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    ASTM E729 - 96(2014)

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

    Active Standard ASTM E729 | Developed by Subcommittee: E50.47

    Book of Standards Volume: 11.06

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    Significance and Use

    5.1 An acute toxicity test is conducted to obtain information concerning the immediate effects on test organisms of a short-term exposure to a test material under specific experimental conditions. An acute toxicity test does not provide information about whether delayed effects will occur, although a post-exposure observation period, with appropriate feeding, if necessary, might provide such information.

    5.2 Results of acute toxicity tests might be used to predict acute effects likely to occur on aquatic organisms in field situations as a result of exposure under comparable conditions, except that (1) motile organisms might avoid exposure when possible, and (2) toxicity to benthic organisms might be dependent on sorption or settling of the test material onto the substrate.

    5.3 Results of acute tests might be used to compare the acute sensitivities of different species and the acute toxicities of different test materials, and to study the effects of various environmental factors on results of such tests.

    5.4 Results of acute toxicity tests 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 (2).

    5.5 Results of acute toxicity tests might be useful for studying the biological availability of, and structure-activity relationships between, test materials.

    5.6 Results of acute toxicity tests will depend on the temperature, composition of the dilution water, condition of the test organisms, exposure technique, and other factors.

    1. Scope

    1.1 This guide (1)2 describes procedures for obtaining laboratory data concerning the adverse effects (for example, lethality and immobility) 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. 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 This guide describes tests using three basic exposure techniques: static, renewal, and flow-through. Selection of the technique to use in a specific situation will depend on the needs of the investigator and on available resources. Tests using the static technique provide the most easily obtained measure of acute toxicity, but conditions often change substantially during static tests; therefore, static tests should not last longer than 96 h, and test organisms should not be fed during such tests. Static tests should probably not be conducted on materials that have a high oxygen demand, are highly volatile, are rapidly transformed biologically or chemically in aqueous solution, or are removed from test solutions in substantial quantities by the test chambers or organisms during the test. Because the pH and concentrations of dissolved oxygen and test material are maintained at desired levels and degradation and metabolic products are removed, tests using renewal and flow-through methods are preferable and may last longer than 96 h; test organisms may be fed during renewal and flow-through tests. Although renewal tests might be more cost-effective, flow-through tests are generally preferable.

    1.4 Acute tests may be performed to meet regulatory data requirements or to obtain time-independent estimates of toxicity.

    1.4.1 If the objective is to obtain data to meet regulatory requirements, it may be necessary to limit the number of observation times based on stipulations of the regulatory agency and cost considerations.

    1.4.2 If the objective of an acute toxicity test is to determine a time-independent (that is, incipient, threshold, or asymptotic) toxicity level, an appropriate number of observations must be taken over an exposure duration of sufficient length to establish the shape of the toxicity curve or allow the direct or mathematically estimated determination of a time-independent toxicity value (1), or both.

    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, and so forth, 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) at the end of the test, but it is desirable to provide information concerning the dependence of adverse effects on both time and concentration. Thus, when feasible, flow-through and renewal tests should be conducted so that LC50s or EC50s can be reported from 6 h to an asymptotic (time-independent, threshold, incipient) value, if one exists. 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:





    Referenced Documents




    Summary of Guide


    Significance and Use






     Special Requirements


     Construction Materials


     Metering System


     Test Chambers








    Dilution Water










    Test Material




     Stock Solution


     Test Concentration(s)


    Test Organisms








     Care and Handling




     Disease Treatment










     Experimental Design


     Dissolved Oxygen






     Beginning the Test




     Duration of Test


     Biological Data


     Other Measurements


    Analytical Methodology


    Acceptability of Test


    Calculation of Results




    1.8 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.

    1.9 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.

    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

    E724 Guide for Conducting Static Acute Toxicity Tests Starting with Embryos of Four Species of Saltwater Bivalve Molluscs

    E943 Terminology Relating to Biological Effects and Environmental Fate

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

    E1191 Guide for Conducting Life-Cycle Toxicity Tests with Saltwater Mysids

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

    E1203 Practice for Using Brine Shrimp Nauplii as Food for Test Animals in Aquatic Toxicology

    E1563 Guide for Conducting Static Acute Toxicity Tests with Echinoid Embryos

    E1604 Guide for Behavioral Testing in Aquatic Toxicology

    ICS Code

    ICS Number Code 07.080 (Biology. Botany. Zoology)

    UNSPSC Code

    UNSPSC Code 77101501(Risk or hazard assessment)

    Referencing This Standard
    Link Here
    Link to Active (This link will always route to the current Active version of the standard.)

    DOI: 10.1520/E0729-96R14

    Citation Format

    ASTM E729-96(2014), Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians, ASTM International, West Conshohocken, PA, 2014, www.astm.org

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