Document KJXnNZ5jo0wn0x2XN2eG6yaqr

ros AR226-009 `A 96-HOUR STATIC ACUTE TOXICITY TEST `WITH THE FRESHWATER MUSSEL (Unio complamatus) FINAL REPORT WILDLIFEINTERNATIONAL LTD. PROJECTNUMBER: 454A-105 3M LAB REQUEST NO. U2723 U.S Enionmentl PrteionAge Series 850O~PEcPoSlogNiucamlbeEfrfe8c5t0s.T1e0s7t5Guidelines hb and (OECD Guideline 203 AUTHORS: HeKrurtKR.eDvrot,tarPhD STUDYINITIATION DATE: My 3, 1999 STUDY COMPLETION DATE: August 11, 1999 AMENDEDREPORT DATE: Apr 26,2000 Submitted to Em3MiConrporaabtioornsry 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife I45n98tCeomrenrsaDtriiveonal Ltd. Easton, Maryland 21601 (410) 822-8600 Page 1 of 43 000505 AMENDED JWIILDLLLIFENINETERRNAATIONALLLmo. e 2. PPRROOJCETCTNNOO.:: 4M54A-A105S `GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT - SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static Acute ToxicityTestwith the Freshwater Mussel (Unio complamatus) `WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-105 STUDY COMPLETION: August 11,1999 AMENDED REPORT: April 26, 2000 `Thisstudy wasconductedincompliancewithGoodLaboratoryPractioeStandardsas publishedbytheUSS. `EnvironmentalProtection Agencyin40 CFR Parts 160and792,17August 1989; OECD PrinciofpGloeosd Laboratory Practice, OCDE/GD (92) 32, Environment Monograph No. 45, Paris 1992; and Japan MAFF, 59 NNohSan,Notification No. 3850, Agricultural Production Bureau, 10 August 1984with thefollowingexceptions: Thetestsubstance wasnot characterizedinaccordancewithfullGLPcompliance; however, the `characterizationwasperformedaccordingto 3MStandardOperatingProceduresandMethods,andallrawdata arebeingmaintained inthe 3M archives.Thetest subisbseint grecaharanctec rizee din accorwdiathnGcLPe. `Thestability of thetestsubstanceunderconditions ofstorageatthetestsitewasnotdeterminedin `accordance with Good Laboratory Practice Standards. = STUDY DIRECTOR: eel] \ Kurt R. Drottar `Senior Biologist 4/2616 DATE L iE 000806 AMENDED WILDLIFE INTERNATIONAL Lo. 3. PROJECT NO.: 454A-105 `QUALITY ASSURANCE STATEMENT `This stwasuexd aminy ed for compliancewithGood LaboratoryPractice Standardsas publisbhytehde U.S. EnvironmentalProtection Agency, 40 CFR Parts 160 and 792, 17 August 1989; OECD PrinciopfGloeosd Laboratory Practice, OCDE/GD (92) 32, Environment Monograph No. 45, Paris 1992; and Japan MAFF, in5s9peNcothiSoanns,anNodtaiufdiciattsiaonn dNto.hed38a5t0e,stAhgartiacunlytfurianldiPnrgosduwcetrieornepBourretaeudt, o1t0heASutguudstyDi19r8e4c.torTahnedLdaabteosraotforalyl Management were as follows: AcmvTy: Test Substance Preparation Aula Semple Preparation `end Observations Biogial DaaendDrat. Report Acaytical Da snd Drs Report DATE CONDUCTED: May27,1999 May28,1999 June 29,1999 Ju 29annde 30, 1999. `Final Report August 11,1999. `AmendedReport. April20, 2000 DATEREPORTED TO: STUDYDIRECTOR: ~~ MANAGEMENT: May27,1999 June1,1999 May28,1999 June 29,1999 Ju3,n19e99 June30,1999 June 30,1999 August 11,1999. April 20, 2000 June30,1999 August 11, 1999. April 24,2000 Zh = Timothy A. Springer, PLD. `Manager, Regulatory and Technical Support Af afoo DATE 000507 AMENDED WILDLIFE INTERNATIONAL Lr. 4 REPORT APPROVAL PROJECTNO.: 454A-105 SPONSOR: 3M Corporation TITLE: PFOS: A 96-HourStaticAcuteToxicity Testwiththe FreshMwusasetl(eUnrio complamatus) `WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-105 STUDYDIRECTOR: Al \ Kurt R. Drottar `Senior Biologist MANAGEMENT: leFD. NoDinre-ctToarr,get Plants Aquatic Toxicology and y/aclo0 "DATE Dal9 2%foo 0008508 AMENDED -- WILDLIFE INTERNTATOIMONAAL Lo. os. TABLE OF CONTENTS PPRROOJJEECCTTNNOO.::44s5t4AA-d100s5 SY 1 OBJOCHVE ccs EXPETENUA]DESIGN... ssssssssssssenseneeessess 8 'RBEASD UMI S SCUSSION... 12 TABLES `Table 1 - SumomfAa nalr ytiy cal CHEMISTYDRA... 15 `Table 2 -Tempe Disr sola vedtOxu ygernae ndp,Hof Wait nthe eTerst Chambers........................oo. 16 `Table 3 -Hardness, AlkalinityandConductivityofWaterintheTest CRATES... 17 Table 4 - CumulativePercentMortality andTreatment-Related ESFSCtS..................verene. 18. 000509 = WT ILDLT IFE E INTERR NATIN ONAL A Lro. TIOMPA PRROOJJKECL CTTNNOO::\ 44s5i4AA-d1 00s5 "6. TAOFBCONLTENETS -Continue-d FIGURE Figure 1. ConcentatonCu-rvRee(9s6-pHOoUnDsBe). 20 ~ APPENDICES Appendix 1 MSepaesciufriecCdoDnrdiucntgatnhcee,-HWaeredokePesr,iAoldkaIlmimnietdyi,aatnedlypPHroecfeWdeinlgltWhatTeert... 21 Appendix I~ iAnnaWlAyIsIesoefPensticaidesi,OrLogdan.inWces,lalMetlalsaWnBEd..O.the Inorganics 22 Appendix I ThteAnealysrisLodfi.PPFrOoajSeicntFlNor:4e54sAi1nhS0u5pwporrat ovtf Wiecdirfve ereeeio23 ADPERSXI=V CRA10IPGRSOOL...rrrsmnsen 40 Appendix V + PersonnelIve ntheSY... 4] 000810 AMENDED W--I_--LDLIFE INTERNATIONeAeL Lm. 7. PPRROOJEECCTTNNOO.:: d4s54A4d-1e0s5 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATIONOFSTUDY, RAW [DATAAND ACOOP FTY HE FINAL REPORT: SUMMARY `3M Corporation Ms. Susan A. Beach Wildlife International Ltd. Easton, Maryland 21601 LTD. PROTECT NUMBER: TEST SUBSTANCE: STUDY: MEANMEASURED TEST (CONCENTRATIONS: `TEST DATES: LENOTHOFTEST: TEST ORGANISM: SOURCEOFTESTORGANISMS: AGEOFTESTORGANISMS: RMEEPARSESUERNETAMTEINVEOTFSMU2S0SELS: `WET WEIGHT (g): TOTAL LENGTH (mm): "96-HOURLCS0: 95%CONFIDENCE LIMITS: NO-MORTALITY CONCENTRATION: 454A-105 PFOS (Perfluorooctane Sulfonic Acid PotassiumSalt) PMuFsOsSe:l A(Un9 to c6 ompS- ltaamtaiH tcuAsc)o ute Tu oxicr ity Testwiththe Freshwater Negative Control, 5.3, 12, 20, 41and 79 mg a.i/L `Experimental St-aMary2t 8, Biological Termination -- June 1999 1, 1999 ExperimentalTermination ~ June3, 1999 96 Hours Freshwater Mussel (Unio complamatus) Carolina Biological Supply Company `Burlington, North Carolina 27215 Unknown Mean =76.5; Mea= 4n8.7; Range =57.51093.5 Ra=4n 4.6g to5e 4.9 Somgaill 516a 8mgnaid /L 20mgai/l 000811 AMENDED WWIILLDDLLIIFFEE IINNTTEERRNNAATTIIOONNAALLLLmm.. pe PPRROOJEECCTTNNOO..:445544AA-d10055 INTRODUCTION `Thisstudywasconducted byWildlifeInternationalLtd.for 3MCorporationattheWildlifeInternational Lud.aquatictoxicologyfacility inEaston, Maryland. Thein-lifephaseofthetestwasconductedfrom May 28to June 1, 1999. RawdatageneratedbyWildlifeInternatioLntadl.and acopyofthefinalreportarefiledunder Project Number454A-105inarchiveslocatedontheWildlifeIntemationalLtd.site. OBJECTIVE "Theobjectiveofthisstudywas to evaluatetheacutetoxicity of PFOS(Perfluorooctane SulfonicAcid PotassiumSalt) to thefreshwatermussel,Unio complamatus, during a 96-hourexposureperiodunderstatictest `conditions. [EXPERIMENTAL DESIGN Freshwater musselswereexposedto geometricseriesof fivetestconcentrations and anegative (dilution water) control. Tworeplicatetestchambersweremaintained incachtreatmentandcontrol group,with 10 freshwatermusselsineachtestchamberfor atotalof 20freshwatermusselspertestconcentration.Nominaltest. concentrationswereselectedinconsultationwiththe Sponsor,andwerebased upontheresultsofanexploratory rangefindingtoxicitytest. Nominaltestconcentrationsselectedwere 5.7, 11,23, 46and 91mgactiveingredient (8. Thetestsolutionswererenewedat approximately 48hoursofthe test. New estsoluwtereiporepnarsed andthemussels weretransferredfromthe oldto thenewsolutions.Meanmeasuredtestconcentratiwoernes. determinedfromsamplesoftestwatercollectedfromeach treatmentandthecontrolgroupatthebeginningofthe test,atappro48hxouris,amndaattetstteermilnatiyon. Freshwatermusselswereindiscriminatelyassignedtoexposurechambersattestinitiation. Observations ofmortalityandotherclinicalsigns oftoxicityweremadeatapproximately6, 24, 48, 72and 96hoursaftertest. initiation. Cumulative percentmortality observedinthetreatmentgroups was usedtoestimateorcalculateLCS valuesat24, 48, 72and96hours. Theno-mortality concentration wasdeterminedbyvisualinterpretationofthe: morality daa 000512 AMENDED -- WILDLIFE INTERNAO TIOA NALiLoe, "9. PPRROOEJCETCTNNOO::4s45e4A4d-100s5 MATEARNI DMA ETL HOS DS Thestudy was conductedbasedon theprocedures outlinedinthe protocol, "PFOS: A96-Hour Static AcuteToxicityTestwiththe Freshwater Mussel". The protocol was basedon proceduresoutlined in U.S. EnvironmentalProtection Agency Series 850 --Ecological Effects TestGuidelines, OPPTS Number 850.1075 (1): OECDGuidelineforTestingofChemicals203: Fish, AcuteToxicityTest (2); and ASTMStandard E729- eo. ' 88a, StandardGuideforConducting Acute. Toxicity Testswith Fishes, Macroinvertebrates.and Amphibians Test Substance `Thetestsubstancewasreceivedfrom 3MCorporation onOctober29,1998andwasassignedWildlife InternationalLtd.identificationnumber4675. `Thetestsubstancewasdescribed as a white powder. Itwas identifiedas FC-95 fromlot nu2m17b (T-e 629r 5).Informationprovidedbythe Sponsorindicated apurity of 98.9%,andanexpirationdataof 2008. `The test substancewas reanalyzedby the Sponsor and the Certificate of temperature. AnalysisdatedMarch9,2000indicated a - purity of90.49%. `Thetest substancewas storedat ambient room reparationofTestConcentrations ' Nominal testconcentrawterieo5.n7s, 11, 23,46and91 mg .i/L, basedon atestsubstancepurityof 90.49%.Allmaterialswhichcameintocontactwiththe testsubstanceduring preparationoftestconceatrations `wereconstructedofplasticorstainless steel. Aprimary stocksolutionwaspreparedindilutionwaterat a concentrationof 91mga.i/L. Theprimary stock solutionwasmixedwith an electric paddlemixer for `approximately24hoursto aid inthesolubilization ofthe test substance.After mixing,theprimarystock solution `was proportionally dilutedwithdilution watertopreparethe four additionaltestconcentrations. Alltestsolutions `appearedclearandcolorless. `Test solutionswere renewedonDay 2ofthe test with freshly prepared solutions. Test Organism The freshwater mussel, Uniocomplamatus,wasselectedasthetestspeciesforthisstudy. The freshwatermusselisrepresentativeofanimportant groupofaquaticinvertebrates andwas selectedforuseinthe testbaseduponcommercialavailability. Freshwater musselsusedinthetestwereobtainedfromCarolina suppl `Biological Supply Company,Burlington,NorthCarolina.Theidentificationofthe specieswas providedbythe AMENDED 000513 W--I_L-- DLIFE INTERNATIONAL Lo, -10- PRPROOEJCETCTNNOO:.:4s454AA-d1005s musselsFwreersehhwealtdefromruaspsperloxsiwmearteehlye1l5ddaatyapspprroioxritmoatteesltyntgh.eDsuarmiengttehmepheorladtiurnegapesruiosdesd,dthuermiungsstehlestsehsot.wedTnhoe dsaigynbsooldfidngipesrieoordapstrsreecsese.dTinhgetmhuestessetl,swwearteentoempfeerddautruirnegstrhaenhgoelddfirnogpmer20i.o3do0rd2u2r.i2nCgt.heTthesetp. HDouftrhetihwena1t4ge.r rwaantgeerdmfome8.a2.0s8.a6aeundedsrdcirsisbeoeldviemndtohxeyeEgnevnnirraonntgmeednfstraolm 7Co.n6d1i0ti8o.8nmsgs/ecLt.ionIofnthsistreprorut.mAatnetdenmsettthaoidttsiosine,dotfnhoer musselswerecollectedfromtheholdingtankandtransferredtothe estchambers. indiscrimTihnaetaevlyersealgeectteodtmaulssleenlgstfhro(mdtihsteasnecemferolomt otfhteesttioprgoafntishmeswuamsbo48t.o7mtuhmewdiitsthalrvaanlgveeofed6g4e.6)to0f524.09 mum.Theaveragewetweightwas76.5gramswith range of$7.5to 3.5grams. TestApparatus depthofTweasttechianmabreeprrsewseenrteat2i5v-etLepsotlcyheatmhbyelernweesaqauaprpiraocxoinmtaatienliyn2ga3p.2prcomx.iaTeasttelchya2m0beLrosfwetreestisnodliusctriiomni.naTtehley ploasbietlieodnweidtihnthaenpreonjveicrtonnmuemnbtearl,cthesatmcboenrcesnettrtatoimoanianntdarienplaitceamtep.eratureof 222C.Thetestchambewerrse `DilutionWater Thewate usedorholdingandtestingwasfreshwaterobtainedfrom awel approximately40meters deep locatedontheWildifeInternatiLtod.nsaitl.Thewell waterischaracterizedas moderatly-whaatrerd, pTehreiosdpiemcimfeidcicaotnedulcytparneccee,dihnagrtdhneetsse,staalkraelpirneistye,natneddipnHAmpepaesnudriexmeIntsofthewellwaterduringthefour-week andpumTpheedwinetlolwat3e7r,w8a00s-pLasstsoerdatghetroaungkhanasdaanedrafitleedrwtiotrhesmporvaeypnaorztzilceless.gPreraitoerrtohuasnea,ptphreoxwiamtaetraelgya2i5n waams, filtered (0.45pm)toremovemicroorgaandnpairtsicmless.Theresultsofperiodicanalysesperformedto measuretheconcentrationsofselectedcontaminantsinwellwaterusedby WildlifeIntemationalLtd.are presineApnpetndeixdI. 000814 bWoIoLDCLImFEtINe TERa NATIONAL Lo. - ON.PR.OJE.CT NO.: 454A-c105B Conti Lightingusedtoilluminatetheholdingtanksandtestchambersduringholding andtestingwasprovided `by fluorescenttubesthatemitted wavelengths similarto natural sunlight (Colortone 50). Aphotoperoifo1d6 `hoursoflightand hoursofdarknesswascontrolledwithanautomatictimer. A 30-minutetransition periodof low lightintensitywasprovided when lightswentonand offto avoidsudden changesinlighting. Lightintensity at testinitiation wasapproximately 369 ux atthe surface ofthewater.Light intensity was measuursiengda SPER ScientificLtdl.ightmeter. `Temperaturewasmeasuredin cachtestchamberatthebeginningandendof thetestusing aliquid-in- glassthermometer. Temperature also was measuredcontinuouslyinonenegativecontrol replicateusing a Fulscope ER/C Recorder. The target test temperatureduring thestudywas 22:2C. Dissolvedoxygen `measurements weremadeon watersamples from allreplicate testchambersof cach treatmentand the control at testinitiationandat approximately24-hourintervals thercafier. MeasuofrpHewemremeadneitnosne altematingreplicateofeachtreatment groupandthecontrol attestinitiationand atapproximately 24 hour intervalsthereafter.Hardness,alkalinity and specificconductancewere measuredinthedilution waterandlow, `middleand high testsubstanceconcentrationsattestinitiationandattesttermination. Measurementsof pHweremade using aFisherAccumet Model915pHmeter,anddissolvedoxygen was `measured using a Yellow Springs Instrument Model 51B dissolved oxygen'meter. Specificconductancewas. `meaussinugarYeelld ow Springs InstruMmodeeln3t3 Salinity-Conductivity-Temperamettuerre.Hardnessand alkalinitymeasurementsweremadebytitrationbasedonproceduresinStandardMethodsfor the Examination of Water andWastewat(e4)r. Observations Observationsweremadetodeterminethenumberofmortalities. Musselswithopenshellsandnot respondingtogentleprodding were considereddeadThe numberofindividualsexhibiting clinicalsigns of toxicityor abnormalbehavioralsowereevaluated. Observationsweremadeapprox6i,2m4,a48,t72eanld9y6 `hoursaftertest initiation. 000815 ize `StatisticalAnalyses `The 24, 48, 72 and96-hour LCS0values andthe 95% confidenceintervalswerecalcuwhlenapotsseiblde. bsyipnrgotbhietcanaloysims,tsphofeutmwoavtrinoegfaCvr5e.raSgeepmheatnh(o8d)onrbhinsomsitauldpsr,oLbaCb3i0livtaylwuiesthcnoounl-dlniontebaerinatlernptoleadtiaotn2(45a,n6d, 74)5 `hoursdue tothe lackof anadequateconcentration-resppatotnesren.However, the binomialmethodwasusedto `evaluatemortalityat 72hoursandthe probitmethodwasusedto evaluatemortality at96hours. Theno-mortality concentrationwasdeterminedbyvisualinterpretation ofthemortalitydata. Analytical Chemistry Water sampleswere collected at mid-depthfromcachreplicate test chamberof each treatmentand controlgroupatthebeginningofthetest, at48hours (oldsolutions)andattestterminationtomeasure concenotftrhetaesttisubostnancse. The samples werecollectedinplastic(Nalgene)bottlesandanalyzed immediately withoutstorage. Analytical procedures usedinthe analysis ofthe samplesareprovided in Appendix IIL At test terminaalltreimaoinnin,g live mussels were sacrifofrcoillcecetiodn oftissue samples. Thetissue sampleswere compobsy rieptliceatde,frozen andshiptpoethde Sponsorfor possible analysis. "REAS NDU DISL CUT SSIS ON Measouf Trese tComncee ntrn atit ons Resultsofanalysestomeasureconcentrations of PFOSinwatersamples collectedduringthetestare `presentedin Table 1and inthe analyticalchemistry report (AppendixI). Nominal concentrsaelteciteodnfosr `useinthisstudywere5.7,11,23,46and 91 mga.i/L. Samplescollectedattestinitiationhadmeasuredvalues thatrangedfrom74to 96% ofnominalvalues. Measured valuesforsamplestakenat 48hoursrangedfrom 81to 9o9 fnomi% nal. Measuredvaluesforsamplestakenat 96hoursrangedfrom 89to 13 ofn0 omin% al. When `measured concentrationsofthe samplesanalyzedattestinitiation,approximately 48hoursandat testtermination `wereaveraged,themeanmeasuredconcentrations forthisstudywere 5.3, 12,20,41and 79mg 2.i/L. Mean `measuredconcentrationswereusedintheestimationorcalculationofLCS0 values. ObseravndaMetasiuroemnensts Measurementsoftemperature,dissolved oxygenand pHarepresentedinTable2. Temperatureswere AMENDED `withinthe222Crangeestablishedforthetest.MeasuorfpeHrmanegen dfrtoms7.9108.5durtihentegst. On 000816 WILDLIFE INTERNATIONAL Lm. 1- PROJECT NO.: 454A-105 Day 1ofthetest,dissolvedoxygenconcentrationshaddroppedaslowas5.0mg/L (7%ofsaturation), Consequently,mildaerationwasinitiatedtoalltestchambers.Aferaration,dissolved oxygenconcentrations remained 25.9 mg/L (68% of saturation) throughout the test. Measurements of hardness, alkalinity and `condarueprcestenitedviniTatbley3 Dailyobseorfmovrtalaitytandiothoercnliniscal signsoftoxicityobservedduringthe testareshownin `Table. Freshwater musselsinthenegativecontrol, 5.3,12and20mg2. /L treatmentgroups appearednormal andhealthyduringthetest. After96-hoursofexposure,mortalityinthe41 and79 mg ../L treatmgreonupts was Sand 90%,respectively. LCSO valuesand 95% confidencelimitsat 24,48,72and96hourswere estimated orcalcultedfromthemortalitydata,andareshownin Table5. Agraphoftheconcentration-rescuprovnesies preseinnFtigeurde 1. CONCLUSIONS `The 96-hour LCS0 value for freshwater mussels (Unio complamatus) exposed to PFOS was 59 mg iL. The 95%confliimitdsweere5n1acnde68mga.i/Landtheslopeoftheconceatration-espcounrsve was 10. The96-hourno-mortalityconcenwtars2a0mtgiaoiLn. : 000817 AMENDED e WILDLIFE INTA ERNATIONAAe L TLme. e 2PRROOJREOCTTHNOO.:d45544AA-010S5 14- REFERENCES 1 US. Environmental Protection Agency. 1996. Series 850 --Ecological Effects Test Guidelines (draft), OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater and Marine. 2 Organisation for Economic CooperationandDevelopment. 1993. OECDGuidefolrTiesntiengosf Chemicals. Guideline 203: Fish, Acute Toxicity Test. Ado byp thet Coue ncild on12 July 1992. 3 ASTM Standard E729-88a. 1994. StandardGuideforConductAicnutgeToxicityTestswith Fishes, Macroinvertebrates,andAmphibians. American Society for Testing and Materials. 4 APHA, AWWA, WPCF. 1985.StandardMethods for the Examination ofWaterand Wastewater. 16th Edition. American Public Health Association. American Water Works Association. Water `Pollution Control Federation, New York. 5 cSotmempuhnainc,aCt.iEon.. 1978. U.S. EPA, EnvironmentalResearchLaboratory, Duluth, Minnesota. Personal 6 Finney, D.J. 1971. Statistical Methods inBiologicalAssay. Second edition. GriffinPress, London. 7 Thompson, W.R. 1947.Bacteriological Reviews. VoIl,N.o. 2. Pp. 115-145. 8 Stephan, CE. 1977. "Methods for Calculating an LCS," Aquatic Toxicology and Hazard Evaluations, American SocietyforTesting and Materials. Publication Number STP 634, pp 65-84. 000518 WILDLIFE INTERNATIONAL Lo. S15. PROJECTNO: 454A-105 Table 1 SummaryofAnalyticalChemistry Data mm----------e TTeeSwuObrsgummiessm:: PFrReOshSwater Mussel, Unio complamaus NDileutimonaWater. _ Wel Water Sap Concentration Tine CMonecemnterastion MCeonmceVntreateiomn Feosf ail) gave Repxl3 T ai) ail) Nom-inal AA8 "i9s% 3 B 9% <0Q 57 A8A "0o BAB 95i%% sis4i7a 53 9 52372406 un A3A 800 11041 12 2 109 A5 8 i59%%s 111005334 8A 0o l1e9s0 5 A8A pi%l 21188977 B % 24 4 A8A "00 w37e2 71 a 8 BA8 5a%% 53is52 0 8AA @0o 480173 EE 5 8A 5i% 7872s -- Be ey ! The limitof quanti(tLaOQt)iweosn0.115mg a.i.L. 000819 AMENDED PROJECTNO: 454A-105 "16 Tabe2 Temperate,DissolvedOsygenand pH ofWieinteTest Chambers _ TToesh FG 705 a TE DToe oOCpgoiemno_ WF eVerM_ r a,Uniom cm omplain Tw o=mn ms = wm TaneiCtoi) pleBAseo4mC8O 5oogwy gi een) pu M Ysaw o awm oz EA = ome 6 aon we mo 5 Asomms oowus oYws odasoow wwo ow7 8wo 0a= ommeoa1 rlom n APomma owus oYw owoe o=w anowz swoooao womowwomY n Apmosm oowus oYsYs o8m oo oa m wFC d wo a oowm mow om- a ABmosE aows oTss onse aD auw2 2 wo0o momeouwom- B A mmows4s o wsG4 u YI w wBooo masoaos a7e 2 Kmpimdcoepaencostsrtaoy5omgfeeek sgeGOGompiimnoa2yC1.a01k2e15rC: To 2 itsntv ltabe. 222 I8] AMENDED W_I--LDLIFE INTERNATIONAL Lo. a7 PRPORIOJCETCTNNOO.::44S544AAr-1l005s Table 3 `Hardness, Alkalinity and ConductivityofWater in the Test Chambers TTpest Substance: DTielsuttOirongaWnaitsemr:: PWROSea WFreelslhwWaatteerrMussel,Unio complamatus `C(omncgeanitrlalt)ion Replicate, Neegaattive Control BA 53 BA 2 AB 7 AB _--m--m--m--m (HToiumrse) %0 %o 9%o 00 (mHgalLrdesnCesasCO) 113420 131366 131464 141346 (mpAllkaalsinCiatbo 117742 171744 117784 117768 C(ounmdbuocst/ievmi) 333000 333000 334200 335400 000821 AMENDED [T meoo nrs te anondGe n copes 1. Ties CamePercent MoralityadTres.Related Eft SS -- CTT tei Dw ppftm EN EE pu pe teow me Meow few ETA E PROJECTNO: 4540-105 oom ime Me TE Bl Cinsami 33e @88 bh 3 Pw PY Sim AMENDED --WI-- LDLIFE ICNT TERNNAATTIIOONANLALLm. 194 PRPOREOJCETCNTON:O4.:si45A4dAl-1o0s5 Table 5 LCS0 Values Sponsor. 3M Corporation `Test Substance: PFOS Test Organism: ~~ Freshwater Mussel Unio complamatus Dilution Water: Well Water Time LCs0 (mg a.i./L) "Tower 95% Confidence Limits (mg ai/L) 24 Hours >79 - Upper 95% Confidence Limits (mg a.i/L) - Statistical Method Visual Interpretation 48 Hours. >79 4 - Visual Interpretation 72 Hours' 79 >41 - `Binomial os __ SL 68 Prob 96 Hours 59 51 68 Probit Confidencelimits could notbecalculated withthemortality dataobtained. *TheusefulnessofthisLCS0isquestionablebecause aconcentration-effect relationshipwasnotdemonstrated over areasonable range (c.g, <3 to >7 63) ofpercent dead. - 000823 AMENDED WILDLIFE INTERNATIONAL Lm. -20- Figure 1. Concentration-Response Curve (96-Hour Data) PROJECT NO.: 454A-105 7 6 5 25 3 <4 25 5 =3 2 1 1 Slope = 10 . . - 10 100 Mean Measured Concentration (mg a.i./L) 0cosz4 AMENDED WILDLIFE INTERNATIONAL Lo. "21 PROJECT NO.: 454-105 APPENDIXI --_-- `Specific Conductance, Hardness, Alkalinity and pHof Well Water Measured `During the 4-Week Period Immediately Preceding the Test "Sponsor: `Test Substance: `Test Organism: "3M Corporation| PFOS Freshwater Mussel Unio complamatus Dilution Water: Well Water -- `Spec(iufuincbCoosn/deumc)tance ve R21IN=4) Res 310-330 Hard(nmeg/sLsas CaC0:) 126 (N=4) 120-132 Alalinity 174(N=4) 170-178 (mg/L as CaC0;) o pH 83(Ne =4) 81-85 000825 WWIILLDDLLIIFFEEIINNTTEERRNNAATTIIOONNAALLLLoo.. 2 PRPOROFJEECCTTNNOO.;: 44s5a4An-i1o05s APPENDIX AnaolfyPessticeidess,Organics, Metals andOtherInorganics _-m_i vaw atvosesvmbcoMEAvSUREcDCom nCaTm RATIm ON oy in Wildlife International Ltd. Well Water" a{n TR foa [SoE3y -- BBEE, EEetne.n FLmn. BuKaahyeel 5RiHs . = ia Gromer? =CCooptper YLaeagd i?; WR Dsoowm oBmm PDPooooEEm PDooomm = DDooomm = CioHE 8 B o o os s e8s=&%& Boom=Em P E m EB mOB < 20 aL <PI om20100 hHoLn "< 1i0s0 : 030 felAl or E= e Potassiung EE m EE662 Emg ee on 000826 WILDLIFE INTERNATIONAL Lm. oe APPENDIX Ill PROJECT NO.: 454A-105 `THE ANALYSIS OF PFOS IN FRESHWATER IN SUPPORT OF `WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-105 000827 WILDLIFE INTERNATIONAL Lm. 24 PROJECT NO. 4544-105 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static Acute Toxicity Test with the Freshwater Mussel `WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-105 PRINCIPALINVESTIGATOR: 4 /.l JUL " Scieatistd. Van Hoven, Ph.D. MANAGEMENT: WMialnlaagredrB,.ANnailxyotni,cail C)hernistry - 4-93-00 DATE oar 000828 AMENDED WILDLIFE INTERNATIONAL Lo. -25- PROJECTNO.: 454A-105 Introduction Freshwatersampleswerecollectedfromanacutetoxicitystudydesignedtodeterminetheeffects of PFOS (PerfluorooctaneSulfonicAcidPotassiumSalt)tothefreshwatermussel (Uniocomplamatus). This Studywasconductedby WildlifeIntemationalLtd.andideatified asProjectNo.: 454A-10S.Theanalyses ofthesewatersampleswereperformed at WildlifeIntemationalLtd.usinghigh performalinqcuied chromatographywithmassspectrometricdetection (HPLC/MS). Sampleswerereceivedforanalysison May 28, 30and June 1,1999. Sampleswereanalyonzeeacdhsamplereceiptday. `S Testubsant d Ina ternn alStc ande ard Thetestsubstanceusedfortheanalyticalportionofthisstudywas WildiifeInternationalLtd. ideatificationnumber4675. Thetestsubstancewasused10preparecalibration standardsand matrix fortificationsamples. `Theintemal standardwasreceivedfrom 3MCorporationon July2, 1998andwasassigned Wildlife InternationalLtd. identificationnumber 4526uponreceipt. Thestnandat rd,ae granr ularmnatea ria,l was identified as: 1H, 1H, 2H,2H Perfluorooctane Sulfonic Acid,ChemicalAbstract Number: 27619-97-2. "Thestandardwasstoredunderambieatconditions. AnalyticalMethod `Themethodusedfortheanalysisofthefreshwater sampleswasdevelopedatWidiifeIntemational Ltd. and entitled "Analytical Method Validationforthe Determinationof PFOS in Freshwater, Saltwater, andAlgalMedia".ThismethodologywasincludedasAppendix IIofWildifeInternationalLtd.protocol `number 454/01 1299/MVALISUB4S4.Itwasbaseduponmethodporolviodegdbyy 3M Corporation. Samples were diluted in a 50% methanol: 50% NANOpure water solution containing 0.mg14H0PF0OS (intemal standard)/L. and 0.05% formic acid (v4)sothatthey fll within the calibration rangeofthe PFOS methodology. Conceatrationsof PFOSinthestandardsandsamplesweredetermined by reverse-phase high performanceliquidchromatographyusing aHewlett-PacMkoadredl 1100HighPerformaLniqcuied Chromatograph (HPLC) with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin- 000829 W--I_L--DL--IFE INTERNATIONAL rLor. -26- PRPROOEJCECTTNNOO.::44s54AAl-1005s Elmer TurbolonSpray ion source. HPLC separations were achieved using a Keystone Betasil Cp analyticalcoluma (100mm x 2mm 1D, 3 pumparticle ize). Theinstrumentparametersaresummarized in Table1. A methodflowchartisprovidedinFigure . CalCi urvb eanr d Lia mitotfQi uanto itatnion Calibrationstandardsof PFOSpreparedin a 50%methanol : 50% NANOpurewatersolution containing0.100mg 4H PFOS (internalstandard) and0.05%formicacid(v/v),ranginginconcentration from 0.00229 to 0.0274 mga:/L wereanalyzedwiththe samples. Linear regression equations were `generatedusingpeakarearesponse ratios(PFOS:internalstandard)versustherespectiveconcentration ratios(PFOS :internalstandard)ofthecalibrationstandards. Atypicalcalibrationcurveispreseatedin Figur2e. TheconcentrationofPOSinthesamples wasdeterminedby substitutingthepeakarea r`easndphoingshecraaltiibroastiinotnotshtaenadpaprldiscaarbelperleisneeaatrerdeignreFsisgiuorneesqu3aatnido4n.,reRspecetivpelyr. eisonechrnomtatoagrtamsiofvloew Themethodlimitof quantitation(LOQ)fortheseanalyseswassetat0.115mga.i.[L calculatedasthe productofthe lowest calibration standard analyzed. (0.00229 mg a.i/L) and the dilution factorof the matrix blank samples (50.0). MaFtroixrBltankianfd icatSiamoplnes `Thrematrixblanksamples wereanalyzedtodeterminepossibleinterference. Nointerferenwceerse observedatorabovetheLOQduringsamplesanalyses(Table2). Arepresentativechromatogram of a `matrixblank is preseinaFitguree5d. Freshwaterwasfortifiedat0.915, 45.7and 110mga.i/Landanalyzedconcurrentlywiththesamples todeterminethe meanproceduralrecovery(Table3). Sampleconcewnertenrotacortreciteodfonrtshe meanprocreecodveruyorf94a.7l%. Arepresentativechromatogramof amatrixfortification ispresented inFigur6e, 000830 AMENDED WILDLIFE INTERNATIONAL Lm. 2 PROJECT NO.: 454A-105 `ExampleCalculations Sample number 454A-105-16, nominal concentration of 5.7 mg a../L in freshwater. Initial Volume: 0.100 mL Final Volume: 25.0 mL Dilution Factor: 250 POS Peak Area: 596872 Internal Standard PeakArea: 401298 Peak Arca Ratio: 1.4874 Calibration curve equation. Slope: 6.30785 Intercept: 0.04889 PFOS (ug ail) atinstument. : = (--Peakarea ratio-(Y-inStloeper.cexp1tS). Concentration (14874 - 0.04889) x 100 pg ai/L 630785 = mBgain Note: LS.= internal standard. PROS(nani (mg iL) insample: PFOS(ug 2.1L)abtienstxrDiulutmioenFancttor 238x250 T1000 = 570mgail 000821 AMENDED a Pent of NominalConcentration. = rPPFFsOOSS(nBgA1Li1L/))Li)nnnoamsipamepl,e Calculatedrecovery: 98.9% `Note: manualcalculmaatydiifofenr. RESULTS `SampleAnalysis Freshwatersampleswerecollectedfromtheacutetoxicitystudywiththefreshwater mussel (Unio complamatus) at test initiation, May 28, 1999 (Hour 0), on May 30, 1999 (Hour48), and at test `termination, June 1, 1999(Hour 96). Themeasuredconcentrations of PFOS inthesamples collectedat initiationofexposure ofthetest organisms (Hour 0) rangedfrom 73.7 to 96.0% ofthenominal `concentrations. `Samplescollected at Hour 48had ameasuredconcentration rangeof 81.2 to 98.9% of `nominalvalues. Samplescollectedattesttermination(Hour96)had ameasuredconcentrationrangeof 88.5 to 130%ofnom vali uesn (Taa blel 4). Arepresentative chromatogramofatestsample is shownin Figure 7. : i 000822 AMENDED WILDLIFE INTERNATIONAL Lo. 29 PROJECT NO.: 454A-105 Table 1 INSTRUMENT: `Typical LC/MS Operational Parameters Howlet-PackardModel 1100FighPerformaLinqcuied Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin-Elmer TurbolonSpray ion source. Operated in selective ion monitoring mode (STM). ANALYTICAL COLUMN: Keystone Betasil Cis column (100 mm x 2 mm LD, 3 pum particle size) OVEN TEMPERATURE: 30C STOP TIME: 10.0 minutes FLOW RATE: 0220 mUminute MOBILE PHASE: 72.0% Methanol : 28.0% NANOpure Watercontaining0.1% Formic Add INJECTION VOLUME: 2504 PFOS RETENTION TIME: Approximately 6.4 minutes INTERNAL STANDARD RETENTION TIME: Approximately 4.4 minutes PFOS MONITORED MASS: 498.6 amu INTERNAL STANDARD MONITORED MASS: 426.7 amu 000833 A WILDLIFE INTERNAO TIOA NAL Lm. "30 |FPRROOKJEECCTTNNOO.:: a45s4Aa-105 Table 2 A Matrix Blanks Analyzed Concurrently During Sample Analysis (`4N5u4mA-b1e0r5) MAB4 Sample Type "Matrix Blank Measured Concentration of (mPgFaOiS/'L) <LOQ MAB2 ~ Matrix Blank <10Q MAB-3 Matrix Blank <LOQ "The limit of quantita(tLiOoQ)n was 0.115`mga.i./Lbaseduponthe productof the lowestcalibration. standard analyzed (0.00229mgai/L) and thedilutionfactorofthematrixblank samples (50.0). 000834 AMENDED i WO ILDLIFE IdNTEiR RNaATlIaOnNAtL Lo... PROJEC. TLNsO..:.4.54A-105SO 31. Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis TT --rt Sa(m4p5l4eA-) N1u05m-b)er MAS-10 MAS4 MAS-7 ConcemiratiomofPFOS FFoouritiffiieedd 0915 (mgai/L) MMeeaswureeds. 0.780 ~ 0915 0.952 0915 0.858 Percent. ReRceocovveerresd 852 104 938 MAS-11 457 437 95.7 MAS-5 457 435 95.0 MAS-8 45.7 42.7 93.4 MAS-12 110 MAS-6 110 --MMAASS-9? m11e0 942 855 3 103 1w0e6 696.77 Mean =94.7 _ Standard Deviation = 6.50 CV =6386 N=29 Note: Resultsandcorrectionsfornewtestsubstancepurityweregeneratedusing MacQuanversion 1.5 softwareand manual calculations. Valueshavebeen rounded for reporting purposes. 000835 AMENDED WILDLIFE INTERNATIONAL Lm. 22. PROJECTNO: 454A-105 Tabled Measured ConFcreensthrwaattieornsMoufssPeFlOAScuitneFTroexsihcwiattyerTeSsatmples from a -- e CToonmc(emmngatarliaftT)ieorn (45SN4euAmm:pb1le0er5e ) EToiumrse) RCoe (SnmcegnMatierlasltui)rone' NFomeoiefntal "12 0 Io a <<1100Q0 <10Q E -= 251 %as 2 % p<1r0yQ <10Q -= 57 i3 00 15 8 sasas 984595 sis 13 216 98% 2 9% 557204 526 550899 512 n ss a 00 . n10a1 8 112 354603 a1 1 9a% 30 9% 010s5 8123 154 130 175 a00 119608 8537 187 83 23 a% 2 9% 21897 s1i0s0 24 580 a 150 00 21 a 031s2 88348 371 52 23 9I%] Fl % 38925 61045 "0s 8s 9 3uun 800 7407 755146 813 589 23 a% 36 56 872s 394684 557 537 oThe el`immaisntuoafl cqEaluTelSaaetnrieotcn.ii(oVLnaasOlTtQuo)eiswhnoaaesvnwe0b.1e81e5nmUgrSouaSnd1RedbpfaoasrenrdeyupopWroetnirnteghGpueppirocsoiedseu.dctwointhgelMoawecsucanlivberratsiioonnst1a3ndsartdaanraelyaznedd (0190229 ng 1)andthlution ctoofthemtx lank samples (50.0) AMENDED 000836 WILDLIFE INTERNATIONAL Lt. eee 3. PPRROOJTEECCTTNNOO.:: 445848A0-010055 METHOD OUTLIINNEFRFEORSHTWHAETAENRALYSIS OF PFOS Preparematrixinftorotfirfeiscahtwiaotnesramupsliensggbayss-ptiikgihntgsyrhiengreeqsuainsidtCelvaoslsumAevoofluPmFeOtSrisctfolacskksso.lutionsdircetly v 4 DiluteCmlaatsrsiAxfvortioficaltionufalnasdmktsewseittshat5mp0l%remsietinhoatnchoelr:an5g0e%ofNtheA calibNratiO ownatsep trasnodlu aurtdisbornycopancrttaiianlilnygfilling `0s.a1m0p0lmeagnd4HbriPnFgOtShef(ilnatsekmatlo vsolutme waithantnhd0ddi.l0ut5ai%ofn osrromlviecdnatc.iyPdr(ovLc/ve)s.sAtdhedmtahteraixpblpanrkosapmvprollieuuasmietnogef thesamedilutionandaliquotvolumeasforth-- e lowestfortification level. Mixwellbyseverrepaealt v . Ampulatesamplesandsubmitfor LC/MSanalysis. Figur1e. Analyticalmethodflowchartfortheanalysisof PFOSinfreshwater 000837 WILDLIFE INTERNATIONAL Lm. EY PROJECT NO.: 454A-105 2.00 o 1.50 : us 030 0.000000 0050 0100 015 0200 0250 0300 Concentration (Ratio) Figure2. Atypical calibcurrvaeftoriPFoOSn. Slop=e 6.30785; Inte=r0c.0e48p89t; r = 0.9986. 000838 AMENDED WILDLIFE INTERNATIONAL Lm. "35. PROJECTNO.: 454A-105 : 10 152 Itans63t51ycp:s s 8 To 5 } . . ] . 2 132 1 Po fe Sh 1W31h 5W1R 1W81y 2Gi1s | TSamn Figur3e. Arepresentativeionchromatogramofalow-level (0.00229mga.i/L)PFOSstandard. 000829 AMENDED WILDLI= FE TINTT-ERRPNAATTIIOONNAALLmLr.. "36. PPRROOJJEECCTTNNOO.::ds454AA-d1o0s5 100 152 Intensity:50611 cps. a a 7 . o . . 4 3 133 % 2 + Tf 44 50 GG 82 8Sr 161 Ww 181 220i81 | San Figured. Arepresentativeionchromatogramof ahigh-level (0.0274mga.i/L) PFOSstandard. 000840 AMENDED WILDLIFE INTERNATIONAL Lm. -37- PROJECTNO.: 454A-105 100--2 90 80 " Iotan1s18tcy. p:s : 152 . 60 50 40 127 2j0ie i a ais i FT ANl A A IT "lll i 10 Iii bf FJN% 2%REsmRIE5NR0Es 6sTARE7TH&ERE7 eTRsI s Tme FigureS. Arepresentativechromatogramof amatrix blank sample (454A-105-MAB-2). "Thearrowindicatesth reteationtimeofPFOS. 000841 T WILDLIrE F=E INR TERNAN TIONAA L Lo. TIONPPRRA OOEJECCTTNNOOL .::d4s54AAd-. l10o5s 3 To o a 7 s 5 4 3 2 1 9_25S1T 42 2T6e:l5 152 138 o3Tm 1sAsIos 1661x Intonsty: 42545cp.s + 17816 1982261111"s | TSaen Figur6e. (Ar4e5pr4eAse-n1ta0t5iv-eMc4Ah)Sr.omatogramof a 1.00mg a.i./L matrix fortification sample 000842 WILDLIFE INTERNATIONAL Lm. "39. PROJECTNO.: 454A-105 10 9 8 7 5 - . 4 174 Intonsty:36233cps B : jo 151 20 jt ff 41 th 06 WW T1E28 181 4181 0! 2WWSan Figure7. Arepresentative chromatogramof a6.3mga.i./Ltestsample (454A-105-16). 000843 WILDLIFE INTERNATIONAL Lo. -40- PROJECTNO.: 454A-105 APPENDIIXV ChatnoPgroteocsol `Thisstudywasconductedinaccordancewiththe approvedProtocolwiththefollowingchanges: 1. Theprotocol wasamendedto addtheproposedexperimentalstartandterminationdates,test `conceanndtetstrsuabsttanicenoumnbesr. 2. Theprotocol wasamendd torenew thetestsolutionsat 48hours. 3. The protocolwasamendedtospecify that GLPanalysesofwellwaterwillbereported. 4. Theprotocolwasamendedtoreducetheloading rate. 5. Theweightandlengthofthemussels wasdeterminedbyweighingandmeasuring 20individualsfromthe `same lot of test organisms. 6. Themussels wereindiscriminatelydistributedtothetestchambers. 000844 WILDLIFE INTERNATIONAL Lm. ae PROJECT NO: 454A-105 APPENDIVXI Report Amendment 1 OriginalReport: Pa1-4g ,6e ands 24. Amendment: T`nheewspiaggneastwuererseacnhdadnagteedstdoiuenctloutdheetahdedaitmieonndoefdtrheerpoerptodrattaem,ernedvimseendptaagseAnpupmebnerdsi,xVaIn.d Reason: To reflectthe issuing of an amended report. 2. OriginalReport: Page2 Amendment: "Thecompliancestatementwasrevised. Reason: Toclarifyhowthetestsubstancewas characterized. 3. OriginalReport: Page `Amendment: Information provided by the Sponsor reflecting the reanalysis of the test substance, includingthereanalysisdateandthepurity, wasadded totheTestSubstancesection. Reason: "Toreflectthecurrenttestsubstanceinformationprovidedbythe Sponsor. 4. OriginalReport: Entirereport `Amendment: All est substanceconcentrations werechangedto reflectthepurityof thetestsubstance `asdeterminedbytheSponsor in a reanalysisofthetestsubstance(FC-95, Lot 217). TestcocnacteeontfraantailoynssiosrdiagtiendaMllayrwcehr9e,b2a0s0e0 dinodnictahteedrea ppourritteydopfur9i0t.y4o9%f.`T9h8e.r9ef%o.rTe,haell certifi fest substance concentrations, including nominal concentrations, `measured conceatrations,and LCS0values,wererecalculatedand reportedas mg .i./Lbasedon. the90.49%purity. Reason: Toreporttheresultsofthetestbasedonthetest substancepurityof90.49%atthe reqoftuheeSposnsotr. 000845 AMENDED WILDLIFE INTERNATIONAL Lm. a3. PROJECT NO.: 454A-105 APPENDIX VI ~ContinuedReport Amendment AMENDMENT SIGNA' SKutrutdy Drotar < DHiernerctyor, AO7qKurautegiecr,ToxiPch.Do. logy andNon-Target Plants 4/26/00 DATE -- BKoh foo REVIEWED BY: Timothy5ofSpringer, P=RD. Quality Assurance DATE+ [2/00 : 000846 AMENDED WILDLIFE INTERNATIONAL LTD. PROJECT NO: 454A-105 Page 1 of2 AMENDMENT TO STUDY PROTOCOL u STUDY TITLE: FPRFEOSS:HWAAT9E-RHMOUUSRSSETLATIC ACUTE TOXICITY TEST WITH THE H PROTOCOL NO.: 454/042199MUSISUB454 AMENDMENT NO: 1 = SPONSOR: 3M Corporation PROJECT NO.: 454A-105 EFFECTIVE DATE: May 20, 1999 m AMENDMENT: Page 2 mo Add: EExxppeerriimmeennttaall TSetarrmtinDaattei:on5D/a2t8e:/996/1/99 Test Concentrations: Negative Control, 63, 13, 25, 50 and 100 mg 2i/L Test Substance No.: 4675 = REASON: The above information was not known when the protocol was signed by the Study Director. AMENDMENT: EXPERIMENTAL DESIGN, Page3 = `Add: aTensdttshoelumtuisosneslwsilwliblebreenterrawnesdfaetrraepdprforxoimmattheeloyl4d8thooturhse.neNwetwesttesstolsuotliuotnis.ons will be prepared = REASON: Test solutions vil be renewed at 48 hours to improve the water quality. u AMENDMENT: Dilution Water, Page 5 HE Change: ASenlaelcytseedsowriglalnbiec paenrdfoirnomregdanaitclecaosntsotintcueenatnsnuoaflltyhetowedleltewramtienreatnhde ctohnecreensturlattsioonfsthoef H analyses will be summarized in the inal report. To: AorngaalnyiscesawnidlibneorpgearnfiocrcmoendstaittlueeansttsoonftcheeanwneualllwyattoerdeatnedrmtiheneretshueltcsoonfctehnteramtoisontsorefcesnetleGctLePd H compliant analyses will be summarized in the final report . REASON: To specify that GLPanalyseswill be reported. H 000847 : Za asatosement H WILDLIFE INTERNATIONAL 11. PPaRgOeJ2EoCfT2NO: 4544-105 AMENDMENT:TestApparatus, Page 5 Ho Change: Tweastterchambers will be 25-L, polyethylene aquaria filed with approximately 15 L of Ho To: Test chambers will be 25-L, polyethylene aquaria filled with approximately 20 L of water. u REASON: To reduce the loading rate. acai, \ ml STUDY DIRECTOR H - l Fr = LABORATORY MANAGEMENT wo Hoa A SPONSOR'S REPRESENTATIVE =/27]79 DATE - 1/97/74 DATE Lliglea ba o Be. 000848 I [| WILDLIFE INETERRNATIOa NAL um. eee PROJECT NO, 4544-105 Page Lof1eee I) DEVIATION TO STUDY PROTOCOL = STUDY TITLE: PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE FRESHWATER MUSSEL PROTOCOL NO.: 454/042199/MUS/SUB454 DEVIATION NO.: 1 Ho SPONSOR: 3M Corporation PROJECT NO.: 454-105 u e-- DeATE--OF--DEF-- ACTO--DEV--IAT-- ION:--June--1, 19--99 ---------------- representative mussels fom th sam ot were measured DEVIATION: `The protocol states that valve heights and weightsofthe individual mussels will |] bemeasured attheendofthetest. The valveheightsandweightsof 20 weighedandmeasuredwerindisrminatlyslciedfrom th Sam lot of es organisms. |] HH REASON: Biologistoversight.The mussels usedinthe test were shippedtothe Sponsor before they `wereweiag ndh meae surd ed. The musselsusedinthetestandthe musselswhichwere Consequently, it is thebest judgmoefnthte Study Director that this deviation did not `adversely affect the resultsofthe study. = AN u STUDY DIRECTOR H Vi / oo LABORATORY MANAGEMENT =I DATE &Li/r DATE un 000849 . PROJECT NO.: 454A-105 I e WILDLIFE INTERNAATOIONNAAe L TLTDD. Pe aoge leofeens ml DEVIATION TO STUDY PROTOCOL ml STUDY TITLE: PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE FRESHWATER MUSSEL PROTOCOL NO.: 454/042199/MUS/SUB454 SPONSOR: 3M Corporation DEVIATION NO.: 2 PROJECT NO.: 454A-105 DATE OF eer--------e-- DEFACTO DEVIATION: May 28, 1999 DEVIATION: Tashseigpnreodtotcoolexstpaotseusrethacthatmobceornstroalt biss, freshwater mussels vill tos initiation. The musscls be randomly were actally mussels) indiscriminately distributed (a random number table was not used to distribute the REASON: Biologist oversight. Itis thebest judgmentofthe Study Director that this deviation did `not adversely affect the resultsofthe study. ERE mi STUDY DIRECTOR DATE LABORATORY MANAGEMENT - reno DATE = 000850 mW wm |] mu om PROTOCOL PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST `WITH THE FRESHWATER MUSSEL SerieUs.S8.5E0n-vEicroolnomgeinctaallEProtfecTtfieosntAcGguiednteclyisnes `OPPTS Number 850.1075 and (OECD Guideline 203 3M Lab Request No. U2723 i Submitted to u 3M Corporation Environmental Laboratory uo 9`B3u5ilBduinsgh2A-v3e6n-0u9e u WiLpLiFe St Paul, Mimesota 55144 INTERNATIONAL Ltp. 3 un pe 8598 Commerce Dive a `Easton, Maryland 21601 nu, April 21,1999 000851 Ho LWYIILLDDLLIIFFEENITNETRENRANATTIIOONNAALL,,0LTD u - PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST Ho `WITH THE FRESHWATER MUSSEL mW `SPONSOR: mu 3M Corporation Environmental Laboratory 9Bu3i5ldBiunsgh2A-v3eEn-0u9e St. Paul, Minnesota 55144 `SPONSOR'S REPRESENTATIVE: Ms. Susan A. Beach u TESTING FACILITY: oH `Wildlife International Ltd. 8598 Commerce Drive Easton, Maryland 21601 u STUDY DIRECTOR: SKeunritoDrrAoqtutaarticBilogist LABORATORYMANAGEMENT: Henry O. Krueger, Ph.D. H Directorof Aquatic Toxicology& Non-Target Plants = -- l BR FOR LABORATORY USE ONLY N rose: _USGAJOS ExSparetrDiamteen:ta_ l _____ TExepmeirniamteinotnaDlate: ____________ p TestConcentrations: __ nu Test SubstanceNo.: Reference Substance No. (ifapplicable): ____ Nem L) PROTOCOL APPROVAL | STUDY DIRECTOR color Ba 0 `ORY {AGEMENT . = SPONSOR'S REPRESENTATIVE ak DATE ln DATE 29/99 DATE 000852 PROTOCOLNO. 454/042199/MUS/SUB454 3M LABREQUEST NO. U2723 i . 1 WILDLIFE INTERNATIONAL, LTD "3. a INTRODUCTION i "Wildlife Intemational, Ltd. will conducta static acute toxicity test with the freshwater mussel for the: Sponsor at the Wildlife Intemational, Ltd. aquatic toxicology facility in Easton, Maryland. The study will a beperformedbasedonproceduresinthe U.S.EnvironmentalProtection AgencySeries 850 -Ecological Effects Test Guidelines OPPTS Number 850.1075 (1); OECD Guideline for TestingofChemicals 203: Fish, Acute Tosicity Test (2); and ASTM Standard E729-88a Standard Guidefor Conducting Acute Toxicity m Tests with Fishes, MacroinvertebratesandAmphibians (3). Raw data for all work performed at Wildiife Intemational, Ltd.and acopyofthefinal reportwillbefiledbyprojectnumberinarchiveslocated on the 1] `Wildlife International, Ltd. site,orat analtemativelocationtobe specifiedin the final report. u PURPOSE "The purposeofthis studyistodeterminetheacuteeffectsof atestsubstanceonthefreshwatermussel a during a 96-hour exposure period understatictestconditions. EXPERIMENTAL DESIGN mn Freshwatermusselswillbeexposedto ageometricseriesofatleastfivetestconcentrations and a ~ negative dilution water) controlfor 96 hours. Two reptlestichcambaertswiell be maintained in cach u treatmeatandcontrolgroup,with 10freshwatermusselsineachchamberfor totalof 20freshwatermussels per test concentration. u `Nominal estconcentrationswillbeselectedinconsultationwith he Sponsor,andwillbebasedupon : information such as the results of exploratory range-finding toxicity data, known toxicity data, physi- || callchemicalpropertiesofth test substanceorothrrelevantinformation. Targetconcentranteiedonnost i exceed 1000mg/Lorthesolubilitylimitofthetestsubstanceinwater(whicheverislower). Generally,cach HN testsubstanceconcentrationusedinthedefinitivetestwillbeatleast 60%ofthenexthigherconcentration unless information concerning the concentration-ffect curve indicates that differntdilation factorwould u be more appropriate. Water samples romeach test chamber will be collected at specified intervals for analysisofthetestsubstance. Resultsofanalysesvillbeusedtocalculatemeanmessuredtest = concentrations. . Tocontrol bis,freshwatermusselswillberandomlyassignedtoexposurechambersat est nition. H Nootherpotentialsourcesofbiasareexpected to affecttheresultsof thestudy. Observoaftmoirtoalnitsy and other clinical signs will be made throughout the 96-hour test period. Cumulative percent mortality u 000853 H PROTOCOL NO. 454/042199/MUS/SUB4S4 3MLABREQUESTNO, U2723 mm . - wm WiLbLIFE INTERNATIONAL, 110 u 4 Nu hoobusrerivnteedrvianlts.he Trheaetnmoe-nmtorgtraoluiptsywcilolnbecsenteradtiotnowcialllubeltd,etwerhmeinnepdo,site, CSO values 24,48, 72and 96 n Test Substance MATERIALS AND METHODS X Information Laboratory Practice oStnantdhearcdhsar(aGcLtPe)r.izaTthieonSpooftnseotricroespnonostribrlreefeforoernplcreovsiudbisntgaWncielsdies rIenqtueirrned by Good n sttuedy.nMveiritficeativoenritfhicaathioen eofsGt LsuPbtsetatncseubhsatsabneceenchchaarraacctteerirziezdaiatccimoorndtipng.rtooGvLtPioSWdiplriedordtIontteastmwiaoteniaolonLaield. n OLeFd,TiEt SwiTllSbeUnBoSteTdAiNntCheEcoBmYplSsPnOceNSstOatRem(eAnptpoefntdhixfiinaltroepboert.seTdhe0atparocvhiedfoirnmfIsorDmEatNiTonInFeIceCsAsaTrIy OfoNr, `GLP compliance. i 1 mused"Ttehset Ssubsptancioesraensndploonrsseisbtloesufborsrtaalnl icnontfainoerrsrermleamtaeadittnoitnihgeoattetnhsetseubsnotafntdcheeasntdya,greesto acosptany 1 PrepoafTrestaContcenitraotionns ofadmiTnihsetrtaettionsuwbasstasncewillbe admin1i0thsettteorrgaeniddirec nto dilationwaterThis route locted because it presets the most key routeofexposure to aquatic organisms ! TestOrganism 1 IepresTehnetirvsohf aancirmpmourstsaenlthgarsobupeeonfasqluactticdinvetrhtectbreatess,eacndshfaovretbiesesntuedlye.ctFerdefsohrwautseerimnutshseetlostaor. I he reaestofthe Sponsor. ofthetest. Mussels vill bo Vave height bined rom and weightsofte individual mussels will a commer upp or hitch,adthe be mess denyofthe the end spin will be verified by the supplicr. I | andatFarpepsrhovxaimtaetselmyutshseelsawmielltbeempheerladtfuroeaatsuesaetdd1u4rdiangytphreiteostt.oAtnheytceshtaningewsaitnerwaftreormttehmpsearmatesuoreuwriclel | orRroeitfeemx0ocrteeeetsdth3nainCtp5ieornd.ayFr.eFsrhewsahtweatreursmsueslsewlislwlioltlbbeehueslddaitntthheetteessttitemepeyrsathuorwe sfioganrsmoifdniisemaasoofrs7 rdagys collected fromhol%didinegdouraicncgltihmeat7idoanytsapnrkisoarndottrhaentsefsetr,reAdtttoetshteintietsitatcihoan,mtbheerfsreshAwtatteesrttmeursmsicnlastviiolnlablel PROTOCOL NO, 454/042199/MUS/SUB4S4 000854 3MLABREQUENSOT. U272 WILDLIFE INTERNATIONAL, LTD "5. o remaining live composited by mussels will be replicate, fozen sacrificed for collection of tissu samples. The and shipped 10 the Sponsor orpossible analysis tissu samples will be N Dilution Water N appro`xWimaatteerluys4e0dmfeoterrtshdeohopldloicnagtaednodntetshteiWngiolfdfereisthewmaattieornmauls,seLilds.wsitle.lbTehoebtwaaitneerdvfirlobme paawsseeldl ntohzrzoluegsh.aPrsiaonrdtoiuesretahnedwpautemrpweidllibneofail3te7r,e8d 0100..4st5oraamgeitaonrkdewrhteorreetmhoevweaftieepwairltibceleasr,atWeadtewriutshesdfpoary N culturing and testing specific conductance is characterizedas are approximately: moderatelyhard. Typical valuesfor ardacss, alkalinity, pH and N AHlalradlnienistsy,,mmgg/L/.LaassCCaaCCOO,, 11%450 N pH Specific Conductance, samhos/em 81 330 n + oftheeHlalrdwnaetsesr,.aMlkeaalinnsitayn, dpHraanngdesspoefctihfiecmceoansduurcetdanpcaerwaimleltbeermefoarsuthreefdouwre-ewkeleyktopemroniiotdoprrehsedcionngsitshteetnecsyt 1 cwoinlclenbterpatironsoofisvneltechiteefdiodnraglaerneipdoarntd.iAnnoarlgyasneicwciolnlsbtietpceatrsfoofrmtheedweatllelawsattornacnedarnensuualtlsloyftohdeeatnearlmyisneestwhilel 1 be summarized in the final report. TestApparatus 1 chamberTseswtilclhbaembpeosristivoinledbien 2a5n-eLn,vpiorloynemtehnytlaelnechaaqmubaeriraofriltleemdpweirtahtuarpep-roonxtiromlalteedlywa1t5erLobafthwattoemra.inTteaisnt 1 & temperatureof22 replicate. + 2C.Testchambers will be labelled with th project number, test concentration and 1 `EnvironmentalConditions I beproviLdigehdtbinyguhsordetsoeinltumtuibneastetthaht cuiltturweasvaenldentgetshtcshsaimmbilearrstdourniantgurahlosludnilni,gahctclei.m5at,iCoonl,aotnodnecst5i0ng).ilAl . I tprhaontsoipteiornipodeiofod16ohfoulorwsiofghitignhtteannsdit8ywhoiulrslboefdpraorvkiwdiedlwbheceonnltrioglhltesd80wtonhanndauotf omtoaatvotiidmesru.ddAen30c-hmainnguetse | oinreliigvhtailnetenntsity. Light intensity willbe messured at est ntatonwith SPER Scientific Li. ight meter000855 | PROTOCOL NO. 454/042199/MUS/SUB454 3M LABREQUESTNO. U2723 | wm W_ ILDLIFE INTERNATIONA-- L, LT0 -- =o -6- m beginniTnhgeantadrgeentdtoeftttheemtpeesrtautsuirnegwailllqubied2i2n-+gl2asCs. thTeermmpoemreatetru.reTweimlplebreatmueraesuarlseodwiinllablerempelaiscautreesdatwitthhe mn inc-ognltaissnotuhesrrmeocmoertdeerripnriaobretaokteerstadijniatcieatnitono. the test. Recordermeasuvilrlbeveerifmiedewitnhatliqusid iW initiatiDonisasnodlvatedapopxryogxeimnawtielllyb2e4-mheoausruirnetdeirvalsaltl hreerpelaifcearteussoifngtaheYetlrleaotwmSepnrtiangnsd IcnosnttroulmegnrtoMuopdselat St1e8s.t mn daipspsroolpvreidaotexyacgteinomnsetweirl,lobreetqaukievnalaefnttr. coInntshueleavteonnt wthiatthtdhiessSoplovnesdoorx.ygMeenalseuvreelmsefnltlsobeflopwH6w0il%lsbaetmuraadteioni,n mn ianltteermvaatlsitngheraeptleicratessiongf.thFeisthreearAtmcecnutmacntdMocodnterol91g5rpoHupsmat etesotreienqiutirivaatiloe,nnta.ndIfaattapprroeximtagtremoluype2r4en-ahchoteusr n i1nt0e0rv%alm,ortthaelnitdyi,scdoinstisnoulevde. oxygen, pH, and temperature measurements will be taken at the net sampling n end higHhartdentesssu,bsaltkaanlcineitcyo,nacenndtsrpaetciiofniscaontdeuscttnatnacteiwoinl abnedmeaattseusrtetdeirnmtihneatdiionl.utioHnawradtneersasnadnodwa,lkmaildindiltey n EmxeaamsiunraetmieonntosfwWilaltbeermaanddeWbaystteawtaiteorn(u8s.inSgppecriofciecdcuornedubcatsaendceonwimletbheomdesssiunrSetdaundsairndaMeYetlhloodwsSfoirngthse n Instrument Model 33Salinity-Conductivity-Temperature meter, orequivalent. BiologicalMeasurements 1 8.2O2d7b96hs2 owesro+ f1vmhooraura.ltiLteyidnodclniinsicsdaelfsiingendsstofhtolxaicciktyofwviilsibbelemmaodveembeetnwteeeng0o-p24enhoourgsa,painndgaste2l4l, | in the musselaftergentleprodding. Al linical observations willbe noted. nl `SamplingforAnalyticalMeasurements 1 andattWheatceardsoafmtphleetsewtitloldbeetceorlmliencetecdofnrceonmircaatcihonessotfcthhamtbeestrsautbtshteabnecgei.Innntihnegeovfetnhte thesatt,d1u0r0i%ngmtohrealteistty, Secmuprslienwainlylbtreecaotlmleencttetdhaetnmsaimdp-ldiengptofftohmactraecshttmeesnttcwhialmbteerrmainnadteanfaolllyozweidnigmtmheedieaxtteslaymoplpilnagciendtienrvaanl 1 i 2ppropristestoragecontainer(cg,polyethyleneor polypropylen anallyzed. Theo samplesample scishsummearim zedbeelow bot)andstoredunder refrigerationunl. 000856 1 PROTOCOL NO. 454/042199/MUS/SUBA454 MLABREQUEST NO, U2723  W-- ILDL_ IFEn Ir NTERNATIONAL, e LD e, 1 7- ----PR-- OPOS-- EDNNUUF MMBBEERRR SSOOFFVO VEERRIIFFF IICCAATTIIO OONNSSAAS MMPPLLEEE SS D N --_-- Experir mental Gp roup. OfOHoowwss HoHowawss HoH9o%wwss n Control 2 2 2 i Level I-Low Concentration Level2 2 2 2 2 2 2 Level 3 i Level 4 2 2 2 2 2 2 Level 5-High Concentration 2 2 2 ] B --T_o--tals B 2 n 12 1 Total NumberofVerification Sample=s 36. 1 controTl h(QeC)absoavmepnlesusumchboafsemsaatmrrpilxsesblarnepkrseasnendtftorhtoisfeiccaotliloencstperdferpoarmetdhaentdeasntaalnydzdedodnuroitnigntchleudaenaqluytailciatly 1 chemistry phaseofthe study. appropriate test chambers will At be ctohleledcitsecdreatnidonoanfatlhyzeedStifuadynDainraelcyttoirc,awlaeteorrsianmpslaemsplfirnogmoronaenaolrysmiosrei.s |] insutshpeecrtaedw.daTthaearnedafsionnaflorerptohreta.dditionalsampleswill bedescribedbytheStudyDirectoranddocumented 1 AnalyticalChemistry methodu(CsheemdiwcialllbaneablyassiesdofutphoenmseatmhpoldeoslwoiglypbreovpiedrfeodrbmyetdhbeySWpiolndsloifreanIdntiedmeanttiiofnieadl,inLidA.ppTehndeiaxna1l.ytiTchael I `methodologyusedtoanalyzethetestsampleswill bedocumentedintherawdataandsummarizedinthe inal report. 1 `Data Analysis `Whentheconcentration-responsepattern allowscalculationofanLCS0value,thedatawillbe avnaalluyezaenddustihnegt9h5%cocmopnuftiedesnocfetiwnatreerovfalCbEy.pSrtoebpihtanan(a5l).ysTish,etphreogmroavmiwnagsadveesriaggneemdteotchaoldcu,loatrebtihneoLmCiSa0l || mproorbtaabliiltiytdywatiatchonlolnelcitneedaarti2n4t,er4p8o,l7at2iaonnd(6976,8h)o.urTsh. eTLhCeSOn0ov-maolrutealwiitlylcboenccaelncturlaattieodn,wwilhlebnepdoestseirblmei,neuds.in0g 00857 . PROTOCOLNO, 454/042199/MUS/SUB454 3M LAB REQUEST NO, U2723 um uN W-I_--LDLIFE INTE0RN0AT\I/ON\ALb,n(--10 x 8 N Records obe maintained REC TOBOEMR AIND TAIS NED for data generated by Widife Intemational, Lid. wil inclu, but mot be limited to: N 1. Acoopftyhe signed protocol. n 2. 3. DaMteesonfitnitiiaatfinodnccahnaardatctteeirirzoamtiinonnooaffttthheeitteoesstnt.substanceifprovidbeydthe Sponsor. 4. Test organism history, holding and acclimation records. 1 5. Stock solution calculation and preparation,ifapplicable. 6. Observations. 7. Waterchemistry results (cg. alkalinityand hardness), . a 8 9. TSthaetimsetitchaoldcsalucsuelatitooanns,alifyzaepptlesitcasbulbestancsconcentrations ndth resultsofanalyticalmeasurements, i 10. Test conditions (light intensity, photoperiod, etc), 1 11. Calcualndaprtepairaotinon oftest concentrations. 12. Copyoffinal report 1 FINAL REPORT fina portofth resusofthesud will bepreparedby Ade Inermationl Lid. Thereport vil include, but not be limited to, the folowing, when applicable: i 1. Name andaddress ofth facilityperformingthestudy. | 2 Datesuponwhich termination dtes thestudy was initiatedandcompleted,andthe definitiveexperimental start and 3 A statement of compliance signed Laboratory Practice Standards. by the Study Director addressing any exceptions to Good I 4 3 OTbhjeetsetsitvesusbasntdanpcreocdeedauirfeisc,ataisonsitnatleddinnthgenaampepr,ocvheedmipcroatloacbols,triancctlmuadimnbgelrol choadneg umobtheerpsrioogchol,. | purity, compoasndoithetriinfoormanti,on probvytihedSpoensodr. . | 6. Stability andsolubility Sponsor. ofth tet substance underth conditionsofadministration, ifprovided by the - 7. Adescription ofthemethodsusedtoconductthe est, | # aAnddwesecirgihpttisoonoffrtehperetessetntoagtainviesgmrso,uipnocflutdeisntgortghaenissomursc,ef,esdciteynpteifsi,clnigatni,angeotreaInfendpshasogteio,peltrenigoytdh.s | 9. A descriptionofthepreparationofthe tet solutions 000858 PROTOCOL NO. 454/042199/MUS/SUB454 3MLABREQUESTNO. U2723 i . 1 WILDLIFE INTERNATIONAL, LTD 1 -5- 10. The methods used 0 allocate organisms totes chambers and beginthetest, he numofobrgaenisrms 1 `and chambers per treatment, and the durationofthe test. 11. A descriptionof circumstances that may have affected the quality or integrityofthe data. [] 12. The name of the Study Director and the namesofother scientists, professionals, and supervisory personnel involved in the study. 1 13. Adescription ofthetransformations,calculations,andoperationsperformedonthe data, asummary and analysisofthe biological data and analytical chemistry dats, anda statement ofthe conclusions drawn from the analyses. 1 14. Statisticalmethodsusedtoevaluat thedata. 15. A graphoftheconcentration-mortality curve at the endofth test. i 16. Thesignedanddatedreportsof eachofthe individualscientist or otherprofessinivoolvnedainltshe study. i 17. The location where raw data and final report are tobe stored. 18. Astatementpreparedbythe QualityAssuranceUnitlistingth date thastudyinspectionsandaudits were made and the datesofany findings reported tothe Study Director and Management 1 19. Ifitisnecessarytomakecorrectionsoradditionsto afinalreportafer ithasbeenaccepted,such `changeswillbe madeintheformofanamendmentissuedbytheStudy Director. Theamendmewnitll |] clearlyidentifythepartofthefinalreportthat ibeingamendedandthereasonsfortheamendment, and will be signed by the Study Director. 1 CHANGING OF PROTOCOL i Plannedchangestotheprotocol willbeintheformofwrittenamendmentssignedbytheStudy Director and the Sponsors Representative. Amendments willbeconsidered as partofthe protocol and will be attached to the final protocol. Any other changes will be inthe foofwrrimtin deviations signed by the i Study Director and fled withth raw data. All changestoth protocol will be indicated in the ina report. 1 GOOD LABORATORY PRACTICES `Thisstudywill be conductedinaccordancewithGoodLaboratoryPracticeStandardsforEPA(40 1 CFR Part 160 and/or Part 792); OECD Principles of Good Laboratory Practice (OCDE/GD (92) 32, v Environment Monograph No. 45); and Japan MAFF (59 NohSan, Notification No. 3850, Agricultural Production Bureau). Each study conducted by Wildlife International, Ld. is routinely examined by the 1 `WildlifeInternationa,Lid.QualityAssuranceUnitfor compliancewithGoodLaboratoryPractices,Standard OperatingProceduresandthespeifiod protocol. A staoftcomepliamnceewitnhGotodLaboratory Practices 1 will be preparedforallportionsofthestudyconductedbyWildlifeInternationalLtd.TheSponwsilolbre: 00085 i PROTOCOL NO. 454/042199/MUS/SUB454 3MLABREQUESTNO. U2723 a WILDLIFE INTERNATIONAL, LTD -10- responsible forcompliancewith Good Laboratory Practice for procedures pecformed by othr laboratorics | (e., residue analyses or pathology). Raw data for all work performed at Wildlife International, Lid. and a `copyofthefinalreportwillbefledbyprojectnumberinarchives locatedontheWildlifeInternational, Ltd o site,oratanaltemative location tobespecifiedinthefinalreport. u: Er PROTOCOL NO, 454/042199/MUS/SUB4S4 000860 3MLABREQUEST NO. U2723 5 WILDLIFE INTERNATIONAL, 110 N "11. REFERENCES 1] bn(raf)p,miOvPoPnTmSenNtuamlbePrro8t5e0c.t1i0o7n5A:gFeinschy.Acu1t99e6.ToxSiceiitsy T8e5st0,.FErceoslhogwiactaelrfmfgosneTses,t Guidlines wu 7 oOfrgCahnemiiscaatliso.n fGouriEdecloinnoem2i0c3:CoFoisphe,rAactuitoen aTonrdiDcevelesotp.meAndto.pt1e9d95,byOtFeCCDoGuuniclei3n3 ortTyesstoinnsg Il PAST Fishes MSatcarnodiamredrEt7b2r5a-t8e8,8.an1d99A4m.phiSbtiaanndsa.rdAGmueirdiecfaonrSoCcoinedtuycftoinTgesAtciuntgeaTndoiitaTrests with w * PPpoleHlutAeioinAnCWo,nWtrAoAmlerWFieRdceaCrna.tPiuob1nl9,i8N5c.eHSweaalYntodhrokA.rssoMceiatthioodns. fAormetrhiecEaanmWiantaetroWnoorfWkastAercaondmWaastewmaeterrc. u 3 rStephian, CnE. 1978. US. EPA,EnviromentalResearchLaboratory, Dut, Minoesos. Porson a Thompson, WR. 1947. BacteriologicalReviews. Vol I, No.2. Pp. 15.145, a 7 vSetleupahtaonsC. EAmer19i7c7a.n S"Mietthoodrs TfeortiCngalanldiMnageri6lhs.LCPSuObl.icAaqtuiaontiNc bTorsicSoTloRgy43a4ndorHamsaerd [ L[ohndmone. DA. 1971. Stttical Methods i Biological Assy. Second eicn. Grin Press, ! i PROTOCOL NO. 454/042199/MUS/SUBAS4 000861 3MLAB REQUEST NO, U2723 i i WILDLIFE INTERNATIONAL, LTD oo "12 i APPENDIX b "IDENTIFICATIOONF TEST SUBSTBYASPNONCSOER TobeCompletedby Sponsor i L Test Substance Identity (nametobe usedinthereport): P(FOPS erfluSoulfroncicAocidcPottassaiumnSaelt Reference Standard (ifapplicable): Analytical Standard: _N/A i Internal Standard: _P1.1e.2.r2HfHHlH uorooSuclfotnicaAcnid e Test Substance Sample Code or Batch Number: __ Lot 217 I `Test Substance Pur(%iActtiyve Ingredient): __989 Expiration Date: 2008 i TR I Test Substance Characterization ermine priorto 5ts i hsstacy secordancewih GLPSaar? i IL Test SubstanceStorageConditions Ye xNo Pleaseindicatethe recommendedstorageconditionsatWildlifeIntemationalLid. ---- S-- HE-- o-- e-- m------------------------------------ ' Em ae ve xe B Otterpertinentstability information: B IV. TestConcentrations: xAbdejsudsutptoesnttcohnecpenutrraityio(n%)0gi1v0e0n%baoir.e. i Dao1 TtesatdGfuostmiactteoanltAeCnIipStionto100% V. TocityInformation: Mammalian: RatLDSO 251 mglky MowseLDSO_N/A i Aquatic: InvertebrateToxicity (EC/LCS0) FishToxicity (LCS0) Daphnia magna: 27 mg/l Rainbow Trout: 11 mg/l 1 DS uphniO amagm na: gll FatheadMinnow:38mg [|] OtPlheearsTosxeisciMtySIDnfSormation (inching findingsofchronicandsubchronictests): a 1 000862 u PROTOCONOL. 454/042199/MUS/SUB454 3MLABREQUENSOT. U2723 a WILDLIFE INTERNATIONAL, LTD "13. APPENDIXI Hu Analytical Method Provided by Sponsor `Samples vil be analyzed based upon procedures provided by the Sponsor inthe following analytical methods: wm 1. Liquid Chromatography Sulfonc Acid Potassium SMaalsts(PSFpOeSc)roImneFlersyhw(aLlCeMrS,)SaMltewtahtoedr afond AthlegaDletMeeedmiinuamtion of Pefluorooctans mm sampleAscvoilplyobfdtohceuambeonvteemdeitnhtohdwrialvlebdeatmaaainntdaisnuemdmianritzheerainwdthatai.nTalhreeapocrttu.almethuoseddtooanallyzoethgeteyst mn =i" PROTOCONOL. 454/042199/MUS/SUB4S4 000863 3M LAB REQUESTNO, U2723