Document 3NM52Z336Mv1drp6p1jopyd5D

PFOS: A Flow-throu SaltwaterMysid (My en, CHRONIC TOXICITY TO THE SALTWATER MYSID TEST SUBSTANCE Identity:Perfluorooctanesulfonatmea;y alsobe referredtoas 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 saltC,AS # 2795-39-3) Remarks: The testsubstance isa whitepowder. Sample was taken from 3M lotnumber 217. Sample was storedunder ambient conditions priortotestingP.uritydeterminedto be 90.49% by LC/MS, 'H-HMR, 'gFNMR and elementalanalyses techniques. METHOD Method: OPPTS 850.1350 Type: Flow-throughchronic GLP: Yes Year completed: Study completed 1999. Report completed 2000 Species: Mysidopsisbahia Supplier:In-houseculturesW,ildlifIenternationaLlt,d.,Easton,MD Analyticalmonitoring:PFOS measured on days 0, 7,14,21, 28,and 35 Exposure period:35 days Statisticamlethods: Survivaldatawas evaluated(priotropairingand afterpairingu)sing2 X 2 contingencytablesto identiftyreatmentgroups thatshowed a statisticalsliygnificandtifference(p@@0.05f)rom the negative controlgroup. Allpontinuous-variabldeata (reproductioannd growth) were evaluatedfornormalityusingShapiro-Wilk'tsestand for homogeneity ofvarianceusing Bartlettt'esst.Analysisofvarianceand Dunnett'stestwere used toevaluatedifferencebsetween treatmentand controlmeans. Allstatistictaelstswere performed usinga personal computer withSPSS/PC Version2.0 or"TOXSTAT Release 3.5"statistical software. Test mysids age: < 24-hoursoldattestinitiation Pretreatment: None Test conditions:Naturalseawaterdilutetdo200/0w0ithwellwater, 0.45@im filtered. Dilutionwater chemistry (duringthe 4-week period immediatelypreceding thetest): Salinity: 20 (20-20)O/oo TOC: < 1.0mg/L Stockand testsolutiopnreparationP:rimarsytockprepareadt0.0895 mg/L and mixed forapproximately24 hours priorto use. Aftermixing,the primarystocksolutionwas proportionalldyilutedwithdilutiownater to preparefiveadditionasltocksolutionsat concentrationsof0.0447, 0.0224, 0.0112,0.00559,and 0.00280 mg/L. The sixstockswere injectedintothe dilutemrixingchambers (ata rateof4.60 muminute) where they were mixed withdilutiownater(ata rateof 150 muminute) to achievethe desiredtestconcentrations. Flow through rate:Approximatelyeleven volume additionsoftest water every24- hours Stabilitoyf the testchemical solutions:Extremelystable Exposure vessels: Priorto pairingm,ysids placed inglass beakers withnylon mesh screen attachedtotwo holeson opposite sides.Afterreachingsexualmaturityp,airsplacedinglasspetri disheswithsidesofnylon mesh screen attachedwithsilicone adhesive.Both pre-paidngand post-paidngexposure vesselswere placed in9L glassaquariafillewdithapproximately5 L oftest solution.The depth was approximately6.2 cm priortopairingand 5.5 cm afterpairing. The testchambers forthe second generationexposure were 2L beakers with1L oftestsolutionwhich was dipped outofa test chamber from the appropriatetreatmentgroup. Number of replicates:four Number of concentrations:sixplusa negativecontrol Number of fishper replicate:Fifteenjuvenilesbeforepaidng,5 pairs(10adults)when possibleafterpairing. Feeding:Fed livebrineshrimp naup[ii3 orfourtimes per day. Not fed the lastday ofthe test. Water chemistry during the study: Dissolved oxygen range (0- 35 days): 6.0- 6.4 mg/L (controelxposure) 5.9- 6.3mg/L (1.3mg/L exposure) pH range (0 - 35 days) 8.2- 8.4 (controelxposure) 8.3- 8.4 (1.3mg/L exposure) Test temperature range (0- 35 days) 24.5- 25.20C (controelxposure) 24.4 - 25.1OC (1.3mg/L exposure) Method of calculatingmean measured concentrations:arithmetic mean RESULTS Nominal concentrations:Bk control0,.086,0.17,0.34,0.69,1.4,2.7 mg/L Measured concentrations: <LOQ, 0.057,0.12,0.25,0.55,1.3,2.6 mg/L Element value: 20-day survival(pre-pairinNgO)EC 0.55 mg/L 35-day (post-paidngs)urvivalNOEC 0.55 mg/L 35-day reproductionNOEC = 0.25 mg/L 35-day growth NOEC = 0.25 mg/L 35-day reprod& growth LOEC = 0.55 mg/L 2 nd generationacute survivalNOEC = 0.85 mg/L (highest concentratiotnested) Allelement valuesbased on mean measured concentrations AnalyticalMethodology: Analyses oftestsolutionswere performed at WildlifIenternationaLltd.using high performance liquidchromatography withmass spectrometridcetection(HPLC/MS). When determiningthe concentrationofthetestsubstance inthetestsolutionst,he same and most prominentpeak response forperfluorooctanesulfonawtaes used. No attemptwas made to quantifyon the basisof individuailsomeric components. The LOQ (limiotfquantitatiowna)s 0.0.0458mg/L inthis study. The mean percentrecoveryof matrixfortificatiaonnaslyzed concurrentlyduringsample analysiswas 92.8.Samples collectedat pretestranged from 57.4 to99.3% of nominal. Samples attestinitiatihoand measured valuesfrom 67.1 to 103% ofnominal.Measured values for samples taken attestterminationranged from 59.8 to 90.0% of nominal. Summary ofanalyticcahlemistrydata: NominalTest Concentration, mg/L Measured DuplicateValues at 0,7, 14, 21, 28, 35, Days, Respectively,mg/L Mean Measured Concentration, mg/L Percentof Nominal Negative Control All< LOQ - - 0.086 0.0694, 0.0578,0.0478, 0.0619, 0.0606, 0.0614, 0.057 66 0.0554, 0.0509,0.0515, 0.0569,0.0580, 0.0514 0.17 0.125, 0.114, 0.0778, 0.125, 0.124, 0.127, 0.0970, 0.12 71 0.112,0.122, 0.128, 0.124, 0.119 0.34 0.289. 0.286,0.231, 0.197,0.276-10.253, 0.227, 0.25 74 0.212, 0.262,0.271, 0.278, 0.251 0.69 0.562,0.659,0.581.0.450,O.M, 0.542,0.516, 0.55 80 0.528, 0.529, 0.544, 0.556, 0.583 1.4 1.23,1.32,1.13,1.20,1.35,1,27,1.23,1.15.1.39, 1.3 93 1.39,1.26,1.20 2.7 2.56, 2.79,2.58,2.30, 2.54, 2.69, allmysids dead 2.6 96 1after14-days exposure Biologicalobservations Survival:Allsurvivinmgysids appeared normal. Survivalinthe 1.3 and 2.6mg@L treatmentswere statisticalsliygnificantdliyfferent from thenegativecontrolgroup. Reproduction: The day offirsbtrood releaseinthisstudywas Day 22. Dunnett'stestshowed thatreproductiorwias significantly reduced inthe 0.55 mg/L treatmentgroup when compared tothe negativecontrol(p @@_0.05)T.he 1.3and 2.6 mg/L treatmentgroups were notincludedinthe statisticaanlalysisofthe reproductiondata due toa statisticaslilgynificandtifferenceinsurvival. Growth: Mysids exposed to PFOS atconcentrations< 0.25 mg/L showed no statisticaslilgynificanrteductionsinlengthor dryweight (p < 0.05). Second Generation Acute Exposure: SurvivalinallPFOS treatmentgroups was > 95% and was notstatisticadlilfyferenftrom the controls.Allsurvivingmysids inthe second generation exposure appeared normal withno overtsignsoftoxicity. Percent Survival Juvenile Adult Mean Measured Pro-PairingPost-Pairing Concentration, Survival. Survival, mg/L Day 20 Day 35 Negabve Control 78 92 0.057 -92 96 0.12 76 90 0.25 82 97 0.65 83 95 1.3 32 57 2.6 0 Second Generation Survival Mean Measured Concentrations,mg/L Total Number Exposed Number Aliveafter 96-hours Percent Survival Negative Control 71 68 96 0.057 65 63 97 0.12 0.25 83 79 95 62 59 95 0.55 13 13 100 Adult Mysid Growth Mean Measured Concentration,mg/L Number of Surviving Mysids/Number Exposed Total Length, Mean + SD, mm Dry Weight, Mean + SD, mg Negative Control 36139 6.43+ 0.0634 0.634 + 0.0510 0.057 44/46 6.43+ 0.599 + 0.0276 0.07:279 0.12 0.25 36/40 36137 6.56+0.105 0.641 + 0.0241, 6.40+ 0.0548 0.622 + 0.0227 0.55 35/37 6.14+ 0.079@4- 0.562+ 0.006f4 1.3 8/14 5.85 +0.178 0.436 + 0.0441 Reproduction Mean Measured Concentration,mg/L Replicate Number of ReproductiveDays Negative Control A 70 B 53 c 70 D 42 0.057 A 60 B 70 c 70 D 56 0.12 A 70 B 46 c 64 D 70 0.25 A 70 B 56 c 61 D 56 0.55 A 54 B 56 c 70 D 56 1.3 A 22 B 14 c 0 D Number of Yourmj Mean Number of Young/Reproductive Day Overall Mean + SD 18 0.257 0.315+ 0.092@ 14 0.264 20 0.286 19 0.452 17 0.283 0.261+ 0.087@ 14 0.200 13 0.186 21 0.375 21 0.300 0.361 + 0.101 22 0.478 22 0.407 18 0.267 19 0.271 0.252+ 0.072:T 12 0.214 21 0.344 10 0.179 3 0.0556 0.0559+ 0.0376F 6 0.107 3 0.0429 1 0.0179 0 - 0 0 0 CONCLUSIONS There were no statisticaslilgynificanetffectson survivalr,eproductionor growth ofmysid shrimp exposed topotassium perfluorooctanesulfonaatte concentrations< 0.25 mg/L for35 days. Reproduction,lengthand dry weightwere the most sensitivebiologicaelndpointsinthisstudy. Second generationmysids exposed to PFOS duringa static96-hourexposure showed no adverse effects. Submifter:3M Company, EnvironmentalLaboratory,P.O. Box 33331,'St. Paul,Minnesota,55133 DATA QUALITY ReliabilityK:lfmischranking= 1 REFERENCES This studywas conducted atWildlifIenternationaLltd.,Easton, MD atthe requestofthe 3M Company. OTHER Last changed: 5/3/00 PFOS: A FLOW -THROUGH LEFE-CYCLE TO)UCITY TEST WITH THE SALTWATER MYSD) (Mysidopsibsahia) FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-107 3M LAB REQUEST NO. U2723 U.S. EnvironmentalProtectioAngency Series850 - EcologicalEffectsTestGuidelines OPPTS Number 850.1350 AUTHORS: KurtF- Drottar Henry 0. Krueger,Ph.D. STUDY INITIATION DATE: May 21,1999 STUDY CONTLETION DATE: April26,2000 Submittedto 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St Paul,MN 55144 Wildl!feInternationaLlt,d. 8598 Commerce Drive Easton,Maryland 21601 (410)822-8600 Page 1 of60 WILDLIFE INTERNATIONAL, LTD. -2- PROJECT NO.: 454A-107 GOOD LABORATORY PRAMCE COMTLLA-NCE STATEM[ENT SPONSOR. 3M Corporation TITLE: PFOS: A Flow-ThroughLife-CyclTeoxicitTyestwiththeSaltwateMrysid (Mysidopsibsahia) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBEK- 454A-107 STUDY COMPLETION: April26,2000 Thisstudywas conductedincompliancewithGood LaboratoryPracticSetandardsaspublishedby the U.S.EnvirorumentParlotectioAngency in40 CFR Parts160 and 792, 17 August 1989;OECD Principleosf Good LaboratoryPractic(eENV/MC/CHEM (98)17);and JapanMAFF, 59NohSan,NotificatiNoon. 3850, AgriculturParloductioBnureau,10August 1984,withthefollowinegxceptions: The testsubstancewas not characterizeidn accordancewithfiffGlLP compliance;how@ver,the characterizatwiaosn performedaccordingto3M StandardOperatingProceduresandMethods,and allraw data arebeingmaintainedinthe3M archives.The testsubstanceisbeingrecharactefiziendaccordancewithGLP. The stabiliotfythetestsubstanceunderconditionosf storageatthetestsitew.as notdeterminedin accordancewithGood LaboratoryPracticSetandards. STUDY DIRECTO KurtF- Drottar SeniorBiologist SPONSOR APPROVAL: Sponsor DATE 7 bkTE WILDLIFE INTERNATIC)NAL, LTD. -3- PROJECTNO.: 454A-107 QUALITY ASSURANCE STATEMEENT Thisstudywas examined forcompliancewithGood LaboratoryPracticSetandardsaspublishedby the U.S.EnvironmentaPlrotectioAngency in40 CFR Parts160 and 792,17August 1989;OECD Principleosf Good LaboratorPyractic(eENV/MC/CHEM (98)17);andJapanMAFF, 59 NohSan,NotificatiNoon. 3850, AgriculturaPlroductionBureau,10August 1984. The datesofallinspectionasndauditsandthedatesthatany findingwsere reportetdotheStudyDirectoarnd LaboratoryManagement were asfollows: AC=Y: Protocol TestSubstancePreparation LightMeterReadingand AnalyticaSlampling SalinitMyeasurements CalibratioSntandards Preparation BiologicaDlataand DraftReport AnalyticaDlataand DraftReport FinalReport DATECONDUCTM: May 24,1999 June 10,1999 June16,1999 June17,1999 July21,1999 October1,4 and 5,1999 October4-7,199 April20,2000 DATE REPORTED TO: STMYDMECTOR: MANAGEbffi"NT: May 24,1999 July16,1999 June14,1999 June 16,1999 June17,1999 June18,1999 Junel8,1999 June18,1999 July21,1999 July21,1999 October6,1999 October7,1999 - October7,1999 October8,1999 April20,2000 April24,2000 KimbcklyA. Hox* DATE QualityAssuranceRepresentative WILDLIFE INTERNATIONAL, LTD. -4- PROJECT NO.: 454A-107 REPORT APPROVAL SPONSOKTITLE: 3M Corporation PFOS: A Flow-ThroughLife-CyclTeoxicitTyestwiththeSaltwateMrysid (Mysidopsibsahia) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBEF- 454A-107 STUDY DIRECTOR-. Kurt Drottar SeniorBiologist. t4 i e--m7-y6.-4u-egeit,h.D. DirectorA,quaticToxicologyand Non-TargetPlants DATE DATE WILDLIFE INTERNATIONAL, LTD. -5- PROJECT NO.: 454A-107 TABLE OF CONTENTS Title/CoPvaegre.......................................................................I................. Good LaboratoryPracticCeomplianceStatement.............................................2................. QualityAssuranceStatement.............................................................3................. ReportApproval.......................................................................4................ TableofContents......................................................................5................ Summary .............................................................................7................ Introducti.o.n.........................................................................8................ Objective...............................................................................................8................................ ExperimentalDesign....................................................................8................ Materialsand Methods ..................................................................9................ Resultsand Discussio.n................................................................1.4................ Conclusions..........................................................................................1..5.......................... References........................................................................................................1.7............... TABLES Table 1 - Summary of AnalyticalChemistry Data ..........................................1.8................ Table2 - SalinitoyfWater intheNegativeControland HighestTreatinenGtroup TestChambers....1.9.. Table3 - pH ofWaterintheTestChambers......I...........................................2.0............... Table4 - Temperature(*C)ofWater intheTestChambers...................................2.1............... Table5 - DissolvedOxygen Content(mg/L)ofWater intheTestChambers .....................2.2............... WILDLIFE INTERNATIONAL, LTD. .6- PROJFCTNO.: 454A-107 TABLE OF CONTENTS -Continue-d Table6 - Survivaolf JuvenilMeysids Exposed toPFOS (Day0 ThroughPairinogn Day 20)..............................................2.3................. Table7 - SurvivaolfAdultMysids Exposed toPFOS (Day20 ThroughTestTerminationon Day 35)....................................2.4................. Table8 - Mean Number ofYoung ProducedPerReproductiveDay ................!.........................2..5............ Table9 - Mean TotalLengthofAdultMysidsatTestTenninatio.n............................2.6................. Table10- Mean Dry WeightofAdultMysidsatTestTerminatio.n........................................2..7................ Table11- SurvivaolfSerond-GeneratioMnysidsExposed toPFOS Duringa 96-Hour StatiEcxposure...............................................2.8................ APPENDICES Appendix I- DilutioWnater SalinitMyeasuredDuringthe 4-Week PeriodImmediatelyPrecedingtheTest.......................................2.9................ Appendix 11- .AnalysesofPesticidesO,rganics,MetalsandOtherInorianics inWildlifIenternationLatld,.Saltwate.r.....................................3.0................ AppendixIII- The AnalysisofPFOS inSaltwater inSupportofWildlifIenternationLatld,.ProjecNto.: 454A- 107 ..................3.1............... Appendix IV - Mysid Reproduction.....................................................5.2................ AppendixV - MysidTotalLength(mm) .................................................5.5................ AppendixVI - MysidDryWeight(mg)...................................................5.7............... Appendix VU - ChangestoProtoco.l.....................................................5.9................ Appendix VUI -PersonnelInvolvedintheStudy.............................................................6.0............... WILDLIFE INTERNATIONAL, LTD. -7- PROJECT NO.: 454A-107 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCANON OF.STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: WRDLHE INTERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STMY:' NOMINAL TEST CONCENTRANONS: MEAN MEASURED TEST CONCENTRANONS: TEST DATES: LENGTH OF FIRSTGENERANONE)CPOSURE: TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: NOEC: LOEC: MATC: SUAEMLARY 3M Corporation SusanA. Beach WH&ffe InternationLatld,. Easton,Maryland 21601 454A-107 PFOS (PerfluorooctaSnuelfoniAccidPotassiumSalt) PFOS: A Flow-ThroughLife-CyclTeoxicitTyestwiththe SaltwateMrysid (Mysidopsibsahia) NegativeControl0,.086,0.17,0.34,0.69,1.4and 2.7mg a.i./L NegativeControl,0.057,0.12,0.25,0.55,1.3and2.6mg a.i./L ExperimentaSl= (OECD) - May 26,1999 ExperimentaSltart(EPA) - June16,1999 BiologicaTlerminatio-n July25, 1999 ExperimentaTlermination- July25,1999 35 DUs SaltwateMrysid (Mysidopsibsahia) WildlifIenternationLatlc,lCultures Easton,Maryland 21601 Juvenile<s24 hoursold 0.25mg a.i./L 0.55mg a.i./L 0.37ES a.i.AL WILDLIFE INTERNATIONAL, LTD. PROJECTNO.: 454A-107 INTRODUCRION Thisstudywas conductebdyWildlifIenternationLatlc,Lfor3M CorporatioanttheWildlifIentmmfionaL Ltd.aquatictoxicologfyaciliitnyEaston,Maryland.The in-lifpehaseofthetestwas conductedfrom June 16, 1999toJuly25,1999.Raw datageneratedby WildlifIenternationLatld,.anda copyofthefinarleportarefiled underProjecNtumber 454A- 107inarchivelsocatedon theWildlifIenternationLatld,.site. OBJECRIVE The objectivoef thisst* was to evaluatetheeffertosf PerfluorooctanSeulfoniAccid PotassiumSalt (PFOS) on thesurvivalg,rowthandreproductioonfthesaltwatemrysid(Mysidopsisbahia)underflow-through testconditions. EXPERIMENTAL DESIGN Mysidopsis bahia neonates,lessthan 24 hours old,were exposed to a geometricseriesof six test concentratioannsd a negative(saltwatecro)ntrolfor35 days.Nominal testconcentr@tiownesre selecteidn consultatiwoinththeSponsor,and-were based.upon known toxicitdyata.Nominal testconcentrationwsere 0.086,0.17,0.34,0.69,1.4and 2.7mg activeingredien(tai.)/LM.ean measuredtestconcentratiownesre determinedfrom samplesoftestwatercollectefdrom eachtreatmenatnd controlgroupatthebeginningofthe testa,tweeklyintervaldsuringthetestand attesttermination. Deliveryofthetestsubstancewas initiataepdproximatel5y1 hourspriortotheintroductionftheneonate mysidstothetestwaterinordertoachieveequilibriuomfthetestsubstancienthetestchambers.Four replicate testchambers,eachcontaininogne compartmentwith15 mysids,weremaintainedforeachtwtrnentandcontrol group.To beginthetestn,eonatemysidswere impartialdliystributiendgroupsofone ortwo among glass beakersuntileachbeakercontained15 mysids.The mysidswerethentransferretdothetestcompartments.A totaolf60 mysidswere exposedineachtreatmenatnd thecontrolgroup. On Day 20 ofthetestf,emaleand male adultswerepaired,andthereproductioonfthepairedmysidswas monitoredthroughDay 35. Observationosfmortalityc,linicaslignsof toxicitya,ndreproductiownere made WILDLIFE INTERNATIONAL, LTD. -9- PROJECTNO.: 454A-107 daily.At testterminationt,helengthsand dryweightsof allsurvivinfgirst-generatniroynsidwseremeasurecAlU young producedinthetestwere removed to a separatetestchamber atthesame testconcentratioonn a daily basis.The second-generatimoynsidswere exposedfor96 hoursunderstatitcestconditions. The no-observed-effect-concent(rNaOfEiCo)n and lowest-observed-effect-concent(rLaOtEiCo)nwere determinedby examinationofthemortalityg,rowthand reproductiodnata.The maximum acceptablteoxicant concentratio(nMATC) was calculateadsthegeometricmean oftheNOEC and LOEC. MATERL4,LS AND METHODS The studywas conductedbasedontheprocedureosutlineidntheprotocol",PFOS: A Flow-ThroughLifeCycleToxicityTestwiththeSaltwateMrysid (Mysidopsisbahia)".The protocol'wabsasedon procedures outlineidnU.S.EnvironmentalProtectioAngency Series850 - EcologicaElffectsTestGuidelinesO,PPTS .Number 850.1350(1);andAS7M StandardEl 191-90StandardGuideforConductingLife-CycleToxicity TestswithSaltwateMrysids (2). TestSubstance The testsubstancewas receivedfrom 3M Corporationon October29, 1998 and was assignedWildlife InternationLatld,.identificatniuomnber 4675. The testsubstancweas &scribedasa whitepowder. Itwas identifiaesdFC-95 fromlotnumber 217 (T-6295).Informatiopnrovidedby theSponsorindicateadpurityof 90.49'/oa,nd an expiratiodnateof2008. The testsubstancewas storedatambientroom temperature. PEWarationof TestConcentrations One stocksolutiownas preparedforeachofthesixconcentratiotnessted.A primarystockwas preparedby dissolvintghetestsubstanciendilutiownaterata concentratioofn0.0895mg a.i./mL.The primarystockwas stirrewdithanelectrisctainlessteetlop-downmixerforapproximatel2y4 hourstoaidinthesolubdizatioont thetestsubstance.Aftermixing,theprimarystockappearedclearand colorleswsitha foam on thesurface. Aliquotsoftheprimarystocksolutiownere proportionaldliylutewdithdilutiownatertopreparefiveadditional stocksatconcentratioonfs0.0447,0.0224,0.0112,0.00559and 0.00280mg a.i./mL.The sixstockswere injecteidntothedilutemrixingchambers(atarateof4.60mL/minute)wheredW we remixedwithchlutiownater WILDLIFE INTERNATIONAL, LTD. -10- PROJECTNO.: 454A-107 (ata rateof 150mIJminute)toachievethedesiretdestconcentrationAsR. testsolutionasppearedclearand colorless. TestOr- ism The saltwatmeyrsid,Mysidopsibsahia(recentrleynamedAme?icamysibsahia)w,as selecteadsthetest speciesforthisstudy.The saltwatemrysid isrepresentatiovfean importantgroupofaquaticorganismsandwas selectefdoruseinthetestbasedupon pasthistoroyfuseand easeofculturinignthelaboratoryM.ysids usedin thetestwere neonateslessthan 24 hoursoldand were obtainedfrom culturemsaintainedby Wildlife InternationLatld,.,Easton,Maryland. Adultmysidswereheldinwaterfromthesame sourceasusedduringthetest.Adultmysidswere fedlive brineshrimp(Artemiasp.)two or6= timesdailyduringholding.Brineshrimpwereperiodicalelnyrichewdith a fattyacidsupplement(ALGAMAC-2000, AquafaunaBio-MarineI,nc.H,awthorne,CaliforniaD)u.ringthe 14-dayholdingperiodprecedingthetestw,atertemperaturersangedfrom 25.0to25.4'C.The pH ofthewater rangedfrom8.0to8.1,salinitryangedfrom 20 to23%o q)artpserthousand)a,nd dissolveodxygen rangedfrom 6.gto7.4mg/L. InstrumentatiounsedforwatermeasurementsisdescribeidntheEnvironmentalConditions sectionofthisreport. At testinitiatitohne,neonatemysidswerecarefullcyollectefdromthecultureasnd transferreodne andtwo ata timetoglassbeakers.Mysids thenwere transferrefdrom thebeakerstothetestcompartments.AR transfers were made usinga wide-borepipettbeelow thesurfacoef thewater.The mysidswere fedlivebrineshrimp nauplitihreetofourtimesa dayduringthetestopreventcannibalis(mexcepton thelastday ofthetest). TestApparatus A continuous-flodwilutewras used to deliveerach concentrationf thetestsubstanceand a negative (saltwatecro)ntrolA. peristaltpiucmp (Cole-ParmeIrnstrumenCtompany, Chicago,IL)was usedtodelivetrhe sixtestsubstancestocksolutionisntomixingchambersassignedtoeachtreatmengtroup.The stocksolutions wereMuted withdilutiownaterinthemixingchambersinor* toobtainthedesiredtestconcentrationTsh.e flowofdilutiownatertothemixingchamberswas controllebdy rotametersR.otameterswere calibratepdriotro testinitiatiaonndatweeklyintervaltshereaftdeurringthetestT.he flowoftestwaterfr6m eachnixingchamber was spliatnd allowedtoflowintoreplicatestchambers.The proportioonf testwaterthatwas spliitntoeach WILDLIFE INTERNATIONAL, LTD. PROJFCT NO.: 454A-107 replicatweas checkedpriortothetestand atweeklyintervaltshereaftedruringthetesttoensurethatflowrates variedby no more than10% ofthemean forthefourreplicates. The dilutewras adjustesdo thateachtestchamber receivedapproximately11volumeaddition6sftestwater every24 hours.The deliverpyump was calibratebdeforethetestandatapproximatelwyeeklyintervaldsuring thetest.The generaloperatioonfthedilutewras checkedvisuallaytleastwo timesperday duringthetestand once attheend ofthetest. Priortosexualmaturitym,ysidswere heldinone comparunentplacedineachreplicatteestchamber (15/compart3nentT)h.e compartmentswere glassbeakerswithnylonmesh screenattachedto two holeson oppositesides.Aftermysids attainedsexualmaturity,reproductivpeairswere placedin reproductive compartments(onepairpercompartment).The reproductivceompartmentswere glasspetridisheswithsidesof nylonmesh screenattachewdithsiliconaedhesive.The testcompartmentswereplacedin9-L glassaquariatest chamberscontaininagpproximatel5yL oftestsolutionP.riortopairingt,hedepthofwaterinarepresentative testcompartmentwas 6.2cm. Afterpairingt,hedepthofwaterina representatitveestcompartmentwas 5.5cm. The testchamberswereimpartiallpyositioneidna temperature-controueendvironmentaclhamber designedto maintaina temperaturoef252'C. Testcompartmentswere uniquelyidentifieadnd thetestchamberswere labeledwiththeprojectnumber,testconcentratioanndreplicateT.he testchambersforthesecondgeneration exposurewere2-LbeakerswithIL oftestsolutiownhich was dippedoutofa testchamber fromtheappropriate treatmengtroup.' DilutioWnater The waterusedforculturinagnd testinwgas naturalseawatercollecteadtIndianRiverInletD,elaware,and was dilutedtoa salinitoyfapproximatel2y00/oowithwellwater.Salinitmyeasurementsduringthefour-weekperiodinunediatelpyrecedingthetestarepresentedinAppendix 1. The freshly-corectsedawaterwas passed througha sand filtetro remove particlegsreaterthan approximatel2y5 gm, andpumped intoa 37,800-Lstoragetankwherethewaterwas aeratewdithspraynozzles. Priortodelivertyothedilutesrystem,thewateragainwas filtere(d0.45pm) toremovemicroorganismsand particleTsh.e resultosfperiodicanalysesperformedtomeasure@fficeoncentratioonfsselectecdontaminantsin saltwateursedby WildlifIenternationaLlt,d.arepresentedinAppendix 11. WILDLIFE INTERNATIONAL, LTD. -12- PROJECT@4O.: 454A-107 EnvironmentaClonditions Lightingusedtoilluminatehecultureasnd testchambersduringculturinagnd testinwgas providedby fluorescetnutbesthatemittedwavelengthsimilatronaturaslunligh(tColortone5*0). A photoperioodf 16 hoursoflighatnd8 hoursofdarknesswas controllewdithan automatictimer.A 30-minutetransitipoenriod of low lightintensitwyas providedwhen lightwsent on and offto avoidsuddenchangesinlightingL.ight intensitryangedfrom 623 to815 luxoverthesurfaceofthenegativecontrolr,eplicatAetestchamber. Light intensitwyas measuredweekly,Wing a SPER ScientifMiocdel 840006C lightmeter f Temperaturewas measuredineachtestchamber atthebeginningand endofthetestand atweeklyintervals duringthetestusinga liquid-in-gltahsesrmometer.Temperaturealsowas measuredcontinuousliynonenegative controlreplicatuesinga FulscopeERIC Recorder.The targetestemperaturdeuringthestudywas 252*C. Dissolvedoxygen and pH measurementswere measuredin alternatreplicateosf eachtreatmentand control groupatthebeginningand end ofthetestand atweeklyintervaldsuringthetest.Salinitwyas measureddailyin alternatreeplicateosfthenegativecontroland thehighesttreatmengtroupwithsurvivinmgysids.- Dissolvedoxygenwas measuredusinga YellowSpringsInstrumentModel 51B dissolveodxygenmeter, and measurementsofpH weremade usinga FisherAccumet Model 915 pH metrr.Salinitwyas measuredusinga Bio-Marine,Inc.A,quafaunarefractometer. BiologicaOlbservationsand Measurements Observationosfthesurvivaalndbehaviorofeachfirst-generatmiyosnidwere made dailythroughouthe test.The criterfioardeathincludedlackofmovement, absenceofrespiratormyovements,and lackofreactiotno gentleprodding.At thetimeofpairing(Day 20),thesexand maturitoyfeachmysidwas determinedby microscopicexaminationa,nd,when possible5,male/femalepairsweremade foreachreplicatteestchamber. Any immaturemysidsorextrafemaleswerediscardeadtthistime.Sexuallymaturemales,whichwereleftover afteprairingweremaintainedina separatceompartmentwithinthatreplicate. Aftermysidswerepairedt,henumber of second-generatimoynsidswerecountedand recordeddailyuntil testterminationS.econd-generatiomnysidswere alsoobservedforabnormaldevelopmentand aberranbtehavior. Aftereachobservations,econd-generatimoynsidswere collecteadnd exposedatthesame testconcentration understatitcestconditionfsor96 hours.Ifa male ina male/femalpeairdied,itwas replacedwitha male,if WILDLIFE INTERNATIONAL, LTD. -13- PROJECTNO.: 454A-107 availablfer,omthepoolofmalesmaintainedinthesame replicatAet. testterminationt,hesexofeachsurviving first-generatmiyosnidwas confirmedand thelengthof eachmysidwas measuredusingcalipersE.achsurviving first-generatwiaosnthenplacedina dryingoven atapproximatel6y0*C forapproximatel2y4 hours.The dry weightof eachsurvivinfgirst-generatmiyosnid was thendeterminedusingan analyticablalance. StatisticAanlalyses Statisticaanlalyseswere-performeodn survivaolfthefirsatnd second-genera@omnysids,thenumber of young releasepderreproductivdeay,andthelengthanddryweightof eachsurvivinfgirst-generatmiyosnid. Survivawlas evaluatepdriortopaiiin(gDay 0 throughDay 20) and afteprairing(Day 20 throughDay 35). Survivaldatawere analyzedusing2 x 2 contingencytablesand theChi-squaretesttoidentiftyreatmengtroups thatwere statisticadlilfyferenftrom thecontroglroup. The analysesofreproductio(nnwnber ofliveyoung producedperreproductivdeay)andgrowth(dryweights andlengthsd)atamcludedthosetreatmentwshichdidnotexhibita statistirceadluction.siunrvivalA.nalyses includetdheevaluatioonfhomogeneityofvarianceussingBartlettt'esstand theassessmentofnormalituysing theShapiro-Wilk'tsest.When datawere deemed normal andhomogeneous,an analysiosfvariancteestwas used todeterminewhetherornot statisticaslilgynificadnitfferencexsistedamong experimentaglroups.Those treatmentsstatisticadlilfyferenftrom thecontrolgroupwere identifiuesdingDunnett'stest.The resultosfthe statistiacnaallyseswereusedtoaidinthedeterminatioonftheNOEC and LOEC.' The MATC was calculataesd thegeometrimcean oftheNOEC and LOEC. Allstatistitceasltwsereperformedusingapersonalcomputerwith SPSS/PC Version2.0(3)or"TOXSTAT Release3.5"statistiscoafltwar(e4). AngL)IicaClhemisgy Priortotestinitiatitowno, setsofpreteswtatersampleswerecollectefdromtwo replicatteestchambersof boththelow and highleveltreat3negnrtoupstodetermineifnominalconcentrationhsadbeen achieved.Water sampleswerealsocollectefdromtwo alternatirnegplicatoens Days 0,7,14,21,28and35. Watersampleswere collectefdrommid-depthofthetestchamber and placedinplasti(cNalgenes)bottles.Sampleswereanalyzedas soon aspossiblewithoutstorage.Analyticaplroceduresusedintheanalysiosf thesamplesareprovidedin AppendixIII. WILDLIFE INTERNATIC)NAL, LTD. -14- PROJECTNO.: 454A-107 RESULTS AND DISCUSSION MeasuremenotfTestConcentrations Resultsof analysestomeasureconcentratioonfs PFOS inwatersamplescollectedduringthetestare presenteidnTableI and theanalyticcahlemistryreport(AppendixHI).Nominal concentratioun@se4inthis studywere0.086,0.17,0.34,0.69,1a.n4d 2.7mg a.i./LW.hen measuredconcentrafioonfssamplescollecteodn Days 0,7,14,21,28and 35 wereaveraged,themean measuredconcentratiownesre 0.057,0.12,0.25,0.55,1.3 and 2.6mg &i.AL,which represente6d6,71,74,80,93 and96% ofthenominalconcentrationrse,spectively. Mean measuredconcentrationwsere usedtoexpresstheNOEC, LOEC and MATC. Pretesstamplescollectetdo verifydiluteprerformance(AppendixIM werenotusedm thecalculatiofnmean measuredconcentrations. Physicaland ChemicalMeasurementsof Water Measurementsof salinitiynthenegativecontroland thehighestreatmengtrouprangedfrom 19to2 1'/oo throughouthetest(Table2). MeasurementsofpH rangedfrom 8.2to8.4(Table3) and temperaturweas maintainedwithinthe252'C rangeestablishefdorthetest(Table4). Dissolvedoxygenconcentrations remained 5.8mg/L (79% ofsaturatiotnh)roughouthetest(Table5). Survival A summary ofsurvivaflromtestinitiatitoonpairingon Day 20 ispresenteidnTable6. Ingenerala,ll survivinmgysidsappearednormal.After20 daysofexposure,survivailnth@negativecontroglroupwas 78%. SurvivalinthePFOS treabnengtroups:g0.55mg a.i./rLangedfrom75 to92% andwerenotstatisticddilfyfmvt from thenegativceontrol.Survivailnthe1.3and 2.6mg a.i./tLreatmengtroupswas 32 and0*/or,espectively, andwas statisticadlilfyferenftromthenegativceontrol(p !@0.05). Observationosfsurvivaalfteprairing(fromDay 20 totesterminatioonn Day 35)arepresenteidnTable7. In generala,llsurvivinmgysidsappearednormal. Survivailnthenegativecontrowlas 92%. Survival percentageisnthePFOS treabnentgroups-@0.55mg a.i./rLangedfrom 90 to97% andwere notstatistically differenftromthenegativecontrol.Survivalinthe1.3mg a.i./tLreatmengtroupwas 571/oandwas statistically differefnrtomthenegativceontro(lp:g0.05). WILDLIFE INTERNATIONAL, LTD. -15- PROJECTNO.: 454A-107 on A summary ofthemean number ofyoung producedperreproductivdeay ispresentedinTable 8. Young productiofnorindividuatlestcompartmentsispresenteidnAppendixIV. For eachfemale,thenumber of reproductivdeayswas definedasthenumber ofdaysthatthefemalewas alivefrom theday of firsbtrbodrelease ofany femaleinthetesttotheendofthetest.The day offirsbtroodreleasienthisstudywas Day 22. The mean number ofyoung producedperreproductivdeay inthenegativecontroglroupswas 0.315.Reproductionrateisn the0.057,0.12,0.25 and 0.55-mga.i./tLreatmentgroupswere 0.261,0.361,0.2@2and 0.0559young per reproductivdeay,respectivelDyu.imett'stestshowed thatreproductiownas significantrleyducedinthe0.55mg a.i./tLreabnengtroupwhen compared tothenegativecontrol(p !@0.05).The 1.3and 2.6mg a.i.AtLreatment groupswerenot includeidnthestatisticaanlalysiosfthereproductiodnatadue toa statisticaslilgynificant differenciensurvival. Growth Summariesof thelengthsand dryweightsofthesurvivinagdultmysidsarepresentedinTables9 and 10, respectivelIyn.dividuamleasurementsareprovidedinAppendicesV and VI. The mean.lengthand mean dry weightinthenegativecontrolgroupwere 6.43mm and 0.63mg, respectivelMyy.sids exposedto PFOS at concentratio!n@0s.25mg a.i./sLhowed no statisticaslilgynificarnetductionisnlengthordryweight(p> 0.05). Mysids exposedto0.55 mg a.i./sLhowed statisticaslilgynificarnetductionisnboth lengthand dryweight (p!@0.05).The 1.3and 2.6mg a.i./tLreatmengtroupswerenotincludeidnthestatistiacnaallyseosfgrowthdue toa statisticaslilgynificadnitfferenciensurvival. SecondGenerationAcute Enosure The resultosfthesecondgeneratioenxposurearepresenteidnTable11.After96-hours,controlsurvival was 96%. SurvivalinallPFOS treatmengtroupswas @!95% and was notstatisticadlilfyferenftromthecontrols. AR survivinmgysidsinthesecondgeneratioenxposureappearednormalwithno overtsignsoftoxicity. CONCLUSIONS There were no statisticaslilgynificanetffectson survivalr,eproductionor gtowth of mysid shrimp (Mysidopsisbahia)exposedtoPFOS atconcentratioonfs!g0.25mg a.i./fLor35 days.Reproductionl,engthand WILDLIFE INTERNATIONAL, LTD. -16- PROJECTNO.: 454A-107 dryweightwerethemost sensitivbeiologicaelndpointsinthisstudy.Mysid shrimpexposedto1.3and2.6mg a.i./hLad significantrleyducedsurvivailncomparisontothenegativecontrolM.ysids exposedto0.55mg '&i./L had significantrleyducedreproductiolne,ngthand dry weightin comparisonto thenegativecontrol. Consequentlyt,heLOEC, basedon reproductionl,engthand dryweight,was 0.55mg a.i./LT.he NOEC was 0.25mg a.i./aLnd theMATC was calculatetdobe 0.37mg a.i./LS.econdgeneratiomnysidsexposedtoPFOS duringa stati9c6-hourexposureshowed no adverseeffects. WILDLIFE INTERNATIONAL, LTD. -17- PROJECT NO.: 454A-107 REFERENCES I U.S.Enviromental ProtectioAngency. 1996.Series850 -EcologicaElffectTsestGuideline(sdraft), OPPTS Number 850.1350:Mysid ChronicToxicitTyest. 2 ASTM Standard E1191-90. 1991. StandardGuideforConductingLife-CycleToxicitTyestswith SaltwateMrysids,AmericanSocietfyorTestinagnd Materials. 3 SPSS Inc. 1988.SPS@@C+ Version2.0.Chicago,Minois. 4 West,Inc.and D. D.GuHey. 1996.TOXSTAT Version3.5.WesternEcoSystemsTechnology,Inc. Cheyenne,Wyoming. .18- Table I Summazy ofAnalyticalChemist*rDyata Sponsor 3M Corpomdon TestSubstance:PFOS TestOrpnism: SaltwateMrysid,Mysidopsisbabia DflufioWnater.-FiltereSdaltwater Nominal Test Concentration (mg a.iJL) NegafiveCont-r-ol ILW <-OQ <LOQ <LoQ 0.086 0.0694 0.0579 0.0478 0.0619 0.17 0.125 0.0778 0.114 0.125 0.34 0.289 0.231 0.286 0.197 0.69 0.562 0.581 0.659 OA50 1.4 1.23 1.13 1.32 1.20 2.7 2.56 2.58 2.79 2.30 ReplicateAs and C measured 2 ReplicateBs and D measured 3 4 LOQ - Limitofquantitafiownas 0.0458 mg a.i./L am lee not collecteddue to I()06/moorwity. Measured Concentmfion(mg ai./L) a Day2l' Day 28' <LOQ <LOQ <-OQ <LoQ <LOQ <I)OQ 0.0606 0.0614 0.0554 0.0509 0.0515 0.0569 0.124 0.127 0.0970 0.112 0.122 0.128 0.276 0.253 0.227 0.212 0.262 0.271 0.543 0.542 0.516 0.528 0.529 0.544 1.35 1.23 1.39 1.27 1.15 1.39 2.54 _4 - 2.69 Day 357 <IOQ <LOQ 0.0580 0.0514 0.124 0.119 0.278 0,251 0.556 0.583 126 1.20 - WILDLIFE INTERNATIONAL, LTD. -19- '@'CTNO.:454A-107 Table2 SalinitoyfWater intheNegativeControland HighestTreatmentGroup TestChambers Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: DilutionWater: SaltwaterMysid,Mysidopsisbahia FiltereSdaltwater NegativeControl 2.6mg a.i.//]L1.3mg a.i./L DE EMlicate 0 A 1 B 2 c 3 D 4 A 5 B 6 c 7 D 8 A 9 B 10 c 11 D 12 A 13 B 14 c 151 D 16 A 17 B 18 c 19 D 20 A 21 B 22 c 23 D 24 A 25 B 26 c 27 D 28 A 29 B 30 c 31 D 32 A 33 B 34 c 35 D s@@@ (%0-) 20 21 20 20 20 20 20 20 20 20 20 20 20 20 21 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 s4inLty(oko) 20 21 20 19 20 20 20 20 20 20 20 20 20 20 20 20 -20' 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 -T @ Days 15-35,salinitwyas measured inthe1.3mg a.i./tLreatmentgroup due to 100% pnftah in.the2.6mg a.i.AtLreatment u2. WILDLIFE INTERNATIONAL, LTD. .20. PROJECTNO.: 454A-107 Table3 pH of Water intheTestChambers Sponsor: 3M Corporation TestSubstance: PFOS TestOTgmAsm:. SaltwateMrysid,Mysidopsisbahia DilutionWater: FiltereSdaltwater Mean Measured Testpa TestConcentration (mgai./L) Replicate 0 7 14 21 28 35 NegativeControl A 8.4 - - - 8.3 - B - 8.2 - 8.3 c - 8.3 - D - - 8.3 - 0.057 A 8.4 - - 8.3 - B - 8.2 - - 8.3 c - 8.3 - D - - 8.3 0.12 A 8.4 - - 8.3 B - 8.2 - c - 8.3 8.3 - D - - 8.3 - 0.25 A 8.4 - - 8.3 - B - 8.2 - - 8.3 c - 8.3 - 8.3 - 0.55 A 8.4 - 8.3 - B - 8.2 - 8.3 c - 8.3 - - D - - 8.3 - 1.3 A 8.4 - - 8.3 - B - 8.3 - - 8.3 c - 8.3 - - D - - 8.3 2.6 A 8.4 - - B - 8.3 - c - 8.3 D 'Mc&swments discontinueddue to100% mortalip@. WILDLIFE INTERNATIONAL, LTD. -21- PROJECT NO.: 454A-107 Table4 Temperature(OC)ofWater intheTestChambers Sponsor 3M Corporation TestSubstance: PFOS TestOrganism: SaltwateMrysid,Mysidopsisbahia DilutioWnater. FgtcrcSdaltwater Mean Measured TestConcentration TestD!y (Mg&iJL) Rcp*atc 0 7 14 21 28 35 NegativCeontrol Al 25.0 252 24.8 24.9 25.2 25.0 B 25.0 25.1 24.8 24.9 25.2 24.9 c 25.0 25.0 24.6 24.9 25.2 25.0 D 25.0 25.1 24.5 24.9 25.1 25.0 0.057 A 25.0 25.2 24.8 25.0 25.1 24.9 B 25.0 25.3 25.0 25.0 25.1 25.0 c 25.0 252 24.9 25.0 25.1 25.1 D 25.0 25.2 25.0 25.0 25.2 25.0 0.12 A 25.0 25.1 24.8 25.1 25.1 24.9 B 24.9 25.1 24.9 25.1 25.0 24.9 c 25.0 25.2 24.9 25.1 25.0 24.9 D 25.0 25.2 24.9 25.0 25.1 24.9 0.25 A 24.8 25.1 24.7 25.1 25.0 25.0 B 24.8 25.0 24.9 25.1 --25'l 25.0 c 24.9 25.0 24.8 25.1 '25'.0 25.0 D 24.9 25.0 24.9 25.1 25.1 24.9 0.55 A 24.9 25.0 24.9 .25.0 25.1 25.0 B 24.7 25.0 24.8 25.0 25.0 24.9 c 24.8 25.0 24.8 25.0 25.0 24.9 D 24.7 25.0 24.8 25.0 25.0 24.9 1.3 A 24.6 24.8 24.4 24.5 24.8 24.8 B 24.7 24.9 24.7 24.5 24.8 24.8 c 24.9 25.0 24.5 24.7 24.9 24.9 D 25.1 25.1 25.1 25.0 25.0 24.9 2.6 A 24.9 25.1 25.0 _2 - - B 24.8 25.0 - c 24.8 25.0 25.1 _2 D 24.8 25.1 25.1 2 -ITem-paatruemeasuredcontinuousldyuringthetestrangedfrom24.5to25.5*C. 2 Measurementsdiscontinudeudeto100% mortality. WILDLIFE INTERNATIONAL, LTD. -22- PROJECT NO.: 454A-107 Table5 DissolvedOxygen Content(mg/L)ofWaterintheTestChambers' Sponsor: 3M Corporation TestSubstance: PFOS TestOrgnnigm: SaltwateMrysid,Mysidopsisbahia DilutionWater: FiltereSdaltwater Mean Measured Test,L)!Y TestConcmtrati6n (mgai./L) ReplicMk 0 7 14 21 28 35 NegativeControl A B c D 6.1 - - - 6.0 - - 6.4 - 6.3 - 6.2 - - - - 6.0 - 0.057 A 6.1 - - 6.0 - B - 6.3 - - 6.2 c - 6.0 - D - - 6.0 0.12 A 6.1 - - 6.0 B - 6.1 - 6.3 c - 6.3 - - D - - 6.0 0.25 A 6.1 - - B - 6.1 - 6.3 c - 6.2 - - D - - 5.8 0.55 A 6.1 - 6.0 - B - 6.2 - - 6.2 c - 6.2 - - D - - 6.0 - 1.3 A 6.1 - - 6.0 - B - 6.2 - - 6.3 c - 6.2 - - D - - 5.9 2.6 A 6.1 - - B - 6.2 - c - 6.3 D A dissolveodxygenconcentratioofn4.4mg/L represent6s0% saturatiaotn25*C insaltwatewritha saliniotfy 20%o. 'Mewurements discontinudeude to 100-/m.2@@- WILDLIFE INTERNATIONAL, LTD. -23- PROJECTNO.: 454A-',L-@'I Table6 SurvivalofJuvenilMeysids ExposedtoPFOS (Day0 ThroughPairingonDay 20) Sponsor. 3M Corporation TestSubstance: PFOS TestOrganism: SaltwaterMysid,Mysidopsisbahia Ddufion Water Filter@Saltwater Mean Measured TestConcentration (mgaijl) Replicate No. Afive/No.@Msed Neptive Control A B c D 14/15 10115 14/15 9/15 Survivalon Pa 20 TOW 47/60 PercentSurvival 78 0.057 A B c D 14/15 13/15 15/15 13/15 55160 92 0.12 A B c D 14/15 lOil5 9/15 12/15 45/60 75 0.25 A B c D 13/15 10/15 14/15 12/15 49/60 82 0.55 A B c D 11/15 13/15 13/15 13/15 50/60 83 1.3 A 7/15 B 4/15 c 4/15 D 4/15 19/60 32* 2.6 A 0/15 0/60 0* B 0/15 c 0115 D 0115 Indicatae@s@cant differenfcreomthenegativceontroulsin2gX 2 contin&ent2aXblcl(LpO-05). WILDLIFE INTERNATIONAL, LTD. -24- PRC)J-ECTNO.: 454A-107 Table7 SurvivaolfAdultMysidsExposed toPFOS (Day20 ThroughTestTerminatioonn Day 35) Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: SaltwateMrysid,Mysidopsisbahia DilutioWnater. FiltereSdaltwater Mean Measured SurvivaaltTestTermination TestConcentration (mg&i./L) Replicate No. Alive/NoE.Uosed Total NegativeControl A B 11/12 7/8 36/39 c 10/11 D 8/8 Percent 92 0.057 A B c D 12/13 12/12 10/11 10/10 44/46 96 0.12 A B c D 12/12 7/9 7/9 10/10 36/40 90 0.25 A B c D 10/10 8/8 9/10 9/9 36/37 97 0.55 A B c D 7/8 10/10 10/10 8/9 35/37 95 1.3 A B c D *Indicateas siE!Ecantdifferencferomth( 3n 4/4 0/0 1/3 31using a 2 X 2 8/14 57* lsk 0.05). WILDLIFE INTERNATIONAL, LTD. -25- PROJECTNO.: 454A-107 Table8 Mean Number ofYoung ProducedPerRzproductiveDay Sponsor: 3M Corporation TestSubstance: PFOS TestOrgqnism: SaltwateMrysid,Mysidopsisbahia DilutioWnater: FiltereSdaltwater Mean Measured Number of TestConcentration Reproductive (mga.i.L ETlicate RM NegativCeontrol A 70 B 53 c 70 D 42 Number of Young Produced 18 14 20 19 Mean Number of YoungateFoductive paX 0.257 0.264 0.286 0.452 overall Mean s 0.315 0.0925 0.057 A 60 17 0.283 0.261 0.0873 B 70 .14 0.200 c 70 13 0.186 D 56 21 0.375, 0.12 A 70 21 0.300 0.361 0.101 B 46 22 0.478 c 54 22 0.407 D 70 18 0.257 0.25 A 70 19 0.271 0.252 0.0723 B 56 12 0.214 c 61 21 0.344 D 56 10 .0.179 0.55 A 54 3 0.0556 0.0559* 0.0376 B 56 6 0.107 c 70 3 0.0429 D 56 1 0.0179 1.3' A 22 0 0.000 0.000 0.000 B i4 0 0.000 c 0 0 0.000 D 11 0 0.000 Thistreatmengtroupwasnotincludeidnthestatistiacnalyseosfreproductidounetoa statisticsailglnyificant differenicnesurvival. IndicataesignificdainftferenfcreomthenegativceontroulsingDunnett'tses(tp:50.05). WILDLIFE INTERNATIONAL, LTD. -26- PROJECTNO.: 454A-107 Table9 Mean TotalLengthofAdultMysids attheEnd ofthe35-Day TestPeriod Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: SaltwateMrysid,Mysidopsisbahia DilutioWnater FiltereSdaltwater Mean Measured Concentration ME ai.l,) LeLheate NegativeControl A B c D ReplicatMeean (MM) 6.45 6.34 6.46 6.48 OverallMean s (MM) 6.43 0.0634 0.057 A B c D 6.39 6.45 6.43t 0.0729 6.52 6.36 0.12 A B c D 6.55 6.65 6.56 0.105 6.62 6.42 0.25 A B c D 6.48 6.38 6.40 0.0548 6.36 6.38 0.55 A B c D 6.05 6.11 6.14*t 0.0794 6.16 6.24 1.3' A 5.93 B 5.98 5.85 0.178 c D 5.65 Thistreatmengtroupwas notincludedinthestatistiacnaallyseosftotalengthduetoa statisticaslilgymficant differenicnesurvival. Indicataesig@EcantdifferenfcreomL!L@e controulsingDunnett'tses@tE:50.05). WILDLIFE INTERNATIONAL, LTD. -27- PROJECTNO.: 454A-107 Table 10 Mean Dry Weight ofAdultMysids attheEnd ofthe35-Day TestPeriod Sponsor: 3M Corporation TestSubstance: PFOS TestOrgnniqrn: SaltwateMrysid,Mysidopsisbahia DilutionWater: FiltereSdaltwater Mean Measured Concentration @Ed a.i.LL) @alicate Negative Control A B c D ReplicatMeean (ME2 0.599 0.706 0.596 0.636 OveralMlean t s f- 0.63.4 0.0510 0.057 A 0.616 0.599 0.0276 B 0.627 c 0.590 D 0.565 0.12 A 0.647 0.641 0.0241 B 0.664 c 0.644 D 0.607 0.25 A 0.644 0.622 0.0227 B 0.601 c 0.603 D 0.639 0.55 A 0.556 0.562* 0.00624 B 0.558 c 0.563 D 0.570 1.31 A 0.440 0.436 0.0441 B 0.478 c D 0.390 Thistreatmengtroupwas notincludedinthestatistiacnaallyseosfdryweightduetoa statisticsailplfyficant differenciensurvival. Indicateassi2i@c4mtdfferencferom theneg!@vecontroulsingDunnett'tsest(p !50.05). WILDLIFE INTERNATIONAL, LTD. -28- PROJECTNO.: 454A-107 Table 11 Survivalof Second-GeneratioMnysidsExposed toPFOS During96-Hour StaticExposures Sponsor: TestSubstance: TestOrganism: 3M Corporation PFOS SaltwateMrysid,Mysidopsisbahia DilutioWnater: FiltereSdaltwater Mean Measured TestConcentration (mgai.U Total No.E)gosed No.AliveAfter 96 HoursofExposure LObscivations) NegativeControl 71 68 (AN) 0.057 65 63 (AN) 0.12 83 79 (AN) 0.25 62 59(AN) 0.55 13 AN AppearsNormal. 13 LAn Percent Survival 96 97 95 9.5 100 WILDLIFE INTERNATIONAL, LTD. -29- PROJECTNO.: 454A-107 APPENDIX I DilutioWnater SalinitMyeasuredDuringthe 4-Week PeriodImmediatelyPrecedingtheTest Sponsor: Test Substance: TestOrganism: DilutioWnater: Salinit(yOAo) 3M Corporation PFOS SaltwateMrysid,Mysidopsisbahia Filtm-eSdaltwater Mean 20 (N 4) Range 20-20 WILDLIFE INTERNATIONAL, LTD. -30- PROJECTNO.: 454A-107 APPENDIXE Analysesof PesticideOsr,ganicsM,etalsandOtherInorganics inWildlifIenternationLatld,.Saltwater' ANALYSIS Miscelmeous Measurements TotalDissolvSeodlids Ammonia Nitrogen TotalOrganicCarbon2 TotalCyanide WASURED < < < CONCENTRATION 23,500 mg/L 0.050 mg@L 1.0 mg/L 10.0 ug/L OrganoMorines and PCBS Aldrin AlphaBHC BetaBHC DeltaBHC Gamma BHC (Lindant) Chlordane DDD, pp' DDE, pp' DDT, pp' Dieldrin PndnsillfaAn, EndosulfanB, EndosulfanSulfate Endrin EndrinAldehyde Heptachlor Methoxychlor Heptac.-oErpoxide Toxaphene PCB-1016 PCB-1221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260, < 0.005 lug/L < 0.005 Ag/L < 0.005 ug/L < 0.005 A481i, < 0.006 mg/L < 0.025 @zgti, < 0.006 lug/L < 0.005 mg/L < 0.008 ug/L < 0.005 mg/L < 0.005 kigIL < 0.005 jug/L < 0.018 lug/L- < 0.010 lug/L < 0.005 Izg/L < 0.005 Aig/L < 0.007 jug/L < 0.005 mg/L < 0.500. ug/L < 0.260 lug/L < 0.260"' pg/L < 0.260 < 0.720 lug/L < 0.720 lug/L < 0.720 4g/L < 0.720 kigIL Metalsand Other Inorganics Aluminym' Arsenic Berylliu3m Cacirmum3 Caiciul@l- ct 'UM3 Co 7 Copper3 Iron Lead3 ma gnestun@3 Manganese m @ io-,Iycqcn3um Nickel Potassiu93 Selcn,,Um sSioldv*e 3 . Im ;@@ey < 100 @ugil < 25.0 mg/L < 0.50 mg/L < 1.0 lu@ 235 mg/L < 2.0 ug/L < 1.0 99/L < 20.0 99/L < 100 lug/L < 10.0 A491L 760 mg/L < 4.0 << 02..200 mg/L < 20.0 UB/L 277 MGIL < 25.0 AA91L < i.0 AlgIL 6,010 mg/l, < 20.0 mg/L =alyses performedbyQST EnvironmentalG,ainesvillFel,oridfaorsamplescollecteodnNovember 3 throughNovember 7, 1997. 23Analysesperformedby WildlifIenternationLatld,.forthesamplecollecteodn November 5,1997. Analysesperformedby WildlifIentemafionaLlt,d.forsamplescollecteodn November 5 through7,1997. WILDLIFE INTERNATIONAL, LTD. -31APPENDIX M PROJECTNO.: 454A-107 THE ANALYSES OF PFOS IN SALTWATER IN SUPPORT OF WILDLIFE INTERNATIONAL PROJECT NO.: 454A-107 WILDLIFE INTERNATIONAL LTD. -32- PROJECTNO.: 454A-107 REPORT APPROVAL SPONSOR- 3M Corporation TrrLE: PFOS: A Flow-ThroughLife-CyclTeoxicityTestwiththeSaltwateMrysid (Mysidopsibsahia) W]ILDLIFE R4nRNATIONAL LTD. PROJECT NO.: 454A-107 PRINCIPAL INVESTIGATOR- Jig,k.PaacGregor,B.S. Scientist MANAGENffi'NT: WillardB.N7,iT xon,Ph.D. Manager,AnalyticaClhemistry DATE DATE WILDLIFE INTERNATIONAL LTD. -33- PROJECTNO.: 454A-107 Introduction Saltwatersampleswere collectefdrom a flow-throughfe-cyclteoxicitsytudydesignedtodetermine the effectosf PFOS (PerfluorooctaSnuelfonicAcid PotassiumSalt)tothesaltwatemrysid (Mysidopsis bahia).Thisstudywas conductedby WildlifIenternationLatld.and identifiaesdProjectNo.: 454A-107. The analysesofthesewatersampleswere performedatWildlifeInteniationLatld.usinghighperformance .liquicdhromatographywithm,4ssspectrometridcetectio(nHPLC/MS).Sampleswere receivedforanalysis betweenJune 15,1999 and July21, 1999 and were analyzedbetweenJune 15,1999 and July22, 1999. TestSubstanceand InternaSltandard The testsubstanceusedforthisstudywas WildlifIenternationLatld.identificatniuomnber 4675. The testsubstancewas usedtopreparecalibratiaond matrixfortificatsiaomnples. The internasltandardwas receivedfrom 3M Corporatioonn July2, 1998 and was assignedWildlife InternationLatld.idenfificatniuomnber 4526 upon receiptT.he internasltandarda,granularmaterialw,as idenfifiaesd: IK IR 2K 2H PerfluorooctaSnuelfoniAccid,ChemicalAbstracNtu mber: 27619-97-2. 'Me standardwas storedunderambientconditions. AWyfical The method used fortheanalysisofthesaltwatesrampleswas developedatWildlifeInternationLatld. and entitle"dAnalyticaMlethod forthe Determinatioonf PFOS in FreshwaterS,altwatera,nd Algal MediurW'.Thismethodologywas includedas AppendixIIofWildlifIenternationLatld.protocolnumber 454/011299/MVAIJSUB454. Itwas basedupon methodologyprovidedby 3M Corporation. Samples were dilutedin a 50% methanol: 50% NANOpure@ water solutioncontaining 0.100mg4HPFOS (internasltandard)/aLnd 0.05% formicacid(v/v)so thattheyfellwithinthe calibratiroanngeofthePFOS methodology. Concentrationosf the PFOS in the standardsand sampleswere determinedby reverse-phasheigh performanceliquidchromatographyusinga Hewlett-PackarMdodel 1100 High PerformanceLiquid Chromatograph(HPLC) witha Perkin-EhneArPI IOOLC Mass Spectrometeerquippedwith a Perkm- WILDLIFE INTERNATIONAL LTD. -34- PROJECT NO.: 454A-107 Elmer TurbolonSprayion source. HPLC separationwsere achievedusinga KeystoneBetasil.Cls analyticaclolumn (50nun x 2 mm I.D.,3 pxnparticlseize).The instrumenptarameterasresummarizedin Table 1. A methodflowcharitsprovidedinFigure1. CalibratioCnurve and LimitofOuanfitation CalibratiosntandardsO@.YFOS preparedin a 50% medmnol: 50% NANOpure(D water solution containin0g.100nig4H PFOS (internasltandard)/aLnd 0.05% formicacid(v/v)r,anginginconcentration from 0.000915to0.00915mg a.i./Lw,ere analyzedwiththesamples.The same and mostprominentpeak responseforPFOS was utilizetdomonitorPFOS inallcalibratioqnu,alitcyontrola,nd studysamples.No attemptwas made toquantifyPFOS on thebasisof individuailsomericomponents.Linearregression equationswere generatedusingpeak arearesponsemtios(PFOS :internasltandardv)ersustherespective concentratiroantio(sPFOS: internasltandardo)fthecalibratisotnandards.A typicaclalibration.cuirsve ..presenteidnFigure2. The concentratioonf PFOS inthesampleswas determinedby substitutitnhgepeak arearesponseratiosintotheapplicablleinearegressioenquation.Representativieonchromatogramsof low and highcalibratiosntandardsarepresentedinFigures3 and 4,respectively. 'Me method limitofquantitati(oLnOQ) fortheseanalyseswas setat0.0458mg a.i./cLalculateads theproductofthelowestcalibratiosntandardanalyzed(0.000915mg a.i./La)nd thedilutiofnactorofthe matrixblankswnples(50.0). MatrixBlankand FortificatiSoanmple-s Six matrixblank sampleswere analyzedto determinepossibleinterferenceN.o interferencweesre observedatorabovetheLOQ duringsamplesanalyses(Table2). A representatiivoenchromatograionfa matrixblankispresentedinFigure5. Saltwatewras fortifiaetd 0.0823,0.366 and 3.66mg a.i./aLnd analyzedconcurrentlwyiththe samplestodeterminethemean proceduralrecovery(Table3). Sample concentratiownesre notcorrected forthemean proceduralrecoveryof92.8%. A representatiivoenchromatogramofa matrixfortificatison presentedinFigure6. WILDLIFE INTERNATIONAL L-rD. -35- PROJECTNO.: 454A-107 ExMple Calculations Sample number 454A-107-3,nominalconcentrationf 0.086mg a.i.AiLnsaltwater. Peak Area Ratio= AnalytePeak Area/IntemalStandardPeak Area ConcentratioRnatio ConcentratioonfAnalyte/ConcentratioofnInternaSltandard InternaSltandardConceptrafion0:.100 mg/L InitiVaollume: 0.500 ML FinalVolume: 25.0mL DilutioFnactor:50.0 PFOS Peak Area: 12963 InternaSltandardPeak Area: 145150 PeakArea Ratio:0.08931 Calibratiocnurveequation. Slope:5.76026 Intercept:0.00940 Curve isweighted (1/x). Peak area ratio- (Y-intercept) PFOS (mg a.i./La)t insh-ument x InternalStandard Concentration Slope 0.08931 - 0.00940 5.76026 x 0.100 0.001387 PFOS (mg a.i./Li)n sample = PFOS (mg a.i.ALa)t instnimentx DilutionFactor = 0.001387 x 50.0 = 0.06935 WILDLIFE INTERNATIONAL LTD. -36- PROJECT NO.: 454A-107 PFOS (mg a.i./Li)nsample PercentofNominal Concentratio-n PFOS (mg a.i.ALn)ominal X 100 -0.06935 0.086 x 100 80.6% RESULTS Sample Agalysis Saltwatersamples were collectedfrom the flow-throughhfe-cycletoxicitystudywith thesaltwater mysid (Mysidopsisbahia)atpre-testJ,une 15,1999,attestinitiatioJnu,ne 16,1999 (Day 0),and weekly duringthetestthroughtestterminatioonn July21, 1999(Day 35).The measuredconcentratioonfsPFOS inthesamplescollecteadtpre-tesrtangedfrom 57.3to 99.3% ofthenominalconcentratio(nTsable4). The measured concentratioonfs PFOS in the samplescollecteadt initiatioofn exposureof @letest organisms(Hour 0)rangedfrom67.1to 103% ofthenominalconcentratio(nTsable5). Samplescollected at Day 7,Day 14,Day 21 and Day 28 had measuredconcentratiornangesof 45.8*to95.6%, 70.5to 99.6%, 57.1 to 87.9% and 59.9 to 99.3% of nominalvalues,respectivelyS.am-plescollecteadt test terminatio(nDay 35) had a measuredconcentratiroanngeof 59.8to 90;0% of nominalvalues.A representatiivoenchromatograrnof a testsarnpleisshown inFigure7. WILDLIFE INTERNATIONAL L-m. -37- PROJECT NO.: 454A-107 INSFRUMENT: Table 1 TypicalHPLC/MS OperationaPlarameters Hewlett-PackarMdodel I100 HighPerformanceLiquid Chromatogmph witha Perldn-ElmeArPI IOOLC Mass Spectrometer equippedwitha Perldn-EhneTrurbolonSprayionsource.Operatedin selectiivoenmonitorinmgode (SM. ANALYTICAL COLUMN: KeystoneBawil Clscolumn(50mm x 2 mm I.D.3, pznparticlseize) OVEN TENDERATURE: 300C STOP TIME: FLOW RATE: MOBELE PHASE: 5.00minutes 0.220mljminute 72.0% Medmol: 28.0% NANOpureg Waterconmining 0.1% FormicAcid INJECTION VOLUME: 25.0 p@L PFOS RETENTION TIME: Approximately3.8to4.5minutes INTERNAL STANDARD RETENTION TUAE: Approximately2.6to 3.0minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 426.7mnu WILDLIFE INTERNATIONAL LTD. -38- PROJECTNO.: 454A-107 Table2 MatrixBlanksAnalyzedConcumntly DuringSaxnpleAnalysis Number (454A-107-) MAB-1 Slmgle Type MatrixBlank Measured Concentratioonf PFOS' (mg a.i.AL) < LOQ MAB-2 MAB-3 NlAB-4 MatrixBlank MatrixBlank MatrixBlank < LOQ < LOQ < LOQ MAB-5 MatrixBlank < LOQ MAB-6 MatrixBlank < LOQ The limiotfquantitati(oLnOQ) was 0.0458mg a.i.AbLasedupon theproductofthelowestcalibrafion standard@@W@zed,(0.000915 a.i./La)ndthedilutiofnitetofrthematrixblanks2m2les(50-2). WILDLIFE INTERNATIONAL LTD. -39- PROJECTNO.: 454A-107 Table 3 MatrixFortificatiAonaslyzedConcurrentlDyuringSample Analysis Sample Number (454A-107-) MAS-1 MAS-4 MAS-7 MAS-10 MAS-13 .MAS-16 MAS-2 MAS-5 MAS-8 MAS-1 1 MAS-14 NUS-17 MAS-3 MAS-6 MAS-9 MAS-12 MAS-15. MAS-18 Concentrationosf PFOS (ME a.i./L) Fortified Measured 0.0823 0.0748 0.0823 0.0828 0.0823 0.0673 0.0823 0.0662 0.0823 0.0693 0.0823 0.0787 Percent Recovered 90.9 101 81.8 80.4 84.2 95.6 0.366 0.350 95.6 0.366 0.359 98.1 0.366 0.314 85.8 0.366 0.303 82.8 0.366 0.360 98.4 0.366 0.287 78.4 3.66 3.29 89.9 3.66 3.54 96.7 3.66 3.39 92.6 3.66 3.88 106 3.66 3.80 104 3.66 3.98 109 Mean = 92.8 StandardDeviation= 9.19 CV = 9.90 N = 18 WILDLIFE INTERNATIONAL LTD. -40- PROJECTNO.: 454A-107 Table4 Measured ConcentrationosfPFOS inPre-tesDtiluterVerificatioSnamplesfrom a I I SaltwateMrysid Life-CyclTeoxicitTyest Nominal Test Concentration (mga.i.[L) 0.086 Sample Number (454A-107@ PT-1 PT-2 PT-5 PT-6 Sainpli-n-sz Time (Hours) -24 -24 -24 -24 PFOS Measured Concentration (ME a.i./L)1.2. 0.0581 0.0493 0.0627 0.0606 Percent of Nominal 67.6 57.3 72.9 70.5 2.7 PT-3 -24 2.21 81.9 PT-4 -24 2.34 86.7 PT-7 -24 2.56 94.8 PT-8 -24 2.68 99.3 1 The limitofquantitatio(nLOQ) was 0.0458 mg a.i./bLasedupon theproductofthelowestcalibration standardanalyzed(0.000915 mg a.i./La)nd thedilutiofnactorofthematrixblanksamples(50.0). 2 Measured valuesofmatrixblanksand matrixfortificatsiaomnplesanalyzedconcurrentlwyithpre-test sampleswere <LOQ, 83.0,95.6,100,<LOQ, 93.4,85.0and 106 percentofnominalconcentrations forpre-tesItand pre-tes2t,respectively. WILDLIFE INTERNATIONAL LTD. -41- PROJECT NO.: 454A-107 Table 5 Measured ConcentrationosfPFOS inSaltwaterSamples from a SaltwateMrysid Life-CyclTeoxicityTest Nominal Test Concentration (mga.i./L) 0.0 Sample Number (454A-107-) 11 2 15 16 29 30 43 44 55 56 67 68 Sampling Time 0 0 7 7 14 14 21 21 28 28 35 35 PFOS Measured Concentration (mga.i./L) < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Percent of Nominal - - 0.086 3 0 0.0694 80.7 4 0 0.0578 67.2 17 7 0.0478 55.6 18 7 0.0619 72.0 31 14 0.0606 70.5 32 14 0.0614 71.4 45 21 0.0554 64.4 46 21 0.0509 59.2 57 28 0.0515 59.9 58 28 0.0569 66.2 69 35 0.0580 67.4 70 35 0.0514 59.8 The limiotfquantitati(oLnOQ) was 0.0458mg a.i./bLasedupontheproductofthelowestcalibration standardanalyzed(0.000915mg a.i./La)nd thedilutiofnactorofthematrixblanksamples(50.0). WILDLIFE INTERNATIONAL LTD. -42- PROJECTNO.: 454A-107 Table5 (continued) Measured ConcentrationosfPFOS inSaltwaterSamples from a SaltwateMrysid Life-CyclTeoxicityTest NominalTest Concentration (mg a.i./L) 0.17 Sample Number (454A-107-) 5 6 19 20 33 34 47 48 59 60 71 72 Sampling Time (Day) 0 0 7 7 14 14 21 21 28 28 35 35 PFOS Measured Concentration (rnga.i./L)I. 0.125 0.114 0.0778 0.125 0.124 0.127 0.0970 0.112 0.122 0.128 0.124 0.119 Percent of Nominal 73.5 67.1 45.8 73.5 72.9 74.7 57.1 65.9 71.8 .75.3 72.9 70.0 0.34 7 0 0.289 85.0 8 0 0.286 84.1 21 7 0.231 67.9 22 7 0.197 57.9 35 14 0.276 81.2 36 14 0.253 74.4 49 21 0.227 66.8 50 21 0.212 62.4 61 28 0.262 77.1 62 28 0.271 79.7 73 35 0.278 81.8 74 35 0.251 73.8 The limiotfquantitati(oLnOQ) was 0.0458mg a.i./bLasedupontheproductofthelowestcalibration standaradnalyzed(0.000915mg a.i.ALa)nd thedilutiofnactorofthematrixblanksamples(50-0). WILDLIFE INTERNATIONAL LTD. -43- PROJECT NO.: 454A-107 Table5 (continued) Measured Concentrationosf PFOS inSaltwaterSamples from a SaltwateMrysid Life-CyclTeoxicitTyest Nominal Test Concentration (m a.i./L) 0.69 Sample Number (454A-107-) 9 10 23 24 37 38 51 52 63 64 75 76 Sampling Time (29-) 0 0 7 7 14 14 21 21 28 28 35 35 PFOS Measured Concentration (mA a.i.AL) 0.562 0.659 0.581 0.450 0.543 0.542 0.516 0.528 0.529 0.544 0.556 0.583 Percent of Nominal 81.4 95.5 84.2 65.2 78.7 78.6 74.8 76.5 76.7 .78.8 80.6 84.5 1.4 11 0 1.23 87.9 12 0 1.32 94.3 25 7 1.13. 80.7 26 7 1.20 85.7 39 14 1.35 96.4 40 14 1.27 90.7 53 21 1.23 87.9 54 21 1.15 82.1 65 28 1.39 99.3 66 28 1.39 99.3 77 35 1.26 90.0 78 35 1.20 85.7 "Me limitofquantitati(oLnOQ) was 0.0458mg a.i./bLasedupon theproductofthelowestcalibration standardanalyzed(0.000915mg a.i./La)nd thedilutiofnactorofthematrixblanksamples(50.0). WILDLIFE INTERNATIONAL LTD. -44- PROJECT NO.: 454A-107 Table5 (continued) Measured ConcentrationosfPFOS in SaltwateSramples from a I SaltwateMrysid Life-CyclTeoxicitTyest Nominal Test Sample Sampling PFOS Measured Percent Concentration Number Time Concentration Of (mB a.i.AL) (454A-107-) (Day) (mg a.i.AL) Nominal 2.7 -'13 0 2.56 94.8 14 0 2.79 103 27 7 2.58 95.6 28 7 2.30 85.2 41 14 2.54 94.1 42 14 2.69 99.6 The limitofquantitati(oLnOQ) was 0.0458mg a.i./bLasedupontheproductofthelowestcalibration standardanalyzed(0.000915mg a.i./La)nd thedilutiofnactorofthematrixblanksamples(50.0). WILDLIFE INTERNATIONAL LTD. -45- PROJECTNO.: 454A-107 MEETHOD OUTLINE FOR THE ANALYSIS OF PFOS IN SALTWATER Preparmeatrxifortificaitsiaomlpilesby spikingtherequisitveolumeofPFOS stocksolutiondsirectly intofilteresdaltwateursinggas-tighstyringeasnd ClassA volumetrifclasks. Dilutematrixfortificatainodntestsamplesintotherangeofthecalibratisotnandardsby partiallfyilling ClassA volumetrifclaskswith50% methanol:50% NANOpureg watersolutiocnontaining 0.100mg 4H PFOS (internsatlandard)/aLnd 0.05% formicacid(v/v)A.dd the Appropriatevolume ofsmnpleand bringtheflasktovolumewiththeOution solvent.Processthematrixblanksampleusingthesame dilutioannd aliquot volumeasforthelowestfortificatlieovnel.Mix wellby several repeatinversions. Ampulate samplesand submitforLCMS analysis. Figure1. Analyticamlethod flowchartfortheanalysiosfPFOS insaltwater. WILDLIFE INTERNATIONAL LTD. -46- PROJECTNO.: 454A-107 0.70-,-, 0.600 0.500 0.40- 0.30- 0.200 10- 0.00 0.000 0.020 0.040 0.060 Q080 Concentratio(nRatio) 0.100 Figure2. A typicaclalibraticounrvefbrPFOS. Slope 5.76026;Intercept0.00940; r 0.9974.Curveisweighted(I/x). WILDLIFE INTERNATIONAL LTD. -47- PROJECTNO.: 454A-107 IF OUbm" Fft 00 Mft 1"*m Pw &M I=LC Mm@,.o@ at@N-1w"as */w L% c"b -a" 6 am". soi. cc a ftk*ft .1 ..kw pma-lel we aftloo Cff InI pftd awd 1: 4@M 01 K 119 424.7 Mdo linl 2 OL.6 7k- o L%L Wim muL waft 6 8@. VAdi% is FrrvArL ONO 2I0 &m..& EW*dAd RT L7$ Am 196289 H.wd 14ods stotTkw L" EndTkm WWih 322 m lb" Two A.22"* ww. 4- It.1"8 16..Is Ice so 5* 4* 30 9 1 29 21 41 *1 BAB 1.72 X" ke.@,.kr.14*" We ii 11 1100,, 112 LU 4.23 vmw mboa@.."Is Pow* lba A- 17. "a nu ewblm" Fftod FM *&ARM pdk AN 1*= NDIb..Ct- IAAIOAI@-16PJNW, 4"'6'$WPP=WWUbOrde-Dsy6l&ARM. PpoiLl" 49?L%4*4C@l am SAMOI 4S? IsI poled FFCB ,, 'skmft* 4ffm kke Am Abu&& rabwdlonr@ 1: 4M OIL% lie so@ 4".# "do iww amtlkvl &%L VAdh Ab& wwa 10.0 1.0 3 BAILVAdk I VAILiono 15 I Prr &17. An@ 1764 Howd 674 am Two &47hn 4j3 wift GA3 a Tbm A La TV" vo W-4 am 11Ltno 14kis 100 00 so To 14 :0 :0 an 1.72 L,n 94 79 L30 P".2.146 ty,No we Figure3. A representatiivoenchromatogramofa low-leve(l0.000915mg a.i.ALP)FOS standard. WILDLIFE INTERNATIONAL LTD. -48- PROJECTNO.: 454A-107 C.S..K- CM "09 I"MW ftftA'N IMC $9"6-CW- C-@ "MKAJ@W PFM Mo.M Lft eyd. - C" 0 "*M. 8-0@ ddr." 00 U..bkft "@,o PMS-Ios wm BMW CR M Ipftd *VFM 426.7 NolaTMM O.Ma Th@ U. vvm Vdft Vdft frrV*L "ce) SWAGO EWWWRT .2-0. 3 6I. gIo L7* AM* MONM 173036 14211 Sm Thm &d Tbm vam , 9PAMnbn Tbu TV* La &22 an I= A.n WOC JM 16.1"9 1&41 47 100 TO 60 4 :*o 17 21 1 62 4 am 411.72 aLla *&1" @ieft14211qm to I*$ 14*.1= Tb" PON.* ThM A. 7.IM OM 020mdkoFlaCidPFOO ISAWN Fft AFI10= MD@ l@lff-"AWW cwmmft 4"&'" "M blowLibCvt%-Ow 6 T&ANM. BwOn dmWmW n oc m emwdw .1M.&W ftp io @fas PPO*-IW WTD am4o f.I pbld '4HPFOO LbeAM Abnko rahmem Tb" 1: 424 ai &k 110 awe 49@A Nobw T%NL amalk@ 10.0 IA MkL v@ VADL WASK som vadeb PRYALOMO as &TAGO 6*90wxr Am so3so H@WK 5"4 ft" T.0 LOO 4= wwm *A YbW bftpomm Two A-3v2a3 W.@t JM 14.IM 14L41 100 70 00 so, 40 10 bo-W- sm ape 94 77.9 @ 4-1 In am Lu 426 To* Figure4. A representatiivoenchromatograronf a high-level(0.00915mg a.i./LP)FOS standard. WILDLIFE INTERNATIONAL LTD. -49- PROJECTNO.: 454A-107 C--ft ow pme %OAOO Pft Am imc wAt..CkPFM *OW La Oob -OW a MMM. &.00 b mUiMg p@ a,WWW MMM.MM Pftg-lfr 4MA-10?- w om I pw" bft-wftow" V#-C 1- 19. it" It:" 106 426.7 "on W*OL a 02 at" Thrm a' L&L W%la 8* :00 "A YAM a a@. vacmh Is To Kr VA& "ON) 20 EWMW AM FIT LTG 17,too MMON 14$SD IM am Ti- &4 TbrA &10 kftwub" vrlm 043 TW4 TWO A.Inas 10 - b*@%Wr. 14NO We of is 20 32 2. 47 21 4I1." lfl Gig L" 87 97 100 g', 141 &30 442 To* P*"* lb.LA- ft%mlm a cmmm.w=ny 7.im "M POW "ANN FMS mvw Pok API"KC NDMue0@ I% opk -ow 0 *LROM. - I*AS"14?-ILLWW dm*ww cc 0 bww p@ a -.00 --Wm@ fti@ 14 PFWIGY 454A-107- 487toI pwbg PFQB tawo a - -44-Fw LbsAft& AbNko PAWmImThm 1'.44401 K litmova 4".@ . Ndwyk,"L 10.0 awe TOML MLVAM VAILYAM 1.0 OrrVft 1 8-046 riwmftwd &W Aft 0 76M so7b" VAM FAMFOAR TV" bftraftnTwo -0-00 -am V144JM 14,It" 14.'64 14 Ica so 30 to 10 L 21 CAN In$ qw es 7411 03 41 4@l ii 101 sm 1.72 In 423 Tws FigureS. A representativoenchromatograionfa matrixblanksample(454A-107-MAB-1). The arrowindicatetsheretentiotnimeof PFOS. WILDLIFE INTERNATIONAL L-m. -50- PROJECTNO.: 454A-107 M..a@ V.." 1.6 ft@ U. i- 'TI.- @ aw,ow ft.ammm "mm Pftm loomN)W--Cb- o-.@ mw&mw mm Wpm Lft*od. - ow a I"Mn. 8-0- *Wr@w m ae akdm O.Pow M nom*Ai ft" 15.1a PF"-M 464A-la?- MAS-1 4L$? 1.1 PWW 4ffm u WdW,,OdWdod Wed. Jm 10. IWO 17M 1: 4.94Ot kk 426.7 Nab$ CMK,OM TThh@@ M" L vrlm 0, vom VtA a Kr V*L ON&) 110SWO 2.0 :.2 9I 20 Eq"c"d RT L7$ 166709 Ar" 1 H-W rAd Tk. 2916 E.dTh" *"Vo" Wkth PAWnftnThm 231 L14 CA3 2.72 10& so so 40 30 14 0r 9::i@ om Is 3 4@4 -64 1.72 L" bd@.W. 12fiS q. 8.1L-r 94-ITOIIG 3m 4.23 Th- Po" WA A. 17.im am CWbmgm.I:Cd PPOS 1"$M Puft APOIODLC NO*IMWMM I.OAWM7-1"NW. AIOTPMMWMLftCVW-DWOIOANM. S..O. d.1pw CIO FM_la 464A@107- MAS-1 4J" InI pwW ". M surd" 4HPFOB the AM AB$WM R.6.OM TWM 1: 4A4 01 &% 1% am* 418.4 Now Th@ 10.0 amtw@ 1*0 S*L vom 3 &&a VAL" SOL VAM 4$ wr V*L in NT i. Am& 14"8 meigm lose am mm Lu cis vom ow ftbnftnr@ &86 T"o A.VB wact im Is.low 1700 00 90 so TO so to 40 10 14 21 am IM qw 92 77 41,2 11 1 &a &1 101 am tu 4.U Th" Figure6. A representativoenchromatogramofa matrixfbrtificatsiaomnple(454A-107-MAS-1). WILDLIFE INTERNATIONAL -51- PROJECT NO.: 454A-107 POK C.1 PPOI IOAMN POt AP4 IOKZ HD*fimWW~ IAA54AIar-ISAOM: "44,."? "08 WON LOS C"k - OW 0 UANM. SmOn ddv"W a CC b WMft P@ r @&A rpm-ll4 s 464A-107- 4wm Pod" 4HPFOS 1 woc ;m IIL low ir-30 1: 4m al #A In 4".T N.%o 7b@ 2.0 Ch" lbft& 03.2 tft VAM V..L VACM 1 e@ TAft Is FrrWIIL ONO 20 amoca I "-"d III.,I L76 Ana 14 10 awl Tbw ow ybm vr"m pA6wlftn WAWEV- T)" lil 3.14 am L72 A-se 100 66 70 so 30 *0 14 0. 6 4@@ 40 48 21 ' 471 f@i OAB 1.72 am "Nft lign cps $247 -IGO lot s"i 101 am 3@w 4m r@ 1.6 PA."* iw " 't. ion 4426 Ft. ad PRO 1"0" pw?c AN to" fc%b..Q- c@ 454&tw"mwpmub*Ab.fto SLUM. a."" 4 CC b *W" P-M d ftm.00- "09-114 a 4S4A@107- .427 b I pww we.& im 14.to" I?= :lm- *VFOB A%Wh& FAiwOm Tbw 1: 4A4 01 Lt.lis 10.0 13 .0 AOL Wkm VAIL W.0 @ML VAdik 4 grrvft ofto SNAG& EVOCkidffr Lp? Am Ilse$ Hdlht 897 a" um Evdlbu ., Who PA&@ rpm as$ 4A2 w SAvGs A 14 100 so me 70 so 40 le 4 1 .. . . . li, om I.M st 76 am &" ftpmoft U" Figure7. A representatiivoenchromatogramofa testsample(454A-107-3). WILDLIFE INTERNATIONAL, LTD. -52- PROJECT NO.: 454A-107 APPENDIX rV Spomnsor TestSubstance: TestOr D iIu tvio ngawmasetmr:: Mean asured TestConcmtration (MR &i.@L) NegativeControl MLsid EEEoduction oration 'PFMOCS7p SaltwateMrysid,Mysidopsisbahia FiltereSdaltwater Test ESEcate c Number of Reproductive Dgs' A 1 14 2 14 3 14 4 14 5 14 B 1 14 2 14 3 14 4 11 Number of Young Produced 3 2 6 5 2 4 8 2 0- Number of Young/Reproductive Dav 0.257 0.264 c 1 2 3 4 5 14 6 14 3 14 4 14 5 14 2 0.286 D 1 2 3 14 9 14 6 14 4 0.452 0.057 A 1 2 3 4 5 14 3 14 6 14 3 14 5 4 0 .0.283 B 1 2 3 4 5 14 2- 14 3 14 5 14 0 14 4 0.200 c 1 2 3 4 5 14 3 14 3 14 3 14 0 14 4 0.186 D 1 2 3 4 14 2 14 10 14 7 14 2 0.375 'Me number ofreproductivdeaysisthenumber ofdaysthathefemalewas alivefromfirsbtroodreleasteothe end ofthetesl WILDLIFE INTERNATIONAL, LTD. -53- PROJECT NO.: 454A- 107 APPENDIX IV (Continued) Mysid PLproduction ?e2 smur@stance: TestOrganism: DilutioWnater: 3M Corporation PFOS SaltwateMrysid,Mysidopsisbahia FiltereSdaltwater TeMsetaCnonMceeansturraetdion Test (mga.i./L) l@gficate C2Martment Num -o? Reproductive DMI A 1 14 2 14 3 14 4 14 5 14 Number of Young Produc6d 6 0 8 6 1 Numb Young/Reproductive Day 0.300 B 1 2 3 4 14 7 14 8 14 5 4 2 0.478 c 1 2 3 4 14 7 14 9 14 4 12 2 0.407 D 1 2 3 4 5 14 9 14 2 14 0 14 5 14 2 0.257 0.25 A 1 2 3 4 5 14 2 14 4 14 2 14 6 14 5 0.271 B 1 2 3 4 14 4 14 1 14 7 14 0 0.214 c 1 2 3 4 5 14 2 5 2 14 7 14 6 14 4 0.344 D 1 2 3 4 5 14 1 14 0 14 3 14 6 0.179 The number ofreproductivdeaysisthenumber ofdaysthatthefemalewas alivefrom firsbtroodreleasteothe Ofthetest. WILDLIFE INTERNATIONAL, LTD. -54- PROJECTNO.: 454A-107 APPENDIX IV (Continued) Mysid Reproduction Sponsor: TestSubstance: TestOrganism: DilutioWnater: Mean Measured TestConcentration Lmg a.i.U 0.55 3M Corporation PFOS SaltwateMrysid@Mysidopsisbahia FiltereSdaltwater Test Balicate CM2artinent Number of Reproductive Do' A 1 14 2 14 3 12 4 14 Number of Young Produced 0 3 0 0 Number of Young/Reproductive D 0.0556 B 1 2 3 4 14 0 14 0 14 2 14 4 .0.107 c 1 2 3 4 5 14 0 14 0 14 0 14 3 14 0 0.0429 D 1 2 3 4 14 0 14 0 14 0 14 1 0.0179 1.3 A 1 2 3 3 0 11 0 8 0 0.000 B 1 14 0 0.000 c D 1 11 0 0.000 ThenumberofMroductiveks isthenumberof@ffsthathefemalewas alivferompairintgotheendofthetesl Mysid Number 1 2 3 4 5 6@ 7 8 9 10 11 12 Mean Rep A 6.80 6.85 6.30 6.45 6.30 6.75 6.15 6.65 6.30 6.50 5.85 - 6.45 Nega6ve Control Rep B 6.20 6.65 6.55 6.95 5.90 6.10 6.00 - 6.34 Rep C 6.50 6.85 6.20 6.60 6.25 6AO 6.15 6.30 6.70 6.65 - 6.46 Rep D 6.55 7.10 6.35 6.10 6.40 6A5 6.30 6.55 6.48 -55APPENDIX V Mysid TotalLength(mm) Rep A 6.15 6.30 6.35 6.15 6.55 6.90 6.20 6.65 6.25 6.40 6.15 6.50 6.38 0.57 mg a.i.AL Rep B 6.35 6.20 6.15 6.70 6.45 6.60 6.30 6.80 6.30 6.40 6.50 6.65 6.45 Rep C 6.05 6.80 6.55 6.70 6.45 6.65 6.50 6.70 6.30 6.50 - 6.52 Rep D 6.30 6.45 6.40 6.90 6.55 6.45 5.90 6.35 5.95 6.35 - 6.36 Rep A 6.25 6.7,0 6.40 6.55 6.60 7.10 6.35 6.40 6.25 7.00 6.55 6.40 6.55 0.]2mga Rep B 6.75 6.60 6.55 6.80 6.45 6.90 6.50 - 6.65 Mysid Number 1 2 3 4 5 6 7 8 9 10 II 12 Mean Rep A 6.55 6.40 6.35 6A5 6.50 6.25 6.55 6.70 6.45 6.60 - 6.48 0.25 mg a.i.AL LRM B 6.25 6.40 6.30 6.15 6.45 6.90 6.35 6.20 - - 6.38 Rep C 5.95 6.35 6.80 6.10 6.75 6.20 6.40 6.35 6.35 - 6.36 EMD 6.20 6.45 6.50 6.35 6.75 6.55 6.05 6.80 5.75 - 6.38 -56- APPENDIX V (Continued) MysidTotalLtno (mm) ReE A 6.05 5.85 6.10 5.90 5.80 6.35 6.30 - 6.05 0.55mg a.i./L Uc B 6.05 6.25 6.35 6.20 6.00 6.20 6.15 6.60 5.05 6.25 - 6.11 Rep C 6.20 6.20 5.85 6.00 6.10 6.35 6.25 6.40 6.25 6.00 - 6.16 Ee2 D 6.20 6.45 5.95 6.10 6.25 6.40 6.10 6.45 - - 6.24 ET A 5.85 6.05 5.90 - - 5.93 Rcg B 5.75 6.25 5.85 6.05 - - 5.98 Sponsor: TestSubstum: DTi-l'ut.to Mysid Rep Number A 3M Corporation PFOS SaltwatMeyrsidM,ysidopsbiashia FiltereSdaltwater NegativeConhol Rep Rep Rep B c D 1 51 0.57 0.49 0.68 2 0.70 0.77 0.78 0.86 3 0.'52 70 0.60 0*54 4 0.64 0.86 0.57 0 59 5 0.56 0.61 0.51 0.'53 6 0.75 0.75 0.72 0.67 7 0.44 0.68 0.50 0.60 8 0.79 - 9 0.58 - 0.67 0.62 0.52 - 10 0.69 - 0.60 - 11 0.41 - - - 12 - - - - mean 0.599 0.706 0.596 0.636 -57- APPENDIX VI Mysid Dry Weight (mg) Rp A 0.53 0.62 0.58 0.64 92 0.79 0.51 0.66 0.49 0.57 0.50 0.69 0.616 0.057mg a.i./L Rp Rep B c 0.65 0.45 0.50 0.78 0.55 0.60 0.69 0.56 0.47 0.63 0.84 0.59 0.52 0.54 0.73 0.71 0.61 0.49 0.72 0.55 0.66 - 0.58 0.627 0.590 Rep D 0.59 0.51 0.46 0.80 0.57 0.52 0.46 0.67 0.49 0.58 - 0.565 Rep A 0.52 0.71 0.65 0.70 0.72 0.80 0.54 0.59 0.59 0.93 0.49 0.63 0.647 0.12 Rep B 0.97 0.62 0.55 0.68 0.64 0.71 0.59 - 0.664 STPZ S-.I@u.: TDTItZW*.t.@ Mysid Rep Nwnbcr A 3M Corpmtion PFOS SaitwatMcyrsid,Mysidopisbahia FiltcmdSaltwawr 0.25mg a.iJL Rep Rep Rep B c D 1 72 0.45 0.50 0.61 2 0-.60 0.68 0.62 0.67 3 0.54 0.62 0.96 0.59 4 0.55 0.53 0.45 0.53 5 63 0.58 0.71 0.84 6 0.48 0.79 0.55 0.73 7 79 0.60 0.51 0.52 0-71 0.56 0.67 0.80 91 0'* 67 - 0.56 0.46 to 0.75 - - II - - - - Me= 0.644 0.601 0.603 0.639 -58- APPENDIX VI (Continued) Mysid Dry Weight(mg) Rep A 62 -00..51 0.54 0.45 0.43 0.71 0.63 - - 0.556 0.55mita.i.AL Rep Rep B c 0.50 0.57 0.48 0.42 0.67 0.39 0.62 0.61 0.57 0.51 0.61 0.60 0.54 0.91 0.59 0.65 0.36 0.48 0.64 0.59 - - 0.558 0.563 Rep D 0.54 0.66 0.52 0.69 0.55 0.60 0.49 0.52 - - 0.570 Rep A 0.36 0.52 0.44 - - - 0.440 1.3m Rep B- 0.40 0.57 0.43 0.51 - - - 0.478 WILDLIFE INTERNATIONAL, LTD. -59- PROJECTNO.: 454A-107 APPENDIXVEI Changes toProtocol Thisstudywas conductedm accordancevaththeapprovedProtocolwiththefollowincghanges: I The proposedexpcnmentalstaratnd t=mation dateswereamended totheprotocol. 2. AnalysisoffeedforPFOS was deletedby amendment. 3. The proportioonfwatersplitoeachreplicatweas notcheckedattheend ofthetest. 4. Temperaturewas notmeasuredintheB replicatoefthe2.7mg a.i./tLreatmengtroupon Day 14 ofthetest. 5. Nominal testconcentratiownesre 0.086,0.17,0.34,0.69,1.4and 2.7mg a.i./L. WILDLIF'E INTERNATIONAL, LTD. -60- PROJECTNO.: 454A-107 APPENDIXVIH PersonnelInvolvedintheStudy The fohowmg keyWildlifIenternationLatld,.personnewleremvolved m theconductormanagement of tlusstudy: 1. Henry0. KruejerP,h.D.,E"r, AquaticToxicologayndNon-TargetPlants 2. WillardB.Nixon,PILD.,Manager,AnalyticaClhemistry 3. JonA. MacGregor,Scientist 4. Mark A. Mank, LaboratorySupervisor 5. KurtR- DrottarS,eniorBiologist PROTOCOL PFOS: A FLO.W-THROUGH LIFE-CYCLE TOMCITY TEST WITH THE SALTWATER MYSID (Mysidopsibsahia) US. EnvironmentaPlrotectioAngency ,,Serie8s50 -EcologicaElffectsTestGuidelines OPPTS Number 850.1350 3M Lab RequestNo. U2723 Submittedto 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St Paul,Minnesota55144 -WILDLIFE INTERNATIONAL 8598 Commerce Drive Easton,Maryland21601 (410)822-9600 LTD. May 13,1999 WILDLIFE INTERNATIONAL, L-ID ,2- PFOS: A FLOW-THROUGH LUTCYCLE TO)UCrrY TEST WITH THE SALTWATER MYSID (Mysidopsibsahia) SPONSOR: 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St PauL Minnesota55144 SPONSOR!S REPRESENTATIVE: Ms. SusanA. Beach TESTING FACILITY: WildlffIenternationLatld,.. 8599 Commerce Drive Easton,Maryland 21601 STUDY DIRECTOR- KurtDrottar SeniorAquaticBiologist LABORATORY MANAGEMENT: Henry0. Krueger,Ph.D. DirectoorfAquaticToxicology& Non-TargetPlants FOR LABORATORY USE ONLY ProposedDates: Experimental StartDate: Experimental TerminationDate: ProjectNo.: TestConcentrationsN:egativeControl0,.094.0.19.0.38.0.75.1a.n5d 3.0mg a.i./L TestSubstanceNo.: 4675 ReferenceSubstancNeo. (ifapplicable)4:526 PROTOCOL APPROVAL STUDY DIRECTOR DATE ORATORY MANAGEMENT ORI Sll NSOR!S REPRESENTATIVE PROTOCOLNO.: 454/051399/MYS-LC/SUB454 DATE DA4 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -3- INTRODUCRION WildlifIentm-nationLatLd.willconducta flowthroughhfe-.cyctloemcitytestwiththesaltwater mysid,Mysidopsisbahia,fortheSponsorattheWildlifIenternationLatLcLaquatitcoxicologfyaciliitny Easton,Maryland. Tlw st* willbe performedbased on proceduresintheU.S.EnvironmentalProtection Agency Series850 -EcologicaElffectsTestGuideh= OPPTS Number 850.1350 (1);andAS7MStandard El 191-90 Standard Giddefor Cond@ctingLife-CycleToxicityTests%ithSakwater Wds (2).Raw data forallwork performedatWdWe InternationaLlt,d.and a copy ofthefinalreportwillbe filedby project number in=hives locatedm theWHME InternationaLlt,d.siteo,r ata'aalternativleocationto be specified inthe finalreporl PURPOSE The purpose of thisstudyisto determinetheeffectsof a testsubstanceon the saltwatermysid, Mysidopms bahia,dunng cbromc exposure.Effectson growth,surrivaland reproductimwillbe evaluated. EXPERIMENTAL DESIGN Mysids willbe exposed to a seriesof sixtestconcentrationas,negative(dilutiownater) control begmmg atthejuvenilestage(<24 hoursold)and contmumg foratleast7 days afterthemedian b= of first brood releaseinthecontroltreatment.The testwillnotbe terminateduntilatleast28 days and may be continuedattherequestof theSponsor. Test concentrationwsillbe chosen inconsultatimwith fficSponsor and willbe based upon known toxicitdyat& Each testconcentratiownillbe 50% of the,next higher treatment. Four replicatteestchambers willbe used foreach ftabnent and controlgroup,and each testchamber willcontainone compartment holding15 mysids. Inordertocontrolbias,mysids willbe indiscriminately assignedto exposurechambers attestinitiatioNrol otherpotentiaslourcesofbiasareexpectedtoaffectthe resultsof the study.Mysids willbe indiscriminatedliystributeidn groups of one to threeamong glass be&crs representmgeach testcompariment untileach beakercontms 15 mymds. The mysids willthen be gentlytransferremdto thetestcompartments. Thus, a totalof 60 mysids willbe exposed in each treatment and controlgroup.Aftermysids attainsexualmatanty (umally 10 to 14 days afterthebeginningof the test), femaleand male adultmysids willbe pafira and thereproductionofthepaurd mysids willbe monitored. PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -4, Observationsformortalityand clinicaslignsof toxicitwyillbe made daily The number of liveyoung produced by eachpairwillbe counted,recorded,and removed to a separatetestcompartment atthesame test concentratioonn a dailybasis.The young willbe exposed understaticonditionsfor96 hours. The dry weightand lengthof first-generatimoynsids aliveattheend of thetestwillbe determined.Data on'survival, growth,and reproductionwillbe analyzedtodetmnine theeffectosf thetestsubstancem theseparameters. MATERL4,LS AND MIETHODS Test Substance Informationon thecharacterizatiofn test,controlor referencesubstancesisrequiredby Good LaboratoryPracticeStandards(GLP). The Sponsor isresponsiblfeorprovidingWildlifeIntemationaLLtcL writtenverificatiotnhatthetestsubstancehas been characterizeadccordingto GLPs priorto itsuse in the study.IfwrittenverificatioofnGLP testsubstancecharacterizatiiosnnotprovidedtoWildlifeIntemationaL Ltd.,itwillbe notedintheempliance statemmt of thefinalreport.The &Adwd form IDENTMCATION OF TEST SUBSTANCE BY SPONSOR (Appendix I)istobe used toprovideinformationnecessaryfor GLP compliance. The Sponsor isresponsiblfeorallinformationrelatedto thetestsubstanceincludingtheretentionof a reservesample of the lotor batch of the testsubstanceused in thisstudy. The Sponsor alsoagreesto acceptany unused testsubstanceand/ortestsubstancecontainersremaining atthe end of thestudy. E=aration ofTestConcentrations The testsubstancewillbe admini@ tothetestorganisminwater. Thisrouteof %,dtnintiration was selectedbecause itrepresentsthemost likelyrouteof exposureto aquaticorganism . Test Organis The nrysidM,)sidopsisbahia,willbe used inthistest.This speciesisrepresentativoef an important group of organisms and was selectedforuse inthe testbased upon past use and ease of handlinginthe laboratory.Mysids willbe obtainedfrom WildlifeW=ational, Ltd.cultures.AU mysids used inthetest willbe from the same brood stock. PROTOCOLNO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -5- Testswillbeinitiatweidthmysidslessdm 24-hoursoldouveniles)C.ulturewsillbe maintaineidn a recirculatisnagltwatesrystemkeptatapproximatel2y5*C. Saltwatewrillbe filteretdhrougha biofilter andUV sterilizperdiortoenterintgherecirculatisnygstealAR mysidswUl be heldinthelaboratorfyorat least14 daysbeforejuvenileasrecollectefdortestingM.ysids willnotbe subjectetdo more thana 30C changeintemperaturoera 3 */,.changein salinitiynany 12-hourperiodduringthe14 daysbeforetest initiatioMny.sidswillbefedlivebrineshrimpnauphi(Arteimasp.)ad libitumdaily.Brineshrimpwillbe periodicalelnyrichewdithALGAMAC-2000 (fattaydd supplement)F.eed(br@ shrimp)providedtothe niysidwsillbe analyzedforPFOS. Excessfoodwillbe removed from thetestchambersdaily. Mysids willbe handledasinfmquentlyaspossiblebutwhen handlingisnecessaryitwillbe done gentlyc,arefullayn,dqmckly A siphoncollectosrystemvnU be usedtocorectneonatemysidsdu=tly fi-om broodtanks.Young mysidswillbe transferruesdinglarge-borpeipettes. DilutioWnater Naturalseawatercoll=W atMan RiverInletD,elawarewillbe thesourceofthedilutiownaterused inthecult= faciliatnydtestsystealThe seawaterwillbe filtertehdrougha sandfiltepriortoitsdehvery toa 37,800-Lholdingtank-The salinitoyftheseawaterwillbe dilutetdoapproximatel2y0 0/.(partpser thousand)withWildlifIenternatioLntadl.weuwaterintheholdingtank 'Me 20 0/.saltwatewrin be aerated usingspraynozzlesandfiltere(d0.45@m) priortoitsdelivertyothetestsystem. Salinitwyillbe measuredweeklytomonitortheconsistencoyf thesaltwaterM.eans and rangesof themeasuredparametersforthefour-weekperiodprecedingthetestwillbe providedinthefinalreporl Analyseswillbe performedtodeterminetheconcentrationosfselecteodrganicand inorganiconstituents oftheseawaterandresultosftheanalyseswillbe summarizedinthefinalreport. TestApparatus A continuous-fldoiwlutewrillbe usedtoprovideeachconcentrationfthetestsubstanceand a negativ(edilutiwoanter)controlA. syringepump, peristaltpiucmp, or a similadrevicewillbe usedto delivetrhetestsubstancteomixingchamberswherethetestsubstancveaU be mixedwithdilutiownater The flowofchlutiownaterintoeachmixingchamberwillbe controlleudsingmtameters.The rotwneterwsillbe calibratepdriortothetestandverifieadnd/orcalibrateadtleasotnce aweek duringthetest.Aftermixing, PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -6- testsolutionswillbe splitoeach replicatcehamber. The proportionofwatersplitoeachrepheac willbe chockepdriortothetest,atapproximatelyweekly thm=fter and attheend of thetesttomm thatthese flow ratesvary by no more than 10% of themean. The Euter willbe adjustedso thateach testchamber receivesatleast5 volume additionsof test solutionevery24 hours. Peristaltpiucmps willbe calibratepdriorto testinitiatioand atapproximately weekly mtervalsth=after and syringepumps willbe calibratepdriortotestuntiationn.e deliveryofthe testsubstanceto thetestchambers willbegin atleast48 hours priorto the testin order to establish equilibriumconcentrationosf thetestsubsta=. The generaloperationof thedilutewrillbe checkedvmwly at leasttwo times per day duringthetest. Testchambers willbe 9-L glassaquariafillewdith approxim@ 5 L of testsolution.Each treaunent and controlgroup willhave fourreplicatteestchambers. Test chambers willbe impartiallpyositionedinan environmentalchamber designedto maintaina tmnperatureof 25 2*C. Test chambers willbe labelledwith theprojectnumber, testconcentrationand replicate. Priortosexualmaturity,n3ysidswillbe heldinone compartment placedin eachreplicatteestchamber (15/comparftnent)T.he compartmmts consistof glassbeakerswithnylonmesh screw attachedtotwo holes on oppositesidesof the beaker.AU attachmentsof thenylon mesh screenand glasswillbe made with siliconeadhesive. Aftermysids aaain sexualmaturity(usually10 to 14 days afterthebeginningofthetest)u,p to5 male and 5 female adultniysidswillbe pairedperreplicatteestchamber. Individuaplairswillbe held in reproductivecompartments (one pairper compartment)placedin each testchamber. The reproductive compartments willbe glasspetd disheswith sidesof nylon mesh screenattachedwith siliconeadhesive. Immature and additionaflemaleswillbe discarded.Additionalmale niysidswillbe mmntamed in a separate testcompartment of each replicatteestchamber. Additionalmales willbe used toreplacedead males inthe same replicatdeuringtheexposure.The testwillnotbe terminatedbefore7 days pastthemedian fi= of firsbtrood releasein the control(s)T.he testwiU continueforatleast29 days and may be extendedlonger atthe requestof the Sponsor. PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LrD -7- EnvironmentaClonditions Ambient room lightwillbe usedtoilluminattehetestsystems.Fluorescentlightubes(ColortonelD 50 orequivalentt)hatemitwavelengthssimilartonaturalsunlighwtillbe controllebdy an automatictimer toprovidea photoperiodof 16hoursoflightand 8 hoursofdarkness.A 15-to30-minutetransitiopneriod willbe providedwhen lightsgo on and offtoavoidsudden changes inlightintensityL.ight intensitWyill be measured attestinitiatioand atweekly intervaltsh=after with a SPER ScientifiLctd.lightmeter or equivalent The targettesttemperaturewillbe 25 :E2*C. Temperaturewillbe monitored and recorded continuouslyina negativecontrolusinga FulscopcER/C Recorder(1900 J SeriesModel A) orequivalent duringtheentiretest Recordermeasurements win be verifiewditha liquid-in-glatshsermometer priorto testinitiatioand atleastweekly thereafterT.emperaturewillalsobe measured in each testchamber atthe beginningand end ofthe testand atweekly intervaldsuringthe testusing a liquid-in-glastshermometer. Dissolvedoxygen and pH willbe measured atthebeginningand theend of thetestand atweekly intervaldsuringthetestinalternatinrgeplicatteestchambers of each treatinmtlevel.,In the event that dissolvedoxygen levelsfalblelow 60*/osaturationa,ppropriataectionswillbe takenift consultatiownith the Sponsor. Salinitwyillbe measured dailyi.n alternatinrgeplicateosf the negativecontroltreatinmtand thehighestPF.OS treatmentgroup.IfI001/omortalityoccursinany treatmentthendissolvedoxygen,pH and temperaturemeasurements willbe made atthattime and then discontinued.Dissolved oxygen willbe measured using a Yellow SpringsInstrumentModel 5 1B dissolvedoxygen meter or equivalentw,ater pH willbe measured usinga FisherAccumet Model 915 pH meter or cquivalm@ and salinitwyillbe measured using a refractometeorr equivalentinstrument. BiologicalMeasurements Observationsof=rtahty and clinicaslignsoftoxicitiynfn-st-gcneratimoynsids willbe made daily. The criterifaordeathincludelackof movement, absenceof respiratormyovements, and lackof reactionto gentleproddmg Live young produced in each compartment willbe counted,recorded,and removed daily. Live young willbe exposed to the same concentratioonf thetestsubstanceunder statictestconditionsfor approximately96 hours.At theend of thetestt,hedry weight and totalbody lengthof each first-gmm-ation PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD .8- mysid willbe determined.Observationsofabnormal developmentand abamt behaviorwillbe made for first-ansdeemd-generationmysids throughoutthetest. S=hng forknwyficalMeasurements Water samples willbe rorectedfrom two replicatteestchambers of both thelow and high level concentrationpsriortoexposure.Water samples alsowillbe coucaed from two alternatinrgeplicatteest chambers of each ftatmentgroup and thecontrolatthebegmning of thetes%atweekly intervaldsuringthe test,and at the end of the testto determineconcentrationosf the testsubstance.In the event that 1000/o mortalityoccursm any treatincnt@hensamplmg of thattreatmentwilltmmmate fouowmg thenext smnplmg interval.Samples willbe collectedatmid-depthfrom each testchamber, and analyzedimmediately. The sample scheme isqlmmaiized below: PROPOSED NUUBMS OF VFRMCANON SAIVH"IM ExperimentaGlroup Control LevelI-Low Concentration Level2 Level3 Level4 Level5 Level6-lEghConcentration Pmtm? Day 0 Day 7 Day 14 Day 21 Day 28 End - 2 2 2 2 2 2 2@ 2 2 2 2 2 2 - 2 2 2 2 .2 2 2 2 2 2 2 2 2 2 2 2 2 2 - 2 2 2 2 2 2 2 2 2 2 2 2 2 Totals 4 14 14 14 14 14 14 Tre-tesstamplesvAD be collectaefdterconditioninogfthediluterM.ore dw one pro-testamplinginterval may be requiredd,ependingupon theSponwes needs,and additionsaalmplingand resultwsillbe documented intheraw dataandincludeidnthefinarleport bone samplefrom theA andB replicatteestchambers. TotalNumber ofVerificatiSoanmples- 88 The above numbers of samples representthosecollectedfrom thetestand do not includequality control(QC) samples such asmatrixblanksand fortifiratiopnrseparedand analyzedduringtheanalytical chemistryphase of thestudy.At thediscretioonf theStudy DirectorW,ater samples alsowillbe collected from atleastone appropriatechamber whenever a malfunctionisdetectedinany partof thetestsubstance deliverysystem. PROTOCOLNO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -9- AngyficalMeasurements Chemical analysisofthe sampleswillbe performedby WildlifehternationalLtd. The analytical method used willbe based upon methodology providedby the Sponsor and identifieidn Appendix U. Modificationsmade to the analyticamlethod willbe documented in theraw data and describedin thefinal report Data AnalyEes Data tobe analyzedmclude the survivalof first-generatino3nysidst,hemunber of young releasedper reproductiveday,thedry weightand lengthof each first-generatimoynsid aliveattheend of thetest.if deemed necessary,clinicaslignsof toxicityin the adultn3ysidsmay alsobe analyzedstatistically. Concentration-responsceurveswillbe fittetdo the adultsurvivalr,eproductionand growth data. To evaluatewhether or not survivalisaffectedby thetreat:Lnen2tsX, 2 contingencytablesand the chi-squaretestwillbe used on survivaldata(i.e.n,umbers of aliveand dead mysids). Analyses of reproductio(nnumber ofyoung produced per survivingadult)and growth (weightand length)datawillbe evaluatedfornormalityand homogeneity of variances.Ifthe dataaredeemed normal with homogeneous variancesh,ypothesistestinugsing analysisof variance(ANOVA) and multiplemeans tests(e.g.D,unnett's, Bonferroni,Scheffe)willbe used. If the datafml the testfor normalityor homogeneity,then data transformationwsillbe triedinan attempttocorrectheconchtiorlWhen thedatatransformationfsml to correctfornon-normalityor heterogeneitoyf variancesn,onparametricprocedureswillbe used to identify statisticalsliygnificancthfferenceasmong theexperimentalgroups. The statisticaanlalysesof sumval growth and reproductiodnatawillaidbitmay notbe exclusively used in the determinationof the no observedeffectconcentratio(nNOEC) and lowest observedeffect c.cmccntrati(oLnOEC). The maxixnum acceptablteoxicantconcentratio(nMATC) willbe calculateadsthe geometricmean of the NOEC and LOEC. AU statisticeavlaluationwsillbe done using commerciai,iv availablceomputer softwareprograms suchas TOXSTAT (3)OR SPSS/PC+ (4). RECORDS TO BE MAINTAINED Records tobe maintainedfordatageneratedby WildlifeInternationaLlt,d.willinclude,but not be limitedto: PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -10- 1. A copy ofthesignedprotocol. 2. Identificatiaonnd characterizatoifonthetestsubstancei,fprovidedby theSponsor. 3. Datesofinitiatiaonndterminatioonf thetest 4. Historyofthetesto@rg ' nL 5. Testorganismweightand lengthmeasurementsattheend ofthetest. 6. Stocksolutiocnalculatiaondprcpuation. 7. Dailyobservations. S. Water chemistrryesult(se.g.s,alinitaynd pH). 9. Ifapplicablet,hemethodsusedtoanalyzetestsubstanceconcentratioannsd theresultosfanalytical 10. Statisticcaallculations. 11. Testcondition(slighitntensitpyh,otoperiode,tc.). 12. Calculatioanndpreparatioonftestconcentrations. 13. Copy offinarleport. FINAL REPORT A finalreportoftheresultosfthestudywillbepreparedby WildlifIenternationLatld,.The report willincludeb,utnotbe limitetdothefollowingw,hen appropriate: 1. Name and addressofthefacilipteyrformingthesb*. 2.. Datesupon whichthestudywas initiateadnd completed.ItistheresponsibiliotfytheSponsorto providethefinaldatethatdataarerecordedforchemistrypathologyand/orsupportingevaluations thatmay be generatedatotherla@oratories. 3. A statementof compliancesignedby the StudyDirectoarddressinagny exceptiontso Good LaboratoryPracticSetandards. 4. Objectivesandproceduresas statedintheapprovedprotocolincludinagnychangesintheoriginal protocol. 5. The teasubstanciedentificatinocnl,udinngame,chemicalabstracntumberorcodenumber,strength, purityc,ompositiona,ndothercharacteristpircosvidedby theSponsor 6. Stabiliatnydsolubiliotfythetestsubstanceundertheconditionosfadministratioinfp,rovidedby dw Sponsor. 7. A descriptionf themethodsusedto conductthetest. 8. A descriptionfthetestorganismsi,ncludintghesourceofthetestorganimm,scientifniac ,age, lifestage,means andrangesofweightsandlengthso,bserveddiseasest,reatmentsh,oldingprocedures,and fwdingregime. PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -11- 9. A descriptionf thepreparatioonf thetestsolutiontsh,emethodsusedtoallocatoerganismstotest chambersandbegn thetestt,henumber oforganismsand chambersperbut3nent,andtheduration ofthetest 10. A descriptiofncircumstancetshatmay have affectetdhequalitoyr integritoyfthedata. 11. The name oftheSh* Directoarnd thenames ofotherscientistpsr,ofessionalasn,d supervisory personnelinvolvedinthestudy. 12. A descriptionfthetransformationcsa,lculationasn,d operationpserformedon thedata,a summary and analysiosf thebi6fogicadlataand analyticaclhemistrydata,and a statementof theconclusions drawn fromtheanalyses. 13. Statisticmaelthods usedtoevaluatethedata. 14. The signedand datedreportsofeachoftheindrviduaslcientisotrsotherprofessionalisnvolvedinthe 15. The locatiownhere raw dataandfirwreportaretobe stored. 16. A "cmmt preparedby theQualityAssuranceUnithstingthedatesthatstudymspmdons and audits weremade andthedatesof anyfindingrseportetdotheStudyDirectorand Management. 17. Ifitisnecessartyomake correctionosr additiontsoa finalreportafterithas been accepteds,uch changeswillbe made intheformofan amendment issuedby theStudyDirectorT.he amendment'will clearliydentiftyhepartofthefinalreporthatisbeingamended and theremons forthealteration. Amendments willbesipo and datedby theStudyDirector. CHANGING OF PROTOCOL Plannedchangestotheprotocolwillbe intheform ofwrittenamendments signedby theSh* DirectoarndtheSponsoesRepresentativAem.endments willbe consideredaspartoftheprotocolandwill be attachetdothefinaplrotocol-A.ny otherchangeswillbe intheform ofwrittendeviationssignedby the SU* Directoarndfilewdiththeraw data.AR changestotheprotocowlillbe indicateidnthefinalreport. GOOD LABORATORY PRACNCES This sbidy willbe conducted in accordance with Good Laboratory Practice Standards for EPA (40 CFR Part160and/orPart792);OECD PrincipleosfGood LaboratorPyractic(eENV/MC/CHEM (98)17)-2 and Japan MAFF (59 NohSan, NofificatioNno. 3850, AgriculturaPlroductionBureau). Each study conductedby WildlifeInternationaLlt,d.isroutineleyxamined by theWildlifeInternationaLlt,d.Quality Assurance Umt forcompliancewith Good LaboratoryPracticesS,tandardOperatingProceduresand the PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 specifiepdrotocol.A statementof compliancewithGood LaboratoryPTacticewsillbe preparedforall portionsof the studyconductedby WildlifeInternationaLlt,d. The Sponsor willbe responsiblefor compliancewith Good LaboratoryPracticefsorproceduresperformedby otherlaboratorie(se.g.r,esidue analysesorpathology).Raw dataforallwork performedatWildlifeIntmmationalL,tdLand a COPY of the finalreportwillbe filedby projecntumber inarchivelsocateodn theWildlffclnt=ational,Ltd.siteo,r at an alternativleocationtobe specifieidnthefinalreport PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LA B REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -13- REFERENCES I U.S.EnvironmentaPlrotectioAngency. 1996.Serie8s50-ErologicalEff"Test Guidelfim (draftO)P,PTS Number 850.1350:MysidChronicToxicitTyest. 2 ASIM StandardE1191-90.1991.StandardGuideforConductinLgife-CXleToxicitTyestsw4th SaltwaterMysidAsm,ericanSocieLfyorTestinagndMaterials. 3 West, Inc.and D. D. Gulley. 1996. TOYSTATRelease 3.5.Westem EcoSystcms Techmlogy, Inc. Cheyenne,Wyoming. 4 Norusis,MJ. 1988.SPSS/PC+ V2.0 basemanual forthe113MPC/XT/AT and PS/2. PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFF- INTERNATIONAL, LTD -14- U)ENMCATION APPENDIXI OF TEST SUBSTANCE BY SPONSOR TobeCcmplftdbySpm" 1. TestSubstanceIdcntit(yname tobe used inthereport)P:FOS(Perfluamod=eSulfonicAcid'PotassiumS ReferenceStandard(ifapplicable)A:nalyticalStandard: N/A InternaSltandard. l.l2.2HJiJ-LHPafluor*octaneSulfonicAcid Test Substance Sample Code or Batch Number Lot 217 Test SubstancePurity(0/AoctiveIngredient): 99.9 ExpiratioDnate: 2008 U. Test Substm= r%jvrictmizafic-m Have de idcnd and ad3erdmadmisfm Znootao'npm'mT= sU=M been kaxrtoitsuseinthisstudyinwcwimw withGLP Stmxlw&? Yes x No U TestSubstonm SWrap Cm&Wm Pkm hxkatethercoommendedstorapomditionastWM& btm@ Ili Ambient Has fiks-tabiuyofihetea mbslmm undertbew stoirwcmfidcw bom ddmm3inEd inaooomlmcr,widiGLP Stmdm-ds? Odxr pmfinels3tabilitny@cn: Yes x No IV. TestConcenh-d&m: V. TwiciLy information: Adjusttatcmm&afim to1000/o&L x basedupon dz purty(I/*g)mm abom Do notadjustatamomtrafimtd 1000/0 &L Test the n-t-ial AS LS. NlmmmH=- AWWV. RatLD50 251 mg&g -Mouse LD50 kmrwn-,ftTodcit(yF=O) Pa@ghniamagna: 27 ma Dapimia magna: 50 m2/L N/A FL@hTwdcky aOO) Rainbow Trout: 11 mgLL Fadiod hfimow: 38 mg/L Odxr TomeityInfamubm Cmdidmg fMm ofdr= andmkbrcm ft* Please see MSDS PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LrD -15- APPENDIX]I AnalyticalMethod Providedby Sponsor Samples willbe ana4-zedbased upon proceduresprovidedby theSponsor inthe folkmw analytical methods: 1. Liquid ChromaU)gWby Mass Spectrometry(LCMS) Method forthe Detmmination of PcrfluorooctamSulfonicAcid Potassium Salt(PFOS) la-Freshwater,Saltwaterand Algal Medimn A copy of theabove method wiU be maintainedintheraw data. The wtad mefliodologyused to analyzethe testsamples willbe documented in theraw data and summmizod infficfinalreport. PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL L-rD. PP,OJECTNO.: 454A-107 Page 1 of I AWNDM[ENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A FLOW-THPOUGH LIFE-CYCLE T03aCITY TEST WITH TIHE SALTWATER MYSID (Mysidopsibsahia) PROTOCOL NO.: 454/051399/MYS-LC/SUB454 SPONSOR: 3M Corporation AMEENDMMNT NO.: I PROJECT NO.: 454A-107 EFTECTIVE DATE: June16,1999 AMENDMENT: PageZ Add: ProposedDates: ExperimentalStartDate: 6/16/99 ExperimentalTerminationDate: 7/21/99 REASON: The above informatiownas not known when theprotocolwas signedby the Study Director. AMENDMENT: TestOrganism Page 5 Delete: Feed (brineshrimp)providedto themysidswillbe analyzedforPFOS. REASON: The feedhasalreadybeen screenedforPFOS. STUDY DIRECTOR DATE LAB&AT6R-@'kkRAGEMENT SPONSOR'S REPRESENTATIVE DATT 7ld7lqq DATE "54110;lanw.jidl Revitwed b@ 0&(-52