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1.1 This guide (1) describes procedures for obtaining laboratory data concerning the adverse effects of a test material added to dilution water, but not to food, on certain species of freshwater and saltwater fishes, macroinvertebrates, and amphibians during 2- to 8-day exposures, depending on the species, using the static, renewal, and flow-through techniques. These procedures will probably be useful for conducting acute toxicity tests with many other aquatic species, although modifications might be necessary.

1.2 Other modifications of these procedures might be justified by special needs or circumstances. Although using appropriate procedures is more important than following prescribed procedures, results of tests conducted using unusual procedures are not likely to be comparable to results of many other tests. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting acute tests.

1.3 Selection of the technique to be used in a specific situation will depend upon the needs of the investigator and upon available resources. Static tests provide the most easily obtained measure of acute toxicity, but should not last longer than 96 h. Renewal and flow-through tests may last longer than 96 h because the pH and the concentrations of dissolved oxygen and test material are maintained at desired levels and degradation and metabolic products are removed. Static tests might not be applicable to materials that have a high oxygen demand, are highly volatile, are rapidly biologically or chemically transformed in aqueous solution, or are removed from test solutions in substantial quantities by the test chambers or organisms during the test. Flow-through tests are generally preferable to renewal tests, although in some situations a renewal test might be more cost-effective than a flow-through test. Shell deposition tests with bivalve molluscs must be flow-through tests.

1.4 These procedures are applicable to most chemicals, either individually or in formulations, commercial products, or known mixtures. With appropriate modifications these procedures can be used to conduct acute tests on temperature, dissolved oxygen, pH, and on such materials as leachates, oils, particulate matter, sediments, and surface waters. See Guide E1192 Procedures for conducting acute tests on aqueous effluents.

1.5 In the development of these procedures, an attempt was made to balance scientific and practical considerations and to ensure that the results will be sufficiently accurate and precise for the applications for which they are commonly used. A major consideration was that the common uses of the results of acute toxicity tests do not require or justify stricter requirements than those set forth herein. Although the tests may be improved by using more organisms, longer acclimation times, etc., the requirements presented herein should usually be sufficient.

1.6 Results of acute toxicity tests should usually be reported in terms of an LC50 (median lethal concentration) or EC50 (median effective concentration). In some situations, it might only be necessary to determine whether a specific concentration is acutely toxic to the test species or whether the LC50 or EC50 is above or below a specific concentration.

1.7 This guide is arranged as follows:

Section Referenced Documents 2 Terminology 3 Summary of Standard 4 Significance and Use 5 Hazards 6 Apparatus 7 Facilities 7.1 Special Requirements 7.2 Construction Materials 7.3 Metering System 7.4 Test Chambers 7.5 Cleaning 7.6 Acceptability 7.7 Dilution Water 8 Requirements 8.1 Source 8.2 Treatment 8.3 Characterization 8.4 Test Material 9 General 9.1 Stock Solution 9.2 Test Concentration(s) 9.3 Test Organisms 10 Species 10.1 Age 10.2 Source 10.3 Care and Handling 10.4 Feeding 10.5 Disease Treatment 10.6 Holding 10.7 Acclimation 10.8 Quality 10.9 Procedure 11 Experimental Design 11.1 Dissolved Oxygen 11.2 Temperature 11.3 Loading 11.4 Beginning the Test 11.5 Feeding 11.6 Duration of Test 11.7 Biological Data 11.8 Other Measurements 11.9 Analytical Methodology 12 Acceptability of Test 13 Calculation of Results 14 Report 15

1.8 This standard may involve hazardous materials, operations, and equipment. This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Specific hazard statements are given in Section 7.