1、Designation: E 2455 06Standard Guide forConducting Laboratory Toxicity Tests with FreshwaterMussels1This standard is issued under the fixed designation E 2455; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revisi
2、on. 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 standard guide describes methods for conductinglaboratory toxicity tests with early life stages of freshwatermussels includ
3、ing 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 cited as poten
4、tially 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 (1); Neves 199
5、7, 2004 (2, 3).2Habitatalteration, introduction of exotic species, over-utilization, dis-ease, predation and pollution are considered causal or contrib-uting factors in many areas of the United States (Neves et al1997) (4). Over the past decade, there have been over 75published studies conducted tha
6、t 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 sensitive specie
7、s 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 previous studie
8、s 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 Unionidae or Marg
9、aritiferidae (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 stage uses
10、 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 parasitic stage o
11、n 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 or the fins
12、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 overgrowth of host t
13、issue.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 mussels on
14、 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-sustained exi
15、stenceas 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 of sediment
16、 for food and oxygen.Newly-transformed juvenile mussels feed using ciliary currentson 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 physiologically diff
17、erent from adult mussels, protec-tion of habitat quality of adult life stages may not be protective1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fate and is the direct responsibility of SubcommitteeE47.03 on Sediment Assessment and Toxicology.Curr
18、ent edition approved April 1, 2006 Published May 2006. Originallyapproved in 2005. Last previous edition approved in 2005 as E 2455-05.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, We
19、st Conshohocken, PA 19428-2959, United States.of 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 effects of contaminants on mussels need to evaluatepotential exposur
20、e 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 mussels.Annex A1 provides guidance for conducting water-only toxi
21、c-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 toconduct an inter-laboratory toxicity test with glochidia andju
22、venile 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 for obtainingglochidia to propagate juvenile mussels using a host
23、 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 E 724 describes procedures for conductingacute 48-h toxicity tests with embryos or larvae of sa
24、ltwaterbivalve mollusks. Endpoints measured in Guide E 724 includesurvival or shell deposition. Procedures outlined in GuideE 724 may be useful in helping to design studies for conduct-ing toxicity tests with freshwater mussels as outlined in AnnexA1.1.4.4 Results of tests, even those with the same
25、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 forconducting toxicity tests with aquatic organisms. If tests are
26、conducted 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 aquaticorganisms; however, modifications may be necessary.1.5 S
27、ummary 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. Freshwater musselstended to be less sensitive in exposures to some p
28、esticides 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 ammoniamay not be protective of freshwater mussels.1.5.2 Augspurg
29、er 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 harmful to mussels in acute and chronic exposures and wereused to
30、 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). The freshwater mussels are at the sensitiveend of the ran
31、ge 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 N at pH 8 at 25C (for applicationwhen salmonids absent). No
32、 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 concentration(CCC), the estimated CCC for mussels was 20 to 75 % lessthan the CCC of 1.24 mg/L total a
33、mmonia as N at pH 8 and25C. Hence, Augspurger et al (2003) (6) concluded that theacute and chronic WQC for ammonia may not 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
34、, Utterbackia imbecillis, Lampsiliscardium, Lampsilis siliquoidea, Megalonaias nervosa, andLigumia subrostrata, and with two commonly-tested organ-isms, Ceriodaphnia dubia and Daphnia magna. Chemicalsselected for testing (carbaryl, copper, 4-nonylphenol, pen-tachlorophenol, permethrin, and 2,4-dichl
35、orophenoxyaceticacid 2,4-D) represented different chemical classes and differ-ent toxic modes of action (Dwyer et al 2005a,b) (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)
36、 concluded that thetoxicity data generated with C. dubia and D. magna wererelatively protective of the range of sensitivities exhibited byglochidia of the mussels species tested. However, toxicity datagenerated with the commonly-tested U. imbecillis were notalways protective of the range of sensitiv
37、ities exhibited by theother mussel species tested.1.6 This standard is arranged as follows:SectionScope 1Referenced Documents 2Terminology 3Summary of Guide 4Significance and Use 5Apparatus 6Hazards 7Dilution Water 8Test Material 9Test Organisms 10Quality Assurance and Quality Control 11Experimental
38、 Design 12Analytical Methodology 13Calculation of Results 14Report 15Precision and Bias 16Keywords 17Guidance for Conducting Water-only Toxicity Testswith Early Life Stages of Freshwater MusselsAnnex A1References1.7 The values stated in SI units are to be regarded as thestandard. The values given in
39、 parentheses are for informationonly.E24550621.8 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 standard to establish appro-priate safety and health practices and determine the applica-bility of regula
40、tory limitations prior to use. Specific hazardstatements are given in Section 7.2. Referenced Documents2.1 ASTM Standards:3D 4447 Guide for Disposal of Laboratory Chemicals andSamplesE 177 Practice for Use of the Terms Precision and Bias inASTM Test MethodsE 691 Practice for Conducting an Interlabor
41、atory Study toDetermine the Precision of a Test MethodE 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 Termino
42、logy Relating to Biological Effects and En-vironmental FateE 1023 Guide for Assessing the Hazard of a Material toAquatic Organisms and Their UsesE 1241 Guide for Conducting Early Life-Stage ToxicityTests with FishesE 1367 Test Method for Measuring the Toxicity ofSediment-Associated Contaminants with
43、 Estuarine andMarine InvertebratesE 1391 Guide for Collection, Storage, Characterization, andManipulation of Sediments for Toxicological Testing andfor Selection of Samplers Used to Collect Benthic Inver-tebratesE 1706 Test Method for Measuring the Toxicity ofSediment-Associated Contaminants with Fr
44、eshwater Inver-tebratesE 1847 Practice for Statistical Analysis of Toxicity TestsConducted Under ASTM GuidelinesE 1850 Guide for Selection of Resident Species as TestOrganisms for Aquatic and Sediment Toxicity TestsIEEE/ASTM SI 10 Standard for Use of the InternationalSystem of Units (SI) (the Modern
45、ized Metric System)3. Terminology3.1 The words “must,” “should,” “may,” “can,” and “might”have very specific meanings in this standard. “Must” is used toexpress an absolute requirement, that is, to state that a testought to be designed to satisfy the specified conditions, unlessthe purpose of the te
46、st requires a different design. “Must” isused only in connection with the factors that relate directly tothe acceptability of a test. “Should” is used to state that thespecified condition is recommended and ought to be met ifpossible. Although the violation of one “should” is rarely aserious matter,
47、 violation of several will often render the resultsquestionable. Terms such as “is desirable,” “is often desirable,”and “might be desirable” are used in connection with lessimportant factors. “May” is used to mean “is (are) allowed to,”“can” is used to mean “is (are) able to,” and “might” is used to
48、mean “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 E 729 and E 1241 and TerminologyE 943 and D 1129. For an expla
49、nation of units and symbols,refer to Practice E 380. A listing of the common and scientificnames of freshwater mussels in NorthAmerica can be found inAFS (1998) (13).3.3 Definitions of Terms Specific to This Standard: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.FIG. 1 Life Cycle of a Freshwater Mussel (Chris Barnhart, Missouri State University, Springfield, MO)E24550633.3.1 acute testa c
