1、Designation: E1563 98 (Reapproved 2012)Standard Guide forConducting Static Acute Toxicity Tests with EchinoidEmbryos1,2This standard is issued under the fixed designation E1563; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the y
2、ear 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 covers procedures for obtaining laboratorydata concerning the acute effects of a test material on em
3、bryosand 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)3and West Coast species (Strongylocentrotus purpuratus, S.dr
4、oebachiensis, 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).These procedures will probably be useful for conducting s
5、taticacute 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 appropriate to a particular species or special needs andcircumstanc
6、es 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 unmodified versions of these procedures mightprovide useful infor
7、mation 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. With appropriate modifications, these proce-dures can be used
8、 to conduct acute tests on temperature,dissolved oxygen, and pH and on such materials as aqueouseffluents (see also Guide E1192), leachates, oils, particulatematter, surface waters and sediments (Annex A1). Renewaltests might be preferable to static tests for materials that havea high oxygen demand,
9、 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 with echinoid embryosshould usually be reported as the 50 %
10、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 below a specificconcentration.1.5 This guide is arranged as fol
11、lows: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 8.1Source 8.2Treatment 8.3Characterization 8.4Test Material
12、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 10.7Procedure 11Experimental Design 11.1Dissolved Oxygen 11.21A Standard Guide is a document, dev
13、eloped 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 Fateand is the direct
14、responsibility of SubcommitteeE47.01 on Aquatic Assessment and Toxicology.Current edition approved Dec. 1, 2012. Published January 2013. Originallyapproved in 1995. Last previous edition approved in 2004 as E1563 98 (2004).DOI: 10.1520/E1563-98R12.3The boldface numbers in parentheses refer to the li
15、st of references at the end ofthis standard.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1Temperature 11.3Beginning the Test 11.4Feeding 11.5Duration of Test 11.6Biological Data 11.7Control Performance 11.8Other Measurements 11.9Ana
16、lytical Methods 12Acceptability of Test 13Calculation of Results 14Report 15Keywords 16AnnexSediment Tests Annex A11.6 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, associated with its use. It is there
17、sponsibility 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:4E380 Practice for Use of the International
18、System of Units(SI) (the Modernized Metric System) (Withdrawn 1997)5E724 Guide for Conducting Static Acute Toxicity TestsStarting with Embryos of Four Species of SaltwaterBivalve MolluscsE729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-iansE
19、943 Terminology Relating to Biological Effects 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 Tes
20、t Method for Measuring the Toxicity of Sediment-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-tebratesE1525 Guide
21、 for Designing Biological Tests with SedimentsE1706 Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater 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 la
22、rva. Theterm “larva” 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 the
23、test ought to be 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
24、 andought to be 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”
25、 is usedto mean “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
26、this guide, referto Guide E729 and Terminology E943. For sediment tests(Annex A1), refer to standards E1391 and E1525. For anexplanation of units and symbols, refer to Practice E380.4. Summary of Guide4.1 Adult sea urchins and sand dollars are brought into thelaboratory and identified to species. If
27、 the gonads 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.
28、In order to 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
29、 on the species 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 theor
30、ganisms and the suitability of the dilution water, testconditions, handling procedures, etc.; and (2) the basis forinterpreting data obtained from the other treatments. In each ofone or more other treatments, the embryos and resulting larvaeare maintained in dilution water to which a selected concen
31、-tration of test material has been added. The EC50 is calculatedbased on the proportion of larvae that develop into normalpluteus larvae in chambers containing the test material relativeto normal larvae in the controls at the termination of the test.5. Significance and Use5.1 An acute toxicity test
32、is conducted to obtain informationconcerning the acute effects of a short-term exposure of4For 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 Sum
33、mary page onthe ASTM website.5The last approved version of this historical standard is referenced onwww.astm.org.E1563 98 (2012)2organisms to a test material under specific experimental con-ditions. An acute toxicity test does not provide informationconcerning whether delayed effects will occur.5.2
34、Because embryos and larvae are usually assumed to bethe most sensitive life stages 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
35、 concentrations to saltwater species in general. Theresults of these acute toxicity tests are often assumed to be animportant consideration when assessing the hazard of materialsto other saltwater organisms (see Guides E724 and E1023)orwhen deriving water quality criteria for saltwater organisms(6).
36、5.3 The results of acute toxicity tests might be used topredict acute effects 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 substrat
37、e.5.4 The 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 factors on the results of such tests.5.5 The results of acute toxicity tests might be useful
38、 forstudying the biological availability of, and structure-activityrelationships 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.6. Apparatus6.1 Facilities:6.1.1 Flow-throu
39、gh troughs with appropriate trays should beavailable for holding and conditioning test animals (7). Thewater-supply system should be equipped for temperature con-trol and aeration (see 8.3) and should contain strainers and airtraps. Air used for aeration should be free of fumes, oil, andwater; filte
40、rs to remove oil and water are desirable. TestFIG. 1 Drawings Exemplifying Key Developmental Stages of Normal Echinoid Larvae Occurring During the First 48 to 96 h of Develop-ment and Examples of Abnormal or Arrested Development (38)E1563 98 (2012)3chambers should be in a constant-temperature room,
41、incubator,or recirculating water bath. A dilution-water tank or headbox,which 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 fur
42、ther reducethe possibility of contamination of the test organisms by testmaterials 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 cleane
43、d. Organisms should beshielded from disturbances, with curtains or partitions, 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 conne
44、cted to auxiliary powersupplies, might be designed to initiate aeration, sound 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 hol
45、ding and conditioning troughs toincrease or decrease more than 2C/day to reduce 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 substancestha
46、t can be leached or dissolved by aqueous solutions inamounts that affect the 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,
47、and fluorocarbon plastics should be used whenever possible tominimize dissolution, 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 tanksand in the water-supply system, but they should be
48、soaked,preferably in flowing dilution water, for a week or more beforeuse (8). Brass, copper, lead, galvanized metal, cast-iron pipe,and natural rubber should not contact the dilution water, stocksolutions, or test solutions before or during the test. Items madeof neoprene rubber and other materials
49、 not mentioned aboveshould not be used unless it has been shown that the embryosand resulting larvae of the test species do not show more signsof stress, such as discoloration, abnormal development, orFIG. 2 (a) Examples of Normal and Abnormal Development ofPurple Sea Urchin (Strongylocentrotus purpuratus) Embryos atthe Conclusion 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 a Bor-derline Abnormal Pluteus with Poor Symmetrical Form and OneMissing Arm. Figs. 2d through 2
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