12th Version Nov 2018 OECD GUIDELINE FOR TESTING OF ...

1 12th Version

Nov 2018

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OECD GUIDELINE FOR TESTING OF CHEMICALS

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Draft revised version

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Fish, Acute Toxicity Test

5 INTRODUCTION

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7 1. OECD Guidelines for Testing of Chemicals are periodically reviewed to incorporate

8 scientific progress, changing regulatory needs, and animal-welfare considerations. The

9 revision of this Guideline (originally adopted in 1981, updated in 1984, 1992), reflects also

10 updates on a series of recommendations from the OECD Fish Toxicity Testing Framework

11 2011 (OECD Guidance Document No. 171) (1), and include:

12 (a) The possibility to use the Threshold Approach for Acute Fish Toxicity Testing

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(Guidance Document No. 126) (2)

14 (b) Consideration of alternative methods, such as Quantitative Structure Activity

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Relationships (QSAR)

16 (c) A specification that testing the minimum concentration causing 100% and the

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maximum concentration causing 0% mortality are not mandatory requirements (e.g. no

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need to test additional concentrations just to demonstrate 0 and/or 100% mortality)

19 (d) Guidance on the circumstances under which a water control is required when solvent is

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used (Guidance Document No. 23) (3)

21 (e) The introduction of estuarine and marine fish species in the recommended species list.

22 (f) The enhanced recording of visible abnormalities (also referred to as sublethal clinical

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signs) that fish may display during the exposure in order to improve our ability to

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predict chemical toxicity and minimise vertebrate suffering of laboratory animals in the

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future (2) analogously to those described in Guidance Document No. 19 on the

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recognition, assessment, and use of clinical signs as humane endpoints for

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experimental animals used in safety evaluation (OECD 2000a) for mammalian studies

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(4)

29 2. In the interest of animal welfare and efficient use of resources, it is important to

30 avoid/reduce the use of animals whenever possible and appropriate. Therefore, before

31 carrying out a fish acute toxicity test according to this guideline, it should be considered

32 whether information on fish acute toxicity could be derived with alternative methods in a

33 weight-of-evidence approach. This would include the use of QSAR, read-across, fish

34 embryos (5), fish cell lines and others. Where testing on fish is required, use of the threshold

35 approach (2) or the limit test as described in ? 31 of this guideline should be considered.

36 37 3.

Definitions used in this Test Guideline are given in Annex 1.

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39 PRINCIPLE OF THE TEST

40 4. The fish are exposed to the test chemical for a period of 96 hours. Mortalities are 41 recorded, and where possible the concentrations estimated to kill 50% of the fish (LC50), are 42 determined.

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43 INITIAL CONSIDERATIONS

44 5. Useful information about chemical-specific properties include the structural formula, 45 molecular weight, purity, stability in water and light, pKa, Koc and Kow, water solubility and 46 vapour pressure, as well as results of a test for ready biodegradability (OECD TG 301) (6) or 47 CO2 in sealed vessels (OECD TG 310) (7). Solubility and vapour pressure can be used to 48 calculate Henry's law constant, which will indicate whether losses due to evaporation of the 49 test chemical may occur. Conduct of this test guideline without the information listed above 50 should be carefully considered as the study design will be dependent on the physicochemical 51 properties of the test chemical and could lead to meaningless or difficult to interpret results. 52 For poorly water soluble test chemicals requiring a longer period of time to be significantly 53 taken up by the test organisms, only long-term tests (preferably OECD 210) should be 54 performed. The steady state conditions are likely not to be reached within the duration of a 55 short-term toxicity test. Other important information, particularly when accompanied with 56 systematic collection of sublethal clinical signs (see Annex 4) includes mode of action (e.g. 57 polar narcosis).

58 6. A validated analytical method, of known accuracy, precision, and sensitivity, for the 59 quantification of the test chemical in the test solution should be available (8), where 60 technically feasible. Performance parameters should be reported (e.g. accuracy, precision, 61 Limit of Detection LoD, Limit of Quantification LoQ, specificity, working range).

62 7. If the Test Guideline is used for the testing of a mixture, a substance of Unknown or 63 Variable composition, Complex reaction products or Biological materials (UVCB) or a multi64 constituent substance, its composition should, as far as possible, be characterised, e.g. by 65 the chemical identity of its constituents, their quantitative occurrence and their chemical66 specific properties (see ? 5). Recommendations about the testing of difficult test chemicals 67 (e.g. mixture, UVCB or multi-constituent substance) are given in Guidance Document No. 23 68 (3). Before use of the Test Guideline for regulatory testing of a mixture, it should be 69 considered whether it will provide results acceptable for the intended regulatory purpose. For 70 the sample preparations of nanomaterials, the relevant Guidance Document on Aquatic and 71 Sediment Toxicological Testing of Nanomaterials should be used (9).

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73 VALIDITY OF THE TEST

74 8. For a test to be valid, the following conditions should be fulfilled:

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- in the control(s) (dilution water control, solvent control), the mortality should not

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exceed 10% (or one fish, if fewer than 10 control fish are tested) at the end of the

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exposure.

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- the dissolved oxygen concentration should be 60% of the air saturation value in all

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test vessels throughout the exposure;

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- analytical measurement of all test concentrations

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- there must be evidence that the concentration of the chemical being tested has been

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satisfactorily maintained, and preferably it should be at least 80% of the nominal

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concentration throughout the test. If the deviation from the nominal concentration is

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greater than 20%, results should be based on the measured concentration (Guidance

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Document No. 23: section 9, particularly ? 178) (3)

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86 9. If a minor deviation from the validity criteria is observed, the consequences should be 87 considered in relation to the reliability of the test data and these considerations should be 88 included in the report.

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90 DESCRIPTION OF THE METHOD

91 Apparatus

92 10. Normal laboratory equipment for the conduct of this assay, with appropriate 93 documentation to validate that the equipment is working correctly, include: 94 (a) oxygen meter; 95 (b) pH meter; 96 (c) light meter; 97 (d) adequate apparatus for temperature control; 98 (e) equipment for determination of hardness of water; 99 (f) equipment for determination of total organic carbon concentration (TOC) and/or chemical 100 oxygen demand (COD); 101 (g) equipment for the determination of concentration of test chemical in test solution; 102 (h) equipment to maintain water temperature and oxygen content as appropriate; 103 (i) tanks made of chemically inert material. 104 105 Test Vessels

106 11. Any glass, stainless steel or other chemically inert vessels can be used. As silicone is 107 known to have a strong capacity to absorb lipophilic chemicals, the use of silicone tubing in 108 flow-through studies and use of silicone seals in contact with water should be minimised. 109 Tubes for dosing should be made of inert material and silicone seals can be avoided by the 110 use of e.g. monoblock glass aquaria. The dimensions of the vessels should be large enough 111 to keep fish free of stress (other than from the tested chemical) and to comply with loading 112 rate criteria given in ? 20. Test vessels should be randomly positioned in the test area and 113 shielded from unwanted disturbance (excessive noise, vibration, light). When testing difficult 114 test chemicals, Guidance Document No. 23 (3) should be consulted and the study design 115 modified appropriately. For example, for some difficult test chemicals (poorly water soluble 116 and adsorptive), pre-conditioning of the flow-through system for a suitable period with 117 appropriate concentrations of the test chemical should be considered in order to ensure 118 stable exposure concentrations (3) prior to the introduction of test organisms. For volatile and 119 other difficult test chemicals, further specific measures should be taken (3). Any silicone 120 materials used in a test should be preferably discarded and not reused for subsequent tests 121 with different test chemicals.

122 Selection of Species

123 12. The selection of species depends on regulatory requirements (industrial chemical, 124 pharmaceutical, biocide or plant protection product) and on environmental exposure 125 scenarios (cold or warm water species, freshwater or estuarine/marine fish). A list of 126 recommended fish species for this test is provided in Annex 2, Table 1. These fish species 127 are readily available, are easy to maintain, and most have historical use in chemical safety 128 testing. They can be bred and cultivated either in fish farms or in the laboratory, under 129 disease-free conditions, providing healthy animals of known provenance for testing. If

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130 species that are not listed in Annex 2, Table 1 are used, the rationale must be reported 131 together with any adaptations to the test guideline's recommendations. Fish used for testing 132 should not be displaying visible signs of disease and stress.

133 Age and Size of Fish

134 13. Fish should be juveniles (see Annex 2, Table 1, for size guidance) and originate from 135 the same source and population to ensure uniformity. The fish should be of the same age (if 136 unknown it can be estimated via the size) and have normal appearance.

137 Holding of Fish

138 14. All fish should be held in the laboratory for at least 9 days before they are used for 139 testing. The first 48 hours constitute a settling-in period. Then, fish should be acclimatised for 140 at least 7 days (48 hours settling-in + 7 days acclimatisation = 9 days) in water similar to test 141 water (see Annex 3 for relevant characteristics) immediately before testing. Holding of fish 142 should be under the following conditions:

143 Photoperiod:

appropriate to the species (see Annex 2, Table 1);

144 Temperature:

appropriate to the species (see Annex 2, Table 1);

145 Oxygen concentration:

at least 80% of air saturation value;

146 Feeding: 147 148 149

three times per week or daily until 24 - 48 hours before the exposure is started. Feed should be given to satiation. Surplus food and faeces should be removed as necessary to avoid accumulation of waste.

150 15. During the acclimatisation period that follows the 48-hour settling-in, mortalities are 151 recorded, and the following criteria applied:

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- mortalities >10% of population in seven days before the start of the test: rejection of

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entire batch;

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- mortalities between 5 and 10% of population in seven days before the start of the

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test: acclimatisation continued for seven additional days;

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- mortalities of ................
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