STRATEGIES FOR USING SHALLOW-WATER BENTHIC FORAMINIFERS AS BIOINDICATORS

STRATEGIESFORUSINGSHALLOW-WATERBENTHICFORAMINIFERSAS BIOINDICATORSOFPOTENTIALLYTOXICELEMENTS:AREVIEW

MICHAEL MARTINEZ-COLON1,3,PAMELA HALLOCK1 AND CARLOS GREEN-RUIZ2

ABSTRACT

Thisreviewexaminesenvironmentalfactorsaffecting potentiallytoxicelements(PTEs)incoastalwaterswiththe goalofproposingwaystoenhancetheuseofbenthic foraminifersasbioindicatorsofsuchpollution.Pollutionof coastalsystemsbyPTEs,oftenreferredtoasheavymetals,is amajorconcernforscientists,resourcemanagers,and regulatoryagencies.Bioavailability,uptakerates,speciation, claymineralogy,pH,complexation,andotherfactorscontrol thebehaviorofPTEsinmarinesystems,especiallyin estuaries.Whilebreakthroughworkhasexaminedincorporationandassimilationofmetalsintomarinemacroinvertebrates,similarresearchonmarineprotistsisstillinthe developmentalstage.Manystudiesassumeorconcludethat foraminiferalassemblagesandthefrequencyofdeformed testsarefirst-lineindicatorsofpollution,butotherspresent confoundingresults.UnderstandingthecomplexgeochemistriesofPTEs,coastalwaters,andsedimentsiscriticaltothe designandinterpretationofmeaningfulstudies.

Applicationsofforaminifersasbioindicatorsrequirestrong scientificmodelsbasedonbothfieldandlaboratory experimentsandwhichspecificallyexaminetheinfluenceof PTEsandotherpollutantsatcommunity,assemblage, population,individual,andgene-expressionlevels.Genomic studiesofkeyforaminiferaltaxawithstrongpotentialas bioindicatorsarecriticallyneededasabasisforstudiesof geneexpressionindicatingexposuretospecificstressors. Thoughmajorchallengesexisttofullyrealizingthepotential forapplicationofforaminifersasenvironmentalindicators, theirglobalimportanceinthepastandpresentargues stronglyforfurtherdevelopmentofthesepromisingtools.

INTRODUCTION

Waterpollutionhasbeenanenvironmentalconcernsince theonsetoftheIndustrialRevolution.Anthropogenic activities(i.e.,agriculture,mining,industrialization,urbanization)havecontaminatedsurface,ground,andcoastal waterswithexcessivenutrientsandpotentialtoxins(Hunter andArbona,1995;Alve,2000;Elberlingandothers,2003; Farkasandothers,2003;Green-Ruı´zandPa´ez-Osuna, 2004;Haywardandothers,2004).Thesepollutantscan alterecosystemsbyreducingthefecundityandsurvivalof organisms,andtheycanpromotediseasesthatcanbe passedtohumans(SmedleyandKinniburgh,2005).

Historically,pointsourcessuchasindustrialeffluentand sewageoutfallswereamongthemajorsourcesofwater pollution.Becausepointsourcescanbemanaged,non-

1 CollegeofMarineScience,UniversityofSouthFlorida,St. Petersburg,FL33701,USA

2 InstitutodeCienciasdelMaryLimnologı´a,UniversidadNacional Auto´nomadeMe´xico,Mazatla´n-Me´xico

3 Correspondenceauthor.E-mail:mmartin8@mail.usf.edu; foram3438@yahoo.com

pointsourcesofpollutionsuchasatmosphericfalloutand storm-waterrunoffarenowquantitativelymoreimportant thanpointsourcesinmanymanagedareas.Thematerial introducedintoaquaticenvironmentsbynon-pointsources istypicallyneitherwelldefinednorreadilymanaged,even wherethepoliticalwillexiststodoso.

Potentiallytoxicelementsinestuarinesedimentshave beenstudiedbothspatiallyandtemporallyformanyyears. WorkbyTessierandothers(1979),Santosandothers (2005),andmanyothershavelaidthegroundworkfor furtherresearchonhowindividualpollutantsaffectspecific organisms,includingmicrobiota.Complicatingfactorsfor understandingresponsesoforganismsinestuarineenvironmentsincludenaturalvariabilityinpH,salinity, sedimenttextureandmineralogy,andorganicproductivity, allofwhicharelinkedtoclimaticandgeographicregions andtothecomplexityofthesubjectorganisms.Avarietyof approacheshasbeendevelopedtoidentifypollutantsin coastalwaters.Pollutantshavebeenmeasuredinboththe watercolumnandsubstrate(e.g.,Kotandothers,1999; Zhangandothers,2002).Theireffectshavebeenstudiedon manymarineorganisms,includingfish,decapods(Rainbow andWhite,1989;Rainbow,1995),plankticcrustaceans, ostracodes(Eagar,2000),andbenthicforaminifers(Coccioni,2000;Elberlingandothers,2003;Farkasandothers, 2003;LeCadreandDebenay,2006;Frontaliniand Coccioni,2008).Benthicinfaunagenerallyarepreferred forbioassaysandasbioindicatorsbecausetheyaretypically sessileoroflimitedmobilityandthereforearedirectly influencedbynaturalorinducedstressesintheirenvironment(Bilyard,1987).

OBJECTIVES

Thispapercomprisestwosectionsthataddresstwo majorobjectives.Thefirstobjectiveistoprovidean overviewofenvironmentalfactorsinfluencingmobility andbioavailabilityofpotentiallytoxicelements(PTEs)in coastalwaters.Thissectioncanprovideresearchersand resourcemanagers,whoareproposingorbeginningto workwithPTEcontamination,withinsightintothe complexrangeoffactorsandenvironmentalconditions thatmustbeconsidered,keepinginmindthatPTEsaretoo diverseandcomplexintheirchemicalbehaviorstopermita comprehensivetreatmentinareviewpaper.

Theinsightprovidedbythefirstsectionisessentialfor thesecondobjective,whichistoencourageresearchonhow PTEsaffectbenthicforaminifersand,inturn,onhow foraminiferscanbeusedasbioindicatorsofpollution.This sectionexaminespreviousstudiesofbenthicforaminifersin coastalwaterscontaminatedbyPTEs.Benthicforaminifers areidealtoolsinenvironmentalresearchbecausetheyare small,abundant,diverse,andincludestress-toleranttaxa. Inaddition,foraminifershaveshellsthatleavearecordof

pastassemblages,andwhichoftenprovidemorphological orgeochemicalevidenceofenvironmentalchange(Yanko andothers,1999;Schafer,2000).Understandingthecomplex geochemistryofPTEs,aswellastheequallycomplex chemistriesofcoastalwatersandsediments,isvitaltothe designandinterpretationofobservationalandexperimental studiesofPTEcontaminationincoastalenvironments.The secondsectionconcludeswithrecommendationsforfuture experimentstoenhanceunderstandingofindividual,populationandassemblageresponsestotheenvironmental stressesresultingfromPTEcontamination.

POTENTIALLYTOXICELEMENTS

Elementscanbeclassifiedasessentialornon-essential dependinguponwhethertheyarerequiredforbiological activities(i.e.,metabolicprocesses).Organismstolerate non-essentialelements(e.g.,As,Cd,Hg,Pb)atlimited concentrations,whereasessentialelements(e.g.,N,P,Ca, Cu,Fe,Zn)arerequiredformetabolism(e.g.,Venugopal andLuckey,1975).Bothnon-essentialandessential elementscanbetoxicathighconcentrations,depending uponbioavailability.Toleranceofindividualorganismsto PTEsdependsonbiologicalfactors,suchasspecies,size, sex,lifestage,detoxificationmechanisms,intakeroute,and physiologicalstate,anduponabioticfactorssuchasthe chemicalspeciesoftheelement,oxidationstateofthe environment,andadsorptionbyclaymineralsororganic matter.

Theterminologyassociatedwithnaturallyoccurring heavyelementsthatarepotentiallytoxictobiotaisvaried andoftenconfusing.Theterm‘‘heavymetal’’iswidelyused torefertotheseelements(Duffus,2002),whichalsoare commonlybutincorrectlyreferredtoastracemetals (RainbowandFurness,1990).Inthiscontext,‘‘heavy’’ conventionallyreferstodensity(.5gcm 3);however, metalsarecommonlyclassifiedasheavyelementsbased onothercharacteristics,includingatomicweight,atomic number,chemicalproperties,andtoxicity(Duffus,2002; Selinusandothers,2005).Duffus(2002)thereforerecommendedavoidingterminologythatclassifiesmetalsas ‘‘heavy,’’andrevisedtheclassificationschemetoreflect

chemicalpropertiesbasedontheLewisacid-behavior scheme(Lewis,1923).Thisclassificationallowspredictions ofhowametalwillcomplexwithorganicsorminerals basedonoxidationstate(Table1):ClassAmetalsarehard (non-polarizable)metals;ClassBaresoft(more-polarizable)metals;andathirdgroupconsistsofintermediate metals.Thelatterareproblematictoclassifybecausethey haveastrongtendencytoformcomplexeswithbothhard andsoftmetals(Duffus,2002).Less-polarizablemetals havehighercharges(higheroxidationstates)whereasmore polarizablemetalshaveloweroxidationstates.

MostcommonPTEsalsocanbecategorizedbasedon chemicalaffinities.AccordingtoGarrett(2005),lithophilic elementshavestrongaffinityforsilicateminerals,oxides, andcarbonates;whilechalcophilicelementshavestrong affinitytosulfides,arsenides,selenides,andtellurides.The sameelementcanbelithophilicorchalcophilic,depending uponitsvalence(e.g.,As+5 islithophilic,As+3 ischalcophilic).

Forsimplicity,thispaperreferstoPTEsaselements havingpotentiallydetrimentaleffectsonorganismsinan ecosystem.ThisuseofPTEisconsistentwithterminology employedbyDavisandothers(1978),Pescod(1992),and AlHwaitiandothers(2005).Table1liststhemostcommon PTEsconsideredinstudiesofaquaticpollution,indicating, theircommonvalencestates,behavior(lithophilicor chalcophilic),classbasedontheLewisacid-behavior scheme,averageconcentrationsinshaleandseawater,and concentrationatwhichtoxiceffectscanbepredicted (ERM).

SOURCES,TRANSPORT,MOBILITYAND BEHAVIOROF PTES

IgneousandmetamorphicrocksintheEarth’scrustare theultimatesourcesofPTEsinnature(Fig.1).While physicalerosionandsedimenttransportcanmovePTEsin solidforms,chemicalweathering(dissolution,oxidation, andhydrolysis)istheprimarymechanismofreleaseand mobilizationofelementsfromrocks(Siegel,2002).Asrocks weathertoformsedimentsandsoils,solubleionsare mobilizedintosurfacewatersorleachedintogroundwater andtransportedtolargerwaterbodies(i.e.,rivers,lakes, oceans;Fig.1).

TABLE 1.AveragemetalconcentrationsofPTEsinshaleandseawater,andsedimentqualitycriteria(ERM).A:Class-Ametal;B:Class-Bmetal; I-Intermediate;*Lithophilic; {Chalcophilic.

MetalDuffus(2002)Garrett(2005)

ConcentrationinShale (ppm)a

ConcentrationinSeawater (ppm)a ERM(ppm)b AlA*80,0000.003— FeA,I*, { 47,2000.003— MnI*8500.0002— CrI*900.0003370 NiI*, { 680.000551.6 ZnI*, { 950.0003150 CuB*, { 450.0002270 CoI*, { 191310-6 PbB*, { 203310-6 218 As- { 130.001770 CdB*, { 0.38310-5 9.6 HgB { 0.44310-7

a FromKrauskopfandBird(1995). b FromLongandothers(1995).ERM:EffectRangeMedian.

FIGURE 1.Simplifieddiagramofpotentiallytoxicelement(PTE)reservoirsandchangesinconcentrationsfromrocksintowaterbodies.Adapted from aKrauskopfandBird(1995)and bSiegel(2002).

AnimportantmechanismforaquatictransportofPTEs iscomplexedwithclaymineralsandorganicmatter,both dissolvedandparticulate.AsnotedinTable1,PTEstend tobefarmoreconcentratedinshalesthaninnaturalwater bodies,inlargepartbecausePTEstendtohavelow solubilitiesinoxygenatedwatersandmorecommonlyare associatedwithclaymineralsandorganicmatterthatmake upestuarinesedimentsandthatmightlithifyasshale.An increaseovernaturalbackgroundconcentrationsofPTEs ineithersedimentsorwaterbodiesiscommonlyassociated withanthropogenicsourcesandactivitiessuchasagriculture,mining,andurbanorindustrialwastes(Table2). SedimentdepositionisthemajorsinkforPTEsentering aquaticecosystem,butsedimentsalsocanbeasource, dependinguponchemicalcharacteristicsofthedepositional environmentandthepotentialforresuspension(Horowitz, 1991;Yuandothers,2001;Fig.2).

Thechemistryofawaterbody,includingporewaters, influencewhatPTEscanbeincorporatedintosedimentsby authigenicanddiageneticprocesses(e.g.,intoFe/Mn/Mg/ Aloxides,sulfides),adsorptiontoclayminerals,oreither adsorptiontoorabsorptionbyorganicmatter.Adsorption isrelatedtotheassociationofaniontoasurface(organic orinorganic)eitherby(1)chemicalattractions(covalentor ionicbonding)or(2)electrostaticattractioninvolving oppositechargesbetweenanionandasolidsurface (NationalResearchCouncil,2003).Thelatterpromotes displacementbyionsoflikecharge,hence,becoming exchangeable(NationalResearchCouncil,2003).Onthe otherhand,absorptionistheuptakeofaspeciesintothe volumeofanothermaterialandistheprimaryretention

mechanismforPTEsinorganicmatter,althoughadsorptionoccursaswell(NationalResearchCouncil,2003;Du Laingandothers,2009b).

Sedimenttexture,claymineralogy,kindsandquantities oforganicmatter,andpHinfluenceratesandprocessesof removalofPTEsfromcoastalwaters.Whenexposedtothe negativesurfacechargeofclays,lithophilicPTEadsorption willvarydependingontypeofclayanditsadsorption capacity:montmorillonite . vermiculite . illite . chlorite . kaolinite(Horowitz,1991;Table2).Becauseclaystend tocomplexandadsorbPTEs,especiallyunderoxidizing conditions(Luoma,1990),concentrationofPTEsin sedimentstendstobeinverselycorrelatedwithgrainsize (Green-Ruı´zandPaez-Osuna,2004).Sinceclaysare stronglyacidic,adsorptionoflithophilicPTEsgenerally increaseswithincreasingpH(FarrahandPickering,1979; Green-Ruı´z,2005).AbovecertainthresholdpHvalues, dependentonclaymineralogy,ionsofmostlithophilic PTEsareremovedfromsolution(FarrahandPickering, 1979;Zhangandothers,2002).Thus,clayscanactually sequesterPTEsastheyfallthroughthewatercolumnorlie insurficialsediments.

Conversely,lithophilicPTEsmaybemobilizedinacidic orreducingconditions.Pore-waterchemistryandsediment mineralogybothplayimportantrolesinPTEmobilization (Daviesandothers,2005;DuLaingandothers,2009a; Table3).Aerobictodysaerobicbreakdownoforganic matter,whichdecreasesaquaticpH,caninduceremobilizationofsomelithophilicPTEs.Forexample,Znremains adsorbedintoclaysandoxidesatpHgreaterthan6but becomesmobileinlowerpHconditions(Siegel,2002).In

Anthropogenica

TABLE 2.SourcesofanthropogenicandnaturalPTE’s.

AsCdCoCrCuFeHgMnMoNiPbSbSeSnTiVZn

Alloysxxxxx——x—xxxx—x—x

Batteries——————xx—xx—x———x Biocides(agricultural,anti-fouling)x—————xx—————x——— Coatings(anti-corrosives)—x—x————x—x——x——x Pharmaceuticals,Dentalxx—xx—x—x—xxxx——x Fertilizersxx—xx—xx—xx—————x FossilFuelCombustionxx————x———xxx———— Mining,Smelting,Metallurgyxx—xx—xx—xxxx—xxx NuclearReactor—x——————————————— Paintsxxxxx——x—xxxx—x—x PetroleumRefiningx—xxx————xx————xx Plastics—x————————x—————x PulpandPaper———xx—x——xx—————— Semi/Super-Conductorsx——————————x—xx—— Pipes,Sheets————xx————x—————— ElectricalEquipment————x—x————x————— Natural HydrothermalProcessesx———xx—x—x——————— Chromite—FeCr2O4 ———x—x——————————— Pyrite—FeS2‘ x—x——x—x—x——————x Cinnabar—HgS——————x—————————— Azurite—Cu3(OH)2(CO3)2 ————x———————————— Pyroxene—(Mg,Fe)SiO3 —————x——————————— Montmorillonites*——xxxx—x—xx———xxx Illites*—————x—x——————x—— MnOxides*——x——x———xx—————x a Siegel(2002). ‘ BierensdeHaan(1991). *PTE’scommonlyfoundadsorbed(Siegel,2002).

addition,suchPTEsinsedimentscanenterthefoodweb wheningestedbydetritusfeederswhoseacidicdigestive fluidscanmobilizethoseions(Siegel,2002).

PTEconcentrationsinsedimentsandsedimentpore waterscanvarydramaticallyinaverticalprofile.For example,Caplatandothers(2005)reportedthatFe,Mn, Cu,andZnconcentrationsvariedverticallyasafunctionof decompositionoforganicmatterandreductionofoxide phases.AdsorptionofPTEsintoclaymineralsorFe/Mn oxides,hydroxides,oroxyhydroxidescanremovesuch elementsfromtheaqueousphase,therebyreducingtheir accessthroughmetabolicprocessesintoorganisms.However,adsorbedPTEsmightbereleasedintosolution, becomingbioavailableifdepositedinorganic-richenvironmentswheresulfate-reducingbacteriaareactivelyproducingsulfides(Santosandothers,2005),unlessthePTEsare chalcophilesthatprecipitateinorganically(e.g.,CdSand FeS).

REDOX POTENTIALAND PTES

Redoxreactions(Eh)alsoinfluencetheincorporationor releaseofPTEsfrommarinesediments.Ofparticular interestasEhchangesarePTEinteractionswithsulfides (Riedelandothers,1997;Siegel,2002),aswellaswithFe/ Mnoxidesandhydroxides(Horowitz,1991).Moreover,Eh andpHinfluencesco-occur.ResponsestochangesinEh dependuponwhetherthePTEislithophilicorchalcophilic (Fig.3).Hydrogensulfideisproducedbysulfate-reducing bacteriaunderanoxicconditionswhenorganicmatteris present.Althoughmostcommonlyre-oxidized,someH2S

reactstoformmetalsulfides(i.e.,pyrite;MorseandLuther, 1999;DuLiangandothers,2009).Sulfideionsbindwith divalentstatesofdissolvedPTEs(i.e.,CdS,FeS,CuS), therebydetoxifyingthem(DiToroandothers,1992). Sulfidesformedinanoxicenvironmentsarerelatively insolublecompoundsthatoftencontainseveralPTEs (LanderandReuther,2004).However,ifinterstitialwaters becomeevenmorereducingandacidic,solubilityofsome PTEswillthenincrease(Fig.3).

OftenmoreproblematicisthebehaviorofPTEsthatare releasedfromsulfideminerals(sphalerite,pyrite,and chalcopyrite)whenexposedtooxidizingconditions.Commonlysulfidesaredividedintothreemaincategories:acid volatilesulfides(AVS;i.e.,FeS),ironpyrite(FeS2),whichis morestable,andorganicsulfides(DiToroandothers, 1992).ThereleasedPTEsmayeitheradsorbontoFe/Mn hydroxides,clayminerals,ororganicmatter(e.g.,Hansen andothers,1996;Siegel,2002)orbecomebioavailable.For example,MorseandLuther(1999)determinedthat,in anoxicconditions,metalconcentrationsinpyriteminerals decrease(Cd , Pb , Zn , Mn , Ni , Co)accordingto theirincreasingwater-exchangereactions(Pb Cd . Zn . Mn . Co . Ni).Theyfoundthatmetalions(Pb,Zn,and Cd)withwater-exchangereactionsfasterthanthatofFe+2 willprecipitateasmetalsulfidesbeforeironmonosulfides (FeS)andpyrite(FeS2),althoughsmallamountsofthese metalionsalsowillprecipitatedirectlyintopyrite.In addition,releasedPTEslikeCdcancompeteanddisplace Fefromless-stableFeS(AVS)(DiToro,andothers,1992). ThisscenarioiscontrolledinpartbyAVSconcentration (Hansenandothers,1996),inwhichminimalFeSoxidation

FIGURE 2.AnthropogenicsourcesofPTEsandgeochemicalcyclesinshallowcoastalwaters. A PTEtransportinaquaticenvironments(modified fromSchnoor,1996). B IdealizedPTEandpHvariabilityineutrophicconditions.Shadedbarrepresentsenvironmentalgradient:oxic(lightgray), hypoxic(shadedgray),andanoxic(black).

willincreasethecompetitionandeventualformationof CdS(themineralgreenockite).

Arsenic(As)isanotherPTEwhosebehaviorandtoxicity isquitecomplicatedbecauseitcanoccurinseveralvalence states.Arsenate(As+5)canbeincorporatedintoiron oxyhydroxidesinoxidizingaquaticenvironments,becomingimmobilized(DeVitreandothers,1991;Siegel,2002; SmedleyandKinniburgh,2005).Arsenite(As+3)ismuch moresolubleandtoxicthanarsenateandcanbemobilized inreducingenvironments.However,undersulfidicconditions,arsenitewillbeincorporatedintosulfidemineralsand therebyimmobilized(MorseandLuther,1999).The greatestenvironmentalconcerniswhereAsconcentrations areelevatedandwaterchemistriesaretransitionalbetween oxidizingandanoxic.Forexample,biotalivingina naturallyAs-richenvironmentcouldbeunaffectedbyhigh concentrationsofAs+5 incorporatedinironhydroxidesin well-oxygenatedsurficialsedimentsandAs+3 insubsurface sulfides(Pichlerandothers,1999a,1999b).Themost bioavailableAs+3 likelywouldbeatthehypoxicinterface.

TABLE 3.Mobilityofselectedelements,includingPTEs(*)asinfluencedbyredoxpotentialandpHoftheaquaticmedium.ModifiedfromDavies andothers(2005).

Oxidizing(ph5-8)Oxidizing(ph ,4)Reducing RelativeMobility

HighMobilityCl,*Br,S,C,N,*MoCl,*Br,*S,C,N,BCl,*Br

ModerateMobilityCa,Na,Mg,*Zn,*As,*Sr,*HgCa,Na,Mg,*Sr,*Zn,*Cd,*Hg, *Cu,*Co,*Ni,*As,Mn,P Ca,Na,Mg,*Sr,Ba,Mn

SlightMobilityK,Rb,Mn,P,*Pb,*Cu,*Ni,*Co,*CdK,RbK,Rb,P,Fe ImmobileAl,*Cr,*CsAl,*As,*MoFe,Al,*Cu,*Pb,*Zn,*Cd,*Hg, *Ni,*Co,*As,*Mo,*Cr

IntheirstudyofaeutrophicfjordinNorway,Abdullahand others(1995)foundbioavailableAs+3 variedsubstantially amongbasinsofvaryingredoxconditionsinresponseto releasefromsulfidesfollowedbyslowremovalby oxidation.

SALINITY,IONIC STRENGTH, AND PTES

Salinitychangesinestuariesalsoinfluencethepartitioningofcontaminantsbetweensedimentsandinterstitial waters.Theinfluenceofsalinityisreflectedbyratesof desorptionduetocomplexationwithseawateranions(Cl andSO4 2)orsorptionwithcations(Na+,K+,Ca+2,Mg+2), aswellascoagulation-precipitation-flocculationofclay minerals(Ribaandothers,2003;WangandLiu,2003).The flocculationofclaymineralsishighlyinfluencedbysalinity, providinganimportantmechanismforremovalofPTEs fromestuarinewaters.Increasingsalinityistypically associatedwithincreasingpHandthereforeincreasing potentialforadsorptionofsusceptiblePTEsbyclay minerals,asnotedabove.Astheclaymineralsflocculate, theirsettlingratesincrease,asdoratesofremovalof adsorbedPTEs.

Ionicstrengthisintimatelyrelatedtosalinity.For example,astheconcentrationofmonatomicions(i.e., Na+ andK+)increases,theionicstrengthofseawateralso increases.Asionicstrengthincreases,adsorptioncapacities

FIGURE 3.pH-Ehdiagramofsomeheavymetalsshowingtheir increasingtrendsinmobility(modifiedandcompiledfrom Siegel,2002).

onorganismsandsedimentcomponents(clayminerals, organiccarbon,carbonates,FeandMnoxidesand hydroxides)arereducedand,hence,thereislessmetal uptake(DuLaingandothers,2008;DuLaingandothers, 2009).ThisisbecauseNa+,K+,Ca+2,andMg+2 willcompete withPTEsforsorptionsitesand,inaddition,somePTEs areimmobilizedaschloridecomplexes(i.e.,CdCl2)(Du Laingandothers,2008).Moutaandothers(2008)noticed thatPTEslikeCu+2 competewithCa+2 foradsorptionsites onsoilparticlesinahighionic-strengthmedium.Similarly, Green-Ruı´z(2009)noticedadecreaseintheadsorption capacityofHg+2 onmontmorillonitewithanincreasein Na+ intheaqueousmedium.

BioavailabilityofPTEscanvarywidelywithinan estuary.ThePTE-ionconcentrationsinestuarinesediments arecommonlyhigherinbrackishareaswherethereis freshwaterinput(Ribaandothers,2003).Kraepieland others(1997)foundaninverserelationshipbetweenthe activityofametalandsalinityinestuariesthatisrelatedto theconservativetosemi-conservativenatureofPTEions. Forexample,theinorganicspeciationofthesemi-conservativetonon-conservativeCu+2 iondoesnotvaryinsaline waterduetoitsweakaffinitytoCl orSO42 (Kraepieland others,1997;Fritioffandothers,2005).Mostcommonly, Cu+2 formsstrongcarbonatecomplexesthatcanremainat equilibriumifpHisrelativelyconstant(Kraepieland others,1997).Incontrast,NiandZn,whicharerelatively conservativeelementsinestuaries,formstrongcomplexes withCl andSO42 ;hence,theirdissolvedconcentrations areexpectedtopeakatpredictablemid-salinities(Kraepiel andothers,1997;Fritioffandothers,2005).Thisstrong affinityresultsindesorptionofNiandZnfromthe particulatefractionandtheircomplexationbyCl as salinityincreases(Kraepielandothers,1997;Ribaand others,2003).

Anapparentcontradictionemergesfromtheobservationsnotedaboveinrelationtotheeffectsofsalinityon PTEbehavior.Thesolutiontotheparadoxisthatboth processes,thatis,flocculationofclayminerals,whichbinds somePTEions,anddesorptionofPTEsthatcomplexwith Cl assalinityincreases,occurinestuarineecosystemsand mustbetakenintoaccountwhenconsideringthepotential behaviorofPTEs.Areasoffreshwaterinputareenrichedin PTEconcentrationwhereflocculationdominates.When sedimentsaretransportedseaward,adsorptioncapacities arereducedanddesorptionprocessescandominate.

PTEASSESSMENT METHODS

Potentiallytoxicelementscanbemeasuredinsediments orwatersamples.Analysesperformedonsedimentscan utilizebulksamplesormudfractions.Becausetheaffinity betweenclaysandPTEsisverystrong,bulksediment analysesgenerallyunderestimateconcentrationsofPTEsas aconsequenceofthecomplexarrayofgrainsizesand compositionsbeinghomogenizedpriortoanalysis.This dilutioneffectcanbeavoidedifonlythefinefractionis studied.Themudfractioncanbeextractedbywetordry sievingusingnon-adsorptivenetmeshesanddeionized watertominimizecontaminationandloss.Thus,researchersmustclearlyreportsedimentgrain-sizedistributionsand whetherbulkorfinefractionswereanalyzedtoreadily enablecomparisonwithdatafromotherstudies.

Anotherapproachistodeterminethe‘‘speciation’’of PTEsinsedimentandwatersamples.Sequentialextraction procedures,usingselectiveleachingmethods,haveshown thatsedimentsarecomposedofseveralfractions:residual (e.g.,lithicminerals),acid-soluble(e.g.,carbonates), reducible(e.g.,Fe/Mnoxides),oxidizable(e.g.,organic matter),andexchangeable(e.g.,clayminerals)(Tessierand others,1979;Perez-Cidandothers,1998).Thistechnique attemptstodifferentiatebetweenformsofPTEandparticle phases(NationalResearchCouncil,2003).Concentrations canvaryinfractionsotherthanthecarbonateandresidual, dependingontheenvironmentalconditionsfromwhicha sampleoriginated.Unfortunately,bioavailabilitycannotbe determinedforeachfractionbecauseofthestrongreactants usedtoextractPTEs.

Analyzingwatersamples(includingporewaters)isas complexasdealingwithsediments.Samplescanbefiltered orleftunfiltered.Theadvantageoffilteringisthatdissolved PTEsgenerallyrepresentthemostreadilybioavailable portion.Samplesmustnotbeacidifiedpriortofiltration becauseacidificationreleasesPTEsintosolution.Filtrate canbeanalyzedbysequentialextractionorbioavailability techniques,oras‘‘bulk’’sedimentormud,dependingon whattypeoffilterisused.Apossibledisadvantageof filteringisthatlargevolumesofwaterareneededtoacquire asufficientamountoffiltratetoanalyze.TheNational ResearchCouncil(2003)providesadetailedexplanationof techniquesandapproaches.

Determiningtheconditionsandconcentrationsunder whichaPTEisdetrimentaltotheenvironmentisessential todevelopingeffectivemanagementstrategies.Several indiceshavebeenproposedandappliedinenvironmental studies.Thosethathavebeenutilizedinforaminiferal studiesarediscussedbelow.

TheEnrichmentFactorIndex(EF)isaratiothatrelates concentrationofaPTEinsedimentsrelativetoits concentrationinthesurroundingregionofthestudyarea (Green-Ruı´zandothers,2005).Valuescalculatedrepresent therelativeconcentrationofthePTEabovebackground level(Green-Ruı´zandothers,2005).ValuesrangefromEF ,1(noenrichment)toEF .50(extremeenrichment) (Acevedo-Figueroa,2006):

EF ~ Mi½ = Ei½ sample ðÞ Mr½ = Er½ background ðÞ ð1Þ

whereMi andMr arethePTEconcentrations,andEi andEr arethenormalizingPTEconcentrations,inthesediment andintheEarth’scrust,respectively.

Thelog-basedGeo-accumulationIndex(GI)takesinto accountanypossiblevariationsinbackgroundvalues obtainedfromtheclayfractionbasedonlithological differences.Indexvaluesranging0–6reflectthedegreeof contamination(Arraes-MoreiraandBoaventura,2003).A valueof6isequivalenttocontaminationlevels100times greaterthanbackgroundvalues.

~ log2 Mi½ sample ðÞ 1:5Mr ½ background ðÞ ð2Þ

whereMi isthePTEconcentrationinthesedimentandMr istheconcentrationintheEarth’scrust.The1.5isa constantusedtominimizebackgroundvariations

Tobetterrelatetheseindexvaluestobackground concentrationsandtodeterminenaturaloranthropogenic toxicity,theycanbenormalizedtoAl,Li,Fe,orgrainsize usingregressionanalysis.Aluminumisusedwidelybecause itisanessentialcomponentofthealumino-silicatemineral group.Inaddition,itisthemostabundantnaturally occurringPTE,itishighlyrefractory,anditsbackground concentrationstypicallyarenotinfluencedbyanthropogenicinputs(SchroppandWindom,1988),exceptunder unusualcircumstancessuchasbauxitemining(Taylorand others,2007).Whenworkingwithglaciallyderived sediments,Liiscommonlyused(Loring1990).Normalizing toFecanbeusefulbecausebackgroundconcentrations tendtobeuniformandFeprimarilyoccursinthefine sedimentfraction(Acevedo-Figueroaandothers,2006). Theaverageconcentrationofthereferenceelementisbased uponvaluesfromdifferentrocktypesinthewatershedor region.However,shalevaluescanbeconsideredasthe globalreferencestandardforunpollutedsediments(Leopoldandothers,2008).

Normalizingconcentrationstograinsizesseemslogical, butinpracticecanbeproblematic.Severalauthors(e.g.,De Groot,1964;Ackermann,1980;Villaescusa-Celayaand others,2000)recommendednormalizingtospecificgrainsizeranges(i.e., , 63 mm, , 20 mm, , 16 mm,or , 2 mm). However,Green-Ruı´z(2000)arguedagainstthisapproach becauseitreflectsneitherthetexturenorthemassofthe entiresample.Thisapproachtendstounderestimateindex values.

RegressionanalysisbetweenaspecificPTEanda normalizingelementhasbeenusedwidelywithprediction limitsof95% (two-standarddeviations).Thisapproachis highlydependentonthecorrelationcoefficientbetween naturallyoccurringconcentrationsofthePTEofinterest andthenormalizingelement(i.e.,Al)inreference environments(SchroppandWindom,1988).Strongcorrelationsprovidenarrowpredictionlimitswithlowthreshold valuesforconsideringwhetherasampleiscontaminated. Sedimentsareconsidered‘‘PTE-enriched’’ifindexvalues exceedthepredictedlimitsor‘‘natural’’iftheyfallwithin thoselimits(SchroppandWindom,1988).

Anotherstrategytoestimatetoxicityinmarineand estuarineenvironmentsistoconsiderestablishedsedimentqualityguidelines.LongandMorgan(1990)proposed

EffectRangeLowandMedium(ERLandERM)criteria for11PTEsandotherpollutantsbasedonsediment analysesandeffectsonspecificbiota(i.e.,amphipods, shrimp,andpolychaetes).Thesetwobenchmarksestablish threeconcentrationrangesforcertainPTEs.ConcentrationslowerthanERLindicatethateffectsofthatPTE likelywillnotbeobserved.ConcentrationsbetweenERL andERMrepresentarangeinwhicheffectsmightoccur. AtconcentrationsgreaterthanERM,thebiotaforwhich thecriteriawereestablishedarelikelytobeimpacted. Similarly,MacDonald(1994)postulatedThresholdEffects Level(TEL)andProbableEffectsLevel(PEL)basedonthe reviseddatasetusedbyLongandothers(1995).Forboth biologicalandgeochemicalreasons,thesebenchmark valuesmightnotbeapplicabletoeverymarineand estuarineenvironment.Therationaleissimple—thephysicochemicalparametersforwhichthevalueswereestablishedaredifferentineachenvironment,andthebiota couldbeverydissimilarandreactdifferentlytotheeffects ofpollutants.Nevertheless,effects-levelcriteriacanbeused asascreeningtoolinenvironmentalassessment.

BIOAVAILABILITYOF PTES

Theterm‘‘bioavailability’’hasbeenusedextensivelybut withvariabledefinitionsthatreflectthecomplexityofhow PTEsinfluencebiota.LanderandReuther(2004)posed severalmeaningsincluding(p.141)‘‘physico-chemical availabilityofmetalsintheexposuremedium’’and ‘‘toxicologicalbehaviorofmetalsinsidetheorganism body.’’Dependingoncontext,theNationalResearch Council(2003,p.21)definedbioavailabilityas‘‘afraction ofachemicalaccessibletoanorganismforabsorption,the rateatwhichasubstanceisabsorbedintoalivingsystem, orameasureofpotentialtocauseatoxiceffect.’’

Severalresearchteamshaveproposedmethodsto estimatetheconcentrationsofPTEsinsedimentsthatare bioavailable.Tessierandothers(1979),Loring(1981),and LuomaandBryan(1981)developedextractiontechniques todeterminefractionationofmetalsindifferentsediment grainsizes.Theirresultsshowedthatconcentrationsof metalsextractedwith1MHClcloselyresembledthose extractedbythedigestionprocessofcertainorganisms. TurnerandOlsen(2000)questionedtheuseofsuch extractantsandarguedthattheuseof1MHCloverestimatesthebioavailabilityofFebyafactorgreaterthan10. Theyalsonotedthatthereleaseofmetalsfromamorphous andcrystallineFe-oxidephases,usingHCl,isnot analogoustoreleaseunderdigestiveconditions.Nevertheless,intheabsenceofabetterextractant,1MHCliswidely usedasauniversalleachingchemicaltoestimatePTE bioavailabilityinsediments(Tessierandothers,1979; MorseandLuther,1999;NationalResearchCouncil, 2003;Green-Ruı´zandPa´ez-Osuna,2004).

Recognizingdifferencesinorganismsusceptibility,Rainbow(1995)tookadifferentapproachtothestudyof bioavailabilitybylookingfororganismsthatbioaccumulate PTEs.Rainbow(1995,p.183)definedtheterm‘‘metal biomonitor’’as‘‘aspecieswhichaccumulatesheavymetals initstissues,andmaythereforebeanalyzedasameasureof thebioavailabilityofthemetalsintheambienthabitat.’’He

assertedthatthistermislesssusceptibletomisinterpretationthantheothers(e.g.,bioindicator,biologicalmonitor). Ithaslimiteduse,however,becausethemetalbiomonitor musteitherbepresentintheenvironmentinquestionor introducedintotheenvironmentorsedimentfromthe environmentforbioassaypurposes.Furthermore,howa bioaccumulatedelementmightinfluenceotherspeciesinthe environmentrequiresfurtherassessment.

Substantialprogressindefiningandassessingbioavailabilityhasbeenmadeusingmacroorganisms(e.g.,Rainbow andWhite,1989;TurnerandOlson,2000)andexperimentalresearchonforaminifershasbecomeincreasingly common.Anexpandingfieldofresearchisinvestigating howasingle-celledforaminiferincorporatesPTEsintoits shellandhowthiscanbeusedasabioindicator(e.g., BreslerandYanko,2000;SamirandEl-Din,2001;Erez, 2003).Thistopicisdiscussedindetailinthesecondsection ofthisreview.

SUMMARYOF PTEBEHAVIORIN COASTAL ENVIRONMENTS

AssurfacewatersorgroundwatercarryingPTEsmix withmarinewatersinestuaries,naturalchangesoccurthat areinherentlyverycomplex.Assalinityrises,sodoesthe potentialforPTEcomplexationwithionsinseawater.And aspHrises,ratesofadsorptionbyclaymineralsincreaseas thoseclaysmineralsflocculateandsettletotheseafloor. Moreover,naturalfreshwaterrunoffalsodeliversnutrients thatstimulatebiologicalproductivity.Theamountof productivitydeterminesthefluxoforganicmattertothe substratumandthereforetheredoxpotentialofthebottom sediments,whichinfluencesPTEbehavior.Asbiotic activityaerobicallybreaksdownorganiccarbon,pH declinesandlithophilicPTEsmobilize.Whenoxygen becomeslimiting,sulfate-reducingbacteriaactivelyproduce H2S.Iftheenvironmentisonlyintermittentlyanoxic,most oftheH2Swillbeoxidized,furtheracidifyingtheimmediate microenvironment.However,theproductionofCO2 during respirationandthedissolutionofcalciumcarbonatein responsetodecliningpHresultsinhighinterstitial alkalinity,whichcaninduceprecipitationofCaCO3 inthe presenceofexcessCa+2.Inmorecalciumlimitedenvironments,suchasestuarieswithpredominantlysiliciclastic sedimentation,precipitationofsulfidesismorelikely, whichremoveschalcophilicPTEs(ScholzandNeumann, 2007).

Theoxic,hypoxic,andanoxiczonesofmicrobialactivity withinthesediments,andtheirinfluencesonPTEremoval ormobilization,areremarkablycomplexeveninpristine systems.Interestingly,PTEstendtobemostmobileinthe hypoxiczone.Theclaysandferricoxidesinoxygenated sedimentsimmobilizelithophilicPTEs.Inanoxicenvironments,thereducedPTEsaresequesteredinsulfides.Any processesthatincreasebothorganicmatterandmixing havethepotentialtoincreasebioavailabilityofPTEs.Thus, sediment-dwellingorganismsplaymultipleroles,including respiringCO 2 ,mixingoxygenatedseawaterintothe sediments,andingestingsediments;allprocessesthat potentiallymobilizePTEs.

Anthropogenicactivitiestendtoincreasevariabilityand complexityofPTEbehaviorinamultitudeofways.

Anthropogenicactivitiesthatchangethenaturalflowof freshwateraccentuatetheseasonalhighsandlowsofinflux toestuaries,resultinginwidersalinityfluctuations. Nutrientandorganicinputareinproportiontothehuman populationofthewatershed(e.g.,Walsh,1984;Vitousek andothers,1997).Asaresult,biologicalproductivity increasesinthesurfacewatersandmoreorganicmatter settlesonthesubstrate,increasingthepotentialforboth hypoxicandanoxiczoneswithinanestuary.Storms, boatingactivities,dredging,andanyothernaturalor anthropogenicactivitiesthatdisturbsedimentscanmix mobilePTEsfromhypoxicporewatersintothewater column,wherethePTEshavegreaterpotentialtoimpact thelocalbiota.Moreover,ifthoseactivitiesmixanoxic sedimentsintooxygenatedenvironments,sulfidesinthose sedimentscanoxidizeandtherebyincreasethebioavailabilityofPTEs.

Thus,monitoringandassessmentoftheriskstocoastal ecosystemsassociatedwithPTEcontaminationarehighly complexproblemsthatrequireinterdisciplinaryteamswith expertiseinbiologyandecologyofestuarineandneritic biotas,biochemistry,geochemistry,andhydrodynamic modeling,WiththiscontextualbackgroundonPTE behavior,toxicity,andmeasurement,wenowexaminethe potentialforusingforaminifersinmonitoringandrisk assessmentofcoastalecosystems.

FORAMINIFERA

ThecharacteristictraitsoftheClassForaminifera enhancetheirpotentialforexposuretoPTEs(i.e.,the extensivesurfaceareaoftheirthread-likegranuloreticulopodia)andtheirpotentialtoprovidearecordofsuch exposure(i.e.,theirprotectivetestorshell).Foraminifers arefoundinvirtuallyallmarineecosystemsthatsupport eukaryoticlife(e.g.,Goldstein,1999).Moreover,afew speciesareamazinglyresilienttoenvironmentalstressesand areoftenamongthelastorganismsfoundlivinginpolluted sites(Schafer,2000).

Awiderangeofenvironmentalfactorsinfluencewhere foraminiferscanliveandwheretheirshellscanaccumu-

late.Theseinfluencesincludetemperature,pH,water motion,salinity,dissolvedoxygen(DO),turbidity,light intensity,foodsupply,substratumtypeincludingsediment texture,bioticinteractions,andtaphonomicprocesses. However,asubstantialnumberofstudieshaveshownthat, givensuitablephysicalandchemicalparameters,food supplyisthepredominantfactorcontrollingbenthic foraminiferalpopulationsandassemblages(Jorissen 1999;Murray2001).

Foodsupply,bothqualityandquantity,notonly providestheenergyandrawmaterialsforpopulation growth,butalsoinfluenceschemicalparameterssuchas DO,pH,alkalinity,andsulfidecontentatthesedimentwaterinterfaceandwithinthesedimentsandporewaters. Jorissen(1999)developedaconceptualmodelofthe distributionofbenthicforaminifersinoligotrophic,mesotrophic,andeutrophicsettingsbasedonfoodsupplyatthe sediment-waterinterface(Fig.4).Heindicatedthat(1)in eutrophicsettings,excessfoodreducesDO,resultingin anoxicandsulfidicporewatersthatlimitinterstitialhabitat depthandmodulateabundance;(2)inoligotrophicsettings, abundanceanddepthdistributionsarelimitedbyfood supply,and(3)inmesotrophicenvironments,organic matterpenetratesdeeperintosedimentsthaninoligotrophicsettings,andoxygenextendsdeeperthanineutrophic settings.Thisexpandsthegradientsinfoodsupply,pHand Ehthatsupportdiverseandabundantepifaunaand infauna,bothshallowanddeep.

Moreover,ifPTEsarepresentinthesediments, mesotrophicconditionscanincreasetheirbioavailability whilealsoincreasingpotentialforexposurebyincreasing theinterstitialdepthrangeinwhichtheforaminiferscan live.Asnotedpreviously,changesinpHandEhinfluence themobilityandbioavailabilityofPTEswithinthe sediments.Forexample,experimentsbyRiedelandothers (1997)determinedthatthedirectionandmagnitudeofMn, As,andCufluxesfromsedimentsarelargelygovernedby oxic–anoxicgradientsinestuarineenvironments.Thus, infaunaltaxainmesotrophictoeutrophicenvironmentsare mostlikelytoencounterbioavailablePTEs.

FORAMINIFERSIN ENVIRONMENTAL ASSESSMENT

Foraminifershavebeenusedinpollutionstudiesin coastalenvironmentsforthepast50years.Therangeof anthropogenicpollutionsourcesexaminedincludesewage outfalls,organicwastes,thermaleffluent,pesticides,oil, agriculture,aquaculture,acidminedrainage,andharbor activities(e.g.,Resig,1960;Seiglie,1968;Yankoandothers, 1994;Alve,1995;SenGuptaandothers,1996;Alveand Olsgard,1999;Yankoandothers,1999;Elberlingand others,2003;Haywardandothers,2004;Tsujimotoand others,2006).Benthicforaminifershaveprovenusefulin assessmentandmonitoringofcoastalandshelfenvironmentsbecauseoftheirtaxonomicdiversity,widedistribution,abundance,relativelysmallsizeandshortreproductivecycles,andbecausetheirshellsareoftenwellpreserved insediments(Yankoandothers,1999).Foraminiferal specieshavespecificecologicalnichesandpopulations reactquicklytoenvironmentalchanges(Hallockand others,2003;Wardandothers,2003).Spatialandtemporal distributionsofbenthicforaminifersvaryinresponseto bothbioticandabioticenvironmentalparameters(Schafer, 2000;ArmynotduChateletandothers,2004).

Nevertheless,numerousresearchershaverecommended cautionwhenusingbenthicforaminifersasproxiesfor environmentalcontaminationbecausethelivingprotistsare respondingtomultipleenvironmentalparameters.Moreover,taphonomicprocessesaltertherecordoftheliving assemblagethatispreservedwithinthesediments.Asnoted intheprevioussection,coastalenvironments,especially estuaries,arecomplexsystemswithmanyparameters simultaneouslyvarying,evenintheabsenceofpollution (Debenayandothers,2000;Murray,2001).Thus,interpretingresponsesofforaminiferstoPTEpollutioninestuaries canbeverychallenging(Alve,1995;Geslinandothers,2000; LeCadreandothers,2003;LeCadreandDebenay,2006). Thesechallengesdonotprecludetheuseofforaminifersin estuaries,butrathernecessitatecarefulformulationof questionsandrecognitionofthepossibilityforinteractions andco-varyingparameters.Moreover,justbecausean organismcanreactinaparticularwaytonaturalvariation doesnotinvalidatethatresponseasanindicatorof anthropogenicstress.Rather,understandinghowapopulationorassemblagerespondsnaturallytoenvironmental stressesprovidesinsightintounderstandingresponseswhen anthropogenicstressesareadded.Thisiswhysampling designshouldincludegradientsandreferencesites,which allowsfornaturalresponsestobecharacterizedand distinguishedfromresponsestoanthropogenicpollutants.

Oneoftheimportantchallengesininterpretingresponses offoraminifersorotherorganismstoPTEpollutionin estuariesishowtomeasurePTEconcentrations.Amajor goalofthefirstsectionwastopresenttherangeof considerations,includingwhethertomeasureporewaters, bulksediments,ormudfractions,andhowtodetermine bioavailability,thresholdsforresponse,andenrichment factors.

ForaminiferalAssemblages

Moststudiesthataddresstheeffectsofpollutionusing foraminifersdosobyexaminingassemblagesofforamin-

ifersinsediments.Presenceorabsenceofkeytaxa,aswell astheirabundanceanddistribution,oftencanbestatisticallylinkedtocontaminantsources.Responsesofforaminiferalassemblagestocontaminantgradientshavebeen describedbymanyauthors(e.g.,Schafer,2000;Samirand El-Din,2001;ArmynotduChateletandothers,2004; Tsujimotoandothers,2006).Suchresponsescanbe representedbydrasticassemblagechanges(Elberlingand others,2003),stepwisefaunalchanges(Haywardand others,2004,2006),orfluctuationsinfaunalassemblages andspeciesabundance(Alve,2000).Characterizationof backgroundconditions,especiallyfoodsupply,isessential tointerpretingresponsestocontaminants.Likewise, thresholdsforinfluence,thoughpoorlyknown,are importanttoconsider.

Ingeneral,anincreaseinpollutantsfirstleadsto decliningspeciesdiversityasthemoresensitivespeciesdie offandmoretoleranttaxaareabletoexploitfoodand spaceresources.Forexample,Yankoandothers(1999) notedthatPTEconcentrationsinsedimentstendedtobe negativelycorrelatedwithforaminiferalabundanceand diversity,andpositivelycorrelatedwithincidencesof deformedtests.Ultimately,evenstress-toleranttaxawill declineindensitiesasthestressintensifies.

However,whenpollutantsincludenutrientsororganic matter(e.g.,fromsewageoragriculturalrunoff),thereis moretypicallyanenrichmenthaloatthemarginof influence.Alve(1995)developedamodeldescribingthe transitionfromoligotrophictoeutrophicconditionsfor foraminiferalassemblagesbasedonorganicpollution. Undernaturalconditions,foodisoftenlimiting,sosome increaseinfoodsupplyactuallyincreasesbothdiversityand abundance.Jorrisen’s(1999)modelexplainshowtheinitial increaseinfoodsupplyincreasesabundanceanddiversity byincreasingnotonlyfoodsupplybutinhabitablerangeof depthwithinthesubstrate.Resig(1960),Seiglie(1971), Mohtahidandothers(2008),andmanyothersreported decreasingspeciesdiversitywithdistancefromsewage outfallsanddisposalsites,suggestingthattrophicresources limitdiversityinnaturalenvironments.However,asapoint sourceisapproached,foodsupplyincreasesandoxygen concentrationsbecomemorevariable,andopportunistic speciesbloomwhilediversitytypicallydeclines(Alve,1995). Ultimately,eventhemoststress-toleranttaxawilldeclinein abundancewhenoxygenbecomeslimiting.Alve’s(1995) modelpredictsafaunalturnover,witha‘‘deadzone’’ devoidofeukaryotesincloseproximitytoapointsource thatischaracterizedbyanoxicporewatersatthesediment surface(MurrayandAlve,2002).

Recognizingthecriticalrolethatfoodsupplyplaysin controllingforaminiferalassemblagesprovidesinsightinto studiesthatreportlittleornodiscernibleresponseto inorganicpollutionsources.Moreover,salinityandpH changesoftenfurthercomplicateinterpretations.For example,intheirmulti-proxystudyonintertidalestuaries inNewZealand,Haywardandothers(2004,2006)found thatstepwiseshiftsindominancefromcalcareousto agglutinatedformswereassociatedwiththearrivaland establishmentofhumans.Differencesinfreshwaterinput andpHwerepredominantfactors.Theyfoundatwo-fold differenceinPTEconcentrations(mudfraction)between

studiedestuarieshadlittletonoeffectontheforaminiferal faunas,whichmightindicatethatbioavailabilitieswere belowthresholdsforresponse.

Intensebiologicalactivityresultingfromsewagepollutioncanleadtohypoxia,whichwilllowerforaminiferal diversityandabundancenearthesource.Othernatural chemicalstressors,suchashypo-andhypersalinity,andpH extremes,alsocanbeamplifiedbyhumanactivities.Thus, ifconcentrationsofPTEsorothercontaminantsare minimallybioavailableorarebeloworneartheirthresholds ofinfluence,assemblageresponsestonaturalenvironmentalvariability,ortoanthropogenicallyrelatedchangesin foodsupplyorfreshwaterinflux,willdominate.Evenin extremecases,suchastheresponsetosulfidicminetailings reportedbyElberlingandothers(2003),determiningthe lethalmechanismcanbeevasive.Inthatstudy,itwasnot readilyapparentwhethertheazoiczonewastheconsequenceofPTEtoxicityorlowpH.

Becausethecombinationoffreshwaterinput,organic matter,andintermittenthypoxiacomplicatestressresponsesandinfluencebioavailabilityofPTEs,itisnotsurprising thatseveralofthestudiesthatmoststronglyimplicatePTEs inimpactingforaminiferalassemblageswereinwarmer coastalwaterswithnear-normalmarinetoslightlyhypersalineconditions.Examplesofsuchstudiesincludethoseby Seiglie(1971)inPuertoRico,Yankoandothers(1999)and SamirandElDin(2001)intheeasternMediterranean,and Carnahanandothers(2008)insouthernFlorida.

Foraminiferalassemblagesclearlyrespondtoenvironmentalchanges,thoughtodatequantitativerelationships arenotwellestablished.Therefore,interpretingthecauses ofthosechangescanbechallengingforresearchersand resourcemanagers.Anthropogenicpollutiontypically involvesmorethanonepotentialstressor.Nevertheless, manystudieshaveestablishedthatmostforaminiferaltaxa tendtobesensitivetoenvironmentalstressandonlyafew taxatendtoberelativelystress-tolerant.Thus,changesin assemblagescanbeusefullow-costbioindicatorsof environmentalchange,eventhoughchemicalanalysesare requiredtodetermineexactlywhatstressorsarepresent.

ControversiesinAssemblageAssessments

Whendesigningastudytoassessforaminiferalassemblages,whatshouldbesampled?Therehavebeenstudies focusedonlyonlivingpopulationsorassemblages(e.g., Hallockandothers,1995;Buzasandothers,2002),studies thatdistinguish,tabulate,andcomparedatabetweenlive specimensanddeadshells(e.g.,Alve,2000),andstudies thatreportonlytotalassemblageswithoutdifferentiating theirliveanddeadcomponents(e.g.,Carnahanandothers, 2008).InthepaperbyAlve(2000),shereportedthatliving foraminiferswerenotabundantinhersamplesandthat deadassemblagestypicallyexhibitedmuchhigherdiversities.Thiskindofsituationforcestheissueofwhethertotal assemblagesprovideusefulinformation.

Unfortunately,theissueofwhattosampleisnotasimple one.Distinguishinglivespecimensisnottrivial(Bernhard, 2000).Avarietyofstainshavebeenutilized,especiallyrose Bengal(Walton,1952)andSudanblackB(Walkerand others,1974).Hallockandcolleagues(Hallockandothers,

1986;Williamsandothers1997;Hallockandothers,2006) utilizedsymbiontcolorandreticulopodialactivityinstudies oflargerforaminifersthathostalgalsymbionts.Bernhard (2000)discussedtheseandavarietyofotherapproaches suchasadenosinetriphosphate(ATP)assaysandultrastructuralstudies,recommendingthatacombinationof techniquesbeapplied,dependingupontheresearchgoals. Examininglivesamplesandverifyingreticulopodialactivity iscertainlythemostreliablemethodofrecognizinglive specimensbut,likeATPassaysandultrastructuralmethods,itisgenerallytoolabor-intensivewhenassessinglarge numbersofsamples.Fortheassemblageanalysesneeded forenvironmentalmonitoring,preservationwithsubsequentstainingisthemostpracticalmethodforhandling largenumbersofsamples.MurrayandBowser(2000) reevaluatedtheroseBengalstainingtechniqueandfoundit toberelativelyreliablewhenproperlyapplied.

Asnotedabove,theliveassemblageatanypointintime doesnotfullyrepresentwhatcanliveintheenvironment. Buzasandothers(2002)monitoredliveforaminiferal assemblagesinrelativelyclosespatialgridsmonthlyover afive-yearperiod,whichrevealedthateachliveassemblage collectedsimplyreflectedalocalpatchintimeandspace, andtheyconcludedthattheaccumulatedassemblagedata bestcharacterizedthefauna.Theproblemoflossof informationinthelive-onlysamplingscenariobecomes evenmoreacutewhendealingwithenvironmentswith phytalsubstratesinhabitedbyasignificantproportionof thelivingassemblage.Deadassemblagesinsurface sedimentsaremostcomparabletoassemblagesincore samples.Whereasaliveassemblagerepresentsanexactspot atapointintime,adeadassemblagethathasnothad substantialandselectivetaphonomiclossbetterreflectsthe widercommunityoftheareaandprovidesatemporal perspective.

Themostcontroversialapproachisthatwhichanalyzes totalassemblages.Countingallintactshellsisbyfarthe easiestandmoststraightforwardassessment,requiring minimalsamplepreparationandtechnicaljudgment. However,taphonomiclosscanbesubstantialinhyposaline, higherlatitude,anddeep-seaenvironmentswheredissolutionoftenprecludespreservationofcalcareoustests (MurrayandAlve,1999a-b;Pattersonandothers,1999; MurrayandPudsey,2004).Theseauthorshaverecommendedagainstreportingtotalassemblages.Ontheother hand,therearemanyenvironmentswheretheliveandtotal assemblagesaresimilarinspeciescompositionandpercentages.Forexample,intropicalestuariesandcoastal regionswheresalinityisnearnormalandthemajorityof taxaliveonphytalorhardsubstrates,liveindividualsinthe sedimentsaretypicallylessthan10 % ofthetotal assemblage(Cockeyandothers,1996;Peeblesandothers, 1997).Moreover,thespeciesrepresentedinliveassemblages areasubsetofthoseinthetotalassemblage.Therefore, justificationforthemuchgreatertimeandthereforecostof live-deadassessmentsversusanalysesoftotalassemblages dependsuponthegoalsoftheparticularstudy.However, whenworkinginareaswhereselectivetaphonomiclosscan beanticipatedorwheretheenvironmentalchangesof concernarelikelytohaveoccurredwithinthepastseveral years,analysesbasedontotalassemblagesshouldbe

avoided.Inpreviouslyunstudiedareas,pilotstudiesshould utilizeavarietyofapproachesasrecommendedby Bernhard(2000)todeterminethemostusefulmethodsof distinguishinglivefromdeadspecimens,whetherlive specimenscomposeasignificantproportionofthetotal assemblage,andtheamountoftaphonomicloss.

Thus,thegoals,environment,andavailableresources dictateiftotalassemblagedatacanprovideameaningful interpretation.AsstatedbyEngle(2000,p.3-1),‘‘Anideal indicatoroftheresponseofbenthicorganismstoperturbationsintheenvironmentwouldnotonlyquantifytheir presentconditioninecosystemsbutwouldalsointegrate theeffectsofanthropogenicandnaturalstressorsonthe organismsovertime.’’Hallockandothers(2003)argued thatthiskindofinformationispreciselywhatthetotal assemblageinasedimentsamplecanprovide.

Thesubjectofavailableresourcespresentsasecond controversialissueofwhetherallspeciesinanassemblage needtobeidentified,whichisoftenadifficultandtimeconsumingtask,orifrecognitionofindicatorspeciesor morphogroupsprovidesufficientenvironmentaldata. Environmentalagenciesandresourcemanagersprefer numericalindicesthatcanbeusedwithothermetricsin environmentalassessments(e.g.,Jacksonandothers,2000). Forexample,Hallockandothers(2003)derivedaformula basedonproportionsoflargerforaminifers,stress-tolerant taxa,andothersmallerbenthictaxainsedimentsamplesas anindicatorofwhethernearshoretropicalwaterscould supportreef-buildingcoralcommunities.SenGuptaand others(1996)founddistinctiveratiosof Ammonia and Elphidium relatedtooxygenlevelsandeutrophication,and proposedahypoxiaindexbasedonthesetaxa.Numerous studiesinmid-tolow-latitudesrevealthatspeciesof

Ammonia arecommonlythemoststress-toleranttaxa (Buzas-StephensandBuzas,2005;Berginandothers, 2006;Buroneandothers,2006;Unluandothers2006; FrontaliniandCoccioni,2008),commonlyexhibitingshell abnormalitiesordissolutionunderextremeconditions. Carnahanandothers(2009)usedboththeFORAMIndex andamodified Ammonia-Elphidium Indexontotalforaminiferalassemblages,findingthattheircombineduse,along withabundanceanddiversity(numberofgenera),provided excellentcharacterizationofzonesofenvironmentalinfluencewithinBiscayneBay.

MorphologicalAbnormalities

Acharacteristicthatmakesforaminifersparticularly usefulasenvironmentalindicatorsistheirtendencyto developmalformedtestsinstressedenvironments(Table4). Doubleapertures,enlargedapertures,twistedchambers, weakenedshellstructure,andstuntedortwinnedshellsare themostcommonmorphologicanomaliesreported(Pl.1; Seiglie,1971,1975;Banerji,1990;Alve,1995;Tolerand Hallock,1998;Stouffandothers,1999;Yankoandothers, 1999;SamirandEl-Din,2001;PolovodovaandSchonfeld, 2008).Abnormaltestgrowthcanbeinducedbydeviations fromthepreferredphysicochemicalenvironment,which includestemperature,salinity,foodsupply,DO,andpH (Boltovskoyandothers,1991;Yankoandothers,1998). Understressedconditions,asexualreproductionmightbe favoredoversexualreproduction(Hallockandothers, 1995),whichcaninfluencethefrequencyofdeformities becauseschizontsmightsurvivegeneticdamagethatcould precludesuccessfulsexualreproduction.Hallockandothers (1995)andTolerandHallock(1998)foundtheincidenceof shelldeformitiesinasexualbroodsof Amphisteginagibbosa

TABLE 4.Commonforaminiferalshelldeformitiesdescribedinseveralstudiesinnaturalandpollutedenvironments.

Deformity

Author(s)

TwistedChamberAlve,1991;Yankoandothers,1999;Samir,2000

AberrantShell/ChambersSeiglie,1975;Alve,1991;Hallockandothers,1995;Yankoandothers,1998;Yankoandothers,1999; Coccioni,2000;Geslinandothers,2000;Samir,2000;Debenayandothers, 2001;SamirandEl-Din, 2001;LeCadreandDebenay,2006;CrevisonandHallock,2007;Romanoandothers,2007; FrontaliniandCoccioni,2008

Non-developedShellYankoandothers,1999;Hallockandothers,1995;Coccioni,2000;Romanoandothers,2007; FrontaliniandCoccioni,2008

TwinningSellierdeCivrieux,1970;Alve,1991;Hallockandothers,1995; Yankoandothers,1998;Stouffand others,1999;Yankoandothers,1999;Coccioni,2000;Geslinandothers,2000;Debenayandothers, 2001;Cearretaandothers,2002;Mericandothers,2004;Ernstandothers, 2006;Frontaliniand Coccioni,2008

TripleShellGeslinandothers,2000;Debenayandothers,2001;Mericandothers,2004 WrongCoilingDirectionYankoandothers,1998;Yankoandothers,1999;Coccioni,2000;SamirandEl-Din,2001;Ernstand others,2006;LeCadreandDebenay,2006;FrontaliniandCoccioni,2008 DoubleApertureSellierdeCivrieux,1970;Alve,1991;Hallockandothers,1995;Yankoandothers,1999;Samir,2000; SamirandEl-Din,2001 MultipleAperturesYankoandothers,1998 AdditionalChambersYankoandothers,1998;Yankoandothers,1999;Coccioni,2000;FrontaliniandCoccioni,2008 ReducedChamber/ShellSizeSeiglie,1975;Banerji,1990;Alve,1991;JayarajuandReddi,1996;Coccioni,2000;Geslinandothers, 2000;Samir,2000;Debenayandothers,2001;FrontaliniandCoccioni,2008 ProtuberancesonOneorMoreChambersAlve,1991;Yankoandothers,1998;Coccioni,2000;Geslinandothers,2000;Samir,2000;Debenay andothers,2001;SamirandEl-Din,2001;FrontaliniandCoccioni,2008 EnlargedAperturesAlve,1991;JayarajuandReddi,1996 LackofShellSculptureSeiglie,1975;Yankoandothers,1998 ProtrudingProloculus/SpiroconvexSeiglie,1975;SamirandEl-Din,2001 EncrustingOffspringHallock,2000 BrittleShells/ExcessiveBreakageHallockandothers,1995;TolerandHallock,1998;Hallock,2000;Tolerandothers,2001

increasedwithstressthatcauseddeathanddigestionof algalsymbiontsinthebroodparent.Characterizing abnormalitiescanbecomplicatedbythedifficultyin differentiatingbetweennormalandabnormalforms(Geslin andothers,2000),andthepotentialforasingletestto exhibitmultipletypesofabnormalities(Yankoandothers, 1998;CrevisonandHallock,2007).Becausemanyepiphytic species,suchas Rosalinaglobularis and Planorbulina mediterranensis,developaberrantteststhatconformwith theirsubstrateofattachment,theirshellmorphologies shouldnotbeusedasindicatorsofstress(Langer,1993; Geslinandothers,2000).

Malformedforaminifersarecommonlyfoundineuryhalineenvironments.Abnormaltestsalsohavebeenproduced incontrolledhypersalinecultures(Stouffandothers,1999) andinnaturalhypersaline(52–65%)conditions(Debenay andothers,2001).Hypersalinitycaninhibitorreduce reticulopodialactivityofyoungindividualsfollowing asexualreproduction,whichcanresultintheirfusionand subsequentgrowthastwinnedtests(Stouffandothers, 1999;MurrayandAlve,2002).Sincethereticulopodia coalescetoproduceatemplateoverwhichtheinnerorganic liningofthenewchamberforms(Angell,1967a,1967b), anymechanismthatinhibitsreticulopodialactivitycould resultinanomalouslyarrangedchambers.LeCadreand others(2003)observedthisphenomenonin Ammonia beccarii transferredfromapH8.0toapH7.0culture medium.ThehypersalineandlowerpHculturesthathad reducedreticulopodialactivitydidnotproducethesame kindsoftestabnormalities.

Texturalabnormalitiesalsooccurinthewallstructureof foraminifers,buttheyarenotnecessarilylinkedtoa reductioninreticulopodialactivity.Potentiallytoxic elementswithionicradiisimilartothoseofCaandMg (i.e.,Cu,Zn)candisruptthecrystallizationprocessduring chamberformation(Debenayandothers,2000).Reportsof highproportionsofCuandZnindeformedtests(Sharifi andothers,1991)andpreferentialabsorptionofsomePTEs (Cu . Zn . Cr . Pb;SamirandEl-Din,2001)supportthe possibilitythatsomeanomaliesingrossmorphologycan resultfromdisruptedcrystalstructure.

Shellabnormalitiesmightalsobeinducedbymetabolic perturbationsresultingfromavarietyofstressesincluding insufficientfood,hypoxia,ortoxicpollutants.Debenayand others(2001)describedanomaliesintheorganicmatrix causedbymetabolicperturbationsthatresultedincavities inchamberwalls.LeCadreandDebenay(2006)determined experimentallythatincreasingconcentrationofCu + 2 inhibitedgrowthof Ammonia spp.,butsurprisinglyresulted inlowpercentagesofdeformitiesamongthestuntedshells.

Anomalouslysmallshells,eithersubadultstages(few chambers)orsmalladults(withsmallchambers),oftenare commoninenvironmentsmarginallyunsuitablefora species.Boltovskoyandothers(1991)concludedthatlow metabolicratesinresponsetolowDOcanstunttests. Anotherhypothesistoexplainthepredominanceofsmall individualsinmarginalconditionsisbasedonobservations byAlveandGoldstein(2003),whosuggestedthat propagulesmightbeimportantinthedispersalofbenthic foraminifersbecausetheycanbetransportedlongdistances beforetheysettleoutofsuspension.Thepropagulesremain

dormantuntilenvironmentalconditionsarefavorable.If, withinashorttimerelativetotheforaminiferalspecieslife span,conditionsagainbecomeunsuitable,thejuvenileswill die,andthiswillbereflectedinthesampledsedimentbya predominanceofsmalldeadshells.

Whetherspecificshellabnormalitiescanindicatespecific stressorsisstilllargelyunresolved.Mostforaminiferal studiesfrompollutedenvironmentshavefocusedonlinking malformedteststoPTEs,withoutdistinguishingtherole playedbythenaturalstressorsinthosesameenvironments thatalsomightalsocaninduceabnormalities(e.g.,Seiglie, 1975;SamirandEl-Din,2001;FrontaliniandCoccioni, 2008).ThecomplexityofnaturalparameterscouldoverprintormagnifytheeffectsofPTEs.Withoutadditional experimentalworkassessingtheeffectsofnaturalparameters(pH,temperature,hypoxia,andsalinity)andPTEson foraminiferalshellconstruction,itwillcontinuetobevery challengingtodeterminewhetheradeformityiscausedby naturaloranthropogenicstressors.

EXPERIMENTAL APPROACHES

Thefullpotentialofforaminifersastoolsinenvironmentalmonitoringandriskassessmentrequiresthetesting ofhypothesesformulatedfromfieldandlaboratory observations.Furthermore,thequalitiesthatmakeforaminifersexceptionalmonitoringtoolsalsoareadvantageousinexperimentalresearch.Comparedtomostinvertebratesandvertebrates,collectingandmaintaininglarge populationsofshallow-waterforaminifersrequireless effort,littlespace,andfewerresources.Becauseforaminiferscanbestudiedasindividuals,populations,or assemblages,experimentalapproachesandstatisticaldesign canbesimilartothoseusedformacroorganisms.Responses canbeenumeratedbyanalyzingaspectsoftheirshellssuch astheirgrowthrates,morphologicfeatures,wallultrastructure,andisotopicchemistry.AdvancesinDNA sequencing,whichallowanalysisofminuteamountsof DNA,willultimatelyenhanceutilizationoftheseprotistsin environmentalstudies.However,othertechnologicalchallengesfaceforaminiferalresearchers.Ofthethousandsof livingspeciesofforaminifers,fewerthan20havebeen maintainedandobservedtoreproduceinaxenicculture experiments(Flakowskiandothers,2005),whichreflects boththechallengesinvolvedintheircultureandthesmall numberoflaboratorieswherethebiologyofforaminifersis beinginvestigated.

Severalapproachescanbeutilizedtotestthehypothesis thatparticularstressorsinfluencetestmorphology.Laboratoryobservation(Ro¨ttgerandHallock,1982;Hallock andothers,1995),ultrastructurestudy(Angell,1967a, 1967b;Debenayandothers,2001),andexperiments(Stouff andothers,1999;LeCadreandDebenay,2006)are essentialtounderstandinganddescribingbothwhatis normalmorphologicvariationandwhatareabnormalities. Hypotheseshavebeenposedbypreviousstudies,for examplethatshellanomaliescanbeproducedbyfactors influencingreticulopodialactivity,organicmatrixproductionandintegrity,orcrystalformation.Experimental testingofthesehypotheses,followedbydetailedultrastructuralstudiesusingtransmissionandscanningelectron

microscopycouldelucidateprocessesproducingatleast someoftheabnormalities.Althoughthestudyofmechanismsbywhichforaminifersprotectthemselvesfrom xenobioticssuchasPTEswaspioneeredbyBreslerand Yanko(1995a,1995b),muchmoreworkisneededtobetter understandhowshellabnormalitiescanresultfrom hypersensitivitytoPTEs.

Avarietyofcytologicaltoolscanbeappliedto understandandanalyzetheresponsesofforaminifersto environmentalchanges.BreslerandYanko(1995a,1995b) werethefirsttoapplycytophysiologicalandcytochemical techniquestodetermineeffectsofPTEs(Cd,Cu,Br,I,and Hg)onepiphyticforaminifers(Pararotaliaspinigera and Rosalinamacropora ).Theyidentifiedseveraldefense mechanismsthatforaminifershaveagainstxenobiotics (seeBreslerandYanko,2000forfurtherexplanation):(1) amucopolysacharidecoatthatservesasadiffusionbarrier thatbindssomecationicpollutants;(2)aplasmamembrane thatformsanimpermeablediffusionbarrieragainstnatural andanthropogenicanionicpollutants;(3)amembranemediatedtransportsystemthatmobilizesanionicpollutants outofthecytoplasm;(4)activeintralysosomalaccumulationthatisolatessomecationicpollutants;(5)peroxidases thatprotectcytoplasmagainstexcessoxygenandperoxides; (6)haloperoxidasesthatprotectcytoplasmagainstBr and I penetrationandtransformthemintohaloderivatives; and(7)metallothioneine-likeCu+2 bindingproteinsthat protectagainstotherPTEs.BreslerandYanko(1995a, 1995b)alsoobservedadecreaseinesterases(hydrolase enzyme)andlysosomalreductionorabsenceinthe cytoplasmassymptomsofPTEcontamination.

Utilizingbothcytologicalandultrastructuralapproaches canfurtherelucidateresponsestoPTEcontamination.Le CadreandDebenay(2006)demonstratedthatCu-contaminatedforaminifershadthickorganiclinings,abnormal lipidvesicles,andanincreasednumberofresidualbodies; thelattertwothoughttobemechanismsofdetoxification. MicroprobeanalysisshowednohighCu+2 concentrationsin thecellularstructuresofforaminiferswithabnormaltests. Theseresearchersspeculatedthatthethickeningofthe organicliningatthebaseoftheporesmightprovide protectionagainstdiffusionofheavymetalsintotheshell. Inaddition,thepossiblepresenceofmetallothionein proteins,whichbindCd,Cu,Zn,Ag,andHg,ledLe CadreandDebenay(2006)topostulatetheirfunctionasa detoxificationmechanism.BreslerandYanko(1995a, 1995b)notethatCu+2 couldbeeliminatedbymetallothioneinproteins,oxidized,convertedintosulfidederivatives, orincorporatedintotheshellsofforaminifers.

Giventhesmallsizeofmostforaminifersandthe potentialforworkingwithindividuals,laboratorymicrocosmapproacheshavetremendouspotentialforhypothesis testing.Microcosmdesignshavealreadybeendevelopedfor experimentallytestingpaleoceanographicproxies(Chandlerandothers,1996;Wilson-Finelliandothers,1998; Hintzandothers,2004;Nooijerandothers,2007). Microcosmbioassayapproachesareincreasinglybeing utilizedtoassessresponsesofsedimentmeiofaunato contaminants(Austinandothers,1994;Lauthandothers, 1996;Lawler,1998;Millwardandothers,2001).Leeand Correa(2005,2007)andLeeandothers(2006)used

microcosmstocorroboratethedetrimentaleffectsofcopper inminetailingsonlittoralmeiofauna,includingforaminifers.

Experimentalresearchstudyingtheeffectsofstressorsis undoubtedlychallenging.WhenworkingwithPTEs,itis essentialtousechemicallyphobicmaterials(e.g.,Teflon)to buildculturedevices,therebyminimizingtheadsorptionof PTEsandotherpollutantsintotheexperimentaldevice. WhenexaminingeffectsofPTEsinmicrocosmsorcultures, experimentalconcentrationsshouldberelatedtofield conditions.Forexample,thebioavailableconcentrations ofPTEsobtainedfrommudfractionsorwater-porewater samplescanbeusedtoselecttestconcentrationsfor experiments,ascantheorganic(oxidizable)fractionin sediments,asdeterminedfromthesequentialextraction procedures.Bothapproachesprovidearationalefor determiningtestconcentrationsandconstrainingthe experimenttotherangeofconcentrationsfound‘‘naturally’’inthespecificenvironment.

Mesocosmexperimentsareequallypromising.For example,Gustafssonandothers(2000)determinedchanges inforaminiferalcommunitystructureswhensedimentswere treatedwithTri-nButyltin(TBT),whichisfoundin antifoulingpaintsusedoncommercialandrecreational boats.Comparedtothecontrolwith0.00nmolofTBT, foraminiferalabundanceincreasedwhentreatedwith0.02 nmolanddecreasedconsiderablywhenexposedtoconcentrationsof2.00nmol.Theincreaseunderthelow concentrationwasinterpretedtoberelatedtoreduction ofpredatorsandcompetitors(i.e.,nematodes,ostracodes, andmolluscs)thatweremoresensitivetothoseconcentrationsthanweretheforaminifers.

Field-basedexperimentsusingPTE’sandnaturalorganic-richsedimentshavedemonstratedthecomplexityof workinginthenaturalenvironment.AlveandOlsgard (1999)reportednegativeeffectsonreproductionand communitystructure,yettheydidnotfindmalformedtests insedimentsinoculatedwithCu+2 concentrationsexceeding 900ppm.Incontrast,culturesexposed20daysto10ppm ofCu+2 producedmalformed Ammonia spp.(LeCadreand Debenay,2006).AconfoundingfactorintheAlveand Olsgard(1999)fieldexperimentwaslikelyorganic-matterin thesediment,whichcanreducePTEtoxicitybycomplexation(LanderandReuther,2004).

Experimentalworkonheavymetalincorporationinto foraminifersisinitsinfancy.Notonlyarephysicochemical parametersimportant,butfeedingstrategiesmustbe considered.Yankoandothers(1998)postulatedthatPTEs areincorporatedintoforaminiferalcytoplasmduring feedingorbydiffusionfromsurroundingwaters.They furthersuggestthatPTEsthenaffectthecytoskeleton, whichinturninfluencesshellmorphology.Asnoted previously,Debenayandothers(2000)postulateduptake ofcopperduringshellcrystallizationasamechanismfor disruptingtestgrowth.Allofthesehypothesescanbetested experimentally.

PTEUPTAKE PATHWAYS

Acommonassumptionisthatforaminiferscanassimilate PTEsbyingestingcontaminateddetritusoralgae.Indeed, trophictransferofPTEsisacommonphenomenonin

FIGURE 5.Simplifiedmodelofmetalintakeintoanorganismthroughthecellmembraneappliedtoaforaminifer(Compiledandmodifiedfrom NationalResearchCouncil,2003,andLanderandReuther,2004).ML—complexedmetalions;M—metalions;DML—diffusioncoefficientforML; DM—diffusioncoefficientforM.

marineenvironments(Reinfelderandothers,1998).Numerousspeciesofalgae(macro-andmicroalgae)can sequesterPTEsthroughadsorptionbyfunctionalgroups (i.e.,carboxylic,amino,hydroxo-carboxylic;Gonza´lezDa´vila,1995).Forexample,thediatom Dunaliellatertiolecta hasbeenfoundwithhighercopperconcentrations thanitssurroundingenvironment(Gonza´lez-Da´vila,1995). However,thesealgaelowerPTEtoxicitybyoxidation,so excretedPTEs(Ag,Se,andZn)arelessbioavailableto diatomsthantheyareintheirinorganicform(Wangand Fisher,1998).

Foraminiferscommonlyingestalgaeandbacteriathrough phagocytosis.PTEswithinthepreyorinclaysthatenter endocytoticvacuolesarelikelymobilizedbylowpHlevels (, 3;TwiningandFisher,2004)andtherebybecome bioavailableduringvacuolization.Thisisanalogousto PTEdesorption,whichcommonlyoccursduringpHchanges orduringdigestionbymarinebivalves(Reinfelderand others,1998).Organiccoatings(i.e.,bacterialextra-cellular polymers)onsedimentstendtoaugmentPTEassimilation bybivalvesduringingestion.Inthecaseofforaminifers, sedimentsororganicmattercontainingPTEscouldbe trappedduringvacuolizationandthechemistrywithinthe vacuolescouldincreasetheirbioavailability.Inaddition, granuloreticulopodiaarecommonlyobservedcoatedwith sedimentparticles,organicdetritus,bacteria,andvarious microalgae(BreslerandYanko,1995a),allofwhichcould containPTEsthatincreasetheriskofcontaminationtothe foraminifer.

AnotherpotentialmechanismofPTEincorporationis diffusionduringcalcification.Erez(2003)developeda modelofthevacuolizationprocessofperforateplanktic foraminifersthroughendocytosis.Accordingtothismodel, vacuolizationofseawaterisfollowedbyactivetransportof

hydrogenionsoutofthevacuoleandcalciumionsin,which raisesthepHwithinthevacuoleandtherebypromotes calcification.Duringthisprocess,arestrictedmicroenvironmentiscreatedinwhichtheincorporationofPTEs couldoccurasabyproductofcalcification.

Asnotedpreviously,theextensivesurfaceareaof granuloreticulopodiaprovidesavastdiffusiveboundary layeroverwhichPTEuptakecanoccurdirectlyfrom seawater.DirectincorporationofPTEshasbeenpostulated byYankoandothers(1998).Free-ion-activitymodels (FIAM)assumethatthebioavailabilityofmetalsisbased onuncomplexedmetalactivityinsteadofthetotalor dissolvedconcentrations(LanderandReuther,2004).Such modelsassumethat(1)themasstransferdoesnot determineflux,(2)complexassociation-dissociationreactionsareatequilibrium,and(3)free-ionactivitycontrols reactionswithsurfacesitesonorganisms(bioavailability; Ribaandothers,2003;LanderandReuther,2004).Sucha modelcouldbeappliedtoPTEintakethroughdiffusionor facilitateddiffusion(NationalResearchCouncil,2003)by foraminifers.BasedonFIAMfundamentals,Figure5isa simplifiedmodelthatshowspathwaysformetalionsand complexedmetalionsfromtheenvironment(bulkmedium) intotheboundarylayerandthroughthemembrane (LanderandReuther,2004;NationalResearchCouncil, 2003).TherateofPTEuptakefromseawaterorporewater byforaminiferslivinginthesediment-waterinterface dependsuponthefluxoftheionsofconcern.Inwell oxygenatedwatersaswellasanoxicporewaters,certain PTEspeciesaresequesteredandionfluxislimited. However,manyinfaunalforaminifersliveatornearthe hypoxicboundarywherethePTEsaremosteasily mobilized.Theseforaminifersaremorelikelyexposedto dissolvedPTEsinporewatersincethesezonesaremore

PLATE1

1–4 Ammonia sp.: 1 undeformed; 2 protrudedproloculus(p); 3 double(twinned)shell; 4 pittedsurfaceandhighlydissolved. 5–14 Quinqueloculina rhodiensis: 5 undeformed; 6 doubleaperture(Ap). 7–8 aberrantformsshowingdissolution; 9–11 changeincoilingdirection, 12 highlydissolvedshell, 13 undeformedwithframboidalpyrite, 14 framboidalpyrite; 15–18 darkenedshells: 15 Broeckina sp.; 16 Archaiasangulatus; 17 Peneroplis sp.; 18 Quinqueloculina cf. Q.multimarginata.ForaminiferswerecollectedfromTorrecillaLagooninPuertoRico.Scalebars 5 200 mmunlessotherwise noted.

susceptibletopHchangesthatcanreleasetheelementsinto solutionorintoamorebioavailablephase(Fig.3).

NEEDFOR FOCUSED EXPERIMENTAL STUDIES

Althoughafewexperimentshaveaddressedtheuseof foraminifersasindicatorsofpollution(seeNigamand others,2006),thereisneedtoelucidatethephysiological andbiochemicalimpactsofPTEsandothercontaminants atthecommunity,population,andindividualphysiological andbiochemical-activitylevels.Addressingtheseneedswill requiremanymorefield-andlaboratory-basedexperiments,rangingfrommicrocosmexperimentstotest responsesofindividualtaxatomesocosmstudiesof foraminiferalassemblages.Additionallyandmostimportantly,thereisneedforstudiesofindividualstodetermine howtheyincorporatePTEsintotheircytoplasmandshells. HowdodifferentPTEsinfluencethebiochemistryand physiologyoftheindividualforaminifer?DoPTEsinterfere withnormalbiochemicalactivityinawaythatimpactsthe functioningofthegranuloreticulopodiaortheproduction ofshellmatrixcompounds,therebypromotingabnormal growth?

Criticaltostudiesofindividualphysiologicaland biochemicalresponsesarethedevelopmentandmaintenanceofaxenicculturesofmoreforaminiferalspecies. Standardizedculturetechniquesanddependablelineagesof representativesofseveralorderswillprovidethepathway towardunderstandingthebiologyoftheseprotistsandtheir diversityofresponses.Ifbiologicalcultureswerereadily available,thedoorswouldopentoawidevarietyof foraminiferalstudies,includinggenetics,biochemistry,and cellularphysiology.Secondonlytocalcareousnannoplanktoninaccountingforbiogeniccalciumcarbonate,foraminiferalshellscoverroughlyonethirdoftheEarth,orhalf theoceanfloor(Kennett,1983).Thus,wecontendthat understandingforaminiferalbiologyisvitaltounderstandingtheoceanspastandpresenttomoreeffectivelypredict theEarth’sfuture.

CONCLUDING REMARKS

Todate,themajorityofstudiesaimedatdeterminingthe effectsofPTEsonforaminiferalassemblagesandmorphologiesincoastalenvironmentshaveutilizedcorrelation approaches,inwhichwaterandsedimentchemistrieshave beencomparedwithparametersbasedonbenthicforaminifers.Whilethiscanbeveryusefulfirst-lineevidence, correlationdoesnotprovecausation.

Thekeytoapplyingforaminifersasenvironmental indicatorsisstrongscientificmodelingbasedonfieldand laboratoryobservationsandexperimentsthatspecifically examinetheinfluenceofpollutantsatthecommunity, assemblage,population,individual,orgene-expression levels.Fieldstudiesshouldincorporatedetailedgeochemicalanalysestoassessbioavailabilityaswellastotal concentrations.Micro-andmesocosmexperimentsmust bedesignedtoidentifyspecificresponsesandvulnerabilities tospecificcontaminants.Perhaps,mostcriticallyneeded aregenomicstudiesofkeyforaminiferaltaxawithstrong potentialasbioindicators.Forexample,genomicanalyses ofwidelydistributedgenera,includinganagglutinategenus

(e.g., Ammobaculites or Trochammina),amiliolid(e.g., Quinqueloculina or Triloculina),androtaliidsatopposite endsofwaterqualitygradients(e.g., Ammonia and Amphistegina),couldleadtobreakthroughapplicationsof foraminiferalbioindicatorsinmid-andlow-latitudecoastal systems.

Realizationofthefullpotentialofforaminifersas environmentalindicatorsfacesmajorchallenges.Their nearlyubiquitouspresenceinmarineandestuarine environments,alongwiththeirglobalimportanceinthe pastandpresent,arguesstronglyforcommittingthe necessaryhumanandmonetaryresourcestofurther developthesepromisingtools.

ACKNOWLEDGMENTS

TheauthorswouldliketothankDr.GuillemMateuVicens,Dr.ValentinaYanko-Hombach,Dr.Charlotte Brunner,ananonymousreviewer,Dr.ElizabethAlve,Dr. KenFinger(JFReditor),JulieRichey,andIniaSotofor insightfulcommentsonthemanuscript.FundingforMMCwasprovidedbytheCollegeofMarineScienceandthe SloanFoundation.Workwaspartiallysupportedby fundingfromtheNationalScienceFoundation(grant numberCHE-0221834),theNationalOceanicandAtmosphericAdministrationthroughtheFloridaHurricane Alliance,NationalOceanicandAtmosphericAdministration-NationalEstuarineResearchReserve(grantnumber NA07N0S4200050),PuertoRicoSeaGrant(grantnumber R-21-1-08),U.S.GeologicalSurveyCooperativeAgreement99HQAG0004,andfromtheU.S.Environmental ProtectionAgencyGulfEcologyDivision(grantnumber X7-96465607-0).

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Received19February2008

Accepted7July2009