Document B8kzEpq6yDbZegX4GRE0qD92j

PFOS: A Se Cladoceran(( . CHRONIC TOXICITYTO FRESHWATER INVERTABRATES(Daphnimaagna) TEST SUBSTANCE IdentitPye:rfluorooctanesulmfaoynatles;obereferrteodas PFOS or FC-95. (1-Octanesulfoniaccid,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-p,otassiumsaltC,AS # 2795-39-3) Remarks: The testsubstanceisa whitepowder. Sample was takenfrom 3M lotnumber 217. Sample was storedunder ambientconditionspriorto testingP.uritydeterminedto be 90.49% by LC/MS, 'H-HMR, 'GF-NMR and elementalanalysestechniques. METHOD Method: OPPTS 850.1300,OECD Guideline211,and ASTM StandardE 1193-87. Type: Semi-StaticLife-CyclTeoxicity GLP: Yes Year completed:Studycompleted1999. Reportcompleted2000 Species:Daphnia magna Supplier:In-houseculturesW,ildlifIenternationLatld,.E,aston,MD Analyticalmonitoring:PFOS measured on days 0,2,11,14,18,and 21. Exposure period:21 days Statisticamlethods: Survivaldatawas evaluatedon flrst-generation daphnids,thenumber ofliveyoung and the lengthand dryweightofthe survivinfgirst-generatdiaopnhnids. Survivaldatawere analyzedusing Fisher'esxacttest.Reproductionand growth(lengthand dryweight)data were evaluatedfornormalityusingShapiro-Wilk'tsestand for homogeneity ofvar* ianceusingBartlettt'esst.Analysisofvarianceand Dunnett'stestwas used to identiftyreatmentgroupsthatwere statistically significanitncomparisonto thenegativecontrol(p :s0.05).Allstatistical testswere performedusinga personalcomputerwithSPSS/PC Version 2.0 or'70XSTAT Release 3.5"statisticsaolftware. Test organism age: < 24-hoursoldattestinitiation Pretreatment:None Test conditions Dilutionwater:0.45 @Lmfilterewdellwaterpassed througha UV sterilizteorremove microorganismsand fineparticles Dilutionwater chemistry (duringthe 4-week period immediately preceding the test): hardness: 124 (120-128)mgll as CaC03 alkalinity: 169 (164-172)mg/L as CaC03 pH: 8.2(8.0-8.3). TOC: < 1.0mg/L Conductivity3:29 (315-340@)Lmhos/cm Ca/Mg ratio:35/13.5 Na/K ratio: 21.3/6.62 Stockand testsolutiopnreparatioPnr:imarystocksolutiwoans prepared indilutiownater at46 mg/L. Itwas stirreudntilalltestsubstance was dissolvedpriortouse. Aftermixing,the primarystocksolutionwas proportionalldyilutedwithUV sterilizeddilutiownater topreparefive additionalstocksolutionsat nominal concentrationsof1.4,2.9,5.7,11, and 23 mg/L. Alltestsolutionsappeared clearand colorless. Renewal rate:Every Monday, Wednesday and Fdday. Exposure vessels:250-mL plasticbeakerscontaining approximately200 mL testsolution.The depth was approximately 5 cm. ': Number ofreplicates1:0 Number oftestorganisms perreplicate1: Number ofconcentrations6:plusa negativecontrol Feeding:Each testchamber was fed0.3mL ofYCT (amixtureof yeast,Cerophyllo,and troutchow at 1800 mg TSS/L) and 0.60 mL ofSelenastrum capticomutum (3.5x 107 cells/mL)once daily. Lighting:Colortone@50 fluorescenltights.Intensitryanged from 329 -383 luxatthewater surface.Photoperiodof 16-hourslight8,hours dark witha 30-minute transitiopneriod. Water chemistry of new and old solutions during the study: Dissolved oxygen range (0- 21 days): 8.3- 8.9 mg/L (negativecontrolexposure) 8.3- 9.0mg/L (12mg/L exposure) 8.4- 8.9 mg/L* (48 mg/L exposure) pH range (0- 21 days) 8.1- 8.4 (negativecontroelxposure) 8.2- 8.5 (12 mg/L exposure) 8.4- 8.5*(48 mg/L exposure) Test temperature range (0- 21 days) 19.4- 20.1OC (negativecontrolexposure) 19.4- 20.1OC (12 mg/L exposure) 19.4- 19.50C*(48mg/L exposure) (Measurements discontinuedatDay 3 due to 100% mortality. Element basis:Survival,reproductionand growth. Effectconcentrations based on survival. Method ofcalculatimnegan measured concentrationasr:ithmetic mean RESULTS Nominal concentrations:Negative control,1.4,2.9,5.7,11,23, 46 mg/L Measured concentrations: <LOQ, 1.5,2.9,5.6,12,24,48 mg/L Element value: 21-day NOEC 12 mg/L 21-day LOEC 24 mg/L 21-day MATC 17 mg/L 2 nd generationacute survivalNOEC = 12 mg/L Allelement valuesbased on mean measured concentrations AnalyticalMethodology: Analyses oftestsolutionswere performed at WildlifeInternationaLltd.using highperformance liquicdhromatography with mass spectrometridcetection(HPLC/MS). When determiningthe concentrationofthetestsubstance inthetestsolutionst,hesame and most prominentpeak response forthe testsubstancewas used. No attemptwas made toquantifyon the basisofindividuailsomeric components. The LOQ (limiotfquantitatiowna)s 0.458 mg/L inthisstudy. The mean proceduralrecoveryofmatrixfortificatiaonnaslyzed concurrentlyduringsample analysiswas 104%. Measured values ofnew samples ranged from 94 to 121% ofnominal. Measured valuesfrom the old solutionsranged from 90 to 108% ofnominalvalues.PFOS was stable throughoutthe renewal periods. Summary ofanalyticcahlemistrdyata: NominalTest Concentration, mg/L Mean Measured Percentof Measured DuplicateValues at0,2,11, 14,iB.and Concentration, Nominal 21 Days, Respectively,mg/L mgil Negafive Control All< LOO - - 1.4 1.78,1.72.1.58,1.56,1.38,1.47,1.36,1.32,1.38. 1.5 107 1.43,1.50,1.45 2.9 3.20,3.05,3.01,3.07,2.75,2.77,2.85,2.71,2.79. 2.9 100 2.81,2.81,.2,82 5.7 5.97,5.87,5.65,5.72,5.63,5.59,5.36,5.39,5.58, 5.6 98 5.75,5.24.5.37 11 11.5, 11.6,11.5,11.3 12 109 23 24.2,23.1,24.0,24.6.22.8,22.5,23.6,23.1,24.8, 24 104 26.0,alldaphnidsdead after18-days exposure 46 47.3,48.0.49.1,49.4,alldaphnids dead after2-days 48 104 exposure I NOTE: Mean measured concentrationwsere determinedfrom new (renewal solutionsa)nd correspondingoldsolutionsduringeach week ofthetest.Days 0, 11,and 18 are"new"and days 2,24,and 21 are "old". Biologicalobservations Survival:Al'lsurvivinfgirsgtenerationdaphnidsappeared normal attestterminationS.urvivalinthe24 and 48 mg/L treatmentswas statisticaslilgynificantdliyfferenftrom thenegativecontrolgroup. Reproduction:Daphnids inthe controland treatmentgroups :512 mg/L startedproducingneonates on Day 9. The Bonferronit-test showed thatreproductiownas notsignificantrleyduced inany treatmentgroup :512mg/L (p>0.05).The 24 and 48 mg/L treatmentgroupswere notincludedinthestatisticaanlalysisofthe reproductiodnatadue toa statisticaslilgynificaenftfecton survival. Growth: The Bonferronit-tessthowed thatmean lengthand dry weightinthetreatmentgroups :512mg/L were notsignificantly reducedincomparisontothenegativecontrol(p> 0.05). Second GenerationAcute Exposure: After48-hoursofexposure, survivailnthenegativecontrolwas 95%. Survivalinthe 1.5,2.9, 5.6,12,and 24 mg/L treatmentgroupswas 100, 100,100,90,and 0% respectivelyS.urvivalinthe24.mg/L treatmentgroup was significantdliyfferenftrom thenegativecontrol(p :50.05). Summar@y Mean Measured Concentration, mg/L PercentMortality Day 7 Day 14 NegativeControl 0 0 1.5 0 0 2.9 0 0 5.6 0 0 12 0 10 24 70 90 48 100 100 Day 21 0 10 10 10 10 100 100 Second GenerationMortality Mean Measured Concentrationsm,gA. Total Number Exposed Number Cumulative Aliveafter Percent 48-hours Dead NegativeControl 20 19 6 1.5 20 20 0 2.9 20 20 0 5.6 20 20 0 12 20 18 10 24 8 0 100 Summary of Length and Dry Weight ofSurviving Individually-ExposeFdirst-GeneratioDnaphnids Mean Measured Concentrationm,g/L Number of TotalLength,Mean DryWeight,Mean + SD, Surviving + SD, mm mg Daphnids NegativeControl 10 1.5 9 2.9 9 5.6 9 12 9 24 0 48 0 4.65+0.111 4.66+0.118 4.62+0.100 4.61+0.124 4.59+0.102 0.695 + 0.100 0.669 + 0.0623 0.724 + 0.110 0.727 + 0.0665 0.723 + 0.0661 Reproduction Mean Measured Concentration,mg/L Number of Surviving Daphnids Mean LiveYoung SurvivingAdult Daphnid (+SD) Negative Control 10 122+19.2 FirstDay of Reproduction TotalNumber of TotalNumber of Dead / Immobile Aborted Eggs Neonates 9 0 0 1.5 9 142+24.7 9 0 0 2.9 9 136+17.9 9 0 0 5.6 9 132+19.6 9 0 0 12 9 119+26.5 9 1 0 24 0 48 1 0 1 11 10 0 None CONCLUSIONS There were no adverseeffectson survivalr,eproductioonr growthof Daphnia magna exposed tothetestsubstanceatconcentratio<n12 mg/L for21 days. Daphnia magna exposed to 24 and 48 mg/L had significantly reduced survival. Authorand/orsubrnifter:3M Company, EnvironmentalLaboratoryP,.O. Box 33331, St.Paul,Minnesota,55133 DATA QUALITY ReliabilityK:imisch ranking1. REFERENCES Thisstudywas conductedatWildlifIenternationaLlt,d.Easton,MD atthe requestof the3M Company. OTHER Last changed: 5/3/00 PFOS: A SENH-STATIC LIFE-CYCLE T03UCITY TEST WITH THE CLADOCERAN (Daphniamagna) FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A- 109 3M LAB REQUEST NO. U2723 U.S. EnvironmentalProtectioAngency Series850 - EcologicaElffects OPPTS Number 850.1300 OECD Guideline211 AUTHORS: KurtR- Drottar Henry 0. Krueger,Ph.D. STUDY INITIATION DATE: July2,1999 STUDY COMPLETION DATE: February28,2000 AMENDED REPORT DATE: April26,2000 Submittedto 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St.Paul,Minnesota 55144 WildlifeInternationalL,td. 8598 Commerce Drive Easton,Maryland 21601 (410)822-8600 Page I of77 AMENDED WILDLIFE INTERNATIONAL LTD. -2- PROJECTNO.: 454A-109 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR- 3M Corporation TITLE: PFOS: A Semi-StatiLcife-CyclTeoxicityTestwiththeCl&ioceran(Daphniamagna) WILDLEFE R4TE RNATIONAL LTD. PROJECT NUMBER. 454A-109 STUDY CONTLETION: Febnjary28,2000 AMENDED REPORT: April26,2000 This study was conductedin compliancewith Good LaboratoryPracticeStandardsas publishedby the U.S. EnvironmentalProtectioAngency in40 CFR Parts160 and 792, 17 kugust1989;OECD Principleosf Good Laboratory Practic(eENV/MC/CHEM (98) 17);and JapanMAFF, 59 NohSan, NofificafionN3o8.50,AgriculhirParloductioBnureau, 10 August 1984,with thefollowingexceptions: The testsubstancewas notcharacterizeidnaccordancewithfiffGlLP compliance;however,thecharacterization was performedaccordingto3M StandardOperatingProceduresand Methods,and allraw dataarebeingmaintainedinthe 3M =hives. The testsubstanceisbeingrecharacteriziendaccordancewithGLP. The stabilitoyfthetestsubstanceunderconditionosfstorageatthetestsitewas notdeterminedinaccordancewith Good LaboratoryPracticeStandards. STUDY DIRECTOR. ,00 1 1 : KurtP, ijrottar DATE SeniorBiologist SPONSO APPROVAL: Sponsor Y197 /,M IYATE AMIENDED WILDLIFE INTERNATIONAL LTD. -3- PROJFCTNO.: 454A-109 QUALITY ASGURANCE STATEMIENT Thisstudywas ommined forcompliancewithGood LaboratoryPracticSetandardsas publishedby theU.S. EnvironmentaPlrotectioAngency in40 CFR Parts160 and792,17 August 1989;OECD Principleosf Good Laboratory Practic(eENV/MC/CHEM (99)17;andJapanMAFF, 59 NohSan,NofificatiNoon. 3850,AgiculturaPlroductioBnureau, 10 August1984. The datesof allinspectionasndauditsandthedatesthatanyfindingwserereportetdotheSb* Dhwtor andLaboratoryManagement were asfollows: ACITV=. Test SubstancePreparation Test DiWation Observafions DraftReport and Data AnalyticalData and Dmft Report FinalReport Amended Report DATE CONDUCTED: July9, 1999 July12,1999 July27, 1999 October 12 and 13, 1999 October 12 and 13, 1999 February28,2000 April25 and 26,2000 DATE REPORTM TO: STUDYDIRECTOR.- NIANAGENEENT: July9, 1999 February23, 2000 July12,1999 July 13,1999 July27, 1999 July28, 1999 October 13, 1999 October 13, 1999 October 14, 1999 October 14, 1994 February2g,2000 February 28,2000 April26,2000 Apr.i2l6,2000 1:@a@ H Jar&6sH. Coleman QualityAssuranceRepresentative 6DATE AMENDED WILDLIFE INTERNATIONAL LTD. .4- PROJFCTNO.: 454A-109 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: P.FOS:A Semi-StatiLcife-CyclTeoxicitTyestwiththeCladoceran(Daphniamagna) WU,DLIFE INTERNATIONAI@,LTD. PROJECT NUMBER: 454A-109 STUDY DIRECTOR: KurtR- Drottar SeniorBiologist MANAGEMENT: Herny0. km@ier,*I)-. DirectorA,quaticToxicologyand Non-TargetPlants DATE DATFE ANffi"NDED WILDLIFE INTERNATIONAL LTD. -5- PROJFCTNO.: 454A-109 TABLE OF CONTENTS Title/CovePrage........................................................................I................... Good LaboratoryPracticCeomplianreStatement..............................................2................... QualityAssuranceStatement..............................................................3................... ReportApproval.......................................................................4................... Tableof Contents.......................................................................5................... Summary ..............................................................................7.................. Introducti.o.n..........................................................................8.................. Objectiv.e.............................................................................8.................. ExpmimentalDesign....................................................................8................... Materialasnd Methods ...................................................................9.................. R.esultasnd Discussion..................................................................1.3.................. Conclusion.s................................................................................1..4........................... References.......................:............................................................................1.5.................. TABLES Table1 - Summary ofAnalyticaClhemistryData.............................................1.6.................. Table2 -TemperatureofWater in,theTestChambers .........................................1.7.................. Table3 -DissolvedOxygen Contentof Water intheTestChambers ..............................1.9................. Table4 -pH ofWater intheTestChambers .................................................2.1.................. WILDLIFE INTERNATIONAL LTD. -6- PROJECTNO.: 454A-109 TABLE OF CONTENTS Continued- TABLES (Continued) Table5 - SpecifiCconductanceH,ardnessandAlkalinitiyntheNegativeControl and HighestTreat3nenGtroup TestChambers .......................................2.3.................. Table6 - Summary of Cumulativ-ePercentMortalityand Treatinent-RelatEefdfects forFirst-GeneratiDoanphnids....................................................2.4.................. Table7 - Summary ofDaphnia magna ReproductionDuringtheLife-CyclTeoxicityTest............2.5......... Table8 - Summary ofLengthand Dry Weight of SurvivingIndividually-ExposFeidrst-Generation Daphnids.....................................................................2.6.................. Table9 - CumulativePercentMoMUty/Immobilityand Treatment-RelateEdffectfsor Second-GeneratioDnaphnids.....................................................2.7.................. APPENDICES Appendix I- SpecifiCconductance,Hardness,AlkalinitayndpH ofWell Water Measured Duringthe4-Week PeriodImmediately'PreceditnhgeTest..............2.8........... Appendix II- AnalysesofPesticideOsr,ganics,Metalsand OtherInorganics inWildlifIenternationLatld.WellWater....................................2.9.................. Appendix III- The AnalysisofPFOS inFreshwaterinSupportof WildlifIenternationLatld.ProjecNto.: 454A- 109............................3.0.................. Appendix IV CumulativePercentMortalitaynd Treatinent-RelatEefdfect-s First-GeneratiD.aopnhnids...............................................4.9.................. Appendix V NeonateProduction.....................................................7.0.................. Appendix VI LengthandDry Weight ofSurvivingFirst-GeneratiDoanphnids.................7.2............... Appendix VU Changes totheProtoco.l.................................................7.4.................. AppendixVIII PersonnelInvolvedintheStudy...........................................7.5.................. Appendix IX ReportAmendment .....................................................7.6.................. AMENDED WILDLIFE INTERNATIONAL LTD. -7- PROJECT NO.: 454A-109 SPONSOR: SPONSOR'S REPRESENTANVE: LOCANON OF STUDY, RAW I DATA AND A COPY OF TBE FINAL REPORT: SUNUDARY 3M Corporation SusanA. Beach WildlifIenternationLatlcL Easton,Maryland 21601 WILDLIFE DnERNAnONAL LTD. PROJECT NUMBER: . TEST SUB STANCE: STUDY: NOhffNAL TEST CONCENTRANONS NEAN NEASURED CONCENTRATIONS: TEST TEST DATES: LENGTH OF TEST: TEST ORGANLSHC SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: NOEC: LOEC: g MATC: 454A-109 PFOS (PerfluorooctaSnuelfoniAccid PotassiumSalt) PFOS: A Semi-StatiLcife-CyclTeoxicitTyestwiththe Cladoceran(Daphniamagna) NegativeControl,1.4,2.9,5.7,11,23 and46 mg a.i.[L NegativeControl,1.5,2.9,5.6,12,24 and48 iiiga.i.AL ExperimentaSltart- July12,1999 BiologicaTlerminatio-nAugust2,-1999 ExperimentaTlerminatio-n August4, 1999 21 Days NeonateCladocera(nDsaphniamagna) WildlifeInternationLatlcLcultures Easton,Maryland 21601 Juvenile<s 24 hoursold 12 mg a.i.AL 24 mg a.i./L 17 mg a.i.AL ANEENDED WILDLIFE INTERNATIONAL LTD. "8 PROJECT NO.: 454A-109 INTRODUCNON ThisstudywasconductebdyWildlifIenternatioLntaclLfor3M CorporatiaotntheWildliWfaemationaLltda.quatic toxicologfyacilitiynEaston,Maryland.The in-lifpehaseofthetestwas conductedfrom July12,1999 toAugust2,1999. R.awdatageneratebdy WildlifeInternationLatld.and acopy ofthefinarleportarefileudnderProjectNumber454A- 109in arcluvelsocateodn theWildlifeInternationLatlcLsite. OBJECTIVE The objectivoefthisstudywas todeterminetheeffectosfPerfluorooctaSnuelfoniAccidPotassiumSalt(PFOS) m thesurvivalg,rowthand reproductioonfthecladoceran(Daphniamagna) duringa 21-dayexposureperiodundersemistatitcestconditions. EXPERIMIENTAL DESIGN Daphnidswereexposedtoa geometricserieosf sixtestconcentratioannsd anegative(dilutiownater)controlT.en replicatteestchamberswere maintainedineach treatmentand controlgroupwithone daphnidin eachtestchamber. Nominal testconcentrationwsere selecteidn consultatiowniththe Sponsor,and werebased upon theresultosf an exploratorryangefindingtoxicittyest.Nominal testconcentratiownesre 1.4,2.95,.7,11,23 and 46 mg a.i.ALM.ean measuredconcentratiownesredeterminedfrom new and correspondinogldsolutiondsuringeachweek oftheteslDaphnids weretransferretdonew solutionesveryMonday, Wednesday andFridayduringthetest. First-generatidoanphnidswere observeddailyforsurvivalt,heonsetofreproductioann,d clinicaslignsoftoxicity. With theonsetofreproductiont,henumber of second-generatinoenonateswerecounted(numberofliveneonatesn,umber of deadorimmobileneonatesand number ofabortedeggs)everyMonday, Wednesday and Fridayduringthetest.If availablet,wentysecond-generationneonatesfrom eachtreatmentand thecontrolwere exposedunderstatitcestconditions for48 hours.At testtemiinationt,helengthand dryweightofeachfirst-generatdiaopnhmd was determined.The noobserved-effect-wneentra(tNiOoEnC) andlowest-observed-effect-concent(.LrOaEtCi)ownere&tmined byexamination ofthemortalitgyr,owthandreproductiodnata.The maxunum acceptablteoxicanctm=tr-&on (MATC) wascalculataesd thegeometricmean oftheNOEC andLOEC. AMENDED WILDLIFE INTERNATIONAL LTD. -9- PROJECTNO.: 454A-109 MATERL4J,SAND METHODS Thestudwyasconductbeadseodntheproceduroeustlinientdheprotoc"oPlFOS:A Semi-StaLtiifce@CyTcolxeicity TeswtiththeCladocer(aDnaphnimaagna)"T.heprotocwoalsbaseodnproceduroeustliniendtheU.S.Environmental ProtectioAngency Series850 - EcologicaElffectTsestGuidelineOsPPTS Number 850.1300(1);OECD Guidelin2e11: Daphnia magna ReproductionTest(2);StandardEvaluationProcedure,Daphnia magna Life-(7),(d2e1-DayRenewal) ChronicToxicitTyest(3);andASTM StandardE 1193-87,StandardGuideforConductingRenewalLife-CyvlTeoxicity TestswithDaphnia magna (4). TestSubstance The testsubstancewas receivedfrom 3M Corporatioonn October29,1998 andwas assignedWildlifIenternational Ltd.idenfificatniuomnber 4675. The testsubstancewas describeadsa whitepowder.Itwas identifiaesdFC-95 fromlot number 217 (T-6295).Informatiopnrovidedby theSponsorindicateadpurityof 98.9%,andan expiratiodnateof2008. The testsubstancewas reanalyzebdy theSponsorand theCertificaotfeAnalysisdatedMarch 9,2000 in&catedapuritoyf 90.49%. The testsubstancewas storedatambientroom temperature. Pnaration ofTestConcentrations A primarystocksolutiownas preparedby addingthetestsubstancteoUV sterilizdeidlutiownaterataconcentration of46mga.i./L.The testsolutiownas stirrewdithan electrsitcainlessteetlop-downmixeruntilallthetestsubstar=was dissolvedT.he primarystocksolutiownas proportionaldliylutewdithUV sterilizdeidlutiownatertopreparetheotherfive testconcentrationNse.w solutionwsere preparedforeachrenewalday.Aftermixing,alltestsolutionasppearedclearand colorlessA.llreportedtestconcentratiownesre adjustefdorthepurityoftheactiveingredienitnthetestsubstance (90.49%). TestOr&am@sm The cladoceran,Daphnia magna, was selectedas the testspeciesfor thisstudy. Daphnids are representativeof an unportantgroupof aquaticinvertebrataensd were selectefdoruseinthetestbasedupon pasthistoryofuseand easeof culturinignt-helaboratoryD.aphnid neonatesusedinthetestwerelessthan2.4-hoursoldand wereobtainedfromcultures maintainedby WildiffheiternationLatld.,Easton,MarylancL AMENDED WILDLIFE INTERNATIONAL LTD. -10- PROJECTNO.: 454A-109 Adultdaphnidswere cultureadtapproximateltyhesame temperaturaesusedduringdw testinWildlifIentm=onaL Ltd.wellwater.Daphnidsintheculturewsereheldforatleast27 dayspriortocollectiofnthejuvenilefsortestingT.he adultshowed no signsofdiseaseorstresdsuringtheholdingperiod.Duringthe14-dayholdingperiodprecedingthews@ watertemperaturersangedfrom 20.0to20.3*C.The pH ofthewaterrangedfrom 9.3to8.5and dissolveodxygenranged from 8.2to 8.8mg/L. Instrumentatioand methods usedforwatermeasurementsaredescribedintheEnvironmental Conditionssectionofthisreport. Neonatedaphnids(<4 hoursold)were obtainedfortestinfgrom sixindividuaaldultdaphnids.At tesitnitiatitohne, juveniledaphmds were collectefdrom thecultureasnd placedin glassbeakersone to two at a timeuntileach beaker contained10 daphnids.The beakerswerethenimpartiallayssignedtotreatmengtroups.One daphnidwas thent-amfmted toeachtestchamber usinga wide-borepipetunderneaththeair/wateirnterfaceD.aphnids inthecultureasnd thoseusedin thetestwere feda mixtureofyeast,CerophyRs,andtroutchow (YCT, 1800 mg TSS/L),as wellasa suspensionofthe freshwategrreenalgaS,elenastrumcapricornutum(3.5x 107 ceUs/mL).D'uringtheteste,achtescthamberwas fed0.3mL ofYCT and 0.60mL ofSelenastrumoncedaily. TestApparatus Testchamberswere250-mL plasti(cNalgene)beakerscontaininagpproximftly200 mL oftestsolutioTrhledepth oftestsolutioinna representatitveestchamber was approximatel5y cnL Test.chamberswere randomlypositionebdy treatmengtroupinan environmentaclhamber settomaintaina temperaturoef20 I'C. The waterbathwas enclosedina plexiglashsood tominimizethepotentiaflorcross-contaminatioTnh.e testchamberswerelabellewdiththeprojecntumber, testconcentratioand replicate. DilutioWnater The waterusedforculturinagndtestinwgas freshwateorbtainedfroma wellapproximately40 metersdeeplocated on theWildlifIenternationLatld.site.The wellwaterischaracterizaesdmoderately-harwdater.The specificconductar=, hardnessa,lkalinitayn,dpH ofthewellwaterduringthefour-weekperiodimmediatelyprecedingthetestarepresenteidn AppendixI. The wellwaterwas passedthrougha sandfiltetroremoveparticlegsreaterdm approximatel2y5 @m, andpumped intoa 37,800-Lstoragetankwherethewaterwas aeratedwithspraynozzlesP.riortouse,thewateragainwas filteretdo WILDLIFE INTERNATIONAL LTD. -11- PROJFCTNO.: 454A-109 0.45pm and passedthroughaUV sterilizteorremove nucroorgamsms and fineparticleTsh.e resultosfperiodicanalyses performedtomeasuretheconcentrationosfselectecdontaminantisnwe:lwaterusedby WildlifeInternationLatlcLare presentedinAppendix ]I. EnvironmentalConditions Lightingusedtodlummate thecultureasndtestchambersdunng culturinagndtestinwgas providedby fluorescent tubesthatemittedwavelengthsimflatronaturalsunligh(tColortone5'0s).A photoperioodf 16hoursoffightand8 hours ofdarknesswas controllewdithan automatictimer.A 30-minutetransitipoenriodoflowlighitntensiwtays providewdhcn lightwsent on andofftoavoidsuddenchangesinlightingL.ightintensitwyas measuredattestinitiatiaonnd atweekly intervaltshereaftweirtha SPER ScienfifLitcd.lightmeter.Lightintensitryangedfrom329 to383 luxatthesurfacoefthc water. Temperaturewas measuredinalternatirnegplicateosfeachtreatmenatndthecontrolgroupattestinitiatiiono,ld andnew testsolutionosn renewaldays,and attestterminatiounsingahquid-in-glatshsermometer.Temperaturewas also measuredcontinuousliynone negativecontrolreplicatuesinga FulscopeER/C Recorder.The targetesttemperature duringthetestwas 20 1*C. Dissolvedoxygenwas measureddailyinalternatirnegplicatdeusringthefirsstevendaysof thetesti,noldandnew solutionosn renewaldays,and attesterminationM.easurementsofpH were made inalternating replicataetstestinitiatioin,oldand new solutionosn renewaldaysand attestwrmination.Hardness,alkaliniatnyd specificconductancewere measuredinthenegativecontroland thehighesttestconcentratiwointhsurvivindgaphnidsattest initiatiaonnd atapproximatelwyeeklyintervaltshereafter. MeasurementsofpH weremade usinga FisherAccumet Model 915 pH meter,and dissolveodxygen was measured usinga YellowSpringsInstrumentModel 5IB dissolveodxygenmeter.Specificonductancweas measuredusingaYellow SpringsInstrumenMtodel 33 Salinity-Conductivity-TemperamWemter.Hardnessand alkalinimteyasuremcntsweremade by titratiboansedon proceduresinStandardMethods for theExaminationof Water and Wastewater(5). BiolggicaOlbservationsand Measurements The firsgteneratiodnaphnidswereobserveddailyduringthetestforsurvivalt,heonsetofreproductiona,nd clinical signsoftoxicityT.he criterifaordeathincludedwhiteopaque coloratiolna,ckofmovement of appendages,absenceof heartbeaatnd lackofresponsetogentleprodding.Immobilizatiownas definedaslackofmovement exceptforminor WILDLIFE INTERNATIONAL LTD. -12- PROFECTNO.: 454A-109 spontaneoursandom movement oftheappendages.The presenceof eggsinthebroodpouch,abortedeggs,malesor ephippiawere alsorecordecl With theonsetofreproductiont,henumber ofsecond-generatinoenonateswere counted(numberofliveneonates, number ofdeador irnmobilneeonatesand number of abortedeggs)andrecordedeveryMonday, Wednesday andFriday duringthetest.At eachobservatiopneriod,thelivefint-generatidoanphnidswere retainedand thesecond-generation daphnidswere discardecOln Day 14,thesecond-generationneonateswere collecteadnd usedforthesecond-generation acuteexposure.At testterminationt,helength(measuredfromtheapexofthehead tothebaseofthespine)andchyweight weredeterminedforeachsurvivinfgfi-st-generadtaipohnniclThe individuadlaphnidswereremovedfromthetestchambers andplacedintotaredaluminum dryingpans. The lengthof eachdaphnidwas determinedusinga caliperT.he panswere thenplacedi'na dryingoven atapproximatel6y0 *C forapproximatel4y3 hours.The panswere removedfrom thedrying oven andplacedina desiccatotrocool.When cool,thepans wereweighedusinga MettlerAE-240 analyticablalance. StatisticAanlalyses Statisticaanlalysewsere performedon survivaolffirst-generatdiaopnhnids,thenumber of liveyoungandthele@gth and dryweightof thesurvivinfgirst-generatdiaopnhnids.Survivaldatawere analyzedusingFisher'esxacttest ReproductonandgrowthOengthanddryweight)datawere evaluatefdornormalityandhomogeneityofvariancuesingthe Shapiro-Wilk'tsestand Bartlettt'esstr,espectivelyD.unnett'stestwas thenusedto identiftyreatmengtroupsthatwere statisticsailglnyificainntcomparisontothenegativecontro(lp !g0.05). AR statistitceasltwsere performedon a personalcomputerusingTOXSTAT Version3.5(6)statistiscoafltware. AnglyficaClhemis Watersampleswere collectefdrom new and oldsolutionesachweek duringthetest.Duplicatesamplesof new solutionwserecollectefdrom thefreshlpyreparedsolutionsD.uplicatesamplesoftheoldsolutionwserecollectefdrom a compositeofthermnainingrephcatteestchambers.Sampleswerecollecteidnplasti(cNalgmes)bottleasndweremialy@ assoonaspossiblewithoutstorage.Analyticaplroceduresused intheanalysiosfthesamplesareprovidedinAppendix Ill. WILDLIFE INTERNATIONAL LTD. -13- PROJECT NO.: 454A-109 RESULTS AND DISCUSSION VerificatoifoTnestConcentrations Resultsofanalysesto measureconcentrationosf PFOS inwatersiunplecsollectedduringthetestarepresentedin TableI andtheanalyticcahlemistryrepor@(AppendixUD. Nominal concentratiounsedinthestudywere1.4,2.9,5.171,, 23 and 46 mg a.i.ALM.easured valuesofnew samplesrangedfrom 94 to121% ofnominalvaluesw,hereasmeasuredvalues fromtheoldsolutionrsangedfrom90 to 108% ofnominalvalues.ConsequentlyP,FOS was stabltehroughoutherenewal periods.When measuredconcentrationosf samples collectedduringeach week of thetestwere averaged,themean measuredconcentratiownesre 1.5,2.9,5.6,12,24 and 48 mg a.i./wLh,ichrepresente1d07,100,98,109P104and 104% of nominal,respectivelTyh.e resultosf thestudywere basedon mean measuredconcentrations. Physicaland ChemicalMeasurementsofWater Measurementsoftemperatured,issolveodxygen and pH arepresentedinTables2,3 and 4,respectivelWya.ter temperaturmeeasurementswere withintherangeof 20 1*C establishefdorthetest.Dissolvedoxygen concentrations remained2!8.3mgtl (91% ofsaturatiotnh)roughoutthetest MeasurementsofpH rangedfrom 8.1to 8.5. Measurements ofspecificconductance,hardnessand alkaliniitnythenegativceontroalndthehighestreatmengtroup arepresenteidnTable5. No apparentdifferencewsere observedforany of theseparametersthroughoutheexposure period. Survivaland BiolopicaOlbservations A summary oftheresultosftheobservationosfmortalitaynd sublethaslignsoftoxicitfyorfa-st-gmeratdiaopnhnids areshown inTable6. DailyobservationasreprovidedinAppendix V. After21 daysofexposure,survivailnthenegative controlgroupwas100%. Survivalinth1e.5,2.9,5.61,2,24and48 mg a.i./tLreatmengtroupswas 90,90,90,90,0 and 0%, respectivelAyt. testterminationa,llsurvivindgaphnidsexposedtoPFOS appearednormal.Fisher'esxacttestshowed thatsurvivailnthe24 and 48 mg a.i./tLreatmentgroupswas statisticadlilfyferenwthen compared tothenegativceontrol (p !@0.05). AMENDED WILDLIFE INTERNATIONAL LTD. -14- PROJECTNO.: 454A-109 RWoducti A summary ofthemean number of liveyoung producedby thefirst-generatdiaopnhnidsispresentedinTable7. A completelistinogfthe number ofneonatesproducedisprovidedinAppendixV. Daphnidsinthecontroland PFOS treatmengtroups:@12 mg a.i./sLtartepdroducingneonateson Day 9. Therewere no observationosfabortedeggsinany treatmengtroup.Reproductionaveraged122young peradultinthenegativceontrolR.eproductioninthe1.5,2.9,5.a6nd 12 mg a.i./tLreatmengtroupsaveraged142,136,132 and 119 young peradultr,espectivelTyh.e Bonferronti-tessthowed thatreproductiownas notsignificantrleyducedinany treatmentgroup:g12 mg &i./L(p> 0.05).The24and48mga.i./L treatmengtroupswerenot includeidnthestatisticaanlalysiosfreproductiodnue toa statisticaslilgynificaenftfecotn survival. Growth A summary of themean lengthsand mean dryweightsofthesurvivinfgirst-generatdiaopnhnidsarepresentedin Table8. IndividuamleasurementsareprovidedinAppendix VI. The mean lengthand dryweightoffirst-generation daphnidsinthenegativceontrolwas 4.65mm and0.695mg, respectivelTyh.e Bonferronit-tessthowed thatmean length anddryweightinthePFOS treatmentgroups 12 mg a.i.AwLere notsignificantrleyducedincomparisontothenegative control(p> 0.05). Ser,ond-GeneratiAocnuteExposure The resultosfthesecond-generatioancuteexposurearepresenteidnTable9. After48-hoursofexposures,urvivailn thenegativecontrowlas 95%. Survivalinthe1.5,2.9,5.6,12 and 24 mg a.i./tLreatmengtroupswas 100,100,100,90 and 0%, respectivelFyi.sher'esxacttestshowed thatsurvivailnthe24 mg a.i./tLreatmengtroupwas significantdliyfferent from thenegativceontrol(p:T0,.05). CONCLUSIONS Therewere no adverseeffectson survivalr,eproductioonr growth of Daphnia magna exposed to PFOS at concentration:s@12 mg a.i./fLor21 days. Daphnia magna exposedto 24 and 48 mg a.i./hLad significantrleyduced survivalC.onsequentlyt,heLOEC forthisstudy,basedon survivawlas 24 piga.i./LT.he NOEC was 12mg a.i./aLndthe MATC was calculatetdobe 17mg a.i./LT.he resultosfthesecond-generatiaocnuteexposurealsoindicateadNOEC of12 mg a.i./L. AhffiNDED WILDLIFE INTERNATIONAL LTD. .15- PROJECT NO.: 454A-109 REFERENCES 1 U.S. Environmental Protection Agency. 1996. Series 850 -Ecological Effects Test Guidelines (draft), OPPTS Number 850.1300: Daphnia Chronic Toxicity Test 2 OECD Guideline for Testing of Chemicals. 1997. Guideline 211: Daphnia magna Reproduction Test 3 U.S. Environmental Protection Agency. 1986. StandardEvaluation Procedure, Daphnia magnaLife-CXle (21Day Renewal) Chronic Toxicity Test. Hazard Evaluation Division. Office of Pesticide Programs, EPA 540/9-86141. 4 American Societyfor Testing and Materials. 1991. Standard Guide for Conducting Renewal Life-Cycle ToxicityTestswithDaphnia magna, ASTM E 1193-87. PhiladelphiaP,A. 5 APE[KAWWAWPCF. 1985. StandardMethodsfor theExamination of Water and Wastewater. 16thEdition, American PublicHealthAssociationA.merican waterWorks AssociatiorWLater PollutioCnontrolFederatioNne,w York. 6 West, Inc.and D.D. Gulley.1996. TOXSTAT, Version3.5.WesternEcoSystemsTechnology,Inc.Cheyenne, Wyoming- PRO TableI Summary ofAnalyticaClhemistryData ponsor: Test Substance: TestOrganism: DilutionWater: 3M Cozpomtioii PFOS CladoceranD,aphnia magna Well Water NominalTest Concentmtion (mg a.i./L) NegativeControl 1.4 2.9 5.7 11 23 46 Day 0 ew <L 1.78 1.72 3.20 3.05 5.97 5.87 11.5 11.5 24.2 23.1 47.3 48.0 D(;oa 2 rd) <<L 1.58 1.56 3.01 3.07 5.65 5.72 11.6 11.8 24.0 24.6 49.1 49.4 D 11 <L88 <L 1.38 1.47 2.75 2.77 5.63 5.59 11.3 11.3 22.8 22.5 2 2 'Ilelimiotfquantitati(oLnOQ) was 0.458mg a.i./L. discontinuid@ude to100% mortality. D 14 @gld) <L <1 1.36 1.32 2.85 2.71 5.36 5.39 11.2 11.6 23.6 23.1 DI eLw' <L82 <L 1.38 1.43 2.79 2.81 5.58 5.75 11.8 11.6 24.8 25.0 Day 21 (Old) < L82 <L 1.50 1.45 2.81 2.82 5.24 5.37 11.5 11.3 -2 _2 Mean Measured Concentration (msta.i.AL) <-OQ 1.5 2.9 5.6 12 24 48 PRO Table2 TemperatureofWater intheTestChambers' Sponsor: TestSubstance: TestOrganism: DilutionWater: 3M Corporation PFOS CladoceranD,aphnia magna Well Water Mean Measured TestConcentration (mg a.i./L) NegativeControl Day 0 New 19.4 Day 2 Old New 19.6 19.5 Day 4 Old New 19.5 19.6 Temperature(*C) Day 7 Old New 19.8 19.8 1.5 19.4 19.6 19.5 19.5 19.6 19.8 19.8 2.9 19.4 19.6 19.5 19.5 19.6 19.8 19.8 5.6 19.4 19.6 19.5 19.5 19.6 19.8 19.8 12 19.4 19.5 19.5 19.5 19.6 19.8 19.8 24 19.4 19.5 19.5 19.5 19.7 19.8 19.8 48 19.4 19.5 19.5 --2 -- -- -- 'TemperaWm measuredrontmuouslyduringthetestrangedfrom 19;0*Cto20.OOC. 2 No Me&%Z=ent takendue to Iooo/MorUhty. Day 9 Old New 20.0 20.1 20.0 20.1 20.0 20.1 20.1 20.1 20.1 20.1 20.1 20.1 -- -- PRO. -18- Table2 (Continued) TemperatureofWater intheTestChambers' Sponsor: TestSubstance: TestOrganism: DilutionWater: 3M Corporation PFOS CladoceranD,aphnia magna Well Water Mean Measured TestConcentration (mg&i./L) NegativeControl Day 14 Old New 19.9 19.8 Temperature C) Day 16 Old New 19.9 19.8 Day 18 Old New 20.1 20.1 1.5 19.8 19.8 19.9 19.8 20.1 20.1 19.8 19.8 19.9 19.9 20.0 20.1 5.6 19.9 19.9 19.9 19.9 20.1 20.1 12 19.9 19.9 19.9 19.9 20.1 20.1 24 19.9 19.9 20.0 19.9 20.1 --2 48 -- -- -- -- -- 'Temperaturemeasuredcontinuousldyuringthetestrangedfi-om19.OOCto20.0-C. 2 Measurementsdiscontmueddue to 1000/.mortality.. PR( -19- Table 3 DissolvedOxygen ContentofWater intheTestChambers' Sponsor: TestSubstance: Test Organism: DilutionWater 3M Corporation PFOS Cladocemn, Daphnia magna Well Water Mean Me&%ued Test Concentration (mg ai./L) NegativeControl Day 0 New 8.3 Day I Old 8.3 DissolvedOxygen Concentration(mg/L) Day 2 Old New 8.6 8.9 Day 3 Old 8.7 Day 4 Old Now 8.6 8.6 1.5 8.3 8.3 8.6 8.9 8.8 8.6 8.8 2.9 8.3 8.3 8.6 8.9 8.8 9.6 8.8 5.6 8.3 8.3 8.6 8.9 8.8 8.6 8.8 12 8.3 8.3 8.6 9.0 8.8 8.6 8.8 24 8.4 8.3 8.6 8.9 8.8 8.6 8.8 48 8.4 8.4 8.6 8.9 - - 'A dissolvedoxygen concentrationof 5.4mg/L mpmmts 2Measurements discontinueddue to 1000/9mortality 60% saturatioant20*C in fi-eshwater Day 5 Old 8.7 8.8 8.8 8.8 8.8 8.8 - Day 6 Old 8.8 8.8 8.9 8.9 8.8 8.9 - PR( @20 Table3 (Continued) DissolvedOxygen ContentofWater intheTestChambers' Sponsor: TestSubstance: TestOrganism: DilutionWater 3M Corporation PFOS Cladocem, Daphnia magna Well Water DissolvedOggen Concentratio(nmg/L) Mean Measured TestConcentration (mg &i./L) NegativeControl D!% 9 Old New 8.7 8.8 Dg 11 Old New 8.4 8.7 D 14 Old New 8.4 8.8 Day 16 Old New 8.8 8.8 1.5 8.7 8.9 8.4 8.8 8.4 8.8 8.8 8.8 2.9 8.7 8.9 8.4 8.8 8.4 8.8 8.8 8.8 5.6 8.7 8.8 8.5 8.8 8.4 8.8 8.8 8.8 12 8.7 8.8 8.4 8.8 8.4- 8.8 8.8 8.8 24 8.7 8.9 8.4 8.8 8.5 8.9 8.9 8.9 48 -- -- 'A dissolvedoxygen concentrationof 5.4mg/L represents60*/osaturatioant20*C infi-eshwater. 'No measurement takendue to1000/9mortality. D Old 8.4 8.4 8.5 8.4 8.4 8.4 PR -21- Table4 pH ofWater intheTestChambers Sponsor: TestSubstance: TestOrganism: DilutionWater: 3M Corporation PFOS CladoceranD,aphnia magna Well Water Mean Measured TestConcentration (mg &i./L) NegativeControl Day 0 New 8.3 Day 2 Old New 8.3 8.1 1.5 8.4 8.4 8.2 2.9 8.4 8.4 8.2 5.6 8.4 8.4 8.2 12 8.4 8.4 8.3 24 8.4 8.4 8.3 48 8.5 8.4 8.4 'Measurementsdl%continueddue to IW% mortality. pH Day 4 Old New 8.4 8.2 Day 7 Old New 8.4 8.4 8.4 8.2 8.5 8.3 8.4 8.2 8.5 8.3 8.5 8.2 8.5 8.4 8.5 8.3 8.5 8.4 8.5 8.4 8.5 8.5 Day 9 Old 8.3 8 8.4 8 8.4 8 8.4 8 8.4 8 8.5 8 -22- Table4 (Continued) pH ofWater intheTestChambers Sponsor: TestSubstance: TestOrganism: DilutionWater: 3M Corporation PFOS CladoceranD,aphnia magna Well Water Mean Measured TestConcentration (mg ai./L) NegativeControl Day 14 Old New 8.3 8.2 1.5 8.3 8.2 2.9 8.3 8.2 5.6 8.3 8.3 12 8.4 8.3 24 8.4 8.3 48 -- 'Measurementsdiscontinueddue to 100% mortality. pH Day 16 Old New 8.3 8.1 8.3 8.1 8.3 8.1 8.3 8.1 8.3 8.2 8.3 8.3 PRO Day 18 Old New 8.3 8.2 8.3 8.2 8.3 8.2 8.3 8.2 8.3 8.3 8.4 Wl LD LlFE NTER NATI 0 NAL LTD. -23- PROJECT NO.: 454A-109 Table5 SpecifiCconductance,Hardnessand AUmlinityintheNegativeControl andHighestTreatmentGroup TestChambers Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: CladoceranD,aphnia magna DilutionWater: WellWater ES&ativeControl Time Parameter SpecifiCconductance (IUMIIOS/CM) RE 0 310 Day 7 320 Dg 14 310 Day@i 315 Hardness 136 128 132 128 (mg/L asCaC03) Alkalinity 178 176 172 170 (mg/L asCaC03) Parameter oi SpecifiCconductance 310 Gzmhos/cm) Hardness 136 (mg/L asCaCO3) Alkalinity 178 (mg/L as CaCO3) 48 mg a.i.//Ll24!!Iga.i./L212 mg a.i./L3 Day 72 320 Time D!l 142 310 128 132 176 174 Day 2 13 320 128 174 ANM'NDED PR -24- Table 6 Summary ofCumulativePercentMortalityand Treatment-RelateEdffectsf'orFirst-GeneratiDoanphr Sponsor: TestSubstance: TestOrganism: DilutionWater: 3M Corporation PFOS CladoreranD,aphnia magna Well Water Mean Measured CumulativeObservationstoDay 7 TestConcentration % % (mga.i.)L) Mortality Immobile Effects NegativeControl 0 0 iOAN CumulativeObservationtsoDay 14 % % Mortaii!x Immobile Effects 0 0 10 AN 1.5 0 0 IOAN 0 0 10 AN 2.9 0 0 10,kN 0 0 10 AN 5.6 0 0 10 AN 0 0 10 AN 12 0 0 IOAN 10 0 9 AN 24 70 0 3 AN 90 0 1 AN 48 I OBDdIi:VtetdE@ec Inicatesa sii@ 100 0 100 0 -N= AppearsNormal. tdiffermcfeorm thenegativecon6l on D!y 21 usingFisher'esxacttest 0.05). Cinnulative % Mortality 0 10 10 10 10 ioo* ioo* PR( -.25- Table 7 Summary ofDaphnia magna ReproductionDuring theLife-CyclTeoxicityTest Sponsor: Test Substance: TestOrganism: DilutionWater: 3M Corporation PFOS Cladoceran,Daphnia magna Well Water Mean Measured Test Concentration (mg &i./L) Number of Surviving Individuafly-Exposed Daphniclg NegativeControl 10 Mean LiveYoung/ SurvivingAdultDaphnid (SD) 122 (19.2) First.Day of Reproduction 9 Total Dead/I 1.5 9 142 (24.7) 9 2.9 9 136(17.9) 9 5.6 9 132(19.5) 9 12 9 119 (26.5) 9 24' 0 11 48' 0 None 'Thistreatmentwas excludedfrom thestatisticaanlalyses.ofreproductiodnue toa statisticaslilgynfflcansturvivaelffect. WILDLIFE INTERNATIONAL LTD. -26- PROJECTNO.: 454A-iuy Table8 Summary ofLengthandDry Weightof Surviving Individually-ExposFeidrst-GeneratiDoanphnids Sponsor: Test Substance: TestOrganism: DilutionWater: 3M Corporation PFOS CladoceranD,aphnia magna Won Water Mean Measured TestConcentration (mg a.i./L) Number of Individually-Exposed SurvivinDgaphnids NegativeControl 10 TotalLength(mm) Mean (SD) 4.65(0.111) 1.5 9 4.66(0.118) 2.9 9 4.62(0.100) 5.6 9 4.61(0.124) 12 9 4.59(0.102) 24 0 48 0 Dry Weight(mg) Mean (SD) 0.695(0.100) 0.669(0.0623) 0.724(0.110) 0.727(0.0665) .0.723(0.0661) AMENDED -27- Table 9 CumulativePercentMortality/hmobilitaynd Treatrnent-RelatEefdfectsf'orSecond-GeneratioDn Sponsor 3M Corporation TestSubstance: PFOS TestOrganism: Cladocemn,Daphnia magna DilufionWater Well Water Mean Measured Concentration Daphnial (mg a.iJL) Replicate Replicate NegativeControl A 10 B 10 Number Dead 0 0 5 Hours Number Immobile - 0 0 Effects 10AN 10AN Number Dead 0 0 24 Hours Number hnmowe 0 0 1.5 A 10 0 0 10 AN 0 0 B 10 0 0 10 AN 0 0 2.9 A 10 0 0 10 AN 0 0 B 10 0 0 IOAN 0 0 5.6 A 10 0 0 10 AN 0 0 B 10 0 0 10 AN 0 0 12 A 10 0 0 10AN 0 0 B 10 0 0 10 AN 0 0 24 A 4 0 0 4 AN I B 4 0 0 4 AN 2 ObservedEffects:AN AppearsNormal, C - Lethargic Indicateas significandtifferencferom thenegativecontrolat48 hours usingFisher'esxacttest(p I 0 0.05). Effects 10 AN 10 AN IOAN 10AN 10 AN 10 AN 10 AN 10 AN 10 AN IOAN IANIC IAN,.IC .,,(Percent immobile and Dead 0 Numbe Dead 0 I 0 0 0 0 0 0 0 0 0 0 1 1 50 4 4 WILDLIFE INTERNATIONAL LTD. -28- PROJECT NO.: APPENDIX I SpecifiCconductance,Hardness,ABudinityand pH of Well Water Measured Duringthe4-Week PeriodImmediatelyPrecedingtheTest Sponsor. 3M Corporation Test Substance: PFOS TestOrganism: CladoceranD,aphnia magna DilutioWnater: WellWater SpecifiCconductance wffios/=) Mean 329 (N = 4) 315-340 Hardness (mg/LasCaC03) 124(N = 4) 120-128 Alkginity (mg/L as CaCO3) 169 (N 4) 164-172 pH 8.2(N 4) 8.0-8.3 WILDLIFE INTERNATIONAL LTD. -29- PROJECTNO.: 454A-109 APPENDIX ]I Analyses of PesticidesO,rganics,Metals and Other Inorganics inWildlifehftmationalLtd.Well Water' ANALYSIS MEEASURED CONCENTRATION MiscellaneoMuesasurements TotalDissolvedSolids Ammonia ITitrogen TotalOrgame CiLrbon2 TotalC)iuiide Organochlorineasnd PCBs Aldrin AlphaBHC BetaBHC DeltaBHC Gumna BHC (Lindane) Chlordane DDD, pp' DDE, pp DDT,pp Dieldriri F.ndosulfqAn, EndosulfanB, 'Fn-ogilifaSnulfate Endrin EndrinAldehyde Heptachlor M6thoxychlor HeptachlorEpoxide Toxaphene PCB@1016 PCB-1221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 286 < 0.050 < 1.0 < 10.0 < 0.005 < 0.005 < 0.005 < 0.005 < 0.006 < 0.025 < 0.006 < 0.005 < 0.008 < 0.005 < 0.005 < 0.005 < 0.018 < 0.010 < 0.005 < 0.005 < 0.007. < 0.005 < 0.500 < 0.260 < 0160 < 0.260 < 0.720 < .0720- < 0.* 720 < 0.720 mg/L MFVL mitl mi@IL )ug/-L Al t4 U JAPUA OA4 IMU 'AUA AUA MU jiA4 iu jiu @U4 AUL p IU p JU Metalsand OtherIuorganics B Ui ipiu.qi cc hrcol?uUqMl3 Cobal Copo itoil Lead3 hugnes iutr3 Niaiigaense mem y Moly enum3 Nickel Potassiu-3 Sol * 9 < 100 < 25.0 y@IL < 0.50 mg/-L < 1.0 ufv-L 35.0 miVL < 2.0 Ufv-L < 1.0 ug/-L < 20.0 uW-L < 100 JAUA < 10.0 JA"4 13.5 m < 1.0 mg/L < 020 < 2.0 iAgl-L ug/L < 2.0 MWL 6.62 mg/L < 25.0 uA- SSaVor'd3 iym Zinc < 1.0 213 < 20.0 uA- mk/L 41 Analym performedby QST EnvirorunentaGla,inesvillFel,oridfaorsamplescollectoend November 3 thmugh November 7,1997. 2 Analysesperformedby WildlifIentcimtionaLltd.forthesamplecollecteodn November 5, 1997. 3 Analysesperformedby WildlifIentemationaLltd.forsamplescollecteodn November 5 thmugh 7,1997. WILDLIFE INTERNATIONAL LTD. -30- PROJECTNO.: 454A-109 APPENDIX M THE ANALYSIS OF PFOS IN FRESHWATER IN SUPPORT OF W'ELDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-109 WILDLIFE INTERNATIONAL LTD. -31- PROJECTNO.: 454A-109 REPORT APPROVAL SPONSOP, 3M Corporation TTRLE: PFOS: A Semi-StaticLife-CycleToxicityTestwiththeCladoceran(Daphnia magna) W]ILDLIFE RQTERNATIONAL LTD. PROJECT NO.: 454A-109 PRINCEPAL RQVESTIGATOR-- Jm&. MacGregor,B.S. Scientist MANAGEhffi'NT: WifludB. Nixon,Ph.D./ Manager,AnalyticaClhermstry DATE. DATE AMENDED WILDLIFE INTERNATIONAL LTD. -32- PROJ-ECT NO.: 454A-109 Freshwatersampleswere collectefdrom a senu-statihcfe-cyclteoxicitsytudydesignedto &-tem-dnedw effectosf PFOS (PerfluorooctaSnuelfonicAcid PotassiumSalt)tothecladoceran(Daphnia magna). This studywas conductedby WildlifbeitemationaLltd.and identlfiaesdProjecNto.: 454A-109. Theanalyses of thesewater sampleswere performedat WildlifeInternationLatld.usinghigh performanceliquid chromatographywithmass gmtometric detectio(nBPLC/MS). Sampleswere receivedfbranalysis betweenJuly12,1999 and Atigust2, 1999 and were analyzedon each samplereceipdtay. TestSubstanceand InternaSltandard The testsubstanceusedforthisstudywas WildlifIentemationaLltd.idenfificatniuomnber 4675. The testsubstancewas usedtD preparecalibratiaond matrixfort&catiosnamples. The mtemal standardwas receivedfrom 3M Corporatioonn July2,1999 and was assipeclWildlife InternationLatld.idenfificatniuomnber 4526 upon receiptT.he internasltandarda,granularmaterialw,as identifiaesd: 1K IK 2K 2H PerfluorooctaSnuelfoniAccid,ChemicalAbstractNumber: 27619-97-2. The standardwas storedunderambientconditions. AAW3!LcalMethod The method.used fbrtheanalysisofthefreshwatersampleswas developedatWildlifeInternational Ltd.and entitle"dAnalyticaMlethod fbrtheDeterminatioonfPFOS inFreshwaterS,altwatera,nd Algal Medium". Thismethodologywas includedasAppendixH ofWildlifIenternationLatld.protocolnumber 454/011299/MVAIJSUB454. Itwas basedupon methodologyprovidedby 3M Corporation. Samples were dilutedin a 50% medmnol : 50% NANOpure(D water solutioncontaining 0.100mg 4H PFOS (internasltandard)AaLnd 0.05% fonnicacid(v/v)so thattheyfellwithinthe calibratiornangeofthePFOS methodology. Concentrationosf the PFOS inthe standardsand sampleswere determinedby reverse-phasheigh performanceliquidchromatographyusinga Hewlett-PackarMdodel 1100 High PerformanceLiquid Chromatograph(HPLC) witha Perkin-ElmeArPI 10OLC Mass Spectrometeerquippedwitha Perldn- WILDLIFE INTERNATIONAL L-m. -33- PROJECTNO.: 454A-109 Ehner TurboIonSpmy ion source. BPLC sepamfionswere acluevedusinga KeystoneBetasilCls analyticcaollumn(50mm x 2 mm I.D.,3 pm particlseize).The ftistrumepnatrametersaresummarizedin Table1. A methodflowcharitsprovidedinFigure1. CalibratioCnurveand LimitofOuantitation Calibratiosntandardsof PFOS preparedin a 50% methanol.5:0% NANOpure(D watersolution containin0g.100mg 4HPFOS (internasltandard)/aLnd 0.05% formicacid(v/v)r,nnsdn inconcentration from 0.00915to 0.0915mg a.i./Lw,ere analyzedwiththesamples.The same and most prominentpeak responseforPFOS was utilizetdomonitorPFOS inallcalibmfionq,ualitycontrola,nd studysamples.No attemptwas made toquantifyPFOS on the,basisofindividuailsomericomponents. Linearregression equationsweregeneratedusingpeak arearesponseratios(PFOS :internasltandard)versustherespective concentratiroantio(sPFOS :internasltandardo)fthecalibratiosntandards.A typicalcalibraticounrveis presenteidnFigure2. The concentratioonfPFOS inthesampleswas determinedby substitutiinhgepeak arearesponseratiosintotheapplicablleinearegressioenquation.Representativieonchromatogmm of low and lughcalibratisotnandardsarepresentedinFigures3 and 4,respectively. 'Me methodlimitofquantitati(oLnOQ) fortheseanalyseswas setat0.458mg a.i./cLalculateadsthe productof thelowestcalibratiosntandardanalyzed(0.00915mg a.i./La-n)d thedilutiofniwtorof the matrixblanksamples(50). MatrixBlankand FortificatiSomnRIes Six matrixblank sampleswere analyzedto determinepossibleinterferenceN.o interferencweesre observedatorabovetheLOQ duringsamplesanalyses(Table2).A representatiivoenchromatograznofa matrixblankispresenteidnFigure5. Freshwaterwas fortifiaetd0.915,9.15and 54.9mg a.i./aLnd analyzedconcurrentlwyiththesamples todetemiinethemean proceduralrecovery(Table3).,Sample concentratiownesre not correctefdorthe mean procedurarlecoveryof 104%. A representatiivoenchromatograionf a matrixfortificatiison presentedinFigure6. ANM'NDED WILDLIFE INTERNATIONAL LTD. -34- PROJECTNO.: 454A-109 ExarMle Calculations Sample number 454A-109-3,nominalconcentmtionof 1.4mg a.i./iLnfi-eshwater. PeakArea Ratio= AnalytePeakArea/IntemalStandardPeakA= Conomt-&on Ratio= ConcentratioonfAiWytelConcentrationfInternaSltandard InternaSltandardConcentration0:.100mgtl InitiaVlolume: 0.500n@L FinalVolume: 25.0mL DilutioFnactor:50.0 PFOS Peak Area: 3091181 InternaSltandardPeakAim: 3169689 PeakArea Ratio:0.97523 Calibratiocnurveequation. Slope:2.42974 hitercept0:.11482 Curveisweighted(Itx) . Peakarearatio-(Y-intercept) PFOS (mg a.i./La)tInstruemnt- Slope x InternaSltandardConcentration PFOS (mg a.iJL)insample= PFOS (mg &iJL)atinstrumenxtDilutioFnactor Calculatecdoncentration1:.78mg a.i./L Note:manual calculatiomnasy chffer. PercentofNominal Conc4ntrafio-nPFOS (mg a.i./Li)nsample x 100 PFOS (mg a.i./Ln)ominal Calculatedrecovery:121% Note:manualcalcumon may differ. ANE'NDED WILDLIFE INTERNATIONAL LTD. -35- PROJECTNO.: 454A-109 RESULTS Smple Analysis Freshwatersampleswere collectefdrom the semi-statihcfe-cyclteoxicitsytudywiththe cladoceran (Daphnia magna) attestinitiatioJnu,ly 12, 1999 (Day 0),on July14,1999(Day 2 old solutions)o,n July23, 1999 (Day II - new solutionso)n,July26,1999(Day 1-4- oldsolutionso)n,July30, 1999 (Day 18 - new solutionsa)ndidtesterminatioonn August2, 1999(Day 21 oldsolutions)n.e measured concentratioonfsPFOS inthesamplescollecteadtinitiatioofnexposureofthetestorganisms(Hour 0) rangedfrom 96.5to 121% ofthenominalconcentration(sTable4)..SamplescollecteadtDay 2,Day 11, Day 14,and Day 18had measuredconcentratiornangesof 97.8to 108*/o9,4.3to 1010/o8,9.8to 103% and 94.5to 109% of nominalvalues,respectivelyS.amplescollecteadt testterminatio(nDay 21) had a measuredconcentratiornangeof91.0to 102% ofnominalvalues.A representatiivoenchromatogramofa testsampleisshown inFigure7. AMENDED WILDLIFE INTERNATIONAL L-m. -36- PROJECTNO.: 454A-109 INSTRUMENT: Table I TypicalBPLC/MS OperationaPlamniewn Hewlett-PackarMdodel I100 HighPerformanceLiquid Chrmnatographwitha Perkin-EbneArPI IOOLC Mass Spectrometer equippedwitha Perldn-EhneTrurboIonSprayionsource.Operatedin selectivieonmonitorinmgode (SIM. ANALYTICAL COLUMN: KeystoneBetasiCliscolumn(50mm x 2 mm I.D.,3 pm particlseize) OVEN TEMPERATURE: 300C STOP TE@E: 5.00minutes FLOW RATE: MOB]ILE PHASE: 0.220mljminute 72.0% MeLhanol 29.0% NANOpurelD Water containing O.I%FomiicAcid INJECTION VOLUME: PFOS RETENTION TIME: .25.0pL Approximately4.3minutes INTERNAL STANDARD RETENTION TIME: Approximately3.0minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6amu 426.7amu WILDLIFE INTERNATIONAL LTD. -37- PROJECTNO.: 454A-109 Table2 MatrixBlanksAnalyzedConcurrentlDyuringSample Analysis Number (454A-109-) MAB-1 MAB-2 S=le Type MatrixBlank MatrixBlank Measured Concentratioonf PFOS' (mg a.i./L) < LOQ < LOQ MAB-3 MatrixBlank < LOQ @MAB-4 MatrixBlank < LOQ MAB-5 MatrixBlank < LOQ MAB-6 MatrixBlank < LOQ The limitofquantitati(oLnOQ) was 0.458mg a.i.AbLasedupon theproductofthelowestcalibration standard@ng@ @0.00915 a.i./La)ndthechlutiofniwtoorfthematrixblanksamples(50). AAffiNDED WILDLIFE INTERNATIONAL LTD. -38- PROJECT NO.: 454A-109 Table 3 MatrixFortLficatiAonaslyzedConcurrentlDyuringSampleAnalysis Sample Number (454A-109-) MAS-1 MAS4 MAS-7 MAS-10 MAS-13 MAS-16 Concentrationosf PFOS (Lnga.i.L) Fortified Measured 0.915 0.915 0.915 0.915 0.915 0.915 0.988 1.02 0.952' 0.942 0.997 0.979 Percent Recovered 108 111 104 103 109 107 MAS-2 9.15 9.33 102 MAS-5 9.15 9.33 102 MAS-8 9.15 9.33 102 MAS-1 1 9.15 9.15 100 MAS-14 9.15 9.15 100 MAS-17 9.15 9.61 105 MAS-3 54.9 59.9 109 MAS-6 54.9 56.7 103 MAS-9 54.9 55.3 101 MAS-12 54.9 55.9 102 MAS-15 54.9 57.3 104 NUS-18. 54.9 55.7 102 Mean = 104 StandardDeviation= 3.32 CV = 3.19% N = 18 Note:Resultsand correctionfsornew testsubstancepurityweregeneratedusingMacQuan version1.5 softwareand manual calculationVsa.lueshave beenroundedforreportinpgurposes. Reportedvaluemresents mean ofLughcatere-analyse(s0.970and 0.933mg a.i.AL). AMENDED WILDLIFE INTERNATIONAL LTD. -39- PROJECT NO.: 454A-109 Table4 Measured Concentrationosf PFOS inFreshwaterSamplesfrom a CladoceranSemi-StatiLcife-CyclTeoxicityTest NominalTest Concentration LM.9a.i.U 0.0 Sample Number L454A-109@ 1 2 15 16 29 30 41 42 53 54 65 66 Sampling Time T Y) 0 0 2 2 11 11 14 14 18 18 21 21 PFOS Measured Concwtrafion' @2!9a.i.LLJ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Percent Of Nominal - - - - - '7 1.4 3 0 1.78 121 4 0 1.72 117 17 2 1.58 108 18 2 1.56 107 31 11 -1.38 94.3 32 11 1.47 101 43 14 1.36 93.1 44 14 1.32 89.8 55 18 1.38 94.2 56 18 1.43 97.4 67 21 1.50 102 68 21 1.45 98.5 Note:Resultsand correctionfsbrnew testsubstancepuritywere generatedusingMacQuan version1.5 softwareand manual calculationVsa.lueshavebeen roundedforreportinpgurposes. The limitofquantitati(oLnOQ) was 0.458mg a.i./bLasedupon theproductofthelowestcalibmfion standaradnalyzed(0.00915mg a.i./La)nd thedilutiofnactorofthematrixblanksamples(50). AMENDED WILDLIFE INTERNATIC)NAL LTD. -40- PROJECTNO.: 454A-109 Table4 (continued) Measured Concentrafionsof PFOS in FreshwaterSamples from a Cladoceran Semi-StaticLifi@-CyclTeoxicityTest Nominal Test Concentmtion Ln!ga.i.@Li 2.9 Sample Number L454A-109:1 5 6 19 20 33 34 45 46 57 58 69 70 Sampling Time TU) 0 0 2 2 11 11 14 14 18 18 21 21 PFOS Measured Concentration' Lm& a.i.2 3.20 3.05 3.01 3.07 2.75 2.77 2.85 2.71 2.79 2.81 2.81 2.82 Percent Of Nominal 113 107 106 108 96.9 97.6 101 95.5 98.3 99.1 .99.0 99.2 5.7 7 0 8 0 21 2 22 2 35 11 36 11 47 14 48 14 59 18 60 18 71 21 72 21 5.97 104 5.87 102 5.65 98.1 5.72 99.1 5.63 97.6 5.59 96.9 5.36 93.0 5.39 93.5 5.58 96.8 5.75 99.6 5.24 91.0 5.37 93.2 Note:Resultsand correctionfsornew testsubstancepuritywere generatedusing MacQuan version1.5 softwareand manual calculationsV.alues have been rounded forreportmg purposes. The limitof quantitatio(nLOQ) was 0.458 mg a.i.ALbased upon theproductof the lowestcalibration standardanalyzed(0.00915mg aiJL) and thedilutio&nctor ofthematrixblanksamples(50). AMENDED WILDLIFE INTERNATIONAL LTD. -41- PROJECTNO.: 454A-109 Table4 (continued) Measured Concentrationosf PFOS inFreshwaterSamplesfrom a CladoceranSemi-StatiLcife-CyclTeoxicityTest NominalTest Concentration LM-9a.i.u 11 Sample Number L454A-109@ 9 10 23 24 37 38 49 50 61 62 73 74 Sampling Time 929) 0 0 2 2 11 11 14 14 18 18 21 21 PFOS Measured Concent-dfion' Lniga.i&j 11.5 11.5 11.6 11.8 11.3 11.3 11.2 11.6 11.8 11.6 11.5 11.3 Percent Of Nominal 96.5 97.0 97.8 99.3 95.0 94.5 94.0 97.5 99.4 97.4 97.1 94.5 23 11 0 12 0 25 2 26 2 39 11 40 11 51 14 52 14 63 18 64 18 24.2 106 23.1 101 24.0 105 24.6 109 22.8 99.6 22.5 98.3 23.6 103 23.1 101 24.8 108 25.0 109 46 13 0 .14 0 47.3 103 48.0 105 27 2 49.1 107 28 2 49.4 108 Note:Resultsand contchons fornew testsubstancepuritywere generatedusingMacQuan version1.5 softwareand manual calculationsV.alueshavebeen roundedforreportinpgurposes. The limitofquantitati(oLnOQ) was 0.458mg a.i./bLasedupon theproductofthelowestcalibration standardanalyzed(0.00915mg a.i.ALa)ndthedilutiofnlictofrthematrixblanksamples(50). AAENDED WILDLIFE INTERNATIONAL LTD. -42- PROJECT NO.: 454A-109 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FRESHWATER Preparmeatrifxoraficatsiaomnplebsyspudngthercqlusviotleu.meofPFOS stocskolutiodnisrectly intofilterefdreshwateursinggas-tigbstyringeasnd ClassA volumetncflasks. Dilutematnx fortlficatainodttiestsamplesintotherangeOfthecalibratisotnandardbsy partay filling ClassA volumetrifclaskwsith50% methanol:50% NANOPureg watersolutiocnontaining 0.100mg 4H PFOS (internsatlandardyland0.05% formicacid(v/v).Addthe Appropriatevolumeofsampleand bringtheflasktovolumewiththedilution solvent.Processthematrixblanksampleusingthesame dilutioannd aliquot volumeasforthelowestfortificatlieovnel.Mix wellby several repeatinversions. Ampulatr,samplesand submitforLCMS analysis. Figure1. AnalyticamlediodflowchartfortheanalysiosfPFOS infi-eshwater. WILDLIFE INTERNATIONAL LTD. -43- PROJECTNO.: 454A-109 2.50-,,, a zoo - 1.50 1.00 0.50- 0.000.000 0.200 0.400 Q600 0.800 Concentratio(nRatio) 1.000 Figure2. A typicaclalibraticounrveforPFOS. Slope 2.42974;Intercept0.11482;r 0.9974. Curveisweighte(dI/x). AMENDED WILDLIFE INTERNATIONAL L-m. -44- PROJECTNO.: 454A-109 PWMK&uniitio.mlaw CaRmSM Mr. Od "M I&PA" FMOC MN I=W Wth@Ckm O.....ftASCI@ima, --" UN olb -ow 9 1&49"10-120AM PM_IM 497SA-004c-l 4M V% I pead *ffm iw ta.lm 1&43 1: 4M 01 IA 2U stim 100 4".7 Now Th@ 0 to Ourt Thom :.2 191LVrAft 8 MtdL Vr4lh -1 ma" qm sea xr WNL 00co 20 so 7 wr SOMCFAD AIM 31ON79 NoWd 244954 2.01 40 stm Tbm firdTWA La $0 V&VW am 20 I is 85 00 7,1.02. 1,91.146 222 250 273 ol ii , "$1 0 111 161 lii* '211 241 271 OW OM ij3 lAs L*4 Ls4 aA& &U 4M 4AG Tbm p*i.t ii.LAd tL ISM IL" odb.o@ FimOd PM ILUM FOC APIIMC A-MO D. @Af@ Lib*Pb OSV 0 NDM&=M@ IAIO""I-ILL4,1 ftpldod 4ffrL%4w-l STO OMM 4" InI pwi0d bomm Owndwt 4MPFW UNAM Aboftft PAWRon I%* I..4.n ai NL ou @we "8.6 Mdolk,W 10.0 amaTkVL 2a.0 um yr4lb it& wim BOLVAM 40 WYAL"* 2I0 amom OPMed AMR ffr 4L7 104'aul Newt ST4" WAltrdft &" WAI%M wm 4A ow 421 U OL Jd IIL190 12143 $74a we as$ so so To 410 40 so 217 L4. lig 1341 . . . .. . . .. iil7 ti,- 241 tfi', am IAS lm I" tm an 4M 4M UM Figure3. A representatiivoenchromatogramofa low-leve(l0.00915mg a.i./LP)FOS standard. AAMNDED WILDLIFE INTERNATIONAL LTD. -45- PROJECT NO.: 454A-109 P*ftt kkk Jd 1& 180 I&V CWb@#- Pft Cd P@ tIA&" o@ft Au,%,Ia & -we M ft& M OMC MAW--Morft - a" 0 1"1" F"$-I" wm also 4." InI pdsd *RFOB 420.7 NdlolbvL almaTHML lAhLVVAM WAAL %VIM BUL WAdlh FtTWWL "no 2.0 .2 :0 a EV*Mdff LOS A," 3214144 263216 ow Tka EIWTbm "M , 0' rawnkn Tbm TO- 2AI &46 027 A-SMM kiM JW IL 1"0 I*M op lei TO so so 40 0i 04 106I 1 1.1 1 226 249 276 $1 97, ' ' ill ii' -161 III '2il' '2il' BW O.U 143 lm 2.04 L4 &" L" 4A$ 4." @Ti" t zm-* P*fttbkkJdIt "W "W ftomm PIKOdPFC4WALM ftftAF%If= MD*%NCh@IAA44AIGO-ILUJW. 0-@ 4K-@*0IL W. Lb.006 -OW 0IZAAAO ftpleao PM-in M Lin 4M thI pwbd PFON smra%h* UnAm I 4jAisc!rNlWkmayTk" Ls ii" ltdol 10.0 GumaTbML Le LOILVAdih MIOL WAM SOLVAdih 4 ff VAL 6"4 2: wr 4I.17 Am 74934*4 454W RE"VAM w4m um Two 4A &a A.42=1 VAk Ad It.ISM 13= low 70 so so 40 ft 4"m cps 0.0 . 28 R7 83 IEG 164 91- III 151 101 gil *41 271 ow OM IM 143 2M 244 LOS &U 4M L" TMW Fipm 4. A representativieo'nchromatogram of a high-leve(l0.0915 mg a.i./LP)FOS standard. AMENDED WILDLIFE INTERNATIONAL LTD. -46- PROJ'ECTNO.: 454A-1-@-"-' hhw* ahmm it in* i&w C-6-2- ft. CA "M iamm ftft*A "Ke mm-.a- c@ D. .V.I"Oob-ow 012JLLn P*p 13d a FMO@uff 464A-IW noil-I 425isIPwW 4*fm am u voww SWW" 1:4ASCnI&M 494.7 Nol"TkVL LO ameitmm .2 L*L 0. LOAW%Mv64m e@ VAL%H :0 FrWrKF*L.* tIo SWAG& Evomdwr LSS AM 3114700 H"M 246140 WmTbm 221 EndTWA &44 YAM om Type A-M Mm JA Ia.tm 1= 100 179 lti24*M cps 70 40 le I,* 32 63 93 122 143 227 248 76 "3-1, 'it - -it - -iii- -iii- 'lil- -2it- 2ii- 'nit' "am OM 1.03 IM 2M L64 2A LE 4M 4A$ r@ Poo* um id CL "me "M Cdb." fftOd PFOBT&ALM PsftAM IQ" NDJA-M- 494&10 IL .@. Libard.-ow 0 ILRL" IAI""IM-12JULft Pp "a m PFOO-UT 464A-IW MAD-1 4AG InI poW bdww Mndwt 4HPFOB UMAM IBVA 4AjUQ!@lit i'g" M M Nobe iwm almitio@ ML VAM MUL WIM o@ VFAM gr VAL ON* 10.0 Lo 4 2: mr 4I.17 A@mi,v 10046 044 WAtt IWO 4A$ &dun 4" vf-4h sm roe A-4=M VAP. JA Ia.19M 1321 1 190 qw lot 90 70 S& 40 so I* 11 all go 171 *at La.. 4i,-Oi * 'lit" "lil' '2il' '2411 271 IA& ISI L04 L&4 SM &U 4M 4M Ibn Figure5. A representatiivoenchromatogramofa matrixblanksample(454A-109-MAB-1). no arrowindicatetsheretentiofni= ofPFOS. WILDLIFE INTERNATIONAL LTD. -47- PROJ,-ECTNO.: 454A-109 ftbat tbftJd St *IN It" oft Cd "M 1&%Lm fte. &" "OLC ickm-.D- 4U&10 01 AW Lft C@ift - CW 0 %SOAM 454A-ift- 4M InI PNiOd of4m &iM.M It lm 13= 1: 4A 01 L% MY OMM 426.7 90 Ndw Tk@ #*L VAIL WIM OWL VAdih 0 I Frrvf4LOMW to RT Los so AMIw 3149869 4 WAlt RJIA 264361 2A' Endlbm L4$ ,, - VACOM am PAIM raw an :0 T"- A-as 0 264NI qo 182 3d12q., _el,IS _91g,o an IA3 IAS 127143 ii. . 224 261 gil, -241- *ail' 22A,I 1L&614' '1LOS LU 4M 4A$ am ybw IA F*fttum Ad tL I"*IMY Caft"m PikCd PPOBIBJMW ftftA14W&C M*bnD@ O.-ft 454&10 0.-W. LIMCpb -OW 0 WAM IA@0"10-IZALW PM-"& 4S4A-IW 428 k%I pothd &AFAM& 4HPFOS AFN 4A (1@IlIWL,o.'Y@ 499.6 ng-Tk@ ame ikvl 10.0 2.0 kft TAM WI& YAM IN& "M 4 "%*Lono .: &.Ago I epmw Rr 4.17 AM losolO 110944 ow Tb" 4AI OdThn VOM 4A$ W444 T"o A-M lot 90 To as so 40 Ila" "0 217 10 too'49 'il 181 OM JAS IA3 2A4 a" IM LO 4= 4se T@ Figure6. A representatiivoenchromatogramofa matrixfordficatisoanmple(454A-109-MAS-1). WILDLIFE INTERNATIONAL L-m. -48- PROMCT NO.: 454A-109 COOK'N" paftt#@lPiInM lk-"m--A! ftft AN UMC M*1-0Cdo -OW 0 ISIO-N I WO"100-IZ"BW 3 Mw, JW 12. IM 14= cn beIpow m as" Sol 10 i..4m ai Lt. 2w 4".? H" THML 1.0 auwtib@ 0.2 NbLVAM VA& VFAV% 7 OWL WAdih 4: PfVfML ANf,fwtt sl$94so 276394 L$3 I" Tbm EndTMW &" YAM o3w= TWO A-= L it $2 31 o 161 eIlA3 IJ3 I2Z.I04 Lu K*WMW. 270M CP 4.1 4.. TWA P".* OAKid Et@ "27 PBKCd M= UALLN PdtCAPIMMO HD&bnCWM I*A"AIGO-IIALOR 4MA4" OLangoUb eph -ow 0ILLOLN PPOIL114 3 No% ikdIILloss14M lw 4M b Ipolbd bftmd 4HPFOB UNAM Abmkftpwbwdm11.9 I-4A al I&3w "6.4 Ndlelk,*L IOA so amwg ikill as &*L WAdb MWL VrAft 70 BMLVAdft 4 RT VibL0=4 a: so RT 4.. 6 Am sooilai 193061 4: am ibm sm 4m WW& am TWO A.4nja lnni cp ais 1-ts s'i iii *'I 'iii lil' lil' 211 241 271 SM 1= IS$ L*4 L&4 LOG &A 4M 4M 7bm Figur7e. A representatiivoenchromatogram ofa testsample(454A-109-3). PROJE 49!- APPENDIX IV CumulativePmmt Mortalityand Treatmbnt-RelateEdffects-'First-GencratiDoanphnids Sponsor. ----@mCor-pomtion TestSubstance: PFOS TestOrganism: Cladocemn, Daphnia DilutionWater WellWater Mean Measured TestConcentration (mgaijl) Replicate magna initial Number EXP=E NegativeControl A 1 B 1 c 1 D 1 E 1 F 1 G 1 H 1 I 1 i 1 1.5 A 1 B 1 c 1 D 1 E 1 F 1 G 1 H 1 1 1 Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2.9 A 1 0 B 1 0 c 1 0 D 1 0 E 1 0 F 1 0 G 1 0 H 1 0 1 1 0 1 0- ObservedEffects:AN AppearedNormal; C-Lethargic. 2 All2!lanismson Ra 0 apeearednormal. Day I Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN Number immobile 0 0 0 0 0 0 0 0 0 0 RU2 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 .0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN Number hnmobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 -50- PROJEC APPENDIX IV (Continued) CumulativePamt Mortalitaynd Treat[nent-RelatEefdferts-'First-GeneratiDoanphnids Sponsor Test Substance: TestOrganism: DilutionWater 3M Corporation PFOS Cladocemn, Daphnia magna Well Water Mean Measured TestConcentration (mg a.iJL) Replicate Initial Number EXPOSCd2 5.6 A 1 B 1 c 1 D 1 E 1 F 1 0 1 H 1 I 1 i 1 Number Immobile 0 0 0 0 0 0 0 0 0 0 pay I ..... Cumulative Dead Effects 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN, 0 1 AN 0 1 AN 0 1 AN 0 1 AN 12 A 1 0 0 1 AN B 1 0 0 1 AN c 1 0 0 1 AN D 1 0 0 1 AN E 1 0 0 1 AN F 1 0 0 1 AN 0 1 0 0 1 AN H 1 0 0 1 AN 1 1 0 0 1 AN i 1 0 0 1 AN 24 A 1 0 0 1 AN B 1 0 0 1 AN c 1 0 0 1 AN D 1 0 0 1 AN E 1 0 0 1 AN F 1 0 0 1 AN G 1 0 0 1 AN H 1 0 0 1 AN 1 1 0 0 1 AN i 1 0 0 1 AN 'robsmed Effects:AN - AppearedNorFnal;C - lzffiargic. 2 AllManisms on Da 0 !g=Ld normal. Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 2 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1c 0 0 1 AN Number hnmobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 PROJE( -51 APPENDIX IV (Continued) CumulativePercentMortalityand Treatindnt-RelatEefdfects-'First-GeneratiDoanphnids Sponsor Test Substance: TestOrganism: DilutionWater. 3m Corporation PFOS Cladocemn, Daphnia Well Water Mean Measured TestConcentmfion (mg a.i./L) Replicate magna Initial Number EXpoWd2 Number Immobile 48 A 1 0 B 1 0 c 1 0 D 1 0 E 1 0 F 1 0 0 1 0 H 1 0 I 1 0 i 1 0 ObservedEffects:AN AppearedNomal; C uflm,gic. 2 Allodwisms on Day 0 apE!EA normal. Day I Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN pal 2 Number Cumulative irnmobile' Dead 0 1 0 1 0 1 0 1 0 0 0 0 0 1 0 1 Effects - Numbe Immobi 0 0 0 0 0 0 0 0 0 0 -52-- PROJE APPENDIX IV (Continued) CumulativePercentMortalityand Treatm'ent-RelatEefdfects-'First-CTmeratiDoanphnids Sponsor 3M Corporation TestSubstance: PFOS TestOrganism: Cladocemn,Daphnia magna DilutionWater Well Water Mean Measured Initial Dav 4 Dav 5 TestConcentration Number Number Cumulafive Number Cumulative Nu (mg a.i./L) Replicate Exposed' Immobile Dead Effects Immobile Dead Effects Itnm NegativeControl A B c D E F G H I i 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 1.5 A 1 0 0 1 AN 0 0 1 AN B 1 0 0 1 AN 0 0 1 AN c 1 0 0 1 AN 0 0 1 AN D 1 0 0 1 AN 0 0 1 AN E 1 0 0 1 AN 0 0 1 AN F 1 0 0 1 AN 0 0 1 AN G 1 0 0 1 AN 0 0 1 AN H 1 0 0 1 AN 0 0 1 AN I 1 0 0 1 AN 0 0 1 AN 1 0 0 1 AN 0 0 1 AN 2.9 A 1 0 B 1 0 c 1 0 D 1 0 E 1 0 F 1 0 G 1 0 H 1 0 I 1 0 i 1 0 ObservedEffects:AN - AppearedNormal; C - Lethargic. 2 Alloglanismson D!l 0 agff!rcdnormal. 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN PRO -53 APPENDIX IV (Continued) CumulativePercentMortalityand Treatrncht-RelatEefdfects-'First-GeneratiDoanpbnids Sponsor: Test Substance: TestOrganism: DilutionWater: 3M Corporation PFOS Cladocemn, Daphnia magna Well Water Mean Measured TestConcentration (mg a.i./L) Replicate Initial Number ExpwO 5.6 A 1 B 1 c 1 D 1 E 1 F 1 0 1 H 1 I 1 i 1 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 4 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 12 A 1 0 0 1 AN B 1 0 0 1 AN c 1 0 0 1 AN D 1 0 0 1 AN E 1 0 0 1 AN F 1 0 0 1 AN 0 1 0 0 1 AN H 1 0 0 1 AN 1 1 0 0 1 AN i 1 0 0 1 AN 24 A 1 0 B 1 0 c 1 0 D 1 0 E 1 0 F 1 0 0 1 0 H 1 0 I 1 0 i 1 0 ObservedEffects:AN - Appon-edNormal; C - Lethargic. 2 AlloLLanismson Day 0 !22MTd nomal. 1 1 1 0 1 AN 1 1 0 1 AN 0 1 AN 1 1 u=mber Immobile 0 0 0 0 0 0 0 0 0 0 Day 5 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 1 0 1 0 1 0 0 1 AN 0 1 0 1 0 0 1 AN 0 0 1 AN 0 1 0 1 Num bnmo 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 -54- PROJE APPENDIX IV (Continued) CumulativePercentMortalityand Treatmeht-RelateEdffects-'First-GeneratiDoanphnids Sponsor Test Substance: TestOrganism: DilutionWater: 3M Corpomtion PFOS Cladomm, Daphnia magna Wen Water Mean Measured TestConcentration (mg a.idl) Replicate Initial Number ExPOseII2 Number Immobile 48 A 1 0 B 1 0 c 1 0 D 1 0 E 1 0 F 1 0 G 1 0 H 1 0 I 1 0 i 1 0 ObservedEffects:AN - AppearedNormal; C Lethargic. 2 All2manisms on P.aZ 0 !Wcared normal. Day 4 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 5 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects PROJE 55 APPENDIX IV (Continued) CumulativePercentmortalityand Treatmcht-RelateEdffects-'First-GeneratiDoanpbnids Sponsor TestSubstance: TestOrganism: 3M Corporation PFOS Cladocemn,Daphnia magna DilutioWnater: Won Water Mean Measured TestConcentration Initial Number (mga.i./L) Replicate ExPOsOd' NegativeControl A 1 B 1 c 1 D 1 E 1 F 1 0 1 H 1 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 7 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1 AN 1 AN 1 AN 1 AN 1 AN 1AN 1AN 1AN 1AN 1 AN 1.5 AB 11 00 00 11 AANN c 1 0 0 1 AN D 1 0 0 1AN E 1 0 0 1AN F 1 0 0 1AN 0 1 0 0 1AN H 1 0 0 1AN 0 0 1 AN 0 0 1 AN 2.9 A 1 0 0 1 AN B 1 0 0 1 AN c 1 0 0 1 AN D 1 0 0 1 AN E 1 0 0 1 AN F 1 0 0 1 AN 0 1 0 0 1AN H 1 0 0 1 AN I 1 0 0 1 AN i 1 0 0 IAN ObservW Effects:AN - App@eareNdormal;C lediargic. 2 Allomisms on L)a0lapff!rcndomal. Number hnmobile 0 0 0 0 0 0 0 0 0 0 Dav 8 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1AN 1AN 1AN 1AN 1AN 1 AN 1AN 1AN 1AN 1AN 0 0 1AN 0 0 1AN 0 0 1AN 0 0 1 AN 0 0 1AN 0 0 1 AN 0 0 1 AN 0 0 1AN 0 0 1AN 0 0 1AN 0 0 1AN 0 0 1AN 0 0 1AN 0 0 1AN 0 0 1AN 0 0 1AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN Num Immobi 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 -56- PROJE( APPENDIX IV (Continued) Cumulatve pc=nt Mortahtyand Treatinent-RelatEefdfects-'First-GeneratiDoanphnids Sponsor 3M Corporation TestSubstance: PFOS TestOrganism: Cladocemn, Daphnia magna DilutionWater: WellWater Mean Measured Initial D!y 7 Day 8 TestConcentration Number Number Cumulative Number Cumulative Nu (mg a.i./L) Replicate Exposed2 Inimobile Dead Effects Immobile Dead Effects im 5.6 A 1 0 0 1 AN 0 0 1 AN B 1 0 0 1 AN 0 0 1 AN c 1 0 0 1 AN 0 0 1 AN D 1 0 0 1 AN 0 0 1 AN E 1 0 0 1 AN 0 0 1 AN F 1 0 0 1 AN 0 0 1 AN 0 1 0 0 1 AN 0 0 1 AN H 1 0 0 1 AN 0 0 1 AN I 1 0 0 1 AN 0 0 1 AN i 1 0 0 1 AN 0 0 1 AN 12 A 1 00 00 11AANN 00 00 1IAANN B 1 c 1 0 0 1 AN 0 0 1 AN D 1 0 0 1 AN 0 0 1 AN E 1 0 0 1 AN 0 0 1 AN F 1 0 0 1 AN 0 0 1 AN 0 1 0 0 1 AN 0 0 1 AN H 1 0 0 1 AN 0 0 1 AN I 1 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 24 A B c D E F G 1 H 1 I 1 i 1 ObservedEffects:AN AppearedNOMRI; C All2ganisms on Day 0 appearednormal. 0 0 0 0 0 0 0 0 0 0 Lcftrgic- 1 1 1 0 1 AN 1 1 0 1 AN 0 1 AN 1 1 0 1 0 1 0 1 0 0 1 AN 0 1 0 1 0 0 1 AN 0 0 1 AN 0 1 0 1 -57- PROJE APPENDIX IV (Continued) CumulativePmmt Mortalityand Treaftn6nt-RelatEefdfects-'First-GeneratiDoanphnids Sponsor. Test Substance: TestOrganism: Dilubon Water. 3M Corporation PFOS Cladocemn, Daphnia magna Well Water Mean Measured TestConcentration (mg a.i./L) Replicate Inifial Number Exposed' Number Inmobile 48 A 0 B 0 c 0 D 0 E 0 F 0 0 0 H 1 0 1 0 1 0 ObservedEffects:AN - AppearedNormal; C lathugic. 2 All2M'sms on Day 0 agE!.a2d normal. )Lay7 Cumulative Dead 1 1 Effects Number lxnmobile 0 0 0 0 0 0 0 0 0 0 Day 8 Cumulative Dead Effects Num Imm 0 0 0 0 0 0 0 0 0 0 -58- PROJE APPENDIX IV (Continued) CumulativePmmt Mortalityand Treatmdnt-RelateEdffects-'First-GeneratiDoanphnids Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: Cladocemn, Daphnia magna Ddutwn Water: Well Water Mean Measured Initial 2910 D!l 11 Ted Concentration Number Number Cumulative -7um=b@er cumulative (mg a.i./L) Replicate Exposed' Immobile Dad Effects immobile Dead Effects Iin NegativeControl A 1 0 0 1 AN 1 0 0 1 AN 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN B c D 1 0 1 0 0 1 AN 0 0 1 AN E 1 0 0 1 AN 0 0 1 AN F 1 0 0 1 AN 0 1 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN H 1 I 1 0 0 1 AN 0 0 1 AN i 1 0 0 1 AN 0 0 1 AN 1.5 A 1 0 0 1 AN 0 0 1 AN 1 AN 1 0 0 1 AN 0 1 AN 0 0 0 0 1 AN B c 1 0 D 1 0 0 1 AN 0 0 1 AN E 1 0 0 1 AN 0 0 1 AN 1 AN 1 0 0 1 AN 1 AN 0 0 0 0 1 AN F 0 1 0 0 H 1 0 0 1 AN 0 0 1 AN 1 0 0 1 AN 0 0 1 AN 1 AN 1 0 0 1 AN 0 0 2.9 A 1 B 1 c 1 D 1 E 1 F 1 0 1 H 1 I 1 i 1 @r-ObservedEffects:AN - AppearedNormal; C 2 Allorganismson Day 0 ao!2.d normal. 0 0 0 0 0 0 0 0 0 0 Lediargic. 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN PROJ -59@ APPENDIX IV (Continued) CumulativePercentMortalityand Treatment-RelateEdffects-'First-GencratiDoanphnids Sponsor: TestSubstance: Ted Organism: DilutionWater 3M Corporation PFOS Cladocemn,Daphnia magna WellWater Mean Measured TestConcentration (mg a.i./L) Replicate initial Number EYPOOed2 5.6 A 1 B 1 c 1 D 1 E 1 F 1 0 1 H 1 I 1 i 1 Number finmobile 0 0 0 0 0 0 0 0 0 0 Day 10 Cumulati7a Dead 0 0 0 0 0 0 0 0 0 0 Effects 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 11 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 12 A 1 0 0 1 AN 0 0 1 AN B 1 0 0 1 AN 0 0 1 AN c 1 0 0 1 AN 0 0 1 AN D 1 0 0 1 AN 0 0 1 AN E 1 0 0 1 AN 0 0 1 AN F 1 0 0 1 AN 0 0 '1AN G 1 0 0 1 AN 0. 0 1 AN H 1 0 0 1 AN 0 0 1 AN I 1 0 0 1 AN 0 0 1 AN i 1 0 0 1 AN 0 0 1 AN 24 A 1 0 1 B 1 0 1 c 1 0 1 D 1 0 0 1 AN E 1 0 1 F 1 0 1 0 1 0 0 1 AN H 1 0 1 I 1 0 1 i 1 0 1 --r-ObserveEdffects:AN Appeared Normal; C - Lethargic. 2 Allorganismson Day 0 appearednormal. 0 1 0 1 0 1 0 0 1 AN 0 1 0 1 0 0 1 AN 0 1 0 1 0 1 PROJ -60- APPENDIX IV (Continued) CumulativePercentMortalityand Treatmeht-RelateEdffects-'First-CmeratioDnaphnids Sponsor 3M CDrpmtion TestSubstance: PFOS TestOrganism: Cladocemn,Daphnia magna DilutionWater WellWater Mean Measured TestConcentration (mg a.iJL) Initial Number Repliclt' ExPOsld2 Number Immobile 48 A 1 0 B 1 0 c 1 0 D 1 0 E 1 0 F 1 0 0 1 0 H 1 0 1 1 0 i 1 0 ObservedEffects:AN -Appeared Normal; c - Leffiargic. 2 Allorganismson Day 0 !gg!arednormal. Day 10 Cumulative Dead 1 1 1 1 1 1 1 1 1 1 Effects Number Immobile 0 0 0 0 0 0 0 0 0 0 Day II Cumulative Dead Effects Number Immobilq 0 0 0 0 0 0 0 0 0 0 PROJE -61 APPENDIX IV (Continued) CumulativePercentMortalityand Treatmdnt-RelateEdffects-'First-GeneratiDoanphnids Sponsor: 3M Corporation Ted Substance: PFOS TestOrganism: cladocemn,Daphnia magna I)ilutioWnater Well Water Mean Measured initial 13 Dav 14 TestConcentration Number Number Cumulative Number Cumulative Nu (mg a.i./L) Replicate ExPoscld2 Irmnobile Dead Effects Immobile Dead Effects hnn NegativeControl A 1 0 0 1 AN 1 AN 0 0 1 AN 0 0 1 AN B 1 0 0 c 1 0 0 1 AN 0 0 1 AN D 1 0 0 1 AN 0 1 AN 0 0 1 AN 0 0 1 AN E F 1 0 1 0 0 1 AN 0 0 1 AN 0 1 0 0 1 AN 0 0 1 AN H 1 0 0 1 AN 0 0 1 AN I 1 0 0 1 AN 0 0 1 AN i 1 0 0 1 AN 0 0 1 AN 1.5 A 1 0 0 11 AANN 00 00 1IAANN B 1 0 0 c 1 0 0 1 AN 0 0 1 AN D 1 00 00 11 AANN 00 00 11AANN E 1 F 1 0 0 1 AN 0 0 1 AN G 1 0 0 1 AN 0 0 1 AN H 1 0 0 1 AN 0 0 1 AN I 1 0 0 1 AN 0 0 1 AN 1 0 0 1 AN 0 0 1 AN 2.9 A 1 0 0 1 AN B 1 0 0 1 AN c 1 0 0 1 AN D 1 0 0 1 AN E 1 0 0 1 AN F 1 0 0 1 AN G 1 0 0 1 AN H 1 0 0 1 AN I 1 0 0 1 AN i 1 0 0 1 AN ObservedEffects:AN Appeared Normal; C Lcffiugic. 2 Allogoisms on Pay 0 !ge=r.!@normal 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN -62- PROJEC APPENDIX IV (Continued) CumulativePercentMortalityand Treatm6nt-RelateEdffects-'First-GeneratiDoanphnids Sponsor 3M Corporation TestSubstance: PFOS TestOrganism: Cladocemn,Daphnia magna DilutioWnater WellWater Mean Measured TestConcentration (mga.iJL) Initial Number Replicate ExPsW2 5.6 A 1 B 1 c 1 D 1 E 1 F 1 0 1 H 1 I 1 i 1 Number Immobile 0 0 0 0 0 0 0 0 0 0 Dgl 13 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1 AN 1 AN 1 AN 1 AN 1AN 1 AN 1AN 1 AN 1 AN 1 AN 12 A 1 0 0 1 AN B 1 0 0 1 AN c 1 0 0 1 AN D 1 0 0 1 AN E 1 0 0 1 AN F 1 0 0 1AN G 1 0 0 1 AN H 1 0 1 I 1 0 0 1AN 1 0 0 1 AN 24 A 1 0 1 B c 1 0 1 0 1 1 - D 1 0 0 1AN E 1 0 1 F 1 0 1 G 1 0 H 1 0 I 1 0 i 1 0 ObservedEffects:AN - AppearedNormal;t@ Lethargic. 2 Allomanismson !2!Z0 appearednormal. Number Immobile 0 0 0 0 0 0 0 0 0 0 pal 14 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1AN 1AN 1AN 1AN 1 AN 1 AN 1 AN 1 AN 1AN 1AN 0 0 1AN 0 0 11AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 1 0 0 1 AN 0 0 1 AN 0 1 0 1 0 1 0 0 1AN 0 1 0 1 0 1 0 1 0 1 0 1 Num finmo 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 .63- PROJE APPENDIX IV (Continued) CumulativePercentMortalitayndTreatm6t-RelateEdff"' -First-GemationDaphnids Sponsor 3M Corporation TestSubstance: PFOS TestOrpnism: Cladocemn,Daphnia magna DilutioWnater: Well Water Mean Menured initial Day 13 Day 14 Test Concentration Number Number Cumulative Number Cumulative Nu (mg a.i./L) Replicate EXP09cd2 Immobile Dead Effects Immobile Dead Effects hnn 48 A 1 0 1 B 1 0 1 c 1 0 1 D 1 0 1 E 1 0 1 F 1 0 1 0 1 0 1 H 1 0 1 I 1 0 1 i 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 ObwrvedEffects:AN -Appeared Normal; C - Lcdwgic. 2 Allomisms on Day 0 mmt.!@ normal. PRO -64- APPENDIX IV (Continued) CumulativePercentMortalityand Treatinent-RelatEefdfects-'First-GeneratiDoanphmds Sponsor 3M Corporation TestSubstance: PFOS TestOrganism: Cladocem, Daphnia magna Dflu6onWater WellWater Mean Measured initial pay 16 17 TestConcentration Number Number Cumulative Number Cumulative Nu (mg a.i./L) Replicate Exposed2 Immobile Dead Effects Immobile Dead Effects im NegativeControl A B c D E F G H 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 1.5 A 1 0 0 IAN 0 0 1 AN B 1 0 0 1 AN 0 0 1 AN c 1 0 0 1 AN 0 0 1 AN D 1 0 0 1 AN 0 0 1 AN E 1 0 0 1 AN 0 0 1 AN F 1 0 0 1 AN 0 0 1 AN G 1 0 0 1 AN 0 0 1 AN H 1 0 0 1 AN I 1 0 0 1 AN 0 0 1 AN 0 0 1 AN i 1 0 0 1 AN 0 0 1 AN 2.9 A 1 B 1 c 1 D 1 E 1 F 1 G 1 H 1 1 1 ObservedEffects-A:N AppearedNormal; C 2 Allogmisms on D!Z 0 app!!Ed normal. 0 0 0 0 0 0 0 0 0 0 Ledwgic. 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN PROI -65,.- APPENDIX IV (Continued) CumulativePercentMortalityand TreatmentRelatedEffects-'FirstGenerationDaphmds Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: Cladocem, Daphnia magna DilutionWater Well Water Mean Measured initial (16 )LX 17 TestConcentration Number Number Cumulative Number Cumulative (mg a.i./L) Replicate ExpoSed2 Immobile Dead Effects Immobile Dead Efrects b 5.6 A 1 0 0 1 AN 0 0 1 AN B 1 0 0 1 AN 0 0 1 AN c 1 0 0 1 AN 0 0 1 AN D 1 0 0 1 AN 0 0 1 AN E 1 0 0 1 AN 0 0 1 AN F 1 0 0 1 AN 0 0 1 AN G 1 0 0 1 AN H 1 0 0 1 AN I 1 0 0 1 AN 0 0 1 AN 0 0 1AN 0 0 1AN i 1 0 0 1 AN 0 0 1 AN 12 A 1 0 0 1 AN 0 0 1 AN B 1 0 0 1 AN 0 0 1 AN c 1 0 0 1 AN 0 0 1 AN D 1 0 0 1 AN 0 0 1 AN E 1 0 0 1 AN F 1 0 0 1 AN 0 0 1 AN 0 @o I AN G 1 0 0 1 AN H 1 0 1 0 0 1 AN 0 1 I 1 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 24 A 1 0 B 1 0 c 1 0 D 1 0 E 1 0 F 1 0 G 1 0 H 1 0 I 1 0 i 1 0 ObservedEffects:AN AppearedNormal; C - Ledargic. 2 AJIo!lanismson Rg 0 !222!ad normal. 1 1 1 0 1 AN 1 1 1 1 1 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 PRO -66- APPENDIX IV (Continued) CumulativePercentMortalityand Treatment-RelateEdffects-'First-GeneratiDoanphmds Sponsor: TestSubstance: TestOTganism: DilufionWater 3M Corporation PFOS Cladocemn, Daphnia magna Well Water Mean Measured initial D!X 16 Day 17 TestConcentration Number Number Cumulative Number Cumulative Nu (mg a.i./L) Replicate EXPO$ed2 Immobile Dead Effects Immobile Dead Effects im 48 A 1 0 1 B 1 0 1 c 1 0 1 D 1 0 1 E 1 0 1 F 1 0 1 0 1 0 1 H 1 0 1 I 1 0 1 i 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 ObservedEffects:AN - AppearedNormal; C Lediargic. 2 Allom2isms on Pal 0 !EE!ad normal. PRO -67- APPENDIX IV (Continued) CumulativePercentMortalityand Treati@ent-RelatEefdfects-'First-GcneratiDoanphnids Sponsor: TestSubstance: TestOrganism: DilutionWater. 3M Corporation PFOS Cladocemn, Daphnia magna Well Water Mean Measured Initial R!Y 19 RU20 TestConcentration Number Number Cumulative Number Cumulative Nu (mg a.iJL) Replicate ExpoSw2 Immobile Dead Effects Immobile Dead Effects im NegativeControl A B c D E F G H I i 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 1 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 1.5 A 1 0 0 1 AN 0 0 1 AN B 1 0 0 1 AN 0 0 IAN c 1 0 0 1 AN 0 0 1 AN D 1 0 0 1 AN 0 0 1 AN E 1 0 0 1 AN 0 0 1 AN F 1 0 0 1 AN 0 0 1 AN 0 1 0 0 1 AN 0 0 1 AN H 1 0 0 1 AN I 1 0 0 1 AN 0 0 1 AN 0 0 1 AN i 1 0 0 1 AN 0 0 1 AN 2.9 A 1 0 B 1 0 c 1 0 D 1 0 E 1 0 F 1 0 G 1 0 H 1 0 1 0 1 0 Observed Effects:AN - AppearedNormal; C - Lethargic. 2 All2Misms on!2al0 ap22!Ednormal. 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN -68- PROJE APPENDIX IV (Continued) CumulativePercentMortalityand Treatm6t-RelatedEffects-'First-GeneratiDoanphnids Sponsor 3M Corporation TestSubstance: PFOS TestOrganism: Cladoccmn,Daphnia magna DilufioWnater: WellWater Mean Measured Initial Day 19 )Ll20 TestConcentration Number Number Cumulative Number cumulative Nu (mga.i./L) Replicate Exposed' Immobile Dead Effects Immobile Dead Effects Imn 5.6 A 1 0 0 1AN 0 0 1 AN B 1 0 0 1AN '0 0 1 AN c 1 0 0 1AN 0 0 1 AN D 1 0 0 1 AN 0 0 1 AN E 1 0 0 1 AN 0 0 1 AN F 1 0 0 1AN 0 0 1 AN G 1 0 0 1AN 0 0 1 AN H 1 0 0 1 AN 0 0 1 AN I 1 0 0 1AN 0 0 1 AN i 1 0 1 0 1 12 A 1 0 0 1 AN 0 0 1 AN B 1 0 0 1 AN 0 0 1 AN c 1 0 0 1AN 0 0 1 AN D 1 0 0 1 AN 0 0 1 AN E 1 0 0 1AN 0 0 1 AN F 1 0 0 1AN 0 0 1 AN 0 1 0 0 1AN 0 0 1 AN H 1 0 1 0 1 I 1 0 0 1 AN 0 0 1 AN i 1 0 0 1AN 0 0 1 AN 24 A 1 0 1 B 1 0 1 c 1 0 1 D 1 0 1 E 1 0 1 F 1 0 1 0 1 0 1 H 1 0 1 I 1 0 1 i 1 0 1 ObservedEffectsA:N AppearedNormal;C - Ixdiugic. 2 Alloglanismosn D!y 0 !EU!2d nomud. 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 PROJB APPENDIX IV (Continued) CumulativePercentMortalityand Treato@ent-RelatEefdfects-'First-GeneratiDoanphnids Sponsor 3M Corporation Ted Substance: PFOS TestOrganism: Cladocom, Daphnia magna DilubonWater WellWater Mean Measured initial Day 19 Day 20 Test(Cmogncae.nit.r/aLt)ion Replicate ENXuPmObSeedr2 bNnummobbeirle CumDuelaadtive Effects INmummobbeirle CumDuelaadtive Effects INmumm 48 A 1 0 1 0 1 0 B 1 0 1 0 1 0 c 1 0 1 0 1 0 D 1 0 1 0 1 0 E 1 0 1 0 1 0 F 1 0 1 0 1 0 G 1 0 1 0 1 0 H 1 0 1 0 1 0 I 1 0 1 0 1 0 i 1 0 1 0 1 0 ObservedEfrects:AN - AppearedNonnaL C Udmgic. 2 All2manismson Day 0 2Ecarednormal. WILDLIFE INTERNATIONAL LTD. PROJFCTNO.:454A-109 -70- APPENDIXV NeonateProduction Sponsw. Ted Substance: TestChpnism: DilutionWater 3M CoMorafion PFOS Cladoctran,Daphnia magna wellwater Mean Measured Test L)ax Concentration 2 5 7 9 11 14 16 19 21 JER LIJLI Rcpficau NegativeControl A 0 0 0 0 27 40 0 25 26 B 0 0 0 9 41 0 45 4 14 c 0 0 0 0 14 43 0 27 10 D 0 0 0 3 29 48 0 39 25 E 0 0 0 9 40 41 0 35 27 F 0 0 0 0 24 37 0 34 31 G @o 00 14 0 38 39 19 20 H 0 00 0 21 29 0. 21 29 1 0 00 9 32 45 0 31 23 1 0 00 0 20 33 34 0 18 1.5 A 0 0 0 0 21 43 0 36 31 B 0 0 0 2 0 39 39 0 26 c 0 0 0 6 37 47 0 38 35 D 0 0 0 0 23 44 0 0 32 E 0 0 0 0 20 33 35 0 31 F 0 0 0 5 38 51 0 36 30 G 0 0 0 0 25 48 31 0 22 H 0 0 0 16 46 44 1 41 34 1 0 0 0 10 37 27 12 40 33 1 0 0 0 3 29 41 0 37 27 2.9 A 0 0 0 0 13 37 35 7 17 B 0 00 7 38 41 4 41 36 c 0 00 11 37 38 0 42 34 D 0 00 0 25 45 33 0 20 E 0 00 0 24 43 34 3 23 F 0 00 19 0 47 36 1 14 G 0 00 0 19 44 35 0 24 H 0 00 0 17 40 43 0 35 1 0 00 15 41 0 38 33 15 1 0 00 0 26 46 29 0 29 Ms valueexr-ludcdfrom c431cuMon of the mean due toadultdeath. Total 118 113 94 143 151 126 129 100 140 105 SD) = 122 (19.2) 131 106 163 99, 119 160 126 182 159 136 SD) = 142 (24.7) log, 167 162 123 127 116 122 135 142 129 SD) - 136(t17.9) AMENDED WILDLIFE INTERNATIC)NAL LTD. PROJECT NO.: 454A- 109 -71- Sponsw. TestSubgance: TestOrganim: DilutioWnater Mean Measured Test Conomtrafion APPENDIX V (Continued) NeonateProduction 3M CoWmfion PFOS Cladocmn. Daphda Wen Water -agna 25 7 9 11 14 16 19 21 5.6 A 00 0 0 21 38 31 0 35 B 00 0 5 34 43 0 31 33 c 00 0 0 22 37 19 0 26 D 00 0 0 27 41 29 0 20 E 00 0 0 19 43 39 0 29 F 00 0 11 36 52 0 37 34 G 00 0 0 17 38 4 34 32 H 00 0 5 29 46 0 38 30 1 0a 0 0 14 39 40 0 34 00 0 0 19 35 19 - - 12 A 00 0 0 19 39 0 0 29 B-- 0 0 0 0 16 40 34 17 20 c 00 0 0 24 36 35 0 34 D 00 0 is 0 39 34 19 0 E 00 0 0 26 36 37 0 34 F 00 0 10 37 0 40 37 15 G 00 0 10 34 41 0 39 41 H 00 0 0 20 - - - - 1 00 0 0 11 30 32 0 30 00 0 0 13 27 29 0 14 24 A 0- - - - - - B 0 c 0 D 00 0 0 0 8 24 E 0 F 0 G 00 0 0 9 H 00 0 0 1 0 1 0 48 A B c D E F G H This valuee=luded from calcumon of themean due to adultdeath. Total 125 146 104 117 129 170 125 148 127 73' SD) - 132 19.5) 86 127 129 109 133 139 165 20 1 103 83 SD) = 119 (26.5) 0 0 0 321 0 0 91 0 0 .0 0 0 0 0 0 0 0 0 0 0 0 rc -0 AMENDED WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454A-109 -72- APPENDIXVI Lengthand Dry Weightof SurvivinFgirst-GeneratiDoanphnids Sponsor. 3M Corporation TestSubstance: PFOS TestOrganimn: Cladoceran,Daphnia magna DilutioWnater- WellWater Mean MeasuredTest Concentration Individual Length NegafiveControl LT!P) 4.70 4.65 4.55 4.65 4.55 4.80 4.@5 4.50 4.75 4.80 Mean Length Lmm SD) 4.65(t0.111) 1.5 4.45 4.66 0.118) 4.60 4.70 4.80 4.55 4.75 4.80 4.60 4.65 2.9 4.62 0.100) 4.-7-5 4.50 4.65 4.70 4.@5 4.45 4.70 4.60 4.65 Individual Dry Weights - @!W 0.73 0.68 0..87 0.65 0.60 0.69 0.75 0.61 0.55 0.82 0.68 0.70 0.63 0.81 0.61 0.67 0.63 0.68 0.61 0.63 0.68 0.70 0.94 0.7-10.80 0.82 0.60 0.63 Mean Dry Weights Lma sD) 0.695(0.100) 0.669 0.0623) 0.724 0.110) ANffi'NDED WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-109 -73- APPENDIX VI (Continued) Lmgth and Dry WeigbtofSurvivingFirst-OmcratioDnaphnids Sponsor- 3M Corporation TestSubstance: PFOS TestOrganism: Clado=un, Daphnia magna DilutioWnater- WellWater Mean MeasuredTest Concentration Individual Length Lmg a.i./L) LIB!P@ 5.6 4.45 4.70 4.50 4.70 4.65 4.8-0 4.45 4.55 4.65 mean Length @mm SD) 4.61(0.124) individual Dry Weights L%f@ 0.71 0.69 0.83 0.75 0.82 0.68 0.62 0.73 0.71 Mean Dry Weights Lms t SD) 0.727(t0.0665) 12 4.70 4.59 0.102) 0.76 0.723 0.0661) 4.60 0.64 4.75 0.76 4.65 0.65 4.45 0.82 4.60 0.65 4.55 0.70 4.45 0.74 4.55 0.79 AMENDED WILDLIFE INTERNATIONAL L-rD. -74- APPENDIXVII Changes toProtocol PROJECTNO.: 454A-109 Thissu* was conductedinaccordancweiththeapprovedProtocolwiththefollowincghanges: I. The proposedexperimentasltaratnd terminatiodnateswmr, amended totheprotocol. 2. Duringthesecondgencrafioenxposure,neonateswere observedat5,24 and 48 hours. WILDLIFE INTERNATIONAL LTD. -75APPENDIX VIH PersonnelInvolvedintheSb* PROJECT NO.: 454A- 109 The followinkgey Wildlifeh*mational Ltd.pmwml Sb*: weremvolved m theconductormanagement offlm I Hemy 0. Krueger,Ph.D.,Dk=Wr, AquaticToxicologyand Non-TargetPlants 2. WiUud B. Nbwn, Ph.D.,Mmu6m, AnalyticaClhemistry 3. Jon A. MacGregor, Scientist 4. Mark A. Mank, LaboratorySupm-visor 5. Kurt P, DrottarS,eniorBiologist WILDLIFE INTERNATIONAL L-rD. PROJECT NO.: 454A-109 -76APPENDIX IX ReportAmendment I. OriginaRleport: Pages 14, 6 and 31 Amendment: The pageswere changedtoincludetheamended reportdate,revisedpagenumbers,and new signatureasnd datesdue totheadditioonfthereportamendment asAppendix DC Reason: To reflecttheissuingof an amended reporl 2. OriginaRleport: Page 2 Amendment: The compliancestatementwas revised. Reason: To clarifhyow thetestsubstancewas characterized. OriginaRleport.-Page 9 Amendment: Informatiopnrovidedby theSponsorreflectitnhgereanalysiosfthetestsubstance, includintghereanalysidsateand thepurityw,as addedtotheTestSubstancesection. Reason: To reflectthecurrentestsubstanceinformatiopnrovidedby theSponsor. 4. OriginaRleport: Entirereport Amendment: Alltestsubstancceoncentratiownesrechangedtoreflectthepiu4 ofthetestsubstance asdeterminedby theSponsorina reanalysiosf thetestsubstance(FC-95,Lot 217). Testconcentratioonrsiginalwleyre basedon thereportepdurityof 98YIo. The certificaotfeanalysidsatedMarch 9,2000 indicateadpurityof90.490/oT.bereforea,ll test substanceconcentrationisn,cludingnominal concentrationsm,easured concentrationasn,dNOEC, LOEC andMATC valuesw,ererecalculateadnd reported asmg a.i./bLasedon the90.49% purity. Reason: To reportheresultosfthetestbasedon thetestsubstancepurityof 90.49% atthe requestoftheSponsor. AMENDED WILDLIFE INTERNATIONAL LTD. -77APPENDIX IX -ContiimedReportAmendment AMENDMENT SIGNATURES: KurtP,)Frottar- DATE ShidyDirector Henr@ 6QKmegei, -Ph.D. DirectorA,quaticToxicologyandNon-TargetPlants REVEEWED BY:' Jaj(96Hs. Coleman QualityA.ssurance DAtLt DATE PROJECTNO.: 454A-109 AMENDED PROTOCOL PFOS: A SENU-STATIC LIFE-CYCLE TOMCRTY T WITH THE CLADOCERAN (Daphniamagna) U.S.EnvironmentaPlrotectioAngency Series850 - EcologicalEffectsTestGuidelines OPPTS Number 850.1300 OECD Guideac 211 3M Lab RequestNo. U2723 WILE)LIFE Submittedto 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St.PauL Minnesota 55144 INTF-RN'ATIONAL LTD. 8598 Commerce Drive Easton,Maryland 21601 (410)822-8600 June 10, 1999- WILDLIFE INTERNATIONAL, LTD -2- PFOS:A SENU-STATILCEFE-CYCLTE03aCnYTEST WnE THECLADOCERAN(Daphnmiaagna) SPONSORSPONSOR!S REPRES 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St.PauL Minnesota55144 Ms. SusanA. Beach TESTING FA WildlifIenternationLatld,. 8598 Commerce Drive FAston,Maryland 21601 STUDY DIRECTOR-- KurtDrottar SeniorAquaticBiologist LABORATORY MANAGEMENT: Henry 0. Kmeger,Ph.D. DirectoorfAquaticToxicology& Non-TargetPlants FOR LABORATORY USE ONLY ProposedDates: Experimental StartDate: Experimental TerminationDate: ProjectNo.: TestConcentrations-:NeizativCeontrol,1.6.3.1.6.3.13,25 and 50 miza.i./L TestSubstanceNo.: 4675. ReferenceSubstancNeo. (ifapplicable):4526 PROTOCOL APPROVAL STUDY DIRECTOR DATE ORATORY MANAGEMENT TOR sPIO OSRRW0!'LS ri RYE IP'R E S iz a"r-finlc- DATE 6 13D DATE PROTOCOL No.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD 3- INTRODUCTION WildlifeIntemationaLLtd.willconducta semi-statilcife-cyctloexicittyestwith the cladoceran, Daphnia nwgna, forthe SponsorattheWildlifeInternationaLlt,d.aquatictoxicologyfacilitiyn Easton, Maryland. The study willbe performed based on proceduresoutlinedinthe U.S. EnvironmentalProtection Agency Series850 - EcologicalEffectsTest GuidelinesOPPTS Number 850.1300 (1);OECD Guideline 211: Daphnia magna ReproductionTest (2);Standard EvaluationProcedure,Daphnia Magna Life-Cycle (21-Day Renewal) Chronic YbxicityTest (3);and ASTM E 1193-87 Standard Guide for Conducting Renewal Life-CycleTodcity TestswithDaphnia magna (4).Raw dataforallwork performed at Wildlife IntemationaLLtd.and a copy of the finalreportwillbe filedby projectnumber inarchiveslocatedo.n the WildlffeIntemationaLLtd.siteo,r atan alternativleocationtobe specifiedinthe finalreport. PURPOSE The purpose ofthisstudyisto determinethe effectsofthetestsubstanceon thesurvivalg,rowth, and reproductionof thecladoceran,Daphnia magna, under seim*-stattiecstconditionsf,ora periodof 21 days. EXPERIMENTAL DESIGN Daphnids willbe exposed to a geometricseriesof sixtestconcentrationsa,nd a negative(dilution water) controlfor 21 days. Individualneonate daphnidswillbe placed in ten replicatteestchambers iinparday assignedto each testconcentrationand control.Individualleyxposed daphnids willbe observed forsurvivalr,eproductionand growth. Nominal testconcentrationwsillbe selectedinconsultatiownith the Sponsor,and willbe based upon informationsuch as the resultsof exploratoryrange-findmgtoxicitydata. known toxicitydata, physical/chemicalpropertiesof the testsubstanceor otherrelevantinformation. Each. testsubstance concentrationwillbe 50% ofthe next higherone. Water sampleswillbe collectedatspecifiedintervalfsor analysisof the testsubstance. Resultsof the analyseswillbe used to calculatemean measured test concentrations.New testsolutionswillbe preparedthreetimes per week (e.g.,Monday, Wednesday, Friday),and thetestorganisms willbe transferrefdrom the oldtothenew solutions. To controlbias,neonate daphnids willbe impartiallayssignedto exposurechambers attestinitiation. No other potentialsourcesof bias are expectedto affectthe resultsof the study. Observationsof the survivalofthefirst-generatidoanphnids willbe made daily.The presenceof eggs inthe brood patch,males PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -4- or wintereggs alsowillbe recordeddaily.With the onsetof reproductiont,henumber of young presentin each testchamber willbe coulted every Monday, Wednesday and Fridayduring the testand at test termination.An acuteexposurewillalsobe performed forany t-eatrnengtroups which produce young. Twenty neonatesfrom each treatrnengtroup and thecontrolwillbe exposed under statictestconditionsfor 48 hours. The resultsof thetestwillbe used to calculatteheno observedeffectconcentration(NOEC) and the lowestobservedeffectconcentratio(nLOEC). ne NOEC isdefinedas the highestconcentrationthat produces no statisticauy-signifiecfafnetcton survivalr,eproductiono,r growth. 'Me LOEC isdefinedas thelowestconcentrationthatproducesa statisticalsliygnificanetffecton survivalr,eproduction,or growth. 'Me maximum acceptabletoxicantconcentration(MATC) willbe calculatedas the geometricmean of the NOEC and LOEC. MATERLKLS AND METHODS Test Substance Informationon the characterizationf test,controlor referencesubstancesis requiredby OECD Principlesof Good LaboratoryPractice.IMe Sponsor isresponsiblfeor providingWildlifeInternational, Ltd.writtenverificatiotnhatthe testsubstancehas been characterizeadccordingto GLPS. If written verificationf GLP testsubstancecharacterizatiiosnnot providedto WildlifeInternationaLlt,d.,itwillbe noted inthe compliance statementof the finalreport.The attachedform EDENTIFICATION OIF TEST SUBSTANCE BY SPONSOR (Appendix I) is to be used to provide informationnecessary for GLP compliance. The Sponsor isresponsibleforallinfomiationrelatedtothe testsubstanceand agrees to accept any unused testsubstanceand/ortestsubstancecontainersremaining at the end of the study. P=aration ofTest Concentrations The testsubstancewillbe mixed directliyntodilutionwater. Ile solutionwsillbe mixed untilthe testmaterialisdissolved.Once dissolvedt,he solutionwillbe distributeidntotestchambers or dilutedwith dilutionwaterto achievethe desiredtestconcentrations. PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M IAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -5- TestOMaWsm ThecladocerDaanp,hnimaagna,hasbeenselectaesdthetesstpecifebsrthisstudyD.aphnidasre representatiovfe an importantgroup of aquaticinvertebrateasn,d have been selectefdoruse inthetest basedupon pastuse historyand easeof culturinignthelaboratoryD.apbnid neonatestobe usedinthetest willbe lessdm 24 hoursoldand willbe obtainedfrom culturemsaintainedatWildlifeInternationaLlt,d., Easton,Maryland. The idenbt@'@btfhe specieswillbe verifieudsingappropriatteaxonomickeys such as Pennak(5). Daphnids willbe calun-edinwaterfrom thesame source.andatapproximateltyhesame temperature as willbe uiedduringthetest.Daphnidsintheculturepsroducingneonatesfbrthetestwillbe heldforat least10 days priorto collectioonf theneonatesfbrtestingN.e.onatesfrom daphnidsthatshow signsof diseaseor streswsillnotbe -usedastestorganisms.Daphnids inholdingthatproduceephippiaalsow.illnot beused tosupplyneonatesfortesting. Daphnidsinthecultxuwwillbe fedonce daily.The dietwillbe a mixtureofyeast,Cerophyll(&a,nd troutchow (YCT), supplementedwith a suspensionof the freshwatergreen alga Selenastrum capricornutum. Specificationfsoracceptablelevelsof contaminantsin daphnid dietshave not beenestablishedH.owever, thereareno known levelsof conluninantsreasonablyexpectedtobe presentinthe dietthatareconsideredtointerferweiththepurposeor conductof thetest. Neonateswillbe obtainedfortestinfgrom atleastffim individualdults.Priortotestinitiatitohne, neonateswillbe collectefdrom cultureasnd transferretdoglassbeakers.Ile daphnidswillbe releasedinto thetestcompartmentsbelow thewatersurfaceusinga wide-borepipette.Daphnidswillbe feddailyduring thetestusingthesame dietas usedinthecult=. Daphnidswillbe fed0.3mL ofYCT and 0.6mL of Selenastruomncedaily.The frequencyoffeedingwillbe documentedintheraw dataandthefinarleport. DilutioWnater Water used fortheculturinagndtestingof daphnidswillbe obtainedfrom a wellapproximatel4y0 metersdeep locatedon theWildlifeInternationaLlt,d.site.Ili6waterwillbe passedthrougha sand filter and pumped intoa 37,800-Lstoragetankwhere thewaterwillbe aeratedwithspraynozzles.Priortouse the waterwillbe filteretdo 0.45 pm and passedthroughan ultraviolsetterilizienrorderto remove PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, L-rD -6- microorganismsand fineparticles.Water used for culturingand testingis characterizedas moderately hard. Typicalvaluesforhardness,allmlinityp,H and specificonductanceareapproximately: Hardness,mg/L as CaCO3 Alkalmity,mg/L as CaC03 pH SpecificConductance,gmhos/cm 145 190 8.1 330 Hardness, alkalinityp,H and specificconductance will be -measured weekly to monitor the consistencyof the well water. Means and ranges of the measured parametersfor the four-week period precedingthetestwillbe providedinthefinalreport.Analyseswillbe performed atleastonce annuallyto determinethe concentrationosf selectedorganicand inorganicconstituentosf the well water and resultsof themost recentGLP compliantanalyseswillbe summarized inthefinalreport. Test Apparatus Test chambers willconsistof 250-mL Nalgene(D beakers containing200 mL of testsolution.Test chambers willbe randomly positionedby treatmentgroup in a temperatut-controllewdater bath or an environmentalchamber. Test chambers willbe labeledwith the projectnumber, testconcentrationand replicate. Environmental Conditions Lightingused to illuminattehe culturesand testchambers duringculturingand testingwill be providedby fluorescenttubesthatemitwavelengthssimilarto naturalsunlight(e.g.C,olortone(D50). A photoperiodof 16 hours of fightand 8 hours of dark willbe controllewdith an automatictimer. A 30minute transitioonf low lightintensitwyillbe providedwhen lightsgo on and offto avoid sudden changes inlightintensity.Lightintensitwyillbe measured justpriorto testinitiatioand at approximatelyweekly intervaltshereaftewrith a SPER ScientifiLctd.lightmeter or equivalent. The targettesttemperaturewillbe 20 10 C. Temperature.willbe measured in one alternating, replicatoef each treatrnenatnd thecontrolgroup at testinitiatioinn,the.oldand new solutionson renewal days,and attestterminationusinga liquid-in-glatshsermometer. Temperature alsowillbe measured with a continuousrecorderin one negativecontrolreplicate.Recorder measurements willbe verifiedwith a liquid-in-glatshsermometer priortotestinitiatioand atleastweekly thermfter. PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -7- Dissolved oxygen willbe measured in one alternatinrgeplicateof each treatmentand the control group dailyduring the firstweek of the tes@ in the old and new solutionson renewal days, and at test terminationusinga Yellow SpringsInstrumentsModel 51B dissolvedoxygen meter,or equivalent.in the eventthatdissolvedoxygen levelsfallbelow 60% saturationd,issolvedoxygen willbe measured in every testchamber and appropriateactionswillbe taken afterconsultatiownith the Sponsor. Measurements of pH willbe made in one alternatiargeplicatoef each treatmentand the controlgroup attestinitiatioinn,the old and new solutionson renewal days,and at testterminationusing'aFisherAccumet Model 915 pH meter,or equivalent.If a treatmentlevelreaches100% mortalityd,issolvedoxygen, pH and temperature measurements willbe takenatdW time,then discontinued. Hardness,alkalinitya,nd specificconductancewillbe measured in the negative(dilutionwater) controland the highesttestconcentrationattestinitiatioannd atapproximatelyweekly intervaltshereafter. Hardness and alkalinitymeasurements willbe made by titratiounsing proceduresbased on methods in Standard Methods for the Examination of Water and Wastewater (6). Specificconductance will be measured using a Yellow Springs InstrumentsModel 33 Salinity-Conductivity-Temperatmuerteer,or equivalent. BiologicalObservations The firstgenerationdaphnidswill be observed dailyduringthe testfor survival,the onset of reproduction,and clinicalsignsof toxicity.The criterifaor death includeabsence of heartbeat@white opaque coloration,lack of movement of appendages and lack of response to gentle prodding. Immobilizationisdefinedas a lack of movement exceptforminor spontaneousrandom movement of the appendages. Examples of clinicaslignsof toxicityincludeinabietyt,o maintainpositionin the water column, uncoordinatedswimming and cessationof feeding. The presenceof eggs in the brood pouch, abortedeggs,males, or ephippiaalsowillbe recorded. Afterthe onsetof reproductiont,wenty neonates from each treatmentgroup and the controlwillbe exposed for48-hoursunder statictestconditions.The neonateswillbe observed at24 and 48 hours formortality/immobilitaynd clinicaslignsoftoxicity. With the onset of reproductiont,he number of second-generatiodnaphnids willbe counted and recordedapproximately threetimes per week. Second generationneonateswillbe observed for clinical signsof toxicity.Second-generationdaphnids not used for the acuteexposure willbe discarded.The PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, L-rD lengthand dryweightofthesurvivingfirst-generatdiaopnhnidswillbe measuredattheend ofthetest.The testwillnotbe considereadcceptabliefcontrolsurvivails<80% or ifthenumber of young producedby eachsurvivindgaphnidlivintghefull21 daysislessdm an averageof60. Sanling forAnal3iicaMleasurements Water sampleswiU be collectefdrom thenew and oldsolutiondsuringeach week of thetestto determineconcentratioonfsth&-tesstubstance.Duplicatesamplesforthenew solutionwsillbe collected from the freshlypreparedsolutionsw,hereassamplesfrom theold solutionwsillbe obtainedfrom a compositeof aU replicatestchambersforeach controland treatmentlevel.In the eventthat100% mortalitoyc.cursin any treatmentt,hensamplingof thatveatmentwdl temunatefollowmgthe next samplingintervalS.ampleswiU be collecteadtmid-depthand analyzedinunediatelyT.he sample scheme issununarizedbelow: PROPOSED NUMBERS Week I OF SANfinM Week 2 week 3 E)q)mimentalGroup NW Old@ Nevv* Old, New* Old@ Control 2 2 LevelI-Low Concentradon 2 2 Level 2 2 2 Level3 2 2 Level4 2 2 Level 5 2 2 Level 6-1-ligChoncentration 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 TOTAIS 14 14 14 14 14 14 ' New solution(sduplicatseamplescollectefdrom mtmmediate chambers) b Old solution(sduplicatseamplesfrom a compositeofallreplicatteestchambers fmm eachcontroland testlevel) The above numbers of samplesrepresentthosecollectefdrom thetestand do not includequality control(QC) samplessuchasmatrixblanksand fortificatipornesparedand analyzedduringtheanalytical chemistryphaseofthestudy. PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -9- AnalyficalChemi Chemical analysisof the samples willbe performedby WildlifeInWmationA Ltd. ne analytical method used willbe based upon methodology provided by the Sponsor and identifieidn Appendix H. Modificationsmade to theanalyticalmethod willbe documented inthe raw data and describedin the final report. Data Analyges Statisticanlalyseswillbe perfbrmed on the survivalof first-generatidoanphnids,the number of live young produced by each individuary-exposeddaphnid,and the lengthand dry weight of the individuallyexposed fira-genemtiondaphnids. To evaluatewhether or not survivalisaffectedby the treatments,2 X 2 contingencytablesa.nd the chi-squaretestwillbe used on survivaldata (i.e.n,umbers of aliveand dead daphnids). Analyses of reproductionand growth (weightand length)data willbe evaluatedfor normalityand homogeneity of variances.Ifthe data aredeemed normal with homogeneous variances,hypothesistestingusing analysisof variance(ANOVA) and multiplemeans tests(e.g.D,unnetes,Bonferron@ Scheffe)willbe used. Ifthe data failthe testsfornormalityor homogeneity, then data b-ansformationwsillbe triedin an attemptto correctthe condition. When the data t-ansfbrmationfsailto correctfor non-normalityor heterogeneityof variances,nonparame tricprocedureswillbe used to identifystatisticalsliygnificandtifferenceasmong the experimentalgroups. Ile statisticaanlalysesof survivalgrowth and reproductiondatawillaidbut may not be exclusively used in the determinationof the no observed effectconcentratio(nNOEC) and lowest observed effect concentratio(nLOEC). Ile maximum acceptabletoxicantconcentratio(nMATC) willbe calculateadsthe geometric mean of theNOEC and LOEC. RECORDS TO BE MAINTAINED Records to be maintained for data generatedat WildlifeInternationalL,td. willinclude,but not be limitedto: I. A copy of the signedprotocol. 2. Identificatioannd characterizatioonf thetestsubstance,ifprovidedby the Sponsor. PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD 10- 3. Dates of initiatioannd terminationof thestudy. 4. Length and dry weight measurements. 5. Reproduction parameters. 6. Test organism culturerecords. 7. Resultsof rangefindingtestsw,hen applicable. 8. Stock solutioncalculatioannd preparation. 9. Daily observations. -I, 10. Water chemistrycalculation(se.g.h,ardnessand alkalinity). 11. Ifapplicablet,he methods used to analyzetestsubstanceconcentmtionsand the resultsof analytical measurements. 12. Statisticcaallculations. 13. Testconditionsand physical/chemicalmeasurements. 14. Calculationand preparationoftestconcentrations. 15. Copy of finalreport. FINAL REPORT A finalreportof theresultsof thestudy willbe preparedby WildlifeInternationalL,td. The report willincludethefollowing,when applicable: 1. Name and addressof thefacilitpyerformingthestudy. 2. Dates upon which the studywas untiatedand completed. Itistheresponsibilitoyf the Sponsor to providethe finaldatethatdataare recordedforchemistry,pathologyand/or supportingevaluations thatmay be generatedatotherlaboratories. 3. A statementof compliancesigned by the Study Directoraddressingany exceptionsto Good laboratory PracticeStandards. 4. Objectivesand procedures,as statedin the approved protocol includingchanges in the original protocol. 5. The test substance identificationi,ncluding name, chemical abstractnumber or code number, strengthp,urity,composition,and otherinformationprovidedby theSponsor. 6. Stabilitaynd solubilitoyfthetestsubstanceunder the conditionsof administrationi,fprovided by the Sponsor or contractedtoWildlifeInternationalL,td. 7. A descriptionof themethods used toconduct thetest. PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD 8. A descriptioonfthetestorganisms,includingthesource,scientifniacme, age,fifestage,feedtypes, lightintensitaynd photoperiod. 9. A descriptioonfthepreparatioonf thetestsolutionst,hemethodsusedto allocatoerganismstotest chambers and beginthetestt,henumber oforganismsand chambers pertreatmenta,nd theduration ofthetest. 10. A descriptioonfcircumstancetshatmay have affeew thequalitoyrintegritoyfthedata. 11. 'Me name of theStudyDirectorand the names of othersmenflstsp,rofessionalasn,d supervisory personnelinvolvedinthestudy. 12. A descriptioonfthetransformationcsa,lculationasn,d operationpserformedon thedata,a summary and analysisofthebiologicadlataand analyticaclhemistrydata,and a statemenotftheconclusions drawn from theanalyses. 13. Statisticmaelthods used toevaluatethedata. 14. 'Me signedand datedreportsof each oftheindividuaslcientisotrsotherprofessionailnsvolvedin thestudy. 15. Ile locationwhere raw dataand finalreportare-tobe stored. 16. A statementpreparedby the QualityAssuranceUnit listintghedatesthatstudyinspectionasnd auditswere made and findingsreportedtotheStudyDirectorand Management. 17. Ifitisnecessarytomake correctionosr additiontso a finalreportafterithasbeen accepted,such changeswillbe made intheform ofan amendmeirtissuedby theStudyDirector.The amendment willclearlyidentiftyhe partof the finalreportthatisbeingamended and the reasonsforthe amendment, and willbe signedand datedby theStudyDirector. CHANGING OF PROTOCOL Plannedchanges to the protocolwillbe inthe form of writtenamendments signedby the Study Directorand theSponsor.Amendments willbe consideredas partof theprotocoland willbe attachedto thefinalprotocol.Any otherchangeswillbe intheform ofwrittendeviationfsiledwiththeraw data.AR changestotheprotocolwillbe indicateidnthefinalreport. PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, L-rD 12- GOOD LABORATORY PRACTICES Thisstudywillbe conducteidnaccordancweithGood LaboratorPyracticSetandardfsorEPA (40 CFR Part160 and/orPart792);OECD (ENV/MC/CBEM (98)17);and JapanMAFF (59 NohSan, NotificatiNoon. 3850,AgriculturParloductionBureau).Each studyconductedby WildlifeInternational, Ltd.isroutineleyxaminedby theWildlifeIntemationaLtd.QualityAssuranceUnit forc.ompliancewith Good LaboratoryPracticesS,tandardOperatingProceduresand thespecifiepdrotocol.A statementof compliancewithGood LaboratoryPracticewsillbe preparedforallportionsof the studyconductedby WildlifeInternationLatld,. The Sponsorwillbe responsiblfeor compliancewith Goo d Laboratory Practicefsbrproceduresperformedby otherlaboratori(ees.g.r,esidueanalysesorpathology).Raw datafor allwork performedatWildlifeInternationLatld,Land a copyof thefinalreportwillbe filedby project number in archiveslocatedon the WildlifehftmationalL,td.siteo,r at an alternatilvoecationto be specifieidnthefinalreport. PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, L-rD 13REFERENCES I U.S. Environmental ProtectionAgency. 1996. Series850- EcologicalEffectsTest Guidelines (draft)O,PPT'S Number 850.1300:Daphnid ChronicToxicityTest. 2 OECD GuidelineforTestingof Chemicals. 1997. Guideline211: Daphnia magna Reproduction Test. 3 U.S. Environmental Protection Agency. 1982. &andard Evaluation ProceAre, Daphnia magna Life-Cycle (21-Day Renewal) Chronic Toxicity Test. EPA 540/9-86-141. Environmental Protection Agency, Officeof PesticidePrograms. Washington, D.C. 4 American Society for Testing and Materials Standard E1193-96. 1997. Standard Guide for Conducting Life-Cycle Toxicity Tests with Daphnia magna. American Society for Testing and Materials.PhiladelphiaP,A. 5 Pennak, XW. 1978. FreshwaterInvertebrateosf the UnitedStates.2nd Ed. 365 p. 6 "HA, AWWA, WPCF. 1985. Standard Methodsfor the Examination of Water and Wastewater. 16th Edition, American Public Health Association. American Water Works Association. Water PollutionControlFederation,New York PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -14- APPENDIXI IDEq"MCAnON OF TM SUBSTANCE BY SPONSOR To be Ccinpletedby Sponsor 1. Test Substanceidentit(yname to be used inthereport):PFOS (PerfluorooctanSeulfonicAcid Potassium Salt ReferenceStandard(ifapplicable)A:nalyticalStandard.-N/A InternalStandard. 1.1.2.2HJLI-LH PerfluorooetaneSulfonicAcid Test SubstanceSample Coctior Batch Number: Lot 21 Test SubstancePunty (0/Aoctive Ingrechent): 98.9 ExpimfionDate: 2008 R TestSubsta= Clmmcumizafian Have iheidentity, tyand composifionoradierchmacwdsfm whichW.m _ pn-a-tr.= = substan@amndmfixe= Mmdard b= ddanifiefdniotrDitusseinthissh* inacomlmm withGLP Swndards? ----Yes x No IEL TestSubstanceStorW Cmdifions Pkm indicatieherwmmended sorw conditionastVadl& hdcmafionalIAi Ambient Has thestabfltoyfthetestsubsta= underihesestorw conm= been ddamin6d inacomd= withGLP Stmdards? Otherpmtinertstabilitny@on: Yes x No IV.TestCaxenftafim: Adjusttmt cmcmtratim to 10OP/,aiL x basedupon dr-panty(*/og)mm above Do notacbustestCMCWtratiM tD IWI, ai Testft n-t-ialAS IS. V. Tmi&y hffimmation: Ntmnnuliam- RatID50 251 mg&g - Maw LD50 - N/A Aquatic- Invmtdwft TodcityT)Cd.C50) Fish'Todcit(yLOO) DgRhnia magna: 27 mgl Rainbow Trout: II nMi DipMa mamm: 50 mgl FatheadMinnovr 38 mpJL OtherTodcityfiffixmati(omncjudinfgindin ofchmnicand sbchronict&): Please see MSDS PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -15- APPENDIX I[[ AnalyticalMethod Providedby Sponsor Samples willbe analyzedbased upon proceduresprovidedby the Sponsor inthe following analyticamlethods: I. Liquid Chromatopaphy Mass Spectrometry (LCMS) Method forthe Determination of PerfluorooctaneSulfonicAcid Potassium Salt(PFOS) In Freshwater,Saltwaterand AlgalMedium A copy oftheabove method willbe mamtamed m theraw data.Ile actualmethodology used to analyzethetestsamples willbe documented m theraw dataand sumniafizedinthefmal report. PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 07/09/99 13:31 '0822 $915 AQUATIC BLDG Z002/002 WILDLIFE INTERNATIONAL L-M. PROJECT NO.: 4S4A-109 PageI ofI AMENDMENT TO STUDY PROTOCOL STUDY TRME: PFOS: A SM-STATIC LIF&CYCLE TO)ACITY TEST WITH THE CLADOCERAN (Daphniamagna) PROTOCOL NO.: 454/061099/DAP-LC3/SU]3454 AMENDMENT NO.: I SPONSOR.- 3M Corpomtion PROJECT NO.: 454A-109 EMCITVE DATE: July12,1999 AMZNDMENT: Page2 Add: ExperimentalStartDate- 7/12/99 ExperimentalTftm@nationDate- 8/2/99 REASON.- The above informatiownas not known when theprotocolwas signedby the Study Director. STUDY DIRECTOR LABORATORY MANAGEMENT SPONSOIUS REPRESENTATIVE DATE DATE DAT