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)

Glass or stainless steel containers with a height of 12 to 20 cm can be used as culture vessels. Each culture container is loaded with a layer of wet artificial sediment prepared as described in Appendix 5. The depth of the sediment layer should allow for natural burrowing behaviour of the worms (2 cm minimum depth for T. tubifex). Reconstituted water is added to the system. Care should be taken to minimise disturbing the sediment. The water body is gently aerated (e.g. 2 bubbles per second with 0,45 μm-filtered air) via a pasteur pipette positioned 2 cm above the sediment surface. The recommended culture temperature is 20 ± 2 °C.

The worms are added to the culture system with a maximum loading of 20 000 individuals/m² sediment surface. A higher loading may cause a reduction in growth and reproduction rates (43).

In artificial sediment cultures, the worms have to be fed. A diet consisting of finely ground fish food, e.g. TetraMin® can serve as additional nutrition (8); Klerks 1994, personal communication. The feeding rates should allow for sufficient growth and reproduction and should keep build-up of ammonia and fungal growth in the culture at a minimum. Food may be administered twice a week (e.g. 0,6 - 0,8 mg per cm² of sediment surface). Practical experience has shown that application of food suspended and homogenised in deionised water may facilitate homogeneous food distribution on the sediment surface in the culture containers.

To avoid accumulation of ammonia, the overlying water should be exchanged using a flow-through system, or, at least once a week, manually. Sediment should be changed every three months in the stock cultures.

Sampling of worms from the culture can be done by sieving the culture sediment through a 1 mm sieve if only adults are required. For retaining cocoons a 0,5 mm mesh, and for juvenile worms a 0,25 mm sieve is suitable. The sieves can be placed into reconstituted water after the sediment has passed through. The worms leave the mesh and can then be picked from the water using a soft steel forceps or a pipette with fire-polished edges.

Only intact and clearly identified specimens of Tubifex tubifex (e.g. (64)) are used to start a test or new cultures. Diseased or injured worms as well as cocoons infested with fungal hyphae have to be discarded.

A synchronised culture can provide worms of a specified age in suitable intervals when desired. New culture vessels are set up in the chosen intervals (e.g. every two weeks), starting with animals of a certain age (e.g. cocoons). At the culture conditions described here the worms are adult after 8 - 10 weeks. The cultures can be harvested, when the worms have laid new cocoons, e.g. after ten weeks. The sampled adults can be used for tests, and new cultures can be started with the cocoons.

Lumbriculus variegatus (MÜLLER), Lumbriculidae, Oligochaeta

Lumbriculus variegatus (Lumbriculidae, Oligochaeta) is also an inhabitant of freshwater sediments worldwide and is widely used in ecotoxicological testing. Information on the biology, culture conditions, and sensitivity of the species can be obtained from (1)(6)(9)(36). Lumbriculus variegatus can also be cultured in the artificial sediment recommended for T. tubifex according to (8) within certain limitations. Since, in nature L. variegatus prefers more coarse sediments than T. tubifex (59), laboratory cultures with the artificial sediment used for T. tubifex may cease after 4 to 6 months. Practical experience has shown that L. variegatus can be held in a sandy substratum (e.g. quartz sand, fine gravel) in a flow-through system using fish food as nutritional source over several years without renewing the substratum. A major advantage of L. variegatus over other aquatic oligochaete species is its quick reproduction, resulting in rapidly increasing biomass in laboratory-cultured populations (1)(6)(9)(10).

Culture methods

Culture conditions for Lumbriculus variegatus are outlined in detail in Phipps et al. (1993) (10), Brunson et al. (1998) (28), ASTM (2000) (1), U.S. EPA (2000) (6). A short summary of these conditions is given below.

The worms can be cultured in large aquaria (57 - 80 l) at 23 °C with a 16L:8D photoperiod (100 - 1 000 lux) using daily renewed natural water (45 - 50 l per aquarium). The substrate is prepared by cutting unbleached brown paper towels into strips, which may then be blended with culture water for a few seconds to result in small pieces of paper substrate. This substrate can then directly be used in the Lumbriculus culture aquaria by covering the bottom area of the tank, or be stored frozen in deionised water for later use. New substrate in the tank will generally last for about two months.

Each worm culture is started with 500 - 1 000 worms, and fed a 10 ml suspension containing 6 g of trout starter food 3 times per week under renewal or flow-through conditions. Static or semi-static cultures should receive lower feeding rates to prevent bacterial and fungal growth. Food and paper substrate should be analysed for the substances to be used in bioaccumulation tests.

Under these conditions the number of individuals in the culture generally doubles in about 10 to 14 d.

Lumbriculus variegatus can be removed from the cultures e.g. by transferring substrate with a fine mesh net, or organisms using a fire polished wide mouth (about 5 mm diameter) glass pipette, to a separate beaker. If substrate is co-transferred to this beaker, the beaker containing worms and substrate is left overnight under flow-through conditions, which will remove the substrate from the beaker, while the worms remain at the bottom of the vessel. They can then be introduced to newly prepared culture tanks, or processed further for the test as outlined in (1) and (6). Injuries or autotomy in the worms should be prevented, e.g. by using pipettes with fire polished edges, or stainless steel picks for handling these worms.

An issue to be regarded critically when using L. variegatus in sediment bioaccumulation tests is its reproduction mode (architomy followed by morphallaxis). This asexual reproduction results in two fragments, which do not feed for a certain period until the head or tail part is regenerated (e.g. (36)(37)). This means that in L. variegatus sediment and contaminant uptake via ingestion may not take place continuously as in tubificids, which do not reproduce by fragmentation.

Therefore, a synchronisation should be performed to minimise uncontrolled reproduction and regeneration, and subsequent high variation in test results. Such variation can occur, when some individuals, which have fragmented and therefore do not feed for a certain time period, are less exposed to the test substance than other individuals, which do not fragment during the test, e.g. (38). 10 to 14 days before the start of exposure, the worms should be artificially fragmented (synchronisation) (65). Large worms should be used, which preferably do not show signs of recent fragmentation. These worms can be placed onto a glass slide in a drop of culture water, and dissected in the median body region with a scalpel. Care should be taken that the posterior ends are of similar size. The posterior ends should then be left to regenerate new heads in a culture vessel containing the same substrate as used in the culture and reconstituted water until the start of exposure. Regeneration of new heads is indicated when the synchronised worms are burrowing in the substrate (presence of regenerated heads may be confirmed by inspecting a representative subsample under a binocular microscope). The test organisms are thereafter expected to be in a similar physiological state. This means, that when regeneration by morphallaxis occurs in synchronised worms during the test, virtually all animals are expected to be equally exposed to the spiked sediment. Feeding of the synchronised worms should be done as soon as the worms are starting to burrow in the substrate, or 7 d after dissection. The feeding regimen should be comparable to the regular cultures, but it may be advisable to feed the synchronised worms with the same food source as is to be used in the test. The worms should be held at test temperature, at 20 ± 2 °C. After regenerating, intact complete worms of similar size, which are actively swimming or crawling upon a gentle mechanical stimulus, should be used for the test. Injuries or autotomy in the worms should be prevented, e.g. by using pipettes with fire polished edges, or stainless steel picks for handling these worms.

When using Lumbriculus variegatus in the test, due to the specific reproduction mode of this species, an increase of the number of worms should occur during the test, if conditions are appropriate (6). A lack of reproduction in a bioaccumulation test with L. variegatus should be recorded, and considered when interpreting the test results.

Branchiura sowerbyi (BEDDARD), Tubificidae, Oligochaeta (not validated in ring test)

Branchiura sowerbyi inhabits a variety of sediment types of reservoirs, lakes, ponds and rivers, originally in tropical areas. They can be also found in warm water bodies of the northern hemisphere. However, they are more abundant in mud-clay sediments with high organic matter content. Furthermore, the worms are living in the sediment layer. Even the posterior end of the worms is usually burrowed. This species is easily identified from the gill filaments on their posterior part. The adults can reach a length of 9 - 11 cm and a wet weight of 40-50 mg. The worms have a high rate of reproduction, show population doubling times of less than 2 weeks and under the conditions of temperature and feeding described below (Aston et al., 1982, (65)). B. sowerbyi has been used both in toxicity and bioaccumulation studies (Marchese & Brinkhurst 1996, (31) Roghair et al. 1996, (67) respectively).

Culture methods

A summary of culture conditions for Branchiura sowerbyi is given below (provided by Mercedes R. Marchese, INALI, Argentina, and Carla J. Roghair, RIVM, The Netherlands).

No single technique for culturing the test organisms is required. The organisms can be cultured using uncontaminated, natural sediment (31). Practical experience showed that a medium consisting of natural sediment and sand improves the condition of the worms compared to pure natural sediment (32)(67). 3 L-beakers containing 1 500 ml sediment/water medium, consisting of 375 ml of natural uncontaminated sediment (about 10 % Total Organic Carbon; about 17 % of the particles ≤ 63 μm), 375 ml of clean sand (M32), and 750 ml of reconstituted or dechlorinated tap water can be used for the culture (31)(32)(67). Paper towels also can be used as a substrate for culturing, but population growth is lower than in natural sediment. In semi-static systems the water layer in the beaker is slowly aerated, and the overlying water should be renewed weekly.

Each beaker contains 25 young worms to start with. After two months the large worms are picked out of the sediment with a pair of tweezers and are put in a new beaker with freshly made sediment/water medium. The old beaker also contains cocoons and young worms. Up to 400 young worms per beaker can be harvested in this way. Adults worms can be used for reproduction for at least one year.

The cultures should be maintained at a temperature of 21 to 25 °C. Variation of temperature should be kept below ± 2 °C. The time required for embryonic development from an egg being laid until the young leaves the cocoon is approximately three weeks at 25 °C. The egg production obtained per surviving worm in B. sowerbyi was found to range from 6,36 (31) to 11,2 (30) in mud at 25 °C. The number of eggs per cocoon ranges from 1,8 to 2,8 (66)(69) or up to 8 (68).

Dissolved oxygen, water hardness, temperature, and pH should be measured weekly. Fish food (e.g. TetraMin®) can be added as suspension two or three times per week ad libitum. The worms can also be fed with thawed lettuce ad libitum.

A major advantage of this species is the high individual biomass (up to 40 - 50 mg wet weight per individual). Therefore this species may be used for testing bioaccumulation of non-radiolabelled test substances. It can be exposed in the systems used for T. tubifex or L. variegatus with a single individual per replicate (11). Replication, however, should then be increased, unless larger test chambers are used (11). Also, the validity criterion related to burrowing behaviour needs to be adjusted for this species.

LITERATURE

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• (22)The following chapters of this Annex:

Chapter A.4, vapour pressure

Chapter A.5, Surface tension

Chapter A.6, Water solubility

Chapter A.8, Partition coefficient, shake flask method

Chapter A.24, Partition coefficient, HPLC method

Chapter C.7, degradation — abiotic degradation: hydrolysis as a function of pH

Chapter C.4 A-F Determination of ready biodegradability

Chapter C.19, Estimation of the adsorption coefficient (Koc ) on soil and on sewage sludge using high performance liquid chromatography (HPLC)

Chapter C.29, Ready biodegradability CO2 in sealed vessels

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• (33)Chapter C.1 of this Annex, Fish, Acute Toxicity Test.

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• (40)Chapter C.8 of this Annex, Toxicity for Earthworms.

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C.47.   FISH, EARLY-LIFE STAGE TOXICITY TEST

INTRODUCTION

• 1.This test method is equivalent to OECD test guideline (TG) 210 (2013). Tests with the early-life stages of fish are intended to define the lethal and sub-lethal effects of chemicals on the stages and species tested. They yield information of value for the estimation of the chronic lethal and sub-lethal effects of the chemical on other fish species.

- 2.Test guideline 210 is based on a proposal from the United Kingdom which was discussed at a meeting of OECD experts convened at Medmenham (United Kingdom) in November 1988 and further updated in 2013 to reflect experience in using the test and recommendations from an OECD workshop on fish toxicity testing, held in September 2010 (1).

PRINCIPLE OF THE TEST

- 3.The early-life stages of fish are exposed to a range of concentrations of the test chemical dissolved in water. Flow-through conditions are preferred; however, if it is not possible semi-static conditions are acceptable. For details the OECD guidance document on aquatic toxicity testing of difficult substances and mixtures should be consulted (2). The test is initiated by placing fertilised eggs in test chambers and is continued for a species-specific time period that is necessary for the control fish to reach a juvenile life-stage. Lethal and sub-lethal effects are assessed and compared with control values to determine the lowest observed effect concentration (LOEC) in order to determine the (i) no observed effect concentration (NOEC) and/or (ii) ECx (e.g. EC10, EC20) by using a regression model to estimate the concentration that would cause a x % change in the effect measured. Reporting of relevant effect concentrations and parameters may depend upon the regulatory framework. The test concentrations should bracket the ECx so that the ECx comes from interpolation rather than extrapolation (see Appendix 1 for definitions).

INFORMATION ON THE TEST CHEMICAL

• 4.Test chemical refers to what is being tested. The water solubility (see chapter A.6 of this Annex) and the vapour pressure (see chapter A.4 of this Annex) of the test chemical should be known and a reliable analytical method for the quantification of the chemical in the test solutions with known and reported accuracy and limit of quantification should be available. Although not necessary to conduct the test, results from an acute toxicity test (see chapters C.1 or C.49 of this Annex), preferably performed with the species chosen for this test, may provide useful information.

• 5.If the test method is used for the testing of a mixture, its composition should as far as possible be characterised, e.g. by the chemical identity of its constituents, their quantitative occurrence and their substance-specific properties (like those mentioned above). Before use of the test method for regulatory testing of a mixture, it should be considered whether it will provide acceptable results for the intended regulatory purpose.

- 6.Useful information includes the structural formula, purity of the substance, water solubility, stability in water and light, pKa, Pow and results of a test for ready biodegradability (e.g. chapters C.4 or C.29 of this Annex).

VALIDITY OF THE TEST

• 7.For a test to be valid the following conditions apply:

• — the dissolved oxygen concentration should be > 60 % of the air saturation value throughout the test;

• — the water temperature should not differ by more than ± 1,5 °C between test chambers or between successive days at any time during the test, and should be within the temperature ranges specified for the test species (Appendix 2);

• — the analytical measure of the test concentrations is compulsory.

• — overall survival of fertilised eggs and post-hatch success in the controls and, where relevant, in the solvent controls should be greater than or equal to the limits defined in Appendix 2.

- 8.If a minor deviation from the validity criteria is observed, the consequences should be considered in relation to the reliability of the test data and these considerations should be included in the report. Effects on survival, hatch or growth occurring in the solvent control, when compared to the negative control, should be reported and discussed in the context of the reliability of the test data.

DESCRIPTION OF THE METHOD

Test chambers

- 9.Any glass, stainless steel or other chemically inert vessels can be used. As silicone is known to have a strong capacity to absorb lipophilic substances, the use of silicone tubing in flow-through studies and use of silicone seals in contact with water should be minimised by the use of e.g. monoblock glass aquaria. The dimensions of the vessels should be large enough to allow proper growth in the control, maintenance of dissolved oxygen concentration (e.g. for small fish species, a 7 L tank volume will achieve this) and compliance with the loading rate criteria given in paragraph 19. It is desirable that test chambers be randomly positioned in the test area. A randomised block design with each treatment being present in each block is preferable to a completely randomised design. The test chambers should be shielded from unwanted disturbance. The test system should preferably be conditioned with concentrations of the test chemical for a sufficient duration to demonstrate stable exposure concentrations prior to the introduction of test organisms.

Selection of species

- 10.Recommended fish species are given in Table 1. This does not preclude the use of other species, but the test procedure may have to be adapted to provide suitable test conditions. The rationale for the selection of the species and the experimental method should be reported in this case.

Holding of the brood fish

- 11.Details on holding the brood stock under satisfactory conditions may be found in Appendix 3 and the references cited (3)(4)(5).

Handling of fertilised eggs, embryos and larvae

• 12.Initially, fertilised eggs, embryos and larvae may be exposed within the main vessel in smaller glass or stainless steel vessels, fitted with mesh sides or ends to permit a flow of test solution through the vessel. Non-turbulent flow-through in these small vessels may be induced by suspending them from an arm arranged to move the vessel up and down but always keeping the organisms submerged. Fertilised eggs of salmonid fishes can be supported on racks or meshes with apertures sufficiently large to allow larvae to drop through after hatching.

- 13.Where egg containers, grids or meshes have been used to hold eggs within the main test vessel, these restraints should be removed after the larvae hatch, according to the guidance in Appendix 3, except that meshes should be retained to prevent the escape of the larvae. If there is a need to transfer the larvae, they should not be exposed to the air and nets should not be used to release larvae from egg containers. The timing of this transfer varies with the species and should be documented in the report. However, a transfer may not always be necessary.

Water

- 14.Any water in which the test species shows suitable long-term survival and growth may be used as test water (see Appendix 4). It should be of constant quality during the period of the test. In order to ensure that the dilution water will not unduly influence the test result (for example by complexation of test chemical), or adversely affect the performance of the brood stock, samples should be taken at intervals for analysis. Measurements of heavy metals (e.g. Cu, Pb, Zn, Hg, Cd, Ni), major anions and cations (e.g. Ca²⁺, Mg²⁺, Na⁺, K⁺, Cl^–, SO4^2–), ammonia, total residual chlorine pesticides, total organic carbon and suspended solids should be made, for example, on a bi-annual basis where a dilution water is known to be relatively constant in quality. If the water is known to be of variable quality the measurements have to be conducted more often; the frequency is dependent of how variable the quality is. Some chemical characteristics of an acceptable dilution water are listed in Appendix 4.

Test solutions

• 15.For flow-through tests, a system which continually dispenses and dilutes a stock solution of the test chemical (e.g. metering pump, proportional diluter, saturator system) is required to deliver a series of concentrations to the test chambers. The flow rates of stock solutions and dilution water should be checked at intervals during the test and should not vary by more than 10 % throughout the test. A flow rate equivalent to at least five test chamber volumes per 24 hours has been found suitable (3). However, if the loading rate specified in paragraph 19 is respected, a lower flow rate of e.g. 2-3 test chamber volumes is possible to prevent quick removal of food.

• 16.Test solutions of the chosen concentrations are prepared by dilution of a stock solution. The stock solution should preferably be prepared by simply mixing or agitating the test chemical in dilution water by using mechanical means (e.g. stirring and/or ultrasonication). Saturation columns (solubility columns) or passive dosing methods (6) can be used for achieving a suitable concentrated stock solution. The use of a solvent carrier is not recommended. However, in case a solvent is necessary, a solvent control should be run in parallel, at the same solvent concentration as the chemical treatments; i.e. the solvent level should preferably be equal across all concentrations as well as the solvent control. For some diluter systems this might be technically difficult; here the solvent concentration in the solvent control should be equal to the highest solvent concentration in the treatment group. For difficult to test substances, the OECD Guidance Document No. 23 on aquatic toxicity testing of difficult substances and mixtures should be consulted (2). If a solvent is used, the choice of solvent will be determined by the chemical properties of the substance. The OECD Guidance Document No. 23 recommends a maximum concentration of 100 μl/l. To avoid potential effect of the solvent on endpoints measured (7), it is recommended to keep solvent concentration as low as possible.

- 17.For a semi-static test, two different renewal procedures may be followed. Either new test solutions are prepared in clean vessels and surviving eggs and larvae gently transferred into the new vessels, or the test organisms are retained in the test vessels whilst a proportion (at least two thirds) of the test solution / control volume is changed.

PROCEDURE

Conditions of Exposure

Duration

- 18.The test should start as soon as possible after the eggs have been fertilised and preferably being immersed in the test solutions before cleavage of the blastodisc commences, or as close as possible after this stage. The test duration will depend upon the species used. Some recommended durations are given in Appendix 2.

Loading

- 19.The number of fertilised eggs at the start of the test should be sufficient to meet statistical requirements. They should be randomly distributed among treatments, and at least 80 eggs, divided equally between at least four replicate test chambers, should be used per concentration. The loading rate (biomass per volume of test solution) should be low enough in order that a dissolved oxygen concentration of at least 60 % of the air saturation value can be maintained without aeration during the egg and larval stage. For flow-through tests, a loading rate not exceeding 0,5 g/l wet weight per 24 hours and not exceeding 5 g/l of solution at any time has been recommended (3).

Light and temperature

- 20.The photoperiod and water temperature should be appropriate for the test species (see Appendix 2).

Feeding

- 21.Food and feeding are critical, and it is essential that the correct food for each life-stage is supplied from an appropriate time and at a level sufficient to support normal growth. Feeding should be approximately equal across replicates unless adjusted to account for mortality. Surplus food and faeces should be removed as necessary, to avoid accumulation of waste. Detailed feeding regimes are given in Appendix 3 but, as experience is gained, food and feeding regimes are continually being refined to improve survival and optimise growth. Live food provides a source of environmental enrichment and therefore should be used in place of or in addition to dry or frozen food whenever appropriate to the species and life stage.

Test concentrations

- 22.Normally five concentrations of the test chemical, with a minimum of four replicates per concentration, spaced by a constant factor not exceeding 3,2 are required. If available, information on the acute testing, preferable with the same species and/or a range finding test should be considered (1) when selecting the range of test concentrations. However, all sources of information should be considered when selecting the range of test concentrations, including sources like e.g. read across, fish embryo acute toxicity test data. A limit test, or an extended limit test, with fewer than five concentrations as the definitive test may be acceptable where empirical NOECs only are to be established. Justification should be provided if fewer than five concentrations are used. Concentrations of the test chemical higher than the 96 hour LC50 or 10 mg/l, whichever is the lower, need not be tested.

Controls

- 23.A dilution-water control and, if needed, a solvent control containing the solvent carrier only should be run in addition to the test chemical concentration series (see paragraph 16).

Frequency of Analytical Determinations and Measurements

• 24.Prior to initiation of the exposure period, proper function of the chemical delivery system across all replicates should be ensured (for example, by measuring test concentrations). Analytical methods required should be established, including an appropriate limit of quantification (LOQ) and sufficient knowledge on the substance stability in the test system. During the test, the concentrations of the test chemical are determined at regular intervals to characterise exposure. A minimum of five determinations is necessary. In flow-through systems, analytical measurements of the test chemical in one replicate per concentration should be made at least once a week changing systematically amongst replicates. Additional analytical determinations will often improve the quality of the test outcome. Samples may need to be filtered to remove any particulate matter (e.g. using a 0,45 μm pore size) or centrifuged to ensure that the determinations are made on the chemical in true solution. In order to reduce adsorption of the test chemical, the filters should be saturated before the use. When the measured concentrations do not remain within 80-120 % of the nominal concentration, the effect concentrations should be determined and expressed relative to the arithmetic mean concentration for flow-through tests (see Appendix 6 of the test method C.20 for the calculation of the arithmetic mean (8)), and expressed relative to the geometric mean of the measured concentrations for semi-static tests (see Chapter 5 in the OECD Guidance Document on aquatic toxicity testing of difficult substances and mixtures (2)).

- 25.During the test, dissolved oxygen, pH, and temperature should be measured in all test vessels, at least weekly, and salinity and hardness, if warranted, at the beginning and end of the test. Temperature should preferably be monitored continuously in at least one test vessel.

Observations

• 1. Stage of embryonic development : the embryonic stage at the beginning of exposure to the test chemical should be verified as precisely as possible. This can be done using a representative sample of eggs suitably preserved and cleaned.

• 1. Hatching and survival

:

observations on hatching and survival should be made at least once daily and numbers recorded. If fungus on eggs is observed early in embryonic development (e.g. at day one or two of test), those eggs should be counted and removed. Dead embryos, larvae and juvenile fish should be removed as soon as observed since they can decompose rapidly and may be broken up by the actions of the other fish. Extreme care should be taken when removing dead individuals not to physically damage adjacent eggs/larvae. Signs of death vary according to species and life stage. For example:

• — for fertilised eggs: particularly in the early stages, a marked loss of translucency and change in colouration, caused by coagulation and/or precipitation of protein, leading to a white opaque appearance;

• — for embryos, larvae and juvenile fish: immobility and/or absence of respiratory movement and/or absence of heartbeat and/or lack of reaction to mechanical stimulus.

• 1. Abnormal appearance : the number of larvae or juvenile fish showing abnormality of body form should be recorded at adequate intervals depending on the duration of the test and the nature of the abnormality described. It should be noted that abnormal larvae and juvenile fish occur naturally and can be of the order of several percent in the control(s) in some species. Where deformities and associated abnormal behaviour are considered so severe that there is considerable suffering to the organism, and it has reached a point beyond which it will not recover, it may be removed from the test. Such animals should be euthanised and treated as mortalities for subsequent data analysis. Normal embryonic development has been documented for most species recommended in this test method (9) (10) (11) (12).

• 1. Abnormal behaviour : abnormalities, e.g. hyperventilation, uncoordinated swimming, atypical quiescence and atypical feeding behaviour should be recorded at adequate intervals depending on the duration of the test (e.g. once daily for warm water species). These effects, although difficult to quantify, can, when observed, aid in the interpretation of mortality data.

• 1. Weight : at the end of the test, all surviving fish are weighed at least on a replicate basis (reporting the number of animals in the replicate and the mean weight per animal): wet weight — (blotted dry) is preferred, however, dry weight data may also be reported (13).

- 31. Length : at the end of the test, individual lengths are measured. Total length is recommended, if however, caudal fin rot or fin erosion occurs, standard length can be used. The same method should be used for all fish in a given test. Individual length can be measured either by e.g. callipers, digital camera, or calibrated ocular micrometer. Typical minimum lengths are defined in Appendix 2.

DATA AND REPORTING

Treatment of results

• 32.It is recommended that the design of the experiment and selection of statistical test permit adequate power (80 % or higher) to detect changes of biological importance in endpoints where a NOEC is to be reported. Reporting of relevant effect concentrations and parameters may depend upon the regulatory framework. If an ECx is to be reported, the design of the experiment and selection of regression model should permit estimation of ECx so that (i) the 95 % confidence interval reported for ECx does not contain zero and is not overly wide, (ii) the 95 % confidence interval for the predicted mean at ECx does not contain the control mean (iii) there is no significant lack-of-fit of regression model to the data. Either approach requires the identification of the percent change in each endpoint that is important to detect or estimate. The experimental design should be tailored to allow that. When the above conditions for determining the ECx are not satisfied, the NOEC approach should be used. It is not likely that the same percent change applies to all endpoints, nor is it likely that a feasible experiment can be designed that will meet these criteria for all endpoints, so it is important to focus on the endpoints, which are important for the respective experiment in designing the experiment appropriately. Statistical flow diagrams and guidance for each approach are available in Appendixes 5 and 6 to guide in the treatment of data and in the choice of the most appropriate statistical test or model to use. Other statistical approaches may be used, provided they are scientifically justified.

- 33.It will be necessary for variations to be analysed within each set of replicates using analysis of variance or contingency table procedures and appropriate statistical analysis methods be used based on this analysis. In order to make a multiple comparison between the results at the individual concentrations and those for the controls, the step-down Jonckheere-Terpstra or Williams' test is recommended for continuous responses and a step-down Cochran-Armitage test for quantal responses that are consistent with a monotone concentration-response and with no evidence of extra-binomial variance (14). When there is evidence of extra-binomial variance, the Rao-Scott modification of the Cochran-Armitage test is recommended (15) (16) or Williams or Dunnett's (after an arcsin-square-root transform) or Jonckheere-Terpstra test applied to replicate proportions. Where the data are not consistent with a monotone concentration-response, Dunnett's or Dunn's or the Mann-Whitney method may be found useful for continuous responses and Fisher's Exact test for quantal responses (14) (17) (18). Care should be taken where applying any statistical method or model to ensure that the requirements of the method or model are satisfied (e.g. chamber to chamber variability is estimated and accounted for in the experimental design and test or model used). Data are to be evaluated for normality and Appendix 5 indicates what should be done on the residuals from an ANOVA. Appendix 6 discusses additional considerations for the regression approach. Transformations to meet the requirements of a statistical test should be considered. However, transformations to enable the fitting of a regression model require great care, as, for example, a 25 % change in the untransformed response does not correspond to a 25 % change in a transformed response. In all analyses, the test chamber, not the individual fish, is the unit of analysis and the experimental unit and both hypothesis tests and regression should reflect that (3) (14) (19) (20).

Test report

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

Test chemical:

Mono-constituent substance

• — physical appearance, water solubility, and additional relevant physicochemical properties;

• — chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI code, structural formula, purity, chemical identity of impurities as appropriate and practically feasible, etc. (including the organic carbon content, if appropriate.

Multi-constituent substance, UVCBs and mixtures:

• — characterised as far as possible, e.g., by chemical identity (see above), quantitative occurrence and relevant physicochemical properties of the constituents

Test species:

• — scientific name, strain, source and method of collection of the fertilised eggs and subsequent handling.

Test conditions:

• — test procedure used (e.g. semi-static or flow-through, loading);

• — photoperiod(s);

• — test design (e.g. number of test chambers and replicates, number of eggs per replicate, material and size of the test chamber (height, width, volume), water volume per test chamber);

• — method of preparation of stock solutions and frequency of renewal (the solubilising agent and its concentration should be given, when used);

• — method of dosing the test chemical (e.g. pumps, diluting systems)

• — the recovery efficiency of the method and the nominal test concentrations, the limit of quantification, the means of the measured values and their standard deviations in the test vessels and the method by which these were attained and evidence that the measurements refer to the concentrations of the test chemical in true solution;

• — dilution water characteristics: pH, hardness, temperature, dissolved oxygen concentration, residual chlorine levels (if measured), total organic carbon (if measured), suspended solids (if measured), salinity of the test medium (if measured) and any other measurements made;

• — water quality within test vessels, pH, hardness, temperature and dissolved oxygen concentration;

• — detailed information on feeding (e.g. type of food(s), source, amount given and frequency).

Results reported individually (or on a replicate basis) and as mean and coefficient of variation, as appropriate, for the following endpoints:

• — evidence that controls met the overall survival acceptability standard of the test species (Appendix 2);

• — data on mortality at each stage (embryo, larval and juvenile) and cumulative mortality;

• — days to hatch, numbers of larvae hatched each day, and end of hatching;

• — number of healthy fish at end of test;

• — data for length (specify either standard or total) and weight of surviving animals;

• — incidence, description and number of morphological abnormalities, if any;

• — incidence, description and number of behavioural effects, if any;

• — approach for the statistical analysis (regression analysis or analysis of the variance) and treatment of data (statistical test or model used);

• — no observed effect concentration for each response assessed (NOEC);

• — lowest observed effect concentration (at p = 0,05) for each response assessed (LOEC);

• — ECx for each response assessed, if applicable, and confidence intervals (e.g. 90 % or 95 %) and a graph of the fitted model used for its calculation, the slope of the concentration-response curve, the formula of the regression model, the estimated model parameters and their standard errors.

Any deviation from the test method.

Discussion of the results, including any influence of deviations from the test method on the outcome of the test.

Table 1

Fish species recommended for testing

| FRESHWATER | ESTUARINE and MARINE |

| --- | --- | | Oncorhynchus mykiss Rainbow trout | Cyprinodon variegatus Sheepshead minnow | | Pimephales promelas Fathead minnow | Menidia sp. Silverside | | Danio rerio Zebrafish | | | Oryzias latipes Japanese ricefish or Medaka | |

LITERATURE:

• (1) OECD (2012), Fish Toxicity Testing Framework, Environmental Health and Safety Publications Series on Testing and Assessment No.171, OECD, Paris.

• (2) OECD (2000), Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, Environmental Health and Safety Publications, Series on Testing and Assessment. No. 23, OECD Paris.

• (3) ASTM (1988), Standard Guide for Conducting Early Life-Stage Toxicity Tests with Fishes. American Society for Testing and Materials, E 1241-88. 26 pp.

• (4) Brauhn, J.L. and R.A. Schoettger (1975), Acquisition and Culture of Research Fish: Rainbow trout, Fathead minnows, Channel catfish and Bluegills, Ecological Research Series, EPA-660/3-75-011, Duluth, Minnesota.

• (5) Brungs, W.A. and B.R. Jones (1977), Temperature Criteria for Freshwater Fish: Protocol and Procedures, Ecological Research Series EPA-600/3-77-061, Duluth, Minnesota.

• (6) Adolfsson-Erici, et al. (2012), A flow-through passive dosing system for continuously supplying aqueous solutions of hydrophobic chemicals to bioconcentration and aquatic toxicity tests, Chemosphere 86, 593-599.

• (7) Hutchinson, T.H. et al. (2006), Acute and chronic effects of carrier solvents in aquatic organisms: A critical review, Aquatic Toxicology, 76, 69-92.

• (8) Chapter C.20 of this Annex, Daphnia magna Reproduction Test.

• (9) Hansen, D.J. and P.R. Parrish (1977), Suitability of sheepshead minnows (Cyprindon variegatus) for life-cycle toxicity tests, In Aquatic Toxicology and Hazard Evaluation (edited by F.L. Mayer and J.L. Hamelink), ASTM STP 634.

• (10) Kimmel, H. B.et al. (1995), Stages of embryonic development of the zebrafish. Developmental Dynamics, 203:253–310.

• (11) Gonzalez-Doncel, M. et al (2005), A quick reference guide to the normal development of Oryzias latipes (Teleostei, Adrinichthydae) Journal of Applied Ichthyology, 20:1–14.

• (12) Devlin, E.W. et al. (1996), Prehatching Development of the Fathead Minnow, Pimephales promelas Rafinesque. EPA/600/R-96/079. USEPA, Office of Research and Development, Washington, D.C..

• (13) Oris, J.T., S.C. Belanger, and A.J. Bailer, (2012), Baseline characteristics and statistical implications for the OECD 210 Fish Early Life Stage Chronic Toxicity Test, Environmental Toxicology and Chemistry 31; 2, 370 - 376.

• (14) OECD (2006). Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application, Environmental Health and Safety Publications Series on Testing and Assessment No.54, OECD, Paris.

• (15) Rao, J.N.K. and A.J. Scott (1992), A simple method for the analysis of clustered binary data, Biometrics 48, 577-585.

• (16) Rao, J.N.K. and A.J. Scott (1999), A simple method for analyzing overdispersion in clustered Poisson data, Statistics in Medicine 18, 1373-1385.

• (17) Dunnett C.W. (1955), A multiple comparisons procedure for comparing several treatments with a control, Journal of Ameican Statistical Association, 50, 1096-1121.

• (18) Dunnett C.W. (1964), New tables for multiple comparisons with a control. Biometrics, 20, 482-491.

• (19) Rand, G.M. and S.R. Petrocelli (1985), Fundamentals of Aquatic Toxicology. Hemisphere Publication Corporation, New York.

- (20) McClave, J.T., J.H. Sullivan and J.G. Pearson (1980). Statistical Analysis of Fish Chronic Toxicity Test Data, Proceedings of 4th Aquatic Toxicology Symposium, ASTM, Philadelphia.

Appendix 1

DEFINITIONS:

Fork length (FL): refers to the length from the tip of the snout to the end of the middle caudal fin rays and is used in fishes in which it is difficult to tell where the vertebral column ends (www.fishbase.org)

Standard length (SL): refers to the length of a fish measured from the tip of the snout to the posterior end of the last vertebra or to the posterior end of the midlateral portion of the hypural plate. Simply put, this measurement excludes the length of the caudal fin. (www.fishbase.org)

Total length (TL): refers to the length from the tip of the snout to the tip of the longer lobe of the caudal fin, usually measured with the lobes compressed along the midline. It is a straight-line measure, not measured over the curve of the body (www.fishbase.org)

Figure 1

Description of the different lengths used

Image omitted; see official source

Text of image

Standard length

Fork length

Total length

Chemical: 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.

Lowest observed effect concentration (LOEC) is the lowest tested concentration of a test chemical at which the chemical is observed to have a statistically significant effect (at p < 0,05) when compared with the control. However, all test concentrations above the LOEC should have a harmful effect equal to or greater than those observed at the LOEC. When these two conditions cannot be satisfied, a full explanation should be given for how the LOEC (and hence the NOEC) has been selected. Appendixes 5 and 6 provide guidance.

No observed effect concentration (NOEC) is the test concentration immediately below the LOEC, which when compared with the control, has no statistically significant effect (p < 0,05), within a stated exposure period.

Test chemical: Any substance or mixture tested using this test method

UVCB: substances of unknown or variable composition, complex reaction products or biological materials.

IUPAC: International Union of Pure and Applied Chemistry.

SMILES: Simplified Molecular Input Line Entry Specification.

Appendix 2

TEST CONDITIONS, DURATION AND SURVIVAL CRITERIA FOR RECOMMENDED SPECIES

SPECIES	TEST CONDITIONS	TEST CONDITIONS	TEST CONDITIONS	RECOMMENDED DURATION OF TEST	Typical minimum mean total length of control fish at the end of the study (mm) [^E0246]	SURVIVAL OF CONTROLS (minimum)	SURVIVAL OF CONTROLS (minimum)
	Temperature (°C)	Salinity (0/00)	Photoperiod (hrs)	RECOMMENDED DURATION OF TEST	Typical minimum mean total length of control fish at the end of the study (mm) [^E0246]	Hatching success	Post-hatch success
Freshwater:	Freshwater:	Freshwater:	Freshwater:	Freshwater:	Freshwater:	Freshwater:	Freshwater:
Oncorhynchus mykiss Rainbow trout	10 ± 1,5 [^E0247]		12 - 16 [^E0248]	RECOMMENDED DURATION OF TEST	Typical minimum mean total length of control fish at the end of the study (mm) [^E0246]	2 weeks after controls are free-feeding (or 60 days post-hatch)	40	75 %	75 %
Pimephales promelas Fathead minnow	25 ± 1,5		16	RECOMMENDED DURATION OF TEST	Typical minimum mean total length of control fish at the end of the study (mm) [^E0246]	32 days from start of test (or 28 days post-hatch)	18	70 %	75 %
Danio rerio Zebrafish	26 ± 1,5		12 - 16 [^E0249]	30 days post-hatch	11	70 %	75 %
Oryzias latipes Japanese Ricefish or Medaka	25 ± 2		12 - 16 [^E0249]	30 days post-hatch	17	80 %	80 %
Estuarine and Marine:	Estuarine and Marine:	Estuarine and Marine:	Estuarine and Marine:	Estuarine and Marine:	Estuarine and Marine:	Estuarine and Marine:	Estuarine and Marine:
Cyprinodon variegatus Sheepshead minnow	25 ± 1,5	15-35 [^E0250]	12 - 16 [^E0249]	32 days from start of test (or 28 days post-hatch)	17	75 %	80 %
Menidia sp. Silverside	22 - 25	15-35 [^E0250]	13	28 days	20	80 %	60 %

Typical minimum mean total length is not a validity criterion but deviations below the figure indicated should be carefully examined in relation to the sensitivity of the test. The minimum mean total length is derived from a selection of data available at the current time.

The particular strain of rainbow trout tested may necessitate the use of other temperatures. Brood stock must be held at the same temperature as that to be used for the eggs. After receipt of eggs from a commercial breeder, a short adaptation (e.g. 1-2 h) to test temperature after arrival is necessary.

Darkness for larvae until one week after hatching except when they are being inspected, then subdued lighting throughout test (12-16 hour photoperiod) (⁴).

For any given test conditions, light regime should be constant.

[^E0250]: (⁵)

For any given test this shall be performed to ± 2 ⁰/00.

Appendix 3

FEEDING AND HANDLING GUIDANCE FOR BROOD AND TEST ANIMALS OF RECOMMENDED SPECIES

SPECIES	FOOD [^E0251]	FOOD [^E0251]	FOOD [^E0251]	FOOD [^E0251]	POST-HATCH TRANSFER TIME	TIME TO FIRST FEEDING
SPECIES	Brood fish	Newly-hatched larvae	Juveniles	Juveniles	POST-HATCH TRANSFER TIME	TIME TO FIRST FEEDING
SPECIES	Brood fish	Newly-hatched larvae	Type	Frequency	POST-HATCH TRANSFER TIME	TIME TO FIRST FEEDING
SPECIES	Brood fish	Newly-hatched larvae	Freshwater:	Freshwater:	Freshwater:	Freshwater:	Freshwater:	Freshwater:	Freshwater:
SPECIES	Brood fish	Newly-hatched larvae	Oncorhynchus mykiss Rainbow trout	trout food	POST-HATCH TRANSFER TIME	TIME TO FIRST FEEDING	None [^E0252]	trout starter BSN	2-4 feeds per day	14-16 days post-hatch or at swim-up (not essential)	19 days post hatch or at swim-up
Pimephales promelas Fathead minnow	BSN, flake food, FBS	BSN	BSN48, flake food	2-3 times a day	POST-HATCH TRANSFER TIME	TIME TO FIRST FEEDING	once hatching is 90 %	2 day post hatch
Danio rerio Zebrafish	BSN, flake food	Commercial larvae food, protozoa [^E0253], protein [^E0254]	BSN48, flake food,	BSN once daily; flake food twice daily	once hatching is 90 %	2 days post hatch
Oryzias latipes Japanese Ricefish or Medaka	flake food	BSN, flake food (or protozoa or rotifers)	BSN48, flake food (or rotifers)	BSN once daily; flake food twice daily or flake food and rotifers once daily	not applicable	6-7 days post spawn
Estuarine and Marine:	Estuarine and Marine:	Estuarine and Marine:	Estuarine and Marine:	Estuarine and Marine:	Estuarine and Marine:	Estuarine and Marine:
Cyprinodon varieqatus Sheepshead minnow	BSN, flake food, FBS	BSN	BSN48	2-3 feeds per day	not applicable	1 day post hatch/swim-up
Menidia sp. Silverside	BSN48, flake food	BSN	BSN48	2-3 feeds per day	not applicable	1 day post hatch/swim-up
FBS frozen brine shrimps; adults Artemia sp BSN brine shrimp nauplii; newly hatched BSN48 brine shrimp nauplii; 48 hours old	FBS frozen brine shrimps; adults Artemia sp BSN brine shrimp nauplii; newly hatched BSN48 brine shrimp nauplii; 48 hours old	FBS frozen brine shrimps; adults Artemia sp BSN brine shrimp nauplii; newly hatched BSN48 brine shrimp nauplii; 48 hours old	FBS frozen brine shrimps; adults Artemia sp BSN brine shrimp nauplii; newly hatched BSN48 brine shrimp nauplii; 48 hours old	FBS frozen brine shrimps; adults Artemia sp BSN brine shrimp nauplii; newly hatched BSN48 brine shrimp nauplii; 48 hours old	FBS frozen brine shrimps; adults Artemia sp BSN brine shrimp nauplii; newly hatched BSN48 brine shrimp nauplii; 48 hours old	FBS frozen brine shrimps; adults Artemia sp BSN brine shrimp nauplii; newly hatched BSN48 brine shrimp nauplii; 48 hours old

Food should be given to satiation. Surplus food and faeces should be removed, as necessary to avoid accumulation of waste

yolk-sac larvae require no food

filtered from mixed culture

[^E0254]: (³)

granules from fermentation process.

Appendix 4

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 5

STATISTICAL GUIDANCE FOR NOEC DETERMINATION

General

The replicate tank is the unit of analysis. Thus, for continuous measurements, such as size, the replicate mean or median should be calculated and these replicate values are the data for analysis. The power of the tests used should be demonstrated, preferably based on an adequate historical database for each lab. The size effect that can be detected with 75-80 % power should be provided for each endpoint with the statistical test to be used.

The databases available at the time of development of this test method establish the power possible under the recommended statistical procedures. An individual lab should demonstrate its ability to meet this power requirement either by conducting its own power analysis or by demonstrating that the Coefficient of Variation (CV) for each response does not exceed the 90th percentile of CVs used in developing the TG. Table 1 provides these CVs. If only replicate means or medians are available, then the within-replicate CV can be ignored.

Table 1

90th Percentile CVs for selected Freshwater Species

Species	Response	CV_Between Replicates	CV_Within Replicates
Rainbow Trout	Length	17,4	9,8
Rainbow Trout	Weight	10,1	28
Fathead Minnow	Length	16,9	13,5
Fathead Minnow	Weight	11,7	38,7
Zebrafish	Length	43,7	11,7
Zebrafish	Weight	11,9	32,8

For almost all statistical tests used to evaluate laboratory toxicology studies, the comparisons of interest are of treatment groups to control. For that reason, it is not appropriate to require a significant ANOVA F-test before using Dunnett's or Williams' test or a significant Kruskal-Wallis test before using the Jonckheere-Terpstra, Mann-Whitney, or Dunn test (Hochberg and Tamhane 1987, Hsu 1996, Dunnett 1955, 1964, Williams 1971, 1972, 1975, 1977, Robertson et al. 1988, Jonckheere 1954, Dunn 1964).

Dunnett's test has a built-in multiplicity adjustment and its false positive and false negative rates are adversely affected by using the F-test as a gatekeeper. Similarly, the step-down Williams and Jonckheere-Terpstra tests using a 0,05 significance level at every step preserve an overall 5 % false positive rate and that rate and the power of the tests are adversely affected by using the F- or Kruskal-Wallis test as a gatekeeper. Mann-Whitney and Dunn's test have to be adjusted for multiplicity and the Bonferroni-Holm adjustment is advised.

A thorough discussion of most of the recommendations on hypothesis testing and verification of assumptions underlying these tests is given in OECD (2006), which also contains an extensive bibliography.

Treatment of Controls when a Solvent is Used