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)

Flow-through or semi-static conditions can be employed (cf. paragraph 4); flow-through conditions are recommended to limit potential exposure of test substance via water as a result of any desorption from spiked food or faeces. The test consists of two phases: uptake (test substance-spiked feed) and depuration (clean, untreated feed) (cf. paragraph 16). In the uptake phase, a ‘test’ group of fish are fed a set diet of a commercial fish food of known composition, spiked with the test substance, on a daily basis. Fish ideally should consume all of the offered food (c.f. paragraph 141). Fish are then fed the pure, untreated commercial fish food during the depuration phase. As for the aqueous exposure method, more than one test group with different spiked test substance concentrations can be used if necessary, but for the majority of highly hydrophobic organic test substances one test group is sufficient (cf. paragraphs 49 and 107). If semi-static conditions are used fish should be transferred to a new medium and/or a new test chamber at the end of the uptake phase (in case the medium and/or apparatus used in the uptake phase has been contaminated with the test substance through leaching). The concentrations of the test substance in the fish are measured in both phases of the test. In addition to the group of fish fed the spiked diet (the test group), a control group of fish is held under identical conditions and fed identically except that the commercial fish food diet is not spiked with test substance. This control group allows background levels of test substance to be quantified in unexposed fish and serves as a comparison for any treatment-related adverse effects noted in the test group(s) (¹⁸¹). It also allows comparison of growth rate constants between groups as a check that similar quantities of offered diet have been consumed (potential differences in palatability between diets should also be considered in explaining different growth rate constants; cf. paragraph 138). It is important that during both the uptake and depuration phases, diets of nutritional equivalency are fed to the test and control groups.

An uptake phase that lasts 7-14 days is generally sufficient, based on experience from the method developers (38) (39). This range should minimise the cost of undertaking the test whilst still ensuring sufficient exposure for most substances. However, in some cases the uptake phase may be extended (cf. paragraph 127). During the uptake phase the substance concentration in the fish may not reach steady-state so data treatment and results from this method are usually based on a kinetic analysis of tissue residues. (Note: Equations for estimating time to steady-state can be applied here as for the aqueous exposure test — see Appendix 5). The depuration phase begins when the fish are first fed unspiked diet and typically lasts for up to 28 days or until the test substance can no longer be quantified in whole fish, whichever is the sooner. The depuration phase can be shortened or lengthened beyond 28 days, depending on the change with time in measured chemical concentrations and fish size.

This method allows the determination of the substance-specific half-life (t 1/2, from the depuration rate constant, k 2), the assimilation efficiency (absorption across the gut; a), the kinetic dietary biomagnification factor (BMFK), the growth-corrected kinetic dietary biomagnification factor (BMFKg), and the lipid-corrected (¹⁸²) kinetic dietary biomagnification factor (BMFKL) (and/or the growth- and lipid-corrected kinetic dietary biomagnification factor, BMFKgL) for the test substance in fish. As for the aqueous exposure method, increase in fish mass during the test will result in dilution of test substance in growing fish and thus the (kinetic) BMF will be underestimated if not corrected for growth (cf .paragraphs 162 and 163). In addition, if it is estimated that steady-state was reached in the uptake phase an indicative steady-state BMF can be calculated. Approaches are available that make it feasible to estimate a kinetic bioconcentration factor (BCFK) from data generated in the dietary study (e.g. (44) (45) (46) (47) (48). Pros and cons of such approaches are discussed in Appendix 8.

The test was designed primarily for poorly soluble non-polar organic substances that follow approximately first order uptake and depuration kinetics in fish. In case a substance is tested that does not follow approximately first order uptake and depuration kinetics, then more complex models should be employed (see references in Appendix 5) and advice from a biostatistician and/or pharmacokineticist sought.

The BMF is normally determined using test substance analysis of whole fish (wet weight basis). If relevant for the objectives of the study, specific tissues (e.g. muscle, liver) can be sampled if the fish is divided into edible and non-edible parts (cf. paragraph 21). Furthermore, removal and separate analysis of the gastrointestinal tract may be employed to determine the contribution to whole fish concentrations for sample points at the end of the uptake phase and near the beginning of the depuration phase, or as part of a mass balance approach.

Lipid content of sampled whole fish should be measured so that concentrations can be lipid-corrected, taking account of lipid content of both the diet and the fish (cf. paragraphs 56 and 57, and Appendix 7).

Fish weight of sampled individuals should be measured and recorded, and be linked to the analysed chemical concentration for that individual (e.g. reported using a unique identifier code for each fish sampled), for the purpose of calculating growth that may occur during the test. Fish total length should also be measured where possible (¹⁸³). Weight data are also necessary for estimating BCF using depuration data from the dietary test.

INFORMATION ON THE TEST SUBSTANCE

Information on the test substance as described in paragraphs 3 and 22 should be available. An analytical method for test substance concentrations in water is not usually necessary; methods with suitable sensitivity for measuring concentrations in fish food and fish tissue are required.

The method can be used to test more than one substance in a single test. However, test substances should be compatible with one another such that they do not interact or change their chemical identity upon spiking into fish food. The aim is that measured results for each substance tested together should not differ greatly from the results that would be given if individual tests had been run on each test substance. Preliminary analytical work should establish that each substance can be recovered from a multiply-spiked food and fish tissue sample with i) high recoveries (e.g. > 85 % of nominal) and ii) the necessary sensitivity for testing. The total dose of substances tested together should be below the combined concentration that might cause toxic effects (cf. paragraph 51). Furthermore, possible adverse effects in fish and the potential for interactive effects (e.g. metabolic effects) associated with testing multiple substances simultaneously should be taken into consideration in the experimental design. Simultaneous testing of ionisable substances should be avoided. In terms of exposure, the method is also suitable for complex mixtures (cf. paragraph 13, although the same limitations in analysis as for any other method will apply).

VALIDITY OF THE TEST

For a test to be valid the following conditions apply (cf. paragraph 24):

• — Water temperature variation is less than ± 2 °C in treatment or control groups

• — Concentration of dissolved oxygen does not fall below 60 % of the air saturation value

• — The concentration of the test substance in fish food before and at the end of the uptake phase is within a range of ± 20 % (based on at least three samples at both time points)

• — A high degree of homogeneity of substance in food should be demonstrated in preliminary analytical work on the spiked diet; at least three sample concentrations for the substance taken at test start should not vary more than ± 15 % from the mean

• — Concentrations of test substance are not detected, or are present only at typical trace levels, in un-spiked food or control fish tissues relative to treated samples

• — Mortality or other adverse effects/disease in both control and test group fish should be ≤ 10 % at the end of the test; if the test is extended for any reason, adverse effects in both groups are ≤ 5 % per month, and ≤ 30 % cumulatively. Significant differences in average growth between the test and the control groups of sampled fish could be an indication of a toxic effect of the test substance.

REFERENCE SUBSTANCES

If a laboratory has not performed the assay before or substantial changes (e.g. change of fish strain or supplier, different fish species, significant change of fish size, fish food or spiking method, etc.) have been made, it is advisable that a technical proficiency study is conducted, using a reference substance. The reference substance is primarily used to establish whether the food spiking technique is adequate to ensure maximum homogeneity and bioavailability of test substances. One example that has been used in the case of non-polar hydrophobic substances is hexachlorobenzene (HCB), but other substances with existing reliable data on uptake and biomagnification should be considered due to the hazardous property of HCB (¹⁸⁴). If used, basic information on the reference substance should be presented in the test report, including name, purity, CAS number, structure, toxicity data (if available) as for test substances (cf. paragraphs 3 and 22).

DESCRIPTION OF THE METHOD

Apparatus

Materials and apparatus should be used as described in the aqueous exposure method (cf. paragraph 26). A flow-through or static renewal test system that provides a sufficient volume of dilution water to the test tanks should be used. The flow rates should be recorded.

Water

Test water should be used as described in the aqueous exposure method (cf. paragraphs 27-29). The test medium should be characterised as described and its quality should remain constant during the test. The natural particle content and total organic carbon should be as low as possible (≤ 5 mg/l particulate matter; ≤ 2 mg/l total organic carbon) before test start. TOC need only be measured before the test as part of the test water characterisation (cf. paragraph 53).

Diet

A commercially available fish food (floating and/or slow sinking pelletised diet) that is characterised in terms of at least protein and fat content is recommended. The food should have a uniform pellet size to increase the efficiency of the feed exposure, i.e. the fish will eat more of the food instead of eating the larger pieces and missing the smaller ones. The pellets should be appropriately sized for the size of the fish at the start of the test (e.g. pellet diameters roughly 0,6-0,85 mm for fish between 3 and 7 cm total length, and 0,85-1,2 mm for fish between 6 and 12 cm total length may be used). Pellet size may be adjusted depending on fish growth at the start of the depuration phase. An example of a suitable food composition, as commercially supplied, is given in Appendix 7. Test diets with total lipid content between 15 and 20 % (w/w) have commonly been used in the development of this method. Fish food with such a high lipid concentration may not be available in some regions. In such cases studies could be run with a lower lipid concentration in the food, and if necessary the feeding rate adjusted appropriately to maintain fish health (based on preliminary testing). The total lipid content of the test group and control group diets needs to be measured and recorded before the start of the test and at the end of the uptake phase. Details provided by the commercial feed supplier of analysis for nutrients, moisture, fibre and ash, and if possible minerals and pesticide residues (e.g. ‘standard’ priority pollutants), should be presented in the study report.

When spiking the food with test substance, all possible efforts should be made to ensure homogeneity throughout the test food. The concentration of test substance in the food for the test group should be selected taking into account the sensitivity of the analytical technique, the test substance's toxicity (NOEC if known) and relevant physicochemical data. If used, the reference substance should preferably be incorporated at a concentration around 10 % of that of the test substance (or in any case as low as is practicable), subject to analysis sensitivity (e.g. for hexachlorobenzene a concentration in the food of 1-100 μg/g has been found to be acceptable; cf. (47) for more information on assimilation efficiencies of HCB).

The test substance can be spiked to the fish food in several ways depending on its physical characteristics and solubility (see Appendix 7 for more details on spiking methods):

• — If the substance is soluble and stable in triglycerides, the substance should be dissolved in a small amount of fish oil or edible vegetable oil before mixing with fish food. In this instance, care should be taken to avoid producing a ration that is too high in lipid, taking into account the natural lipid content of the spiked feed, by adding the minimum known quantity of oil required to achieve distribution and homogeneity of the test substance in the food, or;

• — The food should be spiked using a suitable organic solvent, so long as homogeneity and bioavailability are not compromised (it is possible that (micro)crystals of the test substance may form in the food as a consequence of solvent evaporation and there is no easy way to prove this has not occurred; cf. (49)), or;

• — Non-viscous liquids should be added directly to fish food but they should be well mixed to promote homogeneity and facilitate good assimilation. The technique for mixing should ensure homogeneity of the spiked feed.

In few cases, e.g. less hydrophobic test substances more likely to desorb from the food, it may be necessary to coat prepared food pellets with a small quantity of corn/fish oil (see paragraph 142). In such cases, control food should be treated similarly and the final prepared feed used for lipid measurement.

If used, the results of the reference substance should be comparable with literature study data carried out under similar conditions with a comparable feeding rate (cf. paragraph 45) and reference substance-specific parameters should meet the relevant criteria in paragraph 113 (3^rd, 4^th and 5^th points).

If an oil or carrier solvent is used as a vehicle for the test substance, an equivalent amount of the same vehicle (excluding test substance) should be mixed with the control diet in order to maintain equivalency with the spiked diet. It is important that during both the uptake and depuration phases, diets of nutritional equivalency are fed to the test and control groups.

The spiked diet should be stored under conditions that maintain stability of the test substance within the feed mix (e.g. refrigeration) and these conditions reported.

Selection of fish species

Fish species as specified for the aqueous exposure may be used (cf. paragraph 32 and Appendix 3). Rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio) and fathead minnow (Pimephales promelas) have been commonly used in dietary bioaccumulation studies with organic substances before the publication of this TM. The test species should have a feeding behaviour that results in rapid consumption of the administered food ration to ensure that any factor influencing the concentration of the test substance in food (e.g. leaching into the water and the possibility of aqueous exposure) is kept to a minimum. Fish within the recommended size/weight range (cf. Appendix 3) should be used. Fish should not be so small as to hamper ease of analyses on an individual basis. Species tested during a life-stage with rapid growth can complicate data interpretation, and high growth rates can influence the calculation of assimilation efficiency (¹⁸⁵).

Holding of fish

Acclimatisation, mortality and disease acceptance criteria are the same as for the aqueous exposure method prior to test conductance (cf. paragraphs 33-35).

PERFORMANCE OF THE TEST

Pre-study work and range-finding test

Pre-study analytical work is necessary to demonstrate recovery of the substance from spiked food/spiked fish tissue. A range-finding test to select a suitable concentration in the food is not always necessary. For the purposes of showing that no adverse effects are observed and evaluating the palatability of spiked diet, sensitivity of analytical method for fish tissue and food, and selection of suitable feeding rate and sampling intervals during depuration phase etc., preliminary feeding experiments may be undertaken but are not obligatory. A preliminary study may be valuable to estimate numbers of fish needed for sampling during the depuration phase. This can result in significant reduction in the number of fish used, especially for test substances that are particularly susceptible to metabolism.

Conditions of exposure

Uptake Phase duration

An uptake phase of 7-14 days is usually sufficient, during which one group of fish are fed the control diet and another group of fish the test diet daily at a fixed ration dependent on the species tested and the experimental conditions, e.g. between 1-2 % of body weight (wet weight) in the case of rainbow trout. The feeding rate should be selected such that fast growth and large increase of lipid content are avoided. If needed the uptake phase may be extended based on practical experience from previous studies or knowledge of the test substance's (or analogue's) uptake/depuration in fish. The start of the test is defined as the time of first feeding with spiked food. An experimental day runs from the time of feeding to shortly before the time of next feeding (e.g. one hour). Thus the first experimental day of uptake runs from the time of first feeding with spiked food and ends shortly before the second feeding with spiked food. In practice the uptake phase ends shortly before (e.g. one hour) the first feeding with unspiked test substance as the fish will continue to digest spiked food and absorb the test substance in the intervening 24 hours. It is important to ensure that a sufficiently high (non-toxic) body burden of the test substance is achieved with respect to the analytical method, so that at least an order of magnitude decline can be measured during the depuration phase. In special cases an extended uptake phase (up to 28 days) may be used with additional sampling to gain an insight into uptake kinetics. During uptake the concentration in the fish may not reach steady-state. Equations for estimating time to steady-state, as an indication of the likely duration needed to achieve appreciable fish concentrations, can be applied here as for the aqueous exposure test (cf. Appendix 5).

In some cases it may be known that uptake of substance in the fish over 7-14 days will be insufficient for the food concentration used to reach a high enough fish concentration to analyse at least an order of magnitude decline during depuration, either due to poor analytical sensitivity or to low assimilation efficiency. In such cases it may be advantageous to extend the initial feeding phase to longer than 14 days, or, especially for highly metabolisable substances, a higher dietary concentration should be considered. However, care should be taken to keep the body burden during uptake below the (estimated) chronic no effect concentration (NOEC) in fish tissue (cf. paragraph 138).

Duration of the depuration phase

Depuration typically lasts for up to 28 days, beginning once the test group fish are fed pure, untreated diet after the uptake phase. Depuration begins with the first feeding of ‘unspiked’ food rather than straight after the last ‘spiked’ food feeding as the fish will continue to digest the food and absorb the test substance in the intervening 24 hours, as noted in paragraph 126. Hence the first sample in the depuration phase is taken shortly before the second feeding with unspiked diet. This depuration period is designed to capture substances with a potential half-life of up to 14 days, which is consistent with that of bioaccumulative substances (¹⁸⁶), so 28 days comprises two half-lives of such substances. In cases of very highly bioaccumulating substances it may be advantageous to extend the depuration phase (if indicated by preliminary testing).

If a substance is depurated very slowly such that an exact half-life may not be determined in the depuration phase, the information may still be sufficient for assessment purposes to indicate a high level of bioaccumulation. Conversely, if a substance is depurated so fast that a reliable time zero concentration (concentration at the end of uptake/start of depuration, C 0,d) and k 2 cannot be derived, a conservative estimate of k 2 can be made (cf. Appendix 7).

If analyses of fish at earlier intervals (e.g. 7 or 14 days) show that the substance has depurated below quantification levels before the full 28-day period, then subsequent sampling may be discontinued and the test terminated.

In few cases no measurable uptake of the test substance may have occurred at the end of the uptake period (or with the second depuration sample). If it can be demonstrated that: i) the validity criteria in paragraph 113 are fulfilled; and ii) lack of uptake is not due to some other shortcoming of the test (e.g. uptake duration not long enough, deficiency in food spiking technique leading to poor bioavailability, lack of sensitivity of the analytical method, fish not consuming food, etc.); it may be possible to terminate the study without the need to re-run it with a longer uptake duration. If preliminary work has indicated that this may be the case, analysis of faeces, if possible, for undigested test substance may be advisable as part of a ‘mass balance’ approach.

Numbers of test fish

Similar to the aqueous exposure test, fish of similar weight and length should be selected, with the smallest fish being no less than two-thirds of the weight of the largest (cf. paragraphs 40-42).

The total number of fish for the study should be selected based on the sampling schedule (a minimum of one sample at the end of the uptake phase and four to six samples during the depuration phase, but depending on the phases' durations), taking into account the sensitivity of the analytical technique, the concentration likely to be achieved at the end of the uptake phase (based on prior knowledge) and the depuration duration (if prior knowledge allows estimation). Five to ten fish should be sampled at each event, with growth parameters (weight and total length) being measured before chemical or lipid analysis.

Owing to the inherent variability in the size, growth rate, and physiology among fish and the likely variation in the quantity of administered diet that each fish consumes, at least five fish should be sampled at each interval from the test group and five from the control group in order to adequately establish the average concentration and its variability. The variability among the fish used is likely to contribute more to the overall uncontrolled variability in the test than the variability inherent in the analytical methodologies employed, and thus justifies the use of up to ten fish per sample point in some cases. However, if background test substance concentrations in control fish are not measurable at the start of depuration, chemical analysis of two-three control fish at the final sampling interval only may be sufficient so long as the remaining control fish at all sample points are still sampled for weight and total length (so that the same number are sampled from test and control groups for growth). Fish should be stored, weighed individually (even if it proves necessary for the sample results to be combined subsequently) and total length measured.

For a standard test with, for example, a 28-day depuration duration including five depuration samples, this means a total of 59-120 fish from test and 50-110 from control groups, assuming that the substance's analytical technique allows lipid content analysis to be carried out on the same fish. If lipid analysis cannot be conducted on the same fish as chemical analysis, and using control fish only for lipid analysis is also not feasible (cf. paragraph 56), an additional 15 fish would be required (three from the stock population at test start, three each from control and test groups at the start of depuration and three each from control and test groups at the end of the experiment). An example sampling schedule with fish numbers can be found in Appendix 4.

Loading

Similarly high water-to-fish ratios should be used as for the aqueous exposure method (cf. paragraphs 43 and 44). Although fish-to-water loading rates do not have an effect on exposure concentrations in this test, a loading rate of 0,1-1,0 g of fish (wet weight) per litre of water per day is recommended to maintain adequate dissolved oxygen concentrations and minimise test organism stress.

Test diet and Feeding

During the acclimatisation period, fish should be fed an appropriate diet as described above (paragraph 117). If the test is being conducted under flow-through conditions, the flow should be suspended while the fish are fed.

During the test, the diet for the test group should adhere to that described above (paragraphs 116-121). In addition to consideration of substance-specific factors, analytical sensitivity, expected concentration in the diet under environmental conditions and chronic toxicity levels/body burden, selection of the target spiking concentration should take into account palatability of the food (so that fish do not avoid eating). Nominal spiking concentration of the test substance should be documented in the report. Based on experience, spiking concentrations in the range of 1-1 000  μg/g provide a practical working range for test substances that do not exhibit a specific toxic mechanism. For substances acting via a non-specific mechanism, tissue residue levels should not exceed 5 μmol/g lipid since residues above this level are likely to pose chronic effects (19) (48) (50) (¹⁸⁷). For other substances care should be taken that no adverse effects occur from the accumulated exposure (cf. paragraph 127). This is especially true if more than one substance is being tested simultaneously (cf. paragraph 112).

The appropriate amount of the test substance can be spiked to the fish food in one of three ways, as described in paragraph 119 and Appendix 7. The methods and procedures for spiking the feed should be documented in the report. Untreated food is fed to the control fish, containing an equivalent quantity of unspiked oil vehicle if this has been used in the spiked feed for the uptake phase, or having been treated with ‘pure’ solvent if a solvent vehicle was used for test group diet preparation. The treated and untreated diets should be measured analytically at least in triplicate for test substance concentration before the start and at the end of the uptake phase. After exposure to the treated feed (uptake phase), fish (both groups) are fed untreated food (depuration phase).

Fish are fed at a fixed ration (dependent on species; e.g. approximately 1-2 % of wet body weight per day in the case of rainbow trout). The feeding rate should be selected such that fast growth and large increase of lipid content are avoided. The exact feeding rate set during the experiment should be recorded. Initial feeding should be based on the scheduled weight measurements of the stock population just prior to the start of the test. The amount of feed should be adjusted based on the wet weights of sampled fish at each sampling event to account for growth during the experiment. Weights and lengths of fish in the test and control tanks can be estimated from the weights and total lengths of fish used at each sampling event; do not weigh or measure the fish remaining in the test and control tanks. It is important to maintain the same set feeding rate throughout the experiment.

Feeding should be observed to ensure that the fish are visibly consuming all of the food presented in order to guarantee that the appropriate ingestion rates are used in the calculations. Preliminary feeding experiments or previous experience should be considered when selecting a feeding rate that will ensure that all food from once-daily feeding is consumed. In the event that food is consistently being left uneaten, it may be advisable to spread the dose over an extra feeding period in each experimental day (e.g. replace once-daily feeding with feeding half the amount twice daily). If this is necessary, the second feeding should occur at a set time and be timed so that the maximum period of time possible passes before fish sampling (e.g. time for second feeding is set within the first half of an experimental day).

Although fish generally rapidly consume the food, it is important to ensure that the substance remains adsorbed to the food. Efforts should be made to avoid the test substance becoming dispersed in water from the food, thereby exposing the fish to aqueous concentrations of the test substance in addition to the dietary route. This can be achieved by removing any uneaten food (and faeces) from the test and control tanks within one hour of feeding, but preferably within 30 minutes. In addition, a system where the water is continuously cleaned over an active carbon filter to absorb any ‘dissolved’ contaminant may be used. Flow-through systems may help to flush away food particles and dissolved substances rapidly (¹⁸⁸). In some cases, a slightly modified spiked food preparation technique can help to alleviate this problem (see paragraph 119).

Light and Temperature

As for the aqueous exposure method (cf. paragraph 48), a 12 to 16 hour photoperiod is recommended and temperature (± 2 °C) appropriate for the test species used (cf. Appendix 3). Type and characteristics of illumination should be known and documented.

Controls

One control group should be used, with fish fed the same ration as the test group but without the test substance present in the feed. If an oil or solvent vehicle has been used to spike the feed in the test group, the control group food should be treated in exactly the same way but with the absence of test substance so that the diets of the test group and control group are equivalent (cf. paragraphs 121 and 139).

Frequency of Water Quality Measurements

The conditions described in the aqueous exposure method apply here also, except that TOC need only be measured before the test as part of the test water characterisation (cf. paragraph 53).

Sampling and Analysis of Fish and Diet

Analysis of Diet Samples

Samples of the test and control diets should be analysed at least in triplicate for the test substance and for lipid content at least before the beginning and at the end of the uptake phase. The methods of analysis and procedures for ensuring homogeneity of the diet should be included in the report.

Samples should be analysed for the test substance by the established and validated method. Pre-study work should be conducted to establish the limit of quantification, percent recovery, interferences and analytical variability in the intended sample matrix. If a radiolabelled material is being tested, similar considerations as those for the aqueous exposure method should be considered with feed analysis replacing water analysis (cf. paragraph 65).

Analysis of Fish

At each fish sampling event, 5-10 individuals will be sampled from exposure and control treatments (in some instances numbers of control fish can be reduced; cf. paragraph 134).

Sampling events should occur at the same time on each experimental day (relative to feeding time), and should be timed so that the likelihood of food remaining in the gut during the uptake phase and the early part of the depuration phase is minimised to prevent spurious contributions to total test substance concentrations (i.e. sampled fish should be removed at the end of an experimental day, keeping in mind that an experimental day starts at the time of feeding and ends at the time of the next feeding, approximately 24 hours later. Depuration begins with the first feeding of unspiked food; cf. paragraph 128). The first depuration phase sample (taken shortly before the second feeding with unspiked food) is important as extrapolation back one day from this measurement is used to estimate the time zero concentration (C 0,d, the concentration in the fish at the end of uptake/start of depuration). Optionally, the gastrointestinal tract of the fish can be removed and analysed separately at the end of uptake and at days 1 and 3 of depuration.

At each sampling event fish should be removed from both test vessels and treated in the same way as described in the aqueous method (cf. paragraphs 61-63).

Concentrations of test substance in whole fish (wet weight) are measured at least at the end of the uptake phase and during the depuration phase in both control and test groups. During the depuration phase, four to six sampling points are recommended (e.g. 1, 3, 7, 14 and 28 days). Optionally, an additional sampling point may be included after 1-3 days' uptake to estimate assimilation efficiency from the linear phase of uptake for the fish while still near the beginning of the exposure period. Two main deviations from the schedule exist: i) if an extended uptake phase is employed for the purposes of investigating uptake kinetics, there will be additional sampling points during the uptake phase and so additional fish will need to be included (cf. paragraph 126); ii) if the study has been terminated at the end of the uptake phase owning to no measurable uptake (cf. paragraph 131). Individual fish that are sampled should be weighed (and their total length measured) to allow growth rate constants to be determined. Concentrations of the substance in specific fish tissue (edible and non-edible portions) can also be measured at the end of uptake and selected depuration times. If a radiolabelled material is being tested, similar considerations as those for the aqueous exposure method should be considered with feed analysis replacing water analysis (cf. paragraph 65).

For the periodic use of a reference substance (cf. paragraph 25), it is preferable that concentrations are measured in the test group at the end of uptake and at all depuration times specified for the test substance (whole fish); concentrations need only be analysed in the control group at end of uptake (whole fish). In certain circumstances (for example if analysis techniques for test substance and reference substance are incompatible, such that additional fish would be needed to follow the sampling schedule) another approach may be used as follows to minimise the number of additional fish required. Concentrations of the reference substance are measured during depuration only on days 1, 3 and two further sampling points, selected such that reliable estimations of time zero concentration (C 0,d) and k 2 can be made for the reference substance.

If possible the lipid content of the individual fish should be determined on each sampling occasion, or at least at the start and end of the uptake phase and at the end of the depuration phase. (cf. paragraphs 56 and 67). Depending on the analytical method (refer to paragraph 67 and to Appendix 4), it may be possible to use the same fish for both lipid content and test substance concentration determination. This is preferred on the grounds of minimising fish numbers. However, should this not be possible, the same approach as described in the aqueous exposure method can be used (see paragraph 56 for these alternative lipid measurement options). The method used to quantify the lipid content should be documented in the report.

Quality of the analytical method

Experimental checks should be conducted to ensure the specificity, accuracy, precision and reproducibility of the substance-specific analytical technique, as well as recoveries of the test substance from both food and fish.

Fish growth measurement

At the start of the test a sample of fish from the stock population need to be weighed (and their total length measured). These fish should be sampled shortly before the first spiked feeding (e.g. one hour), and assigned to experimental day 0. The number of fish for this sample should be at least the same as that for the samples during the test. Some of these can be the same fish used for lipid analysis before the start of the uptake phase (cf. paragraph 153). At each sampling interval fish are first weighed and their length measured. In each individual fish the measured weight (and length) should be linked to the analysed chemical concentration (and lipid content, if applicable), for example using a unique identifier code for each sampled fish. The measurements of these sampled fish can be used to estimate the weight (and length) of fish remaining in the test and control tanks.

Experimental Evaluation

Observations of mortality should be performed and recorded daily. Additional observations for adverse effects should be performed, for example for abnormal behaviour or pigmentation, and recorded. Fish are considered dead if there is no respiratory movement and no reaction to a slight mechanical stimulus can be detected. Any dead or clearly moribund fish should be removed.

DATA AND REPORTING

Treatment of results

Test results are used to derive the depuration rate constant (k 2) as a function of the total wet weight of the fish. Growth rate constant, k g, based on mean increase in fish weight is calculated and used to produce the growth-corrected depuration rate constant, k 2g, if appropriate. In addition, the assimilation efficiency (a; absorption from the gut), the kinetic biomagnification factor (BMFK) (if necessary growth corrected, BMFKg), its lipid-corrected value (BMFKL or BMFKgL, if corrected for growth dilution) and feeding rate should be reported. Also, if an estimate of the time to steady-state in the uptake phase can be made (e.g. 95 % of steady-state or t 95 = 3,0/k 2), an estimate of the steady-state BMF (BMFSS) can be included (cf. paragraphs 105 and 106, and Appendix 5) if the t 95 value indicates that steady-state conditions may have been reached. The same lipid correction should be applied to this BMFSS as to the kinetically-derived BMF (BMFK) to give a lipid-corrected value, BMFSSL (note that no agreed procedure is available to correct a steady-state BMF for growth dilution). Formulae and example calculations are presented in Appendix 7. Approaches are available that make it feasible to estimate a kinetic bioconcentration factor (BCFK) from data generated in the dietary study. This is discussed in Appendix 8.

Fish weight/length data

Individual fish wet weights and lengths for all time periods are tabulated separately for test and control groups for all sampling days during the uptake phase (stock population for start of uptake; control group and test group for end of uptake and, if conducted, the early phase (e.g. day 1-3 of uptake) and depuration phase (e.g. days 1, 2, 4, 7, 14, 28, for control and test group). Weight is the preferred measure of growth for growth dilution correction purposes. See below (paragraphs 162 and 163) and Appendix 5 for the method(s) used to correct data for growth dilution.

Test substance concentration in fish data

Individual fish test substance residue measurements (or pooled fish samples if individual fish measurements are not possible), expressed in terms of wet weight concentration (w/w), are tabulated for test and control fish for individual sample times. If lipid analysis has been conducted on each sampled fish then individual lipid-corrected concentrations, in terms of lipid concentration (w/w lipid), can be derived and tabulated.

• — Test substance residue measurements in individual fish (or pooled fish samples if individual fish measurements are not possible, cf. paragraph 66) for the depuration period are converted to their natural logarithms and plotted versus time (day). If a visual inspection of the plot shows obvious outliers, a statistically valid outlier test may be applied to remove spurious data points as well as documented justification for their omission.

• — A linear least squares correlation is calculated for the ln(concentration) vs. depuration (day) data. The slope and intercept of the line are reported as the overall depuration rate constant (k 2) and natural logarithm of the derived time zero concentration (C 0,d) (cf. Appendix 5 and Appendix 7 for further details). Should this not be possible because concentrations fall below the limit of quantification for the second depuration sample, a conservative estimate of k 2 can be made (cf. Appendix 7).

• — The variances in the slope and intercept of the line are calculated using standard statistical procedures and the 90 % (or 95 %) confidence intervals around these results evaluated and presented.

• — The mean measured fish concentration for the final day of uptake (measured time zero concentration, C 0,m) is also calculated and compared with the derived value C 0,d. In case the derived value is lower than the measured value, the difference may suggest the presence of undigested spiked food in the gut. If the derived value is very much higher than the measured value, this may be an indication that the value derived from the depuration data linear regression is erroneous and should be re-evaluated (see Appendix 7).

Depuration rate and biomagnification factor

To calculate the biomagnification factor from the data, first the assimilation efficiency (absorption of test substance across the gut, α) should be obtained. To do this, equation A7.1 in Appendix 7 should be used, requiring the derived concentration in fish at time zero of the depuration phase (C 0,d), (overall) depuration rate constant (k 2), concentration in the food (C food), food ingestion rate constant (I) and duration of the uptake period (t) to be known. The slope and intercept of the linear relationship between ln(concentration) and depuration time are reported as the overall depuration rate constant (k 2 = slope) and time zero concentration (C 0,d = e^intercept), as above. The derived values should be checked for biological plausibility (e.g. assimilation efficiency as a fraction is not greater than 1). (I) is calculated by dividing the mass of food by the mass of fish fed each day (if fed at 2 % of body weight, (I) will be 0,02). However, the feeding rate used in the calculation may need to be adjusted for fish growth (this can be done using the known growth rate constant to estimate the fish weight at each time-point during the uptake phase; cf. Appendix 7). In cases where k 2 and C 0,d cannot be derived because, for example, concentrations fell below the limit of detection for the second depuration sample, a conservative estimate of k 2 and an ‘upper bound’ BMFk can be made (cf. Appendix 7).

Once the assimilation efficiency (α) is obtained, the biomagnification factor can be calculated by multiplying α by the ingestion rate constant (I) and dividing by the (overall) depuration rate constant (k 2). The growth-corrected biomagnification factor is calculated in the same way but using the growth-corrected depuration rate constant (k 2g; cf. paragraphs 162 and 163. An alternative estimate of the assimilation efficiency can be derived if tissue analysis was performed on fish sampled in the early, linear phase of the uptake phase; cf. paragraph 151 and Appendix 7. This value represents an independent estimate of assimilation efficiency for an essentially unexposed organism (i.e. the fish are near the beginning of the uptake phase). The assimilation efficiency estimated from depuration data is usually used to derive the BMF.

Lipid Correction and Growth-Dilution Correction

Fish growth during the depuration phase can lower measured chemical concentrations in the fish with the effect that the overall depuration rate constant, k2, is greater than would arise from removal processes (e.g. metabolism, egestion) alone (cf. paragraph 72). Lipid contents of test fish (which are strongly associated with the bioaccumulation of hydrophobic substances) and lipid contents of food can vary enough in practice such that their correction is necessary to present biomagnification factors in a meaningful way. The biomagnification factor should be corrected for growth dilution (as is the kinetic BCF in the aqueous exposure method) and corrected for the lipid content of the food relative to that of the fish (the lipid-correction factor). Equations and examples for these calculations can be found in Appendix 5 and Appendix 7, respectively.

To correct for growth dilution, the growth-corrected depuration rate constant (k 2g) should be calculated (see Appendix 5 for equations). This growth-corrected depuration rate constant (k 2g) is then used to calculate the growth-corrected biomagnification factor, as in paragraph 73. In some cases this approach is not possible. An alternative approach that circumvents the need for growth dilution correction involves using mass of test substance per fish (whole fish basis) depuration data rather than the usual mass of test substance per unit mass of fish (concentration) data. This can be easily achieved as tests according to this method should link recorded tissue concentrations to individual fish weights. The simple procedure for doing this is outlined in Appendix 5. Note that k 2 should still be estimated and reported even if this alternative approach is used.

To correct for the lipid content of the food and fish when lipid analysis has not be conducted on all sampled fish, the mean lipid fractions (w/w) in the fish and in the food are derived (¹⁸⁹). The lipid correction factor (Lc) is then calculated by dividing the fish mean lipid fraction by the mean food lipid fraction. The biomagnification factor, growth corrected or not as applicable, is divided by the lipid correction factor to calculate the lipid-corrected biomagnification factor.

If chemical and lipid analyses were conducted on the same fish at each sampling point, then the lipid-corrected tissue data for individual fish may be used to calculate a lipid-corrected BMF directly (cf. (37)). The plot of lipid-corrected concentration data gives C 0,d on a lipid basis and k

lipid) and the dietary concentration on a lipid basis (C food-lipid). Lipid corrected parameters are similarly then used to calculate BMF (note that growth rate constant correction should also be applied to the lipid fraction rather than the fish wet weight to calculated the lipid-corrected, growth corrected BMFKgL).

Interpretation of results

Average growth in both test and control groups should in principle not be significantly different to exclude toxic effects. The growth rate constants or the growth curves of the two groups should be compared by an appropriate procedure (¹⁹⁰)).

Test report

After termination of the study, a final report is prepared containing the information on Test Substance, Test Species and Test Conditions as listed in paragraph 81 (as for the aqueous exposure method). In addition, the following information is required:

Test Substance:

• — Any information on stability of the test substance in prepared food;

Test Conditions:

• — Substance nominal concentration in food, spiking technique, amount of (lipid) vehicle used in food spiking process (if used), test substance concentration measurements in spiked diet for each analysis (at least in triplicate before study start and at end of uptake) and mean values;

• — If used, type and quality of carrier oil or solvent (grade, supplier, etc.) used for food spiking;

• — Food type employed (proximate analysis (¹⁹¹), grade or quality, supplier, etc.), feeding rate during uptake phase, amount of food administered and frequency (including any adjustments based on sampled fish weight);

• — Time at which fish were collected and euthanised for chemical analysis for each sample point (e.g. one hour before the following day's feeding);

Results:

• — Results from any preliminary study work;

• — Information on any adverse effects observed;

• — Complete description of all chemical analysis procedures employed including limits of detection and quantification, variability and recovery;

• — Measured lipid concentrations in food (spiked and control diet), individual, mean values and standard deviations;

• — Tabulated fish weight (and length) data linked to individual fish, both for control and exposure groups (for example using unique identifiers for each fish) and calculations, derived growth rate constant(s) and 95 % confidence interval(s);

• — Tabulated test substance concentration data in fish, mean measured concentration at end of uptake (C 0,m), and derived (overall) depuration rate constant (k 2) and concentration in fish at start of depuration phase (C 0,d) together with the variances in these values (slope and intercept);

• — Tabulated fish lipid contents data (listed against specific substance concentrations if applicable), mean values for test group and control at test start, end of uptake and end of depuration;

• — Curves (including all measured data), showing the following (if applicable, concentrations may be expressed in relation to the whole body of the animal or specified tissues thereof): — growth (i.e. fish weight (and length) vs. time) or natural logarithm transformed weight vs. time; — the depuration of the test substance in the fish; and — natural logarithm transformed concentration (ln concentration) vs. depuration time (including the derived depuration rate constant k 2, and natural logarithm derived concentration in fish at start of depuration phase, C 0,d).

• — If a visual inspection of a plot shows obvious outliers, a statistically valid outlier test may be applied to remove spurious data points as well as documented justification for their omission.

• — Calculated growth-corrected depuration rate constant and growth-corrected half-life.

• — Calculated assimilation efficiency (α).

• — ‘Raw’ dietary BMF, lipid and growth-dilution corrected kinetic BMF (‘raw’ and lipid-corrected based on whole fish wet weight), tissue-specific BMF if applicable.

• — Any information concerning radiolabelled test substance metabolites and their accumulation.

• — Anything unusual about the test, any deviation from these procedures, and any other relevant information.

• — A summary table of relevant measured and calculated data, as hereafter: Substance Depuration Rate constants and Biomagnification Factors (BMFK) k g (growth rate constant; day^{– 1}): Insert Value (95 % CI) [^E0114] k 2 (overall depuration rate constant, day^{– 1}): Insert Value (95 % CI) k 2g (growth-corrected depuration rate constant; day^{– 1}): Insert Value (95 % CI) [^E0114] C 0,m (measured time zero concentration, the concentration in fish at end of uptake) (μg/g): Insert Value ± SD [^E0115] C 0,d (derived time zero concentration of depuration phase; μg/g): Insert Value ± SD [^E0115] I (set feed ingestion rate; g food/g fish/day): Insert Value I g (effective feeding rate, adjusted for growth; g food/g fish/day) [^E0115]: Insert Value ± SD [^E0115] C food (chemical concentration in the food; μg/g): Insert Value ± SD [^E0115] α (substance assimilation efficiency): Insert Value ± SD [^E0115] BMFK (kinetic dietary BMF): Insert Value (95 % CI) [^E0114] BMFKg (growth-corrected kinetic dietary BMF): Insert Value (95 % CI) [^E0114] t 1/2g (growth-corrected half-life in days): Insert Value ± SD [^E0115] Lc (lipid correction factor): Insert Value BMFKgL (lipid-corrected growth-corrected kinetic BMF): Insert Value BMFSS-L (indicative lipid-corrected steady-state BMF) [^E0115]: Insert Value ± SD [^E0115] (¹) CI: confidence interval (where possible to estimate). (²) SD: Standard deviation (where possible to estimate).

LITERATURE:

• (1) Chapter C.13 of this Annex, Bioconcentration: Flow-through Fish Test.

• (2) Chapter A.6 of this Annex, Water Solubility

• (3) Li A, Doucette W.J. (1993), The effect of cosolutes on the aqueous solubilities and octanol/water partition coefficients of selected polychlorinated biphenyl congeners. Environ Toxicol Chem 12: 2031-2035

• (4) Chapter A.8 of this Annex, Partition Coefficient (n-octanol/water): Shake Flask Method.

• (5) Chapter A.24 of this Annex, Partition Coefficient (n-octanol/water), HPLC Method.

• (6) Chapter A.23 of this Annex, Partition Coefficient (1-Octanol/Water): Slow-Stirring Method.

• (7) Chapter C.7 of this Annex, Hydrolysis as a Function of pH.

• (8) (OECD (1997), OECD Environmental Health and Safety Publications Series on Testing and Assessment Number 7: Guidance Document on Direct Phototransformation of Chemicals in Water OCDE/GD(97)21. Organisation for Economic Co-operation and Development (OECD), Paris, France.

• (9) Chapter A.5 of this Annex, Surface Tension of Aqueous Solutions.

• (10) Chapter A.4 of this Annex, Vapour Pressure.

• (11) Chapter C.4 of this Annex, Ready Biodegradability.

• (12) Chapter C.29 of this Annex, Ready Biodegradability — CO2 in sealed vessels

• (13) Connell D.W. (1988), Bioaccumulation behaviour of persistent chemicals with aquatic organisms. Rev. Environ. Contam. Toxicol. 102: 117-156.

• (14) Bintein S., Devillers J. and Karcher W. (1993), Nonlinear dependence of fish bioconcentration on n-octanol/water partition coefficient. SAR QSAR Environ. Res. 1: 29-39.

• (15) OECD (2011), QSAR Toolbox 2.1. February 2011. Available from: http://www.oecd.org/document/54/0,3746,en_2649_34379_42923638_1_1_1_1,00.html.

• (16) Brown R.S., Akhtar P., Åkerman J., Hampel L., Kozin I.S., Villerius L.A. and Klamer H.J.C. (2001), Partition controlled delivery of hydrophobic substances in toxicity tests using poly(dimethylsiloxane) (PDMS) films. Environ. Sci. Technol. 35: 4097-4102.

• (17) Fernandez J.D., Denny J.S. and Tietge J.E. (1998), A simple apparatus for administering 2,3,7,8-tetrachlorodibenzo-p-dioxin to commercially available pelletized fish food. Environ. Toxicol. Chem. 17: 2058-2062.

• (18) Nichols J.W., Fitzsimmons P.N., Whiteman F.W. and Dawson T.D. (2004), A physiologically based toxicokinetic model for dietary uptake of hydrophobic organic compounds by fish: I. Feeding studies with 2,2′, 5,5′-tetrachlorobiphenyl. Toxicol. Sci. 77: 206-218.

• (19) Parkerton T.F., Arnot J.A., Weisbrod A.V., Russom C., Hoke R.A., Woodburn K., Traas T., Bonnell M., Burkhard L.P. and Lampi M.A. (2008), Guidance for evaluating in vivo fish bioaccumulation data. Integr. Environ. Assess. Manag. 4: 139-155.

• (20) Verbruggen E.M.J., Beek M., Pijnenburg J. and Traas T.P. (2008). Ecotoxicological environmental risk limits for total petroleum hydrocarbons on the basis of internal lipid concentrations. Environ. Toxicol. Chem. 27: 2436-2448.

• (21) Schlechtriem C., Fliedner A. and Schäfers C. (2012), Determination of lipid content in fish samples from bioaccumulation studies: Contributions to the revision of OECD Test Guideline 305. Environmental Sciences Europe 2012, 24:13. published: 3 April 2012.

• (22) Chapter C.47 of this Annex, Fish, Early-Life Stage Toxicity Test.

• (23) Chapter C.15 of this Annex, Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages

• (24) Chapter C.14 of this Annex, Fish, Juvenile Growth Test.

• (25) OECD (2000), OECD Environmental Health and Safety Publications Series on Testing and Assessment No. 23: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures ENV/JM/MONO(2000)6. Organisation for Economic Co-operation and Development (OECD), Paris, France.

• (26) US-EPA (1994), Great Lake water quality initiative technical support document for the procedure to determine bioaccumulation factors 822-R-94-002. US EPA, Office of Water, Office of Science and Technology, Washington, DC, USA.

• (27) US-FDA (1999), Pesticide analytical manual (PAM). Vol.1. US Food and Drug Administration, Rockville, MD, USA.

• (28) US-EPA (1974), Section 5, A (1) Analysis of Human or Animal Adipose Tissue, in Analysis of Pesticide Residues in Human and Environmental Samples, Thompson, J.F., Editor. US-EPA, Research Triangle Park, NC, USA

• (29) Bligh E.G. and Dyer W.J. (1959), A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911-917.

• (30) Gardner W.S., Frez W.A., Cichocki E.A. and Parrish C.C. (1985), Micromethod for lipids in aquatic invertebrates. Limnol. Oceanogr. 30: 1099-1105.

• (31) Smedes F. (1999), Determination of total lipid using non-chlorinated solvents. Analyst. 124: 1711-1718.

• (32) OECD (2006), OECD Environmental Health and Safety Publications Series on Testing and Assessment No. 54: Current approaches in the statistical analysis of ecotoxicity data: a guidance to application. ENV/JM/MONO(2006)18. Organisation for Economic Co-operation and Development (OECD), Paris, France.

• (33) Springer T.A., Guiney P.D., Krueger H.O. and Jaber M.J. (2008), Assessment of an approach to estimating aquatic bioconcentration factors using reduced sampling. Environ. Toxicol. Chem. 27: 2271-2280.

• (34) Springer T.A. (2009), Statistical Research Related to Update of OECD Guideline 305. Wildlife International, Ltd, Easton, MD, USA.

• (35) Arnot J.A., Meylan W., Tunkel J., Howard P.H., Mackay D., Bonnell M. and Boethling R.S. (2009), A quantitative structure-activity relationship for predicting metabolic biotransformation rates for organic chemicals in fish. Environ. Toxicol. Chem. 28: 1168-1177.

• (36) Parkerton T., Letinski D., Febbo E., Davi R., Dzambia C., Connelly M., Christensen K. and Peterson D. (2001), A practical testing approach for assessing bioaccumulation potential of poorly water soluble organic chemicals (presentation). in SETAC Europe 12th Annual Meeting: Madrid, Spain.

• (37) Fisk A.T., Cymbalisty C.D., Bergman Ã. and Muir D.C.G. (1996), Dietary accumulation of C12- and C16-chlorinated alkanes by juvenile rainbow trout (Oncorhynchus mykiss). Environ. Toxicol. Chem. 15: 1775-1782.

• (38) Anonymous (2004), Fish, dietary bioaccumulation study — Basic protocol, document submitted to the TC-NES WG on PBT.

• (39) Anonymous (2004), Background document to the fish dietary study protocol, document submitted to the TC-NES WG on PBT.

• (40) Bruggeman W.A., Opperhuizen A., Wijbenga A. and Hutzinger O. (1984), Bioaccumulation of super-lipophilic chemicals in fish, Toxicol. Environ. Chem. 7: 173-189.

• (41) Muir D.C.G., Marshall W.K. and Webster G.R.B. (1985), Bioconcentration of PCDDs by fish: effects of molecular structure and water chemistry. Chemosphere. 14: 829-833.

• (42) Thomann R.V. (1989), Bioaccumulation model of organic chemical distribution in aquatic food chains. Environ. Sci. Technol. 23: 699-707.

• (43) Nichols J.W., Fitzsimmons P.N. and Whiteman F.W. (2004), A physiologically based toxicokinetic model for dietary uptake of hydrophobic organic compounds by fish: II. Stimulation of chronic exposure scenarios. Toxicol. Sci. 77: 219-229.

• (44) Gobas F.A.P.C., de Wolf W., Burkhard L.P., Verbruggen E. and Plotzke K. (2009), Revisiting bioaccumulation criteria for POPs and PBT assessments. Integr. Environ. Assess. Manag. 5: 624-637.

• (45) Sijm D.T.H.M. and van der Linde A. (1995), Size-dependent bioconcentration kinetics of hydrophobic organic chemicals in fish based on diffusive mass transfer and allometric relationships. Environ. Sci. Technol. 29: 2769-2777.

• (46) Sijm D.T.H.M., Verberne M.E., de Jonge W.J., Pärt P. and Opperhuizen A. (1995), Allometry in the uptake of hydrophobic chemicals determined in vivo and in isolated perfused gills. Toxicol. Appl. Pharmacol. 131: 130-135.

• (47) Fisk A.T., Norstrom R.J., Cymbalisty C.D. and Muir D.G.G. (1998), Dietary accumulation and depuration of hydrophobic organochlorines: Bioaccumulation parameters and their relationship with the octanol/water partition coefficient. Environ. Toxicol. Chem. 17: 951-961.

• (48) McGrath J.A., Parkerton T.F. and Di Toro D.M. (2004), Application of the narcosis target lipid model to algal toxicity and deriving predicted-no-effect concentrations. Environ. Toxicol. Chem. 23: 2503-2517.

• (49) Poppendieck D.G. (2002), Polycyclic Aromatic Hydrocarbon Desorption Mechanisms from Manufactured Gas Plant Site Samples. Dissertation. Department of Civil, Architectural and Environmental Engineering, University of Texas, Austin, TX, USA.

• (50) McCarty L.S. and Mackay D. (1993), Enhancing ecotoxicological modelling and assessment: body residues and modes of toxic action. Environ. Sci. Technol. 27: 1718-1728.

• (51) OECD (2012), OECD Environmental Health and Safety Publications Series on Testing and Assessment No. 175: Part I — Validation Report of a ring test for the OECD TG 305 dietary exposure bioaccumulation fish test. Part II — Additional Report including comparative analysis of trout and carp results ENV/JM/MONO(2012)20. Organisation for Economic Co-operation and Development (OECD), Paris, France.

Appendix 1

DEFINITIONS AND UNITS:

The assimilation efficiency (α) is a measure of the relative amount of substance absorbed from the gut into the organism (α is unitless, but it is often expressed as a percentage rather than a fraction).

Bioaccumulation is generally referred to as a process in which the substance concentration in an organism achieves a level that exceeds that in the respiratory medium (e.g. water for a fish or air for a mammal), the diet, or both (1).

Bioconcentration is the increase in concentration of the test substance in or on an organism (or specified tissues thereof) relative to the concentration of test substance in the surrounding medium.

The bioconcentration factor (BCF or K B) at any time during the uptake phase of this accumulation test is the concentration of test substance in/on the fish or specified tissues thereof (C f as mg/kg) divided by the concentration of the substance in the surrounding medium (C w as mg/l). BCF is expressed in l·kg^{– 1}. Please note that corrections for growth and/or a standard lipid content are not accounted for.

Biomagnification is the increase in concentration of the test substance in or on an organism (or specified tissues thereof) relative to the concentration of test substance in the food.

The biomagnification factor (BMF) is the concentration of a substance in a predator relative to the concentration in the predator's prey (or food) at steady-state. In the method described in this test method, exposure via the aqueous phase is carefully avoided and thus a BMF value from this test method cannot directly be compared to a BMF value from a field study (in which both water and dietary exposure may be combined).

The dietary biomagnification factor (dietary BMF) is the term used in this test method to describe the result of dietary exposure test, in which exposure via the aqueous phase is carefully avoided and thus the dietary BMF from this test method cannot directly be compared to a BMF value from a field study (in which both water and dietary exposure may be combined).

The depuration or post-exposure (loss) phase is the time, following the transfer of the test fish from a medium containing test substance to a medium free of that substance, during which the depuration (or the net loss) of the substance from the test fish (or specified tissue thereof) is studied.

The depuration (loss) rate constant (k 2) is the numerical value defining the rate of reduction in the concentration of the test substance in the test fish (or specified tissues thereof) following the transfer of the test fish from a medium containing the test substance to a medium free of that substance (k 2 is expressed in day^{– 1}).

Dissolved organic carbon (DOC) is a measure of the concentration of carbon originating from dissolved organic sources in the test media.

The exposure or uptake phase is the time during which the fish are exposed to the test substance.

The food ingestion rate (I) is the average amount of food eaten by each fish each day, relative to the estimated average fish whole body weight (expressed in terms of g food/g fish/day).

The kinetic bioconcentration factor (BCFK) is the ratio of the uptake rate constant, k 1, to the depuration rate constant, k 2 (i.e. k 1/k 2 — see corresponding definitions in this Appendix). In principle the value should be comparable to the BCFSS (see definition above), but deviations may occur if steady-state was uncertain or if corrections for growth have been applied to the kinetic BCF.

The lipid normalised kinetic bioconcentration factor (BCFKL) is normalised to a fish with a 5 % lipid content.

The lipid normalised, growth corrected kinetic bioconcentration factor (BCFKgL) is normalised to a fish with a 5 % lipid content and corrected for growth during the study period as described in Appendix 5.

The lipid normalised steady-state bioconcentration factor (BCFSSL) is normalised to a fish with 5 % lipid content.

A multi-constituent substance is defined for the purpose of REACH as a substance which has more than one main constituent present in a concentration between 10 % and 80 % (w/w).

The octanol-water partition coefficient (K OW) is the ratio of a substance's solubility in n-octanol and water at equilibrium (Methods A.8 (2), A.24 (3), A.23 (4)); also expressed as P OW. The logarithm of K OW is used as an indication of a substance's potential for bioconcentration by aquatic organisms.

Particulate organic carbon (POC) is a measure of the concentration of carbon originating from suspended organic sources in the test media.

Solid-phase microextraction (SPME) is a solvent-free analytical technique developed for dilute systems. In this method, a polymer coated fibre is exposed to the gas or liquid phase containing the analyte of interest. Generally, a minimum analysis time is imposed so that equilibrium conditions are established between the solid and fluid phases, with respect to the measured species. Subsequently the concentration of the analyte of interest can be determined directly from the fibre or after extracting it from the fibre into a solvent, depending on the determination technique.

A steady-state is reached in the plot of test substance in fish (C

f made on samples taken at intervals of at least two days are within ± 20 % of each other, and there is no significant increase of C f in time between the first and last successive analysis. When pooled samples are analysed at least four successive analyses are required. For test substances which are taken up slowly the intervals would more appropriately be seven days.

The steady-state bioconcentration factor (BCFSS) does not change significantly over a prolonged period of time, the concentration of the test substance in the surrounding medium being constant during this period of time (cf. Definition of steady-state).

Total organic carbon (TOC) is a measure of the concentration of carbon originating from all organic sources in the test media, including particulate and dissolved sources.

The uptake rate constant (k 1) is the numerical value defining the rate of increase in the concentration of test substance in/on test fish (or specified tissues thereof) when the fish are exposed to that substance (k 1 is expressed in l kg^{– 1} day^{– 1}).

Substances of Unknown or Variable composition, Complex reaction products and Biological materials are known as UVCB

Chemical is a substance or a mixture.

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

LITERATURE:

• (1) Gobas F.A.P.C., de Wolf W., Burkhard L.P., Verbruggen E. and Plotzke K. (2009), Revisiting bioaccumulation criteria for POPs and PBT assessments. Integr. Environ. Assess. Manag. 5: 624-637.

• (2) Chapter A.8 of this Annex, Partition Coefficient (n-octanol/water): Shake Flask Method

• (3) Chapter A.24 of this Annex, Partition Coefficient (n-octanol/water), HPLC Method.

• (4) Chapter A.23 of this Annex, Partition Coefficient (1-Octanol/Water): Slow-Stirring Method.

Appendix 2

SOME CHEMICAL CHARACTERISTICS OF AN ACCEPTABLE DILUTION WATER

Component	Limit concentration
Particulate matter	5 mg/l
Total organic carbon	2 mg/l
Un-ionised ammonia	1 μg/l
Residual chlorine	10 μg/l
Total organophosphorous pesticides	50 ng/l
Total organochlorine pesticides plus polychlorinated biphenyls	50 ng/l
Total organic chlorine	25 ng/l
Aluminium	1 μg/l
Arsenic	1 μg/l
Chromium	1 μg/l
Cobalt	1 μg/l
Copper	1 μg/l
Iron	1 μg/l
Lead	1 μg/l
Nickel	1 μg/l
Zinc	1 μg/l
Cadmium	100 ng/l
Mercury	100 ng/l
Silver	100 ng/l

Appendix 3

FISH SPECIES RECOMMENDED FOR TESTING