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PFOS: ARX. `WITH TAH9E6E-AHSOTUERRSNHOELYLSTDEEPRO(SCIraTsIsOosNtrTeEaSvTirginica) FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER:454A-106 3M LAB REQUEST NO. U2723 U.S Environmental Protection Agency Series 850 ~Ecol Effo ectg sTei stGc uida elil nes (OPPTS Number 850.1025 AUTHORS: Kut Drotiar - HenryO. Krueger, Ph.D. STUDY INITIATION DATE: May 3, 1999 - STUDY COMPLETION DATE: August 11,1999 : AMENDED REPORT DATE: April 26,2000 `Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue. St. Paul, Minnesota 55144 Wildlife International Ltd. 410) 522.8600 8598 Commerce Drive `Easton, Maryland 21601 a 000745 AMENDED bO WIoLDm LIFtEbiImiNbTiEO nRcNnATIOtNnALtLm... PROJEC. TNO.: i4o5n4sAs-1t06AR 2. (GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PROS: A 96-Hour Shell Deposition Test vith the Eastern Oyster (Crassostrea virginica) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-106 STUDY COMPLETION: August 11, 1999 AMENDED REPORT: Aprit26, 2000 Envir`oTnhmiesnsttauldPyrwotaescctoinondAucgteendciyncionm4p0lCiaFnRcPeawritthsG1o6o0adLnadb7o9r2a,to1r7yPAruacgtuiscte 1S9t8a9nd;aOrdEsCasDpuPbrliinschiepdlebsyotfhGeoUoS.d `LNaobhoSraant,orNyotPirfaicctaitcieo,nONCo.D3E8/5G0,DAg(r9i2c)ul3t2u,rEanlviPrroondumcetnitoMnoBnuoregarua,p1h0NAou.gu4s5t, 1P9a8ri4swi1t9h9t2h;eafnodlloJwaipnagneMxcAeFptFi,on5s9: `Thetestsubstancewasnotcharacterizedinaccordancewithfull GLPcomplisnce;however, the characterizationwasperformedaccordingto 3MStandardOperatingProceduresandMethods,andallawdata are beingmainitnthae3iManrcheivdes. Thetest substanceisbeingrecharacterizedinaccordancewith GLP. "Thestabilityofthetestsubstanceunderconditionsofstoragea thetestsitewasnotdeterminedin accordance vith Good Laboratory Practice Standards. ER STUDYDIRECTOR: Kurt Drottar Seaior Biologist pee DATE `SPONSOR APPROVAL: . 5; Y)37 Jeo DATE 000746 AMENDED WILDLIFE INTERNATIONAL Lm. oto PROJECTNO.: 454A-106 QUALITY ASSURANCE STATEMENT U.S. E"nTvhiirssontmuednytwaalPsroetxeacmtiinoendAfgoernccoym,p4l0iaCnFcRewPiatrthsGo16o0daLnadbo7r9a2,to1r7yAPruagcutsitce1S9t8a9n;dOarEdCsDaPsrpuibnlicshieodpbflyGoteohsde Laboratory Practice, OCDE/GD (92) 32, Environment Monograph No. 45, Paris 1992; and Japan MAFF, in5s9peNcothiSoanms,anNodtaiufdiicattsiaonndNto.he3d8a5t0e,s tAhgartiacunlytufrianldiPnrgosdwucetrieonrepBourretaeud,tot10heAuSgtuusdtyD1i9r8e4c.torTahnedLdaatbeosraotfoarlyl Management were as follows: AcTvTy: TestSubsancePreparation FoTretsitfinciattiioonnPraenpdarMaattioinx. RBeipoolrogtical Data and Dent DAnnatlytRiecpaolrDtutaand `FinalReport AmendedReport DATE CONDUCTED: May21, 1999 May24,1999 iy, 1599 Jy1,1999 August 11,1999. Api 25,2000 DATE REPORTED TO: STUDYDIRECTOR: MANAGEMENT: May21,1999 May21, 1999 May24,1999 May26,199 Jy1,1999 Juy2,1999 Jy1,1999 August 11,1999 Api 25,2000 July2,1999 August 11,1999 i 26,2000 NH. Coban `wQuarlityHA.sCsourlaenmcaenRepresentative y-26-00 DATE 000747 AMENDED WILDLIFE INTERNATIONAL Lm. vhs REPORT APPROVAL PROJECT NO.: 454A-106 SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Shell Deposition Test with the Easter Oyster (Crassasirea virginica) WILDLIFE INTERNATIONAL LTD, PROJECT NUMBER: 454A-106 Al STUDYDIRECTOR: Kurt R Drottar Senior Biologist : MANAGEMENT: Heary O PhD. Director, Aquatic Toxicology and Non-Target Plants pee - DATE . oh foo DA 000748 AMENDED WILDLIFE INTERNATIONAL Lo. $= `TOAFCBONTLENETS PROJECTNO.: 454A-106 Tab OFCOMERS... DB mmmmmmmsmmm--------S---------- MBCGT1 MIEISBOLS.....ns sss TABLES "Table 1 - SummaryofAnalytical CRETIStY Dat... `Table 2 - Temperature,DissolvedOxygenandpHofWaterintheTest `Table:3 -ShellGrowthand Shel GrowthInhibitionat Test Table 4 -IndividualOysterShellMeasuremets.......... : 15 CATIDES....c.crvr 16 TEMEEHOR........evre 11 msn 000749 e WILDLIFE INTERNATIONAAL Lm. R tte DPRROOTJEECCTINNOO.:: 455441-12066 -6- `TOAFCBONTLENETS - Continue-d APPENDICES Appendix. I~ SalinityandpHofUnfilteredSaltwater MeasuredDuringthe 4-WeekPeriodImmediatelyPrecedingthe Test Appendix II AnalysesofPesticides, Organics, MetalsandOtherInorganics ...........cc. 19 AppendIix- TheAnalysisof PFOSin UnfilteredSaltwaterin SupportofWildife IntemaLttd.iPROoJGnGtaNOl: 454-10... 21 Appendix V - Personnel IVOIVEdinthe SKY.. I. 000750 AMENDED e WILDDLLIPFEE ITNETERRNNAATTIOONNAAe LLLLTm,. e EPRDOJEECCTTHNOO::a4S54UA1-10066 7. SPONSOR. SPONSOR'S REPRESENTATIVE: DLAOTCAATAINODNOAFCSOTPUYDOYF,TRHAEW FINALREPORT: "WILDLIFEINTERNATIONAL LTD, PROTECTNUMBER: TESTSUBSTANCE: stooy: MEANMEASURED TEST CONCENTRATIONS: TESTDATES: LENOTHOFTEST: TEST ORGANISM SOURCE OFTESTORGANISMS: OYSTERLENGTH(N = 20). TEHOURECS: 95%CONFIDENCELIMITS: C'ONNOC-OEBNSTERRAVTEIDO-ENF:FECT- LCOoWnEcSeTn-TORBASTEIROVNED-EFFECT SUMMARY IM Corporation Ms. SusanA. Beach E`asWtiodne,MIantreyrlnaantidon2al1L6t0d1. 454A-106 PFOS(PerfluorooctaneSulfonic AcidPotassiumSalt) PFOS: A 96-Hour Shell Deposition TestwiththeEasternOyster (Crassostrea virginica) NegativeControl, 0.36,0.40, 13, 1.9 and 3.0 mgail Experimental Star-t May 24, 1999 Biological Termination - May 28, 1999 Experimental Termination - June 14, 1999 96 Hours Eastern Oyster (Crassostrea virginica) P.CumminsOysterCompany, Inc. B6a1l8tiWmeosrte,3M3a%ryStlraencdt 21211 Me=3a3.8mnm, Range =2t7041..5m8m >30mgail `Not Caleulable L9mgaiA L 30mgai: n 000751 AMENDED LWIO LDLE IFE INNITEERRNNAATTIIOONNAALLLLDm,. eee -8- PPRROOTJEECCTTNNOO: 540540A0-10066 INTRODUCTION "ThsstudywasconductedbyWildlifeInternationalLd.for 3MCorporationattheWiklifeInternational Lid.aquatictoxicologyfacilityinEaston,Maryland.Thein-life phaseofth testwasconducted romMay240 May 28, 1999. Rawdatageneratedby WildlifeInternationalLtd.and acopyofth finareportarefiledunder ProjectNumber454A-106inarchiveslocatedon theWildlifeInternational Ltd. site. . OBJECTIVE `Theobjeofcthitsstiudvywaestoevaluatetheacute toxiocfiPFtOyS(Perfluorooctane SulfonicAcid Potassium Salt) on shell depositionoftheeaster oyster, Crassostrea virginica, during a 96-hour exposure, `periodunderstatictestconditions. EXPERIMENTAL DESIGN Eastemoysterswereexposedto ageometri series of ivetestconcentrationsand anegative (dilution `water) control. Onereplicate test chamberwas maintainedineach treatment and controlgroup, with 20 oystersin eachtestchamber. NominaltestconceatrationswereselectedinconsultationwiththeSponsor, endwerebased upontheresultsofanexploratoryrangefindingtoxicitytest. Nominaltestconcenstelerctaedwterie1o2,n2.s0, 33,55and9.1mgactiveingredient(a..VL.. Meanmeasuredtestconcentrationsweredeterminedfromsamples oftestwatercollectedfromeachtreatmentandthecontrolgroupat thebeginningofthetet, a approximately48 hours,andattesttermination. `Theoysters were indiscriminatelyassignedto exposurechambersattestinitiation.Algalcells (Thalassiosira pseudonana, Skeletonema sp., Chaetoceros sp.,and Isochrysis sp) were provided to supplement naturally occuringalgaeandto maximizeoystergrowthratesduringtheest. Measurementsofshelldepositionforcachoysterweremadeat 96 hoursandwereusedtoestimate the ECS0 value, the no-observed-cffect-concentration (NOEC) and the Jowest- observed-ffect-concentration (LOEC). TheECS0 istheconcentrationoftestsubstanceinwaterthat isestimatedtoinhibitshelldepositonby 50%,relative othe control. 000752 AMENDED WILDLIFE INTERNATIONAL Lm. -9- PROJECTNO.: 454A-106 MATERIALS AND METHODS "The study was conducted based on the procedures outlined in the protocol, "PFOS: A 96-Hour Shell Deposition Test with the Eastern Oyster (Crassostrea virginica)". The protocol was based on procedures outlined in U.S. Environmental Protection Agency Series 850 -- Ecological Effects Test Guidelines, OPPTS Number 850.1025 (1); Standard Evaluation Procedure, Acute ToxicityTest for Estuarine and Marine Organisms (Mollusc 96-Hour Flow-Through Shell Deposition Study) (2)andASTM Standard E729-88a `Standard Guidefor Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians (3). TestSubstance `Thetestsubstance was receivedfrom 3MCorporationOctober29, 1998andwasassignedWildlife International Ltd. identification number 4675. The testsubstancewas described as a white powder. It was. identifiaesd FC-95 fromlotnumber 217 (T-6295).InformationprovidedbytheSponsorindicated a purityof 98.9%, andanexpirdaatetoif2o00n8.Thetestsubstancewasreanalyzedbythe SponsorandtheCertificateof Analysisdated March9, 2000indicated apurity of 90.49%.Thetestsubstance wasstored atambientroom temperature. - PrepoafTerstaContcenitraotionns Notem stcoi ncenn tratia onswl ere 1.2,2.0,3.3,5.5 and 9.1 mg.i/L, basedonatestsubstpaurnitcyoef 90.49%. Allmaterialswhichcameintocontactwiththetestsubstanceduringpreparationoftestconcentrations wereconsoft plasrticu orsc tait nlese sstd eel. A primarystocksolution waspreparedin dilutionwaterat a concentraotfi9o.n1 mgai/L.Theprimarystocksolutionwasmixedwithanelectricpaddlemixerfor `approximately24 hoursto aidinthesolubilization ofthe testsubstance. After mixing,theprimary stocksolution. appearedclearandcolorlesswithsomewhiteparticulatematerialsuspendedthroughoutthesolution. The `primary stock wasproportionally dilutedwithdilutionwaterto preparethefouradditetstcioncoentnrataionls. All test solutions appeared clear and colorless. TestOrganism `The eastern oyster, Crassostrea virginica, was selectedasthe test speciesforthis study. The Eastem Oysterisrepresentativeofanimportantgroup of aquaticorganismsand wasselectedforuseinthetestbased `uponpasthistoryof useand easeofhandlinginthelaboratory. Easternoystersusedinthetestwereobtained 000753 AMENDED WILDLIFE INTERNATIONAL Lm. "10- PROJECT NO.: 454A-106 froPm. CumminsOyster Company,Baltimore,Maryland. Theoysters wereheldinwater fromthesamesource `asuseddurtihentesgt.The oysterswere suppliedunfilterednaturalseawater duringholding andwereheldfor 12 dayspriortotestinitiation.Tosupplementthedietoftheoystersandenhancetheirconditionandgrowth,the oysters wereprovidedwith an algal suspensionofThalasstosira sp., Skeletonema sp., Chaetoceros sp., and Isochryssips. continuously during holadnditesntingg. During the12-dayholding periodprecedingthetest,theoystersshowednosignsofdiseaseorstress. Duringtheholdingperiod,watertemperaturesrangedfrom 21.8t022.9C.ThepHofthewaterrangedfrom7.9 108.1,salinityremainedat20/%o(parts perthousand)anddissolved oxygen ranged from 7.0 to7.6 mg/L. Instrumentation usedforwatermeasurementsisdescribed intheEnvironmentalConditions sectionofthisreport `Priortotestinitiation,recently deposited shellattherounded(ventral)endwasremovedusing asmall electricgrinder.Care wastakentoremovetheshellrimuniformlytoproduce asmooth,rounded, bluntprofil. `Thelengths of20impartially-selectedoystersweredeterminedbymeasuringthelongestdistancefromtheumbo totheedgeof the shell.Measurementwasmade tothenearest0.05mmtoconfirmthatthe oystersfellwithinthe 25to 50mmsizecriterion.Theaverage lengthof the oysterswas 33.8mmwith arangeof27.8to41.5mm. At chambers test initiation, the oysters were collected fromtheholdingtankandindiscriminatelytransferredto thetest DilutionWater : `Thewaterused forholdingandtesting wasnaturalseawatercollectedatIndianRiverInlet,Delaware,and dilutedto asalinityofapprox20i%emwiathwtelelwl ateyr. Thedilution waterwasstoredin a19,000-Ltank whereitwasacral byrecirculation. Salinityand pHmeasurements takenduringthe four-weekperiod immediatelyprecedingthetestarepresentedin Appendix I. Theresultsofanalysesperformedto measuredthe. AppendiIxL concentrationsofselectedcontaminantsinsaltwaterusedbyWildlifeInternationalLtd.arepresentiedn TestApparatus `Test chambers were 52-L polyethyleneaquariacontainingapproximately40 Loftestsolution.Eachtest chamberwascontinuouslystirredtocirculatethesupplementalalgaeusinganelectric paddlemixer.Inaddition, `eachtestchamberwasgentlyaerated.Thedepthofwaterin a representativetestchamber wasapproximately21 cm.Test chamberswereimpartially positionedinanenvironmentalchambersettomaintain atemperatureof 221C.Thetestchamberswerelabeledwiththeprojectnumber andtestconcentration. 000754 WILDLIFE INTERNATIONAL Lo. -1- PROJECT NO: 454A-106 `EnvironmentalConditions Lightingusedtoilluminatetheholdingtanksandtestchambersduring holdingandtesting wasprovided. byfluorescenttubesthatemitted wavelengthssimilartonaturalsunlight(Colorton 50). Aphotoperoifo1d6 hoursoflightand 8hoursofdarknesswescontrolledwithanautomatictimer. A 30-minutetransition periodof lowlightnteasitywasprovidedwhe lightswenton andofftoavoidsuddenchangesinlighting. Lightintensity attestinitiationwas approximately 498huxatthesurfaceofthewater. Lightintweasmneassuriedutsinyga SPER ScieLntdt. liighftmeitecr. `Temperaturewasmeasiredincachtestchamberatthebeginningandeadofthetestusing a liquid-in- glassthermometer. Temperaturealsowasmeasuredcontinuouslyinthenegativecontroltestchamberusing a FulEsR/cCRoecoprdeer. Thetargettesttemperatureduringthestudywas221C.Dissolvedoxygenwas. mea ineaschtu estchrambeerdadily.Measurementsof pH weremadeincachtestchamberattestniaton,afer appr4o8hoxursianmdatatheetndeoftlhe tyest.Dilutionwatersalinitywasmeasuredat testinititionandtest termination. MeasuremeatsofpHweremadeusing aFisherAccumetModel 915 pHmeter, an dissolvedoxygenwas measuredusingaYellowSpringsInstrumentModel51Bdissolvedoxygenmeter. Salvasimenasuiredtusiynga Bio-MarinIen,c.Aquafauna refractometer. Observations : Oysterswereobserveddailyduringthetestformortalityandclinica sigsoftoxicity.Attheendofthe test, thelongestfingerofnewshellgrowthwasmeasuredtothenearest0.05 mm. Si `Shellgrowthinhibition wascalculatedforeachtreatmentgroupa thepercentreduction inshellgrowth relativetomeanshellgrowth in the negative control. Thefollowingformulawas used: Soliton MeanShell`GMreoawnthSuheeslnlG~rMoewathneShmellGrowthomemes 000755 WILDLIFE INTERNATIONAL Lo. 2. PROJECTNO.: 454A-106 `The ECS0valuewasestimatedbyvisualinspectionoftheshellgrowthinhibition data.Theshellgrowth data ws evaluatedfornormanad hlomiogetneiytyof variancesusingtheChi-Squaterset and Bartlett's test, respectively. Dunnett'stestwasthenusedtoidentifytreatmentgroupswhichhad a statisticallysignificant (50.05)reductioninshellgrowthascomparedtothecontrol (4), AnalyticalChemistry `Watersampleswerecollectedatmid-depthfromeachtreatmentandthecontrolgroupat th beginningof thetest,atapproximately48hours andat testterminationtomeasureconcentrationsofth testsubstance. The `sampleswerecollectedinplastic(Nalgene)botlesandanalyzedimmediatelywithoutstorage. Afterrevioefw theanalyticalresults, thesampleswerereanalyzedinduplicate.Analyticalproceduresusedintheanalysisofthe `samples areproivnAippdendeixIdL. RESANU DDILSCUTSSISON MeasurementofTestConcentrations : ResultsofanalysestomessureconcentrationsofPFOSinwater samplescollectedduringthetestare: presentedin Table 1andintheanalyticalchemistryreport (AppendixI). Nominalconcentrsaletciteodfnosr useinthisstudywere 12,20, 3.3, 5.5and9.1 mgaL.Samplescollectedattestinitiationhadmeasuredvalues thatrangedfrom 28to46%ofnominal values. After24hoursofmixing,theprimarystocksolutionwas `measuretod be 32 to 38% ofnominalandwas probaabtlthye liofmsoluibilittyinunfiltered saltwater, The recoveriesintheothertestconccatrationsweresiilarbecausetheywereproportionaldilutionsoftheprimary | stock. Measuredvaluesforsamplestakenat48hoursrangedfrom 15t0 41%ofnominal. Measured valuesfor `samplestakenat96 hoursrangedfrom<LOQto 52%ofnominal.When measuredconceroafttheisaompnless analyzeda est initiation,approximately48 hoursandattest terminationwereaveraged,themeanmeasured concefonrthtissrtudaywterei0.o36,n0.4s0, 1.3, 1.9 and3.0mg aL. Meanmeasuredconcentrationswere usedintheestiomftahetECSi0voalnue. ObseravndaMetasiuroemnensts Measurementsoftemperature,dissolvedoxygenandpHarepresentedin Table2. Temperatureswere withinthe221Crangeestablishedforthetest. Dissolvedoxygenconcentrreamatinieod2n6.s1mg/L (79 percentofsaturation)throughoutth testandpHrangedfrom7.5to38.1.Dilution watersalinitymeasuredattest initiation andtestterminationwas20and 21%o, respectively. 000756 AMENDED WILDLIFE INTERNATIONAL Lo. 13. PROJECTNO: 454A-106 Oystersinthenegativecontrolandall PFOStreatmentgroups appearednormal andhealthythroughout. thetestperiod.After96hoursofexposure,oystershellgrowthi thenegativecontrolwas2.67mm(Table3). Meanshellgroiwthet0.h36,0.40,1.3, 1.9and 3.0mg aLtreatment groups was2.50, 2.40, 251,2.13and 1.91mm,respectively. Whencomparedtothenegativecontrolgroup,shellgrowthinhibition rangedfrom6.4% inthe 0.36mga.i/Ltreatmeatgroupto 28%inthe 3.0mga.i/L treatmentgroup. Dunntesetsthotwed'thsat oystershellgrowthwassignificantlyreducedinthe3.0mg .i/Ltreatmentgroupincomparisontothenegative: control.IndividualmeasuremeatsofoystershellgrowtharcpresentedinTable 4. CONCLUSIONS `The96-hourECS0 valueforeasternoysters (Crassostrea virginica) extpoPFoOSwsas>e3.0dmg 41,thehighestconcentrationtestedandthepracticallimitofsolubilityinunfiltered saltwater. Basedon a statisticallysignificantreduction inshelldepositioni the3.0mga../L treatmentgroup,theLOECwas3.0mg 1/LandtheNOEweCs 1.9mgai/L. 000757 AMENDED WILDLIFE INTERNATIONAL Lo. Su. REFERENCES PROJECT NO: 454A-106 1 U`OSP.PTESnvNiurmobnemern8t5a0l.P1r0o2t5e:cOtyisotnAegreAncuctye. T1ox9i9c.itySeTreisets(S8h50e-lElcDoelpoogsiictailonE)f.fects Test Guidelines (draf), 2 US.EnvironmentalProtection Agency. 1985.StandardEvaluationProcedure,Acute Toxicity Test JorEstuarineandMarine Organisms (Mollusc96-HourFlow-ThroughShellDeposition Studs). EPA '540/9-85-011. Washington, D.C. 3 ASTMStandard ET29-885. 1994.StandardGuideforConductAicuntge ToxicityTestswithFshes, Macroinvertebrates, andAmphibians. American Society for Testing and Materials. 4 West, Inc. and D. D. Gulley. TOXSTAT Version 35. Copyright 1996. Westem EcoSystems Technology, Ic., Cheyeane, Wyoming. 000758 WWIILLDDLLIIFFEEIINNTTEERRNNAATTIIOONNAALLLLSo.. se PPRROOMJCECTTNNOO.:: s45e4At-t10s6 Table 1 i Toisas: PFOe St of Analy e Ci Data e DiDTosNtiioOnorgeWnmeeiTiramrg_ UEUnaSsaflmeipOeitrnsgSstv,eeCrrsasosea Vvigiiicar VeVi Fa Coenen Te Conan Commision or Wegaos Cone 5 Ere Frog 0 48, <L0Q <LoQ 48 <LOQ % <L0Q 9% <L0Q 12 ii00 9% 9% 000o3333i11 036 30 <L0Q <LoQ 20 @b00d 0o0o6e2%n2 040 20 %= 003355 3 a00 01J33i36]4 13 . PF 5ix% S1i5o% ss ioo 28i0 1s ss 45x a31s%o 01 a00 ss3au%t 50 5 85x 3ii5h% Thelimitofquantitation (LwasO 0.115Qmga) ... Meanmeasuredconcentration doesnotinclude values <LOQ. 000759 AMENDED PROIECTNO: 481A-105 "16 Tae Tempers,DissvedOry ndpH ofWtineTesChambers: _ e Toa op E7n7t08eOt,G-- nns ---- DTe-- itCootemrinUT nietB e Tbo e ywi mi%ro To Cr TtoCpyoi C203 og7y61womo on wi mwt a Sem om aw wom a @ om os wm oe wm om aw" " om @ wm oe Boome wow " w om @ mw vo ome wow " @ om @ ma as ww wow w PE a mee a TRempr e croene mynoTioSBeeteeed erie 70 TC 2 832 AMENDED WILDLIFE INTERNATIONAL Lm. 17. PROJECT NO.: 454A-106 Table 3 Shell Deposition and Shell Growth InhibitionatTest Termination Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Eastern Oyster, Crassostrea virginica Dilution Water: Unfiltered Saltvater `Mean Measured Concentration ~~ Shell Deposition (sum) (mgaill) Mean + SD' "NegativeCoutrol 26720826 -- Shell Growth Inhibition (%) = 036 25040933 64 040 2400820 10 13 25120919 60 19 21340804 2 30 1900501 2 "The96-hourEC50 valuewes >3.0 mg a1/1. -- Mean and standard deviation for 20 oysters. indicates a significant difference from the negative control using Dunnett's test (50.05). 000761 AMENDED MWLIDLDLLIIFFEE IINNTTEERRNNAATTIIOONNAALLtLrmo.. -18- PPRROOEJECCTTNNOO.:: 445544AA-110066 Tabled Individual Oyster Shell Measurements TSpomor 3M Coporstion -- `Test Substance: PFOS `Test Organism: Easter Oyster, Crassostrea virginica Dilution Water: Unfiltered Saltwater Mean Measured Concentrations (mg a.i./L) T 2 3 4 5 6 7 8 9 10 -1 12 13 14 15 176 18 19 20 Mean SD Range Negati4ve00Control 332050 255 250 275 270 2.70 185 220 4.40 205 155 3.05 245 412450 175 285 2.20 267 0.824 1454.40 200356 115800 365 255 1.90 240 4.00 145 255 355 1.80 335 230 3.70 237650 090 285 095 2.50 0.933 090-400 0.40 232250 039100 235 2.10 225 2.80 175 245 285 3.00 445 3.05 195 117200 205 1.65 2.90 240 0.820 090-455 13 218500 222655 240 2.70 2.90 445 1.80 3.08 . Leo 08s 4.10 2.70 245 31.8050. 295 095 3.30. 2.51 0919 085-445 19 2124s0 316800 225 230 3.10 0.65 0.80 2.40 2.70 1.65 1.80 220 170 03.9105 220 2.90 2385 2.13 0.804 065-360 30 018605 212705 3.05 1.60 0.90 240 225 180 2.70 110 235 170 215 21..6600 145 2.00 220 191 0.591 080-305 000762 AMENDED WILDLIFE INTERNATIONAL Lm. "19- PROJECT NO.: 454A-106 APPENDIX Salinity and pHofUnfiltered Saltwater Measured `Duringthe4-WeekPeriod ImmediatelyPrecedingtheTest `TSepsotnsSourb:stance: 3PFMOCSorporation `DTielsuttOirongaWnaitsemr:: _ `UEnafsitletrenrOedysStaelrt,waCtrearssostrea virginica -- Mem Rep Sali0ni%t)y 21(N=4) 20-21 pH 8.1 (N=4) 80-82 000763 PWd AIlLDoLeIFbEiIdNd eTiEnRhNiATIONAd L Lo. .. PROJECT N. O.s : 454iA-106J -20- - AnalysesofPesticides, OArPgaPnEicNsD,IMXetIals and Other Inorgarics in Wildiffe International Lid. Saltwater! ANALYSES MEASURED COE NCENT TRATI ION Ee -- TeoOeDpitCcseoiht 3< nwPB o=n To Sow wo Organochlorinaneds PCBs ~v aa som wi AGDmeiaimicmie) Se ISuowoomem wwee ii B30 Soe i foB[reemr4 Erin I Sm d ou ojwrae um i Se Sou we E ie Sooom =Jo . HTeoeaorrpie pe La PI Emdu]ee iijoam FRiiiama Ri . oEumme ojjnaa Im je =i RE] ja Mots m8 terlaorpncs Jm-- r a SO ) wm Sa Srr a O < W =Th 0B [iW4Lw ja S2e SSooea iwr fF{riregia { PE E iEE1 a xi Sie S<<an5mTe =hwwte Sbe=i < aon"Bo =wt r optoaWadEom r nl Li.fo ioceehdooRvoevn Wp ag 197. r 000764 WILDLIFE INTERNATIONAL Lm. a1. PROJECT NO.: 454A-106 APPENDIX Il `THE ANALYSIS OF PFOS IN UNFILTERED SALTWATER IN SUPPORT OF `WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-106 ~ . 000765 WILDLIFE INTERNATIONAL Lm. 2. PROJECT NO.: 454A-106 `REPORT APPROVAL SPONSOR: 3M Corporation TITLE: 'PFOS: A 96-Hour Shell DepositionTestwith the Eastern Oyster (Crassostrea virginica) `WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-106 PRINCIPAL INVESTIGATOR: gL Ray `Van Hoven, Ph.D. Scientist dibuldGp `Willard B. Nixon, Ph.D? Manager, Analytical Chemistry 4-25-00 DATE # Jit DATE' 000766 AMENDED WILDLIFE INTERNATIONAL Lm. 23. PROJECTNO.: 454A-106 Introduction Unfilteredsaltwater sampleswerecollectedfromanacutetoxicitystudydesignedtodeterminethe effoefcPtFOsS(Perfluorooctane SulfonicAcidPotassiumSalt)ontheshelldepositionoftheeastern oyster (Crassostrea virginica). ThisstudywascondubycWitldelidfe InternationalLtd.and identifiaesd ProjectNo.: 454A-106. TheanalysesofthesewatersampleswereperformedatWildlife International Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). `SampleswerereccivedforanalysisonMay24,26and28, 1999. Sampleswereanalyzedoneachsample receiptday. AllsampleswerereanaolnyJzunee6d,1999toconfirm originalanalyses. Thesecond anawlaysrespoirtesd. `TestSubstance andInteralStandard | Thetestsubstanceusedfortheanalyticalportionofthisstudywas WildiifeInternationalLid. identification umber4675. Thetestsubstancewasusedtopreparecalibrationstandardsandmatrix fortificationsamples. Theinternalstandardwasreceivedfrom 3MCorporation on July2, 1998andwas assignedWildlife InternationalLtd.identificationmumber 4526 uponreceipt.Theinternalstandard, agranularmaterial,was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2, "Thestandardwasstoredunderambientconditions. Analytical Method Themethodusedfortheanalysisoftheunfilteredsaltwatersampls wasdevelopedatWildife Intemational Ltd. andeatitid"AnalyticalMethodValidationfortheDeterminationof PFOSin Freshwater, SaltanwdAalgtaleMedria,". Thismethodologywasincludedas AppendixIIof Wildlife Intemational Lid. protocol number 454/011299/MVAL/SUB4S4. It was based upon methodology probyv3MiCordporeatidon. Samples were dilutedin a 50% methanol : 50%NANOpure watersolutioncontaining 0.mg14H0PF0OS (internal standard and 0.05% formic acid (vi) so that they fel within the calibrationrangeofthe PFOSmethodology. 000767 WILDLIFE INTERNATIONAL Lm. -2- PROJECT NO.: 454A-106 ConcentrationsofPFOSinthestandardsand samplesweredeterminedbyreversed-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chro(mHPaLC)twitohagPerrkin-aElmper AhPI 100LCMass SpectequrippoedmwitehatPeerkirnEimer TurbolonSpray ion source. HPLC separations were achicved using a Keystone Betasil Cie analyticalcolumn(100mm x 2mm LD, 3 jumparticlesize). Theinstrumentparametersaresummarized inTabl1e. AmethodflowchartisprovidedinFigure1. Calibration standardsof PFOS prepared in a 50% methanol : 50% NANOpure water solution containing0.100mg 4H PFOS(internalstandard) and0.05% formicacid(v),ranginginconcentration from 0.00229 to 0.0274 mg a/L. wereanalyzedwiththe samples. Linear regression equations were `generatedusingpeakarearesponseratios (FOS :internalstandard)versustherespectiveconcentration ratios (PFOS : internal standard)ofthe calibration standards. A typical calibration curve is preseated in Figure 2. The concentration of PFOS in the samples was determined by substituting the peak area responseratiosintotheapplicablelinearregressionequation. Repreisonechnromtataogrtamisovfloew andhighcalibrationstandardsar preseatedin Figures 3and4,respectively. "Themethodlimitofquantitation(LOQ)fortheseanalyseswassetat 0.115mg .i/Lcalculatedasthe productofthe lowest calibration standard analyzed (0.m0ga0.i/L2)a2ndt9he dilution factorofthe. `matrix blanksamp(l50e.0s). `MFatorixrBltankianfd icatiSamoplnes. Onomatrix blank samplewasanalyzedto determine possibleinterference. Nointerferenwceerse observedatorabovethe LOQduring samplesanalyses(Table2). Thematrixblankchromatogriams presienFnigturee d5. Unfilteredsaltwaterwasfortifiedat 0.457, 4.57and11.0mg a.i/Landanalyzedconcurrentlywiththe samples (Table2). Sampleconcentrationswerenotcorrectedfor meanprocedural recovery. High recoverieswereobtainedforthelow-level (0.457mga.i./L)matrixfortificsaatmipolen. Previous analyses insaltwater verified that the methodiscapable ofproducing quantitative recoveries atthisconcentration level. Arepresentativechromatogramof amatrixfortificationispresentedinFigure6. 000768 AMENDED WILDLIFE INTERNATIONAL Lo. 2s. PROJECT NO.: 454A-106 ExampleCalculations `Samplenumber 454A-106-5B, nominal concentrationof 5.5 mg a.i./L in unfiltered saltwater. Initial Volume: 0.100 mL Final Volume: 25.0 mL Dilution Factor: 250 PFOS Peak Area: 71220 Internal Standard Peak Area: 74271 Peak Area Ratio: 0.95892 Calibration curve equation. : Slope: 7.78372. Intercept: 0.17175 Curve is weighted (1/x). PFOS (mgaif)at nstrmens = ook 0"CERO) eakarearatio-- intercept) xx11S.S,.CCoonncceennttrraatticion Slope 77872 (0.95892 - 0.17175) x 0.100 mg ai/L. = 00101mgai/l " Note: LS. = internal standard. [-- (ngaill) insample PFOS(mg2.i/L)atinstrument xFinalVolume (mL) Lita Volume (nl) 0.0101 x250 0.100 = 253mgaill 000769 AMENDED WILDLIFE INTERNATIONAL Lm. %6- PROJECTNO: 4564-106 PercentofNominal Concentrati-on. = '= PPFFOOSS((mmgg a 23T./LI)e L inRonIsmaiT) mnBpaElle, 1g Calculatedrecovery: 46.1% Note: manual calulmaayiiofsn. RESULTS `Sample Analysis Unfilterdsaltwatersampleswerecollotedfrom theshelldepositiontestwiththe caste oyster (Crassostrea virginica) at est iition, May 24, 1999 (our 0), on May 26, 1999 (H48)o, anudatrest termMiany28a,1t99i9(oHounr9,6).Themeasuredconcentrationsof PFOS inthesamplescolecadat initiationof exposureofthetet organisms (Hou0r) rangedfom 279to 46.1%ofthe nominal concentrations. SamplescollectedatHour 48had ameasuredconocatraton rangeof14.9to40.9%of nominalvalues. Samplescollectedattesttermination (Hour 96)had ameasuredconocntrtionrangeof <LOQo52.2%ofnominalvalues(Table3). Arepresentativechromofaatettsaomplgeirshaownmin Figur7e. 000770 AMENDED WIrTLDLIFE INTERNATIONAL Lm. 27. IPRROOJCECTTNNOO.:: M454AA-N10S6 INSTRUMENT: ANALYTICAL COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: Table 1 `Typical LC/MS Operational Parameters "Howlet-Packard Model 1100HighPerformaLinqcuied `qCuhiprpoemdawittohgawrPieatrphkaihnP-eErlkmienr-TElumerrAbPoIl1o0n0iLoSCnpsMoarusrascyeS.peOctpreormeatteeridn selective ion monitoring mode (SIM). KeystoneBetasilCiscolum (50mm x 2 mm LD. 3 pumparticlesize) 30C 5.00 minutes 0.220 mLiminute MOBILE PHASE: INJECTION VOLUME: PFOS RETENTION TIME: INTERNAL STANDARD RETENTIONTIME: PFOS MONITORED MASS: INTERNAL STANDARD _M--ONImTOmRED MASS: A72.c0%iMd ethanol:28.0% NANOWatpercu ontar inine g 0. 1% Formic 250 - Approximately 3.6 minutes Approxi2m.a6tmienulteys ~ 498.6 amu 4267 amu _--_ 000771 WILDLIFE INTERNATIONAL Lo. 28- PROJECT NO.: 454A-106 Table 2 Matrix Blank and Fortifications Analyzed Concurrently During Sample Analysis Sample Number (854-106) Type MAB `Matrix Blank `ConcentrationsofPFOS (mg a:i/L) Fortified 00 Measured <L0Q Percent Recovered = MAS-10 Matrix Fortification 0.457 0760 166 MAS-10A' Matrix Fortification 0457 0812 177 MAS-10B Matrix Fortification 0.457 0769 168 MAS-11 MatrixFortification 457 457 98 MAS-12 Matrix Fortification 110 130 19 "Note:Rsesulotsafannddcmtoarnreuwcatliocanaslcfurolrantieeownst.eVsatlsuuebsshtaavnecebpeuernitryouwnedreodgfoernreerpaotretdiunsgipnugrMpoascesQ.uanversion 1.5 * The limitofquantitation (LOQ) was standard analyzed (0.00229m a.L/L) 0a.n1d1t5hemdgilau.tii,o/nLfbaactsoerdoufptohnetmhaetrpirxodbulcataok fsatmhpelleosw(e5s0.t0c)a.libration 2 Sample 454-106-MAS-10 vas rediluted in duplicate and analysis confirmed original result; suspect fortification error - 000772 AMENDED WILDLIFE INTERNATIONAL Lm. -29- PROJECTNO.: 454A-106 Tabl3e Measured Concentrationsof PFOSinUnfiltered Saltwater SamplesfromanOysterShellDeposition Test. To T Cm onceata ruionTeSm Npumbeer (main) (454.106) 00 iAB j7y 11338A SawTimse (oun) 0 aa 9% 9% PRO `ConceS ntratioM n' emFen owf d (mgaif) Nominal <<1I00QQ =- <<1100QQ -- <10Q z <10Q - 12 P8N 0 00338331 22779 . rby aa 00344219 23671 114BA %% <<1100QQ -- 20 - 3nB 00 99BA a" 115588 %% 0062323 33019s 00321939 115469 00225479 112284 33 a8n o - 111356 a3449 101048 a". [os2 22607 11668 %% 115m8 022 ss 5AB o 1uBA aa" 7EAD %% 228432 p6r1 22204 3"6099 o1s4m5 264 01 A 3=39 EZ-) oFcy 00 3304 13260 . 112248 aa. 33sm "30930 113848 9%% 129199 22379 "ote: Rceavluclallsaiaonnsd.cVoarlruoecsiboanvseTboerennreowufnodsedsfuobaepnocreipoguprulrpawserees.generaied wing Macuanversion1.5 sofvare ad manual * T(h0e.00l2i2m5ittaogf1q.u4e1a1t)iantdiotnhe(dLilOuQti)ownfaasct0or.o1f1t5hemmagtraixLbabka:sseadmuplpeosn(tSh0e0)p.roductofthelowestcalibrationstandardanalyzed *Rtheseesafmospslaemsucpolnefl6iArm(e6td:r2e1smulgtfoirLsa)mwplaesGnBo.t cludedinthetable. Sample 6wasreilted iduplicate (6C and 6D)andresults of 000773 AMENDED WILDLIFE INTERNATIONAL Lm. -30- PROJECT NO.: 454A-106 METHODIONUUTNLFIINLETFEORREDTHSEALATNAWLAYTSEIRS OF PFOS `Preparematrixfortificationsamplesbyspikingtherequisitevolumeof PFOSstocksolutionsdirectly intounfilteredsaltwaterusinggas-tightsyringesandClass Avolumetricflasks. i Dilutematrixfortificationandtestsamplesintotherangeofthe calibrationstandardsbypartiallyfilling Class Avolumetricflaskswith50% methanol : 50% NANOpurewatersolutioncontaining `0s.a1m0p0lmeagn4dHbriPnFgOtSh(inftlearsnkatlosvtoalnduamredw)i/Ltahnthd0d.i0l5ut%if onsoolvernatcm .iPdr(iovc/c ev)s.stAhdedmtahtreiaxpbplroapnrkisaatmepvloeluusmiengo.f thesame dilutionandaliquot volumeasforthe lowestfortification level. Mixwellbyseveralrepeat inversions. + Ampulatesamplesand submitfor LC/MSanalysis. Figure1. Analyticalmethodflowchartfortheanalysisof PFOS inunfilteredsaltwater. 000774 WILDLIFE INTERNATIONAL Lr. "31. PROJECT NO.: 454A-106 300 250 _ 20 gz g 150 2 100 2 050 000 0000 0050 0100 0150 0200 0250 0300 Concentration (Ratio) Figur2e. AtypicalcalibrationcurveforPFOS. Curve is weighted (I). Sl=o7.p7837e2; Intercep=t 0.17175;1= 0.9825. 000775 AMENDED WILDLIFE INTERNATIONAL Lm. 32 PROJECTNO.: 454A-106 Inanshy: 1102 cps 100 os 90 80 70 60 so . : A 40 30 7 10 g 2B1h 4i1m 821s 8s1w 10U1n Scmaen 1 Figure3. Areprescatativeionchromatogramof a low-level (0.00229mga.i/L) PFOSstandard. 000776 AMENDED WILDLIFE INTERNATIONAL Lm. 33. PROJECTNO.: 454A-106 0 8s Intonsty: 6397 cps a a 7 a A . + 78 a 2 1 o2z1s EaE 2o5t5 3a13 a10z1 TSiomne Figured. Arepreionscheromnatotgraamotfahiighv-leevel (0.0274mg 2.i/L) PFOS standard. 000777 AMENDED WILDDLLIPFEEIINNETERRNAATOINONAALLLLDm., eee 34. PROJECTA NO.: 454A-106 [TE . Intent: 280 cp of 80 70 60 ' 50 - 40 0 20 * 10 34 R50 AerPer 81924 A WU A Ty i 2b1w aim 21s aI1 B4ax TSnone FigureS. The matrix blank ion chromatogram (454A-106-MAB-4). The arrow indicates the retetinmetofiPFoOSn. 000778 WILDLIFE INTERNATIONAL Lm. -35- PROJECT NO.: 454A-106 10 86 ntnsty: 9001 cps a 8 7 6 5 4 )3 .7 A : 1 2h1 hpil f 1 f8 B Figure6. Areprescatativeionchromatogram of a 4.57mga.i/Lmatrixfortificationsample: (454A-106-MAS-11). 000779 AMENDED WWIILLDDLLIIFFEEIINNTTEERRNNAATTIIOONNAALLLLmo.. ~~~~~ = _36- PPRROOEJECCTTNNOO.:: 445544AA-106 Intensity: 5239'cps 10 87 of sof 7 o 5 4 73 5 2 , 12 ; 7I r 51 1I 1 o4m Figure7. A representative ion chromatogram ofa 5.5 mg .i/Ltest sample (454A-106-5B). 000780 AMENDED WILDLIFE INTERNATIONAL Lm. 3. PROJECTNO: 454A-106 APPENDIIXV Chatno Pgroetocsol `Thisstudywasconductedinaccordancewiththe approvedProtocolwiththefollowingchanges: 1. Theprotocolwasamendedto addthe proposedexperimentalstartandterminationdates,test `conceannd ttestrsuabsttanicenoumnbesr. 2. Theprotocolwasamended tospecifythatGLPanalysesofsaltwaterwillbereported. 3. Theprotocolwasamendedtochangethe analyticalsampling schedule. 4. Salinity wasmeasuredinthe negativecontrolattestinitiationandtermination. 000781 WILDLIFE INTERNATIONAL Lm. ETN PROJECTNO.: 454A-106 APPENDIX V Personnel Involvedi the Study `ThefollowingkeyWildlifeInternationalLtd.personnelwereinvolvedintheconductor management ofthis study: 1. Heary O. Krueger, Ph.D, Director, Aquatic Toxicology and Noo:TargetPlants 2. WillardB. Nixon,Ph.D,Manager, AnalyticalChemistry 3. Mark A. Mank, Laboratory Supervisor 4. Timothy Z Kendall, Laboratory Supervisor 5. Kurt R.Drotta,SeniorBiologist . 6. Raymond L. VanHoven, Ph.D, Scientist 000782 WILDLIFE INTERNATIONAL Lm. 39. PROJECTNO.: 454A-106 APPENDIVXI Report Amendment 1. OriginalReport: Pa1-g 4,6e ands 22 Amendment: `Thepageswere changed toincludethe amended reportdate, revised page numbers, and `new signatures anddates dueto the additionofthe report amendment asAppendixVL Reason: Toreflecttheissuingofanamendedreport. 2. OriginalReport: Page? Amendment: `Thecompliancestatementwas revised. : Reason: Toclarify howthetestsubstancewascharacterized. 3. OriginalReport: Page `Amendment: Information provided by theSponsorrefltheerecanatlysiisonftghe test substance, includingthe reanalysis dateandthepurity,wasaddedtotheTestSubstancesection. Reason: Toreflectthecurrenttestsubstance informationprovidedbytheSponsor. 4. OriginalReport: Entire report `Amendment: Alltest substance concentrations were changed toreflectthepurityof the testsubstance asdeterminedbytheSponsor in areanalysisofthetestsubstance(FC-95,Lot217). Test concentrationsoriginally were basedonthereportedpurity of 98.9%. The . certificateof analysisdatedMarch9, 2000 indicated apurity of 90.49%. `Thereforael,l fest substance concentrations, including nominal concentrations, measured concentrations,andLCS0values,wererecalculatedand reportedasmg a.1/L basedon `the90.49% purity. Reason: Toreporttheresults ofthetestbasedonthetestsubstancepurity of90.49%atthe. req ofu theeSposnsotr. 5. OriginalReport: Page15 `Amendment: Addfootnotes 1 and 2to Table1, SummaryofAnalytical Chemistry Data. Reason: To clarify themeasuredconcentrartepioortnesd. 000783 AMENDED WILDLIFE INTERNATIONAL Lm. -40- APPENDIVXI -ContinuedReport Amendment PROJECT NO.: 454A-106 6. Original Report: Page 17 `Amendment: Changethe 96-hourECS0value of >10mg 2.i/Lto>3.0 mga.i/L. Reason: TtohacnortrheecntotmhienraelpocrotnceednEtrCaSti0ovna.luetoreflectthemeanmeasuredconcentration,rather AMENDMENT SIGNATURES: KSutrutdyR.DiDrreoctttoarr HeDinrrecytoZr,KArquueagteirc,To-- xicology and Noo-Target Plants REVIEWED BY: Spero[ H: I J`eQualityH.AsCsourlaenmcaen, . Hae DATE _- 4-26-00 DATE 000784 AMENDED |] WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-106 Page 10f2 N: AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR SHELL DEPOSITION TEST WITH THE EASTERN un OYSTER (Crassostrea virginica) PROTOCOL NO.: 454/042199/0YS-DEP/SUBA454 BH ovon svcopmion AMENDMENT NO.: 1 PromCr 0s 500106 un EFFECTIVE DATE: May 20, 1999 AMENDMENT: Page 2 nu Add: Experimental Start Date: 5/24/99 ETexspterCiomnecnetnatrlaTteiornmsi:natNieognatDiavtee:Co5n/t2r8o/l,991.3, 22, 3.6, 6.0 and 10 mgai/L u Test Substance No. 4675 REASON: The above information was not known when the protocol was signed by the Study ou Director o AMENDMENT: Dilution Water, Page 5 Change: Analyses will be performed at least once annually to determine the concentrations of I] selected organic and inorganic constituents of the seawater and the results of the analyses will be summarized in the final report ou To: Analyses wil be performed at least once annually to determine the concentrationsofselected organic and inorganic constituentsofthe seawater and the resultsofthe most recent GLP `compliant analyses will be summarized in the final report un REASON: To specify that GLP analyses will be reported. u AMENDMENT:SamplingforansivicalMeasurements, Page 7 o Change: Woautresr(sa1mphloeusr)wdiulrlibnegcotlhleetcetsetdtfordoemteeramcihnteesctonccheanmtbraetrioatnstoefttinhititaetsiotnsuabnsdtanecvee.ry 24 a To: tWeastte(raspapmrpolxeismawtielllybe48cohloleucrtse)dafnrdomateatecshttteestrmcihnaamtbioenrtao dteetseirnmiitinaeticoonn,caetnttrhaetmiiodnpsooifntthoefttehset substance. un REASON: The Sponsor requested a change in the analytical sampling schedule. 000785 a-- Reviewed by GAED5a i q WILDLIFEINTERNATIONALto. a a Nu atl un ; STUDY DIRECTOR ] 4A LABORATORY MANAGEMENT N. uN SPONSOR'S REPRESENTATIVE u n na n n |_| u = |] B I ea PROJECT NO.: 454A-106 Page2of2 ~~ S784 (79 DATE IR 1/25 DAT? b17/99 DATE - 000786 HM WWuIDLLDLIIFFEE IINNTTEERRNNAATTIIOONNAALLVtDm. ee Regi oi PROJECT NO. 454A-106 n DEVIATION TO STUDY PROTOCOL u STUDY TITLE: POFYOSS:TERA(C9r6as-sHoOstUrReaSvHirEgLinLicDa)EPOSITION TEST WITH THEEASTERN PROTOCOL NO.: 454/042199/0YS-DEP/SUB4S4 DEVIATION NO.: 1 un SPONSOR: 3M Corporation PROJECT NO: 454A-106 n D-A_T--E OF DEFACTO DEVIATION: May 24, 1999 un DEVIATION: `Thecprhotaoatcotlemssttbiantietesiatthriaotn dainldutaionthweaetnedrosfatlihneityteswti.llDibleutmieoanswuarteedrsailnineiatcyhwtaesst `onlymeasuredinthenegativecontrolatthebeginninganden oftd he test. n REASON: ici cvesiht. Wii sis 4 sors und svt $000 gjauldlgomnetnatnkowfhithcehiSsturdyeDircectoirtotrmhaaicnthtiasuidneclvoinastatiaonnttsdailedinnoidttya.dvCeornsseelqyueafnftelcyt,hsetreshultbseostf A eumm nu the study. pr _glofp n STUDY DIRECTOR 1 LABORATORY MANAGEMENT DATE DAT] 1 xo 1 000787 PROTOCOL WITPHFOTS:HEAEA9S6-THEORUNROSYHSETLELRD(ECPraOsSsIoTstIrOeNa vTiErgSiTnica) US, Environmental Protection Agency Series 850-OPEPcoTlSogNiucamlbEefrfe8c5ts0.T1e0s2t5Guidelines 3M Lab Request No. U2723 Submitted to ; Envir3onMmeCnotraplorLaatbioornatory 9B3ui5ldBiunsgh2A-v3eEn-0u9e St. Paul, Minnesota 55144 AR WILDLIFE INTERNATIONAL LTD. 3 8598 Commerce Drive i| Easto(n4,10M)ar8y2l2a.n8d60201601 i | April 21,1999 000788 i WILDLIFE INTERNATIONAL, LTD 2. Ho WITPHFOTS:HEAEA96S-THEORUNROSYHSETLELRD(ECPrOasSsIoTstIrOeNa vTiErgSiTnica) SPONSOR: 4| | 3EnMviCroornpmoernattailonLaboratory B9u3i5ldBiunsgh2A-v3e5n-0u9e St Paul, Minnesota 55144 SPONSOR'S REPRESENTATIVE: Ms. Susan A. Beach =H TESTINGFACILITY: W8i5l9d8iCfeomInmteermcateioDnrailv,eLid. Easton, Maryland 21601 H STUDY DIRECTOR: SKeunritoDrrAoqtuiaartic Biologist H LABORATORY MANAGEMENT: HDeinrry Oe.ofcKAruqteugaoetrir,cPTh.oDx.icology & Non-Target Plants Hl FORLABORATORY USE ONLY o | Proposed Dates: : EStxapretrDiamten:tal ETexrpmeirniamteiaotnalDate: H Project No:__ASUA- [Os . Test Concentrations: sse-------- Ho Test Substance No. Reference Substance No. if applicable): u ---- H a% \ d STUDY DIRECTOR <A DATE -- HB CABORATORY MANAGEMENT DATE A SPONe SOR'S REPR0 ESENT[AToIVEud DA4T/E29) 99 000789 Ho PROTOCOL NO, 454/042199/0YS-DEPISUB4S4 3M LAB REQUEST NO, U2723 u N WILDLIFE INTERNATIONAL, LTD 3. n un Wildlife International, Ltd. wil coInNduTcRtOaDUstCatTicIOacNute toxicity test with the Easier oyster (Crassostrea virginica) for the Sponsor at the Wildlife International, Ltd. aquatic toxicology facility in un Easton, Maryland.Thestudyvil bepecformedbasedon proceduresinthe U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1025 (1);StandardEvaluation u Procedure, Acute Toxicity Testfor Estuarine and Marine Organisms (Mollusc 96-Hour Flow-Through `ShellDeposition Study) (2) andASTMStandardE729-88a Standard GuideforConducting Acute Toxicity Tests with Fishes, Macroinverteratesand Amphibians (3). Raw data for all work performed st Wildlife ua InternationLatld,.and acopyofthefinalreportwillbefiledbyproject numberinarchiveslocated onthe a `WildlifeIntemational, Ltd.site,oratanaltermative locationtobespecifiedinthefinalreport. PURPOSE un Thepurposeof thisstudy istodeterminetheeffectsofatestsubstanceontheshelldepositionofthe u `casteroyster (Crassostrea virginica) durinag 96-hourexpo undsersu tatr icte est conditions. un ou . om MH Ha > |: - Oysers illbe expos 3ErXPoEmReIMsEeNsTAfLDaESe]stIfGNv test conseaationsad 3negative (diwal teru) ct onti rolo forn96 hours. Onetestchambweilrlbemaintainedforeach treaatnmdceonntrotl groupwith 20oystersineach chamber. Nominaltestconcentrationswillbeselectedin consultationwiththe `Spaondnwisllboe bras,ed upon informationsuchastheresultsofexploratoryrange-finding toxicity data, knowntoxicitydata, physical/chemicalpropertiesofthetestsubstanceorotherrelevantinformation. Target concentrations will not exceed 120 mg/L or the solubility limitofthe test substance in water (whichever is lower). Generally,eachtestsubstanceconcentrationusedinthedefinitivetestwillbeat least60%ofthe next higher concentration unless information concerning the concentration-effect curve indicates that a differentdilutionfactorwouldbemoreappropriate. Watersamplesfromappropriatetestchamberswill be collected atspecifiedintervalsforanalysisofthetestsubstance.Resultsofanalyseswillbe usedtocalculate: `meanmeatesstcuoncerntreatiodns. i Oysters approximately 25to 50mmlongwill bemaintainedatWildlifeInternational,Ltd.in unfiltered saltwaterfor aperiodof atleast 10days. Tocontrolbias, oystersusedinthetestwillbe a indiscriminatelydistributed. Noothrpotential sourcesofbiaar expectedtoaffcttheresults ofthestudy. . 000779 B PROTOCOL NO. 454/042199/0YS-DEP/SUB454 3MLABREQUEST NO. U2723 I i WILDLIFE INTERNATIONAL, LTD 1 ae Justprior tothe initoftiheatestt,irecoentnly deposited hell will be removed by grinding the periphery of I the oysters againstanelectricdiscgrionsdimeilarr apparatus. Theinitial shell length (afer grinding) of twentyindiscriminatelyselectedoysterswillbemeasuredand20indiscriminatelyselectedoysterswillbe I `placed incachtestchamber. Attheconclusionofthe 96-hour test,depositionofnewshellwill be measured forcach oyster. Since shell depositioin oftenasymmettrheilcenagtlhofthelongest "finger"ofnewshell 1 willbemeasured using calipers. Shell growth inhibition for eachtreatment group vill be determined by calcmeuanlshealldtepoisitniongas aperceofnthtecaongtroel. Moanshellgrowihinhibiitnicaocnh I treatmentgroupwillbe expressedas apercentageofmeancontrolshelldeposition.AnECS0valuewillbe calculated,whenpossible,to expresstheconcentrationoftestsubstancethtinduces a 50%reductinisohenll 1 deposition. I MATEARNDIMAETLHODSS TestSubstance 1 Informationonthe characterizationoftest,controlof referencesubstancesisrequiredby Good LaboPracricsaStatndaordsr(GLyP).TheSponsorisresponsiblefo providingWildl Internationa,Lid 1 `writtenverificationtha th tetsubstancehasbeencharacterizedaccordingto GLPSprior to itsus inthe study. Ifwritenverification ofGLPtestsubstancecharacterizationisnotprovidedtoWildeIntemational, 1 Lid, it willbenotedinthecompliancestatement ofthe finalreport.Theattached form IDENTIFICATION OFTEST SUBSTANCE BYSPONSOR(AppendixI) i tobeusedtoprovideinformation necessaryfor i GLP compliance. "The Sponsorisresponsibleforal informationrelatedtothetes substanceandagreestoacocptany | `unusedtestsubstanceandortestsubstan containersremaining atthecadofthestudy. | PreparationofTestConcentrations "Thetestsubstancewillbeadministered oth tetorganisminwater,Thisroutsof administration L `wasselectedbecauseitrepresentsthemostlikelyrout ofexposuretoaquaticorganisms. l | 000791 L PROTOCONOL, 454/042199/0YS-DEP/SUB454 3MLABREQUEST NO. U2723 ~-- WILDLIFE INTERNATIONAL, LTD a - a radia, TestOrganism A "The castem oyster (Crassostrea virginica) willbeusinethistdest. This species is representative of animportantgroupoforganisms and was selectedforuscinthetestbased uponpastuse andesseof Aa Testorganismswillbe obtainedfrom acommercial suppl orresearchfacility. Allorganismswill beobtainedfromthesamesourceandheldinthelaboratoryforat east 10daysprior o esting.Oysterswill H notbeusedinthetest fthey showsignsofdisease orstressorif morethan5% die duringthe 7daysprior tothetest. `Saltwatersuppliedto theoystersduringholdingandtesting wil beunfiltered. Asaltwaalgtaseucrh a as Thalasssp,iSaklsetionremaa sp., Chastoceros sp. andox Isochrysis sp.willbeaddedtothewater for supplemental nutition. a | gH I] d Dilution Water . `Wateruse fox holdingandtestingwillbenaturalunfilteredseawatercollectedatIndianRiver Inlet, Delaware. Theseawaterwillbepumpedito a19,000-Lholdingtankwherethsalinityoftheseawaterwill be diluted to approximately 20 %s (parts per thousand) with freshwater from a well on the Wildlife InternationLatdl., ste. Thesaltwaterintheholdingtankwill be aeratedbyrecircpuriloratotdeilivoerny tothetestchambers. Analyseswillbeperformedatleastonce annuallytodeterminetheconcentrations of selectedorganicandinorganicconstituentsoftheseawaterandtheresults oftheanalysesvillbesummarized inthefinal report.Dilution water salinityshouldbe20+ 1%,andthepHshouldnot varybymorethen 1 nitdurtihentesgt. Ho ei 000792 = Test Apparatus, L "Testchambersvillbe 52-Lpolyethylenechambersfilledwithapproximately 40 Loftestsolution. a Testchamberswillbeimpartiallypositionedinanenvironmentalchambero atemperature controlled water . bathtomaintain a temperatureof 22 +1C. Anelectricmixerwillbe placedincachtestchamberto a `maintainthesuspeofnth saligalodinet.Inaddition,eachtestchamberwillbemildly aeratedto redethe . biologicaloxygendemandduetothefeed.Testchamberswilbe labeledwiththeprojectnumber andtest a PROTOCOL NO. 454/042199/0YS-DEP/SUB4S4 3MLABREQUESTNO, U2723 i . i WILDLIFE INTERNATIONAL, LTD 1 "6- |} Environmental Conditions Lightingusedtoillumtihenhiadtinegand test chamberswill beprovidebyd fluorescent tubes that 1 Tits1. Tp techs |} `emit wavelengthssimilartonaturalsunlight (e.g.,Colortone 50). A photoof1p 6hoeurr sofiligohtadnd 8 hoursofdark will be controlled with an automatic timer. A 30-minute transition periodof low light 1 intensity will be provided when lights go on andoff to avoid sudden changes in light intensity. Light intensity willbe measatutesrtinietiatidon witha SPER Scientific Ltd. light meortequievalrent 1 the beginning and cadofth tetusing a iquid-n-glass thermometer. Temperature also will be measured with acontinuous recordienrthenegativecontrolchamber. Recordermeasurements willbeverified with 1 aliquid-in-glass therpmrioor tmo teetitnitieatiron. 1 orient. Messen ofp wil bo mde wing Fier Acne Mot 915 i me i Dissolvedoxygenwillberecordedineachtestchamberdaily. ThepH willbemeasuinrceacdh treaandtconmtreolgnrotup a est initiation, midpoint (approximately 48 hours),andtheendofth tes. Dissolvedcxygenwillbe measuredusing YellowSpringsInstrumentModel $1 dissolvedoxygenmeter, equivalent.Intheeventhta dissolved oxygen concentrationsfal below 60% saturation,dissolvedoxygen 1 measurementswillbemadein everytestchamberand appropriate actions willbetakenafteconsultation : `iththe Sponsor. Dilution watersalinitywillbemeasuredineachchamberattest ntiaion andattheend 1 ofthetest.Salinitywill be measuredusinga Bio-Marin,Inc.AquaFeunarefractometer,orequivalent. If a treatment group reaches 100% mortality, dissolved oxygen, pHand temperature measurements will be 1 takenatthat time,thendiscontinued. 1 BiologicalMeasurements i Oysterswillbeinspectedvisuallyatapproximately0-24hoursandat24,48,72,and 96hours(= 1 I hor)afer testinitiation. Oystershavingopenshellsandnotrespondingtogentleprodding villbo . `condseaidadnderermoevedd. Anyshell abnorobmseravedlviillbteriecoerdsed. Whtheetesntis I terminated,sheldeposition willbe recordedforeach oysterby measuringthe lengthoftelongest fingerof newshell. Shelgrowthinhibitionfor eachtreatmentgroupwil bedeterminedbycalculatingmeanshell I deposition as apercentageofthecontrol. 000793 I PROTOCOLNO. 454/042199/0YS-DEP/SUBAS4 3MLABREQUEST NO. U2723 ||[ WILDLIFE INTERNATIONAL, (0 | 7. iVi `SamplingforAnalytical Measurements I `Water samples willbecollected fromeachtest chambeart test initiation and every 24 hours (+ 1 hourduringth tettodetermineconcenrations ofthe est substance.Intheeventthat 100% mortality i occursinanytreatment,thensamplingofthattreatmentwillterminatefollowingthenextsamplinginterval. ; Sampleswill becollectedat mid-depthfrom eachtestchamber, analyzed immediatelyorplacedinan ] appropriatestoragecontainer (glassorpolypropylenebottle)andstoredunderrefrigeration (~4C)until i analyzed. The sumpleschemeis summarized below: : --_-- PROPOSED NUMBERS OaFuVERIFICATION SAMn PLESw 3i Experimental Group 0 Hours Hours Hours Hours Hours 13 Control 1 1 1 1 1 ti Level 1-Low Concentration 1 1 1 1 1 ! Level 2 1 1 1 1 1 ; Love 3 1 1 TE 1 Lowel4 1 1 1 11 Level 5. 1 1 1 1 1 6 6 6 6 6 i ToulNumberofSamples =30 Theabovemanbersofsamplesrepresentthosecollectedfrom the test and do nt include quality control (QC) samples such as matrix blanks and fortifications prepared and analyzed during the analytical chemistry phaseofthe study. i Ansivical Chemisty i Chemical anaolftyhessamiplses willbeperformed byWildlife InternationLatld,.Theanalytical I methodusedwillbebaseduponmethodologyprovidedbytheSponsorandidentifiedin AppendixI. The | methodologyusedtoanalyzethetestsampleswillb documentedinthera dataandsummarized ithefinal | report. | PROTOCOL NO. 454/042199/0YS-DEP/SUB454 000794 3M LABREQUEST NO. U2723 | 1 1 WILDLIFE INTERNATIONAL, LTD 1 -8- DataAnalysis nl Mean shell growth inhibitionineach treatment group will be expressed as a percentageofcontrol growth deposition. An ECS0 value wil be calculated when possible to express the concentration of test i Substance thatinduced a 50% inhibitioninshelldeposition. The following forwmillubelusead: n setnhibition=C=T x00 Where C=Mean growthofnegativecontrols c T=Mean groofwtrteathed u Tei db ne tion ng CP pote vs rs) `The no-observed-effect-concentration (NOEC) and lowest-observed-ffct-concentration (LOEC) will be: u determinedbased (whenpossible)on astatisticalanalysisofthedose-response dataandanassessmentof 1 the dose-response pattem. REC TOBOEMR AIND TAIS NED un RecordstobemaintainedfordatageneratedbyWildiie InternatLitdo.nwailllinclude, but notbo limited to - 1 1. Copyofsigned protocol. 2. Identaindfchairaccteraiztatiionooftnhe test substance,ifprovbiysdponesodr. i 3. Datesofinitiationandterminationofthetest. 4. Holding records || 5. Calculation and preparationoftest concentrations. 6. Stock solution calculations and preparation, ifapplicable. n 7. Observations 8. Ifapplicable,themethodsusedtoanalyzetestsubstanceconcentrationsandtheresultsofanalytical n measuremeats. 9. Statistical calculations,if applicable. i 10. Test conditions and physicallchemical measurements 11. Measuremeatsofshell deposition. R 12. Copyoffinal report. a 000795 i PROTOCOL NO. 454/042199/0YS-DEP/SUBAS4 3M LAB REQUEST NO. U2723 R n WILDLIFE INTERNATIONAL, LTD -9- n FINAL REPORT un A report of the results ofthe study will be prepared by Wildlife International Ltd. The report will include, but not be limited to, the following, when applicable: ou 1. Name and addressofthefacilityperformingthestudy. 2. Dates uponwhichthe studywasinitiatedandcompleted. Itsthe responsibiolfitthye Sponsotro nu provthiefdinealdatsthat dataae recorded for chemistry, pathology and/or supporting evaluations that may be generated at other laboratories un 3. A statement of compliance signed by the Study Dicctor addressing any exceptions to Good Laboratory Practice Standards. un 4. Objectivesandproceduresasstateintheapprovedprotocol,includinganychangesinth original protocol un 5. Thetestsubstanceidentificdbyname,chemical absmutmbersoracodcenutmbesr,strength,purity, and composition or other appropriate characteristics,if provided by the Sponsor. on 6. Stabilityandthesolubilityofthetstsubstancesunderthe conditions ofadministration,ifprovided by the Sponsoor contracted to Wildlife IntertionalLtd un 7. Adescriptionofthemethodsusedtoconductthetest. 8. Adescriptionofthotest system,inchingthesourceofthe testorganisms,scientificname,lfstage, n meansandranges of lengths, observed discases,treatmentsand holdingprocedures. 9. A descriptionofthepreparationofthe test solutions, methods used to allocate organisms 10 test ; chambersandbeginthetest,numbersoforganismsandchambersper treatment,allocationof n organismsto testchambersandduration ofthetest. 10. Adescriptionof circumstancesthatmayhaveaffectedthequalityo integrityofthedata. = 11. The naomfteheStudyDiraendcthtenoamersof therscientists, professionals,andsupervisory personnel involved in the study. n 12. Adescorfthietrpanstforimatoionns,calculations,oroperationsperformedonthedata, a summary un adnadwannafloysmisotftehebaiolsogicaldataandanalyticalchemistrydata,and astatementoftheconclusions . 13. Statistical methodsusedtoevaluatethedata,if applicable. om 14. Thesignedanddatedreportsofcachoftheindividualscientistsorothe professionalsinvolvedinthe study,ifapplicable. nm 15. Thelocationwhererawdataandfinal reportar obostored. 000796 [3 PROTOCOL NO. 454/042199/0YS-DEP/SUBA4S4 3MLAB REQUEST NO. U2723 a N WILDLIFE INTERNATIONAL, LTD x -10- 16. A statementpreparedbytheQualityAssuranceUnitlistingthedatesthat studyinspectionsandaudits un "wer made and thedatesofany findings reportedtotheStudyDirectorand Management. 17. Ifitis necessary to make corrections or additions to final report after it has been accepted, such nu changeswil bemadeintheformofanamendmentissuedbytheStudy Director.The amendmewnitll clearlyidentifythepart ofthe inal reportthatisbeingamendedand thereasonsfortheamendments. Nn AmendmentswillbesignedanddatedbytheStudyDirector. nu CHANGING OF PROTOCOL Planned changes to the protocol will be in the form of written amendments signed by the Study un Directorandthe Sponsor'sRepresentative. Amendmentswillbeconsideredaspartoftheprotocol andvil beatttoathecfinahlpreotocdol. Anyotherchangeswillbei theformofwrittendeviationssignedbyth. I" Study Director and filed with therawdata. All changes to the protocol will be indicated in the final report. n (GOOD LABORATORY PRACTICES "This studywillbe conducted in accordance with Good Laboratory Practice Standards for EPA (40 ou CFR Part 160 andor Part 792); OECD Principles of Good Laboratory Practice (OCDE/GD (92) 32, Environment Monograph No. 45); and Japen MAFF (59 NohSan, Notification No. 3850, Agricultural = Production Bureau). Each study conducted by Wildlife Intemational Lid. is routinely examined by the Wilde International Ltd.QualityAssurance Unit forcompliancewith Good Laboratory Practices, Standard u Operating Proceduresandth specifiedprotocol. AstatementofcompliancewithGoodLaboratoryPractices willbepreparedforallportionsofthestudyconductedbyWildifeIntemationalLtd.The Sponwsilolbre . responsibleforcompliance with Good Laboratory Practice for procedures perfobrymotehedr laboratories nN (e:., residueanalorpyathsoleogys).RawdataforallworkperformedatWildiifeInternational Ltd. anda copyofthefinalreportvil befiledbyproject numberinarchiveslocatedonthe WideIntemationalLtd. n site,oratan altemative location tobespecifiedinthefinalreport. |B = a 000797 hy PROTOCOLNO. 454/042199/0YS-DEP/SUBAS4. 3MLAB REQUEST NO. U2723 a WILDLIFE INTERNATIONAL, LTD "1. REFERENCES EH 1 US. EnvironmentalProtection Agency. 1996. Series 850- EcologicalEffects Test Guidelines (dra), OPTS Number 850.1025: OysterAcute Toxicity Test (Shell Deposition). H 2 US. Environmental Protection Agency. 1985. Standard Evaluation Procedure, Acute Toxicity Testfor Estuarine and Marine Organisms (Mollusc 96-Hour Flow-Through Skell Deposition Study). EPA-540/9-85-011. Ho 3 ASTM Standard E729-88a. 1994. Standard Guidefor Conducting Acute Toxicity Tests with Fishes, Macroinveriebrates, and Amphibians. American Society for Testing and Materials. H 4 NorbergKing, T.J. 1953. ALincarsroMlethaodftorSoilnt Tsey. TheInitio Concentration (ICp) Approach. Version 2.0. U.S. Environmental Protection Agency. National H Effluent Toxicity Assessment Center. Duluth, Minnesota. Technical Report 03-93. i Ho0 PROTOCOL NO. 454/042199/0YS-DEP/SUBA454 000798 3MLAB REQUEST NO, U2723 B 0 WILDLIFE INTERNATIONAL, LTD 12. 8 APPENDIX B IDENTIFICATIONOFTEST SUBSTANCEBY SPONSOR "TobeComplbyeSptonesodr B 1Test Substance Identity (nametobeused inthereport): P(EOPS erfluSoulfroniocAocidcPottassaiunmSaelt ReferenceStandard (ifspplicable): Analytical Standard: NA B Internal Standard: _P1,1e.2s2H,fHlHuoroocSutlfoanicnAceid TestSubstanceSampleCodeorBatch Number:__Lot 217 0 TestSubstance Purity (%ActiveIngredient): _98.9 Expiration Date: _2008 IL TestSubstanceCharacterization B GB etermatidpce 5us nis styE insecawih GoLPoStatnda:rd? Ys _x No | IL TestSubsStotragaeConndictioens PleasindicatetherecommendedsiorageconditionsatWH IntemationalLid. HB bb ISESRCENEERETER E vw x B Oberptistaoblyienfornmatiton: Bo rece V. ToxiiyInformation: RB Mammalian: RatLDSO Aquatic: . Mmpmmenwi, J 251mpg MowseLDSO_N/A InvertebrateToiity(ECLCS0) Fish Toric (LCS0) B D2 ophn7 iamam gna:e/l. RainbowTrout: Lime Daphniamagna: SOmgfl FatheadMimow:38mgll, HB Other ToxicityInformation (inchingfindingsofchronicandsubchronictests): . Pleasesoe MSDS ~~ B B 000799 B PROTOCOL NO. 454/042199/0YS-DEP/SUBAS4 3MLAB REQUESNTO,U2723 E WILDLIFE INTERNATIONAL, LTD "13 APPENDIX I E Analytical Method Provided by Sponsor method`sS:amplesvillbeanalyzedbaseduponprocedures providedbythe Sponsorinthefollowinganalytical u 1. Liquid Chromatography Mass Spectrometry (LCMS) Method for the Determination of HN Periluorooctane Medium Sulfonic Acid Potassium Salt (PFOS) In Freshwater, Saltwater and Algal Acopyofthe abovemethodwillbemaintainedintherawdata."Theactual methodologyused to HN analyzethetest sampleswillbe documentedintheraw data and summarized in thefinalreport. u H PROTOCOL NO, 454/042199/0YS-DEP/SUB4S4 000800 3M LAB REQUEST NO. U2723