1、Designation: E2591 07 (Reapproved 2013)Standard Guide forConducting Whole Sediment Toxicity Tests withAmphibians1This standard is issued under the fixed designation E2591; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of
2、 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 standard covers procedures for obtaining labora-tory data concerning the toxicity of test material (for example,
3、sediment or hydric soil (that is, a soil that is saturated, flooded,or ponded long enough during the growing season to developanaerobic (oxygen-lacking) conditions that favor the growthand regeneration of hydrophytic vegetation) to amphibians.This test procedure uses larvae of the northern leopard f
4、rog(Rana pipiens). Other anuran species (for example, the greenfrog (Rana clamitans), the wood frog (Rana sylvatica), theAmerican toad (Bufo americanus) may be used if sufficientdata on handling, feeding, and sensitivity are available. Testmaterial may be sediments or hydric soil collected from thef
5、ield or spiked with compounds in the laboratory.1.2 The test procedure describes a 10-d whole sedimenttoxicity test with an assessment of mortality and selectedsublethal endpoints (that is, body width, body length). Thetoxicity tests are conducted in 300 to 500-mL chamberscontaining 100 mLof sedimen
6、t and 175 mLof overlying water.Overlying water is renewed daily and larval amphibians are fedduring the toxicity test once they reach Gosner stage 25(operculum closure over gills). The test procedure is designedto assess freshwater sediments, however, R. pipiens can toler-ate mildly saline water (no
7、t exceeding about 2500 mg Cl-/L,equivalent to a salinity of about 4.1 when Na+is the cation) in10-d tests, although such tests should always include a con-current freshwater control. Alternative test durations and sub-lethal endpoints may be considered based on site-specificneeds. Statistical evalua
8、tions are conducted to determinewhether test materials are significantly more toxic than thelaboratory control sediment or a field-collected referencesample(s).1.3 Where appropriate, this standard has been designed tobe consistent with previously developed methods for assessingsediment toxicity to i
9、nvertebrates (for example, Hyalella az-teca and Chironomus dilutus toxicity tests) described in theUnited States Environmental ProtectionAgency (USEPA, (1)2freshwater sediment testing guidance, Test Methods E1367 andE1706, and Guides E1391, E1525, E1611, and E1688. Testsextending to 10 d or beyond,
10、and including sublethal measure-ments such as growth, are considered more effective inidentifying chronic toxicity and thus delineating areas ofmoderate contamination (1-3).1.4 Many historical amphibian studies, both water andsediment exposure, have used tests of shorter duration (5 daysor less) (fo
11、r example, 4-7) and, although both survival andsublethal endpoints were often assessed, there is substantiveevidence that tests of longer duration are likely to be moresensitive to some contaminants (8, 9). Research performed todevelop and validate this test protocol included long-term(through metam
12、orphosis) investigations and other researchershave also conducted long-duration tests with anurans (7-11).Inthe development of these procedures, an attempt was made tobalance the needs of a practical assessment with the importanceof assessing longer-term effects so that the results will demon-strate
13、 the needed accuracy and precision. The most recentsediment toxicity testing protocols for invertebrates haveencompassed longer duration studies which allow the measure-ment of reproductive endpoints (1, 12). Such tests, because ofincreased sensitivity of the sublethal endpoints, may also behelpful
14、in evaluating toxicity. Full life-cycle studies withanurans (including reproduction) are usually not feasible fromeither a technical or monetary standpoint. However, if site-specific information indicates that the contaminants present arelikely to affect other endpoints (including teratogenicity), t
15、henthe duration of the toxicity test may be increased throughmetamorphosis or additional sublethal endpoints may be mea-sured (for example, impaired behavior, deformities, time-to-metamorphosis). The possible inclusion of these endpoints andextension of test length should be considered during develo
16、p-ment of the project or study plan (see 8.1.1).1.5 The methodology presented in this standard was devel-oped under a Department of Defense (DoD) research programand presented in a guidance manual for risk assessment staff1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffec
17、ts and Environmental Fate and is the direct responsibility of SubcommitteeE47.03 on Sediment Assessment and Toxicology.Current edition approved March 1, 2013. Published March 2013. Originallyapproved in 2007. Last previous edition approved in 2007 as E259107. DOI:10.1520/E2591-07R13.2The boldface nu
18、mbers in parentheses refer to the list of references at the end ofthis standard.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1and state/federal regulators involved in the review and ap-proval of risk assessment work plans and report
19、s (13).Todevelop this method, a number of tests with spiked sedimenttests were conducted (13, 14). Since development of themethodology it has been used operationally to evaluate field-collected sediments from several state and federal environmen-tal sites (15, 16). For most of these studies the pref
20、erred testorganisms, Rana pipiens, was used. At a lead-contaminatedstate-led site, operated by the Massachusetts HighwayDepartment, Xenopus laevis(African clawed frog) was used inthe sediment test system because of availability problems withRana pipiens (17), The test method was also used to evaluat
21、esediment toxicity at a cadmium-contaminated USEPA Region4-led site in Tennessee (18). The methodology was used to helpcharacterize potential effects of contaminants on amphibiansand to help develop preliminary remedial goals, if warranted.All tests evaluated survival and growth effects after 10 d o
22、fexposure in accordance with the methods presented in thisstandard.1.6 The use of larval amphibians to assess environmentaltoxicity is not novel. Researchers have used tadpoles toexamine toxicity of metals and organic compounds. Most ofthese studies have been through water exposure, usually in amann
23、er similar to fish or invertebrate exposure as described inGuide E729 (19-29). Fewer studies have focused on exposureof anuran larvae to sediments, and the methods employed varywidely, from in situ enclosures (30) to laboratory tests usingvariable exposure conditions and organism ages (4, 8, 31-33).
24、No studies were identified that used the same test conditions asdescribed in this standard. However, several laboratory-basedevaluations of sediment effects on amphibians are described inthe following subsections.1.6.1 Sediment toxicity tests conducted in the laboratorywith amphibians were performed
25、 over a range of test durationsfrom4d(4, 31, Guide E1439-98 Appendix X2) to 12 d (33)and through metamorphosis (8, 32). Sediment toxicity testswith anurans native to North America were started with larvaltadpoles between Gosner stages 23 and 25 (8, 32, 33). Testtemperatures were between 21 and 23C a
26、nd feeding beganafter tadpoles reached Gosner stage 25. Food sources wereTetramin (8), boiled romaine lettuce (32), or boiled romainelettuce and dissipated rabbit food pellets (33). Tests wereconducted in static renewal mode with water replacementsconducted at varying rates (daily (31, 33), weekly (
27、8), every 3to5d(32). Test design (number of replicates, test vessel size,number of organisms per replicate) varied depending on theobjective of the study with several tests conducted in aquaria(32), large bins (8), or swimming pools (33). Endpointsevaluated at test termination included survival (4,
28、8, 31-33),growth (8, 31-33), bioaccumulation of metals (8), developmen-tal rates (8, 32), deformities (31, 32), swimming speed (33) andforaging activity levels (32).1.6.2 To assess the effect of direct contact with the sedi-ments containing PCBs, Savage et al. (32) exposed larvaltadpoles (Gosner sta
29、ge 23 to 25; wood frogs (R. sylvatica) tofield-collected sediments under conditions that allowed bothdirect contact with the sediment and separation from thesediment with a 500 m mesh barrier. The study found thatlethal and sublethal effects on tadpoles observed throughmetamorphosis were more pronou
30、nced when direct contactwith the sediment was allowed. The test conditions describedin this standard allow tadpoles to maintain direct contact withthe sediment.1.6.3 Sediment toxicity testing with Xenopus laevis hasfocused on evaluating the developmental effects of sedimentextracts, as opposed to wh
31、ole sediments, on frog embryos.Methods have been developed which expose blastula stageembryos to sediment by enclosing the embryos in a Teflonmesh insert that rests over the top of the sediment in thesedimentwater interface region (31, Guide E1439-98 Appen-dix X2). These studies are conducted evalua
32、te survival,growth, and physical malformations of the embryos after a 4-dexposure period. The test conditions described in this standardallow more direct contact with the sediment, using older testorganisms, and a longer exposure duration.1.7 Sediment toxicity tests are an effective means forevaluat
33、ing the impact of sediment contamination on amphib-ians in a multiple lines of evidence paradigm. The evaluation ismost powerful when toxicity testing sampling stations areco-located with sediment analytical chemistry samples andecological surveys, allowing for a detailed evaluation of theco-occurri
34、ng data in the ecological risk assessment. The spatialand temporal co-location of toxicity testing and analyticalsamples is particularly important for establishing contaminant-specific effects and assessing contaminant bioavailability.1.8 In order for a sediment toxicity test to be sensitive itmust
35、be of sufficient duration to measure potential toxicity andit must be conducted during the appropriate developmentalstage of the test organisms life cycle. Using recently hatchedtadpoles and conducting the sediment exposure test for 10 d toallow the evaluation of growth endpoints meets both of these
36、sensitivity requirements.1.9 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.10 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this
37、standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:3D4447 Guide for Disposal of Laboratory Chemicals andSamplesE177 Practice for Use of the Terms Precision and Bias inASTM Te
38、st MethodsE691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test MethodE729 Guide for Conducting Acute Toxicity Tests on Test3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of
39、 ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.E2591 07 (2013)2Materials with Fishes, Macroinvertebrates, and Amphib-iansE943 Terminology Relating to Biological Effects and Envi-ronmental FateE1367 Test Method for Measuring the Toxicity of Sediment
40、-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-tebratesE1439 Guide for Conducting the Frog Embryo Teratogen-esis
41、Assay-Xenopus (FETAX)E1525 Guide for Designing Biological Tests with SedimentsE1611 Guide for Conducting Sediment Toxicity Tests withPolychaetous AnnelidsE1688 Guide for Determination of the Bioaccumulation ofSediment-Associated Contaminants by Benthic Inverte-bratesE1706 Test Method for Measuring t
42、he Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates3. Terminology3.1 The words “must”, “should”, “may”, “can” and “might”have very specific meanings in this guide. “Must” is used toexpress an absolute requirement, that is, to state that the designof a test ought to be in a
43、manner that satisfies the specifiedconditions, unless project goals dictate needed alterations inorder to address the study hypotheses. “Should” is used to statethat the specified condition is recommended and ought to bemet if possible.Although the violation of one “should” is rarelya serious matter
44、, violation of several could render the resultsquestionable. Terms such as “is desirable”, “is often desirable”and “might be desirable” are used in association with lessimportant factors, the alteration of which will probably nothave substantive effects on test outcome. “May” means “is(are) allowed
45、to,” “can” means “is (are) able to” and “might”means “could possibly.” In this manner, the classic distinctionbetween “may” and “can” is preserved and “might” is neverused as a synonym for either “may” or “can.”3.2 DefinitionsFor definitions of general terms related totoxicity testing and used in th
46、is guide, refer to Guide E943.3.3 Definitions of Terms Specific to This Standard:3.3.1 IC25 (25 % inhibition concentration),nconcentration at which there is a 25 % reduction in organ-ism performance, relative to the control. Performance may besurvival or a sublethal measurement such as growth.3.3.2
47、overlying water, nwater that is placed over thesediment for the duration of the study. Overlying water may besurface water collected from the project site or from a cleanlake or reservoir, or may be reconstituted water prepared in thelaboratory (for example, moderately hard water; (34).3.3.3 referen
48、ce-toxicant test, na test conducted with areagent-grade reference chemical to assess the sensitivity of thetest organisms. Deviations outside an established normal rangemay indicate a change in the sensitivity of the test organismpopulation. Reference-toxicity tests are most often performedin the ab
49、sence of sediment.3.3.4 test sediment or test material, nsediment that maycontain contaminants, which is being evaluated using this testprocedure.4. Summary of Guide4.1 Each test consists of eight replicates of the test material(for example, field-collected sediment or spiked sediment) andoverlying water with five test organisms (recently-hatchedtadpoles) per replicate. A laboratory control sediment (some-times called a negative control) is used to provide (1) ameasure of the acceptability of the test by indicating the qualityof tadpoles, test conditions and handling
