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 Wastewater 

 
INTRODUCTION 

 This guideline is one of a series of test guidelines that have been 

developed by the Office of Prevention, Pesticides and Toxic Substances 

(OPPTS), United States Environmental Protection Agency for use in the testing 

of pesticides and toxic substances, and the development of test data to meet the 

data requirements of the Agency under the Toxic Substances Control Act (TSCA) 

(15 U.S.C. 2601), the Federal Insecticide, Fungicide and Rodenticide Act 

(FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food, Drug and 

Cosmetic (FFDCA) (21 U.S.C. 346a). 

 OPPTS developed this guideline through a process of harmonization of 

the testing guidance and requirements that existed for the Office of Pollution 

Prevention and Toxics (OPPT) in Title 40, Chapter I, Subchapter R of the Code 

of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) in 

publications of the National Technical Information Service (NTIS) and in the 

guidelines published by the Organization for Economic Cooperation and 

Development (OECD). 

 For additional information about OPPTS harmonized guidelines and to 

access this and other guidelines, please go to http://www.epa.gov/oppts and 

select “Test Methods & Guidelines” on the left side menu.
1 

OPPTS 835.3280–Simulation tests to assess the primary and ultimate 

biodegradability of chemicals discharged to wastewater: biodegradation in 

wastewater, activated sludge, anaerobic digester sludge, mixing zone for 

treated effluent and surface water and mixing zone for untreated 

wastewater and surface water. 

(a) Scope—(1) Applicability. This guideline is intended for use in testing 

pursuant to the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. This OPPTS test guideline is based on Proposed Guideline 

OECD 314. Simulation tests to assess the primary and ultimate biodegradability of 

chemicals discharged to wastewater: biodegradation in wastewater, activated sludge, 

anaerobic digester sludge, mixing zone for treated effluent and surface water and mixing 

zone for untreated wastewater and surface water. October 2007 Revised Draft Test 

Guideline, annex to OECD communication ENV/EHS/MCH/cm/2007.08, Paris 2 

November 2007. 

(b) Purpose. (1) This guideline describes methods for determining the extent and 

kinetics of primary and ultimate biodegradation of organic substances whose route of 

entry into the environment begins with their discharge to wastewater. Personal care, 

household cleaning and laundry products are typically discarded down the drain as part of 

their normal use and become common components of domestic wastewater. Likewise, 

pharmaceuticals are excreted or in some cases disposed down the drain. Other substances 

may be episodically or continuously discharged to wastewater as a result of 

manufacturing processes. 

(2) This guideline consists of five separate but related simulation tests for 

assessing the primary and ultimate biodegradation of chemicals in wastewater during 

transit in the sewer, secondary treatment in an activated sludge treatment system, 

anaerobic digestion of sludge as well as treated effluent in the mixing zone of surface 

water and untreated wastewater that is directly discharged to surface water. 

Biodegradation in each compartment can play an important role in determining chemical 

exposure in interconnected aquatic and terrestrial habitats. 

 (c) General Considerations. (1) Figure 1 shows the most common transport 

pathways for chemicals discharged to wastewater. Wastewater initially enters a sewer, 

where it may remain for hours or a few days, during its transport to a wastewater 

treatment plant (WWTP) or environmental release site. In most situations, the wastewater 

is treated before release, but in some situations the wastewater is released to surface 

water directly or with only minimal primary treatment. In a typical WWTP, a portion (40-

60%) of the solids is removed during primary treatment. The resulting effluent is then 

subjected to biological treatment and the solids are removed in a final clarifier. The final 

effluent is subsequently released to surface water. The sludge solids removed during 

primary treatment and final clarification are most commonly digested under anaerobic 

conditions if the sludge disposal involves land application. 
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 function of its partitioning behavior and its 

biodegradation rate, and other processes such as volatilization. Due to chemical 

residence time and the level of biological activity, the critical opportunities for significant 

removal through biodegradation are 1) in the sewer, 2) during aerobic secondary 

treatment and 3) during anaerobic digestion of the sludge. Consequently, these three 

systems are the most important to simulate for quantifying biodegradation losses during 

wastewater transport and treatment. Furthermore, the effects of treatment processes 

extend into the environment at the time of release. Thus, biodegradation in the mixing 

zones, in the water and in biosolids, as the chemical moves away from the point of 

release, is key to understanding downstream dispersion and exposure. 

(3) The five simulation test methods described in this guideline use an open batch 

system or closed flow-through batch system (an example of the latter is shown in Figure 

2), and include elements from Organization for Economic Cooperation and Development 

(OECD) guidelines 301, 303A, 309, 310 and 311 (see paragraphs (o)(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 rate of mineralization, and 3) follow the 

formation and decay of major transformation products when appropriate. In addition, 

characterization and quantification of major transformation products may be possible if 

suitable analytical methods are available. 
3 

Figure 2-. Example of flow-through test apparatus

Example of Flow-Through Test Set-Up

Air

Test

Mixture (1 L)

CO2 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 biodegradation of both 

new and existing substances released to wastewater (see paragraphs (o)(6) through 

(o)(11) of this guideline). In some cases, the resulting kinetic constants can serve as input 

constants for exposure models used for risk assessment. These tests are intended as 

higher tier tests for assessing the biodegradation of chemicals that do not biodegrade in 

OECD screening tests, or for refining biodegradation rates used for an exposure 

assessment. 

(d) General principle of the tests. (1) Typically, a test substance, radiolabeled in 

an appropriate position, is incubated with an environmental sample that has been freshly 

collected from a representative field site or maintained in the laboratory under conditions 

realistically simulating some future environmental condition. Abiotic and biotic test 

systems are prepared for each test substance and condition. Biological activity is 

inhibited in the abiotic control, which is used for estimating mineralization by difference, 

establishing extraction efficiency and recovery of the parent molecule, and quantifying 

other loss processes such as hydrolysis, oxidation, volatilization or sorption to test 

apparatus. 

(2) If an analytical method with the required sensitivity is identified, the rate of 

parent degradation or transformation can be determined using a non-radiolabeled test 

substance or by following the disappearance of a substance already in an environmental 

sample. However, ultimate biodegradation of non-radiolabeled substances cannot be 
4 

determined unless the biodegradation pathway is well understood and analytical methods 

with required sensitivity are available for potential metabolites. 

(3) An environmentally relevant concentration of the test substance is dosed to 

both abiotic and biotic test systems. The prepared test systems are incubated at a relevant 

temperature with continuous mixing when appropriate. Samples are periodically removed 

for determination of mineralization and primary biodegradation. The duration of the tests 

is based upon the time necessary to establish whether ultimate degradation takes place 

and the rate at which it occurs, and thus, it does not necessarily reflect the hydraulic 

residence time relevant for a chemical in a specific compartment in the environment. 

When the results of the tests are used for environmental risk assessment, the relevant 

hydraulic retention times in the compartments covered by these tests have to be 

determined according to the scenarios within the specific risk assessment. 

(4) Tests can be performed using an open batch system or a closed flow-through 

batch system where traps are used to capture evolved 14CO2 or 

14CH4. A closed flowthrough system is necessary for volatile test materials. It is also usually preferred for 14Clableled test chemicals. Open systems are appropriate for nonvolatile 3

H-labeled test 

chemicals and for refining the biodegradation kinetics of nonvolatile 14C test materials, 

whose ability to be mineralized has been established previously. In the open system, 

mineralization to 14CO2 (14CH4) can be determined indirectly by measuring the difference 

in residual radioactivity between samples from the biotic and abiotic test systems 

following acidification. Similarly, mineralization to 3H2O can be determined indirectly by 

measuring the difference in residual radioactivity following drying. The open system is 

not appropriate for use with volatile test substances. In a flow-through system, evolved 

14CO2 is measured directly in the base traps. In addition, dissolved 14CO2 is determined 

by acidifying samples in a sealed vessel and measuring radioactivity in a base trap 

contained in the vessel. Under anaerobic conditions, the evolved 14CO2 and 14CH4 are 

collected in tandem. The 14CO2 is trapped in base and 14CH4 is combusted and converted 

to 14CO2, which is subsequently trapped in a similar manner. In the case of volatile test 

substances, an additional in-line organic trap (e.g. activated charcoal) is placed before the 

gas traps to capture volatilized parent and metabolites. The choice of test design depends 

on the type of radiolabel (14C or 3H), the environmental compartment and the properties 

of the test substance. 

(5) Samples from both test systems are analyzed for total radioactivity, extractable 

parent and metabolites and radioactivity associated with the extracted solids. The level of 

parent and metabolites is determined using chromatographic separation and radioanalytical detection methods. The solids remaining from the extraction process are 

combusted to estimate incorporation into biomass by difference or can be further 

fractionated to determine uptake into various components of biomass. A complete mass 

balance of the test system is obtained from the sum total of all fractions at each sampling. 

(6) The level of parent remaining with time can be analyzed using various decay 

models to estimate primary biodegradation rates. Likewise, the level of cumulative 

mineralization can be analyzed using various models to estimate mineralization rates (see 
5 

paragraph (i)(2) of this guideline). 

(e) Applicability of tests— (1) General. The various tests are designed to assess 

biodegradation during key phases of wastewater transit as well as treatment and 

environmental release. The choice of tests should be based on the release scenarios and 

anticipated properties of the substance of interest. In the case of volatile test substances, 

appropriate modification should be made to quantify losses due to volatilization. 

(2) 314A: Test for biodegradation in a sewer system. The purpose of this 

test is to evaluate biodegradation in raw wastewater under conditions normally found in 

sewer systems. It is useful to perform this test if there is sufficient time in a sewer for the 

substance to undergo significant biodegradation and loss. Hence, it is usually most useful 

for relatively labile substances discharged to large municipal sewer systems. In addition, 

the test provides data that may be used to determine the concentration of a substance 

sorbed to primary sludge. Under the conditions of the test, the concentration of test 

substance is at its expected level in wastewater with the biomass level being that 

normally present in a representative wastewater sample. While oxygen is present, the 

system is minimally aerated to simulate dissolved oxygen (DO) conditions in sewers. 

(3) 314B: Test for biodegradation in activated sludge. (i) The purpose of the 

activated sludge test is to evaluate biodegradation during a widely used form of 

biological sewage treatment. It is applicable to any substance subjected to such treatment 

and is key to estimating final effluent concentrations. This test is generally performed 

first among the tests in this guideline and is the most important test in the series. This test 

is characterized by a high level of biomass and a relatively low level of test substance 

under well-aerated conditions. 

(ii) The activated sludge test can compliment or be a lower cost alternative to 

OECD 303A, a dynamic simulation of a wastewater treatment plant that can be used to 

determine the removal of a test substance under a specific set of operating conditions (i.e. 

hydraulic retention time, solids residence time, solids level, etc.). OECD 303A can 

generate a percentage removal or a comprehensive picture of biodegradation and sorption 

that occur at steady state during treatment. However, as an alternative to the expense and 

complexity of running a full scale system, the activated sludge test can generate firstorder rate constants for loss of parent and mineralization, and these can be used as inputs 

to a variety of wastewater simulation models to estimate removal under any set of 

operating conditions. 

(4) 314C: Test for biodegradation in anaerobic digester sludge. The purpose 

of this test is to evaluate biodegradation during anaerobic sludge digestion. It is 

particularly relevant for sorbing substances, which partition to primary and secondary 

sludge. This test is useful for refining the concentration of a substance present in the 

sludge leaving a treatment plant as well as demonstrating the potential for anaerobic 

biodegradation. The test is characterized by reducing conditions, a high level of anaerobic 

biomass, and a level of test substance based on expected wastewater concentrations and 

partitioning behavior. 
6 

(5) 314D: Test for biodegradation in treated effluent-surface water mixing 

zone. The purpose of the effluent mixing zone test is to evaluate the biodegradation of 

that portion of a substance that passes through treatment and is released in effluent to 

surface water; and it can be used to demonstrate that biodegradation occurring in the 

treatment plant continues in the receiving environment. It is based on the principle that 

both the substance and microbes degrading that substance are discharged together in 

effluent. The results of this test can be used to estimate the reduction in a substance 

concentration as a result of biodegradation as a volume of water moves downstream from 

a wastewater treatment plant. This test is characterized by very low levels of both test 

substance and biomass under well-aerated conditions. This test differs from OECD 309 in 

that the surface water is amended with treated effluent, and it can be used to evaluate 

volatile substances. Also, OECD 309 focuses largely on mineralization whereas this test 

is designed to evaluate primary and ultimate biodegradation as well as metabolite 

formation and disappearance of chemicals discharged to wastewater. 

(6) 314E: Test for biodegradation in untreated wastewater-surface water 

mixing zone. The purpose of this test is to evaluate biodegradation in untreated 

wastewater that is directly discharged to surface freshwater. This test is useful for 

determining the relative biodegradation rate for a substance compared to other organic 

components of wastewater. Under the conditions of this test, the levels of test substance 

and biomass are based on their expected concentrations in wastewater-surface water 

mixing zones. Oxygen is present but at reduced levels, due to the high level of organic 

loading.

(f) Information on the test substance. (1) In most cases, 14C or 3

H radiolabeled 

test substances should be used in these tests. For radiolabeled substances, additional 

unlabeled substance may be necessary to achieve the needed test concentration. For 

substances with low specific activities, the sensitivity of the method can be improved in 

part by increasing the volume of the analytical samples. 

(2) For 14C, the radiolabel should be located in the most recalcitrant portion of the 

molecule to monitor comprehensively metabolite formation and decay. For large or 

complex substances, it may be appropriate to repeat the study using test substance labeled 

in a different position. In other cases, it may be more appropriate to position the label in 

a portion of the molecule whose fate is poorly understood. Regardless, interpretation of 

the results should consider the position of the label as it relates to mineralization and the 

metabolites observed. 

(3) For nonvolatile substances, tritiated (3

H-labeled) substances can be an 

alternative to 14C-labeled substances, for reasons of cost or practicality of the synthesis. 

Tritium labeling often results in random or uniform distribution of tritium atoms in the 

molecule. It is also important to consider that isotopic tritium can exchange with 

hydrogen in water (see paragraph (o)(26) of this guideline). These two facts should be 

taken into account in interpreting mineralization and metabolite patterns. 
7 

(4) Nonlabeled test substances can be used to determine the rate of parent 

degradation or transformation if an analytical method with the required sensitivity is 

available. 

(5) The information in paragraphs (f)(5)(i) through (f)(5)(x) of this guideline, on 

the test substance, is helpful for designing a test: 

(i) Solubility in water [OECD 105] (see paragraph (o)(12) of this guideline); 

(ii) Solubility in organic solvent (applicable to substances applied with solvent or 

having low water solubility); 

(iii) Dissociation constant (pKa) if the substance is subject to protonation or 

deprotonation [OECD 112] (see paragraph (o)(13) of this guideline); 

(iv) Vapor pressure [OECD 104] (see paragraph (o)(14) of this guideline) and/or 

Henry’s law constant; 

(v) Substance stability in water and in the dark; i.e. susceptibility to hydrolysis 

[OECD 111] (see paragraph (o)(15) of this guideline); 

(vi) Environmental concentration, if known or estimated; 

(vii) Toxicity of the test substance to microorganisms [OECD 209] (see paragraph 

(o)(16) of this guideline); 

(viii) Ready biodegradability [OECD 301] (see paragraph (o)(1) of this guideline) 

and/or inherent biodegradability [OECD 302] (see paragraphs (o)(17) through (o)(19) of 

this guideline). 

(ix) Susceptibility to direct and indirect photolysis; 

(x) Partitioning behavior.

(g) Reference substance. A substance that is normally easily degraded under the 

test conditions may be useful as a reference substance. The purpose of such a reference 

substance is to ensure that the microbial community in the test system is active. 

Alternatively, a substance, whose fate in the environment is well understood, may be 

included as a standard, the results for which can be compared to those for the test 

substance. While the use of a reference substance is not required, it may provide useful 

information for the interpretation of the test results. 

 (h) Quality criteria—(1) Validity of the test. (i) The mass balance from the 

abiotic test system is used to confirm the recovery of parent from the system. It is 

recommended that an abbreviated pilot die-away study be conducted prior to the 

definitive test to establish the appropriate extraction system for parent and metabolites. 
8 

Target recoveries from the test matrix should be 85%-110%; however, these ranges 

should not be used as the sole basis for acceptance of the test. If parent recoveries from 

initial samples taken from the abiotic control are in the targeted range, the sample 

preparation procedures are suitable for the recovery of the parent compound from the test 

matrix. Lower-than-targeted recoveries in the abiotic test system could be due to poor 

extraction efficiency, sorption to glassware, or abiotic degradation (see paragraph 

(h)(1)(iii) of this guideline). 

 (ii) Total recovery of radioactivity in both abiotic and biotic systems should 

normally range from 75 to 115% for individual samples, and average total recovery for 

all samples within a treatment should normally range from 85 to 110%. However, these 

ranges should not be used as the sole basis for acceptance of the test. If mass balances 

from the abiotic system are in the targeted range but those in the biotic test system are 

significantly below this range, the lower recovery likely results from the inability to 

efficiently trap 14CO2 and/or recover metabolites, or the loss of metabolites to glassware 

or volatilization. 

(iii) If chemical analysis from the abiotic control samples reveals that parent 

remained intact throughout the experiment, degradation in the biotic system can be 

attributed to microbial activity. If the abiotic system indicates degradation of parent over 

time, interpretation of these results may include a description/explanation of the likely 

abiotic process that occurred. Comparison between the extent of parent degradation and 

metabolite formation observed in the two systems will provide an estimate of the extent 

of biological versus chemical degradation in the biotic system, assuming loss is not an 

artifact of sample preparation.

(2) Sensitivity of analytical methods. The limit of detection (LOD) of the 

analytical method for the test substance and for the transformation products should be ≤

1% of the initial amount added to the test system, if possible. The limit of quantification 

(LOQ) should be ≤ 3% of the added concentration. 

(3) Results with reference substance. When a reference substance is included, 

the results for the reference substance should approximate those anticipated based on the 

reasons for its selection. 

 (i) Data and reporting—(1) Plot of data. For each sample, the exact time of 

incubation including the time needed to terminate biological activity if applicable is 

reported. Also, for each sampling point, the percentage of the dosed radioactivity 

recovered as parent, metabolites and associated with solids, as well as the cumulative 

amount of mineralization and the total mass balance, are reported. These percentages are 

plotted against time for both the biotic and abiotic systems, when appropriate.

 (2) Kinetic analyses (Optional). (i) In some cases, it may be desirable to fit the 

results from these tests to kinetic models. These models could include decay models for 

parent and production models for mineralization (e.g. 14CO2 or 3

H2O). The most common 

and useful models for this purpose employ first-order kinetics. Most exposure models 

(e.g. EUSES, SimpleTreat) utilize first-order 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 concentration exceeds saturation, the data may 

still fit a first-order function, but these quasi first-order rates will be slower than a true 

first-order rate. Such quasi first-order kinetics may arise from a need to test a higher 

concentration than that occurring in situ due to analytical constraints, or they may reflect 

the actual in situ situation. 

 (iii) When degradation occurs in an exponential manner and the onset of this 

degradation is not preceded by a lag period during which little or no degradation occurs, 

it may be possible to fit decay or production data to a first-order model. Under such 

circumstances, the percentage of parent remaining as a function of time may be fitted to a 

simple (one-compartment) or two-compartment first-order loss function using nonlinear 

regression methods. Such equations have the form: 

k t

y Ae = − 1

y (Ae )+(Be ) k t k t − 1 − 2 =

where y equals the percentage of parent remaining at time (t), A equals the percentage 

degraded at first-order rate constant k1, and B equals the percentage degraded at the firstorder rate constant k2. Such curve fitting can be achieved using nonlinear methods found 

in commercially available statistical or curve-fitting software. The two-compartment 

model is useful when biodegradation is biphasic, consistent with the existence in the test 

system of two different pools of test material (e.g. dissolved and sorbed) that exhibit 

different rates of biodegradation. 

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

y A( e ) k t1 1 − = −

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

where y equals the percentage of the material mineralized at time (t), A equals the 

percentage mineralized at first-order rate constant k1, and B equals the percentage 

mineralized at the first-order rate constant k2. 

 (v) In some situations, biodegradation, particularly loss of parent, may occur so 

rapidly that a true zero time point cannot be measured in the biotic system. In such 

situations, data from the abiotic system may be used to represent time zero for the kinetic 

analyses. 

 (vi) When first-order kinetics are observed, half-lives (T1⁄2) can be calculated 
10 

from the estimated first-order rates (k1 or k2) for each pool (A or B) using the following 

equation: 

T1⁄2 = -ln2/k 

 (vii) In some circumstances, the observed data can be fit to a variety of other 

models, such as Monod or other growth models, which is beyond the scope of the current 

guideline. Additional detail on biodegradation kinetics can be found in a report from the 

FOCUS Work Group on Degradation Kinetics (see paragraph (o)(20) of this guideline). 

Half-life is only relevant for materials exhibiting first-order degradation patterns. In the 

absence of first-order kinetics, it may be appropriate to report degradation times for 50% 

(DT50) and 90% (DT90) of the test material, if these levels of degradation are observed 

during the course of the study. These values can be determined directly or estimated 

using standard interpolation procedures. 

 (viii) When data are fit to a model, the model equation and the software used to fit 

the model should be reported. The correlation coefficient (r2

), F value, if available, and a 

plot of the fitted curve with the actual data should be provided. The estimated rate 

constants (k1 or k2) and other parameters (A, B) should be reported with their standard 

errors.

(3) Test report. (i) The type of study, i.e. wastewater, activated sludge, mixing 

zone or anaerobic digester sludge test, should be clearly stated in the test report. 

 

(ii) The test report should also contain the information in paragraphs (i)(3)(ii)(A) 

through (i)(3)(ii)(D), when appropriate.

(A) Test substances: 

(1) Common names, substance names, Chemical Abstracts Service (CAS) 

numbers, structural formulas and relevant physical/chemical properties of test and 

reference substances; 

(2) Substance names, CAS numbers, structural formulas and relevant 

physical/chemical properties of substances used as standards for identification of 

metabolites; 

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

(4) Radiochemical purity and specific activity of radiolabeled substances; 

(5) Positions within the molecule of radiolabeled atoms.

(B) Environmental samples: 

(1) Source of environmental samples including geographical location and relevant 
11 

data regarding known prior or existing exposure to the test substance and related 

substances; 

(2) Logic used to estimate relevant environmental concentration; 

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

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

(5) Suspended solids level, biological oxygen demand (BOD), chemical oxygen 

demand (COD) and total organic carbon (TOC) as appropriate; 

(6) Time between collection and use in the laboratory test, sample storage 

conditions and any pretreatment of the sample prior to initiating the test. 

(C) Experimental conditions: 

(1) Dates of performance for the study; 

(2) Amount of test substance applied, and test and reference substance 

concentrations; 

(3) Method of application of the test substance and associated logic for selection; 

(4) Incubation conditions including lighting, aeration type, temperature; 

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

measurements; 

(6) Number of replicates. 

(D) Results: 

(1) Precision and sensitivity of the analytical methods including the LOD and 

LOQ; 

(2) Recovery for each analyte and disposition of dosed radioactivity for each 

sampling time and system, in tabular form; 

(3) Average mass balance with standard deviation across all time points for each 

system; 

(4) Procedures and models used to estimate biodegradation rates from the data; 

(5) Biodegradation rates and related parameters with relevant standard errors 

along with correlation coefficients (R2

) and F statistics for the selected models; 
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 for a substance in raw 

wastewater during its time in a sewage conveyance system. It is based on a procedure 

originally published in Matthijs et al. (see paragraph (o)(6) of this guideline). The test 

duration is typically kept short (≤96 hr) in order to simulate the residence time in a sewer, 

but it can be extended to assess the extent to which a substance can be degraded by 

wastewater microbes. It is useful to perform this test if there is sufficient time in a sewer 

for the substance to undergo significant biodegradation and loss. Hence, it is most useful 

for relatively labile substances discharged to large municipal sewer systems. Aeration 

levels in a sewer can vary widely. To be conservative and simulate the more typical 

conditions in a sewer, the test is conducted under low DO conditions (≤1 mg/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 regarding the current load of the substance. For 

substances not consistently present in wastewater, sufficient test substance (radiolabeled 

and unlabeled) should be added to approximate the expected concentration in wastewater 

during an episodic release or following commercialization of a new substance. This 

concentration would reflect the total mass released and the volume of wastewater in 

which the release is diluted. Approaches for estimating wastewater concentration can be 

found in Holman (see paragraph (o)(21) of this guideline) and the European Technical 

Guidance Document (see paragraph (o)(22) of this guideline). In most situations, the 

substance and its degrader populations will not be at steady state and the observed 

kinetics will be pseudo first-order or second-order Monod.

(2) Principle of the test. (i) Test substance is incubated with abiotic and biotic 

wastewater over a period of time under low DO conditions. Biological activity is 

inhibited in the abiotic control, which is used for estimating mineralization by difference, 

determining extraction efficiency and recovery of the parent molecule, and quantifying 

other loss processes such as hydrolysis, oxidation, volatilization or sorption to test 

apparatus. 

(ii) If an analytical method with the required sensitivity is available, the rate of 

parent degradation or transformation can be determined using a non-radiolabeled test 
13 

substance or by following the disappearance of a substance already in wastewater. 

However, ultimate biodegradation cannot be determined unless the biodegradation 

pathway is well understood and analytical methods with required sensitivity are available 

for potential metabolites. 

(iii) Test substance at an environmentally relevant concentration is dosed to both 

abiotic and biotic test systems, which are incubated with continuous slow mixing. The 

biotic samples are incubated in such a way that DO levels remain at or below 1 mg/l, 

which is typical for sewage. Samples are periodically removed for determination of 

mineralization and primary biodegradation. 

(iv) Tests can be performed using an open batch system or a sealed, flow-through 

batch system where traps are used to capture evolved 14CO2. The closed flow-through 

system should be used for volatile test substances and is usually preferred for 14C-lableled 

test substances. Open systems are appropriate for nonvolatile 3

H-labeled test substances 

and for refining the biodegradation kinetics of nonvolatile 14C test materials, whose 

ability to be mineralized has previously been established. In the open system, 

mineralization to 14CO2 can be determined indirectly by measuring the difference in 

residual radioactivity between samples from the biotic and abiotic systems following 

acidification. Similarly, mineralization to 3

H2O can be determined indirectly by 

measuring the difference in radioactivity in a sample following drying. In the flowthrough systems, evolved 14CO2 is measured directly in the base traps. In addition, 

dissolved 14CO2 is determined by acidifying samples in a sealed vessel and measuring 

radioactivity in a base trap contained in the vessel. 

(v) Samples from both systems are analyzed for total radioactivity, extractable 

parent and metabolites, and radioactivity associated with the extracted solids. The levels 

of parent and metabolites are determined using chromatographic separation and when 

appropriate, radio-analytical detection methods. The solids remaining from the extraction 

process are combusted to estimate incorporation into biomass by difference, or can be 

further fractionated to determine uptake into various components of biomass. A complete 

mass balance of the test system is obtained from the sum of all fractions at each 

sampling.

(3) Applicability of the test. The method is readily applicable to water-soluble 

and poorly water-soluble substances that are nonvolatile. It also can be adapted for 

volatile substances. Typically, 14C or 3H labeling of substances is required for the 

assessment of mineralization. Either radiolabeled or nonlabeled substance can be used for 

the assessment of primary biodegradation. 

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

wastewater in the test vessels is determined based on the number and volume of the 

samples needed for the test. Typically, 1-2 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 controller connected to an actuator valve, which monitors and 
14 

controls the aeration of the wastewater (see Figure 3). This aeration is balanced against 

continuous sparging with nitrogen to achieve the targeted DO level. Alternatively, the 

wastewater can be incubated with stirring but minimum aeration to keep DO levels low, 

and nitrogen or air can be added periodically to maintain the DO level. In this case, DO 

readings should be reported at regular intervals.

(B) An open test is open to the atmosphere but incubated under conditions that 

maintain DO levels at the desired level. Flow-through systems are sealed with an 

appropriate closure containing a sampling port with a valve for removing wastewater 

samples and connections for influent and effluent gas lines. The closure can be a rubber 

stopper, but an alternative type of closure may be necessary when working with a volatile 

hydrophobic substance. When testing volatile substances, it is recommended that gas 

lines and sampling tubes consist of inert materials (e.g. polytetrafluoroethylene, stainless 

steel, glass). 

Figure 3. Example of controlled dissolved oxygen flow-through apparatus 

(C) The headspace of the test vessel is continuously purged with gas at a rate 

sufficient to maintain the wastewater at the desired DO level but not too fast to prevent 

efficient trapping of CO2. The test vessel is connected to a series of traps containing 

potassium hydroxide (e.g. 1.5 N) or other appropriate CO2 absorbent. An empty trap is 

usually included and positioned in front of the absorbent in the trapping train, as a 

precaution against backflow or condensation. 
15 

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

used: 

(1) Miscellaneous glassware and pipettes; 

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

(3) Centrifuge; 

(4) pH meter; 

(5) Solid CO2/acetone or liquid nitrogen bath; 

(6) Freeze dryer (lyophilizer); 

(7) Oven or microwave oven for dry weight determinations; 

(8) Membrane filtration apparatus; 

(9) Autoclave; 

(10) Facilities to handle radiolabeled substances; 

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

liquid scintillation counter); 

(12) Equipment to quantify 14Cand 3

H in solid samples (e.g. sample oxidizer); 

(13) Equipment to trap volatilized 14C and 3H from gas trapping system (in-line 

activated charcoal trap or equivalent); 

(14) Equipment for thin layer chromatography (TLC) or high performance liquid 

chromatography (HPLC); 

(15) Equipment to quantify 14C and 3

H for TLC (scanner) or HPLC (in-line 

detector); 

(16) Analytical equipment for the determination of the test (and reference) 

substance if specific substance analysis is used (e.g. gas chromatograph, 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 probe and actuator valve; 

(3) COD digestion vials; 

(4) Nitrogen ammonia reagent set; 

(5) Spectrophotometer. 

(iii) Selection of wastewater. The source of wastewater should be consistent with 

the objective of the simulation test. For a site-specific assessment, the wastewater should 

be obtained from the specific sewer system of interest. For a generic assessment samples 

should be predominantly derived from domestic sources. Although difficult to duplicate 

in practice, the European Technical Guidance Document (TGD) procedure uses 450 mg 

suspended solids/l and 270 mg BOD/l as default levels for wastewater (see paragraph 

(o)(22) of this guideline). In North America, a typical wastewater contains 100 to 350 mg 

suspended solids/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 should be noted at collection. Collection 

containers should allow for adequate ventilation and measures should be taken to prevent 

the temperature of the wastewater from significantly exceeding the temperature used in 

the test. The wastewater is typically stored at test temperature with continuous slow 

mixing. Samples should not be stored frozen. 

(v) Preparation of test systems. (A) The freshly collected wastewater should be 

largely free from coarse particles. Total suspended solids (TSS), COD, pH and NH3 

(optional) in the wastewater should be determined. 

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

combination of chemical and heat sterilization. A proven approach is to add mercuric 

chloride (1 g/l) to the wastewater, which is then autoclaved for at least 90 min. Typically 

the volume of medium is <= half the volume of the container being autoclaved (e.g. 500 

ml activated sludge in a 1-liter container). After cooling, the pH of the abiotic system 

should be measured and adjusted to match that of the biologically active system. 

Alternative approaches to deactivate the system can also be used.

(vi) Test substance preparation. (A) Ideally, distilled water should be used to 

prepare stock solutions of the test and reference substances. When appropriate, an 

alternative method may be used to solubilize or disperse the test substance in a manner 

consistent with its normal entry into the environmental compartment of interest. Watermiscible nontoxic solvents may be used when necessary, but attention should be paid to 

the associated organic load involved with adding organic solvents. Alternatively, the 

sample may be added in a neat form (i.e. without water) to the test system in a manner 

that maximizes its even and rapid distribution in the sludge. For substances that are 
17 

poorly soluble and typically associated with suspended solids in wastewater, it may be 

appropriate to adsorb the test substance onto an inert solid carrier, which is then dosed to 

the test system. If the test substance cannot be evenly distributed in the test system prior 

to the initial sampling point, individual test systems can be prepared that are destructively 

sampled at each sampling interval. 

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

and even distribution of the test substance in the system and accurate administration of 

the dose between like systems. Ideally, when dosing with aqueous solutions, the added 

volume should be ≥2 ml and <10 ml; for nontoxic solvents, <0.1 ml/l. If appropriate, 

dosing solutions may be prepared in advance and refrigerated. The activity of the stock 

solution should be checked by liquid scintillation counting (LSC).

(vii) Test conditions—(A) Test temperature. Incubation should take place in the 

dark (preferred) or in diffuse light at a controlled temperature, which may be the field 

temperature or a standard laboratory temperature of 20-25°C. Depending on location, 

mean annual wastewater temperature ranges from 10 to 20.1°C, with 15.6°C being 

representative (see paragraph (o)(23) of this guideline).

(B) Agitation. To keep the solids in suspension, test vessels are minimally 

agitated by means of continuous mixing or stirring.

(viii) Test duration. The duration of the test should be sufficient to assess the 

biodegradation of the test substance during its normal residence time in the sewer system. 

However, it may be extended to obtain additional data points to estimate kinetic 

constants, or to assess the completeness of degradation under the conditions of the test. 

Conversely, it may be ended before this time if degradation has plateaued. 

(ix) Number of test vessels. At a minimum, there should be a single abiotic and 

a single biotic test vessel for each test substance concentration. While replicates can be 

prepared for each system, more useful kinetic information usually can be gained by 

increasing the number of time points at which samples are collected within a system.

(5) Procedure—(i) Dosing. At test initiation, the chamber closure is removed and 

the test substance is quantitatively added directly with constant mixing. It is 

recommended that the dose be administered in a gradual fashion below the air-water 

interface, to ensure uniform distribution of the test substance into the wastewater. The 

biotic and abiotic systems are dosed in an identical manner. Generally, the biotic systems 

are dosed first, followed by the abiotic systems. Exact timing is typically more critical for 

kinetic analyses.

(ii) Sampling schedule. Sampling times are selected based on existing 

biodegradation data or the results of a pilot study, as no fixed time schedule for sampling 

is universally applicable. A recommended sampling schedule for a rapidly degraded 

substance is 15, 30 and 60 min, with additional samplings after 2, 5, 8, 12 and 24 hr and 

days 2, 3 and 4. The sampling schedule for a slowly degrading substance should be 
18 

adjusted so that a sufficient number of measurements are made during the degradation 

phase. 

(iii) Measurement of mineralization—(A) Indirect measurement of 14CO2. (1) 

Individual replicate samples (e.g. 1 ml) are collected from each system and deposited in 

separate vials, located in a fume hood, that contain sufficient acid (e.g. 1 ml of 0.1 N 

HCl) to lower the sample pH to <2.

(2) The samples are bubbled with air for several hours or allowed to stand 

overnight to allow the dissolved 14CO2 to diffuse from the samples. The samples are 

combined with a scintillation cocktail that is suitable for the sample matrix and analyzed 

by LSC. The percentage of 14CO2 is calculated based on the difference between the total 

counts in the biotic and abiotic samples.

(B) Direct measurement of 14CO2. (1) Evolved 14CO2: The first base trap in the 

series is removed and quickly capped. The remaining traps are moved forward in the 

same order and a fresh trap placed behind the existing traps, and the trapping system 

reconnected as quickly as possible. Replicate subsamples (e.g. 1 ml) are removed from 

the base trap and transferred to scintillation vials, combined with a scintillation cocktail 

that is suitable for the sample matrix, and analyzed by LSC.

(2) Dissolved 14CO2: Samples (e.g. 10 to 25 ml) are removed through the 

sampling port of the test flask. They are then placed in vessels (e.g. Bellco Glass 

Biometer 2556-10250) containing a compartment with an appropriate CO2 absorbent (e.g. 

1.5 N KOH). The vessels are sealed 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 (e.g. overnight) to allow 

CO2 to diffuse from solution and be trapped from the headspace by the sorbent. Samples 

of the sorbent are combined with a scintillation cocktail that is suitable for the sample 

matrix and analyzed by LSC. 

Figure 4. System for recovering dissolved 14CO2

 

Sample from test 

vessel (10 - 20 ml) 

1.5 N KOH 

(1 - 2 ml) 
19 

(C) Indirect measurement of 3

H2O. (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) Half of the samples are immediately analyzed directly by LSC for a wet 

measurement. The remaining samples are allowed to dry completely to remove the 3H2O. 

The samples are combined with a scintillation cocktail that is suitable for the sample 

matrix and analyzed by LSC. The percentage 3H2O is calculated based on the difference 

between the total counts in the wet and dry samples and the initial level of radioactivity 

dosed to the samples.

(iv) Measurement of total radioactivity in wastewater. Replicate small-volume 

samples (e.g. 1 ml) are analyzed directly by LSC to quantify the radioactivity remaining 

in each system over time. These measurements are used to confirm that the recovery of 

radioactivity from the extracted samples is acceptable and to monitor for volatilization. 

The total solids in these samples should not exceed 30 mg dry weight in order to avoid 

counting efficiency problems.

(v) Measurement of parent and metabolites—(A) Extraction. (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 concentrating and extracting the samples. 

A proven approach for nonvolatile test substances involves flash freezing the samples, 

followed by lyophilization and extraction of the dried residue with appropriate solvent(s) 

for parent and metabolites. Flash freezing quickly stops biological activity without 

hydrolyzing or otherwise altering labile test substances. Flash freezing is a quick process 

if there is sufficient depth in the dry ice/acetone or liquid nitrogen bath to submerge the 

sample tube. The level of the bath should be above the sample level in the tube. The 

dried solids are extracted and the resulting extracts can be concentrated through 

evaporation. The total radioactivity in each extract is then determined by LSC.

(3) For volatile test substances, the sample can be passed through a filter and solid 

phase extraction (SPE) column or SPE disk placed in tandem, which are subsequently 

eluted with appropriate solvents to recover parent and metabolites. Alternatively, samples 

can be centrifuged, and parent and metabolites can be extracted from the liquor by solid 

phase or liquid/liquid extraction. The solids can then be extracted directly or mixed with 

a drying agent (e.g. sodium sulfate) and allowed to dry prior to extraction with an 

appropriate solvent system. An alternative is to extract the solids and dry the extract by 

running the solvent through a column containing a drying agent. In some cases, it may be 

possible to directly extract the entire aqueous sample with an appropriate solvent system 

and then filter it to recover biomass solids. The total radioactivity in all extracts is 

determined by LSC. Care should be taken in concentrating extracts containing volatile 
20 

test materials or metabolites. 

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

document recoveries and consider the time involved in terminating biological activity and 

factor it into the sample times used for kinetic analyses.

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

metabolites in the extracts can be determined using TLC, HPLC, or other separation 

techniques with radioactivity detection.

(2) If sensitive specific analytical methods are available, primary biodegradation 

can be assessed by measuring the total residual concentration of test substances and 

metabolites instead of using radioisotope techniques. Generally, for nonlabeled 

substances, it will only be possible to track parent disappearance from the aqueous phase.

(C) Characterization of metabolites. Whenever possible, the chromatographic 

behavior of unknown peaks should be compared to that of predicted metabolites, if 

authentic standards exist. Usually, the quantity and purity of metabolites generated in this 

test make definitive identification by other direct means impossible. Depending on 

chromatographic behavior, it is usually possible to determine if a metabolite is more or 

less polar than the parent. This information, combined with known biochemical reactions 

and knowledge of when a metabolite appears and disappears in the sequence of 

biodegradation, can form an additional basis for inferring its identity. If necessary, the 

Kow of major metabolites can be estimated by HPLC (e.g. using OECD 117; see 

paragraph (o)(24) of this guideline) using an on-line radioactivity detector.

(vi) Measurement of extracted solids and incorporation into biomass. If the 

extracted samples are filtered, the filter will retain carbonate salts as well as 

microorganisms from the test system. The filter containing the biosolids is placed in a 

scintillation vial and acidified to pH ≤2 by submerging it in a weak acid solution (e.g. 1 

ml of 0.1 N HCl). The samples are allowed to sit for sufficient time (e.g. overnight) for 

the dissolved 14CO2 to diffuse from the samples. The samples are analyzed by LSC. In 

the case of non-filtered extracted solids, they are combusted to determine the level of 

activity remaining with the solids. The level of radioactivity in the biotic solids above 

that in solids from the abiotic control typically represents incorporation of radioactivity 

into biomass. The distribution of this radioactivity among various components of biomass 

(i.e. nucleic acids, protein, cell wall, etc.) can be determined using a modified Sutherland 

and Wilkinson procedure (see paragraphs (o)(7) and (o)(25) of this guideline).

(vii) Measurement of volatilized radioactivity. For volatile test substances, 

traps are extracted with appropriate solvents and the radioactivity in the extracts analyzed 

by LSC. The relative abundance of parent and metabolites in the extract(s) can be 

determined as described above in paragraph (j)(5)(v)(B)(1) of this guideline.

(k) 314B: Biodegradation in activated sludge—(1) General. (i) This 

test is designed to assess the extent to which a substance can be degraded in activated 
21 

sludge and to provide rates of primary and ultimate biodegradation under the conditions 

of the test. It is based on a procedure originally published by Federle & Itrich (see 

paragraph (o)(7) of this guideline). Activated sludge in its various configurations is the 

most common secondary wastewater treatment process. The usefulness of the measured 

rates for predicting removal in actual treatment using wastewater treatment models (e.g. 

SimpleTreat) will be a function of the fidelity of the simulation to actual conditions in an 

activated sludge wastewater treatment plant. 

(ii) Four factors determine the test substance concentration in this test: 1) whether 

the material is released continuously or episodically; 2) current presence in the 

environment; 3) expected presence in the environment for a new chemical; and 4) 

analytical sensitivity. 

 

(iii) When a chemical substance is already present in the environment in a 

continuous fashion, the most accurate kinetics are obtained by adding a tracer level of the 

radiolabeled substance to freshly obtained environmental samples. Under these 

circumstances, the normal ratio of substance to degraders is not disrupted and the 

observed biodegradation rates reflect in situ rates. 

 

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

most accurate rates are obtained when the substance and degrader populations are in a 

steady state balance. This situation can be achieved by adding a tracer level of test 

substance to activated sludge that has been exposed to the substance under expected 

loading and operating conditions in a laboratory continuous activated sludge system (e.g. 

OECD 303 A). 

 

(v) When an existing or new substance enters the environment in an episodic 

manner, there is not a normal steady state ratio of biomass to test chemical to disrupt, so 

the test substance is dosed to freshly collected samples at the level in wastewater 

expected for a release event. This concentration should reflect the total mass released and 

the volume of wastewater in which the release is diluted. Approaches for estimating 

wastewater concentration can be found in Holman (see paragraph (o)(21) of this 

guideline) and the European Technical Guidance Document (see paragraph (o)(22) of this 

guideline). 

 

(vi) Superseding the previous considerations is analytical sensitivity. When it is 

not possible to use ideal (e.g. tracer) levels of test substance due to analytical 

consideration, the lowest possible concentration is employed. At high test substance 

concentrations, biodegradation may be associated with lag periods related to second–

order processes (i.e. growth), which complicate the kinetic analysis. When this standard 

for simulation is not achieved, observed biodegradation rates may not be fully 

representative, which should be considered in the interpretation of results. This factor is 

particularly important for continuously released substances, which often reach steady 

state conditions in wastewater systems. 

(2) Principle of the test. (i) The test substance is incubated with abiotic and 
22 

biotic activated sludge over a period of time. Biological activity is inhibited in the abiotic 

control, which is used for estimating mineralization by difference, establishing extraction 

efficiency and recovery of the parent molecule, and quantifying other loss processes such 

as hydrolysis, oxidation, volatilization or sorption to test apparatus.

(ii) If an analytical method with the required sensitivity is available, the rate of 

parent degradation or transformation can be determined using a non-radiolabeled test 

substance or by following the disappearance of a substance already in wastewater. 

However, ultimate biodegradation cannot be determined unless the biodegradation 

pathway is well understood and analytical methods with required sensitivity are available 

for potential metabolites. 

(iii) Test substance at an environmentally relevant concentration is dosed to both 

abiotic and biotic test systems, which are incubated at a relevant temperature with 

continuous mixing when appropriate. Samples are periodically removed for 

determination of mineralization and primary biodegradation. 

(iv) Tests can be performed using an open batch system or a sealed, flow-through 

batch system where traps are used to capture evolved 14CO2. The closed flow-through 

system should be used for volatile test substances and usually is preferred for 14C-lableled 

test substances. Open systems are appropriate for nonvolatile 3

H-labeled test substances 

and for refining the biodegradation kinetics of nonvolatile 14C-labeled test substances, 

whose ability to be mineralized has previously been established. In the open system, 

mineralization to 14CO2 can be determined indirectly by measuring the difference in 

residual radioactivity between samples from the biotic and abiotic systems following 

acidification. Similarly, mineralization to 3H2O can be determined indirectly by 

measuring the difference in radioactivity in a sample following drying. In the flowthrough systems, evolved 14CO2 is measured directly in the base traps. In addition, 

dissolved 14CO2 is determined by acidifying samples in a sealed vessel and measuring 

radioactivity in a base trap contained in the vessel. 

 

(v) Samples from both systems are analyzed for total radioactivity, extractable 

parent and metabolites and radioactivity associated with the extracted solids. The levels 

of parent and metabolites are determined using chromatographic separation and when 

appropriate radio-analytical detection methods. The solids remaining from the extraction 

process are combusted to estimate incorporation into biomass by difference or can be 

further extracted using a modified Sutherland and Wilkinson procedure (see paragraphs 

(o)(7) and (o)(25) of this guideline) to determine uptake into various components of 

biomass. A complete mass balance of the test system is obtained from the sum total of all 

fractions at each sampling. 

 (3) Applicability of the test. The method is readily applicable to water-soluble or 

poorly water-soluble substances that are nonvolatile. It can also be adapted for volatile 

materials. Typically, 14C or 3

H radiolabelling is required for the assessment of 

mineralization. Both either radiolabelled or nonlabelled substance 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 port with a valve for removing mixed liquor 

suspended solids (MLSS) samples and connections for influent and effluent gas lines (see 

Figure 2). This closure can be a rubber stopper, but glass is recommended when working 

with a volatile hydrophobic test material. When testing volatile substances, it also is 

recommended that gas lines and sampling tubes consist of inert materials (e.g. 

polytetrafluoroethylene, stainless steel, glass). 

(B) The headspace of the test vessel is continuously purged with air or CO2-free 

air at a rate sufficient to maintain the activated sludge in an aerobic condition, but not too 

fast to prevent efficient trapping of CO2. The test vessel is connected to a series of traps 

containing a potassium hydroxide (e.g. 1.5 N) or other appropriate CO2 absorbent. An 

empty trap is usually included and positioned in front of the absorbent in the trapping 

train, as a precaution against backflow or condensation. 

(ii) Equipment. 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 CO2/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 radiolabeled substances; 

(K) Equipment to quantify 14C and 3

H in liquid samples and solid samples (e.g. 

liquid scintillation counter); 
24 

(L) Equipment to quantify 14C and 3

H in solid samples (e.g. sample oxidizer); 

(M) Equipment to trap volatilized 14C and 3

H from gas trapping system (in-line 

activated charcoal trap or equivalent); 

(N) Equipment for TLC or HPLC; 

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

detector); 

(P) Analytical equipment for the determination of the test (and reference) 

substance if specific substance analysis is used (e.g. gas chromatograph, high 

performance liquid chromatograph, mass spectrometer).

(iii) Selection of activated sludge source. (A) The source of activated sludge 

should be consistent with the objective of the simulation test. For a site-specific 

assessment, the activated sludge should be obtained from the specific wastewater 

treatment plant of interest. For a generic assessment activated sludge should be obtained 

from a typical wastewater treatment plant receiving predominantly domestic wastewater. 

If the substance is currently a component of wastewater entering the wastewater 

treatment facility or is episodically released to wastewater, freshly collected activated 

sludge will be ideal for the test.

(B) For a new substance that will be continuously released to wastewater, 

activated sludge ideally should be obtained from a laboratory-scale treatment system 

[OECD 303A] (see paragraph (o)(2) of this guideline), which has been fed wastewater 

amended with unlabeled test substance. The source of the starting sludge, wastewater 

(influent) and the operating conditions (influent concentration, hydraulic retention time, 

solids retention time) for the laboratory unit should accurately reflect site-specific or 

generic conditions. In the case of the latter, the TGD specifies a hydraulic retention time 

(HRT) of 6.9 hr and a solids retention time (SRT) of 9.2 d in its generic scenario for 

wastewater treatment (see paragraph (o)(22) of this guideline). The TGD also provides 

guidance on estimating wastewater concentration based on expected usage volumes. In 

general, steady state will be reached within 2 to 3 SRTs after which point the sludge can 

be used for testing.

(iv) Collection, transport and storage of activated sludge. The activated 

sludge should be collected from a well-mixed region of the aeration basin. The pH and 

temperature of the sample should be noted at collection. Collection containers should 

allow for adequate ventilation and measures should be taken to prevent temperature of 

the sludge from significantly exceeding the temperature used in the test. The activated 

sludge is typically stored at test temperature with continuous aeration. Samples should 

not be stored frozen.

(v) Preparation of the test systems. (A) The activated sludge should be sieved 

through a 2-mm screen. The TSS concentration should be measured and if necessary 
25 

adjusted to the target concentration. The TGD uses a default level of 4000 mg/l in its 

generic scenario (see paragraph (o)(22) of this guideline). However, 2500-3000 mg/l may 

be more typical for North America (see paragraph (o)(23) of this guideline). The sludge 

can be diluted with liquor or tap water if the solids concentration is too high. 

Alternatively, if the solids concentration is too low, the solids can be allowed to settle and 

some of the liquor can be decanted. A final TSS level and pH should then be determined. 

(B) Abiotic sludge is typically prepared using a combination of chemical and heat 

sterilization. A proven approach is to add mercuric chloride solution (1 g/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 abiotic sludge should be measured and adjusted 

to match that of the biologically active sludge. Alternative approaches to deactivate the 

sludge can also be used.

(vi) Test substance preparation. (A) Ideally, distilled water should be used to 

prepare stock solutions of the test and reference substances. When appropriate, an 

alternative method may be used to solubilize or disperse the test substance in a manner 

consistent with its normal entry into the environmental compartment of interest. Watermiscible nontoxic solvents may be used when necessary, but attention should be paid to 

the associated organic load involved with adding organic solvents. Alternatively, the 

sample may be added in a neat form to the test system in a manner that maximizes its 

even and rapid distribution in the sludge. For materials that are poorly soluble and 

typically associated with suspended solids in wastewater, it may be appropriate to adsorb 

the test material onto an inert solid carrier, which is then dosed to the test system. If the 

test material cannot be evenly distributed in the test system prior to the initial sampling 

point, individual test systems can be prepared that are destructively sampled at each 

sampling interval. 

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

and even distribution of the test substance in the system and accurate administration of 

the dose between like systems. Ideally, when dosing with aqueous solutions, the added 

volume should be ≥2 ml and <10 ml; for nontoxic solvents, <0.1 ml/ L. If appropriate, 

dosing solutions may be prepared in advance and refrigerated. The activity of the stock 

should be checked by LSC.

(vii) Test conditions—(A) Temperature. Incubation should take place in the 

dark (preferred) or in diffuse light at a controlled temperature, which may be the field 

temperature or a standard laboratory temperature of 20-25°C.

(B) Agitation. To keep the sludge well mixed and in suspension, the test vessels 

are agitated by means of continuous shaking or stirring. Agitation also facilitates oxygen 

transfer from the headspace to the liquid so that aerobic conditions can be adequately 

maintained.

(viii) Test duration. The duration of the test should be sufficient to assess the 
26 

biodegradation of the test substance during its normal residence time in an activated 

plant, or its completeness of degradation under such conditions. Normally, the test period 

will be 28 d. However, it may be extended to obtain additional data points in order to 

estimate kinetic constants or to assess the completeness of degradation under the 

conditions of the test. Conversely, it may be ended before this time if degradation has 

plateaued. 

(ix) Number of test vessels. At a minimum, there should be a single abiotic and 

a single biotic test vessel for each test substance concentration. While replicates can be 

prepared for each system, more useful kinetic information usually can be gained by 

increasing the number of time points sampled within a system.

(5) Procedure—(i) Dosing. At test initiation, the chamber closure is removed 

and the test substance is quantitatively added directly to the activated sludge with 

constant mixing. It is recommended that the dose be administered gradually below the 

air-water interface, to ensure uniform distribution of the test substance in the sludge. The 

biotic and abiotic systems are dosed in an identical manner. Generally, the biotic systems 

are dosed first, followed by the abiotic systems. Exact timing is typically more critical for 

kinetic analyses.

(ii) Sampling schedule. Sampling times are selected based on existing 

biodegradation data or the results of a pilot study, as no fixed time schedule for sampling 

is universally applicable. A recommended sampling schedule for a rapidly degraded 

substance would be 5, 15, 30, 45, 60 and 90 min, with additional samplings after 2, 3, 5, 

8, 12 and 24 hr. Subsequent samples could be taken after 2, 3, 4, 5, 6 and 7 d and weekly 

until day 28. The sampling schedule for a slowly degrading substance should be adjusted 

so that a sufficient number of measurements are made during the degradation phase. 

(iii) Measurement of mineralization—(A) Indirect measurement of 14CO2. (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 total solids in the samples should not exceed 30 

mg dry weight.

(2) The samples are bubbled with air for several hours or allowed to stand 

overnight to allow the dissolved 14CO2 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 14CO2 is calculated based on the difference between the total 

counts in the biotic and abiotic samples.

(B) Direct measurement of 14CO2. (1) For rapidly degrading substances, it can be 

difficult to measure accurately the rate of 14CO2 evolved due to the rate of the mass transfer 

of 14CO2 from the headspace into the base trap. Under these conditions, it is recommended 

that indirect measurement of 14CO2 be conducted simultaneously with direct 

measurement.
27 

(2) Evolved 14CO2: The first base trap in the series is removed and quickly 

capped. The remaining traps are moved forward in the same order and a fresh trap placed 

behind the existing traps, and the trapping system reconnected as quickly as possible. 

Replicate subsamples (e.g. 1 ml) are removed from the base trap and transferred to 

scintillation vials, combined with a scintillation cocktail that is suitable for the sample 

matrix, and analyzed by LSC.

(3) Dissolved 14CO2: Samples (e.g. 10 to 25 ml) are removed through the 

sampling port of the test flask. They are then placed in vessels (e.g. Bellco Glass 

Biometer 2556-10250) containing a compartment with an appropriate CO2 absorbent (e.g. 

1.5 N KOH). The vessels are sealed 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 (e.g. overnight) to allow 

CO2 to diffuse from solution and be trapped from the headspace by the sorbent. Samples 

of the sorbent are combined with a scintillation cocktail that is suitable for the sample 

matrix and analyzed by LSC.

(C) Indirect measurement of 3

H2O. (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 total 

solids in the samples should not exceed 30 mg dry weight.

(2) Half of the samples are immediately analyzed directly by LSC for a wet 

measurement. The remaining samples are allowed to dry completely to remove the 3H2O. 

The samples are combined with a scintillation cocktail that is suitable for the sample 

matrix and analyzed by LSC. The percentage 3H2O is calculated based on the difference 

between the total counts in the wet and dry samples and the initial level of radioactivity 

dosed to the samples.

(iv) Measurement of radioactivity in MLSS. Small-volume samples of MLSS 

(e.g. 1 ml) are analyzed directly by LSC to quantify the radioactivity remaining in each 

system over time. These measurements are used to confirm that the recovery of 

radioactivity from the extracted samples is acceptable, and to monitor for volatilization. 

To avoid counting efficiency problems, the total solids in these samples should not 

exceed 30 mg dry weight. 

(v) Measurement of parent and metabolites—(A) Extraction. (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 concentrating and extracting the samples. 

A proven approach for nonvolatile test substances involves flash freezing the samples, 

followed by lyophilization and extraction of the dried residue with appropriate solvent(s) 

for parent and metabolites. Flash freezing quickly stops biological activity without 

hydrolyzing or otherwise altering labile test substances. Flash freezing is a quick process 
28 

if there is sufficient depth in the dry ice/acetone or liquid nitrogen bath to submerge the 

sample tube. The level of the bath should be above the sample level in the tube. The 

resulting extracts can be concentrated through evaporation and the total radioactivity in 

each extract determined by LSC.

(3) For volatile test substances, MLSS can be passed through a filter and solid 

phase extraction (SPE) column or SPE disk placed in tandem, which are subsequently 

eluted with appropriate solvents to recover parent and metabolites. Alternatively, samples 

can be centrifuged, and parent and metabolites extracted from the liquor by solid phase or 

liquid/liquid extraction. The solids can then be extracted directly, or mixed with a drying 

agent (e.g. sodium sulfate) and allowed to dry prior to extraction with an appropriate 

solvent system. An alternative is to extract the solids and then remove the water from the 

solvent by running it through a column containing a drying agent. In most cases, it is not 

efficient to use liquid/liquid extraction to recover parent and metabolites from MLSS. 

The total radioactivity in all extracts is determined by LSC. Care should be taken in 

concentrating extracts containing volatile test substances or metabolites.

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

document recoveries and consider the time involved in terminating biological activity, 

and factor it into the sample times used for kinetic analyses.

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

and metabolites in the extracts can be determined using TLC, HPLC or other separation 

techniques with radioactivity detection.

(2) If sensitive specific analytical methods are available, primary biodegradation 

can be assessed by measuring the total residual concentration of test substances and 

metabolites instead of using radioisotope techniques.

(C) Characterization of metabolites. Whenever possible, the chromatographic 

behavior of unknown peaks should be compared to that of predicted metabolites, if 

authentic standards exist. Usually, the quantity and purity of metabolites generated in this 

test make definitive identification by other direct means impossible. Depending on 

chromatographic behavior, it is usually possible to determine if a metabolite is more or 

less polar than the parent. This information, combined with known biochemical reactions 

and knowledge of when a metabolite appears and disappears in the sequence of 

biodegradation, can form an additional basis for inferring its identity. If necessary, the 

Kow of major metabolites can be estimated by HPLC (e.g. using OECD 117; see 

paragraph (o)(24) of this guideline) using an on-line radioactivity detector.

(vi) Measurement of extracted solids and incorporation into biomass. The 

extracted solids are combusted to determine the level of activity remaining with the 

solids. The level of radioactivity in the biotic solids above that in solids from the abiotic 

control typically represents incorporation of radioactivity into biomass. The distribution 

of this radioactivity among various components of biomass (i.e. nucleic acids, protein, 

cell wall, etc.) can be determined using a modified Sutherland and Wilkinson procedure 
29 

(see paragraphs (o)(7) and (o)(25) of this guideline).

(vii) Measurement of volatilized radioactivity. For volatile test substances, 

traps are extracted with appropriate solvents and the radioactivity in the extracts analyzed 

by LSC. The relative abundance of parent and metabolites in the extract(s) can be 

determined as described in paragraph (k)(5)(v)(B)(1) of this guideline.

(l) 314C: Mineralization and transformation in anaerobic digester sludge—

(1) General. (i) This test is designed to assess the extent to which a substance can be 

degraded during anaerobic digestion. It also provides rates of primary and ultimate 

biodegradation under the conditions in a digester. Anaerobic digestion is commonly used 

to stabilize and reduce the mass of sludge generated by wastewater treatment plants. 

Biodegradation during anaerobic digestion is particularly relevant for substances with a 

high tendency to partition to primary and secondary sludge. Removal during anaerobic 

digestion can significantly decrease the level of a substance present in sludge used as a 

soil amendment. The test is also easily adaptable for septage, to evaluate anaerobic 

biodegradation in septic tanks. The 314C test should be carried out after having 

conducted a 314B test (and sometimes a 314A test) since this (these) test(s) would help 

provide perspective on both the presence and levels of parent and potential degradation 

products associated with the primary and secondary sludge entering anaerobic digestion. 

(ii) Given that many digesters are operated as batch or plug-flow systems, which 

have long residence times (30 - 60 d), it is not essential that the substance and its 

degrader populations be at steady state at the initiation of a test, to generate useful 

degradation rates for exposure assessments.

(iii) To simulate conditions associated with episodic release of a substance, 

freshly collected digester sludge can be incubated with the maximum concentration of 

test substance expected to occur in sludge as a result of periodic releases. Approaches for 

estimating expected sludge concentrations can be found in Holman (see paragraph 

(o)(21) of this guideline) and the European Technical Guidance Document (see paragraph 

(o)(22) of this guideline). For existing substances continuously discharged to wastewater, 

freshly collected digester sludge can be incubated with radiolabeled test substance at a 

tracer level, or the concentration expected to occur in digester sludge. Usually, sufficient 

time is available within the time frame of the test for acclimation of the sludge to a new 

(test) substance. However, a laboratory anaerobic reactor operated in a draw-and-fill 

mode, amended with the substance at its expected concentration in sludge, can be 

considered as an option for generating acclimated sludge. This type of sludge should 

yield the most accurate kinetic data for a new substance that will be continuously exposed 

to wastewater.

(2) Principle of the test. (i) The test substance is incubated with abiotic and 

biotic digester sludge over a period of time. Biological activity is inhibited in the abiotic 

control, which is used for estimating mineralization by difference, establishing extraction 

efficiency and recovery of the parent molecule, and quantifying other loss processes such 

as hydrolysis, oxidation, volatilization or sorption to test apparatus.
30 

(ii) If an analytical method with the required sensitivity is available, the rate of 

parent degradation or transformation can be determined using a non-radiolabeled test 

substance or by following the disappearance of a substance already in wastewater. 

However, ultimate biodegradation of non-radiolabeled substances cannot be determined 

unless the biodegradation pathway is well understood and analytical methods with 

required sensitivity are available for potential metabolites.

(iii) Test substance at an environmentally relevant concentration is dosed to both 

abiotic and biotic test systems, which are incubated at a relevant temperature under static 

conditions with occasional or continuous mixing. Samples are periodically removed for 

determination of mineralization and primary biodegradation.

 (iv) Tests can be performed using an open batch system or a sealed, flow-through 

batch system where traps are used to capture evolved 14CO2 and 14CH4. The closed flowthrough system is should be used for volatile test substances and usually is preferred for 

14C-labeled test substances. Open systems are appropriate for nonvolatile 3

H-labeled test 

substances and for refining the biodegradation kinetics of nonvolatile 14C-labeled test 

substances, whose ability to be mineralized has previously been established. In the open 

system, mineralization to 14CO2 and 14CH4 can be determined indirectly by measuring the 

difference in residual radioactivity between samples from the biotic and abiotic systems 

following acidification. Similarly, mineralization to 3H2O can be determined indirectly by 

measuring the difference in residual radioactivity between samples following drying. In 

the closed batch system, gas sampling bags may also be used to trap gas from anaerobic 

digester sludge. In the flow-through systems, evolved 14CO2 is measured directly in the 

base traps, and 14CH4 is combusted to 14CO2 which is measured directly in a second set of 

base traps. As an option, dissolved 14CO2 can be determined by acidifying samples in a 

sealed vessel and measuring radioactivity in a base trap contained in the vessel. 

(v) Samples from both systems are analyzed for total radioactivity, extractable 

parent and metabolites and radioactivity associated with the extracted solids. The level of 

parent and metabolites is determined using chromatographic separation and, when 

appropriate, radio-analytical detection methods. The solids remaining from the extraction 

process are combusted to estimate incorporation into biomass by difference, or can be 

further fractionated to determine uptake into various components of biomass. A complete 

mass balance of the test system is obtained from the sum of all fractions at each 

sampling.

(3) Applicability of the test. The method is readily applicable to water-soluble or 

poorly water-soluble substances that are nonvolatile. It also can be adapted for volatile 

substances. Typically, 14C or 3

H labeling of substances is required for the assessment of 

mineralization. Either radiolabeled or nonlabeled substance can be used for the 

assessment of primary biodegradation.

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

of digester sludge in the test vessels is based on the number and volume of the samples 

needed for the test. Typically, 0.25 to 1 liter of digester sludge, diluted with an anaerobic 
31 

salts medium, is placed in 0.5 to 2-l containers. The sludge is diluted to facilitate 

quantitative removal of subsamples during the study. Anaerobic sludge should be 

protected from oxygen at all times including setup and sampling.

(B) Open batch systems are generally closed with a foam or cotton stopper to 

minimize evaporative loss of water and are incubated inside an anaerobic chamber under 

a reducing atmosphere. Flow-through test vessels are sealed with an appropriate closure 

containing a sampling port with a valve for removing sludge samples, and connections 

for influent and effluent gas lines. This closure can be a rubber stopper, but glass is 

recommended when working with a volatile hydrophobic test substance. When testing 

volatile substances, it also is recommended that gas lines and sampling tubes consist of 

inert materials (e.g. polytetrafluoroethylene, stainless steel, glass).

(C) The flow-through system is a modification of that originally described by 

Steber and Wierich (see paragraph (o)(10) of this guideline) and later refined by Nuck 

and Federle (see paragraph (o)(11) of this guideline). An example of a typical system is 

shown in Figure 5. The test vessels are continuously purged with a flow of nitrogen and 

connected to a series of traps containing potassium hydroxide solution (1.5 N) or other 

appropriate CO2 absorbent. An empty trap is usually included and positioned in front of 

the absorbent in the trapping train, as a precaution against backflow or condensation. The 

effluent gases from these traps are mixed with oxygen and passed through a quartz 

column packed with cupric oxide and maintained at approximately 800°C in a tube 

furnace, to combust methane to CO2. The gas exiting the combustion column is then 

passed through another series of base traps.

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

used:

(1) Miscellaneous glassware and pipettes; 

(2) Magnetic stirrers or shaker for continuous mixing of the test flasks; 
32 

Figure 5. Example of flow-through anaerobic test apparatus 

(3) Centrifuge; 

(4) pH meter; 

(5) Solid CO2/acetone or liquid nitrogen bath; 

(6) Freeze dryer (lyophilizer); 

(7) Oven or microwave oven for dry weight determinations; 

(8) Membrane filtration apparatus; 

(9) Autoclave; 

(10) Facilities to handle radiolabeled substances; 

(11) Equipment to quantify 14C and 3

H in liquid samples and solid samples (e.g. 

liquid scintillation counter); 

(12) Equipment to quantify 14C and 3

H in solid samples (e.g. sample oxidizer); 

(13) Equipment to trap volatilized 14C 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 14C and 3H for TLC (scanner) or HPLC (in-line 

detector); 

(16) Analytical equipment for the determination of the test (and reference) 

substance if specific substance analysis is used (e.g. gas chromatograph, 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 simulation test. For a site-specific assessment, the 

sludge should be obtained from the specific digester system of interest. For a generic 

assessment digester sludge should be obtained from a typical single-stage or first-stage 

digester receiving primary and secondary sludge from a wastewater treatment plant, 

receiving predominantly domestic wastewater. If the substance is currently a component 

of the wastewater entering the treatment facility or is episodically released to wastewater, 

freshly collected digester sludge will be ideal for the test. For a new substance that will 

be continuously released to wastewater, acclimated sludge from a laboratory anaerobic 

reactor may be more appropriate. For a generic assessment, this reactor should simulate 

the operation of a single-stage anaerobic digester, and be semi-continuously fed sludge 

that consists of combined primary and secondary sludge solids from a wastewater 

treatment plant receiving predominantly domestic wastewater, which has been amended 

with test substance at its expected sludge concentration for approximately 60 days. 

 (iv) Collection, transport and storage of digester sludge. The digester sludge 

should be collected from the digester in a manner that protects it from oxygen. The use of 

wide-mouth bottles, constructed from high-density polyethylene or a similar material that 

can expand, is recommended for the collection of digester sludge. The temperature of the 

sample should be noted at collection. Sample containers should be tightly sealed. During 

transport, the temperature of the sample should not significantly exceed the temperature 

used in the test. The digested sludge is typically stored under the exclusion of oxygen at 

test temperature. Storage containers should be vented in a manner that releases excess 

biogas but does not allow ambient air into the container.

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

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

test initiation. This solution is autoclaved for 30 min with slow exhaust, and allowed to 

cool overnight in an anaerobic chamber or under an anaerobic atmosphere.
34 

Ingredient Amount

Potassium phosphate monobasic, KH2PO4 ...................................... 8.5 mg/l 

Potassium phosphate dibasic, K2HPO4 .......................................... 21.8 mg/l 

Sodium phosphate dibasic heptahydrate, Na2HPO4• ......... 7H2O.. 50.3 mg/l 

Ammonium chloride, NH4Cl ......................................................... 20.0 mg/l 

Magnesium sulfate heptahydrate, MgSO4•7H2O............................. 2.2 mg/l 

Calcium chloride anhydrous, CaCl2................................................. 2.8 mg/l 

Ferrous chloride, FeCl2•4H2O........................................................ 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 protect the sludge from exposure to oxygen. The 

digester sludge should be sieved through a 2-mm screen. The total solids concentration 

should be measured. 

(2) A workable solids level that can be sampled during the study is approximately 

25,000 mg/l. If the solids are too high, they can be diluted with the dilution media. 

Alternatively, if the solids concentration is too low, the solids can be allowed to settle, the 

liquor decanted and the sludge resuspended in the dilution medium. A final solids level 

and pH should then be determined.

(3) The preparation of the abiotic sludge is typically performed using a 

combination of chemical and heat sterilization. A proven approach is to add mercuric 

chloride solution (1 g/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 abiotic 

sludge should be measured and adjusted to match that of the biologically active sludge. 

Alternative approaches to deactivate the sludge can also be used. When preparing the 

abiotic sludge it should be stored and manipulated in an anaerobic chamber, or another 

approach utilized to protect it from exposure to oxygen. 

(vi) Test substance preparation. (A) Distilled water should be used to prepare 

stock solutions of the test and reference substances. When appropriate, an alternative 

method may be used to solubilize or disperse the test substance in a manner consistent 

with its normal entry into digester sludge. When practical, the dosing solutions should be 

equilibrated overnight in a reducing atmosphere to remove DO prior to use. The volume 

of added stock should be of sufficient capacity to ensure rapid and even distribution of 

the test substance in the system and accurate administration of the dose between like 

systems. Ideally, when dosing with aqueous solutions, the added volume should be ≥1 

ml and <10 ml; for nontoxic solvents, <0.1 ml/ L. The activity of the stock should be 

checked by LSC. For substances that are poorly soluble and typically associated with 

sludge solids, it may be appropriate to adsorb the test material onto an inert solid carrier, 

which is then dosed to the test system. If the test material cannot be evenly distributed in 

the test system prior to the initial sampling point, individual test systems can be prepared 

that are destructively sampled at each sampling interval. 
35 

(B) As an alternative, the test substance can be applied to dried inactive sludge 

solids, which can be mixed into the test system. Water-miscible nontoxic solvents may be 

used when necessary, but attention should be paid to the associated organic load involved 

with adding organic solvents. In addition, the test substance may be added in a neat form 

in a manner that maximizes its even and rapid distribution in the sludge.

(vii) Test conditions—(A) Test temperature. Incubation should take place in the 

dark (preferred) or in diffuse light at a controlled temperature, which may be the typical 

operating temperature for an anaerobic digester (35°C +/- 3°C), a field temperature, or a 

standard laboratory temperature of 20-25°C.

(B) Agitation. To simulate the static conditions that occur in a digester, the test 

vessels are usually not continuously mixed. During sampling, they should be kept well 

mixed to ensure a representative sample. Additionally, they can be gently agitated for a 

few minutes, 2 or 3 times per week.

 (viii) Test duration. The duration of the test should be sufficient to assess the 

biodegradation of the test substance during its normal residence time in an anaerobic 

sludge digester. Normally, the test period will be approximately 60 d. However, it may be 

extended to obtain additional data points, in order to estimate kinetic constants or assess 

the completeness of degradation under the conditions of the test. Conversely, it may be 

ended before this time if degradation has plateaued. 

 

(ix) Number of test vessels. At a minimum, there should be a single abiotic and 

a single biotic test vessel for each test substance concentration. Replicates can be 

prepared for specific substance analysis. These are maintained under anaerobic 

conditions but typically not connected to the mineralization apparatus, and can be 

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 exposure to oxygen. It is 

recommended that the dose be administered gradually below the air-water interface, to 

ensure uniform distribution of the test substance in the sludge. The biotic and abiotic 

systems are dosed in an identical manner.

(ii) Sampling schedule. Sampling times are selected based on existing 

biodegradation data or the results of a pilot study, as no fixed time schedule for sampling 

is universally applicable. A recommended sampling schedule for a rapidly degraded 

substance would be 30, 60, and 120 min, with additional samplings after 4, 8 and 24 hr. 

Subsequent samples could be taken after 2, 4 and 7 d and weekly until day 56. The 

sampling schedule for slowly degrading substances should be adjusted so that a sufficient 

number of measurements are made during the degradation phase. 

(iii) Measurement of mineralization—(A) Indirect measurement of 14C Gases 
36 

(

14CO2 and 14CH4). Direct LSC counting of samples is not possible due to the high solids 

levels in the samples. Therefore, samples are centrifuged and the supernatant analyzed for 

total radioactivity by LSC, and the solids combusted and then analyzed for radioactivity 

to determine the total radioactivity in the sample. Individual replicate samples (e.g. 1 ml) 

of digester sludge are collected from each system and placed in centrifuge tubes that 

contain sufficient acid (e.g. 0.025 ml of HCl) to lower the sample pH to <2. The samples 

are centrifuged and the supernatant transferred to a scintillation vial, which is allowed to 

stand overnight for the dissolved 14CO2 to diffuse from the samples. The samples are 

combined with a scintillation cocktail that is suitable for the sample matrix and analyzed 

by LSC. The solids remaining in the centrifuge tube are combusted using a sample 

oxidizer prior to LSC. The percentage of total 14C gas produced is calculated based on the 

difference between the total counts in the biotic and abiotic samples.

(B) Direct measurement of 14CO2 and 14CH4. (1) Evolved 14CO2 and 14CH4: 

Direct measurement of 14CO2 and 14CH4 is possible only in a sealed flow-through batch 

system with connected base traps. For 14CO2, the first base trap in the first trapping train 

is removed and quickly capped. The remaining traps are moved forward in the same 

order and a fresh trap placed behind the existing traps, and the trapping system 

reconnected as quickly as possible. Replicate subsamples (e.g. 1 ml) from the removed 

base trap are transferred to scintillation vials, combined with a scintillation cocktail that is 

suitable for the sample matrix, and analyzed by LSC. This process is repeated for the 

second trapping train to determine 14CH4.

(2) Dissolved 14CO2 (optional): Sludge samples (e.g. 10 ml) are removed through 

the sampling port of the test flask. They are then placed in vessels (e.g. Bellco Glass 

Biometer 2556-10250) containing a compartment with an appropriate CO2 absorbent (e.g. 

1.5 N KOH). The vessels are sealed and sufficient acid (e.g. 6N 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 CO2 to diffuse from 

solution and be trapped from the headspace by the sorbent. Samples of the sorbent are 

combined with a scintillation cocktail that is suitable for the sample matrix and analyzed 

by LSC.

(C) Indirect measurement of 3

H2O. Samples (e.g. 8 ml) of sludge are collected 

from each system and placed in centrifuge tubes, located in a fume hood, that contain 

sufficient acid (e.g. 1 ml of 0.1 N HCl) to lower the sample pH to <2. The tubes are 

mixed and centrifuged. Individual replicate samples (e.g. 1 ml) of the supernatant are 

placed in separate vials. Half of the samples are immediately analyzed directly by LSC 

for a wet measurement. The remaining samples are allowed to dry completely to remove 

the 3H2O. The samples are combined with a scintillation cocktail that is suitable for the 

sample matrix and analyzed by LSC. The percentage 3H2O is calculated based on the 

difference between the total counts in the wet and dry samples and the initial level of 

radioactivity dosed to the samples.

(iv) Measurement of radioactivity in digester sludge. Direct LSC counting of 

samples is not possible due to the high solids levels in the samples. Therefore, samples 
37 

are centrifuged and the supernatant analyzed for total radioactivity by LSC, and the solids 

combusted and then analyzed for radioactivity to determine the total radioactivity in the 

sample. Individual replicate samples (e.g. 1 ml) of digester sludge are collected from each 

system and placed in centrifuge tubes. The samples are centrifuged and the supernatant 

transferred to a scintillation vial. The samples are combined with a scintillation cocktail 

that is suitable for the sample matrix and analyzed by LSC. The solids remaining in the 

centrifuge tube are combusted using a sample oxidizer prior to LSC.

(v) Measurement of parent and metabolites—(A) Extraction. (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 concentrating and extracting the samples. 

A proven approach for nonvolatile test substances involves flash freezing the samples, 

followed by lyophilization and extraction of the dried residue with appropriate solvent(s) 

for parent and metabolites. Flash freezing quickly stops biological activity without 

hydrolyzing or otherwise altering labile test substances. Flash freezing is a quick process 

if there is sufficient depth in the dry ice/acetone or liquid nitrogen bath to submerge the 

sample tube. The level of the bath should be above the sample level in the tube. The 

resulting extracts can be concentrated through evaporation and the total radioactivity in 

each extract determined by LSC. 

(3) For volatile test substances, samples can be centrifuged, and parent and 

metabolites extracted from the liquor by solid phase or liquid/liquid extraction. The solids 

can then be extracted directly or mixed with a drying agent (e.g. sodium sulfate) and 

allowed to dry prior to extraction with an appropriate solvent system. An alternative is to 

extract the solids and then remove the water from the solvent by running it through a 

column containing a drying agent. The total radioactivity in all extracts is determined by 

LSC. Care should be taken in concentrating extracts containing volatile test substances or 

metabolites. 

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

document recoveries and consider the time involved in terminating biological activity, 

and factor it into the sample times used for kinetic analyses. 

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

and metabolites in the extracts can be determined using TLC, HPLC or other separation 

techniques with radioactivity detection. 

(2) If sensitive specific analytical methods are available, primary biodegradation 

can be assessed by measuring the total residual concentration of test substances and 

metabolites instead of using radioisotope techniques. 

(C) Characterization of metabolites. Whenever possible, the chromatographic 

behavior of unknown peaks should be compared to that of predicted metabolites, if 
38 

authentic standards exist. Usually, the quantity and purity of metabolites generated in this 

test make definitive identification by other direct means impossible. Depending on 

chromatographic behavior, it is usually possible to determine if a metabolite is more or 

less polar than the parent. This information, combined with known biochemical reactions 

and knowledge of when a metabolite appears and disappears in the sequence of 

biodegradation, can form an additional basis for inferring its identity. If necessary, the 

Kow of major metabolites can be estimated by HPLC (e.g. using OECD 117; see 

paragraph (o)(24) of this guideline) using an on-line radioactivity detector.

(vi) Measurement of extracted solids and incorporation into biomass. The 

extracted solids are combusted to determine the level of activity remaining with the 

solids. The level of radioactivity in the biotic solids above that in solids from the abiotic 

control typically represents incorporation of radioactivity into biomass. The distribution 

of this radioactivity among various components of biomass (i.e. nucleic acids, protein, 

cell wall, etc.) can be determined using a modified Sutherland and Wilkinson procedure 

(see paragraph (o)(25) of this guideline). 

(vii) Measurement of volatilized radioactivity. For volatile test substances, the 

volatility traps are extracted with appropriate solvents and the radioactivity in the extracts 

analyzed by LSC. The relative abundance of parent and metabolites in the extract(s) can 

be determined as described in paragraph (l)(5)(v)(B)(1) of this guideline. 

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

General. (i) This test is designed to evaluate the biodegradation of the portion of a 

substance that passes through treatment and is released in effluent to surface water. It can 

be used to demonstrate that biodegradation occurring in the treatment plant continues in 

the receiving environment. It also is useful for determining the extent of biodegradation 

as well as rates of primary and ultimate biodegradation in this environmental 

compartment. The results can be used to estimate the reduction in substance 

concentration resulting from biodegradation as a volume of water moves downstream 

from a wastewater treatment plant outfall. The test medium consists of freshly collected 

surface water and effluent. The usefulness of the measured rates for predicting 

downstream exposure will be a function of the fidelity of the simulation to actual 

conditions in the mixing zone. Along with test substance concentration, factors to 

consider in the designing of this test include the level of solids in effluent and the degree 

to which effluent is diluted in receiving water. 

(ii) To simulate conditions associated with episodic release of a substance, an 

appropriately chosen mixture of surface water and effluent is incubated with the 

concentration of test substance expected to occur in effluent diluted into surface water 

during a release event. In these situations, the substance and its degrader populations will 

usually not be at steady state and the observed kinetics will be pseudo first-order, or 

second-order Monod. Approaches for estimating effluent concentrations can be found in 

Holman (see paragraph (o)(21) of this guideline) and the European Technical Guidance 

Document (see paragraph (o)(22) of this guideline). 
39 

(iii) For substances that are or will be continuously released to wastewater, 

degrader populations in the treatment plant will become acclimated to the substance. For 

existing substances continuously discharged to wastewater, surface water with the 

expected concentration of test substance and freshly collected effluent under a given 

discharge scenario will provide the most realistic kinetic parameters. For new substances 

that will be continuously discharged to wastewater, the use of effluent that was exposed 

to the substance under simulated activated sludge conditions in the laboratory (e.g. in an 

OECD 303A test) will provide the most accurate kinetics. It should be noted that use of 

303A may lead to an overestimation of degradation in treatment, as compared to a real 

environmental release situation. 

 (iv) In most circumstances, due to analytical considerations, it will be impossible 

to test at actual surface water concentrations. Consequently, observed biodegradation rates 

may not be fully representative of those under actual environmental conditions. This should 

be considered in the interpretation of results. 

(2) Principle of the test. (i) The test substance is incubated with abiotic and 

biotic mixtures of surface water and effluent over a period of time. The ratio of these 

components is based on specific or generic scenarios for release of treated effluent to 

surface water. Biological activity is inhibited in the abiotic control, which is used for 

estimating mineralization by difference, establishing extraction efficiency and recovery 

of the parent molecule, and quantifying other loss processes such as hydrolysis, 

oxidation, volatilization or sorption to test apparatus. 

(ii) If an analytical method with the required sensitivity is available, the rate of 

parent degradation or transformation can be determined using a non-radiolabeled test 

substance or by following the disappearance of a substance already in wastewater. 

However, ultimate biodegradation cannot be determined unless the biodegradation 

pathway is well understood and analytical methods with required sensitivity are available 

for potential metabolites. 

(iii) Test substance at an environmentally relevant concentration is dosed to both 

abiotic and biotic test systems, which are incubated at a relevant temperature with 

continuous mixing when appropriate. Samples are periodically removed for 

determination of mineralization and primary biodegradation. 

(iv) Tests can be performed using an open batch system or a sealed, flow-through 

batch system in which traps are used to capture evolved 14CO2 (see Figure 2). The closed 

flow-through system should be used for volatile test substances and usually is preferred 

for 14C-lableled test substances. Open systems are appropriate for nonvolatile 3

H-labeled 

test substances and for refining the biodegradation kinetics of nonvolatile 14C-labeled test 

substances, whose ability to be mineralized has previously been established. In the open 

system, mineralization to 14CO2 can be determined indirectly by measuring the difference 

in residual radioactivity between samples from the biotic and abiotic systems following 

acidification. Similarly, mineralization to 3H2O can be determined indirectly by 

measuring the difference in radioactivity in a sample following drying. In the flow-
40 

through systems, evolved 14CO2 is measured directly in the base traps. In addition, 

dissolved 14CO2 is determined by acidifying samples in a sealed vessel and measuring 

radioactivity in a base trap contained in the vessel. 

(v) Samples from both systems are analyzed for total radioactivity, extractable 

parent and metabolites and radioactivity associated with the extracted solids. The level of 

parent and metabolites is determined using chromatographic separation and, when 

appropriate, radio-analytical detection methods. The solids remaining from the extraction 

process are combusted to estimate incorporation into biomass by difference, or can be 

further fractionated to determine uptake into various components of biomass. A complete 

mass balance of the test system is obtained from the sum of all fractions at each 

sampling. 

(3) Applicability of the test. The method is readily applicable to water-soluble or 

poorly water-soluble substances that are nonvolatile. It also can be adapted for volatile 

substances. Typically, 14C or 3H labeling of substances is required for the assessment of 

mineralization. Either radiolabeled or nonlabeled substance can be used for the 

assessment of primary biodegradation. 

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

surface water-effluent mixture in the test vessels is based on the number and volume of 

the samples needed for the test. Typically, 1-2 l of surface water are placed in 2- or 4-l 

flasks. Open batch systems are generally closed with a foam or cotton stopper to 

minimize evaporative loss of water. Flow-through systems are sealed with an appropriate 

closure containing a sampling port with a valve for removing samples and connections 

for influent and effluent gas lines. This closure can be a rubber stopper, but glass is 

recommended when working with a volatile hydrophobic test substance. When testing 

volatile substances, it also is recommended that gas lines and sampling tubes consist of 

inert materials (e.g. polytetrafluoroethylene, stainless steel, glass). 

(B) The headspace of the test vessel is continuously purged with air or CO2-free 

air at a rate sufficient to maintain the system in an aerobic condition but not fast enough 

to prevent efficient trapping of CO2. The test vessel is connected to a series of traps 

containing a potassium hydroxide (1.5 N) or other appropriate CO2 absorbent. An empty 

trap is usually included and positioned in front of the absorbent in the trapping train, as a 

precaution against backflow or condensation. 

(ii) Equipment. 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; 
41 

(D) pH meter; 

(E) Solid CO2/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 radiolabeled substances; 

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

liquid scintillation counter); 

(L) Equipment to quantify 14C and 3

H in solid samples (e.g. sample oxidizer); 

(M) Equipment to trap volatilized 14C and 3

H from gas trapping system (in-line 

activated charcoal trap or equivalent); 

(N) Equipment for TLC or HPLC; 

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

detector); 

(P) Analytical equipment for the determination of the test (and reference) 

substance if specific substance analysis is used (e.g. gas chromatograph, highperformance liquid chromatograph, mass spectrometer). 

(iii) Selection of environmental samples. (A) The source of surface water, 

activated sludge or effluent should be consistent with the objective of the simulation test. 

For a site-specific assessment, activated sludge or effluent should be obtained from the 

specific wastewater treatment plant of interest. Likewise, the surface water should be 

obtained upstream from that treatment plant’s outfall. The ratio of these components 

should be chosen to simulate a specific flow scenario (e.g. low flow or mean flow). 

Alternatively, if hydraulic conditions below the outfall are known, the test medium can 

simply be water samples obtained downstream from the outflow. However, such 

conditions are variable and hard to reproduce. 

(B) For a generic assessment activated sludge or effluent should be obtained from 

a typical wastewater treatment plant receiving predominantly domestic wastewater. 

Likewise, the surface water should be typical of surface waters into which effluent is 

released. If the substance is currently a component of wastewater entering the wastewater 

treatment facility or is episodically released to wastewater, freshly collected activated 
42 

sludge or effluent will be ideal for the test. WWTP effluent consists of activated sludge 

liquor and biosolids, but it can be variable in its solids level and impacted by chlorination 

or other processes. The best method of simulating this scenario is using filtered effluent 

and surface water combined at the targeted dilution ratio and separately adding activated 

sludge biosolids at a targeted concentration. If the effluent is difficult to obtain, the 

activated sludge can be filtered or centrifuged to remove biosolids, and the liquor and 

biosolids can then be added at a defined ratio to the test system. For a generic assessment, 

the TGD assumes that effluent containing 30 mg biosolids/l is diluted 10-fold into surface 

water containing 15 mg suspended solids/l (see paragraph (o)(22) of this guideline). 

Hence, 3 mg of biosolids in 100 ml of filtered effluent or activated sludge liquor and 900 

ml of surface water approximates this generic scenario. Additionally, a scenario based on 

10 mg of biosolids and 330 ml of liquor per l may be considered to simulate critical low 

flow conditions that might occur during dry seasons. 

(C) For a new substance that will be continuously released to wastewater, the 

activated sludge or effluent ideally should be obtained from a laboratory-scale treatment 

system such as a porous pot or continuous activated sludge test (e.g. an OECD 303A test) 

(see paragraph (o)(2) of this guideline), which has been fed wastewater amended with 

unlabeled test substance. The source of the starting sludge, wastewater (influent), and 

operating conditions (influent concentration, hydraulic retention time, solids retention 

time) for the laboratory unit should accurately reflect site-specific or generic conditions. 

In the latter case, the TGD specifies an HRT of 6.9 hr and an SRT of 9.2 d in its generic 

scenario for wastewater treatment. The TGD also provides guidance for estimating 

concentration of a substance in wastewater based on expected use volumes. In general, 

steady state will be reached within 2 to 3 SRTs, after which the biosolids or effluent can 

be used for testing. 

(iv) Collection, transport and storage of environmental samples. The activated 

sludge should be collected from a well-mixed region of the aeration basin. Surface water 

should be collected from a site with known inputs of wastewater. The temperature of the 

samples should be noted at collection. Collection containers should allow for adequate 

ventilation and measures should be taken to prevent the temperature of the sample from 

significantly exceeding the temperature used in the test. The samples are typically stored 

at test temperature with continuous aeration. Samples should not be stored frozen. 

(v) Preparation of test systems. (A) The surface water should be characterized 

by measuring TSS, total hardness and pH. A standard plate count and organic carbon 

analysis are optional. When using activated sludge to represent WWTP effluent, the 

MLSS is sieved through a 2mm screen, blended (optional) and allowed to settle. The TSS 

concentration of the liquor is measured. The liquor is added to the surface water at a 

volume sufficient to achieve the targeted biosolids concentration. If more volume is 

needed to reach the targeted dilution ratio in the test, filtered or centrifuged activated 

sludge liquor is added to reach the necessary dilution. If using treated effluent and a 

targeted activated sludge biosolids concentration, the effluent is filtered and mixed with 

the surface water at the targeted dilution ratio. The MLSS is prepared as described 

previously. The pH and TSS of the prepared surface water mixture should be measured. 
43 

An optional standard plate count and organic carbon analysis can also be performed. 

(B) The abiotic system is typically prepared using a combination of chemical and 

heat sterilization. A proven approach is to add mercuric chloride solution (0.1 g/l) to the 

mixture, which is then autoclaved for at least 90 min. Typically the volume of medium is 

<= half the volume of the container being autoclaved (e.g. 500 ml activated sludge in a 1-

liter container). After cooling, the pH of the abiotic system should be measured and 

adjusted to match that of the biologically active system. Alternative approaches to 

deactivate the surface water mixture can also be used. 

(vi) Test substance preparation. (A) Ideally, distilled water should be used to 

prepare stock solutions of the test and reference substances. When appropriate, an 

alternative method may be used to solubilize or disperse the test substance in a manner 

consistent with its normal entry into the environment. Water-miscible nontoxic solvents 

may be used when necessary, but attention should be paid to the associated organic load 

involved with adding organic solvents. Alternatively, the sample may be added in a neat 

form to the test system in a manner that maximizes its even and rapid distribution in the 

test medium. For materials which are poorly soluble and typically associated with 

suspended solids in effluent, it may be appropriate to adsorb the test material onto an inert 

solid carrier, which is then dosed to the test system. If the test material cannot be evenly 

distributed in the test system prior to the initial sampling point, individual test systems can 

be prepared that are destructively sampled at each sampling interval. 

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

and even distribution of the test substance in the system and accurate administration of 

the dose between like systems. Ideally, when dosing with aqueous solutions, the added 

volume should be ≥2 ml and <10 ml; for nontoxic solvents, <0.1 ml/ L. If appropriate, 

dosing solutions may be prepared in advance and refrigerated. The activity of the stock 

should be checked by LSC. 

(vii) Test conditions—(A) Test temperature. Incubation should take place in the 

dark (preferred) or in diffuse light at a controlled temperature, which may be the field 

temperature or a standard laboratory temperature of 20-25°C. 

(B) Agitation. To keep the test medium in suspension, the test systems are 

agitated by means of continuous shaking or stirring. Agitation also facilitates oxygen 

transfer from the headspace to the liquid so that aerobic conditions can be adequately 

maintained. 

 (viii) Test duration. The duration of the test should be sufficient to assess the 

biodegradation of the test substance during its normal residence time in the WWTP 

effluent-surface water mixing zone, or its completeness of degradation under such 

conditions. Normally, the test period will be 28 d. However, it may be extended to obtain 

additional data points, in order to estimate kinetic constants or to assess the completeness 

of degradation under the conditions of the test. Conversely, it may be ended before this 

time if degradation has plateaued. 
44 

(ix) Number of test vessels. At a minimum, there should be a single abiotic and a 

single biotic test vessel for each test substance concentration. While replicates can be 

prepared for each system, more useful kinetic information usually can be gained by 

increasing the number of time points sampled within a system. 

(5) Procedure—(i) Dosing. At test initiation, the test vessel is opened and the 

test substance is quantitatively added directly to the system with constant mixing. It is 

recommended that the dose be administered gradually below the air-water interface, to 

ensure uniform distribution of the test substance in the test medium. The biotic and 

abiotic systems are dosed in an identical manner. Generally, the biotic systems are dosed 

first, followed by the abiotic systems. Exact timing is typically more critical for kinetic 

analyses. 

(ii) Sampling schedule. Sampling times are selected based on existing 

biodegradation data or the results of a pilot study, as no fixed time schedule for sampling 

is universally applicable. A recommended sampling schedule for a rapidly degraded 

substance would be 5, 30, 60 min, with additional samplings after 3, 5, 8, 12 and 24 hr. 

Subsequent samples could be taken after 2, 3, 4, 5, 6 and 7 d and weekly until day 28. 

The sampling schedule for slowly degrading substance should be adjusted so that a 

sufficient number of measurements are made during the degradation phase. 

(iii) Measurement of mineralization—(A) Indirect measurement of 14CO2. (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 14CO2 to diffuse from the samples. The samples are combined with a 

scintillation cocktail that is suitable for the sample matrix and analyzed by LSC. The 

percentage of 14CO2 is calculated based on the difference between the total counts in the 

biotic and abiotic samples.

(B) Direct measurement of 14CO2. (1) For rapidly degrading substances, it can be 

difficult to measure accurately the rate of 14CO2 evolved due to the rate of the mass transfer 

of 14CO2 from the headspace into the base trap. Under these conditions, it is recommended 

that indirect measurement of 14CO2 be conducted simultaneously with direct 

measurement. 

(2) Evolved 14CO2: The first base trap in the series is removed and quickly 

capped. The remaining traps are moved forward in the same order and a fresh trap placed 

behind the existing traps, and the trapping system reconnected as quickly as possible. 

Replicate subsamples (e.g 1 ml) from the removed base trap are transferred to 

scintillation vials, combined with scintillation cocktail that is suitable for the sample 

matrix, and analyzed by LSC. 
45 

(3) Dissolved 14CO2: Samples (e.g. 25 to 50 ml) are removed through the 

sampling port of the test flask. They are then placed in vessels (e.g. Bellco Glass 

Biometer 2556-10250) containing a compartment with an appropriate CO2 absorbent (e.g. 

1.5 N KOH). The vessels are sealed 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 (e.g. overnight) to allow 

CO2 to diffuse from solution and be trapped from the headspace by the sorbent. Samples 

of the sorbent are combined with a scintillation cocktail that is suitable for the sample 

matrix and analyzed by LSC.

(C) Indirect measurement of 3

H2O. (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) Half of the samples are immediately analyzed directly by LSC for a wet 

measurement. The remaining samples are allowed to dry completely to remove the 3

H2O. 

The samples are combined with a scintillation cocktail that is suitable for the sample 

matrix and analyzed by LSC. The percentage 3H2O is calculated based on the difference 

between the total counts in the wet and dry samples and the initial level of radioactivity 

dosed to the samples.

(iv) Measurement of total radioactivity in surface water. Small-volume 

samples (e.g. 1 ml) are analyzed directly by LSC to quantify the radioactivity remaining 

in each surface water over time. These measurements are used to confirm that the 

recovery of radioactivity from the extracted samples is acceptable and to monitor for 

volatilization. 

(v) Measurement of parent and metabolites—(A) Extraction. (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 concentrating and extracting the samples. 

A proven approach for nonvolatile test substances involves flash freezing the samples, 

followed by lyophilization and extraction of the dried residue with appropriate solvent(s) 

for parent and metabolites. Flash freezing quickly stops biological activity without 

hydrolyzing or otherwise altering labile test substances. Flash freezing is a quick process 

if there is sufficient depth in the dry ice/acetone or liquid nitrogen bath to submerge the 

sample tube. The level of the bath should be above the sample level in the tube. The 

extract is filtered to recover the solvent and solids separately. The filter must be 

compatible with the solvent type (e.g. aqueous or non-aqueous). The resulting extracts can 

be concentrated through evaporation and the total radioactivity in each extract determined 

by LSC. 

(3) For volatile test substances, the sample can be passed through a filter and solid 

phase extraction (SPE) column or SPE disk placed in tandem, which are subsequently 
46 

eluted with appropriate solvents to recover parent and metabolites. Alternatively, the 

aqueous samples can be extracted with an appropriate solvent system and then filtered to 

recover biomass solids, assuming sufficient extraction efficiency. The total radioactivity 

in all extracts is determined by LSC. Care should be taken in concentrating extracts 

containing volatile test substances or metabolites. 

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

document recoveries and consider the time involved in terminating biological activity, 

and factor it into the sample times used for kinetic analyses.

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

metabolites in the extracts can be determined using TLC, HPLC or other separation 

techniques with radioactivity detection. 

(2) If sensitive specific analytical methods are available, primary biodegradation 

can be assessed by measuring the total residual concentration of test substances and 

metabolites instead of using radioisotope techniques.

(C) Characterization of metabolites. Whenever possible, the chromatographic 

behavior of unknown peaks should be compared to that of predicted metabolites, if 

authentic standards exist. Usually, the quantity and purity of metabolites generated in this 

test make definitive identification by other direct means impossible. Depending on 

chromatographic behavior, it is usually possible to determine if a metabolite is more or 

less polar than the parent. This information, combined with known biochemical reactions 

and knowledge of when a metabolite appears and disappears in the sequence of 

biodegradation, can form an additional basis for inferring its identity. If necessary, the 

Kow of major metabolites can be estimated by HPLC (e.g. using OECD 117; see 

paragraph (o)(24) of this guideline) using an on-line radioactivity detector. 

(vi) Measurement of extracted solids. Since the filters will retain carbonate salts 

as well as microorganisms from the test system, the filter containing the biosolids is 

placed in a scintillation vial and acidified to pH ≤2 by submerging it in a weak acid 

solution (1 ml of 0.1 N HCl). The samples are allowed to sit for sufficient time (e.g. 

overnight) for the dissolved 14CO2 to diffuse from the samples. The samples are 

combined with a scintillation cocktail that is suitable for the sample matrix and analyzed 

by LSC. The level of radioactivity in the biotic solids above that in solids from the 

abiotic control typically represents incorporation of radioactivity into biomass. 

(vii) Measurement of volatilized radioactivity. For volatile test substances, the 

volatility traps are extracted with appropriate solvents and the radioactivity in the extracts 

analyzed by LSC. The relative abundance of parent and metabolites in the extract(s) can 

be determined as described in paragraph (m)(5)(v)(B)(1) of this guideline. 

(n) 314E Biodegradation in untreated wastewater-surface water mixing 

zone—(1) General. (i) In developing regions lacking wastewater treatment 

infrastructure, it is common for wastewater to be directly discharged to surface waters. 
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 biodegradation as well as rates of primary and 

ultimate biodegradation under such direct discharge conditions. The results can be used 

to estimate the reduction in substance concentration resulting from biodegradation as a 

volume of water moves downstream from a wastewater outfall. As an option, this 

reduction can be compared to decreases in other wastewater components such as BOD, 

COD or TOC. The test medium consists of freshly collected wastewater and surface 

water. The usefulness of the measured biodegradation rates for predicting downstream 

exposure will be a function of the fidelity of the simulation to actual conditions in the 

mixing zone. Along with test substance concentration, factors to consider in designing 

this test include DO concentration and the degree to which effluent is diluted into surface 

water. 

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

wastewater and surface water incubated with radiolabeled test substance at a tracer level 

will provide the most realistic kinetic parameters regarding the current substance load. 

For substances not consistently present in wastewater, sufficient test substance 

(radiolabeled and unlabeled) should be added to approximate the expected concentration 

in wastewater diluted into surface water during an episodic release or following 

commercialization of a new substance. Approaches for estimating such an expected 

wastewater concentration can be found in Holman (see paragraph (o)(21) of this 

guideline) and the European Technical Guidance Document (see paragraph (o)(22) of this 

guideline). 

(iii) For low dilution situations, it is best to incubate the mixtures under reduced 

DO conditions (1-4 mg/l) to simulate the DO level below a wastewater outfall. In this 

test, the substance and its degrader populations usually are not at steady state and the 

observed kinetics will be pseudo first-order, or second-order Monod. 

(iv) The test can be run using a single or two-phase design. In the former, 

wastewater is diluted into surface water from a clean or wastewater-impacted source. In 

the latter, wastewater is sequentially diluted into clean and wastewater-impacted surface 

waters. In this test design, the test substance is dosed into wastewater diluted in clean 

surface water and disappearance of test substance and conventional pollutants (BOD, 

COD, etc) are monitored over time (phase 1). Subsequently, a second dose of test 

substance and wastewater is added to the same system to simulate dilution of wastewater 

into surface water previously polluted by wastewater (phase 2). 

(2) Principle of the test. (i) The test substance is incubated with abiotic and 

biotic mixtures of wastewater and surface water, usually under reduced DO conditions 

(1-4 mg/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 used for estimating mineralization by difference, 

establishing extraction efficiency and recovery of the parent molecule, and quantifying 

other loss processes such as hydrolysis, oxidation, volatilization or sorption to test 
48 

apparatus. 

(ii) If an analytical method with the required sensitivity is available, the rate of 

parent degradation or transformation can be determined using a non-radiolabeled test 

substance or by following the disappearance of a substance already in wastewater. 

However, ultimate biodegradation cannot be determined unless the biodegradation 

pathway is well understood and analytical methods with required sensitivity are available 

for potential metabolites. 

(iii) Test substance at an environmentally relevant concentration is dosed to both 

abiotic and biotic test systems, which are incubated at a relevant temperature with 

continuous mixing. The biotic samples are incubated in such a way that DO levels remain 

at a reduced level (1-4 mg/l), characteristic of the situation below a wastewater outfall. 

Samples are periodically removed for determination of mineralization and primary 

biodegradation and, as an option, the level of other wastewater components (e.g. COD, 

TOC and ammonia) can be determined concurrently. 

(iv) In the two-phase test design, once biodegradation of the test substance and 

wastewater components have leveled off, a second dose of test substance and fresh 

wastewater is mixed into the existing test system to simulate wastewater being diluted 

into previously polluted surface water, and the sampling process is repeated. 

(v) Tests can be performed using an open batch system or a sealed, flow-through 

batch system in which traps are used to capture evolved 14CO2 (see Figure 2). The closed 

flow-through system should be used for volatile test substances and usually is preferred 

for 14C-lableled test substances. Open systems are appropriate for nonvolatile 3H-labeled 

test substances and for refining the biodegradation kinetics of nonvolatile 14C-labeled test 

substances, whose ability to be mineralized has previously been established. In the open 

system, mineralization to 14CO2 can be determined indirectly by measuring the difference 

in residual radioactivity between samples from the biotic and abiotic systems following 

acidification. Similarly, mineralization to 3

H2O can be determined indirectly by 

measuring the difference in radioactivity in a sample following drying. In the flowthrough systems, evolved 14CO2 is measured directly in the base traps. In addition, 

dissolved 14CO2 is determined by acidifying samples in a sealed vessel and measuring 

radioactivity in a base trap contained in the vessel. 

(vi) Samples from both systems are analyzed for total radioactivity, extractable 

parent and metabolites and radioactivity associated with the extracted solids. The level of 

parent and metabolites is determined using chromatographic separation and when 

appropriate, radio-analytical detection methods. The remaining solids from the extraction 

process are combusted to estimate incorporation into biomass by difference. A complete 

mass balance of the test system is obtained from the sum of all fractions at each sampling. 

(3) Applicability of the test. The method is readily applicable to water-soluble 

and poorly water-soluble substances that are nonvolatile. It also can be adapted for 

volatile substances. Typically, 14C or 3

H-radiolabeling of substances is required for the 
49 

assessment of mineralization. Either radiolabeled or nonlabeled substance can be used for 

the assessment of primary biodegradation. 

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

wastewater-surface water mixture in the test vessels is based on the number and volume 

of the samples needed for the test. Typically, 1-2 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 controller connected to an 

actuator valve, which controls the aeration of the wastewater (see Figure 3). This aeration 

is balanced against continuous sparging with nitrogen to achieve the targeted DO level. 

Alternatively, the wastewater can be incubated with stirring but minimum aeration, to 

keep DO at desired levels, and nitrogen or air can be added periodically to maintain the 

DO level. In this case, DO readings should be reported at regular intervals. 

(B) Open systems are generally closed with a foam or cotton stopper to minimize 

evaporative loss of water. Flow-through systems are sealed with an appropriate closure 

containing a sampling port with a valve for removing samples, and connections for 

influent and effluent gas lines. This closure can be a rubber stopper, but glass is 

recommended when working with a volatile, hydrophobic test substance. When testing 

volatile substances, it also is recommend that gas lines and sampling tubes consist of inert 

materials (e.g. polytetrafluoroethylene, stainless steel, glass). 

(C) The headspace of the test vessel is continuously purged with air or CO2-free 

air at a rate sufficient to maintain the system in an aerobic condition, but not so fast as to 

prevent efficient trapping of CO2. The test vessel is connected to a series of traps 

containing a potassium hydroxide (1.5 N) or other appropriate CO2 absorbent. An empty 

trap is usually included and positioned in front of the absorbent in the trapping train, as a 

precaution against backflow or condensation. 

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

used: 

(1) Miscellaneous glassware and pipettes; 

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

(3) Centrifuge; 

(4) pH meter; 

(5) Solid CO2/acetone or liquid nitrogen bath; 

(6) Freeze dryer (lyophilizer); 

(7) Oven or microwave oven for dry weight determinations; 
50 

(8) Membrane filtration apparatus; 

(9) Autoclave; 

(10) Facilities to handle radiolabeled substances; 

(11) Equipment to quantify 14C and 3

H in liquid samples and solid samples (e.g. 

liquid scintillation counter); 

(12) Equipment to quantify 14C and 3

H in solid samples (e.g. sample oxidizer); 

- Equipment to trap volatilized 14C and 3

H from gas trapping system (in-line 

activated charcoal trap or equivalent); 

(13) Equipment for TLC or HPLC; 

(14) Equipment to quantify 14C and 3

H for TLC (scanner) or HPLC (in-line 

detector); 

(15) Analytical equipment for the determination of the test (and reference) 

substance if specific substance analysis is used (e.g. gas chromatograph, 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 probe and actuator valve; 

(3) COD digestion vials; 

(4) Nitrogen ammonia reagent set; 

(5) Spectrophotometer. 

(iii) Selection of environmental samples. (A) The source of wastewater and 

surface water should be consistent with the objective of the simulation test. For a 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 specific flow scenario (e.g. 

low flow or mean flow). Alternatively, if hydraulic conditions below the outfall are 

known, the test medium can simply be water samples obtained downstream from the 

outflow. However, such conditions are variable and hard to reproduce. 

(B) For a generic assessment wastewater samples should be predominantly 

derived from domestic sources, and the surface water should be typical of surface waters 
51 

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 North America, typical wastewaters 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 storage of environmental samples. Wastewater 

should be collected from a sewer access point or at the head of a wastewater treatment 

plant. The temperature of the sample should be noted at collection. During transport, the 

temperature of the sample should not significantly exceed the temperature used in the 

test. The wastewater is typically stored at test temperature with low mixing. No samples 

should ever be stored frozen. 

(v) Preparation of test systems. (A) The freshly collected wastewater should be 

largely free of coarse particles. TSS, pH and COD should be determined for the 

wastewater. NH3, organic carbon and standard plate count are optional analyses. The 

surface water should be characterized by measuring TSS, total hardness and pH. A 

standard plate count and organic carbon analysis are optional. The wastewater is added to 

the surface water at a volume sufficient to achieve the targeted dilution of wastewater 

into surface water. The pH, COD and TSS of the prepared surface water mixture should 

be measured. Optionally, standard place count, NH3 and organic carbon analyses can also 

be performed on the mixture. 

(B) The abiotic system is typically prepared using a combination of chemical and 

heat sterilization. A proven approach is to add mercuric chloride (HgCl2 = 0.5 g/l) to the 

mixture, which is then autoclaved for at least 90 min. Typically the volume of medium is 

<= half the volume of the container being autoclaved (e.g. 500 ml activated sludge in a 1-

liter container). After cooling, the pH of the abiotic system should be measured and 

adjusted to match that of the biologically active system. Alternative approaches to 

deactivate the surface water mixture can also be used. 

(vi) Test substance preparation. (A) Ideally, distilled water should be used to 

prepare stock solutions of the test and reference substances. When appropriate, an 

alternative method may be used to solubilize or disperse the test substance in a manner 

consistent with its normal entry into the environment. Water-miscible nontoxic solvents 

may be used when necessary, but attention should be paid to the associated organic load 

involved with adding organic solvents. Alternatively, the sample may be added in a neat 

form to the test system in a manner that maximizes its even and rapid distribution in the 

test medium. For substances that are poorly soluble and typically associated with 

suspended solids in wastewater, it may be appropriate to adsorb the test material onto an 

inert solid carrier, which is then dosed to the test system. If the test material cannot be 

evenly distributed within the test system prior to the initial sampling point, individual test 

systems can be prepared that are destructively sampled at each sampling interval. 

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

distribution of the test substance in the system, and accurate administration of the dose 
52 

between like systems. Ideally, when dosing with aqueous solutions, the added volume 

should be ≥ 2 ml and <10 ml; for nontoxic solvents, <0.1 ml/l. If appropriate, dosing 

solutions may be prepared in advance and refrigerated. The activity of the stock should 

be checked by LSC. 

(vii) Test conditions—(A) Test temperature. Incubation should take place in the 

dark (preferred) or in diffuse light at a controlled temperature, which may be the field 

temperature or a standard laboratory temperature of 20-25°C. 

(B) Agitation. To keep the solids in suspension, the test vessels are minimally 

agitated by means of continuous mixing or stirring. 

 (viii) Test duration. The duration of the test should be sufficient to assess the 

biodegradation of the test substance during its normal residence time in the 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 estimate kinetic constants or to 

assess the completeness of degradation under the conditions of the test. Conversely, it 

may be ended before this time if degradation has plateaued. 

(ix) Number of test vessels. At a minimum, there should be a single abiotic and 

a single biotic test vessel for each test substance and test substance concentration. While 

replicates can be prepared for each system, more useful kinetic information usually can 

be gained by increasing the number of time points sampled within a system. 

(5) Procedure—(i) Dosing. At test initiation, the test vessel is opened and the 

test substance is quantitatively added directly to the system with constant mixing. It is 

recommended that the dose be administered gradually below the air-water interface, to 

ensure uniform distribution of the test substance in the test medium. The biotic and 

abiotic systems are dosed in an identical manner. Generally, the biotic systems are dosed 

first, followed by the abiotic systems. Exact timing is typically more critical for kinetic 

analyses. 

(ii) Sampling schedule. Sampling times are selected based on existing 

biodegradation data or the results of a pilot study, as no fixed time schedule for sampling 

is universally applicable. A recommended sampling schedule for a rapidly degraded 

substance would be 15, 30 and 60 min, with additional samplings after 2, 5, 8, 12 and 24 

hr, and day 2, 3 and 7 and weekly thereafter. The sampling schedule for slowly degrading 

substances should be adjusted so that a sufficient number of measurements are made 

during the degradation phase. 

(iii) Measurement of mineralization—(A) Indirect measurement of 14CO2. (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 
53 

to allow the dissolved 14CO2 to diffuse from the samples. The samples are combined with a 

scintillation cocktail that is suitable for the sample matrix and analyzed by LSC. The 

percentage of 14CO2 is calculated based on the difference between the total counts in the 

biotic and abiotic samples. 

(B) Direct measurement of 14CO2. (1) Evolved 14CO2: The first base trap in the 

series is removed and quickly capped. The remaining traps are moved forward in the 

same order and a fresh trap placed behind the existing traps, and the trapping system 

reconnected as quickly as possible. Replicate subsamples (e.g. 1 ml) from the base trap 

are removed and transferred to scintillation vials, combined with a scintillation cocktail 

that is suitable for the sample matrix, and analyzed by LSC. 

(2) Dissolved 14CO2: Samples (e.g. 10 to 25 ml) are removed through the 

sampling port of the test flask. They are then placed in vessels (e.g. Bellco Glass 

Biometer 2556-10250) containing a compartment with an appropriate CO2 absorbent (e.g. 

1.5 N KOH). The vessels are sealed 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 (e.g. overnight) to allow 

CO2 to diffuse from solution and be trapped from the headspace by the sorbent. Samples 

of the sorbent are combined with a scintillation cocktail that is suitable for the sample 

matrix and analyzed by LSC. 

(C) Indirect measurement of 3H2O. (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) Half of the samples are immediately analyzed directly by LSC for a wet 

measurement. The remaining samples are allowed to dry completely to remove the 3H2O. 

The samples are combined with a scintillation cocktail that is suitable for the sample 

matrix and analyzed by LSC. The percentage 3H2O is calculated based on the difference 

between the total counts in the wet and dry samples and the initial level of radioactivity 

dosed to the samples. 

(iv) Measurement of total radioactivity in wastewater-surface water mixture. 

Replicate small volume samples (e.g. 1 ml) are analyzed directly by LSC to quantify the 

radioactivity remaining in each system over time. These measurements are used to 

confirm that the recovery of radioactivity from the extracted samples is acceptable and to 

monitor for volatilization. 

(v) Measurement of parent and metabolites—(A) Extraction. (1) A sample is 

collected from both the abiotic and biotic systems. The sample volume is typically >10 

ml, but the size will depend on the test concentration, specific activity and the sensitivity 

of the analytical procedures. 

(2) Various approaches can be used for concentrating and extracting the samples. 

A proven approach for nonvolatile test substances involves flash freezing the samples, 
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. Flash freezing is a quick process 

if there is sufficient depth in the dry ice/acetone or liquid nitrogen bath to submerge the 

sample tube. The level of the bath should be above the sample level in the tube. The 

resulting extracts can be concentrated through evaporation and the total radioactivity in 

each extract determined by LSC. 

(3) For volatile test substances, the sample can be passed through a filter and solid 

phase extraction (SPE) column or SPE disk placed in tandem, which are subsequently 

eluted with appropriate solvents to recover parent and metabolites. Alternatively, it may 

be possible to directly extract aqueous samples with an appropriate solvent system and 

then filter them to recover biomass solids. The total radioactivity in all extracts is 

determined by LSC. Care should be taken in concentrating extracts containing volatile 

test substances or metabolites. 

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

document recoveries and consider the time involved in terminating biological activity, 

and factor it into the sample times used for kinetic analyses. 

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

and metabolites in the extracts can be determined using TLC, HPLC or other separation 

techniques with radioactivity detection. 

(2) If sensitive specific analytical methods are available, primary biodegradation 

can be assessed by measuring the total residual concentration of test substances and 

metabolites instead of using radioisotope techniques. 

(C) Characterization of metabolites. Whenever possible, the chromatographic 

behavior of unknown peaks should be compared to that of predicted metabolites, if 

authentic standards exist. Usually, the quantity and purity of metabolites generated in this 

test make definitive identification by other direct means impossible. Depending on 

chromatographic behavior, it is usually possible to determine if a metabolite is more or 

less polar than the parent. This information, combined with known biochemical reactions 

and knowledge of when a metabolite appears and disappears in the sequence of 

biodegradation, can form an additional basis for inferring its identity. If necessary, the 

Kow of major metabolites can be estimated by HPLC (e.g. [OECD 117](see paragraph 

(o)(24) of this guideline) using an on-line radioactivity detector. 

(vi) Measurement of extracted solids and incorporation into biomass. The 

extracted solids are combusted to determine the level of activity remaining with the 

solids. The level of radioactivity in the biotic solids above that in solids from the abiotic 

control typically represents incorporation of radioactivity into biomass. 

(vii) Measurement of volatilized radioactivity. For volatile test substances, the 

volatility traps are extracted with appropriate solvents and the radioactivity in the extracts 
55 

analyzed by LSC. The relative abundance of parent and metabolites in the extract(s) can 

be determined as described in paragraph (n)(5)(v)(B)(1) of this guideline. 

(o) References.

(1) OECD (1992). Ready Biodegradability, no. 301, adopted 17 July 1992. 

Organization for Economic Cooperation and Development, Paris. 

(2) OECD (2001). Simulation Test - Aerobic Sewage Treatment. 303A: Activated 

Sludge Units. 303B: Biofilms, no. 303, adopted 22 January 2001. Organization for 

Economic Cooperation and Development, Paris. 

(3) OECD (2002). Aerobic and Anaerobic Transformation in Aquatic Sediment 

Systems, no. 308, adopted 24 April 2002. Organization for Economic Cooperation and 

Development, Paris. 

(4) OECD (2004). Aerobic Mineralization in Surface Water – Simulation 

Biodegradation Test, no. 309, adopted 13 April 2004. Organization for Economic 

Cooperation and Development, Paris. 

(5) OECD (2006). Anaerobic Biodegradability of Organic Compounds in 

Digested Sludge: by Measurement of Gas Production, no. 311, adopted 23 March 2006. 

Organization for Economic Cooperation and Development, Paris. 

(6) Matthijs, E., G. Debaere, N.R. Itrich, P. Masscheleyn, A. Rottiers, M. 

Stalmans and T.W. Federle (1995). The fate of detergent surfactants in sewer systems. 

Water Sci. Technol. 13, 321-328. 

(7) Federle, T.W. and N.R. Itrich (1997). A comprehensive approach for assessing 

the kinetics of primary and ultimate biodegradation of chemicals in activated sludge: 

Application to linear alkylbenzene sulfonate (LAS). Environ. Sci. & Technol. 31, 3597-

3603. 

(8) Itrich, N.R. and T.W. Federle (2004). Effect of ethoxylate number and alkyl 

chain length on the pathway and kinetics of linear alcohol ethoxylate biodegradation in 

activated sludge. Environ. Toxicol. Chem. 23, 2790-2798. 

(9) Federle, T.W. and N.R. Itrich (2006). Fate of free and linear alcohol 

ethoxylate derived fatty alcohols in activated sludge. Ecotox. Environ. Safe. 64, 30-41. 

(10) Steber, J. and P. Wierich (1987). The anaerobic degradation of detergent 

range fatty alcohol ethoxylates. Studies with 14C-labeled model surfactants. Water Res. 

21, 661-667. 

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

mineralization of 14C radiolabeled compounds under realistic anaerobic conditions. 
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Environ. Sci. Technol. 30, 3597-3600. 

(12) OECD (1995). Water Solubility, no. 105, adopted 27 July 1995. Organization 

for Economic Cooperation and Development, Paris. 

(13) OECD (1981). Dissociation Constants in Water, no. 112, adopted 12 May 

1981. Organization for Economic Cooperation and Development, Paris. 

(14) OECD (2006). Vapor Pressure, no. 104, adopted 23 March 2006. 

Organization for Economic Cooperation and Development, Paris. 

(15) OECD (2004). Hydrolysis as a function of pH, no. 111, adopted 13 April 

2004. Organization for Economic Cooperation and Development, Paris. 

(16) OECD (1984). Activated sludge, respiration inhibition test, no. 209, adopted 

4 April 1984. Organization for Economic Cooperation and Development, Paris. 

(17) OECD (1981). Inherent Biodegradability: Modified SCAS Test, no. 302A, 

adopted 12 May 1981. Organization for Economic Cooperation and Development, Paris. 

(18) OECD (1992). Inherent Biodegradability: Zahn-Wellens/EMPA Test, no. 

302A, adopted 17 July 1992. Organization for Economic Cooperation and Development, 

Paris. 

(19) OECD (1981). Inherent Biodegradability: Modified MITI Test (II), no. 302C, 

adopted 12 May 1981. Organization for Economic Cooperation and Development, Paris. 

(20) Guidance Document on Estimating Persistence and Degradation Kinetics 

from Environmental Fate Studies on Pesticides in EU Registration (2006). Report of the 

FOCUS Work Group on Degradation Kinetics, EC Document Reference 

Sanco/10058/2005 version 2.0. 434 pp. 

(21) Holman, W.F. (1981). Estimating the Environmental Concentrations of 

Consumer Product Components. Aquatic Toxicology and Hazard Assessment -4th 

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(22) European Commission (2003). Technical Guidance Document on Risk 

Assessment in support of Commission Directive 93/67/EEC on Risk Assessment for new 

notified substances, Commission Regulation (EC) No 1488/94 on Risk Assessment for 

existing substances, and Directive 98/8/EC of the European Parliament and of the 

Council concerning the placing of biocidal products on the market. Part II (Chapter 3), 

Environmental risk assessment, EUR 20418 EN/2. European Commission, Joint Research 

Centre, Ispra, Italy. 

(23) Tchobanoglous, G. and F.L. Burton (1991). Wastewater Engineering: 
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Treatment, Disposal and Reuse/Metcalf & Eddy, Inc. McGraw-Hill, New York, NY. 

(24) OECD (2004). Partition Coefficient (n-octanol/water), High Performance 

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for Economic Cooperation and Development, Paris. 

 

 (25) Sutherland, I.W. and J.F. Wilkinson (1971) In (J.R. Norris and D.W. 

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