Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (Text with EEA relevance)

• 1.This test method is equivalent to OECD test guideline (TG) 209 (2010). This test method describes a method to determine the effects of a chemical on micro-organisms from activated sludge (largely bacteria) by measuring their respiration rate (carbon and/or ammonium oxidation) under defined conditions in the presence of different concentrations of the test chemical. The test method is based on the ETAD (Ecological and Toxicological Association of the Dyestuffs Manufacturing industry) test (1) ( 2), on the previous OECD TG 209 (3) and on the revised ISO Standard 8192 (4). The purpose of the test is to provide a rapid screening method to assess the effects of chemicals on the microorganisms of the activated sludge of the biological (aerobic) stage of waste-water treatment plants. The results of the test may also serve as an indicator of suitable non-inhibitory concentrations of test chemicals to be used in biodegradability tests (for example Chapters C.4 A-F, C.9, C.10, C12 and C.29 of this Annex, OECD TG302C). In this case, the test can be performed as a screening test, similar to a range-finding or limit test (see paragraph 39), considering the overall respiration only. However, this information should be taken with care for ready biodegradability tests (Chapter C.4 A-F and C.29 of this Annex) for which the inoculum concentration is significantly lower than the one used in this test method. Indeed, an absence of inhibition in this respiration test does not automatically result in non-inhibitory conditions in the ready biodegradability test of Chapters C.4 A-F or C.29 of this Annex.

• 2.Overall, the respiration inhibition test seems to have been applied successfully since it was first published, but on some occasions spurious results were reported, e.g. (2) (4) (5). Concentration related respiration curves are sometimes bi-phasic, dose-response plots have been distorted and EC50 values have been unexpectedly low (5). Investigations showed that such results are obtained when the activated sludge used in the test nitrifies significantly and the test chemical has a greater effect on the oxidation of ammonium than on general heterotrophic oxidation. Therefore, these spurious results may be overcome by performing additional testing using a specific inhibitor of nitrification. By measuring the oxygen uptake rates in the presence and absence of such an inhibitor, e.g. N-allylthiourea (ATU), the separate total, heterotrophic and nitrification oxygen uptake rates can be calculated (4) (7) (8). Thus, the inhibitory effects of a test chemical on the two processes may be determined and the EC50 values for both organic carbon oxidation (heterotrophic) and ammonium oxidation (nitrification) may be calculated in the usual way. It should be noted that in some rare cases, the inhibitory effect of N-allylthiourea may be partially or completely nullified as a result of complexation with test chemicals or medium supplements, e.g. Cu⁺⁺ ions (6). Cu⁺⁺ ions are essential for Nitrosomonas, but are toxic in higher concentration.

• 3.The need for nitrification in the aerobic treatment of wastewaters, as a necessary step in the process of removing nitrogen compounds from wastewaters by denitrification to gaseous products, has become urgent particularly in European countries; the EU has now set lower limits for the concentration of nitrogen in treated effluents discharged to receiving waters ([^E0077]).

• 4.For most purposes, the method to assess the effect on organic carbon oxidation processes alone is adequate. However, in some cases an examination of the effect on nitrification alone, or on both nitrification and organic carbon oxidation separately, are needed for the interpretation of the results and understanding the effects.

PRINCIPLE OF THE TEST METHOD

• 5.The respiration rates of samples of activated sludge fed with synthetic sewage are measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. Under consideration of the realistic exposure scenario, longer contact times could be appropriate. If the test chemical is rapidly degraded e.g. abiotically via hydrolysis, or is volatile and the concentration cannot be adequately maintained, additionally a shorter exposure period e.g. 30 minutes can be used. The sensitivity of each batch of activated sludge should be checked with a suitable reference chemical on the day of exposure. The test is typically used to determine the ECx (e.g. EC50) of the test chemical and/or the no-observed effect concentration (NOEC).

• 6.The inhibition of oxygen uptake by micro-organisms oxidising organic carbon may be separately expressed from that by micro-organisms oxidising ammonium by measurement of the rates of uptake of oxygen in the absence and presence of N-allylthiourea, a specific inhibitor of the oxidation of ammonium to nitrite by the first-stage nitrifying bacteria. In this case the percentage inhibition of the rate of oxygen uptake is calculated by comparison of the rate of oxygen uptake in the presence of a test chemical with the mean oxygen uptake rate of the corresponding controls containing no test chemical, both in the presence and absence of the specific inhibitor, N-allylthiourea.

• 7.Any oxygen uptake arising from abiotic processes may be detected by determining the rate in mixtures of test chemical, synthetic sewage medium and water, omitting activated sludge.

INFORMATION OF THE TEST CHEMICAL

• 8.The identification (preferably CAS number), name (IUPAC), purity, water solubility, vapour pressure, volatility and adsorption characteristics of the test chemical should be known to enable correct interpretation of results to be made. Normally, volatile chemicals cannot be tested adequately unless special precautions are taken (see paragraph 21).

APPLICABILITY OF THE TEST METHOD

• 9.The test method may be applied to water-soluble, poorly soluble and volatile chemicals. However, it may not always be possible to obtain EC50 values with chemicals of limited solubility and valid results with volatile chemicals may only be obtained providing that the bulk (say > 80 %) of the test chemical remains in the reaction mixture at the end of the exposure period(s). Additional analytical support data should be submitted to refine the ECx concentration when there is any uncertainty regarding the stability of the test chemical or its volatility.

REFERENCE CHEMICALS

• 10.Reference chemicals should be tested periodically in order to assure that the test method and test conditions are reliable, and to check the sensitivity of each batch of activated sludge used as microbial inoculum on the day of exposure. The chemical 3,5-dichlorophenol (3,5-DCP) is recommended as the reference inhibitory chemical, since it is a known inhibitor of respiration and is used in many types of test for inhibition/toxicity (4). Also copper (II) sulphate pentahydrate can be used as a reference chemical for the inhibition of total respiration (9). N-methylaniline can be used as a specific reference inhibitor of nitrification (4).

VALIDITY CRITERIA AND REPRODUCIBILITY

• 11.The blank controls (without the test chemical or reference chemical) oxygen uptake rate should not be less than 20 mg oxygen per one gramme of activated sludge (dry weight of suspended solids) in an hour. If the rate is lower, the test should be repeated with washed activated sludge or with the sludge from another source. The coefficient of variation of oxygen uptake rate in control replicates should not be more than 30 % at the end of definitive test.

• 12.In a 2004 international ring test organised by ISO (4) using activated sludge derived from domestic sewage, the EC50 of 3,5-DCP was found to lie in the range 2 mg/l to 25 mg/l for total respiration, 5 mg/l to 40 mg/l for heterotrophic respiration and 0,1 mg/l to 10 mg/l for nitrification respiration. If the EC50 of 3,5-DCP does not lie in the expected range, the test should be repeated with activated sludge from another source. The EC50 of copper (II) sulphate pentahydrate should lie in the range of 53-155 mg/l for the total respiration (9).

DESCRIPTION OF THE TEST METHOD

Test vessels and apparatus

• 13.Usual laboratory equipment and the following should be used:

• (a) Test vessels — for example, 1 000 ml beakers to contain 500 ml of reaction mixture (see 5 in Fig.1);

• (b) Cell and attachments for measuring concentration of dissolved oxygen; a suitable oxygen electrode; an enclosed cell to contain the sample with no headspace and a recorder (e.g. 7, 8, 9 in Fig.1 of Appendix 2); alternatively, a BOD bottle may be used with a suitable sleeve adaptor for sealing the oxygen electrode against the neck of the bottle (see Fig. 2 of Appendix 3). To avoid loss of displaced liquid on insertion of the oxygen electrode, it is advisable first to insert a funnel or glass tube through the sleeve, or to use vessels with flared-out rims. In both cases a magnetic stirrer or alternative stirrer method, e.g. self-stirring probe, should be used;

• (c) Magnetic stirrers and followers, covered with inert material, for use in measurement chamber and/or in the test vessels;

• (d) Aeration device: if necessary, compressed air should be passed through an appropriate filter to remove dust and oil and through wash bottles containing water to humidify the air. The contents of vessels should be aerated with Pasteur pipettes, or other aeration devices, which do not adsorb chemicals. An orbital shaker operated at orbiting speeds between 150 and 250 rpm with flasks of, for example, 2 000 ml capacity, can be used to satisfy the oxygen demand for the sludge and overcome difficulties with chemicals that produce excessive foam, are volatile and therefore lost, or are difficult to disperse when aerated by air sparging. The test system is typically a number of beakers aerated continuously and sequentially established (e.g. at ca. 10 - 15 minute intervals), then analysed in a sequential manner. Validated instrumentation that allows the simultaneous aeration and measurement of the oxygen consumption rate in the mixtures may also be used;

• (e) pH-meter;

• (f) Centrifuge, general bench-top centrifuge for sludge capable of 10 000 m/s².

Reagents

• 14.Analytical grade reagents should be used throughout.

Water

• 15.Distilled or deionised water, containing less than 1 mg/l DOC, should be used except where chlorine free tap water is specified.

Synthetic sewage feed

• 16.The medium should be prepared to contain the following constituents at the stated amounts:

—  peptone	16 g
—  meat extract (or a comparable vegetable extract)	11 g
—  urea	3 g
—  sodium chloride (NaCl)	0,7 g
—  calcium chloride dihydrate (CaC12, 2H2O)	0,4 g
—  magnesium sulphate heptahydrate (MgSO4, 7H2O)	0,2 g
—  anhydrous potassium monohydrogen phosphate (K2HPO4)	2. 8g
—  distilled or deionised water to 1 litre

• 17.The pH of this solution should be 7,5 ± 0,5. If the prepared medium is not used immediately, it should be stored in the dark at 0 °C to 4 °C, for no longer than 1 week or under conditions, which do not change its composition. It should be noted that this synthetic sewage is a 100 fold concentrate of that described in the OECD Technical Report ‘Proposed method for the determination of the biodegradability of surfactants used in synthetic detergents’June 11, 1976, with moreover dipotassium hydrogen phosphate added.

• 18.Alternatively, components of the medium can be sterilised individually prior to storage, or the peptone and meat extract can be added shortly before carrying out the test. Prior to use, the medium should be thoroughly mixed and the pH adjusted if necessary to pH 7,5 ± 0,5.

Test chemical

• 19.A stock solution should be prepared for readily water soluble test substances up to the maximum water solubility only (precipitations are not acceptable). Poorly water soluble substances, mixtures with components of different water solubility and adsorptive substances should be directly weighed into the test vessels. In these cases, use of stock solutions may be an alternative if dissolved concentrations of the test chemicals are analytically determined in the test vessels (prior to adding activated sludge). If water accommodated fractions (WAFs) are prepared, an analytical determination of the dissolved concentrations of the test chemicals in the test vessels is also essential. Using organic solvents, dispersants/emulsifiers to improve solubility should be avoided. Ultrasonication of stock solutions and pre-stirring suspensions, e.g. overnight, is possible when there is adequate information available concerning the stability of the test chemical under such conditions.

• 20.The test chemical may adversely affect pH within the test system. The pH of the test chemical-treated mixtures should be determined prior to the test set up, in a preliminary trial, to ascertain whether pH adjustment will be necessary prior the main test and again on the day of the main test. Solutions/suspensions of test chemical in water should be neutralised prior to inoculum addition, if necessary. However, since neutralisation may change the chemical properties of the chemical, further testing, depending on the purposes of the study, could be performed to assess the effect of the test chemical on the sludge without pH adjustment.

• 21.The toxic effects of volatile chemicals, especially in tests in which air is bubbled through the system, can result in variable effect levels occurring owing to losses of the substance during the exposure period. Caution should be exercised with such substances by performing substance specific analysis of control mixtures containing the substance and modifying the aeration regime.

Reference chemical

• 22.If 3,5-dichlorophenol is used as reference chemical, a solution of 1,00 g of 3,5-dichlorophenol in 1 000 ml of water should be prepared (15). Warm water and/or ultrasonication should be used to accelerate the dissolution and make the solution up to volume when it has cooled to room temperature. However, it should be ensured that the reference chemical is not structurally changed. The pH of the solution should be checked and adjusted, if necessary, with NaOH or H2SO4 to pH 7 - 8.

• 23.If copper(II)sulphate pentahydrate is used as a reference chemical, concentrations of 58 mg/l, 100 mg/l and 180 mg/l (a factor of 1,8) are used. The substance is weighed in directly into the test vessels (29 - 50 - 90 mg for 500 ml total volume). It is then dissolved with 234 ml of autoclaved tap water. Copper(II)sulphate pentahydrate is easily soluble. When the test is started, 16 ml of synthetic sewage and 250 ml of activated sludge are added.

Specific inhibitor of nitrification

• 24.A 2,32 g/l stock solution of N-allylthiourea (ATU) should be prepared. The addition of 2,5 ml of this stock solution to an incubation mixture of final volume of 500 ml results in a final concentration of 11,6 mg ATU/l (10^{– 4} mol/l) which is known to be sufficient (4) to cause 100 % inhibition of nitrification in a nitrifying activated sludge containing 1,5g/l suspended solids.

Abiotic control

• 25.Under some rare conditions, a test chemical with strong reducing properties may cause measurable abiotic oxygen consumption. In such cases, abiotic controls are necessary to discriminate between abiotic oxygen uptake by the test chemical and microbial respiration. Abiotic controls may be prepared by omitting the inoculum from the test mixtures. Similarly, abiotic controls without inoculum may be included when supporting analytical measurements are performed to determine the achieved concentration during the exposure phase of the test, e.g. when using stock solutions of poorly water soluble chemicals with components with different water solubility. In specific cases it may be necessary to prepare an abiotic control with sterilised inoculum (e.g. by autoclaving or adding sterilising toxicants). Some chemicals may produce or consume oxygen only if the surface area is big enough for reaction, even if they normally need a much higher temperature or pressure to do so. In this respect special attention should be given to peroxy substances. A sterilised inoculum provides a big surface area.

Inoculum

• 26.For general use, activated sludge should be collected from the exit of the aeration tank, or near the exit from the tank, of a well-operated wastewater treatment plant receiving predominantly domestic sewage. Depending on the purpose of the test, other adequate types or sources of activated sludge, e.g. sludge grown in the laboratory, may also be used at suitable suspended solids concentrations of 2 g/l to 4 g/l. However, sludges from different treatment plants are likely to exhibit different characteristics and sensitivities.

• 27.The sludge may be used as collected but coarse particles should be removed by settling for a short period, e.g. 5 to 15 minutes, and decanting the upper layer of finer solids or sieving (e.g. 1 mm² mesh). Alternatively, the sludge may be homogenised in a blender for a ca. 15 seconds or longer, but caution is needed regarding the shear forces and the temperature change which might occur for long periods of blending.

• 28.Washing the sludge is often necessary, e.g. if the endogenous respiration rate is low. The sludge should first be centrifuged for a period to produce a clear supernatant and pellet of sewage solids e.g. 10 minutes at ca. 10 000 m/s². The supernatant liquid should be discarded and the sludge re-suspended in chlorine-free tap water, with shaking, and the wash-water should then be removed by re-centrifuging and discarding again. The washing and centrifuging process should be repeated, if necessary. The dry mass of a known volume of the re-suspended sludge should be determined and the sludge concentrated by removing liquor or diluted further in chlorine-free tap water to obtain the required sludge solids concentration of 3 g/l. The activated sludge should be continuously aerated (e.g. 2 l/minute) at the test temperature and, where possible used on day of collection. If this is not possible, the sludge should be fed daily with the synthetic sewage feed (50 ml synthetic sewage feed/l activated sludge) for two additional days. The sludge is then used for the test and the results are accepted as valid, provided that no significant change in its activity, assessed by its endogenous heterotrophic and nitrification respiration rate, has occurred.

• 29.Difficulties can arise if foaming occurs during the incubation to the extent that the foam and the sludge solids carried on it, are expelled from the aeration vessels. Occasionally, foaming may simply result from the presence of the synthetic sewage, but foaming should be anticipated if the test chemical is, or contains, a surfactant. Loss of sludge solids from the test mixtures will result in artificially lowered respiration rates that could mistakenly be interpreted as a result of inhibition. In addition, aeration of surfactant solution concentrates the surfactant in the foam layer; loss of foam from the test system will lower the exposure concentrations. The foaming can be controlled by simple mechanical methods (e.g. occasional manual stirring using a glass rod) or by adding a surfactant-free silicone emulsion antifoam agent and/or use the shake flask aeration method. If the problem is associated with the presence of the synthetic sewage, the sewage composition should be modified by including an antifoam reagent at a rate of e.g. 50 μl/l. If foaming is caused by the test chemical, the quantity needed for abatement should be determined at the maximum test concentration, and then all individual aeration vessels should be identically treated (including those, e.g. blank controls and reference vessels where foam is absent). If antifoam agents are used, there should be no interaction with inoculum and/or test chemical.

TEST PROCEDURE

• 30.The inhibition of three different oxygen uptakes may be determined, total, heterotrophic only and that due to nitrification. Normally, the measurement of total oxygen uptake inhibition should be adequate. The effects on heterotrophic oxygen uptake from the oxidation of organic carbon, and due to the oxidation of ammonium are needed when there is a specific requirement for such two separate end-points for a particular chemical or (optionally) to explain atypical dose-response curves from inhibition of total oxygen uptake.

Test conditions

• 31.The test should be performed at a temperature within the range 20 ± 2 °C.

Test mixtures

• 32.Test mixtures (FT as in Table 1) containing water, synthetic sewage feed and the test chemical should be prepared to obtain different nominal concentrations of the test chemical (See Table 1 for example of volumes of constituents). The pH should be adjusted to 7,5 ± 0,5, if necessary; mixtures should be diluted with water and the inoculum added to obtain equal final volumes in the vessels and to begin the aeration.

Reference mixtures

• 33.Mixtures (FR) should be prepared with the reference chemical, e.g. 3,5-dichlorophenol, in place of the test chemical in the same way as the test mixtures.

Blank controls

• 34.Blank controls (FB) should be prepared at the beginning and end of the exposure period in tests in which the test beakers are set up sequentially at intervals. In tests performed using equipment which allows simultaneous measurements of oxygen consumption to be made, at least two blank controls should be included in each batch of simultaneous analysis. Blank controls contain an equal volume of activated sludge and synthetic medium but not test or reference chemical. They should be diluted with water to the same volume as the test and reference mixtures.

Abiotic control

• 35.If necessary, for example if a test chemical is known or suspected to have strong reducing properties, a mixture FA should be prepared to measure the abiotic oxygen consumption. The mixture should have the same amounts of test chemical, synthetic sewage feed and the same volume as the test mixtures, but no activated sludge.

General procedure and measurements

• 36.Test mixtures, reference mixtures and the blank and abiotic controls are incubated at the test temperature under conditions of forced aeration (0,5 to 1 l/min) to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flocs in suspension. Stirring the cultures is also necessary to maintain sludge flocs in suspension. The incubation is considered to begin with the initial contact of the activated sludge inoculum with the other constituents of the final mixture. At the end of incubation, after the specified exposure times of usually 3 hours, samples are withdrawn to measure the rate of decrease of the concentration of dissolved oxygen in the cell designed for the purpose (Fig.2 of Appendix 3) or in a completely filled BOD bottle. The manner in which the incubations begin also depends on the capacity of the equipment used to measure oxygen consumption rates. For example, if it comprises a single oxygen probe, the measurements are made individually. In this case, the various mixtures needed for the test in synthetic sewage should be prepared but the inoculum should be withheld, and the requisite portions of sludge should be added to each vessel of the series. Each incubation should be started in turn, at conveniently timed intervals of e.g. 10 to 15 minutes. Alternatively, the measuring system may comprise multiple probes that facilitate multiple simultaneous measurements; in this case, inoculum may be added at the same time to appropriate groups of vessels.

• 37.The activated sludge concentration in all test, reference and blank (but not abiotic control) mixtures is nominally 1,5 g/l of suspended solids. The oxygen consumption should be measured after 3 hours of exposure. Additional 30-minute exposure measurements should be performed as appropriate and previously described in paragraph 5.

Nitrification potential of sludge

• 38.In order to decide whether sludge nitrifies and, if so, at what rate, mixtures (FB) as in the blank control and additional ‘control’ mixtures (FN) but which also contain N-allylthiourea at 11,6 mg/l should be prepared. The mixtures should be aerated and incubated at 20 °C ± 2 °C for 3 hours. Then the rates of oxygen uptake should be measured and the rate of oxygen uptake due to nitrification calculated.

Test designs

Range-finding test

• 39.A preliminary test is used, when necessary, to estimate the range of concentrations of the test chemical needed in a definitive test for determining the inhibition of oxygen consumption. Alternatively, the absence of inhibition of oxygen consumption by the test chemical in a preliminary test may demonstrate that a definitive test is unnecessary, but triplicates at the highest tested concentration of the preliminary test (typically 1 000 mg/l, but dependent on the data requirement) should be included.

Table 1

Examples of mixtures for the preliminary test

Reagent	Original Concentration	Original Concentration	Original Concentration	Original Concentration	Original Concentration
Test chemical stock solution	10 g/l	10 g/l	10 g/l	10 g/l	10 g/l
Synthetic medium stock solution	See paragraph 16	See paragraph 16	See paragraph 16	See paragraph 16	See paragraph 16
Activated sludge stock suspension	3 g/l of suspended solids	3 g/l of suspended solids	3 g/l of suspended solids	3 g/l of suspended solids	3 g/l of suspended solids
Components of mixtures	Dosing into test vessels [^E0078]	Dosing into test vessels [^E0078]	Dosing into test vessels [^E0078]	Dosing into test vessels [^E0078]	Dosing into test vessels [^E0078]
Components of mixtures	FT1	FT2	FT3-5	FB1-2	FA
Test chemical stock solution (ml) (paragraphs 19 to 21)	0,5	5	50	0	50
Synthetic sewage feed stock solution (ml) (paragraph 16)	16	16	16	16	16
Activated sludge suspension (ml) (paragraphs 26 to 29)	250	250	250	250	0
Water (paragraph 15)	233,5	229	184	234	434
Total volume of mixtures (ml)	500	500	500	500	500
Concentrations in the mixture
Test suspension (mg/l) Activated sludge	10	100	1 000	0	1 000
(suspended solids) (mg/l)	1 500	1 500	1 500	1 500	0

The same procedure should be followed with the reference chemical, to give flasks FR1-3

• 40.The test should be performed using at least three concentrations of the test chemical, for example, 10 mg/l, 100 mg/l and 1 000 mg/l with a blank control and, if necessary, at least three abiotic controls with the highest concentrations of the test chemical (see as example Table 1). Ideally the lowest concentration should have no effect on oxygen consumption. The rates of oxygen uptake and the rate of nitrification, if relevant, should be calculated; then the percentage inhibition should be calculated. Depending on the purpose of the test, it is also possible to simply determine the toxicity of a limit concentration, e.g. 1 000 mg/l. If no statistically significant toxic effect occurs at this concentration, further testing at higher or lower concentrations is not necessary. It should be noted that poorly water soluble substances, mixtures with components of different water solubility and adsorptive substances should be directly weighed into the test vessels. In this case, the volume reserved for the test substance stock solution should be replaced with dilution water.

Definitive test

Inhibition of total oxygen uptake

• 41.The test should be carried out using a range of concentrations deduced from the preliminary test. In order to obtain both a NOEC and an ECx (e.g. EC50), six controls and five treatment concentrations in a geometric series with five replicates are in most cases recommended. The abiotic control does not need to be repeated if there was no oxygen uptake in the preliminary test, but if significant uptake occurs abiotic controls should be included for each concentration of test chemical. The sensitivity of the sludge should be checked using the reference chemical 3,5-dichlorophenol. The sludge sensitivity should be checked for each test series, since the sensitivity is known to fluctuate. In all cases, samples are withdrawn from the test vessels after 3 hours, and additionally 30 minutes if necessary, for measurement of the rate of oxygen uptake in the oxygen electrode cell. From the data collected, the specific respiration rates of the control and test mixtures are calculated; the percentage inhibition is then calculated from equation 7, below.

Differentiation between inhibition of heterotrophic respiration and nitrification

• 42.The use of the specific nitrification inhibitor, ATU, enables the direct assessment of the inhibitory effects of test chemicals on heterotrophic oxidation, and by subtracting the oxygen uptake rate in the presence of ATU from the total uptake rate (no ATU present), the effects on the rate of nitrification may be calculated. Two sets of reaction mixtures should be prepared according to the test designs for ECx or NOEC described in paragraph 41, but additionally, ATU should be added to each mixture of one set at a final concentration of 11,6 mg/l, which has been shown to inhibit nitrification completely in sludge with suspended solids concentrations of up to 3 000 mg/l (4). The oxygen uptake rates should be measured after the exposure period; these direct values represent heterotrophic respiration only, and the differences between these and the corresponding total respiration rates represent nitrification. The various degrees of inhibition are then calculated.

Measurements

• 43.After the exposure period(s) a sample from the first aeration vessel should be transferred to the oxygen electrode cell (Fig. 1 of Appendix 2) and the concentration of dissolved oxygen should immediately be measured. If a multiple electrode system is available, then the measurements may be made simultaneously. Stirring (by means of a covered magnet) is essential at the same rate as when the electrode is calibrated to ensure that the probe responds with minimal delay to changing oxygen concentrations, and to allow regular and reproducible oxygen measurements in the measuring vessel. Usually, the self-stirring probe system of some oxygen electrodes is adequate. The cell should be rinsed with water between measurements. Alternatively, the sample can be used to fill a BOD bottle (Fig. 2 of Appendix 3) fitted with a magnetic stirrer. An oxygen probe with a sleeve adaptor should then be inserted into the neck of the bottle and the magnetic stirrer should be started. In both cases the concentration of dissolved oxygen should continuously be measured and recorded for a period, usually 5 to 10 minutes or until the oxygen concentration falls below 2 mg/l. The electrode should be removed, the mixture returned to the aeration vessel and aerating and stirring should be continued, if measurement after longer exposure periods is necessary.

Verification of the test chemical concentration

• 44.For some purposes, it may be necessary to measure the concentration of the test chemical in the test vessels. It should be noted that if stock solutions of:

• — poorly water soluble substances,

• — mixtures with components with different water solubility, or

• — substances with good water solubility, but where the concentration of the stock solution is near the maximum water solubility,

• are used, the dissolved fraction is unknown, and the true concentration of the test chemical that is transferred into the test vessels is not known. In order to characterise the exposure, an analytical estimation of the test chemical concentrations in the test vessels is necessary. To simplify matters, analytical estimation should be performed before the addition of the inoculum. Due to the fact that only dissolved fractions will be transferred into test vessels, measured concentrations may be very low.

• 45.To avoid time-consuming and expensive analytics, it is recommended to simply weigh the test chemical directly into the test vessels and to refer to the initial weighed nominal concentration for subsequent calculations. A differentiation between dissolved, undissolved or adsorbed fractions of the test chemical is not necessary because all these fractions appear under real conditions in a waste water treatment plant likewise, and these fractions may vary depending on the composition of the sewage. The aim of the test method is to estimate a non inhibitory concentration realistically and it is not suitable to investigate in detail which fractions make a contribution to the inhibition of the activated sludge organisms. Finally, adsorptive substances should be also weighed directly into the test vessels; and the vessels should be silanised in order to minimise losses through adsorption.

DATA AND REPORTING

Calculation of oxygen uptake rates

• 46.The oxygen uptake rates should be calculated from the mean of the measured values, e.g. from the linear part of the graphs of oxygen concentration versus time, limiting the calculations to oxygen concentrations between 2,0 mg/l and 7,0 mg/l, since higher and lower concentrations may themselves influence rates of consumption. Excursion into concentration bands below or above these values is occasionally unavoidable and necessary, for example, when respiration is heavily suppressed and consequently very slow or if a particular activated sludge respires very quickly. This is acceptable provided the extended sections of the uptake graph are straight and their gradients do not change as they pass through the 2,0 mg/l or 7,0 mg/l O2 boundaries. Any curved sections of the graph indicate that the measurement system is stabilising or the uptake rate is changing and should not be used for the calculation of respiration rates. The oxygen uptake rate should be expressed in milligrammes per litre per hour (mg/lh) or milligrammes per gramme dry sludge per hour (mg/gh). The oxygen consumption rate, R, in mg/lh, may be calculated or interpolated from the linear part of the recorded oxygen decrease graph according to Equation 1:

R = (Q1 – Q2)/Δt × 60	(1)

• where:

• Q1 is the oxygen concentration at the beginning of the selected section of the linear phase (mg/l);

• Q2 is the oxygen concentration at the end of the selected section of the linear phase (mg/l);

• Δt is the time interval between these two measurements (min.).

• 47.The specific respiration rate (Rs) is expressed as the amount of oxygen consumed per g dry weight of sludge per hour (mg/gh) according to Equation 2:

Rs= R/SS	(2)

• where SS is the concentration of suspended solids in the test mixture (g/l).

• 48.The different indices of R which may be combined are:

• S specific rate

• T total respiration rate

• N rate due to nitrification respiration

• H rate due to heterotrophic respiration

• A rate due to abiotic processes

• B rate based on blank assays (mean)

Calculation of oxygen uptake rate due to nitrification

• 49.The relationship between total respiration (RT), nitrification respiration (RN) and heterotrophic respiration (RH) is given by Equation 3:

RN = RT – RH	(3)

• where:

• RN is the rate of oxygen uptake due to nitrification (mg/lh);

• RT is the measured rate of oxygen uptake by the blank control (no ATU; FB) (mg/lh).

• RH is the measured rate of oxygen uptake of the blank control with added ATU (FN) (mg/lh).

• 50.This relationship is valid for blank values (RNB, RTB, RHB), abiotic controls (RNA, RTA, RHA) and assays with test chemicals (RNS, RTS, RHS) (mg/gh). Specific respiration rates are calculated from:

RN S = RN/SS	(4)
RTS = RT/SS	(5)
RHS= RH/SS	(6)

• 51.If RN is insignificant (e.g. < 5 % of RT in blank controls) in a preliminary test, it may be assumed that the heterotrophic oxygen uptake equals the total uptake and that no nitrification is occurring. An alternative source of activated sludge would be needed if the tests were to consider effects on heterotrophic and nitrifying micro-organisms. A definitive test is performed if there is evidence of suppressed oxygen uptake rates with different test chemical concentrations.

Calculation of percentage of inhibition

• 52.The percentage inhibition, IT, of total oxygen consumption at each concentration of test chemical, is given by Equation 7:

IT = [1 – (RT – RTA)/RTB] × 100 %	(7)

• 53.Similarly, the percentage inhibition of heterotrophic oxygen uptake, IH, at each concentration of test chemical, is given by Equation 8:

IH = [1 – (RH – RHA)/RHB] × 100 %	(8)

• 54.Finally, the inhibition of oxygen uptake due to nitrification, IN, at each concentration, is given by Equation 9:

IN = [1 – (RT – RH)/(RTB – RHB)] × 100 %	(9)

• 55.The percentage inhibition of oxygen uptake should be plotted against logarithm of the test chemical concentration (inhibition curve, see Fig.3 of Appendix 4). Inhibition curves are plotted for each aeration period of 3 h or additionally after 30 min. The concentration of test chemical which inhibits the oxygen uptake by 50 % (EC50) should be calculated or interpolated from the graph. If suitable data are available, the 95 % confidence limits of the EC50, the slope of the curve, and suitable values to mark the beginning of inhibition (for example, EC10 or EC20) and the end of the inhibition range (for example, EC80 or EC90) may be calculated or interpolated.

• 56.It should be noted that in view of the variability often observed in the results, it may in many cases be sufficient to express the results additionally in order of magnitude, for example:

EC50	< 1 mg/l
EC50	1 mg/l to 10 mg/l
EC50	10 mg/l to 100 mg/l
EC50	> 100 mg/l

Interpretation of results

ECx

• 57.ECx-values including their associated lower and upper 95 % confidence limits for the parameter are calculated using appropriate statistical methods (e.g. probit analysis, logistic or Weibull function, trimmed Spearman-Karber method or simple interpolation (11)). An ECx is obtained by inserting a value corresponding to x % of the control mean into the equation found. To compute the EC50 or any other ECx, the per-treatment means (x) should be subjected to regression analysis.

NOEC estimation

• 58.If a statistical analysis is intended to determine the NOEC, per-vessel statistics (individual vessels are considered as replicates) are necessary. Appropriate statistical methods should be used according to the OECD Document on Current Approaches in the Statistical Analysis of Ecotoxicity Data: a Guidance to Application (11). In general, adverse effects of the test chemical compared to the control are investigated using one-tailed (smaller) hypothesis testing at p ≤ 0,05.

Test report

• 59.The test report should include the following information:

Test chemical

• — common name, chemical name, CAS number, purity;

• — physico-chemical properties of the test chemical (e.g. log Kow, water solubility, vapour pressure, Henry's constant (H) and possible information on the fate of the test chemical e.g. adsorption to activated sludge);

Test system

• — source, conditions of operation of the wastewater treatment plant and influent it receives, concentration, pre-treatment and maintenance of the activated sludge;

Test conditions

• — test temperature, pH during the test and duration of the exposure phase(s);

Results

• — specific oxygen consumption of the controls (mg O2/(g sludge × h);

• — all measured data, inhibition curve(s) and method for calculation of EC50;

• — EC50 and, if possible, 95 per cent confidence limits, possibly EC20, EC80; possibly NOEC and the used statistical methods, if the EC50 cannot be determined;

• — results for total, and if appropriate, heterotrophic and nitrification inhibition;

• — abiotic oxygen uptake in the physico-chemical control (if used);

• — name of the reference chemical and results with this chemical;

• — all observations and deviations from the standard procedure, which could have influenced the result.

LITERATURE

• (1)Brown, D., Hitz, H.R. and Schäfer, L. (1981). The assessment of the possible inhibitory effect of dyestuffs on aerobic waste-water bacteria, Experience with a screening test. Chemosphere 10 (3): 245-261.

• (2)King, E. F. and Painter H. A. (1986). Inhibition of respiration of activated sludge; variability and reproducibility of results. Toxicity Assessment 1(1): 27-39.

• (3)OECD (1984), Activated sludge, Respiration inhibition test, Test Guideline No. 209, Guidelines for the testing of chemicals, OECD, Paris.

• (4)ISO (2007). ISO 8192 Water Quality- Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation, International Organization for Standardization.

• (5)Bealing, D. J. (2003). Document ISO/TC147/WGI/N.183, International Organization for Standardization.

• (6)Painter, H A, Jones K (1963). The use of the wide-bore dropping-mercury electrode for the determination of the rates of oxygen uptake and oxidation of ammonia by micro-orgranisms. Journal of Applied Bacteriology 26 (3): 471-483.

• (7)Painter, H. A. (1986). Testing the toxicity of chemicals by the inhibition of respiration of activated sludge. Toxicity Assessment 1:515-524.

• (8)Robra, B. (1976). Wasser/Abwasser 117, 80.

• (9)Fiebig S. and Noack, U. (2004). The use of copper(II)sulphate pentahydrate as reference substance in the activated sludge respiration inhibition test — acc. to the OECD guideline 209. Fresenius Environmental Bulletin 13 No. 12b: 1556-1557.

• (10)ISO (1995). ISO 10634 Water Quality — Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in aqueous medium, International Organization for Standardization.

• (11)OECD (2006). Current approaches in the statistical analysis of ecotoxicity data: a guidance to application, Series on testing and assessment No. 54, ENV/JM/MONO(2006)18, OECD, Paris.

Appendix 1

Definitions

The following definitions are applicable to this test method.

Chemical means a substance or a mixture.

ECx (Effect concentration for x % effect) is the concentration that causes an x % of an effect on test organisms within a given exposure period when compared with a control. For example, an EC50 is a concentration estimated to cause an effect on a test end point in 50 % of an exposed population over a defined exposure period.

NOEC (no observed effect concentration) is the test chemical concentration at which no effect is observed. In this test, the concentration corresponding to the NOEC, has no statistically significant effect (p < 0,05) within a given exposure period when compared with the control.

Test chemical means any substance or mixture tested using this test method.

Appendix 2

Fig. 1:   Examples for measuring unit

Image omitted; see official source

Key:

1 activated sludge 2 synthetic medium 3 test chemical 4 air 5 mixing vessel	6 magnetic stirrer 7 oxygen measuring cell 8 oxygen electrode 9 oxygen measuring instrument 10 recorder

Appendix 3

Fig. 2:   Example of measuring unit, using a BOD bottle

Image omitted; see official source

Key:

• 1 Test vessel

• 2 oxygen electrode

• 3 oxygen measuring instrument

Appendix 4

Fig. 3:   Example of inhibition curves

Image omitted; see official source

Key:

• X concentration of 3,5-dichlorophenol (mg/l)

• Y inhibition (%)

• Image omitted; see official source inhibition heterotrophic respiration using a nitrifying sludge

• Image omitted; see official source inhibition nitrification using a nitrifying sludge.

C.12.   BIODEGRADATION

MODIFIED SCAS TEST

1. METHOD

1.1.   INTRODUCTION

The purpose of the method is the evaluation of the potential ultimate biodegradability of water-soluble, non-volatile organic substances when exposed to relatively high concentrations of micro-organisms over a long time period. The viability of the microorganisms is maintained over this period by daily addition of a settled sewage feed. (For weekend requirements, the sewage may be stored at 4 ^oC. Alternatively, the synthetic sewage of the OECD confirmatory test may be used.)

Physico-chemical adsorption on the suspended solids may take place and this must be taken into account when interpreting results (see 3.2).

Because of the long detention period of the liquid phase (36 hours), and the intermittent addition of nutrients, the test does not simulate those conditions experienced in a sewage treatment plant. The results obtained with various test substances indicate that the test has a high biodegradation potential.

The conditions provided by the test are highly favourable to the selection and/or adaptation of micro-organisms capable of degrading the test compound. (The procedure may also be used to produce acclimatised inocula for use in other tests.)

In this method, the measure of the concentration of dissolved organic carbon is used to assess the ultimate biodegradability of the test substances. It is preferable to determine DOC after acidification and purging rather than as the difference of Ctotal-Cinorganic.

The simultaneous use of a specific analytical method may allow the assessment of the primary degradation of the substance (disappearance of the parent chemical structure).

The method is applicable only to those organic test substances which, at the concentration used in the test:

• — are soluble in water (at least 20 mg dissolved organic carbon/litre),

• — have negligible vapour pressure,

• — are not inhibitory to bacteria,

• — do not significantly adsorb within the test system,

• — are not lost by foaming from the test solution.

The organic carbon content of the test material must be established.

Information on the relative proportions of the major components of the test material will be useful in interpreting the results obtained, particularly in those cases where the results are low or marginal.

Information on the toxicity to microorganisms of the substance may be useful to the interpretation of low results and in the selection of an appropriate test concentration.

1.2.   DEFINITIONS AND UNITS

CT	=	concentration of test compound as organic carbon as present in or added to the settled sewage at the start of the aeration period (mg/litre),
---	---	---
Ct	=	concentration of dissolved organic carbon found in the supernatant liquor of the test at the end of the aeration period (mg/litre),
---	---	---
Cc	=	concentration of dissolved organic carbon found in the supernatant liquor of the control at the end of the aeration period (mg/litre).

The biodegradation is defined in this method as the disappearance of the organic carbon. The biodegradation can be expressed as:

• 1. The percentage removal Dda of the amount of substance added daily: Image omitted; see official source [1] where Dda = degradation/daily addition.

• 1. The percentage removal Dssd of the amount of substance present at the start of each day: Image omitted; see official source [2 (a)] Image omitted; see official source [2 (b)] where Dssd = degradation/substance start of day; the indices i and (i + 1) refer to the day of measurement. Equation 2(a) is recommended if effluent DOC varies from day to day, while equation 2(b) may be used when effluent DOC remains relatively constant from day to day.

1.3.   REFERENCE SUBSTANCES

In some cases, when investigating a new substance, reference substances may be useful; however, no specific reference substance is recommended here.

Data on several compounds evaluated in ring tests are provided (see Appendix 1) primarily so that calibration of the method may be performed from time to time and to permit comparison of results when another method is employed.

1.4.   PRINCIPLE OF THE TEST METHOD

Activated sludge from a sewage treatment plant is placed in a semi-continuous activated sludge (SCAS) unit. The test compound and settled domestic sewage are added, and the mixture is aerated for 23 hours. The aeration is then stopped, the sludge allowed to settle and the supernatant liquor is removed.

The sludge remaining in the aeration chamber is then mixed with a further aliquot of test compound and sewage and the cycle is repeated.

Biodegradation is established by determination of the dissolved organic carbon content of the supernatant liquor. This value is compared with that found for the liquor obtained from a control tube dosed with settled sewage only.

When a specific analytical method is used, changes in the concentration of the parent molecule due to biodegradation can be measured (primary biodegradability).

1.5.   QUALITY CRITERIA

The reproducibility of this method based on removal of dissolved organic carbon has not yet been established. (When primary biodegradation is considered, very precise data are obtained for materials that are extensively degraded).

The sensitivity of the method is largely determined by the variability of the blank and to a lesser extent by the precision of the determination of dissolved organic carbon and the level of test compound in the liquor at the start of each cycle.

1.6.   DESCRIPTION OF THE TEST PROCEDURE

1.6.1.   Preparations

A sufficient number of clean aeration units, alternatively, the original 1,5 litre SCAS test unit may be used, and air inlet tubes (Figure 1) for each test substance and controls are assembled. Compressed air supplied to the test units, cleaned by a cotton wool strainer, should be free of organic carbon and pre-saturated with water to reduce evaporation losses.

A sample of mixed liquor, containing 1 to 4 g suspended solids/litre, is obtained from an activated sludge plant treating predominantly domestic sewage. Approximately 150 ml of the mixed liquor are required for each aeration unit.

Stock solutions of the test substance are prepared in distilled water; the concentration normally required is 400 mg/litre as organic carbon which gives a test compound concentration of 20 mg/litre carbon at the start of each aeration cycle if no biodegradation is occurring.

Higher concentrations are allowed if the toxicity to microorganisms permits it.

The organic carbon content of the stock solutions is measured.

1.6.2.   Test conditions

The test should be performed at 20 to 25 ^oC.

A high concentration of aerobic microorganisms is used (from 1 to 4 g/litre suspended solids), and the effective detention period is 36 hours. The carbonaceous material in the sewage feed is oxidised extensively, normally within eight hours after the start of each aeration cycle. Thereafter, the sludge respires endogenously for the remainder of the aeration period, during which time the only available substrate is the test compound unless this is also readily metabolised. These features, combined with daily re-inoculation of the test when domestic sewage is used as the medium, provide highly favourable conditions for both acclimatisation and high degress of biodegradation.

1.6.3.   Performance of the test

A sample of mixed liquor from a suitable predominantly domestic activated sludge plant or laboratory unit is obtained and kept aerobic until used in the laboratory. Each aeration unit as well as the control unit are filled with 150 ml of mixed liquor (if the original SCAS test unit is used, multiply the given volumes by 10) and the aeration is started. After 23 hours, aeration is stopped and the sludge is allowed to settle for 45 minutes. The tap of each vessel is opened in turn, and 100 ml portions of the supernatant liquor are withdrawn. A sample of settled domestic sewage is obtained immediately before use, and 100 ml are added to the sludge remaining in each aeration unit. Aeration is started anew. At this stage no test materials are added, and the units are fed daily with domestic sewage only until a clear supernatant liquor is obtained on settling. This usually takes up to two weeks, by which time the dissolved organic carbon in the supernatant liquor at the end of each aeration cycle approaches a constant value.

At the end of this period, the individual settled sludges are mixed, and 50 ml of the resulting composite sludge are added to each unit.

95 ml of settled sewage and 5 ml of water are added to the control units, and 95 ml of the settled sewage plus 5 ml of the appropriate test compound stock solution (400 mg/litre) are added to the test units. Aeration is started again and continued for 23 hours. The sludge is then allowed to settle for 45 minutes and the supernatant drawn off and analysed for dissolved organic carbon content.

The above fill-and-draw procedure is repeated daily throughout the test.

Before settling, it may be necessary to clean the walls of the units to prevent the accumulation of solids above the level of the liquid. A separate scraper or brush is used for each unit to prevent cross contamination.

Ideally, the dissolved organic carbon in the supernatant liquors is determined daily, although less frequent analyses are permissible. Before analysis the liquors are filtered through washed 0,45 μm membrane filters or centrifuged. Membrane filters are suitable if it is assured that they neither release carbon nor absorb the substance in the filtration step. The temperature of the sample must not exceed 40 ^oC while it is in the centrifuge.

The length of the test for compounds showing little or no biodegradation is indeterminate, but experience suggests that this should be at least 12 weeks in general, but not longer than 26 weeks.

2. DATA AND EVALUATION

The dissolved organic carbon values in the supernatant liquors of the test units and the control units are plotted against time.

As biodegradation is achieved, the level found in the test will approach that found in the control. Once the difference between the two levels is found to be constant over three consecutive measurements, such number of further measurements as are sufficient to allow statistical treatment of the data are made and the percentage biodegradation of the test compound is calculated (Dda or Dssd, see 1.2).

3. REPORTING

3.1.   TEST REPORT

The test report shall, if possible, contain the following:

• — all information on the kind of sewage, the type of unit used and the experimental results concerning the tested substance, the reference substance if used, and the blank,

• — the temperature,

• — removal curve with description, mode of calculation (see 1.2),

• — date and location where the activated sludge and the sewage were sampled, status of adaptation, concentration, etc.,

• — scientific reasons for any changes of test procedure,

• — signature and date.

3.2.   INTERPRETATION OF RESULTS

Since the substance to be tested by this method will not be readily biodegradable, any removal of DOC due solely to biodegradation will normally be gradual over days or weeks, except in such cases where acclimatisation is sudden as indicated by an abrupt disappearance occurring after some weeks.

However, physico-chemical adsorption can sometimes play an important role; this is indicated when there is complete or partial removal of the added DOC at the outset. What happens subsequently depends on factors such as the degrees of adsorption and the concentration of suspended solids in the discarded effluent. Usually the difference between the concentration of DOC in the control and test supernatant liquors gradually increases from the initial low value and this difference then remains at the new value for the remainder of the experiment, unless acclimatisation takes place.

If a distinction is to be drawn between biodegradation (or partial biodegradation) and adsorption, further tests are necessary. This can be done in a number of ways, but the most convincing is to use the supernatant liquor, or sludge, as inoculum in a base-set test (preferably a respirometric test).

Test substances giving high, non-adsorptive removal of DOC in this test should be regarded as potentially biodegradable. Partial, non-adsorptive removal indicates that the chemical is at least subject to some biodegradation.

Low, or zero removals of DOC may be due to inhibition of microorganisms by the test substance and this may also be revealed by lysis and loss of sludge, giving turbid supernatants. The test should be repeated using a lower concentration of test substance.

The use of a specific analytical method or of ¹⁴C-labelled test substance may allow greater sensitivity. In the case of ¹⁴C test compound, the recovery of the ¹⁴CO2 will confirm that biodegradation has occurred.

When results are also given in terms of primary biodegradation, an explanation should, if possible, be given on the chemical structure change that leads to the loss of response of the parent test substance.

The validation of the analytical method must be given together with the response found on the blank test medium.

4. REFERENCES

• (1)OECD, Paris, 1981, Test Guideline 302 A, Decision of the Council C(81) 30 final.

Appendix 1

SCAS test: example of results

Substance	CT (mg/l)	Ct - Cc (mg/l)	Percentage biodegradition, Dda	Test duration (days)
4-acetyl aminobenzene sulphonate	17,2	2,0	85	40
Tetra propylene benzene sulphonate	17,3	8,4	51,4	40
4-nitrophenol	16,9	0,8	95,3	40
Diethylene glycol	16,5	0,2	98,8	40
Aniline	16,9	1,7	95,9	40
Cyclopentane tetra carboxylate	17,9	3,2	81,1	120

Appendix 2

Example of test apparatus

Figure 1

Image omitted; see official source

Text of image

Air

Volume 50 ml

C.13.   BIOACCUMULATION IN FISH: AQUEOUS AND DIETARY EXPOSURE

INTRODUCTION

This test method (TM) is equivalent to OECD test guideline (TG) 305 (2012). The major goal of this revision of test method is two-fold. Firstly, it is intended to incorporate a dietary bioaccumulation ([^E0079]) test suitable for determining the bioaccumulation potential of substances with very low water solubility. Secondly, it is intended to create a test method that, when appropriate, utilises fewer fish for animal welfare reasons, and that is more cost-effective.

In the years since adoption of the consolidated test method C.13 (1), numerous substances have been tested, and considerable experience has been gained both by laboratories and by regulatory authorities. This has led to the conviction that the complexity of the test can be reduced if specific criteria are met (cf. paragraph 88), and that a tiered approach is possible. Experience has also shown that biological factors such as growth and fish lipid content can have a strong impact on the results and may need to be taken into account. In addition, it has been recognised that testing very poorly water soluble substances may not be technically feasible. In addition, for substances with very low water solubility in the aquatic environment, exposure via water may be of limited importance in comparison to the dietary route. This has led to the development of a test method in which fish are exposed via their diet (cf. paragraph 7-14 and 97 onwards). Validation (ring test) of the dietary exposure test was conducted in 2010 (51).

The main changes include:

• — The testing of only one test concentration can be considered sufficient, when it is likely that the bioconcentration factor (BCF) is independent of the test concentration.

• — A minimised aqueous exposure test design in which a reduced number of sample points is possible, if specific criteria are met.

• — Fish lipid content should be measured so that BCF can be expressed on a 5 % lipid content basis.

• — Greater emphasis on kinetic BCF estimation (when possible) next to estimating the BCF at steady state.

• — For certain groups of substances, a dietary exposure test will be proposed, where this is considered more suitable than an aqueous exposure test.

• — Fish weight should be measured so that BCFk can be corrected for growth dilution.

Before carrying out any of the bioaccumulation tests, the following information about the test substance should be known:

• (a) Sensitivity of the analytical technique for measuring tissue and aqueous or food concentrations of both the test substance and possible metabolites (cf. paragraph 65).

• (b) Solubility in water [TM A.6; (2)]; this should be determined in accordance with a method that is appropriate for the (estimated) range of the solubility to obtain a reliable value. For hydrophobic substances, this will generally be the column elution method.

• (c) n-Octanol-water partition coefficient, K OW ([^E0080]) [TMs A.8 (4), A.24 (5), A.23 (6)]; or other suitable information on partitioning behaviour (e.g. sorption to lipids, K OC); this should be determined in accordance with a method that is appropriate for the (estimated) range of the K OW to obtain a reliable value. For hydrophobic substances, this will generally be the slow-stirring method [TM A.23 (6)];

• (d) Substance stability in water (hydrolysis [TM C.7 (7)]);

• (e) Substance stability in food (specifically when a dietary exposure test approach is chosen);

• (f) Information on phototransformation relevant for the irradiation conditions in the test (8);

• (g) Surface tension (i.e. for substances where the log K OW cannot be determined) [TM A.5 (9)];

• (h) Vapour pressure [TM A.4 (10)];

• (i) Any information on biotic or abiotic degradation in water, such as (but not restricted to) ready biodegradability [TMs C.4 parts II to VII (11), C.29 (12)], where appropriate;

• (j) Information on metabolites: structure, log K OW, formation and degradability, where appropriate;

• (k) Acid dissociation constant (pK

Independent of the chosen exposure method or sampling scheme, this test method describes a procedure for characterising the bioaccumulation potential of substances in fish. Although flow-through test regimes are much to be preferred, semi-static regimes are permissible, provided that the validity criteria (cf. paragraphs 24 and 113) are satisfied. In the dietary exposure route, the flow-through system is not necessary to maintain aqueous concentrations of the tested substance, but will help maintain adequate dissolved oxygen concentrations and help ensure clean water and remove influences of e.g. excretion products.

Independent of the chosen test method, sufficient details are given in this test method for performing the test while allowing adequate freedom for adapting the experimental design to the conditions in particular laboratories and for varying characteristics of test substances. The aqueous exposure test is most appropriately applied to stable organic substances with log K OW values between 1,5 and 6,0 (13) but may still be applied to strongly hydrophobic substances (having log K OW > 6,0), if a stable and fully dissolved concentration of the test substance in water can be demonstrated. If a stable concentration of the test substance in water cannot be demonstrated, an aqueous study would not be appropriate thus the dietary approach for testing the substance in fish would be required (although interpretation and use of the results of the dietary test may depend on the regulatory framework). Pre-estimates of the bioconcentration factor (BCF, sometimes denoted as K B) for organic substances with log K OW values up to about 9,0 can be obtained using the equation of Bintein et al. (14). The pre-estimate of the bioconcentration factor for such strongly hydrophobic substances may be higher than the steady-state bioconcentration factor (BCFSS) value expected to be obtained from laboratory experiments, especially when a simple linear model is used for the pre-estimate. Parameters which characterise the bioaccumulation potential include the uptake rate constant (k 1), loss rate constants including the depuration rate constant (k 2), the steady-state bioconcentration factor (BCFSS), the kinetic bioconcentration factor (BCFK) and the dietary biomagnification factor (BMF) ([^E0081]).

Radiolabelled test substances can facilitate the analysis of water, food and fish samples, and may be used to determine whether identification and quantification of metabolites will be necessary. If total radioactive residues are measured alone (e.g. by combustion or tissue solubilisation), the BCF or BMF is based on the total of the parent substance, any retained metabolites and also assimilated carbon. BCF or BMF values based on total radioactive residues may not, therefore, be directly comparable to a BCF or BMF derived by specific chemical analysis of the parent substance only. Separation procedures, such as TLC, HPLC or GC ([^E0082]) may be employed before analysis in radiolabelled studies in order to determine BCF or BMF based on the parent substance. When separation techniques are applied, identification and quantification of parent substance and relevant metabolites should be performed ([^E0083]) (cf. paragraph 65) if BCF or BMF is to be based upon the concentration of the parent substance in fish and not upon total radiolabelled residues. It is also possible to combine a fish metabolism or in vivo distribution study with a bioaccumulation study by analysis and identification of the residues in tissues. The possibility of metabolism can be predicted by suitable tools (e.g. OECD QSAR toolbox (15) and proprietary QSAR programs).

The decision on whether to conduct an aqueous or dietary exposure test, and in what set-up, should be based on the factors in paragraph 3 considered together with the relevant regulatory framework. For example, for substances, which have a high log K OW but still show appreciable water solubility with respect to the sensitivity of available analytical techniques, an aqueous exposure test should be considered in the first instance. However it is possible that information on water solubility is not definitive for these hydrophobic types of substances, so the possibility of preparing stable, measurable dissolved aqueous concentrations (stable emulsions are not allowed) applicable for an aqueous exposure study should be investigated before a decision is made on which test method to use (16). It is not possible to give exact prescriptive guidance on the method to be used based on water solubility and octanol-water partition coefficient ‘cut off’ criteria, as other factors (analytical techniques, degradation, adsorption, etc.) can have a marked influence on method applicability for the reasons given above. However, a log K OW above 5 and a water solubility below ~ 0,01 - 0,1 mg/l mark the range of substances where testing via aqueous exposure may become increasingly difficult.

Other factors that may influence test choice should be considered, including the substance's potential for adsorption to test vessels and apparatus, its stability in aqueous solution versus its stability in fish food (17) (18), etc.

Information on such practical aspects may be available from other completed aqueous studies. Further information on the evaluation of aspects relating to the performance of bioaccumulation studies is available in the literature (e.g. (19)).

For substances where the solubility or the maintenance of the aqueous concentration as well as the analysis of these concentrations do not pose any constraints to the realization of an aqueous exposure method, this method is preferred to determine the bioconcentration potential of the substance. In any case, it should be verified that the aqueous exposure concentration(s) to be applied are within the aqueous solubility in the test media. Different methods for maintaining stable concentrations of the dissolved test substance can be used, such as the use of stock solutions or passive dosing systems (e.g. column elution method), as long as it can be demonstrated that stable concentrations can be maintained and the test media are not altered from that recommended in paragraph 27.