United States Prevention, Pesticides EPA712-C-08-006
Environmental Protection And Toxic Substances October 2008
Agency (7101)
&EPA Fate, Transport and
Transformation Test
Guidelines
OPPTS 835.3280
Simulation Tests to
Assess the Primary and
Ultimate Biodegradability
of Chemicals Discharged
to Wastewater
-------
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.
-------
OPPTS 835.3280-Simulation tests to assess the primary and ultimate
biodegradability of chemicals discharged to wastewater: biodegradation in
wastewater, activated sludge, anaerobic digester sludge, mixing zone for
treated effluent and surface water and mixing zone for untreated
wastewater and surface water.
(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 Proposed Guideline
OECD 314. Simulation tests to assess the primary and ultimate biodegradability of
chemicals discharged to wastewater: biodegradation in wastewater, activated sludge,
anaerobic digester sludge, mixing zone for treated effluent and surface water and mixing
zone for untreated wastewater and surface water. October 2007 Revised Draft Test
Guideline, annex to OECD communication ENV/EHS/MCH/cm/2007.08, Paris 2
November 2007.
(b) Purpose. (1) This guideline describes methods for determining the extent and
kinetics of primary and ultimate biodegradation of organic substances whose route of
entry into the environment begins with their discharge to wastewater. Personal care,
household cleaning and laundry products are typically discarded down the drain as part of
their normal use and become common components of domestic wastewater. Likewise,
Pharmaceuticals are excreted or in some cases disposed down the drain. Other substances
may be episodically or continuously discharged to wastewater as a result of
manufacturing processes.
(2) This guideline consists of five separate but related simulation tests for
assessing the primary and ultimate biodegradation of chemicals in wastewater during
transit in the sewer, secondary treatment in an activated sludge treatment system,
anaerobic digestion of sludge as well as treated effluent in the mixing zone of surface
water and untreated wastewater that is directly discharged to surface water.
Biodegradation in each compartment can play an important role in determining chemical
exposure in interconnected aquatic and terrestrial habitats.
(c) General Considerations. (1) Figure 1 shows the most common transport
pathways for chemicals discharged to wastewater. Wastewater initially enters a sewer,
where it may remain for hours or a few days, during its transport to a wastewater
treatment plant (WWTP) or environmental release site. In most situations, the wastewater
is treated before release, but in some situations the wastewater is released to surface
water directly or with only minimal primary treatment. In a typical WWTP, a portion (40-
60%) of the solids is removed during primary treatment. The resulting effluent is then
subjected to biological treatment and the solids are removed in a final clarifier. The final
effluent is subsequently released to surface water. The sludge solids removed during
primary treatment and final clarification are most commonly digested under anaerobic
conditions if the sludge disposal involves land application.
-------
Figure 1. Pathways for chemicals discharged to wastewater
Pathways for Chemicals
Discharged to
Wastewater
MIXING ZONE**
Treated Effluent with Surface Water
SURFACE
WATERS
WASTEWATER TREATMENT
PLANT
Final
Effluent
Aerobic**
Biological
Treatment
Sludge
Digestion**
(Anaerobic)
Final
Clarification
Secondary
Sludge
MIXING ZONE**
Untreated (PoorlyTreated)
Wastewater with Surface Water
Digested
Sludge
SLUDGE AMENDED SOIL
•"Simulated in
the New Guideline
(2) The fraction of the chemical released to the environment in the final effluent
or associated with sludge solids is a function of its partitioning behavior and its
biodegradation rate, and other processes such as volatilization. Due to chemical
residence time and the level of biological activity, the critical opportunities for significant
removal through biodegradation are 1) in the sewer, 2) during aerobic secondary
treatment and 3) during anaerobic digestion of the sludge. Consequently, these three
systems are the most important to simulate for quantifying biodegradation losses during
wastewater transport and treatment. Furthermore, the effects of treatment processes
extend into the environment at the time of release. Thus, biodegradation in the mixing
zones, in the water and in biosolids, as the chemical moves away from the point of
release, is key to understanding downstream dispersion and exposure.
(3) The five simulation test methods described in this guideline use an open batch
system or closed flow-through batch system (an example of the latter is shown in Figure
2), and include elements from Organization for Economic Cooperation and Development
(OECD) guidelines 301, 303A, 309, 310 and 311 (see paragraphs (o)(l) through (o)(5) of
this guideline). The principal objectives of the methods are to 1) measure the rate of
primary biodegradation, 2) measure the rate of mineralization, and 3) follow the
formation and decay of major transformation products when appropriate. In addition,
characterization and quantification of major transformation products may be possible if
suitable analytical methods are available.
-------
Figure 2-. Example of flow-through test apparatus
Example of Flow-Through Test Set-Up
Air
CO2 Traps
Containing
100 ml of 1.5NKOH
(4) These tests can be designed to assess accurately the biodegradation of both
new and existing substances released to wastewater (see paragraphs (o)(6) through
(o)(ll) of this guideline). In some cases, the resulting kinetic constants can serve as input
constants for exposure models used for risk assessment. These tests are intended as
higher tier tests for assessing the biodegradation of chemicals that do not biodegrade in
OECD screening tests, or for refining biodegradation rates used for an exposure
assessment.
(d) General principle of the tests. (1) Typically, a test substance, radiolabeled in
an appropriate position, is incubated with an environmental sample that has been freshly
collected from a representative field site or maintained in the laboratory under conditions
realistically simulating some future environmental condition. Abiotic and biotic test
systems are prepared for each test substance and condition. Biological activity is
inhibited in the abiotic control, which is used for estimating mineralization by difference,
establishing extraction efficiency and recovery of the parent molecule, and quantifying
other loss processes such as hydrolysis, oxidation, volatilization or sorption to test
apparatus.
(2) If an analytical method with the required sensitivity is identified, the rate of
parent degradation or transformation can be determined using a non-radiolabeled test
substance or by following the disappearance of a substance already in an environmental
sample. However, ultimate biodegradation of non-radiolabeled substances cannot be
-------
determined unless the biodegradation pathway is well understood and analytical methods
with required sensitivity are available for potential metabolites.
(3) An environmentally relevant concentration of the test substance is dosed to
both abiotic and biotic test systems. The prepared test systems are incubated at a relevant
temperature with continuous mixing when appropriate. Samples are periodically removed
for determination of mineralization and primary biodegradation. The duration of the tests
is based upon the time necessary to establish whether ultimate degradation takes place
and the rate at which it occurs, and thus, it does not necessarily reflect the hydraulic
residence time relevant for a chemical in a specific compartment in the environment.
When the results of the tests are used for environmental risk assessment, the relevant
hydraulic retention times in the compartments covered by these tests have to be
determined according to the scenarios within the specific risk assessment.
(4) Tests can be performed using an open batch system or a closed flow-through
batch system where traps are used to capture evolved 14CO2 or 14CH4. A closed flow-
through system is necessary for volatile test materials. It is also usually preferred for 14C-
lableled test chemicals. Open systems are appropriate for nonvolatile 3H-labeled test
chemicals and for refining the biodegradation kinetics of nonvolatile 14C test materials,
whose ability to be mineralized has been established previously. In the open system,
mineralization to 14CC>2 (14CH4) can be determined indirectly by measuring the difference
in residual radioactivity between samples from the biotic and abiotic test systems
following acidification. Similarly, mineralization to 3H2O can be determined indirectly by
measuring the difference in residual radioactivity following drying. The open system is
not appropriate for use with volatile test substances. In a flow-through system, evolved
14CC>2 is measured directly in the base traps. In addition, dissolved 14CC>2 is determined
by acidifying samples in a sealed vessel and measuring radioactivity in a base trap
contained in the vessel. Under anaerobic conditions, the evolved 14CC>2 and 14CH4 are
collected in tandem. The 14CC>2 is trapped in base and 14CH4 is combusted and converted
to 14CC>2, which is subsequently trapped in a similar manner. In the case of volatile test
substances, an additional in-line organic trap (e.g. activated charcoal) is placed before the
gas traps to capture volatilized parent and metabolites. The choice of test design depends
on the type of radiolabel (14C or 3H), the environmental compartment and the properties
of the test substance.
(5) Samples from both test systems are analyzed for total radioactivity, extractable
parent and metabolites and radioactivity associated with the extracted solids. The level of
parent and metabolites is determined using chromatographic separation and radio-
analytical detection methods. The solids remaining from the extraction process are
combusted to estimate incorporation into biomass by difference or can be further
fractionated to determine uptake into various components of biomass. A complete mass
balance of the test system is obtained from the sum total of all fractions at each sampling.
(6) The level of parent remaining with time can be analyzed using various decay
models to estimate primary biodegradation rates. Likewise, the level of cumulative
mineralization can be analyzed using various models to estimate mineralization rates (see
-------
paragraph (i)(2) of this guideline).
(e) Applicability of tests— (1) General. The various tests are designed to assess
biodegradation during key phases of wastewater transit as well as treatment and
environmental release. The choice of tests should be based on the release scenarios and
anticipated properties of the substance of interest. In the case of volatile test substances,
appropriate modification should be made to quantify losses due to volatilization.
(2) 314A: Test for biodegradation in a sewer system. The purpose of this
test is to evaluate biodegradation in raw wastewater under conditions normally found in
sewer systems. It is useful to perform this test if there is sufficient time in a sewer for the
substance to undergo significant biodegradation and loss. Hence, it is usually most useful
for relatively labile substances discharged to large municipal sewer systems. In addition,
the test provides data that may be used to determine the concentration of a substance
sorbed to primary sludge. Under the conditions of the test, the concentration of test
substance is at its expected level in wastewater with the biomass level being that
normally present in a representative wastewater sample. While oxygen is present, the
system is minimally aerated to simulate dissolved oxygen (DO) conditions in sewers.
(3) 314B: Test for biodegradation in activated sludge, (i) The purpose of the
activated sludge test is to evaluate biodegradation during a widely used form of
biological sewage treatment. It is applicable to any substance subjected to such treatment
and is key to estimating final effluent concentrations. This test is generally performed
first among the tests in this guideline and is the most important test in the series. This test
is characterized by a high level of biomass and a relatively low level of test substance
under well-aerated conditions.
(ii) The activated sludge test can compliment or be a lower cost alternative to
OECD 303 A, a dynamic simulation of a wastewater treatment plant that can be used to
determine the removal of a test substance under a specific set of operating conditions (i.e.
hydraulic retention time, solids residence time, solids level, etc.). OECD 303A can
generate a percentage removal or a comprehensive picture of biodegradation and sorption
that occur at steady state during treatment. However, as an alternative to the expense and
complexity of running a full scale system, the activated sludge test can generate first-
order rate constants for loss of parent and mineralization, and these can be used as inputs
to a variety of wastewater simulation models to estimate removal under any set of
operating conditions.
(4) 314C: Test for biodegradation in anaerobic digester sludge. The purpose
of this test is to evaluate biodegradation during anaerobic sludge digestion. It is
particularly relevant for sorbing substances, which partition to primary and secondary
sludge. This test is useful for refining the concentration of a substance present in the
sludge leaving a treatment plant as well as demonstrating the potential for anaerobic
biodegradation. The test is characterized by reducing conditions, a high level of anaerobic
biomass, and a level of test substance based on expected wastewater concentrations and
partitioning behavior.
-------
(5) 314D: Test for biodegradation in treated effluent-surface water mixing
zone. The purpose of the effluent mixing zone test is to evaluate the biodegradation of
that portion of a substance that passes through treatment and is released in effluent to
surface water; and it can be used to demonstrate that biodegradation occurring in the
treatment plant continues in the receiving environment. It is based on the principle that
both the substance and microbes degrading that substance are discharged together in
effluent. The results of this test can be used to estimate the reduction in a substance
concentration as a result of biodegradation as a volume of water moves downstream from
a wastewater treatment plant. This test is characterized by very low levels of both test
substance and biomass under well-aerated conditions. This test differs from OECD 309 in
that the surface water is amended with treated effluent, and it can be used to evaluate
volatile substances. Also, OECD 309 focuses largely on mineralization whereas this test
is designed to evaluate primary and ultimate biodegradation as well as metabolite
formation and disappearance of chemicals discharged to wastewater.
(6) 314E: Test for biodegradation in untreated wastewater-surface water
mixing zone. The purpose of this test is to evaluate biodegradation in untreated
wastewater that is directly discharged to surface freshwater. This test is useful for
determining the relative biodegradation rate for a substance compared to other organic
components of wastewater. Under the conditions of this test, the levels of test substance
and biomass are based on their expected concentrations in wastewater-surface water
mixing zones. Oxygen is present but at reduced levels, due to the high level of organic
loading.
(f) Information on the test substance. (1) In most cases, 14C or 3H radiolabeled
test substances should be used in these tests. For radiolabeled substances, additional
unlabeled substance may be necessary to achieve the needed test concentration. For
substances with low specific activities, the sensitivity of the method can be improved in
part by increasing the volume of the analytical samples.
(2) For 14C, the radiolabel should be located in the most recalcitrant portion of the
molecule to monitor comprehensively metabolite formation and decay. For large or
complex substances, it may be appropriate to repeat the study using test substance labeled
in a different position. In other cases, it may be more appropriate to position the label in
a portion of the molecule whose fate is poorly understood. Regardless, interpretation of
the results should consider the position of the label as it relates to mineralization and the
metabolites observed.
(3) For nonvolatile substances, tritiated (3H-labeled) substances can be an
alternative to 14C-labeled substances, for reasons of cost or practicality of the synthesis.
Tritium labeling often results in random or uniform distribution of tritium atoms in the
molecule. It is also important to consider that isotopic tritium can exchange with
hydrogen in water (see paragraph (o)(26) of this guideline). These two facts should be
taken into account in interpreting mineralization and metabolite patterns.
-------
(4) Nonlabeled test substances can be used to determine the rate of parent
degradation or transformation if an analytical method with the required sensitivity is
available.
(5) The information in paragraphs (f)(5)(i) through (f)(5)(x) of this guideline, on
the test substance, is helpful for designing a test:
(i) Solubility in water [OECD 105] (see paragraph (o)(12) of this guideline);
(ii) Solubility in organic solvent (applicable to substances applied with solvent or
having low water solubility);
(iii) Dissociation constant (pKa) if the substance is subject to protonation or
deprotonation [OECD 112] (see paragraph (o)(13) of this guideline);
(iv) Vapor pressure [OECD 104] (see paragraph (o)(14) of this guideline) and/or
Henry's law constant;
(v) Substance stability in water and in the dark; i.e. susceptibility to hydrolysis
[OECD 111] (see paragraph (o)(15) of this guideline);
(vi) Environmental concentration, if known or estimated;
(vii) Toxicity of the test substance to microorganisms [OECD 209] (see paragraph
(o)(16) of this guideline);
(viii) Ready biodegradability [OECD 301] (see paragraph (o)(l) of this guideline)
and/or inherent biodegradability [OECD 302] (see paragraphs (o)(17) through (o)(19) of
this guideline).
(ix) Susceptibility to direct and indirect photolysis;
(x) Partitioning behavior.
(g) Reference substance. A substance that is normally easily degraded under the
test conditions may be useful as a reference substance. The purpose of such a reference
substance is to ensure that the microbial community in the test system is active.
Alternatively, a substance, whose fate in the environment is well understood, may be
included as a standard, the results for which can be compared to those for the test
substance. While the use of a reference substance is not required, it may provide useful
information for the interpretation of the test results.
(h) Quality criteria—(1) Validity of the test, (i) The mass balance from the
abiotic test system is used to confirm the recovery of parent from the system. It is
recommended that an abbreviated pilot die-away study be conducted prior to the
definitive test to establish the appropriate extraction system for parent and metabolites.
-------
Target recoveries from the test matrix should be 85%-110%; however, these ranges
should not be used as the sole basis for acceptance of the test. If parent recoveries from
initial samples taken from the abiotic control are in the targeted range, the sample
preparation procedures are suitable for the recovery of the parent compound from the test
matrix. Lower-than-targeted recoveries in the abiotic test system could be due to poor
extraction efficiency, sorption to glassware, or abiotic degradation (see paragraph
(h)(l)(iii) of this guideline).
(ii) Total recovery of radioactivity in both abiotic and biotic systems should
normally range from 75 to 115% for individual samples, and average total recovery for
all samples within a treatment should normally range from 85 to 110%. However, these
ranges should not be used as the sole basis for acceptance of the test. If mass balances
from the abiotic system are in the targeted range but those in the biotic test system are
significantly below this range, the lower recovery likely results from the inability to
efficiently trap 14CC>2 and/or recover metabolites, or the loss of metabolites to glassware
or volatilization.
(iii) If chemical analysis from the abiotic control samples reveals that parent
remained intact throughout the experiment, degradation in the biotic system can be
attributed to microbial activity. If the abiotic system indicates degradation of parent over
time, interpretation of these results may include a description/explanation of the likely
abiotic process that occurred. Comparison between the extent of parent degradation and
metabolite formation observed in the two systems will provide an estimate of the extent
of biological versus chemical degradation in the biotic system, assuming loss is not an
artifact of sample preparation.
(2) Sensitivity of analytical methods. The limit of detection (LOD) of the
analytical method for the test substance and for the transformation products should be <
1% of the initial amount added to the test system, if possible. The limit of quantification
(LOQ) should be < 3% of the added concentration.
(3) Results with reference substance. When a reference substance is included,
the results for the reference substance should approximate those anticipated based on the
reasons for its selection.
(i) Data and reporting—(1) Plot of data. For each sample, the exact time of
incubation including the time needed to terminate biological activity if applicable is
reported. Also, for each sampling point, the percentage of the dosed radioactivity
recovered as parent, metabolites and associated with solids, as well as the cumulative
amount of mineralization and the total mass balance, are reported. These percentages are
plotted against time for both the biotic and abiotic systems, when appropriate.
(2) Kinetic analyses (Optional), (i) In some cases, it may be desirable to fit the
results from these tests to kinetic models. These models could include decay models for
parent and production models for mineralization (e.g. 14CC>2 or 3H2O). The most common
and useful models for this purpose employ first-order kinetics. Most exposure models
(e.g. BUSES, SimpleTreat) utilize first-order rates as critical input parameters.
-------
(ii) A first-order model assumes that the rate constants of a reaction depend solely
upon the concentration of the test material. True first-order conditions exist when the test
material is below the concentration at which the biodegradative capacity of a system
becomes saturated. As the test substance concentration exceeds saturation, the data may
still fit a first-order function, but these quasi first-order rates will be slower than a true
first-order rate. Such quasi first-order kinetics may arise from a need to test a higher
concentration than that occurring in situ due to analytical constraints, or they may reflect
the actual in situ situation.
(iii) When degradation occurs in an exponential manner and the onset of this
degradation is not preceded by a lag period during which little or no degradation occurs,
it may be possible to fit decay or production data to a first-order model. Under such
circumstances, the percentage of parent remaining as a function of time may be fitted to a
simple (one-compartment) or two-compartment first-order loss function using nonlinear
regression methods. Such equations have the form:
y =
where y equals the percentage of parent remaining at time ft), A equals the percentage
degraded at first-order rate constant kj, and B equals the percentage degraded at the first-
order rate constant k2. Such curve fitting can be achieved using nonlinear methods found
in commercially available statistical or curve-fitting software. The two-compartment
model is useful when biodegradation is biphasic, consistent with the existence in the test
system of two different pools of test material (e.g. dissolved and sorbed) that exhibit
different rates of biodegradation.
(iv) In a similar manner, mineralization data can be fit to a simple or two-
compartment first-order production model. Such models have the form:
y = A(l-e~klt)
where y equals the percentage of the material mineralized at time (f), A equals the
percentage mineralized at first-order rate constant k\, and B equals the percentage
mineralized at the first-order rate constant &2.
(v) In some situations, biodegradation, particularly loss of parent, may occur so
rapidly that a true zero time point cannot be measured in the biotic system. In such
situations, data from the abiotic system may be used to represent time zero for the kinetic
analyses.
(vi) When first-order kinetics are observed, half-lives (Ty2) can be calculated
-------
from the estimated first-order rates (k} or k2} for each pool (A or B) using the following
equation:
Ti/2 = -Irillk
(vii) In some circumstances, the observed data can be fit to a variety of other
models, such as Monod or other growth models, which is beyond the scope of the current
guideline. Additional detail on biodegradation kinetics can be found in a report from the
FOCUS Work Group on Degradation Kinetics (see paragraph (o)(20) of this guideline).
Half-life is only relevant for materials exhibiting first-order degradation patterns. In the
absence of first-order kinetics, it may be appropriate to report degradation times for 50%
(DT50) and 90% (DT90) of the test material, if these levels of degradation are observed
during the course of the study. These values can be determined directly or estimated
using standard interpolation procedures.
(viii) When data are fit to a model, the model equation and the software used to fit
the model should be reported. The correlation coefficient (r2), F value, if available, and a
plot of the fitted curve with the actual data should be provided. The estimated rate
constants (kj or £2) and other parameters (A, B) should be reported with their standard
errors.
(3) Test report, (i) The type of study, i.e. wastewater, activated sludge, mixing
zone or anaerobic digester sludge test, should be clearly stated in the test report.
(ii) The test report should also contain the information in paragraphs (i)(3)(ii)(A)
through (i)(3)(ii)(D), when appropriate.
(A) Test substances:
(1) Common names, substance names, Chemical Abstracts Service (CAS)
numbers, structural formulas and relevant physical/chemical properties of test and
reference substances;
(2) Substance names, CAS numbers, structural formulas and relevant
physical/chemical properties of substances used as standards for identification of
metabolites;
(3) Purities of and nature of known impurities in test and reference substances;
(4) Radiochemical purity and specific activity of radiolabeled substances;
(5) Positions within the molecule of radiolabeled atoms.
(B) Environmental samples:
(1) Source of environmental samples including geographical location and relevant
10
-------
data regarding known prior or existing exposure to the test substance and related
substances;
(2) Logic used to estimate relevant environmental concentration;
(3) Time, date and field conditions relevant to collection;
(4) Temperature, pH, DO and redox potential as appropriate;
(5) Suspended solids level, biological oxygen demand (BOD), chemical oxygen
demand (COD) and total organic carbon (TOC) as appropriate;
(6) Time between collection and use in the laboratory test, sample storage
conditions and any pretreatment of the sample prior to initiating the test.
(C) Experimental conditions:
(1) Dates of performance for the study;
(2) Amount of test substance applied, and test and reference substance
concentrations;
(3) Method of application of the test substance and associated logic for selection;
(4) Incubation conditions including lighting, aeration type, temperature;
(5) Information on analytical techniques and the method(s) used for radiochemical
measurements;
(6) Number of replicates.
(D) Results:
(1) Precision and sensitivity of the analytical methods including the LOD and
LOQ;
(2) Recovery for each analyte and disposition of dosed radioactivity for each
sampling time and system, in tabular form;
(3) Average mass balance with standard deviation across all time points for each
system;
(4) Procedures and models used to estimate biodegradation rates from the data;
(5) Biodegradation rates and related parameters with relevant standard errors
along with correlation coefficients (R2) and F statistics for the selected models;
11
-------
(6) Additional characterization or identification of any of major metabolites,
where appropriate and possible;
(7) Proposed pathway of transformation (optional);
(8) Discussion of results.
(j) 314A: Biodegradation in a sewer system—(1) General, (i) This test
is designed to provide rates of primary and ultimate biodegradation for a substance in raw
wastewater during its time in a sewage conveyance system. It is based on a procedure
originally published in Matthijs et al. (see paragraph (o)(6) of this guideline). The test
duration is typically kept short (<96 hr) in order to simulate the residence time in a sewer,
but it can be extended to assess the extent to which a substance can be degraded by
wastewater microbes. It is useful to perform this test if there is sufficient time in a sewer
for the substance to undergo significant biodegradation and loss. Hence, it is most useful
for relatively labile substances discharged to large municipal sewer systems. Aeration
levels in a sewer can vary widely. To be conservative and simulate the more typical
conditions in a sewer, the test is conducted under low DO conditions (<1 mg/1). In order
to achieve this but avoid anoxic conditions (e .g. DO levels < 0.2 mg/L), DO, or the
corresponding oxygen concentration in the test vessel headspace, should be monitored
periodically. Air, oxygen, or nitrogen may be added periodically to the test vessels to
maintain DO in this range.
(ii) For existing substances consistently present in wastewater, freshly collected
wastewater incubated with a tracer level of radiolabeled test substance will provide the
most realistic kinetic parameters regarding the current load of the substance. For
substances not consistently present in wastewater, sufficient test substance (radiolabeled
and unlabeled) should be added to approximate the expected concentration in wastewater
during an episodic release or following commercialization of a new substance. This
concentration would reflect the total mass released and the volume of wastewater in
which the release is diluted. Approaches for estimating wastewater concentration can be
found in Holman (see paragraph (o)(21) of this guideline) and the European Technical
Guidance Document (see paragraph (o)(22) of this guideline). In most situations, the
substance and its degrader populations will not be at steady state and the observed
kinetics will be pseudo first-order or second-order Monod.
(2) Principle of the test, (i) Test substance is incubated with abiotic and biotic
wastewater over a period of time under low DO conditions. Biological activity is
inhibited in the abiotic control, which is used for estimating mineralization by difference,
determining extraction efficiency and recovery of the parent molecule, and quantifying
other loss processes such as hydrolysis, oxidation, volatilization or sorption to test
apparatus.
(ii) If an analytical method with the required sensitivity is available, the rate of
parent degradation or transformation can be determined using a non-radiolabeled test
12
-------
substance or by following the disappearance of a substance already in wastewater.
However, ultimate biodegradation cannot be determined unless the biodegradation
pathway is well understood and analytical methods with required sensitivity are available
for potential metabolites.
(iii) Test substance at an environmentally relevant concentration is dosed to both
abiotic and biotic test systems, which are incubated with continuous slow mixing. The
biotic samples are incubated in such a way that DO levels remain at or below 1 mg/1,
which is typical for sewage. Samples are periodically removed for determination of
mineralization and primary biodegradation.
(iv) Tests can be performed using an open batch system or a sealed, flow-through
batch system where traps are used to capture evolved 14CC>2. The closed flow-through
system should be used for volatile test substances and is usually preferred for 14C-lableled
test substances. Open systems are appropriate for nonvolatile 3H-labeled test substances
and for refining the biodegradation kinetics of nonvolatile 14C test materials, whose
ability to be mineralized has previously been established. In the open system,
mineralization to 14CO2 can be determined indirectly by measuring the difference in
residual radioactivity between samples from the biotic and abiotic systems following
acidification. Similarly, mineralization to 3H2O can be determined indirectly by
measuring the difference in radioactivity in a sample following drying. In the flow-
through systems, evolved 14CO2 is measured directly in the base traps. In addition,
dissolved 14CO2 is determined by acidifying samples in a sealed vessel and measuring
radioactivity in a base trap contained in the vessel.
(v) Samples from both systems are analyzed for total radioactivity, extractable
parent and metabolites, and radioactivity associated with the extracted solids. The levels
of parent and metabolites are determined using chromatographic separation and when
appropriate, radio-analytical detection methods. The solids remaining from the extraction
process are combusted to estimate incorporation into biomass by difference, or can be
further fractionated to determine uptake into various components of biomass. A complete
mass balance of the test system is obtained from the sum of all fractions at each
sampling.
(3) Applicability of the test. The method is readily applicable to water-soluble
and poorly water-soluble substances that are nonvolatile. It also can be adapted for
volatile substances. Typically, 14C or 3H labeling of substances is required for the
assessment of mineralization. Either radiolabeled or nonlabeled substance can be used for
the assessment of primary biodegradation.
(4) Description of the test method—(i) Test apparatus. (A) The volume of
wastewater in the test vessels is determined based on the number and volume of the
samples needed for the test. Typically, 1-2 1 of wastewater is placed in 2- or 4-1 flasks.
Ideally, the wastewater is incubated under controlled DO conditions (typically 0.2-1.0
mg/1). This condition can be achieved using an oxygen probe immersed in the wastewater
attached to an oxygen controller connected to an actuator valve, which monitors and
13
-------
controls the aeration of the wastewater (see Figure 3). This aeration is balanced against
continuous sparging with nitrogen to achieve the targeted DO level. Alternatively, the
wastewater can be incubated with stirring but minimum aeration to keep DO levels low,
and nitrogen or air can be added periodically to maintain the DO level. In this case, DO
readings should be reported at regular intervals.
(B) An open test is open to the atmosphere but incubated under conditions that
maintain DO levels at the desired level. Flow-through systems are sealed with an
appropriate closure containing a sampling port with a valve for removing wastewater
samples and connections for influent and effluent gas lines. The closure can be a rubber
stopper, but an alternative type of closure may be necessary when working with a volatile
hydrophobic substance. When testing volatile substances, it is recommended that gas
lines and sampling tubes consist of inert materials (e.g. polytetrafluoroethylene, stainless
steel, glass).
Figure 3. Example of controlled dissolved oxygen flow-through apparatus
Trapping System
(optional)
DO Controller
CO? Trap*
Co-rtoinir^
100mloM 5NKCW
Air
Solenoid
Valve
c
Wastewater
Mixture
(C) The headspace of the test vessel is continuously purged with gas at a rate
sufficient to maintain the wastewater at the desired DO level but not too fast to prevent
efficient trapping of CO2. The test vessel is connected to a series of traps containing
potassium hydroxide (e.g. 1.5 N) or other appropriate CO2 absorbent. An empty trap is
usually included and positioned in front of the absorbent in the trapping train, as a
precaution against backflow or condensation.
14
-------
(ii) Equipment. (A) The standard laboratory equipment listed in this paragraph is
used:
(1) Miscellaneous glassware and pipettes;
(2) Magnetic stirrers or shaker for continuous mixing of the test flasks;
(3) Centrifuge;
(4) pH meter;
(5) Solid CCVacetone or liquid nitrogen bath;
(6) Freeze dryer (lyophilizer);
(7) Oven or microwave oven for dry weight determinations;
(8) Membrane filtration apparatus;
(9) Autoclave;
(10) Facilities to handle radiolabeled substances;
(11) Equipment to quantify 14C and 3H in liquid samples and solid samples (e.g.
liquid scintillation counter);
(12) Equipment to quantify 14C and 3H in solid samples (e.g. sample oxidizer);
(13) Equipment to trap volatilized 14C and 3H from gas trapping system (in-line
activated charcoal trap or equivalent);
(14) Equipment for thin layer chromatography (TLC) or high performance liquid
chromatography (HPLC);
(15) Equipment to quantify 14C and 3H for TLC (scanner) or HPLC (in-line
detector);
(16) Analytical equipment for the determination of the test (and reference)
substance if specific substance analysis is used (e.g. gas chromatograph, high-
performance liquid chromatograph, mass spectrometry).
(B) The laboratory equipment listed in this paragraph is useful but not essential:
(1) Oxygen meter;
15
-------
(2) Oxygen controller with probe and actuator valve;
(3) COD digestion vials;
(4) Nitrogen ammonia reagent set;
(5) Spectrophotometer.
(iii) Selection of wastewater. The source of wastewater should be consistent with
the objective of the simulation test. For a site-specific assessment, the wastewater should
be obtained from the specific sewer system of interest. For a generic assessment samples
should be predominantly derived from domestic sources. Although difficult to duplicate
in practice, the European Technical Guidance Document (TGD) procedure uses 450 mg
suspended solids/1 and 270 mg BOD/1 as default levels for wastewater (see paragraph
(o)(22) of this guideline). In North America, a typical wastewater contains 100 to 350 mg
suspended solids/1 and 110 to 400 mg BOD/1 depending on its strength (see paragraph
(o)(23) of this guideline).
(iv) Collection, transport and storage of wastewater. The wastewater should be
collected from a sewer access point or at the head of a wastewater treatment plant. The
pH, DO and temperature of the sample should be noted at collection. Collection
containers should allow for adequate ventilation and measures should be taken to prevent
the temperature of the wastewater from significantly exceeding the temperature used in
the test. The wastewater is typically stored at test temperature with continuous slow
mixing. Samples should not be stored frozen.
(v) Preparation of test systems. (A) The freshly collected wastewater should be
largely free from coarse particles. Total suspended solids (TSS), COD, pH and NH3
(optional) in the wastewater should be determined.
(B) The preparation of the abiotic system is typically performed using a
combination of chemical and heat sterilization. A proven approach is to add mercuric
chloride (1 g/1) to the wastewater, which is then autoclaved for at least 90 min. Typically
the volume of medium is <= half the volume of the container being autoclaved (e.g. 500
ml activated sludge in a 1-liter container). After cooling, the pH of the abiotic system
should be measured and adjusted to match that of the biologically active system.
Alternative approaches to deactivate the system can also be used.
(vi) Test substance preparation. (A) Ideally, distilled water should be used to
prepare stock solutions of the test and reference substances. When appropriate, an
alternative method may be used to solubilize or disperse the test substance in a manner
consistent with its normal entry into the environmental compartment of interest. Water-
miscible nontoxic solvents may be used when necessary, but attention should be paid to
the associated organic load involved with adding organic solvents. Alternatively, the
sample may be added in a neat form (i.e. without water) to the test system in a manner
that maximizes its even and rapid distribution in the sludge. For substances that are
16
-------
poorly soluble and typically associated with suspended solids in wastewater, it may be
appropriate to adsorb the test substance onto an inert solid carrier, which is then dosed to
the test system. If the test substance cannot be evenly distributed in the test system prior
to the initial sampling point, individual test systems can be prepared that are destructively
sampled at each sampling interval.
(B) The volume of added stock should be of sufficient capacity to ensure rapid
and even distribution of the test substance in the system and accurate administration of
the dose between like systems. Ideally, when dosing with aqueous solutions, the added
volume should be >2 ml and <10 ml; for nontoxic solvents, <0.1 ml/1. If appropriate,
dosing solutions may be prepared in advance and refrigerated. The activity of the stock
solution should be checked by liquid scintillation counting (LSC).
(vii) Test conditions—(A) Test temperature. Incubation should take place in the
dark (preferred) or in diffuse light at a controlled temperature, which may be the field
temperature or a standard laboratory temperature of 20-25°C. Depending on location,
mean annual wastewater temperature ranges from 10 to 20.1°C, with 15.6°C being
representative (see paragraph (o)(23) of this guideline).
(B) Agitation. To keep the solids in suspension, test vessels are minimally
agitated by means of continuous mixing or stirring.
(viii) Test duration. The duration of the test should be sufficient to assess the
biodegradation of the test substance during its normal residence time in the sewer system.
However, it may be extended to obtain additional data points to estimate kinetic
constants, or to assess the completeness of degradation under the conditions of the test.
Conversely, it may be ended before this time if degradation has plateaued.
(ix) Number of test vessels. At a minimum, there should be a single abiotic and
a single biotic test vessel for each test substance concentration. While replicates can be
prepared for each system, more useful kinetic information usually can be gained by
increasing the number of time points at which samples are collected within a system.
(5) Procedure—(i) Dosing. At test initiation, the chamber closure is removed and
the test substance is quantitatively added directly with constant mixing. It is
recommended that the dose be administered in a gradual fashion below the air-water
interface, to ensure uniform distribution of the test substance into the wastewater. The
biotic and abiotic systems are dosed in an identical manner. Generally, the biotic systems
are dosed first, followed by the abiotic systems. Exact timing is typically more critical for
kinetic analyses.
(ii) Sampling schedule. Sampling times are selected based on existing
biodegradation data or the results of a pilot study, as no fixed time schedule for sampling
is universally applicable. A recommended sampling schedule for a rapidly degraded
substance is 15, 30 and 60 min, with additional samplings after 2, 5, 8, 12 and 24 hr and
days 2, 3 and 4. The sampling schedule for a slowly degrading substance should be
17
-------
adjusted so that a sufficient number of measurements are made during the degradation
phase.
(iii) Measurement of mineralization—(A) Indirect measurement of 14COi. (7)
Individual replicate samples (e.g. 1 ml) are collected from each system and deposited in
separate vials, located in a fume hood, that contain sufficient acid (e.g. 1 ml of 0.1 N
HC1) to lower the sample pH to <2.
(2) The samples are bubbled with air for several hours or allowed to stand
overnight to allow the dissolved 14CC>2 to diffuse from the samples. The samples are
combined with a scintillation cocktail that is suitable for the sample matrix and analyzed
by LSC. The percentage of 14CO2 is calculated based on the difference between the total
counts in the biotic and abiotic samples.
(B) Direct measurement of 14COi. (1) Evolved 14CC>2: The first base trap in the
series is removed and quickly capped. The remaining traps are moved forward in the
same order and a fresh trap placed behind the existing traps, and the trapping system
reconnected as quickly as possible. Replicate subsamples (e.g. 1 ml) are removed from
the base trap and transferred to scintillation vials, combined with a scintillation cocktail
that is suitable for the sample matrix, and analyzed by LSC.
(2) Dissolved 14CC>2: Samples (e.g. 10 to 25 ml) are removed through the
sampling port of the test flask. They are then placed in vessels (e.g. Bellco Glass
Biometer 2556-10250) containing a compartment with an appropriate CO2 absorbent (e.g.
1.5 N KOH). The vessels are sealed and sufficient acid (e.g. 6N HC1) is added to lower
the pH of the samples to < 2 without opening the vessels to the atmosphere (see Figure
4). The samples are allowed to sit for a sufficient length of time (e.g. overnight) to allow
CC>2 to diffuse from solution and be trapped from the headspace by the sorbent. Samples
of the sorbent are combined with a scintillation cocktail that is suitable for the sample
matrix and analyzed by LSC.
14,
Figure 4. System for recovering dissolved CC>2
Valvad Port for
Acidifying
Sample from test
vessel (10-20 ml)
1.5 N KOH
(1 -2ml)
18
-------
(C) Indirect measurement of 3H2O. (7) Individual replicate samples (e.g. 1 ml)
are collected from each system and placed in separate vials, located in a fume hood, that
contain sufficient acid (e.g. 1 ml of 0.1 N HC1) to lower the sample pH to <2.
(2) Half of the samples are immediately analyzed directly by LSC for a wet
measurement. The remaining samples are allowed to dry completely to remove the 3H2O.
The samples are combined with a scintillation cocktail that is suitable for the sample
matrix and analyzed by LSC. The percentage 3H2O is calculated based on the difference
between the total counts in the wet and dry samples and the initial level of radioactivity
dosed to the samples.
(iv) Measurement of total radioactivity in wastewater. Replicate small-volume
samples (e.g. 1 ml) are analyzed directly by LSC to quantify the radioactivity remaining
in each system over time. These measurements are used to confirm that the recovery of
radioactivity from the extracted samples is acceptable and to monitor for volatilization.
The total solids in these samples should not exceed 30 mg dry weight in order to avoid
counting efficiency problems.
(v) Measurement of parent and metabolites—(A) Extraction. (7) A sample is
collected from both the abiotic and biotic systems. The sample volume is typically >10
ml, but will depend on the test concentration, specific activity and the sensitivity of the
analytical procedures.
(2) Various approaches can be used for concentrating and extracting the samples.
A proven approach for nonvolatile test substances involves flash freezing the samples,
followed by lyophilization and extraction of the dried residue with appropriate solvent(s)
for parent and metabolites. Flash freezing quickly stops biological activity without
hydrolyzing or otherwise altering labile test substances. Flash freezing is a quick process
if there is sufficient depth in the dry ice/acetone or liquid nitrogen bath to submerge the
sample tube. The level of the bath should be above the sample level in the tube. The
dried solids are extracted and the resulting extracts can be concentrated through
evaporation. The total radioactivity in each extract is then determined by LSC.
(3) For volatile test substances, the sample can be passed through a filter and solid
phase extraction (SPE) column or SPE disk placed in tandem, which are subsequently
eluted with appropriate solvents to recover parent and metabolites. Alternatively, samples
can be centrifuged, and parent and metabolites can be extracted from the liquor by solid
phase or liquid/liquid extraction. The solids can then be extracted directly or mixed with
a drying agent (e.g. sodium sulfate) and allowed to dry prior to extraction with an
appropriate solvent system. An alternative is to extract the solids and dry the extract by
running the solvent through a column containing a drying agent. In some cases, it may be
possible to directly extract the entire aqueous sample with an appropriate solvent system
and then filter it to recover biomass solids. The total radioactivity in all extracts is
determined by LSC. Care should be taken in concentrating extracts containing volatile
19
-------
test materials or metabolites.
(4) Other approaches can be utilized, but with all approaches it is important to
document recoveries and consider the time involved in terminating biological activity and
factor it into the sample times used for kinetic analyses.
(B) Analysis of parent and metabolites. (7) The relative abundance of parent and
metabolites in the extracts can be determined using TLC, HPLC, or other separation
techniques with radioactivity detection.
(2) If sensitive specific analytical methods are available, primary biodegradation
can be assessed by measuring the total residual concentration of test substances and
metabolites instead of using radioisotope techniques. Generally, for nonlabeled
substances, it will only be possible to track parent disappearance from the aqueous phase.
(C) Characterization of metabolites. Whenever possible, the chromatographic
behavior of unknown peaks should be compared to that of predicted metabolites, if
authentic standards exist. Usually, the quantity and purity of metabolites generated in this
test make definitive identification by other direct means impossible. Depending on
chromatographic behavior, it is usually possible to determine if a metabolite is more or
less polar than the parent. This information, combined with known biochemical reactions
and knowledge of when a metabolite appears and disappears in the sequence of
biodegradation, can form an additional basis for inferring its identity. If necessary, the
Kow of major metabolites can be estimated by HPLC (e.g. using OECD 117; see
paragraph (o)(24) of this guideline) using an on-line radioactivity detector.
(vi) Measurement of extracted solids and incorporation into biomass. If the
extracted samples are filtered, the filter will retain carbonate salts as well as
microorganisms from the test system. The filter containing the biosolids is placed in a
scintillation vial and acidified to pH <2 by submerging it in a weak acid solution (e.g. 1
ml of 0.1 N HC1). The samples are allowed to sit for sufficient time (e.g. overnight) for
the dissolved 14CC>2 to diffuse from the samples. The samples are analyzed by LSC. In
the case of non-filtered extracted solids, they are combusted to determine the level of
activity remaining with the solids. The level of radioactivity in the biotic solids above
that in solids from the abiotic control typically represents incorporation of radioactivity
into biomass. The distribution of this radioactivity among various components of biomass
(i.e. nucleic acids, protein, cell wall, etc.) can be determined using a modified Sutherland
and Wilkinson procedure (see paragraphs (o)(7) and (o)(25) of this guideline).
(vii) Measurement of volatilized radioactivity. For volatile test substances,
traps are extracted with appropriate solvents and the radioactivity in the extracts analyzed
by LSC. The relative abundance of parent and metabolites in the extract(s) can be
determined as described above in paragraph (j)(5)(v)(B)(7) of this guideline.
(k) 314B: Biodegradation in activated sludge—(1) General, (i) This
test is designed to assess the extent to which a substance can be degraded in activated
20
-------
sludge and to provide rates of primary and ultimate biodegradation under the conditions
of the test. It is based on a procedure originally published by Federle & Itrich (see
paragraph (o)(7) of this guideline). Activated sludge in its various configurations is the
most common secondary wastewater treatment process. The usefulness of the measured
rates for predicting removal in actual treatment using wastewater treatment models (e.g.
SimpleTreat) will be a function of the fidelity of the simulation to actual conditions in an
activated sludge wastewater treatment plant.
(ii) Four factors determine the test substance concentration in this test: 1) whether
the material is released continuously or episodically; 2) current presence in the
environment; 3) expected presence in the environment for a new chemical; and 4)
analytical sensitivity.
(iii) When a chemical substance is already present in the environment in a
continuous fashion, the most accurate kinetics are obtained by adding a tracer level of the
radiolabeled substance to freshly obtained environmental samples. Under these
circumstances, the normal ratio of substance to degraders is not disrupted and the
observed biodegradation rates reflect in situ rates.
(iv) When a new substance will be released continuously at some future time, the
most accurate rates are obtained when the substance and degrader populations are in a
steady state balance. This situation can be achieved by adding a tracer level of test
substance to activated sludge that has been exposed to the substance under expected
loading and operating conditions in a laboratory continuous activated sludge system (e.g.
OECD 303 A).
(v) When an existing or new substance enters the environment in an episodic
manner, there is not a normal steady state ratio of biomass to test chemical to disrupt, so
the test substance is dosed to freshly collected samples at the level in wastewater
expected for a release event. This concentration should reflect the total mass released and
the volume of wastewater in which the release is diluted. Approaches for estimating
wastewater concentration can be found in Holman (see paragraph (o)(21) of this
guideline) and the European Technical Guidance Document (see paragraph (o)(22) of this
guideline).
(vi) Superseding the previous considerations is analytical sensitivity. When it is
not possible to use ideal (e.g. tracer) levels of test substance due to analytical
consideration, the lowest possible concentration is employed. At high test substance
concentrations, biodegradation may be associated with lag periods related to second-
order processes (i.e. growth), which complicate the kinetic analysis. When this standard
for simulation is not achieved, observed biodegradation rates may not be fully
representative, which should be considered in the interpretation of results. This factor is
particularly important for continuously released substances, which often reach steady
state conditions in wastewater systems.
(2) Principle of the test, (i) The test substance is incubated with abiotic and
21
-------
biotic activated sludge over a period of time. Biological activity is inhibited in the abiotic
control, which is used for estimating mineralization by difference, establishing extraction
efficiency and recovery of the parent molecule, and quantifying other loss processes such
as hydrolysis, oxidation, volatilization or sorption to test apparatus.
(ii) If an analytical method with the required sensitivity is available, the rate of
parent degradation or transformation can be determined using a non-radiolabeled test
substance or by following the disappearance of a substance already in wastewater.
However, ultimate biodegradation cannot be determined unless the biodegradation
pathway is well understood and analytical methods with required sensitivity are available
for potential metabolites.
(iii) Test substance at an environmentally relevant concentration is dosed to both
abiotic and biotic test systems, which are incubated at a relevant temperature with
continuous mixing when appropriate. Samples are periodically removed for
determination of mineralization and primary biodegradation.
(iv) Tests can be performed using an open batch system or a sealed, flow-through
batch system where traps are used to capture evolved 14CC>2. The closed flow-through
system should be used for volatile test substances and usually is preferred for 14C-lableled
test substances. Open systems are appropriate for nonvolatile 3H-labeled test substances
and for refining the biodegradation kinetics of nonvolatile 14C-labeled test substances,
whose ability to be mineralized has previously been established. In the open system,
mineralization to 14CC>2 can be determined indirectly by measuring the difference in
residual radioactivity between samples from the biotic and abiotic systems following
acidification. Similarly, mineralization to 3H2O can be determined indirectly by
measuring the difference in radioactivity in a sample following drying. In the flow-
through systems, evolved 14CC>2 is measured directly in the base traps. In addition,
dissolved 14CC>2 is determined by acidifying samples in a sealed vessel and measuring
radioactivity in a base trap contained in the vessel.
(v) Samples from both systems are analyzed for total radioactivity, extractable
parent and metabolites and radioactivity associated with the extracted solids. The levels
of parent and metabolites are determined using chromatographic separation and when
appropriate radio-analytical detection methods. The solids remaining from the extraction
process are combusted to estimate incorporation into biomass by difference or can be
further extracted using a modified Sutherland and Wilkinson procedure (see paragraphs
(o)(7) and (o)(25) of this guideline) to determine uptake into various components of
biomass. A complete mass balance of the test system is obtained from the sum total of all
fractions at each sampling.
(3) Applicability of the test. The method is readily applicable to water-soluble or
poorly water-soluble substances that are nonvolatile. It can also be adapted for volatile
materials. Typically, 14C or 3H radiolabelling is required for the assessment of
mineralization. Both either radiolabelled or nonlabelled substance can be used for the
assessment of primary biodegradation.
22
-------
(4) Description of the test method, (i) Test Apparatus. (A) The volume of
activated sludge in the test systems is determined based upon the number and volume of
the samples needed for the assessment. Typically, 1 to 2 liters of sludge are placed into 2-
or 4-liter flasks. Open batch test systems are generally closed with a foam or cotton
stopper to minimize evaporative loss of water. Flow-through systems are sealed with an
appropriate closure containing a sampling port with a valve for removing mixed liquor
suspended solids (MLSS) samples and connections for influent and effluent gas lines (see
Figure 2). This closure can be a rubber stopper, but glass is recommended when working
with a volatile hydrophobic test material. When testing volatile substances, it also is
recommended that gas lines and sampling tubes consist of inert materials (e.g.
polytetrafluoroethylene, stainless steel, glass).
(B) The headspace of the test vessel is continuously purged with air or CO2-free
air at a rate sufficient to maintain the activated sludge in an aerobic condition, but not too
fast to prevent efficient trapping of CO2. The test vessel is connected to a series of traps
containing a potassium hydroxide (e.g. 1.5 N) or other appropriate CC>2 absorbent. An
empty trap is usually included and positioned in front of the absorbent in the trapping
train, as a precaution against backflow or condensation.
(ii) Equipment. The standard laboratory equipment listed in this paragraph is
used:
(A) Miscellaneous glassware and pipettes;
(B) Magnetic stirrers or shaker for continuous mixing of the test flasks;
(C) Centrifuge;
(D) pH meter;
(E) Solid CCVacetone or liquid nitrogen bath;
(F) Freeze dryer (lyophilizer);
(G) Oven or microwave oven for dry weight determinations;
(H) Membrane filtration apparatus;
(I) Autoclave;
(J) Facilities to handle radiolabeled substances;
(K) Equipment to quantify 14C and 3H in liquid samples and solid samples (e.g.
liquid scintillation counter);
23
-------
14
(L) Equipment to quantify C and H in solid samples (e.g. sample oxidizer);
(M) Equipment to trap volatilized 14C and 3H from gas trapping system (in-line
activated charcoal trap or equivalent);
(N) Equipment for TLC or HPLC;
(O) Equipment to quantify 14C and 3H for TLC (scanner) or HPLC (in-line
detector);
(P) Analytical equipment for the determination of the test (and reference)
substance if specific substance analysis is used (e.g. gas chromatograph, high
performance liquid chromatograph, mass spectrometer).
(iii) Selection of activated sludge source. (A) The source of activated sludge
should be consistent with the objective of the simulation test. For a site-specific
assessment, the activated sludge should be obtained from the specific wastewater
treatment plant of interest. For a generic assessment activated sludge should be obtained
from a typical wastewater treatment plant receiving predominantly domestic wastewater.
If the substance is currently a component of wastewater entering the wastewater
treatment facility or is episodically released to wastewater, freshly collected activated
sludge will be ideal for the test.
(B) For a new substance that will be continuously released to wastewater,
activated sludge ideally should be obtained from a laboratory-scale treatment system
[OECD 303 A] (see paragraph (o)(2) of this guideline), which has been fed wastewater
amended with unlabeled test substance. The source of the starting sludge, wastewater
(influent) and the operating conditions (influent concentration, hydraulic retention time,
solids retention time) for the laboratory unit should accurately reflect site-specific or
generic conditions. In the case of the latter, the TGD specifies a hydraulic retention time
(HRT) of 6.9 hr and a solids retention time (SRT) of 9.2 d in its generic scenario for
wastewater treatment (see paragraph (o)(22) of this guideline). The TGD also provides
guidance on estimating wastewater concentration based on expected usage volumes. In
general, steady state will be reached within 2 to 3 SRTs after which point the sludge can
be used for testing.
(iv) Collection, transport and storage of activated sludge. The activated
sludge should be collected from a well-mixed region of the aeration basin. The pH and
temperature of the sample should be noted at collection. Collection containers should
allow for adequate ventilation and measures should be taken to prevent temperature of
the sludge from significantly exceeding the temperature used in the test. The activated
sludge is typically stored at test temperature with continuous aeration. Samples should
not be stored frozen.
(v) Preparation of the test systems. (A) The activated sludge should be sieved
through a 2-mm screen. The TSS concentration should be measured and if necessary
24
-------
adjusted to the target concentration. The TGD uses a default level of 4000 mg/1 in its
generic scenario (see paragraph (o)(22) of this guideline). However, 2500-3000 mg/1 may
be more typical for North America (see paragraph (o)(23) of this guideline). The sludge
can be diluted with liquor or tap water if the solids concentration is too high.
Alternatively, if the solids concentration is too low, the solids can be allowed to settle and
some of the liquor can be decanted. A final TSS level and pH should then be determined.
(B) Abiotic sludge is typically prepared using a combination of chemical and heat
sterilization. A proven approach is to add mercuric chloride solution (1 g/1) to the sludge,
which is then autoclaved for at least 90 min. Typically the volume of medium is <= half
the volume of the container being autoclaved (e.g. 500 ml activated sludge in a 1-liter
container). After cooling, the pH of the abiotic sludge should be measured and adjusted
to match that of the biologically active sludge. Alternative approaches to deactivate the
sludge can also be used.
(vi) Test substance preparation. (A) Ideally, distilled water should be used to
prepare stock solutions of the test and reference substances. When appropriate, an
alternative method may be used to solubilize or disperse the test substance in a manner
consistent with its normal entry into the environmental compartment of interest. Water-
miscible nontoxic solvents may be used when necessary, but attention should be paid to
the associated organic load involved with adding organic solvents. Alternatively, the
sample may be added in a neat form to the test system in a manner that maximizes its
even and rapid distribution in the sludge. For materials that are poorly soluble and
typically associated with suspended solids in wastewater, it may be appropriate to adsorb
the test material onto an inert solid carrier, which is then dosed to the test system. If the
test material cannot be evenly distributed in the test system prior to the initial sampling
point, individual test systems can be prepared that are destructively sampled at each
sampling interval.
(B) The volume of added stock should be of sufficient capacity to ensure rapid
and even distribution of the test substance in the system and accurate administration of
the dose between like systems. Ideally, when dosing with aqueous solutions, the added
volume should be >2 ml and <10 ml; for nontoxic solvents, <0.1 ml/ L. If appropriate,
dosing solutions may be prepared in advance and refrigerated. The activity of the stock
should be checked by LSC.
(vii) Test conditions—(A) Temperature. Incubation should take place in the
dark (preferred) or in diffuse light at a controlled temperature, which may be the field
temperature or a standard laboratory temperature of 20-25°C.
(B) Agitation. To keep the sludge well mixed and in suspension, the test vessels
are agitated by means of continuous shaking or stirring. Agitation also facilitates oxygen
transfer from the headspace to the liquid so that aerobic conditions can be adequately
maintained.
(viii) Test duration. The duration of the test should be sufficient to assess the
25
-------
biodegradation of the test substance during its normal residence time in an activated
plant, or its completeness of degradation under such conditions. Normally, the test period
will be 28 d. However, it may be extended to obtain additional data points in order to
estimate kinetic constants or to assess the completeness of degradation under the
conditions of the test. Conversely, it may be ended before this time if degradation has
plateaued.
(ix) Number of test vessels. At a minimum, there should be a single abiotic and
a single biotic test vessel for each test substance concentration. While replicates can be
prepared for each system, more useful kinetic information usually can be gained by
increasing the number of time points sampled within a system.
(5) Procedure—(i) Dosing. At test initiation, the chamber closure is removed
and the test substance is quantitatively added directly to the activated sludge with
constant mixing. It is recommended that the dose be administered gradually below the
air-water interface, to ensure uniform distribution of the test substance in the sludge. The
biotic and abiotic systems are dosed in an identical manner. Generally, the biotic systems
are dosed first, followed by the abiotic systems. Exact timing is typically more critical for
kinetic analyses.
(ii) Sampling schedule. Sampling times are selected based on existing
biodegradation data or the results of a pilot study, as no fixed time schedule for sampling
is universally applicable. A recommended sampling schedule for a rapidly degraded
substance would be 5, 15, 30, 45, 60 and 90 min, with additional samplings after 2, 3, 5,
8, 12 and 24 hr. Subsequent samples could be taken after 2, 3, 4, 5, 6 and 7 d and weekly
until day 28. The sampling schedule for a slowly degrading substance should be adjusted
so that a sufficient number of measurements are made during the degradation phase.
(iii) Measurement of mineralization—(A) Indirect measurement of 14COi. (7)
Individual replicate samples (e.g. 1 ml) are collected from each system and placed in
separate vials, located in a fume hood, that contain sufficient acid (e.g. 1 ml of 0.1 N
HC1) to lower the sample pH to <2. The total solids in the samples should not exceed 30
mg dry weight.
(2) The samples are bubbled with air for several hours or allowed to stand
overnight to allow the dissolved 14CC>2 to be stripped from the samples. The samples are
combined with a scintillation cocktail that is suitable for the sample matrix and analyzed
by LSC. The percentage of 14CC>2 is calculated based on the difference between the total
counts in the biotic and abiotic samples.
(B) Direct measurement of 14COi. (-/) For rapidly degrading substances, it can be
difficult to measure accurately the rate of 14CC>2 evolved due to the rate of the mass transfer
of 14CC>2 from the headspace into the base trap. Under these conditions, it is recommended
that indirect measurement of 14CC>2 be conducted simultaneously with direct
measurement.
26
-------
(2) Evolved 14CC>2: The first base trap in the series is removed and quickly
capped. The remaining traps are moved forward in the same order and a fresh trap placed
behind the existing traps, and the trapping system reconnected as quickly as possible.
Replicate subsamples (e.g. 1 ml) are removed from the base trap and transferred to
scintillation vials, combined with a scintillation cocktail that is suitable for the sample
matrix, and analyzed by LSC.
(3) Dissolved 14CO2: Samples (e.g. 10 to 25 ml) are removed through the
sampling port of the test flask. They are then placed in vessels (e.g. Bellco Glass
Biometer 2556-10250) containing a compartment with an appropriate CC>2 absorbent (e.g.
1.5 N KOH). The vessels are sealed and sufficient acid (e.g. 6N HC1) is added to lower
the pH of the samples to <2 without opening the vessels to the atmosphere (see Figure 4).
The samples are allowed to sit for a sufficient length of time (e.g. overnight) to allow
CC>2 to diffuse from solution and be trapped from the headspace by the sorbent. Samples
of the sorbent are combined with a scintillation cocktail that is suitable for the sample
matrix and analyzed by LSC.
(C) Indirect measurement of 3H2O. (7) Individual replicate samples (e.g. 1 ml)
are collected from each system and placed in separate vials, located in a fume hood, that
contain sufficient acid (e.g. 1 ml of 0.1 N HC1) to lower the sample pH to <2. The total
solids in the samples should not exceed 30 mg dry weight.
(2) Half of the samples are immediately analyzed directly by LSC for a wet
measurement. The remaining samples are allowed to dry completely to remove the 3H2O.
The samples are combined with a scintillation cocktail that is suitable for the sample
matrix and analyzed by LSC. The percentage 3H2O is calculated based on the difference
between the total counts in the wet and dry samples and the initial level of radioactivity
dosed to the samples.
(iv) Measurement of radioactivity in MLSS. Small-volume samples of MLSS
(e.g. 1 ml) are analyzed directly by LSC to quantify the radioactivity remaining in each
system over time. These measurements are used to confirm that the recovery of
radioactivity from the extracted samples is acceptable, and to monitor for volatilization.
To avoid counting efficiency problems, the total solids in these samples should not
exceed 30 mg dry weight.
(v) Measurement of parent and metabolites—(A) Extraction. (7) A sample of
MLSS is collected from both the abiotic and biotic systems. The sample volume is
typically >10 ml, but will depend on the test concentration, specific activity and the
sensitivity of the analytical procedures.
(2) Various approaches can be used for concentrating and extracting the samples.
A proven approach for nonvolatile test substances involves flash freezing the samples,
followed by lyophilization and extraction of the dried residue with appropriate solvent(s)
for parent and metabolites. Flash freezing quickly stops biological activity without
hydrolyzing or otherwise altering labile test substances. Flash freezing is a quick process
27
-------
if there is sufficient depth in the dry ice/acetone or liquid nitrogen bath to submerge the
sample tube. The level of the bath should be above the sample level in the tube. The
resulting extracts can be concentrated through evaporation and the total radioactivity in
each extract determined by LSC.
(3) For volatile test substances, MLSS can be passed through a filter and solid
phase extraction (SPE) column or SPE disk placed in tandem, which are subsequently
eluted with appropriate solvents to recover parent and metabolites. Alternatively, samples
can be centrifuged, and parent and metabolites extracted from the liquor by solid phase or
liquid/liquid extraction. The solids can then be extracted directly, or mixed with a drying
agent (e.g. sodium sulfate) and allowed to dry prior to extraction with an appropriate
solvent system. An alternative is to extract the solids and then remove the water from the
solvent by running it through a column containing a drying agent. In most cases, it is not
efficient to use liquid/liquid extraction to recover parent and metabolites from MLSS.
The total radioactivity in all extracts is determined by LSC. Care should be taken in
concentrating extracts containing volatile test substances or metabolites.
(4) Other approaches can be utilized, but with any approach it is important to
document recoveries and consider the time involved in terminating biological activity,
and factor it into the sample times used for kinetic analyses.
(B) Analysis of parent and metabolites. (7) The relative abundance of parent
and metabolites in the extracts can be determined using TLC, HPLC or other separation
techniques with radioactivity detection.
(2) If sensitive specific analytical methods are available, primary biodegradation
can be assessed by measuring the total residual concentration of test substances and
metabolites instead of using radioisotope techniques.
(C) Characterization of metabolites. Whenever possible, the chromatographic
behavior of unknown peaks should be compared to that of predicted metabolites, if
authentic standards exist. Usually, the quantity and purity of metabolites generated in this
test make definitive identification by other direct means impossible. Depending on
chromatographic behavior, it is usually possible to determine if a metabolite is more or
less polar than the parent. This information, combined with known biochemical reactions
and knowledge of when a metabolite appears and disappears in the sequence of
biodegradation, can form an additional basis for inferring its identity. If necessary, the
Kow of major metabolites can be estimated by HPLC (e.g. using OECD 117; see
paragraph (o)(24) of this guideline) using an on-line radioactivity detector.
(vi) Measurement of extracted solids and incorporation into biomass. The
extracted solids are combusted to determine the level of activity remaining with the
solids. The level of radioactivity in the biotic solids above that in solids from the abiotic
control typically represents incorporation of radioactivity into biomass. The distribution
of this radioactivity among various components of biomass (i.e. nucleic acids, protein,
cell wall, etc.) can be determined using a modified Sutherland and Wilkinson procedure
28
-------
(see paragraphs (o)(7) and (o)(25) of this guideline).
(vii) Measurement of volatilized radioactivity. For volatile test substances,
traps are extracted with appropriate solvents and the radioactivity in the extracts analyzed
by LSC. The relative abundance of parent and metabolites in the extract(s) can be
determined as described in paragraph (k)(5)(v)(B)(7) of this guideline.
(1) 314C: Mineralization and transformation in anaerobic digester sludge—
(1) General, (i) This test is designed to assess the extent to which a substance can be
degraded during anaerobic digestion. It also provides rates of primary and ultimate
biodegradation under the conditions in a digester. Anaerobic digestion is commonly used
to stabilize and reduce the mass of sludge generated by wastewater treatment plants.
Biodegradation during anaerobic digestion is particularly relevant for substances with a
high tendency to partition to primary and secondary sludge. Removal during anaerobic
digestion can significantly decrease the level of a substance present in sludge used as a
soil amendment. The test is also easily adaptable for septage, to evaluate anaerobic
biodegradation in septic tanks. The 314C test should be carried out after having
conducted a 314B test (and sometimes a 314A test) since this (these) test(s) would help
provide perspective on both the presence and levels of parent and potential degradation
products associated with the primary and secondary sludge entering anaerobic digestion.
(ii) Given that many digesters are operated as batch or plug-flow systems, which
have long residence times (30 - 60 d), it is not essential that the substance and its
degrader populations be at steady state at the initiation of a test, to generate useful
degradation rates for exposure assessments.
(iii) To simulate conditions associated with episodic release of a substance,
freshly collected digester sludge can be incubated with the maximum concentration of
test substance expected to occur in sludge as a result of periodic releases. Approaches for
estimating expected sludge concentrations can be found in Holman (see paragraph
(o)(21) of this guideline) and the European Technical Guidance Document (see paragraph
(o)(22) of this guideline). For existing substances continuously discharged to wastewater,
freshly collected digester sludge can be incubated with radiolabeled test substance at a
tracer level, or the concentration expected to occur in digester sludge. Usually, sufficient
time is available within the time frame of the test for acclimation of the sludge to a new
(test) substance. However, a laboratory anaerobic reactor operated in a draw-and-fill
mode, amended with the substance at its expected concentration in sludge, can be
considered as an option for generating acclimated sludge. This type of sludge should
yield the most accurate kinetic data for a new substance that will be continuously exposed
to wastewater.
(2) Principle of the test, (i) The test substance is incubated with abiotic and
biotic digester sludge over a period of time. Biological activity is inhibited in the abiotic
control, which is used for estimating mineralization by difference, establishing extraction
efficiency and recovery of the parent molecule, and quantifying other loss processes such
as hydrolysis, oxidation, volatilization or sorption to test apparatus.
29
-------
(ii) If an analytical method with the required sensitivity is available, the rate of
parent degradation or transformation can be determined using a non-radiolabeled test
substance or by following the disappearance of a substance already in wastewater.
However, ultimate biodegradation of non-radiolabeled substances cannot be determined
unless the biodegradation pathway is well understood and analytical methods with
required sensitivity are available for potential metabolites.
(iii) Test substance at an environmentally relevant concentration is dosed to both
abiotic and biotic test systems, which are incubated at a relevant temperature under static
conditions with occasional or continuous mixing. Samples are periodically removed for
determination of mineralization and primary biodegradation.
(iv) Tests can be performed using an open batch system or a sealed, flow-through
batch system where traps are used to capture evolved 14CO2 and 14CH4. The closed flow-
through system is should be used for volatile test substances and usually is preferred for
14C-labeled test substances. Open systems are appropriate for nonvolatile 3H-labeled test
substances and for refining the biodegradation kinetics of nonvolatile 14C-labeled test
substances, whose ability to be mineralized has previously been established. In the open
system, mineralization to 14CO2 and 14CH4 can be determined indirectly by measuring the
difference in residual radioactivity between samples from the biotic and abiotic systems
following acidification. Similarly, mineralization to 3H2O can be determined indirectly by
measuring the difference in residual radioactivity between samples following drying. In
the closed batch system, gas sampling bags may also be used to trap gas from anaerobic
digester sludge. In the flow-through systems, evolved 14CO2 is measured directly in the
base traps, and 14CH4 is combusted to 14CO2 which is measured directly in a second set of
base traps. As an option, dissolved 14CO2 can be determined by acidifying samples in a
sealed vessel and measuring radioactivity in a base trap contained in the vessel.
(v) Samples from both systems are analyzed for total radioactivity, extractable
parent and metabolites and radioactivity associated with the extracted solids. The level of
parent and metabolites is determined using chromatographic separation and, when
appropriate, radio-analytical detection methods. The solids remaining from the extraction
process are combusted to estimate incorporation into biomass by difference, or can be
further fractionated to determine uptake into various components of biomass. A complete
mass balance of the test system is obtained from the sum of all fractions at each
sampling.
(3) Applicability of the test. The method is readily applicable to water-soluble or
poorly water-soluble substances that are nonvolatile. It also can be adapted for volatile
substances. Typically, 14C or 3H labeling of substances is required for the assessment of
mineralization. Either radiolabeled or nonlabeled substance can be used for the
assessment of primary biodegradation.
(4) Description of the test method—(i) Test apparatus. (A) The volume
of digester sludge in the test vessels is based on the number and volume of the samples
needed for the test. Typically, 0.25 to 1 liter of digester sludge, diluted with an anaerobic
30
-------
salts medium, is placed in 0.5 to 2-1 containers. The sludge is diluted to facilitate
quantitative removal of subsamples during the study. Anaerobic sludge should be
protected from oxygen at all times including setup and sampling.
(B) Open batch systems are generally closed with a foam or cotton stopper to
minimize evaporative loss of water and are incubated inside an anaerobic chamber under
a reducing atmosphere. Flow-through test vessels are sealed with an appropriate closure
containing a sampling port with a valve for removing sludge samples, and connections
for influent and effluent gas lines. This closure can be a rubber stopper, but glass is
recommended when working with a volatile hydrophobic test substance. When testing
volatile substances, it also is recommended that gas lines and sampling tubes consist of
inert materials (e.g. polytetrafluoroethylene, stainless steel, glass).
(C) The flow-through system is a modification of that originally described by
Steber and Wierich (see paragraph (o)(10) of this guideline) and later refined by Nuck
and Federle (see paragraph (o)(ll) of this guideline). An example of a typical system is
shown in Figure 5. The test vessels are continuously purged with a flow of nitrogen and
connected to a series of traps containing potassium hydroxide solution (1.5 N) or other
appropriate CC>2 absorbent. An empty trap is usually included and positioned in front of
the absorbent in the trapping train, as a precaution against backflow or condensation. The
effluent gases from these traps are mixed with oxygen and passed through a quartz
column packed with cupric oxide and maintained at approximately 800°C in a tube
furnace, to combust methane to CC>2. The gas exiting the combustion column is then
passed through another series of base traps.
(ii) Equipment. (A) The standard laboratory equipment listed in this paragraph is
used:
(1) Miscellaneous glassware and pipettes;
(2) Magnetic stirrers or shaker for continuous mixing of the test flasks;
31
-------
Figure 5. Example of flow-through anaerobic test apparatus
Anaerobic Sludge
Mineral Salts
"C Test Material
WATER BATH 35° C
4L4E
C03 Trans
™TO Containing
Tr*P 100 ml of U H
KOH'or-'CO,
Cupric Oxide
5?\o :wj-£>
vaire TUBE FURNACE
TTL
KOH'or-'CH,
(%) Centrifuge;
(4) pH meter;
f5j Solid CO2/acetone or liquid nitrogen bath;
(6) Freeze dryer (lyophilizer);
(7) Oven or microwave oven for dry weight determinations;
(8) Membrane filtration apparatus;
(9) Autoclave;
(10) Facilities to handle radiolabeled substances;
(11) Equipment to quantify 14C and 3H in liquid samples and solid samples (e.g.
liquid scintillation counter);
(12) Equipment to quantify 14C and 3H in solid samples (e.g. sample oxidizer);
(13) Equipment to trap volatilized 14C and 3H from gas trapping system (in-line
activated charcoal trap or equivalent);
32
-------
(14) Equipment for TLC or HPLC;
14
(15) Equipment to quantify C and H for TLC (scanner) or HPLC (in-line
detector);
(16) Analytical equipment for the determination of the test (and reference)
substance if specific substance analysis is used (e.g. gas chromatograph, high-
performance liquid chromatograph, mass spectrometer).
(B) The specialized equipment listed in this paragraph may be used:
(1) Anaerobic chamber;
(2) Tube furnaces;
(3) Redox probe and mV meter.
(iii) Selection of digester sludge source. The source of digester sludge should be
consistent with the objective of the simulation test. For a site-specific assessment, the
sludge should be obtained from the specific digester system of interest. For a generic
assessment digester sludge should be obtained from a typical single-stage or first-stage
digester receiving primary and secondary sludge from a wastewater treatment plant,
receiving predominantly domestic wastewater. If the substance is currently a component
of the wastewater entering the treatment facility or is episodically released to wastewater,
freshly collected digester sludge will be ideal for the test. For a new substance that will
be continuously released to wastewater, acclimated sludge from a laboratory anaerobic
reactor may be more appropriate. For a generic assessment, this reactor should simulate
the operation of a single-stage anaerobic digester, and be semi-continuously fed sludge
that consists of combined primary and secondary sludge solids from a wastewater
treatment plant receiving predominantly domestic wastewater, which has been amended
with test substance at its expected sludge concentration for approximately 60 days.
(iv) Collection, transport and storage of digester sludge. The digester sludge
should be collected from the digester in a manner that protects it from oxygen. The use of
wide-mouth bottles, constructed from high-density polyethylene or a similar material that
can expand, is recommended for the collection of digester sludge. The temperature of the
sample should be noted at collection. Sample containers should be tightly sealed. During
transport, the temperature of the sample should not significantly exceed the temperature
used in the test. The digested sludge is typically stored under the exclusion of oxygen at
test temperature. Storage containers should be vented in a manner that releases excess
biogas but does not allow ambient air into the container.
(v) Preparation of the test systems—(A) Dilution medium. An appropriate
volume of the mineral salts solution listed in this paragraph should be prepared prior to
test initiation. This solution is autoclaved for 30 min with slow exhaust, and allowed to
cool overnight in an anaerobic chamber or under an anaerobic atmosphere.
33
-------
Ingredient Amount
Potassium phosphate monobasic, KH2PO4 8.5 mg/1
Potassium phosphate dibasic, K2HPO4 21.8 mg/1
Sodium phosphate dibasic heptahydrate, Na2HPO4» 7H2O.. 50.3 mg/1
Ammonium chloride, NFLtCl 20.0 mg/1
Magnesium sulfate heptahydrate, MgSO4»7H2O 2.2 mg/1
Calcium chloride anhydrous, CaQ2 2.8 mg/1
Ferrous chloride, FeCl2'4H2O 0.25 mg/1
Deionized water to volume
(B) Digester sludge. (7) The digester sludge ideally should be stored and
manipulated inside an anaerobic chamber or under an anaerobic atmosphere. However,
other approaches may be utilized to protect the sludge from exposure to oxygen. The
digester sludge should be sieved through a 2-mm screen. The total solids concentration
should be measured.
(2) A workable solids level that can be sampled during the study is approximately
25,000 mg/1. If the solids are too high, they can be diluted with the dilution media.
Alternatively, if the solids concentration is too low, the solids can be allowed to settle, the
liquor decanted and the sludge resuspended in the dilution medium. A final solids level
and pH should then be determined.
(3) The preparation of the abiotic sludge is typically performed using a
combination of chemical and heat sterilization. A proven approach is to add mercuric
chloride solution (1 g/1) to the sludge, which is then autoclaved for at least 90 min.
Typically the volume of medium is <= half the volume of the container being autoclaved
(e.g. 500 ml activated sludge in a 1-liter container). After cooling, the pH of the abiotic
sludge should be measured and adjusted to match that of the biologically active sludge.
Alternative approaches to deactivate the sludge can also be used. When preparing the
abiotic sludge it should be stored and manipulated in an anaerobic chamber, or another
approach utilized to protect it from exposure to oxygen.
(vi) Test substance preparation. (A) Distilled water should be used to prepare
stock solutions of the test and reference substances. When appropriate, an alternative
method may be used to solubilize or disperse the test substance in a manner consistent
with its normal entry into digester sludge. When practical, the dosing solutions should be
equilibrated overnight in a reducing atmosphere to remove DO prior to use. The volume
of added stock should be of sufficient capacity to ensure rapid and even distribution of
the test substance in the system and accurate administration of the dose between like
systems. Ideally, when dosing with aqueous solutions, the added volume should be >1
ml and <10 ml; for nontoxic solvents, <0.1 ml/ L. The activity of the stock should be
checked by LSC. For substances that are poorly soluble and typically associated with
sludge solids, it may be appropriate to adsorb the test material onto an inert solid carrier,
which is then dosed to the test system. If the test material cannot be evenly distributed in
the test system prior to the initial sampling point, individual test systems can be prepared
that are destructively sampled at each sampling interval.
34
-------
(B) As an alternative, the test substance can be applied to dried inactive sludge
solids, which can be mixed into the test system. Water-miscible nontoxic solvents may be
used when necessary, but attention should be paid to the associated organic load involved
with adding organic solvents. In addition, the test substance may be added in a neat form
in a manner that maximizes its even and rapid distribution in the sludge.
(vii) Test conditions—(A) Test temperature. Incubation should take place in the
dark (preferred) or in diffuse light at a controlled temperature, which may be the typical
operating temperature for an anaerobic digester (35°C +/- 3°C), a field temperature, or a
standard laboratory temperature of 20-25°C.
(B) Agitation. To simulate the static conditions that occur in a digester, the test
vessels are usually not continuously mixed. During sampling, they should be kept well
mixed to ensure a representative sample. Additionally, they can be gently agitated for a
few minutes, 2 or 3 times per week.
(viii) Test duration. The duration of the test should be sufficient to assess the
biodegradation of the test substance during its normal residence time in an anaerobic
sludge digester. Normally, the test period will be approximately 60 d. However, it may be
extended to obtain additional data points, in order to estimate kinetic constants or assess
the completeness of degradation under the conditions of the test. Conversely, it may be
ended before this time if degradation has plateaued.
(ix) Number of test vessels. At a minimum, there should be a single abiotic and
a single biotic test vessel for each test substance concentration. Replicates can be
prepared for specific substance analysis. These are maintained under anaerobic
conditions but typically not connected to the mineralization apparatus, and can be
sub sampled or sacrificed at a particular sampling point.
(5) Procedure—(i) Dosing. At test initiation, the test substance is
quantitatively added directly to the digester sludge with constant mixing. Dosing should
be done in such a manner that the test system is protected from exposure to oxygen. It is
recommended that the dose be administered gradually below the air-water interface, to
ensure uniform distribution of the test substance in the sludge. The biotic and abiotic
systems are dosed in an identical manner.
(ii) Sampling schedule. Sampling times are selected based on existing
biodegradation data or the results of a pilot study, as no fixed time schedule for sampling
is universally applicable. A recommended sampling schedule for a rapidly degraded
substance would be 30, 60, and 120 min, with additional samplings after 4, 8 and 24 hr.
Subsequent samples could be taken after 2, 4 and 7 d and weekly until day 56. The
sampling schedule for slowly degrading substances should be adjusted so that a sufficient
number of measurements are made during the degradation phase.
(iii) Measurement of mineralization—(A) Indirect measurement of 14C Gases
35
-------
(14COi and 14CH4). Direct LSC counting of samples is not possible due to the high solids
levels in the samples. Therefore, samples are centrifuged and the supernatant analyzed for
total radioactivity by LSC, and the solids combusted and then analyzed for radioactivity
to determine the total radioactivity in the sample. Individual replicate samples (e.g. 1 ml)
of digester sludge are collected from each system and placed in centrifuge tubes that
contain sufficient acid (e.g. 0.025 ml of HC1) to lower the sample pH to <2. The samples
are centrifuged and the supernatant transferred to a scintillation vial, which is allowed to
stand overnight for the dissolved 14CO2 to diffuse from the samples. The samples are
combined with a scintillation cocktail that is suitable for the sample matrix and analyzed
by LSC. The solids remaining in the centrifuge tube are combusted using a sample
oxidizer prior to LSC. The percentage of total 14C gas produced is calculated based on the
difference between the total counts in the biotic and abiotic samples.
(B) Direct measurement of 14CO2 and 14CH4. (7) Evolved 14CO2 and 14CH4:
Direct measurement of 14CO2 and 14CIL; is possible only in a sealed flow-through batch
system with connected base traps. For 14CO2, the first base trap in the first trapping train
is removed and quickly capped. The remaining traps are moved forward in the same
order and a fresh trap placed behind the existing traps, and the trapping system
reconnected as quickly as possible. Replicate subsamples (e.g. 1 ml) from the removed
base trap are transferred to scintillation vials, combined with a scintillation cocktail that is
suitable for the sample matrix, and analyzed by LSC. This process is repeated for the
second trapping train to determine 14CH4.
(2) Dissolved 14CO2 (optional): Sludge samples (e.g. 10 ml) are removed through
the sampling port of the test flask. They are then placed in vessels (e.g. Bellco Glass
Biometer 2556-10250) containing a compartment with an appropriate CO2 absorbent (e.g.
1.5 N KOH). The vessels are sealed and sufficient acid (e.g. 6N HC1) is added to lower
the pH of the samples to <2 without opening the vessels to the atmosphere (see Figure 4).
The samples are allowed to sit for a sufficient length of time to allow CO2 to diffuse from
solution and be trapped from the headspace by the sorbent. Samples of the sorbent are
combined with a scintillation cocktail that is suitable for the sample matrix and analyzed
by LSC.
(C) Indirect measurement of 3H2O. Samples (e.g. 8 ml) of sludge are collected
from each system and placed in centrifuge tubes, located in a fume hood, that contain
sufficient acid (e.g. 1 ml of 0.1 N HC1) to lower the sample pH to <2. The tubes are
mixed and centrifuged. Individual replicate samples (e.g. 1 ml) of the supernatant are
placed in separate vials. Half of the samples are immediately analyzed directly by LSC
for a wet measurement. The remaining samples are allowed to dry completely to remove
the 3H2O. The samples are combined with a scintillation cocktail that is suitable for the
sample matrix and analyzed by LSC. The percentage 3H2O is calculated based on the
difference between the total counts in the wet and dry samples and the initial level of
radioactivity dosed to the samples.
(iv) Measurement of radioactivity in digester sludge. Direct LSC counting of
samples is not possible due to the high solids levels in the samples. Therefore, samples
36
-------
are centrifuged and the supernatant analyzed for total radioactivity by LSC, and the solids
combusted and then analyzed for radioactivity to determine the total radioactivity in the
sample. Individual replicate samples (e.g. 1 ml) of digester sludge are collected from each
system and placed in centrifuge tubes. The samples are centrifuged and the supernatant
transferred to a scintillation vial. The samples are combined with a scintillation cocktail
that is suitable for the sample matrix and analyzed by LSC. The solids remaining in the
centrifuge tube are combusted using a sample oxidizer prior to LSC.
(v) Measurement of parent and metabolites—(A) Extraction. (7) A sample of
digester sludge is collected from both the abiotic and biotic systems. The sample volume
is typically >10 ml, but the size will depend on the test concentration, specific activity
and the sensitivity of the analytical procedures.
(2) Various approaches can be used for concentrating and extracting the samples.
A proven approach for nonvolatile test substances involves flash freezing the samples,
followed by lyophilization and extraction of the dried residue with appropriate solvent(s)
for parent and metabolites. Flash freezing quickly stops biological activity without
hydrolyzing or otherwise altering labile test substances. Flash freezing is a quick process
if there is sufficient depth in the dry ice/acetone or liquid nitrogen bath to submerge the
sample tube. The level of the bath should be above the sample level in the tube. The
resulting extracts can be concentrated through evaporation and the total radioactivity in
each extract determined by LSC.
(3) For volatile test substances, samples can be centrifuged, and parent and
metabolites extracted from the liquor by solid phase or liquid/liquid extraction. The solids
can then be extracted directly or mixed with a drying agent (e.g. sodium sulfate) and
allowed to dry prior to extraction with an appropriate solvent system. An alternative is to
extract the solids and then remove the water from the solvent by running it through a
column containing a drying agent. The total radioactivity in all extracts is determined by
LSC. Care should be taken in concentrating extracts containing volatile test substances or
metabolites.
(4) Other approaches can be utilized, but with all approaches it is important to
document recoveries and consider the time involved in terminating biological activity,
and factor it into the sample times used for kinetic analyses.
(B) Analysis of parent and metabolites. (7) The relative abundance of parent
and metabolites in the extracts can be determined using TLC, UPLC or other separation
techniques with radioactivity detection.
(2) If sensitive specific analytical methods are available, primary biodegradation
can be assessed by measuring the total residual concentration of test substances and
metabolites instead of using radioisotope techniques.
(C) Characterization of metabolites. Whenever possible, the chromatographic
behavior of unknown peaks should be compared to that of predicted metabolites, if
37
-------
authentic standards exist. Usually, the quantity and purity of metabolites generated in this
test make definitive identification by other direct means impossible. Depending on
chromatographic behavior, it is usually possible to determine if a metabolite is more or
less polar than the parent. This information, combined with known biochemical reactions
and knowledge of when a metabolite appears and disappears in the sequence of
biodegradation, can form an additional basis for inferring its identity. If necessary, the
Kow of major metabolites can be estimated by HPLC (e.g. using OECD 117; see
paragraph (o)(24) of this guideline) using an on-line radioactivity detector.
(vi) Measurement of extracted solids and incorporation into biomass. The
extracted solids are combusted to determine the level of activity remaining with the
solids. The level of radioactivity in the biotic solids above that in solids from the abiotic
control typically represents incorporation of radioactivity into biomass. The distribution
of this radioactivity among various components of biomass (i.e. nucleic acids, protein,
cell wall, etc.) can be determined using a modified Sutherland and Wilkinson procedure
(see paragraph (o)(25) of this guideline).
(vii) Measurement of volatilized radioactivity. For volatile test substances, the
volatility traps are extracted with appropriate solvents and the radioactivity in the extracts
analyzed by LSC. The relative abundance of parent and metabolites in the extract(s) can
be determined as described in paragraph (l)(5)(v)(B)(7) of this guideline.
(m) 314D Biodegradation in treated effluent-surface water mixing zone—(1)
General, (i) This test is designed to evaluate the biodegradation of the portion of a
substance that passes through treatment and is released in effluent to surface water. It can
be used to demonstrate that biodegradation occurring in the treatment plant continues in
the receiving environment. It also is useful for determining the extent of biodegradation
as well as rates of primary and ultimate biodegradation in this environmental
compartment. The results can be used to estimate the reduction in substance
concentration resulting from biodegradation as a volume of water moves downstream
from a wastewater treatment plant outfall. The test medium consists of freshly collected
surface water and effluent. The usefulness of the measured rates for predicting
downstream exposure will be a function of the fidelity of the simulation to actual
conditions in the mixing zone. Along with test substance concentration, factors to
consider in the designing of this test include the level of solids in effluent and the degree
to which effluent is diluted in receiving water.
(ii) To simulate conditions associated with episodic release of a substance, an
appropriately chosen mixture of surface water and effluent is incubated with the
concentration of test substance expected to occur in effluent diluted into surface water
during a release event. In these situations, the substance and its degrader populations will
usually not be at steady state and the observed kinetics will be pseudo first-order, or
second-order Monod. Approaches for estimating effluent concentrations can be found in
Holman (see paragraph (o)(21) of this guideline) and the European Technical Guidance
Document (see paragraph (o)(22) of this guideline).
38
-------
(iii) For substances that are or will be continuously released to wastewater,
degrader populations in the treatment plant will become acclimated to the substance. For
existing substances continuously discharged to wastewater, surface water with the
expected concentration of test substance and freshly collected effluent under a given
discharge scenario will provide the most realistic kinetic parameters. For new substances
that will be continuously discharged to wastewater, the use of effluent that was exposed
to the substance under simulated activated sludge conditions in the laboratory (e.g. in an
OECD 303A test) will provide the most accurate kinetics. It should be noted that use of
303A may lead to an overestimation of degradation in treatment, as compared to a real
environmental release situation.
(iv) In most circumstances, due to analytical considerations, it will be impossible
to test at actual surface water concentrations. Consequently, observed biodegradation rates
may not be fully representative of those under actual environmental conditions. This should
be considered in the interpretation of results.
(2) Principle of the test, (i) The test substance is incubated with abiotic and
biotic mixtures of surface water and effluent over a period of time. The ratio of these
components is based on specific or generic scenarios for release of treated effluent to
surface water. Biological activity is inhibited in the abiotic control, which is used for
estimating mineralization by difference, establishing extraction efficiency and recovery
of the parent molecule, and quantifying other loss processes such as hydrolysis,
oxidation, volatilization or sorption to test apparatus.
(ii) If an analytical method with the required sensitivity is available, the rate of
parent degradation or transformation can be determined using a non-radiolabeled test
substance or by following the disappearance of a substance already in wastewater.
However, ultimate biodegradation cannot be determined unless the biodegradation
pathway is well understood and analytical methods with required sensitivity are available
for potential metabolites.
(iii) Test substance at an environmentally relevant concentration is dosed to both
abiotic and biotic test systems, which are incubated at a relevant temperature with
continuous mixing when appropriate. Samples are periodically removed for
determination of mineralization and primary biodegradation.
(iv) Tests can be performed using an open batch system or a sealed, flow-through
batch system in which traps are used to capture evolved 14CC>2 (see Figure 2). The closed
flow-through system should be used for volatile test substances and usually is preferred
for 14C-lableled test substances. Open systems are appropriate for nonvolatile 3H-labeled
test substances and for refining the biodegradation kinetics of nonvolatile 14C-labeled test
substances, whose ability to be mineralized has previously been established. In the open
system, mineralization to 14CC>2 can be determined indirectly by measuring the difference
in residual radioactivity between samples from the biotic and abiotic systems following
acidification. Similarly, mineralization to 3H2O can be determined indirectly by
measuring the difference in radioactivity in a sample following drying. In the flow-
39
-------
through systems, evolved CC>2 is measured directly in the base traps. In addition,
dissolved 14CC>2 is determined by acidifying samples in a sealed vessel and measuring
radioactivity in a base trap contained in the vessel.
(v) Samples from both systems are analyzed for total radioactivity, extractable
parent and metabolites and radioactivity associated with the extracted solids. The level of
parent and metabolites is determined using chromatographic separation and, when
appropriate, radio-analytical detection methods. The solids remaining from the extraction
process are combusted to estimate incorporation into biomass by difference, or can be
further fractionated to determine uptake into various components of biomass. A complete
mass balance of the test system is obtained from the sum of all fractions at each
sampling.
(3) Applicability of the test. The method is readily applicable to water-soluble or
poorly water-soluble substances that are nonvolatile. It also can be adapted for volatile
substances. Typically, 14C or 3H labeling of substances is required for the assessment of
mineralization. Either radiolabeled or nonlabeled substance can be used for the
assessment of primary biodegradation.
(4) Description of the test method—(i) Test apparatus. (A) The volume of the
surface water-effluent mixture in the test vessels is based on the number and volume of
the samples needed for the test. Typically, 1-2 1 of surface water are placed in 2- or 4-1
flasks. Open batch systems are generally closed with a foam or cotton stopper to
minimize evaporative loss of water. Flow-through systems are sealed with an appropriate
closure containing a sampling port with a valve for removing samples and connections
for influent and effluent gas lines. This closure can be a rubber stopper, but glass is
recommended when working with a volatile hydrophobic test substance. When testing
volatile substances, it also is recommended that gas lines and sampling tubes consist of
inert materials (e.g. polytetrafluoroethylene, stainless steel, glass).
(B) The headspace of the test vessel is continuously purged with air or CO2-free
air at a rate sufficient to maintain the system in an aerobic condition but not fast enough
to prevent efficient trapping of CC>2. The test vessel is connected to a series of traps
containing a potassium hydroxide (1.5 N) or other appropriate CC>2 absorbent. An empty
trap is usually included and positioned in front of the absorbent in the trapping train, as a
precaution against backflow or condensation.
(ii) Equipment. The standard laboratory equipment listed in this paragraph is
used:
(A) Miscellaneous glassware and pipettes;
(B) Magnetic stirrers or shaker for continuous mixing of the test flasks;
(C) Centrifuge;
40
-------
(D) pH meter;
(E) Solid CCVacetone or liquid nitrogen bath;
(F) Freeze dryer (lyophilizer);
(G) Oven or microwave oven for dry weight determinations;
(H) Membrane filtration apparatus;
(I) Autoclave;
(J) Facilities to handle radiolabeled substances;
14
(K) Equipment to quantify C and H in liquid samples and solid samples (e.g.
liquid scintillation counter);
(L) Equipment to quantify 14C and 3H in solid samples (e.g. sample oxidizer);
(M) Equipment to trap volatilized 14C and 3H from gas trapping system (in-line
activated charcoal trap or equivalent);
(N) Equipment for TLC or HPLC;
(O) Equipment to quantify 14C and 3H for TLC (scanner) or HPLC (in-line
detector);
(P) Analytical equipment for the determination of the test (and reference)
substance if specific substance analysis is used (e.g. gas chromatograph, high-
performance liquid chromatograph, mass spectrometer).
(iii) Selection of environmental samples. (A) The source of surface water,
activated sludge or effluent should be consistent with the objective of the simulation test.
For a site-specific assessment, activated sludge or effluent should be obtained from the
specific wastewater treatment plant of interest. Likewise, the surface water should be
obtained upstream from that treatment plant's outfall. The ratio of these components
should be chosen to simulate a specific flow scenario (e.g. low flow or mean flow).
Alternatively, if hydraulic conditions below the outfall are known, the test medium can
simply be water samples obtained downstream from the outflow. However, such
conditions are variable and hard to reproduce.
(B) For a generic assessment activated sludge or effluent should be obtained from
a typical wastewater treatment plant receiving predominantly domestic wastewater.
Likewise, the surface water should be typical of surface waters into which effluent is
released. If the substance is currently a component of wastewater entering the wastewater
treatment facility or is episodically released to wastewater, freshly collected activated
41
-------
sludge or effluent will be ideal for the test. WWTP effluent consists of activated sludge
liquor and biosolids, but it can be variable in its solids level and impacted by chlorination
or other processes. The best method of simulating this scenario is using filtered effluent
and surface water combined at the targeted dilution ratio and separately adding activated
sludge biosolids at a targeted concentration. If the effluent is difficult to obtain, the
activated sludge can be filtered or centrifuged to remove biosolids, and the liquor and
biosolids can then be added at a defined ratio to the test system. For a generic assessment,
the TGD assumes that effluent containing 30 mg biosolids/1 is diluted 10-fold into surface
water containing 15 mg suspended solids/1 (see paragraph (o)(22) of this guideline).
Hence, 3 mg of biosolids in 100 ml of filtered effluent or activated sludge liquor and 900
ml of surface water approximates this generic scenario. Additionally, a scenario based on
10 mg of biosolids and 330 ml of liquor per 1 may be considered to simulate critical low
flow conditions that might occur during dry seasons.
(C) For a new substance that will be continuously released to wastewater, the
activated sludge or effluent ideally should be obtained from a laboratory-scale treatment
system such as a porous pot or continuous activated sludge test (e.g. an OECD 303A test)
(see paragraph (o)(2) of this guideline), which has been fed wastewater amended with
unlabeled test substance. The source of the starting sludge, wastewater (influent), and
operating conditions (influent concentration, hydraulic retention time, solids retention
time) for the laboratory unit should accurately reflect site-specific or generic conditions.
In the latter case, the TGD specifies an HRT of 6.9 hr and an SRT of 9.2 d in its generic
scenario for wastewater treatment. The TGD also provides guidance for estimating
concentration of a substance in wastewater based on expected use volumes. In general,
steady state will be reached within 2 to 3 SRTs, after which the biosolids or effluent can
be used for testing.
(iv) Collection, transport and storage of environmental samples. The activated
sludge should be collected from a well-mixed region of the aeration basin. Surface water
should be collected from a site with known inputs of wastewater. The temperature of the
samples should be noted at collection. Collection containers should allow for adequate
ventilation and measures should be taken to prevent the temperature of the sample from
significantly exceeding the temperature used in the test. The samples are typically stored
at test temperature with continuous aeration. Samples should not be stored frozen.
(v) Preparation of test systems. (A) The surface water should be characterized
by measuring TSS, total hardness and pH. A standard plate count and organic carbon
analysis are optional. When using activated sludge to represent WWTP effluent, the
MLSS is sieved through a 2mm screen, blended (optional) and allowed to settle. The TSS
concentration of the liquor is measured. The liquor is added to the surface water at a
volume sufficient to achieve the targeted biosolids concentration. If more volume is
needed to reach the targeted dilution ratio in the test, filtered or centrifuged activated
sludge liquor is added to reach the necessary dilution. If using treated effluent and a
targeted activated sludge biosolids concentration, the effluent is filtered and mixed with
the surface water at the targeted dilution ratio. The MLSS is prepared as described
previously. The pH and TSS of the prepared surface water mixture should be measured.
42
-------
An optional standard plate count and organic carbon analysis can also be performed.
(B) The abiotic system is typically prepared using a combination of chemical and
heat sterilization. A proven approach is to add mercuric chloride solution (0.1 g/1) to the
mixture, which is then autoclaved for at least 90 min. Typically the volume of medium is
<= half the volume of the container being autoclaved (e.g. 500 ml activated sludge in a 1-
liter container). After cooling, the pH of the abiotic system should be measured and
adjusted to match that of the biologically active system. Alternative approaches to
deactivate the surface water mixture can also be used.
(vi) Test substance preparation. (A) Ideally, distilled water should be used to
prepare stock solutions of the test and reference substances. When appropriate, an
alternative method may be used to solubilize or disperse the test substance in a manner
consistent with its normal entry into the environment. Water-miscible nontoxic solvents
may be used when necessary, but attention should be paid to the associated organic load
involved with adding organic solvents. Alternatively, the sample may be added in a neat
form to the test system in a manner that maximizes its even and rapid distribution in the
test medium. For materials which are poorly soluble and typically associated with
suspended solids in effluent, it may be appropriate to adsorb the test material onto an inert
solid carrier, which is then dosed to the test system. If the test material cannot be evenly
distributed in the test system prior to the initial sampling point, individual test systems can
be prepared that are destructively sampled at each sampling interval.
(B) The volume of added stock should be of sufficient capacity to ensure rapid
and even distribution of the test substance in the system and accurate administration of
the dose between like systems. Ideally, when dosing with aqueous solutions, the added
volume should be >2 ml and <10 ml; for nontoxic solvents, <0.1 ml/ L. If appropriate,
dosing solutions may be prepared in advance and refrigerated. The activity of the stock
should be checked by LSC.
(vii) Test conditions—(A) Test temperature. Incubation should take place in the
dark (preferred) or in diffuse light at a controlled temperature, which may be the field
temperature or a standard laboratory temperature of 20-25°C.
(B) Agitation. To keep the test medium in suspension, the test systems are
agitated by means of continuous shaking or stirring. Agitation also facilitates oxygen
transfer from the headspace to the liquid so that aerobic conditions can be adequately
maintained.
(viii) Test duration. The duration of the test should be sufficient to assess the
biodegradation of the test substance during its normal residence time in the WWTP
effluent-surface water mixing zone, or its completeness of degradation under such
conditions. Normally, the test period will be 28 d. However, it may be extended to obtain
additional data points, in order to estimate kinetic constants or to assess the completeness
of degradation under the conditions of the test. Conversely, it may be ended before this
time if degradation has plateaued.
43
-------
(ix) Number of test vessels. At a minimum, there should be a single abiotic and a
single biotic test vessel for each test substance concentration. While replicates can be
prepared for each system, more useful kinetic information usually can be gained by
increasing the number of time points sampled within a system.
(5) Procedure—(i) Dosing. At test initiation, the test vessel is opened and the
test substance is quantitatively added directly to the system with constant mixing. It is
recommended that the dose be administered gradually below the air-water interface, to
ensure uniform distribution of the test substance in the test medium. The biotic and
abiotic systems are dosed in an identical manner. Generally, the biotic systems are dosed
first, followed by the abiotic systems. Exact timing is typically more critical for kinetic
analyses.
(ii) Sampling schedule. Sampling times are selected based on existing
biodegradation data or the results of a pilot study, as no fixed time schedule for sampling
is universally applicable. A recommended sampling schedule for a rapidly degraded
substance would be 5, 30, 60 min, with additional samplings after 3, 5, 8, 12 and 24 hr.
Subsequent samples could be taken after 2, 3, 4, 5, 6 and 7 d and weekly until day 28.
The sampling schedule for slowly degrading substance should be adjusted so that a
sufficient number of measurements are made during the degradation phase.
(iii) Measurement of mineralization—(A) Indirect measurement of 14COi. (/)
Individual replicate samples (e.g. 1 ml) are collected from each system and placed in
separate vials, located in a fume hood, that contain sufficient acid (e.g. 1 ml of 0.1 N
HC1) to lower the sample pH to <2.
(2) The samples are bubbled with air for several hours or allowed to sit overnight
to allow the dissolved 14CC>2 to diffuse from the samples. The samples are combined with a
scintillation cocktail that is suitable for the sample matrix and analyzed by LSC. The
percentage of 14CC>2 is calculated based on the difference between the total counts in the
biotic and abiotic samples.
(B) Direct measurement of 14COi. (7) For rapidly degrading substances, it can be
difficult to measure accurately the rate of 14CC>2 evolved due to the rate of the mass transfer
of 14CC>2 from the headspace into the base trap. Under these conditions, it is recommended
that indirect measurement of 14CC>2 be conducted simultaneously with direct
measurement.
(2) Evolved 14CC>2: The first base trap in the series is removed and quickly
capped. The remaining traps are moved forward in the same order and a fresh trap placed
behind the existing traps, and the trapping system reconnected as quickly as possible.
Replicate subsamples (e.g 1 ml) from the removed base trap are transferred to
scintillation vials, combined with scintillation cocktail that is suitable for the sample
matrix, and analyzed by LSC.
44
-------
(3) Dissolved 14CC>2: Samples (e.g. 25 to 50 ml) are removed through the
sampling port of the test flask. They are then placed in vessels (e.g. Bellco Glass
Biometer 2556-10250) containing a compartment with an appropriate CC>2 absorbent (e.g.
1.5 N KOH). The vessels are sealed and sufficient acid (e.g. 6N HC1) is added to lower
the pH of the samples to <2 without opening the vessels to the atmosphere (see Figure 4).
The samples are allowed to sit for a sufficient length of time (e.g. overnight) to allow
CC>2 to diffuse from solution and be trapped from the headspace by the sorbent. Samples
of the sorbent are combined with a scintillation cocktail that is suitable for the sample
matrix and analyzed by LSC.
(C) Indirect measurement of 3H2O. (7) Individual replicate samples (e.g. 1 ml)
are collected from each system and placed in separate vials, located in a fume hood, that
contain sufficient acid (e.g. 1 ml of 0.1 N HC1) to lower the sample pH to <2.
(2) Half of the samples are immediately analyzed directly by LSC for a wet
measurement. The remaining samples are allowed to dry completely to remove the 3H2O.
The samples are combined with a scintillation cocktail that is suitable for the sample
matrix and analyzed by LSC. The percentage 3H2O is calculated based on the difference
between the total counts in the wet and dry samples and the initial level of radioactivity
dosed to the samples.
(iv) Measurement of total radioactivity in surface water. Small-volume
samples (e.g. 1 ml) are analyzed directly by LSC to quantify the radioactivity remaining
in each surface water over time. These measurements are used to confirm that the
recovery of radioactivity from the extracted samples is acceptable and to monitor for
volatilization.
(v) Measurement of parent and metabolites—(A) Extraction. (7) A sample is
collected from both the abiotic and biotic systems. The sample volume is typically >25
ml. However, the size will depend on the test concentration, specific activity and the
sensitivity of the analytical procedures.
(2) Various approaches can be used for concentrating and extracting the samples.
A proven approach for nonvolatile test substances involves flash freezing the samples,
followed by lyophilization and extraction of the dried residue with appropriate solvent(s)
for parent and metabolites. Flash freezing quickly stops biological activity without
hydrolyzing or otherwise altering labile test substances. Flash freezing is a quick process
if there is sufficient depth in the dry ice/acetone or liquid nitrogen bath to submerge the
sample tube. The level of the bath should be above the sample level in the tube. The
extract is filtered to recover the solvent and solids separately. The filter must be
compatible with the solvent type (e.g. aqueous or non-aqueous). The resulting extracts can
be concentrated through evaporation and the total radioactivity in each extract determined
by LSC.
(3) For volatile test substances, the sample can be passed through a filter and solid
phase extraction (SPE) column or SPE disk placed in tandem, which are subsequently
45
-------
eluted with appropriate solvents to recover parent and metabolites. Alternatively, the
aqueous samples can be extracted with an appropriate solvent system and then filtered to
recover biomass solids, assuming sufficient extraction efficiency. The total radioactivity
in all extracts is determined by LSC. Care should be taken in concentrating extracts
containing volatile test substances or metabolites.
(4) Other approaches can be utilized, but with all approaches it is important to
document recoveries and consider the time involved in terminating biological activity,
and factor it into the sample times used for kinetic analyses.
(B) Analysis of parent and metabolites. (7) The relative abundance of parent and
metabolites in the extracts can be determined using TLC, HPLC or other separation
techniques with radioactivity detection.
(2) If sensitive specific analytical methods are available, primary biodegradation
can be assessed by measuring the total residual concentration of test substances and
metabolites instead of using radioisotope techniques.
(C) Characterization of metabolites. Whenever possible, the chromatographic
behavior of unknown peaks should be compared to that of predicted metabolites, if
authentic standards exist. Usually, the quantity and purity of metabolites generated in this
test make definitive identification by other direct means impossible. Depending on
chromatographic behavior, it is usually possible to determine if a metabolite is more or
less polar than the parent. This information, combined with known biochemical reactions
and knowledge of when a metabolite appears and disappears in the sequence of
biodegradation, can form an additional basis for inferring its identity. If necessary, the
Kow of major metabolites can be estimated by HPLC (e.g. using OECD 117; see
paragraph (o)(24) of this guideline) using an on-line radioactivity detector.
(vi) Measurement of extracted solids. Since the filters will retain carbonate salts
as well as microorganisms from the test system, the filter containing the biosolids is
placed in a scintillation vial and acidified to pH <2 by submerging it in a weak acid
solution (1 ml of 0.1 N HC1). The samples are allowed to sit for sufficient time (e.g.
overnight) for the dissolved 14CC>2 to diffuse from the samples. The samples are
combined with a scintillation cocktail that is suitable for the sample matrix and analyzed
by LSC. The level of radioactivity in the biotic solids above that in solids from the
abiotic control typically represents incorporation of radioactivity into biomass.
(vii) Measurement of volatilized radioactivity. For volatile test substances, the
volatility traps are extracted with appropriate solvents and the radioactivity in the extracts
analyzed by LSC. The relative abundance of parent and metabolites in the extract(s) can
be determined as described in paragraph (m)(5)(v)(B)(7) of this guideline.
(n) 314E Biodegradation in untreated wastewater-surface water mixing
zone—(1) General, (i) In developing regions lacking wastewater treatment
infrastructure, it is common for wastewater to be directly discharged to surface waters.
46
-------
This test is designed to simulate these situations and evaluate the biodegradation of a
substance that is discharged to surface water as a component of untreated wastewater. It
is useful for determining the extent of biodegradation as well as rates of primary and
ultimate biodegradation under such direct discharge conditions. The results can be used
to estimate the reduction in substance concentration resulting from biodegradation as a
volume of water moves downstream from a wastewater outfall. As an option, this
reduction can be compared to decreases in other wastewater components such as BOD,
COD or TOC. The test medium consists of freshly collected wastewater and surface
water. The usefulness of the measured biodegradation rates for predicting downstream
exposure will be a function of the fidelity of the simulation to actual conditions in the
mixing zone. Along with test substance concentration, factors to consider in designing
this test include DO concentration and the degree to which effluent is diluted into surface
water.
(ii) For existing substances consistently present in wastewater, freshly collected
wastewater and surface water incubated with radiolabeled test substance at a tracer level
will provide the most realistic kinetic parameters regarding the current substance load.
For substances not consistently present in wastewater, sufficient test substance
(radiolabeled and unlabeled) should be added to approximate the expected concentration
in wastewater diluted into surface water during an episodic release or following
commercialization of a new substance. Approaches for estimating such an expected
wastewater concentration can be found in Holman (see paragraph (o)(21) of this
guideline) and the European Technical Guidance Document (see paragraph (o)(22) of this
guideline).
(iii) For low dilution situations, it is best to incubate the mixtures under reduced
DO conditions (1-4 mg/1) to simulate the DO level below a wastewater outfall. In this
test, the substance and its degrader populations usually are not at steady state and the
observed kinetics will be pseudo first-order, or second-order Monod.
(iv) The test can be run using a single or two-phase design. In the former,
wastewater is diluted into surface water from a clean or wastewater-impacted source. In
the latter, wastewater is sequentially diluted into clean and wastewater-impacted surface
waters. In this test design, the test substance is dosed into wastewater diluted in clean
surface water and disappearance of test substance and conventional pollutants (BOD,
COD, etc) are monitored over time (phase 1). Subsequently, a second dose of test
substance and wastewater is added to the same system to simulate dilution of wastewater
into surface water previously polluted by wastewater (phase 2).
(2) Principle of the test, (i) The test substance is incubated with abiotic and
biotic mixtures of wastewater and surface water, usually under reduced DO conditions
(1-4 mg/1) over a period of time. The ratio of these components is based on specific or
generic scenarios for release of wastewater to surface water. Biological activity is
inhibited in the abiotic control, which is used for estimating mineralization by difference,
establishing extraction efficiency and recovery of the parent molecule, and quantifying
other loss processes such as hydrolysis, oxidation, volatilization or sorption to test
47
-------
apparatus.
(ii) If an analytical method with the required sensitivity is available, the rate of
parent degradation or transformation can be determined using a non-radiolabeled test
substance or by following the disappearance of a substance already in wastewater.
However, ultimate biodegradation cannot be determined unless the biodegradation
pathway is well understood and analytical methods with required sensitivity are available
for potential metabolites.
(iii) Test substance at an environmentally relevant concentration is dosed to both
abiotic and biotic test systems, which are incubated at a relevant temperature with
continuous mixing. The biotic samples are incubated in such a way that DO levels remain
at a reduced level (1-4 mg/1), characteristic of the situation below a wastewater outfall.
Samples are periodically removed for determination of mineralization and primary
biodegradation and, as an option, the level of other wastewater components (e.g. COD,
TOC and ammonia) can be determined concurrently.
(iv) In the two-phase test design, once biodegradation of the test substance and
wastewater components have leveled off, a second dose of test substance and fresh
wastewater is mixed into the existing test system to simulate wastewater being diluted
into previously polluted surface water, and the sampling process is repeated.
(v) Tests can be performed using an open batch system or a sealed, flow-through
batch system in which traps are used to capture evolved 14CO2 (see Figure 2). The closed
flow-through system should be used for volatile test substances and usually is preferred
for 14C-lableled test substances. Open systems are appropriate for nonvolatile 3H-labeled
test substances and for refining the biodegradation kinetics of nonvolatile 14C-labeled test
substances, whose ability to be mineralized has previously been established. In the open
system, mineralization to 14CO2 can be determined indirectly by measuring the difference
in residual radioactivity between samples from the biotic and abiotic systems following
acidification. Similarly, mineralization to 3H2O can be determined indirectly by
measuring the difference in radioactivity in a sample following drying. In the flow-
through systems, evolved 14CO2 is measured directly in the base traps. In addition,
dissolved 14CO2 is determined by acidifying samples in a sealed vessel and measuring
radioactivity in a base trap contained in the vessel.
(vi) Samples from both systems are analyzed for total radioactivity, extractable
parent and metabolites and radioactivity associated with the extracted solids. The level of
parent and metabolites is determined using chromatographic separation and when
appropriate, radio-analytical detection methods. The remaining solids from the extraction
process are combusted to estimate incorporation into biomass by difference. A complete
mass balance of the test system is obtained from the sum of all fractions at each sampling.
(3) Applicability of the test. The method is readily applicable to water-soluble
and poorly water-soluble substances that are nonvolatile. It also can be adapted for
volatile substances. Typically, 14C or 3H-radiolabeling of substances is required for the
48
-------
assessment of mineralization. Either radiolabeled or nonlabeled substance can be used for
the assessment of primary biodegradation.
(4) Description of the test method.—(i) Test apparatus. (A) The volume of the
wastewater-surface water mixture in the test vessels is based on the number and volume
of the samples needed for the test. Typically, 1-2 1 of sample is placed in 2- or 4-1 flasks.
Ideally, the wastewater-surface water mixture is incubated under one or more controlled
DO conditions (e.g. 1 and 4 mg DO/1). This condition can be achieved using an oxygen
probe immersed in the wastewater attached to an oxygen controller connected to an
actuator valve, which controls the aeration of the wastewater (see Figure 3). This aeration
is balanced against continuous sparging with nitrogen to achieve the targeted DO level.
Alternatively, the wastewater can be incubated with stirring but minimum aeration, to
keep DO at desired levels, and nitrogen or air can be added periodically to maintain the
DO level. In this case, DO readings should be reported at regular intervals.
(B) Open systems are generally closed with a foam or cotton stopper to minimize
evaporative loss of water. Flow-through systems are sealed with an appropriate closure
containing a sampling port with a valve for removing samples, and connections for
influent and effluent gas lines. This closure can be a rubber stopper, but glass is
recommended when working with a volatile, hydrophobic test substance. When testing
volatile substances, it also is recommend that gas lines and sampling tubes consist of inert
materials (e.g. polytetrafluoroethylene, stainless steel, glass).
(C) The headspace of the test vessel is continuously purged with air or CO2-free
air at a rate sufficient to maintain the system in an aerobic condition, but not so fast as to
prevent efficient trapping of CO2. The test vessel is connected to a series of traps
containing a potassium hydroxide (1.5 N) or other appropriate CO2 absorbent. An empty
trap is usually included and positioned in front of the absorbent in the trapping train, as a
precaution against backflow or condensation.
(ii) Equipment. (A) The standard laboratory equipment listed in this paragraph is
used:
(1) Miscellaneous glassware and pipettes;
(2) Magnetic stirrers or shaker for continuous mixing of the test flasks;
(3) Centrifuge;
(4) pH meter;
(5) Solid CO2/acetone or liquid nitrogen bath;
(6) Freeze dryer (lyophilizer);
(7) Oven or microwave oven for dry weight determinations;
49
-------
(8) Membrane filtration apparatus;
(9) Autoclave;
(10) Facilities to handle radiolabeled substances;
(11) Equipment to quantify 14C and 3H in liquid samples and solid samples (e.g.
liquid scintillation counter);
(12) Equipment to quantify 14C and 3H in solid samples (e.g. sample oxidizer);
- Equipment to trap volatilized 14C and 3H from gas trapping system (in-line
activated charcoal trap or equivalent);
(13) Equipment for TLC or HPLC;
(14) Equipment to quantify 14C and 3H for TLC (scanner) or HPLC (in-line
detector);
(15) Analytical equipment for the determination of the test (and reference)
substance if specific substance analysis is used (e.g. gas chromatograph, high-
performance liquid chromatograph, mass spectrometer).
(B) The laboratory equipment listed in this paragraph is not essential but useful:
(1) Oxygen meter;
(2) Oxygen controller with probe and actuator valve;
(3) COD digestion vials;
(4) Nitrogen ammonia reagent set;
(5) Spectrophotometer.
(iii) Selection of environmental samples. (A) The source of wastewater and
surface water should be consistent with the objective of the simulation test. For a site-
specific assessment, the wastewater should be obtained from the specific sewer system of
interest and the surface water should be obtained upstream from the wastewater outfall.
The ratio of these components should be chosen to simulate a specific flow scenario (e.g.
low flow or mean flow). Alternatively, if hydraulic conditions below the outfall are
known, the test medium can simply be water samples obtained downstream from the
outflow. However, such conditions are variable and hard to reproduce.
(B) For a generic assessment wastewater samples should be predominantly
derived from domestic sources, and the surface water should be typical of surface waters
50
-------
into which wastewater is released. Although difficult to duplicate in practice, the TGD
uses 450 mg suspended solids/1 and 270 mg BOD/1 as default levels for wastewater (see
paragraph (o)(22) of this guideline). In North America, typical wastewaters contain from
100 to 350 mg suspended solids/1 and 110 to 400 mg BOD/1 (see paragraph (o)(23) of this
guideline).
(iv) Collection, transport and storage of environmental samples. Wastewater
should be collected from a sewer access point or at the head of a wastewater treatment
plant. The temperature of the sample should be noted at collection. During transport, the
temperature of the sample should not significantly exceed the temperature used in the
test. The wastewater is typically stored at test temperature with low mixing. No samples
should ever be stored frozen.
(v) Preparation of test systems. (A) The freshly collected wastewater should be
largely free of coarse particles. TSS, pH and COD should be determined for the
wastewater. NH3, organic carbon and standard plate count are optional analyses. The
surface water should be characterized by measuring TSS, total hardness and pH. A
standard plate count and organic carbon analysis are optional. The wastewater is added to
the surface water at a volume sufficient to achieve the targeted dilution of wastewater
into surface water. The pH, COD and TSS of the prepared surface water mixture should
be measured. Optionally, standard place count, NH3 and organic carbon analyses can also
be performed on the mixture.
(B) The abiotic system is typically prepared using a combination of chemical and
heat sterilization. A proven approach is to add mercuric chloride (HgCb = 0.5 g/1) to the
mixture, which is then autoclaved for at least 90 min. Typically the volume of medium is
<= half the volume of the container being autoclaved (e.g. 500 ml activated sludge in a 1-
liter container). After cooling, the pH of the abiotic system should be measured and
adjusted to match that of the biologically active system. Alternative approaches to
deactivate the surface water mixture can also be used.
(vi) Test substance preparation. (A) Ideally, distilled water should be used to
prepare stock solutions of the test and reference substances. When appropriate, an
alternative method may be used to solubilize or disperse the test substance in a manner
consistent with its normal entry into the environment. Water-miscible nontoxic solvents
may be used when necessary, but attention should be paid to the associated organic load
involved with adding organic solvents. Alternatively, the sample may be added in a neat
form to the test system in a manner that maximizes its even and rapid distribution in the
test medium. For substances that are poorly soluble and typically associated with
suspended solids in wastewater, it may be appropriate to adsorb the test material onto an
inert solid carrier, which is then dosed to the test system. If the test material cannot be
evenly distributed within the test system prior to the initial sampling point, individual test
systems can be prepared that are destructively sampled at each sampling interval.
(B) The volume of added stock should be sufficient to ensure rapid and even
distribution of the test substance in the system, and accurate administration of the dose
51
-------
between like systems. Ideally, when dosing with aqueous solutions, the added volume
should be > 2 ml and <10 ml; for nontoxic solvents, <0.1 ml/1. If appropriate, dosing
solutions may be prepared in advance and refrigerated. The activity of the stock should
be checked by LSC.
(vii) Test conditions—(A) Test temperature. Incubation should take place in the
dark (preferred) or in diffuse light at a controlled temperature, which may be the field
temperature or a standard laboratory temperature of 20-25°C.
(B) Agitation. To keep the solids in suspension, the test vessels are minimally
agitated by means of continuous mixing or stirring.
(viii) Test duration. The duration of the test should be sufficient to assess the
biodegradation of the test substance during its normal residence time in the wastewater-
surface water mixing zone. Normally the test period will be 28 d. However, it may be
extended to obtain additional data points, in order to estimate kinetic constants or to
assess the completeness of degradation under the conditions of the test. Conversely, it
may be ended before this time if degradation has plateaued.
(ix) Number of test vessels. At a minimum, there should be a single abiotic and
a single biotic test vessel for each test substance and test substance concentration. While
replicates can be prepared for each system, more useful kinetic information usually can
be gained by increasing the number of time points sampled within a system.
(5) Procedure—(i) Dosing. At test initiation, the test vessel is opened and the
test substance is quantitatively added directly to the system with constant mixing. It is
recommended that the dose be administered gradually below the air-water interface, to
ensure uniform distribution of the test substance in the test medium. The biotic and
abiotic systems are dosed in an identical manner. Generally, the biotic systems are dosed
first, followed by the abiotic systems. Exact timing is typically more critical for kinetic
analyses.
(ii) Sampling schedule. Sampling times are selected based on existing
biodegradation data or the results of a pilot study, as no fixed time schedule for sampling
is universally applicable. A recommended sampling schedule for a rapidly degraded
substance would be 15, 30 and 60 min, with additional samplings after 2, 5, 8, 12 and 24
hr, and day 2, 3 and 7 and weekly thereafter. The sampling schedule for slowly degrading
substances should be adjusted so that a sufficient number of measurements are made
during the degradation phase.
(iii) Measurement of mineralization—(A) Indirect measurement of 14COi. (7)
Individual replicate samples (e.g. 1 ml) are collected from each system and placed in
separate vials, located in a fume hood, that contain sufficient acid (e.g. 1 ml of 0.1 N
HC1) to lower the sample pH to <2.
(2) The samples are bubbled with air for several hours or allowed to sit overnight
52
-------
to allow the dissolved 14CC>2 to diffuse from the samples. The samples are combined with a
scintillation cocktail that is suitable for the sample matrix and analyzed by LSC. The
percentage of 14CC>2 is calculated based on the difference between the total counts in the
biotic and abiotic samples.
(B) Direct measurement of 14CC>2. (1) Evolved 14CC>2: The first base trap in the
series is removed and quickly capped. The remaining traps are moved forward in the
same order and a fresh trap placed behind the existing traps, and the trapping system
reconnected as quickly as possible. Replicate subsamples (e.g. 1 ml) from the base trap
are removed and transferred to scintillation vials, combined with a scintillation cocktail
that is suitable for the sample matrix, and analyzed by LSC.
(2) Dissolved 14CC>2: Samples (e.g. 10 to 25 ml) are removed through the
sampling port of the test flask. They are then placed in vessels (e.g. Bellco Glass
Biometer 2556-10250) containing a compartment with an appropriate CC>2 absorbent (e.g.
1.5 N KOH). The vessels are sealed and sufficient acid (e.g. 6N HC1) is added to lower
the pH of the samples to <2 without opening the vessels to the atmosphere (see Figure 4).
The samples are allowed to sit for a sufficient length of time (e.g. overnight) to allow
CC>2 to diffuse from solution and be trapped from the headspace by the sorbent. Samples
of the sorbent are combined with a scintillation cocktail that is suitable for the sample
matrix and analyzed by LSC.
(C) Indirect measurement of 3H2O. (7) Individual replicate samples (e.g. 1 ml)
are collected from each system and placed in separate vials, located in a fume hood, that
contain sufficient acid (e.g. 1 ml of 0.1 N HC1) to lower the sample pH to <2.
(2) Half of the samples are immediately analyzed directly by LSC for a wet
measurement. The remaining samples are allowed to dry completely to remove the 3H2O.
The samples are combined with a scintillation cocktail that is suitable for the sample
matrix and analyzed by LSC. The percentage 3H2O is calculated based on the difference
between the total counts in the wet and dry samples and the initial level of radioactivity
dosed to the samples.
(iv) Measurement of total radioactivity in wastewater-surface water mixture.
Replicate small volume samples (e.g. 1 ml) are analyzed directly by LSC to quantify the
radioactivity remaining in each system over time. These measurements are used to
confirm that the recovery of radioactivity from the extracted samples is acceptable and to
monitor for volatilization.
(v) Measurement of parent and metabolites—(A) Extraction. (7) A sample is
collected from both the abiotic and biotic systems. The sample volume is typically >10
ml, but the size will depend on the test concentration, specific activity and the sensitivity
of the analytical procedures.
(2) Various approaches can be used for concentrating and extracting the samples.
A proven approach for nonvolatile test substances involves flash freezing the samples,
53
-------
followed by lyophilization and extraction of the dried residue with appropriate solvent(s)
for parent and metabolites. Flash freezing quickly stops biological activity without
hydrolyzing or otherwise altering labile test substances. Flash freezing is a quick process
if there is sufficient depth in the dry ice/acetone or liquid nitrogen bath to submerge the
sample tube. The level of the bath should be above the sample level in the tube. The
resulting extracts can be concentrated through evaporation and the total radioactivity in
each extract determined by LSC.
(3) For volatile test substances, the sample can be passed through a filter and solid
phase extraction (SPE) column or SPE disk placed in tandem, which are subsequently
eluted with appropriate solvents to recover parent and metabolites. Alternatively, it may
be possible to directly extract aqueous samples with an appropriate solvent system and
then filter them to recover biomass solids. The total radioactivity in all extracts is
determined by LSC. Care should be taken in concentrating extracts containing volatile
test substances or metabolites.
(4) Other approaches can be utilized, but with all approaches it is important to
document recoveries and consider the time involved in terminating biological activity,
and factor it into the sample times used for kinetic analyses.
(B) Analysis of parent and metabolites. (7) The relative abundance of parent
and metabolites in the extracts can be determined using TLC, UPLC or other separation
techniques with radioactivity detection.
(2) If sensitive specific analytical methods are available, primary biodegradation
can be assessed by measuring the total residual concentration of test substances and
metabolites instead of using radioisotope techniques.
(C) Characterization of metabolites. Whenever possible, the chromatographic
behavior of unknown peaks should be compared to that of predicted metabolites, if
authentic standards exist. Usually, the quantity and purity of metabolites generated in this
test make definitive identification by other direct means impossible. Depending on
chromatographic behavior, it is usually possible to determine if a metabolite is more or
less polar than the parent. This information, combined with known biochemical reactions
and knowledge of when a metabolite appears and disappears in the sequence of
biodegradation, can form an additional basis for inferring its identity. If necessary, the
Kow of major metabolites can be estimated by HPLC (e.g. [OECD 117](see paragraph
(o)(24) of this guideline) using an on-line radioactivity detector.
(vi) Measurement of extracted solids and incorporation into biomass. The
extracted solids are combusted to determine the level of activity remaining with the
solids. The level of radioactivity in the biotic solids above that in solids from the abiotic
control typically represents incorporation of radioactivity into biomass.
(vii) Measurement of volatilized radioactivity. For volatile test substances, the
volatility traps are extracted with appropriate solvents and the radioactivity in the extracts
54
-------
analyzed by LSC. The relative abundance of parent and metabolites in the extract(s) can
be determined as described in paragraph (n)(5)(v)(B)(l) of this guideline.
(o) References.
(1) OECD (1992). Ready Biodegradability, no. 301, adopted 17 July 1992.
Organization for Economic Cooperation and Development, Paris.
(2) OECD (2001). Simulation Test - Aerobic Sewage Treatment. 303A: Activated
Sludge Units. 303B: Biofilms, no. 303, adopted 22 January 2001. Organization for
Economic Cooperation and Development, Paris.
(3) OECD (2002). Aerobic and Anaerobic Transformation in Aquatic Sediment
Systems, no. 308, adopted 24 April 2002. Organization for Economic Cooperation and
Development, Paris.
(4) OECD (2004). Aerobic Mineralization in Surface Water - Simulation
Biodegradation Test, no. 309, adopted 13 April 2004. Organization for Economic
Cooperation and Development, Paris.
(5) OECD (2006). Anaerobic Biodegradability of Organic Compounds in
Digested Sludge: by Measurement of Gas Production, no. 311, adopted 23 March 2006.
Organization for Economic Cooperation and Development, Paris.
(6) Matthijs, E., G. Debaere, N.R. Itrich, P. Masscheleyn, A. Rottiers, M.
Stalmans and T.W. Federle (1995). The fate of detergent surfactants in sewer systems.
Water Sci. Technol. 13, 321-328.
(7) Federle, T.W. and N.R. Itrich (1997). A comprehensive approach for assessing
the kinetics of primary and ultimate biodegradation of chemicals in activated sludge:
Application to linear alkylbenzene sulfonate (LAS). Environ. Sci. & Technol. 31, 3597-
3603.
(8) Itrich, N.R. and T.W. Federle (2004). Effect of ethoxylate number and alkyl
chain length on the pathway and kinetics of linear alcohol ethoxylate biodegradation in
activated sludge. Environ. Toxicol. Chem. 23, 2790-2798.
(9) Federle, T.W. and N.R. Itrich (2006). Fate of free and linear alcohol
ethoxylate derived fatty alcohols in activated sludge. Ecotox. Environ. Safe. 64, 30-41.
(10) Steber, J. and P. Wierich (1987). The anaerobic degradation of detergent
range fatty alcohol ethoxylates. Studies with 14C-labeled model surfactants. Water Res.
21, 661-667.
(11) Nuck, B.A. and T.W. Federle (1996). Batch test for assessing the
mineralization of 14C radiolabeled compounds under realistic anaerobic conditions.
55
-------
Environ. Sci. Technol. 30, 3597-3600.
(12) OECD (1995). Water Solubility, no. 105, adopted 27 July 1995. Organization
for Economic Cooperation and Development, Paris.
(13) OECD (1981). Dissociation Constants in Water, no. 112, adopted 12 May
1981. Organization for Economic Cooperation and Development, Paris.
(14) OECD (2006). Vapor Pressure, no. 104, adopted 23 March 2006.
Organization for Economic Cooperation and Development, Paris.
(15) OECD (2004). Hydrolysis as a function of pH, no. Ill, adopted 13 April
2004. Organization for Economic Cooperation and Development, Paris.
(16) OECD (1984). Activated sludge, respiration inhibition test, no. 209, adopted
4 April 1984. Organization for Economic Cooperation and Development, Paris.
(17) OECD (1981). Inherent Biodegradability: Modified SCAS Test, no. 302A,
adopted 12 May 1981. Organization for Economic Cooperation and Development, Paris.
(18) OECD (1992). Inherent Biodegradability: Zahn-Wellens/EMPA Test, no.
302A, adopted 17 July 1992. Organization for Economic Cooperation and Development,
Paris.
(19) OECD (1981). Inherent Biodegradability: Modified MITI Test (II), no. 302C,
adopted 12 May 1981. Organization for Economic Cooperation and Development, Paris.
(20) Guidance Document on Estimating Persistence and Degradation Kinetics
from Environmental Fate Studies on Pesticides in EU Registration (2006). Report of the
FOCUS Work Group on Degradation Kinetics, EC Document Reference
Sanco/10058/2005 version 2.0. 434 pp.
(21) Holman, W.F. (1981). Estimating the Environmental Concentrations of
Consumer Product Components. Aquatic Toxicology and Hazard Assessment -4th
Conference, pp. 159-182. Branson, D.R. Dickson, K.L., eds. STP 737. American Society
for Testing and Materials, Philadelphia, PA.
(22) European Commission (2003). Technical Guidance Document on Risk
Assessment in support of Commission Directive 93/67/EEC on Risk Assessment for new
notified substances, 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. Part II (Chapter 3),
Environmental risk assessment, EUR 20418 EN/2. European Commission, Joint Research
Centre, Ispra, Italy.
(23) Tchobanoglous, G. and F.L. Burton (1991). Wastewater Engineering:
56
-------
Treatment, Disposal and Reuse/Metcalf & Eddy, Inc. McGraw-Hill, New York, NY.
(24) OECD (2004). Partition Coefficient (n-octanol/water), High Performance
Liquid Chromatography (HPLC) Method, no. 117, adopted 13 April 2004. Organization
for Economic Cooperation and Development, Paris.
(25) Sutherland, I.W. and J.F. Wilkinson (1971) In (J.R. Norris and D.W.
Ribbons, eds.) Methods in Microbiology, Vol. 5B, pp 345-383. Academic Press, New
York, NY.
(26) Waterfield, W.R., J.A. Spanner and F.G. Stanford (1968). Tritium exchange
from compounds in dilute aqueous solutions. Nature 218, 472-473.
57
------- |