Document V3YYrvGG94MZOj07DeNDrp8Z4

4 I il PFOS: A 48-Hour StaticAcute ToxicityTeSt with the Cladoceran (Daphnia magna) ACUTE TOXICITY TO AQUATIC INVERTEBRATES (E.G.,DAPHNIA) TEST SUBSTANCE Identity:Perfluorooctanesulfonatmea;y alsobe referredtoas PFOS or FC-95. (I-Octanesulfoniaccid,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-p,otassium salt,CAS # 2795-39-3) Remarks field:The testsubstance isa whitepowder. Sample was taken from 3M lotnumber 217. Sample was storedunder ambient conditions priortotestingP.uritydeterminedto be 90.49% by LC/MS, 'H-HMR, 'gFNMR and elementalanalysestechniques. METHOD Method: OECD 202 and OPPTS 850.1010 Test type: Staticacute GLP: Yes Year Completed: Study completed1999. Reportcompleted2000 Species: Daphniamagna Analyticalmonitoring:PFOS measured at0,24,48-hours Statisticamlethods: EC50 valuescalculatedw,hen possibleb,y probit analysis,moving average method orbinomialprobabilitwyithnon-lin.ear interpolatiounsingthe computer softwareofC.E. Stephan. Test daphnid source: Obtained from culturesmaintainedby Wildlife InternationaLltd.,Easton,MD. Identificatioofnthe originablrood stock was verifiebdy the Academy ofNaturalSciences,PhiladelphiaP,A. Test daphnid age at study initiation<: 24-hours Test conditions Dilutionwater: 0.45 gm filterewdellwater Dilutionwater chemistry (duringthe 4-week period immediately preceding the test): hardness: 132 (128-136)mg/L as CaC03 alkalinity: 178 (176-178)mg/L as CaC03 pH: 8.3(8.2-8.3) TOC: < 1.0mg/L Conductivity: 313 (310-315)pmhos/cm Ca/Mg ratio: 35/13.5 Na/K ratio: 21.3/6.62 Lighting:Colortone@)50 fluorescentlightsi,ntensitaypproximately 359 lux.Photopedod of 16-hourslight8,-hoursdarkwitha 30minute transitiopneriod. StockandtestsolutionpsreparatioAn:primarsytocskolution was preparedindilutiownater at 91 mg/L. Itwas mixed for-19.5 hours priortouse. Aftermixing,theprimarystockwas proportionalldyilutedwithdilutiownater topreparethe four additionatlestconcentrations.Alltestsolutionsappeared clearand colorless. Exposure vessels: 250 mL plasticbeakers containing 240 mL of testsolution.The approximate depth oftestsolutionwas 6.4 cm. Number of replicatest:wo Number of daphnids per replicatet:en Number of concentrations:fiveplusa negativecontrol Water chemistry during the study: Dissolved oxygen range (0- 48 hours): 8.6- 8.9mg/L (controelxposure) 8.6- 9.1mg/L (91mg/L exposure) pH range (0- 48 hours) 8.2- 8.5 (controelxposure) 8.5- 8.6 (91mg/L exposure) Test temperature range (0- 48 hours) 19.5- 20.20C (controelxposure) 19.3- 20.1OC (91 mg/L exposure) Element basis:mortalitaynd immobilization Method of calculatingmean measured concentrations: arithmetimcean RESULTS Nominal concentrations: Bk control,12,20,33, 55,91 mg/L Measured concentrations: <LOQ, 11,20,33, 56,91 mg/L Element value:24-hourECo = 82 (81-83)mg/L 24-hour ECso = >91 mg/L (C.I.notcalculable) 24-hour ECw = >91 mg/L (C.I.notcalculable) 48-hour EC,o = 53 (<I1->91)mg/L 48-hour ECw = 61 (33-91)mg/L 48-hour ECw = 63 (<I1->91)mg/L Allelement valuesbased on mean measured concentrations StatisticaElvaluation:The EC5o valuesand 95% confidenceintervals were calculatewdhen possibleby probiatnalysist,hemoving average method or binomialprobabilitwyithnon-linearinterpolatiuosning the computer softwareofC.E. Stephan. The EC,o and ECgo valueswere calculatedwhen possibleusing theBruce-Versteeg method because there were lessthan two concentrationswithpartiaml'ortalitoyrimmobility. AnalyticaMlethodology:Analysesoftestsolutiownesre performeadt WildlifIenternationaLltd.usinghigh performance liquidchromatography withmass spectrometridcetection(HPLC/MS). When determiningthe concentrationofthe testsubstance inthe testsolutionst,he same and most prominentpeak response forperfluorooctanesulfonawtaes used. No attemptwas made to quantifyon the basisofindividuailsomeric components. The LOQ (limiotfquantitatiowna)s 4.58 mg/L inthisstudy. The mean percentrecoveryofmatrixfortificatiaonaslyzed concurrently duringsample analysiswas 96.2.Samples collectedattestinitiatihoand measured valuesfrom 85.5 to 112% ofnominal. Measured values for samples taken at24-hours ranged from 92.2 to 115% ofnominal. Measured valuesforsamples taken at48-hours ranged from 91.6 to 106% of nominal. Summary of analyticalchemistry data. NominalTest Measured DuplicatedValues at0,24, Mean Percentof Concentration, and 48-hours,Respectivelym,gtl Measured Nominal mgA- Concentrabon, mgA- NegativeControl All-cLOO <LOQ - 12 10.5,10.6,11.5,12.5,-10.91,2.0 11 92 20 17.2,18.1.22.8,21.6,21.4,18.8 20 100 33 30.2,34.1.34.0.36.1,31.3,34.0 33 100 55 50.5.49.9.57.0,63.0.56..8.56.4 56 102 91 87.6,102,90.1.84.4,W 7,92.4 91 ioo Biologicalobservations after48-hours: Daphnids inthe negative controlt,he 11 and the 20 mg/L treatmentsappeared healthyand normal throughoutthe testwithno mortalityi,mmobilityorovertclinicaslignsof toxicityF.ivepercentmortalitwyas observed at48-hours inthe negative control.The effectsnoted inthisstudy were mortalityn;o immobilization was noted atany testconcentration. Cumulative ercent mortality: Mean Measured Test Conc.,mgtl 24-houre 48-hours Neg. Control 0 5 i1 0 0 20 0 0 33 0 0 56 0 91 35 100 Controrlesponses:atisfactory CONCLUSIONS The potassiumperfluorooctanesulfon4a8t-ehourEC50 forDaphnia magna was determinedtobe 61 mg/L witha 95% confidenceintervaolf 33-91 mg/L. The 48-hournoimmobilizatioannd no observedeffect concentratiownas 33 mg/L. Submitter:3M Company, EnvironmentalLaboratoryP,.O.Box 33331, St. Paul,Minnesota,55133 DATA QUALI-R@ Reliability:Klimischranking I REFERENCES This studywas conducted atWildlifIenternationaLlt,d.Easton,MD atthe requestofthe3M Company. OTHER Last changed: 5/3/00 PFOS: A 48-HOUR STATIC ACUTE TO)ACITY TEST WITH THE CI-ADOCERAN (Daphniamagna) FINAL REPORT WI]LDLIFE INTERNATIONAL LTD. PROJECT NUMBER- 454A- 104 3M LAB REQUEST NO. U2723 U. S EnvironmentalProtectioAngency Series850 - EcologicalEffectsTestGuidelines OPPTS Number 850.1010 and OECD Guideline202 AUTHORS: KurtR- Drottar Henry 0. Krueger,Ph.D. STUDY INITIATION DATE: December 10,1998 STUDY COWLENON DATE: June4,1999 ANM'NDED REPORT DATE: April26,2000.? Submittedto 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St.PauL Minmota 55144 Wildl!feInternationaLltd. 8598 Commerce Drive Easton,Mmyland 21601 (410)822-8600 - Page 1 of43 ANENDED WILDLIFE INTERNATIONAL L-rD. -2- PROJECT NO.: 454A-104 GOOD LABORATORY PRACRICE COMEPLLKNCESTATEMENT SPONSOR- 3M Corporation TITLE: PFOS: A 48-HourStatiAccuteToxicitTyestwiththeCladocera(nDaphniamagna) WILDLEFE INTERNATIONAL LTD. PROJECT NUMBER-- 454A-104. STUDY CONTLETION DATE: June4,1999 ANM'NDED REPORT DAT]T" April26,2000 Thisstudywas conductedm compliancewithGood LaboratoryPracticSetandardsaspublishedby the U.S.EnvironmentalProtectioAngency in40 CFR Parts160 and 792, 17 August 1989;OECD Principleosf Good LaboratoryPracticeO,CDE/GD (92)32,EnvironmentMonograph No. 45,Paris1992;andjapanMAFF, 59 NohSan,NotificatiNoon. 3850,AgriculturParloductionBureau,10 August 1984,withthefollowing exceptions: The testsubstancewas not charactmuedm accordancevnthfullGLP comphance;.howevert,he charactefintiwoans perfomcieadccordintgo3M StandardOperatingProceduresand Methods,and allraw data arebeingmaintainedinthe3M archivesT.he testsubstanceisbeingrecharactmizeidnaccordancewithGLP. The stabiliotfy thetestsubstanceundercon(htionosf storageatthetestsitewas notdetermmed m accordancewithGood LaboratoryPracticSetandards. STUDY DIRECTOR: KurtF- Drottar SeniorBiologist >6 DATE PONSOR A ROVAL: A-.t@ SAPn 7 -1a) 15ArE ANEENDED WILDLIFE INTERNATIONAL LTD. -3- PROJECTNO.: 454A-104 QUALITY ASSURANCE STATEMEENT Thisb* wasexaminefdorcomplianwciethGoodLaboratoPrryactiScteandaradsspublishbeydthe U.S.EnvirorumentParlotectioAngency in40 CFR Parts160 and 792,17 August 1999;OECD Principleosf Good LaboratoryPracticeO,CDEIGD (92)32,EnvironmentMonograph No. 45,Paris1992;andJapanMAFF, 59 NohSan,NotificatiNoon. 3850,AgriculturParloductioBnureau,10 August 1984. The datesof all inspectionasnd auditsand thedatesffidany findingwsere reportetdotheStudyDirectorand Laboratory Management were asfollows: ACTIVITY: DATE CONDUCTED: TestSubstancePreparation February15,1999 DATE REPORTED TO: STUDY DIRECTOR: MANAGFIAENT: Febnuay 15,1999 February16,1999 AnalyticaSlample Preparation February16,1999 February17,1999 February 19, 1999 Water Chemistry Measurements Fobnimy17,1999 February17,1999 Fcbnmy 19,1999 BiologicaDlataand DraftReport March 17 and18,1999 March 18,1999 March 29,1999 AnalyticaDlataand DraftReport FinalReport Amended Report March 23 - 25,1999 June4,1999 April20 and25,2000 March 25,1999 June4, 1999 April25,2000 March 26,1999 June4, 1999 April25,2000 Ja@@ H. Coleman QualityAssuranceRepresentative Lk- C)o DATE ANIENDED WILDLIFE INTERNATIONAL LTD. -4- REPORT APPROVAL PROJECT NO.: 454A-104 SPONSOR: 3M Corporation TITLE: PFOS: A 48-HourStatiAccuteToxicitTyestwiththeCladocem (Daphniamagna) WILDLEFE INTERNATIONAL LTD. PROJECT NUMBEK- 454A-104 STUDY DIRECTO KurtF, Drottor SeniorBiologist MANAGENEW Henry-6.*ruejer,Pb-'b. Dk=Wr, AquatirT-oxicologyand Nm-Target Plants DATE DATE ANMNDED WILDLIFE INTERNATIONAL L-m. -5- PROJECTNO.: 454A-104 TABLE OF CONTENTS Title/CovePrage.........................................................................I............. Good LaboratoryPracticCeomplianceStatement...............................................2............. QualityAssuranceStatement...............................................................3............. ReportApproval.........................................................................4............. Tableof Contents........................................................................5............. Summary ...............................................................................7............. Introducti.o.n...........................................................................8............. Objectiv.e..............................................................................8............ ExperimentalDesign............................................................................8.................. Materialsand Methods ....................................................................9............. Resultsand Discussion....................................................................1.2............. Conclusions.......................................................*.....................1.3............. Refertmecs..............................................................................1.4.................... TABLES TableI- Summary ofAnalyticaClhemistryData..............................................1.5............. Table2 -Tempo-om, DissolvedOxygen, and pH ofWater infficTestChambers ....................1.6............ Table3 -CumulativeP=cnt Mortahty/ImmobiliatnydTreatment-RelatEefdfect.s...................1.7............ I Table4 - EC 10,EC50 andEC90 Values.....................................................1.8............. WILDLIFE INTERNATIONAL LTD. -6- PROJECT NO.: 454A-104 TABLE OF CONTENTS -ContinuedFIGURE Fig= 1.Concentration-RespoCnusreve(49Hour Data)........................................1.9.............. APPENDICES Appendix I- SpecifiCconductance,Hardness,Alkalinitya,ndpH of WeU Water Measured Duringthe4-Week PeriodImmediatelyPrecedingtheTest.................2.0.............. Appendix ]I- Analym ofPesticideOsr,ganicsM,etalsand OtherInorganieisn WildlifIenternationLatld.WellWater...............................................2..1............... AppendixIH- The AnalysisofPFOS inFreshwaterinSupportof WildlifIenternationLatld.ProjecNto.:454A-104 ................................2.2............. AppendixIV - Changes toProtocol........................................................4.0.............. AppendixV - PersonnelInvolvedintheStudy.........................f...@.........................................4.1................ AppendixVI - ReportAmendment .........................................................4.2............. AMENDED WILDLIFE INTERNATIONAL LTD. -7- PROJECT NO.: 454A-104 SPONSOR: SPONSOR'S PXPPRESENTAME: LOCATION OF STMY, RAW DATA AND A COPY OF TBE FINAL REPORT: LTD. PROJECT NLTMBEP,: TEST SIJBSTANCE: STMY: MEAN NEASURED TM CONCENTRATIONS: TEST DATES: LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANISW: AGE OF TEST ORGANOM: 49-HoURECSO: 95% CONFIDENCE IXWTS: NO MORTA=/IUMOBUlTY CONCENTRANON: NO-OBSERVED-EFFECTCONCENTRATION: SUNEMLARY 3M Corporation Ms. SusanA. Beach WildlifhedematimalLt& Faston,Maryland 21601 454A-104 PFOS (PerfluorooetaSnuelfoniAccid PotassiumSalt) PFOS: A 49-HourStatiAccuteToxicitTyestwiththe Cladoceran(Daphniamagna) NegativeControL11,20,33,56 and91 mg &L/L Expaimmtal Start- February16,1999 BiologicaTlerminatio-n February18,1999 ExperimentaTlomination- February18,1999 48 Hours Neonatecladocera(nDsaphniianagan) WilcUe IntmiiafionLatld.CulUm Easton,Maryland 21601 < 24 hours attestinitiation 61 mg &i./L 33 and 91 mg a.i./L 33 mg &ijL 33 mi &i.AL AMENDED WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A- 104 INTRODUCTION Tinssu* was conductedby WildlifIenternationLatld.for3M CorporatioanttheWildlifIenternational LtcLaquatictoxicologfyaciliitnyEaston,Maryland. The m-hfephaseof thetestwas conductedfrom February16,1999 toFebruary18,1999. Raw datageneratedby WildlifIentmmationaLltd.and a copy ofthe finarleportarefileudnderProjecNtumber 454A- 104 inarchivelsocatedon theWildlifeIntm-nationLatld.site. OBJECTIVE The objectivoefthissb* was toevaluatteheacutetoxicitoyfPFOS tothecladoceranD,aphnia magna, duringa 48-hourexposureperiodunderstatitcestconditions. EXPERIWNTAL DESIGN Daphmds were exposedto a geometricseriesoffiveWA concentrationasnd a negative(chlutiownater) controlT.wo replicatteestchambersweremaintainedineachtreatineanntd controlgroup,w*ith10daphmds in eachtestchamber fora totaolf20 daphnidspertestconcentrationN.ominal testconcen:tratiownesre selecteidn consultatiwoinththeSponsor,and were basedupon theresultosf an exploratorryangefindintgoxicittyest Nominal testconcentratiosneslectewdere 12,20,33,55 and91 mg activeirigredie(nat.i.)/MLe.an measured testconcentratiownesre determinedfrom samplesoftestwatercoReeW fi-omeachftabnentandcontroglroupat thebeginningofthetesta,tapproximatel2y4 hours,and attesttermination. Daphnids were asspw to exposurechambersattestuutiationO.bservationosf mortality/itmnobialnidtoytherclinicaslips oftoxicitwyeremade atapproximatel3y,24 and48 hoursaftetrest initiatioCnu.mulativepercentmortalitaynd mmnobihtyobservedinthetreatmengtroupswereusedtoestimate orcalculatEeCIO, EC50 andEC90 valuesat24 and48 hours.The no mortality/immobihctoyncentratioand theno-observed-effect-concent(rNaOtEiCo)n were determinedby visualinterpretatioofnthemortality, immobilitayndclinicaolbservatiodnat& AMENDED WILDLIFE INTERNATIC)NAL LTD. -9- PROJECTNO.: 454A-104 MATERL4,LSAND METHODS ThestudwyasconductaecdcorditnogtheproceduroeutslineidntheprotocolP,FOS: A 48-HourStatic AcuteToxicitTyestwiththeCladocera(nDaphniamagna). The protocowlas basedon procedureosutlineidn U.S.Environn=talProtectioAngency Series850 - EcologicaElffectsTestGuidelineOsPPTS Number 950.1010(1),OECD GuidelinfeorTestingofChemicals2,02: Daphnia sp.AcuteImmobilizatioTnestand ReproductioTnest(2)andASTM StandarEd-729-88aS,tandardGuideforConductingAcuteToxicjtTyests withFishesM,acroinvertebrata-nedsamphibians(3). TestSub The testsubstancewas receivedfrom 3M Corporatioonn October29, 1998 was assignedWildlife InternationLatlcLidentificatniuomnber 4675. The testsubstancewas describeads a whitepowder. Itwas identifiaesdFC-95 fromlotnmnber217 (T-6295).Informatiopnrovidedby theSponsorindicateadpuritoyf 98.9% and an expiratiodnateof2008. The testsubstancewas reana4-mdby theSponsorand theCtrtificaotfe AnalysisdatedMarch 9,2000 indicateadpurityof 90.490/oT.he testsubstancewas storedatambientroom temperature. )LrgarafioonfTestConcentrations Nominal testconcentratiownesre 12,20,33,55 and 91 mg &i./L,bisedon a testsubstancepurityof 90.49%. AU materialwshichcame intocontactwiththetestsubstancedurinipreparatioonftestconcentrations wereconstructeodf plastiocrstainlesssteelA. primarystocksolutiownas preparedincwutionwaterata concentratioofn91 mg &i./L.The primarystocksolutiownas mixed withan electridcrum mb= witha stainless steepladdleforapproximatel1y9.5hourstoaidinthesolubffizatoifotnhetestsubstance.Aftermixing,the primarystocksolutiownas proportionaldliylutewdithdilutiownaterto preparethefourad(htionatlest concentrationAsl.ltestsolutionasppearedclearand colorless. TestOr am'sm The clado=an,Daphnia magna, was selecteadsthetestspeciefsorthisb*. Dapbnidsam representative ofanimportantgroupofaquaticinvertebrataensd wereselectefdoruseinthetestbasedupon pag hist@ ofuse and easeofculturinignthelaboratoryD.apbnid neonatesusedinthetestwerelessthan24-hoursoldand were obtainedfromculturemsamftmed by WildlifIenternationLatld.,Euton,Maryland.IdentLficatoifmdw ongmal broodstockwas verifiebdy theAcademy ofNaturalSciencesP,luladelphiPaA,. AMENDED WILDLIFE INTERNATIONAL LTD. -10- PROJECT NO.: 454A-104 Adultdaphmds were cultureidnwaterfromthesame sourceand atapproxnnateltyhesame temperaturaes usedduringthetest.Adultdaphnidsintheculturewsereheldforatleast28 dayspriortocollectionfthe juvenilefsortestingT.he adultshowed no signsofdiseaseorstresdsuringtheholdingperiod.Duringthe 14-dayperiodprecedingthetestw,atertemperaturesrangedfrom 19.9to20.4'OC.The pH ofthewaterranged from 8.1to 8.3,and dissolveodxygen rangedfrom 8.3to 8.8mg/L. Instrumentatiuosned forwater measurementasredescribeidntheEnwronmentalConditionsectioonfthisreport. Neonate daphmds were obwmed fortestinfgrom mchvidualadultdaphmds which wereobservedtohaveno neonatespresentlessOm 24 hourspriortotestinitiatioTrhle progenyfrom 10 adultswereusedinthetestAt testinitiatitohne,juvenildeaphnidswerecollectefdrom thecultureasndindiscriminatterlaynsferretdo 10-mL glassbeakers.The dapbmds thenweretransferrefdromthebeakerstothetestcompartments.AllMmsfm were performedbelow theair/wateirnterfacuesinga wide-borepipet.Daphmds intheculturewserefeda mixtureof yeast,CerophyUs, and troutchow, as well as a suspensionof the fi-eshwategreen alga,Selenastrum capricornutum.The adultswere fedpriortotestinitiatibount,neonateswerenotfedduringthetest. -TestApparatus Testchambersconsisteodf250-mL plasti(cNalgene(Db)eakerscontainin2g40 niLoftestsolutionT.he depthoftestsolutioinnarepresentatitveestchamberwas 6.4cm Testchamberswerein@@ly placedin atemperature-.ontrouweadterbathsettomaintaina tempmv= of20 10C-.Testchambersw= labellewdith thepmjectnumber,testconcentratioand replicate. DilutioWnater The waterusedforculturinagndtestinwgas firshwateorbtainedfroma wellapproxim@ 40 nidm deep locatedon theWildhfeInternationLatld.site.The wellwaterischm=Wind asmoderately-hawradter.The specificconduct&=, hardnessa,lkalinitayn,d pH ofthewellwaterduringthefour-weekperiodimmediately precedingthetestarepresentedinAppendix I. The wellwaterwas passedthroughasandfiltetroremove particlegsreatetrhanapproximatel2y5 ;zm,and pumped intoa 37,800-Lstoragetankwhere thewaterwas aeratedwithspraynozzles.Priortouse,thewater againwas filtm-e(d0.45pm) toremovemicroorganismsandparti@lesT.he resultosfpefiodimcmlym performed WILDLIFE INTERNATIONAL LTD. -11- PROJFCT NO.: 454A-104 tomeasuretheconcentratioonfs selectecdontaminantsinwellwaterusedby WildlifIenternationLatld.are presentedinAppendix H. Conditions Lightingusedtoilluminattehecultureasnd testchambersduringculturinagndtestinwgas providedby fluorescenttubes thatemittedwavelengthsimilatro naturaslunligh(tColortone5c0). A photoperioodf 16 hoursoflighatnd8 hoursofdarimesswas coitrouedwithan automatictimer.A 30-minutetransitipoenriod oflow lightintensitwyas providedwhen lightwsent on and offtoavoidsuddenchangesinlightingL.ight intensitayttestinitiatiwoans approximately359 luxatthesurfaceofthewater.Lightintensiwtays measured usinga SPER ScientifLitcd.lightmeter. Temperaturewas measuredineachtestchamber atthebeghming and end ofthetestusingaliquid-in-glm thermometer.Temperaturealsowas measuredcontinuousliynone negativecontrorleplicatuesinga Fulscope ER/CRecorder.The targetestemperaturdeuringthesu* was 20 1*C.ThepH anddissolveodxygm content ofthewaterwere measuredineachtestchamber attestinitiatioanta,pproximatel2y4 hoursaftetrestinitiation and attheend ofthetesl Hardness,alalinityg,)*.ificconduciancaendtotalorganicarbon(TOC) were measuredinthedilutiownaterattestinitiatiaonnd attesttermination. MeasurementsofpH weremade usinga FisherAccumet Model 915 pH meter,anddissolveodxygenwas measuredusinga Yellow SpringsInstnment Model 5 IB dissolveodxygenmeter.Specificconductancweas measuredusinga Yellow SpringsInstrumentModel 33 Salinity-Conductivity-Tempermaettuerre.Hardnessand a&dinitymewu==ts weremade by titrafiboansedon proceduresinStandardMethodsfortheExamination of Waterand Wastewater(4).Totalorganiccarbonwas measumd usingalb*rnadaMlodel 5000TOC analym. Observations Observationwseremade todeterminethenumbers ofmortalitiaensdimmobileorgmm. Themunbersof individualesxlubitincglinicaslignsoftoxicitoyrabnormalbehavioralsowereevaluatedO.bservationwsere made approximatel3y,24,and 48 hoursaftertestinitiatiorl WILDLIFE INTERNATIONAL LTD. -12- PROJECTNO.: 454A-104 StafisticalAnal,ges The 24 and 48-hourEC50 valuesand the95% confidencientmvalsw= calculatewdhen possiblbeyprobit analysist,hemoving averagemethodorbinomialprobabiliwtiythnon-lineairnterpolati(o5n,6,7)usingthe computersoftwareofC.E.Stephan(8).The no mortality/immobihctoyncentratiaondNOEC weredetermined by inspectioonfthemortalityi,mmobilityandclinicaolbservatiodnata.Inthissu*, ECIO andEC90 values were calculatewdhen possibluesingtheBnice-VersteeMgethod (9)becausetherewere lessdam two concentratiownisthpartiamlortalitoyrimmobility(i.et.h,epmbitmethod couldnotbe used). Ang3@dcalOMLSM Sampleswere collectefdrom mid-depthof eachreplicatteestchamber atthebeginningofthetest,at approximately24 hoursand attesterminatiotnomeasureconcentratioonfsthetestsubstance.The samples werecollecteidnplasti(cNalgeneQDb)ottleasnd analyzedimmediatelwyithoutstorageA.nalyticaplrocedures usedintheanalysiosfthesamplesareprovidedinAppendix M. RESULTS AND DISCUSSION MeasurementofTestConcentrations Resultsof analysestomeasureconcentratioonfs PFOS inwatersaniplecsollecteddunng thettstare presenteidnTableI andintheanalyticcahlemistrryeport(AppendixIII)N.ominalconcenftvfisoenlectefdoruse inthisstudywere 12,20,33,55 and91 mg &i./L.Samplescolleew attestinitiati(o0nHours)had measured valuesthatrangedfrom 85 to 112% ofnondne. Samplescoueded at24 hourshad measuredvaluesthatranged from92 to113% ofnominal whereasmeasuredvaluesforsamplestaim at48 hoursrangedfrcm92 to1060/oof nominal.When measuredconcenbstonsof die samplescollecteadttestmitmtlon2,4 hours,and attest terminatioWn= averagedt,heme= measuredconcentratiofnosrthissft* were11,20,33,56and91 mg &iA,. Mean measuredconcentratiownesreusedintheestimatioonrcalculationfEC 10,EC50 and EC90 values. ANffi'NDED WILDLIFE INTERNATIONAL LTD. -13- PROJECT NO.: 454A-104 Observationsand Measurements Measurementsoftemperatured,issolvedoxygenand pH arepresentedinTable2. Temperaturesremained withinthe20 1*C rangeestablishefdorthetest.Dissolvedoxygen concentratiornesmafiied8@.:6mg/L(95% of saturatiotnh)roughouthetestMeasurementsofpH rangedfrom 8.2to8.6duringthetest. Dailyobservationosfmortalitiym,mobilityandotherclinicaslips observedduringthetestarcshown in Table3. Daphnidsinthenegativecontrolappearedhealthyand nomud throughouthetest,although5% mortalitwyas observedat48 hours.Daphnidsinthe11 and 20 mg a.i/Ltreatmengtroupsalsoappearednomud withno mortality/immobiloirtoyvertclinicsailps oftoxicityA.fter48 hoursofexposurem,ortalityfmunobility in the 33, 56 and 91 mg aiJL treatmengtroupswas 0, 35 and 100%, respectivelAyl.thoughno mortality/immobiloictcyurredinthe33 mg a.i./tLreatmentgroup,onedaphnidwas observedtobe lethargic. Based on the5% contromlortalitoybservedt,hisleduwgywas notconsideretdobe treatinent-relaEtCeIdO., EC50 andEC90 valuesand 95% confidencelimitsat24 and 48 hourswere estimateodr calculatefdrom the mortality/immobildiattya,andareshown inTable4. CONCLUSIONS The 48-hourEC50 valueforLiaphniamagna exposedtoPFOS was 61 m *ga.i./wLith95% confidence .limitosf33 and91 mg a.i./LT.he no mortalityfm=obilictoyncentratiaondNOEC were33 mg a.i./L. AMENDED WILDLIFE INTERNATIONAL LTD. -14- PROJECTNO.: 454A-104 REFERENCES I U.S.Environm ntalProtectionAgency. 1996. Series8SO - EcologicaElffectsTestGuidelines (draftO)P,PTS Number 850.1010.AquaticinvertebraAtceuteToxicitTyestF,reshwater.Daphnids. 2 Organisationfor Economic Cooperationand Development. 1984. Daphnia sp.Acute ImmobilizatiToenstand ReproduchonTest OECD GuidelinfeorTestinogfChemicals.Guideline 202. Paris. 3 ASTM Standard Eii9-&8a. 1994. Standard Guidefor Conducting Acute ToxicityTestsiothFishes, Macroinvertebrates, and Amphibians. American SocietyforTesting and Materials. 4 APHA, AWWA, WPCF. 1985. StandardMethodsfor theFxaminationof Waterand Wastewater. 16th Edition,American PublicHealthAssociation.American Water Works Association.Water PollutimControlFederauon,New York. 5 Stephan,CE. 1977. "Medx)ds for Calculatingan LC50," Aquatic Toxicologyand Hazard EvaluationsA.mericanSocietfyorTestingandMaterialsP.ublicatioNnumber STP 634,pp 65-94. 6 Stephan,C.E. 1978.U.S.EPA, EnvironmentaRlesearchLaboratoryD,uluth,Minnesota.Personal communication. 7 Thompson, WJL 1947, BacteriologicalResdews. Vol. IL No. 2. Pp. 115-145. 8 Fmney,D.J. 1971. StatisticaMlethods inBiologicalAssay. Secondedition. GfiffinPress,LondorL 9 Bruce,R.D.and DJ. Versteeg.1992.A statistipcraolcedureforinodelincgontinuoutsoxicitdyata. EnviromnmtalToxicologyand Chemistry,Vol.11p,p 1485-1494. WILDLIFE INTERNATIONAL LTD. -15- PROJECTNO.: 454A-104 Table1 Summiuy ofAnalyticaClhemistryData Sponsor, TestSubstance: TestOrganism: Dilution Water: Nominal Test Concent-adon (mg a.i.@2 NegativeControl 3M Corporation PFOS Cladoceran, Daphnia Wen Water amh@cate A-,! B A B A B magna S;-Vm Time 2L.u@rr, 0 24 24 48 48 12 A 0 B 0 A 24 B 24 A 48 B 48 20 A 0 B 0 A 24 B 24 A 48 B 48 33 A 0 B 0 A 24 B 24 A 48 B 48 55 A .0 B 0 A 24 B 24 A 48 B 48 91 A 0 B 0 A 24 B .24 A 48 B 48 The limitofquantitati(mLOQ) was 4.58mg ai/L. Measured Concenmildon LME&I < LOQAu <LOQ <LOQ <LOQ <LOQ <LOQ 10.5 10.6 11.5 12.5 10.9 12.0 17.2 18.1 22.8 21.6 21.4 18.8 30.2 34.1 34.0 36.1 31.3 34.0 50.5 49.9 57.0 63.0 56.8 56.4 87.6 102 90.1 84.4 88.7 92.4 Mean Measured Concentrafion (mg ai.@Lj <LOQ 11 20 33 56 91 Percent Of Nominal 92 100 100 102 100 ANMNDED WILDLIFE INTERNATIONAL L-m. -16- PROJECTNO.: 454A-104 Table2 Temperawm, DissolvedOxygen and pH of Water intheTestChambers Sponsor TestSubstance: TestOrganism: ])HutionWater: Mean Measured Concentration (mgaijl) NegativeControl 3M Corporation PFOS Cladooem, Daphnia magna Well Water 0 Hour' Replicate TemV D03 (*C) (mgtl) pH A 19.6 8.9 82 B 19.5 9.9 83 24 Hours DO (mg/L) pH 8.6 SA 8.6 8.5 48 Ikuft4 Temp DO (-C) _iMgIL_gH_ 202 8.6 8.5 19.9 8.6 8.5 11 A 19.5 8.9 83 9.6 8.5 20.1 8.7 8.5 B 19.4 8.9 8.3 8.6 8.5 20.1 8.6 8.5 20 A 19A 8.9 8.3 8.6 8.5 20.1 8.6 8.5 B 19A 8.9 8.3 9.6 8.5 20.0 8.7 8.6 33 A 19A 9.0 83 8.6 8.5 20.0 8.6 8.6 B 19A 9.0 83 8.6 9.5 20.1 8.6 8.6 56 A 19.3 9.1 8.4 8.6 8.5 20.0 8.7 8.6 B 19.3 9.1 8A 8.6 8.5 @20.0 8.7 8.6 91 A 19.4 9.1- 8.5 8.6 8.5 20.1 8.6 9.6 B 19.3 9.1 8.5 8.6 -9.5 20.0 8.7 8.6 Theo-hourdflutiownatermeasurementsforhardnessa,&ahnity,spedficoonducftnceand TOC were 136nig/LasCaCD3,178mgLas CaCO3,315,umhos/cm and < I mgC/L, respectively. 2 Temperaturemeasuredcdntinuousldyuringthetog rangedfrom approximately19.5to20.5*C. ' A dissolovxeydgecnoncentraotfi5oAnmg/Lmpmmts 600/a9wmtionat20*Cinfieshwater. 4 The testftnnmaton ddutionwatermeasurements forhardncs%allmbmty,spmfficconductanceand TOC were 140 mg/L asCACC@, 190 mgtl asCaCO-%,315 Annhoolcm and < I mgCAL, respoefively. ANffi'NDED WILDLIFE INTERNATIONAL -17- Table3 CumulativePmmt Mortality/fimnobialnidtTyreatment-RelatEefdferts' Sponsor 3M Corporation Ted Substance: PFOS TestOrganimn: Ckdooemn, Daphnia magna DilufioWnater WellWater Mean Menured Concentration DaphniA/ (mg&i./L) Replicate Replicate Number Dad NWtive Control A B 10 0 10 0 3-Hours Number Immobile 0 0 Effects 10 AN 10 AN II A 10 0 0 10 AN B 10 0 0 10AN 20 A 10 0 0 10 AN B 10 0 0 IOAN 3@3 A 10 0 0 10 AN B 10 0 0 10 AN 56 A 10 0 0 10 AN B 10 0 0 ..IOAN 91 Obmved A 10 0 0 B 10 0 0 Effects:AN - Appears NoniW. C IxthaW. 10 AN IOAN Number Dead 0 0 0 0 0 0 0 0 0 0 3 4 24 Hours Number Immobile 0 0 Effects 10 AN IOAN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 9ANIC 0 10 AN 0 5AN,2C 0 !!!2c Percent Immobile and Dead 0 0 0 0 0 35 Number Dead 1 0 0 0 0 0 0 0 5 2 10 10 WILDLIFE INTERNATIONAL LTD. -18- PROJFCTNO.: 454A-104 Table4 ECIO, EC50 andEC90 Values Sponsor: TestSubstance: TestOrganism: DilutionWater: Time 24 Hours 3M Corporation PFOS CladoceranD,aphnia magna WellWater ECIO (mg&i./L) 82 Lower 95% Confidence Limits 81 Upper 95% Confidence Limits 83 statistical Method Bruce-Versteeg 48 Hours Time 24 Hours 53 EC50 (mg a.i.AL) > 91 <11 Lower 95% Confidence Limits -1 > 91 Upper 95% Confidence Limits -1 PnELlersteZeL_ Statistical Method Visual IhnLspewction 48 Hours Time 24 Hours 61 EC90 (mga.i./L) > 91 33 Lower 95% Confide= Limits 91 Upper 95% Confidence Limits Binomial Statistical Method VisualInspection 48 Hours 63 <11 > 91 Confidencelumtscouldnotbe calculatewdiththeM2a@L /i@mmo@bi!!td:ataobtained. ANEENDED WILDLIFE INTERNATIONAL LTD. -19- Figure1. Concentration-ResponCsuerve(48Hour Data) PROJECT NO.: 454A-104 8 7- 0 6- 0 5E E 4m 0 3- 2- 10 100 1000 Mean Measured Concentration(mg a.i./L) AMENDED WILDLIFE INTERNATIONAL LTD. -20- PROJECT NO.: 454A-104 APPENDDCI SpemficConductance,Hardness,A&ahmty andpH ofWell Water Measured Duringthe4-Week PeriodImmediatelyPrecedingtheTest Sponsor: TestSubstance: TestOrganism: DilutionWater: 3M Corporation PFOS CladoceranD,aphnia magna Well Water Mean SpecifiCconductance (,umhos/cm) 313(N = 4) 310-315 Hardness (mg/L asCaCO3) 132(N= 4) 128-136 ,Mkalinity (mg/Las CaC03) 178(N = 4) 176-178. pH 8.3(N = 4) 8.@- 8.3 WILDLIFE INTERNATIONAL LTD. -21- PROJECT NO.: 454A-104 APPENDIX 11 Analysm ofPesticideOsr,ganicsM,eWs andOtherlaorganics m WildlffIenternationLatld.WeU Water' ANALYSIS MEASURED CONCENTRATION AlismflaneousMensurownts TotalDissolvedSolids Ammonia Nitrogen TotalOrgatue C-arbon2 TotalCyinide Orpnoch'o " and PCBs Ald% BHBCHC W DeltaBHC Gamma BHC (Undam) Chlordane DDD, pp' DDE, pp' DDT,pp' Dicidnn Fndo.gulfan,A Endomffan, B EndosWfan SuMft Endrin EndrinAMehyde Hqftchlor McthoxycWor HeptachlorEpoxide Toxaphene PCX1016 PCB-1221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 Metab and Other Inorganies ,muffi@nym Arsenic 3 c c coppw Iron i,zm3 Nfw mu*ni;3 Nmpnew ;M;;;;num3 Nickel 3 PSoolt&s-iuriu ssoadzipm 3 =YM 296 < 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 < 0.019 < 0.010 < 0.005 < 0.005 < 0.007 < 0.005 < 0.500 < 0.260 < 0.260 < 0260 < 0.720 < 0.720 < 0.720 < 0.720 < 100 < 25.0 < 0.50 < 1.0 35.0 < 2.0 < 1.0 < 20.0 < 100 < 10.0 13.5 < 1.0 < 0.20 < 2.0 < 2.0 6.62 < 25.0 < 1.0 21.3 < 20.0 mg/L miV_L miVL uitL P41-L jug/-L mil-L A[Y-L gfv-L ui/-L miv-L A41-L jufy-L 4 -& Afy-L Ufy-L gio-L ufv-L 4/-L A@V-L A4,-L Atl-L ;4w-L Afy-L pfy-L IUAAit-L pfv-L Ail-L /4fyL PB/-L mfv-L uAAIVLniipl Y4/-L 41-L A41-L giv-L 4&m@fL ui@-L Ail-L gfp-L A41-L rniVL gfv-L Agt-L mivl A4L 2 A,6n,anla-yYiSs-eeas .grmedby T E.nvkonmentaL GainesvillFel,oridaforumples coUceW on November 3 %mugh November 7,1997. 3 prmad b.y- %Dl a lnwmfiorw Ltd.forthe samplecoflectio@ndNovember 5, 1"7. Analym *formed by Wd(UifeIntcmfiondLtd.forsamplescofleew on November 5 thmugh 7,1997. WILDLIFE INTERNATIONAL LTD. -22- APPENDDC M PROJECT NO.: 454A- 104 THE ANALYSIS OF PFOS IN FRESHWATER IN SUPPORT OF WILDIHE DMRNATIONAL LTD. PROJECT NO.: 454A-104 WILDLIFE INTERNATIONAL L-m. -23- PROJECTNO.: 454A-104 REPORT APPROVAL SPONSOP- 3M Corporation TIME: PFOS: A 48-HourStatiAccuteToxicityTestwiththeCladoceran (Daphniamagna) WELDLIFE PMRNATIONAL LTD. PROJECT NO.: 454A-104 PRINCIPAL DIVESTIGATOR: RayiWnA. Van Hoven,Ph.D. Scientist MANAGENE'TR: WiUwd B.Nixon,Ph.D" Manager,AnalyticaClh=dstry 4-2s-oo DATE DAIT@ AMENDED WILDLIFE INTERNATIONAL LTD. -24- PROJECTNO.: 454A-104 Introdu Freshwatersampleswere collectefdrom a statiaccuteaquatictoxicitsytudydesignedtodeterminethe effectosfPFOS (PerfluorooctaSnuelfonicAcid PotassiumSalt)tothecladoceran(Daphnia magna). This studywas conductedby WildffiInternationLatld.and identifiaesdProjecNto.: 454A-104. The analyses of thesewatersampleswere performedat WildlifebftmationalLtd.usinghighperformancehcllnd chromatographwyithmass spectrometrdiectectio(nIPLC/MS). Sampleswere receivefdoranalysiosn February16,17 and 18,1999and were analyzedon each samplereceipdtay. TestSubstanceand hdemal Standard The testsubstanceused forthe analyticaplortionof tlnsstudywas WildlifeInternationLatld. identificatniuomnber 4675. The testsubstancewas usedtopreparecahbrationand matrixfortification samples. The internasltandardwas receivefdrom 3M Corporationon July2, 1998 and was assignedWildlife InternationLatld.idenlaficatniuomnber 4526 upon receiptI.le-mtemalstandarda,granularmaterialw,as identifiaesd: II-LIK 2K 2H PerfluorooctaSnuelfoniAccid,ChemicalAbstractNumber: 27619-97-2. The standardwas storedunderambientconditions. AnOyficalMethod The method usedforthe analysiosfthefi-eshwatesrampleswas developedatWildlifeInternational Ltd.and entitle"dAnalyticaMlethod fortheDeterminatioonf PFOS inFreshwaterS,altwatera,nd Algal Medium". Thismethodologywas includedas AppendixH ofWildlffIenternationLatld.protocolnumber 454/011299/MVAIJSUB454. Itwas basedupon methodologyprovidedby 3M Corporation. Samples were dilutedin a 50% methanol: 50% NANOpure* water solutiocnontifiiing 0.100mg4HPFOS (internsatlandard)/aLnd 0.05% fonnicacid(v/v)so thattheyfellwithinthe calibratiornangeofthePFOS methodology. Concmtrafionsofthe PFOS in the standardsand sampleswere determinedby reverse-phasheigh performanceliquidchromatographyusinga Hewlett-PadcarMdodel 1100 IfighPerformanceLiquid Chromalograph(BPLC) witha Peridn-ElmeArPI 10OLC Mass Spectrometeerquippedwith a Peridn- WILDLIFE INTERNATIONAL L-m. -25- PROJECT NO.: 454A-104 Elmer TurboIonSpmy ion source. BPLC sepamfionswere achievedusinga KeystoneBetasilCis analyticcaollumn (100nun x 2 min I.D.,3 pm particle.sizTeh)e.instrumenptarametersaresummarized inTable1.A methodflowcharitsprovidedinFigure1. CalibratioCnurve and LimitofQuanfitation Calibratiosnumdardsof PFOS preparedin a 50% methanol: 50% NANOpureg watersolution tainin0-.9100 mg 4H PFOS (internaslmndard)/Land 0.05% formicacid(v/v)r,anginginconcentration fi-om0.00915to 0.0457 ingai./Lwere analyzedwiththesamples.Linearregressioenquationswere generatedusingpeak arearesponseratios(PFOS :internasltandard)versustherespectivceoncentrations ofthecalibratisotnandardsA. typicaclalibratioxnrvvispresentedinFigure2. The concentrationf PFOS inthesampleswas &-tmminedby substitutitnhgepeak arearesponseratiosintotheapplicablleinear regressioenquation.Representativieonchromatogramsoflow and highcalibratiosntandardsarepresented inFigures3 and 4,respectively. The method hrmtofquant=on (LOQ) fortheseanalysewsas setat4.59mg a.i.AcLalculateadsthe productof thelowestcalibratisotnandardanalyzed(0.00915mg a.i./La)nd the'dilutifoanctorof the matrixblanksamples(500). MatrixBlankand Fortificat:Simopnlea Threematrixblanksampleswere analyzedtodetermme possiblmeftrferenceN.o mter&renceswere observedator abovetheLOQ duringsamplesanalyses(Table2). A representaticvheromatogramof a matrixblankispresenteidnFig= 5. Freshwaterwas foftMW at9.15,45.7and 91.5mg a.i./aLnd analyzedconcurrentlwyiththesamples todetermmethemean proceduralrecovery(Table3). Sample concentratiownesre not correctefdorthe mean procedurarlecoveryof96.2%. A representaticvheromatogramofa matrixfortificatisopnresented inFigure6. AAM'NDED WILDLIFE INTERNATIONAL LTD. -26- PROJIECTNO.: 454A-104 ExainpleCalculations Sample number 454A-104-27,nominalconcentrafionf 12 mg a.i./iLnfi-eshwater. InitiVaollume: 0.loonill FinalVolume: 50.0mL DilutioFnactor 500 PFOS PeakArea: 3027 InternaSltandardPeak Area: 585991 Peak AreaRatio:0.5167 Calibratiocnurveequation. Slope:0.02117 Intercept0:.05525 Curve islinear. PFOS (tLga.i./]aLt)histrment Peak arearatio- (Y-kteFcept) Slope 0.5167- 0.05525 0.02117 21.8 PFOS (mg a.iJL)insample PFOS (pga.i./La)tinshumentx DilutioFnactor 1000 21.8x 500 1000 10.9 ABEENDED WILDLIFE INTERNATIONAL LTD. -27- PROJECT NO.: 454A-104 PercentofNominal Concentration PFOS (mg a.i./iLn)sample x 100 PFOS (mg a.i.ALn)ominal Calculatedrecovery9:1.6% Note:manual calculatimoany differ. RESULTS SMIE Analgis Freshwatersampleswere collectefdrom theacutetoxiatystudywiththecladocera(nDaphniamagna) attestinitiatioFne,bruary16, 1999 (Hour 0),on February17, 1999 (Hour 24),and attesttermination, February18,1999 (Hour 48).The measuredconcoitmtionosfPFOS inthesamplescollecteadtinitiation of exposureof thetestorganisms(Hour 0) rangedfrom 85.5to 112% of thenominalconcentrations. SamplescollecteadtHour 24 had a measuredconcentratiornangeof 92.2to 115% of nominalvalues. -Samplescollecteadttestterminatio(nHour 48) had a measuredconcentratiornangeof 91.6to 106% of nominalvalues(Table4).A representaticvheromatogramofa testsampleisshown inFigure7. AMENDED WILDLIFE INTERNATIONAL LTD. -28- PROJECTNO.: 454A-104 INSTRUMENT: TableI TypicalLC/MS OperationaPlarameters Howlen-PackardModel I100High PerformanceLiquid Chromatographwitha Peridn-ElmeArPI IOOLC Mass Spectrometer equippedwitha Peridn-EhneTrurbolonSprayionsource.Operatedin selectiivoenmonitorinmgode (SM. ANALYTICAL COLUMN: OVEN TEMPERATURE: STOP TIME: KeystoneBetasilCigcolumn(100mm x 2 nun I.D.,3 pinparticle size) 300C 10.0minutes FLOW RATE: 0.220mljniinute MOBILE PHASE: 72.0% Mcflmol: 28.0% NANOpure(D Watercontaining 0.1% FormicAcid INJECTION VOLUMEE: PFOS RETENTION TIME: .50.0pL Approximaftl7y.1min@ INTERNAL STANDARD RETENTION TIME: Approximately4.9minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6wm 426.7amu WILDLIFE INTERNATIONAL LTD. -29- PROJECTNO.: 454A-104 Table 2 Matnx BLmks AnalyzedConcurrentlDyunng SampleAnalysis Number (454A-104-) NLAB- 1 S2MIe Type MatrixBlank Measured Concentratioonf PFOS' (niga.i.AL) .<LOQ MAB-2 MatrixBlank < LOQ MAB-3 MatrixBLuk < LOQ 'Me limitofquanfitafi(oLnOQ) was 4.58mg a.iJLbasedupon theproductofthelowestcalibration standard (0.00915 a.iJL)and thedilutiofnactorofffimcatrixblanks!Mles @500). AMENDED WILDLIFE INTERNATIONAL LTD. -30- PROJFCT NO.: 454A-104 Table 3 MatrixFortificatiAonaslyzedConcurrentlDyuringSampleAnalysis Sample Number (454A-104-) MAS-1 MAS-4 MAS-7 Concentrationosf PFOS LM.9a.i.u Fortified Measured 9.15 8.42 9.15 9.72' 9.15 9.15 Percent Recovered 92.0 106 100 MAS-2 45.7 40.3 88.1 MAS-5 45.7 48.1 105 MAS-8 45.7 45.1 98.7 MAS-3 91.5 78.7 86.0 MAS-6 91.5 84.0 91.8 MAS-9 91.5 89.7 98.0 Mean = 96.2 StandardDeviation = 7.11 - CV - @= 7.39 N= 9 Note:Resultsand coffectionfsornew testsubstmcepunty were genemtedusingMacQuan version1.5 softwareand manual calculationVsa.lueshave been roundedforrepprtinpgurposes. 'Mean resulotfduplicatmejectiono*fstherecwutedonpw sample.Originarlesul(t5.48mg a.i./Lw)as notincludedinthestatistiacnaallysisofthedata. Pu-21icartesultfsor454A-104-MAS-4 are9.24and 10.2. ANEENDED WILDLIFE INTERNATIONAL LTD. -31- PROJECTNO.: 454A-104 Table 4 Measured ConcentrationosfPFOS inFreshwaterSamples from a CladoceranStatiAccuteToxicityTest Nominal Test Concentration (ME a.i./L) 0.0 Sample Number (454A-104-) @i!l 2 13 14 25 26 Sampling Time ffo-urs) 0 0 24 24 48 48 PFOS Measured Concentrationi (a a.i.g < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Percent of Nominal - 12 3 0 4 0 15 24 16 24 27 48 28 48 10.5 88.5 10.6 89.5 11.5 96.@ 12.5 105 10.9 91.6 12.0 101 20 5 0 6 0 17 24 18 24 29 48 30 48 17.2 85.5 18.1 90.2 22.8. 113 21.6. 107 21.4 106 18.8 93.6 33 7 0 30.2 8 0 34.1 19 24 34.0 20 24 36.1 31 48 31.3 32 48 34.0 Note:Resultsand correctionfsornew testsubstancepuritywere generatedusingMacQuan 91.7 104 103 110 95.1 103 version1.5 softwareand manualcalculationVsa.lueshavebeenroundedforreportinpgurposes. 'The limitof quanfitafi(oLnOQ) was 4.58mg a.i./bLasedupon theproductofthelowestcalibration standardanalyzed(0.00915mg a.i./La)nd thedilutiofnitctorfthematrixblanksamples(500). 2 Me= resulotfduplicatmejectionosftherwduwd ongmal sample.'Originarlesul(t113mg a.i.[Lw)as notincludedinthestatistiacnaallysisofthedata. Duplicateresultfsor454A-104-23are90.6and 89.6. ANENDED WILDLIFE INTERNATIONAL LTD. -32- PROJECTNO.: 454A-104 Table4 (Continued) Measured ConcentrationosfPFOS inFreshwaterSamplesfrom a CladoceranStatiAccuteToxicityTest N inaTlest Concentration (-M- a.i.L) 55 Sample Number (454A-104@ 9 10 21 22 33 34 Sampling Time aour-s) 0 0 24 24 48 48 PFOS Measured Concentrationi (M a.i./L) 50.5 49.9 57.0 63.0 56.8 56.4 Percent of Nominal 92.1 90.9 104 115 104 103 91 11 0 12 0 23 24 87.6 95.7 102 112 90.12 98.5 24 24 35 48 36 48 84.4 92.2 88.7 96.9 92.4 101 Note:Resultsand correctionfsornew testsubstancepuritywere generatedusingMacQuan version1.5 softwareand manual calculationVsa.lueshave been roundedforreportinpgwposes. 'The limitof quantitati(oLnOQ) was 4.58 mg a.i./bLasedupon theproductofthelowestcalibration standardanalyzed(0.00915mg a.i.ALa)nd thedilutiofnactorofthematiixblanksamples(500). 2Mean resulotfduplicatienjectioonfstherediluteodriginaslample.Originarlesul(t113mg a.i.ALw)as notincludedinthestatisticaanlalysiosfthedata. 2uplicateresultfsor454A-104-23are90.6and 89.6. ANffiNDED WILDLIFE INTERNATIONAL L-M. -33- PROJECTNO.: 454A-104 MEETHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FRESHWATER PreparmeatrifxordficatsoanmplebsyspikintgherequisviotleumeofPFOS stocskolutiodnisrectly intofi-eshwatuesringgas-tighstyringesand ClassA volumetricflasks. Dilutematrixfortificatainodntestsamplesintotherangeofthecalibratisotnandardbsy partialfliyun ClassA volumetrifclaskswith50% methanol:50% NANOpure(D watersolutiocnontaining 0.100 mg 4H PFOS (internsatlandard)/aLnd 0.05% formicacid(vtv).Add theappropriatveolumeof sampleand bringtheflasktovolume withthedilutiosnolvent.Processthematrixblanksampleusing thesame ddutim and aliquovtolume as forthelowestfortificaflaeovnel.Mix wellby severalrepeat inversions. Ampulatesamplesand submitforLC/MS analysis. Figure1. Analyticamlethod flowcharftortheanalysiosf PFOS infreshwater. WILDLIFE INTERNATIONAL -34- PROJECTNO.: 454A-104 1.20- 1.00- 0.80 0 0.60 0.40 0.20 000 0.000 0.010 0.020 0.030 0.040 Concentratio(nmg &i/L) 0.050 Fig= 2. AtypicalcalibraticounrveforPFOS. Slope= 0.02117;Intm=%=0.05525; r= 0.9961. AMENDED WILDLIFE INTERNATIONAL LTD. -37- PROJECTNO.: 454A-104 100. go. 807060- intensw. 1772 cps 169 40@ 30- 20- 25 149 0ow 31 el 91 iii iii iii 211 sm 1.30 2.55 3.81 5.08 &32 7.67 sm Tkne F"igum5. A representatcihvreomatogmm ofa matrixblanksample(454A-104-MAB-3). lle arrowindicatetsheretentiotnimeofPFOS. WILDLIFE INTERNATIONAL LTD. -40- PROJFCTNO.: 454A-104 APPENDDC IV Changes toProtocol Tlusstudywas conductedm accordancewrfftiheapprovedProtocolvnththefollowmgchanges: I The protocolwas amended to add theproposedoqmimcntal startand terminatiodnates,test concentratioannsd testsubstancenumber 2. The protocolwas amendod to deletesmmialfeed analym. 3. The protocolwas amended toadd dw analyticamlothodl 4. The protocowlas amended tochangethestattsbeaalnalym. WILDLIFE INTERNATIONAL L-rD. -42- PROJECT NO.: 454A-104 APPENDIXVI ReportAmendment I. OriginalReport: Pages 1-4,6 and 23 Amendment: The pageswere changedtoincludetheamended reportdate,revLRedpagenumbers,and new signatureasnd datesdue totheadditioonfdm reportamendment asAppendixVI Reason: To reflecttheissuingof an amended reporl 2. OriginaRleport: Page 2 Amendment: The compliancestatementwas revised. Reason: To claifyhow thetestsubstancweas chmwtmzecl 3. OriginaRleport: Page 9 Amendment: Informatiopnrovidedby theSponsorreflectitnhgereanalysiosfthetestsubstance, includintghereanalysidsateand thepurityw,as addedtotheTestSubstancesection. Reason: To reflectthecurrentestsubstanceinformatiopnrovidedby theSponsor. 4. Ongind Report: Entirereport Amendment: Alltestsubstanceconecatrafaown=s changedtoreflectthepuritoyfthetestsubstance asdeterminedby theSponsorina reanalysiosfthetestsubstance(FC-95,Lot 217). Testcmcentrationosri&ay w= basedon thereportepduritoyf 98.9%. The certificaotfeanalysidsatedMm-ch 9,2000 indicateadpurityof90.49*/oT.hereforea,ll test substanceconcentrationsi,ncludingnominal concentrationmse,asured concentrationasn,d EC values,wererecalculataendd reportedasmg aijlbasedondie 90.490/pourity. Reason. To reportheresultosf thetestbasedon thetestsubstancpeurityof 90.49% atthe requestoftheSponsor. ANffiNDED WILDLIFE INTERNATIONAL LTD. -43- APPENDIXVI -Continued- ReportAmendment PROJECT NO.: 454A- 104 KurtR- Drottar stu@Lr,7@ HefirO.Knieku,-Ph.D. DirectorA,quaticToxicologyandNon-TargetPlants REVEEWED BY: 0.G&wo@ Coleman QualityAssurance eolo6 DATE @/6 DAT8 @-@ 6-0 DATE AMENDED WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-104 Pagel of2 AMENDMEENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 48-HOUR STATIC ACUTE TOXICITY TEST WITH THE CLADOCERAN (Daphniamagna) PROTOCOL NO.: 454/120198/DAP-48HI/SUB454 AMENDMENT NO.: I SPONSOR: 3M Corporation PROJECT NO.: 454A-104 EFFECTIVE DATE: December 21,1998 AMENDMENT: Page2 Add: ExperimentaSltartDate: 2/16/99 ExperimentaTlerminationDate: 2/18/99 TestConcentrationsN:egativeControl,13,22,36,60 and 100mg a.i./L TestSubstanceNo.: 4675 REASON: The above informatiownas not known when theprotocolwas signedby the Study Director. AMENDMENT: TestOrgani Page 5 Delete: Feed (YCT) providedto daphi@dsintheculturewsillbe analyzedatleastonce annuallyto ensurethatthereareno contaminantastlevelksnown tobe capableof interferiwnigththestudy. REASON: I-Estoricaanlalysesof WildlifIenternationLatld.aquaticfeedhaveshown thatno contaminantsarepresentatlevelksnown tobe capableofinterferiwnigththestudy. AMENDMENT: APPENDIX IL Page14 Add: LiquidChromatographyMass Spectrometr(yLCMS) Method fortheDeterminatioonf PerfluorooetaSnuelfoniAccid,PotassiumSalt(PFOS) inFreshwaterF,iltereSdaltwatearnd AlgalMedium. REASON: To add theanalyticmaelthod tobe usedinthestudy. a45411"antendl 24A.*"W WILDLIFE INTERNATIONAL LTD. STUDY DIRECTOR LABORATORY MANAGEI@ENT SPOI@S--OW S REPRESENTATIVE PROJECTNO.: Page 2 of 2 454A-104 DATE 01,f7 DA@IE DATE a454llO4iammdl WILDLIFF- INTERNATIC)NAL L-rD. PROJECTNO.: Page I of I 454A-104 AMIENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 48-HOUR STATIC ACUTE TO)GCITY TEST WITH THE CLADOCERAN (Daphniamagna) PROTOCOL NO.: 454/120198/DAP-48Hl/SLTB454 AMEENDMENT NO.: 2 SPONSOR: 3M Corporation PROJECT NO.: 454A- 104 EFFECTIVE DATE: March 9,1999 AMMNDMEENT: Data AnalysisP,age 8 Change: When the dose-responsepatternallowscalculatioonfECIO, EC50 and EC90 values, the datawillbe analyzedusingthe computer softwareof C.E. Stephan (6). To: When thedose-responsepatternallowscalculatioonfECIO, EC50 and EC90 values,the data willbe analyzedusingthe computer softwareof C.E. Stephan (6),or anotherappropriate methodology. REASON: The computer softwareof C.E. Stephan does not calculatEeCIO orEC90 values. STRY DIRECTOR L OARATORY MANA SPONSOR'S REPRESENTATIVE DATE DATE DATE a45,AI041amema Aw PROTOCOL PFOS: A 48-HOUR STATIC ACUTE TO)UCITY TEST WITH THE CLADOCERAN (Daphniamagna) U.S.EnvironmentaPlrotectioAngency Series850 -EcologicalEffectsTestGuidelines OPPTS Number 850.1010 and OECD Guideline202 3M Lab RequestNo. U2723 WILDLIFE Submittedto 3M Corporafion Envkomncntal Laboratory P.O.Box 33331 St Paul,Minnesota 55133 INTERNATIONAL LTD. 8598 Commerce Drive Easton,Maryland 21601 (410)822-8600 December 1,1998 PROTOCOL NO.: 454/12019&/DAP-48Hl/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -2- PFOS: A 48-HOUR STATIC ACUTE TO)GCITY TEST WITH Tiff CLADOCERAN (Daphnia magna) SPONSO 3M Corporation EnvirorumentalLaboratory P.O.Box 33331 St Paul,Minnesota 55133 REPRESENTATIVE: Ms. Susan A. Beach TESTING FACILITY: WildlifeInternationaLltcL 8598 Con=erce Drive Easton,Maryland 21601 STUDY DIRECT KurtDrottar SeniorAquatic Biologist LABORATORY MANAGEMENT: Hemy 0. Krueger,Ph.D. DirectorofAquaticToxicology& Non-TargetPlants FOR LABORATORY USE ONLY Proposed Dates: Expmimental StartDate: ProjectNo.: 4(!5qq- loel Expain=tal Tamination Date: Test Concentrations: Test SubstanceNo.: ReferenceSubstanceNo. (ifapplicable): PROTOCOL APPROVAL STUDY DIRECTOR )1,ze_ - QJtATORY-IWAGENffiNT SPONSOR!S REPUStITrA= DATE DATE/ 171qk DATE PROTOCOL NO.: 454/120199/DAP-48Hl/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -3- INTRODUCRION WildlifIentmmatimalLtd.willconducta statiaccutetoxicittyestwiththecladoceranD,aphnia magna, fortheSponsorattheWildlifIent=ationaLltd.aquatitcoxicologfyaciliitnyEaston,MarylancL The studywillbe performed based on proceduresinthe U.S. EnvirorunentalProtectionAgency Series 850 - EcologicaElffectsTestGuidelinesOPPTS Nmnber 850.1010 (1),in OECD GuidelineforTesting of Chemicals, 202: Daphnia sp.Acute ImmobilizationTestand Reproduction Test(2)and ASTM StandardE-729-88a,Standard Guide for Conducting Acute ToxicityTestswithFishes,Macroinvertebratesandamphibians (3).Raw dataforallwork performedatWildlifeInternationaLltd.and a copy ofthe@fmalreportwillbe filedby projectnumber in archiveslocatedon theWildlifeInternationaLltcL site,or atan alternativleocationtobe specifiedinthefinalreport. OBJECRIVES The objectivoef tlissW4 isto determinethe acuteeffectsof a testsubstanceon thecladoceran, Daphnia magna, under statictestconditionsfora periodof 48 hours. EXPERIM Daphnids willbe exposedto a geometricseriesof atleastfivetestconcentrationasnd a negative (dilutiownater)controlfor48 hours. Two replicatteestchambers willbe maintainedineach treatment and controlgroup,with 10 neonatedaphnids in each chamber so thata totalof 20 neonatedaphnids are exposed 'ineach treatmentand controlgroup. Nominal testconcmitrationwsillbe selectedinconsultatiownith the Sponsor,and-willbe based upon informationsuch astheresultsof exploratorryange-findmgtoxicitdyata,known toxicitydata, physical/chemicaplropertiesof thetestsubstanceor otherrelevantinformationT.argetconcentrations need notexceed 1000 mg/L or thesolubilitlyimitof thetestsubstanceinwater(whicherveirslower). Generallye,ach concmtmbm oftestsubstai=used in thedefinitivteestvnn be atleast600/oof thenext higherbmhnezt, unlessinformationconcerningtheconcentration-effeccutrveindicatetshata different dilutimfactorwould be more appropriateW.ater samplesfrom eachtestchamber willbe collecw at specifiedintervalfsoranalysisof thetestsubstance.Resultsof the analyseswillbe used to calculate mean measured testconcentrations. is- PROTOCOL No.: 454/120198/DAP-48HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -4- To controlbias neonatedaphnidswillbe impartiallayssignedto exposurechambers attest initiatioNno. otherpote'ntiaslourcesofbiasareexpectedtoaffectheresultsof thestudy.ECIO,EC50 and EC90 valueswillbe calculate-dw,henpossibleb,ased on thentunbcrof dead or immobilized daphnidsobservedineachtestconcentratioanftereach24-hourintervaolfexposure.The no-mortality concentratioannd theno-observed-effeccotncentratio(nNOEC) wiU be determined. MATERIALS AND METHODS Test Substance Informationon thecharacterizatioofntes@controlorreferencesubstancesisrequiredby Good LaboratoryPracticSetandards(GLP). The SponsorisresponsiblfeorprovidingWildlifeInternational LtcLwrittenverificatitohnatthetestsubsta= hasbeen characterizeadccordingto GLPs priortoitsuse inthestudy.Ifwrittenvmificatioonf GLP testsubstancecharacterizatiosnotprovidedtoWildlife InternationaLltcL,itwillbe notedinthecompliancestatementof thefinalreport.The attachedform IDENTMCATION OF TEST SUBSTANCE BY SPONSOR (AppendixI)istobe usedtoprovide informationnecessaryforGLP compliance. The Sponsorisresponsibleforallinformatiornelatedtothetestsubstanceand agreestoaccept any unused testsubstanceand/ortestsubstancecontainerrsemainingattheend ofthesbidy. P=aration of TestConcentrations The testsubstancewillbe administeretdothetestorganismdirectliyntodilutiownater.This routeof atirninistmfiwoans selectebdecauseitrepresenttshemost likelryouteof exposuretoaquatic organisms. TestQrgm@sm The cladoceranD,aphnia magna, has been selectedasthetestspeciesforthisstudy.Daphnids arerepresentativoefan importantgroup ofaquaticinvertebrateasn,d have been selectefdoruse inthe testbased upon pastusehistoryand easeof culhuinginthelaboratoryD.apbnid neonatestobe used in thetestwillbe lessdm 24 hoursoldand wiD be obtainedfrom culturemsaintainedatWildlffe InternationLatld.,Easton,Maryland. The identitoyfthespecieswin be vmifiedby thesupplierofthe PROTOCOL NO.: 454/12019&/DAP-48HI/SUB454 3M LAB REQUEST NO. U272@3 WILDLIFE INTERNATIONAL LTD. -5- originaclultureor by WildlifeInternationaLltd.personnelusingappropriatetaxononuc keys such as Peimak (4). Daphnids willbe culturedin water from the same source and at approximatelythe same temperatureasvnU be used duringthetest.Daphnids inthe culturesproducing neonates forthe testwill be heldforatleast10 days priorto collectioonf theneonatesfortesting.Adult daphnids inthe culture willproduce an averageof atleast3 young per adultperday overthe 7 day periodpriorto thetest. Neonates from daphnids thatshow signsof diseaseor stresswillnot be used as testorganisms. Dapliiiidisn holdingthatproduce ephippiaalsowillnot be used to supplyneonatesfortesting. Daphnids intheculturewsillbe fed once daily.The dietwillbe a mixture of yeast,Cerophyll(g, and troutchow (YCT), supplemented with a suspensionof the freshwatergreen alga Selenastrum capncornutum. Adults arefed duringthe24-hourperiodpriorto testinitiatiobnu,t neonateswillnot be fedduringthetest Feed (YCT) providedtodaphnidsintheculturewsillbe analyzedatleastonce annuallyto ensurethatthereareno contaminantsatlevelsknown tobe capableof interferinwgith the sft*. Specificatiofnosracceptablelevelsof contaminantsindaphnid dietshave not been established. However, ffi= areno known levelsof contaminantsreasonablyexpectedto be presentinthe dietthat are consideredto interferweith the purpose or conductof thetest. Neonateswillbe obtainedfortestinfgrom at leastthreeindividualadults.Priorto testinitiation, the neonateswillbe collectedfrom culturesand transferretdo smallcontainers.The daphnids willbe releasedintothetestcompartments below the watersurfaceusinga wide-borepipette. DilutionWater The water used for cultarmgand testingwillbe obtainedfrom a wellapproxnnately40 meters deep locatedon theWildlifeht=abonal Ltd.site.The waterwillbe passedthrougha sand filtearnd pumped intoa 37,800-L storagetankwhere thewater willbe aeratedwith spray nozzles.Priorto use thewaterwillbe filteretdo0.45,um inordertoremove fineparticlesW.ater used forculturingand testingischaractmizedas moderatelyhard. Typicalvaluesforhardness,alkalinitpyH, and specific conductanceare approximftly: PROTOCOLNO.: 454/120199/DAP-48Hl/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -6- Hardness,mg/L as CaC03 'Alkalinitmyg,/L as CACO, pH SpecificConductance,ymhos/cm 145 190 8.1 330 Hardness, alkalinitpyH, and specificonductancewillbe measured weekly to monitor the consistencoyf thewellwater. Means and rangesof themeasured parametersforthe four-weekperiod precedingthetestwillbe providedinthefinalreport.Analyseswillbe performed at leastonce annually to detemiinethe concentrationosf selectedorganicand inorganicconstituentisnthe water and results of theanalyseswillbe summarized inthe&W reporl Test A12paratus Test chambers willconsistofpolyethylenebottles.The sizeof bottleand thevolume of test solutionwillbe sufficientto-meet the requirementsof sampling foranalyficavlerificationf test substanceconcentrationasnd routinewaterchemistryanalyses.Test chambers willbe indiscriminately positionedina tempmahze-contronedwater bath or an environmentalchamber. Test chambers willbe labelledwith the projectnumber, testconcentrationand replicate. -EnvironmentalConditions Lightingused to-illuminatehe culturesand testchambers duringculturingand testingwillbe provided by fluorescentubesthatemitwavelengthssimilarto naturalsunlight(e.g.C,olortoneOD50). A photopmiod of 16 hoursof lightand 8 hours of darknesswillbe controllewdith an automatictimer. A 30-mimft Uwktion periodoflow lightintensitwyillbe providedwhen lightgso on and offtoavoid sudden changes in lightintensity.Lightintensitwyillbe measured attestinitiatioand at test terminationwith a SPER ScientifiLctd.lightmeter orequivalent. The targetestt=pem= willbe 20:k 1 -C. Temperaturewillbe measured in each testchamber atthebeginningand end ofthetestusinga hand-heldthermomete r. Temperaturealsowillbe measured with a cmtmuous recorderinone negativecontrolchamber. Recordermeasun=ents Willbe vmfied with a hand-heldthermometerpriorto testinitiation. PROTOCOL NO.: 454/120198/DAP-48Hl/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -7- Dissolved oxygen willbe measured in allreplicateosf the treatmentand controlgrpups attest initiatioand atapproximately24-hourintervaltshereafteursinga Yellow SpringsInstrumentModel 5 1B dissolvedoxygen meter,orequivalent Intheeventthatdissolvedoxygen levelsfallbelow 60% sawration,appropriateactionswillbe taken afterconsultatiownith the Sponsor. Measurements of pH willbe made in allreplicateosf thetreatmentand controlgroups attestinitiatioannd atapproximately 24-hourintervaldsereafterusinga FisherAerumet Model 915 pH meter,orequivalentIfa treatment group reaches 100% mortalityd,issolvedoxygen, pK and temperaturemeasurements willbe taken at thenext sampling intervalt,hendiscontinued. Hm-dms, pI-La&anily,specificconductanceand totalorganiccarbon(TOC) willbe measured inthedilutiownateratthebeginningand end of thetest.Hardness and alkalinitmyeasurements willbe made by titratiounsing proceduresbased on methods in Standard Methods for the Examination of Water and Wastewater (5).Specificconductancewillbe measured usinga Yellow SpringsInstrument Model 33 Salinity-Conductivity-Temperamteutrer,or equivalent Totalorganiccarbon willbe measured on a Shimadzu Model 5000 TOC analyzer,or equivalent. BioloeicalObservations Observationsof mortalityi,mmobilizationand clinicaslignsof toxicitwyillbe made between 0- 24 hours, and at24 and 48 hours:LI hour. Daphnids not ableto swim within15 secondsaftergentle agitatioonf the testchamber willbe consideredimmobile. SwnpliniiforAn"cal Measurements Water sampleswillbe collectefdrom eachtestchamber atthebeginningof thetestand at24 and 48 hours(:h1hour)toddmmw concenuationsofthetestsubstance Intheeventthat100% mortality occursin any treatmentthen samplingofthattreatmentwillterminatefollowingthenext sampling mterval Samples willbe collectedatnud-depth from each testchamber and analyzedunmo&atebr, or placedinan appropriatsetoragecontainer(e.g.p,olyethyleneor polypropylenebottle)and storedunder refrigeratiountilana4rzrA The sample scheme issinnm below: PROTOCOLNO.-. 454/12019&/DAP-48HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -8- PROPOSED ExperimentalGroup Control SolventControl(ifneeded) Level I-Low Concentration Level 2 Level 3 Level 4 Level5-High Concentration Totals NUMBERS OF VERIFICATION SAMPLES 0 Hours 24 Hours 2 2 2 2 2 2 2 2 2 2 2 2 2 2 14 14 48 Hours 2 2 2 2 2 2 2 14 TotalNumber ofVerificatioSnamples 42 The above nwnbers of samplesrepresentthosecollectedfrom thetestand do not includequality' control(QC) samples such as matrixblanksand fortificatiopnrseparedand analyzedduringthe analyticaclhemistryphase of the study. AnWyfical Chemist[y Chemical analysiosfthesampleswillbe performedby WildlffeInternationaLltd. The analytical method used willbe basedupon mzthodolou providedby theSponsor and identifieidnAppendix ]I. The methodologyused to analyzethetestsamples willbe documented in theraw data and sunmimized in thefinalreport. Data Anabmis V&m fficdose-responspeattemallowscaladatim ofECIO, EC50 and EC90 values,thedata will be analyzedusingthecomputer softwareof C.E. Stephan (6).The program was designedto calculate the EC valuesand the95% confidenceintervalbsy probitanalysist,hemoving averagemethod, or bfixxniaplrobabilitwyithnonlinearinterpolatio(n7,8 and 9)..The EC valueswillbe calculateudsing mortality/immobilitdyatacollecteadt24 and 48 hours- The no-mortalitcyoncentratioannd thenoobserved-effecctoncentratio(nNOEC) willbe determined. PROTOCOL NO.: 454/120198/DAP-48Hl/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -9- RECORDS TO BE MAINTAINED Recordstobe maintainedfordatageneratedby WildlifeInternationaLltd.willincludeb,utnot be limitedto: 1 . A copy of thesignedprotocol. 2. Identificatiaond characterizationfthe testsubstance,ifprovidedby the Sponsor. 3. Dates of initiatioannd terminationof thetest. 4. Historyof the testorganism,cultureand holdingrecords. 5. Stock solutioncalculatioannd preparation. 6. Daily observations. 7. Water chemistryresults(e.g.h,ardnessand alkalinity). 8. If applicable,the methods used to analyzetestsubstanceconcentrationasnd the resultsof analyficamleasurements. 9. Statisticcaallculations. 10. Testconditions(lightintensitpyh,otoperiode,tc.). 11. Calculationand preparati'oonf testconcentrations. 12. Copy of finalreport. FINAL REPORT A finalreportoftheresultosfthestudywillbe preparedby WildlifIenternationaLltcL The report willinclude,butnotbe limitedtothefollowingw,hen applicable: 1 . Name and addressof thefacilitpyerformingthe study. 2. Datesupon vAn6 thesh* was nutiateadnd completed,and thedefinitiveexperunentalstartand terminationdates. 3. A statementof compliancesignedby theSb* Directoraddressingany exceptionsto Good LaboratoryPracticeStandards. 4. Objectives and procedures,as statedinthe approved protocol,includingallchanges to the protocol. 5. The testsubstanceidentificatiioncludingname, chemicalabstractnumber or code number, strengthp,unty,composition,and otherinformationprovidedby the Sponsor. 6. Stabilitaynd solubilitoyf thetestsubstanceunder the conditionsof administrationi,fprovided by the Sponsor. PROTOCOLNO.: 454/120198/DAP-4gHl/SU]3454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -10- 7. A descriptionof the methods used to conduct thetest, 8. A descriptimof thetestorganisms,includingthe source,scientifincame, age or lifestage,feed types,resultsof latestfeedanalyses,lightintensitaynd photoperiod. 9. A descriptionof thepreparationof the testsolutions. 10. The me thods used to alloratoerganisms to testchambers and beginthe testt,he number of organisms and chambers per treatment,and the durationof thetest. 11. A descriptionof circumstancesthatmay have affectedthequalityor integritoyf the data. 12. 71bename of theSb* Directorand thenames of otherscientistpsr,ofessionalsa,nd supervisory personnelinvolvedin the study 13. A descriptionof thetransformationsc,alculationsa,nd operationsperformed on the data,a mmmimy and analysiosfthebiologicadlataand analyticaclhemistrydata,and a statementof the conclusiondsrawn fiom thean*=. A graphplottmgtheconcentratiornesponsecurveforeach periodan EC50 iscalculatedw,hen sufficiendtata exists.Ifthedataisconduciveto evaluation by probitanalysist,he slopeof theconcentration-rrsponsceurvewillbe reportea 14. Stafisticmaelthods used to evaluatethedata. 15. The signedand datedreportsof each of theindividualscientistosr otherprofessionalisnvolved in the study. 16. The locationwh= raw dataand finalreportaretobe stored. 17. A statementpreparedby fficQualityAssurance Unit listintghedatesthatstudyinspectionsand auditswere made and thedatesof any findingsreportedto the Study Directorand Management. 18. Ifitisnecessaryto make correctionosr additionsto a finalreportafterithas been accepted,such changeswillbe made intheform of an anvmdment issuedby theSh* Du-ector.The amendment willclearlyidentiftyhepartofthe finalreportthatisbeing amended and thereasonsforthe amendment, and willbe signedby theSu* Duwtor CHANGING OF PROTOCOL Phmned changestD theprotocolwillbe intheform ofwrittenamendments signedby theStudy Directorand theSponsoes Representative.Amendments willbe consideredas partof theprotocoland willbe attachedtothefinalprotocol.Any otherchanges win be in theform of writtendeviationssigned by the StudyDirectorand filedwiththeraw dat& Allchangestotheprotocolwillbe mchcatedindw finalreporl PROTOCOLNO.: 454/120198/DAP-48Hl/SLJB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. GOOD LABORATORY PRAMCES ThisstudwyilbleconductiendaccordanwciethGoodLaboratoPrryactiScteandarfdosrEPA (40CFR Part160 and/orPart792);OECD PrincipleosfGood LaboratoryPractic(eOCDE/GD (92) 32, EnvironmentMonograph No. 45);and Japan MAFF (59 NohSan, NotificatiNoon. 3850, AgncultaralProductionBureau).Each studyconductedby WildlifeInternationLatld.isroutinely examined by the WildlifeInternationLatld.QualityAssuranceUnit forcompliancewith Good LaboratoryPracticesS,tandardOperatingProceduresand thespecifiepdrotocol.A statementof compliancevnthGood LaboratoryPracticewsillbe preparedforallportionsofthestudyconductedby WildfifeInternationLatld. The Sponsorwillbe responsiblfeorcompliancewithGood Laboratory Practicefsorproceduresperformedby otherlaboratori(ees.g.r,esidueanalysesorpathology).Raw data forallwork performedatWildliffeitmnationLatld.and a copy ofthefinalreportwillbe filedby project number in archiveslocatedon theWildlifeInternationLatlcLsiteor atan alternatilvoecationtobe specifieidnthefinalreport. PROTOCOLNO.: 454/120198/DAP-48HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -12- REFERENCES 1 US. EnvironmentalProtectionAgency. 1996. Series850-FmlogicalEffectsTest Guidelines (draft),OPPTS Nmnber 850.1010: Aquatic InvertebrateAcute ToxicityTest,Freshwater Daphnjds. 2 Organisation for Economic Cooperation and Development 1984. Daphnia sp.Acute ImmobilizationTest and Reproduction Test. OECD Guidelinefor Testingof Chemicals. Guideline202. Paris. 3 ASTM Standard E729-88a. 1994. Standard Giddefor Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, andamphibians. American Society for Testing and Materials. 4 Pennak, ILW. 1978. FreshwaterInvertebrateosf theUnitedStates.2nd EcL 365 p. 5 APHA, AWWA, WPCF. 1985. Standard Methods for the F-tamination of Water and Wastewater. 16th Edition, American Public Health Associatiom American Water Works Association.Water PollutionControlFederationN,ew York-- 6 Stephan, C.E. 1978. U.S. EPA, EnvironmentalResearchLaboratory,Duluth,Minnesota. Personalcommunication. 7 Finney, D.J. 197 1. StatisticaMlethods in Biological Assay. Second edition.GriffinPress, I.ondom 8 Thompson, W.R. 1974. Bacteriological ReWews. Vol. IL No. 2. Pp. 115-145. 9 Stephan,C.E. 1977. Methods for Calculatingan LC50, Aquatic Toxicology and Hazard Evaluations. American SocietyforTesting and Materials.PublicationNumber ST? 634. Pp 65-84. nn^'rr%i@f%T 'kTf% Ale A 11 ^^1 f%n ff% A rt AnTY-1 ICIT Tn A C A ')'KST An 'nTr%TTT'Cl@rkTr% TY'1'7o'0'2 WILDLIFE INTERNATIONAL LTD. -13- APPENDIX I IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR To be Completed by Sponsor 1. Test SubstanceIdentit(ynarm tobe used inthereport):PFOS (PerfluorooetanSexflfoniAcc,id Potassium Sl* ReferenceStandard(ifapplicable)A:nalyticaSltandard: N/A InternaSltandard: I-I-2-2H.FLH.HPtffluorooctanSeulforfiAcciL- Test Substance Sample Code or Batch Number: Lot 217 Test SubstancePurity(*/AoctiveIngrechent): 98.9 ExpiratioDnate: 2008 ]I. Test SubstanceCharactmintion Have the identitys, and compositionor othercharacteristics which appropriateb@dv@me gem@tmystubstanceand referencestandard bew determift6dpriort6 itsuse in thisstudyin accordancewith GLP Standards? Yes No X Ill. Test Substance StorageConditions Pleaseindicatethe recommended storageconditionsat WildlifeInternationaLltd. Ambient room tgmpcm=s Has the stabilitoyf the testsubstanceunder thesestoragecondifions been determinedin accordancewith GLP Standards? Yes No X Other pertinentstabilitiynformation: IV. Test Concentrations: Adjusttestconcentratiotno 100*/*&i. X based upon thepurity(*/og)iven above. Do not adg'usttestconcentrationto I008/o &i. Tea themateriaAlM. V. ToxicityInfor-znation: Mannnalian: RatLD50 251mgft - Mouse LD50 N/A Aquatic: InvertebratTeoxicity(EC/LC50) FishToxicity(LC50) DVIaliamagn: 27 mL-/L Rainbow Trout: II me/L DopWa magm: 50 mL-/L Fathead Mimiow, 38 mprJL Other ToxicityInformation(mcluchngfm(hngsofchronicand subcbromc tmU): Pleasesee MSDS PROTOCOL NO.: 454/120198/DAP-48HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTE:RNATIONAL LTD. -14- APPENDIX Il AnalyticaMlethod tobe Providedby Sponsor PROTOCOL NO.: 454/120198/DAP-48Hl/SUB454 3M LAB REQUEST N.0. U2723