Document YjnzjzeXBvD2BQNwoyJkw2wqD

I I PFOS: A 96-Hour StaticAcute ToxicityTest with the Freshwater Mussel (Unio complamatus) I ACUTE TOXICITY TO AQUATIC INVERTEBRATES MUSSEL) (FRESHWATER TEST SUBSTANCE Identity:Perfluorooctanesulfonatmea;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 saltC,AS # 2795-39-3) Remarks: The testsubstance isa whitepowder. Sample was taken from 3M lotnumber 217. Sample was storedunder ambient conditionspriorto testing.Puritydeterminedtobe 90.49% by LC/MS, 'H-HMR, 'GF-NMR and elementalanalyses techniques. METHOD Method: The studywas conducted usinga protocolbased on procedures outlinedinU.S. EnvironmentalProtectionAgency Series850 - Ecological EffectsGuidelines,OPPTS Number 850.1075; OECD 203: Fish,Acute ToxicityTest;and ASTM Standard E729-88a, Standard Guide for Conducting ToxicityTestswithFishes,Macroinvertebrateasnd Amphibians. Test type: Semi-staticRenewal G LP: Yes Year Completed: Study completed 1999. Reportcompleted2000 Species: Unio complamatus Analyticalmonitoring:Test substance concentrationmseasured by LCMS at0,48, 96-hours Statisticamlethods: LCso valuescalculatedw,hen possible,by probit analysis,moving average method or binomialprobabilitwyithnon-linear interpolatiounsingthe computer softwareofC.E.Stephan. Test organism source: Obtained from CarolinaBiologicaSlupply Company, BurlingtonN,orth Carolina.Carolinacollectedfrom the wild. Test organism age at study InitiationU:nknown Test conditions Dilutionwater:0.45 pm filterewdellwater Dilutionwater chemistry (duringthe 4-week period Immediately preceding the test): hardness: 126 (120-132)mg/L as CaC03 alkalinity: 174 (170-178)mg/L as CaC03 pH: 8.3(8.1-8.5) TOC: < 1.0mg/L Conductivity: 321 (310-330)pmhos/cm Ca/Mg ratio: 35/13.5 Na/K ratio: 21.3/6.62 LightingC:olortone5@0 fluorescelnitghtisn,tensiatpyproximately 369 lux.Photopedod of 16-hourslight8,-hoursdark witha 30minute transitiopneriod. Stock and testsolutionspreparation: A primarystocksolution was prepared indilutiownater at91 mg/L. Itwas mixed for approximately24 hours priorto use. Aftermixing,the primarystock was proportionalldyilutedwithdilutiownaterto preparethefour additionatlestconcentrations.Alltestsolutionsappeared clearand colorless. Exposure vessels: 25 liteprolyethyleneaquada containing approx@mtely 20 L oftestsolution.The approximatedepth oftest solutionwas 23.2 cm. Number of replicates:two Number of testorganisms per replicatet:en Number of concentrations:fiveplusa negativecontrol Water chemistry during the study: Dissolved oxygen range (0- 96 hours): 5.8- 8.5mg/L (controelxposure) 5.0- 8.6 mg/L (79mg/L exposure) pH range (0- 96 hours) 8.0- 8.4 (controelxposure) 7.9- 8.5 (79 mg/L exposure) Test temperature range (0- 96 hours) 21.4- 21.80C (controelxposure) 21.8- 23.70C (79 mg/L exposure) Element Basis: MortalityM.usselswithopen shellsand not resp6ndingtogentleproddingwere considereddead. The number ofindividualesxhibitincglinicaslignsoftoxicitoyr abnormal behavioralsowere evaluated. Method of calculatingmean measured concentrations: arithmetimcean RESULTS Nominal concentrations: <LOQ, 5.7,11,23,46, 91 mg/L Measured concentrations: <LOQ, 5.3,12,20,41, 79 mg/L Element value: 96-hourLC5o = 59 mg/L (51-68mg/L) StatisticaElvaluation:The LC50 valuesand 95% confidenceintervals were calculatedwhen possibleby probitanalyssi,the moving average method or binomialprobabilitwyithnon-lineairn' terpolatiounsingthe computer softwareofC.E. Stephan. AnalyticMaelthodologyA:nalyseosftesstolutiownesreperformeadt WildlifIenternationLatld.usinghighperformanceliquicdhromatography withmass spectrometridcetection(HPLC/MS).When determiningthe concentratioonfthetestsubstanceinthetestsolutionst,hesame and most prominentpeak responseforperfluorooctanesulfonawtaes used. No attemptwas made toquantifyon thebasisofindividuailsomeric components. The LOQ (limiotfquantitatiowna)s 0.115mg/L inthisstudy. The mean percentrecoveryofmatrixfortificatiaonnaslyzedconcurrently duringsample analysiswas 94.7%. Samples collecteadttestinitiatihoand measured valuesfrom 73.7% to96.0% ofnominal.Measured valuesior samples takenat48-hoursranged from 81.2to98.9% ofnominal. Measured valuesforsamples takenat96-hoursranged from 88.5 to 130% of nominal. Summary of nalyticalchemistry data: Nominal Test Concentration, mg/L Measured DuplicatedValues at0, 48, Mean and 96-hours,Respectively, mg/L Measured Concentration, g/L Percent of Nominal Negative Control All< LOO -CLOO - 5.7 5.47,4.93,5.18, 5.70,5.24, 5.26 5.3 93 11 11.4,10.1, 11.2,10.5,10.9,16.4 12 109 23 19.0,16.8,18.7, 18.7,22.9.22.4 20 46 37.2,40.6,37.1. 39.6,48.2,40.5 41 87 1 89 91 69.0, 74.7, 81.3, 77.6. 88.2. 85.7 79 87 Biologicalobservationsafter96-hours:Musselsinthenegativecontrol, the5.3,12 and the20 mg/L.treatmentasppeared healthyand normal throughoutthetestwithno mortalitoyrovertclinicaslignsoftoxicityF.ive percentmortalitwyas observedat96-hoursinthe41 mg/L treatmentand 90% mortalitwyas observedinthe79 mg/L treatment.No abnormal behaviorwas noted inthese concentrations. Cumulative Mean Measured Test Cone., mg/L Neg. Control percentmortal 24 Hours 0 48 Hours 0 5.3 0 0 12 0 0 20 0 0 41 0 0 30 40 72 Hours 0 0 0 0 1 0 50 96 Hours 0 0 0 0 5 90 ControrlesponseS:atisfactory CONCLUSIONS The potassiumperfluorooctanesulfona9t6e-hourLC50 fortheFreshwater Mussel,Unio complamatus was determinedtobe 59 mg/L witha 95% confidenceintervaolf51-68 mg/L. The 96-hourno mortalitcyoncentration was 20 mg/L. Submitter: 3M CorporationE,nvironmentalLaboratoryP,.O. Box 33331, St.Paul,Minnesota,55133 DATA QUALITY ReliabilityK:limischrant(in1g. REFERENCES Thisstudywas conducted atWildlifIenternationaLlt,d.Easton,MD atthe requestofthe3M Company. OTHER Last changed: 5/3/00 PFOS: A 96-HOUR STATIC ACUTE TO)ACITY TEST WITH THE FRESHWATER MUSSEL (Uniocomplamatus) FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER. 454A-105 3M LAB REQUEST NO. U2723 U. S EnvironmentalProtectioAngency Series850 - EcologicaElffectsTestGuidelines OPPTS Number 850.1075 and OECD Guideline203 AUTHORS: KurtR. Drottar Henry 0. Kmeger, Ph.D. STUDY INITIATION DATE: May 3,1999 STUDY COMPLETION DATE: August 11,1999 AMENDED REPORT DATE: April26,2000 Submittedto 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St.PauL Minnesota 55144 WildlifeInternationalLtd.' 8598 Commerce Drive Easton,Maryland 21601 (410)822-8600 Page 1 of43 AMENDED WILDLIFE INTERNATIONAL LTD. -2- PROJECTNO.: 454A-105 GOOD LABORATORY PRAMCE COMEPLLANCE STATEMEENT SPONSOR: 3M Corporation TITLE: PFOS: A 96-HourStatiAccuteToxicitTyestwiththeFreshwateMrussel(Uniocomplamatus) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER- 454A-105 STUDY COMPLETION: AVgust 11,1999 AMENDED REPORT: April26,2000 Thisstudywas conductedm comphancevnth Good LaboratoryPracticSetand@ aspubhshodbytheUS. EnvironmentaPlrotectioAngency in40 CFR Parts160 and 792,17 August 1989;OECD PrinciploefsGood LaboratorPyracticeO,CDEIGD (92)32,EnvironmentMonogWh No. 45, Paris1992;and JapanMAFF, 59 NohSan,NofificatiNoon. 3850,AgriculturParloductionBureau,10August 1984 withthefollowinagcceptions: The testsubstancewas not characterizmed accordancewithfidlGLP compliance;however,the characterizatiwoans performedaccordingto3M StandardOperatingProceduresand Methods,and allraw data -.arebeingmaintainedinthe3M archives.The testsubstanceisbeingrecharactefiziendaccordancewithGLP. The stabiliotfythetestsubstanceunderconditionosf storageatthetestsitewas notdeterminedin accordancewithGood LaboratoryPracticSetandards. STUDY DIRECTOR. KurtP,Drona SeniorBiologist SPONSOR KPPROVAL.. I'A SP@ nsor 6 /66 DATE IYATE AMENDED WILDLIFE INTERNATIONAL LTD. -3- PROJECT NO.: 454A-105 QUALITY ASSURANCE STATEMIENT Tinssbxlywas exanunedforcompliancewithGood LaboratoryPracticSetandardsaspublishedby the U.S.EnvirorunentParlotectioAngency,40 CFR Parts160 and792,17 August 1989;OECD PrincipleosfGood LaboratoryPracticeO,CDE/GD (92)32,EnvironmentMonographNo. 45, Paris1992;and JapanMAFF, 59 NohSan,NotificatiNoon. 3850,AgriculturaPlroductionBureau,10 August 1984. The datesof all inspectionasnd auditsand thedatesthatany findingwsere reportetdo theStudyDirectorand Laboratory Management were asfollows: ACnVrrY: Test Substance Preparation DATE CONDUCTED: May 27,1999 AnalyticalSample Preparation and Observations May 28,1999 BiologicalData and Draft Report Jime29,1999 Analytical Data and Draft Report June29 and 30,1999 FinalReport Amended Report August 11,1999 April20,2000 STUDY DATE REPORTED TO: DIRECTOR: MANAGENEENT: May 27,1999 June 1, 1999 May 29,1999 June 29,1999 June 50, 1999 August 11,1999 April20,2000 June 3,1999 June 30,1999 June 30, 1999 August 11,1999 April24,2000 Timothy A. Sp@inge@,Ph.D. Manager,Regulatoryand TechnicalSupport DATE 4-/o o AMENDED WILDLIFE INTERNATIONAL LTD. -4- REPORT APPROVAL PROJECTNO.: 454A-105 SPONSOR: 3M Corporation NTLE: PFOS: A 96-HourStatiAccuteToxicitTyestwiththeFreshwateMrussel(Uniocomplamatus) W]ILDLIFE R4TERNAnONAL LTD. PROJECT NUMBER.- 454A-105 STUDY DIRECT Kurt& Drottar SeniorBiologist' MANAGENffi'NT: HenriO'&tiiW,QPh.D. Dkector,AquaticToxicologyand Non-TargetPlants TI DATE DAtf, AMENDED WILDLIFE INTERNATIONAL LTD. -5- PROJECT NO.: 454A-105 TABLE OF CONTENTS TitleJCovePrage.........................................................................1............... Good LaboratoryPracticCeomplianceStatement.......................................................2.................... QualityAssuranceStatement...............................................................3................ ReportApproval......................................................:.................................4............................... Tableof Contents........................................................................5............... Summary ...............................................................................7............... Introducti.o.n...........................................................................8............... Objectiv.e..............................................................................8............... ExperimentdDesign......................................................................8............... Materialsand Methods ....................................................................9............... -Resultasnd Discussion..................................@r.......................................................................1..2............... Conclusion.s...........................................................................................1.3................... References................................................................................................1.4............... TABLES TableI- Summary ofAnalyticaClhemistryData..............................................1.5............... Table2 -TemperatureD,issolvedOxygm andpH ofWater intheTestChambers ....................1.6............... Table3 -Hardness,Alkalinitaynd ConductivitoyfWater intheTestChambers......................1.7............... Table4 -CumulativePercentMortalitaynd Treatment-RelaWEffect.s............................1.8.............. Table5 -LC50 Values...................................................................1.9............... WILDLIFE INTERNATIONAL .6- PROJECTNO.: 454A-105 TABLE OF CONTENTS -Continued- FIGURE Figure1. Concentmtion-ResponsCeurve(96-HourData).......................................2.0............... APPENDICES Appendix I- SpecifiCconductanceH,ardness,AUcalinitya,ndpH of WellWater Measured Duringthe4-Week PeriodImmediatelyPrecedingtheTest..................2.1.............. Appendm R - AnalysesofPesticideOsr,ganicsM,etalsand OtherInorgamcs inWildlifIenternationLatld.WellWater........................................2.2.............. AppendixIII- Tbc AnalysisofPFOS inFrwhwaterinSupportofWildlife IntmnationaLltd.ProjedNo.: 454A- 105.................................:......2.3.............. AppendixIV - Changes toProtocol........................................................4.0.............. AppendixV - PersonneIlnvolvedintheSh* ..............................................4.1.............. AppendixVI - ReportAmendment ........................................................4.2.............. AMENDED WILDLIFE INTERNATIONAL LTD. -7- PROJECT NO.: 454A- 105 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCANON OF SRMY, RAW DATA AND A COPY OF THE FINAL REPORT: WMDLIFE Un-ERNAnONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STMY: MEAN IVIEASUREDTEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST: TEST ORGANISM: SOURCE OF TESTORGANISM: AGE OF TEST ORGANME: b.@@@s REPRESENTATIVE OF 20 MUSSELS: WET WEIGHT (g): TOTAL LENGTH (mm): 96-HouRLc5O: 95% CONFIDENCE LIMITS: NO-MORTALrrYCONCENTRAnON: SUBBLARY 3M Cowation Ms. SusanA. Beach Wildlifleatm-natioUn&al Easton,Maryland 21601 454A-105 PFOS (PufluorooctaSmulfoniAccidPotassiumSalt) PFOS: A 96-HourStatiAccuteT("dcitTyestwiththeFreshwater Mussel(Uniocomplamatus) NegativeControL5.3,12,20,41 and79 mg a.i./L ExpmimenW Start- May 28,1999 BiologicaTlmnination- June1,1999 ExperimentdTerminatio-n June3,1999 96 Hours FreshwatMeurssel(Uniocomplamatus) CarolinaBiologicaSlupplyCompany BurlingtonN,orthCarolina27215 unknown Mean = 76.5; Mean = 48.7; Range = 57.5to93.5 Range = 44.6to54.9 59 mg &iAL 51 and68 mg &iJL 20 mg ai./L ANffiNDED WILDLIFE INTERNATIONAL LTD. -8- PROJECT NO.: 454A-105 INTRODUC]RION ThistudwyasconductbeydWildliIfneternatiLotndaf.lor3M CorporataitotnheWildlihfaemational Ltd.aquatictoxicologfyaciliitnyEaston,MarylancLThe in-hfephaseofthetestwas conductedfromMay 28 to June 1,1999. Raw datageneratebdy WildlifeInternationLatlcLand a copy ofthefinarleportarefileudnder ProjectNumber 454A- 105inarchivelsocatedon theWildlifIenternationLatld.site. OBJECIM The objectivoefthissh* was toevaluatetheacutetoxicitoyfPFOS (PerfluorooctaSnuelfoniAccid PotassiumSaltt)othefreshwatemrussel,Unio complamatus,duringa 96-hourexposureperiodunderstatitcest conditions. EXPERUAENTAL DESIGN Freshwatermusselswereexposedtoa geometricserieosffivetestconcentratioansdanegativ(ecWWon water)control.Two replicatestchambersweremaintainedineachtreatmentand controglroup,with10 freshwatemrusselsineachtestchamber fora totaolf20 freshwatemrusselspertestemcentratiorNlominaltea concentratiownesre selecteidnconsultatiowniththeSponsor,and were basedupon*theresultosfanexploratory rangefindingtoxicittyeslNominal testconcentrationsselectewdere5.7,11,23,46 and 91 mg activheigedient -(a.i.)/TLh.e testsolutionwsererenewedatapproxunatel4y8 hoursofthetestN.ew testsolutionwsereprepared andthemusselswere transferrefdrom theoldto thenow solutionsM.ean measuredtestconcentratiownesre determinedfrom samplesoftestwatercollectefdi-omeachtreatmentand thecontroglroupatthebeginninogfthe w4 atapproxknatel4y8 hours,and attestemiinatiorl Freshwatermusselswere indiscriminatealsysignedtoexposurechambersattesitmtmfiorlObservations ofmortalitayndotherclinicaslignsoftoxicitwyeremade atapproximatel6y,24,48,72 and96 hoursaftetrest initiatioCnu.mulativepercentmortalitoybservedinthetreatmengtroupswas usedtoestimatoercakulatLeC50 valuesat24,48,72 and96 hours.The no-mortalitcyoncentmtionwas determinedby vlmal interpretatoifotnhe mortalitdyata. ANffiNDED WILDLIFE INTERNATIONAL LTD. -9- PROJECT NO.: 454A-105 MATERLALS AND MEETHODS Thestudwyasconductbeadseodntheproceduroeustlinientdheprotoc"oPlFOS:A 96-HouSrtatic Acute ToxicityTestwiththeFreshwaterMusser'. The protocolwas basedon proceduresoutlineidnU.S. EnvironmentaPlrotectioAngency Series850 - EcologicalEffectsTestGuidelinesO,PPTS Number 850.1075 (1):OECD GuidelineforTestingof Chemicals203: Fish,AcuteToxicitTyest(2);andASTM StandardE72988a,StandardGiddefor ConductingAcute ToxicitTyestswithFishes,Macroinvertebrateasndamphibians (3). Tog Substance The testsubstancewas receivefdrom 3M Corporationon October29,1998 and was assignedWildlife InternationLatlcLidentificatniuomnber 4675. The testsubstancewas describedasa wlutepowder Itwas idenfifiaesdFC-95 fromlotnumber217 (T-6295).Informatiopnrovidedby theSponsorindicateadpurityof 98.9%,and an expiratiodnataof2008. The testsubstancewas reanalyzebdy theSponsorand theCerfificaotfc AnalysisdatedMarch 9,2000 indicateadpurityof 90.490/oT.he testsubstancewas storedatambientroom temperature. Pnaration ofTestConcentrations Nominaltestconcentratiownesre 5.7,11,23,46 and91 mg &i/L,basedon a testsubstancpeurityof 90.490/oA.llmaterialwshichcame intocontactwiththetestsubstanceduringpreparatioonftestconcentrations wereconstructeodf plastiocrstainlessteel A primarystocksolutiownas preparedinchlutiownaterata conemuifionof 91 mg a.L/L.The primarystocksolutiownas mixed withan electripcaddlemixerfor approximatel2y4 hoursto-aiidnthesolubffizatioofnthetestsubstance.Aftermbdn& theprimarystocksolution was proportionaldliylutewdithdilutiownatertopreparethefouradditionatlestconcentrvhonsA.lltestsowbons appearedclearandcolorlessT.estsolutionwsererenewedon Day 2 ofthetestwithfiwmy preparedsolutions. TestOr- * The freshwatemrusselUnio complamatus,was selecteadsthetestspeciefsorthissb*. Ilm freshwatmeursselisrepresentatoifvaenimportangtroupofaquatiicnvertebraatnedswas selectfeodruseinthe testbasedupon commercialavailabilitFyr.eshwatermusselsused inthetestwereobtainedfi-omCarolina BiologicaSlupplyCompany, BurlingtonN,orthCarolina.The identificatoifotnhespeciewsas providedby the supplier- AMENDED WILDLIFE INTERNATIONAL LTD. -10- PROJECT NO.: 454A-105 Freshwatermusselswere heldatapproximateltyhesame temperatureas used duringthetest.The musselswere heldforapproximatel1y5dayspriorto testingD.uringtheholdingperiodst,hemusselsshowedno signsofdiseaseorstressT.he musselswerenotfedduringtheholdingperiodorduringthetest.Duringthe14dayholdingperiodprecedintghetestw,atertemperaturersangedfrom 20.3to 22.2*C. The pH of thewater rangedfrom 8.2to8.6and dissolveodxygenrangedfrom 7.6to8.8mg/L. hmftumentafimandmedxklsusedfor watermeasurementsaredescribeidntheEnidronmentalCondinons sectioonftlusreporlAt testinitiatitohne, musselswerecollectefdrom t4choldingtankandt=fmted tothetestchambers. i The averagetotalength(dmta= from ft tipofthemnbo to thedmW valveedge)of 20 indiscriminatesleylectemdusselsfromthesame lotoftestorganismswas 48.7mm witha rangeof44.6to54.9 rnm The averagewetweightwas 76.5gramswitharangeo,f 57.5to93.5grams. TestApparatus Testchamberswere 25-L polyethylenaequariacontaininagpproximatel2y0 L of testsolutionT.he depthofwaterinarepresentatitveestchamber was approximatel2y3.2 caL Testchamberswere 1* positioneidnan environmentaclhambersettotnaintaiantenipm-ai=of222'C. The.tescthamberswere labeledwiththeprojectnumber,testconcentratioand replicate. DilutioWn The waterusedforholdingandtestinwgas fivshwateorbtainedfrom a wellapproximatel4y0 meters deepIMAW an theWildlifIenternatioLntadl.siteThe wellwateris&uww=d asmoderately-hawradter. The specificconductanceh,ardnessa,llmlinitayn,d pH measurementsof thewellwaterduringthefour-week periodimmediatelyprecedingthetestam presenteidnAppendix I. The wellwaterwas passedthrougha sandfiltetroremove particlegsmaterdm approxfinate2l5y pan, and pumped intoa 37,900-Lstoragetankand aeratedwithspraynozzles.Priorto use,thewateragainwas filtere(d0.45pm) toremove microorgwiismand particlesT.he resultosf periodicanalysesperformedto measurethemmulhons of selectecdontaminantms wellwaterusedby WildlifIenternationLatld.are presenteidnAppendixK WILDLIFE INTERNATIONAL LTD. -11- PROJECT NO.: 454A-105 EnvironmentalConditions Lightingusedtoilluminatteheholdingtanksand testchambersduringholdingand test3nwgas provided by fluorescenttubesthatemittedwavelengthsimilatronaturaslunligh(tColortone5*0).A photoperioodf16 hoursoflightand 8hoursofdarknesswas controllewdithanautomatictimer.A 30-minutetransitipoenriodof low lighitntensitwyas providedwhen lightwsenton andofftoavoidsuddenchangesinhotmg. Lightintensity attestinitiatiwoans approximatel3y69 luxatthesurfaceofthewater.Lightintensitwyas measuredusinga SPER ScientifLitcd.lightmeter. Temperaturewas measuredineachtestchamber atthebeginningand end ofthetestusinga liquid-inglassthermometer.Temperaturealsowas measuredcontinuousliyn one negativecontrolreplicatuesinga FulscopeER/C Recorder The targetesttemperaturdeuringthesft* was 2220C. Dissolvedoxygen measurementswere made on watersamplesfrom allreplicatteestchambersofeachtreatmentand thecontrolat testinitiatiaonnd atapproximatel2y4-hourintervalUsmvaftL-r.MeasurementsofpH weremade inone altemat3nrgeplicatoefeachtreatn=tgroupand thecontrolattestinitiatiaonnd atapproximatel2y4 hour intervaltshereafterH.ardness,alladiniatnyd specificconductanceweremeasuredinthedilutiownaterand low, nuddleand hightestsubstanceconcentratioantstestinitiatiaond attesttamfination.. MeasurementsofpH weremade usinga FisherAccmnet Model 915 pH meter,anddwived oxygenwas measuredusinga Yellow Springsbstrummt Model 51B dissolveodxygen'meterSpecificconductancewas measuredusinga YellowSpringshmtrumentModel 33 Salinity-Conductivity-Teppammteutrer.Hardnessand alkalinimteyasurementsweremade by titratiboansedon proceduresinStandardMethodsfor theExamination ofWater and Wastewater(4). Observations Observationswere made to detmmme thenumber ofmortalitiesM.ussels withopen shellsand not respondingtogmde prodchngwere consideredead The number of mchvidualsexlubitincglinicaslignsof toxicitoyrabnormalbehavioralsowereevaluatecOlbservationws=ma& approximat*6,24,48,72 md96 hoursaftetrestnutiation. WILE)LIFE INTERNATIONAL LTD. -12- PROJF.CTNO.: 454A-105 StatisticAanlalyses The 24,48, 72 and 96-hourLC50 valuesand the95% confidencientervawlesrecalculatewdhen possible by probitanalysist@hemoving averagemethod or binomialprobabilitwyithnon-hnearinterpolati(o5n,6,7) usingthecomputersoftwareofC.E.Stephan(8).Inthisstudy,LC50 valuescouldnotbe calculateadt@4 and48 hoursduetothelackofan adequateconcentrafion-respopnasne= However,thebinomialmethodwas usedto evaluatmeortalitayt72 hoursand theprobitmethod was usedtoevaluatemortalitayt96hours.The no-mortality concentratiwoans determinedby visualmterpretafaonf themortalitdyata. AnglyficaClhemiLUX Water sampleswere collecteadtnud-depthfrom each replicatteestchamber of each treatmenatnd controglroup atthe beginningof thetes@at48 hours(oldsolutionsa)nd attesttmmiin,afitoonmeasure concentratioonfsthetestsubstance.The sampleswere collecteidnplasti(cNalgeneS)bottleasnd analyzed immediatelywithoutstorage.Analyticaplroceduresused intheanalysiosfthesamplesareprovidedinAppendix M. At testerminatioanl,lremaininglivemusselswere sacrificfeodrcollectiofntissuseamples'The tissue -sampleswerecompositedby replicatfer,ozenand shippedtotheSponsorforpossibleanalysis. RESULTS AND DISCUSSION Measurementof TestConcmtrations ResWts of analysestomeasure concentrationosf PFOS inwatersamplescollectedduringthetestare presenteidnTableI andintheanalyticcahlemistryreport(AppendixIU).Nominal concentratiosneslectefdor useinthissa* were5.7,11,23,46 and91 mg a.i./LS.amplescollecteadttestinitiatihoandmeasuredvalues thatrangedfrom 74 to960/oofnominalvalues.Measured valuesforsamplestakenat48 hoirsrangedfmm 81 to 990/of nominal.Measured valuesforsamplestakenat96 hoursrangedfi-om89 to 130% of nominal.When measuredconcentratioonfsthesamplesanalyzedattestinitiatioanp,proximatel4y8 hoursand attest=limfion were averagedt,hemean measuredconcentratiofnosrthissh* were 5.3,12,20,41 and79 mg a.i./LM.ean measuredconcentratiownesreusedintheestimatioonr calculatiofnLC50 values. Observationasnd Measurements Measurementsoftemperatured,issolveodxygen and pH arepresentedinTable2. Temperahm were withinthe22*20C rangeestablishefdorthetest MeasurementsofpH rangedfrom 7.9to8.5duringthetestO.n AMENDED WILDLIFE INTERNATIONAL LTD. -13- PROJECT NO.: 454A-105 Day I of thetestdissolveodxygenconcentratiohnasd droppedas low as 5.0mg/L (57% of saturation). Consequentlym,ildaeratiownas initiatteodalltestchambers.Afteraerationd,issolveodxygenconcentrations remained;>-5.m9g/L (68% of saturatimt)hroughouthe test.Measurementsofhardness,alkaliniatnyd conductivitayrepresentedinTable3. Dailyobservationosfmortalitayndotherclinicaslignsoftoxicitoybservedduringthetestareshown in Table4. Freshwatermusselsin'thneegativecontrol5,.3,12 and 20 mg ai./Ltreatmengtroupsappearednonnal andhealthydunng thetest After96-hoursofexposurem,ortalitiynthe41 and 79 mg a.i./bLwbnent groups was 5 and90%, respectivelLyC.SO valuesand95% confide= limitast24,49,72 and96 hourswereestimated orcalculatefdromthemortalitdyata,and areshown inTable5. A graphofthecmcentration-responcsuerveis presentedinFigure1. CONCLUSIONS The 96-hourLC50 valueforfreshwatemrussels(Unio complamatus)exposedto.PFOS was 59 mg a.i./LT.he 95% confidencleimitwsere 51 and 68 mg a.i./aLndtheslopeoftheconcentration-respocnusreve was 10.The 96-hourno-mofti* concentratiwoans 20 mg &iJL. AHEENDED WILDLIFE INTERNATIONAL LTD. -14- PROJECT NO.: 454A-105 REFERENCES 1 U.S.EnvirorumentaPlrotectionAgency. 1996. Series850 - EcologicaElffectTsestGuidelines (draftO)P,PTS Number 850.1075:FishAcuteToxicitTyestF,reshwaterandMaHne. 2 OrganisationforEconomic Cooperationand Development 1993.OECD GuidelinefsorTestinogf Chemicals.Guideline203: Fish,AcuteToxicitTyest.AdoptedbytheCouncilon 12July1992. 3 ASTM Standard E729-88a. 1994. Standard Giddefor ConductingAcute To=c7ty Testsw7thFishes, Macroinvertebrates, andamphibians. American SocietyforTesting and Materials. 4 APHA, AWWA, WPCF. 1985. StandardMethodsfor theExaminationof Waterand Wastewater. 16th Edition.American PublicHealthAssomation. American Water Works AssociatioiLWater -PollutiCoonntrolFederatioNne,w Yo& 5 Stephan,CE. 1978.U.S.EPA, EnvironmentaRlesemrh LaboratixyD,uluth,Minnesot& Personal on. 6 Finney,DJ. 1971. StatisHcalMethods in BiologicalAssay. Secondedition.GfiffinPress,London. -7 Thompson,W.R. 1947.BacteriologRiecwaelws.Vol4.1,No.2.Pp.115-145. 8 Stephan, C.E. 1977. "Methods for Calculating an LC50," Aquatic Toxicology and Hazard Evaluations,American SocietyforTesting and Materials.PublicationNumber STP 634, pp 65-84. WILDLIFE INTERNATIONAL LTD. -15- PROJECT NO.: 454A-105 TableI Summmy ofAnalyticaClhemistryData Sponsor: Test SubstAmee: Test Organism: DilutionWater-* Nominal Test Concenftfion (mg ai./L) NegativeControl 3M Corporation PFOS FreshwaterMusseL Umo complamatus Well Water Rcp@catc, A B A B A B Ssn-mling T *u'cn al@,m) 0 0 48 48 96 96 Measured Concentmfion L%g ai./L) <LQQ<LOQ <LO <LOQ <LOQ 5.7 A 0 B 0 A 48 B 49 A 96 B 96 5.47 4.93 5.18 5.70 5.24 5.26 A 0 B 0 A 48 B 48 A 96 B 96 11.4 10.1 11-2 10.5 10.9 15.4 23 A 0 B 0 A 48 B 48 A 96 B 96 19.0 16.8 18.7 18.7 22.9 22.4 46 A 0 B 0 A 48 B 48 A 96 B 96 37.2 40.6 37.1 39.5 49.2 40.5 91 A 0 B 0 A 48 B 48 A 96 B 96 The limitof quantitatio(nLOQ) was 0.115 mg ai./L. 69 0 74.* 7 81.3 77.6 88.2 85.7 Nl= Measured Concentration (mg &i./L) <LOQ 5.3 12 20 41 79 PM=t Of Nominal 93 109 87 89 87 AMENI)ED -16- Table2 Temperature,DissolvedOxygen and pH of Water intheTestChambers spmmr 3M Corporation TestSubstance: PFOS Ted Organism: FreshwaterMu3ml. Uniocmnpkwwtus DilutioWnater: wallwater Mean Measured 0 Hour TestConcentration Tempi D"O (mga.i./L) Replicatr- (oC) (mg&) pH NegativeControl A B 21.4 8.5 8.4 21.4 8.5 - 24 Hours D& (MWL) pH 5.8 - 6.1 8.0 48Hours(Old) DO (MGIL) EH 8.2 8.3 8.1 - 5.3 A 21.5 8.5 9.5 5.7 - B 21.5 8.5 - 6.1 8.0 9.2 9.3 8.2 - 12 A 21.7 8.5 8.4 6.0 - B 21.8 8.5 - 5.6 9.0 8.2 8.3 8.1 - 20 A 21.8 8.5 8.5 5.8 - 8,1 8.3 B 21.8 8.5 - 5.7 8.0 8.2 - 41 A 22.5 8.5 8.5 5.4 - 8.2 8.3 B 22.5 8.5 - 5.6..@, 8.0 8.2 - 79 A 23.7 9.6 8.5 5.0 - B 23.6 8.5 - 5.2 8.1 8.1 8.2 8.0 - Temperaturemwmned continuousldyuringthetestrangedfrom approximatel2y1@.0to21-50C.. 2 A dwolved oxygenconcenhidm of5.2mgtlrepresent6s0'Ysoaturatiaotn220C infitshwater. 3 Wd aeratiownas initiatteodalltescthmbm. 72 Hours DO (mg/L) pH 8.2 - 8.1 8.2 8.0 - 8.1 8.2 8.1 - 8.1 8.2 8.2 - 8.2 8.2 8.1 - 8.2 8.2 7.8 - 7.7 8.1 WILDLIFE INTERNATIONAL LTD. -17- PROJECTNO.:454A1-05 Table3 Hardness,AlkalinitayndConductivitoyfWater intheTestChambers Sponsor* TestSubstance: TestOrganism:. DilutioWnater. Mean ; eam=n-ed Concentration L%I a.i./L) NegativeControl 5.3 20 79 3M Corporation PFOS FreshwateMrusseL U?docomplamams WeRWaa Replicate. Sampling Time (Houm) Hardness (MWL asCaCO3) A 0 140 B 96 132 A 0 136 B 96 136 A 0 144 B 96 136 A 0 144 B 0 136 AUmlini (mll asCaIZ03) 172 174 174 174 174 178 178 176 Conductivity (omhos/cm) 300 330 300 330 320 340 340 350 ANFENDED Table4 CumWative PercentMortafityand Treatinent-RelaWEffects BPOMW. Ted Subdanoo: TedOrgmian: Ddation Water Mean Meanuvd Ted C4watmtion (MGALM) 3M Cmpomtion PFOS Frodnvdw Munat Untocomplwwtus Well Water Repliede No. EqmW 6 Hours No. Dm& FirectO N%Wvc Control A 10 0 10 AN B 10 0 10 AN 24 Hours No Dead 0 0 Effects 10 AN 10 AN 48 Hours- No. Dead 0 0 Effects 10 AN 10 AN 72 Ham No. Dead 0 0 Effwu 10 AN 10 AN 53 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN B 10 0 10 AN 0 IOAN 0 10 AN 0 10 AN 12 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 20 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 41 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN B 10 0 10 AN 0 10 AN 0 IOAN 0 10 AN 79 A 10 0 B 10 0 Cumubti"mm*wofdkmdmuml& ObowvW Effbou.AN - Appom Norrml 10 AN 10 AN 3 7AN 3 7AN. 3 7 AN 5 5 AN 4 6 AN 6 4AN 9 No. Dead 0 0 0 0 0 0 0 0 I 0 9 9 WILDLIFE INTERNATIONAL LTD. -19- PROJECT NO.: 454A-105 Table5 LC50 Values Sponsor: Tea Substance: TestOrganism: DilutionWater: Time 24 Hours 3M Corporation PFOS FreshwaterMussel Unio complamatus Well Water Lower 95% Confidence LC50 Limits @2!9ai./L) (mg &i./L) > 79 Upper 95% Confidence Limits (mgai./L) Statistical Method VisualInterpretation 48 Hours > 79 VisualInterpretation 72 Hoiiri? 79 >41 Binomial 96 Hours 59 51 68 Probit IConfidencehnutscouldnotbe calculatewdithft mortalitdyataobumed. 2 The USefUlneSoSfthisLC50 isquestionablbecausea concentration-effedmlatimwsabsinpotdmmstra@ overa reasonablreange(e.g.-,*7 to>63) of deacl ANTENDED WILDLIFE INTERNATIONAL LTD. -20- Figure1. Concentration-ResponCsuerve(96-HourData) 7 6- 5:cto ! .jo 2 a. 4- 0 23 Slope 10 PROJECT NO.: 454A-105 10 100 Mean Measured Concentrabon (mg a.i./L) ANffiNDED WILDLIFE INTERNATIONAL LTD. -21- PROJFCTNO.: 454A-105 APPENDIX I SpecifiCconductanceH,m-dms, Alkalinitaynd PH ofWell WaterMeasured Duringthe4-Week PeriodlimmediatelPyrecedingtheTest Sponsor TestSubstance: TestOrgani-.m: DilutionWater: 3M Corporation PFOS FreshwaterMussel Unto complamatus Well Water SpecificConductance (pmhos/cm) mean 321(N= 4) 310 -330 Hardnbss (mg/L asCaC03) 126(N = 4) 120-132 A"inity (mg/L asCaCO3) 174(N = 4) 170-178 pH 8.3(N 4) 8.1 -8.5 WILDLIFE INTERNATIONAL L-m. -22- PROJECT NO.: 454A-105 APPENDDC]L AnalysesofPesticideOsr,ganics,Metalsand OtherInorganics inWildlifIenternationLatld.WellWater' ANALYSIS NOPASURED CONCENTRATION Mimnaneous Measurumuts TotalDissolveSdolids Animonia1-@"en TotalOrganicuuwn 2 TotalCyiAde Organochlorineasnd PCBs Aldrin AlphaBHC BftBHC DeltaBHC Gumna BHC (Undane) Chlordane DDD, pp" DDE, pp: DDT,pp Dieldrfii T7ndmtil A Endos B End fan'sulfate Endrin EndrinAidehyde Heptachlor M6thoxycWor HoptachlorEpoxide Toxaphene PCW1016 PCB-1221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 286 < 0.050 < 1.0 < 10.0 < 0.005 < 0.005 < 0.005 < 0.005 < 0.006 < 0.025 < 0.006 < 0.005 < 0.008 < 0.005 < 0.005 < 0.005 < 0.018 < 0.010 < 0.005- < 0.005 < 0.007 < 0.005 < 0.500 < 020 < 0.260 < olm < 0.720 < 0.720 < 0.720 < 0.720 mg/L mfpl MIYL WL PB/-L 4/-L ilw-L PW-L Oiv-L W-L pfv-L AW-L pfv-L Aiv-L i4/-L i*-L pfy-L Pi/-L 41-L 41-L 4/-L 14,L PW-L PW-L Metalsand OtherIuorganics Mum@nymi Arsenic c ccaicnit h00UT"P um3 co 3 ifignem,unx h4afiganese merd 3 moEFenum potaS6U_Tq3 sel ' sn=R'-3 WCYM < 100 PW-L < 25.0 itA- < 0.50 iLA- < 1.0 itA- 35.0 MA < 2.0 t4l-L < 1.0 < 20.0 < 100 < 10.0 I < 1.0 < 0.20 PW-L < 2.0 < 2.0 6.62 n*/-L < 25.0 4-L < 1.0 21-3 < 20.0 QST EnviroamentaL GainesviUa Floridafor samples ooBeeW on November 3 through November Anayl'=*f _Mned b-y VrdWe lnkmfional Ltd.for& sample c4DUoc*don November 5,1997. Analym performed by WildlifelatmmfaonalLtd.forsamplescohectodon November 5 thmugh 7, 1997. 7,1997. WILDLIFE INTERNATIONAL L-rD. -23- APPENDIX M PROJECT NO.: 454A-105 THIEANALYSIS OF PFOS IN FRBHWATER IN SUPPORT OF WILDLIFE INTEPNANONAL LTD. PROJECT NO.: 454A-105 WILDLIFE INTERNATIONAL LTD. -24- PROJECTNO.: 454A-105 REPORT APPROVAL SPONSOR- 3M Corporation TRME: PFOS: A 96-Hour StatiAccuteToxicityTestwiththeFreshwaterMussel WELDLEFE BqTERNAnONAL LTD. PROJECT NO.: 454A-105 -PRINCIPAL RQVESTIGATOR- Raygo'nd9.Van Hoven,Ph.D. scientist MANAGEMENT: KAWI WMud B.Nixon,Ph.D/ Manager.AnalyticaClhmnistry DATE DATL( AMENDED WILDLIFE INTERNATIONAL LTD. -25- PROJECTNO.: 454A-105 Introducti Freshwatersampleswere collectefdiun an acutetoxicitsytudydesignedtodetemimetheeffectosf PFOS (PerfluorooctaSnuelfoniAccid PotassiumSalt)tothefreshwatemrussel(Uniocomplamatus).This studywas conductedby WildlifeInternationLatld.and identifiaesdProjecNto.: 454A-105. The analyses of thesewatersampleswere performedat WildlifeJntemationaLltd.usinghigh performanceliquid chromatographywithmass spectrometridcetectio(nBPLC/MS). Sampleswere.receivedforanalysison May 28,30 and June 1,1999.'Sampleswere analyzedan eachsamplereceiptday. TestSubstanceand InternaSltandard The testsubstanceused forthe analyticaplortionof thisstudywas WildlifeInternationLatld. identificatniuomnber 4675. 'Me testsubstancewas used to preparecalibratiosntandardsand matrix fortificatsiaomnples. 'fheinternasltandardwas receivedfrom 3M Corporationon July2, 1998 and was assignedWildlife InternationLatld.idenfificatniuomnber 4526 upon receiptM.ie-utemalstandarda,granularmaterw,was idmfifieads: 1K 1K 2K 2H PerfluorooctaSnuelfonicAcid,ChemicalAbsb-adNumber: 27619-97-2. ne standardwas storedunderambientconditions. ARAL)tcal ne method usedfortheanalysiosf thefi-eshwatsearmpleswas developedatWildlifbeftmational Ltd.and entitle"dAnalyticaMlethod ValidatiofnortheDeterminatioonfPFOS inFreshwaterS,gWdter, and AlgalMedW'. Thismethodologywas includedas AppendixH ofWildliflenwxnationaLltd.protocol number 454/011299/MVAIJSUB454. Itwas basedupon methodologyprovidedby 3M Corporation. Sampleswere dilutedin a 50% methanol: 50% NANOpure(D water solutiocnontibiing 0.100mg 4H PFOS (internasltandard)AaLnd 0.05% fonnicacid(vtv)so thatthey fellwithinthe calibratiroanngeofthePFOS methodology. Concentrationosf PFOS in the standardsand sampleswere detenmned by reverse-phasheigh performanceliquidchromatographyusinga Hewlett-PackarMdodel 1100 High PerformanceLiquid Chromatograph(BPLC) witha Peridn-EhneArPI IOOLC Mass Spectrometeerquippedwitha Perldn- WILDLIFF- INTERNATIONAL L-m. -26- PROJECTNO.: 454A-105 Elmer TurbolonSpmy ion source. IHPLC separationwsere achievedusing a KeystoneBetasflCls analyficaclolumn(100mm x 2 nun I.D.,3 pm particlseize).The instnunenptarametersaresununarized inTable1. A meffiodflowcharitsprovidedinFigureI- CalibratioCnurveand Limitof Calibrafiosntandardsof PFOS preparedin a 50% methanol: 50% NANOpurcS water solution tainin0i.z100 mg 4H PFOS (internaslUndard)/Land 0.05% formicacid(v/v)r,anginginconcentration from 0.00229to 0.0274mg a.i./wLere analyzedwiththesamples.Linearregressioenquationswere generatedusingpeak arearesponseratios(PFOS :internasltandard)versustherespectivceoncentration ratio(sPFOS :internasltandardo)fthecalibratisotnandardsA. typicalcalibraticounrveispresentedin Figure2. 'Me concentratiofn PFOS inthesampleswas determinedby substitutitnhgepeak area responseratiosintotheapplicablleinearegressioenquatim. Representativieonchromatogmms of low and highcalibratisotnandardasrepresenteidnFigures3 and 4,m%=fively. "Me methodlimitofquanttaton(LOQ) fortheseanalysewsas setat0.115 mg a.iJL calculateadsthe productof the lowestcalibratisotnandardanalyzed(0.00229mg a.i./La)nd,thedilutio&nctor of the matrixblanksamples(50.0). MatrixBlankand FortLficatiSon Threematnx bink sampleswere analyzedto&-t== possiblmet,erferenceN.o interferencweesre observedator abovethe LOQ duringsamplesanalyses(Table2). A representaticvheromatogramof a matrixblankispresenteidnFigure5. Freshwaterwas fordfiedat0.915,45.7and 110 mg a.i.ALand analyzedconcurrentlwyiththesamples to determinethemean procedurarlecovery(Table3). Sample were notcorrectefdbrthe mean procedurarlecoveryof94.7%. A representatcihvreonutw= ofa matrixfortificatiisopnr=god inFigure6. AMENDED WILDLIFE INTERNATIONAL LTD. -27- PROJECTNO.: 454A-105 ExamalCealculations Sample number 454A-105-16,nominalconcentrationf 5.7mg a.i.AiLnfi-eshwater. InitiVaollume:0.100mL FinalVolume: 25.0mL DilutioFnactor.250 PFOS PeakArea. 596972 InternaSltandardPeak Area: 401298 Peak Area Ratio:1.4874 Calibratiocnurveequation. Slope:6.30785 Intercept0:.04889 PFOS (ILga.iJL)athwftunumt (Peakarearati-o (Y-intmr-epxt)L)S. Concentration SIOPO-1 (1.4974- 0.04889)x 100pg a.i./L 6.30785 22.8pg aiJL Note:I.S. internasltandard. PFOS (mg a.i.ALi)nsample PFOS (pga.iJL)atkabument x DilutioFnactor 1000 4mg 22.8x 250 1000 5.70mg a.iJL AMEOED WILDLIFE INTERNATIONAL LTD. -28- PROJECTNO.: 454A-105 PercentofNominal Concentration Calculaterdecovery9-8.9% Note:manualcalculatiomnay dffer. PFOS (mg a.i./Li)nsample x 100 PFOS (mg a.i.ALn)ominal RESULTS .SMP-lgA!Wmis Freshwatersampleswere collectefdrom the acutetomcitystudywiththe freshwatemrussel(Unio complamatus)at testinifmfionM,ay 28, 1999 (Hour 0),on May 30, 1999 (Hour48),and at test temiftmdonJ,une 1,1999 (Hour 96). The measuredconcentratioonfsPFOS inthesamplescollqcteadt mifafionof exposureof the testorganisms(Hour 0) rangedfi-om73.7 to 96.0% of the nominal concentrationsS.amples collecteadtHour 48 had a measuredconcenuafionrangeof .81.2to98.9% of nominalvalues.Samplescollecteadttestwnnination(Hour 96)had a measuredconcentratiornangeof 88.5to 130% ofnominalvalues(Table4). A representaticvheromatq;mm ofa testsampleisshown in Figure7. AMENDED WILDLIFE INTERNATIONAL L-rD. -29- PROJECT NO.: 454A-105 INSTRUhffi'NT: Table I TypicalLC/MS OperationaPlarameters Hewlett-PackarMdodel I100High PerfomianceLiquid Chromatographwitha Perldn-EhneArPI IOOLC Mass Spectrometer eqiuppedwitha Perkin-EhneTrurbolonSprayionsource.Operatedin selectivieonmonitorinmgode (SM. ANALYTICAL COLUMN: OVEN TEMPERATURE: STOP TIME: KeystoneBetasiCliscolumn(100mm x 2 nun I.D.,3 pm particlseize) 300C 10.0minutes FLOW RATE: 0.220mljminute MOBELE PHASE: 72.0% Methanol:28.0% NA:NOpuree Water containin0g.1% Fonnic Acid INJECTION VOLUME: PFOS RETENTION TIME: INTERNAL STANDARD RETENTION TIME: -25.0ILL Approxiinaftl6y.4n-dnutes Approxfinatel4y.4minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6anm 426.7amu WILDLIFE INTERNATIONAL LTD. -30- PROJECTNO.: 454A-105 Table2 MatrixBLuks AnalyzedConcurmfly DuringSampleAnalysis Number (454A'105-) MAB-4 SMI.E Type MatrixBlank Measured ConcentrafioOnf PFOS' (mg a.i.AL) <LOQ MAB-2 MatrixBlank < LOQ MAB-3 MatrixBlank < LOQ The limitofquanfitati(oLnOQ) was 0.115 mg a.i./bLasedupon theproductofthelowestcalibration standar!d2!bZ!d 0.00229Mg a.i.2andthedilutiofnactoorfthematrixblank!!ELles(50.0). ANEOED WILDLIFE INTERNATIONAL LTD. -31- PROJECTNO.: 454A-105 Table 3 MatrixFortificatioAnsalyzedConcurrentlDyuringSampleAnalysis Sample Number (454A-105-) MAS-10 MAS-4 MAS-7 Concentrationosf PFOS (mga.i.L) Fortified Measured 0.915 0.780 0.915 0.952 0.915 0.858 Percent Recovered 85.2 104 93.8 MAS-1 1 45.7 43.7 95.7 MAS-5 45.7 43.5 95.0 MAS-8 45.7 42.7 93.4 MAS-12 110 94.2 85.5 MAS-6 110 113 103 MAS-9 110 106 96.7 Mean = 94.7 StandardDeviation= 6.50 CV' = 6.86 @. N = 9 Note:Resultsand correctionfsornew testsubstancepuritywere generatedusingMacQuan version1.5 softwareand manual calculationsV.alueshave been roundedforMarl@,ng2u!poses. A Aff'iNDED WILDLIFE INTERNATIONAL LTD. -32- PROJECT NO.: 454A-105 Table 4 Measured ConcentrationosfPFOS inFreshwaterSamples from a FreshwaterMusselAcute ToxicitTyest Nominal Test Concentrafion @Mi a.i./L) 0.0 Sample Number (454A-105-) 1 "'@'2 13 14 25 26 sampling Time 2LOur.5) 0 0 48 49 96 96 PFOS Measured Concentrationi LMJa.i.iL@ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Percent of Nominal 5.7 3 0 4 0 15 48 16 48 27 96 28 96 5.47 4.93 5.18 5.70 5.24 5.26 94.9 85.5 89.8 98.9 90.9 91.-2 11 5 0 6 0 17 48 18 48 29 96 30 96 11.4 96.0 10.1 84.3 11.2 94.1 10.5 88.2 10.9 91.3 15.4 130 23 7 0 8 0 19 48 20 48 31 96 32 96 -19.0 83.3 16.8 73.7 18.7 81.8 18.7 81.8 22.9 100 22.4 98.0 46 9 0 10 0 21 48 22 48 33 96 34 96 37.2 81.4 40.6 88.9 37.1 81.2 39.5 86A 48.2 105 40.5 88.5 91 11 12 23 24 35 36 Note: Resultsand con-ectionfsornew 0 0 49 48 96 96 testsubstancepuritywere 69.0 74.7 81.3 77.6 88.2 85.7 generatedusingNiacQuan 75.4 81.6 88.9 84.9 96.4 93.7 version1.5 softwareand manual calculationVsa.lueshavebeenrounded forreportinpgurposes. The limitofquantitatio(nLOQ) was 0.115 mg a.i./bLasedupon theproductofthelowestcalibratiosntandardanalyzed (0.00229mg a.i./La)nd thedilutiofnactorofthematdx blanksamples(50.0). ANIENDED WILDLIFE INTERNATIONAL LTD. -33- PROJECT NO.: 454A-105 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FRESHWATER Preparematrixfortificatsiaomnplesby spikintgherq*ite volumeofPFOS stocksolutiondsirectly mto fi-eshwatuesrmg gas-tigbsiynngesand ClassA volumetdcflasks. Ddutr,matnx fortificataondtestsamplesuto therangeofthecalibratisotnandardbsy partiallfyillmg ClassA volumetdcflaskwsith50% methanol:50% NANOpwa watersomon containing 0.100mg 4H PFOS (btmmalstandard)/aLnd 0.05% fonnicacid(v/v).Add theappropriatveolumeof . leand bringtheflasktovolumewiththedilutiosnolveirPtr.ocessthematrixblanksunpleusing thesame chlutioannd WWat volumeasforthelowestfortificatloenvel.Mix wellby severalrepeat inversions. AmpuL-de samplesand submitforLC/MS analysis. Figure1. AnalyticamlethodflowchartfortheanalysiosfPFOS infreshwater..@, WILDLIFE INTERNATIONAL LTD. -34- PROJECTNO.: 454A-105 2.000 1.50- 1.00'. 0.50- 0.00 0.000 ...... 0.050 0.100 0.;5,0,'0.200 Concentmfim(Rado) 0.250 0.300 Flgure2. A typicaclalibraticounrveforPFOS. Slope 6.30785;Intercept0.04889;r 0.9986. ANEMED WILDLIFE INTERNATIONAL L-m. -35- PROJECT NO.: 454A-105 100.90, 80, lntensft6r3.51 cps 152 60r so. 40- 3& 20- 132 315 39481-*Y- 'Y-7- 96 395 OVA 0 . ".r-5- P 61 91 121 151 181 2il soon 1.30 2.55 &81 &06 6.32 7.67 8.83 Tkne Figure3. A representatiivoenchi 'ogramofa low-lowl(0.00229inga.iJL)PFOS sbmdard. AMEMED WILDLIFE INTERNATIONAL -36- PROJECTNO.: 454A.105 lntensft5l0r6.11 cps 100- 152 go, so@ 70, 6& 4(@ 133 20, 1& 20 .44 59 - 82 113 187. 208 12, 151 'lil 211 soon I.SO 2.55 3.61 6.06 6.32 7.67 8.83 Tkne Mgum 4. A--- veionchromatogramofa high-leve(l0.0274mg a.i.ALP)FOS standarcl AND41DED. WILDLIFE INTERNATIONAL LTD. -37- PROJECTNO.: 454A-105 2S go. so, 70- .60,- lntenslt1y1.8 cps 162 40 40- 30- 20" 0 127 97 as 1 115 215 vi 31 al ol 121 151 lil 211 S@wn 1.30 2.55 3.81 &06 6.32 7.67 OAS Tkm Figure5. A representatiIve ofa matrixblanksample(454A-105-MAB-2). The arrowindicatetsheretentiotninmofPFOS. WILDLIFE INTERNATIONAL LTD. -38- PROJECTNO.: 454A-105 100. go, so. 706050@ 403020, - Intensity4*2.545 cPs 152 133 9 0ol 25 42 1.30 63 67, 2.55 87 . 9 1. 3.81 113 iii 5m 161 6.32 ISO I.OT82r.1-I1 I lil 7ZT 211 SAS Tkne Fipre 6. A representativcehromatogram of a 1.00 mg a.i./Lmatrix fortificatiosnample (454A-105-MAS-4). WILDLIFE INTERNATIONAL -39- PROJECT NO.: 454A-105 lntensft@3*9233 cps 100- 174 90, so, 70- 6(@ 60. 40- 30. 151 20- 10, ol 26 41 . .9.1.1.06..1.26. 20a 31 al ol 121 151 181 2il sm 1.30 2.55 3.81 5.06 6.32 7.67 sm Tkhe F"igure7. A representaticvheromatogramofa 6.3mg a.i.AtLestsainple(454A-105-16). WILDLIFE INTERNATIONAL LTD. -40- PROJECTNO.: 454A-105 APPENDIX IV Changes toProtocol Thisstudywas conductedm accordancewiththeapprovedProtocolwiththefollowmgchanges: I. The protocolwas amended to add the proposedexpminientasltartand terminatiodnates,test concentratioannsd testsubstancenumber 2. The protocolwas amencl6dtorenew thetestsolutionast48 hours. 3. The protocolwas amended tospeciftyhatGLP awaysm ofwellwaw willbe reported 4. The protocolwas amended toreducetheloadingrate. 5. The weightand lengthofdw musselswas detmmiinwby weighingandmeasuring20 individualfsromam same lotoftestorganisms. 6. The musselswm-e &tnbuted tothetestchambers. WILDLIFE INTERNATIONAL LTD. -42- PROJECT NO.: 454A-105 APPENDIXVI ReportAmendment I. OriginalReport: Pages 1-4,6 and 24 Amendment: The pageswere changedtomclude theamended reportdate,revlscpdagenumbers,and new signatureasnd datesdue totheadditioonfthereportamenchnentasAppendixVI. Reason: To reflecttheissuingof an amended reporl 2. OriginaRleport: Page 2 Amendment: The compliancestatementwas mvise& Reason: To clarifhyow thetestsubstancewas charactmized. 3. OriginaRleport: Page 9 Amcnchncnt: Informatiopnrovidedby theSpon" reflectitnhgereanalysiosfthetestsubstance, includintghereanalysidsateand thepurityw,as addedtotheTestSubstancesection. Reason: To reflectthecurrentestsubstanceinformafiopnrovidedby thc.Sponsor. 4. Ongmal Report: Entirereport Amendment: AU testsubstanceconcentrationwsere changedtoieflectthepwityofthetesstwastonm asdeterminedby theSponsorina reanalysiosfthetestsubstanc(eFC-95,Lot217). Testconcentratioonrsiginallwym basedon thereportepdurityof 98.9*/oT.he =tificateofanalysidsatedMarch 9,2000indicateadpurityof90.49"/Toh.mvkm, all test substa= concentrationisn,cludingnominal concentrationmse,asured concentrationasn,d LC50 valuesw,ere recalculateadnd reportedasmg &i./Lbasedon the90.49% purity. Reason: To reporttheresultosf thetestbasedon thetestsubstanceptv4 of 90.490/aotthe requestoftheSponsor. AMENDED Wi LDLI FE INTERNATI ONAL LTD. -43- PROJECT NO.: 454A-105 APPENDIXVI -Continued- ReportAmenchnent AMENDMENT SIGNATUWS: .KsutrtuF4-Drottar . Djxpetor dlla 2 DATE Hemy OvKrucier,Ph.D. Dkeetor,AquaticToxicologyandNon-TargetPlants DATLF REVMWED BY: TiinothFy@p@er, Ph.D. QualityAssurance @/c t9 DATE AMENDED WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454A-105 Page I of2 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR STATIC ACUTE TO)GCITY TEST WITH TIIE FRESHWATER NFUSSEL PROTOCOL NO.: 454/042199/MUS/SLJB454 SPONSOR: 3M Corporation EF'FECTIVE DATE: May 20,1999 AMENDMENT: Page 2 AMENDMENT NO.: I PROJECT NO.: 454A- 105 Add: ExperimentalStartDate: 5/28/99 ExperimentalTerminationDate: 6/l/99 Test Concentrations:Negative Control,6.3,13,25, 50 and 100 mg Test Substance No.: 4675 a.i./L REASON: The above informationwas not known when theprotocolwas signedby the Study Director. AMEENDMENT: EXPERINLENTAL DESIGN Page 3 Add: Testsolutionwsillbe renewed atapproximately48 hours. New testsolutionswillbe prepared and the mussels willbe transferrefdrom the old to the new testsolutions. REASON: Test solutionswillbe renewed at48 hours to improve the water quality. AMENDMENT: DilutionWater,Page 5 Change: Analyses willbe performed atleastonce annuallyto determinethe concentrationsof selectedorganic and inorganicconstituentsof the well water and the resultsof the analyseswillbe sununarizedinthe finalreport. To: Analyseswillbe performed atleastonce annuallyto determinethe concentrationosf selected organicand inorganicconstituentosf the well water and the resultsof the most recentGLP compliantanalyseswillbe sununarizedinthe finalreport. REASON: To specifythatGLP analyseswillbe reported. a45411051&mendl WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454A-105 Page 2 of2 In AMENDMENT: TestApparatusPage 5 Change: Test chamberswillbe 25-L,polyethylenaequariafillewdithapproximatel1y5 L of water To: Testchamberswillbe 25-L,polyethylenaequariafillewdith approximatel2y0 L ofwater. REASON: To reducetheloadingrate. STUDY DIRECTOR DATE LABORX-TORY MARkdEMENT DATE sp6NsoR,s REPREsENTATivE DA a4MIgsiammdl WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-105 Page I of I DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR STATIC ACUTE TO)UCITY TEST WITH THE FRESHWATER MUSSEL PROTOCOL NO.: 454/042199/MUS/SU]3454 SPONSOR: 3M Corporation DATE OF DEFAC-RO DEVIATION: June1,1999 DEVIATION NO.: I PROJECT NO.: 454A- 105 DEVIATION: The protocosltatetshatvalveheightsand weightsoftheindividuamlusselswill be measuredattheend of thetest.The valveheightsand weightsof 20 representativmeusselsfrom thesame lotwere measurecl REASON: BiologisotversightThe musselsusedinthetestwere shippedtotheSponsorbeforethey were weighed and measured. The musselsused inthetestand themusselswhich were weighedandmeasuredwereindiscriminatseellyectedfrom thesame lotoftestorganisms. Consequentlyi,tis thebestjudgment of theStudy Directorthatthisdeviatiodnidnot adverselyaffect heresultosf thestudy- STUDY DIRECTOR 19Z LABORATORY MANAGEMENT KRD4-Sft\105*dl DATE ol DATE WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454A-105 Page I of I DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR STATIC ACUTE T03UCITY TEST WITH THE FRESHWATER MUSSEL PROTOCOL NO.: 454/042199/MUS/SUB454 SPONSOR: 3M Corporation DEVIATION NO.: 2 PROJECT NO.: 454A-105 DATE OF DEFACTO DEVIATION: May 28,1999 DEVIATION: The protocolstatesthatto controlbias,freshwatermusselswillbe randomly assignedto exposurechambers attestinitiationT.he musselswere actually in@@ly distribut(eadrandom number tablewas notusedtodistributthee mussels). REASON: BiologisotversightI.tisthebestjudginenotftheStuclyDirectotrhatthisdeviatiodnid not adverselyaffectheresultsof thestudy. STUDY DIRECTOR LABORATORY MANAGENEENT KPM4--uW\105\prC DATE DATE PROTOCOL PFOS: A 96-HOUR STATIC ACUTE TO)ACITY TEST WITH THE FRESHWATER MUSSEL U.S.EnviromnentalProtectioAngency Series850 -EcologicaElffectsTestGuidelines OPPTS Number 850.1075 and OECD Guideline203 3M Lab RequestNo. U2723 WILDLIFE Submittedto 3M Corporation EnvironmentalLaboratory Building2-3E-09 935 Bush Avenue St,PauL Minnesota 55144 NTERNATIONAL LTD. 8598 Commerce Drive r Easton,Maryland 21601 (410)822-8600 April21, 1999 WILDLIFE INTERNATIONAL, L-rD go -2- PFOS: A 96-HOLJR STATIC ACUTE TO)UCITY TEST WITH THE FRESHWATER MUSSEL SPONSO 3M Corporation EnvironmentalLaboratory Building2-3E-09 935 Bush Avenue St.Paul,Minnesota 55144 SPONSOR!S REPRESENTATIVE: TESTING FACILITY: STUDY DIRECTO LABORATORY MANAGEMENT: Ms. Susan A. Beach WildlifeInternationLatld. 8598 Commerce Drive Easton,Maryland 21601 Kurt DrotLar SeniorAquatic Biologist Henry 0. Krueger,Ph.D. DirectorofAquaticToxicology& Non-TargetPlants FOR LABORATORY USE ONLY Proposed Dates: Experimental StartDate: ProjectNo.: q!54A Test Concentrations: Test SubstanceNo.: - /0!@- Experimental TerminationDate: ReferenceSubstanceNo. (ifapplicable): PROTOCOL APPROVAL STUDY DIRECTOR /3 DATE L 0 k&OPY MANAGEMENT SPOVSO-R!SREPRESENTATIVE PROTOCOLNO. 454/042199/MUS/SUB454 DATE qbq lqq DATE 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -3- INTRODUCTION WildlifeInternationaLlt,d.willconducta statiaccutetoxicittyestwith thefreshwatermusselforthe Sponsor attheWildlifeInternationaLlt,d.aquatictoxicologyfacilitiynEaston,Maryland. The studywill be performed based on proceduresintheU.S. EnvironmentalProtectionAgency Series850 - Ecological EffectsTest GuidelinesOPPTS Nwnber 850.1075 (1);OECD GuidelineforTestingof Chemicals203:Fish, Acute ToxicityTest (2);and ASTM StandardE729-88a Standard Guide for Conducting Acute Toxicity Tests with Fishes,Macroinvertebratesandamphibians (3).Raw dataforallwork performedatWildlife InternationalL,td.and a copy of thefinalreportwillbe filedby projectnumber inarchiveslocatedon the WildlifeInternationaLlt,d.siteo,r atan alternativleocationtobe specifieidnthefinalreporl PURPOSE The purposeofthisstudyistodeterminetheacuteeffectosfatestsubstanceon thefreshwatemrussel during a 96-hour exposureperiodunder statitcestconditions. EXPERIMENTAL DESIGN Freshwatermussels-wilble exposed to a geometricseriesof at leastfivetestconcentrationasnd a negative(dilutiownater)controlfor96 hours. Two replicattre,stchambers willbe maintainedineach treatmentand controlgroup,with 10 freshwatermusselsineach chamber fora totalof 20 freshwatermussels per testconcentration. Nominal testconcentrationwsillbe selectedinconsultatimwiththeSponsor,and willbe basedupon informationsuch as the resultsof exploratorryange-findintgoxicitydata,known toxicitydata,physical/chemicalpropertieosf thetestsubstanceor otherrelevantinformation.Targetconcentrationnseed not exceed 1000 mg/L orthesolubilitlyin-Loiftthetestsubstanceinwater(whicheverislower).Generallye,ach testsubstanceconcentratiounsed inthedefinitivteestwillbe at least60*/oof thenexthigherconcentration unlessinformationconcerningtheroncentration-effceucrtveindicatetshata dffcrentchlutiofnactorwould be more appropriate.Water samplesfrom each testchamber willbe collecteadtspecifieidntervalfsor analysisof the testsubstance.Resultsof analyseswillbe used to calculatmeean measured test concentrations. To controlbias,firshwatermusselswillbe randon-Ayassignedtoexposurechambers attestinitiatiom No otherpotentiaslourcesof biasareexpectedto affecttheresultosf thesb*. Observationsof mortality and otherclinicaslignswillbe made ffimghout the 96-hour testperiod.Cumulativepercentmortality PROTOCOL NO. 454/042199/MUS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -4- observed in thetreatmentgroups-,Nible]used to calculatew,hen possible,LC50 valuesat24, 48, 72 and 96 hour intervalsT.he no-mortalitcyoncentratiownillbe determined. MATERIALS AND METHODS Test Substance Informationon thecharacterizatiofn test,controlor referencesubstancesisrequiredby Good LaboratoryPracticeStandards(GLP). The SponsorisresponsiblfeorprovidingWildlifeInternationaLlt,d. writtenverificatiotnhatthetestsubstancehas been characterizeadccordingto GLPs priorto itsuse inthe study.Ifwrittenverificatiomf GLP testsubstancecharacterizatiiosnnot providedtoWildlifeInternational, Ltd.,itwillbe notedinthecompliancestatementofthefinalreport.The attachedform IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR (Appendix 1)isto be used.to provideinformationnecessaryfor GLP compliance. The Sponsor isresponsibleforallinformationrelatedto thetestsubstanceand agreesto acceptany unused testsubstanceand/ortestsubstancecontainersremainingat theend of the study. Preparationof Test Concentrations The testsubstancewillbe administeredtothetestorganism directliyntodilutiownater. This route of administratiownas selectedbecauseitrepresentsthemost likelryouteof exposure to aquaticorganisms. Test Organism The freshwatermussel has been selectedas thetestspeciesforthisstudy. Freshwatermussels are representativoef an importantgroup of aquaticinvertebrateasn,d have been selectedforuse inthe testat therequestof the Sponsor. Valve heightand weightsof theindividuamlusselswillbe measured attheend of thetest.Mussels willbe obtainedfrom a commercial supplieror hatchery,and theidentitoyf thespecies willbe verifiedby thesupplier. Freshwatermussels willbe heldforatleast14 days priorto thetestin water from the same source and at approximatelythesame t=iperatureas used duringthetest.Any changes in watertemperaturewin not exceed 3 'C per day. Freshwatermussels willbe held atthetesttemperaturefora minimum of 7 days priorto testinitiationF.reshwatermusselswillnotbe used inthetestiftheyshow signsof diseaseor stress or ifmore than 5% dieduringthe7 days priorto thetest.At testinitiatiotnh,e freshwatermussels willbe collectedfrom holdingor acclimatiotnanksand transferretdothetestchambers. At testterminationall PROTOCOL NO. 454/042199/MUS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, L-rD -5- remaininglivemussels willbe sacrificefdorcollectioonf tissue@amples. The tissuesamples willbe composited by replicatef,rozenand shippedto theSponsor forpossibleanalysis. DilutionWater Water used for the holdingand testingof freshwatermussels willbe obtainedfrom a well approximately40 metersdeep locatedon theWildlifeIiitemationaLlt,d.site.The waterwillbe passed through a sand filtearnd pumped intoa 37,800-L storagetank where thewater willbe aeratedwith spray nozzles.-Priorto use thewater willbe fdtoredto 0.45,vm inordertoremove fineparticlesW.ater used for culturingand testingischaracterizeadsmoderatelyharcl Typicalvaluesforhardness,alkaliinty,pH and specificonductanceareapproximately: Hardness,mg/L as CaCO3 Alkalinitym,g/L as CaCO3 pH SpecificConductance,gmhos/cm 145 190 8.1 330 Hardness,allWmity,pH and sp=fic conductancewillbe measured weekly tomonitortheconsistmcy of thewellwater. Means and ranges.of themeasured parwncteisforthe four-weekperiodprecedingthetest willbe providedinthe finalreport.Analyses willbe performed atleastonce annuallytodeterminethe concentrat.toorfmselectedorganicand inorganicconstituentosfthewellwater and resultosfthe analyseswill be summarized inthe finalreport. TestAnar Test chambers willbe 25-L, polyethylenaequariafillewdith approximately15 L of water. Test chambers willbe positionedin an environmentalchamber or temperature-controllweadterbath to maintain a temperatureof 22 2 *C. Test chambers willbe labelledwiththeprojectnumber, testconcentratioannd replicate. EnvironmentalConditions Lightingused to illuminattehecult= and testchambers duringholding,acclimationa,nd testingwill be providedby fluorescenttubesthatcmitwavelengthssunilartonaturalsunlight(e.g.C,olortonel5l0). A photopmiod of 16 hours of lightand 8 hoursof darkwillbe controllewdith an automatictimer.A 30-n3inutc transitiopneriodof low lightintensitwyillbe providedwhen lightsgo on and offto avoidsudden changes inlightintensityL.ightintensitwyillbe measured attestinitiatiwointha SPER ScientifiLctd.lightmeter or equivalent. PROTOCOL NO. 454/042199ftAUS/SU3454 3M LAB REQXJEST NO. U2723 FMF.MM WILDLIFE INTERNATIONAL, LTD -6- The targettesttemperaturewillbe 22 2*C. Temperature willbe measured in allreplicateast the beginningand end of thetestusing a liquid-in-glatshsermometer.Temperature alsowillbe measured with a continuousrecorderina beakeradjacenttothetest.Recordermeasurements willbe verifiewdith a liquidin-glassthermometer priorto testinitiation. Dissolvedoxygen willbe measured in allreplicateosf the treatrnenatnd controlgroups at test initiatioannd at approximately24-hour intervaltshereafteursing a Yellow SpringsInstrumentModel 5 1B dissolvedoxygen meter,or equivalent.Intheeventthatdissolvedoxygen levelsfallbelow 60% saturation, appropriateactionswillbe takenafterconsultatiowniththe Sponsor. Measurements of pH willbe made in alternatinrgeplicateosfthe treatmentand controlgroups attestinitiatioand atapproximately24-hour intervaltshereafteursinga FisherAccwnet Model 915 pH meter,or equivalent.Ifa treatmentgroup reaches 100% mortalityd,issolvedoxygen,pH, and temperaturemeasurements willbe taken atthenext sampling intervalt,hendiscontinued. Hardness,alkalinitayn,d specificonductancewillbe mgasured inthedilutiownaterand low,middle and high testsubstanceconcentrationasttestinitiatioand attesttermination.Hardness and alkalinity measurements willbe made by titratiounsingproceduresbased on methods inStandard Methods for the Examination of Water and Wastewater(4).Specificonductancewillbe measuredusinga Yellow Springs InstrumentModel 33 Salinity-Conductivity-Temperatmuerteer,orequivalent. BiologicalMeas Observationsof mortalityand clinicaslignsof toxicitwyillbe made between 0-24 hours,and at 24, 48, 72 and 96 hours I hour.Lethalitiysdefmed as thelackof visiblmeovement (eg open orgaping shell) inthemussel aftergentleprodding.Allclinicaolbservationswillbe notecl SmRIing forAngLyticalMeasurements Water samples willbe collectefdrom each testchamber atthebeginningof ffictestd,uringffictest and atfficend of thetea w downnine concentrationosfthe testsubstance.In theeventihgd100% mortality occursin any breauncntthensamplingof thathwbnent willterminatefollowingthenext samplinginterval. Samples willbe collectedatmid-depth from each testchamber and analyzediimnediateloyr placedin an appropriatestoragecontainer(e.g.p,olyethylenoer polypropylenebottle)and storedunderrefrigeratiuontil analyzed. The sample scheme issummarized below: PROTOCOL NO. 454/042199/MUS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, L-rD -7- PROPOSED NUMBERS ExperimentalGroup Control LevelI-Low Concentration Level2 Level 3 Level4 Level5-HighConcentration Totals OF VERIFICATION SAMPLES 0 Hours 48 Hours 96 Hours 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 12 12 12 TotalNumber ofVerificatioSnamples 36 The above numbers of samplesrepresenthosecollectefdrom thetestand do not includequality control(QC) samplessuch asmatrixblanksand fortificatipornesparedand analyzedduringtheanalytical chemistryphase ofthe study.At thediscretioonfthe Studyf)irectowra,tersamplesfrom one or more appropriatteestchambers willbe collecteadnd analyzedifan analyticaelrrorinsamplingor analysisis suspected.The reasonfortheadditionaslampleswillbe describedby theStudy Directorand documented intheraw dataand finalreport. AngLyficalChemis Chemicalanalysisofthesampleswillbe performedby WildlifeInternationaLlt,d- The analytical method used willbe basedupon methodologyprovidedby theSponsor and identfiiedinAppendix 11.The methodologyusedtoanalyzethetea sampleswillbe documaiw intheraw dataand summarizedinthefinal report. Data Anal)@5is When the concentration-respopnasteternallowscalculatioonf an LC50 value,the datawillbe analyzedusingthecomputersoftwareofC.E.Stephan(5).The program was designedtocalculatteheLC50 value and the 95% confidenceintervably probitanalysist,hemoving averagemethod,or binomial probabilitwyithnonlineairnterpolati(o6n,7,8)T.he LC50 valuewillbe calculatewdh,en possibleu,sing mortalitydatacollecteadt24,48,72 and 96 hours.The no-mortalitcyoncentratiownillbe determined. PROTOCOL NO. 454/042199/MUS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -8- RECORDS TO BE MAINTAINED Recordstobemaintainefdordatageneratebdy WildlifIenternatioLntadl.,willincludbeu,tnotbe limitedto: I. A copy of the signedprotocol. 2. Identificatiaond characterizatioofnthe testsubstance,ifprovidedby the Sponsor. 3. Dates of initiatiaond terminationofthetesl 4. Test organism historyh,oldingand acclimationrecords. 5. Stock solutioncalculatioannd preparationi,fapplicable. 6. Observations. 7, Water chemistryresults(e.g.a,lkalinitaynd hardness). 8. The methods used toanalyzetestsubstanceconcentrationasnd theresultsof analyticamleasurements. 9. Statisticcaallculationsi,fapplicable. 10. Testconditions(lighitntensityp,hotoperiodd,e.). 11. Calculationand preparationof testconcentrations. 12. Copy offinalreport. FINAL REPORT A finalreportof theresultosfthestudywillbe preparedby WildlifeInternationaLlt,d. The report willinclude,butnotbe limitedto,thefollowingw,hen applicable: 1. Name and addressof the facilitpyerformingthe study. 2. Dates upon which the studywas initiateadnd completed,and the defufitiveexperimentalstartand terminationdates. 3. A statementof compliancesignedby the Sb* Directoraddressingany exceptionsto Good Laboratory PracticeStandards. 4. Objectivesand procedures,as statedinthe approved protocol,includingallchangesto theprotocol. 5. The testsubstanceidentificatiionncludingname, chenficalabstractnumber or co& number, strength, purity,composition,and otherinformationprovidedby the Sponsor. 6. Stabilitaynd solubilitoyf thetestsubstanceunderthe conditionsof administratioinf,providedby the Sponsor. 7. A descriptioonf themethods used toconductthe test. 8. A descriptioonf thetestorganisms,includingfficsource,scientifincame, age or lifestage,lengths and weightsof a representativgeroup of testorganisms,feedtypes,lightintensitaynd photoperiod. 9. A descriptioonf the preparationof thetestsolutions. PROTOCOL NO. 454/042199/MUS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -9- 10. The methods used toallocatoerganismstotestchambers and begin thetest,thenumber of orgaristns and chambers pertreatment,and the durationof the test. 11. A descriptioonf circwnstancesthatmay have affectedthe qualityor integritoyf the data. 12. The name of the Study Directorand the names of otherscientistsp,rofessionalsa,nd supervisory personnelinvolvedin thestudy. 13. A descriptioonf thetransformationsc,alculationasn,d operationsperformedon thedata,a summary and analysisof thebiologicadlataand analyticaclhemistrydata,and a statementof theconclusions drawn from theanalyses. 14. Statisticamlethods used to evaluatethe data. 15. A graph of theconcentration-mortalciutryve atthe end of thetest 16. The signedand datedreportsofeach oftheindividualscientistosr otherprofessionalisnvolvedinthe study. 17. The locationwhere raw dataand finalreportareto be stored. 18. A statementpreparedby theQualityAssurance Unit hstingthedatesthatstudyinspectionasnd audits were made and thedatesof any findingsreportedtothe Study Directorand Management. 19. Ifitisnecessaryto make correctionosr additionsto a finalreportafterithas been accepted,such changes willbe made intheform of an amendrnent issuedby theStudyDirector.'Me amendment will clearlyidentiftyhepartof thefinalreportthatisbeing amended and thereasonsforthe amendment, and wiU be signedby theStudy Director. CHANGING OF PROTOCOL Planned changes to the protocolwillbe in the form of writtenamendments signedby the Study Directorand the Sponsoes RepresentativeA.mendments wiU be consideredaspartoftheprotocoland will be attachedto thefinalprotocol.Any otherchanges willbe intheform of writtendeviationssignedby the Study Directorand filedwiththeraw data.Allchanges totheprotocolwillbe indicatedinthefinalreport. GOOD LABORikTORY PRACTICE This studywiU be conductedin accordancewith Good LaboratoryPracticeStandardsforEPA (40 CFR Part 160 and/orPart792);OECD Principleosf Good LaboratoryPractice(OCDE/GD (92) 32, EnvirorunentMonograph No. 45); and Japan MAFF (59 NohSan, NotificatioNno. 3850, Agricultural ProductionBureau).Each studyconductedby Wildlifebftmational,Ltd.isroutineleyxamined by the Wildhfe IntemationaLLtd.QualityAssuranceUnit forromphuice withGood LaboratoryPracticesS,tandard OperatingProceduresand thespecifiepdrotocol.A statementofcompharr-ewithGood LaboratoiyPractices willbe preparedforallportionsofthestudyconductedby WildlifeIntemationaLltd. The Sponsorwillbe PROTOCOL NO. 454/042199/MUS/SUB454 3M LAB REQTJEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -10- responsibleforcompliancewith Good LaboratoryPracticesforproceduresperformed by othexlaboratories (e.g.r,esidueanalysesorpathology).Raw dataforallwork performedatWildlifeInternationaLlt,d.and a copy of thefmal reportwillbe filedby projectnumber inarchiveslocatedon theWildlifeInternationaLlt,d. site,or atan alternativleocationto be specifiedin thefinalreport. PROTOCOL NO. 454/042199/MUS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFFR INTE:RNATIONAL, LTD REFERENCES I U.S.EnvironmentalProtectioAngency. 1996. Series850-EcologicaElffectTsest Guidelines (draft)O,PPTS Number 850.1075:FishAcuteToxicitTyestF,reshwaterandmarine. 2 OrganisationforEconomic Cooperationand Development 1993.OECD GuidelinefsorTesting ofChemicals.Guidelin2e03:Fish,AcuteToxicitTyest.Adoptedby theCouncilon 12 July1992. 3 ASTM Standard E729-88a. 1994. Standard Guide for Conducting Acute Toxicity Tests with Fishes,Macroinvertebrates, andamphibians. American Society forTesting and Materials. 4 APRA, AWWA, WPCF. 1985.StandardMethodsfor theExaminationof WaterW7d Wastewater. 16th EditionA,merican PublicHealthAssociation.American Water Works Association.Water PollutioCnontrolFederatioNne,w York. 5 Stephan,C.E. 1978.U.S.EPA, EnvironmentaRlesearchLaboratoryD,uluth,Minnesota.Personal conununication. 6 Thompson, W.R. 1947. BacteriologicaRleviews. Vol.IL No. 2. Pp. 115-145. 7 Stephan, C.E. 1977. "Methods for CalculatingA LC50," Aquatic Toxicology and Hazard Evalual7ons.American SocietyforTestingand Materials.PublicationNumber STP 634, pp 65-84. 9 Finney,DJ. 1971. StatisticMaelthods inBiologicaAlssay. Secondedition.GriffinPress, London. PROTOCOL NO. 454/042199/MUS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD k APPENDIXI D)ENTIHCATION OF TEST SUBSTANCE BY SPONSOR To be CorWkftd by Spmsm I. TestSubstanceIdentit(yname tobe used inthereport):PFOS (PerfluorooctanSeulfonicAcid PotassiumSalt ReferenceStandard(ifapplicable)A:nalyticaSltandard: N/A InternaSltandard: l.l.2.2H.H.H.HPerfluorooctanSeulfonicAcid Test Substance Sample Code or Batch Nwnber: Lot 217 TestSubstancePurity(0/AoctiveIngredient): 98.9 ExpirationDate: 2008 ]I. TestSubstancelamactmizatim Have thekkntitys,trm%n tmIasnud4= 2:onm=odffcbmwtaisfics wlich appropriately. st@ been ddmnifi6d liriotroitsusem thisstudym aaxrdance vathGLP Smndards? Yes IH. TestSubstanceSWrage Cm&fiom Pleaseindicatteherecomnxmded stmge cmmons atWildlifIentmnadonallAd x No Ambient Has thestabiliotfythetestsubstanceunderfl= sbrageconditions bem detmmmed m aaadance vadiGLP SWndards? Offiapm*W stabiliitnyfmnafial: Yes x No IV. TestConcmtrafior)s: AdjusttestcmicmtratimtD 100'/ao.L x basedWm thepumy Clo)grvm above Dp notad4g testcm=nft-afimtD 1000/o &L TesttheniatmiaAlS IS. V. ToxicitIynfonnatim: MmmnaharL RatLD50 251mg/kiz MoiwLD50-N/A Agjafic: hwatebratTeoxicitTy=O) FishTo)d* (LC50) DgRbnia magna: 27 ma Rainbow Trout: II mp-IL Dgphnia magga: 50 mWL Fathead Minnow: 3 8 mgJL Od= To)dcitIynfamuhm (ichxbg fin&W ofchroniacndmbduaic tests): Pleasesee MSDS PROTOCOL NO. 454/042199/MUS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -13- APPENDIX H OEM AnalyticalMethod Providedby Sponsor Samples willbe analyzedbased upon proceduresprovidedby theSponsor in thefollowinganalyticamlethods: 1. Liquid Chromatography Mass Spectrometry(LCMS) Method forthe Determmation of Perfluorooctane SulfonicAcid PotassiumSalt(PFOS) InFreshwater,Saltwaterand Algal Medium A copy of the above method willbe maintainedin theraw data.The actualmethodology used to analyzethetest samples willbe documented intheraw dataand summmized inthefmal report. PROTOCOL No. 454/042199/MUS/SUB454 3M LAB REQUEST NO. U2723