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    ASTM E1604 - 20

    Standard Guide for Behavioral Testing in Aquatic Toxicology

    Active Standard ASTM E1604 | Developed by Subcommittee: E50.47

    Book of Standards Volume: 11.06

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    Significance and Use

    5.1 Protection of a species requires the prevention of detrimental effects of chemicals on the survival, growth, reproduction, and health of that species. Behavioral toxicity provides information concerning sublethal effects of chemicals and signals the presence of toxic test substances.

    5.1.1 The behavioral responses of all organisms are adaptive and essential to survival. Major changes in the behavioral responses of fish, amphibians, and macroinvertebrates may result in a diminished ability to survive, grow, or reproduce and cause significant changes in the natural population (8).

    5.2 The results from behavioral toxicity tests may be useful for measuring injury in the assessment of damages resulting from the release of hazardous materials (9) .

    5.3 Behavioral toxicity test methods may be useful for long-term monitoring of effluents (10) .

    5.4 The results from behavioral toxicity data can be used to predict the effects of exposure on fish, amphibians, and aquatic invertebrates likely to occur in field situations as a result of exposure under similar conditions, including the avoidance of exposure by motile organisms (11).

    5.5 The results from behavioral toxicity tests might be an important consideration for assessing the hazard of materials to aquatic organisms. Such results might also be used when deriving water quality criteria for fish and aquatic invertebrate organisms.

    5.6 The results from behavioral toxicity tests can be used to compare the sensitivities of different species, relative toxicity of different chemical substances on the same organism, or effect of various environmental variables on the toxicity of a chemical substance.

    5.7 The results from behavioral toxicity tests can be used to predict the effects of long-term exposure.

    5.8 The results of behavioral toxicity tests can be useful for guiding decisions regarding the extent of remedial action needed for contaminated aquatic and terrestrial sites.

    5.9 The behavioral characteristics of a particular organism must be understood and defined before a response can be used as a measure of toxicity. The range of variability of any behavioral response of unexposed organisms is influenced by genetic, experiential, physiological, and environmental factors. Thus it is important to avoid selecting test organisms from populations that may vary significantly in these factors.

    5.10 The results of behavioral toxicity tests will depend on the behavioral response measured, testing conditions, water quality, species, genetic strain, life stage, health, and general condition of test organisms. Therefore, the behavioral response may be affected by the test environment.

    1. Scope

    1.1 This guide covers some general information on the selection and application of behavioral methods useful for determining the sublethal effects of chemicals to fish, amphibians, and macroinvertebrates.

    1.2 Behavioral toxicity occurs when chemical or other stressful conditions, such as changes in water quality or temperature, induce a behavioral change that exceeds the normal range of variability (1).2 Behavior includes all observable, recordable, or measurable activities of a living organism and reflects genetic, neurobiological, physiological, and environmental determinants (2).

    1.3 Behavioral methods can be used in biomonitoring, the determination of no-observed-effect and lowest-observed-effect concentrations, and the prediction of hazardous chemical impacts on natural populations (3).

    1.4 Behavioral methods can be applied to fish, amphibians, and macroinvertebrates in standard laboratory toxicity tests, tests of effluents, and sediment toxicity tests.

    1.5 The various behavioral methods included in this guide are categorized with respect to seven interdependent, functional responses that fish, amphibians, and macroinvertebrates must perform in order to survive. These functional responses include respiration, locomotion, habitat selection, feeding, predator avoidance, competition, and reproduction (4). These responses can be documented visually or through video or acoustic imagery. Electronically recorded information can be derived through manual techniques or through the use of digital image analysis software (5, 6, 7).

    1.5.1 The functional responses are not necessarily mutually exclusive categories. For instance, locomotion, of some form of movement, is important to all behavioral functions.

    1.6 Additional behavioral methods for any category may be added when new tests are developed as well as when methods are adapted to different species or different life stages of an organism.

    1.7 This guide is arranged as follows:


    Section Number



    Referenced Documents




    Summary of Guide


    Significance and Use




    Test Facility


    Water Supply


    Safety Precautions


    Test Material


    Test Organisms


    Responses Measured


    Behavioral Test Method Selection Criteria


    Experimental Design


    Acceptability of Test


    Calculation of Test Results




    1.8 The values stated in SI units are to be regarded as the standard. For an explanation of units and symbols, refer to IEEE/ASTM SI 10.

    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, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Specific precautionary statements are given in Section 9.

    1.10 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

    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

    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

    E1241 Guide for Conducting Early Life-Stage Toxicity Tests with Fishes

    E1383 Guide for Conducting Sediment Toxicity Tests with Freshwater Invertebrates

    E1711 Guide for Measurement of Behavior During Fish Toxicity Tests

    E1733 Guide for Use of Lighting in Laboratory Testing

    E1847 Practice for Statistical Analysis of Toxicity Tests Conducted Under ASTM Guidelines

    ICS Code

    ICS Number Code 07.060 (Geology. Meteorology. Hydrology)

    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/E1604-20

    Citation Format

    ASTM E1604-20, Standard Guide for Behavioral Testing in Aquatic Toxicology, ASTM International, West Conshohocken, PA, 2020, www.astm.org

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