1、Designation: E724 98 (Reapproved 2012)Standard Guide forConducting Static Acute Toxicity Tests Starting withEmbryos of Four Species of Saltwater Bivalve Molluscs1This standard is issued under the fixed designation E724; the number immediately following the designation indicates the year oforiginal a
2、doption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This guide describes procedures for obtaining laboratorydata concerni
3、ng the acute effects of a test material on embryosand the resulting larvae of four species of saltwater bivalvemolluscs (Pacific oyster, Crassostrea gigas Thunberg; easternoyster, Crassostrea virginica Gmelin; quahog or hard clam,Mercenaria mercenaria Linnaeus; and blue mussel, Mytilusedulis Linnaeu
4、s) during static 48-h exposures. These proce-dures will probably be useful for conducting static acutetoxicity tests starting with embryos of other bivalve species(1)2although modifications might be necessary.1.2 Other modifications of these procedures might be justi-fied by special needs or circums
5、tances. Although using proce-dures appropriate to a particular species or special needs andcircumstances is more important than following prescribedprocedures, results of tests conducted by using unusual proce-dures are not likely to be comparable to results of many othertests. Comparison of results
6、 obtained by using modified andunmodified versions of these procedures might provide usefulinformation concerning new concepts and procedures forconducting 48-h acute tests starting with embryos of bivalvemolluscs.1.3 These procedures are applicable to most chemicals,either individually or in formul
7、ations, commercial products, orknown mixtures. With appropriate modifications these proce-dures can be used to conduct acute tests on temperature,dissolved oxygen, and pH and on such materials as aqueouseffluents (see also Guide E1192), leachates, oils, particulatematter, sediments, and surface wate
8、rs. Renewal tests might bepreferable to static tests for materials that have a high oxygendemand, are highly volatile, are rapidly biologically or chemi-cally transformed in aqueous solution, or are removed from testsolutions in substantial quantities by the test chambers ororganisms during the test
9、.1.4 Results of acute toxicity tests with embryos of bivalvemolluscs should usually be reported as the EC50 based on thetotal incompletely developed and dead organisms. It might alsobe desirable to report the LC50 based only on death. In somesituations, it might only be necessary to determine whethe
10、r aspecific concentration is acutely toxic to embryos or whetherthe EC50 is above or below a specific concentration.1.5 This guide is arranged as follows:SectionReferenced Documents 2Terminology 3Summary of Guide 4Significance and Use 5Hazards 6Apparatus 7Facilities 7.1Construction Materials 7.2Test
11、 Chambers 7.3Cleaning 7.4Acceptability 7.5Dilution Water 8Requirements 8.1Source 8.2Treatments 8.3Characterization 8.4Test Material 9General 9.1Stock Solution 9.2Test Concentration(s) 9.3Test Organisms 10Species 10.1Age 10.2Source 10.3Handling 10.4Brood Stock Source and Condition 10.5Spawning and Fe
12、rtilization 10.6Quality 10.7Procedure 11Experimental Design 11.1Dissolved Oxygen 11.2Temperature 11.3Beginning the Test 11.4Feeding 11.5Duration of Test 11.6Biological Data 11.7Other Measurements 11.81This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental F
13、ateand is the direct responsibility of SubcommitteeE47.01 on Aquatic Assessment and Toxicology.Current edition approved Dec. 1, 2012. Published December 2012. Originallyapproved in 1980. Last previous edition approved in 2004 as E74 98 (2004). DOI:10.1520/E0724-98R12.2The boldface numbers in parenth
14、eses refer to the list of references at the end ofthis guide.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1Analytical Methods 12Acceptability of Test 13Calculation of Results 14Report 15Annex Annex A11.6 This standard does not purpo
15、rt to address all of thesafety concerns, if any, associated with its use. It is 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 hazardstatements are given in Section
16、 6.2. Referenced Documents2.1 ASTM Standards:3E380 Practice for Use of the International System of Units(SI) (the Modernized Metric System)4E729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-iansE943 Terminology Relating to Biological Effects
17、and Envi-ronmental FateE1023 Guide for Assessing the Hazard of a Material toAquatic Organisms and Their UsesE1192 Guide for Conducting Acute Toxicity Tests on Aque-ous Ambient Samples and Effluents with Fishes,Macroinvertebrates, and AmphibiansE1367 Test Method for Measuring the Toxicity of Sediment
18、-Associated Contaminants with Estuarine and Marine In-vertebratesE1391 Guide for Collection, Storage, Characterization, andManipulation of Sediments for Toxicological Testing andfor Selection of Samplers Used to Collect Benthic Inver-tebratesE1706 Test Method for Measuring the Toxicity of Sediment-A
19、ssociated Contaminants with Freshwater Invertebrates3. Terminology3.1 Definitions:3.1.1 The words “must,” “should,” “may,” “can,” and“might” have very specific meanings in this guide. “Must” isused to express an absolute requirement, that is, to state that thetest ought to be designed to satisfy the
20、 specified condition,unless the purpose of the test requires a different design.“Must” is only used in connection with factors that directlyrelate to the acceptability of the test (see 13.1). “Should” isused to state that the specified condition is recommended andought to be met if possible.Although
21、 violation of one “should”statement is rarely a serious matter, violation of several willoften render the results questionable. Terms such as “isdesirable,” “is often desirable,” and “might be desirable” areused in connection with less important factors. “May” is usedto mean “is (are) allowed to,” “
22、can” is used to mean “is (are)able to,” and “might” is used to mean “could possibly.” Thusthe classic distinction between “may” and “can” is preserved,and “might” is never used as a synonym for either “may” or“can.”3.1.2 For definitions of other terms used in this guide, referto Guide E729, Terminol
23、ogy E943, and Guide E1023. For anexplanation of units and symbols, refer to Practice E380.3.2 Definitions of Terms Specific to This Standard:3.2.1 embryoused herein to denote the stages between thefertilization of the egg and the trochophore (2) (Figs. 1 and 2).3.2.2 larvaused herein to include the
24、trochophore and thestraight hinge stage (2) (Figs. 1 and 2).4. Summary of Guide4.1 Adult saltwater bivalve molluscs are brought into thelaboratory, cleaned of detritus and fouling organisms, andidentified to species. If the gonads are not ripe, the bivalves areconditioned to bring them into a suitab
25、le reproductive state.Bivalves with ripe gonads are maintained under conditions thatkeep the gonads ripe without inducing undesired spontaneousspawning or resorption of gametes. In order to start a test,spawning is induced by using one or more stimuli which maybe physical (for example, temperature),
26、 biological (forexample, heat-killed bivalve sperm), or chemical (for example,serotonin).4.2 In each of two or more treatments, embryos and theresulting larvae of one species are maintained for 48 h. In eachof one or more control treatments, the embryos and resultinglarvae are maintained in dilution
27、 water to which no testmaterial has been added in order to provide (a) a measure ofthe acceptability of the test by giving an indication of thequality of the organisms and the suitability of the dilutionwater, test conditions, handling procedures, and so forth, and(b) the basis for interpreting data
28、 obtained from the othertreatments. In each of one or more other treatments, theembryos and resulting larvae are maintained in dilution waterto which a selected concentration of test material has beenadded. The 48-h EC50 is calculated based on the proportion oflive larvae with completely developed s
29、hells in chamberscontaining the test material to live larvae with completelydeveloped shells in the controls at the termination of the 48-htest.5. Significance and Use5.1 An acute toxicity test is conducted to obtain informationconcerning the acute effects of a short term exposure oforganisms to a t
30、est material under specific experimental con-ditions. An acute toxicity test does not provide informationconcerning whether delayed effects will occur.5.2 Because embryos and larvae are usually assumed to bethe most sensitive life stages of these bivalve mollusc speciesand because these species are
31、commercially and recreationallyimportant, results of these acute tests are often considered to bea good indication of the acceptability of pollutant concentra-tions to saltwater molluscan species in general. Results of theseacute toxicity tests are often assumed to be an importantconsideration when
32、assessing the hazard of materials to other3For referenced ASTM 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. The las
33、t approved version of this historical standard is referencedon www.astm.org.E724 98 (2012)2saltwater organisms (see Guide E1023) or when deriving waterquality criteria for saltwater organisms (3).5.3 Results of acute toxicity tests might be used to predictacute effects likely to occur to aquatic org
34、anisms in fieldsituations as a result of exposure under comparable conditions,except that toxicity to benthic species might depend onsorption or settling of the test material onto the substrate.5.4 Results of acute tests might be used to compare theacute sensitivities of different species and the ac
35、ute toxicities ofdifferent test materials, and to determine the effects of variousenvironmental factors on results of such tests.5.5 Results of acute toxicity tests might be useful forstudying biological availability of, and structure activity rela-tionships between, test materials.FIG. 1 Drawings E
36、xemplifying Five Key Developmental Stages of Bivalve Larvae Occurring During the First 48 h of DevelopmentE724 98 (2012)35.6 Results of acute toxicity tests will depend ontemperature, composition of the dilution water, condition of thetest organisms, and other factors.6. Hazards6.1 Many materials ca
37、n adversely affect humans if precau-tions are inadequate. Therefore, skin contact with all testmaterials and solutions of them should be minimized by suchmeans as wearing appropriate protective gloves (especiallywhen washing equipment or putting hands in test solutions),laboratory coats, aprons, and
38、 glasses. Special precautions, suchas covering test chambers and ventilating the area surroundingthe chambers, should be taken when conducting tests onvolatile materials. Information concerning toxicity to humans(4), recommended handling procedures (5), and chemical andphysical properties of the tes
39、t material should be studied beforea test is begun. Special procedures are necessary with radiola-beled test materials (6) and with materials that are, or aresuspected of being, carcinogenic (7).FIG. 2 Photomicrographs (450) Exemplifying Five Key Developmental Stages of Bivalve Larvae Occurring Duri
40、ng the First 48 h of De-velopmentE724 98 (2012)46.2 Although in most cases disposal of stock solutions, testsolutions, and test organisms poses no special problems, healthand safety precautions and applicable regulations should beconsidered before beginning a test. Removal or degradation ofthe test
41、material might be desirable before disposal of stock andtest solutions.6.3 Cleaning of equipment with a volatile solvent such asacetone should be performed only in a well-ventilated area inwhich no smoking is allowed and no open flame, such as a pilotlight, is present.6.4 WarningAn acidic solution s
42、hould not be mixed witha hypochlorite solution because hazardous fumes might beproduced.6.5 To prepare dilute acid solutions, concentrated acidshould be added to water, not vice versa. Opening a bottle ofconcentrated acid and adding concentrated acid to water shouldbe performed only in a fume hood.6
43、.6 PrecautionUse of ground fault systems and leakdetectors is strongly recommended to help prevent electricalshocks because salt water is a good conductor of electricity.6.7 To protect hands from being cut by sharp edges of oystershells, cotton work gloves should be worn over appropriateprotective g
44、loves (see 6.1), if necessary, when oysters arehandled. When an oyster knife is used to open adults of anybivalve species, heavy rubber work gloves should be worn toprotect hands from puncture.7. Apparatus7.1 Facilities:7.1.1 Flow-through troughs with appropriate trays (8)should be available for hol
45、ding and conditioning the broodstock. The water-supply system should be equipped for tem-perature control and aeration (see 8.3), and should containstrainers and air traps. Air used for aeration should be free offumes, oil, and water; filters to remove oil and water aredesirable. Test chambers shoul
46、d be in a constant-temperatureroom, incubator, or recirculating water bath. A dilution-watertank or headbox, which might be used to prepare reconstitutedwater, is often elevated so that dilution water can be gravity fedinto holding and conditioning troughs and test chambers. Thefacility should be we
47、ll ventilated and free of fumes. To furtherreduce the possibility of contamination of brood stock and testorganisms by test materials and other substances, especiallyvolatile ones, holding and conditioning troughs should not bein a room in which toxicity tests are conducted, stock solutionsor test s
48、olutions are prepared, or equipment is cleaned. Duringholding, conditioning, and testing, organisms should beshielded from disturbances with curtains or partitions toprevent unnecessary stress.7.1.2 It is probably desirable to include some safeguards inthe system that supplies water to holding and c
49、onditioningtroughs. Monitors, possibly connected to auxiliary powersupplies, might be designed to initiate aeration, sound alarms,or activate telephone auto-dialing alarms if water flow ortemperature deviates from preset limits. If temperature be-comes too high or low, corrective action should not cause thetemperature of the water in holding and conditioning troughs toincrease or decrease more than 2C/day to reduce the chancesof spontaneous spawning.7.2 Construction MaterialsEquipment and facilities thatcontact stock solutions, test solutions, or any water into whichbrood
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