1、Designation: E 1604 94 (Reapproved 2007)Standard Guide forBehavioral Testing in Aquatic Toxicology1This standard is issued under the fixed designation E 1604; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revisio
2、n. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide covers some general information on theselection and application of behavioral methods useful fordetermining the sublet
3、hal effects of chemicals to fish, amphib-ians, and macroinvertebrates.1.2 Behavioral toxicity occurs when chemical or otherstressful conditions, such as changes in water quality ortemperature, induce a behavioral change that exceeds thenormal range of variability (1).2Behavior includes all observ-ab
4、le, recordable, or measurable activities of a living organismand reflects genetic, neurobiological, physiological, and envi-ronmental determinants (2).1.3 Behavioral methods can be used in biomonitoring, thedetermination of no-observed-effect and lowest-observed-effect concentrations, and the predic
5、tion of hazardous chemicalimpacts 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 guideare categorized
6、 with respect to seven interdependent, func-tional responses that fish, amphibians, and macroinvertebratesmust perform in order to survive. These functional responsesinclude respiration, locomotion, habitat selection, feeding,predator avoidance, competition, and reproduction (4).1.5.1 The functional
7、 responses are not necessarily mutuallyexclusive categories. For instance, locomotion, of some formof movement, is important to all behavioral functions.1.6 Additional behavioral methods for any category may beadded when new tests are developed as well as when methodsare adapted to different species
8、 or different life stages of anorganism.1.7 This guide is arranged as follows:Section NumberScope 1Referenced Documents 2Terminology 3Summary of Guide 4Significance and Use 5Interferences 6Test Facility 7Water Supply 8Safety Precautions 9Test Material 10Test Organisms 11Responses Measured 12Behavior
9、al Test Method Selection Criteria 13Experimental Design 14Acceptability of Test 15Calculation of Test Results 16Report 171.8 The values stated in SI units are to be regarded as thestandard.1.9 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is
10、 theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use. Specific precau-tionary statements are given in Section 9.2. Referenced Documents2.1 ASTM Standards:3E 729 Guide for Conducti
11、ng Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-iansE 1241 Guide for Conducting Early Life-Stage ToxicityTests with FishesE1383 Guide for Conducting Sediment Toxicity Tests withFreshwater Invertebrates43. Terminology3.1 DefinitionsThe words “must,” “should, “may,
12、” “can,”and “might” have very specific meanings. “Must” is used toexpress an absolute requirement, that is, to state that the testought to be designed to satisfy the specified condition, unlessthe purpose of the test requires a different design. “Must” isused only in connection with the factors that
13、 directly relate tothe acceptability of the test. “Should” is used to state that thespecified condition is recommended and ought to be met if1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fate and is the direct responsibility of SubcommitteeE47.01
14、on Aquatic Assessment and Toxicology.Current edition approved Oct. 1, 2007. Published October 2007. Originallyapproved in 1994. Last previous edition approved in 2002 as E 1604-94(2002).2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.3For referenced AS
15、TM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.4Withdrawn.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, W
16、est Conshohocken, PA 19428-2959, United States.possible. Although the violation of one “should” is rarely aserious matter, violation of several will often render the resultsquestionable. Terms such as “is desirable,” “is often desirable,”and “might be desirable” are used in connection with lessimpor
17、tant factors. “May” is used to mean “is (are) allowed to,”“can” is used to mean “is (are) able to,” and “might” is used tomean “could possibly.” Thus the classic distinction between“may” and “can” is preserved, and “might” is never used as asynonym for either “may” or “can.”3.2 Definitions of Terms
18、Specific to This Standard:3.2.1 behaviorthe complex of observable, recordable, ormeasurable activities of a living organism.3.2.2 behavioral toxicitythe phenomenon observed whena behavioral response varies beyond the range of normal as aresult of exposure to chemical or other stressors.4. Summary of
19、 Guide4.1 The potential toxicity of chemical substances in water,food, or sediments is assessed by measuring the behavior offish, amphibians, and macroinvertebrates during exposure,using static, flow-through, or food exposure systems. Thebehavioral response of organisms exposed to chemical sub-stanc
20、es in water, effluents, food, or sediments is compared withthe behavioral responses of control organisms. The behavioralresponses measured during toxicity tests are highly sensitive tosublethal exposure. The behavioral measures are relevant toessential life functions that fish, amphibians, and macro
21、inver-tebrates often must perform in order to survive and includerespiration, locomotion, habitat selection, feeding, predatoravoidance, competition, and reproduction. Data are obtained todetermine the effects of toxic substances on behavior fromshort (for example, 1 h) or long-term (partial to full
22、 life cycle)exposures.5. Significance and Use5.1 Protection of a species requires the prevention of detri-mental effects of chemicals on the survival, growth, reproduc-tion, health, and uses of individuals of that species. Behavioraltoxicity provides information concerning sublethal effects ofchemic
23、als and signals the presence of toxic test substances.5.1.1 The behavioral responses of all organisms are adaptiveand essential to survival. Major changes in the behavioralresponses of fish, amphibians, and macroinvertebrates mayresult in a diminished ability to survive, grow, or reproduce andcause
24、significant changes in the natural population (5).5.2 The results from behavioral toxicity tests may be usefulfor measuring injury in the assessment of damages resultingfrom the release of hazardous materials (6).5.3 Behavioral toxicity test methods may be useful forlong-term monitoring of effluents
25、 (7).5.4 The results from behavioral toxicity data can be used topredict the effects of exposure on fish, amphibians, and aquaticinvertebrates likely to occur in field situations as a result ofexposure under similar conditions, including the avoidance ofexposure by motile organisms (8).5.5 The resul
26、ts from behavioral toxicity tests might be animportant consideration for assessing the hazard of materials toaquatic organisms. Such results might also be used whenderiving water quality criteria for fish and aquatic invertebratesorganisms.5.6 The results from behavioral toxicity tests can be used t
27、ocompare the sensitivities of different species, relative toxicityof different chemical substances on the same organism, oreffect of various environmental variables on the toxicity of achemical substance.5.7 The results from behavioral toxicity tests can be used topredict the effects of long-term ex
28、posure.5.8 The results of behavioral toxicity tests can be useful forguiding decisions regarding the extent of remedial actionneeded for contaminated aquatic and terrestrial sites.5.9 The behavioral characteristics of a particular organismmust be understood and defined before a response can be useda
29、s a measure of toxicity. The range of variability of anybehavioral response of unexposed organisms is influenced bygenetic, experiential, physiological, and environmental factors.Thus it is important to avoid selecting test organisms frompopulations that may vary significantly in these factors.5.10
30、The results of behavioral toxicity tests will depend onthe behavioral response measured, testing conditions, waterquality, species, genetic strain, life stage, health, and generalcondition of test organisms. Therefore, the behavioral responsemay be affected by the test environment.6. Interferences6.
31、1 A number of factors can suppress, elicit, or alterbehavioral responses and thus influence behavioral test resultsand complicate data interpretation. The following factorsshould be considered in the experimental protocol or in thediscussion of results when measuring behavioral responsesduring toxic
32、ity tests:6.1.1 The pretest handling of test organisms resulting fromcollection, transfer, and maintenance of the culture environ-ment can affect the response observed during exposure to toxicsubstances.6.1.2 The health, nutritional state, and physical condition ofthe organism can influence the test
33、.6.1.3 Behavioral responsiveness may vary by species, ge-netic strain, population, gender, and developmental stage of theorganism.6.1.4 Prior exposure to hazardous materials, environmentalstresses, and pathogens can affect the behavioral responses.6.1.5 Social status, such as dominance or sex of the
34、 indi-viduals tested, and experiential factors, such as prior experi-ence with predator or prey species, can influence the behavioralresponse. Individuals tested in isolation may respond differ-ently than when tested in groups.6.1.6 Cyclical changes (circadian, seasonal, annual, andreproductive) in
35、behavioral responses can occur.6.1.7 The behavioral response can be affected by apparatusdesign and by the procedural sequence of the measurementmethod.6.1.8 Behavioral responses will vary according to the extentto which test organisms acclimate to the physical variables ofthe testing environment, i
36、ncluding water quality, temperature,E 1604 94 (2007)2water flow, light, cover, and substrate, as well as their recoveryfrom handling, acceptance of diet, and adjustment to noveltesting chambers.6.1.9 Behavioral responses to toxic substances may subsideover time.7. Test Facility7.1 FacilitiesThe faci
37、lity should include a constant tem-perature area for culturing and testing. Test and culturechambers may be placed in a temperature-controlled recircu-lating water bath or in a constant-temperature area.Air used foraeration should be free of fumes, oil, and water and can requirefilters to remove oil
38、, water, and bacteria. The test facilityshould be well-ventilated and free of fumes. Enclosures may benecessary to ventilate test chambers.7.1.1 Culture and animal care facilities should not be in aroom in which toxicity tests are conducted, stock solutions ortest solutions are prepared, or equipmen
39、t is cleaned.7.1.2 A timing device should be used to provide a light-:darkness cycle. A15 to 30-min transition period, allowing fora gradual change in light intensity when the lights are turned onor off, may be desirable for reducing stress caused by instan-taneous illumination or darkness.7.2 Const
40、ruction MaterialsConsistent with specificationsdelineated, for example, in Guide E 1241, equipment andfacilities that come into contact with stock solutions, testsolutions, food, sediment, air, or water, into which the testorganisms are placed, should not contain substances that can beleached or dis
41、solved in amounts that affect the test organismsadversely. The materials should be chosen to minimize sorptionof test materials.7.3 Water and Air Delivery SystemsThe water deliverysystem used in flow-through testing can be one of severaldesigns. The system should be capable of delivering equalvolume
42、s of water at an equal rate of flow to each replicatetreatment container. Various metering systems, using differentcombinations of siphons, pumps, solenoids, valves, etc., havebeen used successfully to control the flow rates of water andtoxic substances (see Guide E 1241).7.3.1 The metering system s
43、hould be calibrated before thetest by determining the flow rate of water and air through eachtest chamber. The general operation of the metering systemshould be visually checked daily throughout the test. The waterdelivery system should be adjusted during the test if necessary.At any particular time
44、 during the test, flow rates through anytwo test chambers should not differ by more than 10 %.7.4 Test ChambersIn a behavioral toxicity test with fish,amphibians, and macroinvertebrates, the measurement of be-havioral response may take place directly in the exposurevessel, or the organisms may be tr
45、ansferred to a specificapparatus or observation chamber for the purpose of measuringa behavioral response (see section 8.1.8). The independentexperimental unit for such tests is based on the smallestphysical exposure unit between which there are no water, airconnections, or common access to sediment
46、 or food. All testchambers must be identical, and the test compartments withineach chamber must be identical and placed in analogouslocations within each test chamber.7.4.1 Test chambers may be constructed in several ways andof various materials, depending on the experimental design andcontaminants
47、of interest. Clear silicone adhesives, suitable foraquaria, should be used sparingly since they sorb some organiccompounds that may be difficult to remove. New test chamberssealed with silicone adhesives should be weathered for at least48 h in water of the same quality as that used in the toxicity t
48、estto leach potentially toxic compounds from the adhesive.7.4.2 Apparatus will vary according to the response beingmeasured and species and life stage being tested. Organismsmay be observed directly in the exposure chamber, or they maybe transferred to specialized apparatus for measurement of theres
49、ponse. Recording of response may require (1) direct visualobservation, (2) video-recorded observation, or (3) electroni-cally recorded observation.7.5 CleaningTest chambers, water delivery systems,equipment used for preparing and storing exposure water, andstock solutions should be cleaned before use. Consistent withGuide E 729, new items should be washed in the followingmanner: (1) detergent wash, (2) tap water rinse, (3) water-miscible organic solvent wash, (4) tap water rinse, (5) acidwash (such as 10 % concentrated hydrochloric acid), and (6)rinse at least twice with distil
