Document QJR8vg4KRZv4MabYJ8GdwjdrR

. PFOS: A 96-Hour StaticAcute ToxicityTest with the Fathead Minnow (Pimephales promelas) ACUTE TOXICITY TO FISH TEST SUBSTANCE Identity:Perfluorooctanesulfonatmea;y alsobe referredto as PFOS or FC-95. (I-Octanesulfoniaccid,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-p,otassiumsalt,CAS # 2795-39-3) Remarks: The testsubstance isa white powder. Sample was taken from 3M lotnumber 217. Sample was storedunder ambient conditionspriorto testingP.uritydetermined tobe 90.49% by LC/MS, 'H-HMR, 'GF-NMR and elemental,analysestechniques. METHOD Method: OECD 203 and OPPTS 850.1075 Type: Staticacute GLP: Yes Year completed: Study completed1999. Reportcompleted2000 Species:Pimephalespromelas Supplier:In-houseculturesW,ildlifIenternationaLlt,d.,Easton,MD Analyticalmonitoring:PFOS measured at0,48,96-hours Exposure period:96-hours Statisticamlethods: LC5o valuescalculatedw,hen possibleb,y probit analysis,moving average method or binomialprobabilitwyithnon-linear interpolatiounsingthe computer softwareofC.E. Stephan. Test fishage: Approximately126 days old Length and weight: 35 (30-38)mm, 0.36 (0.21-0.49g) Loading: 0.24 g fish/L Pretreatment: None Test conditions: Dilutionwater: 0.45 gm filterewdellwater Dilutionwater chemistry (duringthe 4-week period Immediately preceding the test): hardness: 131 (128-136)mg/L as CaC03 alkalinity: 177 (176-178)mg/L as CaC03 pH: 8.3 TOC: < 1.0mg/L Conductivity: 311 (310-315) gmhos/cm Stock and testsolutionpreparation: Primarystockprepared in dilutiownater at27 mg/L and mixed for-22 hours priortouse. Aftermixing,primarystocksolutiownas proportionaldliylutewdith dilutiownaterto preparethefouradditionatlestconcentrations. Concentrations dosing rate:Once Stabilitoyf the testchemical solutions:Extremelystable Exposure vessels: 25L polyethylenaequariacontaining approximately15L oftestsolutionw;aterdepth approximately17.6 CM. Number of replicates:two Number of fish per replicate:ten Number of concentrations: fiveplus a negative control Water chemistry during the study: Dissolved oxygen range (0 - 96 hours): 7.8- 8.8mg/L (controelxposure) 7.7- 9.0mg/L (28mg/L exposure) pH range (0- 96 hours) 8.3- 8.6 (controelxposure) 8.4- 8.5 (28mg/L exposure) Testtemperaturerange (0- 96 hours) 20.4- 22.1OC (controelxposure) 21.3- 22.30C (28mg/L exposure) Method ofcalculatingmean measured concentrationsa:rithmetimcean RESULTS Nominal concentrations:Bk control3,.6,5.9,9.9,16,27 mg/L Measured concentrations:<LOQ, 3.3,5.6,9.5,17,28 mg/L Element value: 24-hourLC5o > 28 mg/L (C.In.otcalculable) 48-hour LCso > 28 mg/L (C.In.otcalculable) 72-hour LCso 27 (22- 41)mg/L 96-hourLC50=9.5 (8.0- 11)mg/L Allelement values.basedon mean measured concentrations StatisticaElvaluationof Mortality: Confidencelimitfsor24 and 48-hourscouldnotbe calculatedue to lack of mortalityT.he 72-hourLCsovalueisquestionablebecause a concentration-effercetlationshwiaps notdemonstratedovera reasonable range ofpercentdead. The 24 and 48-hourLC50valueswere determined by visualinterpretatioPnr.obitwas used tocalculattehe72-hourLC5oand Moving Average forthe 96-hourLC5o. AnalyticalMethodology: Analyses oftestsolutionwsere performedat WildlifIenternationLatld.usinghighperformanceliquicdhromatography withmass spectrometridcetection(HPLC/MS).When determiningthe concentrationofthetestsubstanceinthetestsolutionst,hesame and most prominentpeak responseforperfluorooctanesulfonwaatse used. No attemptwas made toquantifyon thebasisofindividuailsomeric components. The LOQ (limiotfquantitatiowna)s 0.458mg/L inthisstudy. The mean percentrecoveryof matrixfortificatiaonnaslyzedconcurrently duringsample analysiswas 97.9.Samples collecteadttestinitiatihoand measured valuesfrom 85.3to 117% ofnominal.Measured valuesfor samples takenat48-hoursranged from 86.3to 101% ofnominal. Measured valuesforsamples takenat 96-hoursrangedfrom 87.6to 98.3% ofnominal. Summary of analyticalchemistry data: Nominal Test Concentration, mg/L M6asured DuplicateValues at0. 48, and 96-hours,Respectively,mgll Mean Measured Percentof Concentration, Nominal mgA. Negative Control AN < LOO <Loa - 3.6 3.16,3.53,3.08,3.22,3.46,3.13 3.3 92 5.9 6.06,6.07,5AS, 5.89,5.70.5.55 6.6 95 9.9 8.99,9.47,9.88,9.33.9.70,9.62 9.5 96 Is 18.2,19.3,15.0,15.6,14.8,16.2 17 106 27_ 28.5,28.5,27.0,Z7.8,26-8.26.6 28 104 Biologicalobservations after96-hours:Fishinthe negativecontroland the3.3mg/L exposure concentratioanppeared normal.Some orallofthe survivinfgishwere observedto be swimming erratical(l4y/16in5.6mg/L exposure,10/10in9.5mg/L,4/4in17 mg/L)attesttermination. Cumulative percentmortality: Mean Measured Test Conc., mg/L 24-hours 48-hours 72-hour3 Neg. Control 0 0 0 3.3 0 0 0 5.6 0 0 0 9,5 0 0 0 1 17 0 1 0 1 is o o 50 96-hours 0 0 20 50 80 I-oo Lowestconcentraticoanusing100% mortalit2y8:mg/L Mortalitoyf controls:None CONCLUSIONS The potassium perfluorooctanesulfonat9e6-hour LC50 forfathead minnow was determined to be 9.5 mg/L with a 95% confidence intervalof 8.0 -11 mg/L. The 96-hour no mortalityand no effectsconcentrationwas 3.@ mg/L. Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability:Klimischranking 1 REFERENCES This study was conducted at WildlifeInternationaLltd.,Easton, MD at the request of the 3M Company. OTHER Last changed: 5/3/00 PFOS: A 96-HOUR STATIC ACUTE TO)GCITY TEST V= THE FATHEAD MINNOW (hmephalespromelas) FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER-- 454A- 102 3M LAB REQUEST NO. U2723 U. S EnvironmentalProtectioAngency Series850 - EcologicalEffectsTestGuidelines OPPTS Number 850.1075 and OECD Guideline203 AUTHORS: KurtF, Drottar Henry 0. Krueger,Pb-D. STUDY INITIATION DATE: December 4,1998 STUDY COMPLETION DATE: July14,1999 AMI@NDED REPORT DATE: April26,2000 Submittedto 3M Corporation Envkomnental Laboratory 935 Bush Avenue St.Pmd, Minnesota 55144 Wildl!feIntemationalLtd. 8598 ConunerceDrive Easton,Maryland 21601 (410)822-8600 Page I of43 AMENDED WILDLIFE INTERNATIONAL LTD. -2- PROJECTNO.: 454A-102 GOOD LABORATORY PRAC]RICECONYLLKNCE STATEMEENT SPONSOK- 3M Corporation TITLE: PFOS: A 96-HourStatiAccuteToxicitTyestwiththeFatheadMinnow (Pimephalepsromelas) WU,DLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-102 STUDY COMPLETION: Jul@114,1999 ANffi'NDEDREPORT: April26,2000 ThisstudywasconducteidncomphancewithGood LaboratorPyracticSetmidardasspubUshedbytheU.S. EnvironmentaPlrotectioAngencyin40 CFR Parts160and792,17August1989;OECD PrinciploefsGood ,LaboratorPyracticeO,CDEIGD (92)32,EnvironmentMonograph No. 45, Paris1992;andJapanMAFF, 59 NohSan, NotificatiNoon. 3850,AgriculturaPlroductionBureau,10August 1984withthefollowinegxceptions: The testsubstancewas not charactmizeidn accordancewithRM GLP complianceh;owever,the charactmutaonwas performedaccordingto3M StandardOperatingProceduresand Methods,and allraw data arebeingmaintainedinthe3M archivesT.he testsubstanceisbeingrecharactmizeidnaccordancweithGLP. The stabiliotfythetestsubstanceunderconditionosf storageatthetestsitewas notdetmmiineidn accordancewithGood LaboratoryPracticeStandards. STUDY DIRECTO KurtP, Drottar SeniorBiologist SPONSOR APPROVAL: Sponsor DATE 7 T)XTE AMENDED WILDLIFE INTERNATIONAL LTD. -3- PROJECTNO.: 454A-102 QUALITY ASSURANCE STATEMIENT TlusstudwyasexammedforcomplianwciethGoodLaboratoPrryactiScteandaradsspublishbeydthe U.S.EnvironmentalProtectioAngency,40 CFR Parts160 and792,17 August 1989;OECD PrincipleosfGood LaboratorPyracticeO,CDE/GD (92)32,EnvironmentMonograph No. 45, Paris1992;andJapatMAFF, 59NohSan, NodficatioNno. 3850,AgriculturParloductioBnureau,10 August 1984. The datesof all mspectionsand auditsand thedatesthatany finchngwsere reportedtotheStudyDirectorand Laboratory Management were as follows: AC=Y: DATE CONDUCTED: TesStubstanRceparation Febnuuy 11,1999 Testinitiation February15,1999 Matnx Fmtfication Preparation FebniM 15,1999 WaterCh emistry Measurements February17,1999 BiologicaDlataand DraftReport March 17,1999 AnalyticaDlata and DraftReport March 22 - 24,1999 FinalReport July14, 1999 Amended Report April19 and 20,2000 DATE REPORTED TO: STUDYDHTECTOR: MANAGEhffNT: Febnuffy11,1999 February11,1999 February15, 1999 February16,1999 February15,1999 February18,'1999 FebrtfM 17,1999 March 17,1999 Febnu" 19,1999 K4imh 24,1999 March 24,1999 July14,1999 April20,2000 March 24, 1999 July14, 1999 April24,2000 TimodiyA. SpringerP,h.D. DATE Manager,RegWatoryand TechmcalSupport AMENDED WILDLIFE INTERNATIONAL LTD. -4- REPORT APPROVAL PROJECT NO.: 454A-102 SPONSOP- 3M Corporation NTLE: PFOS: A 96-HourStatiAccuteTomcityTestwiththeFadieadMmnow (Pimephales promelas) WELDLEFE INTERNATIONAL LTD. PROJECT NLTMBER. 454A-102 STUDYDIRECTO KurtR. Drottar SeniorBiologist MANAGENffiNT: Hemy O.Ua6gei,'@bD. M=tor, AquaticToodcologyand Non-TargetPlants DATE DA!t ANENDED WILDLIFE INTERNATIONAL LTD. -5. PROJECT NO.: 454A-102 TABLE OF CONTENTS Title/CovePrage.........................................................................I................ Good LaboratoryPracticCeomplianceStatemen.t..............................................2................. QualityAssuranceStatement...............................................................3................ ReportApproval..................... .......................4................ Tableof Contents.......................................................................5................ Summary ...............................................................................7............... Introducti.o.n..........................................................................8............... Objwfive..............................................................................8............... ExperimentaDlesip ................................................................:..........8.................... MaterialasndMethods...................................................................9................ Resultsand Discussion.....................................................?..................1..2.......................... Conclusions...........................................................................1.3............... Refamces ............................................................................1.4............... TABLES TableI -Summary ofAnalyticaClhemistryData..............................................1.5............... Table2 -Tempm-at=, DissolvedOxygen andpH ofWaterindieTestChambers....................1.6............... Table3 -CumWative PercentMortalitaynd Treaumt-RelatedEffect.s............................1.7............... Table4 -LC50 Values....................................................................1.8.............. WILDLIFE INTERNATIONAL LTD. -6- PROJECT NO.: 454A-102 TABLE OF CONTENTS Continued- FIGURE Figure1. Concentration-ResponCsuerve(96-HourData)......................................1.9................ APPENDICES Appendix I- SpecifiCconductanceH,ardnessA,lkalinitayn,dpH ofWellWater Measured Duringthe4-Week PeriodImmediatelyPrecedingtheTest..................2.0............... Appendix II- AnalysesofPesticideOsr,ganics,MeWs and OtherInorganics inWildlifIentmuationaLltd.WellWater.......................................2.1............... AppendixIII- The AnalysisofPFOS inFreshwateirnSupportofWildlife InternationLatld.ProjecNto.:454A- 102 .....................................................2..2.................... AppendixIV - Changes toProtocol..............................................................................4.0............... Appendix V - PersomelInvolvedintheStudy...............................................4.1............... AppendixVI - ReportAmendment ........................................................4.2............... AMENDED WILDLIFE INTERNATIONAL LTD. -7- PROJECT NO.: 454A-102 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCANON OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: WILDIME RfrERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: NEAN NEASURED TEST CONCENTRANONS: TEST DATES: LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANMM: AGE OF TEST ORGANBW: MEASUREMENTS OF 10 NEGATIVE CONTROL FISH: WEICTHT (g): TOTAL LENGTH (MM): 96-HOUR LC50: 95% CONFIDENCE Lnvm: NO MORTALITY CONCENTRATION: NO-OBSERVED-EFFECTCONCENTRATION: SUAUMLARY 3M Corporation Ms. SusanA. Beach WilMe InternationLatld. Easton,Maryland 21601 454A-102 PFOS (PerfluorooctanSeulfonicAcid Potassium Salt) PFOS: A 96-Hour StaticAcute ToxicityTestwith theFathead Minnow (Pimephalespromelas) NegativeControl,3.3,5.6,9.5,17 and 28 mg a.i./L ExperimentalStart- Fqbnuffy 15,1999 BiologicaTlermination- Febmary 19, 1999 ExperimentalTermination- Febmary 19,1999- 96 Hours Fathead Minnow (Pimephales promelas) Wll&ffe hitemationalLtcLcultures Easton,Maryland 21601 Juveniles Mean = 0.36; Mean = 35; Range = 0.21 to 0.49 Range = 30 to 39 9.5 mg a.i.AL 9.0and 11 mg &i./L 3.3mg a.iJL 3.3mg a.i./L AMENDED WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A- 102 DITRODUCRION Thisstudywas conductedby WildlifIentm-nationLatld.for3M CorporatioanttheWildlifhei.t=ational Ltd.aquauc toxicologfyacilitiynEaston,Maryland. The m-hfcphase of thetestwas conductedfrom Febmary 15 toFebnmry 19,1999. Raw datageneratebdy WildlifeInternationLatld.anda copy ofthefinal reportarefileudnderProjecNtumber 454A- 102 inarchivelsocatedon theWildlifIentm-nationLatld.site. OBJEc,nw .Theobjectivoefthisstudywas toevaluatteheacutetoxicitoyf PFOS (PerfluorooetaSnuelfoniAccid PotassiumSalt)tothefatheadminnow,Pimephalespromelas,duringa 96-hourexposureperioudnderstatitcest conditions. EXPERIMEENTAL DESIGN Fatheadminnows were exposedtoa geometricserieosffivetestconcentratioannsd a negative(dilution water)controlT.wo replicatteestchambersweremaintainedineachbwbnent andcontroglroup,with10f;lthead minnows ineach testchamberfora totalof 20 fatheadminnows per testconcentmfionN.ominal test concentratiownesre selecteidnconsultatiowniththeSponsor,and werebasedupon theresultosfan exploratory rangefindingtoxicittyest.Nominal testconcentratiosneslectewdere 3.6,5.9,9.9,16 and 27 mg active ingredien(tai.)ALM.e= measuredtedconcentratiownesre&tmmined fmm samplesofteswtatercollectferdom eachtreaunentand thecontrolgroup atthebegimmg of theum@ atapproximatel4y8 hours,and attest tmmiination. Fatheadmmnows were indiscriminatealsysignedtoexposurechan*crsattestimfiafioO[bLservationosf mortalitayndotherclinicaslignsoftoxicitwyere made atapproximatel2y.5,24,48,72 and96 hoursaftetrest initiatioCnu.mulativepercenmtortalitoybservedinthetreatmengtroupswas usedtomfimateorcalok&LC50 valuesat24,48,72 and 96 hours.The no mortalitcyoncentratioand theno-observed-effect-concentraUon (NOEC) were determinebdy visualinterpretatoifotnhemortalitayndclinicaolbservatiodnata. AMEMED WILDLIFE INTERNATIONAL LTD. -9- PROJECTNO.: 454A-102 MATERL4J,SAND METHODS ThesU* wasconductbeadsedontheproceduroeustlinientdheprotoco"lP,FOS:A 96"HouSrtatic AcuteToxiciTteystwiththeFatheaMdinnow(PimephalespromelTahse)p"r.otocwoalsbaseodnprocedures outlineidnU.S.EnvironmentaPlrotectioAngency Series850 - EcologicaElffectsTestGuidelinesO,PPTS Number 850.1075(1):OECD GuidelineforTestingofChemicals203: Fish,AcuteToxicitTyest(2);and I ASTM Standard E729-88a,@Sltandard Guide for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates andamphibians (3). Test Substance The testsubstancweas receivedfrom 3M Corporationon October29,1998 and was assignedWildlife InternationLatlcLidentificatniuomnber 4675. The testsubstancweas describeads a whitepowder. Itwas identifiaesdFC-95 fromlotnumber 217 (T-6295).Informatiopnrovidedby theSponsorindicateadpuritoyf 98.90/oa,nd an expiratiodnataof2008. The testsubstancewas reanalyzebdy theSponsorand theCertificaotfe AnalysisdatedMarch 9,2000 indicateadpurityof 90.490/oT.he testsubstancweas storedatambientroom temperature- PLeparatioonf TestConcentrations Nominal testconcentrations'we3r.e6,5.9,9.9,16 and27 mg &i./L,basedon atestsubstancpeurityof 90.490/oA.llmaterialwshich came intocontactwiththetestsubstanceduringpreparatioonftestconcentrations were constructeodfplastiocr stainlesssteel.A primarystocksolutiownas preparedindilutiownaterata concentratiofn27mg a.i./LT.he pfimarystocksolutiownas mixedwithanelectnrcm= forapproximate2l2y hoursto aidin thesolubdmbon of thetestsubstance.Aftermixm& theprunarystocksolutiownas proportionaldliylutewdithcwulaonwatertopreparethefouradditionatlestconcentrationAsl.ltestsolutions appearedclearand colorless. TestOrThe fatheadminnow,Pimephalespromelasw,as selecteadsthetestspeciefsorthissh*. The fiwrad mmnow isrepresentatiovfean importantgroupofaquaticvertebrateasnd was selectefdoruseinthetestbased upon pasthistoryofuseinthelaboratoryF.atheadminnows usedinthetestwereobtainedfromculmm at WildlifbeitmmtionaLltd.,Easton,Maryland. AMENDED WILDLIFE INTERNATIONAL LTD. -10- PROJECT NO.: 454A-102 Fatheadmmnows wereheldatapproxnnateltyhesame temperatureasused duringthetest.The fish were heldforapproximatel1y26 dayspriorto testing.The fishwere acclimatetdo testconditionfsor approxunatel4y8 hourspriortotestinitiatioDnu.ringtheholdingand acclimatiopneriodst,hefishshowed no signsof diseaseorstressD.uringthe 14-dayholdingperiodprecedingthetestw,atertemperaturersangedfrom 22.0to22.8*C.The pH ofthewaterrangedfrom 8.2to8.5anddissolveodxygenrangedfrom 7.7to8.4mg/L. instrumentatioand methods used forwatermeasurementsaredescribedin theEnwronmental Conditions sectionof thisreport.At testinitiatitohne,fadieadminnows were collectefdrom theacclimatiotnankand transferrteodthetestchambers. .Duringtheholdingperiod,fatheadmmnows werefeda commercially-prepardeidet The fishwerenot fedduringtheacclimatiopneriod(atleast48 hourspriortothetest)orduringthetesl Allfishusedinthetestwere fromthesame sourceandyearclassa,nd thetotalengthofthelongestflsh was no more thantwicethelengthoftheshorteslThe averagetotalengthof 10 negativceontrolM measured attheend of thetestwas 35 mm witha rangeof 30 to 38 mrn The averagewet weight(blotteddry)of 10 negativceontrolfishattheendofthetestwas 0.36grams witharangeof 0.21to0.49grams.Loadingwas 0.24g fish/Loftestwaterpresentinthetestchambersatanygiventime. TestAgparatus Testchamberswere 25-L polyethylenaequariacontaininagpproximately15 L of testsolutionT.he depthofwaterina representatitveestchamberwas approximately17.6cnx Testchamberswere impartially positioneidnan environmentaclhambersettomaintaina temperaturoef22-+2*C.The testchambersW= labelewdiththeprojecntumber,testcm=hmbm and replicate. DilutioWnater The waterusedforcidbirinagndtestinwgas freshwateorbtainedfrom a wellapproximatel4y0 meters deep locate(dm theWildlifbetemationalLtcLsite.The wellwaterischmwtmized asmoderately-harwdater. The specificconduct&=, hardnessa,lkalinitayn,d pH measurementsof thewellwaterduringthefour-week periodimmediatelyprecedingthetestarepresenteidnAppendix 1. WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454A-102 The wellwaterwas passedthrougha sandfiltetroremove particlegsreatedrm approximatel2y5 gm@ and pumped intoa 37,800-Lstorageumk and aeratedwithspraynozzles.Priortouse,thewateragainwas filtere(d0.45pm) toremovemicroorganismasnd particlesT.he resultosfperiodiacnalysesperformedto measuretheconcentratioonfs selectecdontaminantisnwellwaterusedby WildlifIenternationLatld.are presentedinAppendix Il. EnvirommcntalConditions Lightingused toilluminattehecultureasnd testchambersduringholdinga,cclimatioannd testinwgas providedby fluorescetnutbesthatemittedwavelengthsunilatro naturaslunligh(tColortone5e0). A photoperioodf 16 hoursof lightand8 hoursofdarknesswas controllewdithan automatictimer.A 30-minute transitipoenriodoflow lightintensitwyas providedwhen fightswent on and offto avoidsuddenchangesin lightingL.ightintensitayttestinitiatiwoans approximatel3y91 luxatffiscurfacoefthewater.Lightintensity was measuredusinga SPER ScientifLitcd.lightmeter. Temperaturewas measuredineachtestchamber atthebeginningof thetestand atapproxanately 24-hourintervaltshereaftuesringa liquid-in-gltahsesrmometer.Tempaium alsowas measuredcontinuousilny one negativecontrorleplicatuesinga FulscopeERIC Rerorder.The targetestemperatureduringthestudywas 22-E2*C.Dissolvedoxygen and pH measurementswere made on watersamplesfrom allreplicatestchambers ofeachtreatmenatnd controlattestinitiatiaonnd atapproximatel2y4-hourintervalfsfimafterH.ardness, alkaliniatnyd specificconductancewere measuredinthedilutiownaterattestuntiatton. MeasurementsofpH weremade usingaFisherAccumet Model 915pH meter,anddissolveodxygenwas measuredusinga YellowSpringsInstrumentModel 5 1B dissolveodxygenmeter.Specificconductancewas measuredusinga YellowSpringsInstrumenMtodel 33 Salinity-Conductivity-Tempermaett=er.Hm-cbm and allWuntymeasurementsweremade by titratiboansedon procedureisnStandardMethodsfortheFxamination of Waterand Wastewater(4). Observations Observationsweremade todetm-mmethenumber ofmortalitieTsh.e number ofmckvidualsexlnbiting clinicaslignsoftoxicitoyrabnormalbehavioralsowereevaluatedO.bservationwseremade approxim@ 2.5, 24,48,72 and96 hoursaftertestinitiation. WILDLIFE INTERNATIONAL L-m. -12- PROJECTNO.: 454A-102 StatisticAanlalyses The 24,48,72and 96-hourLC50 valuesandthe95% confide= intmalswm calculatwehden possible by probitanalysist,hemoving averagemethod orbinomialprobabilitwyithnon-lineairnterpolati(o5n,6,7) usingthecomputersoftwareofC.E.Stephan(8).Inthisstudy,LC50 valuescouldnotbe calculataetd24 and48 hoursdue tothelackof an adequateconcentration-respopnastewn, however,thepmbit method was usedto evaluatemortalitayt72 hoursand themoving averagemethod was usedtoevaluatmeortalitayt96 hours.1"hem mortalitcyoncentratioand 140EC were determinedby visualmtmpretationof themortalitaynd clinical observatiodnata. AgalyficaClhemi Water sampleswere collecteadtnud-depthfrom eachreplicatteestchamber of eachtreaunenatnd controglroupatthebeginningofthews@ at48 hoursand attesttmmimtion tomeasurecm=umfions ofthetest substance.The sampleswere collecteidnplasti(cNalgene(Dv)ialsand analyzedassoon aspossiblweithout storage.AnalyticaplroceduresusedintheanalysiosfthesamplesareprovidedinAppendix M. RESULTS AND DISCUSSION Measurement ofTestConcentrations Resultsof analym tomeasureconcentrationosfPFOS inwatersamplescollectedduringthetestare presenteidnTableI and intheanalyticcahlemistrryeport(AppendixIH).Nominalconcentratiosneslectefdor useinthissu* were 3.6,5.9,9.91,6 and27 mg &i.AL.Samplescollecteadttestinitiatihoandmeasuredvahies thatrangedfrom 85 to I17*/of nominalvalues.Measured valuesforsamplestakenat48 hoursrangedfrom 86 to 101% ofnominal.Measuredvaluesforsamplestd= at96 hoursrangedfrom88 to98% ofnominal. When measuredconcentrationosf thesamplesanalyzedattestinitiatioanp,proximatel4y8 hoursand attest tmmmtion were averagedt,hemean measuredconcentratiofnosrtlusSh* were 3.3,5.6,9.5,17 and28 mg a.i.ALM.ean measuredconcentratiownesre usedintheestimatioonrcalculationfLC50 values. Observationsand Measurements oftemperatured,issolveodxygm and pH -arepresenteidnTable2. Temperatureswcre withinthe22-+20Crangeestablishefdorthetest.Dissolvedoxygenconcenftfionrs=ained2:7.7mg/L(880/oof saun-atiotnh)roughouthetest.measurementsofpH rangedfrom 8.3to 8.6durmg thetest. ANIENDED WILDLIFE INTERNATIONAL LTD. -13- PROJECTNO.: 454A--102 Dailyobservationosfmortalitayndotherclinicaslignsoftoxicitoybservedduringthetestareshown in Table3. Fatheadminnows inthenegativecontrolandthe3.3mg ai.ALtreatmentgroup appearednormaland healthyduringthetest.After96-hoursofexposure,mortalitiynthe5.6,9.5,17 and 28 mg a.i./tLrleatment groupswas 20,50,80 and 1000/or,espectivelLyC.50 valuesand 95% confidencelimitsat24,48,72 and96 hourswere esbmted or calculatefdrom themortalitdyata,and areshown m Table4. A graphof the concentration-respocnusreveispresenteidnFigure1. CONCLUSIONS The 96-hourLC50 valueforfatheadmimows exposedto,PFOSwas 9.5mg a.i./wLith95% confidence limitosf8.0and 11mg ai./L.The 96-hourno mortalitcyoncentratiaond theNOEC were3.3mg a.i./L. AMENDED WILDLIFE INTERNATIONAL L-m. -14- PROJECTNO.: 454A-102 REFERENCES I U.S.EnvironmentalProtectioAngency. 1996. Series850 - EcologicaElffectTsestGuidelines (draftO)P,PTS Number 850.1075:FishAcuteToxicitTyestF,reshwaterandmarine. 2 OrganisationforEconomic Cooperationand Development 1993.OECD GuidelinefsorTestinogf Chemicals.Guideline203: Fish,AcuteToxicitTyest.AdoptedbytheCouncilon 12July1992. 3 ASTM Standard E729-98a. 1994. Standard Guidefor ConducdngAcute Toxidty TestswithFishes, Macroinvertebrates, andamphibians. American Society forTesting and Materials. 4 APHA, AWWA, WPCF. 1985. SttmdardMethodsfor theFxaminationof Water and Wastewater. ,16thEditiorLAmerican PublicHealthAssociatiorLAmerican Water Works Association.Water PollutioCnontrolFederationN,ew Yorl,- 5 Stephan,CX. 1978.U.S.EPA, EnvironmentaRlesearchLaboratoryD,uluth,Minnesota.Personal connnum on. 6 Fmney,D.J. 1971. StatisticalMethodsinBiologicalAssay.Secondeditiom GriffinPress,London. 7 Thompson, W.R. 1947.'BacteyiologicaRlewews. Vol.-IIN,o. 2. Pp. 11'5-145'. 8 Stephan, C.E. 1977. -Methods for Calculating an LC50," Aquatic Toiicologv and Hazard Evaluations,Americim SocietyforTestingand Materials.PublicationNumber STP 634, pp 65-84. WILDLIFE INTERNATIONAL LTD. -15- PROJECTNO.: 454A-102 Table 1 Summary ofAnalyticaClhemistryData Sponsor: Test Substance: TestOrganimn: DilutionWater: OM=MBI Test Concentration LMA ai. NegativeControl 3M Corporation PFOS Fathead Niinnow,Pimephales promelm Well Water Replicaid A B A B A B Sampling Time (Ho!,m) 0 0 48 48 96 96 Measured Concentration (maai.u <LOQ<LOQ <LOQ :LOQ <LOQ <LOQ 3.6 A 0 3.16 B 0 3.53 A 48 3.08 B 48 3.22 A 96 3.46 B 96 3.13 5.9 A 0 B 0 A 48 B 48 A 96 B 96 6.05 5.(Y7 5.48 5.89 5.70 5.55 9.9 A 0 8.99 B 0 9.47 A 48 9.98 B 49 9.33 A 96 9.70 B 96 9.52 16 A 0 18.2 B 0 19.3 A 48 15.0 B 48 15.6 A 96 14.9 B 96 16.2 27 A 0 28.5 B 0 28.5 A 48 27.0 B 49 27.8 A 96 26.8 B 96 26.6 The limitofquantitafi(oLnOQ) was 0.458mg ai./L. Mean Measured Concentration LUai.Q <LOQ 3.3 5.6 9.5 17 28 M==t Of Nominal 92 95 96 106 104 ANgRiDED p -16Table 2 Temperature,DissolvedOxygen and pH ofWater intheTestChambers Sponsor: 3M Corporation TestSubstance: PFOS TestOrpnisin: FatheadMinnow, Pimephalepsrotnelm DilutioWnater: WellWater Mean Measured 0 Hour TestConcentration Terne D03 (mg&i./L) Replicate (*C) (mgAL) pH NegativeControl A B 20.5 8.8 8.6 20.4 8.8 8.6 24 Hours TCMP (OC) 21.7 21.7 PO (mg/L) pH 7.8 8.5 7.8 8.5 48 Hours Temp DO (OC) (mg/L) pH 21.9 8.4 8.4 21.4 8.2 8.4 72 Hours Temp DO (OC) (mg/L) pH 22.1 8.2 8.4 21.6 8.0 8.4 3.3 A 20.5 8.8 8.6 21.7 .7.8 8.5 21.3 8.6 8.5 21.6 8.0 8.4 B 20.4 8.8 8.6 21.6 7.9 8.5 21.3 8.4 8.5 21.5 9.0 8.4 5.6 A 20.5 8.8 8.6 21.4 7.8 8.5 21.2 8.6 8.4 21.5 8.0 8.4 B 20.3 8.8 8.6 21.1 7.8 8.5 21.0 8.4 8.4 21.3 8.0 8.4 9.5 A 20.5 9.0 8.5 21.2 7.9 8.5 21.0 8.4 8.4 21.2 8.0 8.4 B 20.7 9.0 8.5 21.2 7.9 8.5 21.0 8.4 8.5 21.2 8.0 8.4 17 A 21.2 9.0 8.5 21.3 7.8 8.5 21.1 8.6 8.4 21.4 8.0 8.4 B 21.4 9.0 8.5 21.4 7.9 8.5 21.2 8.5 8.4 21.5 8.0 8.4 28 A 22.2 9.0 8.5 21.4 7.7 8.5 21.3 8.6 8.4 21.6 8.0 8.4 B 22.3 9.0 8.5 21.7 7.7 8.4 21.7 8.4 8.4 21.9 8.0 8.4 I'he0-hourdilutiownatermmmments forhudness.WW=ty and specificconductancewere 136 mg/L RS CaC03,178 mg/L asCaC03and 315 pmhos/cm, 2 Temperaturmealuuredcontinuousldyuringthetestrangedfi= approximtely20.5to22.OOC. 3 A dissolveodxygenconcentratioonf5.2mg/L represent6s00/osaturatioant22*C infie-shwater. -17Table 3 CumulativePercentMortalityand Treatinent-RelaWEdffects SPOMW- Ted Substam: Ted Orgafism: DilutionWdw 3M Corporation PFOS FathandMinnow, Pimephales promelas WellWea Mean Meamuvd Ted ConomUiSon (mg ILL/L) No. Replicdo EVo-d 2.5 Houm No. Dad' EffacW NegativeCoatrol A B 10 0 10 AN 10 0 10 AN 24 Hours No. Dead 0 0 Effects 10 A14 10 AN 49 Hom No. Dmd Effwb 0 10 AN 0 10 AN 72 Hours No. Dead Effects 0 10 AN 0 10 AN cunndative pac=t MMUW 0 3.3 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 5.6 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 9.5 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 17 A 10 0 10 AN 0 10 AN, 0 10 AN I 2AN-,7E 15 B 10 0 10 AN 0 10 AN, 0 10 AN 2 3AN;5E 28 A 10 0 10 AN 0 10 AN 0 10 AN 4 5E;IN 50 B 10 0 10 AN 0 10 AN 0 10 AN 6 3E-IN Cumulative mmiber ofdoad fok ObgavedEffo*: AN-AppemNoniW; E-Effaficxwhnming N-LA=ofaquilibriuaL 96 No. Dead 0 0 0 3 I 5 5 9 7 10 10 WILDLIFE INTERNATIONAL LTD. -18- PROJECT NO.: 454A-102 Table4 LC50 Values Sponsor: TestSubstance: TestOrgan'Lqm: DilutionWater: Time 24 Hours 3M Corporation PFOS FatheadMimow, Pimephalespromelas WellWater LC50 @2m a.i.2 Lower 95% Confidence Limits ai.2 >28 Upper 95% Confidence Limits a.i.2 Statistical Method VisualInterpretation 48 Hours >29 VisualInterpretation 72 Hour? 27 22 41 Probit 96 Hours 9.5 8.0 11 MLymS Average ConfidenceEmitscouldnotbe calculatewdiththemoftal4 dataobtained. 2 The usefidms ofthisLC50 isquestionablbeecauseaconcentration-effrecltationshwiaps.notdemonstrated overa reasonabl2eBLe (e.g.<,37 to>631 ofpercentdeacl AN[ENDED WILDLIFE INTERNATIONAL -19- Figure1. ConcmtratLon-ResponsCeurve (96-HourData) 8 7- 2 a. 6- 0 5E E 0 PROJECTNO.: 454A-102 10 100 Mean Measured Concentrabon(mg a.i./L) ANENDED WILDLIFE INTERNATIONAL LTD. -20- PROJECT NO.: 454A- 102 APPENDIXI SpecifiCconductanceH,ardness,AUmlinityandpH ofWell Water Measured Dmmg the4-Week Penod ImmediatelPy=Wmg theTest Sponsor. Test Substance: TestOrganisrw DilutionWater: 3M Corporation PFOS FatheadMimow, Pimephalespromelas WellWater SpecifiCconductance Ganhos/cm) mean 311 (N = 4) Range 310-315 Hardness (mg/L asCaCO3) 131(N = 4) 128-136 A&dinity (mgtlasCaCO3) 177 (N= 4) 176-178 pH 8.3(N = 4) 8.3 WILDLIFE INTERNATIONAL L-m. -21- PROJECTNO.: 454A.102 APPENDIX 11 Analym ofPestimdes,Orgames,Metalsand Otherlaorgamcs m Wddhfe IntemationaLltd.WellWaterl ANALYSIS NM"URIED CONCFZfrRATION MiscellaneousMeasurements TotalDissolvedSolids Animonia ToW Orgamilc=M 2 TotalC5inide 286 < 0.050 < 1.0 < 10.0 Orgomocb'o and PCBs AIZ@s aha BHBCHC DeltiiBHC Ganuna BHC (Undane) Chlordane DDDDED,, ppppI' DDT,pp' Dieldriti Pndo.qWfan,A Endasulfan,B EndosuffanSWfate Endrin EndrinAidehyde Hcpbwblor M6&oxychlor HeptachlorEpo)ddc Toxaphtne PCB@1016 PCB-1221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 < 0.005 < 0.005 < 0.005 < 0.005 < 0.006 < 0.025 < 0.006 < 0.005 < 0.008 < 0.005 < 0.005 < 0.005 < 0.018 < 0.010 < 0.005 < 0.005 < 0.007 < 0.005 < 0.500 < 0.260 < 0.260 < 0.260 < 0.720 < 0.720 < 0.720 < 0.720 Metalsand Other l.u1organk3 ,mutri@nyin Ar3miic B c Calcium Chro ' 3 co!7' co 3 mapesiur< Maiigancw' m 3 MO'.MMI@num Potassiur3g Sol * sil=p-m Sodi 3 M YM < 100 < 25.0 < 0.50 < 1.0 35.0 < 2.0 < 1.0 < 20.0 < 100 < 10.0 < 1.0 < 0.20 < 2.0 < 2.0 6.62 < 25.0 < 1.0 21.3 < 20.0 AnaPpm r Enviromnenbd, GainesvfflFcl,oridaforsamplescoU onWo-v-a'nlm- Analyses*fprmad b.y- VrddliflenftmationaLltd.forthesamplecoffeefoodn November 5,1997. A@Wy;ii performedby WddVe InWnatLorW Ltd.forsamplescollecteodn.November 5 #Lmgh 7, 1997. mg/L Mi/L mgtl 1"91L pg/-L AIVL mg/L mg/L AV-L ug/L Awi, 4/L pgt-L 41L AW-L 4/-L 4AL aiv-L 41-L ui/-L m@v-L mg/L AGIL A4-L UWL 4)-L juiv-L 4@L A4@-L 4/-L A4-1 mi/-L nliv-L 4t-L MFV-L A* -& 4/-L A4fyim m PV-L WILDLIFE INTERNATIONAL LTD. -22- APPENDIXM PROJFCT NO.: 454A-102 THE ANALYSIS OF PFOS IN FRESHWATER IN SUPPORT OF WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-102 WILDLIFE INTERNATIONAL LTD. -23- PROJECT NO.: 454A-102 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-HourStatiAccuteToxicityTestwiththeFatheadMinnow (Pirnephalepsrol@telas) WILDLEFE INTERNATIONAL LTD. PROJECT NO.: 454A-102 PRINCIPAL RNESTIGATOP- RayinendC Van Hoven,Ph.D. Scientist MANAGENIENT: WillardB. Nixon,Ph.15. Manager,AnalyticaClhemistry DATE DAT;:;' AMENDED WILDLIFE INTERNATIONAL LTD. -24- PROJECTNO.: 454A-102 Introduction Freshwatersampleswere collectefdrom a statiaccuteaquatictoxicitsytudydesignedto determinethe effectosf PFOS (PerfluorooctaSnuelfonicAcid PotassiumSalt)to thefatheadminnow (Pimephales promelas).Tlusstudywas conductedby WildlifheftmationalLtd.andidentifiaesdProjecNto.: 454A102. The analysesof dme water sampleswere performedat WildlifIenternationLatld.usinghigh performanceliquidchromat*Vhy with mass spectrometrdiectectio(nHPLC/MS). Sampleswere receivedforanalysiosn February15,17 and 19,1999 and were analyzedon eachsamplereceiptday. TestSubstanceand InternaSltandard The testsubstanceused forthe analyticaplortionof thisstudywas WildlifeInternationLatld. identificatniuomnber 4675. The testsubstancewas usedto preparecalibratioand matrixfortification samples. The internasltandardwas receivedfrom 3M Corporatioann July2, 1998 and was assignedWildlife InternationLatld.idenfificatniuomnber 4526 upon receipt".lbeinternasltandarda,.granulamratmial,was idenfifiaesd: IK 1K 2K 2H PerfluorooctmSeulfoniAccid,ChemicalAbstracNtumber: 27619-97-2. The standardwas storedunderambientconditions. Ang.)IicaMlethod The method used fortheanalysisofthe firshwatesrampleswas developedatWildlifeInternational Ltd.and entitle"dAnalyticaMlethod fordm Determinatioonf PFOS inFreshwaterS,altwatera,nd Algal Medium". Thismethodologywas includeadsAppendixIlofWildlifIentematoinalLtd.protocolnumber 454/011299/MVAIJSUB454. Itwas basedupon methodologyprovidedby 3M Corporation. Samples were dilutedin a 50% methanol: 50% NANOpureg water solutiocnontaining 0.100mg4HPFOS (bitemasltandard)/aLnd 0.05% fonnicacid(v/v)so thattheyfellwithinthe calibratiornangeofthePFOS methodology. Concentrationosf the PFOS in the standardsand sampleswere detemiinedby reverst.-phahsiegh perfbrmanceliquidchromatographyusinga Hewlett-PadmrdModel 1100 I-ligPherformanceLiquid Chromatograph(H]?LC)witha Perkin-Ehm API 10OLC Mass Spectrometeerquippedwith a Peridn- WILDLIFE INTERNATIONAL LTD. -25- PROJECTNO.: 454A-102 Eh= TurboIonSpmy ion source.BPLC separationwsere achievedusinga Keystme BetasilCis analyticcaollurrm(100 nun x 2 I.D.,3 pm particlseize).The instrumenptarametersaresununarized inTable 1.A method flowcharitsprovidedinFigure1. CalibratioCnurve and Lmat of Calibratiosntandardso:tlPFOSpreparedin a 50% methanol: 50% NANOpure(D watersolution containin0g.100 mg 4H PFOS (internasltandard)AaLn,d 0.05% fonnicacid(vtv)r,anginginconcentration from 0.00915to 0.0457 mg a.iJLwere analyzedwiththesamples.Linearregressioenquationswere generatedusingpeak arearesponseratio(sPFOS :mt=al standardv)ersustherespectivceonomb-ations of thecalibratisotnandardsA. typicalcalibraticounrveispresenteidnFigure2. The concentrationf PFOS inthesampleswas dewnninedby subsfitifitnhgepeak arearesponseratiosintotheapplicablIemear regressioenquation.Representativieonchromatogramsoflow and lughcalibratiosntandardsarepresented inFigures3 and 4,respectively. Mm method limitofquanfi=on (LOQ) fortheseanalyseswas setat0.458mg a.i.AcLalculateadsthe productof thelowestcalibratisotnandardanalyzed(0.00915mg ai.AL)and thedilutioEnwWr of the matrixblanksamples(50.0). MatrixBlankandForfificatSi=oInes Threematrixblanksampleswere analyzedtoddermme possiblienterferencNeo. mter&mm were observedat or above theLOQ duringsamplesanalyses(Table2). A representaticvheromalograronf a matrixblankispresentedinFigure5. Freshwaterwas fortifieadt0.915,9.15and 45.7mg a.i./aLnd analyzedconcurrentlwyiththesamples to&tenninethemean procedurarlecovery(Table3). Sample concentratiownesre notcorrectefdordw mean procedurarlecoveryof97.9%. A representaticvheromatog= ofa matrixfortificatiisopnresented inFigure6. AMENDED WILDLIFE INTERNATIONAL LTD. -26- PROJECTNO.: 454A-102 E&wnple Calculations Samplenumber 454A-102-27,nominalconcentrationf 3.6mg a.i./iLnfreshwater. InitiVaollume: 0.250mL FinalVolume: 25.0mL DilutioFnactor:100 PFOS Peak Area: 359685 InteniaSltandardPeakArea: 415648 PeakArea Ratio:0.865 Calibratiocnurveequation. Slope:0.023 Intercept0:.062 Curve islinear. PFOS (jiag.i./La)tinstrumentPeakarearati-o -inter t Slope' PFOS (mg a.i./Li)nsample PFOS (gga.i./La)tinstnunenxtDilutioFnactor 1000 Calculatecdoncentratio3n.:46mg a.i./L Note:manual calculatisomnay chffer. PercentofNominalConc. PFOS (mg a.i./]iLn)sMRIe x 100 PFOS (mg a.i.ALn)ominal Calculatedrecovery9:6.9% Note:manual calculatimoany differ. AMENDED WILDLIFE INTERNATIONAL LTD. -27- PROJECTNO.: 454A-102 RESULTS S=le AgWysis Freshwatersampleswere collectefdromtheacutetoxicitsytudywiththeffitheamdinnow (Pimephales promelasa)ttestinitiatiFoenb,ruary15,1999 (Hour 0),on February17,1999 (Hour 48),and attest terminatioFne,bniary19,1999 (Hour 96).The measuredconcentratioonfsPFOS inthesamplescollected at initiatioofnexposureof thetestorganisms(Hour 0) rangedfrom 85.3to 117% of the nominal concmtrationsS.amplescollecteadtHour 48 had a measuredconcentratiornangeof 86.3to 101% of nominalvalues.Samplescollecteadttestenninafio(nHour 96)had a mmured concentratiornangeof 87.6% to98.3% ofnominalvalues(Table4).A representaticvheromatogramofa testsampleisshown in Figure7. AUURQDED WILDLIFE INTERNATIONAL LTD. -28- PROJECT NO.: 454A-102 INSTRUNM'NT: TableI TypicalBPLC OperationaPlarameters Hewlett-PackarMdodel 1100 High PerfomianceLiquid Chromalographwitha Peridn-EhneArPI IOOLC Mass Spectrometer equippedwitha Perldn-EhneTrurbolmSpray ionsource.Operatedin selectiivoenmonitorinmgode (SM. ANALYTICAL COLUMN: OVEN TEMPERATURE: KeystoneBetasiCllgcolumn (100mrn x 2 mm I.D.,3 pm particle size) 300C STOP TIME: 10.0minutes FLOW RATE: 0.220mljminute MOBELE PHASE: 72.0% Mcdianol:28.0% NANOpureM Water conwning 0.1% FomiicAcid INJECT10N VOLUME: 50.0liL PFOS RETENTION TIME: INTERNAL STANDARD RETENTION TIME: ApproxiinaWly7.1minutes Approximately4.9minutes PFOS MONITORED MASS: RQTERNAL STANDARD MONITORED MASS: 498.6qrm, 426.7amu WILDLIFE INTERNATIONAL LTD. -29- PROJECTNO.: 454A-102 Table2 MatrixBLmks AnalyzedConcurrentlDyuringSampleAnalysis Number (454A-102-) NIAB-1 S=le TMX MatrixBlank Measured Concentratioonf PFOS' (mga.i./L) < LOQ NIAB-2 NL-ttdxBlank < LOQ MAB-3 MatrixBlank < LOQ ne limitofquantitati(oLnOQ) was 0.458mg ai./Lbasedupon theproductofthelowestcalibration standar!d2!!Z!L4(0.00915Mg a.i@Ljandthedilutiofnactoorfthematrixblanks=les (50.0). AMEMED WILDLIFE INTERNATIONAL LTD. -30- PROJECTNO.: 454A-102 Table 3 Matrix FortLficatioAnnsalyzedConeunwdy DuringSample Analysis Sample Number (454A-102-) MAS-1 MAS-4 MAS-7 ConcentrationsofPFOS LM.9a..i.2 Fortified Measured 0.915 0.915 0.915 1.03 0.842 0.970 Percent Recovered 113 92.0 106 MAS-2 @-5 MAS-8 9.15 8.65 94.5 9.15 8.75 95.6 9.15 8.74 95.5 MAS-3 45.7 48.3 106 MAS-6 45.7 37.6 82.2 MAS-9 45.7 44.0 96.2 Mean 97.9 StandardDeviation 9.12 CY 9.32 -N 9 Note: Resultsand correctionfsornew tea substancepuritywere generatedusingMacQuan version1.5 softwareand manual calculationVsa.lues havebeenroundedforrepo!@M LuM2=-. ANEENDED WILDLIFE INTERNATIONAL LTD. -31- PROJECTNO.: 454A-102 Table4 Measured ConcentrationosfPFOS inFreshwaterSamples from a FatheadNtmow StatiAccuteToxicityTest Nominal Test Concentration (ME a.i./L) 0.0 Sample Number (454A-102-) 1 2 13 14 25 26 Sampling Time (Houg) 0 0 48 48 96 96 PFOS Measured Concentration' (mZ a.i./L) < LOQ < LOQ < LOQ < LOQ' < LOQ < LOQ Percent of Nominal - 3.6 3 0 4 0 15 48 16 48 27 96 28 96 3.162 3.53 3.08 3.22 3.46 3.13 88.6 .98.9 86.3 90.3 96.9 87.6 5.9 5 0 6 0 17 48 18 48 29 96 30 96 6.052 102 5.07 85.3 5.48 92.2 5.89 99.0 5.70 95.8 5.55 93.5 9.9 7 0 8 0 48 20 48 31 96 32 96 8.992 89.3 9.472 94.0 9.88 97.8 9.33 92.8 9.70 96.3 9.52 94.3 Note:Resultsand correctionfsornew testsubstancepuritywere generatedusingMacQuan version1.5 softwareand manual calculationsV.alueshave been roundedforreportingpurposes. 'The limiotf quantitati(oLnOQ) was 0.458mg a.i.AbLasedupon theproductofthelowestcalibration standardanalyzed(0.00915mg a.i.ALa)ndthedilutiofnlictorfthematrixblanksamples(50.0). 2Mean resulotfduplicatreedilutioonfstheoriginaslample. Duplicateresult(sinparenthesesf)orthefollowin4g54A-102 samplenumbersare:3 (3.07and 3.25), 5 (6.11and 5.99),7 (9.01and 8.96)8, (9.70and 9.24)and 14 (<0.459and <0.458).The original resultfsorthesesamples(4.44,7.98,21.2,2.21,and 0.74,respectivelayr)enot includedin the statistiacnaallysiosfthedatadue to suspectdilution(se.g.contamination). AMENDED WILDLIFE INTERNATIONAL LTD. -32- PROJECT NO.: 454A-102 Table4 (Continued) Measured Concentrationosf PFOS inFreshwaterSamplesfrom a FatheadMinnow StatiAccuteToxicityTest NominalTest Concentration (TZa.i./L) 16 Sample Number (454A-102-) 9 10 21 22 33 34 Sampling Time QLOU-rs) 0 0 48 48 96 96 PFOS Measured Concentration' (M a.i.L 18.2 19.3 15.0 15.6 14.8 16.2 Percent of Nominal 110 117 90.9 94.5 90.2 98.3 27 11 0 28.5 104 12 0 28.5 23. 48 27.0 98.4 24 48 27.8 101 35 96 26.8 97.6 36 96 26.6 97.0 Note:Resultsand correctionfsornew testsubstancepuritywere generatedusingr&cquan version1.5 softwareandmanual calculationVsa.lueshave been roundedforreportinpgurposes. 'Tlielimitofquantitati(oLnOQ) was 0.458Mg a.i.AbLa,sedupon theproductofthelowestcalibration standaradnalyzed(0.00915mg a.i.ALa)nd thedilutiofnactorofthematrixblanksamples(50.0). 2Men resulotfduplicatreebutionsoftheoriginaslample. Duplicatreesult(sinparenthesesf)orthefollowin4g54A-102 samplenumbers are:3 (3.07and 3.25), 5 (6.11 and 5.99)7, (9.01and 8.96)8, (9.70and 9.24)and 14 (<0.458and <0.458).The original resultfsorthesesamples(4.44,7.98,21.2,2.21,and 0.74,respectivelayr)enot includedin the statistiacnaallnisofthedataduetosuspecdtilutioLnse-sc.onta@2nJ. AMENDED WILDLIFE INTERNATIONAL L-m. -33- PROJECTNO.: 454A-102 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FPJESHWATER PreparmeatrifxortificastaimopnlebsyspddngtherequisivtoelumeofPFOS stocksolutiodnisrectly mto fiwhwaterusinggas-tlgbstyringeasnd ClassA volumetncflasks. Ddutematnx fortificatainodntestsamplesintotherangeofthecalibratisotnandardbsy pwtauy filling ClassA volumetrifclaskswith50% medmnol :50% NANOpure(D watersolutiocnontaining 0.100mg 4H PFOS (fiftmasltandard)/aLnd0.05% fonnicacid(v/v).Add theappropriatveolumeof sampleand bringtheflasktovolumewiththedilutiosnolventP.roms thematrixblanksampleusing thesame dilutioannd aliquovtolume asforthelowestfortificatlieovnel.Mix wellby severarlepeat inversions. AmpuL-ttesamplesand submitforLCMS analysis. Figure1. Analyticamlethod flowcharftortheanalysiosfPFOS infi-.eshwater. WILDLIFE INTERNATIONAL LTD. -34- PROJECTNO.: 454A-102 1.20- 1.00- Q80 - 0 0 ce 0.60 0.40- 0.20 0 QOO 0.000 0.010 Q020 Q030 Q040 Concer&atim (mg ai/L) 0.050 Figure2. A typicaclalibraticounrveforPFOS. Slope 0.023;Intercept0.062;r 0.996 AMENDED WILDLIFE INTERNATIONAL -35- PROJECT NO.: 454A.102 100. go. 807060- intensit)8r361 cps 169 4& 30- 148 20- 2 94 iii ol 3*1 61 * 91' * 'lil lii le'l 2ii OF Scan 1.30 2.55 3.81 6.06 6.32 7.57 8.83 Time Figure 3. A representativieon of a low-level(0.00915mg a.i./LP)FOS sludud. AMENDED WILDLIFE INTERNATIONAL -36- PROJECT NO.: 454A-102 10()90, 80. 7060so, 4 3020, 10. 1 0. intensfty2:9671 cps 169 147 24 53AP 7RRA 10-8; 124 Pig 31 i@i q'i iii 151 181 2i I Sm 1.30 2.55 3.81 5.06 6.32 7.57 8.83 Time Figure4. A representatiivoen ofa high-lev(e0l.0457rngai./L)PFOS standard. ANEMED WILDLIFE INTERNATIONAL LTD. -37- PROJECTNO.: 454A-102 I0()r go. 80, 7060. 50r 40302010. 0. 26 4656 91 75 31 61 91 .1.30 2.55 3.81 intenstty4:26 cps 131 174 k 228 121 1 1 181 211 Scan 6.06 6.32 7.57 8.83 Time Figure 5. A representative of a matrixblanksample (454A- I02-MAB-3). The arrow indicatedsm retentio6n= ofPFOS. WILDLIFE INTERNATIONAL LTD. -38- PROJECTNO.: 454A-102 100, 90. 80. 70r 6050, 403& 2010'. A intensftyi:3084 cps 170 148 20 42 31 1.30 ;1;! 4na 125 61 91 iii 2.55 3.81 5.06 iii 6.32 404 200222 iii 7.67 2il 8.83 Scan nme Figure 6. A representative of a matrixfortificatisoanmple (454A-102-MAS-7). WILDLIFE INTERNATIONAL LTD. -39- PROJECT NO.: 454A-102 to(). 9 ()P. 8 70- 60- so. 40- 30- 20@ 10. 26 An o. F;---T=. 3'1 1.30 intensity2:3428 cps 170 148 SA 6'1 2.55 AA ln3 9*1 3.181 125 lil 5.06 lil 6.32 11 7.57 pis 2ii 8.83 Scan Time I I Figure 7. A representativcehiamatogram of a testsample (454A-102-27). WILDLIFE INTERNATIONAL LTD. -40- PROJECTNO.: 454A-102 APPENDIX IV Changes toProtocol Tlusstudywas conductedm accordancewiththeapprovedProtocolwiththefohowmg changes: I. The protocowlas amended toaddtheproposedoq)mimentalstaratndtmmunationdates,sb* room,test substa= informatioanndtestconcentrations. 2. The protocolwas amended toremove annualfeedanalysis. 3. The protocolwas amended toremove thecalculationf an incipienLtC50 value. 4. The protocolwas amended tospecifytheanalyticmaelthocl WILDLIFE INTERNATIONAL L-m. -41- PROJECT NO.: 454A- 102 APPENDIXV PersonneIlnvolvedintheSu* The followmgkeyWildlifIenternationLatldLpemmel weremvolvedm theconductormanagementoftbis study: 1. Henry0. Krueger,Ph.D,PirectorA,quaticTo)dcologyandNon-TargrtPlants 2. WillardB.Nbwn, Ph.D.,Manager,AnalyticaClhemistry 3. Mark A. Mank, LaboratorSyupavisor 4. TimothyZ.KendalLLaboratorySupovisor 5. KurtI;LDrottarS,eniorBiolo& 6. Raymond L.VanHoven,Ph.D.,Scientist WILDLIFE INTERNATIONAL L-rD. -42- PROJECTNO.: 454A-102 APPENDIX VI ReportAmendment 1 . OriginaRleport: Pages 1-4,6 and 23 Amendment: The pageswere changed toincludet,heamended reportdate,revisepdagenumbers,and new signatureasnd datesdue totheadditioonfthereportamendment asAppendix VI. Reasow. To reflecttheissuingof an amended reporl 2. OrigiiWReport: Page 2 Amendment: The compliancestatementwas revised. Reason: To clm* how thetestsubstancewas charactedzecl 3. OriginaRleport: Page 9 Amendment: Informatiopnrovidedby theSponsorreflectitnhgereanalysiosf thetestsubstance, includintghereanalysidsateand thepurityw,as addedtotheTestSubstancesection. Reason: To reflectthecurrentestsubstanceinformatiopnrovidedby theSponsor. 4. Ongmd ReporL Entirereport Amendment: AU testsubstanceconcentratiownesre changedtor6flect'tphueritoyfdw testsubstance asdeterminedby dw Sponsorina reanalysiosfthetestsubstance(FC-95,Lot 217). Testconcentratioonrsiginalwleyre basedon thereportepdurityof 98.9%. The certificaotfeanalysidsatedMarch 9,2000 indicateadpuritoyf90.490/ol.bereforeasll test substanceconcentrationisn,cludingnominal concentrationmse,asured concentrationasn,d LC50 valuesw,ere recalculateadnd reporteadsmg a.i./bLasedon the90.4V/opurity. Reason: To reporttheresultosf the.tesbtased on thetestsubstancepurityof 90.49% at-the requestof theSponsor. AMENDED WILDLIFE INTERNATIC)NAL LTD. -43- APPENDIX VI -Continued- ReportAmendment PROJECT NO.: 454A-102 ANENDNENT SIGNATURES: KurtR-Drottar " 7/ Henry'Oq*uoier'P,IL-b' Dweetor,AquaticToxicologyand Non-TargetPlants REVIEWED Y: TimothyA. Spd;g"aP,-h.D. QualityAssurance dIlOZ6100 DATE @ DATE DATE AMENDED WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-102 Page I of 2 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOLTR STATIC ACUTE TO)UCITY TEST WITH THE FATHEAD MINNOW (Pimephalespromelas) PROTOCOL NO.: 454/110998/FAT-96HI/SUB454 AMENDMENT NO.: I SPONSOP,-. 3M Corporation PRO.TECT NO.: 454A-102 EFTECTIVE DATE: December 21, 1998 AMENDAMNT: Page 2 Add: ExperimentalStartDate: 2/15/99 ExperimentalTerminationDate: 2/19/99 Test Concentrations:Negative ControL 3.9,6.5,11, 18 and 30 mg a.i./L Test SubstanceNo.: 4675 REASON: The above informationwas not known when the protocolwas.signedby the Study Director. AMIENDMIENT: TestOrganis Page 5 D elete: Feed providedto the fishwillbe analyzedat leastonce annuallyto ensurethatthere are no contaminantsatlevelsknown tobe capableofinterferinwgith the study. REASON: lestoricaalnalysesof WildlifeInternationaLltd.aquaticfeedhave shown thatno contaminantsare presentatlevelsknown tobe capableofinterferinwgith thestudy. AMEENDMEENT: Data Anal@sis Page 8 Change: The LC50 valuewillbe calculatedw,hen possibleu,singmortalitydatacollectedat24, 48, 72 and 96 hours,as wellasthe incipienLtC50. To: The LC50 valuewillbe calculatedw,hen possibleu,singmortalitdyatacollecteadt24,48, 72 and 96 hours. REASON: The incipienLtC50 cannotbe determinedina 96-hour staticacutetest. a45411021anwndl WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454A-102 Page 2 of 2 AMENDMENT: APPENDIX IE[P,age 13 Add: Liquid Chromatography Mass Spectrometry (LCMS) Method for the Determinationof PerfluorooetaneSulfonicAcid, Potassium Salt(PFOS) inFreshwater,FilteredSaltwaterand Algal Medium. REASON: To add the analyticamlethod to be used in the study. STUDY DIRECTOR DATE LABUF&TOIV AAI@-AGEMENT a. ,A SPP40 --S,O1-wR0S@'-sS:; RIF-PR SENTATIVE DAA ---- @- I I s i DA a454l]O2iamndl PROTOCOL PFOS: A 96-HOUR STATIC ACUTE T03UCITY TEST WITH THE FATHEAD MINNOW (Pimephalespromelas) U.S.EnvirormentaPlrotectioAngmq Series850 -EcologicaElffectsTestGuidelines OPPTS Number 850.1075 and OECD Guideline203 3M Lab RequestNo. U2723 WILDLIFF- Submittedto 3M Corporatim EnvironmentalLaboratory P.O. Box 33331 St PmiL Mhmesota 55133 INTF.RNATIONAL. 8598 Commerce Drive Easton,Maryland 21601 (410)822-8600 November 9, 1999 LTD.. PROTOCOL NO.: 454/110998/FAT-96HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -2- PFOS: A 96-HOUR STATIC ACUTE T03aCITY TEST WITH THE FATHEAD MINNOW (Pimephalespromelas) SPONSO 3M Corporation EnvironmentalLaboratory P.O. Box 33331 St Paul,Minnesota 55133 SPONSOR!S REPRESENTATIVE: Ms. Susan A. Bewh TESTING FACILITY: WildlffcInternationLatld. 8598 Commerce Drive Easton,Maryland 21601 STUDY DIRECTO KurtDrottar SeniorAquaticBiologist LABORATORY MANAGEMENT Henry 0. Krueger,Ph.D. DirectorofAquaticToxicology& Non-TargetPlants FOR LABORATORY USE ONLY Proposed Dates: Experimental StartDate: ProjectNo.: L464,4 Test Concentrations: Test SubstanceNo.: Experimental TanninationDate: Refemce SubstanceNo. (ifapplicable): PROTOCOL APPROVAL STUDY DIRECTOR IcAlql? e DATE LABORATORY MANAGEMENT 2!rni SP'ONSOR!S REPRESENTATIVE DATEF I /1 -D-A-1-i PROTOCOLNO.: 454/110998/FAT-96HI/SU]3454 3M LAB REQUEST NO. U2723 VILDLIFE INTERNATIONAL LrD. -3- INTRODUCFION WildlifeInternationaLltcLwillconducta staticacutetoxicittyestwith thefatheadminnow (Pimephalespromelas)forthe SponsorattheWildlifeInternationLatld.aquatictoxicologyfacilitiyn Easton,Maryland. The study willbe performedbased on proceduresin the US. Environmental ProtectionAgency Series850 - EcologicalEffectsTest GuidelinesOPPTS Number 850.1075 (1); OECD GuidelineforTestingof Chemicals203:Fish,Acute ToxicityTest(2);and ASTM Standard E729-88a StandardGiddeforConductingAcute TojdcityTestswithFishes,Macroinvertebratesand Amphibians (3).Raw dataforallwork performedatWildlifelatcmationaLltd.and a copy of thefinal reportwillbe filedby projectnumber in archiveslocatedon theWildlifelatcmatimal LtcLsite,or at an alternativleocationtobe specifieidnthefinalreporl PU"OSE The purpose of thissh* isto determinethe acuteeffectsof a testsubstanceon the fathead minnows (Pimephalespromelas) duringa 96-hourexposureperiodunder statitcestconditions. EXPERIMENTAL DESIGN Faffieamdinnows willbe exposed toa geometricseriesofatleastfivetestconcentrationasnd a negative(dilutiownater)controlfor96 hours.Two replicatteestchambers willbe maintainedineach ftatnent and controlgroup,with 10 fidrad mkmows ineachchamber fora totalof 20 faffieamdinnows per testconcentratiorl Nominal testconcentrationwsillbe selectedin consultatiownith the Sponsor,and willbe based upon informationsuch astheresultsofexploratoryrange-findintgoxicitydata,known toxicitdyata, pbysical/chenucaplropertieosf thetestsubstanceor otherrelevantmformatiom Targetconcentrations need not exceed 1000 mg/L orthesolubilitlyimitofthetestsubstanceinwater(whicheverislower). Generally,each testsubstanceconcentratiounsed in thedefinitivteestvaR bc higher concentratiounnlessinformationconcerningthe concentradon-effe differmt chlufaonfactorwould be more appropriate.Water samples fi-om collectedat specifiedintervalfsoranalysisofthetestsubstance.Resultst calculatemean measured testconcentrations. To controlbias,fatheadminnows willbe impartiallayssignedto imt,at,mlNo offiapotrntiaslourcesofbiasareoq=W toaffectheresult, of mortalityand odm clinicaslignswillbe made throughoutthe96-hour test PROTOCOLNO.: 454/110998/FAT-96HI/SUB454 3M -;,QUEST NO. U2723 $WILDLIFE INTERNATIONAL LTD. -4- mortak observedinthetreatmentgroups willbe used to calculatew,hen possible,LC50 valuesat24, 48,72 and 96 hour intervalsT.he no-mortalitcyoncentrationand the no-observed-effecctoncentration (NOEC) willbe determined. MATERIALS AND METHODS Test Substance Informationon thecharactmizationof test,controlor referencesubstancesisrequiredby Good LaboratoryPracticSetandards(GLP). The Sponsor isresponsiblfeorpmvidmg WildlifeInternational Ltd.writtenvmficatim thatthetestsubstancehas been charwter@ accordingto GLPs priorto itsuse inthestudy.Ifwrittenvenficafamof GLP testsubstancecharactmmiaon isnot providedtowildlife InternationaLltd.,itwillbe notedinthecompliancestatementofthefinalreport.The attachedform IDENTMCATION OF TM SUBSTANCE BY SPONSOR (Appendix I)istobe used toprovide mformattm necessaryforGLP compliance. The Sponsor isresponsibleforallinformationrelatedto the testsubstanceand agreesto ac=pt any unused testsubstanceand/ortestsubstancecontainersremaining attheend of thestudy. P=aration ofTestConcentrations The testsubstancewillbe administeredtothetestorganismdirectliyntodilutiownater. This routeOf on was selectedbecause itreprwmu themost likelyrouteof exposureto aquatic organisms. TestOrgom The fidmd nifimaw(P@nwphalepsromelas)hasbeenselecteadsthetestspeciefsorthissb*. Fad=d minnows arerepresentatiovfean m4xrtmt group ofaquaticvertebratesa,nd have been selected foruse inthetestbased upon pastuse histm in thelaboratory.Fish willbe from thesame sourceand year class,and thestandardlengthofthelmgest fishmeasured willbe no more d= twicethatofthe shorteslFishwillweigh between 0.1 g and 3.0g each and the totalweight ineach testchamber willnot exceed0.8vmw fish/Lof solutionT.otallengft and wet weightsof the individuaflishinone negative controlreplicatweillbe measured attheend of thetestand willbe consideredrepresentativoef thelength and weightof allfishused in thestudy.Fish willbe obtainedfrom a commercial supplieror hatchery, and theidentitoyf thespecieswillbe vmifiedby thesupplieror by WildlifeInternationaLltd.persomel usingappropriatetaxonomickeys,such asEddy (4). PROTOCOL No.: 454/110998/FAT-96Hl/SUB454 3M LAB REQUEST NO. U2723 IWILDLIFE INTERNATIONAL LTD. Fadieadminnows willbe heldforatleast14 days priorto the testinwater from the same source and atapproximatelythe same temperatureas used duringthetest Any changes in water temperature willnotoxmd 3*C perday. Fatheadminnows willbe heldatthetesttemperaturefora minimum of7 days priorto testinitiationF.athead minnows willnotbe used inthe testiftheyshow signsof disease or stressor ifmore than 5% dieduringthe48 hours priortothetest At testinitiatiotnh,efathead mmnows willbe corertedfrom holdingor acclimatiown" and transferretdothetestchambers. During theholdingperiodffitcestfishwillbe fed atleastonce daily.The dietwillconsistof live orfrozenbrineshrimp nauplii(Artemiasp.),and/orcommercial food. Fish willnot be fedfor atleast two days priorto thetestor duringthetest.Feed providedto thefishwillbe analyzedatleastonce amiuallyto ensurethatthereareno contaminantsatlevelsknown to be capableof interferinwgith the study. Specificationfsoracceptablelevelsof contaminantsin fishdietshave not been establishe& However, thereareno known levelsof contaminantsreasonablyexpectedto be presentin thedietthat areconsideredto interferweith fficpurpose or conductof the test DilutionWater Water used forthe culturinagnd testingof fatheadminnows willbe obtainedfrom a well approximftly40 metersdeep locatedon theWildlifeintmuationalLtd.site.The waterwillbe passed dirougha sand ffitearnd pumped intoa 37,800-L storagetankwhere the waterwillbe aeratedwith spray nozzles.Priorto usethewaterwillbe filteretdo 0.45 @= inordertoremove fineparticlesW.ater used formdftumg and testingischmzdm=d as moderatelyharcl Typicalvaluesforhardness,a&almity,pH and specificonductanceareappro=mwly- Hardness,mg/L as CaC03 Alkalinity,mg/L as CaC03 pH SpecificConductance,kmhos/cm 145 190 8.1 330 Hardness, alladinitpyH, and specificonductancewillbe measured weekly to monitor the consist@ ofthewellwater.Me= and rangesofthemeasured parametersfordw four-weekperiod precedingthetestwillbe providedinthefinalreporl Analyses willbe performed atleastonce annually to determinethe concentrationosf selectedorgmue and inorganicconstituentosf thewellwater and resultsofthe analyseswillbe summarized inthefmal reporl PROTOCOL NO.: 454/11099&/FAT-96HI/SUB454 3M LAB REQUEST NO. U2723 IWILDLIFE INTERNATIONAL LTD. -6- Test Apl2aratus Testdiamberswillbe 25-L,polyethyleneaquariafillewdithapproximately15 L ofwater.Test chambers willbe positionedin an environmentalchamber or temperature-controllweadter bath to maintain a temperatureof 22 d:2*C. Testchambers willbe labelledwith theprojectnumber, test concentrationand replicate. EnvironmentalConditions Ligbbng used toilluminattehecultureasnd testchambers dunng holding,=I=ation, and testing willbe providedby fluor=ent Um dud enutwavelengthsskrulatonaturalsunlight(e.g.C,olortonelD 50).A photopum of 16hoursoflightand 8 hoursof darkwillbe controllewdith an automatictimer. A 30-mmute transitipoenriodoflow lightintensitwyillbe providedwhen lightsgo m and offtoavoid sudden changes in lightintensityL.ightintensitwyillbe measured attestinitiatiwointh a SPER ScientifiLctcLlightmeter orequivalent The targetesttmnpcmturewiu be 22 1 20C. Temperature willbe measured allreplicateastthe beginningof the testand at approximately24-hour intervalfsl=caft6rusing a liquid-in-glass thermometer. Temperature alsowillbe measured with a conti@uousrecorderin one negativecontrol chamber. Recorder measurements willbe verifiedwith a fiquid-in-glatshsermometerpriorto test initiation. Dissolvedoxygen willbe measured in allreplicateosf the treatmentand controlgroups attest initiatioannd atapproxitnat* 24-hourintervaltshereafteursinga Yellow SpringsInstrumentModel 51B dissolvedoxygen mew, or equivalmitIntheeventthatdissolvedoxygen levelsfallbelow 60% saturationa,ppropriateactionswillbe takenafterconsultatiownith the Sponsor. Measurements of pH willbe made inallreplicateosftheftabnent and controlgroupsattestinitiatiaond atapproximately 24-hourinterval&s=after usinga Fisha Accumet Model 915 pH meter,oreqmvalent.Ifa ftatment' group reaches 10(r/omortalityd,issolvedoxygen,piL and temperaturemeasurements willbe taken at thenod samplingfiiwval,thendisconfinued. Hardness, dkabmty, and specificonductancewin be measured inthedlutlonwater attest untiation-Hardness and allW=tY measurements willbe made by titratiounsingproceduresbased on meffiodsinStandardMethodsfor theExamunanon of Water and Wastewater (5). Specificconductance willbe measured usinga Yellow SpringsksMm3ent Model 33 Sdinity-Conduedvity-Ternperatumreter, or equivalent. PROTOCOLNO.: 454/110998/FAT-96Hl/SUB454 3M LAB REQUEST NO. U2723 OWILDLIFE INTERNATIONAL LTD. -7- Biolop-icaMleasurements Observationosfmorwity and clinicaslignsoftoxicitwyillbe made between0-24 hours,and at 24,48,72 and 96 hours I hour.Lethalitiysdefinedas thelackofvisiblmeovement (e.g.lackoffin oropmzdar movement) inthefishaftergentleprodding.AR clinicaolbservationisncludingabnormal behaviorwillbe noted. S&=ling forAn"cal Measurements Water sampleswillbe collectefdrom each testchamber atthebeginningof thetestd,uringthe uA and attheendofthetesttodet=ine concentrationosfthetestsubstance.Intheeventthat1000/o mortalitoyccursinany treatmm@ thensamplingofthattreat3newnitllt=mte followintghenext sampling interval.Samples willbe collecteadtmid-depthfrom each testchamber and analyzed immediatelyorplacedinan appropriatsetoragecontah= (e.g.p,olyethylenoerpolypropylenebottle) and storedunder refrigerafiuonntilanalyzed.The sample scheme issummarized below: PROPOSED NUMBERS ExperimentalGroup Control SolventControl(ifnwded) Level 1-Low Concentration Level2 Level3 Level4 Level5-HighConcentratim Totals OF VEREFICATION SAWLES 0 Hours 48 - Hours 2 2 2 2 2 2 2 2 2 2 2 2 2 2 14 14 96 Hours 2 2 2 2 2 2 2 14 TotalNumber ofVerificatioSnamples 42 The abovenumbersofsmwles rqxwmt thoseconectedfrom thetestand do not mclude quality control(QC) samplessuch as matrixblanlcasnd fmtificatiopnrseparedand analyzedduringthe analytca&lMmstry phaseofffiscft* At thechsmvftonoftheStudyDffoctorw,atersamplesfrom one Ormore aPPropnatetestchambers willbe collectedand analyzedifan analyfacaml-rorm samplmg or analysiissMsPectei The reasonfordc additionaslampleswillbe describedby dw Study Directorand documented m theraw dataand finalreport PROTOCOLNO.: 454/110998/FAT-96Hl/SUB454 3M LAB REQUEST NO. U2723 IWILDLIFE INTERNATIONAL LTD. And.)IilCahlemiM Chemical analysiosfthesamplesva be performedby WildlifeInternationaLltd. The analytical method used willbe based upon methodology providedby the Sponsor and identifieidnAppendix II. The methodolou used to analyzethe testsamples willbe documented in theraw dataand summarized inthe finalreport. DaQ An*lil When the dose-responsepatternallowscalculatioonf an LC50 value,thedata willbe ana4,md usingthecomputersoftwareofC.E. Stephan(6).Ile program was designedto calculattehe LC50 value and the95% cmfidence intervably probitanalysist,he moving averagemethod, or binomialprobability vathnonfinewintaplatim(7,8,9)T.he LC50 valuew.illbe calculatedw,hen possibleu,singmortality datacollecteadt24,48,72 and 96 hours,as wellas theincipientLC50. The no-moaaw concentration and theno-observed-offecctoncentratio(nNOEC) willbe determine& RECORDS TO BE MARAAMB RecordsW be maintainedfordaM generatedby WildlffeInternationaLltd.willincludeb,utnot be limitetdo: 1 - A copy of the signedprotocol. 2. Identifa'catainodn charactmizationof thetestsubstance,ifprovidedby the Sponsor. 3. Dates ofinitiatioand tmmiinatioonf thetest 4. Test organism historyh,oldingand acclimationrecords. 5. Sto& solutioncalculatioanndpreparationf,fapplicable. 6. Observations. 7. Water chemistryresultr(.e.g.W,kalinityand hardness). 8. The methods used to analyzetestsubsw= concentrationasnd the resultsof mudytical Luements. 9. SMfisticaclalculationsi,fapplicable. 10. Testconditims(lighitntensitpyh,otopmiod,dc.). II. Calculationand preparationof testconccnbvfions. 12. Copy of finalreporl PROTOCOLNO.: 454/110998/FAT-96HllSUB454 3M LAB REQUEST NO. U2723 !WILDLIFE INTERNATIONAL LTD. -9- FINALREPORT A finraelpoorftthreesulotftshesb* wilblepreparbeyWddMe InternatLitodn.Tahlereport willincludeb,utnotbe limitetdo,thefollowingw,hen applicable: I. Name and addressofthefacilipteyrformingthestudy. 2. Datesqm wlnchthestudywas initiataenddcompleteda,nd thedefinitievxeperimentasltartand terminatiodnates. 3. A statementofcompliancesignedby theStudyDirectoraddressinagny exceptiontso Good LaboratoryPracticSetandards. 4. Objectivesand proceduresa,s statedintheapprovedprotorolincludinagllchangestothe protocol. 5. The testsubstanceidentificatinocnludinngame,chermed abstid nmnber orcodenumber, strengthp,urityc,ompositiona,ndothermformafam providedby thesponsor 6. Stabilitayndsolubiliotfythetestsubstancuendertheconditionosfadministratiofnfp,rovided by theSponsor. 7. A descriptioonfthemethodsusedtoconductthetest 8. A descriptioonfthetestorganismsi,ncludintghesource,scienti:nfaimce, age or lifestage, lengthsand weightsof a representatigvreoup oftestorganisms,feedtypes,lighitntensitaynd. photoperiod. 9. A descriptioonfthepreparatioonfthetestsolutions. 10. The methods usedtoallocatoergm3iS=to testchambersand beginthetestt,henumber of organism and chamberspertreatmenta,nd thedurationofthetest. 11. A descriptioonfcircumstancetshatmay have affeew thequalitoyrintegfitoyfthedata. 12. Ilm nam oftheSu* Dkectorandthenames ofotherscientistpsr,ofessionalasn,dsupervisory personneilnvolvedinthesu*. 13. A descriptioonfthetmdormations,calculationasn,d operationpserformedon thedata,a summary and mm4sis ofthebiologicdaaltaand analyticaclhemistrydata,and a statementofthe conclusiondsrawn from theanalyses. 14. Statisticmaelthods usedtoevaluatethedata. 15. A graphoftheconcmtration-mortalciutryveattheendof thetesl 16. The signedanddatedreportosfeachof ft individuaslcientisotrsotherprofessionailnsvolved inthestudy. 17. The locatiownhere raw dataandfinarleportaretobe stored. 18. A stawnienptreparedby theQualityAssuranceUnitlistintghedatesthatstudyinspectionasnd auditwseremade andthedatesof any findingrseportedtotheStudyDirectorand Management. PROTOCOLNO.: 454/110998/FAT-96Hl/SU]3454 3M LAB REQUEST NO. U2723 L@'WILDLIFE INTERNATIONAL L-rD. -10- 19. U itisnecessaryto make correctionosradditionsto a finalreportafterithas been accepted,such changeswillbe made intheform ofan amendment issuedby theStudyDirector.The amendment willclearliydentiftyhepartofthefinalreporthatisbeingamended andthereasonsforthe amendment,and willbe signedby theStudyDirector. CFIANGING OF PROTOCOL Plmled changestotheprotocolwillbe intheform ofwrittenamendments signedby theStudy DirectoarndtheSponscesRepresentativeA.mendments Willbe considereads partof theprotocoland willbe aff3d'etdothefinaplrotowl AW otherchangeswillbe intheform ofwrittendeviationssigned by theStudyDirectoarnd filedwiththeraw data.AU changestotheprotocowlillbe indicateidnthe finarleport. GOOD LABORATORY PRACRICES Thisstudywillbe conductedinaccordancweithGood LaboratoryPracticSetandardsforEPA (40CFR Part160 and/orPart792);OECD PrinciploefsGood LaboratorPyractic(eOCDF,/GD (92) 32, Enviro=ent Monograph No. 45);and JapanMAFF (59 NohSan,NotificatiNoon. 3850, AgriculturParloductioBnureau).Fach sU* conductedby lwddhfeInternationLatld.isroutinely examined by theWildlifehitem&onalLtd.QualityAssuranceUnit forcompliancewith Good LaboratoryPracticesS,tandardOperatingPmccduresand the specifiepdrotocol.A statementof compliancewithGood LaboratoryPracticewsillbe preparedforallportionsofthestudyconductedby WildlifehitmmationaLltd. The SponsorvnU be responsiblfeorcompliancewithGood Laboratory Pn@ forP=e&m perfmiedbyotberWxratories(e.g.r,esiduaenalyseosrpathology)R.aw data forallworkpuftmed atWildlifIent=&mal Ltd.anda copyofthefinamlpaftwillbe filebdy projed number in archivelsocatedon theWildlifIentmmadonalLtcLsiteo,ratan dtrmativelocatiotnobe specifieidnthefindreport. PROTOCOLNO.: 454/11099&/FAT-96Hl/SUB454 3M LAB REQUFST NO. U2723 WILDLIFE INTERNATIONAL LTD. REFERENCES I U.S.Environmental ProtectionAgency. 1996. Series850-EcologicalEffectsTest Guidelines (draft)O,PPTS Number 850.1075: FishAcute ToxicityTest,Freshwater andmatine. 2 Organisation forEconomic Cooperation and Development 1993. OECD Guidelinesfor TestingofChemicals.Giddeline203:Fish,Acute ToxicityTest.Adopted by theCouncilon 12 July 1992. 3 ASTM Standard E729-88a. 1994. Standard Guidefor Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, andamphibians. American Society for Testing and Materials. 4 Eddy, S. 1974. Yhe FreshwaterFi'shesW.m. C. Brown Company Publishers,Dubuque, Iowa. 5 APHA, AWWA, WPCF. 1985. Standard Methods for the Examination of Water and Wastewater. 16th Edition,American Public Health Association. American Water Works AssociatiorLWater PollutionControlFederationN,ew York. 6 Stephan, C.E. 1978. U.S. EPA, EnvironmentalResearchLaboratory,Duluth,Nlinnesota. Personalcommunicatiorl 7 Thompson, W.R. 1947. BacteHological ReWews. Vol. IL No. 2. Pp. 115-145. 8 Stephan, CX. 1977. "Methods for Calculating an LC50,"Aquatic Toxicology andhazard Evaluations. American SocietyforTestingand Niatmials. Publication Number STP 634, pp 6584. 9 Finney, D.J. 197 1. StatisticaMlethods in BiologicalAssay. Second edition.GfiffinPress, London. PROTOCOLNO.: 454/1109991FAT-96HIISUB454 3M LAB REQUEST NO. U2723 TWILDLIFE INTERNATIONAL LTD. -12- APPENDIX I IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR To be Completed by Sponsor I. Test SubstmiceIdentit(yname tobe used inthereport):PFOS (PerfluorooetanSeulfonicAcid Potassium Sa ReferenceStandard(ifapplicable)A:nalyticaSltandard: N/A InternaSltandard: 1,1.22H.H-H-H PerfluoroortanSeulfonicArid Test Substance Sample Code or Batch Number: Lot 217 Test Substance PurityCO/oActiveIngredient): 98.9 ExpiratioDnate: 2008 11. Test Substance Charactmization Have theiden#ty,stm d compositionor othercharacteristics which appmpriatelydege4=bstance and referencsetandardbeen detmmiffi6Odriorto itsuse inthisstudy inaccordancewith GLP Standards? Yes No X ni. Test Substance StorageConditions Pleaseindicatetherecommended storageconditionsatvrddlifeInternationaLltd. Ambient room I=erature Has the stabilitoyf thetestsubstanceunder@tmhez:sdess?torageconditions been determineiiinaccordancewith GLP S Yes No X Other pertinentstabilitiynformation: IV. Test Concentrations: Adjust testconcentrationto 1000/oa.i. x based upon the purity(0/og)ivenabove. Do not adiusttestconcentrationto 1000/0 a.i.TestthemateriaAlM. V. ToxicityInformation: Mammalian: Rat ILD50 251 mg/kg - Mouse LD50 N/A Aquatic: InvertebraTt(e"ddV (EC/LC50) FishToxicity(LC50) Diggli@amagm: 27 nrL Rainbow Tmut: 11 miyJL Dapbnia magon- 50 mgl Fathead Minnow* 38 mprJL Other ToxicityInformation(includinfgindingsofchronica,nd subau.0lictests): - Pleasesee MSDS PROTOCOLNO.: 454/110998/FAT-96HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -13- APPENDIX ]I AnalyticaMlethod tobe Providedby Sponsor PROTOCOL NO.: 454/110998/FAT-96Hl/SUB454 3M LAB REQUEST NO. U2723