United States Prevention, Pesticides EPA712-C-08-016
Environmental Protection And Toxic Substances EPA 712-C-08-017
Agency (7101) October 2008
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.4100
Aerobic Soil
Metabolism
OPPTS 835.4200
Anaerobic Soil
Metabolism
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances
(OPPTS), United States Environmental Protection Agency for use in the testing
of pesticides and toxic substances, and the development of test data to meet the
data requirements of the Agency under the Toxic Substances Control Act (TSCA)
(15 U.S.C. 2601), the Federal Insecticide, Fungicide and Rodenticide Act
(FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and
Cosmetic (FFDCA) (21 U.S.C. 346a).
OPPTS developed this guideline through a process of harmonization of
the testing guidance and requirements that existed for the Office of Pollution
Prevention and Toxics (OPPT) in Title 40, Chapter I, Subchapter R of the Code
of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) in
publications of the National Technical Information Service (NTIS) and in the
guidelines published by the Organization for Economic Cooperation and
Development (OECD).
For additional information about OPPTS harmonized guidelines and to
access this and other guidelines, please go to http://www.epa.gov/oppts and
select "Test Methods & Guidelines" on the left side menu.
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OPPTS 835.4100 Aerobic soil metabolism
OPPTS 835.4200 Anaerobic soil metabolism
(a) Scope—(1) Applicability. This guideline is intended for use in meeting testing
requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et
seq.) and for testing pursuant to the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, etseq.)
It describes procedures that, if followed, would result in data that would generally be of scientific
merit for the purposes described in paragraph (b) of this guideline.
(2) Background. The source materials used in developing this OPPTS test guideline are
OPP 162-1 Aerobic soil metabolism studies, OPP 162-2 Anaerobic soil metabolism studies, OPP
160-4 General test standards (Pesticide Assessment Guidelines, Subdivision N - Chemistry:
Environmental Fate, EPA report 540/09-82-021, October 1982); and OECD Guideline for the
Testing of Chemicals 307 Aerobic and Anaerobic Transformation in Soil (OECD, adopted April
2002).
(b) Purpose. (1) The method described in this guideline is designed for evaluating aerobic
and anaerobic transformation of chemicals in soil. The experiments are performed to determine the
rate of transformation of the test substance and the nature and rates of formation and decline of
transformation products to which plants and soil organisms may be exposed. Such studies are used
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) The method is applicable to all chemical substances (non-labeled or radiolabeled) 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.
(c) Definitions.
Aerobic transformation involves reactions occurring in the presence of molecular
oxygen (see paragraph (j)0) of this guideline)).
Anaerobic transformation involves reactions occurring under exclusion of molecular
oxygen(see paragraph (j)(l) of this guideline).
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 should 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 (see paragraph (j)(2) of this
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guideline) (modified from IUPAC 1984 (see paragraph (j)(3) of this guideline).
DTso_(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 to.s 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%.
(Disappearance Time 90) is the time within which the concentration of the test
substance is reduced by 90%.
Half-life, to.s 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.
Mineralization is the complete degradation of an organic compound to CC>2 and H^O under
aerobic conditions, and CH4, CC>2 and H2O under anaerobic conditions. In the context of this
guideline, when 14C-labeled compound is used, mineralization means extensive degradation during
which a labeled carbon atom is oxidized with release of the appropriate amount
of 14CC>2 (see paragraph (j)0) of this guideline).
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; or (b) disturbed, as it is usually found in
arable fields or as occurs when samples are taken by digging and used in this guideline (see
paragraph (j)0) of this guideline).
Test substance is any substance, whether the parent compound or relevant transformation
products.
Transformation products are all substances resulting from biotic or abiotic transformation
reactions of the test substance including CO2 and products that are in bound residues.
(d) General considerations. — (1) Principle of the test. 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 analyzed for the parent substance and for transformation
products. Volatile products are also collected for analysis using appropriate adsorption devices.
Using 14C-labeled material, the various mineralization rates of the test substance can be measured by
trapping evolved 14CC>2 and a mass balance, including the formation of soil bound residues, can be
established. Existing guidelines are found at paragraphs (j)(4) through (j)(10) of this guideline.
(2) Soils. The types of soils tested should be representative of the environmental conditons
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where release or use will occur. Aerobic and anaerobic studies with one soil type are generally
sufficient for the evaluation of transformation pathways. Rates of transformation should be
determined in at least three additional soils (see paragraphs (j)(9), (j)0 IX 0)02) of this guideline).
(3) Additional information. Before carrying out a test on aerobic and anaerobic
transformation in soil, the following information on the test substance should be available: solubility
in water; solubility in organic solvents; vapour pressure and Henry's law constant; n-octanol/water
partition coefficient; chemical stability in dark (hydrolysis); and pKa if a molecule is liable to
protonation or deprotonation.
(e) Test procedure.—(1) Test Substance, (i) Use labeled material for studying the pathway
of transformation and for establishing a mass balance. 14C-labeling is recommended but the use of
other isotopes, such as 13C, 15N, 3H, 32P, may also be informative. As far as possible, the label
should be positioned in the most stable part(s) of the molecule. For example, if the test substance
contains one ring, this ring should be labeled; if the test substance contains two or more rings,
separate studies may be necessary to evaluate the fate of each labeled ring and to obtain suitable
information on formation of transformation products. The purity of the test substance should be at
least 95 %.
(ii) 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 should be used for the characterization and/or identification of transformation products
by spectroscopic and chromatographic methods.
(2) Soils.—(i) Soil selection. (A) 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 (see paragraph (j)(13) of this guideline)) 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
(see paragraph (j)(l 1) of this guideline).
(B) 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 (see paragraph (j)(l 1) of this guideline). Soil from foreign sources may be
used, providing the foreign soil will have the same characteristics as soil in the United States
common to the proposed use area. Additional information on use of foreign soils may be obtained
from the document "Guidance for Determining the Acceptability of Environmental Fate Studies
Conducted with Foreign Soils," at the U. S. Environmental Protection Agency' s Environmental Fate
and Effects Division, Office of Pesticides (see paragraph (j)(14)).
(C) All soils should be characterized, at least, for texture (% sand, % silt, % clay) (according
to FAO and USDA classification (see paragraph (j)(13) of this guideline)), pH, cation exchange
capacity, organic carbon, bulk density, water retention characteristic 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 paragraphs
(j)(15),through (j)(20) of this guideline can be used. Microbial biomass should be determined by
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using the substrate-induced respiration (SIR) method (see paragraphs (j)(21), Q)(22) of this
guideline) or alternative methods (see paragraph (j)(16) of this guideline).
(D) 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.
(ii) Collection, handling and storage of soils. (A) 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 fertilizers,
additions of biological materials or other contamination. If soils have been treated with the test
substance or its structural analogues within the previous four years, these should not be used for
transformation studies (see paragraphs Q)(ll), G)(23) of this guideline).
(B) 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 (see paragraph (j)(23) of this guideline). Samples should be transported
in a manner which minimizes 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.
(C) 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 (see paragraph (j)(23) of this guideline).
(D) 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
should be adequate and for a limited time only (4 ± 2 °C for a maximum of three months) to
maintain microbial activity. Recent research results indicate that soils from temperate zones can also
be stored at -20 °C for more than three months (see paragraphs Q)(24), (j)(25) of this guideline)
without significant losses of microbial activity. Detailed instructions on collection, handling and
storage of soils to be used for biotransformation experiments can be found in paragraphs (j)(9),
0)(H), G)(23), (j)(26), and 0)(27) of this guideline.
(E) 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 (see paragraph (j)(23) of this guideline). Storage and pre-
incubation time together should not exceed three months.
(3) Equipment and chemical reagents, (i) Incubation systems consist of static closed
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systems or suitable flow-through systems (paragraphs (j)(8) and (j)(28) of this guideline). Examples
of suitable flow-through soil incubation apparatus and biometer-type flask are shown in Figures 1
and 2 respectively. (paragraph (e)(4)(iii) of this guideline. Both types of incubation system have
advantages and limitations (paragraphs (j)(8) and (j)(28) of this guideline).
(ii) Standard laboratory equipment is required and especially that listed in paragraphs
(e)(3)(ii)(A) through (e)(3)(ii)(I) of this guideline:
(A) Analytical instruments such as GLC, HPLC, TLC-equipment, including the appropriate
detection systems for analyzing radiolabeled or non-labeled substances or inverse isotopes dilution
method,
(B) Instruments for identification purposes (e.g. MS, GC-MS, HPLC-MS, NMR, etc.),
(C) Liquid scintillaltion counter,
(D) Oxidizer for combustion of radioactive material,
(E) Centrifuge,
(F) Extraction apparatus (for example, centrifuge tubes for cold extraction and Soxhlet
apparatus for continuous extraction under reflux),
(G) Instrumentation for concentrating solutions and extracts (e.g. rotating evaporator),
(H) Water bath,
(I) Mechanical mixing device (e.g. kneading machine, rotating mixer),
(iii) Chemical reagents used include, for example (see paragraphs (e)(3)(iii)(A) through
(A) NaOH, analytical grade, 2 mol-dm"3, or other appropriate base (e.g. KOH, ethanolamine),
(B) H2SO4, analytical grade, 0.05 mol-dm"3,
(C) Ethylene glycol, analytical grade,
(D) Solid absorption materials such as soda lime and polyurethane plugs,
(E) Organic solvents, analytical grade, such as acetone, methanol, etc.,
(F) Scintillation liquid.
(4) Application of the test substance to the soil sample, (i) For addition to and
distribution in soil, the test substance can be dissolved in water (deionized or distilled) or, when
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necessary, in minimum amounts of acetone or other organic solvents (see paragraph (j)(7) of this
guideline) 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 paragraph
(e)(4)(vii). The use of solvents which inhibit microbial activity, such as chloroform,
dichloromethane and other halogenated solvents, should be avoided.
(ii) The test substance can also be added as a solid, e.g., mixed in quartz sand (6) or in a
small sub-sample of the test soil which has been air-dried and sterilized. 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.
(iii) About 50 to 200 g of soil (dry weight basis) are placed into each incubation flask (see
Figures 1 and 2) and the soil treated with the test substance by one of the methods described in
paragraph (e)(2) of this guideline. 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 analyzed for the test substance to check for uniform distribution.
For alternative methods, see paragraph (e)(4)(v) of this guideline.
Figure 1—Example of a flow-thru apparatus to study transformation of chemicals in soil (see
paragraphs G)(12), G)(28) of this guideline).
4; soil mcl.ihnliMii ll,i\k tu.ilu- "", X, fulium tndiitxutc H.ip l"i
li>".j.Yll u|ll\ lul" JIUK'O'hu Mill ( "(>_ ,V iitlld Jv kill Vtll(lk\
paJils i. •million O
J tf.iv \\jiiluni; Ivtik1 owtiuniiijL! wak'i 5 «lli\li'iie uhcul ujp lot IMUMIIU u. ll«>\v nulci
M'klllk (.olnpiUHKls
i tllllillHCIllhlJlil' Islv'llk tiUltJIIlnlis « Mllplllllk add tljjl lui .tiljhtH.
• Hll\ I, pi'10 MA' 0 1 J.IIH Mil.illk' iilini>iHItnK
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Figure 2—Example of biometer-type flask for studying the transformation of chemicals in soil (see
paragraph (j)(21) of this guideline.
So da lime to absorb CCh
Oil - treated glass wool or
polyurethane foam to absorb
organic volatiles
Soil + test substance
(iv) 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 layer of soil). Calculate the initial concentration on an area basis using the
following equation:
sml
l[m]»104[m2/ha]»d[kgs
where
CSoii = Initial concentration in soil [mg/kg];
A = Application rate [kg/ha];
1 = thickness of field soil layer [m];
d = dry bulk density of soil [kg/m3].
As a rule of thumb, an application rate of 1 kg/ha results in a soil concentration of
approximately 1 mg/kg in a 10 cm layer (assuming a bulk density of lg/cm3). 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 paragraph (e)(4)(i) of this guideline).
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(v) Alternatively, a larger batch (i.e., 1 to2kg)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 analyzed 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.
(vi) 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.
(vii) 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.
(viii) 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.
(ix) 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 (e)(4)(iv)).
(5) Sampling and measurements, (i) Duplicate incubation flasks are removed at
appropriate time intervals and the soil samples extracted with appropriate solvents of different
polarity and analyzed 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 analyzed 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.).
(ii) When using 14C-labeled test substance, non-extractable radioactivity will be quantified by
combustion and a mass balance will be calculated for each sampling interval.
(iii) In the case of anaerobic and paddy incubation, the soil and water phases are analyzed
together for test substance and transformation products or separated by filtration or centrifugation
before extraction and analysis.
(6) Optional tests, (i) 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
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rates of transformation of a test substance and/or its transformation products in soil.
(ii) A further characterization of non-extractable radioactivity can be attempted using, for
example, supercritical fluid extraction.
(f) Test conditions.—(1) General, (i) 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.
(ii) For chemicals applied or released in colder climates (e.g., during autumn/winter
periods), additional soil samples should be incubated but at a lower temperature (e.g., 10 ± 2 °C).
(iii) Water retention of soil. Water retention characteristic of a soil can be measured as field
capacity (FC), as water holding capacity (WHC) or as water suction tension (pF). Water tension is
measured in cm water column or in bar. Due to the large range of suction tension it is expressed as
pF value which is equivalent to the logarithm of cm water column. FC 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. 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 (see paragraph (j)(19) of this guideline). It is also experimentally easier to determine
than reliable FC values. For explanation see Table 1.
Table 1. Water Tension, Field Capacity (FC) and Water Holding Capacity (WHC)
Height of Water Column(cm)
107
1.6 -104
104
103
6- 102
3.3 -102
102
60
33
10
1
pF(a)
7
4.2
4
3
2.8
2.5
2
1.8
1.5
1
0
bar(b)
104
16
10
1
0.6
0.33(c) ~\
0.1 I
0.06 {
0.033 J
0.01
0.001
Remarks
Dry Soil
Wilting point
Range of
Field capacity(d)
WHC (approximation)
Water saturated soil
(a) pF = log of cm water column.
(b) lbar=105Pa.
(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.
(iv) 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
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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 (see paragraph (j)(19) of this guideline). It is also
experimentally easier to determine than reliable FC-values.
(v) Moisture content. For transformation tests under aerobic conditions, the soil moisture
content should be adjusted to and maintained at a pF of between 2.0 and 2.5 (see paragraph (j)(4) of
this guideline). 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% WHC and from 0.1-0.33 bar (see paragraph (j)(7) of this guideline). The latter range is
equivalent to a pF range of 2.0-2.5. 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 minimize losses of test substance and/or transformation
products by volatilization and/or photodegradation (if any) during moisture addition.
(vi) For transformation tests under anaerobic and paddy incubation, the soil is water-
saturated by flooding.
(vii) 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.
(viii) To obtain information on the relevance of abiotic transformation of a test substance,
soil samples may be sterilized (for sterilization methods see paragraph (j)(25) of this guideline),
treated with sterile test substance (e.g., addition of solution through a sterile filter) and aerated with
humidified sterile air as described in paragraph (f)(l)(vii) of this guideline. For paddy soils, soil and
water should be sterilized and the incubation should be carried out as described in paragraph
(f)(l)(x) of this guideline.
(ix) 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). The test system should allow for measurements such as pH, oxygen
concentration and redox potential and include trapping devices for volatile products. The biometer-
type system should be closed to avoid entrance of air by diffusion.
(x) 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 (see paragraph (j)(10) of this guideline). A
soil depth of at least 5 cm is recommended. The system is ventilated with air as under aerobic
conditions. pH, oxygen concentration and redox potential of the aqueous layer should be monitored
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and reported. A pre-incubation period of at least two weeks is necessary before commencing
transformation studies (see paragraph (e)(2)(ii)(E) of this guideline).
(2) Test duration. The rate and pathway studies should normally not exceed 120 days (see
paragraphs (j)(4), G)(7X (j)(9) of this guideline) because thereafter a decrease of the soil microbial
activity with time would be expected in an artificial laboratory system isolated from natural
replenishment. Aerobic studies might be terminated much before 120 days provided that ultimate
transformation pathway and ultimate mineralization 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% CC>2 is formed. When necessary to characterize 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) (see paragraph (j)(9) of this guideline). Longer incubation periods
should be justified in the test report and accompanied by biomass measurements during and at the
end of these periods.
(g) Quality factors. (1) 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 labeled chemicals (see paragraph (j)(9) of this guideline) and
from 70% to 110% for non-labeled chemicals (see paragraph (j)(4) of this guideline).
(2) 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.
(3) 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.
(4) 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.
(h) Calculations, (i) The amounts of test substance, transformation products, volatile
substances (in % only), and non-extractable 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 DTso. 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.
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(ii) The volatile products trapped give some indication of the volatility potential of a test
substance and its transformation products from soil.
(iii) 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 favored unless r2 < 0.7. If
appropriate, the calculations should also be applied to the major transformation products. Examples
of appropriate models are described in paragraphs (j)(29) through (j)(33) of this guideline.
(iv) In the case of rate studies carried out at various temperatures, the transformation rate
should be described as a function of temperature within the experimental temperature range using
the Arrhenius relationship of the form:
k = A»e~BIT or lnk = lnA-—,
T
where In A and B are regression constants from the intercept and slope, respectively, of a best fit line
generated from linearly regressing In 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.
(i) Test report. The report should include:
(1) Test substance, (i) Common name, chemical name, CAS number, structural formula
(indicating position of label(s) when radiolabeled material is used) and relevant physical-chemical
properties (see paragraph (d)(3).
(ii) Purity (impurities) of test substance.
(iii) radiochemical purity of labeled chemical and specific activity (where appropriate).
(2) Reference substances. Chemical name and structure of reference substances used for the
characterization and/or identification of transformation products;
(3) Test soils, (i) Details of collection site.
(ii) Date and procedure of soil sampling.
(iii) Properties of soils, such as pH, organic carbon content, texture (% sand, % silt, % clay),
cation exchange capacity, bulk density, water retention characteristic and microbial biomass.
(iv) Length of soil storage and storage conditions (if stored).
(4) Test conditions, (i) Dates of the performance of the studies.
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(ii) Amount of test substance applied.
(iii) Solvents used and method of application for the test substance.
(iv) Weight of soil treated initially and sampled at each interval for analysis.
(v) Description of the incubation system used.
(vi) Air flow rates (for flow-through systems only).
(vii) Temperature of experimental set-up.
(viii) Soil moisture content during incubation.
(ix) Microbial biomass initially, during and at the end of the aerobic studies.
(x) pH, oxygen concentration and redox potential initially, during and at the end of the
anaerobic and paddy studies.
(xi) Method(s) of extraction.
(xii) Methods for quantification and identification of the test substance and
transformation products in soil and absorption materials.
(xiii) Number of replicates and number of controls.
(5) Results (i) Result of microbial activity determination.
(ii) Repeatability and sensitivity of the analytical methods used.
(iii) Rates of recovery (% values for a valid study are given in paragraph (g)(l).
(iv) Tables of results expressed as % of applied initial dose and, where appropriate, as mg/kg
soil (on a dry weight basis).
(v) Mass balance during and at the end of the studies.
(vi) Characterization of non-extractable (bound) radioactivity or residues in soil.
(vii) Quantification of released CC>2 and other volatile compounds.
(viii) Plots of soil concentrations versus time for the test substance and, where
appropriate, for major transformation products.
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(ix) Half-life or DTso , DTys and DTgo for the test substance and, where appropriate, for
major transformation products including confidence limits.
(x) Estimation of abiotic degradation rate under sterile conditions.
(xi) An assessment of transformation kinetics for the test substance and, where
appropriate, for major transformation products.
(xii) Proposed pathways of transformation, where appropriate.
(xiii) Discussion and interpretation of results.
(xiv) Raw data (i.e., sample chromatograms, sample calculations of transformation rates and
means used to identify transformation products).
(6) Interpretation and evaluation of results.—(i) 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
(see paragraphs G)(34), G)(35) of this guideline).
(ii) 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.
(j) References. The following references should be consulted for additional information on
this guideline:
(1) OECD (1981). OECD Guideline for the Testing of Chemicals 304A Inherent
Biodegradability in Soil ( adopted 12 May 1981).
(2) DFG: Pesticide Bound Residues in Soil. Wiley-VCH (1998).
(3) T.R. Roberts: Non-extractable pesticide residues in soils and plants. Pure Appl.
Chem. 56, 945-956 (IUPAC 1984).
(4) 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.
(5) 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.
(6) BBA (1986). Richtlinie fur die amtliche Priifung von Pflanzenschutzmitteln, Teil IV,
4-1. Verbleib von Pflanzenschutzmitteln im Boden - Abbau, Umwandlung und Metabolismus.
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(7) ISO/DIS 11266-1 (1994). Soil Quality -Guidance on laboratory tests for
biodegradation of organic chemicals in soil - Part 1: Aerobic conditions.
(8) ISO 14239 (1997). Soil Quality—Laboratory incubation systems for measuring the
mineralization of organic chemicals in soil under aerobic conditions.
(9) SET AC (1995). Procedures for Assessing the Environmental Fate and Ecotoxicity of
Pesticides. Mark R. Lynch, Ed.
(10) MAFF - Japan (2000). Draft Guidelines for transformation studies of pesticides in
soil -Aerobic metabolism study in soil under paddy field conditions (flooded).
(11) OECD (1995). Final Report of the OECD Workshop on Selection of
Soils/Sediments. Belgirate, Italy, 18-20 January 1995.
(12) Guth, J.A. (1980). The study of transformations. In Interactions between
Herbicides and the Soil (R.J. Hance, Ed.), Academic Press, 123-157.
(13) Soil Texture Classification (US and FAO systems): Weed Science, 33, Suppl. 1
(1985) and Soil Sci. Soc. Amer. Proc. 26:305 (1962)
(14) U.S. Environmental Protection Agency (2006). Guidance for Determining the
Acceptability of Environmental Fate Studies Conducted with Foreign Soils. Environmental Fate
and Effects Division, Office of Pesticide Programs, USEPA. Washington DC. This document
can be found at: http://www.epa.gov/oppefedl/ecorisk_ders/soils_foreign.htm
(15) Methods of Soil Analysis (1986). Part 1, Physical and Mineralogical Methods (A.
Klute, Ed.). Soil Science Society of America Book Series 5.
(16) Methods of Soil Analysis (1994). Part 2. Microbiological and Biochemical
Properties (R.W. Weaver, S. Angle and P. Bottomley, Eds.). Soil Science Society of America
Book Series 5.
(17) Methods of Soil Analysis (1996). Part 3. Chemical Methods (D.L. Sparks, Ed.). Soil
Science Society of America Book Series 5.
(18) ISO Standard Compendium Environment (1994). Soil Quality - General aspects;
chemical and physical methods of analysis; biological methods of analysis. First Edition.
(19) Miickenhausen, 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
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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) 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.
(24) 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).
(25) Stenberg B., Johansson M., Pell M., Sjodahl-Svensson K., Stenstrom 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).
(26) 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.
(27) Kato, Yasuhiro. (1998). Mechanism of pesticide transformation in the environment:
; and bio-transformation of pesticides in aqueous envir
Symposium on Environmental Science of Pesticide, 105-120.
Aerobic and bio-transformation of pesticides in aqueous environment. Proceedings of the 16th
(28) 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.
(29) 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.
(30) 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.
(31) 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.
(32) Timme, G., Frehse, H., Laska, V. (1986). Statistical interpretation and graphic
representation of the degradational behaviour of pesticide residues. II. Pflanzenschutz -
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Nachrichten Bayer 39, 188-204.
(33) Timme, G., Frehse, H. (1980). Statistical interpretation and graphic representation
of the degradational behaviour of pesticide residues. I. Pflanzenschutz - Nachrichten Bayer 33,
47-60.
(34) 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.
(35) Hurle K., Walker A. (1980). Persistence and its prediction. In Interactions between
Herbicides and the Soil (RJ. Hance, Ed.), Academic Press, 83-122.
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