1、Designation: E 1563 98 (Reapproved 2004)e1Standard Guide forConducting Static Acute Toxicity Tests with EchinoidEmbryos1,2This standard is issued under the fixed designation E 1563; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, t
2、he 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.e1NOTETable 1 was editorially corrected in March 2006.1. Scope1.1 This guide covers procedures for obtaining laborator
3、ydata concerning the acute effects of a test material on embryosand the resulting larvae of echinoid embryos (sea urchins andsand dollars) during static 48- to 96-h exposures. Theseprocedures have generally been used with U.S. East Coast(Arbacia punctulata and Strongylocentrotus droebachiensis)(1)3a
4、nd West Coast species (Strongylocentrotus purpuratus, S.droebachiensis, and Dendraster excentricus) (2). The basicprocedures described in this guide first originated in Japan andScandanavia (3), and parallel procedures have been used withforeign species, especially in Japan and the Mediterranean (4)
5、.These procedures will probably be useful for conducting staticacute toxicity tests with embryos of other echinoid species,although modifications might be necessary.1.2 Other modifications of these procedures might be justi-fied by special needs or circumstances. Although using proce-dures appropria
6、te to a particular species or special needs andcircumstances is more important than following prescribedprocedures, the results of tests conducted by using unusualprocedures are not likely to be comparable with those of manyother tests. The comparison of results obtained by usingmodified and unmodif
7、ied versions of these procedures mightprovide useful information concerning new concepts andprocedures for conducting acute tests starting with embryos ofechinoids.1.3 These procedures are applicable to most chemicals,either individually or in formulations, commercial products, orknown mixtures. Wit
8、h 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, surface waters and sediments (Annex A1). Renewaltests might be preferable t
9、o static tests for materials that havea high oxygen demand, are highly volatile, are rapidly trans-formed biologically or chemically in aqueous solution, or areremoved from test solutions in substantial quantities by the testchambers or organisms during the test.1.4 Results of acute toxicity tests w
10、ith echinoid embryosshould usually be reported as the 50 % effect concentration(EC50) based on the total abnormally developed embryos andlarvae. In some situations, it might only be necessary todetermine whether a specific concentration is acutely toxic toembryos or whether the EC50 is above or belo
11、w a specificconcentration.1.5 This guide is arranged as follows:SectionScope 1Referenced Documents 2Terminology 3Summary of Guide 4Significance and Use 5Apparatus 6Facilities 6.1Construction Materials 6.2Test Chambers 6.3Cleaning 6.4Acceptability 6.5Safety Precautions 7Dilution Water 8Requirements 8
12、.1Source 8.2Treatment 8.3Characterization 8.4Test Material 9General 9.1Stock Solution 9.2Test Concentration(s) 9.3Test Organisms 10Species 10.1Age 10.2Source of Embryos 10.3Handling 10.4Test Animal Source and Condition 10.5Spawning and Fertilization 10.6Quality 10.71A Standard Guide is a document, d
13、eveloped using the consensus mechanismsof ASTM that provides guidance for the selection of procedures to accomplish aspecific test, but which does not stipulate specific procedures.2This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fate and is the dire
14、ct responsibility of SubcommitteeE47.01 on Aquatic Assessment and Toxicology.Current edition approved April 1, 2004. Published April 2004. Originallyapproved in 1995. Last previous edition approved in 1998 as E 1563 98.3The boldface numbers in parentheses refer to the list of references at the end o
15、fthis standard.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.Procedure 11Experimental Design 11.1Dissolved Oxygen 11.2Temperature 11.3Beginning the Test 11.4Feeding 11.5Duration of Test 11.6Biological Data 11.7Control Performance 1
16、1.8Other Measurements 11.9Analytical Methods 12Acceptability of Test 13Calculation of Results 14Report 15Keywords 16AnnexSediment Tests AnnexA11.6 The values stated in SI units are to be regarded as thestandard.1.7 This standard does not purport to address all of thesafety concerns, if any, associat
17、ed 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. Specific precautionary state-ments are given in Section 7.2. Referenced Documents2.1 ASTM Standards:4E 380 Practice
18、for Use of the International System of Units(SI)5E 724 Guide for Conducting Static Acute Toxicity TestsStarting with Embryos of Four Species of SaltwaterBivalve MolluscsE 729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-iansE 943 Terminology
19、Relating to Biological Effects and En-vironmental FateE 1023 Guide for Assessing the Hazard of a Material toAquatic Organisms and Their UsesE 1192 Guide for Conducting Acute Toxicity Tests onAqueous Ambient Samples with Effluents with Fishes,Macroinvertebrates, and AmphibiansE 1367 Test Method for M
20、easuring the Toxicity ofSediment-Associated Contaminants with Marine and Es-tuarine InvertebratesE 1391 Guide for Collection, Storage, Characterization, andManipulation of Sediments for Toxicological Testing, andfor Selection of Samples Used to Collect Benthic Inverte-bratesE 1525 Guide for Designin
21、g Biological Tests with Sedi-mentsE 1706 Method for Measuring the Toxicity of Sediment-Associated Contaminants with Fresh Water Invertebrates3. Terminology3.1 Definitions:3.1.1 The term “embryo” is used herein to denote the stagesbetween the fertilization of the egg and the pluteus larva. Theterm “l
22、arva” is used herein to refer to the pluteus larvacharacteristic of all echinoids (5) (Fig. 1 and Fig. 2 ).3.1.2 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 b
23、e designed to satisfy the 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
24、 met if possible.Although 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
25、 “is (are) allowed to,” “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.”3.1.3 For definitions of other terms used in this guide, ref
26、erto Guide E 729 and Terminology E 943. For sediment tests(Annex A1), refer to standards E 1391 and E 1525. For anexplanation of units and symbols, refer to Practice E 380.4. Summary of Guide4.1 Adult sea urchins and sand dollars are brought into thelaboratory and identified to species. If the gonad
27、s are not ripe,the sea urchins or sand dollars should be held and fed until thegonads are brought into a suitable reproductive state. Echinoidswith ripe gonads are maintained under conditions that keep thegonads ripe without inducing undesired spontaneous spawningor resorption of gametes. In order t
28、o start a test, spawning isinduced by using one or more stimuli, which may be physical(for example, electrical current) or chemical (for example,potassium chloride).4.2 In each of two or more treatments, embryos and theresulting larvae of one species are maintained for 48 to 96 h,depending on the sp
29、ecies and test temperature. In each of oneor more control treatments, the embryos and resulting larvaeare maintained in dilution water to which no test material hasbeen added in order to provide (1) a measure of the accept-ability of the test by giving an indication of the quality of theorganisms an
30、d the suitability of the dilution water, test condi-tions, handling procedures, etc.; and (2) the basis for interpret-ing data obtained from the other treatments. In each of one ormore other treatments, the embryos and resulting larvae aremaintained in dilution water to which a selected concentratio
31、nof test material has been added. The EC50 is calculated basedon the proportion of larvae that develop into normal pluteuslarvae in chambers containing the test material relative tonormal larvae in the controls at the termination of the test.4For referenced ASTM standards, visit the ASTM website, ww
32、w.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.5Withdrawn.E 1563 98 (2004)e125. Significance and Use5.1 An acute toxicity test is conducted to obtain informationconce
33、rning the acute effects of a short-term exposure oforganisms to a test 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 st
34、ages of these echinoid species, andbecause some of these species are commercially and recre-ationally important, the results of these acute tests are oftenconsidered to be a good indication of the acceptability ofpollutant concentrations to saltwater species in general. Theresults of these acute tox
35、icity tests are often assumed to be animportant consideration when assessing the hazard of materialsto other saltwater organisms (see Guides E 724 and E 1023)orwhen deriving water quality criteria for saltwater organisms(6).5.3 The results of acute toxicity tests might be used topredict acute effect
36、s likely to occur to aquatic organisms infield situations as a result of exposure under comparableconditions, except that toxicity to benthic species might dependon sorption or settling of the test material onto the substrate.5.4 The results of acute tests might be used to compare theacute sensitivi
37、ties of different species and the acute toxicities ofdifferent test materials, and to determine the effects of variousenvironmental factors on the results of such tests.5.5 The results of acute toxicity tests might be useful forstudying the biological availability of, and structure-activityrelations
38、hips between, test materials.5.6 The results of acute toxicity tests will depend ontemperature, composition of the dilution water, condition of thetest organisms, and other factors.FIG. 1 Drawings Exemplifying Key Developmental Stages of Normal Echinoid Larvae Occurring During the First 48 to 96 h o
39、fDevelopment and Examples of Abnormal or Arrested Development (38)E 1563 98 (2004)e136. Apparatus6.1 Facilities:6.1.1 Flow-through troughs with appropriate trays should beavailable for holding and conditioning test animals (7). Thewater-supply system should be equipped for temperature con-trol and a
40、eration (see 8.3) and should contain strainers and airtraps. Air used for aeration should be free of fumes, oil, andwater; filters to remove oil and water are desirable. Testchambers should be in a constant-temperature room, incubator,or recirculating water bath. A dilution-water tank or headbox,whi
41、ch may be used to prepare reconstituted water, is oftenelevated so that dilution water can be gravity-fed into holdingand conditioning troughs and test chambers. The facilityshould be well ventilated and free of fumes. To further reducethe possibility of contamination of the test organisms by testma
42、terials and other substances, especially volatile ones, hold-ing and conditioning troughs should not be in a room in whichthe toxicity tests are conducted, stock solutions or test solutionsare prepared, or equipment is cleaned. Organisms should beshielded from disturbances, with curtains or partitio
43、ns, toprevent unnecessary stress during holding, conditioning, andtesting.6.1.2 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 to initiate aeration, so
44、und alarms,or activate telephone autodialing alarms if the water flow ortemperature deviates from preset limits. If the temperaturebecomes 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 re
45、duce the chancesof spontaneous spawning.6.2 Construction MaterialsEquipment and facilities thatcontact stock solutions, test solutions, or any water into whichthe test organisms will be placed should not contain substancesthat can be leached or dissolved by aqueous solutions inamounts that affect th
46、e test organisms adversely. In addition,equipment and facilities that contact the stock solutions or testsolutions should be chosen to minimize the sorption of testmaterials from water. Glass, Type 316 stainless steel, nylon,and fluorocarbon plastics should be used whenever possible tominimize disso
47、lution, leaching, and sorption, except that stain-less steel should not be used when testing metals. Concrete andrigid plastics may be used for holding and conditioning tanksFIG. 2 (a) Examples of Normal and Abnormal Development ofPurple Sea Urchin (Strongylocentrotus purpuratus ) Embryos atthe Conc
48、lusion of a 72 to 96-h Toxicity Test. Figs. 2a and 2bShow Examples of Normal Echinopluteus with Four DistinctArms and Good Symmetrical Development. Fig. 2c Shows aBorderline Abnormal Pluteus with Poor Symmetrical Form andOne Missing Arm. Figs. 2d through 2j Show Examples ofIncreasing Abnormal and/or
49、 Retarded (Considered Abnormal)Development. Other Species of Sea Urchins and Sand DollarsWill Have the Same General Larval Form, But Will Vary in Size,Conformation, and Number of Larval Arms. Each InvestigatorUsing a Different Species Should Carefully Compare Well-Developed Embryos from Controls with Gradations of AbnormalDevelopment in a Toxicant to Identify Distinctions betweenNormal and Abnormal for Their Given SpeciesFIG. 2 (b) (continued)E 1563 98 (2004)e14and in the water-supply system, but they should be soaked,preferably in flowing dilution water, for a week or more b
