5 Laboratory Toxicity Testing with Freshwater Mussels Christopher G. Ingersoll, Nicola J. Kernaghan, Timothy S. Gross, Cristi D. Bishop, Ning Wang, and AndyRoberts INTRODUCTION Numerous laboratory toxicity studies have been conducted with freshwater mussels in an attempt to understand the role of contaminants in the decline of field populations of mussels(Chapter 7). In these studies, early life stages of musselsofseveral species were highly sensitive to somemetals and ammoniainwater exposures when comparedtomany of the most sensitive species of other invertebrates, fish, or amphibians that are commonly used to establish U.S. Environmental Protec- tion Agency (USEPA)Water QualityCriteria(WQC) (Augspurger et al.2003; USGS 2005a, 2005b). Importantly, results of thesestudies indicate WQC for individual chemicals established for the protectionofaquatic organisms may not be adequately protectiveofsensitive stages of freshwater mussels. This chapter provides asummary of methods from over 75 laboratory toxicity studies conducted with freshwater mussels and alsoprovides an overview of astandardized method for conducting water-only acute and chronic laboratory toxicity tests with glochidia and juvenile freshwater mussels (ASTM 2006a). Three life stages (glochidia,juveniles, and adults) have been used to conductlaboratory toxicity tests with mussels. Withinthis chapter,toxicity studies are separated according to the medium of exposure (aqueous, sediment, and host fish). Each section begins with areview of the methodsused to conduct toxicity tests (e.g., obtaining organisms, duration of exposure, exposure chambers,and toxicity endpoints). Each section also discusses issues that have been identified regarding the routine application of the methods (e.g., to generate data for the derivationofWQC)and discussesresearchneeds.The final sectionofthischapter reviews the use of the Asian clam ( Corbicula fluminea)asasurrogate for assessing effects on native unionids. Finally, asummary of future research needs for improving methods used to conductacute and chronic toxicity tests with freshwater mussels is provided. AQUATIC TOXICITY TESTING WITH GLOCHIDIA, JUVENILE, AND ADULT LIFE STAGES OF FRESHWATER MUSSELS M ETHODS FOR C ONDUCTING A CUTE W ATER-ONLY T OXICITY T ESTS WITH G LOCHIDIA OF F RESHWATER M USSELS ReviewofMethods Conditions that have been used to conductacute toxicity tests with glochidia of freshwater mussels are summarized in Table 5.1including the testconditions recommendedinASTM (2006a). The procedures outlined in Table 5.1 are consistent with acutetoxicity testing methods for fish, macro- invertebrates, and amphibians (ASTM 2006c)and with acute toxicity testing methods for saltwater 4284x—CHAPTER 5—19/10/2006—19:50—CRCPAG—XML MODEL C–pp. 95–134 95 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) TABLE 5.1 SummaryofTest ConditionsUsed to Conduct Toxicity Tests with Glochidia of Freshwater Mussels Conditions Johnson et al. (1990, 1993) Lasee (1991) Huebner and Pynnonen (1992) a Goudreau, Neves, and Sheehan(1993) Jacobson et al. (1997) Keller and Ruessler (1997) McCann (1993) Klaine, Warren, and Summers (1997) USGS (Unpub- lished Data) Recommended Test Con- ditions in ASTM (2006a) 1Species tested Utterbackia imbecillis b Lampsilis cardium c Anodonta cygnea, Anodonta anatina Villosairis Multiple species d Multiple species e V. iris U. imbecillis Multiple species f NA g 2Test type Static Static Static Renewal Static Static Static Static Static, renewal, flow-through Static, renewal, or flow-through (depending on chemical tested) 3Test duration (hours) 24 48 24, 48, 72, 144 24 24, 48 4, 24, 48 24 24, 48 6, 24, 48 6, 24 (up to 48 depending on viability of glochidia) 4Temperature ( 8 C) 20 21 13 22 10–25 25 20 25 20 20 5Light quality Ambient lab light NR h NR NR NR NR NR Ambient lab light Ambient lab light Ambient lab light 6Light intensity NR NR NR NR NR NR NR NR 200 lux 100–1000 lux 7Photoperiod 16L:8D 24D Natural regime16L:8D 16L:8D 12L:12D NR 16L:8D 16L:8D 16L:8D 8Test chamber 100-mL beaker 250-mL or 300- ml beaker 400-mL beakerBasket of mesh netting in 4-L chamber 12-well plate 6-well plate 12-well plate 12-well plate 200-mL dish or 300-mL beaker 100-mL glass chamber (minimum) 9Test solution volume (mL) 50 200 200 NR 3.5 NR 53.5 100 75 (minimum) 10 Glochidia collection Shake piece of cut gill in water Flush gills with syringe Cut gills and press out glochidia using forceps Flush gills with syringe Cut gills and separate glochidia from marsupia NR Flush gills with syringe Flush gills with syringe Flush gills with syringe Flush gills with syringe 11 Age of test organisms (hours) NR NR 3–24 NR NR NR ! 2NR ! 2to ! 24 ! 24 12 Number of organismsper test chamber 10 10 1000–3000 Several hundreds 50–75 50–100 40 50–100 About 1000 About 500 (1000 for repeated sampling during atoxicity test) 13 Number of replicate chambers per treatment 232, Counting 3 samples with about 100 glochidia 2, Counting 3 samples with about 100 glochidia 33or 43 33,Counting a subsample with about 100 glochidia from each replicate 3, Counting asubsample with about 100 glochidia from each replicate 14 Feeding None None None None None None None None None None 15 Aeration None None Yes None None NR NR NR None None, if dissolved oxygen is maintained above acceptable concentration Freshwater BivalveEcotoxicology96 4284x—CHAPTER 5—19/10/2006—19:51—CRCPAG—XML MODEL C–pp. 95–134 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) 16 Dilution water Reconstituted water, hardness 40–50 mg/L as CaCO 3 Hardness 150 mg/L as CaCO 3 Tap water Dechlorinated effluent water Dechlorinated tap water or Clinch River water, VA Reconstituted water, hardness 47– 76 mg/L as CaCO 3 Sinking Creek water, VA Hardness 99–107 mg/L as CaCO 3 Reconstituted water, hardness 170 mg/L as CaCO 3 Depends on experimental design 17 Water quality DO, pH, hardness, alkalinity, conductivity DO, pH, hardness, alkalinity, conductivity pH, Ca, Cu,ZnDO, pH, hardness, alkalinity, conductivity DO, pH, hardness, alkalinity, conductivity DO, pH, hardness, alkalinity, conductivity DO, pH, hardness, alkalinity, conductivity DO, pH, hardness, alkalinity, conductivity DO, pH, ammonia, hardness, alkalinity, conductivity DO, pH, ammonia, hardness, alkalinity, conductivity 18 Endpoint Survival (valve closure with culture medium) Survival (valve closure with NaCl) Survival (valve closurewith KCl) Survival (valve closure with NaCl) Survival (valve closurewith NaCl) Survival (valve closure with NaCl) Survival (valve closure with salt solution) Survival (valve closurewith saline solution) Survival (valve closure with NaCl) Survival (valve closure with NaCl) 19 Control survival (%) O 95 O 90 O 80 80 O 90 O 80 O 80 80 O 90 O 90 (must) The Last Column Provides aSummary of Recommended Conditions That Can be Used to Conduct Toxicity Tests with Glochidia Based on ASTM (2006a) a See also Pynnonen (1995); Hansten et al. (1996). b Formerly Anodonta imbecillis.See also Weinstein (2001). c Formerly Lampsilis ventricosa. d V. iris, A. pectorosa, Pyganodon grandis, L. fasciola, Medionidus conradius.See also Jacobson (1990); Cherry et al. (2002). e Villosaosa lienosa, Villosa villosa, U. imbecillis, Megalonaias nervosa, Lampsilis teres, L. siliquoidea.See also Jacobson (1990), McCann (1993), V. iris, A. pectorosa, M. conradius. f Actinonaias ligamentina, Alasmidonta heterodon, Epioblasma capsaefotmis, Lampsilis siliquoidea, Lampsilis fasciola, Lampsilis abrupta, L. rafinesqueana, Potamilus ohiensis, Pleurobema plenum, Quadrula quadrula, Quadrula pustulosa, Leptodea fragilis, Leptodea leptodon, Venustaconcha ellipsiformis, V. iris. g NA, not applicable; NR, not reported. h USGS unpublished data, Columbia, MO. Laboratory Toxicity Testing with Freshwater Mussels 97 4284x—CHAPTER 5—19/10/2006—19:51—CRCPAG—XML MODEL C–pp. 95–134 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) bivalve mollusks (ASTM 2006c). Gravidfemale mussels are usually collected from the field and held in the laboratory before isolatingglochidia to start atoxicity test (ASTM 2006a,Chapter4). Alternatively, Zimmerman andNeves (2002) suggested glochidiaofsomespecies (including Villosairis and Actinonaias pectorosa )couldbeextracted in the field from afemale and transported back to the laboratory in cool water where glochidia can remainviable for several days without a reduction in ability to successfully attachtoahost fish. This procedure may be particularly useful whenglochidia of endangered species are extracted in the field and the female musselsare then immediately returnedtotheir habitat. Mature glochidiaare typicallyflushed from the marsupium of afemalemussel usingasyringe filled with water.Glochidiahave alsobeen isolated by cutting a sectionofgill from the femalemussel and then teasingout the glochidia in water. (Thistechnique is destructivetothe adultfemaleand maynot be appropriatefor useinisolating glochidiafor conducting toxicity tests.)Nostudies were identified where glochidia were isolated for toxicity testing from conglutinates released intothe water by femalemussels. Before starting an exposure, the viability of glochidia is typicallyevaluated by aresponse to the addition of aconcentratedsolution of NaCl or KCl.Mature and healthy glochidia will snap shut in response to the addition of asaline solution. Immature glochidia isolated from the marsupium of afemale will often still be enclosed in the egg membrane and will be fragile andtend to fracture(ChrisBarnhart,Missouri StateUniversity,Springfield, MO,personal communication). Tests are usually started if greater than 80 to greater than 90% viability of the glochidia is observed (Huebner and Pynnonen 1992; Jacobson et al. 1997; Klaine, Warren, and Summers 1997; ASTM 2006a). If immature glochidia are isolated from afemale mussel, these glochidia should not be used for testing. Exposures are usually started the same daythat glochidia are isolated from afemalebypooling glochidia from at least three females without an extended acclimation period in the exposure water beforethe start of atoxicity test (ASTM 2006a). Theviability of glochidia isolated from each female should be evaluated before they are pooled together. Toxicity tests can be conducted with glochidia obtained from one female (e.g., when alimited number of endangered species are available for testing); however, the results of tests conducted with alimited number of mussels should be interpreted with caution. Additional research is needed to determinethe minimum number of females that should be sampledtoobtain glochidia to start atoxicity test. This research might include an evaluation of the variability in sensitivity of glochidia obtainedfrom individual females usingavariety of toxicants. ASTM (2006a)provides alist of recommended test conditions for conducting toxicity tests with glochidia isolated from femalemussels. The list of recommended test conditions is based on the various methods outlined in Table 5.1 and on the conditions used to conduct an inter-laboratory toxicitytest with glochidia(ASTM2006a). ASTM (2006a)recommends that toxicity tests with glochidiashouldbeconductedat20 8 Cwith a16L:8D photoperiod at an illuminance of about 100–1000 lux (Table 5.1). The endpoint measured in toxicity tests with glochidia is survival (viability)asdeterminedbythe response of organismstothe addition of asolutionofNaCl. Glochidia that close their valves with the addition of asalt solution are classified as alive (viable) in atoxicity test. For mostspecies, the duration of atoxicity test conducted with glochidia shouldbe up to 24 hours with survival measured at 6and 24 hours.Control survival is typically greater than 90% at the end of 24-hourtoxicity tests conducted with glochidia. Longer duration toxicity tests with glochidia (e.g., 48 hours) can be conducted as long as control survival greater than 90% is achieved. However, toxicity tests conducted for greater than 24 hours with glochidia may not be as ecologi- cally relevantgiven the short period of time betweenrelease of glochidia from afemale mussel until encystment on ahost fish (ASTM 2006a;Chapter 4). Effect concentrations are typicallycalculated based on the percentage of viable glochidia in the control at aparticular sampling time. ASTM (2006a) recommends the use of glass test chambers for conducting toxicity tests with glochidia. Test chambersshouldbeaminimum volume of 100 mL containing aminimum of 75 mL of dilution water.Static, renewal, or flow-throughconditionscan be used depending on the Freshwater BivalveEcotoxicology98 4284x—CHAPTER 5—19/10/2006—19:51—CRCPAG—XML MODEL C–pp. 95–134 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) chemical beingtested.Glochidia are not fed during the toxicity test, and aeration of dilution water is typicallynot necessary. Dilution water shouldbeasourceofwater that has been demonstrated to supportsurvival of glochidia for the duration of the toxicity test. For site-specific evaluations,the characteristics of the dilution water should be as similar as possibletothe site of interest.The number of replicates and concentrations tested dependsinpart on the significance levelselected and the type of statisticalanalysis. ASTM (2006a)recommendsaminimum of three replicates should be tested,each replicate containing about at least 500 glochidia (preferably 1,000glochidia/repli- cate if survival is to be evaluated in subsamples of glochidia collected during the toxicity test). Survival can be determined throughout the toxicity testbysubsamplingeach replicate (e.g., by subsampling about 100 glochidia at 6and 24 hours and then placing these organisms into one well of amulti-wellplate to determinesurvival with the addition of asaltsolution). Toxicity tests with glochidia have been conducted for up to 144 hours,but 24 and 48-hour exposuresare most oftenused (Table 5.1). Therelatively short durationoftoxicity tests with glochidiaisbased on the relatively short duration betweenthe releaseofglochidia into the water column and encystment on the host and on the relatively short survival time of glochidia after isolationfrom the female mussel (Table 5.2). If the life history of the glochidia for aparticular species is not known(e.g., the host required for encystment or how long glochidia released from a female mussel can remain in the water column before encysting on ahost), it might be appropriate to conducttoxicity tests with glochidia for longer than 24 hours as long as 90% control survival can be achieved at the end of the test (ASTM 2006a). IssuesRegarding the Use of Methods Glochidia and juvenile musselsofseveral genera have been found to be highly sensitive to some metals and to ammoniainwater exposures compared to many of the most sensitive genera of other invertebrates,fish,oramphibiansthatare commonlytested (Chapter 7, Cherry et al.2002; Augspurger et al. 2003; USGS 2006a, 2006b). However, concerns have been expressedregarding the useoftoxicity data generated with glochidiainthe derivationofWQC (CharlesStephan, USEPA,Duluth, MN;personal communication). These concerns mainly include:(1) the duration of the toxicity tests, (2) the quality of the glochidia at the start of atest, and (3) the test acceptability criteria. The following section provides information that attempts to address these concerns. Areas of ongoing researchorneed for future researchare alsoidentified. Duration of the Toxicity Test 1. How long shouldacute tests with glochidia be conducted (i.e., based on the life historyof the species)? There are nearly 300 species of freshwater musselsinNorth America, and the length of time that glochidia remain viable after release from the marsupium of afemale into the environment dependsonthe life history of the species and the temperature of the water (Chapter 3). Longevity of glochidia after release and before attachment to ahost may exceed one weekand may be dependent on temperature (Zimmerman and Neves 2002); however, some reports are anecdotal (Murphy 1942;Matterson1948; Tedlaand Fernando 1969). Glochidia of some species released in conglutinates remainviable for days or weeks after releaseinto the environment (Chris Barnhart, personal communi- cation). Glochidiaofseveral species,including Anodonta spp., remainviable while free in the environment for 7–14 days (Howard and Anson 1922; Mackie 1984; Huebner and Pynnonen 1992; Pynnonen 1995). Table 5.2 provides asummary of laboratory studies that have evaluated survival times of glochidia after removal from the marsupium of the female or survival time based on results reported in toxicity tests conducted with glochidia. For example, Zimmerman and Laboratory Toxicity Testing with Freshwater Mussels 99 4284x—CHAPTER 5—19/10/2006—19:51—CRCPAG—XML MODEL C–pp. 95–134 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) TABLE 5.2 SurvivalTime of Glochidia after Removal from Female Unionid Mussels Duration of Viability Species Temperature ( 8 C) Day(%Survival) Reference Actinonaias ligamentina 20 7(O 90); 8(O 75); 9(O 50) USGS (2004) Actinonaias pectorosa 10 13 ( O 75) Zimmermanand Neves (2002) 25 5(O 75) Zimmermanand Neves (2002) 20 O 2(O 90) a Jacobson et al. (1997) Alasmidonta heterodon 20 2(O 90); 2(O 75); 2(O 50) USGS (2004) Anodonta anatine 13 O 3(O 90) Huebner and Pynnonen(1992) Anodonta cataracta 10 O 14 ( O 90) Jacobson (1990) Anodonta cygnea 13 O 3(O 90) Huebner and Pynnonen(1992) Anodonta grandis 10 O 14 ( O 90) Jacobson (1990) Elliptio complanata 57NR b Matterson (1948) 20 ! 1(O 90); 3(O 75) Bringolf et al. (2005) Elliptio dilatata 20 ! 1(O 90); 1(O 75); ! 2(O 50) Bringolf et al. (2005) Epioblasma capsaeformis 20 0.3 ( O 90) Wang et al. (2003) Lampsilis abrupta 20 2(O 90); 5(O 75); 7(O 50) USGS (2004) Lampsilis cardium 21 O 2(O 90) a Lasee (1991) Lampsilis fasciola 20 6(O 90); 7(O 75); 8(O 50) Wang et al. (2003) 20 O 2(O 90) a Jacobson et al. (1997) 20 1(O 90); 2(O 75); 3(O 50) Bringolf et al. (2005) 20 2(O 90; 4(O 75); 5(O 50) Bringolf et al. (2005) Lampsilis rafinesqueana 20 6(O 90); 6(O 75); 6(O 50) USGS (2004) Lampsilis siliquoidea 10 9NRTedla and Fernado(1969) 20 8(O 90); 9(O 75); 10 ( O 50) Wang et al. (2003) 25 O 2(O 80) a Keller and Ruessler (1997) 20 1(O 90); 3(O 75); 4(O 50) Bringolf et al. (2005) Limpsilis teres 25 0.2 ( O 80) Keller and Ruessler (1997) Leptodea fragilis 20 1(O 90); 3(O 75); 4(O 50) Wang et al. (2003) Leptodea leptodon 20 1(O 90); 2(O 75) Bringolf et al. (2005) Leptodea leptodon 20 0.25 ( O 90); 1(O 75); 2(O 50) USGS (2004) Margaritifera falcate 11 11 NR Murphy (1942) Medionidus conradius 20 O 2(O 90) a Jacobson et al. (1997) Megelonaias nervosa 25 1(O 80) a Keller and Ruessler (1997) Potamilus alatus 20 6(O 90) 6(O 75); 6(O 50) Wang et al. (2003) Potamilus ohiensis 20 5(O 90); 6(O 75); 7(O 50) Wang et al. (2003) Pyganodon grandis 20 O 1(O 90) 0 Jacobson et al. (1997) Quadrula quadrula 20 1(O 90); 1(O 75); 2(O 50) Wang et al. (2003) Quadrula pustulosa 20 ! 1(O 90); 1(O 75); 1(O 50) Wang et al. (2003) Utterbackia imbecillis 21 10 ( O 80); 14 ( O 50) Fisher and Dimock (2000) 25 O 2(O 80) 0 Keller and Ruessler (1997) 25 O 2(O 80) a Klaine, Warren, and Summers (1997) 20 O 1(O 90) a Johnson, Zam, and Keller (1990, 1993) Venustaconcha ellipsiformis 20 2(O 90); 3(O 75); 3(O 50) Wang et al. (2003) Villosa iris 10 8(O 75) Zimmermanand Neves (2002) 20 5(O 90); 5(O 75); 6(O 50) Wang et al. (2003) 25 2(O 75) Zimmermanand Neves (2002) (continued) Freshwater BivalveEcotoxicology100 4284x—CHAPTER 5—19/10/2006—19:51—CRCPAG—XML MODEL C–pp. 95–134 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) Neves (2002) report that the viabilityofglochidia of V. iris was greater than 75% for 8days at 108 Cand 2days at 258 C, and viability of glochidia of A. pectorosa was greater than 75% for 13 days at 108 Cand 5days at 258 C(Table 5.2). Similarly, glochidia of Utterbackiaimbecillis maysurviveupto19daysbut exhibit50% mortalitywithin 13.5 days (Fisherand Dimock 2000).Survivalofisolated glochidiafrommany species listedinTable 5.2istypically greater than 90%after twotothree days; however, theviability of glochidiafor aparticular speciesshouldbedetermined before the start of an exposure. For example, glochidia of L. teres and E. capsaeformis were viable for only four to six hours and glochidiaof M. nervosa and Q. quadrula were viable for one day after removal from the marsupium of the female (Table 5.2). There- fore, 24 hours is areasonable time period to conducttoxicity tests with glochidia of many species at 208 C, although shorter or longertests might be neededfor aparticular species depending on glochidia survival time and the life history characteristics of the species (i.e., survival of glochidia in the control mustbegreater than 90% at the toxicity test) (ASTM 2006a). Thetime between the release of glochidia from the marsupium of the female mussel to attachment of these glochidia on ahost may only take afew seconds for some species, but hours are requiredfor the gill tissue of afish to migrate to form acyst aroundthe glochidia. Duringthat time,the glochidiamay be exposedtowater-borne toxicants. Anodontinae speciesreleasesglochidia directly into water, whichremain viable for days in order to effectively infest their host fish. Therefore, aprolonged glochidial test would have ecological relevance for these species.Other species releaseglochidia in mucus strands that coat the bottom or remain suspended on vegetation,waiting for their hosts to swim by, and still otherspecies package glochidia in conglutinates that serve as a lure to host fish.Hence, glochidia of these species may also be in water for extended periods of time;however,itisnot known how exposure to water-borne contaminants would be influenced by the mucus or conglutinatesurrounding the glochidia. Toxicity testsconducted for24hourswithglochidia maynot be as ecologicallyrelevantas toxicity tests conducted with juvenile mussels, but they may be useful for somepurposes such as deriving concentrations of achemical that may be protectiveofthe species. Use of glochidia to evaluatethe relative sensitivity of aparticular mussel species to chemicals would be particularly useful when evaluating species where only alimited number of adult mussels are available for methodsdevelopment or for producing juvenile mussels for toxicity testing. Moreover,the host fish for somespecies of mussels or techniques for transforming juvenile musselsinthe laboratory may be unknown (Chapter 4). TABLE 5.2 (Continued) Duration of Viability Species Temperature ( 8 C) Day(%Survival) Reference 22 O 1(O 80) a Goudreau,Neves, and Sheehan (1993) 20 O 1(O 80) a Scheller (1997) 20 O 2(O 90) a Jacobson et al. (1997) Villosaosa lienosa 25 O 2(O 80) a Keller and Ruessler (1997) Villosa nebulosa 20 O 2(O 90) a Jacobson (1990) Villosa villosa 25 O 2(O 80) a Keller and Ruessler (1997) a The value based on control survival in 24- or 48-hour toxicity tests. b NR, not reported. Laboratory Toxicity Testing with Freshwater Mussels 101 4284x—CHAPTER 5—19/10/2006—19:51—CRCPAG—XML MODEL C–pp. 95–134 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) Therelatively short duration of toxicity tests with glochidia is based on the relatively short duration betweenreleaseofglochidia into the water column and encystment on the host and on the relatively short survival time of glochidia after isolation from the female mussel. If the life historyofaparticularspecies is not known (e.g., the host requiredfor encystment or how long glochidia released from afemale mussel can remaininthe water column beforeencysting on ahost), it might be appropriatetoconducttoxicity tests with glochidia for longerthan 24 hours as long as 90% control survival can be achieved at the end of the test. 2. How long can glochidia survive and still be able to attach to ahost? Glochidia of somespecies can still attachtoahost for several days after release from a female depending on temperature (Chris Barnhart,personalcommunication).The maximum time at which greater than 50% of U. imbecillis metamorphosed in atissue culture medium was nine days after isolation from afemale(Fisher and Dimock 2000). Zimmerman and Neves (2002) reported that glochidia can successfully attachtoahost one to two weeks after isolation from afemale. Afuture research project could be to conducta series of toxicity teststodetermine if thereisachangeinsensitivityovertimeafter glochidia have been released into the environment.Sensitivity of L. siliquoidea glochidia held for 24 hours after isolationfrom afemale was similar to newly-released glochidia in exposures to copper (Wang et al. 2003). The sensitivity of glochidia held in an extra piece of the marsupium in arefrigerator overnight was similar to the sensitivity of glochidia tested immediately after isolation from afemale in toxicity tests conducted with zinc or copper (Jerry Farris, Arkansas State University, State University, AK; personal communi- cation). Ultimately, it is more practical to base duration of exposure on survival of control organisms in the laboratory rather than on an estimate of the length of time glochidia can survive and still attachtoahost (e.g., Table 5.2). 3. Are there data that indicate that effect concentrations do not change very muchduring the last half of atoxicity test (i.e., does the EC50 at 6, 24, 48, or 96 hours differ)? There are limited studies with glochidia that have comparedchangesintoxicity over this timeframe.The toxicity of copper (Jacobson et al. 1997; Wang et al. 2003), ammonia (Wang et al. 2003), and chlorine (Wang et al. 2003)decreasedover 48–96-hour exposures. In contrast, no change in the toxicity of several pesticides was observed in 24–48-hour exposures (Keller and Ruessler 1997; Bringolf et al. 2005). If glochidia for aparticular species are able to survive for more than 24 hours, then a24-hourtoxicity test shouldbe considered.Importantly,researchers areencouraged to design studies that generate toxicity data throughout the exposure period (e.g., reporting 6, 24, and 48-hourresponses) (ASTM 2006b). However, generating data for asix-hour exposure period is logistically difficult in an eight-hour day. Quality of Glochidia at the Star tofaToxicity Test 1. How shouldthe quality of glochidia be determined at the start of atoxicity test? Is the use of asolution of NaCl (or KCl)todeterminethe percentage of glochidia exhibiting valve closure an appropriate method to judge the acceptability of glochidia used to start atoxicity test? Does the response of glochidia to asolution of NaCl (or KCl)relate to the ability of glochidia to attachtoahost?Isthere an independent way of determining if glochidia are alive or healthy at the start (or end) of atoxicity test? Valve closure is an ecologically relevantendpoint that is criticalfor glochidia to successfully transform on the host.Ifglochidia do not snap shut, the glochidia should be considered ecologically dead (Huebner and Pynnonen 1992; Goudreau, Neves, and Sheehan 1993;McCann 1993;Jacobsonetal. 1997). Theresponse of glochidiain Freshwater BivalveEcotoxicology102 4284x—CHAPTER 5—19/10/2006—19:51—CRCPAG—XML MODEL C–pp. 95–134 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) toxicity tests was similar wheneither KCl or fish plasma was used to makeglochidia close at the end of an exposure (Huebner and Pynnonen 1992). Decreased response to KClwas considered an indication of reduced glochidiaviabilityand thus reduced capabilitytoattachtothe fishhost(Pynnonen 1995). Asignificantcorrelation was observed betweenthe response of glochidia to KCl and the ability of glochidia of U. imbecillis to metamorphose to the juvenile life stage (Fisher and Dimock 2000). Zimmermanand Neves(2002) reported acorrespondencebetween theresponseof glochidiaof V. iris and A. pectorosa to NaCl and theabilitytoinfest ahostfish. Jacobson et al. (1997) reportedglochidiaof V. iris that responded to the addition of NaCl followinganexposuretocopperwere able to attach to ahostfish with no impairmentofsubsequent metamorphosistojuvenile mussels. Results of these studies indicatethat addition of asolution of NaCl or KCl can be used to estimate the condition of glochidia. While either asolution of salt or fish plasma could be used to determine thepercentageoforganisms closing,itiseasiertoworkwith NaCl comparedtoKCl or fish plasma. 2. Should therebeaholding time for glochidia after harvesting but beforeapplication of a saline solution to determineifglochidia that are initially closed might open? Mature glochidia are not typically closed after being isolated from afemale mussel. Glochidiathat are closed after isolationfrom afemale may reopenafter being held in clean water afew hours (Goudreau, Neves, and Sheehan 1993;Dick Neves, Teresa Newton, USGS, LaCrosse, WI; personal communications). 3. Will immature,stressed, or unhealthy glochidiaclose when exposedtoasaline solution?Couldglochidiabealive andsuccessfully attach to ahostbut not close when exposed to asaline solution? Are brokenglochidia frequently observed at the start of atest? Wouldthe presence of brokenglochidiabeindicative of stress during harvesting? Immature glochidiathatare free of theegg membrane or mature andhealthy glochidiawill closewhenexposed to asalinity challenge. However, immature glochidiaare generally enclosed in an eggmembraneand arefragile andtendto fracture, thus should not be used for toxicity testing. The best approach for avoiding the use of immatureglochidia in toxicity testing is to sample female mussels at atime of the year whenthe organisms would be expected to be releasing matureglochidia (JessJones, US GeologicalSurvey, Blacksburg,VA; personalcommunication). Stressed or unhealthy glochidiacouldeither be opened or closed before the start of a test. If stressed or unhealthy glochidia weretoclosewhenexposedtoasalinity chal- lenge, then theseindividuals would be used in atoxicity test. Measurementofthe viability of glochidia in the control at the end of atoxicity testwould help to identify stressed or unhealthy glochidia. Results of reference-toxicant tests shouldalso be used to evaluate the health of the glochidia used to conductthe test(ASTM 2006a). Broken glochidia have not been observed at the start of atest (Chris Barnhart, Jerry Farris, Dick Neves, TeresaNewton, Ning Wang, USGS, Columbia, MO; personal communications). The presence of broken glochidia may indicate that the glochidia are immature and should not be used for testing. Test Acceptability Criteria for Toxicity Tests with Glochidia 1. What criteria should be used to judge acceptability of atoxicity test conducted with glochidia? ASTM (2006a)recommends that the age of glochidia should be less than 24 hours old at the start of the toxicity test. Viability of glochidia isolated at the beginning of a Laboratory Toxicity Testing with Freshwater Mussels 103 4284x—CHAPTER 5—19/10/2006—19:51—CRCPAG—XML MODEL C–pp. 95–134 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) toxicity testmust be greater than or equal to 80% (preferablygreater than or equal to 90%). Average survival of glochidia in the control at the end of atest mustbegreater than or equal to 90%. ASTM (2006a)also recommends that subsamples of each batch of test organisms used in toxicity tests shouldbeevaluated usingareference toxicant (e.g., NaCl or CuSO 4 ). Data from thesereference-toxicant tests can be used to assess genetic strain or life-stage sensitivity of test organisms to select chemicals. 2. Should glochidia be rinsed before use in atoxicity test? Would rinsing glochidia before the start of atest be stressful to the organisms? Glochidiashould be rinsedwithculture or dilution waterafter removalfrom marsupia to (1)eliminate tissuesorexcessmucus from theexcised glochidia that haveahigh potential forfungal growth and subsequently could affect thesurvival (toxicity tests) or transformation of glochidia(propagation) and(2) reduce the number of protozoans that may be present in the excised gill that could also affect glochidiasurvivalortransformation(ASTM2006a). Rinsed glochidiahavebeen observed to successfully transformonfish or in artificial mediaand high control survivalintoxicity testshas been reportedusing glochidiathathavebeen rinsed (Huebner and Pynnonen 1992; Johnson, Keller, and Zam 1993; Myers-Kinzie 1998; Milam et al. 2005). 3. Should glochidia be acclimated to test conditions before the start of atoxicity test? Glochidia are not typically acclimated to the water-quality characteristics of the dilution water beforethe start of atoxicity test(Table 5.1). Most of these exposures are started the sameday that glochidia are isolated from marsupia of the females. Therefore, minimaltimeisavailable to acclimateglochidia to thedilution water before thestartofatest.Inorder to maintain organismsingood condition and avoid unnecessary stress,ASTM (2006a)recommends that organisms shouldnot be subjected to rapid changesintemperature or water quality beforethe start of atest. Glochidiacan be acclimated in amixture of 50% culturewater and 50% test water and gradually adjustedtothe test temperature within about two hours beforethe start of an exposure(ASTM2006a). Investigators have held adultmussels undertest conditions before isolationofglochidia (e.g., Huebner and Pynnonen 1992), which would result in acclimating glochidia to the selected exposure temperatureinthe toxicity test. However, brooding glochidia in the marsupium are in contact with the hemolymph of the femalethat is physically isolated from direct contact with water (Silverman, McNeil, and Dietz1987). In addition, glochidia are typically released instantaneously into the surrounding water from the marsupium of the femalemussel. Therefore, holding the female mussels in the dilution water beforeisolatingglochidia fortoxicity testing wouldprobablyhaveaminimal influence on theability of glochidia to acclimate to the conditions of the dilution water. M ETHODS FOR C ONDUCTING W ATER-ONLY T OXICITY T ESTS WITH J UVENILE F RESHWATER M USSELS ReviewofMethods ASTM (2006a)provides alist of recommendedtest conditions for conducting toxicity tests with juvenile mussels. The list of recommendedtest conditions is based on the various methods outlined in Table 5.3 and on the conditions used to conductaninter-laboratory toxicity test with juvenile mussels (ASTM 2006a). ASTM (2006a)recommends that toxicity tests with juvenile mussels beconducted at 208 Cwith a16L:8D photoperiod at an illuminance of about 100–1,000lux (Table 5.3). Toxicity tests are typicallystarted with newly-transformed juvenile mussels less than five days after the release Freshwater BivalveEcotoxicology104 4284x—CHAPTER 5—19/10/2006—19:51—CRCPAG—XML MODEL C–pp. 95–134 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) [...]... 16L:8D 16L:8D Petri dish 12-well plate Petri dish 120-mm diam tub with mesh bottom in 4-L chamber 132 by 90 by 130 mm chamber Covered 250 -mL crystallizing dish 50 -mm diam glass tub with mesh bottom in 250 -mL Chamber Dish covered with mesh 30-mL beakers submerged in a 1-L glass beaker 5 0- or 300-mL beaker 300-mL beaker Static or renewal: 50 -mL beakers (minimum); flow-through: 300-mL beakers (minimum) Static... !3, 5, 9 !10 7–10 3 5 0 6–10 1–3 60 3 5, 60 60 10 10 10 10–20 1 5 NR 10 20 50 15 15 5 5 10 12 h Villosa nebulosa, Villosa iris, Anodonta grandisc Static 8 11 Recommended Test Conditions ASTM (2006a) Laboratory Toxicity Testing with Freshwater Mussels 4284x CHAPTER 5 19/10/2006—19 :51 —CRCPAG—XML MODEL C – pp 95 134 TABLE 5. 3 Summary of Test Conditions Used to Conduct Toxicity Tests with Juvenile Freshwater. .. 4284x CHAPTER 5 19/10/2006—19 :51 —CRCPAG—XML MODEL C – pp 95 134 1 2 3 4 5 6 7 8 9 10 11 122 4284x CHAPTER 5 19/10/2006—19 :51 —CRCPAG—XML MODEL C – pp 95 134 TABLE 5. 6 (Continued) Farris et al (1994) Belanger, Meiers, and Bausch (19 95) Laboratory 15 and 25 Ambient 9 NRf 9-L glass tanks Laboratory 15 NRf 460 167 L/min Artificial streams in situ 18–27 Ambient NAf NAf NAf New River, VA Laboratory 25 NRf... hours, 10, 20, 30 days 29 NR 7 months 30 days 56 days 4, 56 days NR NR 16–20 NR 17 NR 7, 14, 21, 30, 60 days 20 Fluorescent NR NR Plastic containers NR NR NR 250 lux 16L:8D 40-L glass aquaria 40 NR Natural conditions 150 0-L plastic containers 150 0 NR 16L:8D 46-L glass aquaria 10 NR 10L:14D 7 5- L fiberglass oval stream 60 30 NR 10 50 6 1 25 30 25 L with a 5- cm layer of gravel 9–10 2 1–6 1 1 1 1 2 4 None... toxicity tests for up to 28 days starting with two- to four-month-old juvenile mussels Valenti et al (20 05) conducted 21-day exposures to mercury starting with two-month old juvenile V iris held in a small amount of sediment and fed algae (Neochloris) USGS (2005a, 2005b, 2005c); Bringolf et al (20 05) conducted toxicity tests starting with two- to four-month-old juvenile A ligamentina, L siliquoidea, or... or renewal: 50 -mL beakers (minimum); flow-through: 300-mL beakers (minimum) Static or renewal: 30 (minimum); flow-through: 200 (minimum) Fish host Test chamber 1 2 5- mL beaker 12-well plate Covered 250 -mL crystallizing dish 9 Test solution volume (mL) 100 3 .5 NR 15 15 5 10 NR 1200 NR 200 150 950 30 or 200 200 10 Procedure for obtaining juveniles Age of test organisms (days) Number of organisms per test... been conducted, and © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) 4284x CHAPTER 5 19/10/2006—19 :51 —CRCPAG—XML MODEL C – pp 95 134 114 4284x CHAPTER 5 19/10/2006—19 :51 —CRCPAG—XML MODEL C – pp 95 134 TABLE 5. 5 Summary of Test Conditions for Conducting Sediment Toxicity Tests with Freshwater Mussels Conditions Keller, Ruessler, and Chaffee (1998) Wade (1992) Newton et al (2003)... Feeding Aeration Dilution water 11 12 13 14 15 Glochidia and newly-transformed Newly-transformed juveniles juveniles 10, 50 –100 15 Newly-transformed juveniles 20 260 mL of overlying water and 15 mL of sieved sediment, two volume additions/day of overlying water Two- to four-month-old juvenile mussels 10 3 3 3–6 4 None Yes Well water, with a hardness of 250 mg/L as CaCO3 Yes None Sediment pore water... movement), growth (shell length) 90– 95 Laboratory Toxicity Testing with Freshwater Mussels 4284x CHAPTER 5 19/10/2006—19 :51 —CRCPAG—XML MODEL C – pp 95 134 16 1 15 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) 116 Freshwater Bivalve Ecotoxicology specific standardized methods have not been developed However, the procedures outlined in Table 5. 5 are generally consistent with the... Fusconaia flava, Amblema plicata 2 Test type Static Static-renewal Flow-through 3 Test duration 72–96 hours 48 hours Flow-through diluter 14, 28 days 4 5 Temperature (8C) Light quality 25, 32 NRb 30–32 NR 21 NR 6 7 8 Light intensity Photoperiod Test chamber NR 12L:12D 23-L aquaria NR NR Plexiglass aquaria Subdued NR 57 -L glass aquaria 9 23 NR 57 5 10 50 10 2–4 1 12 Test solution volume (L) Number of organisms . mm chamber Covered 250 -mL crystall- izing dish 50 -mm diam. glass tub with mesh bottom in 250 -mL Chamber Dish covered with mesh 30-mL beakers submer- ged in a 1-L glass beaker 5 0- or 300-mL beaker 300-mL beaker Static. chamber 100-mL beaker 250 -mL or 30 0- ml beaker 400-mL beakerBasket of mesh netting in 4-L chamber 12-well plate 6-well plate 12-well plate 12-well plate 200-mL dish or 300-mL beaker 100-mL glass. 10 8(O 75) Zimmermanand Neves (2002) 20 5( O 90); 5( O 75) ; 6(O 50 ) Wang et al. (2003) 25 2(O 75) Zimmermanand Neves (2002) (continued) Freshwater BivalveEcotoxicology100 4284x CHAPTER 5 19/10/2006—19 :51 —CRCPAG—XML