United States

Environmental Protection Agency Prevention, Pesticides

And Toxic Substances (7101) EPA 712-C-08-006

October 2008 

# Fate, Transport and
Transformation Test
Guidelines

OPPTS 835.3280
Simulation Tests to Assess the Primary and Ultimate Biodegradability of Chemicals Discharged

to WastewaterINTRODUCTION

This guideline is one of a series of test guidelines that have been developed by the Office of Preventi on, Pesticides and Toxic Substances (OPPTS), United States Environmental Pr otection Agency for use in the testing of pesticides and toxic substances, and the development of test data to meet the data requirements of the A gency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601), t he Federal Insecticide, Fu ngicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq. ), and section 408 of t he Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).

OPPTS developed this guideline th rough 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 (C FR), the Office of Pest icide Programs (OPP) in publications of the Nationa l Technical Information Service (NTIS) and in the guidelines published by the Organiza tion 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 & Gui delines” on the left side menu.1 OPPTS 835.3280–Simulation tests to assess the primary and ultimate biodegradability of chemicals discharged to wastewater: biodegradation in wastewater, activated sludge, anaerobi c digester sludge, mixing zone for

treated effluent and surface wate r 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 Co ntrol 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 pr imary and ultimate biodegradability of chemicals discharged to wastewater: biodeg radation in wastewater, activated sludge, anaerobic digester sludge, mixing zone for tr eated effluent and surface water and mixing

zone for untreated wastewater and surf ace water. October 2007 Revised Draft Test Guideline, annex to OECD communi cation 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 th eir discharge to wastewater. Personal care, household cleaning and laundry prod ucts are typically discarded down the drain as part of their normal use and become common compone nts of domestic wastewater. Likewise, pharmaceuticals are excreted or in some cases disposed down the drain. Other substances may be episodically or continuously disc harged 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 treat ed effluent in the mi xing 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 aqua tic and terrestrial habitats.

(c) General Considerations. (1) Figure 1 shows the most common transport pathways for chemicals discharged to wastew ater. 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 releas e site. In most situations, the wastewater is treated before release, but in some situa tions 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 tr eatment. The resulting effluent is then subjected to biological treatment and the solids are removed in a final clarifier. The final effluent is subsequently rel eased to surface water. The sludge solids removed during primary treatment and final clarification are most commonly digested under anaerobic conditions if the sludge dispos al involves land application.2 SURFACE

WATERS

SOIL Wastewater

Sewer **

Direct Discharge Primary

Solids

Removal

Sludge Digestion ** (Anaerobic ) Aerobic ** Biological

Treatment Final

Clarification SURFACE

WATERS

MIXING ZONE ** Untreated (Poorly Treated)

Wastewater with Surface Water Primary

Effluent

Digested Sludge

SLUDGE AMENDED SOIL MIXING ZONE **

Treated Effluent with Surface Water

Final

Effluent

Primary Sludge Secondary Sludge WASTEWATER TREATMENT

PLANT 

### Pathways for Chemicals Discharged to

Wastewater

**Simulated in

the New Guideline Figure 1. Pathways for chemicals discharged to wastewater

(2) The fraction of the chemical released to the environment in the final effluent or associated with sludge solids is a f unction of its partitioning behavior and its biodegradation rate, and other processes su ch as volatilization. Due to chemical residence time and the level of biological ac tivity, the critical opportuni ties 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. Furthe rmore, the effects of treatment processes

extend into the environment at the time of re lease. Thus, biodegrad ation in the mixing zones, in the water and in biosolids, as the chemical moves away from the point of release, is key to understanding downs tream dispersion and exposure.

(3) The five simulation test methods describe d in this guideline use an open batch system or closed flow-through batch system (a n example of the latter is shown in Figure 2), and include elements from Organizati on for Economic Cooperation and Development (OECD) guidelines 301, 303A, 309, 310 and 311 (see paragra phs (o)(1) through (o)(5) of this guideline). The principal objectives of the methods are to 1) measure the rate of primary biodegradation, 2) measure the ra te of mineralization, and 3) follow the formation and decay of major transformation products when appropriate. In addition, characterization and quantifica tion of major transformation products may be possible if suitable analytical methods are available.3 Figure 2-. Example of flow-thr ough test apparatus



## Example of Flow-Through Test Set-Up

Air

Test Mixture (1 L) CO 2 Traps Containing

100 ml of 1.5 N KOH Sampling

Port with

Valve

Volatile Trap

Empty Trap

(4) These tests can be designed to assess accurately the biod egradation of both new and existing substances released to wa stewater (see paragraphs (o)(6) through (o)(11) of this guideline). In some cases, th e 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 biodegrada tion of chemicals that do not biodegrade in OECD screening tests, or for refining bi odegradation rates used for an exposure

assessment.

(d) General principle of the tests. (1) Typically, a test substance, radiolabeled in an appropriate position, is in cubated with an environmental sample that has been freshly collected from a representative field site or maintained in the la boratory under conditions realistically simulating some future envir onmental condition. Abio tic and biotic test systems are prepared for each test substa nce and condition. Biological activity is inhibited in the abiotic control, which is us ed for estimating mineralization by difference, establishing extraction efficiency and recove ry of the parent molecule, and quantifying other loss processes such as hydrolysis, oxi dation, volatilization or sorption to test

apparatus.

(2) If an analytical method w ith the required se nsitivity 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 be4 determined unless the biodegradation pathwa y is well understood and analytical methods with required sensitivity are av ailable for potential metabolites.

(3) An environmentally relevant concentra tion of the test subs tance is dosed to both abiotic and biotic test systems. The prepar ed test systems are incubated at a relevant temperature with continuous mixing when appr opriate. Samples are periodically removed for determination of mineralization and primar y biodegradation. The duration of the tests is based upon the time necessary to establis h 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 compartmen ts 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 ar e used to capture evolved 14 CO 2 or 14 CH 4. A closed flowthrough system is necessary for volatile test materials. It is also usually preferred for 14 Clableled test chemicals. Open sy stems are appropriate for nonvolatile 3 H-labeled test chemicals and for refining the biode gradation kinetics of nonvolatile 14 C test materials, whose ability to be mineralized has been established previously. In the open system,

mineralization to 14 CO 2 ( 14 CH 4 ) can be determined indirec tly by measuring the difference in residual radioactivity between samples from the biotic and abiotic test systems following acidification. Sim ilarly, mineralization to 3 H 2 O can be determined indirectly by measuring the difference in residual radioa ctivity following drying. The open system is not appropriate for use with volatile test substances. In a flow-through system, evolved

14 CO 2 is measured directly in the ba se traps. In addition, dissolved 14 CO 2 is determined by acidifying samples in a sealed vessel and measuring radioactivit y in a base trap

contained in the vesse l. Under anaerobic c onditions, the evolved 14 CO 2 and 14 CH 4 are collected in tandem. The 14 CO 2 is trapped in base and 14 CH 4 is combusted and converted

to 14 CO 2 , which is subsequently tra pped 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 ( 14 C or 3 H), 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 associat ed with the extracted solids. The level of parent and metabolites is determined us ing chromatographic separation and radioanalytical detection methods. The solids re maining 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 ti me 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 (see5 paragraph (i)(2) of this guideline).

(e) Applicability of tests— (1) General. The various tests are designed to assess biodegradation during key phase s 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 intere st. In the case of vol atile test substances, appropriate modification shoul d 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 biodegr adation in raw wastewater unde r 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 a nd 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 condition s of the test, the concentration of test substance is at its expected level in wast ewater with the biomass level being that normally present in a representative wastew ater 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 leve l of biomass and a relatively low level of test substance

under well-aerated conditions.

(ii) The activated sludge test can complim ent or be a lower cost alternative to OECD 303A, a dynamic simulation of a wastewat er 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 ti me, solids level, etc.). OECD 303A can

generate a percentage remova l or a comprehensive picture of biodegradation and sorption that occur at steady state durin g treatment. However, as an al ternative to the expense and complexity of running a full scale system, th e activated sludge test can generate firstorder rate constants for loss of parent and mi neralization, and these can be used as inputs to a variety of wastewater simulation mode ls 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 evalua te biodegradation during anaero bic 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 subs tance present in the sludge leaving a treatment plan t 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 base d on expected wastewater concentrations and partitioning behavior.6 (5) 314D: Test for biodegradation in t reated effluent-surface water mixing

zone. The purpose of the effluent mixing zone te st is to evaluate the biodegradation of that portion of a substance that passes thr ough treatment and is rele ased in effluent to surface water; and it can be used to demons trate that biodegradation occurring in the treatment plant continues in the receiving envi ronment. 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 us ed 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 condi tions. This test differs from OECD 309 in

that the surface water is amended with treate d 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 chem icals discharged to wastewater.

(6) 314E: Test for biodegradation in untreated wastewater-surface water mixing zone. The purpose of this test is to ev aluate biodegradation in untreated wastewater that is directly discharged to surface freshwater. This test is useful for determining the relative biode gradation rate for a substanc e 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 co ncentrations 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, 14 C or 3 H radiolabeled

test substances should be used in these te sts. For radiolabeled substances, additional unlabeled substance may be necessary to ach ieve 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 14 C, the radiolabel should be located in the most r ecalcitrant portion of the molecule to monitor comprehensively metabo lite formation and decay. For large or complex substances, it may be appropriate to re peat the study using te st 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 poorl y 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 subs tances, tritiated ( 3 H-labeled) substances can be an

alternative to 14 C-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 consid er that isotopic tritium can exchange with hydrogen in water (see paragraph (o)(26) of th is guideline). These two facts should be taken into account in interpreting mi neralization and metabolite patterns.7 (4) Nonlabeled test substances can be used to determine the rate of parent degradation or transformation if an analyt ical 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 (applicab le to substances applied with solvent or having low water solubility);

(iii) Dissociation constant (pK

a ) if the substance is s ubject to protonation or deprotonation [OECD 112] (see paragr aph (o)(13) of this guideline);

(iv) Vapor pressure [OECD 104] (see paragr aph (o)(14) of this guideline) and/or Henry’s law constant;

(v) Substance stability in wa ter and in the dark; i.e. susceptibility to hydrolysis [OECD 111] (see paragraph (o)(1 5) 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 biodegrad ability [OECD 301] (see paragra ph (o)(1) of this guideline) and/or inherent biodegradabil ity [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 referen ce 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 subs tance 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.8 Target recoveries from th e test matrix should be 85% -110%; however, these ranges should not be used as the sole basis for accepta nce of the test. If parent recoveries from initial samples taken from the abiotic contro l are in the targeted range, the sample preparation procedures are suitable for the r ecovery of the parent compound from the test matrix. Lower-than-targeted recoveries in th e abiotic test system could be due to poor extraction efficiency, sorption to glasswar e, or abiotic degradation (see paragraph (h)(1)(iii) of this guideline).

(ii) Total recovery of radioactivity in both abiotic and biotic systems should normally range from 75 to 115% for individua l samples, and averag e 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 basi s for acceptance of the test. If mass balances from the abiotic system are in the targeted ra nge but those in the biotic test system are significantly below this range, the lower reco very likely results from the inability to efficiently trap 14 CO 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 bi otic system can be attributed to microbial activity. If the abioti c system indicates degr adation of parent over time, interpretation of these results may incl ude a description/expl anation of the likely abiotic process that occurred. Comparison betw een 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 a nd for the transformation products should be ≤
1% of the initial amount added to the test system, if possibl e. 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 ba lance, are reported. Th ese 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. 14 CO 2 or 3 H 2 O). The most common and useful models for this purpose employ fi rst-order kinetics. Most exposure models (e.g. EUSES, SimpleTreat) utilize first-orde r rates as critical input parameters.9 (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 conc entration 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 dur ing 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-compartmen t first-order loss function using nonlinear regression methods. Such e quations have the form:

t k 

### Ae y 1 − 

### =

) )+(Be (Ae y t k t k 2

1 − − 

### =

where y equals the percentage of parent remaining at time (t), A equals the percentage degraded at first-order rate constant k 1 , and B equals the percentage degraded at the firstorder rate constant k 2 . 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 biodegrada tion is biphasic, consistent with the existence in the test system of two different pools of test materi al (e.g. dissolved and sorbed) that exhibit different rates of biodegradation.

(iv) In a similar manner, mineralizatio n data can be fit to a simple or twocompartment first-order production mode l. Such models have the form:



## ) e ( A y t k 1 

## 1 −



## − =

) e ( )+B e ( A y t k t k 2 1 

### 1 1

− −



### − − =

where y equals the percentage of the material mineralized at time ( t ), A equals the percentage mineralized at first-order rate constant k 1, and B equals the percentage

mineralized at the first-order rate constant k 2

.

(v) In some situations, biodegradation, pa rticularly 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 (T 1⁄2 ) can be calculated10 from the estimated first-order rates ( k 1 or k 2 ) for each pool ( A or B ) using the following equation:

T 1⁄2 = - ln 2/ k

(vii) In some circumstances, the observed data can be fit to a variety of other models, such as Monod or other growth mode ls, 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 Degradatio n 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 a ppropriate to report de gradation times for 50% (DT 50 ) and 90% (DT 90 ) of the test material, if these levels of degradation are observed during the course of the study. These values can be determ ined 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 (r 2 ), F value, if available, and a plot of the fitted curve with the actual data should be provided. The estimated rate constants ( k 1 or k 2 ) and other parameters ( A, B ) should be reported with their standard

errors.

(3) Test report. (i) The type of study, i.e. wast ewater, activated sludge, mixing zone or anaerobic digester sl udge test, should be clearly stated in the test report.

(ii) The test report should al so 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 pr operties of test and reference substances;

(2) Substance names, CAS numbers, st ructural formulas and relevant physical/chemical properties of substances used as standards for identification of metabolites;

(3) Purities of and nature of known impuritie s in test and reference substances;

(4) Radiochemical purity and specific activ ity of radiolabeled substances;

(5) Positions within the molecu le of radiolabeled atoms.

(B) Environmental samples:

(1) Source of environmental samples incl uding geographical location and relevant11 data regarding known prior or existing expo sure to the test substance and related substances;

(2) Logic used to estimate relevant environmental concentration;

(3) Time, date and field conditi ons relevant to collection;

(4) Temperature, pH, DO and redox potential as appropriate;

(5) Suspended solids level, biologica l 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 in itiating 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 substa nce and associated logic for selection;

(4) Incubation conditions including li ghting, aeration type, temperature;

(5) Information on analytical techniques a nd the method(s) used for radiochemical

measurements;

(6) Number of replicates.

(D) Results:

(1) Precision and sensitivity of the an alytical methods including the LOD and LOQ;

(2) Recovery for each analyte and dispos ition 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 estim ate biodegradation rates from the data;

(5) Biodegradation rates and related parame ters with relevant standard errors along with correlation coefficients (R 2 ) and F statistics for the selected models;12 (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 fo r a substance in raw wastewater during its time in a sewage c onveyance 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 substan ce can be degraded by wastewater microbes. It is useful to perform th is test if there is sufficient time in a sewer for the substance to undergo significant biodegr adation 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/l). 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 regardi ng 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 comm ercialization of a new substance. This

concentration would reflect th e total mass released and th e volume of wastewater in which the release is diluted. Approaches for estimating wastewater concentration can be found in Holman (see paragraph (o)(21) of th is 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 firs t-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 lo w DO conditions. Biological activity is inhibited in the abiotic control, which is us ed for estimating mineralization by difference, determining extraction efficiency and recovery of the parent molecule, and quantifying other loss processes such as hydrolysis, oxi dation, volatilization or sorption to test

apparatus.

(ii) If an analytical method with the requi red sensitivity is available, the rate of parent degradation or transformation can be determined using a non-radiolabeled test13 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 an alytical methods with requir ed sensitivity are available for potential metabolites.

(iii) Test substance at an environmentally relevant con centration is dosed to both abiotic and biotic test systems, which are incubated with conti nuous slow mixing. The biotic samples are incubated in such a way that DO levels remain at or below 1 mg/l, which is typical for sewage. Samples are periodically removed fo r determination of mineralization and primary biodegradation.

(iv) Tests can be performed using an ope n batch system or a sealed, flow-through batch system where traps are used to capture evolved 14 CO 2 . The closed flow-through system should be used for volatile test substances and is usually preferred for 14 C-lableled test substances. Open systems are appropriate for nonvolatile 3 H-labeled test substances and for refining the biodegrad ation kinetics of nonvolatile 14 C test materials, whose ability to be mineralized has previously been established. In the open system,

mineralization to 14 CO 2 can be determined indirectly by measuring the difference in residual radioactivity between samples from the biotic and abiotic systems following

acidification. Similarl y, mineralization to 3 H 2 O can be determined indirectly by measuring the difference in radioactivity ina sample following drying. In the flowthrough systems, evolved 14 CO 2 is measured directly in th e base traps. In addition,

dissolved 14 CO 2 is determined by acidifying sample s 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 associ ated with the extracted solids. The levels of parent and metabolites are determined using chromatographic separation and when appropriate, radio-analytical detection methods. The solids re maining from the extraction process are combusted to estimate incorpora tion 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 obtaine d 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 ar e nonvolatile. It also can be adapted for volatile substances. Typically, 14 C or 3 H labeling of substances is required for the

assessment of mineralization. Eith er radiolabeled or nonlabeled substance can be used for the assessment of pr imary biodegradation.

(4) Description of the test method—(i) Test apparatus. (A) The volume of

wastewater in the test vessels is determ ined based on the number and volume of the samples needed for the test. Typically, 1-2 l of wastewater is placed in 2- or 4-l flasks. Ideally, the wastewater is incubated under controlled DO conditions (typically 0.2-1.0 mg/l). This condition can be achieved using an oxygen probe immersed in the wastewater attached to an oxygen controlle r connected to an actuator valve, which monitors and14 controls the aeration of the wastewater (see Fi gure 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 adde d 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 atmosphe re but incubated unde r conditions that

maintain DO levels at the desired level. Flow-through systems are sealed with an appropriate closure containi ng a sampling port with a valve for removing wastewater samples and connections for influent and effl uent gas lines. The closure can be a rubber stopper, but an alternative type of closure ma y be necessary when wo rking with a volatile hydrophobic substance. When testing volatile s ubstances, it is recommended that gas lines and sampling tubes consist of inert materials (e.g. polytetrafluoroe thylene, stainless steel, glass).

Figure 3. Example of controlled dissolv ed oxygen flow-through apparatus

(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 CO 2 . The test vessel is connected to a series of traps containing potassium hydroxide (e.g. 1.5 N) or other appropriate CO 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.15 (ii) Equipment. (A) The standard laboratory equipm ent listed in this paragraph is

used:

(1) Miscellaneous glassware and pipettes;

(2) Magnetic stirrers or shaker for c ontinuous mixing of the test flasks;

(3) Centrifuge;

(4) pH meter;

(5) Solid CO 2 /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 14 C and 3 H in liquid samples and solid samples (e.g. liquid scintillation counter);

(12) Equipment to quantify 14 C and 3 H in solid samples (e.g. sample oxidizer);

(13) Equipment to trap volatilized 14 C and 3 H from gas trapping system (in-line activated charcoal trap or equivalent);

(14) Equipment for thin layer chromatogr aphy (TLC) or high performance liquid chromatography (HPLC);

(15) Equipment to quantify 14 C and 3 H for TLC (scanner) or HPLC (in-line detector);

(16) Analytical equipment for the determin ation of the test (and reference) substance if specific substance analysis is used (e.g. gas chromatograph, highperformance liquid chromatograph, mass spectrometry).

(B) The laboratory equipment listed in this paragraph is useful but not essential:

(1) Oxygen meter;16 (2) Oxygen controller with prob e 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 si te-specific assessment, the wastewater should be obtained from the specific sewer system of interest. For a generic assessment samples should be predominantly derived from dome stic sources. Although difficult to duplicate in practice, the European Technical Guid ance Document (TGD) procedure uses 450 mg suspended solids/l and 270 mg BOD/l as defau lt levels for wastewater (see paragraph (o)(22) of this guideline). In North America, a typical wastewater contains 100 to 350 mg suspended solids/l and 110 to 400 mg BOD/l 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 s hould be noted at co llection. Collection

containers should allow for ade quate ventilation and measures should be taken to prevent the temperature of the wastewater from signifi cantly 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. To tal suspended solids (TSS), COD, pH and NH 3 (optional) in the wastewater should be determined.

(B) The preparation of the abiotic sy stem is typically performed using a

combination of chemical and heat sterilizat ion. A proven approach is to add mercuric chloride (1 g/l) to the wastew ater, 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-lite r container). After cooling, th e pH of the abiotic system should be measured and adjusted to match that of the biologic ally active system. Alternative approaches to deactivat e the system can also be used.

(vi) Test substance preparation. (A) Ideally, distilled wa ter should be used to prepare stock solutions of the test and re ference substances. When appropriate, an alternative method may be used to solubilize or disperse th e test substance in a manner consistent with its normal entry into the e nvironmental compartment of interest. Watermiscible nontoxic solvents may be used when necessary, but attenti on 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. w ithout water) to the test system in a manner that maximizes its even and rapid distributi on in the sludge. For substances that are17 poorly soluble and typica lly associated with suspended so lids 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 sy stems 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 solution should be checked by liq uid scintillation counting (LSC).

(vii) Test conditions—(A) Test temperature. Incubation should take place in the dark (preferred) or in diffuse light at a c ontrolled temperature, which may be the field temperature or a standard laboratory temperature of 20- 25°C. Depending on location, mean annual wastewater temperature ra nges 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 con tinuous mixing or stirring.

(viii) Test duration. The duration of the test shoul d be sufficient to assess the biodegradation of the test subs tance during its normal residence time in the sewer system. However, it may be extended to obtain ad ditional 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 subs tance concentration. While replicates can be prepared for each system, more useful kine tic information usually can be gained by increasing the number of time points at whic h samples are collected within a system.

(5) Procedure—(i) Dosing. At test initiation, the cham ber closure is removed and the test substance is quantitatively added directly with consta nt 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 id entical manner. Generally, the biotic systems are dosed first, followed by the abiotic systems. Exact timing is typical ly more critical for kinetic analyses.

(ii) Sampling schedule. Sampling times are selected based on existing biodegradation data or the results of a pilo t 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 additiona l samplings after 2, 5, 8, 12 and 24 hr and days 2, 3 and 4. The sampling schedule for a slowly degrading substance should be18 adjusted so that a sufficient number of m easurements are made during the degradation phase.

(iii) Measurement of mineralizati on—(A) Indirect measurement of 14 CO 2. ( 1 ) Individual replicate samples (e.g. 1 ml) are co llected 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 HCl) to lower the sample pH to <2.

( 2 ) The samples are bubbled with air fo r several hours or allowed to stand overnight to allow the dissolved 14 CO 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 14 CO 2 is calculated based on the difference between the total counts in the biotic and abiotic samples.

(B) Direct measurement of 14 CO 2. ( 1 ) Evolved 14 CO 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. Replic ate subsamples (e.g. 1 ml) are removed from the base trap and transferred to scintillation vi als, combined with a scintillation cocktail that is suitable for the sample matrix, and analyzed by LSC.

( 2 ) Dissolved 14 CO 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 comp artment with an appropriate CO 2 absorbent (e.g. 1.5 N KOH). The vessels are seal ed and sufficient acid (e.g. 6N HCl) 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 suffi cient length of time (e.g. overnight) to allow

CO 2 to diffuse from solution and be trapped from the headspace by the sorbent. Samples

of the sorbent are combined with a scintillat ion cocktail that is suitable for the sample matrix and analyzed by LSC.

Figure 4. System for recovering dissolved 14 CO 2

Sample from test vessel (10 - 20 ml) 1.5 N KOH (1 - 2 ml)19 (C) Indirect measurement of 3 H 2 O. ( 1 ) Individual replicate samples (e.g. 1 ml) are collected from each system and placed in se parate vials, located in a fume hood, that contain sufficient acid (e.g. 1 ml of 0.1 N HCl) 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 al lowed to dry completely to remove the 3 H 2 O. The samples are combined with a scintillatio n cocktail that is suitable for the sample matrix and analyzed by LSC. The percentage 3 H 2 O is calculated based on the difference between the total counts in the wet and dry sa mples 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 a nd to monitor for volatilization. The total solids in these samp les should not exceed 30 mg dr y weight in order to avoid counting efficiency problems.

(v) Measurement of parent and metabolites—(A) Extraction. ( 1 ) 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 c oncentrating and extracting the samples. A proven approach for nonvolatile test substa nces 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. Flas h freezing is a quick process if there is sufficient depth in the dry ice/acetone or liquid n itrogen 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 result ing 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 sulf ate) and allowed to dry pr ior 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 volatile20 test materials or metabolites.

( 4 ) Other approaches can be utilized, but with all approaches it is important to

document recoveries and consider the time invol ved in terminating biol ogical activity and factor it into the sample times used for kinetic analyses.

(B) Analysis of parent and metabolites. ( 1 ) The relative abundance of parent and

metabolites in the extracts can be determin ed using TLC, HPLC, or other separation techniques with radioactivity detection.

( 2 ) If sensitive specific an alytical methods are availa ble, primary biodegradation can be assessed by measuring th e total residual concentrati on 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 othe r direct means impossible. Depending on chromatographic behavior, it is usually possibl e 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 app ears and disappears in the sequence of biodegradation, can form an additional basis for inferring its identity. If necessary, the

K
ow of major metabolites can be estimat ed 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 a nd acidified to pH ≤ 2 by submerging it in a weak acid solution (e.g. 1 ml of 0.1 N HCl). The samples are allowed to sit for sufficient time (e.g. overnight) for

the dissolved 14 CO 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 typical ly represents incorporation of radioactivity

into biomass. The distribution of this radi oactivity among various co mponents 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 an d the radioactivity in the extracts analyzed by LSC. The relative abundance of parent a nd metabolites in the extract(s) can be determined as described above in paragraph (j)(5)(v)(B)( 1 ) of this guideline.

(k) 314B: Biodegradation in acti vated sludge—(1) General. (i) This test is designed to assess the extent to which a substance can be degraded in activated21 sludge and to provide rates of primary and ultimate biodegradation under the conditions

of the test. It is based on a procedure originally publishe d by Federle & Itrich (see paragraph (o)(7) of this guideli ne). Activated sludge in its va rious configurations is the most common secondary wastewater treatment process. The usefulne ss of the measured rates for predicting removal in actual treatm ent 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 al ready 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 obtai ned environmental samples. Under these circumstances, the normal ratio of substanc e to degraders is not disrupted and the observed biodegradation rates reflect in situ rates.

(iv) When a new substance will be releas ed continuously at some future time, the

most accurate rates are obtained when the s ubstance 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 environm ent 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 reflec t the total mass released and

the volume of wastewater in which the rel ease is diluted. Approaches for estimating wastewater concentration can be found in Holman (see paragraph (o)(21) of this guideline) and the European T echnical Guidance Document (see paragraph (o)(22) of this guideline).

(vi) Superseding the previous considerati ons 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 concentrat ion is employed. At high test substance concentrations, biodegradation may be associ ated 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 co nsidered in the interpretation of results. This factor is particularly important for continuously re leased substances, which often reach steady state conditions in wastewater systems.

(2) Principle of the test. (i) The test substance is incubated with abiotic and22 biotic activated sludge over a period of time. Biological activity is inhibited in the abiotic control, which is used for estimating minera lization by difference, establishing extraction efficiency and recovery of the parent molecu le, and quantifying other loss processes such as hydrolysis, oxidation, volatilization or sorption to test apparatus.

(ii) If an analytical method with the requi red 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 an alytical methods with requir ed sensitivity are available for potential metabolites.

(iii) Test substance at an environmentally relevant con centration is dosed to both abiotic and biotic test systems, which are incubated at a relevant temperature with continuous mixing when appropriate. Sa mples are periodically removed for

determination of mineralizati on and primary biodegradation.

(iv) Tests can be performed using an ope n batch system or a sealed, flow-through batch system where traps are used to capture evolved 14 CO 2 . The closed flow-through system should be used for volatile test substances and usually is preferred for 14 C-lableled test substances. Open systems are appropriate for nonvolatile 3 H-labeled test substances and for refining the biodegradation kinetics of nonvolatile 14 C-labeled test substances, whose ability to be mineralized has previ ously been established. In the open system,

mineralization to 14 CO 2 can be determined indirectly by measuring the difference in residual radioactivity between samples from the biotic and abiotic systems following

acidification. Similarl y, mineralization to 3 H 2 O can be determined indirectly by measuring the difference in radioactivity ina sample following drying. In the flowthrough systems, evolved 14 CO 2 is measured directly in th e base traps. In addition,

dissolved 14 CO 2 is determined by acidifying sample s 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 associ ated with the extracted solids. The levels of parent and metabolites are determined using chromatographic separation and when appropriate radio-analytical detection methods. The solids re maining from the extraction process are combusted to estimate incorpor ation into biomass by difference or can be further extracted using a modified Sutherla nd and Wilkinson procedure (see paragraphs (o)(7) and (o)(25) of this gui deline) 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, 14 C or 3 H radiolabelling is required for the assessment of

mineralization. Both either ra diolabelled or nonlabelled su bstance can be used for the assessment of primary biodegradation.23 (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 por t 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 stoppe r, but glass is recommended when working with a volatile hydrophobic test material. When testing volat ile substances, it also is recommended that gas lines and sampling tubes consist of in ert materials (e.g. polytetrafluoroethylene, stai nless steel, glass).

(B) The headspace of the test vessel is continuously purged with air or CO 2 -free

air at a rate sufficient to maintain the activ ated sludge in an aerobic condition, but not too fast to prevent efficient trapping of CO 2 . The test vessel is connect ed to a series of traps containing a potassium hydroxide (e.g. 1.5 N) or other appropriate CO 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 CO 2 /acetone 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 ra diolabeled substances;

(K) Equipment to quantify 14 C and 3 H in liquid samples and solid samples (e.g. liquid scintillation counter);24 (L) Equipment to quantify 14 C and 3 H in solid samples (e.g. sample oxidizer);

(M) Equipment to trap volatilized 14 C and 3 H from gas trapping system (in-line activated charcoal trap or equivalent);

(N) Equipment for TLC or HPLC;

(O) Equipment to quantify 14 C and 3 H for TLC (scanner) or HPLC (in-line detector);

(P) Analytical equipment for the determ ination of the test (and reference) substance if specific substa nce 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. Fo r a generic assessment activat ed 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 303A] (see paragraph (o)(2 ) of this guideline), which has been fed wastewater

amended with unlabeled test substance. Th e source of the starti ng sludge, wastewater (influent) and the operating conditions (influ ent concentration, hydrau lic 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 concentr ation based on expected usage volumes. In general, steady state will be reached within 2 to 3 SRTs after whic h point the sludge can be used for testing.

(iv) Collection, transport and storage of activated sludge. The activated sludge should be collected fr om a well-mixed region of th e aeration basin. The pH and temperature of the sample should be noted at collection. Collect ion containers should allow for adequate ventilation and measures should be taken to prevent temperature of the sludge from significantly exceeding the temp erature used in the test. The activated sludge is typically stored at test temperat ure 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 concentratio n should be measured and if necessary25 adjusted to the target con centration. The TGD uses a de fault level of 4000 mg/l in its generic scenario (see paragr aph (o)(22) of this guideline). However, 2500-3000 mg/l may be more typical for North America (see paragr aph (o)(23) of this guideline). The sludge can be diluted with liquor or tap water if the solids concen tration 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 T SS level and pH should then be determined.

(B) Abiotic sludge is typically prepared us ing a combination of chemical and heat sterilization. A proven approach is to add merc uric chloride solution (1 g/l) 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 ab iotic 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 wa ter should be used to prepare stock solutions of the test and re ference substances. When appropriate, an alternative method may be used to solubilize or disperse th e test substance in a manner consistent with its normal entry into the e nvironmental compartment of interest. Watermiscible nontoxic solvents may be used when necessary, but attenti on 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 solven ts, <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 c ontrolled temperature, which may be the field temperature or a standard la boratory temperature of 20-25°C.

(B) Agitation. To keep the sludge well mixed an d 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 the26 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 th e 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 subs tance concentration. While replicates can be prepared for each system, more useful kine tic information usually can be gained by increasing the number of time poi nts sampled within a system.

(5) Procedure—(i) Dosing. At test initiation, the chamber closure is removed and the test substance is quantitatively a dded directly to the activated sl udge with constant mixing. It is recommended that the dose be administered gradually below the air-water interface, to ensure uniform distribu tion of the test substance in the sludge. The biotic and abiotic systems are dosed in an id entical manner. Generally, the biotic systems are dosed first, followed by the abiotic systems. Exact timing is typical ly more critical for kinetic analyses.

(ii) Sampling schedule. Sampling times are selected based on existing biodegradation data or the results of a pilo t 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 14 CO 2. ( 1 ) 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 HCl) to lower the sample pH to <2. The to tal solids in the samples should not exceed 30 mg dry weight.

( 2 ) The samples are bubbled with air fo r several hours or allowed to stand overnight to allow the dissolved 14 CO 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 14 CO 2 is calculated based on the difference between the total counts in the biotic and abiotic samples.

(B) Direct measurement of 14 CO 2. ( 1 ) For rapidly degrading substances, it can be difficult to measure accurately the rate of 14 CO 2 evolved due to the rate of the mass transfer

of 14 CO 2 from the headspace into the base trap. Under these conditions, it is recommended

that indirect measurement of 14 CO 2 be conducted simultaneously with direct

measurement.27 ( 2 ) Evolved 14 CO 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 syst em reconnected as qu ickly as possible. Replicate subsamples (e.g. 1 ml) are removed from the base trap and transferred to scintillation vials, combined with a scintillat ion cocktail that is suitable for the sample matrix, and analyzed by LSC.

( 3 ) Dissolved 14 CO 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 comp artment with an appropriate CO 2 absorbent (e.g. 1.5 N KOH). The vessels are seal ed and sufficient acid (e.g. 6N HCl) 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 suffici ent length of time (e.g. overnight) to allow

CO 2 to diffuse from solution and be trapped from the headspace by the sorbent. Samples

of the sorbent are combined with a scintillat ion cocktail that is suitable for the sample matrix and analyzed by LSC.

(C) Indirect measurement of 3 H 2 O. ( 1 ) Individual replicate samples (e.g. 1 ml) are collected from each system and placed in se parate vials, located in a fume hood, that contain sufficient acid (e.g. 1 ml of 0.1 N HCl) 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 al lowed to dry completely to remove the 3 H 2 O. The samples are combined with a scintillatio n cocktail that is suitable for the sample matrix and analyzed by LSC. The percentage 3 H 2 O is calculated based on the difference between the total counts in the wet and dry sa mples 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. ( 1 ) 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 c oncentrating and extracting the samples. A proven approach for nonvolatile test substa nces 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. Flas h freezing is a quick process28 if there is sufficient depth in the dry ice/acetone or liquid n itrogen 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 to tal radioac tivity 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 dr y 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 colu mn containing a drying agent. In most cases, it is not efficient to use liquid/liquid extraction to r ecover parent and metabolites from MLSS. The total radioactivity in all extracts is determined by LSC. Care should be taken in concentrating extracts containing volat ile test substances or metabolites.

( 4 ) Other approaches can be utilized, but with any approach it is important to

document recoveries and consider the time in volved in terminating biological activity, and factor it into the sample times used for kinetic analyses.

(B) Analysis of parent and metabolites. ( 1 ) The relative abundance of parent

and metabolites in the extract s can be determined using TLC, HPLC or other separation techniques with radioactivity detection.

( 2 ) If sensitive specific an alytical methods are availa ble, primary biodegradation can be assessed by measuring th e total residual concentrati on 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 othe r direct means impossible. Depending on chromatographic behavior, it is usually possibl e 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 app ears and disappears in the sequence of biodegradation, can form an additional basis for inferring its identity. If necessary, the

K
ow of major metabolites can be estimat ed by HPLC (e.g. using OECD 117; see paragraph (o)(24) of this guideline) using an on-line radioactivity detector.

(vi) Measurement of extracted solid s 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 procedure29 (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 an d the radioactivity in the extracts analyzed by LSC. The relative abundance of parent a nd metabolites in the extract(s) can be determined as described in paragraph (k)(5)(v)(B)( 1 ) of this guideline.

(l) 314C: Mineralization and transfor mation 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 dige ster. Anaerobic digestion is commonly used

to stabilize and reduce the ma ss of sludge generated by wast ewater treatment plants. Biodegradation during anaerobic di gestion 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 le vels of parent and potential degradation products associated with the primary and s econdary 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 st eady 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 sl udge can be incubated with th e maximum concentration of test substance expected to occu r in sludge as a resu lt of periodic releases. Approaches for estimating expected sludge concentrations can be found in Holman (see paragraph (o)(21) of this guideline) a nd the European Technical Guidance Document (see paragraph (o)(22) of this guideline). For existing subs tances continuously disc harged to wastewater, freshly collected digester sludge can be incuba ted 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 th e test for acclimation of the sludge to a new (test) substance. However, a laboratory an aerobic reactor operated in a draw-and-fill mode, amended with the substance at its expected concentratio n in sludge, can be considered as an option for generating accl imated sludge. This type of sludge should yield the most accurate kinetic data for a ne w 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. Biologi cal activity is inhibi ted in the abiotic control, which is used for estimating minera lization by difference, establishing extraction efficiency and recovery of the parent molecu le, and quantifying other loss processes such as hydrolysis, oxidation, volatilization or sorption to test apparatus.30 (ii) If an analytical method with the requi red 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-radiol abeled substances cannot be determined unless the biodegradation pathway is well understood and analyt ical methods with required sensitivity are available for potential metabolites.

(iii) Test substance at an environmentally relevant con centration is dosed to both abiotic and biotic test systems, which are in cubated at a relevant temperature under static

conditions with occasional or continuous mi xing. Samples are periodically removed for

determination of mineralizati on 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 14 CO 2 and 14 CH 4 . The closed flowthrough system is should be used for volatile te st substances and usua lly is preferred for

14 C-labeled test substances. Open sy stems are appropriate for nonvolatile 3 H-labeled test substances and for refining the bi odegradation kinetics of nonvolatile 14 C-labeled test substances, whose ability to be mineralized has previously been established. In the open system, mineralization to 14 CO 2 and 14 CH 4 can be determined indirectly by measuring the difference in residual radioactivity between samples from the biotic and abiotic systems following acidification. Sim ilarly, mineralization to 3 H 2 O can be determined indirectly by measuring the difference in residual radioac tivity between samples following drying. In the closed batch system, gas sampling bags ma y also be used to trap gas from anaerobic digester sludge. In the flow-through systems, evolved 14 CO 2 is measured directly in the base traps, and 14 CH 4 is combusted to 14 CO 2 which is measured directly in a second set of base traps. As an option, dissolved 14 CO 2 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 associat ed with the extracted solids. The level of parent and metabolites is determined us ing chromatographic separation and, when appropriate, radio-analytical detection methods. The solids re maining from the extraction process are combusted to estimate incorpora tion 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 obtaine d 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, 14 C or 3 H labeling of substances is required for the assessment of

mineralization. Either radiolabeled or non labeled substance can be used for the assessment of primary biodegradation.

(4) Description of the test me thod—(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, dilu ted with an anaerobic31 salts medium, is placed in 0.5 to 2-l cont ainers. The sludge is diluted to facilitate quantitative removal of subsamples dur ing the study. Anaerobic sludge should be protected from oxygen at all time s including setup and sampling.

(B) Open batch systems are generally cl osed with a foam or cotton stopper to minimize evaporative loss of water and are in cubated 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 th at gas lines and sampling tubes consist of inert materials (e.g. polytetrafluoroe thylene, stainless steel, glass).

(C) The flow-through system is a modifica tion 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)(11) of this guide line). An example of a typical system is shown in Figure 5. The test ve ssels are continuously purged with a flow of nitrogen and connected to a series of traps containing potassium hydroxide solu tion (1.5 N) or other appropriate CO 2 absorbent. An empty trap is usually included and posit ioned in front of the absorbent in the trapping train, as a pr ecaution against backflow or condensation. The effluent gases from these traps are mi xed with oxygen and passed through a quartz column packed with cupric oxide and main tained at approximately 800°C in a tube furnace, to combust methane to CO 2 . The gas exiting the combustion column is then passed through another series of base traps.

(ii) Equipment. (A) The standard la boratory equipment listed in this paragraph is

used: (1) Miscellaneous glassware and pipettes;

(2) Magnetic stirrers or shaker for con tinuous mixing of the test flasks;32 Figure 5. Example of flow-through anaerobic test apparatus

(3) Centrifuge;

(4) pH meter;

(5) Solid CO 2 /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 14 C and 3 H in liquid samples and solid samples (e.g. liquid scintillation counter);

(12) Equipment to quantify 14 C and 3 H in solid samples (e.g. sample oxidizer);

(13) Equipment to trap volatilized 14 C and 3 H from gas trapping system (in-line activated charcoal trap or equivalent);33 (14) Equipment for TLC or HPLC;

(15) Equipment to quantify 14 C and 3 H for TLC (scanner) or HPLC (in-line detector);

(16) Analytical equipment for the determin ation of the test (and reference) substance if specific substance analysis is used (e.g. gas chromatograph, highperformance 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 simulatio n 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 an d secondary sludge from a wa stewater 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, ac climated sludge from a laboratory anaerobic reactor may be more appropriate. For a generi c 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 dom estic 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 t ypically stored under the exclusion of oxygen at test temperature. Storage cont ainers should be vented in a manner that releases excess biogas but does not allow ambi ent air into the container.

(v) Preparation of the test sy stems—(A) Dilution medium. An appropriate

volume of the mineral salts solu tion listed in this paragraph should be prepared prior to

test initiation. This solution is autoclaved fo r 30 min with slow e xhaust, and allowed to cool overnight in an an aerobic chamber or under an anaerobic atmosphere.34 Ingredient Amount Potassium phosphate monobasic, KH 2 PO 4 ...................................... 8.5 mg/l Potassium phosphate dibasic, K 2 HPO 4 .......................................... 21.8 mg/l Sodium phosphate dibasic heptahydrate, Na 2 HPO 4 • ......... 7H 2 O.. 50.3 mg/l Ammonium chloride, NH 4 Cl ......................................................... 20.0 mg/l Magnesium sulfate heptahydrate, MgSO 4 •7H 2 O............................. 2.2 mg/l Calcium chloride anhydrous, CaCl 2 ................................................. 2.8 mg/l Ferrous chloride, FeCl 2 •4H 2 O........................................................ 0.25 mg/l

Deionized water ............................................................................ to volume

(B) Digester sludge. ( 1 ) 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 prot ect the sludge from exposure to oxygen. The digester sludge should be si eved 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/l. If the solids are too high, they can be diluted with the dilution media. Alternatively, if the solids concentration is t oo 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 sl udge is typically performed using a

combination of chemical and heat sterilizat ion. A proven approach is to add mercuric chloride solution (1 g/l) 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 contai ner). After cooling, th e pH of the abiotic sludge should be measured and adjusted to ma tch that of the biologically active sludge. Alternative approaches to d eactivate the sludge can also be used. When preparing the abiotic sludge it should be st ored 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 s ubstances. When appropr iate, an alternative method may be used to solubili ze or disperse the test substance in a manner consistent with its normal entry into digester sludge. Wh en practical, the dosing solutions should be equilibrated overnight in a re ducing 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 accura te administration of the dose between like systems. Ideally, when dosing with aque ous solutions, the a dded 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 poo rly soluble and typica lly 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.35 (B) As an alternative, the test substan ce can be applied to dried inactive sludge solids, which can be mixed into the test sy stem. Water-miscible nontoxic solvents may be used when necessary, but atten tion should be paid to the asso ciated organic load involved with adding organic solvents. In addition, the te st 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 temperat ure, which may be the typical operating temperature for an anaerobic digester (35°C +/- 3°C), a fiel d temperature, or a standard laboratory temperature of 20-25°C.

(B) Agitation. To simulate the static conditions th at occur in a digester, the test vessels are usually not continuously mixed. During sampling, they should be kept well mixed to ensure a representativ e 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 s hould be sufficient to assess the biodegradation of the test substance duri ng 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 or der to estimate kinetic constants or assess the completeness of degradati on 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 mineraliza tion apparatus, and can be subsampled 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 expos ure 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 pilo t 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 a dditional 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 substanc es should be adjusted so that a sufficient

number of measurements are made during the degradation phase.

(iii) Measurement of mineralization—(A) Indirect measurement of 14 C Gases36 ( 14 CO 2 and 14 CH 4 ). Direct LSC counting of samples is not possible due to the high solids levels in the samples. Therefore, samples ar e centrifuged and the supernatant analyzed for total radioactivity by LSC, and the solids co mbusted and then analyzed for radioactivity to determine the total radioactivity in the sa mple. Individual repli cate 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 HCl) to lower the sa mple pH to <2. The samples are centrifuged and the supernatant transferred to a scintillation vial, which is allowed to stand overnight for the dissolved 14 CO 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 solids remaining in the centrif uge tube are combusted using a sample oxidizer prior to LSC. The percentage of total 14 C gas produced is calculated based on the difference between the total counts in the biotic and abiotic samples.

(B) Direct measurement of 14 CO 2 and 14 CH 4. ( 1 ) Evolved 14 CO 2 and 14 CH 4 : Direct measurement of 14 CO 2 and 14 CH 4 is possible only in a sealed flow-through batch system with connected base traps. For 14 CO 2 , the first base trap in the first trapping train is removed and quickly capped. The remaini ng 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. Replic ate subsamples (e.g. 1 ml) from the removed base trap are transferred to scin tillation 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 14 CH 4

.

( 2 ) Dissolved 14 CO 2 (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 comp artment with an appropriate CO 2 absorbent (e.g. 1.5 N KOH). The vessels are seal ed and sufficient acid (e.g. 6N HCl) 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 CO 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 3 H 2 O. Samples (e.g. 8 ml) of sludge are collected from each system and placed in centrifuge t ubes, located in a fume hood, that contain sufficient acid (e.g. 1 ml of 0.1 N HCl) to lower the sample pH to <2. The tubes are mixed and centrifuged. Individua l replicate samples (e.g. 1 ml ) of the supernatant are placed in separate vials. Half of the samp les are immediately analyzed directly by LSC for a wet measurement. The remaining samples are allowed to dry completely to remove

the 3 H 2 O. The samples are combined with a scintillation cocktail that is suitable for the sample matrix and analyzed by LSC. The percentage 3 H 2 O 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, samples37 are centrifuged and the supernatant analyzed fo r 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 sample s 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 usi ng a sample oxidizer prior to LSC.

(v) Measurement of parent and metabolites—(A) Extraction. ( 1 ) 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 c oncentrating and extracting the samples. A proven approach for nonvolatile test substa nces 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. Flas h freezing is a quick process if there is sufficient depth in the dry ice/acetone or liquid n itrogen 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 to tal radioac tivity 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 ph ase or liquid/liquid extraction. The solids can then be extracted directly or mixed w ith 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 wate r from the solvent by running it through a column containing a drying agent. The total radi oactivity in all extracts is determined by LSC. Care should be taken in concentrating extr acts 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 in volved in terminating biological activity, and factor it into the sample times used for kinetic analyses.

(B) Analysis of parent and metabolites. ( 1 ) The relative abundance of parent

and metabolites in the extracts can be determ ined using TLC, HPLC or other separation techniques with radioactivity detection.

( 2 ) If sensitive specific an alytical methods are availa ble, primary biodegradation can be assessed by measuring th e total residual concentrati on 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, if38 authentic standards exist. Usually, the quantity and purity of metabolites generated in this test make definitive identification by othe r direct means impossible. Depending on chromatographic behavior, it is usually possibl e 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 app ears and disappears in the sequence of biodegradation, can form an additional basis for inferring its identity. If necessary, the

K
ow of major metabolites can be estimat ed by HPLC (e.g. using OECD 117; see paragraph (o)(24) of this guideline) using an on-line radioactivity detector.

(vi) Measurement of extracted solid s 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 so lvents 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)( 1 ) of this guideline.

(m) 314D Biodegradation in treated effl uent-surface water mixing zone—(1)

General. (i) This test is designe d to evaluate the biodegra dation of the portion of a substance that passes through trea tment and is released in efflue nt to surface water. It can be used to demonstrate that biodegradation oc curring 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 ultima te biodegradation in this environmental compartment. The results can be used to estimate the reduction in substance concentration resulting from biodegradati on as a volume of water moves downstream from a wastewater treatment pl ant outfall. The test medium c onsists of fres hly 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 te st 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 wa ter 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 stat e 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 guide line) and the European Technical Guidance Document (see paragraph (o)(22) of this guideline).39 (iii) For substances that are or will be continuous ly released to wastewater, degrader populations in the treatment plant will become acclimated to the substance. For existing substances continuous ly discharged to wastewat er, surface water with the expected concentration of te st substance and freshly colle cted effluent under a given discharge scenario will provide the most realistic kinetic pa rameters. For new substances that will be continuously discharged to wastew ater, the use of efflue nt that was exposed

to the substance under simulated activated sl udge 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 degrad ation in treatment, as compared to a real

environmental release situation.

(iv) In most circumstances, due to anal ytical 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 sc enarios for release of treated effluent to surface water. Biological activity is inhibited in the abiotic control, which is used for estimating mineralization by difference, estab lishing extraction efficiency and recovery of the parent molecule, and quantifying other loss processes such as hydrolysis, oxidation, volatilization or so rption to test apparatus.

(ii) If an analytical method with the requi red 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 an alytical methods with requir ed sensitivity are available for potential metabolites.

(iii) Test substance at an environmentally relevant con centration is dosed to both abiotic and biotic test systems, which are incubated at a relevant temperature with continuous mixing when appropriate. Sa mples are periodically removed for

determination of mineralizati on and primary biodegradation.

(iv) Tests can be performed using an ope n batch system or a sealed, flow-through batch system in which traps are used to capture evolved 14 CO 2 (see Figure 2). The closed flow-through system should be used for volatil e test substances and usually is preferred

for 14 C-lableled test substances. Open systems are appropriate for nonvolatile 3 H-labeled test substances and for refining the biodegradation kine tics of nonvolatile 14 C-labeled test substances, whose ability to be mineralized has previously been established. In the open system, mineralization to 14 CO 2 can be determined indirectly by measuring the difference in residual radioactivity between samples from the biotic and abiotic systems following

acidification. Similarl y, mineralization to 3 H 2 O can be determined indirectly by measuring the difference in radioactivity in a sample following drying. In the flow-40 through systems, evolved 14 CO 2 is measured directly in th e base traps. In addition,

dissolved 14 CO 2 is determined by acidifying sample s 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 associat ed with the extracted solids. The level of parent and metabolites is determined us ing chromatographic separation and, when appropriate, radio-analytical detection methods. The solids re maining from the extraction process are combusted to estimate incorpora tion 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 obtaine d 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, 14 C or 3 H labeling of substances is required for the assessment of

mineralization. Either radiolabeled or non labeled 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 l of surface water are pl aced in 2- or 4-l flasks. Open batch systems are generally cl osed with a foam or cotton stopper to minimize evaporative loss of water. Flow-throug h 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 th at gas lines and sampling tubes consist of inert materials (e.g. polytetrafluoroet hylene, stainless steel, glass).

(B) The headspace of the test vessel is continuously purged with air or CO 2 -free air at a rate sufficient to maintain the syst em in an aerobic condition but not fast enough to prevent efficient trapping of CO 2 . The test vessel is connected to a series of traps containing a potassium hydroxide (1.5 N) or other appropriate CO 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 con tinuous mixing of the test flasks;

(C) Centrifuge;41 (D) pH meter;

(E) Solid CO 2 /acetone or liquid nitrogen bath;

(F) Freeze dryer (lyophilizer);

(G) Oven or microwave oven for dr y weight determinations;

(H) Membrane filtration apparatus;

(I) Autoclave;

(J) Facilities to handle radi olabeled substances;

(K) Equipment to quantify 14 C and 3 H in liquid samples and solid samples (e.g. liquid scintillation counter);

(L) Equipment to quantify 14 C and 3 H in solid samples (e.g. sample oxidizer);

(M) Equipment to trap volatilized 14 C and 3 H from gas trapping system (in-line activated charcoal trap or equivalent);

(N) Equipment for TLC or HPLC;

(O) Equipment to quantify 14 C and 3 H for TLC (scanner) or HPLC (in-line detector);

(P) Analytical equipment for the determ ination of the test (and reference) substance if specific substance analysis is used (e.g. gas chromatograph, highperformance liquid chromatograph, mass spectrometer).

(iii) Selection of environmental samples. (A) The source of surface water, activated sludge or effluent should be consiste nt with the objective of the simulation test. For a site-specific assessment, activated sludg e or effluent should be obtained from the specific wastewater treatment plant of inte rest. Likewise, the surface water should be obtained upstream from that treatment plan t’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 cond itions below the outfall are known, the test medium can simply be water samples obtained downs tream 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 rece iving predominantly domestic wastewater. Likewise, the surface water should be typical of surface waters into which effluent is released. If the substance is currently a com ponent of wastewater en tering the wastewater treatment facility or is episodically released to wastewater, freshly collected activated42 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 simula ting 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 filtere d or centrifuged to remove biosolids, and the liquor and

biosolids can then be ad ded at a defined ratio to the test system. For a generic assessment, the TGD assumes that effluent containing 30 mg biosolids/l is diluted 10-fold into surface water containing 15 mg suspended solids/l (s ee 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 l ma y be considered to si mulate critical low flow conditions that might occur during dry seasons.

(C) For a new substance that will be c ontinuously 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 act ivated sludge test (e.g. an OECD 303A test) (see paragraph (o)(2) of this guideline), wh ich has been fed wastewater amended with

unlabeled test substance. The source of th e starting sludge, wastewater (influent), and operating conditions (influent concentration, hydraulic rete ntion time, solids retention time) for the laboratory unit should accurately re flect 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 st orage of environmental samples. The activated sludge should be collected from a well-mix ed region of the aera tion basin. Surface water

should be collected from a site with known in puts of wastewater. The temperature of the samples should be noted at collection. Coll ection 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 aerat ion. Samples should not be stored frozen.

(v) Preparation of test systems. (A) The surface water should be characterized by measuring TSS, total har dness and pH. A standard plat e 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 allowe d to settle. The TSS concentration of the liquor is measured. The liquor is added to the surface water at a

volume sufficient to achieve th e targeted biosolids concen tration. 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 bioso lids 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.43 An optional standard plate c ount and organic carbon analysis can also be performed.

(B) The abiotic system is ty pically prepared using a combination of chemical and heat sterilization. A proven approach is to a dd mercuric chloride solution (0.1 g/l) to the mixture, which is then autoclaved for at leas t 90 min. Typically the volume of medium is <= half the volume of the container being auto claved (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 wa ter should be used to prepare stock solutions of the test and re ference substances. When appropriate, an alternative method may be used to solubilize or disperse th e test substance in a manner consistent with its normal entry into the environment. Water-miscible nontoxic solvents may be used when necessary, but attention shou ld be paid to the associated organic load involved with adding organic so lvents. Alternatively, the samp le 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 appropriat e 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 in itial 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 solven ts, <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 c ontrolled temperature, which may be the field temperature or a standard labor atory 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 ki netic 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.44 (ix) Number of test vessels. At a minimum, there should be a single abiotic and a single biotic test vessel for each test subs tance concentration. While replicates can be prepared for each system, more useful kine tic information usually can be gained by increasing the number of time poi nts sampled within a system.

(5) Procedure—(i) Dosing. At test initiation, the te st vessel is opened and the test substance is quantitativel y 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 subs tance in the test me dium. 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 pilo t 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 additio nal 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 ar e made during the degradation phase.

(iii) Measurement of mineralizati on—(A) Indirect measurement of 14 CO 2. ( 1 ) 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 HCl) 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 14 CO 2 to diffuse from the samples. Th e samples are co mbined with a

scintillation cocktail that is suitable for th e sample matrix and analyzed by LSC. The percentage of 14 CO 2 is calculated based on the differen ce between the total counts in the biotic and abiotic samples.

(B) Direct measurement of 14 CO 2. ( 1 ) For rapidly degrading substances, it can be difficult to measure accurately the rate of 14 CO 2 evolved due to the rate of the mass transfer

of 14 CO 2 from the headspace into the base trap. Under these conditions, it is recommended

that indirect measurement of 14 CO 2 be conducted simultaneously with direct

measurement.

( 2 ) Evolved 14 CO 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 syst em reconnected as qu ickly as possible. Replicate subsamples (e.g 1 ml) from the removed base trap are transferred to scintillation vials, combined with scintillat ion cocktail that is suitable for the sample matrix, and analyzed by LSC.45 ( 3 ) Dissolved 14 CO 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 comp artment with an appropriate CO 2 absorbent (e.g. 1.5 N KOH). The vessels are seal ed and sufficient acid (e.g. 6N HCl) 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 suffici ent length of time (e.g. overnight) to allow

CO 2 to diffuse from solution and be trapped from the headspace by the sorbent. Samples

of the sorbent are combined with a scintillat ion cocktail that is suitable for the sample matrix and analyzed by LSC.

(C) Indirect measurement of 3 H 2 O. ( 1 ) Individual replicate samples (e.g. 1 ml) are collected from each system and placed in se parate vials, located in a fume hood, that contain sufficient acid (e.g. 1 ml of 0.1 N HCl) 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 a llowed to dry completely to remove the 3 H 2 O. The samples are combined with a scintillatio n cocktail that is suitable for the sample matrix and analyzed by LSC. The percentage 3 H 2 O is calculated based on the difference between the total counts in the wet and dry sa mples and the initial level of radioactivity dosed to the samples.

(iv) Measurement of total radi oactivity 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 meas urements 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. ( 1 ) 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 c oncentrating and extracting the samples. A proven approach for nonvolatile test substa nces 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. Flas h freezing is a quick process if there is sufficient depth in the dry ice/acetone or liquid n itrogen 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 solven t and solids separately. The filter must be compatible with the solvent type (e.g. aque ous or non-aqueous). The resulting extracts can be concentrated through evapor ation 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 subsequently46 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 ex traction efficiency. The total radioactivity in all extracts is determined by LSC. Care should be taken in c oncentrating 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 in volved in terminating biological activity, and factor it into the sample times used for kinetic analyses.

(B) Analysis of parent and metabolites. ( 1 ) The relative abundance of parent and

metabolites in the extracts can be determ ined using TLC, HPLC or other separation techniques with radioactivity detection.

( 2 ) If sensitive specific an alytical methods are availa ble, primary biodegradation can be assessed by measuring th e total residual concentrati on 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 othe r direct means impossible. Depending on chromatographic behavior, it is usually possibl e 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 app ears and disappears in the sequence of biodegradation, can form an additional basis for inferring its identity. If necessary, the

K
ow of major metabolites can be estimat ed by HPLC (e.g. using OECD 117; see paragraph (o)(24) of this guideline) usi ng an on-line radioactivity detector.

(vi) Measurement of extracted solids. Since the filters will retain carbonate salts as well as microorganisms from the test syst em, the filter containing the biosolids is placed in a scintillation vi al and acidified to pH ≤ 2 by submerging it in a weak acid solution (1 ml of 0.1 N HCl). The samples are allowed to sit for sufficient time (e.g. overnight) for the dissolved 14 CO 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 incor poration of radioactiv ity into biomass.

(vii) Measurement of volatilized radioactivity. For volatile test substances, the volatility traps are extracted with appropriate so lvents 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)( 1 ) of this guideline.

(n) 314E Biodegradation in untreated wastewater-surface water mixing zone—(1) General. (i) In developing regions l acking wastewater treatment infrastructure, it is common for wastewater to be directly discharged to surface waters.47 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 bi odegradation as well as rates of primary and ultimate biodegradation under such direct disc harge conditions. The results can be used

to estimate the reduction in substance con centration 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 bi odegradation 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 concen tration, factors to consider in designing

this test include DO concentrati on and the degree to which effl uent is diluted into surface

water.

(ii) For existing substances consistently present in wastewater, freshly collected

wastewater and surface water incubated with radi olabeled test substance at a tracer level will provide the most realistic kinetic parameters regarding the current substance load.

For substances not consiste ntly present in wastewater, sufficient test substance (radiolabeled and unlabeled) should be added to approximate the expected concentration

in wastewater diluted into surface wate r during an episodic release or following commercialization of a new substance. Appr oaches for estimating such an expected wastewater concentration can be found in Holman (see paragraph (o)(21) of this guideline) and the European T echnical Guidance Document (see paragraph (o)(22) of this guideline).

(iii) For low dilution situations, it is be st to incubate the mixtures under reduced DO conditions (1-4 mg/l) to simulate the DO level below a wastewater outfall. In this test, the substance and its degrader populati ons 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 singl e 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 dilute d into clean and wastewater-impacted surface waters. In this test design, the test substan ce is dosed into wastew ater diluted in clean surface water and disappearance of test s ubstance 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/l) 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 us ed for estimating mineralization by difference, establishing extraction efficiency and recove ry of the parent molecule, and quantifying other loss processes such as hydrolysis, oxi dation, volatilization or sorption to test48 apparatus.

(ii) If an analytical method with the requi red 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 an alytical methods with requir ed sensitivity are available for potential metabolites.

(iii) Test substance at an environmentally relevant con centration is dosed to both abiotic and biotic test systems, which are incubated at a relevant temperature with continuous mixing. The biotic samples are incuba ted in such a way that DO levels remain at a reduced level (1-4 mg/l), characteristic of the situation below a wastewater outfall. Samples are periodically removed for dete rmination 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 bi odegradation 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 sy stem to simulate wastewater being diluted into previously polluted surface water, and the samp ling process is repeated.

(v) Tests can be performed using an ope n batch system or a sealed, flow-through batch system in which traps are used to capture evolved 14 CO 2 (see Figure 2). The closed flow-through system should be used for volatil e test substances and usually is preferred

for 14 C-lableled test substances. Open systems are appropriate for nonvolatile 3 H-labeled test substances and for refining the biodegradation kine tics of nonvolatile 14 C-labeled test substances, whose ability to be mineralized has previously been established. In the open system, mineralization to 14 CO 2 can be determined indirectly by measuring the difference in residual radioactivity between samples from the biotic and abiotic systems following

acidification. Similarl y, mineralization to 3 H 2 O can be determined indirectly by measuring the difference in radioactivity ina sample following drying. In the flowthrough systems, evolved 14 CO 2 is measured directly in th e base traps. In addition,

dissolved 14 CO 2 is determined by acidifying sample s in a sealed vessel and measuring radioactivity in a base trap contained in the vessel.

(vi) Samples from both systems are analy zed for total radioactivity, extractable parent and metabolites and radioactivity associat ed with the extracted solids. The level of parent and metabolites is determined us ing chromatographic separation and when appropriate, radio-analytical detection methods. The remaining solids from the extraction process are combusted to estimate incorpora tion 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 ar e nonvolatile. It also can be adapted for volatile substances. Typically, 14 C or 3 H-radiolabeling of substa nces is required for the49 assessment of mineralization. Eith er radiolabeled or nonlabeled substance can be used for the assessment of pr imary 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 l of sample is placed in 2- or 4-l flasks. Ideally, the wastewater-surface water mixture is incubated under one or more controlled DO conditions (e.g. 1 and 4 mg DO/l). This condition can be achieved using an oxygen probe immersed in the wastewater attached to an oxygen controlle r 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 incuba ted with stirring but minimum aeration, to keep DO at desired levels, and nitrogen or ai r can be added periodical ly to maintain the DO level. In this case, DO readings should be reported at regula r intervals.

(B) Open systems are generally closed w ith a foam or cotton stopper to minimize evaporative loss of water. Flow-through system s are sealed with an appropriate closure containing a sampling port with a valve fo r removing samples, and connections for influent and effluent gas lines. This cl osure 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 CO 2 -free air at a rate sufficient to maintain the system in an aerobic condition, bu t not so fast as to prevent efficient trapping of CO 2 . The test vessel is connected to a series of traps containing a potassium hydroxide (1.5 N) or other appropriate CO 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. (A) The standard laboratory equipm ent listed in this paragraph is

used:

(1) Miscellaneous glassware and pipettes;

(2) Magnetic stirrers or shaker for con tinuous mixing of the test flasks;

(3) Centrifuge;

(4) pH meter;

(5) Solid CO 2 /acetone or liquid nitrogen bath;

(6) Freeze dryer (lyophilizer);

(7) Oven or microwave oven for dry weight determinations;50 (8) Membrane filtration apparatus;

(9) Autoclave;

(10) Facilities to handle radiolabeled substances;

(11) Equipment to quantify 14 C and 3 H in liquid samples and solid samples (e.g. liquid scintillation counter);

(12) Equipment to quantify 14 C and 3 H in solid samples (e.g. sample oxidizer); - Equipment to trap volatilized 14 C and 3 H from gas trapping system (in-line activated charcoal trap or equivalent);

(13) Equipment for TLC or HPLC;

(14) Equipment to quantify 14 C and 3 H for TLC (scanner) or HPLC (in-line detector);

(15) Analytical equipment for the determin ation of the test (and reference) substance if specific substance analysis is used (e.g. gas chromatograph, highperformance liquid chromatograph, mass spectrometer).

(B) The laboratory equipment listed in this paragraph is not essential but useful:

(1) Oxygen meter;

(2) Oxygen controller with prob e 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 sitespecific 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 specifi c flow scenario (e.g. low flow or mean flow). A lternatively, if hydraulic conditions be low the outfall are known, the test medium can simply be wa ter samples obtained downstream from the outflow. However, such conditions are variable and hard to reproduce.

(B) For a generic assessment wastewat er samples should be predominantly derived from domestic sources, and the surfac e water should be typical of surface waters51 into which wastewater is released. Although difficult to duplicate in practice, the TGD uses 450 mg suspended solids/l and 270 mg BOD /l as default levels for wastewater (see paragraph (o)(22) of this guideline). In Nort h America, typical wast ewaters contain from 100 to 350 mg suspended solids/l and 110 to 400 mg BOD/l (see paragraph (o)(23) of this guideline).

(iv) Collection, transport and stor age 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 signif icantly exceed the temp erature 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. NH 3 , organic carbon and standard plat e count are optiona l analyses. The surface water should be characterized by m easuring TSS, total hardness and pH. A standard plate count and organi c carbon analysis are optional. The wastewater is added to

the surface water at a volume sufficient to achieve the targeted di lution of wastewater into surface water. The pH, COD and TSS of the prepared surface water mixture should be measured. Optionally, standard place count, NH 3 and organic carbon analyses can also be performed on the mixture.

(B) The abiotic system is ty pically prepared using a combination of chemical and heat sterilization. A proven approach is to add mercuric chloride (HgCl 2 = 0.5 g/l) to the mixture, which is then autoclaved for at leas t 90 min. Typically the volume of medium is <= half the volume of the container being auto claved (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 wa ter should be used to prepare stock solutions of the test and re ference substances. When appropriate, an alternative method may be used to solubilize or disperse th e test substance in a manner consistent with its normal entry into the environment. Water-miscible nontoxic solvents may be used when necessary, but attention shou ld be paid to the associated organic load involved with adding organic so lvents. Alternatively, the samp le 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 poor ly soluble and typically associated with suspended solids in wastewater, it may be approp riate 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 te st system prior to the initial sampling point, individual test systems can be prepared that are destructiv ely sampled at each sampling interval.

(B) The volume of added stock should be sufficient to ensure rapid and even

distribution of the test subs tance in the system, and accurate administration of the dose52 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 c ontrolled temperature, which may be the field temperature or a standard labor atory temperature of 20-25°C.

(B) Agitation. To keep the solids in suspension, the test vessels are minimally agitated by means of con tinuous mixing or stirring.

(viii) Test duration. The duration of the test should be sufficient to assess the biodegradation of the test substance during it s normal residence time in the wastewatersurface water mixing zone. Normally the test period will be 28 d. However, it may be

extended to obtain additional data points, in order to estima te 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 subs tance 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 te st vessel is opened and the test substance is quantitativel y 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 subs tance in the test me dium. 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 pilo t 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 af ter 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 14 CO 2. ( 1 ) 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 HCl) to lower the sample pH to <2.

( 2 ) The samples are bubbled with air for several hours or allowed to sit overnight53 to allow the dissolved 14 CO 2 to diffuse from the samples. The samples are combined with a

scintillation cocktail that is suitable for th e sample matrix and analyzed by LSC. The percentage of 14 CO 2 is calculated based on the difference between the total counts in the biotic and abiotic samples.

(B) Direct measurement of 14 CO 2. ( 1 ) Evolved 14 CO 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 possi ble. 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 14 CO 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 comp artment with an appropriate CO 2 absorbent (e.g. 1.5 N KOH). The vessels are seal ed and sufficient acid (e.g. 6N HCl) 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 suffici ent length of time (e.g. overnight) to allow

CO 2 to diffuse from solution and be trapped from the headspace by the sorbent. Samples

of the sorbent are combined with a scintillat ion cocktail that is suitable for the sample matrix and analyzed by LSC.

(C) Indirect measurement of 3 H 2 O. ( 1 ) Individual replicate samples (e.g. 1 ml) are collected from each system and placed in se parate vials, located in a fume hood, that contain sufficient acid (e.g. 1 ml of 0.1 N HCl) 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 al lowed to dry completely to remove the 3 H 2 O. The samples are combined with a scintillatio n cocktail that is suitable for the sample matrix and analyzed by LSC. The percentage 3 H 2 O is calculated based on the difference between the total counts in the wet and dry sa mples 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) ar e analyzed directly by LSC to quantify the radioactivity remaining in each system ove r 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. ( 1 ) A sample is collected from both the abiotic and biotic sy stems. The sample vol ume 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 c oncentrating and extracting the samples. A proven approach for nonvolatile test substa nces involves flash freezing the samples,54 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. Flas h freezing is a quick process if there is sufficient depth in the dry ice/acetone or liquid n itrogen 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 to tal radioac tivity 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 recove r 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 in volved in terminating biological activity, and factor it into the sample times used for kinetic analyses.

(B) Analysis of parent and metabolites. ( 1 ) The relative abundance of parent

and metabolites in the extracts can be determ ined using TLC, HPLC or other separation techniques with radioactivity detection.

( 2 ) If sensitive specific an alytical methods are availa ble, primary biodegradation can be assessed by measuring th e total residual concentrati on 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 othe r direct means impossible. Depending on chromatographic behavior, it is usually possibl e 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 app ears and disappears in the sequence of biodegradation, can form an additional basis for inferring its identity. If necessary, the

K
ow 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 solid s 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 incorpora tion of radioactivity into biomass.

(vii) Measurement of volatilized radioactivity. For volatile test substances, the volatility traps are extracted with appropriate so lvents and the radioactivity in the extracts55 analyzed by LSC. The relative abundance of parent and metabolites in the extract(s) can be determined as described in paragr aph (n)(5)(v)(B)(1) of this guideline.

(o) References.

(1) OECD (1992). Ready Biodegradab ility, no. 301, adopted 17 July 1992. Organization for Economic Cooperation and Development, Paris.

(2) OECD (2001). Simulation Test - Aer obic 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. Or ganization for Economic Cooperation and Development, Paris.

(4) OECD (2004). Aerobic Mineraliza tion in Surface Water – Simulation Biodegradation Test, no. 309, adopted 13 April 2004. Organization for Economic Cooperation and Development, Paris.

(5) OECD (2006). Anaerobic Biodegradab ility of Organic Compounds in Digested Sludge: by Measurement of Ga s 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 su rfactants in sewer systems. Water Sci. Technol. 13, 321-328.

(7) Federle, T.W. and N.R. Itrich (19 97). A comprehensive approach for assessing the kinetics of primary and ultimate biodegr adation of chemicals in activated sludge: Application to linear alkylbenzene sulfona te (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 ethoxylat e 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 alcohol s in activated sludge. Ec otox. Environ. Safe. 64, 30-41.

(10) Steber, J. and P. Wierich (1987). The anaerobic degradat ion of detergent range fatty alcohol etho xylates. Studies with 14 C-labeled model surfactants. Water Res. 21, 661-667.

(11) Nuck, B.A. and T.W. Federle ( 1996). Batch test for assessing the

mineralization of 14 C radiolabeled compounds under realistic anaerobic conditions.56 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 Consta nts in Water, no. 112, adopted 12 May 1981. Organization for Economic Coopera tion and Development, Paris.

(14) OECD (2006). Vapor Pressu re, no. 104, adopted 23 March 2006. Organization for Economic Cooperation and Development, Paris.

(15) OECD (2004). Hydrolysis as a function of pH, no. 111, adopted 13 April 2004. Organization for Economic Coopera tion and Development, Paris.

(16) OECD (1984). Activate d sludge, respiration inhibi tion test, no. 209, adopted 4 April 1984. Organization for Economic Cooperation and Development, Paris.

(17) OECD (1981). Inherent Biodegradab ility: Modified SCAS Test, no. 302A, adopted 12 May 1981. Organization for Economic Cooperation and Development, Paris.

(18) OECD (1992). Inherent Biodegra dability: Zahn-Wellens/EMPA Test, no. 302A, adopted 17 July 1992. Organization fo r Economic Cooperation and Development,

Paris.

(19) OECD (1981). Inherent Biodegradabil ity: Modified MITI Test (II), no. 302C, adopted 12 May 1981. Organization for Economic Cooperation and Development, Paris.

(20) Guidance Document on Estimating Pe rsistence and Degradation Kinetics

from Environmental Fate Studies on Pesticid es in EU Registration (2006). Report of the FOCUS Work Group on Degradation Ki netics, EC Document Reference Sanco/10058/2005 version 2.0. 434 pp.

(21) Holman, W.F. (1981). Estimating th e 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). Tec hnical Guidance Document on Risk Assessment in support of Commission Direc tive 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. Burt on (1991). Wastewater Engineering:57 Treatment, Disposal and Reuse/Metcalf & Eddy, Inc. McGraw-Hill, New York, NY.

(24) OECD (2004). Partition Coefficien t (n-octanol/water), High Performance Liquid Chromatography (HPLC) Method, no. 11 7, 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, Vo l. 5B, pp 345-383. Academic Press, New York, NY.

(26) Waterfield, W.R., J.A. Spanner a nd F.G. Stanford (1968). Tritium exchange from compounds in dilute aqueous solutions. Nature 218, 472-473.