Document xDz4x1yZznBzjEnELrY7mqyb

i---- I PFOS@ A 96-Hour StaticAcute ToxicityTest with the I SaliwateTMysid (Mys@dopsisbahia) i f@@j - II PFOS: A 96-HOUR STATIC ACUTE T03UCITY TEST WITH THE SALTWATER MYSID Wysidopsis bahia) FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 3M LAB REQUEST NO. U2723 454A-101 U.S. EnvironmentalProtectionAgency Series850 - EcologicalEffectsTest Guidelines OPPTS Number 850.1035 AUTHORS: Kurt R. Drottar Henry 0. Knieger,Ph.D. STUDY R4n"lATION DATE: December 4,1998 STUDY COWLETION DATE: June24,1999 AN&-NDED REPORT DATE: April26,2000 Submittedto 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St.PauL MN 55144 Wildl!fIenternatioLntadl. 8598 Commerce Drive Euton, Maryland 21601 (410)822-8600 Page 1 of 41 AMENDED WILDLIFE INTERNATIONAL LTD. -2- PROJECTNO.: 454A-101 GOOD LABORATORY PRACRICE COWLLKNCE STATEMEENT SPONSOR-- 3M Corporation TITLE: PFOS: A 96-HourStatiAccuteToxicitTyestwiththeSaltwateMrysid (Mysidopsibsahia) W]ILDLIFE INTERNATIONAL LTD. PROJECT NUMBEK- 454A-101 STUDY COWLETION: June.24,1999 AMENDED REPORT: April26,2000 Thisstudywas conductedincompliancewithGood LaboratoryPracticSetandardsaspublishedby the U.S.EnvironmentaPlrotectioAngency in40 CFR Parts160 and 792, 17 August 1989;OECD Principleosf Good LaboratorPyrwfice,OCDE/GD (92)32,EnvironmentMonograph No. 45,Paris1992;andJapanMAFF, 59 NohSan, NotificatiNoon. 3850,AgriculturParloductioBnureau,10 August 1984,withthefollowing exceptions: The testsubstancewas not characwdzedinaccordancewithfullGLP compliance;however,the characterizatwiaosn performedaccordingto3M StandardOperatingProceduresand Methods,and allraw data arebeingmaintainedinthe3M archivesT.he testsubstanceisbeingrecharacteriziendaccordancewithGLP. The stabiliotfythetestsubstanceunderconditionosf storageatthetestsitewas notdeterminedin' accordancewithGood LaboratoryPracticSetandards. STUDYDIRECTOR. ------------- KurtP, Drottar DATE SeniorBiologist SPONSOR APPROVAL: Spo%sor /'I 17L L AMENDED .WILDLIFE INTERNATIONAL LTD. -3- PROJECT NO.: 454A- 101 QUALITY ASSURANCE STATEMENT Thisstudywas examinedforcompliancevathGood LaboratoryPracteeStandardsaspublishedby the U.S.EnvironmentaPlrotectioAngency in40 CFR Parts160 and 792,17 August 1989;OECD Principleosf Good LaboratoryPracticeO,CDEIGD (92)32,EnvironmentMonograph No. 45,Paris1992;andJapanMAFF, 59 NohSan,NotificatiNoon. 3850,AgriculturParloductioBnureau,10 August 1984. The datesof all inspectionasnd auditsand thedatesffidany findingws= reportetdotheStudyDirectorand Laboratory Management w= asfollows: ACTrVrrY: TestSubstmm hmmmadon l@&tnxFortLficahoannd Sample Preparation BiologicaDlataand DraftReport AnalyticaDlataand DraftReport FinalRxpcirt AmendedReport DATE CONDUCTED: Mu& 5,1999 mmril 10,1999 March 25, 1999 March 24 and 25,1999 Junc3,1999 April19,2000 DATE REPORTM TO: STUDY DUMCTOIL- MANAGENEEW: March 8,1999 March 9,1999 March 10,1999 March 12,1999 March 25,1999 Mm@h 26,1999 March 25,1999 line 3,1999 April19,2000 March 25,1999 Jmm 3.1999 Apdl20,2000 -.toe jw= ELcoi=an DATE QualityAssuranceRepresentative AMENDED WILDLIFE INTERNATIONAL LTD. -4- REPORT APPROVAL PROJECTNO.: 454A-101 SPONSOR: 3M Corporation TITLE: PFOS: A 96-HourStatiAccuteToxicitTyestwiththeSaltwateMrysid (Mysidopsibsahia) W]ILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-101 STUDY DIRECTO KurtR. Drottar SeniorBiologist MANAGENIENT: Henry0.Kmger, PrD. DirectorA,quaficToxicologyand Non-TargetPlants DATE DAlt AMENDED WILDLIFE INTERNATIONAL L-m. -5- PROJECTNO.: 454A-101 TABLE OF CONTENTS Title/CovePrage........................................................................1.............. Good LaboratozyPracticCeomplianceStatement...............................................2............. QualityAssuranceStatement..............................................................3.............. ReportApproval........................................................................4.............. Tableof Contents...................................................................................................5............. Summmy ................................* .............................................7.............. Introducti.o.n..........................................................................8............. Objectiv.e.............................................................................8............. ExperimentaDlesign.....................................................................8............. materialsand Methods...................................................................9.............. Resultsand Discussion..................................................................1.2............. Conclusions...........................................................................1.2............. References............................................................................1.3............. TABLES Table1 -Summary ofAnalyticaClhemistryData.............................................1.4............. Table2 -T=pcrature,DissolvedOxygen andPH ofWaterintheTestam*= ......................1.5............ Table3 --CunudativPeercentMmtalityand Treatment-RdatedEffect.s...........................1.6............. Table4 -LC50 Values...................................................................17............. WILDLIFE INTERNATIONAL -6- TABLE OF CONRENTS Continued- PROJECTNO.: 454A-101 APPENDICES Appendix I- Sdb4 andpH ofSaltwateMreasuredDuringthe4-Week Period InunediatelPyrecedingtheTest..............................................1.8.............. Appendix H - AnalysesofPesticideOsr,ganics,Metalsand OtherInorgmics inWildlffIentemationaLltcLSaltwate.r.......................................1.9.............. AppendixIII- The AnalysisofPFOS inSaltwateirnSupportofWildlife IntmmationalLtd.ProjectN4o5.4:A-101 .....................................2.0.............. AppendixrV - ChangestoProtoco.l......................................................3.8............. AppendixV - Pmomel InvolveidntheSb* .............................................3.9.............. AppendixVI - ReportAmendmat .......................................................4.0............. ANEENDED WILDLIFE INTERNATIONAL L-m. .7- PROJECTNO.: 454A-101 SPONSOR: SPONSOR" S REPRM=AUVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FA-ML REPORT: WADLEE INTERNATIONAL LTD. PROJECT NUMBEP.: TEST SUBSTANCE: STUDY: NT-AN MEASURED TEST CONCENTRANONS: TEST DATES: LENGTH OF MST: TM ORGANIM SOURCE OF TEST ORGANISMS: SLMUKARY 3M Caporation Ms. Susan A. Beach W-ddUfo InternationLatl4L E&sM Maryland2160 454A-101 PFOS (Paflummtane $ulfoniAccidPotassimnSalt) PFOS: A 96-HourStatiAocuteToxicitTyestwiththe SaltwateMrysid (Mysid@psibsahia) NegativeControL0.57,1.1,1.9,3a.n0d 5.4mg ai./L EVab=W Star-tM@r& 8,1999 BiologicaTlaminadon 10M-arch 12,1999 Expaime@ T=inatio - Mm-ch 12,1999 96 Hours SdMda MysidWdopso bahla) vdme intmnatioIn'a@ci&uitmw Euton,Marylwd 21601 AGE OF TM ORGANOM: 96-9H65F-010H01OC3URORWLKCL5C0: 95% CONFMENCE 12MS: NOMORTAlXrY CONCENTRATION: NC)-OBSERVM)-EFFECT. CONCENTRATION: iuveam 3.6mg aijl 3.0and4.6Mg LIJL 1.1Mg LLAL 1.1!@l LI.AL ANffiNDED WILDLIFE INTERNATIONAL LTD. .8- P ROJECTNO.: 454A-101 INTRODUCRION Thisstudywas conductedby WildlifIentmmationaLltd.for3M CorporatioanttheVtrildhIfneternational Ltd.aquatictoxicologfyaciliitnyEaston,Maryland.The in-lifpehaseofthetestwas conductedfrom March 8, 1999 toMarch 12,1999. Raw datageneratedby W-ildliIfnetmnationaLltd.and a copy ofthefinalreportare filedunderProjecNtumber 454A-101 inmrjiveslocatedon theWildlifheitm-nationLatld.site. OBJECNW The objectivoeftlusstudywas toevaluatteheacutetoxicitoyfPFOS (PerfluorooctaSnuelfomeAcid PotassiumSalt)tothesaltwatemrysid,Mysidopsisbahia,duringa 96-hourexposureperiodunderstatitcest conditions. EXPEREMENTAL DESIGN Saltwatemrysidswerecqmed toa geometricserieosffivetrstconcentratioannsd anegative(saltwater) controlT.wo replicatestchambersw= maintainedineachtreatmenatnd controlgroup,with 10 mysidsin eachtestchamberfora totaolf 20 mysidspertestcmcentrafiomNominaltest - were selecteidn consultatiwoinththeSponsor,and w= basedupon theresultosfan evioratoryrangefindintgoxicittyest. Nominal testconcentratiosneslectewdere 1.1,1.8,3.0,4.9and8.2mg activeingredien(t&L)/L Mean measured testconcentratiownesre determinedfi-amsamplo oftrstwatercollectefdromewh btabnmitandcontroglmq at thebeginningofthetesta,t 48 @om and attestermination. The mysidswere mdtsaimmatelyassignedtoexpos@rechambersattestinitiatioOnb.servafionosf mortalitaynd otherclinicaslignswem made atapproximatel4y.5,.244,8,72,and 96 hoursaftertestinitiation. Cumulativepercentmortalitoybservedinthebrabnentgroupswas usedtoestimateorcalculatLeC,50 valuesat 24,48,72 and 96 hours.The no modalityconcentratiaond no-observed-effect-concentr(aNtOiEoCn) were detennmedby visualinterpretatioofnthemodalityand clinicaolbservatiodnata. ANIENDED WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454A-101 MATEPJAIS AND METHODS The sb* was conductedbasedon theproceduresoutlineidnffipcrotocol"PFOS: A 96-HourStatic Acute ToxicitTyestwiththeSaltwateMrysid (Mysidopsisbahiay'.The protocowlas basedon procedures outlineidnU.S.EnvironmentaPlrotectioAngency Series850 EcologicaElffwb TestGuidelinesO,PPTS Number 850.1035(1);U.S.EnvironmentaPlrotectioAngmq, StandardEvaluatioPnrocedureA,cuteToxicity Testfor&tuayineandmaiine Organisms(2);andASTM StandardE729-ggaStandardGiddeforConducting AcuteToxicitTyestswithFishesM,acroinvertebrateasndamphibians(3). TestSubstance The testsubstancweas receivedfrom 3M Corpomtim on October29,1998 andwas assigiteWdildlife internationLatld.identificatmiuonnber 4675. The testsubstancweas describeads a whitepowder. Itwas idmififiaesdFC-95 fromlotnumber 217 (T-6295).Informatiopnrovidedby theSponsorindicateadpurityof 98.90/oa,nd anexpindon dataof2008. The testsubstancweas reanalyzebdy theSponsorandtheCertificaoWf AnalysisdatedMarch 9,2000 indicateadpurityof 90.490/aI.lm testsubstancewas storedatambientroom temperature. EmRarationofTestConcentmdons Nominaltest were 1.1,1.8,3.0,4.9and 8.2mg aiJL,basedon a testsubstancepurityof 90.490/cA.R materialwshich came intocontactwiththetestsubstancdeuringpreparatioonftestconcentmtims were constructeodfplastiocrstainlessste@LA'prmar'ystocksolutiownas preparedindilutiownaterata concentratioofn8.2Mg LIJL. The primarystocksolutiownas mixedwithaneleewenib= forapradma* 22 hoursto aidin thesolubffizadoofnft testsubstance.Aft= mbdn& theprimarystocksolutiownas proportionaldliylutewdithdilutiownatertoprepareft fouradmang testconcmbmdons. AU testsolutions appearedclearand colorless. TestOmanism The saltwatemrysidwdopsis bahia,was selecteadsft testsped= forthistudy.Mr saltwatemrysid isrepresentatiovfean unportantgroupofaquaticmarineinvertebrataensdwas selectefdoruseinthetestbased upon pasthistoroyfuse and easeofculuninginthelaboratoryM.ysidsusedinthetestw= obtainedas juvenilesfrom culturesmaintainedby V*rjldhfIentmmafionaLltcL, Easton, Maryland. ANMNDED WILDLIFE INTERNATIONAL LTD. -10- PROJECTNO.: 454A-101 Adultmysidswereheldatapproximateltyhesame temperaturaesusedduringtheteslDuringtheholding and acclimatiopneriodst,headultsshowed no signsofdiseaseorstressD.uringthe14-dayholdingperiod precedingthetestw,atertemperaturersangedfimm25.0to25.5*C.IlepHof thewaterrangedfrom7.9to8.1, salinitwyas maintainedat20%o (partsperthousand)and dissolveodxygenrangedfrom 7.0to 7.2 mg/L InstrumentatiuosnedforwatermeasurementsisdesaibedintheEnwronmentalCon&hons sectioonfthirseporl At testmitaton,thejuvmulemysidswere carefullcyollectefdromthecultureasndtransfenedtoglassbeakers. Mysidswere transferrfetdom thebeakerstothetestcompulm@ usmg avnde-borepipetteJ.uvenilmeysids arerelativeflryagdeandwere,th=fore,handledaslittlaespossibleT.he juvenilmeysidswerefedlivebr= shrimp(Artemiasp.)nauplidiailyduringthetesttopreventcaimbali-qm TestApparatus Testchamberswere2-L polyethylenbeucketsRUzd withapproximatel1y000mL oftestwater.The depth ofthetestwaterina representatitveestchamberwas approximatel6y.6cia Testchamberswere impartially positioneidnanenviromnmtalchamberdesignedtomaintainatemperaturoef25:k2'C.The testdm*crs wem labeledwiththeprojecntumber,testconczntrafiamndrephcateandw= coveredwithpleidglutsroughoutthe test. Dilution The waterusedforholdmg,acelimatmaandtestmgwas nata-asleawatercollecteadtIndianRiverInK Delaware,anddilutetdoa salinitoyf approximatel2y0%o withwellwater.SdfiiitayndpH measurementstaken duringthefour-weekperiodimmediatelyprecedmgthetestarepresenteidnAppenchx L The fiwgy-wllectedseawaterwas passedthrougha sandfiltetro r=ove particlegsreatetrhin approximatel2y5 ^ andpumped fifta 37,800-Lstomp tank The fihmrdseawate&rm was dilutewdith freshwatefrroma wellon theVtrildhhftommationalLtd.siteandam*ed withslnw nozzles.Pnortouse,the wateragainwas filtere(d0.45@=) toremove and. particlesT.he resultosfperiodicanalyses performedtomeasuretheconcentratioonfsselectecdont=kmu in saltwateursedby VMdife IntemationaLltd. arepresentedinAppendix IL Conditions Lightingusedto diwaunatethecultureasndtestchambersdurmg cultormgandtestmgwas providedby fluorescetnutbesthatemittedwavelengthsim@ tonaturaslunligh(tColortone50). A photopcrioodf 16 hoursoflighatnd 8 hoursofdarknesswas controllewdithanautomatifcu=. A 30-minutotransitiPoMnW WILDLIFE INTERNATIC)NAL LTD. PROJECT NO.: 454A- 101 oflow lightintensitwyas providedwhen lightwsent on and offtoavoidsuddenchangesinhghting.Light intensitayttestinitiatiwoans approximatel4y37 luxatthesurfacoefthewater.Lightintensitwyas measured usinga SPER ScientifLitccLlightmeter. Temperaturewas measuredineachtestchamber atthebeginningand end ofthetestusinga liquid-in-glass thermometer.Temperaturealsowas measuredcontinuousliynonenegativecontrolreplicatuesinga Fulseope ER/C Recorder.The targetesttemperaturdeuringthestudywas 252'C. Dissolvedoxygen and pH measurementswere made on watersamplescollectefdrom allreplicatteestchambers of each treatmentand controalttesitnitiatiaonnd,atapproximatel2y4-hourintervalUsmufter. Salinitwyas measuredintheduutim waterattestinitiation. MeasurementsofpH were made usinga FisherAccumet Model 915 pH meter,and dissolveodxygen was measuredusinga YellowSpfiM InstrumenMtodel 5IB dissolveodxygenmeter Semtywasnrmuredusmga Bio-marineI,nc.Aquafaunarefractometer. Observations Observationwseremade todeterminethemimber ofmomffitiesT.he munber ofindividualesxhibiting clinicsailgnsoftoxicitoyrabnormalbehavioralsowere evaluatedO.bservationwseremade appram:b@ 4.5, 24,48,72 and96 hoursaftetrestinitiation. StatisticAaaldy-ses 1'he24,48,72and 96-hourLC50 valuesandthe95% confidenchedmvalswerecdculatewdhen possible bypmbit analysitsh,emovingaveragemedwd orbinomidprobabiliwtiythnon-linmfitapdatim(4,5,6u)sing thecomputersoftwareofC.E.Stephan(7).Inthissh*, LC50 valuescouldnotbe calculateadt24 and48 hours due tothelackof an adequateconcentradon-respowpeau=n, however,thepmbit method was usedtoevaluate mortalitayt72 and 96 hours.The no mortalitcyoncentratioandNOEC weredet=ninedby visuahltmpretation ofthemoruhty andclinicaolbservatiodnata. AnW3@ficaUlmdM Water sampleswere collecteadtrmd-depthfromeachreplicatestchamber ofeachtvatnentand controglroupatthebeginningofthetes@at48 hoursand attest=fiaationtomeasureconcmtaticw ofthe= substance.The sampleswere collecteidnplasti(cNaigme(D)vialsand analyzedas soon aspossiblewithout storage.Analyticaplrocedureussedintheanalysiosfthesamplesut providedinAppendix M. WILDLIFE INTERNATIONAL LTD. -12- PROJECTNO.: 454A-101 RESULTS AND DISCUSSION Me&surmnentOf TestConcentratims Resultsof analym tomeasureconcentrationosf PFOS inwatersamplescollectedduringthetestarc presenteidnTableI andintheanalyticcahlemistrryeport(AppendixM). Nominalconcentratiosneslectefdor .useinthissb* were 1.1,1.8,3.0,4.9and 8.2mg aiJL. Samples collecteadttestinitiatihoand measuredvalues thatrangedfrom 52 to71% of nominalvalues.After22 hoursofmbdn& theprimarystocksolutiownas measuredtobe 70 to71% ofnominaland was probablyatdw limiotf solubiliitnyfilteresdaltwaterT.he recoverieisntheothertestconcentrationwse= similabrecausetheywereproportionadldutlonsoftheprimary stock.Measuredvaluesforsamplestakenat49 hoursrangedfrom 44 to71% ofnominal.Measuredvaluesfor samplesW= at96 hoursrangedfrom 36 to 71% ofnomind. When measuredconcentratioonfsthesamples analyzedattestinitiatioanp,proximately48 hoursand attesttmminationwereaveraged,fficmean-measured concentratiofnosrthistudywere0.57,1.1,1.9,3.0and5.4mg &i./L.Mean measuredconcwtrafionwsen used intheestimatioonr calculationf LC50 values. Observationsand Measurements Measurem=ts oftemperatured,issolveodxygenandpH arepresenteidnTable2. Temperatureswere -withitnhe2520C rangeestablishefdorffitcestDissolvedoxygenconcentratiorn=sdned a 6.6mg/L (900/oof sawrationt)hroughou&ta test.Measur=ents ofpH rangedfr= 8.1to8.2duringffitcest Dailyobservationosfmortahtyandotherclinicaslignsoftoxicitoybservedduringdw testam shown in Table3. Mysids inthenegativecontro@anddie0.57and 1.1mg &idL ftwunentgroupsappearednormaland healthy&jringthetoslAfter96-hoursofopmum mortalitiynthe1.9,3.0and 5.4mg &iJL treatmengtroups was 10,40 and 750/or,espectivelyL.C50 valuesand 95% confidencleimitsat24,48,72 and 96 hourswere estimateodrcalculatefdrom themortalitdyata,and areshown inTable4. CONCLUSIONS The 96-hourLC50 valueforsaltwatemrysidsmq)osedtoPFOS was 3.6mg &i./L The 95% confidence -limitwsere 3.0and4.6mg &i./La,nd theslopeoffficoncmtration-responcsuervewas 4.5. The 96-hourno mortalitcyoncentratioand theNOEC were 1.1mg &iJL. ANffiNDED WILDLIFE INTERNATIONAL LTD. -13- PROJECTNO.: 454A-101 REFERENCES 1 U.S. Environmental Protection Agency. 1996. Series 850 -Ecological Effects Test Guideihics (draft), OPPTS Number 950.1035: MysidAcute Toxicio Test. 2 U.S. Environmental Protection Agency. 1985. Standard Evaluation Procedure, Acute Toxicity Test for Estua?lne and Marine Organisim (Shrimp'96-Hour Acute Toxicity Test). Hazard Evaluation Division.Officeof PesticidPerograms. EPA 540/9-85-010.Washington,D.C. 3 ASTM Standard E729-88a. 1994. Standard Guidefor ConductingAcute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. American Society forTwtmg and Materials. 4 Thompson, WIL 1947. BacteriologicalReviews,Vol.ILNo. 2. Pp. 115-145. 5 Stephan,.C.E. 1977. "Methods forCalmdating an LC50", Aquatic Toxicology andHazardEvaluations. American Society for Testing and Materials. Pubhcatim Number STP 634, pp 65-84. 6 Finney, D.J. 1971. StatisticaMlethods In BiologicalAssay. Second edition.GriffinPress,LondorL 7 Stephan,C.E. 1979. US. EPA, EnvironmentalRwearch LaboratoryD,uluth,Mfimesota.Personal communication. WILDLIFE INTERNATIONAL LTD. -14- PROJECTNO.: 454A-101 Table 1 Summary ofA@d@cal Ch=istryDda ?e@ S@u@eLam: TestOrganism: DilutioWnatm Naminal Test Concentration Lmg aiA,) Negahve control on 3PMFOSC"Pora" SaltwateMrysid,Mysidopsisbahla FiltereSdaltwater Ret)licate A B A B A B. S!li@ng Ti*une 0 0 48 48 96 96 A 0 B 0 A 48 B 48 A 96 B 96 1.8 A 0 B 0 A 48 B 48 A 96 B 96 3.0 A 0 B 0 A 48 B 48 A 96 B 96 4.9 A 0 B 0. A 48 B 48 A 96 B 96 8.2 A 0 B 0 A 48 B .48 A 96 B 96 iim limiotfTmfitafim (LOQ) was 0.115mg aiJL. Concena-atim (MffLijL) <L < Loq 0.575 0.622 0.605 0.640 0.391 0.580 1.12 1.19 1.10 1.09 1.04 1.13 1.92 1.99 1.92 1.91 1.79 1.91 3.05 2.66 2.96 3.35 3.11 3.11 5.82 5.78 3.58 5.85 5.22 5.86 clo@@on (mA ai./L) < LUQ 0.57 1.1 1.9 3.0 5,4 Percent Of I Nomind 52 61 63 61 66 AMENDED Table 2 Temperature,DissolvedOxygm and pH ofWater intheTestChambers Sponsw. 3M Corporation Test Substance: Tom Organism: DilutionWater PFOS Sdhvatcr Mysid, AlyddopAs bohia FiltereSdaltwater Mean Measured 0 HZ Ted Concentration Tamp Dd (mgaijl) RepU"to (-C) (alu cH NegativeControl A B 24.2 7A .1 25A 7.2 8.1 24 Hours DO LMIU LH 7.1 8.1 7.0 8.1 48 Hours DO (mg/L) pH 6.9 8.2 6.8 8.2 0.57 A 24.1 7A 9.1 7.0 S.1 6.8 9.2 B 25.3 72 9.2 7.0 9.1 6.8 8.2 1.1 A 24.0 7A 82 7.0 9.1 6.9 92 B 24A 73 82 7.0 9.1 8.9 92 1.9 A 24.0 7A 82 7.1 9.1 6.9 82 B 23.8 72 $1 7.0 8.1 6.8 82 3.0 A 23.6 7.3 92 7.0 9.1 6.8 92 B 23.5 7A 82 7.0 9.1 6.9 92 5.4 A 23.8 73 82 7.1 8.1 6.9 92 B 24.0 72 92 7.0 9.1 6.9 82 The 0-bourdilutiownw (tiegww controlm)easurementformfinity20%o. 2 TeMpaMtUre MM=VD WntlnUOU* during the testranged from apxmdmately 23.5 to 26.OOC. A dissolveodxygen wnmftfion of4A mg/L repumb 60% saturatioant250C insaltwatewri& a s&Wty of20OAo. 72 Hours DO (m&/L) LH 6.8 8.1 6.8 9.1 6.9 9.1 6.8 82 6.8 9.1 6.9 6.9 82 6.9 9.1 6.7 82 6.8 8.2 6.9 92 6.8 82 Tamp (-C) 24.7 24.7 24.7 24.7 24.7 24.8 24.7 24.6 24.5 24A 24.5 24.5 p -16- Table3 CumulativePcr=t Mortalitaynd Treatinent-RelatEefdfects Sponsor Ted Subdawe: Ted Orruism DilutioWnater. 3M Cwpogiflon PFOS Sakwat- Mynd.Wdopsis Fifteregdahvidw bahia Mean Meamutd 4.5 Hours Ted Conoentmtion N& (mgLIJL) Repficdo EVOW NO. % Do& Effect? MW Nepfive Contmi A B 10 0 10 AN 0 10 0 10 AN 0.57 A 10. 0 10 AN 0 B 10 0 10 AN 1.1 A 10 0 10 AN 0 B 10 0 10 AN 1.9 A 10 0 10 AN 0 B 10, 0 10 AN 3.0 A 10 0 10 AN 0 B 10 a 10 AN 5.4 A io 0 10 AN 0 B 10 0 10 AN CamW&tive mmgw of &ad mysidl Obseriad Effwb: AN Appem NonnaL 24 Hom No. % Dead Effccb Dead 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 5 1 9 AN 2 8 AN 15 I 9AN 49 Hours No. % Dead Effects Dead 0 10 AN 0 0 10 AN 0 10 AN 0. 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 2 8 AN 15 I 9AN 4 6 AN 45 5 5 AN 72 Hours No. % Dead Effects Dead 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 5 @l 9 AN 4 6 AN 30 2 BAN 6 4 AN 60 6 4AN WILDLIFE INTERNATIONAL LTD. -17- PROJECTNO.: 454A-101 Table4 LC50 Values Sponsor Test Substance: TestOrganism: DilutioWnater Time 24 Hours 3M Corporation PFOS SaltwateMrysid,Mysidopsisbahla FiltereSdaltwater Lower 95% Confid=cc LC50 Limits Lin . > 5.4 (mgaiu upper 95% Confidence Timi (mx&iJL) 48 Hours > 5.4 72 Hours 4.4 3.6 6.2 96 Hours 3.6 3.0 4.6 Iconfide= limitcsouldnotb@.c-alculatewdithffimcortalitdyataobtainc& Statistical Method Visualhiwpretation VisualInterpretation Probit Probit ANffiNDED WILDLIFE INTERNATIONAL L-m. PROJECT NO.:454A-101 Sponsor: TestSubstw=: TestOrgani= DilutionWater: sdwty (%0) APPENDIXI Salinityand pH of SaltwaterMeasured During the4-Week Penod b=ahately Pmcedmg theTest 3M Corporation PFOS SaltwaterMysid,Mysidopsis bahla FiltereSdaltwater megm 20(N=4) EMLE 20-20 pH 9.2(N 4) 8.1-8.2 WILDLIFE INTERNATIONAL L-m. -19- PROJECTNO.: 454A-loi ANALYS]RS APPENDIXII AnalysesofPesticideOsr,ganicsM,etalsandOtherInorg 'cs inV*rddliIfneternationLatl& Saltwater' NOEASURED CONCENTRATION MisceBantousMeasurounts TotaDliswlvcdSolids Ammonia Mitrogen TotalOrpnic C@@n 2 TotalCyanide 23,500 mg/L < 0.050 mg/L < 1.0 MOA. < 10.0 Organochlorineasnd PCBs Akhin AlphaBHC BetaBHC DeltaBHC Gamma BHC (Undme) Chlordane DDD, pp' DDE, pp' DDT,.pp' Dickbiri Radnm lfi A Endwil&n@ B Rndosulfa-nRillfs Endrin EndrinAlddiyde HeptacNor I&dxmychlor Hqtwldor Epo)iide Twmphene PCB-1016 PCB-1221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 < 0.005 AzgL < 0.005 AW < 0.005 UA < 0.005 U,,- < 0.006 ugL < 0.025 U- < 0.006 A4E < 0.005 A4vL < 0.008 i4g/L < 0.005 AWL < 0.005 PA < 0.005 AIL < 0.018 A41L < 0.010 PA < 0.005 ug/L < 0.005 A/L < 0.007 us/L << 00..050050 mg/L < 0260 A& < 0260 lAg/L < 0260 AW@L < 0.720 AA < 0.720 MB/L < 0.720 A41L < 0.720 MA Metab and Other Ilorpnics Alumin Arwnicym 3 2 Calcium Chmmjum 3 Cobatr CO-PP03 map em*ulk3 Mmearncgumraye MoM*num 3 PNo*twaLs-dr 3 set U silzpm3 W CYM < 100 AA < 25.0 lis/L < 0.50 ;4/L < 1.0 235 9/L << 21..00 IVL < 20.0 < 100 /Ag/L < 10.0 AQVL < 746.00 mUgW/LL < 0.20 < 2.0 < 20.0 /jg& 277 mg/L < 25.0 A41L < 1.0 A4& 6,010 m < 20.0 Analym performedbyQST EaviromentdGaintsviUc,Floridfaorsamplesc@ouecteodnNovemba3 diroughRo-v-=-7=7I,W7. Analym performedbyWildlifleiltetmtioLntadl.forthesunplecogeew on November 5,1997. 3 Analym performedby WildlifIenternationIaAld.forsamplescollecteodn November 5 thmugh 7. 1997. WILDLIFE INTERNATIONAL -20APPENDIXIII PROJECT 140.:454A-101 THE ANALYSIS OF PFOS IN FILTERED SALTWATER IN SUPPORT OF WILDLIFE DnERNAnONAL LTD. PROJECTNO.: 454A-101 WILDLIFE INTERNATIONAL L-m. -21- PROJECT NO.: 454A-101 REPORT APPROVAL SIPONSOP, 3M Corporation TTRLE: PFOS: A 96-HourStaticAcuteToxicityTestwiththeSaltwateMrysid (Adysidopsbiashia) W]ILDLIFE R4TERNATIONAL LTD. PROJECT NO.: 454A-101 -PRINCIPAL WWSTIGATOILRa]@n@:Van Hoven,Ph.D. scientist .Wa-trdB.Nbwn, Ph.D.' NlanagerA,nalyticaClhemistry DATE DAT]G AA4ENDED WILDLIFE INTERNATIONAL L-m. -22- PROJECTNO.: 454A-101 Introdu Saltwatersampleswere collectefdrom a statiaccuteaquatictoxicitsytudydesignedtodeterminethe effectsof PFOS (PerfluorooetanSeulfonicAcid Potassium Salt)to the saltwatermysid (Afysidopms baina).This studywas conductedby WildlifIenternationLatld.and identifiaesdProjectNo.: 454A-101. The analysesofgme watersaznplewsereperformedatWildlifIenternationLatld.usinghighperformance liquicdhromatographwyithm2 s spectunctridcetectioWnLC/MS). Sampleswere receivefdoranalysis on March 8, 10 and 12,1999 and were analyzedon each samplereceipdtay. TestSubstanceand InternaSltandard Ile testsubstanceused forthe analytealportionof tlnsstudywas WildlifeInternationLatld. idenff=fionnumber 4675.'Ile testsubstancweas usedto preparecalibratiaonnd matrixfortification samples. Tle internasltandardwas receivefdrom 3M Corporatioonn July2, 1998 and was assigaedWildlife InternationLatld.identificatniuomnber 4526 upon receiptT.he fid=al standarda,granularnmftrialwas idenfifiaesd: In. IK 2K 2H PerfluorooctaSmulfaniAccid,ChemicalAbsund Number- 27619-97-2. The standardwas storedunder ambumt candrtions. AR93ILW Method The method used fortheanalysisofthesaltwatesrampleswas developedatWddh& InternationLatlci: and entitle-dAnalyticaMlediod fortheDd=ination- of PFOS in FreshwaterS,&water, and A%d Mediu&'. Thismethodologywas indudedas AppendixH ofVrddUk hu=ationalLtd.protocomlmber 454/011299ft"AIJSUB454. Itwu WLud upon methodologyprovidedby 3M Corporation- Samples were dilutedin a 50% nicdkanot: 50% NANOpure(D water solutioncontdning 0.100mg 4H PFOS (intmnalstandard)AaLnd 0.05% fbrmicacid(v/v)so thatthey fellwithinthe calibratiornangeofthePFOS methodology. Concentrationosf the PFOS inthe standardsand sampleswere determinedby reverse-phasleugh perfomianceliquidchromatographyusinga Hewlett-PadmrdModel 1100 FEgh PerformanceLiquid WILDLIFE INTERNATIONAL L-M. -23- PROJECTNO.: 454A-101 ChromatogWh (BPLC) witha Peridn-EimeArPI IOOLC Mass Spectrometeerquippedwitha PerkinElmer TurbolonSpmy ion source. BPLC separationwsere achievedusinga KeystoneBetasilClg analyticaclolumn (100nun x 2 mm I.D.,3 M particlseize).The hatument parametersaresummarized inTable1. A methodflowcharitsprovidedinFigureI- CalibratioCnurve and Limitofguanfitation calibratiosntandardsof PFOS preparedin a 50% methanol:50% NANOpure(& water solution ining0-.100mg 4H PFOS (internsatlandard)/aLnd 0.05% formicacid(v/v)r,@@ inconcentration from 0.00229to 0.0457 mg a.i./Lw,ere analyzedwiththesamples.Linearregressioenquationswere generatedusingpeak arearesponseratios(PFOS :internasltandardv)ersusthermwzfive cmcentrafions ofthe cahbratlonstandardsA. typicalcalibraticoanm isprwmw inFigure2. The concentrationf PFOS inthesampleswas determinedby subsbtrtmgthepeak arearesponseratiosintotheapplicablleinear regressioenquation.Repraentatmeionchromatogmms oflow and highcallratlosntandardsarepresented inFigures3 and 4,respectively. The method limitofquant=on (LOQ) fortheseanalyseswas setat0.115 mg aiJL calculateadsthe productof thelowestcalibratisotnandardanaly=d (0.00229mg a.idl)and thedilutiofnactorof the matrixblanksamples(50.0). MatrixBlwk andFortificatSi=opnlm T@rm matrixblanksampleswere analyzedto determinepossiblmewrferc=. No mtmfemces were observeda.tor above do LOQ duringsamplesanalyses(Table2). A representatidv=ematogram Ofa matrixblankispresentedinFigure5. Saltwatewras fortifiaetd0.457,4.57and 9.15mg a.iJLand analyzedconcurrentlwyiththesamples to determindec mean procedurarlecovery(Table3). Sample were notcomxftd forthe mean procedurarlecoveryof97.4%. A representaticvheromauwm ofa matrixfortificatiismpreseaw inFigure6. ANENDED WILDLIFE INTERNATIONAL LTD. -24- PROJECTNO.: 454A-101 ExaMle Calculations Sample number 454A-101-3,nominalconcentrationf 1.1 mg &iJL insaltwater. TnitiaVlolume: 0.500 mL FinalVolume: 25.0mL DilutioFnactor:50.0 PFOS PeakArea: 235466 hftmal StandardPeak Are&- 449012 Peak Area Ratio:0.5244 Calibratiocnurveequation. Slope:0.04044 htercept0.05941 Curveisweighted(1/x). PFOS (tLga.i./La)tftishument PFOS (mg a.iJL)insample Peakarearati-o (Y-hftTcept) Slope 0.5244- 0.05941 0.04044 11.5 PFOS (pgaiJL)atbobument x DilutioFnactor 1000 11.5x 50.0 1000 0.575 ANIENDED WILDLIFE INTERNAT(ONAL LTD. -25- PROJECTNO.: 454A-101 PercentofNominal Concentration PFOS (mg a.iJL)insample x 100 PFOS (mg a.i./Ln)ominal Calculatedrecovery5:2.4% Note:manualcalculatimoany differ. RESULTS SgMIe Angysis Saltwatesrampleswere collectefdrom,the staticacutetomcitystudyvnth the saltwatemrysid (A,fysidopbsaihsia)attestinitiatiMoanr,ch 8, 1999(Hour0),on March 10,1999 (Hour48),and attest terminationM,arch 12,1999 (Hour 96).The measuredconcentratioonfsPFOS inthesamplescollecteadt initiatioofnexposureof the testorgammus (Hour 0) rangedfrom 52.4 to 70.7% of the nominal concentrationsS.amplescoreded atHour 48 had a measuredconcentratiroanngeof 43.5to 71.0% of nominalvalues.Samplescollecteadttestterminatio(nHour96)had a measuredconcentratiornangeof 35.5to71.1% ofnominalvalues(rable4).A representaticvheromatogramof a testsampleisshown in Figure7. AMENDED WILDLIFE INTERNATIONAL L-m. -26- PROJECTNO.: 454A-101 INSTRUNHNT: TableI TypicalIHPLC OperationaPlarameters Hcwlctt-PncksaMroddel 1100 High PerformanceLiquid Chromatographwitha Pcridn-El= API 1OOLC Mass Spectrometer equippedwitha Perldn-Ehw TurboIonSpmy ionsource.Operatedin selectiivoenmonitorinmgode (SM. ANALYTICAL COLUMN: KeystoneBctasiCllicolumn(100mm x 2 mm I.D.,3 pznparticle size) OVEN TEWERATURE: STOP TIME: 300C 10.0minutes FLOW RATE: 0.220mlhninute MOBILE PHASE: INJECNON VOLUME: 72.0% Medmnot: 28.0% NANOpureO Water containing 0.1% FonnicAcid 25.0p.L PFOS RETENTION TIME: Appraanately7.2minutes INTERNAL STANDARD RETENTION TIME: ApproxffiuWy4.9minutes PFOS MONITORED MASS: D4TERNAL STANDARD MONITORED MASS: 498.6amu 426.7amu WILDLIFE INTERNATIC)NAL LTD. -27- PROJECTNO.: 454A-iol Table2 M=k BlanksAnaly=d Coneunwdy DuringSampleAnalysis Number L454A-101-) MAB-1 SMIC Type MatrixBlank Measured Concenu-afioonf PFOS, (mg a.iJL) < LOQ MAB-2 MatrixBlank < LOQ MAB-3 MatrixBlank <LOQ The limiotfquanfitafi(oLnOQ) was 0.115mg aidl basedupontheproductofthelowestcalibration anal@@ (0.00229 a.iJL)andthedilutiofniwwrofthematrixblank!Mles L50.0). ANCDMED WILDLIFE INTERNATIONAL LTD. -28- PROJECTNO.: 454A-101 Table3 MatrixFortificatioAnsalyzedConeudentlyDuringSampleAnalysis Sample Number (454A-101-) MAS-1 MAS-4 MAS-7 Co=entrationsofPFOS a.i.u Fortified Measured 0.457 0.457 0.537 -1 0.457 0.396 Percent Recovered 117 86.5 MAS-2 4.57 4.27 93.3 MAS-5 4.57 4.43 96.8 MAS-8 4.57 4.36 95.2 MAS-3 9.15 8.18 89.4 MAS-6 9.15 9.42 103 MAS-9 9.15 9.93 97.6 Mean = 97.4 StandardDeviation = 9.41 CV = 9.67 N= 8 NoteR:esultasndcorrectifobnrnsewtesstubstanpcueritwye= generatuesdmgMacQuanversio1n.5 softwaraendrmniiaclalculatioVnasl.uehsavebeenroundefdorreportipnugrposes. 'Resultwas 0.942mg a.iJLor205% ofthenominalconcentratiolnl.iisresulwtas notusedtodetermine themean ELo@@uralMEZVM duetoE!tLble fortificatainodn/ordilutioenrror. AMENDED WILDLIFE INTERNATIONAL L-m. -29- PROJECTNO.: 454A-101 Table4 Measured Concentrationosf PFOS inSaltwateSramples from a SaltwateMrysid StatiAccuteToxicityTest Nominal Test Concentration (-MAa.i. 0.0 Sample Number (454A-101-) 1 2 13 14 25 26 Sampling Time 2iLuris 0 0 48 48 96 96 PFOS Measured Concentration' Ltr!ag.iJL) < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Percent of Nominal - - 3 0 4 0 15 48 16 48 27 96 28 96 0.575 52.4 0.622 56.6 0.605 55.1 0.640 58.2 0.391 35.5 0.580 52.9 1.8 5 0 6 0 17 48 18 48 29 96 30 96 1.12 64.4 1.19 68.3 1.10 63.2 1.09 62.7 1.04 59.8 1.13 64.5 3.0 7 0 1.92 8 0 1.99 19 48 1.92 20 48 1.91 31 96 1.79 32 96 1.91 Note:Resultsand coffeettonfsornew testsubstancepunty were generatedusingMacQuan 65.7 67.9 65.8 65.2 61.2 65.4 version1.5 softwareand manual calculationsV.alueshavebeen munded forreportmgpurposes. The limitofquanfitafio(nLOQ) was 0.115 mg a.i.AbLasedupon theproductof ttil6owestcalibration standardanalyzed(0.00229mg a.i.ALa)nd thedilutiofnactorofthematrixblanksmpka (50.0). 2 Me=Tesult.ofduplicatreeoutionsof dw originaslample.Originarlesul(t12.0mg a.i./Lw)as not includeidntheMfisticaalnalysiosf&e data. Duplicateresultfsbr454A-101-23 are3.55and 3.61. ANIENDED WILDLIFE INTERNATIONAL LTD. -30- @ROJECTNO.: 454A-,101 Table4 (Continued) Measured Concentrationosf PFOS inSaltwateSramples from a SaltwateMrysid StatiAccute ToxicitTyest Nominal Test Concentration M a.i.LL) 4.9 Sample Number L454A-IOI-) 9 10 21 22 33 34 Sampling Time 0. aur.is 0 0 48 48 96 96 PFOS Measured Concentration' LMA a.iJL) 3.05 2.66 2.96 3.35 3.11 3.11 Percent of Nominal 61.6 53.8 59.9 67.8 63.0 62.9 8.2 11 0 5.82. 70.7 12 0 23 48 24 49 35 96 36 96 5.78 70.2 3.582 43.5 5.85 71.0 5.22 63.3 5.86 71.1 Note:Resultsand correctionfsornew testsubstancepurityweregeneratedusingMacQuan version1.5 softwareand maniialcalculilionVsa.lueshavebeenroundedforreportinpgmpwes. The limitof quanfitafi(LmOQ) was 0.115 r4ga.i./bLasedupon theproductofthelowestcalibration standaradnalyzed(0.00229mg ai./L)andthedilutiofnitaoorfdw matrixblanksamples(50.0). 2Mean resulotf duplicatreedilutioonfstheoriginaslample.Originarlesult(12.0mg a.i./Lw)as not includedinthestatistiacnaallysisof thedata. Ralicateresultfsor454A-101-23are3.55and 3.61. AMENDED WILDLIFE INTERNATIONAL LTD. -31- PROJECRNO.: 454A-101 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN SALTWATER Preparematrixfortlflcatisoanmplesby spfldngthe requisitveolume of PFOS stocksolutiondsirectly mto filteresdaltwateursinggas-tlgbstyringesand ClassA volumetne flasks. Ddute matnx fortificaliaonnd testsamplesuto therangeofthecalibratisotnandardsby partiallfyilling ClassA volumeticflaskswith50% medianol:50% NANOpureg watersolutiocnontaining 0.loo mg 4H PFOS (fitbmmaslundard)/Land 0.05% fonnicacid(vtv).Add theappropriatveolume of sample and bringtheflasktovohum withthe dilutiosnolvent.Processthematrixblanksample using thesame dilutioannd aliquotvolume as forthelowestfortlficatiloenvel.Mix wellby severalrepeat inversions. Ampulate samplesand submitforLCMS analysis. Figure1. Analyticamlediod flowchartfortheanalysisof PFOS insakwatcr. WILDLIFE INTERNATIONAL LTD. -32- PROJECTNO.: 454A-101 2,50200'42 1.50 1.00 0 0 0 0 0.50- 0.00 0.000 0.010 0.020 0.030 0.040 Concentratim(mg ai/L) 0.050 Figure2. Atypicalcalibraticoanm forPFOS. Slope= 0.04044;Intercept0=.05941;r= 0.9930. Curveisweigbted(1/x). ANUMED WILDLIFE INTERNATIONAL LTo. -33- PROJECTNO.: 454A-101 100. 9(). 8 ()r 70so. 5 403020- Intensity3.215 cps 175 151 3,1 1.30 0 G*i 2.55 q'i II 3.81 &08 iii 6.32 iii 7.57 -213 211 8.83 @scan Ilme Figure3. A MP ve im chromatog= ofa low-lmcl(0.00229mg ai./L)PFOS standard. AMENDED WILDLIFE INTERNATIONAL -34- PROJECTNO.: 454A-101 intensity3-2743 cps 175 10& go. so7& 60" so. 40- 30- 151 20- 25 3*1 1.30 55 sa 6*1 2.55 106 127 9 05 2g2 lil lil Iii 181 2i I Scan 3.81 5.06 6.32 7.57 8.83 Time irigure4. A representativieonchromatogram ofa high-leve(l0.0457 mg a.iJL)PFOS standard. AMENDED WILDLIFE INTERNATIONAL LTD. -35- PROJECTNO.: 454A-101 132 100, 90. 29 so 70- 60 so. 40r 30 20, 10 h 31 1.30 69 82 99 rAA Cp@z@ 1%1--V. &(I 6,1 2.50 91 3.81 122-- 121 5.07 Intensity1:287 cps 173 159 194 59 151 6.32 I 181 7.58 i L4 211 8.83 scm Thns F'igure5. A representativcehromatogram of a matrixblanksample (454A-101-MAB-1). The arrow indicatetsheretentiofnm ofPFOS. WILDLIFE INTERNATIONAL -36- PROJECTNO.: 454A-101 10 9 8 70- 60- 50 4& 30 20, 28 L"63 53 1.30 2.65 intensit1f1.119 cps 173 150 105 94 105 .119 3.81 5.06 192 230 181 211 6.32 7.57 8.83 'thne Figure6. A repmentafivcehrmatw= ofa matrixfbrtificatsiaomnple(454A-101-MAS-1). WILDLIFE INTERNATIC)NAL L-rD. -37- PROJECTNO.: 454A-iol 100, 90, so 7(@ 60, so 40 30. 2010 16 34 A A^ 4 31 1.36 Intensity1-2998 cps 173 iso 7 91 112 4nn= 9"1 2.55 3.81 5.06 iii 6.32 lei 7.67 - -- - 211 8.83 scan Time rigure7. A representaticvheromatogmm of a testsample(454A-101-3). WILDLIFE INTERNATIONAL LTD. .39- PROJECTNO.: 454A-101 APPENDIX IV Chang= toProtocol Thisstudywas conductedinaccordancewiththeapprovedProtocowliththefollowmgchanges: 1 . The protocolwas amended toadd theproposedexpmimentalstaratnd terminatiodnates,testsubstance informatioannd testconcentrations. 2. The protocolwas amended toremove annualfoodanalysis. 3. The protocolwas amended tospecifytheanalyticamlediod tobe usect 4. The testchamberswere plastibcuckets. 5. pH was measureddagy inalltestchambers. WILDLIFE INTERNATIONAL LTD. -39- PROJECTNO.: 454A-101 APPENDIXV PersonnelInvolvedintheStudy The followmgkeyWildlifheftmtionalLtd.pmw=l weremvolvedm thecmdactormanag=ent ofthis study. I. Henry 0. Kmeger,PLD., DirectorA,quaticToxicologyand Non-TargetPlants 2. WillardB.Nixon,PLD., Manager,AnalyticaClhemistry 3. Mark A.Mank, LkmVory Supwvisor 4. TimothyZ.KM&IL LaboratorSyqm-viwr 5. KurtP, DrottarS,eniorBiologist 6. Raymond L.VanHoven, PILD.,Scientist WILDLIFE INTERNATIONAL LTD. -40- PROJECTNO.: 454A-101 APPENDDC VI, ReportAmendment I. OriginalReport: Pages 14, 6 and21 Amendment: The pageswere changedtoincludetheamended reportdate,revisepdagenumbers,and new signatureasnd datesdue totheadditioonfthereportamendment asAppendixVi. Reason: To reflecttheissuingof an amended report. 2. OriginalReport: Page 2 Amendment: The compliancestatemenwtas revisecl Reason: To clm* how thetestsubstancweas charactm=d. 3. OriginaRleport: Page 9 Amendment: Informatiopnrovidedby theSponsorreflectitnhgereanalysiosf thetestsubstance, mzludmg thereanalysidsateand thepunty,was addedtotheTestSubstancesection. Reason: To reflectthecurrenttestsubstancemfa=tion providedby theSponsor. 4. OnpW Report: Entirereport Amendment: Alltestsubstanceconcentratiownesre changedtoreflectthepuntyofthetestsubsta= asdeterminedby ffiScponsorina reanalysiosfthetestsubstanc(eFC-95,Lot217). Testconcentratioonrsiginalwleyre basedon thereportedpurityof 98.9*/o.The certificoaftaenalysidsatedMu& 9,2000indicateadpuritoyf90-49'/T*h.atfoM all' test substanceemeentrationsi,ncludingnominal concentrationmse,asured concentradonsa,nd LC50 values,wererecalculataendd reportedasmg aiJL basedon the90.49% pin*. Reisom- To reportheresultosf thetestbasedon thetestsubstancepurityof 90.49% atthe requestof theSponsor. 5. OriginalReport: Page 7 Amendment: ExperimentaTlerminatio-n March 12,1999 Reason: To reporttheexperuncntatlerminatiodnateforthestudy. AMENDED WILDLIFE INTERNATIONAL LTD. -41- PROJECTNO.: 454A-101 AMENDMENT SIGNATURES: KurtR.Drottar St* Director APPENDIX VI -Continued- ReportAmendment .,wllog6l6o DATE Henif i3.ckruige-rP,h.D. DirectorA,quaticToxicologyandNon-Targd Plants REVIEWED BY: a5@,@ 14- JaxW H. Coleman DATE QualityAssurance AMENDED WILDLIFE INTF-RNATIONAL LTD. PROJECT NO Page I of2 454A-101 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE SALTWATER MYSID (Mysidopsibsahia) PROTOCOL NO.: 454/110998/WS-96HI/SUB454 AAMNDMENT NO.: I SPONSOR: 3M Corporation PROJECT NO.: 454A-101' EFFECTIVE DATE: December 21, 1998 AMEENDMENT: Page 2 Add: Experimental StartDate: .3/8/99 ExperimentalTerminationDate: 3/12/99 Test Concentrations:Negative Control,1.2,1.9,3.2,5.4 and 9.0mg Test SubstanceNo.: 4675 a.i.AL REASON: The above informationwas not known when the protocolwas signed by the Study Director. AMEENDMEENT: Test Organis Page 5 Delete: Feed provided to the mysids in the cultureswillbe analyzed at leastonce annuallyto ensurethatthereare no contaminants atlevelsknown to be capable of interferinwgith the study. REASON: I-fistoricaanlalysesof WildlifeInternationaLltd. aquaticfeedhave shown thatno contaminants are present atlevelsknown to be capable of interferinwgith the study. AMENDAIENT: APPENDIX H, Page 13 Add: Liquid *Chromatography Mass Spectrometry (LCMS) Method for the Determination of PerfluorooctaneSulfonicAcid, Potassium Salt(PFOS) inFreshwater,FilteredSaltwaterand AlgalMedium. REASON: To add theanalyticamlethod to be used inthe study. a45411021amendl b@ &A- .3 4 WILDLIFE INTERNATIONAL LTD. STUDY DIRECTOR LABORAYOKY N4-AN-AGEMENT SPONSOR'S RFPRLPS ATIVE PROECT NO.: 454A-101 Page 2 of 2 DATE f IF D AfE DKTE a454llO2lantendl ILDLIFE INTERNATIONAL L-rD. PROJECTNO.: 454A-101 Page I of I DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR STATIC ACUTE T03aCITY TEST WITH THE SALTWATER MYSID (Mysidopsibsahia) PROTOCOL NO.: 454/110998/MYS-96HI/SUB454 DEVIATION NO.: I SPONSOR- 3M Corporation PROJECR NO.: 454A-101 DATE OF DEFAC-RO DEVIATION: March 8,1999 DEVIATION: The protocosltatetshatthetestchamberswillconsistofpolyethylenebottles.The testchambers actuallcyonsistedofplasticbuckets. REASON: BiologisotversightItis thebestjudgiuentof theStudyDirectotrhatthisdeviatiodnid not adverselyaffectheresultsofthestudy. STUDY DIRECTOR LABORATORY MANAGEMENT 45UIOI*dl DATE DATE WILDLIFF- INTF-RNATIONAL LTD. PROJECTNO.: 454A-101 Page I of I DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR STATIC ACUTE T03aCITY TEST WITH THE SALTWATER MYSID (Mysidopsibsahia) PROTOCOL NO.: 454/110998/MYS-96HI/SUB454 DEVIATION NO.: 2 SPONSOR: 3M Corporation PROJECRNO.: 454A-101 DATE OF DEFACRO DEVIATION: March 8,1999 DEVIATION: The protocolstatesthatmeasurementsof pH willbe measured in alternate replicatoefseachtw=ent andcontroglroup.Measurementsof pH were actually made inalltestchambers. REASON: BiologisotversightItis thebestjudgmentoftheStudyDirectotrhatthisdeviatiodnid not adverselyaffectheresultosf thestudy. STUDY DIRECTOR LABORATORY MANAGEMENT 454kl0l%prd2 DATE DATE PROTOCOL PFOS: A 96-HOUR STATIC ACUTE T03aCITY TEST WITH THE SALTWATER MYSID (Mysidopsisbahia) U.S.EnvironmentaPlrotectioAngency Series850 -EcologicalEffectsTestGuidelines OPPTS Number 850.1035 3M Lab RequestNo. U2723 Submittedto 3M Corporation EnvironmentalLaboratory P.O. Box 33331 St PauL Minnesota 55133 WILDLIFF- INTERNATIONAL LTD. 8598 Commerce I)rive Easton,Maryland 21601 (410)822-8600 November 9,1998 PROTOCOLNO.: 454/110998/MYS-96HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -2- PFOS: A 96-HOUR STATIC ACUTE T03GCITY TEST WITH THE SALTWATER MYSID (Mysidopsisbahia) SPONSO 3M Corporation EnvirorumentalLaboratory P.O. Box 33331 St Paul,Minnesota 55133 SPONSOR!S REPRESENTATIVE: MS. Susan A. Beach TESTING FACIL WildlifeInternationLatld. 8598 Commerce Drive Easton,Maryland 21601 STUDY DIRECTO Kurt Drottar SeniorAquaticBiologist LABORATORY MANAGEMENT Henry 0. Kmeger, Ph.D. Directorof AquaticToxicology& Non-TargetPlants FOR LABORATORY USE ONLY Proposed Dates: Experimental StartDate: ProjectNo.: q5q A -/0/ Experimental TerminationDate: Test Concentrations: Test Substance No.: ReferenceSubstanceNo. (ifapplicable): PROTOCOL APPROVAL STUDY DIRECTOR DATE AGENIENT DAT9 SPONSOR!S REPRESENTATIVE BDA4 PROTOCOL NO.: 454/110998/MYS-96HI/SUB454 3M LAB REQUEST NO. U2723 /VILDLIFE INTERNATIONAL LTD. -3- INTRODUCRION WildlifIenternatioLntadl.willconducta statiaccutetoxiciteystwiththesaltwatemrysid (Mysidopsibsahia)fortheSponsoratdieWildlifIenternatioLntadl.aquatitcoxicologfyaciliitny Easton, Maryland. The studywillbe performed based on the guidelinepsrovidedin the U.S. EnvironmentalProtectionAgency Series850 - EcologicalEffectsTest GuidelinesOPPTS Number 850.1035 (1);U.S.EnviromnentalProtectioAngency, StandardEvaluationProcedure,Acute Toxicity Testfor Estuarineandmarine Organisms (2);and AS7M Standard E729-88a Standard Guidefor ConductingAcute ToodcitTyestswithFishes,Macroinvertebratesandamphibians (3).Raw datafor allwork performed atWildlifeInternationLatld.and a copy ofthefinalreportwillbe filedby project number in archiveslocatedon theWildlifeInternationLatld.siteo,ratan alternativleocationto be specifiedinthe finalreporl PURPOSE The purpose ofthisstudyistodeterminetheacuteeffectosf a testsubstanceon thesaltwater mysid (Mysidopsisbahia)duringa 96-hourexposureperiodunder statitcestconditions. EXPERIMENTAL DESIGN Saltwatermysids willbe exposed toa geometricseriesof atleastfivetestconcentrationasnd a negative(dilutiownater)controlfor96 hours.Two replicatteestchambers willbe mmntained ineach treatmmt and controlgroup,with 10 mysids m eachchamber fora totalof 20 mysids per testconcentra- tion. NomilW testconcentrationwsillbe selectedinconsultatiownith theSponsor,and Vnu be based upon theresultsof exploratoryrange-findintgoxicitydata,known toxicitydata,physical/chemical propertiesof the testsubstanceor otherrelevantinformatiorlTargetconcentrationnseed not exceed 1000 mgtl -orthesolubilitlyimitofthetestsubstanceinwater(whicheverislower).Generally,each testsubstanceconcentratiounsed in the definitivteestwillbe at least60% of the next higher concentratiounnlessinformationconcerningtheconcentration-effeccutrveindicatetshata diffm-ent dilutiofnactorwould be more appropriate.Water samples from each testchamber willbe collectedat specifieidntervalfsoranalysisof the testsubstance.Resultsof analyseswillbe used to calculatemean measured testconcentrations. PROTOCOLNO.: 454/110998/MYS-96HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -4- To controlbias,juvenilenrysidswillbe impartiallayssignedto exposurechambers at test initiatioNrol otherpotmtialsourcesof bias areexpectedto affecttheresultsof the study.Each group of 10 willthenbe transferretdothetestchamber. Observationsof mortalityand otherclinicaslignswill be made throughoutthe96-hourtestperiod.Cumulativepercentmortalitoybservedinthetreatment groupswillbe used to calculatew,hen possible,LC50 valuesat24, 48, 72 and 96 hour intervals.The no-mortaiityconcentratioannd theno-observed-effecctoncentratio(nNOEC) willbe determined. MATERIALS AND METHODS Test Substance Informationon thecharacterizationf test,controlor referencesubstancesisrequiredby Good LaboratoryPracticSetandards(GLP). The Sponsor isresponsiblfeorprovidingWildlifeInternational Ltd.wntten venficatlotnhatthetestsubstancehas been charactenzedaccordmg to GLPs pnor to itsuse inthe study.Ifwrittenverificatiofn GLP testsubstancecharacterizatiiosnnotprovidedtoWildlife InternationaLltd.,itwillbe noted inthecompliancestatementofthefmal report.The attachedform IDENTMCATION OF TEST SUBSTANCE informationnecessaryforGLP compliance. BY SPONSOR (AppendixI)istobe used toprovide The Sponsorisresponsibleforallinformationrelatedto thetestsubstanceand agreesto accept -anyunused testsubstanceand/ortestsubstancecontainersremainingatthe end of the study. P=aration ofTestConcentrations The testsubstw= w& administeretdothetestorgmm directlmyto dilutiownater Tins route of administratiownas selectedbecause itrepresentsthe most likelyrouteof exposure to aquatic organisms. Test Organism The saltwatermysid (Mysidopsisbahia) has been selectedas the testspeciesforthissU*. Mysi&q arerepresentatiovfean finportangtroup of aquaticmarine invertebratesa,nd have been selected foruse inthetestbasedupon pastuse historyand ease of culturinginthe laboratory.Mysids to be used in the testwillbe lessthan24 hours old and willbe obtainedfrom culturemsaintainedat Wildlife PROTOCOLNO.: 454/110998/MYS-96HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. InternationLatld.,Easton,Maryland. The identitoyf thespecieswillbe verifiebdy thesupplierofthe originalcultureor by WildlifeInternationaLltcLpersonnelusingappropriatteaxonomic keys such as Price(4). Mysids willbe cultureidnwaterfrom thesame sourceand atapproximatelythe same temperature as willbe used duringthetest.Mysids thatshow signsof diseaseor stresswillnot be used as test organisms. Mysids inthecultureasnd duringtestinvgnU be fedatleastonce daily.The dietwillbe livebrine shrimpnauplii(Artemia sp.).Feeding duringculturingmay be supplementedwith saltwateralgaeand rotifersas needed. Feed providedto mysids inthe cultureswillbe analyzedat leastonce annuallyto ensure thatthereareno contaminantsatlevelsknown to be capableof interferinwgith the study. Specificatiofnosracceptablelevelsof contaminantsinmysid chetshave not been established.However, thereare no known levelsof contaminantsreasonablyexpectedto be presentin the dietthatare consideredto interferweith the purpose or conduct of thetest. DilutionWater NaturalseawatercollecteadtInd*anRiverInle@Delawarewillbe thesourceofthechlutiownater used in the culturefacilitaynd testsystem. The seawaterwillbe futm-edthrough a sand filteprriorto itsdelivertyo a 37,800-Lholdingtank The salinitoyf theseawaterwillbe dilutedto approximately20 0/. (partsper thousand)with WildlifeInternationLatld.wellwaterintheholdingtank- The 20 1. saltwaterwillbe aeratedusing spraynozzlesand filtm-e(d0.45idm) priorto itsdeliveryto the test system- Sdimty and pH willbe niewured ww* to monitor theconsistencyof thesaltwater.Means and ranges of themeasured parameters forthe four-week periodprecedingthe testwillbe provided inthe finalreport. Analyseswillbe performed atleastonce annuallyto determinetheconcentrationosf selectedorganic and inorganicconstituentosf the seawaterand resultsof the analyseswillbe summarized inthe finalreport. PROTOCOL NO.: 454/110998/MYS-96Hl/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -6- Test Apl2aratus Test chambers willconsistof polyethylenbeottles.The sizeof bottleand thevolume of test solutionwillbe sufficientto meet the requirementsof samplingforanalyticavlerificationf test substanceconca&ationsand routinewater chemistryanalyses.Testchambers willbe indiscriminately positioneidna tmnpmmhn-controlled waterbath or an environmentalchamber. Test chambers willbe labelledwiththe projectnumber, testconcentrationand replicate. EnvironmentalConditions Lightingused to illuminatteheculturesand testchambers duringholdingand testingwillbe providedby fluorescenttubesthatemitwavelengthssimilartonaturalsunlight(e.g.C,olortone4D50). A photopcriodof16 hoursoflightand 8hoursofdarkwillbe controllewdithan automaticfimr. A 30rnimfttransitipoenriodoflow lightii@ willbe providedwhen lightsgo on and offto avoidsudden changesinlightintmmity.Lightintensitwyillbe measured attestinitiatiownith a SPER ScientifiLctcL lightmeteror equivalent The targetesttemperaturewillbe 25:k 2*C. Temperat=wiR be measured to each testchamber atthe beghuiing and end of thetestusing a liquid-in-glatshsermometer.Temperature alsowillbe measured witha continuousrecorderinone negativecontrolreplicateR.ecordermeasurements willbe verifiedwith a hquid-in-@'astshermometer priorto testinitiation. Dissolvedoxygen willbe measured in allreplicateosf the treatmentand controlgroups attest initiatioannd atapproximately24-hourintervaltshereafteursinga Yellow SpringsInstrumentModel 5 IB dissolvedoxygen meter,orequivalent Intheeventthatdissolvedoxygen levelsfallbelow 60% saturatioanp,propriataectionswillbe tdm afterconsultatiowniththeSponsor. Measurements ofpH will be made in alternatreeplicateosf each treatmentand controlgroup at testinitiatioannd at approximately24-hourintervaltshereafteursing a FisherAccumet Model 915 pH meter,or equivalent. Ifa treatmentgroup reachesI001/omofti*, dissolvedoxygen,pH, and temperaturemeasurements will be takm atthenextsamplingint=al thendisc@ed. Dilutionwatersalinitwyillbe measured attest initiatiuosninga Bio-Marine,Inc.Aquafauna refractometero,requivalent. PROTOCOLNO.: 454/110998/MYS-96HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -7- BiologicalMeasurements Observationsof mortalityand clinicaslignsof toxicitwyillbe made between 0-24 hours,and at 24, 48, 72 and 96 hours 1 hour. The criterifaordeathincludelack of movement, absence of respiratorymovements, and lackofreactiontogentleprodding.Sublethaleffectss,uch as abnormal locomotion,willalsobe noted. Dead organismswillbe removed from the testchambers ateach observationtime. S=Iing forAnglyficaMleasurements Water samples willbe collectedfrom each testchamber atthebeginningof thetest,duringthe testand attheend ofthetestto determineconcentrationsof thetestsubstance.In the eventthat I000/o mortalityoccursinany treatment,then samplingof thattreatmentwillterminatefollowingthenext samplingintervalS.amples willbe collecteadtmid-dcpthfrom each testchamber, analyzedimmediately or placed inan appropriatestoragecontainer(e.g.p,olyethylenoer polypropylenebottle)and stored under refrigeratiountilanalyzed.The sample scheme issumnuwized below: PROPOSED NUMBERS ExperimentalGroup Control SolventControl(ifneeded) Level 1-Low Concentration Level 2 Level 3 Level 4 Level5-High Concentration Totals OF VEFJIFICATION SANTLES 0 Hour 48 Hours 96 Hours 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 14 14 14 TotalNumber ofVerificatioSnamples 42 The above numbers of samplesrepresentthosecollectedfrom the testand do not includequality control(QC) samples such as matrixblanksand fortificatiopnrseparedand analyzedduringthe analyticaclhemistryphase of the study. PROTOCOL NO.: 454/110998/MYS-96Hl/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -8- Angl3iicalChen-fist(y Chemicalmialys@of thesampleswillbe performedby WildlifeInternationaLltd. The analytical method used willbe based upon methodology providedby the Sponsor and identifieidn Appendix II. The methodologyused to analyzethe testsamples willbe documented in theraw data and summarized in thefinalreport. DataAn&sis When thedose-responsepatternallowscalculatioonf an LC50 value,thedatawillbe analyzed usingthecomputersoftwareof C.E.Stephan(5).The program was designedto calculattehe LC50 value and the95% confidenceintervably probitanalysist,hemoving averagemethod, or binomialprobability withnonlin= kdapolation(6,7,8).The LC50 valuewillbe calculatedw,hen possibleu,singmortality data collectedat 24, 48, 72 and 96 hours. The no-mortalityconcentratioannd the no-observed-effect concentratio(nNOEC) willbe determinedby visuallyinterpretintgheclinicaolbservationdata. RECORDS TO BE MAINTAINED Recordstobe mmntamed fordatageneratedby WildlifeinternationLatld.willinclude,but not be limitedto: I. A copy of thesignedprotocol. 2. Identificatiaond characterizationf the testsubstance,ifprovidedby the Sponsor. 3. Dates ofinitiatiaond terminationofthetest 4. Mysid historyand culturerecords. 5. Stock solutioncalculationand preparationi,fapplicable. 6. Observations. 7. Water chemistryresult(se.g.d,issolvedoxygen). 8. The methods used to analyzetestsubstanceconcentrationasnd the resultsof analytical measurements. 9. Statisticcaallculationisf,applicable. 10. TestconditionsOightintensityp,hotoperiod,etc.). 11. Calculationand preparationof testconcentrations. 12. Copy offinalreport. PROTOCOLNO.: 454/110998/MYS-96Hl/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -9- FINAL PORT A finarleportoftheresultosfthest* willbe preparedby WilcUifeInternationaLltd. The report willinclude,butnotbe limitedto thefollowingw,hen applicable: 1 . Name and addressof the facilitpyerfomiingthestudy. 2. Datesupon which thestudywas initiataendd completed,and the definitiveexperimentalstartand terminationdates. 3. A statementof compliancesignedby the Study Directoraddressingany exceptionsto Good LaboratoryPracticeStandards. 4. Objectives and procedures,as statedin the approved protocol,includingallchanges to the protocol. 5. The testsubstanceidentificatiioncludingname, chemicalabstractnumber or code number, strengthp,urity,composition,and otherinformationprovidedby the Sponsor. 6. Stabilitaynd solubilitoyf thetestsubstanceundertheconditionosf administrationi,fprovided by the Sponsor. 7. A descriptioonf the methods used to conductthetest. 8. A descriptionof the testorganism,includingthesource,scientifincame, age or lifestage,feed types,fightintensitaynd photoperiocl 9. A descriptioonf thepreparationof thetestsolutions. 10. The methods used to allocateorganisms to testchambers and begin thetest,the number of orgmusms and chambers pertreab=@ and ft durationofthetest. 11. A descriptioonf circumstancesthatmay have affectedthe qualityor integritoyf the data. 12. The n oftheSU* Dkectorand thenames of otherscientistpsr,ofessionalsa,nd supervisory personnelinvolvedinthe study. 13. A descriptionof the transformationsc,alculationasn,d operationsperformed on the data,a summary and analysiosf thebiologlcadlataand analyticaclhenustrydata,and a statementof the conclusionsdrawn from the analyses. 14. Statisticmaelthods used to evaluatethedata. 15. The signedand datedreportsof each of theindividualscientistosr otherprofessionalsinvolved in the study,ifapplicable. 16. The locationwhere raw data and finalreportareto be stored. IPROTOCOLNO.: 454/110998/MYS-96HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL L-rD. -10- 17. A statementpreparedby theQualityAssurance Unit listintghe datesthatstudyinspectionsand auditswere made and thedatesof any findingsreportedto the Study Directorand Managmnent. 18. ffitisnecessarytomake correctionsor additionsto a finalreportafterithas been accepted,such changeswillbe made intheform of an amendment issuedby theStudy Director.The amendment willclearlyiden* thepartofthe finalreportthatisbeing amended and the reasonsforthe amendment, and willbe signedby the Study Director. CHANGING OF PROTOCOL Plannedchanges to theprotocolwillbe in theform of writtenamendments signedby the Study Directorand theSponsces Representative.Amendments willbe consideredas partof theprotocoland willbe attachedtothefinalprotocolAny otherchanges willbe in theform of writtendeviationssigned by theStudy Directorand filedwiththeraw data.AU changestotheprotocolwillbe hichcatedinthe finalreport. GOOD LABORATORY PRACNC]ES This studywillbe conductedinaccordancewithGood LaboratoryPracticeStandardsforEPA (40 CFR Part160 and/orPart792);OECD PrincipleosfGood LaboratoryPractic(eOCDE/GD (92) 32, Environment Monograph No. 45); and Japan MAFF (59 NohSan, NotificatioNno. 3850, AgriculturaPlroductionBureau). Each studyconductedby WildlifeInternationaLltd.isroutinely examined by the WildlifeInt=&onal Ltd.QualityAssuranceUnit forcompliancewith Good Laboratory PracticesS,tandardOperating Proceduresand the specifiedprotocol. A statementof compliancewith Good LaboratoryPracticeswillbe preparedforallportionsof the studyconducted by WildlifeInternationaLltd. The Sponsor willbe responsibleforcompliancewith Good Laboratory Practicefsorproceduresperformedby otherlaboratorie(se.g.r,esidueanalysesor pathology).Raw data forallwork perfomicdatVtrildhfIenternationLatlcLand a copy of the finalreportwillbe filedby project number in archiveslocatedon theWildlifeInternationLatld.siteo,r atan alternativleocationtobe specifiedin thefinalreport. PROTOCOLNO.: 454/110998/MYS-96HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. REFERENCES I U.S.Environmental ProtectionAgency. 1996. Series850-EcologicaElffectsTest Guidelines (draft)O,PPTS Nwnber 850.1035: MysidAcute ToxicityTest. 2 U.S. Environmental ProtectionAgency. 1985. Standard Evaluation Procedure, Acute ToxicityTestfor Estuarineand Marine Organisms (Shrimp 96-Hour Acute ToxicityTest). Hazard EvaluationDivision.OfficeofPesticidPerograms. EPA-540/9-85-010. Washington, DC. 3 ASTM Standard E729-88a 1994. Standard Guidefor Conducting Acute Toxicity Tests with Fishes,Macroinvertehrates, andamphibians. Ammican Society for Testing and Materials. 4 Price,W.W. 1982. Key to the Shallow Water Mysidacea of the Texas Coast withNotes on theirEcology. Hydrobiologia 93, 9-21. Pr.W Juk Publishers,The Hague. Printedin The Netherlands. 5 Stephan,C.E. US.EPA. 1978. Environmental Research Laboratory,Duluth, Minnesota. Personalcommunicatiorl 6 Finney,D.J. 1971. StatisticalMethods in Biological Assay. Secondeditiom GriffmPress, London. 7 Thompson, W.R. 1947. BacteriologicalReidews. Vol. ]I,No. 2. Pp. 115-145. 8 Stephan,C.E. 1977. "Methods for Calculating an LC50," Aquatic Toxicology and Hazard Evaluations. American Somety for Testmg and Nlatmials. Pubhcafaon Number STP 634, pp 6584. PROTOCOL NO.: 454/110998/MYS-96HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -12- APPENDDC I IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR To be Completed by Sponsor I. Test SubstanceIdentit(yname to be used inthereport):PFOS (PerfluorooctanSeulfonicAcid Potassium Sali ReferenceStandard(ifapplicable)A:nalyticaSltandard: N/A InternaSltandard: 1-1-2.2H.H.H-HPerfluorooctaSnuelfonicAcid Test SubstanceSample Code or Batch Number: Lot 217 Test SubstancePurity(0/AoctiveIngredient): 98.9 ExpirationDate: 2008 11. Test SubstanceCharacterization Have theidentitsyt,V e gemt'tyand compositionor othercharacteristics which appropriateldye e etestsubstanceand referencestandardbeen determifiipdriorto itsuse in thisstudyin accordancewith GLP Standards? Yes Ill. Test Substance StorageConditions Pleaseindicatetherecommended storageconditionsat WildlifeInternationaLltd. No X Ambient room =Uerature Has thestabilitoyf the testsubstanceunder thesestorageconditions been determinedin accordancewith GLP Standards? Yes No X Other pmtinentstabilitiynformation: IV. Test Concentrations: Adjusttestconcentratiotno 100% ai. X baied Wm thepurity(0/,g-i)venabove. Do not adjusttestconcentratiotno 100% - a.i.Iestft material V. ToxicityInformatiow. Mammalian: Aquatic: Rat LD50 251 mg&g Mouse LD50 InvertebratTeoxicity(EC/LC50) N/A FishToxicity(LC50) DV]iWa ma=: 27 mz'L - RainbowTrout:11mgl DApMa mama: 50me/L - F&dnd Minnown 38 mgl Other ToxicityInformation(includinfgmdings ofchronicand subehronictests): Pleasesee MSDS PROTOCOLNO.: 454/110998/MYS-96Hl/SUB454 3M LAB REQUEST NO. U2723 WILDLIF*E INTERNATIONAL LTD. -13- APPENDIX ]I AnalyticalMethod to be Providedby Sponsor PROTOCOLNO.: 454/110998/MYS-96Hl/SUB454 3M LAB REQUEST NO. U2723 ACUTE TOXICITY TO THE SALTWATER MYSID TEST SUBSTANCE ldent4: Perfluorooctanesulfonamtaey; alsobe referretdoas PFOS or FC-95. (1-Octanesulfoniacid,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-p,otassiumsaltC,AS # 2795-39-3) Remarks: The testsubstanceisa whitepowder. Sample was taken from 3M lotnumber 217. Sample was storedunder ambientconditions priortotestingP.uritydeterminedto be 90.49% by LC/MS, 'H-HMR, 'gFNMR and elementalanalysestechniques. METHOD Method: OPPTS 850.1035 Type: Staticacute GLP: Yes Year completed:Study completed1999. Reportcompleted2000 Species:Mysidopsisbahia Supplier:In-houseculturesW,ildlifIenternationLatld,.E,aston,MD Analyticalmonitoring:PFOS measured at0,48,96-hours Exposure period:96-hours Statisticamlethods: LC5ovaluescalculatewdh,en possibleb,y probit analysis,moving average method or binomialprobabiliwtiythnon-linear interpolatiuosningthecomputer softwareofC.E.Stephan. Test fishage: < 24-hoursold Pretreatment:None Test conditions: Dilutionwater: Naturalseawaterdilutetdo200/0w0ithwellwater, 0.45gm filtered. Dilutionwater chemistry (duringthe4-week period Immediately preceding thetest): Salinity: 20 (20-20)Oloo pH: 8.2(8.1-8.2) TOC: < 1.0mg/L Stock and testsolutionpreparation:Primarystockpreparedat 8.2mg/L and mixed for-22 hours priotro use. Aftermixing, primarystocksolutionwas proportionaldliylutewdithdilutiownater topreparethe fouradditionatlestconcentrationsA.lltestsolutions appeared clearand colorless. Concentrations dosing rate:Once Stabilitoyf the testchemical solutions:Extremelystable Exposure vessels:2L polyethylenaequariacontaining approximately1OOOML oftestsolutionw;aterdepthapproximately 6.6 cm. Number ofreplicatest:wo Number ofmysids per replicatet:en Number ofconcentrationsf:iveplusa negativecontrol Feeding:Livebrineshrimpnauplidiaily Water chemistryduringthe study: Dissolvedoxygen range(0- 96 hours): 6.8- 7.4mg/L (controelxposure) 6.8- 7.3mg/L (5.4mg/L exposure) pH range(0- 96 hours) 8.1- 8.2 (controelxposure) 8.1- 8.2 (5.4mg/L exposure) Testtemperaturerange (0- 96 hours) 24.2- 25.40C (controelxposure) 23.8- 24.50C (5.4mg/L exposure) Method ofcalculatinmgean measured concentrations: arithmetimcean RESULTS Nominal concentrations:Bk control1,.1,1.8,3.0,4.9,8.2mg/L Measured concentrations:<LOQ, 0.57,1.1,1.9,3.0,5.4mg/L Element value:24-hourLCso= > 5.4mg/L (Clnotcalculable) 48-hourLCw = > 5.4mg/L C.I.notcalculable) 72-hourLC5o = 4.4(3.6-6.2m)g/L 9"our LCro= 3.6(3.0-4.6m)g/L Allelement valuesbased on'mean measured concentrations StatisticaElvaluationof Mortality:LC5ovaluescouldnotbe calculated for24 and 48-hoursofexposuredue tothelackan an adequate concentration-responspeattern.The probitmethod was used to evaluate mortalitayt72 and 96 hours. AnalyticalMethodology:Analysesoftestsolutionwsere performedat WildlifIenternationLatld.usinghighperformanceliquicdhromatography withmass spectrometridcetection(HPLC/MS). When determiningthe concentratioonfthetestsubstanceinthetestsolutionst,hesame and most prominentpeak responseforperfluorooetanseulfonatewas used. No attemptwas made toquantifyon thebasisofindividuailsomeric components. The LOQ (limiotfquantitatiowna)s 0.115mg/L inthisstudy. The mean percentrecoveryofmatrixfortificatiaonnaslyzedconcurrently duringsample analysiswas 97.4.Samples collecteadttestinitiatihoand measured valuesfrom 52.4 to70.7% ofnominal. Measured valuesfor samples taken at48-hours ranged from 43.5 to71.0% ofnominal. Measured valuesforsamples taken at 96-hoursranged from 35.5 to 7 1.1% of nominal. Summary of analyticalchemistry data: Nominal Test Concentration, mg/L Measured DuplicatbValues at0, 48, and 9Shoum, Respectively,mgfl Mean Measured Percentof Concentration, Nominal mgA. Negative Control AD -cLOO <LOQ - . i.1 0.575,0.62Z0.606,0.640,0391, 0.57 52 0.58D 1.8 1.12,1.19,1.10,1.09,1.04,1.13 1.1 61 3.0 1.92,1.99,I.gZ 1.91,1.79,1.91 1.9 63 4.9 3.05.2.66,2.96.3.36,3.11,3.11 3.0 1 61 8.2 5.82,6.78,3.68, 5.85,5.22,5.86 5A 66 Biologicalobservations after96-hours: Mysids inthe negativecontrol, and the 0.57 and 1.1mg/L (mean measured concentrationst)reatment groups appeared normal and healthyduringthetest. Cumulative percent mortality: Mean Measured Test Cone., mg/L 244%ours 48-hours 724ioum WAWUM Neg. Control 0 0 0 0 1 0.67 0 0 0 0 1.1 0 0 0 0 1.9 1 0 0 5 10 3.0 5 Is 30 40 5.4 Is 45 60 75 Mortal4 ofcontrols:None CONCLUSIONS The potassium perfluorooctanesulfona9t6e-hour LCsoforsaltwatermysids was determinedtobe 3.6mg/L witha 95% confidenceintervaolf 3.0- 4.6 mg/L. The 96-hourno mortalitaynd NOEC concentratiownas 1.1mg/L. Submifter:3M Company, EnvironmentalLaboratoryP,.O. Box 33331,-St. Paul,Minnesota,55133 DATA QUALITY ReliabilityK:Rinischranking= I REFERENCES Thisstudywas conductedatWildlifIenternationLatld.,Easton,MD atthe requestofthe3M Company. OTHER Last changed: 5/3/00