping condition a better tear
resistance can be reached.
In Tables I—IV details about pulping conditions, the results of pyrolysis of phenol lignin
and data showing pulp quality are presented.
From the results shown in Table IV it can be seen that phenolysis of lignocellulosic material
offers the possibility of converting the lignin extracted to a simple mixture of phenols with a
yield of 40%. This yield is far higher than that obtained in pyrolysis or hydrogenolysis of sulphite
or sulphate lignins as reported in the literature [13, 14].

REFERENCES

[1 ] O LSON, J.S., “Carbon cycles and tem perate w oodlands”, E cological Studies (REICHLE, D .E ., E d.),
Chapman & H all, L on d on (1 9 7 0 ) 226.
[2 ] SCHWEERS, W., PER EIRA , H .N ., Über den Holzaufschluss m it Phenolen, 1. Mitt.: Tränkung verschiedener
H ölzer m it Phenol, H olzforschung 2 6 (1 9 7 2 ) 51.
[3 ] SCHWEERS, W., BEH LER , H ., B E IN H O FF, O ., Über den H olzaufschluss m it Phenolen, 2 . M itt.: V or­
läufige U ntersuchungen über die Phenolbilanz, H olzforschung 2 6 (1 9 7 2 ) 103.
[4] SCHWEERS, W., RECHY, M., Über den H olzaufschluss m it Phenolen, 3. Mitt.: Über den A ufschluss von
Kiefern- und B u ch en h olz, Papier (D arm stadt) 27 (1 9 7 3 ) 6 3 6 .
[5] SCHWEERS, W., RECHY, M., BEINH O FF, 0 ., Phenol pulping, Chem. T ech. (A m sterdam ) (1 9 7 4 ) 4 9 0 .
[6] RECHY, M., U ntersuchungen über die M öglichkeiten des A ufschlusses von H olz m it P h en olen, D octorate
Thesis, Hamburg (1 9 7 3 ).
[7] VORHER, W., E ntw icklung eines Kontinuierlichen Pyrolyseverfahrens zum Abbau von Phenollignin m it
dem Ziel der Ligninverwertung unter besonderer Berücksichtigung der Rückgewinnung von Phenol aus
Ablaugen eines P h en olzellstoffp rozesses, D octorate Thesis, Hamburg (1 9 7 6 ).
[8] V ORHER, W., SCHWEERS, W.H.M., U tilization o f phenol lignin, Appl. P olym . Sym p. 2 8 (1 9 7 5 ) 2 77.
[9] B R A U N S, F .E ., The Chem istry o f Lignin, A cadem ic Press, N ew York (1 9 5 2 ) 4 79.
[1 0 ] B R A U N S, F .E ., B R A U N S, D .A ., The Chemistry o f Lignin, Suppl. V ol., A cadem ic Press, N ew York (1 9 6 0 ) 4 7 7 .
[1 1 ] KRATZL, K., Z A U N ER , L , C LAUS, P., R eaktionen m it m arkierten Ligninen und M odellen, 1. Mitt.:
K ondensation m it Phenol, H olzforschung 18 (1 9 6 4 ) 47.
 IA EA -SM -211/45 89

[12] NIMZ, H., Kondensationsreaktionen des Lignins, Holzforschung 23 (1969) 84.

[13] GOHEEN, D.W., MARTIN, J.B., Preparation o f guajacol from spent pulping liquors, Canadian Patent
791.772, 1968.
[14] GOHEEN, D.W., “ Low molecular weight chemicals” , Lignins —Occurrence, Formation, Structure, and
Reactions (SARKANEN, K.V., LUDWIG, C.H., Eds), Wiley Interscience, New York, London, Sidney,
Toronto (1971) 575.

DISCUSSION

M.H.B. HAYES: Your phenolysis mechanism suggests cleavage of an ether linkage, release
of an alkoxide, formation of a carbonium ion and finally condensation with the 4-position of
the phenol. Such reactions can have very considerable implications in humic acid structure deter­
minations (Jackson, Swift, Posner, S oil Sei, and Piper and Posner, S o il Sei, so I would appreciate
a more detailed description of this mechanism.
W.H.M. SCHWEERS: We do not add acidic phenol, but hydrochloric or oxalic acid. The
reaction mechanism has not been studied in our laboratory, but there are publications on it by
Kratzl and co-workers [11] and Nimz [12]. They studied reactions of phenols with lignin or
lignin model compounds, and also looked at degradation products, in particular those obtained
from the oxidation of the condensation products with nitrobenzene. They succeeded in showing
that condensation takes place between the first carbon of the side chain, i.e. the one next to the
aromatic ring, and the ortho or para position of the phenol. Cations react easily with a phenol in
either position; this is a well-known substitution reaction. When the cation is formed by splitting
of the ether, a proton, H+, is added to the ether bridge, which is split, and the resulting cation
reacts with the phenol.
B.R. NAGAR: What type of pyrolyser did you use, and what was the pyrolysis time?
W.H.M. SCHWEERS: We used hydrochloric or oxalic acid as catalysts for phenolysis; for
some reason sulphuric and acetic acid did not work in this way. We did not use catalysts at all
for pyrolysis; we simply heated the material. The pyrolysis time was rather short; we did not
measure it accurately, since the raw material was introduced into a tube heated to 550°C with a
device that moved the raw material along this tube. However, it was approximately 30—60 s.
The main problem is that one must use a sweeping gas, water vapour or nitrogen, because the
monomeric phenols —the degeneration products —have to be removed from the hot zone promptly,
before they are further degraded. Thus the monomeric material remains in the hot tube for
seconds only, whereas the phenol lignin is heated for 30—60 s.
 IA E A -SM -211 /4 6

O X ID A T IO N O F S O M E P H E N O L IC S U B S T A N C E S
A S IN F L U E N C E D B Y C L A Y M IN E R A L S

Z. FILIP
Institut für Landwirtschaftliche
Mikrobiologie,
J ustus-Liebig-Universität,
Giessen

W. FLAIG, E. RIETZ
Institut für Biochemie des Bodens
der Forschungsanstalt für Landwirtschaft,
Braunschweig-Völkenrode,
Federal Republic of Germany

Abstract

O X IDA TIO N OF SOME PHENOLIC SUBSTA N CES A S INFLUENCED B Y CLAY M INERALS.

In m od el experim ents, the oxygen uptake o f 5-m ethylpyrogallol, 2 ,3,4-trihyd roxyb en zoic acid, 2 ,3 ,5 -
trihyd roxytoluene, as w ell as o f the m ixtures o f these p henols w ith protocatechu ic acid, caffeic acid and orcine,
was n o t at all or to a small ex ten t influenced by the presence o f m ontm orillon ite in the water suspension buffered
at pH 7 or 8. Furtherm ore, m ontm orillon ite and kaolinite w hen saturated w ith 1 Г , K +, Ba2 + , Ca2 +, A l3 + or
F e3 + ion s, and added to the pyrogallol solu tion buffered at pH 6 .0 , influenced neither the speed o f the colour
changes, nor the in ten sity o f the brow n colour, evaluated as an extin ction at 3 0 0 nm . H ow ever, b oth phenom ena
were influenced b y m uscovite. N o direct relationship has been found betw een the valence o f the cations in the
exchange p osition s o f m uscovite used and the catalytic effect o f this clay mineral.

INTRODUCTION

Soil humic acids consist of more than 45% humus-like fungal melanins and more than 20%
of different phenolic substances [if. According to Haider and co-workers [2], the oxidative
reactions of phenolic substances, sometimes without any participation by microbial phenoloxidases,
play an important role in the humification processes. As the production of fungal melanins is
markedly accelerated and enhanced after the addition of clays to the culture media [3], tests were
made to establish the possible effect of some clay minerals on the oxygen uptake of phenols and
their mixtures, and also on the browning reaction of pyrogallol.

MATERIAL AND METHODS

For the respirometric tests, 20 /nmol of protocatechuic acid, caffeic acid or orcinol were dis­
solved in phosphate buffer at pH 7.0 or 8.0 and were placed in the main container of the Warburg
vessels with or without addition of 0.5% (wt/vol.) of montmorillonite (Wyoming). In the side arm
of the Warburg vessels, 5.0 pmol of 2,3,5-trihydroxytoluene, 2,3,4-trihydroxybenzoic acid or
5-methylpyrogallol dissolved in 0.5 ml H20 were placed. In the central container of the vessels,
0.2 ml of 10% NaOH solution was present. After heating to 28°C both reagents were mixed together
and the oxygen uptake of the mixtures was measured for 3 to 4 h. In some experiments 10 ppm
of Fe2+ ions were also added to the reaction mixtures.

91
92 FILIP e t al.

TABLE I. OXYGEN UPTAKE FROM REACTION MIXTURES OF PHENOLS WITH AND

WITHOUT MONTMORILLONITE

R eaction m ixtures д т о 1 0 2 at pH 7 д т о 1 0 2 at pH 8
Conrol M ontm. Control M ontm .

5 д т о 1 5-MPG a 5 .6 5.5 6.1 6.2

+ 20 Mmol PCA 6 .0 5 .8 6.4 6.2
+ 20 jUmol CA 6 .5 6 .5 9.3 9.2
4- 20 jUmol Ore, 6 .0 5.8 6.3 6.5

5 Mmol 2,3,4-TH BA 3 .3 2.3 6.0 6.4

+ 20 Mmol PCA 3 .5 3 .6 6.9 7.0
+ 20 д т о 1 CA 5 .0 5 .6 13.0 11.3
+ 20 д т ol Ore, 3 .6 3 .5 11.5 11.4

5 Mmol 2,3,5-TH T 5.5 5 .6 5.9 6.4

+ 2 0 Mmol PCA 6 .4 6.8 8:0 7.5
+ 2 0 д т о 1 CA 8 .0 7.0 10.4 10.0
+ 20 д т о 1 Ore. 6 .5 6.2 6.8 6.2

5 д т о 1 5-MPG +
10 ppm F e3 + 8.6 7.8 11.2 12.4
+ 20 Mmol PCA 8.6 6.6 12.6 13.5
+ 20 д т о 1 CA 10.4 8.1 15.8 16.2
+ 20 д т о ! Ore. 9.7 8.6 13.9 14.0

a 5-MPG — 5-m ethylpyrogallol; PCA = protocatechu ic acid; CA = caffeic acid; Ore. = orcinol;
2,3,4-T H B A 2 ,3,4-trihyd roxy ben zoic acid; 2,3,5-T H T = 2,3,5-trihyd roxytoluene; F eJ + = FeSC).-,.

For the spectrophotometric tests, 100.0 mg of pyrogallol were dissolved in 40 ml of 0.1N

natrium acetate solution buffered at pH 6.0. With the exception of control variants, 40.0 mg of
fine ground quartz, or the same quantity of clay minerals (a fraction < 2 pm) were added. The clay
minerals used (kaolinite, muscovite, montmorillonite) were saturated with H+, K+, Ba2+, Ca2+,
Al3+ or Fe3+ ions before use. The flasks with the reaction mixtures were shaken at 100 rev/min
under a light intensity of 600 W/l m2 at a constant temperature of 22°C. After 4, 24 and 48 hours
respectively, parallels were examined. First the mixtures were centrifuged at 12 000 g for 20 min
and then the pyrogallol solution free from minerals was measured in the range of 250—600 nm using
a SP 1800 Phillips/Unicam spectrophotometer. As well as recording the extinction curves, the
extinction values at 300 nm were determined for the evaluation of colour intensity.

RESULTS AND DISCUSSION

The data in Table I show that the oxygen uptake of phenol mixtures has in fact not been
positively influenced by the presence of montmorillonite. In some cases it was lower than in the
controls. As the sorption of the phenols used can be omitted at the given pH values according to
previous tests [4], the suitable conditions for a heterogenic catalysis at the clay surface are
perhaps neglected. After the addition of Fe2+ ions, the oxygen uptake increased a little at pH 8.0,
but this effect disappeared at pH 7.0, probably because the cations became strongly fixed by clay.
 IA E A -SM -211/46 93

F I G .l. A b s o r p tio n s p e c tr a o f r e a c tio n m ix tu r e s : F I G .2 . A b s o r p tio n s p e c tr a o f r e a c tio n m ix tu r e s :

p y r o g a llo l. p y r o g a llo l + k a o lin ite .

F I G .3 . A b s o r p tio n s p e c tr a o f r e a c tio n m ix tu r e s : F I G .4 . A b s o r p tio n s p e c tr a o f r e a c tio n m ix tu r e s :

p y r o g a llo l + m o n tm o r illo n ite . p y r o g a llo l + m u s c o v ite .

94 FILIP e t al.

These results indicate that montmorillonite alone does not act as a catalytic factor by auto-
oxidative changes of phenolic mixtures which are common in the young cultures of soil fungi
producing dark-coloured melanins.
As the humification must be regarded as to some extent a browning phenomenon of organic
substances, further experiments were carried out using pyrogallol, a trihydric phenol, which can
be oxidized by molecular oxygen to a dark-coloured polymer product. It is also known that
pyrogallol can be formed during oxidative changes of more simple phenols of biological origin [2].
The oxidative changes of pyrogallol were estimated spectrophotometrically in the u.v. range,
where a newly prepared colourless solution very strongly absorbs at 266 nm only. With the lapse
of time the pyrogallol solution became yellow, reddish brown and finally brown. In Fig.l one
can see a strong absorption shoulder at 300 nm and a weak one at 370 nm after 4 h. The first
appeared as a strong absorption band after 24 hours and became a little weaker after 48 hours but
the 370 nm shoulder disappeared almost completely at the same time.
Whereas the molecular oxygen only caused the oxidation and the respective spectral changes
in the pure pyrogallol solution, a secondary oxidation by electron transfer could additionally be
taken into consideration when clay minerals are present in the reaction mixtures. However, the
absorption spectra were only very little changed by kaolinite and montmorillonite. The absorption
at 300 nm and at 370 nm is a little weaker in Figs 2 and 3, but an additional absorption band at
340 nm appeared, particularly when Fe3+ or Al3+ were the exchangeable cations. A blue-colour
reaction, probably caused by reduction of trivalent cations, was responsible for these spectral
changes, which are no longer detectable after 24 hours.
Among the mineral substances used, the muscovite influenced most extensively the absorption
spectra of the pyrogallol solution. In Fig.4 the spectral curve of the 48-hour reaction solution
shows only a very low absorption shoulder at 266 nm instead of a strong band, which is characteristic
for pyrogallol. Below the 300-nm absorption shoulder, the spectral curve shows no longer any
clear-cut absorption bands, similar to soil humus and humus-like substances [5].
Considering that the absorption band or shoulder at 300 nm is very characteristic of oxidative
changes of pyrogallol under the experimental conditions, the optical density of pyrogallol solutions
measured at that wavelength has been chosen for some comparative evaluation of the degree of
pyrogallol oxidation. Other authors also [6,7] recommend the use of the optical density at 300 nm
for estimating the amount of oxidized material formed by a polymerization of polyhydric phenols.
The data in Table II show a general increase in the optical density at 300 nm in dependence on a
reaction time from 4 to 48 h. Compared with the pure pyrogallol solution, the extinction values
are only very little influenced by kaolinite and montmorillonite, but they are distinctly influenced
by muscovite. Therefore o n ly the muscovite, a mica clay, enhanced the formation of a brown
polymer substance in a buffered pyrogallol solution, whereas both kaolinite and montmorillonite
were only a little or not at all active. Measured according to the end effect, i.e. after 48 h of the
reaction time, even the pure quartz (Merck) exerted more influence than either kaolinite or
montmorillonite. It is also of interest to note that mono- and divalent cations in exchangeable
positions of clay minerals were more active than Fe3+ and Al3+. Therefore trivalent metal ions,
when in exchange sites, do not need to increase the oxidation, which can be brought about much
more by the activation of oxygen through chemisorption on or polarization by a solid surface.
Similarly Goodman and Siegel [8] have demonstrated the effect of solids on the oxidation of pyro­
gallol when cellulose was present in the reaction mixture. Other authors observed the oxidation of
hydroquinone to p-benzoquinone even in a nitrogen atmosphere, when Fe3+ or Cu2+ were
exchangeable cations [6], but the opposite reaction, namely the reduction of Prussian brown, was
also observed to be enhanced in the presence of di- and trivalent cations in exchange sites of
bentonite [9]. One can also assume that the possible oxidation power of trivalent metal ions,
which is based on electron transfer reactions, could be made ineffective with regard to the
experimental substance when some oxidizable impurities (e.g. organic materials) are present in the
clays added. In some accordance with our results Kumada and Kato [10], using different natural
 IA EA -SM -211/46 95

TABLE II. INFLUENCE OF QUARTZ AND CLAY MINERALS ON THE BROWNING

REACTIONS OF PYROGALLOL IN A NATRIUM ACETATE SOLUTION (pH 6.0)

E xtin ction values at 3 0 0 п т

R eaction m ixtures after 4 h after 2 4 h after 4 8 h

Pyrogallol (P) 0 .0 6 3 0 .5 0 0 0 ,5 9 2

P + Quartz 0 .075 0 .6 2 0 0 .7 9 5

P + K aolinite-H 1+ 0 .055 0 .5 8 7 0 .6 8 5

P + K aolinite-K 1+ 0 .0 5 8 0 .4 1 7 0 ,6 2 2

P + K aolinite-Ba2+ 0.051 0 .6 4 5 0 .6 4 5

P + Kaolinite-Ca2+ 0 .0 6 6 0 .5 8 7 0 .7 1 5

P + K aolinite-A l3+ 0 .0 5 6 0 .5 5 0 0 ,5 6 5

P + K aolinite-F e3+ 0.0 5 6 0 .4 1 7 0 .6 3 0

P + M uscovite-H1+ 0 .0 7 6 0 .7 5 0 1.2 6 0

P + M uscovite-K 1+ 0 .0 8 6 0.7 5 5 1.1 3 0

P + M uscovite-Ba2+ 0.0 7 3 0 .6 1 2 0 .6 7 0

P + M uscovite-Ca2+ 0 .0 7 0 0 .7 4 0 1.2 5 0
P + M uscovite-A l3+ 0 .0 7 0 0 .4 8 0 1 .2 2 0

P + M uscovite-Fe3+ 0.0 6 3 0 .5 8 0 1.0 4 0

P + M ontm orill.-H 1+ 0 .0 5 0 0.3 5 5 0 .7 2 0

P + M ontm orill.-K l+ 0 .0 6 0 0 .5 2 2 0 .7 2 7

P + M ontm orill.-Ba2"1" 0 .0 6 0 0 .5 2 0 0 .7 1 0

P + M ontm orill.-Ca2+ 0.0 5 5 0 .4 3 2 0 .5 4 0

P + M ontm orill.-A l3+ 0.0 6 5 0 .4 4 0 0 .4 4 0

P + M ontm orill.-Fe3+ 0.2 8 5 0 .4 6 0 0 .6 1 0

and ore clays, refer to their influence on the speed of browning reactions but not to spectral
changes in the pyrogallol solution.
From the results achieved in our experiments, and also from the results of other authors,
it can be concluded that both clays and other solid particles can act as catalytic factors in the
oxidation of йоте phenolic substances important in humification processes. However, a positive
or negative clay effect depends on specific conditions that are not yet exactly determined.
Because the effects mentioned are not only important for the theory of humus-forming processes,
but could also be of practical importance for the alteration of phenolic substances in waste water,
they will be further studied in detail.

REFERENCES

[1 ] SCHNITZER, M ., N E Y R O U D , J.A ., Soil B iol. B iochem . 7 (1 9 7 5 ) 365.

[2 ] H A ID ER , K „ M ARTIN, J.P., FILIP, Z., “Hum us biochem istry”, Soil B iochem istry IV (PA U L , E.A .,
McL a r e n , А Л ., Eds), M. D ekker, N ew York ( 1 9 7 5 ) 196.
[3 ] FILIP, Z ., H A ID ER , K ., M ARTIN, J.P., Soil B iol. B iochem . 4 (1 9 7 2 ) 147.
[4 ] FILIP, Z., “W echselbeziehungen zw ischen Mikroorganismen und Tonm ineralen und ihre Auswirkung auf
die B odendynam ik”, H abilitationsschrift, University o f Giessen (1 9 7 5 ).
96 FILIP e t al.

[5 ] FILIP, Z., SEM OTÄN, J., KUTILEK, M ., Geoderm a 15 (1 9 7 6 ) 131.

[6 ] THOMPSON, T .D ., MOLL, W .F., Jr., Clays Clay Miner. 21 (1 9 7 3 ) 337.
[7] THOMPSON, T .D ., T SU N A SH IN A , A ., Clays Clay Miner. 21 (1 9 7 3 ) 3 51.
[8] GOODM AN, N .S ., SIEGEL, S.M., N ature (L ondon) 184 (1 9 5 9 ) 53.
[9 ] Y A R IV , S., Israel J. Chem. 7 (1 9 6 9 ) 4 5 3 .
[1 0 ] K U M A D A, К ., К А Т О , H ., Soü Sei. Plant Nutr. 16 (1 9 7 0 ) 1 95.
 B IT U M E N S IN
S O IL O R G A N IC M A T T E R
(S e ssio n 7a)
 IAEA-SM-211/47

LIPIDS OF MICROBIAL ORIGIN

IN SOIL ORGANIC MATTER*
, G.H. WAGNER, E.I. MUZOREWA
University of Missouri,
Columbia,
Missouri,
United States of America

Abstract

LIPIDS OF MICROBIAL ORIGIN IN SOIL ORGANIC MATTER.

Fats, waxes and resins occur in soil organic matter, and may arise from microbial synthesis or accuihulate
as resistant remnants of plant residues undergoing humification. Lipids have been reported to account for about
5% of the organic matter in mineral soils. The significance of micro-organisms in contributing to soil lipids was
investigated by determining the amount and distribution of 14C in lipid fractions after incubating various labelled
substrates in the soil. Amendments were glucose, dextran, and cell wall and cytoplasmic fractions of fungal
sclerotia. After humification of glucose, the l4C recovered using benzene-ethanol to extract lipids equalled 7%
of the residual 14C in soil from a fertilized corn plot, and nearly 8% in soil from an untreated timothy plot.
Lipid 14C extracted from soil in which dextran had been incubated was only about one-half that recovered from
the soil incubated with glucose. Production of l4C microbial tissue was probably less in dextran-treated soil,
and an alternate quantity of 14C may have occurred as polysaccharide. The lipid 14C was distributed among
waxes, resins and asphalt in a similar pattern for all substrates investigated. Cell wall and cytoplasmic components
of sclerotia showed considerable resistance to decomposition; however, recovery of 14C in lipids following
incubation of these substrates was very much less than the amount in parallel extractions of the materials before
incubation. The discrepancy may indicate that added microbial lipids became incorporated into soil humus
without undergoing major degradation, and this condensation rendered them inaccessible to extraction.

INTRODUCTION

Lipids are among the least studied components of soil organic matter. These materials
comprise 1 to 5% of the organic matter in mineral soils, and up to 20% of that in organic soils [ 1].
Lipids are found in fossils of soil-forming rocks, in sediments, and in residues of plants, animals
and micro-organisms. Soil lipids can exercise a considerable effect on soil properties. Waxy
substances may waterproof soil particles to prevent movement of nutrients from mineral surfaces
into solution for plant uptake. They may similarly retard mineralization of organic nitrogen.
Low molecular weight components may behave as organic nutrients, and are likely to produce
harmful effects on plant growth. Numerous lipid components could exert stabilizing forces upon
soil aggregates by cementing mineral particles together, and by imparting water repellency to
loosely held aggregates.
The object of this investigation was to evaluate the significance of micro-organisms in
contributing to soil lipids. It was proposed to determine the amount and distribution of I4C in
several lipid fractions after incubating various labelled substrates in the soil.

* Contribution from the Department of Agronomy, Missouri Agricultural Experiment Station, Journal
Series No.7599.

99
100 WAGNER and MUZOREWA

TABLE I. ANALYTICAL DATA FOR SOILS AND RECOVERY OF CARBON-14 IN

EXTRACTED LIPIDS

pCi of 14C/100 g soil

Soil plota,
Organic C Extracted by
incubation period, Lipid
(%) p Initially After
and amendment incubation ^ Benzene- 14C
added
HCI ethanol (% of total)

Timothy 1.47 5.0

5 months
Glucose 3.98 0.959 0.195 0.074 7.7

Corn 1.33 6.0

5 months
Glucose 3.98 0.616 0.134 0.044 7.1

Corn 1.24 6.0

3 months
Dextran 3.27 0.851 0.092 0.030 3.5

Sclerotial
cytoplasm 7.44 4.82 0.156 0.186 3.8
Sclerotial
cell wall 7.32 6.54 0.096 0.067 1.0

Plot of Sanborn Field [4].

EXPERIMENTAL

The soil under study was a Mexico silt loam (Alfisol) from the upper 15 cm of Sanborn
Field. One plot cultivated continuously to com with adequate fertilization had an organic carbon
content of 1.3% and a pH of 6.0. A contrasting plot for study was cultivated continuously to
timothy with no fertilizer treatment, and had an organic carbon content of 1.5% and a pH of 5.0.
The soils were labelled with 14C because of previous incubations for periods up to 5 months with
labelled amendments including glucose [2], dextran, cell wall and cytoplasmic fractions of
sclerotia of M acroph om in a phaseoli [3]. Analytical data for the soils under study and activity
associated with the amendments are summarized in Table I.
Soils were first leached with 0.1N HC1 to remove basic cations and improve extractability
of lipid components [5]. An extraction-fractionation scheme as shown in Fig. 1, and similar to
that suggested by Morrison and Bick [6], was followed. Lipids were extracted from 20 g units
of soil using 130 ml of 2:1 benzene-ethanol in a Soxhlet apparatus for 20 to 21 h. The extract
was concentrated in a rotary evaporator under reduced pressure at 40°C, and an aliquot taken
for 14C determination. The remainder of the lipid-containing solution was taken to dryness,
re-suspended in 20 ml of light petroleum (b.p.63° - 75°C), and extracted in a micro-Soxhlet for
20 to 21 h to yield a crude wax fraction in solution and an insoluble asphalt. The soluble crude
wax fraction was reduced to a fixed volume, an aliquot taken for 14C counting, and the remainder
dried in vacuo. Crude waxes were further fractionated by refluxing for 1 h in 1:1 methanol-iso­
propanol after which any insoluble materials in the hot solvent were added to the previously
separated asphalt. Upon cooling the methanol-iso-propanol, the extracted matter separated into
 IAEA-SM-211/47 101

SOIL

Pre-Treatment
0.1N HCI

Soxhlet
Extraction
2:1 Benzene:Ethanol

Soxhlet
Extraction

F I G .l. S c h e m a tic d ia g r a m o f p r o c e d u r e f o r e x tr a c tio n a n d fr a c tio n a t io n o f s o il lip id s .

a refined wax precipitate and a soluble resin fraction. Aliquots were taken before and after
cooling for 14C determination.
Aliquots for 14C measurement were added to a PPO-POPOP scintillation cocktail [7], and
radioactivity was measured using a Packard Tri-Carb liquid scintillation counter. A standard
14C benzoic acid solution was used to determine counting efficiency.
Total carbon was determined by combusting samples in an electric furnace, trapping the
evolved C0 2 in a scrubbing tower containing NaOH, precipitating the C0 2 with BaCl2 and
titrating with HCI. Radioactivity in the precipitated BaC03 was counted by the liquid scintillation
method [ 8].

RESULTS AND DISCUSSION

The incubation of 14C glucose in soil for a period of 5 months to label soil organic matter
by microbial synthesis yielded residual 14C in the soil, as reported in Table I. The major portion
of the activity initially added to soil had been liberated as C02, and 24% and 15% remained in
the timothy and com soils respectively. Previous investigations have shown that glucose is very
rapidly utilized in these soils [ 8, 9]. During the prolonged incubation it must be assumed that
a number of successive generations of micro-organisms had metabolized the labelled carbon.
102 WAGNER and MUZOREWA

TABLE II. ACTIVITY IN LIPID FRACTION OF M acroph om in a phaseoli

SCLEROTIA

Fraction Total 14C Lipid I4C Waxes Resins Asphalt

(pCi/200 mg) (Percentage of lipid l4C)

Cytoplasm 7.44 3.85 2.2 65.1 15.3

Cell wall 7.32 0.68 1.5 68.2 11.4

F IG . 2. D is tr ib u tio n o f lip id 14C a m o n g r e s in s , w a x e s a n d a s p h a lt f o r s o ils in c u b a te d w ith d iff e r e n t s u b s tr a te s .

At the end of the incubation, glucose carbon had become humified to the extent that the
decomposition rate for 14C was approaching that of much of the native organic matter present
in the soil. A number of investigations have dealt with the distribution of 14C in these soils, and
have included classical fractions of soil organic matter [ 10], amino components [ 11] and
carbohydrates [ 12].
The preliminary leaching of each soil sample with dilute mineral acid removed from 15 to
20% of the residual 14C. This leachate and associated 14C was not investigated further, but it
would include a variety of simple organic components.
The quantity of 14C in the lipid fraction extracted using benzene-ethanol is reported in the
last two columns of Table I. Lipid I4C relative to total residual 14C after incubating glucose was
 IAEA-SM-211/47 103

slightly but significantly higher for the timothy soil than for the corn soil. The timothy soil has
a higher total organic matter content and a lower pH, and the latter parameter would indicate
that fungi had been relatively more significant than bacteria in the humification of the glucose.
Results for extraction of lipids after incubation of dextran in the corn soil are also reported
in Table 1. The dextran-treated soil yielded less lipid 14C than soil incubated with glucose. The
shorter incubation period was not assumed to be the major cause, and rate of decomposition
of dextran at the end of the incubation had declined similarly to that for glucose in a parallel
incubation [12]. A study that compared glucose and dextran during an incubation extending
for three months showed some resistance to decomposition for the polysaccharide [12]. The
lower proportion of 14C in extracted lipid from the dextran-treated soil may reflect a different
population or less 14C microbial biomass with an amount of 14C alternately occurring as
polysaccharide.
The 14C in lipids extracted from soil incubated with cell wall and cytoplasmic components
of sclerotia is shown in Table I. These amendments displayed considerable resistance to
decomposition [3], and the 14C in the extracted lipid must be considered to have arisen from
unaltered or only partially altered amendment as well as from newly synthesized microbial
materials. Extraction of the cell wall and cytoplasmic material before their incorporation into
the soil showed lipid 14C to equal 9% and 52% of the respective totals. Activity associated with
these materials and the distribution of this activity among the lipid fractions is reported in
Table II. After incubating the sclerotial materials in soil, the proportion of residual 14C that
could be extracted as lipid was greatly reduced. Even though decomposition of the amendments
was limited, a marked resistance to extraction with benzene-ethanol developed.
The distribution of the 14C among the several fractions obtained from the extracted lipid
material is reported in Fig.2. Soils incubated with diverse substrates give a similar distribution
pattern for waxes, resins and asphalt. Results for glucose which had been completely utilized
to yield in situ microbial products and tissues were found to be similar to results for highly
resistant sclerotial tissues incubated in soil. Some difference was observed in refined waxes which
were relatively higher for the glucose-treated soils at the expense of asphalts. The latter were
higher for the sclerotial treatments and for dextran also.
The fractionation procedure was not highly refined, and more elaborate procedures
apparently will be needed to determine lipid differences associated with differing microbial
populations in the soil.
The resin fraction constituted the largest proportion of lipid material for all treatments
under study. Some materials were lost during the fractionation procedure, and it is not known
if this loss was equally distributed among the fractions obtained. More likely the loss occurred
during concentration of the initial extract by rotary-vacuum evaporation after which material
adhered tightly to the walls of the flask. Total recovery in the fractions ranged from 75 to 85%
of the crude lipid fraction.
Results of this investigation show that lipids extracted from soil organic matter in mineral
soils may be of microbial origin. Microbially synthesized products of a lipid nature in soil may
become incorporated into soil humus without undergoing major degradative modification.
This presumed condensation imparts significant resistance to extraction by organic solvents to
the lipid materials.

REFERENCES

[1] BRAIDS, O.C., MILLER, R.H., “ Fats, waxes, and resins in soil” , Ch. 6, Soil Components I, Organic
Components (GIESEKING, J.E., Ed.), Springer Verlag, New York (1975).
[2] CHAHAL, K.S., WAGNER, G.H., Decomposition of organic matter in Sanborn Field soils amended with
C-14glucose, Soil Sei. 100(1965) 96.
104 WAGNER and MUZOREWA

[3] WAGNER, G.H., MEDYNSKA-RABINSKA, H., The contribution of melanic fungal tissues to soil organic
matter formation, Humus Planta V (1971) 27.
[4] WAGNER, G.H., “ Significance of microbial tissues to soil organic matter” , Isotopes and Radiation in Soil
Organic-Matter Studies (Proc. Symp. Vienna, 1968), IAEA, Vienna (1968) 197.
[5] HANCE, R.J., ANDERSON, G., Extraction and estimation of soil phospholipids, Soil Sei. 96 (1963) 94.
[6] MORRISON, R.I., BICK, W., The wax fraction of soils: Separation and determination of some components,
J. Sei. Food Agric. 18(1967) 351.
[7] DOBBS, H.E., Oxygen flask method for the assay of tritium, carbon-14 and sulfur-35 labeled compounds,
Anal. Chem. 35(1963) 783.
[8] WAGNER, G.H., “ Microbial growth and carbon turnover” , Ch.6, Soil Biochemistry 3 (PAUL, E.A.,
McCLAREN, A.D., Eds), Dekker, New York (1975).
[9] BEHERA, B., WAGNER, G.H., Microbial growth rate in glucose-amended soil, Soil Sei. Soc. Am., Proc.
38(1974) 591.
[10] MUTATKAR, V.K., WAGNER, G.H., Humification of carbon-14 labeled glucose in soils of Sanborn Field,
Soil Sei. Soc. Am., Proc. 31 (1967) 66.
[11] WAGNER, G.H., MUTATKAR, V.K., Amino components of soil organic matter formed during humification
of 14C glucose, Soil Sei. Soc. Am., Proc. 32 (1968) 683.
[12] OADES, J.M., WAGNER, G.H., Biosynthesis of sugars in soils incubated with 14C glucose and 14C dextran,
Soil Sei. Soc. Am., Proc. 35(1971) 914.
 IAEA-SM-211/48

ANALYSE ET ROLE DES BITUMES

DANS LES SOLS SABLEUX ACIDES

E. FUSTEC-MATHON*, P. JAMBU*,
G. JOLY**, R. JACQUESY**
Universite de Poitiers,
Poitiers,
France

АЫггаа-Яёвитё

ANALYSIS AND ROLE OF BITUMENS IN ACID SANDY SOILS.

In the acid sandy soils of the M6doc Landes (France), the podzols are observed to have a much higher bitumen
content than the hydromorphic type. Study of the acid and neutral fractions of the bitumens extracted from
surface vegetation and various horizons of these soils shows that the acid fractions and the hydrocarbons undergo
less transformation and less degradation in the Ai horizons of the podzols than in the corresponding hydromorphic
soil horizons. The neutral fractions are for the most part only slightly transformed, but are more rapidly degraded,
in the soils. The incubation of soil samples in the presence of 14C-labelled maize straw indicates that the soil
bitumens originate mainly from plants, but that microbial synthesis products are incorporated into this fraction,
more especially in the podzols. The bitumens have a toxic effect on the soil microflora as a whole. The intensity
of this effect varies with the concentration of the extracts, pH of the soil and origin of the microflora. The acid
fraction and the hydrocarbons are the strongest inhibiting agents. The depressant action depends to some extent
on the length of the chains and the nature of the functions. Acidity seems to play the main part in accumulation
of bitumens in the podzols studied. Generally speaking, the bitumens appear to take part in the formation of mor
type humus.

ANALYSE ET ROLE DES BITUMES DANS LES SOLS SABLEUX ACIDES.

Dans les sols sableux acides des Landes du Medoc, on note un taux de bitumes beaucoup plus 61ev6 dans les
podzols que dans les sols hydromorphes. L’etude des fractions acides et neutres des bitumes extraits de la v6getation
superficielle et de divers horizons de ces sols montre que les fractions acides et les hydrocarbures sont moins
transform ^ et moins d6grad6s dans les horizons A | des podzols que dans les horizons correspondants des sols
hydromorphes. La plupart des fractions neutres sont peu tra n sfo rm ^ mais plus rapidement degraddes dans les
sols. L’incubation d’6chantillons de sol en presence de paille de mais marquee au l4C montre que les bitumes
du sol proviennent pour la plus grande part des vegetaux, mais des produits de synthese microbienne s’incorporent
ä cette fraction, en particulier dans les podzols. Les bitumes exercent une action toxique ä l’egard de la microflore
globale des sols. L’intensite de cette action varie en fonction de la concentration des extraits, du pH du sol et de
l’origine de la microflore. La fraction acide et les hydrocarbures sont les plus inhibiteurs. L’action depressive
depend pour une part de la longueur des chaines et de la nature des fonctions. L’acidite semble jouer le role
principal dans Гaccumulation de bitumes au niveau des podzols 6tudies. D’une maniere generale, les bitumes
interviendraient dans la formation des humus de type mor.

1 - INTRODUCTION
Sous le terme de ’‘bitumes" ou simplement de " lip id es du s o l ” on d^signe un
ensemble de produits directem ent e x tr a its du so l par des so lv en ts organiques et
notamment par un melange §thanol - benzene. Cette fra ctio n heterogene (1 ), essen-
tiellem en t lip id iq u e , en e f f e t , n’a ete que peu etu d iee. Cette d esa ffe ctio n est
probablement due au f a i t que le s bitumes ne representent qu'une fra c tio n n e g li-
geable de la m atiere organique dans de nombreux types de s o ls (21. Ces c o n s ti­
tuents sont cependant tr e s abondants dans le s s o ls pauvres, biologiquement peu

* Laboratoire de pedologie — P6dologie des pays atlantiques.

** Laboratoire de chimie organique (XII).

105
106 FUSTEC-MATHON et al.

a c t if s t e l s que le s s o ls acldes e t le s tourbes (1, 3) ou leur ro le e s t certa in e-

ment Important. I ls agira ien t sur le s proprietes physiques du so l et exerceraient
une action toxique sur la germination et la croissan ce des Organes v6getaux sou-
terra in s ; i l s in tervien d raien t egalement sur l ' a c t i v i t e microbienne des s o ls
(1, 4, 5 ).
Au cours de notre etude sur 1 'evolution des s o ls sableux acides de la re­
gion des Landes du M§doc [France), nous avons ete frappes par le s d ifferen ces
importantes des taux de bitumes le long des sequences d ' hydromorphie cro issa n -
te (6 ). Les s o ls de o e tte region sont a ffe c te s par une nappe phreatique peu
profonde e t se rep a rtissen t en fonction du degre d’hydromorphie : des s o ls hy-
dromorphes peu humiferes se developpent dans le s zones b asses, humides alors
que des podzols humiques se d iffer en cien t dans le s zones le s plus e le v e e s. Dans
le s horizons A des s o ls hydromorphes, le taux de bitume represente 0,4 % du sol
e t 6 %du carbone t o t a l. Ce taux augmente tandis que 1 'hydromorphie diminue et
a t te in t dans le s horizons A des podzols 1,7 %du so l e t de 25 ä 30 %du carbo­
ne t o t a l. Les horizons В des podzols en sont depourvus.
Nous avons done essaye de p reciser le s conditions de c e tte accumulation de
bitumes dans certa in s horizons en analysant leur com position, leur evolution et
leur action sur le s p rop rietes b iologiq ues des s o ls .

2 - NETHODES 0 ’ETUDE
Les bitumes ont ete e x tr a its des horizons Aj e t A2 d'un podzol e t de 1 'ho­
rizon A d'un so l hydromorphe au soxh let pendant 0 heures, s o it par un melange
ethanol - benzene, s o it par un melange ether de p etro le - a ceta te d ’eth y le 4 /1 ,
ce dernier permettant d 'e v ite r 1 'e s te r if ic a tio n des acides lib r e s au cours de
1 ’extraction [7 ). Les bitumes ont e te egalement e x tr a its de la vegetation pre-
lev ee en t o t a l i t e ä la surface de ces s o ls , broyee e t homogSneisee La zo­
ne etu diee n 'e st pas actuellem ent p lantee en p ins, mais e i l e l ' e t a i t ä une P e ­
riode relativem ent recente.
Les e x tr a its ont 6te sgpares en une fra ctio n neutre e t une fra c tio n acide.
Dans chaque fr a c tio n , p lu sieu rs fa m ilie s de composes ont ete is o le e s selon le
protocole u t i l i s e par Albrecht (0 ). Les co n stitu en ts de chaque fa m ille ont ete
id e n t if ie s par Chromatographie en phase gazeuse avec programmation de tempera­
tu re. L 'estim ation q u an tita tiv e de chaque substance separee a ete deduite de la
surface du pic correspondent sur le s chromatogrammes. Nous avons e ta b li le s rap­
ports correspondant au "Carbon P re feren tia l Index" [9) : le C .P .I. qui trad uit
la dominance des composes a nombre impair d'atomes de carbones dans le s hydro-
carbures e t le C .P .I.a qui trad u it c e lle des chaines paires dans le s acides et
le s a lc o o ls . Pour 'les auteurs qui ont etudie le s co n stitu a n ts organiques de se ­
diments anciens, le s v ariation s de ces in d ices indiquent une evolution des
con stitu a n ts sous l ' e f f e t de la diagenese (0, 10).
Afin de p reciser l'im portance r e la tiv e des produits d 'o rig in e v eg eta le ou
microbienne dans le s bitumes, des ech a n tillo n s d 1horizon Aj de podzol e t de sol
hydromorphe maintenus ä la cap acite au champ ont ete en rich is en p a ille d e m a is
marquee uniformement au carbone 14 ä raison de 100 mg de p a ille broyee pour 100
g de so l se c . Des ech a n tillo n s de s o l identiques ont ete en rich is en glucose 1**C
ä raison de 2 %par rapport au carbone t o ta l du s o l. Les ec h a n tillo n s ont ete in ­
cubes 3 semaines ä 28°C. L'atmosphere e t a it renouvelee par balayage d ’a ir humi-
d if i § . Apres une extraction ргёа1аЫе des co n stitu a n ts hydrosolubles, le s b itu ­
mes ont ete e x tr a its comme precedemment. La r a d io a c tiv ite de ces fra c tio n s a e t e

La vegetation e s t con stitu ee essen tiellem en t de M o t i n i a e a e r u l e a , E r i c a s c o -

p a r ia , E. t e t r a l i x , E. a i l i a r i s , C a llu n a v u l g a r i s , U le x e u r o p e n s , U. n a n u s
en proportions variab les selon le degre d ’hydromorphie.
 IAEA-SM-211/48 107

TABLEAU I. DISTRIBUTION DES DIFFERENTS TYPES DE CONSTITUANTS DES BITUMES

DANS LES HORIZONS A, ET A2 DE PODZOL ET A! DE SOL HYDROMORPHE [2]
(en mg/100 g de sol sec) I

Podzol Sol hydro-

C onstituants des bitumes morphe
Horizon Aj Horizon A2 Horizon Aj
F r a c tio n a c id e (l )
Honoacides 10,6 3,7 10,4
□ iacides 11,5 2,1 5,2
Acides plus p ola ires 53,0 n.d. 21,4
F r a c tio n n e u tr e
Hydrocarbures 6,1 1,2 2,3
Cetones 10,6 2,1 4,5
A lcools 30,3 1,4 11,0
Esters longs 4,5 0,3 1,0
Glycerides 15,9 2,9 6,5

tl) A c id e s l i b r e s

mesuree en s c in t illa t io n liq u id e dans un melange Toluene - PPO - Р0Р0Р. Les b itu ­
mes ont ete egalement e x tr a its de la p a ille de mais non incubee apres ex tra c­
tio n des composes hydrosolubles.
L 'influ en ce des bitumes sur la m icroflore du so l a ete estim£e ä l'a id e
d'un t e s t de croissan ce de micropopulations t o t a le s . C e lle s -c i provenaient d’un
so l brun le s s iv e [pH 7 ,3 ), des horizons d'un podzol [pH 3,5) e t d ’un so l hy-
dromorphe [pH 5 ,0 ) . Aux m ilieux de cu lture t£moins, 0 ,5 ml d’ethanol-benzene
e ta ie n t ajoutes pour 25 ml de m ilieu , aux m ilieux t e s t s , 0,5 ml de so lu tio n s de
bitumes ä d iffe r e n te s con centration s. 5 a 10 b o ite s ont ete in ocu lees pour cha-
que e s s a i. Les r e s u lta ts ont ёЬ ё exprimes sous forme d'un in d ice de croissan ce :
jc = nombre de colo n ies sur m ilieu t e s t x -|qq
nombre de colo n ies sur m ilieu tSmoin
Un in d ice superieur a 100 trad u it un e f f e t stim ulant de la substance etu -
d iee , un in d ice in fe r ie u r a 100 indique un phenomene d 'in h ib itio n (11, 5 ).

3 - RESULTATS
3.1 - Etude des con stitu an ts
L’ importance r e la tiv e des d iffe r e n ts types de co n stitu a n ts des bitumes e s t
donnee dans le tableau I . Dans le s t r o is horizons, nous retrouvons le s memes f a ­
m ilie s de composes organiques separables en une fra ctio n acide et une fra ctio n
neutre. La fra c tio n acide represente de 55 ä 60 % de 1 ’e x tr a it t o t a l. Bien que
le taux global de bitumes varie d'un horizon a 1 'autre, le s proportions r e l a t i ­
ves des d iffe r e n te s fa m ilie s de composes sont a peu pres id en tiqu es dans chaque
horizon.
3.1.1 - F r a c t i o n a c i.d e
La fra c tio n acide lib r e con tien t des mono et des d ia cid es lin £ a ir e s satures
dans le sq u els dominent le s composes ä nombre pair d'atomes de carbone. E lle ren-
ferme Sgalement des hydroxyacides qui ne sont pas examines dans ce tr a v a il.
1 08 FUSTEC-MATHON et al.

A Plantes

B-C Podzol Ai

D-E Podzol ^2

F -G Sol hyd romorphe A

25 30 nb.de С

M O N O A C I DES DI A C I D E S
F I G .l. H is to g r a m m e s d e r e p a r titio n d e s m o n o a c i d e s ( A , B , D , F ) e t d e s d ia c id e s (С , E , G ).

La fig u re 1 permet de comparer le s histogrammes de re p a r titio n des monoaci­

des et des d iacid es en fon ction du nombre d’atomes de carbone dans la vegetation
e t dans le s t r o is horizons de s o l. Le tableau II indique quels sont le s composes
le s plus abondants dans ces fra c tio n s e t le s valeurs du CPIa.
On con state que pour le s monoacides la longueur des chaines diminue dans le
so l par rapport ä la vägetation de meme que le CRIa. La diminution du CPIa e st
n ette entre le s deux horizons de podzol. Les d ia cid es ne sont pas rencontrSs
dans le s p lantes et leur evolution dans le s divers horizons de so l e s t v o isin e
de c e lle des monoacides.
 IAEA-SM-211/48 109

TABLEAU 11. LONGUEURS DE CHAINES DOMINANTES ET VALEURS DU

C A R B O N P R E F E R E N T IA L IN D E X DANS LES MONO- ET LES DIACIDES

Monoacides D iacides

Maximum CPI. a Maximum CPI.a

»
Vegetation c 30 4,60 Neant Neant

Podzol horizon Aj C2t 3,44 c26 _ c28 2,40

Podzol horizon A2 C20 ' C28 2,22 c24 1,75

Sol hydromorphe c24 ” C28 3,20 c24 2,34

3.1.2 - F r a c tio n n e u tr e
Cette fra c tio n e s t con stitu ee de fa m ilie s de composes lin e a ir e s sa tu res, a
1 'exception des a lc o o ls lib r e s e t des cetones, d erives terpeniques dont la com­
p o sitio n e st pratiquement identique dans le s plantes et dans le s bitumes des
d iffe r e n ts horizons.
La re p a r titio n des hydrocarbures dans le s p lantes e t le s horizons de sol
e s t representee sur la figu re 2 , le tableau III indiquant quels sont le s com­
poses le s plus abondants e t le s valeurs du CPI. Pour ces composes anombre im­
pair d'atomes de carbone dominant, la longueur des chaines tend egaiement ä d i-
minuer dans le s o l. Le CPI, Sieve dans le s vegetaux, diminue tr e s nettement de
1 ’horizon Aj ä 1 'horizon A2 du podzol, sa valeur etant interm ediaire dans le
so l hydromorphe. II convient de noter que le s hydrocarbures du pin, ä chaine
relativem ent courte [in fer ie u r e ä C2o) ne se retrouvent pas en quantite nota­
b le dans le s bitumes des s o ls .
Les g ly cerid es co n stitu en t une part importante de la fra ctio n neutre des
c ir e s e x tr a ite s de la vegetation et egaiement des bitumes des d iffe r e n ts h o ri­
zons a lo r s q u 'ils son t, en general, consideres comme peu abondants dans le s s o ls .
On trouve des mono-, d i- e t tr ig ly c e r id e s , ces derniers en proportion r e la t iv e ­
ment f a ib le . L1hydrolyse basique de ces gly cerid es conduit ä des monoacides
dont le s histogrammes sont tr e s d iff6 r e n ts de ceux des monoacides lib r e s : l ' a -
cide en C2g e s t un element important de la d istrib u tio n des acides d erives des
g ly cerid es dans le s s o ls , a lors que le maximum se s itu e a Cjj pour le s p lantes
de su rface. D'une fapon gen erale, ces histogrammes et le s CPI n'indiquent pas
d 'ev o lu tio n . De meme, dans le cas des este rs ä longue chaine, le s histogrammes
des a lc o o ls et des acid es lin e a ir e s derivant de 1 'hydrolyse basique sont pra­
tiquement identiques pour le s s o ls et la vögetation .

3.2 - Drigine des bitumes

Le tableau IV montre qu'apres incubation en presence de p a ille de mais 1I+C,
le taux de r a d io a c tiv ite obtenu dans le s bitumes e x tr a its des deux types de so l
e st plus elev e que c e lu i present au depart dans la p a ille de mais ll*C. Ce taux
e s t plus elev e pour le podzol que pour le so l hydromorphe. Une p a rtie de c e tte
a c t iv it e provient du m ateriel vegetal incorpor§, mais i l semble qu'une fra c tio n
peut provenir de syntheses m icrobiennes.
Les pourcentages de r a d io a c tiv ite obtenus dans le s bitumes apres incubation
de ces s o ls en presence de glucose 11+C montrent qu’en e f f e t , le s microorganismes,
sont su sce p tib les d'61aborer des quan tites notables de produits de nature l i p i -
dique qui s ' incorporent dans la fra c tio n "bitumes". La production de ces compo­
ses e s t plus importante pour l ’horizon Aj de podzol.
по FUSTEC-MATHON et al.

F H ',.2 . H is to g r a m m e s d e r e p a r titi o n d e s k y d r o c a r b u r e s lin e a ir e s .

3.3 - Influence des bitumes sur la m icroflore des so ls

Nous avons mis en evidence un e f f e t d äp ressif des so lu tio n s de bitumes sur
la croissan ce des microorganismes du so l ( 5 ] . Cet e f f e t augmente avec la con­
centration de l'e x t r a it [Figure 3 ).
La t o x ic it e de l'e x t r a it augmente egalement lorsque le pH du m ilieu d ecroit
(Figure 4 ). La m icroflore d’un horizon Aj de podzol parait la plus r e s is ta n te ,
mais on con state qu'ä la valeur du pH du so l considere, l'in d ic e de croissance
e s t de 80 pour la m icroflore du so l brun le s s iv e , de 40 pour c e lle du so l hydro-
morphe e t de 20 seulement pour c e lle du podzol.
 IAEA-SM-211/48

TABLEAU III. LONGUEURS DE CHAINES DOMINANTES

ET VALEURS DU C A R B O N P R E F E R E N T IA L IN D E X
DANS LES HYDROCARBURES

Hydrooarbures

Maximum CPI

Vdgfitation c29 ' C31 " c 33 13,15

Podzol horizon A2 c29 * C31 ' c 33 8,15

Podzol horizon Aj c2 7 “ c 31 c 33 2,94

Sol hydromorphe c29 ' C31 ‘ C33 5,50

TABLEAU IV. DISTRIBUTION DE LA RADIOACTIVITE DANS

LES BITUMES DANS LES SOLS ENRICHIS EN PAILLE DE MAIS
ET EN GLUCOSE APRES TROIS SEMAINES DTNCUBATION
(en %de la radioactivite introduite)

Sol hydro­
Podzol
morphe

P a ille de mals 1!*C

2,6 2,6
non incubee

P a ille de mals 14С

incubde 3,2 2,9

Glucose ll*C 11,8 B, 5

% Bitumes

F I G .3 . I n f l u e n c e d e la c o n c e n t r a t i o n d e s e x t r a i t s d e b i t u m e s s u r l a c r o i s s a n c e d ’u n e m i c r o p o p u l a t i o n de

so i b ru n le s s iv e .
112 FUSTEC MATHON et al.

F I G .4 . V a r ia tio n d e V e f f e t d e p r e s s i f d u n e s o lu t io n d e b itu m e s d 1 % e n fo n c tio n d u p H s u r d e s m ic r o p o p u la tio n s

d e d iff e r e n t e s o r ig in e s .

F I G .5 . I n f l u e n c e d e s d i f f e r e n t e s f r a c t i o n s d e s b i t u m e s s u r l a c r o i s s a n c e d ’u n e m i c r o p o p u l a t i o n d e s o l b r u n l e s s i v e .

F IG .6 . I n f l u e n c e d e l a l o n g u e u r d e s c h a i n e s e t d e s f o n c t i o n s a l c o o l e t a c i d e s u r la c r o i s s a n c e d ’u n e m i c r o -

p o p u l a t io n d e s o l b r u n le s s iv e .
 IAEA-SM-211/48 113

L’e f f e t in h ib iteu r varie en fonction des d iffe r e n te s fa m ilie s de composes

presents dans le s bitumes [Figure 5 ). Certains con stitu a n ts sont peu inhibiteur.s
(ceton es, e s te r s , a lc o o ls ) . La fra ctio n acide et le s hydrocarbures sont le s plus
toxiqu es.
Enfin, s i l'o n compare l'a c tio n d 1hydrocarbures de d iffe r e n te s longueurs,
d 'a lc o o ls et d 'acid es correspondents (Figure 6 ), on con state qu ’ un hydrocarbure
en Cj7 peut exercer un e f f e t stim ulant tandis que l'a lc o o l et surtout 1 'acide
correspondent ont un e f f e t d e p r e ssif. Pour le s composes ä chaine plus longue
[C28 ■ C261, 1 'in h ib itio n e s t nettement plus f o r te , mais la presence de fonc-
tio n s ne semble pas a lors in te rv e n ir.

4 - DISCUSSION ET CONCLUSIONS
Les taux de bitumes mesures ä un moment donne dans un so l representent un b i-
lan entre le s apports par le s debris vegetaux et animaux plus ou m oins|transform es,
l'a d d itio n de produits d’o rig in e microbienne e t la decomposition de ces d ivers cons­
titu a n ts . II e s t generalement admis que le s produits lip id iq u e s se decomposent len -
tement dans le s s o ls , la degradation etant surtout fre in e e par 1 'anaerobiose
dans le s s e ls engorges e t par l'a c id it e du m ilieu .
Dans la region sableuse des Landes du liedoc, on con state que le s podzols ren-
ferment des q uantites de bitumes nettement plus e lev e es que le s s o ls hydromorphes.
Au niveau des deux types de s o l, la composition de la vegetation e s t tr e s v o isin e ,
la nature des produits apportes au so l par le s debris ne peut done pas etre tre s
d iffe r e n te . Les d iffer en ce s peuvent apparaitre s o it au niveau de la transformation
e t de la decomposition de ces produits dans le s o l, s o it au niveau de la production
de composes d 'o rig in e microbienne.
Dans le s horizons s u p e r fic ie ls des podzols, ce rta in es fr a c tio n s s'accumulent
e t sont moins transformees que dans le s horizons correspondants des s o ls hydromor­
phes. C 'est le cas notamment de la fra ctio n acide e t des hydrocarbures a in s i que
le trad uisen t clairem ent le s valeurs du "Carbon P re feren tia l Index". Par contre,
la plupart des fr a c tio n s neutres e x tr a ite s du so l ( a lc o o ls , ceto n es, e s te r s) sem-
blent peu transformees par rapport aux fra c tio n s correspondantes dans le s plantes
mais e l l e s sont plus rapidement biodegradees. La fa ib le t o x ic it e de ces c o n s ti­
tuants a 1 'egard de la m icroflore glob ale du so l peut expliquer c e tte degradation
p r e fe r e n tie lle .
Par a ille u r s , nous avons egalement pu mettre en Svidence la p o s s ib ilit e d'une
production de composes d 'o rig in e microbienne plus importante au niveau des horizons
s u p e r f ic ie ls des podzols. Cette production e st certainement due pour la plus grande
part aux champignons abondants dans ces s o ls , 1 'accumulation d ’acides gras fa v o ri-
sant elle-meme le developpement des champignons (2 ).

En conclusion, i l apparait que dans ces s o ls sableux l'a c id it e du m ilieu joue

un ro le plus important que 1 ’hydromorphie. La texture permet, en e f f e t , une c e r ta i-
ne c ir c u la tio n des nappes e t une aeration sü ffisa n te du m ilieu . Au niveau des pod­
z o ls , on note, d'une part, une transformation et une decomposition moins poussees
des produits d’orig in e vegetale e t , d'autre part, une production plus importante
de composes d 'o rig in e microbienne. Dans ces s o ls , l'a c id it e du m ilieu a cc ro it 1 'e f ­
f e t toxique des composes lip id iq u e s et reduit l ’a c t iv it e biologique g lo b a le.
II semble que le s bitumes puissen t jouer un ro le tr e s important dans la forma­
tio n des humus de type mor, 1 ’a c id ific a tio n progressive du m ilieu a la su ite du le s -
sivage en tra in era it 1 ’accumulation de produits toxiques su sce p tib les de r a le n tir
l ' a c t i v i t e microbienne e t l ’evolution generale de la m atiere organique. II у aurait
notamment p ersistan ce dans le so l d 'acid es organiques sim ples qui fa v o risen t le pro­
cessu s de p o d z o lisa tio n .
1 14 FUSTEC-MATHON et al.

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[2] BRAIDS, O., MILLER, R., in Soil Components, I — Organic components, Springer-Verlag, Berlin (1975) 343.
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des Mines, Serv. Labo., Qu6bec R.P. 281 et 282 (1953).
[4] WANG, T., Int. Rice Commn Newsl. 18 2 (1969) 23.
[5] FUSTEC-MATHON, E„ RIGHI, D., JAMBU, P., Rev. Ecol. Biol. Sol. 12 1 (1975) 393.
[6] JAMBU, P., RIGHI, D„ Sei. Sol, Bull. A.F.E.S. 3(1973) 207.
[7] ARPINO, P., OURISSON, G., Anal. Chem. 43(1971) 1656.
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[9] COOPER, J., BRAY, E„ Geochim. Cosmochim. Acta 27 (1963) 1113.
[10] ARPINO, P., Les lipides de sediments lacustres Eocene, These Sei. Strasbourg (1973) 107 p.
[11] BECK, G., DOMMERGUES, Y„ VAN DEN DRIESSCHE, R., Rev. Ecol. Biol. Sol 12 1 (1975) 393.

DISCUSSION

B. GUILLET: I noted in your first slide that in horizon A2 there were considerable percentages
of carbon coming from bitumen compared with total carbon. Do you think that this is an effect
originating with the roots? In other words, do the plant roots have a larger bitumen content
than the aerial parts?
E. FUSTEC-MATHON: That is a possibility, but we do not think that the figure is very
significant because this horizon is very poor in organic matter, and as a result of the fractionation
it may also be that errors have been introduced. The level is indeed very low.
J. CORTEZ: To what reason do you attribute the fact that the monoglycerides and
diglycerides are inhibitors of the microflora?
E. FUSTEC-MATHON: I have repeatedly observed this in experiments, and have myself
been surprised that, in particular, they are more active inhibitors than the long esters. I cannot
explain it at present.
CHARACTERIZATION
OF HUMIC ACIDS
(S e ssio n 7 b )
 IAEA-SM-211/7

R e v ie w p a p er

R E C E N T F IN D IN G S O N T H E C H A R A C T E R IZ A T IO N
O F H U M IC S U B S T A N C E S E X T R A C T E D F R O M S O IL S
F R O M W ID E L Y D IF F E R IN G C L IM A T IC Z O N E S

M. SCHNITZER
Soil Research Institute,
Agriculture Canada,
Ottawa, Ontario,
Canada

Abstract

RECENT FIN DIN G S ON THE C H ARACTERIZATION OF HUMIC SUBSTANCES EXTRACTED FROM SOILS
FROM WIDELY D IFFER IN G CLIMATIC ZONES.
T o ob tain inform ation o n the effec t o f clim ate o n the analytical characteristics and chem ical structure o f
hum ic substances, h um ic and fulvic acids w ere extracted from soils form ed under A rctic, co o l tem perate, subtropical
and tropical clim ates. A ll hum ic m aterials were extracted, fractionated, purified and analysed b y the same m ethods.
T hese included elem entary and fu nction al group analyses, sp ectrop h otom etry in th e ultra-violet, visible and
i.r. regions, Electron Spin R esonance (E SR ) spectrom etry and chem ical degradation w ith alkaline К М п 04 and
alkaline CuO. The oxid ation products w ere separated b y solvent extraction and chrom atographic m eth od s, and
iden tified b y mass spectrom etry and micro-i.r. spectrop h otom etry. T he analytical, spectrophotom etric and
spectrom etric data, and the in form ation provided b y chem ical degradation, show ed that hum ic substances form ed
under w id ely differing clim atic con d ition s had essentially similar chem ical structures and characteristics. Major
hum ic ‘building b lock s’ were in all instances com p lex phenolic and benzene-carboxylic structures. M ost o f the
aliphatics w ere n-fatty acids w hich appeared to be esterified to phenolic OH groups. R ecent p hysico-chem ical
studies d one in ou r laboratory show that hum ic and fulvic acids behave like linear, flexib le p oly electro ly tes that
are readily aggregated, m ost lik ely w ith th e aid o f H -bonding, at lo w pH but dispersed, because o f increased dissocia­
tio n o f fu nction al groups, at higher pH . Thus, it b ecom es apparent at this tim e that hum ic and fulvic acids are
n o t single m olecules b u t m olecular associations o f p henolic and benzene-carboxylic com p ou nd s ( ‘building blocks’)
o f m icrobiological, p olyp h en olic, lignin and condensed lignin origins. The ‘building b lock s’ appear to be held
togeth er b y weak linkages, m ainly H-bonds.

INTRODUCTION

It is generally accepted by soil scientists that climate exerts profound effects on the genesis as
well as on the physical and chemical characteristics of soils. Considerably less, however, is known
on how this same environmental factor affects the chemical structure and reactivity of organic
matter. Since humic substances, that is, humic acids (HAs), fulvic acids (FAs) and humins, consti­
tute the bulk of the organic matter in all inorganic and in highly humified organic soils, we focused
our efforts on these materials. The question that we wanted to answer was how similar or different
were humic substances extracted from soils formed under widely differing climates such as that
prevailing in the Arctic, the cool temperate, the subtropical and the tropical zones. To provide
the desired information, we extracted, purified, fractionated, analysed and degraded all HAs and
FAs by the same methods. Chemical analyses included elementary analysis (С, H, N, S, O), func­
tional group analysis (C0 2H, phenolic and alcoholic OH, quinonoid and ketonic C = 0, OCH3)
and oxidative degradation with alkaline KMn04 and alkaline CuO. The oxidation products were
separated by solvent extraction, thin-layer and preparative gas chromatography, and identified on

117
1 18 SCHNITZER

TABLE I. CLIMATIC ZONES AND SOILS

FROM WHICH HAs AND FAs WERE EXTRACTED

(a) A r c tic clim a te

Brunic Turbic C ryosol, Ahb horizon, Northern Canada

fb j Cool, te m p e ra te clim ate, acid soils

V olcanic A sh, horizon, Japan

Diluvial, A ! horizon, Japan
Spod osol, 0 2 and Bh horizons, Eastern Canada
Gray Luvisol, A i and B2 horizons, Western Canada

fc) Cool, te m p e ra te clim ate, n eutral soils

Haploboroll, A i horizon, Western Canada
Glossic U dic N atriboroll, A , horizon, Western Canada
U dic N atriboroll, A 2 horizon, Western Canada

(d) S u b tro p ic a l clim a te

Aridic Boroll, A , horizon, Central Argentina

G lossic N atriboroll, A j horizon, Central Argentina
N atriboroll, A t horizon, Central Argentina
A ridic Haploboroll, A 2 and B2 horizons, Central Argentina

(e) T ropical clim ate

Inceptisols, A i and В horizons, D om inica, West Indies

(including Hydrandepts and Dysdrandepts)

a gas chromatographic-mass spectrometric-computer system and by micro-infra-red spectrophoto­

metry. In addition, all humic samples were analysed by ultra-violet, visible and infra-red spectro­
photometry as well as by electron spin resonance spectrometry.
We were hoping that the results of this investigation would throw light on (a) the effect of
climate on the chemical structure and properties of humic materials that occur in soils, and
(b) advance our knowledge of the structural chemistry of these materials, a problem that has
occupied soil scientists for more than 200 years [ 1, 2].

EXPERIMENTAL

Materials

Climatic zones under which the soils examined were formed, and the classification and
geographic origin of soil samples from which HAs and FAs were extracted, are listed in Table I.
In some cases soil samples were collected from both surface and subsurface horizons, but most
soil samples used in this investigation came from surface horizons.

Extraction, separation and purification of humic substances

Alkaline soil samples were first decalcified with dilute HC1 solution, washed and air-dried.
HAs and FAs were extracted from finely ground, air-dry soil samples with 0.5N NaOH under N2
at room temperature, using a solution:soil ratio of 10:1. Following separation of the dark-coloured
supernatants from the residual soils by centrifugation, the alkaline extracts were acidified to pH 2.0
 IAEA-SM-211/7 1 19

and allowed to stand at room temperature for 24 h. The soluble materials (FAs) were separated
from the coagulates (HAs) by centrifugation. The HAs were redissolved in 0.5N NaOH linder N2
and reprecipitated with 2N HC1. Following centrifugation, the HAs (coagulates) were transferred
to dialysis bags, dialysed against distilled water until free of СГ, and then freeze-dried. When
additional purification was needed, this was done by shaking the HAs at room temperature for
36 h with a large excess of 0.5% (vol./vol.) HC1-HF, centrifuging, washing the residues (HAs) with
distilled water until free of СГ, and then freeze-drying. The FAs were passed repeatedly| over
H-resin contained in large glass columns. In some instances further purification such as dissolution
in methanol, high-speed centrifugation and additional resin treatments was necessary to lower
the ash content of the FAs [3].

Analytical methods

C and H were determined by dry-combustion, N by the automated Dumas method,! S by

oxygen-flask combustion, and О was calculated by subtracting %C+ %H+ %N + %S from 100.
The OCH3 content was determined by the Zeisel method. Total acidity and carboxyl groups were
determined by the method described by Schnitzer and Gupta [4], total C =0 by oximation [5]
and quinonoid groups according to Schnitzer and Riffaldi [6 ]. Ketonic C= О groups were con­
sidered as being equal to the difference between total C = 0 and quinonoid C=0. Phenolic OH
groups were assigned to the difference between total acidity and C02H groups. Total OH groups
were measured by acetylation [7]. Alcoholic OH groups were taken as the difference between
total and phenolic OH groups. j
Moisture was determined by heating samples at 105°C for 24 h, and ash by igniting; samples
at 750°C for 4 h. All data are expressed on a moisture-and ash-free basis.
E4/E 6 ratios were determined by dissolving 2—3 mg of HA and 5 mg of FA in 10 ml of
0.05N NaHC03 solution (pH ±9.0), and measuring optical densities at 465 and 665 nm on a
Beckman model В spectrophotometer. The ratio of the optical densities at the two wavelengths
was the E4/E 6 ratio.
Infra-red spectra were recorded on KBr pellets (± 1 mg of HA or FA per 400 mg of KBr) on
a Beckman IR-12 spectrophotometer. Electron Spin Resonance (ESR) spectra were recorded on
1%(wt/vol.) solutions in 0.1N NaOH on a Varian Associates E3 spectrometer, employing 100 KHz
modulation and a nominal operating frequency of 9.5 GHz [8].

Alkaline KMn04 oxidation of methylated HAs and FAs

One gram samples of each humic material were first suspended in 10 ml of methanol and
methylated with an ether solution of diazomethane, generated from Diazald. The methylation
procedure was repeated with fresh diazomethane until the OCH3-content remained constant.
One gram of each methylated humic material was refluxed for 8 h with 250 ml of 4% (wt/vol.)
aqueous KMn04 (pH = 10). Following oxidation, the excess KMn04 was destroyed by the careful
addition of small volumes of methanol. The insoluble Mn02 was removed by filtration and washed
with small aliquots of hot water. The filtrate and washings were acidified to pH 2 with 6N H2S0 4
solution, transferred to a liquid-liquid extractor and extracted for 72 h with 500 ml of ethyl acetate.
The extract (= oxidation product) was first dried over anhydrous Na2S04, then in a rotary
evaporator and weighed.

Alkaline CuO oxidation

To 500 mg of HA or FA, dissolved in 50 ml of 2N NaOH solution in a stainless-steel auto­

clave, surrounded by a heating mantle, and equipped with a magnetic stirrer, 2.5 g of CuO was
added. The system was heated to 170°C with constant stirring over a period of 1 h, and then
120 SCHNITZER

maintained at that temperature for 2 h. Following cooling to room temperature, suspended CuO
was removed by centrifugation and washed with distilled water. The supernatant solution was
acidified first to pH 6 and then to pH 2, and each time extracted for 24 h in a liquid-liquid extraction
with 250 ml of ethyl acetate. The latter extract (= oxidation product) was dried and weighed
as described above.

Separation and identification of oxidation products

Each oxidation product was first methylated to make it sufficiently volatile for subsequent
gas chromatographic and mass spectrometric analyses. As a second step, each methylated fraction
was dissolved in benzene and examined by analytical gas chromatography. Compounds represented
by well-defined peaks were isolated by preparative gas chromatography under similar conditions
except that more concentrated solutions were injected. Materials eluting from the gas chromato­
graph were collected in capillary tubes and analysed by mass spectrometry and micro-infra-red
spectrophotometry. Each fraction was also analysed directly on a gas chromatographic-mass
spectrometric-computer system (Finigan 3100D GC-MS, interfaced with a model 6000 data
system) [9].
In general, preliminary identification of gas chromatographic peaks was first made by
recording ‘mass chromatograms’ for fragments characteristic of specific compounds or groups
of compounds expected to occur in the mixture and searching through stored spectral data for
specific m/e ratios [10]. The identity of the compound in each peak was then confirmed by
( 1) running its mass spectrum; (2) eluting it from the gas chromatograph and recording its micro-
infra-red spectrum; (3) matching its mass and micro-infra-red spectrum with the known com­
pound to which it corresponded; and (4) co-chromatographing (on the gas chromatograph) of
known and unknown.
The general procedure is summarized in Fig. 1.

D e g r a d a t io n

S o lv e n t e x t r a c t io n

M e t h y la t io n

GLC-MS

S e p a r a t io n o v e r
and t . 1 . c . I

G LC-MS-com puter

F IG .l. P rocedure u sed fo r th e chem ical degradation o f H A s a n d F A s, a n d fo r th e id e n tific a tio n o f o x id a tio n p ro d u cts.
 IAEA-SM-211/7 121

RESULTS AND DISCUSSION

Elementary analyses

Data for elementary analyses of HAs and FAs are shown in Tables II and III. When more
than one set of data was available, the results are presented as ranges. In general, ranges for
HAs were narrower than those for FAs, indicating greater homogeneity for the former. The HAs
contained more С, H and N, but less S and О than the FAs. We were unable to detect any distinct
effect of climate on the elementary composition of the HAs and FAs which we analysed.

Functional groups

Data for functional groups in the different HAs and FAs are shown in Tables IV and V. The
total acidity and C0 2H content of the FAs were in all instances considerably higher than those of
HAs. There were variations in the total C = 0 content, especially among HAs. The OCH3 content
was relatively low for all HAs and FAs. E4/E6 ratios of FAs (Table V) were appreciably higher
than those of HAs in accordance with previous findings published in the literature [ 1]. Again,
climate did not appear to significantly affect the distribution of oxygen-containing functional
groups in the HAs and FAs that we examined.

TABLE II. ELEMENTARY ANALYSIS OF HAs EXTRACTED

FROM SOILS FROM WIDELY DIFFERING CLIMATIC ZONES
C lim atic zone

Elem ent
C ool, tem perate
(%)
A rctic Sub Li upicdl Tropical
A cid soils N eutral soils

C 56.2 5 3 .8 - 5 8 .7 5 5 .7 - 5 6 .7 5 3 .6 - 5 5 .0 5 4 .4 - 5 4 .9

H 6 .2 3 .2 —5.8 4 .4 —5.5 4 .4 —5.0 4 .8 —5.6

N 4.3 0 .8 —2 .4 4 .5 —5.0 3 .3 —4.6 4 .1 —5.5

S 0.5 0 .1 - 0 .5 0 .6 —0.9 0 .8 - 1 . 5 0 .6 —0.8

о 3 2 .8 3 5 .4 - 3 8 .3 3 2 .7 - 3 4 .7 3 4 .8 - 3 6 .3 3 4 .1 - 3 5 .2

TABLE III. ELEMENTARY ANALYSIS OF FAs EXTRACTED

FROM SOILS FROM WIDELY DIFFERING CLIMATIC ZONES

C lim atic zon e

Elem ent
C ool, tem perate
(%)
Arctic Subtropical Tropical
A cid soils N eutral soils

c 4 7 .7 4 7 .6 - 4 9 .9 4 0 .7 - 4 2 .5 4 2 .2 - 4 4 .3 4 2 .8 - 5 0 .6

H 5.4 4 .1 - 4 .7 5 .9 —6.3 5 .9 —7 .0 3 .8 —5.3

N 1.1 0 .9 —1.3 2 .3 —2.8 3 .1 —3.2 2 .0 —3.3

s 1.6 0 .1 —0.5 0 .8 - 1 .7 2.5 1 .3 - 3 .6

о 4 4 .2 4 3 .6 - 4 7 .0 4 7 .1 - 4 9 .8 4 3 .1 - 4 6 .2 3 9 .7 - 4 7 .8
122 SCHNITZER

TABLE IV. FUNCTIONAL GROUP ANALYSIS AND E4/E6 RATIOS OF HAs

EXTRACTED FROM SOILS FROM WIDELY DIFFERING CLIMATIC ZONES

Climatic гопе

Functional group
(m eq /g) C ool, tem perate
A rctic Tropical
A cid soils N eutral soils

T otal acidity 5.6 5 .7 —8.9 6 Л - 6 .6 6 .3 —7.7 6 .2 - 7 .5

C 0 2H 3.2 1 .5 - 5 .7 3 .9 - 4 .5 4 .2 —5.2 3 .8 - 4 .5

Phenolic OH 2.4 3 .2 - 5 .7 2 .1 - 2 .5 2 .1 —2.5 2 .2 - 3 .0

A lcoh olic OH 4 .9 2 .7 - 3 .5 2 .4 - 3 .2 2.9 0 .2 - 1 .6

Q uinonoid C = 0 2.3 1 .4 - 2 .6
|o .l - 1 .8 -{ 4 . 5 —5.6 |o .8 - l .S
K eton ic C = 0 0 .3 - 1 .4
1.7

vq
0

0
00
OCH3 0 .4 0 .4 0.3 0 .3 —0.5

1
e 4/ e 6 5.3 3 .8 —5.0 4 .0 —4.3 3 .9 - 5 .1 5 .0 - 5 .8

TABLE V. FUNCTIONAL GROUP ANALYSIS AND E4/E6 RATIOS OF FAs

EXTRACTED FROM SOILS FROM WIDELY DIFFERING CLIMATIC ZONES

Climatic zo n e

Fun ction al group

C ool, tem perate
(m eq /g)
Arctic Subtropical Tropical
A cid soils N eutral soils

T otal acidity 11.0 8 .9 - 1 4 .2 ND 6 .4 - 1 2 .3 8 .2 - 1 0 .3

C 0 2H 8.8 6 .1 - 8 .5 ND 5 .2 —9.6 7 .2 - 1 1 .2

Phenolic OH 2.2 2 .8 —5.7 ND 1 .2 —2.7 0 .3 - 2 .5

A lcoh olic OH 3.8 3 .4 —4 .6 ND 6 .9 —9.5 2 .6 —5.2

Q uinonoid C = 0 2 .0 0.3 —1.5

■J1.7—3.1 ND i 1.2—2.6
K etonic C - 0 2 .0 l l 1.6—2.7
d

0 .9 —1.2
d

OCH3 0 .6 ND 0 .8 —0.9
1
i

VO
OO
jb

e 4/ e 6 11.5 9 .0 ND 7 . 6 - 1 1 .2

Variations in elementary composition and functional group content

Ranges in elementary composition (Tables II and III) and functional group content (Tables IV
and V) for all HAs and FAs are summarized in Table VI. From these data means were calculated
and these are presented in Table VII. The data in this table may be considered as approximations
of the elementary composition and functional group content of the ‘ideal’ or model HA and FA.
A more detailed inspection of the data shows that (a) the ‘ideal’ HA contains approximately 10%
more C but 10% less О than does the ‘ideal’ FA; (b) there is relatively little difference between
 IAEA-SM-211/7 123

TABLE VI. RANGES AND MEANS FOR ELEMENTARY COMPOSITION,

FUNCTIONAL GROUPS AND E4/E6 RATIOS FOR FAs EXTRACTED
FROM SOILS FROM WIDELY DIFFERING CLIMATIC ZONES

Elem ent
HAs FA s
(%)

c 5 3 .6 - 5 8 .7 ( 5 6 .2 ) a 4 0 .7 - 5 0 .6 (4 5 .7 )

H 3 .2 —6 .2 (4 .7 ) 3 .8 —7 .0 (5 .4 )

N 0 .8 —5.5 (3 .2 ) 0 .9 —3.3 (2 .1 )

S 0. 1 - 1 .5 (0 .8 ) 0 .1 - 3 .6 (1 .9 )
0 3 2 .7 - 3 8 .3 (3 5 .5 ) 3 9 .7 - 4 9 .8 (4 4 .8 )

F u n ction al group
HAs FA s
(m eq /g)

T otal acidity 5 .6 —7.7 ( 6 .7 ) a 6 .4 - 1 4 .2 (1 0 .3 )

C 02H 1 .5 - 5 .7 (3 .6 ) 5 .2 - 1 1 .2 (8 .2 )

Phenolic OH 2 .1 —5.7 (3 .9 ) 0 .3 —5.7 (3 .0 )

A lcoh olic OH 0 .2 —4 .9 (2 .6 ) 2 .6 —9.5 (6 .1 )

Q uinonoid C = 0
j o . 1 - 5 . 6 (2 .9 ) jl.2 - 4 .2 (2 .7 )
K etonic C —0

OCH3 0 .3 —0 .8 (0 .6 ) 0 .3 —1.2 (0 .8 )

e 4/ e 6 3 .8 —5.8 (4 .8 ) 7 .6 - 1 1 .5 (9 .6 )

a Means are in brackets.

the two materials in H, N and S contents; (c) the total acidity and C02H content of the FA are
appreciably higher than those of the HA; (d) both materials contain approximately the same
number of phenolic OH, total C = 0 and OCH3 groups per unit weight, but the FA is richer in
alcoholic OH groups than is the HA; (e) as expected, the E4/E6 ratio of the ‘ideal’ FA is much
higher than that of the ‘ideal’ HA. This indicates that the FA has a lower particle or mplecular
weight than does the HA [11], and it also reflects the abundance of C0 2H groups in the FA.

Major oxidative degradation products

Major compounds produced by the oxidation of methylated and unmethylated HÄs and FAs
are usually aliphatic, phenolic and benzene-carboxylic acids [12]. In addition, smaller amounts
of n-alkanes substituted furans and dialkyl phthalates are also isolated. The most abundant aliphatic
degradation products consist of n-fatty acids, especially the n-C16 and n-Cl 8 acids, and di- and
tri-carboxylic acids (Fig.2). Prominent benzene-polycarboxylic acids are the tri-, tetra-!, penta-
and hexa-forms (Fig.3). Major phenolic acids isolated include those with between 1 and 3 OH
groups and between 1 and 5 C0 2H groups per aromatic ring (Fig.4). Chemical structures of major
products formed by the KMn04 oxidation of methylated and unmethylated HAs and FAs extracted
from tropical volcanic soils are shown in Fig.5 [3]. In this investigation 52 degradation products
were identified. In some of these structures OCH3 groups are attached to the rings and must have
124 SCHNITZER

TABLE VII. ANALYSIS OF ‘IDEAL’ HA AND FA

(fro m m eans o f all data)

Elem ent
HA FA
(Jo )

c 56.2 4 5 .7
H 4 .7 5.4
N 3 .2 2.1
s 0.8 1.9
0 3 5 .5 4 4 .8

1 0 0 .4 9 9.7

F u n ction al groups
(m eq /g)

T otal acidity 6 .7 10.3

C 0 2H 3 .6 8.2
Phenolic OH 3 .9 3 .0
A lcoh olic OH 2 .6 6.1
Q uinonoid C = 0
■^2 . 9 |г .7
K etonic C = 0

ocH 3 0 .6 0.8

E4/E 6 4 .8 9 .6

CH3(CH2)i4COaH
CH3(CH2)i6 с о г н

COgH
(CHg)n n = 0 —8

COgH

Rs4 ^0 ^ .Ra
CHg-COgH
CH-COgH
F IG .2 . M ajor a lip h a tic degradation p ro d u cts.
C H g-co2H r4 R3

occurred in this form in the initial HAs and FAs because these compounds were produced by
oxidation of both methylated and unmethylated HAs and FAs [3]. The structures listed in Figs 2—5
may be considered to constitute the major humic ‘building blocks’. These are similar in HAs,
FAs and humins [12].

Products resulting from the КМПО4 oxidation of methylated HAs and FAs

Major types of products resulting from the KMn04 oxidation of 1.0 g of methylated HAs
and FAs are shown in Tables VIII and IX. The Arctic HA (Table VIII) produced considerably
higher yields of aliphatic compounds than did any of the other HAs. Weight ratios of benzene-
 125

F I G .3 . M a jo r b e n z e n e -c a r b o x y lic d e g r a d a tio n p r o d u c ts .

F I G .4 . M a jo r p h e n o lic d e g r a d a tio n p r o d u c ts .

carboxylic to phenolic compounds, which may be considered to reflect the interrelationship

between major humic ‘building blocks’, were, however, of the same general magnitude, except
that the ratio for HAs from subtropical soils was found to be higher than the remaining ratios.
Aliphatic compounds accounted for only a small proportion of the total products resulting from
the degradation of FAs (Table IX). Weight ratios of benzene-carboxylic to phenolic compounds
were similar for all FAs, and were of the same magnitude as those for most HAs. With one exception,
we were unable to detect any significant effect of climate on the weight ratios of major aromatic
structures for the different HAs and FAs. The only distinct effect of climate that we were able
to observe was that the Arctic HA appeared to contain considerably greater amounts of aliphatic
structures than did the other HAs.

Products resulting from the alkaline CuO oxidation of HAs and FAs

Major types of products resulting from the alkaline CuO oxidation of 1.0 g of HA and FA
are shown in Tables X and XI. Again, the Arctic HA produced larger amounts of aliphatic com­
pounds than did any of the other HAs. Aside from the Arctic HA, the most abundant compounds
 F IG . 5. M a jo r c h e m ic a l s tr u c tu r e s in tr o p ic a l v o lc a n ic H A s a n d F A s . C s ta n d s fo r C O 2H g ro u p or g ro u p

fo r m in g C O 2H on К М пО ц o x id a tio n s .

TABLE VIII. MAJOR TYPES OF PRODUCTS (mg) RESULTING FROM THE KMn04
OXIDATION OF 1.0 g OF METHYLATED HA EXTRACTED FROM SOILS FROM
VARIOUS CLIMATIC ZONES

Clim atic zon e

T yp e o f product C ool, tem perate

A rctic Subtropical Tropical
A cid soils N eutral soils

129.2
©

9.8-51.6
7
0

A liphatic 7.0-15.8 8.3-116.7

Phenolic 36.8 70.4-79.0 68.1-96.5 16.2-31.2 32.3-111.7
B en zene-carboxylic 50.8 80.2-122.4 147.5-173.5 9.2-164.1 49.6-183.3

T otal id en tified 224.2 194.4-282.4 248.6-281.3 118.1-198.5 100.2-350.4

Weight ratio
b en zene-carboxylic
1.4
OO
<N
<4

phenolic 1.1-1.6 5.3-6.1 1.2-2.4

1
 IAEA-SM-211/7 127

TABLE IX. MAJOR TYPES OF PRODUCTS (mg) RESULTING FROM THE KMn04
OXIDATION OF 1.0 g OF METHYLATED FA EXTRACTED FROM SOILS FROM
VARIOUS CLIMATIC ZONES

Climatic zon e

T yp e o f product C ool, tem perate

Subtropical Tropical
A cid soils N eutral soils

A liphatic 9.2 6 .8 - 8 .9 0 ^ 1 .6 5 .7 - 2 7 .1
Phenolic 30 .5 4 3 .1 - 4 6 .5 7 .4 - 6 0 .0 1 5 .9 - 2 6 .3
B en zene-carboxylic 46 .9 7 7 .6 - 9 5 .4 2 0 .6 - 8 6 .9 3 7 .9 - 6 8 .4

T o ta l iden tified 8 6 .6 1 2 9 .6 - 1 4 7 .8 2 8 .0 - 1 4 8 .1 6 4 ,6 - 1 0 8 .0

Weight ratio
benzene-carboxylic |
JO

JO
bo
to
1
00

1
phenolic 1.5 114—3.5

TABLE X. MAJOR TYPES OF PRODUCTS (mg) RESULTING FROM THE CuO-NaOH

OXIDATION OF 1.0 g OF HA EXTRACTED FROM SOILS
FROM VARIOUS CLIMATIC ZONES

Climatic zon e

T yp e o f product C ool, tem perate

A rctic Tropical
A cid soils Neutral soils

A liphatic 104.2 2 6 .2 4 6 .2 1 9 .6 - 5 5 .3
Phenolic 63.6 5 1 .0 6 8 .1 6 2 .7 - 9 8 .0
B en zene-carboxylic 24.8 28.4 2 0 .2 2 0 .9 - 3 4 .9

T otal identified 192.6 105.6 135 .0 1 0 3 .2 - 1 8 8 .2

Weight ratio
benzene-carboxylic
phenolic 0 .4 0 .6 0.3 0 .3 —0.5

formed by the alkaline CuO oxidation of HAs and FAs were phenolics. This confirms earlier
findings [13] that alkaline cupric oxide oxidation was an especially efficient method for releasing
phenolic structures. Relatively low yields of benzene-carboxylic acids resulted from the alkaline
CuO oxidation of the HAs and FAs. This is also indicated by the low benzene-carboxylic to phenolic
weight ratios (Tables X and XI). Again, we failed to observe any effect of climate on yields of
phenolic and benzene-carboxylic oxidation products.
]
Maximum yields of principal structures

In order to uncover which method of oxidation would produce the highest yields of major
products, we oxidized a HA, extracted from the A! horizon of a Haploboroll in Western Canada
128 SCHNITZER

TABLE XI. MAJOR TYPES OF PRODUCTS (mg) RESULTING

FROM THE CuO-NaOH OXIDATION OF 1.0 g OF FA
EXTRACTED FROM SOILS FROM VARIOUS CLIMATIC ZONES

Clim atic zone

Type o f product
C ool, tem perate
Tropical
acid soils .

A liphatic 51.6 1 5 .6 - 4 4 .2

Phenolic 4 5 .2 2 2 .7 - 5 5 .4

B enzene-carboxylic 2 4 .0 1 9 .5 - 4 3 .8

Total identified 1 2 0 .8 5 7 .8 - 1 4 3 .4

Weight ratio
benzene-carboxylic
phenolic 0.5 0 .8 —0.9

TABLE XII. MAJOR CHEMICAL STRUCTURES IN

IN ‘IDEAL’ HA AND FA

HA FA
Major products M ethod
(%) (%)

A liphatic 24.0 2 2 .2 C u O -N a O H + K M n 0 4

Phenolic 20.3 30.2 C u O -N a O H + K M n 0 4

B en zene-carboxylic 32 .0 23 .0 CH 3 CO 2 O H ,K M n 0 4

Total ■76.3 75 .4

„ . benzene-carboxylic
R a t io ----------- ;------ n—®—
phenolic 1 .6 0 .8

A rom aticity 69 71

(cool, temperate zone, neutral soil), and a FA, extracted from the Bh horizon of a Spodosol in
Eastern Canada (cool, temperate climate, acid soil), by the following methods or combination of
methods [13]: (a) alkaline CuO oxidation; (b) alkaline CuO and KMn04 oxidation; (c) alkaline
CuO and KMn04 and H20 2 oxidation; (d) alkaline KMn04 oxidation, and (e) peracetic acid
oxidation. All methods were used on methylated as well as on unmethylated HAs and FAs.
Alkaline CuO oxidation of unmethylated HAs and FAs was found to be especially efficient for
releasing phenolic structures, but was relatively inefficient for degrading aromatic structures bonded
by C-C bonds and which were poor in oxygen. The latter types of structures were more readily
degraded by the more drastic peracetic acid oxidation or oxidation of methylated HAs and FAs
with alkaline KMn04. Maximum yields of major oxidation products resulting from the degradation
of the HA and the FA referred to above, and the methods that were most efficient for this purpose,
are listed in Table XII. The data in the table were multiplied by two because we assumed that
losses during the lengthy oxidation, separation and isolation procedures were 50%. Thus, the
data in Table XII show that the ‘ideal’ HA contains about equal proportions of aliphatic and phenolic
structures, but a greater percentage of benzene-carboxylic structures or structures producing
 IAEA-SM-211/7 129

benzene-carboxylic acids on oxidation. By contrast, the ‘ideal’ FA contains more phenolic than
benzene-carboxylic and aliphatic structures. It is interesting that both the HA and FA contain
approximately equal proportions of aliphatic structures. Aromaticity values were approximated
by expressing yields of phenolic and benzene-carboxylic acids as percentages of total yields. The
aromaticity calculated in this manner is about 70% for both materials. Thus, as far as chemical
structure is concerned, the ‘ideal’ HA and FA are quite similar, except that the FA is richer in
phenolic but poorer in benzene-carboxylic structures than the HA. The main difference between
the two materials is that the ‘ideal’ HA contains more C and N, fewer C02H groups per unit weight,
but has a higher particle or molecular weight than the ‘ideal’ FA, and is thus less soluble in aqueous
solutions at pH < 7.0. 1

Spectrophotometric analyses

Ultra-violet and visible spectra of all HAs and FAs examined were featureless, with optical
densities decreasing as the wavelength increased. Major bands in infra-red spectra were at
3400 cm-1 (hydrogen-bonded OH), 2900 cm-1 (aliphatic С—H stretch), 1720 cm" 1 (C=0 of
C0 2H, C =0 stretch of carbonyl), 1620 cm" 1 (aromatic C=C, hydrogen-bonded C =0 of carbonyl,
COO"), 1400 cm" 1(C=0 stretch or OH-deformation of C0 2H) and near 1200 cm" 1 (С—О stretch
or OH-deformation of C0 2H). Infra-red spectra of HAs and FAs that were low in ash showed
troughs in the 2700 to 2400 cm" 1 region, which were due to hydrogen-bonded C02H groups [14].

ESR spectrometry

ESR spectra of all HAs and FAs were single, symmetrical lines devoid of hyperfine splitting.
Free radical concentrations of HA and FA solutions were of the order of I0 17—1018 spins/g,
spectroscopic splitting factors (g-values) ranged from 2.0032 to 2.0050, and line widths from
2.5 to 4.0 G.

The chemical structure of HAs and FAs

The chemical, spectrophotometric and spectrometric data presented here show that humic
substances formed under widely differing climatic conditions have similar chemical structures.
Recent work [15] has indicated that at least 50% of the aliphatic structures consist of n-fatty
acids esterified to phenolic OH groups (Fig.6). The remaining aliphatics are made up of more
‘loosely’ held fatty acids and n-alkanes that appear to be physically adsorbed on the humic
materials and are not structural components. The principal ‘building blocks’ of all HAs and FAs
that we have so far examined are, however, complex phenolic and benzene-carboxylic acids. The
major questions that need to be answered at this time is how do the ‘building blocks’join together
and what type(s) of structural arrangement is produced?
Some clues as to what structural arrangements may be involved here are provided by recent
work done in our laboratory [ 16] on HAs and FAs with the aid of a Scanning Electron Microscope.

F I G .6 . S tr u c tu r e o f p h e n o l- f a tty a c id e s te r s in H A s a n d F A s .
130 SCHNITZER

ОН

F IG . 7. P a r tia l s tr u c tu r e f o r F A .

We found that at low pH (2-3) FA occurred mainly as elongated fibres or bundles of fibres, forming
a relatively open structure. With increase in pH (4—7), the fibres tended to mesh into a finely
woven network to yield a sponge-like structure. At pH 8, the FA formed sheets which tended to
thicken at pH 9. At pH 10, fine, homogeneous grains were visible. The effect of pH on the HA
structure was similar to that observed on FA, except that because of low solubility in water at
pH < 6, the pH range at which the major transitions could be observed had to be narrowed to
between 6 and 10. In a subsequent study [ 11 ] we observed similar distinct effects of pH on the
viscosity, particle shapes and dimensions and on the polyelectrolytic behaviour of HAs and FAs.
At low pH, HA and FA behaved like uncharged polymers, at higher pH they both exhibited
characteristics typical of linear, flexible polyelectrolytes. This suggests that humic substances
are not made up of condensed rings but contain structures with bonds about which free rotation
can occur. Similar results have been obtained from X-ray studies [17] which show loose, open
structures for humic materials. Other considerations that enter into this discussion are long-term
observations that indicate that HAs and FAs are readily aggregated and dispersed not only by
changes in pH but also by changes in salt concentrations or valence of cations. A meaningful
concept of the chemical structure of HAs and FAs must take all available evidence into account.
It becomes more and more apparent that humic substances are not single molecules but rather
associations of molecules of microbiological, polyphenolic, lignin and condensed lignin origin.
These are the benzene-carboxylic and phenolic compounds that we have isolated, and which appear
to be held together by relatively weak linkages such as hydrogen-bonding, Van der Waals forces
and 7r-bonding. In higher molecular weight humic fractions, that is, HAs and FAs, associations
between ‘building blocks’ appear to be more stable than in FAs, and may in addition to the weaker
linkages mentioned above also involve more energetic bonding via C -0 and C-C bonds. Our data
suggest that aggregation of HA and FA particles at low pH results from intermolecular interactions,
mostly likely via hydrogen-bonds, whereas dispersion at higher pH arises from intermolecular
repulsion due to increased ionization of oxygen-containing functional groups.
A chemical structure that is in harmony with most of the requirements listed above has been
proposed by the author [2]. This molecular arrangement (Fig.7) may account for a significant
portion of the FA structure. Each of the compounds that make up the structure has been isolated
from FA without chemical degradation [2]. Bonding between ‘building blocks’ is exclusively
by hydrogen-bonding, which makes the structure flexible, permits the ‘building blocks’ to aggregate
and disperse reversibly, depending on environmental conditions, and also allows the FA to react
 IAEA -SM -211/7 131

with other inorganic and organic soil constituents either via oxygen-containing functional groups
on the large external and internal surfaces, or by trapping them in internal voids, provided that
the molecular sizes are of the appropriate dimensions and that charges are complementary.

ACKNOWLEDGEMENTS

I am grateful to G. Ogner, S.U. Khan, M.I. Ortiz de Serra, R. Riffaldi, K. Matsuda, J.A. Neyroud,
S.M. Griffith, N. Senesi, Y. Chen, S.I.M. Skinner, D. Skinner and E. Vendette for valuable colla­
boration and assistance with the work reported in this paper.

REFERENCES

[1] KONONOVA, M.M., Soil Organic Matter, Pergamon Press, New York (1966) 13, 101.
[2] SCHNITZER, M., KHAN, S.U., Humic Substances in the Environment, Marcel Dekker, New York (1972) 1, 196.
[3] GRIFFITH, S.M., SCHNITZER, M„ Can. J. Soil Sei. 55 (1975) 251.
[4] SCHNITZER, M., GUPTA, S.U., Soil Sei. Soc. Am., Proc. 29 (1965) 274.
[5] FRITZ, J.S., YAMAMURA, S.S., BRADFORD, E.C., Anal. Chem. 31 (1959) 260.
[6] SCHNITZER, M„ RIFFALDI, R., Soil Sei. Soc. Am., Proc. 36 (1972) 301.
[7] OGG, C.L., PORTER, W.L., WILLITS, C.O., Ind. Eng. Chem. (Anal. Ed.) 17 (1945) 394.
[8] RIFFALDI, R„ SCHNITZER, M., Geoderma 8 (1972) 1.
[9] NEYROUD, J.A., SCHNITZER, M„ Can. J. Chem. 52 (1974) 4123.
[10] SKINNER, S.I.M., SCHNITZER, M„ Anal. Chim. Acta 75 (1975) 207.
[11] CHEN Y., SCHNITZER, M., Soil Sei. Soc. Am., Proc. (submitted for publication).
[12] SCHNITZER, M., “The chemistry o f humic substances”, Environmental Biogeochemistry (NRIAGU, J.O., Ed.),
Ann Arbor Science Publishers Inc., Ann Arbor, Michigan (1976) 89.
[13] SCHNITZER, M., ORTIZ DE SERRA, M.I., Can. J. Chem. 51 (1973) 1554.
[14] SCHNITZER, M., GRIFFITH, S.M., Can. J. Soil Sei. 55 (1975) 491.
[15] NEYROUD, J.A., SCHNITZER, M., Soil Sei. Soc. Am., Proc. 38 (1974) 907.
[16] CHEN, Y., SCHNITZER, M., Soil Sei. Soc. Am., Proc. (in press).
[17] KODAMA, H., SCHNITZER, M., Fuel 46 (1967) 87.

DISCUSSION

W. FLAIG (S cien tific S ecretary): We must all thank Mr. Schnitzer for his outstanding talk.
He succeeded in giving us a remarkably clear summary of a great many data. We acknowledge
that his work represents an enormous step forward in humus chemistry.
According to the data on carboxyl groups in HA (3.6) and FA (8.2), one might perhaps
conclude that FAs are formed by oxidative cleavage of HA. Another explanation might be that
the part of the initial material with aromatic structure is decarboxylated during the polycondensation
processes. What do you think?
M. SCHNITZER: It is not clear yet whether HA is formed from FA or whether the reverse
reaction occurs. I am inclined to favour your first suggestion, although I have no experimental
evidence for either mechanism. My answer is based more or less on a feeling I have about this
matter.
B.R. NAGAR: The work you have reported is most interesting and impressive. Have you
done or are you planning to do work on the ‘nitrogen fraction’ of humus extracted from different
types of soil?
132 SCHNITZER

M. SCHNITZER: Yes, we are now starting work on the ‘unknown’ nitrogen in soils and humic
materials.
T. WEICHELT: In your model structure of humic substance, you have only shown hydrogen
bonds between the various components (units) of the system. What do you think about the
chemical importance of the covalent bonds and Van der Waals bonds in the nature of the substance
in question.
M. SCHNITZER: Although I showed only H-bonding, I believe that Van der Waals inter­
actions, 7Г-7Г bonding and covalent bonding also occur. But I wanted to emphasize that H-bonding
was very important.
E. FUSTEC-MATHON: When the extraction of the humic components from a soil after
extraction of the bitumens with ethanol-benzene is compared with the direct extraction of the
humic components from the same soil, one finds, in the second case, that almost all the bitumens
appear in the HA fraction. Do you not think that the preliminary extraction of products soluble
in ethanol-benzene would modify some of the relationships present, particularly after oxidative
degradation of the HAs?
M. SCHNITZER: In your soils, bitumens appear to be associated with the HA fraction.
This association appears to be relatively loose because you can extract the bitumens with organic
solvents. In order to study the ‘true’ structural HA components, it would be a good thing to
remove the bitumens before oxidative degradation.
 IA E A -SM -211/39

STRUCTURE ET GENESE DES

ACIDES FULVIQUES DES SOLS
DES LANDES DU MEDOC (FRANCE)

D. RIGHI, P. JAMBU, T. DUPUIS

Laboratoire de pedologie,
Universite de Poitiers,
Poitiers,
France

Abstract-Risum£

STRUCTURE AND GENESIS OF FULVIC ACIDS IN THE MEDOC LANDES SOILS (FRANCE).
The structure o f fulvic acids formed in sandy soils with permanent ground water, ranging from podzols to
hydromorphic soils, was studied. The acids fall into three classes: Type I corresponds to А ц surface horizons;
it is characterized by a high polysaccharide content. The nitrogen is present chiefly in the alpha-amino form;
the total acidity is lower than in the following types and the phenol functions predominate. Type II applies to
the hard pans (B2h) in the best-drained podzols; it is differentiated by the absence of polysaccharides and a lower
C/N ratio, since the nitrogen is distributed mainly in two forms: alpha-amino and heterocyclic nitrogen; the total
acidity is strong and the COOH functions predominate. Type III, which comes somewhere between the other
two, relates to the most moist podzol pans.

STRUCTURE ET GENESE DES ACIDES FULVIQUES DES SOLS DES LANDES DU MEDOC (FRANCE).
Nous avons cludie la structure des acides fulviques formes dans des sols sableux ä nappe permanente allant
des podzols aux sols hydromorphes. Ces acides se classent selon trois types: Le type I correspond aux horizons
superficiels A n ; il est caracterise par une forte teneur en polysaccharides; l’azote у est principalement sous forme
Q-ainmce, l’acidite totale est plus faible que dans les types suivants et les fonctions phenols dominent. Le type II
correspond aux horizons d’accumulation indures (B2h) des podzols les mieux draines; il se differencie par
Tabsence de polysaccharides et un rapport C/N plus faible, l’azote se repartissant en deux formes principales:
o-aminee et heterocyclique; Tacidite totale est forte et les fonctions COOH dominent. Le type III, intermediaire
entre les deux precedents, correspond aux horizons d’accumulation des podzols les plus humides.

I NTRO DU CT I O N

Nous avons сЬегоИё ä preciser 1 'influence d ’ un engorgem ent plus ou moins

p r o n o n c e s u r l a n a t u r e d e s a c i d e s f u l v i q u e s (AF) p r o d u i t s a u c o u r s d e s p r o c e s s u s
1
d h u m i f i c a t i o n d a n s l e s s o l s a c i d e s d e s L a n d e s du Medoc. Ces s o l s s o n t r e p a r t i s
selon des sequences d ' hydromorphie c r o is s a n te dont le s po les extrem es sont des
p o d z o l s b i e n d i f f e r e n c i e s ( h o r i z o n B2 h i n d u r e ) e t d e s s o l s h y d r o m o r p h e s p e u h u -
m i f e r e s . Les te rm e s i n t e r m e d i a i r e s s o n t d e s p o d z o l s h u m i q u e s ä h o r i z o n B2 h
m e u b le . Ces s o l s s e d e v e l o p p e n t s u r l e meme s a b l e g r o s s i e r q u a r t z e u x e t p o r ­
tent une lande humide ä sem i-hum ide , uniform em ent plant^e en pins m aritim es.

□ans ces sequences, 1 ' evolution de la m atiere organique est beaucoup plus
d ire c te m e n t c o n d itio n n S e p a r l e rögim e h y d r i q u e q u e p a r l a v e g e t a t i o n И ) . Des
ötudes a n te rie u re s ont m ontre que la tra n sfo rm a tio n des d e b ris vegetaux s u it
deux voies distinctes aux extrem ites des sequences : dans le s podzols, la frag­
m entation de la lignine serait lim itee et celle-ci se transform erait directe­
ment en humine ( 2) ; dans les sols hydromorphes, ou l'a c tiv ite biologique est
p lu s f o r t e , la l i g n i n e se f r a c t i o n n e r a i t en u n ites plus sim ples qui sont a l'o -
r i g i n e , en p a r t i c u l i e r , d ' a c i d e s h u m i q u e s peu e v o lu e s (3).

133
 134
TABLEAU I. CARACTERISTIQUES ANALYTIQUES DES HORIZONS ETUDIES

Podzols Podzols
Sol Sol
Types de sols
ä Bh hydrom. a Bh hydrom.
a Bh indure ä Bh indure
meuble m euble

H orizons
3 LAG 1 3 LAG 5 3 LAG 6 4 LAG 3 1 LAG 1 1 BER 1 1 BER 5 1 BER 6 4 BER 2 6 BER 1
(Ац ) (B h) 22 (B3) 2
(B h) (А ц 1 CAn ) (B 22
h) (B3) 2
[B h] (А ц)

pH e a u 4,2 4,8 5,0 5,4 4,5 3,5 4,5 5,1 4,6 4,9

RIGHI et al.
S/T ( %) 12,3 4,5 9,9 7,5 18,2 17,2 1,5 1,1 2,1 23,8

A rgiles [< 2 y) (%) 1,1 1,4 0,8 1,6 2,8 1,9 0,9 0,8 3,1 6,5

C to tal ( %. ) • 31,7 15,1 15,1 14,1 16,7 66,0 13,0 5,6 21,9 32,2

C/N 40 30 30 20 17 34 26 - 18 16

C AH/ C t o t a l (%) 25,4 29,2 22,9 33,6 27,1 30,6 37,9 9,1 49,5 19,8

C AF/C total C%) 2,1 59,7 64,6 39,5 14,3 1,7 60,5 69,7 25,3 13,3

C humine/C total (%) 21,3 - - - 6,9 12,8 - - - 3,0

 IA E A -SM -211/39 135

M AT E R I E L ET METHOBES D ’ ETUDE

Nous avons compare les AF d e cinq types d 'h o rizo n s rS partis sur deux se­
quences :

- horizons Aj i d e s p o d z o l s l e s m i e u x d r a i n e s [ 1 BER 1 , 3 LAG 1 ) ;

- horizons Aj j d es s o l s hydrom orphes peu h u m ife re s BER 1 , 1 LAG [6 1) ;
- horizons B2 2 h i n d u r e s d e s p o d z o l s l e s m o i n s humides (1B E R 5, 3LAG5) ;
- horizons B 3
d e s p o d z o l s p r e c e d e n t s [ 1 BER 6 , 3 LAG ) ; 6 ;
- horizons B2 h des podzols les plus humides [4 BER 2 ; 4 LAG 3 ) . |

Les caractSristiques analytiques des horizons, donnees au tableau I, m et-

t e n t en e v i d e n c e u n c e r t a i n n o m b r e d e d i f f e r e n c e s . De p l u s , l e s h o r i z o n s A n d e s
podzols se d i s t i n g u e n t des h o r iz o n s А ц d es s o l s hydrom orphes peu h u m ife re s p a r
le u r regim e hydrique e t le u r a c t i v i t e b io lo g iq u e. Pour le s prem iers, le point
de f l e t r i s s e m e n t perm anent e s t a . t t e i n t au c o u rs de l ’e t e , a lo rs que le s deu-
xiem es r e s t e n t p lu s hum ides. L ' a c t i v i t e m i n e r a l i s a t r i c e du c a r b o n e ( m e s u r e e p a r
re s p ir o m e tr ie je s t de 1,5 a 2 f o i s p lu s f o r t e pour le s s o ls hydromorphes (4).

L 'etude m icrom orphologique permet £galement de diffS rencier les divers

2
h o r i z o n s B h. Ceux d e s p o d z o l s l e s m i e u x d ra in e s p r e s e n te n t une m i c r o s tr u c t u re
en re v e te m e n ts fo rm e s p a r ' illuviation 1 e t le depot de la m a tie re organique
soluble ou pseudo-soluble tandis que les horizons B2 h d e s podzols plus humi­
des ont des m icroagregats dans le sq u e ls une p a r t ie de la m atiere organique
p r o v ie n d r a it de la fra g m e n ta tio n e t de 1
' hum ification s u r p la c e de ra c in e s
(5) (61.

Nous a v o n s d i s p e r s e l e s AF d e c e s d i v e r s h o r i z o n s s e l o n l a m e t h o d e d e
Flubert e t G o n z a l e z ( 7 ) , q u i u t i l i s e u n e r e s i n e e c h a n g e u s e d ' i o n s DOWEX 5 0 W -
X 8 f o r m e Fl+ e n m i l i e u aqueux. La t e n e u r en cendres des produits obtenus est
de 3 a 5 p. c e n t.

L 'analyse а ete effectuee en u tilisa n t les techniques dejä em ployees pour

les a c i d e s h u m i q u e s d e c e s memes s o l s ( 3 ) . N e a n m o i n s , q u e l q u e s m o d i f i c a t i o n s
ont e t e a p p o r t e e s . L ' a z o t e a am in e a e t e d o s e p a r l a m S thode de Moore e t S t e i n
( ) 8 q u i u t i l i s e l a r e a c t i o n ä l a n i n h y d r i n e . Une p a r t i e de l 'a z o t e s o lu b le
apres hydrolyse par HC1 6N n 'est i d e n t i f i a b l e n i comme a z o t e d is tilla b le (en
p r e s e n c e d e m a g n ö s i e o u meme d e s o u d e ) , n i c o m me a z o t e a a m i n e . N o u s c o n s i d e -
r o n s , comme G o n z a l e z e t a l. ( 9 ) , q u 'il s ’a g i t d 'u n e forme h e te ro c y c liq u e .
L 'h y d ro g e n e a e t e d ose en d e t e r m i n a n t l ' e a u o b te n u e a p r e s o x y d a tio n d e s AF
ä /1 0 0 0 ° C e n p r i s e n c e d e c a t a l y s e u r .

L e s c o u r b e s d ’ ATD s o u s a z o t e o n t e t e e n r e g i s t r § e s selon une te c h n iq u e

d S j a d e c r i t e ( 1 0 ) . Le d o s a g e d e s p o l y s a c c h a r i d e s а e f f e c tu e p a r la m etho­
d e d e B r i n k et a l. ( 1 1 ) .

RES U L T A T S ANALYTI QUES ET C A R A C T E R I S A T I O N DES AF

Les r£sultats sont regroupgs dans les tableaux II et III et sur;les figu­
res 1 et 2.

- Spectres infrarouges (Figure 1)

Ils perm ettent de classer les AF s e l o n tro is types distin cts :

T y p e I - AF d e s h o r i z o n s A i 1
des podzols e t des so ls hydromorphes. I l s sont
c a r a c t § r i s e s p a r u n e t r e s f o r t e a b s o r p t i o n e n t r e 1150 e t 1000 cm- 1 , d u e a u x
v i b r a t i o n s de v a l e n c e C-0 d e s p o l y s a c c h a r i d e s e t d e s b a n d e s d ' i n t e n s i t i mo-
yenne a 2850, 2930 et 2960 cm- 1 , relatives aux vibrations de valence C-H d e s
g r o u p e m e h t s CH 2 3
e t CFI . On r e l e v e S g a l e m e n t d e s b a n d e s faibles a 1540 e t 1650
сгтг* a t t r i b u e e s a la p resen ce de li a is o n s p e p tid e s .

Т у р е I I - AF d e s h o r i z o n s B 22h e t B 3 d e s p o d z o l s l e s m o i n s h u m i d e s . Ils dif-

fä re n t des precedents par 1 ' absence des bandes c a ra c tö ris tiq u e s des polysac­
charides et des liaisons peptides. C ependant, la bande d 'ab so rp tio n des OH
 u>
04

TABLEAU II. EVALUATION DU DEGRE D’AROMATICITE - ANALYSE ELEMENTAIRE ET DOSAGE DES POLYSACCHARIDES

Podzols Podzols
Sol Sol
Types de sols
ä Bh hydrom. ä Bh hydrom.
a Bh indure a Bh indure
m euble m euble

H orizons
3 LAG 1 3 LAG 5 3 LAG 6 4 LAG 3 1 LAG 1 1 BER 1 1 BER 5 1 BER 6 4 BER 2 6 BER 1
(Ai i ) (ВзгЮ (B3) 2
CB h ) (Ац ) (А ц) (B 22h ) (B3 ) 2
(B h) (Ai i )

D.O. 2920 cm -1 1,79 1,48 1,62 1,47 2,04 1,66 1,61 1,45 1,61 1 ,B0
D.O. 1510 cm “1
Räsidu apres chauffage sous

RIGHI et al.
40,2 42,5 41,4 43,0 40,2 40,2 41,5 41,9 40,3 40,7
N 2 ä B00°C

Carbone total ( %) 45,0 45,2 45,1 44,6 46,4 46,3 45,7 44,3 44,1 42,9

Hydrogene (%) 5,0 3,6 3,8 3,9 4,6 4,9 3,6 3,5 3,9 4,8

Azote to tal (%) 0,90 0,97 0,87 0,98 1,14 1,07 1,08 0,75 0,98 1,03

Rapport C/N 50,0 46,6 51,9 45,4 40,6 43,3 42,2 59,0 45,0 41,7

P olysaccharides ten mg
33 749 2 055 1 985 5 969 21 625 30 151 2 694 1 843 7 846 29 6B 0
p . c e n t g AF)

C polysaccharides ,
C AF
31,4 1,9 1,8 5,6 19,4 26,8 2,4 2,0 7,4 29,1
 IA E A -SM -211/39 137

v e r s 3 4 0 0 cm “1 s'e la rg it vers les hautes fröquences avec de nombreux pointös

a 3220, 3180 e t 3 0 8 0 cm“ 1 , ce qui in d iq u e la p resen ce de g r o u p e s a m i d e s ou
am ines l i e s . Contrairem ent au type p reced en t, le s bandes de v ib r a tio n de v a­
l e n c e C-H v e r s 2 8 5 0 - 2 9 5 0 cm '1
sont toujours tre s f a i b l e s . L ' a b s o r p t i o n d e s OH
fortem ent И ё э d e s a c i d e s e n t r e 2 7 0 0 e t 2 4 0 0 cm “1 est tre s intense.
Type I I I - AF d e s h o r i z o n s B ? h d e s p o d z o l s t r e s h u m i d e s . C ' e s t u n t y p e i n t e r ­
m ed iate e n t r e l e s d e u x p r e c e d e n t s . L e u r s p e c t r e e s t c e l u i du t y p e I I a v e c , en
plus, les bandes faibles caracteristiques des polysaccharides.

- Courbes ATO s o u s atm osphere inerte (Figure 2)

T o u t e s l e s c o u r b e s d e b u t e n t p a r un p i c e n d o t h e r m i q u e d e d e p a r t d e 1 ' ea u
h y g ro sc o p iq u e , s u i v i d 'u n e f f e t e x o th erm iq u e e t a l § e t com plexe d o n t l e m axi-
mu n s e s i t u e e n t r e 3 7 0 e t 5 4 0 ° C . S u r c e m a s s i f e x o t h e r m i q u e s e g r e f f e n t p l u -
sieurs pics endothermiques dont le prem ier, maximum ä 150°C, est reduit ä un
Spaulem ent. A plus h a u t e t e m p e r a t u r e , on o b s e r v e d i f f e r e n t s p i c s e n d o t h e r m i­
ques qui sont lie s ä la d e s tr u c tio n des d iv e r s groupem ents oxygenös s ta b l e s .
Ces pics perm ettent de distinguer tro is types de courbes correspondent aux
tro is types d'AF differencies par infrarouge.

Type I - Leurs courbes sont caracterisees par la prösence d'un öpaulem ent a
235-250°C, suivi d'un pic endotherm ique brutal et assez fo rt, maximumä 490°C.
L 'i n t e n s i t e de ce d e r n ie r d i f f e r e avec l 'o r i g i n e des AF : il est beaucoup plus
i n t e n s e p o u r l e s AF d e s o l s h y d r o m o r p h e s .

Type II - Leurs courbes prösentent un accident endotherm ique assez faible cul­
m inant ä 450°C, s u iv i d'un p ic tres faible, maximum ä 490°C. Ce dernier n ’ap-
parait pas dans tous les cas.

Type I I I - Leurs co u rb es sont i n t e r m e d i a t e s e n tr e le s deux ty p e s p r£ c£ d en ts.

De p l u s , e l l e s p r e s e n t e n t t o u j o u r s un p i c e n d o t h e r m i q u e s u p p l e m e n t a i r e c u l m i n a n t
ä 515°C.

- Poids m oleculaire apparent

Les c o u rb e s o b te n u e s a p r e s p a s s a g e s u r g e l S e p h a d e x G 25 e t G 50 s o n t
tres s e m b l a b l e s d ' u n AF ä l ' a u t r e . S eu le une p e t i t e f r a c t i o n e s t retenue sur
le gel G 25. Par contre, la quasi to ta litö des AF e s t retenue sur le gel G 50,
c e q u i ö t a b l i t un p o i d s m o l e c u l a i r e a p p a r e n t c o m p r i s e n tre 5.000 et 10.000. Ces
r e s u l t a t s sont proches de ceux obtenus p a r H ubert e t G onzalez (7) p o u r d e s AF
de p o d z o l s du Q uebec.

- Degrä d ' arom aticite (Tableau II)

I I p e u t e t r e e s t i m e d e p l u s i e u r s m a n i e r e s . Le r a p p o r t D . 0 . 2 9 2 0 c m " 1/
D. Ü. 1 5 1 0 cm -1
m e s u r e s u r l e s s p e c t r e s i n f r a r o u g e s (C-H a l i p h a t i q u e s / C = C a r o -
m a t i q u e s ) d o n n e u n d e g r e d ' a r o m a t i c i t e p l u s ё 1 е у ё p o u r l e s AF d e s h o r i z o n s
s p o d i q u e s . Le t a u x du " r e s i d u f i x e " ( e s s e n t i e l l e m e n t a r o m a t i q u e ) o b t e n u a p r S s
c h a u f f a g e s o u s a z o t e ä 6 0 0 ° C c o n d u i t a u meme r ö s u l t a t , m a i s l ' e c a r t e n t r e l e s
AF r e s t e faible. Les difförences observees doivent etre liees ä 1 ' im portance
de la fraction polysaccharidique prösente dans les AF d e s horizons А ц.

- Taux de polysaccharides (Tableau II)

Les dosages refleten t les differences observöes sur les spectres infra-
r o u g e s . La t e n e u r en p o l y s a c c h a r i d e s v a r i e dans d 'a sse z grandes pro p o rtio n s
e t d i f f ö r e n c i e n e t t e m e n t l e s AF s e l o n l e u r p r o v e n a n c e . Le r a p p o r t c a r b o n e d e s
p o l y s a c c h a r i d e s / c a r b o n e t o t a l d e s AF d o n n e :
- 20 a 30 p . cent pour les AF d e s horizons А ц (Type I)
5 a 7 p. cent pour les AF d e s 2
h o riz o n s B h meubles (Type III)
2 p. cent environ dans les horizons 2
B h indures et B 3 (Type II)
 u>
00

TABLEAU III. REPARTITION DES FORMES DE L’AZOTE ET DOSAGE DES FONCTIONS ACIDES

Podzols Sol Podzols Sol

Types de sols hydrom. hydrom.
ä Bh ä Bh
ä Bh indure ä Bh indure
m euble m euble
C
D 3 LAG 5 3 LAG 6 4 LAG 3 1 LAG 1 1 BER 5 1 BER В 4 BER 2 6 BER 1
H
4
□
(B 22h ) CB3 ) 2
(B h) (Ац ) (B 22h ) (B3 ) (B h)2 (Au )
I

LD
NJ
О
N to tal (%) 0,97 0,87 0,98 1,14 1,08 0,98 1,03

N non hydrolysable (1) 21,0 10,5 20,2 21,9 20,3 11,0 15,2 12,7

RIGHI et al.
N hydrolysable tl) 79,0 89,5 79,8 78,1 79,7 88,9 84,8 87,3

N a amine (1) 31,9 28,1 43,2 54,5 24,8 30,1 36,4 57,5

N d istilla b le Cl) 17,8 14,0 22,9 25,6 12,6 18,2 19,3 31,8

N heterocyclique tl) 29,3 47,4 13,7 - 42,3 40,6 29,1 -

A cidite totale COOH + OH ( 2) 909 881 959 B 00 955 893 962 827

COOH ( 2) 674 705 616 488 6B7 708 644 483

OH p h e n o l i q u e s ( 2) 235 176 343 312 268 185 318 344

tl) R esultats exprimds en p. cent de 1 'azote total

(2) R e s u lta ts exprimes en meq p . cent g d 'acid e s f u l v i q u e s _s a n S cendres
s e c h e s ä 80°C
 I AE A-SM -211 /3 9 139
tr a n s m is sio n

P o u r l e s h o r i z o n s Ац , ces c h i f f r e s s o n t s u p ö r ie u r s a oeux o b te n u s p ar
G u c K s r t C 1 2 ) p o u r d e s AP d e p o d z o l s . Au n i v e a u d e s h o r i z o n s E ^ h , l e s c h i f f r e s
sont assez sem blables.

- A nalyse elSm entaire (Tableau II)

L a t e n e u r e n h y d r o g e n e e s t n e t t e m e n t p l u s S l e v S e p o u r l e s AF d e s h o r i ­
zons А ц q u i c o n t i e n n e n t u n e f r a c t i o n p o l y s a c c h a r i d i q u e i m p o r t a n t e . Le t a u x
d e c a r b o n e v a r i e t r e s p e u d ' u n AF a 1 ' a u t r e a l o r s q u e l ’ on o b t i e n t d e s d i f -
f ö r e n c e s a s s e z s e n s i b l e s p o u r l ' a z o t e . Le r a p p o r t C/N e s t t o u j o u r s t r e s ё 1 е -
уё (40 ä 6 0 ) . I I e s t maximum d a n s l e s h o r i z o n s B 3 d e s p o d z o l s e t minimum p o u r
l e s AF d e s h o r i z o n s А ц d e s s o l s h y d r o m o r p h e s .

- Formes de l'azo te (Tableau III)

Apr s 6 hydrolyse, environ 80 p. cent de 1 ’a z o te se trouve dans la fraction

hydrolysable. La proportion d ’a z o t e a aminö, d i s t i l l a b l e et hötörocyclique de
cette fraction varie avec le type d'AF considörö :
- pour les AF d u type I, l ’azote а аггйпё d o m i n e largem ent alors que l'azo te
hötörocyclique n 'est pas representö j
- p o u r l e s AF d u t y p e I I , l ' a z o t e а а п и п ё e t l ’ a z o t e h ö tö ro c y c liq u e prödomi-
n e n t d a n s l e s h o r i z o n s В г г Ь i n d u r ö s ; p o u r l e s AF d e s 3
h o riz o n s B , la forme
hötörocyclique en solution represente presque la m oitiö de l'a z o te to tal ;
140 RIGHI e t al.

F I G .2 . C o u r b e s d ’a n a l y s e t h e r m i q u e d i f f e r e n t i e l l e s o u s a t m o s p h e r e i n e r t e .

- pour les AF d u type III, les formes a amine e t d istilla b le augm entent aux
depens de 1 ' azote heterocyclique.

C e s r e s u l t a t s d i f f e r e n t d e c e u x o b t e n u s p a r G o n z a l e z e t H u b e r t [91 q u i
o n t o b s e r v i s u r d e s AF d e p o d z o l s d u Q u e b e c u n e r e p a r t i t i o n i d e n t i q u e d e s f o r ­
mes d e l ’a z o t e q u e l q u e s o i t l e p r o f i l ou l ' h o r i z o n e t u d i e .

- Fonctions oxygenees (Tableau III)

La r e p a r t i t i o n des fonctions С00Н e t OH p h e n o l i q u e s est differente dans

c h a q u e t y p e d ' A F . L e s AF d e s h o r i z o n s A 31
des s o ls hydromorphes (type I) ont
1 ' a c i d i t e c a rb o x y liq u e la p lu s f a i b l e , mais so n t le s p lu s r i c h e s en f o n c t io n s
p h e n o l s . L e s AF d e s h o r i z o n s E ^ h i n d u r e s e t B 3
(type II) ont l 'a c i d i t § carb o ­
x y l i q u e l a p l u s f o r t e , m a i s p e u d e f o n c t i o n s p h e n o l . L e s AF d e s h o r i z o n s B h 2
m e u b l e s ( t y p e I I I ) o n t ä l a f o i s u n e f o r t e a c i d i t ö p h e n o l i q u e comme c e u x d u
type I et une f o r t e a c i d i t e c a r b o x y l i q u e comme c e u x du type II. C 'est done a
ce niveau que 1 ’a c i d i t e t o t a l e e s t l a p l u s f o r t e .

CONCLUSI ON

L ' e t u d e d e s AF p r o v e n a n t d e d i v e r s h o r i z o n s d e p o d z o l s e t d e s o l s h y d r o ­
m orphes peu h u m i f e r e s ne s em b le p a s m e t t r e en S v id e n c e de d i f f e r e n c e s f o n d a -
m e n t a l e s d a n s l a s t r u c t u r e d e s AF p r o p r e m e n t d i t s . L e s d i f f e r e n c e s o b s e r v e e s
apparaissent essentiellem ent liees ä la ргёэепсе en proportions variables se-
lon l ' o r i g i n e d e s AF d e s u b s t a n c e s n o n specifiquem ent humiques, te lle s que
les p o l y s a c c h a r i d e s ou l e s p r o t e i n e s .

La rS partition des groupes fonctionnels azotfis et oxygenes differencie

nettem ent les AF. L 'azo te "höterocyclique" domine dans les AF d e s horizons
spodiques les mieux draines alors que l'a z o te а ат!пё devient plus im portant
 IA E A -SM -211/39 141

dans les profils plus humides et q u 'il est maximum d a n s les sols hydromor-
phes. De т ё т е , dans les podzols, l'a c id ite est principalem ent due adx grou-
pem ents c a rb o x y liq u e s a l o r s que dans le s s o l s hydromorphes l 'a c i d i t e pheno-
l i q u e e s t m a j o r i t a i r e . C e s c a r a c t e r e s c o n f e r e n t a u x AF d e s h o r i z o n s !s p o d i -
ques i n d u r e s u n c a r a c t e r e p l u s " m a t u r e " q u ' a u x AF d e s s o ls hydromorphes.
Ceci e s t en a c c o rd avec l ' a g e a p p a re n t e le v e (= 3.0 0 0 ans B .P.) donne par
la d a ta tio n des horizons spodiques in d u r e s (1 3 ). D 'a u t r e p a r t , c e t t e "matu-
r i t e " a p p a r a i t p l u s g r a n d e du f a i t de 1 ' e l im i n a ti o n p r e f e r e n t i e l l e p a r la
nappe des petites molecules hydrosolubles.

La teneur plus elevee en groupem ents phenoliques des AF d e s sols hydro­

morphes peu hum iferes tra d u irait un caractere d'hum us jeune, mais aussi un
a u t r e mode d ’ h u m i f i c a t i o n : d a n s c e s s o l s , l a lignine serait p lu s fragm en-
t e e (3) e t s e s monomeres p a r t i c i p e r a i e n t p l u s largem ent ä la Synthese des
p r o d u its humiques.

REFERENCES

[1 ] JA M B U , P ., R IG H I, D ., Sei. Sol 3 ( 1 9 7 3 ) 207.

[2 ] D U PU IS, T ., JA M B U , P ., R IG H I, D ., T rans. In t. S ym p. (H u m u s e t P lan ta VI> P rague (1 9 7 5 ) 4 4 1 .
[3 ] R IG H I, D ., JA M B U , P ., M A T H O N , E ., D U PU IS, T ., C.R. I erColl. in t. (B io d e g rad atio n e t H u m ificatio n !,
N an cy , 1974, P ierro n e d . (1 9 7 5 ) 3 5 8 .
[4 ] K H O X A Y O .C ., T hese U niv. P o itiers (1 9 7 5 ) 68 p.
[5 ] R IG H I, D ., D E C O N IN C K , F ., P roc. 4 th In t. W orking M eeting o n Soil M icro m o rp h ., K in g sto n , ;1973, Soil
M icroscopy (R U T H E R F O R D , G .K ., E d .), K ingston, C anada (1 9 7 4 ) 567.
[6 ] R IG H I, D ., Sei. Sol 4 (1 9 7 5 ) 315.
[7 ] H U B E R T , G ., G O N Z A L E Z , A ., Can. J . Soil Sei. 51 (1 9 7 1 ) 157.
[8 ] M O O R E , S „ S T E IN , W .,J . B iol. C hem . 211 ( 1 9 5 4 ) 9 0 7 .
[9 ] G O N Z A L E Z , A ., H U B E R T , G ., C an. J . Soil Sei. 52 (1 9 7 2 ) 1.
[1 0 ] D U P U IS, T ., JA M B U , P ., D U P U IS , J ., A n n . A gron. 21 1 (1 9 7 0 ) 75.
[1 1 ] B R IN K , R „ D U B A C H , P ., LY N C H , D ., Soil Sei. 89 (1 9 6 0 ) 157.
[1 2 ] G U C K E R T , A ., T hese D o c t. E ta t U niv. N an cy (1 9 7 3 ) 124 p .
[1 3 ] R IG H I, D ., G U IL L E T , B ., (D a ta tio n s p a r le ca rb o n e -1 4 n a tu re l de la m a tie re o rg an iq u e d ’h o riz o n s sp o d iq u es de
p o d z o ls d es L a n d es d u M edoc (F ra n c e )!, p re se n ts c o m p te s re n d u s , IA E A -SM -211 /7 0 .

DISCUSSION

P. L O SS A IN T : It is w ell k n o w n th a t th e fulvic acids are ra th e r u n sta b le sub stan ces w hose

p ro p e rtie s an d c o n c e n tra tio n in soils change fro m season to season. D oes y o u r p a p e r re la te to a
single sam pling? D o y o u in te n d to stu d y th e p ro p e rtie s o f y o u r fulvic acids as a fu n c tio n o f
g eo clim atic v ariatio n s? j
P. JA M B U : A ll th e sam plings w ere d o n e a t th e sam e tim e a t th e en d o f spring. T h e seasonal
v a ria tio n s in th e p ro p e rtie s o f fulvic acids have n o t y e t b e en stu d ied . It is a n in te restin g su b ject,
an d w e in te n d to u n d e rta k e th is w ork.
 IAEA -SM -211 /7 2

AGGREGATION-DISPERSION PHENOMENA
IN HUMIC SUBSTANCES
N. SE N E SI
I s titu to d i C him ica A graria,
F a c o ltä di A graria,
U n iversitä di Bari,
Bari,
Italy

Y. C H E N
D e p a rtm e n t o f Soil an d W ater,
F a c u lty o f A g riculture,
H e b rew U n iv ersity,
R e h o v o t,
Israel

M. SC H N IT Z E R
Soil R esearch In stitu te ,
A g ricu ltu re C anada,
O tta w a , O n ta rio ,
C anada

R a p p o rteu r: M. S ch n itzer

Abstract

A G G R E G A T IO N -D IS P E R S IO N P H E N O M EN A IN H UM IC SUBSTA N CES.
H u m ic su b stan ce s te n d to aggregate o r disperse in aqueous so lu tio n , d ep e n d in g o n th e e n v iro n m e n ta l
c o n d itio n s, especially th e pH . T hese p h e n o m e n a a ffe c t th e reac tiv ity o f h u m ic su b stan ce s to w a rd s o th e r organics
an d in o rg an ics in soil. S canning E le c tro n M icroscopy (SEM ) was used to ex p lo re th e e ffe c t o f v ary in g th e p H o n
shapes, d im ensions, e x te n t o f aggregation a n d disp ersio n o f h u m ic an d fulvic acid. T h e h u m ic m a terials changed
fro m fibres an d b u n d le s o f fibres a t lo w p H to a fin e ly w oven n e tw o rk a t in te rm e d ia te p H , an d to p la stic-ty p e sh eets
an d fin e grains a t th e h ig h e st p H . E le c tro n S pin R esonance (E S R ) w as a n o th e r te c h n iq u e u sed to assess aggregation-
d isp ersio n p h e n o m e n a . R egardless o f s o lu tio n p H (2 .2 —12.5), all E S R sp e c tra w ere sim ple, s y m m e tric al lin es w ith o u t
h y p e rfin e sp littin g . S pin c o n te n t an d g-values increased w ith th e increase in p H , b u t lin e w id th s decreased . F ro m
th e m a g n itu d e o f th e g-values it ap p e ared th a t th e free radicals w ere s u b stitu te d sem iq u in o n es w h ich , in alkaline :
so lu tio n , w ere stab ilized as se m iq u in o n e io n s. T he low spin c o n te n t an d b ro a d lin e w id th s o b serv ed in th e sp e c tra a t
low p H levels, o r a fte r stan d in g a t ro o m te m p e ra tu re fo r several days, signalized th e fo rm a tio n o f hig h ly p o ly m eriz ed
h u m ic s tru c tu re s . O n th e o th e r h a n d , a t h ig h pH values, o r a fte r irra d ia tio n w ith lig h t in th e visible reg io n , ESR
sp e c tra sh ow ed stro n g increases in free rad ical c o n te n t an d n arro w er line w id th s, suggesting ex ten siv e d ispersion
o f h u m ic p articles. V isco sity m e asu rem en ts w ere d o n e o n these m a terials to o b ta in a d d itio n a l in fo rm a tio n o n
p a rtic le d im en sio n s and o n p o ly e le c tro ly tic b eh a v io u r in aqueous so lu tio n s. H u m ic acid a t n e u tra l p H an d fulvic
acid a t v ery low p H e x h ib ite d sim ilar p a rtic le d im ensions. As th e pH w as in c reased , fu lv ic acid p a rtic le s first
b ecam e sm aller, a tta in in g m in im u m sizes a t pH 3, an d th e n increased b u t slig h tly w ith th e rise in p H , b ecause o f
h y d ra tio n o f d isso cia ted c a rb o x y l groups. A ggregation o f fulvic acid an d h u m ic acid p articles a t lo w p H ap p ears to
resu lt fro m e ith e r in te rm o le c u la r in te ra c tio n s o f th e Van d e r Waal ty p e , in te ra c tio n s b e tw e e n t:-e le c tro n sy stem s o f
a d ja c e n t m olecules, s tro n g h y d ro g e n b o n d in g a n d /o r h o m o ly tic re a c tio n s b etw e en free rad icals. A s th e p H in creases,
th e se fo rces b eco m e w eak er, an d because o f increasing io n izatio n o f acidic fu n c tio n a l g ro u p s, p articles s ep arate and
b egin to rep el ea ch o th e r ele c tro sta tic a lly , so th a t th e m olecu lar arran g e m en ts b ec o m e sm aller an d sm aller, bringing
a b o u t th e d ispersion o f h u m ic m aterials.

143
144 SENESI e ta l.

IN T R O D U C T IO N

It h as b een k n o w n fo r som e tim e th a t h u m ic sub stan ces te n d to aggregate o r disperse in

a q u eo u s so lu tio n s, d ep en d in g o n th e e n v iro n m en ta l co n d itio n s, especially th e pH . I t is likely th a t
sim u lta n eo u sly o rganic m olecules such as pesticides, c a rb o h y d ra te s, p ro tein s, p e p tid e s, am in o
acids, as w ell as in o rg an ic substan ces (m e ta l ions, m eta l h y d ro x id es, m inerals an d clays), aggregate
w ith o r disperse o r disengage fro m h u m ic ‘building b lo ck s’. T hus, a b e tte r u n d e rsta n d in g o f these
re ac tio n s is n o t o n ly o f academ ic b u t also o f co n sid erab le p ra ctic a l in te re st. We a p p ro a c h e d the
p ro b le m w ith th e aid o f tw o relatively re c e n t tec h n iq u es: Scanning E le c tro n M icroscopy (SEM )
an d E le c tro n S p in R eso n an ce (E S R ) sp e c tro m e try , and an o ld e r te c h n iq u e, viscom etry.
T h e S canning E le c tro n M icroscope can pro v id e m agnified view s o f m in eral an d organic
surfaces w ith a d e p th o f focus th a t is o f th e o rd e r o f ten s o f m icrons. A n u m b e r o f p u b licatio n s
describe th e a p p lic atio n o f th e SEM to soils an d clay m inerals [1 —4 ], b u t th e m e th o d h as been
little u sed in in v estig atio n s on soil organic m a tte r. B ecause fre q u e n tly fine s tru c tu re is revealed
w ith u n u su a l clarity a n d p a rticle b o u n d a ry relatio n sh ip s can be ex am in ed in great d etail, we p ro ­
ceed ed to ex p lo re th e u sefulness o f SEM fo r investigating e ffe c ts o f varying th e pH o n th e e x te n t o f
ag gregation a n d d isp ersio n o f a h u m ic acid (H A ) an d a fulvic acid (F A ). We w ere especially co n ­
cern ed w ith using sam ple p re p a ra tio n tec h n iq u es th a t w ould n o t dam age th e fabrics n o r p ro d u c e
a rtifac ts. Since all sam ples w ere p re p a re d in aq u eo u s so lu tio n s o r suspensions, it was necessary
to rem ove th e w a te r du rin g th e p re p a ra tio n o f sam ples. T h e tec h n iq u e th a t we fo u n d a fte r
co n sid erab le e x p e rim e n ta tio n m o st sa tisfac to ry fo r th is p u rp o se was u ltra ra p id freezing [5].
T h e relativ ely high c o n te n t o f stable free radicals o f soil h u m ic substan ces has b e en a su b ject
o f co n sid erab le in te re s t to soil scien tists in re c e n t y ears [6 —10]. It is likely th a t free radicals play
im p o rta n t ro les in p o ly m e riza tio n -d e p o ly m e riz atio n re ac tio n s o f h u m ic substances, in re ac tio n s
w ith o rg an ic c o m p o u n d s such as p esticid es a n d to x ic p o llu ta n ts , an d in th e p hysiological effects
th a t th ese su b stan ces are k n o w n to e x ert. In sp ite o f th e co n siderable a m o u n t o f research th a t
soil sc ien tists have so fa r d o n e in th is field, m u ch rem ains to be learn ed on h o w aggregation-
d isp ersio n p h e n o m e n a o f h u m ic sub stan ces are a ffe c te d b y free radicals, an d h o w th e la tte r resp o n d
to chan g es in p H , e x p o su re to long re a c tio n tim es an d to irrad iatio n .
V isco sity m easu rem en ts can provide im p o rta n t in fo rm a tio n on p article dim en sio n s and
p o ly e le c tro ly tic b e h av io u r o f m acro m o lecu les in aq u eo u s so lu tio n s. T he m e th o d h as b e en applied
to h u m ic acids b y a n u m b e r o f w o rk ers w hose c o n trib u tio n s have re ce n tly b een su m m arized by
F laig e t al. [11 ]. T h ere is, how ever, co n siderable d isag reem en t in th e U terature on v iscom etric
m ea su re m e n ts o n h u m ic sub stan ces [11 —14]. O ne m ajo r re aso n fo r th is u n sa tis fa c to ry situ a tio n
is th a t d iffe re n t w o rk ers have u sed w id ely d iffering m e th o d s fo r e x tra c tio n , se p a ra tio n an d
p u rific a tio n o f h u m ic m aterials. A cco rd in g to th e d e fin itio n c u rre n tly a cc ep te d b y m o st soil
scien tists, H A is th e fra c tio n o f soil organic m a tte r th a t is e x tra c te d b y d ilu te base b u t w hich
b eco m es in so lu b le w hen th e alkaline e x tra c t is acidified, w hereas F A is soluble u n d e r these
co n d itio n s. T h is d e fin itio n , how ever, has n o t alw ays b e en ad h ered to , so th a t fre q u e n tly w hat
are really fulvic acids (F A s) are re fe rre d to in th e lite ra tu re as H A s, o r m ix tu re s o f H A s and
F A s are d esig n ated as HAs.

E X P E R IM E N T A L

M aterials a n d m e th o d s

H A a n d FA

T h e H A w as e x tra c te d fro m th e k x h o riz o n (0 —25 cm ) o f th e Beaverhills soil, a H a p lo b o ro ll

(6.1% C, p H (H 2 0 ) 6 .4 ) in C e n tra l A lb e rta , C anada. T h e F A o rig in a ted from th e Bh h o riz o n
(1 5 —21 cm ) o f th e A rm adale soil, a p o o rly d ra in e d S p o d o so l (4.2% C, p H (H 2 0 ) 4 .0 ) in
 IAEA -SM -211 /7 2 145

Prince E d w ard Islan d , C anada. B o th th e H A an d F A w ere e x tra c te d w ith 0 .5 N N aO H so lu tio n

u n d e r N 2 . M eth o d s o f e x tra c tio n , fra c tio n a tio n an d p u rific a tio n o f th e H A an d F A have been
d escrib ed p rev io u sly [1 5 , 16]. T h e p u rifie d H A c o n ta in e d 56.4% C, 5.5% H, 4.1% N , 1.1% S and
33.0% O. O ne gram o f d ry , ash-free H A c o n ta in e d 4.5 m eq C 0 2 H , 2.1 m eq p h e n o lic O H , 2 .8 m eq
alco h o lic O H , 2 .5 m eq k e to n ic C = 0 , 1.9 m eq q u in o n o id C = 0 and 0.3 m eq O C H 3 g ro u p s [ 17].
U ltim a te a n d fu n c tio n a l g ro u p s analyses o f th e p u rifie d F A w ere as follow s: 50.9% C, 3.3% H,
0.7% N , 0.3% S, 4 4 .7 % O, 9.1 m eq C 0 2 H, 3.3 m eq p h e n o lic O H , 3.6 m eq alco h o lic O H , 2.5 m eq
k e to n ic C = 0 , 0 .6 m eq q u in o n o id C = 0 a n d 0.1 m eq O C H 3 g ro u p s p e r g [18].

P re p a ra tio n o f sam ples

In case o f th e F A , it w as p ossible to vary th e pH fro m a b o u t 2 to 10 b y th e a d d itio n o f d ilu te

N aO H so lu tio n ; th e n a tu ra l p H o f a 1% (W /V ) F A so lu tio n in H 20 w as 2.1 . T h e lo w est p H a t
w h ich th e H A w as so lu b le in H 20 w as 6 .0 , w h ich lim ited th e range fo r investigating p H e ffe c ts o n
th ese m aterials to b e tw ee n 6 .0 a n d 10. F o r SEM a n d E S R in v estigations, 1% (W /V ) aq u eo u s
so lu tio n s o f each m aterial w ere p re p are d . F o r viscosity m ea su re m e n ts, 0.6% (W /V ) so lu tio n s in
H 20 w ere e m p lo y e d o v er th e sam e p H range.

S canning E le c tro n M icroscopy

O n e d ro p o f each so lu tio n o r susp en sio n w as sp o tte d o n glass slides, 10 m m in diam eter.

T h e sp ecim en s w ere ra p id ly im m ersed in m o lte n F re o n -1 2 (a t -1 5 5 °C ), w h ich w as c o n ta in e d in
a stain less steel cu p , su b m erged in liquid n itro g e n in a D ew ar flask. A fte r quick-freezing, th e
sp ecim en s w ere tra n s fe rre d to a Speedivac P earse T issue d ry e r, m o d el 1, p laced o n a co ld stage
m ain ta in e d a t -80°C a n d freeze d ried. T h e d ry sp ecim ens w ere th e n a tta c h e d w ith th e aid o f
co llo id al silver p a in t to A l-stubs. T o m ak e th e specim en s su rfa ce -c o n d u ctin g , th e y w ere c o ated
in a S p eedivac C o atin g U n it, m o d el 2 1 E 6 /1 2 5 8 , first w ith a th in lay er o f c arb o n a n d th e n w ith
a la y e r o f gold -p allad iu m . F o llo w in g th e o p e ra tio n , th e specim ens w ere ex am in ed o n a C am bridge
In stru m e n ts S te reo sca n M ark 2A SEM o p e ra te d a t 2 0 kV.

E le c tro n S p in R eso n an ce m ea su re m e n ts

E S R m ea su re m e n ts w ere d o n e o n 1% (W /V ) so lu tio n s as describ ed by R iffald i and

S c h n itz e r [1 9 ]. E S R sp e c tra w ere re c o rd e d a t ro o m te m p e ra tu re o n a V arian A ssociates E-3
s p e c tro m e te r, e m p lo y in g 1 00 kH z m o d u la tio n a n d a n o m in a l o p e ra tin g freq u e n c y o f 9.5 G H z.
S p in c o n c e n tra tio n s , line w id th s an d g-values w ere c o m p u te d fro m th e sp ectra a cco rd in g to
R iffald i a n d S c h n itz e r [1 9 , 20].
Sam p les w ere irra d ia te d in situ th ro u g h slo ts in th e sp e c tro m e te r cavity. L ight sources
w ere 150 an d 5 0 0 W tu n g ste n lam ps m o u n te d 2 0 cm fro m th e cavity. T o p ro d u c e filte red
lig h t, a p p ro p ria te n a rro w b a n d pass in te rfe re n c e filte rs (m a n u fa c tu re d by G ru b b P arsons,
N ew castle u p o n T y n e ) w ere in se rte d in th e lig h t p a th . Irra d ia tio n in creased th e te m p e ra tu re in th e
cav ity b y a b o u t 5 —8 degC.

Viscosity measurements

A m o d ifie d O stw ald v isc o m e ter (C a n n o n -F e n sk e), having a flow tim e o f 150 s f o r 3 m l o f
d istilled w a te r a t 2 5 °C , w as u se d fo r th e v iscosity m easu rem en ts. O u r flo w tim es re p re se n t
averages o f five read in g s m ad e in a c o n s ta n t te m p e ra tu re b a th a t 25 ± 0 .0 5 °C . V a riatio n s b e tw ee n
re p lica te flo w tim e s w ere ± 0.5% .
 146
ШШ

SENESI et al.
F I G .l. S E M m ic r o g r a p h s o f F A a t v a r io u s p H le v e ls : a ~ p H 2 ; b ~ p H 4 ; c ~ p H 6 ; d - p H 7; e - p H 7;
f — p H 8 ; g - p H 9 ; h - p H l O .
 IAEA -SM -211 /7 2 147

T h e relativ e v iscosity (т?г) is d efin e d as:

w h e re 17, = th e v iscosity o f th e so lu tio n

r?0 = th e v iscosity o f th e p u re solvent

T h e sp ecific visco sity ( 17^ ) is:

hsp = - ^ — = 4r - l

T h e re d u c e d visco sity (Tjred) is:

hsp _ hr ~ l
^ re d — с — c

w h e re c is th e c o n c e n tra tio n (W /V )% o f th e solute.

R E SU L T S A N D D IS C U SSIO N
■

p H e ffe c t ob serv ed b y SEM |

T h e e ffe c t o f varying th e p H o n th e shape an d p article a rra n g e m e n t o f F A is shownj in Fig. 1.

A t p H 2 (Fig. 1a), th e F A co n sisted o f elo n g ated fibres a n d b u n d les o f fib res (b u n d le s w ere
p a rtic u la rly p ro m in e n t a t lo w er m ag n ificatio n , w h ich are n o t p re sen te d ). T h e fib res w ere usually
curved, o f te n en d in g in p ro tru s io n e x te n d in g fro m th e p lan e o f th e fibres. T h e fib re th ic k n e ss w as
estim a te d t o ran g e fro m 0.1 to 0 .4 p m . T h e s h o rt, th ic k p ro tru sio n s w ith ro u n d e d h ead s w ere
0 .2 5 —0 .4 5 p m lo n g a n d a p p ro x im a te ly 0 .1 5 p m th ick . A t pH 4 (Fig. 1b), th e fibres te n d e d to
b eco m e th in n e r. T h is te n d e n c y w as m o re p ro m in e n t a t p H 6 ( F ig .lc ) , and a g re ater p r o p o rtio n o f
th e F A o c cu rre d in b u n d les o f closely k n it fibres. T h e tra n s itio n c o n tin u e d a t p H 7 (Fig. Id
an d F ig .le ), w h en a fin e n e tw o rk o f tig h tly m esh ed fibres w ith parallel o rie n ta tio n , w h ich te n d e d
to coalesce, b ecam e visible. A t p H 8 (Fig. If), th e F A p articles fo rm ed a sheet-like s tru c tu re o f
v arying th ick n ess. T h e m icro g rap h s show a d is tin c t change in s tru c tu re a n d im p ro v ed o rie n ta tio n
b e tw ee n p H 7 an d 8 . A t p H 9 (F ig .lg ), th e plastic-like sh eets te n d e d to th ic k e n , especially near
th e edges. A t p H 10 (Fig. lh ) , th e p a rticle s ap p ea red to be fine-grained, w ith a high degree o f
h o m o g en e ity .
T h e e ffe c t o f p H o n th e s tru c tu re o f H A (F ig .2 ) w as sim ilar to th a t show n o n th e F A stru c tu re .
A s sh o w n in F ig .2 a, th e H A s tru c tu re a t p H 6 co n sisted o f fibres and b u n d les o f fibres, w ith m o st
o f th e fib res b eing th ic k e r th a n th o se c o n stitu tin g F A a t th e sam e pH (F ig .lc ). A t p H 8 (F ig .2 b ),
th e fib res a n d coalescing s tru c tu re s becam e less p ro m in e n t. A t p H 10 (F ig .2 c), th e H A s tru c tu re
d iffered co n sid erab ly fro m th a t a t p H 8 . T h e H A particles ap p ea red to align them selves lo n g itu d ­
in ally to fo rm th in sh eets, w ith som e o f th e edges curling u p w ard s. T h e b eh av io u r o f H A a t p H 10
w as sim ilar to th a t e x h ib ite d b y F A a t pH 8 (F ig .If).
M icrographs o b ta in e d o n th e SEM show th a t th e pH h a d a m ark ed e ffe c t o n th e trid im en sio n al
a rra n g e m e n t o f F A p a rticle s (Fig. 1). We w itn ess a grad u al tra n s itio n fro m a m o re ra n d o m s tru c tu re
a t lo w p H to a m o re o rg an ized and o rie n te d o n e a t higher pH . S im u lta n eo u sly , th e p a rticle s becam e
sm aller as th e p H increased.
F o r th e case o f H A (F ig .2 ), a sim ilar tra n s itio n fro m fibres to a m o re hig h ly o rie n te d stru c tu re
co u ld be ob serv ed , a lth o u g h th e p H range w as, b y necessity, n arro w er. T h e m ain d iffere n ce w as th a t
fo r th e H A th e tra n s itio n o c c u rre d a t a h ig h er pH th a n th a t fo r FA .
 148
SENESI e ta l.
F I G .2 . S E M m ic r o g r a p h s o f H A a t v a r io u s p H le v e ls : a —p H 6; b - p H 8; c - p H 10.
 IAEA -SM -211 /7 2 149

TABLE I. ESR PARAMETERS FO R FA AND HA SOLUTIONS AT VARIOUS pHs

Spins/g (X 10_17) Line width (G ) g-values

Material pH -
U ntreated Irradiated Untreated Irradiated Untreated Irradiated
i
1
FA 2 .0 1.44 1.69 2.5 2.5 2 .0 0 3 8 2 .0 0 3 8
FA 5.0 1.50 1.75 3.0 3.2 2 .0 0 3 7 2 .0 0 3 7
FA 7.0 1.56 1.82 3.0 3.0 2 .0 0 3 9 2 .0 0 3 9
FA 8.0 1.51 1.97 2.5 2.5 2 .0 0 4 0 2 .0 0 4 2
FA 9 .0 1.69 2 .6 6 2.0 2.0 2 .0 0 4 4 2 .0 0 4 4
FA 10.6 2.19 7.77 2.0 2.0 2 .0 0 4 4 2 .0 0 4 4
FA 11.6 2.5 6 15.43 2.1 2.2 2 .0 0 4 4 2 .0 0 4 3
FA 12.5 12.06 17.20 2.2 2.2 2 .0 0 4 5 2 .0 0 4 4

HA 6 .0 23.51 28 .5 8 3.3 3.3 2 .0031 2 .0 0 3 2

HA 8 .0 2 6 .2 4 3 0 .5 2 3.5 3.5 2 .0 0 3 5 2 .0 0 3 6
HA 9.3 3 5 .0 0 3 4 .5 9 3.5 3.5 2 .0 0 3 5 2 .0 0 3 6
HA 10.3 3 7 .0 0 2 7 .55 3.6 3.6 2 .0 0 3 9 2.0Ö42

HA 11.1 3 7 .43 2 7 .8 9 3.5 3.2 2 .0041 2 .0 0 4 4

The sponge-like structure revealed by SEM for HA and FA at pH values likely to be encountered
in m ost soils supports the structural concepts proposed earlier by one o f the authors [21] for these
materials. It was suggested th a t hum ic substances had sponge-like structures containing m any
voids th a t could trap inorganic and organic m olecules, including toxic pollutants, provided th at
these had the appropriate m olecular sizes to fit into the voids and th a t the charges were
com plem entary.
The reactivity o f hum ic substances is profoundly affected by the degree o f aggregation or
dispersion o f the constituent molecules. Thus, up to about pH 7 for FA and slightly higher for HA,
sponge-like structures are form ed w ith large ‘outside’ and ‘inside’ surfaces and thus large adsorption
capacities. This situation prevails in practically all soils used for agriculture.

Effect of pH on ESR parameters

I
I
E ffects o f pH on ESR param eters o f FA and HA in aqueous solutions are listed in Table I and
ESR spectra are presented in Fig.3. Regardless o f pH o f the system , all ESR spectra consisted of
single, sym m etrical lines devoid o f hyperfine splitting. Running the ESR spectra in the absence of
oxygen (air) dis not bring ab o u t hyperfine splitting but reduced signal intensities.
Spin concentration in b o th FA and HA increased m arkedly w ith increase in pH (Fig.3 and
Table I). This was also true for g-values, b u t line widths, in the case o f FA, were narrow er at high pH
than at low pH. T he narrow est line w idths ( 2 .0 -2 .2 ) were observed in solutions at p H 3*9.
Because o f m olecular com plexity, we did n o t observe any hyperfine splitting in o u r ESR
spectra. T herefore g-values were the only characteristics th a t we could use to identify the radicals.
Judging from th e m agnitude o f g-values, w hich ranged from 2.0032 to 2.0050, w ith m ost falling
into the 2.0 0 3 8 —2.0042 range, the free radicals in our preparations w ere m ost likely sem iquinones
o r substituted sem iquinones [22]. T he increase in free radical concentrations w ith increase in pH
is m ost likely due to the form ation o f the stabilized, sym m etrical sem iquinone ions, resulting
from dehydrogenation o f phenolic hydroxyl groups and sim ultaneous hydrogenation o f quinonc
150 SENESI e ta l.

F I G .3 . E S R s p e c tr a o f F A a t d iffe r e n t p H le v e ls .

oxygens. T his is in accord w ith similar observations on biologically occurring quinones [2 3 ],

lignin and hum ic substances [24—27]. Steelink [26] considers this behaviour typical o f quinhydrone
systems.
Line w idths narrow ed w ith increase in pH and after allowing solutions o f FA a t high pH to
stand for several hours. The narrow est line w idths resulted from greater tum bling speeds, greater
freedom o f ro tatio n and less association (possibly by breaking H-bonds) w ith neighbouring
molecules. A nother possible mechanism th a t can explain the narrowing o f line w idths a t high pH
is exchange o f electrons betw een orbitals o f d ifferent m olecules, even w hen there is negligible
chemical bonding betw een the molecules [28]. O n the o ther hand, broadening o f line w idths at
neutral and acid pH m ay be due to increasing m olecular association, m ost likely involving
H-bonding and dipolar spin-spin interactions betw een electron spins.
 IA E A -S M -2 U /7 2 151

F I G .4 . E ffe c t o f tim e o n th e p H a n d fr e e r a d ic a l c o n t e n t o f F A . N u m e r a ls o n c u rv e r e fe r to tim e in m in u te s .

Effects o f reaction tim e on pH and ESR param eters

When 10 mg o f FA were dissolved in 1.0 ml o f 0.1 N NaOH, the pH, im m ediately after dis­
solution, was 11.7. A fter 20 m inutes o f standing a t room tem perature under air, the pH dropped to
11.4 (Fig.4). The pH continued to drop w ith tim e o f standing until it had attained a value o f
9.0 after 8640 min. Free radicals first tended to increase, reaching a m axim um value after 30 min
and th en decreased gradually and, betw een 120 and 270 min, rapidly (from 10.90 to
1.99 X 1017 spins/g). Beyond th a t tim e, the decrease in free radical concentration was relatively
slow. T hroughout the experim ent g-values rem ained more or less constant, but tended to increase
above 2.0040 after 1260 min. Line w idths narrow ed sharply betw een 120 and 270 min, and
then rem ained constant.
When the initial pH o f the FA was either 7.0 o r 2.2, spin concentrations decreased pnly very
slowly w ith tim e, b u t the pH rem ained constant for up to 8640 m inutes.
We have observed th a t free radicals in FA -powders were stable over a p eriod o f at least
eight years, b u t w hen in aqueous solutions, free radical concentrations decreased w ithin a short
tim e, depending o n the pH, to values th a t were low er than those for solid samples, indicating the
occurrence o f polym erization reactions under these conditions.

Irradiation experim ents

Solutions o f FA and HA at various pHs were irradiated for 5 m inutes first w ith m onochrom atic
light (480-800 nm ) o f 150 W intensity. No significant changes in ESR param eters from those for
non-irradiated samples were observed. When the same samples were irradiated w ith w hite light o f
greater intensity (500 W), increases in spin concentrations were observed on FA solutions, especially
at p H s > 9.0 (Figs 5 and 7 and Table I). A t pH 11.5, FA solution showed a significant increase in
spin concentration o f a bout 6X, after irradiation. In the case o f HA (Figs 6 and 7 and Table I),
an increase in spin concentrations at p H s < 9.0, and a decrease at pHs o f 9.0 and 10.0, were
observed. Finally w hen the light was turned off, free radical concentrations gradually returned to
 SENESI et al.

pH

F I G .5 . E ffe c t o f p H o n t h e fr e e r a d ic a l c o n t e n t o f F A b e fo r e a n d a f t e r ir r a d ia tio n .
( а - 0 1 х) 6/SNidS

6 7 8 9 10 11 12

pH

F I G .6 . E ffe c t o f p H o n f r e e r a d ic a l c o n t e n t o f H A b e fo r e a n d a fte r ir r a d ia tio n .

 IA E A -SM -211/72 153

pH

F IG . 7. E f f e c t o f ir r a d ia tio n o n r e la tiv e s p in c o n c e n tr a tio n o f H A a n d F A a t v a r io u s p H s .

values equal to those before irradiation. It is notew orthy th a t relative changes in spin concen­
trations for HA were m uch smaller th an for FA (Fig. 7). We also used filtered light a t wavelengths
ranging from 428 to 656 nm to irradiate FA solutions at various pHs. We obtained the! highest
concentrations o f free radicals (especially at pH > 9.0) w hen light betw een 550 and 600 nm
(green-yellow-orange) was used for this purpose. T hroughout the irradiation experim ents, g-values
and line w idths rem ained practically unaffected (Table I), so th at, although the num bers o f radicals
increased, their chemical structure rem ained unaltered.
A lthough irradiation is m ore efficient at high pH than at low pH, w hat is im portant in the
context o f soil chem istry is th a t these reactions also take place, even if at a slower rate, at pH levels
norm ally found in soils (pH 3.5—8.5). Thus, while studying reactions th a t proceed sm oothly at
high pH, we can uncover m echanism s and principles th a t are also relevant a t lower pH levels.

E ffect o f pH on reduced viscosity

Figure 8 shows the relationship betw een r)red and pH at a constant FA concentration o f
0.6 g/100 ml. The curve had a m inim um at pH 3.0, and the same general shape as the 7jred versus
pH curves for HAs extracted from Japanese soils by Kum ada and Kawamura [14], except th at
minima in their curves occurred a t a slightly higher pH. The initial decrease in 7jred w ith increase
in pH (Fig.8), was probably caused by a reduction in FA particle size. With further neutralization
o f acidic functional groups, small increases in 7?red were observed, a behaviour w hich is typical o f
linear polyelectrolytes [14].
T he reduced viscosity o f HA at pH 7, 8.5 and 10.5 showed only small variations, and the values
were similar to those o f FA at pH 1.0, suggesting similarities in particle sizes.
F or FA the m inim um in 7jred a t pH 3.0 appears to signalize a m inim um in particle size at th at
pH. As the pH is lowered, aggregation or association o f particles occurs, so th a t the viscosity
increases; th e FA particles are practically uncharged under thses conditions, and electrostatic
repulsion is n o t an im portant factor. As pH is raised to above 3.0, increased dissociation o f
154 SENESI et al.

pH

F IG . 8. E ffe c t o f p H o n V re (j o f F A a n d H A , a t a c o n s ta n t c o n c e n tr a tio n o f 0 .6 g /1 0 0 m l.

oxygen-containing functional groups takes place, w hich leads to increased electrostatic repulsion o f
FA particles. This is accom panied by an increasing hydration o f the ionized functional groups,
so th a t the n e t result is a gradual increase in viscosity as the pH increases, as we have observed.

CONCLUDING COMMENTS

The aggregation o f FA and HA particles at low pH can be explained by hydrogen bonding,

Van der Waal’s interactions and interactions betw een 7r-electron systems o f adjacent molecules, as
well as by hom olytic reactions betw een free radicals. As the pH increases, these forces becom e
weaker, and because o f increasing ionization o f carboxylic acid and phenolic hydroxyl groups,
particles separate and begin to repel each o th er electrostatically, so th a t the m olecular arrangements
becom e smaller and smaller b u t b e tte r oriented.
The increased free radical content at high pH o r a fter irradiation also signalizes extensive
dispersion o r disaggregation o f hum ic particles. In particular, the considerable, reversible increase
in free radical concentration after irradiation w ith w hite light suggests th at w hen sunlight falls on
surface soils high in hum ic substances (organic m atter), large concentrations o f free radicals should
be form ed in these m aterials, so th a t hum ic substances m ay act as photosensitizers for bonded or
sorbed substances. F o r exam ple, sorbed herbicides m ight be detoxified by free radicals w hose
form ation is stim ulated by light and oxygen (air).
T he HA and FA th a t we investigated behave like flexible, linear, synthetic polyelectrolytes,
so th at we are n o t dealing w ith structures exclusively com posed o f condensed rings, but there must
be num erous linkages a bout w hich relatively free ro tatio n can occur. Aggregation o f HA and FA
molecules at low pH results from interm olecular attraction, whereas dispersion at high pH derives
largely from interm olecular repulsion.
 IAEA -SM -211 /7 2 155

REFERENCES

[1 ] BOHOR, B .F ., HUGES, R .E ., Clays Clay M iner.19 (1 9 7 1 ) 49.

[2 ] GILLOT, J.E., J. Sedim ent. Petrol. 39 (1 9 6 9 ) 90.
[3 ] BORST, R .L., KELLER, W.D., “ Scanning electron micrographs o f API reference clay m inerals and
other selected sam ples” (Proc. Int. Clay C onf. T o k y o , 1969) I, Israel Univ. Press, Jerusalem (1 9 6 9 ) 871.
[4 ] KELLER, W .D., H A N SO N , R .F ., Clays Clay Miner. 23 (1 9 7 5 ) 2 0 1 .
[5 ] BOYDE, A ., WOOD, C., J. Microsc. 9 0 3 ( 1 9 6 9 ) 2 4 1 .
[6 ] STEELINK , C , TOLLIN, G., “ Free Radicals in S oil” , Soil B iochem istry I (M cL A R E N , A .D .,
PETERSON, G.H ., Eds), Marcel D ekker, N ew York (1 9 6 7 ) 147.
[7 ] A TH ER TO N , N.M ., CRANWELL, P .A ., FLO YD , A .G ., HAWORTH, R .D ., Tetrahedron 2 3 (1 9 6 7 ) 1653.
[8 ] SCHNITZER, M., KH AN , S.U ., H um ic Substances in the Environm ent, Marcel D ekker, N ew Y ork (1 9 7 2 )
86, 220.
[9 ] LISANTI, L.E., TESTINI, C., SENESI, N „ A grochim ica 18 (1 9 7 4 ) 134.
[1 0 ] SLAW INSKA, D ., SLAWINSKI, J., SA R N A , T., J. Soil Sei. 2 6 (1 9 7 5 ) 93.
[1 1 ] FL A IG , W., BEUTELSPACH ER, H ., RIETZ, E ., “ Chem ical C om position and Physical Properties o f Hum ic
Substances” , S oil C om ponents I (GIESEKING, J.E ., Ed.), Springer Verlag, N ew Y ork (1 9 7 5 ) 1.
[1 2 ] OR LOV , D .S., G O RSHK OV A, Y .I., N auch. D okl. Vyssh. Shk., Biolog. N auki I ( 1 9 6 5 ) 2 0 7 .
[1 3 ] PIRET, E .L ., WHITE, R .G ., W ALTER, H.C., Jr., M ADDAN, A .J., Jr., Som e p hysico-ch em ical properties o f
peat h um ic acids (S cien tific Proc.), R oyal D ublin S oc., Series A (1 9 6 0 ) 69.
[1 4 ] KU M A D A, K., KAW AM URA, Y ., Soil Sci.Plant Nutr. 14 (1 9 6 8 ) 190.
[1 5 ] SCHNITZER, M., SKINNER, S.I.M ., S oil Sei. 105 (1 9 6 8 ) 392.
[1 6 ] KH AN , S.U., SOWDEN, F.J., Can. J. S oil Sei. 51 (1 9 7 1 ) 185.
[1 7 ] KHAN, S.U ., SCHNITZER, M., Can. J. S oil Sei. 5 2 (1 9 7 2 ) 43.
[1 8 ] OG NER, G„ SCHNITZER, M., Can. J. Chem. 4 9 (1 9 7 1 ) 1053.
[1 9 ] R IF FA LD I, R ., SCHNITZER, M., Soil Sei. Soc. A m ., Proc. 36 (1 9 7 2 ) 772.
[2 0 ] R IF FA L D I, R., SCHNITZER, M., G eoderm a 8 (1 9 7 2 ) 1.
[21 ] SCHNITZER, M., A gronom y Abs. (1 9 7 1 ) 77.
[2 2 ] BLOIS, M.S., Jr., BROWN, H.W., MALING, J.E., “Precision g-value m easurem ents on free radicals o f
b iological interest” , Free Radicals in B iological System s (BLOIS, M.S., Jr., BROWN, H.W., LEMMON, R.M.,
LINDBLOM , R .O ., W EISSBLUTH, M., Eds), A cadem ic Press, N ew Y ork, L ondon (1 9 6 1 ) 117.
[2 3 ] BLOIS, S., B iochim . B iophys. A cta 18 (1 9 5 5 ) 165.
[2 4 ] STEELINK, C , REID, T ., TOLLIN, G ., J. Am . Chem. Soc. 85 (1 9 6 3 ) 4 0 4 8 .
[2 5 ] TOLLIN, G„ REID, T., STEELINK, C., B iochim . Biophys. A cta 6 6 ( 1 9 6 3 ) 4 47.
[2 6 ] STEELINK, C., G eochim . C osm ochim . A cta 28 (1 9 6 4 ) 1615.
[2 7 ] STEELINK, C., “ E lectron param agnetic resonance studies o f hum ic acid and related m od el com p ou nd s” ,
Coal Chem istry, Advances in Chemistry Series N o .5 5 , American Chem ical S o ciety (1 9 6 6 ) 80.
[2 8 ] INGRAM, D .J.E., Free Radicals as Studied b y E lectron Spin R esonance, Butterw orth S cientific Publ.,
L ondon (1 9 5 8 ) 102.
 IA E a |-SM-2 1 1 /7 3

STRUCTURE CHIMIQUE DES ACIDES

HUMIQUES ET FULVIQUES DU SOL
J.-A. NEYROUD
Station föderale de recherches
agronom iques de Changins,
Nyon,
Suisse

M. SCHNITZER
Soil Research Institute,
Agriculture Canada,
Ottawa,
Canada

R a p p o rteu r: M. S ch n itzer

Abstract-Rösumö

CHEMICAL STRU C TUR E OF HUMIC A N D FULVIC ACIDS IN THE SOIL. j

V arious agents o f increasing strength are u sed to degrade hum ic substances in fractions w ith increasing
co m p lex ity. Each fraction , how ever, con tain s th e sam e basic compounds', b en zen e p oly ca rb o x y lic acids, phenol
derivatives and aliphatic com pounds. These building b locks, w hich are present b o th in th e hum ic and fulvic
acid extracts, are form ed in to a heterop olycon densate representing th e stable phase fo r any hum ic substance.
The stability o f this phase is explained by the strong m icrobial degradation resistance o f the individual com ­
pounds, and the form ation o f additional chem ical and p hysico-chem ical bonds. Partial degradation b^ milder
agents removes th e new er com pounds that have n o t y e t been incorporated in to the hum ic structure and still
retain characteristics o f their vegetal or m icrobial origins. These com p ou nd s are similar to th o se id en iified by
a num ber o f investigators in studies on f u n g a l h u m i c a c i d s . It m ay be assum ed that such com pounds probably
undergo m ore changes before acquiring a stable and resistant form.

STRUCTURE CHIMIQUE DES ACIDES HUMIQUES ET FULVIQ UES DU SOL. j

Divers agents d’agressivite croissante degradent les substances hum iques en fractions de com p lexite
croissante. Chaque fraction con tien t cependant les m em es com poses de base: acides benzen e-p olycatboxyliq ues,
derives p henoliques e t derives aliphatiques. Ces briques elem entaires, presentes aussi bien dans les extraits d’AH
que dans les extraits d’A F, son t a s se m b le s en un heterop olycon densat form ant la phase stable de to u te substance
hum ique. Les proprietes de stability de ce tte phase son t dues a la forte resistance ä la degradation m icrobienne
des com p oses individuels et ä Retablissem ent de liens chim iques et physico-chim iques additionnels. D es degrada­
tion s partielles par des agents peu agressifs arrachent des com p oses jeunes qui ne so n t pas encore incorpores a
la charpente hum ique e t p orten t la marque de leur origine vegetale ou m icrobienne; ils so n t sem blables aux
com p oses id en tifies par plusieurs chercheurs etudiant des a c i d e s h u m i q u e s d ’o r i g i n e f o n g i q u e . On petit supposer
que ces com p oses son t appeles ä subir encore quelques transform ations avant d’acquerir une form e stable et
resistante. j

INTRODUCTION j

La transform ation des residus vegetaux e t anim aux en hum us et la lente m ineralisation de
celui-ci participent au processus de form ation du sol, d ont de nom breuses phases restent obscures.
L’hum us du sol - acide hum ique, acide fulvique, hum ine - est un ensemble de com poses de
form ules chim iques mal connues, lies ä la fraction minerale du sol, et dont la principale carac-
teristique est la resistance rem arquable ä la degradation m icrobienne.

157
 Ul
00

TABLEAU I. PROCEDES DE DEGRADATION UTILISES (substance humique utilisee: 1,0 g)

Procede O xydation O xyd ation du O xyd ation ä O xyd ation ä O xyd ation O xyd ation H ydrolyse Degradation
K M n0 4 prod, m eth yle 80°C , acide 4 0 ° C, acide CuO/NaOH CuO/NaOH NaOH p hysico-chim ique
K M n0 4 peracetique peracetique + K M n 0 4 sur
residu

R eference [3] [4] [5] [6 ] [8 ] [8 ] [9] [1 0 ]

N E Y R O U D et SCHNITZER
A gent 2 5 0 ml K M n0 4 M ethylation CH 3 CO 3 H 110 m l CH 3 C 0 2H 100 m l 2N NaOH 1 0 0 m l 2N NaOH 100 m l 2N NaOH 1 0 0 m l H20

2 5 0 m l K M n0 4 80 m l 4 0 m l H2 0 2 30% 5 ,0 g CuO 5 ,0 g CuO

Condi- Chauffer a reflux, 8 h 4 h , 80°C 192 h, 4 0°C 3 h, 170°C 3 h ,1 7 0 ° C 3 h , 170°C a: 3h , 1 7 0 °C

tions b: ultrasons

c: a + b

Extraction A cidifier a pH 2, extraire Filtrer, reprendre dans A cidifier a pH 2, extraire dans l ’acetate d’eth y le
dans l’acetate d’eth yle un solven t organique
R epeter la reaction sur les Separer dans
residue insolubles ' solvants de
O xyder residu 1 polarites
par K M n 0 4 1 croissantes

... , ,

<------------------
 IAEA -SM -211/73 159

Les travaux les plus recents [1,2] accordent aux organismes vivants du sol la part pre-
ponderante dans la transformation des residus en humus stable: ceux-ci subissent une Serie de
profondes degradations et fournissent aux organismes du sol Tenergie et les elements necessaires
ä Tedification de leur propre substance vivante. Certains composes intermediates peuvent
condenser ou polymeriser sous l’action d’enzymes microbiennes ou de facteurs puremeht
chimiques, et acquerir une plus grande resistance ä la degradation. Ce lent processus de matura­
tion donne naissance ä Thumus.
II est malaise d’isoler les substances humiques sensu str ic to des residus vegetaux partielle-
ment humifies et des particules minerales, car le sol est un milieu ouvert, soumis aux influences
climatiques, chimiques et biologiques: aucun modele purement statique ne saurait rendre compte
de la structure de Thumus.
La m o lecu le humique etant trop volumineuse pour une analyse chimique directe, le
chercheur est contraint de la briser en fragments plus petits. L’objet du present memoire est
de passer en revue les diverses techniques de degradation des acides humiques (AH) et fulviques
(AF), et d’avancer une hypothese sur la maniere dont sont assembles les composes de base
(b u ilding sto n e s ) en une structure coherente.

METHODES DE DEGRADATION ET DTDENTIFICATION

Afin de faciliter (’interpretation des resultats on a juge souhaitable de concentrer les

travaux sur un nombre restreint d’echantillons. Ainsi un grand nombre d’informations ont ete
recueillies sur un AH provenant d’un horizon Ah de Chernozem en Alberta (Canada) et sur
un AF provenant d’un horizon Bh de Podzol de Tile du Prince Edouard (Canada).
Le choix d’une technique de degradation brutale peut provoquer la destruction ou
Taltcration de nombreux composes; neanmoins la totalite du materiel est attaquee et les
rendements en composes chimiques simples atteignent environ 25% du poids initial. Avec
des degradations menagees, les rendements sont plus faibles mais la probabilite de formation
d’artefacts est reduite. Une Serie d’agents de degradation ä agressivite decroissante a ete utilisee
sur deux echantillons AH et AF. Les techniques analytiques, brievement passees en revue au
tableau I, sont les suivantes:

— Oxydation au permanganate de potassium (KMn04, pH 10) de la substance humique

brute, qui est entierement degradee en composes facilement identifiables: la predominance
de certains groupes de composes (acides benzene-polycarboxyliques) indique que plusieurs
structures chimiques resistent mal ä Teffet cumule de la temperature, du milieu alcalin et du
pouvoir oxydant du permanganate; les derives phenoliques, par exemple, subissent un clivage
de Tanneau aromatique et sont souvent detruits; la presence frequente d’acide oxalique montre
egalement que des composes ont ete oxydes jusqu’ä un stade proche de la destruction totale
en C02 et H20 [3].
— Oxydation KMn04 de la substance humique methylee: les groupes carboxyle et
OH-phenolique sont proteges de Tattaque electrophile une fois transformes en ester et ether
methyliques respectivement; les rendements sont alors plus eleves et qualitativement plus
varies [4].
— Oxydation ä Tacide peracetique: la reaction d’oxydation est conduite ä une temperature
plus basse (40—80°C) et ä un pH acide; ces conditions sont un peu moins drastiques que les
precedentes [5,6].
— Oxydation par Toxyde de cuivre en milieu alcalin (NaOH) ä 170°C: les oxydes de
metaux lourds ont ete utilises pour Tetude de la structure de la lignine [7]; Toxyde de cuivre est
peu agressif et ne degrade que partiellement les substances humiques, laissant un residu insoluble;
le residu peut etre degrade ä son tour par KMn04 ; les composes identifies lors d’une degrada-
 160
TABLEAU II. ACIDES BENZENE-CARBOXYLIQUES IDENTIFIES DANS 1,0 g D’AF (mg)

КМПО4 sur K M n0 4 sur Acide Acide CuO/NaOH CuO/NaOH NaOH Degradation

AF brut A F m ethyle peracetique peracetique + KM 11O4 p hysico-chim ique
80°C 4 0 °C

R eference [3] [4] [5] [6 ] [8] [8 ] [9] [1 0 ]

A cide benzene 1,2-dicarboxylique 0,3 6,5 1,6 5,5 4 ,9 1 1 ,8 1,0

A cide benzene 1,3-dicarboxylique ■ 2 ,6 3,0 3,0 0,7
A cide benzene 1,4-dicarboxylique

A cide benzene 1,2,3-tricarboxylique 0,4 11,2 4,6 3,3

N EY R O U D e t SCHNITZER
A cide benzene 1,2,4-tricarboxyLique и ■ 3,3 2,9 0,8 15,2 1 2 ,8 2 ,1

A cide benzene 1,3,5-tricarboxylique 1 ,5 0,8

A cide benzene 1,2,3,4-tetracarboxylique 2,0 ] 12,0 7,2 10,2 7,5 12,2
A cid e benzene 1,2,3,5-tetracarboxylique 2,4 , 3 1 ,7 6,5 0,2 4,8 2 ,8

A cide benzene 1,2,4,5-tetracarboxylique 1,1 J 7,9 5,2 6,2 4,1 15,1 2,6
A cide benzene-pentacarboxylique 3,9 4 4 ,6 10,3 22,2 2,9 10,5
A cid e benzene-hexacarboxylique 3,8 3 2 ,7 15,2 41,1 1,0
A cid e 3,4-dicarboxy, l-b en zylcarb oxyliqu e 4,4 4,4
T o ta l 16,5 114 ,9 72,5 8 3 ,7 53,2 39,5 2 6 ,9 3 2 ,4

R ecap itu latif

A cides benzene-carboxyliques 17 115 73 84 53 40 27 32

Derives phenoliques 0 90 45 122 150 146 142 9
Derives aliphatiques 24 0 27 26 115 108 278 24

T o ta l 41 205 145 232 318 294 447 65

N .B.: II s’agit dans chaque cas d’esters m ethyliques.

 IA E A -SM -211/73 161

tion partielle permettent d’avancer certaines hypotheses sur l’assemblage des composes de base
des matieres examinees [8].
— Hydrolyse alcaline (NaOH) ä 170°C: l’absence d’agent oxydant dans la bombe auto­
clave reduit encore le risque de formation d’artefacts lors de la reaction de degradation; la
rupture des liaisons chimiques les plus faibles est assuree par la seule hydrolyse [9].
— Traitement hydrothermal ä 170°C, traitement par les vibrations ultrasoniques: |
l’attaque des produits initiaux est de nature physico-chimique; les quantites de composes
identifies sont naturellement faibles, mais ceux-ci n’ont pas ete modifies par un agent
chimique [10].

Apres l’oxydation ou l’hydrolyse, on extrait les fractions acidifiees dans un solvant1

organique. Un traitement de methylation des groupes fonctionnels carboxyle et hydroxyle
phenolique rend les composes plus volatile et ameliore leur solubilite dans les solvants organiques.
Б est frequent que certaines fractions soient encore d’une grande complexite et presentent des
diagrammes chromatographiques (en phase gazeuse) insatisfaisants. Dans un tel cas une etape
de purification sur colonne ou sur plaque est necessaire. Les nombreuses manipulations ont
malheureusement pour consequence la perte d’une fraction appreciable de l’echantillon.

NATURE DES COMPOSES CHIMIQUES IDENTIFIES

L’identification des diverses structures chimiques avec un degre eleve de certitude est
desormais possible grace aux recents developpements des techniques analytiques: un compose est
identifiepar: 1) son spectre de masse, 2) son spectre infrarouge, 3) l’identite des spectres de
masse et i.r. du compose et de ceux d’un compose standard, 4) la comparaison des temps de
retention du compose inconnu et du compose standard sur le chromatographe en phase gazeuse.
Si les quatre tests sont positifs, on considere l’identite du compose comme certaine.
Quelle que soit la technique de degradation utilisee, chaque fraction separee au terme des
manipulations de purification contient 25 ä 35 composes representes par autant de pics sur un
diagramme chromatographique. Le calcul de la surface delimitee par chaque pic permet une
identification quantitative. Ainsi, les listes de composes presentees dans les experiences de
Schnitzer et al. indiquent toujours: 1) le pourcentage de la fraction initiale extrait dans le
solvant organique apres Tattaque, 2) le pourcentage de la fraction extraite identifiee avec certitude,
3) les quantites de composes identifies dans 1,0 g du produit de depart.
De recents travaux [11-15] ont permis l’identification d’une centaine de composes dans
le meme echantfllon d’AH ou d’AF. Ceux-ci se repartissent en acides benzene-polycarboxyliques,
derives phenoliques et composes aliphatiques (les groupes ester et ether methyliques sont dus ä
l’operation de methylation car la faible teneur en groupes methoxyles du produit initial nous
autorise ä supposer que les composes existent sous forme d’acides et de phenols). Les
tableaux II, III et IV presentent la liste des composes identifies dans un echantillon d’AF.
L’AH s’est comporte de maniere analogue et la liste des composes identifies est qualitativement
semblable.
A l’exception de l’acide benzoi'que, les douze acides benzene-polycarboxyliques ont ete
identifies (tableau II). Si les formes di- et tricarboxyliques apparaissent dans toutes les
degradations, les formes tetra-, penta- et hexacarboxyliques sont d’autant plus abondantes que
l’agent de degradation est plus violent. Elles peuvent meme faire totalement defaut avec des
agents peu agressifs. Des substitutions de type Ar-CH2C 02CH3 remplacent parfois un groupe
carboxylique.
Les derives phenoliques presentent une grande variete (tableau III). II s’agit d’anneaux
aromatiques substitues par des groupes methoxyle, carboxyle, methyle, cetone ou de courtes
chaines aliphatiques. Les combinaisons possibles et leurs isomeres sont nombreux. La plupart
 Оч
TABLEAU III. DERIVES PHENOLIQUES IDENTIFIES DANS 1,0 g DE AF (mg) Ю

K M n04 sur KMnC>4 sur Acide Acide CuO/NaOH CuO/NaOH NaOH Degradation
A F brut A F methyle peracetique peracetique + K M n04 physico-chimique
80°C 40° C

Reference [3] 14] [5] [6] [8] [8] [9] [10]

5-acetyle furfural 0,2

acide methyle 3,4-furane-dicarboxylique 6,2
Methyle benzyle sulfonate 1,4

N EYR O U D et SCHNITZER
N-methyle benzyle sulfonamide 7,7

1,2-dimethoxy benzene 0,8 0,8

1,3-dimethoxy benzene 1,0 1,0
Acide 3-methoxy benzoique 17,5 17,5
Acide 4-methoxy benzoique 8,1 8,1 13,9 5,2

Acide 3,5-dimethoxy benzoique 23,0 23,0 44,7 0,7

Acide 3,4-dimethoxy benzoique 8,8 8,8 32,2 0,7

Acide 3,4,5-trimethoxy benzoique 28,4 28,4 i6,6

Acide 2-methoxy, benzene-1,3-dicarboxylique и

Acide 2-methoxy, benzene-1,5-dicarboxylique 6,2 5,4 0,2
Acide 3-methoxy, benzene-1,2-dicarboxylique i,6 1,0
Acide 4-methoxy, benzene-1,2-dicarboxylique 9,2
Acide 4-methoxy, benzene-1,3-dicarboxylique 30,8 30,4
TABLEAU III. (suite)

Acide 3,4-dimethoxy, benzene-dicarboxylique

Acide 2-methoxy, benzene-l,3,5-tricarboxylique 6 ,0

Acide 3-methoxy, benzene-l,2,4-tricarboxylique 11,9

Acide 5-methoxy, benzene-l,2,4-tricarboxylique
Acide 5-methoxy, benzene-l,2,3-tricarboxylique 4,6

Acide 4,5-dimethoxy, benzene-l,2,3-tricarboxylique 4,2

Acide 2-methoxy, benzene-l,3,4,5-tetracarboxylique 0 ,8

Acide 5-methoxy, benzene-l,2,3,4-tetracarboxylique 47,6 17,0

Acide dimethoxy, benzene-tetracarboxylique 0,4

Acide methoxy, benzene-pentacarboxylique 24,6 4,4

Acide dehydrodiveratrique 5,5

Acide 3,4-dimethoxy, benzyle-carboxylique

3,4-dimethoxy, acetophenone
3,4,5-trimethoxy, acetophenone

Dibutyle phtalate

Acide 4-m ethoxy, 2-carboxy, l-benzyle-carboxylique

T O T A L 0,0 89,6 45,0

 0,8
3,4 7,1
20,2 14,5 11,2
2,8

8,6
54,2

0,4

IAEA-SM-211/73
18,4 3,4

2,2 2,2
1,8
5,1

3,6 3,5

10,9 10,9

1 2 1 ,6 150,0 145,8 141,7 9,1

o\
OJ
 Os
TABLEAU IV. DERIVES ALIPHATIQUES ET AUTRES IDENTIFIES DANS 1,0 g DE AF (mg) 4^

KMn0 4 sur KMn0 4 sur Acide Acide CuO/NaOH CuO/NaOH NaOH Degradation
AF brut AF methyle peracetique peracetique + KMnO, physico-chimique
80°C 40°C

Reference [3] [4] [5] [ei [8 ] [8 ] [9] [10 ]

Serie d’alkanes de n-C12 ä n-C38 17,0

Acide acetique 15,8

Acide oxalique 8 ,2

Serie d’acides gras 4,0

NEYROUD et SCHNITZER
Acide gras n-Ci2 5,3 5,3
Acide gras П-С13 1,3
Acide gras n-C i4 4,2 4,0 1 ,0

Acide gras П-С15 4 ,8 4,8 2,9

Acide gras n-Cjg 0 ,2 30,5 28,5 42,0

Acide gras n-C 17 21,7

Acide gras n-C18 0 ,2 14,1 13,5 15,6

Acide gras П-С19 3,0 2 ,8 2 ,0

Acide gras п-С29 U 9,5

Acide gras n-C2 1 1,4 1 ,2

Acide gras n-C22 1,7

Acide gras n-C24 0 ,2

Esters aliphatiques 13,2 5,6 5,6 7,2

TABLEAU IV. (suite)

Succinate de methyle
Glutarate de methyle
Adipate de methyle
Pimelate de methyle 3,3

Acide propane-tricarboxylique
Acide butane-tetracarboxylique

Phtalate de dibutyle

Adipate de dioctyle 3,8

Composes azotes non identifies 6 ,8

TOTAL 24,0 0,0 26,9

 6,8 28,2 27,6 3,0
6,6
1,8
1,0
4,4 11,4 11,4
4,8

3,6 3,5

174,2

IAEA-SM-211/73
25,8 114,7 108,2 277,8 24,0
166 NEYROUD et SCHNITZER

des derives phenoliques sont formes par la substitution de un ou deux groupes methoxyles sur un
acide benzene-poly carboxy lique. La degradation violente est souvent nefaste ä ces composes
peu resistants. L’action de l’oxyde de cuivre en milieu alcalin est ä ce jour celle qui donne le
meilleur rendement en composes phenoliques, mais il est fort probable que cet agent detruise
aussi plusieurs composes.
Les derives aliphatiques isoles (tableau IV) se composent d’hydrocarbures et d’acides gras
satures, d’acides dicarboxyliques et de quelques autres composes. Seules des degradations
menagees de la substance humique permettent d’isoler ces composes. La egalement, on peut
aisement imaginer que d’autres composes soient presents dans le produit initial.
Plusieurs autres structures chimiques ont ete isolees en quantites minimes, mais leur
presence n’autorise pas de speculations trop hardies: en verite, seule la poursuite des experiences
confirmera leur existence. II en est ainsi des composes azotes que Гоп ne peut isoler en quantites
süffisantes ou qui semblent avoir ete synthetises durant la manipulation de methylation au
diazomethane (tableau IV).

DISCUSSION

Apres avoir identifie les composes elementaires des substances humiques, il convient
d’etudier la maniere dont ils sont arranges pour former les macromolecules humiques. On se
souviendra toutefois que les seuls composes identifies sont ceux qui ont resiste aux diverses
attaques et qui possedent des proprietes de volatility favorables; leurs quantites et proportions
relatives peuvent avoir ete modifiees.
Les composes identifies sont en nombre relativement limite —une centaine —et ils apparais-
sent aussi bien dans les extraits d’AF que dans les extraits d’AH. On peut done les considerer
comme les briques elementaires des macromolecules. La plupart de nos analyses ont ete
concentrees sur un AF de Podzol et un AH de Chernozem, afin de limiter l’erreur due ä la
variability de l’echantillon, mais des travaux effectues dans nos laboratoires sur des substances
humiques d’autres provenances [16,17], et par d’autres auteurs [18,19] semblent confirmer
la nature des composes elementaires que nous avons identifies.
Il est interessant de noter que la degradation chimique d’un AF ou d’un AH par un agent
donne produit souvent les memes composes principaux, les differences visibles se reduisant ä
l’aspect quantitatif et ä la presence ou l’absence de composes mineurs. Ce point met en evidence
la parente pedogenetique des deux fractions humiques et rappelle une fois de plus le caractere
partiel d’une differenciation entre AF et AH basee seulement sur un critere de solubilite en
milieu acide.
L’extraction des fractions AF et AH et l’attaque de l’agent degradant qui a suivi ont eu pour
premiere consequence l’elimination de composes peu resistants ou faiblement fixes sur la charpente
humique. Les composes azotes sont particulierement vulnerables ä cet egard, puisqu’il est peu
frequent de les retrouver apres les travaux de separation chromatographique, bien que la teneur
en azote des substances humiques varie de 1 a 6% [11]. Ils subissent vraisemblablement une
hydrolyse partielle, passent dans la phase aqueuse et ne sont pas repris dans les solvents
organiques utilises par la suite. Selon plus d’un chercheur, les composes azotes ne sont pas des
elements constitutifs des substances humiques, mais bien plutöt des composes adsorbes qui
peuvent facilement etre arraches puis detruits. L’adsorption sur la charpente humique par
l’intermediaire de groupes fonctionnels ou de metaux leur a confere une longevite apparente dans
le sol, mais ces memes produits ne peuvent se maintenir longtemps sous forme libre dans le sol.
Les degradations partielles par des agents peu agressifs arrachent des composes jeunes,
fraichement deposes, qui n’ont pas encore acquis la forme stable des substances humiques vraies;
ces composes portent encore la marque de leur origine vegetale ou microbienne. Ils sont en cela
tres semblables aux composes chimiques identifies par plusieurs auteurs dans des acides hu m iques
 IAE A-SM-211/73 167

[20]. Ces chercheurs ont etudie des substances synthetisees par des champignons
d ’origine fo n g iq u e
et ont observe une similitude de leurs produits avec les acides humiques: niemes proportions de
С, О, H, N, memes diagrammes infrarouges, predominance des derives phenoliques parmi les
composes chimiques identifies. II nous semble que ces acides h u m iques portent un nom mal
choisi, car leurs composes de base sont pr6cis6ment ces composes ä stabilste limitee, pas encore
incorpores ä la charpente humique [21 ]. Leur bas äge ne nous autorise d’ailleurs pas ä les assi-
miler ä des AH dont certains travaux ont montre qu’ils sont vieux de quelques centaines
d’annees [22].
Les degradations successives de la phase stable ä l’aide d’agents differents [23], ainsi que
des degradations de fractions de complexity croissante separees sur un т ё т е echantillon [12]
resultent en l’identification des memes composes de base: la difference entre fractions extraites
reside done moins dans la nature des composes que dans les forces de liaison les maintenant
solidaires. Malheureusement, ces forces sont difficiles ä apprecier (liaison par des cations metalliques,
des ponts H, forces de Van der Waals, liaison ir, etc.) et le chercheur est confronte ä une phase
organique heterogene dans laquelle il n’y a peut etre pas deux sites reactionnels identiques.
Une macromolecule humique serait done formee de deux phases: l’une, Гёсогсе, compren-
drait des composes adsorbes et des composes en voie de transformation en humus stable, alors que
l’autre, le coeur, serait formte ä parts sensiblement egales des trois grands groupes de composes
identifies a ce jour. Cet hetero-polymere possederait une structure poreuse et de nombreux sites
favorisant l’adsorption de composes organiques [4,14].
Le choix des techniques d’extraction et de recolte des fractions determine, dans une large
mesure, la nature des composes qui seront identifies: les procedures d6crites dans ce memoire
sont des contributions ä la connaissance de la phase stable des substances humiques, et causent
la destruction ou la perte d’une partie des composes (composes azotes, phenoliques, etc.).
La controverse sur la nature des substances humiques perd une partie de son acuite des
lors que certains chercheurs ont analyse des fractions contenant essentiellement des composes
transitoires formant Гесогсе des substances humiques (N ährhum us ), et que d’autres ont analyse
les fractions stables (D auerhum us ) qu’ils ont degradees selon des techniques entrainant la perte
des composes les moins resistants.

CONCLUSION

Les quelques considerations qui precedent nous conduisent ä echaffauder une hypothese
sur la structure des substances humiques. Dans les cas les plus favorables, nous identifions
20 ä 30% de la substance humique, ce qui equivaut ä environ 50% du produit initial, compte
tenu des inevitables pertes survenant au cours des manipulations. A cela s’ajoute la part des
composes que nous n’avons pas reussi ä identifier en raison de leur plus grande stabilite
(condensats, polycycles, etc.). Le reste a ete perdu ou detruit: il etait constitue de composes
adsorbes sur la charpente humique et que nos methodes de degradation et de collection ont
detruits ou n’ont pu mettre en evidence.
Apres avoir ete debarrassee de son ecorce - formee des composes les moins resistants -
une substance humique consiste en une association moieculaire d’acides benzene-polycarboxyliques
et de derives phenoliques unis par des liens relativement faibles. Des composes aliphatiques peuvent
egalement etre incorpores dans cette structure.
Divers agents d’activite croissante degradent les substances humiques en fractions, mais
chaque fraction extraite contient les memes composes de base. Les differences entre fractions
sont dues ä une plus grande stabilite resultant de liens plus energiques ou plus nombreux de
type C-0 et C-C. La figure 1 presente notre hypothese sous forme de diagramme: une
substance humique peut etre s6paree en plusieurs fractions de complexite croissante, comme en
temoignent leurs diagrammes i.r. et chromatographiques et leurs poids moieculaires. Les
168 NEYROUD e t SCHNITZER

Adsorb6 ou a l'etat libre:

II — Divers composes a faible poids moleculaire

— Alkanes
a>
э III — Acides gras
c

IV Composes elementares:
Q.
<s>

V — Acides benzene-carboxyliques
— Därives pt^noliques
VI — Derives aliphatiques et divers
c
<ц X
C
О VII

Intensite croissante des

forces de liaison (liens,
,, ponts H, Van der Waals, etc.)

F I G .l. S t r u c t u r e d ’u n e s u b s t a n c e h u m i q u e .

premieres fractions contiennent encore des composes faiblement adsorbes —alkanes, acides gras et
quelques derives phenoliques —alors que les fractions suivantes contiennent toutes les memes
composes de base. L’agent de degradation utilise opere bien entendu une certaine selection
quantitative: les derives phenoliques, par exemple, sont partiellement detruits par le KMn04,
mais bien conserves apres attaque au CuO en milieu alcalin.
Aucune regle ne preside ä l’arrangement des unites de base entre eiles, et la formation de
l’hetero-polycondensat est le resultat du hasard. Ceci explique qu’aucun AF ou AH n’est
identique ä un autre. Les composes organiques ont tres lentement evolue vers des formes stables
(le noyau des substances humiques) qui sont semblables dans la plupart des sols. La diversite des
substances humiques s’explique surtout par la nature des composes transitoires (1’ёсогсе),
dependant fortement des conditions du milieu (pH, presence de cations, oxygene, eau, micro-
flore et microfaune, nature des residus vegetaux, etc.), et moins par la nature des composes
stables.
De nombreuses questions n’ont cependant pas encore regu de reponse et solliciteront
encore l’attention soutenue des chercheurs dans les annees ä venir: quelles sont les forces qui
maintiennent solidaires les elements d’un AF ou d’un AH? Comment expliquer une si extra­
ordinaire resistance de ces produits ä la degradation dans les sols?

REFERENCES

[1] HAIDER, К., MARTIN, J.P., PHILIP, Z., in Soil Biochemistry 4, (PAUL and MacLAREN, Eds),
M. Dekker Inc., New York (1975).
[2] FLAIG, W., BEUTELSPACHER, H., RIETZ, E., “Chemical composition and physical properties o f humic
substances”, Soil Components 1 (GIESEKING, J.E., Ed.), Springer, Berlin (1975).
[3] SCHNITZER, M., DESJARDIN, I.G., Alcaline permanganate oxidation of methylated and unmethylated
fulvic acid, Soil. Sei. Soc. Am., Proc. 34 (1970) 1.
[4] KHAN, S.U., SCHNITZER, M., Further investigations in the chemistry of fulvic acid, a soil humic fraction,
Can. J. Chem. 4 9 (1 9 7 1 ) 13.
[5 ] ■SCHNITZER, M., SKINNER, S.I.M., The peracetic acid oxidation of hum ic substances, Soil Sei. 118 5
(1974).
 IAEA-SM-211/73 169

[6 ] S C H N IT Z E R , M., S K IN N E R , S.I.M ., T h e low te m p e ra tu re o x id a tio n o f h u m ic su b stan ce s, C an. J.

C hem . 5 2 ( 1 9 7 4 ) 7.
[7 ] P E A R L , I.A ., T h e C h em istry o f Lignin, M. D ek k er In c ., N ew Y ork (1 9 6 7 ).
[8 ] N E Y R O U D , J.A ., S C H N IT Z E R , M ., T h e exh au stiv e alcaline cu p ric o x id e o x id a tio n o f h u m ic ac id and
fulvic acid, Soil Sei. Soc. A m ., P roc. 38 6 (1 9 7 4 ).
[9 ] N E Y R O U D , J.A ., S C H N IT Z E R , M ., T h e alcaline h y d ro ly sis o f h u m ic su b stan ce s, G eo d erm a 13
(1 9 7 5 ) 1 7 1 - 8 8 .
[1 0 ] N E Y R O U D , J.A ., S C H N IT Z E R , M ., T h e m ild d eg rad a tio n o f h u m ic su b stan ces, A g ro ch im ica 19 2 (1 9 7 5 ).
[1 1 ] S C H N IT Z E R , M ., K H A N , S.U ., H um ic S ubstances in th e E n v iro n m en t, M. D ek k er In c ., N ew Y o rk (1 9 7 2 ).
[1 2 ] N E Y R O U D , J.A ., S C H N IT Z E R , M ., T h e c h e m istry o f high m o lecu lar w eig h t fulvic acid fractio n s,
C an. J . C hem . 5 2 ( 1 9 7 4 ) 24.
[ 1 3 ] K H A N , S.U. S C H N IT Z E R , M ., P erm a n g an ate o x id a tio n o f h u m ic acids e x tra c te d fro m a gray w o o d ed
soil u n d e r d iffe re n t c ro p p in g sy stem s an d fertilizer tre a tm e n ts , G eo d erm a 7 (1 9 7 4 ) 113 —120. '
[1 4 ] O G N E R , G ., S C H N IT Z E R , M., C hem istry o f fulvic acid, a soil h u m ic fra c tio n an d its re la tio n to lignin,
Can. J. C hem . 49 (1 9 7 1 ) 7. !
[ 15] S C H N IT Z E R , M., In v estig atio n s o n th e chem ical s tru c tu re o f h u m ic su b stan ce s b y gas c h ro m ato g rap h y -
m ass sp e c tro m e try , T rans. 1 0 th In t. Soil Sei. C ongress, M oscow , 19 7 4 , ä p a ra itre . |
[1 6 ] K H A N , S.U ., S C H N IT Z E R , M., P erm an g an ate o x id a tio n o f h u m ic acids, fulvic acids an d h u m ih s e x tra c te d
fro m h o riz o n s o f a black C h ern o z em , a b la ck S olod an d a b la ck S o lo n etz soil, C an. J . Soil Sei. 5 2 (1 9 7 2 ) 43.
[1 7 ] O R T IZ D E S E R R A , M .I., S C H N IT Z E R , M., C h em istry o f h u m ic and fulvic acids e x tra c te d fro m A rg en tin e
soils: I & II, S oil Biol. B iochem . 5 (1 9 7 2 ) 28 1 .
[1 8 ] O G N E R , G ., P erm a n g an ate o x id a tio n o f m e th y la te d an d u n m e th y la te d fulvic acid, h u m ic an d h u m in
iso la ted fro m raw h u m u s, A cta C hem . S cand. 27 (1 9 7 3 ) 1601.
[1 9 ] R A N D A L L , R .B ., B E N G E R , M., G R O O C O C K , C.M ., P roc. R. Soc. A 165 (1 9 3 8 ) 4 3 2 .
[2 0 ] M A R T IN , J.P ., R IC H A R D S , S .J., H A ID E R , K ., P ro p erties an d d e c o m p o sitio n an d b in d in g a c tio n in soil
o f “ h u m ic a c id ” sy n th esiz ed b y “E p i c o c c u m n i g r u m ” , Soil Sei. Soc. A m ., P ro c. 31 (1 9 6 7 ) 657.
[2 1 ] S C H N IT Z E R , M ., N E Y R O U D , J.A ., F u rth e r inv estig atio n s o n th e c h e m istry o f fu n g al “ h u m ic acid s” ,
Soil Biol. B iochem . 7 (1 9 7 5 ) 365.
[2 2 ] P A U L , E .A ., C A M PBELL, C.A ., R E N N IE , D .A ., M cCA LLU M , K .J., T rans. 8 th In t. Soil Sei. C ongress,
B u ca rest (1 9 6 4 ).
[2 3 ] S C H N IT Z E R , M., O R T IZ D E S E R R A , M .I., T h e chem ical d eg rad a tio n o f a h u m ic acid, C an. J. C hem . 51
(1 9 7 3 ) 10.
 IAEA-SM-211/49

APPLICATIONS OF COMPUTER TECHNIQUES

IN HUMUS RESEARCH
B.R. NAGAR
Institut für Biochemie des Bodens
der Forschungsanstalt für Landwirtschaft,
Braunschweig-Völkenrode,
Federal Republic of Germany

Abstract

A PP L IC A T IO N S O F C O M PU TER T E C H N IQ U E S IN HUM US R E S E A R C H .
A c ritic al review is p re s e n te d o f th e ap p licatio n o f c o m p u te r te c h n iq u e s in h u m u s research f o r stu d ie s on
p ro b le m s such as th e c h a ra c te riz a tio n o f s o ü organic m a tte r by in s tru m e n ta l m e th o d s o f an alysis in relatio n , to
so il p ro d u c tiv ity , stu d ie s o n th e origin a n d s tru c tu re o f soil h u m ic acids b y p y ro ly sis m ass s p e c tro m e try an d a
c o m p u te r, stu d ie s to d iscrim in a te b etw e en m u ll a n d m o r ty p es o f h u m u s, an d n itro g en tra n sfo rm a tio n s in soils,
especially s im u la tio n o f n itro g e n b eh a v io u r in soils. T h e a p p licatio n s are d escrib ed o f c o m p u te r te c h n iq u e s in
h u m u s research w o rk in a n u m b e r o f la b o rato rie s b y m a k in g use o f F o u rie r T ra n sfo rm In frared S p ectro sc o p y ,
F o u rie r T ra n sfo rm N u cle ar M agnetic R eso n an ce S p ectro sc o p y a n d p y ro ly sis te ch n iq u es in c o m b in a tio n w ith gas
ch ro m a to g ra p h y o r gas ch ro m ato g rap h y -m ass s p e c tro m e try or m ass sp e c tro m e try (PY G C , PYGC-M S o r PY-M S)
a n d a co m p u te r. I t is suggested th a t th e ap p lic a tio n o f c o m p u te r te c h n iq u e s in h u m u s research o ffers m a n y
in te re s tin g possibilities fo r significant progress. A n u m b e r o f p ro b le m s are p ro p o s e d , in p a rtic u la r th e e lu cid atio n
o f th e s tru c tu re o f th e so-called h e tro c y c lic n itro g en co m p o u n d s b y p y ro ly sis gas ch ro m ato g rap h y -m ass s p e c tro m e try
an d a c o m p u te r sy stem ; th is p ro b le m is co n n e c te d w ith th e slow N -releasing p r o p e rty o f soil h u m u s, j I t h as
also b een suggested th a t a co m p u te riz e d In te rn a tio n a l D o c u m e n ta tio n S ystem fo r S oil H um us R esearch sh o u ld be
esta b lish ed w hich w o u ld serve as th e in te rn a tio n a l so u rce o f in fo rm a tio n .

During the past 25 years, the application of physical methods such as gas chromatography,
ultra-violet, visible and infra-red spectroscopy, nuclear magnetic resonance, electron spin resonance,
X-ray analysis, and ultra centrifuge in humus research have contributed significantly to research in
this field. In 1960s chemists realized that in order to cope with the vast amount of data and
information produced at a very fast rate by physical methods such as mass spectrometry, it was
essential to use computers in combination with them. By using computers in physical methods,
analysis has become faster and more precise. Furthermore, it is most significant that by using
computer techniques in chemical research, it is possible to solve a number of problems that cannot
be solved so far by known methods of research. A critical review is here presented of the application
of computer techniques in humus research, which also incorporates suggestions for the application
of computer techniques in humus research offering interesting possibilities for significant progress
in humus research.

APPLICATIONS OF COMPUTER TECHNIQUES IN CHEMICAL RESEARCH

Computer techniques have been used in chemical research for the following purposes [1 —5]:

(1) Searching recent literature on any topic.

(2) Time-consuming and tedious calculations of the experimental results and for statistical
analysis of the data.
(3) Display of molecular structures.

171
172 NAGAR

(4) Simulation of a chemical system, e.g. nitrogen transformations in soils.

(5) Determining optimum pathways for synthesis of a compound.
(6) Improvement and refinement of modern techniques of research (physical methods of
chemical analysis).
(7) Processing and interpretation of the results obtained from modern instrumental methods
of analysis.

APPLICATIONS OF COMPUTER TECHNIQUES IN HUMUS RESEARCH

Applications of computers for characterization of soil organic matter by modem instrumental

methods of analysis

For the past seven or eight years, Salfeld, Süchtig and Flaig [6—8] have been using computer
techniques for the characterization of soil organic matter by modern instrumental methods of
analysis. Computers have been extensively used for the statistical analysis of the results of
characterization of soil organic matter obtained on a suitable medium. A detailed report of this
work is given in another paper at this symposium.’

Studies on the origin and structure of soil humic acids by curie point pyrolysis-low voltage
ionization mass spectrometry

Recently, Nagar, Waight, Meuzelaar and Kistemaker [9] used a computer system in their
preliminary studies on the origin and structure of humic acids by Curie-point pyrolysis in direct
combination with low voltage ionization mass spectrometry: m/e 16-230 spectra were scanned
128 times at a rate of 5 scan/s. The summed spectra were fed into a PDP-15 computer; normalized
and a calcomp plotted, and the degrees of correspondence between the various spectra were calculated
by the computer. It is noteworthy that computers are capable of producing summation spectra
(the average spectrum for one series) obtained from a large number of soil humic acid samples or
from microbial humic acid samples or from a large number of degraded or original lignin samples
which may be compared. Thus this application of computers in humus research seems to be a very
promising one. A detailed report of the application of this technique in humus research is given
in another paper at this symposium.12

Nitrogen transformation in soils: simulation of nitrogen behaviour in the soil

Beek and Frissel [10] carried out preliminary studies on simulation of nitrogen behaviour
in soils by using a IBM 360 computer, and published a small booklet entitled S im u lation o f
N itrogen B ehaviou r in Soils. According to the authors, the aim of this publication was ‘to present a
summary of the work carried out so far, to provide an exchange of ideas about this approach and
to inform soil microbiologists which kind of experiments have to be carried out to provide model
builders with the data needed to develop and test such an approach. It is stressed that this
description of the simulation model only represents an intermediate step in its developments so
that no definite conclusions can be drawn.’ This seems to be a very useful approach, and the studies
on the simulation of nitrogen transformation by computers are likely to produce significant
results.
1 J.-C hr. S A L F E L D , H. SÖ C H TIG , “ C o m p o sitio n o f th e soil org an ic m a tte r sy stem d ep e n d in g o n soil ty p e
an d la n d u se” , th ese P roceedings, IA E A -SM -211/24.
2 K. H A ID E R , B .R . N A G A R , C. SA IZ , H .L.C. M E U Z E L A A R , J.P . M A R T IN , “ S tu d ies o n soil h u m ic
c o m p o u n d s, fu n g al m elanins an d m o d e l p o ly m ers by p y ro ly sis m a ss-sp ectro m etry ” , th ese Proceedings,
IA E A -SM -211/53.
 IAEA-SM-211/49 173

Studies to discriminate between mull and mor types of humus

Bracewell and Robertson [11] used pyrolysis low resolution mass spectrometry (at 70 eV) to
discriminate between mull and mor types of humus. They used a large number of samples, and the
discriminator value was calculated by multiplying peak intensities with weight factors (the weight
factors were previously determined by using spectra of clearly distinguishable mull and mor types
of humus from a computerized learning machine). In this manner, they obtained a scale which
contained at the one end a 20 X 102 value for mull humus, and for mor humus a negative value up
t o -1.5 X102. The intermediate types of humus have intermediate values on this scale. In another
recent publication on the same subject, Bracewell, Robertson and Stephen [12] simplified the
calculation of the ‘Discriminator Value’; they used only 13 normalized peak heights instead of
110 peaks previously used for the calculation.

Fourier transform infra-red spectroscopy

A considerable amount of work has been carried out on humic fractions by using infra-red
spectroscopy, and it has been mainly possible to indicate the presence of a number of groups in
humic fractions by using this technique. Furthermore, this technique has been able to throw light
on a number of aspects of humus research, namely the influence of chemical extractants, changes
occurring on methylation, saponification, acetylation, oxidation, pyrolysis, formation of metal-humus
complexes, clay-humus complexes and soil pesticides interactions, etc. Recently, significantly with
the use of mini-computers, Fourier Transform Infra-red Spectroscopy has been developed. By
using this technique it has been possible to obtain high resolution on a wide range, and the
experiments can be performed on transient or weak samples very quickly. MacCarthy, Mark and
Griffiths [13] have reported preliminary results on the direct measurement of the infra-red spectra
of humic substances in water by Fourier Transform Infra-red Spectroscopy. To obtain the
spectrum of a humic substance, they stored the spectrum of it obtained in suspension or solution
in digital form, and substracted from it, with a computer, the spectrum of water previously
recorded and stored in the digital form. This method seems to be a promising one for soil humus
research.

Fourier Transform Nuclear Magnetic Resonance Spectroscopy

As humic fractions are mostly insoluble in organic solvents, so far investigations have been
carried out by using Nuclear Magnetic Resonance (N.M.R.) either on the methylated humic
fractions or on the degradation products of the humic fractions; so far this technique has failed
to produce very significant results. However, with the use of digital mini-computers, it has been
possible to develop Fourier Transform Nuclear Magnetic Resonance Spectroscopy. By using
F.T.N.M.R., the sensitivity of the technique has been considerably improved, and there is a gain
in the signal/noise ratio. Consequently, it is possible to perform N.M.R. studies in dilute solutions,
particularly solutions of macro-molecules. Furthermore, it has been possible to carry out investiga­
tions on 29Si, I5N, 2H, 170 and particularly 13C. During the past few years, 13C N.M.R. has been
extensively used in biochemistry. It is most significant that N.M.R. can be gainfully utilized for
biosynthetic studies by enriching the samples with 13C. Currently, research work on the use of
N.M.R., including 13C N.M.R. in humus research, is in progress at the Chemistry Departement,
Imperial College, London, and the Institut für Biochemie des Bodens, Braunschweig-Völkenrode.
If significant results are obtained, they will be reported.
174 NAGAR

Pyrolysis techniques in combination with gas chromatography or gas chromatography-mass

spectrometry or mass spectrometry, including field desorption mass spectrometry, and a computer
system

Pyrolysis techniques in combination with gas chromatography, or gas chromatography-

mass spectrometry, or mass spectrometry including field desorption mass spectrometry and a
computer, are promising techniques for solving the problem of the structures of the components
of soil organic matter, especially the so-called heterocyclic nitrogen compounds. Currently,
research work is in progress in this field at the Chemistry Department, Imperial College, London,
the Institut für Biochemie des Bodens, Braunschweig-Völkenrode and the Institut für Physikalische
Chemie, Bonn, and significant progress in elucidating the structures of the components of soil
organic matter may be possible within a few years.

POSSIBILITIES OF SIGNIFICANT PROGRESS IN HUMUS RESEARCH BY USING

COMPUTER TECHNIQUES

The applications of computer techniques in solving the following problems of humus research
offer many interesting possibilities for significant progress:

(1) Studies on the characterization of the dynamics of soil organic matter in relation to soil
productivity.
(2) Studies on the elucidation of the structures of the so called heterocyclic nitrogen fraction
of soil humus.
(3) Studies on the structures of the components of soil organic matter by Fourier Transform
Nuclear Magnetic Resonance Spectroscopy, Fourier Transform Infra-red Spectroscopy and
pyrolysis techniques-pyrolysis gas chromatography, pyrolysis gas chromatography-mass
spectrometry and pyrolysis mass spectrometry, including field desorption mass spectrometry.
(4) Simulation studies, especially simulation of nitrogen transformations in soils.

COMPUTERIZED DOCUMENTATION SYSTEM FOR SOIL HUMUS RESEARCH

A computerized international documentation system for soil humus research should be

established. The experimental data and all information required for critical evaluation such as type
of soil and methods used for analysis, should be registered on the punched cards or another
acquisition system, and stored in the computer. From this data bank, the original experimental
values and other experimental parameters may be retrieved if and when desired. In this connection
it is desirable that research workers in humus research should use standardized methods of analysis.
The development of this documentation system as the international source of information on
humus research is highly desirable, especially from the point of view of exchange of information
and international co-operation in humus research.

REFERENCES

[1] CARRINGTON, R.A.G. (Ed.), Computer for Spectroscopits, Adam Hilger, London (1974).
[2] DEVOE, J.R., SHIDELLER, R.W., RUEGG, F.C., ARONSON, JR , SHOENFELD, P.S., Anal. Chem. 46
(1974) 509.
[3] HEPPLE, P. (Ed.), The Applications of Computer Techniques in Chemical Research, Institute of Petroleum,
London (1972).
 IAEA-SM-211/49 175

[4] PERONE, S.P., Anal. Chem. 43 (1971) 1288.

[5] ZIEGLER, E., HENNEBERG, D., SOCHOMBERG, G., Angew. Chem. Int.Ed. Engl. 11 (1972) 348.
[6] SALFELD, J. Chr., SÖCHTIG, H., FLAIG, W., Pochvovedenie 8 (1975) 99.
[7] SALFELD, J. Chr., Telma 4 (1974) 235.
[8] SÖCHTIG, H„ SALFELD,. J. Chr., Soils BuU. No.27, FAO, Rome (1974) 71.
[9] NAGAR, B.R., WAIGHT, E.S., MEUZELAAR, H.L.C., KISTEMAKER, P.G., Plant Soil 43 (1975) 681.
[10] BEEK, J., FRISSEL, M.J., Simulation of Nitrogen Behaviour in Soils, Pudoc, Wageningen (1973).
[11] BRACEWELL, J.M., ROBERTSON, G.W., J. Soil Sei. 24 (1973) 421.
[12] BRACEWELL, J.M., ROBERTSON, G.W., STEPHEN, G.J.M., J. Soil Sei. 26 (1975) 62.
[13] MacCARTHY, P., MARK, H.B., Jr., GRIFFITHS, P.R., J. Agric. Food Chem. 23 (1975) 600.
C A R B O N D A T IN G
(S e s s io n 7 c )
 IAEA-SM-211/69

M E S U R E S D ’A C T IV IT E S P E C IF IQ U E D E
F R A C T IO N S D E M A T IE R E O R G A N IQ U E
A P P L IQ U E E S A L ’E T U D E D E L ’E V O L U T IO N
D ES SO LS D E G U Y A N E

J.L. RAPAIRE
Centre scientifique de Monaco,
Principaute de Monaco

J.F. TURENNE
Office de la recherche scientifique et
technique Outre-mer,
Centre des Antilles,
Pointe-ä-Pitre,
Guadeloupe,
Antilles frangaises (DOM)

R a p p o rteu r: B. G u illet

Abstract-R£sum£

MEASUREMENT OF THE SPECIFIC ACTIVITY OF ORGANIC MATTER FRACTIONS AS APPLIEE)

TO STUDIES ON THE EVOLUTION OF GUYANA SOILS.
The soil cartography of the French Guyana coastal plain reveals a gradual transition, over very short
distances, from ferralitic soils at the lower part of the sequences to podzols at the upper part. Analysis of the
specific activity (14C) of three organic matter fractions -- fulvic acids, humic acids and humin — is applied
in the present case to the progressive differentiation of the humic В horizon, characteristic of podzols, on the
basis of the ferralitic soils. The results are given by a mathematical model enabling the residence time to be
calculated, and a renewal rate for the organic fractions to be deduced from it. The analysis makes it possible
to determine the direction and the rate of podzolic evolution, and confirms the gradual penetration of the
landscape by the aquifer: podzolization begins at the higher points in the soil chain and progresses in a down­
ward direction; slow maturation and biodegradation have different effects on all the fractions studied, the
humic acids appearing to undergo the least renewal as a result of insolubilization.

MESURES D’ACTIVITE SPECIFIQUE DE FRACTIONS DE MATIERE ORGANIQUE APPLIQUEES A

L’ETUDE DE L’EVOLUTION DES SOLS DE GUYANE.
La cartographie pedologique de la Plaine cötiere de Guyane fran$aise met en evidence le passage progressif,
en de tres courtes distances, de sols ferralitiques, а Гaval des sequences, ä des podzols, ä l’amont. L’analyse de
l’activit6 specifique en carbone-14 de trois fractions de la matiere organique — acides fulviques, acides humiques,
humine — est ici appliquee ä la differentiation progressive de l’horizon В humique, caracteristique des podzols,
ä partir des sols ferralitiques. Les r£sultats sont donn6s par un modele mathematique qui permet de calculer le
temps de residence et d’en deduire un taux de renouvellement des fractions organiques. Cette analyse permet
de determiner le sens et la vitesse de revolution podzolique et confirme l’envahissement progressif du paysage par
la nappe: la podzolisation а со ттеп сё par les points hauts de la chaine de sols et progresse vers Гaval; une
maturation et une biodegradation lentes affectent inegalement toutes les fractions £tudiees, les acides humiques
paraissant subir le renouvellement le plus faible, ä partir de leur insolubilisation.

I 179
180 RAP A IRE e t TURENNE

INTRODUCTION

La cartographie pedologique de la Plaine cötiere ancienne de Guyane franqaise met en

evidence le passage progressif en de tres courtes distances de sols ferralitiques fortement
dessatures ä morphologie de sols lessiv6s, ä l’aval des sequences, ä des podzols hydromorphes,
ä l’amont.
L’analyse de l’activite specifique en HC de trois fractions de la matiere organique — acides
fulviques, acides humiques et humine — est ici appliquee ä la differenciation progressive de
l’horizon В humique ä partir de l’horizon Bt ferralitique.
Cette differenciation progressive peut etre observde dans des sequences de sols developpees
sur materiau ferralitique (sables fins argileux), sous vegetation de savane; ces sequences pr^sentent
un certain nombre de caracteres:

—Le climat est du type tropical humide ä saison seche (1 ä 3 mois), avec temperature
moyenne de 26°2.
—La nappe, en relation avec la topographie, affleure au centre des formes de relief et
varie saisonnierement (fig. 1a).
—La differenciation pedologique s’exerce sur un materiau homogene.
—Le stade devolution podzolique se situe ä l’amont des sequences.
—Les variations laterales le long de la pente sont importantes et rapides, le passage d’une
morphologie ferralitique ä la morphologie podzolique s’effectue en quelques metres de maniere
continue.

Nous avons montre dans un travail p r u d e n t [ 1] le role fondamental de la matiere organique

solubilis^e en milieu hydromorphe, dans l’hydrolyse intense des mineraux non seulement pri-
maires mais aussi secondaires (kaolinite) qui intervient au cours de cette podzolisation.
Le developpement continu de la sequence de sol foumit le moyen d’apprecier la validity
des mesures de 14C et de leur attribuer une signification pedogenetique: moins que les valeurs
absolues, c’est revolution laterale de ces valeurs qui est prise en consideration.

1. MATERIEL ET METHODES

Les fractions humiques sont extraites au pyrophosphate de sodium pH 10 qui assure un

taux d’extraction superieur ä 50% dans les horizons Bj, etudies.
Cinq echantillons, preleves en 1974, sont analyses: un echantillon, PAR 91, correspond
ä l’horizon de surface A j du podzol le plus en amont. Quatre echantillons correspondent ä
l’horizon profond dans lequel on note ä la fois un maximum de matiere organique, un taux
d’extraction au pyrophosphate de sodium ä pH 10 maximal, ainsi qu’une teneur maximale en
acides fulviques. Ces echantillons concernent, de l’aval vers l’amont, un sol ferralitique inter­
grade podzolique, un podzol peu difference et deux podzols differences. Ces horizons sont
situes au sommet d’un ancien horizon В argilique, peu permeable.

1.1. Distribution laterale et verticale de la matiere organique (fig. 1)

La repartition de la matiere organique dans les profils ferralitiques ou intergrades

podzoliques montre une diminution reguliere avec la profondeur. L’individualisation de la
morphologie podzolique se produit par l’apparition d’un horizon Bh de differenciation maximale
qui se forme au-dessus de l’ancien horizon argilique, qui disparait progressivement par hydrolyse
acide; le degre d’induration de l’alios augmente vers l’amont lie ä la plus grande amplitude de
l’hygroperiodisme saisonnier.
 IAEA-SM-211/69 181

P o d z o ls h y d ro m o rp h e s

A ltitu d e S o l s fe rra lltiq u e s l e s s lv e s

F I G .l. R e p a r titio n l a t e r a l e e t v e r t i c a l e d e la m a t i e r e o r g a n i q u e .

1.2. Analyse de l’activite sp6cifique en 14C, calcul du coefficient de renouvellement

Les mesures d’activite specifique en 14C des differentes fractions sont appliquees ä la
r6solution d’un modele mathematique [2-4] supposant constante pour un horizon donnd
la quantite Q de matiere organique presente dans cet horizon:

dQ
=0
dt
 00
to

TABLEAU I. ANALYSE DE L’ACTIVITE SPECIFIQUE - TAUX DE RENOUVELLEMENT

A M O N T -------------------------------------------------------------------------------------------------------------------------------------------------------------------------- > A V A L
Sol ferralitique
Podzols integrade p odzoliq u e
t ----------------------- --------------------------------------------*------------------------------------------------------------------------------- »
Horizon de surface Horizon В hum ique Horizon Bt
t ------------ \
Echantillon PA R 91 PA R 95 PA R 85 PAR 54 PAR 4 3

RAPAIRE et TU RENNE
AF AH HMN AF AH HMN AF AH HMN AF AH HMN AF AH HMN

Age BP - - - 7160 11 3 0 0 7250 3300 5270 3560 2500 Mo- 2270 1030 270 570

±120 ±270 ±100 + 160 ±460 ± 90 + 100 d em e ±100 ±90 ±150 ±210

A ge m oyen BP - 7530 3560 2110 715

Q* 1,427 1,128 1,217 0 ,4 1 0 0 ,2 4 5 0 ,4 0 5 0 ,6 6 4 0 ,5 1 9 0 ,6 4 2 0 ,7 3 2 1,028 0 ,7 5 4 0 ,8 8 0 0 ,9 6 7 0 ,9 3 0

TR 9 42 25 11 6 0 0 25 0 0 0 11 8 0 0 4100 7500 4500 2940 - 2600 1100 270 600

taux de renouvellem ent 8,4 4 35 0 ,1 6 0,01 0 ,0 2 0 ,2 5 0 ,0 2 0 ,0 8 0 ,1 4 - 0 ,1 7 0 ,3 0 0 ,3 2 0 ,7 4

(g- m'2 ‘ a'1)

A F = A cides fulviques — AH — A cides hum iques - HMN = Hum ine

Par d efin ition Q* = 1 correspond ä 6 l 4 C % o = 0 (Q * = 1 ± ( 6 14 C%?)* 10’3 )

TR = Tem ps de residence
Taux de renouvellem ent pour une epaisseur de sol de 10 cm , d = 1,5 (d = 1,3 pour PA R 91).
 IAEA-SM-211/69 183

avec

F - quantite de matiere organique presente dans la litiere ou dans l’horizon immediatement

superieur
a = pourcentage de F pdnetrant dans l’horizon considere par unitd de temps
b = pourcentage de Q sortant de l’horizon considers par unite de temps.

La тёш е equation s’applique au nombre d’atomes de 14C :

— = а ^ -^ (Ь + Х )
dt

avec X = constante de disintegration.

Les activites specifiques F*, Q*, supposees constantes jusqu’en 1950, debut de l’ere
atomique, permettent alors de calculer le temps de residence:

J _ = J_ F* _
b “ X Q* ~ 1

avec

Le taux de renouvellement q = b • Q, exprime en g ■m“2 • a'1, s’en deduit.

Pour l’horizon de surface, on suppose que cette matiere organique provient uniquement
de la litiere qui se decompose dans 1’аппёе; eile a dans ces conditions l’activite specifique de
l’air de 1’аппёе de sa chute. Les valeurs de l’activite specifique trouvdes pour l’horizon etudie
sont peu elevees et laissent prevoir un temps de residence inferieur ä 80 ans: dans ces conditions
la courbe de variation d’activite de divers composes est donnee par la formule recurrente suivante,
ä calculer ä partir de 1950:

n» Q * fT -n (T R -D + F¥
Qt tr

TR: temps de residence

Q j: activite specifique de la matiere organique Гаппёе T
Fj.: activite spёcifique de Pair Гаппёе T ä la latitude du point de prelevement
(5°10'N, 52°39'W).
Les activit£s specifiques sont exprimees sous la forme:

Q* = 1 + (614C%0)- 10'3
184 RAPAIRE et TURENNE

2. RESULTATS

Les hypotheses proposees pour le calcul du temps de residence permettent d’obtenir une
valeur exprimant en fait la labilite de la fraction organique consideree ( tem p s d e vie), valeur
egale ä l’inverse du coefficient de renouvellement. Par ailleurs, rappelons que Tacceptation du
modele math6matique conduit ä ne pas tenir compte des variations possibles dans le temps des
quantity de carbone presentes dans 1’horizon, ni de la vitesse de descente depuis la surface.

3. TAUX DE RENOUVELLEMENT - ESSAI DTNTERPRETATION

Les ages reportes dans le tableau I sont ceux calcules ä partir de l’equation representant
la decroissance d’un element radioactif (periode du 14C = 5570 ans). Nous les donnons pour
comparaison avec les valeurs mentionnees dans d’autres travaux.
A notre avis, ces äges ne sont significatifs que lorsqu’ils se rapportent ä des composes tres
stables, autrement dit lorsqu’un phenomene d’accumulation continue existe. Dans ces conditions,
la date du debut de ce phenomene serait le double de l’äge attribue ä Tensemble des fractions
ainsi stockees [5]. L’interpretation tient compte, par ailleurs, des resultats donnes par d’autres
auteurs [6].

3.1. Taux de renouvellement de Thorizon superieur des podzols

Les taux de renouvellement calcules a partir des activites specifiques mesurees sont faibles,
10 fois inferieurs aux moyennes citees [7] pour des sols tropicaux; ceci laisse supposer
l’existence de fractions residuelles et la migration rapide en profondeur des composes recents
entrames par le battement saisonnier de la nappe. De plus, les conditions hydromorphes ne
sont pas favorables a des transformations tres rapides. Par ailleurs, le taux de renouvellement
et le temps de residence de Thumine, compards ä ceux des acides humiques, laissent prevoir
Texistence, dans Thumine, d’une part heritee (debris vegetaux peu transformes, recents) [ 1].

3.2. Taux de renouvellement et differentiation de Thorizon В humique

a) L e so l ferra litiq u e intergrade p o d zo liq u e

Le profil PAR 4 montre une fraction fulvique accumulee au niveau etudie (sommet de
Thorizon B) presentant un temps de residence 2 к 4 fois plus grand que celui des autres composes.
11 est done probable que les acides fulviques ne participent que faiblement ä la formation des
autres fractions dans ce niveau. Par ailleurs, Thumine presente le coefficient de renouvellement
le plus dleve. Elle serait done constituee d’une part preponderante de fractions insolubilisees
susceptibles d’etre alimentees par des composes recents [1].

b ) L es p o d zo ls

Le profil PAR 5 montre un vieillissement simultane des fractions humine et acides

fulviques avec un taux de renouvellement similaire. Par contre, les acides humiques seraient
d’äge moderne: ils correspondraient alors a Tinsolubilisation directe des composes jeunes en
provenance de la surface.
On observe enfin dans les profils PAR 8 et PAR 9, vers l’amont, des coefficients de
renouvellement tres faibles pour les acides fulviques et humiques et Texistence d’un rapport
constant entre les temps de residence des acides humiques, d’une part, et des acides fulviques
ou de Thumine, d’autre part, traduisant un vieillissement simultane.
 IAEA-SM-211/69 185

L’augmentation du contraste climatique saisonnier vers le haut de la sequence (fig. 1) peut

etre responsable de la maturation des composes organiques: il у a formation d’un horizon
de type alios avec cimentation et induration.
II faut cgalement admettre devant les ages apparents tres anciens que Гоп est en presence
d’une fossilisation des horizons etudies. Plusieurs caracteres favorables ä celle-ci s’accentuent
de l’aval vers l’amont:

—Ralentissement de l’activite biologique dü ä l’existence d’un taux de plus en plus eleve

d’acides fulviques (35% de C total dans PAR 43 ä 75% dans PAR 95) [8].
—Persistance de l’humidite et presence de produits amorphes [9] pouvant entrainer la
possibility de maintien de certaines fractions organiques; c’est le cas ici ой Гоп observe les
teneurs suivantes en fer et aluminium amorphes correspondent ä l’attaque des mineraux argileux:

PAR 95 PAR 85 PAR 54 PAR 43

A1 libre (pg/g) 1390 1350 720 510

Fe libre (pg/g) 390 660 380 220

Le debut de la podzolisation correspondrait alors, au plus tard, au double de l’äge le plus

ancien observe, soit 22 000 ans. Enfin, l’horizon PAR 95 (le plus ancien) serait passe successive-
ment par les stades represents par PAR 85, PAR 54 et PAR 43. Ceci induit un envahissement
progressif du paysage par la nappe (remontee du niveau marin, augmentation de la pluviometrie
en periode holocene) et la progression de la podzolisation vers l’aval.

CONCLUSION

L’appUcation d’un modele mathematique simple permet, dans une certaine mesure,
d’apprecier le renouvellement des fractions organiques lors de la transformation du sol
ferralitique en podzol. A partir de valeurs theoriques, l’observation d’une sequence de sol
permet d’attribuer une signification pedogenetique aux calculs du taux de renouvellement
des composes organiques. Les acides humiques paraissent subir, apres leur insolubilisation,
le renouvellement le plus faible. Dans les conditions pedoclimatiques de la Plaine cötiere de
Guyane (hygroperiodisme passant ä hydromorphie permanente), on aboutit ä une insolubilisation
efficace des composes organiques, puis ä leur fossilisation.

REFERENCES

[1 ] T U R EN N E, J.F., M odes d a m n ifica tio n e t d ifferen tiation p odzoliq u e dans deux toposequences guyanaises,
T hese Univ. N ancy (1 9 7 5 ) 181.
[2 ] LOBO, P .F .S., U tilisation du 14C atm ospherique com m e traceur de la m atiere organique des sols, These
Univ. federale, Salvador, Bahia (1 9 7 2 ) 63.
[3 ] FL EX O R , J.M., Medida da radioactividade especifica С 14/C 12, ap p lican t) ao estu d o do com portam ento
dinam ico do carbono n o so lo , Tese Inst. Ffsica da Univ. de Säo Paulo (1 9 7 2 ) 93.
[4 ] LOBO, P .F .S., FLEX O R , J.M., R APAIR E, J.L., SIEFFER M A NN , G., Essai de determ ination du tem ps
de residence des fractions hum iques de deux sols ferralitiques par l’utilisation du radiocarbone naturel et
therm onucteaire, Cah. ORSTOM, ser. pedol. 12 1 (1 9 7 4 ) 1 1 5 - 1 2 3 .
186 RAPAIRE et TURENNE

[5 ] GUILLET, B ., R elation entre Thistoire de la vegetation et la podzolisation dans les V osges, These Univ.
N ancy (1 9 7 2 ) 112.
[6 ] SCHARPENSEEL, H.W., R O N ZAN I, C , PIETIG, F ., {Comparative age determ ination on different
hum ic-m atter fractions), Isotop es and Radiation in Soil Organic-Matter Studies, (C .R. C oll. V ienne, 1 968),
A IEA , V ienne (1 9 6 8 ) 6 7 - 7 3 .
[7 ] BOISEZON, P. de, M O REAUX, C., BOQUEL, G ., BACHELIER, G ., Les sols ferralitiques, IV - La
matiere organique e t la vie dans les sols ferralitiques, ORSTOM, Paris (1 9 7 3 ) 146.
[8] M ATHUR, S.P., Can. J. M icrobiol. 15 (1 9 6 9 ) 677.
[9] SIEFFER M A NN , G., Les sols de quelques regions volcaniques du Cam eroum , ORSTOM, Paris (1 9 7 3 ) 183.
 IAEA-SM-211/70

D A T A T IO N S P A R L E C A R B O N E -1 4 N A T U R E L
D E L A M A T IE R E O R G A N IQ U E D H O R IZ O N S S P O D IQ U E S
D E P O D Z O L S D E S L A N D E S D U M E D O C (F R A N C E )

D. RIGHI
Laboratoire de pedologie,
University de Poitiers,
Poitiers

B. GUILLET
CNRS, Centre de pedologie biologique,
Vandoeuvre-les-Nancy,
France

Rapporteur: B. Guillet

\b s tn a -K isu m 6

N A T U R A L C AR BO N -14 D ATING OF ORGANIC MATTER FROM SPODIC HORIZONS OF PODZOLS IN

THE MEDOC LA ND ES (FR A N C E).
The M edoc Landes o f France are characterized by a covering o f quartz sand and the presence o f ground-
water very close to the surface. The soils that develop there are fairly hydrom orphic podzols. The best-drained
podzols have a cem ented spodic horizon. The organic m atter present takes the form o f coatings, and consists
m ainly o f fulvic acids that are on ly slightly biodegradable. The m ost hydrom orphic p odzols have a lo o se B2h
h orizon form ed from mineral particles and rather hum ified organic m atter. The latter is labile and has a
relatively short mean residence tim e.

D ATATIONS PAR LE C A R BO N E-14 N A TU R EL DE LA M ATIERE O RGANIQ UE D ’HORIZONS SPODIQUES

DE PODZOLS DES LA ND ES D U MEDOC (FR A N C E ).
Les Landes du M edoc (France) son t caractensees par un recouvrem ent de sable quartzeux e t la presence
d’u ne nappe pbreatique tres superficielle. Les sols qui s’y dcveloppent son t des p od zols plus ou m oin s hydro-
m orphes. Les p od zols les m ieux draines o n t un h orizon spodique cim ente. La m atiere organique у est sous form e
de revetem ents e t est con stitu ee essen tiellem en t d ’acides fulviques peu biodegradables. Les p od zols les plus hydro-
m orphes o n t un horizon B2h m euble, form e de particules m inerales e t de m atiere organique plus ou m oin s
h um ifiee. Cette m atiere organique est labile e t m ontre un tem ps m oyen de residence relativem ent court.

INTRODUCTION

Les ages apparents ou temps moyens de residence de la matiere organique d’horizons В

spodiques des podzols d’une courte toposequence des Landes du Medoc ont ete determines par
datation ä l’aide du radiocarbone naturel.
Les Landes du Medoc sont caracterisees par un recouvrement de sables grossiers quartzeux
dans lesquels circule une nappe phreatique toujours tres proche de la surface du sol.
Le relief general est plat, mais il existe un microrelief superficiel forme de buttes allongees,
hautes seulement de quelques decimetres et larges d’une quinzaine de metres. Ce microrelief est
fondamental car il determine la position du sol par rapport au toit de la nappe et, en consequence,
la micromorphologie des profils et Revolution pedogenetique.

187
188 RIGHI et GUJLLET

Plili'll alios indure

[■
. -1 horizon Bh meuble
F I G .l. E m p l a c e m e n t s d e s p r o fi ls e t d e s p r e l e v e m e n t s — A g e a p p a r e n t e t r a p p o r t C / N d e la m a tie r e o r g a n iq u e .

Les sommets des buttes portent des podzols humiques а horizon spodique (B22h) indure
(alios). Vers l’aval, on passe progressivement ä des podzols tres hydromorphes ä horizon spodique
meuble. Enfin, les depressions sont occupees par des sols hydromorphes peu humiferes, sans
horizons spodiques (figure 1). La microstructure des horizons spodiques meubles est differente
de celle des horizons indures: les premiers montrent des agregats comprenant des grains de
quartz, des elements vegetaux figures plus ou moins humifies et de la matiere organique
amorphe [1 ], alors que les seconds ont essentiellement des revetements de matiere organique
amorphe [2]. De plus, l’horizon B22h indure des podzols les mieux draines est surmonte d’un
horizon meuble (B2Ih) essentiellement forme d’agregats.
Les deux types d’horizons spodiques (meuble et indure) different egalement par la nature
de la matiere organique humifiee qu’ils contiennent. Les horizons B22h indures sont formes
presque uniquement avec des acides fulviques ä C/N eleve (40 ä 50), alors que dans les horizons
spodiques meubles, les acides humiques (C/N = 20) represented 30 ä 50% du carbone organique
total [3,4].
Nous avons pense que le niveau d’activite biologique et, par consequent, le temps moyen de
residence de la matiere organique devaient etre differents pour chaque type d’horizon spodique.
Nous avons cherche ä les mesurer avec des datations par le radiocarbone naturel.

MATERIEL ET METHODES

Neuf echantillons d’horizons spodiques ont ete preleves. L’emplacement des profils et
des prelevements est indique sur la figure 1. Les echantillons sont traites selon leur microstruc­
ture. Deux cas sont ä considerer:
 IAEA-SM-211/70 189

— Pour les horizons spodiques indures, aucun fractionnement n’a ete tente.
— Dans le cas des horizons spodiques meubles, une separation squelette mineral-agregats
a ete effectuee, suivie d’un fractionnement par taille des agregats eux-memes.

Les agregats sont isoles du squelette mineral de la faqon suivante: 1

— Un maximum de debris vegetaux peu transformes est prcalablement elimine par une
agitation energique de l’echantillon dans l’eau; les debris peu transformes flottent; ils sont
exclus de la datation.
— Apres ce traitement, les agregats sont separes du squelette mineral par flottation dans
l’eau et agitation du type «batee»; les agregats se concentrent ä la surface du liquide et s’ecoulent
lorsque l’on incline le recipient.
— Les agregats isoles sont separes par classes de taille (> 100 pm; 50—100 pm) apres
tamisage ou centrifugation (> 50 pm).

Au total, pour chacun des horizons spodiques meubles, quatre fractions ont ete recueillies
et datees:
— le squelette mineral avec, eventuellement, ses revetements;
— les trois classes d’agregats definies ci-dessus.

TABLEAU I. RAPPORT C/N ET AGES APPARENTS

Echantillons C C/N 5 14Ca Age apparent

(%) (%) (BP)
(a)

3LAG B21h
Total 1,80 1.9,4 - 16,40 1440± 80
R evetem ents 0,13 n.d. - 15,06 1 3 1 0 ± 170

f > 1 0 0 pm 28,65 20,5 - 16,14 1410 ± 70

Agregats 50 ß — 100 ц т 2 3 ,1 0 17,6 - 15,61 Ц 60± 70
< 50 ц т 29,95 17,4 - 16,93 1490± 70

2 —4LA G B2h

Total 1,90 22 ,2 - 14,09 1220± 70

R evetem ents 0,6 0 29 ,4 - 15,91 1390± 70

> l 'O O ß m 17,84 21,0 - 12,75 1100+ 80

Agregats 50 ц - 100 Mm 18,87 17,5 - 13,65 1180 ± 60
< 50 Alm 19,02 18,8 - 12,89 1110 ± 80

4LA G B2h

Total 1,90 21,6 - 9 ,1 7 y 0 ± 80

R evetem ents 0,3 0 25 ,0 - 10,05 850± 80

> 100 Alm 16,30 19,1 - 7 ,4 8 620 ± 60

Agregats 50 Ai - 100 Aim 15,90 18,8 - 10,22 860± 80
< 50 Alm 15,37 18,0 - 9 ,2 2 780± 80

Variation de Pactivite specifique du carbone par rapport au standard.

190 RIGHI et GUILLET

Pour tous les echantillons, le carbone est dose au Carmograph et le C/N determine avec
l’analyseur CHN + О (Carlo Erba) sur des echantillons broyes et homogeneises. Les mesures
de datations ont ete faites selon la methode par scintillation liquide [5,6].

RESULTATS

Les principaux resultats sont donnes au tableau I et ä la figure 1.

— Les ages apparents des horizons В sont plus eleves dans les alios des podzols-amonts que
dans les horizons meubles des podzols-avals.
— Les valeurs des ages apparents augmentent vers la profondeur dans les horizons В des
podzols ä alios (B21h: 1440 ± 70 BP -*■B22h aliotique: 2810 ± 70 BP ->■B3 sous Talios:
3390 ±80 BP1).
— Un gradient d’äge apparent decroissant de la matiere organique des B2h apparait lorsque
Ton passe lateralement des podzols humiques ä alios aux podzols sans alios (2810 ± 70 BP -*•
2380 ± 70 BP ^ 2000 ± 70 BP -*■ 1220 ± 70 BP ^ 770 ± 80 BP).
— II n’existe pas de gradient d’äge apparent dans les horizons spodiques meubles (B2h
et B3) du podzol hydromorphe aval (770 ± 80 BP).
— II n’y a aucune difference significative de temps moyen de residence du carbone dans
chacune des fractions des agregats dans les horizons B21h meubles et dans les B2h des profils
situes ä Taval. Ce resultat peut paraitre surprenant, surtout dans la fraction des agregats
> 1 0 0 pm, bien pourvue en matiere organique peu transformee.
— Pour les horizons 3LAG B21h et 4LAG B2h, il n’y a pas de difference significative de
temps moyen de residence entre les agregats et les revetements des grains du squelette, alors
que pour l’horizon meuble intermediate (2—4LAG B2h) les revetements presentent un temps
moyen de residence un peu plus eleve que celui des agregats qui, par ailleurs, ont un C/N plus
faible.

DISCUSSION

Les mesures d’äges apparents separent nettement deux types d’horizons spodiques: d’une
part des horizons spodiques meubles dont Tage apparent est relativement faible; d’autre part,
les alios indures, dont Tage apparent est beaucoup plus eleve. Ceci ne peut s’interpreter que
par un turn o v e r de la matiere organique plus rapide dans les premiers que dans les seconds [7].
En d’autres termes, au niveau des horizons spodiques, 1’activite biologique serait limitee aux
seuls horizons B2h meubles.
L’aliotisation par des complexes acides fulviques-aluminium [ 1] provoque un ralentisse-
ment du tu m o v e r ä un point tel qu’il у a «fossilisation» des horizons spodiques profonds des
podzols-amonts.
La difference d’activite biologique peut etre mise en relation avec la nature des composes
organiques et des liaisons qu’ils contractent avec les cations mineraux. II existe d’ailleurs une
excellente correlation lineaire entre Tage apparent et le rapport C/N de la matiere organique
(fig-2).
Dans les horizons spodiques meubles, l’ensemble de la matiere organique a un C/N relative­
ment bas et le taux d’acides humiques est egal ou superieur ä celui des acides fulviques. Dans
les alios, au contraire, les acides fulviques ä C/N eleve complexant de fortes quantites d’aluminium

1 BP = B e fo r e P r e s e n t, 1950 dtant le m illesim e de reference.

 IAEA-SM-211/70 191

F I G .2 . C o r r e l a t i o n e n t r e l e r a p p o r t C / N e t l ’ä g e a p p a r e n t d e l a m a t i e r e o r g a n i q u e — y = age a p p a r e n t;

x = r a p p o r t C /N ; r = c o e f f i c i e n t d e c o r r e la tio n lin e a ir e ; a = r is q u e .

predominent. Les complexes semblent peu biodegradables comme en temoigne leur temps
moyen de residence et les mesures experimentales de Juste et al. [8].
La correlation entre le C/N et l’äge apparent semble montrer que:

— l’induration des horizons spodiques se fait avec des composes peu riches en azote, comme
l’indiquerait la valeur du C/N de la matiere organique des revetements en voie de formation autour
des grains du squelette de l’horizon 2—4LAG B2h;
— le «vieillissemento de la matiere organique des alios indures se fait avec une diminution
relative du taux d’azote. II est possible que, ä la maniere de revolution diagenetique des
sediments organiques, l’azote soit elimine des horizons B22h et B3 sous forme de composes
azotes solubles evacues par la nappe.

CONCLUSION

Les differences observees tant dans la microstructure que dans la composition de la matiere
organique des horizons spodiques des podzols des Landes du Medoc se retrouvent dans les
resultats des mesures du temps moyen de residence de la matiere organique de ces horizons.
Les horizons meubles des podzols tres hydromorphes et l’horizon B21h surmontant l’alios
indure ont une matiere organique labile et renouvelable alors que la matiere organique de l’alios
est beaucoup plus stable.
192 RIGHI et GUILLET

REFERENCES

[1 ] RIGHI, D ., Sei. Sol 4 (1 9 7 5 ) 315.

[2 ] RIGHI, D ., DE CONINCK, F ., “M icrom orphological aspects o f hum ods and haplaquods o f the L a n d e s
du M ed o c(F rance)”, (Proc. 4 th Int. Working M eeting on Soil M icrom orphology, K ingston, 197 3 ),
Soil M icroscopy (R U T H E R F O R D , G.K ., Ed.), K ingston, Ontario, Canada (1 9 7 4 ) 567.
[3] JAMBU, P., RIGHI, D ., Sei. Sol 3 (1 9 7 3 ) 207.
[4 ] RIGHI, D ., JAM BU, P., FUSTEC-M ATHON, E., D UPUIS, T ., «Structure e t genese des acides hum iques des
sols des L a n d es d u M ed o c (France)» (R apport Ier Coll. Int. Biodegradation e t H um ification, N ancy, 1974),
Pierron, Sarreguemines (1 9 7 5 ) 358.
[5 ] SCHARPENSEEL, H.W., PIETIG, F ., Geoderm a 2 (1 9 6 9 ) 273.
[6] GUILLET, B„ Bull. EN SA IA (N ancy) X IV 1 (1 9 7 2 ) 117.
[ 7] GUILLET, B., R elation entre lliisto ir e de la vegetation dans les V osges, T hese doctorat es sciences,
N ancy (1 9 7 2 ) 112 p.
[8 ] JUSTE, C., DELAS, J., LANGON, M., C.R. Acad. Sei., Paris 281 D (1 9 7 5 ) 1685.
 IAEA-SM-211/71

T H E S E A R C H F O R B IO L O G IC A L L Y IN E R T
A N D L IT H O G E N IC C A R B O N
IN R E C E N T S O IL O R G A N IC M A T T E R

H.W. SCHARPENSEEL
Ordinariat für Bodenkunde der
Universität Hamburg,
Reinbek,
Schloss,
Federal Republic of Germany

R a ppo rte u r: В. G uillet

Abstract

TOE SEARCH FO R BIOLOGICALLY INER T A N D LITHOGENIC CARBON IN RECENT SOIL ORGANIC

MATTER.
T he search for the existen ce o f biologically inert carbon in recent soil sam ples, as suggested by Gerasimov,
led to soil fraction dating, follow ed by various fractionation techniques. 6N H C l-hydrolysis residues were m ost
con sisten tly superior in age to the untreated soil sam ple. The age gradient from repeated steps o f 6N HCl-hydrolysis
indicates that by an exten d ed con tin u ou s treatm ent o f soil samples w ith synchronous replacem ent o f the acid
m edium the starting p osition for recent soil dating can be considerably im proved. T he gap b etw een the radiocarbon
dates o f recent soil sam ples, representing AM RT (apparent mean residence tim e) values and the true age o f the
soil form ation , can be further m inim ized. A collection o f 26 soü sam ples from soils w ith free carbonates and bi­
carbonate dynam ics did n o t indicate a deviation from the A 13C-range, characteristic for Calvin-type carbon. On
the basis o f Д 13С this exclu des aging o f such soils b y introduction o f lithogenic dead carbon in to th e soil organic
m atter.

INTRODUCTION

The limitations of applying radiocarbon dating techniques to organic carbon of recent soils
are mainly as follows:
1. Root growth, animal transport and percolation are factors constantly transferring young
carbon into the soil profile. The rate is unsteady, although correlated with climatic changes, and
can be roughly estimated only. Unless one succeeds in producing an organic C-sample that is inert
to contemporary biodynamics, the resulting 14C-date will be rejuvenated, and will have at best
the quality of a minimum age (age here interpreted as the time lapsed since the first humus formation
in a regolith).
2. In the case of soils with bicarbonate dynamics, the question arises whether some of the
organic carbon could not originate from the soil carbonates. The result would be an age increase
due to the fossil carbon contribution.
The first complex of the expected rejuvenating effect on the soil age demands C-fractionation
and fraction dating in order to scrutinize the total soil organic carbon for a fraction of biologically
inert carbon that Gerasimov [1,2] claims exists.
The second question can be decided by measuring A13C-values in organic carbon species of
those soils with excessive carbonate reserves and a definitely intact bicarbonate dynamics (Л13С is
obtained by comparing the sample 13C/12C ratio with the Chicago Belemnite Standard [3] or a
secondary standard).

I 193
194 SCHARPENSEEL

F I G .l. S e p h a d e x g e l fr a c tio n a t io n o f h u m ic a c id s u n d e r in e r t g a s.

EXPERIMENTAL

In order to select a soil fraction whose organic matter is mainly composed of biologically
inert carbon sources, soil samples of various origins were subjected to fractionation procedures.
The fractions produced were then combusted with 0 2 in quartz tubes, synthesized into benzene
samples and measured for their natural radiocarbon concentrations in a liquid scintillation spectro­
meter as described by Scharpenseel and Pietig [4,5]. The following modes of fractionation were
applied and the respective dating results compared:
(1) Fractionation into fulvic acid, hymatomelanic acid, brown humic acid, grey humic acid,
humin and humus coal [6].
(2) Fractionation according to particle size of the admixed mineral substance (sod texture
fractions) [7].
(3) Fractionation by extraction with organic solvents of increasing polarity.
 IAEA-SM-211/71 195

(4) Subfractionation of humic acid and humin samples by gel permeation into samples of
various particle sizes (Fig.l).
(5) Fractionation by step-wise interruption of a continuous alkali extraction [7].
(6) Fractionation by repeated 6N HC1 hydrolysis.

RESULTS

The evaluation of earlier results [7] revealed in a loessic hapludalf a slight superiority of age,
associated with the 60 to 1 pm grain size fraction, compared with the coarser and finer Soil textures.
Intermittent samples from a continuous alkali extraction of humic acids gave dates inconclusive
with regard to the age increase with the ensuing extractive exhaustion of the humic acids.
The classical humic matter fractions followed the age sequence fulvic acid -»• humic acid -*•
humin in the case of biologically active terrestrial soils. Hydromorphic soils had the sequential
order fulvic acid -> humin ->■ humic acid, as younger non-humic materials (e.g. cellulose) are
preserved under hydromorphic conditions, and appear as a rejuvenating contamination in the
humin or humus coal fraction.
Table I reflects these trends. It also shows that by Sephadex gel permeation (Fig.l) the
fraction of larger particles passing was always younger than the delayed fraction of smaller particle
size. This is unexpected, but could indicate a certain contribution by larger primary polymeric
substances of more recent origin. The difference between the Sephadex fractions is, however,
insufficient to account for a dynamic relative enrichment of biologically inert carbon in the older
fraction. The same holds true for the soil extracts with organic solvents of increasing polarity.
The sudden age increment in the case of dimethylformamide and dimethylsulphoxide extracts
(column 10) is without replication because of the unbearable smell that developed, in spite of
powerful exhausters, when dimethylsulphoxide reacts with soil organic matter. It is presumed
that the age gap with the other fractions is caused by dead carbon of the extractants despite
thorough vacuum drying of the extracted sample as well as the remaining soil after the extraction
step. This was confirmed recently by A13C-measurements through the courtesy of H. Willkomm,
Institute of Nuclear Physics, University of Kiel.
The most consistent gain in age is observed in the unhydrolysates after 6N HC1 treatment, which
was carried out already in the early days of soil dating by Paul and co-workers [8]. Later a
25-to-30-cm-deep sample of an Argiudoll from Asel and an Argiaquoll from Landshut with sample
depth 50 to 60 cm were repeatedly subjected to eight steps of 6N HC1 hydrolysis over a period
of 48 h each. The soil was also dated without such pretreatment, and was further coked (degassed)
under N2-atmosphere and subsequently combusted with 0 2 in a quartz tube as usual. Sixteen
individual hydrolysis repetitions of 1 kg soil in 3 litres of 6N HC1 were required for each soil sample
(Fig.2).
Table II indicates clearly a tendency towards increasing fraction ages from the unhydrolysed
soil sample to the higher replications. Coking pretreatment by combustion in a quartz tube under
N2-flow did not produce a marked effect, neither in the terrestric soil from Asel nor in the hydro-
morphous soil from Landshut. The Argiudoll (Asel) produced up to the first hydrolysis step
results >100% modern, due to bomb carbon. The disappearance of this effect under the conditions
of the continued hydrolysis indicates that most of the bomb carbon containing young organic
carbon is removed by the action of the 6N HC1. Also the Argiaquoll from Landshut indicates a
gradient of increasing age parallel to the repetitions of hydrolysis.
The age increase of the fractions is almost steady. It indicates that, by continuous treatment
with 6N HC1 over longer periods with concurrent replacement of the acid and solution, we might
arrive at a soil preparation method that delivers a sample closer to the required standard which
Gerasimov calls ‘biologically inert C’.
TABLE I. RESULTS OF FRACTION DATING

196
Frauiloct Podzol, Podzol, Podzol, Chernozem, Chemozern, Pseudogley- Fossil Fossil Nethermoor Kalkarer
Scherpenseel, Scherpenseel Flaesheim, Aseler Söllingen, Chernozem, paparendsina Chernozem, Koislhof, Nethermoor
Ah, nearGeilenkirchen, 0/Ah, Forest, Ah, Ap, Adlum, SwAj below Michelsberg, fA, overlaying Hn,
15-25 cm Bh, 40-55 cm 10—20 cm 40— 50 cm 15-25 cm 40-50 cm Trachyt tuff 140cm Isargravel, 80-90 cm
of Laach, fA, Hn, 80 cm
200cm
Total soil 2470±70 2470 +70 10600 ± 120 7200± 110
Petroleum
benzeneextract 3290±320
Benzene extract 3220 ±80 4130 ± 100 3290± 120
Methanol extract 6380± 90
Acetone extract 4020±360
Acetonitril
extract 2240 ±440
Dimethylformamide
extract 10760 ± 130

SCHARPENSEEL
Dimethylsulphoxide
extract 13 140+200
Hydrolysis residue 3160 ±70 11360 ±150 9730± 170
6NHC1hydrolysate 104.4 2510 ± 100 7270± 140
0.6%mod.
Fulvic acids(after 2930 ±40 140.1% 370 ±70 104.3% 1800 ±60 1140 ±200 6860±250 4270 ±80
organicextraction) mod. mod.
Fulvicacids dialysed 380 ±70 4310 ±210 7060± 110
Hymatomeianic acid 1580±80 114.1% 1390 ±70 4510± 80
acid mod.
Total humic acid 2940±60 2100± 70 6970± 210 8810 ±120
Humicacids 2230±70 1480±60 6110± 100 7590±120
Sephadex 50
fraction I (passing
fraction)
Humic acids 5410±90 2940 ±90 6830± 130 7820± 90
Sephadex 50
fraction 2
(delayed fraction)
Brownhumic acids 2530±60 920 ±50 1560±70 4890± 50 7600± 220 5380±80
Greyhumicacids 2980±70 1140± 70 5970 ±40
Humine 2850±70 117.2% 2460 ±60 2275 ±80 2910+80 10320± 140 6930±80 7110± 110 3490 ±70
mod.
Humuscoal Traces 2920 ±70 2810 +60 9940±140 6830+ 100 7230±110 4460 ±80
Charcoal 10330± 120
 IAEA-SM-211/71 197

10thdate HYDROLYSIS

treated units of 1kg soil each >

F 1 G .2 . F r a c tio n d a tin g : 0 2 -c o m b u s tio n ; N 2 fo llo w e d b y 0 2 -c o m b u s tio n ; 6 N H C h h y d r o ly s is s te p s .

TABLE II. FRACTION DATES FROM HYDROLYSIS REPLICATIONS

T reatm ent Soil from A sel (A rgiudoll) Soil from Landshut (Argiaquoll)

0 2-com bustion > 100% modern 46 4 0 ± 90 56% m odern

N 2-coking + 0 2-com bustion > 100% modern 4 5 7 0 + 110 56.6% Modern

1st hydrolysis step > 100% modern 5610± 80 49% m odern

2nd hydrolysis step 2530± 80 73% modern 5 8 2 0 ± 110 48.5% m odern

3rd hydrolysis step 2410± 70 74% modern 5 7 0 0 ± 100 49.2% m odern

4 th hydrolysis step 2340± 80 74.7% modern 5 5 4 0 ± 110 50.1% m od em

5th hydrolysis step 2770± 80 70.8% modern 6110 ± 90 46.8% m odern

6 th hydrolysis step 2560± 90 72.8% modern 5790± 90 48.6% m odern

7th hydrolysis step 2960± 80 69.2% modern
8th hydrolysis step 3 2 6 0 ± 100 67.2% modern
198 SCHARPENSEEL

TABLE III. CARBON-13 DEVIATION FROM STANDARD OF CARBON WITH VARIOUS

ORIGINS

Carbon source Д 13С Proposed correction Reference

for 14C-age (yr)

Carbonate, Chicago Belem nite 0% 0 [3]

Standard

A tm ospheric CO 2 — 9% o ”

Terrestrial plants, w ood — 25% o

Coal — 23% o "

Petrol —29%o ”

Sedim ent rock, shale - 2 9 - 30%o

14C-dating standard,
N B S-oxalic acid (95%
m odern) —19.6%0
P h otosynth esis, Calvin - 2 3 - —27 % 0 -3 0 t o +30 J.C. Lerman, Proc. 8th Int. Conf.
m echanism , m ost plant R adiocarbon D ating, W ellington,
species New Zealand (1 9 7 2 ) 6 1 3
P h otosynth esis, Hatch - 1 0 - -13% o + 2 0 0 to 240
and Slack m echanism , M.D. H atch, C.R. Slack, B iochem . J.
different graminees, sugar 1 0 1 (1 9 6 6 )1 0 3
cane, corn, sorghum

P h otosynth esis, CAM

m echanism , m ixed form —17%o about 130 P.N. Avadhani, C.B. O sm ond, K.K. Tan
P h otosynth esis and Photorespiration,
W iley Interscience, N ew Y ork (1 9 7 1 )
2 88.
(see also J.C. Lerman, above)

The question whether organic matter of calcareous soils with bicarbonate dynamics might
be aged by lithogenic carbon of the carbonates required Д13С tests of carbon samples derived from
such soils. Should root-uptake of bicarbonate-C occur, this introduction of dead carbon would
age the sample. In consequence the 13C/12C ratio would be shifted from the Calvin-type of
Д13С (—23 to —27%o) towards the Д13С (0 %o) of the Craig Belemnite Standard.
Table III shows the 13C-deviations of C from various sources. Tables IV indicates the Д13С
and Д14С characteristics of 26 soils with intact or earlier bicarbonate dynamics.1
Based on Д13С, all samples except No. 1080 and No. 1082 are free from lithogenic C-contribution.
The whole profile with samples 1080 to 1084 and the samples of this profile only have Д13С levels
near the lowest boundary of Calvin-C. This profile-specific phenomenon suggests that in the past
of this typic steppe soil a type of vegetation with Hatch-Slack photosynthesis might have been
present (Tables III and IV, also Lerman [9]).
In principle, the results of Table IV exclude an intermixture of lithogenic carbon with the
biogenic carbon species, manifested in the soil organic matter. If these results are generalized,
they confirm the'normality’of the natural carbon isotope ratio in organic matter of soils with free
carbonates and bicarbonate dynamics.
1 T.A. Rafter, J.R . H ulston and M.A. Cox, Nuclear Research Centre, Lower H utt, New Zealand, carried out
the 13C /12C-measurements, which are gratefully acknowledged.
TABLE IV. NATURAL CARBON-14 MEASUREMENTS AND CARBON-13/CARBON-12 RATIOS OF ORGANIC CARBON FROM SOILS WITH FREE CaC03 (BICARBONATE
DYNAMICS)
Al3C w a s m e a s u r e d a t t h e I n s t i t u t e o f N u c l e a r S c i e n c e s , L o w e r H u t t , N e w Z e a l a n d

Lab.No. Sample material, origin, soil group Horizon Profile depth %

С Д|3С Д14С AMRT, В.Р.
%
843 HoloceneHapludoll, Sedlec, CSSR Ahca, 40 —60cm 1.5 -33.2 ±0.1 58.7 4280± 60
845 ” * Ah/Cca 80 - 90cm 0.8 -27.9 ±0.1 48.5 5810± 60
846 PleistocenePaleudoll, Sedlec, CSSR fAh, 150 - 160cm 1.5 -30.0 ±0.2 3.06 28000 ±700
847 fAh 340 - 350 cm 4.3 -26.7 ±0.1 4i06 25 730 ±550
854 * fAh Lowest-zone 3.2 -28.2 ±0.1 4.11 25630 ±710
1075 Argiudoll, Bulgaria Ah2 36 - 47 cm 2.6 -24.0 ±0.1 89.6 880± 70
1076 - BAh2 55 - 65 cm 1.2 -26.8 ±0.1 74.7 2340± 70
1077 ” AhB 85 - 95 cm 0.9 -24.3 ±0.1 60.6 4020± 90

IAEA-SM-211/71
1078 * Cl 120 - 130cm 0.5 -24.2 ±0.1 41.6 7040± 80
1079 ClfA 200 - 210cm 1.1 -26.0 ±0.1 25.1 11 100± 90
1080 Pseudomycel-Hapludoll, Bulgaria Ahl 28 - 32 cm 2.5 -18.6 ± 0.1 83.2 1480± 70
1081 e Ah2 37 - 43 cm 1.4 -22.6 ±0.1 83.2 1480± 80
1082 - Ah3 66 —73 cm 0.8 -19.9 ±0.1 69.3 2950± 80
1083 Ah4 102 - 108cm 0.3 —21.2 ±0.1 59.8 4130± 70
1084 " AC 145 - 155 cm 0.6 -24.6 ±0.1 48.8 5760± 90
1108 Samonitsa, Pelludert, Bulgaria Ahl 20 —30 cm 3.0 -24.6 ±0.1 88.4 990 ± 50
1110 » Ah3 75 - 85 cm 2.5' -25.6 ±0.1 69.3 2940± 70
1111 ” BtAh 115 - 125 cm 1.0 -24.4 ±0.1 61.9 3850± 80
1133 Hapludalf, Inden, FRG, particle fractions 2000 - 60дт 0.3 -25.0 ±0.1 67.4 3170± 80
1134 ” 60 - 2дт 0.8 -26.4 ±0.1 65.0 3450± 80
1135 " 2- 1дт 0.5 -24.3 ±0.1 67.1 3280 ± 80
1136 ” 1- 0.5 дт 0.7 -25.0 ±0.1 70.6 2790± 70
1137 " 0.5 - 0.25дт 0.9 -25.9 ±0.1 73.2 2500± 70
1355 - - -Chromoxerert, HaciendaSporacia, Sicily Ah3 ----------- 45 —65'ст 0.8 -26.2 ±0.1 8179 ' 1600 ±‘ 70 _ "
1356 " Ah4 65 —85 cm 0.6 -26.2 ±0.1 78.3 1970± 70
1359 ” AC1 145 - 165 cm 0.3 -28.4 ±0.1 29.6 9790±160
200 SCHARPENSEEL

REFERENCES

[1 ] GERASIM OV, I.P., “Nature and origin o f paleosols”, P aleop ed ology, Origin, Nature and D ating o f Paleosols,
Jerusalem (1 9 7 1 ) 15.
[2] G ERASIM O V, I.P., T he age o f recent soils, Geoderm a 12 1 /2 ( 1 9 7 4 ) 17.
[3] CRAIG, H., G eochim . C osm ochim . A cta 3 (1 9 5 3 ) 60.
[4] SCHARPENSEEL, H.W., PIET1G, F ., Geoderma 2 (1 9 6 8 /1 9 6 9 ) 2 73.
[5] SCHARPENSEEL, H.W., PIETIG, F ., A tom praxis 16 3 (1 9 7 4 ) 1.
[6 ] SCHARPENSEEL, H.W., R O N ZA N I, C , PIETIG, F ., “Comparative age determ ination o n d ifferent hum ic-
m atter fractions”, Isotop es and R adiation in Soil Organic-Matter Studies (Proc. Sym p. V ienna, 1 9 6 8 ), IAEA ,
V ienna (1 9 6 8 ) 67.
[7] SCHARPENSEEL, H.W., Paleop ed ology, Origin, Nature and D ating o f Paleosols, Jerusalem (1 9 7 1 ) 77.
[8] PA U L, E .A ., CAMPBELL, C .A ., R ENN IE, D .A ., McCALLUM, K .J., Trans. 8th Int. Cong. S oil Sei. Bucharest
111(1964) 201.
[9] LERM AN, J.C., 8th Int. C onf. R adiocarbon D ating, W ellington, N ew Zealand (1 9 7 2 ) 6 1 2 .
 D IS C U S S IO N
(on the previous three papers)

P. BOTTNER: I should like to ask a question of a practical nature regarding the paper sub­
mitted by H.W. Scharpenseel (SM-211/71). To date an organic fraction, a relatively large amount
of carbon is necessary. How do you go about separating the fractions on Sephadex gel for dating
purposes?
B. GUILLET: Perhaps Mr. Scharpenseel would answer that question.
H.W. SCHARPENSEEL: We used 150-cm-high tubes of 10-cm diameter. After repeating the
process from 8 to 10 times, we had obtained a sufficiently large amount of carbon to synthesize
more than 3 ml benzene.
B. GUILLET: I would also like to ask Mr. Scharpenseel a question. How sure are you that
there is no contamination by the dead carbon of extractants other than dimethylformamide and
dimethylsulphoxide ?
H.W. SCHARPENSEEL: The fractions, extracted by organic solvents of increasing polarity,
did not cause a major breakthrough in fraction age. The increment associated with thib dimethyl­
formamide and dimethylsulphoxide fractions is caused by the petrochemical dead carbon of the
solvents. This was revealed by Д13С measurement with the mass-spectrometer. The same tests
on the other fractions confirmed normality and purity.

201
 M O D E R N T E C H N IQ U E S
A N D T H E IR IM PA C T
O N S O IL O R G A N IC M A T T E R R E S E A R C H
(S e ssio n 8a)
 IAEA-SM-211/8

R e v ie w p a p er

A D V A N T A G E S A N D L IM IT A T IO N S O F
M A S S - A N D P H O T O -S P E C T R O M E T R Y IN
N IT R O G E N -1 5 -A ID E D S T U D IE S

V. MIDDELBOE
Physics Laboratory,
The Royal Veterinary and
Agricultural University,
Copenhagen,
Denmark

Abstract

A D V A N T A G ES A ND LIMITATIONS OF MASS-AND PHOTO-SPECTROMETRY IN N IT R O G E N -15-AIDED

STUDIES.
In tracer studies aided by th e use o f natural variations or artificial alterations in the iso to p ic com p osition o f
stable nitrogen, 15N /14N analyses are an integral part o f th e investigations. H istorically, op tical spectroscopy was
the first m ethod used for th e assessment o f 1SN abundance, and mass spectroscop y was the secon d , but m ore
rapidly d eveloped, m ethod. A brief, critical review is presented, giving an ou tlin e o f (1 ) th e principles junderlying
m easurem ent o f nitrogen isotop e ratios b y mass- and photo-spectrom etry, (2 ) th e associated procedures for the
preparation o f dinitrogen sam ples, and (3 ) th e various lim its involved in iso to p ic analysis o f tracer nitrogen.
Finally, the'relative m erits o f mass- and p hoto-spectrom etry are surveyed w ith regard to applications Of 1SN
enrichm ent or d ep letion in bioresearch. T w o main conclusions are drawn: p h oto-sp ectrom etry is unique in its
ability to cop e d irectly w ith nanom ole am ounts o f total nitrogen, and m ass-spectrom etry is indispensable in
dealing w ith the extrem ely small variations in 1SN abundances found in nature. T he typ e o f inform ation presented
here is required primarily in the opening stages o f ls N-aided studies.

INTRODUCTION

Naude discovered about 50 years ago that natural nitrogen consists of two isotopds, namely
nitrogen-15 as well as the previously known nitrogen-14 [ l].1 The 1SN/14N ratio is 0.0037:1, so
that approximately four out of every 1000 nitrogen atoms in nature are 1SN. More exactly, the
recognized value at present for the abundance of 15N in atmospheric nitrogen is 0.366 atom per
cent [2].
Small differences in natural 15N-abundances exist [3, 4]. Traditionally, these differences are
stated in terms of the relative deviation of the 1SN/14N ratio of the sample from that of'a selected
standard (see Eq.(4) below). Observed differences are the order of plus or minus a few parts per
thousand.
Nitrogenous compounds with differences in 15N-abundance are produced today oh a commercial
scale. The present limits are up to 99 atom per cent 15N-enrichment or down to 0.35 atom per
cent 15N-depletion (see Eqs (5) and (6) below). Any compound containing nitrogen with an altered
lsN-abundance is in principle labelled and hence it can be used as a tracer.
All nitrogen-15 tracer studies give rise to a number of N-containing samples or fractionations
in each of which the nitrogen composition is to be determined. The analysis is carried out by the
_________
.
The list o f references is n ot m eant to be com prehensive; it cites predom inantly original or recent publications.

205
206 MIDDELBOE

aid of mass- or photo-spectrometry, and in both cases dinitrogen gas (N2) is used to measure
isotopic abundance.
The nitrogen contained in the sample to be analysed is rarely in the form of N2. In fact, the
liberation of pure N2, known as sample preparation [5—10], is often the most difficult and time-
consuming part of the complete lsN-analysis. The common method of sample preparation is
either (1) transformation of all nitrogen into ammonium and liberation of N2 by hypobromite,
or (2) reduction/oxidation of all nitrogen to N2 by hot copper/copper oxide.
A brief review will be presented of isotopic analysis of nitrogen, using mass- or photo­
spectrometry, with particular reference to the inherent limitations and advantages. Such information
is required in the planning and designing of studies that make use of 15N-enrichment or -depletion
relative to normal.

MEASUREMENT OF THE ISOTOPIC COMPOSITION OF DINITROGEN

The best-known method for measuring the isotopic composition of N2 is, no doubt, mass-
spectrometry [11]. An earlier, but only recently developed, method is that of optic
emission spectroscopy [12], here abbreviated to ‘photo-spectrometry’. These two methods differ
in one fundamental respect. In the former, N2 molecules themselves are consumed during measure­
ment. In the latter, light photons emitted by N2 molecules are consumed during measurement.
This difference is reflected in many of the respective advantages and limitations of the two methods.
In mass-spectrometry, a continuous stream of N2 molecules leak into the highly evacuated
machine, where they undergo ionization, separation according to mass, and collection. The
ionization is effectuated by electron bombardment of the N2 molecules. The N2 ions so produced
are separated by high-voltage acceleration followed by magnetic deflection. The beams of
14N14N+, 14N1sN+, and 1SN15N+ ions are collected separately in a Faraday cup. On collection, the
ions discharge, and the intensity of the beam focused on the collector is measured by the quantity
of charge delivered per unit of time.
In photo-spectrometry, light emitted by N2 molecules is optically dispersed, and a small
section of the spectrum so produced is recorded. During measurement the N2 molecules, con­
tained in a previously evacuated and de-gassed tube of glass or silica, are brought to emit light
(electrodelessly) with the aid of an electromagnetic short-wave generator. Light is admitted
through an entrance slit and dispersed by a slowly rotating prism or grating. The monochromatic
light emerging from the exit slit at any given moment is detected by a photo-multiplier tube.
In the N2 spectrum obtained, any given band appears in three separate replicates corresponding
to emission by 14N14N, 14N15N and 15N1SN molecules respectively. It is assumed that the relative
intensities of the three replicates of the bandhead correspond accurately to the amounts (in moles)
of the three species of N2 molecule in the discharge tube [12].
The final step, using either mass- or photo-spectrometry, is the evaluation of an appropriate
measure for the isotopic composition of the sample nitrogen. This evaluation is based on the
observed amounts of at least two of the three types of N2 in the sample, that is 14N14N, I4N15N
and 1SN15N. Any one of the following (interrelated) measures is used to specify the isotopic
composition of nitrogen in a given sample:

moles of 1SN
Iso to p ic abundance: A15 — 100 at.% (1)
moles of N
moles of 14N
A 14 = ---------- —— 100 at.% (2)
moles of N
 I AE A-SM-211/8 207

moles of 1SN A1
Iso to p ic ratio: 1SN/14N = (3)
moles of 14N A14 100 - A15
(15N/14N)sample - (15N/14N)ai
S1SN: 1000 per mille (4)
(15N /14N)air

N -en rich m en t . Agg^pj^ ^natural (5)

N -d ep letio n . A^ltural —^-sample (6)

Assuming that the 14N- and lsN-atoms in the N2 sample are randomly paired, the following
formulas (7 -9 ) are valid for the calculation of numerical values from observed intensities:

'00 „ I28
Als = ---------at.%, where R = — (7)
2R + 1 129

,, 100 I30
A14 = -------- at.%, where R = — (8)
2R + 1 j29

(9)

In formulas (7-9), I28,129 and I30 are the observed relative intensities of the I4N14N, 14N1SN
and 15N1SN mass- or photo-peaks respectively. In formula (9), I29/I28 could also be thb directly
observed ratio between the respective number of N2 molecules of mass 29 and 28, for example
in a double-focusing mass-spectrometer.
If there is doubt concerning the random distribution of the 14N- and 15N-atoms, and the
lsN-abundance is neither very low nor very high, it is safer to base the calculation of isotopic
abundance on one of the following equations (see Eqs (1) and (2)):

I29 + 2 I 3
100 at .9 (10)
2(128 + J29 + J30)

2-I28 + I29
A14 = ---------------------- 100 at.% (11)
2(128 + J29 + J30)

where the I’s have the same meaning as in formulas (7) and (8).

SAMPLE PREPARATION FOR ISOTOPIC ANALYSIS OF NITROGEN

Fiedler and Proksch [13] have recently published a detailed review covering the diversity
of technicalities involved in the preparation of N2 and its isotopic analysis by mass- or photo­
spectrometry. In practice, there are two main approaches; these are termed the ‘Rittenberg
technique’ and the ‘Dumas technique’ respectively.

’N -deviation can be used as a com m on nam e.

208 MIDDELBOE

In the Rittenberg technique, the sample nitrogen is converted to the ammonium form by
the well-known Kjeldahl method, and subsequently N2 is liberated by the hypobromite procedure
according to the following reaction:

2NH3 + ЗВгСГ -»• N2 + 3H20 + 3Br~

In the Dumas technique, the nitrogen is oxidized and/or reduced to N2 by baking the sample
in the presence of copper oxide3 and/or copper. The following are two examples of the reactions
occurring:

CO(NH2)2 + 3CuO ->• N2 + C 02 + 2H20 + 3Cu

2KN03 + 5Cu -►N2 + K20 + SCuO

In both the Rittenberg and the Dumas techniques, unwanted gases, such as H20 or C 02,
are evolved. These gases are either frozen out (liquid air), absorbed chemically (Ca0/Al20 3), or
adsorbed physically (molecular sieves). Unfortunately, none of these procedures trap CO, which
is a practically unavoidable contamination in both mass- and photo-spectrometry.
The Rittenberg technique, the Dumas technique, and intermediary techniques can all be
combined with mass-spectrometry as well as photo-spectrometry. Thus, the limitations and
advantages imposed by the sample preparation procedure are almost independent of the method
used for determining the isotopic composition of the N2 prepared.

LIMITS INVOLVED IN ISOTOPIC ANALYSIS OF TRACER NITROGEN

The limits involved in the determination of the isotopic composition of nitrogen are primarily
(1) amount of N2 required, (2) precision and accuracy, and (3) rapidity.
The amount of N2 needed for a routine isotope analysis using mass-spectrometry is
100 —1000 pg. The corresponding figure for photo-spectrometry is 1 — 100 jug, which is of the
order of 10 — 100 times less.
In considering precision and accuracy, small deviations of 15N-abundance close to that found
in nature are best treated separately. In the case of natural variations, the long-term accuracy of
a dual collector mass-spectrometer is about one 5-unit [15], which is equivalent to 0.0004 atom
per cent 1SN (see Eq.(4)), whereas the accuracy of a high resolution photo-spectrometer in determin­
ing near natural abundances is about 0.01 atom per cent [16], indicating a 25 times greater
probable error. In the case of routine analysis of samples from 15N-aided bioresearch, the precision
is 0.2 —0.5% rel. for mass-spectrometry and 2 —5% rel. for photo-spectrometry [13]. However,
the variation in replicate samples from bioresearch is often 5 —10%4 before making the measure­
ments of 15N-abundance [17].
The time required for a routine determination of the isotopic composition of nitrogen by
mass- or photo-spectrometry is about the same, namely 5 —10 minutes, which is normally less
than the time needed for sample preparation. However, if the samples contain only NH4-N
(originally, or after total-N analysis) little further time is required for the production of N2 in
mass-spectrometry or in automatic photo-spectrometry using the Statron Isonitromat 5200
instrument. Otherwise, if the Statron NOI-4 or NOI-5 instrument is used, or in any case if

3 N ickel o x id e at 1000°C is also used [14].

4 With good subsampling this is presumably due to ‘field-variation’, hence for m ost studies it is the 1SN-
deviation (see F o o tn o te 1) w hich varies 5—10%, and n ot necessarily the ls N -abundance.
 IAEA-SM-211/8 209

the sample nitrogen is not in the form of ammonium, about an extra half man-hour is needed for
sample preparation (digestion time excluded).

MASS- VERSUS PHOTO-SPECTROMETRY IN NITROGEN-15-AIDED STUDIES: CONCLUSION

The most distinguished quality of the mass-spectrometer is its superior accuracy. This is used
to full advantage in studies involving the measurement of natural variations in lsN-abundänce, since
these differences correspond to only a few 5-units, i.e. only some parts per thousand.
In field experiments using labelled nitrogen, the purchase of material that is lsN-depleted or
only slightly 15N-enriched (see Eqs (5) and (6)) can amount to substantial current savings; but the
subsequent reduction in the lsN-deviation (see Footnote 4) to be analysed could necessitate a
somewhat higher precision than can be attained in routine photo-spectrometry [ 18 ], in which case
the use of mass-spectrometry would be advantageous.
The larger sample of N2 required for mass-spectrometry compared with photo-spectrometry
can be a real advantage when relatively large N-containing objects of investigation have to be sub­
sampled for 15N-analysis; however, this disadvantage in photo-spectrometry can be overcome
by Kjeldahl or Dumas digestion of a larger sample followed by subsampling the NH^-solution or
N2-gas produced.
Finally, mass-spectrometry has the advantage that the measurements are normally considered
absolute, whereas photo-spectrometry can only be exploited to the limit of its precision by the
use of calibration standards.
The price of the commercially available automatic 15N-analyser based on photo-spectrometry
is comparable with that of a mass-spectrometer having the general range of specifications mentioned
in this report, whereas the price of the non-automatic 15N-analyser based on photo-spectrometry
is about four times less.
A major advantage of a photo-spectrometer is its simplicity and ease of maintenance relative
to a mass-spectrometer [19].
The use of photo-spectrometry to determine isotopic composition is essential when the
total amount of nitrogen in the available sample (e.g. in a chromatographic fraction) is extremely
restricted. In a 2-cm3 standard discharge tube 10 Mg of N2 is optimal for analysis. This amount
can be routinely reduced to 1 Mg N2 by the use of argon as carrier gas [20], and by the use of
miniature discharge tubes as well as carrier gases the limit can be lowered to 0.2 Mg N2 [21].

ACKNOWLEDGEMENT

The writer is grateful to Gunter Ewald, who has kindly supplied translations of some recent
Russian literature on the optical nitrogen-15 method and its applications in agriculture.

REFERENCES

[1 ] N A U D E, S.M., An isotop e o f nitrogen, mass 15, Phys. Rev. 34 (1 9 2 9 ) 1498.

[2 ] JUNK , G., SVEC, H .J., The absolute abundance o f the nitrogen isotop es in the atm osphere and com pressed
gas from various sources, G eochim . C osm ochim . A cta 14 (1 9 5 8 ) 234.
[3 ] HOERING, T ., Variations o f nitrogen-15 abundance in naturally occurring substances, Science 122 (1 9 5 5 ) 1233.
[4 ] R ENN IE, D .A ., PA U L, E .A ., JOH NS, L.E., Natural nitrogen-15 abundance o f soil and plant sam ples, Can.
J. S oil Sei. 5 6 ( 1 9 7 6 ) 43.
[5 ] R ITTENBERG, D ., KESTON, A .S., RO SEBUR Y , F ., SCHOENHE1MER, R „ Studies in protein m etabolism :
II. The determ ination o f nitrogen isotop es in organic com p ou nd s, J. B iol. Chem. 127 (1 9 3 9 ) 2 91.
210 MIDDELBOE

[6 ] HOCH, M., W EISSER, H .-R ., R eaktionen m it ls N: 11. E ine spektroskopische M ikrom ethode zur Bestim m ung
von N -l 5, Helv. Chim. A cta 33 7 (1 9 5 0 ) 2128.
[7] ROLLE, W., Eine einfache M ethode zur Umarbeitung von Salpetersäure ( N - l 5) and Nitrat (N -15) in
elem entarischen S tick sto ff zur m assenspektrom etrischen Isotopenanalyse, Z. Chem . 4 (1 9 6 4 ) 3 96.
[8] BREM NER, J.M., “Isotope-ratio analysis o f nitrogen in nitrogen-15 tracer investigations”, M ethods o f Soil
A nalysis, Part 2 (BLA CK , C .A ., E d .), American S ociety o f A gronom y, M adison, W isconsin (1 9 6 5 ) 1256.
[9] ROSS, P.J., M ARTIN, A .E., A rapid procedure for preparing gas sam ples for nitrogen-15 determ ination,
A nalyst 95 (1 9 7 0 ) 817.
[1 0 ] FA U ST, H., R EIN H A R D T, R ., Zur Verwendung von M olekularsieb als A bsorbens für N ebenprodukte bei
der N -15 Probenchem ie, Isotopenpraxis 11 (1 9 7 5 ) 321.
[1 1 ] N IER , A .O ., A mass spectrom eter for routine isotop e abundance m easurem ents, Rev. Sei. Instrum. 11 (1 9 4 0 )
212.
[ 12] HERZBERG, G ., D as S tick stoffisotop der Masse 15, Z. Phys. Chem. В 9 (1 9 3 0 ) 43.
[1 3 ] FIED LER, R ., PROKSCH, G ., The determ ination o f nitrogen-15 by em ission and mass spectrom etry in
b iochem ical analysis, Anal. Chim. Acta 78 (1 9 7 5 ) 1.
[1 4 ] LA ZEEVA , G .S., PETROV, A .A ., STOLBO VA, E.P., Prim enenie stabil’nogo izotopa N -l 5 issledovanijach
p o zem ledeliju, K olos, M oscow ( 1 9 7 3 ) 16.
[1 5 ] SH EA RER , G .B., KOHL, D .H ., COMMONER, B., T he precision o f determ inations o f.th e natural abundances
o f nitrogen-15 in soils, fertilizers and shelf chem icals, Soil Sei. 118 5 (1 9 7 4 ) 306.
[1 6 ] M IDDELBOE, V ., High resolution op tical ls N-analysis, Appl. Spectrosc. 2 8 3 (1 9 7 4 ) 2 74.
[1 7 ] FIED LER, R ., PROKSCH, G., Em ission spectrom etry for routine analysis o f n itro g en -i5 in agriculture,
Plant Soil 3 6 ( 1 9 7 2 ) 371.
[1 8 ] R ENN IE, D .A ., FR IED , M., An interpretive analysis o f the significance, in soil fertility and fertilizer
evaluation, o f N -15 labelled fertilizer experim ents con d ucted under field con d ition s, Int. Sym p. Fertilizer
Evaluation, I.S.S.S. Com II and IV , N ew Delhi (1 9 7 1 ) 639.
[1 9 ] LLOYD-JONES, C.P., H U D D , G .A ., HILL-COTTINGHAM, D .G ., The determ ination o f nitrogen-15 in plant
m aterial w ith an em ission spectrom eter, A nalyst 99 (1 9 7 4 ) 580.
[2 0 ] K EEN EY , D .R ., TEDESCO, M.3., Sam ple preparation for and nitrogen iso to p e analysis b y the NOI-4 em ission
spectroscope, Anal. Chim. A cta 6 5 ( 1 9 7 3 ) 19.
[2 1 ] GOLEB, J.A ., MIDDELBOE, V ., O ptical nitrogen-15 analysis o f small nitrogen sam ples w ith a m ixture o f
helium and xen on to sustain the discharge in an electrodeless tube, Anal. Chim. A cta 4 3 (1 9 6 8 ) 229.

DISCUSSION

T. WEICHELT: Was absorption spectroscopy or emission spectroscopy used for the measure­
ments you described?
V. MIDDELBOE: As I recall, the original discovery by Naude was made using absorption
spectroscopy on NO. Nowadays, we use an electronic vibrational transition (in the second positive
system) of molecular nitrogen in emission. Normally, a bandhead in the near ultra-violet region
(300—400 nm) is used.
K. STÜHMEIER: As you have mentioned, photo-spectrometry cannot give absolute results;
calibration standards have to be used. How are the standards themselves measured?
V. MIDDELBOE: The 1SN abundances of the calibrated standards are determined by mass-
spectrometry.
K. STÜHMEIER: In photo-spectrometric assay, you make use of a calibration curve obtained
from the standards. Is there linearity or not? I would think there is no linearity, and that this
is therefore not a very exact method.
V. MIDDELBOE: In a recent publication in the International Journal of Isotopes and Radiation
by two French authors (whose names, I regret, I do not remember), it is found that the whole range
from natural to 99 atom per cent 1SN can be covered by 3—4 linear calibration curves.
K. STÜHMEIER: If amounts of nitrogen are low, it is possible to mix the sample with natura
nitrogen from laboratory stock and then carry out mass-spectrometric work without difficulty.
V. MIDDELBOE: In principle, I agree.
 IAEA-SM-211/8 211

К. STUHMEIER: Are you aware that it is possible to measure pg-quantities of nitrogen by

mass-spectrometry using an electron multiplier instead of a Faraday cup?
V. MIDDELBOE: Yes, I am aware that an electron multiplier (SEV in German) can be used
as the collector, and that the sensitivity of detection is thereby increased. However, it is my
impression that the accuracy is simultaneously decreased.
K. STÜHMEIER: You say that the precision of the two methods differs by a factor of 25.
When comparing the two methods, this should be considered rather high.
V. MIDDELBOE: If you feel that I have somewhat underestimated photo-spectrometry vis-ä-vis
mass spectrometry I have no objection because I am a photo-spectrometry fan. I can assure you
that the optical emission spectrometer is one of my pet instruments.
 IAEA-SM-211/53

S T U D IE S O N S O IL H U M IC C O M P O U N D S ,
F U N G A L M E L A N IN S A N D M O D E L P O L Y M E R S
B Y P Y R O L Y S IS M A S S -S P E C T R O M E T R Y

K. HAIDER, B.R. NAGAR, C. SAIZ

Institut für Biochemie des Bodens der
Forschungsanstalt für Landwirtschaft,
Braunschweig-Völkenrode

H.L.C. MEUZELAAR
Institute for Atomic and Molecular Physics,
Amsterdam,
The Netherlands
J.P. MARTIN
Department of Soil and Environmental Sciences,
University of California,
Riverside,
California,
United States of America

Abstract

STUDIES ON SOIL HUMIC COMPOUNDS, FU N G A L M ELANINS A ND MODEL POLYM ERS B Y PYROLYSIS

MASS-SPECTROMETRY.
B iopolym ers or synth etic polym ers are frequently characterized by pyrolysis in com b in ation w ith gas
chrom atography, m ass-spectrom etry or gas chrom atography and m ass-spectrom etry. Curie-point pyrolysis technique
in conjunction w ith low -voltage electron im pact ion ization m ass-spectrom etry was used to characterize several soil
hum us fractions including w hole soil, and to com pare them w ith h um ic acid-like m elanins from so il fungi, m odel
phenolic polym ers and several biopolym ers such as polysaccharides, lignins or proteins. H um ic acids from different
soil typ es gave com p lex pyrolysis spectra. A lthough th e chem ical structure o f individual com p ou nd s can n ot be
assigned w ith certainty, the spectra show a marked qualitative and even quantitative correspondence through the
presence o f similar series o f h om ologou s ions. T hey are probably related to protein-like m aterials, arom atic and
phenolic com pounds and to polysaccharides. Similar series were observed in the spectra o f w hole so il and w ith
hum ins, but m ore prom inent peaks corresponding to com p lex polysaccharide materials were present in addition.
Spectra from fulvic acids so far investigated d iffered largely from th ose o f h um ic acids, and show ed a greater
relation to polysaccharides. With som e excep tion s spectra from hum ic acid-like m elanins from fungi were similar’to
those from soil h um ic acids, and show ed a similar series o f h om ologou s ions. M odel oxidative polym ers, prepared by
phenolase oxid ation o f phenols in a neutral buffer m edium , w ith and w ith o u t the addition o f am ino acids or a peptide
m ixture, show ed marked d ifferences from m elanins and hum ic acids if on ly sim ple p henols together w ith am ino acids
were used. However, the spectra becam e m ore and m ore similar to the m elanins if a m ore com p lex p h en ol m ixture
was o x id ized togeth er w ith a com p lex peptide m ixture. M ost soil hum ic acids or even soil show ed in addition ion
series w ith characteristics o f lignin-type phenols. H ow ever, these were in general w eak signals or som etim es absent.

Polymer materials can be characterized by the pyrolysis technique in combination with mass
spectrometry or gas chromatography and mass spectrometry. These techniques allow identification
of polymers by their specific pyrograms or by specific pyrolysis products which are related to their
structure.
During the last few years, pyrolysis techniques have been considerably improved, particularly
through the development of curie-point pyrolysis [1,2]. By the coupling of this pyrolyser to a fast

213
214 HAIDER e t al.

F I G .l. S c h e m e o f th e c u r ie - p o in t p y r o ly s is m a ss s p e c tr o m e te r s y s te m .
% T O T A L ION IN T E N S IT Y

m/e

F I G .2 . L o w -v o lta g e p y r o ly s is m a s s s p e c tr a o f c e llu lo s e a n d lig n in f r o m w h e a t s tr a w .

 IAEA-SM-211/53 215

scanning quadrupole mass filter and recording the mass spectra by means of a multi-channel signal
averager in conjunction with low voltage electron impact ionization, characteristic spectra of
widely different synthetic and natural polymers, and even whole bacteria with prominent molecular
ions, have been reported by Meuzelaar and co-workers [3—5]. Their device, used in the studies
reported here, is shown in Fig. 1.
A quartz reaction tube is connected with a ferromagnetic Fe/Ni wire of 0.5 mm diameter
(curie temperature at 610°C). This wire is coated with about 40 to 50 pg of the sample,;and is
centred in a high frequency coil which can be energized by a high frequency generator (1.5 kW,
1.1 MHz, Fischer Labortechnik). The temperature rise time is about 150 ms, and the total pyrolysis
time about 1 s. The pyrolysis reactor is tightly connected to an expansion chamber maintained at
150°C to prevent condensation of pyrolysis products. From there the products enter the electron
impact ionization region consisting of an open crossbeam-type ionizer operating at 10—15 eV
electron energy. In spite of the relatively low ion yield obtained with this low electron impact
ionization, the resulting mass spectrum is more characteristic because of the presence of large
molecular ions and the absence of a bulk of small fragmented ions.
Non-ionized molecules are trapped on a liquid N2-cooled metal shield which prevents a
general pressure build-up outside the beam of pyrolysis products, thus maintaining an optimal
signal/background ratio. The quadrupole mass filter (Riber, Q 156) is scanned repeatedly through a
mass range from m/e 15 to about 180 at a rate of 5 to 10 spectra per second. The ion arrival
pulses from the electron multiplier are amplified, shaped and fed to the pulse-counting input of a
1024-channel signal averager (Fabri-tek, model 1072). All successive mass spectra are summed in the
memory of the signal averager. The relative ion intensities of the summed spectrum therefore
represent the average abundance of the corresponding pyrolysis products at the ionizer throughout
the scanning period.
Some examples of pyrolysis mass spectra from biopolymers, soil organic matter fractions and
fungal products are shown in the figures. An attempt was made to co-ordinate the main peaks to
characteristic compounds. It is recognized, however, that the chemical identity may be tentative as
ions of different structure may have the same mass-to-charge ratio.
Figure 2 provides pyrolysis mass spectra of the characteristic plant polymers, cellulose and
lignin which constitute more than 80% of the total plant mass. Both polymers were isolated from
wheat straw. Cellulose shows high peaks with even numbers indicating the absence of nitrogen-
containing compounds and of small fragmentated ions produced during the ionization process.
A series of homologous ions at m/e 60, 68, 82, 96, 98, 110 and 126 are possibly related to acetic
acid(m/e 60), furan (m/e 68), furfural (m/e 96), furfuryl alcohol (m/e 98), and their partly
methylated derivatives as they were identified during pyrolysis studies of polysaccharides by
Schulten and co-workers [6]. Spectra of more complex polysaccharides, consisting of hexoses
and pentoses as constituent units, show in addition prominent peaks at m/e 114 and 128.
According to Posthumus [7], these peaks are probably related to fragments of pentose sugar
units, and were also shown to originate from the ribose units of ribonucleic acids [7].
Lignin, on the other hand, shows pyrolysis fragments with relatively big molecular ions.
Prominent ion series at m/e 94, 108, 120 and 122, at m/e 124, 138, 150 and 164, and at m/e 154
and 168 are evident. These ion series suggest the presence of the phenol and the p-coumäryl, the
guaiacyl and possibly the syringyl units which are the characteristic building blocks of grass
lignins [8]. Lignins from conifers (with mainly coniferyl alcohol building blocks) or from
beech wood (with coniferyl and sinapyl alcohol building blocks), show the typical ion series related
to their respective units. Peaks, indicative of benzene, and related peaks at m/e 78 and 92 (toluene),
are very faint, and cellulose and protein-related peaks are missing.
Recently, studies on the pyrolysis spectra from whole soils of different origins, fractions of
the isolated soil organic matter, several model polymers prepared from phenols, either phenols or
glucose with amino-acid compounds, from plant materials at different rotting stages as well as from
several fungal melanins, have been published by Nagar and co-workers [9], Meuzelaar and
216 HAIDER e t al.

о
ä?

20 40 60 80 100 120 140 160 180

F I G .3 . L o w - v o l t a g e m a s s s p e c t r a o f a w h o l e p o d z o l s o i l {A } , t h e h u m i c a c i d f r o m th e p o d z o l s o il (В ), th e h u m ic

a c id fr o m a c h ern o zem s o il (C j a n d th e fu lv ic a c id fr o m th e p o d z o l s o il (D ). A r r o w s in s p e c tr u m C p o in t to

p o s s ib le lig n in -ty p e p h e n o ls .

co-workers [10] and Saiz and co-workers [11, 12]. The present paper summarizes some of the
findings and provides further data on pyrolysis mass spectra from additional samples. Figure 3
shows the pyrolysis mass spectra of a whole podzol soil (‘Hohes Moor’ podzol near Stolzenberg/
Weser) and from the humic and fulvic acids isolated from this soil, and from the humic acid of a
chernozem soil (Asel near Hildesheim). The spectrum from the whole soil shows.prominent
peaks at m/e 68, 82, 96, 98 and 126 which, as indicated above (Fig.2), are related to polysaccharides.
However, additional peaks at m/e 78, 92, 94 and 108 are evident, and indicate probably the
presence of benzenoid and phenolic compounds. These peaks are more evident in the pyrolysis
 IAEA-SM-211/53 217

mass spectra of the humic acids isolated from the podzol and the chernozem soil. Peaks related
to the methoxylated phenols of lignin (Fig.2) at m/e 124, 138, 150, 152 and 164 are not prominent
in the pyrolysis products from the podzol soil nor in the humic acid from this soil. However, they
are somewhat more distinct in the spectra from the humic acid from the chernozem soil. But they
occur also here only as small signals, and are indicated by arrows. Compared with the prominent
peaks obtained by pyrolysis of lignin, humic acids so far analysed [9—11 ] show generally only
weak or doubtful signals in relation to lignins. Humic acids from not completely composted
straw [10], however, or from some forest soils [12], have more prominent lignin-related signals.
Peaks that are typical for carbohydrate fragments, and that are. well represented in the1spectrum
from the whole soil, are less prominent in the soil humic acids. On the other hand, fragments
related to sulphur- and nitrogen-containing compounds [13] are highly significant, and indicate the
presence of proteinaceous materials [13] . The presence of sulphur-containing compounds is
indicated by peaks at m/e 34, 48, 62 and nitrogen-containing compounds at m/e 67, 81 and 95
(‘pyrrols’) and m/e 117, 131 and possibly at 145 (‘indoles’). Generally, humic acids from different
soils [9—12] and from peats [10] give similar, highly complex spectra with characteristic ion
series of sulphur- and nitrogen-containing compounds, benzene and phenolic compounds and
smaller amounts of polysaccharide-related materials. Peaks related directly to lignin were not found
to be prominent or were sometimes dubious. The intensity of the latter peaks varied, however, with
the soil type from which the humic acids were isolated. |
The general appearance of the fulvic acid spectrum from the podzol soil differs from that of
the humic acids. This dissimilarity was also noted with fulvic acid fractions from othej- soils
of widely different geographical origins [11,12]. It should be noted that the fulvic acid fraction,
shown in Fig.3, contributes to only about 3% of the total soil organic matter. Peaks related to
aromatic or phenolic compounds (m/e 78, 92, 94, 108 and 122) or to nitrogen-containing
compounds (m/e 67, 81, 95, 117, 131) are either lower as in the humic acids or can be hardly
detected. The low representation of protein-related peaks could be expected from the low
nitrogen content of this fulvic acid. The most prominent peaks observed in the pyrolysis mass
spectrum of the fulvic acid resemble somewhat those from polysaccharides.
Briefly it should be indicated that spectra from isolated polysaccharides differ from the
fulvic acid fraction by prominent peaks at m/e 114 and 128, and resemble therefore more those of the
the whole soil [11, 12]. Polysaccharides have also more prominent peaks at m/e 67, 81 and 95,
which probably indicates that nitrogen-containing units are also present. The humin fractions
resembled largely humic acids. However, peaks related to polysaccharides were higher,! and peaks
related to lignin were of similar low intensity in both the humic acids and the humins.
Figure 4 shows an example of the spectrum from a humic acid-like melanin from E urotiu m
echinulatum , a fungus isolated previously from soils [14] . This fungus was cultivated in a liquid
culture medium containing glucose and asparagine as a carbon and nitrogen source. During
growth the fungus synthesized numerous phenols and anthraquinones which were partly
incorporated into the melanin [14] . The pyrolysis spectrum of the melanin is compared with
that from a model polymer, prepared by enzymatic oxidation of a mixture of phenols and peptides,
which had a similar composition to the phenolic units synthesized by the fungus [15]. It is
remarkable that the pyrolysis mass spectrum of the fungal melanin resembles in the major ion
series to a great extent the spectrum of the soil humic acids [ 10]. However, it should be noted that
in the spectrum of the melanin, prominent signals at m/e 67, 81, 95, 117, 131 and 145 suggest a
higher content of proteinaceous materials. Compared with the humic acid from the chernozem
soil, lignin-derived peaks at m/e 124, 138 and 150 are insignificant. This can be expected, since the
fungus does not synthesize phenols that are related to those of lignins. Melanins from bther
fungi, such as E pico ccu m nigrum , H endersonula toru loidea or A spergillu s niger, are rather similar
to the spectrum shown in Fig.4 [10]. However, the spectrum of the melanin from S ta c h y b o trys
chartarum differs markedly from the others by more prominent peaks and a more regular pattern
218 HAIDER et al.

F I G .4 . L o w -v o lta g e m a s s s p e c tr a o f th e h u m ic -a c id - lik e m e la n in fr o m Eurotium echinulatum ( A ) , a m o d e l o x id a tiv e

p o ly m e r fr o m a m ix tu r e o f p h e n o ls a n d p e p to n e (B ), a 6 N H C l h y d r o ly s e d h u m ic a c id fr o m r e n d z in a s o il (C )

a n d th e 6 N H C l h y d r o ly s e d m e la n in fr o m Eurotium echinulatum (D ).

in the higher mass range. This probably indicates that this melanin contains more carbohydrate, and
possibly also alkane-related materials.
The pyrolysis mass spectrum of the model phenolic polymer shows a striking similarity with
the spectrum obtained from the fungal melanin. Both contain all the characteristic ion series and
a relative abundance of pyrolysis products which seem to fall within the normal range of variations
encountered in the melanins. Spectra obtained from polymers prepared from a less complex
phenolic mixture, or even from the complex phenolic mixture with amino acids as nitrogen-
containing units, differed markedly from the spectrum shown in Fig.4. However, it is interesting
 IAEA-SM-211/53 219

to note that oxidation products from catechol or hydroquinone together with glycine or leucine
show ion series that indicate the possible presence of heterocyclic nitrogen compounds [10] .
The next two spectra in Fig.4 show examples of a 6N HC1 hydrolysed humic acid and a
hydrolysed fungal melanin. Both spectra show a decrease of the peaks at m/e 17 and 67, 81, 95
and 117, indicating a release of nitrogen-containing compounds during hydrolysis. Furthermore,
the peaks related to polysaccharides were decreased, especially in the humic acid sample. Both
spectra show prominent ion series related to phenolic compounds at m/e 94, 108, 110, 120, 122
and 124. Furthermore, in the hydrolysed soil humic acid, the peaks at m/e 138, 150, 154, 162
and 164 have increased, which indicates a relative enrichment of the lignin-like residuesJ Both
hydrolysed polymers give the impression of a mainly phenolic framework with the presence of
traces of lignin-related phenols in the soil humic acid. Even upon hydrolysis, the spectra of both
hydrolysed polymers still show peaks of nitrogen-containing compounds, although the original
signal intensity decreased.
The results of this study indicate that the method of pyrolysis mass spectrometry as it was
used here is appropriate for the characterization of soil organic polymers. Although this method
has certain limitations, especially with respect to the identification of distinct fragments released
during pyrolysis, and it is restricted to fragments of a mass number of about m/e 180 because of the
sensitivity of the quadrupole mass filter, the scanned pyrograms are highly specific, and jallow
definitions and distinctions of polymers from different origins. By the appearance of ion series
in the pyrogram it is also possible to elucidate at least with some probability the structural relations
of the pyrolysis products to the constitutional units of the polymers. If used in connection with
other methods, for example, chemical degradation, gas chromatography, NMR-analysis, jtandem or
field desorption mass spectrometry, this method should be highly informative with respect to the
chemical characterization of soil organic polymers.

REFERENCES

[1] SIMON, W„ KRIEMLER, P., VOELLMIN, J.A ., STEINER, H., J. Gas Chromatogr. 5 (1 9 6 7 ) 52.
[2] BÜHLER, C , SIMON, W., J. Chromatogr. Sei. 8 (1 9 7 0 ) 323.
[3] M EUZELAAR, H.L.C., In’t’VELD, R .A ., J. Chromatogr. Sei. 10 (1 9 7 2 ) 2 13.
[4 ] M EUZELAAR, H.L.C., KISTEMAKER, P.G ., Anal. Chem. 4 5 (1 9 7 3 ) 5 87.
[5 ] M EUZELAAR, H.L.C., KISTEM AKER, P.G., POSTHUM US, M .A., B iom ed. Mass Spectrom . 1 (1 9 7 4 ) 3 12.
[6] SCHULTEN, H .R ., BECKEY, H .P., BOERBOOM, A .J.H ., M EUZELAAR, H .L.C ., Anal. C hem . 4 5 (1 9 7 3 ) 2 3 5 8 .
[7] POSTHUMUS, M .A., N IBBERING , N .N.M ., BOERBOOM, A .J.H ., SCHULTEN, H .R ., B iom ed. Mass Spectrom .
1 (1 9 7 4 ) 352.
[8] SA R K A N EN , K .V ., LUDWIG, D .H ., Lignins, W iley-Interscience, N ew York (1 9 7 1 ).
[9] N AG AR , B.R ., WAIGHT, E.S., M EUZELAAR, H.L.C., KISTEMAKER, P.G., Plant Soil 4 3 (1 9 7 5 ) 6 81.
[10] M EUZELAAR, H .L.C ., H A IDER , K., N A G A R , B .R ., M ARTIN, J.P., Geoderm a (in press). !
[П] SAIZ, C , H A ID ER , K., M EUZELAAR, H .L.C ., Geoderm a (in preparation). J
[12] SAIZ, C., M ARTIN, F ., H A ID ER , K., M EUZELAAR, H.L.C., G eochim . C osm ochim . A cta (in preparation).
[1 3 ] POSTHUM US, M .A ., BOERBOOM, A .J.H ., M EUZELAAR, H .L.C ., Adv. Mass Spectrom . 6 (1 9 7 4 ) 3 97.
[14] SAIZ, C., H A IDER , K„ M ARTIN, J.P., Soil Sei. Soc. A m ., Proc. 39 (1 9 7 5 ) 6 4 9 .
[1 5 ] FILIP, Z., H A IDER , K„ BEUTELSPACH ER, H., M ARTIN, J.P., Geoderm a 11 (1 9 7 4 ) 37.

DISCUSSION

T. WEICHELT: You also used straw lignin for your investigations. From which plant and by
what method did you isolate this lignin?
J.P. MARTIN: We used Bjorkman lignin from wheat straw.
M. SCHNITZER: Your masses are relatively low, which indicates considerable degradation
during pyrolysis. Would you please comment on this point?
220 HAIDER et al.

J.P. MARTIN: The procedure apparently breaks the large aromatic molecules into small
benzenoid units, but it does not break the ring. Polysaccharides are fragmented into smaller units,
probably including acetic acid. It is possible that the phenolic units are overemphasized. Lipid-type
substances either escape ionization and are not detected or else are present in such small amounts
that they do not produce prominent signals. Changing the pyrolysis temperature might alter the
size of the fragments.
B.R. NAGAR: As one of the authors of the paper, I should like to add that the peaks up to
150 are natural to the technique. However, we are working on pyrolysis field desorption mass
spectrometry, and we shall be able to report higher-range mass peaks and their elemental composition
later.
M. SCHNITZER: How much of your total material is volatilized during pyrolysis?
J.P. MARTIN: I do not know how much of the sample is actually pyrolysed.
B.R. NAGAR: We made it clear in our first publication on this technique that we were
reporting only on the pyrolysable fraction. It is our experience that some material remains
unpyrolysed even at 700°C.
 IA EA-SM /211/54

C H R O M A T O G R A P H Y O F H U M IC S U B S T A N C E S
ON C O N TRO LLED PO R E G LASS

O.H. DANNEBERG
Institut für Landwirtschaft am Forschungszentrum
Seibersdorf der Österreichischen
Studiengesellschaft für Atomenergie G.m.b.H.,
Vienna,
Austria

Abstract

CHROMATOGRAPHY O F HUMIC SUBSTANCES ON CONTROLLED PORE GLASS.

To check the suitability o f controlled pore glass (CPG) for the chrom atography o f hum ic substances, a soil
extract from an Austrian ch ernozem and hum ic fractions prepared from the extract w ere chrom atographed using a
colum n filled w ith CPG o f a pore diam eter o f 156 Ä. The chromatograms obtained w ere reproducible,!and show ed
the ex p ected sequence o f elu tion , grey hum ic acids (G H A ) being eluted before brow n hum ic acids (B H A ) and fulvic
acids (F A ). Chromatograms o f com plex hum ic system s agreed w ell w ith the com p u ted sum s o f the chrom atogram s
o f all its com p on en ts. This means that all hum ic substances moved through the colum n in d ep en d en tly Jof the
presence or absence o f others. From these findings, the suitability o f CPG fo r chrom atography o f hum ic substances
w as concluded. A structural alteration o f GHA during the preparation was detected. This was accom panied by
a decrease in m olecular w eigh t and an increase in colour intensity.

INTRODUCTION

Humic substances form an extremely polydisperse system [1], which can be characterized by
methods separating according to molecular weight or to molecular dimensions. Moleculajr exclusion
chromatography on porous media offers a simple, rapid and cheap method of this type, hnd will
be the method of choice if certain conditions are met.
Mehta and co-workers [2] were probably the first who used gel chromatography on Sephadex
to characterize humic substances. The method was successful in the case of fulvic and brpwn
humic acids but failed in the case of grey humic acids because of a strong adsorption of these high
molecular substances on the gel. This was confirmed by our unpublished experiments in jwhich
the humic system of an Austrian chernozem (Fuchsenbigl soil) was chromatographed on
Sephadex G 75 and G 100; grey humic acids, which form a considerable part of this hurric
system [3], showed a marked chemical interaction with the Sephadex gels, and this interaction
was complicated by some inorganic salts normally present in extracts of this soil.
Haller [4] introduced the use of controlled pore glass (CPG) into chromatographic techniques.
Its advantages over organic gels are a rigid structure, which permits a five to ten times higher
flow-rate. Furthermore, its matrix, consisting of almost pure silica, is chemically inert against
many classes of substances. The material is commercially available with pore diameters from
70 up to 2000 Ä. It has been used successfully in many biochemical applications [5]. The aim of
the present work was to check the suitability of CPG for the chromatography of humic substances.
The criteria to be fulfilled in molecular exclusion chromatography of humic substances
are as follows:12

(1) Reproducible chromatograms.

(2) Complete elution of all humic substances within the fractionation range of the column.

221
222 DANNEBERG

(3) Grey humic adds (GHA) should be eluted before brown humic acids (BHA) and fulvic
acids (FA). The colour quotient

should therefore rise continuously from the value characteristic for GHA to that of BHA
and FA.
(4) Any humic substance should move through the column independently of the presence or
absence of any other humic substance. The chromatogram of a complex humic system
should therefore be identical with the sum of the chromatograms of all its components.
An independence from charge, counter ions, etc., can hardly be expected with humic
substances.

All these criteria except the first have been found to fail in the Sephadex experiments.

MATERIAL AND METHODS

The soil used was an Austrian chernozem from Fuchsenbigl, Marchfeld. It has been described
by Zeller and co-workers [6], and its humic system has been characterized in an earlier
publication [3].
The soil was extracted with a chelating resin and water (Chelex 100, obtained from Bio-Rad,
California) using a ratio of soil : resin : water = 1 : 2.5 : 2.5. The extract was centrifuged, and
10 ml samples were taken to prepare freeze-dried preparations that were later used in chroma­
tography. Similarly, the humic fractions FA, BHA and GHA and a humic acid (HA) were
prepared and stored in a freeze-dried state until use.
Fulvic acid was prepared after precipitation of 10 ml extract with HC1 at pH = 1. Humic acid
was centrifuged and washed with 0. IN HC1. The FA was precipitated from the supernatant with
Ba(OH)2 at pH = 6. The resulting precipitate was dissolved by shaking with Chelex and water,
filtered and freeze-dried.
Humic acid was obtained from the acidic precipitation, the residue was dissolved by shaking
with Chelex and water, filtered and freeze-dried.
Grey humic acid was precipitated from 10 ml extract by addition of an equal volume of a
24% solution of NaCl and subsequent centrifugation [7]. The residue was dissolved in water,
re-precipitated with NaCl, re-dissolved, precipitated with acid and washed with 0.1N HC1 until it was
free of excess N a\ It was then dissolved by shaking with Chelex and water, filtered and freeze-
dried.
From the supernatant of the NaCl-precipitation, brown humic acid was precipitated with
HC1 at pH = 1. The resulting precipitate was washed several times with 0.1N HC1, dissolved by
shaking with Chelex and water, and freeze-dried.
A glass column with a dimension of 18 X 1000 mm, filled with CPG of a pore diameter
of 156 A, was used for chromatography. The column, already filled with CPG, was obtained
commercially from Holzel, German Federal Republic. The column was equilibrated with distilled
water and ‘exclusion’ and ‘salt’-volume were determined with Blue Dextran 2000 (Pharmacia,
Sweden) and benzyl-alcohol respectively [8, 9]. Thereafter, the column was equilibrated with the
eluant, a solution 0.02M in Na2B40 7 and 0.05M in NaCl. Because of its rigid structure, CPG does
not swell or shrink during this change in the ionic strength of the medium.
Two types of chromatographic experiments were carried out:
Type 1: The soil extract was chromatographed to obtain a series of chromatographic fractions.
Any of these was precipitated with 2M NaCl and with HC1 at pH = 1. For this purpose, 100 pi of
concentrated HC1 were added to one series of chromatographic fractions and 2.5 ml of 24%
 IAEA-SM-211/54 223

Ax
+ ■F7

F I G .l. R e p r o d u c ib ility o f c h r o m a to g r a m s o f th e o r ig in a l e x tr a c t. F o u r e x p e r im e n ta l r u n s ( s y m b o ls ) a n d c a lc u la te d

m e a n -c h r o m a to g ra m ( s o lid lin e ). A l l c h r o m a t o g r a m s c o n s t i t u t e p l o t s o f E 400 v e r s u s K ^ . \

C>
0
>
0
0

F I G .2 . M e a n - c h r o m a to g r a m s o f th e o r ig in a l e x tr a c t ( s o lid lin e ) a n d c o lo u r q u o t i e n t (tr ia n g le s ). F o r th e c o lo u r

q u o tie n t, th e d is ta n c e o n th e o r d in a te a x is m u s t b e m u ltip lie d b y 10. T h e c h r o m a t o g r a m s c o n s t i t u t e p l o t s o f E 40Q

versu s K d.
224 DANNEBERG

NaCl-solution to a second series. After centrifugation, the humic fractions FA and FA + BHA
respectively remained in solution. GHA, precipitated with NaCl, was re-dissolved by adding
2.5 ml of eluant solution to the residue. Thus the amount of GHA, BHA and FA in the chromato­
graphic fractions was determined.
Type 2: The soil extract was fractionated by precipitation and the humic fractions GHA,
BHA, FA and HA were prepared as outlined above. These humic fractions were chromatographed
separately.
For chromatography, the freeze-dried preparations were dissolved in 10 ml of eluant solution
to obtain the original concentration. One millilitre of this solution was injected into the top of
the column with a syringe and eluted descendingly using a flow-rate of 1 ml/min. One hundred
chromatographic fractions of 2.5 ml were collected, and extinctions were read at 400 and 600 nm
on a Zeiss spectrophotometer. E400 was taken as a measure for the relative concentration of
humic substances, and the colour quotient was calculated from both extinctions. A computer
program was used to convert the results to normalized peak positions (Kd), to calculate mean
chromatograms from several runs and sums of chromatograms from several humic fractions.

RESULTS AND DISCUSSION

When the original extract was chromatographed, a small part of the humic system was excluded
from the pores of CPG, forming a peak at a Kd of 0. Most of the humic system, however, penetrated
the pores and was eluted as a broad plateau, ranging up to a Kd of 0.55; thereafter, the concen­
tration of humic substances decreased to zero at a Kd of 0.8 (Fig.l). Thus there was no part
of the humic system which had been retarded for more than the total pore volume of the column.
The elution was always complete, from 95 to 103% of coloured substances being found in the
eluant. A fairly good reproducibility was obtained, as can be seen from Fig. 1.
The colour quotient (Q4 0 0 / 6 0 0 ) started at a value of approximately 3, which is characteristic of
GHA; from a ^ of 0.3 the colour quotient increased and reached a value of 9 at the end of the
chromatogram (Fig.2). Thus the colour quotient showed the pattern that was to be expected if
GHA were eluted before BHA and FA.

FIG.3. Chromatograms offulvic acids, obtained by Type 1 experiments (solid line), and by Type 2 experiments
(triangles). The chromatograms constitute plots of E400versus Kd.
 IA E A -SM -211/54 225

F I G .4 . C h r o m a to g r a m s o f b r o w n h u m ic a c id s o b ta in e d b y T y p e 1 e x p e r im e n ts ( tr ia n g le s ) a n d b y T y p e 2 e x p e r i­

m e n t s (s o lid lin e ). T h e c h ro m a to g ra m s c o n s titu te p lo ts o f versu s K A .

Fulvic acids were eluted at a Kj between 0.5 and 0.6 (Fig.3). When determined after experi­
ments of Type 1 or Type 2, the peak positions were almost identical. The peak areas, however,
were different. In a Type 1 experiment, higher concentrations of FA were always obtained than in
an experiment of Type 2. The reason for this is probably twofold: The precipitation of FA with
Ba(OHh is probably not complete, and in a Type 1 experiment some breakdown of BHA might
have occurred which resulted in an increase of FA [3, 10]. Accordingly, the brown humic
acids gave a smaller peak area after a Type 1 than a Type 2 experiment (Fig.4). The peak positions,
however, were identical also in the case of BF1A, the Kd being near 0.5.
In the case of GHA, the two types of experiments produced different peak positions and
different peak areas. A Type 1 experiment resulted in a peak around a of 0.1, and a Type 2
experiment gave a much larger peak, situated around a Kd of 0.4 (Fig.5).
When a sum curve of the humic fractions obtained after a Type 1 experiment is compared with
the original extract (Fig.6), a fairly good agreement can be noticed. A loss of substance occurred in
in the region of a Kd of 0, but this is surely due to the fact that part of the GHA was rendered
insoluble by the NaCl-precipitation. An insoluble residue was noticed in the fractions of this
region, when the GHA were re-dissolved. On the other hand, when a sum curve of the humic
fractions GHA and BHA obtained after a Type 2 experiment is plotted (Fig.7), one can see a
good agreement of this sum with the chromatogram of HA.
Summarizing these results, one arrives at the conclusion that all the criteria postulated have
been fulfilled in these chromatographic experiments. In both types of experiments, the chromato­
grams of more complex systems agreed well with the sums of the chromatograms of all their
subfractions. Furthermore, the expected sequence of elution was regularly confirmed. Thus, the
chromatography of humic substances on CPG seems to be governed solely by a molecular exclusion
mechanism without a chemical or physical interaction of humic substances with the matrix.
GHA gave different chromatograms after the two types of experiments. Also in this case,
however, the sum of the chromatograms of two subfractions agreed well with the chromatogram
of the original mixture, the HA. Therefore these differences cannot be caused by artefacts produced
during the chromatographic procedure; they must come from the preparation of GHA and HA.
226 DANNEBERG

F I G .5 . C h r o m a to g r a m s o f g r e y h u m ic a c id s o b ta in e d b y T yp e I e x p e r i m e n t s (s o l i d l i n e ) a n d b y T y p e 2 e x p e r im e n ts

( tr ia n g le s ) . T h e c h ro m a to g ra m s c o n s titu te p lo ts o f E w о v ersu s K ^.

F I G .6 . C o m p a r is o n o f t h e s u m c u r v e (s o lid lin e ) o b t a in e d f r o m a ll T y p e 1 e x p e r i m e n t s w i th t h e c h r o m a to g r a m
o f th e o r ig in a l e x tr a c t. T h e c h r o m a to g r a m s c o n s ti tu t e p l o t s о /Ё ю о v e rs u s K j .
 IA E A -SM -211/54 227

F IG . 7. C o m p a r is o n o f th e s u m cu rve (s o lid lin e ) o f b r o w n a n d g r e y h u m ic a c id o b ta in e d b y T y p e 2 e x p e r im e n ts

w ith th e c h r o m a to g r a m o f h u m ic a c id . T h e c h r o m a t o g r a m s c o n s t i t u t e p l o t s o f Е 400 v e r s u s К д .

Some of the preparative steps used (re-precipitation, drying and re-dissolving) are known to reduce
the ash content of humic substances [1 j. This might be one reason for the lower molecular weight
observed, since sub-units can be split off when metal ions are separated from humic substances [10].
The structural change that happened with the GHA must have resulted in an increase in colour
intensity. This hyperchromic effect is well-known in nucleic acid chemistry [11]. The nature of
this structural change, however, is beyond the scope of this work and will require further
investigations.

REFERENCES

[1] DUBACH, D., MEHTA, N.C., The chemistry o f soil humic substances, Soils and Fertilizers 26 (1963) 293.
[2] MEHTA, N.C., DUBACH, P., DEUEL, H., Untersuchungen über die Molekulargewichtsverteilung von
Huminstoffen durch Gelfiltration an Sephadex, Z.Pflanzenemaehr.Bodenkd. 102 (1963) 128. !
[3] DANNEBERG, O.H., SCHAFFER, K., Eine einfache kolorimetrische Analyse des Huminstoffsyst!ems,
Bodenkultur 25 (1974) 360.
[4] HALLER, W., Chromatography on glass o f controlled pore size, Nature (London) 206 (1965) 693.
[5] HALLER, W. (Ed.), Bibliography on Controlled Pore Glass Chromatography and Related Subjects, Pooks Hill,
Bethseda, Maryland (1974).
[ 6 ] ZELLER, A., OBERLÄNDER, H.E., ROTH, K., A field experiment on the influence of cultivation practices
on the transformation o f l4 C-labelled farmyard manure and 1 4 C-labelled straw into humic substances,
Isotopes and Radiation in Soil Organic-Matter Studies (Proc. Symp. Vienna, 1968), IAEA, Vienna (1968) 265.
[7] FLAIG, W., SCHEFFER, F., KLAMROTH, B., Zur Kenntnis der Huminsäuren: VIII. Zur Charakterisierung
der Huminsäuren des Bodens, Z.Pflanzenemaehr.Bodenkd. 71 (1955) 33.
[ 8 ] GSCHWENDER, H.H., HALLER, W., HOFSCHNEIDER, P.H., Large scale preparation of viruses by steric
chromatography on columns o f controlled pore glass, Biochim. Biophys. Acta 190 (1969) 460.
228 D ANNEBERG

[9] FRISCH-NIGGEMEYER, W., HEINZ, F., STEMBERGER, H., Eine schnelle und verlässliche Methode zur
Erkennung frischer bzw. älterer Infektionen mit Rubella-Virus, Immunität Infektion 2 (1974) 231.
[10] DANNEBERG, O.H., Über die Extraktion von Huminstoffen aus Schwarzerde, Bodenkultur 24 (1973) 111.
[11] SHUGAR, C., “Photochemistry o f nucleic acids”, The Nucleic Acids 3 (CHARGAFF, E., DAVIDSON, J.N., Eds),
Academic Press, New York, London (1960) 54.

DISCUSSION

B.R. NAGAR: Have you done any basic work with standard substances so that you are
sure that no phenomena of adsorption or the like are taking place?
O.H. DANNEBERG: Except for Blue Dextran and benzyl alcohol, which were used to
determine the ‘exclusion’ and ‘salt’-volume respectively, no standard substances were used. But
as we found a 100% recovery of the coloured substances, we are quite sure that no great adsorption
has taken place.
B.G. VOLK: What was the percentage of ash in the material you were analysing?
Do you have any idea of the Fe, Al, or metal content of the ash, and what effects do you think
these metals have on the separations you obtained?
O.H. DANNEBERG: The methods of extraction used produced a material with a high ash
content, some 50%. Furthermore, some carbonate was always co-extracted from the soil containing
carbonate. We recorded the electrical conductivity of the eluant, and found no influence of ash and
salt content of the sample on the elution behaviour, in contrast to the results obtained with
Sephadex.
 IAEAJ s M -211/9

R e v ie w p a p er
I
I
R O L E O F O R G A N IC M A T T E R
IN V O L A T IL IZ A T IO N O F S U L P H U R
A N D N IT R O G E N F R O M S O IL S *

J.M. BREMNER
Department of Agronomy,
Iowa State University,
Ames, Iowa,
United States of America

Abstract

ROLE OF ORGANIC MATTER IN VOLATILIZATION OF SULPHUR AND NITROGEN FROM SOILS.

The importance o f organic matter in volatilization o f sulphur and nitrogen from soils has'been demonstrated
by work supporting the following conclusions: (1) Most o f the sulphur volatilized from soils under aerobic or
waterlogged conditions is in the form o f dimethyl sulphide, dimethyl disulphide and methyl mercaptan produced
by microbial decomposition o f methionine in organic matter; (2) Volatilization of nitrogen from soils through
denitrification o f nitrate under anaerobic conditions is controlled largely, if not entirely, by the supply [>f readily
decomposable organic matter; (3) Organic matter plays a key role in the processes responsible for gasedus loss of
nitrogen through chemical decomposition o f nitrite in soils (chemidenitrification) and volatilization of ammonium
from soils treated with urea. Work reviewed indicates the need for reassessment o f the role of soils in the atmospheric
sulphur and nitrogen cycles, and for consideration o f the possibility that soil may be an important natural sink for
sulphur and nitrogen gases identified as important atmospheric pollutants.

INTRODUCTION
R e c e n t a r t i c l e s c o n c e r n in g th e r o l e o f s o i l s i n a tm o s p h e r ic p o l l u t i o n
h a v e e m p h a s iz e d t h e in a d e q u a c y o f o u r k n o w le d g e o f p r o c e s s e s le a d i n g jto
v o l a t i l i z a t i o n o f n i t r o g e n a n d s u l f u r fr o m s o i l s , and t h e n e e d f o r much
b e t t e r i n f o r m a t i o n c o n c e r n in g t h e a m o u n ts and fo r m s o f N and S r e le a s e d
fr o m s o i l s t o th e a tm o s p h e r e .

T h e p u rp o s e o f t h i s p a p e r i s t o s u m m a riz e r e c e n t w o r k a t Io w a S t a t e
U n i v e r s i t y s h o w in g t h a t o r g a n i c s o i l c o n s t i t u e n t s p l a y a k e y r o l e i n !th e
p r o c e s s e s le a d i n g t o v o l a t i l i z a t i o n o f S an d N fr o m s o i l s , and t o draWj
a t t e n t i o n t o w o r k i n d i c a t i n g t h a t s o i l may be a s i n k as w e l l a s a s o u r c e
o f a t m o s p h e r ic S a n d N g a s e s i d e n t i f i e d as m a jo r a i r p o l l u t a n t s .

VOLATILIZATION OF SULFUR FROM SOILS

A lt h o u g h many s t u d i e s o f v o l a t i l i z a t i o n o f N fr o m s o i l s h a v e b e e n
r e p o r t e d , fe w a t t e m p t s h a v e b e e n made t o s t u d y v o l a t i l i z a t i o n o f S fi^om
s o i l s , a n d i t i s d i f f i c u l t t o f i n d a n y j u s t i f i c a t i o n i n th e l i t e r a t u r e
f o r c u r r e n t c o n c e p ts o f t h e r o l e o f s o i l s i n th e a tm o s p h e r ic S c y c l e . !
T h e s e c o n c e p ts a r e b a s e d o n t h e a s s u m p tio n t h a t v o l a t i l i z a t i o n o f S fr o m
s o i l s o c c u r s t h r o u g h f o r m a t i o n o f H2S b y m i c r o b i a l r e d u c t i o n o f s u l f a t e

»
* Work was supported in part by the Rockefeller Foundation, and by the US Energy Research arid
Development Administration (Contract E(1 l-l)-2 5 3 0 ).

229
230 BREMNER

TABLE I. VOLATILE S COMPOUNDS SEPARATED BY GAS CHROMATOGRAPHIC

TECHNIQUES USED IN WORK REPORTED

Name Formula

Hydrogen sulfide H2S

Sulfur dioxide s o 2

Carbon disulfide c s 2

Carbonyl sulfide c o s

Sulfur hexafluoride SF 6
Methyl mercaptan (methanethiol) CH3 SH
Ethyl mercaptan (ethanethiol) CH3CH2SH
n -Propyl mercaptan ( 1 -propanethiol) CH3 CH2 CH2SH
л-Butyl mercaptan (1-butanethiol) CH3 CH2 CH2 CH2SH
й о -Butyl mercaptan (2-methyl-l-propanethiol) (CH jIjCHCHjSH
Dimethyl sulfide (methylthiomethane) CHjSCHj
Dimethyl disulfide (methyldithiomethane) CH3 SSCH3
Ethyl methyl sulfide (methylthioethane) CH3 CH2 SCH3
Diethyl sulfide (ethylthioethane) CH3CH2 SCH2 CH3
Diethyl sulfide (ethyldithioethane) CH3CH2 SSCH2 CH3

o r d e g r a d a t i o n o f o r g a n ic S com pounds, and t h a t a s u b s t a n t i a l am ount o f

t h e SOo fo u n d i n t h e a tm o s p h e re i s p ro d u c e d b y a tm o s p h e r ic o x i d a t i o n o f
H2S r e l e a s e d fro m s o i l s . A tte m p ts t o v a l i d a t e t h e s e a s s u m p tio n s h a v e
b e e n g r e a t l y h i n d e r e d b y t h e l a c k o f s e n s i t i v e and s p e c i f i c m eth o d s f o r
i d e n t i f i c a t i o n and e s t i m a t i o n o f v o l a t i l e S com pounds. T h is p ro b le m
h a s b e e n overcom e b y t h e r e c e n t d e v e lo p m e n t (1 ) o f g a s c h ro m a to g ra p h ic
te c h n i q u e s t h a t p e r m i t i d e n t i f i c a t i o n and e s t i m a t i o n o f t r a c e (n an o g ram )
am ounts o f m e r c a p ta n s , a l k y l s u l f i d e s and o t h e r v o l a t i l e s u l f u r
com pounds known t o be p ro d u c e d b y m ic ro o rg a n is m s (T a b le I ) . T hese
te c h n i q u e s in v o lv e u s e o f T e f l o n colum ns and a fla m e p h o to m e tr ic
d e t e c t o r f i t t e d w i t h a s u l f u r f i l t e r , and th e y a r e n o t s u b j e c t t o
i n t e r f e r e n c e b y n o n s u l f u r g a s e s e v o lv e d fro m s o i l s u n d e r a e r o b i c o r
a n a e r o b ic c o n d i t i o n s . F o llo w in g i s a summary o f r e c e n t w o rk i n w h ic h
t h e s e te c h n i q u e s w ere u se d t o s tu d y v o l a t i l i z a t i o n o f S fro m s o i l s and
amended s o i l s u n d e r a e r o b i c and w a te r lo g g e d c o n d i t i o n s .

V olatilization o f sulfur from unam ended soils

The p o s s i b i l i t y t h a t s i g n i f i c a n t v o l a t i l i z a t i o n o f S c a n o c c u r from
unam ended s o i l s h a s b e e n s u g g e s te d by S b a la n c e s t u d i e s r e p o r t e d by
N ic o ls o n ( 2 ), and b y t h e d e t e c t i o n o f d im e th y l s u l f i d e i n g a s e s e v o lv e d
from unam ended s o i l s ( 3 ) . To t e s t t h i s p o s s i b i l i t y , we s t u d i e d r e l e a s e
o f v o l a t i l e S fro m 25 unam ended s o i l s s e l e c t e d t o o b t a i n a w id e ra n g e i n
p r o p e r t i e s ( 4 ) . No r e l e a s e o f v o l a t i l e S w as d e t e c t e d w hen 11 o f t h e s e
s o i l s w e re in c u b a t e d u n d e r a e r o b i c o r w a te r lo g g e d c o n d i t i o n s . F o u r te e n
s o i l s r e l e a s e d v o l a t i l e S com pounds when in c u b a ta d u n d e r w a te r lo g g e d
c o n d i t i o n s , b u t o n l y f o u r o f t h e s e s o i l s r e l e a s e d v o l a t i l e S compounds
w hen in c u b a t e d u n d e r a e r o b i c c o n d i t i o n s . W here r e l e a s e o f v o l a t i l e S
 IAEA -SM -211/9 231

w as o b s e r v e d , t h e a m o u n t o f S v o l a t i l i z e d a t 30 °C i n 6 0 d a y s u n d e r a e r o b i c
o r w a t e r lo g g e d c o n d i t i o n s d i d n o t a c c o u n t f o r m o re t h a n 0.0 17* o f t h e t o t a l
S i n th e s o i l s s tu d ie d . T h e v o l a t i l e S d e t e c t e d w as i d e n t i f i e d a s ]
d i m e t h y l s u l f i d e , o r a s d i m e t h y l s u l f i d e a s s o c ia t e d w i t h s m a ll e r am ounjts
o f c a r b o n y l s u l f i d e , c a r b o n d i s u l f i d e , m e t h y l m e rc a p ta n a n d ( o r ) d i m e t h y l
d is u lfid e . H y d ro g e n s u l f i d e c o u ld n o t be d e t e c t e d i n a n y o f th e s o i l ;
a tm o s p h e re s a n a ly z e d .

Volatilization of sulfur from amended soils

T o d e te r m in e i f v o l a t i l i z a t i o n o f S fr o m s o i l s i s p ro m o te d b y a d d i t i o n
o f S - c o n t a i n i n g m a t e r i a l s , a n d t o i d e n t i f y s o u r c e s o f v o l a t i l e S r e le a s e d
fr o m s o i l s , we r e c e n t l y s t u d i e d v o l a t i l i z a t i o n o f S fr o m Io w a s o i l s
i n c u b a t e d u n d e r a e r o b ic and w a t e r lo g g e d c o n d i t i o n s a f t e r t r e a t m e n t w i t h
v a r i o u s i n o r g a n i c and o r g a n i c fo r m s o f S ( 4 - 6 ) . The i n o r g a n i c fo r m s o f
S used in c lu d e d s u l f a t e , s u l f i t e , s u l f i d e , t h i o s u l f a t e , t e t r a t h io n a t e
and e le m e n t a l S , a n d th e o r g a n i c fo r m s in c l u d e d S - c o n t a i n i n g a m in o a c id s
and p r o t e i n s , t h io c y a n a t e s and i s o t h i o c y a n a t e s , s u l f a t e e s t e r s ( o r g a n i c
s u l f a t e s ) , p l a n t r e s id u e s , a n im a l m a n u re s and sew age s lu d g e s .

O u r c o n c lu s io n s fr o m th e s e s t u d i e s c a n b e s u m m a riz e d as f o l l o w s :
1) V o l a t i l i z a t i o n o f S fr o m unam ended o r am ended s o i l s o c c u r s th r o u g h
f o r m a t i o n o f CH SH, CH3SCH3 , CH3SSCH3 , CS2 and COS b y m i c r o b i a l
d e c o m p o s it io n o r o r g a n i c S c o m p o u n d s .
2) M o s t o f t h e S v o l a t i l i z e d fr o m s o i l s u n d e r a e r o b ic o r w a t e r lo g g e d
c o n d i t i o n s i s i n t h e f o r m o f C H .S H , CH SCH an d CH„SSCH„ d e r i v e d
fr o m m e t h io n in e . The r e m a in d e r J i s i n ?he f o r m o f 3so ÖS2 d e r i v e d
fr o m c y s t i n e ( o r c y s t e i n e ) and o f COS, w h ic h i s p r o b a b ly d e r i v e d
fr o m t h io c y a n a t e s o r i s o t h io c y a n a t e s i n p l a n t r e s id u e s . V e ry l i t t l e ,
i f a n y , o f th e S v o l a t i l i z e d fr o m s o i l s i s i n th e fo r m o f H 2S .
3) V o l a t i l i z a t i o n o f S fr o m s o i l s i s i n s i g n i f i c a n t c o m p a re d w i t h
v o la t iliz a t io n o f N.

I t s h o u ld be n o te d t h a t , a lt h o u g h th e s e c o n c lu s io n s a r e b a s e d o n
s t u d i e s w i t h a w id e ra n g e o f s o i l s , t h e y may n o t a p p ly t o some t y p e s o f
s o ils . F o r e x a m p le , o u r c o n c lu s i o n t h a t v e r y l i t t l e , i f a n y , S i s
v o l a t i l i z e d fr o m s o i l s a s H 2S may n o t a p p ly t o h i g h l y o r g a n i c o r s u l f i d e -
r ic h s o ils .

T y p i c a l r e s u l t s o f t h e w o r k d is c u s s e d a r e r e p o r t e d i n T a b le I I ,
w h ic h show s th e fo r m s and a m o u n ts o f v o l a t i l e S e v o lv e d w h e n s a m p le s o f
a s a n d y D i c k i n s o n s o i l w e re in c u b a t e d (3 0 ° C ) u n d e r a e r o b i c o r w a t e r lo g g e d
c o n d i t i o n s f o r 6 0 d a y s a f t e r t r e a t m e n t w i t h v a r io u s i n o r g a n i c and
o r g a n i c fo r m s o f S (4 0 0 p g S /g s o i l ) . Unam ended s a m p le s o f t h i s s o i l
d i d n o t r e le a s e d e t e c t a b l e a m o u n ts o f v o l a t i l e S . I t i s n o te w o rth y t h a t
n o r e le a s e o f v o l a t i l e S c o u ld b e d e t e c t e d fr o m th e s a m p le s am ended W it h
i n o r g a n i c fo r m s o f S ,a n d t h a t H 2S c o u ld n o t b e d e t e c t e d i n a n y o f th e
s o i l a tm o s p h e re s a n a ly z e d .

O u r f a i l u r e t o d e t e c t r e le a s e o f H2S fr o m unam ended s o i l s o r fr o m

s o i l s t r e a t e d w i t h i n o r g a n i c o r o r g a n i c fo r m s o f S i s o f p a r t i c u l a r
i n t e r e s t b e c a u s e , a s n o te d p r e v i o u s l y , c u r r e n t c o n c e p ts o f th e r o l e o f
s o i l s i n t h e a t m o s p h e r ic S c y c l e a r e b a s e d o n th e a s s u m p tio n t h a t S i s
v o l a t i l i z e d fr o m s o i l s as H2S ,a n d t h a t a s u b s t a n t i a l a m o u n t o f t h e SO2
fo u n d i n t h e a tm o s p h e re i s p r o d u c e d b y a t m o s p h e r ic o x i d a t i o n o f H2S
r e le a s e d fr o m s o i l s . T h e re i s n o d o u b t t h a t H2S i s p r o d u c e d i n s o i l s
b y m i c r o b i a l r e d u c t i o n o f s u l f a t e and d e g r a d a t i o n o f o r g a n i c S
c o m p o u n d s , b u t o u r w o r k i n d i c a t e s t h a t H 2S th u s p r o d u c e d i s s o r b e d
v e r y r a p i d l y b y s o i l c o n s t i t u e n t s a n d d o e s n o t e s c a p e t o th e a tm o s p h e re .
232 BREMNER

TABLE II. VOLATILE S COMPOUNDS EVOLVED FROM A SANDY SOIL INCUBATED

UNDER AEROBIC OR WATERLOGGED CONDITIONS AFTER TREATMENT WITH
DIFFERENT FORMS OF S

Volatile S compounds evolved S evolved

calculated
Form o f S added H2S CH3SH CH3SCH3 CH3SSCH3 cos cs2 as % of S added

Inorganic forms a - - - - - - 0

Organic forms:
Methionine + + + 4 9 -5 0
Cystine - - - - - + 1 -2
Cysteine - - - - - + 1 -2
Sulfate esters - - - - - - 0
Thiocyanates and
_ _ _ _ _

©
isothiocyanates +
Plant proteins - + + + + + 2—3C
Plant residues - +b + +b - +b 0 .1 -0 .3 °
Animal manures - 4- + + - - 0 .1 -0 .5
Sewage sludges - +b +b + +b + 0.1 -0 .4 °

a Sulfate, sulfide, sulfite, th iosulfate, tetrathionate and elem ental S.

b N ot d etected under aerobic conditions.
c 9 2 —99% o f the S evolved was in the form o f CH3SH, CH3SCH3 or CH3SSCH3.

S u p p o r t f o r t h i s c o n c lu s i o n h a s b e e n p r o v id e d b y r e c e n t w o r k i n o u r
l a b o r a t o r y ( 7 , 8) s h o w in g t h a t s o i l s s o r b H2S and S0 2 v e r y r a p i d l y and
h a v e l a r g e c a p a c i t i e s f o r s o r p t i o n o f th e s e g a s e s . S o i l s a l s o h a v e th e
c a p a c i t y t o s o r b CH3SCH3, CH3SSCH3, CH3SH, CS2 and COS, b u t t h e i r
c a p a c i t y f o r s o r p t i o n o f th e s e g a s e s i s much s m a ll e r t h a n t h e i r c a p a c i t y
f o r s o r p t i o n o f H2 S o r S0 2 ( 8) . S o r p t i o n o f CH3SH, CH3SCH3, CH3SSCH3,
CS2 and COS b y s o i l s i n v o l v e s m i c r o b i a l p r o c e s s e s t h a t a r e much s lo w e r
th a n th e n o n b i o l o g i c a l p r o c e s s e s r e s p o n s i b l e f o r s o r p t i o n o f I ^ S and

The m o s t im p o r t a n t o v e r a l l c o n c lu s i o n fr o m o u r s t u d i e s o f th e
a b i l i t y o f s o i l s t o r e le a s e an d s o r b S g a s e s i s t h a t t h e r e i s go od
r e a s o n t o q u e s t i o n t h e v a l i d i t y o f th e c u r r e n t b e l i e f t h a t s o i l i s an
i m p o r t a n t s o u r c e o f a t m o s p h e r ic H2S and S02 . I t seems much m ore l i k e l y
t h a t s o i l i s a n i m p o r t a n t n a t u r a l s i n k f o r th e s e g a s e s .

VOLATILIZATION OF NITROGEN FROM SOILS

R e s e a rc h o n v o l a t i l i z a t i o n o f N fr o m s o i l s h a s b e e n g r e a t l y
s tim u la te d b y : 1) i n t e r n a t i o n a l a p p r e c i a t i o n o f t h e k e y r o l e o f
f e r t i l i z e r N i n w o r l d fo o d p r o d u c t io n ; 2 ) a c c u m u la t io n o f e v id e n c e
t h a t a s u b s t a n t ia l am ount o f th e f e r t i l i z e r N a p p lie d to s o i l s i s l o s t
t o th e a tm o s p h e r e ; 3 ) r e c e n t s u b s t a n t i a l in c r e a s e s i n t h e c o s t o f
f e r t i l i z e r N ; a n d 4 ) r e c e n t c la im s t h a t N v o l a t i l i z e d fr o m s o i l s as
n i t r o u s o x id e (N 20 ) may h a v e a s e r io u s e f f e c t o n th e e a r t h ' s o z o n e
s h ie ld .
 IA EA -SM -211/9 233

U n t i l a b o u t I9 6 0 , i t w as g e n e r a l l y assum ed t h a t v o l a t i l i z a t i o n o f
N fr o m s o i l s o c c u r s l a r g e l y , i f n o t e n t i r e l y , b y d e n i t r i f i c a t i o n o f
n i t r a t e an d v o l a t i l i z a t i o n o f am m onium . S in c e t h a t t i m e , th e p o s s i b i l i t y
t h a t s i g n i f i c a n t g a s e o u s l o s s o f f e r t i l i z e r N may o c c u r t h r o u g h c h e m ic a l
d e c o m p o s it io n o f n i t r i t e i n s o i l s ( i . e . c h e m o d e n i t r i f i c a t i o n ) h a s
r e c e iv e d a t t e n t i o n . I n r e c e n t y e a r s , h o w e v e r, r e s e a r c h o n v o l a t i l i z a t i o n
o f N fr o m s o i l s h a s c e n t e r e d l a r g e l y o n d e n i t r i f i c a t i o n , b e c a u s e i t i s
now g e n e r a l l y b e li e v e d t h a t m i c r o b i a l r e d u c t i o n o f n i t r a t e t o N 2 and N2O
b y d e n i t r i f y i n g b a c t e r i a i s th e m a jo r m e c h a n is m o f g a s e o u s l o s s o f N
fr o m s o i l s . Im p e tu s t o r e s e a r c h o n d e n i t r i f i c a t i o n h a s b e e n a d d e d b y
t h e r e c e n t h y p o t h e s is t h a t N 2O r e le a s e d t o th e a tm o s p h e re th r o u g h
d e n i t r i f i c a t i o n o f n i t r a t e i n s o i l s and n a t u r a l w a t e r s may t r i g g e r
r e a c t i o n s i n th e s t r a t o s p h e r e l e a d i n g t o p a r t i a l d e s t r u c t i o n o f th e
o z o n e l a y e r p r o t e c t i n g th e e a r t h fr o m b i o l o g i c a l l y h a r m f u l e f f e c t s 6f
u l t r a v i o l e t r a d i a t i o n fr o m th e s u n ( 9 ) . T h is h y p o t h e s is h a s c r e a t e d
i n t e r n a t i o n a l c o n c e r n t h a t in c r e a s e d u s e o f N f e r t i l i z e r s may in c r e a s e
r e le a s e o f N 2O t o th e a tm o s p h e re b y d e n i t r i f i c a t i o n o f f e r t i l i z e r -
d e r i v e d n i t r a t e i n s o i l s and n a t u r a l w a te r s , and t h e r e b y p ro m o te
d e s t r u c t i o n o f th e o z o n e l a y e r ( 10) .

D enitrification

A lt h o u g h t h e r e a r e r e p o r t s i n th e l i t e r a t u r e o f t h e o c c u r r e n c e o f
d e n i t r i f i c a t i o n i n s o i l s u n d e r a p p a r e n t l y a e r o b i c c o n d i t i o n s , i t i s now
g e n e r a l l y a c c e p te d t h a t t h e d e n i t r i f i c a t i o n o b s e rv e d u n d e r s u c h c o n d i t i o n s
o c c u r r e d a t a n a e r o b ic s i t e s , a n d t h a t d e n i t r i f i c a t i o n d o e s n o t o c c u r u n t i l
th e o x y g e n s u p p ly i s s o r e s t r i c t e d t h a t t h e d e n i t r i f y i n g b a c t e r i a c a n n o t
o b t a i n e n o u g h o x y g e n t o m e e t t h e i r r e q u ir e m e n t s . T h e re i s g o o d r e a s o n t o
b e l i e v e t h a t th e s u p p ly o f r e a d i l y d e c o m p o s a b le o r g a n i c m a t t e r i s a l s o a
c r i t i c a l f a c t o r b e c a u s e a n o x i d i z a b l e s u b s t r a t e i s r e q u i r e d f o r th e
g r o w th o f d e n i t r i f y i n g b a c t e r i a , a n d i t i s w e l l e s t a b l i s h e d t h a t a d d i t i o n
o f r e a d i l y d e c o m p o s a b le o r g a n i c m a t e r i a l s t o s o i l s in c r e a s e s th e
m i c r o b i a l demand f o r o x y g e n an d t h e r e b y in d u c e s d e v e lo p m e n t o f a n a e r o b ic
c o n d i t i o n s p r o m o t in g th e a c t i v i t y o f d e n i t r i f y i n g b a c t e r i a .

B re m n e r a n d Shaw ( 1 1 ) fo u n d t h a t t h e e f f e c t s o f o r g a n i c m a t e r i a l s o n
d e n i t r i f i c a t i o n i n w a t e r lo g g e d s o i l s v a r i e d w i t h t h e i r r e s i s t a n c e t o
d e c o m p o s it io n , e a s i l y d e c o m p o s a b le s u b s ta n c e s s u c h a s g lu c o s e and
m a n n i t o l h a v in g much g r e a t e r e f f e c t s th a n substances decom posable w ith
d if f ic u lt y s u c h a s l i g n i n an d s a w d u s t. T h e y a l s o fo u n d t h a t th e a b i l i t y
o f w h e a t o r o a t s t r a w t o p ro m o te d e n i t r i f i c a t i o n i n w a t e r lo g g e d s o i l s
w as g r e a t l y r e d u c e d w h e n th e s e m a t e r i a l s w e re e x t r a c t e d w i t h w a t e r o r
a llo w e d t o de c o m p o s e b e f o r e u s e as e n e r g y s o u r c e s f o r d e n i t r i f y i n g
b a c te r ia . The d e d u c t io n fr o m th e s e o b s e r v a t io n s t h a t d e n i t r i f i c a t i o n i n
s o i l s u n d e r w a t e r lo g g e d c o n d i t i o n s may be c o n t r o l l e d b y t h e s u p p ly o f
r e a d i l y d e c o m p o s a b le o r w a t e r - s o l u b l e o r g a n i c m a t t e r i s s u p p o r t e d b y
r e c e n t w o r k i n o u r l a b o r a t o r y ( 1 2 ) s h o w in g t h a t t h e c a p a c i t y o f Io w a
s u r f a c e s o i l s f o r d e n i t r i f i c a t i o n o f n i t r a t e u n d e r a n a e r o b ic c o n d i t i o n s
i s v e r y s i g n i f i c a n t l y c o r r e l a t e d ( r = 0 .99***) w i t h t h e i r c o n t e n t o f
w a t e r - s o l u b l e o r r e a d ily - d e c o m p o s a b le o r g a n i c m a t t e r .

A c o m p a r is o n now i n p r o g r e s s ( 1 3 ) o f th e e f f e c t s o f m o re th a n 50
o r g a n i c s u b s ta n c e s o n d e n i t r i f i c a t i o n o f n i t r a t e i n s o i l s h a s sho w n I t h a t
w a t e r - s o l u b l e s u b s ta n c e s o f l o w - m o le c u la r w e i g h t h a v e m uch g r e a t e r e f f e c t s
th a n h i g h - m o l e c u l a r w e i g h t s u b s ta n c e s h a v in g lo w s o l u b i l i t y i n w a t e r .
T h is c o m p a r is o n h a s a l s o sho w n t h a t o r g a n i c s u b s ta n c e s h a v in g a lm o s t
id e n t ic a l e f f e c t s on th e r a te o f d e n i t r i f i c a t i o n o f n i t r a t e in s o ils can
h a v e v e r y d i f f e r e n t e f f e c t s o n t h e r a t i o o f N 2O /N 2 i n th e g a s e s p r o d u c e d
b y d e n i t r i f i c a t i o n , a n d t h a t t h i s r a t i o i s g r e a t ly a f f e c t e d b y th e am ount
234 BREMNER

TABLE III. NITROGEN ISOTOPE DISCRIMINATION IN DENITRIFICATION OF NITRATE

IN SOILS a

Incubation tim e Nitrate N in soil

Soil (days)
A m ount (m g) A1SN value

N icollet 0 1 0 .0 0
1 8 .7 +3
3 6.0 +5
5 3.3 + 1 6

Webster 0 10.0 0
1 8.6 + 9

3 3.0 +19

5 2.2 +20

Harpster 0 10.2 0
1 6.4 +7
3 3 .6 +15
5 3.2 +15

S oil (1 0 g) treated w ith 6 ml w ater containing 10 mg N as K N 0 3 and 7.5 mg C as glucose was incubated
(3 0 °C ) under anaerobic con d ition s (helium atm osphere) for 0, 1, 3 and 5 days.

o f r e a d i l y d e c o m p o s a b le o r g a n i c m a t t e r a d d e d . T h e s e f i n d i n g s h a v e an
o b v io u s b e a r i n g o n c u r r e n t a t t e m p t s t o a s s e s s t h e N 2O /N 2 r a t i o i n th e
g a s e s r e le a s e d b y d e n i t r i f i c a t i o n i n s o i l s u n d e r n a t u r a l c o n d i t i o n s
b e cause th e y in d ic a t e t h a t t h i s r a t i o w i l l depend t o a la r g e e x te n t on
th e a m o u n ts and fo r m s o f r e a d i l y d e c o m p o s a b le o r g a n i c m a t t e r i n th e
s o ils s tu d ie d .

I t i s a p p r o p r i a t e t o d ra w a t t e n t i o n h e r e t o r e c e n t w o r k i n o u r
la b o r a t o r y (1 4 ) on N is o to p e d i s c r i m i n a t i o n i n d e n i t r i f i c a t i o n o f n i t r a t e
i n s o i l s b e c a u s e t h i s w o r k h a s p r o v id e d i n f o r m a t i o n n e e d e d t o a s s e s s th e
p o t e n t i a l v a lu e o f N i s o t o p e - r a t i o a n a ly s e s o f a tm o s p h e r ic N 2O f o r
r e s e a r c h o n s o u r c e s o f t h i s g a s , and t o f u r t h e r e v a lu a t e th e m e th o d
p r o p o s e d b y K o h l, S h e a r e r and Commoner ( 1 5 ) f o r a s s e s s m e n t o f th e
r e l a t i v e c o n t r i b u t i o n s o f s o i l s an d f e r t i l i z e r s t o n i t r a t e e n r ic h m e n t
o f s u r fa c e w a te rs . B r i e f l y , t h e m e th o d p r o p o s e d b y K o h l e t a l . i n v o l v e s
c a l c u l a t i o n s b a s e d o n t h e 15N c o n t e n t o f t h e n i t r a t e i n th e w a t e r u n d e r
s t u d y , and th e p r e d i c t e d c o n te n ts o f th e s o il- d e r iv e d and f e r t i l i z e r -
d e r iv e d n i t r a t e - N e n t e r in g t h i s w a te r . I t in v o lv e s a v a r ie t y o f
a s s u m p tio n s t h a t a r e o p e n t o c r i t i c i s m ( 1 6 , 1 7 ) , one b e in g t h a t
d e n i t r i f i c a t i o n o f n i t r a t e i n s o i l s and s u r f a c e w a t e r s i s n o t
a c c o m p a n ie d b y s i g n i f i c a n t N is o t o p e d i s c r i m i n a t i o n .

To s tu d y N is o to p e d i s c r i m i n a t i o n d u r in g d e n i t r i f i c a t i o n i n s o i l s
o f n i t r a t e c o n t a i n i n g n a t u r a l l e v e l s o f ^^N and ■*■%, we d e te r m in e d th e
a m o u n t and t h e c o n t e n t o f n i t r a t e - N and n i t r i t e - N i n n i t r a t e - t r e a t e d
s o i l s i n c u b a t e d u n d e r a n a e r o b ic c o n d i t i o n s ( h e liu m a tm o s p h e re ) a f t e r
t r e a t m e n t w i t h g lu c o s e t o p ro m o te d e n i t r i f i c a t i o n . A n a ly s e s p e rfo r m e d
show ed t h a t t h e n i t r a t e - N l o s t o n i n c u b a t i o n o f th e s e s o i l s c o u ld l a r g e l y
b e a c c o u n te d f o r a s p r o d u c t s o f d e n i t r i f i c a t i o n ( n i t r i t e , NO, N 2O and
 IA EA -SM -211/9 235

N 2) . T h e d a t a o b t a in e d show ed t h a t t h e r e i s m a rk e d d i s c r i m i n a t i o n
b e tw e e n and 1 5 ^ d u r i n g d e n i t r i f i c a t i o n o f n i t r a t e i n s o i l s ( T a b l^
i n l a n d t h a t s i g n i f i c a n t N is o t o p e e f f e c t s c a n o c c u r b o t h i n r e d u c t i o n
o f n i t r a t e t o n i t r i t e a n d i n r e d u c t i o n o f n i t r i t e t o g a s e o u s fo r m s o|f
N, T h e y a l s o show ed t h a t th e o v e r a l l N i s o t o p e e f f e c t d u r i n g
d e n i t r i f i c a t i o n o f n i t r a t e i n s o i l w i l l d e p e n d o n th e te n d e n c y o f th e
s o i l t o a c c u m u la te n i t r i t e u n d e r c o n d i t i o n s t h a t in d u c e d e n i t r i f i c a t i o n .
T h is w o r k h a s p r o v id e d c l e a r e v id e n c e t h a t d i s c r i m i n a t i o n b e tw e e n
an d d u r in g d e n i t r i f i c a t i o n i n s o ils o f n i t r a t e c o n ta in in g n a t u r a l
l e v e l s o f th e s e is o t o p e s i s o f s u f f i c i e n t m a g n itu d e t o i n v a l i d a t e th e
u s e o f N i s o t o p e - r a t i o a n a ly s e s f o r a s s e s s m e n t o f th e c o n t r i b u t i o n s o f
s o i l and f e r t i l i z e r N t o n i t r a t e i n s u r f a c e w a t e r s o r t o n i t r o u s o x id e
i n t h e a tm o s p h e r e .

R e c e n t a r t i c l e s c o n c e r n in g th e p o t e n t i a l e f f e c t s o f d e n i t r i f i c a t i o n
i n s o i l s on s t r a t o s p h e r ic ozone ha ve s tre s s e d th e need f o r re s e a rc h to
i d e n t i f y s o u r c e s and s i n k s o f a tm o s p h e r ic N20, and h a v e d ra w n a t t e n t i o n
t o c a l c u l a t i o n s i n d i c a t i n g t h a t a m a jo r n a t u r a l s i n k f o r a tm o s p h e r ic
N2O h a s n o t b e e n i d e n t i f i e d ( 1 8 - 2 0 ) , We r e c e n t l y o b t a in e d e v id e n c e [ t h a t ,
a lt h o u g h s o i l h a s b e e n c o n s id e r e d o n ly a s a s o u r c e o f a tm o s p h e r ic N 2^),
th e p o s s i b i l i t y t h a t i t may be an im p o r t a n t n a t u r a l s i n k f o r t h i s g a s
d e s e rv e s a t t e n t io n ( 2 1 ) , T h is e v id e n c e em e rg e d fr o m ga s c h r o m a t o g r a p h ic
s t u d i e s o f th e c a p a c i t y o f s o i l s f o r s o r p t i o n o f N 2O, B r i e f l y , th e s e
s t u d i e s h a v e show n t h a t s o i l s c a n s o r b N2O v e r y r a p i d l y u n d e r a n a e r o b ic
c o n d i t i o n s , and t h a t s o r p t i o n o f N 2O b y s o i l s i s a m i c r o b i a l p r o c e s s
i n v o l v i n g r e d u c t i o n o f N2O t o N 2* T h e y h a v e a l s o d e m o n s tr a te d t h a t t h i s

TABLE IV. EFFECTS OF VARIOUS TREATM ENT OF FIELD-MOIST IOWA SOILS ON

UREASE ACTIVITY

Treatm ent o f soil E ffect on urease activity

Dried at 3 0 , 4 0 , 50 or 60°C for 24 h N one

Dried at 75°C for 24 h Partial loss o f activity

Dried at 105°C for 24 h C om plete loss o f activity
A utoclaved ( 1 20°C ) for 2 h C om plete loss o f activity
L eached w ith water N one
Stored at - 2 0 ° , - 1 0 ° , 5°, 10°, 2 0 °, 3 0 ° or N one
40°C for 6 m onths
Incubated at 30° or 4 0 C under aerobic or w aterlogged N one
co n d ition s for 6 m onths
1
Air-dried and stored at 2 1 —23°C for 2 years N one J
Incubated at 30°C after addition o f jackbean urease Increase follow ed by [decrease
to original activity [
Incubated at 30°C after treatm ent w ith glucose and Increase follow ed by [decrease
other organic materials to original activity [
Incubated at 30°C after treatm ent w ith proteolytic N one 1

en zym es (pronase or trypsin)

Incubated at 30°C after treatm ent w ith urea or N one I

am m onium sulfate 1
1
Treated w ith urease inhibitors (catech ol Marked decrease in activity
h ydroquinone, p -benzoquinone, etc.)
236 BREMNER

TABLE V. CORRELATIONS BETWEEN SOIL UREASE ACTIVITY

AND OTHER SOIL PROPERTIES

Soil property Correlation

co efficien t (r)

Organic carbon 0 .7 2 » * *
T otal nitrogen 0 .7 1 * * *

C ation-exchange capacity 0 .6 7 * * *
Surface area 0 .4 5 *
Clay 0 .5 3 *
Sand - 0 .4 7 *
Silt 0.39
pH - 0 .0 1
C aC 03 equivalent - 0 .1 1

* Significant at 5% level.

*** Significant at 0.1% level.

r e d u c t i o n p r o c e s s i s p ro m o te d b y a n a e r o b ic c o n d i t i o n s and b y o r g a n ic
s u b s ta n c e s t h a t p ro m o te g r o w th o f s o i l m ic r o o r g a n is m s , and i s r e t a r d e d
b y n i t r a t e and b y a c e t y l e n e , w h ic h a l s o i n h i b i t s b i o l o g i c a l f i x a t i o n o f
N2 b y s o i l m ic r o o r g a n is m s . E v id e n c e t h a t s o i l s may r e p r e s e n t a n i m p o r t a n t
n a t u r a l s i n k f o r a t m o s p h e r ic N 2O h a s b e e n o b t a in e d fr o m s t u d i e s s h o w in g
1 ) t h a t t h e c a p a c i t y o f s o i l s f o r s o r p t i o n o f N2O u n d e r c o n d i t i o n s
f a v o r a b le f o r d e n i t r i f i c a t i o n o f n i t r a t e i s much g r e a t e r th a n t h e i r
c a p a c i t y f o r r e le a s e o f t h i s g a s , a n d 2) t h a t w a t e r lo g g e d s o i l s h a v e th e
c a p a c i t y t o re m o v e N^O fr o m a tm o s p h e re s c o n t a i n i n g 20% o x y g e n . The
p o s s i b i l i t y t h a t s o i l s may r e p r e s e n t a n im p o r t a n t n a t u r a l s i n k f o r
a tm o s p h e r ic NO2 a l s o d e s e r v e s a t t e n t i o n b e c a u s e we h a v e fo u n d t h a t
s o i l s s o rb t h i s gas v e r y r a p id ly , a n d ha ve a la r g e c a p a c ity f o r s o r p t io n
o f N 02- I n th e U n it e d S t a t e s a lo n e , s e v e r a l m i l l i o n to n s o f N a s N02
a r e r e le a s e d a n n u a l l y t o t h e a tm o s p h e re b y a u t o m o b ile s a n d b y c o a l
b u r n in g .

C hem odenitrification

The te r m c h e m o d e n i t r i f i c a t i o n w as p ro p o s e d b y C l a r k ( 2 2 ) t o
d e s ig n a t e g a s e o u s l o s s o f N fr o m s o i l s t h r o u g h c h e m ic a l d e c o m p o s it io n
o f n it r it e . I n t e r e s t i n t h i s p r o c e s s w as g e n e r a te d b y w o r k i n 19 58
( 2 3 ) a n d 19 60 ( 2 4 ) i n d i c a t i n g t h a t s u b s t a n t i a l g a s e o u s l o s s o f
f e r t i l i z e r N a d d e d t o s o i l s a s u r e a c a n o c c u r t h r o u g h c h e m ic a l
d e c o m p o s it io n o f n i t r i t e fo rm e d b y n i t r i f i c a t i o n o f t h e ammonium
p ro d u c e d o n h y d r o l y s i s o f u r e a b y s o i l u r e a s e .

D u r in g t h e p a s t 10 y e a r s we h a v e s t u d i e d n i t r i t e d e c o m p o s it io n i n
s o i l s b y a v a r i e t y o f m e th o d s , i n c l u d i n g 15 ц t r a c e r a n d gas
c h r o m a t o g r a p h ic t e c h n iq u e s ( 2 5 - 2 8 ) . B e s id e s s h o w in g t h a t c h e m ic a l
d e c o m p o s it io n o f n i t r i t e i n s o i l s i s a c c o m p a n ie d b y f i x a t i o n o f n i t r i t e -
N a n d f o r m a t i o n o f N 0 2 , N 2 a n d N 20, th e s e s t u d i e s h a v e p r o v id e d e v id e n c e
t h a t t h i s p r o c e s s i s c o n t r o l l e d l a r g e l y b y s o i l pH a n d o r g a n i c - m a t t e r
c o n te n t,a n d in v o lv e s s e lf - d e c o m p o s itio n o f n it r o u s a c id and r e a c t io n o f
n i t r i t e w i t h s o i l o r g a n ic m a t t e r . T h e y h a v e a l s o show n t h a t o r g a n ic
 IA EA -SM -211/9 237

s o i l c o n s t it u e n t s a r e r e s p o n s ib le f o r th e f i x a t i o n o f n i t r i t e - N and
p r o d u c t io n o f N2 a n d N30 o b s e r v e d o n t r e a t m e n t o f n e u t r a l o r a c i d i c s o i l s
w i t h n i t r i t e , a n d h a v e p r o v id e d e v id e n c e t h a t c h e m o d e n i t r i f i c a t i o n
i n v o l v e s r e a c t i o n o f p h e n o l i c s u b s ta n c e s w i t h n i t r o u s a c i d t o f o r m
n i t r o s o p h e n o l s t h a t a r e de c o m p o s e d b y n i t r o u s a c i d w i t h f o r m a t i o n o f N2
a n d N 2O. S u p p o r t f o r t h e c o n c lu s i o n t h a t p h e n o l i c c o n s t i t u e n t s o f s o i l
o r g a n i c m a t t e r p l a y a k e y r o l e i n c h e m o d e n i t r i f i c a t i o n w as o b t a in e d fro m
w o r k s h o w in g t h a t , o f m o re t h a n 100 o r g a n i c s u b s ta n c e s t e s t e d , o n ly
th o s e c o n t a i n i n g p h e n o l i c h y d r o x y l g r o u p s h a d t h e a b i l i t y t o d e com po se
n i t r i t e r a p i d l y , a n d t o b o t h f i x n i t r i t e - N a n d c o n v e r t n i t r i t e - N t o N2
a n d N2O u n d e r m i l d l y a c i d i c c o n d i t i o n s .

A lt h o u g h th e w o r k d is c u s s e d le a v e s n o d o u b t t h a t s o i l o r g a n i c m a t t e r
p r o m o te s c h e m ic a l d e c o m p o s it io n o f n i t r i t e i n s o i l s , we h a v e b e e n u n a b le
to c o n fir m r e p o r t s t h a t s u b s t a n t ia l v o l a t i l i z a t i o n o f N c a n o c c u r th r o u g h
c h e m o d e n itr ific a tio n in s o ils tr e a te d w ith u re a . P r e v io u s w o r k h a s
in d ic a t e d t h a t f e r t i l i z e r N added t o s o i l s as u re a i s p a r t i c u l a r l y
s u s c e p t i b l e t o g a s e o u s lo s s b y c h e m o d e n i t r i f i c a t i o n u n d e r a e r o b i c
c o n d i t i o n s , a n d t h a t lo s s o f u r e a N b y t h i s p r o c e s s i s m o s t e x t e n s i v e i n
s o i l s t h a t te n d t o a c c u m u la te n i t r i t e w hen t r e a t e d w i t h u r e a ( 2 3 , 2 4 ) . We
h a v e fo u n d , h o w e v e r, t h a t t h e 'n i t r o g e n d e f i c i t s ' o b s e r v e d i n s t u d i e s o f
th e f a t e o f u re a N i n s o i l s u n d e r a e r o b ic c o n d it io n s a r e n o t re d u c e d by
a d d i t i o n o f n i t r i f i c a t i o n i n h i b i t o r s s u c h a s N - S e rv e e v e n w hen t h e s o i l s
s t u d i e d a c c u m u la te s u b s t a n t i a l a m o u n ts o f n i t r i t e i n th e a b s e n c e o f
n i t r i f i c a t i o n i n h i b i t o r s , a n d a d d i t i o n o f th e s e i n h i b i t o r s g r e a t l y r e t a r d s
c o n v e r s io n o f u r e a N t o n i t r i t e a n d e l i m i n a t e s n i t r i t e a c c u m u la t io n ( 2 9 ) .
I f , a s h a s b e e n a s s u m e d , t h e 'n i t r o g e n d e f i c i t s ' o b s e r v e d i n s u c h s t u d i e s
r e s u l t fr o m g a s e o u s lo s s o f N th r o u g h c h e m ic a l d e c o m p o s it io n o f n i t r i t e ,
t h e y s h o u ld b e m a r k e d ly r e d u c e d b y a d d i t i o n o f com poun ds t h a t g r e a t l y
r e d u c e f o r m a t i o n a n d a c c u m u la t io n o f n i t r i t e .

V olatilization o f am m onium

A lt h o u g h i t i s w e l l e s t a b l i s h e d t h a t a p p r e c i a b l e l o s s o f f e r t i l i z e r
N c a n o c c u r th r o u g h v o l a t i l i z a t i o n o f ammonium fr o m s o i l s t r e a t e d w i t h
am monium o r am m onia f e r t i l i z e r s , m o s t r e c e n t r e s e a r c h c o n c e r n in g
v o l a t i l i z a t i o n o f am monium fr o m s o i l s h a s b e e n c o n c e rn e d w i t h th e
am monium v o l a t i l i z a t i o n p r o b le m e n c o u n t e r e d w hen f e r t i l i z e r N i s a p p l i e d
as u re a . I n t e r e s t i n t h i s p r o b le m h a s b e e n s t i m u l a t e d b y t h e g r o w in g
im p o r ta n c e o f u r e a f e r t i l i z e r i n w o r l d fo o d p r o d u c t io n , a n d b y th e
a c c u m u la t io n o f e v id e n c e t h a t s u b s t a n t i a l g a s e o u s l o s s o f u r e a N a s
NH3 c a n o c c u r fr o m s o i l s f e r t i l i z e d w i t h u r e a .

T h e re a r e n o i n d i c a t i o n s i n t h e l i t e r a t u r e t h a t s o i l o r g a n i c m a t t e r
p r o m o te s v o l a t i l i z a t i o n o f am m onium fr o m s o i l s t r e a t e d w i t h am m onium o r
am m onia f e r t i l i z e r s . In d e e d , a l l i n d i c a t i o n s a r e t h a t s o i l o r g a n i c
m a t t e r d e c r e a s e s v o l a t i l i z a t i o n o f ammonium fr o m s u c h s o i l s b y v i r t u e
o f i t s c a p a c i t y f o r s o r p t i o n a n d f i x a t i o n o f am m onium . T h e re i s ,
h o w e v e r, s u b s t a n t i a l e v id e n c e t h a t s o i l o r g a n i c m a t t e r h a s a n i m p o r t a n t
e f f e c t o n v o l a t i l i z a t i o n o f am monium fr o m s o i l s t r e a t e d w i t h u r e a .

I t i s w e l l e s t a b l i s h e d t h a t t h e am monium v o l a t i l i z a t i o n p r o b le m
e n c o u n t e r e d i n u s e o f f e r t i l i z e r u r e a r e s u l t s fr o m t h e r a p i d e n z y m a tic
h y d r o l y s i s o f u r e a t o am monium c a r b o n a te b y s o i l u r e a s e , w h ic h i s a
com pone nt o f s o i l o r g a n ic m a t t e r . I t i s e q u a lly w e ll e s ta b lis h e d , i
h o w e v e r, t h a t u r e a s e a d d e d t o s o i l s i s i n a c t i v a t e d v e r y r a p i d l y , so i t
i s d i f f i c u l t to e x p la in w hy p r a c t i c a l l y a l l s o i l s e x h ib it s u b s t a n t ia l
u re a s e a c t i v i t y . I n a n a t t e m p t t o a n s w e r t h i s q u e s t i o n , we h a v e r e c e n t l y
s tu d ie d th e s t a b i l i t y o f u re a s e i n s o i l s and f a c t o r s a f f e c t i n g s o i l
238 BREMNER

u re a s e a c t i v i t y (3 0 -3 2 ). T h is w o r k h a s sho w n t h a t t h e n a t i v e u r e a s e i n
Io w a s o i l s i s r e m a r k a b ly s t a b l e , a n d h a s i n d i c a t e d t h a t d i f f e r e n t s o i l s
h a v e d i f f e r e n t s t a b l e l e v e l s o f u r e a s e a c t i v i t y d e te r m in e d b y t h e a b i l i t y
o f t h e i r c o n s t it u e n t s t o p r o t e c t u re a s e a g a in s t m ic r o b ia l d e g r a d a tio n and
o t h e r p r o c e s s e s le a d i n g t o i n a c t i v a t i o n o f e n z y m e s . Some o f t h e e v id e n c e
f o r t h e s e c o n c lu s io n s i s r e p o r t e d i n T a b le I V . Two f i n d i n g s m e r i t s p e c ia l
a tt e n tio n . One i s t h a t , a lt h o u g h ja c k b e a n u r e a s e w as i n a c t i v a t e d v e r y
r a p i d l y w hen a d d e d t o t h e s o i l s s t u d i e d , p r o lo n g e d i n c u b a t i o n o f unam ended
s a m p le s o f th e s e s o i l s u n d e r a e r o b i c o r w a t e r lo g g e d c o n d i t i o n s h a d n o
e f f e c t on t h e ir u re a s e a c t i v i t y . T h e o t h e r i s t h a t , a lt h o u g h a m a rk e d
in c r e a s e i n u r e a s e a c t i v i t y w as o b s e r v e d i n s o i l s t r e a t e d w i t h g lu c o s e and
o t h e r o r g a n i c m a t e r i a l s t h a t p ro m o te p r o d u c t io n o f u r e a s e b y s o i l
m ic r o o r g a n is m s , t h i s i n c r e a s e w as te m p o r a r y a n d c o u ld n o t b e d e t e c t e d
a f t e r a fe w w e e k s . W it h e a c h s o i l s t u d i e d , u r e a s e a c t i v i t y in c r e a s e d
a f t e r a d d i t i o n o f ja c k b e a n u r e a s e o r o r g a n i c m a t e r i a l s p r o m o t in g m i c r o b i a l
a c t i v i t y , b u t s u b s e q u e n t ly d e c re a s e d a n d s t a b i l i z e d a t t h e l e v e l o b s e rv e d
b e f o r e a d d i t i o n o f th e s e s u b s ta n c e s . The o n ly a p p a r e n t e x p la n a t i o n o f
th e s e f i n d i n g s i s t h a t t h e u r e a s e a c t i v i t y o f unam ended s o i l s r e f l e c t s t h e i r
c a p a c ity f o r p r o t e c t io n o f u re a s e , and t h a t u re a s e i n e x c e s s o f t h i s
c a p a c it y i s decom posed o r in a c t iv a t e d .

I n an a tte m p t to i d e n t i f y th e s o i l c o n s t it u e n t s t h a t p r o t e c t u re a s e
a n d d e t e r m in e t h e s t a b l e l e v e l s o f u r e a s e a c t i v i t y i n d i f f e r e n t s o i l s , we
r e c e n t l y s t u d i e d t h e r e l a t i o n s h i p s b e tw e e n u r e a s e a c t i v i t y i n 21 Io w a
s u r fa c e s o i l s and o th e r p r o p e r t ie s o f th e s e s o i l s ( 3 3 ) . The s o i l s used
w e re s e l e c t e d t o o b t a i n a w id e ra n g e i n p r o p e r t i e s , i n c l u d i n g pH, t e x t u r e
and o r g a n ic - m a tte r c o n te n t. S im p le c o r r e l a t i o n a n a ly s e s show ed t h a t
u r e a s e a c t i v i t y w as c o r r e l a t e d v e r y s i g n i f i c a n t l y w i t h o r g a n i c c a r b o n
c o n t e n t , t o t a l n i t r o g e n c o n t e n t , a n d c a t io n - e x c h a n g e c a p a c i t y ( T a b le V ) ,
w h ic h a r e in d e x e s o f o r g a n i c - m a t t e r c o n t e n t , and m u lt ip l e r e g r e s s io n
a n a ly s e s show ed t h a t o r g a n i c - m a t t e r c o n t e n t a c c o u n te d f o r m o s t o f t h e
v a r i a t i o n i n u re a s e a c t i v i t y i n th e s o i l s s tu d ie d . T h is w o r k i n d i c a t e s
t h a t o r g a n ic s o i l c o n s t it u e n t s c o n t r ib u t e s u b s t a n t i a ll y t o p r o t e c t io n o f
u re a s e a n d e s ta b lis h m e n t o f s t a b le le v e ls o f u re a s e a c t i v i t y i n s o i l s .
O th e r r e c e n t w o r k h a s p r o v id e d e v id e n c e t h a t o r g a n i c s o i l c o n s t i t u e n t s
p r o t e c t u r e a s e ( 3 4 , 3 5 ) , a n d t h e r e seem s v e r y l i t t l e d o u b t t h a t
p r o t e c t i o n o f u re a s e b y o r g a n ic m a tte r i s a k e y f a c t o r i n v o l a t i l i z a t i o n
o f am monium fr o m s o i l s t h r o u g h h y d r o l y s i s o f u r e a b y s o i l u r e a s e .
F u r t h e r r e s e a r c h i s n e e d e d t o e s t a b l i s h t h e m e c h a n is m s b y w h ic h o r g a n ic
s o i l c o n s t i t u e n t s p r o t e c t u r e a s e , a n d t o a c c o u n t f o r t h e r e m a r k a b le
s t a b i l i t y o f n a tiv e s o i l u re a s e .

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[2 6 ] BREM NER, J.M ., N ELSO N , D.W ., “Chem ical d ecom p osition o f nitrite in soils”, Trans. 9 th Int. Congr.
Sod Sei. 2 ( 1 9 6 8 ) 495.
[2 7 ] N ELSO N, D.W., BREM NER, J.M ., R ole o f sod minerals and m etallic cations in nitrite d ecom p osition and
chem odenitrification in sods, Sod Biol. B iochem . 2 (1 9 7 0 ) 1.
[2 8 ] N ELSO N, D.W ., BREM N ER, J.M ., G aseous products o f nitrite d ecom p osition in sods, S od B iol. B iochem .
2 ( 1 9 7 0 ) 203.
[2 9 ] B U N D Y , L .G ., BREM NER, J.M., E ffects o f nitrification inhibitors o n transform ations o f urea nitrogen in
sods, Sod Biol. B iochem . 6 (1 9 7 4 ) 369.
[3 0 ] Z A N TU A , M .I., BREM NER, J.M ., Preservation o f sod samples for assay o f urease activity, Sod B iol.
B iochem . 7 ( 1 9 7 5 ) 297.
[3 1 ] Z A N TU A , M .I., BR EM N ER, J.M ., Production and persistence o f urease activity in sods, Sod B iol. B iochem .
(in press).
[3 2 ] Z A N T U A , M.I., BREM NER, J.M ., Stability o f urease in sods, Sod B iol. B iochem . (in press).
[3 3 ] Z A N TU A , M .I., DUM ENIL, L.C., BREM NER, J.M ., R elationships betw een sod urease activity and other
properties, Sod Sei. S oc. A m ., Proc. (in press).
[3 4 ] B U R N S, R .G ., PUKITE, A .H ., MCLAREN, A .D ., Concerning the location and persistence o f sod urease,
Sod Sei. Soc. A m ., Proc. 36 ( 1 9 7 2 ) 308.
[3 5 ] B U R N S , R .G ., EL-SAY ED , M .H., M cLAR EN , A .D ., Extraction o f an urease-active organo-com plex from
sod, Sod Biol. B iochem . 4 ( 1 9 7 2 ) 107.
240 BREMNER

DISCUSSION

I. C. REM Y : T he use o f n a tu ra l A15N is n o t an ideal m e th o d o f d e te rm in in g th e origin o f

n itra te s in th e soil. H ow ever, I th in k th a t a A1SN close to 0 m ak es it alm o st c ertain th a t th e soil
n itra te s co m e fro m m ineral fertilizers.
J. M. B R E M N E R : I certain ly agree th a t A 1SN values are useless fo r stu d ies o f th e origin o f
n itra te in soils. I d o n o t agree w ith th e second p a rt o f y o u r s ta te m e n t.
J.C . R E M Y : T h e n itro g e n d e fic its are h ig h er w ith th e u re a th a n w ith th e am m o n ia fertilizers.
W hat d o m in a n t p h e n o m e n o n can ex p lain th is behaviour?
J.M . B R E M N E R : O u r w o rk in d ic a te s th a t gaseous loss o f n itro g e n fro m soils tre a te d w ith
u rea o c cu rs largely th ro u g h am m o n ia (N H 3). We have b een u n ab le to o b ta in evidence fo r gaseous
loss th ro u g h ch em o d en itrific atio n .
T. W E IC H E L T : H ow d o y o u ex p lain th e very m ark ed e ffe c t o f lignins an d ta n n in s on n itrite
d eco m p o sitio n ?
J.M . B R E M N E R : Several investigations have in d ic a te d th a t p h e n o lic sub stan ces such as
lignins an d ta n n in s re a c t w ith n itro u s acid to fo rm n itro so p h en o ls, an d th a t th ese tau to m e riz e to
q u in o n e o x im es w hich are d eco m p o sed by n itro u s acid w ith fo rm a tio n o f N 2 an d N 20 . F o r possible
re a c tio n m ech an ism s, see K ainz and H u b er, M ikrochim . A cta (1 9 5 9 ) 8 9 1 , and A u stin , Science
P rogress 4 9 (1 9 6 3 ) 619.
D. L O PE Z H E R N A N D E Z : W hich soil fa cto rs are involved in H2S so rp tio n ? D o y o u th in k
th a t th is so rp tio n p rocess could be a ffe c te d b y pH ? (L o p ez H ern an d ez an d B u rn h am , J. Soil
Sei. 3 2 (1 9 7 4 ); H ingston e t al. (1 9 7 2 ) J. Soil Sei. 3 0 (1 9 7 2 ).
J.M . B R E M N E R : We have n o t th u s far stu d ied th e fa cto rs affectin g so rp tio n o f H 2S by soils.
T he lim ited d a ta available suggest th a t th e soil c o n te n t o f easily re d u cib le F e 3+ m ay be an
im p o rta n t fa c to r, and th a t so rp tio n o f H 2S b y soils involves fo rm a tio n o f iro n su lp h id es and
ele m en tal su lp h u r. T o m y kn o w led g e, no in fo rm a tio n is available co n cern in g th e e ffe c t o f soil
p H o n so rp tio n o f H 2S.
W. F L A IG (Scientific Secretary) : Y o u also m e n tio n SF6 as a volatile c o m p o u n d . Is th is o f
in d u stria l origin? If so, fro m w hich tech n ical process?
J.M . B R E M N E R : Y es, SF6 is p ro d u c e d by in d u stria l processes. T his has v itia te d th e use o f
th is gas as a tra c e r fo r a tm o sp h e ric research. I believe th a t S F 6 is m a n u fa c tu re d fo r several p u r­
poses, b u t I am n o t sure a b o u t th e m ajo r in d u stria l uses o f th is gas.
R .S. SW IFT: F ro m y o u r w o rk w o u ld y o u be p re p are d to express a n o p in io n w h e th e r u rb an
o r ag ric u ltu ra l so urces are th e m ain cause o f n itro u s o x id e p o llu tio n o f th e atm o sp h ere?
J.M . B R E M N E R : It is generally assum ed th a t th e n itro u s o x id e in th e a tm o sp h e re is o f
b iological origin an d th a t th is gas is n o t p ro d u c ed in significant a m o u n ts b y in d u stria l processes.
H ow ever, a re c e n t p a p e r by P ie ro tti an d R asm ussen in G eo p h y sical R esearch L e tte rs (R ef.[ 18]
in th e p a p e r) in d ic a te s th a t n itro u s o x id e is p ro d u c e d by c o m b u stio n processes. T h ere is a clear
need fo r research c o n cern in g a n th ro p o g e n ic sources o f n itro u s o x id e. N o reliable d a ta are available
co n cern in g th e a m o u n ts o f N 20 evolved fro m soils an d th e oceans.
 IA E A -SM -211/55

A P P L IC A T IO N O F H IG H -P R E S S U R E
L IQ U ID C H R O M A T O G R A P H Y T O S T U D IE S
O F E X T R A C T A B L E S O IL O R G A N IC M A T T E R
P o ro u s silic a p a ck in g s

R.H. L O E P P E R T , B.G. V O LK
Soil Science D e p artm en t,
U niv ersity o f F lo rid a,
G ainesville, F lo rid a,
U n ited S ta te s o f A m erica

A bstract

APPLICATION OF HIGH-PRESSURE LIQUID CHROMATOGRAPHY TO STUDIES OF EX TRACTABLE

SOIL ORGANIC MATTER: POROUS SILICA PACKINGS.
A variety o f new high-efficiency porous packing materials, which may be useful for size fractionation
and characterization o f soil organic matter, are now available through the development o f the high-pressure liquid
chromatograph. The object here was to investigate the effect of solvent, saturating cation and excess electrolyte on
elution o f extractable soil organic matter and organic acid standards from one o f the new porous silica |packing
materials (Porasil series) and the de-activated analogues (Porasil X series). Fulvic acid prepared in the H-, Na-,
N(CH3 ) 4- , or N(C4 H9)4 - saturated form was excluded and organic acid standards were partially excluded from the
pores o f the Porasil C packing material, and eluted at V0 when water was used as the eluting solvent. This phenomenon
was partly attributed to electrostatic repulsion o f negatively charged organic matter by negative charge' sites on the
silica surface. Acetone, 2-propanol and methanol-extractable fractions were partially excluded from tile pores when
methanol or DMF was used as the eluting solvent, and adsorbed when THF or acetone was used as the [eluting solvent.
In the presence o f excess electrolyte, cation-saturated samples entered the porous matrix because o f suppression of
charge and decreased electrical double-layer thickness o f the negatively charged solute molecules and the negatively
charged silica surface. As electrolyte concentration was increased, however, adsorption phenomena became more
prevalent as a result o f the decreased electrical double-layer thickness which permitted direct interaction between
active sites on the silica surface and oxygen-containing functional groups o f the organic solute.

IN T R O D U C T IO N

Since th e d e v elo p m en t o f S ep h ad ex th e re has been co n siderable in te re st in th e use o f gel

filtra tio n f o r fra c tio n a tio n an d c h a ra c te riz a tio n o f soil organic m a tte r. A p p lica tio n s o f gel
filtra tio n to stu d ies o f h u m ic an d fulvic acids have been review ed b y S w ift and P o sn er [ ].
D u rin g th e p a st d ecad e, rap id advances have been m ad e in liq u id c h ro m a to g ra p h y . T hese
ad vances have b een d u e p rim arily to th e d e v elo p m en t o f th e high-pressure liq u id c h ro m a to g ra p h
w h ich , in tu rn , has m ad e possible th e use o f sm all-diam eter p acking m aterials and high-efficiency
colu m n s. F o r ex am p le, w ith th e n ew jam -packing m aterials, co lu m n efficiencies as high as
5 0 0 0 th e o re tic a l p late s p e r fo o t are c o m m o n ly a tta in e d .
T h e p ra ctic e o f high-pressure Uquid c h ro m a to g ra p h y (H P L C ) is describ ed b y K irk lan d [2]
an d in review s b y Zw eig a n d S herm a [3] an d G ay lo r, Jam es a n d W eetall [4]. C h aracteristics
o f p ack in g m ate ria ls are su m m arized b y D ark a n d L im p e rt [ 5 ] , K irk lan d [2] an d L au b [ 6 ] .
Pack in g m aterials are gen erally divided in to th re e m ajo r classes: non-rigid gels, semi-rigici gels,
an d rigid gels o r glasses. T h e non-rigid gels are lig h tly cross-linked (e.g. S ep h a d ex G gels) an d
are n o t su itab le fo r H PLC as th e y will d is to rt u n d e r pressure, resu ltin g in a n a lte re d p o re stru c tu re .
T he sem i-rigid gels (p o ly sty ren e -d iv in y lb en z en e ) are highly cross-linked, d o n o t d is to rt u n d e r

241
242 LOEPPERT and VOLK

pressu re, an d are th e re fo re su itab le fo r HPLC. T h e th ird g roup, th e rigid gels, are actu ally p o ro u s
glasses o r silicates, an d are su itab le fo r HPLC.
T h e o b je c t here is to r e p o rt th e effe c ts o f solvent, sa tu ratin g c a tio n and excess n e u tra l salt
o n e lu tio n o f e x tra c ta b le soil organic m a tte r fro m th e p o ro u s silica pack in g m aterials (Porasil
series) an d th e d e-activated analogues (Porasil X series) using HPLC.
T h e p ro p e rtie s o f Porasil have b e en exam ined b y C o o p e r an d B arrall [ 7]. T h e packing
e x h ib its stro n g a d so rp tio n p ro p e rtie s [5, 8, 9] w hich are a ttrib u te d to h y d ro x y l gro u p s on th e
su rface an d L ew is acid sites p re se n t fro m th e m a n u fa ctu rin g process. A d so rp tio n effe c ts m ay
b e red u ced th ro u g h d e-activ atio n o f h y d ro x y l gro u p s; how ever, th e L ew is acid sites are n o t
d e-activ ated b y these p ro c ed u re s [5]. D e-activation p ro c ed u re s in clu d e chem ical tre a tm e n t
w ith p o ly e th y le n e glycol [10, 11], an d p e rm a n e n t d e-activ atio n b y sily atio n w ith h e x am eth y l-
d isilazane [12].

M A T E R IA L S A N D M ETH O D S

S am ple p re tre a tm e n t a n d e x tra c tio n

T h e soil selected fo r s tu d y was th e surface h o riz o n ( 0 —25 cm ) o f T e rra Ceia m u ck , a T y p ic

M edisaprist. C h em ical p ro p e rtie s o f T erra Ceia m u ck are p re sen te d elsew here [13]. A sh
(in o rg an ic) c o n te n t w as lo w ered to less th a n 1 .0% using th e dialysis p ro c e d u re described by
L o ep p e rt a n d V o lk [14].
T h e e x tra c tio n p ro c ed u re is o u tlin e d in F ig .l. Soil e x tra c ts w ere o b ta in e d b y th ree
successive 2 4 -h o u r tre a tm e n ts w ith th e a p p ro p ria te e x tra c ta n t. T h e e x tra c t was cen trifu g ed
fo r 2 h o u rs a t 16 0 0 0 tim es gravity (G ), filtered a n d p u rifie d to less th a n 0.5% ash an d c o n sta n t
n itro g e n c o n te n t.

H EXANE

BE N ZE N E

1E T H Y L ACETATE]
I
lA C E T O N E I

I2 -P R 0 P A N Ö J

I m eth a n o l I

F IG .l. Extraction and fractionation scheme.

 IA EA -SM -211/55 243

Organic matter extracted with dimethylformamide (DMF) was concentrated in a ro :ary

evaporator under vacuum at 40°C, suspended in de-ionized water, and dialysed alternately
against de-ionized water and 0.1N HC1 until a sample with constant nitrogen content and less
than 0.5% ash was obtained.
The NaOH extract was neutralized with 6N HC1, and concentrated in a rotary evaporator
under vacuum at 30°C. The sample was purified similarly to the DMF extract. A portion of
the NaOH extract was separated into humic and fulvic acid fractions by the procedure oiitlined
by Stevenson [15], and purified as above.
Soxhlet fractions were obtained by successive 48-hour extractions with each solvent (Fig.l).
Extracts were concentrated under vacuum at 25°C, dried under a dry-nitrogen jet, re-dissolved
in the extracting solvent and centrifuged. Samples were re-concentrated and purified as above.
All purified samples were lyophilized and stored at 0°C.

Packing materials and solvents

The Porasil and Porasil X packing materials (Table I) were obtained in bulk form from Waters
Associates, and dry-packed into 0.317 cm o.d. X 1 m stainless-steel columns.
Solvents used were de-ionized water and spectro-quality methanol, 2-propanol, tetrahydrofuran
(THF), DMF and acetone. Salt solutions were prepared in water and methanol at concentrations
of 0.001N, 0.01N and 0.05N with the appropriate sulphate salts, and neutralized to pH 7.0.

Sample preparation and liquid chromatography

Samples were dissolved in the eluting solvent to give a 0.1% concentration (wt/vol.), unless
otherwise specified, and centrifuged at 16 000 X G. Samples in salt solutions were neutralized
to pH 7.0 with 1.0N NaOH, (CH3) 4NOH, or (C4H9)4NOH. Injection volume was 10 pi.
The Waters’ ALC liquid chromatograph, equipped with the Model 6000 solvent delivery
system and U6K universal injector, was used for all separations. Solvent flow was maintained
at 0.5 ml/min, and the differential refractive index and u.v. absorption at 254 or 280 nm were

TABLE I. COLUMN PARAMETERS

Packing Pore Vo3 V a Nb D escription

T
material size (A) (m l) (m l)

Porasil A 60 1.82 3.35 360 Porous silica

Porasil C 250 1.76 3 .3 6 340 Porous silica

Porasil E 1500 1.62 3 .4 2 290 Porous silica

Porasil AX 60 1.84 3 .3 2 340 Porous silica de-activated w ith p o ly eth y l :ne

glycol

Porasil CX 250 1.88 3 .4 2 280 Porous silica de-activated w ith p o ly eth y l me

glycol

Porasil EX 1500 1.67 3.49 230 Porous silica de-activated w ith p o ly eth y l sne
glycol

a T otal volum e, from injector to detector.

b Theoretical plate cou n t o f 1 m colum n.
244 LOEPPERT and VOLK

monitored continuously. Column parameters, V0, the elution volume of a non-reactive high
molecular weight solute, and VT, the elution volume of a non-reactive low molecular weight
standard, were determined by elution of acetone or benzene and Blue Dextran 2000 or
2 300 000 molecular weight polystyrene respectively.

RESULTS AND DISCUSSION

Elution characteristics of packed columns are summarized in Table I. In all cases the
column efficiencies, indicated by theoretical plate count, N, decreased with increasing internal
pore size of the packing material. For example, the column efficiencies of Porasil AX, CX and
EX columns were 340, 280 and 230 N respectively.

Effect of solvent

Elution patterns of fulvic acid and 2-propanol extractable organic matter by selected solvents
are shown in Figs 2 and 3 respectively. Organic matter samples were completely soluble at 0.1%
concentration in each of the solvents shown.
The fulvic acid sample was eluted at V0, the elution volume of a non-reactive high-molecular
weight solute, on the Porasil A column when water (Fig.2) or DMF was used as the eluting solvent,
and partially eluted at V0 when methanol (Fig.2) was used as the eluting solvent. Likewise, the
2-propanol organic matter extract was eluted at V0 on Porasil A when DMF was used as the eluting
solvent, and partially eluted at V0 with methanol (Fig.3). In all cases, the quantity of organic solute
eluted at V0 was larger with DMF than with methanol. With each of these solvents the organic
solute was eluted before VT, indicating negligible adsorption. However, when 2-propanol, acetone
or THF (Fig.3) was used to elute the 2-propanol extractable material on the Porasil A column,
a portion of the organic solute was eluted past VT, indicating an adsorptive interaction with
the silica packing material.

F I G .2 . E lu tio n o f H -s a tu r a te d fu lv ic a c id o n P o r a s il A a n d P o r a s il A X w ith s e le c te d s o lv e n ts .
 IA E A -SM -211/55 245

F I G .3 . E lu ti o n o f H -s a tu r a te d 2 - p r o p a n o l e x tr a c t o f D M F - e x tr a c ta b le m a te r ia l o n P o r a s il A a n d P o r a s il A X

w ith s e le c te d s o lv e n ts .

Elution patterns obtained on Porasil AX, compared with Porasil A, indicated reduced
exclusion of fulvic acid from the gel matrix when either water or methanol was used as the
eluting solvent (Fig.2). The use of de-activated Porasil AX resulted in reduced exclusion of the
2-propanol extract when methanol was used as the eluting solvent (Fig.3). When THF (Fig.3)
or acetone was used as the eluting solvent on Porasil AX compared with Porasil A, adsorptive
interaction between the packing material and the organic solute was reduced but not eliminated.
Elution patterns of organic acid standards on Porasil A and Porasil AX showed interesting
similarities to the elution patterns of extractable soil organic matter. In water and DMF, elution
peaks of 1,2,4,5-tetracarboxybenzene and 3,5-dihydroxybenzoic acid occurred at V0 or slightly
greater than V0. In THF, both organic compounds exhibited elution volumes slightly greater
than VT with slightly tailing peaks.
Based on the molecular weights of tetracarboxybenzene and dihydroxybenzoic acid and
the working molecular weight ranges of the gels suggested by the manufacturer, one would
expect that the solute would elute at, or slightly before, VT. Major deviations from this elution
pattern can be attributed to adsorption, electrostatic exclusion or molecular association.
The exclusion of organic solute from Porasil A in methanol and DMF may be attributed to
(1) porous structure of the gel, (2) association of solute molecules, and/or (3) electrostatic
exclusion from the porous matrix. The first explanation is unlikely since the non-reactive
solute, acetone, produced a symmetrical peak at VT with negligible skewing, indicative of
free entrance into the porous gel matrix. Association of the solute molecules in methanol and
DMF may be questionable since aggregation of the molecular units should be greatest in the
least polar and/or least basic solvent. Comparison of the individual solvents shows that DMF
and methanol have stronger basic character, and are considerably more polar than THF, with
dielectric constants of 36.7, 32.6 and 7.58 respectively. Also, de-activation of the porous
silica resulted in increased elution volumes, which should not have been the case if the skewing
was entirely due to aggregation of the solute molecules. Partial exclusion of the organic solutes
from the porous structure of the silica probably resulted from electrostatic repulsion of the
negatively charged solute molecules from the negatively charged silica surface. The greater
246 LOEPPERT and VOLK

apparent exclusion in DMF than in water was caused by the stronger basic character of DMF
solvent and the resulting greater negative charge density of the solute.
Acidic functional groups, COOH and phenolic OH, on the organic solute may be more
highly dissociated in DMF than in methanol, resulting in a greater negative charge density in the
former solvent. In water [2], methanol or DMF, the silica packing material assumed a negative
charge because of dissociation of surface Si(OH) groups. The exclusion of organic material
from the porous matrix of Porasil A may be at least partially attributed to the expanded electrical
double layer of the charged silica in the absence of excess neutral salt, and the resulting electro­
static repulsion between the charged solute molecules and the charged silica surface.
The skewed tailing ends and larger elution volumes of 1,2,4,5-tetracarboxybenzene and
3,5-dihydroxybenzoic acid elution peaks in THF on Porasil A provided strong evidence of
adsorption. Strongly polar DMF and methanol were able to compete with the active Si(OH)
sites on the silica packing material for the acidic solute molecules. Less polar THF could not
compete as effectively for the reactive sites; therefore the solute was adsorbed. Also, the greater
charge density of the solutes in water, methanol and DMF than in THF may have resulted in less
interaction between the negatively charged solute and the negatively charged silica surface.
De-activation of the silica surface with polyethylene glycol would block the reactive sites
[5], and result in reduced negative charge density of the silica surface in water and DMF. There­
fore, electrostatic exclusion of charged solute from the porous matrix was reduced on Porasil AX
in comparison with Porasil A. The evidence of adsorption and electrostatic exclusion interactions
between the solute and the Porasil AX indicated that the packing material was not completely
de-activated.

Effect of saturating cation

Fulvic acid in which the acidic functional groups were saturated to pH 7.0 with Na+, K*,
N(CH3 ) 4 or N(C4H9 ) 4 were eluted at V0 on Porasil C when water was used as the eluting
solvent (Fig.4). De-activation of the porous silica decreased the relative quantity of solute
eluted at V0 and shifted the elution peaks towards VT (Fig. 5).
When Na saturated to pH 7.0, both the 1,2,4,5-tetracarboxybenzene and 3,5-dihydroxybenzoic
acid in methanol and water were excluded from the Porasil A and Porasil AX gel matrix to a greater
extent than corresponding H-saturated samples.
The pronounced exclusion of Na-, K-, N(CH3)4- and N(C4H9) 4 - saturated fulvic acid or
organic acid standards from the Porasil C gel matrix may be attributed to electrostatic repulsion
of the negatively charged solute molecule and the negatively charged sites on the silica surface.
Similar exclusion phenomena have been observed during elution of cation-saturated humic acid
with distilled water on Sephadex [ 1] . Electrostatic exclusion phenomena have been reported
for elution of acidic amino acids [16], aromatic acids [17], inorganic ions [18] and lignosulphonate
[19] on the Sephadex G-gels, and were attributed to electrostatic repulsion between fixed charges
on both the gel and solute molecules.

Effect of excess neutral salt

Elution patterns of Na-saturated fulvic acid in the presence of excess neutral salt on Porasil A
and Porasil AX (Figs 4 and 5 respectively) indicated that the electrolyte resulted in decreases in
the relative quantity of solute excluded from the gel matrix. The presence of neutral salt also
affected elution patterns of low-molecular weight organic acids on Porasil A and Porasil AX
(Figs 6 and 7 respectively), and resulted in a reduction in the relative quantity of material
eluted before VT. Similar phenomena have been observed with Sephadex during the elution
of soil organic matter extracts [1, 20], acidic amino acids [16], aromatic acids [17] and ligno-
 IA E A -SM -211/55 247

F I G .4 . E lu tio n o f N a - s a tu r a te d fu lv ic a c id o n P o r a s il A w ith w a te r a n d N a ^ S O * s o lu tio n s .

F I G .5 . E lu tio n o f N a - s a tu r a te d fu lv ic a c id o n P o r a s il A X w ith w a te r a n d N a ^ S O * s o lu tio n s .

sulphonates [19]. At electrolyte concentrations of 0.05N, a portion of the organic solute was
eluted past VT, indicating adsorption on the silica packing material.
The excess electrolyte suppressed the negative charge and reduced the thickness ofj the electrical
double layer of the negatively charged gel surface and the organic solute molecules. Alsb excess
salt decreases the effective size of solute anions due to reduction in thickness of the eledtrical
double layer. As double-layer thickness decreased with resulting reduction in electrostatic
exclusion, adsorption increased due to direct H-bonding interactions of Si(OH) and Si(OH2)
sites on the packing material and oxygen-containing groups on the organic solute. Adsorption effects
were reduced on the Porasil X series of packing materials (Figs 5 and 7), but were not completely
eliminated. This evidence indicated that the silica surface was not completely de-activated and/or
solute molecules were interacting directly with the de-activating agent.
248 LOEPPERT and VOLK

F J G .6 . E lu tio n o f N a - s a tu r a te d 1 ,2 ,4 ,5 - te t r a c a r b o x y b e n z e n e o n P o r a s il A w ith w a te r a n d N a 2 S O 4 s o lu tio n s .

ELUTION VOLUME (ml)

F IG . 7. E lu tio n o f N a - s a tu r a te d 1 ,2 ,4 ,5 - te t r a c a r b o x y b e n z e n e o n P o r a s il A X w ith w a te r a n d N a 2 S O 4 s o lu tio n s .

CONCLUSIONS

Solvent, saturating cation and excess electrolyte influenced elution of organic solutes from
Porasil A and Porasil AX. Extractable soil organic matter and organic acid standards were
excluded from the Porasil A packing material when eluted with water, methanol or DMF.
Each of these solvents has a significant basic character that promotes dissociation of acidic
functional groups of the organic solute and surface Si(OH) and Si(OH2) groups of the silica
packing material. The exclusion effect arose from electrostatic repulsion of negatively charged
solute molecules and the similarly charged silica surface. Solvents THF, ethyl acetate and acetone
 IA E A -SM -211/55 249

permitted both the packing material and solute molecules to exist as neutral species. Because of
the weakly basic properties of these solvents, the exclusion phenomenon no longer played an
important role. On the other hand, in these solvents, aggregation of acidic solute molecules
may influence elution patterns or adsorption may be severe.
The exclusion effect was greatly reduced but not eliminated on the de-activated Porasil AX
packing material. Excess electrolyte resulted in reduced exclusion from the Porasil A arid AX
packing materials due to suppression of charge and reduction in thickness of the electrical double
layers of charged solute molecules and silica surfaces. Excess electrolyte resulted in increased
adsorption to the Porasil AX packing material.
Alkaline conditions must be avoided with the porous silica packing materials to prevent
stripping of the adsorbed de-activating agent and/or damage of the silica surface. Also, under
alkaline conditions the Si(OH) and Si(OH2) sites at the silica surface would be highly dissociated,
resulting in greater charge density of the silica surface and enhanced exclusion of negatively
charged solute molecules.
From these studies, we can conclude that electrostatic and adsorption phenomena may
prevent accurate separations according to molecular size. The conditions which retard electro­
static exclusion of solute molecules from the packing materials are also the conditions that promote
adsorptive interactions.
Further studies of the elution properties of extractable soil organic matter on porous silica
packing materials by organic solvents having weakly basic properties (e.g. THF) may give improved
results. Also, with the use of HPLC we can gain an insight into properties of soil organic matter
on silica surfaces.

REFERENCES

[ I ] SWIFT, R .S., PO SNER, A.M ., J. Soil Sei. 2 2 2 ( 1 9 7 1 ) 2 3 7 .

[ 2 ] KIR KLA ND , J.J., M odem Practice o f Liquid Chrom atography, W iley-Interscience, N ew Y ork (1 9 7 1 ).
[3 ] ZWEIG, G., SHERMA, J„ Anal. Chem. 4 6 5 (1 9 7 4 ) 73R .
[4 ] GAYLOR, V .F ., JAMES, H .L., WEETALL, H.H ., Anal. Chem. 4 8 5 (1 9 7 6 ) 44 R .
[5 ] D AR K , W .A., LIMPERT, R .J., J. Chromatogr. Sei. 11 3 (1 9 7 3 ) 114.
[ 6 ] LAUB, R .J., R es./D ev. 1974 7 ( 1 9 7 4 ) 2 4 .
[ 7] COOPER, A .R ., BA RR A LL, E.M., J. A ppl. Polym . Sei. 17 4 (1 9 7 3 ) 1253. J
[ 8 ] COOPER, A .R ., JOHNSON, J.F ., PORTER, R .S., Am . Lab. 1973 5 (1 9 7 3 ) 12.
[9 ] SPATORICO, A .L ., BEY ER, G .L., J. Appl. Polym . Sei. 19 11 (1 9 7 5 ) 2 9 3 3 .
[1 0 ] HIATT, C.W., SHELOKOV, A ., ROSENTH AL, E.J., GALIMORE, J.M ., J. Chromatogr. 56 (1 9 7 1 ) 362.
[ I I ] HAWK, G.L., CAM ERON, J.A ., D U FA U L T , L.B., Prep. B iochem . 2 2 ( 1 9 7 2 ) 193.
[1 2 ] COOPER, A .R ., JOHNSON, J.F ., J. Appl. Polym . Sei. 13 7 ( 1 9 6 9 ) 1487.
[1 3 ] VOLK, B.G., SCHNITZER, M., Soil Sei. Soc. A m ., Proc. 37 6 (1 9 7 3 ) 886.
[1 4 ] LOEPPERT, R .H ., VOLK, B.G ., Soil Sei. Soc. Fla., Proc. 33 (1 9 7 4 ) 160.
[1 5 ] STEVENSO N , F.J., “ Chem ical fractionation o f organic m atter” , M ethods o f S oil A nalysis (BLACK , C .A ., Ed.),
Am. Soc. A gronom y, M adison, W isconsin (1 9 6 5 ).
[1 6 ] GELOTTE, B., J. Chromatogr. 3 ( 1 9 6 0 ) 3 3 0 .
[1 7 ] DEM ETRIOU, J.A ., M ACIAS, F.M ., M cARTHUR, M.J., BEATTIE, J.M., J. Chromatogr. 3 4 ( |l9 6 8 ) 3 42.
[1 8 ] NEDD ER M EY ER, P .A ., ROGERS, L.B., Anal. Chem. 4 0 ( 1 9 6 8 ) 7 5 5 .
[1 9 ] FO R SS, K.G., STEN L U N D , B .G ., J. Polym . Sei., Part C 42 (1 9 7 3 ) 951.
[2 0 ] POSNER, A.M., Nature (L on d on ) 198 (1 9 6 3 ) 1161.

DISCUSSION

O.H. DANNEBERG: I can confirm your results regarding the influence of the salt concentra­
tion of the eluant. We have also noticed that humic substances were completely excluded when
eluted with distilled water.
250 LOEPPERT and VOLK

T. WEICHELT: What were the chemical reasons or criteria (polarization, yield, content, etc.)
for using the various organic solvents to extract and, primarily, to fractionate humic substances?
R.H. LOEPPERT: Our reasons for using organic solvents for extraction and fractionation
of soil humic substances are threefold.
(1) Several dipolar aprotic solvents (e.g. DMF, DMSO) have been shown to be excellent solvents
for extracting large quantities of soil organic matter from organic soils previously treated with
dilute HC1 to lower the ash content [14]. Humic acid and fulvic acid fractions extracted from
this soil are 100% soluble in DMF or DMSO at 0.1% concentration.
(2) The porous glass or porous silica packing materials are compatible with aqueous and organic
solvents. The tendency of the silica surface to assume a negative charge and the exclusion on
anions in aqueous solvent systems has been well documented [2]. Therefore we wished to
determine whether the exclusion phenomena would occur in various organic solvent systems.
(3) Many of the monomers of the humic acid or fulvic acid polymers suggested by Schnitzer,
Flaig, Ogner and others, are soluble in cyclic ethers or protic organic solvents. Therefore, we
are interested in determining the adsorption and exclusion behaviour of standard compounds in
various solvent systems on the porous glass packing materials.
T. WEICHELT: What can you say about the importance of the sequence of the organic
solvents used to extract the substances in question?
R.H. LOEPPERT: With the sequence of organic solvents used in the fractionation scheme
we were successful in obtaining fractions with components of greater chemical similiarity. There­
fore, we were able to evaluate interactions between the chromatography packing materials and
soil humic compounds on a variety of fractions with more definable characteristics.
 SLUDGE
(S e s s io n 8 b )
 IA E A -SM -211/74

M U N IC IP A L S L U D G E A S O R G A N IC F E R T IL IZ E R
W ITH S P E C IA L R E F E R E N C E T O
T H E H E A V Y M E T A L S C O N S T IT U E N T S

N. EL-BASSAM, C. TIETJEN
Institut für Pflanzenbau und Saatgutforschung
der Forschungsanstalt für Landwirtschaft,
Braunschweig-Völkenrode,
Federal Republic of Germany

R a p p o rteu r: F. Jacquin

i
Abstract I

MUNICIPAL SLUDGE AS ORGANIC FERTILIZER WITH SPECIAL R EFERENCE TO THE H EAVY

METALS CONSTITUENTS.
Municipal sludge is a p oten tial source o f plant nutrients, but the presence o f to x ic elem ents and com pounds
m ay pose a lim itation for its exten sive use. Experim ents on sludge-treated fields have show n that am ounts up
to 3 0 0 0 m 3 digested sludge/ha did n o t reduce crop yield. The residual effec t o f even lo w rates ( 1 4 0 m 3/h a) is
still visible in the fifth year after application. In addition, the investigations indicated that rates o f
1 0 0 —2 0 0 m 3/h a yearly influenced on ly slightly the soil-water at 1 m or deeper. Heavier application rates
led to an increase in nitrate co n ten ts o f the soil-water and to som e ex ten t o f Ca, Mg, Cl, К as w ell as Zn and Cd.
It is proposed that the am ount o f sludge applied annually on agricultural land should be determ ined tjy the
nitrogen con ten t. The requirem ents o f plants for nutrients also m ust be considered. The lim it for nitrogen
m ay be 2 0 0 m 3 digested sludge ( 5 % dry m atter)/ha annually, w hich is equivalent to about 2 0 0 —4 0 0 k g N /ha.
A t this rate also the needs o f plants for phosphorus w ill be covered. A form ula for determ ining the to ta l
sludge load from the m etallic con ten t is suggested.

INTRODUCTION

The recycling of municipal wastes for soil productivity has recently become necessary
because of the increase in commercial fertilizer prices as a result of shortages and the energy crisis.
Municipal wastes contain plant nutrient elements that can contribute to ensure food production.
Apart from pathogenic bacteria and virus, these wastes may contain toxic elements and
compounds that impair the soil-water-plant system.
Efforts should be intensified to prevent the discharge of heavy metals into the sewage
systems by industry. Until this can be achieved we must endure the heavy metals, and find the
means to protect the environment from contamination.
Our prime concern with waste recycling here is essentially its application to agricultural
land. The soil has to fulfil a double function, namely to be a medium for plant production, and
to act as a physical, chemical and biological filter. Therefore, it is essential that phytotoxic
effects should be eliminated and also the ground-water must be protected from contamination.
The accumulation of toxic elements in soils should consequently not be allowed to exceed
certain tolerable levels. Work has been undertaken by several researchers [1—6] to establish
procedures for controlling the use of sludge on land.
We report here the results of our study on the influence of digested sludge on plant growth,
especially in relation to the extent of soil-water contamination.

253
254 EL-BASSAM and TIETJEN

TABLE I. CONCENTRATION OF NITROGEN

COMPOUNDS IN THE SLUDGE

Material Total-N NH3-N N 0 3-N N 0 2-N

(% dry w t) ■

D igested sludge 4 .2 0 1 .6 6 0 .03 0 .003

Dried sludge 1.84 0.16 0.01 -

TABLE II. INFLUENCE OF DRYING ON CARBON:

NITROGEN RATIO OF THE SLUDGE

Material Total-N Total-C C :N

(% dry w t)

D igested sludge 4 .2 0 3 1 .4 2 7.5

Dried sludge 1.84 3 0 .0 2 16.3

TABLE III. ELEMENTAL COMPOSITION OF SLUDGE

Percentage Parts per

dry weight m illion
dry weight

Рг0 5 2 .9 7 Fe 26 0 0 0

K20 0.44 Pb 27.3

N ajO 0.36 Zn 1 242

CaO 6 .2 7 Cu 291

MgO 0.76 Ni 158

S04 3.16 Cd 4

a 0 .6 7 Hg 3

PROCEDURE

The experiments were conducted in the field of the Institute of Plant Production in 1972.
From July 1972 to May 1973, a municipal digested sewage sludge was uniformly distributed
by a nozzle sprinkler on a sledge. The amounts of digested sludge supplied to the field were 0,
140, 1050 and 3050 m3/ha in 0, 1,7 and 25 applications respectively.
In order to follow the migration of some compounds and elements, 48 suction units [7]
were installed, at depths of 1 and 2 m in the soil. The soil solution was collected every two weeks
by means of continuous suction [8] for analysis. The plants grown were rape, fodder maize and
wheat.
 IA E A -SM -211/74 255

Е С

F I G .l. T h e s p e c ific e le c tr ic a l c o n d u c tiv ity o f th e s o il s o lu tio n .

SLUDGE ANALYSIS

The nature and concentration of various constituents of sludges should be ascertained

before the sludge is applied. The analysis showed that most of the nitrogen in the sludge was
in the ammoniacal form, which is rapidly nitrified by bacteria [9]. Drying of sludge leads to loss
of nitrogen (Tables I and II). The ratio of the major nutrient elements, especially with К which
is low, is unbalanced.
The most predominant metal in the sludge is iron, followed by zinc, copper, nickel and lead
(Table III). Their amounts can be considered representative of municipal sludge having less
industrial wastes.

MIGRATION OF THE SLUDGE COMPONENT IN SOIL

The analysis indicated that sludge application increased the electrical conductivity (Fig. 1)
and thus the salt concentration of the soil solution at the 1 —2 m depth. The electrical con­
ductivity reached 3 mmhos/cm with a rate of application of 3050 m3/ha at the end of 1974.
The nitrate content of the soil-water was enriched up to 600 ppm (Fig. 2).
Heavier application (more than 1000 m3/ha) increased the zinc content of the soil-water
(Fig.3) also. A slight but significant increase in the cadmium concentration in the first year of
application has been observed. A slight rise in the concentration of Ca, Mg, Cl, К was connected
with higher application rates. Migration of Cu and Ni was not observed. '

CROP RESPONSE

Several treatments and trials were conducted, and many crops were grown to investigate
the initial and residual effect of the sludge. Only a few results can be given and discussed here
(Table IV).
It is clear from Table IV that the higher rates above 1000 m3/ha did not produce yield
surplus at the beginning, whereas in the second year the residual effects were observable. It is
interesting to note that the residual effects of even low rates (140 m3/ha) were significantly
high by the end of the fourth year. The work relating to the effects of high concentrations of
these compounds on crop yield is still in progress.
256 EL-BASSAM and TIETJEN

F I G .2 . N itr a te c o n c e n tr a tio n o f th e s o il s o lu tio n .

F I G .3 . Z in c c o n c e n tr a tio n o f th e s o il s o lu tio n .

TABLE IV. YIELD OF RAPE (GREEN), MAIZE (GREEN)

AND WHEAT (GRAIN + STRAW) AS INFLUENCED BY
DIFFEREN T RATES OF MUNICIPAL SLUDGE

Sludge Y ields (t/h a dry m atter)

applied 1 9 7 2 —73
1973 1974 1975
(m 3/ha)
Rape Maize Wheat

0 1.54 4 .5 5 3.67

140 3.35 6 .5 0 5.08

1050 3.2 9 8 .18 6.45

3050 3.46 10.47 11.01

 IAEA-SM-211/74 257

TABLE V. TOLERABLE AMOUNTS OF SOME

ELEMENTS IN SOILS WITH REGARD TO THEIR
PLANT TOLERANCE

Element Tolerable ^
(ppm)

B ea 10
В 100
F 500
Cr 100
Ni 100
Co 50
Cu 100
Zn 300
As 50
Se 10
Mo 10
C da 5
Hga 5
Pb 100

a Special reservation.
b Proposed amounts.

APPLICATION TO AGRICULTURAL LAND

The use of sewage sludge on agricultural land has a big potential for improving soil fertility
and crop production. The only drawback in the sludge is the presence of pollutants that may
have a deleterious effect, particularly over a long period of time.
However, our studies over the last few years reveal that the heavy use of sludge did not
reduce the yield because the whole amount was not supplied in one charge but in several
applications. On the other hand, heavier applications increased the concentration of some com­
pounds and elements, namely nitrates, Ca, Mg and Zn in the soil solution.
On the basis of our investigations and those of others [1—6, 10—12], it is proposed that
the amount of sludge to be applied annually to farm land should be assessed and fixed on the
basis of the nitrogen content of the sludge and the plant needs. The limit for N may be about
200 m3 (5% dry matter)/ha yearly, supplying 200-400 kg N/ha. We have found that the
N content in this range did not contaminate the soil-water. The amount of phosphorus added
at this rate will cover the needs of plants for this element.
A formula for arriving at the total duration of sludge application, given the concentration
of any metal in the sludge, is:

(TCms —IQns) X 4.2 X 106 I

n=
Cm w X Ww
258 EL-BASSAM and TIETJEN

where
n = number of years until the tolerable levels have been achieved
TCms = tolerable metal concentration in soil in ppm according to Table V
ICms = initial metal concentration in soil in ppm
Cmw = concentration of the metal in waste material (e.g. sludge) in ppm in the dry matter
Ww = weight of waste material in kg dry matter/ha yearly.
The number of years in which the sludge can be used as organic fertilizer without
exceeding the tolerable limits with several heavy metals (cumulative effect) may be calculated
as follows:

(TCzn ~ ICzn) X 4.2 X 106

Cznew X Ww
where
ТСи, = tolerable concentration of zinc in the soil (ppm) according to Table V
ICZn = initial zinc concentration in soil (ppm)
Cznew= concentration of zinc equivalent in waste material (= (ppm Zn) + 2 (ppm Cu)
+ 4 (ppm Ni) + 100 (ppm Cd).
The tolerable limits for some toxic elements in soil with regard to their plant compatibility
have been summarized in a proposal of the Board of Public Health of the Federal Republic of
Germany (Table V) [13].
These equations hold good for medium soils of pH 6.5 and up to 30 cm depth, and can
also be used with other types of soil and depth, with appropriate corrections.

REFERENCES

[ 1] CHUMBLEY, C.G., Permissible Levels o f Toxic Metals in Sewage Sludge used on Agricultural Land,
A.D.A.S. Advisory Paper No. 10, Ministry o f Agriculture, Fisheries and Food, Wolverhampton,
England (1971).
[2] LEEPER, G.W., Reactions o f Heavy Metals with Soil with Special Regard to their Application in Sewage
Wastes, Dept, o f Army, Corps o f Eng., Cont. DACW 73-73-C-0026 (1972).
[3] CHANEY, R.L., “Crop and food chain effects o f toxic elements in sludges and effluents”, Proc. Joint
Conf. Recycling Municipal Sludges and Effluents on Land, Champaign, III, EPA, USD A, NASULGC,
Washington, D.C. (1973) 129.
[4] DE HAAN, S., Die chemische Zusammensetzung von Gewächsen auf mit Klärschlamm behandelten
Böden, Landwirtsch. Forsch. 31 1 (1975) 220.
[5] WALKER, J.M., Sewage sludges — Management aspects for land application, Compost Sei. 16 2
(1975) 12.
[6] SHIPP, R.F., BAKER, D.E., Pennsylvania’s sewage sludge research and extension program, Compost
Sei. 16 2 (1 9 7 5 ) 6.
[7] CZERATZKI, W., Saugvorrichtung für kapillar gebundenes Wasser, Landbauforsch. Völkenrode 21
(1971) 13.
[8] EL-BASSAM, N., Aussagewert der chemischen Zusammensetzung einer durch Saugvorrichtung gewonnenen
Bodenlösung, Landbauforsch. Völkenrode 22 (1972) 37.
[9] EL-BASSAM, N., TIETJEN, C., MERTENS, F., Nitrat-, Nitrit- und Ammoniak-N im Boden und im
Bodenwasser sowie Aufnahme durch Markstammkohl und Zuckerrüben bei Zufuhr hoher Klärschlamm­
gaben, Landwirtsch. Forsch. 30 2 (1974) 29.
[10] TIETJEN, C., Principal problems o f the use o f city wastes for crop production and soil conservation,
Soils Bull. No. 27, FAO, R om e(1975) 211.
[11] KLOKE, A., Beeinträchtigung der Qualität von Nahrungs-und Futterpflanzen durch Umweltchemikalien,
insbesondere durch Schwermetalle, Qual. Plant. 24 1/2 (1974) 137.
[ 12] KICK, H., Anwendung von behandelten Siedlungsabfällen auf land- und forstwirtschaftlichen Nutzflächen,
Ber. Landwirtsch. 50 1 (1972) 69.
[13] BUNDESGESUNDHEITSAMT, INSTITUT FÜR WASSER-, BODEN- UND LUFTHYGIENE,
Fachliche Unterlagen für Rechtsverordnung nach § 15, Abfallgesetz (1974).
 IAEA-SM-211/75

E P A N D A G E D E B O U E S R E S ID U A IR E S :
R E P E R C U S S I O N S S U R L ’A Z O T E D U S O L
E T L E P R E L E V E M E N T P A R D U R A Y -G R A S S
D E C d , C r, H g E T Z n

J.C. FARDEAU, G. GUIRAUD,

Jocelyne JAPPE, Gisele LLIMOUS
Departement de biologie,
Service de radioagronomie,
CEA, Centre d’etudes nucldaires
de Cadarache,
St-Paul-lez-Durance,
France

R a p p o rteu r: F. Jacquin

Abstract-Rdsumd

APPLICATION OF RESIDUAL SLUDGE: EFFECTS ON SOIL NITROGEN AND RETENTION OF Cd, Cr, Hg
AND Zn BY RAY GRASS.
In pot studies on the application o f urban sewage rich in organic matter as fertilizer (residual sludges from
sewage purification stations), an investigation was made o f the yield in dry matter and retention o f Zn, Cd, Cr and
Hg by ray grass, followed by an examination o f the mineralization and organization o f soil nitrogen or added
nitrogen fertilizers. All the experiments described were conducted with tracers —radioactive in the case o f Hg, Cd,
Cr and Zn, and stable in the case o f nitrogen. The main results obtained after six months’ cultivation can be
summarized as follows: (1) the suppressing effect produced immediately after application o f a lime-treated sludge
falls o ff rapidly (2 —3 months), whereas the effect produced by straw persists even after six months. The sludges
subsequently exert a positive effect on the yield o f dry matter; (2) in the plant the transfer of the trace
elements studied is not altered by adding residual sludges whereas the addition of 30 ppm of these elements
to the soil in ionic form increases the plant content; (3) although the incorporation o f straw into the soil
considerably increases the organization o f the nitrogen in the fertilizer applied, the changes are limited in the
case o f other forms o f treatment.

EPANDAGE DE BOUES RESIDUAIRES: REPERCUSSIONS SUR L’AZOTE DU SOL ET LE PRELEVEMENT

PAR DU RAY-GRASS DE Cd, Cr, Hg ET Zn.
Dans des etudes en vases de vegetation sur les epandages ä but agricole de d£chets urbains riches en matieres
organiques (boues residuaires de stations depuration d’eaux usees) on a suivi d’une part le rendement en mattere
seche et le pr616vement de Zn, Cd, Cr et Hg par du ray-grass et d’autre part la mineralisation et l’organisation de
l’azote du sol ou des engrais nitriques ajoutes. Toutes les experiences decrites ont ete conduites avec traceur,
radioactif pour Hg, Cd, Cr et Zn, stable pour l’azote. Les principaux resultats obtenus aptes 6 mois de culture
peuvent etre resumes comme suit: 1) L’effet depressif cause immediatement apres ltepandage d’une boue chautee
s’attenue rapidement (2 a 3 mois) alors que celui provoqu6 par la paille se poursuit encore apres 6 mois. Par la
suite les boues provoquent un effet positif sur le rendement en mattere seche. 2) Le transfert dans la plante des
oligo-eiements etudtes n’est pas modifte par l’apport de boues residuaires alors que l’addition de 30 ppm de ces
elements au sol sous forme ionique augmente la teneur des veg6taux. 3) Alors que l’enfouissement de paille
accroit considerablement l’organisation de l’azote de l’engrais apporte, les modifications restent limitees pour les
autres traitements.

259
260 FARDEAU etal.

INTRODUCTION

Des composts urbains et des boues residuaires sont epandus de maniere empirique dans les
zones proches de leur production. Cette pratique, moyen simple pour faire «disparaftre» les
dechets produits, est en general interessante au plan economique pour l’agriculteur, dont la
motivation la plus frequente est celle d’une recherche de matiere organique pour ses sols. On
dispose lä de melanges variables au cours du temps pour lesquels de multiples inconnues subsistent
quant aux causes de leur valeur reelle et aux risques que leur emploi intensif durable presenterait
du fait des differents constituants qu’ils recelent et plus particuliercment des metaux lourds
associes aux matieres organiques. Dans ce memoire, on examine d’une part la valeur fertilisante,
au moyen de quelques donnees relatives au turn o v e r de Tazote en presence des dechets et, d’autre
part, les risques encourus, par Tobservation du transfert du melange sol-dechets vers la plante avec
des isotopes radioactifs de Hg, Cr, Cd et Zn.

1. MATERIEL ET METHOD ES

1.1. Les boues residuaires

Les boues etudiees sont de deux types tres differents:

— La premiere (B2) est une boue digeree anaerobie de la station d’epuration de Maxeville
pres de Nancy stabilisee par le sulfate d’alumine et la chaux. La teneur en carbone organique
est de 15,78% par rapport ä la matiere seche, le rapport C/N de 11, le pH de 11,75 et la teneur en
eau de 73%. Les concentrations en ppm par rapport ä la matiere seche de Zn, Cr, Cd et Hg sont
respectivement de 3200, 290, 40 et 10. La caracteristique agronomique essentielle de cette boue
est un pH tres eleve qui a rendu son epandage equivalent ä un chaulage.
— La seconde (B4) est issue d’une laiterie des environs de Nancy dont les eaux sont epurees
sur lit bacterien. Elle est conditionnee par un polydlectrolyte: la teneur en eau est de 96.6%, la
teneur en carbone de 35,7% par rapport ä la matiere seche, le rapport C/N de 6 et le pH de 6,5.
Les teneurs en oligo-elements: Zn, Cr, Cd et Hg, sont de 16 500, 75, 10 et 2 ppm par rapport ä la
matiere seche. Ses caracteristiques agronomiques principales sont done une forte teneur en eau qui
pourra limiter l’apport de matiere seche dans le sol et un rapport C/N tres bas.

1.2. Le sol

II est calcaire (C03Ca = 22%) et limono-argileux. Le pH est de 7,9, la teneur en azote total
de l%o.

1.3. E tude de Hg, Cd, Zn e t Cr: Emploi des isotopes

Nous avons fait appel aux deux techniques mises au point au laboratoire.

1.3.1. A v e c p la n te

La methode consiste ä introduire le traceur [ 1] dans la fraction assimilable du sol puis ä

melanger le sol radioactif ä la substance dont on veut mesurer les repercussions [2]. Pour chacun
des oligo-eldments on a marque 7 kg de sol [3] permettant de realiser les traitements suivants:

—sol marqud seul servant de reference

- sol marqud additionnd de 30 ppm de Telement
 IAEA-SM-211/75 261

—sol marque additionne de 5% de boues B2

— sol marque additionne de 5% de boues B2 + 30 ppm de l’element
— sol marque additionne de 10% de boues B4
— sol marque additionne de 10% de boues B4 + 30 ppm de l’element
— sol marque + paille ä raison de 2%.

Les pots de culture de 200 g sont semes de ray-grass. Les recoltes sont regulierement analysees.

1.3.2. Sans p la n te

La methode consiste ä etudier les indices caracteristiques des cinetiques de dilution isotopique
des ions dans un melange de 10 g de sol et 100 ml de solution [4]. On rappelle seulement que si Гоп
introduit dans un tel Systeme une quantite de radioactivite R, la radioactivite r restant eri solution au
temps t (exprime en minutes) est donnee par la formule r = r2 X tn, r! etant la radioactivite
presente ä l’instant unite (1 min). Cette formule est äquivalente ä Log r = Log r; + n Log t. Elle est
repräsentee par une droite en coordonnees logarithmiques.

1.4. Etude du turn over de l’azote

On melange 100 ppm d’azote nitrique contenant 32,37% d’azote-15 en exces au sol seul, au
sol additionne des boues B2 ä 5% et B4 a 10%, au sol additionne de paille ä 1%, au sol adiiitionne de
boues B2 et B4 et de paille. Ces melanges sont ensuite mis ä incuber ä 22°C environ et ä humidite
süffisante. Des prälevements effectues au cours du temps permettent de suivre revolution de
l’incorporation de l’azote mineral dans la fraction organique [5].

2. RESULTATS

2.1. Effet sur la matiere seche produite (tableau I)

On peut remarquer que:

— aucun des apports de boues n’a provoque d’effet depressif; qui plus est, un effet positif se
manifeste des le debut sur la vegetation en presence de la boue de laiterie et ä partir de la seconde
coupe avec la boue stabilisee ä la chaux;
— le chrome sous forme de chromate ä la dose de 30 ppm inhibe toute germination; on doit
noter que la presence des boues limite cette toxicitä et permet l’implantation de la vegetation.

2.2. Evolution de l’azote

L’azote du sol apres 94 jours d’incubation a ete separe en 2 fractions: les nitrates extraits ä
l’eau et l’azote restant. On donne dans le tableau II les valeurs des exces isotopiques de ies deux
formes ainsi que la fraction de l’azote introduit retrouvee dans lesdites formes.
On peut faire les remarques suivantes:

— la comparaison des exces isotopiques des nitrates montre que l’apport des boues a favorise
la mineralisation puisque les exces sont plus faibles avec les boues qu’en leur absence;
— la comparaison des pourcentages d’azote presents dans l’azote residuel ainsi que |celle des
exces isotopiques montre une limitation de (’organisation de l’azote et ce plus particulierement
avec la boue chaulee; ces resultats semblent confirmer que si des apports de matiere orgajnique
ont un rapport C/N inferieur ä 25, la mineralisation globale l’emporte sur l’organisation [6].
262 FARDEAU etal.

TABLEAU I. MATIERE SECHE RECOLTEE (m g /k g d e sol)

Sol Sol + Sol + Sol + Sol + Sol + Sol + Sol + S0l +

seul РаШе 30 ppm de 30 ppm B2 в4 B2+ B4+ B4+ Cr
Hg, Zn ou Cd de Cr 30 ppm 30 ppm
Hg, Zn Hg, Zn
ou Cd ou Cd

1ere coupe 1636 6 i6 1450 0 1725 1615 1680 1500 336

2 e coupe 1750 1174 1638 0 3840 3863 4014 3240 3543

TABLEAU II. EVOLUTION DE L’AZOTE INTRODUIT (N t = a zo te restan t)

Sol Sol + Sol + B2 Sol + B2 Sol + B4 Sol + B4

seul paille + paille + paille

Exces no3 21,48 4,26 1 2 ,4 3 1,91 13,35 5,55

isotopique 0,38 2,13 0 ,7 7 0,54 0,059 1,36
Nr

Azote N 03 93,3 1 ,9 64,4 1,87 62,9 13,7

introduit 67,4 41,4
N, 1 0 ,1 0 ,2 2 0 ,2 1 ,8
(%)

TABLEAU III. ACT1VITE SPECIFIQUE ET TENEUR EN Zn DES PLANTES

Sol S0l + Sol + Sol + Bj + Sol + Sol + B 4 + SOl +

seul 30 ppm Zn B2 30 ppm Zn B4 30 ppm Zn paille

Activite specifique 113 29,8 105 28,6 125 36,3 100

de Zn
Teneur des plantes 63,8 112,5 53,7 121,2 70 11,7 80
(ppm Zn/MS)

F I G .l. D i l u t i o n i s o t o p i q u e d e H g 1' e r i p r e s e n c e e t e n V a b s e n c e d e b o u e s c h a u l e e s .
 IAEA-SM-211/75 263

2.3. Effets sur certains oligo-dlements

2 .3 .1 . E ffets au niveau d e la reco lte

On donne comme exemple les resultats obtenus dans le cas du zinc (tableau III).
On peut faire les commentaires suivants:

— seuls les apports d’elements sous forme ionique marquent de maniere significative et se
repercutent sur les teneurs dans les plantes;
— les resultats sont similaires pour les trois autres radioelements utilises;
— des contröles sur la teneur en manganese des plantes au cours de la troisieme con pe
indiquent que celle-ci est constante pour les differents traitements.

2 .3 .2 . E ffets au niveau du p o o l iso to p iq u e m e n t diluable

Afin de nous assurer que la plante ne masque pas certains effets transitoires, ces donnees
ont dte completees par quelques cinetiques de dilution isotopique. Le mercure a foumi les
resultats graphiques les plus interessante (figure 1).
II ressort que l’addition de boue B2 a provoque une diminution considerable de la retention
du mercure par le sol dont le pH a atteint 10,8. On pourrait conclure ä l’existence d’une
discordance avec les resultats sur plantes qui ne font pas apparaftre d’augmentation significative
des teneurs. Si Гоп realise sur le sol incube 94 jours avec des boues des cinetiques de dilution
isotopique, on constate un retour i la courbe du sol temoin et au pH initial. On peut enjdeduire
que le pH du sol a repris sa valeur d’equilibre avant que les plantes n’aient eu la possibilite de prelever
en quantite importante le mercure. II у a lieu de noter que pour Cd la boue chaulee provoque
initialement le phbnomene inverse, ä savoir un accroissement de la retention.

CONCLUSION

Ces resultats preliminaires montrent combien il est difficile de prevoir les effets ä long terme
de l’epandage de boues residuaires. On constate qu’un apport sur une periode limitee ä 3 mois,
d’une part favorise la mineralisation de l’azote et de la sorte augmente les risques de lessiyage et
les pertes par denitrification, et d’autre part ne conduit pas dans les plantes ä des modifications
sensibles des teneurs en oligo-elements lourds, du moins pour les systemes sol-dechets etudies ici.
Mais il est evident que ces seules donnees n’autorisent pas ä prevoir les repercussions d’apports
cumules sur une longue pdriode. Cependant tous ces oligo-elements sont fortement reteJius par les
particules de sol comme le montrent les cinetiques de dilution isotopique de ces ions dans les systemes
sol-solution. Dans la mesure oü ce comportement est identique ä celui des ions phosphate il est
possible de preconiser les memes methodes et de prevoir les memes conclusions. Il n’en feste pas
moins indispensable d’obtenir des mesures directes ä partir d’echantillons de sol recevant de tres
longue date des residue urbains.

REMERCIEMENTS

Ce travail a ete partiellement finance par le Ministern fran9 ais de la Qualite de la Vie dans le
cadre d’une action concertee sur les sols et dechets solides (Action SDS n° 74 039).
264 FARDEAU etal.

REFERENCES

[1] FARDEAU, J.C., GU1RAUD, G., Phosphore assimilable du sol et des engrais determine par la m6thode de
dilution isotopique, Phosphore et Agriculture 60 (1972) 2 3 -3 0 .
[2] GACHON, L., «Appr6ciation de l’efficacite relative de differentes formes chimiques d’engrais phosphates;
application aux sols andiques du Massif Central Frangais», (C.R. Congr. int. des engrais chimiques,
2 1 - 2 7 juin 1976, Moscou), Moscou (1976) 3 8 7 -9 6 .
[3] FARDEAU, J.C., TRIBOI, E., Mesure au champ du phosphore disponible l V , Plant Soil 41 2 (1976) 2 93—302.
[4] FARDEAU, J.C., GUIRAUD, G .,“Use o f 32P04 isotopic dilution kinetics: expectation of fertilizing effect of
some phosphatic fertilizer” , (C.R. Congr. int. des engrais chimiques, 2 1 —27 juin 1976, Moscou),
Moscou (1976) 3 6 2 -7 0 .
[5] GANRY, F., GUIRAUD, G., DOMMERGUES, Y., “Effect o f straw incorporation on the yield and nitrogen
balance in the sandy soil-pearl millet cropping system o f Senegal” (C.R. Coll. int. Jerusalem, 1976), a paraftre.
[6] VAN VEEN, J.A., FRISSEL, M.J., Computer simulation model for the behaviour of nitrogen in soil and of
leaching to ground water, Association ITAL-EURATOM, External Rep. No.30 (1975).
 IA E A jSM -211/76

D E T E R M IN A T IO N D E U A P T I T U D E A L A
B I O D E G R A D A T IO N D E B O U E S R E S I D U A I R E S
D ’O R I G IN E S D IV E R S E S
A c tio n s u r le s p r o p r ie te s p h y s ic o - c h im iq u e s d u s o l

J.L. MOREL, F. JACQUIN,

Laboratoire Matiere organique et environnement,
Ecole nationale superieure d’agronomie
et des industries alimentaires,
Nancy,
France

R a p p o rteu r: F. Jacquin

Abstract-Risumö

DETERMINATION OF THE BIODEGRADATION CAPACITY OF RESIDUAL SLUDGES OF VARIOUS

ORIGINS: EFFECT ON THE PHYSICAL AND CHEMICAL PROPERTIES OF THE SOIL.
In the course o f incubation experiments we observed the biodegradation in the soil o f 12 residual sludges
and four forms o f organic matter taken as reference substances. The sludges enhance the biological activity o f the
soil, though they do so in different ways, depending on the type in question. There is no close relationship between
the C/N ratio and the mineralization level in the sludges. Those originating from dairy water purification systems
are biodegraded more easily than urban sludges; among the latter those that undergo lime flocculation treatment
are the hardest to biodegrade. Study o f the humification o f three o f the sludges shows a favourable effect on the
amount o f bound organic matter, more especially the humins. Decomposition o f the sludges in the sdil is
accompanied by an improvement in its structural stability. The chemical properties of the soil undergo changes,
characteristic o f the basic composition o f the sludge (pH, exchangeable elements, assimilable phosphorus, total
nitrogen). Mineralization o f the sludges is accompanied by a release o f quite considerable quantities c>f nitric
nitrogen, which may present pollution problems. This test is relatively reliable and reproducible; the data obtained
can be used for locating a sample on a reference scale as a function o f capacity for mineralization in the soil and
fertilizing potential.

DETERMINATION DE L’APTITUDE A LA BIODEGRADATION DE BOUES RESIDUAIRES D’ORIGINES

DIVERSES - ACTION SUR LES PROPRIETES PHYSICO-CHIMIQUES DU SOL.
Au cours d’experiences d’incubation, nous avons suivi la biodegradation dans le sol de douze boues
residuaires et de quatre substances organiques prises comme reference. Les boues augmentent l’activi^e biologique
du sol, mais de fa?on differente selon le type considere. II n’existe pas de relation etroite entre le rapport C/N et
le niveau de mineralisation des boues. Celles qui sont issues de Imputation des eaux de laiterie sont biodegradees
plus facilement que les boues urbaines; parmi ces demieres celles qui ont subi un traitement de floculation ä la
chaux sont plus difficilement biodegradables. L’etude de l’humification de trois d’entre elles montre Un effet
favorable sur la quantite de matiere organique Иёе, en particulier sur les humines. La decomposition Jdes boues
dans le sol s’accompagne d’une amelioration de la stabilite structurale de ce dernier. Les propri6tes chimiques
du sol subissent des modifications propres a la composition elementaire de la boue (pH, elements echangeables,
phosphore assimilable, azote total). La mineralisation des boues s’accompagne d’une liberation d’azote nitrique non
negligeable, ce qui risque de poser des problemes de pollution. Ce test est Telativement fidele et reproductible;
par ses resultats, il permet de situer un echantillon au sein d’une echelle de reference en fonction de l’aptitude ä la
mineralisation dans le sol et des potentialites fertilisantes. !

265
266 MOREL et JACQUIN

INTRODUCTION

Afin de contribuer ä leur elimination, l’epandage agricole des boues residuaires constitue une
possibility interessante. En effet, leur forte teneur en matiere organique et en certains elements
fertilisants permettrait de les assimiler ä un amendement organique. Pour cette raison il nous
a paru ndcessaire de verifier si les boues residuaires constituaient un materiau metabolisable par les
micro-organism es du sol, et de comparer leur comportement ä celui de substances organiques
classiques (glucose, paille de bie, foin de luzerne, tourbe acide).
Nous avons done entrepris Гetude in vitro de la biodegradation et de l’humification d’une
douzaine de boues residuaires d’origines et de traitements varies, incorporees ä un sol agricole de
reference. Nous avons egalement determine l’influence de ces mdcanismes sur les proprietes
physiques du sol, notamment sur la stabilite structural sur laquelle l’effet de la matiere organique
est maintenant bien connu [1,2]. De plus nous avons voulu verifier si l’apport de boues dans le
sol s’accompagnait de modifications de ses proprietes chimiques, en particulier l’etat du complexe
absorbant, le pH, les concentrations en phosphore assimilable et en azote total et mineral.

TABLEAU I. CARACTERISTIQUES DES STATIONS D’EPURATION

Traitement

Origin e Traitement des eaux Stabilisation Conditionnement - sechage

Boues urbaines
Longuyon Lit bacterien Digeree anaerobie Epaississement gravitaire
Strasbourg Decantation primaire Digeree апаёгоЫе Lit de sechage
Mer&ville Boues activees - Epaississement gravitaire
Selestat Boues activ6es Digeree аёгоЫе Lit de sechage
Fleville Boues activ6es Digeree anaerobie Lit de s6chage
St-Die Boues activ6es Digeree anaerobie Lit de sechage
f Roculation CaC03 + AI2 SO4
Nancy Boues activöes Dig6ree anaerobie
L Sechage filtre sous vide
Metz Boues activees - Boues autoclavees

Boues industries
alimentaires
L a ite r ie s

St-Hubert Boues activees Stabilisation Floculation polyelectrolyte

par contact cationnique
Marcillat3 Boues activees Stabilisation -
par contact
U.L.V. Boues activees Epaississement gravitaire
Bulgneville
B ra s se rie

Obernai Boues activees Digeree апаёгоЫе Floculation CaC03 + FeCl3

R efoit Egalement des effluents urbains.

 IAEA-SM-211/76 267

TABLEAU II. ANALYSE DES BOUES ETUDIEES

Eau A br6viation
O rigine pH C o rg an iq u e3 N ,a C /N
(%)
B oues u rb ain es

Longuyon 89,7 6,6 3 0 ,66 2 ,54 12,07 Lo

S trasbourg 64,8 7,0 2 3 ,13 1,83 12,63

st
M ereville 98,7 7,5 2 2 ,53 3 ,2 0 7 ,0 4 Mv

S elestat 73,1 6 ,7 2 4 ,9 2 3 ,1 0 8 ,0 4 Se

Fleville 47 ,5 7,35 2 9 ,38 4 ,7 7 6 ,1 6 F

St-D ie 7 1 ,0 6,2 3 2 ,6 4 3 ,3 9 9 ,63 SD

N ancy 73,3 12,7 15,88 1,54 10,31 N

M etz 4 5 ,9 6,15 3 7 ,7 6 1,38 2 7 ,3 6 Me

1

B oues in d u stries
alim en taires

L o tte r ie s

S t-H u b ert 9 6 ,4 6,7 4 0 ,4 8 6 ,5 0 6 ,23 SH

M arcillat 98,3 6,8 3 9 ,1 0 8,97 4 ,3 6 mJ

U .L .V . Bulgneville 96,5 6 ,6 5 3 2 ,3 2 8 ,2 4 3 ,9 2 Bu

B ra sserie

O b em a i 79,5 12,0 9 ,1 4 1,61 5 ,6 7 Ob

/

a E n % du p o id s sec ä 105°C.

1. MATERIEL ET METHODES

1.1. Materiel

Le sol et les differentes substances incubees ont ete decrits dans une publication recente [3]
aussi nous contenterons-nous de rappeier les caracteristiques d’origine des boues ainsi que leur
analyse (tableaux I et II).
1.2. Methodes

1.2.1. Incu bation s

Le dispositif respirometrique et les conditions experimentales sont dgalement decrits

dans [3].

1.2.2. M eth o d es d e dosage

1.2.2.1. Evolution de la matiere organique

L’aptitude des amendements ä contribuer ä la constitution de composes humiques a ete

test6e par les methodes classiques:
— separation densimetrique par le melange bromoforme/alcool de densite 1,8;
— extraction selon la methode Duchaufour-Jacquin ä l’aide des r6actifs alcalins suivants:
pyrophosphate et sulfate de sodium ä pH 7, et NaOHN/lO;
268 MOREL et JACQUIN

- fractionnement de la solution d’extraction par precipitation des acides humiques ä pH 1;

- dosage du carbone par voie seche au Carmhograph 12 Wösthoff.

1.2.2.2. Stabilite structural

Elle a 6te evalu6e selon la m£thode Henin [4] apres 21 jours puis apres 6 mois d’incubation.

1.2.2.3. Proprietes chimiques du sol

- pH: mesure par methode 61ectrom6trique apres melange du sol avec H20 dans la proportion
1/2,5;
— Complexe absorbant: pour la capacite totale d’echange, percolation ä Tacetate d’ammonium
neutre et distillation ä l’appareil de Kjeldahl; pour les bases dchangeables, percolation au
ClNaN/2 et dosage en spectromttrie d’absorption atomique;
— Azote total: methode Kjeldahl;
— Azote mineral: extraction ä KCl N et dosage colorimbtrique;
— Phosphore assimilable: methode Truog.

2. APTITUDE A LA BIODEGRADATION

La degradation des boues räsiduaires dans le sol ob£it aux memes lois que celles qui s’appliquent
aux substances de reference; en effet, on distingue toujours sur les courbes de mineralisation les
trois phases caracteristiques (fig. 1):
— un pic de debut d’incubation,
— une phase de diminution de l’activite biologique consecutive ä Tepuisement des substances
facilement metabolisables,
— enfin, une phase de mineralisation lente.
Dans nos conditions experimentales, et apres 21 jours d’incubation, il est possible de classer
les differentes boues [3] et de les situer par rapport aux substances de reference en fonction de leur
efficacite sur Tactivite mineralisatrice du sol (tableau III). Cette derniere, exprimbe par le taux de
mineralisation globale.

est toujours significativement positive pour l’ensemble des boues. Cependant, l’effet est variable
selon le type de materiau teste, et, d’apres les resultats, il est possible de distinguer:

— Les boues ä forte biodegradation telles que celles qui sont issues de l’epuration des eaux
usees des industries laitieres; il s’agit lä effectivement de residue tres riches en matieres organiques
facilement biodegradables, comme Tatteste Tamplitude des pics obtenus lors des premiers jours
d’incubation.
— Les boues moyennement biodegradables qui sont constitutes exclusivement d’echantillons
provenant de stations depuration urbaines ne pratiquant pas la floculation ä la chaux; au sein de
cet ensemble, il ne nous est pas encore possible de discerner l’influence de chacun des traitements
de Stabilisation; tout au plus constatons-nous que les digestions en ana£robiose et surtout en
aerobiose semblent diminuer la mineralisation des boues dans le sol.
 I AE A-SM-211/76 269

— Les boues peu biod£gradables parmi lesquelles on trouve celles issues de decantation
primaire (St)1 et celles ayant subi un traitement de floculation ä la chaux (N, OB). Pour ces
dernieres, il semble que la floculation ait provoque une forte transformation de la matiere
organique de la boue anterieurement ä l’incorporation dans le sol.
Sans l’utilisation de 14C, il est impossible de determiner d’une facon rigoureuse le taux de
min6ralisation propre de chaque substance. Nous avons neanmoins calculi le taux de min6ralisation
complementaire
I
C degage (sol enrichi) —C degage (sol temoin)
~~ ; X 100
C mtroduit
Les ab rev iatio n s s o n t d o u n ce s d an s le ta b le a u II.
270 MOREL et JACQUIN

TABLEAU III. CLASSEMENT DES DIFFERENTES SUBSTANCES EN FONCTION DE

LEUR APTITUDE A LA MINERALISATION
(apres 21 jou rs d'in cu bation )

T a u x de
T aux de
S ubstances m in eralisatio n G ro u p es
m in eralisatio n
incubees co m p lS m en taire ho m o g en es
globale
p o u r 21 jo u rs

G lucose 8,38 53

L uzerne 8,17 50

B oues de Bulgneville 7 ,0 6 42 2

B oues de M arcillat 6,21 35

3
Paille d e Ыё 5,80 30

B oues de L o n guyon 5,45 29 4

B oues de M ereville 4 ,8 4 25
5
B oues de S t-H u b ert 4 ,6 9 24

B oues de St-D ie 4 ,3 0 22

B oues de M etz 4 ,2 9 21 6
B oues de Fleville 4,25 21

B oues de S elestat 3,4 6 14

7
B oues de N ancy 3,2 2 13

B oues de S trasbourg 2,83 9

8
B oues de O b em a i 2 ,6 4 8

Sol t6 m o in 1,81 -
9
T o u rb e 1,70 0,7

a D ’apres le te st de la plus p e tite a m p litu d e significative de N ew m an e t K euls.

permettant d’6valuer la vitesse de disparition des matieres organiques ajoutees au sol (tableau III).
Ainsi, en 21 jours, ces disparitions s’dchelonnent entre 8% et 42% selon le type de boue ajoute.
Dans le meme temps, 50% environ de glucose et de luzerne ont 6t6 mineralises contre seulement
30% de paille; pour cette derniere, l’absence d’un apport d’azote a certainement constitue le
facteur limitant [5].
A l’oppose des amendements organiques classiques, nous ne constatons pas de correlation
etroite entre la biodegradation des boues residuaires et leur rapport C/N. Ce dernier, generalement
bas, favorise certainement leur biodegradation.
Dans les conditions culturales classiques, il est probable que les phenomenes de decomposition
interviennent de faqon plus moderee; en effet, ils dependent etroitement de la temperature, de
l’humidite et de l’homogeneite du contact boue-sol. Tous ces facteurs sont contrölables avec
notre dispositif experimental, ce qui nous a permis d’etablir pour un sol donne une 6chelle de
reference permettant de prevoir pour chaque boue son aptitude ä etre assimiiee par les micro-
organismes dans le milieu naturel.
 IAEA-SM-211/76 271

TABLEAU IV. REPARTITION DU CARBONE DU SOL APRES 6 MOIS DTNCUBa |tION

( m g /1 0 0 g so l)

S u b stan ces C c c c C C c A F /A H
in cu bees to ta l libre lie ex tra it AF AH h u m in e

T em o in 1439 255 1184 215 22 193 969 0 ,1 1 4

Paille d e ble 1542 321 1221 224 28 196 997 0 ,1 4 3
F o in de lu zern e 1556 268 1288 223 26 197 1065 0 ,1 3 2
T o u rb e acide 1686 395 1291 230 28 202 1061 0 ,1 3 8
B oues d e N ancy 1611 294 1317 223 25 198 1094 0 ,1 2 6
B oues de Fleville 1604 357 1247 225 24 201 1022 0 ,1 1 9
B oues de S t-H ubert 1615 347 1268 226 24 202 1042 i 0 ,1 1 8

3. ETUDE DE LA REPARTITION DE LA MATIERE ORGANIQUE DU SOL APRES

INCUBATION

Afin de limiter le nombre des resultats, nous reproduirons uniquement les effets ä long terme
de trois types de boues residuaires. Nous les comparerons ä ceux des trois substances de reference.
Nous avons retenu les boues suivantes:
— une boue urbaine activde, digeree anaerobie, floculee ä la chaux et au sulfate d’alumine (N);
— une boue urbaine activde, digeree anaerobie, sdchee sur lit (F);
— une boue activee de laiterie-fromagerie (SH).
D’apres les resultats consignee au tableau IV, nous pouvons faire un certain nombre de
remarques quant ä Tinfluence des diverses substances organiques sur la repartition du carbone
du sol. I
Comme de nombreux auteurs Tont ddjä souligne, Tapport de boues rdsiduaires au^mente
toujours le taux global du carbone du sol. Cette augmentation est plus importante avec! les
traitements boues qu’avec ceux des references paille et luzerne, la mineralisation du carbone, dans
ces deux derniers cas, etant beaucoup plus importante.
L’intensite de la transformation par mineralisation et humification des substances organiques
ajoutdes a ete dvalude par le taux suivant:

C introduit —C libre sol enrichi —C libre sol temoin

------------------------- —----- ------------------------------ X 100
C mtroduit

II est ä noter que ce taux ne tient pas compte des stimulations intervenant sur la degradation de la
matiere organique libre preexistant dans le sol. Pour les boues, il varie de 57% (F) et 61% (SH) ä
83% (N), ce qui indique, en plus de la mineralisation, une participation relativement importante
ä Tedification de la matiere organique humifide. II est evident qu’il ne s’agit pas exclusivement
d’une neosynthese microbienne mais dgalement d’accumulation de matiere organique rdsiduelle
plus ou moins transformde.
En ce qui conceme la matiere organique lide ä la fraction mindrale, on observe peu de
variations des quantitds de C extraites par les rdactifs alcalins en fonction des traitements. Ces
quantites sont cependant toutes ldgerement supdrieures au tdmoin. L’augmentation semble se
repartir dgalement entre les acides fulviques et les acides humiques. Neanmoins, le rapport AF/AH
du sol enrichi en boues est peu different de celui du sol temoin, ce qui denote une incorporation
TABLEAU V. AGREGATS STABLES APRES 6 MOIS DTNCUBATION

272
S u b stan ce s incubees T em o in B oues de Fleville T o u r be B o u es d e S t-H u b ert F o in de lu z em e Paille de Ыё B o u es de N an cy

A gregats 27,85 30 ,0 3 3 0 ,1 4 3 1 ,8 3 3 2 ,4 8 3 2 ,8 1 33,3 1

stab les (% )a (a) (ab) (b ) (b c) (c) (O (c)

P o u rcen tag es d ’agregats stables ä l’eau apres les tro is p re tra ite m e n ts alco o l-b en zen e-eau [4 ]; les v aleurs suivies de la m em e le ttr e n e s o n t p as sig n ificativ em e n t d iffe re n tes
au niveau 5%.

TABLEAU VI. INFLUENCE D’UN APPORT DE BOUES RESIDUAIRES SUR LES PROPRIETES CHIMIQUES DU SOL

M O R E L e t JA C Q U IN
(apres 21 jours d ’in cu bation )

A zote C o m p lex e a b s o rb a n t P2O 5

S ubstances pH (m e q / 1 0 0 g sol sec) (%)
in cu b ees N -N 0 3 T Ca Mg К Na A pres A pres
N,
(%) (m g / 1 0 0 g so l sec) 21 jo u rs 6 m o is

T em o in 7,5 1,67 6 ,8 1 2 ,8 8 14,0 1 ,1 0 0 ,6 9 0 ,3 0 0,41 0,41

Paille 7,5 1,75 2,3 1 2 ,9 5 14,1 1,08 0,71 0 ,3 0 0 ,3 8 0 ,3 9

L u zern e 7,3 2,05 22,3 1 3 ,1 0 14,5 1,17 1,05 0 ,2 9 0,41 0 ,4 5

L o n guyon 7,4 ND ND 13,2 8 1 5,0 1,08 0,67 0 ,2 9 ND ND

S trasbourg 7,4 1,94 8,3 13,0 6 15,5 1,13 0,68 0,31 0,48 ND

Fleville 7,1 2,13 2 5 ,8 13,17 14,6 1 ,1 6 0 ,6 9 0 ,2 7 0,51 0 ,5 2

St-D ie 7,2 2,1 7 15,7 12,66 15,0 1,17 0,69 0 ,2 8 0 ,4 9 -

S elestat 7,4 ND ND 13,1 6 14,3 1,24 1,06 0 ,3 2 ND ND

N ancy 7,8 1,97 13,7 13,1 4 2 0,7 1,17 0 ,6 8 0 ,31 0 ,6 6 0 ,63

O b ern ai 7,9 1,92 11,4 13,16 2 1 ,6 1,20 0 ,6 6 0 ,3 0 0 ,3 8 -

S t-H u b ert 6,8 2,0 8 3 5 ,4 13,5 9 14,5 1 ,3 5 0 ,6 9 0 ,4 6 0 ,73 0 ,6 7

M arcillat 6 ,9 ND ND 13,31 14,4 1,32 1,14 0 ,7 8 ND ND

 IAEA-SM-211/76 273

au sein des matieres humiques de composes relativement condensds, contrairement ä ce qüe Гоп
observe avec les substances de ref6rence.
C’est au sein de l’humine que Гоп retrouve la plus grande partie de la matiere organitjue
transformee. Les boues se revelent done etre particulierement efficaces vis-ä-vis de l’augmentation
des differentes formes d’humine, en particulier la boue floculee ä la chaux (N). II s’agirait pour
cette derniere d’une matiere organique dejä tres evoluee, peu biodegradable et certainement
protegee par le carbonate de chaux [6].

4. MODIFICATION DES PROPRIETES PHYSICO-CHIMIQUES DU SOL

4.1. Stabilite structurale

Comme le signalent certains auteurs [7,8], l’effet des boues r6siduaires sur la stabilite
structurale des sols peut etre сотрагё ä celui d’une matiere organique classique. Apres 21 jours
d’incubation, le pourcentage moyen d’agregats stables ä l’eau apres les divers pretraitements est
dans tous les cas sup6rieur ä celui du temoin. Pour six boues testdes, on obtient le classement
suivant:

Temoin < Tourbe < St < F < SH < Ob < N < SD < Luzerne < Paille < Glucose

Cet ordre peut correspondre ä celui de l’intensite de la liberation de produits transitoires, tels les
polysaccharides, qui joueraient un röle essentiel sur l’agregation [2]. La stabilite structurale etant
etroitement liee ä l’intensit6 de revolution du carbone [1,2,9] pendant la periode de forte
mineralisation, nous nous trouvons ainsi dans des conditions privitegiees de structure. Neanmoins,
si Гоп observe les r6sultats obtenus apres six mois d’incubation (tableau V), ces derniers restent
superieurs au temoin, pour tous les traitements, dans des rapports sensiblement identiques ä ceux
obtenus apres trois semaines. Ainsi l’analyse d’agr6gats effectude ä 21 jours donnerait un reflet
interessant de l’influence des boues sur la structure du sol.
De plus, la nature du traitement de floculation dont font l’objet certaines boues intervient
certainement dans les phdnomenes d’agregation. En effet, la presence de fortes quantites de
calcium (Ob, N) ou de polyelectrolyte (SH) joue un role non negligeable sur la structure du sol
qui les re^oit.

4.2. Proprietes chimiques (tableau VI)

4 .2 .1 . p H

L’apport de boues residuaires dans le sol modifie le pH de ce dernier. Les effets les plus
importants sont ressentis apres apport de boues floculees ä la chaux (Ob, N) et de boues issues de
laiteries (SH, Ma, Bu). A l’oppose des premieres, les boues de laiterie provoquent une forte
diminution du pH du sol. Dans ce cas, il est probable que revolution rapide de la matiere
organique provoque une augmentation de l’aciditd par production de groupements carboxyliques.
Hormis ces cas particuliers, dans nos conditions experimentales, un apport de boues est accompagne
d’une legere diminution du pH du sol. ;

4 .2 .2 . C o m p lex e a b sorban t

Apres trois semaines d’incubation, la capacite totale d’echange (T) presente peu de modifi­
cations avec les sols enrichis par rapport au sol temoin. L’augmentation de T n’est vraiment
significative qu-’avec les boues de laiterie et se situe aux environs de 1 meq/100 g de sol. En ce qui
concerne les bases £changeables, on observe les variations suivantes:
274 IAEA-SM-211/76

Ca+* : Seules les boues floculees ä la chaux elevent la concentration des ions Ca*+ echangeables;
ainsi que nous l’avons dejä observe [10], ces boues doivent etre considerees comme un vdritable
amendement calcaire, ce dont il faut tenir compte au moment de l’utilisation agricole.
Mg++: Ce cation subit partout une augmentation; neanmoins, les seuls effets notables
proviennent des boues issues de laiteries et des boues floculees ä la chaux; dans ce dernier cas on
peut supposer que le materiel de floculation serait ä base de calcaire magnesien.
K+: Les boues residuaires urbaines sont bien connues pour leur faible teneur en
potassium [11,12] et, effectivement, nous ne notons aucune modification de К* au sein du
complexe absorbant; par contre, l’apport dans le sol de boues de laiteries-fromageries est suivi
d’une augmentation de К ^changeable.
Na+: La teneur en sodium echangeable ne varie significativement que sous l’effet des boues
de laiteries-fromageries; les quantites de sodium präsentes dans ces boues etant parfois tres
importantes [13] leur epandage pourrait provoquer, ä long terme, des concentrations anormales
de cet dlement dans le sol.

4 .2 .3 . P hosph ore assim ilable

Si Гоп compare les resultats obtenus en 21 jours d’incubation ä ceux obtenus en 6 mois, nous
constatons qu’ils sont pratiquement identiques. Ils indiquent un effet nettement favorable des
boues sur la quantite de phosphore assimilable dans le sol, car ä une exception pres (Ob) l’aug-
mentation est importante. La encore les effets les plus interessante sont obtenus avec les boues
issues de l’epuration des eaux de laiterie. Notons toutefois l’effet negatif d’un apport massif de
boues floculees ä la chaux (N) qui a provoque, en meine temps qu’une remontde importante du pH,
une diminution de la quantite de phosphore assimilable.

4 .2 .4 . A z o te

4.2.4.1. Azote total

Quelques auteurs [14, 15] considerent l’apport de boues rdsiduaires sur les sols agricoles
comme un veritable apport d’azote; dans nos conditions experimentales nous no tons une augmen­
tation importante de N total pour les divers traitements.

4.2.4.2. Azote mineral

Avec l’epandage de boues residuaires se posent deux problemes en relation avec leur teneur en
azote: la nutrition des vegetaux et les risques de pollution des nappes par les nitrates issus de la
mineralisation [16—18]. Les quantites d’azote nitrique retrouvdes apres incubation sont tres
elevees pour certaines boues, en particulier pour celles qui sont issues de laiteries-fromageries.
Ainsi que le signalent certains auteurs, il doit etre tenu compte, dans les calculs des doses d’apport,
des risques d’entrainement par les eaux de drainage.

CONCLUSION

Nos rdsultats montrent que les boues residuaires participent activement ä Involution de la
fertility du sol, et ceci ä plusieurs niveaux. •
Tout d’abord, au cours de leur mineralisation, en libdrant des produits transitoires, elles
favorisent les processus d’agregation et accroissent la stabilite structurale des sols. De plus la
presence dans certaines boues de fortes quantitds de cations ou de polyelectrolytes intervient
 IAEA-SM-211/76 275

egalement sur l’amelioration de la structure. Enfin, les composes humifids issus de revolution de
la matiere organique des boues semblent plus fortement polymerises que ceux provenant de
Гevolution d’amendements organiques classiques.
Les teneurs des boues en azote et phosphore liberes sous forme assimilable contribiient ä la
creation d’une bonne potentiality chimique du sol. Cependant, bien qu’au niveau de l’azote des
phenomenes de reorganisation microbienne puissent intervenir, il subsite des risques de pollution
des eaux par les nitrates dans le cas d’apports de boues trop eleves. II est ä noter que sei les les
boues residuaires issues de laiteries apportent du potassium au sol. Ces dernieres semble it les plus
favorables vis-ä-vis de la fertilisation, hormis toutefois leur effet acidifiant et leur teneur en sodium.
Enfin le comportement de chaque boue residuaire dans le sol de reference constitue un cas
particulier dependant de l’origine et de la technologie d’obtention de la boue. Ce test de laboratoire,
d’une duree de trois semaines, peut refleter utilement ce comportement et permettre la compa-
raison avec des amendements organiques classiques.

REMERCIEMENTS

Ce travail a ete partiellement finance par le Ministere de la Qualite de la Vie (France) dans le
cadre d’une action concertee sur les sols et dechets solides.

REFERENCES

[1] M O N N IER , G ., A ctio n des m a tie res organiques su r la stab ility stru c tu ra le des sols, T hese U niv. Paris (1 9 6 5 ).
[2] G U C K E R T , A ., C o n trib u tio n a l’e tu d e des po ly sac ch arid e s dans les sols e t d e le u r ro le d an s les m ecanism es
d ’ag reg atio n , T hese U niv. N an cy (1 9 7 3 ).
[3] M O R E L , J .L ., JA C Q U IN , F ., E tu d e de la m in eralisatio n dans u n sol agricole d e b o u es resid u aires d ’origines
diverses, B ull. E N S A IA , N an cy (1 9 7 6 ), ä p ara ftre .
[4] H E N IN , S., M O N N IE R , G ., CO M BEA U , A ., M ethode p o u r T e tu d e de la stab ility s tru c tu ra le des sols, A nn.
agron. 1 (1 9 5 8 ) 73.
[5] SIM O N -SY L V E S T R E , G ., L ’en fo u issem en t des pailles dans le sol. E tu d e generale e t rep ercu ssio n s su r la
m icro flo re d u sol, A nn. agron. 11 (1 9 6 0 ) 177.
[6] C H O U L IA R A S, N ., E v o lu tio n de la in at iere org an iq u e dans u n e re n d z in e , T hese de d o c te u r Ingenieur.
N ancy (1 9 7 6 ).
[7] L U N T , H .A ., D igested sew age sludge fo r soil im p ro v em e n t, Bull. C o n n . A gric. E x p . S tn . 6 2 2 (1 9 5 9 ) 30.
[8] E P S T E IN , E., E ffe c t o f sew age sludge o n som e soil p h y sical p ro p e rtie s , J . E n v iro n . Q ual. 4 1 (1 9 7 5 ) 139.
[9] G R A F F IN , P h., E tu d e in teg ree de la d e c o m p o sitio n d ’a p p o rts organ iq u es d an s le sol, A n n . A gron. 2 2 2
(1 9 7 1 )2 1 3 .
[10] M O R E L , J .L ., G U C K E R T , A ., SIB O U T, V ., JA C Q U IN , F ., E tu d e de la v alo risatio n des b o u es resid u aires en
ag ricu ltu re - II. In cid en ce sur les p r o p r ie ty du sol, B ull. E N S A IA N an cy , (1 9 7 6 ), ä p araftre.
[11] K IC K , H ., P O L E T S C H N Y , H ., A n b au la n d w irtsch aftlich e r K u ltu rp fla n z e n a u f en tw ä sserte m A b v asse r­
k lärsch lam m , L a n d w irtsch . F o rsc h ., S o n d e rh e ft 27 1 ( 1 9 7 2 ) 67.
[12] K IN G , L .D ., M O R R IS , H .D ., L and d isposal o f liq u id sew age sludge — IV . E ffe c t o n soil p h o sp h o rus,
p o ta ssiu m , calcium , m agnesium , an d so d iu m , J . E n v iro n . Q ual. 2 3 ( 1 9 7 3 ) 4 1 1 .
[1 3 ] G R A S , R ., L ’ep an d ag e des ea u x resid u aires des in d u stries agricoles e t alim en taires, G enie R ur. 6 —7 (1 9 7 4 ).
[1 4 ] H A A N , S. d e, Ergebnisse aus V ersu ch en m it A bw asserklärschlam m , L a n d w irtsch . F o rsc h ., S o n d e rh e ft
27 1 (1 9 7 2 ) 102.
[1 5 ] K IN G , L .D ., M ineralisation an d gaseous loss o f n itro g e n in soil-applied liq u id sew age sludge, J . E n v iro n . Q ual.
2 3 (1 9 7 3 )3 5 6 . '
[1 6 ] B R A ID S , O .C ., SO B H A N -A R D A K A N I, M ., M O L IN A , J.A ., L iq u id d igested sew age sludge gives field cro p s
n ecessary n u trie n ts , 111. R es., 12 3 ( 1 9 7 0 ) 6. I
[17] R Y A N , J .A ., K E E N E Y , D .R ., W A LSH , L.M ., N itro g e n tra n sfo rm a tio n s an d availab ility o f an an aero b ically
d igested sew age sludge in soil, J . E nviron. Q u ality , 2 4 (1 9 7 3 ) 4 8 9 . I
[1 8 ] E L BA SSA M , N ., T IE T JE N , C., M E R T E N S , F ., N itra t-, N itrit-, u n d A m m o n iak -N im B o d en u n d 1B o d en w asser
sow ie A u fn ah m e d u rc h M ark stam m k o h l u n d Z u c k e rrü b e n b ei Z u fu h r h o h e r K lärsch lam m g ab en , jL andw irtsch.
F o rsc h ., S o n d e rh e ft, 30 2 (1 9 7 4 ) 29. |
>
 IAEA-SM-211/77

E V A L U A T IO N O F M U N IC IP A L R E F U S E
F R O M D A H O M E Y (B E N IN ) A S A N O R G A N I C M A N U R E

F.X. KOMA ALI MU,* I.E. SOE AGNIE +

B.H. JANSSEN
Department of Soils and Fertilizers,
Agricultural University,
Wageningen,
The Netherlands

R a p p o rteu r: F. Jacquin

Abstract

EVALUATION OF MUNICIPAL R E FU SE FROM DAHOMEY (BENIN) AS AN ORGANIC MANURE-

In a pot experiment the release o f nutrients by municipal refuse from Dahomey was followed during
150 days for three separate refuse components: mould, partly decayed and non-decayed material. Crops were
Amaranthus and maize. Initially there was a negative effect o f refuse on yields, but gradually it changed into a
positive one. The main cause o f yield reduction was probably chlorine toxicity. The organic components o f the
refuse caused also nitrogen and phosphorus immobilization. The toxicity diminished because chlorine was
removed with successive leaf harvests o f Amaranthus. The positive effect o f refuse was primarily brought about
by potassium, and at a later stage also by remineralized nitrogen and phosphorus. It is calculated that negative
effects are not to be expected in the field, as chlorine is easily leached out and the nitrogen and phosphorus
immobilization by the organic compounds is compensated by release from the mould fraction.

INTRODUCTION

Grubben [1] and Schelhaas [2] have described how chemically poor sandy soils in the
neighbourhood of Porto Novo and Cotonu (Dahomey) have been greatly improved by continued
application of so-called ‘gadoue’, an organic manure gathered from markets and streets. In a
number of field experiments the beneficial influence of this municipal refuse on yields of various
tropical leaf vegetables and on the efficiency of fertilizers was shown [1 ]. Comparison of
municipal refuse with compost led to the conclusion that composting was unnecessary, and in
fact was to be discouraged because of costs and loss of nutrients. From the fact that initial
crop growth was not retarded when uncomposted or only slightly composted municipal refuse
was worked into the soil immediately before planting, it was deduced that there was no microbial
immobilization of nitrogen, or, if any, remineralization soon started because of the high
temperature. Rapid remineralization under tropical conditions has been reported also by, for
instance, Oliver and Ngo Chan Bang [3]. The present experiment was set up to verify the absence
of immobilization, and more generally to investigate the rate of nutrient release for three
different components of municipal refuse from Dahomey.

* Present address: Serere Research Station, P.O. Soroti, Uganda,

t Present address: Agricultural Experiment Station, P.O.B. 160, Paramaribo, Surinam.

277
278 KOMA ALIMU et al.

MATERIALS

The municipal refuse studied was sampled from five truck loads in Dahomey (Benin).
After drying in the air for 14 days, such substances as stones, glass and plastics were removed.
The remaining refuse was divided into three fractions:

1. Mould (70%), the earthy fraction, passed through a 2-mm sieve and consisting mainly of
street sweepings.
2. Partly decayed organic materials (17%).
3. Non-decayed organic materials (13%).

The organic materials were leaves, organic wastes, and tissues, papers, peels, wood, ropes, etc.
These components will henceforth be referred to as mould, pardec and nondec.
The treated material was sent to Wageningen in the Netherlands. There the organic fractions
were dried at 50°C, stripped of remaining hard objects, and ground to pass through a 2-mm sieve.
The soil used in the experiment was also from Dahomey. It was a sandy ‘sol ferralitique’ [1,2].
Analytical data of soil and refuse components are shown in Table I. Mould was analysed as
soil, and pardec and nondec as soil as well as plant sample. Not all soil analyses proved appropriate
for pardec and nondec. The sum of determined exchangeable cations exceeded the cation
exchange capacity of the refuse components, which, together with the high pH, indicates the
presence of salts. The contents of K, Na, Cl, N 03 and S04 in pardec were lower than in nondec,
indicating that during decomposition a part of the soluble salts had been leached already.

DESIGN

The experimental design was a dummy factorial [4]. Treatments were control (soil alone),
and single and double applications of each of the refuse components. Half of the pots received
nutrient solution, half did not. There were four replicates and in each replicate there were two
control pots.
The calculation of the rates of refuse application was based on the following considerations.
In the field about 50 t of refuse is applied which amounts to about 45 t of dry material. This
can be divided roughly into 38 t of mould, 4 t of partly decayed and 3 t of non-decayed material.
It was supposed that pardec contained twice and nondec three times as much available nutrients
as mould. Therefore the fertilizing value of 50 t of refuse equals that of 38 + (2 X 4) + (3 X 3) = 55 t
of mould. Assuming a soil weight of 2 300 000 kg per ha, this is 24 g mould per kg soil. The pots
used in the experiment contained 2.8 kg of soil. Hence, to be comparable with field conditions,
a single application of mould should amount to 67 g and of pardec and nondec to 34 and 22 g
per pot respectively.
The nutrient solution consisted of a mixture of NH4N 03, KH2P04, K2S04 and micronutrients
(the soil was supposed to supply sufficient calcium and magnesium). The solution was applied four
times, on days 8, 40, 95 and 125 (planting time is reference), each time supplying 420 mg N, 77.5 mg
P and 487.5 mg К per pot. The amounts would compensate for the expected withdrawals of
normally growing plants.
The treatments and additions of nutrients by the refuse components are shown in Table II
(figures are based on results of Table I).

EXPERIMENTAL

The test crops were A m aran thu s cruentus L., a tropical leaf vegetable, and maize successively.
Amaranthus had been sown previously; the seedlings were 18 days old when they were trans-
 IAEA-SM-211/77 279

TABLE I. ANALYTICAL DATA OF SOIL AND MUNICIPAL REFUSE COMPONENTS

Analysed as soil Soil Mould Pardee Nondec

p H (l : 2.5 H jO ) 6.3 8 .8 7.3 6 .6

p H (l : 2.5 1NKC1) 5.8 8 .8 7.3 6.5

CEC (meq per 100 g) 3.3 2.5 17.1 32.3

E x c h a n g e a b le c a tio n s

Ca 2.3 10.3 31.9 18.8

Mg 0.5 2 .0 6 .2 14.4
j(meq per 100 g)
К 0.13 2.9 17.6 18.9

Na 0.03 1.6 7.8 13.3

Pw (mg P jO j per litre soil) 12 149 a a

Р-Вгау I (ppm P) 24 126 240 k l9

P in 2N H N 0 3 (ppm P) 241 842 5 1 8 0 (?) 1612

C-Kurmies (%) 0.5 1.1 2 0 .1 39.9

N-total (%) 0.06 0 .1 1

a a

Cl (ppm) 23 416 See below

NO 3 -N (ppm) 11 11 66 2 1 2

A n a ly s e d a s p la n t m a te r ia l (% o f d r y m a tte r )

N P Ca Mg К Na Cl N 0 3 -N S 0 4-S

Pardee 0.60 0.27 2.47 0.18 0.55 0 .2 1 0.26 0 .0 2 0 .0 2

Nondec 1.59 ®^ 1.08 0.25 1.24 0.32 0.54 0.04 0.07

a Analysis was not possible or results were not reliable.

planted to the pots at the start of the experiment. Leaves were harvested after 35, 63 and 95 days.
At the first two harvests the plants of one replicate were uprooted, at the third harvest the plants
of the other two replicates were uprooted. After the third harvest from each pot 300 g of soils
were sampled. Then the pots were refilled with the remaining soil and sown with maize (day 100).
One replicate was harvested at day 135, the other at day 150.
The pot experiment was carried out in a conditioned greenhouse of the Department of
Tropical Crop Science of the Agricultural University at Wageningen between August 1974 and
February 1975.
Day and night temperatures were 30° and 20°C for Amaranthus and 25° and 20°C for
maize. Relative humidity was 70-80%; day/night length 13/11 h for Amaranthus and 14/10 h
for maize. Extra light was supplied by HLRG bulbs.
Leaves, stems and roots of Amaranthus and shoots and roots of maize were analysed
separately. Amaranthus was analysed for N, P, K, Mg, Ca and Na after digestion according to Lindner and
Harley [5], and for N 03, Cl and S04 after extraction with water. In maize only N, P and К were
determined, and Cl, N 03 and S04 in the maize of harvest 4.
280 KOMA ALIMU et al.

TABLE II. AMOUNTS OF NUTRIENTS PRESENT AT THE START OF THE EXPERIMENT

(mg per pot)
N u trien ts a d d e d w ith th e solu tion are n o t in clu ded

Mould Pardec
Nondec
Component Soil alone
(g per pot) 2800 67 134 34 68 22 44

Organic N 1680 1754 1827 1884 2088 2030 2380

n o 3-n 31 32 32 33 35 35 40

P-Bray I 67 76 84 75 84 76 86

P3 - - - 188 375 274 547

Kb 142 218 293 330 517 416 689

Cl 64 92 120 153 241 183 302

a P according to the plant analysis procedure (see Table I).

К according to plant analysis procedure for pardec and nondec and exchangeable К for soil and mould.

x = s o w in g o f m a iz e h a rv e s t num ber

F I G .l. E ffe c ts o f r e fu s e c o m p o n e n ts o n c u m u la tiv e d r y m a tte r y ie ld s o f a b o v e g r o u n d p a r ts o f A m a r a n th u s a n d

m a iz e .

Soil samples of the harvested pots were analysed for N 03, P-Bray I, exchangeable К and
pH-KCl. The analytical procedures for soils were: cation exchange capacity and exchangeable
cations: ammonium acetate, pH 7; Pw, water-extractable phosphate according to Sissingh [6],
P-Bray I as described by Bray and Kurtz [7], and P after extraction with 2N HN03; N total,
digestion in a mixture of sulphuric acid and salicylic acid; N 03, potentiometric in a 0.01M CuS04
extract; Cl, potentiometrically in a water extract. К and Na were determined by flame photo­
metry, P by colorimetry, and Ca and Mg by atomic absorption spectrophotometry.
 IAEA-SM-211/77 281

TABLE III. DRY WEIGHT (G PER POT) AND NITROGEN, PHOSPHORUS, POTASSIUM
AND CHLORIDE CONTENTS (% OF DRY MATTER) OF AMARANTHUS LEAVES FROM
THREE HARVESTS

Mould Pardee Nondec

Harvest Nutrient Soil
No. solution alone
Single Double Single Double Single Double

1 Without Dry weight 2.26 2.74 3.23 1.82 1.45 0.81! 0.20
N 3.86 3.83 3.42 3.56 3.89 4.56, 5.42
P 0.60 0.45 0.47 0.50 0.58 0.46 0.45
К 4.10 5.35 6.09 6.93 7.14 8.12 7.44
Cl 1.47 1.40 1.68 1.60 2.03 1.45 n.d.

With Dry weight 5.04 4.13 3.01 3.43 2.76 4.52 3.54
N 5.06 5.43 5.46 5.21 4.71 5.16 '5.05
P 0.55 0.51 0.53 0.53 0.51 0.50 0.42
К 8.32 8.51 8.52 8.76 7.80 8.75 8.07
Cl 0.56 0.48 0.34 0.85 1.36 0.90 0.78

2 Without Dry weight 0.75 1.54 0.92 1.02 1.56 1.33 1.33
N 2.79 2.88 2.80 3.32 3.23 4.40 4.46
P 0.46 0.43 0.41 0.35 0.33 0.37 0.28
К 3.04 4.69 4.52 6.43 6.93 7.33 7.28
Cl 0.93 1.41 1.75 2.52 2.57 1.78 1.89

With Dry weight 6.95 7.81 8.44 7.28 6.04 6.72 7.03
N 3.03 3.13 3.32 3.12 3.53 3.34 3.97
P 0.54 0.59 0.64 0.60 0.49 0.6l| 0.51
К 6.36 6.94 7.16 7.06 7.32 7.37 7.50
Cl 0.26 0.73 0.98 1.07 1.48 1.30 1.63

3 Without Dry weight 0.62 0.97 1.12 1.53 2.23 1.89] 1.98
N 3.29 2.97 2.76 2.70 2.53 3.38 4.33
P 0.62 0.54 0.49 0.42 0.36 0.32| 0.28
К 2.32 3.20 3.41 4.95 6.13 5.96 5.95
Cl 0.35 1.09 0.85 2.14 2.49 2.27 1.08

With Dry weight 4.73 5.15 5.89 5.44 5.27 5.51 7.21
N 3.12 3.00 2.33 2.70 2.79 2.61 2.87
P 0.75 0.86 0.74 0.70 0.52 0.59 0.44
К 5.44 5.41 5.69 6.02 6.31 6.12 6.28
Cl 0.11 0.22 0.30 0.35 1.18 0.34 0.88
282 KOMA ALIMU et al.

C I (m g p e r p o t )

RESULTS AND DISCUSSION

The effects on dry matter yield differed with the refuse component and changed with time
(Fig. 1). At the first harvest all treatments had a negative influence on yield, except for mould
in the pots without nutrient solution. Gradually the effects changed from negative to positive.
At the end, after 150 days, the non-decayed material produced the highest response, followed
by the partly decayed material, whereas there was hardly any effect left from the mould.
At first glance these results seem to be due to nitrogen immobilization followed by
remineralization. The results of chemical crop analysis, however, showed that other factors
were involved too. The Amaranthus leaves had rather high chlorine contents, particularly in
the pots not receiving a nutrient solution (Table III). The N 03 and S04 ions in the nutrient
solution probably depressed the uptake of Cl by the plants receiving this solution. Chlorine
toxicity might therefore be the main cause of the negative effects on yield of the first harvest.
The reversal of the effects can be attributed to the removal of the chlorine by the successive leaf
cuttings. Figure 2 shows that after the third harvest all pots, with one exception, contained less
than 100 mg Cl. As will be shown below, this was a critical value for Amaranthus. This explains
why there was no yield reduction for maize and why the chlorine contents of maize were low.
The positive influence of the refuse components was chiefly a potassium effect. This is
indicated by the potassium contents of the leaves, particularly after the treatments without
nutrient solution (Tables III and IV).
Since the refuse components with the highest potassium content had also the highest chlorine
content, the response to refuse was always a combined response to potassium application and
chlorine toxicity. To study this interrelation the objects were divided into five classes of amounts
of chlorine present in soil + not harvested plant parts: < 40, 40-80, 80—130, 130-200 and
> 200 mg Cl, with rounded averages of 25, 60, 110, 160 and 260 mg Cl per pot. The amounts
 IAEA-SM-211/77 283

TABLE IV. DRY WEIGHT (G PER POT) AND NITROGEN, PHOSPHORUS, POTASSIUM
AND CHLORIDE CONTENTS (% OF DRY MATTER) OF MAIZE SHOOTS, FOR t Wo
HARVESTS

M ould Pardee Nondec

Harvest Nutrient
Control
No. solution
N orm al Double N orm al Double N orm al Double

Without D ry weight 1.67 1.62 1.92 2.57 4.41 3.85 6.64

N 1.66 1.55 1.75 1.46 1.12 1.21 0.96

P 0.41 0.38 0.31 0.32 0.24 0.29 0.22
К 0.77 0.87 0.77 1.45 3.92 2.80 3.89

Cl 0.16 0.08 0.12 0.12 0.31 0.77 0.92

With D ry weight 13.56 16.53 18.13 12.80 16.83 15.77 19.80

N 4.43 4.27 4.19 4.51 4.26 4.31 3.95
P 0.50 0.50 0.49 0.57 0.57 0.47 0.49

К 6.10 6.80 6.45 6.82 6.92 6.36 6.95

Cl 0.07 0.07 0.07 0.10 0.09 0.06 0.07

Without D ry weight 3.10 3.85 3.23 5.14 6.97 6.23 12.69

N 1.21 0.88 1.06 0.76 0.71 0.82 0.63
P 0.41 0.33 0.38 0.25 0.28 0.32 0.21
К 0.45 0.44 0.53 0.58 2.12 1.76 2.27
Cl N ot determined

With D ry weight 36.02 40.84 40.86 52.82 49.04 48.47 57.05

N 2.02 1.81 1.78 1.28 1.51 1.50 1.31
P 0.25 0.26 0.31 0.29 0.27 0.21 0.21
К 2.88 2.67 2.77 2.25 3.21 2.35 2.56
Cl N ot determined

of chlorine and potassium were found by subtracting the amounts removed with cut leaves from
the amounts initially present (Table II), taking into account potassium application by the nutrient
solution. Since the leaf yields were of the same order of magnitude, the results of the three
harvests could be brought together in one graph (Fig. 3). The toxic effects became pronounced
when more than 100 mg Cl was present. The pots containing 2800 g of soil and 400 ml of water,
which reduces to about 35 ppm Cl on a dry soil basis or 7 meq Cl per litre of soil solution. These
values are rather low, characterizing Amaranthus as a chlorine-sensitive crop. (Total salt concen­
trations may be higher, e.g. the salt concentration in the soil solution of the pots receiving nutrient
solution was about 70 meq per litre.)
Tables III and IV show also that the refuse components decreased leaf phosphorus contents
and sometimes leaf nitrogen contents. To find out whether these decreases should be attributed
to dilution in the plant caused by the increased dry matter production, or immobilization, yields
of dry matter, nitrogen, phosphorus and potassium were calculated relative to the control yield
(soil alone) as 100%. Figure 4 shows relatively low phosphorus withdrawals for pardec and
especially for nondec. For pardec also nitrogen withdrawal was lower than that for the control.
284 KOMA ALIMU et al.

FIG.3. Relations between dry matter yields o f Amaranthus leaves and the amounts o f potassium and chlorine
present in soil + not harvested parts o f plants.

r e la tiv e y ie ld ( % ) ------------ d r y m a tte r

•280

2L.Q
NON DECAYED

■200

■160

■120

■80

22 w
r e tu s e (g p e r p o t)

■120
100
80
■60
67 13Д ' 3l 68 22 u*
r e f u s e (g p e r p o t)

FIG.4. Effects of refuse components on relative yields of dry matter, nitrogen, phosphorus and potassium,
cumulative for three harvests of Amaranthus. Above without, below with, nutrient solution. Soil alone is 100%.
 IAEA-SM-211/77 285

2087

% %

0 67 134 0 34 68 0 22 44
r e f u s e (g p e r p o t )

F I G .5 . E ffe c t s o f r e fu s e c o m p o n e n ts o n r e la tiv e y ie ld s o f d r y m a tte r , n itr o g e n , p h o s p h o r u s a n d p o ta s s iu m fo r

th e s e c o n d h a r v e s t o f m a iz e . A b o ve w ith o u t, b e lo w w ith , n u tr ie n t s o lu tio n . S o i l a lo n e is 1 0 0 % .

TABLE V. UPTAKE OF NITROGEN PHOSPHORUS AND POTASSIUM AFTER 95 AND

150 DAYS, EXPRESSED AS PERCENTAGES OF THE AMOUNTS PRESENT AT THE START
OF THE EXPERIMENT (N AS ORGANIC N, P ACCORDING TO PLANT ANALYSIS
PROCEDURE, К ACCORDING TO PLANT ANALYSIS PROCEDURE FOR PARDEC AND
NONDEC AND AS EXCHANGEABLE К FOR SOIL AND MOULD)
R e a d f o r refuse co m p o n en ts: u p ta k e m inus c o n tro l u p ta k e as percen tage o f to ta l a m o u n t p resen t
m inus a m o u n t p re se n t in co n tro l
Data are of treatments without soil solution

Mould Pardee Nondec

Nutrient Days Control
67 134 34 68 22 44

N 95 9 24 17 -4 5 8 4
150 12 27 18 -1 10 13 11
(C/N 8 10 33 25 )
P 95 - - - -3 -2 -2 4 -2 0
150 - - - -3 5 —2 ; 0
К 95 92 181 89 95 71 64 28
to
О

150 113 183 113 104 110 82

286 KOMA ALIMU e t al.

According to the data of Table I, the C/N and C/P ratios were 33 and 74 for pardec
and 25 and 225 for nondec. These values make it plausible that pardec caused nitrogen and that
nondec caused phosphorus immobilization. At the end of the experiment refuse treatments
yielded more nitrogen and phosphorus than the control (Fig. 5). Probably remineralization of
immobilized nitrogen and phosphorus had begun.
Both Figs 4 and 5 show again that potassium was the principal yield stimulator. Mould
contained too little potassium to maintain a rise in yield. These results are in agreement with
those of Grubben [1], who found that municipal refuse increased potassium uptake more than
nitrogen and phosphorus uptake (e.g. his tables 46, 47 and 49).
An attempt was made to calculate the mineralized amounts of nitrogen, phosphorus and
potassium as the difference between the sum of nutrient taken up + left in the soil, and the sum
of initially present nutrient + additions with nutrient solution [8, 9]. However, since the amounts
of mineralized nutrients were of the same size as the variation between replicates and the
analytical errors, results were not very reliable, certainly not for the pots with nutrient solution.
More information is being obtained by calculating the ratio of the withdrawal of nutrients and
the amount of nutrients initially present (Table V). The results for nitrogen were rather high;
differences in nitrogen utilization corresponded with the differences in C/N ratios. There was
hardly any net phosphorus uptake from the organic refuse components after 150 days. From
soil and mould more potassium was taken up than was present as exchangeable potassium,
probably because of release by potassium-bearing minerals. The potassium present in the organic
components was used or almost used after 150 days.
Some of the findings of the present experiment seem to contradict the results obtained by
Grubben [ 1]. The difference between the greenhouse and field experiments can be explained as
follows:

1. In the field complete municipal refuse instead of separate components is applied. This
lowers the risk of chlorine toxicity. The Cl content of complete refuse can be calculated
as: 0.70 X 416 + 0.17 X 2600 + 0.13 X 5400 = 1435 ppm. Application of 45 t of dry
refuse to 2 300 000 kg of soil containing 23 ppm Cl results in an average Cl content of
50 ppm. This is higher than the above-mentioned critical value of 35 ppm.
2. However, in the field chlorine will easily be leached, either by rain or by pouring water
(pouring is common practice for leaf vegetables in Dahomey).
3. The immobilization of nitrogen and even the more severe immobilization of phosphorus
by the organic components can be compensated by release from the mould fraction.
According to the data of this experiment, the phosphorus withdrawal from the refuse
components after 35 days was: 2.6 mg P per 134 g mould, -0 .5 mg P per 68 g pardec and
-16.7 mg P per 44 g nondec. This results in -3.7 mg P per 100 g complete refuse or 1.7 kg P
per 45 t dry refuse. The stimulating influence of potassium in the municipal refuse will
mask any negative effect of this low phosphorus immobilization.

CONCLUSION

The initial negative effect of the organic refuse components on yield was caused by chlorine
toxicity, and phosphorus and nitrogen immobilization. The change to positive effects was due
to removal of chlorine by successive leaf harvests and to a gradual remineralization of nitrogen
and phosphorus. The positive influence of the refuse components was primarily a matter of
potassium.
In the field negative effects are not to be expected because chlorine is leached out, and
release of phosphorus and nitrogen from mould compensates initial immobilization by the
organic components.
 IAE A-SM-211/77 287

Problems might arise when the organic components constitute considerably more than 30%
of the refuse. According to data of Grubben and Schelhaas [1,2], the organic fraction varies
from 2—35%. Therefore it might be concluded that the results of the present experiment support
the findings of Grubben [ 1], and justify the practice of the gardeners in Dahomey who use
municipal refuse without composting.

A C K N O W LED G EM EN TS

The authors are much indebted to Miss A. Hoogendijk, A. van den Berg, C. Vermeer and
co-workers for technical assistance, and to the Department of Tropical Crop Science for the
readiness to place facilities at their disposal.

REFER EN C ES

[1] GRUBBEN, G .J.H ., La culture de Г Amarante, Legume - Feuffles Tropicales: Avec reference speciale au
Sud-Dahomey, Mededelingen Landbouwhogeschool Wageningen, particularly Chapter 8, «Sol et fertilisation»
(1975) 75 (English edition to be published).
[2] SCHELHAAS, R.M., De Tuinbouwgronden van Zuidoost Dahomey, Dep. Agric. Res. Royal Trop. Inst.
Amsterdam (1974).
[3] OLIVER, R ., NGO CHAN BANG, Etude de la reorganisation de Гazote dans un sol ferralitique sur gneiss
de Madagascar (Am batobe — Tananarive), Agron. Trop. (Paris) 25 12 (1970) 1068.
[4] YATES, F., “ The Design and Analysis o f Factorial Experiments” , Imp. Bureau Soil Sei. Techn.lcom m .,
Chapter 15, “ Dummy treatments” , (1937) 35.
[5] LINDNER, R.C., H ARLEY, C.P., A rapid method for the determination o f nitrogen in plant tissues, Science
9 6 (1 9 4 2 ) 565.
[6] SISSINGH, H.A., Analytical procedure o f the Pw method, used for the assessment o f the phosphate status
o f arable soil in the Netherlands, Plant Soil 34 (1971) 483. j
[7] BRAY, R.H., KURTZ, L.T., Determination o f total, organic and available phosphorus in soils, Soil Sei.
5 9 (1 9 4 5 ) 39. I
[8] KOMA ALIMU, F.X ., Evaluation o f Municipal Refuse o f Domestic Origin as an Organic Manure* MSc
Thesis, Soil Science and Water Management, Wageningen (1975).
[9] SOE AGNIE, I.E., Evaluatie van stadsvuil uit Dahomey als organische m eststof in een potproef met mais,
Verslag ingenieursonderzoek Bodemvruchtbaarheid, Wageningen (1975).
 DISCUSSION
(on the previous four papers)

Ch. JUSTE: With reference to paper SM-211/74, what method was used for determining
the maximum acceptable heavy metal concentrations in a given soil?
F. JACQUIN: I shall ask Mr. El-Bassam, one of the authors of that paper, to answer your
question.
N. EL-BASSAM: The limits were included in a proposal of the German Bundesgesundheits­
amt (Federal Office of Public Health) after a review of the literature.
F. JACQUIN: The authors of paper SM-211/74 have proposed a table showing the maximum
heavy metal contents of soils; the proposed figures are expressed in ppm of the total element.
In the present state of knowledge about the dynamics and exchange of various organo-cationic
complexes they could not have done better. We shall propose this formula to the French
authorities in the Ministry for the Quality of Life.
P. MARKOU: I wonder whether in addition to Cl , boron could also have exerted a
negative influence on the effect of the compost on the maize yield. Our experience in Cyprus
indicates that boron is as important as chlorine, and has an equally (if not more) negative
influence. It is thus evident that in semi-arid climates one must, in addition to heavy metal
toxicities, seriously consider salts as well —chlorides, boron, etc.
A. BEN MILOUD: Mr. Janssen has reported (SM-211/77) that he used fresh urbar refuse.
In Morocco farmers used fresh urban waste before composting plants were installed. We noted
that it was harmful to plants. Have any field experiments been made with such waste?
F. JACQUIN: The aim of the laboratory experiment was essentially to confirm results
obtained in the field; however, this urban refuse came mainly from matter intended for! human
consumption (fruit and vegetables) containing a lot of soil.
R.A. SOBULO: In connection with paper SM-211/7 7 ,1 would like to mention that in
Nigeria the cost of transporting urban refuse from the site of production to the point of use is
a major prohibitive factor. Could you please explain the economics of using the urban refuse
on food crops? Is it used on both vegetable and cereal crops in Dahomey?
F. JACQUIN: In this particular case, no, because we are dealing with residues rich! in dry
matter. This report gives the essential scientific facts about the use of residual sludge, j^pplying
the formulae in the field would require careful adaptation to each problem.

289
 IAEA-SM-211/31

VARIATION IN CONTENT OF POLYCYCLIC AROMATIC

HYDROCARBONS IN SOIL AND PLANTS BY
USING MUNICIPAL WASTE COMPOSTS IN AGRICULTURE

P.-Chr. ELLWARDT
Institut für Biochemie des Bodens
der Forschungsanstalt für Landwirtschaft,
Braunschweig-Völkenrode,
Federal Republic of Germany

Abstract

VARIATION IN CONTENT OF POLYCYCLIC AROMATIC HYDROCARBONS IN SOIL AND PLANTS BY

USING MUNICIPAL WASTE COMPOSTS IN AGRICULTURE.
An investigation into the variation in the content o f various polycyclic aromatic hydrocarbons by the
utilization o f municipal waste compost as soil conditioner for increasing the content of soil organic matter is
reported. Polycyclic aromatic hydrocarbons with carcinogenic effects are o f particular interest. Sewage sludge,
waste composts, untreated soil, soil treated with sewage sludge and waste compost, and plants (potatoes, oats and
rye) grown on these soils have been analysed to determine the concentration o f Various polycyclic aromatic
hydrocarbons. More than 160 different compounds can be obtained by extraction and gas chromatographic
separation. A quantitative analysis has been made o f eight o f them: benzo(b, k, j)fluoranthene, benzo(e)-,
benzo(a)pyrene, perylene, indeno(l, 2, 3-cd)pyrene, dibenz(a, h)-, dibenz(a, c)anthracene and benzo(ghi)perylene.
After using municipal waste compost (100 t/ha) the content o f polycyclic aromatic hydrocarbons increases by
65—75%. In contrast to authors who have studied selected polyarenes in model experiments, the reported
experiments have not shown an uptake by the plants from the soil. There are bigger differences in content of
polycyclic aromatic hydrocarbons between different kinds o f plant. In plants with a large leaf surface (potatoes),
there is a higher amount than in plants with a smaller leaf surface (cereals), so that a contamination of polycyclic
aromatic hydrocarbons by uptake from the atmosphere is suggested.

The re-utilization of organic materials as fertilizers, especially for nitrogen, is necessary through­
out the world. The increasing amounts of municipal and industrial wastes could therefore be
used for this purpose, and help to solve the problem of disposal of household waste and sewage
sludge. Municipal waste can also be disposed of by combustion, but this involves additional con­
tamination of the atmosphere, as well as the problem of the residue disposal.
Mostly municipal waste contains a large amount of organic materials which could be used as
an organic soil conditioner on arable land in agriculture after composting.
With the use of municipal waste compost harmful substances are introduced into the soil,
such as heavy metals, polychlorinated biphenyls and polycyclic aromatic hydrocarbons with
carcinogenic effects. Uptake of these substances by plants and consequently dangers for the health
of humans must be considered. It is therefore important to ascertain the variation in the con­
centration of harmful substances in soils treated with municipal waste compost and in plants
grown on these soils. The widespread utilization of municipal waste compost cannot be considered
before these questions are'answered.
In numerous investigations benzo(a)pyrene was used as an indicator for the extent of con­
tamination with carcinogenic polycyclic aromatic hydrocarbons. However, the limitation to a
special polycyclic aromatic hydrocarbon does not seem to be the appropriate way to evaluate
the carcinogenic effect. Von Brune [ 1] found that in the condensate of car exhausts only 7—9%
of the carcinogenic effect comes from benzo(a)pyrene. Furthermore, Druckrey and Schildbach
[2] and Lazar and co-workers [3] demonstrated that in the condensate of the smoke of cigarettes
only 1-2% of the carcinogenic effect can be attributed to this substance.

291
292 ELLWARDT

TABLE I. AMOUNTS (in pplO9) OF VARIOUS POLYCYCLIC AROMATIC

HYDROCARBONS IN SOILS
L o a m y sand in the area o f the Forschungsanstalt f ü r Landwirtschaft, Braunschweig-Völkenrode

Polyarene Sample I Sample II Sample III Sample IV Mean value

Benzo(b,k,j)fluoranthene 104 106 120 118 112

Benzo(e)pyrene 48.7 49.7 50.5 44.3 48.3
Benzo(a)pyrene 22.7 23.8 24.4 21.8 23.2
Perylene 5.2 3.1 5.0 3.5 4.2
Indeno( 1}2,3-cd)pyrene 16.0 14.5 21.4 18.2 17.5
Dibenz(a,h)anthracene 38.4 36.5 43.7 33.8 38.1
Dibenz(a,c)anthracene 16.2 10.9 15.1 11.2 13.4
Benzo(ghi)perylene 31.6 33.6 31.9 31.4 32.1

Sam ple (w e ig h t: 20 o r 50 g)

2N K O H (m e th a n o l-w a te r, 9 + 1 ,3 0 0 mO in te rn a l standard

filte r in g
j

s o lu tio n o f sam ple in s o lu b le m aterials

+ m e th a n o l-w a te r (8 + 2 ,4 0 0 m l) c y c lo h e x a n e (2 X 8 0 0 m l)
]
2 X c y c lo h e x a n e m e th a n o l-w a te r

e v a p o ra tio n t o 3 0 ml

+ D M F -w a te r (9 + 1 , 2 X 60 m l)

I
D M F -w a te r ( 9 + 1 , 120 m l) c y c lo h e x a n e

+ w a te r ( 1 2 0 m l) + c y clohe xane (2 4 0 m l)

I
cyclo h e x a n e D M F -w a te r

+ waiter (2 X 1 0 0 m l)

cyclo h e x a n e w a te r

S iO j-C o lu m n

10 -1 0 0 m l c y c lo h e x a n e 0 - 1 0 ml

S E P H A D E X L H 2 0 co lu m n

Г I l
6 5 — 190 ml ( fra c tio n II) 3 8 — 6 5 ml ( fra c tio n I) 0 — 3 3 ml

G L C (2 5 0 ° is o th e rm ) G L C (1 2 0 ° — 2 4 0 ° C)

F lG .l. S c h e m e f o r c o n c e n tr a tio n o f p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s in s o il s , p l a n t s a n d s e w a g e s lu d g e c o m p o s t s .
 IAEA-SM-211/31 293

Hence it follows that it is mainly other polycyclic aromatic hydrocarbons that givq rise to
the carcinogenic effects, or the strong effect first sets in with combination of some polyarenes.
With most of the polycyclic aromatic hydrocarbons the extent of these carcinogenic effects is
still unknown as it is very difficult to determine them. Therefore all determinable polycyclic
aromatic hydrocarbons are measured, even if their identity is not yet known.
By analysing soils we have found more than 160 different polycyclic aromatic compounds.
A great many of these are still unidentified, so only ten components were first selected. These
are three benzofluoranthenes, the two benzopyrenes, indeno(l ,2,3-cd)pyrene, two dibenzanthracenes
and benzo(ghi)perylene. Table I shows the concentration of these compounds found in four
different soils from the experimental fields of the Forschungsanstalt für Landwirtschaft,
Braunschweig-Völkenrode. The three benzofluoranthenes are not separated into single components,
so the values in this table show the total concentration of them. The values in the tables are
parts per thousand million (ppl 09), which is дg per kg of sample or g per 1000 t. To determine
these compounds in soil and plants it is necessary to make a time-consuming enrichment, because
the concentration is very low. Grimmer and co-workers [4] have described a useful method to
isolate and determine polycyclic hydrocarbons in plants and food (see Fig.l). Twenty or 50 g
of the sample are mixed with 300 ml of aqueous-methanolic 2N KOH and an internal standard,
and the mixture is refluxed for 2 h. After this saponification the residual solids are filtered off
and washed with methanol. Then the liquid phase is extracted with cyclohexane by shaking it
in a separatory funnel. Washing the cyclohexane-phases with a mixture of water and methanol
is followed by a liquid-liquid partition in a dimethylformamide-water-cyclohexane-system. The
cyclohexane-phase is evaporated to about 0.5 ml under reduced pressure. Then comes a chroma­
tographic purification on silica gel, and after evaporation to 0.5 ml a separation into two fractions
on Sephadex LH 20 with isopropanol is made. These two solutions are evaporated to microlitres.
To prevent evaporation to dryness, 5 д1 dimethylformamide were previously mixed. Then the
fractions are separated into their components by a gas chromatographic measurement. For these
gas chromatographic separations a glass column (length 10 m) packed with 5% wt/wt OV 101 on
Gaschrom Q 100 —120 mesh is used.
In the first fraction are all the aromatic hydrocarbons with one to three rings, and all their
methyl- and dimethyl substituted derivatives. The second fraction contains all the aromatic
hydrocarbons with three or more rings. Most of the aromatic compounds with well-known
carcinogenic effects are in this fraction, for example benz(a)anthracene, benzo(a)pyrene and all
the dibenzanthracenes. The gas chromatogram is shown in Fig.2.
For about 20 years the Forschungsanstalt für Landwirtschaft, Braunschweig-Völkenrode, has
been carrying out experiments with municipal waste composts. A sampling was made from the
experiments covering two years to analyse the concentrations of polycyclic aromatic hydrocarbons
in the municipal wastes. The municipal waste composts, the data of which are shown in Table II,
were produced in a compost factory in Bad Kreuznach. The decomposed compost was deposited
for about six months before it was used in the test. Sewage sludge with a content of 98% water
was applied to the experimental field. The values in Table II therefore refer to the sample with
98% water.
The relatively high content of polycyclic aromatic hydrocarbons in the municipal wastes
leads to a considerable increase in them in the soil. The samples in Table III are from soils treated
with 100 t/ha sewage sludge and municipal waste compost. The values show a clear increase after
treatment. After using municipal waste compost on arable land the content of polyarenes increases
by about 65—75% in soil. It is therefore necessary to know what happens to these compounds in
soil, such as the following consequences:
1. Increase of the concentration of polyarenes by repeated applications of municipal waste
compost.
2. Leaching of polycyclic aromatic compounds to the groundwater.
3. Degradation by chemical and biochemical reactions.
 294
ELLWARDT
F I G .2 . G a s c h r o m a to g ra p h (F r a c tio n I I ) o f p o ly c y c li c a r o m a tic h y d r o c a r b o n s . C o lu m n : le n g th 1 0 m , i.d . 2 m m ,

5% w tfw t О V 101 on G a sch ro m Q 1 0 0 -1 2 0 m esh .

 IAEA-SM-211/31 295

4. Loss of the carcinogenic effects through chemical reactions.

5. Uptake of polyarenes by plants.
6. Transfer or metabolization of these compounds in plants.
Only variations in the concentration of polycyclic aromatic hydrocarbons in some soils and
plants through the use of municipal waste compost in agriculture can answer these problems.
For this purpose potatoes, oats and rye were analysed. A sample was taken from each of
these plants from a test plot, a plot treated with fresh municipal waste compost and
from a plot treated with decomposed municipal waste compost. The tubers and haulms
of the potatoes were analysed in particular and the grain and straw from oats and rye
also. Table IV shows the results of the concentration of polycyclic aromatic hydrocarbons
found in these plants. No simultaneous increase of polyarenes in plants grown on soil treated
with municipal waste compost is recorded. The content in tubers of the potatoes seems therefore
to be constant in all three plots. In the potato haulms the content is of considerably higher value,
but in all three plots there are almost no differences. The higher concentration of polycyclic
aromatic hydrocarbons in the haulms of potatoes compared with the tubers can be explained by
uptake of these substances from the atmosphere. Straw of oats and rye, equally in contact with
the atmosphere, will be contaminated with lower concentrations of polyarenes, because the
leaves are of considerably smaller surface. In the samples of oats and rye there are great oscillations

TABLE II. AMOUNTS (in pplO9) OF VARIOUS POLYCYCLIC AROMATIC

HYDROCARBONS IN SEWAGE SLUDGE AND WASTE COMPOSTS FROM BAD KREUZNACH

Polyarene Sewage sludge Fresh waste compost Decomposed waste compost

Benzo(b,kj)fluoranthene 2962 3400 3270

Benzo(e)pyrene 1247 1750 1520
Benzo(a)pyrene 806 680 655
Perylene 250 131 109
Indeno( 1,2,3-cd)pyrene 281 457 311
Dibenz(a,h)anthracene 684 785 847
Dibenz(a,c)anthracene 257 134 133
Benzo(ghi)perylene 610 802 823

TABLE III. AMOUNTS (in pplO9) OF VARIOUS POLYCYLIC AROMATIC

HYDROCARBONS IN SOILS TREATED WITH SEWAGE SLUDGE AND WASTE COMPOSTS

Plot treated Plot treated Plot treated

Polyarene Test plot with with with
sewage sludge fresh compost decomposed compost

Benzo(b,kj)fluoranthene 112 129 144 176

Benzo(e)pyrene 48.3 52.4 78.0 88.0
Benzo(a)pyrene 23.2 31.5 89.2 40.7
Perylene 4.2 5.9 7.2 7.0
Dibenz(a,h)anthracene 17.5 17.1 28.3 29.6
Dibenz(a,c)anthracene 38.1 43.6 57.5 63.4 j
Benzo(ghi)perylene 13.4 9.3 24.4 27.5 !
i
TABLE IV. AMOUNTS (in pplO9) OF VARIOUS P O L Y C Y C L I C H Y D R O C A R B O N S IN POTATOES, OATS AND RYE tO
40
On
Benzo- Benzo(e)- Benzo(a)- Perylene Indeno- D ibenz- Dibenz- Benzo-
(b ,k j)- pyrene (1,2,3-cd )- (a,h)- (a ,c)- (ghi)-
tluoranthene pyrene anthracene perylene

P o ta to e s Test plot 4.6 2.0 0.8 3.5 1.7 2.5 4.2

Tubers Plot with

fresh 5.4 2.6 0.8 6.2 1.7 5.0 5.0
compost

Plot with
decomp. 5.1 2.5 0.9 5.5 8.0 3.5 5.7
compost
Test plot 25.4 7.8 5.6 11.0 3.4 9.7

ELLWARDT
Haulms 35.0 6.5

Plot with
fresh 42.4 24.8 7.9 5.0 n.d.a 10.0 8.8 11.8
compost

Plot with
decomp. 50.0 33.4 8.4 6.8 3.2 9.5 4.9 9.2
compost

O a ts Test plot 15.2 5.8 1.2 4.8 n.d. 16.5 n.d. 4.6

Grain Plot with

fresh 21.2 7.5 1.5 13.1 5.4 20.2 n.d. 26.6
compost

Plot with
decomp. 16.2 7.0 1.2 n.d. 3.7 5.2 10.3 7.4
compost
TABLE IV. (cont.)

Benzo- B en zo(e)- Benzo(a)- Perylene Indeno- Dibenz - Dibenz- Benzo-

(b ,k j) - pyrene :(l,2,3-cd ) - (a ,h )' (a ,c)- (ghi) -
fluoranthene pyrene anthracene perylene

Straw Test plot 16.2 3.5 1.3 3.0 n.d. 1.4 1.0 6.4

Plot with
fresh 31.5 5.3 8.2 5.6 n.d. n.d. 13.3 17.7
compost

Plot with
decomp. 14.6 15.2 6.1 9.3 n.d. 5.1 10.9 17.1
compost

IA E A -SM -211/31
R y e Test plot 10.7 4.9 2.7 2.8 n.d. 1.9 2.3 0.5

Grain Plot with

fresh 3.5 4.0 2.3 1.5 n.d. 0.5 3.2 9.7
compost

Plot with
decomp. 1.5 5.5 2.5 9.9 n.d. 1.5 4.1 7.9
compost

Straw Test plot 11.3 2.3 3.3 1.0 n.d. и 1.3 6.8

Plot with
fresh 11.4 3.5 1.5 2.5 1.3 3.4 3.6 4.8
compost

Plot with
decomp. 10.8 3.3 1.4 4.8 0.5 1.9 2.4 2.9
compost

n.d. = not determinable.

298 ELLWARDT

in the concentrations, especially of the polyarenes with an upper boiling point such as the
dibenzanthracenes and benzo(ghi)perylene. These differences in content are not causally connected
with the different contents of polyarenes in the three plots. Wagner and Siddiqi [5] could show
that plants take up polycyclic aromatic hydrocarbons if these compounds are applied in very
high concentrations. In model experiments with nutrient solutions Harms [6] found that a strong
sorption occurred in the root system. Accordingly it seems that the utilization of municipal
waste compost for increasing soil organic matter does not cause an increase in the content of
carcinogenic polycylic aromatic hydrocarbons in plants. Before a final decision is made on the
use of municipal waste composts as source of nutrients or for soil conditioning, it is necessary to
ascertain whether the polycyclic aromatic hydrocarbons are degraded to water-soluble compounds
with carcinogenic effects, or if additive enrichment occurs, whereby concentrations are reached
that affect the plant uptake. *

REFERENCES

[ 1] BRUNE VON, Aerztl. Praxis XXVIII 16(1976).

[2] DRUCKREY, H., SCHILDBACH, A., Quantitative Untersuchungen zur Bedeutung des Benzpyrens für die
carcinogene Wirkung von Tabakrauch, Z. Krebsforsch. 6 5 (1 9 6 3 ) 465.
[3] LAZAR, P., CHOUROULINKOV, I., LIBERMANN, C., GUERIN, M., Benzo(a)pyrene carcinogenicity of
cigarette smoke condensate —results o f short-term and long-term tests, J. Natl. Cancer Inst. 37 5 (1966) 573.
[4] GRIMMER, G., HILDEBRANDT, A.,"BÖHNKE, H., Profilanalyse der polycyclischen aromatischen Kohlen­
wasserstoffe in proteinreichen Nahrungsmitteln, Ölen und Fetten (gaschromatographische Bestimmungs­
methode), Dtsch. Lebensm.-Rundsch. 71 3 (1975) 93.
[5] WAGNER, K.H., SIDDIQI, I., Der Stoffwechsel von 3,4-Benzpyren und 3,4-Benzfluoranthen im Sommerweizen,
Z. Pflanzenernaehr. Bodenkd.127 (1970) 211.
[6] HARMS, H., Metabolisierung von Benzo(a)pyren in pflanzlichen Zellsuspensionskulturen und Weizenkeim­
pflanzen, Landbauforsch. Völkenrode 25 2 (1975) 83.
 SOIL ORGANIC MATTER
COMPONENTS AND PLANT METABOLISM
(Session 9a)
 IAEA-SM-211/56

METABOLISM OF SOIL-RELATED
PHENOLIC COMPOUNDS IN PLANTS
AND CELL SUSPENSION CULTURES

H. HARMS
Institut für Biochemie des Bodens
der Forschungsanstalt für Landwirtschaft,
Braunschweig-Völkenrode,
Federal Republic of Germany

Abstract

METABOLISM OF SOIL-RELATED PHENOLIC COMPOUNDS IN PLANTS AND CELL SUSPENSION CULTURES.

Various specifically 14C-labelled benzoic and cinnamic acids were added to the nutrient solutions o f sterile-
cultured seedlings and cell suspension cultures o f different plant species, and tested for metabolic reaction. On the
basis o f 14C 0 2-formation and isolated 14C-labelled metabolites, the decarboxylation and O-demethylation reactions
were shown to be restricted to specific substituted groups o f the aromatic ring. Decarboxylation o f substituted
phenolic acids could only be observed when the aromatic acids possessed a hydroxyl group in the para position.
The O-demethylation reactions were shown to be specific for para m ethoxyl groups. The ring cleavage reaction was
found to be specific for ortho dihydroxy compounds. The methoxyl group in the 4-position was also split if the
carboxyl group in the para position was modified to a Cj-side chain or even to an alcohol group. The Cз-side chain
had been split much faster than the alkyl-aryl-ether bonds by demethylation reaction. In addition, various polycyclic
hydrocarbons specifically labelled with 14C were added to the nutrient solutions o f seedlings and cell suspension
cultures o f plants, and tested for metabolic reaction. These polyaromatic compounds are absorbed only by the roots.
As autoradiographic studies show, there is no transport into the sprouts, apart from anthracene which can be detected
in the upper organs also. Experiments with different cell cultures indicate that the absorbed polyaromatic hydro­
carbons are metabolized to a less extent. Most o f the absorbed activity can be isolated as the applied compound.
Compared with other cell cultures tested, those o f C h e n o p o d i u m r u b r u m behaved quite differently. Absorbed
benzo(a)pyrene was not extractable with organic solvents but was mainly detected in the form o f water-soluble
compounds. By means o f high-pressure liquid chromatography these oxygenated derivatives were detected and
analysed.

During microbial degradation of lignin and humic substances, phenols and phenolic acids
are formed. These compounds are present either in the free form or can be easily hydrolysed
from soil organic matter. As various experiments [1,2] show, phenols and phenolic derivatives
are assimilated by plants and have an influence on plant metabolism. Initially our interest was
aimed at the uptake and translocation of these different phenolic compounds by plants under
aseptic conditions. In further experiments we followed the metabolism and catabolism of the
compounds. For these studies we used cell suspension cultures of various plant species as well as
sterile-grown plants. Cell suspension cultures can be expected to circumvent some difficulties
known from experiments with plants, such as low concentrations of the metabolites formed as
well as extensive binding of the phenolic substrates to insoluble polymeric structures.
Figure 1 provides a recapitulation of the tests applied. Various benzoic and cinnamic acids
14C-labelled in carboxyl, in the para and the meta methoxyl groups as well as in ring-C-atoms,
were added to the nutrient solutions of sterile-cultured seedlings and cell suspension cultures, and
tested for metabolic reaction.
On the basis of 14C02-formation the decarboxylation and the O-demethylation reactions
were shown to be restricted to specific substituted groups of the aromatic ring. Decarboxy­
lation of substituted benzoic acids could be observed only when the aromatic acids

301
302 HARMS

possessed a hydroxyl group in the para position. Further hydroxy or methoxy groups in
the ortho position to the hydroxy group in the para position increased the release of C02
from the labelled carboxy group.
The O-demethylation reactions were shown to be specific for methoxyl groups in the p-position.
The products formed by O-demethylation of these methoxyl groups could be identified as
p-hydroxybenzoic acid from anisic acid, vanillic acid from veratric acid, and syringic acid from
trimethoxybenzoic acid. These 4-hydroxybenzoic acids are normally decarboxylated to a great
extent after being fed to cultures. When they are formed in the cells and in the plants by
O-demethylation, no decarboxylation reaction can be observed. These observations and some
other indications give evidence of a possible compartmentalization of plant cells.
The ring cleavage reaction was found to be specific for orthodihydroxy compounds such as
protocatechuic or caffeic acid. To follow the metabolites formed by ring cleavage reactions, in-vitro
experiments are necessary. We have been unable to do these, but as other investigators [3] show,
metabolites like a-carboxy-cis, cis-muconic acid are formed.
The experiments show that there is no difference between seedlings and cell suspension cultures
of plants in the metabolic reactions.
In further experiments we tested how the observed demethylation reaction is influenced by
other substituents of the ring. For this account the carboxyl group of specifically 14C-labelled
veratric acids was modified to a C3-side chain to get the corresponding cinnamic acids. Furthermore
the corresponding veratryl alcohols were synthesized. These compounds were added to the nutrient
solutions of cell cultures and tested for metabolic reaction (Table I).
 IAEA-SM-211/56 303

TABLE I. PERCENTAGE DISTRIBUTION OF THE ACTIVITY IN ISOLATED COMPOUNDS

FROM CELL SUSPENSION CULTURES OF MUNG- AND SOYBEAN AFTER ADDITION OF
DIMETHOXY CINNAMIC ACID AND VERATRYL ALCOHOL CARBON-14-LABELLED
IN DEFINED POSITIONS :

Percentage o f the absorbed activity! in

Cell suspension Applied Labelled co2 dimethoxy ferulic veratric vanillic

culture compound in cinnamic acid acid acid
acid

P h a s e o lu s Dimethoxy 3-methoxy 1.0 32.9 0.6 7.9 17.6

aureu s cinnamic 4-methoxy 23.5 35.7 - 7.0 -
(mung-bean) acid Сз-side 0.5 32.4 - 9.1 13.4
chain
G ly c in e m a x . Dimethoxy 3-methoxy 0.7 39.5 — 3.1 1.5
(soybean) cinnamic 4-methoxy 5.5 36.9 - 2.7 -
acid Сз-side 0.6 39.0 2.1 0.6
chain "
veratryl- veratric vanillic
alcohol acid acid

G ly c in e m a x . Veratryl 3-methoxy 0.9 23.8 33.6 24.0

(soybean) alcohol 4-methoxy 18.6 24.7 32.4 -

As already known for benzoic acids the O-demethylation reaction is also specific for methoxy
groups in p-position of cinnamic acids. In this case the methoxy group is split to nearly the same
extent. The same amount of activity released as C02 from 4-methoxy-labelled dimethoxy cinnamic
acid is detectable in the form of vanillic acid when 3-methoxy-labelled dimethoxy cinnamic acid
has been added to the cell cultures. The binding in /З-position of the C3-side chain is split to a much
quicker extent than the methoxy group in the 4-position by demethylation. Therefore most of the
activity was isolated in form of the corresponding benzoic acids. If the carboxyl group of the
specifically labelled veratric acids is reduced to the corresponding alcohol group, and these compounds
are added to cell cultures, a release of C02 from the ether group in the 4-position is detectable.
From veratric alcohol labelled in the 3-methoxy group, 14C-labelled vanillic acid can be isolated. The
alcohol group first in return seems to be oxydized to a carboxy group.
Kinetics of the reactions —(3-splitting of the C3-side chain, O-demethylation and decarboxylation
of cinnamic acids - show that the C3-side chain is split much faster than the alkyl-aryl-ether bonds by
demethylation reaction. This is also the reason why only small amounts of fcrulic acid could be
isolated when dimethoxy cinnamic acid was applied to cell cultures.
Another subject of our investigations with phenolic compounds deals with the metabolism
of polyaromatic hydrocarbons [4]. These compounds are ubiquitous pollutants of the atmosphere,
waterways and soil, and are present in the food chain. Some of these compounds can cause cancer
in experimental animals and are therefore suspected of causing also cancer in humans. In order to
understand how plants and food are contaminated it is thus necessary to know the rate of uptake
of the polyaromatic hydrocarbons and the metabolites that are possibly formed.
Various polyaromatic hydrocarbons such as benzo(a)pyrene, anthracene, benzanthracene and
dibenzanthracene specifically labelled with l4C were added to the nutrient solution of Sterile
cultures of seedlings and cell suspension cultures of plants. The absorption rate and metabolic
reaction were tested. Autoradiographic studies of the plants show that the activity in most cases is
TABLE II. DISTRIBUTION OF THE ACTIVITY IN THE TEST PLANTS AFTER THE ADDITION OF CARBON-14-LABELLED POLYAROMATIC

304
HYDROCARBONS

Applied Test plant Fractions Distribution of ppm

compound applied activity

Benzo(a)pyrene T r itic u m s a tiv u m Applied activity/plant 100 % - 400 fig

(wheat) Remainder o f activity in culture medium 84.7 % - 338.8 pg

^ ^ sprout (22.34 mg/plant) 0.2 % - 0.84 pg 37.6
Dry weight root (1Q 95 mg/plant) 865.8
2.4 % - 9.48 Mg

Benzo(a)pyrene B e ta v u lg a r is Applied activity/plant 100 % — 163 Mg

(sugar-beet) Remainder of activity in culture medium 88.5 % - 144.2 Mg
^ A sprout (106.0 mg/plant) 0.26% - 0.43 Mg 4.1
Dry weight root ( 29 8 mg/plant) 614.1
11.2 % - 18.3 Mg

Benzo(a)pyrene A t r ip le x h o r te n s is Applied activity/plant 100 % -1 6 0 Mg

(orach) Remainder o f activity in culture medium 94 % — 151 Mg

HARMS
^ . , sprout (93.08 mg/plant) 0.3 %- 0.48 Mg 5.2
loot (26.26 mg/plant) 5.7 %- 9.15MS 348.4

Benzo(a)pyrene A t r ip le x h o r te n s is Applied activity/plant 100 % ~ 66 Mg

(orach) Remainder of activity in culture medium 85.1 % - 56.2 Mg
_ . , . sprout (74.38 mg/plant) 0.2 % - 0.13 Mg 1.8
Dry weight root ( 2 2 2 4 mg/plant) 391.2
13.2 % - 8.7 Mg

Dibenz(a,h) B e ta v u lg a r is Applied activity/plant 100 % — 173 Mg

anthracene (sugar-beet) Remainder of activity in culture medium 95 % - 164.3 Mg
^ A sprout (122.7 mg/plant) 0.15% — 0.26 Mg 2.1
Dry weight root ( 32.2 mg/plant) 4.8 % - 8.4 Mg 260.7

Dibenz(a,h) A tr ip le x h o r fe n sis Applied activity/plant 100 % — 175 Mg

anthracene (orach) Remainder o f activity in culture medium 94.9 % - 165.9 Mg
л x sprout (96.12 mg/plant) 0.07% — 0.12 Mg 1.2
ГУ weig r0Qt (30.95 mg/plant) 5.0 % - 8.74 Mg 282.4

Anthracene A t r ip le x h o r te n s is Applied activity/plant 100 % — 215 Mg

(orach) Remainder o f activity in culture medium 92.5 % — 199 Mg
_ . . . sprout (60.79 mg/plant) 0.34% - 0.73 Mg 12.0
Dry weight roof (2 1 . 17 mg/plant) 5.6 % - 1 2 . 1 f i g 570.6
 IAEA-SM-211/56 305

absorbed only by the roots. There is no transport of the added polycyclic aromatics into the sprouts.
The only exception is anthracene, which can be detected in the upper green organs also. A distribu­
tion of the activity in the different plant species after addition of various polycyclic hydrocarbons is
summarized in Table II.
The experiments with plants show that only small amounts of the added polycyclic aromatic
hydrocarbons can be detected in the roots, and only 0.2% is located in the sprout material.
Depending upon the plant species used, growth of the cell cultures was inhibited to a varying
extent. The cells of the various plant cell cultures absorbed 60—90% of the added polyaromatic
hydrocarbons. Most of this quantity could be extracted from the plant material with organic
solvents in the form of the added compound, and only 1—3% were isolated as metabolites.
Compared with other plants tested, those of the family Chenopodiaceae behaved quite differently.
Absorbed benzo(a)pyrene was not extractable with organic solvents, but was mainly detected in the
form of water-soluble compounds. By means of high-pressure liquid chromatography these
compounds were detected and analysed as oxygenated derivatives. They seem to be similar to
derivatives known from animal experiments [5] like epoxides which undergo a number of further
subsequent reactions to form phenols and even dihydrodiols of benzo(a)pyrene. These oxygenated
derivatives are much more potent carcinogens.

REFERENCES

[1] HARMS, H., SÖCHTIG, H., HAIDER, K., Aufnahme und Umwandlung von in unterschiedlichen Stellungen
C14-markierten Phenolcarbonsäuren in Weizenpflanzen, Z. Pflanzenphysiol. 64 (1971) 437.
[2] HARMS, H., Pflanzliche Zellsuspensionskulturen — ihr Leistungsvermögen für Stoffwechseluntersuchungen,
Landbauforsch. Völkenrode 23 (1973) 127.
[3] SHARMA, H.K., JAMALUDDIN, M., VAIDYANATHAN, C.S., An enzyme system clearing the aromatic ring
o f 2,3-dihydroxybenzoic acid from leaves o f T e c o m a s t a r t s , FEBS Lett. 28 (1972) 41.
[4] HARMS, H., Metabolisierung von Benzo(a)pyren in pflanzlichen Zellsuspensionskulturen und Weizenkeim­
pflanzen, Landbauforsch. Völkenrode 25 (1975) 83.
[5] SELKIRK, J.K., Determination o f metabolic products o f benzo(a)pyrene by LC, Chromatographic Rev. 2
(1975) 1.
 IAEA-SM-211/57

ESTUDIO DE LA ACCION EJERCIDA

SOBRE LA PLANTA DE MAIZ POR
DOS TIPOS DE ACIDO HUMICO

V. HERNANDO, B.C. ORTEGA, C. FORTUN

Instituto de Edafologfa у Biologia Vegetal,
Madrid,
Espana

Abstract-Resumen

STUDY OF THE ACTION OF TWO TYPES OF HUMIC ACID ON THE MAIZE PLANT.
The action exerted on maize cultivation by two humic acids obtained from manure with different extract­
ants was studied. It was found that the acids, when applied in identical doses, had different effects on the
physiological processes o f the plant, leading to variations in the production of vegetable matter and in the mineral
nutrition and moisture content. It was also shown that the major alteration produced in the molecular structure
of the humic acid by the extractant 0.1N NaOH had a favourable physiological effect on the production of
vegetable matter by the maize plant.

ESTUDIO DE LA ACCION EJERCIDA SOBRE LA PLANTA DE MAIZ POR DOS TIPOS DE ACIDO HUMICO.
Se ha estudiado la influencia que ejercen dos äcidos hümicos, obtenidos de un estiercol con diferentes
extractantes, sobre el cultivo del malz. Se ha comprobado que la aplicacion de ambos acidos en dosis identicas
actüa sobre los procesos fisiolögicos de la planta en forma distinta, dando lugar a variaciones en la production
de materia vegetal, en las alimentaciones minerales у en el contenido de humedad de la misma. Por otro lado,
se ha puesto de manifiesto que la mayor alteration de la estructura molecular del äcido humico provocada por el
extractante NaOH 0,1N ejerce un efecto fisiolögicamente favorable sobre la production de materia vegetal en
la planta de mafz.

INTRODUCTION

La explication del efecto que ejercen los acidos hümicos sobre el desarrollo de las plantas
se ve dificultada por una serie de inconvenientes, entre los que es preciso destacar la election del
extractante mas adecuado para obtener dicho äcido humico, tal у сото se encuentra enjel material
orgänico humificado.
Estudios realizados por otros investigadores [ 1] у por nosotros mismos [2], ban puesto de
manifiesto que las resinas de intercambio ionico son, entre todos los estudiados, los extractantes
que menos afectan la integridad estructural del äcido humico, por lo que se puede suporier que
el äcido humico extrafdo con resinas se comporte de una forma mas natural frente a las plantas
que los acidos hümicos obtenidos con el resto de los extractantes comünmente empleados.
Segun lo expuesto, pretendemos con este trabajo estudiar el comportamiento que, frente a
las plantas de max'z, tienen los acidos hümicos extrafdos de estiercol con NaOH 0,1N у resina
Lewatit-S-100, ya que comprobamos [2] que con estos dos extractantes se obtienen acidos hümicos
estructuralmente diferentes.

MATERIALES Y METODOS

Se Uevo a cabo un experimento de invernadero con plantas de mafz en cultivo hidroponico

sobre cuarzo triturado. Se utilizaron dosis de 0, 6, 12, 24, 48 у 96 mg de äcido hümico por kg
de cuarzo, realizando tres repeticiones de cada tratamiento. El cultivo se mantuvo en invernadero
durante dos meses, permaneciendo präcticamente constantes las condiciones iniciales. :

307
 308
CUADRO I. ACCION DE LOS ACIDOS HUMICOS S Y R SOBRE EL PESO DE LA PARTE AEREA DE LA PLANTA DE MAIZ

Plantas tratadas con S Plantas tratadas con R

Tratamientos
Peso %Д Peso %Д Humedad Peso %Д Peso % A Humedad
AH/kgCz)
humedo Peso seco Peso % humedo Peso seco Peso %
humedo seco humedo seco

HERNANDO et al.
0 339,6 33,4 91,9 3 3 9 ,6 33,4 91,9
6 319,6 - 5,9 41,5 + 24,3 87,0 315,9 - 7 ,0 35,1 + 5,1 88,8
12 238,1 - 30,0 22,6 - 32,3 94,8 359,7 + 6,0 44,4 + 33,0 87,6
24 298,4 - 12,1 34,1 + 7,1 88,7 339,0 -0 ,2 40,3 + 20,7 88,1
48 289,4 - 14,8 36,1 + 8,1 87,7 299,7 - 11,8 30,8 - 7,8 89,7
96 307,8 - 9,4 37,7 + 12,9 87,7 284,4 - 16,3 21,7 -3 5 ,0 92,4

mg AH/kgCz = miligramos de äcido hiimico рог kg de cuarzo, + Д = incremento. - Д = disminucion.

CUADRO II. ACCION DE LOS ACIDOS HUMICOS S Y R SOBRE EL PESO DE LA RAIZ DE LA PLANTA DE MAIZ

Plantas tratadas con S Plantas tratadas con R

Tratamientos
(mg AH/kgCz) Peso % Д Peso %Д Humedad Peso % Д Peso % A Humedad
humedo Peso seco Peso % humedo Peso seco Peso %

IAEA-SM-211/57
humedo seco humedo seco

0 97,0 13,1 85,4 97,0 13,1 85,4

6 85,1 - 12,5 9,1 - 30,5 89,3 79,7 - 17,8 8,6 - 34,4 89,2
12 113,5 ■+ 17,0 13,3 + 1,5 88,2 118,1 + 21,8 14,6 + 11,5 87,4
24 133,6 + 37,7 17,7 + 35,1 86,8 110,8 + 14,2 12,6 — 3,8 88,6
48 96,6 -0 ,4 1 9,6 - 26,7 90,0 65,4 - 32,6 6,6 -4 9 ,6 88,9
96 121,6 + 24,9 13,9 + 6 ,i 88,5 89,7 - 7,5 11,2 - 14,5 87,5

mgAH/kgCz = miligramos de acido humico рог kg de cuarzo, + Д = mcremento, - Д = disminucion.

 310
CUADRO Ш. AC'CION DE LOS ACIDOS HUMICOSS y R SOBRE EL CONTENIDO MINERAL DE LA PARTE AEREA DE LA PLANTA DE
MA1Z

Plantas tratadas con S Plantas tratadas con R

Tratamicntos
{mg AH/kg Cz) N P к Ca Mg Na Fe Mn Zn Al N P К Ca Mg Na Fe Mn Zn Al

HERNANDO et al.
(%) (ppm) (% ) (ppm)

0 2,10 0,44 4,4 0,54 0,53 0,005 69 164 37 100 2,10 0,44 4,4 0,54 0,53 0,005 69 164 37 100
6 1,80 0,27 2,8 0,50 0,45 0,004 61 127 23 50 1,80 0,36 3,6 0,62 0,57 0,003 63 163 31 66
12 2,10 0,41 4,6 0,47 0,44 0,004 58 122 35 66 1,40 0,32 2,9 0,53 0,50 0,005 65 110 20 50
24 1,80 0,34 3,7 0,53 0,49 0,004 70 135 38 50 2,60 0,47 4,2 0,61 0,51 0,004 95 190 35 83
48 1,50 0,40 4,1 0,56 0,52 0,004 53 137 35 50 2,10 0,45 4,5 0,65 0,48 0,004 72 158 35 83
96 1,40 0,30 3,0 0,48 0,47 0,004 55 108 28 50 2,20 0,49 4,7 0,61 0,49 0,004 65 173 28 66

mg АН/kg Cz = miligramos de äcido humico por kilogramo decuarzo.

 IAEA-SM-211/57 311

Se separö, en cada una de las plantas, la parte aerea de la raiz у se desecaron a 60°C. Se
determino peso hümedo у seco, tanto de raiz сото de parte aerea, macro у oligoelementos у se
realizo un estudio estadistico de los resultados, que fueron significativos al nivel de probabilidad
del 5%.
Denominaremos S a los äcidos hümicos obtenidos con NaOH 0,1N у R a los obtenidos con
resina.
En los cuadros I у II podemos observer la acciön de los äcidos hümicos S у R sobre los
rendimientos, tanto de materia hümeda сото seca, de la parte aerea у de la raiz de la planta de
mafz.
Se puede apreciar que el äcido humico S produce, para todas las dosis de aplicacion, un
descenso en la production de la parte aerea que incluso llega a alcanzar el valor del 30% para
la dosis de 12 mg. Sin embargo, al considerar los pesos secos, unicamente la dosis de 12 mg
es la quedalugar a una disminuciön, mientras que las demäs experimentan un incremento, siendo
el mas alto el de la dosis de 6 mg (24,3%).
Con respecto a la produccion de la raiz es preciso senalar que la dosis de 6 mg de äcido
humico S es la que produce los incrementos mäs bajos en los rendimientos (—12,5 у —30,5) con
respecto a los pesos tanto humedos сото secos, mientras que la dosis de 24 mg es la que produce
unos aumentos mayores (37,7% у 35,1%).
Рог otro lado, el äcido humico R ocasiona un descenso de peso hümedo de la parte aerea para
todas sus dosis de aplicacion, excepto para la de 12 mg, que da lugar a un incremento del 6%.
En lo que se refiere a peso seco, ünicamente las dosis de 48 у 96 mg producen una disminuciön
en el mismo. En el caso de la raiz, son las dosis de 12 у 24 mg de äcido humico R las que dan
lugar a un aumento en el rendimiento de peso hümedo, mientras que tan solo la dosis de 12 mg
produce un aumento de peso seco у las demäs dosis aplicadas ocasionan un efecto depresivo.
Es muy interesante hacer notar que todas las dosis aplicadas de los dos äcidos hümicos pro­
ducen, en general, un incremento en el porcentaje de humedad tanto de la raiz сото de la
parte aerea.

CONTENIDO MINERAL DE LA PLANTA

En el cuadro III podemos observar que, en general, las dosis crecientes de aplicacion de
äcido humico S producen un descenso en el contenido de macro у oligoelementos de la parte
aerea. En esta misma parte de la planta, la aplicacion de äcido humico R ocasiona una disminuciön
en el porcentaje de elementos mayores (N, P, К у Ca) en las dosis de 6 у 12 mg; sin embargo,
se produce un aumento en las restantes dosis aplicadas.
En las plantas tratadas con äcido humico R у para la parte aerea de las mismas, las distintas
dosis aplicadas no producen präcticamente variaciön en los contenidos de Fe, Mn у Zn; j sin
embargo, si producen una sensible disminuciön en el contenido de Al.
En el caso de la raiz (cuadro IV), tambien las dosis crecientes de aplicacion de äcido humico
S producen, en general, un descenso en el contenido de macro у oligoelementos.
La aplicacion de äcido humico R da lugar a una disminuciön en el porcentaje de N, P у К en
las tres primeras dosis de aplicacion (6, 12 у 24 mg) produciendose un aumento en las otras dos
dosis, mientras que los porcentajes de Ca у Mg no varian präcticamente.
Los contenidos de Fe у Al de la raiz de las plantas tratadas con äcido humico R disminuyen
con el aumento de las dosis aplicadas, mientras que aumenta con las mismas el contenido de Mn,
permaneciendo präcticamente constantes los contenidos de Zn у Cu. Este hecho nos demuestra
que a pesar de que el äcido humico R tiene mayor proporciön de Fe у Al que el äcido humico ,S[2],
östos no han sido absorbidos рог las plantas estudiadas ya que presentan un menor contenido de
dichos elementos en estos tratamientos.
 312
CUADRO IV. ACCION DE LOS ACIDOS HUMICOS S Y R SOBRE EL CONTENIDO MINERAL DE LA RAIZ DE LA PLANTA DE MAIZ

Plantas tratadas con S Plantas tratadas coni?

Tratamientos
(mg АН/kg CzJ
N P К Ca Mg Na Fe Mn Zn Cu Al N P К Ca Mg Na Fe Mn Zn Cu Al
(%) (ppm) (%) (ppm)

HERNANDO e, al.
0 1,04 0,24 2,1 0,76 0,79 0,061 3030 980 43 15 1885 1,04 0,24 2,1 0,76 0,79 0,061 3030 980 45 15 1885
6 0,90 0,30 2,8 0,80 0,70 0,060 1750 1000 47 15 1275 0,81 0,22 1,7 0,76 0,74 0,072 1243 866 40 10 850
12 0,81 0,22 1,9 0,60 0,69 0,054 1550 760 50 15 1025 0,63 0,19 L7 0,72 0,65 0,049 1300 700 32 12 950
24 0,98 0,20 2,1 0,59 0,64 0,052 2033 766 58 20 1466 0,79 0,24 1,7 0,68 0,64 0,047 1200 860 33 17 1280
48 0,95 0,27 2,4 0,71 0,89 0,065 1300 966 63 17 1150 1,20 0,28 2,5 0,63 0,68 0,052 2250 1160 42 18 1350
96 0,64 0,17 1,7 0,60 0,69 0,050 1200 568 42 17 1100 1,45 0,32 3,4 0,76 0,78 0,061 560 1100 50 12 650

mg АН/kg Cz = miligramos de acido humico рог kilogramo de cuarzo.

 IAEA-SM-211/57 313

DISCUSION

La distinta naturaleza de los äcidos hümicos aplicados a plantas de maiz se pone de manifiesto
en un efecto diferente sobre las mismas. En los cuadros V у VI podemos observar una Serie de
relaciones que nos expresan claramente estos diferentes efectos.
El concepto de Agm (alimentaciön global de macroelementos) indica el contenido total de
macroelementos expresados en tanto porciento,y el de Ag0 el contenido total de oligoelementos
expresados en partes por millön.
Dado que el contenido mineral de la soluciön nutritiva empleada en el cultivo es e.'. calculado
сото öptimopara el desarrollo de la planta de maiz, hemos de suponer que las dos alimentaciones
globales correspondientes a las plantas testigo serän tambien las öptimas. Сото vemos en el
cuadro V, la acciön de los äcidos hümicos se refleja tanto sobre las alimentaciones globales сото
sobre las variaciones en los rendimientos en materia vegetal seca. Esta acciön de los äcidos
hümicos sobre la producciön de materia vegetal serä tanto mäs efectiva si debido a su aplicaciön
se obtienen incrementos en el rendimiento con alimentaciones minerales mäs bajas. Esto es, las
plantas testigo necesitan de una alimentaciön global de macroelementos de 8 у de oligoelementos
de 370 para la producciön de 100 g de materia vegetal seca; sin embargo, рог la acciön de 6 mg
de äcido hümico S por kg de cuarzo solo son necesarias alimentaciones de 5,8 у 261 para la
obtenciön de 100 g de materia vegetal seca. Lo expuesto anteriormente equivale a decir que la
dosis de 6 mg de äcido hümico S produce 27,4% de descenso en la alimentaciön global de macro­
elementos de la planta de maiz у 29,5% en la de oligoelementos, causando sin embargo un
incremento de 24,3% en la producciön de materia vegetal seca.
Esta misma dosis de 6 mg de äcido hümico R da lugar a un descenso en la alimentaciön
global de macroelementos del 13,3% у a un descenso en la de oligoelementos de 12,7%, asi сото
a un aumento del 5,1% en la producciön de materia vegetal seca.
Esto nos muestra el diferente comportamiento de los dos äcidos hümicos S у R en su acciön
sobre el desarrollo de la planta de maiz.
En trabajos que hemos realizado anteriormente [3,4], encontramos que la aplicaciön al
cultivo de maiz del äcido hümico no produce efectos proporcionales a las dosis aplicadas, sino
que hay sucesivos aumentos у disminuciones dependiendo de la cuantia de la dosis. Eri el presente
trabajo podemos comprobar que el äcido hümico extraldo con NaOH 0,1N se comporta de manera
identica que en los trabajos antes citados, esto es, da lugar a un mäximo en la producciön de
materia vegetal seca para la dosis de 6 mg у a un minimo de producciön para la dosis de 12 mg.
A partir de esta dosis, la producciön aumenta con la dosis aplicada.
Esto nos confirms la hipötesis sostenida en los citados trabajos, segün la cual atribufamos
el efecto del äcido hümico sobre el desarrollo de las plantas a su acciön, tanto sobre los procesos
enzimäticos сото sobre los respiratorios.
Vemos (cuadro V) que por la acciön de los dos äcidos hümicos S y R disminuyen muy
acusadamente las alimentaciones globales de oligoelementos; es decir, la planta necesita una menor
concentraciön de oligoelementos para su desarrollo.
Esto se podrfa explicar si los dos äcidos hümicos facilitaran, en el metabolismo fisiolögico de
la planta, la acciön de los oligoelementos, permitiendoles por ejemplo una mayor movüidad, con
lo cual las plantas necesitarfan para su desarrollo menores concentraciones de los mismos.
Excepto para la dosis de aplicaciön de 12 mg de äcido hümico S, la acciön de este äcido hümico
se manifiesta por una disminuciön del % AAgm у del % AAgo у un aumento del % Д de peso seco
de parte aerea (cuadro II); esto es, la acciön del äcido hümico da lugar a un incremento en la
producciön de peso seco de materia vegetal, con una disminuciön del contenido en los elementos
minerales analizados que aparecen en el cuadro IV, lo que indica la acciön directa del äcido hümico
S sobre la smtesis de compuestos orgänicos que,formando parte de la materia vegetal, önicamente
contienen С, H у O, esto es sustancias orgänicas del tipo de la celulosa, almidön, azucares, etc.
 314
CUADRO V. ACCION DE LOS ACIDOS HUMICOS S Y R SOBRE LAS ALIMENTACIONES MINERALES Y PESOS DE LA PARTE AEREA

Plantas tratadas con S Plantas tratadas con R

Tratamientos
(mg АН/kg Cz)
Agm %AAgm Ago % AAg0 % A peso % A peso Agm AAgm Ago %A Ag0 %Apeso %Apeso
humedo seco humedo seco

HERNANDO et al.
0 8,0 370 8,0 370
6 5,8 - 27,4 261 - 29,5 - 5,9 + 24,3 6,9 - 13,3 323 - 12,7 - 7,0 + 5,1
12 8,0 0 281 - 24,1 - 30,0 - 32,3 5,7 - 29,6 245 - 33,8 + 6,0 + 33,0
24 6,9 - 14,5 293 - 20,8 - 12,1 + 2,1 8,4 + 4,6 403 + 8,9 - 0,2 + 20,7
48 7,1 - 11,7 275 - 25,7 - 14,8 + 8,1 8,2 + 2,0 348 - 5,9 - 11,8 - 7,8
96 5,7 - 29,6 241 - 34,9 - 9,4 + 12,9 8,5 + 5,9 332 - 10,3 - 16,3 -3 5 ,0

mgAH/kg Cz = miligramos de äcido humico рог kilogramo de cuarzo.

Agm = alimentacion global de macroelementos.
Ag0 = alimentacion global de oligoelementos.
 IAEA-SM-211/57 315

Este hecho nos confirms la hipötesisde Sladsky [5] segun la cual uho de los efectos mäsjimpor-
tantes del äcido hümico es su acciön sobre los procesos metabölicos que intervienen en la funciön
clorofilica.
En el mismo cuadro podemos ver el comportamiento de las distintas dosis de äcido hümico R
у comprobar la diferente acciön de los dos äcidos hümicos, tanto sobre las alimentaciones minerales
сото sobre la production de materia vegetal. Nos parece importante destacar el hecho de que
mientras la dosis de 12 mg de äcido hümico S es la ünica que produce un notable descenso (32,3%)
en la production de parte aerea seca, sea esta misma dosis de 12mg,pero de äcido hümico R , la
que produce mayor incremento (33,0%) en la production de parte aerea seca.
Tarn bien podemos comprobar que todos los tratamientos con los äcidos hümicos S у R pro-
ducen una disminuciön en el contenido de humedad de la parte aerea, lo cual nos muestra la
acciön del äcido hümico sobre el regimen hfdrico de la planta.
En el caso de la raiz (cuadro VI), la dosis optima de aplicaciön del äcido hümico S es la de
24 mg ya que da lugar al mäximo incremento en la producciön de peso seco de raiz (35,1%) con
disminuciones en las alimentaciones globales (% AAgm = 8,6 у % AAg0 = 27,0).
Para el äcido hümico R la dosis optima corresponde а 12 mg con un incremento de peso seco
del 11,5% у disminuciones del % Д А ^ =21,Oy del%AAg0 = 49,7. La dosis mäs peijudicial de
este äcido hümico R es la de 48 mg con una disminuciön de peso seco de 49,6%, un incremento
% AAgjn = 7,0 у una disminuciön % A A g0 = 32,6. Es interesante hacer notar que tres (12, 24 у
96 mg) de las 5 dosis de äcido hümico S aplicadas producen incrementos de peso seco de raiz,
mientras que solamente una (12 mg) de äcido hümico R da lugar a un incremento de peso seco.
Anteriormente pusimos de manifiesto [2 ] que los äcidos hümicos S contienen mäs grupos
funcionales carboxilicos у fenölicos que los R , у que los äcidos hümicos R estan mäs pölimerizados
que los S; por lo tanto, los äcidos hümicos S retendrän mäs Fe у Al de la solution nutritiva por
formation de complejos entre estos cationes у los grupos COOH у ОН у, a su vez, las moleculas
de estos complejos son mäs pequenas que las de los complejos formados por los äcidos hümicos R ;
por lo tanto, los complejos S-Fe у .5-Al penetrarän con mäs facilidad en la raiz, lo que nos explica
la mayor concentration de Fe у Al en las raices de las plantas tratadas con äcido hümico S.
Ademäs, el que la moläcula de äcido hümico S este menos polimerizada у tenga un mayor
contenido fenölico implica que este äcido es mäs fäcilmente absorbido por las plantas, actuando
mäs intensamente sobre los procesos respiratorios.
Ahora bien, en los cuadros ya mencionados vemos сото las plantas tratadas con ambos
äcidos tienen, para todas las dosis de aplicaciön, alimentaciones globales de oligoelementos inferio­
res a las no tratadas. Esta disminuciön en el contenido de oligoelementos por la raiz tiene una
clara tendencia a aumentar con las dosis de aplicaciön de los dos äcidos hümicos, llegando a
alcanzar valores del 50,8 у del 60,2%.
El aumento de las dosis aplicadas de los dos äcidos hümicos implica el hecho de la formation
de una cantidad mayor de ciertos complejos S у R -oligoelementos, con lo cual aumentarä la
dificultad de absorciön de algunos oligoelementos por las raices у, сото consecuencia, disminuirä
en ellas la concentraciön de los mismos al aumentar las dosis de aplicaciön de los äcidos hümicos.
Por otra parte, el contenido en determinados oligoelementos es menor en las raices tratadas
con äcido hümico R que con äcido hümico S, lo cual es debido a que los complejos 7?-oligo­
elementos, al tener un mayor volumen molecular que los S-oligoelementos, penetran con mayor
dificultad en la raiz. I
De lo expuesto anteriormente deducimos que los extractantes empleados en la funciön de
las fracciones orgänicas humificadas modifican sustancialmente las moleculas de los äcidos hümicos
extraidos, de tal forma que al estudiar la acciön de dos de estos äcidos hemos podido observar
efectos totalmente diferentes en cuanto a su acciön sobre el desarrollo у la absorciön de nutrientes
por la planta de mafz.
Tanto en parte aerea сото en raiz las mismas dosis de äcido hümico extrafdo con diferentes
extractantes у aplicadas al cultivo del mafz aetüan sobre los procesos fisiolögicos de la planta
 316
CUADRO VI. ACCION DE LOS ACIDOS HUMICOS S Y R SOBRE LAS ALIMENTACIONES MINERALES Y PESOS DE LA RAIZ

Plantas tratadas con 5 Plantas tradadas con R

Tratamientos
(mg АН/kg Cz) % Д Ago % A peso A peso %A A ^ Ag0 %A peso $A pesc
Agm °?° A Agm Ag0 % Agm Ago % A

humedo seco humedo seco

HERNANDO et al.
0 5,0 5933 5,0 5953
6 5,6 + 11,4 4087 - 31,3 - 12,5 - 30,5 4,3 - 13,8 3009 -4 9 ,5 - 17,8 - 34,4
12 4,3 - 14,4 3400 - 4 2 ,9 + 17,0 + 1,5 3,9 - 21,0 2994 - 4 9 ,7 + 21,8 + 11,5
24 4,6 - 8,6 4343 - 27,0 + 37,7 + 35,1 4,1 - 18,0 3390 -4 3 ,1 + 14,2 -3 ,8
48 5,3 + 5,8 3496 -4 1 ,3 -0 ,4 - 26,7 5,3 + 7,0 4820 - 19,0 -3 2 ,6 -4 9 ,6
96 3,8 - 22,8 2927 -5 0 ,8 + 24,9 + 6 ,i 6,8 + 35,7 2372 - 6 0 ,2 - 7,5 - 14,5
(

mgAH/kgCz = miligramos de acido humico рог kilogramo de cuarzo.

A gm=alinientaciön global de macroelementos.
Ago = alimentacion global de oligoelementos.
 IAEA-SM-211/57 317

en forma distinta, dando lugar a variaciones en la production de materia vegetal, en las alimentaciones
minerales у en el contenido de humedad.
En resumen, los extractantes utilizados en nuestro trabajo para la obtenciön de äcido hümico
ejercen una action que afecta de forma distinta a su estructura molecular, lo cual implica un diferente
comportamiento de los dos äcidos hümicos con respecto a la planta. Esto es, al quedarmäs
despolimerizada la molecula del äcido hümico por efecto del extractante, su action fisiologica serä
mäs efectiva, ya que, lögicamente, al ser mäs pequenas las moleculas degradadas, serän absorbidas
mäs fäcilmente por las rafces de las plantas.

REFERENCIAS

[1] ROSELL, R.A., ORTIZ, M.I., Rev. Invest. Agropecu. INTA, Serie 3, Vol IV, n-4 (1969) 41.
[2] FORTUN, C., Rev. Real Acad. Cienc. Exactas, Fis. у Nat., Tomo LXVI, cuademo 3- (1972) 333.
[3] ORTEGA, C„ Tesis Doctoral, Univ. Madrid (1966).
[4] ORTEGA, C., HERNANDO, V., SANCHEZ CONDE, Marla P., “Diferencias entre las acciones del äcido
hümico extraldo de un estiercol у el extraido de una turba sobre las plantas de malz”, Isotopes and radiation
in Soil Organic Matter Studies (Actas Simp. FAO/OIEA, Viena, 1968), OIEA, Viena (1968) 541.
[5] SLADSKY, Z„ Biol. Plant. 7 (1951) 598.

DISCUSSION

R.A. ROSELL: Did you take the different ash content (and possibly micronutrient content)
of the two humic acids into account? We have observed that the resin HA always contains more
ash (some 10 times more) than the NaOH HA, even after treatment with acids.
C. FORTUN: .Yes, because the doses of humic acid added were a function of the micro­
nutrient content of the particular humic acid applied.
R.W. ALDAG: Do you have any explanation of why the percentage nitrogen content of
the plants which were treated with R humic acid (see Tables III and IV) is higher than that of
the plants which were treated with 5 humic acid, although the R acid used is supposed to be of
higher molecular weight than S acid?
C. FORTUN: The differences in the nitrogen content of the aerial part of the plant when
S humic acid or R humic acid was applied are very small (considering that it is a macronutrient),
and for that reason are not grounds for saying that the content is a result of nitrogen having been
transferred from one or the other humic acid; there is reason for thinking that other factors have
an influence. In any event, if your idea is that there may have been a transfer from part of the
humic acid molecule to the aerial part of the plant, that is not the case, seeing that the determina­
tions of the aerial part were carried out with the aim of confirming that the absorption of ‘micro-
nutrients’ and of part of the humic acid molecule was better when the extractant utilized for obtain­
ing the humic acids was 0. IN NaOH.
R.A. ROSELL: Since, in general, both humic acids contain similar amounts of total
nitrogen, I think that the observed differences may be due to physiological factors.
R.W. ALDAG: Can you tell us, up to what molecular weight organic compounds can be
taken up by plant roots?
C. FORTUN: No, I cannot. But I think it must be the parts of low-molecular-weight humic
acid, namely the fractions in which amino acids, phenols, etc., are to be found (and particularly
the latter), which can affect the respiration of the plant.
 IAEAjSM-211/58
I
I
EFFECT OF ORGANIC MATTER AND SALTS ON THE
ACTIVITY OF SOME SOIL ENZYMES

A.S. ABDEL-GHAFFAR, M.H.A. EL-SHAKWEER, M.A. BARAKAT

Department of Soil and Water Science,
Faculty of Agriculture, J
Alexandria University
and
Soil Salinity Laboratory,
Agriculture Research Center,
Alexandria,
Egypt

Abstract

EFFECT OF ORGANIC MATTER AND SALTS ON THE ACTIVITY OF SOME SOIL ENZYMES.j
The activities o f dehydrogenase, catalase, protease, cellulase, invertase and amylase, as well as C 0 2 evolution,
were measured periodically for 28 days using a clay soil with and without the addition of clover straw] and NaCl,
CaCl2 and Na2C 0 3. The mere addition o f the plant material greatly increased the activities o f all the soil enzymes
tested even in the presence o f salts. The only exception to this trend occurred in the CaCl2-straw-treated soil with
protease whose activity was lower than in the untreated soil. With CaCl2, dehydrogenase, catalase, protease and
amylase activities decreased whereas those of invertase and cellulase increased. With Na2C 0 3, dehydrogenase,
catalase and protease activities increased whereas those o f invertase and cellulase decreased. Sodium chloride
increased only the activities o f cellulase and invertase whereas other enzymes were depressed. The simple corre­
lation test between C 0 2-C values and the average corresponding activity value for each enzyme showed that
dehydrogenase, catalase, protease and amylase had positive and cellulase and invertase had negative correlation
coefficients.

INTRODUCTION

Recently, much attention has been devoted to soil enzymology, and a wealth of data have
been accumulated [1—4]. Obviously, enzymes in the soil are involved in the decomposition of
organic matter and many other chemical transformations in the soil. It is well known that
generally the addition of organic matter to soil improves its physical, chemical and biological
properties, and that the activities of soil enzymes are usually connected with soil organic matter [4].
In another paper in this Symposium [5] it was demonstrated that salts affected the rate and
extent of the decomposition of plant residue added. The decomposition process was favoured by
Na2C 03 and depressed by other salts tested.
The present work is an attempt to study the effect of Na2C 03, NaCl and CaCl2 on the activi­
ties of some soil enzymes. Amylase, invertase, cellulase, dehydrogenase, catalase and protease
are some of the important enzymes in soil responsible for the rate and course of decomposition
of organic materials. Shehata [6], Abd-el-Malek [7] and Shady [8] found that salinity reduced the
activity of soil enzymes.

MATERIALS AND METHODS

A top soil (0 —30 cm) sample was obtained from a fertile field. The soil was clay, and
contained 0.53% organic carbon and 0.08% total nitrogen. Its pH was 7.8 (in 1:5 H20 suspension).

319
320 A BDEl^GHAFFAR et al.

The soil sample was air-dried and passed through a 2-mm sieve before using. Powdered clover
straw was added to the soil at the 1% rate. The organic carbon and total N contents of this plant
residue were 38% and 2.2% respectively. The salts, NaCl, CaCl2 and Na2C 03, were used in concen­
trations equal to 25 meq/100 g soil.
Experiments were conducted at 29 ± 1°C, using 100-g portions of the soil with and without
either clover straw or salt or both, and the moisture content adjusted to 60% WHC. Carbon dioxide
evolved and the activities of six soil enzymes were determined periodically for 28 days.
The evolved C 02 was absorbed by NaOH solution and the Na2C 03 formed was precipitated
as BaC03. Excess NaOH was titrated with HC1 [9]. Results were reported as mg C 02 —C/100 g
soil.
The methods used for determining amylase and invertase activities were those of Ross [10],
and cellulase was determined by the method described by Pancholy and Rice [11]. The reducing
sugar was determined by Nelson’s method and the results were recorded as pmol glucose produced/g
soil per 24 h. The titrimetric method outlined by el-Essawi and co-workers [12] was used for
determining catalase activity, which was expressed as mmol of H20 2 decomposed/g soil per 15 min.
Dehydrogenase activity was determined as described by Pancholy and Rice [11], and the results
were recorded as pg formazon/g soil per 24 h. Protease activity was determined using gelatin as
the substrate and the amount of hyrolysed gelatin was measured spectrophotometrically, using
the cupric phosphate reagent [13]. The results were expressed as percentage of gelatin hydrolysed.
All reported data represent the average of three replicates.

RESULTS AND DISCUSSION

The addition of clover straw caused striking and remarkable increases in the activities of the
six soil enzymes tested regardless of the salt added, as shown in Tables I—VI. Similar results have
been reported by other workers [1,2,6—8,11,14,15]. The only exception was the very low
protease activity in the CaCl2-treated soil (Table III). Pancholy and Rice [11] stated that the
type of organic matter added to the soil determined the activity of the soil enzymes, but they
did not find any correlation between soil enzymatic activity and amount of organic matter.
Generally, the activities of dehydrogenase (Table I) and catalase (Table II) were highest in
the soils incubated for 4 days, and then decreased gradually. Other enzymes (protease, cellulase,
invertase and amylase), particularly cellulase, showed a gradual increase throughout the experi­
mental period (28d). This trend was found in nearly all treatments.
The addition of NaCl, CaCl2 and Na2C 03 (25 meq/100 g soil) to the soil treated with 1%
clover straw caused different changes in the activities of the six soil enzymes under investigation.
The extent and magnitude of the salt effect on soil enzyme activity varied with incubation time,
type of salt and enzyme. Similar findings have been reported by Egyptian workers [6—8].
Dehydrogenase activity values are shown in Table I. They were highest in the presence of
Na2C 03 and lowest with CaCl2. Upon incubation of the soil-straw mixture for 4 days, the
amounts of formazan formed, in ug/g soil per 24 h, were 242.7,240.7,95.8 and 268.7 for the
salt-free soil and soils treated with NaCl, CaCl2 and Na2C 03 respectively. These data indicate
that CaCl2 inhibited and NaC03 stimulated dehydrogenase activity. Sodium chloride slightly
depressed the formation of formazan.
Catalase activity showed a trend similar to dehydrogenase activity (Table II). The amount
of H20 2 decomposed by the soil-straw mixture that had been incubated for 4 days was 1.7 mmol/g
soil per 15 min. The corresponding values in the presence of NaCl, CaCl2 and Na2C 03 were 1.5,
1.3 and 1.8 respectively. The catalase activity was depressed more by CaCl2 than by NaCl and
it was increased by Na2C 03. It is of interest to note that catalase activity of the untreated soil
(no salts or clover straw) did not change during the experimental period.
 IAEA-SM-211/58 321

TABLE I. EFFECT OF SALTS ON DEHYDROGENASE ACTIVITY

Figures are in pg fo rm azan ( TPF)/g so il p e r 2 4 h

D ays Soil tre a te d w ith clover straw and

U n tre a te d
of
soil
in c u b a tio n N one NaCl C aC l2 N a2C 0 3

0 4 3 .9 2 1 4 .8 24 2 .7 160.9 2 5 8 .0

4 57.2 242.7 240.7 95.8 2 6 8 .7

12 23 .9 228.1 89.1 99.1 2 3 4 .8

20 16.0 63.8 69 .2 5 7.9 147.0

28 18.0 61 .2 93.1 6 3 .2 131.0

TABLE II. EFFECT OF SALTS ON CATALASE ACTIVITY

Figures are in m m o l H 20 2 d e co m p o se d /g so il p e r 15 m in

D ays Soil tre a te d w ith clover straw and

U n treated
of
soil
in c u b a tio n N one N aCl C aC l2 N a2C 0 3

0 0.8 0.8 0.6 0.6 0.8

4 0.8 1.7 1.5 1.3 1.8
12 0.8 i.6 1.4 1.3 1.8
20 0.8 1.4 1.3 1.1 1.4
28 0.8 1.2 1.4 1.2 1.4

TABLE III. EFFECT OF SALTS ON PROTEASE ACTIVITY

Figures are in p ercen tage o f gelatin h y d ro ly se d

D ays Soil tre a te d w ith clover straw and

U n treated
of
soil
in c u b a tio n N one NaCl C aC l2 N a2C 0 3

0 0.3 0.7 0.7 0.3 0.7

4 0.5 5.5 5.5 0.3 2 0.5

12 0.5 10.3 8.9 0.3 21.1

20 0.8 10.8 10.9 0.3 2 1 .2

28 1.1 10.7 10.1 0.5 2 1.8

Table III shows the activity of protease, expressed as percentage gelatin hydrolysed under
experimental conditions. After 28 days’ incubation, Na2C03 increased greatly the protease
activity whereas CaCl2 severely depressed it. The CaCl2-treated soil had 25 times less protease
activity than the salt-free soil. With Na2C03, proteolysis was about twice as high as in the control
soil and about 73 times as high as in the CaCl2-treated soil. Protease activity of the CaCl2-treated
soil did not change with the addition of clover straw or during the 28 days of incubation. The ability
of the soil to hydrolyse gelatin was slightly depressed by NaCl.
322 ABDEL-GHAFFAR e t al.

TABLE IV. EFFECT OF SALTS ON CELLULASE ACTIVITY

Figures are in p m o l glu co se/1 0 0 g soil p e r 2 4 h

D ays Soil tre a te d w ith clover straw and

U n treated
of
soil
in c u b a tio n N one NaCl C aC l2 N a2C 0 3

0 9.8 170.3 181.4 192.1 145.1

4 10.5 183.6 188.6 268.6 168.9
12 9.8 192.6 247.3 312.5 193.1
20 10.3 198.3 281.6 335.3 201.6
28 9.4 210.9 338.1 392.4 234.9

TABLE V. EFFECT OF SALTS ON INVERTASE ACTIVITY

Figures are in p m o l glu cose/g so il p e r 2 4 h

D ays Soil tre a te d w ith clover straw and

U n treated
of
soil
in c u b a tio n N one NaCl C aC l2 N a2C 0 3

0 48.1 69.1 66.3 63.1 44.1

4 38.5 60.9 71.2 88.4 26.9
12 30.2 64.3 77.3 79.4 42.7
20 32.7 66.9 79.6 100.7 43.5
28 32.7 73.2 81.3 103.7 46.1

TABLE VI. EFFECT OF SALTS ON AMYLASE ACTIVITY

Figures are in p m o l glu cose/g so il p e r 2 4 h

D ays Soil tre a te d w ith clover straw and

U n treated
of
soil
in c u b a tio n N one N aCl C aC l2 N a2C 0 3

0 1.9 9.3 7.3 7.8 10.6

4 2.7 19.7 18.2 16.4 17.4
12 2.2 19.6 18.8 16.7 17.8
20 3.7 19.6 18.8 16.6 18.1
28 6.5 19.3 18.9 16.8 18.4

Cellulase activity was greatly increased by the addition of clover straw, and increased more
in the presence of NaCl and CaCl2 (Table IV). Generally the activity in the clover-straw-treated
soil was 20—40 times higher than in the untreated soil. The activity of cellulase was slightly
affected by Na2C03. In all treatments, the activity was highest in the soil incubated for 28 days.
Invertase activity generally showed trends similar to cellulase (Table V). Invertase was
stimulated by NaCl and CaCl2 but definitely depressed by Na2C03 compared with the control
(soil treated with clover straw). Also, maximum invertase activity was attained after incubation
of the soil for 28 days.
 1AEA-SM-211/58 323

TABLE VII. CORRELATION COEFFICIENT (r) BETWEEN ENZYME

ACTIVITIES AND C02-C EVOLVED FROM SOIL TREATED WITH
CLOVER STRAW

E n z y m e activ ity w ith treatm ent®

Enzym e
C o n tro l NaCl CaCl2 N a2C 0 3

D ehydrogenase 162.1 147.0 95 .4 2 0 7 .9 + 0 .9 6

C atalase 1.3 1.2 1.1 1.4 + 0 .95

P rotease 7.6 7.1 0.3 17.1 + 0.91

C ellulase 191.1 24 7 .4 3 0 4 .2 188.7 - 0 .9 7

Invertase 6 6 .9 75.1 87.1 4 0 .7 - 0 .9 4

A m ylase 17.5 16.4 14.9 16.5 + 0 .4 6

C u m u lativ e C 0 2-C 91.1 66.3 4 6 .7 103.2 -

m g /100 g soil p er 28 d

a E n zy m e activities are expressed as in previous tab les. E a ch fig u re in th e ta b le

re p re se n ts th e average ac tiv ity (5 in c u b a tio n p erio d s X 3 rep licates) o f th e en zy m e.

It is of interest to note that high invertase and cellulase activities are associated with low
dehydrogenase and catalase activities and vice versa. Also, Skujins [1 ] reported a similar associ­
ation between invertase and catalase.
The values for amylase activity are presented in Table VI. Addition of clover straw increased
the amylase activity five times or more. The salts (NaCl, CaCl2 and Na2C03), especially CaCl2,
lowered the activity of amylase but the activity was consistently much higher than in the untreated
soil.
The correlation coefficients between C02-C evolved in the case of clover-straw-amended soil
and the corresponding activity values for each enzyme are shown in Table VII. It is evident that
dehydrogenase, catalase, protease and amylase had positive whereas cellulase and invertase had
negative correlation coefficients.
These data indicate that Cad2 depressed the activity of the enzymes that were positively
correlated with C02-C evolution, and enhanced the enzymes that were negatively correlated
with it. Na2C03 behaved quite contrary to CaCl2, at least with respect to dehydrogenase,
catalase, protease and invertase. NaCl increased the activity of cellulase and invertase but it was
not as effective as CaCl2.

REFERENCES

[1 ] S K U JIN S , J . “E n z y m es in soils” , Soil B io c h em istry 1 (M cL A R E N , A .D ., P E T E R S O N , C .H ., E d s ), M arcel

D ek k er, N ew Y o rk (1 9 6 7 ) 371.
[2 ] K U P R E V IC H , V .F ., SH C H ER B A K O V A , T .A ., “ C o m parative e n z y m a tic activ ity in diverse ty p e s o f so il” ,
S oil B io c h em istry 2 (M cL A R E N , A .D ., S K U JIN S , J ., Eds), M arcel D ek k er, N ew Y o rk (1 9 7 1 ).
[3 ] G A L A S T Y A N , A .S h., E n z y m a tic ac tiv ity o f soils, G eo d e rm a 1 2 (1 9 7 4 ) 43.
[4 ] K ISS, S., e t al., B iological significance o f en z y m es ac c u m u la te d in soil, A dv. A gron. 2 7 (1 9 7 5 ) 25.
[5 ] EL -SH A K W EER , M .H .A ., G O M A H , A .M ., B A R A K A T , M .A ., A B D E L -G H A F F A R , A .S ., “E ffe c ts o f salts
o n d ec o m p o sitio n o f p la n t residues” , th e se P roceedings, IA E A -SM -211 /2 2 , V ol. I.
[6 ] S H E H A T A , S.M ., E v a lu atio n o f som e B iological T e sts as P a ram eters fo r M icrobial A ctiv ities re la te d to
S oil F e rtility , P hD T hesis, C airo U niversity (1 9 7 2 ). ]
324 ABDEL-GHAFFAR et al.

[7] A B D -EL-M A LEK , S.M., E n z y m atic A ctivities in Soils as In d ic a to rs o f F e rtility , M Sc T hesis, C airo
U niversity (1 9 7 3 ).
[8] SH A D Y , M .A.M ., S tudies o n som e M icrobiological E n zy m es in Soils, P hD T hesis, A in-S ham s U niversity
(1 9 7 5 ).
[9 ] BLA CK , C.A . (E d .), M eth o d s o f Soil A nalysis, P a rt 2, A m . Soc. A g ro n o m y , M adison (1 9 6 5 ).
[ 10] R O SS , D .J., A survey o f ac tivities o f en zy m es hy d ro ly sin g sucrose a n d starc h in soils u n d e r p a s tu re , J.
S oil Sei. 1 7 ( 1 9 6 6 ) 1.
[1 1 ] P A N C H O L Y , S.K ., R IC E , E .L ., Soil enzym es in re la tio n to o ld field succession: A m ylase, cellulase,
in v ertase, d eh y d ro g en a se an d u rease, Soil Sei. Soc. A m ., P ro c. 3 7 (1 9 7 3 ) 47.
[12] EL-ESSA W I, T .M ., e t al., P relim in ary stu d ie s o n som e soil en zy m es A lex. J . A gric. R es. 21 (1 9 7 3 ) 125.
[1 3 ] S K U JIN S , J ., D ehydrogenase: A n in d ic a to r o f biological activ ities in arid soils, B ull Ecol. Res.
C om m . (S to c k h o lm ) 17 (1 9 7 3 ) 235.
[1 4 ] K H A N , S.U ., E n z y m a tic activity in gray w o o d ed soil as in flu en ced by cro p p in g sy stem s an d fertilizers,
Soil B iol. B iochem . 2 (1 9 7 0 ) 137.
[1 5 ] EL-ESSA W I, T.M ., S tudies o n Soil E n zy m es w ith Special R efe re n ce to Soil P h o sp h a tase (M eth y lp arath io n
H y d ro lase), P hD T hesis, A lex a n d ria U niversity (1 9 7 2 ).

DISCUSSION

W. ROCHUS: The activity of the different enzymes depends on the pH. If you add Na2C03,
you are perhaps working nearer the pH optimum. Could this be a reason for your results?
A.S. ABDEL-GHAFFAR: In all treatments, every enzyme was tested with a buffer solution
of recommended pH. In the paper on the effect of salts on the decomposition of plant residues [5],
it was seen that Na2C03 and CaC03 promoted the decomposition process whereas MgC03 depressed
it.
R.A. SOBULO: Do you think the activity of the enzymes is due to the anion or the cation
of the salts added to the system?
A.S. ABDEL-GHAFFAR: I cannot say that it is the cation or the anion; probably it is both.
K.A. MALIK: Did you estimate cellulase activity at higher concentrations of salts, especially
NaCl? We found increased activity of cellulase at lower concentrations of NaCl.
A.S. ABDEL-GHAFFAR: No, I did not test the activity of cellulase in the presence of salt
concentrations higher than 25 meq/100 g soil.
 IN T E R A C T IO N B E T W E E N
A G R O C H E M IC A L S A N D O R G A N IC M A T T E R
(S e ssio n 9 b )
 IAEA-SM-211/78

B L O C A G E D E M O L E C U L E S S -T R IA Z IN IQ U E S
P A R L A M A T IE R E O R G A N IQ U E

M. SCHIAVON, F. JACQUIN, C. GOUSSAULT

Laboratoire Matiere organique
et envirormement,
Ecole nationale superieure d’agronomie
et des industries alimentaires,
Nancy,
France

R a p p o rteu r: F. Führ

Abstract-Risum£

B LO C K IN G O F S -T R IA Z IN E M O L EC U LES BY O R G A N IC M A T TER .
S tu d y o f th e v ariatio n in th e ra te o f e x tra c tio n o f atra z in e by m e th a n o l o r w a te r in ex p e rim e n ts o n th e
in c u b a tio n o f soil tre a te d w ith th is h e rb ic id e is a m eans o f d eterm in in g th e active ro le o f org an ic m a tte r in th e
d isap p earan ce o f p h y to to x ic ity ; 3 0 —40% o f th e I4C-labelled p ro d u c t is very rap id ly rem o v ed fro m th e a m o u n t
ap p lied an d th e re fo re possibly fro m its h erb icid al fu n c tio n . In th e ‘d e to x ific a tio n ’ process th e fulvic acids and
th e h u m in s play a p re d o m in a n t p a rt. H ow ever, w hereas th e h u m ic acids fo rm stab le b o n d s w ith s-triazine
m o lecu les, th e fulvic acids an d h u m in s in d ic a te th e ex isten ce o f low -energy b o n d s, suggesting th a t certain
m o lecu les fix e d by th e organic m a tte r m ay c o n trib u te , by a p ro cess o f release, to n ew sta te s o f eq u ilib riu m in
th e so il so lu tio n .

B L O C A G E D E M O L EC U LES S -T R IA Z IN IQ U E S PA R LA M A T IE R E O R G A N IQ U E .
L ’e tu d e de In v o lu tio n d u ta u x d’e x tra c tio n de l’atrazin e p a r le m e th a n o l o u l’ea u , au co u rs d ’exp 6 rien ces
d ’in c u b a tio n de sol tra its p a r c e t h erb icid e, p e rm e t de c o n s ta te r le rö le a c tif de la m a tie re o rg an iq u e dans le
p ro cessu s de d isp aritio n de la p h y to to x ic ite ; e n e ffe t, tre s ra p id e m e n t, 30 a 40% d u p r o d u it m a rq u e au 14C est
s o u stra it au dosage e t d o n e ev e n tu ellem e n t ä so n ro le herb icid e. D ans ce p ro cessu s d e « d eto x ific atio n » , les acides
fu lviques e t les h u m in e s jo u e n t un ro le p re p o n d e ra n t. C e p e n d a n t, ta n d is q u e les acides h u m iq u es 6 tab lissen t
avec les m o lecu les s-triaziniques des liaisons stab les, les acides fulviques e t les h u m in e s m e tte n t en evidence
l’ex iste n c e de liaisons de faible energie p e rm e tta n t d e p en ser q u e ce rtain e s m o lecu les fixees p a r la m a tie re
o rg an iq u e p e u v e n t, p a r lib e ra tio n , c o n trib u e r ä de n o u v ea u x eq u ilib re s d an s la s o lu tio n d u sol.

1. INTRODUCTION

L’etude de la persistance des herbicides dans le sol, vu son importance agronomique, a fait
l’objet de nombreux travaux. En effet, plusieurs auteurs [1-8 ] ont essaye de relier la duree de vie des
matteres actives ä differents facteurs: texture, teneur en matiere organique ou argile, humidite,
techniques culturales, dose d’application, etc., et de connaitre les voies preponderates qui con-
duisent ä la disparition de l’herbicide, entre autres: degradation par voie physico-chimique et
biologique, lessivage [9—15].
Compte tenu des liaisons que contracte l’herbicide avec les constituants du sol, on conclut
d’une mantere generate ä la disparition de la substance lorsque le dosage chimique donne un
resultat negatif. L’absence de residus dans les extraits de sols n’a de valeur que sur le plan agronomique.
Kruglov [ 16], travaillant sur la simazine, a demontre que la majeure partie de l’herbicide contractait,
apres 4 mois d’incubation, des liens solides avec la matiere organique et que seule l’extraction de
celle-ci par des reactifs alcalins permettait de recuperer, en partie, les residus s-triaziniques non
extraits par le chloroforme ou l’eau.

327
328 SCHI AVON et al.

Nos travaux ont essentiellement pour but de suivre la repartition des residus s-triaziniques,
non extractibles par le methanol ou par l’eau, dans les differentes fractions organiques d’un sol
(ces travaux sont en partie similaires ä ceux tout recents de Kruglov [16]). A partir de nos
resultats, nous nous attacherons ä mettre en 6vidence les processus de fixation «irreversible» et
les differences d’affinitc de l’atrazine et ses metabolites vis-ä-vis des diverses fractions organiques.

2. MATERIEL ET METHODES

2.1. La matiere active

Les experiences ont ete realisees avec de l’atrazine marquee au 14C sur son heterocycle, apres
dilution isotopique avec une matiere active d’un degre de purete egal ä 99,8%.
La radioactivite introduite dans chaque echantillon de sol a ete de 1,5-106 desint/min,
correspondant ä une concentration en herbicide dans le sol de 15 ppm.

2.2. Le sol

II s’agit d’un sol brun lessive, prcleve ä la ferme experimentale de l’ENSAIA, dont les
principales caracteristiques physico-chimiques sont les suivantes:

A LF LG SF SG pH C N C/N s T S/T
24,2 32,5 15,5 5,3 15 6,7 2,54 0,25 10 9,9 10,5 94,7

2.3. Preparation des echantillons

Apres prelevement, le sol est tamisc ä l’etat frais ä 2 mm, puis homogeneise. Une partie
est stockee ä - 18°C pour les repetitions de l’experience, l’autre partie est placee, par fractions
equivalentes ä 100 g de sol sec, dans les erlenmeyers du dispositif d’incubation decrit par chone et al. [ 17].
Chaque echantillon est amene ä 75% de sa capacite au champ par la solution d’enrichissement
en atrazine. L’incubation du sol est realisee pendant une duree de 31 jours ä la temperature de
28°C. Au cours de l’incubation, des echantillons sont preleves et soumis ä Tanalyse; Tintervalle
de temps scparan’t chaque prelevement d’echantillon a ete determine par failure de la courbe de
degagement journalier de C02.

2.4. Extraction et dosage des molecules s-triaziniques «libres»

L’equivalent ä 40 g de sol sec est extrait:

— soit au Soxhlet pendant 2 h 30 min par 250 ml de methanol,
— soit par agitation pendant 24 h par 250, 150 puis 100 ml d’eau distillde.
La radioactivite extraite est directement mesuree par scintillation liquide en presence d’lnstagel.

2.5. Extraction de la matiere organique et dosage de la radioactivitd dans les c№^rentes fractions

Apres epuisement du sol par l’eau ou par le methanol, une fraction de 5 g est prelevee pour
extraction de la matiere organique suivant la methode de Duchaufour et Jacquin, modifiee
comme suit:
— extractions au pyrophosphate ä 1%
— extractions ä la soude N/10.
 IAEA-SM-211/78 329

Dans les deux cas les extractions sont poursuivies jusqu’ä l’obtention d’un liquidej clair.
Les extraits alcalins obtenus sont ensuite regroupes, puis surune partie aliquote de] 20 ml on
precede ä la separation des acides fulviques et acides humiques par abaissement du pH,I
jusqu’a la valeur de 1. Apres centrifugation, les acides humiques sont solubilises dans de la
soudeN/10.
La teneur en carbone de chaque extrait obtenu, ainsi que du culot, est dosee par combustion
au Carmhograph Wösthoff 12.
La radioactivite des extraits organiques aqueux est directement mesuree par scintillation
liquide, tandis que pour le culot, on precede ä sa combustion et le 14C02 degage est pidge dans de
la soude N/2, puis compte par scintillation liquide.

3. RESULTATS

3.1. Evolution du taux d’extraction de la radioactivite «libre» au cours de l’incubation

Le tableau I donne les resultats obtenus aprös differents temps d’incubation.

Apres contröle du phenomene de volatilisation (inferieure a 2%), nous avons pu constater
que la somme de la radioactivite extraite et residuclle atteignait 98 a 99,5% de la radio­
activite introduite.

3.2. Repartition de la radioactivite residuelle dans les diff£rentes fractions organiques

Le tableau II donne le pourcentage de radioactivite residuelle fixee respectivement sur chaque

fraction de la matiere organique. Pour faciliter Interpretation des resultats, nous donnons
toujours ä l’ensemble de la radioactivite residuelle la valeur 100%.
TABLEAU I. TAUX D’EXTRACTION DE LA RADIOACTIVITE
DU SOL UTILISE A DIFFERENTS TEMPS DTNCUBATION

R ad io activ ite e x tra ite

DurSe de (%)
l’in c u b a tio n S o lv an t: Solvant:
(d) eau m e th a n o l

0 92 98
2 88,5 93
4 84,5 9 1,5
12 74 81,5
31 6i 69,5

TABLEAU II. REPARTITION (en %) DE LA RADIOACTIVITE RESIDUELLE SUR LES

DIFFERENTES FRACTIONS DE LA MATIERE ORGANIQUE

D ur£e de l’in c u b a tio n S olvant: eau S o lvant: m e th a n o l

(d ) AF AH H um ine AF AH H u m in e

2 75 ,2 _ 24,8 7 7 ,0 - 23
4 64,1 6 ,9 29,0 57,6 11,9 30,5
12 59,6 8,5 3 1 ,7 54,3 12,5 3 3 ,2

31 50,3 13,6 86,1 4 7 ,8 13,9 ! 3 8,4

330 SCHI AVON e t al.

TABLEAU III. AFFINITE DES MOLECULES S-TRIAZINIQUES VIS-A-VIS DES

DIFFERENTES FRACTIONS ORGANIQUES2

D ur6e d’in c u b a tio n Solvant: eau Solvant: m e th a n o l

(d) AF AH H um ine AF AH H u m in e

2 37 ,0 - 5,6 23,5 _ 3,2

4 64,5 4,1 8,0 25,3 3,7 5,9
12 82 ,0 7,8 15,4 5 3,2 8,2 11,3
31 121,0 i6 ,i 27,5 90,8 12,7 2 3,0

T a u x de rad io activ ity

D’ap res la relatio n : --------------------------------- X 100.
T a u x de ca rb o n e

3.3. Repartition de la radioactivity residuelle dans les differentes fractions organiques en

fonction de leur teneur en carbone

Pour mieux saisir l’affinitc dans la liaison molecules radioactives — matiere organique, il
convient de relier le taux de radioactivity mesure au niveau de chaque fraction au taux de carbone.
Le tableau III donne la relation au moment des differentes analyses,

Taux de radioactivity
--------------------------- X 100
Taux de carbone

3.4. Origine de la radioactivity compiymentaire extraite par le mythanol

En combinant les rcsultats des tableaux I et II nous pouvons visualiser l’origine du complement
de radioactivity extraite au methanol par rapport a Геаи (fig. 1).4

4. DISCUSSION ET CONCLUSION

Nos resultats concernant la variation du taux d’extraction de l’atrazine par le methanol ou

Геаи sont conform es ä ceux de Fusi et Franci [3] et de Kruglov [16]. Dans nos conditions d’incubation,
apres 31 jours, 30 ou 40% des molecules s-triaziniques ne sont plus extractibles par le methanol
ou Геаи (tableau I). Cette radioactivite residuelle, dans les meines conditions experimentales, est
encore plus importante pour des sols plus organiques et l’ecart entre le pouvoir d’extraction de
Геаи et du methanol s’accentue dans ce cas [18].
Paraliyiement ä une diminution du pouvoir d’extraction des differents solvants, on assiste ä
une augmentation en valeur absolue de la radioactivite liee aux differentes fractions organiques.
Si nous tenons compte des valeurs relatives (tableau II), nous constatons une diminution pour
les acides fulviques. Neanmoins, il faut souligner qu’au cours de l’incubation, les processus
d’humification et de mineralisation modifient l’importance relative des 3 fractions: acides fulviques,
acides humiques et humines. Aussi, seul le tableau III peut rendre compte de l’affinite de chaque
fraction organique vis-ä-vis des molecules s-triaziniques. Nous remarquons alors que les acides
fulviques manifestent le maximum d’affinite vis-ä-vis des molecules s-triaziniques, suivis par
l’humine et les acides humiques.
Une forte adsorption des molecules s-triaziniques par les acides fulviques semble en accord
avec leur richesse en groupements fonctionnels carboxyliques et phenoliques et avec le mode de
 IAEA-SM-211/78 331

R a d io a c tiv ite
in t r o d u it e
M E M E

9 0 _

8 0 _

s o lv a n ts ; ID: r a d io a c tiv ite Н ее a u x a c id e s fu lv iq u e s ; Я : r a d io a c tiv ite Н ее a u x a c id e s h u m iq u e s ; 0: r a d io a c tiv ity

lie e a u x h u m in e s ; M : m e th a n o l; E : eau.

fixation propose par Sullivan et Felbeck [19]. En ce qui conceme les humines, l’importance de la radio­
activite trouvee a ce niveau et la possibility d’une desorption des molecules s-triaziniques fixees sur ces
substances permettent de penser que deux types de processus interviennent dans la fixation
des molecules herbicides:
— liaisons par l’intermediaire de groupements carboxyliques;
— incorporation directe de la molecule s-triazinique au moment de la polycondensation.
Nous remarquerons enfin l’existence, aussi bien au niveau des acides fulviques que des
humines, de liaisons de faible energie qui permettent au methanol l’extraction d’un cdmplement
de residus. En effet, comme l’indique la figure 1, seule la valeur du taux de radioactivite fixee
par les acides humiques n’est pas affectee, quel que soit le solvant d’extraction (methanol ou eau).
En conclusion, nos resultats indiquent un röle particulierement actif de la matiere organique
dans l’abaissement de la teneur en molecules s-triaziniques utilisables pour la plante. Dans ce
processus, les acides fulviques et, avec une moindre intensite, les humines jouent un role
preponderant. La possibilite d’une extraction complementaire au methanol met en evidence
l’existence de liaisons de moindre energie entre les 2 composants et done la possibility pour
certaines molecules s-triaziniques de repasser dans la solution du sol ä l’etat libre, ce qui pourrait
etre ä l’origine de faction herbicide prolongec de ccs molecules.
332 SCHIAVON et ai.

REFERENCES

[1 ] BO U C H ET , F ., D A G N EA U D , J.P ., H A U C O U R T , P., «A ctivity des triazin es en fo n c tio n de le u r localisatio n

e t du sens d u m o u v e m e n t de l’eau dans le sol», S y m p o siu m o n H erbicides an d th e Soil, EW RC (1 9 7 3 ) 91.
[2] C O U V R E U R , F ., BO U C H ET , F ., M A U M EN E, J., « E tu d e des po ssib ilit6 s de re im p la n ta tio n d ’u n e p arcelle
de сёгёа1е d 6 tru ite apr£s u n tra ite m e n t h erb icid e d’au to m n e» , C .R . 7 e C o n f. d u C O LU M A , T .l (1 9 7 3 ) 53.
[3] F U S I, P., F R A N C I, M., Sulla p ersiste n za d’a tra z in a n ei te rre n i sterili e n o n sterili a v ario g rad o d i u m id itä ,
A g rochim ica 15 (1 9 7 1 ) 557.
[4] H A N C E, R .J., In flu e n ce o f p H , ex changeable ca tio n an d th e p resence o f o rganic m a tte r o n th e ad so rp tio n
o f som e herb icid es by m o n tm o rillo n ite , Can. J. Soil Sei. 4 9 (1 9 6 9 ) 357.
[5] SO U LA S, G ., «In flu en ce d u ta u x d ’argiie su r la persistance d e l’atrazin e d an s le sol», C .R . 8 e C onf. du
CO LU M A , T .l ( 1 9 7 5 ) 3 .
[6] STEC K O , V ., “ C o m p ariso n o f th e p ersiste n ce an d m o v e m en t o f th e soil ap p lied h erb icid es sim azin e and
b ro m a c il” , Proc. 1 0 th B ritish W eed C o n tro l C onf. 1 9 70, V ol. I (1 9 7 1 ) 3 03.
[7] W EBER, J.B ., “ M echanism s o f a d s o rp tio n o f s-triazines by clay co llo id s an d facto rs affec tin g p la n t
availability” , R esidue R eview s (G U N T H E R , E d.) Springer-V erlag 3 2 (1 9 7 0 ) 93.
[8] W A LK ER , A ., C R A W F O R D , D .V ., “ T h e ro le o f organic m a tte r in a d s o rp tio n o f triazin e h erb icid es b y soils” ,
Iso to p es an d R a d ia tio n in Soil O rganic-M atter S tudies (P ro c. S y m p . V ien n a, 1 9 6 8 ), IA E A , V ien n a (1 9 6 8 ) 91.
[9] A R M S T R O N G , D .E., C H E S T E R S , G ., H A R R IS , R .F ., A trazin e h y d ro ly sis in soil, Soü Sei. Soc. A m .,
P roc. 31 (1 9 6 7 ) 6 1 .
[1 0 ] A R M S T R O N G , D .E ., C H E S T E R S , G ., A d so rp tio n -cataly sed chem ical h y d ro ly sis o f atrazin e , E nviron. Sei.
T e ch n o l. 2 ( 1 9 6 8 ) 683.
[1 1 ] BEY N O N , K .I., STO Y D IN , G ., W R IG H T, A .N ., A com p ariso n o f th e b reak d o w n o f th e triazin e h erb icid es,
P estic. B iochem . P hysiol. 2 (1 9 7 2 ) 153.
[1 2 ] M O Y ER , J.R ., H A N C E , R .S ., M cK O N E, C .E ., T h e e ffe c t o f a d so rb en ts o n th e ra te o f d e g rad a tio n o f h erb icid es
in c u b a te d w ith soil, Soil Biol. B iochem . 4 (1 9 7 2 ) 307.
[1 3 ] S C H IA V O N , M ., E v o lu tio n c o m p a r e de Г atrazin e dans u n p eio so l e t u n sol b ru n lessiv6, T h ese d e d o c to ra t
de sp6cialite en ag ro n o m ie, Univ. N ancy I (1 9 7 4 ).
[1 4 ] S K IP P E R , H .D ., V O L K , V .V ., Biological an d chem ical d eg rad a tio n o f atrazin e in th re e O regon soils, W eed
Sei. 2 0 ( 1 9 7 2 ) 3 4 4 .
[15] ZIM D A H L, R .L ., F R E E D , V .H ., M O N TG O M E R Y , M .L., e t al., T h e d eg rad a tio n o f triazin e an d u racil h erb icid es
in soil, W eed Sei. 10 (1 9 7 0 ) 18.
[16] K R U G L O V , L a d ec o m p o sitio n e t la re p a rtitio n de la sim azine, m a rq u ee au 14C, d an s le sol, A grochim ie 6
(1 9 7 5 ) 112.
[1 7 ] C H O N E, T h ., JA C Q U IN , F ., Y A G H I, M ., E m p lo i de 14C e t 45Ca co m m e elem en ts tra ceu rs d e lT iu m ificatio n ,
Bull. E N S A IA 1 5 ( 1 9 7 3 ) 6 9 .
[18] PIN A U D , P ., E tu d e de l’in c o rp o ra tio n e t d e la d eg rad a tio n de l’atrazin e d an s u n sol de ty p e ren d zin e,
D iplom e d ’e tu d e s a p p ro fo n d ie s, E N S A IA , N ancy (1 9 7 4 ).
[1 9 ] S U L L IV A N , J.D ., F E L B E C K , G .T ., A s tu d y o f th e in te ra c tio n o f s-triazine h erb icid es w ith h u m ic acids fro m
th re e d iffe re n ts soils, S oil Sei. 106 1 (1 9 6 8 ) 42.
 IAEA+SM-211/79

BO U N D O R A D SO R BED
M E T H A B E N Z T H IA Z U R O N R E S ID U E S IN S O IL

F. FUHR, W. MITTELSTAEDT
Arbeitsgruppe Radioagronomie
der Kemforschungsanlage Jülich GmbH,
Jülich
К. HAIDER
Institut für Biochemie des Bodens
der Forschungsanstalt für Landwirtschaft,
Braunschweig-Völkenrode,
Federal Republic of Germany
R a p p o rteu r: F. Führ

Abstract

B O U N D O R A D S O R B E D M E T H A B E N Z T H IA Z U R O N R E S ID U E S IN SOIL.
T h e e x tra c ta b ility o f m e th a b e n z th ia z u ro n residues fro m soil decreases w ith tim e a fte r ap p lic a tio n . E m p lo y in g
a tw o -ste p e x tra c tio n p ro c e d u re (a c e to n e /c h lo ro fo rm /w a te r follow ed b y 0.1 N N aO H ), 93% o f th e 14C -labelled
m e th a b e n z th ia z u ro n resid u e s could b e rem o v ed fro m th e soü. A ccording to th e resu lts o f T L C an d c o c h ro m a to ­
g ra p h y , m o re th a n 94% o f th e e x tra c te d ra d io c a rb o n re p resen ted u n a lte re d m e th a b e n z th ia z u ro n . I n an a d d itio n a l
e x p e rim e n t, 14C-labelled m e th a b e n z th ia z u ro n w as in c u b a te d w ith tw o h u m ic acid-like m elan in -p ro d u cin g soil
fu n g i. T h e m elanins co n ta in e d o n ly sm all a m o u n ts o f rad io activ ity . T h e resu lts in d ic a te th a t th e n o n -e x tra c ta b le
resid u e p o rtio n o f m e th a b e n z th ia z u ro n seem s t o b e r a th e r stro n g ly ad so rb ed b y soil o rg an ic m a tte r b in d in g sites
th a n in c o rp o ra te d in to o r assim ilated b y so ü o rg an ic m a tte r.

The urea-derivative methabenzthiazuron (Tribunil®)1 is used as a broad spectrum herbicide

in cereal crops [1 ]. In soil the compound is very resistant to degradation or mineralization [2,3].
In a field study with [benzene ring-U-14C] methabenzthiazuron [3,4], about 80% of the applied
radiocarbon was still found at harvest time mainly in the upper S-cm soil layer. Furthermore, the
total water or organic extractable soil residues decreased with time after application [4]. In the
crop plants the radiocarbon decreased to one tenth in the rotational rye crop compared with the
residue found in the treated spring wheat.
The question is therefore whether the residues in soil are methabenzthiazuron, highly adsorbed
by soil constituents, or whether they are incorporated by soil organic matter during chemical and
microbial degradation processes thus rendering a persistent and biologically inactive fraction.
Attempts were made to characterize these bound or incorporated methabenzthiazuron residues in
the soil humus using extraction methods, or employing humic acid-like melanin-forming soil fungi
to follow residue incorporation during melanin formation.

1 B ayer A G , L everkusen, F ed eral R ep u b lic o f G erm any.

333
334 FUHR et al.

MATERIALS AND METHODS

Incubation

In a laboratory experiment under constant temperature (22°C) and moisture (65% of the
water-holding capacity), 200 g of a sandy soil were incubated with 10 ppm [benzene ring-U-14C]
methabenzthiazuron for 98 days. The test soil had the following characteristics: pH 6.3,
carbon 1.15%, clay 3.7%, silt 8.2% and sand 88.1%.

Extraction

Soil aliquots were shaken at room temperature for 35 h either with H20 , methabenzthiazuron
solution (25 ppm), 0.1N NH4-oxalate, or 0.1N NaOH. Other aliquots were exhaustively extracted
with acetone/chloroform/H20 [5,6], followed by a 0.1N NaOH extraction. The 0.1N NaOH-
extracts were separated into fulvic and humic acids by acidification.

TLC

All extracts were partitioned between chloroform and water. According to Jarczyk [5],
methabenzthiazuron and related substances quantitatively partition into the chloroform fraction.
Two-dimensional TLC was performed on silica-gel plates (Macherey, Nagel & Co., Sil G 100 UV254)
using benzene/ethyl acetate/methanol (70/20/10) and benzene/methanol (90/100) as developing
solvents.

Fungal studies

Two melanin-forming fungi (E u rotiu m echin ulatu m and H elm in th osporiu m spec.), isolated
previously from soil [7, 8], were grown in glass flasks with a liquid culture medium containing
glucose and asparagine as carbon and nitrogen sources [7]. The melanin formation of
E. ech in ulatu m occurred primarily in the culture solution and that of H elm in th osporiu m spec,
primarily in the mycelial mats. Seven days after inoculation, 20 ppm [2'-benzthiazolyl-14C]
methabenzthiazuron, dissolved in 1 ml acetone, were added per flask. After 4 weeks, the cells were
removed by filtration, repeatedly washed and lyophilized. AUquots of the culture solution and the
cells were exhaustively extracted with ethyl-acetate or with 0.1N NaOH (cells). Melanins from the
culture media or extracted from the cells were precipitated by acidification, separated by centrifu­
gation, redissolved, and precipitated.

Radioactivity

X-ray film (Kodak Regulix BB 14) was used for the detection of 14C on TLC plates. Radio­
active TLC spots and radioactivity in extract aliquots were quantitated by liquid scintillation
assay (Packard 3375).

RESULTS AND DISCUSSION

During the soil incubation, only 0.3% of the [benzene ring-U-14C] methabenzthiazuron was
mineralized to 14C02. Water extraction removed only 36% of the residual soil radiocarbon (Table I).
Extraction with a 25 ppm methabenzthiazuron solution or 0.1N NH4-oxalate, a solution normally
used to determine the exchange capacity of soil, removed 59% of the residual radiocarbon.
 IAE A-SM-211/79 335

TABLE I. THE EXTRACTABILITY OF METHABENZTHIAZURON

RESIDUES FROM SOIL AFTER INCUBATION OF
[BENZENE RING-U-14C] METHABENZTHIAZURON UNDER
STANDARDIZED CONDITIONS
2 0 0 g o f soil, 1 0 p p m m eth aben zth iazu ron , 2 2 ° C, 65% w ater-holdin g
cap a city, f o r 9 8 days
T otal iAC-residue in the so il = 1 0 0

E x tra c ta n t R ad io carb o n

H 20 36.5
M eth a b en zth iazu ro n
so lu tio n (2 5 p p m ) 59.0
0.1 N N H 4-oxalate 59.5

A c e to n e /C h lo ro fo rm /H 20 8 0 .4
0.1 N N aO H 12.8
S u p e rn a ta n t I 8.7
H um ic acids I 3.6

O .IN N a O H 7 2 .2
S u p e rn a ta n t II 5 4.9
H u m ic acids II 16.9

TABLE II. RELATIVE DISTRIBUTION OF RADIOCARBON IN THE CHLOROFORM

FRACTIONS (2-DIMENSIONAL TLC) OF DIFFERENT SOIL EXTRACTS AFTER A
98-DAY INCUBATION OF [BENZENE RING-U-14C] METHABENZTHIAZURON IN SOIL
T otal 14C in th e ch lo ro fo rm fra ctio n s = 100

T o ta l ex tra c te d R elat ve p erce n tag e d istrib u tio ri

14C in th e
F r a c tio n /e x tra c t M eth a b en zth ia zu ro n M etab o lite N o.
ch lo ro fo rm -fractio n
1 2 3 4 5
(% )a

H 20 33.1 99 .0 0.3 0.6 b b

M eth a b en zth iazu ro n
s o lu tio n (2 5 p p m ) 57.8 98 .7 0.3 0 .7 0 .3 0.3 0.1
A c e to n e /c h lo ro fo rm /H 20 80.3 98.6 b 0 .4 0.7 0.2
С Н О з -ex tract fro m
su p e rn a ta n t I 6.3 94.5 3.6 0 .6 0 .6 0 .2 0.2
С Н О з-e x tra c t fro m
su p e rn a ta n t II 43 .4 93.7 5.0 0 .2 0 .4 0.1 0.1

T o ta l 14C -residue in th e soil = 100.

ь <0.1%.
336 FUHR et al.

TABLE III. DISTRIBUTION AND EXTRACTABILITY OF RADIOACTIVITY AFTER

INCUBATION OF E urotiu m echin ulatu m AND H elm in th osporiu m SPEC. WITH
[2'-BENZTHIAZOLYL-14C] METHABENZTHIAZURON IN LIQUID CULTURE MEDIA
3 0 0 m l cu ltu re m ediu m w ith 2 0 p p m m eth aben zth iazu ron a d d e d 1 w e ek a fter inoculation;
a p p lie d ra d io a c tiv ity ~ 5 0 0 00 0 d p m jfla sk ; in cu bation a t 2 4 ° C f o r 5 w eeks

P ercentage o f a pp lied ra d io a c tiv ity 3

in cells or e x tra c ta b le w ith e x tra c ta b le w ith s u p e rn a ta n t u p o n

in cu ltu re organic solvents 0 . IN N aOH p re c ip ita tio n o f th e
m ed iu m m elanin

E u r o t iu m e c h in u l a tu m

Cells 4 6 .4 ± 1.6 3 1 .2 ± 1.5 4 3 .0 3 9.8

C u ltu re m ed iu m 3 5 .6 ± 0.8 25.1 ± 0.5 3 4.7

H e l m i n th o s p o r i u m spec.

Cells 36.1 ± u 8 .0 ± 0 .8 33.5 2 7.2

C u ltu re m edium 50.1 ± 0.5 4 9 .8 ± 0.6 4 9 .8 b

a M ean values an d stan d a rd d ev iatio n o f th re e flasks.

b N o m elanin w as p recip itated .

The exhaustive acetone/chloroform/H20 extraction [6] removed 80% of the radiocarbon from
the soil, and an additional 13% was found in the following NaOH-extract (Table I). The radio­
activity in the acetone/chloroform/H20-extract was found to be almost exclusively unaltered
methabenzthiazuron (Table II). In the NaOH-extract, two thirds of the radiocarbon remained
upon precipitation of the humic acids in the supernatant and was shown to be predominantly
methabenzthiazuron. In contrast to the results from experiments with organochlorine pesticides [9]
and s-triazine herbicides [10], ultrasonic extraction did not increase the extraction efficiency of
the non-extractable methabenzthiazuron residues.
More than 70% of the residual radiocarbon was dissolved by direct extraction with 0.1N NaOH
(Table I). Methabenzthiazuron was shown to be stable in this drastic extractant. The greater
portion remained also in the supernatant, and 94% of the chloroform-soluble radiocarbon was found
to be methabenzthiazuron.
These results were supported by data obtained after a 77-day incubation of [carbonyl-I4C]
methabenzthiazuron in the same soil. Also similar results were found after spray application of
[benzene ring-U-14C] methabenzthiazuron to a degraded loess soil (В-horizon) in a lysimeter
experiment under field conditions [3,4].
Incubation studies of the two melanin-forming soil fungi with [2'-benzthiazolyl-14C]
methabenzthiazuron showed an appreciable adsorption of the added radioactivity to cells (Table III).
The recovery of the radioactivity in this experiment was about 80 to 86%, and the possibility
cannot be excluded that some of the methabenzthiazuron was degraded to C02 or other volatile
compounds.
Exhaustive extraction of the culture media or cells from E. echinulatum with organic solvents
yielded only about two thirds of the radioactivity. Nearly all of it could be recovered from the
yellowish-coloured culture medium of H elm in th osporiu m spec., whereas from the highly melanized
cells less than 25% was obtained. NaOH-extraction, however, was more effective, and removed
from the cells more than 90% of the adsorbed radioactivity. Upon acidification of these NaOH-
extracts or of the dark-coloured culture solution from E. ech in ulatu m , most of the radioactivity
 IAEA-SM-211/79 337

remained in the supernatant, and was apparently not combined with the melanins. From these
supernatants, almost 100% of the radioactivity could be extracted with organic solvents before
precipitation of the melanins. Thin-layer chromatography has shown that the radioactivity in
these organic extracts was found predominantly in the form of unaltered methabenzthiazuron, and
only a small portion of the radioactivity was distributed in a few metabolites, just as found in the
various extracts of the soil experiments (Table II).
The results indicate that the methabenzthiazuron applied and its residues are strongly
adsorbed by humic compounds, but only a rather small fraction seems to be possibly incorporated
into soil organic matter. From plant experiments Schmidt [11] concluded that methabenzthiazuron
is highly adsorbed by native soil exchangers leading to biological inactivation. On the basis of
results of an experiment using cation exchange resin he suggests soil binding by protonation
or cation exchange reactions, since methabenzthiazuron exhibits weak basic properties. In our
studies, TLC-cochromatography showed that over 90% of the extracted soil-14C was unaltered
methabenzthiazuron (Table II). However, the adsorption mechanism between pesticides and
organic fractions is in general not yet well understood [12]. It seems that adsorption of urea
herbicides to soil organic matter is more of a physical type than chemisorption, or even incorporation
into humic-acid-type molecules [12,13]. On the other hand, experiments by Mathur and Morley [14]
have indicated that methoxychlor was incorporated into humic material by the fungus A spergillus
versicolor. They, as well as Hsu and Bartha [15], also have employed fungi capable of Utilizing
humic acids as a carbon source. These fungi depolymerize humic acid herbicide complexes. It
would be interesting to study the turnover rates of the humic-acid-adsorbed or linked pesticide
molecules. Where high incorporation rates are observed, the bio-availability of these non-extractable
residues should be tested in plant experiments using pre-extracted soil.

ACKNOWLEDGEMENTS

The authors thank Dr. Ecker, Bayer AG, PH-E Isotopenlabor Chemie im Institut für
Pharmakokinetik, for synthesizing the 14C-labelled methabenzthiazuron.

REFERENCES

[1 ] H A CK , H ., P flan z en sch u tz-N ac h rich te n B ayer 2 2 ( 1 9 6 9 ) 34 1 .

[2] C H EN G , H .H ., F U H R , F ., M IT T E L S T A E D T , W ., E n v iro n m en tal Q u ality an d S afety : P esticides,
(K Ö R T E , F ., C O U L ST O N , F „ E ds) (1 9 7 5 ) 271.
[3 ] F Ü H R , F ., M IT T E L S T A E D T , W ., L a n d w irtsch . F o rsc h , (in press).
[4 ] F Ü H R , F ., Jü l-R ep o rt N o . 1 2 3 4 (1 9 7 5 ).
[5 ] JA R C Z Y K , H .J., P flan z en sch u tz-N ac h rich te n B ayer 25 (1 9 7 2 ) 21.
[6 ] C H EN G , H .H ., F Ü H R , F ., A gric. F o o d C hem . 2 4 (1 9 7 6 ).
[7 ] S A IZ -JIM E N E Z , C ., H A ID E R , K „ M A R T IN , J.P ., S oil Sei. Soc. A m ., P ro c. 3 9 (1 9 7 5 ) 6 4 9 .
[8 ] M A L IK , K .A ., H A ID E R , K ., Soil Sei. Soc. A m ., P roc. (in p re p a ra tio n ).
[9 ] JO H N S E N , R .E ., S T A R R , R .J .,J . A gric. F o o d C hem . 2 0 ( 1 9 7 2 ) 4 8 .
[1 0 ] H IL L , B .D ., S TO B B E, E .H ., J . A gric. F o o d C hem . 2 2 ( 1 9 7 4 ) 1143.
[1 1 ] SC H M ID T, R .R ., Z. P flk ran k h . P flsc h u tz 79 (1 9 7 2 ) 59.
[1 2 ] W EED , S.B., W E B ER , J .B ., “ P esticide-organic m a tte r in te ra c tio n s ” , P esticid es in S oil an d W ater
(G U E N Z I, W .D ., E d .), S oil Sei. Soc. A m ., M adison, W isconsin (1 9 7 4 ) 39.
[1 3 ] K H A N , S.U ., M A Z U R K E V IC H , R ., S oil Sei. 118 (1 9 7 4 ) 339.
[1 4 ] M A T H U R , S.P., M O R L E Y , H .V ., S oil Sei. 1 2 0 ( 1 9 7 5 ) 238.
[1 5 ] H SU , Т .-S., B A R T H A , R „ Soil Sei. 118 (1 9 7 4 ) 21 3 .
 D IS C U S S IO N
(on the previous two papers)

J. P. MARTIN ( Chairman): I would like to comment on Mr. Fiihr’s paper (SM-211/79). In

contrast with your results with methabenzthiazuron, we have noted that the ring portions of
chlorpropham and 2,4—D were incorporated into the melanins of certain fungi to a great; extent,
and were stabilized against biodegradation. The side chain carbons, on the other hand, were
metabolized to C02 or utilized for synthesis of cell constituents that readily decomposed.
F. FUHR: I too think that the portion of methabenzthiazuron found to be adsorbed by the
soil organic matter is stabilized against biodegradation, since we observe very little degradation
of this compound in the soil.
L.A. DOUGLAS: Looking at your results, and in the light of present thinking, do you
consider that the herbicide you have been using persists for too long a time?
F. FUHR: This compound is being used as a herbicide with long-lasting action and since it
came into use it has been proved that it is relatively persistent. On the other hand, however, it is
also highly adsorbed, and its biological activity decreases as a consequence. We have studied the
uptake by untreated plants, and the results showed that the uptake rates decreased to one tenth
compared with the treated plants.
K. A. MALIK: We have also isolated some similar species of H elm in th o sp o riu m , but we are
unable to extract any melanin from its dark coloured mycelium with alkali. Do you have any
comment on that?
F. FUHR: The part of the work concerned with this aspect was carried out in the laboratory
of Mr. Haider who, unfortunately, is not here to answer this question. However, as you:will have
seen, our paper was not concerned with the extraction of the melanin. We were only interested
in obtaining information about the cell-adsorbed herbicide.

339
 PEAT
(S e s s io n 9 c )
 IAEA-&M-211/81

C O M P O SIT IO N A N D C O N T E N T O F
A M IN O A C ID S IN P E A T -F O R M IN G
P L A N T S A N D IN P E A T S

F. MACIAK
Warsaw Agricultural University,
Warsaw,
Poland

H. SÜCHTIG, W. FLAIG
Institut für Biochemie des Bodens,
Braunschweig-Völkenrode,
Federal Republic of Germany

R a p p o rteu r: В. S ch effer

Abstract

C O M P O S IT IO N A N D C O N T E N T O F A M IN O A C ID S IN P E A T - F O R M IN G P L A N T S A N D IN P E A T S .
Investigations have been carried out on the contents and com position o f amino acids in different kinds
of peats and peat-forming plants as well as in lignin isolated from them. Results revealed quantitative and small
qualitative variations in the contents and com position o f am ino acids in the various kinds of peats and plant
species that were compared. A m o n g various factors the kind and the degree o f peat decom position had a large
influence on the quality and quantity o f am ino acids.

INTRODUCTION

Among the chemical properties of organic soils, particularly peats and mucks, the nitrogen
basic importance. Variation in nitrogen give these soils a specific character [1—4].
c o n te n t is o f
The nitrogen content in organic soils is important, as any quantitative and qualitative changes in its
content are distinctly reflected in chemical and also indirectly in the physical properties of soil
formations that exert a decisive influence on their fertility.
From the large number of works on the peat soil nitrogen [5—8] it appears that the organic
nitrogen occurs in forms that are both easily decomposed and resistant to decomposition.
In the course of peat formation there occur both decomposition of fresh peat-forming
plants and synthesis of various new organic nitrogen compounds.
The species of peat-forming plants have a strong influence on the above, particularly the
content and character of the nitrogen bound in the plants. Among the various forms of peat
nitrogen it seems that amine nitrogen is more important, taking into consideration that this
form of nitrogen as well as large quantities of microbial tissue are synthesized and are relatively
easily decomposed by microbes during peat formation and humification.
The aim of the present investigations was to determine the total nitrogen content as well
as the content and composition of amine nitrogen in different species of peat-forming plants and
corresponding kinds of peat.
Particular attention has been paid to determining the influence of the kind and the degree
of decomposition of peats on the composition of amino nitrogen.

343
 344
TABLE I. GEOBOTANICAL CHARACTERISTICS OF PEATS

Sample Depth of Site Botanical composition K in d of Decom position A sh ( % of D.M.) pH

No. borings (% ) peat degree
(cm) (% ) Crude Pure

C arex sp. 75
B r y a le s 15 Sedge
la 0 -2 0 Wizna 40 18.74 16.83 6.8
S a lix sp. 7 peat
M e n y a n th e s tr ifo lia ta 3

C arex sp. 65
Below ” D rуa le s 25 Sedge-moss 20 7.15 3.16 6.4
lb
50 M e n y a n th e s tr ifo lia ta 3 peat
E r io p h o r u m a n g u s tif 2

MACIAKet al.
C a rex sp. 80
Sedge-moss
2a 0 -1 7 Kosify B r y a le s 15 45 15.05 11.55 6.0
peat
E r io p h o r u m a n g u s tif. 5

C a rex sp. 65
Below ” B r y a le s 30 Sedge-moss 15 10.26 5.49 6.4
2b
50 M e n y a n th e s tr ifo lia ta 3 peat
E r io p h o r u m a n g u s tif 2

P h r a g m ite s c o m m . 50 M uck
0 -1 7 Biebrza A ln u s g lu tin o s a 15 developed Over 13.39 8.51 5.5
3a
field 44 C a rex sp. 5 from 60
G r a m in e a e 30 reed peat

P h r a g m ite s c o m m . 65
Below •• Reed peat 45 13.25 11.42 6.1
3b A ln u s g lu tin o s a 25
50 C a rex sp. 10
TABLEI. (cont.)

A in u s g lu tin o s a 15 M u ck
0 -1 9 Biebrza P h r a g m ite s c o m m . 25 developed Over 10.88 5.8
4a 15.21
field 9 C arex sp. 10 from 70
G r a m in e a e 50 reed peat

P h r a g m ite s c o m m . 90
Below - 10.85 7.06 6.2
4b C arex sp. 2 Reed peat 50
50
A in u s g lu tin o s a 8

P h r a g m ite s c o m m . 5 M u ck
0 -1 7 A in u s g lu tin o s a 10 developed 70 17.34 15.59 6.1
5a Modzelöwka
C arex sp. 10 from
G r a m in e a e 75 reed peat

IA E A -SM -211/81
Below P h r a g m ite s c o m m . 75
5b •• C arex sp. 20 Reed peat 70 10.56 6.37 6.3
50
A in u s g lu tin o s a 5

A in u s g lu tin o s a 50 M u ck
6a 0 -1 7 Kosfty C a rex sp. 10 developed Over 18.12 10.71 6.6
field 2 G r a m in e a e 40 from 60
w ood peat

A in u s g lu tin o s a 60
Below S a lix sp. 10 14.67 8.54 6.1
6a A ld er peat 50
50 C a rex sp. 15
P h r a g m ite s c o m m .

•Lamane A in u s g lu tin o s a 25 M u ck
7a " 0 -1 7 P h r a g m ite s c o m m . 10 developed - 50 16.74 10.42-------- - 6.2
Grady
G r a m in e a e 65 from
wood peat

345
 346
TABLEI. (cont.)

Sample Depth o f Site Botanical composition K in d of Decom position A sh ( % o f D .M .) pH

No. borings (% ) peat degree
(cm) (%> Crude Pure

A ln u s g lu tin o s a 65
Below •Lamane S a lix sp. 17
7b Alder peat 60 15.30 13.24 5.9
50 Grady C a r e x sp. 3
P h r a g m ite s c o m m . 15

S p h a g n u m sec. P a lu s tr ia 3 85

” sec . C u s p id a ta b 10 Sphagnum 5
8 0 -1 5 Ram sloh 1.40 - 3.7
E r ic a c e a e d 2 peat
E r io p h o r u m v a g in a tu m 3

MACIAK et al.
S p h a g n u m s e c .P a lu s tr ia a 58
” sec . C u s p id a ta b 30
9 1 5 -3 0 •• E r ic a c e a e d 7 ” 5 1.24 - 3.7
E r io p h o r u m v a g in a tu m 4
P ic e a e x c e ls a 1

S p h a g n u m sec. P a l u s t r i a a 65
” sec , C u s p i d a t a b 15
10 1 5 0 -1 6 5 ” ” $ e c .A c u tifo lia c 5 18 1.14 - 4.6
E r ic a c e a e d 8 ■>
E r io p h o r u m v a g in a tu m 10

Sphagnum sec. Pa lustria a 42

" s e c .C u s p id a ta b 20
11 1 6 5 -1 8 0 ” " s e c .A c u tifo lia c 10 - 28 1.12 - 4.2
E r ic a c e a e d 12
E r io p h o r u m v a g in a tu m 16
TABLE I. (cont.)

Spha g n u m s e c .P a lu s tr ia * 5
12 3 0 0 -3 1 5 Ram sloh ” sec . C u s p id a ta b 15 Eriophorum 50 0.92 4.1
-
E r ic a c e a e d 10 peat
E r io p h o r u m v a g in a tu m 70

S p h agnum sec. P a l u s t r i a a 5
” sec, C u s p i d a t a b 20
13 3 1 5 -3 3 0 ” sec. A c u tifo lia 0 3 •• 55 0.96 - 4.1
E r ic a c e a e d 15
E r io p h o r u m v a g in a tu m 65

sec. P a lu s tr ia : Sphagnum im b r ic a tu m , S . c y m b ifo liu m , S. p a p illo s u m .

IAEA-SM-211/81
sec. C u s p id a ta : S p h a g n u m c u s p id a tu m , S. recurvum .

sec. A c u tifo lia : S p h a g n u m a c u tifo liu m , S . r u b e llu m .

E r ic a c e a e : L e d u m p a lu s t r e , O x y c o c c u s q u a d r ip e ta lu s , C a llu n a v u lg a r is .

347
 348
MACIAK et al.
F I G .l. H u m u s fr a c tio n o f p e a t in th e la y e r s o f 0 - 2 0 cm (a ) a n d b e lo w 5 0 cm (b ); 5 — 70 d eg rees o f p e a t

d e c o m p o s itio n (a c c o r d in g to m ic r o s c o p ic a n a ly s is ).
 IAEA-SM-211/81 349

MATERIAL AND METHODS

The low-peat samples were taken from the upper 0 —20 cm layer, and in the layer below
50 cm, from deposits of the peat-bog Biebrza (Poland). \
The high-peat samples were taken from the profile of the high-moor at Ramsloh (Federal
Republic of Germany).
The peat-forming plants were collected from low- and high-moors in Warsaw province.
Botanical composition and the degree of peat decomposition were determined by the micro­
scopic method.
Humus compounds in air-dry peat samples (after decalcification with 2% HC1) were deter­
mined by means of soil extraction with an alcohol: benzol mixture (1:1).
Humic and fulvic acids were determined as follows: after determining bitumens, Ithe soil was
extracted with 0.IN NaOH and then the alkaline solution was acidified with 2N H2S04. The
precipitate obtained consisted of humic acids and filtrate; the fulvic acids.
pH value of peats (in H20 ) was obtained by the potentiometric method using a glass
electrode; ash was determined by ignition of samples at a temperature of 550°C.
Acid hydrolysis of peat-forming plants and peat, and lignin separated from peat-forming plants
and peat was carried out in a solution of 6N HC1 at a temperature of 120°C for 18 h.
Analyses of total and amine nitrogen were accomplished by Bremner’s [9] method. The
amount of lignin in peat-forming plants and in peats was estimated by hydrolysis of the air-dry
samples in 72% H2S04, without pre-treatment with organic solvents [10].
The content and composition of amino acids were determined in the hydrolysate obtained
on hydrolysis of peats, plants and their lignin with 6N HC1.
The quantitative analysis of amino acids in the hydrolysates was accomplished by liquid
chromatography using an analysator apparatus (Biotronik model LC 4010).
The amount of the amino acids was calculated as nitrogen in amino acid, as percentage of
total amino acid nitrogen.

RESULTS OF INVESTIGATIONS

Geobotanical properties of various kinds of peat

The peat samples collected represented almost all the more important kinds of low and high
peats. The highest proportion of residue of a particular peat-forming plant determines the kind
of peat. The low peats are represented by sedge, sedge-moss, reed, wood and alder peats, and the
high peats by sphagnum and eriophorum peats (Table I).
The investigated peats are characterized by different degrees of decomposition. The degree
of decomposition ranges from 15 to over 70% in low peats and from 5 to 55% in high peats.
The degree of peat decomposition has in particular a marked influence on the content of
ash and humic substances, which increase with the increasing degree of decomposition1.
As is clear from the data presented in Fig. 1, all kinds of strongly decomposed peats contain
higher amounts of humic and fulvic acids; however, the content of bitumens in these is often
smaller compared with weakly decomposed peats, and depends rather on the kind of peat.

Content of total and amine nitrogen in peats and peat-forming plants

Tables II and III show the total and amine nitrogen in peats and peat-forming plants, and
the nitrogen bound with lignin. When comparing simultaneously various kinds of peats and
species of peat-forming plants, one can state that all kinds of low peat contain higher amounts of
total nitrogen than peat-forming plants from low-moors, but the situation is the reverse with
350 MACIAK et al.

TABLE II. CONTENT OF LIGNIN AND NITROGEN IN PEATS

Sample Depth of Decom position Contents of Total N In % of peat total N Total N

No. borings degree ash-free in lignin N bound Am ine-N of peat
(cm) <%) lignin ( % o f D.M.) with ( % of D.M.)
( % of D.M.) lignin

la 0 -2 0 40 50.78 4.53 69.98 27.82 3.83

lb > 50 20 61.35 1.16 73.02 24.54 3.52
2a 0 -1 7 45 51.21 3.99 64.35 22.10 3.59
2b > 50 15 65.33 4.24 75.82 24.63 3.68
3a 0 -1 7 > 60 57.13 4.96 72.60 29.63 4.16
3b > 50 45 69.32 4.53 92.11 27.55 3.42
4a 0 -1 9 >70 52.08 4.93 67.46 26.47 4.24
4b > 50 50 68.55 4.68 81.73 26.11 3.93
5a 0 -1 7 70 57.26 3.99 64.99 22.60 3.79
5b > 50 70 72.94 3.96 87.95 24.15 3.40
6a 0 -1 7 >60 58.77 2.97 48.32 23.63 3.87
6b > 50 50 66.74 2.83 55.66 19.83 3.45
7a 0 -1 7 50 45.41 4.09 57.15 20.68 3.57
7b > 50 60 71.02 4.35 89.61 21.02 3.56

8 0 -1 5 5 35.84 2.04 83.52 37.40 0.91

9 1 5 -3 0 5 36.78 1.99 89.88 33.59 0.79
10 1 5 0 -1 6 5 18 40.70 2.88 97.54 30.91 1.22
11 1 6 5 -1 8 0 28 65.06 1.76 97.46 24.60 1.18
12 3 0 0 -3 1 5 50 67.35 1.33 99.47 16.54 0.87
13 3 1 5 -3 3 0 55 68.76 1.12 100.00 22.75 0.76

high peats, i.e. peat-forming plants of high-moor have higher content of total nitrogen compared
with their high peats.
The peat-forming plants investigated contain usually about 40% of amine nitrogen in
relation to the total nitrogen (Table III). The higher values of 40 - 44% occur both in peat­
forming plants of low-moors and high-moors. Various kinds of peat (Table II) contain from 16
to 33% of amine nitrogen. The peats and peat-forming plants investigated contain large amounts
of lignin and nitrogen bound with lignin. The nitrogen bound with lignin is up to 83 to 100%
of total nitrogen in high peats.
In the low peats the nitrogen bound with lignin varies from low (48%) to very high (92%)
percentage of total nitrogen. On the contrary, the individual species of peat-forming plants,
both from low- and high-moors, contain mostly smaller amounts of nitrogen bound with lignin.
The hydrolysis of this material with 6N HC1 at the temperature of 120°C demonstrated
that the nitrogen bound with lignin is in considerable part in the form of amine nitrogen
(Table IV).
 IAEA-SM-211/81 351

TABLE III. CONTENT OF LIGNIN AND NITROGEN IN PEAT-FORMING PLANTS

Sample Plants Content o f Total N % o f plant total N Total N

No. ash-free in lignin N bound Am ine-N o f plant
lignin { % of D.M.) •with ( % o f D.M.)

( % o f D .M.) lignin

N ot
i C a r e x r o s tr a ta 23.49 5.60 60.24 analysed 2.24
2 ” g r a c ilis 26.96 3.80 65.02 40.19 1.62

3 ” p seu d o cyp eru s 25.43 3.99 88.38 42.96 1.28

4 ” p ilo s a 22.02 6.68 43.34 - 3.90

5 G lic e r ia a q u a tic a 18.74 4.82 28.79 - 3.34

6 P h r a g m ite s c o m m u n is 31.39 5.98 67.09 37.92 3.35

7 S a lix c in e r e a 59.15 4.34 94.23 44.74 2.74

8 E q u is e tu m p a lu s tr e 24.42 5.13 66.47 44.09 2.38

9 V a le r ia n a o ffi c in a l is 22.57 4.45 55.00 44.15 2.87

10 F ili p e n d u la u lin a r ia 34.85 5.46 68.60 43.22 2.90

11 C o m arum p a lu s tr e 36.61 2.70 62.20 - ■ 1.64

12 T yp h a la tifo lia 22.01 4.19 49.82 44.07 ; i.87

13 Sph a g n u m p a lu s tr is 14.55 3.17 35.61 - 1.32

14 ” c u s p id a tu m 18.62 4.96 70.74 42.37 1.47
15 C a llu n a v u lg a r is 43.88 1.69 91.75 40.55 1.52
16 P in u s s ilv e s tr is 38.06 3.48 93.07 40.72 1.43
17 L e d u m p a lu s tr e 47.14 3.23 81.77 35.69 ! 1.92
18 O x y c o c c u s q u a d r ip e ta lu s 51.02 2.41 88.51 41.45 1.40
19 V a c c in iu m u lig in o s u m 50.40 2.51 75.21 33.29 : i.7 o
20 a E r io p h o r u m v a g in a tu m 30.65 2.93 64.87 - 11.48
21b •• 28.16 5.01 71.22 42.11 1.99
22 " a n g u s tifo liu m 28.41 3.79 83.15 44.62 1.36

k Collected from different high-moors.

Composition of amino acids in peats and peat-forming plants

As shown in Table V, there were 15 amino acids in low and high peats. About 60% of the
amino-acid nitrogen in low peats is in the form of neutral amino acids, and about 20% each as
basic and acidic amino acids. However, in the high peats about two-thirds of the amino-acid
nitrogen is in the form of neutral amino acids, about 20% in the basic amino acids and only
about 15% of its amino-acid nitrogen (11—16%) is in the form of acidic amino acids.
Further differences in amino-acid content can be found in different peats. There are some
quantitative variations when the degree of decomposition and the depth of peat profile is taken
into consideration.
More strongly decomposed low peats taken from the upper layer of a peat-moor are
usually richer in the amount of basic and acidic amino acids compared with the deeper layer.
 352
TABLE IV. DISTRIBUTION OF AMINO-ACID NITROGEN IN LIGNIN FROM PEATS AND PEAT-FORMING PLANTS

Nitrogen in am ino acid as percentage of total amino-acid nitrogen

A m in o acids L o w peats a High peats 3 Plants b

8 11 2 6 21
a 1 b a 2 b a 3 b a 4 b a 5 b a 6 b a 7 b

A c id ic

l. Aspartic acid 6.2 9.0 13.3 13.3 3.5 10.4 10.7 6.9 13.9 14.7 7.6 9.9 12.6 12.7 9.1 8.6 6.0 7.3 4.7

2. Glutamic acid 9.8 3.0 9.4 6.6 12.3 9.8 6.9 9.4 7.5 7.3 11.9 8.8 8.0 7.8 6.7 5.5 11.0 8.4 10.7

B a s ic

1. Arginine 9.0 11.2 10.8 11.5 8.2 10.7 10.3 9.9 8.9 8.4 - 10.4 10.2 8.8 9.3 9.8 11.3 14.4 9.3

2. Histidine - - - - - - - - 3.5 - - - - - 5.9 - 4.3 4.7 3.9

3. Lysine 10.1 11.7 11.1 10.5 8.5 11.4 11.0 10.0 12.0 11.1 12.5 11.8 11.7 11.8 14.3 13.2 15.1 12.5 10.7

MACIAKetal.
N e u tr a l

1. Alanine 14.4 14.8 11.9 9.6 15.8 10.7 8.8 11.9 7.1 9.2 14.6 10.9 10.1 11.3 9.9 9.3 7.8 6.2 10.5

2. Cystine 4.4 4.3 3.2 0.9 5.0 2.2 - 2.4 - - 3.7 - 0.6 - - - 0.8 - 2.1

3. Glycine 11.2 12.2 11.2 10.8 12.5 7.9 11.8 11.1 7.7 7.4 12.2 10.6 13.0 10.9 8.3 8.2 6.8 7.3 7.2

4. Isoleucine 4.9 4.1 4.0 3.7 6.2 4.6 4.1 4.9 3.7 4.7 6.0 4.5 3.9 4.4 4.1 4.4 3.8 3.5 3.7

5. Leucine 6.6 6.4 5.7 5.1 8.4 7.3 5.7 8.0 5.1 6.5 9.5 8.0 6.3 6.6 7.2 6.9 7.5 7.6 8.9

6. Methionine 0.9 0.8 0.9 0.9 - 1.3 1.1 1.2 1.2 1.0 1.3 1.3 1.1 1.0 - - 1.4 1.4 l.i

7. Phenylalanine 3.7 3.8 3.1 3.8 - 3.4 3.4 3.4 3.3 4.1 - 3.7 2.9 3.4 3.9 4.4 3.8 3.9 4.0

8. Proline 5.8 5.8 5.8 5.1 0.6 6.9 4.6 6.8 5.6 5.2 6.8 4.3 4.2 4.0 4.2 3.9 4.2 4.7 5.7

9. Serine 0.5 0.5 1.3 4.6 1.4 1.5 6.6 1.8 5.6 4.9 1.0 2.4 3.0 3.1 5.8 5.8 7.9 7.7 9.0

10. Threonine 1.1 1.2 2.3 5.0 1.0 2.3 6.3 2.2 5.2 5.5 1.9 4.1 4.7 5.0 8.0 8.2 1.5 3.7 0.4

11. Tyrosine 1.3 1.3 0.8 1.3 - 1.2 2.0 1.2 3.2 2.3 - 1.5 1.1 1.3 3.7 5.2 4.0 1.4 0.9

12. Valine 10.1 10.0 8.1 7.3 12.5 8.5 6.7 8.9 6.5 7.9 10.9 7.8 6.6 7.9 7.3 6.4 5.7 5.3 6.3

For details of samples see Table 1.

For details of samples see Table III.
TABLE V. DISTRIBUTION OF AMINO-ACID NITROGEN IN PEATS

Nitrogen in amino acid as percentage o f total amino-acid nitrogen

Amino acids Low peats8 High peats 8

1 2 3 4 5 6 7 8 9 10 11 12 13
a b a b a b a b a b a b a b

A c id ic

1. Aspartic acid 13.9 11.9 15.1 12.1 15.0 11.9 14.9 3.4 13.8 14.1 13.9 10.3 15.4 19.3 8.8 7.4 6.1 10.2 7.2 5.7
2. Glutamic acid 6.9 6.6 5.1 5.8 6.3 6.7 7.8 2.0 8.1 6.8 6.9 8.2 6.5 6.9 7.8 7.0 7.0 4.1 5.6 5.3
B a sic

1. Arginine 6.2 6.7 6.0 6.0 5.9 5.8 6.0 7.9 4.8 5.2 4.1 4.8 4.5 trace 8.5 8.9 7.8 8.7 6.0 5.6

IAEA-SM-211/81
2. Histidine 4.3 3.3 3.9 2.8 4.7 4.8 3.7 4.1 3.6 7.5 4.0 5.8 6.3 10.5 2.0 1.7 1.4 1.6 1.8 2.1
3. Lysine 15.4 14.6 14.1 15.3 10.4 11.8 7.7 15.5 14.5 10.1 12.9 11.6 7.9 13.5 11.0 9.5 7.9 9.1 10.0 11.5
N e u tr a l

1. Alanine 9.7 10.6 10.5 9.9 11.1 10.4 9.2 20.5 9.3 9.1 11.1 7.7 7.6 8.0 6.1 7.2 8.1 6.1 9.3 8.5
2. Glycine 12.7 12.2 12.4 12.7 10.7 11.8 18.5 12.8 17.8 17.4 13.8 18.1 19.4 28.8 10.1 10.2 10.5 8.1 8.7 8.4
3. Isoleucine 2.7 2.5 2.4 2.3 2.5 2.0 1.7 3.4 1.7 1.2 1.9 1.5 1.7 trace 2.8 3.1 2.8 2.6 3.7 3.7
4. Leucine 3.6 3.9 3.8 3.4 4.2 4.5 3.9 6.2 4.2 3.3 4.8 3.9 3.9 trace 4.8 5.9 5.2 4.8 6.5 6.1
5. Phenylalanine 3.3 4.7 4.9 4.4 5.0 4.2 4.4 3.4 4.1 4.9 4.6 3.8 3.8 trace 4.8 3.4 3.6 4.2 4.3 4.0
6. Proline 6.0 5.3 5.3 8.8 4.6 10.0 5.1 10.0 4.8 4.0 4.0 5.4 7.4 9.1 8.8 10.0 6.7 13.8 4.7 7.1
7. Serine 4.5 5.2 4.4 4.8 6.2 5.0 5.5 1.7 5.3 5.7 5.6 6.9 7.1 3.6 4.9 4.9 6.1 4.6 6.3 6.0
8. Threonine 4.5 4.9 4.7 4.8 5.7 4.7 5.4 2.7 5.3 7.4 5.9 7.1 5.0 4.6 5.9 5.8 7.5 6.4 8.4 9.3
9. Tyrosine 0.9 1.2 0.7 1.0 1.6 1.0 1.0 1.4 1.0 1.2 ’ 1.2 0.9 1.0 trace 5.7 7.5 11.2 10.2 ил 10.5
10. Valine 5.6 6.4 6.7 5.9 5.7 5.5 5.2 5.2 3.8 4.3 5.2 3.9 3.7 tm cr 8.0 7.6 8.0 5.6 6.4 6.3

For details o f samples see Table I.

353
 354
TABLE VI. DISTRIBUTION OF AMINO-ACID NITROGEN IN PEAT-FORMING PLANTS

Nitrogen in amino acid as percentage of total amino-acid nitrogen

Low-moor plants High-moor plants a

4 5 6 7 8 9 10 11 12 13 14 IS 16 17 18 19 20 21 22

1. Aspartic acid 10.9 7.8 15.4 16.4 8.1 7.7 8.3 7.8 8.5 11.2 6.9 13.7 10.9 18.0 19.2 10.1 6.2 4.2 12.2 14.3 - 9.5

2. Glutamic acid 11.5 8.6 6.7 9.3 8.4 8.3 8.6 16.0 14.4 7.7 5.7 16.4 9.0 10.1 6.3 7.4 6.1 7.8 10.3 8.2 - 7.9

Basic
1. Arginine 9.6 11.4 9.6 8.1 14.3 14.5 13.7 12.4 14.5 12.4 10.7 7.0 10.3 13.7 9.7 14.6 11.9 12.5 8.7 10.2 - 10.6

2. Histidine 9.4 6.1 Not 6.2 5.7 - - 8.0 9.5 trace trace 8.6 - 2.0 - 6.1 9.1 - 3.0
- - -

M A C IA K etal.
analysed
3. Lysine 13.3 13.5 26.9 8.7 13.2 13.0 13.4 9.4 12.0 14.1 10.9 8.0 11.9 11.1 9.9 13.3 30.2 32.7 11.4 12.4 - 26.1

Neutral
1. Alanine 5.8 7.9 6.3 6.6 8.0 8.3 7.4 8.3 6.5 6.6 4.2 5.3 9.0 7.5 7.8 5.9 5.7 6.8 6.6 5.7 - 7.3

2. Glycine 7.4 7.8 6.9 7.7 7.6 7.7 8.4 6.9 6.8 8.0 8.2 6.9- 9.8 8.0 5.5 6.4 6.8 7.8 6.4 12.4 - 6.7

3. Isoleucine 3.5 3.1 2.8 2.3 3.2 3.6 3.9 3.9 4.0 3.6 6.3 1.6 3.4 2.9 2.2 3.7 3.3 3.1 3.4 1.2 - 3.2

4. Leucine 6.6 7.2 6.6 4.9 4.6 7.0 7.7 6.2 5.6 6.3 16.8 4.1 7.1 5.5 4.3 6.1 5.9 6.3 6.6 3.4 - 6.6

1.5 1.8 1.0 0.4 2.0 1.4 1.6 1.5 1.6 1.2 trace trace trace 0.6 1.5 2.5 1.8 1.1 1.7 trace - 1.1
S. Methionine
3.6 3.5 2.7 3.4 3.4 3.5 3.6 3.6 3.3 3.1 - 1.6 3.2 2.9 3.2 3.8 3.2 3.0 3.5 - - 2.8
6. Phenylalanine
7.0 4.3 1.4 11.3 4.3 9.7 7.0 7.3 6.4 9.9 12.5 14.2 6.7 3.7 7.4 9.4 1.7 0.6 6.3 10.8 - 2.1
7. Proline
8. Serine 5.2 5.0 4.5 5.2 5.3 4.8 5.2 5.8 5.3 4.4 4.3 6.2 5.9 5.1 4.5 4.8 4.3 5.0 5.1 4.7 - 4.0

5.0 5.3 4.3 4.1 5.5 3.6 3.9 4.5 4.1 4.4 3.6 5.6 5.5 5.6 4.2 5.0 4.0 3.5 4.9 4,1 - 3.4
9. Threonine
1.3 1.5 1.1 1.1 1.5 1.9 2.0 1.6 2.0 1.7 - 0.8 1.8 1.6 1.1 2.0 1.9 1.2 1.4 - - 1.2
10. Tyrosine
5.7 5.2 3.8 4.6 5.0 4.9 5.2 5.1 5.1 5.4 1.8 3.9 5.5 4.9 4.6 4.9 4.2 4.4 5.5 3.5 - 4.4
11. Valine

Fo r details of samples see Table III.

 IAEA -SM -211/81 355

The deeper layers of the peat profile in both low and high peats contain a distinctly higher
quantity of neutral amino acids. Among the basic amino acids lysine appears in the largest amounts
in the acidic aspartic acid and in neutral glycine. The quantitative composition of amino iacids
in the low peats studied (Table V, Nos 1—7) is in the following order: Lys>Asp>Gly> Ala>Glu,
Pro, Arg, Val>Ser, Thr, His, Leu>Phe> lie > Tyr. j
For the high peats (Table V, Nos 8-13) it is as follows: Lys>Gly>Asp, Arg, Val, Pro,
Ala, Glu, Tyr>Leu, T hr> S er> Phe> Ile> H is.
The present data on the amounts of amino acids liberated by hydrolysis of low and high peats
indicate that low peats characterize themselves by higher amounts of such amino acids asj aspartic
acid, glycine, lysine and histidine; on the other hand, the high peats are richer in tyrosine.
Data obtained on the analysis of peat-forming plant hydrolysates (Table VI) show that the
amino acids composition does not differ considerably from peats. The peat-forming plants always
contain methionine, but this is absent in peats. There are also differences in the amounts of the
various amino acids released by hydrolysis of peats and plants. It can be seen that most plants
contain far more amino acids in basic form, but on the contrary plants have less neutral forms
of amino acids. Among the basic forms of amino acids the peat-forming plants have almost the
same amounts of arginine and lysine (over 10%). However, peats have mostly the same value
of lysine but the level of arginine in peats is about half that in plants. The amounts of aspartic
and glutamic acids are about the same in plants, which together amount to about 20% of the
amino-acid nitrogen. However, peats have two or three time more aspartic acid compared with
glutamic acid. Among the neutral amino acids in plant hydrolysates, thyrosine and methionine
appear in the smallest amounts (1—2%). The contents of the remaining amino acids in plants
are mostly between 3 to 8% of the total amino-acid nitrogen. The peats contain also distinctly
more alanine and glycine compared with peat-forming plants.
It is necessary to add that some peat-forming plants analysed are characterized by a high
amount of certain amino acids, for instance L edu m palu stre and O x yco ccu s quadripetalu s
have over 30% of the total amino nitrogen in the form of lysine, and C om arum palu stre and
T yph a latifolia are very rich in proline. In addition to proline, T yph a latifolia has also the
highest content of acidic amino acids.
The composition and content of amino-acid nitrogen in hydrolysates of lignin complexes
obtained from peats and some peat-forming plants are presented in Table IV. ;
From the analytical data contained in Tables V and VI as well as in IV, it appears that the
hydrolysates obtained from lignin differ somewhat according to the content and composition of
amino acid from hydrolysates obtained from peats and peat-forming plants. \
The hydrolysates from lignin are richer in cystine and methionine. Although methionine
was found also in hydrolysates from peat-forming plants, it was absent in hydrolysates from peats.
There are some quantitative differences also. Larger amounts of alanine and valine appear in
hydrolysates of lignin than in peat and plant hydrolysates; however the content of proline and
threonine is somewhat less.

DISCUSSION

The results have proved that the peat-forming process leads to quantitative and some quali­
tative changes in amine nitrogen components. Marked differences occur between various kinds
of peat and the corresponding species of peat-forming plants. In all species of peat-forming plants
the amine nitrogen occurs in higher quantities than in peats. The differences in amount of
amine nitrogen even reach over 100% (when P hragm ites com m u n is is compared with the dif­
ferent kinds of reed peat, Salix cinerea with woody peats and E rioph oru m vaginatum with
eriophorum peats).
356 MACIAK e t al.

Smaller variations occur in the contents of amine fractions, between Carex species of plants
and Carex peats or Carex-moss peats, the smallest one occurring between sphagnum plants and
sphagnum peats.
The degree of peat decomposition or humification has a decisive influence on the quantita­
tive relation. The decomposition of plant residue leads to an increase in total nitrogen and a
decrease in the amine fraction [11 —13]. It is caused by the decomposition and conversion of
the amine nitrogen in a form more resistant to microbial activity. Differences in amino-acid
composition revealed by the occurrence of certain amino acids in some kinds of peat and by
their absence in comparable species of peat-forming plants, or vice versa, have been found in our
investigation. For instance, methionine was found in the peat-forming plants but was absent in
all peat hydrolysates. In earlier research [14] also the methionine was noted in hydrolysates
of reed whereas it was absent in reed peat. Individual species of peat-forming plants contain, as
a rule, higher quantities of glutamic acid than comparable kinds of peat. On the other hand,
high contents of aspartic acid occur in plants from high-moor and distinctly less in high peats.
There are also large quantitative differences when the amount of basic amino acids
(lysine, arginine) and some neutrals (glycine, alanine) are taken into consideration in peats and
in peat-forming plants.
These differences may result from the activity of soil micro-organisms which, in the peat­
forming process, can promote the decomposition of certain amino acids occurring in peat-forming
vegetation, or cause their accumulation or their synthesis [15-17].
It is commonly assumed that the amino acids reported in soils or in plant material are in
the form of protein or peptides, but many workers suggest that some of the amino-acid nitrogen
in soils may be in complexes formed by the reaction of quinones or phenols with amino acid
or peptides [18—20].
The analysis of lignin isolated earlier from peats or peat-forming plants shows that a large
part of the amino-acid nitrogen is bound with lignin.
From the data described above, it is clear that there are some qualitative and quantitative
differences in the composition and contents of amino-acid nitrogen in isolated lignin compared with
peats and plants.
The hydrolysates from lignin are richer in methionine and cystine compared with those
obtained from peats and peat-forming plants. It is known that these amino acids are unstable
towards acid hydrolysis, and their absence in some hydrolysates can be the result of acid
hydrolysis.

REFERENCES

[1] BATURO, V.A., RAKOVSKIJ, V.E., Kimya Torfoobrazovatelej, Tr. Inst. Torfa 6 (1957) 52.
[2] BREMNER, J.M., Studies on soil humic acids: II. Observation on the estimation of free aminogroups:
Reaction o f humic and lignine preparations with nitrous acid, J. Agric. Sei. 48 (1957) 352.
[3] SOWDEN, F.J., Estimation o f amino acids in soil hydrolysates by the Moore and Stein method, Soil
Sei. 8 0 (1 9 5 5 ) 181.
[4] SOWDEN, F.J., Distribution o f amino acids in selected horizons of soil profiles, Soil Sei. 82 (1956) 491.
[5] KOJIMA, R.T., Soil organic nitrogen: I. Nature o f the organic nitrogen in a muck soil from Geneva,
New York, Soil Sei. 64 (1947) 157.
[6] KOJIMA, R.T., Soil organic nitrogen: II. Some studies on the amino acids of protein material in a muck
soil from Geneva, New York, Soil Sei. 64 (1947) 245.
[7] MACIAK, F., “Effect o f fertilization and tillage on content of organic forms o f nitrogen in peat soil and
its humus fractions”, Proc. 4th Int. Peat Congress, Otaniemi, Finland, IV (1972) 105.
[8] WAKSMAN, S.A., STEVENS, K.F., Chemical composition of peat, Soil Sei. 26 (1928) 113, 239.
[9] BREMNER, J.M., Methods o f Soil Analysis: Chemical and Microbiological Properties 2, American Society
o f Agronomy, Madison, Wisconsin (1965) 49.
 IA EA -SM -211/81 357

[10] RITTER, G.J., SEBORG, R.M., MITCHELL, R.L., Factors affecting quantitative determination of lignin
by 72 percent sulfuric acid method, Ind. Eng. Chem. Analyt. Edn 4 (1932) 202.
[П] MACIAK, F., Bilans azotowy w roslinnosci torfotworczej i torfach (Badania nad formani azotu w torfach),
Rocz. Nauk Roln., Ser. A4 (1963) 87, 563.
[12] MACIAK, F., Formy azotu w glebie torfowej i frakcjach humusowych gleby przy wieloletnim uzytkowaniu
takowym, polowym i przemiennym, Rocz. Glebozn. 2 (1973) 399.
[13] SÖCHTIG, H., MACIAK, F., Bindung des Stickstoffes und Vorkommen phenolischer Verbindungen im
Torf, T elm a l (1 9 7 1 )4 9 .
[14] MACIAK, F., Relationship between total and amine nitrogen content and the composition of ammo acids
in peat-forming plants and in peats, Ekol. Pol. S-A 2 (1966) 193.
[15] BREMNER, J.M., The chemical nature o f soil organic nitrogen (Studies on soil organic matter),
J . Agric. Sei. 3 9 (1 9 4 9 ) 183.

[16] STEFENSON, E.J., Isolation and identification o f some amino compounds in soils, Soil Sei. Am.,
Proc. 2 0 (1 9 5 6 ) 201.
[17] STEFENSON, E.J., Effect o f some long rotation on the amino acid composition of the soil, Soil Sei.
Am., Proc. 2 0 (1 9 5 6 )2 0 4 . \

[18] COULSON, C.B., DAVIES, R.J., KHAN, E.J.A., Chemical studies on upland peat in North Wales; J. Sei.
Food Agric. 10 (1959) 209. !
[19] FLAIG, W., Zur Bildungsmöglichkeit von Huminsäuren aus Lignin, Holzforschung 9 (1955) 1.
[20] FLAIG, W., “Über die biochemische Bildung von Humusstoffen aus Lignin”, Biochemistry o f Wood 2
(4th Int. Congress o f Biochemistry), Pergamon Press, New York (1959).
 IAEA -SM -211 /8 2

STABILIZATION OF ORGANIC MATTER

IN SAND MIXED CULTURES
B. SCHEFFER j
Niedersächsisches Landesamt für
Bodenforschung,
Ausseninstitut für Moorforschung
und angewandte Bodenkunde,
Bremen,
Federal Republic of Germany
R a p p o rteu r: В. S ch effer

Abstract

STABILIZATION OF ORGANIC MATTER IN SAND MIXED CULTURES.

For 40 years drained peat soils have been deep-ploughed. After this soU improvement, which is called
‘German sand mixed culture’, stable arable land is developed from bogs. The new soil will be formed by processes
o f soil formation with phases o f settling, homogenization and humification. Intensive decomposition and
transformation o f the organic matter take place in the topsoüs. Chemical analyses with soils o f different old sand
mixed cultures prove that in the course o f time the organic matter will decrease and the nitrogen content increase.
The content o f the > 1.59 specific gravity fraction increases. With the increasing age of sand mixed cultures we
found carbon and nitrogen, especially in the > 1 .5 9 specific gravity fraction. Therefore the mineral components,
and also quartz sand, form stable organic-mineral compounds with the organic matter, and improve also the stability
of the soil texture. After 100—130 years the soils o f sand mixed cultures are similar to Plaggenesch soils.

1. SOIL FORMATION AND DEVELOPMENT OF SAND MIXED CULTURES

For 40 years the peat layer to 1.40 m of drained peat soils in West Germany has been deep-
ploughed. Under these soils sand and peat are in sloping layers and only in the topsoil are peat
and sand mixed after liming and fertilizing [ 1]. ;
The physical conditions, including air and water and thermal conditions [2], and also the
chemical conditions, are improved. For the first time land use with liming and fertilizing promotes
the decomposition of peat.
Kuntze [3] has deduced the process of soil development of sand mixed cultures from data
of yield. He differentiates phases of settling, homogenization and humification, combined with
intensive decomposition and transformation of the peat matter in the topsoil (Fig.l). [With
increasing age of a sand mixed culture the organic matter will be changed from peat to humus.
The new organic matter is no longer easily decomposed, and no separation of the sand and humus
components is possible. The last phase of these processes is an anthropogenic soil, almost like the
Plaggenesch soil.
The soil development of sand mixed cultures can be measured by the changes in the organic
matter. We made experiments on seven different old sand mixed culture soils from Königsmoor,
in the Harburg district, with financial subsidy from the Deutsche Forschungsgemeinschaft
(Ku 224/25). We hope to discover whether the organic and inorganic soil components form stable
combinations and so improve the soil structure and stability of the soil texture and reduce soil
erosion.

359
360 SCHEFFER

Yield
(dt/ha grain equivalent)

F I G .l. Y ie ld a n d s o il d e v e l o p m e n t o f s a n d m i x e d c u ltu r e s [3].

Therefore we made gravity separations (with carbon tetrachloride) with various old sand
mixed cultures. It is an interesting question whether the stability of the organic matter with
increasing age of sand mixed cultures is caused by the formation of organic-mineral compounds in
the soil in spite of the loss of clay components.

2. DESCRIPTION OF THE EXPERIMENTS

2.1. Material used

In Königsmoor there are various old sand mixed cultures with the same conditions (soil,
climate) [4]. In addition, soil samples of a Plaggenesch soil from nearby Lauenbrück were analysed.

1. Sand mixed culture 0 years old

2. Sand mixed culture 8 years old
3. Sand mixed culture 15 years old
4. Sand mixed culture 20 years old
5. Sand mixed culture 36 years old
6. Sand mixed culture 58 years old, deep-ploughed Ericapodsol
7. Plaggenesch 250 years old

2.2. Description of the methods

We used the following methods to characterize these soils:

1. pH-value (0.01 N CaCl2)
2. Volume weight (g/1)
3. Loss on ignition (%)
4. C content of the loss on ignition (Wösthoff)
5. N content of the loss on ignition (Kjeldahl)
6. r-value (Keppler)
7. Gravity separation with CC14 [5,6]
 IAEA -SM -211 /8 2 361

TABLE I. CHEMICAL SOIL DATA

Soil Age Volume Ash C N C:N r-value >H

(yr) weight (% DM) {% organic DM) (%) CaCl2)
(g/о

Sand mixed culture 0 1043 64.5 55.3 0.59 95 57.1 3.0

Sand mixed culture 8 1179 92.3 50.1 1.44 34 58.3 4.0
Sand mixed culture 15 1362 91.7 52.6 1.68 31 74.1 4.5
Sand mixed culture 20 1447 90.3 50.0 1.65 30 56.7 4.6
Sand mixed culture 36 1580 94.6 47.2 2.03 23 74.4 4.7
Sand mixed culture 58 1529 93.2 45.6 2.00 23 73.7 4.2
Plaggenesch soil 250 1318 97.7 53.0 4.35 13 60.4 4.9

3. RESULTS AND DISCUSSION

Table I shows the chemical data of the soils analysed. With increasing age of sand mixed
cultures the volume weight of the soils increases, also the pH-value increases conditionally by
liming and land use, the ash content increases also and the humus content decreases. The C:N ratio
becomes smaller with increasing age of these soils, as more nitrogen is fixed in the organic matter.
The oligotrophic raised bog peat as original organic matter contains only a little nitrogen (< 1% N).
The decomposition and transformation of the organic soil matter is produced by liming and
nitrogen-fertilizing and also by the soil aeration.
The r-values, and also the contents of organic matter insoluble in 72% H2S04, are Usually
higher in sand mixed cultures than in the Plaggenesch soil. The r-values increase with the
decomposition of the light soluble peat components with the increasing age of a sand mixed cul­
ture. These components, which are very strongly decomposed, have no influence on the soil
development. They may be humic substances or coal, which are formed by peat formation.
After drying out, the soil of a young sand mixed culture is easily separated into sand and
organic matter. On the other hand, old sand mixed cultures and also sandy soils rich in humus
can hardly or only with great difficulty be separated mechanically into sand and organic matter.
Therefore the humus of these soils is very stably fixed to the mineral components.
We carried out experiments with carbon tetrachloride to find out the content of the organic
components fixed on mineral components in various old sand mixed cultures, and to discover
whether this content increases with the soil development of sand mixed cultures. The gravity
separation with carbon tetrachloride (CC14) (d = 1.59) is suitable as the organic matter has a
specific density of 1.5 g/cm3, and therefore it is easy to separate it from the mineral components.
The <1.59 specific gravity fraction (Table II) decreases in favour of the >1.59 specific
gravity fraction with increasing age in sand mixed cultures. If the increase in the > 1.59 specific
gravity fraction is linear after eight years, we have the following equation: у = 0.05 x + 94.77;
г = 0.88* (n = 5, у = > 1.59 specific gravity fraction, x = time). According to this equation, the
sand mixed cultures have after 100 years the same content of < 1.59 and > 1.59 specific gravity
fractions as the Plaggenesch soil (0.3% <1.59 specific gravity fraction; 99.7% >1.59 specific
gravity fraction) [7]. ;
The C : N ratio of the < 1.59 specific gravity fraction decreases from 58 of the peat sand
mixture (0 year old) to 2 5 ^ к Ь increasing age of the sand mixed cultures; only the 58 years’ old
sand mixed culture —a deep-ploughed Ericapidsol —has a C:N ratio of 30. The < 1.59 specific
gravity fraction contains also the remains of plants and roots, which cannot be separated
362 SCHEFFER

T A B L E II. G R A V IT Y S E P A R A T IO N W ITH CC14

< 1.59 specific gravity fra c tio n > 1.59 sp ecific grav ity fra c tio n
Soil Age C o n te n t C N C :N C o n te n t C N C :N
(y r) (%) ( % organic DM) (% ) (% org an ic DM)

Sand m ixed c u ltu re 0 10.3 55.5 1.0 58 89.7 5 5.9 0 .8 78

Sand m ixed cu ltu re 8 4.6 5 5 .4 1.7 33 9 5 .4 5 0.8 1.9 26
Sand m ixed c u ltu re 15 5.5 5 3 .4 1.8 30 94.5 53.5 2.0 27
S and m ixed c u ltu re 20 3.7 55.5 1.9 29 9 6.3 5 4.8 2.3 23
Sand m ixed c u ltu re 36 3.3 53.8 2.2 25 9 6 .7 5 0 .4 2.3 22
Sand m ixed c u ltu re 58 2.3 58.2 2.0 30 9 7.7 5 3.2 2.3 25
Plaggenesch 250 0.3 56.7 2.1 28 9 9 .7 4 1 .4 3.1 13

prev io u sly b y sifting. In th e > 1.59 specific g rav ity fra c tio n also th e C :N ra tio decreases w ith th e
age o f a san d m ix ed cu ltu re.
A n a tte m p t to sep arate th e organic m a tte r w ith 2M p h o sp h o ric acid fro m th e m in eral c o m p o ­
n e n ts was u n su c c essfu l.1
T h e n itro g e n favours also in th ese soils th e fo rm a tio n o f su c h h u m u s c o m p o n e n ts, w hich are
fix ed m ain ly in th e > 1 .5 9 sp ecific gravity fra c tio n . F ig u re 2 show s th e tra n s fo rm a tio n o f th e
o rg an ic m a tte r in sand m ix ed c u ltu res; 70% o f c arb o n an d n itro g e n are in th e < 1.59 specific
grav ity fra c tio n in y o u n g sand m ix ed cu ltu res. A fte r 58 y ears o n ly 2 0 —30% o f c arb o n an d n itro g e n
are fix ed in th e < 1.59 specific g rav ity fra c tio n an d 7 0 —80% o f C an d N in th e > 1.59 specific
grav ity fra c tio n . M ath em atical e q u a tio n s show th a t a fte r 130 y ears th e d istrib u tio n o f th e organic
m a tte r in < 1 .59 an d > 1.59 specific gravity fra c tio n s is th e sam e as in a P laggenesch soil [7].

1 J.C h r. Salfeld a n d H . S üchtig o f th e In s titu t für B iochem ie d es B o d en s d e r F o rsc h u n g sa n stalt für

L a n d w irtsc h a ft, B raunschw eig-V ölkenrode, are th a n k e d fo r m aking th e se analyses.
 IA E A -SM -211/82 363

In th e first 2 0 y ears a fte r deep-ploughing w e fo u n d th e m ain tra n s fo rm a tio n s in th e phase

o f h o m o g en iz atio n ; th is p rocess slow s d o w n in th e p hase o f h u m ific atio n . T h e g rav ity se p a ra tio n
show s th a t th e o rg an ic m a tte r o f sand m ix ed c u ltu res will be stab ilized w ith increasing age by
fix a tio n o n th e m in eral soil c o m p o n e n ts. T h e new organic su b stan ces fo rm ed b y th e d e c o m p o sitio n
o f th e o rg an ic m a tte r a n d fix a tio n o f n itro g e n are m ore closely co m b in e d o n th e m in eral soil
c o m p o n e n ts w ith increasing soil d ev elo p m en t. O rganic-m ineral c o m p o u n d s are also fo rm ed .
O rganic-m ineral c o m p o u n d s are fo rm ed by chem ical, bio ch em ical an d p hysical re ac tio n s in
th e soil. T h ey are very re sista n t to b a cteria and also have an in flu e n ce o n th e high sta b ility o f th e
soil te x tu re [8]. A lso th e fluvial sands o f th e W üm m esander, w h ich have little iro n and a lu m in iu m ,
c o n ta in in th e silt fra c tio n free O H -groups and c atio n s to w h ic h organic sub stan ces are fixed.
O lig o tro p h ic raised bog p e ats p ro d u c e n o re a c tio n a fte r a long p erio d w ith clay m inerals [8];
th e re fo re th e p e ats in sand m ix ed c u ltu res m u st be first d e co m p o sed b y m icro-organism s in th e
p hase o f soil d e v elo p m en t. T h e n th e n ew org an ic sub stan ces rich in n itro g e n fo rm stable
organ ic-m in eral c o m p o u n d s, especially w ith th e in flu e n ce o f soil m icro-organism s. In p a rtic u la r
th e n itro g e n fav o u rs th e fixing o f organic substan ces in th e > 1.59 specific g rav ity fra c tio n w ith
in creasin g age o f san d m ixed cu ltu res.
O rganic su b stan ces also can fix m in eral c o m p o n e n ts an d fo rm stab le m in eral h u m u s com plexes.
F u rth e r chem ical a n d p hy sical e x p e rim e n ts will be able to c h aracterize th e linkage b e tw ee n sand
an d h u m u s, and th e in flu e n ce o f soil b a c te ria in th ese processes.

REFEREN CES

[1] BA D E N , W., D eu tsch e S an d m isc h k u ltu re n (T ie fp flu g k u ltu re n ) n u r u n te r e n tsp re c h e n d e n V o rau sse tzu n g en !,
W asser B oden 1 0 ( 1 9 5 8 ) 349.
[2 ] K U N T Z E , H ., D JA C O V IC , B ., E influss m in eralisch er u n d org an isch er K o m p o n e n te n a u f p h y sik alisch e
E ig en sch afte n von S an d m isc h k u ltu re n , Z. K u ltu rte c h n ik F lu rb erein ig u n g 11 (1 9 7 0 ) 72.
[3 ] K U N T Z E , H ., D ie T o rfk o m p o n e n te in d er B o d en b ild u n g a u f S an d m isc h k u ltu re n , M itt. D tsch . B o d en k d .
Ges. 15 (1 9 7 2 ) 155.
[4 ] K U N T Z E , H ., M o o rb ö d en N o rd d e u tsc h la n d s, M itt. D tsch. B o d en k d . Ges. 13 (1 9 7 1 ) 105.
[5 ] M cK E A G U E , J .A ., O rganic m a tte r in p article-size an d specific grav ity fractio n s o f so m e A h-h o riz o n s,
Can. J. Soil Sei. 51 ( 1 9 7 1 ) 4 9 9 .
[6 ] S C H E F F E R , B., K ritisch e S ich tu n g k o n v e n tio n e lle r M eth o d e n d e r H u m u su n tersu ch u n g in d e r M oor- u n d
B o d en k u n d e , T E L M A 4 (1 9 7 4 ) 175.
[7 ] S C H E F F E R , В., H ID D IN G , H ., B o d en c h em isch e U n tersu ch u n g en v ersch ied en a lte r S an d m isc h k u ltu re n
(in p re p a ra tio n ). !
[8 ] S C H E F F E R , F ., SCH A C H TSC H A B E L, P ., L e h rb u c h d e r B o d en k u n d e , 9 th E d n , E nke-V erlag, S tu ttg a rt (1 9 7 6 ).
 IAE A -SM -211 /8 3

ORGANIC NITROGEN CONTENT OF PEAT SOILS

COMBINED WITH DIFFERENT FRACTIONS OF
HUMIC SUBSTANCES

W. R O C H U S
In te rfa k u lta tiv e s L eh rg eb iet C hem ie
d er U n iv ersität G ö ttin g e n ,
G ö ttin g e n ,
F e d e ral R e p u b lic o f G erm any

R a p p o r te u r : В. S c h e ffe r

Abstract

O R G A N IC N IT R O G E N C O N T E N T O F P E A T SO IL S CO M BIN ED W ITH D IF F E R E N T F R A C T IO N S O F
H U M IC SU BSTA N CES.
T h e c o n te n t o f fulvic an d h u m ic acids, silicic-hum ic acids, clay h u m u s co m p lex es a n d to ta lly e x tra c te d
resid u e w as d e te rm in e d in a series o f p e a tla n d lo ts farm ed fo r 10, 15 o r 31 y ea rs w ith farm y ard m a n u re o r
clover-grass as organic fertilizers. T h ese c o m p o n e n ts give in c o n n e c tio n w ith th e ir n itro g e n c o n te n t sig n ifican t
in fo rm a tio n o n soil fo rm in g processes. T h e investigations w ere carried o u t o n sand-m ix an d fen cu ltiv a tio n soils.
I t co u ld be sh o w n th a t, d ep e n d in g o n th e ap p lied o rg an ic m a n u re , th e to ta l o rg an ic n itro g en c o n te n t in soil
o rganic m a tte r varies in th e sam e soil ty p e an d age; it decreases w ith increasin g age, b u t it is g reater in farm y ard -
m a n u red soils, w h ere also th e c o n te n t o f organic m a tte r is higher. W ith increasing age it d ecreases m o re slow ly
th a n in g reen-m anured soils. T h e N c o n te n t o f th e h u m u s fra c tio n s is d iffe re n t in th e tw o sand-m ix and th e fen
cu ltiv a tio n soils also. G en erally in th e farm y ard -m an u re d soils th e n itro g e n c o n te n t is h ig h er in th e H A , SHA ,
CHC an d T E R fra c tio n s and lo w er in th e F A fra c tio n . C o m paring th e tw o ty p e s o f soils, it is ev id en t th a t th e
o rganic m a tte r c o n te n t g en erally , an d also th e N c o n te n t, is h ig h e r in th e fe n cu ltiv a tio n soils. W ith increasing
age, th e c o n te n t o f soil organic m a tte r increases b u t th e N c o n te n t decreases. H ow ever, in regard to th e to ta l
soil, th e N c o n te n t increases in c o rre la tio n w ith increasing organic m a tte r.

IN T R O D U C T IO N

In v estig atio n s o n m an u rin g o f p e a t soils have b e en carried o u t a t th e S ta te P e a t R esearch

S ta tio n in B rem en fo r m o re th a n 60 years. In th is p ro g ram m e th e n itro g e n su p p ly f o r th ese soils
h as b e en stu d ied to o using d iffe re n t k in d s o f org an ic n itro g e n fertilizers.
F o r p ed o lo g ical reaso n s, p a rtic u la rly fo r stu d ies o n soil d e v elo p m en t, it is o f in te re s t n o t
o n ly to ex am in e th e to ta l n itro g e n b alan ce b u t also to discover h o w m u ch n itro g e n is co m b in ed
w ith o rg an ic soil c o m p o n e n ts a t c ertain tim e s [ 1 ].
A re p o rt w ill be given h e re o f a m e th o d to e lu c id ate th is q u e stio n using sam ples o f sand-
m ix a n d fe n c u ltiv atio n soils fro m p lo ts o f th e K oen ig sm o o r research sta tio n . T hese p lo ts w ere
fe rtiliz ed as u su al, b u t p a r t o f th e n itro g e n w as ap p lied b y fa rm y a rd m an u re o r, in som e p lo ts,
b y g reen m an u rin g , in o rd e r to stim u la te th e biological a c tiv ity o f p e a t soils, as w as d o n e fo rm e rly ,
a n d to e n ric h th e h u m u s m a tte r [2,3].
T h e o rig in al n itro g e n o f p e a t m a tte r is lib e rate d in p a rts b y bio ch em ical tran sfo rm a tio n s.
I t can b e u sed to g e th e r w ith n itro g e n o f fe rtiliz ers b y m icro-o rganism s o r p lan ts, an d also e n te rs
in to th e p ro cesses o f soil dev elo p m en t.
In th e co u rse o f tim e , fro m th e sand and p e a t m ix tu re , in th e presen ce o f o th e r biological
an d m in e ral m ate ria ls, soils are su b s e q u e n tly fo rm ed m ain ly consisting o f h u m ific a tio n p ro d u c ts
in th e ir o rg an ic m a tte r in clu d in g n itro g e n fro m vario u s sources.

365
366 ROCHUS

IN V E S T IG A T E D SO ILS

A ll sam ples w ere ta k e n fro m p lo ts o f th e e x p erim e n tal sta tio n in K o enigsm oor. T h ey can
b e co n sid ered to b e o f th e sam e ty p e as regards th e genesis, n a tu re an d u tiliz a tio n . T h ey w ere
c u ltiv ated a n d u sed as a rab le lan d fo r 10, 15 or 31 years.
O n e p a rt o f th e soils w as cu ltiv ated as sand -mix, and a n o th e r p a rt as D u tc h fen cu ltiv atio n .
O n e h a lf o f th e tw o soil ty p e s w as m an u re d w ith farm y a rd m an u re , th e o th e r h a lf w ith green
m an u rin g in th e cro p p in g sequence. M oreover, all th e soils w ere farm ed in th e sam e m an n er.
T o m ak e a c o m p ariso n w ith a re c e n tly c u ltiv ated p e a t soil, a n artificial soil w as m ixed in
th e sam e w ay a n d fro m th e sam e m aterials (ta k e n fro m a neig h b o u rin g virgin raised bo g se c tio n )
as used fo r th e cu ltiv ated soils. I t w as investigated to g e th e r w ith th e p e a t lan d soils.
T h e p lo ts w ere as follow s (n u m b e r o f years o f cultiv atio n -in brack ets):
I (1 0 ) S an d -m ix c u ltiv atio n , d eep p lo u g h ed , used as arable lan d , w ith o u t fa rm y a rd m anuring.
II (1 5 ) S an d -m ix c u ltiv atio n , deep plo u g h ed , used as arable lan d , cro p p in g seq uence w ith
clover grass w ith o u t fa rm y a rd m anuring.
III (3 1 ) T h e sam e as II (1 5 ), b u t w ith 31 y ears o f cu ltiv atio n .
IV (1 5 ) S an d -m ix c u ltiv atio n , d e ep plo u g h ed , used as a rab le land w ith farm y a rd m an u rin g
w ith o u t clover grass.
V (3 1 ) T h e sam e as IV (1 5 ), b u t w ith 31 y ears o f cu ltiv atio n .
VI (3 1 ) D u tc h fe n c u ltiv atio n , used as arable lan d , cro p p in g seq uence w ith clover grass w ith o u t
fa rm y a rd m an u rin g .
V II (3 1 ) T h e sam e as VI (3 1 ), b u t w ith fa rm y a rd m a n u re w ith o u t clover grass.
V III (0 ) A rtific ial soil o f th e sam e co m p o sitio n as th e o th e r soils a fte r cu ltiv atio n .

M ETH O D S

T h e o rg an ic c o m p o n e n ts w ere o b ta in e d fro m sam ples o f th e soils described (a b o u t 1.5 kg

fro m each ) b y to ta l an d fra c tio n a tin g e x tra c tio n in glass co lu m n s (1 5 0 cm long, 5.5 cm dia.)
w ith aq u eo u s solv ents a t various pH .
T h e c o n stitu e n ts so lu b le in w a te r an d d ilu te d h y d ro c h lo ric acid (0 .1 N ) w ere first elim in ated .
T h e n th e soil w as e x tra c te d in th e co lu m n by d ilu te d alk ali (0 .1 N K O H ). T h e e x tra c t w as acidified
w ith d ilu te d HC1 t o a p H o f 6.5.
A fte r stan d in g 15—20 h , th e p re c ip ita te d silicic-hum ic acids (SH A ) w ere se p a ra ted , w ashed
w ith w a te r a n d th e n w ith 0 .1 N a m m o n iu m h y d ro x id e, an d d ried a t 105°C .
T o c o m p le te th e e x a m in a tio n o f significant c o m p o u n d s o f soil-form ing processes, th e h u m ic
acids (H A ) o f th e sam ples w ere d e te rm in e d . T h ey w ere iso lated b y acidifying th e so lu tio n rem aining
a fte r p re c ip ita tio n o f silicic-hum ic acids an d th e w ash-w aters w ith d ilu te d HC1 u p to pH 1, separating
a n d w ashing th e p re c ip ita te d h u m ic acids an d d ry in g a t 105°C .
T h e fulvic acids w ere iso lated fro m th e filtra te an d u n ite d w ith th e fulvic acid fra c tio n o f th e
HC1 e x tra c tio n . S u b seq u e n tly th e resid u e in th e c o lu m n w as w ashed w ith w a ter. A fte r rem oving
th e alk ali th e e x tra c tio n o f clay-hum us com p lex es (C H C ) fo llow ed. T h e y w ere flo cc u la ted fro m
th e su sp en sio n b y acid ify in g w ith d ilu te d HC1 u p to pH 2 , sep arated an d d ried a t 105°C .
T h e a m o u n ts o f fulvic acids, h u m ic acids, silicic-hum ic acids, clay h u m u s com p lex es and
to ta lly e x tra c te d residues o b ta in e d w ere d e te rm in e d gravim etrically, an d also assessed as percen tag e
o f th e w h o le soil organic m a tte r free fro m w a ter and ash.
T h e to ta lly e x tra c te d org an ic p a rt o f th e resid u e w as finally d e te rm in e d .
T h e n itro g e n c o n te n t o f th e soils an d th e ir fra c tio n s w as d e te rm in e d b y th e K jeldahl m e th o d .
T h e n itro g e n w as c alcu lated as p erce n ta g e o f soil organic m a tte r a n d as %o o f n itro g e n in then-
co m p o n e n ts.
F o r fu r th e r d e ta ils o f th e m e th o d s see R e f .[ 4].
 IA EA -SM -211/83 367

TABLEI. CONTENT OF ORGANIC MATTER (ОМ ) (%) IN SAND-MIX AND FEN

CULTIVATION SOILS AT THE BEGINNING, AND AFTER 10, 15 AND 31 YEARS OF
AGRICULTURE SHOWING THE DISTRIBUTION OF ORGANIC MATTER IN THE silNGLE
COMPONENTS (IN PERCENTAGE OF TOTAL OM) !

P eat-sand S and-m ix F en M a lu re d
m ix tu re cu ltiv a tio n soils w it l
Clover
Age a fte r c u ltiv a tio n (years) grass

0 10 15 31 31 В = F arm y a rd
m an u re

%OMa 15.0 7.1 8.0 5.2 5.4 A

in soil
n.d. 9 .2 7 .2 7.5 В

FA 28.2 19.7 12.5 17.3 18.5 A

% o f OM
n.d. 13.0 19.4 21.3 В

HA 27 .6 25.3 28.2 38.5 18.5 A

% o f OM
n.d. 51.1 40.3 18.7 В

SHA 2.5 31 .0 3 3 .8 15.4 2 9 .6 A

% o f OM
n.d. 12.0 12.5 29.3 В

CHC 1.1 16.9 15.0 25.0 27.8 A

% o f OM
n.d. 14.1 2 2 .2 2 4 .0 В

TER 40 .6 7 .0 10.0 3.8 5.5 A

% o f OM
n.d. 9.8 5.6 6 .7 В

a
OM = O rganic m a tte r I
FA = Fulvic acids !
HA = H um ic acids i
SHA = Silicic-hum ic acids
CHC — C lay h u m u s com plexes
TER -- T o ta lly e x tra c te d residue
n .d . — n o t d eterm in ed .

R E SU L T S

T h e o rg an ic m a tte r gen erally decreased in all th e soils investigated w ith increasing age o f
u tiliz a tio n as a rab le lan d . O n e th ird o f th e org an ic m a tte r w as d eco m p o sed a fte r 31 y ears o f
cu ltiv atio n . T h e d ecrease w as less in fa rm y ard -m an u red soils. In fe n c u ltiv atio n soils th e decrease
o f o rg an ic m a tte r w as less th a n in green -m an u red soils. See T ab le I.
T h e n itro g e n c o n te n t also d ecreased gen erally w ith th e increasing age o f th e soils. T h e
fa rm y a rd -m a n u red soils h ad a d istin c tly sm aller N c o n te n t th a n th e green -m an u red soils.
T h e fe n c u ltiv atio n soils h a d a h ig h er N c o n te n t th a n th e sand-m ix cu ltiv atio n soils. See
T ab les II an d III.
368 ROCHUS

T A B L E II. C O N T E N T (%) O F N IT R O G E N IN O R G A N IC M A T T E R (OM ) A N D ITS

D IS T R IB U T IO N IN T H E SIN G L E C O M PO N EN TS O F T H E HUM US C O M PLEX ES (%) IN
SA ND-M IX A N D F E N C U L T IV A T IO N SO ILS M A N U R E D W ITH C L O V E R G R A SS (A ) AN D
F A R M Y A R D M A N U R E (B ) A F T E R 0, 10, 15 A N D 31 Y E A R S O F A G R IC U L T U R E

P eat-sand S and-m ix F en M anured

m ix tu re cu ltiv a tio n soils w ith :
A — Clover
Age a fte r cu ltiv a tio n (years) grass

0 10 15 31 31 В = F arm y a rd
m an u re

% OMa 15.0 7.1 8.0 5.2 5.4 A

in soil
n.d. 9.2 7.2 7.5 В

% N total 1.02 1.98 2.05 1.87 2 .04 A

in OM
n.d. 1.83 i.6 i 1.76 В

% of N 45.1 4 2 .0 37.3 3 2.8 3 4 .7 A

in FA
n.d. 2 9 .0 26.6 2 7.6 в

% of N 38 .6 24.8 28.6 4 0.3 2 4.8 A

in HA
n.d. 35.3 41.1 2 6 .9 В

% of N 3.9 26.3 26.6 18.4 28.8 A

in SHA
n.d. 27.5 22.4 3 2 .0 В

% of N 0 .6 4.8 5.2 6.3 9.5 A

in CHC
n.d. 5.8 7.6 11.1 В

% of N 11.8 2.1 2.3 2.2 2.2 A

in T E R
n.d. 2.4 2.3 2.4 В

% of N 83.7 66 .8 65 .9 73.1 59.5 A

in F A +
HA n.d. 64.3 6 7 .7 54.5 В

a
OM = O rganic m a tte r
FA = F ulvic acids
HA = H um ic acids
SHA = Silicic-hum ic acids
CHC — C lay h u m u s com plexes
TER = T o ta lly e x tra c te d residue
n.d. = n o t d e te rm in e d .
 IA E A -SM -211/83 369

T A B L E III. A M O U N T (%o) O F N IT R O G E N (N)' IN T H E HU M US CO M PL EX ES A N D T H E IR

CO M PO N EN TS IN SA ND-M IX A N D F E N C U L T IV A T IO N SO ILS M A N U R E D W ITH C L O V E R
G R A S S (A ) A N D F A R M Y A R D M A N U R E (B ), A F T E R 0 , 10, 15 A N D 31 Y E A R S O F A G R IC U L T U R E

P eat-sand S and-m ix F en M anured

m ix tu re cu ltiv a tio n soils w ith :

Age a fte r cu ltiv a tio n (y ears) grass

0 10 15 31 31 В = F arm y a rd
m a n u re

%OMa 15.0 7.1 8.0 5.2 5.4 A

in soil
n.d. 9.2 7.2 7.5 В

«•Nw 1.53 1.41 1.64 0.9 7 1.10 A

in soil
n.d. 1.68 1.16 1.32 В

% „N f a 0 .6 9 0 .5 9 0.61 0.3 2 0 .3 8 A
in soil
n.d. 0.49 0.31 0 .3 6 B 1

*» V 0 .5 9 0.35 0.4 7 0.3 9 0 .27 A

n.d. 0.59 0.4 7 0 .35 В

^ o N SHA 0 .0 6 0.37 0.4 4 0.1 8 0 .3 2 A

in soil
n.d. 0.4 6 0.26 0 .43 В

^°^C H C 0.01 0.07 0.0 8 0.0 6 0.11 A

n.d. 0 .1 0 0.09 0 .15 В

% o N TE r 0 .1 8 0.03 0.0 4 0.0 2 0 .0 2 a ;

in soil
n.d. 0.0 4 0.03 0 .03 в

a OM = O rganic m a tte r
FA = Fulvic acids
HA = H u m ic acids
SHA = Silicic-hum ic acids
CHC = C lay h u m u s com plexes
TER = T o ta lly e x tra c te d residue
n .d . = n o t d eterm in ed .

T h e d is trib u tio n o f th e n itro g e n am o n g single fra c tio n s o f th e h u m u s com p lex es is very

d iffe re n t. I t d ecreases in th e fulvic acid fra c tio n w ith decreasing age, p a rtic u la rly in farm y ard -
m an u re d soil.
T h e fe n c u ltiv atio n soil show s, a fte r 31 y e ars’ u tiliz a tio n , a higher N -c o n te n t in th e fulvic
acid fra c tio n . In th is fra c tio n th e free o r o n ly w eak ly b o u n d c o m p o u n d s can be fo u n d , lib erated
a fte r m ild acid ic o r alk alin e hy d ro ly sis.
In th e H A fra c tio n th e N c o n te n t decreases a t first stro n g ly , an d a fte rw a rd s increases. A fte r
31 y e ars’ u tiliz a tio n if is sig n ifican tly h ig h er th a n a t th e beginning. R em a rk ab ly , in fe n cu ltiv atio n
soils n o in crease fo llo w ed th e decrease.
370 ROCHUS

In th e F A and HA frac tio n s, a b o u t tw o th ird s o f N total is in te g rate d . In th e fresh ly m ixed

soil, th e m ain a m o u n t w as fo u n d in T E R . D uring soil d ev elo p m en t, th e N w as b o u n d increasingly
in th e SH A fra c tio n ( 2 6 —30% ). T hese p ro d u c ts w ere rem o v ed again w ith tim e . T h is a c c o u n ts fo r
a d ecrease o f th e N c o n te n t in th e farm y ard -m an u red soils.
T h e n itro g e n b o u n d in th e CHC fra c tio n a fte r u tiliz a tio n w as fo u n d to be m o re th a n 10
tim es h ig h er th a n th e sta rtin g a m o u n t, a n d in fa rm y a rd m an u re soils h ig h er th a n in green-m anured.
T h e fe n c u ltiv atio n soils have a b o u t o n e th ird m o re N th a n th e sand-m ix soils a fte r 31 y ears o f
u tiliz a tio n .
T h e n itro g e n b o u n d in th e T E R is less significant.
I t is re m a rk ab le th a t farm y ard -m an u red p lo ts had a h ig h er c o n te n t o f soil OM an d H A , and
a lo w er c o n te n t o f org an o m in eralic su bstances and n itro g e n th a n g reen-m anured soils

REFERENCES

[1] N A U C K E , W ., “T o rf” , U llm anns E n cy clo p ad ie 1 7 (1 9 6 6 ) .

[2] B A D E N , W., B eu rteilu n g von D üngung von M oor an d A n m o o r, L a n d w irtsch . S c h rifte n re ih e B o d en u n d
P flan z en d er R u h r-S tic k sto ff A G, 10 (1 9 6 1 ).
[3] BA D E N , W., B ew irtsc h aftu n g u n d L eistung des G rünland es a u f d e u tsc h e r H o ch m o o r-K u ltu r, M itt. u.
A rb eiten d. staa tl. M oo rv ersu ch sstatio n B rem en, 9. Ber. (1 9 6 6 ).
[4 ] RO C H U S, W ., G ew in n u n g u n d S tabüisierung von H u m in s to ff-F ra k tio n e n , M itt. D tsch . B o d en k d . G es. 4
(1 9 6 5 ) 301.
 CHAIRMEN OF SESSIONS

Session 1 H. Söchtig Federal R epublic o f Germany

Session 2 O.T. R otin i AGROCHIMICA

Session 3 L. De Leenheer Belgium

Session 4 E .A . Paul Canada

Session 5 D .R. Sauerbeck Federal R epublic o f Germ any

Session 6 F. J.acquin France

Session 7 A .S. Abdel-Ghaffar E gypt

Session 8 M. Schnitzer AGROCHIMICA

Session 9 J.P. Martin U nited S tates o f America

SECRETARIAT
S cientific Secretaries: Y. B A R R A D A Join t F A O /IA E A Division
o f A to m ic Energy in
F ood and Agriculture,
IA E A , V ienna
H. MATSUO F o o d and Agriculture Organization
o f the U nited N ations,
R om e
W. FLAIG AGROCHIMICA

A dm inistrative Secretary: Gertrude SEILER Division o f External

R elations, IAEA

Editor: S.M. FREEM AN Division o f Publications,

IAEA

Records Officer: L.S. LIEBERMAN D ivision o f Languages,

IAEA

371
 LIST OF PARTICIPANTS
ALGERIA

F eraga,A . Centre universitaire de recherches,

d’etu d es e t de realisations,
U niversite de C onstantine,
Tour A dm inistrative,
route A in El B ey,
Constantine

AUSTRALIA

Ladd, J.N . CSIRO, D ivision o f Soils,

B.P. 2 , Glen O sm ond, S.A . 5 0 6 4

Martin, J.K. CSIRO, Division o f Soils,

B.P. 2, Glen O sm ond, S.A . 5 0 6 4

AUSTRIA

Danneberg, O.H. österreich isch e Studiengesellschaft für

A tom energie GsmbH,
Institut für Landwirtschaft,
Lenaugasse 10,
A -1082 Vienna

BELGIUM

Beum er, J.M.J. U niversite Libre de B ruxelles,

Ecole de Sant6 Publique,
rue Belüard 100,
B -1040 Brussels

Cappaert, I.M.J. Laboratory o f S o il Physics,

Faculty o f Agricultural Sciences,
University o f G hent,
Coupure Links 5 33,
B -9 0 0 0 Ghent

De L eenheer, L. Laboratory o f Agricultural Pedology,

Faculty o f Agricultural Sciences, j
U niversity o f G hent,
Coupure Links 533,
B -9 0 0 0 G hent

Taslim, H. Laboratory o f S oil Fertility and S o il B iology ,

Faculty o f A gronom y,
C atholic U niversity o f Leuven,
Kardinal Mercierlaan 9 2 ,
B -3 0 3 0 Heverlee
374 LIST OF PARTICIPANTS

V erdonck, O. Laboratory o f Soil Physics,

Faculty o f Agricultural Sciences,
U niversity o f G hent,
Coupure Links 5 3 3 ,
B -9000 Ghent

Verstraeten, L.M.J. Laboratory of Soil Fertility and Soil Biology,

Faculty o f A gronom y,
Catholic U niversity o f Leuven,
Kardinal Mercierlaan 9 2 ,
B -3030 Heverlee

CANADA

B iederbeck, V .O . Research S tation ,

Agriculture Canada,
S w ift Current,
Saskatchew an S9H 3X 2

Paul, E.A. D epartm ent o f S oil Science,

University o f Saskatchew an,
Saskatoon,
Saskatchew an S7N 0W0

COSTA RICA

G on zalez, M.A. Centro de Investigaciones A gronom icas,

Universidad de C osta Rica,
San Jose

CYPRUS

Markou, P. D epartm ent o f Agriculture,

Ministry o f Agriculture and Natural R esources,
Soil and Plant N utrition S ection,
N icosia

CZECHOSLOVAKIA

Krälovä, M. Institute o f E xperim ental B otan y,

C zechoslovakian A cadem y o f Sciences,
K e dvoru 1 6 /1 5 , 1 6 6 3 0 Prague 6

Pospisil, F. Institute o f E xperim ental B otan y,

C zechoslovakian A cadem y o f Sciences,
Na Karlovce 1, 1 6 6 0 0 Prague 6

DENMARK

M iddleboe, V. Physics Laboratory,

Veterinary and Agricultural University,
Thorvaldsenvej 4 0 ,
Copenhagen

Sorensen, L.H. Danish A to m ic Energy C om m ission,

Research Establishm ent Ris&
D K -4000 R oskilde
 LIST OF PARTICIPANTS 375

EGYPT

Abdel-Ghaffar, A.S. D epartm ent o f Soil and Water Science,

Faculty o f Agriculture,
U niversity o f A lexandria,
A lexandria

R izk, S.G. Soils and Water Research Institu te, j

Agricultural M icrobiological Research S ection,
Agricultural Research Centre,
Gamaa Street,
Giza

FRANCE

A ndreux, F. Centre de p ed ologie b iologique du CNRS,

17 rue Notre-Dam e-des-Pauvres,
B.P. 5, F -5 4 5 0 0 V andoeuvre-les-Nancy

ВаШу, J.-R. Service de ped ologie, B ätim ent 4R3,.

U niversite Paul Sabatier,
118 rue de N arbonne, j
F -3 1 4 0 0 Toulouse j

B elo t, Y .A . CEA, Centre d’etu d es nucleaires de Fontenay-aux-R oses,

B .P .6, F -9 2 2 6 0 Fontenay-aux-R oses !

Berthelin, J. Centre de p ed ologie biologique du C NRS,

17 rue Notre-Dam e-des-Pauvres,
B.P. 5, F -5 4 5 0 0 Vandoeuvre-les-Nancy

B ottner, P. CEPE-CNRS,
B.P. 5 0 5 1 ,
F -3 4 0 3 3 M ontpellier Cedex

C atroux, G. IN R A , Laboratoire de m icrobiologie des sols,

B .V . 15 4 0 , F -2 1 0 3 4 D ijon Cedex

Chabalier, P.-F. IRAT,

110 rue de l’U niversite,
F -7 5 0 0 7 Paris

Chassin, P. C N R A , Institut national de la recherche agronom ique,

route de St Cyr,
F -7 8 0 0 0 V ersailles

C ortez, J. CEPE-CNRS,
B.P. 5 0 5 1 , F -3 4 0 3 3 M ontpellier C edex

D abin, B.R .B. O ffice de la recherche scientifique et

tech n iq u e outre-m er,
24 rue Bayard,
F -7 5 0 0 8 Paris

D upuis, Th.L. Laboratoire de ped ologie,

Faculte des sciences, ;
U niversite de Poitiers,
4 0 avenue du R ecteur Pineau, |
F -8 6 0 2 2 Poitiers C edex !
376 LIST OF PARTICIPANTS

Fardeau, J.-C. CEA, D B /S R A ,

CEN Cadarache,
B.P. 1, F -l 31 1 5 St Paul-lez-Durance

Fustec-M athon, E. Laboratoire de ped ologie,

Faculty d es sciences,
Universite de Poitiers,
4 0 avenue du R ecteur Pineau,
F -8 6 0 2 2 Poitiers Cedex

G um et, B. Centre de pedologie biologique du CNRS,

17 rue Notre-Dame-des-Pauvres,
B.P. 5, F -5 4 5 0 0 Vandoeuvre-les-Nancy

Jacquin, F. Laboratoire m atiere organique et environnem ent.

Ecole nationale superieure d ’agronom ie et
des industries alim entaires,
38 rue Ste Catherine,
F -5 4 0 0 0 N ancy

Jam bu, P. Laboratoire de ped ologie,

Faculte des sciences,
Universite de Poitiers,
4 0 avenue du R ecteur Pineau,
F -8 6 0 2 2 Poitiers C edex

Juste, Ch. IN R A , Station d’agronom ie,

Centre de recherches de B ordeaux,
D om aine de la Grande Ferrade,
F -3 3 1 4 0 Pont-de-Ia-Maye

Lossaint, P. CEPE'CNRS,
B.P. 5 0 5 1 , F -3 4 0 3 3 M ontpellier Cedex

M etche, M. Ecole nationale superieure d’agronom ie

et des industries alim entaires
38 rue Ste Catherine,
F -5 4 0 0 0 Nancy

Morel, J.L. Ecole nationale superieure d ’agronom ie

e t des industries alim entaires,
38 rue Ste Catherine,
F -5 4 0 0 0 N ancy

N go, B.C. G ERDAT-IRAT,

B.P. 5 0 3 5 , F -3 4 0 3 2 M ontpellier Cedex

Pichot, J.P. GER D AT-IR AT,

B.P. 5 0 3 5 , F -3 4 0 3 2 M ontpellier Cedex

R em y, J.C. Station agronom ique de l ’Aisne,

rue Fernand Christ,
F -0 2 0 1 1 Laon

Thom ann, C.L. S ociete du canal de Provence

et d ’am enagem ent de la region proven$ale,
Le T h olon et,
F -l 3 1 0 0 Aix-en-Provence
 LIST OF PARTICIPANTS

T outain, F. Centre de p ed ologie biologique du CNRS,

17 rue Notre-Dame-des-Pauvres,
B.P. 5, F -5 4 5 0 0 V andoeuvre-les-Nancy

VeUy, J. GER D AT-IR AT,

B.P. 5 0 3 5 , F -3 4 0 3 2 M ontpellier C edex

GERMAN DEMOCRATIC REPUBLIC

Faust, H. Central Institute for Isotop e and

R adiation Research,
Permoserstrasse 15,
D D R -705 Leipzig

Markgraf, G. H um boldt-Universität zu Berlin,

S ek tion Pflanzenproduktion,
Bereich Bodenkunde und Pflanzenernährung,
Invalidenstrasse 4 2 ,
D D R -104 Berlin

Aldag, R.W. Institut für Bodenkunde der U niversität G öttingen,

von Sieboldstrasse 4 , j
D -3400 G öttingen |

GERMANY, FEDERAL REPUBLIC OF

El-Bassam, N. Institut für Pflanzenbau und Saatgutforschung,

Forschungsanstalt für L andwirtschaft,
Bundesallee 50,
D -3 3 0 0 Braunschw eig-Völkenrode

Ellwardt, P.Chr. Institu t für B iochem ie des Bodens

der Forschungsanstalt für Landwirtschaft,
Bundesallee 50,
D -3300 Braunschweig-Völkenrode

FUip, Z. D epartm ent o f Agricultural M icrobiology,

Justus-Liebig U niversity,
Senckenbergstr. 3,
D -6301 Giessen

Friedrichsen, J. D eu tsche G esellschaft für Technische

Zusammenarbeit (G Z ) GmbH,
Postfach 51 8 0 ,
D -6236 Eschborn 1

Friesei, P. Institut für Pflanzenernährung und B odenkunde

der Christian A lbrechts-Universität Kiel,
Ohlhausenstrasse 4 0 - 6 0 ,
D -23 Kiel

Führ, F. A rbeitsgruppe R adioagronom ie,

Kernforschungsanlage Jülich,
Postfach 1913,
D -5 17 Jülich
378 LIST OF PARTICIPANTS

Haider, K. Institut für B iochem ie des Bodens

der Forschungsanstalt für Landwirtschaft,
Bundesallee 50,
D -3 3 0 0 Braunschweig-Völkenrode

Harms, H.H. Institut für B iochem ie des Bodens

der Forschungsanstalt für L andwirtschaft,
Bundesallee 5 0 ,
D -3300 Braunschweig-Völkenrode

Herzel, F. Institut für Wasser-, B oden- und L ufthygiene

des Bundesgesundheitsam tes,
Corrensplatz 1,
D -1000 Berlin 33

Jarczyk, H.J. Bayer AG Pflanzenschutz-A nw endungstechnik,

Biologische Forschung,
D -5090 Leverkusen-Bayerwerk

Johnen, B.G. I.C.I. Plant P rotection D ivision,

J ealott’s Hill Research Station,
Bracknell, Berks., R G 12 6E Y , England

K eppel, H. Isotopenlaboratorium ,
Forschungsanstalt für Landwirtschaft,
Bundesallee 50,
D -3 3 0 0 Braunschw eig-Völkenrode

Kerpen, W. Arbeitsgruppe R adioagronom ie,

Kernforschungsanlage Jülich,
Postfach 1913,
D -5 17 Jülich

M ittelstaedt, W. Arbeitsgruppe R adioagronom ie,

Kernforschungsanlage Jülich,
Postfach 191 3 ,
D -5 17 Jülich

Nagar, B.R . Institut für B iochem ie des B odens

der Forschungsanstalt für L andwirtschaft,
Bundesallee 50,
D -3 3 0 0 Braunschw eig-Völkenrode

O ttow , J.C.G. Institut für B odenkunde und Standortslehre,

U niversität H ohenheim ,
Emil-W olffstrasse 27,
D-7 Stuttgart-H ohenheim

R ochus, W. Interfakultatives Lehrgebiet Chem ie,

U niversity o f G öttingen,
von Sieboldstrasse 2,
D -34 G ottingen

Saalbach, £ . R uhr-Stickstoff AG,

Landwirtschaftliche Forschung H anninghof,
H anninghof 35,
D -4408 D ülm en/W estfalen
 LIST OF PARTICIPANTS 379

Salfeld, J.-Chr. Institut für Biochem ie des Bodens

der Forschungsanstalt für L andwirtschaft,
Bundesallee 50,
D -3300 Braunschweig-Völkenrode i
1
j
Sauerbeck, D .R . A grikulturchem isches Institu t der U niversität B onn
M eckenheim er A llee 176,
D -5300 Bonn

Scharpenseel, H.W. Ordinariat für Bodenkunde,

Universität von Hamburg,
R einbeck, D -2 0 5 7 Schloss

Scheffer, B. Niedersächsisches Landesam t für B odenforschung,

A usseninstitut für M oorforschung und A ngew andte
B odenkunde,
Friedrich-Misslerstr. 4 6 —4 8 ,
D -2 8 0 0 Bremen

Schweers, W.H.M. Federal Research Centre for Forestry

and Forest Products,
Leuschnerstr.9,
D -205 Hamburg 80

S ochtig, H. Institut für B iochem ie des B odens

der Forschungsanstalt für L andwirtschaft,
Bundesallee 50,
D -3300 Braunschw eig-Völkenrode

Som m er, C. Institut für B iochem ie des B odens

der Forschungsanstalt für L andwirtschaft,
Bundesallee 50,
D -3300 Braunschw eig-Völkenrode

Still, G. M et. and Rad. Res. Laboratory U SD A -A .R .S.,

P.O .B. 5 6 7 4 , U niversity Station,
Fargo, N orth D akota 5 8 1 0 2 ,
U nited S tates o f America

Stühm eier, К. Isotopenlaboratorium der Forschungsanstalt

für Landwirtschaft,
Institut Für B iochem ie des B odens,
Bundesallee 50,
D -3300 Braunschw eig-Völkenrode

T ietjen, C. Institut für Pflanzenbau und Saatgutforschung,

Forschungsanstalt für L andwirtschaft,
Bundesallee 50,
D -3300 Braunschw eig-Völkenrode j

W eichelt, T. Interfakultatives L ehrgebiet C hem ie, j

U niversity o f G öttingen, j
von Sieboldstrasse 2 , [
D -34 G öttingen
380 LIST OF PARTICIPANTS

HUNGARY

D ebreczeni, B. U niversity o f Agriculture,

Pater K.U.I.,
H -2100 G ödöllö

Timar, E. Institut o f Soil Science and Agricultural Chem istry,

Herman-Otto u t-4 1 5 ,
H r l0 2 2 Budapest

IRELAND

H ayes, M.H.B. D epartm ent o f Chem istry,

U niversity o f Birm ingham , Edgbaston,
Birmingham B 15 2TT, England

ITALY

Brunetti, N. D ivisione A pplicazioni R adiazioni, C .S.N .,

Casaccia,
1-00100 R om e

Nigro, C. Istituto Sperim entale per la Nutrizione delle Piante,

via Navicella 2,
R om e

LUXEMBOURG

Mersch, J. Services techniques de l ’agriculture,

Laboratoires de controle e t d’essais,
avenue Salentiny,
Ettelbruck

MOROCCO

Ben M iloud, A. Ministere de Г agriculture

et de la reform e agraire,
Rabat

MEXICO

L*A nnunziata, M .F. Instituto N acional de Energfa Nuclear,

Insurgentes Sur N o. 1 0 7 9 , Apartado Postal N o .2 7 —190,
M exico 18, D .F .

NETHERLANDS

D e Haan, S. Institute for S oil F ertility,

Oosterweg 92,
Haren/Groningen

Halma, G. Soil Science D epartm ent,

Agricultural U niversity,
10, Duivendaal,
W ageningen-6140
 LIST OF PARTICIPANTS 381

Janssen, B.H. D epartm ent o f Soils and Fertilizers,

Agricultural U niversity,
D e D reyen 3,
W ageningen-б 140

NIGERIA

A dejunji, S.A. Agricultural Research C ouncil o f Nigeria,

P.O .B. 5 3 82,
M oor Plantation,
Ibadan

Kadeba, O. Savanna Forestry Research S tation ,

P.O .B. 1039,
Samaru

S obu lo, R .A. Institute o f Agricultural Research and Training,

U niversity o f Ife,
P.O.B. 5 0 2 9 ,
Ibadan
NORWAY

Bergseth, H. D epartm ent o f S oil Science,

Agricultural University o f N orw ay,
P.O.B. 27,
1432 Äs-NLH

Ogner, G. T he Norwegian Forest Research Institute,

B oks 61,
1432 Äs-NLH

PAKISTAN

Malik, K.A. N uclear Institute fo r Agriculture and B iology,

P.O .B ox 12 8 , Thang R oad,
Lyallpur

PHILIPPINES

Francisco, L.C. S oil Research D ivision,

Bureau o f Soils,
M .Y. Orosa S treet,
Ermita, Manila

POLAND

Maciak, F. Warsaw Agricultural U niversity

(S ykola G low na Gospodarstw a W iejskiego-A.R .),
ul. R akow iecka 2 6 /3 0 , |
Warsaw

MySkow, W. Institu te o f Soil Science and C ultivation o f Plants,

Izabella 9 /2 0 ,
24 -1 0 0 Pulawy

SPAIN

Fortun Garcia, C. Instituto de E d afologfa у B iologfa V egetal,

Serrano 11 5 , Madrid-6
382 LIST OF PARTICIPANTS

SRI LANKA

Nagarajah, S. D ivision o f Agricultural C hem istry,

Central Agricultural Research Institute,
Gannoruwa, Peradeniya
SWITZERLAND

Blaser, P. Eidgenössische A nstalt für das forstl.

V ersuchsw esen (E A F V ),
Zürcherstr. 111,
C H -8903 Birm ensdorf

Kühni, H.R. CIBA-GEIGY Lim ited,

AC 7 .3 2 , R-K 15.5 .0 2 ,
C H -4002 Basle

N eyroud, J.A. Station federate de recherche agronom ique de Changins,

C H -1260 N yon

Schüepp, H. Eidgenössische Forschungsanstalt,

C H -8820 W ädenswil

Sticher, H.J. Eidgenössische T echnische H ochschule,

ETH Zürich,
C H -8092 Zürich

TURKEY

Evliya, Z.H. Soil Science D epartm ent,

Faculty o f Agriculture,
£ukurova University,
Adana

Ö ib e k , H . S oil S cience D epartm ent,

Faculty o f Agriculture,
Qukurova U niversity,
Adana

UGANDA

Sserunjoji, J.M.S. T setse C ontrol Division,

D epartm ent o f Veterinary Service and Anim al Industry,
P.O. B o x 7 1 4 1 ,
Kampala

UNION OF SOVIET SOCIALIST REPUBLICS

K ruglov, L.B. Byelorussian S cientific Institute

for Soil and A grochem istry,
Minsk

Lavrova, l.A . A ll-U nion S cientific Institu te for S o il A m elioration,

Pryanishnikov Street 31,
M oscow

L ukosiuniene, E.I. Lithuanian Institute o f Agriculture,

D otnuva A cadem y, V ilnyus
 LIST OF PARTICIPANTS 383

Matveeva, V.I. Byelorussian S cientific Institu te for Soil

and A grochem istry,
Minsk

Mazur, G .A . Ukrainian Research Institu te o f Agriculture,

Kiev

Shevtsova, L.K. A ll-U nion S cientific Institu te for S o il A m elioration,

Pryanishnikov Street 3 1 ,
M oscow

T ishchenko, A .T. A ll-U nion S cientific Institu te for S oil A m elioration,

Pryanishnikov Street 31,
M oscow

UNITED KINGDOM
M uzorewa, E.I. D epartm ent o f A gronom y,
University o f Missouri,
C olum bia, Missouri 6 5 2 0 1 ,
U nited States o f America

O’Callaghan, M.R. Soil Research Group,

Chemistry D epartm ent,
U niversity o f Birm ingham , E dgbaston,
Birmingham В 15 2TT

Parsons, J.W. D epartm ent o f S o il S cience,

U niversity o f A berdeen, M eston Walk,
A berdeen A B 9 2U E ,
Scotland

S w ift, R .S. Edinburgh S ch ool o f Agriculture,

W est Mains R oad,
Edinburgh EH9 3JG,
Scotland

UNITED STATES OF AMERICA

Bremner, J.M. D epartm ent o f A gronom y,

Iowa State University,
A m es, Iow a 50011

D uxbury, J.M. Cornell University,

D epartm ent o f A gronom y,
9 1 7 Bradfield Hall,
Ithaca, N Y 14853

Frederick, L.R. U S Energy Research and D evelopm ent A dm inistration,

W ashington D .C. 2 0 5 4 5

L oeppert, R.H. S oil Science D epartm ent,

University o f Florida,
2 1 6 9 McCarty Hall,
G ainesville, FI 32611

Martin, J.P. D epartm ent o f Soil Science and

Agricultural Engineering,
University o f California,
R iverside, California 9 2 5 0 7
384 LIST OF PARTICIPANTS

V olk, B.G. S oil Science D epartm ent,

University o f Florida,
2 1 6 9 McCarty Hall,
Gainesville, FI 32611

Wagner, G.H. D epartm ent o f A gronom y,

U niversity o f M issouri,
135 Mumford Hall,
C olum bia, Missouri 65201

VENEZUELA

L opez H ernandez, D . Instituto de Z oologfa Tropical,

Facultad de Ciencias,
Universidad C entral d e V enezuela,
Los Chaguaramos,
Caracas

Santiago, P.A . Universidad del Zulia, Apartado 5 2 6 ,

Maracaibo

ORGANIZATIONS

AGROCHIMICA

Altemüller, H.J. Institut für B iochem ie des B odens

der Forschungsanstalt für Landwirtschaft,
Bundesallee 50,
D -3 3 0 0 Braunschw eig-Völkenrode,
Federal R epublic o f Germany

Arcara, P.G . ls titu to Sperim entale per lo S tu d io e la D ifesa del S u o lo ,

Piazza d’A zeglio N o .30,
1-50121 F lorence, Italy

B uchholz, H.E. Forschungsanstalt Für Landwirtschaft,

Bundesallee 50,
D -3300 B raunschw eig-Völkenrode,
Federal R epublic o f Germany

C escatti, G. lstitu to Agrario S. M ichele Alladige,

Trento, Italy

Cheng, B.T. D epartm ent o f Agriculture,

G overnm ent o f Quebec,
P.O.B. 174, Sillery, Quebec G1T 2R I^
P.Q ., Canada

D om s, E. C hem isches Untersuchungslaboratorium ,

Institut für B iochem ie des B odens,
Forschungsanstalt für Landwirtschaft,
Bundesallee 50,
D -3300 Braunschw eig-Völkenrode,
Federal R epublic o f Germany
 LIST OF PARTICIPANTS 385

Domsch, K. Institut für Biochemie des Bodens der

Forschungsanstalt für Landwirtschaft,
Bundesallee 50,
D-3300 Braunschweig-Volkenrode,
Federal Republic o f Germany

Douglas, L.A. School of Agriculture and Forestry,

University o f Melbourne,
Parkville, Victoria 3052,
Australia

Im, J.N. Forschungsanstalt für Landwirtschaft,

Bundesallee 50,
D-3300 Braunschweig-Volkenrode,
Federal Republic of Germany

Nannipieri, P. Laboratorio per la Chimica del Terreno,

C.N.R.,
56200 Pisa, Italy

Orsini, D. S.C.A.M.,
Via Bellaria 164,
Modena, Italy

Reverte, L. Centro de Edafologfa у Biologia Aplicada del Segura,

Avda 18 de Julio N o .l,
Apartado 195,
Murcia, Spain

Rietz, E. Institut für Biochemie des Bodens der

Forschungsanstalt für Landwirtschaft,
Bundesallee 50,
D-3300 Braunschweig-Volkenrode,
Federal Republic of Germany

Rosell, R.A. Instituto de Edafologfa e Hidrologia,

Alem 925,
Bahfa Blanca, Argentina

Rotini, O.T. Istituto di Chimica Agraria,

Universitä degli Studi,
Lungamo Simonelli 9,
1-56100 Pisa, Italy

Schnitzer, M. Soil Research Institute,

Agriculture Canada,
Central Experimental Farm,
Ottawa, Ontario K l A 0C6, Canada

Sequi, P. Laboratory for Soil Chemistry, C.N.R.,

Via Corridoni 78,
1-56100 Pisa, Italy

Testini, C. Istituto di Chimica Agraria

dell’ Universitä di Sassari,
Facoltä di Agraria,
Via Enrico de Nicola,
1-17100 Sassari, Italy
386 LIST OF PARTICIPANTS

INTERNATIONAL SOCIETY OF SOIL SCIENCES (ISSS)

Carballas, T.M. Instituto de Investigaciones

Agrobiologicas de Galicia,
Consejo Superior de Investigaciones Cientificas,
Apartado 122, Santiago de Compostela,
Spain
 AUTHOR INDEX
Roman numerals are volume numbers.
Italic numerals refer to the first page o f a paper by the author concerned.
Upright numerals denote the page numbers o f comments and questions in discussions.
Literature references are not indexed.

Abdel-Ghaffar, A.S.: I 2 0 5 , 212,213,394; Flaig, W.: I 8,116,138,169,213,252,

II 3 1 9 , 324 299, 383, 411; II 41, 83, 91, 131,
Abd-el-Malek, Y.: I 1 8 3 240, 3 4 3
Agbeko, К.: II 3 3 Fortun, С : II 307, 317
Aldag, R.W.: I 96, 280, 2 9 3 , 298, 299; Francisco, L.C.: I 387, 394
II 57,317 Friedrichsen, J.: I 383
Al Zahawe, F.: I 8 3 Führ, F.: II 333, 339
Amarasiri, S.L.: I 9 7 Fustec-Mathon, E.: II 105, 114, 132
Amato, M.: I 301 Gabriels, D.: I 1 1 7
Andreux, F.: II 32,41,45,57 Gohar, M. R.: I 1 8 3
Antoun, G.G.: I 1 8 3 Golebiowska, D.: II 4 3
Arcara, P.G.: I 3 9 5 Gomah, A.M.: I 2 0 5
Bailly, J.-R.: II 55,41 Gonzalez, M.A.: I 1 5 9
Barakat, M.A.: I 2 0 5 ; II 3 1 9 Gonzalez I, J.: I 2 3 9
Ben Miloud, A.: II 289 Goussault, С: II 3 2 7
Berthelin, J.: I 4 1 3 , 423,424 Guckert, A.: I 4 0 3
Biederbeck, V.O.: I 115,169,281,383, Guillet, В.: II 114, 187, 201
423 Guiraud, G.: II 2 5 9
Bottner, P.: I 147, 26 3 , 273, 402; II 201 Haider, К.: I 2 1 5 ; II 23, 213, 333
Bremner, J.M.: II 22 9 , 240 Harms, H. : II 301
Calcinai, M.: I 63 Hayes, M.H.B.: I 137,138,411; II 89
Cappaert, I.M.J.: I 12 3 , 131, 137, 138 Hera, G: I 3 1 5
Chabalier, Р.-F.: I 8 3 , 116 Hernando, V.: II 3 0 7
Cheikzadeh-Mossadegh, D.: I 4 1 3 Jacquesy, R.: II 1 0 5
Chen, Y.: II 1 4 3 Jacquin, F.: I 170, 403, 411; II 4 3 ,2 6 5 ,
Cheng, B.T.: I 31, 39 289, 327
Chone, T.: II 4 3 Jaiyeola, K.E.: I 1 0 5
Cortez, J.: I 2 6 3 ; II 114 Jambu, P.: II 105, 133, 141
Dabin, B.R.B.: I 68 Janssen, B.H.: II 277
Danneberg, O.H.: I 280,291; II 221, Jappe, J.: II 259
228, 249 Johnen, B.G.: I 141, 147, 148 203,394
DeBoodt, M.: I 1 1 7 ,1 2 3 ,1 3 1 Joly, G.: II 1 0 5
De Haan, S.: I 21 Joshi, O.P.: I 3 6 5
De Leenheer, L.: I 9, 19, 147, 170 Juste, Ch.: I 138; II 21,289
Douglas, L.A.: II 339 Koma Alimu, F.X.: II 277
Dupuis, T.: II 1 3 3 Ladd, J.N.: I 157, 301
El-Bassam, N.: II 25 3 , 289 V Annunziata, M.F.: I 137, 239, 252, 253
Ellwardt, P.-Chr.: II 291 Lapid, F.M.: I 3 8 7
El-Shakweer, M.H.A.: I 205, II 3 1 9 Lavrova, I.A.: I 331
Fardeau, J.C.: II 2 5 9 Lenvain, J.: I 1 1 7
Filip, Z.: I 182,213; II 91 Livens, J.: I 3 5 9

387
388 AUTHOR INDEX

Llimous, G.: II 2 5 9 Rochus, W.: II 324, 3 6 5

Loeppert, R.H.: II 241, 250 Rosell, R.A.: I 280; II 75,21,317
Longueval, G: I 69 Rotini, O.T.: I 3
Lopez Hernandez, D.: I 255; II 240 Ruiz, J.: I 2 5 5
Lossaint, P.: I 263, 281; II 141 Sachdev, M.S.: I 365, 371
McGill, W.B.: I 1 4 9 Saiz, G; II 2 1 3
Maciak, F.: II 3 4 3 Salfeld, J.-Chr.: I 227, 2 8 5
Malik, K.A.: I 104, 212, 2 1 5 , 225; Sauerbeck, D.R.: I 18, 137, 747, 148,
II 324, 339 157, 159, 169, 170, 212, 394;
Markou, P.: II 289 II 14
Martin, J.K.: I 197, 203 Scharpenseel, H.W.: I 104, 138, 203, 225
Martin, J.P. : I 181,299,394; II 23, II 4 1 , 193, 201
32, 21 3 , 219, 220, 339 Scheffer, В.: II 3 5 9
Metche, M.: I 402, 423, 424; II 43, 83 Schiavon, M.: II 3 2 7
Meuzelaar, H.L.G: II 2 1 3 Schnitzer, M.: I 68, 182, 292, 298, 411;
Middelboe, V.: II 2 0 5 , 210, 211 II 21, 777, 131, 132, 143, 157,
Miglierina, A.M.: II 1 5 219, 220
Mittelstaedt, W.: II 3 3 3 Schweers, W.H.M.: II 85, 89
Monib, M.: I 1 8 3 Senesi, N.: II 1 4 3
Morel, J.L.: II 2 6 5 Sequi, P.: I 63, 68,411
Muzorewa, E.I.: II 99 Shevtsova, L.K.: I 339, 347
Mysköw, W.: I 169,225,311,424 Sobulo, R.A.: I 39, 104, 105, 116, 383;
Nagar, B.R.: I 39, 169; II 89, 131,772, II 289,324
2 1 3 , 220, 228 Süchtig, H.: I 8, 2 2 7 , 2 8 5 , 291, 292;
Nagarajah, S.: I 97, 104, 383 II 3 4 3
Nannipieri, P.: I 395, 402 Soe Agnie, I.E.: II 2 7 7
Neyroud, J.-A.: II 1 5 7 Sorensen, L.H.: II 3, 14
Nkundikije-Desseaux, V.: II 3 3 Sserunjoji, J.M.S.: I 213
Novilla, L.Q. de: II 1 5 Stühmeier, К.: II 210,211
Ortega, B.C.: II 3 0 7 Subbiah, B.V.: I 365, 371
Ottow, J.C.G.: I 402,424 Swift, R.S.: I 6 8 , 171, 182, 275, 280,
Oza, A.M.: I 371 281,411; II 240
özbek, H.: II 59 Taslim, H.: I 3 4 9
Parsons, J.W.: I 301 Tietjen, G: II 2 5 3
Paul, E.A.: I 147, 148, 149, 157, 169, 273, Ток, H.H.: 1 4 0 3
311 Tombesi, L.: I 4 3
Pedrazzini, F.: I 3 9 5 Turenne, J.F.: II 17 9
Pichot, J.: I 83, 96 Velly, J.: I 69, 81
Piovanelli, G: I 3 9 5 Verdonck, О.: I 123, 131
Posner, A. M.: I 171 Verstraeten, L.M.J.: I 349, 359, 383
Pospisil, F.: 1 252,311 Volk, B.G.: II 228, 241
Rapaire, J.L.: II 1 7 9 Vorher, W.: II 85
Remy, J.C.: II 240 Wagner, G.H.: II 99
Rietz, E.: II 91 Weichelt, T.: I 81,104,292; II 67,
Righi, D.: II 1 3 3 ,1 8 7 83, 132, 210, 219, 240, 250
Rizk, S.G.: I 1 8 3

TRANSLITERATION INDEX

Л ав р о в а, И .A . Lavrova, I. А.
Ш евцова, Л . К . Shevtsova, L.K.
T h e fo llo w in g c o n v e r s io n ta b le is p r o v i d e d f o r th e c o n v e n ie n c e o f r e a d e r s a n d t o e n c o u r a g e th e u s e o f S I u n its .

F A C T O R S F O R C O N V E R T IN G U N IT S TO S I S Y S T E M E Q U IV A L E N T S *
SI base units are the metre (m), kilogram (kg), second (s), ampere (A), kelvin (K), candela (cd) and mole mol).
[For further information, see International Standards ISO 1000 (1973), and ISO 31/0 (1974) and its ;everal parts]

M u ltip ly by to ob ta in

M ass

pound mass (avoirdupois) 1 Ibm = 4.536 X 10“ 1 kg

ounce mass (avoirdupois) 1 ozm = 2.835 X 101 g
ton (long) (= 2240 Ibm) 1 ton = 1.016 X 103 kg
ton (short) (= 2000 Ibm) 1 short ton = 9.072 X 102 kg
tonne (= metric ton) 1t = 1.00 X 103 kg

L e n g th

statute mile 1 mile = 1.609 X 10° km

yard 1 yd = 9.144 X 10' 1 m
foot 1 ft = 3.048 X 10' 1 m
inch 1 in = 2.54 X 10-2 m
mil (= 1 0 '3 in) 1 mil 2.54 X 10~2 mm

A rea

hectare 1 ha = 1.00 X 104 m2

(statute mile)2 1 mile2 = 2.590 X 10° km2
acre 1 acre = 4.047 X 103 m2
yard2 1 yd2 * 8.361 X 10' 1 m2
foot2 1 ft 2 = 9.290 X 10~2 m2
inch2 1 in 2 6.452 X 102 mm2

V o lu m e

yard3 1 yd3 = 7.646 X 10“ ' m3

foot3 1 ft3 = 2.832 X 10“ 2 m3
inch3 1 in3 = 1.639 X 104 mm^
gallon (Brit, or Imp.) 1 gal (Brit) - 4.546 X 10‘ 3 m3
gallon (US liquid) 1 gal (US) = 3.785 X 10' 3 m3
litre 11 = 1.00 X 10' 3 m3

F orce

dyne 1 dyn = 1.00 X 10" 5 N

kilogram force 1 kgf = 9.807 X 10° N
poundal 1 pdl = 1.383 X 10"‘ N
pound force (avoirdupois) 1 Ibf = 4.448 X 10° N
ounce force (avoirdupois) 1 ozf = 2.780 X 10' 1 N

P ow er

British thermal unit/second 1 Btu/s = 1.054 X 103 w

calorie/second 1 cal/s = 4.184 X 10° W
foot-pound force/second 1 ft-lbf/s = 1.356 X 10° w
horsepower (electric) 1 hp 7.46 X 102 w
horsepower (metric) (= ps) 1 ps = 7.355 X 102 w
horsepower (550 ft •Ibf/s) 1 hp = 7.457 X 102 w

Factors are given exactly or to a maximum of 4 significant figures

M u ltip ly by to o b ta in

D e n s ity

pound mass/inch3 1 lbm/in 3 = 2.768 X 104 kg/m3

pound mass/foot3 1 lbm/ft 3 = 1.602 X 101 kg/m3

E n erg y

British thermal unit 1 Btu 1.054 X 103 j

calorie 1 cal = 4.184 X 10° j
electron-volt 1 eV 1.602 X 10‘ 19 j
erg 1 erg 1.00 x io - 2 j
foot-pound force 1 ft-lb f 1.356 X 10° j
kilowatt-hour 1 kW-h 3.60 X 106 j

P ressure

newtons/metre2 1 N/m 2 1.00 Pa

atmosphere* 1 atm 1.013 X 10s Pa
bar 1 bar - 1.00 X 105 Pa
centimetres of mercury (0°C) 1 cmHg = 1.333 X 103 Pa
dyne/centimetre2 1 dyn/cm2 = 1.00 X 10" 1 Pa
feet of water (4°C) 1 ftH20 = 2.989 X 103 Pa
inches of mercury (0°C) 1 inHg = 3.386 X 103 Pa
inches of water (4°C) 1 inH20 = 2.491 X 102 Pa
kilogram force/centimetre2 1 kgf/cm2 = 9.807 X 104 Pa
pound force/foot2 1 Ib f/ft 2 = 4.788 X 10' Pa
pound force/inch2 (= psi\ b 1 lbf/in 2 6.895 X 103 Pa
torr (0°C) (= mmHg) 1 torr 1.333 X 102 Pa

V e lo c ity , a c c e le r a tio n

inch/second 1 in/s = 2.54 X 10‘ mm/s

foot/second (= fpsl 1 ft/s 3.048 X 10' 1 m/s
foot/minute 1 ft/min = 5.08 X 10‘ 3 m/s
[4.470 X 10" 1 m/s
mile/hour {= mph) 1 mile/h =-
[1.609 X 10° km/h
knot 1 knot 1.852 X 10° km/h
free fall, standard (= g) = 9.807 X 10° m/s2
foot/second2 1 -ft/s2 3.048 X 10' 1 m/s2

T e m p e r a tu r e , th e r m a l c o n d u c tiv ity , e n e r g y /a r e a - tim e

Fahrenheit, degrees — 32 °F -3 2 \ 5
°R f 9 i° c
Rankine 1 к
1 Btu-in/ft 2 -s- °F 5.189 X 102 W/m К
1 Btu/ft-s-°F = 6.226 X 10l W/m-К
1 cal/cm-s-°C = 4.184 X 102 W/m К
1 Btu/ft 2 s = 1.135 X 104 W/m2
1 cal/cm2-min = 6.973 X 102 W/m2

M is c e lla n e o u s

foot3 /second 1 f t 3/s 2.832 X 10‘ 2 m3/s

foot3 /mi nute 1 f t 3/min = 4.719 X 10-4 m3/s
rad rad = 1.00 X 10' 2 J/kg
roentgen R 2.580 X 10-4 C/kg
curie Ci 3.70 X Ю 10 disintegration/s

*atm abs: atmospheres absolute; b lbf/in 2 (g) (« psig): gauge pressure;

atm (g): atmospheres gauge. lbf/in 2 abs (- psia): absolute pressure.
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