1、Designation: E2455 06 (Reapproved 2013)Standard Guide forConducting Laboratory Toxicity Tests with FreshwaterMussels1This standard is issued under the fixed designation E2455; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the yea
2、r 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 standard guide describes methods for conductinglaboratory toxicity tests with early life stages of freshwate
3、rmussels including glochidia and juvenile mussels in water-onlyexposures (Annex A1). Future revisions to this standard maydescribe methods for conducting toxicity tests with (1) adultfreshwater mussels and (2) contaminated sediments usingvarious life stages of freshwater mussels.1.2 Many factors are
4、 cited as potentially contributing to thedecline of freshwater mussel populations in NorthAmerica. Ofthe nearly 300 taxa of freshwater mussels in NorthAmerica, 70species (23 %) are listed as endangered or threatened andanother 40 species (14 %) are candidates for possible listing(Williams et al 1993
5、 (1); Neves 1997, 2004 (2, 3).2Habitatalteration, introduction of exotic species, over-utilization,disease, predation and pollution are considered causal orcontributing factors in many areas of the United States (Neveset al 1997) (4). Over the past decade, there have been over 75published studies co
6、nducted that have evaluated the role ofcontaminants in the decline of populations of freshwatermussels (Kernaghan et al 2005) (5). In these studies, early lifestages of mussels of several species are highly sensitive tosome metals and ammonia in water exposures when comparedto many of the most sensi
7、tive species of other invertebrates,fish, or amphibians that are commonly used to establish U.S.Environmental Protection Agency Water Quality Criteria(WQC; Augspurger et al 2003 (6), Keller et al 2005 (7),Kernaghan et al 2005 (5); USGS (2005a,b) (8, 9) section 1.5).Importantly, results of these prev
8、ious studies indicate WQC forindividual chemicals established for the protection of aquaticorganisms may not be adequately protective of sensitive stagesof freshwater mussels.1.3 Summary of Life History of Freshwater Mussels:1.3.1 Freshwater mussels are bivalve mollusks belonging tothe family Unioni
9、dae or Margaritiferidae (section 10.1).Adultsare sedentary animals, spending their entire lives partially orcompletely burrowed in the bottoms of streams, rivers, orlakes. Adult mussels are filter feeders, using their gills toremove suspended particles from the water column. Themicroscopic, juvenile
10、 stage uses foot (pedal) feeding to somedegree for the first several months of their lives, feeding ondepositional materials in pore water of sediment, includingbacteria, algae, and detritus. Freshwater mussels have anunusual and complex mode of reproduction, which includes abrief, obligatory parasi
11、tic stage on fish or other host organismscalled glochidia (Fig. 1).1.3.2 The successful transfer of mature glochidia to asuitable host constitutes a critical event in the life cycle of mostfreshwater mussels. Once the glochidia are released from thefemale, the glochidia need to attach to the gills o
12、r the fins of anappropriate fish host and encyst to complete development.Although glochidia may survive for months during brooding inthe female mussel, glochidia typically survive for only a fewdays after release unless the glochidia reach a compatible host.Encystment on the host occurs by overgrowt
13、h of host tissue.Metamorphosis of juvenile mussels on the fish host occurswithin days or weeks, depending on species and temperature.Host fish specificity varies among mussels. While some musselspecies appear to require a single host organism, other speciescan transform their glochidia into juvenile
14、 mussels on severalspecies of host fish. Following proper host infestation,glochidia transform into microscopic juveniles and excyst(drop off) and settle into suitable habitat to survive. Thetransformation of glochidia to juveniles results in the develop-ment of internal organs necessary for self-su
15、stained existenceas a benthic organism.1.3.3 Newly-transformed juvenile mussels have a life styledifferent from adult mussels. Transformed juvenile musselsmay be at the sediment-water interface or may burrow severalcentimeters into sediment and rely on water percolating be-tween substrate particles
16、of sediment for food and oxygen.Newly-transformed juvenile mussels feed using ciliary currents1This guide is under the jurisdiction ofASTM Committee E50 on EnvironmentalAssessment, Risk Management and Corrective Action and is the direct responsibil-ity of Subcommittee E50.47 on Biological Effects an
17、d Environmental Fate.Current edition approved March 1, 2013 Published May 2013. Originallyapproved in 2005. Last previous edition approved in 2006 as E2455-06. DOI:10.1520/E2455-06R13.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.Copyright ASTM Inter
18、national, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1on the foot and mantle. Older juvenile and adult mussels likelyuse different food types when living in different microenviron-ments. Given that glochidia and juvenile mussels are ecologi-cally and physiolog
19、ically different from adult mussels, protec-tion of habitat quality of adult life stages may not be protectiveof glochidia or juvenile life stages of freshwater mussels.Distributions of adult mussels are dependent both on thepresence of host fish and on microhabitat conditions. Efforts toassess effe
20、cts of contaminants on mussels need to evaluatepotential exposure to host fish in addition to exposure to eachunique life stage of freshwater mussels.1.4 Summary of Toxicity Testing Conditions:1.4.1 Section 4 provides a summary of conditions forconducting toxicity tests with glochidia and juvenile m
21、ussels.Annex A1 provides guidance for conducting water-only toxic-ity tests with glochidia and juvenile mussels. Recommendedtest conditions for conducting these toxicity tests are based onvarious published methods outlined in Table A1.1 and TableA1.4 in Annex A1 and are based on the conditions used
22、toconduct an inter-laboratory toxicity test with glochidia andjuvenile mussels (section 16.5). Glochidia and juvenile musselsare only available on a seasonal basis. Section 10 describesprocedures for collecting adult female mussels from the field toobtain glochidia for conducting toxicity tests or f
23、or obtainingglochidia to propagate juvenile mussels using a host organism.1.4.2 In the field, mussels may be exposed to contaminantsin water, sediment, or food. This standard only addresseseffects associated with exposure of mussels to contaminants inwater.1.4.3 Guide E724 describes procedures for c
24、onducting acute48-h toxicity tests with embryos or larvae of saltwater bivalvemollusks. Endpoints measured in Guide E724 include survivalor shell deposition. Procedures outlined in Guide E724 may beuseful in helping to design studies for conducting toxicity testswith freshwater mussels as outlined i
25、n Annex A1.1.4.4 Results of tests, even those with the same species,using procedures different from those described in Annex A1may not be comparable. Comparison of results obtained usingmodified versions of these procedures might provide usefulinformation concerning new concepts and procedures forco
26、nducting toxicity tests with aquatic organisms. If tests areconducted with procedures different from those described inthis standard, additional tests are required to determine com-parability of results. General procedures described in thisstandard might be useful for conducting tests with other aqu
27、aticorganisms; however, modifications may be necessary.1.5 Summary of Results of Toxicity Tests Conducted withFreshwater Mussels:1.5.1 Keller et al (2005) (7) summarized results of acutelaboratory toxicity tests conducted with glochidia and juvenilemussels described in 16 published studies. Freshwat
28、er musselstended to be less sensitive in exposures to some pesticides andother organic compounds compared to other commonly-testedaquatic organisms. In contrast, Keller et al (2005) (7) con-cluded that U.S. Environmental Protection Agency (USEPA)water quality criteria (WQC) for some metals and ammon
29、iamay not be protective of freshwater mussels.1.5.2 Augspurger et al (2003) (6) evaluated ammonia tox-icity data generated for glochidia and juvenile of freshwatermussels in laboratory toxicity tests. Specifically, these toxicitydata were used to estimate concentrations that would not likelybe harmf
30、ul to mussels in acute and chronic exposures and wereused to evaluate the protectiveness of the WQC for ammonia.Results of acute toxicity tests (24 to 96 h) for 10 species in 8genera were used to calculate genus mean acute values(GMAVs) ranging from 2.56 to 8.97 mg/L(total ammonia as Nat pH 8 at 25C
31、). The freshwater mussels are at the sensitiveend of the range when added to the GMAVs from the databaseused to derive the acute WQC for ammonia. Recalculation ofthe criteria maximum concentration (CMC) including thesemussel data resulted in a CMC 75 % lower than the CMC of5.62 mg/L total ammonia as
32、 N at pH 8 at 25C (for applicationwhen salmonids absent). No chronic ammonia toxicity data(for example, 21 to 28-d exposures) were available for fresh-water mussels; however, when a range of acute to chronicratios were used to estimate a criteria continuous concentrationFIG. 1 Life Cycle of a Freshw
33、ater Mussel (Chris Barnhart, Missouri State University, Springfield, MO)E2455 06 (2013)2(CCC), the estimated CCC for mussels was 20 to 75 % lessthan the CCC of 1.24 mg/L total ammonia as N at pH 8 and25C. Hence, Augspurger et al (2003) (6) concluded that theacute and chronic WQC for ammonia may not
34、be protective offreshwater mussels.1.5.3 Milam et al (2005) (10) conducted a series of 24-hacute toxicity tests with glochidia of six freshwater musselspecies, Leptodea fragilis, Utterbackia imbecillis, Lampsiliscardium, Lampsilis siliquoidea, Megalonaias nervosa, andLigumia subrostrata, and with tw
35、o commonly-testedorganisms, Ceriodaphnia dubia and Daphnia magna. Chemi-cals selected for testing (carbaryl, copper, 4-nonylphenol,pentachlorophenol, permethrin, and 2,4-dichlorophenoxyaceticacid 2,4-D) represented different chemical classes and differ-ent toxic modes of action (Dwyer et al 2005a,b)
36、 (11, 12).Nosingle chemical elicited consistently high or low toxicity;however, carbaryl and 2,4-D were generally the least toxic tothe species tested. Milam et al (2005) (10) concluded that thetoxicity data generated with C. dubia and D. magna wererelatively protective of the range of sensitivities
37、 exhibited byglochidia of the mussels species tested. However, toxicity datagenerated with the commonly-tested U. imbecillis were notalways protective of the range of sensitivities exhibited by theother mussel species tested.1.6 This standard is arranged as follows:SectionScope 1Referenced Documents
38、 2Terminology 3Summary of Guide 4Significance and Use 5Apparatus 6Hazards 7Dilution Water 8Test Material 9Test Organisms 10Quality Assurance and QualityControl11Experimental Design 12Analytical Methodology 13Calculation of Results 14Report 15Precision and Bias 16Keywords 17Guidance for Conducting Wa
39、ter-only Toxicity Testswith Early Life Stages ofFreshwater MusselsAnnex A1References1.7 The values stated in SI units are to be regarded as thestandard. The values given in parentheses are for informationonly.1.8 This standard does not purport to address all of thesafety concerns, if any, associated
40、 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 7.2. Referenced Documents2.1 ASTM Standards:3D4447 Guide f
41、or Disposal of Laboratory Chemicals andSamplesE177 Practice for Use of the Terms Precision and Bias inASTM Test MethodsE691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test MethodE724 Guide for Conducting Static Acute Toxicity TestsStarting with Embryos of Four Sp
42、ecies of SaltwaterBivalve MolluscsE729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-iansE943 Terminology Relating to Biological Effects and Envi-ronmental FateE1023 Guide for Assessing the Hazard of a Material toAquatic Organisms and Their Us
43、esE1241 Guide for Conducting Early Life-Stage Toxicity Testswith FishesE1367 Test 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 Testin
44、g andfor Selection of Samplers Used to Collect Benthic Inver-tebratesE1706 Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater InvertebratesE1847 Practice for Statistical Analysis of Toxicity TestsConducted Under ASTM GuidelinesE1850 Guide for Selection of Resi
45、dent Species as TestOrganisms for Aquatic and Sediment Toxicity TestsIEEE/ASTM SI 10 Standard for Use of the InternationalSystem of Units (SI) (the Modernized Metric System)3. Terminology3.1 The words “must,” “should,” “may,” “can,” and “might”have very specific meanings in this standard. “Must” is
46、used toexpress an absolute requirement, that is, to state that a testought to be designed to satisfy the specified conditions, unlessthe purpose of the test requires a different design. “Must” isused only in connection with the factors that relate directly tothe acceptability of a test. “Should” is
47、used to state that thespecified condition is recommended and ought to be met ifpossible. Although the violation of one “should” is rarely aserious matter, violation of several will often render the resultsquestionable. Terms such as “is desirable,” “is often desirable,”3For referenced ASTM standards
48、, 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.E2455 06 (2013)3and “might be desirable” are used in connection with lessimportant factors.
49、 “May” is used to mean “is (are) allowed to,”“can” is used to mean “is (are) able to,” and “might” is used tomean “could possibly.” Thus, the classic distinction between“may” and “can” is preserved, and “might” is never used as asynonym for either “may” or “can.”3.2 DefinitionsFor definitions of other terms used in thisstandard, refer to Guides E729 and E1241 and TerminologyE943 and D1129. For an explanation of units and symbols,refer to Practice E380. A listing of the common and scientificnames of freshwater mussels in NorthAmerica can be found inAFS (1998) (13).3.3 Definiti
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