United States Prevention, Pesticides EPA712-C-08-008
Environmental Protection And Toxic Substances October 2008
Agency (7101)
4>EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.0001
Principles and Strategies
Related to
Biodegradation Testing
of Organic Chemicals
under the Toxic
Substances Control Act
(TSCA)
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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 835.0001: Principles and strategies related to biodegradation
testing of organic chemicals under the Toxic Substances Control Act
(TSCA).
(a) Scope—(1) Applicability.. This guideline is intended for use in testing
pursuant to the Toxic Substances Control Act (TSCA) (15) U.S.C. 2601).
(2) Background The source material used in developing this harmonized
OPPTS guideline is the Revised Introduction to the Organization for Economic
Cooperation and Development (OECD) Guidelines for Testing of Chemicals, Section 3.
Part 1: Principles and strategies related to the testing of degradation of organic chemicals
(adopted 23 March 2006), available from Source OECD at http://masetto.sourceoecd.org.
(b) Overview—(1) Degradation tests that may be useful for assessment.
Information on the degradability of organic chemicals may be used for hazard assessment
or for risk assessment under TSCA. Hazard assessment or risk in general, and aquatic
hazard classification in particular, are normally based on data obtained in standardized
tests for ready biodegradability, but results of tests simulating biodegradation in water,
aquatic sediment and soil may also be used for these purposes. Other types of test data
that may be considered in an assessment of the potential environmental hazard or risk
include sewage treatment plant (STP) simulation data, inherent biodegradability,
anaerobic biodegradability, biodegradability in seawater and abiotic transformation.
(2) Other information that may be useful for assessment. In order to assess
the environmental risk of a particular chemical, information allowing the estimation of its
likely concentrations in the environment is necessary. Such an estimate should initially be
based on knowledge of the likely use and disposal patterns of the chemical, its physical-
chemical properties and the characteristics of the receiving environment.
(3) Simulation tests. Degradation of organic chemicals in the environment
influences exposure and, hence, it is a key parameter for estimating the risk of long-term
adverse effects on biota. Degradation rates, or half-lives, are preferably determined in
simulation biodegradation tests conducted under conditions that are realistic for the
particular environmental compartment (e.g. STP, surface water, sediment or soil).
Simulation tests aim at mimicking actual environmental conditions such as redox
potential, pH, temperature, microbial community, concentration of test substance and
occurrence and concentration of other substrates.
(4) Purpose of this guideline. Factors such as pH and temperature, in
combination with the intrinsic properties of the chemical, are important in determining
the environmental degradation of organic chemicals. The purpose of this guideline is to
describe the principles of different types of degradation tests and to present guidance for
the interpretation and use of degradability data.
(c) Biodegradation in water, soils and sediments—(1) Introduction, (i)
Because of the large number of chemicals that are being used in society, an approach is
needed that provides adequate knowledge for decision making as regards environmental
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protection, but which at the same time enables costs for testing to be kept to a minimum.
Ideally, a system is needed that allows preliminary screening of chemicals, using
relatively simple tests of ultimate biodegradability, with the identification of those
chemicals for which more detailed, and hence more costly, studies may be needed. It is
possible to organize the examination of the biodegradability of chemicals into a general
testing strategy, consisting of tests of varying complexity, environmental realism and
cost.
(A) First, aerobic biodegradability should be examined in a screening test for
ready biodegradability.
(B) In the case of a negative result in a test for ready biodegradability,
biodegradation of the chemical may be examined in a simulation test to obtain data to be
used for assessing the biodegradation rate in the environment or in a biological STP. This
would also be the case when a positive result for ready biodegradability has been
obtained but when a more precise biodegradation half-life or DTso is needed for risk
assessment. Alternatively, or as a supplement, a screening test for inherent
biodegradability may be conducted for generation of data describing the potential
biodegradability under optimized aerobic conditions, such as those which may potentially
occur in STPs at long sludge ages.
(C) In addition, potential biodegradability under anoxic conditions may be
examined in a screening test for anaerobic biodegradability.
(ii) Reserved.
(2) Definitions - (i) Ready biodegradability tests. (A) Stringent screening tests,
conducted under aerobic conditions, in which a high concentration of the test substance
(in the range of 2 to 100 mg/L) is used and biodegradation is measured by non-specific
parameters like Dissolved Organic Carbon (DOC), Biochemical Oxygen Demand (BOD)
and CO2 production. Domestic sewage, activated sludge or secondary effluent is the
typical source of microorganisms (inoculum) in tests for ready biodegradability. The
inoculum should not have been pre-adapted to degradation of the test substance by
previous exposure to the test substance or structurally related chemicals. A positive result
in a test for ready biodegradability can be considered as indicative of rapid and ultimate
degradation in most environments including biological STPs, where ultimate degradation
is the degradation of the substance to CO2, biomass, H2O and other inorganic substances
like NH3.
(B) A chemical attaining the pass level in these tests at a certain rate after
termination of the lag phase may be classified as readily biodegradable. The pass level
depends on the analytical parameter measured.
(ii) Simulation tests. (A) Aerobic and anaerobic tests that provide data for
biodegradation under specified environmentally relevant conditions. These tests simulate
degradation in a specific environment by use of indigenous biomass, media, relevant
solids (i.e. soil, sediment, activated sludge or other surfaces) to allow sorption of the
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chemical, and a typical temperature which represents the particular environment. A low
concentration of test substance is used in tests designed to determine the biodegradation
rate constant whereas higher concentrations are normally used for identification and
quantification of major transformation products for analytical reasons.
(B) A low concentration of chemical in these types of tests refers to a
concentration (e.g. less than 1 ug/L to 100 ug/L), which is low enough to ensure that the
biodegradation kinetics obtained in the test reflect those expected in the environment
being simulated. Biodegradation is measured either by radiolabelling techniques or by
specific chemical analyses. Tests of these types may be subdivided according to the
environment that they are designed to simulate, e.g.: a) soil, b) aquatic sediments, c)
surface water and d) STPs.
(iii) Inherent biodegradability tests. (A) Aerobic tests that possess a high
capacity for degradation to take place, and in which biodegradation rate or extent is
measured. The test procedures allow prolonged exposure of the test substance to
microorganisms and a low ratio of test substance to biomass, which offers a better chance
to obtain a positive result compared to tests for ready biodegradability. Some of these
tests may be conducted using microorganisms that have previously been exposed to the
test substance, which frequently results in adaptation leading to a significant increase of
the degradation rate.
(B) A substance yielding a positive result in a test of this type may be classified
as inherently biodegradable, which, preferably, should be qualified by one of the terms
with pre-adaptation or without pre-adaptation as appropriate. Because of the favorable
conditions employed in these tests, rapid biodegradation in the environment of inherently
biodegradable chemicals cannot generally be assumed.
(iv) Anaerobic biodegradability screening tests. Screening tests, conducted
under anoxic conditions, in which a high concentration of the test substance (mg/L) is
used and biodegradation is measured by non-specific parameters like total inorganic
carbon (TIC), CC>2 and CIL; production. These tests are used for the evaluation of
potential anaerobic biodegradability in an anaerobic digester at a given range of
concentration of microorganisms.
(v) Monod kinetics. The rate of degradation of the test substance in a laboratory
study where the substance is the sole source of carbon and energy may be described by: -
dS/dt = [(umax/Y)BS]/[Ks + S]; where: -dS/dt is the degradation rate, umax is the maximum
specific growth rate, Y is the yield coefficient, B is the biomass concentration, Ks is the
half saturation constant, and S is the concentration of the test substance.
(vi) Pseudo-first order. The rate of degradation is proportional to the
concentration of the test substance and biomass, i.e.: -dS/dt = ki SB; where ki is the first
order rate constant. It is assumed that the concentration of biomass (B) is constant during
the experiment.
(vii) First order kinetics. I.e.-dS/dt = ki S, may be used when the degradation of
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the test substance is independent of the concentration of biomass.
(viii) Half-life, (to.s(d)) is characteristic of the rate of a first-order reaction and
corresponds to the time interval for the concentration to decrease by a factor of two.
(ix) 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 tO.5 when
transformation does not follow first order kinetics.
(3) Ready biodegradability tests, (i) Ready biodegradability tests are designed
so that positive results are unequivocal. Given a positive result in a test of ready
biodegradability, it may be assumed that the chemical will undergo rapid and ultimate
biodegradation in the environment. In such cases, no further investigation of the
biodegradability of the chemical, or of the possible environmental effects of
transformation products, is normally required. However, the fact that the chemical is
found to be readily biodegradable does not preclude concern about biodegradation rate
constants and the transformation products in cases of high influx into a receiving
environment. Realizing that ready biodegradability tests may sometime fail because of
the stringent test conditions, consistent positive test results from test(s) should generally
supersede negative test results. However, when conflicting test results are reported, it is
recommended to check the origin of the inoculum in order to check whether or not
differences in the adaptation of the inoculum may be the reason.
(ii) When the risk of adverse effects cannot be excluded, as is the case for some
high production volume chemicals, it is recommended that the biodegradation rate of the
parent substance in a relevant simulation test be determined. If necessary, a risk
assessment including the parent substance and possible major transformation products
may be performed.
(iii) A negative result in a test for ready biodegradability does not necessarily
mean that the chemical will not be degraded under relevant environmental conditions, but
it means that the next level of testing, i.e. either a simulation test or an inherent
biodegradability test, should be considered.
(iv) The OECD tests that can be used to determine the ready biodegradability of
organic chemicals include the six test methods described in the OECD Test Guidelines
No. 301 A-F: DOC Die-Away Test (TG 301 A), CO2 Evolution Test (TG 301 B),
Modified MITI Test (I) (TG 301 C), Closed Bottle Test (TG 301 D), Modified OECD
Screening Test (TG 301 E) and Manometric Respirometry Test (TG 301 F). The pass
levels in this paragraph, obtained within 28 days, may be regarded as evidence of ready
biodegradability: 70% DOC removal (TG 301 A and TG 301 E); 60% theoretical carbon
dioxide (ThCO2) (TG 301 B); 60% theoretical oxygen demand (ThOD) (TG 301 C, TG
301 D and TG 301 F). Suggestions to decrease the pass level of the respirometric tests of
TG301 from 60% to 50% have been put forward by contract laboratories and in the
literature. Such a change has, however, not yet taken place.
(v) These pass levels have to be reached in a 10-day window within the 28-day
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period of the test. The 10-day window does not apply to TG 301 C. The 10-day window
begins when the degree of biodegradation has reached 10% DOC removal, ThOD or
ThCO2 and has to end before or at day 28 of the test. The pass levels of either 60% ThOD
(or ThCO2) or 70% DOC removal practically represent complete ultimate degradation of
the test substance, as the remaining fraction of 30-40% of the test substance is assumed to
be assimilated by the biomass or present as products of biosynthesis.
(vi) Another test for ready biodegradability, which represents an alternative to the
CO2 Evolution Test (TG 301 B), is the Headspace Test (Ready Biodegradability - CO2 in
sealed vessels; TG 310). In this test, the CO2 evolution resulting from the ultimate
aerobic biodegradation of the test substance is determined by measuring the inorganic
carbon (1C) produced in sealed test bottles, and the pass level has been defined as 60% of
theoretical maximum 1C production (ThIC).
(vii) As all ready biodegradability tests (see paragraphs (c)(3)(iv) and (c)(3)(vi)
of this guideline) pertain to conditions in fresh waters, screening test procedures suitable
for marine environments have also been developed: The OECD TG 306 on
Biodegradability in Seawater includes seawater variants of the Closed Bottle Test (TG
301 D) and of the Modified OECD Screening Test (TG 301 E). Degradation of organic
chemicals in seawater has generally been found to be slower than that in freshwater,
activated sludge and sewage effluent, and, therefore, a positive result obtained during 28
(Closed Bottle Method) or 60 days (Shake Flask Method) in the biodegradability in
Seawater test can be regarded as evidence of a chemical's potential for biodegradation in
the marine environment. For example, a result of >20% ThOD or DOC removal is
indicative of potential for primary biodegradation in the marine environment, whereas a
result of >60% ThOD or 70% DOC removals is indicative of potential for ultimate
biodegradation in the marine environment.
(viii) Test guidelines for ready biodegradability and biodegradation in marine
environments (see paragraph (c)(3)(vii) of this guideline) are similar in several respects:
in all the tests, the test substance providing the sole source of organic carbon (except for
carbon associated with the biomass) is diluted in a test medium containing a relatively
low concentration of biomass. In all the tests, a non-specific analytical method is used to
follow the course of biodegradation. This has the advantage that the methods are
applicable to a wide variety of organic substances and there is no need to develop specific
analytical procedures. Since these methods also respond to any biodegradation residues
or transformation products, an indication of the extent of ultimate biodegradation is
provided.
(ix) The standardized test duration is 28 days although tests may be prolonged
beyond 28 days if the biodegradation has started but not yet reached a plateau. However,
only the extent of biodegradation achieved within 28 days should be used for the
evaluation of ready biodegradability. Degradation after 28 days would allow the test
substance to be classified as inherently biodegradable (see paragraph (c)(5)(iii) of this
guideline.
(x) It has been recognized that standardization of the inoculum might also
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improve the comparability of the methods. However, it was concluded that this is not
possible without significantly reducing, at the same time, the number of species present
in the test system. A mixed inoculum is therefore test is per definition no longer a test for
ready biodegradability, and a positive result may then be used to classify the test substance
as inherently biodegradable with pre-adaptation (see paragraph (c)(2)(iii)(B) of this
guideline). An OECD inter-laboratory comparison exercise (ring test) (see paragraph
(e)(l) of this guideline) took place in 1988 in order to ensure the practicability and
validity of the tests.
(4) Simulation tests—(i) Objective. Simulation tests aim at assessing the rate
and extent of biodegradation in a laboratory system designed to represent either the
aerobic treatment stage of STP or environmental compartments, such as fresh or marine
surface water.
(ii) Sewage treatment (A) The fate of chemicals in STPs can be studied in the
laboratory by using the Simulation Test - Aerobic Sewage Treatment: Activated Sludge
Units (TG 303 A) and Biofilms (TG 303 B). The removal of the test substance is
determined by monitoring the changes in DOC and/or Chemical Oxygen Demand (COD).
The basic test procedures (TG 303 A and TG 303 B) recommend addition of the test
substance at a concentration of DOC between 10 mg/L and 20 mg/L. However, many
chemicals are normally present at very low concentrations, even in waste water, and
procedures for testing biodegradation at suitably low concentrations (<100 |ig/L) are
presented in Annex 7 to the TG 303 A.
(B) No specific pass levels have been defined for the elimination of chemicals in
aerobic sewage treatment simulation tests. It is noted that such a level would have to be
related to the specific operating conditions and plant design. The test results may be used
to estimate the removal in STPs, and the resulting effluent concentrations for prediction
of the concentration in the treatment plant and the receiving aquatic environment.
(C) Monod kinetics (originally proposed for pure cultures and single substrate
systems only) may be used for description of the degradation of a substance when it is
assumed that growth is a continuous process, and that the biomass is produced during
utilization of the test substance. It follows from Monod kinetics that the effluent
concentration is independent of the influent concentration, whereas this is not the case
where the test substance serves as a secondary substrate for the degrading biomass. The
presence of the test substance as a secondary substrate represents a low concentration
scenario, which implies that the degradation rate may be expressed by pseudo-first order
or first order kinetics (see paragraphs (e)(2) and (e)(3) of this guideline).
(iii) Soil, sediment and water. (A) The following tests can be used to
simulate the biodegradation of organic chemicals under environmentally realistic
conditions in soil, sediment or surface water: Aerobic and Anaerobic Transformation in
Soil (TG 307); Aerobic and Anaerobic Transformation in Aquatic Sediment Systems (TG
308); and Aerobic Mineralization in Surface Water - Simulation Biodegradation Test
(TG 309).
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(B) Aerated soils are aerobic, whereas water-saturated or water-logged soils are
frequently dominated by anaerobic conditions. The surface layer of aquatic sediments can
be either aerobic or anaerobic, whereas the deeper sediment is usually anaerobic. These
conditions in soil or sediment may be simulated by using aerobic or anaerobic tests
described in the test guidelines (TG 307 and TG 308).
(C) Generally, a low concentration of the test substance is used in tests designed
to determine biodegradation. A low concentration in these types of tests means a
concentration (e.g. from 1 |ig/L to 100 |ig/L in TG 309), which is low enough to ensure
that the biodegradation kinetics (first order or pseudo-first order) obtained in the test
reflect those expected in the environment.
(D) The temperature dependence of the kinetic constants should be considered. It
is recommended to perform the test at a temperature characteristic of the environment
that is simulated.
(E) When using radiolabelled chemicals, the label should be located in the most
recalcitrant part of the molecule when total mineralization is assessed. If the most stable
structure does not include the functional or environmentally relevant part of the molecule,
it may be appropriate to use a test chemical with a different labeling.
(F) Measuring disappearance of the parent compound by chemical analysis does
not imply mineralization. Simulation tests are especially useful if it is known from other
tests that the test substance can be mineralized and that the degradation, which is
measured, covers the rate determining process.
(G) The results of simulation tests may include first order or pseudo-first order
rate constant; degradation half-life or DT50 half-saturation constant; maximum specific
growth rate; fraction of mineralized label, and, if specific analyses are used, the final
level of primary degradation; mass balance during and at the end of the study;
identification and concentration of major transformation products, where appropriate; and
a proposed pathway of transformation, where appropriate. For a complete overview of the
result parameters in relation to the simulation degradation test guidelines please refer to
these guidelines (TG 303, TG 304, TG 307, TG 308 and TG 309).
(5) Inherent biodegradability tests, (i) Using favorable conditions, the
tests of inherent biodegradability have been designed to assess whether the chemical has
any potential for biodegradation under aerobic conditions. Tests for inherent
biodegradability vary in their degradation capacity (see paragraph (c)(5)(ii) of this
guideline), and the differences in test conditions should be considered if the results are
used as an indication of potential biodegradation or environmental persistence. Inherent
biodegradability can be measured by specific analysis (primary biodegradation) or by
non-specific analysis (ultimate biodegradation).
(ii) The tests that can be used to determine the inherent biodegradability of
organic chemicals include three methods described in the OECD Test Guidelines No. 302
A-C: Modified SCAS Test (TG 302 A), Zahn-Wellens/EMPA Test (TG 302 B) and
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Modified MITI Test (II) (TG 302 C). The biodegradation capacity in these tests generally
increases in the order TG 302 C < TG 302 B < TG 302 A.
(iii) Since inherent biodegradability can be considered to be a specific property of
a chemical, it is not necessary to define limits on test duration or biodegradation rates.
Biodegradation above 20% of theoretical (measured as BOD, DOC removal or COD)
may be regarded as evidence of inherent, primary biodegradability, whereas
biodegradation above 70% of theoretical (measured as BOD, DOC removal or COD)
may be regarded as evidence of inherent, ultimate biodegradability. When results of
ready biodegradability tests indicate that the pass level criterion is almost fulfilled (i.e.
ThOD or DOC slightly below 60% or 70% respectively), such results can be used to
indicate inherent biodegradability. This is also the case when the pass level criterion is
fulfilled but the 10-day window criterion is not. Such application of ready
biodegradability tests, which may include their incubation beyond 28 days, may in some
cases eliminate the need for additional testing of biodegradability in inherent or
simulation tests.
(iv) When the results indicate that inherent, ultimate biodegradability does occur,
it indicates that the substance has a potential for degradation under favorable conditions,
e.g. in well-operated STPs. When a negative result is obtained in a test of inherent
biodegradability, it may lead to a preliminary conclusion of environmental persistence
and to an evaluation of potential adverse effects of transformation products. An
alternative is to examine ultimate biodegradation at environmentally realistic low
concentrations of the chemical in a simulation test.
(6) Anaerobic biodegradability screening tests, (i) The potential anaerobic
biodegradability of organic chemicals under methanogenic conditions can be determined
by using OECD 311, Anaerobic Biodegradability of Organic Compounds in Digested
Sludge/By Measurement of Gas Production. The test substance, which is the sole added
organic carbon in the test, is exposed to diluted anaerobically digested sludge of a
relatively low concentration. Biodegradability of the test substance is followed by
measurements of the increase in headspace pressure resulting from the evolution of CO2,
CH4 and TIC.
(ii) A test duration of 60 days is recommended but the test may be prolonged
beyond 60 days or terminated earlier if degradation has reached a plateau, indicating a
sufficient degree of biodegradation (>60% of theoretical gas production). No formal
decisions on criteria for anaerobic biodegradability have been made but, tentatively, the
lowest value (60%) for ready aerobic biodegradability (60% ThOD or 60% ThCO2) has
been adopted.
(iii) OECD 311 is designed to assess the ultimate anaerobic biodegradability of
organic chemicals in heated digesters for anaerobic sludge treatment. The test is,
therefore, not necessarily applicable to environmental compartments such as anoxic
sediments and soils.
(7) Interpretation of results—(i) Ready biodegradability tests. (A) In
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order to interpret the results of a test, the full biodegradation curve should be considered
so that the duration of the lag phase, slope and plateau level can be identified. The
duration of 28 days in the ready biodegradability tests was defined in order to allow for
sufficient time for the microorganisms to adapt to the chemical (lag phase) by an increase
in the number of active degrading microorganisms that results in detectable degradation.
(B) While the test duration of 28 days allows time for adaptation, chemicals that
degrade slowly after a short lag phase should not be considered as readily biodegradable
in tests employing the 10-day window (the 10-day window does not apply to TG 301 C).
The 10-day window is the 10-day period that starts when biodegradation exceeds 10%. A
chemical can only be described as readily biodegradable if the pass level for ready
biodegradability is reached within the 10-day window.
(C) Although these tests are intended for pure chemicals, it is sometimes relevant
to examine the ready biodegradability of mixtures of structurally similar chemicals like
oils and surface-active substances (surfactants). Such substances often occur as mixtures
of constituents with different chain lengths, degree and/or site of branching or stereo-
isomers, even in their most purified commercial forms. Testing of each individual
component may be costly and impractical. If a test on the mixture is performed and it is
anticipated that a sequential biodegradation of the individual structures is taking place,
then the 10-day window should not be applied to interpret the results of the test. A case
by case evaluation should however take place on whether a biodegradability test on such
a complex mixture would give valuable information regarding the biodegradability of the
mixture as such (i.e. regarding the degradability of all the constituents) or whether instead
an investigation of the degradability of carefully selected individual components of the
mixture is would be more useful.
(D) It should be noted that such mixtures are here regarded as technical materials
of similar types of chemicals (e.g. homologues of surfactants composed of fatty alcohols
of varying chain length, or poly(oxyalkylene) polyol materials having defined molecular
weight distributions). Tests for ready biodegradability are not generally applicable for
complex mixtures containing different types of chemicals.
(E) The results of a ready biodegradability test may be used for aquatic hazard
classification of chemicals. According to the principles described in the "Harmonized
Integrated Classification System for Human Health and Environmental Hazards of
Chemical Substances and Mixtures" (see paragraph (e)(4) of this guideline), a positive
result in one of the OECD tests for ready biodegradability can be considered as indicative
of rapid degradation in most environments. Positive results obtained by the TG 306,
which is more suitable for marine environments, can also be considered as evidence of
rapid degradability.
(F) Results from ready biodegradability tests may be used for assessment of
biodegradation in a specific environmental compartment, when no data from tests
simulating the conditions in that compartment are available (see paragraphs (e)(5) and
(e)(6) of this guideline). First order rate constants may be derived with the purpose of
modeling biodegradation in STPs, surface water, sediment and soil by using pragmatic
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principles (see examples in paragraphs (c)(7)(i)(G) and (c)(7)(i)(H) of this guideline).
(G) For example, the European Commission Technical Guidance Document on
Risk Assessment (see paragraph (e)(5) of this guideline) prescribes that a rate constant
(k) of 1.0 hour"1, and a half-life of 0.69 hours, may be assigned to readily biodegradable
chemicals (fulfilling the pass level and the 10-day window) in models for estimating the
elimination of chemicals in STPs (STP models). A lower rate constant of 0.3 hour"1,
equivalent to a half-life of 2.3 hours, may be used in STP models, if a chemical reaches the
pass level during the 28-day period, but fails the 10-day window (see paragraph (e)(5) of
this guideline).
(H) The same objective is addressed in a U.S. Environmental Protection Agency
guidance document describing the use of biodegradability data for multimedia models
and STP models (see paragraph (e)(6) of this guideline). This document describes how
results of ready biodegradability tests may be used to derive activated sludge half-lives as
indicated in this paragraph:
Readily degradable chemicals: 1 hour (k = 0.69 hour"1);
Chemicals attaining 40% degradation: 3 hours (k = 0.23 hour"1);
Chemicals attaining 20 but <40% degradation: 10 hours (k = 0.069 hour"1).
(I) If the biodegradability of the chemical does not reach the pass level, it is
recommended to examine whether it was inhibitory to microbial activity at the
concentration used in the test. If the test substance was inhibitory, it may be re-tested at
low, non-inhibitory concentrations in a relevant simulation test (TG 303, TG 307, TG 308
or TG 309). Re-testing in a modified ready biodegradability test at a much lower
concentration (i.e. more than 10 times lower than prescribed) cannot generally be
recommended, as suitable simulation test methods are available (see paragraphs (e)(4)
and (e)(5) of this guideline).
(ii) Simulation tests. (A) In simulation tests, a chemical that fails to meet
the criteria for ready biodegradability, or even inherent biodegradability, may be rapidly
degradable when present at low concentrations in the environment. Simulation tests may
be used to examine the biodegradation of organic chemicals in STPs (TG 303 A and TG
303 B), soil (TG 307), aquatic sediment (TG 308) and surface water (TG 309). If it can
be demonstrated that the chemical is ultimately degraded by more than 70% in 28 days
under realistic conditions in the aquatic environment (e.g. by using TG 308 or TG 309),
then the definition of rapid degradability in relation to aquatic hazard classification will
have been met (see paragraph (e)(4) of this guideline).
(B) The results of a simulation test may show a rapid transformation of the parent
compound, whereas ultimate degradation, measured by e.g. CC>2 gas production, is
limited due to the formation of recalcitrant transformation products. It is, therefore,
necessary to distinguish between primary and ultimate biodegradation, when the rate and
extent of degradation are calculated.
(C) If first-order kinetics are assumed, which is reasonable at the low substance
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concentrations prevailing in most aquatic environments, the definition of rapid
degradability in relation to aquatic hazard classification (see paragraph (e)(4) of this
guideline) will have been satisfied when a chemical is ultimately degraded in a
simulation test with a half-life of less than 16 days. Results of aquatic simulation tests
(e.g. TG 308, TG 309) may be used directly for aquatic hazard classification purposes,
when realistic environmental conditions are simulated; i.e. when the following conditions
are met: the substance concentration is realistic for the general aquatic environment
(often in the low |ig/L or |ig/kg range); the inoculum is from a relevant aquatic
environment; the inoculum concentration is realistic (e.g. 103-106 cells/mL in surface
water); the temperature is realistic (e.g. 5°C to 25°C); and ultimate degradation is
determined (i.e. determination of the mineralization rate or the individual degradation
rates of all relevant transformation products).
(D) If no data are available from aquatic simulation or screening tests, the
degradation rate of a chemical in surface water may be estimated by using results of a
simulation test for degradation in soil (e.g. TG 307). In relation to aquatic hazard
classification, a chemical may be considered rapidly degradable in the aquatic
environment, if it is ultimately degraded in soil with a half-life of less than 16 days
provided that no pre-exposure has taken place and that a realistic concentration of the test
chemical has been employed (see paragraph (e)(4) of this guideline). In relation to risk
assessment, similar approaches have been proposed for extrapolation of results from
biodegradation tests to rate constants in surface water, sediment and soil (see paragraph
(e)(5) of this guideline).
(E) Whenever possible, assessment of biodegradation in the environment should
be based on results from tests simulating the conditions in the relevant environmental
compartment. Degradation half-life and kinetic constants determined in a simulation test
should be corrected to the average outdoor temperature or it should be documented that
the difference between test temperature and outdoor temperature is negligible.
(F) It should be noted that results of a simulation test should only be extrapolated
to degradation in the real environment if the concentrations used in the test were in the
same order of magnitude as the concentrations expected in the environment.
(G) Man-made organic chemicals will normally be present at low concentrations
(i.e. low |ig/L level) in the environment compared to the total mass of biodegradable
carbon substrates. This implies that the anticipated biodegradation kinetics are first-order
(non-growth kinetics). If a higher concentration is used in a test (e.g. to examine
transformation products), biodegradation of the chemical will frequently support growth
of the degrading microorganisms.
(H) Degradation kinetics in soil or sediments may often deviate from first-order
kinetics because sorption/desorption processes take place simultaneously with
degradation processes. In such cases expert judgment should be applied in estimating a
degradation half-life or half-lives for various sub-compartments (see paragraph (e)(7) of
this guideline).
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(I) When results from more than one simulation test are available, a suitable half-
life or DT50 in the higher end of the observed range should be used for estimating
environmental degradation, taking into account the realism, relevance, quality and
documentation of the studies in relation to the environmental conditions (see paragraph
(e)(5) of this guideline).
(iii) Inherent biodegradability tests. (A) Inherent biodegradability tests are
used to assess whether a chemical has any potential for biodegradation. The European
Commission Technical Guidance Document (see paragraph (e)(5) of this guideline)
proposes that results of the Zahn-Wellens/EMPA Test (TG 302 B) and the Modified
MITI Test (II) (TG 302 C) may be used for extrapolation to a rate constant in models for
estimation of the elimination of chemicals in STPs. However, this extrapolation is only
allowed, if the inherent biodegradability tests fulfill specific criteria. The pass level of
70% degradation in the Zahn-Wellens/EMPA Test must be reached within 7 days,
including the lag-phase and the log-phase, the log-phase should be no longer than 3 days,
and the percentage removal in the test before biodegradation occurs should be below
15%. The pass level of 70% in the Modified MITI Test (II) must be reached within 14
days, including the lag-phase and the log-phase, and the log-phase should be no longer
than 3 days. During the lag-phase, growth of specific microorganisms may be
exponential, but due to their small number, it takes several doubling times before
biodegradation becomes detectable above the background value of the inoculum. During
the log-phase, exponential growth of specific microorganisms continues and the
maximum growth rate |imax remains constant. The log-phase ends when depletion of the
parent substance leads to a decrease of the growth rate below |imax.
(B) Also the approach of the U.S. Environmental Protection Agency and
Environment Canada to derive input data for multimedia models and STP models (see
paragraphs (e)(6), (e)(8) and (e)(9) of this guideline) includes principles for extrapolation
of inherent biodegradability test results to activated sludge half-lives in STP models and
surface water. A negative result in tests for inherent biodegradability may lead to a
preliminary conclusion of environmental persistence, but it should not be regarded as a
definitive evidence of persistence, as the high concentration of the test substance may
impede ultimate biodegradability by inhibiting the degrading microorganisms.
(C) If the microorganisms in the test are inhibited by the test substance, it is
recommended that ultimate biodegradability of the chemical be examined in a simulation
test by using a realistic source of inoculum, realistic temperature and a realistic low
concentration of test substance.
(iv) Anaerobic biodegradability test. (A) The screening test for potential
anaerobic biodegradability (TG 311, Anaerobic Biodegradability of Organic Compounds
in Digested Sludge/By Measurement of Gas Production) is performed at a high
incubation temperature (35°C), which approximates the temperature in heated digesters
for anaerobic sludge treatment. This temperature favors anaerobic biodegradation of
chemicals with low or moderate toxicity to anaerobic bacteria. On the other hand,
because this test uses a high concentration of test substance, negative results may be
observed for some chemicals that would otherwise be biodegradable at lower,
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presumably more realistic concentrations.
(B) The test temperature implies that the results obtained are not necessarily
representative of what might be observed in other anoxic environments such as aquatic
sediments, as does the use of digester sludge as the inoculum. If the test substance was
inhibitory in the screening test, it may be re-tested at low, non-inhibitory concentrations
in a relevant simulation test, e.g., TG 308, which can be conducted under strictly
anaerobic conditions.
(d) Abiotic transformation — (1) Introduction, (i) Chemicals in aquatic
environments, soil and air may be transformed by abiotic processes such as hydrolysis,
oxidation and photolysis. Abiotic transformation can be an important step in the pathway
for degradation of anthropogenic chemicals in the environment. In the atmosphere,
abiotic transformation (oxidation by hydroxyl radicals) is the most important process for
the complete destruction of airborne chemicals. Although abiotic transformation in water,
sediments and soil in itself usually is only primary degradation, the products formed by
such abiotic processes may be biodegraded further by microorganisms.
(ii) Generally, the most important processes for the degradation of most
chemicals in the aquatic environment are biodegradation and combined degradation by
hydrolysis and subsequent biodegradation. In aquatic systems, sediments and soil, even
slow hydrolysis or biodegradation is likely to be more important than
phototransformation, because of the limited possibility for exposure to sunlight.
Depending on environmental conditions, time of the year and latitude,
phototransformation may be an important step in the initial transformation of some
chemicals, which may lead to biodegradable transformation products.
(2) Hydrolysis, (i) Abiotic hydrolytic transformation of chemicals in aquatic
systems may be examined at pH values normally found in the environment (pH 4-9) by
use of available hydrolysis test guidelines (see paragraphs (e)(10) and (e)(ll) of this
guideline. These methods are generally applicable to chemical substances (14C-labeled or
non-labeled) for which an analytical method with sufficient accuracy and sensitivity is
available. The results of a test of hydrolysis may include repeatability and sensitivity of
the analytical methods; recoveries; mass balance during and at the end of the study (when
14C-labelled test substance is used); and half-life or
(ii) Most hydrolysis reactions follow apparent first-order reaction rates and,
therefore, half-lives are independent of the concentration. This usually permits the
extrapolation of laboratory results determined at 10"2 to 10"3 M to environmental
conditions (<10"6 M) (see paragraph (e)(12) of this guideline).
(3) Phototransformation. (i) The potential effects of solar irradiation on the
fate of chemicals in surface water may be examined by use of guidelines referenced in
paragraphs (e)(13) and (e)(14) of this guideline.
(ii) The rate of photolysis of a chemical in the environment depends on several
factors. These include the attenuation of solar light in natural water bodies and the
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intensity of solar radiation, the latter of which is itself dependent on factors such as
latitude and season. Any data on half-lives or DT50, DT75 and DT90 values should be
reported along with calculations associated with these data, and the results of any outdoor
experiments, if the latter have been conducted. Where possible, information on
transformation products should be provided as well.
(e) References.
(1) OECD (1998). OECD Ring-test of Methods for Determining Ready
Biodegradability.
(2) Berg, U.T. and N. Nyholm (1996). Biodegradability simulation studies in
semicontinuous activated sludge reactors with low (|ig/l range) and standard (ppm range)
chemical concentrations. Chemosphere 33, 711-735.
(3) Nyholm, N., F. Ingerslev, U.T. Berg, J.P. Pedersen and H. Frimer-Larsen
(1996). Estimation of kinetic rate constants for biodegradation of chemicals in activated
sludge wastewater treatment plants using short-term batch experiments and |ig/l range
spiked concentrations. Chemosphere 33, 851-864.
(4) OECD (2001). Harmonised integrated classification system for human health
and environmental hazards of chemical substances and mixtures. OECD Series on
Testing and Assessment, No. 33.
(5) European Commission (2003). Technical Guidance Document on risk
assessment in support of Commission Directive 93/67/EEC on risk assessment for new
notified substances and commission regulation (EC) No. 1488/94 on risk assessment for
existing substances and Directive 98/8/EC of the European Parliament and of the Council
Concerning the Placing of Biocidal Products on the Market.
(6) U.S. Environmental Protection Agency (2000). Interim guidance for using
ready and inherent biodegradability tests to derive input data for multimedia models and
wastewater treatment plants (WWT) models, http://www.epa.gov/oppt/exposure/pubs/
halflife.htm
(7) Wolt, J.D, H.P. Nelson Jr., C.B. Cleveland and IJ. van Wesenbeeck (2001).
Biodegradation kinetics for pesticide exposure assessment. Rev. Environ. Contam.
Toxicol. 169, 123-164.
(8) BEC (Bonnell Environmental Consulting) (2001). Conducting the multi-media
exposure assessment of new substances in Canada. Final report. Prepared under contract
for the New Substances Division, Environment Canada; Ottawa, Canada. July, 2001.
(9) Webster, E., D. Mackay, F. Wania, J. Arnot, F. Gobas, T. Gouin, J. Hubbarde
and M. Bonnell (2005). Development and application of models of chemical fate in
Canada. Final report to Environment Canada under Contribution Agreement 2004-2005.
Canadian Environmental Modelling Network, Peterborough, Ontario, Canada. March
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2005.
(10) OECD (2007). Hydrolysis as a function of pH, no. Ill, adopted 13 April
2004. Organization for Economic Cooperation and Development.
(11) U.S. Environmental Protection Agency (2007). Hydrolysis studies. OPPTS
835.2120.
(12) Mabey, W. and T. Mill (1978). Critical review of hydrolysis of organic
compounds in water under environmental conditions. J. Phys. Chem. Ref. Data 7, 383-
415.
(13) U.S. Environmental Protection Agency (2007). Direct photolysis rate in
water by sunlight. OPPTS 835.2210.
(14) U.S. Environmental Protection Agency (2007). Indirect photolysis screening
test: sunlight photolysis in waters containing dissolved humic substances. OPPTS
835.5270.
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