Document OJE8J3jmx6gLv66dbergZNN1j

ARYIG-00T sros AN EARLY LIFE-STAGE TOXICITY TEST WITH THE FATHEAD MINNOW (Pimephales promelas) FINAL REPORT WILDLIFE INTERNATIONLTADL,PROJECTNUMBER: 4544-105 3M LAB REQUEST NO. U2723 GFPTS Number 850.1400 U.S Environmental Protection Agency Series 850 Ecological Effects Test Guidelines (OECD Guideline 210 AUTHORS: HeyK,t.KeDeost,anPAD STUDY INITIATION DATE: July 2, 1999 STUDY COMPLETION DATE Ape 26,200 `Submitted to Environ Labs 3M Corporation 935 Bush Avenue. St. Paul, Minnesota 55144 Wildlife International, Ltd. t1o59n%,Caommamna D2i16v0e1 page tar2 (410) 822-8600 00307 WILDLIFE INTERNATIONAL Lo. -2- PROJECT NO.: 454A-108 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: An EarlyLifeStage Toxicity Test with the Fathead Minnow (Pimephalespromelas) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-108 STUDY COMPLETION: April 26,2000 US. Ea"vTihriosnsmteundtyalwaPrsoctoencdtiuocnteAdgiennccoympinli4a0ncCeFwRitPhaGrtoso1d6L0aabonrda7t9o2r,y1Pr7aAcutgiuesStta1n9d8a9r;dsOaEpCuDblPirsihnecdipblyesthoef `AgGroiocdulLtaubroarlaPtroordyuPcrtaicotnicBeur(eEaNu,V/10MACu/gCuHstEM19(8948,)wi1t7h and Japan MAFF, 59NohSan, the following exceptions: Notification No. 3850, charTahcetteeswrtsaiusbzpseatratfnocireomewndaascncoortdcihnagrtacot3erMiSzteadindnaarcdcOopredraantcienwgPirtohcfeudulrleGsLaPndcoMmeptlhioadnsc,ea;nhdowsellvrearw,dtahtea arebeingmainitnthae3iManrcheivdes.The estsubstanceisbeingrecharacterizedinaccordancewith GLP. `Thestabilityofthetestsubstance underconditions ofstorageatthetestsite Wasnotdeterminedin accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: JZ KurtR Drottar Seaior Biologist R VAL: 5 yfaloo DATE La % -- 0603898 WWIILLDDLLIIFFEEIINNTTEERRNNAATTIIONAALLLLmm.. ~~ PPRROOJJEECCTNNO..:445544AA-1088 3. QUALITY ASSURANCE STATEMENT `Thisstudywasexaminedfor compliancewith Good LaboratoryPractice Standardsaspublishedbythe USS. Environmental Protection Ageninc4y0CFRParts 160 and 792, 17 August 1989; OECD Principles of AgGroiocudlLtaubroarlPartoordyucPtriaocntiBcuer(eaEuN,V1/0MACu/gCusHtEM198(49.8)Th17e)d;aatnedsoJfapaalnl iMnAspFeFct,io5n9s aNnodhSaaudni,tsNaotnidftichaetdiaotneNsot.h3a8ta5n0y, findingswerereportedtothe StudyDirector andLaboratory Managementwereasfollows: AcvITy: TotSubstance repanion AMSenaeasulrpyetmueSnoturrsTnnetptdiIiEnngtgDogaO,ionn Obsertons ADrvffaReypaorDtet nd DaBicnlogRieepaloDrtt 0d Fi Report "DATECONDUCTED: August 2,1999 DATEREPORTED TO: STUDYDIRECTOR: ~~ MANAGEMENT: Aug, 1999 Auguns, 1999 Auge, 1999 September, 1999 August, 1999 Sepemters, 1999 December,64347, 1999 December, 1969 Jauary 35,2000 AI2A425,2000 Janay 2000 Apei26,2000 Augus, 1999 September 10,199 December 15,1999 : Juasy7,2000 Apil26,2000 Marshall&, Hynson Quality Assurance Program #4. DATE 724 000399 WILDLIFE INTERNATIONAL Lo. " REPORT APPROVAL PROJECT NO.: 454A-108 SPONSOR: 3M Corporation TITLE: PFOS: An Early Life-Stage Toxicity Test with the Fathead Minnow (Pimephales promelas) `WILDLIFEINTERNATIONAL LTD. PROJECTNUMBER: 454A-108 STUDYDIRECTOR: om KSeunritorRB.iDorlootgtiasrt : MANAGEMENT: J 44 Henry O. r, PAD. Director, Aquatic Toxicology and Non-Target Plants. Fer DATE Y2tfec DA 001000 WILDLIFE INTERNATIONAL Lo. -5- `TOA FCB ONTLENE TS PROJECTNO: 454A-108 REPO ADDIOVAL sss TableOFCOMEDcNoS erst 4 --. TABLES Table 1- Summary ofAnalyChEtMSitrcy al DBA........ovwwssmmssnmssisssmmmmmmmsss 11 Table2- Specific Conductance,Hardness and Alkalinity ofthe Test SOIHONS............... --1 Table3- pHofWaterintheTest Chambers............o sneneremensmsssnsssesermrenernens 1 Table 4- Dissolved OxygenContent (mg/L) of Waterinthe Test Chambers.... ------ 01601 WILDLIFE INTERNATIONAL Lm. te PROJECTNO.: 454A-108 TABLE OF CONTENTS - Continue-d TABLES (Cont'd) Table - Temperature (C) OfWate i the Test CRAIETS vr 21 `Table 6 - Hatching Successof FatheadMinnow EmbryosExposed 10the Test SUbSLADGE ror mmm------ `Table 7 -Survivalof FatheadMinnowLarvaeExposedtotheTest Substance: FRIED PIRI R csmmmmmesmsmmmmmsm-- smn 3h Table 8-TotalLength,WetWeightandDryWeightofFatheadMinnowLarvae attheEndofthe 42-Day Post-Hatch Observation Period... 2 APPENDICES : Appendix 1- Dilution Water Chemistry Parameters MeasuredDuringthe `4-Weck PeriodImmedPrieceadintgtehely Test... 25 Appendix I~ AnalysesofPesticides,Organics,MetalsandOtherInorganics ~~ - ny WildlifeInternational Lid. WellWBE... 26 Appendix 1. TIhenAtnaelyrsinsLoatfdt.BPFiROOSoJiEncntaNFoil.e2hw4ae5rin4SupApor-of1Wi0de8....vs 21 Appendix IV- CumulativePercentMortalityandTreatment-ReElSRaCtteSd rv 41 Appendix V= FishTotal Legh(I)... 36 Appendix VI- FishWet WEIght (Mg). 61 Appendix VII- FishDry Wei(g18)ht rvs 66 Appendix IX- PersonnelInVOIVedi he SOY... 2 001002 WILDLIFE INTERNATIONAL Lm. 7. PROJECT NO.: 454A-108 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OFSTUDY, RAW DATAAND ACOPYOFTHE FINALREPORT: SUMMARY 3M Corporation Susan A. Beach `Wildife International Lid. Easton, Maryland 21601 LTD, PROJECT NUMBER: TEST SUBSTANCE: sTupy: NOMINALTEST CONCENTRATIONS MEANMEASUREDTEST CONCENTRATIONS: TESTDATES 454A-108 PFOS (Pecfluorooctane Sulfonic Acid Potassium Salt) PFOS: An Early Life-Stage Toxicity Test with the Fathead Minnow (Pimephalespromelas) NegativeControl, 014,029,0.57, 11a,nd 42.6m.ga3L. NegContarol,t0.15i,0.3v0,0.e60, 12, 2.4 and 4.6 mgaL. EBioxlopgiecalrTierSmmtianerattn-ioAtnuga-uSsletpt5,em1b9e9r921, 1999 Experimental Termination-September22, 1999 LENOTH OFTEST: 47Dy B . TEST ORGANISM: SOURCE OFTEST ORGANISMS: AGE OFTEST ORGANISMS: FathcadMinnow(Pimephales promelas) `WEialsdtioinf,eMIanrteyrlnaantdion2a1l6L0i1d. Cultures Eggs 24hoursoldat test initiation. "CNOON-COEBNSTERRAVTEIDO-ENF:FECTCLOONWCEESNTT-ROABTSIEORVNE:D-EFFECTMATOXXIIMCAUNMT ACOCNCCEEPNTTARBALTEION 030mgaill 060mg ai/l 042 mgai/l 01603 WILDLIFE INTERNATIONAL Lm. -3- PROJECTNO.: 454A-108 INTRODUCTION "ThisstudywasconductedbyWildlifeInternational Ltd.for 3M CorpatotherWildalifteIntiemaotionnal Ltd aquatiofatciliotyiinEcastoonl,Moaryglanyd.Thein-life phaseofthe testwascondfuromcAtugeusdt, 1999toSepte2m1,b1e99r9. Ravw datageneratedbyWildifeInternationalLtd.and acopyofthefinalreportare. FedunderProjectNumber454A-108inarchiveslocated ontheWildifeIntemationalLtd.site. v OBJECTIVE "Theobjectiveofthi studywastoevaluatetheeffects ofPFOSoncarlylife-stagedevelopmentofthe: fathead minnow, Pimephalespromelas. Titomhatech,hatching success, survival andgrowthwereevaluated duthre47i-danystugdy, EXPERIMENTAL DESIGN Fatheadminnowembryoswereexposed to ageometricseriesofsixtestconcentrationsand anegative. (dilutionwater)controlunder flow-throughconditions.Fourreplicatetestchambersweremaintainedineach reaantdthemconetronlgrotup.Eachtestchambercontainedoneincubationcupwith 20embryos, sultingina totalof 80embryos pertreatment.Theexposureperiodincluded a 5-dayembryo hatchingperiod,and a 42-day `posthatch juvenilegrowthperiod.The Sponsorrequestedthatthestudybeextendedpastthenormal 28-day `posthatchgrowthperiodtodetermineif survivorshipwouldbeaffected. `Nominaltestconcentrationswereselectedi consultationwiththe Sponsor, endwerebaseduponacute: toxicitydata. Nomina testconcenwetrre0a.14t,0i.2o9,n0.5s7, 11,23and4.6mgactive ingredient (3.1. Meanmeasuredtestconcentrationsweredeterminedfromsamplesoftestwater collectedfromeachtreatmentand {thecontrolgroup tthebeginning ofthetest,onDay4, atweeklyintervalsduringthetest,andattesttermination. Tostartthetest,embryosless than24hoursoldwerecollectedfromculturesandgroupsof 1and 2 individualswereimpartiallydistributed among incubationcupsuntilcachcupcontained 20embryos.Onecup asthenplacedincachtreatmentandcontro testchamber. Twiceduringthenexttwentyhou-rsfanodduilry thereafter,alldeadembryoswerecountedandremovedfrom thecupstoavoidcontaminatingviableembryos.All 01004 WILDLIFE INTERNATIONAL Lo. -9- PROJECT NO.: 454A-108 gs that remained were considered viable. Dead embryos continued to be removed daily. After hatching, the larvaewerecountedandreleasedintoth testchambers,whereexposurecontinueduntilesttermination. Observationsofmortalityandotherclinicalsigns weremadedailyduringthetest. Titomhatech, hatchingsuccess,growth,andsurvivalweremonitoredi eachtreatmentandcontrolgroup. Datawereevaluated to determinetheno-observed-efect-concentration (NOEC), the lowest-observed-effect-concentration (LOEC) andthe maximumscceptabletoxicantconcentration (MATC). MATERIALS AND METHODS "Thestudywasconductedaccordingtothe proceduresoutlinedintheprotocol, "PFOS: AnEarlyLifeStageToxicity Testwiththe Fathead Minnow (Pimephalespromelas)". Theprotocol was baonsproecedudres outlined in U.S. Environmental Protection Agency Series 850 ~ Ecological Effects Test Guidelines, OPPTS `Number 850.1400 (1); OECD Guidelinefor TestingofChemicals, 210: Fish Early-Life Stage Toxicity Test (2); ASTM Standard 1241-88, SandardGuidefor ConduEarclytLifie-nStaggeToxicityTests with Fish 3) andStandardEvaluation Procedure, Fish EarlyLife-Stage Test (4). TestSubstance `Thetestsubswtaasrenceciveedfrom 3MCorpoornaOcttoibero29n, 1998andwasassignedWildlife InteLtdm. idaentitficaitiononumnbear46l75.Thetestsubstancewasdescribedas awhitepowder. Itwas identified as FC-95 from lot number 217 (T-6295). Information provided by the Sponsor indicatedapurity of 90.49%, and an expiration daoft200e8, The test substance was stored at ambientroomtemperature. `Preopf Taesrt Caonctentiratoionns Onestocksolution waspreparedforeachofthe ixconcentrationstested. Apristomck saluiron wyas preparedbydissolving PFOSindilutionwaterat aconceatration of 88.4mga.i/L.Thepristmockasorlutiyon seasstirredwithanelectri stainlesssteel top-downmixeruntilalthetestsubstance vsdissolved.Theprimary stocksolutionwasproportionallydilutedwith dilutionwatertopreparefivesecondarystocksolutions at concentroaft44i.o2,ns 2.1, 11.0, 5.52and2.76mg a../L.Stocksolutionswere preparedeverythre tofour daysdurtih ntesgt.Thefivestockswereinjectedntothedilutesmixingchambers (at a ateof 6.0mLminute) 01005 WILDLIFE INTERNATIONAL Lm. -10- PROJECT NO: 454A-108 `wheretheyweremixedwithdilutionwater(at arateof 116mL/minute)toachievethedesiredtestconcentrations. `Theresultanttestconcentrationswereadjustedforpurityoftheactiveingredientinthetestsubstance (90.49%). TestOrganism Thefatheadminnow, Pimephales promelas, wassclectedas the test speciesforthis study. Fathead `minnowsarerepresentativeofanimportantgroupofaquaticvertebratesandwereselectedforuseinthestudy `based uponpasthistoryofuse and ease of culturing inthelaboratory.Fatheadminnowembryos used inthistest originated fromcultures maintainedbyWildlifeInternationalLtd., Easton,Maryland.The embryoswere removedfromspawningsubstratesandexaminedunderthe dissectingmicroscope to selecthealthyspecimens at `apprtho e saxmei stamgea of dteveeloplmenyt. Embryos collected for useinthetestwere from six individual `spawnsandwere lessthan 24hours oldwhenthetestwasinitiated. `Newly-hatched larvae were fedlivebrine shrimpnauplii (Artemia sp.) 3timesper dayduring the first 7 dayspost-hatch.OnDays 8through 40post-hatch,allfish werefed livebrineshrimpnauplii 3timesdaily on `weekdaysand2times dailyon weekends.Brineshrimpnaupliiwereobtainedby hatchingcystspurchasedfrom `Bon Artn emieaInvteri natl ional lIne c.,SaltLakeCity,Utah. Fishwerenotfedforatleast 48 hourspriortothe terminationof thetesttoallow forclearanceof thedigestivetractsbeforeweightmeasurementsweremade. To ensure thatthefeeding rateperfishremained constant,rationswere adjusted each week fo accountforlosses due. to mortality. : The averagewet weightof16fishinthenegativecontrol(Replicate A) measuredatthe endofthe test was 0.16g. Biomass loading (thetotalwet weightofthe fishperliter oftestwater) atthe end ofthe test was 0.062gof fishperliteroftestwaterthat passedthrough hetestchamber during a 24-hourperiod.Instantaneous loading(thetotalwetweightoffishperliterofwaterinthetank)attheendofthetest was 0.37goffishperliter oftestwater. TestApparatus Acontinuous-flowdiluter wasused to deliver cach concentration of the test substance and a negative: (dilutionwater)control. Aperistalticpump (Cole-ParmerInstrumentCompany)wasusedtodeliverthesixtest `subsstotckasolnutcioensintomixingchambersassignedtocachtreatmentgroup. Thestocksolutiwoenres. diluted withwell waterin themixingchambers inorder to obtainthe desired testconcentrations. Theflow of 010606 WWIILLDDLLIIFEIINNTTEERRNNAATTIIONALLLom.. BIR PPRROOJJEECTNNOO..:445544AA-10088 dilution watertothemixingchambers wascontrolledbyrotameters. Rotwaeremcaliebrattedperiorrtotsest initiation andatweeklyintervalsthereafter.Theflowoftestwaterfromcachmixingchamber wassplitand allowedtoflowintofourreplicatetestchambers. Theproportionofthetestwaterthatwassplitintocach replicate wascheckedpriortothetest,andatweeklyintervalsthereafiertoensurethat flow ratesvariedbyno morethan +10%ofthemeanofthefourreplicates. `Thediluterwasadjustedsothateachtestchamberreceivedapproximately6.0volumeadditionsofest `watereve24hrourys. Thetestsubstancedeliverypumpwascalibratedbeforethetestand atweeklyintervals thereafter.Thegeneraloperationofthediluterwascheckedvisuallyatleasttwotimesperdayduring thetestand atleastonceat thebeginningand endofthetest. `Thetest chamberswere9-L glas aquariafilledwithapproximately 7Loftestsolution.Thedepthofthe testwaterin arepresentativetest chamber wasapproximately 17cm. Thetestchamwerbeimeparrtialsly positionedin atemperature-controlled environmentalchamberdesignedto maintain atemperoaft25u2rCe. `Testchamberswere labeledwiththeproject number,testconcentarndartepliicoaten. `Theembryoincubationcupsweresuspendedinthe watercolumnofeachtestchamberandattachedtoa rockerarm.Thereciprocatingmotion oftherockerarm (approximatel2yrpm)facilitatedcirculationoftestwater aroundtheembryosduringincubation.Theincubationcupswereconstructedfrom glasscylinders approximately 50mmindiameterwith 425umnylonscreenmeshattachedtothebottomwith siliconesealant. `DilutionWater `Thewaterusedforculturingandtesting wasfreshwaterobtainedfrom awel approximately40meters deeplocate ontheWildlifeInternationLtadl. site. Thewellwaterischaracterizedasmoderatlywa-tehra.Trhde: specific conductance, hardness, alkalinity,andpH ofthe welwaterduringth four-veek period immediately precedingthetestarepresentedin AppendixI. `Thewellwaterwaspassedthrough asandfiltertoremoveparticlesgreaterthanapproximately25 am, andpumpednto a37,800-Lstoragetankwherethewaterwasaeratedwithspraynozzles.Priortodeliverytothe dilutersystem,the wateragainwasfiltered (0.45 jim),thenpassedthrough aUVsterilizer toremove 01607 WILDLIFE INTERNATIONAL Lo. "n- PROJECTNO.: 454A-108 `microoragnadnpairtsicmless. Theresoufpleritodsic analysesperformedtomeasuretheconcentratioonfs selectedcontaminantsinwellwaterusedbyWildife InternationalLd.arepresentedin AppendixIL EnvironmentalConditions Lightingusedtoilluminatetheculturesandtest chambersduringculturingandtestingwasprovidedby fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone 50). A photoperiod of 16hoursoflightand 8hoursofdark wascontrolledwithanautomatictimer. A 30-minutetransitionperiodof Towlightintensitywasprovidedwhenlightsweatonandofftoavoidsuddenchanges inlighting.Lightintensity wasmeasuredatthesurface ofthenegativecontrol (repA)latitescinaititatieon,at testterminaatnidoant `weeklyintedurrivngatheltesst.Lightintensityrangedfrom 58110 619 lux. `Temperaturewasmeasuredineachtestchamberatthebeginningandendofthe testandatweekly intervalsduringthetestusing liquid-itnhe-rgmolmaetsesr.Temaplsoweasmerasua redct ontinuuousrlyineone negativecontrolreplicateusing aFulscopeER/CRecorder. Thetarget esttemperatureduringthestudywas. 25:2C. ThepH wasmeasuredatthebeginningandendofthetestandatweeklyintervalsduringthetestin altemating replicatesof cach treaandtthecmonterolgnrotup.Dissolvedoxygenconcentwrearetmeiaosunresd daily in alermatingreplicatesofeachtreatmentandthecontrolgroupduringthefrst 7days ofthetest,atweekly intervalsduringth test,anda tettermination. Hardness,alkalinityandspecificconductanceweremeasuredin thenegativecontrolandthehighesttreatmentgroupwithsurvivingorganismsatthebeginningofthetest, ncea `weekduringth test,andat tettermination. Measurementsof pH weremadeusing aFisherAccumetModel915 pH meteranddissolvedoxygenvas `measuredusinga YellowSpringsInstrumentModel S1Bdissolvedoxygenmeter.Specificconductancewas, `meaussinug rYeellodw SpringsInstrumentModel 33Salinity-Conductivity-Temperaturemeter. Hardocssand alkalinity were measured based on procedures in Standard Methods for the Examination of Water and Wastewat(e5)r `OBiolbogiscal ervaantd Mieasourenmensts Observationsofmortalityweremadetwiceduringthe fist 24 hoursoftheembryoexposureperiod and dailyuntil hatch. Afterhatching, larvaewereobserveddalytoevaluatethenumberofmortalitiesandthemumber ofindividuals exhibiting clinical signsoftoxicityor abnormalbehaviorduringthe 42-daypost-haichexposure 010608 WILDLIFE INTERNATIONAL Lo. BTR PROJECTNO.: 454A-108 period. Fromtheseobservations,hatchingsuccess,timetohatch,andpost-hatchgrowthandsurvivalwere evaluated.Hatchingsuccesswasestimatedasthepercentageofviableembryosthat heichedsuccessfully. Posthatchsurvivalwasestimatedasthemumberoflarvaesurvivingtotettermination dividedbythe numberof embryoswhichhatchedsuccessfully. Posthatchgrowthofthefatheadminnowswasevalusteda theconclusion ofthe 42-daypostexhposaurtepecrihod. Totallengthsforeachsurviving fishweredeterminedusing a digitizingtabletwiththe SIGMA SCANTMscientific measurementsystem andwetanddryweightswere measuredusingananalytbialcaancle. `DataAnalyses Hatchingsuccess, post-hatchsurvival,wetweight,dyweightandtotallengthofthe juvenileish were testendpointsthatwereanalyzedstatistically. Datacollectedduringthi tetwereethercontinuous-verisdbatlae (e8, weightandlength)oriscrete-varidaatbale (c.g,mortalityproportions). Discrete-variabledatawereanalyzedusing 2 X 2contingencytablestoidentifytreatmentgroupsthat shoa stwatiseticadlly significant difference(p<0,05)fromthenegcaonttroilgvrouep. Allcontinuous-variable datawereevaluatedfornormalityusing Shapirteosta-ndWforiholmogken'eistyofvarusiingBaartnlettc' teest. AnalysisofvarianceandDunnett' testwereusedtoevaluatedifferencesbetweentreatmentandcontrol means. `Theresultsofthestatisticalanalyseswereusedtoaidinthedeterminationofthe NOEC,LOECand MATC.TheMATCwasestimatedasthegeometricmeanoftheNOECand LOEC.Allstatistetstiscwearle perfoonarpmerseondal computer using TOXSTAT 3. (6)and SPSS/PCVersion2.0 7) statisticalsoftware. ge Ga Priortotes initiation,oneset ofpretestwatersampleswascollectedfromtworeplicatetestchambersof eachtreatmentgroupandthenegativecontroltoevaluatediluterperformance.On Days0,4,7,14,21,28,35, 42andattesttermination, sampleswerecollctedfromtwoaltematingreplicatetestchambersofeachtreatment andthecontrolgrouptomeasureconcentrationsofth testsubstance.Sampleswerecollectedatmid-depthfrom cachtetchamber.Sampleswerecollectedin30-mL Nalgenebottlesandanalyzedimmediately. Analytical `proceduresusedintheanalysisofthesamplesareprovidedin AppendixIIL. 01009 WWIILLDDLLIFEEIINNTTEERRNNAATTIIOONALLLDr.. ~~ PRPROOJJEECCTNNO:4454AA--1088 1- RES ANU DDIL SCUT SSIS ON } . Resultsofanalysesto measureconcentrationsof PFOSinwatersamplescollectedduringthetestare preseinTtaeblde 1andintheanalyticalchemistryreport (AppendixI), Nominalconcentrsaclteciteodfnosr useinthisstuwedrey0.14,0.29,0.57, 1.1, 2.3and4m. ga.6 i/L. Whenmeasuredconcentroaftsaimopnless `analyzedduringthetestwereaveraged, themeanmeasuredconcentrationswere0.15,0.30,0.60,12,24and4.6 `mg a.i/L,whichrepresented 107, 103, 105, 109, 104and 100% ofthenominal concentrations,respectively. Meanmeasuredconcentrationswereusedtoexpressthe NOEC,LOECandMATC.Pre-testsamplescollectedto `verifydiluteroperation(AppendixIIT) werenotused tocalculatemeanmeasuredvalues. PMhysiecalaandsCheumicral emenotfWaster Meas oftu he sr pecie ficcm ondue ctann ce,ht ardns essandalkalinity madeonthenegativecontroland treatmentwaterarepreseinnTtabeled2.Noapparentdifferences intheseparametersexistedbetweenthe treaandtthmcoentrnolgtrosup. MeasuofrpHe,dimssoelvendoxtygesn,and tempearreapretseuntredien `Tables 3, 4, and 5, respectively. Water pH measurements were consistent over time and shonowobe viodus differencesbetweencontrolwaterandthetreatmentgroups. Measou fpHrrae ngem dfre om8n.0t to 8s .4. Dissolvedoxygenconcentrationsremained 27.6mg/L (92% ofsaturation). Alltemperaturemeasurementswere `withinthedesiredrangeof 25+2C. HatchingSuccess ndTimetoHatch AllviablefatheadminnowembryoshatchedonDay 4or 5of thetest(Table6). Th were eno r appae rent `differencesbetweenthetimetohatchinthe negativecontrolandthePFOS treatmentgroups.Hatching successin thenegativecontrolwas 93%.HatchingsuccessinthePFOStreatmentgroupswas >90%.Hatchingsuccesswas `notsignificantlydifferentin any PFOStreatmentgroupincomparisontothenegativecontrol(p > 0.05). `After 42daysofpost-hatchexposure,survivalinthenegativecontrol was 88%(Table7). Post-hatch. survival in the 0.15, 0.30, 0.60, 1.2, 2.4 and 4.6 mg a.i./L treatment groups was 79, 81, 66, 5.4, 0 and 0%, respectively. Analysisofpost-hatch survival data by 2 X 2 contingency tables showed that survival was significantlydifferentinthe0.60, 1.2, 24 and 4.6mg a/Ltreatmentgroupsincomparison to thenegative 01010 WILDLIFE INTERNATIONAL Lo. S15. PROJECT NO.: 454A-108 control p< 0.05). Allfatheadminnowssurvivingto estterminationappearednormalwithno overtsignsof `sublethaltoxicity. Fatheadminnowswhichdidnotsurvivegenerallyappearedobeswimmingeratcallypriorto death (AppendIiVx). Growth GrowthdataforeachtreatmentandthecontrolgrouparesummarizedinTable 8. Theindividual `measurementsareprovidedinAppendices V,VI andVIL. Growthwasevaluated atestterminationbymeasuring thetotal length,wetweightandyweightofeachsurviving ish.Thestatisticalanalysesofgrowthdatadidnot includePFOS treatmentgroupswhichhada statisticallysignificantfect onsurvival. Fatheadminnowsexposed to BFOSatconcentrationsof0.15or0.30mg aL for42daysposthatch showednostatisticallysignificant reductionsin totallength,wetweightordryweightincomparison tothenegativecontrol(p>0.05) CONCLUSIONS Fathead minnows (Pimephales promelas) exposed to PFOS at concentrations $0.30 mg 2/1, for 42 dayspost-hatchshowedno statistically sigificant reductionsintimetohatch,hatchingsuccess,survival or growth. Themostsensitiveendpointin thisstudy waspost-hatchsurvival. Fatheadmizexmposoed two PFsOS atconcentrations 0.60mga.i./Lhadstatistically significant differencesinpost-hatchsurvivalincomparison to thenegativecontrol. ConseqtuheeLOnEtCfloryth,is study, basedonpost-hatchsurvival, was0.60mga.i/L. The NOECwas0.30 mga./L andthe MATCwascalculatedtobe 0.42mg aL. 001011 WILDLIFE INTERNATIONAL Lm, -16REFERENCES PROJECTNO. 454A-108 1 U(dSra.ftE),nvOiPrPoTnSmeNnutamlbePrro8t5e0c.t1i4o0n0:AgFeinschy.Earl1y9-9L6i.feSSetraiegse 850 -- Ecological Toxicity Test. Effects Test Guidelines 2 OECD Guideline for Testing of Chemicals. 1992. Guideline 210: Fish Early-Life Stage Toxicity Test. 3 ASTM Standard E1241-88. 1988.StandardGuideforConduEarclytLiife-nStagge Toxicity Tests with Fish. AmericanSociety for Testing and Materials. 4 US. Environmental Protection Agency. 1986. StandardEvaluation Procedure, Fish EarlyLife- Stage Test. Officeof Pesticide Programs. Hazard Evaluation Division. EPA 540/9-86-138. s APHA, AWWA, WPCF. 1989.Standard Methodsforthe Examinationof Water and Wastewater. 17th Edition, American Public Health Association. American Water Works Association. Water Pollution Control Federation, New York. 6 `CWheesyte,nnIenc,.WyanodmiDn.gD. Gulley. 1996. TOXSTAT 35. Westen EcoSystems Technology, Inc. 7 SPSS Inc. 1988. SPSS/PC+ Version 2.0. Chicago, Illinois. 061012 n- Tae SummaryofAnalCyhetmiiscDaatla PROJECTNO: 454A-108 T EE e FUEMe,Pps rots NSoRos mOTeet bo Dut Dut Dui Deal Deis Dux bud: puoCiiiebsa No7da [---- WOW IW WW IW <AR IY WW a = ou o3rg aGm8 oHuE MoEme UB M8 aNmR aaw3 auwg os 0 om o3m7 oOUmR oMmR oHwZ oOwR o8%w oamm oBwye oMmN ow 0 o0 Somm 8ems Gomm MosWe ooums 0oes ous BamRs 8os8s om 10s u HR OBO NOm oE m oE om oEm E 0 namBmoom@ow3m 2 o--o - o= -=oo= oa or us i om ogg 2 = = = = =u 0 REECE Er rE 222 22 o PROJECTNO: 454-108 ue Tae? SpecificContuctance, HardocssandAllyoftheTestSlutions TT --- rou7s C70ain DTiviOerpWkar WFeeldWMear,Pincphprloemst . em Pees orTeot, 0 1 um om xs ow a Et=e) PgROSTeamted no Bwe0 we Wwmw mw e w Xo Bwmw ma0 (mHguwdsCsac0) NPeRpOiSveTrCeuanelnt WBo owue uwge wbee niWe w meww n6e (Aoignteye) ToC PNeEgOuSiTneeCaomol pwe vems Ab vmse vome as woem nme ewmmne ca 85 C -- 2A4mg-- eir ---------- Aman 3g 2 8 WILDLIFE INTERNATIONAL Lo. 194 PROJECT NO.: 454A-108 Tabl3e PH of WaitntheeTerst Chambers STepsSrubstamee: M PFOS C DiTleusttiOorngWiastme:r._ FWaetlhleaWdaMteirnow,Pimephales promelas T"MoeC anMesnired onve Ten gno gain) 0 2 1 2 2 3s 2a NegiveCowol 81% 82 82 01s 52 52 s2 030 82 82 82 060 82 82 82 12 82 82 82 24 2 82 2 4 52 82 Fee A Db A Messurementsdiscontinueddue 0TORO. 83 84 83 sa 83 84 83 84 83 84 --= 5_c oo 80 81 80s 81 81 ss 80 82 -- -A BB 81 8 82 om wm - : 01015 20. PROJECTNO: 454A-108 Tables DissolvedOxygenContent (mg/L)of Wotei teTestChambers" TSopnoSurbstance: SPRHOCSo.porsion TDeistoOrngaWntiesmr:__FWaetlhlsWodtMeir sa, Pinepphroameelass TeNsteaCrMceeansturrneidon a) 0 1 2 3 Tey a 0s 6 7 wi7n ow 3 ew NemiveCowol ois 030 060 12 2 6 81 80 81 81 80 80 80 82 81 81 82 78 76 80 8180 81 81 80 80 80 32 82 81 82 76 78 80 81 m1 82 81 80 80 75 81 82 81 82 76 78 80 S180 82 81 m1 80 80 81 81 81 82 78 76 80 52 30 sso 80 79 80 82 82 so 82 76 79 82 80 80 81 80 S081 80 82 SF - - . = = G1 80 81 81 81 80 80 81 Fo. oo = E Wee A BC 5 AmB 5e _Ca Db A BC A _B_C_ "Messmer iscontinseddoe 100% mil 223 22 WILDLIFE INTERNATIONAL Lo. a1. PROJECT NO.: 454A-108 Table TemperaturCe)of Waterinthe TestChambers "STpeosntsSourbstace: P3MFOCorporation, TDielsuttOirongmWaitsemr:. _ FWaetlhleWaadtMeirnnow,Pimephales promelas TMTeesmtMCoenacemntrraetdion Tey --gO Nae(gmvigeaCio)ln)rol| RReepplilAceavtee. 7 Z0A 377 21447 2I241L3 B 0 22845 3 23457 8 42425 42478 BC262444 224477 226476 224465 226455 224477 224454 22455 D245 247 267 245 245 247 245 245 01s AB 22645 22476 220477 224466 22445 2U478 2256 220444 C244 D265 22477 224476 224465 224455 226477 224454 u24s4 030 B A 22444 224477 C244 247 224477 246 224465 245 224455 244 224487 247 22444422444 2s 244 D245 247 246 245 245 247 244 244 060 AB 224044 224476 224456 224445 226044 224476 224434 22034 DC224433 224466 224665 224454 224444 224466 42634 224444 12 AB 226s4 224456 224s5 224445 22445 A2u4s6 U2434 M2434 DC226454 24467 22466 22455 224455 224466 224445 22444 24 A 245 247 2 - - - - --- B 246 247 2 - - - - - Cc 246 247 2 - - - - --- D 245 247 2 - - - - - 46 A 245 246 - - - - - --- B 244 246 -2 - - - - - c 245 245 -2 - - - - - rcor ntin Tor coring of DBenpr 2245henega2i4v5e somrol=replete =A owedG=a betre m--pertrse rienga omn 2-40 1025.0C throughouthetest. "Measurementsdiscontinued due to 100% morality 001017 WILDLIFE INTERNATIONAL Lm. 2. PROJECT NO. 454A-108 Table 6 THate ching Successof FatheadMimows ExpootsheeTedst Substance TTDesixoOSurbrsWtgaanacser: _PFWRealOlhSWdatMeirnnow,Pinhole promelas Tes Cohncveern Nameof me NumTboedr op(mwaiClo)n RepAliwc Ewi=Boowd DoTl oDey Da0d Da1ys Dem ys wHabel c55 x2x o0ooo0 &2sm=mom os AB< Tomx2 oooooo o o 0 z s2 m wm m > oo 3 on A8 23 o oo o 0oo 41 wonm =n cb 23 oooo 00 21 wmom 00 8A 2 00 00 00s om w 7 c5 Fd o0 o0 11 2 12 A5 c xx 2 oo o0oo0074 om omm m 5 oo sm 24 AB 220 oooo 0oo 73 wwmm cb F2l o0 ooo0 42. m ." A8 22 00 00 FE 5c 32 o0 _o0 ForenFathiog Svs -mo EggsTl ahal X 100 Numbof eEgrgs Exposed 00 43 u wm m o0 s1m1 TFeemare Huhing5Sue' 9 0 os = El = 601613 WILDLIFE INTERNATIONAL Lr. 3 PROJECT NO.: 454A-108 Table? SurvivalofFatheadMinnowLarvaeExposedtothe Test Substancefor42DaysPost-Hatch TSextSpubstrance: MPFCOeSpenmien DLTiMeliseuttoaiOnonrMngWeaWanaisttsuemrr:,e:d_WFWaetlhlWeWaadMtE eirnnow,TPoitmaepliales pro"mAeplparsoximateLiveCounts Test Concentration Numberof Replicsle Larvae 7 14(Days2P1ostH2a8tch) 35 (mgail) NegativeCoatral AB 1T B m15 115h 105 1157 16s Dc 2180 11851188 1188 U18 717 TPepelcnatie 4 Suvil 1156 8 1708%s TTreeatment SPeurcvenit E] 01s AB 1 Bo1w18ows1m 1o1ss 11ss 115 587 " Dc 0 11 116 1166 11s5 105s 11s5 7795 030 AB Bmo1oa8s a1sm 11s6 11s6 116s 116 5s8 "81 c D 1B Is 1155 1158 1158 1136 16 o1u 6s6 060 AB Bv8o1 1m6 ol16 u15 a1B 1B2-e6 or c0 D1 1198 11581618 1133 1O3B 12 368 12 BA 8 1oo2n oowr 47 23 32 21 s13 sa c D 18107 067 20 01 01 -01 0560 24 AB 18 00 00 00 00 00 00 0000 00 Dc 08 00 o0 o0 00 0o 00 0000 46 AB 71 00 00 00 00 00 00 0000 00 e ivecountsmadeonDey Dc2 rough 2a 111p9osthatch00were 00variabl00e Gu to00hede i00ficult00in counti00n00g1rge aumbers of li"viingnidsh.iTsichgenaicfoitucnaenttsmdaidfferoenncDeafryo4m2twheerneeegaxtaicvteccoonutnrsoowfialnlgfiashsXurv2icvoinntgintgtehnecyentdaoblfet(pe5es0t05) 001019 WWIILLDDLLIIFFE IINNTTEERRNNAATTIIOONNALtLom. Su. ~~ PRPROOJJEECCTNOO.::454544AA--10088 Table "Total Leantgtthhe,EWnedtofWtehiegh4t2a-nDdaDyrPyosWte-iHgathcthofObFsaetrhveaatdioMninPenroiowdL. arvae TSepsotwSsuobrs:tance: P3FMOCSorporation DTielsuttOirognaWnaitsemr:: _ WFaetlhleWadatMeirnnow, Pimephalespromelas "Mean Measured "Number of Test(Cognaceiotlr)ation ~~ LSuarrvivvaineg ToMielaLneng(t:hSDu)m) Negative Control 6 2650721) 01s LC) 266 (:0208) 030 8 266(:0813) 060" 50 265 (:0399) 12! ` 267(:200) 24 0 - -- WMeetaWenig(h:tSD()mg) 158 (9.10) 160(:3.10) 167(:119) 166(2113) 185 (:338) - 46 0 - - Not analyzed statisticallydue ta significant(p< 0.03)effonesurcvivtal -- DMrey aWenig(h:tSD(m)g) 325 (:120) 333 (:0900) 342(:270) 15270) 354(:666) - -- 01020 WILDLIFE INTERNATIONAL Lo. -25- PROJECTNO.: 454A-108 APPENDIX 1 Diluti4o-nWWeaetekrPeCrhieomdisItmrmyedPiaartaemleytePrrsecMeedaisnugrtehdeDTuersting the Spomsor: 3M Corporation TTeesstt SOurbgsatnainscme::~~ FPaFtOhSeadMinnow,Pimephales promelas Dilution Water: __Well Water Specific Conductance. ~; (uamhos/em) Mean EINE) Range 315-330 Hardness (mg/L as CaCO3) 126 (N=4) 124-128 Alkalinity (mg/L as CaCO) 120-4) 170-172 oH 8208-4) 82-83 01021 WIWLILDDLLIIFFEEININTTEERRNNAATTIIOONNAALLLoo.. -26- PRPROOJJEECCTTNNOO..: 44S5i4AA--l1o0g8 APPENDIIXT Analyses ofPesticides, Organics, Metalsand OtherInorganics --------------in W-- ildli-- fe Int-- ernati--onal -- Ltd. W--ell W--ater'-------------- A ANALYSIS ee MEASUREDC CONCRENTARATION ee Mech RT, oa om Ry < 188 mo ~ a EmmiLindon) BR Chlordane. BrBEriiann, `EndosulfanSulfate. Be `Endrin Aldehyde Eo, [RRSEEIi Jost RPBEIE mgt Eoininu)? SSern' f= MLNeoaarde ps Be fed FSol l iEBm =z PE =r <som 0025 -gL 8 3 S Sa Bm E 4= < 0018 S Sa g < 0.005 igaLr igrl : 2007 ar : $$ 0 auuee iiiuartrr :SM0720 it a . - ps : 24 z 030 farLo <<Soaki7]8 iaarr4 R= :<< o510t0 apurga < 38 < #5 =at <= a3if p=rA Ss Som byWis Inston Lo rp se Svan th, 1997. 001022 WILDLIFE INTERNATIONAL Lo. 21. APPENDIX II PROJECTNO.: 454A-108 `THE ANALYSIS OF PFOS IN FRESHWATER IN SUPPORT OF "WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454A-108 001023 WILDLIFE INTERNATIONAL Lo. L238. PROJECTNO.: 454A-108 : REPORT APPROVAL SPONSOR: 3M Corporation TITLE: 'PFOS: An EarlyLife-Stage Toxicity Test with the Fathead Minnow (Pimephaes promelas) WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-108 PRINCIPAL INVESTIGATOR: ffon Aa. MgactGregoor,gBS.: Scientist : MANAGEMENT: 7.747) `Willard B. Nixon, Ph.D/ `Manager, Analytical Chemistry BATE faafbe ah DATE' 001024 WILDLIFE INTERNATIONAL Lm. 29. PROJECT NO. 454A-108 Introduction Freshwater samples were collected from an carly lfe-stage toxicity study designed to determine the effects of PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) to the fathead minnow (Pimephales promelas). This study was conducted by Wildlife International Ltd. and identified as Project No. 454A-108. The analysesofthese water samples were performedatWildlife International Lid. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). Samples were received foranalysisbetween August 4,1999andSeptemb2e1r, 1999andweretypicallyanalyzedoneach `sample receipt day. TSestubsantd InaternnalStcandeard "The test substanceusedfor this studywas WildlifeInternational Ltd.identificationnumber4675. The test substance was used to prepare calibration and matrix fortification samples. `The internal standard was received from 3M Corporation on July 2, 1998 and was assigned Wildlife International Ltd. identification number 4526 upon receipt. Theinternal standard, agranularmaterial, was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2. "Thestandardwas stored under ambient conditions. Analytical Method . `Themethodusedfortheanalysisofthe freshwatersampleswasdevelopedatWildlifeInternational Ltd. and entitled "Analytical Method for the Determinationof PFOS in Freshwater, Saltwater, and Algal Medium". Thismethodologywasincludedas AppendixII ofWildlife InternationalLtd.protocol number 454/011299/MVAL/SUB454, It was based upon methodology provided by 3M Corporation. Samples were diluted in a 50% methanol : 50% NANOpure water solution containing 0.10 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v) so that they fell within the calibration rangeofthe PFOS methodology. Concentrations ofthe PFOSinthestandardsandsamples weredeterminedbyreverse-phasehigh `performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin- 001025 SWILL DLIFE INTEARNOATINONAAL Le m. ee "30. PTRROOJIEECCTTNNOO.:: 445554A8-010088 Elmer TurbolonSpray ion source. HPLC separations were achieved using a Keystone Betasil Cy `analyticalcolumn(50mm x 2 mm 1.D., 3pmparticlesize). Theinstrumentparametersaresummarizeidn `Tabl1e. AmethodflowchartisprovidedinFigure1. CalibratioCnurve and Limit of Quantitation Calibration standards of PFOS prepared in a 50% methanol : 50% NANOpure water solution `containin0g.100mg 4H PFOS (internal standard)/Land0.05%formic acid (v/v),ranging inconcentration from 0.000915to0.00915mga.i./L,wereanalyzedwiththesamples.Thesame andmostprominepenatk responsefor PFOS wasutilizedto monitor PFOS in all calibration,quality control,andstudy samples. No attemptwasmadetoquantify PFOSonthebasisofindividualisomericcomponents.Linearregression `equationsweregeneratedusingpeakarearesponseratios(PFOS :internal standard)versustherespective concentration ratios (PFOS internal standard)ofthe calibration standards. A typical calibration curve is presiennFigtureed2.Theconcentrationof PFOSinthesampleswasdeterminedbysubstitutingthepeak area response ratios into the applicable linear regression equation. Representative ion chromatograms of low andhighcalibrationstandardsare presentedinFigures 3and4, respectively. `Themethod limitofquantitation(LOQ)fortheseanalyseswassetat 0.0458mga.i/L calculatedas `theprodoufcthte lowest calibrationstandardanalyzed (0.000915mga.i./L)andthedilufacttoiroofthne `matrix blanksamples (50). `MFatroixrBltankianfd icatiSamoplnes Ninematrixblanksampleswereanalyzedto determinepossibleinterference. Nointerferenwceeres observedatorabovethe LOQduringsamplesanalyses(Table 2). Arepresioencnhrtomaatt ogriamvofea `matrixblank ispresiennFitguered5. Freshwater was fortified at 0.0915, 0.915 and 7.32 mg a.i/L and analyzed concurrently with the `samplestodeterminethe meanprocedural recovery(Table 3). Sampleconcentwerreanottciororenctsed forthemeanprocedural recoovf1e0r2%y. A representativeionchromatogramof amatrixfortificationis presienFnigturee 6d. 001026 WILDLIFE INTERNATIONAL Lm. 31 PROJECT NO. 454A-108 `ExampleCalculations `Sample number 454A-108-3, nominal concentrationof 0.14 mg a. /L in freshwater. PeakAreaRatio =AnalytePeakArea/Internal StandardPeakArca . Concentration Ratio = ConcentrationofAnalyte/Concentration ofInternal Standard Internal Standard Concentration: 0.100 mg/L. Initial Volume: 0.500 ml. Final Volume: 25.0 mL Dilution Factor: 50.0 POS Peak Area: 244330 Internal Standard Peak Area: 2316753 Peak Area Ratio: 0.10546 Calibration curve equation. Slope: 3.44236 Intercept: 0.00427 Curve is weighted (14%) PFOS (mga.iD/La)attiinstrumen=t Peak area raStiloop-e(V-intercept) xIntemmalStandardConcentrati.on 0= .10546- 0.00427 x 0.100 =000294 PFOS (mg a.i/L)insample = PFOS (mg a.i/L)at instrument x Dilution Factor =000294 x 50.0 =0147 019027 WILDLIFE INTERNATIONAL Lo. 32 PROJECT NO.: 454A-108 _PFOS (mgaiin/saLmp)le PercentofNominal Concentratio=n PFOS (mg a.i/L) x 100 =0.a 147 *100 =105% v RESULTS SampleAnalysis Freshwatersampleswerecollectedfromtheearly life-stagetoxicitystudywiththefatheadminnow (Pimephales promelas) at pretest, August 4, 1999, at test initiation, August 5, 1999 (Day 0), on August 9,199 (Day 4), on August 12, 1999 (Day 7) and weekdy throughoutthetest unil test termination on September 21, 1999 (Day 47). The measured concentrations of PFOS in the samples collectaetd pre- testrangedfrom 91.4 to 105%ofthe nominalconcentrations(Table 4). Themeasuredconcentrationsof PFOS in the samples collectaetd initiationofexposureofthetestorganisms (Hour 0) ranged from 95.5 to 114%ofthe nominal concentrations (Tabl5e). Samples collected at Day 4, Day.7, Day 14, Day 21, Day28, Day 35 andDay42 hadmeasuredconcentrationranges of93.1 to 116%, 97.0 to 113%, 91.7 to 100%, 102 to 113%, 108 to 119%, 107 to 128% and 101 to 114% of nominal values, respectively. `Samples collected at test termination (Day 47) had a measured concentration rangeof95.2 to 111% of `nominalvalues. Arepresentative ionchromatogram of atestsampleisshowninFigure7. 01028 WILDLIFE INTERNATIONAL Lm. EN PROJECT NO: 454A-108 INSTRUMENT, Table 1 `Typical HPLC/MS Operational Parameters "Hewlet-Packard Model 1100High Performance Liquid Chromatograph witha Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin-Elmer TurbolonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: KeystoneBetasil Cys col(u50mmmixn2 mm LD. 3 pumparticlesize) OVEN TEMPERATURE: 0c STOP TIME: 5.00 minutes FLOW RATE: 0.220 mUminute MOBILE PHASE: 72.0% Methanol :28.0% NANOpure Watercontaining 0.1% Formic Acid INJECTION VOLUME: 2504 PFOS RETENTION TIME: Approximately 4.3 minutes INTERNAL STANDARD RETENTION TIME: Approximatel3y.0minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 4267 amu 01029 WILDLIFE INTERNATIONAL Lo. ER PROJECT NO.: 454A-108 Table2 Matrix Blanks Analyzed Concurrently During Sample Analysis `Number (454A-108) MAB-1 Sample Type "Matrix Blank `Measured Concentration of . PFOS' (mgai/l) <L0Q MAB-2 MAB-3 MAB4 MAB-5 MAB. MAB7 MAB: ~ Matrix Blank Matrix Blank Matrix Blank Matrix Blank Matrix Blank Matrix Blank Matrix Blank <LOQ <L0Q <10Q <LOQ <10Q <LoQ | <oQ MAB-9 Matrix Blank <L0Q "The limitof quantitation (LOQ) was 0.0458 mg a.1/L. based upon the productofthe lowest calibration standard analyzed (0.000915 mg a.i./L) and the dilution factorofthe matrix blanksamples (50) 061030 WILDLIFE INTERNATIONAL Lo. ER PROJECT NO.: 454A-108 Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis `Sample Number (454A-108-) MAS-1 MAS-4. MAS-7 MAS-10 MAS-13 MAS-16 MAS-19 MAS-22 MAS-25 Concentrations of PFOS -- (mgai/l) Fortified Measured. 0.0915 0.0915 ~ 0.0915 0.0915 0.0915 0.0915 0.0915 0.0915 0.0915 0.0908 0.0924 0.0893 0.0942 0.0884 0.0886 0.103 0.0933 0.0970 Percent Recovered 992 101 97.6 103 96.6 96.8 13 102 106 MAS2 091s 0894 97.7 MAS-S 0915 0.908 99.2 MAS-8 0915 0.899 98.3 MAS-11 0915 0.961 105 MMAASS--1147 00991155 00.591828 919078 MMAASS-2230 00991155 00.990898 919038 MAS26 L015 0933 102 MAS3 73 MMAASS.-96 773322 MMAASS--1152 773322 MAS-18 732 MMAASS-2241 773322 MAS-27 132 759 104 77.7553 110036 173636 110050 736 101 77.4571 110023 713 97.4 `Mean = 102 Standard Deviation= 3.99 CV =391% N=27 01021 WILDLIFE INTERNATIONAL Lo. "36 PROJECTNO.: 454A-108 Tabled Measured ConcentrationsoEfarPlFyLOiSfeinSPtraegteesTotxDiiclituyerTeVsetrification Samples from an CoNnocmeinntarlatTieostn (mgai/L) (Negativ0e0Control) 014 NSoammbpleer (54A108) PT2L PT34 SamTpilmieng (Hours) 2244 2244 PCFonOcSenMteraastuiroend Perocfent (mgai/)? `Nominal <<1100QQ - 00114417 101 105 029 PPTTs6 224 00226756 952 914 057 PP1T7 22 00536651 9.1 98.4 11 PPTT-10 22 111009 100 9.1 23 PPTTL 2244 2a1m3 9256 943 46 PPT-1143 224 444478 997742 s"tTahnedlairmidatnoafqluyaznetidt(a0t.i0o0n0(91L5mOg) wiasL0).0a4n5d8th1gdial.u1t/iLobnafascetdouropfotntheemaptrroidxubcltaonfktshaemplolweesst(5c0a)l.ibration s*aMmepalsuersewdevraelu<esOoQf,mat10r1i,x9b7l.a7naknsdan1d2maptriex rfoorctfinfeiocmanitinotanlsacomnpcleenstraantailoynsz.ed concurrentlywith pre-test 01032 WILDLIFE INTERNATIONAL Lm. 37. PROJECT NO.: 454A-108 Table 5 Measured Concentrationsof PFOS in Freshwater Samples from a Fathead Minnow Early Life-Stage Toxicity Test TNT ommaiTSameplex Con(cmegnatir/aLt)ion 00 (Negative Control) (45N4u7m-b1e0r8) 1 "12s 216 350 "5 54 ont "5 98 % Sampling Ti0m)e 04 74 17 21 22 23 30 a2 a PFOSMewwFeemd Con(cegnatiralt)ion" Nomoifnal <<1100QQ =z <<1100QQ z- <<1100QQ zz <<1100QQ zz <<1100QQ z- <<1100QQ zz <<1100QQ Ez <<1100QQ zz <<1L00QQ -- os 3s 00 118" ss 312 77 i4s% 11 556 22 6& F2 77s% 33s5 88 2 996s @a 00.116407 110154 G014410 11000 00114484 110035 o01s34 99%547 0011533 110092 00.115680 111134 0o1r7s3 112s3 00..115670 11112s 00114575 1u0r5 stTahnedalrimdiatnaolfyqzueadnt(i0t0at0i0o9n15(LmOgQ)2/wa1s)a0n.0t4h5e8dimlgutia.oinf/aLctboarseodftuhpeomnattihexbplraondukcstaomfptlhese l(5o0)w.est calibration 661033 WILDLIFE INTERNATIONAL Lo. 3. PROJECT NO.: 454A-108 `Table (continued) Measured Concentrationsof PFOS in Freshwater Samples from a Fathead Minnow Early Life-Stage Toxicity Test CoNnocmeinntarlatTieosnt (mg ai) 025 SNaummpbleer (54-108, 63 "129 33 "w 557 a& iId k8g 0%7 SamTpilmieng x) 00 44 77 11 22 22 33ss 22 aa "PCFoOnSceMnetarastuiroend' Fetoofent mg ail) Nominal 00227877 955550 00227809 993.71 00229%2 110012 00226669 192387 00331057 110069 00338431 1n1ss 00331215 1fr0e9} 00331139 111008 002196 - 915022 057 37 00 0[x576 110010 22 P4 60661599 110196 3365 77 00369872 110153 590 11 00553359 994369 65 2211 0065088 110072 60 2Py 0066137 i108 750 33ss 0066446 11133 980 22 00557%5 110011 1090 aa1 00538435 995563 st"Tahnedalrimdiatnaolfyqzueadn(ia0io.n0(0mLO0gQ92)/1w15a)sa0n.d0t4h5e8dimlgutiaonLfactboarsoefdthuepomanttrhiexbplraondkucstamopfletshe(l5o0w)estcalibration 01034% WILDLIFE INTERNATIONAL Lo. 39. PROJECT NO.: 454A-108 Table 5 (continued) Measured Concentrationsof PFO in Freshwater Samples from a Fathead Minnow Early Life-Stage Toxicity Test CoNnocmeinntarlatTieostn (mgaill) iT SNaummpbleer (454A-108,) 150 "pr3 E3l8 5512 61 772 8812 21 110012 SaTmipmieng ) 0 44 77 11 221 28% 33ss 22 aa P`RCoOnSceMnetarastuiroend' Fefoofent (mg ail) Nominal 11113s 110038 112251 1n0s 111233 110132 110130 91060 112149 110138 113301 111189 113310 11198 1Li 110068 110s9 - 91.013 23 n 0 225 04 2 4 309 77 222271 9968.71 22456 110170 224338 110036 4s 11 00 444306 599.51 7 27 44 447799 110046 2a 17 444766 917030 s"tTahnedaTridamnoiaflqyuzeadn(i0a.t0i0o09n15(LmOgQ2):w1a)sa0n.d0t4h5e8dimlgutibon aa1c/LtsreofdtuhpeomnatthexbplraondkucstamopfletsheT(o50w)e.stcalibration 061035 WILDLIFE INTERNATIONAL Lo. a0 PROJECTNO.: 454A-108 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FRESHWATER Prepare matrix fortification samples by spiking the requisite volumeof PFOS stock solutions directly intofiltered freussinghgasw -tiga htsytrine gesarnd Class Avolumetric flasks. ~ 4 Dilutematrix fortification andtestsamplesintothe range of thecalibrationstandards bypartiallyfilling Class A volumetric flasks with 50% methanol: 50% NANOpurewatersolutioncontaining 0.100 mg 4H PFOS (internal standard)/L and 0.05%formicacid (v/v). Addthe Appropriate volume ofsample and bring the flask to volume with the dilution solvent. Process the matrix blank sampleusingthe same dilution and aliquot `volume as for the lowesrtefpoerattifiincvaetrisoinonlse.vel. Mix well by several . . Ampulate samples and submit for LCMS analysis. Figure 1. Analyticalmethodflowchartforthe analysisof PFOS infreshwater. 01036 WILDLIFE INTERNATIONAL Lm. Lar. PROJECTNO.: 454A-108 040 1+ 035 030 30% ELI om 010 0.05 * 000 . - 0000 0020 000 0060 0080 0100 Concentration(Ratio) Figure 2. Atypical calibrationcurvefor PFOS. Sl=o3.4p423e6; Interce=pt 0.00427; = 0.9994. Curveis weighted (1x). 061037 WILDLIFE INTERNATIONAL Lo. a2. PROJECTNO.: 454A-108 | EERE | TTT ERE ET | mm ws | | em | | Ea | Fo : | | EEE EER ES" } e | e]| Figure3. Arepresentativeionchromatogorfam low-level (0.000915 mg a../L) PFOS standard. 010338 WIWILLDDLLIIFFEEIINNTTEERRNNAATTIIOONNAALLLLoo.. 5. seiner PRPROOJJEECCTTNNOO.::d4s5i4aA-i1o0s8 | --| | BT | A | TEE ee | . e-- Figure4. Arepresentative ionchromatogramof a high-level (0.00915mga.i./L) PFOSstandard. 01029 WILDLIFE INTERNATIONAL Lo. ae | oe E-- PROJECT NO.: 454A-108 | | | | EL. K + | |Po EB CA ETTE RET TRsBe | i FigureS. A representative ion chromatogramofa matrix blank sample (454A-108-MAB-1). Thearrowindicatestheretention timeof PFOS. 01010 A WILDLIFEN INTR ERNATOIONNAAL Lm. eect -45- PPRBOOJRERCTTNNOD.;: 44354540-010088 | EEE | i A_ TEC EERE bas | eee | Figure 6. A representative ion chromofaat mato rixg fortrifia catimon sample (454A-108-MAS-1). 01041 ZWDN ILLDILT PIFEEE INTER RNATN IONAA L Lm.T~~IONAPPL RROOJJEECCL TTNNOO.::4m 455e4AAd-1l. 0o8s ~46- | mT |. EE: vv | E End d : || | TT TT i Figure 7. A representative ionchromaotfaotegstrsaa mplme (454A-108-3). 01042 oer: sus H Be e Ee cote sn posse e BEEE en e a oo a PER OTF GOED FOIE Eprom of o PSEEPPEREoOFF8 TEE AE R FEA RE TopPPpEirrboomo F+or F8o PPEEE oOn EEoE f gg rePt EPe E e OE: g oy 1 fF 1 ODoFF b1 ooF i BEos OE PE LEFE og oo+r fFoB 3oIxFF obomg# OBEr oDopFzE or oc: FEE ae a fr ComniveMAaPPlENyDIsXndT(rCosntmineuend)tReElfifd" II CD tet tie ou thee tow ee tow fhe en wees ROTOR TN HE 3 PROJECTNO: 454-108 I RI the way me FH] v wn a re 222 2= PE -- T fo l 08 OUD" ew Memes 0 ComeAPPgEaNtDIn XIV (Coe ntesRt)edm Els' msi m oo y pe m wo te e m tow e mma RW sm 5 aw FROJCTNO: 454A-108 m te te n memw wme 3 aN 3N 82e 2a 0r com J REACn BSre EE mres-- ormin = == EEfeeFwnTn1 a8 o1m me rwtT m3e mocTyo. son anwo= m a == owe 1: 1 = g22g TE EEE EEESSTE METSRSSERCANRS------------------------e--t ----o ------ 28 sie PROJECTNO: 454A-108 ComiativeAMoPrPalEiNtyDsIdXVTr(sCiomneinntaRcte)led Ef" _ a TEale Moa a el pie hase io pies ips oe ow oe RN HE] I] hat the ines ime 3 w 3 wn 2 8 SS 52 PROJECTNO 454-108 CumsiveMorAaPliPtEyNsDnIXTIrVem(CoonnttiRnouleid)e Efe" [ESrRaI Ettte sons tee ee CD tet tsoiw fren ios tee ios nem wt tne ios ume TEE Ee ey Tree 238 S% = Br Ere coms ARE pre APPENDIXIV (Continued) SEeE we tee on=me on wx e on =me == fr 1 % 1 & 1 % om ==wn 1& : an = we 12 To pe Cy Ee pee 32 2 she CumstivAMPorPaEliNtDyIaXnIVT(reCmoonntnRe)l Efe" [TEHrOOaET Flaten pple LFro EE meme me OR AN TON HE PROJECTNO: 454A-108 otmeme 3N inm es mome 3 FH] Er re -- 232 a3S WWIILLDDLLIIFFEEIINNTTEERRNNAATTIIOONNAALLLLom.. _s5- PPRROOJJEECCTTNNOO.::445544AA--110088 TTTootoOrgeuiesn: Diie erWie er Ten(Co6n0c)ern a ois ax oa 12 2 APPENDIX IV (Continued) TPRHOCS CoupmaulnativePercent Mortality and TreatmentRelated Effects' FWeulleVeMirmaw,Paphos promt BRE BRE Roaow Wand goes Bice Be5 oomm133 awww A5eoY 0 es14 ~wnN 5 ox oN A5 wuw ma2 wan c 5 w 73 w A Bcom m s37 ~Nwn 5 0 w A [I mn ~wn c 5w ooww ~= A5 m Bw B -z 5 =k m W=m zz DLdT Ems 55 w ! 3i: a~WwN 5w 2i7 aaWwN 3w `75 ~wwnn `w 1n aw 7 w= 8 -2 fH= z2 tetr ud omen 33 nwN iw 5ii w~nN 5N 273 naN 3w s7 aWN 7a 1i aw i1s w~ 1 -z fHx =: " A [I ww -- n -: 1n -z AC E e El 01 AppBcaioemras,ABmC= wm ery, =eErdez enag,hR= u y gaborozneoi ---- : 01051 WWIILLDDLLIIFEIINNTTEERRNNAATTIIOONALLLoo.. -56- PRPOROJJEECCTNNOO::445564AA-l1088 APPENDIXV Fish Total Length (mm) SNpegoantsiovre:Control `Test Substance: `Test Organism: Dilution Water: 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Fish Number r EEm ee cceceecgm-- A B c D 1 ~ 26.0 2 286 3 265 54 33LL10 76 225550 98 2277.21 110 2275.31 2 24 13 246 14 268 15 212 16 214 17 .o- 18 - 19 - 20 = 296 317 278 26.1 257 216 283 283 282 235029 7218.7 235014 22632 228360 227830 22675 225663 228026 310824 228547 224269 290 152 25 289 20.1 256 29.1 237 312 268 281 245 - 282 255 - 21 207 - - - - - - = = = Replicate Mean OverallMean(is) 271 27.1 257 260 26.5 (0.721) Note: Totallengthvalueswereroundedforpresentation. Manual calculaotfimeoanns and standarddeviation may differ slightly from those generated using TOXSTAT3.5. 61052 WILDLIFE INTERNATIONAL Lo. 57. PROJECT NO.: 454A-108 APPENDIXV (Continued) Fish Total Length (mm) 0.15 mgai/l. `Sponsor: `Test Substance: Test Organism: Dilution Water: `Fish Number 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water --S A B C D 1 ~ 29.0 29 219 268 23 227652 226530 229686 22567 45 21948 22.810 2327..10 226582 6 2%3 233 279 253 78 237219 227649 2253.70 220.11 5 181 307 2338 273 10 239 26.1 27.7 290 1 285 249 23 276 12 295 28.1 21.7 258 1143 2273.16 226.01 22462 8 22914 11s6 2-78 3~04 2-0 3a00 7 13 - - 2 " - 19 Replicate M2e0an - - 6 = 26=3 - - "368 2 = 6 = OverallMean (is) 26.6(+ 0.208) Note: Totallength valueswereroundedforpresentation. Manualcalculoaftmeianosannsd standarddeviationmaydifferslightlyfrom thosegeneratedusing TOXSAT3.5 01053 WILDLIFE INTERNATIONAL Lo. oss PROJECT NO.: 454A-108 APPENDIX V (Continued) Fish Total Length (mm) 030mgaisl. `Sponsor: 3M Corporation Test Substance: PFOS Test Organism: ~~ Fathead Minnow, Pimephales promelas Dilution Water: Well Water Fish Number ------ Ee-- esmsme-- srgr-- A B c D 1 N259 308 278 278 2 27.0 252 272 259 43 261924 2274.60 2295.01 225646 5 6 228507 226718 22759 2337.01 7 280 29 283 280 8 9 219957 22672 22853 228918 10 255 263 256 259 1121 2380.15 228723 25-4 221739 13 210 303 - 304 14 20.6 250 - 23.1 15 273 25.1 - 24 176 18 -- 2-42 -- 28-.0 - = - 19 - = - - 20 = = = = Replicate Mean 255 264 27.1 273 OverallMean (5) 26.6 (03813) Note: _Totallength values wereroundedforpresentation. Manualcalculoaftmeianosannsd. standarddeviationmay differslightlyfromthosegenerated using TOXSTAT3.5. G105% WILDLIFE INTERNATIONAL Lo. -59. PROJECT NO.: 454A-108 APPENDIX V (Continued) Fish Total Length (mim) 060 mgaill Sponsor: 3M Corporation TTeesstt OSurbgsatnainscme:: PFaFtOhSeadMinnow,Pimephales promelas Dilution Water: _ Well Water Fish Number RNS = 5 CT 5 T 271 302 280 254 2 27 28 290 246 3 25 302 27 211 4 24 253 281 279 5 28 27 23 308 6 270 247 258 259 7 271 289 25 25 3 291 250 250 274 9 10 284 20 27 275 269 300 256 231 il 237 269 251 234 12 313 291 264 22 13 14 - 263 - 25-4 -- 1s 16 - - -- -- 17 13 - - - - - - - 19 - - - - 20 - = - - Replicate Mean 263 262 271 %3 OverallMean (45) 265(20399) sNottaen:dardTodetvaialtieonngtmhayvadliufefsewresrleigrhotuynfdreodmfotrhporseesgeennteartaitoend.MuasinnugaTlOcXSaTlATc3u.5loafmteianosannsd 01055 WILDLIFE INTERNATIONAL Lo. -60- PROJECT NO.: 454A-108 APPENDIX V (Continued) Fish Total Length (mm) 12mgaill, `Sponsor: SM Corporation Test Substance: PFOS Test Organism: Fathead Minnow,Pimephalespromelas Dilution Water: _ Well Water Fish Number x ) Replicate T 5 T 2 S262 21 267 - 287 = = - 3 4 - - - - 56 -- -. - - 78 - -- - Ee - 9 10 - ~ - - - - - - 1 2 - - - - 13 14 - - - - z .n 1s 16 - - - - - E 17 18 - - - - 19 20 = = - - . Replicate Mean 247 267 nT - OverallMean (45) 267202) "Note: Totallength valueswere roundedforpreseatation. Manualcalculoaftmeianosnasnd standarddeviation maydiffer lightlyfromthosegeneratedusing TOXSTAT 3.5 01056 WILDLIFE INTERNATIONAL Lo. -61- PROJECT NO: 454A-108 APPENDIX VI Fish WetWeight (mg) Negative Control Sponsor: 3M Corporation TTeesstt SOurbgsatnainscme::~~ FPaFtOhSead Minnow, Pimephalespromelas Dilution Water: _ WellWater Fish Number --_-- x 5 Repliate T po) T= 481 79 841 729 2 2005 1582 1829 2014 3 1489 1876 2062 1645 4 2086 279 1628 250 5 2310 1430 1739 1424 6 129.1 1375 1249 2092 7 1301 824 2104 1650 8 1511 1278 1399 1739 9 10 1531 1690 1546 2387 1815 1420 787 1046 i 2 1326 645 275.1 1801 2156 285 983 1242 13 14 1355 2022 1479 1963 604 1459 1247 2868 1s 16 1476 1638 8.1 - 1650 1820 499 1440 17 - - 774 569 18 - - A - 19 - - - - 20 - ~ = - Replicate Mean 1612 1685 1520 a4 Overall Mean (25) 158 (49.10) dNoetvei:ationTomtaayldilfenfgetrhslviaglhutelsywreormertohuonsdeegdenfeorraptreedscuastiantgioTnO.XMSaTnAuTal3c.5a. lcuofmleaanstanidsotanndasrd 01057 WILDLIFE INTERNATIONAL Lo. -62- PROJECT NO.: 454A-108 APPENDIX VI (Continued) Fish WetWeight (mg) OlSmgaill STpeosntsSourb:stance: 3M Corporation PFOS TestOrganism:~~ FatheadMinnow,Pimephales promelas DilutionWater: WellWater Fish Number eee -- = 5Roploae < Ds T= 2 1525 1397 TI80 1339 2001 2024 55 1229 3 4 1666 676 1244 1998 1604 1480 2047 1924 5 6 28 2059 9.0 190 290.1 1672 1253 1730 7 8 2400 1768 1650 1959 366 1282 S84 1466 9 10 558 1040 2008 180.1 766 1817 1773 1987 1 12 1982 218 1391 2103 700 1846 1775 1234 13 14 1030 1568 1550 105.0 1867 1356 2021 ms is 16 1523 - 2143 - 1999 2193 ~ - 17 18 ~ - - - - - 19 20 = -~ - - - - ROevpelrialclasMeMaenan(45) 1603 1566 169 1586 160 (+310) Note: Totallength valueswere roundedforprescriation.Manualcalculoafmteianosnansd standard deviation may diffe slightly from those generated using TOXSTAT35. 01053 WILDLIFE INTERNATIONAL Lo. 6 PROJECT NO.: 454A-108 APPENDIX VI (Continued) Fish Wet Weight (mg) 030mgai/t Sponsor: 3M Corporation Test Substance: Test Organism: FPaFtOhSe.ad Minnow, Pimephalespromelas Dilution Water: _ Well Water Fish Number JEN x 5 T 5 To 2 14s 1616 2088 181.1 2067 2084 1624 1553 3 4 624 1843 1355 1932 1920 1471 2036 1358 5 6 2037 1267 1656 1856 2618 2119 209.1 1642 7 8 1631 756 1238 201.7 157.0 1807 201 1932 9 10 2563 1256 1929 175.1 1265 1454 1783 1240 1 12 1989 2634 1997 2022 1509 - 1927 1516 13 14 84 537 246 1221 - 247 656 1s 16 1898 - 1063 1492 - 9.0 1792 7 18 - - -- -- 19 2 ~ - ~ - = Replicate Mean 52 701 13 1627 Overall Mean (45) 167119) Nsottaen:dard Tdoetvailatlieonngmthavyaldiufefsewresrlieghrtoluynfdreodfmotrhporseesgeennteartaitoend.MuasinnugaTlOcXSaTlATcu3.l5o.afmteianosannsd 01053 WILDLIFE INTERNATIONAL Lo. 64 PROJECT NO.: 454A-108 . APPENDIX VI (Continued) Fish Wet Weight (mg) 060mg ai/l. `Sponsor: 3M Corporation `Test Substance: PFOS `TestOrganism: FatheadMinnow, Pimephales promelas Dilution Water: __Well Water mmm REE `Fish Number A B C D 1 ~206.7 2237 180.5 1612 2 962 122.1 2254 1256 3 1132 221.0 200.6 85.1 4 1310 160.7 189.0 2013 5 93.3 1033 1749 2727 6 169.8 1411 156.7 1328 7 163.2 2053 1482 1693 8 2315 1306 1725 1753 9 10 223.1 2012 84.0 1529 2314 2360 1372 88.0 3 11 1362 159.4 151.8 859 12 1763 212.0 1773 2518 13 - 177.1 125.1 - 14 - - - - 15 - - - # 11768 --- --- --- --- 129 =- a- =- =- : Replicate Mean 161.8 161.0 1823 1572 OverallMean (4s) 166(+ 11.3) Note. Total lengthvalueswereroundedforpresentation. Manualcalculoafmteianosannsd, 01060 WILDLIFE INTERNATIONAL Lm. -65- PROJECT NO: 4544-108 APPEVIN(CDontIinuXed) FishWetWeig(hmgt) L2mgaill "TSepsotnSsuobrstance: SM Corporation| PFOS DiTleusttiOorngaWnaitsemr:: _ FatheadMinnow,Pimephales promelas Well Water Fish Number -- ry Ree 5 T > T 2 S1137337 a - 20-6 -- 3 4 - - - - 5 6 - -- - - 87 2- -- -- -- 9 10 1 - - - - - - - . 1123 - - ee - - 115 - - - - 16 7 r - -- - 1198 = - - - - 20 Replicate Mean 535 TI - . me - Overall Mean (25) 185 (2338) sNtoatned:ardTdoetvailatlieoangmihayvdaliufesfewselriegrhtoluyndreodmftohropsreegseennteartaitoend.uMsainnguaTlOcXSaTlATc3u.l5o.afmteianosannsd 61061 WILDLIFE INTERNATIONAL Lo. "66 PROJECTNO.: 454A-108 APPENDIX VII Fish Dry Weight (mg) Negative Control `Sponsor: 3M Corporation `Test Substance: Test Organism: FaPtFhOeSadMinnow,Pimephales promelas. Dilution Water: __Well Water Fish Number -- Replicate A B C D 1 ~ 303 512 408 388 2 45 327 40.5 24 3 320 399 403 329 4 430 452 336 504 55 7 2445.66 254 22%826 157 2347.95 476 36023 340 3 331052 235618 303727 317504 10 30.0 457 216 210 1 216 118 436 20.1 12 55.0 39.7 06.5 26.1 113 15 226.46 27 3a718 308 301083 27 22552 L093 16 194 - 395 299 17 - - 168 us 18 - - - - 19 - - - - M Te2n 0 0 m=T = 8 = a=4 Overall Mean (5) 32.5 (+ 1.20) Note: Total length values were rou fornpred sene tatid on. Manual calculaotfimoeannss and standarddeviation may differ slightly from thosegenerated using TOXSTAT3.5. 001062 WILDLIFE INTERNATIONAL Lm. -61- PROJECT NO.: 454A-108 APPENDIX VII (Continued) Fish Dry Weight (mg) 01S mgaisl `Sponsor: 3M Corporation "Test Substance: PFOS Test Organism: ~~ Fathead Minnow, Pimephalespromelas Dilution Water: __Well Water Fish Number x 5 Replicate T 5 1 ~ 390 23 43.6 39.4 2 215 258 556 262 3 35.1 25.1 336 40.5 4 139 398 276 323 5 46.7 169 619 272 6 415 220 34.0 387 7 493 3438 164 135 8 36.1 411 263 321 9 109 412 146 389 10 194 375 350 46.7 1 443 303 139 383 ; 12 503 453 389 230 13 219 327 38.1 412 14 284 20.7 269 233 15 322 46.6 432 484 16 - - - C- 17 - - - - 18 - - - - 19 - - - - 20 = = = = Replicate Mean 331 EX 340 340 OverallMean (#5) 33.3 (0900) Note: _ Totallength vawleru e roe unds ed forpresentation."Manualcalculaotfimeoannss and standarddeviationmaydifferslightlyfrom thosegeneratedusing TOXSTAT3.S. 01963 WILDLIFE INTERNATIONAL Lo. 68 PROJECT NO.: 454A-108 APPENDIX VII (Continued) Fish Dry Weight (mg) 030mgai Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Fathead Minow,Pimephalespromelas -- er ------------ Fish Number --_-- x B Reem CT 5 T = 20 a2 399 1 2 39 373 48 21 3 109 262 401 67 4 400 393 305 27.1 5 3 319 390 514 5 258 25 452 341 7 33 238 441 31 8 156 353 467 397 9 22 238 24 347 10 258 343 22 242 i 27 04 2s 287 12 560 395 - 321 13 182 458 - 501 14 n7 22 - 145 15 a0 201 - 190 16 17 - 2-71 -- 3~64 13 19 - - ~ - = ~ > 2 - - - a Replicate Mean 318 356 380 354 Overall Mean (4) 342270) Note: Totallengthvalues wereroundedforpresentation.Manualcalculoafmteianosnansd standard deviation may differ slightlyfromthose generated using TOXSTAT3.5. 01064 WILDLIFE INTERNATIONAL Lm. -69- PROJECT NO.: 454A-108 APPENDIX VII (Continued) `Fish Dry Weight (mg) 060mg ail Sponsor: 3M Corporation Test Substance: Test Organism: FPaFtOhSeadMinnow,Pimephales promelas Dilution Water, _Well Water Replicate -- Fish Number A B CT D T 2 = a9 188 wT 231 385 463 288 29 3 4 21 251 444 327 399 369 158 400 5 6 182 206 351 542 330 253 301 26 7 8 313 486 1 269 274 25 3s 358 9 10 490 29 143 304 468 491 279 17.1 il 12 270 598 317 24 296 379 163 511 13 14 - 2-4 2~0 -- 1s 16 2 - - ~ 7 18 Ca - - - 19 - - - - -- ReplicateMean 1 316 365 308 Overall Mean (+5) 3352270) sTtoatned:arddTeovtiaaltlieonngmtahyvdailfufeesrwesrliegrhtoluyfndreodmftohropsreegseennteartaitoend.uMsainnugaTlOcXSaTlATcu3.l5oa.ftmeianosnesnd 01055 WILDLIFE INTERNATIONAL Lr. -70- PROJECT NO.: 454A-108 . APPENDIX VII (Continued) Fish Dry Weight (mg) L2mgaifl `Sponsor 3M Corporation Test Substance: PFOS Test Organism: ~~ Fathead Minnow,Pimephalespromelas Dilution Water: _ Well Water Fish Number m x e5 m SE Te ee s >e 2T <7 23536 342 ne - - - - 3 4 - - - ~ - 5 5 - - - ue - 78 -- - - - 9 - - = 10 - - - - 1 12 - -- -E - 13 - - - - 1 15 - - -- Le- 16 17 - - - ha - - 13 - - - - 19 20 = -= -z -- Replicate Mean 55 342 26 = OverallMean (45) 35.4 (6.66) "Note: Totallength valueswere roundedforpreseataton. Manualcalculoafmteianosnansd standard deviation maydiffer lightly rom those generated using TOXSTAT 3.5. 01056 WILDLIFE INTERNATIONAL Lo. 7. PROJECTNO.: 454A-108 APPENDIXVIII ChatnoPgroteocsol . `Thisstudywasconductedinaccordance withtheapprovedProtocolwiththe folowingchanges: 1. Theprotocolwasamendedtoaddtheproposedexperimentalstatandterminationdats. 2. Thepost-hatchgrowthperiodwasexteadedto 42days. 3. Nominaltestconcentrativhswere0.14,0.29,0.57,1.1, 2.3 and 4.6mg ai/L. 01067 WWIILLDDLLIIFFEIINNTTEERRNNAATTIIOONALLLm.. ~~ PPRROOJJEECTNNOO..: 445544AA--108 n- APPENDIX IX Personnel Involved in the Study studyThefollowingkeyWildlifeInternatioLnda.l persomnlwereinvolvedntheconductormanagementofhis 1. Heary O. Krueger, Ph.D, Director, Aquatic ToxicologyandNon-Target Plants 2. Willard B. Nixon,Ph.D Manager, Analytical Chemistry 3. Timothy Z. Kendall, Supervisor 4. KurtR Drottr, Senior Biologist 5. Jon A. MacGregor, Scientist 6. Mark A. Mank, Laboratory Supervisor 01063 PROTOCOL PROS: AN EARLY LIFE-STAGE TOXICITY TEST WITH THE FATHEAD MINNOW (Pimephales promelas) . - U.S. Environmental Protection Agency `Series 850 - EcoElffeo ctsg TesitGc uida elil nes (OPPTS Number 850.1400 - 3M Lab Request No. U2723 Submitted to 3M Corporation Envi9r3o5nmBeuntsahlALvabeomreatory St. Paul, Minnesota 55144 :. 2 WILDLIFE INTERNATIONAL LTD. 3H Ea8s5t9o8n,CMoamrmyelracnde D2r1i6v0e1 . 10)822:8600 June 10, 1999 01069 WILDLIFE INTERNATIONAL, LTD 2. PFOS: ANEARLYLIFE-STAGE TOXICITY TEST WITH THE 'FATHEAD MINNOW (Pimephales promelas) SPONSOR: `SPONSOR'S REPRESENTATIVE: 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 Ms. Susan A. Beach TESTNGFACIITY: `Widelaternational,Ltd. 8598 Commerce Drive Easton, Maryland 21601 STUDY DIRECTOR: Kurt Drottar Senior Aquatic Biologist LABORATORYMANAGEMENT: Henry O. Krucger, PRD. Directorof Aquatic Toxicology & Non-Target Plants FORLABORATORY USE ONLY `Proposed Dates: Experimental `Start Date: `Experimental `Termination Date: Project No.: "Test Concentrations: NegativeControl016,031,063,13.2.5 0dSOmgaifl `Test Substance No.: 4675 Reference SubstanceNo. (if applicable): 4526 PROTOCOLAPPROVAL STUDY DIRECTOR DATE )RATORY MANAGEMENT . SPONSOR'S REPRES} DATE 6, / 30/99 DA 01070 PROTOCOLNO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD 3. INTRODUCTION `Wildlife International, Ltd.will conductanearly life-stage toxicitytestwith fatheadminnow (Pimephales promelas) for the Sponsor at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland.ThestudywillbeperformedbasedonproceduresintheUS, Environmental Prolection Agency Series850 - EcologicalEffects Test Guidelines OPPTS Number 850.1400(1);ASTMStandard [E1241-88 Standard Guidefor ConducEatrliyLnifge Stage Toxicity Tests with Fish (2);andStandard EvaluationProcedure, FishEarlyLife-StageTest(3). RawdataforallworkperformedatWildlifeInterna`tional, Ltd. and a copyofthefinalreportwillbefiledbyproject numberinarchiveslocatedonthe Wildlife International,Ltd.site,oratanalternative locationtobespecifiedinthe finalreport. PURPOSE `Thepurposeofthisstudyistodeterminetheeffectsof a testsubstanceonthe timetohatch,hatching success, survival, and growth of fathead minnows (Pimephales promelas) during carly Life stage development. EXPERIMENTALDESIGN Fatheadminnow embryoswillbeexposedto aseries ofsixtestconcentrations and a negative (Dilutionwater) control.Testconcentrations willbechosenbythe StudyDirector inconjunction withthe Sponsorbasedonacutetoxicitydata.Eachtetconcentration willbe S0%ofthe ext highertreatmentlevel. "Thetestwillbeginwhengroupsofnewly-fetilizedembryosareplacedinincubationcupsand ! exp too tess tsoleutid on. Incubationcups,eachcontaining20embryos,willbeplacedineachoffour replicatetestchamberspertreatment(totalof 80embryospertrestmentandcontrol). Afterhatching, larvae `willbereleasedfromtheincubationcups intothe largertestchamberswhereexposurewillcontinueand growthandmortality willbe observedover a28-dayperiod. `Observations ofthe effectsofthetestsubstanceonhatchingsuccess,growth, andsurvivalwillbe: `usedtocalculatethe no observedeffectconcentration(NOEC)andthelowestobservedeffect concentration (LOEC). The NOECandLOECwillbeusedtocalculatethemaximumacceptabletoxicantconcentration MATO). 01071 "PROTOCOLNO.: 454/061099/FAT-ELS/SUB454 3MLABREQUESNOT. U2723 WILDLIFE INTERNATIONAL, LTD 4 MATEARNI DMA ETL HOS DS TestSubstance Infoorntmheachatraciteriozatnion oftest,controlorreferencesubstancesisrequiredbyGood LaboratoryPractice Standards (GLP).The Sponsorisresponsible forproviding Wildlife International, Lid. `writtenverification thatthetetsubstancehasbeencharacterizedaccording to GLP priortotsuseinthe study. IfwrittenverificationofGLPtestsubstancecharacterizationisnotprovidedto WildlifeIntemational, Ltd,itwillbenotedinthecompliancestatemeatofth finalreport.TheattachedformIDENTIFICATION `OFTEST SUBS BYSTPOA NSONR(ACppenEdix itobeusetoprovide informationnecessafroyr GLPcompliance. `The Sponsarisrespofonralsliinfobrmlatieonrelatedtoth testsubstanceincludingthereeation of areservesampleofthelotorbatchofthetestsubstanceusedinthisstudy.The Spoalnsoasgreoesrto accept anyunused test substanceand/ortestsubstancecontainersremainingatthe codofthestudy, f-- a. "Thetestsubstancewillbeadministeredtothetestorganisminwater. Thisrouteofadministration `wasselectedbecause itrepresentsonepossible routeofexposuretoaquaticorganisms.Thetestsubstance willbemixeddirectlywithdilutionwater. TestOrganism Fatheadminnows (Pimephalespromelas)willbeusedinthistest.Thisspei rcepreiscateativseof animportantgroup oforganisms and was selectedfor useinthetestbasedupon past useandeaseof `handlinginthelaboratory. Embryosusedtostartthe estvillbeapproximately 2to 24-hoursoldandwill beobtainedfromculturesmaintainedinthelaboratoryat WildlifeIntemational,Ltd.or acommercial supplier Embryoswillbe removedfromspawningsubstratesusiag ageatl rollingmotionwiththeindex fingerorwith aclean razorbladeandexaminedunder amicroscopetoselecthealthyspecimensi approx- imatelythesamestage ofdevelopment.Embryosfrom aminimumofthreespawnswillbecombinedand usedto initiatetesting.1fmorethan50% oftheeggsfrom givensubstratearefungusedorhavenotbeen fertilized, 00embryosfrom that substrate willbeusedin thetest. 001072 PROTOCOLNO: 454/061099/FAT-ELS/SUB454 3MLABREQUESNTO.U2723 WILDLIFE INTERNATIONAL, LTD -5- `Newly-hatchedlarvaewillbefedlivebrineshrimpnauplii (Artemiasp.)3timesperday duringthe frst7dayspost-hatch. Thereafter,thyslbefedlivebrineshrimpnauplii 3timesdailyonweekdaysand atleast 2timesdailyonweekendsuntilthetestisterminated.To casure thatth feeding ateperfishremains constant,theamountoffoodsuppliedtoeachreplicat testchambervil beadjustedon aweeklybasisto accountforlossesduetomortality. Fishwillnotbefedforapproxianately 48hourspriortothetermination ofthetesttoallowthe fish to cleartheirdigestivetractsbeforemeasurementsofweightaremade. Specificationsforacoeptable levelsof contaminantsin fishdietshavenotbeeaestablished.However,there aren0knownlevelsofcontaminantsreasonablyexpectedtobepresentinthedie thatareconsideredto interferewiththepurposeoFconductof thetest. Loading,definedasthe otalwetweightoffishperlite oftestsolution, willnotexceed0.5gramsof fishperlite ofsolutionthatpassesthrough achamberin 24hours. Instalonadtingawinllcnotoexucesed 5gramsoffishperltrofestsolutionpresentinthetes chamberatanygiveatime. DilutionWater `Waterusedfortheculturingandtestingoffatheadminnowswillbeobtained from awell approximately 40metersdecplocatedontheWildlifeInternational,Lid. site.Thewavit llbeepasrsed through asandfilterandpumpedinto a 37,800-Lstoragetankwherethewaterwil beaeratedwithspray nozzles.Priortousethewaterwillbe ilteredto0.45 uminordertoremovefineparticlesandthenpassed through aUVsterilizerinordertoremovemicroorganisms. Waterusedforculturingandtestingis characastmoederratielzyheardd.Typicalvaluesfor hardness,alkalinity,pHandspecificconductanceare. approximately: HAalrkdanliensist,y,mmgg//LLaassCCaaCCOOys 11405, pSpHecificConductance,pushos/cm 330. `Hardness, alkalinity,pHandspecificconductancewillbemeasuredweeklytomonitortheconsistency ofthewelwater. Meansandrangesofthemeasuredparametersfo thefour-wockperiodprecedingthetest willbeproivnthiefidnalreepodrt. Analysesvillbeperformedatleastonceanmuallytodeterminethe concentrationsofselectedorganic andinorganicconstituentsofthewellwaterandresusofthemostrecent `GLPcompliantanalyseswillbesummarizedinthe finalreport. 601073 PROTOCOLNO: 454/061099/FAT-ELS/SUB4S4 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD age `TestApparatus `A continuous-flowdiluterwillbeusedtoprovidecachconcentration ofthetest substanceand a negative (dilwautetr)cionotronl. Aperistalticpumpwillbeusedtodeliverthetestsubstancetomixing chamberswhere thetestsubstancevil bemixedwithdilutionwater,Theflow ofdilution waterintoeach `mixingchamberwill be controlled using rotameters. Therotameterswill be calibrated priortotestnation andatapprowexekily mintearvatlsteherleafyer. Aftermixing,testsolutionswillbe split to cach replicate chamber, The proportion ofwatersplittocach replicate will be checkedpriortothetestand at approximatelyweeklyinterval thereaftertoensurethattheseflowratesvarybynomorethan 10%ofthe `mean flowrateofthefour replicates. "Thediluterwil beadjustedsothateachtestchamberreceivesatleast volumeadditionsoftest solution every24hours. Peristaltic pumpswillbecalibratedbeforeeachstudyandatapproximatelyweekly intervalsthereafer, Thedelivery oftestsubstancetotetchambersvillbeginatleast48hoursprior tothe testinordertoestablishequilibriumconcentrationsofthe estsubstance. Thegeneraloperoafttheidioliner willbecheckedvisuallyatleas twotimesperdayduringthetestandatleastonceat thebeginningandend ofthetest. Embryoincubationcupswillbeconstructedfrom glasscylindersapproximately 50mmindiameter, with425-um nylonor Teflonscree attachedtothebottomusingsiliconesealant. Testchambweilrlbse 9:glassaquariafilledwithapproxi7mLaotfweatlery. Testchambersvillbepositionedin teperature`controledenvironmentalchambertomaintain temperatureof25 + 1C. Testchamwilblbeelabrellsed withprojectnumber,replicateandtestconcentration. Pavi Conti `Ambient roomlightwillbeusedto illuminatethetestsystems. Fluortuebesstchaetenmitt `wavelengthssimilartonaturalsunlight (c.g,Colortone 50)wilbecontrolledbyaaautomatictimerto provide aphotoperiodof 16hours oflightand 8hoursofdarkness. A 30-minute transofiltowilioghnt intensity willbeprovided whenlightsgoonandoffto avoidsuddenchangesin light intesy. Light intensityvillbemeasured atest intao ,atweeklyintervalsduringthetest,andatthecadofthetest.with a SPERScientific Lu.lightmeterorequivalent. Thetestwillbeconductedatatargettemperatureof25 2C. Tempewilrlbeamotnituorerdaend recordedcontinuouslyduringth entir estinonecontrolreplicateusing a FulscopeER/C Recarder(1900 JSeries ModelA)or equivalent. Recordermeasurementswillbeverified with aliquid-in-glassthermomeizr priortotestinitiationandatlastweeklythereafter.Thetemperaturein cachtestchamberalsovillbe PROTOCOLNO.: 454/061099/FAT-ELS/SUB454 3MLABREQUNOE.US27T23 01074 WILDLIFE INTERNATIONAL, LTD 7. measuredusing aliquid-in-glassthermometeratthebeginningofthetest,atweeklyintervalsduringthe est, andattheendofthetest. Dissolvedoxygenwillbemeasuredinalternatereplicatesofeach treatmentandcontrolgroup atthe beginningofthetest,dailyduring thefirst 7daysofthetes,atweeklyintervalsduringthetestandattest terminationusing aYellowSpringsInstrumentModel S1Bdissolvedoxygenmeter,ofequivalent.Inthe eventthatdissolved oxygenlevelsfalbelow 60%saturation,dissolvedoxygenmeasurementsvil bemade dailyineverytestchamberadappropriateactionswillbetakenaterconsultationwiththe Sponsor. Measurementsof pHvillbemadein siternatereplicatesofeachtreatmeatandcontrolgroupattestinitiation, at weeklyintervalsduringthetestandattestterminationusing aFisherAccumetModel 915 pHmeter,or equivalent. IF atrealteveml ereanchtes 100% mortality,temperature,dissolvedoxygen and pH. `measurementswillbetakeaattha time,thendiscontinued. Hardness,alkalinity,andspecificconductancewillbemeasuredinthedilution waterandinthe highesttreatment level a tetinitiation,atweeklyintervalsduring th testandattesttermination. Hardness andalkalinitymeasurementswillbe madebytitrationusingproceduresbasedonmethodsinStandard Methodsfor the ExaminationofWaterand Wastewater (4). Specific condwuillcbemteaasurnedcusieng aYellow SpringsInstrumMeodnetl 33 Salinity-TCempoernatduruemcetetr,iorveqiuitvaylen-t. `Procedures Prior totestinifiation,embryoincubationcupswillbeimpartiallyplacedinglassbeakerscontaining. dilution water within 3Cofthetesttemperature. Inordertocontrolbias,groupsofone,twoorthree. embryoswillbeindiscriminatelydistributedamong thecupsuntileachcontains20embryos. Noother `potentialsourcesofbiasareexpectedtoaffectth resultsofthestudy.Twoincucubpsawiltlbiepolacned ineachduplicatetestchambertoachieve atotalof40embryosperreplicateand 80embryospertreatment. Incubationcupswillbeattachedto 8rockerarm andsuspendedinthewatercolumnofthetestchambers. "Thereciprocatingmotion oftherockeram (approximately 2rmvillfacilitatecirculationoftestsolution aroundtheembryosduringtheincubationperiod. Duringthefirs dayofexposure,embryosvillbeobservedtwiceformortalityandeggswithfungus. `Thereafter,until hatchingi complete,embryomortalityandtheremovalofdead eambryosandorembryos withfunguswillbeperfoonrcemdaeildy. Afterhatch,larvaewillbecountedandreleasedintothetest chamber.Thetestwillberepeatedif thepercentageofembryosinth controlgroupthathatchessuccessfully islessthan66%,orif thepost-hatchsurvivalinanycontrolchamberislessthan 70%. 01075 PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO, U2723 WILDLIFE INTERNATIONAL, LTD "se `Whenhatchinghasreached >909%inthecontrolgroup,larvaewillbereleased the respectivetest cehmabmbreyrosoc.cAurnsy.uAnfbearttehhedemtibarlyroeslveiaslelobftkheepltairnvateh,etehgegexcpupossunrtevlithlely choanvtienhueaftocrheadolreanstl28ddeaaytsh.ofthe willbeDcatooln tlidmueertoicnhtatchhee,tehsdatt.chDiungestuocehsisg,hsnuartvuirvaall,moanraglritoywtthha(tvaertiwseesighr,odmyfuwnegiaglhitnfaenctitootnasldlnugrtihn)g bemebcroynosniidecrdeedvnealtouprmaelmnto,rtalallfng(uDs.Ae.dBcebray,ospetrhsaotnarlecroemmmouviecdadturoinn)g.thDeaifliyrsbts2c4rhvoautirosnosfoifnfcuybmatairontwail,l {ucnrumsiunaaltbieohna,vailors,uravnivdionvgerfailslhwaiplpelaberraentcaeiwnieldlfboermmeaadseudruermienngttshoef2w8e-tdawyepiogsht,dariychwegirgohwtt,hapnedrtiootda.lAItctehs.t SampliPnrgifoorrtoAneaxlpyotsiucraelwMaetaesrusraemmpelnetss il becollectedfomtworepicat estchambersofeachtreatment cShraomubpearnsdatththnebgeagtiinveicnognotfrotlhe. teWsatt,eornDseaympl4eosaflthseotvesitl,labfeapcporloextimeadtferloywmetewkolyalitneterrnvaatlisndgurrepilnictahteteesstt, oacncduartsithneacnaydtorfeatthemetnets,tth0ednestaemrpmliingcoonfctenhtarartaitonmseonfttiheltetsetrsmuibnsattaenfcoel.lIonwitnhgethveennxttthsatam1p0l0i%ngmionrttearlviatly Sampleswillbecollectedat mid-depthfromeachtest chamber andanalyzedimmediately.Thesample schemeissummaribzeleowd: PROPOSED NUMBERS OF VERIFICATION SAMPLES _ : pmimmalGrow__Peit_Duy0 Dud Du? Dwld Dual Dum Ed Con > 2: 2 2 2 2 2 LodllovConminion 2 2 2 2 2 2 2 2 Lowi 2 2 2 2 2 2 a2 2 Lowi3 2 2 2 2 2 2 a2 2 Leva I SE Levis 2 2 2 2 2 2 a2 2 LoGHghConomimin 2 2 22 2 2 2 2 Tos Ww aw ww uw reDcirvesd,mdelpeendiingllpboocnlhelSponssoercdos,inndadodfitonialtes,plMiengrseadhelnsrlsbt ospuimennedenrb ybe us dn inth ol uprt TodNumberofVeSipen =112 01076 PROTOCOL NO: 454/061099/FAT-ELS/SUB4S4 MLAB REQUESTNO, U2723 WILDLIFE INTERNATIONAL, LTD "9. `Theabove numbersof samplesrepresentthosecollectedfromthetestanddonotincludequality control (QC)samplessuchasmatrixblanks andfortificationspreparedandanalyzedduringtheanalytical chepmhaiseosftthesrtudyy. At thediscretionoftheStudyDirector,water samplesalsowillbecollected fromatleastoneappropriatechamber whenever amalfunctionisdetectedinanypartof thetestsubstance deliverysystem. AnalyticalMeasurements Chemical analysisofthesampleswillbeperformedbyWildlifeIntemational,Ltd.The analytical `methodusedwillbebased uponmethodologyprovided by theSponsorand identifiedin AppendixII. The `methodology usedtoanalyzethetestsampleswillbedocumentedinthe rawdataandsummarizedinthe final report. DataAnalyses "Theendpointsusedtodeterminethe statisticalsignificanceoftoxicant-relatedeffects onfathead `minnowsinclude hatchingsuccess, survivalofjuvenilefish, otallength,wetweight anddryweightof survivingfish Thedatasetswillbeevaluatedfornormality andhomogeneityofvariances. Data transformationswillbeemployed,ifnecessary,tocorrectthedatafor non-normalityorheterogeneous `variances. `Wheathe datameettheconditions ofnormalityandhomogeneityofvariances,testsforsignificant differenceswilluse 2 X 2contingencytablesandanalysisofvariance (ANOVA)procedurewithan `appropriatemeanstest(e.g, Dunnett'stest,Duncan'stest, Tukey'stest,etc.). Whentheconditioonfs `normalityandhomogeneityofvariancesarenotmet,andno transformations are successfulincorrectingthe `condition,thennonparametricstatisticalprocedureswillbe used.Theresultsofthestatiasnatlyisecswailll aidinthe deteromf tihen NOaECt anid LoOEnC. Themaximumacceptabletoxicantconcentrawtililobne calculatedas thegeometricmeanofthe NOEC and LOEC. RECTOBOEMR AIND TAIS NED N 01077 limited to: . RecordstobemaintainedfordatageneratedatWildlifeInternational,Ltd.willincludebutnotbe 1. Acopy ofthe signedprotocol. PROTOCOLNO.: 454/061099/FAT-ELS/SUB4S4 3MLABREQUNOE.US27T 23 WILDLIFE INTERNATIONAL, LTD -10- 2. Identificationandcharacterizationof thetestsubstance,ifprovidedby the Sponsor. 3. Dates ofinitiation andterminoaftthietoesnt. 4. History ofthetestorganism. 5. Weight and lengthmeasurements. 6. Results of rangetesfts,iwhnend appilicnablge. 7. Stocksolutioncalcuanldapretpairatoionn. . 8. Obsermvadae dturiingtohentesst. 9. Waterchemistrycalculations (c.g,hardnessandalkalinity). 10. Ifapplicable,themethodsusedtoanalyze testsubstanceconcentrationsandtheresults ofanalytical `measurements 11. Statisticalcalculations. 12. Testconditionsandphysical/chemicalmeasurements. 13. Calculationandpreparation oftestconcentrations. 14. Copyoffinalreport. FINALREPORT Afinalreportof theresultsof the studywillbepreparedbyWildlifeInternational,Ltd.Thereport willinclude,butnotbelimitedto, thefollowing,whenapplicable: 1. Nameandaddressofthefacility performingthe study. 2. Datesuponwhichthe studywasinitiatedandcompleted. ItistheresponsofithbeSiplonisotrtyo providethe inaldatsthatdataarerecordedforchemistrypathologyand/orsupportingevaluations that maybegeneratedatotherlaboratories. 3. Astatementofcompliancesignedbythe StudyDirectoraddressing anyexceptionstoGood LaboratoryPractice Standards. 4. Objectivesandproceduresas statedintheapprovedprotocol,includinganychangesintheoriginal protocol. 5. Thetestsubstance identification,includingname,chemicalabstractnumberorcodenumber,strength, purity,composition,andothercharacteristics providedbythe Sponsor. 6. Stability andsolubilityofthetestsubstanceundertheconditionsofadministration,ifprovided bythe Sponsor. 7. Adescriptionofthemethodsusedtoconductthetest. 619078 PROTOCOLNO.: 454/061099/FAT-ELS/SUB454 3MLABREQUNOE. US27T 23 WILDLIFE INTERNATIONAL, LTD n- 8. Adescription ofthetestorganisms,includingthe sourceof thetestorganisms,scientificname,age, life stage, meansandrangesofweightsandlengths, observeddiseases and treatments. 9. Adescription ofthepreparationofthetestsolutions,themethodsusedtoallocateorganismstotest chambers andbeginthetest,thenumberoforganismsandchamberspertreatment,andtheduration ofthe test. 10. Adescription ofcircumstancesthatmayhaveaffectedthequalityorintegrityofthedata. 11. Thename ofthe StudyDirectorandthenamesofotherscientists,professionals,and supervisory `personnelinvolvedinthestudy. 12. Adescriptionofthetransformations,calculations,andoperationsperformedonthedata, asummary andanalysisofthebiologicalandanalyticalchemistrydata, and statementoftheconclusionsdraw. fromtheanalyses. 13. Thesignedanddatedreportsof eachoftheindividualscientistsorotherprofessionalsinvolvedinthe: `stif auppdlicyabl,e. 14. Statisticalmethods employedforanalyzingthedata. 15. The locationwhere rawdata andfinalreportareto be stored. 16. AstatementpreparedbytheQuality AssuranceUnitlistingthedatesthatstudyinspectionsandaudits `weremade andthe dates ofany findingsreported to theStudy Directorand Management. 17. Ifitis necessarytomakecorrectionsoradditionsto afinalreportafterithasbeenaccepted,such changeswillbemadeintheform ofanamendmentissued by the StudyDirector. Theamendmentwill clearlyideatifythepartofthe finalreportthatisbeing amendedandthe reasonsforthealteration. `Amendmentswillbesignedanddatedbythe StudyDirector. CHANOFGPRIOTNOCG OL Plannedchangesto theprotocolwillbeinthe formofwrittenamendmentssignedbytheStudy Directorandthe Sponsor'sRepresentative. Amendmentswillbeconsideredaspartoftheprotocolandwill be attachedtothe finalprotocol. Anyotherchangeswillbeintheformofwrittendeviationssignedbythe `StudyDirectorand filedwiththerawdata. Allchangestotheprotocolwillbeindicatedinthefinalreport. GOODLABORATORY PRACTICES `This studywillbeconducted in accordancewithGoodLaboratoryPractice Standardsfor EPA (40 CFR Part160and/or Part792); OECDPrinciplesofGoodLaboratoryPractice(ENV/MC/CHEM(98) 17); PROTOCOLNO.: 454/061099/FAT-ELS/SUB454 3MLABREQUNOE.US27T23 01079 E We ILE DLIFE INTERNAA e TIONAL, LTD e. 12- and Japan MAFF (59 NobSan, Notification No. 3850, Agricultural Production Bureau). Each study `conducted by Wildlife International,Ltd.isroutinely examined by theWildlifeInternational,Ltd.Quality AssuranceUnit forcompliancewith Good LaboratoryPractices, StandardOperating Procedures andthe specifiedprotocol. Astatementofcompliance with GoodLaboratoryPractices willbepreparedforall `portionsof the studyconductedby Wildlife International, Ltd. The Sponsor willberesponsibfloer `compliancewithGood LaboratoryPractioes forproceduresperformedby otherlaboratories (.g.,residue. analysesorpathology). Raw.dataforallworkperformedatWildlifeInternational,Ltd.anda copyofthe final reportwillbe filedbyprojectnumberinarchives locatedonthe WildlifeInternationalLtd.site,or at `20altemativelocationtobespecifiedinthefinal report. 7 PROTOCONOL.: 454/061099/FAT-ELS/SUB4S4 01080 3MLABREQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD "13 REFERENCES 1 U.S. Environmental Protection Agency. 1996. Series 850- Ecological Effects Test Guidelines (draft), OPPTS Number 850.1400: Fish Early-Life Stage Toxicity Test. 2 ASTM Standard E1241-92. 1977. Standard Guidefor ConductingEarlyLife-Stage Toxicity TestswithFishes. AmericanSocietyfor TestingandMaterials. 3 United States Eavironmental Protection Agency. 1986. StandardEvaluation Procedure, Fish Ea13r8l.yLife-Stage Test. OfficeofPesticide Programs, Hazard Evaluation Division. EPA 540/9-86- 4 APHA, AWWA, WPCF. 1985. Standard Methods for the Examination ow Water and Wastewater. 16" Edition, American Public Health Association. Ame Watr erWoi rksc Assoa ciatn ion. `Water Pollution Control Federation, New York. PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 01081 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD 14. APPENDIX IDENTOFITEFST SIUBCSTAANCETBYISPOONSNOR TobeCornpblySptonesodr I Test SubstanceIdentity (nametobeuseinthereport): PEOS(PerflucroastaneSulfonicAcidPotassiumSalt RefercoceStandard (fapplicable): Analytical Standard: NA Internal Standard:_1.1.2.2HHHHPerluorooctaneSulforieAcid Test Substance SamCopdeloreBa.ch Number: _Lot217 TestSubstancePurity(%ActiveIngredient): _ 98.9 `Expiration Date: _2008 IL TestSubstanceCharacterization Hi tphreodperniat sgh,ptye mSeucobmpsotsmie txionefeoreercSsctheaadrS ocridbseiyces Yo _x No IL Test SubsStetragaeConnccioens Pleas indicatetheecernmendedstorageconditionsofWifeIntemational,Ld. Abe Hasthe sta ably substance nertheseSstyageconditions Ys x No IV. TestConoentrations: V. TonicityInformation: : xbeeAddjustpteastncotn(o4ie)negtavpiaosrntbioon1y0.0% ai DaoinTetsatSsortoactetricaol cAmSiSc.nto 10%. Mma: RatLDSO 251mg/kg MouseLDSO N/A Aquatic: Invertebrate Taiciy(EC/LCS0) Fish Toxicity(LCS0) `OtherToicyInformation ochling findingsofchronicandsubchronic ests: B0 lesse0 seMs0 DS 0000000000 PROTOCONOL.: 454/061099/FAT-ELS/SUBAS4 01082 3MLABREQUESTNO. U2723 WILDLIFE INTERNATIONAL, LD "154 APPENDIIXI `AnMaethlodyProtvideidbycSpaonslor Sampleswillbeanalyzedbaseduponproceduresprovided by the Sponsorinthefollowing analytical 1. LiquidChromatographyMassSpectrometry (LCMS)Methodforthe Determination of Perfluorooctane Sulfonic Acid PotassiumSalt(PFOS)InFreshwater, SaltwaterandAlgal Medium Acopyoftheabovemethodwillbemaintainedinthe rawdata.Theactualmethodoulsoedgtyo `analyzethetestsampleswillbedocumentedintheraw data andsummarizedin thefinalreport. PROTOCOLNO.: 454/061099/FAT-ELS/SUB4S4 01083 3MLABREQUENSOT. U2723 --N WI--LD--LT I--FE E INTER RNATN IONALA LTD.TIONA PaPPaRggOeeJL l1EooCfTf1tNOt .: 454D A-108. * AMENDMENT TO STUDY PROTOCOL, STUDY TITLE: FPAFOTSH:EAAND EMAIRNLNYOLWI(FPEi-mSepThAaGlEesTpOroXmIeClaIsT)Y TEST WITHTHE PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 SPONSOR: 3M Corporation EFFECTIVE DATE: August 2, 1999 AMENDMENT NO.: 1 PROJECT NO.: 454A-108 AMENDMENT: Page? Add: Proposed Dates: Experimental Start Date: 8/5/99 Experimental Termination Date: 9/6/99 REASON: The above Director. information was not known when the protocol was signed by the Study STUDY DIRECTOR Off DATE L. ORY MANAGEMENT DATE SPONSOR'S REPRESENTATIVE DA clsattnnend 0108% Roniomod hras/ad R309