祝你成功

2007;73:1078–1086

EffectsofheavymetalsinriverwatersinJapanonimmobilityandmortalityofDaphniamagnaand

Oryziaslatipeslarvae

HirotatsuMURANO,1aKanaeMATSUZAKI,2HiroakiSHIRAISHI2ANDMeikoWAKABAYASHI3*

1

GlobalEnvironmentalForum,Tsukuba,Ibaraki305-8506,2ResearchCenterforEnvironmentalRisk,NationalInstituteforEnvironmentalStudies,Tsukuba,Ibaraki305-8506,and3CollegeofCross-CultureCommunicationandBusiness,ShukutokuUniversity,Iruma,Saitama354-8510,Japan

ABSTRACT:Samplesofriverwaterscontaininghighconcentrationsofzincandotherheavymetalsbutlowconcentrationsofotheranthropogeniccontaminantswerecollectedtoinvestigatetherela-tionshipbetweentoxicityofheavymetalsandnaturallypresentorganicmattersorhardness,aswellastheeffectsofheavymetalsonaquaticorganisms.AcutetoxicitytestswereconductedforthewatersamplesusingDaphniamagnaandmedakaOryziaslatipes.AlmostalltheD.magnadiedinriverwaterscontaininghighconcentrationsofzinc,butO.latipesinthesamewaterswerehardlyaffected.Sincethetestorganismswerenotonlyexposedtozincbutalsootherheavymetalsintheriverwaters,weexaminedthetoxicityusingtoxicunitscomposedofzinc,copper,lead,andcadmium.TheresultsofabioassaywiththeriverwatersshowedthatthemortalityofD.magnadidnotdependsolelyonthetotalnumberoftoxicunitsofheavymetals.TheorganicmattersandthehardnessoftheriverwaterscoulddecreasetheacutetoxicityofzincandotherheavymetalstoD.magna.KEYWORDS:

Daphniamagna,hardness,heavymetals,organicmatter,rivers,zinc.

INTRODUCTION

Numerousinvestigationshaveshownthatseveralchemicalsinriverandseawatersimmobilize,1,2kill,3,4inhibitthegrowthof,5orreducereproduc-tionof6,7aquaticorganisms.TheMinistryoftheEnvironmentofJapanhascarriedoutriskassess-mentstudiestoidentifythosehazardouschemi-calswhichwillleadtofutureriskmanagementinJapanesesurfacewaters.8Theriskisassessedbycomparingthemeasuredconcentrationsoftoxicchemicalsinthewaterswiththetoxicitiesofthechemicalstoaquaticorganismsasdeterminedinthelaboratory.Theresultsofthesestudieshaveshownthatzincandotherchemicalsposeahighrisktoaquaticorganisms.Onthebasisofthesestudiesin2003,theMinistryoftheEnvironmentofJapanpublishedenvironmentalqualitystandards

*Correspondingauthor:Tel:81-49-274-1511.

Fax:81-49-274-1525.Email:mwak@ccb.shukutoku.ac.jpa

Presentaddress:GraduateSchoolofLifeandEnvironmentalSciences,UniversityofTsukuba,1-1-1Tennoundai,Tsukuba-shi,Ibaraki305-8572,Japan.

Received6October2006.Accepted14May2007.forzincinriverandseawatersdesignedtoprotectaquaticorganisms.9

Generally,theresultsofbioassaysconductedinlaboratoriesshowonlythetoxicityofacertainchemical.Aquaticorganismsinnaturalwaters,however,areexposedtomorethantwochemicalssimultaneously.Therefore,itisdifficulttodeter-minetheriskthatchemicalsposetoaquaticorgan-ismsinnaturalwatersonthebasisoftoxicitydataofasinglechemicalmeasuredinthelaboratory.Moreover,thetoxicityofachemicaltoaquaticorganismsdependsonnumerousfactors,suchasthedissolvedorganicmatters,hardness,andthesuspendedparticleloadofriverorlakewaters.4,10–13Toevaluatethetoxicityofmixturesofchemicalstoaquaticorganisms,severalstudieshaveusedthesumoftheirtoxicunits.14–16Thenumberoftoxicunitsequalstheratioofthemeasuredconcentra-tionofachemicaltoitseffectiveconcentration,i.e.theconcentrationthatistoxictoaquaticorgan-isms(e.g.concentrationofachemicalcausingdeathin50%ofthetestorganisms[LC50],andcon-centrationofachemicalcausingimmobilizationin50%oftheorganisms[EC50]).However,therehasbeenlimitedinvestigationontheeffectsoforganic

doi:10.1111/j.1444-2906.2007.01439.x

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mattersorhardnessonthecombinedtoxicityofheavymetalstoaquaticorganisms.

Zincisahighlytoxicchemicaltoaquaticorgan-isms.4,5RiverandseawatersinJapanandinmanyothercountriescontainhighconcentrationsofzincandotherheavymetals.17–19Moreover,highconcentrationsofzinchavebeenobservedintissuesofamphipodsandfishlivingincontami-natedriversorseas.17,20,21Therefore,itisimportanttoascertaintheenvironmentaltoxicityofzinctoaquaticorganismswithabioassayusingnaturalriverwater.

Theaimsofthisstudyaretoconfirmconcentra-tionsofheavymetalsinriverwaters,andtheirtox-icitytoDaphniamagnaandOryziaslatipes.Itwasevaluatedwhethertotalorganiccarbon(TOC)andhardnessinriverwaterswouldaffectthetoxicityofzincandotherheavymetalstoaquaticorganisms.MATERIALSANDMETHODSSampling

WatersampleswerecollectedfromfiveriversystemsinJapan(Fig.1).Sampleswerecollectedfromknowncontaminatedanduncontaminatedareasofeachriversystem.Fourriversystems,theNamari(A),Kaishu(B),Miyata(C)andWatarase(D),havemineraloredepositsorsmeltingplants

intheircatchmentbasins.Up-anddownstreamofsamplingsite1oftheNamariRiver,therearemineraloredepositsandaleadsmeltingplant,respectively.Thedepositsincludesphalerite,galena,wurtzite,andpyrite.21InthecatchmentbasinoftheKaishuRiver,therearemineraloredepositsthatincludepyrite,chalcopyrite,andsphalerite.22Upstreamofsamplingsite2oftheMiyataRiver,thereisasmeltingslagwastedump.OnthewestbankoftheWataraseRiver,mineraloredepositsextend.Thedepositsincludechalco-pyrite,bornite,pyrite,sphalerite,galena,arseno-pyrite,andtinstone.23,24TheItadoriRiver(E),incontrast,isuncontaminated.Theserivershavelittleinflowfromhouseholdsorfactories.Surfacewatersampleswerecollectedinpolyethylenebottlesandstoredat-20°Cuntilbioassaywasperformed.Chemicalanalysis

Concentrationsofheavymetals,TOC,andhard-nessweremeasuredaccordingtotheJapaneseIndustrialStandardmethods.25Theorganiccon-tentsofthewatersampleswereanalyzedfordiffer-encesbetweenunfilteredandfiltered(0.45-mmfilters)samplesinapreliminaryexperiment.Asta-tisticallysignificantdifferencebetweentheorganiccontentsinunfilteredwatersandfilteredwaters

ABC

DE

Fig.1SamplingsitesinsixriversystemsinJapan.NamariRiver(A),KaishuRiver(B),MiyataRiver(C),WataraseRiver(D),andItadoriRiver(E),showingsamplingsites(᭺)andoredeposits(󰀂).

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HMuranoetal.

wasnotobserved.Thus,TOCconcentrationiseffectivelyequivalenttodissolvedorganiccarbon(DOC)fortheserivers.Theconcentrationofzincandcopperwasmeasuredbyinductivelycoupledplasmaatomicemissionspectroscopy(ICP-AES).Flamelessatomicabsorptionspectrophotometrywasusedformeasurementofcadmium,lead,andhardness(asCaCO3).TOCwasdeterminedbyhigh-temperaturecombustion.Electricalconductivity(EC)wasmeasuredinsitubythefour-alternatingcurrent(4-AC)electrodemethod.Bioassay

Bioassaysoftheriverwatersampleswerecon-ductedusingD.magnaandO.latipes.Tapwater,suppliedbythemunicipalwaterworksofTsukuba,Japan,wasdechlorinatedbyacolumnofgranularactivatedcarbonandusedasthecontrol.Eachbioassaywascarriedoutfor48handtoxiceffectswereobservedat24handattheendoftheassay.Immobilizationordeathofthetestorganismswasconfirmedunderastereomicroscope.Ifatestorganism’sheartdidnotpumpfor15s,weassumedthatitwasdead.WemeasuredthepHandDOCofthewateratthestartandendofeachtest.ThepHandDOCweremeasuredbyglasselectrodeandpolarographmethods,respectively.IncaseswherethepHwasoutsidetherange6.0–8.5atthestartofthetestandsomeorganismswerefounddeadorimmobilizedattheendofthetest,anaddi-tionaltestwascarriedoutafteradjustingthepHto6.0–8.5with0.1NHClor0.1NNaOH.Ifallorganismsdied,thetestwaterwasdilutedwithdechlorinatedtapwaterandanotherbioassaywasperformed.TheLC50andEC50valueswerecalculatedbasedontheprobitmethod26,27usingEcoTox-Staticsv2.4software(YoshiokaY,pers.comm.,2004;http://www.intio.or.jp/jset/ecotox.htm).

Daphniamagna

AnacuteimmobilizationtestforD.magnawascarriedoutinaccordancewithtestguideline202oftheOrganizationforEconomicCooperationandDevelopment(OECD).27CulturesofDaphniamagnahavebeenmaintainedatthetestinglabo-ratory(NationalInstituteforEnvironmentStudy[NIES],Tsukuba,Japan)sincethemid-1980s.TheoriginofthecolonywastheUnitedStatesofAmericaEnvironmentalProtectionAuthority(US-EPA).Theparentsofthedaphnidsusedinthetestswereacclimatedat20Ϯ1°Cundercoolwhitefluo-rescentlights(<1000lx)withan8:16hdark–light

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periodforatleast28days.Theyweremaintainedindechlorinatedtapwater,andthewaterwasrenewedtwiceaweek.Thedechlorinatedwaterwaswellaeratedbeforeusingforcultivation.Duringacclimation,parentdaphnidswerefeddailywithgreenalgaeScenedesmussp.Offspringthatwerebornafterthethirdlarvaewereusedforthetest.Allparentdaphnidsproducedmorethan60larvaewithin21daysanddidnotproduceanyrestingeggs.TheD.magnalarvaewereusedforbioassaywithin24hposthatch.Twentydaphnids,dividedintofourgroupsoffive,wereusedforeachtestconcentrationandthecontrol.Eachgroupwasplacedin20-mLglassvesselsfilledwith15mLoftestwaters,i.e.riverwater,riverwaterdilutedwithdechlorinatedtapwater,ordechlorinatedtapwater.Thebioassaywasconductedbasedonthestaticmethodunderthesameconditionsofaccli-mationdescribedabove,anddechlorinatedtapwaterwasusedforthecontrol.Duringthetestperiod,thetestorganismswerenotfedandtestsolutionswerenotaeratedtopreventtheforma-tionofmetaloxides.Oryziaslatipes

AnacutetoxicitytestformedakaO.latipeslarvaewascarriedoutaccordingtoOECDtestguideline203.26Fishwereobtainedfromcommercialfisher-iesandhavebeenculturedattheNIEStestinglaboratorysincemid-1980s.Parentfishwereaccli-matedusingaflow-throughchamberat24Ϯ1°Cundercoolwhitefluorescentlights(<1000lx)withan8:16hdark–lightperiodforatleast28days.Theywereculturedindechlorinatedtapwater.Duringacclimation,parentfishwerefeddailywithbrineshrimpArtemiasalinaandtheculturewaterwasaeratedbypump.LarvaeofO.latipesthathadproducedmanyeggsduringacclimationwereusedforbioassaywhentheywerelessthan24hold(<24hposthatch).Thebioassayswerecarriedoutusingthestaticmethodunderthesameconditionsasforacclimationin200-mLglassbeakers,eachcontaining100mLofriverwaterordechlorinatedtapwater.Duringthetestperiod,testorganismswerenotfedandtestsolutionswerenotaerated.Tenfishwereplacedineachvessel.Thebioassayswereperformedinduplicate.Sensitivitytest

Toconfirmthesensitivityofthetestorganisms,asensitivitytestwascarriedoutinduplicate.Themethodforthesensitivitytestwasthesameasforthebioassay.DaphniamagnaandO.latipeswere

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exposedtozincsulfate(CASno.7733-02-0,WakoPureChemical,Osaka,Japan),for48h.Zincsulfateconcentrationswere180,320,580,1000,and1800mg/LinthesensitivitytestofD.magnaand1800,3200,5800,10000,and18000mg/LinthesensitivitytestofO.latipes.Thesensitivitytestswereperformedpriortothebioassaysoftheriverwater.Toxicunits

Todeterminethetoxicityofthecombinedheavymetals,theexpectedtoxicitiesoftheheavymetalswereexpressedastoxicunits(TU)andthetoxicunitsumwascalculatedbyequation1:15,16

∑a

TU=∑[Mei],(1)

i=1

MeLC

50,i

where

[∑TU=sumoftoxicunits

Me]=concentrationofaheavymetalinriver

water(μgL)MeLC50=meantoxicityvaluecalculated

usingpublished48-hLC50valuesofeachheavymetal(μgL)a=thenumberofdifferentheavymetals

includedinthesum

TheheavymetalconcentrationsineachriverwatersampleweredividedbythecalculatedmeantoxicityofeachheavymetaltoD.magna.Themeantoxicitywascalculatedusingknown48-hLC50valuesdescribedinpublishedpapers.4,5,7,30–33Iftheconcentrationofaheavymetalwasbelowthequantificationlimit(Zn1,Cu1,Pb1,andCd0.25mg/L),avalueofhalfthequantificationlimitwasused.28

RESULTSANDDISCUSSION

Heavymetalconcentrationsandothercharacteris-ticsoftheriverwatersandtheresultsoftheriverwaterbioassayswithD.magnaandO.latipesareshowninTable1.Duringthetests,thewatertem-peratureofthetestsolutionsforD.magnaandO.latipesweremaintainedat20.1–20.8and23.4–24.9°C,respectively.Nocontrolorganismsinthedechlorinatedtapwaterdiedduringthetestperiod.

Thewatersofriversystemswithmetaloredepositsintheircatchmentscontainedrelativelyhighconcentrationsofzinc.Thesewatersalsocon-

tainedrelativelyhighconcentrationsofcopper,lead,andcadmium.Organicmatterconcentra-tionswererelativelylowintheserivers.TheTOCranged<0.5–1.7mgcarbon/L(mgC/L)(Table1).SamplesfromthelowerreachesoftheMiyataRiver,whichflowsthroughpopulatedareas(sites1and2,Fig.1),containedhigherlevelsofTOCthanotherriverwatersamples.ThewatersoftheItadoriRiver,forwhichthecatchmentisfreefromcon-taminantsources,containedonlyasmallamountofheavymetals.ComparedwithtypicalriversinJapan,theNamariRiverandMiyataRivershowedrelativelyhighhardnessvalues,2Ϯ140(stan-darderror[SE])and270Ϯ80mg/L(asCaCO3),respectively,whilethehardnessoftheotherriverswasintherange31Ϯ4mg/L(Table1).

The48-hLC50valuesofzincsulfateforD.magnaandO.latipeswere280–400and6500–7700mgZn/L,respectively(Table2).TheLC50valuesforD.magnawerereportedas151mgZn/L4and752mgZn/L.5TherewasnoreportonLC50forO.latipes.The96-hLC50sofzincsulfateforjuvenileOncorhynchusmykissinsoftandhardwaterswerereportedtobe430and7210mgZn/L,respectively.29

TheresultsoftheacutetoxicitytestsforD.magnaandO.latipesexposedtotheriverwatersareshowninTable1.Daphniamagnawereimmobilizedordeadin12ofthe18riverwatersamples,whereasO.latipesweredeadinonly4ofthe18riverwatersamples(Table1).

DaphniamagnaandO.latipeswereaffectedbyvariousheavymetalspresentinthetestedriverwaters(Table1).Therefore,thecombinedtoxi-citiesoftheheavymetalsintheriverwatertotheSTU.testorganismswereevaluatedbasedon15,16Oryziaslatipeswaslittleaffectedbytheheavymetalscontainedintheriverwatersinthisstudy.Therefore,thecombinedtoxicitiesofheavymetalstoO.latipeswerenotanalyzed.

TheLC50valuesusedtodetermineTUwereobtainedfrompublishedwork:thereported48-hLC50valuesforD.magnaexposedtozinc,copper,lead,andcadmiumare151–752,4,5thisstudy7–54,4,73610–4400,7,30and20–118mg/L.7,31–33Weusedmeantoxicityvaluesofzinc(340Ϯ130mg/L),copper(38Ϯ16mg/L),lead(4000Ϯ400mg/L),andcadmium(49Ϯ10mg/L)calcu-latedfromthosevaluesbyassumingthatallheavymetalswerepresentasions.TUwerecalculatedbydividingtheheavymetalconcentrationofeachheavymetalinthewaterofeachriverbyitscalcu-latedmeantoxicitytoD.magna(Fig.2).InFigure2,themortalityofD.magnaandSTUarecompared.Theoretically,whenSTUisgreaterthanone,halfofthetestorganismsshouldbedead.Inourresults,themortalityofD.magnatendedtobehighwhentheywereexposedtoriverwaterswith

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Table1ChemicalandphysicalpropertiesofriverwatersandbioassayresultsforDaphniamagnaandOryziaslatipesHeavymetalconcentration(mg/L)TOC(mgC/L)EC(mS/cm)24h201002020141300920000000000000202000072000020200000072011042037.97.77..07.37..08.47.97.67.4–8.07.97.77.97.57.77.87.57.58.07.62020202020201202020207.06.76.97.07.27.07.37.48.07.16.87.67.87.78.37.48.38.07.37.48.28.32061020000202006.86.97.47.17.37.27.67.36.76.77.37.39.69.29.39.38.58.68.38.68.68.38.57.97.68.57.77.78.38.37.6–8.67.4–8.47.6–8.848h24h48h0h48h0h48h8753563447717061742719919131037313772827534<0.511.41.20.50.70.61.70.80.81.20.6<0.5<0.5<0.5<0.5<0.5<0.5822323232925133403601104543226360043450ImmobilityDeadpHoftheD.magnatestsolution24h1000200000000200000CumulativenumberofimmobilizedordeadD.magnaDOofCumulativepHofDOoftheD.magnanumberoftheO.latipestheO.latipestestsolutiondeadO.latipestestsolutiontestsolution48h100019000005009000000h6.86.97.47.17.06.76.97.08.97.68.08.38.57.08.98.18.28.448h7.0–7.17.17.0–7.17.57.27.27.3–7.47.48.07.6–7.77.87.67.67.7–7.67.67.77.87.70h6.76.77.37.37.67.16.87.67.17.96.48.17.98.07.87.66.87.048h7.6–7.77.97.9–8.08.0–8.37.7–7.87.7–7.98.08.07.8–7.98.37.47.6–7.78.07.8–8.07.5–7.77.87.5–7.77.5–7.67.3–7.87.5–8.07.4–8.37.3–8.0©2007JapaneseSocietyofFisheriesScience

Cu21190141133042423824031<1<1<1<1<0.2511<0.25<0.25<111<1<1<0.250.44.1<0.25<0.251212.63.8<0.2538621231.73.24.421211127.3<0.254.3PbCdHardnessasCaCO3(mg/L)11<1RiversystemSiteSamplingdate†ZnNamari123429/5/200229/5/200229/5/200229/5/2002260091054400Kaishu123428/5/200228/5/200228/5/200228/5/200249001300700430Miyata12316/10/200316/10/200316/10/200383140240FISHERIESSCIENCE

Watarase1234510/6/20039/6/20039/6/20039/6/200310/6/200351323073Itadori129/11/20039/11/2003DechlorinatedtapwaterDO,dissolvedoxygen;EC,electricalconductivity;TOC,totalorganiccarbon.†Day/month/yearHMuranoetal.

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Table2LC50valuesofzincsulfateforDaphniamagnaandOryziaslatipes

D.magna

48-hLC50(mgZn/L)

(95%CL)400(270–560)280(240–300)

O.latipes

48-hLC50(mgZn/L)

(95%CL)6500(5400–7800)7700(6300–11000)

ChemicalZnSO4

CL,confidencelimits.

% Mortality100100 100 100 100100 100 100100100 0 20 152000 0 0 0017.2Lower histogram15ZnCuPbCd UT 107.97.15 4.22.92.4 1.31.41.11.61.800.20.1 0.30.10.00.00.0123423 12341iiii 1234512 rrrraaaauuuuaaateeeeeiirrmmmmhhhhttaaasssssooaaaaaddaaaaisisisisaaaaiyiyiyrrrrraaNNNNKKKKMMMaaaaattIItttttaaaaaWWWWWFig.2RelationshipsbetweenSTUandtoxicityto

Daphniamagna.TheupperbargraphshowspercentmortalityandthelowerhistogramshowsSTUforheavymetals.

highTUvaluesforzinc(e.g.NamariRiver1andKaishuRiver1–4).Also,whenSTUwasgreaterthanone,mortalitywashigh,eveniftheTUofzincwaslessthanone(e.g.WataraseRiver3).However,insomecases,thebioassayresultsdidnotshowtheexpectedSrelationshipbetweenRiverTU.Specifically,1and3watersNamarihadSRiverTUhigher2theandmortalitythan4andone,Miyataandbutmortalitywaslessthan50%.WataraseRiver2hadlowSTU,butthemortalitywas100%.Itissug-gestedthatWataraseRiver2containedotherheavymetals,whichwedidnotmeasure.

TherelationshipbetweenthemortalityofD.magnaandSTUindicatesthatthetoxicityofheavymetalsinriverwaterstoD.magnadidnotdependsolelyonheavymetalconcentrations.Itisgenerallyknownthatwhenorganicchemicalsformachelatewithheavymetalions,thetoxicityoftheheavymetalstoaquaticorganismsisreduced.Hardnessinwateralsoreducesthetoxicityofheavymetalstoaquaticorganisms.PaulaskisandWinner11studiedtheacute(72-h)toxicityofzinctoD.magnainwaterwithdifferenthardnessvalues(50–200mg/LasCaCO3)andhumicacidconcentrations(0.00–1.50mg/L),andfoundthatincreasesineitherthehumicacidconcentrationorhardnessresultedinproportionaldecreasesinacutezinctoxicity.Oikarietal.4foundthatthetoxic

effectsofzinc,copper,andleadonD.magnainnaturalwatersdecreasedinthepresenceofhumicacids.Pettinenetal.12studiedtheinteractionbetweendissolvedorganicmattersandwaterhard-nessandtheireffectsontheacutetoxicityofcadmiumtoD.magna.Theyfoundthatdissolvedorganicmattersinsoftwaterhadaprotectiveeffectagainstcadmiumtoxicityandthatcalciumionsinterferedwiththeuptakeofcadmiumions,eitherbycompetingwiththemfortransportthroughmembranesorbyreducingmembranepermeabil-ity.BecauseTOCisknowntocontainorganicchemicals(e.g.humicacids)thatformchelateswithmetals,wemeasuredTOCintheriverwatersinthisstudy.Toexaminewhetherthetoxicityofheavymetalswasaffectedbytheorganicmatterscon-tainedinriverwaters,wecomparedTOC,whichisanS18TU.indexbioassaysBecauseofwater(Fig.allD.pollution2),magnawecarriedwerebyorganicdeadmatter,withoutadditionalineightofbio-theassaysforthoseriverwatersbygraduallydilutingtheriverwatersampleswithdechlorinatedtapwater(Table3andFig.3).TwodistinctivefeaturescanbefoundinFigure3.First,inriverwatercol-lectedfromthesamesite,asthedegreeofdilutionincreasedorSTUdecreased,mortalityofD.magnadecreased.Second,forsimilarSTU,regardlessofsamplingsitesorrivers,mortalityofD.magnadecreasedasTOCorhardnessincreased.Weobservethisfeatureespeciallyinareas(i),(ii),and(iii)ofFigure3.Inareas(i)and(ii),althoughthehighestSTUwasinwatersfromtheMiyataRiver,mortalitywaslowestinthosewatersbecauseoftheirhighTOCandhardness.Inareas(i)and(ii),bothorganicmatter(TOC)andhardnesscanbeconsideredimportantcontributorstothede-creasedtoxicityofheavymetalstolivingorganismsincludingD.magna.Inarea(iii),theKaishuRiverwatersshowedthelowestmortalityofD.magnadespitehavingthehighestSTU.ThehardnessofeachriverwaterwasalmostthesameandmaynothavecontributedtothedecreaseinthetoxicityofheavymetalstoD.magnainarea(iii).TOCoftheKaishuRiverwaterwashigherthanforotherriverwatersinarea(iii).Thisresultindicatesthatinarea(iii),organicmattersintheriverwaterdecreasesthetoxicityofheavymetalstoD.magna.

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Table3CumulativenumberofdeadorimmobilizedD.magnaImmobilizedSTU24h0150020592008201315200020592006201315207.87.87.67.07.06.7162001516207.06.948h24h48h0h2.65.30.210.1.91.42.84.2DeadpHoftheD.magnatestsolution48h7.37.07.77.67.67.37.17.0TOC(mgC/L)0.250.250.250.260.280.40.550.78281807057457669HardnessasCaCO3(mg/L)ResultsofbioassaysusingDaphniamagnaforriverwaterdilutedwithdechlorinatedtapwaterDOoftheD.magnatestsolution0h7.27.58.07.87.86.76.87.148h9.29.49.08.98.38.58.28.6©2007JapaneseSocietyofFisheriesScience

0.430.620.80.250.250.250.250.250.25<1175267360634526817871820.531.11.60.090.170.260.260.792.44150190020056200718062003010170020043100416052007.37.47.68.08.37.37.87.77.67.5–7.87.77.77.78.38.18.37.97.87.77.8–8.07.98.07.98.38.18.38.17.88.17.4–8.48.68.38.47.87.57.68.88.78.67.6–8.8RiversystemSiteDilutionratioNamariNamari111/32/3KaishuKaishuKaishuKaishuKaishuKaishu1112221/811/271/91/32/3FISHERIESSCIENCE

1/32/3MiyataMiyataMiyataWataraseWataraseWataraseWataraseWataraseWataraseDechlorinatedtapwater2222223331/32/31/271/91/3TOCandhardnessofdilutedtestwaterswereestimatedfrommeasuredTOCandhardnessvaluesoftheriverwatersampleandthoseofdechlorinatedtapwater.HMuranoetal.

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(a)1000.700.280.250.25800.250.250.4060(ii)0.55(iii)0.80400.26y200.430.25(i)ti0.250.62ltta0.25r0.25o0M0.25 (b)fo10045 %807169768026705760(ii)(iii)3604081207845175267(i)06381082123456Sum of toxic unitsFig.3RelationshipsbetweenSTUandmortalityofDaphniamagnafordifferentlevelsof(a)TOCand(b)hardnessfortheNamariRiver(᭺),KaishuRiver(᭝),MiyataRiver(ᮀ),andWataraseRiver(¥).ItalicizednumbersshowestimatedTOC(mgC/L)orhardness(mg/L)oftestsolutionsdilutedwithdechlorinatedtapwater(TOC<0.5mgC/L,hardnessasCaCO3L).BoldnumbersshowTOC(mgC/L)or=hardness82mg/(mg/L)ofundilutedtestsolutions.Ifaheavymetalcon-centrationorTOCorhardnessofasamplewasbelowthequantificationlimit,avalueofhalfthelimitwasusedfortheanalysis.(i),(ii),and(iii)indicategroupsofdistincttrends.

Ithasbeenreportedthatthecombinedtoxi-cityofheavymetalstoaquaticorganismsissynergistic–antagonistic,34–36andpH37,38isanimportantfactorfortoxicity.Furtherstudiesarerequiredtodefinethecombinedtoxicityofheavymetalsinnaturalwater.CONCLUSION

Riverwaterscontainedmorethantwoheavymetals.ThecombinedtoxicityofheavymetalstoD.magnadidnotdependonlyontheheavymetals.Organicmattercontent(TOC)andhard-nessofriverwatersreducedthetoxicityofheavymetalstoD.magna.ACKNOWLEDGMENTS

WethankY.Fujiwara(NIES)andT.Nakajima(GlobalEnvironmentalForum)fortechnicalassis-tanceinthisstudy.

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