10 Biomarker Responses of Unionid Mussels to Environmental Contaminants Teresa J. Newton and W. Gregory Cope INTRODUCTION Unionidmussels areecologicallyand economically importantinaquatic ecosystems.The biomassof unionids canexceedthe biomassofall otherbenthic organismsbyanorder of magnitude(Negus1966; Layzer,Gordon, andAnderson1993),and production (range,1–20gdrymass/m 2 /yr) canequal that by allother macrobenthos in many streams(Strayeretal. 1994). Thus,unionidsmay play importantroles in particle processing,nutrientrelease,and sediment mixing (Vaughnand Hakenkamp2001; Strayer et al.2004).Mussels also serveasfoodfor aquaticmammals,including raccoons,muskrats, andotters (Van derSchalie andVan derSchalie 1950). Historically,unionidsprovidedasupplemental food source to indigenous peoplesprior to European settlement, butlarge-scale commercialinterestin unionidsdid notdevelop until themid-1800s (Claassen 1994).Soon thereafter, unionids were extensivelyharvestedfor theproductionofpearl buttons, andpresently,unionid shells areusedin themultimilliondollarAsian cultured-pearl industry (Anthony andDowning2001). Overharvesting,widespread habitatdestruction, pollution, land-usechange,and invasive speciesintroductionshave caused manyunionid populationstodecline or disappear. In North America, mostspecies are now extinct or imperiled, and unionids are widely recognized as one of themostimperiled plants or animalsonthe continent(Master et al. 2000).Although not sufficiently documented, exposure to toxic contaminants may alsobecontributing to these declines. There are few instances where chemical spillsand other point sources of contaminants have caused localized mortality(Sheehan,Neves,and Kitchel 1989;Fleming, Augspurger,and Alderman 1995); however,widespread decreasesindensity and diversity are more likely to result from the subtle, pervasive effects of chronic, low-levelcontamination (Naimo1995). There is convincing evidence that unionids,and glochidial and juvenile life stages in particular, are sensitive to manycontaminants relative to otheraquatic species (e.g., Newton et al. 2003, Chapter5andChapter 7).However, foragiven chemical, toxicity canvary by an orderof magnitude among life stage and species (Cherry et al. 2002; Augspurger et al. 2003). This is not surprising given the diversity of life history adaptationspresent in this faunalgroup. For example, differencesinlongevity (30–130years), habitat requirements (silt to gravel),feeding strategies (filter-, deposit-, and pedal-feeding), and reproduction (hermaphrodites, dioecious) all contribute to this diversity. Although there are about 1,000 species of unionids worldwide, and about 300 species in North America, only about 11 species have been reported in the peer-reviewed literature to assess theeffects of contaminants on biomarkerresponses in this imperiledfaunalgroup.Clearly, 4284X—CHAPTER 10—19/10/2006—15:11—KARTHIA—XMLMODEL C–pp. 257–284 257 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) ecotoxicological research on unionids needs to expand to encompassthe breadth of life histories found in this group. Thedevelopment of physiological and biochemical tests or “biomarkers” of sublethal exposure are critical in assessing the condition of unionids. Several textbooksand review articles have been writtenonthe use of biomarkers in awide range of aquatic organisms (e.g., McCarthy and Shugart 1990; Huggett et al.1992; Vander Oost,Beyer,and Vermeulen 2003)—many of thesehave application in unionids. Freshwater bivalve ecotoxicology gained momentum in the early 1970s with potassium and copper bioassays with unionids (Imlay 1971). Onceinitial studies revealed thatunionids were indeed sensitive to avariety of contaminants relative to otherinvertebrates and fishes, there has been growing interest in using unionids to evaluate the toxicity of chemicals. Most studies in freshwater bivalve ecotoxicology have either been short-term laboratory tests with single chemicals (Chapter 5and Chapter 7) or monitoringofbiomarker responses in thefield(Chapter 6and Chapter 9).Bothofthese approaches arenecessary to understandbasic biological responses. However, methods had to be, and are continuing to be, developed to culture and maintain adults and juveniles in suitablephysiological condition before and during testing and to evaluatesuitable sublethal response endpoints(ASTM 2006). The field has made substantial progress in many of theseareas in thepast twodecades,but it could furtherbenefitfromlessonslearned in other ecotoxicological areas of study, such as with marine bivalves(ASTM 2002). Although thereare substantial differencesinreproductivestrategiesbetweenunionidsand marinebivalvesthat may confound certain direct comparisons, the field of unionid ecotoxicology could benefitfrom some of the approaches commonlyused in marine studies.For example, a frameworkusedinmarine bivalveecotoxicology recognizedthatthisfieldneededtomove beyond simple acute tests to multidisciplinary studies that gather knowledge at several levels of biological organization,conduct persistent andsystematic field studies,and provideiteration betweenexperiment and field observations (Luoma 1996). Further, this framework stressed that standardized “simple”ecotoxicologicapproaches lack power in explainingimplications of contaminants in complicated circumstances. Additionallessons from marine bivalvesthat should be incorporated into afuture framework for unionid ecotoxicologyinclude the following (from Luoma 1996): 1. Viewing contaminants as just one of the several influential physical,chemical, or bio- logical variables in manyaquatic systems. 2. Recognizingthatcontaminants aredistributed amongsolution, suspendedparticles, sediments,porewaters, and food resources and that each species or life-history stage may “sample” differently from this complex matrix. 3. Toxicity databases derivedfromwater-onlyexposures were developedtosupport regulatory criteria, but theseprobablyunderestimate theexposures of bivalves in many circumstances. 4. Biological responses to contaminants in nature can be muchmore complex than the responses observed in the laboratory. 5. When conducted alone, simplistic toxicity tests, asingle biomarker, whole organism analysis,orstudies that excludevariables otherthancontaminantswillprobablybe insensitivetoall but the mostextremeinfluences of contaminants. Contaminants caninfluence unionids at many differentlevelsofbiologicalorganization (Figure 10.1). Usually, the most robustapproach is the use of anumber of different indices that span thelevelsofbiologicalcomplexity, such as biochemical,cytological, physiological, and autecological. In theory,acontaminantfirstexertsits effectsatthe molecular level,and the changesatthis level lead to changes in organelles and cellular structures, and so forth. However, Freshwater BivalveEcotoxicology258 4284X—CHAPTER 10—19/10/2006—15:11—KARTHIA—XMLMODEL C–pp. 257–284 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) in nature,thingsare rarelythissimplebecause at each levelofbiologicalorganization,there probably existshomeostatic mechanismstocounteract these disruptive effects. Also,because organisms are rarely exposed to asingle contaminantatatime, certain combinations of contami- nants may act in an additive or synergistic manner to amplify the effects of one another. Lastly, this approach implies alinear response to contaminants that may not include adetectablethreshold effect. Certain contaminants, like essential metals, may be requiredfor basic biological functions at low concentrations, whereasaboveathreshold level, they may become toxic. This concept should not be overlookedinstudies of the effects of contaminants on unionids. This review of biomarker responsesinunionidsexposed to environmental contaminants focuses on studies that (1) reportedmeasured contaminant concentrations;(2) had robustexperi- mentaldesigns,including the replication of controland contaminant treatments;and (3)were published in the peer-reviewed literature. These criteria effectively removed about 50% of the paperspublished in this area,primarilybecause many authorsreportedonlynominal concen- trations. However, we believe thesecriteria are criticalfor the objective evaluation of the effects of contaminants on unionids.For example, if only nominal concentrations are reported, the actual concentration of agivencontaminant that is available for uptake is basically unknown. In some studies that only report nominal concentrations, the exposure concentrations are so high that they exceed theknown solubility of thecontaminant. In these instances, theactual amount of the contaminant that is available for uptake may be asmall fraction of the nominal concentration, which couldseriously underestimatetoxicity. Also, manycontaminants may adhere to the walls of exposure chambersorcan be lost to volatilization—both of which may effectivelyreducethe actual exposure concentration. Similarly, treatments need to be replicatedtoget an estimate of the variation associated with the exposure. Future studies should strive to report measured contaminant concentrations(wheneverpossible) andreplicate contaminantand control treatmentstoensure arobust design and analysis. BIOMARKER CONCEPT Abiomarker is achange in abiological response (at the molecular, cellular,biochemical, physio- logical, or behavioral level) that can be related to exposure to, or toxiceffects of, environmental Levelofecological relevance Levelofecological complexity High High Low Low Molecular Population Organelle Cellular Tissue Organism FIGURE 10.1 Ageneralized approach of biological organization that illustrates how agiven contaminant may exert its influence on unionids (Modified from Stebbing A. R. D., The Effects of Stress and Pollution on Marine Animals,Praeger Scientific, New York, 1985.With permission.) Biomarker Responses of Unionid Mussels to Environmental Contaminants 259 4284X—CHAPTER 10—19/10/2006—15:11—KARTHIA—XMLMODEL C–pp. 257–284 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) chemicals (Peakall 1994). As defined, biomarkers can span several levels of biological organization (Figure 10.1), but biomarkers that have been investigated most extensively have been enzymes involved in the detoxification of xenobiotics and their metabolites(biotransformation and antiox- idantenzymes). Biomarkers are generally classified into those that indicate exposure, effects, or susceptibility. Although connections must be established between exposure to contaminants and effectsonbiota, biomarkers show promise as indicators demonstrating that contaminantshave entered organisms, have been distributed among tissues, and are eliciting toxic effects at critical targets(McCarthy and Shugart1990). In arecent review of biomarkers in the fisheries literature, Van der Oost, Beyer, and Vermeulen (2003) proposed six criteriathat should be established for each candidate biomarker: 1. The assay should be reliable,relatively inexpensive, and easy to perform. 2. The response shouldbesensitive to pollutant exposure and/oreffects in order to serve as an early warning parameter. 3. Baseline data of the biomarker should be well defined in order to distinguish between natural variability and contaminant-induced stress. 4. The impacts of the confounding factorsshouldbewell established. 5. The underlying mechanism of the relations between the response and pollutant exposure should be established. 6. The relation betweenthe biomarker response and its long-term impact to the organism should be established. Although there are numerous classification systems for biomarkers, we will follow the one recently used by Van der Oost, Beyer, and Vermeulen (2003) that groups biomarkers into 1of10 categories (Table 10.1). Many of thesebiomarker types have already been applied to unionids, but additional studies with multiplespecies are needed. The available data suggest that this is apromising avenue for future research. BIOTRANSFORMATION ENZYMES To our knowledge, there have only been two studies that have examined the effects of contaminants on enzymes associated with Phase Iofbiotransformation. This first phase of metabolism involves the exposing or adding of reactive functional groups, through oxidation, reduction, or hydrolysis. The activity of 7-ethoxyresorufin O-deethylase (EROD) has been used as abiomarker in fish, and these datasuggest that EROD activitymay notonly indicate chemicalexposure(primarily to organic contaminants such as polycyclic aromatic hydrocarbons[PAHs], polychlorinated biphenyls [PCBs], polychlorinated dibenzo-p -dioxins[PCDDs] and polychlorinateddibenzofurans), but may also precede effects at various levels of biological organization (Whyte et al. 2000; Van der Oost, Beyer, and Vermeulen 2003). In unionids,a1.5-fold increaseinEROD activity was observed in the digestive glandof Elliptiocomplanata deployed for 62 days downstream of amunicipal waste effluent, relative to musselsdeployed upstream(Gagne ´ et al. 2002). These findings are consistent with elevated levels of PAHs—a common constituent in sewage effluent and urban runoff. These initial data suggestthat EROD activitymay be avaluable indicator of exposure to organic contami- nantsinunionids (Table 10.1). More recently, exposure of Unio tumidus to diethylhexylphthalate induced the expression of CYP4 (a cytochrome P450 enzyme),however, no CYP1A sequence was amplified in Aroclor-treated mussels(Chaty, Rodius, and Vasseur 2004). Thebulkofthe publisheddata on biotransformationenzymes in unionids involves those enzymesassociated with thesecondphase of biotransformation. Thisphase involvesthe conjugation(theadditionoflarge andoften polar chemical groups)ofthe xenobiotic parent compound or its metabolitewith an endogenousligand. Theaddition of more polargroups Freshwater BivalveEcotoxicology260 4284X—CHAPTER 10—19/10/2006—15:11—KARTHIA—XMLMODEL C–pp. 257–284 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) TABLE 10.1 Categories of Biomarkers with Known or Potential Application to Unionids CategoryofBiomarker Examples Reference in Unionids Biotransformation enzymes A. Phase ICytochrome P450 Chaty, Rodius, and Vasseur (2004) Ethoxyresorufin O-deethylase (EROD)Gagne ´ et al. (2002) Aryl hydrocarbon hydroxylase (AHH) NA a B. Phase II Reduced (GSH) and oxidized (GSSG) glutathione Cossu et al. (1997, 2000), Doyotte et al. (1997) Glutathione S-transferases (GST) Ma ¨ kela ¨ ,Lindstro ¨ m-Seppa ¨ ,and Oikari (1992) UDP-glucuronyl transferases NA a Oxidative stress Superoxide dismutase (SOD) Cossu et al. (1997), Doyotte et al. (1997) Catalase (CAT) Cossu et al. (1997), Doyotte et al. (1997) Glutathione peroxidase (GPOX) Cossu et al. (1997, 2000), Doyotte et al. (1997) Glutathione reductase (GRED) Cossu et al. (1997, 2000), Doyotte et al. (1997) Lipid peroxidation (LPOX) Cossu et al. (1997, 2000), Doyotte et al. (1997) Biotransformation products Polyaromatic hydrocarbon metabolites in bile NA a Amino acids and proteins Amino acids Gardner, Miller, and Imlay (1981), Day, Metcalfe, and Batchelor (1990) Stress proteins NA a Metallothioneins (MT) Holwerda (1991),Couillard et al. (1993, 1995a, 1995b),Malley et al. (1993), Wang et al. (1999), Gagne ´ et al. (2002), Perceval et al. (2002) Hematological Serum transaminases NA a Alterations in the heme pathway Chamberland et al. (1995) Immunological Cell- and humoral-mediated immunity NA a Phagocytosis Blaise et al. (2002) Lysosomalactivity NA a Reproductive and endocrine Imposex NA a Vitellogenin Gagne ´ et al. (2001a, 2001b, 2001c), Riffeser and Hock (2002), Blaise et al. (2003) Sexual competence NA a Neuromuscular Cholinesterases Doran et al. (2001) Genotoxic DNA damage Black et al. (1996), Gagne ´ et al. (2002), Rodius, Hammer, and Vasseur (2002) Irreversiblegenotoxic events NA a Physiological and morphological Histopathology Lasee (1991) Osmotic and ion regulation Malley, Huebner, and Donkersloot (1988), Hemelraad et al. (1990) Digestive processes Ma ¨ kela ¨ ,Lindstro ¨ m-Seppa ¨ ,and Oikari (1992), Naimo, Atchison,and Holland- Bartels (1992), Milam and Farris (1998) (continued) Biomarker Responses of Unionid Mussels to Environmental Contaminants 261 4284X—CHAPTER 10—19/10/2006—15:11—KARTHIA—XMLMODEL C–pp. 257–284 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) generally facilitates the excretion of these chemicals by biota. Many biotransformation enzymes can be induced or inhibited upon exposure to contaminants. Enzyme induction is an increaseinthe amount or activity of these enzymes (or both), while inhibition refers to the blocking of enzymatic activity, usually because of binding or complex formation with the inhibitor (Van der Oost, Beyer, and Vermeulen 2003). The primary Phase II enzymes that have been examined in unionids include reduced (GSH) and oxidized(GSSG) glutathione and glutathione S-transferase (GST, Table 10.1). Reduced glutathione is atripeptide whosemajor functions are to conjugateelectrophilic inter- mediatesand to serveasanantioxidant.Thisenzymeensures thereductionofoxidants, the quenching of free radicals, the neutralization of organic peroxides, and the elimination of hydro- carbons by conjugation (Cossu et al. 1997). Most of the researchonthe utility of Phase II enzymes as potential biomarkers in unionids comes from research on U. tumidus in which reduced levels of GSH in cytosolic and particulate fractions of the gills and digestive gland werereported after 15- and30-daydeploymentatsites near anddownstreamfromthe outfall of acokery (primarily contaminated with PCBs and PAHs; Cossu et al. 1997; Doyotte et al. 1997). For example, GSH concentrations were reduced in the cytosolic fraction by 79% in thegillsand by 59% in the digestive glandatthe most polluted site(Cossu et al.1997). Thesedecreases paralleled lipid peroxidation in thegills (see OxidativeStress section),which reflectedcellinjuryand toxic effects in this tissue. Similar resultswerefoundwhen U. tumidus were transplantedtoother areas contaminated by effluents from alaundry and afoundry(Cossu et al. 2000). One of the detoxification enzymes that has been assayed in unionids is GST. This is afamily of enzymes that catalyze the initial step of mercapturic acid synthesis—the conjugation of GSH with TABLE 10.1 (Continued) Category of Biomarker Examples Reference in Unionids Condition indices Ma ¨ kela ¨ ,Lindstro ¨ m-Seppa ¨ ,and Oikari (1992), Naimo, Atchison,and Holland- Bartels (1992),Couillard et al. (1995a, 1995b),Hickey,Roper, and Buckland (1995),Hyo ¨ tyla ¨ inen, Karels, and Oikari (2002), Blaise et al. (2003) Energetics Ma ¨ kela ¨ ,Lindstro ¨ m-Seppa ¨ ,and Oikari (1992), Naimo, Atchison,and Holland- Bartels (1992), Hickey, Roper, and Buckland (1995),Gagne ´ et al. (2002, 2001b),Hyo ¨ tyla ¨ inen, Karels, and Oikari (2002) Valve activity Balogh and Salanki (1984); Huebner and Pynno ¨ nen (1992), Englund and Heino (1996), Ka ´ da ´ retal. (2001), Markich, (2003) Growth Manly and George (1977); Foster and Bates (1978), Muncaster, Hebert, and Lazar (1990), Lasee (1991); Couillard et al. (1995a), Beckvar et al. (2000), Gagne ´ et al. (2001b), Bartsch et al. (2003), Newton et al. (2003) a Not available. Source:Modified from Van der Oost, R., Beyer, J., and Vermeulen, N. P. E., Environ Toxicol Pharmacol,13, 57–149, 2003. With permission. Freshwater BivalveEcotoxicology262 4284X—CHAPTER 10—19/10/2006—15:11—KARTHIA—XMLMODEL C–pp. 257–284 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) xenobiotics and their metabolites. Their primary roles are defense against oxidative damage and peroxidative products of DNA and lipids. GST activitywas significantlyreduced (~20%) in the digestive glandof Anodontaanatina deployed for4months at asite20kmdownstreamofa bleached kraft pulp and paper mill, but not at asite 5kmdownstream, compared to control sites (Figure 10.2;Ma ¨ kela ¨ ,Lindstro ¨ m-Seppa ¨ ,and Oikari 1992). Given that the spatialextent of this effect was limited, the authors concluded that organically bound chlorine (a major constituentofthe effluentfrombleached kraft pulpand papermills) didnot consistentlyinduceGST activity in unionids. OXIDATIVE STRESS Many environmental contaminants have been shown to exerttoxic effects through oxidative stress. Antioxidant defenses include antioxidant enzymes (superoxide dismutase [SOD],catalase [CAT], glutathione peroxidase [GPOX], and glutathione reductase [GRED])and freeradical scavengers (vitamins Cand E, carotenoids, and glutathione), whose function is to remove reactive oxygen species thus protectingorganisms from oxidative stress (Doyotte et al. 1997). SOD catalyzes the conversion of reactive superoxide anions into hydrogen peroxide, which in turnisdetoxified by CAT. Hydrogen peroxide and hydroperoxides are destroyedbyGPOXs in the presence of GSH. Glutathione is regenerated by GRED.When antioxidant systems are impaired, oxidative stress may also producelipid peroxidation (LPOX), or the oxidation of polyunsaturated fatty acids. Catalaseisoften one of the earliest antioxidant enzymes to be induced and has been shown to be induced in Mytilus sp. exposedtoorganic pollution (Porte et al. 1991). Catalaseand SOD activity were measured in U. tumidus after in situ deployment at sites upstream and downstream of effluent from acokery. In one study, CAT and SOD (Figure 10.3)were significantly reduced at the most polluted site, relative to musselsdeployed upstream(Cossu et al. 1997). In the second study,SODand CAT weremarkedly unchanged upon exposure to the cokery effluent,suggesting that theseenzymes were not sensitive to short-termexposure to the chemicals contained therein (Doyotte et al. 1997). Interestingly, the experimental design for these two studies were nearly identical, with the exception that musselsinthe latterstudy were deployedfor 7days, whereas musselsinthe Cossu et al. Distance (km) from pulp mill 0 100 50 150 200 250 − 35 − 15 520 ∗ Glutathione S-transferase activity (nmol/mg protein/min) FIGURE 10.2 Glutathione S-transferase activity in the digestive gland of A. anatina after a4-month deploy- ment to sites upstream ( K 35 and K 15 km) and downstream (5 and 20 km) from ableached kraft pulp and papermill. Asterisk indicatesasignificant difference fromthe upstreamreferencesites(Adaptedfrom Ma ¨ kela ¨ ,T.P., Lindstro ¨ m-Seppa ¨ ,P., and Oikari A. O. J., Aqua. Fenn.,22, 49–55, 1992.) Biomarker Responses of Unionid Mussels to Environmental Contaminants 263 4284X—CHAPTER 10—19/10/2006—15:11—KARTHIA—XMLMODEL C–pp. 257–284 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) (1997) study weredeployedfor 15 and 30 days. Thesedata suggestthatthe temporal variation in theseoxidativeenzymes maymakeitdifficulttodetecttreatmenteffects at shorter exposure durations. Selenium-dependent GPOX,GRED,and GSH levels appear to be early biomarkersofexposure to pollutants in unionids. For example, deployment of U. tumidus to sites upstreamand downstream of effluentfromacokery resulted in significant decreasesinSe-dependentGPOXand GRED activities (Cossu et al. 1997; Doyotte et al. 1997). Similar exposure of U. tumidus to effluents from othersources (a laundry and afoundry) also resulted in significant reductions in Se-dependent GPOX and GRED activities (by 70 and 80%, respectively) (Cossu et al. 2000). However, in the latter study, arelation among the antioxidant response and the degreeand type of contamination in sediments was not consistently observed, suggesting that these effects couldresult from unidenti- fied contaminants and/or issues associated with contaminantbioavailability. Lipidperoxidationisanimportantoutcome of oxidativestress becauseitcan demonstrate theability of asingleradical speciestopropagate anumberofadverse biochemical reactions (Van derOost, Beyer, andVermeulen2003).Studies suggest that LPOXhas considerable potential as abiomarker forenvironmentalriskassessment (Stegemanetal. 1992), although it canresultasaconsequenceofcellular damage becauseofavariety of stressorsother than exposuretocontaminants.Numerousstudies in thefisheries literature have demonstratedan enhancementinLPOX as afunction of contaminantexposure(see references in Vander Oost, Beyer, andVermeulen 2003),and thepreliminary data on unionids arepromising. In thefirstof threestudies on antioxidant enzymesin U. tumidus ,LPOX(as expressed by malonaldehyde content, MDA) didnot differ betweenreference unionids andthose exposed to acomplex industrialeffluent (Doyotte et al.1997).Itispossiblethatthe lack of an effect in this study wasafunction of the7-day deploymentperiod; in subsequent studies, unionids were deployedfor at least15days. In afollow-up study, decreases in Se-dependent GPOX andGRED activitiesand GSH levels were associatedwithathree-fold increase in MDAcontentinthe gillsand with a high level of contamination of sedimentsbyPAHsand PCBs (Cossu et al.1997).Similarly, Superoxide dismutase activity 0 20 40 60 80 100 120 15 days 30 days Site ASite BSite CSite D ∗ FIGURE 10.3 Superoxide dismutase activity in the cytosolic fraction of the gills of Unio tumidus deployed for 15 or 30 days to four sites along the Fensch River, France. Site Awas areference, Site Bwas upstream of a complex cokery effluent, Site Cwas downstream near the outfall, and Site Dwas about 2.5 km downstream. Asterisk indicates asignificant difference from control (Adapted from Cossu, C., Doyotte, A., Jacquine, M. C., Babut, M., Exinger, A., and Vasseur, P., Ecotoxicol. Environ. Saf., 38, 122–131, 1997.) Freshwater BivalveEcotoxicology264 4284X—CHAPTER 10—19/10/2006—15:11—KARTHIA—XMLMODEL C–pp. 257–284 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) Cossu et al.(2000) foundelevatedlevelsofMDA in unionids deployedinfour rivers in France with various pollutionsources.Inparticular, in oneriver primarily contaminatedbymetals, deficiencies in antioxidant defenses resulted in dramaticlipid peroxidation—MDAconcen- trations rangedfrom29to85ng/mg proteindownstreamofthe source,while thecontrolsdid not exceed 5ng/mg protein. TheelevatedlevelsofMDA,coupled with decreasesinSe-depen- dent GPOX andGRED activities andGSH levels,suggest that theseantioxidant parameters may be usefulbiomarkersofexposure. AMINO ACIDS AND PROTEINS Changes in the concentrations of freeamino acids in the gill, mantle, and adductor muscle in marine bivalveshave been used as biomarkers of exposure to contaminated environments(Livingstone 1985 and references therein). For example, the ratio of taurine to glycine has been used in marine bivalvestoindicate biochemical responses to hydrocarbons(Widdowsetal. 1982). To our knowl- edge, two studies have measured free amino acidconcentrations in unionids and neither measured this ratio; thus,the utilityofthis ratio in unionids is unclear but deserves future study. Both unionid studies suggestthat increases or decreasesintotal-free amino acidsinsome tissues (especially the mantle andadductormuscle) maybeindicative of generalizedstressinduced by avariety of environmental factors(e.g., starvation or increased temperature)and may be useful as an in situ biochemical indexoftoxicity (Gardner,Miller, and Imlay 1981; Day, Metcalfe,and Batchelor 1990). As with many aspects of unionid biochemistry and physiology, the concentrations of most free amino acids vary seasonally. Thus, the baseline seasonal variation needs to be characterized in agivenpopulation prior to attributing changesinamino acid concentrations to contaminant- induced effects (biomarker criterion 3inBiomarker Concept). The molecules that probably offer the greatestpotential for monitoring biological effects and have attracted the most attention are enzymes and other functional proteins such as metallothio- neins (MT) (Livingstone 1985). Freshwater and marine molluscs are knowntoaccumulatemetals from theirenvironment. Thetolerance of the resulting body burden hasbeen attributedtothe existence of an effective detoxification system (Viarengo 1989). Metallothioneins are low molecu- lar weight,cysteine-richmetal binding proteins that functionasadetoxificationmechanism by sequestering divalent metals through specific ligands present in the cytosol.They are thought to provide one of two protective functions (1) interception and binding of free metal ions that are initially taken up by the cell and (2) removal of metals from non-thionein ligands that include cellular targets of toxicity (Van der Oost, Beyer, and Vermeulen 2003). One of the first studies to examinethe role of metal-binding proteins in unionids ( A. cygnea ) found that Cd and Cu were bound to different fractions upon laboratory exposure. For example, Cu was generally bound to the high molecular weight fraction, whereas Cd was mainly bound to aspecific metal-binding, carbohydrate-containing protein fraction of M r ~11,000 (Holwerda 1991).The Cd-binding proteinwas similartothe metal-binding proteins observed in Mytilus (Roesijadiand Hall 1981) and Crassostrea (Ridlington andFowler1979).Inone of thefirst reports of MT-inductioninunionids, Malley et al. (1993) showed that a22-dayexposure of A. grandisgrandis to waterborneCdinduced MT in the gills.Although MT was measured in the mantle, gills, foot,kidney, visceral mass, and whole body,only MT in the gills increased significantly with increasing exposure to Cd. More recently, the time coursefor MT induction has been examined. Before induction of MT (~14 d) in Pyganodongrandis,Cdwas primarily bound to the high molecular weight fraction, and after induction (~90 d) all the Cd had apparently shifted to fractions of moderate molecular size (15 to 3kDa)—which corresponds to the expected MT-fraction (Couillard et al. 1995a). Field studies supportexperimental data that MTs sequesterheavy metals and that MT levels correlate with tissue levels of heavy metals. Induction of MT in unionids has been observed in field Biomarker Responses of Unionid Mussels to Environmental Contaminants 265 4284X—CHAPTER 10—19/10/2006—15:11—KARTHIA—XMLMODEL C–pp. 257–284 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) locations where mussels were exposed to Cd (Malleyetal. 1993; Couillard et al. 1995b)and urban effluents (Gagne ´ et al. 2002). Concentrations of MT generally correlate with tissue Cd concen- trations, but not with tissue concentrations of Cu or Zn (Figure10.4;Couillard, Campbell, and Tessier 1993; Wang et al. 1999). Variation in MT concentrations among field locations is strongly correlated with free Cd 2 C (Couillard, Campbell,and Tessier 1993; Wangetal. 1999). Most recently, Perceval et al. (2002) examined the relative influence of limnological and geochemical confounding factorsonMTsynthesis in natural populations of P. grandis in lakes alongaCd concentration gradient (biomarker criterion 4inBiomarker Concept). Predictive models found that dissolved Ca ( K )and freeCd 2 C ( C )explained 62% of the variation in MT. These data can be used in monitoring programstoselect field sites to reduce the relative influence of factorsthat confound MT concentrations (Perceval et al. 2004). 0500 1000 1500 2000 2500 0 100 200 300 400 Cadmium p <0.01 01000 2000 3000 4000 5000 6000 0 100 200 300 400 Metal concentration in gills (nmol/g drywt) 4000 6000 8000 10000 12000 Metallothionein (nmol metal binding sites/g drywt) 0 100 200 300 400 Copper p >0.05 Zinc p >0.05 FIGURE 10.4 Concentration of metallothionein as afunction of Cd, Cu, and Zn in the gills of A. grandis from 11 lacustrine sites along ageochemical gradient of pH, Cd, Cu, and Zn in an area influenced by mining and smelting (Adapted from Couillard, Y., Campbell, P. G. C., and Tessier, A., Limnol. Oceanogr.,38, 299–313, 1993.) Freshwater BivalveEcotoxicology266 4284X—CHAPTER 10—19/10/2006—15:11—KARTHIA—XMLMODEL C–pp. 257–284 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) [...]... 0.5 0.4 0.3 0 50 100 150 200 250 Cadmium concentration (μg/L) 300 FIGURE 10. 6 Respiration rate in L ventricosa as a function of cadmium exposure during a 28-day laboratory study (Adapted from Naimo, T J., Atchison, G J., and Holland-Bartels, L E., Environ Toxicol Chem., 11, 101 3 102 1, 1992.) © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) 4284X CHAPTER 10 19 /10/ 2006—15:12—KARTHIA—XML... of exposure to contaminants (Table 10. 2) These studies have had variable results For example, Naimo, Atchison, and Holland-Bartels (1992) reported that condition indices in L ventricosa © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) 4284X CHAPTER 10 19 /10/ 2006—15:11—KARTHIA—XML MODEL C – pp 257–284 272 Freshwater Bivalve Ecotoxicology TABLE 10. 2 Summary of the Effects of Environmental... (SETAC) 4284X CHAPTER 10 19 /10/ 2006—15:12—KARTHIA—XML MODEL C – pp 257–284 284 Freshwater Bivalve Ecotoxicology Rodius, F., Hammer, C., and Vasseur, P., Use of RNA arbitrarily primed PCR to identify genomic alterations in the digestive gland of the freshwater bivalve Unio tumidus at a contaminated site, Environ Toxicol., 17, 538–546, 2002 Roesijadi, G and Hall, R E., Characterization of mercury-binding... Chemistry (SETAC) 4284X CHAPTER 10 19 /10/ 2006—15:11—KARTHIA—XML MODEL C – pp 257–284 270 Freshwater Bivalve Ecotoxicology methods development, concentration-response studies with model chemicals, and documentation that genotoxic effects on individuals ultimately influence populations are still required (biomarker criterion 6 in Biomarker Concept) PHYSIOLOGICAL AND MORPHOLOGICAL From the mid-1970s, aquatic ecotoxicology... Chemistry (SETAC) 4284X CHAPTER 10 19 /10/ 2006—15:12—KARTHIA—XML MODEL C – pp 257–284 Ratio of taurine:glycine Porphyrin profile Phagocytosis, lysosomal destabilization Vitellogenin Cholinesterases DNA damage (comet assay), micronucleus assay Histopathology, osmotic and ion regulation, scope for growth 280 Freshwater Bivalve Ecotoxicology use in unionids or their use in marine bivalves (Table 10. 5) In addition,... estradiol competitors that were able to compete with estradiol-binding sites in gonad cytosols and induced © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) 4284X CHAPTER 10 19 /10/ 2006—15:11—KARTHIA—XML MODEL C – pp 257–284 268 Freshwater Bivalve Ecotoxicology ´ vitellogenin (Gagne et al 2001a) Moreover, the estrogen-competing potential of the extracts was significantly correlated... Lindstrom-Seppa, and Oikari (1992) Increase (digestive gland) Increase (gonads) —b Note: Tissues sampled shown in parentheses a b Polycyclic aromatic hydrocarbons Not measured © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) 4284X CHAPTER 10 19 /10/ 2006—15:12—KARTHIA—XML MODEL C – pp 257–284 ´ Gagne et al (2002, 2001b) Hickey, Roper, and Buckland (1995) 274 Freshwater Bivalve Ecotoxicology... and Vasseur, P., Glutathione reductase, selenium-dependent glutathione peroxidase, glutathione levels, and lipid peroxidation in freshwater bivalves, Unio tumidus, as biomarkers of aquatic contamination in field studies, Ecotoxicol Environ Saf., 38, 122–131, 1997 © 2007 by the Society of Environmental Toxicology and Chemistry (SETAC) 4284X CHAPTER 10 19 /10/ 2006—15:12—KARTHIA—XML MODEL C – pp 257–284... 5–17, 2002 ` Perceval, O., Couillard, Y., Pinel-Alloul, B., Giguere, A., and Campbell, P G C., Metal-induced stress in bivalves living along a gradient of Cd contamination: Relating sub-cellular metal distribution to population-level responses, Aquat Toxicol., 69, 327–345, 2004 ´ Porte, C., Sole, M., Albaiges, J., and Livingstone, D R., Responses of mixed-function oxygenase and antioxidase enzyme system... (SETAC) 4284X CHAPTER 10 19 /10/ 2006—15:11—KARTHIA—XML MODEL C – pp 257–284 Acetylcholinesterase activity (μmole substrate hydrolyzed/min/g protein) Biomarker Responses of Unionid Mussels to Environmental Contaminants 1200 269 Amblema plicata 100 0 800 600 400 200 0 0.0 0.2 0.4 0.6 0.8 1.0 Chlorpyrifos concentration (mg/L) 1.2 FIGURE 10. 5 Acetylcholinesterase activity in Amblema plicata after 96-hour exposure . Naimo, T. J., Atchison, G. J., and Holland-Bartels, L. E., Environ. Toxicol. Chem.,11, 101 3 102 1, 1992.) Freshwater BivalveEcotoxicology274 4284X CHAPTER 10 19 /10/ 2006—15:12—KARTHIA—XMLMODEL C–pp Reference: 7.91 G 0.37 Cd-exposed: 5.94–6.78 Naimo, Atchison, and Holland-Bartels (1992) a Measured in males only. Freshwater BivalveEcotoxicology272 4284X CHAPTER 10 19 /10/ 2006—15:12—KARTHIA—XMLMODEL. in freshwater bivalve ecotoxicology have either been short-term laboratory tests with single chemicals (Chapter 5and Chapter 7) or monitoringofbiomarker responses in thefield (Chapter 6and Chapter