Document kD4bk3xYN8zjRmQvYBoQQkyw0

PFOS: A 96-Hour ShellDepositionTestwiththeEastern Oyster (Crassostreavirginica) I ACUTE EASTERN OYSTER SHELL DEPOSITION TEST SUBSTANCE Identity:Pertluorooctanesulfonatmea;y alsobe referredtoas PFOS or FC-95. (1-Octanesulfoniaccid,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-p,otassium salt,CAS # 2795-39-3) Remarks: The testsubstance isa whitepowder. Sample was taken f@om 3M lotnumber 217. Sample was storedunder ambientconditionspriorto testing.Puritydetermined tobe 90.49% by LC/MS, 'H-HMR, 19F-NMR and elementalanalyses techniques. METHOD Method: OPPTS 850.1025 Type: Staticacute GLP: Yes Year completed: Study completed1999. Reportcompleted2000 Species: Crassostreavirginica Supplier:P. Cummins OysterCompany, Inc.B,altimoreM,D Shellgrinding:Priortotestinitiatiroenc,entldyepositedshellatthe rounded (ventrale)nd was removed usinga smallelectrigcrinder.Care was taken toremove theshellrimuniformlytoproduce a smooth, rounded, bluntprofile. Analyticalmonitoring:PFOS measured at0,48,96-hours Exposure period:96-hours Statisticamlethods: Shellgrowth inhibitiownas calculatedforeach treatmentgroup as the percentreductioninshellgrowthrelativteomean shellgrowth inthe negativecontrol.The EC50 valuewas estimatedby visualinspectionofshellgrowth inhibitiodnata.The shellgrowth datawas evaluatedfornormalityand homogeneity ofvariancesusingtheChiSquare testand Bartlettt'esst,respectivelyD.unneft'stestwas used to identiftyreatmentgroups which had a statisticaslilgynifica(n@t0.05) reductioninshellgrowthas compared tothe control. Test oyster age: unknown Length: 33.8 (27.8-41.5m)m, Pretreatment: None Test conditions: Dilutionwater:Naturalseawater dilutedtoa salinitoyf200/oowith wellwater Dilutionwater chemistry (duringthe 4'-weekperiod immediately preceding the test): salinity: 21 (20-21)O/oo pH: 8.1(8.0-8.2) TOC: < 1.0mg/L Stock and testsolutionpreparation:Primarystockpreparedin dilutiownaterat9.1mg/L and mixed for-24 hourspriortouse. Aftermixing,primarystocksolutioanppeared clearand colorless withsome whiteparticulatmeaterialsuspended throughoutthe solution.Itwas proportionaldliylutewdithdilutiownaterto prepare thefouradditionatlestconcentrationsA.lltestsolutionasppeared clearand colorless.Due tothe relativeloyw solubiliotfyPFOS@ in naturalseawater,the highestconcentratioanttainablweiththis matrixisapproximately3.3 mg/L. Concentrationsdosing rate:Once Exposure vessels: 52L polyethyieneaquariacontaining approximately40L oftestsolutionw;aterdepthapproximately21 cm. Each chamber was continuouslsytirretdocirculattehe supplementalalgaedietusingan electripcaddlemixer. Feeding:Algalcells(Thalassiosirpaseudonana, Skeletonema sp.,Chaetocerossp.,and lsochrysisp.)were providedto supplementnaturalloyccurringalgaeand tomaximize oyster growth ratesduringthe test. Number of replicateso:ne Number ofoysters per replicatet:wenty Number of concentrations:fiveplusa negativecontrol Water chemistry during the study: Dissolved oxygen range (0- 96 hours): 6.6- 7.6mg/L (controelxposure) 6.1- 7.7mg/L (3.0mg/L exposure). pH range (0- 96 hours) 7.6- 8.1 (controelxposure) 7.6- 8.1 (3.0mg/L expos' ure) Testtemperaturerange (0- 96 hours) 22.2- 22.30C (controelxposure) 21.8- 22.70C(3.0mg/L exposure) Method ofcalculatingmean measured concentrations: arithmetimcean RESULTS Nominal concentrations:Bk control1,.2,2.0.3.3,5.5,9.1mg/L Measured concentrations:<LOQ, 0.36,0.40,1.3,1.9,3.0mg/L Element value: 96-hourEC50 =>3.0 mg/L (C.I.notcalculable) 96-hourNOEC = 1.9mg/L ' Allelementvaluesbased on mean measured concentrations Statisticeavlaluatioonfshellgrowth: ECsovaluescouldnotbe calculatdeudetoinsufficisehnetlglrowth inhibitioantthe highestattainableconcentration. AnalyticalMethodology: Analyses oftestsolutionswere performed at WildlifIenternationaLltd.usinghighperformance liquicdhromatography withmass spectrometridcetection(HPLC/MS). When determiningthe concentrationofthe testsubstance inthetestsolutionst,hesame and most prominentpeak response forperfluorooctanesulfonawtaes used. No attemptwas made toquantifyon thebasisofindividuailsomeric components. The LOQ (limiotfquantitatiowna)s 0.115 mg/L inthisstudy. Samples collecteadttestinitiatihoand measured valuesfrom 28 to46% ofnominal. Measured valuesforsamples takenat48-hoursranged from 15 to41 % ofnominal.Measured valuesforsamples takenat 96-hours ranged from <LOQ to52% ofnominal. Summary of analyticalchemistry data: Nominal Test Measured DuplicateValues at 0.48, Concentration, and 96-houm, Respectivelym,g/L mgll Mean Measured Percentof Concentration, Nomlnal mgA. Negabve Control All< LOO <LOQ - 1.2 0.331,0.353,0.341,0.429,<LOQ, 0.36 30 <Loa 2.0 0.622,0.633,0.299,0.313,0.249, 0.40 20 0.267 3.3 1.36,1.15,0.924,0.878,1.58,1.72 1.3 39 5.5 2.42,2.63,2.02,2.24,1.460,.970 1.9 35 9.1 3.39,3.44,3.01-,3.74,3.57,1.99, 3.0 33 1 1 2.19 1 1 *3 replicateasn2lyzed attime 0 Biologicalobservations after96-hours: Oystersinthe negativecontrol. and allPFOS treatmentgroups appeared normal and healthythroughout theexposure period. Shelldepositioannd shellgrowthInhibitiaotntesttermination: Mean Measured ShellDeposition PercentInhibition Concentration, Mean + SD, mm in ShellGrowth mg/L Negative Control 2.67+0.824 - 0.36 2.50+0.933 6.4 0.40 2.40+0.820 10 1.3 2.51 +0.919 6.0 1.9 2.13 + 0.804 20 3.0 .1.91+ 0.591 28 Mortalityof controls:None CONCLUSIONS The potassium perfluorooctanesulfonat9e6-hourEC50 forthe Eastern Oysterwas determinedtobe > 3.0mg/L, the highestconcentratiotnested and thepracticallimiotfsolubilitiynunfilteresdeawater.The 96-hourno effectconcentrationwas 1.9mg/L. Submitter:3M Company, EnvironmentalLaboratory,P.O. Box 33331, St. Paul,Minnesota,55133 DATA QUALITY ReliabilityK:limis@hranking= 1 REFERENCES Thisstudywas conducted at WildlifIenternationLatld.,Easton,MD atthe requestofthe3M Company. OTHER Last changed: 5/3100 PFOS: A 96-HOUR SHELL DEPOSITION TEST WITH THE EASTERN OYSTER (Crassostreva7rginica) FINAL REPORT WU,DLEE INTERNATIONAL LTD. PROJECT NUMBER-- 454A-106 3M LAB REQUEST NO. U2723 U S EnvironmentalProtectioAngency Series850 - EcologicaElffectsTestGuWcHm OPPTS Number 850.1025 AUTHORS: KurtP, Drottar Henry 0. Krueger,Ph.D. STUDY R4MnON DATE: May 3,1999 STUDY COWLENON DATE: August 11,1999 AMENDED REPORT DATE: April26,2000 Submittedto 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St.PauL Nihmesota 55144 Wildl!feInternationalLtd.. 8598 Commerce Ikive Easton,Maryland 21601 (410)822-9600 Page I of40 AMENDED WILDLIFE INTERNATIONAL L-m. -2- PROJECTNO.: 454A-106 GOOD LABORATORY PRACFICE COWLLANCE STATEMENT SPONSOF- 3M Corporation TITLE: PFOS: A 96-HourShellDepositioTnestwiththeEasternOyster(Crassostriedarginica) -WELDLIFE INTERNATIONAL LTD. PROJECT NUMBEI;L-454A-106 STUDY COMPLETION: August 11,1999 ANM'NDED REPORT: Aprir26,2000 Thisstudywas conductedm compha= vnthGood LaboratoryPrwticeStandm-dasspublishebdy theU.S. EnvironmentalProtectioAngency in40 CFR Parts160 and792,17 August 1989;OECD PrincipleosfGood LaboratorPyracticeO,CDE/GD (92)32,EnvironmentMonograph No. 45, Paris1992;andJapanMAFF, 59 NohSan,NotificationNo3.850,AgdculturaPlroductionBureau,10August 1984withthefollowinmgceptions: The testsubstm= was not chmwtmmd in accordancweithfidlGLP complim=; however,the charactmizafiownas performedaccordingto3M StandardOperatingProceduresand Methods,and all.radwata arebeingmaintainedinthe3M archivesT.he testsubstanceisbeingrechanctmizedinaccordancweithGLP. The stabilitoyf thetestsubstanceunderconditionosf stqrageatthetestsitewas notdeterminedin accordancewithGood LaboratoryPracticSetandards. STUDY DIRECTOR-- KurtI;LDrottar SeniorBiologist SPONSOR APPROVAL: A Lr- -Sp&sor DATE 7 DATE ANffiNDED WILDLIFE INTERNATIONAL LTD. -3- PROJIECTNO.: 454A-106 QUALITY ASSURANCE STATEMENT ThLssb* was exammed forcompliancewithGood LaboratoryPracticSetandardsas publishebdy the U.S.EnvirorumentParlotectioAngency,40 CFR Parts160 and 792,17August 1999;OECD PrinciploefsGood .LaboratorPyracticeO,CDE/GD (92)32,EnvironmentMonograph No. 45,Paris1992;and JapanMAFF, 59 NohSan,NofificatiNoon. 3850,AgriculturParloductioBnureau,10 August 1984. The datesof all mspectionsand auditsand thedatesthatany finchngwsere reportetdo dw StudyDurctorand Laboratory Management were asfollows: ACTIVITY: DATE CONDUCTED: TesStubstancPereparation May 21,1999 Testh@tiatioannd ]K&trix Fortifica@ohnweration l@by 24,1999 BiologicaDlataand Draft Report July1,1999 AnalyticaDlata and DraftReport July1,1999 FinalReport Amended Report August 11,1999 April25,2000 STUDY DATE REPORTED TO: DIRECTOR: MANAGENIENT: I&y 21,1999 May 21,1999 I&y 24,1999 May 26,1999 July1.1999 July2, 1999 July1, 1999 August 11,1999 April25,2000 July2, 1999 August 11,1999 April26,2000 JanWs H. Coleman DATE QualityAssuranceRepresentative ANENDED WILDLIFE INTERNATIONAL LTD. -4- REPORTAPPROVAL PROJECTNO.: 454A-106 SPONSOR- 3M Corporation TITLE: PFOS: A 96-HourShellDepositioTnestwiththeEasternOyster(Crassostrevairginica) WILDLIFE R4TERNAnONAL LTD. PROJECT NUMBER. 454A-106 STUDY DIRECTOK- KurtF- Drottar SeniorBiologist MANAGEMENT: Henry 6"4K=g@T, @h.D. Dfiww, AquaticToxicologaynd Non-TargetPlants DATE DATT AUMNDED WILDLIFE INTERNATIONAL LTD. -5- PROJECTNO.: 454A-106 TABLE OF CONTENTS Title/CovePrage.........................................................................1................ Good LaboratoryPracticCeomplianceStatemen.t..............................................2................. QualityAssuranceStatemen.t..............................................................3................. ReportApproval.........................................................................4................ TableofContents.....................................................1.....................................5.................................. Summary ..............................................................................7................. Introducti.o.n..........................................................................8................. Objective..............................................................................8................. EVerimentalDesip ..................................................*............................8................. Materialasnd Modwds ....................................................................9................ Resultsand Discussion..................................................................1.2................. Conclusions...........................................................................1.3............................. Refm=ces ..........................................................................................................1..4.................................. TABLES TableI - Summary ofAnalydW ChemistryData..............................................1.5................ Table2 - Temperatm, DissolvedOxygm and pH ofWater inffiTcestChambers ....................1.6................. Table3 -ShellGrowth andSheU Growth InhibitioantTestTerminatio.n...........................1.7................ Table4 -IndividuaOlysterShellMewmanents ...............................................1.8................ WILDLIFE INTERNATIONAL LTD. ,6, TABLE OF CONTENTS -Continued- PROJFCTNO.: 454A-106 APPENDICES Appendix I- SaliniatnydpH ofUnfilterSeadltwater .Mewured Duringthe4-Week PeriodbmuxhatelyPrecedingtheTest..................1.9.............. Appendix ]I- Analym ofPesticideOsr,ganicsM,etalsand OtherInorgamcs inWildlifIenternationLatl& Saltwate.r........................................2.0............. AppendixM - The AnalysiosfPFOS inUnffitereSdaltwateirnSupportofWildlife InternationLatlcLProjecNto.: 454A-106 .......................................21.............. AppendixIV - Changes toProtocol.......................................................3.7.............. AppendixV - PersonneIlnvolvedintheSb* .........................................................3..8...................... AppendixVI - ReportAmendment ........................................................3.9.............. ANCENDED WILDLIFE INTERNATIONAL LTD. -7- PROJECTNO.: 454A-106 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF TBE FINAL REPORT: SUBEMLARY 3M Corporation Ms. SusanA- Beach WildlifIenternationLatld. Easton,Maryland 21601 WILDLEER4TERNAnONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: NMAN NEASURIED TEST CONCENTRANONS: TEST DATES: LENGTH OF TEST: TEST ORCTANIBC SOURCE OF TEST ORCTANL%B: OYSTER LENGTH (N 20): 96-HouREc5O: 95% CONFIDENCE LXMTrs: NO-OBSERVED-=CTCONCENTRANON: LOVIEST-OBSERVED-EFFECT CONCENTRANON: 454A-106 PFOS (PerfluorooctmSeulfoniAccid PotassiumSalt) PFOS: A 96-HourShellDeposifionTestwiththeEasternOyster (CrassostreWarginica) NegafiveControL0.36,0.40,1.3,1.9and3.0mg &iJL ExpaknentalStart- May 24,1999 BiologicaTlerminatio-nMay 28,1999 ExperimentaTlerminatio-n June14,1999 96 Hours - Eastern Oyster (Crassostrea wrginica) P.Cummins OysterCompgmy, kib. 618 West 33d Sbvd BaltimoreM,aryland 21211 Mean = 33.8mm, 27.8to41.5mm >3.0mg &i.AL Not Calculable 1.9mg a.i./L 3.0mg &LAL AMEENDED WILDLIFE INTERNATIONAL LTD. -8- INTRODUCRION PROJECT NO.: 454A-106 Thisstudywas conductedby WildlifleatmmationaLltd.for3M CorporatioanttheWildUz Int=atiolW Ltd.aquatictoxicologfyaciliitnyEaston,Maryland.The in-hfephaseofthetestwas conductedfrcmMay 24 to May 28,1999. Raw datageneratebdy WildlifIenternationLatld.anda copyofthefinarleportarefileudnder ProjecNtmnber 454A- 106 inarchivelsocatedon theWildlifIentmmationaLltd.site. OBJECIIW The objectivoefthissh* was toevaluatetheacutetoxicitoyfPFOS (PerfluorooctmSulfoniAccid PotassiumSalt)on shelldepositioonf theeastemoyster,Crassostreaidrginicad,uringa 96-hourexposure period@@ statitcestconditions. EXPFITHWENTAL DESIGN Easternoysterswere exposedtoa geometricserieosffivetestconcentratioannsd a negative(dilution water)control.One replicatteestchamber was maintainedineacb:"Ueatmenatndcontroglro*upw,ith20 oysterisn eachtestchamber.Nominal testconcentratiownesre selecteidnconsultatiowniththeS'ponsora,ndwerebased upontheresultosfanexploratorryangefindingtoxicittyestN.ominal testconcentratiosneslectewdere1.2,2.0, 3.3,5.5and9.1mg activiengredien(ta.L)/L.M.ean measuredtestconcentratiownesre&t=ined fromsamples oftestwatercollectefdromeachftatnentand thecontroglroupatthebe&ning ofthetesta,tVFoid@ 48 hours,and attesttermination. The oysterswere . disaiminatel-Y assigned to exposure chambers at test mitmuon. Algal cefls (Yhalassios7ra pseudonana, Skeletonema sp., Chaetoceros sp., and Isochrysis sp.) were provided to supplement naturally o==g algaeandtommmm oystegrowth rate&srmg thetesl Measurementosfsheldlepositifoonreachmystewreremade at96 hoursandwereusedtoesfi=tethe EC50 value,theno-observed-effect-concentr(aNfOiEoCn) and the'lowesto-bserved-offect-concenumfion (LOEC). The EC50 istheconcentratioofntestsubstancienwaterthatiswfimftd toinhibistheldlq)osidonby 50-1/roe,lativteothe control. AMENDED WILDLIFE INTERNATIONAL LTD. -9- PROJECTNO.: 454A-106 MATERL4,LSAND MEETHODS Thestudwyasconductbeadsedontheproceduroeustliniendtheprotoc"oPlFOS:A 96-HouSrhell DepositiToenstwiththeEasterOnyste(rCrassostvrieraginicaT)h"e.protocwoalsbasedonprocedures outlineidnU.S. EnvironmentaPlrotectioAngency Series850 - EcologicaElffectsTestGuidelinesO,PPTS Number 850.1025 (1);StandardEvaluationProcedure,Acute ToxicityTestfor Estuarineand Ma?ine Organisms (Mollusc96-HourFlow-Yhrough ShellDepositionStudy)(2)and AS7M StandardE729-88a StandardGuideforConductingAcuteToxicitTyestswithFishesM,acroinvertebrateasndamphibians (3). Tea Substance The testsubstancewas receivefdrom 3M Corporatioonn October29,1998 andwas assignedWildlife InternationLatld.identificatniuomnber 4675. The testsubstancweas describeads a whitepowder. Itwas identifiaesdFC-95 fromlotnwnber 217 (T-6295).Informatiopnrovidedby theSponsorindicateadpuritoyf 98.90/oa,nd an expiratiodnateof2008. The testsubstancewas reanalyzebdy theSponsorandtheCertificaOtfe AnalysisdatedMarch 9,2000 indicateadpurityof 90.49*/oT.he testsubstancewas storedatambientroom -tmnperatw*e. P=arafion of TestConcentrations Nominal testconcentratiownesre 1.2,2.0,3.3,5.5and 9.1mg ai./L,basedon atestsubstancepuritoyf 90.49*/oA.llmaterialwshichcame intocontactwiththetestsubstancdeurinipreparatioonftestconcentrations wereconstructeodfplastiocrstainlessteel.A primarystocksolutiownas preparedindilutiownaterata concentratiofn9.1mg a.i./LT.he primarystocksolutiownas mixed withan electripcaddleml= for 'approximatel2y4 hourstoaidinthesolubilizatioofnthetestsubstanceA.ftermbdng thepfimarystocksolution appearedc.learand colorleswsithsome wluteparticulamtaeterialsuspendedthroughouthesolutiolTlhe prunzy stockwas proportionaldliylutewdithcwutionwatertopreparethefouradditiontaelstconcenuidons. Alltestsolutionasppearedclearand colorless. TestOrgam@sm The easternoyster,Crassostrea virginica,was seleew asthe testspeciesforthisstudy. The Eastern Oysterisrepresentatiovfean unportantgroupof aquaficorganismsand was selectefdoruse inthetestbased upon pasthistoryofuse and easeof handlinginthelaboratoryE.asternoystersusedinthetestwereobtained AMENDED WILDLIFE INTERNATIONAL LTD. -10- PROJECT NO.: 454A-106 fromP.Ciunmin OysterCompany, BaltimoreM,aryland.The oysterwsereholdinwaterfrom thesame source asusedduringtheteslThe oysterwsere suppliedunfilterendaturasleawaterduringholdingandwereheldfor12 dayspriortotestimtiatioiTlo supplementthedietoftheoystersandenhancetheicronditioannd growth,the oysterswereprovidedwithan algalsuspensionofYhalassiosirsap.,S7celetonemsap.,Chaetocerossp.,and Isochrysisp.continuousldyuringholdingand testing. Duringthe12-* holdingperiodprecedingthetestt,heoystershowed no signsofdiseneor stress. Duringtheholdingperiodw,atertemperaturersangedfi-am21.8to22.90C.The pH ofthewawrmWdfmm7.9 to8.1,salinitryemainedat20@%o qwts perthousand)and dissolveodxygenrangedfi-om7.0to 7.6mg/L. InstrumentatiuosnedforwatermeasurementsisdescribeidntheErmronmentalCon&tionssectioonfthirseport .Priortotestuntiauonr,ecentldyepositedshellattherounded(ventrale)ndwas removedusinga smdl ele=c grinderC.arewas takentoremovetheshellrununiformltyoproducea smooth,romukd, bluntprofile. The lengthsof20 unpmtmhy-selecteodysterwseredetmmmed by measuringthelongestdistm= fimmtheumbo totheedgeoftheshell.Measurementwas made tothencmvd 0.05mm toconfum thattheoysterfselwlithinthe 25 to50 mm sizecriterioTnh.e averagelengthoftheoysterwsas 33.8mm witha rangeof27.8to41.5mm At -testnutiationt,heoysterswerecoBeeW from theholdingtank-.anmdchsmmmately transferrteodthetest chambers. DilutioWnater The waterusedforholdingand Wstmg was naturalseawatercorertedatInch=Rim bK Delawarea,nd dilutetdoa salinitoyfapproximatel2y0%o withwellwater.The dilutiownaterwas storedina 19,000-Ltank where itwas aeratebdy rcc=dation.Salinitaynd pH mewm=mits takenduringthefour-weekperiod m=echat@lyprecedintghetestarepresentedm Appendix I.The resultosfanalysespcrfm2cd tomeasuredthe - of selectecdontaminantms saltwateursed by VrddhfeInternationLatld.= presentemd AppendixIL TestA am-atm Testchamberswere52-Lpolyethylenaequariacontaininagpproximatel4y0 L oftestsohticilEachtest chamberwas cmtmwwly shrredtocuudatethesupplementaallgaeusingan electripcaddlemv= Inaddition, eachtestchamber was gentlyamted. The depthofwaterinarepresentatitveestchamberwas approximatel2y1 cm. Testchamberswere impartiallpyositionedm an envwonmmtzd chamber settomaintaina t=pmzbm of 22-+IOC.The testchamberswerelabeledwiththeprojecmtimber and testconcentration. WILDLIFE INTERNATIONAL L-m. PROJECTNO.: 454A-106 EnvironmentaClonditions Liofang usedtoilluminateheholdingtanksand testchambersdunng holdingand tmtmg was pmvided by fluorescenttubesthatemittedwavelengthsimilatronafta@dsunligh(tColortone5*0).A photoperioodf 16 hoursoflighatnd 8hoursofdarknesswas controllewdithan automatictima. A 30-minutetransitipoenriodof low lighitntensitwyas providedwhen lightwsent on and offtoavoidsuddenchangesinlightingL.igbtintensity attestinitiatiwoans approximatel4y98 luxatthesurfacoef ffiwcater.Lightintensitwyas memured usinga SPER ScientifLitcd.lighmteter Temperaturewas memared ineachtestchamber atthebeginningand end ofthetestusingahquid-inglassthermometer.Temperaturealsowas measuredcmfinuouslyinffincegativeomtroltestchamberusinga FulscopeER/C Rworda. The targetestemperaturdeuringthesb* was 22+-10C.Dissolvedoxygen was memrod ineachtestchamber dailyM.eummenu ofph w= ma& inewh testchamber attestkftiaucma,fter approximatel4y8 hoursand attheendoftbetesl Dilutiownatersalinitwyas measuredattestinitiatiaonndtest termination. Measurementsofph weremade usinga FisherAccumet Model 915 pH mew, anddl%sohedoxygenwas -measuredusinga YellowSpringsInstrumenMtodel 5IB dissolvodowgm meter.Salinitywansx=m)d usinga Bio-Mmine,Inc.Aquafaunareftcwmeter. Observations OysterswereobservedailyduringthetestformortalitayndclinicsailgnsoftoxicityA.t theendofft testt,helongestfingerofnew shellgmwth was measuredtodw nearest0.05mnL StatistiAcnaallMes Shellgmwth mhibitlownas calculatefdoreachbtabnentgroupasthepmmt reductioinnshelglrowth relativteomean shellgrowthinthenegativceontrolThe followinfgormulawas used. Mean ShellCcowdit,.,- Mean ShellCwowdkw...t % Inhibiti-on Mean ShellCnuwdit,., WILDLIFE INTERNATIONAL LTD. -12- PROJECTNO.: 454A-106 The EC50 valuewas estimatebdy visualinspectioonftheshelglrowthinhibitidoanta.The shelglrowth datawas evaluatefdornormalityandhomogeneityof varianceussingtheChi-SquaretestandBartlett'tses@ respectivelDyu.mea's testwas dm usedtoidentiftyreatmengtroupswhichhad a statisticaslilgynificant (:s0.05)reductioinnshellgrowthascomparedtothecontro(l4). An4Lyfical Watersampleswerecollecteadtnud-*th from eachftatinenatndthecontroglroupatthebegmrangof thewA atapproximatel4y8 hoursandattesterminatiotnomeasureconcmftfionsofthetestsubstance.The sampleswerecollecteidnplasti(cNalgeneO)bottleasndma4-md mmuxhatelywithoutstorageA.ftermviewof theanalyticraelsulttsh,esampleswereM=47,ed induplicateA.nalyticaplrocedureussedinthean*= ofthe samplesareprovidedinAppendixIIL RESULTS AND DISCUSSION MeasurementofTestConcentrations Resultsofanalym tomeasurecon=bzfions ofPFOS inwatersamplescollecwduringthetestare presenteidnTableI andintheanalyticcahlemistrryeport(AppendixIII)N.omiradconcmftfionsselectefdor useinthisb* were 1.2,2.0,3.35,.5and9.1mg ai.AL.Samplescollecteadttestin@mhad measuredvahies thatrangedfrom 28 to460/ofnominalvalues.After24 hoursofmixing,the stocksolutiownas measuredtobe 32 to38% ofnomihd andwas probablyatthe.limoiftsolubiliitnyunfilterswaltwaterT.he recoverieisntheothertestconcentratiownesre sundarbecausedM wereproportionadlilutionosftheprimary stock Measuredvaluesforsamplestakenat48 hoursrangedfi-om15 to41% ofnominal.Measuredvaluesfor samplestakenat96 hoursrangedfi-om<LOQ to52% ofnominal.When measuredconcentratioonfsthesa=l ana4,mdattestwitaatloanp,proxmuftly48 hoursand attestterminatiownereavan4 themean measured concentratiofnosrthissb* were 0.36,0.40,1.3,1.9and 3.0mg ai.AL.Me= measuredconcentmtionwsere usedintheestimatioonftheEC50 value. Observationasnd Measurements Measurementsoftemperatured,issolveodxygenand pH arepresenteidnTable2. Temperatureswere vnthm the2210C rangeestablishefdorthetest Dissolvedoxygenconcentratiornesmainod;->6m.g1/L (79 percentofsaurationt)hroughouthetestand pH rangedfrom 7.5to8.1.Dilutionwatersalinitmyeasuredattest initiatiaonnd testterminatiownas 20 and 2 I%o,respectively. AAENDED WILDLIFE INTERNATIONAL LTD. -13- PROJECT NO.: 454A-106 OystersinthenegativceontrolandallPFOS uvth=t groupsappearednormalandhealthythroughout thetestpmiocl Afta 96 hoursofexposurro,ystershelglrowthinthenegativecontrowlas 2.67mm (Table3). Mean shelglrowthinthe0.36,0.40,1.3,1.9and 3.0mg a.L/Ltreatmentgroupswas 2.50,2.40,2.512,.13and 1.91mm, respectivelWyh.en comparedtodw negativecontroglroup,shellgrowthinlu'bitriaongedfrom6.40/o inthe0.36mg &i.ALtreatinegnrtoupto28% inthe3.0mg &iJL treatmengtroup.Dunnett'stestshowed that oystershelglrowthwas sigafficantrleyducedinthe3.0mg &iJL uwtinmt groupincomparisontothenegative controlIndividuamleasurementsofoystershellgrowtharepresentedinTable4. CONCLUSIONS The 96-hourEC50 valueforeasternoysters(CrassostreaWrginica)exposedtoPFOS was >3.0 mg &i.ALt,hehighestconcmtratiotne-stedand thepracticallimitof solubiliitnyunfilterseadltwaterB.asedon a statisticaslilgynificarnetductioinnsheldlepositioinnthe3.0mg &i.ALU=Unent group,theLOEC was 3.0mg &i./LandtheNOEC was 1.9mg &iJL. AN[ENDED WILDLIFE INTERNATIONAL LTD. -14- REFERENCES PROJECT NO.: 454A-106 1 U.S.EnvironmentalProtectioAngency. 1996.Series850-EcologicEaflfectTsestGuideline(rd,raft), OPPTS Number 850.1025:OysterAcuteToxicitTyest(ShelDleposition). 2 U.S.EnvirommentalProtectioAngency. 1985.StandardEvaluatioPnrocedureA,cuteToxicitTyest forFstua?ineandmapine Organisms(Mollusc96-HourF7ow-Arough ShellDepositioSntudy).EPA 540/9-85-011.Washington,D.C. 3 ASIMSUmdardE729-Ma. 1994. Standard Guidefor ConductingAcute ToxicityTestswith Fishes, Macroinvertebrates,andamphibians. American SocietyforTesting and Materials. 4 West,Inc.and D. D. Gulley.TOXSTAT Version3.5. Copyright1996. WesternEcoSystems TechnologyI,nc,Cheyem, Wyommg WILDLIFE INTERNATIONAL L-m. -15- PROJECT NO.: 454A-106 spomr. TestSubstance: TestOrganism: DilutioWnater- NominalTest Concentration @29&iJL) NegativeControl Table1 Summmy ofAnalytiedChemistryData 3M Corporation PFOS EasternOysterC,rassosovawrginica UnfiltmedSaltwater Sampling Time (Hours) Measm-ed Concentration (mgai.!U McimMeasured Concentration (MgLi./L) 0 <LOQI <LOQ 0 <LOQ 49-, <LOQ 48 <LOQ 96 <LOQ 96 <LOQ 1.2 0 0 48 48 96 96 0.331 0.353 0.341 0.429 <OQ <LOQ 0.362 2.0 0 0 48 48 96 96 0.622 0.40 0.633 0.299 0.313 0.249 0.257 3.3 0 0 48 48 96 96 1.36 1.3 1.15 0,924 0.879 1.58 1.72 5.5 0 0 48 48 96 96 2.42 1.9 2.53 2.02 2.24 1.45 0.970 9.1 0 0 48 48 96 96 3.44 3.0 3.01 3.74 3.57 1.99 2.19 'Thelimiotf on (LOQ)was 0.115Mg Li./L. 2]K4C4Mmc&qurecdoncentratidoonesnotinrludevaluescLOQ. Peroot Of Nominal 30 .20' 39 35 33 ANMNDED -16- Table2 Temperature,DissolvedOxygen and pH ofWater intheTestChambers Sponsor: 3M Corporation TestSubstemm: PFOS TestOrganimn: EasternOyda, Crasiostreva7rginica DilutionWater: UnfiltereSdtwater Mean Meanned 0 Hour 24 Hours 48 Hours 72 Hours TestC4n=tmtion Teml D& DO DO DO Ten (mgaiJL) (*C) (mg/L) pH (MGIL) (mg/L) pH (MWL) (-C NegatiVeControl 22.3 7.6 8.1 6.8 6.9 7.7 6.6 22. 0.36 22.3 7.6 8.1 6.6 6.7 7.7 6.5 22. 0.40 22.4 7.6 8.1 6.4 6.6 7.7 6.3 22. 1.3 22.6 7.6 8.1 6.4 6.6 7.7 6.3 22. 1.9 22.6 7.6 8.1 6.4 6.7 7.7 6.3 22. 3.0 22.7 7.7 8.1 6.4 6.S 7.7 6.1 21. 2 Tempmture mmmed continwuslyduringdw tesrtangedfromapproidmtel2y2.0to22.50C. A disgohmdoxygenconcentmtionof4.7mg/L represent6s0% sahnadoil WILDLIFE INTERNATIONAL LTD. -17- PROJECTNO.: 454A-106 Table3 ShellDepositioanndShellGrowth InhibitioantTestTmunatim Sponsor: 3M Corporation TestSubstance:PFOS TestOrganism:EasternOyster,CrassostreaWrginica DilutioWnater: Unfiltm-eSdaltwater Mean Me&un-ed Concentration ShellDepositio(nmm) (mg&iJL) mcim sd NWfive Control 2.67 0.824 ShellGrowth Inhibiti(ocn/o) 0.36 2.50 0.933 6.4 0.40 2.40 0.820 10 1.3 2.51 0.919 6.0 1.9 2.13 0.804 20 3.0 1.912 0.591 28 The 96-hourEC50 valuewas >3.0mg &iA,. 'Mean andstanduddeviatimfor20 mystm. 2Indicateassigmficancthffcrencfei-omtheElLti@vecontrolusingDumctt's test(P--'@0.05). AHMMED WILDLIFE INTERNATIONAL LTD. -18- PROJECT NO.: 454A-106 Table4 IndividuOaylsterShellMewum=ts Sponsor: 3M Corporation TestSubstance:PFOS TestOrganLsm: EasternOyster,Crassostreawrginica DilutionWater: Unfiltm-eSdaltwater Mcim Measmed Co@tions (mg &i./L). NegativeContml 0.36 0.40 1.3 1.9 3.0 1 4.00 2.05 3.20 1.50 2.20 1.65 2 3.25 1.80 2.25 2.80 1.45 0.80 3 3.00 1.90 3.10 2.65 3.60 2.20 4. 2.55 3.65 0.90 2.25 1.80 1.75 5 2.50 2.55 2.35 2.40 2.25 3.05 6 2.75 1.90 2.10 2.70 2.30 1.60 7 2.70 2.40 2.25 2.90 3.10 0.90 8 2.70 4.00 2.90 4.45 0.65 2.40 9 1.85 1.45 1.75 1.80 0.90 2.25 10 2.20 2.55 2.45 3.05 2.40 1.80 11 4.40 3.55 2.85 1.60 2.70 2.70 12 2.05 1.80 3.00 0.85 1.65 1.10 13 1.55 3.35 4.45 4.10 1-.80 2.35 14 3.05 2.30 3.05 2.70 i'.Io 1.70 15 2.45 3.70 1.95 2.45 1.70 2.15 16 4.20 3.75 1.20 3.00 3.15 2.60 17 1.45 2.60 1.70 1.85 0.90 1.60 18 1.75 0.90 2.05 2.95 2.20 1.45 19 2.85 2.85 1.65 0.95 2.90 2.00 20 2.20 0.95 2.90 3.30. 2.85 2.20 Mean 2.67 2.50 2.40 2.51 2.13 1.91 SD 0.824 0.933 0.820 0.919 0.804 0.591 RaW 1.45-4.40 0.90-4.00 0.90-4.55 0.85-4.45 0.65-3.60 0.90-3,05 AMENDED WILDLIFE INTERNATIONAL LTD. -19- PROJECTNO.: 454A-106 APPENDIXI SalinitayndpH ofUnfiltereSdaltwateMreasured Duringthe4-Week PeriodImmediatelyPrwAxhngtheTest Spomsm: TestSubstance: TestOrganism: DilutioWnater: Salinity (0/00) pH 3M Cmporation PFOS Eastmn Oyster,Crassostreavlrginica Unfiltm-eSdaltwater MeAm 21 (N = 4) 8.1(N = 4) Range 20-21 8.0-8.2 WILDLIFE INTERNATIONAL LTD. -20- PROJECT NO.: 454A-106 APPENDIXII AnalysesofPesticidesO,rganics,Metals and OtherInorganics inWildlifIenternationLatld.Saitwaterl ANALYSIS bULtSURED CONCENTRATION bnsceumneom MCBMulmemb ToW Dissohvd Solids Ammoni, N&M@@ Tow Orpak TOW CYR" Orgenochlorhm and PCBO Aldrin AlphaBHC BD&BHC Doha BHC Gamm BHC (LWm) Chlordane DDD. pp' DDE.W DDT. pp' DiddritL End=,w- A End=W- B FAdoviihnSuffift F-irw F-hin Aldthy& HoptacWMWWXYCWor He$addw Epo3d& Touphme PCB-1016 PCB-1221 PCB-1232 PCB-1242 PCB-1249 PCB-1254 PCB-1260 Metab and00or laorganies Ahnnfiniii? Arvage BaAk-' cadMb.n3 Cddtm? awomhm@ Cobae Macmy mobtdomm, )Ci" Pd=hM? Sdaubm? Suva? SO&& zhe 23,500 < 0.050 < 1.0 < 10.0. < 0.005 < 0.005 < 0.005 < 0.005 < 0.006 < 0.025 < 0.006 < 0.005 < 0.009 < 0.005 < 0.005 < 0.005 < 0.019 < 0.010. < 0.005 < 0.005 < 0.007 < 0.005 < 0.500 < 0.260 < 0.260-- < 0.260 < 0.720 < 0.720 < 0.720 < 0.720 < 100 < 25.0 < 0.50 < 1.0 235 < 2.0 < 1.0 < 20.0 < 100 < 10.0 760 < 4.0 < 0.20 < 2.0 < 20.0 277 < 25.0 < 1.0 6,010 < 20.0 n*/L mg/L MSIL pg/L P91L pg/L MIL PSIL PSIL PWL PVL M& M& MIL P91L M& M/L WL pg/L P91L P91L P91L P91L M& M& PSIL "S MIL P91L 99/L PWL "1 pg/L "L MWL Pff/L M/L M& P91L PWL MWL MIL pg/L MIL usdl 'Analym pafornwd by QST EuvrommaL GamwwUc6 Flon& forsampla coUacW on November 3 *sough November 7,1997 'Anaiym pafmw by vrddeh int-amiceaii& forihewm*h wUecW on November 5,1997. 'Anaiysapa&=d by vrddbh hdanwwnat Lt& firumpia conededon November 5 ftm& 7,1997 WILDLIFE INTERNATIONAL -21APPENDIXIII PROJECT NO.: 454A- 106 THE ANALYSIS OF PFOS IN UNFILTERED SAL TWATER IN SUPPORT OF WILDTIFE RMRNANONAL LTD. PROJECT NO.: 454A-106 WILDLIFE INTERNATIONAL L-m. -22- PROJECTNO.: 454A-106 REPORT APPROVAL SPONSOR. 3M Corporation TITLE: PFOS: A 96-HourShellDepositioTnestwiththeEasterOnyster(Crassostreiadrginica) WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-106 PRINCIPAL RQVESTIGATOR- Rayn%'Kd Ll Van HovM Ph.D. Scientist MANAGENffiNT; WillardB.Nixon,Ph.D/ Manager,AnalyticaClhemistry DATE DATV ANUI@MED WILDLIFE INTERNATIONAL L-m. -23- PROJECTNO.: 454A-106 Unfifteresdaltwatesrampleswere collectefdrom an wot toxicitsyh* designedto determintehe effectosf PFOS (PerfluorooetaSnuelfoniAccid PotassiumSalt)on theshelldepositioonf theeastern oyster(Crassostre-awrginica)I.bisstudywas conductedby Wddhfr,kftmationalLtd.and identlfiaesd ProjecNto.: 454A-106. The analysesoffl= watersampleswere performedatWildlifIenternational Ltd. usinghigh performanceliquidchromatographywith mass spectrometri&c-tecfio(nBPLC/MS). Sampleswere receivefdoranalysiosn May 24,26 and 28, 1999. Samples were analyzedon eachsample receipdtay. AU sampleswere reanalyzeodn June 6, 1999 to confirmoriginaalnalyses.The second analysiwsas reported. TestSubstanceand Internal The testsubstancuesedfordw analyticpaolrtionof tlussu* was WddA InternatioLntadl. identificatniuomnber 4675. The testsubstancewas used to preparecalibratiosntandardsand matrix fortificatsiaomnples. The internasltandardwas remved from 3M Corporatioonn July2, 1998 and was assignedWildlife InternationLatld.identificatniuomnber 4526 upon receiptT.he internaslundard,a g=ulu materialwas idenfifiaesd: IK IIi,2K 2]HPerfluorooetaSnuelfoniAccid,ChemicalAbsftd Number: 27619-97-2. The standardwas storeudnderambientconftons. Ang)EficaMlethod The method used fortheanalysiosf theunfikm-edsaltwatesrantij@weass developedat WMife InternationLatld.and entitle"d'AnalyticMaelthod Validatiofnor the Ddmmimtion of PFOS in FreshwaterS,altwatearn,d Mgal MeM'. 11@smethodologwyas includeads AppendixIlofWildlife InternationLatld.protoconlumber 454/011299/MVAUSUB454. It was based upon methodology providedby 3M Corporation. Sampleswere dilutedin a 50% methanol: 50% NANOpure(D water solutioncontaining 0.100mg4HPFOS (internsatlandard)AaLnd 0.05% fomiicacid(v/v)so thattheyfellwithinthe calibratirodnngeofthePFOS methodology. WILDLIFE INTERNATIONAL LTD. -24- PROJECTNO.: 454A-106 Concentrationosf PFOS in the standardsand sampleswere determinedby reversed-phahsiegh performanceliquidchromatographyusinga Hewlett-PackardModel 1100 High PerformanceLiqtud Chromatograph(HPLC) witha Peridn-EhneArPI IOOLC Mass Spectrometeerquippedwitha PerlanElmer TurboIonSprayion source.HPLC separationwsere achievedusinga KeystoneBetasilCls analyticacloluirm(100mm x 2 nun I.D.,3 pm particle.sizTeh)e.finuumentparametersaresummarized inTable1. A methodflowcharitsprovidedinFigure1,. CalibratioCnurve and Lmut ofOumrhtation Calibratiosntandardsof PFOS preparedin a 50% methanol 50% NANOpure(D watersolution containin0g.100mg 4H PFOS (internsatlandard)/aLnd 0.05% formicacid(vtv)r,anginginconcentration from 0.00229to 0.0274mg a.i.AwLere analyzedwiththe samples.Linearregressioenquationwsere generatedusingpeak am responseratios(PFOS :internasltandardv)ersustherespectivceoncentration ratios(PFOS :internasltandardo)fthe calibratisotnandardsA. typicaclalibraticounrveispresenteidn Figure2. The concentratiofn PFOS in thesampleswas determinedby substitutitnhgepeak area responseratiosintotheapplicablIemearmw=sion equation.Plzpresentatiovnechronutagmms oflow and highcalibratiosntandardasrepresentedinFigures3 and 4,respectively. The method limitofquanttatio(nLOQ) fortheseanalyseswas setat0.115mg a.i.AcLalculateadsthe productof thelowestcalibratisotnandardanalyzed(0.00229mg ai.AL)and thedilutiofnactorof the matrixblanksamples(50.0). MatrixBlankand FortLficatSiamnles One matrixblank samplewas analyzedto &-tmmine possibleinterfermiceN.o interferencweesre observedator above theLOQ duringsamplesanalysesCrable2). The matrixblankchromatogramis presentedinFigure5. Unfilteresdaltwatewras forfifiaetd0.457,4.57and 11.0mg &iJL and analyzedconcuffadywiththe samples(rable2). Smnple conomt-dfionwsere not con=W formean procedurarlecovery.High recoveriewsereobtainedforthelow-leve(l0.457mg a.i./Lm)atrixfortificatsiaomnple.Previousanalyses insaltwatevrerifietdhatthemethodiscapableofproducingquanumm recoverieastthisconcmtr@on level.A representaticvheromatogramofa matrixfortificatiospnresenteidnFigure6. AMENDED WILDLIFE INTERNATIONAL LTD. -25- PROJECT NO.: 454A-106 E&=Ic Calculations Samplenumber 454A-106-5B,nminal concentratioonf5.5mg a.i.AiLnunfilteresdakwater. InitL-dVolumeO:.IOOML FinalVolume: 25.0mL DilutioFnactor:250 PFOS Peak Area: 71220 InternaSltandardPeak Area.-74271 Peak Area Ratio:0.95892 Calibratiocnurveequation. Slope:7.78372Intercept0:.17175 Curveisweighted(1/x). PFOS (mg a.i./La)thabument Note:I.S. internasltandard. PFOS (mg a.i.ALi)nsample (Peakarearati-o (Y-intereepxt)I).S.Concentration Slope (0.95892- 0.17175)x 0.100mg a.i.AL 7.78372 0.0101mg a.i.AL PFOS (raga.i.ALa)tinstrumenxtFinalVolume,(mL) hlww Volume(mL) 0.0101x 25.0 0.100 2.53mg a.i.AL AAIENDED WILDLIFE INTERNATIONAL LTD. -26- PROJECTNO.: 454A-106 PercentofNominal Concentration Calculatedrecovery4:6.1% Note:manualcalcumon may differ. PFOS (mg a.i.ALi)nsample x 100 PFOS (inga.iJLn)ominal RESULTS S@Mle Analysis Unfiheredsaltwatesrampleswere collectefd3romthe shelldepositiotnestwith the easternoyster (CrassostreaWrginicat)testifi@ May 24, 1999 (Hour0),on @by 26,1999(Hour48),and attest terminatioMna,y 28,1999 (Hour96). The measuredconcentmtionosfPFOS inthesamplescollecteadt initiatioofnwqx)sureof the testorganisms(Hour 0) rangedfrom 27.9to 46.1% of the nominal concentrationSsa.mplescollecteadtHour 48 had a measuredconcentratiornangeof 14.9to40.9% of nominalvalues.Samplescollecteadttestterminatio(nHour 96)had a measuredconcentratiornangeof. LOQ.to 52.2% ofnominalvalues(rable3).A representaticvheiomalogramofa testsampleisshown in Figure7. AUMNDED WILDLIFE INTERNATIONAL LTD. -27- PROJECTNO.: 454A-106 INSTRUMENT: ANALYTICAL COLUMN: OVEN TEMPERATURE: STOP TIMEE: FLOWRATE: -MOBILE PHASE: Table 1 TypicalLC/MS OperationaPlarameters Hewlett-PackarMdodel 1100High PerfomianceLiquid Chromatographwitha Peridn-ElmerAPI 10OLC Mass Spectrometer eqwpped witha Perian-Ehm TurboIonSprayionsource.Operatedm selectiivoenmonitorinmgode (SM. Keystme BetuffC,8column(50mm x 2 nun I.D.3, pm particlseize) 300C 5.00minutes 0.220mljniinute 72.0% Medmol: 28.0% NA@40pureM Water containin0g.1% Fonnic Acid INJECTION VOLUMEE: 25.0 IAL PFOS RETENTION TIME: Approxftnatel3y.6minutes R4TERNAL STANDARD RETENTION TIME: Approxhately2.6mkutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 426.7 WILDLIFE INTERNATIONAL LTD. -28- PROJECTNO.: 454A-106 Table2 MatrixBlankand FortLficataoAnsalyzedConcurrentlDyuringSample Analysis Sample Number (454-106-) Type MAB-4 MatrixBlank ConcentmfionosfPFOS (mg a.i.AL) Fortified 0.0 Measuredi <LOQ Percent Recovered - MAS-10 MatrixFortification 0.457 0.760 166 MAS-IOA@ MatrixFortification 0.457 0.812 177 MAS-10B 2 MatrixFortification 0.457 0.769 168 MAS-1 1 MatrixFortification 4.57 4.57 99.8 MAS-12 MatrixFortification 11.0 13.0 119 Note:Rmft and correctionfsornew testsubstancepurityweregeneratedusingMacQuan version1.5 softwareand manual calculationsV.alueshave,beenroundedforreportinpgurposes. 'I'hleimitofquazifitat(iLoOnQ) was 0.115 mg a.i.AbLasedupon the productofthelowestcalibration standardanalyzed(0.00229mg a.i.ALa)nd thedilutiofnlictofrthematrixblanksamples(50.0). 2 Sample 454-106-MAS-10 was rediluteidn duplicataend analysicsonfmnedoriginarlesults;uspect -fortificateirornor. AMENDED WILDLIFE INTERNATIONAL -29- PROJECTNO.: 454A-106 Table3 Measured Concentrationosf PFOS inUnfiltm-eSdakwater Samples from an OysterShellDepositionTest NomirW Tcd Conowtafion (mitaiJL) o.o Sampk Number (454A-106-) IA IB 7A 7B 13A 13B Sampling Time (Hours) 0 0 48 48 96 96 PFOS Measured Concentration' (mR aijl) < LOQ < LOQ < LOQ <LA)Q < LOQ <LA)Q PCM=T Of Nominal - 12 2A 0 2B 0 &A 49 BB 49 14A 96 14B 96 0331 27.9 0.353 29.7 0341 29.7 0.429 36.1 < LA)Q - < LOQ 2.0 3A 0 3B 0 9A 49 9B 48 ISA 96 15B 96 0.622 0.633 o_lw 0313 0.249 0.257 30.9 31.5 14.9 15.6 12A 12.8 33 4A 0 4B 0 10A 49 IOB 48 16A 96 16B 96 136 1.15 0.924 0.979 1.58 1.72 41A 34.9 28.0 26.7 48.0 522 5.5 5A 0 5B 0 IIA 49 IIB 49 17A 96 17B 96 2.42 2.53 2.02 2.24 IA5 o.@yo 44.1 46.1 36.9 40.9 26.4 17.6 9.1 6A 0 6B 0 339 37.1 6C 0 3.44 37.6 6D 0 12A 48 3.01 32.9 3.74 40.9 12B 49 3.57 39.0 ISA 96 1.99 21.7 IBB 96 2.19 23.9 Note ResWb and cmywuons for now Ug substancepunty were generatedusingblacqun vemon 1.5softwaraend rnAnuW calculatioVnasl.ueshavebm rounded fi)rreportingpurposes. The limitof quenfiftf(iLoDnQ) was 0.115mg aiJL basedupon thepmductof thelowestcalibratisotnandaradnab-nd (0.00229mg a.iJL)andthedilutiofnilotoofrthematrixblanksamples(50.0). 2 Romh forsample6A (621 mg aiJL)was notincludeidnthetable.Sample6 was remuted induplic4o (6Cand6D) andresultosf thew samplesconfirmedrmh forsample6B. AMENDED WILDLIFE INTERNATIONAL L-m. -30- PROJECTNO.: 454A-106 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN UNFTLTERED SALTWATER Preparematrixforttficatsiaomnplesby sp&% therevisftvolumeofPFOS stocksolutiondsirectly intounfilterseadltwateursinggas-tgk syringesand ClassA volumetneflasks. Dilutematrixfortificataonndtestsamplesintotherangeofthecalibratisotnandardbsy pubay filling ClassA volunietifilcaskwsith50% medawl :'5(r/NoANOpure(D watersolutiocnontaining 0.100 mg 4H PFOS (internsatlandarclyalnd 0.05% formicacid(v/y).Add theappropriatveolumeof sampleandbringtheflasktovolume with&c dklutiosnolventProcessthematrixblanksampleusing thesame dilutioannd aliquovtolumeas forthelowestfortificatlieovnel.Mm wellby severarlepeat inversions. Ampulatesamplesand submitforLC/MS analysis. F-igure1. AnalyticamlethodflowcharftortheanalysisofPFOS inunflheresdaltwater. WILDLIFE INTERNATIONAL L-m. -31- PROJECTNO.: 454A-106 3.00- 2.50- 0 2.00- 0 0 .1,-1.50 co 1.00- 0 0 0,50- 0.00 0.000 0.050 ...... 0.100 0.150 0.200 0.250 0.300 Concentrati(mRafio) Figum 2. A typicaclalibmtiocnurveforPFOS. Slope 7.78372;hdercept 0.17175;r 0.9825. Curveisweighted(I/x). ANENDED WILDLIFE INTERNA-nONALL-m. -32- PROJECT NO.: 454A-106 lntens1f1t0r2 .cps. 100. 86, 90- 80- 70- Go. 40, so. 73 20- 15 28 - 44 5268 1 1 109 21 471 6*1 0.88 1.72 2.65 329 423 7kne Figure3. A representatiivoenchromatogramofa low-lmd (0.00229mg A.LAL)PFOS smndarcl AMENDED WILDLIFE INTERNATIONAL -33- PROJECT NO.: 454A-106 lntermftWr97 cps 100- 86 90. 80- 70- so- so@ 4& 73 3& 2(@ 0 A 0 902 ii 471 6*1 ii OA8 1.72 2.65 3.39 423 Tkne, F"igure4. A reprmtadve ion ofa high-leve(l0.0274mg a.iJL)PFOS swadard. ANMNDED WILDLIFE INTERNATIONAL LTD. -34- PROJECTNO.: 454A-106 14 100- 90. fntensftr2.80 cps 70. 60. 5040-30, 20- 14 14X 1. f\!@,-A 3 50 61 67 A K!SCU yy xi- 4*1 ii 0.88 1.72 2.55 86 3.39 104 AA--.@= i v 1*61 sm 423 Time Figum 5. The matrix blank ion chr=atogram (454A-106-MAB4). The arrow indicatetshe retentiotnime of PFOS. WILDLIFE INTERNATIONAL LTD. -35- PROJECTNO.: 454A-106 100. kftmltr.9001 cps 86. 80. 7 60, so, 40 30- 73 20- 14 98 38 48 SS ft grin 0 . 8- 0 1. 21 41 67, 81 ioi sew 0.88 1.72 2ss 3.39 4.23 Thm F'igur6e. A representatiivoenchromatogramofa 4.57mg a.i./mLatrixfortificatsiaomnple (454A-106-MAS-11). AMENDED .WILDLIFE INTERNATIONAL -36- PROJECTNO.: 454A-106 100. 90. 80. 70- S& 40so. 20- -2 OM lntenskr.6239@cps 87 73 4Z 471 871 st ioi sm, 1.72 2.65 sm i,423 Tkne Figure 7. A representativieon ofa 5.5 inga.i./Ltestsample (454A-106-5B). AMENDED WILDLIFE INTERNATIONAL LTD. -37- PROJECTNO.: 454A-106 APPENDIX IV ChangestoProtocol ThLssh* was conductedm accordancvenththeapprovedProtocowli& thefouowmg changes: I- The protocolwas amended to a& the proposodexpmimentidstartand taminationdates,test concentrationasnd testsubstancenumber. 2. 1'heprotocolwas amendedtospeciftyhatGLP analym ofsdtwaterwillbe repaft& 3. The protocolwas amended tochangetheanalyticsaalmplingschedule. 4. Salinitwyas measuredinthenegativecontrolattestnutiatioannd tmmination. WILDLIFE INTERNATIONAL LTD. -38- PROJECT NO.: 454A-106 APPENDDCV PersonneIlnvolvedintheSh* The followmgkeyArildhfIontmmauonalLtd.pmwnnd w= mvolvedm fficonductormanagementofthis study: 1. Henry0.Kruega,Ph.D.,DirectorA,quaticT("dcologyandNon-Targd Plants 2. WillardB.Nixcm,Ph.D.,Manager,AnalyticaClhemistry 3. Mark A. Mank, LaboratorSyupmvisor 4. TimothyZ.KmdalL LaboratorSyupavisor 5. KurtI;LDmttar,SeniorBiologist -6. Raymond L.VanHo%@en,Ph.D.,Scientist WILDLIFE INTERNATIONAL L-m. -39- PROJECTNO.: 454A-106 APPENDIXVI ReportAmendment I. OriginaRleport: Pages14, 6 and22 Amendment: Rcimn: Ile pageswerechangedtoincludetheamended reportdate,revisepdagenumb=, and new sigiiatm=anddatesduetotheadditioonfthereportamendment asAppendixVL To reflecttheissuingofan amended reporl 2. OriginaRleport: Page 2 Amendment: Reason: The.compliancestatementwas revised. To clarifhyow thetestsubstancewas characterized- 3. Ori&W Report: Page 9 Amendment: informatiopnrovidedby theSponsorreflectitnhgeremudysisof thetestsubstance, includintghereanalysidsateand thepunty,was addedtotheTestSubstancesection. Reason: To reflectthecurrentestsubstancemformatlonprovidedby theSponsor 4. OnpW Report:Entirreeport Amendmmt:. AU testsubstancecono=Umbous were changedtoreflectthepuritoyfthetesstubstance asdeterminedby theSponsorina reanalysiosfthetestsubstanc(eFC-95,Lot217). Testcdncatrafionosriginallwym basedon thereportepdwityof 98.9*/oT.he certificaotfeanalysidsaed March 9,2000 indicateadpuritoyf90.49'/*T.hereforael,l testsubstanceconcentrationisn.c,ludingnominal cm=trations, measured concentrafionasn,d LC50 values,were recalculateadndreportedasmg ai.ALbasedon the90.490/pouzity. Reason: To reporttheresultosf thetestbasedon thetestsubstancpeurityof 90.490/aotthe requestof theSponsor. 5. OriginaRleport: Page 15 Amendment: Add footnoteIsand 2 toTable1,Summary ofAn*fical ChemistryData. Reum: To clariftyhemeasuredconcentratiornVsortecL ANM'NDED r WILDLIFE INTERNATIONAL LTD. -40- PROJECTNO.: 454A-106 APPENDIXVI -Continued- ReportAmendment 6. OriginaRleport: Page 17 Amendment: Changethe96-hourEC50 valueof>10 mg &i./Lto>3.0mg a.L/L. Reason: To correcthereportedEC50 valuetoreflectthemean measuredconcentratiorna,ther dm thenominalconcmtratiom AMENDMENT SIGNATURES: KurtR. Drottar Sb* Dffwtor Hedry10.Krueger,?h.1). .D=W, AquaticToxicologyandNon-TargetPlants REVMVED BY: JaifiiHs..Coleman QualityAssurance TIV- DATE DA-T -L7 q-,A6-00 DATE AMENDED WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 4S4A-106 Page I of 2 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR SHELL DEPOSITION TEST WITH THE EASTERN OYSTER (Crmsostreavirgi?iica) PROTOCOL NO.: 454/042199/OYS-DEP/SL-B454 AMENDMENT NO.: I SPONSOR: 3M Corporation PROJECT NO.: 4S4A- 106 EFFECTIVE DATE: May 20,1999 AMENDMENT: Page2 Add: ExperimentalStartDate: 5/24/99 ExperimentalTerminationDate: 5/28/99 TestConcentrationsN:egativeControl,l.'72,.2,3.6,6.0and 10 mg a.i./L TestSubstanceNo.: 4675 REASON: The above informatiownas not known when the protocolwas signedby theStudy Director. AMENDMENT: DilutioWnater,Page 5 Change: Analyseswillbe performedatleastonce annuallytodeterminetheconcentrationosf selectedorganicand inorganicconstituentosf the seawaterand theresultosf the analyseswillbe sun-Lmarizeidnthe finalreport. To: Analyseswillbe performedatleastonce annuallyto determinetheconcentrationosf selected organicand inorganicconstituentosf the seawaterand the resultosf themost recentGLP compliantanalyseswillbe summarizedinthefinalreport. REASON: To speciftyhatGLP analysewsillbe reported. AMENDMENT: Samplinpf,oranalzicaMleasurements,Page 7 Change: Water sampleswillbe collectefdrom each testchamber attestinitiatiaonnd every24 hours (l hour)duringthetestto determineconcentrationosfthetestsubstance. To: Water sampleswiU be codectedfrom eachtestchamber attestinitiatioant,themidpointofthe test(approximatel4y8 hours)and attestterminationto determineconcentrationosfthetest substance. REASON: The Sponsor requesteda change inthe analyticaslamplingschedule. .45#11061ammdl Pip-vitwebdq GA(g WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A- 106 Page 2 of 2 STUDY DIRECTOR LABOICA:TORY MANAGEMENT SPWSOR'S REPRFSENTATRVE DATE DAT@DATE a45411061amen& WILDLIFE INTERNATIONAL L-rD. PROJECTNO.: 454A-106 Page I of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR SHELL DEPOSITION TEST WITH THE EASTERN OYSTER (Crassostrewarginica) PROTOCOL NO.: 454/042199/OYS-DEP/SUB454 DEVIATION NO.: I SPONSOR: 3M Corporation PROJECT NO.: 454A-106 DATE OF DEFACTO DEVIATION: May 24,1999 DEVIATION: The protocolstatesthatdilutiownatersalinitwyillbe measured ineach test chamber attestinifiatiaonnd attheend of thetest.Dilutionwatersalinitwyas ordymeasured inthenegativecontrolatthebeginningand endof thetest. REASON: BiologisotversightW.ildlifIenternationLatld.storesunfilterseadltwateirna 5,000 gallontankwluchisromrcwatedtomamtmn const= sabmty. Consequently,itisthebest judgmentoftheStudyDirectotrhattiusdeviatiodnidnot adverselayffectheresultosf thestudy. STUDY DIRECTOR L o@ TORY MANAGENffiNT KRD454ahlO6Vrd) DATE DAT@ PROTOCOL PFOS: A 96-HOUR SHELL DEPOSITION TEST WITH THE EASTERN OYSTER (Crassostrewarginica) U.S.EnvironmentaPlrotectioAngency Series850 -EcologicalEffectsTestGuidelines OPPTS Number 850.1025 3M Lab RequestNo. U2723 Submittedto 3M Corporation Envkonmcntal Laboratory Building2-3F,09 935 Bush Avenue St.PaA Minnesota 55144 WILDLIFE INTERNATIONAL LTD. 8598 Commerce I)zive Easton,Maryland 21601 (410)922-8600 April21, 1999 arm LM WILDLIFE INTERNATIONAL, LTD . -2 PFOS: A 96-HOUR SHELL DEPOSITION TEST WITH THE EASTERN OYSTER (CrassostreaVirginica) SPONSOR: 3M Corporation EnvironmentalLaboratory Building2-3E-09 935 Rush Avenue St Paul,Minnesota 55144 SPONSOR'S REPRESENTATIVE: Ms. Susan A. Beach TESTING FACILIT WildlifeInternationaLlt,d. 8598 Commerce Drive Easton,Maryland 21601 STUDY DIRECT Kurt Drottar SeniorAquatic Biologist LABORATORY MANAGENffi-NT: Henry 0. Krueger,Ph.D. Directorof AquaticToxicology& Non-TargetPlants FOR LABORATORY USE ONLY ProposedDates: Experimental StartDate: ProjectNo.: q54A-/0& Experimental TerminationDate: Test Concent-rations: Test SubstanceNo.: ReferenceSubstanceNo. (ifapplicable): PROTOCOL APPROVAL STUDY DIRECTOR ULAgBOR-YifOaRAYG-FJ GENENT /CY17 DATE 13?f DATE SPO@ @OR!S REPRESENTATIVE PROTOCOL No. 454/042199/OYS-DEP/SUB454 DATE 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, L-rD -3- INTRODUCRION WildlifIenternatioLntadl.w,illconducat statiaccutteoxicitteystwiththeEasteronyster (Crassostrveiarginicfao)rtheSponsoarttheWildliIfneternatiLotncaLla,quatitcoxicolofgaycilinty Easton,MarylancLThe studywillbe performedbasedon proceduresintheU.S.EnvironmentaPlrotection Agency Series850 -EcologicaElffectTsestGuidelineOsPPTS Nmnber 850.1025(1);StandardEvaluation Procedure,Acute ToxicityTestforFstuarineandmarine Organisms (Mollusc96-Hour F7ow-Yhrough ShellDepositionStudy)(2)andAS7MStandard E729-88aStandardGitidefoCronductingAcuteToxicity TestswithFishes,Macroinvertebrateasndamphibians (3).Raw dataforallwork pmformed atWildlife InternationLatld,.and a copyofthefinarleportwillbe filebdy projectnumber inarchivelsocatedon the WildlifheitemationaLlt,d.siteo,ratan alternatilvoecatiotnobe specifieidnthefinarleporl PURPOSE The purposeofthisstudyistodeterminteheeffedsofa testsubstanceon theshelldepositioonfthe easternoyster(Crassostrevairginicad)uringa 96-hourexposureunderstatitcestconditions. EXPERIMENTAL DESI Oysterswillbe exposedto a geometricseriesof atleastfivetestconcentrationasnd a negative (dilutiownater)controflor96 hours.One testchamberwillbe maintainedforeachtreatinmatndcontrol groupwith20 oysterisneachchamber.Nominal testconcentratiownisllbe sclwtedinconsultatiwmiththe Sponsor,and willbe basedupon informatiosnuchastheresultosf exploratorryange-findintgoxicitdyata, known toxicitdyata,pbysical/chenupcraolpertieosfthetestsubstancoerotherrelevanitnformationT.arget concentratiownisllnotexceed120mg/L orthesolubililtiymitofthetestsubstancienwater(whicheveris lower).Generallye,achtestsubstanceconcentratiuosnedinthedefinititvestwillbe atleast600/of the nexiIngherconcentratiounlessinformatiocnoncernintgheconcentration-efcfuercvteindicatetshata chffcmtddutim factowrould be more appropriateWater samplesfrom appropriatestchambersVAR be collecteadtspecifieidntervaflosrw*sis ofthetestsubstanceR.esultsofanalysewsillbe usedto calculate mean measuredtestconcentrations. Oystersapproximatel2y5 to 50 mm longwillbe maintainedatWildlifIentemationaLLtd.in =Mtered saltwatefrora periodofatleast10 days. To controblias,oysterussedinthetestwillbe mdlsmmmately distributeNdo.otherpoWntLalsourcesofbiasarewq=W toaffecttheresultosftheSU*. PROTOCOL No. 454/042199/OYS-DEP/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -4- Justpriorto themiltiationf thetest,recentlydepositedshellwillbe removed by gfincbngtheperipheryof theoystersagainstan electridciscgrinderorsfinflaarpparatus.The initiaslhelllength(aftegrrinding)of twentyinchscrin@telyselectedoysterswillbe measured and 20 indiscriminatesleylectedoysterswillbe placedineach testchamber. At theconclusionof the96-hourtestd,epositionof new shellwillbe measured foreach oyster.Sinceshelldepositionisoftenarynmwtricalthelengthof thelongest"finger"of new shell willbe measured usingcalipers.Shellgrowth inhibitiofnoreachtreatmentgroupwiU be determinedby calculatinmgean shelldepositionas a percentageof the control.Mean shellgrowth inhibitioinneach treatmentgroup willbe expressedas a percentageof mean controlshelldepositiomAn EC50 valuewillbe calculatedw,hen possiblet,oexpresstheconcentratioonftestsubstancethatinducesa 50*/oreductionin shell deposition. MATERL4kLS AND METHODS Test Substan Informationon thecharacterizatioofn test,controlor referencesubstancesisrequiredby Good LaboratoryPracticeStandmds (GLP). The SponsorisresponsiblfeorprovidingWildlifeIntmmationalL,td. writtenverificatiotnhatthetestsubstancehas been characterizeadccordingto GLPs priorto itsuse inthe study.IfwrittenverificatioofnGLP testsubstancecharacterizatiiosnnotprovidedtoWildlifeIntemadomd, Ltd.,itwiU be notedinthecompliancestatementofft finalreport.The anacbedform IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR (Appendix 1)istobe used to provideinformationecessaryfor GLP compliance. The Sponsor isresponsibleforallinformationrelatedtothetestsubstanceand agreesto acceptany unused testsubstanceazid/otrestsubstancecontainersremainingattheend of thesu*. P=aration of TestConcentrations The tea substancewillbe administeredtothe testorganismin water. This routeof admmatrauon was selcow becauseitrepresenttshemost likelymute of exposureto aquaticorgamsms. PROTOCOL NO. 454/042199/OYS-DEP/SLIB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -5- TestOrganis Theeasteronyste(rCrassostriedarginicwai)lbleusedinthisteslThisspecieissrepresentatoifve an unportantgroup of organismsand was selectedforuse inthe testbased upon pastuse and easeof handlingin thelaboratory. Test organismswillbe obtainedfrom a commercialsupplieror researchfacilityA.llorganismswill be obtainedfrom thesame sourceand heldinthelaboratoryforatleast10 dayspriortotestingO.ysterswin not be used inthe testiftheyshow signsof diseaseor stressor ifmore than5% dieduringthe7 days prior to the test Saltwatersuppliedtotheoystersduringholdingand WstingwiU be mffltered.A saltwateralgasuch as nalassiosirasp.,S7celetonemsap.,Chaetocerossp.and/orIsoch?ysisp.willbe added to thewater for supplementalnutritioil DilutionWater Water used forholdingand testingwin be naturalunfilteresdeawatercollecteadtMan River Uet, Delaware. The seawaterwillbe pumped hitoa 19,000-Lholdingtankwhere thesalinitoyf theseawaterwill be dilutedto approximately20 O/oo(p@ per thousand)with freshwaterfrom a well on the Wildlife IntmnationalL,td.site.The saltwateirntheholdingtankwillbe aeratedby recirculatiporniortodelivery to the testchambers. Analyseswillbe performedatleastonce annuauy to determinetheconcentrationosf selectedorganicand inorganicconstituentosfthe seawaterand theresultsof theanalyseswillbe summmized inthe finalreporl Dilutionwatersalinitsyhouldbe 20 I%o,and thepH shouldnot varyby more than 1 unitduringthetest. Test ARparatus Testchambers willbe 52-L po4vkleno chambers fillewdith approximately40 L of testsolution. Testchambers willbe impartiallpyositioneidn an environmentalchamber or a wmperatom-controlledwater bath to mstintaina temperatureof 22 I*C. An electrimcixer win be placedin each testchamber to maintainthe suspensionof thealgal&et. In adfton, eachtestchamber willbe mildlyaeratedtoreducethe biologicaolxygen demand due to thefeed.Test chambers willbe labeledwith theprojectnumber and test roncentration- PROTOCOL NO. 454/042199/OYS-DEP/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD EnvironmentalConditions Lightingused to illuminatteheholdingand testchambers willbe providedby fluorescenttubesthat emitwavelengthssimilartonaturalsunlight(e.g.C,olortones50). A photoperiodof 16 hoursoflightand 8 hoursof dark willbe controllewdith an automaticffim. A 30-minutetransitiopneriodof low light intensitwyillbe providedwhen fightsgo on and offto avoid sudden changes inlightintensityL.ight intensitwyillbe measured attestinitiatiwointha SPER ScientifiLctd.lightmew orequivalent. The targettesttemperaturewillbe 22 I *C. Tcmperabm willbe measured ineach testchamber at thebeginningand end of thetestusinga liquid-in-glatshsermometer.Temperature alsowillbe measured with a continuousrecorderinthenegativecontrolchamber. Recordermeasurements willbe verifiewdith a liquid-in-glatshsermometerpriorto'tesitnitiation. Dissolvedoxygen willbe recordedin eachtestchamber daily.The pH willbe measured ineach treat3nenatnd controlgroup attestinitiatiomni,dpoint@appro@dmtely 48 hours),and theend of the tesl Dissolvedoxygen willbe measured usinga Yellow SpringsbishimaentModel 5 IB dissolvedoxygen meter, or equivalent Measurements of pH willbe made using a FisherAccumet Model 915 pH meter,or equivalent In theeventthatdissolvedoxygen concentrationfsallbelow 60*/osaturationd,issolvedoxygen measurements willbe made in everytestchamber and appropriateactionsvaU be takenafterconsultation with theSponsor. Dilutionwater salinitwyillbe measured in each chamber at testmitiatimand atthe end of thetest.Salinitwyillbe measured usinga Bio-Matine,Inc.AquaFauna refractometero,r equivalent If a treatmentgroup reaches 1001/omortalityd,issolvedoxygen,pH and tmnperaturemeasurements willbe takenatthattime,d= discontinued. BiologicalMeasurements Oysterswillbe in@ visuallaytapproximately0.24hoursand at24,48,72,and 96 hours 1 hour) after test mitiatiorl Oysters havmg opm shells and not responding to gentle prodding will be considereddead and removed. Any sha abnormalitieosbservedwillbe recorded.When thetestis tmmmated, shelldepositiownillberecordedforeachoysterby measuringthelengthofthelongestfingerof new shell.Shellgmwth inhibitiofnoreachtreatmentgroup willbe determinedby calculatinmgean shell depositionas a percentageof thecontrol. PROTOCOL NO. 454/042199/OYS-DEP/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -7- Smpling forAnalyticalMeasurements Water samples willbe collectefdrom each testchamber at testinitiatioand every24 hours (:t hour)duringthetesttodetermineconcentratioonfsthe testsubstance. In the eventthat100% mo i rtalty occursinany treatmentt,hensamplingof thattreatmentwillterminatefollowingthenextsamplinginterval. Samples willbe collecteadt mid-depthfrom each testchamber, analyzedimmediatelyor placed in an appropriatestoragecontainer(e.g.g,lassor polypropylenebottle)and storedunder refrigerati(o-n4oC)until analyzed.The sample scheme issummarized below: PROPOSED NUMBERS ExperimentalGroup 0 Hours OF VEREFICATION 24 Hours 48 Hours SAMPLES 72 Hours 96 Hours Control Level I-Low Concentration Level 2 Level 3 Level 4 Level 5 6 6 6 6 6 TotalNumber of Samples 30 The above numbers of samples representthose collectedfrom the testand do not includequality control(QC) samples such as matrixblanksand fortificatiopnrseparedand analyzedduringtheanalytical chmnistryphase of the study. AngLyficalChemistry Chemical analysisof the smnpleswillbe performed by WildlffeInternationaLlt,d. The analytical method used willbe based upon methodologyprovidedby the Sponsor and.idenfifiiendAppendix II.The methodology used to analyzethetestsampleswillbe documented intheraw dataand ==uized inthefinal report. PROTOCOL NO. 454/042199/OYS-DEP/SUB454 3M LAB REQUEST NO. U2723 I @j WILDLIFE INTERNATIONAL, L7-D -8- Data Analysis Mean shellgrowth inhibitioinneach-treatmengtroup willbe expressedas a percentageof control growth depositionA.n EC50 valuew.illbe calculatewdhen possibletoexpresstheconcentratioonf test substancethatinduceda 50% inhibitioinshelldepositionT.he followingformulawillbe used: %Inhibition=c -T x 100 c Where C = Mean growth ofnegativecontrols T = Mean growth of treated The datawiU be ana47zd by lineairnterpolatiuosningICPIN computersoftware(when possible()4). 'Me no-observed-effect-wncentra(tNiOoEnC) andlowest-observed-effert-roncent(rLaOtEiCo)n wiu be determinedbased (when possibleo)n a statisticaanlalysisofthedose-responsedataand an assessmentof thedose-responsepatterm RECORDS TO BE MAINTAINED Records tobe maintainedfordatageneratedby WildlifeInternationaLltd.willinclude,butnot be limitedto: 1. Copy ofsignedprotocol. 2. Identificatiaonnd charactmizationfthetestsubstancei,fprovidedby sponsor. 3. Datesofinitiatiaonnd terminatioonf thetesl 4. Holdingrecords. 5. Calculatioannd preparatioonftestconcentrations. 6. Stocksolutiocnalculationasndpreparationi,fapplicable. 7. Observations. Ifapplicablet,hemethodsusedtom*ze testsubstanceconcentrationasnd theresultosfanalytical measurements. 9. Statisticcaallculationisf,applicable. 10. Testconditionasnd pbysical/chemicmaelasurements. 11. Measurements of shelldepositiorl 12. Copy of finarleporl PROTOCOL NO. 454/04219910YS-DEP/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -9- FINAL REPORT A reporotftheresulotfsthestudwyilblepreparebdyWildliIfneternatiLotnda_lThereporwtin includeb,ut notbe limitedto,thefollowingw,hen applicable: 1. Name and addressofthefacilipteyrformingtheSb*. 2. Datesupon whichthestudywas initiateadnd completed.ItistheresponsibiliotfytheSponsorto providethefinaldatethatdataarerecordedforchemistryp,athologyand/orsupportingevaluations thatmay be generatedatotherlaboratories. 3. A statementof compliancesignedby the Sb* Directoraddressingany excepfionsto Good LaboratoryPracticeStandards. 4. Objecfivesand proceduresasstatedintheapprovedprotocoli,ncludingany changesintheoriginal protocol. 5. The testsubstanceidentifiebdy name,chemicalabstractnsumber orcode number,str,,mgtphu,rity, and compositionorotherappropriatceharacteristiicfsp,rovidedby theSponsor. 6. Stabilitaynd thesolubiliotfythetestsubstancesundertheconditionosfadministratioinf,provided by theSponsororcontractetdoWildlifeinternationLatlcL 7. A descriptioonfthemedio& usedtoconductthetest 8. A descriptioonfthetestsystem,includintghesourceofthetestorganisms,scientifniacme, lifestage, means and rangesoflengthso,bserveddiseasest,reatmentsand holdingprocedures. 9. A descriptioonfdw preparatioonf thetestsolutionsm,ethods used to allocatoerganismsto test chambers and beginthe testn,umbers of organismsand chambers per treatmenta,llocatioonf organismstotestchambers and durationofthetest 10. A descriptioonfcircumstancetshatmay have affectetdhequalitoyr integritoyfthedata. 11. The name ofthe Sb* Directorand thenames of otherscientistpsr,ofessionalasn,d supervisory personnelinvolvedinthesh*. 12. A descriptioonfthetransformationcsa,lculationosr,operationpserformedon thedata,a summary and analysisofthebiologicadlataand analyticaclhemistrydata,and a statementoftheconclusions drawn from theseanalyses. 13. Statisticmaelthods usedtoevaluatethedata,ifapplicable. 14. The signedand datedreportsofeachoftheindividuaslcientisotrsotherprofessionalmsvolved inthe study,ifapplicable. 15. The locationwhere raw dataand finalreportaretobe stored. PROTOCOL NO. 454/042199/OYS-DEP/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -10- 16. A statementpreparedby theQualityAssuranceUnithsfingthedatesthatstudyinspectionasnd audits were made and thedatesofany findingsreportedtotheStudy Directorand Management. 17. Ifitisnecessarytomake correctionosr additiontso a finalreportafterithas been accepteds,uch changeswillbe made intheform ofan amaidment issuedby theStudyDirector.The amendment will clearliydentiftyhepartofthefinarleportthatisbeingamended and thereasonsfortheamendments. Amendments willbe signedand datedby theStudy Director. CHANGING OF PROTOCOL Planned changesto theprotocolwillbe inthe form of writtenamendments signedby the Study Directorand theSponsoes RzpresentativeA.mendments willbe consideredaspartoftheprotocoland will be attachedtothefinalprotocolA.ny otherchangeswillbe intheform ofwrittendeviationssignedby th6 Study Directorand filedwiththeraw data.Allchangestotheprotocowlillbe indicateidnthefinalreport. GOOD LABORATORY PRAC]RICES Thisstudywillbe conductedinaccordancewithGood LaboratoryPracticSetandardsforEPA (40 CFR Part160 and/orPart792);OECD Principleosf Good LaboratoryPractic(eOCDEIGD (92)32, EnvironmentMonograph No. 45);and Japan MAFF (59NohSan, NotificatioNno. 3850, Agricultural ProductionBureau).Each studyconductedby WildlifeInternationLatld.isroutineleyxaminedby the WildlifIentmmationaLltd.QualityAssuranceUnitforcomplm= withGood LaboratorPyracticeSst,andard Opm-dbng Proceduresand thespecifiepdrotocol.A statementofcompliancewithGood LaboratoryPractices willbe preparedforallportionosfthestudyconductedby WildlifIentmmationaLltd. The Sponsorwillbe responsiblfeorcomplianerw.ithGood LaboratoryPracticefsorproceduresperformedby otherlaboratories (e.g.r,esidueanalysesorpathology)R.aw dataforallwork performedatWildlifeInternationLatld.and a copy ofthefinalreportwillbe filebdy projeanumber inarchivelsocatedon theWildlifIenternationLatlcL siteo,r atan alternatilvoecatiotnobe specifieidnthefinalreporl PROTOCOL NO. 454/042199/OYS-DEP/SUB454 3M LAB REQUEST NO. U2723 El WILDLIFE INTERNATIONAL, LTD REFERENCES 1 U.S.EnvironmentalProtectionAgency. 1996. Series850-EcologicaElffectTsest Guidelines (draft)O,PPTS Nwnber 850.1025:OysterAcuteToxicitTyest(ShelDleposition). 2 U.S.EnvironmentalProtectioAngency. 1985.StandardEvaluationProcedureA,cute Toxicity Testfor Estuarineand Marine Organisms (Mollusc96-Hour FZow-Yhrough ShellDeposition Study).EPA-540/9-85-011. 3 ASTM Standard E729-88a. 1994. Standard Guide for Conducting Acute Toxicity Tests with Fishes,Macroinvertebrates, andamphibians. American Society for Testing and Matmials. 4 Norberg-IGng, T.J. 1993. A LinearInterpolatioMnethodfor SublethalToxicity:Yhe Inhibition Concentration aCp) Approach. Version 2.0. U.S. Environmental PmtecfionAgency. National EffluentToxicityAssessmentCenter.Duluth,Niimesot& TechnicalReport03-93. PROTOCOL NO. 454/042199/OYS-DEP/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -12- IDENTMCANON APPENDIXI OF TEST SUBSTANCE BY SPONSOR To be Ccrnpletebdy Sponsor I.TestSubstanceIdentit(yname tobe used inthereport):PFOS (PerfluorooctaSnuelfonicAcid PotassiumSalt ReferenceStandard(ifapplicableA)n:alyticaSltandard:-N/A InternaSltandard: 1.1.2.2FLH.H.PHerfluorooctanSeulfonicAcid TestSubstanceSample Code orBatchNumber: Lot 217 TestSubstancePurity(0/AoctiveIngredient): 98.9 I TesStubstm= ExpiratioDnate: 2008 HavtehWeei.y&2 which and offimchmzcbmistics = Z@@and = Mmdardbeen debmnffi8ldiritooritsim inft sb* inaccordancweithGLP Standm-ds? --_@Yes a TestSubsLmm StorW Cai&bm x No Pleamfixhcateherecmm3midedstomp cmdifionastWM& hftmahaw W Ambimt Has thesWnl4 oftheteswtbamm under&= swrap ameibom beenddmmin6d inaanilmm wkh GLP Standards? Offifpfmfixmtstabilitnyfamafion: ---LYCS x No IV. TestC"Emdrafions: Ad-jus_ttestmmmtmfimtolOOO/oai A--baged Wm thepmty CO/oS)m above V. To;dcitIynfmnafim Do notadjustestcmcmhzfim to1000/0 &i TestthemateriaAlS IS. ManmuhmAquafic: RatLD50 251mpJkg Mm=LD50-N/A kwatebift Twd&y TZA@C50) D@Rhnia map-na: 27 mg/L DgphAia mazna: 50 mgLL FLqhTwxiiy=O) Rainbow Trout: 11 mp-,/L FatheadMinnow: 38mpjL Odzr ToxicitIynfmnafimCmckxbg Enffmpofchmnicmd mbcbranitcem): Pleasesee MSDS PROTOCOL NO. 454/042199/OYS-DEP/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -13- APPENDIX R AnalyticalMethod Providedby Sponsor Samples willbe analyzedbasedupon proceduresprovidedby theSponsor inthe followinganalytical methods: 1 Liquid Chromatography Mass Spectrometry(LCMS) Method for the Determinationof PerfluorooctanSeulfonicAcid PotassiumSalt(PFOS) In Freshwater,Saltwaterand Algal Medium A copy of theabove method willbe maintainedintheraw data. The actualmethodology used to analyzethe testsamples willbe documented intheraw dataand sumnimized inthe flnalreport. PROTOCOL NO. 454/042199/OYS-DEP/SUB454 3M LAB REQUEST NO. U2723