1、Designation: E 724 98 (Reapproved 2004)Standard Guide forConducting Static Acute Toxicity Tests Starting withEmbryos of Four Species of Saltwater Bivalve Molluscs1This standard is issued under the fixed designation E 724; the number immediately following the designation indicates the year oforiginal
2、 adoption or, in the case of revision, the year of last revision. 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 describes procedures for obtaining laboratorydata conce
3、rning 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 Linn
4、aeus) 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 circ
5、umstances. 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 resu
6、lts 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 for
7、mulations, 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 E 1192), leachates, oils, particulatematter, sediments, and surface
8、waters. 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
9、test.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 wh
10、ether 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 7.2
11、Test 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 an
12、d Fertilization 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 11.8Analytical Methods 12Acceptability of Test 13Calculation of Results 14Report 15Annex Annex A11Th
13、is guide is under the jurisdiction of ASTM Committee E-47 on BiologicalEffects and Environmental Fate and is the direct responsibility of SubcommitteeE47.01 on Aquatic Assessment and Toxicology.Current edition approved April 1, 2004. Published April 2004. Originallyapproved in 1980. Last previous ed
14、ition approved in 1998 as E 74 98.2The boldface numbers in parentheses refer to the list of references at the end ofthis guide.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.1.6 This standard does not purport to address all of thesa
15、fety 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 6.2. Referenced Documents
16、2.1 ASTM Standards:3E 380 Practice for Use of the International System of Units(SI) (the Modernized Metric System)4E 729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-iansE 943 Terminology Relating to Biological Effects and En-vironmental Fate
17、E 1023 Guide for Assessing the Hazard of a Material toAquatic Organisms and Their UsesE 1192 Guide for Conducting Acute Toxicity Tests onAqueous Ambient Samples and Effluents with Fishes,Macroinvertebrates, and AmphibiansE 1367 Test Method for Measuring the Toxcity ofSediment-Associated Contaminants
18、 with Marine and Etau-rine InvertebratesE 1391 Guide for Collection, Storage, Characterization, andManipulation of Sediments for Toxilogical Testing, and forSelection of Samples Used to Collect Benthic InvertebratesE 1706 Test Methods for Measuring the Toxicity ofSediment-Associated Contaminants wit
19、h Fresh Water In-vertebrates3. 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 specified condition,unl
20、ess 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 violation of one “shou
21、ld”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,” “can” is used to mean “i
22、s (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 E 729, Terminology E 943, and Guide E
23、 1023. For anexplanation of units and symbols, refer to Practice E 380.3.2 Descriptions 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 trochophore and th
24、estraight 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 suitable reproductive st
25、ate.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), biological (for e
26、x-ample, 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 water to which
27、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 obtained from t
28、he 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 shells in chamber
29、scontaining 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 test material und
30、er 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 commercially and
31、 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 assessing the ha
32、zard of materials to othersaltwater organisms (see Guide E 1023) or when derivingwater quality criteria for saltwater organisms (3).5.3 Results of acute toxicity tests might be used to predictacute effects likely to occur to aquatic organisms in fieldsituations as a result of exposure under comparab
33、le conditions,3For 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.E 724 98 (2004)2except that toxicity
34、 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 acute toxicities ofdifferent test materials, and to determine the effects of variousenvironmental fa
35、ctors 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.5.6 Results of acute toxicity tests will depend on tempera-ture, composition of the dilution water, condition of th
36、e testorganisms, and other factors.6. Hazards6.1 Many materials can 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 (especiallyFIG. 1 Drawings Exempl
37、ifying Five Key Developmental Stages of Bivalve Larvae Occurring During the First 48 h of DevelopmentE 724 98 (2004)3when washing equipment or putting hands in test solutions),laboratory coats, aprons, and glasses. Special precautions, suchas covering test chambers and ventilating the area surroundi
38、ngthe 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 test material should be studied beforea test is begun. Special procedures are necessary with radio
39、la-beled test materials (6) and with materials that are, or aresuspected of being, carcinogenic (7).6.2 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
40、beginning a test. Removal or degradation ofthe test 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 pi
41、lotlight, is present.FIG. 2 Photomicrographs (3450) Exemplifying Five Key Developmental Stages of Bivalve Larvae Occurring During the First 48 h ofDevelopmentE 724 98 (2004)46.4 WarningAn acidic solution should not be mixed witha hypochlorite solution because hazardous fumes might beproduced.6.5 To
42、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.6.6 PrecautionUse of ground fault systems and leakdetectors is strongly recommended to help prev
43、ent 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 gloves (see 6.1), if necessary, when oysters arehandled. When an oyster knife is used to open ad
44、ults 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 holding and conditioning the broodstock. The water-supply system should be equipped for tem-peratu
45、re 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 should be in a constant-temperatureroom, incubator, or recirculating water bath. A dilution-watertan
46、k 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 well ventilated and free of fumes. To furtherreduce the possibility of contamination of brood sto
47、ck 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 solutions are prepared, or equipment is cleaned. Duringholding, conditioning, and testing, organ
48、isms 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 conditioningtroughs. Monitors, possibly connected to auxiliary powersupplies, might be designed
49、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 stock or test organisms will be placed should not containsubstances that can be leached or disso
