Commission Regulation (EC) No 761/2009Show full title

1. METHOD

This method is equivalent to OECD TG 309 (2004) (1).

1.1. INTRODUCTION

The purpose of this test is to measure the time course of biodegradation of a test substance at low concentration in aerobic natural water and to quantify the observations in the form of kinetic rate expressions. This simulation test is a laboratory shake flask batch test to determine rates of aerobic biodegradation of organic substances in samples of natural surface water (fresh, brackish or marine). It is based on the ISO/DIS 14592-1 (2) and it also includes elements from the testing methods C.23 and C.24 (3)(4). Optionally, with long test times, semi-continuous operation replaces batch operation in order to prevent deterioration of the test microcosm. The principal objective of the simulation test is to determine the mineralisation of the test substance in surface water, and mineralisation constitutes the basis for expressing degradation kinetics. However, an optional secondary objective of the test is to obtain information on the primary degradation and the formation of major transformation products. Identification of transformation products, and if possible quantification of their concentrations, are especially important for substances that are very slowly mineralised (e.g. with half-lives for total residual ¹⁴C exceeding 60 days). Higher concentrations of the test substance (e.g. > 100 μg/l) should normally be used for identification and quantification of major transformation products due to analytical limitations.

A low concentration in this test means a concentration (e.g. less than 1 μg/l to 100 μg/l) which is low enough to ensure that the biodegradation kinetics obtained in the test reflect those expected in the environment. Compared to the total mass of biodegradable carbon substrates available in the natural water used for the test, the test substance present at low concentration will serve as a secondary substrate. This implies that the anticipated biodegradation kinetics is first order (‘non-growth’ kinetics) and that the test substance may be degraded by ‘cometabolism’. First order kinetics implies that the rate of degradation (mg/L/day) is proportional to the concentration of substrate which declines over time. With true first order kinetics the specific degradation rate constant, k, is independent of time and concentration. That is, k does not vary appreciably during the course of an experiment and does not change with the added concentration between experiments. By definition, the specific degradation rate constant is equal to the relative change in concentration per time: k = (1/C) · (dC/dt). Although first order kinetics are normally expected under the prescribed conditions, there may be certain circumstances where other kinetics are more appropriate. Deviations from first order kinetics may e.g. be observed if mass transfer phenomena such as the diffusion rate, rather than the biological reaction rate, is limiting the rate of biotransformation. However, the data can nearly always be described by pseudo first order kinetics accepting a concentration dependent rate constant.

Information on biodegradability of the test substance at higher concentrations (e.g. from standard screening tests) as well as information on abiotic degradability, transformation products and relevant physico-chemical properties should be available prior to the test to help establish the experimental planning and interpret the results. The use of ¹⁴C labelled test substances and the determination of the phase distribution of ¹⁴C at the end of the test, enable ultimate biodegradability to be determined. When non-labelled test substance is used, ultimate biodegradation can only be estimated if a higher concentration is tested and all the major transformation products are known.

1.2. DEFINITIONS

Primary biodegradation: The structural change (transformation) of a chemical substance by microorganisms resulting in the loss of chemical identity.

Functional biodegradation: The structural change (transformation) of a chemical substance by microorganisms resulting in the loss of a specific property.

Ultimate aerobic biodegradation: The breakdown of a chemical substance by microorganisms in the presence of oxygen to carbon dioxide, water and mineral salts of any other elements present (mineralisation) and the production of new biomass and organic microbial biosynthesis products.

Mineralisation: The breakdown of a chemical substance or organic matter by microorganisms in the presence of oxygen to carbon dioxide, water and mineral salts of any other elements present.

Lag phase: The time from the start of a test until adaptation of the degrading micro organisms is achieved and the biodegradation degree of a chemical substance or organic matter has increased to a detectable level (e.g. 10 % of the maximum theoretical biodegradation, or lower, dependent on the accuracy of the measuring technique).

Maximum level of biodegradation: The degree of biodegradation of a chemical substance or organic matter in a test, recorded in per cent, above which no further biodegradation takes place during the test.

Primary substrate: A collection of natural carbon and energy sources that provide growth and maintenance of the microbial biomass.

Secondary substrate: A substrate component present in such a low concentration, that by its degradation, only insignificant amounts of carbon and energy are supplied to the competent microorganisms, as compared to the carbon and energy supplied by the degradation of main substrate components (primary substrates).

Degradation rate constant: A first order or pseudo first order kinetic rate constant, k (d^–1), which indicates the rate of degradation processes. For a batch experiment k is estimated from the initial part of the degradation curve obtained after the end of the lag phase.

Half-life, t_1/2 (d): Term used to characterise the rate of a first order reaction. It is the time interval that corresponds to a concentration decrease by a factor 2. The half-life and the degradation rate constant are related by the equation t_1/2 = ln2/k.

Degradation half time, DT₅₀ (d): Term used to quantify the outcome of biodegradation tests. It is the time interval, including the lag phase, needed to reach a value of 50 % biodegradation.

Limit of detection (LOD) and limit of quantification (LOQ): The limit of detection (LOD) is the concentration of a substance below which the identity of the substance cannot be distinguished from analytical artefacts. The limit of quantification (LOQ) is the concentration of a substance below which the concentration cannot be determined with an acceptable accuracy.

Dissolved organic carbon (DOC): That part of the organic carbon in a sample of water which cannot be removed by specified phase separation, for example by centrifugation at 40 000 ms^–2 for 15 min. or by membrane filtration using membranes with pores of 0,2 μm-0,45 μm diameter.

Total organic ¹⁴C activity (TOA): The total ¹⁴C activity associated with organic carbon.

Dissolved organic ¹⁴C activity (DOA): The total ¹⁴C activity associated with dissolved organic carbon.

Particulate organic ¹⁴C activity (POA): The total ¹⁴C activity associated with particulate organic carbon.

1.3. APPLICABILITY OF THE TEST

This simulation test is applicable to non-volatile or slightly volatile organic substances tested at low concentrations. Using flasks open to the atmosphere (e.g. cotton wool plugged), substances with Henry’s law constants less than about 1 Pa·m³/mol (approx. 10^–5 atm·m³/mol) can be regarded as non-volatile in practice. Using closed flasks with a headspace, it is possible to test slightly volatile substances (with Henry’s law constants < 100 Pa·m³/mol or < 10^–3 atm·m³/mol) without losses from the test system. Loss of ¹⁴C-labelled substances may occur, if the right precautions are not exercised, when the CO₂ is stripped off. In such situations, it may be necessary to trap CO₂ in an internal absorber with alkali or to use an external CO₂ absorber system (direct ¹⁴CO₂ determination; see Appendix 3). For the determination of biodegradation kinetics, the concentrations of the test substance must be below its water solubility. It should be noted, however, that literature values of water solubility may be considerably higher than the solubility of the test substance in natural waters. Optionally, the solubility of especially poorly water-soluble test substances may be established by use of the natural waters being tested.

The method can be used for simulating biodegradation in surface water free of coarse particles (pelagic test) or in turbid surface water which, e.g. might exist near a water/sediment interface (suspended sediment test).

1.4. PRINCIPLE OF THE TEST

The test is performed in batch by incubating the test substance with either surface water only (pelagic test) or surface water amended with suspended solids/sediment of 0,01 to 1 g/L dry weight (suspended sediment test) to simulate a water body with suspended solids or re-suspended sediment. The suspended solids/sediment concentration in the lower range of this interval is typical for most surface waters. The test flasks are incubated in darkness at an environmental temperature under aerobic conditions and agitation. At least two different concentrations of test substance should be used in order to determine the degradation kinetics. The concentrations should differ from each other by a factor of 5 to 10 and should represent the expected range of concentrations in the environment. The maximum concentration of the test substance should not exceed 100 µg/L, but maximum test concentrations below 10 µg/L or less are preferred to ensure that the biodegradation follows first order kinetics. The lowest concentration should not exceed 10 µg/L, but lowest test concentrations of 1-2 μg/L or less than 1 μg/L are preferred. Normally an adequate analysis of such low concentration can be achieved by use of commercially available ¹⁴C-labelled substances. Because of analytical limitations, it is frequently impossible to measure the concentration of the test substance with the required accuracy, if the test substance is applied at a concentration ≤ 100 µg/L (see second paragraph in section 1.7.2). Higher concentrations of test substance (> 100 µg/L and sometimes > 1 mg/L) may be used for the identification and quantification of major transformation products or if a specific analysis method with a low detection limit is not available. If high concentrations of test substance are tested, it may not be possible to use the results to estimate the first order degradation constant and half-life, as the degradation will probably not follow first order kinetics.

Degradation is followed at appropriate time intervals, by measuring either the residual ¹⁴C or the residual concentration of test substance when specific chemical analysis is used. ¹⁴C labelling of the most stable part of the molecule ensures the determination of the total mineralisation, while ¹⁴C labelling of a less stable part of the molecule, as well as the use of specific analysis, enable the assessment of only primary biodegradation. However, the most stable part does not necessarily include the relevant functional moiety of the molecule (that can be related to a specific property such as toxicity, bioaccumulation, etc.). If this is the case, it may be appropriate to use a test substance, which is ¹⁴C-labelled, in the functional part in order to follow the elimination of the specific property.

1.5. INFORMATION ON THE TEST SUBSTANCE

Both radiolabelled and non-labelled test substances can be used in this test. ¹⁴C-labelling technique is recommended and labelling should normally be in the most stable part(s) of the molecule (see also section 1.4). For substances containing more than one aromatic ring, one or more carbons in each ring should preferably be ¹⁴C-labelled. In addition, one or more carbons on both sides of easily degradable linkages should preferably be ¹⁴C-labelled. The chemical and/or radiochemical purity of the test substance should be > 95 %. For radiolabelled substances, a specific activity of approx. 50 μCi/mg (1,85 MBq) or more is preferred in order to facilitate ¹⁴C measurements in tests conducted with low initial concentrations. The following information on the test substance should be available:

• solubility in water [Method A.6],

• solubility in organic solvent(s) (substances applied with solvent or with low solubility in water),

• dissociation constant (pKa) if the substance is liable to protonation or deprotonation [OECD TG 112] (5),

• vapour pressure [Method A.4] and Henry’s law constant,

• chemical stability in water and in the dark (hydrolysis) [Method C.7].

When poorly water-soluble substances are being tested in seawater, it may also be useful to know the salting out constant (or ‘Setschenow constant’) K^s, which is defined by the expression: log (S/S’) = K^s C_m, where S and S’ are the solubility of the substance in fresh water and seawater, respectively, and C_m is the molar salt concentration.

If the test is carried out as a ‘suspended sediment test’ the following information should also be available:

• n-octanol/water partition coefficient [Method A.8],

• adsorption coefficient [Method C.18].

Other useful information may include:

• environmental concentration, if known or estimated,

• toxicity of the test substance to microorganisms [Method C.11],

• ready and/or inherent biodegradability [Methods C.4 A-F, C.12, C.9, OECD TG 302 (5)],

• aerobic or anaerobic biodegradability in soil and sediment/water transformation studies [Methods C.23, C.24].

1.6. REFERENCE SUBSTANCE

A substance, which is normally easily degraded under aerobic conditions (e.g. aniline or sodium benzoate) should be used as reference substance. The expected time interval for degradation of aniline and sodium benzoate is usually less than 2 weeks. The purpose of the reference substances is to ensure that the microbial activity of the test water is within certain limits; i.e. that the water contains an active microbial population.

1.7. QUALITY CRITERIA

1.7.1. Recovery

Immediately after addition of the test substance, each initial test concentration should be verified by measurements of ¹⁴C activity, or by chemical analyses in the case of non-labelled substances, in at least duplicate samples. This provides information on the applicability and repeatability of the analytical method and on the homogeneity of the distribution of the test substance. Normally, the measured initial ¹⁴C activity or test substance concentration is used in the subsequent analyses of data rather than the nominal concentration as losses due to sorption and dosing errors thereby are compensated. For ¹⁴C-labelled test substance, the level of recovery at the end of the experiment is given by mass balance (see last paragraph in section 1.8.9.4). Ideally, the radiolabelled mass balance should range from 90 % to 110 %, whereas the analytical accuracy should lead to an initial recovery of between 70 % and 110 % for non-labelled test substances. These ranges should be interpreted as targets and should not be used as criteria for acceptance of the test. Optionally, the analytical accuracy may be determined for the test substance at a lower concentration than the initial concentration and for major transformation products.

1.7.2. Repeatability and sensitivity of analytical method

Repeatability of the analytical method (including the efficiency of the initial extraction) to quantify the test substance, and transformation products, if appropriate, should be checked by five replicate analyses of the individual extracts of the surface water.

The limit of detection (LOD) of the analytical method for the test substance and for the transformation products should be at least 1 % of the initial amount applied to the test system if possible. The limit of quantification (LOQ) should be equal to or less than 10 % of the applied concentration. The chemical analyses of many organic substances and their transformation products frequently require that the test substance is applied at a relatively high concentration, i.e. > 100 μg/L.

1.8. DESCRIPTION OF THE TEST METHOD

1.8.1. Equipment

The test may be conducted in conical or cylindrical flasks of appropriate capacity (e.g. 0,5 or 1,0 litre) closed with silicone or rubber stoppers, or in serum flasks with CO₂-tight lids (e.g. with butyl rubber septa). Another option is to perform the test by use of multiple flasks and to harvest whole flasks, at least in duplicate, at each sample interval (see last paragraph in section 1.8.9.1). For non-volatile test substances that are not radiolabelled, gas-tight stoppers or lids are not required; loose cotton plugs that prevent contamination from air are suitable (see second paragraph in section 1.8.9.1). Slightly volatile substances should be tested in a biometer-type system with gentle stirring of the water surface. To be sure that no bacterial contamination occurs, optionally the vessels can be sterilised by heating or autoclaving prior to use. In addition, the following standard laboratory equipment is used:

• shaking table or magnetic stirrers for continuous agitation of the test flasks,

• centrifuge,

• pH meter,

• turbidimeter for nephelometric turbidity measurements,

• oven or microwave oven for dry weight determinations,

• membrane filtration apparatus,

• autoclave or oven for heat sterilisation of glassware,

• facilities to handle ¹⁴C-labelled substances,

• equipment to quantify ¹⁴C-activity in samples from CO₂-trapping solutions and, if required, from sediment samples,

• analytical equipment for the determination of the test (and reference) substance if specific chemical analysis is used (e.g. gas chromatograph, high-pressure liquid chromatograph).

1.8.2. Stock solutions of test substance

Deionised water is used to prepare stock solutions of the test and reference substances (see first paragraph in section 1.8.7). The deionised water should be free of substances that may be toxic to microorganisms, and dissolved organic carbon (DOC) should be no more than 1 mg/L (6).

1.8.3. Collection and transport of surface water

The sampling site for collection of the surface water should be selected in accordance with the purpose of the test in any given situation. In selecting sampling sites, the history of possible agricultural, industrial or domestic inputs must be considered. If it is known that an aquatic environment has been contaminated with the test substance or its structural analogues within the previous four years, it should not be used for the collection of test water, unless investigation of degradation rates in previously exposed sites is the express purpose of the investigator. The pH and temperature of the water should be measured at the site of collection. Furthermore, the depth of sampling and the appearance of the water sample (e.g. colour and turbidity) should be noted (see section 3). Oxygen concentration and/or redox potential in water and in the sediment surface layer should be measured in order to demonstrate aerobic conditions unless this is obvious as judged from appearance and historic experience with the site. The surface water should be transported in a thoroughly cleansed container. During transport, the temperature of the sample should not significantly exceed the temperature used in the test. Cooling to 4 °C is recommended if transport duration exceeds 2 to 3 hours. The water sample must not be frozen.

1.8.4. Storage and preparation of surface water

The test should preferably be started within one day after sample collection. Storage of the water, if needed, should be minimised and must in any case not exceed a maximum of 4 weeks. The water sample should be kept at 4 °C with aeration until use. Prior to use, the coarse particles should be removed, e.g. by filtration through a nylon filter with about 100 μm mesh size or with a coarse paper filter, or by sedimentation.

1.8.5. Preparation of water amended with sediment (optional)

For the suspended sediment test, surface sediment is added to the flasks containing natural water (filtered to remove coarse particles as described in section 1.8.4) to obtain a suspension; the concentration of suspended solids should be between 0,01 and 1 g/L. The surface sediment should come from the same site as that from which the water sample was taken. Dependent on the particular aquatic environment, the surface sediment may either be characterised by a high organic carbon content (2,5-7,5 %) and a fine texture or by a low organic carbon content (0,5-2,5 %) and a coarse texture (3). The surface sediment can be prepared as follows: extract several sediment cores using a tube of transparent plastic, slice off the upper aerobic layers (from surface to a depth of max. 5 mm) immediately after sampling and pool them together. The resulting sediment sample should be transported in a container with a large air headspace to keep the sediment under aerobic conditions (cool to 4 °C if transport duration exceeds 2-3 hours). The sediment sample should be suspended in the test water at a ratio of 1:10 and kept at 4 °C with aeration until use. Storage of the sediment, if needed, should be minimised and must not in any case exceed a maximum of 4 weeks.

1.8.6. Semi-continuous procedure (optional)

Prolonged incubation (several months) may be necessary if a long lag time occurs before a significant degradation of the test substance can be measured. If this is known from previous testing of a substance, the test may be initiated by using a semi-continuous procedure, which allows periodical renewal of a part of the test water or suspension (see Appendix 2). Alternatively, the normal batch test may be changed into a semi-continuous test, if no degradation of the test substance has been achieved during approximately 60 days of testing using the batch procedure (see second paragraph in section 1.8.8.3).

1.8.7. Addition of the test (or reference) substance

For substances with high water solubility (> 1 mg/L) and low volatility (Henry’s law constants < 1 Pa·m³/mol or < 10^–5 atm·m³/mol), a stock solution can be prepared in deionised water (see section 1.8.2); the appropriate volume of the stock solution is added to the test vessels to achieve the desired concentration. The volume of any added stock solution should be held to the practical minimum (< 10 % of the final liquid volume, if possible). Another procedure is to dissolve the test substance in a larger volume of the test water, which may be seen as an alternative to the use of organic solvents.

If unavoidable, stock solutions of non-volatile substances with poor water-solubility should be prepared by use of a volatile organic solvent, but the amount of solvent added to the test system should not exceed 1 % v/v and should not have adverse effects on the microbial activity. The solvent should not affect the stability of the test substance in water. The solvent should be stripped off to an extremely small quantity so that it does not significantly increase the DOC concentration of the test water or suspension. This should be checked by substance-specific analysis or, if possible, DOC analysis (6). Care must be taken to limit the amount of solvent transferred to what is absolutely necessary, and to ensure that the amount of test substance can dissolve in the final volume of test water. Other techniques to introduce the test substance into the test vessels may be used as described in (7) and (8). When an organic solvent is used for application of the test substance, solvent controls containing the test water (with no additions) and test water with added reference substance should be treated similarly to active test vessels amended with test substance in solvent carrier. The purpose of the solvent controls is to examine possible adverse effects caused by the solvent towards the microbial population as indicated by the degradation of the reference substance.

1.8.8. Test conditions

1.8.8.1. Test temperature

Incubation should take place in the dark (preferred) or in diffuse light at a controlled (± 2 °C) temperature, which may be the field temperature or a standard temperature of 20-25 °C. Field temperature may be either the actual temperature of the sample at the sampling time or an average field temperature at the sampling site.

1.8.8.2. Agitation

Agitation by means of continuous shaking or stirring must be provided to maintain particles and microorganisms in suspension. Agitation also facilitates oxygen transfer from the headspace to the liquid so that aerobic conditions can be adequately maintained. Place the flasks on a shaking table (approx. 100 rpm agitation) or use magnetic stirring. Agitation must be continuous. However, the shaking or stirring should be as gentle as possible, while still maintaining a homogeneous suspension.

1.8.8.3. Test duration

The duration of the test should normally not exceed 60 days unless the semi-continuous procedure with periodical renewal of the test suspension is applied (see section 1.8.6 and Appendix 2). However, the test period for the batch test may be extended to a maximum of 90 days, if the degradation of the test substance has started within the first 60 days. Degradation is monitored, at appropriate time intervals, by the determination of the residual ¹⁴C activity or the evolved ¹⁴CO₂ (see section 1.8.9.4) and/or by chemical analysis (section 1.8.9.5). The incubation time must be sufficiently long to evaluate the degradation process. The extent of degradation should preferably exceed 50 %; for slowly degradable substances, the extent of degradation must be sufficient (normally greater than 20 % degradation) to ensure the estimation of a kinetic degradation rate constant.

Periodic measurements of pH and oxygen concentration in the test system must be conducted unless previous experience from similar tests with water and sediment samples collected from the same site make such measurements unnecessary. Under some conditions, the metabolism of primary substrates at much higher concentrations within the water or sediment could possibly result in enough CO₂ evolution and oxygen depletion to significantly alter the experimental conditions during the test.

1.8.9. Procedure

1.8.9.1. Preparation of flasks for pelagic test

Transfer a suitable volume of test water to the test flasks, up to about one third of the flask volume and not less than about 100 ml. If multiple flasks are used (to allow harvesting of whole flasks at each sampling time), the appropriate volume of test water is also about 100 ml, as small sample volumes may influence the length of the lag phase. The test substance is added from a stock solution as described in sections 1.8.2 and 1.8.7. At least two different concentrations of test substance differing by a factor of 5 to 10 should be used in order to determine degradation kinetics and calculate the kinetic degradation rate constant. Both of the selected concentrations should be less than 100 µg/L and preferably in the range of < 1-10 µg/L.

Close the flasks with stoppers or lids impermeable to air and CO₂. For non-¹⁴C-labelled non-volatile test chemicals, loose cotton wool plugs that prevent contamination from air are suitable (see section 1.8.1) provided that any major degradation products are known to be non-volatile, and if indirect CO₂ determination is used (see Appendix 3).

Incubate the flasks at the selected temperature (see section 1.8.8.1). Withdraw samples for chemical analysis or ¹⁴C measurements at the beginning of the test (i.e. before biodegradation starts; see section 1.7.1) and then at suitable time intervals during the course of the test. Sampling may be performed by withdrawal of sub-samples (e.g. 5 ml aliquots) from each replicate or by harvest of whole flasks at each sampling time. The mineralisation of the test substance may either be determined indirectly or directly (see Appendix 3). Usually, a minimum of five sampling points are required during the degradation phase (i.e. after ended lag phase) in order to estimate a reliable rate constant, unless it can be justified that three sampling points are sufficient for rapidly degradable substances. For substances that are not rapidly degraded more measurements during the degradation phase can easily be made and, therefore, more data points should be used for the estimation of k. No fixed time schedule for sampling can be stated, as the rate of biodegradation varies; however the recommendation is to sample once a week if degradation is slow. If the test substance is rapidly degradable, sampling should take place once a day during the first three days and then every second or third day. Under certain circumstances, such as with very rapidly hydrolysing substances, it may be necessary to sample at hourly intervals. It is recommended that a preliminary study is conducted prior to the test in order to determine the appropriate sampling intervals. If samples have to be available for further specific analysis, it is advisable to take more samples and then select those to be analysed at the end of the experiment following a backwards strategy, i.e. the last samples are analysed first (see second paragraph in section 1.8.9.5 for guidance on stability of samples during storage).

1.8.9.2. Number of flasks and samples

Set up a sufficient number of test flasks to have:

• test flasks; at least duplicate flasks for each concentration of test substance (preferably a minimum of 3) or multiple test flasks for each concentration, if whole flasks are harvested at each sampling time (symbolised F_T),

• test flasks for mass balance calculation; at least duplicate flasks for each test concentration (symbolised F_M),

• blank control, no test substance; at least one blank test flask containing only the test water (symbolised F_B),

• reference control; duplicate flasks with reference substance (e.g. aniline or sodium benzoate, at 10 µg/l) (symbolised F_C). The purpose of the reference control is to confirm a minimum of microbial activity. If convenient, a radiolabelled reference substance may be used, also when the degradation of the test substance is monitored by chemical analyses,

• sterile control; one or two flasks containing sterilised test water for examining possible abiotic degradation or other non-biological removal of the test substance (symbolised F_S). The biological activity can be stopped by autoclaving (121 °C; 20 min.) the test water or by adding a toxicant (e.g. sodium azide (NaN₃) at 10-20 g/l, mercuric chloride (HgCl₂) at 100 mg/l or formalin at 100 mg/l) or by gamma irradiation. If HgCl₂ is used, it should be disposed of as toxic waste. For water with sediment added in large amount, sterile conditions are not easy to obtain; in this case repeated autoclaving (e.g. three times) is recommended. It should be considered that the sorption characteristics of the sediment may be altered by autoclaving,

• solvent controls, containing test water and test water with reference substance; duplicate flasks treated with the same amount of solvent and by use of the same procedure as that used for application of the test substance. The purpose is to examine possible adverse effects of the solvent by determining the degradation of the reference substance.

In the design of the test, the investigator should consider the relative importance of increased experimental replication versus increased number of sampling times. The exact number of flasks required will depend on the method used for measuring the degradation (see third paragraph in section 1.8.9.1; section 1.8.9.4 and Appendix 3).

Two subsamples (e.g. 5 ml aliquots) should be withdrawn from each test flask at each sampling time. If multiple flasks are used to allow harvesting of whole flasks, a minimum of two flasks should be sacrificed at each sampling time (see first paragraph in section 1.8.9.1).

1.8.9.3. Preparation of flasks for suspended sediment test [optional]

Add the necessary volumes of test water and sediment, if required, to the test vessels (see section 1.8.5). The preparation of flasks for suspended sediment test is the same as for the pelagic test (see sections 1.8.9.1 and 1.8.9.2). Use preferably serum bottles or similar shaped flasks. Place the closed flasks horizontally on a shaker. Obviously, open flasks for non-¹⁴C-labelled, non-volatile substances should be placed in upright position; in this case magnetic stirring and the use of magnetic bars coated with glass are recommended. If necessary, aerate the bottles to maintain proper aerobic conditions.

1.8.9.4. Radiochemical determinations

The evolved ¹⁴CO₂ is measured indirectly and directly (see Appendix 3). The ¹⁴CO₂ is determined indirectly by the difference between the initial ¹⁴C activity in the test water or suspension and the total residual activity at the sampling time as measured after acidifying the sample to pH 2-3 and stripping off CO₂. Inorganic carbon is thus removed and the residual activity measured derives from organic material. The indirect ¹⁴CO₂ determination should not be used, if major volatile transformation products are formed during the transformation of the test substance (see Appendix 3). If possible, the ¹⁴CO₂ evolution should be measured directly (see Appendix 3) at each sampling time in at least one test flask; this procedure enables both the mass balance and biodegradation process to be checked, but it is restricted to tests conducted with closed flasks.

If the evolved ¹⁴CO₂ is measured directly during the test, more flasks should be set up for this purpose at the start of the test. Direct ¹⁴CO₂ determination is recommended, if major volatile transformation products are formed during the transformation of the test substance. At each measuring point the additional test flasks are acidified to pH 2-3 and the ¹⁴CO₂ is collected in an internal or external absorber (see Appendix 3).

Optionally the concentrations of ¹⁴C-labelled test substance and major transformation products may be determined by use of radiochromatography (e.g. thin layer chromatography, RAD-TLC) or HPLC with radiochemical detection.

Optionally the phase distribution of the remaining radioactivity (see Appendix 1) and residual test substance and transformation products may be determined.

At the end of the test the mass balance should be determined by direct ¹⁴CO₂ measurement using separate test flasks from which no samples are taken in the course of the test (see Appendix 3).

1.8.9.5. Specific chemical analysis

If a sensitive specific analytical method is available, primary biodegradation can be assessed by measuring the total residual concentration of test substance instead of using radiolabelling techniques. If a radiolabelled test substance is used (to measure total mineralisation), specific chemical analyses can be made in parallel to provide useful additional information and check the procedure. Specific chemical analyses may also be used to measure transformation products formed during the degradation of the test substance, and this is recommended for substances that are mineralised with half-lives exceeding 60 days. The concentration of the test substance and the transformation products at every sampling time should be measured and reported (as a concentration and as percentage of applied). In general, transformation products detected at ≥ 10 % of the applied concentration at any sampling time should be identified unless reasonably justified otherwise. Transformation products for which concentrations are continuously increasing during the study should also be considered for identification, even if their concentrations do not exceed the limit given above, as this may indicate persistence. Analyses of transformation products in sterile controls should be considered, if rapid abiotic transformation of the test substance (e.g. hydrolysis) is thought possible. The need for quantification and identification of transformation products should be considered on a case by case basis, with justifications being provided in the report. Extraction techniques with organic solvent should be applied according to directions given in the respective analytical procedure.

All samples should be stored at 2 to 4 °C and air-tight if analysis is carried out within 24 hours (preferred). For longer storage, the samples should be frozen below – 18 °C or chemically preserved. Acidification is not a recommended method to preserve the samples, because acidified samples may be unstable. If the samples are not analysed within 24 hours and are subject to longer storage, a storage stability study should be conducted to demonstrate the stability of chemicals of interest under – 18 °C storage or preserved conditions. If the analytical method involves either solvent extraction or solid phase extraction (SPE), the extraction should be performed immediately after sampling or after storing the sample refrigerated for a maximum of 24 hours.

Depending on the sensitivity of the analytical method, larger sample volumes than those indicated in section 1.8.1 may be necessary. The test can easily be carried out with test volumes of one litre in flasks of 2-3 litre volume, which makes it possible to collect samples of approx. 100 ml.