Document e7pgZMLMedBX81NdEY7QZVNGE

= AR226-0099 ASWEIMTIHSTTHAETCILCALDIOFPCEFECORSY:ACNLEDaTpOhXnIiCaITmiYgTrEa)ST FINAL REPORT WILDLIFEINTERNATIONALLTD, PROJECTNUMBER: 454A-109 3M LAB REQUEST NO. U2723 U.S. Environmental Protection Agency Series 850 -- Ecological Effects ~ (OPPTS Number 850.1300 `OECD Guideline 211 AUTHORS: Kurt R. Drottar Henry O. Krueger, Ph.D. STUDY INITIATION DATE: July2, 1999 STUDYCOMPLETIONDATE: Feary26,200 AMENDED REPORT DATE: April 26,2000 Submitted to - 555 Buh Avene 3M Corporation `Environmental Laboratory St. Paul, Minnesota 55144 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1077 01091 AMENDED WILDLIFE INTERNATIONAL Lm. 2. PROJECT NO.: 454A-109 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Semi-Statc Life-Cycle Toxicity Testwith the Cladoceran (Daphnia magna) `WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-109 STUDY COMPLETION: February 28, 2000 AMENDED REPORT: April 26,2000 Envir"oTnhmiesnsttaulPdryowteacsticoonnAdguecntceydiinnc4o0mCplFiRanPacretwsi1t6h0Gaonodd7L9a2,bo1r7aAtuogruysPtra1c9t8i9c;e SOtEaCndDarPdrisnacisppluebsolfisGhoeoddbyLatbhoeratUoSr.y P1r0acAtuigcues(tE1N9V84/,MwCi/thCtHEhMfo(l9l8o)wi1n7)g;eaxncdepJtaiponasn:MAFF, 59NokSan,NotificationNo. 3850,Agricultural Production Bureau, `wasperf`oTrhmeetdeasctcsuobrsdtianngcteow3aMsnStoatncdhaarrdaOcpteerriazteidnignaPcrcoocreddaunrceeswaintdhMfeltlhoGdLsP,acnodmpalliaanwced;athaoawreevebre,itnhgemcahianratcatienreidzianttiohne 3Marchives.Thetestsubstanceisbeingrecharacterizedinaccordancevith GLP. "Thetabiltyofthe testsubstanceunderconditionsof storageatthetest stewasnotdeterminedinaccordancewith `Good Laboratory Practice Standards. STUDYDIRECTOR: Kurt R Drottar DATE Seaior Biologist "J - Sponsor yz ? 001092 AMENDED WILDLIFE INTERNATIONAL Lo. -3. PROJECTNO.: 454A-109 `QUALITY ASSURANCE STATEMENT ThisstudywasexaminedforcompliancewithGoodLaboratoryPracticeStandardsaspublishedbythe U.S. `Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD PrinciplesofGoodLaboratory Practice (ENV/MC/CHEM (98) 17;and Japan MAFF, 59 NohSan,Notification No. 3850, AgriculturalProductionBureau, 10August 1984.Thedatesofalinspectionsand audits andthedatesthatanyfindingswerereported totheStudyDirector `and Laboratory Management were as follows: AcmviTy: Test Subst Preprsion Tentitaion Observations DafRepotendDus ADs ussndDao tReprt Fis Report. Amended Rept ~DATECONDUCTED: uy, 1999 Jy12,1999 say21,199 Octobe 120d13,1999 October 120d13,1999 February 28,2000 p2522426,2000 STUDYDIR[EDCATTOERRE:PORTMEADNATGOE:MENT: uy,1999 February 23,2000 Jy12,1599 Jay13,1999 ly 27,1599 Juiy2s,1999 Ocber13,1999 October 13,1999 coer 14,1999 October16,1999 Fbruy28,2000 February 28,2000 p25,2000 Api26,2000 SponH. Cloan Jz `H. Coleman QualityAssurance Representative Hoo DATE 001093 AMENDED WILDLIFE INTERNATIONAL Lm. 4. PROJECT NO.: 454A-109 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Semmi-Statc Life-Cycle Toxicity Testwith the Cladoceran (Daphnia magna) WILDLIFE INTERNATIOLNTADL. PROJECT NUMBER: 454A-109 STUDYDIRECTOR: en) / \ Kurt R Drottar Senior Biologist MANAGEMENT: 44 HDiernercyto0r, AquatKriuecger,Toxicology and `Non-Target Plants 4fpe/oo DATE Yet DA 01094 AMENDED WILDLIFE INTERNATIONAL Lm. 5. PROJECTNO.: 454A-109 `TOAFCBONTLENETS THICRAHE U rsmmsmmammmmsmmmmmmmmommms------ TRACI mreinmmsmsmmmm-------------- EXPE)DESIGN. Materialsand Methods 8 ------------------ COREMISIONS. ots sss - 14 TABLES Table 2-TemperatureofWater i the TestCRADETS vr 11 `Table 3 -DissolvedOxygenContent ofWateri the TestCRAIDETS vv: 19 J DO --| 01095 WILDLIFE INTERNATIONAL Lo. -6- PROJECTNO.: 454A-109 TAOFBCONLTENETS -Continued- `TABLES (Continued) Tabl5e- SapnedciHfiigcheCsotnTdrucetaatnmceen,tHGaIrOdUnepsTseasntdAlkalinityinCthReANeMgaBtivEeCTonStr.ol.....rvrvrn23 Tabl6e- Summaryof CumulativPercentMortality andTreatment.Related Effects 00First:GEDOTBion DPBS... 20 Tabl7e SummaryofDaphniamagnaReproductionDuringtheLife-Cycle Toxicity Test... 25 Table 8 SummaryofLengthandDryWeightof Surviving Individually-ExposedFirst-Generation Tabl9e Cumulative Percent Mortality/lmmobilaintdyTreatment:RelatedEffectsfor APPENDICES Appendix 1+ MSepeacsiufriceCdoDnudruicntgantchee,4H-aWredeneksPse,rAilokdaIlmimnietdyiaantedlypPHroefceWdeilnlgtWhateer Test................28 Appendix II - AinnaWliylsdelsiofefIPnestticeidres,nOLragiadtniWicesol, lMnWetLaaEllTsanv dOtherInorganics 2 Appendix TI - T`WhieldlAinfaelIysnistofePrFOnSLiatndt.FPrireosojhewncatteaNroli.:n Supportof 4S4A-109....cvv 30 Appendix IV - CumulativePercentMortalityandTreatment-RelatedEffects Frst-GEDOrBtion DAPHAAS.....rrr 49 APPEOEX V = Neon PrOBUGHON vss 10 Appendix VI - LengthandDry WeightofSurviving First-GeneDraapthiiiodns ne T2 ADPEAiX VII = CHA10 tNhePGROEMO.S.. 14 Appendix VII-I PersonnelInOIVEdinthe SEY... 7S Appendix TX - ReportAMERATEDL cc wmmmmmmmmne 8 AMENDED 01096 WILDLIFE INTERNATIONAL Lo. "7. PROJECTNO.: 454A-109 sponsor: SPONSOR'SREPRESENTATIVE: LOCATION OFSTUDY, RAW "DATAAND ACOPYOFTHE FINAL REPORT: WILLTDD.LIPFREOTIENCTTERNNUAMTBIEORN:AL `TEST SUBSTANCE: STUDY: NOMINAL TEST CONCENTRATIONS MEANMEASURED TEST (CONCENTRATIONS: TESTDATES: LENOTHOR TEST: "TESTORGANISM: SOURCEOFTEST ORGANISMS: AGEOFTESTORGANISMS: NOE, LoEC: Naar: SUMMARY 3M Corporation SusanA. Beach WildlifeInternationalLtd. Easton, Maryland 21601 4544-109 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A Semi-Static Life-Cycle ToxicityTestwiththe Cladoceran (Daphnia magna) `NegativeControl, 1.4,2.9,5.7,11,23and46mga.i/L. Negative Control, 1.5,2.9, 5.6, 12,24 and 48mga.i/L. ExperiSmtaret-nJtulya12l, 1999 Biological Termination -- August 2,-1999 Experimental Terminatio--n Augus4t, 1999 210s "Neonate Cladocerans (Daphnia magna) `Wildife IntemationalLid. cultures `Easton, Maryland 21601 Juve<n24iholuresosld Tmgail 24mgaill mg ail, AMENDED 01697 WILDLIFE INTERNATIONAL Lo. -8- PROJECTNO.: 454A-109 INTRODUCTION "Thisstudy wasconductedby WildlifeInternationalLid.for 3MCorporationattheWideInternationalLud.aquatic: toxfaicilictyinoEaslton,oMargylaynd.The n-lifephaseofth testwasconductedfrom July 12, 19toA9ugus9t2, 199. RawdatageneratedbyWildiieInternation Ltd.and acopyofthe fina reportarefledunderProjectNumber454A-109in archiveslocatedontheWildlifeInternationalLid. ite. OBJECTIVE "Theobjectiveofthisstudy wastodeterminetheeffectsofPerluorooctaneSulfonicAcidPotaSsalts(PiFOuS)mon the survival, growthandreproductionofthecladoceran (Daphniamagna) durinag21-dayexppoeriosdunuderrsemeistatic testconditions. EXPERIMENTAL DESIGN `Daphnidswere exposedtoageometricseriesofsix testconcentrationsand anegative (dilutionwater)control.Ten replicatetestchambersweremaintainedincachtreatmentandcontrolgroupwithonedaphnidi cachtestchamber. Nominaltestconcentrationswereselectedinconsultation viththe Sponsor,andwerebasedupontheresultsofan explranogerfinadingttoxoicitryteyst. Notm stcoincenn tratiaonswlere 1.4,2.9,5.7, 11, 23and 46mg ai/L. Mean `measuredconcentrationswere determinedfromnewandcorrespondingoldsoutioasduringcachweekofthett. Depnids verotransferredtonewsolutionsevery Monday, WednesdayandFridayduringthe est. First-generationdaphidswereobserveddailyforsurvival,theonsetofreproduction,and clinicalsignsof toxicity. `Withthe onset ofreproduction,thenumberofsecond-generationneonateswere counted (aurmberofliveneonates,number ofdeadorimmobileneonatesandnumberofabortedeggs)every Monday,WednesdayandFridayduringth test. If availabletwentysecond-generationneonatesfromeachtreatmentandthecontrolwereexposedunderstati testconditions for48hours. Attesttermination,thelengthanddryweightof eachfirst-generation daphnidwasdetermined.Thenobserved-ffect-concentration (NOEC)andlowest-observed-ffct-concentration(LOEC)were deterbymexiaminnaetidon ofthemortality,growthandreproductdiatoan.Themaximumacceptabletoxicantconcentration (MATC)wascalculatedas thegeometricmeanofthe NOEC and LOEC. AMENDED 001098 WILDLIFE INTERNATIONAL Lo. -9- PROJECT NO.: 454A-109 MATEARNDIMAETLHOSDS "Thestudywasconducted basedontheproceduresoutlinedintheprotocol, "PFOS: ASemni-Stati Life-Cycle Toxicity TestwiththeClad(Doapchneiamraganayn'.Theprotocol wasbasedonproceduresoutlinedi the UsS. Environmental Protection AgencySeries 850 EcologicalEffectsTestGuideOPlPTiSNnumebesr 850.1300 (1); OECDGuideline211 Daphnia magnaReproduction Test (2);Standard Evaluation Procedure,DaphniamagnaLife-Cycle(21-DayRenewal) ChronicToxicityTest(3; and ASTM StandarEd 1193-87,StandardGuideforConductingRenewalLife-CycleTosicity Tests withDaphmangnia(a9. TestSubstance "Thetestsubstancewasreceivedfrom 3MCorporationonOctober29, 1998andwasassignedWildiieInternational Lid.identificationnumber 4675. Thetestsubstancewasdescribedas awhitepowder. Itwas idenastFCi-9f5firoemldot number217 (T-6295). Informationprovidedbythe Sponsorindicated apuriy of98.9%,and anexpirationdate of2008. `Thetestsubstancewasreanalyzedbythe SponsorandtheCertificateofAnalysisdatedMarch9, 2000indicated apurityof 90.49%. Thetestsubstancewasstoredatambient room temperature. `Preparation of Test Concentrations J AprimarystocksolutionwaspreparedbyaddingthetestsubstancetoUVsterilizeddilution wetera concentration of46mga.i/L.Thetet olutionwasstirredwithanelectricstainlesssteltop-downmixeruntilallthetestsubstancevas dissolved.Theprimary stocksolutionwasproportionallydilutedwithUVsterilizeddilutionwatert preparetheotherfive testconcentrations.Newsolutionswereprepared foreachrenewalday.Aftermixing,al test solutionsappesred clearand colorless. Al reportedtestconcentrationswereadjustedforthepurityoftheactiveingrodicntinthe testsubstance (00.49%). `Test Organism "Thecladoceran,Daphniamagna,wasselectedasthetestspeciesforthisstudy. Daparhreprnesenitatidveofsan importantgroupof aquaticinvertebrates andwereselected foruseinthetestbaseduponpasthistoryofuseandeaseof culitntuherlabiorantorgy. Daphnid neonatesusedinth testwerelessthan24-hours ldandwere binedfrom cultures `maintainedbyWildlifeInternationalLtd,Easton,Maryland. 01099 AMENDED WILDLIFE INTERNATIONAL Lm. -10- PROJECT NO.: 454A-109 Adultdaphnidswereculturedst approximatelythesametemperature asusedduringthetestinWildlifeIntemational, Lid.well water. Daphnidsinthecultureswere heldforatleast 27dayspriortocollection ofthe juvenilesfortesting. The adults showednosignsofdiseaseorstressduringtheholdingperiod.Duringthe 14-dayholdingperiodprecedingthetest, `atertempreangredfaromt20u.00r20e.3%sC.The pHofthewaterrangedfrom8.3t03.5anddissolvedoxygen ranged fr8.2otom8.8 mg/L. Instrumentationandmethodsusedfor watermeasuremeatsaredescribedintheEnvironmental Conditions secoftthiisreopornt. `Neonate daphnids (<24hoursold)were obtainedfortestingfromsixindividualadultdaphnids.Attest nition,the juveniledaphnidswerecollectedfromthecultures andplacedinglassbeakersone10twoatatimeuntileachbeaker cont1a0idspnhneidsd. Thebeakerswerethenimpartiallyassignedtotreatmentgroups. Onedapwashthenntrainsfedrred toeachtestchamberusing awide-borepipetundemeaththeair/waterinterface.Daphnidsintheculturesandthoseusedin thetestworefed 8mixtureof east,Cerophylle,andtroutchow(YCT,1800mgTSS/L),aswellas asuspensionofthe freshwatergreenalga, Selenastrum capricornutu(3m.5 x 10"cells/mL). Duringth test,cachtestchamberwasfod0.3mL ofYCT and0.60mLofSelenaosncterdaiulym. Test Apparatus "Testchamberswere250-mLplastic(Nalgene)beakerscontainingapproximately200m oftestsolution.Thedepth oftestsolutionin arepresentativetestchamberwas approximately 5 cm.Testchamberswererandomlypositionedby treatmentgroupinanenvironmentalchambersttomaintain atemperature of20:1C. Thewaterbathwasenclosienda plexiglasshoodtominimizethepotential forcross-contamination.Thetetchamberswerelabelledwiththeprojctuber, testconcentanrdaretpliicoatne. DilutionWater "Thewate usedforculturingandtestingwesfreshwater obtainedfrom awellapproximately 40metersdecp located ontheWildiieIntematiLtod.nsiatel. Thewellwaterischaracterizedas moderately-hardwate.Thespecificconductance, hardness,alkalinity,andpHofthevell waterduringthefour-weekperiodimmediatelyprecedingthetestarepresentedin AppendiLx "Thewel water waspassedthrough a sandfiler to removeparticlesgreaterthan approximately254am,and pumped into 837,800-Lstoragetankwherethe waterwasaeratedwithspraynozzles.Priortouse,thewateragainwesfilteredto 01100 WILDLIFE INTERNATIONAL Lm. -1- PROJECTNO.: 454A-109 0.45jumandpassedthrough aUVsterilizertoremovemicroorganismsandfine particles.Theresults ofperiodicanalyses performedtomeasuretheconcentrationsofselectedcontaminantsinwellwaterusedby WildlifeInternationalLid. are: presineApnpetndeixIdL `Environmental Conditions Lightingusedto illuminatetheculturesandestchambersduringculturingandtetingwasprovidedbyfluorescent tubesthatemittedwavelengthssartonaturasunlight(Colortone 50). Aphotof1o6hopurseoflrightiando8odurs ofdarknesswascontrolledwithanautomat timer. A 30-minutetransitionperiod oflowlight intensitywasprovidedwhen lightswentonandoff 0avoidsuddenchangesinlighting.Lightintensitywasmeasuredattestinitiation andatweekly intervalsthereaftervitha SPERScientificLid.lightmeter.Lightintensityrangedfrom 329t0 383huxatthesurfaceofthe water. Temperaturewasmeasuredinalternatingreplicatesofeachtreatmentandthecontrolgroupa test nition,in old andnewtest solutionsonremevwaldays,andattestterminationusing a iquidin-glassthermometer, Temperature wasalso measuredcontinuouslyinonenegativecontrolreplicateusing aFulscope ER/CRecorder. Thetargettesttemperature. duringth testwas 202 1C. Dissolvedoxygenwasmeasureddailyinateratingreplicatesduringthefirstsevendaysof thetest,inoldandnewsolutionsonrenewaldays,andattesttermination. MeasoufpHrweeremmadeeinnaltetmatsing. replicatesattestinitiation,inoldandnewsolutionsonrenewaldaysandattesttermination. Hardness,alkeliity and sperificconductanceweremeasuredinthenegativecontrol andthehighestestconcentrationwithsurviving aphids afst initiationandatapprowexekilyimntearvatls teherleafyer `MeasurementsofpHweremadeusing FisherAccumetModel915pHmeter,anddissolvedoxygenwasmeasured using a YellowSpringsInstrument Model 5Bdissolvedoxygenmeter. Specificconductancewasmeasuredusing Yellow SpringsInstrumentModel 33 Salnity-Conducivity-Temperature meter.Hardnessandalkalinity measurewmereenmatdse by titration based on proceduresinStandardMethodsfor the Examination ofWaterand Wastewater (5) BOiolobgicsal ervaantdMieasourenmensts "Thefirstgenerationdaphnidswereobserveddailyduringthetes forsurvival,theonsetofreproduction,andclinical Siofgtonxicisty. Thecriteriafordeathincludedwhiteopaquecoloration, lack ofmovementof appendages,absenceof heartbeatandlackofresponsetogentleprodding. Immobilization wasdefineda lack ofmovementexceptforminor 061101 WILDLIFE INTERNATIONAL Lo. "12. PROJECTNO. 454A-109 spontancousrandommovementoftheappendages.Thepresenceofeggsinthebroodpouch,abortedeggs,males or ephweireaplsopreciordaed. Withtheonset ofreproduction,the numberofsecond-generationneonateswerecounted(umberofliveneonates, `umberofdeadorimmobileneonatesandnumberofabortedeggs)andrecordedevery Monday, WednesdayandFriday duringthetest. Ateachobservationperiod,thelivefirst-generation daphnidswereretainedandthesecond-generation dapwhernediiscadrdesd. OnDay 14,thesecond-generation neonatesverecollectedandusedfo thesecond-generation acuteexposure.Attesttermination,thelength (measuredfromtheapexoftheheadtothebase ofthespine)anddryweight weredeterminedforeach surviving first-genedraapthiniodn.Theindividualdaphnidswerereovedfromthetest chambers andplacedintotaredaluminumdryingpans.The lengthofcachdaphnid wasdeterminedusing acaliper.Thepanswere thenplacedin adryingovenatapproximately60Cforapproximately 43hours. The panswereremovedfromthedrying ovenandplacedin adesicctaotcooorl.Whencool, thepanswereweighedusing a MettlerAE-240analytical balance. `StatisticalAnalyses Statistical analyseswereperformedonsurvivalof first-generation daphnids,the numberof liveyoungand thelerigth anddryweightofthesurvivingfirst-genedraapthniiodns. SurvivaldatawereanalyzedusingFisher'sexacttest. Reproduction andgrowth(lengthanddryweight) datawere evaluatedfornormalityandhomogeneityofvarianceusingthe `Shapiro-Wilk'stestandBartlet' test, respectively. Dunnettstestwasthenusedtoidentifytreatmentgroupsthat were statisticallysignificantincomparisontothenegativecontrol (p< 0.05). Alstatisticaltestswereperformedon apersonalcomputerusing TOXSTATVersion3.5 (6)statisticalsoftware, ges Chen `Watersampleswerecollectedfromnevandoldsolutionseachweekdurtihetnesgt. Duplicatesamplesofnew Solutionswerecollectedfrom th freshlypreparedsolutions.Duplicatesamplesofthe odsolutionswerecollectedfrom a `compositeoftheremainingreplicatetestchambers. Sampleswerecollectedinplastic(Nalgene)bottlesandvereanalyzed assoonasposwsithioubtstloraege. Analyticalproceduresusedi theanalysisofthesamplesaeprovidednAppendix II. 001192 WILDLIFE INTERNATIONAL Lm. -n. PROJECT NO.: 454A-109 RES ANU DDIL SCUT SSIS ON `VeroifTefsteCosnceintroationns Resultsofanalysestomeasureconcentrationsof PFOS inwatersamplescollectedduringthetestarepresentedin `Table 1 andtheanalyticalchemistry report (AppendixIIT). Nominalconcentrationsusedinthestudywere 1.4,2.9,5.7, 11, 23and46 mga.i./L. Measured valuesofnewsamples rangedfrom94to 121%ofnominalvalues,whereasmeasuredvalues fromtheoldsolutions rangedfrom 90to 108% of nominalvalues. ConsPFeOSwqassutabe lethnrougthoutltheyrene,wal periods. Whenmeasured concentrationsofsamplescollectedduringeachweekofthetestwere averaged,themean `measuredconcentwerrea1.t5,i2.o9,n5.s6, 12, 24and48mga.i./L,whichrepresented 107,100,98, 109, 104and 104%of `nominal,respectively. Theresultsofthestudy werebasedon meanmeasuredconcentrations. PMhysiecalaandsCheumicral emenoftWaster Measurementsoftemperature, dissolvedoxygenandpHarepresentedin Tables2, 3and4,respectively. Water temperaturemeasurementswerewithintherangeof 20+1Cestablished forthetest.Dissolved oxygen concentrations `remained 28.3mg/L(91%of saturation)throughoutthetest. MeasuorfpeHrmanegen dfrtoms 8.1to8.5. Measurementsofspecificconductance,hardnessandalkalinityinthenegativecontrolandthehighesttreatmentgroup arepreseinnTtabeledS. No apparent differenceswere observed foranyof theseparametersthroughouttheexposure: period. Stata Ce Asummary oftheresultsoftheobservations ofmortalityandsublethalsigns oftoxicityforfirst-generation daphnids are shionTawbln e6. Dailyobservationsare proivnAippd endeixd V.After21daysofexposure,survivalinthe negative `controlgroupwas 100%.Survivalinthe 1.5,2.9,5.6,12,24and 48mga.i/L treatmentgroups was90,90, 90,90, 0and 0%,respectively. At testtermination,allsurvivingdaphnidsexposedto PFOSappearednormal. Fisexhactetesrtsh'owesd. thatsurvivalinthe 24and 48mga.i./L treatmentgroupswasstatisticallydifferentwhencompared tothenegativecontrol (p 50.05). 01103 AMENDED WILDLIFE INTERNATIONAL Lm. S14 PROJECTNO.: 454A-109 Reproduction Asummaryofthemeannumberof iveyoung producedbythefirst generationdaphnidsispresentedi Table 7. A completelisingofthe. numofbneoenatresproducedisproivniAppdendeixdV. Daphidisnthecontroland PFOS treatmentgroups <12mga./L statedproducingneonatesonDay9.Therewerenoobserofvabaorttedeiggosinnansy treatmentgroup.Reproductionaveraged 122youngperadultinthenegativecontrol. Reproductinitohne 1.5,29,5.6and 12mga./Ltreagrtoupmsaevernagted 142, 136,132and 119youngperadultrespectively. TheBonfteestrshoowned thatreproduction wasnotsignificantlyreducedin any treatmentgroup <12 mg aL (> 0.05). The24and 48mg ai/L rcatmentgroupswerenotincludedinthestatisticalanalysisofreproductiondueto astatisticallysignificanteffecton survival. Growth Asummaryofthemeanlengthsand meandryweightsofthesurviving first-generation daphnidsare prescatedin Table8. IndividualmeasaureprroveidemdineApnpentdixVsI. Themeanlengthanddryweightoffirst-generation aphintihenedgatsivecontrolwas 4.65mmand0.695mg,respectively. TheBonferronit-testshowedthatmeanlength anddryweightinthe POStreatmentgroups <12 mga../Lwerenotsgrificantyreducedincomparison to thenegative control(p>0.05). `GSece ond:neraActutei Expoosunre `Theresultsofthesccond-generationacuteexposureare presentedin Table 9. After48-bowrsofexpossuurvirvaeli,n thenegativecontrolwas95%. Surinvthei1.5v,2.a9, 5l.6, 12and 24mg aL treatmentgroupswas 100,100,100, 0and 0%,respectively.Fisher'sexacttetshowedthatsurvivalinthe24mg ./Ltreatmentgroupwassignificantlydiffrent fromthenegativecontrol(7 5 0.05). CONCLUSIONS There were no adverse effectsonsurvival, reproductionorgrowthof Daphniamagnaexposed to PFOSat `concentrations<12mg .i/Lfor21 days. Daphniamagnaexposedto 24and 48 mga.i/Lhadsignificantlyreduced survival. Consequently,theLOECforthsstudy,basedonsurvival was 24 mg aL.TheNOECwas12mgaian/dtLhe MATCwascalctuobel1a7mtgae../dL.Theresultsofthesecond-generation acuteexposurealsoindicatedaNOECof 12 mgaill. 01104% AMENDED WILDLIFE INTERNATIONAL Lm. -15- PROJECTNO.: 454A-109 REFERENCES 1 US. Environmental Protection Agency. 1996. Series 850 ~Ecological Effects Test Guidelines (dra), OPPTS `Number 850.1300: Daphnia Chronic Toxicity Test. 2 OECD Guideline for TestingofChemicals. 1997. Guideline 211: Daphniamagna Reproduction Test. 3 US.Environmental Protection Agency. 1986. StandardEvaluationProcedure,Daphniamagna Life-Cycle(21- DayRenewal) Chronic Toxicity Test. Hazard Evaluation Division. OfficeofPesticidePrograms, EPA 540/9-86- 141 ~ 4 American Society for Testing and Materials. 1991. Standard Guidefor Conducting Renewal Life-Cycle Toxicity Tests with Daphnia magna, ASTM E 1193-87. Philadelphis, PA. 5 APHA,AWWA, WPCF. 1985. StandardMethodsfor theExaminationofWaterand Wastewater. 16thEditon, `American Public HealthAssociation. American waterWorksAssociation. WaterPollutionControlFederatiNoenw, York. 6 West, Inc. and D.D. Gulley. 1996. TOXSTAT, Version 3.5. Wester EcoSystems Technology, Inc. Cheyenne, Wyoming. 01195 . mogervo: sin. Tot Senne ydChDien a Bem Eee e . E SmEEE omme mp own omy T omy oa gem a NegativeControl wBwooWmomoBwmWoommoomoowm o<tamw aowe " B2 omom omomomoomoomw oo#om ow w i wom omomo@moo@m v Boomowomowow ov " @womogomoi.momL30ex - A EEC } 2 gi Jr 1. PROJECTNO: 454A-109 ~ Tae? TempofWeaterinatheTtestuCharmbeers" TE TeotSOovmpoeE n PCRlOa) Sas,Dophiamgn DivsonWat:_ Well Member gail) TewGm is SDT New OF Wer BABE BS wa ms ms ToS Dwi Der OW Wer OW New BS Be Bs BE ws 96 ws Bs ows oem Od Wer OW Wer mo mi mi oh m0 21 20 18 2 a 196 1s ws 16 1s bs mo W120 198 s6 Ba ws ss 1s we bs 1s Wm mo ws n wa es wes ds we ws ms a1 mi mo ws u we ms ws ws 7 ws Bs m1 ww 1 -- m " p we a ws etg wrsa-- gedbom213e 0C OT-C = = r a No essen endeo 100% scrlty 9 g 3 AMENDED Es PROJECTNO: 4544-109 Tale (Comme) TempofWeaterrinathe TtestuCharmbeers" -- TTootrOrspomineen:. FCOSlaDepemasgna -- DisonWar: WellVer a[ Bote T BTreimcpe CO, W Bote---- Som ani) Toa We oi We od Ww oi Tem BEBE ms me mmm 1s 198 198 199 18 a mr 20 2 18 18 199 99 20 1 20 s6 199 199 19 195 om 1 20 n 199 9s 19 19 m m1 20 u 199 99 mo 199 = - " - - - - - - - iTemposmdemii da 1S0%gaByR DU CRIOC 228 B @ AMENDED 1. PROJECTNO: 454A-105 ie Ties DissolvedOrygenContentof Wate itheTestChambers TT--oo_tp--ee----C--10t5 ,Dephiamgns BeDorr Weta TMeGeasemsiieon Doi Termy eBw BOd TCO oe Dwi ows ot OWFw NB ew w0404wwNew CT Ow 04 mOu -- 7 Ow I wNe wom ow ow ow ow om om ow wom 6 wow ow ow ow ow ow om ow wom Boom ow ow ow ow wom ow wom BY Wom ow ow ow ow wom ow wow - RR A RE - TN R ieieerimtiee o 4 S4 s lyea i TCs &2 28 AMENDED "n- PROIECTNO: 454A109 . Ta3 bConltien) Dison OsynContentof Water he Test Chambers - TTobpe Cols Doi_ mae ---- ey mem bys Dyn Doi byte byw oon R i Oi e Ne M O04 w New Oh e 4 mNew onu mNem ow u w New wou 1s EE 2 wm me ss sa as a ss wm ss ss wom ms om ow om om wm om ow a u wom ow wm sw wm wm sm mwas u wow wm mows ww wm wm 2 ie.oy A te errno J -- 23 AMENDED a PROTECTNO: 454-109 e Ties HofWterTiesntChtamhbeers TTTorShmmcs WOSome TDotoOpsian_CWinwcrem,Dpismena ToNtCgnrhieon a Doo e BT -- ot -- eDpo-- Tm---- T-- os------ -- o--n -- oni) or OW Fe Of Wer Ou fer Od Ne OF Te TeamsCroet Tom mw wm www ww 1s Mom om wm om os om uw om om ow 2 LE I YY 5 Mom om as om ss om a om om wm n MoM mas mss MM om om om u Mom mms mM ss 8 8s mom ow E i-- mmr e"-- or no-- Go w TpO-- rrm ay --wee-- m S --o ---- e-- e-- e -- 288 # AMENDED a. PROIECTNO: d5tA-109 . Tda(Coniic) Hf WaTottCruh nbers eTEotEompe 1CE5a0t3Eer--opienna Dan, ven pOeS Soi oe .. ww om w Team w wea e w w e mw a 1s " w wow ow I. w www wu ss wow ow Is w se " wom ow u w www - - A --re-- " EerE---- ZSrp--z---- z --- ------- - ----- 28 NEED WILDLIFE INTERNATIONAL Lo. a. PROJECTNO: 454A-109 Tables Specific Conduc`tanadncHei,ghHeasrtdTnreesastamnedntAlGkraoliunpitTyesitnthCheaNmebgeartsive Control STpeosntsSourbstance: 3M Corporation PFOS Test Organism: Cladoceran, Daphnia magna Dilution Water, _Well Water --r Negative Control 3 Time Parameter Dey0 Day? Day 14 Day 31 Specific Conductance 310 320 310 BY (anhos/cm) Harduess 136 128 132 128 (mg/L as CaC0y) Alkalinity 178 176 mn 170 (mg/L a5 CaCO) EE Parameter Specific Conductance (umhos/em) Day 0' 310 Time Day 7 Day 14 320 310 Day 21' 320 Harduess 136 128 132 128 (mg/L as CaCls) Alkalinity 178 176 17 174 I Wm(gmag/iL/als C2aC40y) mgai// 12mg ail AMENDED 001113 ue PROJECTNO: 454A-109 . Tiles. `Summcyof CumlaivePercentMoralitysdTretmot.RelatedEfe FirstGersonDephoids SW TeokSamce o PROS me DTieseOnrWgiaes:_ Clealdlcenree,Dopomagna TI e Corin. 55 we Co0meGrwee Day E GST)a Mew im0me BfoocAs Mooy lomoCEble Efe aC)o Gr0eoe OTT Mew moCE ve Bris 1s o oom 0 oom 0 oa 2 o oan o oa 0 0 ean ss o oom 0 TM 0 0 ean n o oom 0 0 ean 0 0 om u am o 1 100 o - " 10 - 10 o - 100 o - T ICornsopaAti=Arpepfeorathlpivi coDay21wing Fci tets 009 2 k w AMENDED Ea PROJECTNO: 454A-105 Table? `SummacyofDaphniamagnaReproduction DisiagthLifCylTovicyTest E Ton SubsmcE s: BFOS. DTiesotOnrgWaesimr:.__CWleadlolcWeerkar, Daphniamagna TMTeTmetMConecewrni NamIboedovfidSialvliExvpioasegd| SuMrveivminlgAvdeuYoDmpyti|d FaODF a ToDweadlimmNobaie mbTooefwArbNoerotweedrl (gain) Depts eso) Reproduction Neonstes Ee TT Nema v meen o 15 5 10am 0 29 9 1366179) 5 o o 56 9 126195 5 o n 5 196265) ' o w o - un 0 0 p Thi tentv"ams ey xofontee adelsoysof eprodocionduc o=szSatsialygfNeowaevid s - - o22 BB @ AMENDED WILDLIFE INTERNATIONAL Lm. -26. PROJECT NO.: 454A-109 Table 8 `SumomfaLenrgtyhandDry WeiofSgurvhivitng. Individually-Exposed First-Generation Daphnids Sponsor. 3M Corporation `Test Substance: PFOS DTielsuttOiorngaWnaitsemr:: ~~ WClealdlocWeartaenr, Daphnia magna Mean Measured `TestConcentration (mg ail) "Negative Control `Number of ~~ Individually-Exposed 10 Surviving Daphnids `Total Length (mm) Mean (SD) 765 GOI) 15 9 4.66 (:0.118) 29 9 4.62 (20.100) 56 9 4.61 (20.124) 12 9 4.59 (20.102) u 0 - E 0 " -- Dry Weight (mg) Mean (+SD) 0.695 (20.100) 0.669 (0.0623) 0.724 (0.110) 0.727 (+0.0665) 0.723 (0.0661) - 01116 AMENDED Ea FROECTNO; 450010 Ties Cumin PeetMoca nity edTsoRel fc SnGeninDis rg -- fCelegehee BDvnCTBE Bm see oTnE Do mee mwohw on bo pe mfi=e T To8 5 Ba 4 e 1 bw eTm o Ra S sagonmoe0 ooRwm ooowww ooSoooww maBonmoo wowR 0 0omww 0 0od0 om oo ssagonmoosoeaw r oooww 0EEE0 ow 0 BaPom ndoooaww o ooomww 0 1 0oemam ow e TR ER sEfe E Ce2EEde owEE pL we t EEfc eo begat 8 er 08) 322 5 mom WILDLIFE INTERNATIONAL Lm. 28. PROJECT NO.: 454A-109 APPENDIX SpecificC`oDnudruincgtatnhece4,-HWaerednkePsesr,iAoldkaIlmimneidtiyaatneldypPHroecfedWienlgltWhaetTeerstMeasured --S_po--nsor 3M Corporation = Test Substance: PFOS Test Organistu: Cladoceran, Daphnia magna Dilution Water: Well Water SpecieConductance (anhos/em) Mean HN-4) Range 315-340 Hard(omegs/sL as CaCOy) 124(N=4) 120-128 Alkal(imngi/tLyas CaCO0y) 169(N=4) 164-172 oH 820N=4) 80-83 01118 WWIILLDDLLIIFFEEIINNTTEERRNNAATTIIOONNAALLLLom.. i. PROJCTNO: PROJECT NO.: 4seado9 454A-109 pr `Analyses of Pesticides,Organics,MetalsandOtherInorganics H swcvs otesTE ssaemlE ws,scose caEv ASLiREioD CmoanmcavTm RATIOooNomm y in Wildlife International Ltd. Well Water" Sio1 toa . Oren Aldrin aad ~ foomm < 0.005 g =oml Pom a EBiDBEDDn,eEpp's, <PPPooommm0.006 gL Fi m Pdoaomom EEa B1aeee HPPPE oooemETE 1@ & Pom 3 Hii PCB-1254 <i PoB m0e720 & oHLF RShe? . Bom Bim vy? 2< 5030 siarr py Calcium' : i350 1mg/L. fore : E iar YLeaadm'in ET < 100 ug a = Bie 1 < 020 <6 gL pra =Ios0B a= A Cee To lm 01119 We ILDE r LIFe EC In NTEORe ANATTe IIOONNr AALL LLe IrD,. eeerneTPe EROOJTECe CTTNNOOe :LS455a AA-N10S9e -30- APPENDIX `THE ANALYSIS OF PFOS IN FRESHWATER IN SUPPORT OF `WILDLIFE INTERNATIONAL LTD. PROJECTNO: 4544-109 001120 WWIILLDDLLIIFFEEIINNTTEERRNNAATTIIOONNAALLLLeo.. we PPRROOEJECCTTNNOO.:: 445s4eAa-d1o0s9 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Semi-Stati Life-Cycle Toxicity Test with the Cladoceran (Daphnia magna) `WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-109 PRINCIPALINVESTIGATOR: Tok. AMacG4repgeh, eBS.Ag Scientist MANAGEMENT: Sint1 -- `Willard B. Nixon, Ph.D. Manager, Analytical Chemistry rE hesbo - ) DATE Ya 001121 AMENDED WILDLIFE INTERNAOTIOANALtLot,. -32- PPRROOJJEECCTTNNOO..: 44s54eAA-10099 ' Introduction Freshwatersamples were collectedfrom asemi-staticlife-cycletoxicitystudydesignedtodetermine the effectsof PFOS (PerfluorooctaneSulfonicAcid PotassiumSalt)tothecladoceran (Daphnia magna). `This study wasconductedby Wildife International Lu. and ideaatsPirofjeicteNod.: 454A-109. Theanalyses ofthese watersamples wereperformedat Wildlife IntemationalLtd.using high performanceliquid chromatographywithmassspectrometricdetection(HPLC/MS). Sampleswerereceivedfor analysis between July12,1999and Avigust2,1999andwereanalyzedoncach samplereceiptday. TSestubsantdInaternalStcandeard Thetest substance used forthis studywas Wildfe InternLatdt.iideontinficaatilon test substancewas used to preparecalibration andmatrix fortificationsamples. number4675. The The intemal standardwasreceived from 3M Corporationon July 2,1998andwasassignedWildlife Intemational Lud. identification nu45m26 ub pone recer ipt. The internalstandard, agranularmaterial was identifiaesd: 1H, IH, 2H,2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: `Thestandardwasstoredunderambientconditions. 27619-97-2. AnaiviealMethod Themethodused fortheanalysis of the freshwater samples was developed at Wildlife International Ltd.andentitled"AnalyticalMethodfortheDeterminationof PFOSinFreshwater,Saltwater,andAlgal Medium".`ThismethodologywasincludedasAppendixIIofWildlifeIntemational Ltd.protocol number 454/011299/MVAL/SUBA4S4. It was basedupon methodology provided by 3MCorporation. Samples were dilutedin a 50% methanol : 50% NANOpurewatersolution containing 0.mg14H0 PF0 OS (internal standard)/L and 0.05% formic acid (v/v) so that they fell within the calirbanrgeoaftthiePoFOnS methodology. Concentrations ofthe PFOSin thestandardsandsamplesweredetermined byreverse-phasehigh performance liquid chromatography using a Hewlett-Packard Model 1100 High PerformanceLiquid Chromatograph (HPLC) with a Perkin-Elmer API 100LC Mass Spectrometer `equipped with a Perkin- 01122 WILDLIFE INTERNATIONAL Lm. 3. PROJECT NO.: 4544-109 Elmer TurbolonSpray ion source. HPLC separations were achieved using a Keystone Betasil Cys analyticalcolumn (50mm x 2mm .D., 3pmparticlesize). Theinstrumentparameters are summarized in Table1. Amethodflowchartisprovidedin Figure1. i. (Calibration standards of PFOS prepared in a 50% metha:n5o0%l NANOpure water solution `containing0.100mg 4HPFOS(internalstandard) and0.05% formic acid (vA), ranincgoncientnratgion from0.00915to0.0915mga.i./L,wereanalyzedwiththesamples. Thesameandmost prominent peak responsefor PFOSwasutilizedtomonitor PFOSinallcalibration, qualitycontrol,andstudysamples. No atwt asme adem to p quant tify PFOSonthebasiofs individual isomeric components. Linear regression equations weregeneratedusingpeakarearesponse ratios(PFOS :internalstandard)versus the respective `concentration ratios(PFOS: internalstandard) ofthe calibration standards. Atypical calibrationcurveis presien nFigturee d2. Theconcentrationof PFOS inthesampleswasdetermined bysubstituting thepeak arearesponseratiosintotheapplicablelinearregressionequation. Represioencnhrtomaatotgriamvsoef lowandhighcalibrationstandardsarepresentedin Figures 3and4,respectively. "Themethodlimitofquantitation(LOQ)for theseanalyseswassetat 0.458mga.i./Lcalculatedasthe prodouftchte lowest calibration standard analyzed (0.00915 mg a.i/L)andthe dilution facotfothre `matrixblanksamples (50). xB -- Sixmatrixblanksampleswereanalyzedto determinepossibleinterference. No interferenwceerse observedatorabovetheLOQduringsamplesanalyses(Table2). Arepresioencnhrtomaattogriamvofea `matrixblank spreseinnFitguered5. Freshwaterwasfortified at0.915,9.15and54.9 mga.i./Landanalyzedconcurrentlywiththesamples todeterminethemeanproceduralrecovery(Table3).Sampleconcewnerte nrota cortreciteo dfonrts he meanproceduralrecoveryof 104%. Arepresentativeionchromatogramof amatrixfortificationis presiennFitguere6d. AMENDED 001123 JI WMILLDDN ULIIFFEET INTE ERNAR TIONN AL LA m. TIONAPPRROL OEJCECTTNL NOO..:443D 564AA-1100. 99 -34- `Example Calculations `Sample umber 454A-109-3, nominalconcentrationof 1.6mga. infreshwater, PeakAreaRatio =AnalytePeakArea/InernalStandardPeakArea Concentration Ratio =ConcentrationofAnalyte/ConcofeInntetrnralaSttanidoarnd InemalStandard Concentr0a.t10i0omgn/:L Initial Volume: 0.500mL Final Volume:25.0mL Dilution Factor: 50.0 PFOSPeakArea: 3091181 Internal StandardPeakArea: 3169689 PeakAreaRatio: 0.97523 Calibcurrvaeetquiatoionn. Slope: 2.42974 Intercept: 0.11482 Cuiswerighvted(e15x) PROS(mg 0.1L) atIstrumene =PES 215 180 CY1M)EtasI pardCo0 ncentr) ation PFOS(mg ./L)insample = PFOS (mga../L)at instrument xDilution Factor Caleulatedconcentration: 1.78mg ai/L Note: manualcalculamtaiydoifnfser Pernt ofNominal Cononration =PFRpOpSo(mPgiaeit/L)imnssaammmpllee Calculatedrecovery: 121% Note: manualcaulamtayidioffner AMENDED 601124 WILDLIFE INTERNATIONAL Lo. .35. PROJECTNO.: 454A-109 RESULTS `SampleAnalysis Freshwatersampleswere collected fromthesemi-static lfe-<ycletoxicity studywiththecladoceran (Daphnia magna) at test initiation, July 12, 1999 (Day 0), on July 14,1999 (Day 2 old solutions), on July 23, 1999 (Day 11 ~ new solutions), on July 26,1999 (Day 14 -- old solutions), on July 30, 1999 (Day18 ~nersolutions)anddttestterminationonAugust2, 1999(Day21oldsolutions).Themeasured concentrationsofPFOSinthesamplescollectedatinitiationofexposureofthe testorganisms(Hour0) rangedfrom96.5 to 121%ofthenominalconcentrations(Table4). Samples collateDacyt2,eDady 11, Day14,andDay18hadmeasuredconcenrtanrgeasotfi97o.8n0 108%, 94.3to 101%, 89.8to 103%and 94.5 10 109%ofnominalvalues, respectively. Samplescollectedattesttermination(Day21)had a `measuredconcenrtarngaeotf9i1.o0tno 102%ofnominalvalues. A represioenchnrotmaatotgriamvofea testsampleisshowninFigure7. : AMENDED 01125 t WfILcDLIFBoE E INi TEd RNATs IONAa L Lm. .. PROJECTN.O. ..: s45s4A-10l9 O -36- Table 1 INSTRUMENT. `Typical HPLC/MS Operational Parameters C"Hhorwoimeattt-oPgarcakpahrdwiMtohdealPe11r0k0iHni-gEhlPmAerePfrIor1m0a0ncLeC LMiaqsuisdSpectrometer equipped with a Perkin-Elmer TurbolonSpray selective ion monitoring mode (SIM). ion source. Operated ia ANALYTICAL COLUMN: Keystone Betasil Cy column (50 mm x 2 rum LD,, 3 um particle size) OVEN TEMPERATURE: 30C STOP TIME: 5.00 minutes FLOW RATE: 0220 mUminute MOBILE PHASE: 072..10%%FMoertmhiacnAoclid: 28.0% NANOpure Watercontaining INJECTION VOLUME: 2504 PFOS RETENTION TIME: Approximately 4.3 minutes INTERNAL STANDARD RETENTION TIME: Approximately 3.0 minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 4267 amu 01126 WILDLIFE INTERNATIONAL Lo. 37 PROJECT NO.: 454A-109 Table2 Matrix BlanksAnalyzed ConcurrentlyDuring Sample Analysis (5N4u-m1be0r9) MAB-1 Sample Type Matrix Blank Measured PCFoOnSo'entratioofn (mgai/L) <10Q MAB2 > MatrixBlank <L0Q MAB3 MatrixBlank <L0Q MAB4 Matrix Blank <10Q MAB-S Matrix Blank <10Q MAB Matrix Blank <10Q s"tTahnedlaimridtaonfalqyuzaendti(a0t.i0o0n9(1L5OmQg) aw.ais/L)0a.n4d58tmh gdialu.t1i/oLnbaascetodruopfotnhetmhaetprrioxdbulcaonkftshaemlpolwees(s5t0c)a.bration AMENDED 01127 A W-- ILDLIF-- E INTER-- NATIONAR L Lo. O rm TR PROJC ECTNNOO.;: 4455T 44A-010090 ETH Table3 Matrix Fortifications Analyzed Concurrently During Sample Analysis --_---- `Concentrationsof PFS Sample Number --) (454A-109) (@gai/L) FoFroritiffeiedd MMeaasurded Percent ReRcecoovveererdd =~ MAST MAS4 0915 oss 0.988 102 108 1 MAS-7 0915 0952! MAS-10 0915 0962 104 103 MAS-13 MAS-16 0915 0915 0997 0979 109 107 MAS2 9.15 MAS-5 915 933 933 102 102 MASS 9.15 933 102 MAS-11 MAS-14 9.15 915 915 9.15 100 100 MAS-17 915 961 105 MASS 549 599 109 MASS 549 567 103 MASS 549 553 101 MAS-12 | 549 559 102 MAS-1S 549 513. 104 MAS-18 549 557 102 - Me=a10n4 Standard Deviation = 3.32 ov =319% N=8 "Note:Resultsandcorrectionsfornewtest substancepurityweregenerated using MacQuanversion 1.5 softwareandmanualcalculations. Valueshavebeen roundedforreportingpurposes !Reported valuerepreseatsmeanofduplicatere-analyses(0.970and 0.933mg a.i/L). 01128 AMENDED WILDLIFE INTERNATIOA NALALm. sri 39. PRIOJBEOCTTNNOO.;:S4554A0-1009 Table 4 MeasureCdlCaodnocceenrtarnatSieomnis-oStfatPiFcOLSifien-CFyrcelsehTwoaxtiecriStaymTpelsets from a Nominal Test Concentration (gai) 00 Sample Number (4544-109) 1 2 1165 29 30 a 5a23 4 65 6 Sampling Time. (ay) 0 0 22 1 n 14 1184 18 21 21 POS Measured Concentration' (mgai/l) <10Q <10Q <<LL00QQ <10Q <10Q <10Q <<1L00QQ <10Q <L0Q <L0Q Percent of Nominal --- 14 3 0 4 0 178 121 172 nr 1" 2 Ls 108 18 2 136 107 31 n 138 943 32 n 147 101 "a 14 4" 14 136 9.1 132 898 55 18 138 92 56 8 143 974 1 21 150 102 & 21 145 985 "Note:sRoefstuwlatsreaannddcomrarneucatliocnaslcfuolrantieownts.esVtalsuubessthaanvceepbuereintyrowuenrdeegdefnocrrraetpeodrutsiinnggpMurapooQseusa.nversion 15 Tshtaenldiamridtoanfaqlyuzaedn(t0i.0t0a9(1tL5iOmoQg)n.wia/sL)0.a4n5d8tmhegdai.liu.t/iLobnafsacetdorupoofnthtehemaptrroidxucbtloanfktshaemlpolweesst(5c0a).libration 001129 AMENDED WILDLIFE INTERNATIONAL Lm. -40- PROJECT NO.: 454A-109 `Table 4 (continued) MeasuredConcentrationsofPFOSinFreshwaterSamplesfrom a Cladoceran Semi-Static Life-Cycle Toxicity Test Nominal Test Concentration (gail) 29 Sample Number (454A-109) NE] "6 19 20 3 34 a 46 57 58 6 70 Sampling Time ay) 0 0 2 2 1 1 1 1 18 18 21 21 PFOS Measured Concentration' (mgai/l) 320 30s 301 307 275 271 285 227719 281 281 28 Percent of Nominal 110173 106 %1098 976 101 9%53.5 99.910 92 57 7 0 597 104 3 0 587 102 21 2 565 98.1 - 2 2 572 9.1 35 In 563 97.6 36 11 559 969 : a 14 536 9.0 8 14 539 925 59 18 558 9%38 0 18 575 96 71 21 524 91.0 7 21 537 92 "Note:Results andcorrectionsfornow testsubstancepuritywere generatedusingMacQuanversion 1.5 softwareandmanualcalculations. Valueshavebeenroundedfo reporting purposes. !Thelimitofquantitation(LOQ) vias0.458mg a.1/Lbasedupontheproductofthelowestcalibration standardanalyzed (0.00915mg a.i/L) andthedilutionfactor ofthematrix blank samples (50). 01130 AMENDED A WILDLIFE INTERNAA TIONAL . Lm. -a1- PPRROOJFEECCTTNNOO.::44s54AAl-1e0y9 `Table 4 (continued) Measured ConcentraotfiPoFOnSs in FreshwaterSamples froam Cladoceran Semi-Static Life-Cycle Toxicity Test `Nominal Test Concentration (mIgnai/L) Sample Number 0 (454A-109-) 10 2 24 37 398 50 61 672 " Sampling `Time (Da0y) 0 2 2 1 1141 14 18 2118 2 PFOS Measured Concentration' (mg1a1i3/L) 113 116 11.8 1n3 1132 116 18 11116s 13 Percent. of No9mi5nal 970 978 993 95.0 9944.0.5 075 99.4 9o77.41 045 2 1n2 00 223412 110016 25 2 240 105 26 2 246 108 39 1 238 996 4501 1n4 22356 918033 2 14 21 101 63 18 248 108 64 18 250 109 46 13 0 413 103 14 0 480 105 27 2 49.1 107 28 2 494 108 "Note:Resultsand correctionsfornew testsubstancepurityweregeneratedusing MacQuanversion 1.5 softwareand manualcalculations.Valueshavebeen rounded forreportingpurposes. $`Thelimitofquantitation(LOQ)was0.458mga.i./Lbased upon theproductofthelowestcalibration standardanalyzed (0.0m0g9a.1i./5L) and the dilution factor of thematrix blanksamples (50). 01131 AMENDED W_I--LDLIFE INTERNATIONAL Lr. a. |PPRROOFJEECCTTNNOO.:: s45a4Al-1e09 METHOD OUTILINNEFRFEOSRHTWHAETAENRALYSIS OF PFOS Preparematirnitxo ffiolrtteirfeidcfartieosnhswaamtpelreussbinygsgpaisk-itniggthhtesyrreiqunigseisatenvdoClluamsesofA vPoFlOumSesttroicckfsloalsuktsi.onsdirectly - 5 DiluteCmlaatsrsiAxvfoolrtuimfeictartiiocnfalansdktsewsittsham5p0l%esmientthoatnhoelra: n5g0e%ofNtAhNeOcaPluibrreatiownasttearnsdolaurtdisobnycopanrttaiianlilnygfiling 0A.p1p0r0omprgia4tHePvFoOlSum(eionftesmaamlpslteaannddabrrdi)ngatnhde0f.l0a5k%tofvoormliucmaecwiidth(vtvh)e.diAldutditohne solvenvt.oPlruomceeasssftohemthaetlroiwxbesltafnoksratmipfliecuasltieinvegoltn.hMesiaxmweedlillubtysieonvaenrdaalliquot repeatinversions. . + AmpulatesamplesandsubiforLCMSanalysis, Figure1. Analyticalmethod flowchartfortheanalysisof PFOSinfreshwater. 01132 WILDLIFE INTERNAo TIOs NAL , Lr. 43. PRPOREOJCETCTNNOO.::44554A4-1A09) 250 5 200 3 150 fi 050 000 000 0200 0400 0600 080 1000 Concentration (Ratio) Figure 2. Atypical calibration curve for PFOS. Curve is weighted (1/x). Slope = 2.42074; Intercept = 0.11482; r = 0.9974. 061133 AMENDED W--I_--LDLIFE INTEe RNATIOr NAL Lm. aa PRPOROFJCECTTNNOO.:: 445544AA-l10099 om 2 a = Figure3. A representative ion chromatogram ofa low-level (0.00915 mg 2.0/L) PFOS standard. AMENDED 001134 WILDLIFE INTERNATIONAL Lm. a5. PROJECT NO. 454A-109 Figured. Arepresentativeionchromatogramofa highvlevel (0.0915mg a.i/L) PFOS standard. AMENDE Go3%as WILDLIFE INTERNATIONAL Lt. 46 PROJECT NO: 454A-109 Figure 5. A represeatative ion chromatogramof a matrix blank sample (454A-109-MAB-1). "Thearrowindicate theretentiontime ofPFOS. : 01136 WILDLIFE INTERNATIONAL Lm. -a7- PROJECTNO.: 454A-109 Figure. A representative ion chromatogramofamatrix fortification sample (454A-109-MAS-1). 01137 WWIIDLLDILFIEFEINITNETRENRANATTIIOONNAALLi Lo. on -48- PROJECT NO.:A 454A-109 --_-- Figure 7. A representative ion chromatogram oaf test sample (454A-109-3). 01138 a momcryo. swim Comb Pc rly Tsrmmontarfc Fo GoinDis e E -- EeSreEE E-- e ene= = e on = m(manie) gRepbPg B[ este oooExooo7rd'oodrirb1le Deroofarfd omfmmofeRch olmw:bfohebbbDe3a:d or ow orb Ef|oB|fechorpreofCrB:roos ems mF om wa SgfPooobloroos oobbodopororbsoooowwmmmWospoooozsff f8sFpbooRomm|Wm bbrFfooorboodro Rm|om EPsPPPooooEohbhnof bboA borp oowmm|I oAofobf rbibffoIo|B RW R bbFbbrfoB'''i wa gfEPoPo ooobolbroobe oyorrprsrppoooooommmmm|obosobofb bobbrsbFooomRoooiRmR os3orobpportorfooo''olnmow TEE IPEosTE sA ff IBF g Ggo mor 2h Aogni gmDey0apposed ora. macro; sai AmB Cnt -- CCraeesMeyTTert SisFFrvOo De BEeE E Ee m= = = o Cpe PE FT ooBpeoE 1 odbT3ET:Rbboomm&EoEEEofECEe:bE f ao&oBfEfm: CuB ibo'i PPBoopbooo rrobboomm foFfF rFbo RE F ob t tR m on E s Co [PI oElr po r r E oror roomomR Aoo3t rbFfBoom EEFWRB fb' PPE ooboofdE opr ow fI bF bFElR ob f oA wom we $fPPPoooo0obls rroonsdFospp momm|oomFrooopft eb bmmopra bw bbbooommbbFLo1y a 3?EP E mm ePPmooo:f bow| ffF bFRRoE8 5 1C ame 2 si BROJECTNO:4540-109 . APPENDIXIV (Coniuet) : CumstivPec ial ad reste RentedEs -FitGainDh Te TOS ImTSokoeoi meeee a w a To Cemum TB = wh7e8 w e BooETedpothsc oowI l os SeE amhsoloaE ne t DaE C 1 i fCeZ n le S :o Pe en PI o S25: 01PP ooo ss8sssomaWe mm s s o st t C t .: : v[THooIE ii o osss Wh m eetC :: 1 Vs JEEe TN eye 222 RBs AEE B ie momen: wi e= EeeF ComePeerysToon lstos CexDi -- BEEEREeeeEE ESheeneeBE =S Ee C= ae EE To] 1 & =E1ao&--Ea R: ou=r a= E IBPo PooEboobb F oodfEbbFbbo oooRE mmw ooofffff fFFbAfF oB|BBoboFFFF bbibbA oom'oiim aPFEPPo s oooobrrboosr Foro sobbbbot oroooommmmmowmbos foodbfo fobFmoBo||o|omobpsbFbobozrforoioo'nmoioonmm' PFPooollodd tbboommoF 8bFB|BFForii oii' wa PPPooppooor E rrosA fomm| or Fob doforFowRoom|oo3frboorroooimmm 2ga P[opEoLo E Aofo@mw obAof o o@ fFffom| & AMENDED. a [rn Come etyAiPtPTEoNDmIeXsIV Bs (Continued) inCeoeps : BEEe Em BSpeB = == == a= EE(mgai) RepetEooA oTseTbbOoEx12SponenB3dR tboDeRrbad meRw& Pood bom JmoeobotSfrdeof___R %FFDexd omaRR &I mbjnm:or AFstle ro eDeoaod s mwmE&ff fF Rf ro HtEbEoolorEEpboomm oEff FFb |IBF ob om ogIPPorooEoobrboddAE broAbosowmmI om rfrpF fFoboomB IRmFF:frEboboooommmi IPPoEoboorE oEdsAo w bfbf oI|R pbroowm 2Polo1 0 b1 az bd1 oza b1ez Pgoolrodbonboamo bob8 oma Ebaw [ 58 m Ey ---- om sa FROIECTNO: 454A.109 . APPENDIXIV Contin) CunmivePenMoraysodTetonReedEft FstGeinDep --Tr_--rec 1W05 e DTbCooarnknviiean_VCebaetDep sm,pe a w -- naih) Re7 wBoEdo E i a De--i Ges oe E Dwi Be----e e 5 3: o 0T Po o 8ss s1z t st e e : n n0aC=z= 55 ooss o o e o z= EE ! -- po--s -- oi MpcDouro o To Ceme leTek Du7 HE= s e oo dr::: :6 0 i :: :iz 222 B AMEE a noscrvo: cnin ConeFev Tmbt APPENDIXIV(Continued) TinCanDri -- EEeoEm : -- EEeeEEeEer Be = sevTEeon E5BT Ea E:EE C=EH% eaTEBEa ES1oEt CE:OE=THBFaEReAme mEea E3AOE o=nfow'oem $ [[PPAA oloobf oopd Popo A bboor ooomwmlm or ffobbr bbA FboRmI||I AAb obooAftnmm obdBPPoroobLoobboof rFFpbrooooRRmmmmoR OFoFfFrb FFFfFFmRE o|||m EbrE o Fr oofffooos 'mmmm bod om ob rom PFPoopoolarr oopr W|W FbbFmmror 8fiWm wsPE SPr opooohforrorFrooswoRRmmmsFoOforFFf FoFEbaoolRdoWmom Eo s3boosoaoosfroomo'mm 2gR 5B E fA PgPm oobotfFfm ofrF |mow FfF e Fbolmomort ofb fTfroosmm@ moscow A e EeBSE-- eConma eresMey Toma eis APPENDIXIV(Continued) Fi Coen ps ol EE=Ee He =E = = CEE teTiTBD odS3 oRrN1 |soeR ':Sfoa f=3S: mo@@|embo: o=e 3hm=oi EL PPo oopoT of PPoolro btrpbA ooooRmwwAmbfbAF ob fbFbbooEmm|E II b bbIfA oroobomo|omm SEFI pPo oooopbrpo % pobof oommmWOoFooofff dFbbFEo oowRE | om bE fbo fooorotoommmiom TE OI g PFoonordoFb B|W bb Ob boFmwEborromoim otto <Poor1 0op p1 om= oh b di m = b 1 = 83 fPoorbobborob oomm f bob bo b osa0 3 TEE oe 57 FROIECTNO: 454A-109 . APPENDIXIV (Contind) ol CumtvPercent Moral andTres ReedEf -FitGeinDepids ---Ta-- re 108 DTboattweareDphimes fNrerei Noe, "oom Tote CoSmE -- --Toc wn= ) Reb75er BueIE lieaE d DoE i en sea soo Di Pleo lesow DiTsBios 5 2E 0o HE s st o e e I z 1c ::: [HII oc Post oz : os T--_oet Tr Cl e fsb= epi 238 53 AMENDED oer: sinio --ConnPe evsAPPTENeDImXIVl(Codnimst)FeOeDoi Bie EEee Em esi) Ree3PEBP PPtro oooTo1blOpoooo2d b piB3beErTCp1tpobFfre e ooomww|oR bpmbbbff eS Ebbbab1B b omwo|oommRrS 2o3bror1 r= oorofommimm=m PPoloo o oe oomm 8FbF Ro rboomw I PE PPBPoooooporroT boEbso3 rAt rfboooommmm|bbofooFb rbbbA FobE oowRwm|EoI@ombEorpIoor rodo3boroooo'mmmim PI PopobrA e FdA Rm ton o FE BdPPoooorrpooooT osd oorAR orrr ommBomF oOsFAFbbbFFI oWRo|o@mm oEo oorr roo:r3tomR'm 38 PP opop o oom ffF FFofmoFrf: OoWm EE re ikia 2 AMENDED "- PROJECTNO: 4540-109 . APPENDIX IV (Continued) : Comminsial odTreat Reed Es Fs Gena Dophics -- ST Tmeemmpe CSola,Dopo meg = edCSonO=ceoTieien Eoo3 e I 1BNumober obf"Fmbmer 5 CoCmuBtive WF | pes im eTbT T tr h sComDuaiaitve pms IN bTamCT eEer Ci oDms--aive mphen SP o oo 1 o 8 8 8 w 38 mmIWN i Poor P$ $ FE os 8r IN sb IN srsmIINNN IN i os Ww HE A Ww u A 5EE o 01 0e 88 Pood r eawm 88 IN o FE 8o 8 mIN $I o $ $ Poe 8 orIoImW Poor S Pioo 1oe8s888nIN i vos 8 IW FF$ bEA oosn Por OW g Fs E o s IIWw N Ww u $ A d5 o 1r 1e 3rb8 = am o sS soo bo.am 1. 8 gsL:aw o dgoo i 8i sr 8Gbior a-m H$ E li z 25 TE T i a C| T $ i : :i : i: % -- nem a: PROIECTNO:4540109 . APPENDIXIV(Coin) : -- Cr CeomemiePerens eral sdTreo Reed flocs FstGatoDep fei tiriaetn w Te ww=e w= Tenn Ea p-- eScam---- Toa-- Ca=n tosT e bord pon ,CDS" me ra i Du,we 3te pepi spew e 5P P0o1o ls oss8 n1nz:z no sobs HI n oz z s so oz t ss o o nz t o : t [I 0ol 8 ahf: esoo :: Potos e rE Rm CL e 232BE a AMENDED La: FROIECTNO: 4500109 . APPENDIXIV (Conic : --we rCumie nPec MoralityadTrim RindEc Fi GeinDips e i JIeonnge PA Caome e -- B ants Ro8B te BSido Ejp oE s "BaS E r m s E Ee 3e "D3a IN N pinti Do o w ps H }3 og PPooorE iop3 FE FE 3 88r roo IlNmmm ss p s5 8 iidf388 8A 888 IomN 1 A ee me oom @ am 2 i 3 FE : FE P g 2Pooe oi s f3 s8r8 omoIN meowe rpp s 8 O daAM 3 2888 8 b 88 8m m mIN Pooodsp rooomm o8s 88 iil 88 88 2 s gpS e oo oe e se s e eer e m mo mr e mpn n o mm a b r 8 srowmIR 88 Pg PP Pooo 1 oe oo r8psrs8be ooeoomWommw e ns 8 p n nn i w Rof p b o8 f88 WN [Cg A EEERr ki E TomEo -a- PROJECTNO: 454-109 . APPENDIXIV (Continued) : --- Cumin PeetMeltysd Tres RestoEc FistenrionDope Te pe IoECmonDinemnCerwemBy od oTeoCeDS y pe fTiot CBoa mea bT= lkoe CB pe = BsT Booooo0 bbb 15e5 s83 aiN 8 8 soos 8sIaN im. rrprF a-- Tiw 8 a ss I P P vobo b sos l8 saWm mm s d 88p 8 oaAwmm Poros or om rw 8s s so s s iiN os 8m 5 A Bss Boooobbbobse 8roosm domai a8 mos 80s88 omm wm g88r sp ss 9 8 m mimIW P p go ooloo l 8 sb b rnmmr sg8 oe r8 aam Pooler om 8 8 mw :s 8 s03 ; mIIiWe 8 8 Wm 3 dS o 1o8 b1hs gambs $rc bam 8 P;o am 328B sig ovoo ori 88 P18 h i1bt tzz 4c P 3e soo iHE s : si 0::: pPi]o oz=:= : Pc BT E ooDay0. oral mmo " PROJECTNO.: 454A-109. ' APPENDIXIV (Contin) CmtePenial so Tres RetoEf Fit.GesctionDep S TBooerrCaTbstO.Epimp eeCaertnen ete Do=oeo,dplT n CwC oBee fem Ca rpm pe bT mi o =Ba me 0 T Forosa or DsEn t h EE 5 oooh : 1 cz o HE iSobPoe Posnos: r 1 y o i 2 JESFE SPN 4S | 8S S }4c:S 0HESU S | : S HoI i : er 388 8 NEED " nomena: aio Com Pes MeyT orm eo sEetcFo t renDl _---------------------------- EEEEEmHamwoameenImEEm ERaa a EE ra EEoe w= pEg I Poroorf o1o:p1 1tfoomm opro 1tFff1 &oom FFfb oo@ :Ffoff: BO&&R Fof obFB@ NPE orET or 8I A fF i sp PFPgEoooEEorf orord iAT opfof oom| T tooFpr offfoo omooA foofpmo& F FFFBi PPPooofnrfy bofboF o FfBFbf oB@ fF fFF F3 EHii gg325 wapyPSEr ooEobnbos oobFfoE b O b frpooooowm|EbEFftoFp orbooI oofobosommo@ F 8sFsE FoopFF oR Bm&oo&m : E ER t yPfL tofto f0F% #o& f| FE w&i i "PROJECTNO: 454A-109 : Jre---- Comat MySenSaSn Sines BE Bg e eea = a -- a If :Fi! Tr |1 {11 1 11IrT% rB%BEF $ia II1}1i &82&8IT E${ E11 !1F%BRB t!i 1{1 1f1i i11F%B! 0{18 i%fBE 1I iIoIt Z#o8 " +1t$ii1!: If1ItI0I!III11E m%&% ! 1 1 1% 1I 3I.8i.I 113R%WRmE 1% |33O1LI131BBB%um iR iIPoh1L1io1r l2om V f1b3I0E2ow @f1I o:i1Bxom 8 E oF I sr T nor RB a i i2:- $ Ei EE a Al rpmscoDey spp ome fT $ i- = -- BROTECTNO: 4540-109 APPENDIXIV Contin) CominPretMalyadTrem Reed Eifects -Fist GeneonDips h T oraee , Rtor EDp a ms SfeCroienss os B=od(a i OoDw pres = EE s E i o or PossstI 8008 mTeoio mT Dai" phen FE E E o o s oa o aot== Tmeole C=Paedm ses e os s so 11 1 sCI --TE p o ---- y Fo-- ard CL 23 a53 -- PROJECT NO.: 454A-109 ot Conn ey ATBoOotCetnt FoOe ls Ewre E --n 2EaE mve BEE a EE mr Ee weetPPP poLoopOoooo1p ffriof rifbO3o o ooTwwRW|m bff f:OfooFrbfroF1F oEmR||& oFfoFFf ftFFfff mm||io@R& Por ot fr o| bf WpPFI CoPoloEEooosrrpTTEEefbR pom oommITofIr F froFBmR|' sAf OIfF oboso''mm oPvoofnrftofo| obfb ffbrB| FFFFFFB'Hi 322 PpE PPo oooolpoF odfffof top oFber oRm|WobFF FfFFbbrf oooBmmmBR FfoOFF FffoBmiaom ffm 85 r FREfi t fo fF 8fF e | Cf0 FEwi "a PROJECTNO: 454A-109 : APPENDIXIV Coins) CuminPctMealyaodrst.Rested Eff Fit.Generation Dips SEiee HoT Ee COD ots Bond Boll CBS pee both CRS pe bot hel" me = 5 82Po 1iiod80 FF 8b E E mm8o8 F Fn E E mIm 0 2 88 FH SPio oo0Ploss8rdsomoiiwm r r 8 r d8omWim w s 2 882 Ww Poors yo oor om rw u Are som ss om sam &B Poo o io o r8 bo 8 homb 3 i 8MIoom 8 8 8 2 AAWw SgPoo ool8Edaroniam obo om d 8 gobroow waonm Hr 8 E s b Ww Wawm u A Eo o earh . o yo y i 3 HI 0 HE i: 385 T $TioLri os3 fHhE ocn e f80 fP= oscf83g bic o : r ee R AEE 6- PROJECTNO: 4544-105 APPENDIXIV (Continsed) CumulativePercentMoralitysdTreat.Reltod Ec -FsGenerationDephids TT e-- TDooOrri:a Caet,e opie TetrCoicite)itien Rhos PNowoordmpbrie "BCDooamte" Bice 0 T T % Ts Toti CBDoaoimGe o Te mTooma ee GDoetm bm % T= D i i z i i z 1 i 3 }= Toen bA p ee m i z i z 0| i z i - i2 22 BaR S AMENDED WILDLIFE INTERNATIONAL Lo. PROJECT NO: 454A-109 =70- APPENDIVX `NeonateProduction ETor aSubsace TFhiOSComprsion DTiotvOrogWuiestme:r_ CWeedloloWeartne,rDaphniamagna MeaVsie,Tet ConGcmenstrianti)on _Repicue 2 05 7 Topi Gontd AB 00 0oo0 DE 000 000 000 GFc oo0 00 00 100 00 00 55 00 oo 9 mu 16 18 2 05 aB& 0 40 5& %1 o3a u3@ o@as ooo om3 1 7 u0w aoo w oo Bumm a 05oomanm moo&m ooom aaoo o1m 1s A5B 00 00 00 20 o0m & o %0 u E5 000 000000 006 mowm 4oom& ooomo o0om =3x: GHFoooo0 000 000 m506 4m3 os4& n01m xo0a 1x= TJ 00 00 00 w3 wom ma ono owom om 2 ABo0 0000 07 oomm oo&m 34 a71 %ow S D o0 o0 oo0 o0m o3wm sosm 3oom o0a x2 GFE oo00 000 000 o0 ma0u 4a# 33u% `300 aB1m HTo00o000 J 58 00 05 10o5 om4% 40 &m o0o m31us om Lhisvee sidedfom oulistion of omens NOMI Tout Tuss ii5sn 11s2 11100080 RGSD) 1226192) Ios wT1oo6 1i1366 1156 26-522047)) 016 11a 1J1267 111325 2st) 001160 AMENDED WILDLIFE INTERNATIONAL Lm. n- PROJECT NO. 45A-109 A`P NeoP natE ePVrN o(dCuoD cnttiiIonnueXd) TRE---- TDoeOiprea: Cwlavre,rDlmapa MCesniiiadnTet or opal mere 20 70VMFFF re ss AB8oo0ooo00 500 AmmommB oovmob o9oao 033B jii12os 5FEo0so0eo0o0om00 xmmoo 8@8 %0% oooono BB ii1nh 6oTooooeoso0 o58d omoWm o%o@R o4ooe NmToOBR iiisi Poses os ow ox oB.IE sem a iss n ABSo0o00o0000 00oo am8w % o03u% oo6no mo3 ii0 . 5FE oS0000oo00 om0wm om 3xYokum oo0pb ooouoi ii13 . STG osooooooo 0 0 d00m moNm ooa202ooo mo12%40 afiia) bose ss on om 3 5 0% 20:TB5o 026)9) A$ 0$o-cz:= = z = I = I II -II -= = o5 3 5PPFoe0g 5s r 5o- a or8 ozom -oCoc:= oso os 5 I I I = 28o 3 yf o8oo2zo 2o 8 8 oo - oo z zo==: ato02o a A mz IsIzI=z:==zz 4TI zo Iz oz=.:= 0 5f Eg t I II II I IIzzII IIzzII :== t SIIIo II o ZIIT o IIz II z= 2 yo zzz zz zz zz t5o T elo e om aiooemn a Se 01151 AMENDED WILDLIFE INTERNATIONAL Lr. 7. PROJECT NO: 454A-109 APPENDIVXI Leagth andDry Weightof Surviving First-Generation Dephids I Tes Substance: Tet Orgmism: =DiluMteiosnseWaetwderT:est `Co(ncgenatirant)ion: Negative Contd PClFaOdSocera, Daphnia magna Well WaterDer dividual (Lemnmgt)h a4s0 4i5s5s is5s0 s5s0 447350 Mem (Loemn:gtShD TE GoD Individual DryfWeyights 007638 008675 00660 "006715 0o5n5 15 4ia4s05 4.66(2 0.118) 000616803 445503 008611 47550 006613 4is6s0 0os6t8 29 47=5 462(: 0100) 0-83 4is5s0 o0s7s0 443750 0o9n4 par0 0o3n0 4is6s0 005630 Mean D(roygW:ei3gDht)s T6550: 0.100) 0.(6 +0.606293) 0724 (:0.110) 01162 AMENDED WILDLIFE INTERNATIONAL Lo. 73- PROJECT NO: 4544-109 APPENDVIIX (Continued) STees rSubstance: TDeisltuiOorngaWniiesrn:. _ MeasMueraednTest C(ongceaniralt)ion 6 LengthandDryWeightofSurvivingFirst-Generation Daphnids FMFOaS pes CWlealdoWcaetrear, Daphnia magna Ldeivnigdtuhal Gam) LMeeagmh (om: SD) DrydiWoveigid)guhatls i53n5 761 (0120) 00761 47500 008735 463s0 o06m8 45a5s 006723 6s or DGrygMW:eeai3ngDht)s 0727(: 006%) 2 445700 459: 0100) 001664 0723: 00661) 4i7s5s 00766s 448s0 006m5 455 070 aissss o0m1 01163 AMENDED WILDLIFE INTERNATIONAL Lm. 4. APPENDIVIXI ChatnoPgroetocsol PROJECTNO.: 454A-109 "Thsstudy wasconductedinaccordancewiththeapprovedProtocolwiththefollowingchanges: 1. Theproposedexperimentalstartandtermination dateswereamendedtotheprotocol. 2. Duringthesecondgeneration exposure,neonateswereobservedat 5, 24and48hours. 01154 WILDLIFE INTERNATIONAL Lm. PROJECT NO.: 454A-109 -75APPENDIX VIII Personnel Involved in the Study studyT:hefollowingkey WildlifeInternationalLtd.personnelwereinvolvedinthe conductormanagementof this 1 HearyO. Krueger, Ph.D,, Director, Aquatic ToxicanodNlono-Tagrgyet Plants. 2. `WillarBd. Nixon,Ph.D, Manager,Analytical Chemistry 3. Jon A. MacGregor, Scientist 4. Mark A. Mak, Laboratory Supervisor s Kurt R. Drotar,SeaorBiologist 01165 WILDLIFE INTERNATIONAL Lr. 76. APPENDIIX Report Amendment PROJECTNO.: 454A-109 1. OriginalReport: Pages 14,6and31 Amendment: DTehvespiaggneastwureerseacnhdadnagteedstdouientcoltudheetahdedaitmieonndoefdtrheeproerptodrtaatm,ernevdimseendptaagseAnpupmebenrdsi,xaInXd. Reason: Toreflecttheissuingofanamendedreport 2. OriginalReport: Page2 Amendment: Thecompliancestatement wasrevised. Reason: Toclarifyhowthetest substancewascharacterized. 3. Original Report. Page Amendment: iInncflourdmaitnigotnheprreoavniadleydsbisydathteeaSnpdonthseoprurreiftleyc,twiangsatdhederedatnoatlyhseiTseosfttShuebtsetsatncseusbestcatnicoen,. Reason: Toreflectthecurrent estsubstanceinformationprovidedbythe Sponsor. 4. OriginalReport: Entirereport Amendment: aAsldlettesetrsmuibnsteadnbcyetchoenScpeaotnrsaotriionnsawereraencalhyasnigseodfttohreetfelesctttshuebpsutraintcyeo(fFtCh-et9e5s,tLsoutbs2t1a7nc)e. TceersttifciocnacteeontfraantailoynssiosdriagtiendalMlayrwcehr9e,b2a0s00eidnodnictahteerdeppourrtietydopfur9i0t.y4o9%f. 9T8h.e9r%e.foTrheale,l ctoensctentsruabtsitoannsc,eancdoNncOeEntCr,atLioOnEs,CainndclMudAiTngC vnaolmuiesn,awlerceornecceanltcrualtaitoendsa,ndmreeapsourrteedd 25mga./Lbasedonthe90.49%purity. X Reason: Toreporttheresultsofthe estbasedo thetestsubstancepurityof90.49% atthe reqoftuheeSposnsotr. 061156 AMENDED D WILDE LIFc E INTEm A RNATO IOm NAAL ELTm. rim-- ePe ROJEe C-- T NO.: 454-- Ae-1y09eee 774 APPENDIX IX - ContinuedReport Amendment AMENDMENT SIGNATURES: ZY \ KSutrutdyRDiDrreoctttoarr [26/52 DATE FDeiarercytor, Aquatic ,ToxiPchDo.logy and Non-Target Plants REVIEWED BY: H. Colman I Quality,H,AsCsoulreamnacen DA 4-26-00 DATE 01157 AMENDED PROTOCOL PFOS: A SEMI-STATICLIFE-CYCLETOXICITY TEST "WITH THE CLADOCERAN (Daphnia magna) `SeriUesS.850En--viErcoonlmogeinctaallEPfrfoetcetcstTioenstAgGueindceylines 'OPPTS Number 850.1300 (OECD Guideline 211 3M LabRequest No. U2723 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 WILDLIFE INTERNATIONAL LTD. 8598 Commerce Drive `Easton, Maryland 21601 (410) 822-8600 June 10, 1999 3 01163 WILDLIFE INTERNATIONAL, LTD -2- PFOS: A SEMI-STATICLIFE-CYCLETOXICITY TEST `WITH THE CLADOCERAN (Daphnia magna) SPONSOR: SPONSORSREPRESENTATIVE: `TESTING FACILITY: STUDYDIRECTOR: LABORATORYMANAGEMENT: 3`EMnviCroornpmoernattailonLaboratory 935 Bush Avenue St. Paul, Minnesota 55144 Ms.SusanA. Beach Wildlife Internationa, Lid. 8598 Commerce Drive `Easton, Maryland 21601 Kurt Drotar Senior Aquatic Biologist Heary O. Krueger, PhD. DirectorofAquatic Toxicology & Non-Target Plants Proposed Dates: `Experimental `Start Date: FOR LABORATORY USE ONLY Experimental `Termination Date: ProjectNo.: Test Concentrations: NegativeControl,16,3.1.63,13.25and50mgai/L Test Substance No.: 4675. `ReferenceSubstanceNo. (ifapplicable): _4526 PROTOCOLAPPROVAL STUDY DIRECTOR DATE ORATORY MANAGEMENT I A Ded. SPONSOR'S REPRESENTATIVE AE L130/99 DATE % 01159 PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -3- INTRODUCTION `Wildlife International, Ltd. will conduct a semi-static life-cycle toxicity test with the cladoceran, Daphnia magna, for the Sponsor at the Wildlife Intemational, Ld. aquatic toxicology facility in Easton, Maryland. Thestudywillbeperformedbasedonproceduresoutlinedin theU.S. EnvironmentalProtection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1300 (1); OECD Guideline 211: Daphnia magna Reproduction Test (2); Standard Evaluation Procedure, Daphnia Magna Life-Cycle (21-Day Renewal) Chronic Toxicity Test (3); and ASTME 1193-87 Standard Guide for Conducting Renewal Life-Cycle Toeity Tests with Daphnia magna (4). Rawdata for all work performed at Wildlife IntemationLa1ld,.and acopyofthefinalreportwillbefiledbyprojectnumberinarchives locatedon the `WildlifeInternational,L1d.site oratanalternative locationtobespecifiedin thefinalreport. PURPOSE The purposeofthisstudy isto determine theeffectsofthetestsubstanceonthesurvival,growth,and reproofthdeculadcocertan,iDaophnniamagna,undersemi-staictestcondition,for aperiofo2d1 days. EXPERIMENTAL DESIGN Daphnids will be exposed to a geometric series of six test concentrations, and a negative (dilution water) controlfor21 days. Individualneonatedaphnidswillbeplacedintenreplicatetestchambers impartiallyassignedtoeachtestconcentratainodn control. Individually exposed daphnidswillbe observed forsurvival, reprodauncdgtriowothn. `Nominaltestconcentrationswillbeselectedinconsultationwiththe Sponsor,andwillbebasedupon. information such astheresults ofexploratory range-findingtoxicity data, knowntoxicitydata, physiprocpertaies loftlhetcestshubsetancmeoriothcerraelevlant information. Each.test substance concwiellbne5t0%orfthaenetxtihighoeronne. Watersampleswillbe collectedatspecifiedintervalsfor anaoftlhetyestssubistansce. Resultsoftheanalyseswillbeusedtocalculatemeanmeasuredtest concentrations. New test solutions will be prepared three times per week (.8, Monday, Wednesday, Friday), andthetestorganismswil betransferredfrom theoldtothenewsolutions Tocontrolbias, neonatedaphnidswillbeimpartiallyassignedtoexposurechambersattestinitiation. Nootherpotentialsourcesofbiasareexpectedtoaffecttheresultsofthestudy. Observationsofthe surviovftahle first-generation daphnids will be made daily.The preseofnecggesinthebroodpatch, males. PROTOCOL NO.: 454/061099/DAP-LC3/SUB4S4 3M LAB REQUEST NO, U2723 WILDLIFE INTERNATIONAL, LD 4 `orwinter eggs also willbe recordeddaily. With theonset of reproduction, the numberofyoung present in cachtest chamberwillbecountedeveryMonday, WednesdayandFridayduringthetestandattest termination. Anacute exposurewillalso be performedforany treatment groupswhichproduceyoung. `Twentyneofrn omea acht tree atms ent gr ando theu contp rol wbieexlposled understatictestconditions for 48hours. Theresultsofthetestwillbeusedtocalculatethenoobserved effectconcentration (NOEC)and thelowestobservedeffectconcentr(aLtOiECo)n. The NOECisdefined as thehighestconcentrattihoant produces no statistically-significant effect on survival, reproduction, or growth. The LOEC is defined as the lowestconcentrationthatproduces astatistically significanteffectonsurvival,reproduction,orgrowth. "Themaximumacceptabletoxicantconcentration (MATC)willbecalculatedasthegeometric meanofthe 'NOEC andLOEC. TestSubstance MATER ANI DMA ETL HOS DS Informationonthecharacterizationoftest, control orreferencesubstancesis requiredby OECD Principles ofGood Laboratory Practice. The Sponsor is responsible for providing Wildlife Intemational, Ltd.writtenverificationthatthe testsubstance hasbeen characterizedaccordingto GLPs. Ifwritten verificationof GLPtestsubstancecharacterizationisnotprovided toWildlife International,Ltd.,itwillbe notedinthecompliancestatementofthe finalreport. TheattachedformIDENTIFICAOTFTIEOSNT SUBSTANCE BY SPONSOR (Appendix I) is to be used to provide information necessary for GLP compliance. "The Sponsorisresponsibleforallinformationrelatedtothe testsubstanceandagreestoacceptany `unusedtest substanceand/ortestsubstancecontainersremainingattheendofthe study. PrepoafTerstaContcenitraotionns "Thetest substancewillbemixeddirectlyintodilutionwater. Thesolwiu llbt emi ixeo dunn tilts he testmateisrdisisoa lveld. Once dissolved,thesolutionwillbe distributedintotestchambersordilutedwith dilutionwatertoachievethe desiredtestconcentrations. PROTOCOLNO.: 454/061099/DAP-LC3/SUB454 01171 3MLABREQUENSOT. U2723 WILDLIFE INTERNATIONAL, LTD "5. `TestOrganism `Thecladoceran,Daphniamagna,basbeenselectedasthetestspeciesforthisstudy. Daphnidsare represeataotfiavne impogroruptofaaqnuattic invertebrates,andhavebeenselecfotreusdeinthetest baseduponpastusehistoryandeaseofculturinginthelaboratory. Daphnidneonatesto beusedinthetest `willbelessthan 24hoursoldandwillbeobtainedfromculturesmaintainedat WildlifeInternational,Lid., Easton,Maryland.The identityofthespecieswillbeverified usingappropriatetaxonomickeyssuchas Pennak(5). Daphaidswillbeculturedinwaterfromthesame sourceandatapproximatelythesametemperature aswillbeusedduringthetest. Daphaidsin theculturesproducingneonatesforthetestwillbeheldforat Teast10dayspriortocollectionoftheneonatesfor esting. Neonatesfromdaphnidsthatshowsigns of diseaseorstresswillnotbeusedastestorganisms.Daphaidsinholdingthatproduceephippiaalsovill not `beusedto supplyneonatesfortesting. 'Daphaidsinthecultureswillbefedoncedaily.Thedietwillbe amixtureof yeast,Cerophyllan,d trout chow (YCT), supplemented with a suspension of the freshwater green alga Selenastrum capricornutum. ~ Specifications for acceptable levels of contaminants in daphnid diets bave fot been established. However,thereare noknownlevelsofcontaminantsreasonablyexpectedtobepreseatinthe dietthatareconsideredtointerfere withthe purposeorconductofthetest. Neonateswillbeobtainedfortestingfromatleasttheeindividualadults. Priortotestinitiatitohne, neonateswillbecollectedfromculturesandtransferredtoglassbeakers.Thedapwihllbnerelieasdedisnto thetestcompartmentsbelowthewatersurfaceusing awide-borepipette. apwih llbefi eddad ilydus ring thetestusingthesamedietasusedinthecultures. Daphnidswillbefed 0.3mLof YCTand 0.6 mLof Selenaosncterdauilmy. Thefrequencyoffeedingwilbedocumentedintherawdata andthe inalreport. Dilution W `Waterusedfortheculturing andtestingofdaphnidswillbeobtainedfrom awellapproximately 40 `metersdeep locaotnethde Wildlife International, Ltd. site. Thewaterwillbe passedthrough asandfilter `and pumpedinto a 37,800-Lstoragetankwherethe aterwillbeaeratedwithspraynozzles. Pritoousre: thewatervillbe filteredto 045 umandpassedthroughanultravioletsterilize inortdo reemorve PROTOCOLNO.: 454/061099/DAP-LC3/SUB4S4 3MLABREQUESNOT. U2723 WILDLIFE INTERNATIONAL, LTD -6- `microoarngdfainnepiarsticmless. Waterusedforculturingandtestingischaracterizedas moderately bard. Typicalvaluesfor hardness, alkalinity,pHandspecific conduarceaptproaxinmatceley: HAalrkadlniensisty,,mmgg//LLaassCCaaCCOO 114950 SppeHcificConductance, mbos/em~~ 33031 Hardacss,alkalinity, pHand specific.conductancewillbemeasuredweekly to monitorthe consiofsthteweelnlwacteyr. Meansandrangesofthemeasured parametersforthefour-weekperiod `precedingthetestwillbeprovidedinthefinalreport.Analyseswillbeperformedat leastonceannuallyto determinethe conceatrationsofselected organic and inorganic constituentsofthewellwater and resalts of the most recent GLPcompliant analyseswillbesummarizedinthe final report. Test Apparatus `Test chambers will consist of 250-mL Nalgene beakers containing 200 mLoftest solution. Test chambers will be randomly positioned by treatment group in a temperature-controlled water bath or an environmental chamber. Testchamberswillbelabeledwiththeprojectnumber,testconcentrationand replicate. : Enis " Ligushedttoiillunminagtetheculturesandtestchambersduringculturingandtestingwill be providedbyfluoresceattubesthatemitwavelengths simtionlataurralsualight(e.8, Colortone 50). A photoperiodof 16hoursoflightand 8hoursofdarkwilbecontrolledwithanautomatictimer. A 30`minutetransitionoflowlightintensitywillbeprovidedwhen lights goonandoffto avoid sudden changes inlightintensity. Lightintensitywill bemeasured justprior otestinitiationandatapproximatelyweekly intervalsthereafterwith a SPERScientificLu.lightmeterorequivaleat. `Thetargettesttemperwialltbeu2r0e4 1C. Temperaturewillbemeasuredinonealternating replicateofeachtreatmeatandthecontrolgroupattest initiation,intheoldandnewsolutionsonrenewal days,andattestterminautsiinogan liquidin-glass thermometer. Temperaturealsowill be measured with 2 continous recorder in one negative control replicate. Recorder measurements will be verified with a liquid-in-glassthermometerpriortotest initiationandatleastweeklythereafter. 001173 PROTOCOLNO.: 454/061099/DAP-LCH/SUB454 3MLABREQUENSOT. U2723 WILDLIFE INTERNATIONAL, LD -1- Dissolvedoxygenwillbemeasured in onealternatingreplicateofeachtreatmentandthe control groupdailyduring the firstweek ofthetest,intheoldandnewsolutions onrenewal days, andattest termination using aYellow Springs Instruments Model 51B dissolved oxygen meter, or equivalent. In the eventthatdissolvedoxygen levelsfallbelow60%saturation,dissolvedoxygenwillbemeasuredinevery testchamberandappropriate actionswillbe takenafterconsultationwiththeSponsor. Measuremenotfs pHwill bemadeinonealternatingreplicate of cachtreatmentandthecontrolgroupattestinitiation,inthe oldandnewsolutionsonrenewaldays,andattestterminationusing aFisher Accumet Model915 pH `meter,orequivalent. If atreatment level reaches 100% mortality, dissolved oxygen, pHand temperature `measwiullr bee takm en aettn hatttimse,thendiscontinued. Hardness, alkalinity, and specific conductance will be measured in the negative (dilution water) controlandthehighesttest concentrationattestinitiationandatapproximatelyweekly intervalsthereafter. `Hardnessand alkalinitymeasurementswill be madeby titrationusingproceduresbasedonmethods in Standard Methods for the Examination of Water and Wastewater (6). Specific conductance will be equivalent. `measured using a Yellow Springs Instruments Model 33 Salinity-Conductivity-Temperature meter, or istogh . Thefirstgenerationdaphnidswill beobserveddailyduringthe testforsurvival,theonsetof repraonddcliu nicc al st ignsiofotoxn ici, ty. Thecriteria fordeathincludeabsence ofheartbeat, white opaque coloration, lack of movement of appendages and lack of response to gentle prodding. `Immobilizationisdefinedas alackof movementexceptforminorspontaneousrandommovementofthe appendages. Examplesofclinical signs of toxicity include inability to maintain position in the water column,uncoordinatedswimmingandcessaoftfeiedoinng. Thepresenceofeggsinthebroodpouch, aborted eggs, males, or ephippia also will be recorded. After the onsetofreproduction, twenty neonates from cachtreatmentgroup andthecontrolwillbe exposedfor 48-hoursunder statictest conditions. The `neonateswillbeobservedat 24 and 48hoursformortality/immobilityandclinicalsigns oftoxicity. `With theonsetof reproduction, the numberof second-generation daphaids will be counted and recorded approximately three times per week. Second generation neonates will be observed for clinical signsoftoxicity. Second-generation daphnids not used for the acute exposure will be discarded. The PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 01174 WILDLIFE INTERNATIONAL, LTD "5. lengthanddryweightofthesurviving first-generationdaphnidswillbemeasured attheendof thetest. The test will notbe consideredacceptable ifcontrol survival is<80%or ifthe numberofyoungproducbeyd eachsurvivingdaphnidliving thefull21daysis lessthanan averageof60. `SamplingforAnalytical Measurements `Watersampleswillbecollectedfrom the new and oldsolutionsduringeach week of thetestto determineconcenotfrtheatetstsiubostnansce.Duplicatesamplesforthenewsolutionswill becollected fromthe freshlypreparedsolutions,whereassamplesfromthe oldsolutionswillbe obtainedfrom a compositeof allreplicatetestchambersforeachcontrolandtreatment level.Intheeventthat 100% `mortality occurs inanytreatment,thensampling ofthattreatmentwillterminatefollowingthenext sampling interval. Samples will be collected at mid-depthand analyzed immediately. The sample scheme issummarized below: TTT ExpeineslGrosp PROPOSED NUMBERS OF SAMPLES Van Wekz Vex Teo veo ver oR Corel 2 2 2 2 2 7. Level1-LowConcentration 2 2 2 2 2 2 Levi2 2 2 2 2 22 Level 22 2222 Levels 22 2222 Levels 2 2 2222 Level 6-HighConcentration 2 2 2 2 2 2 TOTALS 11 111 TOedwssooilnossd(pflaiphccttcespipelsesfcroomlecomproosmitEenoellmraepslicBa atbeetchamber rom achcmt adtestlevel) "Theabovenumbersofsamplesrepresentthosecollected fromthetest anddonotincludequality control (QC)samples suchasmatrixblanksandfortificationspreparedandanalyzed duringtheanalytical chepmhai seosftt he srtuy dy. PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 01175 3M LABREQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -9- fel Ghent Chemical analysisofthe samples will be performed by Wildlife Intemational, Ltd. The analytical `method used will be based upon methodology provided by the Sponsor and identified in Appendix II. Modificationsmadetotheanalytical methodwillbedocumentedin therawdataanddescribedin the final report. . `DataAnalyses Statisticalanalyseswillbeperformedonthesurvivaloffirst-generationdaphnids, the numberoflive `young produced by each individually-exposed daphnid, andthe length and dry weightofthe individually exposed first-generation daphnids. Toevaluatewhetheror notsurvivalisaffectedbythe treatments, 2 X 2contingency tables andthe chi-square test will be used on survival data (i.c., numbers of alive and dead daphnids). Analyses of reproduction and growth (weight and length) data will be evaluated for normality and homogeneity of variances. Ifthedataaredeemednormalwithhomogeneousvariances, hypothesistestingusing analysisof variance (ANOVA) and multiple means tests (.g., Dunnett's, Bonferroni, Scheffe) will be used. Ifthedata failthetestsfornormalityorhomogeneity,thendatatransformationswillbetriedin anattempttocorrect the condition. Whenthedata transformations failtocorrectfornon-normalityorheterogeneityof variances, nonparametricprocedureswillbeusedtoidentifystatisticallysignificantdifferencesamongthe `experimental groups. Thestatisticalanalysesofsurvival,growthand reproductiondatawillaid butmay notbeexclusively usedinthedeterom fthiennooabsetrvi edeoffenct conc(Ne OECn )at ndlr owea stot bsei rvedo effn ect concentration (LOEC). Themaximumacceptable toxicant concentration (MATC) will be calculated as the geometric meanofthe NOEC and LOEC. REC TOBOEMR AIND TAIS NED RecordstobemaintainedfordatageneratedatWildlifeInternational, Ltd.willinclude,butnotbe limited to: 1 Acopyofthesignedprotocol. 001176 2. Identification and characterizationofthe test substance,if provided by the Sponsor. PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD "104 3. Datesofinitiationandtermination ofthestudy. 4. Length and dry weight measurements. 5. Reproduction parameters 6. Test organism culture records. 7. Resultsofrangetesfts,iwhnendappilicnablge. 8. Stock solution calculation and preparation. 9. Daily observations. ~~ 10. Waterchemistrycalculations (..,hardnessandalkalinity). 11. Ifapplicable,the methodsusedtoanalyzetestsubstanceconcentrationsandtheresultsofanalytical `measurements. 12. Statistical calculations. 13. Test conditions and physical/chemical measurements. 14. Calculationandpreparationof testconcentrations. 15. Copy offinal report FINAL REPORT Afinalreportoftherestsofthestudy will beprepared byWildlifeIntemational, Lid. Thereport `will include the following, when applicable: : 1. Naandmadde sessofthefacilityperformingthe study. 2. Datesuponwhichthestudy wasinitiatedandcompleted. ItistheresponosfithbeSipolnisotrtyo `providethefinal datethatdataaerecordedforchemistry,pathology and/orsupporting evaluations that may be generated at other laboratories. 3. Astatementofcompliancesignedby the StudyDirectoraddressing anyexceptionstoGood Laboratory Practice Standards. 4. Objectivesandprocedures,asstatedintheapprovedprotocol,includingchangesintheoriginal protocol 5. The test substance ideatification,includingname, chemical abstract mumber or codemumber, strength, purity, composition, and other information provided by the Sponsor. 6. Stability and solubilityofthe test substanceunderthe conditionsofadministration,if provided by the `Sponsororcontractedto WildlifeIntemational, Ltd 7. Adescroiftphetmeithoodnsuse tocondtuh ctetst, 01177 PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 3M LAB REQUEST NO, U2723 WILDLIFE INTERNATIONAL, LTD Te 8. Adescriptionofthetestorganisms,includingthesource,scientificname,age,liestage,feedtypes, lightinteanndphsotiopetrioyd. 9. Adescriptionofthepreparation ofthetestsolutions,themethodsusedtoallocateorganismstotest chambersandbeginthetest,thenumberoforganist andchamberspertreatment,andtheduration ofthetest. 10. Adescriptionofcircumstancesthatmayhaveaffectedthequalityorintegrityofthedata. 11. Thenameof the Study-Directorandthenamesofotherscientists,professionals,andsupervisory `personnelinvolvedin thestudy. 12. Adescriptionofthetransformations,calculations,andoperationsperformedon thedata, asummary andanalysisofthebiologicaldataandanalytical chemistrydata,and astatementoftheconclusions drawnfrom theanalyses. 13. Statisticalmethodsusedtoevaluatethedata. 14. Thesignedanddatedreportsofeachoftheindividualscientist orotherprofessionalsinvolvedin thestudy. 15. Thelocationwhererawdataandfinalreportaretobestored. 16. A statementpreparedbytheQualityAssuranceUnitlistingthedates thatstudyinspectionsand `auditsweremadeandfindings reportedtothe StudyDirectorandManageme. 17. fitis necessarytomakecoprectionsoradditionsto afinalreportafterithasbeenaccepted,such changeswillbemadeintheformofanamendmentissuedbythe StudyDirector. Theamendment. will clearly identifythepartofthefinalreportthatisbeingamendedandthereasonsforthe `amendment,andwillbesignedanddatedby theStudy Director. CHANOFGPRIOTNOCGOL Plannedchangestotheprotocolwilbeintheform ofwrittenamendmentssignedbythe Study DiraendtchetSpoonsorr. Amendmentswilbeconsideredaspartoftheprotocol andwillbeattachedto thefinal protocol. Anyotherchangeswillbeintheformofwrittendeviationsfiledwiththerawdata. All changestotheprotocolwillbe indicatedin thefinal report. 01178 PROTOCOLNO.: 454/061099/DAP-LC3/SUB454 3MLABREQUENSOT.U2723 WILDLIFE INTERNATIONAL, LTD -n2- GOODLABORATORY PRACTICES This studywillbe conductedinaccordancewith GoodLaboratory PracticeStandardsfor EPA(40 CFR Part 160 and/or Part 792); OECD (ENV/MC/CHEM (98) 17); and Japan MAFF (59 NohSan, Notification No. 3850, Agricultural Production Bureau). Each study conducted by Wildlife International, Ltd.isroutinelyexaminedby the WildlifeInternational,Ltd. QualityAssuranceUnitfor.compliance with GoodLaboratoryPractices,StandardOperatingProceduresandthespecifiedprotocol. Astatemeonft compliancewith GoodLaboratoryPractices will be preparedfor allportionsofthe studyconductedby `Wildlife International, Ltd. The Sponsor will be responsible for compliance with Good Laboratory Practices for procedures perfobrymotehedr laboratories (.g., residue anaolrpyathsole ogys). Rawdatafor allworkperformedatWildlifeInternational,Ltd. and acopyofthefinalreportwillbefiledbyproject numberinarchiveslocatedonthe WildlifeInternational,Ltd.site,oratanalterlnocaatitonitovbee specified inthe finalreport. | PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 0179 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAaL, LTD eee eeeies -1- REFERENCES 1 (UdSra.ft)E,nOviPrPoTnSmeNnutmablePrro8t5e0c.t1i3o0n0:ADgaepnchyn.id1C9h9r6o.nicSerTioexsici8t5y0-TeEsctological Effects Test Guidelines 2 OTeEstC. D Guideline for Testing of Chemicals. 1997. Guideline 211: Daphnia magna Reproduction 3 LiUfSe.CEyncvlier(o2n1m-eDnatyalRePnreowtaelc)tiCohnroAngiecncTyox.ic1i9t8y2.TestS.taEndPaArd54E0v/a9l-u8a6t-i1o4n1.ProEcnevdiurroen,meDnatpahlniPraotmeactginoan Agency, OfficeofPesticide Programs. Washington, D.C. 4 American Society for Testing and Materials Standard E1193-96. 1997. Standard Guide for CMaotnedriuacltsi.ngPLhiiflea-CdyecllpehTiPoaAx,i.city Tests with Daphnia magna. American Society for Testing and 5 Pennak, RW. 1978. FreshwaterInvertebratesof theUnitedStates.2ndEd. 365 p. 6 A16PtHhAE,ditAiWonW, AAm,erWiPcCanF.Pub1l9i8c5.HeSatlatnhdaAsrsdocMieatthioond.sfoArmetrheicEaxnamWiantaetiroWnoofrkWsatAessroacniadtiWoans.tewWaattere.r Pollution Control Federation, New York. PROTOCOL NO.: 454/061099/DAP-LC3/SUB4S4 01180 3M LAB REQUEST NO. U2723 WILDLIFE INTERNAATOIONNAA L, LTD eee Sue eee APPENDIXI IDENTOFI TEF ST SI UBSCTAA NCET BYI SPO ONSN OR "TobeComplbyeSptonesodr 1. TestSubstanceIdentity (nametobeusedi thereport): P(FOPS erfluSoulfroniocAocidcPottassaiunmSaelt Reference Standard(ifapplicable): Analytical Standard: _N/A InternalStandard:_P1.e1.2r2HfHLHlLHuoroocSutlfoanicnAceid `Test Substance Sample Codeor Batch Number: __Lot217 `TestSubstancePurity (%ActiveIngredient): _989 Expiration Date: _2008 1. TestSubstanceCharacterization Hayethe duty,srengh,purity and compesiion orobercharacieristics: `whichappropriately testsubstance and reference standard been `determined prior10 itsuseinthisstudy in accordancewith GLPStandards? IIL TestSubsStotragaeConndictioens Yes x No Abet Please indicatethe recommended storage conditions at WildlifeIntemational Led. JHaassteheS:abiy ofthe tesstubsStaangceeuinldesrthesestorageconditions: Yes o Otherpertinent stability information: i IV.Test Concentrations: Adjusttestconcenttroa10t0i%oain. xbasedpuuripty (o%)ngivten ahbovee. 'V.Toxicity Information: _rrelnmone Donot adjust testconcentrtaot1i00o%n Mammalian: RatLDS0 251 Moum seg LDSh O Ng /A Aquatic: Invertebrate Toxicity(EC/LCS0) Daphniamagna:27mg/l. Daphniamagna:SOmg/l. `Fish Tandcity(LCS0) Rainbow Trout: 11mg/l Fathead Minnow:38mg/l `OtherToxicity Information(including findings of chronic and subchronictests): Pleasesee MSDS PROTOCOLNO.: 454/061099/DAP-LC3/SUB454 01181 3MLABREQUENSOT. U2723 A We ILDLIFE INTERNATIONAL, LTD r [TR APPENDIIXI `AnMaetlhodyProtvidiedbcy Spaonslor analytiScaalmpmleetshowdisl:beanalyzedbaseduponproceduresprovidedbythe Sponsorinthefollowing 1. LiPeqrufilduoCrhorooctmaanteogSrulafpohnyiMcaAscsidSpPeocttarsosmieutmryS(alLtC(MPSF)OMSe)tIhnodFfreosrhtwhateeDre,teSramlitnwaatteironaonfd Algal Medium toanalAyczoetphyeotfetshtesaabmoplveesmweitlhlbodedwoilclubmeenmtaeindtianitnhedeirnatwhdeartaawandadtsau.mTmahreiazcteudailnmtheetfihnoaldreoplourots.gedy PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 01182 3M LAB REQUEST NO. U2723  otoese 13:31 maz sais AQUATIC BLOG @o0z/002 WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-109 Page 1 of1 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A SEMI-STATIC LIFE-CYCLE TOXICITY TEST WITH THE ~ CLADOCERAN (Daphnia magna) PROTOCOL NO.: 454/061099/DAP-LC3/SUB454 AMENDMENT NO.: 1 SPONSOR: 3M Corporation PROJECT NO.: 4544-109 EFFECTIVE DATE: July 12,199 AMENDMENT: Page -- Add: Experimental Start Date: 7/12/99 Experimental Termination Date: 8/2/99 REASON: The above information was not known when the protocol was signed by the Study Director. STUDY DIRECTOR DATE LABORATORY MANAGEMENT DATE SPONSOR'S REPRESENTATIVE DATE asatosamendl 001183