Document evXmLg4EO9azXYy2a41xR5Ome

PFOS: Fathead 1. 1 I CHRONIC TOXICITY TO EARLY LIFE STAGE OF FISH TEST SUBSTANCE Identity:Perfluorooctanesulfonatmea;y alsobe referredto as PFOS or FC-95. (1-Octanesulfoniaccid,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-p,otassium salt,CAS # 2795-39-3) Remarks field: The testsubstance isa whitepowder. Sample was taken from 3M lotnumber 217. Sample was storedunder ambient conditionspriortotesting.Pudty determinedtobe 90.49% by LC/MS, 'HHMR, 'GF-NMR and elementalanalyses techniques. METHOD Method: OECD 210, OPPTS 850.1400 Type: Flow-throughchronic GLP: Yes Year completed: Study completed1999. Reportcompleted2000 Species: Pimephalespromelas Supplier:In-houseculturesW,ildlifIenternationaLlt,d.,Easton,MD Analyticalmonitoring:PFOS measured on days 0,4, 7,14,21,28,35, 42, and 47 Exposure period:47 days Statisticamlethods: Discrete-variabldeatawere analyzed using 2 X 2 contingencytablestoidentiftyreatmentgroups thatshowed a statistically significandtifferenc(ep<0.05)from the negativecontrolgroup. All continuous-vadablodata were evaluatedfornormalityusingShapiroWilk'stestand forhomogeneity ofvarianceusing Bartlettt'esst.Analysis ofvarianceand Dunnett'stestwere used toevaluatedifferencebsetween treatmentand controlmeans. Test fishage: eggs < 24-hours oldattestinitiation Pretreatment: None Test conditions: Dilutionwater: 0.45 @im filterewdellwater Dilutionwater chemistry (duringthe 4-week period immediately preceding the test): hardness: 126 (124-128)mg/L as CaC03 alkalinity: 172 (170-172)mg/L as CaC03 pH: 8.2 (8.2-8.3) TOC: < 1.0mg/L Conductivity: 321 (315-330)@imhos/cm Stockand testsolutiopnreparationP:rimarsytockprepareidndilution water at88.4 mg/L and mixed untilalltestsubstancedissolvedpriorto use. Aftermixing,the primarystocksolutionwas proportionalldyiluted withdilutiownater to preparefiveadditionasltocksolutionsat concentrationsof44.2,22.1,11.0,5.52,and 2.76 mg/L. Stock solutions were prepared everythreetofourdays duringthetest.The sixstocks were injectedintothe dilutemrixingchambers (ata rateof6.0 muminute) where theywere mixed with dilutiownater (ata rateof 116 muminute) to achievethe desiredtestconcentrations. Flow through rate:Approximatelysixvolume additionsoftest water every 24-hours Stabilityof the testchemical solutions:Extremelystable Exposure vessels: 9L glassaquariafillewdithapproximately7 L oftestsolutionwitha depth ofapproximately17 cm. Embryo incubationcups were constructedfrom glasscylinders approximately50 mm indiameterwith425 @imnylonscreen mesh attachedtothe bottom withsiliconesealant.The cups were suspended inthe water column ofeach 9L glassaquarium and attachedtoa rockerarm witha reciprocatinmgotion of approximately2 rpm. Number of replicates:four Number offishper replicate:twenty Number of concentrations: sixplusa negativecontrol Feeding: Livebrineshrimp nauplii.Fed 3 times perday duringthe firs7t days post-hatch.On days 8 through40 post-hatchf,ed 3 times dailyon weekdays and 2 timesdailyon weekends. Not fed foratleast48 hours priortotheterminationoftesttoallowforgut clearancepriortoweight measurements. Water chemistry during the study: Dissolved oxygen range (0- 47 days): 7.6 - 8.2mg/L (controelxposure) 7.6- 8.2mg/L (1.2mg/L exposure) pH range (0- 47 days) 8.0- 8.4 (controelxposure) 8.0- 8.4 (1.2mg/L exposure) Test temperature range (0- 47 days) 24.4 - 24.70C (controelxposure) 24.3 - 24.70C (1.2mg/L exposure) Method of calculatingmean measured concentrations:arithmetic mean RESULTS Nominal concentrations: Bk control,0.14,0.29,0.57,1.1,2.3,4.6 mg/L Measured concentrations: <LOQ, 0.15,0.30,0.60,1.2,2.4,4.6 mg/L Elementvalue:5-dayhatchabilNiOtEyC 4.6mg/L 42-day post-hatchsurvivaNlOEC = 0.30mg/L 42-day post-hatchgrowthNOEC 0.30 mg/L 42-day post-hatchsurvivaLlOEC 0.60mg/L Allelementvaluesbased on mean measured concentrations StatisticaElvaluationof Mortality: The statisticdailfferencfeorgrowthat concentrationesqual toand higher than 0.60mg/L was notevaluateddue toa significanetffecton survival. No statisticaslilgynificadnitfferencbeetween thenegativecontroland the highestconcentratiotnestedwas seen forhatchability. AnalyticalMethodology: Analyses oftestsolutionwsere performedat WildlifIenternationLatld.usinghighperformanceliquicdhromatography withmass spectrometridcetection(HPLC/MS). When determiningthe concentratioonfthetestsubstanceinthetestsolutionst,hesame and most prominentpeak responseforperfluorooctanseulfonatewas used. No attemptwas made to quantifyon thebasisof individuailsomeric components. The LOQ (limiotfquantitatiowna)s 0.0458mg/L inthis study.The mean percentrecoveryof matrixfortificatiaonnaslyzed concurrentldyuringsample analysiswas 102.Samples collecteadt pretestrangedfrom 91.4to 105% ofnominal.Samples attestinitiatihoand measured valuesfrom 95.5 to 114% ofnominal. Measured valuesfor samples takenattestterminatiornanged from 95.2to 111% ofnominal. Summary of analyticalchemistry data: Nominal Test Mean Measured Percentof Concentration, Measured DuplicateValues at0,4,7. 14,21, 28,35, Concentration, Nominal mg/L 42, and 4@ Days, Respecbvely, mg/L mg/L Negafive Control All< LOQ - - 0.14 0.147,0.160,0.141, 0.140,0.144,0.148,0.134, 0.15 107 0.135,0.153,0.143,0.160,0.158,0.179,0.173, 0.157,0.160,0.147,0.155 0.29 0.287,0.277,0.270,0.289,0.292,0.296,0.269, 0.30 103 0.266,0.307,0.315,0.343,0.341,0.311,0.325, 0.319,0.313,0.296,0.276 0.57 0.571,0.576,0.619,0.659,0.597,0.642,0.539, 0.60 105 0.535,0.608,0.580,0.639,0.617,0.646,0.644, 0.575,0.676,0.545,0.643 1.1 1.14,1.13,1.21,1.25,1.13,1.23,1.03,1.10,1.19, 1.2 109 1.24,1.30,1.31,1.30,1.31,1.14,1.19,1.13,1.09 2.3 2.21,2.27,2.52,2.46,2.43,2.38,fishalldead at 2.4 104 Day 7 4.6 4.56,4.40,4.79,4.79,4.46,4.76,fishalldead at 4.6 100 Day 7 Biologicaolbservations Hatchingsuccess and time to hatch:Allviablefatheadminnow embryos hatched on Day 4 or 5. There were no apparent differencesbetween the time to hatch inthe negative controland the PFOS treatmentgroups. Survival:Allfishsurvivingto testterminationappeared normal with no overtsigns of sublethaltoxicityF.ish which did not survive generallyappeared to be swimming erraticallpyriorto death. Growth: Fish exposed to PFOS at concentrationsof 0.15 or 0.30 mg/L for42 days post-hatchshowed no statisticalsliygnificant reductionintotallength,wet weight or dry weight incomparison to the negativecontrol. Mean Measured Concentration,mgA- Number of Eggs Exposed Negabve Control 80 0.15 80 0.3 80 0.6 80 1.2 80 2.4 80 4.6 80 Hatchability Number Hatched, Day 3 Number Hatched, Day 4 0 20 0 18 0 14 0 28 0 25 0 16 0 14 Number Hatched, Day 5 54 58 58 48 49 59 60 Total Number Hatched 74 76 72 76 74 75 74 Percent Hatching Success 93 95 90 95 93 94 93 Larval Survival Mean Measured Percent Concentration, SurvivalD,ay mg/L 42 Negabve Control 88 0.15 79 0.3 81 0.6 66 1.2 5.4 2.4 0 4.6 0 Growth Mean Measured Concentrationm,g/L Number of TotalLength Surviving Mean + SD, mm Larvae Wet Weight Mean + SD, mg Dry Weight Mean + SD, mg NegaUve Control 65 26.5+0.721 158+9.10 32.5+1.20 0.15 60 26.6+0.208 160+3.10 33.3+0.900 0.30 0.60 1.2 2.4 4.6 58 26.6+0.813 167+11.9 34.2+2.70 50 26.5+0.399 166+11.3 33.5+2.70 4 26.7+2.02 185+33.8 35.4+6.66 0 - CONCLUSIONS Fathead minnows exposed to potassium perfluorooctanesulfonaatte concentrations< 0.30 mg/L for42 days post-hatchshowed no statistically significanrtedui@tionisntimeto hatch,hatchingsuccess,survivalor growth. The most sensitivendpointinthisstudywas post-hatchsurvival. Submifter:3M Company, EnvironmentalLaboratory,P.O. Box 33331, St. Paul,Minnesota,55133 DATA QUALITY ReliabilityK:limischranking= 1 REFERENCES Thisstudywas conducted atWildlifIenternationaLltd.,Easton,MD atthe requestofthe 3M Company. OTHER Last changed: 5/3/00 PFOS: AN EARLY LIFE-STAGE TO)UCITY TEST WITH THE FATHEAD MINNOW (Pimephalespromelas) FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER- 454A- 108 3M LAB REQUEST NO. U2723 US EnvironmentalProtectioAngency -Series850 - EcologicalEffectsTestGuidelines OPPTS Number 850.1400 OECD Guideline2 10 AUTHORS: KurtR- Drottar Henry 0. Krueger,Ph.D. ST'LJDYMTIATION DATE: July2,1999 STUDY COMPLETION DATE: April26,2000 Submittedto 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St.Paul,Minnesota 55144 Wildl@feInternational,Ltd. 8598 Commerce Drive Easton,Maryland 21601 (410)822-8600 Page Iof 72 WILDLIFE INTERNATIONAL LTD. -2- PROJECT NO.: 454A-108 GOOD LABORATORY PRACRICE COMEPLLKNCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: An EarlyLife-StagTeoxicitTyestwiththeFatheadMinnow (Pimephalespromelas) .WILDLIFE INTERNATION@AL LTD. PROJECT NUMBER: 454A-108 STUDY COMPLETION: April26,2000 Thisstudywas conductedincompliancewithGood LaboratoryPracticSetandardsaspublishedby the U.S.EnvironmentaPlrotectioAngency in40 CFR Parts160 and 792,17August 1989;OECD Principleosf Good LaboratoryPractic(eENV/MC/CHEM (98)17);andJapanMAFF, 59 NohSan,NotificatiNoon. 3850, AgriculturParloductionBureat410August 1984,withthefollowinegxceptions: The testsubstancewas not characterizeidn accordancewithfullGLP complianceh;owever,the characterizatiwoans performedaccordingto3M StandardOperatingProceduresand Methods,and allraw data arebeingmaintainedinthe3M archives.The testsubstanceisbeingrecharactefiziendaccordancewithGLP. The stabilitoyfthetestsubstanceunderconditionosf storageatthetestsitewas notdeterminedin accordancewithGood LaboratoryPracticSetandards. STUDY DIRECTOR- KurtR- Drottar SeniorBiologist SPONSOR, @PPROVAL: sp Lsor DATE b/,TE WILC)LIFE INTERNATIONAL LTD. -3- PROJECTNO.: 454A-108 QUALITY ASSURANCE STATEMENT Thisstudywas examinedforcompliancewithGood LaboratoryPracticSetandardsas publisiebdy the U.S.EnvironmentaPlrotectioAngency in40 CFR Parts160 and 792,17 August 1989;OECD Priicipleosf Good LaboratoryPractic(eENV/MC/CHEM (98)17);andJapanMAFF, 59 NohSan,NotificatiNoon. 3850, AgriculturParloductionBureau,10 August 1984. The datesofallinspectionasndauditsand thedatesthatany findingwserereportedtotheStudyDirectorandLaboratoryManagement were asfollows: ACTIVITY: @-I)ATE CONDUCTED: DATE REPORTED TO: STUDY DMCTOR-- MANAGFIVENT: Test Substance Preparation AnalyticalSampling,DO Measurements and Egg SeparationforTest Initiation Observations AnalyticaDlata and DraftReport BiologicaDlata and DraftReport FinalReport August 2, 1999 August 2, 1999 August 5,1999 September 9, 1999 August 5,1999 September 9, 1999 December 3,6 and 7,1999 December 7, 1999 January3-5,2000 April24 and 25,2000 January 5,2000 April26, 2000 August 4,1999 August 9,1999 September 10, 1999 December 15,1999 Januar@ 7, 2000 April26,2000 Marsh@t?ayalAlstsuHHry:nsAonlon@ DATE Qualit1yA1s@suranceProgramS ervisor 'WILDLIFE INTERNATIONAL LTD. -4- REPORT APPROVAL PROJECTNO.: 454A-108 SPONSOR- 3M Corporation TITLE: PFOS: An EarlyLife-StagTeoxicitTyestwiththeFatheadMinnow (Pimephalepsromelas) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-108 STUDY DIRECTOR: KurtR- Drottar SeniorBiologist MANAGEMENT: I/ - /@nHenr-@0&,uejer,A.'D. DirectorA,quaticToxicologyandNon-TargetPlants DATE Y/.-@) DATIE WILDLIFE INTERNATIONAL LTi). PROJECTNO.: 454A-108 TABLE OF CONTENTS Title/CovePrage........................................................................1................. Good LaboratoryPracticCeomplianceStatement..............................................2................. QualityAssuranceStatement..............................................................3................. ReportApproval........................................................................4................. TableofContents.......................................................................5................. Summary ..............................................................................7................. Introducti.o.n..........................................................................8................ Objective..............................................................................8................ ExperimentaDlesign.....................................................................8................. Materialasnd Methods...................................................................9................. Resultsand Discussio.n.................................................................1.4................. Conclusions...........................................................................15................. References...................................................t................................................1..6.................................. TABLES Table1 - Summary ofAnalyticaClhemistryData...........................................1.7................. Table2 - SpecifiCconductanceH,ardnessandAlkdinityoftheTestsolutio.n.s..................1.8................. Table3 - pH ofWater intheTestChambers...............................................1.9................ Table4 - DissolveOdxygen Content(mg/L)ofWaterintheTestChambers.....................2.0................. WILDLIFE INTERNATIONAL L-m. -6- PROJECTNO.: 454A-108 TABLE OF CONTENTS -Continued- TABLES (Cont'd.) Table5 -Temperatur(e'C)ofWaterintheTestChambers.....................................2.1................. Table6 -HatchingSuccessofFatheadMinnow Embryos Exposed totheTestSubstanqe...........................................................2.2................. Table7 -Survivalof FatheadMinnow LarvaeExposedtotheTestSubstance for42 Days Post-Hatch.........................................................2.3................. Table8.-TotalLength,Wet Weight and Dry Weightof FatheadMinnow Larvae attheEnd ofthe42-Day Post-HatchObservationPeriod...............................2.4................ APPENDICES Appendix I- DilutionWater ChemistryParametersMeasuredDuringthe 4-Week PeriodImmediatelyPrecedingtheTest...............................2.5................. Appendix H - AnalysesofPesticidesO,rganics,Metalsand OtherInorganies inWildlifIenternationLatld.WellWater...........................................2..6................... Appendix M - The AnalysisofPFOS inFreshwaterinSupportofWildlife InternationLatld.ProjectNo.:454A-108....................................2.7................ Appendix IV - CumulativePercentMortalitayndTreatment-RelateEdffects....................4.7................ Appendix V - FishTotalLength(nun)..................................................5.6................ Appendix VI - FishWet Weight(mg)....................................................I.....6.1................ Appendix VU - FishDryWeight(mg)...................................................6.6................ AppendixVIII- Changes toProtocol...........................................................7..1................... Appendix IX - PersonnelInvolvedintheStudy...........................................7.2................ WILE)LIFE INTERNATIONAL LTD. -7- PROJECT NO.: 454A-108 SPONSOR: SPONSOR'S REPR.ESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: WU,D= RQTFRNATIONAL LTD. PROJECT NUMBER: TEST SU13STANCE: STUDY: NObk[NAL TEST CONCENTRATIONS: MEAN NEASURED CONCENTRATIONS: TEST TEST DATES: p LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: NO-OBSERVED-EFFF,CTCONCENTRATION: LOWEST-OBSERVED-EFFECTCONCENTRATION: NiA)MvfUM ACCEPTABLE TO)ACANT CONCENTRATION: SUNEMLARY 3M Corporation SusanA. Beach WildffeInternationLatld. EastonM,nland 21601 454A-108 PFOS (PerfluorooctaSnuelfoniAccid PotassiumSalt) PFOS: An EarlyLife-StagTeoxicityTestwiththeFathead Minnow (Pimephalespromelas) NegativeControl0,.14,0.29,0.57,1.1,2.3and4.6mg a.i./L NegativeControl0,.15,0.30,0.60,1.2,2.4and4.6mg a.i./L ExperimentalStart- August5,1999 BiologicaTlermination- September21, 19@99 ExperimentaTlermination- September22,1999 47 bays FatheaMdinnow(Pimephalpersomelas) WildlifIenternationLatld.Cultures Easton,Maryland 21601 Eggs <24 hoursoldattestinitiation 0.30mg a.i./L 0.60mg a.i./L 1-4_2mg ai.AL WILL)LIFE INTERNATIONAL LTD. -8- PROJECTNO.: 454A-108 INTRODUCRION Thisstudywas conductedby WildlifIenternationLatld.for3M CorporatioanttheWildlifIenternational Ltd.aquatictoxicologfyacilitiynEaston,Maryland.The in-lifpehaseofthetestwas conductedfromAugust5, 1999 toSeptember2 1,1999. Raw datageneratebdy WildlifeInternationLatld.and a copyofthefinarleporatre fileudnderProjecNtumber 454A-108 inarchivelsocatedon theWildlifIenternationLatld.site. OBJECTIVE The objectivoef thisstudywas toevaluatteheeffectosfPFOS on earlylife-stadgeevelopmentofthe fatheadminnow, Pimephalespromelas.Time tohatch,hatchingsuccesss,urvivaalndgrowthwereevaluated duringthe47-daystudy. EXPERIMEENTAL DESIGN Fatheadminnow embryos were exposed toa geometricserieosfsixtestconcentrationasnd a negative (dilutiownater)controulnderflow-througchonditionsF.ourreplicatteestchambersweremaintainedineach treatmenatnd thecontroglroup.Each testchamber containedone incubatiocnup with20 embryos,resultiinnga totaolf 80 embryos pertreatmentT.he exposureperiodincludeda5-dayembryo hatchingperioda,nd a42-day post-hatcjhuvenilgerowthperiod.The Sponsorrequestedthatthestudybe extendedpastthenormal28-day post-hatcghrowthperiodtodetermineifsurvivorshiwpouldbe affected. Nominal testconcentratiownesreselecteidnconsultatiowniththeSponsor,and werebasedupon acute toxicitdyata.Nominal testconcentratiownesre 0.14,0.29,0.57,1.1,2.3and4.6mg activeingredien(ta.i.)/L. Mean measuredtestconcentrationwsere determinedfrom samplesoftestwatercollectefdromeachtreatmenatnd thecontrolgroupatthebeginningofthetesto,n Day 4,atweeklyintervaldsuringthetesta,ndattesterminatiorl To starthetes@embryos lessthan24 hoursoldwere collectefdrom cultureasnd groupsof I and2 individualwsere impartialldyistributeadmong incubatiocnupsuntielachcup containe2d0 embryos. One cup was thenplacedineachtreatmenatndcontroltestchamber.Twice duringthenexttwenty-fouhroursanddaily thereaftearl,ldead embryoswere countedand removed from thecupstoavoidcontaminatinvgiablembryos.AU WILDLIFE INTERNATIONAL LTD. -9- PROJECTNO.: 454A-109 eggsthatremainedwere considerevdiable.Dead embryos continuedtobe removed daily.Afterhatchingt,he larvaewerecountedand releaseidntothetestchambers,where exposurecontinueduntiltesttermination. Observationosfmortalitaynd otherclinicaslignsweremade dailyduringthetest.Time tohatch, hatchingsuccessg,rowth,and survivawleremonitoredineachtreatmenatnd controlgroup.DataW= evaluated todeterminteheno-observed-effect-concent(rNaOtEiCo)n, thelowest-observed-effect-concent(rLaOtEiCo)n and themaximum acceptablteoxicanctoncentrati(oMnATC). NUTERLALS AND MIETHODS The studywas conductedaccordintgotheprocedureosutlineidntheprotocol",PFOS: An EarlyLifeStageToxicitTyestwiththeFatheadMinnow (Pimephalespromelas)"T.he protocowlas basedon procedures outlineidnU.S.EnvironmentaPlrotectioAngency Series850 - EcologicaElffectsTestGuidelinesO,PPTS Number 850.1400(1);OECD GuidelinefoTrestingofChemicals,210: FishEarly-LifSetageToxicitTyest (2);ASTM StandardE 1241-88,StandardGuideforConductingEarlyLife-StagTeoxicitTyestswithFish(3) andStandardEvaluatioPnrocedure,FishEarlyLife-StagTeest(4). TestSubstance The testsubstancewas receivedfrom 3M Corporationon October29, 1998 and was assignedWildlife InternationLatld.identificatniuomnber 4675. The testsubstancewas describedas a whitepowder. Itwas identifiaesdFC-95 fromlotnumber217 (T-6295).Informatiopnrovidedby theSponsorindicateadpurityof 90.49%,and an expiratiodnateof 2008.The testsubstancewas storedatambientroom temperature. Pnaration ofTestConrentrations One stocksolutiownas preparedforeachofthesixconcentratiotnessted.A primarystocksolutiownas preparedby dissolvinPgFOS indilutiownaterata concentratioonf88.4mg a.i./]TLh.e primarystocksolution was stirrewdithanelectrisctainlessteeltop-downmixeruntialllthetestsubstancewas dissolvedT.he primary stocksolutiownas proportionaldliylutewdithdilutiownaterto preparefivesecondarystocksolutionast concentratioonfs44.2,22.1,11.0,5.52and 2.76mg a.i./LS.tocksolutionwsere preparedeverythreetofour daysduringthetest.The fivestockswere injecteidntothedilutemri.xingchambers(atarateof6.0 mL/niinute) WILE)LIFE INTERNATIONAL LTD. -10- PROJECTNO.: 454A-108 where theywere mixed withdilutionwater (ata rateof 116 muminute) toachievethedesiredtestconcentrations. The resultantestconcentrationswere adjustedforpurityof theactiveingredienitnthetestsubstance(90.491/o). Test Organism The fatheadminnow, Pimephales promelas, was selectedas the testspeciesfor thisstudy. Fathead minnows arerepresentativoef an importantgroup of aquaticvertebrateasnd were selectedforuse inthe study based upon pasthistoryof use and ease of culturinginthelaboratory.Fatheadminnow embryos used inthistest originatedfrom culturesmaintainedby WildlifeInternationLatld.,Easton,Mary land. The embryos were -removed from spawning substratesand examined under thedissectinmgicroscope toselecthealthyspecimensat approximatelythe same stageof development. Embryos collectefdoruse in thetestwere from sixindividual spawns.and were lessthan 24 hours old when thetestwas iziitiatecl Newly-hatched larvaewere fedlivebrineshrimp nauplii(Artemiasp.)3 timesper day duringthefirs7t days post-hatch.On Days 8 through 40 post-hatch,allfishwere fed livebrineshrimp nauplii3 timesdailyon weekdays and 2 timesdailyon weekends. Brineshrimp naupliiwere obtainedby hatchingcystspurchasedfrom BonnevilleArtemia InternationaIlnc.,SaltLake City,Utah. Fish were not fedforatleast-4h8ours priorto the terminationof thetestto allowforclearanceof thedigestivteractsbeforeweight measurements were made. To ensure thatthefeedingrateper fishremained constant,rationswere adjustedeach week toaccountforlossesdue to mortality. The averagewet weight of 16 fishinthenegativecontrol(ReplicateA) measured attheend of thetest was 0.16 g. Biomass loading(thetotalwet weightofthefishperfiteorftestwater)attheend ofthetestwas 0.062 g of fishper hterof testwater thatpassed throughthetestchamber duringa 24-hourperioctInstantaneous loading(thetotalwet weightof fishperliteorf waterin thetank)attheend of thetestwas 0.37 g of fishperliter of testwater. Test Apparatus A continuous-flowdiluterwas used to delivereach concentratioonf the testsubstanceand a negative (dilutiownater)control.A peristaltpiucmp (Cole-ParmerInstrumentCompany) was usedtodelivetrhesixtest substancestock solutionsintomixing chambers assignedto each treatmentgroup. The stocksolutionswere dilutedwithwellwaterinthemixing chambers inordertoobtainthedesiredtestconcentrationsT.he flow of WILDLIFE INTERNATIONAL LTD. -11- PROJECTNO.: 454A-108 dilutiownatertothemixingchamberswas controllebdy rotaincterRso.tarneterwsere calibratepdriortotest initiatiaonnd atweeklyintervaltshereafteTrh.e flowoftestwaterfrom eachmixingchamber was splitand allowedtoflowintofourreplicatestchambers.The proportioonfthetestwaterthatwas spliitntoeach replicatweas checkedpriortothetesta,nd atweeklyintervaltshereaftteorensurethatflowratesvariedby no more thant 10% ofthemean ofthefourreplicates. The dilutewras adjustedso thateachtestchamber receivedapproximatel6y.0volume additionosftest waterevery24 hours.The testsubstancedeliverpyump was calibratebdeforethetestand atweeklyintervals thereafteTrh.e generaloperatioonfthedilutewras checkedvisuallaytleastwo timesperdayduringthetestand atleastonce atthebeginningand end of thetest. The testchamberswere9-L glassaquariafillewdithapproximatel7y L oftesstolutionT.he depthofthe testwaterina representatitveestchamber was approximatel1y7 crn.The testchamberswereimpartially positioneidna temperature-controbendvironmentaclhamber designedtomaintaina temperaturoef25 2'C. Testchamberswere labeledwiththeprojectnumber,testconcentratioannd replicate. The embryo incubatiocnups were suspendedinthewatercolumn ofeachtestchamber and attachetdoa rockerarm.The reciprocatimnogtionoftherockerarm (approximatel2yrpm)facilitactierdculatiooftnestwater aroundtheembryos duringincubatiohT.he incubatiocnupswereconstructefdromglasscylinderaspproximately 50 mm indiameterwith425 @4m nylonscreenmesh attachedtothebottomwithsiliconseealant. DilutioWnater The waterusedforculturinagndtestinwgas freshwateorbtainedfrom a wellapproximatel4y0 meters deeplocatedon theWildlifeInternationLatld.site.The wellwaterischaracterizaesdmoderately-hawradterT.he specificconductanceh,ardnessa,lkalinitayn,d pH of thewellwaterduringthefour-weekperiodimmediately precedingthetestarepresentedinAppendix 1. The wellwaterwas passedthrougha sandfiltetroremove particlegsreatetrhanapproximatel2y5 i..,-m, and pumped intoa37,800-L storagetankwhere thewaterwas aeratedwithspraynozzles.Priortodelivertyothe dilutesrystem,thewateragainwas filtere(d0.45[tm),thenpassedthrougha UV sterilizteorremove WILDLIFE INTERNATIC)NAL LTD. -12- PROJECT NO.: 454A-108 microorganisms and particles.The resultsof periodicanalysesperformed to measure theconcentrationosf selectedcontaminantsin wellwater used by WildlifeInternationaLltd.arepresentedinAppendix H. EnvironmentalConditions Lightingused toilluminatetheculturesand testchambers duringculturingand testingwas providedby fluorescenttubesthatemittedwavelengthssimilarto naturalsunlight(Colortonem50). A photoperiodof 16 hours oflightand 8 hours ofdark was controllewdithan automatictimer.A 30-minutetransitiopneriodof low lightintensitwyas provided@when lightswent on and offto avoidsudden changesinlighting.Lightintensity was measured at the surfaceof the negativecontrol(replicatAe) attestinitiatioant,testterminationand at weekly intervaldsuringthetest.Lightintensitryangedfrom 581 to619 lux. Temperature was measured in each testchamber atthe beginning and end of the testand atweekly intervaldsuringthetestusing a hquid-in-glastshermometer. Temperature alsowas measured continuousliynone negativecontrolreplicateusing a Fulscope ER/C Recorder. The targettesttemperatureduringthestudywas 252'C. The pH was measured atthebeginningand end ofthetestand atweekly intervaldsuringthetestin alternatinrgeplicateosf each treatmentand thecontrolgroup. Dissolvedoxygen concentrationwsere measured dailyinalternatinrgeplicateosf each treatmentand thecontrolgroup duringthe firs7t days of thetesta,tweekly intervaldsuringthetest,and attesttermination.Hardness,alkalinitaynd specificonductancewere measured'm thenegativecontroland thehighesttieatmentgroup with survivingorganisms atthebeginningof thetesto,nce a week duringthetest,and attesttermination. Measurements of pH were made usinga FisherAccumet Model 915 pH meter and dissolvedoxygen was measured usinga Yellow SpringsInstrumentModel 5IB dissolvedoxygen meter. Specificconductancewas measured usinga Yellow SpringsInstrumentModel 33 Salinity-Conductivity-Temperatmuerteer.Hardnessand alkalinitwyere measured based on procedures in Standard Methods for the Examination of Water and Wastewater (5). Biolop-icaOlbservationsand Measurements Observationsof mortalitywere made twiceduringthefirst24 hoursof theembryo exposureperiodand dailyuntilhatch.Afterhatching,larvaewere observeddailytoevaluatethenumber ofmortalitieasnd thenumber ofindividualesxhibitingclinicaslignsoftoxicitoyr abnormal behaviorduringthe42-daypost-hatchexposure WILDLIFE INTERNATIONAL LTD. -13- PROJECTNO.: 454A-108 period.From theseobservationsh,atchingsuccess,time to hatch,and post-hatchgrowth and survivalwere evaluated.Hatching successwas estimatedas thepercentageofviableembryos thathatchedsuccessfullyP.osthatchsurvivalv7asestimatedas thenumber of larvaesurvivingtotestterminationdividedby thenumber of embryos which hatchedsuccessfifflPyo.st-hatchgrowth ofthe fatheadminnows was evaluatedattheconclusion of the 42-day post-hatchexposureperiod. Totallengthsforeach survivingfishwere determinedusing a digitizintgabletwith the SIGMA SCANrm scientifimceasurement system and wet and dry weightswere measured usingan analyticablalance. Data AnWyses Hatchingsuccess,post-hatchsurvivalw,et weight,dry weightand totallengthof thejuvenilefishwere testendpointsthatwere analyzedstatisticallDya.ta collecteduringthistestwere eithecrontinuous-variabdlaeta (e.g.w,eightand length)or discrete-variabdlaeta(e.g.m,ortalitpyroportions). Discrete-variabldeatawere analyzedusing 2 X 2 contingencytablesto identifytreatmentgroups that showed a statisticaslilgynificandtifferenc(ep!@0.05f)rom thenegativecontrolgroup. Allcontinuous-variable datawere evaluatedfornormalityusing Shapiro-Wilk'stestand forhomogeneity of varianceusingBartlettt'essl Analysisof varianceand Dunnett'stestwere used toevaluatedifferencebsetween treatmentand controlmeans. The resultsof the statisticaanlalyseswere used to aidin thedeterminationof the NOEC, LOEC and MATC. The MATC was estimatedas thegeometricmean of theNOEC and LOEC. Allstatistictaelstswere performed on a personalcomputer usingTOXSTAT 3.5(6)and SPSS/PC Version2.0(7)statisticsaolftware. AnalygcalChemisgy Priortotestinitiatioonn,e setof pretestwater sampleswas collectedfrom two replicatteestchambers of each treatmentgroup and the negativecontrolto evaluatediluteprerformance.On Days 0, 4,7, 14,21,28, 35, 42 and attestterminations,wnples were collectedfrom two alternatinrgeplicatteestchambers of each treatment and thecontrolgroup tomeasure concentrationosf thetestsubstance.Samples were collecteadtmid-depthfrom each testchamber. Samples were collectedin30-mLNalgene@z bottlesand analyzedimmediately.Analytical proceduresused in the analysisof the samples areprovidedin Appendix HI. WILDLIFE INTERNATIONAL LTD. -14- PROJECT NO.: 454A-108 RESULTS AND DISCUSSION VerificatoifoTnestConcentrations Restdtsof analysestomeasureconcentrationosf PFOS inwatersamplescollectedduringthetestare presenteidnTableI andintheanalyticcahlemistryreport(AppendixHI).Nominal concentratiosneslectefdor useinthisstudywere 0.14,0.29,0.57,1.1,2.3and 4.6mg a.i./LW.hen measuredconcentratioonfssamples analyzedduringthetestwere averaged,themean measured concentrationwsere 0.15,0.30,0.60,1.2,2a.n4d 4.6 mg a.i./Lw,hich represented1,07,103,105,109, 104 and 1000/o.otfhenominalconcentrationrse,spectively. Mean measuredconcentrationwsere used toexpresstheNOEC, LOEC and MATC. Pre-testamplescollectteod verifdyiluteorperatio(nAppendixIM were notusedtocalculatmeean measuredvalues. Physicaland ChemicalMeasurementsof Water Measurementsof thespecificconductanceh,ardnessand alkalinimtayde on thenegativecontroland treatrnewnatterarepresentedinTable2. No apparentdifferenceisntheseparametersexistedbetweenthe treatmentasnd thecontrolgroup. Measurements of pH, dissolveodxygen,and temperaturearepresenteidn Tables3,4, and 5, respectivelyW.ater pH measurementswere consistenotvertimeand,showed no obvious differencebsetweencontrolwaterand thetreatmentgroups.Measurementsof pH rangedfrom 8.0to8.4. Dissolvedoxygen concentratiornesmained2!7.6mg/L (92% ofsaturationA)R. temperaturmeeasurementswere withinthedesiredrangeof 252'C., HatchingSuccessand Time toHatch Allviablefatheadminnow embryos hatchedon Day 4 or5 ofthetest(Table6).Therewereno apparent differencebsetweenthetimetohatchinthenegativecontroland thePFOS treatmengtroups.Hatchingsuccesisn thenegativecontrolwas 93%. HatchingsuccessinthePFOS treatmentgroupswas @9:0%. Hatchingsuccesswas notsignificantdliyfferenitnany PFOS treatmentgroup incomparisontothenegativecontrol(p> 0.05). Post-HatchSurvivaland ClinicaOlbservations After42 daysofpost-hatcehxposure,survivailnthenegativecontrowlas 88% (Table7).Post-hatch survivailnthe0.15,0.30,0.60,1.2,2.4and 4.6mg a.i./tLreatmengtroupswas 79,81,66,5.4,0 and 0%, respectivelyA.nalysisofpost-hatchsurvivadlataby 2 X 2 contingenctyableshowed thatsurvivawlas significantdliyfferenitnthe0.60,1.2,2.4and 4.6mg a.i./tLreatmengtroupsincomparisontothenegative WILDLIFE INTERNATIONAL LTD. -15- PROJECTNO.: 454A-108 control(pg 0.05).Allfatheadminnows survivintgotestterminatioanppearednormalwithno overtsignsof sublethatloxicityF.atheadminnows which didnotsurvivegenerallayppearedtobe swirr@g erraticaplrliyotro death(Appendix Growth Growth dataforeachtreatnenatnd therontroglrouparesilmmari7eidnTable8. The individual measurementsareprovidedinAppendicesV, VI andVU. Growth was evaluateadttesterminatiboynmeasuring thetotalengthw,et weightand-dryweightofeachsurvivinfgish.The statistiacnaallysesofgrowthdatadidnot includePFOS treatmengtroupswhichhad a statisticaslilgynificaenftfecotn survivalF.atheadminnows exposed toPFOS atconcentratioonfs0.15 or0.30mg a.i./fLor42 dayspost-hatcshhowed no statisticaslilgynificant reductionisntotalengthw,et weightordryweightincomparisontothenegativecontro(lp> 0.05). CONCLUSIONS Fatheadminnows (Pimephalespromelas)exposedto PFOS atconcentrationfs,0.30mg a.i./fLor 42 dayspost-hatcshhowed no statisticaslilgynificarnetductionisntimetohatch,hatchingsuccesss,urvivaolr growth.The most sensitiveendpointinthisstudywas post-hatcshurvivalF.atheadmipnows exposedtoPFOS atconcentration2st0.60mg a.i./hLad statisticaslilgynificadnitfferenceisnpost-hatcshurvivailncomparisonto thenegativecontrolC.onsequentlyt;heLOEC forthistudy,basedon post-hatcshurvivalw,as 0.60mg a.i./L. The NOEC was 0.30mg a.i./aLnd theMATC was calculatetdobe 0.42mg a.i./L. WILE)LIFE INTERNATIONAL LTD. -16- PROJECTNO.: 454A-108 REFERENCES 1 U.S.EnvironmentaPlrotectioAngency.1996.Serie8s50- EcologicEaflfectTsestGuidelines (draftO)P,PTS Number850.1400F:ishEarly-LiSfteageToxiciTteyst. 2 OECD GuidelinfeorTestinogfChemicals1.992.Guidelin2e10:FishEarly-LiSfteageToxicity Test. 3 ASTM StandardE1241-88.1988.StandarGduidefoCronductinEgarlyLife-StaTgoexiciTteysts withFish.AmericanSocieftoyrTestinagndMaterials. 4 U.S. Environmental Protection Agency. 1986. Standard Evaluation Procedure, Fish Early Life- Stage Test. Officeof PesticidePrograms. Hazard EvaluationDivision.EPA 540/9-86-138. 5 APHA, AWWA, WPCF. 1989. StandardMethodsfor theExamination of Water and Wastewater. 17th Edition,American Public Health Association. American Water Works Association. Water PollutioCnontrolFederationN,ew York. 6 West, Inc. and D.D. Gulley. 1996. TOXSTATO 3.5. Western EcoSystems Technology, Inc. Cheyenne,Wyoming. 7 SPSSInc. 1988. SPSS/PC+ Version 2.0. Chicago, Illinois. 17- Table I Summary ofAnalyticaClherfiistDiayta Sponsor: Test Substance: Test Organism: DilutionWater: 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Nominal Test Concentration (mg a.i./L) Day 0 Dg 4_ Day 7 NegativeControl 0.14 <LO Q I <LOQ 0.147 0.160 <LOQ <LOQ 0.141 0,140 <L OQ <LOQ 0.144 0.148 0.29 0.287 0.270 0.292 0.277 0.289 0.296 0.57 0.571 0.619 0.597 0.576 0.659 0.642 1.1 1.14 1.21 1.13 1.13 1.25 1.23 2.3 2.21 2.52 2.43 2.27 2.46 2.38 4.6 4.56 4.79 4.46 4.40 .4.79 4.76 2'Thelimitof quantitafio(nLOQ) was 0.0458mg a.i.. Measurements discontinueddue to 100% mortality. Day 14 <LOQ <LOQ 0.134 0.135 0.269 0.266 0.539 0.535 1.03 1.10 -2 -2 -2 -2 Da 21 <LOQ <LOQ 0.153 0.143 0.307 0.315 0.608 0.580 1.19 1.24 - - Day 28 <LOQ < LOQ O@160 0.158 0.343 0.341 0.639 0.617 1.30 1.31 - - Day 35 <LOQ <LOQ 0.179 0.173 0.311 0.325 0.646 0.644 1.30 1.31 - - Da 4Z <LOQ <'LOQ 0.157 0.160 0.319 0.313 0.575 0.576 1.14 1.19 - - Day 47 <LOQ <L 0.147 0.155 0.296 0.276 0.545 0.543 1.13 1.09 - - m Conce (mg 0. 0. 0. 1 2 4 18- Table2 SpecificConductance,Hardnessand AlkalinitoyftheTestSolutions Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: FatheadMimow, Pimephalespromelas DilutionWater: WellWater Parameter SpecificConductance Omffioslein) Hardness (mg/LasCaC03) Alkalinity Lmg! asCaC03) 'TestChamber Replicate 14.6mg a.i./L 22.4mg a.i./L 31 .2mg a.i./L Test Group NegativeControl PFOS Treatment NegativeControl PFOS Treatment NegativeControl PFOS Treatment TestDa, 0 7 14 21 28 315 330 300 300 290 3301 3 102 3153 3003 3003 136 140 140 136 144 140' 1402 1443 1403 1363 176 178 178 178 178 1781 178' 1763 1783 1763 A D A B c WILDLIFE INTERNATIONAL LTD. -19- Table3 pH ofWater intheTestChainbers Sponsor: Test Substance: Test Organism: Dilution Water: MeanMeasured Test Concentration (mga.i./L) 3M Corporation PFOS Fathead Nfumow, Well Water 0 Pimephales promelas 7 14 TestDay 21 28 NegativeControl 8.1 8.2 8.2 8.3 8.4 0.15 8.2 8.2 8.2 8.3 8.4 0.30 8.2 8.2 8.2 8.3 8.4 .0.60 8.2 8.2 8.2 8.3 8.4 1.2 8.2 8.2 8.2 8.3 8.4 2.4 8.2 8.2 -1 - - 4.6 8.2 8.2 -1 Replicate A D A B c 'Measurement.d-iscontinuedue to100% mortality. PROJF,@---@',14-4O5.4:A-108 35 42 47 8.0 8.1 8.1 8.0 8.1 8.1 8.1 8.1 8.2 8.1 8.1 8.2 8.0 8.2 8.2 - - - A B c -20- Table4 DissolvedOxygen Content(mg/L)ofWater intheTestChambers' Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: FatheadMinnow, Pimephalespromelas DilutionWater: Well Water Mean Measured TestConcentration TestDav (mg a.i./L) 0 1- 2 3 4 5 6 7 14 21 28 NegativeControl 8.1 8.0 8.1 8.1 8.0 8.0 8.0 8.2 8.1 8.1 8.2 0.15 8.1 8.0 8.1 8.1 . 8.0 8.0 8.0 8.2 8.2 8.1 8.2 0.30 8.1 8.1 8.2 8.1 8.0 8.0 7.9 8.1 8.2 8.1 8.2 0.60 8.1 8.0 8.2 8.1 8.1 8.0 8.0 8.1 8.1 8.1 8.2 1.2 8.2 8.0 8.1 8.0 8.0 7.9 8.0 8.2 8.2 8.0 8.2 2.4 8.0 8.0 8.1 8.0 8.0 8.1 8.0 8.2 .-2 -- 4.6 8.1 8.0 8.1 '8.1 8.1 8.0 8.0 8.1 --2 Ret)licate A B c D A B c D A B olvedoxygen tratioonf4.9mg/L represent6s0% saturatioant25 C infreshwater. 'Measurementsdiscontinueddue to 100% mortaii WILDLIFE INTERNATIONAL LTD. -21- PROJECT NO.: 454A-108 Table5 Temperature('C)ofWater intheTestChambers Sponsor: TestSubstance: TestOrganism: DilutioWnater: 3M Corporation PFOS FatheadMinnow, Pimephalespromelas WeR Water Mean Measured TestDay TestConcentration (mg a.i./L) Replicate., 0 7 14 21 28 35 42 47 NegativeControl A' B c D 24.4 24.7 24.7 24.5 24.5 24.7 24.5 24.6 24.4 24.7 24.7 24.5 24.5 24.7 24.4 24.5 24.4 24.7 24.6 24.6 24.5 24.7 24.5 24.5 24.5 24.7 24.7 24.5 24.5 24.7 24.5 24.5 0.15 A 24.4 24.7 24.7 24.6 24.4 24.7 24.5 24.4 B 24.5 24.6 24.7 24.6 24.5 24.8 24.6 24.4 c 24.4 24.7 24.7 24.6 24.5 24.7 24.5 24.5 D 24.5 24.7 24.6 24.5 24.5 24.7 24.4 24.4 0.30 A 24.4 24.7 24.7 24.6 24.5 24.8. 24.4 24.4 B 24.4 24.7 24.7 24.5 24.5 24.7 24.4 24.4 c 24.4 24.7 24.6 24.5 24.4 24.7 24.5 24.4 D 24.5 24.7 24.6 24.5 24.5 24.7 24.4 24.4 0.60 A 24.4 24.7 24.6 24.5 24.4 24.6 24.4 24.4 B 24.4 24.6 24.5 24.4 24.4 24.7 24.3 24.3 c -24.3 24.6 24.6 24.5 24.4 24.6 24.3 24.4 D 24.3 24.6 24.5 24.4 24.4 24.6 24.4 24.4 1.2 A 24.5 24.5 24.5 24.4 24.4 24.5 24.3 24.3 B 24.4 24.6 24.5 24.5 24.5 24.6 24.4 24.4 c 24.4 24.6 24.6 24.5 24.5- 24.6 24.4 24.4 D 24.5 24.7 24.6 24.5 24.5 24.6 24.5 24.4 2.4 A 24.5 24.7 --2 -- -- -- -- -- B 24.6 24.7 --2 c 24.6 24.7 --2 D 24.5 24.7 --2 4.6 A 24.5 24.6 --2 B 24.4 24.6 --2 c 24.5 24.5 --2 D 24.5 24.5 --2 IA continuousrecordingoftemperatureinthenegativecontrolreplicatAe showed thatthetemperaturerangedfrom24.0 to25.OOC throughoutthetest. 'Measurementsdiscontinueddue to 100% mortality. WILDLIFE INTERNATIONAL LTD. -22- PROJECT NO.: 454A-108 Table 6 HatchingSuccessofFatheadMirmows Exposed totheTestSubstmce Sponsor. 3M Corporation Test Sub:-.tance:PFOS TestOrgLnism: Fathead Nfinnow, Ptnwphales pronwias DilufionWater Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Number of Eggs Exposed Number Hatched Day 1 RU2_ Dal 3 Day 4 Day 5 Total Number Hatched NegativeControl A B c D 20 0 0 0 7 18 74 20 0 0 0 5 18 20 0 0 0 6 20 20 0 0 0 2 18 0.15 A 20 0 0 0 8 18 76 B 20 0 0 0 5 19 c 20 0 0 0 2 19 D 20 0 0 0 3 20 0.30 A 20 0 0 0 4 17 72 B 20 0 0 0 1 19 c 20 0 0 0 2 18 D 20 0 0 0 7 19 0.60 A 20 0 0 0 5 18 76 B 20 0 0 0 6 19 c 20 0 0 0 10 20 D 20 0 0 0 7 19 1.2 A 20 0 0 0 9 18 74 B 20 0 0 0 7 19 c 20 0 0 0 4 18 D 20 0 0 0 5 19 2.4 A 20 0 0 0 7 18 75 B -20 0 0 0 3 19 c 20 0 0 0 4 20 D 20 0 0 0 2 18 4.6 A 20 0 0 0 3 19 74 B 20 0 0 0 4 17 c 20 0 0 0 1 -19 D 20 0 0 0 6 19 PercentHatching Success Number of EstgsHatched X 100 Number of Eggs Exposed Treatment Percent Hatching Success' 93 95 90 95 93 94 93 WILDLIFE INTERNATIONAL LTD. -23- PROJECT NO.: 454A-10@s Table7 SurvivaolfFatheadMinnow LarvaeExposed totheTestSubstancefor42 Days Post-Hatch Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: FatheadMinnow, Pimephales promelas DilutionWater: Well Water Mean Measured Test Initial Number of ApproximateLive Countsi (DaysPost-Hatch) Concentration Replicate Larvae 7 14 21 28 35 42 (mg a.i./L) NegativeControl A B c D 18 17 17 17 17 16 16 18 15 15 15 15 15 15 20 19 19 18 17 17 17 18 18 18 18 18 17 17 Replicate Treatment Percent Percent Survival Survival 89 88 83 85 94 0.15 A 18 17 17 16 15 15 15 83 79 B 19 18 18 17 15 15 15 79 c 19 17 16 16 15 15 15 79 D 20 17 17 16 15 15 15 75 0.30 A 17 15 15 15 15 15 15 88 -81 B 18 18 17 16 16 16 16 89 c 18 15 15 15 13 11 11 61 D 19 19 18 18 16 16 16 0.60 A 18 17 16 16 15 13 12 67 66* B 19 17 17 16 14 14 13 68 c 20 19 19 18 13 13 13 65 D 19 18 18 16 13 1 3 12 63 1.2 A 18 12 12 7 3 3 2 11 5.4* B 19 11 10 4 2 2 1 5.3 c 18 10 7 2 1 1 1 5.6 D 19 7 60000 0.0 2.4 A 18 000000 0.0 0.0* B 19 00000 0 0.0 c 20 00 000 0 0.0 D 18 0 0 000 0 0.0 4.6 A 19 0 0 0 0 00 0.0 0.0* B 17 000000 0.0 c 19 0 0 0 0 00 0.0 D 19 0 00000 0.0 'Livecountsmade on Day 2 through21 post-hatchwere variabledue tothedifficulitnycountinglargenumbers of livingfish.The countsmade on Day 42 were exactcountsofallfishsurvivingattheend ofthetest. *Indicateassignificandtifferencferom thenegativecontrolusinga 2 X 2 contingencytable(p 0.05). WILDLIFE INTERNATIONAL LTD. -24- PROJET- i NO.: 454A- 108 Table8 TotalLength,Wet Weight andDry Weight ofFatheadMinnow Larvae attheEnd ofthe42-Day Post-HatchObservationPeriod Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: FatheadMinnow, Pimephalespromelas DilutioWnater: WellWater Mean Measured TestConcentration (mga.i./L) Number of Surviving Larvae TotalLength(mm) Mean (tSD) NegativeControl 65 26.5(0.721) 0.15 60 26.6(0.208) 0.30 58 26.6(0.813) Wet Weight(mg) Mean (SD) 158(9.10) 160(3.10) 167(11.9) DryWeight(mg) Mean (SD) 32.5(1.20) 33.3(0.900) 34.2(2.70) 0.60' 50 26.5(0.399) 166(11.3) 33.5(2.70) 1.2' 4 26.7(2.02) 185(33.8) 35.4(.6.66) 2.4' 0 4.6' 0 - Not analyzesdtatisticadluleytoasipffican(tp 0.05)effecotn survival. WILDLIFE INTERNATIONAL LTD. -25- PROJECTNO.: 454A-108 APPENDIX I DilutionWaterChemistryParameterMseasuredDuringthe 4-Week PeriodImmcdiatelyPrecedingtheTest Sponsor: Test Substance: TestOrganism: DilutioWnater: 3M Corporation PFOS FatheadMinnow, Pimephalespromelas WeH Water SpecifiCconductance (@4mhos/cm) Mean 321(N = 4) Range 315-330 Hardness (mg/LasCaC03) 126(N = 4) 124-128 Alkalinity (mg/LasCaC03) 172(N = 4) 170-172 pH 8.2(N= 4) 8.@- 8.3 WILDLIFE INTERNATIC)NAL LTD. -26- PROJECTNO.: 454A-108 APPENDIX E Analysesof PesticideOsr,ganics,Metalsand OtherInorganics inWildlifIenternationLatld.WellWater' ANAILYSIS ANASURED CONCENUUTION MiscellaneousMeasurements TotalDissolvedSolids AmmoniaNitrogen TotalOrganic Cirbon2 TotalCyanide 286 < 0.050 < 1.0 < 10.0 mg/L mg/L mg/L /.Ig/L Organochlorines and PCBs Aldrin Alpha BHC Beta BHC DeltaBHC Gamma BHC (Lindane) CWordane DDD, pp' DDE, pp' DDT, pp' Dieldnn Fndosulfnn,A Endosulfan,B Endosulfan Sulfate Endrin Endrin Aldehyde Heptachlor Methoxychlor HeptachlorEpoxide Toxaphcne PCB-1016 PCB-1221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 < or.005 < 0.005 < 0.005 < 0.005 < 0.006 < 0.025 < 0.006 < 0.005 < 0.008 < 0.005 < 0.005 < 0.005 < 0.018 < 0.010 < 0,005 < 0.005 < 0.007 < 0.005 < 0.500 < 0.260 < 0.260 < 0.260 < 0.720 < 0.720 < 0.720 < 0.720 jai JU a )LU ug/L 9/L 9/L 9/L 9/L 99//LL gg/L Ag/L Metals and Other organics Aluminym11 Arsenic .3 BeryU!um, CadrnjuT' Calcium Chroir4um3 Cobalt' copiw n Lead3 NiWesiun 3 Manganese m MNi,.cl.ky.ue;'lFe@num3 potaSSiu 3 Scien.4.i-m9 Silver' Sodiym 3 Zinc < 100 < 25.0 ug/L < 0.50 < 1.0 35.0 mg/L < 2.0 Ag/L < 1.0 Ag/L < 20.0 A < 100 < 10.0 13.5 mg/L < 1.0 ug/L < 0.20 ug/L < 2.0 Aglj- < 2.0 u@VL 6.62 m 9/L < 25.0 9/L < 1.0 9/L 21.3 m 9/-L < 20.0 JU 2'Analysepse yQ@ST EnvironmentalC,TainesviUeFloridaforsamplescoll on November 3 throughNovember 7, 1997. Analyses@cdgnned by UdlifIentemationaLltd.forth@sampleroUect6don November 5,1997. 3Analyses@edonned by WildlifIentemationaLltd.forsamplesrollecteodn November 5 through7,1997. WILDLIFE INTERNATIONAL LTD. -27- APPENDIX III PROJECTNO.: 454A-108 THE ANALYSIS OF PFOS IN FRESHWATER IN SUPPORT OF WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-108 WILDLIFE INTERNATIONAL LTD. -28- PROJECTNO.: 454A-108 REPORT APPROVAL SPONSOR-- 3M Corporation TITLE: PFOS: An EarlyLife-StagTeoxicitTyestwiththeFatheadMinnow (Pimephalepsromelas) WILDLIFE R@TERNATIONAL LTD. PROJECT NO.: 454A-108 PRINCIPAL R-;VESTIGATOR: kfonA. MacGregor,B@S. Scientist MANAGEMENT: WillardB. Nixon,Ph.D/ Manager,AnalyticaClhemistry DATE. DATE' WILDLIFE INTERNATIONAL LTD. -29- PROJECTNO.: 454A-108 Introduction Freshwatersampleswere collectefdrom an earlylife-stagteoxicitsytudydesignedto determinethe effectsof PFOS (PerfluorooctanSeulfonicAcid PotassiumSalt)to the fatheadminnow (Pimephales promelas). This study was conductedby Wildlif@InternationaLltd. and identifieads ProjectNo.: 454A-108. The analysesof thesewatersampleswere performedatWildlifeInternationLatld.usinghigh performanceliquidchromatographywith mass spectxometridcetection(HPLC/MS). Samples were receivedforanalysisbetween,August4, 1999 and September21, 1999 and were typicallaynalyzedon each sample receiptday. Test Substanceand InternaSltandard The testsubstanceused forthisstudywas WildlifeInternationLatld.idenfificatinounmber 4675. The testsubstancewas used topreparecalibratioand matrixfortificatisoanmples. The internasltandardwas receivedfrom 3M Corporationon July2, 1998 and was assignedW*ildlife InternationLatld.identificatniuomnber 4526 upon receipt.The internasltandard,a granularmaterialw,as identifiaesd: IH, IH, 2H, 2H PerfluorooctanSeulfonicAcid,ChemicalAbstractNufiiber:27619-97-2. The standardwas storedunder ambientconditions. Anal3iicaMlethod The method used fortheanalysisof the freshwatersampleswas developedat WildlifeInternational Ltd.and entitle"dAnalyticaMlethod fortheDeterminationof PFOS inFreshwater,Saltwatera,nd Algal Medium". TMs methodologywas includedas Appendix H of WildlifeInternationLatld.protocolnumber 454/011299/MVAL/SUB454. Itwas basedupon methodologyprovidedby 3M Corporation. Samples were dilutedin a 50% methanol : 50% NANOpure(D water solutioncontaining 0.100 mg 4H PFOS (internasltandard)AaLnd 0.05% forn-@cacid (v/v)so thatthey fellwithinthe calibratiornangeofthePFOS methodology. Concentrationosf the PFOS in thestandardsand sampleswere determinedby reverse-phasheigh performanceliquidchromatographyusing a Hewlett-PackardModel 1100 High PerformanceLiquid ChromatographWLC) witha Perkin-EhnerAPI 10OLC Mass Spectrometerequippedwitha Perkin- WILDLIFE INTERNATIONAL LTD. -30- PROJECTNO.:454A-108 Elmer TurboIonSprayion source.BPLC separationwsere achievedusinga KeystoneBetasilCis analyticaclolumn(50mm x 2 mm I.D.,3 pm particlseize).The instrumenptarametersaresununarizeidn Table1. A methodflowcharitsprovidedinFigure1. CalibratioCnurveand LimitofQuantitation Calibratiosntandardsof PFOS preparedin a 50% methanol:50% NANOpure(D water solution contahiin0g.100 mg 4H PFOS (internasltandard)/aLnd 0.05% formicacid(v/v)r,anginginconcentration from 0.000915to0,00915mg a.i./Lw,ere analyzedwiththesamples.The same and most pron-dnenpteak responseforPFOS was utilizetdomonitorPFOS inallcalibratioqnu,alityr-ontroaln,d studysamples.No attemptwas made to quantifyPFOS on thebasisof individuailsomericomponents. Linearregression equationswere generatedusingpeak arearesponseratios(PFOS :internasltandard)versustherespective concentratiornatios(PFOS :internasltandardo)fthecalibratisotnandards.A typicaclalibratiocnurveis presentedinFigure2. The concentratioonf PFOS inthesampleswas determinedby substituti@nhgepeak arearesponseratiosintotheapplicablleinearegressioenquation.Representativieon chromatogramsof low and highcalibratiosntandardasrepresentedinFigures3 and 4, respectively. The method limitof quantitati(oLnOQ) fortheseanalysewsas setat0.0458mg a.i./cLalculateads theproductofthelowestcalibratisotnandardanalyzed(0.000915mg a..i/L)andthedilutiofnactoorfthe matrixblanksamples(50). MatrixBlankand FortificatiSognmples Nine matrixblanksampleswere analyzedtodeterminepossibleinterferenceN.o interferencweesre observedatorabovetheLOQ duringsamplesanalyses(Table2). A representatiivoenchromatogramofa matrixblankispresente'dinFigure5. Freshwaterwas fortifieadt 0.0915,0.915and 7.32 mg a.i./aLnd analyzedconcurrentlwyith the samplestodeterminethemean proceduralrecovery(Table3). Sample concentrationwsere notcorrected forthemean proceduralrecoveryof 102%. A representatiivoenchromatogramofa matrixfortificatiison presentedinFigure6. WILDLIFE INTERNATIONAL LTD. -31- PROJECTNO.: 454A-108 Exgmple Calculations Sample number 454A-108-3,nominalconcentratioonf 0.14mg a.i./iLnfreshwater. Peak Area Ratio= AnalytePeak Area/MternalStandardPeak Area ConcentratioRnatio= ConcentratioonfAnalyte/Concentratiofn InternaSltandard InternalStandardConcentration0:.100 mg/L InitiaVlolume: 0.500mL FinalVolume: 25.0ML DilutioFnactor:50.0 PFOS Peak Area: 244330 Intemal StandardPeak Area: 2316753 Peak Area Ratio:0.10546 Calibratiocnurve equation. Slope: 3.44236 Intercept0:.00427 Cur-veisweighted(I/x) PFOS (mg a.i./La)tinstnunent Peak arearatio-(Y-interceptxiInternaSltandardConcentration Slope 0.10546 -0.00427 3.44236 x 0.100 0.00294 PFOS (mg a.i./Li)nsample PFOS (mg a.i.ALa)tinstrumenxt DilutionFactor 0.00294 x 50.0 0.147 WILDLIFE INTERNATIONAL LTD. -32- PROJECTNO.: 454A-108 PFOS (mg a.i./Li)nsample PercentofNominal Concentration PFOS (mg a.i./Ln)ominal x 100 0.147 0.14 A iuv 105% RESULTS Sample AnLlysis Freshwaterswnpleswere collectefdrom the earlylife-statgoexicitystudywith thefatheadminnow (Pimephalespromelas) at pretest,August 4, 1999, at testinitiatioAnu,gust 5, 1999 (Day 0),on August 9,1999(Day 4),on August 12,1999 (Day 7) and weeklythroughoutthetestuntiltesttermination on September21, 1999 (Day 47). 'Me measured concentrationosf PFOS inthesamplescollecteadt pretestrangedfrom 91.4to 105% of thenominalconcentration(sTable4). The measured concentrationosf PFOS inthesamplescollecteadtinitiatiofnexposureof thetestorganisms(Hour 0) rangedfrom 95.5to 114% of thenominalconcentration(sTable5). Samples collecteadtDay 4, Day -7,Day 14,Day 2 1, Day 28, Day 35 and Day 42 had measured concentratiornangesof 93.1 to 116%, 97.0 to 113%, 91.7to 100%, 102 to 113'/o,108 to 119%, 107 to 128% and 101 to 114% of nominal values,respectively. Samples collecteadt testterminatio(nDay 47) had a measured concentratiornange of 95.2to III% of nominalvalues.A representatiivoenchromatogram ofa testsample isshown inFigure7. WILDLIFE INTERNATIONAL LTD. -33- PROJECT NO.: 454A-108 TableI INSTRUMENT: TypicalBPLC/MS OperationaPlarameters He lett-PackaMroddel 1100High PerformanceLiquid Chroniatograpwhitha Perkin-ElmeArPI IOOLC Mass Spectrometer equippedwitha Perkin-ElmeTrurboIonSprayionsource.Operatedin selectiivoenmonitorinmgode (SM. ANAL'YTICAL COLUMN: KeystoneBetasilCIBcolumn(50 nun x 2 mm I.D.,3 pznparticlseize) OVEN TEMPERATURE: 300C STOP TIME: 5.00minutes FLOW RATE: 0.220mL/minute MOBELE PHASE: 72.0% Methanol:28.0% NANOpure@ Water containing 0.1% Formic Acid INJECTION VOLUME: 25.0liL PFOS RETENTION TIME: Approximately4.3minutes INTERNAL STANDARD RETENTION TIME: Approximately3.0minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6amu 426.7 amu WILDLIFE INTERNATIONAL LTD. -34- PROJECTNO.: 454A-108 Table2 MatrixBlanksAnalyzedConcurrentlDyuringSwnple Analysis Number (454A-108-) MAB-1 Sample Type MatrixBlank Measured Concentratioonf PFOS' (mga.i./L) < LOQ MAB-2 MatrixBlank < LOQ MAB-3 MatrixBlank < LOQ MAB-4 MAB-5 MAB-6 MatrixBlank MatrixBlank MatrixBlank < LOQ < LOQ < LOQ MAB-7 MatrixBlank <LOQ MAB-8 MatrixBlank <LOQ MAB-9 MatrixBlank <LOQ The limiotfquantitati(oLnOQ) was 0.0458mg a.i./bLasedupon theproductofthelowestcalibration standardanalyzed(0.000915mg a.i./La)nd thedilutiofnactorofthematrixblanksamples(50). WILDLIFE INTERNATIONAL LTD. -35- PROJECT NO.: 454A-108 Table 3 MatrixFortificatiAonaslyzedConcurrentlDyuringSampleAnalysis Sample Number (454A-108-) MAS-1 N4AS4 MAS-7 MAS-10 MAS-13 MAS-16 NIAS-19 NIAS-22 MAS-25 MAS-2 MAS-5 MAS-8 NIAS-1 1 NIAS-14 NIAS-17 MAS-20 NIAS-23 MAS-26 MAS-3 MAS-6 NIAS-9 NIAS-12 MAS-15 NiAS-19 NiAS-21 MAS-24 MAS-27 ConcentrationosfPFOS (mga.i./L) Fortified Measured 0.0915 0.0908 0.0915 0.0924 0.0915 0.0893 0.0915 0.0942 0,0915 0.0884 0.0915 0.0886 0.0915 0.103 0.0915 0.0933 0.0915 0.0970 Percent Recovered 99.2 101 97.6 103 96.6 96.8 113 102 106 0.915 0.994 97.7 0.915 0.908 99.2 0.915 o@899 98.@ 0.915 0.961 105 0.915 0.988 108 0.915 0.912 99.7 0.915 0.988 108 0.915 0.909 99.3 0.915 0.933 102 7.32 7.59 104 7.32 7.53 103 7.32 7.75 106 7.32 7.33 100 7.32 7.66 105 7.32 7.36 101 7.32 7.51 103 7.32 7.47 102 7@32 7.13 97.4 Me= 102 StandardDeviation=3.99 CV = 3.91% N=27 WILE)LIFE INTERNATIONAL LTD. -36- PROJECTNO.: 454A-108 Table4 Measured ConceitrationosfPFOS inPre-tesDtiluteVrerificatiSoanmplesfrom an EarlyLife-StagTeoxicitTyest Nominal Test Concentration (mg a.i./L) 0.0 (NegativCeontrol) Sample Number (454A-108-) PT-1 PT-2 Sampling Time (Hours) -24 -24 PFOS Measured Concentration (mg a.i./L)'-2 < LOQ < LOQ Percent of Nominal -- 0.14 PT-3 -24 PT4 -24 0.141 101 0.147 105 .0.29 PT-5 -24 PT-6 -24 0.276 95.2 0.265 91.4 0.57 PT-7 -24 PT-8 -24 0.565 99.1 0.561 98.4 1.1 PT-9 -24 PT-10 -24 1.10 100 1.09 99.1 2.3 PT-1 1 -24 PT-12 -24 2.13 92.6 2.17 94.3 4.6 PT-13 -24 4.48 97.4 PT-14 -24 4.47 97.2 The limiotfquantitati(oLnOQ) was 0.0458mg a.I./Lbasedupontheproductofthelowestcalibration standardanalyzed(0.000915mg a.i./La)nd thedilutiofnactorofthematrixblankswnples(50). 2 Measured valuesofmatrixblanksand matrixfortificatsiaomnplesanalyzedconcurrentlwyithpre-test sampleswere <LOQ, 101,97.7and 112 percentof nominalconcentrations. WILDLIFE INTERNATIONAL LTD. -37- PROJECT NO.: 454A-108 Table 5 Measured ConcentrationosfPFOS inFreshwaterSamples from a FatheadMinnow EarlyLife-StagTeoxicityTest Nominal Test Concentration (mg a.i./L) 0.0 (NegativeControl) Sample Number (454A-108-) 1 2 '115 16 29 30 43 44 53 54 63 64 73 74 83 84 93 94 Sampling Time (Day) 0 0 4 4 7 7 14 14 21 21 28 28 35 35 42 42 47 47 PFOS Measured Concentration' (mg a.i./L) < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Percent of Nominal 0.14 3 0 0.147 105 4 0 0.160 114 17 4 6.141 101 18 4 0.140 100 31 7 0.144 103 32 7 0.148 106 45 14 0.134 95.7 46 14 0.135 96.4 55 21 0.153 109 56 21 0.143 102 65 28 0.160 114 66 28 0.158 113 75 35 0.179 128 76 35 0.173 124 85 42 0.157 112 86 42 0.160 114 95 47 0.147 105 96 47 0.155 111 The limitof quantitation(LOQ) was 0.0458 mg a.i./bLased upon the productof the lowestcalibration standardanalyzed(0.000915mg a.i./La)nd thedilutiofnactorofthe matrixblanksamples(50). WILI)LIFE INTERNATIONAL LTD. -38- PROJECTNO.: 454A-108 Table5 (continued) Measured ConcentrationosfPFOS inFreshwaterSamples from a FatheadMinnow EarlyLife-StagTeoxicityTest Nominal Test Concentration (mg a.i./L) 0.29 Sample Number (454A-108-) 5 6 @il9 20 33 34 47 48 57 58 67 68 77 78 87 88 97 98 Sampling Time (Day) 0 0 4 4 7 7 14 14 21 21 28 28 35 35 42 42 47 47 PFOS Measured Concentrationi (mg a.i./L) 0.287 0.277 0.270 0.289 0.292 0.296 0.269 0.266 0.307 0.315 0.343 0.341 0.311 0.325 0.319 0.313 0.296 0.276 Peicent of Nominal 99.0 95.5 93.1 99.7 101 102 92.8 91.7 106 109 118 118 107 112 110 108 102 95.2 0.57 7 0 0.571 100 8 0 0.576 101 21 4 0.619 109 22 4 35 7 0.659 116 0.597 105 36 7 0.642 113 49 14 0.539 94.6 50 14 59 21 60 21 0.535 93.9 0.608 107 0.580 102 69 28 0.639 112 70 28 79 35 0.617 108 0.646 113 80 35 0.644 113 89 42 0.575 101 90 42 0.576 101 99 47 0.545 95.6 100 47 0.543 95.3 The limitof quantitati(oLnOQ) was 0.0458 mg a.i./Lbased upon the product;f the lowestcalibration standardanalyzed(0.000915mg a.i./La)nd thedilutiofnactorofthe matrixblank samples(50). WILE)LIFE INTERNATIONAL LTD. -39- PROJECI'NO.: 454A-108 Table 5 (continued) Measured Concentrationsof PFOS inFreshwater Samples from a FatheadMinnow Early Life-StageToxicityTest Nominal Test Concentration (mg a.i./L) 1.1 Sample Number (454A-108-) 9 10 '-@23 24 37 38 51 52 61 62 71 72 81 82 91 92 101 102 Sampling Time (Day) 0 0 4 4 7 7 14 14 21 21 28 28 35 35 42 42 47 47 PFOS Measured Concentration' (mg a.i./L) 1.14 1.13 1.21 1.25 1.13 1.23 1.03 1.10 1.19 1.24 1.30 1.31 1.30 1.31 1.14 1.19 1.13 1.09 Peicent Of Nominal 104 103 110 114 103 112 93.6 100 108 113 118 119 118 119 104 108 103 99.1 2.3 11 0 12 0 25 4 26 4 39 7 40 7 2.21 96.1 2.27 98.7 2.52 110 2.46 107 2.43 106 2.38 103 4.6 13 0 14 0 27 4 28 4 41 7 42 7 4.56 99.1 4.40 95.7 4.79 104 4.79 104 4.46 97.0 4.76 103 The limitof quantitatio(nLOQ) was 0.0459 mg a.i./Lbased upon the productof the lowestcalibration standardanalyzed(0.000915mg a.i./La)nd the dilutiofnactorof thematrixblank samples(50). WILDLIFE INTERNATIONAL LTD. -40- PROJECTNO.: 454A-108 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FRESHWATER Preparematrixfortificatsiaomnplesby spikintgherequisivtoelumeofPFOS stocksolutiodnisrectly intofilterefdreshwaterusinggas-tightsyringesand ClassA volumetricflasks. Dilutematrixfortificatiaonnd testsamples intotherange ofthecalibratiosntandardsby partiallfyilling Class A volumetricflaskswith 50% methanol: 50% NANOpure(D watersolutioncontaining 0.100 mg 4H PFOS (internasltandard)/aLnd 0.05% fonnicacid(v/v).Add the Appropriatevolume of sample and bringtheflaskto volume withthedilution solvent.Processthematrixblank sample usingthesame dilutioannd aliquot volume as forthelowestfortfficatiloenvel.Mix wellby several repeatinversions. Ampulate samples and submit forLCMS analysis. Figure 1. Analyticalmethod flowchartfortheanalysisof PFOS infreshwater. .WILDLIFE INTERNATIONAL LTD. -41- PROJECTNO.: 454A-108 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 0.000 0.020 ...0..04.0 ...0..06.0...0..0.80...0..1.00 Conccntration (Ratio) Figure2. A typicaclalibraticounrveforPFOS. Slopc= 3.44236;Intercept0.00427;r 0.9994. Curveisweighted(I/x). WILDLIFE INTERNATIONAL LTD, -42- PROJECTNO.: 454A-108 pros_ioi ................. .................. 9= G.Ww Tt- Aug 0. l@ 3t.-.@m ol W. m a- a.a 8-. VA@ 40 MT 2.." ", BO 0.1 M- T@ E- T@ 3.2 wn.- r@ M V....Agr a,", 40 3, pi iai igi isi til 241 2,, a- O.U 1.03 l.U $Ad LB. 3AS LU 4LOG 4." Y- . ....... . ...... "os SID 4." M I p@ PKS W@ S@ 4HPFOS p@ -7 T@ ta.0 T@ .D 400 @T @ t...O 2 E@d Tt.. @.v- Ty" A M Tt- AW .... 19" 1.:" .............. IOU P. 90 me so 4 all .1 el 01 fil''lil'li.' "Mil''241 271 aD.U I.Ob ILS3 LM Z44 &" LSS 41)9 4." 'M- Figure3. . .... ................................. A representatiivoenchromatogramofa low-leve(l0.000915mg a.i./LP)FOS standard. WILDLIFE INTERNATIONAL LTD. -43- PROJECTNO.: 454A-108 ... ... .................. "Osi1w 467 A@ p@ . ............ orp 0.0,W T@- L ---- ----- ol 207 Ice T@ Its", I" W Wm E@- OW.* 20 AT 243a4SO '4"81 3-M 3.w I- A-M 70 go so 40 I* 23 39 mm 1. 1 or '34 lAt el .2, Is, M 2@ @244 .0t 21, *4, 27, 0- 3." 4@ 4AS Tk- .. .... ............... 46@SA-004C@@ AT W..(-l .0 760929 r, 4.... -W.@ yyp. 41 0.81 A-60 0 sp 40- 210 :w 31 7$ el a 12, ,.I ,.I all 241 271 e- 4@ L . .............. .. ................ ...... Figure4. A representativoenchromatogramofa high-leve(l0.00915mg a.i./LP)FOS standard. WILDLIFE INTERNATIONAL LTD. -44- PROJECTNO.: 454A-108 ........... ........ T-. 20 @ll" 1*1173 W",.- Tp. 40 @7 31 -- ------- --- PIC -I- T@- p@ IKS S.-@3 12.0 3 C-) :o E-1 T @Tmwg,,V,n Ty" . ..................... ...... . ...... ...... 0. m" 1.03 l@ L $AS ..... . ..... .... .......... . .. ..................... ........... Figure5. A representativoenchromatogramofa matrixblanksample(454A-108-NLkB-1). The arrow 'indicattehseretentiotnimeof PFOS. WILDLIFE INTERNATIONAL LTD. -45- PROJECTNO.: 454A-108 z ... ......------------ ....-... ---------------------------.---.-.--.-.-.-. .................. 1: 01 ..267 a-. 40 w_ 1-@ to .T 2 2340074 @om 171642 F- T@ A-m 13 3* a& olso 143 1 1" 24. 2.1 2it 4" .. .... .......................... ........... PPOX ia. . @i A@ 4HPFI= ...... IS- ........................ ........ 4. to 2 1112*2 me 7. to 34 .1 .1 .1 an I.m I.n 'il' '2il a4l *11 &" . ............... . .. .... . .. .................... Figure6. A representatiivoenchromatogramofa matrixfortificatsiaomnple(454A-108-NIAS-1). .WILDLIFE INTERNATIONAL LTD. -46- PROJECT NO.: 454A-108 -----.---- ........................ ........... I- . .... .... .... ... . 2 T@@ .o 6. Ito& ............. ............................. 14 : 4@ 01 W. 2@ 26.7 N@ T.- 3 L@. w- aa 31,,-" IT -om 6-. am k-A.,.,@T,@ 3.12 "7 A. 85 so is a I It' III- -Is, all a- 3.w . .... ..... .....-.- -------------------------- . ............... PFos-I 1. 3 T@, @o 6. PKD GI 01- I- -Pfl@s 221 2.0 PIT 2 6-@ 2o EP- T@ . ................................... low a. so 2. 3. ., 1 01_. 1a21" lil' 642 1*3 *Is all 2-1 271 s- 446 . .......-....-.-.-- ---.-- ......... . .................... Figure7. A representatiivoenchromatograinof a testsample (454A-108-3). -47- APPENDIX IV Sponsor: Test Substance: Test Organism: DilutionWater: 3M Corporation PFOS Fathead Minnow, Pinwphalespronwlas Well Water CumulativeMortalityand Treatment-RelatedEffects' Mean Measured Test Concentration (mg a.iA,) NegativeControl Replicate A B c D Hatched 18 18 20 18 Dax 5 Dead Effects 0 18AN 1 17AN 0 18AN 0 18AN Day 6 #Dead 0 0 0 0 Effects AN AN AN AN Dayl_ Dead Effects I AN 2 AN 0 AN 0 AN ______Day_g #Dead I 2 I 0 0.15 A 18 0 18AN 0 AN I AN I B 19 0 19AN 0 AN 1 AN I c 19 0 19AN 0 AN I AN I D 20 0 20AN 0 AN I AN I 0.30 A 17 0 17AN 0 AN 0 AN I B 18 0 18AN 0 AN 0 AN 0 c 18 0 18AN 0 AN I AN I D 19 0 19AN 0 AN 0 AN 0 0.60 A 18 0 18AN 0 AN 0 AN 0 B 19 0 19AN 0 AN 0 AN I c 20 0 20AN 0 AN 0 AN 0 D 19 0 19AN 0 AN I AN 1 1.2 A 18 0 18AN 1 AN 5 AN 5 B 19 0 19AN 3 AN 7 AN 7 c 18 0 1 8AY4 2 AN 6 AN 8 D 19 0 19AN 2 AN 9 AN 9 2.4 A 18 0 18AN 14 4E 18 - 18 B 19 0 19AN .18 1E 19 - 19 c 20 0 18AN, 2C 12 6AN, 2E 20 - 20 D 18 0 17AN, IC 17 IE 18 - 18 4.6 A 19 0 19E,C 19 19 - 19 B 17 0 17E,C 17 17 - 17 c 19 0 19E,C 19 19 - 19 D 19 2 17E,C 19 19 - 19 ObservedEffects:AN Appears Normal; C Lethargy;E = Erratics%vimming; R Lying on bottom. -48- APPENDIX IV (Continued) CumulativeMortalityand Treatinent-RelatEefdfects' Sponsor: Test Substance: TestOrganism: DilutionWater: 3M Corporation PFOS FadicadMinnow, Pimephalespromelas Well Water Mean Measured DaLl 0 DayII_ Day 12 TestConcentration (mg ai./L) Replicate Hatched Dead Effects Dead Effects Dead Effects NegativeControl A 18 1 AN B is 2 AN c 20 1 AN D 18 0 AN I AN 3 AN I AN 0 AN I AN 3 AN I AN 0 AN 0.15 A 18 1 AN I AN I AN B 19 1 AN I AN I AN c 19 2 AN 2 AN 2 AN D 20 3 AN 3 AN 3 AN 0.30 A 17 1 AN 2 AN 2 AN B 18 0 AN 0 AN 0 AN c 18 3 AN 3 AN 3 AN D 19 0 AN 0 AN 0 AN 0.60 A 18 1 AN 1 AN I AN B 19 2 AN 2 AN 2 AN c 20 1 AN I AN I AN D 19 1 AN I AN I AN -1.2 A 18 5 AN 6 AN 6 AN B 19 7 AN 8 AN 8 AN c 18 8 AN 8 AN 8 AN D 19 11 AN 12 AN 12 AN 2.4 A 18 18 18 18 - B 19 19 - 19 - 19 - c 20 20 - 20 - 20 - D 18 18 - 18 - 18 - 4.6 A 19 19 - .-19 - 19 - B 17 17 - 17 - 17 - c 19 19 - 19 - 19 - D 19 19 - 19 - 19 - ObservEefdfectAsN:-&E!mNonnal; C- L9@@Ll E- Erratsiwcitr@g;R = Lyinognbottom. Day I Mortality I 3 I 0 I I 2 3 2 0 3 0 I 2 I 1 6 8 8 12 18 19 20 18 19 17 19 19 -49- APPENDIX IV (Continued) CumulativeMortalityand Treatment-RelateEdffects' Sponsor: Test Substance: Test Organism: DilutionWater: 3M Corporation PFOS Fathead Minnow, Pimephales promelas Well Water Mean Measured Test Concentration Day 15 Day 16 (-g a-i./L) Replicate Hatchod Dead NegativeCortrol A 19 1 B 18 3 c 20 1 D 18 0 Effects AN AN AN AN Dead I 3 I 0 Effects AN AN AN AN Dead EffeM I AN 3 AN I AN 0 AN 0.15 A 18 1 AN I AN I AN B 19 1 AN I AN I AN c 19 2 AN 2 AN 2 AN D 20 3 AN 3 AN 3 AN 0.30 A 17 B 18 c 18 D 19 2 AN 0 AN 3 AN 0 AN 2 AN 0 AN 3 AN 0 AN 2 AN 0 AN 3 AN 0 AN 0.60 A 18 B 19 c 20 D 19 1 AN 2 AN 1 AN 1 AN I AN 2 AN I AN I AN I AN 2 AN I AN I AN 1.2 A 18 6 AN 6 AN 6 AN B 19 8 AN 8 AN 8 AN c 18 8 AN 8 AN 8 AN D 19 12 AN 12 AN 12 AN 2.4 A 18 18 18 - 18 B 19 19 19 - 19 - c 20 20 20 - 20 - D 18 18 - 18 - 18 - 4.6 A 19 19 - '19 - 19 - B 17 17 - 17 - 17 - c 19 19 - 19 - 19 - D 19 19 - 19 - 19 - Observed Effects:AN Appears Normal; C Lethargy, E Erraticswhmnin&r, R Lying on bottom. Da Dead I 3 I 0 I I 2 3 2 0 3 0 I 2 I I 6 9 9 12 18 19 20 18 19 17 19 19 -50- Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration (Mg Li./L) Negative Control 3M Corporation PFOS Fathead Mirmow, Well Water Pimcphales promelas Day 20 APPENDIX IV (Continued) CumulativeMortalityand Treatment-RelatedEffects' Day 21 D!y 22 Replicate A B c D Hatched 18 18 20 18 Dead 1 3 2 0 Effects AN AN AN AN Dead I 3 2 0 Effects AN AN AN AN Dead I 3 2 0 Effects AN AN AN AN 0.15 A is I AN I AN 2 AN B 19 1 AN I AN I AN c 19 3 AN 3 AN 3 AN D 20 3 AN 3 AN 4 AN 0.30 A 17 2 AN B 18 1 AN c 18 3 AN D 19 1 AN 2 AN I AN 3 AN I AN 2 AN 1 AN 3 AN I AN 0.60 A 18 2 AN B 19 2 AN c 20 1 AN D 19 1 AN 2 AN 2 AN I AN I AN 2 AN 3 AN I AN I AN 1.2 A B c D 2.4 A B c D 4.6 Observed Effects:A A B c D Ap= 18 6 AN 19 9 AN 18 11 AN 19 13 AN 6 AN 9 AN 12 AN 13 AN 18 18 19 19 - 20 20 - 18 18 - 18 - 19 - 20 - 18 - 19 17 19 19 Nomial; C 19 17 19 19 Lethugy; E - Erraticswimming-, R 19 - 17 - 19 - 19 - Lying on bottom. 6 AN 9 AN 12 AN 13 AN 18 - 19 - 20 - 18 - 19 - 17 - 19 - 19 - D!y 2 Dead I 3 2 0 2 I 3 4 2 I 3 1 2 3 I I 6 10 12 13 18 19 20 18 19 17 19 19 -51- Sponsor: Test Substance: Test Organism: DilutionWater. Mean Measured Test Concentration (mg ai./L) NegativeControl 3M Corporation PFOS Fathead Minnow, Pimephales,@romelas Well Water D!y 25 APPENDIX IV (Continued) CumulativeMortalityand Treatment-RelatedEffects Day 26 Day 27 Replicate Hatched Ded A 18 1 B 18 3 c 20 2 D 18 0 Effects AN AN AN AN Dead Effects I AN 3 AN 2 AN 0 AN Dead Effects I AN 3 AN 2 AN 0 AN 0.15 A 18 2 AN 2 AN B 19 1 AN 2 AN c 19 3 AN 3 AN D 20 4 AN 4 AN 2 AN 3 AN 3 AN 5 AN 0.30 A 17 2 AN B 18 2 AN c 18 3 AN D 19 1 AN 2 AN 2 AN 3 AN I AN 2 AN 2 AN 3 AN I AN 0.60 A 18 2 AN B 19 3 AN c 20 1 AN D 19 2 AN 2 AN 3 AN 2 AN 3 AN 2 AN 3 AN 2 AN 4 AN 1.2 A 18 9 AN 11 AN, 2E 13 AN B 19 13 AN 15 AN 15 AN c 18 14 AN 16 AN 16 AN D 19 18 IE 19 - 19 - 2.4 A 18 18 18 - 18 - B 19 19 - 19 - 19 - c 20 20 - 20 - 20 - D 18 18 - 18 - 18 - 4.6 A 19 19 - '19, B 17 17 - 17 c 19 19 - 19 D 19 19 - 19 19 - 17 - 19 - 19 - ObservedELe @AN =@ars Normal; C Letharg)rE Erratiscwinuning;R = Lying on bottom. Day 2 Dead I 3 2 0 3 3 3 5 2 2 4 3 2 3 2 4 13 15 16 19 18 19 20 18 19 17 19 19 -52- Sponsor: Test Substance: Test Organism: DilutionWater: Mean Measured Test Concmtration (Mg LIJL) NegativeControl 3M Corporation PFOS FatheadMinnow, Pimephales promelas Well Water D!y 30 APPENDIX IV (Continued) CumulativeMortalityand Treatment-Relat'Eefdfects Day 31 D Replicate Hatched Dead A 18 1 B 18 3 c 20 2 D 18 0 Effecta AN AN AN AN Dead Effeas I AN 3 AN 2 AN 0 AN Dead Effects I AN 3 AN 2 AN 0 AN 0.15 A 18 3 AN 3 AN B 19 3 AN 3 AN c 19 3 AN 3 AN D 20 5 AN 5 AN 3 AN 3 AN 3 AN 5 AN 0.30 A 17 2 AN B 18 2 AN c 18 4 AN D 19 3 AN 2 AN 2 AN 4 AN 3 AN 2 AN 2 AN 4 AN 3 AN 0.60 A 18 2 AN 2 AN B 19 4 AN 4 AN c 20 3 AN 3 AN D 19 4 AN 4 AN 3 AN 4 AN 4 AN 4 AN -1.2 A 18 15 AN 15 AN 15 AN B 19 17 AN 17 AN 17 AN c 18 16 AN 17 AN 17 AN D 19 19 19 19 - 2.4 A 18 18 18 B 19 19 19 c 20 20 - 20 D 18 18 - 18 18 - 19 - 20 - 18 - 4.6 A 19 19 - 19 B 17 17 - 17 c 19 19 - 19 D 19 19 19 19 - 17 - 19 - 19 - Observed Effects:AN Appems Normal; C Ledwgy-, E Erratiscw4nniing,R Lying on bottom. Day Dead I 3 3 0 3 4 4 5 2 2 5 3 3 5 7 6 15 17 17 19 18 19 20 18 19 17 19 19 -53- Sponsor: Test Substance: Test Organism: DilutionWater: Mean Measured Test Concentration (mg &i./L) Negative Control 3M Corporation PFOS Fathead Mitmow, Pimephales promelas Well Water Day 35 APPENDIX IV (Continued) CurnulativeMortalityand Treattnent-RelateEdffects' Day 36 D Replicate Hatched Dead A 18 1 B 18 3 c 20 3 D 18 0 Effects AN AN AN AN Dead Effects I AN 3 AN 3 AN 0 AN Dead Effects 2 AN 3 AN 3 AN 1 AN 0.15 A 18 3 AN B 19 4 AN c 19 4 AN D 20 5 AN 3 AN 4 AN 4 AN 5 AN 3 AN 4 AN 4 AN 5 AN 0.30 A 17 2 AN 2 AN 2 AN B 18 2 AN 2 AN 2 AN c 18 5 AN 5 AN,IR 7 AN D 19 3 AN 3 AN 3 AN 0.60 A 18 3 AN B 19 5 AN c 20 7 AN D 19 6 AN 4 AN 5 AN 7 AN 6 AN 4 AN 5 AN 7 AN 6 AN .1.2 A 18 15 AN 15 AN 15 AN B 19 17 AN 17 AN 17 AN c 18 17 AN 17 AN 17 AN D 19 19 19 - 19 - 2.4 A 18 18 18 - 18 - B 19 19 - 19 - 19 - c 20 20 - 20 - 20 - D 18 18 - 18 - 18 - 4.6 A 19 19 - 19 - 19 - B 17 17 - 17 - 17 - c 19 19 - 19 - 19 - D 19 19 - 19 - 19 - Observed Effecu: AN Appears Normal; C Lethargy; E Erratiscwinuning,R = L42L2E.!!2MM. Day Dead 2 3 3 I 3 4 4 5 2 2 7 3 4 5 7 6 15 17 17 19 18 19 20 18 19 17 19 19 -54- APPENDIX IV (Continued) CumulativeMortalityand Treatrnent-RelatEefdfects' Sponsor: Test Substance: Test Organisin: DilutionWater: 3M Corporation PFOS FatheadM irinowP,imepholespromelas WellWater Mean Measured TestConomtration D!y 40 Day 41 Day 42 ______Day 43 (mg a-iJL) Replicate Hatched Dead Effects Dead Effects Dead Effeds Dead NegativeControl A 18 2 AN 2 AN 2 AN 2 B 18 3 AN 3 AN 3 AN 3 c 20 3 AN 3 AN 3 AN 3 D 18 1 AN I AN I AN I 0.15 A 19 3 AN 3 AN 3 AN 3 B 19 4 AN 4 AN 4 AN 4 c 19 4 AN 4 AN 4 AN 4 D 20 5 AN 5 AN 5 AN 5 0.30 A 17 2 AN 2 AN 2 AN 2 B 18 2 AN 2 AN 2 AN 2 c 18 7 AN 7 AN 7 AN 7 D 19 3 AN 3 AN 3 AN 3 0.60 A 18 5 AN 5 AN 5 AN 5 B 19 5 AN 5 AN 5 AN 5 c 20 7 AN 7 AN 7 AN 7 D 19 6 AN 6 AN 6 AN 6 -1.2 A 18 15 AN 15 AN 15 AN 15 B 19 17 AN 17 AN 17 AN 17 c 20 17 AN 17 AN 17 AN 17 D 19 19 19 - 19 - 19 2.4 A 18 18 B 19 19 c 20 20 D 18 18 18 - 18 - 18 19 - 19 - 19 20 - 20 - 20 18 - 18 - 18 4.6 A 19 19 @l9 - 19 19 B 17 17 17 17 17 c 19 19 19 19 19 D 19 19 19 19 19 Observed,EffectsA:N Appears Nonnal; C Lethargy-,E Erratiscwimming; R = Lying on bottom. WILDLIFE INTERNATIONAL LTD. -55- PROJECT'NO.: 454A-108 APPENDIX IV (Continued) CumulativePercentMortalitayndTreatment-RelateEdffects Sponsw. Teg Subma=: Tea OWniumDilutionWater- 3M Corporation PFOS FatheadMinnow,Pimephale-psromelas WellWata Mean Measured TestConcentration Day 4 Day 46 (mg &i./L) Replicate Hatched Dead NegativeControl A 18 2 B 18 3 c 20 3 D 18 1 Effects AN AN AN AN Dead Effects 2 AN 3 AN 3 AN I AN 0.15 A 18 3 AN B 19 4 AN c 19 4 AN D 20 5 AN 3 AN 4 AN 4 AN 5 AN 0.30 A 17 2 AN 2 AN B 18 2 AN 2 AN c 18 7 AN 7 AN D 19 3 AN 3 AN 0.60 A 18 5 AN 6 AN B 19 5 AN 5 AN c 20 7 AN 7 AN D 19 6 AN 6 AN 1.2 A 18 15 AN 15 AN B 19 17 AN 17 AN c 18 17 AN 17 AN D 19 19 - 19 - 2.4 A is 18 - 19 - B 19 19 - 19 - c 20 20 - 20 - D is 18 - 18 - 4.6 'ObservedEffects:AN A 19 B 17 c 19 D 19 AppearsNonnal, C 19 17 19 19 Lcffiugy,E - Erratiscwim@g-, R 19 - 17 - 19 - 19 - Lying on bottom. Day 47 Dead Effects 2 AN 3 AN 3 AN I AN 3 AN 4 AN 4 AN 5 AN 2 AN 2 AN 7 AN 3 AN 6 AN 6 AN 7 AN 7 AN 16 AN 18 AN 17 AN 19 - 18 - 19 - 20 - 18 - 19 - 17 - 19 - 19 - WILDLIFE INTERNATIONAL LTD. -56- PROJECT NO.: 454A-108 APPENDIX V FishTotalLength(mm) NegativeControl Sponsor: Test Substance: TestOrganism: DilutionWater: 3M Corporation PFOS FatheadMinnow, Pimephales promelas Well Water Leplicate FishNumber A B c 1) 1 26.0 29.6 31.7 27.8 2 28.6 26.1 25.7 27.6 3 26.5 28.3 28.3 28.2 4 31.0 30.9 27.7 30.4 5 31.1 25.2 27.8 25.1 6 25.0 26.2 23.0 28.3 7 25.5 22.3 28.6 27.0 8 27.1 26.5 26.3 28.2 9 27.2 29.7 26.6 20.6 10 27.1 30.2 25.7 22.9. 11 25.3 18.4 28.4 24.6 12 32.4 29.0 15.2 24.5 13 24.6 28.9 20.1 25.6 14 26.8 29.1 23.7 31.2 15 27.2 26.8 28.1 24.5 16 21.4 -- 28.2 25.5 17 -- 22.1 20.7 18 -- 19 20 -- -- -- -- ReplicateMean 27.1 27.1 25.7 26.0 OverallMean (+-s) 26.5 0.721) Note: Totallengthvalueswere roundedforpresentationM.anual calculatioonfsmeans and standarddeviatiomnay diffesrlightlfyrom thosegeneratedusingTOXSTAT* 3.5. WILDLIFE INTERNATIONAL LTD. -57- PROJECT NO.: 454A-108 APPENDIX V (Continued) FishTotalLength(mm) 0.15mg a.i./L Sponsor: 3M Corporation Test Substance: PFOS TestOrganism: DilutionWater: FatheadMinnow, Pimephalespromelas Well Water Replicate FishNumber A B c D 1 29.0 23.9 27.9 26.El 2 26.2 25.0 29.8 25.,7 3 27.5 26.3 26.6 29.(*) 4 19.8 28.0 27.0 26.El 5 29.4 23.1 32.1 25..,! 6 28.3 23.3 27.9 25.Et 7 32.1 26.9 23.0 20. 8 27.9 27.4 25.7 26.1, 9 18.1 30.7 23.8 27.-3 10 23.9 26.1 27.7 29.0 11 28.5 24.9 22.3 27.(') 12 29.5 28.1 27.7 25.E@ 13 23.6 26.1 26.8 29.z@ 14 27.1 24.0 24.2 24.1. 15 27.8 30.4 29.0 30.0 16 -- -- -- -- 17 18 19 20 ReplicatMeean OverallMean (s) 26.6 26.3 26.8 26.6 0.208) 26.(') Note: Totallengthvalueswere rounded forpresentationM.anual calculationosfmeans and standardeviatiomnay diffesrlisltiflrom thosegeneratedusingTOXSTAT" 3.5. WILDLIFE INTERNATIONAL LTD. -58- PROJECT NO.: 454A-108 APPENDIX V (Continued) FishTotalLength(mm) 0.30mg a.i./L Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: FatheadMinnow, Pimephalespromelas DilutioWnater: WellWater Replicate FishNumber A B c D 1 25.9 30.8 27.8 27.8 2 27.0 25.2 27.2 25.9 3 19.4 24.0 29.1 26.6 4 26.2 27.6 25.0 25.4 5 28.0 26.1 27.9 33.1 6 25.7 27.8 28.5 27.0 7 28.0 22.9 28.3 28.0 8 19.7 26.2 28.8 29.8 9 29.5 24.7 24.5 28.1 10 25.5 26.3 25.6 25.9 11 28.1 28.2 25.4 .27.3 12 30.5 27.8 -- 27.9 13 21.0 30.3 310.4 14 20.6 25.0 23.1 15 27.3 25.1 22.4 16 -- 24.2 28.0 17 -- -- 18 19 20 ReplicateMean 25.5 26.4 27.1 27.3 OverallMean (:F-s) 26.6 0.813) Note: TotallengthvalueswereroundedforpresentationM-anual calculatioonfsmeans and standardeviatiomnay diffesrlightlfyrom thosegeneratedusingTOXSTATO 3.5. WILDLIFE INTERNATIONAL LTD. -59- PROJECTN@O.: 454A-108 APPENDIX V (Continued) FishTotalLength(mm) 0.60mg a.i./L Sponsor: TestSubstance: TestOrganism: DilutionWater: 3M Corporation PFOS FatheadMinnow, Pimephalespromelas Well Water Replicate FishNumber A B c D 1 27.1 30.2 28.0 25.4 2 22.7 22.8 29.0 24.6 3 24.5 30.2 28.7 21.1 4 24.4 25.3 28.1 27.9 5 22.8 22.7 27.3 30.8 6 27.0 24.7 25.8 25.9 7 27.1 28.9 24.5 28.5 8 29.1 25.0 25.0 27.4 9 28.4 22.0 28.7 25.6 10 27.5 26.9 30.0 23.1 11 23.7 26.9 25.1 23.4 12 31.3 29.1 26.4 @2.2 13 -- 26.3 25.4 -- 14 -- -- 15 16 17 18 19 20 -- -- - ReplicatMeean 26.3 26.2 27.1 26.3 OveralMlean (-s) 26.5 0.399) Note: TotallengthvalueswereroundedforpresentationM.anual calculatioonfsmeans and standardeviatiomn@y diffesrlightlfyrom thosegenerateudsinLTOXSTAT" 3.5. WILDLIFE INTERNATIONAL LTD. -60- PROJECTNO.: 454A-108 APPENDIX V (Continued) FishTotalLength(mm) 1.2mg a.i. Sponsor: TestSubstance: TestOrganism: DilutionWater: 3M Corporation PFOS FatheadMinnow, Pimephalespromelas Well Water Replicate FishNumber A B c D 1 26.2 26.7 28.7 2 23.1 -- -- 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 -- - -- ReplicatMeean 24.7 OverallMean (+-s) 26.7 28.7 26.7 2.02) Note: Totallengthvalueswere roundedforpresentationM.anual calculatioonfsmeans and standardeviatiomnay diffesrlightlfyrom thosegeneratedusingTOXSTATO 3.5. WILDLIFE INTERNATIONAL LTD. -61- PROJECT NO.: 454A-108 APPENDIX VI FishWet Weight (mg) NegativeControl Sponsor: Test Substance: Test Organism: DilutionWater: 3M Corporation PFOS Fathead Minnow, Pimephalespromelas Well Water Replicate Fish Number A B c D 1 148.1 247.9 184.1 1'72.9 2 200.5 158.2 182.9 2,D1.4 3 148.9 187.6 206.2 1154.5 4 205.6 227.9 162.8 2'25.0 5 231.0 143.0 173.9 1.42.4 6 129.1 137.5 124.9 209.2 7 130.1 82.4 210.4 165.0 8 151.1 127.8 139.9 173.9 9 153.1 169.0 181.5 78.7 10 154.6 238.7 142.0 104.6 11 132.6 64.5 215.6 98.3 12 275.1 180.1 28.5 124.2 13 135.5 202.2 60.4 124.7 14 147.9 196.3 145.9 296.8 15 147.6 163.8 165.0 49.9 16 89.1 -- 182.0 144.0 17 -- 18 77.4 56.9 -- -- 19 20 ReplicateMean OverallMean (=Es) -161.2 -- 168.5 158 -152.0 9.10) -1.48.4 Note: Totallengthvalueswere rounded forpresentationM.anual calculationosf means and standard deviationmay differslightlfyrom thosegeneratedusing TOXSTATO 3.5. WILDLIFE INTERNATIONAL LTD. -62- PROJECT NO.: 454A-108 APPENDIX VI (Continued) FishWet Weight (mg) 0.15 mg a.i./L Sponsor: Test Substance: Test Organism: DilutionWater: 3M Corporation PFOS Fathead Minnow, Pimephalespromelas Well Water Replicate Fish Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 A 182.5 139.7 166.6 67.6 222.8 205.9 240.0 176.8 55.8 104.0 198.2 231.8 103.0 156.8 152.3 -- B 118.0 133.9 124.4 199.8 89.0 119.0 165.0 195.9 200.8 180.1 139.1 210.3 155.0 105.0 214.3 -- c 200.1 242.4 160.4 148.0 290.1 167.2 86.6 128.2 76.6 181.7 70.0 184.6 186.7 135.6 199.9 -- D 195.5 122.9 204.7 142.4 125.3 173.0 581.4 14('P.6 17-)'.3 19E;.7 17 il5. 12-3.4 2 0,',1!. 11,!.5 21@).3 17 18 19 20 ReplicateMean OverallMean (s) -160.3 -156.6 160 -163.9 3.10) -158.6 Note: Totallengthvalueswere rounded forpresentation.Manual calculationosf means and standarddeviationmay differslightlfyrom those generatedu@@g TOXSTAT* 3.5. WILDLIFE INTERNATIONAL LTD. -63- PROJECTNO.: 454A-108 APPENDIX VI (Continued) FishWet Weight(mg) 0.30mg a.i./L Sponsor: TestSubstance: TestOrganism: DilutionWater: 3M Corporation PFOS FatheadMinnow, Pimephalespromelas Well Water Replicate FishNumber A B c D 1 @I'144.5 208.8 206.7 162.4 2 161.6 181.1 208.4 155.3 3 62.4 135.5 193.2 203.6 4 184.@ 192.0 147.1 135.8 5 203.7 165.6 185.6 261.8 6 126.7 211.9 209.1 164.2 7 163.1 123.8 201.7 157.0 8 75.6 180.7 220.1 193.2 9 256.3 142.9 126.5 178.3 10 125.6 175.1 145.4 124.0 11 198.9 199.7 150.9 142.7 12 268.4 202.2 -- 151.6 13 83.4 224.6 234.7 14 53.7 122.1 65.6 15 189.8 106.3 93.0 16 -- 149.2 179.2 17 -- 18 19 20 ReplicatMeean OveralMlean (s) -153.2 -- 170.1 167 -181.3 11.9) -162.7 Note: Totallengthvalueswere roundedforpresentationM.anual c culationosfmeans and standardeviatiomnay diffesrlightlfyrom thosegenerate!d@@TOXSTAT" 3.5. WILDLIFE INTERNATIONAL LTD. -64- PROJECT NIO.:454A-108 APPENDIX VI (Continued) FishWet Weight (mg) 0.60mg a.i./L Sponsor: Test Substance: Test Organism: DilutionWater: 3M Corporation PFOS Fathead Minnow, Pimephalespromelas WeH Water Replicate Fish Number B c D 1 -206.7 223.7 180.5 161.2 2 96.2 122.1 225.4 125.6 3 113.2 221.0 200.6 85.1 4 131.0 160.7 189.0 201.3 5 93.3 103.3 174.9 272.7 6 169.8 141.1 156.7 132.8 7 163.2 205.3 148.2 169.3 8 231.5 130.6 172.5 175.3 9 223.1 84.0 231.4 137.2 10 201.2 152.9 236.0 88.0 11 136.2 159.4 151.8 85.9 12 176.3 212.0 177.3 2@1.8 13 -- 177.1 125.1 - 14 -- -- 15 16 17 18 19 20 ReplicateMean OveraffMean (=Ls) 161.8 161.0 166 182.3 11.3) 157.2 Note: Totallengthvalueswere rounded forpresentationM-anual calculationosf means and standarddeviationmay differslightlfyrom thosegeneratedusing_LOXSTAT'o 3.5. WILDLIFE INTERNATIONAL LTD. -65- PROJECTNO.: 454A-108 APPENDIX VI (Continued) FishWet Weight(mg) 1.2mg a.i. Sponsor: 3M Corporation Test Substance: PFOS TestOrganism: FatheadMinnow, Pimephalespromelas DilutionWater: WellWater Replicate FishNumber A B c D 1 173.3 179.3 220.6 2 133.7 -- 3 4 5 6 7 8 9 10 11 12 13 14 15 16 -- 17 7- 18 -- 19 20 -- -- -- ReplicatMeean OverallMean (:Ls) 153.5 179.3 220.6 185 33.8) Note: Totallengthvalueswere roundedforpresentationM.anual calculatioonfsmeans and standardeviatiomnay diffesrlightlfyrom thosegenerateudsingTOXSTAT* 3.5. Wl LD LiFE INTE R NATI 0 NAL LTD. -66- PROJECT NO.: 454A-108 APPENDIX VII FishDry Weight(mg) NegativeControl Sponsor: Test Substance: TestOrganism: DilutionWater: 3M Corporation PFOS FatheadMinnow, Pimephalespromelas Well Water Replicate FishNumber 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 ReplicatMeean OveralMlean (s) A B c D 30.3 51.2 40.8 38.8 42.5 32.7 40.5 42.4 32.0 39.9 40.3 32.9 43.0 45.2 33.6 50.4 45.6 28.6 37.5 30.3 24.6 26.2 24.9 46.2 25.4 15.7 47.6 34.0 31.5 25.1 30.2 37.0 30.2 36.8 37.7 15.4 30.0 45.7 27.6 21.0 27.6 11.8 43.6 20.1 55.0 39.7 26.1 26.6 37.8 10.3 25.2 29.4 43.1 30.8 .62.9 29.7 30.8 32.7 09.3 19.4 -- 39.5 -29.9 -- 16.8 11.5 -- -- -- -- -- -- 32.7 34.0 31.8 31.4 32.5 1.20) Note: Totallengthvalueswere roundedforpresentationM.anual calculatioonfsmeans and standarddeviatiomnay diffesrhg@t1l from thoseStmeratedus@a TO XSTAT" 3.5. WILDLIFE INTERNATIONAL LTD. -67- PROJECTNO.: 454A-108 APPENDIX VU (Continued) FishDry Weight(mg) 0.15mg ai./L Sponsor: TestSubstance: TestOrganism: DilutioWnater: 3M C rporation PFOS FatheadMinnow, Pimephalespromelas WeR Water Replicate FishNumber A B c D 1 39.0 2 27.5 3 35.1 4 13.9 5 46.7 6 41.5 7 49.3 8 36.1 9 10.9 10 19.4 11 44.3 12 50.3 13 21.9 14 28.4 15 32.2 16 -- 17 18 19 20 -- ReplicatMeean 33.1 OveraUMean (s) 22.3 43.6 25.8 55.6 25.1 33.6 39.8 27.6 16.9 61.9 22.0 34.0 34.8 16.4 41.1 26.3 41.2 14.6 37.5 35.0 30.3 13.9 45.3 38.9 32.7 38.1 20.7 26.9 46.6 43.2 -- -- -- 32.1 33.3 -- 34.0 0.900) 39.4 26.2 40.5 32.3 27.2 38.7 13.5 32.1 39.9 46.7 38.3 23.0 41.2 23.3 -.48.4 -34.0 Note: TotallengthvalueswereroundedforpresentationM.anual calculatioonfsmeans and standardeviatiomnay diffesrlightlfyrom thosegeneratedusingTOXSTATO 3.5. WILDLIFE INTERNATIONAL LTD. -68- PROJECT@4O.: 454A-108 APPENDIX VU (Continued) FishDryWeight(mg) 0.30mg a.i./L Sponsor: TestSubstance: TestOrganism: DilutioWnater: 3M Corporation PFOS FatheadMimow, Pimephalespromelas WellWater FishNumber 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Replicate A B c 1) 27.1 41.2 39.9 33.1 33.9 37.3 47.8 32.1 10.9 26.2 40.1 43.7 40.0 39.3 30.5 27.1 42.3 31.9 39.0 51.4 25,8 42.5 45.2 34.1 33.3 24.8 44.1 33.1 15.6 35.3 46.7 39.7 52.2 28.8 24.4 34.7 25.8 34.3 28.2 24.2 42.7 40.4 32.5 28 7 56.0 39.5 -- 32.' 1 18.2 45.8 50.1 11.7 23.2 14.5 41.0 20.1 19.0 -- 27.1 36.4 -- ReplicatMeean OveralMlean (s) 31.8 33.6 38.0 33 .4 34.2 2.70) Note: Totallengthvalueswere roundedforpresentatiorMlanual calculationosfmeans and standardeviatiomnay diffesrlightlfyromthosegenerateudsingToxsTAr* 3.5. WILDLIFE INTERNATIONAL LTD. -69- PROJECTNO.: 454A-108 APPENDIX VII (Continued) FishDry Weight (mg) 0.60mg a.i./L Sponsor: Test Substance: Test Organism: DilutionWater: 3M Corporation PFOS Fathead Mimow, Pimephalespromelas Well Water Replicate FishNumber A B c D 1 43.9 44.7 38.5 28.8 2 18.8 23.1 46.3 23.9 3 23.1 44.4 39.9 15.8 4 25.1 32.7 36.9 40.0 5 18.2 20.6 35.1 54.2 6 33.0 25.3 30.1 24.6 7 31.3 42.1 27.4 34.5 8 48.6 26.9 32.5 35.8 9 49.0 14.3 46.8 27.9 10 42.9 30.4 49.1 17.1 11 27.0 31.7 29.6 16.3 12 59.8 42.4 37.9 51.1 13 -- 32.4 24.0 -- 14 -- -- 15 16 17 18 19 20 ReplicateMean OverallMean (s) 35.1 31.6 36.5 30.8 33.5 2.70) Note: Totallengthvalueswere rounded forpresentationM.anual calculationosf means and standarddeviationmy differslightlfyrom thosegenerated1!@@TOXSTAT" 3.5. WILE)LIFE INTERNATIONAL LTD. -70- PROJECT@@O.: 454A-108 APPENDIX VII(Continued) FishDry Weight(mg) 1.2mg a.i./L Sponsor: TestSubstance: TestOrganism: DilutioWnater: 3M Corporation PFOS FatheadMinnow, Pimephalespromelas Well Water Replicate FishNwnber A B c D 1 33.3 34.2 42.6 2 25.6 - -- 3 4 5 6 7 8 9 10 12 13 14 15 16 17 18 19 20 -- -- -- ReplicatMeean 29.5 34.2 42.6 OverallMean (+-s) 35.4 6.66) Note: Totallengthvalueswere roundedforpresentationM.anual calculatioonfsmeans and standardeviatiomnay diffesrliahgyfrom thosegenerateudsingTOXSTAT" 3.5. WILDLIFE INTERNATIONAL LTD. -71- PROJECT NO.: 454A-108 APPENDDC VRI Changes to Protocol ILI, This studywas conducted in accordancewith the approved Protocolwith thefollowingchani:yes: 1 . The protocolwas amended to add theproposed experimentalstartand terminationdates. 2. The post-hatchgrowth periodwas extended to 42 days. 3. Nominal testconcentra6ohswere 0.14,0.29,0.57,1.1,2.3.and4.6 mg a.i./L. WILDLIFE INTERNATIONAL LTD, -72- PROJECT14O.: 454A-108 APPENDIX IX PersonnelInvolvedintheSbA@y The followinkgey WildlifIenternationLatld.personnelwere involveidntheconductormanal,,erneonfthis study: 1. Henry0.Krueger,Ph.D.,DirectorA,quaticToxicologyandNon-TargetPlants 2. WillardB.Nixon,Pb-D','ManagerA,nalyticaClhemistry 3. TimothyZ.Kendall,Supervisor 4. KurtR- DrottaiS@eniorBiologist 5. JonA. MacGregor,Scientist 6. Mark A. Mank, LaboratorySupervisor PROTOCOL PFOS: AN EARLY LIFE-STAGE TO)GCITY TEST WITH THE FATHEAD NffNNOW (Pimephalespromelas) U.S.EnvironmentaPlrotectioAngency Series850 -EcologicalEffectsTestGuidelines OPPTS Number 850.1400 3M Lab RequestNo. U2723 Submittedto 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St.PauL Nfhmesota 55144 WILDLIFE INTERNATIONAL LTD. 8598 Commerce Drive Easton,Maryland 21601 (410)822-8600 June 10, 1999 WILDLIFE INTERNATIONAL, LTD -2- PFOS: AN EARLY LIFE-STAGE TOMCITY TEST WITH THE FATHEAD MINNOW (Pimephalespromelas) SPONSOK- 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St Paul,Minnesota 55144 SPONSOR!S REPRESENTATIVE: Ms. Susan A. Beach TESTING FAC WildlifeInternationaLlt,& 8598 Commerce Drive Easton,Maryland 21601 STUDY DIRECTO Kurt Droam SeniorAquatic Biologist LABORATORY MANAGEMENT: Henry 0. Krueger,Ph.D. DirectorofAquatic Toxicology& Non-Target Plants FOR LABORATORY USE ONLY Proposed Dates: Experimental I S= Date: ProjectNo.: Experimental TerminationDate: Test Concentrations:NeizativeControl,0.16.0.31.0.63.1.3.2.a5nd 5.Omg a.i./L Test SubstanceNo.: 4675 ReferenceSubstanceNo. (ifapplicable):4526 PROTOCOL APPROVAL STUDY DIRECTOR DATE ORATORY MANAGEMENT e;@', 0 SPORSOR!S REPRESEN'TA-TI-V-E PROTOCOL NO.: 454/061099NAT-ELSISUB454 DATE DATE 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, L-rD -3- INTRODUCRION WildlifIenternatioLntaclL,willconductan earlyhfe-stagteoxicityestwithfatheadminnow (Pimephalpersomelas)fortheSponsorattheWildlifIenternatioLntadl.,aquatitcoxicologfyaciliitny Easton,MarylancL The studywillbe performedbased on proceduresin theU.S. Environmental Protection Agency Series850 - EcologicalEffectsTest GuidelinesOPPTS Number 850.1400 (1);AS7M Standard E]241-88 Standard Guidefor Conducting Early LifeStage To)dcityTests with Fish (2);and Standard EvaluationProcedure,Fish-EarlyLife-StageTest (3).Raw dataforallwork performed atWildlifeIntemationalLtctand a copy ofthefinalreportwillbe filedby projectnumber inarchiveslocatedon theWildlife InternationaLlt,cLsiteo,ratan alternativleocatiotnobe specifieidnthefinalreporl PURPOSE The purposeofthisstudyistodeterminetheeffectosfa testsubstanceon thetimetohatch,hatching success,survival,and growth of fatheadminnows (Pimephales promelas) during early lifestage development. EXPERIMENTAL DESIGN Fathead minnow embryos willbe exposed to a seriesof six testconcentrationsand a negative (Dilufionwater)control.Testconcentrationwsillbe chosen by theStudy Directorinconjunctionwith the - Sponsor based on acutetoxicitdyat& Each testconcentratiownillbe 50*/oof thene)ilhighertreatmentlevel The testwillbegin when groups of newly-ferdhzedembryos are placed in incubationcups and exposed to testsolution.Incubationcups,each containing20 embryos, willbe placed in each of four replicatteestchambers pertreatment(totaolf 80 embryos pertreatmentand control).Afterhatching,larvae willbe releasedfrom theincubationcups intothelargertestchambers where exposure willcontmue and growth and mortalitywillbe observed over a 28-day period. Observationsof theeffectsof thetestsubstanceon hatchingsuccess,growth, and survivalwillbe used to calculatteheno observed effectconcentratio(nNOEC) and thelowestobserved effectconcentration (LOEC). The NOEC and LOEC willbe usedtocalculattehemaximum acceptabletoxicantconcentration (MATC). PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, L-rD -4- TestSubstance MATERIALS AND METHODS Informationon the characterizationf test,controlor referencesubstancesisrequiredby Good LaboratoryPracticeStandards(GLP). The Sponsor isresponsiblfeorprovidingWildlifeInternationaLlt,d. writtenverificatiotnhatthetestsubstancehas been characterizeadccordingtoGLPs priortoitsuse inthe study.Ifwrittenverificatiofn GLP testsubstancecharacterizatiiosnnotprovidedtoWildlifeIntemationaL Ltd.,itwillbe notedinthecompliancestatementof thefinalreport.The attachedform IDENTIFICATION OF TEST SUBSTANCE GLP compliance. BY SPONSOR (Appendix 1)istobe used toprovideinformationecessaryfor The Sponsor isresponsibleforallinformationrelatedto thetestsubstanceincludingthe retentioonf a reservesample of the lotor batch of thetestsubstanceused inthisstudy. The Sponsor alsoagreesto acceptany unused testsubstanceand/ortestsubstancecontainersremaining atthe end of the study. Proaration ofTest Concentrations The testsubstancewillbe aciministeretdothetestorganism inwater. Thisrouteof administration was selectedbecause itrepresentsone possiblerouteof exposure to aquaticorganisms. The testsubstance willbe mixed directlwyith dilutiownater. Test Organism Fatheadminnows (Pimephalespromelas) willbe used inthistest Thisspeciesisrepresentativoef an importantgroup of organisms and was selectedforuse in the testbased upon pastuse and ease of handlinginthelaboratory.Embryos used to starthetestwillbe approximately2 to24-hoursoldand will be obtainedfrom culturesmaintainedin the laboratoryat WildlifeIntemationaLLtd.or a ronirnercial supplier. Embryos willbe removed from spawning substrateussing a gentlerollingmotion with the index fingeror with a cleanrazorblade and examined under a microscope to selecthealthyspecimens in approxitnateltyhesame stageof development. Embryos from a minimum of threespawns willbe combined and used to initiatteesting.Ifmore than 50% of the eggs from a givensubstratearefimgused or have not been fertilizendo, embryos from thatsubstratewillbe used inthetest. PROTOCOL NO.: 454/061099/FAT-ELS/SLTB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -5- Newly-hatchleadrvaweillbefedlivberinsehrimpnauplf(iArtemisap.)3 timesperdayduringthe firs7t dayspost-hatcb.nemafta, theywillbe fedlivebrineshrimp nauphi3 timesdailyon weekdays and atleast2 fims dailyon weekends untilthetestiswrmir@ To ensurethatthefeedingrateperfishremains constan@theamount of food suppliedto each replicatteestchamber willbe adjustedon a weekly basisto accountforlossesdue tomortality.Fishwillnotbe fedforapproximately48 hourspriortothetermination of the testto allowthe fishto cleartheirdigestivetractsbefore measurements of weight are made. Specificationfsoracceptable-leveolfscontaminantsin fishdietshave notbeen established.However, there areno known levelsof contaminantsreasonablyexpectedto be presentinthe dietthatareconsideredto interferweith the purpose or conduct of thetest. Loading,definedas thetotalwet weightof fishper hterof testsolutionw,illnot exceed 0.5 gram-qof fishperhterofsolutionthatpassesthrougha chamber in24 hours. Instantaneousloadingwillnotexceed 5 grams of fishper liteorf testsolutionpresentinthe testchamber at any giventime. DilutionWater Water used for the culturingand testingof fatheadminnows willbe obtainedfrom a well approximately4.0metersdeep locatedon theWildlifeInternationaLlt,d.site.Thd water willbe passed througha sandfiltearnd pumpe4 intoa 37,800-L storagetankwhere thewater willbe aeratedwith spray nozzles.Priortouse thewaterwillbe filteretdo0.45 pm inordertoremove fineparticleasnd thenpassed, througha UV sterilizeirn orderto remove microorganisms.Water used for culturingand testingis characterizeads moderatelyhard. Typicalvaluesforhardness,aBWinity,pH and specificonductance are appro)dmately: Hardness,mg/L as CaC03 Alkalinitym,g/L as CaC03 pH SpecificConductance, tLmhos/cm 145 190 8.1 330 Hardness,allWinityp,H and specificonductancewillbe measured weeldy to monitortheconsistency of thewellwater. Means and rangesof the measured parametersforthe four-wcekperiodprecedingthetest willbe providedinthe finalreport.Analyseswillbe performed atleastonce annuallyto determinethe concentrationosf selectedorganicand inorganicconstituentosf thew eU water and resultsof the most recent GLP compliantanalyseswillbe summarized inthefinalreporl PROTOCOLNO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -6- Test ARDaratus A continuous-flowdiluterwillbe used to provide each concentratioonf thetestsubstanceand a negative(dilutiownater)control.A peristaltpiucmp willbe used todeliverthe testsubstancetomixing chambers where thetestsubstancewiU be mixed with dilutiownater. The flow ofdilutiownater intoeach mixing chamber willbe controlleudsingrotameters.The rotameterswillbe calibratepdriortotestinitiation and atapproximatelyweekly intervaltshereafterA.ftermixing,testsolutionwsillbe splitoeachreplicate chamber. The proportionof water splitto each replicatweillbe chocked priorto the testand at approximatelyweekly inter%@atlhsereaftetro ensure thattheseflow ratesvary by no more dm 10% of the mean flowrateof thefourreplicates. The dilutewrillbe adjustedso thateach testchamber receivesatleast5 volume additionsof test solutionevery24 hours. Peristaltpiucmps willbe calibratebdeforeeach study and atapproximatelyweekly intervaltshereafterT.he deliveryof testsubstanceto testchambers willbegin atleast48 hours priortothe testin ordertoestablisehquilibriumconcentrationsof thetestsubstance.The generaloperationof thediluter willbe checked visuallyat leasttwo timesper day during thetestand atleastonce atthebeginningand end of thetest. Embryo incubationcups willbe constructedfrom glasscylinderaspproximately50 mm indiameter, with 425 -pm nylon or TeflonIDscreenattachedto the bottom using siliconseealant.Test chambers willbe 9-L glassaquariafiflewdith approximately7 L of water. Test chambers willbe positionedin a temperaturecontrolleednvironmentalchamber tomaintaina temperatureof 25 J:1'C. Testchambers willbe labelled with projectnumber, replicataend testconcentration. EnvironmentalConditions Ambient room lightwiU be used to illinninatehe testsystems. Fluorescenttubesthatemit wavelengthssimilarto naturalsunlight(e.g.C,olortone(t50) willbe.controllebdy an automatictimerto providea photoperiodof 16 hours of lightand 9 hours of darkness.A 30-minutctransitioonf low light intensitwyillbe providedwhen lightsgo on and offto avoidsudden changes in lightintensityL.ight intensitwyillbe measured attestinitiationa,tweekly intervaldsuringthetest,and attheend ofthetest.with a SPER ScientifiLctd-lightmeter or equivalent The testwillbe conducted ata targettemperatureof 25 2'C. Temperaturewillbe monitoredand recordedcontinuouslyduringthe entiretestinone controIreplicatuesing a FulscopeER/C Recorder(1900 J SeriesModel A) or equivalentRecordermeasurements* willbe verifiewditha hquid-in-glastshermometer priortotestinitiatioannd atleastweekly thereafter.The temperaturein each testchamber alsowillbe PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -7- measured usinga hquid-in-glastshermometer atthe beginningof the test,atweekly intervalsduringthetes@ and at the -.ndof thetest. ,Dissolved oxygen willbe measured inalternatereplicateosf each treatmentand controlgroup atthe beginningofthetestd,ailyduringthefirs7t days ofthetesta,tweekly intervaldsuringthetestand attest terminationusinga Yellow SpringsInstrumentModel 51B dissolvedoxygen meter,or equivalent.Inthe eventthatdissolvedoxygen levelsfallbelow 60% saturationd,issolvedoxygen measurements willbe made dailyin every testchambera@d appropriateactionswillbe taken afterconsultatiownith the Sponsor. Measurements of pH willbe made inalternatreeplicateosf each treabnentand controlgroup attestinitiation, atweekly intervaldsuringthetestand attestterminationusinga FisherAccumet Model 915 pH meter,or equivalent.If a treatmentlevelreaches 100% mortalityt,emperature,dissolvedoxygen and pH measu rements willbe taken atthattime,then discontinued. Hardness,alkalinitayn,d specificconductancewillbe measured in the dilutionwater and in the highesttreatmentlevelattestinitiatioant,weekly intervaldsuringthe testand attesttermination.Hardness and alkalinitmyeasurements willbe made by titratiounsing proceduresbased on methods in Standard Methodsfor theExamination of Water and Wastewater (4).Specificconductancewillbe measured using a Yellow SpringsInstrumentModel 33 Salinity-Conductivity-Temperatmuerteer,or equivalenl Procedures Priorto testinitiatioenm,bryo incubationcups willbe impartiallpylacedinglassbeakerscontaining dilutiownaterwithin3'C of thetesttemperature.In orderto controlbias,groups of one,two or three embryos willbe indiscriminatedliystributeadmong the cups untileach contains20 embryos. No other potentialsourcesof biasareextectedto affecttheresultsof thestudy. Two incubationcups willbe placed ineach duplicatetestchamber to achievea totalof 40 embryos per replicataend 80 embryos per treatment. Incubationcups willbe attachedto a rockerarm and suspended in the.watercolumn of the testchambers. The reciprocatinmgotionof therockerarm (approximately2 rpm) willfacilitactierculatioonftestsolution around the embryos duringthe incubationperiod. During the firsdtay of exposure,embryos willbe observedtwiceformortalityand eggs with ftmgus. Thereafteru,ntilhatclungiscomplete,embryo mortalityand the removal of dead embryos and/orembryos with ftmgus willbe performed once daily.Afterhatch,larvaewillbe counted and releasedintothe test chamber. The testwillbe repeatedifthepercentageof embryos inthecontrolgroup thathatchessuccmsfidly islessdm 66%, or ifthepost-hatchsurvivalinany controlchamber islessthan70%. PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD When hatchinghas reached>90% inthe controlgroup,larvaewillbe releasedto theirrespectivteest chambers. Any unhatched embryos willbe kept in theegg cups untilthey have hatchedor untildeathof the embryo occurs. Aftertheinitiarleleaseof thelarvae,theexposurewillcontinueforatleast28 days. Biolop-icaMleasurements Data on 6= tohatch,hatchingsuccess,survivaland gmwth (wetweight,dryweightand totalength) willbe collecteduring thetest Due tohigh naturalmortalitythatarisesfrom fungalinfectiondsuring embryonic development allfimgusedembryos thatareremoved duringthefirs2t4 hoursof incubationwill be considerednaturalmortality(D.A.Benoi@ personalcommunication).Dailyobservationsof frymortality, unusual behavior,and overallappearancewillbe made duringthe 28-day post hatch growth perioclAt test termination,allsurvivingfishwillbe retamed formeasurements ofwet weigh@ dry weight and totallength. SAMpILmizforAnWyfical Measurements Priorto exposure water samples willbe collectefdrom two replicatteestchambers of each treatment group and thenegativecontrol.Water samples alsowillbe collectedfrom two alternatinrgeplicatteest chambers atthe beginningof thetes@on Day 4 of thetest,at approximatelyweekly intervaldsuringthe test, and at theend of the testto determineconcentrationosf thetestsubstance.In theeventthat100% mortality occursinany treatmentt,hensamplingofthattreatinmtwillterminatefollowingthenextsamplinginterval. Samples willbe collectedatmid-depth from each testchamber and analyzedimmediately.The sample scheme is summarized below: PROPOSED NUMBERS OF VERFICATION SAWLES ExperimentaGlroup Pmtee Day 0 Day 4 Day 7 Day 14 Day 21 Day 28 End Control 2 2 2 2 2 2 2 2 LevelI-Low Concentration 2 2 2 2 2 2 2 2 Level2 2 2 2 2 2 2 2 2 Level3 2 2 2 2 2 2 2 2 Level4 2 2 2 2 2 2 2 2 Level5 2 2 2 2 2 2 2 2 Level6-I-IigChoncentration 2 2 2 2 2 2 2 2 Totals 14 14 14 14 14 14 14 14 *Pre-tcsstampleswillbe collecteadfterconditioninogf thediluterM.ore thanone pro-tesstamplingintmal may be requiredd,ependingupon theSponsorsneeds,and additionaslamplingand resultvsvifble documentedintheraw dataand includedinthefinarleporl TotalNumber ofVerificatiSoanmples- 112 PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -9- The above numbers of samples representthosecollectedfrom the testand do not includequality control(QC) samplessuch asmatrixblanksand fortificatiopnrsepared and analyzedduringtheanalytical chemistryphase ofthestudy.At thediscretioonf theStudy Director,watersamples alsowillbe collected from atleastone appropriatechamber whenever a malfunctionisdetectedin any part of the testsubstance deliverysystem. Ang3!ficalMeasurements Chemical analysisofthesamples willbe performed by WildlifeInternationaLlt,d. The analytical method used willbe based upon methodology providedby the Sponsor and identifieidnAppendix II.-The methodology used to analyzethetestsampleswillbe documented intheraw dataand summarized inthe final report. Data Analyses The endpointsused to determinethestatisticsailgnfflcancoef toxicant-relatedffectson fathead minnows includehatchingsuccess,survivalofjuvenilefish,totallength,wet weight and dry weight of survivingfish.The data setswillbe evaluatedfor normalityand homogeneity of variances. Data transformationwsillbe employed, ifnecessary,to correctthe data for non-normalityor heterogeneous variances. When thedatameet theconditionsof normalityand homogeneity of variances,testsforsignificant differencewsilluse 2 X 2 contingencytablesand analysisof variance(ANOVA) procedurewith an appropriatmeeans test(e.g,D,unnett'stest,Dunc&s testTukey's test,etc.).When the conditionsof normalityand homogeneityof variancesarenot met,and no transformationsaresuccessfulincorrectingthe conditiont,hen nonparametricstatisticparlocedureswillbe used. The resultsof the statisticaanlalyseswill aidinthedeterminatioonftheNOEC and LOEC. The maximum acceptabletoxicantconcentratiownillbe calculatedas the geometricmean of the NOEC and LOEC. RECORDS TO BE MAINTAINED Records to be maintainedfor datageneratedat WildlifeInternationalL,tcL willincludebut not be limitedto: I. A copy of the signedprotocol. PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, L-rD -10- 2. Identificatiaonnd characterizatioofnthetestsubstancei,fprovidedby theSponsor. 3. Datesofinitiatiaonnd terminatioonfthetest. 4. Historyofthetestorganism@ 5. Weight and lengthmeasurements. 6. Resultsofrangefindintgestsw,hen applicable. 7. Stocksolutiocnalculatioanndpreparation. 8. Observationsmade d4ringthetest9. Water chemistrycalculatio(nes.g.h,ardnessand alkalinity). 10. Ifapplicablet,hemethods usedtoanalyzetestsubstanceconcentrationasndtheresultosf analytical measurements. 11. Statisticcaallculations. 12. Testconditionasndphysical/chemicmaelasurements. 13. Calculatioanndpreparatioonftestconcentrations. 14. Copy offinalreport. FINAL REPORT A finalreportoftheresultosfthestudywillbepreparedby WildlifIenternationLatld,.The report willincludeb,utnotbe limitetdo,thefollowingw,hen applicable: 1. Name and addressofthefacilitpyerformingthestudy. 2. Datesupon which thestudywas initiataendd completeclItistheresponsibiliotfytheSponsorto providethefinaldatethatdataarerecordedforchemistrypathologyand/orsupportinegvaluations thatmay be generatedatotherlaboratories. 3. A statementof compliancesignedby the StudyDirectoraddressingany exceptiontso Good LaboratoryPracticSetandards. 4. Objectivesand proceduresasstatedintheapprovedprotocoli,ncludinganychangesintheoriginal protocol. 5. The testsubstanciedentificationnc,ludinngame,chemicalabstracntumber orcodenumber,strength, purityc,ompositiona,nd othercharacteristpircosvidedby theSponsor. 6. Stabilitaynd solubiliotfythetestsubstanceundertheconditionosfadministratioinfp,rovidedby the Sponsor. 7. A descriptioonfthemethods used toconductthetest. PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD 8. A descriptioonf thetestorganisms,includingthe sourceof thetestorgani,.g,mscientifincame, age, lifestage,means and ranges of weights and lengths,observed diseasesand treatments. 9. A descriptioonf the preparationof thetestsolutionst,he methods used to allocateorganisms to test chambers and begin thetest,thenumber of organisms and chambers per treat3nenta,nd theduration of thetest. 10. A descriptioonf circumstancesthatmay have affectedthequalityor integritoyfthedata. 11. The name of theStudy Directorand the names of otherscientistpsr,ofessionalsa,nd supervisory personnelinvolvedinthestudy. 12. A descriptioonf thetransformationsc,alculationsa,nd operationsperformed on thedata,a summary and analysisof thebiologicaalnd analyticaclhemistrydata,and a statementof theconclusionsdrawn from the analyses. 13. The signedand datedreportsof each of theindividualscientistosr otherprofessionalisnvolvedin the study,ifapplicable. 14. Statisticmaelthods employed for analyzingthe data. 15. The locationwhere raw data and finalreportareto be stored. 16. A statementpreparedby the QualityAssurance Umt hstlngthe datesthatstudyinspectionsand audits were made and thedatesof any findingsreportedto the Study Directorand Management. 17. Ifitisnecessaryto make correctionosr additionsto a finalreportafterithas been accepted,such changeswillbe made intheform of an amenchncntissuedby the StudyDirector.The amendment will clearliydentiftyhepartof thefinalreportthatisbeing amended and thereasonsforthealteration. Amendments willbe signedand datedby the Study Director. CHANGING OF PROTOCOL Planned changes to the protocolwillbe in the form of writtenamendments signed by the Study Directorand the Sponsoes RepresentativeA.mendments willbe consideredas partof theprotocoland will be attachedtothefinalprotocol.Any otherchanges willbe intheform ofwrittendeviationssignedby the StudyDirectorand filedwiththeraw data.AR changes totheprotocolwillbe indicatedinthefinalreport. GOOD LABORATORY PRACTICES Thisstudywillbe conductedinaccordancewith Good LaboratoryPracticeStandardsforEPA (40 CFR Part160 and/orPart792);OECD Principleosf Good LaboratoryPractic(eENV/MC/CHEM (98) 17); PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -12- and Japan MAFF (59 NohSan, NotificatioNno. 3850, AgriculturdProductionBureau). Each study conductedby Wjl&ife InternationaLlt,cLisroutmelyexamined by theWildlifeInternationaLlt,cLQuality Assurance Unit forcompliancewith Good LaboratoryPractices,StandardOperatingProceduresand the specifiepdrotocol.A statementof compliancewith Good LaboratoryPracticeswillbe preparedforall portionsof the studyconductedby WildlifeIntemationaLLtd- ne Sponsor willbe responsiblfeor compliance with Good LaboratoryPractcesforproceduresperformed by otherlaboratorie(se.g.r,esidue analysesor pathology).Ra%@.dataforallwork performed atWildlifeEftmationaL Ltcland a copy ofthe finalreportwillbe filedby projectnumber inarchiveslocatedon theWildlifeInternationaLltd.siteo,r at an alternativleocationtobe specifiedinthefinalreport. PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -13- REFERENCES 1 U.S.EnvironmentalProtectionAgency. 1996.Series850-EcologicaElffectTsest Guidelines (draftO)P,PTS Number 850.1400:FishEarly-LifSetageToxicitTyest. 2 ASTM StandardE1241-92. 1977.StandardGuideforConductingEarlyLif@e-StaTgoexicity TestswithFishes.AmericanSocietyforTestingand Materials. 3 United States Environm ntal Protection Agency. 1986. Standard Evaluation Procedure, Fish Early Life-Stage Test. Office of PesticidePrograms, Hazard Evaluation Division- EPA 540/9-86138. 4 APHA, AWWA, WPCF. 1985. Standard Methods for the Examination ow Water and Wastewater. 16dEdition,American PublicHealthAssociation.American Water Works Association. Water PollutioCnontrolFederatiorN@ew York- PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -14- APPENDIXI D)ENMCANON OF TEST SUBSTANCE BY SPONSOR To be CaVletedby Sponsor 1. TestSubstanceIdentit(yname tobe used inthereport):PFOS (PerfluorooctanSeulfonicAci@ PotassiumSalt ReferenceStandard(ifapplicable)A:nalyticaSltandard: N/A InternaSltandard: 1.1.2.21-LH.1-PLgHftuoroo_ctanSeulfonicAcid Test Substance SampleCode or Batch Number: Lot 217 Test SubstancePurity(0/AoctiveIngredient): 98.9 ExpiratioDnate: 2008 IL TestSubstmm Chxwtmization Have flyi,d-m& y, Purityandconvosibon orOffifdfimactais6cs vAk h T. propn-a-tciy% Ur t6stsubstarfeaend rekn= stmxlxdbeen dd=iE,Ed @nm toitsusem thissuxlyinaa=dance withGLP Stmdar&? Yes x No a Testsubslmm swraw con&ficw Pl= in@ ffi=eo==ded st=w cm&ms atWMife kkm,-dona Ud Ambiat Has thestabilkoyfthetestsibsLT= underffi= sbrdw condifions been detaTaii3iidnaworda= withGLP Standards? Offiapatnat stabiliitnyformation: -Yes x No IV. TestCzncmbafims: Adjusttestcmcwt-afiontoIW/o ai x -basedwm ft pwityC/o)gmn abom Do notadjusttestCOOCCDU-dt= to 1000/o a.LTestthematerialAS IS. V. TodcityWom3afico: MmmWi&L RatLD50 251 mg/kg - Mmm LD50 N/A Aquatic: InvatebrfTtmi&y (ECW50) DgpbAiamagna: 27mg/L FishTmi&y(LC50) Rainbow Trout: II mpJL DgphWa magna: 50 mg/L Fathead Minnow: 38 mg/L Odxx TomcityInformatLoCnmdudmg fimm ofcbrooc and mbchmc tests): Pleasesee MSDS PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD APPENDIXII AnalyticalMethod Providedby Sponsor Samples willbe analyzedbased upon prmodures methods: provided by the Sponsor in the following analytical 1. Liquid Chromutpgraphy Mass Spectrometry (LCMS) Method for the Determination of Pafluorooetane Sulfonic Acid Potassium Salt (PFOS) In Freshwater. Saltwater and Algal Medium A copy of the above method willbe maintained in the raw data. The actud methodology used to analyze the testsamples will be documented in the raw data and siimma6zed in the finalreport. PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-108 Page I ofI AWNDMIENT TO STUDY PROTOCOL STUDY TITLE: PFOS: AN EARLY LIFE-STAGE T03aCITY TEST WITH TBE FATHEAD NEENNOW (Pimephalespromelas) PROTOCOL NO.: 454/061099/FAT-ELS/SUB454 AMEENDMMNT NO.: I SPONSOR: 3M Corporation PROJECT NO.: 454A-108 EFFECTIVE DATE: August 2,1999 AM:ENDMIENT: Pag62 Add: Proposed Dates: Experimental StartDate: 8/5/99 Experimental TerminationDate: 9/6/99 REASON: The above informationwas not known when the protocolwas signedby the Study Director. STUDY DIRECTOR 9. LAAC'WATOR'Y MANAGEMENT SPO@;SOR'S REPRESENTATIVE DATE Lqi DAH a5411081amendl