Document JNKpev1aoEbxqmjL3aO9RZyoB

PFOS: A WHour ToxicitTyest withthe FreshwaterAlga (Selenastrumcapticomutum) TOXICITY TO AQUATIC PLANTS (E.G.,ALGAE) TEST SUBSTANCE Identity:Perfluorooctanesulfonatmea;y alsobe referredtoas PFOS or FC-95. (1-Octanesulfonicacid,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-p,otassium salt,CAS # 2795-39-3) Remarks: The testsubstanceisa whitepowder. Sample was taken from 3M lotnumber 217. Sample was storedunder ambient conditionspriorto testing.Puritydetermined tobe 90.49% by LC/MS, 'H-HMR, 19F-NMR and elementalanalyses techniques. METHOD Method: OECD 201, OPPTS 850.5400,ASTM 1218-90E Test: Staticacute GLP: Yes Year completed: Study completed 1999. Report completed 2000 Species: Selenastrum capricomutum Source: Originallfyrom The CultureCollectioonfAlgae atthe University ofTexas atAustin,maintainedinculturemedium atWildlifIenternational Ltd.,Easton, MD Analyticalmonitoring: PFOS measured at0, 72,96-hours Element basis: Reported threeways: number of cells/mia,rea under the growth curve and growth rate Exposure period:96-hours Startdate: 4/12/99 End date:4/16/99 Analyticalmonitoring: Test concentrationsmeasured at0,72,and 96hours. Test organisms laboratoryculture:Algae cultureshad been actively growing infreshwateralgalculturemedium foratleasttwo weeks priorto testinitiatioSnt.ock nutrienstolutionswere preparedby adding reagentgrade chemicalsto reverseosmosis-purifiewdellwater. Test Conditions: Test temperature range: 23.6- 25.BOC Growthmedium:ASTM StandarGduide1218-90E1,990 Compound MgCl2,6H20 CaCl2,2H20 H3B03 MnC12-4FI20 ZnC12 FaCii6H20 COCl2,6H20 Na2M0042H20 CuCii2H20 Na2EDTA'2H20 NaN03 MgSO47H20 K2HP04 INaHCO3 Nominal Concentration Units 12.16 mg/L 4.40 mgA- 0.1856 mg/L 0.416 mg/L 3.28 ug/L 0.1598 mg/L 1.428 ugIL 7.26 ug/L 0.012 ug/L 0.300 mgA- 25.50 MgA. 14.70 MgA- 1.044 mg/L 15.0 1 mg/L Dilutiownater source:WildlifIenternationLatld.wellwater purifiebdy reverseosmosis. The testmedium was preparedby addingtheappropriatevolumes ofstocknutrienstolutiontso purifiewdellwater.The pH ofthemedium was adjustedto7.5+ 0.1using10% HCI and themedium was sterilizebdy filtration (0.22Wn)priortouse. Stock and testsolutionpreparation:A primarystocksolution was preparedinalgalmedium ata concentratioonf 183 mg/L. The primarystocksolutionwas stirrewditha magneticstirplatefor approximately24 hours. Aftermixing,theprimarystocksolution was proportionaldliylutewdithalgalmedium to preparethefive additionatlestconcentrationsA.llfinatlestsolutionasppeared clearand colorless. Exposure vessels: Steril2e50 mL polycarbonateEdenmeyer flasksplugged withfoam stopperscontainin1g00 mL oftest solution. Agitation:Shaken continuouslyat 100 rpm Number of replicates:three Initiaallgalcellloading:1.0 X 104 cells/mL Number of concentrations:seven plusa negativecontrolplusan abioticontrolatthe highestconcentratiotnested Water chemistry: pH range (0- 96 hours) 7.5- 8.1 (controelxposure) 7.4- 7.5 (179mg/L exposure) Test temperature range (0- 96 hours) 23.6 - 25.80C Lightlevels:(0- 96 hours) 3870 - 4610 luxfrom continuouscool-white fluorescenltighting Method ofcalculatingmean measured concentrations: a(ithmetimcean RESULTS Nominal concentrations:Bk control5,.7,11,23, 46,91, 183 mg/L plus 183 mg/L abioticontrol. Measured concentrations: <LOQ, 5.5,11,21, 44, 86, 179,169 mg/L Element value: 24-hourECw (celdlensity)= 163 (74-191)mg/L 24-hourEBCSO (areaunder curve)= 122 (19-176)mg/L 24-hourEC5o (growthrate) 136 (30 -204) mg/L 48-hourEC5o (celdlensity) 81 (72- 90)mg/L 48-hourEBCSO (areaundercurve)= 84 (67- 146)mg/L 48-hour ErC50(growthrate) 142 (107- 185)mg/L 72-hourECIO (celdlensity) 37 (<O - 64) mg/L 72-hourEbClO (areaunder curve)= 46 (<O- 56) mg/L 72-hour ERCIO(growthrate) 53 (23- 64)mg/L 72-hourECso (celdlensity) 70 (44- 78) mg/L 72-hourEbC50 (areaundercurve)= 74 (55- 82)mg/L 72-hour ErC5o(growthrate) 120 (103- 132)mg/L 72-hourEC90 (celdlensity) 153 (130- 165)mg/L 72-hourEbC90 (areaunder curve)= 165 (145- 176) mg/L 72-hour ErCgo(growthrate) >179 mg/L (C.I.notcalculable) 96-hourEC,o (celdlensity) 49 (43- 50)mg/L 96-hourEBCIO (areaundercurve)= 49 (40- 50)mg/L 96-hourErClo(growthrate) 59 (54- 63)mg/L 96-hourECso (celdlensity) 71 (66- 73)mg/L 96-hourEbC50 (areaundercurve)= 71 (67- 74)mg/L 96-hourErCso(growthrate) 126 (115- 138)mg/L 96-hourEC90 (celdlensity) 137 (105- 153)mg/L 96-hourEbC90 (areaunder curve)= 145 (125- 155)mg/L 96-hourErCso(growthrate)= >179 mg/L (C.I.notcalculable) 72-hourNOEC (growthrate,celldensity,area under thecurve):44 mg/L 96-hourNOEC (growthrate,celldensity,area under thecurve):44 mg/L Allelement valuesbased on mean measured concentrations Statisticamlethods: Celldensitiesa,rea under the growth curve values, growth ratesand percentinhibitiovnalueswere calculatedusing"The SAS System forWindows", Release 6.12.These valueswere then analyzed by linearinterpolatiounsingTOXSTAT Version 3.5to estimatethe ECIO, EC5o,and ECgo valuesand 95% confidencelimitast72 and 96. Cell densitiesa,reas under the growth curveand growth ratesat 72 and 96 hours were alsoevaluatedfornormalityand homogeneity ofvariances usingtheShapiro-Wilks'tsestand Badett'stest,respectivelyT.he treatmentgroups were then compared to the controlusing Dunnett'stest. Resultsofthestatisticaanlalyses were used todeterminethe NOEC values. AnalyticalMethodology: Analyses oftestsolutionswere performed at WildlifIenternationaLltd.using high performance liquicdhromatography withmass spectrometridcetection(HPLC/MS). When determiningthe concentrationofthe testsubstance inthetestsolutionst,hesame and most prominentpeak response forperfluorooctanesulfonawtaes used. No attemptwas made to quantifyon the basisof individuailsomeric components. The LOQ (limiotfquantitatiowna)s 0.115 mg/L inthisstudy. The mean percentrecoveryofmatrixfortificatioansalyzed concurrently duringsample analysiswas 99.1.Samples collectedattestinitiatihoand. measured valuesfrom 82.0 to98.1% ofnominal. Measured valuesfor samples takenat72-hours ranged from 91.7 to 105% ofnominal. Measured valuesforsamples taken at96-hours ranged from 90.3 to 102% ofnominal. Forthe abioticcontrolsm,easured valuesforsamples taken at72-hoursranged from 81.9 - 105% ofnominaland forsamples taken at 96-hours,90.3- 103% of nominal. Summary of analyticalchemistry data: Nominal Test Concentration, mg/L Measured Values at0, 72, and 96hours,Respectively,mgfl Mean Measured Percentof Nominal Concentration, mg/L Negative Control All< LOQ <LOQ 5.7 4.73,6.04,5.84 5.5 96 11 10.7,11.2,12.1 11 100 23 46 91 183 183 (abiodc) 19.8.23.1,20.7 42.7,41.9,46.3 83.3,86.0,88.3 179,186,172 notana@@d, 150,188 21 91 44 96 86 95 179 1 98 169 92 Controrlesponses:atisfactory Biologicalobservationsafter96-hours: Mean Measured Mean Number Concentration, ofCellsper mg/L mL Percent Percent Percent Inhibitiovnia lnhibiuonvia Inhibitivoina Density Area Under Growth Rate theCurve NegativeControl 2,740,000 5.5 3,040,000 11 2,880,000 21 .-3,240.000 44 3.080,000 86 626,667 179 33,667 -11 -5.1 -18 -12. 77 99 -8.5 -3.3 -13 -5.3 76 98 -1.9 -0.84 -3.0 -2.0 27 79 Observations: After96 hours ofexposure,therewere no signsof aggregation,flocculatioonradherence ofthe algaetothe flasksinthe negativecontrolorany testtreatmentgroup. Inadditiont,herewere'no noticeablechanges incellcoloror morphology when compared tothe negativecontrola,lthougha few cellsappeared enlargedinthe 86 and 179 mg/L treatmentgroups. ReversibilityofGrowth InhibitionT:he 179 mg/L treatmentgroup was maximallyinhibiteadfter96-hours.Aliquotsofthe testsolutionwere dilutedwithalgalmedium and culturedforfivedays. Based on the growth observed intherecoveryphase, the effecton algalgrowth was found to be algistatic. CONCLUSIONS The potassium perfluorooctanesulfona9t6e-hourECso and 95% confidenceintervaflorSelenastrum capficornutumwas determined using threecalculatiomnethods. By celldensity,itwas 71 (66-73)mg/L, by area under thegrowth curve itwas 71 (67-74)mg/L and by growth rate 126 (115-138)mg/L. The 96-hourNOEC was determinedby Dunnett's procedure(p < 0.05)to be 44 mg/L usingallthreemethods. No-signsof aggregation,-ffocculatiorn,adherence were noted inany ofthe test solutionsorthe controls.This testsubstance was determinedtobe algistatic. Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St. Paul,Minnesota,55133 DATA QUALITY RellabliftyK:limischranking 1 REFERENCES Thisstudywas conductedat WildlifIenternationLatld.,Easton,MD atthe requestofthe3M Company. OTHER Las,tchanged: 5/3/00 PFOS: A 96-HOUR TO)UCrrY TEST WITH THE FRESHWATER ALGA (Selenastrucmapncornutum) FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-103A 3M LAB REQUEST NO. U2723 U.S. EnvironmentalProtectioAngency Series850 - EcologicalEffectsTest Guidelines OPPTS Number 850.5400 and OECD Guideline201 AUTHORS: KurtR- Drottar Henry 0. Krueger,Ph.D. STUDY INITIATION DATE: December 4,1998 STUDY CONTLETION DATE: -June4,1999 AMENDED REPORT DATE: April26,2000 Submittedto 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St.Paul,Minnesota 55144 Wildl!feInternationaLltd. 9598 Commerce Drive Easton,Maryland 21601 (410)822-8600 Page 1 of58 - AMENDED WILDLIFE INTERNATIC)NAL LTD. -2- PROJECT NO.: 454A-103A GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR. 3M Corporation TITLE:PFOS:A 96-HouTroxiciTteystwiththeFreshwatAelrga(Selenastcraupmncornutum) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER-- 454A-103A STUDY COMPLETION: June4,1999 REPORT AMENDED: April26,2000 Tlusstudywas conductedm compliancewithGood LaboratoryPracticSetandardsaspublishedby theU.S. EnvironmentaPlrotectioAngency in 40 CFR Parts160 and 792, 17 August 1989;OECD PrincipleosfGood LaboratoryPracticeO,CDE/GD (92)32,EnvironmentMonograph No. 45,Paris1992 and JapanMAFF, 59 NohSan, Noti:ficatNio.n 3850,AgriculturaPlroductionBureau,10 August 1984 withthefbllowinegxceptions: The testsubstancewas notcharactenzedm accordancewithfiffGlLP compliance;however,the characterizatiwoans performedaccordingto 3M StandardOperatingProceduresand Methods,and allraw dataarebemg niafiitainiendthe3M archives.The testsubstanceisbeingrecharacteriziendaccordance withGLP. The stabiliotfythetestsubstanceunderconditionosfstorageatthetestsitewas notdeunninedin accordancewithGood LaboratoryPracticeStandards. STUDY DIRECTO KurtR. Drottar SeniorBiologist SPONSOR-. SS ioAorricz@ DATE 7 /4ZC@ I@ATE AMENDED WILDLIFE INTERNATIONAL L-m. -3- PROJECTNO.: 454A-103A QUALITY ASSURANCE STATEMEENT Thisstudywas exanunedforcompliancewithGood LaboratoryPracticeStandardsas publishedby the U.S.EnvironmentaPlrotectioAngency in 40 CFR Parts160 and 792, 17 August 1989;OECD PrincipleosfGood LaboratoryPracticeO,CDE/GD (92)32,EnvironmentMonograph No. 45,Paris1992; and JapanMAFF, 59 NohSan, NotificatiNoon. 3850,AgriculturaPlroductioBnureau,10 August 1984. The datesof allmspectons and audrtsand thedatesthatany findingwserereportedtotheStudyDirector and LaboratoryManagement were asfollows: ACTIVrrY: InitialTrial 454A-103 Test SubstancePreparation InstrumentSet-up DraftReportand Data DefinitiTvreial454A-103A Test SubstancePreparation DraftReportand Data AnalyticaDlata and DraftReport FinalReport Amended Report DATE CONDUCTED: DATE REPORTED TO: STUDY DERECTOR: MANAGEMENT: February 18,1999 February22 and 23, 1999 March 29,1999 February18,1999 February23, 1999 March 29,1999 February19,1999 February24, 1999 March 30,1099 April9, 1999 May 10 and 11,1999 Aprif9,1999 May 11,1999 April12,1999 May 12,1999 may 10 and 11,1999 June4, 1999 April19 and 20,2000 May 11,1999 June 4,1999 April20,2000 May 11,1999 June 4,1999 April24,2000 TimothyiAASp-rte=r,Ph.D. Manager,Regulatoryand TechnicalSupport DATE ANEENDED WILDLIFE INTERNATIONAL LTD. -4- PROJFCTNO.: 454A-103A REPORT APPROVAL SPONSOR- 3M Corporation TTI'LE: PFOS: A 96-HourToxicitTyestwiththeFreshwateArlga (Selenastrucmapricornutum) WILDLEFE RITERNATIONAL LTD. PROJECT NUMBER-- 454A-103A STUDY DIRECTOR- Kurtk Drottar SeniorBiologist Z//i>2 DATE MANAGENEENT: Henry 6-.Kruegtr,Ph.D. DirectorA,quaticToxicology andNon-TargetPlants DATL( ANM'NDED Wi'LDLIFE INTE'RNATIONAL LTD. -5- PROJECT NO.: 454A-103A TABLE OF CONTENTS Title/CovePrage ..................................................................1.................. Good LaboratoryPracticCeomplianceStatement.........................................2.................. QualityAssuranceStatement.........................................................3.................. ReportApproval..................................................................4.................. TableofContents..........@z................................................................5.................. .Summary ........................................................................7................. Introductio.n....................................................................9.................. Objective.......................................................................9.................. ExperimentalDesign...............................................................9................. Materialasnd Methods..............................................................10.................. Resultsand Discussion.............................................................1.4.................. Conclusions.....................................................................1.7................. References...................................................................................1.8............................ TABLES Table I - summary of AnalyticalChemistry Data .......................................1.9.................. Table2 - TemperatureMeasurements................................................2.0.................. Table3 - LightIntensitMyeasurements...............................................2.1.................. Table4 - pH Measurements.......................................................2.2................. Table 5 - M2e4a-nHoCuerlIlnDteenrsviatDliuersainndgPtehrecTeensttI.n.l.u.b.i.ti.O.fn.o.r.E.a.c.h...................................2..3................... Table6 - Mean AreasUndertheGrowth Curveand PercentInhibitifoonrEach 24-HourIntervaDluringtheTest................................................2..4.................. Table7 - Mean Growth Ratesand PercentInhibitiofnorEach 24-Hour Interval DuringtheTest...............................................................2..5................... AMENDED WILDLIFE INTERNATIONAL LTD. -6- PROJECTNO.: 454A-103A TABLE OF CONTENTS -Continued- Tableg- EC10, EC50 and EC90 ValuesBasedon CellDensityOverthe96-Hour ExposurePeriod.........................................................2.6................ Table9- EC10, EC50 and EC90 ValuesBasedon Area UndertheGrowth Curve Over the96-Hour ExposurePeriod................................................2.7................ Table 10- EC 10,EC50 and tC-'9V0aluesBased on Growdi RateOver the96-Hour ExposurePeriod.........................................................2.8................ Table 11 -CellDensitiefsortheRecoveryPhase .........................................2.9................ FIGURES Figure1 - NegativeControlAlo Growth,ExpressedinCellDensity, Duringthe96-HourExposure...............................................3.0................ Figure2 - Concentration-ResponCsuerve,ExpressedinCellDensity.....................*.........3..1................ -Figure3 - RecoveryPhaseResponseCurve,ExpressedinCellDensity........................3.2................ APPENDICES AppendixI - FreshwaterAlgalMedium .....................................................3.3............... Appendix H - AnalysesofPesticidesO,rganics,Metalsand OtherInorganics inWildlifIenternationLatld.WellWater..................................3.4............... AppendixM - The AnalysisofPFOS inFreshwaterAlgalMedium in Supportof WildlifIenternationLatld.ProjecNto.: 454A-103A .........................3.5............... AppendixIV - CellDensityfbrEach ReplicatPeerTreatmentOver the 96-Hour ExposurePeriod..............................................5.2................ AppendixV - Area UndertheGrowth Curve forEach ReplicatPeerTreatmentOver the 96-HourExposurePeriod.............................................5.3................ AppendlxVI - Growth RateforEach ReplicatPeerTreatmentOver the96-Hour ExposurePeriod.54 AppendixVlg- Changesto Protoco.l........................;................................................5.5................ Appendix VM - PersonnelInvolvedintheStudy.........................................5.6............... AppendixIX - ReportAmendment ..................................................5.7............... AMENDED WILDLIFE INTERNATIONAL LTD. -7- PROJECT NO.: 454A-103A SPONSOR: SPONSOleS REPRESENTATIVE: LOCA-NON OF STUDY, RAW DATA AND A COPY OF TBE FINAL REPORT: WILD11FE R4TERNATIONAL,, LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: Is,@ MEASURED TEST CONCENTRANONS: TEST DATES: LENGTH OF EXPOSURE: TEST ORGANISM: SOURCE OF TEST ORGANISMS: CELL DENSITY 72-HouR EC50: 95% coNFiDfNcE unffrs: 96-HOUR EC 10: 95% CONFIDENCE LUAM: 96-HOUR EC50: 95% CONFMENCE 12&TS: 96-HOUR EC90: 95% CONFMENCE 1DZTS: 72-HOURNOAEC: 96-HOUR NOAEC: SUNUMLARY 3M r-orpo"roant"io-n Ms. SusanA. Beach WildlifIenternationLatld. Easton,MD 21601 454A-103A PFOS (PerfluoroocataSnuelfoniAccid PotassiumSalt) PFOS: A 96-HourToxicityTestwiththeFreshwaterAlga (Selenastrucmappicornutum) NegativeControL 5.5,11,21,44,86 and 179 mg a.i.AL ExperimentalStart-April12,1999 ExposureTermination-April16,1999 ExperimentaTlermination-April21, 1999 96 Hours Freshwate=rga (Selenastrcuamp?icornutum) WildlifIenternationLatld. ALWL M!Uland 21601 70 mg a.i.AL 44 and 78 mg a.i.AL 49 mg a.i./L 43 and 50 mg a.i.AL 71 miza.iJL 66 iZd 73 mg a.i./L 137 mg a.i./L 105 and 153 mg a.i.AL 44 mg a.i.AL 44 Mg a.iA, ANENDED WILDLIFE INTERNATIC)NAL LTD. -8- AREA UNDER THE GROWTH CURVE 72-HOUR EC50: 95% CONFIDENCE LBETS: 96-HOURECIO: 95% CONFIDENCE IJNM: 96-HOUREC50: 95% CONFIDENCE I2&n: 96-HOUR EC90: 95% CONFIDENCE LUvgrS: 72-HOURNOAEC: 96-HOTJR NOAEC: GROVRM RATE 72-HouR EC50: 95% CONFIDENCE LRZM: 96-HOURECIO: 95% CONFIDENCE 12drrS: 96-HOUREC50: 95% CONFIDE NCE LRvflTS: 96-HOUR FC90: 95% CONFIDENCE LmTs: 72-HOURNOAEC: 96-HOURNOAEC: SUbD4ARY (Continued) 74 mg a.i.AL 55 and82mg a.i.AL 49 ragai.AL 40 and 50 mg a.iJL 71 mg a.i.AL 67 and74 mg aiJL 145 mg a.i.AL 125 and 155 mg a.i.AL 44 mg a.i.AL 44 mg a.i.AL 120 mg a.i./L 103 and 132 mg ai.AL 59 mg a.iJL 54 and 63 mg a.i./L 126 mg a.i.AL 115 and 138 mg a.i./L >179 mg a.i.AL Not Calculable 44 mg a.i./L 44 mg a.i./L PROJECT NO.: 454A-103A ANCENDED WILDLIFE INTERNATIONAL LTD. -9- PROJECTNO.: 454A-103A INTRODUCTION Ths studywas conductedby WildlffeInternationLatld.for 3M Corporauonat theWildlife InternationLatld.aquatc toxicologfyilcdrtiyn Easton,MarykncL The m-hfe phaseof thetestwas conductedfromApril12,1999toApril21, 1999. Raw datageneratebdy WildlifIenternationLatld and a copy of thefinalreportarefiledunderProjectNumber 454A-103A inarchivelsocatedon theWildlife InternationLatld.site. OBJECTTVE The objectivoef thestudywas to evaluatethetoxicitoyf PFOS (PerfluorooctaSnuelfonicAcid .PotassiuSmalt)to thegrowthof the freshwatearlga,Selenastrumc-apneornutumd,uringa 96-hour exposureperiod. EXPEPU*IENTAL DESIGN 'Me freshwatearlgk Selenastrucmapricornutumw,as exposedto a goom@tficserieosf sixtest concentratioannsd a negative(culwm mechum) controlunder s=c conditionfsor96 hours. Three replicatestchamberswere maintainedforeachtutrnentand controglroup.One additionarleplicatweas alsoniahtainedforanalyticaslamplingon Day 3 of thetest.Inadditiono,ne "abioticr"eplicat(etest solutiownrthoutalW) was preparedforthelughestestconcentratioNno.minaltestconcentratiownesre selecteidnconsuhatim withfficSponsorand were basedupon theresultosfrangefindingand definitive .testsT.he nominaltestconcenu-atiosneslectewdere 5.7,11,23, 46, 91 and 183 mg activeingredient (a.i.)/Lm.ean measuredtestconcentrationwsere determinefdromsamplesoftestmedium collectefdrom eachtreatmentand thecontroglroupattestinitiatioant,approximatel7y2 hours,and attesttermination. At testin@on, an inoculumof thealgalccuswas preparedata concentratiofnapproxinately 1.0X 106ceUs/mL. The concentratimof algalcells*intheinoculumwas verifieadnd 1.0mL was addedto eachtestchambertoachievea nominalconcet&ationofapproxiinawl1y.0X 10'ceUs/mL. Sampleswere AMENDED WILDLIFE INTERNATIONAL LTD. -10- PROJECTNO.: 454A-103A collectefdrom eachreplicatestchamberatapproximatel2y4-hourintervaldsuringthetesttodetermine ceR densitiesC.eR densitiewsere measuredforeach replicataend were used to calculataereasunderthe growthcurveand growthrates.Percentmlnbitiovnaluesrelativteothecontrolwere calculatefdoreach parameterover the 96-hourexposureperiod.EC50 valuesbased upon cefldensitieasr,easunderthe growthcurveand growthrateswere calculatefdoreach24 hourintervalE.C10 and EC90 valueswere calculatedi,fpossiblef,orthe72 and 96 hour intervalsT.he no-observed-adverse-effect-wncentrafion (NOAEC) was determinebdasedupon slifistiecvaalluatioonfthe72-hourand 96-hourresultsA.t theend ofthe96-hourexposure,algistafeifcfectwsere differenfiaftreodm algicidaelffects. MATERLALS AND METHODS The studywas conductedaccordingto theproceduresoudimedintheprotocol",PFOS: A 96-Hour ToxicityTest with the FreshwaterAlga (Selenastrumcapncornutum)". The protocolwas based on proceduresoutlinedin theU.S. EnvironmentaPlrotectioAngency Series850-EcologicaElffectsTest GuidelinesO,PPTS Number 850.5400:AlgalToxicitTyiersland LT(dmft)(1a)ndOECD Guidelinefsor Testingof Chemicals2,01 Algal,GrowthInhibitioTnest(2). TestSubstance The testsubstancewas receivedfrom 3M Corporationon October29, 1998 and was assigned WildlifeInternationLatld.idenfificatniuomnber 4675. 'Me testsubstancewas describeads a white powder. Itwas idenfifiaesdFC-95 fromlotnumber 217 (T-6295).Informatiopnrovidedby theSponsor indicatead purityof 99.9% and an expiratiodnateof 2008. The testsubstancewas reanalyzebdy the Sponsorand theCertificaotfeAnalysisdatedMarch 9, 2000 indicatead purityof 90.49%. The test substancewas storedatambientroom temperature. PLeparatioonf TestConcentrations Nominal testconcentrationwsere 5.7,11,23,46, 91 and 183 mg a.i.ALb,ased on a testsubstance purityof90.49%. Allmaterialwshichcame intocontactwiththetestsubstanceduringpreparatioonftest concentrationwsere constructeodfplastiocr stainlesssteelA. primarystocksolutiownas preparedinalgal AHEENDED WILDLIFE INTERNATIONAL LTD. -11- PROJECT NO.: 454A-103A medium ata concentrationf 183 mg a.i.ALT.he primarystocksolutiownas stirrewditha magneticstir plateforapproxfinate2l4y hoursto aidin thesolubffizatoifonthetestsubstance.Aftermixing,the primarystocksolutiownas proportionaldliylutewdith algalmedium to preparethefiveadditionatlest concentrationAsl.lfinaltestsolutionasppearedclearand colorless. Test Organi ThefreshwaatlegraS,eignastrcuamppicornutwuams,selectaesfdt tesstpecifeosrthisstudyT.he speciesisrepresentatiovfean importantgroup of fi-eshwataelrgae,and was selectefdoruse inthetest basedupon a pasthistoryofuse and easeof culturinignthelaboratoryO.riginalalgalcultxuwwere obtainedfromUTEX --Me CultureCollectioonfAlgaeattheUmversrtyofTexas atAustinand havebeen maintainedinculturemedium atWildlifIenternationLatld.,Easton,Maryland.Algalcellussedinthistest wereobtainedfrom WildlifIenternationLatld.culturetshathad beenactivelgyrowinginculturemedium foratlea@two weeks priortotestinitiatioTnh.e negativecontrolorganismswere expectedtQ exhibit exponentiaglrowthoverthe96-hourexposureperiod.Exponentialgrowth,definedas theperiodofgrowth where thealgalceusaredividinagta constantrate,isindicatebdy thelinearsectionofthegrowthcurve (Figure1). OECD guidelinersecommend thatcontrolcellgrowthshouldhave increasebdy a factorofat least16 within3 days. CultureMedium The algalceuswere cultureadnd testedin freshwateWrgg medium (3).Storknutrienstolutions were preparedby addingreagent-gradcehemicalsto WildlifeInternationLatld.WellwaterPurifiebdy reverseosmosis.The testmedium was preparedby addingtheappropriatveolumes of stocknutrient solutiontsopun:Ecdwellwater(Appmdix 1).The pH ofthemedium was adjustetdo7.5 0.1using10% HCI and themedium was sterilizbeydfilwafia(n0.22ym) priortouse.Analyseswereperformedatleast once annuallyto determinetheconcentrationosf selecteodrganicand inorganiconstituentisnthe well water.The resultosfanalysesperformedtomeasuretheconcentratioonfsselectecdontammantsinwell waterusedby WildlifherwmationalLtd.arepresentedinAppendixIl. AMENDED WILDLIFE INTERNATIONAL LTD. -12- PROJECTNO.: 454A-103A TestApparatus Testchamberswere stffff2e5,0-mL polycarbonatEerlentneyefrlaskspluggedwithfoam stoppers and contained100 mL oftestorcontrolalgalmedium. The testchamberswere labelewdiththeproject number,concentratioand replicataen,d were indiscriminateploysitioneodn a mechanicalshakertablein an environmentaclhamber designedtomaintainthedesiredtesttmnpemturethroughoutthetest.Ile test chamberswere shakencontinuouslayt 100 rpm. EnvironmentalConditions Testflaskswereheldinan environmentaclhamber ata temperaturoef24*2*C. The tmnperatureof a containeorf wateradjacentothetestflasksintheenvironmentaclhamber was recordedtwicedaily duringthetestusinga hqlnd-m-glastshermometer. The algaewereheldundercontinuouscool-wlntfeluorescenltightintghroughouthetest.The target fightintensitwyas 4300 430 lux.Lightintensitwyas measuredatthefburcomers andthemiddleofthe shakertabledaily.Lightfiftisiwtays measuredusingan SPER ScientifMiocdel 840006 lighmteter. The pH ofthemedium preparedfbreach treatmenatnd controglroupwas me @suredattestinitiation and terminatiounsinga FisherAccumet Model 915 pH meter. Sam@les'forpH measurementat test initiatiwoenre collectefdrom theindividuablatchesoftestsolutiopnreparedforeachtreatmenatnd control group. At testtermmaton,samplesoftestsolutiownere collectefdrompooledreplicateosfthetreatment andcontrolgroupsforpH measurement. AlgalGrowth Testmechurnsampleswere collectefdrom thetreatmentand controlgroupsforthedetermmahon of algalcelldensitiesS.inglesampleswere collectefdrom each of thethree"biologicalr"eplicatepser treatmenatnd controlgroup at 24-hourintervaldsuringthe 96-hourexposure,and were counted unmediately.Cellcountswere conductedusinga hmnacytometerand microscope.Each samplewas diluteudsingan electrolystoelutio(nIsotonl*a)s,needed,tomaintaincountingaccuracy.A smallamount ofeach samplewas loadedontoa hanacytometerand 10 gnds were counted."Me mean number ofcells WILDLIFE INTERNATIONAL L-m. -13- PROJECT NO.: 454A-103A pergnd was estnuiw and tlusvaluewas used to calculattehecelldensitoyf thesample. Usingthis techniquet,heminimum quanfifiabclelldensitwyas 1.0 X 10'ceUs/mL. All96 hour cellcountsampleswere examinednucroscopicaffloyratypicaclellmorphology(e.g., changesincellshape,sizeorcolor).Growth of cellsinthereplicatteestchambers alsowas assessedfor aggregationosr flocculabonosfceflsand adherenceof theceusto thetestchamber. StatisticAanlalyses Celldensitieasr,eaunderthegrowthcurvevalues,growthratesand percentinhibitiovnalueswere calculateudsing,The SAS SystemforWindows",Release6.12(4). Area underthegrowthcurvewas calculatfeodrthecontroalndtwt=t groupusingthefollowinfgormula: A = ((Ni-N.)/2)(ti)+((N,+N2-2N.)/2)(t2-ti)+((N..,+N.-2N.)/2)(t.-iti) where: A = Area N. = Nominalnumber ofceUs/mL att. Ni = Mean measurednumber of CeUs/mL atti N2= Mean measurednumber of ceUs/mL att2 N. = Mean measurednumber of ceUs/mL att. ti= timeoffimtmeasuranmt aftebreginninogf= (hours) t2= timeofsecondmenuranent aftebreginninogftest(hq@in) t,=, timeofe measurementafterbeginningoftest(hours) Growth rateswere calculatefdorthecontroland eachtreatmentgroupusingthefollowinfgormula: Growth Rate In(N.IN.) tu where:N, = Mean measurednumber ofceUs/mL att, N,,= Mean measurednumber of ceUs/mL att@ t,= Tune ofe measurementaftebreginningoftest(hours) Percentmlubium valueswere calculatefdoreach ftatrnmtgroupas thepercentreductioinncell densitya,reaunderthegrowthcurveand growthraterelativteothecontrorleplicatesT.he following formulawas used: WILDLIFE INTERNATIONAL LTD. -14- PROJECTNO.: 454A-103A PercentInhibiti=onMean CeU DensLty,@ -Mean CellDensiWz,,== X 100 Mean CeU Densityc..,d CeU densitieasr,easunderthegrowth curveand growth rateswere analyzedstatisticatloleystftnate theECIO, EC50 and EC90 values(i.e.t,hetheoretictaelstconcentratiotnhsatwould producea'10,50 or 90% reductioinn each parainctere,spectivelayn)d 95% confidencleimitsat72 and 96 hours. EC50 valueswere alsocalculatefdorthe24 and 48 hour timeintervalsT.he EC valuesand 95% confidence lirmtwserecalcul-itbeydlmeayuiterpolatiuosningTOXSTAT Version3.5(5).CeU densitieasr,easunder ..thgerowthcurveand growth ratesat72 and 96 hourswere evaluatefdornormalityand homogeneityof variancesusingthe Shapiro-WiWs testand Bartletf%s-s@respectivelyT.he treatmengtroupswereffim comparedtothecontrolusingDunnett'stest.Resultsofthestatistiacnaallyseswere usedtodetermintehe NOAEC values. Age3lLcaalmiw Samples of testmedium were collectefdrom thenegativecontroland eachtreatmentgroupattest initiatiaotn,approximatel7y2 hours and at testtenninati(tmo measureconcentratioonfsthe test substance.Samplesoftestmedium collecteadttestinitiatiwoenre takenfrom theindividuablatchesof test solutiopnreparedforeachtvatinentand thecontrolgroup.Samplescollecteadt72 hourswere collected from theadditiona"lanalyticarle"plicatesS.amples collecteadttesttenninatiownere a compositeofthe -remumng replicatefsoreach treatmentand the controlgroup. The sampleswere placedin plastic (NalgeneO)bottlesand were analyzedmunech@ly withoutstorage.Analyticaplrocedureussed inthe analysiosfthesamplesarepresenteidnAppendixIII. RESULTS AND DISCUSSION MeasurementofTestConczntrafions ResultsofanalysestomeasureconcentrationosfPFOS inthetestsolutionasrepresenteidnTableI and AppendixM. Nominal concenu-atiosneslectefdoruse inthisstudywere 5.7,11,23,46, 91 and 183 mg a.i.AL.Samples collecteadtthebeginningofthetesthad measuredconcentratiotnhsatrangedfrom 82 to98% ofnominal.Samplescollecteadt72 hoursand test-terminatihoand recoverietshatrangedfrom ANE'NDED WILDLIFE INTERNATIONAL LTD. -15- PROJECTNO.: 454A-103A 82 to 105% and 90 to 103% of nominal,respectivelWyh.en thevaluesobtainedfortestinitiatiaotn, 72 hoursand attestterminatiownere averaged,themean measuredtestconcentratiownesre 5.5,11,21, 44,86 and 179 mg a.i.AL. Observationsand Measurements Measurementsoftemperatureand lightintensitayrepresenteidnTables2 and 3,respectivel'yM.e temperaturersangedfrom 23.6.t2o5.80C and werewithintherangeestablishfeodrthetest(242*C). Ile lightintensitryangedfrom 3870 to4540 luxand was withinthedesiredrangeforthetest(approximately 3870 to4730 lux)(Table3).MeasurementsofpH were 7.4to7.5on Day 0 and rangedfrom7.5to8.4at 96 hours(Table4).The pH ofthe"abioticr"eplicataettesterminatiownas 7.4. The effectofPFOS upon Selenastrumcapricornutumwas determinebdy evaluatindgifferenceisn celldensitieasr,easunderthegrowth curveand growthrates.Mean valueswere usedto calculatgerowth mlubrttonforeach 24-hourperiod.Mean celldensitieasr,easunderthegrowthcurveand growthratesand theircorrespondinpgercentmlubitiovnaluesarepresentedinTables5,6 and 7,respectivelCye.lldensity, areaunderthe growth curveand growth ratevaluesforeach individuarleplicataerepresentedin AppendicesIV, V and VL respectivelyE.CIO, EC50 and EC90 valuesand 95% confidencleimits calculatefdoreach 24-hourintervablasedon celldensitya,reaunderthegrowthcurveand growthrateare presentedinTables8,9 and 10,respectively. Changes incelldensityindicatetdhatexponentiaglrowth occurredinthenegativecontrolreplicates (Figure1).The coefficieonftvariatiofnorcontroclelldensitywas 4%. After72 hoursofexposure,cdl densitypercentinhibitiointhe5.5,11,21,44,86 and 179mg a.i.AtLreatmengtroupswas -5.2,-1.9,-5.5, 11,72 and 970/or,espectivelDyu.mwft'stestshowed OW celldensitywas significantrleyducedinthe86 and 179 mg a.i.AtLreatmengtroupsincomparisontothenegativecontrol(p:r,0.05C)o.nsequentlyt,he NOAEC for72 hour celldensitywas 44 mg a.i./L.After96 hoursof exposurec,elldensitpyercent inhibitiionthe5.5,11,21,44,86 and 179 mg a.i./tLreatmengtroupswas -11,-5.1,-18,-12,77 and 990/or,espectivelDyu.nnetVstestshowed thatcelldensitywas s4nificantlryeducedinthe86 and 179 mg a.i./tLreatnengtroups(p--.0.05C)o.nsequentlyt,heNOAEC for96 hourcelldensitwyas 44 mg a.i.AL. ANENDED WILDLIFE INTERNATIC)NAL LTD. -16- PROJECT NO.: 454A-103A After72 hoursofexposure,areaunderthegrowthcurvepercentinhibitiointhe5.5,11,21,44,86 and 179inga.i./tLreatmengtroupswas -2.9,1.3,-3.8,5.8,68 and 940/or,espectivelyD.unnett'stest showed thatareaunderthegrowthcurvewas significantrleyducedinthe86 and 179 mg a.i./tLreatment groups(p--.0.05C)o.nsequentlyt,heNOAEC for72 hourareaunderthegrowthcurvewas 44 inga.i.AL. After96 hoursofexposure,areaunderthegrowthcurvepercentmlubitioinnthe5.5,11,21,44, 86 and 179 mg a.i./tLreatnengtroupswas -8.5,-3.3,-13,-5.3,75 and 98*/or,espectivelDyu.nnett'tsestshowed thatareaunderthegrowthcurvewas sigmficantlryeducedinthe86 and 179 mg a.i.AtLreatmentgroups (p-,.0.05C)o.nsequentlyt,heNOAEC for96 hourareaunderthegrowthcurvewas 44 mg a.i.AL. After72 hoursof exposureg,rowthratepercentinhibitiionthe5.5,11,21,44,86 and 179 mg a.i./tLreatmengtroupswas -1.1,-0.68,-0.72,3.4,31 and 820/or,espectivelDyu.nnett'stestshowedthat growth ratewas sigmficardryeducedin the 86 and 179 mg a.i./tLmftent groups(p:50.05). Consequentlyt,heNOAEC for72 hourgrowthratewas 44 mg a.i.ALA.fter96 hoursofexposure,growth ratepercentinhibitiionthe5.5,11,21,44,86 and 179 mg a.i.AuLwtnent groupswas -1.9,-0.84,-3.0 -2.0,27 and 79%, respectivelDyu.nnett'stestshowedthatgrowthratewas significantrleyducedinthe86 and 179 mg a.i./tLreatmengtroups(p-q0.05).Consequentlyt,heNOAEC for96.hourgrowthratewas 44 mg a.i.AL. VisualandMcrosg@Mic Observations After96 hoursofexposure,therewere no signsofaggregationf,locculatiornadherenceofthealgae tothetestflaskisnthenegativcemtrotorany PFOS bmtrnmt group.Inaddition*,= were no noticeable changesincellcolorormorphologywhen comparedtothenegativecontrolalthougha few cellasppeared enlargeidnthe86 and 179mg ai./Luwb=t group. ReversibALtoyfGrowth Inlub The 179 mg a.i.AtLreatmengtroupwas maximallyinhibiteadttheend of the96-hourexposure period.Ahquotsofthetestsolutiowneredilutewdithalgalmedium and culturedforfivedays. Based on thegrowthobservedintherecoveryphase,theeffecton algalgrowthwas foundto alesmfic.Celldensities fortherecoveryphasearepresenteidnTableIIand areillustratgerdaphicalliynFigure3. AMENDED WILDLIFE INTERNATIC)NAL L-m. -17- PROJFCTNO.: 454A-103A CONCLUSIONS T"neconclusionfsthistudywerebasedonthemostsensitievne4poinmteasure(di.ec.e,ldlensity, areaunderthegrowthcurveand/orgrowth rate).'Me 72-hourEC50, basedon celldensityw,as 70 mg a.i.AwLith95% confidenchemitsof 44 and 78 mg a.i.ALT.lle96-hourECIO, basedon celldensityw,as 49 mg a.i.AwLith95% confidencelimitosf 43 and 50 mg a.i.ALT.he 96-hourEC50, basedon ceR density, was 71 mg a.i./wLith95%-confidencleimitosf 66 and 73 mg a.i.ALT.he 96-hourEC90, basedon cell densityw,as 137 mg a.i./wLith95% confidencleimitosf 105 and 153 mg a.i./LThe 72-hourand 96-hour NOAEC values,basedon celldensitya,reaunderthegrowth curveand growth ratew,ere 44 mg a.i.AL. Based on thepresenceof visiblWegal growth at recoveryterminationP,FOS was consideretdo be algi@tic,rathetrhanalgicidaalt,theconcentrationtsested. AMEMED WILDLIFE INTERNATIONAL LTD. -18- PROJECTNO.: 454A-103A REFERENCES I U.S. EnvironmentalProtectionAgency. 1996. Series850 - EcologicaElffectsTestGuidelines (draftO)P,PTS Number 850.5400:AlgalToxicityT,iersI and ff. 2 OrganizatioonfEconomic Cooperationand Development 1984.Algal,GrowthInhibitioTnest. OECD GuidelinfeorTestingofChemicals.Guidelin2e01. Paris. 3 ASTM Standard Guide 1218-90E, Standard Guide for Conducting Static 96-Hour Toxicity Tests withMicroalgae. Aui6a 1990. 4 The SAS System forWindows. 1996. Release6.12,TS Level0020. SAS InstituItnec.,Cary, NorthCarolina. 5 West, Inc-and D.D. Gulley.TOXSTAT Version3.5. Copyright1996. WesternEcoSystems Technology,Inc..Cheyenne.Wyoming. WILDLIFE INTERNATIONAL LTD. -19- PROJECT NO.: 454A-103A Table 1 Summary ofAnalyticaClhemistryData Sponsor: Test Substance: Test Orgamsrw DilutionWater: Nominal ConcenU-ation @Mg a.i./L) Negative Control 3M Corporation PFOS Freshwater Alga, Selenastrum cappicornutum FreshwaterAlgalMedium Sampling Time 2Logw oi 723 964 Measured Concentration' (mg a.1.2 < LoQ < LOQ <IOQ 5.7 0 4.73 72 6.04 96 5.84 Mean Measured Concentration (mg a.i.2 - 5.5 Percent Of Nominal - 96 11 0 10.7 72 11.2 96 12.1 11 100 23 0 19.8 72 23.1 96 20.7 21 91 46 0 42.7 72 41.9 96 46.3 44 96 91 0 83.3 72 86.0 96 88.3 86 95 183 0 179 72 186 96 172 179 98 183 725 150 169 92 96' 188 llimit of Quantitation a@M was 0.115 mg ai.AL. 20-hour samples were collectedfrom mdmdual batches of testsolutionpreparedfor the Uratmmt and control 3goups attestinitiation. 72-hour sampleswere collectefdrom the adchtional(D) replicat(ebiotic). 496-hour samples were composita of testsolutioncollectedfiromeach of the ftm replicatepser treatmentand controlgroup- 72 and 96 hour sample collectedf3romthe additional2 MLhcate @ o@tic - AAffi'NDED WILDLIFE INTERNATIONAL LTD. -20- PROJECT NO.: 454A-103A Table2 TemperatureMmsurmnents Sponsor: Test Substance: TestOrganism: DilutionWater: 3M Corporation PFOS FreshwaterAlga,Selenastrumcappicornutum FreshwaterAlgalMedium Time Q?Ul Measurement 1 0 23.6 1 24.8 2 24.8 3 25.1 4 25.8 5 25.7 6 25.6 7 24.0 8 23.9 9 23.9 Measurement 2 24.2 24.2 24.0 25.3 24.9 24.4 24.9 24.0 23.8 23.9 WILDLIFE INTERNATIONAL LTD. -21- PROJFCTNO.: 454A-103A Table 3 LightIntensitMyeasurements Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: FreshwaterAlga,Selenastrumcapncornutum DilutionWater: Freshwater@l@ Medium LightfiftnsitMyeasurementsOux) Test Day No. I No. 2 No. 3 No. 4 No.5 0 3940 3880 4300 3900 3870 1 3910 4270 4440 4610 4010 2 4000 4290 4310 4540 4170 3 4120 4400 4350 4160 4240 4 4200 3990 3890 4070 4140 5 3910 4120 4180 4200 4340 6 4180 4200 4120 4200 4370 7 4300 4180 3950 4430 4080 8 4010 4230 4180 4230 4450 9 4150 4090 4240 4350 4270 WILDLIFE INTERNATIONAL L-m. .-22- PROJFCTNO.: 454A-103A Table4 pH Mewurements Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: FreshwaterAlga,Selenastrumcappicornutum DilutioWnater: FreshwaterAlgalMedium Mean Measured Concentration LH Mmmwmts (mga.i./L) 0 Hours' NegativeControl 7.5 5.5 7.4 96 Hours2 8.1 8.2 11 7.5 8.2 21 7.5 8.4 44 7.4 8.3 86 7.4 7.8 179 7.4 7.5 169 (Abi2@cj 7.4 0-hoursampleswere coReeW from thebatchesof testsolutio'npreparefdorthetreatnentand controglroupsattestinifialiorl 2 96-hoursampleswere collectefdrom thepooledreplicatepsertreatnenatnd controlgroup. AACRQDED -23Table 5 Mea n CellDensitiesand PercentInhibitiofnorEach 24-Hour IntervaDluringtheTest' Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: FreshwaterAlga,Selenastrumcapricomutum DilutionWater: FreshwaterAlgalMedimn Mean Measured Conoentration (mg &i.AL) Neptive Control 24Mrs Mean Cell Percent Density Inhibition 32,667 - Mean Cell Densi@L 129,000 46 Ho Percent Inhibition - Mean Cell D22!i:!X 603,333 22 Hom Percent Inhibition - 5.5 29,000 11 129,333 -0.26 635,000 -5.2 11 29,333 10 121,000 6.2 615,000 -1.9 21 28,000 14 133,667 -3.6 636,667 -5.5 44 29,667 9.2 140,667 -9.0 535,000 11 86 25,667 21 56,000 57 17 1,()002 72 179 14,333 56 27t667 79 1 ValuescalculateudsingSAS 6.12.Manual calculatiopni@aydfferslightly 20,6672 97 2 Indicateassigmficanctliffem= fromthenegativecontroalt72 hoursusingDunneWs test(p 0.05). 3 Indicateassigmficandtfferencefromthenegativecontroalt96 hoursusingDunneWs test(p:g0.05). -24Table 6 Mean Areas Under theGrowth Curve and PercentInhibitiofnorEach 24-HourIntervaDluringti Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: FreshwaterAlga,Selenastrumcapncomulum DilutioWnater: FreshwaterAluW Medium Mean Measured 2illan TestConcentration mosm Percent me4m (mg a.iJL) Area Inhibition Area NegativeControl 272,000 1,972,000 Percent Inhibition - 0 -22 HQM Mean .,Percent m Area hihibition 10,520,000 -- 50,4 5.5 228,000 16 1,888,000 4.3 10,820,000 -2.9 54,6 11 232,000 15 1,796,000 8.9 10,388,000 1.3 52,0 21 216,000 21 1,916,000 2.8 10,920,000 -3.8 57,2 44 236,000 13 2,040,000 -3.4 9,908,000 5.8 86 188,000 31 928,000 53 3,412'0002 68 179 52,000 81 316,000 84 656 '0002 94 ValuescalculateudsingSAS 6.15.Manual calculationmsay diff-selrightly 2 Indicatesa sigmficantdfferencefrom thenegativecontrolat 72 hours usingDunnett'stat (D :g0.05). 3 Mcam a significadnitfferencferom thenegativecontrolat96 hoursusingDunnett'stest(p :50.05). 53,0, 12,74 1,06 -25Table 7 Mean Growth Ratesand PercentInhibitiofnorEach 24-Hour IntervaDluringtheTest' Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: DilutionWater: FreshwaterAlga,Selenasimm capricornutum FreshwaterAlgalMedium Mean Measured 0-2 TestConcentration Mean (mg a.i./L) Growth Rate 0 -46 HQM Percent Mean Percent Inhibition Growth Rate Inhibition NegativeControl 0.0493 -- 0.0533 - 0 -ZZHQJO Mean ..Pa=nt Growth Rate Inhibition 0.0568 -- IV Grov 0. 5.5 0.0438 11 0.0533 0.0 0.0574 -1.1 0. 11 0.0448 9.1 0.0518 2.7 0.0572 -0.68 0. 21 0.0428 13 0.0540 -1.4 0.0572 -0.72 0. 44 0.0452 8.3 0.0551 -3.3 0.0549 3.4 0. 86 0.0373 24 0.0357 33 0.03922 31 0. 179 0.0138 72 0.0208 61 0.01002 82 0.0 ValuescalculateudsingSAS 6.12.Manual caladationmiay diffesrlightly - 2 Indicateassignificadnitfferencferomthenegativecontroalt72 hoursusingDunnetfstest(p:g0.05). 3 Indicateassignificadnitfferencferomthenegativecontroalt96 hoursusingDunnetestest(p 0.05). -26Table 8 ECIO, EC50 and EC90 Values Based on CellDensityOver the96-Hour ExposurePeriod Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: FreshwaterAlga,Selenastrumcapticornutum DilutionWater- FreshwaterAlgalMc&um 95% Confidence ECIO Limits Time (mg a.i./L) @mi a.i./L) 24 Hours Not Detmmiined 48 Hours Not Determined 72 Hours 37 <0.0and 64 96 Hours 49 43 and 50 EC50 @ma a.i./L) 163 81 70 71 95% Confidence Limits LMA a.i.!L 74 and 191 72 and 90 44 and 78 66 and 73 EC9( LM.A a.i. Not Not Deten 153 137 -27Table 9 EC 10,EC50 and EC90 Values Based on Area Under theGrowth Curve Over the 96-Hour Exposur Sponsor: TestSubstance: TestOrganism: 3M Corporation PFOS FreshwaterAlp, Selenastrumcapricor?wtum DilutionWater: FreshwaterAlgalMedium 95% Confidence ECIO Lblits Time (maa.i./L) (inga.i./L) 24 Hours Not Ddemiined 49 Hours Not Detmmined 72 Hours 46 <0.0and 56 96 Hours 49 40 and 50 EC50 (mg a.i.AL) 122 84 74 71 95% Confidence Limits (mg a.i./L) 19 and 176 67 and 146 55 and 82 67 and 74 EC90 (ma a.i. Not Detmri Not Deteni 165 145 -28Table 10 EC 10,EC50 and EC90 Values Based on Growth Rate Over the96-Hour Exposure Period Sponsor: 3M Corporation TestSubstance: PFOS TestOrganism: FreshwaterAlga,Selenaitrumcapricornutum DilutionWater. FreshwaterAlgalMedium 95% Confidence ECIO Time Ln!ga.i.AL) Limits (mR a.i./L) 24 Hours Not Determined 48 Hours Not Detmm:kined 72 Hours 53 23 and 64 96 Hours 59 54 and 63 'Confidencelimitcsouldnotbe calculatewdiththedataobtained. EC50 (mg a.i.AL). 136 142 120 126 95% Confidence Limits a.i./L) 30 and 204 107 and 185 103 and 132 115 and 138 EC90 Lmg a.i.@Lj Not Detmmine Not Detmmine >179 >179 WILDLIFE INTERNATIONAL LTD. -29- PROJECTNO.: 454A-103A TableII CellDensilaefsortheRewvery Phase Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Freshwater Alga, Seknastrum DilutionWater. FreshwaterAW Me&un Mean Measured TestConcentration (mgai./L) Day 0 NegativeControlRep. A 10,000 capricornutum CellDensitie(sL"m@L Day 3 1,220,000 Pal 5 6,420,000 . @179' 1,000 35,000- 1,180,000 The t=tment group was dilutetdoa concentrationfthetestsubstanctehattheoreticalwloyuld notinhibigtrowth. 2 Due to themethod usedto preparemovmy phase testsolutionsi,ni@ celldensitiewsere not equivalentthroughouttheuwtnent groups. Ahffi'NDED WILDLIFE INTERNATIONAL LTD. -30- PROJECTNO.: 454A-103A Figure1. NegativeControlAlgalGrowtb,ExpressedinCellDensityD,unng the96-HourExposure. 100000001000000-- 100000-10000 1000 0 Mean Cell Density (Colls/mL) NegativeControl 2 24 48 72 96 ANENDED WILDLIFE INTERNATIONAL LTD. -31- Figure2. Concentration-ResponCsuerve,ExpressedinCellDensity PROJECTNO.: 454A-103A 10000000-1000000 100000-- 100001 iooo-. 0 Mean Cell Densfty (Colls/mL) NegativeControl 5.5 mg/L 11.mg/L 21 mg/L 44 mg/L -86 rTig-/L 179 mg/L 24 48 72 96 ExposureDuration(Houm) ANIENDED WILDLIFE INTERNATIONAL LTD. -32- Fig= 3 - Fe=veq Ph= ResponseCurve,ExpressedinCeU Density PROJECT NO.: 454A-103A 100000001000000-- 100000-. 100001 10001 ioo-. 0 CellDensity(Colls/mL) -*-Negative Control ib 179 mg/L 1 2 3 4 5 Exposure Duration (Days) AMENDED WILDLIFE INTERNATIONAL L-m. -33- PROJIECTNO.: 454A-103A APPENDIX I FreshwaterAlgalMedium' Sponsor Test Substance: TestOrgmism: 3M Corporation PFOS FreshwaterAlga,Selenastrumcaphcornutum DilutioWnater-. Freshwater Medium Compound Nominal Concentration MgCI206H20 CaCI202H20 H3BO3 MnCI2.04H20 ZnCI2,.T FCC13i6H20 COCl2*6H20 Na2MOO492H20 CUC1202H20 Na2EDTA02H20 NaN03 MgSO497H20 K2]HP04 NaHC03 12.16 4.40 0.1856 0.416 3.28 0.1598 1.428 7.26 0.012 0.300 25.50 14.70 i.o44 15.0 mg/L mg/L mg/L mg/L AgIL mg/L AGIL AGIL AGIL mg/L mg/L mg/L mg/L mg/L The pH was adjustedto7.5 0.1 using10% HC 1. WILDLIFE INTERNATIONAL L-m. -34- PROJECTNO.: 454A-103A APPENDIX ]I AnalysesofPesticideOsr,ganics,Metalsand Ofl= laorganics inWildlifbeftmafionaLltd.WellWaterl ANALYSIS MiscellaneousMeasurements TotalDL-@wlvedSolids Ammonia Nitmen - TotalOrgamc C'wbon@ Totd Cy7auide RAEASURFD CONCENTRATION 286 < 0.050 < 1.0 < 10.0 ddorines and PCBs 0Airmo Alpha BHC Bela BHC DeltaBHC Gamma BHC (Lindane) Chlordane DDD, pp0' DDE, pp DDT pp yef&9 P.ndosul A B Sulfate Endrin Endda Aldehyde Hep*hlor MEthoxychlor HeptacliloErpo)dde Tokaphene PCB-1016 PCB-1221 PCB-1232 PCB-1242 PCB-1249 PCB-1254 PCB-1260 < O.W5 < O.W5 < 0.005 < 0.005 < 0.006 < 0.025 < 0.006 < 0.005 < 0.008 < 0.005 < 0.005 < 0.005 < 0.018 < 0.010 < O.W5 < 0.005 < 0.007 < 0.005 < 0.5w < 0.260 < 0.260 < 0.260 < 0.720 < 0.720 < 0.720 < 0.720 Metals ani Other Inarganics Al!m Arsenic M un@3 < 100 < 25.0 < 0.50 < 1.0 Calciu]3-9 cb0 W Cobal 35.0 < 2.0 < 1.0 < 20.0 < iw 3 < 10.0 map esiuli3k hla-ngenese' IMM7&O;ILgpum 3 N LIC@D Sel 3 siive Sodium 3 @ziue 13.5 < 1.0 < 0.20 < 2.0 < 2.0 6.62 < 25.0 < 1.0 21.3 < 20.0 A@419 pefl WQSETnvir@eGnituanle,sYvlioUrfcios,draame=scp-of(lmN= 7 19;'7. 23 Ai@@ perpfrmed by WildlifeintannfionaLlt& forthesmple collecteodn Novemba 5,1997. ArAyses imformed by WildlifIent=ahonal Lt& forsmples collecteodnNovemba 5 ftm& 7,1997. MV-L n4 -& mfy-L AilL A91-L mi/-L Ail-L tzw-L Pt/-L Mi/-L Ail-L pi/-L P4/-L 941-L JA@- 144 -& 4 -& lAfy-L AW-L mix_ Ail-L pt/-L Mi&%-XL AW-L n3@WL 41L WILDLIFE INTERNATIONAL L-m. -35- APPENDIX M PROJECTNO.: 454A-103A THE ANALYSIS OF PFOS IN FRESHWATER ALGAL MIEDRUM IN SUPPORT OF WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-103A WILDLIFE INTERNATIONAL LTD. -36- PROJFCTNO.: 454A-103A REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-HourToxicitTyestwithffiFcreshwateArlga(Selenastrurmapricomutum) WILDLIFE BITERNATIONAL LTD. PROJECT NO.: 454A-103A PP,JNC@IPALR4VESnGATORRayrrWd L@ Van Hoven,Ph.D. Scientist WillardB. Nixon,Ph.D. Manager,AnalyticaClhwdstry DATE DATIff AMENDED WILDLIFE INTERNATIONAL LTD. -37- PROJECTNO.: 454A-103A Introduction Freshwateralgalmedium sampleswere collectefdrom an acutetoxicitsytudydesignedto detemune the effectsof PFOS (PerfluorooctanSeulfonicAcid Potassium Salt)to the freshwateralga (Selenastrum cap?icornutum).This studywas conductedby WildlifeInternationaLltd.and identifieads ProjectNo.: 454A-103A. The analyseosfthesewatersampleswereperformedatWildlifIenternationLatld.usinghigh performanceliquidchromatographywith mass spectrometridcetectio(nHPLC/MS). Sampleswere receivedforanalysiosn April12,15 and 16,1999 and were analyzedan eachsamplereceiptday. TestSubstanceand Internal T'hetestsubstanceused forthe analyticaplortionof thisstudywas WildlifeInternationLatld. identificatniummber-4675. The testsubstancewas used to preparecalibratisotnandardsand matrix fortificatsiaomnples. The internasltandardwas receivedfrom 3M Corporationon July2, 1998 and was assignedWildlife internationLatld.idmitificatniuomnber 4526 upon receiptT.he internasltandarda,granularmaterialwas identifiaesd: IK 1K 2K 2H PerfluorooetaSnuelfoniAccid,ChemicalAbstractNumber: 27619-97-2. T'hestandardwas storedunderambientconditions. Agg3ILcalMethod The methodusedfortheanalysiosfthefreshwatearlgalmedium sampleswas developedatWildlife InternationLatld.and entitle"dAnalyticaMlethod Validatonfor the,DeWrminaton of PFOS in FreshwaterS,altwatera,nd AlgalMedia!'.This methodologywas includedas AppendixIlof Wildlife InternationLatld.protocolnumber 454/011299/MVAUSUB454. It was based upon methodology providedby 3M Corporation. Sampleswere dilutedin a 50% methanol: 50*/oNANOpure(D water solutioncontaining 0.100mg4HPFOS (internasltandard)/aLnd 0.05% fonnicacid(v/v)so thattheyfellwithinthe calibratiornangeofthePFOS methodology. ConcenU-ationosf PFOS in the standardsand sampleswere dkunnined by reverse-phasheigh performanceliquidchromatographyusinga Hewlett-PackarMdodel 1100 legh PerformanceLiquid WILDLIFE INTERNATIONAL LTD. -38- PROJECT NO.: 454A-103A Chromatograph(EPLC) witha Peridn-EhneArPI IOOLC Mass Spec=neter equippedwith a PerldnEhner TurbolonSpmy ion source. IHPLC separationwsere achievedusinga KeystoneBetasilCis analyticaclolumn(100nun x 2 nun I.D.,3 pm particlseize).The hishumentparametersaresummarized inTable1. A methodflowcharitsprovidedinFigure1. CalibratioCnurve and LimitofOuanfitation Calibratiosntandardosf PFOS preparedin a 50% methanol: 50% NANOpureg watersolution containin0g.100mg4HPFOS (internsatlandard)/aLnd 0.05% formicacid(v/v)r,angininconcentrafion from 0.00229to 0.0457mg a.i".w/eLre analyzedwithdw smwles. Linearregressioenquationswere generatedusingpeak arearesponseratios(PFOS :mtemw standard)versustherespectivceoncentration ratio(sPFOS :hiternisdtandardo)fthecalibratisotnandardsA. typicaclalibraticounrveispresmtedin Figure2. The concwtrafionof PFOS in thesampleswas determinebdy substitutitnhgepeak area responseratiosintotheapplicablleinearegressioenquation.Representativieonchromatogramsof low and high.calibratsitoanndardsarepresentedinFigures3 and 4,respectively. The method limitofquantitati(oLnOQ) fbrtheseanalysewsas setat0.115 mg a.iJLcalculateadsthe productof thelowestcalibratisotnandardanalyzed(0.00229mg a.i./La)nd theMutim EwWr of the matrixblanksamples(50.0). MatrixBlankand FortificatiSoMn21cs Threematrixblanksampleswere analyzedto determinepossiblmeterfe=ce. No mterfermceswere observedator above theLOQ duringsamplesanalyses(Table2). A representaticvheromawgram of a matrixblankispresenteidnFigure5. Freshwateralgalmedium was fortifiaetd0.915,45.7and 220 mg a.iJLand analyzedconcurrently withthesamplestodeterminethemean procedurarlecovery(Table3). Sampleconcentratiownesre not comcted forthe mean procedurarlecoveryof 99.1%. A representaticvheromatograionf a matrix fortificatisopnresenteidnFigure6. AMENDED WILDLIFE INTERNATIONAL LTD. -39- PROJECTNO.: 454A-103A ENmale Calculations Samplenumber 454A-103A-2,nominalconcentratiofn5.7mg a.i./iLnfreshwatearW medium. InitiVaollume: 0.100 mL FinalVolume: 25.0mL DilutioFnactor.250 PFOS Peak Area- 164427 InternaSltandardPeak Area: 240533 Peak Area Ratio:0.6836..- Cahb=on curveequauon. Slope:3.43926 Iuftrcept0:.03327 Curveisweighted(I/x). PFOS (pga.i./La)tinstrument (Peakarearati-o (Y-fiftrcepxtI).)S.Conc=trafion Slope (0.6836- 0.03327)x 100pg a.i.AL 3.43926 18.9pg a.i-/L Noie:I.S.= kwmal standard. PFOS (mg a.i./Li)nsample PFOS (pga.i./La)tinsbwnentx DilutioFnactor 10004mg 18.9x 250 1000 AMENDED WILDLIFE INTERNATIONAL LTD. -40- PROJFCTNO.: 454A-103A 4.73mg a.i./L PercentofNominal Concentration PFOS (mg a.i.ALi)nsample x 100 PFOS (mg a.i./Ln)ominal Calculatedrecovery8:2.0% Note:manual calculatimoany differ. RESULTS Simple Analysis Freshwateralgalmedium sampleswere collectefdrom theacutetoxicitsytudywiththefreshwatearlga I (Selenastmmcapricornutum)attestinitiatiAopnr,il12,1999 (Hour 0),on April15,1999 (Hour72),and attestterminationA,pril16,1999 (Hour 96). The measuredconcentratioonfsPFOS inthesamples collecteadtinitiatioofnexposureofthetestorganisms(Hour 0)rangedfrom 82.0to98.1% ofthenominal concentrationsS.amplescollecteadtHour 72 had a measured concentratiornangeof 91.7to 105% of nominalvalues.The Hour-72 abioticsamplewas 81.9% ofthenominalconcentrationS.amplescollected attestterminatio(nHour 96)had a measuredconcentratiornangeof90.3t@ 102% ofnominalvalues.The Hour-96 abioticsamplewas 103% ofthenominalconcentratio(nTable4). A representaticvheromatogram ofa testsampleisshown inFigure7. AMENDED WILDLIFE INTERNATIONAL LTD. -41- PROJFCTNO.: 454A-103A INSTRUNMQT: TableI TypicalLC/MS OperationaPlarameters Hewlett-Pa&ardModel 1100High PerfomianceLiquid Chromatographwitha Peridn-ElmerAPI IOOLC Mass Spectrometer equippedwitha Peridn-EhneTrurboIonSprayionsource.Operatedin seleefiivoenmonitorinmgode (SM. ANALYTICAL COLUMN: KeystoneBeWil C,8column(100mm x 2 mm I.D.,3 pm particlseize) OVEN TEMPERATURE: STOP TIME: 300C 10.0minutes FLOW RATE: 0.220mljminuto MOBILE PHASE: 72.0% Medkanol:28.0% NANOpure(D Water containin0g.1% Fomiic Acid INJEcnON VOLUME: 25.0pL PFOS RETENTION TIME: Approxi@ly 6.6minutes INTERNAL STANDARD RETENTION TIME: Appro7dn@y 4.5n-,dnutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6amu 426.7 amu WILDLIFE INTERNATIONAL L-rD. -42- PROJECT NO.: 454A- 103A Table2 MatrixBLuks AnalyzedConeun=Wy DuringSample Analysis Number (454A-103A-) MAB-1 S!Mle Type MatrixRia Measured Concentratioonf PFOS' (mga.i./L) < LOQ MAB-2 MatrixBlank < LOQ MAB-3 MatrixBlank < LOQ Ile limitof quanfitatim(LQQ) was 0.115 mg a.iJLbased upon the productof the lowestcalibration standard!2!!Xzed(0.00229ifiag.i./La)ndthedilutiofnilctorfthematrixblank les(50.2). AMENDED WILDLIFE INTERNATIONAL LTD. -43- PROJECTNO.: 454A-103A Table3 MatrixFortificatiAonaslyzedConcurrentlDyuringSample Analysis Sample Number (454A-103A-) MAS-1 MAS4 MAS-7 Concentrationosf PFOS Fortified 0.915 0.915 0.915 Measured 0.884 1.10 1.02 Percent Recovered 96.6 120 112 MAS-2 45.7 41.3 90.2 MAS-5 45.7 48.4 106 MAS-8 45.7 41.4 90.6 MAS-3A 220 210 95.2 MAS-3B 220 201 91.7 NlAS-3C 220 209 95.0 MAS-6 220 221 101 -MAS-9 220 202 91.9 Mean = 99.1 StandardDeviation= 9.75 CV = 9.84 N = 11 Note:Resultsand correctionfsornew testsubstancepuritywere generatedusingMacQuan version1.5 softwareandmanualcalculationVsa.lueshavebeenroundedforE22rtin g!aoses. AAffi'NDED WILDLIFE INTERNATIONAL LTD. -44- PROJECTNO.: 454A-103A Table4 Measured ConcentrationosfPFOS inFreshwaterAlgalMedium Samples from a FreshwaterAlga Acute ToxicityTest NomhW Test Concentration mA a.i./L) 0.0 Sample Number (454A-103L- 1 8 15 Sampling Time Eau-rs) 0 72 96 PFOS Measured Concentration' (Am ai.AL) < LOQ < LOQ < LOQ Percent Of Nominal 5.7 2 0 9 72 16 96 11 3 0 10 72 17 96 23 4 0 11 72 18 96 4.73 82.0 6.04 105 5.84 101 10.7 89.8 11.2 93.6 12.1 102 19.8 86.2 23.1 101 20.7 90.3 46 5 0 12 72 19 96 91 6 0 13 72 20 96 183 7 0 14 72 21 96 42.7 93.4 41.9 91.7 46.3 101 8@.3 91.0 8@.-O 94.0 88.3 96.5 179 99.1 186 102 172 94.0 183 14A 72 150 81.9 (Abiotic) 22 96 188 103 Note:Resultsand correctionfsornew testsubstancepuritywere generatedusingMacQuan version1.5 softwareand manual calculationVsa.lueshave beenroundedforreportinpgurposes. 'The limitof quantitati(oLnOQ) was 0.115 mg a.i./bLasedupon theproductofthelowestcalibration standardanalyzed(0.00229mg a.i./La)nd thedilutiofnactorofthematrixblanksamples(50.0). AMENDED WILDLIFE INTERNATIONAL LTD. -45- PROJECT NO.: 454A-103A MF,THOD OUTLINE FOR THE ANALYSIS OF PFOS IN FRESHWATER ALGAL MFDIUM Preparematrixfortificatisoanmplesby spikintghereqaisitveolume of PFOS stocksolutiondsirectly intofreshwatearlgalmedium usinggas-fi& syringeasnd ClassA volumetncflwks. Dilutematrixfortificatainodntestsamplesintotherangeofthecalibratiso=ndards by partialfliylling ClassA volumetdcflaskwsith50% niedmol :50% NANOpureg watersolutiocnontaining 0.100mg 4H PFOS (inten*standard)AaLnd 0.05% formicacid(v/v.)A.dd theappropriatveolumeof sampleand bringtheflasktovolume withdw dilutiosnolvent.Processthematrixblanksampleusing thesame dilutioannd aliquovtolumeasforthelowestfortificatlieovnel.Mix wellby severarlepeat inversions. Anipulatesamplesand submitforLC/Ms analysis. Figure1. Analyticamlethod flowcharftortheanalysiosfPFOS infreshwatearlgalmechum. WILDLIFE INTERNATIONAL L-rD. -46- PROJECTNO.: 454A-103A 2.00- 0 -1.50- 1.00 Q50 0.00 0.000 0.100 0.200 0.300 Q400 Cmcentratim (Ratio) 0.500 Figure2. A typicaclalibraticounrveforPFOS. Slope 3.43926;Intercept0.03327;r 0.9986. Curveisweighted(I/x). AME-4DED WILDLIFE INTERNATIONAL L-rD. -47- PROJECTNO.: 454A-103A intensit2y8:49 cps 1 OC)@ 157 go8(@ 706(@ 40, 30- 2 0* 10. -v 0. . 24 2:s 31 1.30 136 el !Oi I .. . 6*1 91 2.55 3.81 121 5.06 151 6.32 181 7.57 Z14 =9=90-- 2 i1 SCan 8.83 Time Figure3. A representafiivoen ULL ofa low-leve(l0.00229mg a.i./LP)FOS standard. AMENDED WILDLIFE INTERNATIONAL L-m. -49- PROJECT NO.: 454A-103A intensit3y2:143 cps 155 100, 90. 70, 6& 504& 30, 135 20. 7 24 0 . . . 3*1' 1.30 63 79 120115 - I id 6'1 91 121 2.55 3.81 5.06 151 6.32 IS9404 224 iii , '21*1 Scan 7.57 8.83 Time Fi*gure4. A representativoenchromatogrunofa high-leve(l0.0457mg a.idl)PFOS sumdard. AMEMED WILDLIFE INTERNATIONAL LTD. -49- PROJECTNO.: 454A-103A 10 25 intensit2y3:4 cps 80, 7 Or 60- so. 40, 30136 156 20- 41 56 el 95 112 10, hA.. A k-@,API I.- yv Lp I v 215 :: v V" 31 6.1 91 121 151 lil 2il 1.30 2.55 3.81 5.06 6.32 7.57 8.83 Scan 'rime Figure5. A representaticvleuotimtograomfa matrixblanksample(454A-103A-MAB-1). Mm arrowindicattehserftntiofnim ofPFOS. WILDLIFE INTERNATIONAL LTD. -50- PROJECTNO.: 454A-103A 10(). go- so. 7 Or- 60, so. 40302 Or 10, A 0-f 24 ng 3'1 1.30 intensit9y1:04 cps 157 137 77 94 115 i,6ngnft 6'1 q'I Ii ''1-61 2ii Scan 2.55 3.81 5.06 6.32 .7.57 8.83 Time Figure6. A representaficvheromatogrmnofa nmtdx fordficatisoannipl(e454A-103A-MAS-1). WILDLIFE INTERNATIONAL LTD. -51- PROJECT NO.: 454A-103A Intensit1y.:1161cps 100- 161 90, 80- 70- 607 5 40, 3(@ 140 20, 10. 7 25 -Ao as ag 1(),3 118 lgg 0 . . . 3*1 6'1 9'1 121 151 lil 2il Sm 1.30 2.55 3.81 5.06 6.32 7.57 8.83 Time Figure7. A representative ofa testsample(454A-103A-2). WILDLIFE INTERNATIONAL L-rD. -52- PROJECTNO.: 454A-103A APPENDIX IV CeH DensityforEach RepheatePerTreatmentOver the96-Hour ExposurePeriod Sponsor. 3M Corporation Test Substance: Test Orpaisni: PFOS FreshwaterAlga,Selenasimm capricomutum DilutionWater FreshwaterAlgal Medium Mean Measured Concentration (mg &i./L) RepUrate 24 Hours Negative Control A B c 34,000 33,000 31,000 5.5 A 29,000 23,000 Ic 35,000 A 30,000 B 27,000 c 31,000 21 A 30,000 B 26,000 c 28,000 44 A 30,000 B 32,000 c 27,000 86 A 37,000 B 18,000 c 22,000 179 A 15,000 B 18,000 c 10,000 Cell Densities(CeUdmL)L 48 Hours 132,000 130,000 125,000 72 Hours 710,000 605,000 495,000 136,000 118,000 134,000 790,000 500,000 625,000 105,000 124,000 134,000 560,000 660,000 625,000 141,000 132,000 129,,000 960,000 450,000 600,000 136,000 151,000 135,000 515,000 700,000 390,000 65,000 57,000 46,000 215,000 136,000 162,000 35,000 22,000 26,000 21,000 18,000 23,000 96 Hours 2,720,000 2,860,000 2,640,000 3,000,000 3,180,000 2,940,000 2,680,000 2,740,000 3,220,000 3,340,000 3,080,000 3,300,000 2,800,000 3,020,000 3,420,000 650,000 760,000 470,000 45,000 30,000 26,000 The =tlal celldensityof the dock culturewas determmed and an moculum volume was admmatm-ed to each test chamber toyielda celldensityofapproxftnatel1y0,000 ceUstmL attestinitiatio(n0 hours). ANIENDED WILDLIFE INTERNATIONAL LTD. -53- PROJECTNO.: 454A-103A APPENDIX V Area UndertheGrowth CurveforEach RepheatePerTreatrnenOtver the96-Hour ExposurePeriod Sponsor Test Substance: TestOrganism: DilutionWater. 3M Corporation PFOS FreshwaterAlga,Selwasirwn capricomutum FreshwaterAlga Medium Me= Measm-ed Concentration CumulativeArea Under theGrowth Curve (mg aiJL) Replir-ate 0 - 24 Hours 0-48Hom 0 - 72 Hours NegativeControl A 288,000 2,040,000 11,904,000 B 276,000 1,992,000 10,572,000 c 252,000 1,894,000 9,094,000 0 -96 Hours 52,824,000 51,912,000 46,464,000 5.5 A 228,000 1,968,000 12,720,000 57,940,000 B 156,000 1,608,000 8,794,000 52,704,000 'C 300,000 2,089,000 10,956,000 53,496,000 A 240,000 1.620,000 9,360,000 48,000,000 B 204,000 1,776,000 10,944,000 51,504,000 c 252,000 1,992,000 10,860,000 56,760,000 21 A 240,000 2,052,000 13,824,000 63,994,000 B 192,000 1,848,000 9,592,000 50,712,000 c 216,000 1,848,000 iowooo 56,904,000 44 A 240,000 1,992,000 9,564,000 49,104,000 B 264,000 2,220,000 12,192,000 56,592,000 c 204,000 1,908,000 7,968,000 53,448,000 86 A 324,000 1,308,000 4,428,000 14,568,000 B 96,000 756,000 2,832,000 13,344,000 c 144,000 720,000 2,976,000 10,320,000 179 A 60,000 420,000 852,000 1,404,000 B 96,000 336,000 576,000 912,000 c 0 192,000 540,000 888,000 AAffiNDED WILDLIFE INTERNATIONAL L-m. -54- PROJECTNO.: 454A-103A APPENDIX VI Growth RateforEach RephcatePerTreatmentOver the96-Hour ExposurePenod Sponsor. 3M Corporation TestSubstanre: PFOS TestOrpnism DilutioWnater FreshwateArlga,Selenastncmapncornutum FreshwateArlgalMedium Mean Measured GrowthRate Concentmfim (ni&gi./L) Replicate 0- 24Hours 0- 48 Hours 0 -72 Hours NegativeControl A B 0.0510 0.0497 0.0538 0.0534 0.0592 0.0570 c 0.0471 0.0526 0.0542 5.5 -A 0.0444 0.0544 0.0605 @cB 0.0347 0.0522 0.0514 0.0541 0.0543 0.0574 II A 0.0458 0.0490 0.0559 B 0.0414 0.0525 0.0582 c 0.0471 0.0541 0.0574 21 A 0.0458 0.0551 0.0619 B 0.0398 0,0538 0.0529 c 0.0429 0.0531 0.0569 44 A 0.0458 0.0544 0.0547 B 0.0485 0.0566 0.0590 c 0.0414 0.0542 0.0509 86 A 0,0545 0.0390 0.0426 B 0.0245 0.0363 0.0363 c 0.0329 0.0318 0.0387 179 A 0.0169 0.0261 0.0103 B 0.0245 0.0164 0.0082 c O.ODOO 0.0199 0.0116 0-96 Hours 0.0584 0.0589 0.0581 0.0594 0.0600 0.0592 0.0582 0.0585 0.0602 0.0605 0.0597 0.0604 0.0587 0.0595 0.0608 0.0435 0.0451 0.0401 0.0157 0.0114 0.0010 AMENDED WILDLIFE INTERNATIONAL L-m. -55- PROJECT NO.: 454A-103A APPENDIX VII Changesto Protocol Tlusstudywas conductedm accordancewiththeappmved Protocolwiththefohowi% changes: I. The protocolwas amended to add the proposedexperimentasltartand terminatiodnates,test concentrationasnd testsubstancenumber. 2. Ile protocowlas amendedtospeciftyheanalyticmaelthod. 3. Ile protocowlas amended torepeatthetestwitha highertestconcenuafion. WILDLIFE INTERNATIONAL LTD. -56- PROJECTNO.: 454A-103A APPENDIX VEII PersonnelInvolvedintheStudy The fohowmg key persomelwere mvolvedm theconductor management oftinsstudy: I. Henry 0. Krueger,Ph.D.,DirectorA,quaticT("dcologyand Non-TargetPlants 2. WillardB.Nbwn, Ph.D.,Manager,AnalyticaClhemistry 3. Raymond L.VanHoven, Ph.D.,Scientist 4. KurtR. DrottarS,eniorBiologist 5. Mark A. Mank, LaboratorySupeniwr WILDLIFE INTERNATIONAL LTD. -57- PROJECTNO.: 454A-103A APPENDIX IX ReportAmendment I. OriginaRleport: Pages 1-4,6 and 36 Amendment: 'Me pages were changedto includethe amended reportdate,revisedpage numbers,and new signatureasnd datesdue to the additionof the report amendment asAppendix IX. Reason: To reflecttheissuingofan amended report. 2. OriginaRleport: Page 2 Amendment: The compliancestatementwas revised. Reason: To ckt;fyhow thetestsubstancewas cmmetenzed. 3. OriginaRleport: Page 10 Amendment: Informatiopnrovidedby theSponsorreflectitnhgereanalysiosfthetestsubstance,. includintghereanalysidsateand thepurity,was addedto theTest Substance section. Reason- To reflectthecurrentestsubstanceinformatiopnrovidedby theSponsor. 4. OriginaRleport: Entirereport Amendment: Alltestsubstanceconcentratiownesre changedto reflectthepurityof thetest substanceasdeterminedby theSponsor ina reanalysiosf thetestsubstance(FC95,Lot217).Testcwccnftafionosriginalwleyre basedon thereportepdurityof 98.9%. The certificaotfeanalysidsatedMarclf9, 2000 indicateadpurityof 90.49%. Therefore,all testsubstanceconcentrationisn,cludingnominal emccamons, nmsured concentrationEsC, values,and NOAEC valueswere recalculataendd reportedas mg a.i./bLasedon the90.49% purity. Reason: To reportheresultosfthetestbasedon thetestsubstancepurityof90.49% atthe requestof theSponsor. AMENDED WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454A-103 Page I of I AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER (Selenastrucmapricomutum) ALGA PROTOCOL NO.: 454/110998/SEL-96HI/SLTB454 AMENDMENT NO.: I SPONSOR: 3M Corporation PROJECT NO.: 454A-103 EFFECTIVE DATE: January4,1999 AMENDMENT: Page 2 Add: Experimental StartDate: 2/19/99 ExperimentalTerminationDate: 2/23/99 Test Concentrations:Negative ControL 6.3,13,25, 50 and 100 mg Test Substance No.: 4675 a.i./L REASON: The above informationwas not known when the protocolwas signedby the Study Director. AMENDMENT: APPENDIX II,Page 16 Add: Liquid Chromatography Mass Spectrometry (LCMS) Method for the Determinationof PerfluorooctandSulfonicAcid, Potassium Salt(PFOS) inFreshwater,FilteredSaltwaterand Algal Medium. REASON: To add the analyticamlethod to be used inthe study. STLJDY DIRECTOR c;tzC7(gq DATE LABoAAT6RY-@@AGEMENT DATP- SPORSOR'S REPRESENTATIVE a454llO3lamendl DATE v(&,bcy@d OWILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454A-103 Page 1 of I AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR TO)ACITY TEST WITH THE FRESHWATER (Selenastrucmapricornutum) ALGA PROTOCOL NO.: 454/110998/SEL-96HI/SUB454 AMENDMENT NO.: 2 SPONSOR: 3M Corporation PROJECR NO.: 454A-103 EFFECRIVE DATE: April8,1999 AMIENDMENT: Page 2 Change:. ExperimentalStartDate: 2/19/99 ExperimentalTerminationDate: 2/23/99 Test ConcentrationsN:egativeControL6.3,13,25, 50 and 100 mg &i.AL To: ExperimentaSltartDate:4/12/99 ExperimentalTerminationDate:4/16/99 TestConcentrationsN:egativeControl,6.3,13,25, 50, 100 and 200 mg a.i./L REASON: The testwillbe repeatedbecausecellcountswere not made witha hemaqwmeter and a microscope.An additionatlestconcentratiownas added inan attempttoobtainEC90 values. AMENDMENT: EXPERIMENTAL DEST Page 3 Change: Target concentrationwsillnotexceed 100 mg/L or thesolubililtiymitofthetestsubstancein water. To: Targetconcentrationwsillnotexceed 1000 mg/L or thesolubilitlyimitofthetestsubstanceinwater. REASON: The new nominalconcentrationesxceed 100 mg a-i-AL. STUDY DIRECTOR 1?7 DATE LABOFATORY kAk.NACiEMENT o. SPONSORR'FSPRESENtk-Tr@V-E bAtE Ilqq - DAIE a454llo3tamena im-n@ PROTOCOL PFOS: A 96-HOUR TO)CCITY TEST WITH THE FRESHWATER ALGA (Selenastrumcapiicornutum) U.S.EnvironmentalProtectioAngency Series850 -EcologicaElffectsTestGuidelines OPPTS Number 850.5400 OECD Guideline201 3M Lab RequestNo. U2723 WILDLIFF- Submittedto 3M Corporatim EnvironmentalLaboratory P.O.Box 33331 St Paul,Minnesota 55133 INTE:RNATIONAL LTD. 8598 Comma= Drive Enton, Maryland 21601 (410)822-8600 November 9, 1998 PROTOCOLNO.: 454/110998/SEL-96HI/SUB454 3M LAB REQUEST NO. U2723 wra w@A I WILDLIFE INTERNATIONALLTD. lu -2- PFOS: A 96-HOUR T03aCITY TEST WITH THE FRESHWATER ALGA (Selenastrumcaphoornutum) SPONSO 3M Corporation EnvironmentalLaboratory P.O. Box 33331 St PauL Minnesota 55133 SPONSOR!S REPRESENTATIVE: TESTINGFA Ms. Susan A. Beach )WildlifIenternationLatld. 8599 Commerce Drive Easton.Maryland 21601 Ems STUDY DIRECTO Kurt Drottar SeniorAquatic Biologist LABORATORY MANAGEMENT: Henry 0. Krueger,Ph.D. DirectorofAquaticToxicology& Non-Target Plants FOR LABORATORY USF, ONLY Proposed Dates: Experimental StartDate: ProjectNo.: '45e4.-4/0,3 Test Concentrations: Test SubstanceNo.: Refcn= Experimental TerminationDate: SubstanceNo. (ifapplicable): PROTOCOLAPPROVAL STUDYDIRECTOR DATE @U P" , LAB kkTSORYPM.ANAGEMENT TORY: /,) DATE SPIOSOR@RR!ESPRESENtk-TfVE /OIZ DAT9 PROTOCOLNO.: 454/110998/SEL-96HI/SU13454 3M LAB REQUEST NO. U2723 IWILDLIFE INTERNATIC)NAL LTD. -3- INTRODUCRIO WildlifeInternatioLntadl.willconducta 96-hourtoxicittyestwiththefreshwatearlga, Selenastrucmapticornutufmo,rtheSponsoarttheWildliIfneternatioLnTaClLaquatitcoxicolofgaycility in Easton,Maryland. The studywill*beperformedbased on proceduresintheU.S. En*omnental ProtectioAngency Series850 -EcologicalEffectsTestGuidelinesOPPTS Number 850.5400 (1)and OECD GuidelineforTestingofChemicals,20 1: Alga,Growth InhibitionTest(2).Raw dataforall work performed atWildlifeInternationLatld.and a copy of thefinalreportwillbe filedby project number in archiveslocatedon theWildlifeInternationLatld.siteor at an alternativleocationto be specifiedin the finalreport. PURI?OSE The purposeofthisstudyisto determinethe toxicitoyf a testsubstancetothe fi-eshwategrreen alga,Selenastrum cap,-icornutum,duringa 96-hourexposure period. EXPERIMENTAL DESI The greenalga,Seknastrum cap?icomutum, willbe =posed to a geometricseriesof atleastfive testconcentrationasnd a negative(culturmeedion) controlfor96.hours.Targetconcentrationwsillnot exceed 100 mg/L or the solubilitlyimitof the testsubstancein water. Generallyt,he nominal concentrationof each testsubstanceused inthe definitivteestwillbe atleast50% of the next higher teaftnen@udess mformatlm concerninfgt concentubon-effeccturvemcheatesthata dffcmt dilution factorwould be more appropriate. Test solutionswillbe inoculatedwith 10,000.ccUs/mL. Three "biologicc'replicatepser experimentalgroup willbe preparedforevaluatingcelldensities.One or more additiona"lanalyticar' replicatewsillbe includedin thetes@as needed,to providetestsohitim foremeentrationverification on Day 3 oftheevosure. An abiotirceplicataetthehighestconcentratiownillbe includedinthetest and willbe swnpled on Day 3 and Day 4. Inorderto controlbias,thepositionoftheflaskswillbe detmmmed by mdwmmnate draw dailyduringthe exposurepenoct No ofl= potentiaslourcesof bias areexpectedto affectheresultosfthesh*. The responseof thealgaewillbe measured in terms of celldensityb,iomass expressedas area underthegmwth curve,and growdirate.An EC50 (i.e.t,he theoreticatlestconcentratiotnhatproduces a 50% reductioninthe measured parameter)valuefor celldensitywillbe calculatefdoreach 24 hour interval.EC10, EC50 and EC90 valuesforcelldensityb,iomass (FbC)and growthrate(EC) willbe PROTOCOL NO.: 454/110998/SEL-96HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL L-rD. -4- calculatedi,fpossible,atthe 72 and 96 hour intervalsT.he no observed adverseeffectconcentration (NOAEC), which isthehighesttestconcentratiotnhatinducesno adverseinhibitoreyffecton growtk willbe dewmined relativteo eachparameterat72 and 96 hours based upon evaluationof the statistical resultsand the dose-responsepatterLr At theend of the96-hour exposure,algistateifcfectswillbe differentiatferdom algicidaelffectsinthosetreatmentwshich aremaxirrwly inhibited. MA17ERIALS AND METHODS Test Substance hffonnationon the characwrizatioonf wA controlor referencesubstancesisrequiredby Good LaboratoryPracticSetandards(GLP). The Sponsor isresponsiblfeorprovidingWildlifeint=ational LtcLwnttenvcnficatiotnhatthetestsubstancehas been charactuizedaccordingto GLPs pnor to itsuse inthestudy.Ifwrittenverifir-atoifonGLP testsubstancechmmctmizationisnotprovidedtoWildlife InternationaLltd.,itwillbe notedinthecompliancestatementof thefinalreporl The attachedform IDDENIMCATION OF TEST SUBSTANCE BY SPONSOR informationnecessaryforGLP compliance. (Appendix 1)istobe used toprovide The Sponsor isresponsibleforallinformationrelatedto the testsubstanceand agreesto accept any unused testsubstanceand/ortestsubstancecontainersremaining attheend of the study. P=aration of TestSolutions The testsubstancewillbe administeretdoSelenastruminan aqueousmediual This routeof admmmtr&on was sekctedb=m itrepresentishemost likelyrouteof exposureto algae,which exists in aquaticenvirorunents. Testsolutionwsillbe preparedby cwutmg one or more stocksolutionswith algalculturemedium to achievethe targetconcentrationsS.torkswillbe preparedinalgalculturemediuwl AU weights, volumes and dilutionussed to prep= thetestsolutionswillbe documented infficraw data. Test Oreanism The freshwatergreen alga,Selenastrumcap?icomutum, willbe used in thistesl This species isrepresentativoefan kvamant group of algaeand was selectedforuse inthetestbased upon pastuse and ease of handlingin the laboratory.Stock cultureso,btainedfrom theCultureCollectionof Algae attheUmvcmty of Texas atAustm or amflw swpher, willbe maintam@ inculturemedium atWildlife hltmmationaLltd.fora minim= oftwo wedcs pnor touse ina toxicittyest.Algae used in toxicittyests PROTOCOL NO.: 454/110998/SEL-96Hl/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL L-rD. -5- willbe in exponentialgrowth phase,which isdefinedas the periodof growth when algalcens are dividingat a constantrate. Justpriortobeginningthetestan inoculum ofthestockculturewillbe preparedso thateach millilitoefrinoculum containsenough rellstoprovidean initiaclelldensityofapproximately10,000 cells/mLineach replicate. CultureMedium Culturemedimn prepareda=rding toWildlifketa@onal Ltd.StandardOperatmg Prm-edures willbe used as dilutionwater. The concentrationosf thecomponents in themedium arepresentedin Table 1 (3).Stock nutrientsolutionwsillbe preparedby addingreagent-gradcehemicalsto Wildiffe InternationaLltd.weflwaterpurifiedby reverse-osmosisA.ppropriatevolumes of thestocknutrient solutionwsillthenbe chlutovdathpurifiewdeU waterto preparethemodiunl The medium willbe filter stmihzed(0.2gm orautoclavedp)riortouse.The finalpH offt medimn shouldbe 7.5 0.1. Analy- ses willbe performed atleastonce amuauy to determinethe concentrationosf selectedorganicand morgamc const:twentosf thewellwaterandresultosftheanalysesvaU be summmued m the fmal report. Specificationfsor acceptablelevelsof contaminantshave not been establishefdorculture mednun Howem, tlm= areno Imb of contaminantsreasonablyexpectedtobe presentinthemedium thatare consideredtD interf= with thepurpose or conduct of thestudy. TestAaaratus Test chambers v@M be sterile2,50-mL polyearbmate Erlennieyerflasksplugged with foam stoppers.Each testchamber willhold 100 mL oftestsolutiorlTest chambers willbe @n&mimin@ positioneodn a mechanicalshakertablein an environmentalchamber and willbe shaken continuously atapproximately100 rpnl Test chambers willbe labeledwith the pmject number, testconcentration, and replicate. EnvironmentalConditions Testflaskwsillbe heldat24:12'C undercontinuousfluorescenltightin(ge.g.c,oolwhitelight tubes)at an intensitoyf approximately4300 :k100/oIwL Light intensitwyillbe measured atfive locationson the shakertabledailyduringdm test.Temperaturewillbe measured twicedailyinthe environmentalchamber using a liquid-in-glatshsermometer. PROTOCOLNO.: 454/110998/SEL-96Hl/SU]3454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -6- The pH ofeachbwftmt andthecontrowlillbe measured attestinitiatiaond temiinati.using a FisherAccumet Model 915 pH meter or equivalent.Samples willbe collectefdrom thebatch of test solutionpreparedattestinitiatiofnoreach treatrnenatnd controlgroup forthemeasurement of pH. At testterminationp,H willbe measured inpooled testsolutionsamples collectedfrom thethreereplicates of each treatmentand controlgroup. BiologicalMeasurements Celldensitlewsillbe momtored duringthe testby conductingcellcountsusing a h=aqwmeter and a nn=scope. One samplewillbe collectefdrom each replicatoef thetreatmentand controlgroups at approximately24 hour intervaldsuringthe exposureperiocl The samples willbe evaluated inu@@y. Each samplewillbe diluteudsingan electrolytseolution(Isoton(g)a,sneeded,tomaintain countingaccuracy.The celldensityof the sample willbe calculatedbased on the mean number of cells per gficl Samples of testsolutionwsillbe collectefdrom each replicatpeertreatn=t and controlgroup at the end of the test Each replicatseample willbe examined microscopicallfyor atypicalcell morphology (e.g.c,hangesinshape,sizeor color).Growth ofcersin theflasksalsowillbe assessed foraggregationsor flocculafionosf cellsand adherenceof the ceus to thetestchamber. At the end ofthetest,algisufiecffectsw,hich resultintheinhibitioonf cellgrowd4 willbe differentiatefdrom algicidaelffectsw,luch resultin the deathof cells.AUquots (0.5mL) of test solutionswillbe takc:fftrom each replicatteestchamber where growth ismaximally inhibited. Maximallyinhibitetdeatnent arethoseinwhich itcannotbe visuallydeterminediflivealgalceus are present(e.g.absenceofcolortothetestsolution)T.hese aliquotswillbe combined and dilutedwith algalmediwn to a concentratioonfthetestsubstancethatdmreficaly shouldnot affectgDwdL A negativecontrolwillbe preparedby dilutinag0.5-mL aliquotfi-omone negativecontrolreplicatteo 100 mL with algalmechual Growth ofthesesubculturewsillbe monitoredforup to9 days todetermine whetherinhibitioonf growth observedduringthetestisreversibleS.amples willbe collectefdorcell countsatrecoveryphaseuntiationt,mmifintionand atapproximately3-dayintervalbsetween uutiatim mid tamunatom The recoveryphase may be terminatedonce algalgrowth issufficientto mcheatethat the algalcellshave fidlyrecoveredfrom effectsdue tothetestsubstance.This willbe determined through visualexaminationof therecoverytestsolutions. PROTOCOL NO.: 454/110998/SEL-96HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -7- Sa=line forAnalyficalMeasurements Samples of theexposure solutionswillbe collectedon Days 0,3 and 4 and analyzedfor test substanceconcentrationsD.ay 0 samples willbe takenfrom each batchof testsolutionpriorto their distributiaomnong thereplicatteestchambers. Day 3 samples willbe collectefdrom the extrareplicat, testch=ber(s) includedinthetesttoprovidesample forthatanalysisand from theabiotirceplicates. Ifmore than one replicatiesincludedforthe Day 3 sample analysist,hen thesolutionfsrom each replicatweillbe con4)ositetdoprovidethesample forthattreatment Day 4 samples willbe composited solutionsfrom the threereplicatersemaining atthe end of thetest. Samples collectedon Days 3 and 4 may be ccntdffigedor filtm-etdoremove algalceflspriorto analysis.Samples willbe analyzedimmediatelyor placed in an appropriatestoragecontainer(e.g., polypropyleneorpolyethylenebottle)and storeduntilanalyzed. The sample scheme issummarized below: PROPOSED NUMBERS OF VERNICATION SAWLES Experimentalg2E Control SolventControl(ifneeded) Level I-Low Concentration Level 2 Level 3 Level 4 Level5-IfighConcenuiWm IFTo-t@; Day 0 I 1 1 1 1 1 I 7 D 33 @@Day@4 D 4 I I 1 1 1 1 1 1 1 1 1 1 I I 7 7 The above mimbers of samplesrepresentthosecollectedfrom the testand do not includequality control(QC) samples such as matrixblanksand fortificatiopnrseparedand analyzedduringft analyticaclhemistryphase of thestudy. PROTOCOL NO.: 454/110998/SEL-96HI/SUB454 3M LAB REQUEST NO. U2723 -OWILDLIFE INTERNATIONAL LTD. An"calChemistry Chemicalanalysiosfthesampleswillbe performedby WildlifeInternationaLltd. The analytical method used willbe based upon methodology providedby theSponsor and identifieidnAppendix II. Modificationsmade to the analyticamlethod willbe documented in theraw data and describedin the finalreport. StatistiAcnagll= Biomass expressedas areaunderft gmvah curvewillbe calculatedforthebzatinentand control groups usingthefollowingformula: A = ((N,-N.)12)(t,)4<(N,+N2-2NJ/2)(t2-@)+...+((N.-,+N.-2NJ/2)(t.-t,,,.,) Where: A = Area underthegrowth curve N. = Measured number ofcelLs/mLatt@ N, =Mmured number of ccUstmL at N2 =MeWUred number ofCEWML at t2 N. =Measured number ofceUdmL att. t,= Tixneoffirsmteasurement afterbeginningof test(hours) t2 = Time of secondmeasurement afterbeginningoftest(hours) t.= Time of nthmeasurement afterbeginningoftest(hours) Growth rates(ju)willbe calculatefdorthetuftnent and controlgroupsusingthefollowing formula- InN.. InNo t.- to iz= Average specifigcrowth rate N. = Measured mimber of ceRs/mL att@ N. = Measured number ofcells/mLatt. to= Time ofbeginningoftest(hours) t,=, Time ofnthmcww=ent afterbeginningof test(hours) PROTOCOLNO.: 454/110998/SEL-96Hl/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -9- Percent inhibitiownillbe calculatedforeach treatmentgroup based upon mean celldensity, biomass and growth rateusingthe followingformula: Mean Control- Mean Treatment PercentInhibitio=n &Zonse Response- Mean ControlResponse x 100 EC values willbe determined,when possibleu,sing linearinterpolatioonr othersuitable techniqueswith treal3nmtresponse(i.e.ecU densitiesb,iomass and grow0a rate)and exposure concentrationdata foreach.24-hourintervaolf thetest. A no observedadverseeffecctoncentratio(nNOAEC) Win be selectedbased on an evaluationof thedose-responsepatternand theresultsof thestatisticaanlalysisusing individuarleplicatvealuesof theceU densityb,iomass and growthroe. Statisticalsliygrlificadnitffmmm between thecontroland the treatmentgroups willbe identifieudsinganalysisof variance(ANOVA) and a testto compare trcaw=t mean responsestothecontrolresponse(e.g.D,unnett'stestorBonfm-roni'st-test)D.ata will be assessedfornormalityand homogeneityof variancespriorto performingtheANOVA. Transforma. tionswillbe usedto correctany conditionofnm-normdity orunequalerrorvariancesi,fpossible.If a solventcontrolisused inadditiontothenegativecontroltheirresponseswillbe compared Student'st-test.Data from thetwo controlgroups willbe pooledforbtabnent levelanalysesifno statisticdailffermcesexist.Othirwise,allcomparisonswillbe made to thesolventcontrolgroup. StatisticcaolmparisonsfortheANOVA and di,-c,omparison of mean responseswillbe carriedoutusing TOXSTAT4D V3.5 software(4)orequivalmt. RECORDS TO BE MALYEALM Records tobe mmntamed fordatageneratedatWildlifeInternationLatlcLwillincludeb,utnot be limitedto: I- Copy of siped protocol' 2. Idenfificatiaond characterizatiofnthetestsubstance,ifprovidedby Sponsor. 3. Dates of initiatioannd terminationof thetest. 4. Source ofalpe. 5. Cultureconditions. PROTOCOL NO.: 454/110998/SEL-96HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -10- 6. Growth measurements. 7. Calculationand preparationof testconcentrations. 8. Observations. 9. The methods used to analyzetestsubstanceconcentrationasnd the resultsof analytical measurements. 10. Statisticcaallculations. 11. Test conditionsand physical/chemicamleasurements. 12. Copy of fmal rcporl FINAL REPORT A finarleportoftheresultosfthesb* willbepreparedby WilcnifIcnternationLatld. The report willincludeb,ut notbe limitedto thefollowingw,hen applicable: 1. Name and addressof thefacilitpyerformingthestudy. 2. Datesupon which thesft* was initiataendd completed,and thedefinitiveexperimentalstartand tMM3inationdates. 3. A statementof compliancesignedby ft SU* Directoraddressingany exceptionsto Good Laboratory PracticeStandards. 4. Objectives and procedures,as statedin the approved protocolincludingallc.hanges to the protocol. 5. The testsubstanceidentificatiionncludingna=, chemicalabstractnumber or CO& number, strengthp,urity,composition,and otherinformationprovidedby the Sponsor. 6. Stabilitaynd solubilitoyf thetestsubstanceundcr fficconditionsof ifprovided by the Sponsor. 7. A descriptioonf themethods used toccmduct thetest. S. A descriptioonfthetestorganisms,includingthesource,scientifniacme, age or lifestage,light intensitaynd photoperiod. 9. A descriptionof thepreparationof thetestsolutions. 10. The methods used to arorateorgudmm to testchambers and begin the&-s@thenumber of Organisms and chambers per treatmmt@ and the durationof ffictest. 11. A descriptioonf circumstancesthatmay have affectedthequalityor integdtyof the data. PROTOCOL NO.: 454/110998/SEL-96HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. 12. The name offfieSu* Diedor and ffineames of otherscientistpsr,ofessionalsa,nd supervisory personnelinvolvedin thestudy. 13. A descriptionof thetransformationsc,alculationsa,nd operationsperfoz-zneodn thedata,a summary and mm4,sisofthebiologicadlataand analyticaclhemistrydata,and a statementof the conclusionsdrawn from theanalyses.A graph plottingtheconcentratiornesponsecurve. 14. Statisticamlethods used to evaluatethe data. 15. The signedand datedreportsof each of the individualscientistosr otherprofessionalisnvolved inthesft*. 16. The locationwhere raw dataand finalreportare tobe stored. 17. A stat@ preparedby theQualityAssuranceUnitlistintghedatesthatsh* inspectionasnd auditswere made and thedatesof any findingsreportedtothe Study Directorand Management. 18. Ifitisnemsmy tom&kr.conwfions or additionsto a finalreportafterithas been accepted,such changeswillbe made intheform of an amendment issuedby theStudy Director.The amendment willclearlyidentiftyhepartof thefinalreportthatisbeing amended and thereasonsforthe amendment, and willbe signedby the Study Director. CHANGDUG OF PROTOCOL Pilinnedchanges to theprotocolwillbe intheform of writtenamendments signedby the Sb* Directorand theSponsor'sRepresentative.Amendments willbe consideredas partof theprotocoland willbe aftachedtD thefnalprot=L Any otherchanges willbe m the form of wnuen deviationsigned by the Study Directorand filedwiththeraw data.Allchangestotheprotocolwillbe indicateidnthe finalreport. GOOD LABORATORY PRACHCES This sft* willbe conductedinaccordancewithGood LaboratoryPracticeStandardsforEPA (40 CFR Part 160 and/orPart792);OECD PrincipleosfGood LaboratoryPractic(eOCDE/GD (92) 32, Enviromumt Monograph No. 45); and Japan MAFF (59 NohSan, NotificatioNno. 3850, AgriculturaPlroductionBureau). Each studyconductedby WildlifeIntemtlmal Ltd.isroutinely examined by the WildlifeInternationLatld.QualityAssuranceUnit for compliancewith Good Laboratory PracticesS,tandard OperatingProceduresand the specifiedprotocol.A statementof PROTOCOLNO.-. 454/110998/SEL-96Hl/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LrD. -12- compliancewith Good LaboratoryPracticeswillbe preparedforallportionsof the studyconductedby WildlifeInternationaLltd. The Sponsor willbe responsiblfeorcompliancewith Good Laboratory Practicesforpror-edurepserformedby otherlaboratorie(se.g.r,esidueanalysesor pathology).Raw data forallwork performedatWildlifektmmationalLtcLand a copy of the finalreportwillbe filedby project number in archiveslocatedon theWildlifeIntematimalLtd.siteor atan alternativleocationto be specifiedin the finalreport. PROTOCOLNO.: 454/110998/SEL-96Hl/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -13- REFERENCES I US. EnvironmentalProtectionAgency. 1996. Series850- EcologicalEffectsTest Guidelines (draft)O,PPTS Number 850.5400: Algal Toxicity,Tim-sI and ]I. 2 OECD Guideline forTesting of Chemicals. 1984. 201: Alga, Growth InhibitionTest. 3 ASTM Standard Guide 1218-90E. 1990. Standard Guide for Conducting Static96-Hour ToxicityTestswith Microalgae. 4 West, Inc.and D. D. GuRey. 1996. TOYSTATRelease 3.5. Western EcoSyste= Technology, Inc.Chq=e, Wyoming. PROTOCOLNO.: 454/110999/SEL-96Hl/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -14- FRESHWATER TABLE I ALGAL MEDIUM CONSTrrUENTS Component Nominal Concentration MgCI2-6H20 CRC'2-2HO H3BO3 NinCI2-4H20 ZnC'2 FeCI3-6H20 CoCI2-6H20 N%MoOe2H20 CUC'2-2H20 Na2F-DTA-2H20 NaN03 MgS04'7H20 K2W04 NAHCO., 12.16 mg/L 4.40 mg/L 0.1856 mgtl 0.416 mg/L 3.28 oug/L 0.1598 mg/L 1.428 gg/L 7.26 iiA 0.012 gA 0.300 nigAL 25.50 mg/L 14.70 mg/L 1.044 mg/L 1-5.00 mg/L The PH ofthemedium willbe adjusteda,s necessaryt,o7.5:10:.1 using0.1 N NAOH or 100/aHCI. PROTOCOLNO.: 454/110998/SEL-96Hl/SUB454 3M LAB REQUEST NO. IJ2723 WILDLIFE INTERNATIONAL LTD. -15- APPENDIX I IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR To be Completedby Sponsor 1. TestSubstancIedentit(yna= tobeusedinthereport)P:FOS (EerfluorooctaSnuelfoWcAcid potassiumSalt ReferenceStandard(ifapplicableA)n:alyticaSltandard: N/A IntmW Standard: 1-1.2-2H-H.H.PHerfluorooetaSnielifonAicid TestSubstanceSample Code or BatchNumber: Lot217 TestSubstancePurity(*/AoctiveIngredient):98.9 ExpiratioDnate:-2008 U. TestSubstanceCharactmization Have theidentity, gmty and compositionorothercharacteristics whichappropriatdeo:me c testsubstazfcaendreferencsetandardbeen det=iifiipdriortoitsuseinthisstadyinaccordancweithGLP Standards? Yes M. TestSubstanceStorageConditions No X Pleaseindicatteherecommended storageconditionastWildlifeInternationLatld. Ambient room I=Xumtm Has thestabiliotfythetestsubstanceunderthesestorage-condifions be= determinciilnaccordancweithGLP Standards? Otherpertinensttabiliitnyformation: Yes No X IV. TestConcentrations: V. Toxicityinformation: Adjusttestconcentratiotno 1000/a.L x basedupon thepunty (0/og)ivenabove. Do notadjustestconcentratiotno I000/o - &i.Testthematww AM. Mammal4m: Aquatic: RatLD50 251mg&g - MouseID50 N/A InvertebraTtoexicit(yECAC50) FishToxicit(yLC50) Dohnia magna: 27mpJL DaWmia magnae 50 mga, Rainbow Trout:II mL,/L Fag=d Minnow* 38 nXl OtherTox3cty Informatio(nmclucbngfindingosfchrome and subchromctests): -Pleme seeMSDS PROTOCOLNO.: I 454/110998/SEL-96HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL L-rD. -16- APPENDIX 11 AnalyticalMethod to be Providedby Sponsor PROTOCOL NO.: 454/110998/SEL-96Hl/SUB454 3M LAB REQUEST NO. U2723