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    ASTM E1295 - 01(2013)

    Standard Guide for Conducting Three-Brood, Renewal Toxicity Tests with Ceriodaphnia dubia

    Active Standard ASTM E1295 | Developed by Subcommittee: E50.47

    Book of Standards Volume: 11.06

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    Significance and Use

    5.1 Ceriodaphnia  was first used as a toxicity test organism by Mount and Norberg (4). Introduced for use in effluent and ambient water evaluations, Ceriodaphnia have also been a valuable addition to single chemical test procedures.

    5.2 Protection of a population requires prevention of unacceptable effects on the number, weight, health, and uses of the individuals of that species, or species for which the test species serves as a surrogate. A three-brood toxicity test is conducted to help determine changes in survival and the number of neonates produced that result from exposure to the test material.

    5.3 Results of three-brood toxicity tests with C. dubia might be used to predict chronic or partial chronic effects on species in field situations as a result of exposure under comparable conditions.

    5.4 Results of three-brood toxicity tests with C. dubia might be compared with the chronic sensitivities of different species and the chronic toxicities of different materials, and to study the effects of various environmental factors on results of such tests.

    5.5 Results of three-brood toxicity tests with C. dubia might be useful for predicting the results of chronic tests on the same test material with the same species in another water or with another species in the same or a different water. Most such predictions are based on the results of acute toxicity tests, and so the usefulness of the results of a three-brood toxicity test with C. dubia might be greatly increased by also reporting the results of an acute toxicity test (see Guides E729 and E1192) conducted under the same conditions. In addition to conducting an acute test with unfed C. dubia, it might also be desirable to conduct an acute test in which the organisms are fed the same as in the three-brood test, to see if the presence of that concentration of that food affects the results of the acute test and the acute chronic ratio (see 10.4.1).

    5.5.1 A 48 or 96-h EC50 or LC50 can sometimes be obtained from a three-brood toxicity test with a known test material, but often all the concentrations in the test will be below the EC50 or LC50. In addition, it is usually desirable to know the EC50 or LC50 before beginning the three-brood test, as a means to determine the concentrations for use in the chronic test (see 10.4.1). It should be noted that results from an acute test may not necessarily correspond to those of a chronic test, due to the addition of food to the chronic test.

    5.6 Three-brood toxicity tests with C. dubia might be useful for studying biological availability of, and structure activity relationships between, test materials.

    5.7 Results of three-brood toxicity tests with C. dubia can vary with temperature, quality and quantity of food, quality of the dilution water, condition of the test organisms, and other factors.

    5.8 Results of three-brood toxicity tests with C. dubia might be an important consideration when assessing the hazards of materials to aquatic organisms (see Guide E1023), or when deriving water quality criteria for aquatic organisms.

    1. Scope

    1.1 This guide describes procedures for obtaining data concerning the adverse effects of an effluent or a test material (added to dilution water, but not to food) on Ceriodaphnia dubia Richard 1894, during continuous exposure throughout a portion of the organism's life. These procedures should also be useful for conducting life cycle toxicity tests with other Cladocera (Guide E1193), although modifications will be necessary.

    1.2 These procedures are applicable to most chemicals, either individually or in formulations, commercial products, or known mixtures, that can be measured accurately at the necessary concentrations in water. With appropriate modifications these procedures can be used to conduct tests on temperature, dissolved oxygen, pH, and on such materials as aqueous effluents (see also Guide E1192), leachates, oils, particulate matter, sediments (see also Guide E1383), and surface waters. Renewal tests might not be applicable to materials that have high oxygen demand, are highly volatile, are rapidly biologically or chemically transformed, or sorb to test chambers. If the concentration of dissolved oxygen falls below 4 mg/L or the concentration of test material decreases by more than 20 % in test solution(s) between renewals, more frequent renewals might be necessary.

    1.3 Other modifications of these procedures might be justified by special needs or circumstances. Results of tests conducted using unusual procedures are not likely to be comparable to results of many other tests. Comparisons of results obtained using modified and unmodified versions of these procedures might provide useful information on new concepts and procedures for conducting three-brood toxicity tests with C. dubia.

    1.4 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 8.

    1.5 This guide is arranged as follows:




    Referenced Documents




    Summary of Guide


    Significance and Use






     Construction Materials


     Test Chambers




    Reagents and Materials




    Dilution Water










    Test Material




     Stock Solution




     Test Concentration(s)




     Sample Containers






    Test Organisms








     Brood Stock










     Demonstration of Feasibility


     Experimental Design


     Dissolved Oxygen




     Preparing Test Solutions


     Conditioning Test Chambers


     Beginning a Test


     Renewing Test Solutions


     Duration of Test


     Biological Data


     Other Measurements


    Analytical Methodology


    Acceptability of Test









    Appendix X1

     Culture Techniques

    Appendix X2

     Test Chambers

    Appendix X3

     Statistical Guidance

    Appendix X4

    2. Referenced Documents (purchase separately) The documents listed below are referenced within the subject standard but are not provided as part of the standard.

    ASTM Standards

    D1193 Specification for Reagent Water

    D3978 Practice for Algal Growth Potential Testing with Pseudokirchneriella subcapitata

    E729 Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians

    E943 Terminology Relating to Biological Effects and Environmental Fate

    E1023 Guide for Assessing the Hazard of a Material to Aquatic Organisms and Their Uses

    E1192 Guide for Conducting Acute Toxicity Tests on Aqueous Ambient Samples and Effluents with Fishes, Macroinvertebrates, and Amphibians

    E1193 Guide for Conducting Daphnia magna Life-Cycle Toxicity Tests

    E1383 Guide for Conducting Sediment Toxicity Tests with Freshwater Invertebrates

    E1706 Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates

    ICS Code

    ICS Number Code 07.100.20 (Microbiology of water)

    UNSPSC Code

    UNSPSC Code 77101501(Risk or hazard assessment)

    Referencing This Standard

    DOI: 10.1520/E1295-01R13

    ASTM International is a member of CrossRef.

    Citation Format

    ASTM E1295-01(2013), Standard Guide for Conducting Three-Brood, Renewal Toxicity Tests with Ceriodaphnia dubia, ASTM International, West Conshohocken, PA, 2013,

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