United States Prevention, Pesticides EPA712-C-08-002
Environmental Protection And Toxic Substances October 2008
Agency (7101)
4>EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.3190
Aerobic Mineralization in
Surface Water -
Simulation
Biodegradation Test
I
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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 835.3190: Aerobic mineralization in surface water -
simulation biodegradation test.
(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. This OPPTS test guideline is based on the OECD
Guideline for the Testing of Chemicals no. 309, Aerobic Mineralization in
Surface Water - Simulation Biodegradation Test (adopted 13 April 2004),
available from Source OECD at http://masetto. sourceoecd.org..
(b) Purpose. The purpose of this test is to measure the time course of
biodegradation of a test substance at low concentration in aerobic natural water
and to quantify the observations in the form of kinetic rate expressions. This
simulation test is a laboratory shake flask batch test to determine rates of aerobic
biodegradation of organic substances in samples of natural surface water (fresh,
brackish or marine). It is based on the ISO/DIS 14592-1 (see paragraph (1)(1) of
this guideline) and it also includes elements from the OECD Guidelines 307 and
308 (see paragraphs (1)(2) and (1)(3) of this guideline). Optionally, with long test
times, semi-continuous operation replaces batch operation in order to prevent
deterioration of the test microcosm. The principal objective of the simulation test
is to determine the mineralization of the test substance in surface water, and
mineralization constitutes the basis for expressing degradation kinetics. However,
an optional secondary objective of the test is to obtain information on the primary
degradation and the formation of major transformation products. Identification of
transformation products, and if possible quantification of their concentrations, are
especially important for substances that are very slowly mineralized (e.g. with
half-lives for total residual 14C exceeding 60 days). Higher concentrations of the
test substance (e.g., >100 jig/1) should normally be used for identification and
quantification of major transformation products due to analytical limitations.
(c) General considerations. (1) A low concentration in this test means a
concentration (e.g. less than 1 |ig/l to 100 |ig/l) which is low enough to ensure
that the biodegradation kinetics obtained in the test reflect those expected in the
environment. Compared to the total mass of biodegradable carbon substrates
available in the natural water used for the test, the test substance present at low
concentration will serve as a secondary substrate. This implies that the anticipated
biodegradation kinetics are first order (non-growth kinetics) and that the test
substance may be degraded by cometabolism. First-order kinetics implies that the
rate of degradation (mg/L/day) is proportional to the concentration of substrate
which declines over time. With true first-order kinetics the specific degradation
rate constant, k, is independent of time and concentration. That is, k does not vary
appreciably during the course of an experiment and does not change with the
added concentration between experiments. By definition the specific degradation
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rate constant is equal to the relative change in concentration per time: k =
(l/C)-(dC/dt). Although first-order kinetics are normally expected under the
prescribed conditions, there may be certain circumstances where other kinetics are
more appropriate. Deviations from first-order kinetics may, for example, be
observed if mass transfer phenomena such as the diffusion rate, rather than the
biological reaction rate, limit the rate of biotransformation. However, the data can
nearly always be described by pseudo first-order kinetics accepting a
concentration-dependent rate constant.
(2) Information on biodegradability of the test substance at higher
concentrations (e.g. from standard screening tests) as well as information on
abiotic degradability, transformation products and relevant physico-chemical
properties should be available prior to the test to help in planning the experiment
and interpreting the results. The use of 14C-labeled test substances and the
determination of the phase distribution of 14C at the end of the test enable ultimate
biodegradability to be determined. When nonlabeled test substance is used,
ultimate biodegradation can only be estimated if a higher concentration is tested
and all the major transformation products are known.
(d) Definitions.
Degradation half time, DT50 (d): Term used to quantify the outcome of
biodegradation tests. It is the time interval, including the lag phase, needed to
reach a value of 50% biodegradation.
Degradation rate constant: A first-order or pseudo first-order kinetic rate
constant, k^"1), which indicates the rate of degradation processes. For a batch
experiment k is estimated from the initial part of the degradation curve obtained
after the end of the lag phase.
Dissolved organic 14C activity (DOA): The total 14C activity associated
with dissolved organic carbon.
Dissolved organic carbon (DOC)'. That part of the organic carbon in a
sample of water that cannot be removed by specified phase separation, for
example by centrifugation at 40000 ms"2 for 15 min or by membrane filtration
using membranes with pores of 0.2 jim - 0.45 jim diameter.
Functional biodegradation: The structural change (transformation) of a
chemical substance by microorganisms resulting in the loss of a specific property.
Half-life, ti/2 (d): Term used to characterize the rate of a first-order
reaction. It is the time interval that corresponds to a concentration decrease by a
factor of 2. The half-life and the degradation rate constant are related by the
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equation ti/2 = In2/k.
Lag phase: The time from the start of a test until adaptation of the
degrading microorganisms is achieved and the biodegradation degree of a
chemical substance or organic matter has increased to a detectable level (often
10% of the maximum theoretical biodegradation, but may be lower depending on
the accuracy of the measuring technique).
Limit of detection (LOD) and limit of quantification (LOQ): The limit of
detection (LOD) is the concentration of a substance below which the identity of
the substance cannot be distinguished from analytical artifacts. The limit of
quantification (LOQ) is the concentration of a substance below which the
concentration cannot be determined with an acceptable accuracy.
Maximum level of biodegradation: The degree of biodegradation of a
chemical substance or organic matter in a test, recorded in per cent, above which
no further biodegradation takes place during the test.
Mineralization: The breakdown of a chemical substance or organic matter
by microorganisms in the presence of oxygen to carbon dioxide, water and
mineral salts of any other elements present.
Paniculate organic 14C activity (POA): The total 14C activity associated
with paniculate organic carbon.
Primary substrate: A collection of natural carbon and energy sources that
provide growth and maintenance of the microbial biomass.
Primary biodegradation: The structural change (transformation) of a
chemical substance by microorganisms resulting in the loss of chemical identity.
Secondary substrate: A substrate component present in a such low
concentration, that by its degradation, only insignificant amounts of carbon and
energy are supplied to the competent microorganisms, as compared to the carbon
and energy supplied by their degradation of main substrate components (primary
substrates).
Total organic 14C activity (TOA): The total 14C activity associated with
organic carbon.
Ultimate aerobic biodegradation: The breakdown of a chemical substance
by microorganisms in the presence of oxygen to carbon dioxide, water and
mineral salts of any other elements present, with the associated production of new
biomass and organic microbial biosynthesis products.
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(e) Principle of the test. (1) The test is performed in batch by incubating
the test substance with either surface water only (pelagic test) or surface water
amended with suspended solids/sediment of 0.01 to 1 g/L dry weight (suspended
sediment test) to simulate a water body with suspended solids or resuspended
sediment. The suspended solids/sediment concentration in the lower range of this
interval is typical for most surface waters. The test flasks are incubated in
darkness at an environmental temperature under aerobic conditions and agitation.
At least two different concentrations of test substance should be used in order to
determine the degradation kinetics. The concentrations should differ from each
other by a factor of 5 to 10 and should represent the expected range of
concentrations in the environment. The maximum concentration of the test
substance should not exceed 100 |ig/L, but maximum test concentrations below
10 |ig/L are preferred to ensure that the biodegradation follows first-order
kinetics. The lowest concentration should not exceed 10 |ig/L, but lowest test
concentrations of 1-2 jig/L or less are preferred. Normally an adequate analysis of
such low concentration can be achieved by use of commercially available 14C-
labelled substances. Because of analytical limitations, it is frequently impossible
to measure the concentration of test substance with the required accuracy, if the
test substance is applied at a concentration <100 jig/L (see paragraph (h)(2)(ii) of
this guideline). Higher concentrations of test substance (>100 |ig/L and
sometimes >1 mg/L) may be used for the identification and quantification of
major transformation products or if a specific analysis method with a low
detection limit is not available. If high concentrations of test substance are tested,
it may not be possible to use the results to estimate the first-order degradation
constant and half-life, as the degradation will probably not follow first-order
kinetics.
(2) Degradation is followed at appropriate time intervals, by measuring
either the residual 14C or the residual concentration of test substance when specific
chemical analysis is used. 14C labeling of the most stable part of the molecule
ensures the determination of the total mineralization, while 14C labeling of a less
stable part of the molecule, as well as the use of specific analysis, enables the
assessment of only primary biodegradation. However, the most stable part does
not necessarily include the relevant functional moiety of the molecule (that can be
related to a specific property such as toxicity, bioaccumulation, etc.). If this is the
case, it may be appropriate to use a test substance, which is 14C-labelled, in the
functional part in order to follow the elimination of the specific property.
(f) Applicability of the test. (1) This simulation test is applicable to
nonvolatile or slightly volatile organic substances tested at low concentrations.
Using flasks open to the atmosphere (e.g. cotton wool plugged), substances with
Henry's law constants less than about 1 Pa-mVmol (approx. 10"5 atm-m3/mol) can
be regarded as nonvolatile in practice. Using closed flasks with a headspace, it is
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possible to test slightly volatile substances (with Henry's law constants <100
Pa-m3/mol or <10"3 atm-m3/mol) without losses from the test system. Loss of 14C-
labelled substances may occur, if the right precautions are not exercised, when the
CO2 is stripped off. In such situations, it may be necessary to trap CO2 in an
internal absorber with alkali or to use an external CO2 absorber system (direct
14CO2 determination; see paragraph (j)(4)(iii) of this guideline). For the
determination of biodegradation kinetics, the concentrations of the test substance
should be below its water solubility. It should be noted, however, that literature
values of water solubility may be considerably higher than the solubility of the
test substance in natural waters. Optionally, the solubility of especially poorly
water-soluble test substances may be established by use of the natural waters
being tested.
(2) The method can be used for simulating biodegradation in surface water
free of coarse particles (pelagic test) or in turbid surface water which, for
example, might exist near a water/sediment interface (suspended sediment test).
(g) Test and reference substances—(1) Test substance. Both
radiolabeled and nonlabeled test substances can be used in this test. 14C-labelling
technique is recommended and labeling should normally be in the most stable
part(s) of the molecule (see paragraph (e)(2) of this guideline). For substances
containing more than one aromatic ring, one or more carbons in each ring should
preferably be 14C-labelled. In addition, one or more carbons on both sides of
easily degradable linkages should preferably be 14C-labelled. The chemical and/or
radiochemical purity of the test substance should be >95%. For radiolabeled
substances, a specific activity of approx. 50 |iCi/mg (1.85 MBq) or more is
preferred in order to facilitate 14C measurements in tests conducted with low
initial concentrations.
(i) The following information on the test substance should be available:
(A) Solubility in water (see paragraph (1)(4) of this guideline),
(B) Solubility in organic solvent(s) (substances applied with solvent or
with low solubility in water);
(C) Dissociation constant (pKa) if the substance is liable to protonation or
deprotonation (see paragraph (1)(5) of this guideline);
(D) Vapor pressure (see paragraph (1)(6) of this guideline) and Henry's
law constant;
(E) Chemical stability in water and in the dark (hydrolysis) (see paragraph
(1)(7) of this guideline),
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(ii) When poorly water-soluble substances are being tested in seawater, it
may also be useful to know the salting out constant (or Setschenow constant) Ks,
which is defined by the expression: log (S/S') = Ks-Cm, where S and S' are the
solubility of the substance in fresh water and seawater, respectively, and Cm is the
molar salt concentration.
(iii) If the test is carried out as a suspended sediment test the following
information should also be available:
(A) n-Octanol/water partition coefficient (see paragraphs (1)(8) and (1)(9)
of this guideline);
(B) Adsorption coefficient (see paragraph (1)(10) of this guideline,
(iv) Other useful information may include::
(A) Environmental concentration, if known or estimated;
(B) Toxicity of the test substance to microorganisms (see paragraph (1)(11)
of this guideline);
(C) Ready and/or inherent biodegradability (see paragraphs (1)(12) and
(1)(13) of this guideline),
(D) Aerobic or anaerobic biodegradability in soil and sediment/water
transformation studies (see paragraphs (1)(2) and (1)(3) of this guideline).
(2) Reference substance. A substance, which is normally easily degraded
under aerobic conditions (e.g. aniline or sodium benzoate) should be used as
reference substance. The expected time interval for degradation of aniline and
sodium benzoate is usually less than 2 weeks. The purpose of the reference
substances is to ensure that the microbial activity of the test water is within certain
limits; i.e., that the water contains an active microbial population.
(h) Quality criteria—(1) Recovery. Immediately after addition of the test
substance, each initial test concentration should be verified by measurements of
14C activity, or by chemical analyses in the case of nonlabeled substances, in at
least duplicate samples. This provides information on the applicability and
repeatability of the analytical method and on the homogeneity of the distribution
of the test substance. Normally, the measured initial 14C activity or test substance
concentration rather than the nominal concentration is used in the subsequent
analyses of data, as losses due to sorption and dosing errors thereby are
compensated. For 14C-labelled test substance, the level of recovery at the end of
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the experiment is given by mass balance (see paragraph G)(4)(vi) of this
guideline). Ideally, the radiolabeled mass balance should range from 90% to
110%, whereas the analytical accuracy should lead to an initial recovery of
between 70% and 110% for nonlabeled test substances. These ranges should be
interpreted as targets and should not be used as criteria for acceptance of the test.
Optionally, the analytical accuracy may be determined for the test substance at a
lower concentration than the initial concentration and for major transformation
products.
(2) Repeatability and sensitivity of analytical method, (i) Repeatability
of the analytical method (including the efficiency of the initial extraction) to
quantify the test substance, and transformation products, if appropriate, should be
checked by five replicate analyses of the individual extracts of the surface water.
(ii) The limit of detection (LOD) of the analytical method for the test
substance and for the transformation products should be at least 1% of the initial
amount applied to the test system if possible. The limit of quantification (LOQ)
should be equal to or less than 10% of the applied concentration. The chemical
analyses of many organic substances and their transformation products frequently
require that the test substance is applied at a relatively high concentration, i.e.
>100
(i) Description of the test method — (1) Equipment. The test may be
conducted using conical or cylindrical flasks of appropriate capacity (e.g. 0.5 or
1.0 liter) closed with silicone or rubber stoppers, or in serum flasks with CCVtight
lids (e.g. with butyl rubber septa). Another option is to perform the test using
multiple flasks and to sacrifice whole flasks, at least in duplicate, at each sample
interval (see paragraph (j)(l)(iii) of this guideline). For nonvolatile test substances
that are not radiolabeled, gas-tight stoppers or lids are not required; loose cotton
plugs that prevent contamination from air are suitable (see paragraph (j)(l)(ii) of
this guideline). Slightly volatile substances should be tested in a biometer-type
system with gentle stirring of the water surface. To be sure that no bacterial
contamination occurs, optionally the vessels can be sterilized by heating or
autoclaving prior to use. In addition, the following standard laboratory equipment
is used:
(i) Shaking table or magnetic stirrers for continuous agitation of the test
flasks;
(ii) Centrifuge;
(iii) pH meter;
(iv) Turbidimeter for nephelometric turbidity measurements;
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(v) Oven or microwave oven for dry weight determinations;
(vi) Membrane filtration apparatus;
(vii) Autoclave or oven for heat sterilization of glassware;
(viii) Facilities to handle 14C-labelled substances;
14,
(ix) Equipment to quantify C-activity in samples from CO2-trapping
solutions and, if required, from sediment samples;
(x) Analytical equipment for the determination of the test (and reference)
substance if specific chemical analysis is used (e.g. gas chromatograph, high-
pressure liquid chromatograph).
(2) Stock solutions of test substance. Deionized water is used to prepare
stock solutions of the test and reference substances (see paragraph (i)(7)(i) of this
guideline). The deionized water should be free of substances that may be toxic to
microorganisms, and dissolved organic carbon (DOC) should be no more than 1
mg/L (see paragraph (1)(14) of this guideline).
(3) Collection and transport of surface water. The sampling site for
collection of the surface water should be selected in accordance with the purpose
of the test in any given situation. In selecting sampling sites, the history of
possible agricultural, industrial or domestic inputs should be considered. If it is
known that an aquatic environment has been contaminated with the test substance
or its structural analogs within the previous four years, it should not be used for
the collection of test water, unless investigation of degradation rates in previously
exposed sites is the express purpose of the investigator. The pH and temperature
of the water should be measured at the site of collection. Furthermore, the depth
of sampling and the appearance of the water sample (e.g. color and turbidity)
should be noted. Oxygen concentration and/or redox potential in water and in the
sediment surface layer should be measured in order to demonstrate aerobic
conditions unless this is obvious as judged from appearance and historic
experience with the site. The surface water should be transported in a thoroughly
cleaned container. During transport, the temperature of the sample should not
significantly exceed the temperature used in the test. Cooling to 4°C is
recommended if transport duration exceeds 2 to 3 hours. The water sample should
not be frozen.
(4) Storage and preparation of surface water. The test should
preferably be started within one day after sample collection. Storage of the water,
if needed, should be minimized and must in any case not exceed a maximum of 4
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weeks. The water sample should be kept at 4°C with aeration until use. Prior to
use, the coarse particles should be removed, for example by filtration through a
nylon filter with about 100 jim mesh size or with a coarse paper filter, or by
sedimentation.
(5) Preparation of water amended with sediment [optional]. For the
suspended sediment test, surface sediment is added to the flasks containing
natural water (filtered to remove coarse particles as described in paragraph (i)(4)
of this guideline) to obtain a suspension; the concentration of suspended solids
should be between 0.01 and 1 g/L. The surface sediment should come from the
same site as that from which the water sample was taken. Depending on the
particular aquatic environment, the surface sediment may either be characterized
by a high organic carbon content (2.5-7.5%) and a fine texture or by a low organic
carbon content (0.5-2.5%) and a coarse texture (see paragraph (1)(2) of this
guideline). The surface sediment can be prepared as follows: extract several
sediment cores using a tube of transparent plastic, slice off the upper aerobic
layers (from surface to a depth of max. 5 mm) immediately after sampling and
pool them together. The resulting sediment sample should be transported in a
container with a large air headspace to keep the sediment under aerobic conditions
(cool to 4°C if transport duration exceeds 2-3 hours). The sediment sample should
be suspended in the test water at a ratio of 1:10 and kept at 4°C with aeration until
use. Storage of the sediment, if needed, should be minimized and should not in
any case exceed a maximum of 4 weeks.
(6) Semi-continuous procedure [optional], (i) Prolonged incubation for
up to several months may be required in order to achieve a sufficient degradation
of recalcitrant substances. The duration of the test should normally not exceed 60
days unless the characteristics of the original water sample are maintained by
renewal of the test suspension. However, the test period may be extended to a
maximum of 90 days without renewal of the test suspension, if the degradation of
the test substance has started within the first 60 days.
(ii) During incubation for long periods, the diversity of the microbial
community may be reduced due to various loss mechanisms and due to possible
depletion of the water sample of essential nutrients and primary carbon substrates.
It is therefore recommended that a semi-continuous test is used to adequately
determine the degradation rate of slowly degrading substances. The test should be
initiated by use of the semi-continuous procedure if, based on previous
experience, an incubation period of three months is expected to be necessary to
achieve 20% degradation of the substance. Alternatively, the normal batch test
may be changed into a semi-continuous test, if no degradation of the test
substance has been achieved during approximately 60 days of testing using the
batch procedure. The semi-continuous procedure may be stopped and the test
continued as a batch experiment, when a substantial degradation has been
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recorded (e.g. >20%).
(iii) In the semi-continuous test, every two weeks, about one third of the
volume of the test suspension is replaced by freshly collected water with the test
substance added to the initial concentration. Sediment is likewise added to the
replacement water to the initial concentration (between 0.01 and 1 g/1), if the
optional suspended sediment test is performed. When carrying out the test with
suspended sediment solids, it is important that the sediment remain suspended
during water renewal, and that the residence time is identical for solids and water,
as otherwise the intended similarity to a homogenous aqueous system with no
fixed phases can be lost. For these reasons, an initial concentration of suspended
sediment in the lower range of the specified interval is preferred when the semi-
continuous procedure is used.
(iv) The prescribed addition of test substance implies that the initial
concentration of test substance is not exceeded by the partial renewal of the test
suspension and, hence, the adaptation, which is frequently seen with high
concentrations of a test substance, is avoided. As the procedure comprises both a
re-inoculation and a compensation of depleted nutrients and primary substrates,
the original microbial diversity is restored, and the duration of the test can be
extended to infinity in principle. When the semi-continuous procedure is used, it
is important to note that the residual concentration of the test substance should be
corrected for the amounts of test substance added and removed at each renewal
procedure. The total and the dissolved test substance concentration can be used
interchangeably for compounds that sorb little. Sorption is generally insignificant
(<5%) under the specified conditions (0.1-1 g solids/1) for substances of log Kow
<3 (valid for neutral compounds that sorb primarily by hydrophobic partitioning).
(7) Addition of the test (or reference) substance, (i) For substances with
sufficient water solubility (>1 mg/L) and low volatility (Henry's law constants <1
Pa-mVmol or <10"5 atm-m3/mol), a stock solution can be prepared in deionized
water (see paragraph (i)(2) of this guideline); the appropriate volume of the stock
solution is added to the test vessels to achieve the desired concentration. The
volume of any added stock solution should be held to the practical minimum
(<10% of the final liquid volume, if possible). Another procedure is to dissolve
the test substance in a larger volume of the test water, which may be seen as an
alternative to the use of organic solvents.
(ii) If unavoidable, stock solutions of nonvolatile substances with poor
water solubility should be prepared by use of a volatile organic solvent, but the
amount of solvent added to the test system should not exceed 1% v/v and should
not have adverse effects on the microbial activity. The solvent should not affect
the stability of the test substance in water. The solvent should be stripped off to an
extremely small quantity so that it does not significantly increase the DOC
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concentration of the test water or suspension. This should be checked by
substance-specific analysis or, if possible DOC analysis (see paragraph (1)(14) of
this guideline). Care must be taken to limit the amount of solvent transferred to
what is absolutely necessary and to ensure that the amount of test substance can
dissolve in the final volume of test water. Other techniques to introduce the test
substance into the test vessels may be used (see paragraphs (1)(15) and (1)(16) of
this guideline). When an organic solvent is used for application of the test
substance, solvent controls containing the test water (with no additions) and test
water with added reference substance should be treated similarly to active test
vessels amended with test substance in solvent carrier. The purpose of the solvent
controls is to examine possible adverse effects caused by the solvent towards the
microbial population as indicated by the degradation of the reference substance.
(8) Test conditions - (i) Test temperature. Incubation should take place
in the dark (preferred) or in diffuse light at a controlled (±2°C) temperature,
which may be the field temperature or a standard temperature of 20-25°C. Field
temperature may be either the actual temperature of the sample at the sampling
time or an average field temperature at the sampling site.
(ii) Agitation. Agitation by means of continuous shaking or stirring
should be provided to maintain particles and microorganisms in suspension.
Agitation also facilitates oxygen transfer from the headspace to the liquid so that
aerobic conditions can be adequately maintained. Place the flasks on a shaking
table (approx. 100 rpm agitation) or use magnetic stirring. Agitation must be
continuous. However, the shaking or stirring should be as gentle as possible,
while still maintaining a homogeneous suspension.
(9) Test duration, (i) The duration of the test should normally not exceed
60 days unless the semi-continuous procedure with periodical renewal of the test
suspension is applied (see paragraph (i)(6) of this guideline). However, the test
period for the batch test may be extended to a maximum of 90 days, if the
degradation of the test substance has started within the first 60 days. Degradation
is monitored, at appropriate time intervals, by the determination of the residual
14C activity or the evolved 14CO2 (see paragraph (j)(4) of this guideline) and/or by
chemical analysis (paragraph (j)(5) of this guideline). The incubation time must
be sufficiently long to evaluate the degradation process. The extent of degradation
should preferably exceed 50%. For slowly degradable substances, the extent of
degradation should be sufficient (normally greater than 20% degradation) to
ensure the estimation of a kinetic degradation rate constant.
(ii) Periodic measurements of pH and oxygen concentration in the test
system should be conducted unless previous experience from similar tests with
water and sediment samples collected from the same site makes such
measurements unnecessary. Under some conditions, the metabolism of primary
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substrates at much higher concentrations within the water or sediment could
possibly result in enough CC>2 evolution and oxygen depletion to significantly
alter the experimental conditions during the test.
(j) Procedure— (1) Preparation of flasks for pelagic test, (i) Transfer a
suitable volume of test water to the test flasks, up to about one third of the flask
volume and not less than about 100 ml. If multiple flasks are used (to allow
sacrifice of whole flasks at each sampling time), the appropriate volume of test
water is also > 100 ml, as smaller sample volumes may influence the length of
the lag phase. The test substance is added from a stock solution as described in
paragraph (i)(7) of this guideline. At least two different concentrations of test
substance differing by a factor of 5 to 10 should be used in order to determine
degradation kinetics and calculate the kinetic degradation rate constant. Both of
the selected concentrations should be less than 100 |ig/L and preferably in the
range of <1-10 |ig/L.
(ii) Close the flasks with stoppers or lids impermeable to air and CC>2. For
non 14C-labelled nonvolatile test chemicals, loose cotton wool plugs that prevent
contamination from air are suitable provided that any major degradation products
are known to be nonvolatile, and if indirect CO2 determination is used (see
paragraph (j)(4) of this guideline).
(iii) Incubate the flasks at the selected temperature. Withdraw samples for
chemical analysis or 14C measurements at the beginning of the test (i.e. before
biodegradation starts; see paragraph (h)(l) of this guideline) and then at suitable
time intervals during the course of the test. Sampling may be performed by
withdrawal of sub-samples (e.g. 5-ml aliquots) from each replicate or by sacrifice
of whole flasks at each sampling time. The mineralization of the test substance
may either be determined indirectly or directly (see paragraph (j)(4) of this
guideline). Usually, a minimum of five sampling points are needed during the
degradation phase (i.e. after ended lag phase) in order to estimate a reliable rate
constant, unless it can be justified that three sampling points are sufficient for
rapidly degradable substances. For substances that are not rapidly degraded more
measurements during the degradation phase can easily be made and, therefore,
more data points should be used for the estimation of k. No fixed time schedule
for sampling can be stated, as the rate of biodegradation varies; however the
recommendation is to sample once per week if degradation is slow. If the test
substance is rapidly degradable, sampling should take place once per day during
the first three days and then every second or third day. Under certain
circumstances, such as with very rapidly hydrolyzing substances, it may be
necessary to sample at hourly intervals. It is recommended that a preliminary
study be conducted prior to the test in order to determine the appropriate sampling
intervals. If samples have to be available for further specific analysis, it is
advisable to take more samples and then select those to be analyzed at the end of
12
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the experiment following a backwards strategy, i.e. the last samples are analyzed
first (see paragraph (j)(5)(iv) of this guideline for guidance on stability of samples
during storage).
(2) Number of flasks and samples, (i) Set up a sufficient number of test
flasks to have:
(A) At least duplicate flasks for each concentration of test substance (but
preferably a minimum of 3); more if whole flasks are sacrificed at each sampling
time (symbolized FT);
(B) For mass balance calculation, at least duplicate flasks for each test
substance concentration (symbolized FM);
(C) At least one blank test flask containing only the test water (i.e. no test
substance) (symbolized FB);
(D) Duplicate flasks with reference substance (e.g. aniline or sodium
benzoate, at 10 |ig/l) (symbolized FC). The purpose of the reference control is to
confirm a minimum of microbial activity;
(E) One or two flasks containing sterilized test water for examining
possible abiotic degradation or other nonbiological removal of the test substance
(symbolized Fs). The biological activity can be stopped by autoclaving (121°C;
20 min) the test water or by adding a toxicant (e.g. sodium azide at 10-20 g/1,
mercuric chloride at 100 mg/1 or formalin at 100 mg/1), or by gamma irradiation.
If HgCb is used, it should be disposed of as toxic waste. For water with sediment
added in large amount, sterile conditions are not easy to obtain; in this case
repeated autoclaving (e.g., three times) is recommended. It should be considered
that the sorption characteristics of the sediment may be altered by autoclaving.
(F) Duplicate flasks for both test water and test water with reference
substance are treated with the same amount of solvent and by the same procedure
as that used for application of the test substance. The purpose is to examine
possible adverse effects of the solvent on degradation of the reference substance.
(ii) In the design of the test, the investigator should consider the relative
importance of increased experimental replication versus increased number of
sampling times. The exact number of flasks required will depend on the method
used for measuring the degradation.
(iii) Two subsamples (e.g. 5-ml aliquots) should be withdrawn from each
test flask at each sampling time. If multiple flasks are used to allow sacrifice of
whole flasks, a minimum of two flasks should be sacrificed at each sampling
13
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time.
(3) Preparation of flasks for suspended sediment test [optional]. Add
the necessary volumes of test water and sediment, if required, to the test vessels.
Prepare flasks for the suspended sediment test as for the pelagic test (see
paragraph (j)0) °f this guideline). Preferably use serum bottles or similarly
shaped flasks. Place the closed flasks horizontally on a shaker. Open flasks for
non 14C-labelled, nonvolatile substances should be placed in upright position; in
this case magnetic stirring and the use of magnetic bars coated with glass are
recommended. If necessary, aerate the bottles to maintain proper aerobic
conditions.
(4) Radiochemical analysis, (i) General. Evolved 14CC>2 can be measured
indirectly or directly. 14CC>2 is determined indirectly by the difference between the
initial 14C activity in the test water or suspension and the total residual activity at
the sampling time as measured after acidifying the sample to pH 2-3 and stripping
off CC>2. Inorganic carbon is thus removed and the residual activity measured
derives from organic material. Indirect 14CC>2 determination should not be used if
major volatile transformation products are formed during the transformation of
the test substance. If possible, 14CO2 evolution should be measured directly at
each sampling time in at least one test flask; this procedure enables both the mass
balance and biodegradation process to be checked, but it is restricted to tests
conducted with closed flasks.
(ii) Indirect 14COi determination. (A) For routine measurements, the
indirect method is normally the least time-consuming and most precise method if
the test substance is nonvolatile and is not transformed into volatile
transformation products. Simply transfer unfiltered samples, for example 5-ml
size to scintillation vials. A suitable activity in samples is 5,000 dpm - 10,000
dpm (80-170 Bq) initially, and a minimum initial activity is about 1000 dpm. The
CC>2 should be stripped off after acidifying to pH 2-3 with 1-2 drops of
concentrated H3PO4 or HC1. The CC>2 stripping can be performed by bubbling
with air for about lA-\ hour. Alternatively, vials can be shaken vigorously for 1-2
hours (for instance on a microplate shaker) or with more gentle shaking be left
overnight. The efficiency of the CC>2 stripping procedure should be checked (by
prolonging the aeration or shaking period).
(B) A scintillation liquid, suitable for counting aqueous samples should
then be added, the sample homogenized on a whirling mixer and the radioactivity
determined by liquid scintillation counting, subtracting the background activity
found in the test blanks (FB). Unless the test water is very colored or contains a
high concentration of particles, the samples will normally show uniform
quenching and it will be sufficient to perform quench corrections using an
external standard. If the test water is highly colored, quench correction by means
14
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of internal standard addition may be necessary. If the concentration of particles is
high it may not be possible to obtain a homogeneous solution or gel, or the
quench variation between samples may be large. In that case the counting method
described below (see paragraph (j)(4)(ii)(C) of this guideline) for test slurries can
be used.
(C) If the test is carried out as a suspended sediment test, the 14CC>2
measurement could be made by taking a homogeneous 10-ml sample of the test
water/suspension and first separating the phases by centrifugation at a suitable
speed (e.g. at 40,000 m/s2 for 15 min). The aqueous phase should then be treated
as described above (see paragraph (j)(4)(ii)(B) of this guideline). The 14C activity
in the paniculate phase (POA) should be determined by resuspending the
sediment into a small volume of distilled water, transferring to scintillation vials,
and adding scintillation liquid to form a gel (special scintillation liquids are
available for that purpose). Depending on the nature of particles (e.g. their content
of organic material), it may be feasible to digest the sample overnight with a
tissue solubilizer and then homogenize on a whirling mixer prior to the addition
of scintillation liquid. Alternatively, the POA can be determined by combustion in
excess of oxygen by use of a sample oxidizer. When counting, internal standards
should always be included, and it may be necessary to perform quench corrections
using internal standard addition for each individual sample.
(iii) Direct 14COi determination. If the evolved 14CC>2 is measured
directly during the test, more flasks should be set up for this purpose at the start of
the test. Direct 14CC>2 determination is recommended if major volatile
transformation products are formed during the transformation of the test
substance. At each measuring point the additional test flasks are acidified to pH 2-
3 and the 14CC>2 is collected in an internal (i.e. placed in each flask at the start of
the test) or external absorber. An absorbing medium, either alkali (e.g. 1 N NaOH
solution, or a NaOH pellet), ethanolamine or ethanolamine-based, or
commercially available absorbers, can be used. For direct measurement of the
14CO2, the flasks should be closed with e.g. butyl rubber septa.
(iv) Parent and degradates. Optionally, the concentrations of 14C-
labelled test substance and major transformation products may be determined by
use of radiochromatography (e.g. thin layer chromatography, RAD-TLC) or
HPLC with radiochemical detection.
(v) Phase distribution. Optionally, the phase distribution of the remaining
radioactivity in the test medium, and of the residual test substance and
transformation products, may be determined. Useful information characterizing
biodegradability behavior can be obtained from measurements of the distribution
of TO A between the dissolved state (dissolved organic 14C activity, DOA) and the
particulate state (particulate organic 14C activity, POA) after separation of
15
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particulates by membrane filtration or centrifugation. POA consists of test
substance sorbed onto the microbial biomass and onto other particles in addition
to the test substance carbon that has been used for synthesis of new cellular
material and thereby incorporated into the particulate biomass fraction. The
formation of dissolved 14C organic material can be estimated as the DOA at the
end of biodegradation (plateau on the degradation versus time curve).
(A) Estimate the phase distribution of residual 14C in selected samples by
filtering samples through a 0.22 jim or 0.45 jim membrane filter made from a
material that does not adsorb significant amounts of the test substance
(polycarbonate filters may be suitable). If sorption of test substance onto the filter
is too large to be ignored (to be checked prior to the experiment), high-speed
centrifugation (2,000 x g; 10 min) can be used instead of filtration.
(B) Proceed with the filtrate or centrifugate as described in paragraph
(j)(4)(ii) of this guideline for unfiltered samples. Dissolve membrane filters in a
suitable scintillation fluid and count, normally using only the external standard
ratio method to correct for quenching, or use a sample oxidizer. If centrifugation
has been used, resuspend the pellet formed from the particulate fraction in 1-2 ml
of distilled water and transfer to a scintillation vial. Wash subsequently twice with
1 ml distilled water and transfer the washing water to the vial. If necessary, the
suspension can be embedded in a gel for liquid scintillation counting.
(vi) Mass balance. At the end of the test the mass balance should be
determined by direct 14CC>2 measurement using separate test flasks from which no
samples are taken in the course of the test.
(5) Specific chemical analysis, (i) If a sensitive specific analytical method
is available, primary biodegradation can be assessed by measuring the total
residual concentration of test substance instead of using radiolabeling techniques.
If a radiolabeled test substance is used (to measure total mineralization), specific
chemical analyses can be made in parallel to provide useful additional
information and check the procedure. Specific chemical analyses may also be
used to measure transformation products formed during the degradation of the test
substance, and this is recommended for substances that are mineralized with half-
lives exceeding 60 days.
16
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Figure 1. Examples of plots of degradation data (as residual activity)
vs. time.
100
lag phase (t)
first order kinetics
"tailing"
s
1
w
£
J3
kg phass
Time
Top: Arithmetic plot. Bottom: Semi-logarithmic plot.
(ii) The concentration of the test substance and the transformation
products at every sampling time should be measured and reported (as a
concentration and as percentage of applied). In general, transformation products
detected at >10% of the applied concentration at any sampling time should be
identified unless reasonably justified otherwise. Transformation products for
which concentrations are continuously increasing during the study should also be
17
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considered for identification even if their concentrations do not exceed 10% of the
applied concentration of parent substance, as this may indicate persistence.
(iii) Analyses of transformation products in sterile controls should be
considered if rapid abiotic transformation of the test substance (e.g. hydrolysis) is
thought possible. The need for quantification and identification of transformation
products should be considered on a case-by-case basis, with justification being
provided in the test report. Extraction techniques using organic solvent should be
applied according to directions given in the respective analytical procedure.
(iv) All samples removed for analysis should be stored at 2 to 4°C and in
air-tight containers if analysis is carried out within 24 hours (preferred). For
longer storage, the samples should be frozen below -18°C or chemically
preserved. Acidification is not recommended because acidified samples may be
unstable. If the samples are not analyzed within 24 hours and are subject to longer
storage, a storage stability study should be conducted to demonstrate the stability
of chemicals of interest under -18°C storage or preserved conditions. If the
analytical method involves either solvent extraction or solid phase extraction
(SPE), the extraction should be performed immediately after sampling or after
storing the sample refrigerated for a maximum of 24 hours.
(v) The size of test flasks and sample volumes (see paragraphs (i)(l) and
(j)(l)(i) of this guideline) may have to be changed depending on the sensitivity of
the analytical method. The test can easily be carried out with test volumes of one
liter in flasks of 2-3-liter volume, which makes it possible to collect samples of
approx. 100 ml.
(k) Data and reporting—(1) Treatment of results, (i) Plot of data.
Round off sampling times to a whole number of hours (unless the substance
degrades substantially in minutes to hours) but not to a whole number of days.
Plot the estimates of the residual activity of test substance (for 14C-labeled
substances) or the residual concentration (for nonlabeled substances), against time
both in a linear and in a semi-logarithmic plot (see Figure 1). If degradation has
taken place, compare the results from flasks FT with those from flasks FS. If the
means of the results from the flasks with test substance (FT) and the sterile flasks
(Fs) deviate by less than 10%, it can be assumed that the degradation observed is
predominantly abiotic. If the degradation in flasks Fs is lower, the figures may be
used to correct those obtained with flasks FT (by subtraction) in order to estimate
the extent of biodegradation. When optional analyses are performed for major
transformation products, plots of their formation and decline should be provided
in addition to a plot of the decline of the test substance.
(ii) Calculation of degradation kinetics. (A) Estimate the lag phase
duration IL from the degradation curve (semi-logarithmic plot) by extrapolating its
18
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linear part to zero degradation or alternatively by determining the time for
approximately 10% degradation (see Figure 1). From the semi-logarithmic plot,
estimate the first order rate constant, k, and its standard error by linear regression
of In (residual 14C activity or test substance concentration) versus time. With 14C
measurements in particular, use only data belonging to the initial linear part of the
curve after the ended lag phase. It may sometimes be relevant to calculate two
different rate constants, if the degradation follows a biphasic pattern. For this
purpose two different phases of the degradation curve are defined. Calculations of
the rate constant, k, and the half-life t/2 = In2/k, should be carried out for each of
the individual replicate flasks, when subsamples are withdrawn from the same
flask, or by using the average values, when whole flasks are sacrificed at each
sampling time. When the first procedure is used, the rate constant and half-life
should be reported for each of the individual replicate flasks and as an average
value with a standard error. If high concentrations of test substance have been
used, the degradation curve may deviate considerably from a straight line (semi-
logaritmic plot) and first-order kinetics may not be valid. In this case it is not
meaningful to define a half-life. However, for a limited data range, pseudo first-
order kinetics can be applied and the degradation half-time DT50 (time to reach
50% degradation) can be estimated. It should be borne in mind that the time
course of degradation beyond the selected data range cannot be predicted using
the DT50, which is merely a descriptor of a given set of data. Analytical tools to
facilitate statistical calculations and curve fitting are easily available and the use
of this kind of software is recommended.
(B) If specific chemical analyses are made, estimate rate constants and
half-lives for primary degradation as above (see paragraph (k)(l)(ii)(A) of this
guideline) for total mineralization. If primary degradation is the limiting process
data points from the entire course of degradation can sometimes be used. This is
because measurements are direct by contrast to measurements of 14C activity.
(C) If 14C-labelled substances are used, a mass balance should be
expressed in percentage of the applied initial concentration, at least at the end of
the test.
(iii) Residual activity. When the 14C-labelled part of an organic substance
is biodegraded, the major part of the 14C is converted to 14CC>2, while another part
is used for growth of biomass and/or synthesis of extracellular metabolites.
Therefore, complete ultimate biodegradation of a substance does not result in a
100% conversion of its carbon into 14CC>2. The 14C built into products formed by
biosynthesis is subsequently released slowly as 14CC>2 due to secondary
mineralization. For these reasons plots of residual organic 14C activity (measured
after stripping off CO2) or of 14CC>2 produced versus time will show a tailing after
degradation has been completed. This complicates a kinetic interpretation of the
data and for this purpose, only the initial part of the curve (after ended lag phase
19
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and before approx. 50% degradation is reached) should normally be used for the
estimation of a degradation rate constant. If the test substance is degraded, the
total residual organic 14C activity is always higher than the 14C activity associated
with the remaining intact test substance. If the test substance is degraded by a
first-order reaction and a constant fraction d is mineralized into CC>2, the initial
slope of the 14C disappearance curve (total organic 14C versus time) will be d
times the slope of the corresponding curve for the concentration of test substance
(or, to be precise, the part of the test substance labeled with 14C). Using
measurements of the total organic 14C activity uncorrected, the calculated
degradation rate constant will therefore be conservative. Procedures for estimating
the concentrations of the test substance from the measured radiochemical
activities based on various simplifying assumptions have been described in the
literature (see paragraphs (1)(1), (1)(17) through (1)(19) of this guideline). Such
procedures are most easily applied for rapidly degradable substances.
(2) Interpretation of results, (i) If k is found to be independent of the
added concentration (i.e. if the calculated k is approximately the same at the
different concentrations of test substance), it can be assumed that the first-order
rate constant is representative of the test conditions. To what extent the results can
be generalized or extrapolated to other systems should be evaluated by expert
judgment. If a high concentration of test substance is used, and the degradation
therefore does not follow first-order kinetics, the data cannot be used for direct
estimation of a first-order rate constant or a corresponding half-life. However,
data derived from a test using a high concentration of test substance may still be
usable for estimating the degree of total mineralization and/or detection and
quantification of transformation products.
(ii) If the rates of loss processes other than biodegradation are known (e.g.
hydrolysis or volatilization), they may be subtracted from the net loss rate
observed during the test to give an approximate estimate of the biodegradation
rate. Data for hydrolysis may, for example, be obtained from the sterile control or
from parallel test using a higher concentration of the test substance.
(iii) The indirect and direct determination of 14CC>2 (see paragraph (j)(4) of
this guideline) can only be used to measure the extent of mineralization of the test
substance to CC>2. Radiochromatography (RAD-TLC) or HPLC may be used to
analyze the concentrations of 14C-labelled test substance and the formation of
major transformation products. To enable a direct estimation of the half-life, it is
necessary that no major transformation products (defined as >10% of the applied
amount of test substance) be present. If major transformation products as defined
here are present, a detailed evaluation of the data is necessary. This may include
repeated testing and/or identification of transformation products (see paragraph
(j)(5)(i) of this guideline). As the proportion of test substance carbon converted to
CC>2 varies, depending largely on the concentration of test substance and other
20
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substrates available, test conditions and the microbial community, this test does
not allow a straightforward estimation of ultimate biodegradation as in a DOC
die-away test. Test results are more comparable to those from a respirometric test.
The degree of mineralization will thus be less than or equal to the minimum level
of ultimate biodegradation. To obtain a more complete picture of the ultimate
biodegradation (mineralization and incorporation into biomass), the analysis of
the phase distribution of 14C should be performed at the end of the test (see
paragraph (j)(4)(v) of this guideline). The 14C in the particulate pool will consist
of 14C incorporated into bacterial biomass and 14C sorbed to organic particles.
(3) Validity of the test, (i) If the reference substance is not degraded
within the expected time interval (for aniline and sodium benzoate, usually less
than two weeks), the test may be invalid and the results should be further verified;
alternatively, the test should be repeated with a new water sample. In an ISO ring-
test of the method in which seven laboratories participated, adapted degradation
rate constants for aniline ranged from 0.3 to 1.7 day"1 with an average of 0.8 d"1 at
20°C and a standard error of ± 0.4 d"1 (t/2 = 0.9 days). Typical lag times were 1 to
7 days. The waters examined were reported to have a bacterial biomass
corresponding to 103 to 104 colony forming units (CPU) per ml. Degradation rates
in nutrient-rich Mid-European waters were greater than in Nordic oligotrophic
waters, which may be due to the different trophic status or previous exposure to
chemical substances.
(ii) The total recovery (mass balance) at the end of the experiment should
be between 90% and 110% for radiolabeled substances, whereas the initial
recovery at the beginning of the experiment should be between 70% and 110% for
nonlabeled substances. However, the indicated ranges should only be interpreted
as targets and should not be used as criteria for acceptance of the test.
(4) Test report. The type of study, i.e. pelagic or suspended sediment test,
should be clearly stated in the test report, which should also contain the following
information:
(i) Test substance and reference substance(s).
(A) Common names, chemical names (IUPAC and/or CAS names are
recommended), CAS numbers, structural formulas (indicating position of 14C if
radiolabeled substance is used) and relevant physical/chemical properties of test
and reference substance (see paragraph (g)(l) of this guideline);
(B) Chemical names, CAS numbers, structural formulas (indicating
position of 14C if radiolabeled substance is used) and relevant physical/chemical
properties of substances used as standards for identification and quantification of
transformation products;
21
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(C) Purity (and known impurities) of test and reference substances;
(D) Radiochemical purity of labeled chemical and specific activity (where
appropriate).
(ii) Surface water.
(A) Location and description of sampling site including, if possible,
contamination history;
(B) Date and time of sample collection;
(C) Nutrients (total N, ammonium, nitrite, nitrate, total P, dissolved
orthophosphate);
(D) Depth of collection;
(E) Appearance of sample (e.g. color and turbidity);
(F) DOC and TOC;
(G) BOD;
(H) Temperature and pH at the place and time of collection;
(I) Oxygen or redox potential (mandatory only if aerobic conditions are
not obvious);
(J) Salinity or conductivity (in the case of sea water and brackish water);
(K) Suspended solids (in case of a turbid sample);
(L) Any other relevant information about the sampling location at the time
of sampling (e.g. actual or historical data on flow rate of rivers or marine currents,
nearby major discharges and type of discharges, weather conditions preceding the
sampling time).
(iii) Suspended sediment test.
(A) Depth of sediment collection;
(B) Appearance of the sediment (such as colored, muddy, silty, or sandy);
22
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(C) Texture (e.g. % coarse sand, fine sand, silt and clay)
(D) Dry weight in g/1 of the suspended solids, TOC concentration or
weight loss on ignition as a measure of the content of organic matter;
(E)pH;
(F) Oxygen or redox potential (mandatory only if aerobic conditions are
not obvious).
(iv) Test conditions.
(A) Delay between collection and use in the laboratory test, sample
storage and pretreatment of the sample dates of performance of the studies;
(B) Amount of test substance applied, test concentration and reference
substance;
(C) Method of application of the test substance including any use of
solvents;
(D) Volume of surface water used and sediment (if used) and volume
sampled at each interval for analysis;
(E) Description of the test system used;
(F) If dark conditions are not to be maintained, information on the diffuse
light conditions;
(G) Information on the method(s) used for establishing sterile controls
(e.g. temperature, time and number of autoclavings);
(H) Incubation temperature;
(I) Information on analytical techniques and the method(s) used for
radiochemical measurements and for mass balance check and measurements of
phase distribution (if conducted);
(J) Number of replicates.
(v) Results.
(A) Percentages of recovery (see paragraph (h)(l) of this guideline);
23
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(B) Repeatability and sensitivity of the analytical methods used including
the LOD and LOQ (see paragraph (h)(2) of this guideline);
(C) All measured data (including sampling time points) and calculated
values in tabular form and the degradation curves. For each test concentration and
for each replicate flask, report the linear correlation coefficient for the slope of the
logarithmic plot, the estimated lag phase and a first-order or pseudo first-order
rate constant (if possible), and the corresponding degradation half-life (or the half-
life period, ti/2);
(D) Report relevant values as the averages of the results observed in
individual replicates, for example length of lag phase, degradation rate constant
and degradation half-life (or tl/2);
(E) Categorize the system as either non-adapted or adapted as judged from
the appearance of the degradation curve and from the possible influence of the
test concentration;
(F) The results of the final mass balance check and results on phase
distribution measurements (if any);
(G) The fraction of 14C mineralized and, if specific analyses are used, the
final level of primary degradation;
(H) The identification, molar concentration and extent of formation, as a
percentage of applied, of major transformation products (see paragraph (j)(5)(i) of
this guideline), where appropriate;
(I) A proposed pathway of transformation, where appropriate;
(J) Discussion of results.
(vi) Optional information.
(A) Microbial biomass (e.g. acridine orange direct count or colony
forming units);
(B) Inorganic carbon;
(C) Chlorophyll-a concentration as a specific estimate for algal biomass.
(1) References.
(1) ISO/DIS 14592-1 (1999). Water quality - Evaluation of the aerobic
24
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biodegradability of organic compounds at low concentrations - Part 1: Shake flask
batch test with surface water or surface water/sediment suspensions. International
Organization for Standardization, Geneva.
(2) OECD (2002). Aerobic and anaerobic transformation in aquatic
sediment systems. No. 308, adopted 24 April, 2002. Organization for Economic
Cooperation and Development, Paris.
(3) OECD (2002). Aerobic and anaerobic transformation in soil. No. 307,
adopted 24 April 2002. Organization for Economic Cooperation and
Development, Paris.
(4) OECD (1995) Water Solubility. No. 105, adopted 27 July, 1995.
Organization for Economic Cooperation and Development, Paris.
(5) OECD (1981) Dissociation Constants in Water. No. 112, adopted 12
May, 1981. Organization for Economic Cooperation and Development, Paris.
(6) OECD (2006) Vapor Pressure. No. 104, adopted 23 March, 2006.
Organization for Economic Cooperation and Development, Paris.
(7) OECD (2004) Hydrolysis as a Function of pH. No. Ill, adopted 13
April 2004. Organization for Economic Cooperation and Development, Paris.
(8) OECD (1995) Partition Coefficient (n-octanol/water), Shake Flask
Method. No. 107, adopted 27 July, 1995. Organization for Economic Cooperation
and Development, Paris.
(9) OECD (2004) Partition Coefficient (n-octanol/water), FIPLC Method.
No. 117, adopted 13 April 2004. Organization for Economic Cooperation and
Development, Paris.
(10) OECD (2000) Test No. 106: Adsorption -- Desorption Using a Batch
Equilibrium Method. No. 106, adopted 21 January 2000. Organization for
Economic Cooperation and Development, Paris.
(11) OECD (1984) Activated Sludge, Respiration Inhibition Test. No. 209,
adopted 4 April 1984. Organization for Economic Cooperation and Development,
Paris.
(12) OECD (1992) Ready Biodegradability. No. 301, adopted 17 July
1992. Organization for Economic Cooperation and Development, Paris.
(13) OECD (1981) Inherent Biodegradability: Modified MITI Test (II)
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No. 302, adopted 12 May 1981. Organization for Economic Cooperation and
Development, Paris.
(14) ISO 8245 (1999). Water quality - Guidelines on the determination of
total organic carbon (TOC) and dissolved organic carbon (DOC). International
Organization for Standardization, Geneva.
(15) ISO 10634 (1995). Water quality - Guidance for the preparation and
treatment of poorly water-soluble organic compounds for the subsequent
evaluation of their biodegradability in an aqueous medium. International
Organization for Standardization, Geneva.
(16) OECD (2000). Guidance Document on aquatic toxicity testing of
difficult substances and mixtures. Environmental Health and Safety Publications,
Series on Testing and Assessment, No. 23. Organization for Economic
Cooperation and Development, Paris.
(17) Simkins, S. and M. Alexander (1984). Models for mineralization
kinetics with the variables of substrate concentration and population density.
Appl. Environ. Microbiol. 47, 394-401.
(18) Ingerslev, F. and N. Nyholm (2000). Shake-flask test for
determination of biodegradation rates of 14C-labeled chemicals at low
concentrations in surface water systems. Ecotoxicol. Environ. Safe. 45, 274-283.
(19) ISO/CD 14592-1 (1999). Ring test report: Water Quality - Evaluation
of the aerobic biodegradability of organic compounds at low concentrations part 1
- report of 1998/1999 ring-test. Shake flask batch test with surface water or
surface water/sediment suspensions. International Organization for
Standardization, Geneva.
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