OECD/OCDE 307
Adopted:
24th
April 2002
1/17
OECD GUIDELINE FOR THE TESTING OF CHEMICALS
Aerobic and Anaerobic Transformation in Soil
INTRODUCTION
1. This Test Guideline is based on existing guidelines (1)(2)(3)(4)(5)(6)(7)(8)(9). The method
described in this Guideline is designed for evaluating aerobic and anaerobic transformation of chemicals in
soil. The experiments are performed to determine (i) the rate of transformation of the test substance, and
(ii) the nature and rates of formation and decline of transformation products to which plants and soil
organisms may be exposed. Such studies are required for chemicals which are directly applied to soil or
which are likely to reach the soil environment. The results of such laboratory studies can also be used to
develop sampling and analysis protocols for related field studies.
2. Aerobic and anaerobic studies with one soil type are generally sufficient for the evaluation of
transformation pathways (8)(10)(11). Rates of transformation should be determined in at least three
additional soils (8)(10).
3. An OECD Workshop on soil and sediment selection, held at Belgirate, Italy in 1995 (10) agreed,
in particular, on the number and types of soils for use in this test. The types of soils tested should be
representative of the environmental conditions where use or release will occur. For example, chemicals
that may be released in subtropical to tropical climates should be tested with Ferrasols or Nitosols (FAO
system). The Workshop also made recommendations relating to collection, handling and storage of soil
samples, based on the ISO Guidance (12). The use of paddy (rice) soils is also considered in this
Guideline.
PRINCIPLE OF THE TEST
4. Soil samples are treated with the test substance and incubated in the dark in biometer-type flasks
or in flow-through systems under controlled laboratory conditions (at constant temperature and soil
moisture). After appropriate time intervals, soil samples are extracted and analysed for the parent
substance and for transformation products. Volatile products are also collected for analysis using
appropriate adsorption devices. Using 14
C-labelled material, the various mineralisation rates of the test
substance can be measured by trapping evolved 14
CO2 and a mass balance, including the formation of soil
bound residues, can be established.
APPLICABILITY OF THE TEST
5. The method is applicable to all chemical substances (non-labelled or radiolabelled) for which an
analytical method with sufficient accuracy and sensitivity is available. It is applicable to slightly volatile,
non-volatile, water-soluble or water-insoluble compounds. The test should not be applied to chemicals
which are highly volatile from soil (e.g. fumigants, organic solvents) and thus cannot be kept in soil under
the experimental conditions of this test.
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INFORMATION ON THE TEST SUBSTANCE
6. Non-labelled or labelled test substance can be used to measure the rate of transformation.
Labelled material is required for studying the pathway of transformation and for establishing a mass
balance. 14
C-labelling is recommended but the use of other isotopes, such as 13
C, 15
N, 3
H, 32
P, may also be
informative. As far as possible, the label should be positioned in the most stable part(s) of the molecule1
.
The purity of the test substance should be at least 95 %.
7. Before carrying out a test on aerobic and anaerobic transformation in soil, the following
information on the test substance should be available:
(a) solubility in water [OECD Guideline 105] (13);
(b) solubility in organic solvents;
(c) vapour pressure [OECD Guideline 104] (13) and Henry’s law constant;
(d) n-octanol/water partition coefficient [OECD Guidelines 107 and 117] (13);
(e) chemical stability in dark (hydrolysis) [OECD Guideline 111] (13);
(f) pKa if a molecule is liable to protonation or deprotonation [OECD Guideline 112] (13).
8. Other useful information may include data on toxicity of the test substance to soil
microorganisms [OECD Guidelines 216 and 217] (13).
9. Analytical methods (including extraction and clean-up methods) for quantification and
identification of the test substance and its transformation products should be available.
REFERENCE SUBSTANCES
10. Reference substances should be used for the characterisation and/or identification of
transformation products by spectroscopic and chromatographic methods.
DEFINITIONS
11. See Annex 1.
QUALITY CRITERIA
Recovery
12. Extraction and analysis of, at least, duplicate soil samples immediately after the addition of the
test substance gives a first indication of the repeatability of the analytical method and of the uniformity of
the application procedure for the test substance. Recoveries for later stages of the experiments are given
by the respective mass balances. Recoveries should range from 90% to 110% for labelled chemicals (8)
and from 70% to 110% for non-labelled chemicals (3).
1
For example, if the test substance contains one ring, labelling on this ring is required; if the test substance contains
two or more rings, separate studies may be needed to evaluate the fate of each labelled ring and to obtain suitable
information on formation of transformation products.
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Repeatability and sensitivity of analytical method
13. Repeatability of the analytical method (excluding the initial extraction efficiency) to quantify test
substance and transformation products can be checked by duplicate analysis of the same extract of the soil,
incubated long enough for formation of transformation products.
14. The limit of detection (LOD) of the analytical method for the test substance and for the
transformation products should be at least 0.01 mg⋅kg-1
soil (as test substance) or 1% of applied dose
whichever is lower. The limit of quantification (LOQ) should also be specified.
Accuracy of transformation data
15. Regression analysis of the concentrations of the test substance as a function of time gives the
appropriate information on the reliability of the transformation curve and allows the calculation of the
confidence limits for half-lives (in the case of pseudo first order kinetics) or DT50 values and, if
appropriate, DT75 and DT90 values.
DESCRIPTION OF THE TEST METHOD
Equipment and chemical reagents
16. Incubation systems consist of static closed systems or suitable flow-through systems (7)(14).
Examples of suitable flow-through soil incubation apparatus and biometer-type flask are shown in Figures
1 and 2, respectively. Both types of incubation systems have advantages and limitations (7)(14).
17. Standard laboratory equipment is required and especially the following:
- Analytical instruments such as GLC, HPLC, TLC-equipment, including the appropriate
detection systems for analysing radiolabelled or non-labelled substances or inverse isotopes
dilution method;
- Instruments for identification purposes (e.g. MS, GC-MS, HPLC-MS, NMR, etc.);
- Liquid scintillation counter;
- Oxidiser for combustion of radioactive material;
- Centrifuge;
- Extraction apparatus (for example, centrifuge tubes for cold extraction and Soxhlet apparatus
for continuous extraction under reflux);
- Instrumentation for concentrating solutions and extracts (e.g. rotating evaporator);
- Water bath;
- Mechanical mixing device (e.g. kneading machine, rotating mixer);
18. Chemical reagents used include, for example:
NaOH, analytical grade, 2 mol • dm-3
, or other appropriate base (e.g. KOH, ethanolamine);
H2SO4, analytical grade, 0.05 mol • dm-3
;
Ethylene glycol, analytical grade;
Solid absorption materials such as soda lime and polyurethane plugs;
Organic solvents, analytical grade, such as acetone, methanol, etc.;
Scintillation liquid.
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Test substance application
19. For addition to and distribution in soil, the test substance can be dissolved in water (deionised or
distilled) or, when necessary, in minimum amounts of acetone or other organic solvents (6) in which the
test substance is sufficiently soluble and stable. However, the amount of solvent selected should not have a
significant influence on soil microbial activity (see paragraphs 8 and 44). The use of solvents which
inhibit microbial activity, such as chloroform, dichloromethane and other halogenated solvents, should be
avoided.
20. The test substance can also be added as a solid, e.g. mixed in quartz sand (6) or in a small subsample
of the test soil which has been air-dried and sterilised. If the test substance is added using a solvent
the solvent should be allowed to evaporate before the spiked sub-sample is added to the original non-sterile
soil sample.
21. For general chemicals, whose major route of entry into soil is through sewage sludge/farming
application, the test substance should be first added to sludge which is then introduced into the soil sample
(see paragraph 41).
22. The use of formulated products is not routinely recommended. However, e.g. for poorly soluble
test substances, the use of formulated material may be an appropriate alternative.
Soils
Soil selection
23. To determine the transformation pathway, a representative soil can be used; a sandy loam or silty
loam or loam or loamy sand [according to FAO and USDA classification (15)] with a pH of 5.5-8.0, an
organic carbon content of 0.5 - 2.5% and a microbial biomass of at least 1% of total organic carbon is
recommended (10).
24. For transformation rate studies at least three additional soils should be used representing a range
of relevant soils. Those soils should vary in their organic carbon content, pH, clay content and microbial
biomass (10).
25. All soils should be characterised, at least, for texture (% sand, % silt, % clay) [according to FAO
and USDA classification (15)], pH, cation exchange capacity, organic carbon, bulk density, water retention
characteristic2
and microbial biomass (for aerobic studies only). Additional information on soil properties
may be useful in interpreting the results. For determination of the soil characteristics the methods
recommended in references (16)(17)(18)(19)(20) can be used. Microbial biomass should be determined by
using the substrate-induced respiration (SIR) method (21)(22) or alternative methods (17).
Collection, handling, and storage of soils
26. Detailed information on the history of the field site from where the test soil is collected should be
available. Details include exact location, vegetation cover, treatments with chemicals, treatments with
organic and inorganic fertilisers, additions of biological materials or other contamination. If soils have
2
Water retention characteristic of a soil can be measured as field capacity, as water holding capacity or as water
suction tension (pF). For explanations see Annex 2. It should be reported in the test report whether water retention
characteristics and bulk density of soils were determined in undisturbed field samples or in disturbed (processed)
samples.
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been treated with the test substance or its structural analogues within the previous four years, these should
not be used for transformation studies (10)(12).
27. The soil should be freshly collected from the field (from the A horizon or top 20 cm layer) with a
soil water content which facilitates sieving. For soils other than those from paddy fields, sampling should
be avoided during or immediately following long periods (> 30 days) of drought, freezing or flooding (12).
Samples should be transported in a manner which minimises changes in soil water content and should be
kept in the dark with free access of air, as much as possible. A loosely-tied polyethylene bag is generally
adequate for this purpose.
28. The soil should be processed as soon as possible after sampling. Vegetation, larger soil fauna
and stones should be removed prior to passing the soil through a 2 mm sieve which removes small stones,
fauna and plant debris. Extensive drying and crushing of the soil before sieving should be avoided (12).
29. When sampling in the field is difficult in winter (soil frozen or covered by layers of snow), it may
be taken from a batch of soil stored in the greenhouse under plant cover (e.g. grass or grass-clover
mixtures). Studies with soils freshly collected from the field are strongly preferred, but if the collected and
processed soil has to be stored prior to the start of the study storage conditions must be adequate and for a
limited time only (4 ± 2°C for a maximum of three months) to maintain microbial activity3
. Detailed
instructions on collection, handling and storage of soils to be used for biotransformation experiments can
be found in (8)(10)(12)(23)(24).
30. Before the processed soil is used for this test, it should be pre-incubated to allow germination and
removal of seeds, and to re-establish equilibrium of microbial metabolism following the change from
sampling or storage conditions to incubation conditions. A pre-incubation period between 2 and 28 days
approximating the temperature and moisture conditions of the actual test is generally adequate (12).
Storage and pre-incubation time together should not exceed three months.
PERFORMANCE OF THE TEST
Test conditions
Test temperature
31. During the whole test period, the soils should be incubated in the dark at a constant temperature
representative of the climatic conditions where use or release will occur. A temperature of 20 ± 2 °C is
recommended for all test substances which may reach the soil in temperate climates. The temperature
should be monitored.
32. For chemicals applied or released in colder climates (e.g. in northern countries, during
autumn/winter periods), additional soil samples should be incubated but at a lower temperature (e.g.
10 ± 2 °C).
3
Recent research results indicate that soils from temperate zones can also be stored at –20°C for more than three
months (25)(26) without significant losses of microbial activity.
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Moisture content
33. For transformation tests under aerobic conditions, the soil moisture content4
should be adjusted to
and maintained at a pF of between 2.0 and 2.5 (3). The soil moisture content is expressed as mass of water
per mass of dry soil and should be regularly controlled (e.g. in 2 week intervals) by weighing of the
incubation flasks and water losses compensated by adding water (preferably sterile-filtered tap water).
Care should be given to prevent or minimise losses of test substance and/or transformation products by
volatilisation and/or photodegradation (if any) during moisture addition.
34. For transformation tests under anaerobic and paddy conditions, the soil is water-saturated by
flooding.
Aerobic incubation conditions
35. In the flow-through systems, aerobic conditions will be maintained by intermittent flushing or by
continuously ventilating with humidified air. In the biometer flasks, exchange of air is maintained by
diffusion.
Sterile aerobic conditions
36. To obtain information on the relevance of abiotic transformation of a test substance, soil samples
may be sterilised (for sterilisation methods see references 13 and 26), treated with sterile test substance
(e.g. addition of solution through a sterile filter) and aerated with humidified sterile air as described in
paragraph 35. For paddy soils, soil and water should be sterilised and the incubation should be carried out
as described in paragraph 38.
Anaerobic incubation conditions
37. To establish and maintain anaerobic conditions, the soil treated with the test substance and
incubated under aerobic conditions for 30 days or one half-life or DT50 (whichever is shorter) is then
water-logged (1-3 cm water layer) and the incubation system flushed with an inert gas (e.g. nitrogen or
argon)5
. The test system must allow for measurements such as pH, oxygen concentration and redox
potential and include trapping devices for volatile products. The biometer-type system must be closed to
avoid entrance of air by diffusion.
Paddy incubation conditions
38. To study transformation in paddy rice soils, the soil is flooded with a water layer of about 1-5 cm
and the test substance applied to the water phase (9). A soil depth of at least 5 cm is recommended. The
4
The soil should neither be too wet nor too dry to maintain adequate aeration and nutrition of soil microflora.
Moisture contents recommended for optimal microbial growth range from 40-60% water holding capacity (WHC)
and from 0.1-0.33 bar (6). The latter range is equivalent to a pF-range of 2.0 – 2.5. Typical moisture contents of
various soil types are given in Annex 3.
5
Aerobic conditions are dominant in surface soils and even in sub-surface soils as shown in an EU sponsored
research project [K. Takagi et al. (1992). Microbial diversity and activity in subsoils: Methods, field site, seasonal
variation in subsoil temperatures and oxygen contents. Proc. Internat. Symp. Environ. Aspects Pesticides
Microbiol., 270-277, 17-21 August 1992, Sigtuna, Sweden]. Anaerobic conditions may occur only occasionally
during flooding of soils after heavy rainfalls or when paddy conditions are established in rice fields.
OECD/OCDE 307
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system is ventilated with air as under aerobic conditions. pH, oxygen concentration and redox potential of
the aqueous layer should be monitored and reported. A pre-incubation period of at least two weeks is
necessary before commencing transformation studies (see paragraph 30).
Test duration
39. The rate and pathway studies should normally not exceed 120 days6
(3)(6)(8), because thereafter
a decrease of the soil microbial activity with time would be expected in an artificial laboratory system
isolated from natural replenishment. Where necessary to characterise the decline of the test substance and
the formation and decline of major transformation products, studies can be continued for longer periods
(e.g. 6 or 12 months) (8). Longer incubation periods should be justified in the test report and accompanied
by biomass measurements during and at the end of these periods.
Treatment and application of test substance
40. About 50 to 200 g of soil (dry weight basis) are placed into each incubation flask (see Figures 1
and 2 in Annex 4) and the soil treated with the test substance by one of the methods described in
paragraphs 19-22. When organic solvents are used for the application of the test substance, they should be
removed from soil by evaporation. Then the soil is thoroughly mixed with a spatula and/or by shaking of
the flask. If the study is conducted under paddy field conditions, soil and water should be thoroughly
mixed after application of the test substance. Small aliquots (e.g. 1 g) of the treated soils should be
analysed for the test substance to check for uniform distribution. For alternative method, see paragraph 42.
41. The treatment rate should correspond to the highest application rate of a crop protection product
recommended in the use instructions and uniform incorporation to an appropriate depth in the field (e.g.
top 10 cm layer7
of soil). For example, for chemicals foliarly or soil applied without incorporation, the
appropriate depth for computing how much chemical should be added to each flask is 2.5 cm. For soil
incorporated chemicals, the appropriate depth is the incorporation depth specified in the use instructions.
For general chemicals, the application rate should be estimated based on the most relevant route of entry;
for example, when the major route of entry in soil is through sewage sludge, the chemical should be dosed
into the sludge at a concentration that reflects the expected sludge concentration and the amount of sludge
added to the soil should reflect normal sludge loading to agricultural soils. If this concentration is not high
enough to identify major transformation products, incubation of separate soil samples containing higher
rates may be helpful, but excessive rates influencing soil microbial functions should be avoided (see
paragraphs 8 and 19).
6
Aerobic studies might be terminated much before 120 days provided that ultimate transformation pathway and
ultimate mineralisation are clearly reached at that time. Termination of the test is possible after 120 days, or when
at least 90% of the test substance is transformed, but only if at least 5% CO2 is formed.
7
Calculation of the initial concentration on an area basis using the following equation:
]/[]/[10][
]/[10]/[
]/[ 324
6
mkgdhamml
kgmghakgA
kgmgC
soil
soilsoil
••
•
=
Csoil = Initial concentration in soil [mg • kg-1
]
A = Application rate [kg • ha-1
]; 1 = thickness of field soil layer [m]; d = dry bulk density of soil [kg • m-3
].
As a rule of thumb, an application rate of 1 kg • ha-1
results in a soil concentration of approximately 1 mg • kg-1
in
a 10 cm layer (assuming a bulk density of 1 g • cm-3
).
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42. Alternatively, a larger batch (i.e. 1 to 2 kg) of soil can be treated with the test substance, carefully
mixed in an appropriate mixing machine and then transferred in small portions of 50 to 200 g into the
incubation flasks (for example with the use of sample splitters). Small aliquots (e.g. 1 g) of the treated soil
batch should be analysed for the test substance to check for uniform distribution. Such a procedure is
preferred since it allows for more uniform distribution of the test substance into the soil.
43. Also untreated soil samples are incubated under the same conditions (aerobic) as the samples
treated with the test substance. These samples are used for biomass measurements during and at the end of
the studies.
44. When the test substance is applied to the soil dissolved in organic solvent(s), soil samples treated
with the same amount of solvent(s) are incubated under the same conditions (aerobic) as the samples
treated with the test substance. These samples are used for biomass measurements initially, during and at
the end of the studies to check for effects of the solvent(s) on microbial biomass.
45. The flasks containing the treated soil are either attached to the flow-through system described in
Figure 1 or closed with the absorption column shown in Figure 2 (see Annex 4).
Sampling and measurements
46. Duplicate incubation flasks are removed at appropriate time intervals and the soil samples
extracted with appropriate solvents of different polarity and analysed for the test substance and/or
transformation products. A well-designed study includes sufficient flasks so that two flasks are sacrificed
at each sampling event. Also, absorption solutions or solid absorption materials are removed at various
time intervals (7-day intervals during the first month and after one month in 14-day intervals) during and at
the end of incubation of each soil sample and analysed for volatile products. Besides a soil sample taken
directly after application (0-day sample) at least 5 additional sampling points should be included. Time
intervals should be chosen in such a way that pattern of decline of the test substance and patterns of
formation and decline of transformation products can be established (e.g. 0, 1, 3, 7 days; 2, 3 weeks; 1, 2, 3
months, etc.).
47. When using 14
C-labelled test substance, non-extractable radioactivity will be quantified by
combustion and a mass balance will be calculated for each sampling interval.
48. In the case of anaerobic and paddy incubation, the soil and water phases are analysed together for
test substance and transformation products or separated by filtration or centrifugation before extraction and
analysis.
Optional tests
49. Aerobic, non-sterile studies at additional temperatures and soil moistures may be useful for the
estimation of the influence of temperature and soil moisture on the rates of transformation of a test
substance and/or its transformation products in soil.
50. A further characterisation of non-extractable radioactivity can be attempted using, for example,
supercritical fluid extraction.
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DATA AND REPORTING
Treatment of results
51. The amounts of test substance, transformation products, volatile substances (in % only), and nonextractable
should be given as % of applied initial amount and, where appropriate, as mg⋅kg-1
soil (based
on soil dry weight) for each sampling interval. A mass balance should be given in percentage of the
applied initial amount for each sampling interval. A graphical presentation of the test substance
concentrations against time will allow an estimation of its transformation half-life or DT50. Major
transformation products should be identified and their concentrations should also be plotted against time to
show their rates of formation and decline. A major transformation product is any product representing ≥
10% of applied dose at any time during the study.
52. The volatile products trapped give some indication of the volatility potential of a test substance
and its transformation products from soil.
53. More accurate determinations of half-lives or DT50 values and, if appropriate, DT75 and DT90
values should be obtained by applying appropriate kinetic model calculations. The half-life and DT50
values should be reported together with the description of the model used, the order of kinetics and the
determination coefficient (r2
). First order kinetics is favoured unless r2
< 0.7. If appropriate, the
calculations should also be applied to the major transformation products. Examples of appropriate models
are described in references 28 to 32.
54. In the case of rate studies carried out at various temperatures, the transformation rates should be
described as a function of temperature within the experimental temperature range using the Arrhenius
relationship of the form:
,
T
B
-AlnklnoreAk T/B
=•= −
where ln A and B are regression constants from the intercept and slope, respectively, of a best fit line
generated from linearly regressing ln k against 1/T, k is the rate constant at temperature T and T is the
temperature in Kelvin. Care should be given to the limited temperature range in which the Arrehenius
relationship will be valid in case transformation is governed by microbial action.
Test report
55. The report must include:
Test substance:
- common name, chemical name, CAS number, structural formula (indicating position of
label(s) when radiolabelled material is used) and relevant physical-chemical properties (see
paragraph 7);
- purity (impurities) of test substance;
- radiochemical purity of labelled chemical and specific activity (where appropriate);
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Reference substances:
- chemical name and structure of reference substances used for the characterisation and/or
identification of transformation products;
Test soils:
- details of collection site;
- date and procedure of soil sampling;
- properties of soils, such as pH, organic carbon content, texture (% sand, % silt, % clay), cation
exchange capacity, bulk density, water retention characteristic, and microbial biomass;
- length of soil storage and storage conditions (if stored);
Test conditions:
- dates of the performance of the studies;
- amount of test substance applied;
- solvents used and method of application for the test substance;
- weight of soil treated initially and sampled at each interval for analysis;
- description of the incubation system used;
- air flow rates (for flow-through systems only);
- temperature of experimental set-up;
- soil moisture content during incubation;
- microbial biomass initially, during and at the end of the aerobic studies;
- pH, oxygen concentration and redox potential initially, during and at the end of the anaerobic
and paddy studies;
- method(s) of extraction;
- methods for quantification and identification of the test substance and transformation products
in soil and absorption materials;
- number of replicates and number of controls.
Results:
- result of microbial activity determination;
- repeatability and sensitivity of the analytical methods used;
- rates of recovery (% values for a valid study are given in paragraph 12);
- tables of results expressed as % of applied initial dose and, where appropriate, as mg⋅kg-1
soil
(on a dry weight basis);
- mass balance during and at the end of the studies;
- characterisation of non-extractable (bound) radioactivity or residues in soil;
- quantification of released CO2 and other volatile compounds;
- plots of soil concentrations versus time for the test substance and, where appropriate, for
major transformation products;
- half-life or DT50 , DT75 and DT90 for the test substance and, where appropriate, for major
transformation products including confidence limits;
- estimation of abiotic degradation rate under sterile conditions;
- an assessment of transformation kinetics for the test substance and, where appropriate, for
major transformation products;
- proposed pathways of transformation, where appropriate;
- discussion and interpretation of results;
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- raw data (i.e. sample chromatograms, sample calculations of transformation rates and means
used to identify transformation products).
Interpretation and evaluation of results
56. Although the studies are carried out in an artificial laboratory system, the results will allow
estimation of the rate of transformation of the test substance and also of rate of formation and decline of
transformation products under field conditions (33)(34).
57. A study of the transformation pathway of a test substance provides information on the way in
which the applied substance is structurally changed in the soil by chemical and microbial reactions.
LITERATURE
(1) US- Environmental Protection Agency (1982). Pesticide Assessment Guidelines, Subdivision N.
Chemistry: Environmental Fate.
(2) Agriculture Canada (1987). Environmental Chemistry and Fate. Guidelines for registration of
pesticides in Canada.
(3) European Union (EU) (1995). Commission Directive 95/36/EC of 14 July 1995 amending Council
Directive 91/414/EEC concerning the placing of plant protection products on the market. Annex II,
Part A and Annex III, Part A: Fate and Behaviour in the Environment.
(4) Dutch Commission for Registration of Pesticides (1995). Application for registration of a pesticide.
Section G: Behaviour of the product and its metabolites in soil, water and air.
(5) BBA (1986). Richtlinie für die amtliche Prüfung von Pflanzenschutzmitteln, Teil IV, 4-1. Verbleib
von Pflanzenschutzmitteln im Boden - Abbau, Umwandlung und Metabolismus.
(6) ISO/DIS 11266-1 (1994). Soil Quality -Guidance on laboratory tests for biodegradation of organic
chemicals in soil - Part 1 : Aerobic conditions.
(7) ISO 14239 (1997). Soil Quality – Laboratory incubation systems for measuring the mineralization of
organic chemicals in soil under aerobic conditions.
(8) SETAC (1995). Procedures for Assessing the Environmental Fate and Ecotoxicity of Pesticides.
Mark R. Lynch, Ed.
(9) MAFF - Japan (2000). Draft Guidelines for transformation studies of pesticides in soil – Aerobic
metabolism study in soil under paddy field conditions (flooded).
(10) OECD (1995). Final Report of the OECD Workshop on Selection of Soils/Sediments. Belgirate,
Italy, 18-20 January 1995.
(11) Guth, J.A. (1980). The study of transformations. In Interactions between Herbicides and the Soil
(R.J. Hance, Ed.), Academic Press, 123-157.
(12) ISO 10381-6 (1993). Soil Quality - Sampling - Part 6: Guidance on the collection, handling and
storage of soil for the assessment of aerobic microbial processes in the laboratory.
307 OECD/OCDE
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(13) OECD (1993). Guidelines for the Testing of Chemicals. Paris. OECD (1994-2000). Addenda 6-11 to
Guidelines for the Testing of Chemicals.
(14) Guth, J.A. (1981). Experimental approaches to studying the fate of pesticides in soil. In Progress in
Pesticide Biochemistry. D.H. Hutson, T.R. Roberts, Eds. J. Wiley & Sons. Vol 1, 85-114.
(15) Soil Texture Classification (US and FAO systems): Weed Science, 33, Suppl. 1 (1985) and Soil Sci.
Soc. Amer. Proc. 26:305 (1962).
(16) Methods of Soil Analysis (1986). Part 1, Physical and Mineralogical Methods. A. Klute, Ed.)
Agronomy Series No 9, 2nd Edition.
(17) Methods of Soil Analysis (1982). Part 2, Chemical and Microbiological Properties. A.L. Page, R.H.
Miller and D.R. Kelney, Eds. Agronomy Series No 9, 2nd Edition.
(18) ISO Standard Compendium Environment (1994). Soil Quality - General aspects; chemical and
physical methods of analysis; biological methods of analysis. First Edition.
(19) Mückenhausen, E. (1975). Die Bodenkunde und ihre geologischen, geomorphologischen,
mineralogischen und petrologischen Grundlagen. DLG-Verlag, Frankfurt, Main.
(20) Scheffer, F., Schachtschabel, P. (1975). Lehrbuch der Bodenkunde. F. Enke Verlag, Stuttgart.
(21) Anderson, J.P.E., Domsch, K.H. (1978). A physiological method for the quantitative measurement
of microbial biomass in soils. Soil Biol. Biochem. 10, 215-221.
(22) ISO 14240-1 and 2 (1997). Soil Quality - Determination of soil microbial biomass - Part 1:
Substrate-induced respiration method. Part 2: fumigation-extraction method.
(23) Anderson, J.P.E. (1987). Handling and storage of soils for pesticide experiments. In Pesticide
Effects on Soil Microflora. L. Somerville, M.P. Greaves, Eds. Taylor & Francis, 45-60.
(24) Kato, Yasuhiro. (1998). Mechanism of pesticide transformation in the environment: Aerobic and
bio-transformation of pesticides in aqueous environment. Proceedings of the 16th
Symposium on
Environmental Science of Pesticide, 105-120.
(25) Keuken O., Anderson J.P.E. (1996). Influence of storage on biochemical processes in soil. In
Pesticides, Soil Microbiology and Soil Quality, 59-63 (SETAC-Europe).
(26) Stenberg B., Johansson M., Pell M., Sjödahl-Svensson K., Stenström J., Torstensson L. (1996).
Effect of freeze and cold storage of soil on microbial activities and biomass. In Pesticides, Soil
Microbiology and Soil Quality, 68-69 (SETAC-Europe).
(27) Gennari, M., Negre, M., Ambrosoli, R. (1987). Effects of ethylene oxide on soil microbial content
and some chemical characteristics. Plant and Soil 102, 197-200.
(28) Anderson, J.P.E. (1975). Einfluss von Temperatur und Feuchte auf Verdampfung, Abbau und
Festlegung von Diallat im Boden. Z. PflKrankh Pflschutz, Sonderheft VII, 141-146.
(29) Hamaker, J.W. (1976). The application of mathematical modelling to the soil persistence and
accumulation of pesticides. Proc. BCPC Symposium: Persistence of Insecticides and Herbicides,
181-199.
OECD/OCDE 307
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(30) Goring, C.A.I., Laskowski, D.A., Hamaker, J.W., Meikle, R.W. (1975). Principles of pesticide
degradation in soil. In “Environmental Dynamics of Pesticides”. R. Haque and V.H. Freed, Eds.,
135-172.
(31) Timme, G., Frehse, H., Laska, V. (1986). Statistical interpretation and graphic representation of the
degradational behaviour of pesticide residues. II. Pflanzenschutz - Nachrichten Bayer 39, 188-204.
(32) Timme, G., Frehse, H. (1980). Statistical interpretation and graphic representation of the
degradational behaviour of pesticide residues. I. Pflanzenschutz - Nachrichten Bayer 33, 47-60.
(33) Gustafson D.I., Holden L.R. (1990). Non-linear pesticide dissipation in soil; a new model based on
spatial variability. Environm. Sci. Technol. 24, 1032-1041.
(34) Hurle K., Walker A. (1980). Persistence and its prediction. In Interactions between Herbicides and
the Soil (R.J. Hance, Ed.), Academic Press, 83-122.
307 OECD/OCDE
14/17
ANNEX 1
DEFINITIONS
Test substance: any substance, whether the parent compound or relevant transformation products.
Transformation products: all substances resulting from biotic or abiotic transformation reactions of the test
substance including CO2 and products that are in bound residues.
Bound residues: ”Bound residues” represent compounds in soil, plant or animal, which persist in the matrix
in the form of the parent substance or its metabolite(s)/transformation products after extraction. The
extraction method must not substantially change the compounds themselves or the structure of the matrix.
The nature of the bond can be clarified in part by matrix-altering extraction methods and sophisticated
analytical techniques. To date, for example, covalent ionic and sorptive bonds, as well as entrapments,
have been identified in this way. In general, the formation of bound residues reduces the bioaccessibility
and the bioavailability significantly (1) [modified from IUPAC 1984 (2)].
Aerobic transformation: reactions occurring in the presence of molecular oxygen (3).
Anaerobic transformation: reactions occurring under exclusion of molecular oxygen(3).
Soil is a mixture of mineral and organic chemical constituents, the latter containing compounds of high
carbon and nitrogen content and of high molecular weights, animated by small (mostly micro-) organisms.
Soil may be handled in two states:
(a) undisturbed, as it has developed with time, in characteristic layers of a variety of soil
types;
(b) disturbed, as it is usually found in arable fields or as occurs when samples are taken by
digging and used in this guideline (3).
Mineralisation is the complete degradation of an organic compound to CO2 and H2O under aerobic
conditions, and CH4, CO2 and H2O under anaerobic conditions. In the context of this guideline, when 14
Clabelled
compound is used, mineralisation means extensive degradation during which a labelled carbon
atom is oxidised with release of the appropriate amount of 14
CO2 (3).
Half-life, t0.5, is the time taken for 50% transformation of a test substance when the transformation can be
described by first-order kinetics; it is independent of the concentration.
DT50 (Disappearance Time 50) is the time within which the concentration of the test substance is reduced
by 50%; it is different from the half-life t0.5 when transformation does not follow first order kinetics.
DT75 (Disappearance Time 75) is the time within which the concentration of the test substance is reduced
by 75%.
DT90 (Disappearance Time 90) is the time within which the concentration of the test substance is reduced
by 90%.
(1) DFG: Pesticide Bound Residues in Soil. Wiley – VCH (1998).
(2) T.R. Roberts: Non-extractable pesticide residues in soils and plants. Pure Appl. Chem. 56, 945-956 (IUPAC 1984).
(3) OECD Test Guideline 304 A: Inherent Biodegradability in Soil (adopted 12 May 1981).
OECD/OCDE 307
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ANNEX 2
WATER TENSION, FIELD CAPACITY (FC) AND WATER HOLDING CAPACITY (WHC)(1)
Height of Water Column
[cm] pF(a)
bar(b)
Remarks
107
7 104
Dry Soil
1.6 ë 104
4.2 16 Wilting point
104
4 10
103
3 1
6 ë 102
2.8 0.6
3.3 ë 102
2.5 0.33(c)
102
2 0.1 Range of
60 1.8 0.06 Field capacity(d)
33 1.5 0.033
10 1 0.01 WHC (approximation)
1 0 0.001 Water saturated soil
(a) pF = log of cm water column.
(b) 1 bar = 105
Pa.
(c) Corresponds to an approximate water content of 10% in sand, 35% in loam and 45% in clay.
(d) Field capacity is not constant but varies with soil type between pF 1.5 and 2.5.
Water tension is measured in cm water column or in bar . Due to the large range of suction tension it is
expressed simply as pF value which is equivalent to the logarithm of cm water column.
Field capacity is defined as the amount of water which can be stored against gravity by a natural soil 2
days after a longer raining period or after sufficient irrigation. It is determined in undisturbed soil in situ in
the field. The measurement is thus not applicable to disturbed laboratory soil samples. FC values
determined in disturbed soils may show great systematic variances.
Water holding capacity (WHC) is determined in the laboratory with undisturbed and disturbed soil by
saturating a soil column with water by capillary transport. It is particularly useful for disturbed soils and
can be up to 30 % greater than field capacity (1). It is also experimentally easier to determine than reliable
FC-values.
(1) Mückenhausen, E. (1975). Die Bodenkunde und ihre geologischen, geomorphologischen,
mineralogischen und petrologischen Grundlagen. DLG-Verlag, Frankfurt, Main.
307 OECD/OCDE
16/17
ANNEX 3
SOIL MOISTURE CONTENTS (g water per 100 g dry soil) OF VARIOUS SOIL TYPES FROM
VARIOUS COUNTRIES
Soil Moisture Content at
Soil Type Country _________________________________________
WHC1
pF = 1.8 pF = 2.5
Sand Germany 28.7 8.8 3.9
Loamy sand Germany 50.4 17.9 12.1
Loamy sand Switzerland 44.0 35.3 9.2
Silt loam Switzerland 72.8 56.6 28.4
Clay loam Brazil 69.7 38.4 27.3
Clay loam Japan 74.4 57.8 31.4
Sandy loam Japan 82.4 59.2 36.0
Silt loam USA 47.2 33.2 18.8
Sandy loam USA 40.4 25.2 13.3
1
Water Holding Capacity
OECD/OCDE 307
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ANNEX 4
Figure 1
Example of a flow-through apparatus to study transformation of chemicals in soil (1)(2)
1: needle valve 4: soil metabolism flask (waterlogged
only for anaerobic and
paddy conditions;)
7, 8: sodium hydroxide trap for
CO2 & other acidic volatiles
2: gas washing bottle containing water 5: ethylene glycol trap for organic
volatile compounds
9: flow meter.
3: ultramembrane (sterile conditions
only), pore size 0.2 µm
6: sulphuric acid trap for alkaline
volatile compounds
Figure 2
Example of a biometer-type flask for studying the transformation of chemicals in soil (3)
Soda lime to absorb CO2
Oil - treated glass wool or
polyurethane foam to absorb
organic volatiles
Soil + test substance
(1) Guth, J.A. (1980). The study of transformations. In Interactions between Herbicides and the Soil
(R.J. Hance, Ed.), Academic Press, 123-157.
(2) Guth, J.A. (1981). Experimental approaches to studying the fate of pesticides in soil. In Progress
in Pesticide Biochemistry. D.H. Hutson, T.R. Roberts, Eds. J. Wiley & Sons. Vol 1, 85-114.
(3) Anderson, J.P.E. (1975). Einfluss von Temperatur und Feuchte auf Verdampfung, Abbau und
Festlegung von Diallat im Boden. Z. PflKrankh Pflschutz, Sonderheft VII, 141-146.