Document 9JENemeEe840N10vxR0wR5VK6

BIOCONCENTRATION - REVISED AR226_ 1108 TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC-95. (1-Octanesulfonic acid, 1,1,2.2.3,3,4,4,5,56.6.7.7,88,8heptadecafluoro-, potassium salt, CAS # 2795-39-3) sRuebmsatraknsc:eisSaamwphlietefrpoomwd3eMr.pPruordiutcytdioentelrotminnuemdbteorb2e178.6.T9h%ebtyesLtC/MS, "H-HMR, "F-NMR and elemental analyses techniques. =2 METHOD: Es Method/guideline followed: US EPA OPPTS 850.1730 and OECD 305 3- TGyLpPe:(YFIlNo):w-thYreosugh exposure with flow-through depuration phase. == YSepaerc:ies2:001, RBelupeogritlla(mLeenpdoemdis2m0a0c2rochirus) 2= Supplier: Osage Catfisheries, Inc., Osage Beach, Missouri Length and weight at test termination: MMeeaann lweeniggthht==622.7m0mg,, rraannggee 256.-06-36 m3.m329 Loading: 0.48 g fish/Liday (based on initial loadingof90 fish per tank, using mean fish weight at the endofthe study and volume of water that Fpiasshseadget:hroAupgphrotxeismt acthealmyb7emroinnt2h4s-haoturtses)t. initiation Analytical monitoring: Concentration of PFOS in water and fish. PNruemtbreeartmoefncto:ncNeontnreations: Two plus a negative control Test concentrations (mean measured): Negative control, 0.086 and 0.87 mglL Uptake period: 62-days (0.086 mg/L exposure) 35-days (0.87 mg/L exposure this exposure ended after 35-days due to fish mortality) Depuration period: 56-days (0.086 mg/L exposure) 2 Test conditions: None (0.087 mg/L exposure) = Dilution water: Moderately-hard well water ~ DilutioSnpweactifeirccchoenmdiuscttrayn:ce: 313 (31-0 315 umhos/cm) 2z ; Hardness: 130 (1--21382 mglL) x Alkalinity: 178 (1-71768) ? MeasurpHe:d d8u.r1in(g8t0h-e842-w)eek period immediately preceding the R test. CONTAIN NO cBI 000283 Test conditions (cont): Stock and test solution preparation: Two stock solutions were pelreecptraircetdoapt-1d0owannmdi1x0er0 tmogaiad..i/nL.thSetsooclukbsiolliuztaitoinosnofsttihrreedtewsitth an substance. After mixing, the stocks appeared clear and colorless. Stocks were prepared at approximately weekly intervals during the uptake phase. Stocks injected into the diluter mixing chambers at a rate of 3.5 mLiminute where they were mixed with dilutionwaterat a rateof 350 mLiminute to achieve the desired test concentrations. Al inal test solutions appeared clear and colorless. Diluter flow rate: Approx. 6.3 volume additions per 24-hours. Exposure vessels: 104 L stainless steel aquaria filled with approximately 80 L solution. Number of replicates: None -- one vessel per concentration Number of fish per vessel: 0 Diet: Flake food, Ziegler Brothers, Inc., Gardners, PA m Watem r chemistry ranges during the study: Photoperiod: 16 hours light and 8 hours dark with a 30 minute transition period. Light intensity: 278 luxat surface of the negative control vessel at test initiation Collectionoftissue samples: Fish were collected from test chambers by random selection at 12 time points during the 62-day uptake phase. `They were euthanized, blotted dry, weighed and measured. Fish then rinsed with dilution water, blotted dry again and dissected into edible and nonedible tissue fractions. The fractions were individually weighed. The head, fins and viscera were considered to be nonedible tissue. The remaining tissue, including skin was considered to be edible tissue. Statistical methods: Whole fish concentwreraectalicuolantesd based on the sumofthe edible and nonedible parts. Steady-state bioconceniration BCF values were originally calculated from the tissue concentrations at apparent steady-state using the BIOFAC model. Upon further investigation, this model was deemed inappropriate for use with `twhiesredantoat ssetta.tisTtihcealtlyisssiugenicfoicnacnetntdriaffteiroennscehsadbertewaecehnedthaeploaisnttthwrheeeresatmhpelree days. However, aplotofthe data clearly shows a trendof increasing concentrations in the tissues. In addition, the BIOFAC program is not accepted as appropriate for surfactants. An amendedreportwas issued with BCFK values calculated as outlined in the draft OPPTS 850.1730 Guidance Document. The kinetic bioconcentration factor (BGFK), uptake arantde w(khio)laenfdisdheepuxrpaotsieodn troat0e.(0k8z6) wmeg/rLe.caTlhceulsaeterdatfeorcotnhseteadnitbslew,enroenetdhiebnle 000284 used to calculate a BCFK (BCFK = Ki/Kz) and half-lives for clearance for each tissue type. The results from this data reanalysis are presented below. RESULTS Nominal concentrations: Negative control, 0.1 and 1.0 mg/L Mean measured concentrations: < 0.05, 0.086 and 0.87 mg/L Kine i ntration fact 0.086 mg/L exposure BCFK: Edible 1124 Time to reach 50% clearance: 86 days ICEK): Nonedible 4013 116 days Whole Fish 2796 112 days 0.87 mg/L. exposure Although BCF values were calculated using the BIOFAC software, the results are not reported here. Allofthe fish had died or been sampledpriorto achieving steady-state. As aresult, the BCF values were underestimated and are not relevant. 000285 PFOS Concentrations in Tissues of Bluegill Exposed to 0.086 mg/L. re[ee Valuesarefrom 4 individual fish at each sample period. alka kg mal [ [01 Gohours) [[ 7570o155m014%o0782m |GAeTSow51s0m 0m41e7i 0w407|o |0m283im 03m51s e0.20| .15| [ia |vaov.arme| oases we | wawozmans| [ [3 2% s |[mmeammrmmssmwis|| wsiomrsemsomi|| wwaawmmsamsssdaas|| [ [sa o ||weoamwaowsmia%wss||wmowwaimmziizsr ||w@aawrsemssvi|| [Owpumetoonbay[| misaean_ e | womm_ sws| me| rmemi| [ [2 is a mmomsswwmimwmms ||wwiemaissrawms|| wswaenosismeisss|| [8 miwemavz|srmsmsen | seesmee | PFOS Concentrations in TissuesofBluegill Exposed to 0.87 mg/L. RETewe Valuesarefrom 4 individual fish at each sample period. nally `malkg akg [ [01 (chours) [|eTmeAwRm.s1m10s73e0 ||9T4u,a4i2a9n0i||e27m13o00o.28z67e89 || [5 |wawewnms| wamosass|wsorwsmi| [ [oi a wmeameriees||wwimamaaeas ||wwoewse || | mmwm |mommow |mmm | | `Sampling of fish stopped after Uptake Day 28 due to mortality. `Test organism mortality: Negative control: None during the uptake phase (62 days) or depuration phase (35 days) 0.086 mg/L exposure: One fish died after 49 days and one after 59 days of exposure in the uptake phase, none during the depuration phase (total of 2.2% mortality during the study). 000286 0.87 mg/L exposure: Mortality first noted on Day 9 and continued through Day 35of the uptake phase at which time allofthe fish had either died or had been sampled Analytical methodology: Analyosfteesst solutions and fish tissues `awnedreanpaelryfzoerdmebdyaHtPWiLlCdiwfiethInstienmgalteioqnuaald,rLuipdo.leWamtaesrsssapmepcltreosmweterriec diluted detection. Tissue samples were homogenized, extracted, diluted and analyzed by HPLC with triple quadrupole mass spectrometric detection. When determining the concentration of the test substance in the samples, wthaessuasmeed.anNdomaotsttempprtowmiansenmtadpeeatkorqeusanptoinfsyeofnorthpeerbfalsuiosroofoicntdainveisduulaflonate isomeric components. The LOQ was 0.05 mg/Lforwater in this study. For tissue samples, the LOQ was calculated on an individual basis for each sample since each entire submitted sample, of differing weight, was extracted without an adjustment to constant weigh. Recovery was excellent in bothwater and fish tissues, ranging from 84.9. to 122%offortification levels. Analytical results were not corrected for procedural recovery. CONCLUSIONS PFOS bioconcentrated in the tissues of bluegill sunfish during this study. `The BCFK values calculatedforthe edible, nonedible and whole fish tissues from the 0.086 mg/L exposurewere calculated to be 1124, 4013, `and 2796, respectively. PFOS depurated slowly. The BIOFAC estimatesfor the time to reach m5g0l%Lcelxepaorsaunrcee fwoerreedi8b6l,e,1n1o6naednidb1le12anddaywsh,orleespfeicsthitvieslys.ues from the 0.086 DATACUAY jG --@---- eee Reliability: Kiimisch ranking = 1 REFERENCES "This study was conductedatWikdife Intemational, Ltd., Easton, MD at the requestofthe 3M Company, Lab Request number U2723. The report was amended and reissued June 6, 2002. OTHER Last changed: 6/11/02 000287 - PPERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): `AFLOW-THROUGH BIOCONCENTRATIONTESTWITH THE `BLUEGILL (Lepomis macrochirus) FINAL REPORT `WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-134 ENVIRONMENTAL LABORATORY REQUEST NUMBER: U2723 US, BaviemmentalProtectionAgency `Series 850`--OEPcoTlSogNicmalbEeffe$c5t0s.T1e7s3t0Guidelines and (OECD Guideline 305 AUTHORS RaymonKduLt.RVanDFiroovern, PhD. Henry O Knege, Pi. STUDY INITIATION DATE: October 31, 2000 STUDY COMPLETION DATE: June 21, 2001 AMENDED STUDY COMPLETION DATE: Je 6, 2002 SUBMITTED TO: Envi3oMmmCeapmolrLsatiboonsiry B5u3i5Bldunsg 23v6e.0e9 StPaul,Minnesota5514451.Pa,Minnesota 55144 Wildlife International, Ltd. E5s3t9o8n,CMoamrmylearncd D21i6v0e1 (410) 822-8600 Page ofi3e AMENDED 000288 Wildlife International, Led. 2 Project Number 454A-134 `GOODLABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: wPiesrtfhlBueorgo(oecptosasPnomteaascshuiolucmhfSiaorln)t(aPtFOeS,):AFlow-ThroughBioconcentrtTieosnt WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-134 STUDY COMPLETION: June 21,2001 AMENDED STUDY COMPLETION: fine, 2002 ThisstudywasconductedincompliancewithGoodLaboratoryPractice Standards as publishedbythe U.S. Environmental ProAtgee ncyc in4t0CiFRoParnts 160 and 792,17August 1989. STUDY DIRECTOR: Lo, / ase Henry O. r, Ph.D. NDoinreTcatroofre Ps Aquatic Toxicology and SPONSOR APPROVAL: A Soom o Repaii rs Borer 4 June 03 Date oBuef fe 2 000289 AMENDED Wildlife International, Ltd. an Project Numb 4S4A-134 `QUALITYASSURANCE STATEMENT "Thisstywasexaminedfor compliancewithGoodLaboratoryPractice Standardsaspublishedbythe: US.EnviPrroteoctinonAgmenceyinn40tCFaRPalrts 160 and792, 17 August1989,Thedatoefasll MiannspaegcetimoensntawndesuriatsFoalnldotwhs:edatesthatany findingswere reported 1theStudyDirectorandLaboratory scm TetSubizmrepion Nati Foresion [-- AonDe sdrtp. Bio Dat ndDr Rept Eni DATECONDUCTED: ~~ STUDYDIRDECATTOERR:EPORTEDMTAON:AGEMENT: Nowmber9,2000 NooleB200 November,200 ---- Dembek200 Dumbell200 Fey20,2000 Fey20,2000 Retry22,2001 NaNoyy2104-31071001 Mayas,301 Sea, 000 Joe113,2000 Soe,2000 ne 1,200 Jne20,2000 soe,2000 ne20,2000 Amended Fa Reon Sue.2002 sm Soe,2002 1. Hynson Qual Program Supervisor sll 2.00, 000290 AMENDED --re--e---------------------------------------- Wildlife International, Ltd. 4 REPORT APPROVAL Project Number454A-134 SPONSOR 3M Corporation "TITLE :PerfluorooctanPoctasssuilumfSaolnt(aPtFOeS,): AFlow-ThroughBioconcentration Testwiththe `Bluegill (Lepomis macrochirus) WILDLIFE INTERNATIONAL, LID, PROJECT NUMBER: 454A-134 STUDYDIRECTOR: Log Le DHeairyrO.ofcAqouatrir,cPTh.oDi.elogy ind `Noo-Target Plants. ny) Date. 2 PRINGRALINVES;TIGATOR: 34 ; Directoofr| A sblifoz AMENDED 000291 --e--ee-------------------------------------- Wildlife International, Ltd. sTAOFBCOLNTEENTS ProjectNumber454A-134 TOfH CORREO S... `TABLES Tabl1e Mosnsan RangesofWeeQuality PACES... 20 Table2 Concentrations ofPFOSin Water SamplesDuring the Uptake Tabled ConcentratofiPoFnOsSin Water SamplesDuringtheDopuiaion Phase.................cue. 21 PRES... 22. Tabled PTFOiSsCsofoBhengillcExipnnoEsdeidtb1l5e,0aN0on6stdiblieanodWhBEoALn.le rsFioshve rs Tables ApparentSteadBCyFV-aluSestforaBitlelExp100o08s6mged aL... 26 Tabled BCFK ModelEstimatesforBlegillExposed 00.086mg aL... _-- AMENDED 000292 _--mm AOorm- Wildlife International, Ltd. 6 "TABLE OFCONTENTS - Continued - Project Number 454A-134 Table? Table 8 Figure 1 Figure Figure Figured `TABLES (Cont'd) PFOSConceinEndibtle NronsaibltesindWoholneFsish TisosfBulueegisll Expose10d 0.87 Mg84/L rrr 28 `Day28 BCFValuesfo BluegillExposed0.0.87mg aL... .30 FIGURES ConcentrationsofPFOSinEdible FishTissuesof BluegillExposed 1C0o0n0c86emngtraaiotffiPoOnSsinNonadibleFishTissuesofBlugiA l ExposedE C0o0n08c6enGtAraLotLfi.Po.On.Ss inWholFish Ti--ssues--ofBluegillExpos es 3 OF Bel ApoSed 10087 GSI rrr rere Concentrationsof PFOS inEdible. Nonedible,andWhole FishTissues AMENDED 000293 fe m-- t------------------------ Wildlife International, Led. PootNumber 4S4A-134 EA TAOFBCOLNTEENTS -Continue-d Appendia Appendix? APPENDICES SpecificConductance,Hardocs,Allin ndpF of WellWater Auayss of Pesticides,Organicssnd eas MeasuredDuring the4-WeekPeriodImmediatelyPrecedingthe Test ................ 35 inWildlifeInternational Ltd.WellWater .......c.coeevmssmsmisssscsssssns 36 Appendix 3 Appendind AppendixS Appendi6x Ll nemaLitoPronject]No ASHA3A ere... `CTohnceeAnntarlaytsiiosnosfiPnFerersfhwlatuerraondoBclutcgailnPloeStuasnsfusiisluhmTfSioasslnute(aiPntFSeOuS,p)portof 38 Temperature and pH ofWot th TetCHIBOGR rr 89 DissolvodOsygenof Wate ithe Test Chambers. 91 Hadoes,Allin,Conductivitysnd TOCof Wate th `Appendix 7 ADPOAXS Appendix 9 `Appeadix10 `Appendix 11 ADDED 12 `CumulativeMortality andTreatment-RelEfaftecetds ........o.ccreromrrrrnnecs 96 CH1G0PSOG. 105 "0 "Protocol,AmendanmdDeEVnIBHtODsS ...........vursrmsnssnssses 106 PersonnelInvolvedinthe SAY .....cwvwvmsssssmmmmmssnssssssssns 130 CalculationsOfTheKineticConcentration Factor (BCFK), `TThheeURptaAkeLRatFeoCroCnlstaaants(,kiA),nTdhTehDeepTuurnaetioTnoRRaetscehCo5n0st%aOnft(Skyw),ay Sate. 131 ReportADGOIEDL ce 133 AMENDED 000294 ------------------------------------------------------ Wildlife International, Ltd. 5 Pusect Number454A134 SUMMARY SPONSOR: 3M Corporation. `BSLPNAOANoSIAOcRDR'SAAORFTCETSPTROUEISDEOYN.OTpTANTaEIwVE: Eason, Ms.SusanA. Beach. VWbs3160s1nationaLs. TDDPLREORTCETRRTOINBOINR,AL pe `TEST SUBSTANCE: PerfluorooctaPnoctasssuilumfSoalnt(aPtFOeS,) STUDY: P"eTrhfrlouuogrhooBcitoacnocnscuelnftornaattieo,nPToetsatswsiituhmtShaelt (PF(ALOeSpFol)moiw:s- Nom Test macrochirus) CMOENCAENNTMREATAISONUSR:TEESDT Negative Control, 0.10 and1.0mg a.i/L concanTeATIONS NegativeControl, 008S6S mdys037 mgeahLorywdEoxporoeto0d87| TesTDATES E`sxapmeprliimnegntal Sat (OBCD) Novae29.2000 Be Sie(OFA) Doser 5 2000 `E`xBipoelnogmiecnaltaTleTrermmiinantaiotnA-pirAipolrn2il,1210,021001 + : LfEreNOr THOF TEST y OuSmgaify I18Dee(62-DayodUps, 56-DDeoyyDepuDrepiroonn TESTORGANG TAT rea) SOURCEOF TEST ORGANISMS: CCaC thie Io AGBOFTESTORGANISMS `IOvsaegietBeesach,Missouri 65065 MSEASRUROEEMENLOTFS 0ANEEGAATTIYVoEr Tw67a30g:1Rior1 am VMeten=2o70, RSaangse 2o20e10332 TESTErS Gtoemif)or cc Tiee Npiade WigShFee TBRERaBIcGT wreundrsttueitomfisthmoaial]dty prt oNTeepschon aslts esaseFrFil AMENDED 000295 _--_--nm Wildlife International, Ltd. 9 Project Number454A-134 [INTRODUCTION Abluegillsunfish,Lepomismacrochirus,bstidoywcasoconnduccteedsfotr 3rM Caortpoiraotinon `attheWildlifeInternational,Ltd. aquatictoxicologyfacilityin Easton,Maryland.Thein-plhasieofftehe LtiesdtwaasndcoancdoupcytoedffrheomfDineaclermebperta5,e20l00dtouAnpdrilP2r,o2j0c0t1.NRuamwbedrat4a5g4enAe-r1a3t4edibnyaWriclhdilviefselIonctaetrendaotniotnhael, `WildlifeInternational,Ltd.site. oBiECTIVE The objective of this studywasto determine the bioconcentration potential of perfluoropootcasstiuamsanlte(PsFOuS)ilntfheoblunega illtsunefi,sh. EXPERIMENTALDESIGN "Thobiosoncentaiontetconsist fa62 uptak phase low by 36-daydepurationphase. Duringtheuptakephase,thetestorganismswereexposedinoneofthreegroups: 1) Anegative (dilution water)control; 2) Anominalconcentrationof0.10 mgactiveingredient(ai);or3) Anominal concenotf1r.a0mtgiaoLn. Atthestartofthedepurationphase,stockflow tothetreadgroupswas Stoppedandthebluegillwereexposedtodilution waterwithout PFOSfo theremainderofthetest. atmEaacdhttehsetpcehgaamtbiverccoonntrtoali,nWead9te0rbsluumepglielslawtetrestoilniotcitaotdioonn,aDnadyo4ne(rre-pleisca)t,eDwaasyt1es(terdefsorte)acoha. uptakeDays 0 0and burs), 1,7, 14,219,5,6ad2628so,nd3epra5tion,Day4s142,28,,82 aTnids5s6dsuarmipnlgesthveetreestlaondcoalneasltyezdedaftsorePtFOeSwuastienrgslaimqupildechorlosmtaitoongprearpihoyd-smasissnpechtertoemsettarny(dnLCl/yMzSe).d ocPEbyOLCISMS.Theroulsoftessanalysesweresd 0callt he BCFvals,uptake oesand cepurion atesin bl tissue, noedible issuandwholefish. 000296 _-- Wildlife International, Ltd. `Project Number 454A-134 "104 MATERANDIMEATHLODSS Thestudy was conductedaccording to the procedures outlined intheprotocol, "Perfluorooctanesulfonate,PotassiumSalt(PFOS)(Appendix 9): AFlow-ThroughBioconcentration Testwith theBluegill(Lepomismacrochirus".Theprotocolwasbasedonproceduresoutline in US, Environmental `ProtectionAgencySecies850 --EcologicalEffectsTestGuidelinesOPPTS Number850.1730 (1);ASTM. Standard E1022-84StandardPractice forConductingBioconcentrarion Testswith FishesandSaltwater BivaeMolluscs (2);and OECDGuidelinefor TestingofChemicals 305,Boconcentration: Flow-Through PashTest (3) "Test andReferenceSubstances "Thetestsubstancewasreceivedfrom 3MCorparation o Octobe 29, 1998andwasassignedWilde InterLdn.idaenttifiicatioonnnumbaerl46,75.Th est substance, white powder,vasidentified esFC-95,Lot. #217.InformationprovidedbytheSponsorindicaedapurtyof869%andanexpirationdateofAugust 31, 2001. The testsubstancewassoredunderambientconditions. R PrepaofrTeasttSoilutoionns "Thenominal testconcewnerte0.r10aantd1i.0mognaLs. Twostocksoluwetrepireoparnedsat conceonft10ranad1t00imoganLs. Theappropristeamount of testsubstancewasweighedoutand disdsilatlion wvateefordeacihsonck.Thestocksolutionswere sidwithan lertop-down mixerto aidinthesolubiolftihezteasttsubistoannce.Afermixing,th stocksolutionsappearedclearandcolorless. Stocksolutionswerepreparedatapproximatelyweeklyinterval duringtheuptake phaseofthetest.Thestock solutionswers injectedinothedilute mixingchambers(starat of3. mL/minute)whereheyweremixedwith dilution water (a atcof350mLminute)toachieveth desiredtestconcentrations.Allfestsolutions appeared clanedcoalorres. TestOrgasismn "Thebluegill,Lepomismacrochirus,wassleceda thetest speicsforthisstudy.The lucgilloinse oftherecommendedfreshwaterfish speciesforuseinbioconeentraion tests (1,2,3). Bluuesegdntihelelst wereoblanedfromOsageCatfisheris,Inc., OsageBeach,Missouri. Thefish wereapproxima7mtoentlhys oldattest ntiaton. Identificationofthespecswasverifiedbyth supplic. 000297 --e--mi------------------------------------A---- -- Wildlife International, Ltd. ue Prsoct Number454A<134 "ThebluegillwereheldinWildlifeInternational,Ltd.wellwaterfor 103dayspriortotesting.Thefish. `wereacclimatedtotestconditions forapproximately 49hourspriortotestinitiation. Dutr he hi oldn ingag nd `acclimation periodstho ish showed no signs ofdiseaseorstress. During the 14-dayholdingperiodpreceding `the test,watertemperaturesrangedfrom22.3 to 22.7C.ThepHofthewater rangedfrom 7.8 to 8.2and tdihsosEolvemdoxiygernraCnoogneddinftrioommn7s.s6ectioon8n.2omftgt/hiLa. pIolrn . As t est t tur astedifu oonr,wm tahteebe rlmueegn aislulrt weemeea ntcsot arledi desfco roimbetn dhien sclmtiontaksnidisciminatlydiscbutod 1102 a 3tin nothetestchambersul achchamber ---- `Duringholdingandduring thetest,thobluogillwerefed lakefoodsupplied byZeigler Brothers,Inc, Gardners,Pennsylvania.Thebluegillwerefeda leastoncedailyduringholdingandoncedailyduringthetest. Feedingandsamplingscheduleswerecoordinate sotat ishweresampleda leas fourhoursaferfeeding. Allfishusedinthetestwerefromthe samesourceandyearclass,andthestandardlengthofthelongest. fish wasnomore than twicethe length of the shortest.Thelength andweightoffishinthe negative controlwere `cmoensaisduerreeddatt ohboecruedporefstchaetaetsivtewaofa6l2lfmismh uistedhianatnhegeteosft.5T6h0e.6meaanmt.otTahlleeanvgetrhaogfe1w0entewgeaitgihvtecbolnttireoldfiesyh) Was 2.70granwitharangeof2.03103.32gis.Losdingwasdefined sthetotalwetweightoffish erie oftestwatcr that passed through th testchamberin 24hours,and wasdetermine tobe0.48 gfish/Liday. The loadingatewasbased he aeragsweightofthefisathcodofthe tanhnil stoking dest00 pe ta), TestApparatus Acontinous.flowdieswas sed delivereachconcniation ofhetestsubstanceandanegative `cSounbtsrtoal.nceAspteorcikstsaolltuictipounsmip(tCoole-iPnagrcmhearImnbsetrrsuamsesnitgCnoemdp1a0ncya,cChhPicFaOgSo,tIrlelaitnmoeinst)w. Tashuestedotkodseloilveurtthiewotenersest mcdwithdilutionwateinthemixing chamberspri todelivery othetestchambers.Theflowofdition `watertothetestchamberswascontrolledbyrotameters. Thedeliveryofwater rom therotameterswaschecked priotheean atsppeoximately cl trv theres. Appr6:3ovolsumeiditeionlsofyest. aterwer delivered othetest chambers every24 ous.Thegeneral persionofthedite wscheckedat leasttwotimes adayduringthetestandonceonthelatdayofthetest. 000298 EEE -- Wildlife International, Ltd. Prec Number454A-134 nTestchambersvere 1041tilesst aquariafilledwithproximately 80 oftestsolution.The depthofthetetwatein areprescatativechamberwasproximally19cm. Tes chambers were indiscriminatelyposionedin temperate<osrlledwaterbathdesignedtomaitsinsconttemperature. "Th vatebathwasclosedin plexiglas ventilationbodinoder miniizeany poteaialfix crus contamination. Test chambersveresiphoneddalyand periodicallycleaneddurintotst 0remove excess foo ndfocalmater. Test chambersvere deniedbythprojctmumbeantetconcration DionWater Thowatersedforbldgandtestiogwessrt cbsiedfom awellproximately 45 cers deeplocatedonthe WiIldntemationida.slie,.Thowellwatei characterized asmodestlyhardwater Thespecificcondoctance,bardoes,alkalinity and pHmessresntsofthewel wate arinthefour-ck periodimmedistelyprocainetestspreeinAppend 1 Thewel waterwaspassedthrong sndfiletoremoveparticles rete th spprocimaelySym, andpumpedino 37800.sora takan seedithsprenoes.Pri (scthwateraginwas ered (0450m)toremovemicroorgnadnpiarstimclse.Theresultsofpcidicmalysesperformedto measuretheconcentrationsofselectedcontaminantsinwellwaterusedbyWife International,Ld.are presinAeppnendeixd2. Environmental Condifions Tho targettempeforrtoatestwaus2r:4e1C, Temwaspenededrctiannoutsyienh negativeconrolwith aFulscopoERICRecorder (1900 Sri modelo.A). Tempewarssasotmuearsreod malletchambers thbogianinad doftheeadatwesklynervesdurinthets,with lui glass thermometer. Dissolvedoxygenwasmeasuredwith aYellowSpringsInstronCopa,os:Mod S1Bdissed onygenmeer.With he cxptonofuptakeDay24,messuenctsweremadedalyincachtest chimber Disolvdoxygenmcasrenntswer otmade onwpaDay2 detobiologist vrsight,Measurementsof PHwermdeineachtetchamber tthe beginningancn ofthetestandatweeklynerval ducingth et. using FisherAccum Model915ph cer. 000299 Wildlife International, Ltd. ProoctNumber454A-134 ne Hardocs alkalny,conductivity ndologni carbon(TOC)weremesrdnthenegativectl. atthebeginning andendofthetest,and atweeklyintervalsduringthetest. Harand d aln kale inits ywes re `measuredbytitration basedonproceduresinStandardMethodsfortheExaminationofWaterandWastewater ). Conductivitywasmeasuredusing aYellowSpringsInstrumcatCompany,Inc. Mo3d3Saeliilty- ConductivityTemperature cer Totaloxganicasbonvasmesred sing ShimadzaodeTOC.5000tl Ambientroomlightwasusedto lluminatethe test systems. Fluotuberstheatemsittcedwaevelnengtths silatonatural sunlight(Colortone50)werecontrolledbyansutomatictimertoprovide photoperiodof16. `hoursoflightand $hoursofdarkness. A30-minutetransitionperiodoflowlightintensitywasprovidedwhen lights wentonandofftoavoidsuddenchangesinlightintensity.Lightintensityatthesurfaceofthewater (over t8h4e0n0e0g6atliivgehtcomnetreol.)was 278luxattestinitiation.Lightintensitywasmeasuredusing aSPERScientificModel Observations Allfish wereobservedonceeachdaytoevaluatethenumberofmortalitiesandthe numberof individuals exibitiogsignsofabnormalbehavior Profocr ExeposdureoufFirshteoPRsOS. ThetestchamberswereconditionedbydeliveringPFOS tothedilutersystem forapproximately Sdays beforeaddingthe ish.Watersamples werecollcte threetinesduringtheprotestperiodtoconfirm that `equilibriumconcentrationsoftestsubstanceinthetest chamberswereachievedpriortoadding thefish. Attheendofthepre-testperiod, heuptakephaseofthetes wasinitiatedonDecersber 5,2000by placing hefishinthetes chambers.Bhosle patilremoved ro theboingtingroupsof 0 2. Thegroupsofbluegillweredistributedamongthetestchambersuntilcachtestchambercontained90fish. `Thedurationof theuptakephasewas 62daysforthelowexposureconcentration(0.1mga.i./L,nominal). The `uptakephaseforthe high concentration (1.0 mga.i/L,nominal) wasterminatedafter35days dueto fish m`mgortialiLty.(Anllsuobseemqxupeoinsturrenefceroaecnecnelrsaitn)ithni.srAeptohrtteonsdaomfptlhienguppearkieodhsaafst,ersDtoacykl35oofoupttaekrearetfmeretnotthreo0.s1 was toppedantelegil wreexpose ition vat wilbot PFOSfo aperiodof 56days. 000300 Wildlife International, Led. ue ProjectNumber454A-134. CollectionandAnalysis ofWaterSamples `Watersampleswerecollctedon Days (0hoursand4hours), 1,37, 14,21,28,35,42,49,56and62 oftheuptakephase. Watersampleswerealsocollected on Days 14,28, 42and 56ofthedepurationphase.At `eachwater samplinginterval twowatersampleswerecollectedfromthenegativecontrolandhressampleswere: `collectedfromeachofthetwoPFOStreatmentgroups. Onenegativecontrolsampleandtwosamplesfromeach ofthe PROStreatmentgroupswereanalyzedforPFO. Thereusingsampleswereheldin reserve asbackup. samples. Allwatersampleswerecollectedfrommid-depthofcachtestchamberusing a glasspipette. The `watersamplesweranalyz forPFOSbyliquidchromatogeapy-massspectrometry(LOS). Procodufrosr analysis ofth atesamplesaeprovidedinAppendix 3. Watesampleswereanalyzedassoon aspossible afer collection withoutstorage. . CollectionandAnalysisof Tissue Samples `TissuesampleswerecolleocntDaeyds 0 (4 hours),1,3,7, 14, 21, 28 35,, 42, 49, 56 a6n 2ofd the `uptakephase.TissuesampleswerealsocollectedonDays14,28, 42and 56 ofthedepurationphase.Ateach tis sampling interval, sufficientmamberof ishwercollected provid tworeplicatesamplesofnegative contolfish ndfo replicatesamplesofeach FOS esmentgroup.Fishwero imparetzonaedlfmlty testchambersandeuthanizedbysevering tespinal codabovethe apecalaregion, The fishwere blottderyd `and measuredfortotallengthand wetweightwithinapproximately15minutesofcollection,whenpossible. `Eachfishwasthenrinsed withdilution water, blotted dry again and dissected into edible andnonedible tissue fractions. Dissectionwasaccomplishedbymakinganincisionfrom justposteriortothebaseofthepectoralfin. dorsally thrtheosiunagorhd. Thehead, fin andviserawere removedfom the bodyandwere considerdto benoncdible tissue. Theremaining tissue(includingskin)wasconsideredthe edibletissue,Tissuesamples `weretransferredtotaredscintillationvials andweighed.Procedures for extraction andanalysisofthe tissue `samplesareproviniAppdenedidx. Allissuesampleswereextractedimmediatelyofstored atapproximately - 14Cuntilextraction. 000301 Wildlife International, Ltd. 1s ProjectNumber 454A 134 `TissueSLeilpeicdCtontiesnhtweecollected toermine idcontent(Appendix3).Thodeterminoafptericoennt pdsprovides theptttoexesBC vals itemsof pidconten.FishweresamonpDalyOdf `uptake,onDay62ofuptakeandonDay56ofdepuration.Allfishcollectedforlipidcontentwere stored at idcots. `appro-x14iCmunatiltaneallysiys.Inthisstudy, no attenptwas made toexpress bioconcentrationinrelation to DataAnWalhyosils ishconamtrationswescloutbased on hes fhe ableandnnd parts. The steady-statebioconcentrationfactor(BCF)valuesweredeterminedfromthe tissueconcentrationsatapparent `steady-state divided bythe averagewater concentration. Tissueconcewen rect onsr idea redtto bi e atoapn pars ent `steady-stateifthreeormoreconsecutivesetsoftissueconcentrationswerenotsignificantlydifferent(p >0.05). `TissueconcentrationswerecvaluatedfornormalityandhomogeneityofvarianceusingtheShapiro-Wilk'stest `andBarttelst,erestpetcti'velsy.Ifthedatadidnotmeettheassumptions,thedatawas transformedinanattempt. wt10corrthee dcatta. Meantissueconcentrationswerethencomparedusing analysisofvarianceandDunnett's BIOFACIwntahseosriegdintaolreesptoirmtattheethtiesksiunevealuecsonwceernetcroansidefrecdtro(bBeaOtFs)te,adhyestautpetaankedrthteccoomnpsuttaenrtpkr)o,gtraem departconstant (1) hecmt ie 0rec 90%indyie, andthe Isforcrane, boughissueconenicuion rompak days 9, 56an62werentsignificantlydiffe (0.0), he concnrations of PROS itissue appearedtotllbeinceasing,Therefor, icanbeardtathe sh id `Dotreachanapparentsteadystateandthatusingthe BIOFAC programmaynothavebeen thebestwayto `estthei sepmaraamet teres. Therefore,the datawerereanalyzed usingtheequationsand graphicalmethods `outlinedinthe draft OPPTS850.1730GuidanceDocument (Appendix 11). Therecalcrautelcaontsteantds `were usedtocalculate a BCFK(BCFK = kL/A2). AMENDED 000302 EE E ES Wildlife International, Ltd. 16 Project Number 454A-134 Water Chemistry RES ANU DDIL SCUT SSIS ON Meansandrangesoftemperature,dissolvedoxygenandpHofthewaterinthetest chambersare preseinaTatblee1d.TheindividualmeasurementsaregiveninAppendicies 4 nd5, Watertemperatuinrtehse: testchamberswerewithinthetemperaturerangeof22 + 1Cestablishedforthetestwithoneexception.OnDay 19ofdepuration,thecontinuoustemperaturerecordermeasured 20 C. Thedurofathitsteimpeoratnure deviation wasapproxima2theolurys.Dissolvedoxygenconcentrations remained>6.4mg/L(74 perceatof saturation)throughoutthetest.MeasurementsofpH rangedfrom7.9to 8.2. Weeklymeasurementsof Appendi6)x. `hardness,alkalinity,conductivityandtotalorganiccarbonremainedconsistentthroughoutthetest(Table 1and ObseofrMorvtalaityatndiCloeanSisgas `ObservationsofmortalityandcliicalsignsareprescatedinAppendix 7. Bluientgheniegaltilve control appearednormaland healthy throughoutthetest. Two bludeiedginithle 0l.1mg a.i/L (nominal) treatmentgroup;allotherfish appearednormal andhealthy. Bluegillinthe 1.0mg a.i./L(nominal)treatment groupstartedto die onDay 9 oftheuptakephase.ByDay 35oftheuptakephase,allfishinthe1.0mg/L treatmentgrouphadeitherdiedorbeensampledfortissueanalyses. Anyfishfounddeadintheaquariawere `storedfrozen. ConcentofrPaFOtSiinoWantesr Concentrationsof PFOS the negativecontrolwere<LOQ(0.0500 ag a.)(Tables2ad3). Measuredconcentrationsof PFOSduringth uptakephaseinthe0.10mg ..Ltreatmentgrouprangedfrom 68to 113%ofthenominaltestconcent(Arpapetndiiox3n).Whenconcenmtearsuaretddiuroingntshe `uptakephasewere averaged,themeanmeasuredconcentration was0.086mg../L.whichrepresented86% ofthenomesitconnecatailon. Messedconciratons of POSduringthuptakephasei the 0mg. 2i/Ltreatmentgrouprangedfrom73to 101%ofthenominaltestconcentration.Whenconcentrations `wmheiacshurrperdedsucraintgetdh8e7u%ptoafktehpsnhoamsiewnealreteasvtecroangceendt,rtahteiomne,aCnmeoasunredcconeocefnnPtEraOttSidorunrwiaansg0t.th87dimepguoraa.it.in/oLn, phweraealsl OeQ. 000303 Wildlife International, Ltd. Project Number454A-134 wr ConcenoftPRrOSaintFiishoTinssses conenatCoinocnenst(rAaptpioennsdoifxP)FTOhSeinctohenneegnatrivaectooniftProoOnltSsissiuetsiasmpeloesfcosnhtacixnpeodsneodq1u0an0t.i0f8i6amblgeaPiFO.S repeaintTaebled.Alboughisuconcerationsfo pteays 49,56and 62vere ot Tsihgenimfeicaanntmlcydaiffecenti(spo>0s.c0o5n)c,tnhreacoonncsefntoramttioenssofsPtFrOSeinutipsaskuesssaapmppelaersewdteorsti4l1l.b6,ei9ncre6ansdin7g3..6 `mhge 2s.i/tKgrfordeadyisblfe,pntonaekdibrlaenagneddfwohomle8f4ishi,respbeclteivselsy.toS1t124ensaeBddCFiyvalt-uiesssceal(tcTualbaaltee5dtf),reom however, heevlc ver bleed ob cllsins sus concenations were il rang with time. Itwasbelivedthatbetterestimatesofthebioconcentrationfactorcouldbeobtainedby `determiningthekineticconcentfarctaort(BiCFoK)n. Inerinreportcomputerprogram BIOFAC was use etinatth ici oncntration factor(BCFK),theuptakerateconstant(k),thedepurationrateconstant (2),theestimatedtimetoreach d9t0c%msitneaeddystthatteu,sainndgthheehBalIfO-lFiAfeCfoprrcolgeraaramncme.yHnoowtehvaevre,abfeterthheebreesptowrtahyaodbceienmfiana ese lizedit was porate. Thro hedatawerreanalyzed sin he apicalmethodsouldthe ra (OPPS150.1730GuidanceDocument(1). ThesenwclosaedescribedinAppend 1. Posof esseconcraton versustnever onstruced ocvlut bedota. Thoraw `datawereplotted aswellas acurve connectingthemean values,and a curve throughthepredicted values Tfrhoem BnIoOfFAhCe.uTphtiaskegrhaapshica(laaypp6ro)acahwsaaskeaysdimiptlteowavyatloeuv,alsuiantce dhaotwwfeollmthiecsudravyesbfaisttahedata. pronouncedinflococson he stmt fhuptakenddestinrtconstants tad), Th plottedresultsinFigures 1-3 showthatthe BIOFACmodelpredicted vafloruDaey6s2arelowerthan theDay 62meansforallthreetissuetypes (whole,edible,andnon-ediblefishtissue). Thus,the. i'BnIeOtFAcComnocdeenlastliiognhtflycun,detrhesetsitmiamtaetdetdhteunpta0keeacnhdd9ep0u%rsattieoandrytattec,onsntdantths,landoeverefsotricmalteadtnhse intheorga! repr.Tercaelted BOFK valuesfri,node, ndwhol sh wer 1124, 4013, and 2796respect(iTavbelel6y AMENDED 000304 Wildlife International, Ltd. 8 Project Number4S4A-134 `Recalculatedestimatesofthetimetoreach90%ofsteadystateinedible tissue,nonedibletissue and `wholefishwere 287, 383, and371days,respectively(Table 6).Duringthedepur phaaseotftiheotesnt, `estimatesofthetimetoreach50%clearanceofPFOSwere 86,116,and 112daysforedibletissue,nonedible tissueand wholefish,respect(Tiavbleel6)y. Th conenratonsof FOS intissues offish exposedto 0.87 mp 1.sreprsetedinTale7. P2F8OFScionrcene4t)r.aBtiyonDsaiyne3d5i,blael,l nosnheidinblee a0n8dw7hmoleLfis.htrisasmueesdnitdngortouapphpedarbtonreascahmsptleaeddyo-swtaetebdyeDaady Consequently,theonly informationonbioconcentrationthatcouldbedeterminedfrom the 0.87mg. ea.dii/tiLsbegir(oTlr uapbwe leaes)at.hBt eaDsm aeyde 2on8n tBeCtFe.oDgahyof28tiBmCeFrveqauliureesdr1angehdfr0om%1o3f6sineedyib.letissueito 308.60i8n6 `mg/L treatmentgroup,these values underestimate the bioconcentration potcatialofPFOS. `CONCLUSIONS : `Perfluorooctanesulfonate, potassiumsalt(PFOS) bioconicntehentistsuresoafbtlueegidll sunfish (Lepomismacrochirus).Bluegill exposedto 0.87mgai werecherdead o hadbeensampledbyDay 35 oftheuptake phase.Consequently,noreliableinformationwasgainedfromthistreatmentgroup.Apparent a`pstpeeaadyr-esdtattoebcoenccenrteraastiionngspwetoreDnaoytr2ea,chheedisnttdhaey0.o0f8e6xmpogsau.ri.e/.LtBrCeFatKmveanltgurouopr. oPiFlOS,cmonccedntratiaonnsd iti, ono wholefishtissues nwedrweh1o12o4f,i4s0h1t3s,aandw27e9r6,8r6e,s1pe1c6t,iv0eldy.1E1s2tdiymsat,erssfoprettivmeltyo:reach 50%clearancefor AMENDED 000305 Wildlife International, Ltd. 1. "REFERENCES Projct Number 4S4A-134 1 US.Environmental ProtectionAgency. 1996.Series850 --EcologicalEffectsTestGuidelines (draft), OPPTS Number 850.1730: Fish BCF. 2 ASTM Standard E1022-84. 1988. StandardPracticeforConductingBioconcentration Tests withFishesandSaltwater BivaiveMolluscs.American SocietyforTesting and Materials. 3 OECD Guideline forTestingofChenicals 305, 1996. Bioconcentraton: low.ThrovghFish Test. 4 APHA,AWWA, WPCF. 1985.StandardMethodsfortheExaminationofWaterand Wastewater. 16thEdition,American PublicHealthAssociation.AmericanWaterWorks Association. WaterPollution Control Federation,New York. 5 OF BIOFAC. 1991. September 19, 1991version.TheDowChemical Company,Midland, 000306 Wildlife International, Ltd. Projoct Number4S4A-134 "0. Table1 Mosas of RangesofWater QuslityPaster TEontOiim 7w h0o8le Lepomismacnn --DNnom--m ip---- wa-- i ------------ iy-------- o e Cwoe mbyner TeT mTEpTel m 00 WET m W CmoenT dny GCo0W0g)n a(Hawem tie)mm SoTpC Co memo ens 912 deem weews obs da a0 miamse eamse uree Beee 1pene aEr daehsedoeer armess imsoenae28-- 72g, 7 tien 52 000307 | a. PujectNember S4A-134 | Table2 Concetaions of PROSinWaterSamplesDuin theUtesPhase [ TTToeteuSOirbpaeane:. BWlFFoOSgeLompomtsmacrchins DioVr _ WelWir Vem Noaind Comin "5 + Deyetpute CMoestroedn paorft eT) Hox Be 13 7 um ow 3% we x a (uO Neal | NCw om 400 400 400 400 <Q 0d 0Q 40Q 0Q 0Q 0Q 40Q 40Q 40Q - 010 O00O8M2 o00o1 GOMo 0O0T7S7 GGOOTEHS 0OG0N0B OOLlIIO3 0000RB2S OOOOSS OOLEIO OOIIDD 0000SS) 0OOGBIYe 00% " 10 oowm: aommo oommo oosms ooswss oowws olwsoomoso oomm ~ o~ o - om o TSRempEleEtoelee mdmetG10O0Wmeoa. [ Nota preted orch plneer twoel sles clitfomexch hsb | 000308 Wildlife International, Led. Project Number 454A-134 2Tabl3e Concentrationsof PFOSinWaterSamplesDuringtheDepurationPhase STeeaSwames DTioCripOaraWatheaos:e MeCaoMmitsese,d @gainy WPROSem wBlauetgiwleLeepomis crack u oysfaian)p = a NegativeControl <woQ <LoQ ome <4000 4<0Q0 TheLit of Quin(OQ wes 000mg aie <0Q <000 ~ ss <0Q 4a000 000309 ProjetNome4544-134 a. --h e i ennl aa 95SConsensus nEble,NonesdWThaobsFetsTissof Blog xpd10086mgi. | TTToloCowrpS: B7T0ei5g cLiepsmacocin ! Dames WelBier . pe CT onti,ET a eT eR CRoeT E elWeT ke CWomrm { Eaonns BOOuoetraearem)y ao0ii 1ss 1a e5d oaosiutrr 2pi7en1tn 3o035ms6 BEsilsinnais |iv En i ooammt j1La5is2t5 ij1nd8 a20igi0s6 ij12en6 050% ibm 0 jd 5 SBian0oo0 333 r0idn 11127am60 Psar o orjemd 0osna EsnEenmioes 3 ;: om iam Ties 2idm io 1062 io mTo0m a jim BBliinn s 77 p@A 11538s 2is iiiiom ii fsEraaennds ii sn7oer ti2o0swm 5B75233 22L50a0mem 22r6r20} Eons i BBoannen a2 i ji iur) afanseh Se, ins Fae 2iome ue nis EBiann aa i12s% tPiaH ia siiino 4a TleFbConranR (biE sot+ t) o0 1X. e | 000310 | Wildlife International, Ltd. ES [------ | $208Connon bl No atWehCn eonFutheTsi )mo w00tsmgain. || TBrree T Eoan ee tPhye =3 anmn TpwraTr T mm=m) ea e oims 5 Ti 5 TH 5s | {| Ene EErviann 2 aa 5 2a5l Tan aT2m08o1 & E3H Ton 3j0=2 2bah fsBrvaans]n 335 naB nosn Ri1ii7 aa8as 2nti0osm0s4 8Pi&Ya os[oomaTmeI 8a@ Fi5nu]ns fe11e250dnm5 ai3wlo j1oirsnt &o5=n EsEimAn 88 Eliana s 3nun 2 i1e5mde ia 501 2 fif2eem] i oaaans Esamnmn #@ piw2f Ban & EH 1fLa8oe% 15 42m fo 2J1fr y hid njawer8a EBsilsmninis Erni aaa& a@@s 5 jr115200 iiwonn Vien st I111 ] on nnmroe a TiFo Commi ES{K elie yie)RKE | | 000311 Pj Namba 540134 ase TabledConn) | FFOSCoss BiblNai ndWhe ishTso BglExpos 008mgail TI-So_HS--B--750g5 Loot chs uae pie T CTm EVRosT NCoodmme Ne Weg oWmeRn sETeinpnis BW0wr parasy Tie@edr pri4rey T2o11m0 pa7aiins EEnin i"i Saie oJneed iWw ikrdo aEds HEsiionnmetso 22 E25S0 aiesm J11] i210an P1aY Bini a = 260 Sa 1 a sBEiiisssnaisisy: aaa Ewfreny iG1aso wn4ws na isie saaus [ Ens a 2 aan a Via a2 |( EEmiisosmais i5o n2wu5e ro1sen pb2il7 jrj17es awass cbeiaincCoonroe ~ EiASESEbeE ssaieettoln)i LKaremde st aos a | 000312 - Wildlife International, Ltd. Project Number4S4A-134 -26- Tables ApparatSteady-State BCF ValosfoBiogilExposed100.086mgai. Tos Substance: PROS A DiTleusttiOorngaWnoitsemr::~_~ Well Wier Bluegill, Lepomis macrochirus Mean Messed ConTcecsurtiion Appar Uptake Days at Fable Tissue Type 3 (mgai/L) S5t,ea5d6y-mStaa6te7 Nonedible 00s 9.56162 iho Fish 00s 9. 56m162 Apparat Sey CSotnacteenTmistsiuoen ns (mg2.i./Kg) 967 ns Apparent ro Steady-State BCF_ ne as 000313 Wildlife International, Ltd. ProjectNunber 4544-134 ar Table 'BCFKEstimates for Bluegill Exposed to 0.086 mg a.i/L "Sponsor: 3M Corporation DionWoe,_ Well Wier `Test Substance: PFOS. Test Organism: ~~Bluegill, Lepomis macrochirus . Bioconcentration UptakeRate Depuration Rate to Reach 90% `Time Factor Constant Constant ofSteady State to Reach 50% `Tissue Type (BCFK) ) (a) (Days) Clearance hNd= eeoFitssh 42T01o09r36 Su13oEs 0To0os06os0 3mwm 1I5T6 -- mene Te-- eee---- eee OEE AMENDED 000314 ||| Wil.dlife Internatiional, Ltd. Fm 4500134 EX EOSContinin leNel WTAaseFri Titsofhg epo097sgat. . FE Traeo Doassh poms | et TT arET he acaTm Ve wi comme sH[rpaeAnweprvoeeen epiiiee pwaiisn na35&ira Tjwiogn p3woornv Han om i i @ jie) on saBa0de8 [= ii\| i"4o i iiB1em0 iiwmnm ii1iamm w5iiwnH fasBfaeddse }:33 maiiB afsiohesr] ana&ns rLi1em} n%iExs EsHnfaateea ;};; aa4a" saJiaemds w2i5 fria nwuwsno | soI nne ui ioEnd L1Hoi5m 44m 2aa15m wiis i oTenamn SE acnsmreR KiamB iha da aw in 000315 Wildlifae Internati'onal,Ltd. > Tab Contin) PROSComins ile,Neri nd WikFi Tiesofhg xpos Fieee 087 i. | a | Te -- De naa Sape v>er oaSneme rMTrGhT yeoenmy TR:EeT aenvne paodr a3 iH Via= 4> sfmod w"s aw a n tom on pr o Te-- inCi BA3 pr 5i KaLe a -- z et Spgs. yy3180 oem pp rv ty et nets | | 000316 Wildlife International, Ltd. ProjoctNumber 4544-134 -%0- Tables Day28BOFValuesfocBlcgil Exposed10037mgai. STpeossSuab:stance: TPFMOCSoporaion DiTlewsitoOnrgaWnaitsemr:: _ BWaeclgliWli,eLerpomismacrochirus MTeesat CoVnecewmsrrateidon TiEsdwiebTlyepe ga0i%n) MeaCnoDuemytr2u8iTone aTiisg) Noa Edible 087 316 NoiWBhFeVaFliusehspresetedBeearobiwasedTow8 toFhmal2xi -- Dry 238 BCF 386 om 000317 Wildlife International, Ltd. a rots | i : Fel, Commins o EGSEkFolk ToorDgBp 0 KS 11. x x x x " . . | i=. x % EEx E x x x Besa --6--Mean Tissueconperirations | oo= n - w | 000318 | | Wildlife International, Ltd. me ProjectNumber4S4A-134. | Fi ConenionsofFOS iNonbl FishTiohlExposed 0.86 11. |: i Ix x IA . 7. . 3 * x 3 ge. x Coma TonsCarri B6oVsentTiecm Constr oo o - = oon 000319 Ee | | Wildlife International, Ltd, resin I. [|| i | gm Fipee3. ---- Commo P70 i VilFi imoBgl Expt 000511. . . g"x 1 ! o Ch x x v4 x x * x xxx C--T--G-- x orotcois --= oanTissueConcentrations aphr w bs a jo me wm . x mm | 000320 new | Wildlife International, Ltd. _ Pr Nes454A134 | rot mA SAAReSmA | i] | 300 ZY 2DO g 2e00 | FA =gn [ream x Whole Fish E f= T g 100 At] 0 0 10 20 30 | Day | 000321 Wildlife International, Ltd. ProjectNumber 454-134 23s- Appendi1x SpeificCDuroingnthd4uWckPtiadAnlnacecyi,aotnlHdypPHarcoefvdWieodlgtlhnWeatTeeertMseassur,ed STospSuebsrt:WCPoROpSerien -- DiTloitoOnrgWaate:r:_vBieolnlgW,eLrepomis macrochirs ree-------------- Neo w _Rano ge [I (umbosm) BN=9 310-315 Har(domssLs CaCO) BOON =4) 28132 AlalimnyiltLyas CaCO) mei-9 176-178 -- oH ------re-- i -- S109)rr--50-- -82 -- ' 000322 Wildlife International, Ltd. 36. AlofePetisdes, Ogi a AMeptpaelnidniW2sIA IteostionalLd.Wel Was PatadoOrnie Cores MestComa Grog Venere Coren EE ETaara pSormieerd T DDiiotbaniic pSoHm necectnt Hpbi pSSorooimms DDDiiniacsbbmtereesh SSSooommm Samii Som Dinition pre `BBBliifaentithkriine Bot 0SS0oo5ppttl pren EDBEnMnotttSoeenTnmttoiirnnhss <pSS0o0or5mtipeugasr BBrreeggrhrecnint SSoumt EEnrdioon site pSorm CBoop tt `Bromopeopylste: Catena pS0r02oepgty pres EB[Ehpiottriauammncnte DSSp0r2ooipvgttyl Cuhtee mes SSoonme [[rrovosmy SSooitn CGCrChiopp roirdaznon pS0Sro05iitgslr FEetEFinemedtfeioiss.en <SSS0.oo0o5imptnel. CCCrrooiinnebs pSSrooemtd FFeov es Epon pSrote Sat pam Cyftuthrin Sam 0.05pg. Fein Fenpropimorph S0o0ptg1 DCDDylpreeotconinnsznole SpS0roo0iSpeetgd FRFFreanesvailgooerpeshtaelsotn <pSS0.aro02moptpidL DDeepsoerniisns fom SSSooionmt FFoRuoomioaen SSSooorttt ffDoirotmseksa pSSrooeutst pFmerpceaypben SSSooorttt fDoermlaornte fon SSononme Sot Fittmaint Line SSoot Som heept TOWen Ferd te moslcdcn oreo 5.20, mit 000323 --i---------------------------------------------------------- Wildlife International, Ltd. a7 Appendix 2 (Continued) of| i Peics eaknsdMAentdaOlspiin WsilPdliefe) tional, Competed Corniion Component Mfhrmi ommme Smosw. oper. Maewaree Neon fNeNoaerimeatin SDoanoe m fVrirogiien r-- Sosy Pic frPFreeanien pprrpooriyineessd RPimoioxp?ns fPPreeudmierivncesl SpSotorty fSSrurrei Freaks Sos Tensor Lid. WellWater" MenardConran SopSeorm DSSootsaw ppSroirvieird pSroims pSSrooissveyyt Phosmet. 002g Terbutryn. <001 ug. FPPhoruspihaotmirdon-cis Fmhormel SSS0ao0torh5eemtgr. TTeTTerebrucetahnhyeleaitzeipennese pppr<0r0or1ogpyls. : PFriins pSraemt Thbonene SLooussta FPPPrrropmooegtnlreyn. PFPrrroogopgamniec PFartioon SpS<o0or0p1tieuaegr.d. SSoos e ZSomt Son TToTTrkolrioifnatoesod-inmnledthyl TTroaeten VTrebneisstonn Vindrsi D0SS0oo1ospsatg SSooyetr SProitnt Sonat T- apgrei Smoagtm Metals CWopme SSoimw pCF=aosmin p3r5Co0met SzMoienrdera SSSarstv BNSeoitrhaagnne <So7rn2aa NSfaameiemyin SpSartaiuvm GCaomm pSriavmy Sion pl Aes tomebyTHO Noon ndFolio lrlcdomNever 5,20 000324 Wildlife International, Ltd. se Appendix "THE `ACNOANLCYESNITSROAFTIPOENRSFILNUFORREOOSCHTWAANTEESRUALNFDONBALTUEE,GIPLOLTASSUNSFIIUSMSH ATILSTS(UPEFOS) IN SUPPORT OF `WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-134 000325 eee -------------------------- Wildlife International, Ltd. "39. SPONSOR: REPORT APPROVAL 3M Corporation Project Number 454A-134 "TITLE: PerfluorooctaPnoetasssuilumfSaolnt(aPtFOeS,): AFlow-Through `BiocTeo stwn ithc thee Blun egilt l(Lr epoa mist maci roco hirn us) `WILDLIFE INTERNATIONAL, LTD, PROJECTNUMBER: 454A-134 PRINCIPAL INVESTIGATOR: Tl VWa/toire PD. Set WI ILDNLIT FE ERNATLITDO .MN ANAAGEL ME, NT Wild 5 Non, i 7: Belala Bueye AMENDED 000326 --------------------------------------------i, Wildlife International, Ltd. 0. Introduction Freshwatersamplesandtissuesampleswerecollected from aflow-throughaquatictestto determinethebioconcentrpaottenitoianlofperfluorooctanesulfonate, potassium salt (PFOinSth)e `bluegill (Lepomis macrochirus). The study was conducted by Wildlife Intemational, Ltd. and identified as Project Number 454A-134. The analyses of freshwater and tissue samples were `performedatWildlife InternationLatld,.byhighperformanceliquidchromatography(HPLC)with massspectrometricdetection. Watersamplesweredilutedandanalyzedby HPLCwithsingle quamd asssr pecu tromp etrio cdel tecte ion (LC/MS). Tissuesampleswere homogenized, extracted, diluted, and analyzed by HPLC with triple quadrupole mass spectrometric detection (LC/MS/MS). Freshwater samweprel colleectsed andanalyzedfrom December 1, 2000 to April 2, 2001. Tissue samples were collected and analyzed from December S, 2000 to April 2, 2001. Additional tissue `sampleswerecollectedandanalyzed gravimetricallyforlipidcontent.Safm or lip pidcl ontee ntws ere collectedonDecember5,2000, February5,2001andApril2, 2001 andanalyzed between April 4 and April 11, 2001. Test Substance and Internal Standard `The test substance, PFOS,wasused to prepare calibration standards and matrix fortification samplesandwasidentified asWildlife International, Ltd. identification number 4675. `Theinternal standardwasreceivedfrom3M Corporatioon Jul2y, 1998andwas assigned `Wildlife InternationalLtd. identification number4526upon receipt. The internal standard, agranular material, wasidentifiaesd: 1H, 1H,2H,2HPerfluorSoulofconticaAcnied,ChemicalAbstract Number: 27619-97-2. The internal standard, referred to hereaftaers 4HPFOS, was stored under ambient conditions. Analytical Method Water and tissue samples were analyzed for PFOS using high performance liquid `chromatography(HPLC)withmassspectrometricdetection. Water sampleswereanalyzed according to the method entitled "Analytical Method Validation for the Determination of Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) in Freshwater, Saltwater and Algal Media" (Wildlife International, Ltd. Project No. 454C-109). Tissue samples were analyzed accordingtothe method entitled "Analytical Method Validation for the Determination of Perflucrooctane Sulfonic Acid, 000327 Wildlife International, Ltd. ae Potassium Slt(PFOS) inFishTissues"(WildlifeInternational,Ltd.ProjectNo.454C-119). Forwateranalyses, `oneminormodificationfromthe validationwas incorporatedintothe presentstudy.Thischwaasn usegofe 10 ppb inplaceof100ppb4HPFOS intemalstandardconcentrationinallcalibrationstandardsandstudysample dilations.Thelowerinternalstandard nominalconcentration wasclosertothe rangeofPFOScalibrationusedin. thepresentwork.Theanalyticalmethodologyimplementedforthedeterminationoflipidcontentinfishtissue is preseonnpatgee4d2. FreshwaterSamples Ameth lowchart fotheanalysisof PFOSinfreshwaterisprsentedinFigure1. Sampleswere dBiFluOteSdiLnanad500.%0m5e%tfhoarnmoilca(cHPL(Cvgv)raodt,h9t9.t9he4y%f):l5l0i%tNiAtNhOpcaulriebrawtaiotnerasonleutoifotnhconLtCaiMninmgth1od0oul. go4y0 H.. Aliquotsofthedilutionsweretransferredto autosamplervialsandsubmittedforanalysisby directinjection. ConcentrationsofPFOSinfreshwatersamplesweredeterminedbyreverse-phasehigh performance liquid inctherotmiactoodgwrialphhyaPuesirnkginaEHmeewlretAtP-P!a3ck0a0r0dLMCmodaeslss11p0s0trHoimgehtePoerpfeorrmanciensLilqcutiidvCehroonmamtonoigtroarpihng((HPSLICM)) ol detectionmode.Themassspectrometerwas equippedwith aPerkin-ElmerTurbolonSprayionsource. ChromatographicseparationswereachievedusingaKeystoneBetasil Cysanalyticalcolumn (50mm x2mm LD, 3pmpartic iz)fedwth aKeystone Javelin iy GuardCaridge 20mm x 2 um).Theinstrument parrme sucmmaicizeedinrTabsle1. Freshwaterqualityontrol (QC)samples (matrixblanksandfortifiwecreaprtociesosendi)n `thesamemannerasthetestsamples.Freshwaterwasfortifiedwiththeappropriate PFOS inmethanol stocksolutionusing agas-tightsyringe. Matrixblanksampleswerenotfortified withthetest `substance. `TiQs C Ssamu plees Bluegillsunfish(Lepomismacrochirus)tissueswereobtainedfrom abreedingstockmaintained at Wildlife International, Ltd. Approximate20l0y indivfiishd(uagaesl3 to 5 monwetreh remsov) ed froma breeding tankandeuthanizedby severing the spinalcordabovetheopercularregion. Heads, finsandviscerawereremovedfromthebodyandwereconsideredtobe nonedibletissue. Thebalance: of the tissue was considered to be edible tissue. The edible and nonedible tissues were separately 000328 -_---- Wildlife International, Ltd. -a- homwo ith agWare ingn staini lessz steele blend der.Anappr numo berpof r 1-griam aaliqt uotse of edibleandnonediblehomogenatewereweighedintoseparate,20-mL.glassscintillationvials. Each vialwasuniquelyidentified andlabeledwith afacilitylogidentificationnumber. ThetissuQCe sampleswerestoredfrozenuntiltheywereusedinthepreparationofmatrixblankand matrix fortificationsamples,orstoragestability samples. Tissue Samples AmethodflowchartfortheanalysisofPFOSinfish tissues ipreseatedinFigure2. Edible `andnonediblefishtissuesamples werecollectedfromthetestin20-mLglassscintillationvialsand storedfrozen,ifnecesunstialanralyysi,s. Uponanalysis,tissuesampleswerebatchedbysampling intaendrtisvsuetayple.Atthattime,threeoftheappropriatetissueQCsamplesalsowere removed frtheofreemzer. Anyfrozensampleswereallowedtothaw.Onetissue QCsamplewasdesignataeds thematblarnksaimplxe.Theother issue QCsamplesweredesignatedasmatixfortificationsamples and were fortified with the appropriate PFOS stock solution(s) in methanol usinggas-tight syringes. TestandQC tissuehomogenateswereextractedasfollows.Teamilloiflmeithtanoelwrerse adtodeaechvdial.Thesampleswerehomogenizedwith atissueshredderforapproximately one `minute, Thesampleswerethensonicatedwithasonicdismembrator or approximately iveminutes `Thesampleswerecapped,shaken,andcentrifigedatapproximately2000rpmforapproximately minutes. Aliquotsoftheextractwerethenvolumetricallydilutedintothecalibrationrangeofthe LCMSmethodology with 50:50methanol: NANOpurewaterdilutionsolvent. Alioqfutheoditlutsed extractswere transferredtoautosamplervialsandsubmittedforanalysis. Back-up tissue samples were collected and stored frozen.Ifnecessary, the back-up tissue sampleswereremoved fromthefreezer,allowedtothaw andprocessedusingthesame procedures describedabove. Tissue StorageStability Samples Stability samples were prepared at test inition to establish test substance stabilityi fish tissuesstoredfrozenduringthestudy. Twotissue QCsamplesofeachfishtissuetype (edible and noneditle)wereremovedfromthefreezerand allotowtehadw. The edible and nonedisih tbislsuees 000329 Wildlife International, Ltd. "ae wereeachfortified at 0100and10,0mg Kgusingthe sppropristePFOSstock solution andagas tightsyringe.Thestabilitysamples werereturnedtothefreezer. Foll 119odaw ysoiffn rozgen storage,th sabiltysampleswereresuovedfromth freeze andanalyzed.Freshfortificatainodn `matrix blanksamplesalsowere preparedandanalyzedatthistime.Thesam (nep wanl dole d)ws ere processedusingthesameproceduresdescribedfor tissuesample analyses. ConcentraotfiPoFnOSsin fish tissueweredeterminedbyreverse-phasehighperformance liquid chromusaintgaHoewlgettr-PaackaprdMhodyel 1100HPLCinterfaced with aPerkin-Elmer APL3000LCMass Spectrometeroperatedinmaltpleionreactionmonitoring (MRM)detectionmode. The mass spectrometer was equipped with a Perkin-Elmer TurbolonSpray ion source. Chromatographicseparationswereachieved using aKeystone Basi C18analyticalcolumn (S0mmx 2mm1D, 3 um particlesize)fittedwith a Keystone Javelin CysGuardCartridge(20mm x 2mm). `Theinstrument parametersaresummarizedinTable2. . Tissue Samples for Lipid Content A method flow-chartforthe analysisoflipid contenint fish tissue is preseated in Figure 3. Edible andnonedible fish tissuesamples to be analyzed for lipid contentwerecollectedfrom thetestat. initiation,onDay62oftheuptakephase(Day 0depuranadattteirmionatnion)(D,ay 56 depuration). OnlyfishfromtheNegative Controland0.10mga.i/Lnominaltest level weresampledfor lipid content. Sampleswerecollectedin20-mLglassscintillationvialsandstoredfrozenuntilanalysis. `Sampleweightswererecordedatthetimeofcollection. Uponanalysis,tissuesamplesdesignatfeodr lipidcontentdeterminationwereremovedfromthefreezer andallowed tothaw. For eachsample, 10 mL ofNANOpurewaterwasaddedtothefishtissue inthevial and thesamplewashomogenizedfor `appor neo minx utei usim ngaatit ssuee shrl eddy er. Each homwo astg ranse ferrn edta oa2t 50-e mL `separatoryfunnelthatcontained 25mL ofchloroformand 50mLofmethanol. Eachsamvpiallwaes rinsedwithan additional 10mLof NANOpurewaterandthe rinse waspouredinto therespective `separatory funnel. The separatory funnels were shakenwithventingfor approximately one minute. Fifty millilitersofchloroform followed by 50 mL of saturated sodium chloride were added to each separatoryfunnel. Theseparatoryfunnelswerebrieflyswirledwithventing.The phwaersealelowsed to separate. For eachsample,the chlolr ayeorwfasodrr ainmed thraopuowgdehr funnel packed with 000330 ee ---------- Wildlife International, Led. au. `lasswooland anhydroussodiumsulfateinto a250-mLround-bottomflask. Anadditional50-mL. aliquot ofchloroformwasaddedtoeach separatoryfunnelandtheextractionanddrainingprocedures wererepeated.Theextractswererotaryevaporatedin awaterbathmaintainedatapproximately40C toneardryness.Eachextractwastransferredto apre-weighedlabeledscintillationvial.Each250-mL. round-bottomflaskwasrinsedwith a smallvolumeofchloroformandtherinsewastransferred tothe: respectivescintillationvial.Theremainingsolventineachvialwasevaporatedunder agentlestream ofnitorrcoleagndrey anir. Thevialswerereweighedand theweightswererecorded.Lipidcontent wascalculatedforeachsampleastheratooflipid tofishtissueweights (mg/Kg). Calibration Standards For wateranalyses, calibration standardsofPFOSpreparedin a 50% methanol : 50% NANOpurewatersolution containing 10.0 pg 4HPFOS/Land 0.05%formicacid (v/v), ranginign concentration rom0.500105.00 aL,wereanalyzedwiththeeachsamplest.PFOS wasfortified into the stanbydapaprroprdiaste dilutions oaf1.00mg a.L/L stock solution ofPFOS in methancl. Five calibration standards (different concentrations) were analyzed with each sample set. The calibration standardserieswasinjectedatthebeginningandendofeachrun,andonestandawrasd. injected, at aminimum, after everyfivesamples. Linearregression equwa eretgenieraotednusisng `peakarearesponseratios(PFOS :intemalstandard)versusth respectiveconcentrationratios(PROS: internalstandard)ofthecalibrationstandards. Atypicalcalibrationcurvefrom wateranalysesis presienFnigturee4d. The concentrationofPFOS inthefreshwatersampleswasdeterminedby substituting thepeakarearesponseratiosntotheapplicable inearregressionequation. Representative onchromatogramsof owandhighcalibrationstandardsusedforfreshwateranalysesare resentedin Figure5s and 6, respectively. `Fortissue analyses,calibration standardswere prepared in 50:50 methanol: NANOpure *water byappropriate dilutionsofether a1.00mg a./L or a 10.0 mg a../L stock soluotfiPFoOnS in `methanol. Two separate PFOS calibration standard sets were employed in the study. For Days 0 through?oftheuptakephase,andforstability sample analyses, PFOScalibrationstandardsangingin concentration rom 0.500 to 5.00 4g 4.1L, wereanalyzedwitheach sample set. For all subsequent sampling intervals, ahigher (5.00 to 50.0 PFOS ig ./L)calibration rangewasrequired tominimize dilution of the sample extracts. For each sample set, five calibration standards (different 000321 Wildlife International, Ltd. ase concentrwaerteianoalnyzsed). Theappropriatecalibration standardserieswasinjectedatthe beginningandendofeachrun andonestandardwasinjected,at aminimum,after everyfivesamples. Linearregressionequationsweregeneratedusing thepeakarea responsesversustherespective conceonfthtecralabattioinstoanndarsds. Atypicalcalibrationcurvefromtissueanalysesis presineFingutree7. Concentrationof PFOSinishtissuesampleswasdeterminedbysubstituting thepeakarearesponseintotheapplicable linearregression equation. Representatioven chromatogramsof owandhigh calibrationstandardsusedfor tissueanalysesarepresentedinFigures 8and, respectively. Forbothwaterandtissueanalysesfor PFOS,thesameandmostprominentpeak responsefor BFOSwasulizedtomonitor PFOSinal calibration,qualitycontrol,andstudysamples.Noattempt wasmade toquantify PFOSonthebess ofindividualisomerlccomponents, Fortification Stocks Freshwaterand fishtissuehomogenateswerefortifiedwiththe appropriatestocksolutionof POS preparedinmethanol. Each stocksolution was assigned aunique identifciodceathtatiwoasn recorded on astock preparlaogtshieoetn. LiomfQuiantittatsion "The methodlitofquantitation (LOQ)for PFOS in freshwatersampleswas0.0500mgaL, calulatedasthe product otfh lowest PFOS calibration standard (0.0005mg a/andtL hedil) ution factorofthematrix laksample (100)analyzedconcurrtlyviththeestsamples. The LOQwascalculatedonanindividualbasisforeachtissuesamplesinceeachentire submitedsample(ofifcingweight)wasextractedwithoutanadjustmentto 8constantweight. The LOQ (mg /Kg) for a given tissue analysis was calculated as the protof the lowest PFOS calibration standard (0.0005 or 0.005 mg &./L)andtheoverall dilution factor (LIKg)of the issue sample. TG llsrae foar 1.207 gram sample analyzed withthe low-level calibration standard set, extraction with 10 mLofmethanol followed bya 10x vohumetic itution (overall dilution factor= 82.85L/Kg),gives 3a LOQ = 0.0414 mg 3./Kg. 000332 --- Wildlife International, Ltd. a6. FreshwaterMatrix Blank andFortificSaatmiploens `Alongwiththeactualfreshwatersampleanalyses,20 freshwatermatrixblanksampleswere analyzedatperiodsthroughoutthestudytodeterminepossibleinterferences(Table3).Nomatrix interferenceswereobservedato abovethelimitofquantitation (0.0500mga.i/L). Arepresentative ionchromatogramofa freshwatermatrixblanksampleis resented inFigure 10. Freshwatersampleswerefortified attwolevels 0.0750and 0.500mga../L)orthreelevels (0.0750,0.25n0d02.00mg.i/L)ateachsamplingintervalusingappropriatestocksolutions ofPFOS prepinamerthaenodl. Freshwatermatrixfortificationswereanalyzedconcurrentlywitheachsample setand theanalyticalresultswerenot cortctedfor mean proceduralrecovery. Recoveries ranged from 84.9 to 12ofn0omi%nal concentration (Table 3). The mean and standard deviation of fortification recoavtethre0i.0e75s0,0.500and2.00mg.L/L fortificationlevelswere94.1% 5.12% (n=20),98.7% 6.60% (a=20),and 96.9% 2.95% (n=12), respectively. Arepresentativoen chromatogramof afreshwatermatrixfortificationispresentedinFigure11. Tissue MatrixBlankand Fortification Samples Along with the actualtissuesample analyses, 16 edible and 16 nonedible fish tissue matrix blanksampleswereanalyzedtodeterminepossible interferences(Tables 4and5, respectively). No intefereaces were observedatorabovetheapplicablelimitsofquantitation duringthetest. Representativeionchromatogramsofedibleandnonediblefishtissuematrixblanksamples Are preseinnFitguereds 12and 13,respectively. Edibleandnonediblefishtissue homogenateswerefortifiedwiththeappropriatestock solutiofnP(FsOS) prepinamerthaenodl. Tissue matrifortifications werepreparedand analyzed concurreatly vith eachsamplesetandtheanalytical resuswerenotcorrectedfor meanprocedural recovery. Thelevelsoffortificationwerechosentobrackettheexpectedconcentrationoftest samples. Becausemeasured concentrations increasedwith imeforthe durationofthe uptakephase, the fortification levels were frequently revised. Low-level fortification levels rangedfrom 0.100 to 5.00mg a../Landhigh-levelfortification levelsrangedfrom 105.000mgtaLo. Edifisbh tlisseue recoveries ranged from 94.8 to 111%ofnominal concentration for the low-levelfortifications, and 000333 Wildlife International, Ltd. ar from92.0to 107%ofnominalconcentrationforthehigh-levelfortifications(Table 4). Themeanand `standarddeviationoffortificationrecoveries atthelow-andhigh-levelfortifications (n =16)forthe edible fish tissue were 102% 4.61% and 100% 4.40%, respectively. Nonedible fish tissue hr ighe tlec velo frorav tnigfee idcfatr rioonmi s9,r3e e.s9ps teocti1 vely1 a(Tnadbf6 lreo5m% )9.0T.1hteome1a22n%aonfdnsotmainndaalrdcdeovniactieonnotfffroorratlitfoiwci-ataoinonnd recoveriesa thelow and ightlevelforifications (n =16)forthenonefidshitisbsuelweere 106%%5.55% and 10+3 8.4% 9%, respectively. Repre ionsche romn atot graa msotfei dibv leae nd `nonediblefishtissuematrixfortificationsamples are presentedinFigures 14 and 15, respectively. Example Calculations The analytical result and percent recovery for freshwater sample 454A-134-68,fromthe 0.10 mg 2.i./L nominal PFOStreatmentgroup,wascalculatedusingthefollowingequations: PPFIenaOtkSAeArlreSaetaRaan=tdia5or9d3=A2Ar4ne0aaly=te1P2e9a5k04A2rca/Intemal StandardPeakArea : Peskararatio= 0.4581 SInlVteoeipmneatleS3rte2ae1np8dt2a=r-d00C1o8n7centration: 10.0 pg a.i/L. R Initialvolume(Vi) =0.100mL. Finalvolume(Vf) =10.0mL Dil facu tor(tVE'iVi)o = 1n 00 PROS 0.1L nt -Fok85. 00 TY) xInternalStandardConcentration _osssi-coon oo ~sam PFOS (ng aL)insample = PFOS (ng 8.1)st Instrument Dilution Factor -00000099113377x 100 Prantof NomilConcemmion=_ PSROOSS(nHgiL0)samppllee _oousTmgaiono Oomgaill oan 000334 EEE EE EE ------_ Wildlife International, Ltd. a `PTFheOaSnatlryetaitcmalernetsgurlotfuopr,weadisbclaeflicsuhltaitsseudeussaimngpltehe4f5o4lAl-o1w3in4g-eEq-u4a3t,ifornosm:the 0.10mga.i./Lnominal Peak Area = 11192 SY-lionpteer=ce6p2t87=.73133.81 Initial Weight (Wi) = 2.2291 g. E`xStercaocntdiaorny DViollutuimone: (Ve) = 10.0 mL Initial Volume (Vi) = 0.0500 mL Final Volume (Vf) = 25.0 mL OverallDilution Factor(Ve/Wi x VEVi) = 2240mL/g =2240 Kg "PFOSatinstrument(guaigl)2.i/L) = @EXA=iintercept) _auo.msy 6287.33 = 1667 pgaill PFOS in sample (mg 2./Kg) = POSa instrument (ug :./L) x overall dilution ctor x uit conversion factor PrOSinsample (ng Ke)=po|LOTSIBLBE RE 2J0ILL tiemg =373mgai/ke RESULTS Freshwater Sample Analysis `Freshwater samples werecollectedand analyzedforPFOS concentraotniDoanys 4and 1 (twice)duringthe pre-uptakephaseofthetestandDays 0 (0and 4hours), 1, 3, 7, 14, 21,28,35, 42, 49, 56,and 62 duringtheuptakephaseofthetest. UptakewassuspeonDdaye6d2ofthe test and depurationbegan. During the depuration phaseofthe test, freshwater samples were collected and `analyzed for PFOS concentrations on Days 14, 28, 42, and 56. Measured concentrations ofPFOS in the pre-test diluter verification (pre-uptake phase) samples were <LOQ in the negative controls Measured concentrations in pre-testsamplesranged from 64.6 to 95.4%ofthe nominal concentration 000335 Wildlife International, Ltd. ~inthe 0.10 mga. treatmentgroupandfrom 71.8to 111% ofthenominalconcentrationinthe 1.0mg/Ltreatmentgroup(Table ). Duringthe uptakephaseofthe test,measuredconcentrations OfPEOSinthenegativecontrol freshwatersampleswere<LOQ. Fressamhplewscoalletcteedfrrom the 0.10and1.0mga. treatment groupsrangedfrom 68.010 113% andfrom 7.410101%ofthe nominalconcentration,respectively (Table7).Duringthedepuration phaseofthetest, measured concentrationsofPOSinallfeswatersampleswere<LOQ. Arepresentativeioncromtogramofs fressahmpwleiasprteseentredinFigure 16. TissueSampleAnalysis Tissuesampleswerecollected onuptakeDays0(4 hours), 1,3, 7, 14,21, 28, 35,42, 49, 56, and62anddepuratDiaoyns 14,28, 42,and 56ofthetest.MeasuredconcentrofaPtFiOSoinnasl negativecontroledibleandnonediblefistissuesampleswere<LOQ(Tables 8and). Theresults of anaolfeydisbleeansdnonediblefish tissuesamplescollectedfomthe0.100and1.00mg JL treatmentgroupsaropresentedin Tes and9, respectively. Representative onchromatogramsof ~ edibleandnonedibletissuesamplesfromthesamestudyfish arepresentedinFigures 17and 18, respectively. Stability SampleAnalysis | Stabilitysampleswerepreparedat test ition (uptakeDey0)andstoredfrozen. Theresuls ofsabitysampleanalysesaepresentedinTable 10. Thesedat indicatethat thetest substancewas stable during relatively long term (119 days) frozen storage at the 0.100 and. 10.0 mg ai/Kg concentrationlevels.Definitive issu samplestoragedidnotexceed 7daysduringtheconductofthe study. Tissue SampleAnalysisforLipid Content "Tisae sampleswerecollected on uptake Days 0 0 hour) and 62anddepuration Day 56 to determine lipid content nfish isson awet-weibgahsst,Theresultsof pid anailnedyiblseasnd nonedibefishissuesarepresentedin Tables 11 nd12,respectively. 000336 FE ---- WildlifeInternational, Ltd. -50Table 1 `Typical HPLC/MS Operations Parametefrors AnalysisofAqueous Samples "STROMENT.| "(lHePwLiCc)-wPiatchkaarPdeMrokidne-lEl1m1e0r0AHPiIgh3P000eMasfsSpoecLtirrgomuedtmCehrreaoqmumianptpoegdcawpihteh P`meorniktionriEnlgmmeordeTu(rSbToMl).onSpray on source. Operinasetlecetidve on ANALYTICAL COLUMN: Keystone Betasil Cscolumn(S0 mmx 2mm LD, 2-43 particle ize) `GUARDCOLUMN: Keystone JavelinCys column(20 am x2mm LD) OVENTEMPERATURE: 40C STOP TIME: 5.00mimics FLOW RATE: 250 pLimimuts MOBILE PHASE: INJECTION VOLUME: PFOSRETENTIONTIME: 7300..00%%MNeAthNaOnpoulx:e Water containing 0.1% Formic Acid 1041 Approximately 3.7 minutes * IRENTTEENRTNIAOLNSTTAEN:DARD PROS MONITOREDMASS: Approximately 25 minutes 499 amex MIONNTIETRONRAELDSTMAASNSD:ARD 427 amu 000337 EEE YWyirladltiyfee `Inmteerrnnaatnioonnaalt,,Ltd4., Jooooooo sie Table2 `TypicalHPLC/MS/MSOperational Parameters for Analysis of Tissue Samples INSTRUMENT: HewleMot det l11- 00HP iga hPercforkmaa nceLriqd uid Chromato-graph (HPLC)with aPerkin-AEPlIm30e0r0MassSpectrometerequippedwitha Pemornkiitno-rEilnmge(rMTRurMb)olmoondSep.rayionsource. Ope inmr ultia plet ionre eactd ion ANALYTICALCOLUMN: GUARDCOLUMN: KeystoneBetasil Ciscolumn (50mm x 2mm LD.,2-p3mparticlesize) Keystone JavelinCyscolumn(20mmx 2mm LD) (OVEN TEMPERATURE: arc STOP TIME: 5.0 minutes : FLOWRATE: 250 pLminute MOBILE PHASE: NECTION VOLUME: 70.0% Methanol: 30.0% NANOWapteu rcor ntaie nin g 0.1% FormicAcid Toop PFOS RETENTIONTIME: PROSMONITOREDMASS. PFOS MONITOREDMASS: Approxim4.a1mtietleys 4990 AMU 499.0 mu 99.1 aa 000338 FE ---- Wildlife International, Led. 52- Table 3 [-- -- aise [ -- -- By -- Foon -- aFomReng 13 -- a Peet MatrixBlanksand FortificationsAnalyzedConcurrentlywith FreshwaterSamples E Fmassi: 44 EeE e o iaror m T - com assaees [maeses 1 -- Samn <0t0 _ mFFaaassssss 141 Ta3n% Ca53e =205 mmFoaasmasss a11 Prt aptrari0a a2oY0e0 bw-ds oWsELs 10 Hewo-Upsie 0P0y0 i0a0 5a w[WreseieB oooo pawrooe oorays a55530 MfWsrzso 0 Hardt 0 rW00oi0 S2cno0m0 a7_ wwess 01 vee aFrn pr9y n-e WMMeersi1 FaWrroo aSo5os n%2e sMweeesn 333 rte aorosnoo p2arv0e0s sa~s wWweeesn n)73 ope a <0Ta0 =_ pr pr] i MMesin 77 PaSn Wione =@ Mwmsissie we ds aaPnroo sWo0oan0 %o~0s "TT00lM0iisoif 1u)sadie )ownaf0o0r5o0f0 aLLrL aslkseSpeolo 0)rot liowes rions22nd catswe gee ngiteverso 1. oll. malctmiydoiesn 000339 Wildlife International, Ltd. "se MatixBlanksandFociaton"sTAabnlael3yz(CeodnCtoinnuceadr)tly vith FritSales -- m -- -- T J--E ean -- FEO ng -- Pret, RE "san134) Msewn 33 Oey) Tortie ayoy ~ Messed' soenos s=as Recovery' Msn wes oaw me wen om aprn] om acio 5"% oo oe Mrmer]Rn ar5y wTorr ETSs MwweeEss 3 3 vm aVainn oY0odm0 5s_o en x wMeemn a8 vss Fr ~ oame pr00 - some os wr 8 wMoenx ume aoVas0 p4r00 _ aon Py re rr es 4 wMwoesny xx ve LRa0n0i0 poSvomsn 4P=o r fwreeenr=]@ ee aoooron 2sSo0ann0s 5p_3d WeMOekUron1 Demin o0W0ro0 aaVocora wi.nw wMMesnsn B3=mm Dye opr0r0y0 d0ooee0n Iwr_s e00s3mwgoifnJpao1.tiaeodn(ow0isQno)nieOGokt3,o0vf0ehegrLsoLr6,sbSllksl.staNmdpsloemB(e10pc0rot to miyowieoeeiralntiyo,n iid 000340 _-- Wildlife International, Ltd. sae `Tabl3e (Continued) -- SugeeNuemMbatrixBlanks-- SanadpForatificationsAnalyz-- edPConcuo rrSenktlPn yRwiSth Fre(smh-- gwaaterSamplesPet SsoioTne a Dioye T0R ) ESoO o = aoria aoe ss Mmsy @ MWiosra % Dome YIe <w0e o-a ar pit sis (00T0e0Mf5aimisgosf1g1i1nomnoxt{hCel0uQt)ioin cor3o0f0he5s1r,calkosate5510)r.otfeGalnoweest raiondseondad "Re wersegeunerl atet dusisng MacQuan, ver1.s6sioftowarneM. anualcalculationsmaydifferslightly. 000341 e-- res-- etrere-- e ---------------- Wildlife International, Ltd. ss. Tabled Cm MatrixBlanksand FortificationsAnalyzedConcurrently withEdible FishTissueSamples `SampleNumber `Sampling Interval PotassiumSalt (PF(mO g2iS/K) g) Peroent (E4BB5aa4EAss-1Ti:34) eOuG ) EBBwaaasnssz 11 Upeke BBEaBpssss 333 pe aForioTtiowfoied. oai0me0 aos00o MeawSsSuoeresemd! Recovery 557s0 0oS0mrs) 5a.3 <su1sn a5.s EBEMrvAaaBssey4 771 Uptake 01W0o%0 1 0G5o500 5P-0A BBBuAaaBssiSso 13W4d Upae 50=%0 a5iwn 10_% SBausmsi a3 Ups 500%0 aaw 0- Basi 3 Cm 3 ot BBuMrsBtTs 2# Upuke o5%m aSaw o-s Basi # Bwms 3 pe 4 00 EaAw 3 Erwostes 3 54% i5o 5T3o wBEwsassnt @@ Up S00000 parows 2_4 BEauuvasseairl a Ue 05i0%0 TaSoaw 01-0 BEaunn x3 Ue oS0o0 P5Y 0_ E (T00i0E0r5amsogat gyan3% (d0i0oWEfcTr0o3f0b0 tECrnsSole 0(120r 0, ooonw e ciioied "Rewseruegelnetratsedusing MacQuan,version1.6software. Manualcalcumlaaydtififerons slightly. 000342 eeererere------------------------ Wildlife International, Ltd. 56+ Toble4 Continued) MatrixBlanksandFortificationsAnalyzedConcurrentlywithEdibleFish TissueSamples S= urpleNumber Suapling eral __Poss Sal : Peres EMBisLe Ge By CmoO mg MaE m ear BBussns @ FSA00 PiEn 6in0 BBaBsLa i14 Depwsion 0S0000 aS3m7 0_ Eis: le = os 0 EEMAuBInS 3 Deion oS0o0 aSims 0i Bsn x Tos Ton BBAuBssS Denon o5m% awwm s-is EMASR 10 20 20 . EBWuAsBsY 53 Deprsion o5m% a>w 2"7 iEsuansvsisco3mp ioquan(00).Fo2rcashsysGo LJOoQwriscolied mist5is pjT rodurowtei ihreep.elroswt eesitnrgaiMfando avderdsic ohn1.o6se, trG,iluMonhackorl of omesmSyn Gkerspo ileh.,Allsample 000343 Wildlife International, Ltd. -s- Table MatrixBlanksand FortificationsAnalyzedConcurratlywithNovedibeFish issu Samples SumpleNumber Semplinglterl C__d _rPoss e Salt e,Parent nize NMABT 0 Tordi pike 000 wa 2050l 0 = NNMaAsSaT 0 0T1omo a5216 21065 NNNaaasspst 111 Upake loaoimo ovl5o3ss 51566 NNaaspsa 33 pate 0050000 o1a0 5- Nase 3 Sao iss sie NNaaapss7 77 Ups 01000 <057ms s-s Nass 7 100 sat 01 NNaAsS 114 puke oS0n0 pion 1.05 NASI 14 ES 25s ia NNaaass 2a11 pe 0S0o0 "o1nm 1- NASD 2 So Sos Tol NNABSTD 328 pe a50 a55m 1_08 NASI 3 EY Sie 103 NNaaAASBTSS 335 Up oS0m0 <5105 w-w NaAsie 3 0 os 108 CNwAsm1> @4 Up oS0m0 a56n 1.s NMASH 4 00 or 0 NNasBal 64 pe 0S0m a50m 1-08 NAS 4 io 08 108 NNMMAASBDz s3s6 Upse 0500 <Sa0t 1-08 CeNstaansvaaiscer6spond oT of gunn(L0G)Fomrewchanys, he L02Q4w5es oid sIot]b pjR rroorcooe mvfaeteyforwl eieadtvoirnsgMaSsaunrd,rvearsihoen vsetalrli,oMn aactnorolfhe omamtykGtseampiloeg,Ahl,l ple 000344 Wildlife International, Ltd. -s8- Tabl$e (Continued) MatrixBlanksand FortificationsAnalyzedConcumeatlywithNonedibleFishTissueSamples TSamTpleNumber Sempling terval C_ oPmointSP aalt(sPFOsS)(o ing 1uKm)eP, eet UNsiBA13Te) @ Opiak)e For0ti0f0ied Mes<su0rod Reco=very" NNMaASsZsE @62 25000 senms 1106 NNMAASBT4 1144 Deporsion o50m0 NMAS28 14 20 <0 Z 50279 Lo10d6 NNMaABsA?S 2288 Depusion 050000 <5a45 1.09 NASH 28 100 nm mn NNMaAsBIa6 4a2 Depron 05000 <w n 519 108 NASR 2 10 EY 589 . NNMMAASB37 5566 Depunaion [500 <0 . 580 11s TEesNsMthaAnvSascor5e6ponetTitofquasition(LO).Fo10c0ohabe, 5L0Q1w2islodesoltihze productothlowestcabbration standodthe overall tionfcrofthemtx banksample.Allsample eRiegmhistw=e1r0e0pgnaermt.ed ing MacQuan, ver1.s6siobowanre.Manual alulmytdifofesnigsht. 000345 ee ee-- Wildlife International, Ltd. 59. Tubes `MeasuredConcentrationsofPrVefrifuicaotiorn SoepoesePottassaiumnSalst(uPFOlS)ifnPoro-nTesttDcilt,er T NomiaT l Test Sample SamplingePeft uroosm menons, Pernt Cogmaeiarls)tin GsNutmnbDeer) Pe OTimn)e Pot(muaSsuilni()0uSm) Nomoifna? QCe6og0ci0vae rFPrETrLO Felpuke aa <<0 00qQ =b>a oi Porraa PPLrII PPrris 3+ 0o00884s40 38404 aa 0000864965 5sis a3 000053s 7 25574 10 1ri7s "3 00931479 9a479 PPrriiss aa o0771388 nnss PPrraaym a3 J1or mor "`TsradeaTrkd(0of00q0u5imngio1n )(LOG)headi0t0i5o0nf0amcge1oftheomdaixeblakBseprlo(t100of).belcoweas lbsion. emtwre grini gMad ou, verso 16sft Mm)cst maydif sight 000346 ee eee eerie Wildlife International, Ltd. a Table7 MeasuredConcentrations of PerfluoroocPottaassinumcSasitu(PlFOfS)oinnFraesthweat,er Ss on BAR Sh Beregmeot r Nominal Test Sample Concentration. Number (454A-134) a (Negative 5 Phase `Sampling Time 04 hours Perfluorooctancsulfonste, PotassiumSalt(PFOS)" 8/1) Percent of Nominal? - 322 3i3 bp<rr0e!Q 5z i: piro %a328 owns a iii n8<0Q0 03ii8: ue CEgSpo;Rmen eoumemp saa 3z2i :;;! psssoooay x: ii soea 22gs3 i:33i fRdpdieoaosn) z3:3 2:z2 pabaoae ay ah Sa ekepa ra "Resultswere generated using MacQuan,version 1.6 software,Manualcalcumlayaditffier olinghtsly. z:: :: c ::= inwai] b5#&l aa &5333 =53[i 000347 p mior------------------ Wildlife International, Ltd. "ai- "T7a (Cobntilnueed) `Measured ConcentrationsofPerfluoroocPottasasinumeSasltu(PlFOfS)oinnFraesthweat,er Samples from a a BluegillSunfish BioVceosPnacsferbneotrdroCaotTsoeiisomotne.nriisme, Pet Ga0il) Concentration. (NSumAbeRr ) 5 DPohpee TSammpeli@ngm 1 pErreeio] `PotassiumSalt (PFOS)" Nosozaifnd i0ol 235 = 8 -== iiit% & @3& 2-z 0 Ws e pe G80a00ommnm 0o45m1 8P1o 52i 2 Sii;me 0005%8% 0324 3 om is EF33 733 oSo83nisls iuuisia 48i aiit ao05e5s0 oiSos a83 a53 as8at5ns5 4o0h3r A Eo oe L%O Wi 0300mg 1 SO3s Berioofambsowe cainmBli SRaaemmapildtin(gw0ea0re0t0sr3pomgetan1/dc1ngeasDeeQyG3t5aUnvpfrtu .oo1f6i00rmHe.nrMalakaylsscphul1n.10t0m0sg)m1a1ydsloemsigehtt,evel 000348 ------------------------------ Wildlife International, Ltd. -a2- Tabet MeasuredCoinscesntSraatmipolnseosffrPomecBloecgSuionflioslh cBitPooctaoasnsncinumetSrasalTtte(issPntRfOSo)innEbaleeFi,sh CNoomreinrotTieosnt SampleNober STpiiens VPPieorfotlloiCoSocacuem(siFiOolSnn)eof (geooil) GA1 T Tppwkee, Gbeos Oupive 2 0 thom gS2a0oi5u1ns0) EEilnes 3iI S3Fo0auy%s EE3do 733 3SSo8el8o %55 aLile 32Si75a 595 iia iSSiiiona Eit55o a33 I 32558 . EEiilo6 i8@o 5 2333 EEliElhss : &i& Sp3ir7m] EEEilss} Dowie a1lt 3Jp9rs]% EEEiiadr i&i 22385% . EElEillsyt a%s 38%or on EEFSsr rte iQOthihoaen G8P1ri5ea%d EEifs ShiiT en 8aG7ri2sz6 EEEi3xs0 3ii boiay- B233 33 iinn pT rcodcstowfetreegoaverosrceabwirniv goasuanma.v4erstioeo1v6Ertrler.iarfoclrcof ThA pClmea(yKiR Do. isghT . 000349 ----- Ww ildlife International, Ltd. -6- `Table 8 (Continued) MeasuredConcentrati`oSnasmopflePs ferorm afBllueguilolSurnfioshoPBoitcoasctsoinuacmSenanltetr(aPstTFeiOusoStn)linfEdoiblneFaishtTiess,ue CNoGnocmeram!rnaTieostn GENAumRber me SeiOiemn PPootfaomriouommSelni>io(PFnst. 0 E43 7 37 EEEd4464s Upke 777 4pp22i45]] EEESs5S7 11i4 2i10t77a EEEeST aaits 11z27o6 EEnn aa 112098 EE2nm1] bF3dd 51183537 EEEs5as3 331s E771d7 EEis5n7 3233 07Fy8%] E110084 22 221523 EEEl10i1z8 : 1 75%%32 EEEll1ii1sd5 p5d 03308 EEEiii2klno 555% 5p588e35 . EEE11122270 aaa 2aa33 EB1U%S Dgusien @ 1 9@2s EEE1113337 1I1t 3f2isr0s EEi16d3 P1 21630 ElEE1ii4ss 2F1S 8524731 EEE11is5st bpiss 3310200 pSio ofebsbketlenngolaScoa, v ndoi ow]vrnSeltMcaclro psoet mt fe sg" h = 000350 i------------------------; TWIilAdlLifSe ITnUtTerRnEatCiIoNnaRlY,LLHede. eerie a4 "Table 8 (Continued) MeasuredConcentrationsofPerfluoroocPtotaasnsicumsSaultl(fPFoOSn)ianEtdiebl,e Fish `Tissue Samplesfrom a Bluegill Sunfish Bioconceatration Test Neil Tost Sapling Patryoocunonestde CoGncretnhitornat)ion SanzpliEeINeSumsber Dweee TOimme `PoetSiaSiansltss(iPFuOSm)" - EEHiiss FH22] i5T5e 10 E5Eiso pode Sf0olt4ihm i1jfr4 Ei32 iii} m ii1s3 E33h5 3ii iSinak E38 333 31is : EEla3s ;73 abar9s] E6l2 : ii;e Ha3A EBEaS7t 3iiis E5Sh3oiHf Eu75r Faid i1io0s Exh] i5 iaio rTEeweroeSnacdrlwtigiosatnfrvern sion r(ol0t0an.FoMrt aerhtcoefabeelBsiisLevOLyCl.esightS. e 000351 eee eee mle ------------------ Wildlife International, Led. "6s. ) MeasuredConcentrationsofPerfluorooctanPoetassusilumfSoalnta(PtFOeS,)in Nonedible Fish `Tissue Samplesfrom 2BluegillSunfishBioconcentratTieosnt T NomiTet T Supls Pefwotrseocancali ConGcerontaortatsion oueive `SanmpiNlNeaiNeumsber Tmreeee, 4pTrOiimmheed PotESTaaooSsamutlstsss(iPFuOSm)' aNi]pe 3!1 JSSooRma NNNMaas 3]3 S00o%%m Ne5ar aiis p3 prro N5NGao aaa S8 Soa NhNiese i33 35Si80n NNNiiotorss iIa 43 pr8] wNNiiittseh . s2a 3pS8ts Nis a 35 NN3imi Dowie JH1i PpSiroa NNNiiigts FiH8o] 3o35R Niisse 22 33%8 - on rJXiaa] Upue Si,in4lboooumms aSaan5ts xJ0ii Gi1 n 8i15r87 Niiae]] 311 iii%n NJiat 33 iso tem rere et ee SEE CPA frome vt cc wigcue, in16 vi. latcoyod sshy "eLenssdtehafnvhaloueescsorrcesaprontdotonldimaintdoefrqusandtitatiovne(rLOQi).tFnoreatchranaflhyseiss,tphelLOLQKw)as,calculatedasthe 000352 ---------------- ee-- ------ -- W0 Weilddlg0 ifee I`nn0 tteerrnnaa0 ttiioonnaa0 ll,, LLted0 d.. 0000000 "6+ MeasuredConcetrton ofefToabrleo(Coonctianuend)eslfoSnlat ePF,OSP)iontNosustiFaisnh Tissue Samples fomBogilSuish ioconomrVeasimTeeoesndtConoonF Noron Tet Suping Peicossincnioots Eonoi Concentration. aNlae `SampleNumber Tmaeke TOi7mme paToisr `Potassium Sait (PFOS)" NNNiaist 777 3i1t0s6 hNNiaEd iii EE#HH3 hiNNo3 3ii 23E3T hhNeiad a33 iibssri . NaNNisE aaa &$13 NN3iw 333 a4a3s : NNNiioaks 333 aGalo NNNiiiodts : 8a3 23Sh86 NNiNNitiilss ib2isd #E5ii11 NNNiiig %5H #0i33 NNNiiis aaa 1iI0 NNNiiIek Demin ai14 2212%%4 i NNiis Fiid iitis NiNNtiiesi aaa i551 . NNNiiissge 3ii 5w7se e neta ctr o roeoe gMoetrophrhetr1c rCsX,l or moe RgiS TO rs no loi ir dig be per 000353 ----ee--e-------------------------------------- LWiHlIdlife SInOtUerRnPatLiIoBnaBlG,TLYtEd,. oeeeeecericeeriemeivemecimnionne, "- `Tabl9e (Continued) TseSales fom Beil Sons Bisons Tost. MeasuredConcentrationsofPerfluorooPoctatssaiunmSaelts(PuFOlS)finoNonnedaibtleeFi,sh e-- Neoi T-- ea ------ Suoplng --Toe-- moomamatb-- ents CoGncoeintaraltion SaampWlieiNsuember Dmoeee TOimme `PoGtaaS5aisltts(iPRuOSm) NNMiigs H2% 82i03 10 wo u abo J NNN3-13ei0 puke d0S,4iiheiooumrns. 42iii3%7 haN3ea%] i3|| J3jiii] NhA3eid 333 ; aMi1i85Hs NNNeeaEld ii]; 8%in NxNaat iiiit 35py 3NX2ad 3i3i 4f0d] NNaS 22 nn retEeoree vbennrse eme, vr ee 1r4ener:toeWanrti cee eGn pl.h ehl 000354 ------------------------------------------------ AWdiEldEliJfeLIEntCeTrTnaUtRiIoNnaGl,JLt,d. reer reiammimeene. "a. Table 10 MeasuredCon`cSetnbrialttiyonSsaompflPes erocmaiBluoegicll SofioshcPBoittcaosanscanncSnastltr(aaPtRTifOeoSsn)otinnTissuetSteora,ge CNooncmeintnraaltion --Caariol Sa(p45o4A-N1u3m4)b __Tisso Na MAB-IS* T Noawdible e PerloroocimT esafonste, -- Pesce PolaSiustsPFiOSo)(nmg Nomiofnal <pr0e z- 0100 EESMTAMSIASSP EBdiibbllee o0u10t9 1105 NNSMTAMSAS3.SS NNooaaeeddbbllee ooanize 11142 100 EESMTAMSA3S6? BEidbdliee 8113 1133 NNSMTAMSA3S6Z__ NNeoendcdbilbee 931103 910 TSps armopdlueco(fvOahDlsLes)osrAeclpaloutndilooenwtsaeoafiqn=gu1(00h.a0000tgn5rmas(gL04G.1).T)hae OosQovweirsl00d5i0l0emongfceTorofchoelme t sb >RTeostasiwrtesgammeprletsewdeeingcMiacdQuoann,Bveercshioenr1,.62s0v00en,dsrreunce rlcs.a mTaoydsnieass iwglehe,eremosved +*fTrThohcmetshopfoccufsoweatnriArgppws,es2P0o0ni1Ae(tPsoef,1o139m0d0oapboermeetn8ss0aongwdie1c2ao0odm%sprleyf.oe ilysperess. 000355 _--_--_-- Wildlife International, Ltd. -o- `Nominal Test `Sample ga0i) Concentration USNuEAmbIIeNr ) OegsiveContr) EL2 EL3 EL4 Lipid CoTnatbelnet i1n1EdibleFish isos UPsee Sampling TOOimOmme)w OOhw 0,0 hour 0,0 hour Lipid `Weight 0O0O3H 0.0453 0.0463 TdF Fish Tissue Tissue W1eA i7g5ht ) W1ei7g9ht0" Zlus 2110 1.8577 1.8335 24,400 25300 EL6 Uptake 6 0.0231 1.5122 Ems7 @@ oouosm oLsesn Bweo ELY 62 0.0162 1.0650 15,300 15,200 BELIITS Demon 556 0on0es 1Iw3os 11857000 ELIS 5 ooiss le lel ELI9 56 0.0256 13m 18,600 on EEiD Upke @ 0o0m@y L1oal z15a6i0 EE1i4 Le@ oosws olwasm 220080 EL21 Depuration 56 Emo2 i55 oommee llawm EL3 56 00172 0.0250 1.2362 13303 "Ratiocalculatedas [lipidweight (g) + fishtissue weight (g) x 1000mg/g x 1000g/Kg 1l3e,n90o0 n18a,8w00 000356 _-----s mM r Wildlife Internationas l, Ltd. . OOOOOO__ 70. _-- NomialTest ~~ Sumple Con(cemntgraat)ion N(uAm-b1e4r) legate3Contra) NN2L NN3A NNsT NNos Table 12 Lipid ContentiaNoncdibleFishTissue Sumpling LipidWeight FishTisme Pe "Time (Day) @ Weight pak 0000Whw O0126 2206658 o0o0mm 002l2%528 210951628 Uptake @ 0o0os ssoml @@ 0oosmis 1177350%2 TTaissruea (WeEigghyt)! 6960040000 19155740000 2167520000 21965500 NnUiS Dopumion 5566 o0o0s3t1ss 1L7o2w6s3 3155700000 NL1I9S 556 0o6o 118518675 523720000 . 010 Nan puke a@ 0o0os 1sa7s6s0 37275000 NNIe @@ ooizs 2l0a1s1s3 934470000 NMz2 Down 5566 o0o0io34 1v7m0s8s5s 210000 N23 5 como RiclcuiedspdwNe2g(6) + ihGusweigh(9 5%100_0 m_f_goo10m00sGCE 17692 use 0200 ass 000357 -- Wildlife International, Ld. a. METHOD OUTLINEFORTEE ANALYSISOF INFRESHWATER 'PERFLUOROOCTANPEOSTAUSSLIUFMOSANLAT(TPFEOS,) Prepat remat frdioccn tsiuomineCl,neeA vFsotreamttBbayehp.kainngihseporegshvpomdof 4 PrTeEbpSahaiintvtsditciPoniosofagstu0d.y0Ca1l0d0QpACLsa1mtplPteRlsOeS0bwrkiltwthiseschaSlOieXdawnnda005m% vet ohraoceasnwmsce.eGAs ea.onF spn m a uvooltamoefo sspfoarrheobwes erg xvilg awnih Tove nti. wel seen ep ir 1 Ampeesmplessesb fcLCMsry. Figure1. MethodflowchartfortheanalysisofPerfluorooctaPontaesssiuumlSaflot(nPaFOtSe)i,n freshwater. CONTAIN NO cp 000358 Wildlife International, Ltd. exnoononeE FT TE AoT n B ET y a -- a to totR e ees :E p S e tn Sess: heii pt pttii2 be e + es LR my uty Ch Ava Bt 0% 0%paANDEE TopeTg pe `asforthelowestfortificationlevelinthatmatrix.Mixwellbyseveralrepeatinversions. EiA ire.Hot echt tissues. Posnnt FiSFE 000359 Wildlife International, Ltd. n- Re domom fc. Aloo bs: rE chsole,sd 1.tofaNANOa]ya t 0 ats tnd cgi oempoto ogggfom acon25mLotcronnd `Rinseeachvialwithanadditional10mLof NANOparewaterandpourrinselatorespective Scspeferpitty oiwi A001 ht rey mo redsd os chpie ty chsry i sg, AtE ohep pa, i ox antonAT apoised idan i.lotd to m chspyrd pe es ct Rymet ec vr adpisACs et het ged Het i Ri cach250iroS ieihsp lleo lor sds A ---- Figure3. Methodlowchrforth alysisofpini sus . 000360 Wildlife International, Ltd. - Area(Rao) " : . p . Conc. (Rati) Figured.SA4it5yp5piiecaml4ca(l4Fi;bOrStat)iro0ndcpur2v7esf0o.mr0P1 (eF=IrP0Ff5O9l9S33ue.oCnrresotioaswdcPe)otti.asgashinutm(eSe1ads.ltu((PmloFnOfiSt)ooirenndfmraeasthswseate=,r. 000361 reese Wirldelife Internatioonnael, Ltd.rere iret Ls. Tio oamma amie frorne wnatwros PETER. EH00 Fmitmd3 EFakrT)ede Sem @ gE ionpeEATB8 Teese et:25000 cu. ;w .. "p: " e BetteitUk iWh AUt s TE to Somme somes rss arctan oBEe5Sm2 222 Fie. 3 Eonar 259 E fR e, a a3 A mesmore erty.25000 x 7 " he = xx To To Th Uh Bh Gb TREN FigureS. Arepresentativeionchromatogramof alow-level (0.500jga.i./L)Perfluorooctanesulfonate, Potassium Salt(PFOS)standard forfreshwater analyses. (monitored masses = 499amu (PFOS ~top) and427amu(4HPFOS internalstandard -bottom). 000362 _--_------ ------__--_--_-- Wildlife International, Ltd. 76. TE Gowan -- SrpE ysrrweeerasreros MEemeEs F209 F FmSaaeAi, l8n to "Ss Eeeye =3 Ee re mmimere . | nny:25000 50 m br y " To Te fe dh Th hE ro om an Temes . stam am it em Ee nt.25000 sSniaoem 1 pwl : BHaiwnae 3#0 iw gm eet ExpucadRT 253 Efe 3 p: : s : [= Toth fe th Hh Us TR Figure 6. Arepresentativeionchromatogramofahigh-level(5.00pg 2.i/L)Perfluorooctanesulfonate, `Potassium Salt(PFOS)standardforfreshwateranalyses. (monitoredmasses =499amu(PFOS -top) and 427amu (4HPFOSinternalstandard -bottom). 000363 ----------er-------------------- Wildlife International, Ltd. 7. 495 ama 99.1 amu. Figur7e. AtypicalcalibrationcurveforPerfluoroocPottaassinumeSasltu(PlFOfS)oinnfiashttiessu,e. `Slope =4552.41;Intercept =69.58; r =0.99940.Curveisweighted (1/x).(monitoredmass = 000364 - Wildlife International, Ltd. 7. noni 2500 cps 0 o . . a 2 220 Th BW184 BE 261 Figur8e.PAorteapsrseisuemntaStalive(iPoFnOcSh)rsotmaandtaorgdrfaomroffi altoiws-slueevaeln(y0.s50.0pFgroa.mil./oLw)Prearnflgueor(o0o.c5t0a0ne0s5u.l0fo0nuatge, ai./L)calibrationset.(monitoredmass =499 amu --99.1 arm). 000365 --------------re------------------------------------ Wildlife International, Ltd. 7- intensity:30000cpa 10 5 200 8 7 s 5 4 3 2 183 1oll0_ 50 ea tze142 267 ASeoTEG3 12301s 1z8o7o7 332017 2a4b1e 2a5r1iSTeem Figure 9. A representativeionchromatogram of a high-Jevel (50.0ug &.i/L)Perflvorooctanesalfonate, Po3ta1s)sciaulmiSbraalttio(nPsFtO.S)(msotnaintdoarrdedfmorafsissh=ti4s9s9useaznalys9e9s..1Farorm)h.igh- (5.r00aton50.g0 e ug. 000366 -- E-- E ---------------- eWiHledUleifee IcnteerPneaEteiAoenTalr,, iLBtRdEr., eeeeeerminecsrmes Twhre toimem grteteroemsoraress ; [S EE2erar,om "p. B rmi3 : Sm de : = roar inlgraion Woah 80 "la -80- PER31 E non EES 1224248 2 Bw mr, wen smi fre mEiwoaorum1 [ EEI| ee eesmmuz " - |: BEE ogiWag O0E50 ho em:20 con i" | Ee item sa-dboao) Figure10. Arepresentativeionchromatogram of afreshwatermatrix blanksample (454A -- 134 --MAB P~o1t,adsisliuutmioSnal=t1(0P0FxO).S)T.he(maornriotwoirneddimcaastsesetshe=r4e9te9natmioun(tPiFmeOSof-Petrfolpu)oarnodoc4t2a7naesmuulfo(n4aHteP,FOS 000367 - WiWlidldlliiffeeIInntteerrnnaattiioonnaall,, LLtedd.. -81- RwihS,rT AE BE =BEE Ere e,ars .: SEBEeefom :: BBR=rEalEL ... He ow : lagrutonWich O47 h03 ami +=% GA m wTm - he aam ze bpi w \ 3imL 40I " an1 igmone mmehaa.rswe masesmons . SE. ding 5S7msmon ; EB=E irBdvLedLhb .s : \ Eae"n5,we SE : aEe Figure 1. Aepm rsativw onchmri ocmoomferr,odimoinr1500,(tpiocnsrpeea(sUSs0A-415343 `amu(PFOS-top)and 427amu (4HPFOS internalstandard -bottom). 000368 - Wildlife International, Ltd. -n- 10 o 8 7 o 5 a :a 'lo 3585 t17h og BtLhe Uh ntonshy:2500cps | 183 193 219 8H)h Bw26) | BWhe 2WhiSamn Figure12. EAMrAepBr-s1e,tavteiavlodncihoronmaftcoogrra=m o1f00nLoKdi)blTe fhiesatmsosuwimnadtircitxbsltaenkrseatlien (ti5m4sAo-f134PerhoroocianSaelts(PaFOSf)(omonnitaortedmeas,s=P49o9eaas-- s99i.1aa)m. 000369 -_ Wildlife International, Ltd. 5 fT Be He BT them To 0 o = Pe a 2 . 18 4 Intensity: 2500 eps. 100 128 teeter 28 ae Figur 13. A repesctive on chromatogram of a noelish asemailnkspe (454A- 134- N-MAB-1, overalldilutionfactor = 100L/Kg). Thearrowindicatestheretentiontime am. ofPerfluorooctaPnoctasssuiulmfSaolnt(aPFtOeS),. (momnasi s=t 499oamru--e99d.1 000370 - Wildlife International, Ltd. nae any: 3500 cps 10 o 8 - + fo TB BW TR . s 22 a 2 + 180 54 7 1: 261 Figure 14. Arepreionscheromnattograamotfian vedieblefistissu mat fortificsaatmipolne 2(40540A-01p34.-E-MAS-6,50.0mg21/Kgnominalconcentorvearatl idiolutnio,n facto=r (monitoredmass = 499amu --+99. 1 am). 000371 --- WWiH ldliRfeTIntAerHnaOtioEna,l,LLtHde. ||. eres = 17o50 intensity:3000cps. 4 210 a 2 u 1028 54 85 11213418; 183 51284 . FtheO)te Uh Th qh UW Yh Figure15. A(r5ep4rese1nt3a4ti-vNeMAoSnc8h,ro1m0a0mtoggraa.1m/oKfg anonmoinndailcboenfciesnhttrastsioonm, aovteralolidfiiluctaioinofanctsoarmp=lSe0000 L(ig)mmoass=o499sru-29e9.1 dcw), 000372 - Wildlife International, Ltd. -86- To ee E Eoeeninevores i =HE EeeenoIomm .: 2YEEJaeg dLh js gESeSW o,n - snon \5 Ee Th TeZh Th Te Un ThE man 1 eoeamens an ue2a renalSura EL5irne --1 EBEFLi E fims EB [ree n= enTe A-83 a "p . bi 1 .p: 2 Tir EERE RY Figure16. Arepresentativeion chromatogramof afreshwatersample (454A-134-3,dilution = 100x) fromthe0.10mga.i.L treatment group.(monitoredmasses =499 amu(PFOS -top) and. 427amu(4HPFOSinternal standard-botiom)). 000373 -- W~ iWialddlliiffeeIInntteerrnnaattiioonnaall,,LLetdd.. ~ ae " tansy: 3500 cps o 7 5 " aa 211 'a 158 24 te The Ph TW UW WER : Figure 17. Arepresentativeionchromatogramofanediblefish issuesample(454A-134-E-30,overall dilutionfactor =1420)fromthe0.10mg a.iLtreatmentgroup).(monitoredmass = 499amu. --+99.1amu). 000374 - Wildlife International, Ltd. ae 10 . 9 8 2 & 5 intensity: 3000 cps 3- 210 2 1 47o5h fhe 131 Wh 7Wo183Rb 262 285 WW Whim PE. 59am i9t9i.v1es:m). 32st of an0o.1n0edmiibleAfLiLs.htiosssueseamGplpe (,45(4A-R134-sN-3m0s, s = 000375 ProtNumer4544-134 9 Appendind Tempers of of Water the TestChambers -- To TWCapon pdetwe | TTookuSnopcr:. BPeFiOSl,Lois macrochins DUpiokoePorwe WellWiDe0 BT Berit Dayar LTE) NCeomnMuensioin Teng' Teg! Teng! Tarp! Tap! Tweeip)e C2Q0 p8i2 C2O19 8pn0 023s 80 0Z3s 8a1 CO ow a9 75 | ows ms 2 ms os As wows wows 7 TCosn meeMoSpse msFE sOoORTu,s 99 ms wm as ow-- MepneMeesnesed D3 bei CoWomeiini)on. TQap n TCeaO! ow epueCorsa 218 $1218 80 mb TeCnOg! ow 29 80 bak T2eng1!581 os a7 wm ms 9 us sw wl TComioosownmesamrFep 2 2 oo pe FROREO Messen dcomineddoe 10% culty 000376 ~ EA op Cont a || -- EPt-- e EE g Se e i r-------- HOST TETYcececeee mr ------ wo ie E)y W a wBm w wWm w LBmoa woow T Pia ? e EmE u T m ma n BW TR e wr m WewBa wEeo. BR ER A 000377 * Wildlife International, Ltd. "1 ProjectNumber 434-134 Appendix DissolvedOxygen (mg/L) of Waterinthe Test Chambers' `TSepoknsSour:ms, D"uTesitOnrgWanaisrm:, Dyo 2! i3 `75 1:5 ni `3M Corporation. PwReOlSWie Bluegill, Lepomismachrochirus. Negi Gal a74 7373 77730 777s i4 GDeoM6nmgaei iI72 7712 i77o3 i77e64 i7s THoEsIL Ibr a73 77a10 777442 7Tso 111s3 77738 71726 &p71A : 1ns 7753 7783 074 n11 2a240 0775s 1770s an 77ss 7768 773 5 is is 75 } 2%F4S pNH7Cs N77C4 N7TaC 7= 77s6 7736 I7ns EF d7o6 I7na 2i5 325 777sss A76 7s 037 73 F3 i7ss Z7es aai2 TR ive yg neo5 mere GP inBER AE * NC-Data inadvernottceolnletctledy. 000378 J WiWlilddlliiffee Intteerrnnaattiioonnaall,,LLttdd., =~ - = `PoroejetcNtNeutmbeeir4i5s4Ac-l13s4 Dissolved | Appendi5x (Continued) of Waterinthe Test Chambers' `SSpoonsoer: . POS `3MCorporation TDesitOorngaenirsm:: _ BwlueelglilWla,tLeerpomismachrochirus Dy3 Negi73Con GSoetsm7gsasit So=TL n3 7 7s 77ss 7s zz -. aa 77%s 74 7i8 72 zz z a" is 743 jr 7740a zzz i" " T5s 1s 772 7s zz z so 5 i7o5 76 022 7s zz z a5 5 07754 75 7 zz z 5555 a 7i78s a 7777a42 -z= z 5@o TA7es 774s5 zzz TR adaco con A m76y esSO hoinAmE TC : * NC-Datainadvertentlynotcollected. 000379 Wildlife International, Ltd. Loe ProjectNumber 454A-134 Dissolved OxygeAnp(pmegn/dLioxfCWoantteiniunetdhe)TestChambers' SSpuebs: Test Orpian: B3PlRMcOCgSaipLoespioomnis macrocins Din Viste: By Wel -- terNegi Col SORE 2T 77ss 3 a3 i76 a 15 a820 ` 30 0a2 0 785 2a84 0a8 1i IH 2005 00io 11 205 709 ;. iise 2a%1 n 2 3502 50 1Iss 2258 n 52 2br0 52 a2FY 3354 55812 23 . % i81 2s 352 Te nB 77ss Fy a2 7A5 a2 S3o 580 2 In abe1) 81 5 8803 bY 5s s5i 55 Hl ei 3 HLTere Ol ron eae TC 000380 - Wildlife International, Ltd. 04. ProjectNumber 454A-134 DissolvedOxygeAn (pmg/pL)eoSf(nWCaotndetriiinnutexhde) TestChambers' TSepaoSmuobrstance: 3PMRCOoSrporation TestOrgans: Blegil Lepomismacrochris Dilution W=ater: WellWater Tega CooeniaDepFra Tonga %3 a321 3 80 581 80 3 10 8802 aps 7778 pr) 82 778 8 a"s 728 % 5 378 81 a 8854 . 8s 8864 86 s5t 8561 2 81 8816 81 55 s82e 5 82 8824 83 56 82 83 TA Gissolved oxygenconcentrationof $2mg/Lrepresents % satitionifeshwter 27C. 000381 ---- e -- ee ------ Wildlife International, Ltd. "os. Project Number4S4A-134 Appendi6x Hardness, Alkalinity,Conductivity andTOCof Waterinthe NegativeControl pr PEest s e ---- `TSpeoxnSsuorb:tmce: PFOS 3MCorporation. TDeistuOPrgoaWnniesskm::_BSleuclgilWla,tLeerpomismacrochirus. pg Te mT E wma sm% wwWo Bw e who alaeCac0" Mamy wm om wm wm ww ww (magsCa/COL ") C(uombmosiecny) M0 ms we mw ms ms wm mw Tc a a a a a a a a a --en Soom WiCoprion Depurasion Phase TTeoxsOSrugbasnie:: PBFuOcSy,Lepomismacrochirus Diu Weer: WellWier Parameter Tem e E T r aTR e E m0S SeO B Se YrV im S r Te ime (ousCgaci" GooMleuCyx" vs ms vs ow mw ms mum CGoutmuibvoy m0 mo ms wm ws ww ws Toc a a a a aa a a mrel imietrersmr---------------------- 000382 - Wildlife International, Ltd. -96- ProjectNumber 4S4A-134. CultivoMoralityA3p2pdeTnredaitxment RestedEffects' -- NSSupebrsge:ti7M0vSGe _ sConrol_UptskePhase TDoioOnrWgese:: _BwlaeclgliwliLeepomismacrochins Dy Observations CNDaebmsetr NReambaienrzg Npaebne]r o? AaN %5 3s 23 aaNN o 5 :c5 aaaNwN o 777s i7 anNy 7os 5 a To 5 i2wno aia Eio 0 o0 11 wwnn 1s an 00 7o& 5 ihiiss aawwNn Ce0 a6 a o a1 iAan 0 a & 25u aiann oo o 33% aaN 0 bs 0o n4 aITM ooo 5 o5 on2 aII oo FH55 553 aaw an 0 EH55 o5 CeBs AN AN p a 000383 eemeem e eeeeeeeeeeemeee e ee Wildlife International, Led. Lo. Project Number 454A-134 Appendix 7 (Continued) Cumulative Mortality andTreatment-RelatedEffects' TSeptoSurbstmoe: 7C505ao Negative Contro--l Uptake Phase D"Tiesst oOrngatnirs,m: Weise Bluegill,Lepomis macrochirus. Nmabieer Nenber Naber Dy3 ObrAas Daoa Rem%ing Semopies 35 FaHN 0 o FH5a aaa 00o o0o aa aaNN " a iiFssy oa5 po aaN i"s oo . @a aaaN riids 0o5 %5 aaN 0 ao 25 5 aaN C0 a i0 00 o s55 aaANN 50 350 55 a~N @ a 33 3 aa aaN F3H I Obed Be Nene 000384 ee Wildlife International, Ltd. 58. Project Number 454A-134 `CumalativeMAorptpaelintdyiaand7Tr(CeoanttmiennuteRde)latedEffects! Regaine ConrolUptakePhase `SSpoanstor:es: PROS 3MCorporation "DTeisst iOrogravniisem:: ve tier Bhaegill, Lepomismacrochirus Ce Naber Number D23aT y Observations aaww ooDead o i`5 aaaw oo0 7|5 aanwN oao [ii3 aa= ooo W aa oo 1Is aa oo iW aaN C0o a aaaNNN ooo %=a aaaNw ooo SE3 aaw oo n4 aaaN 0oo 52 aaawww a0o p51o aaaww 0o BEinE choAn e pt eie dnisdoy. 3bSd Remaining aas a3 3=3 333 FHF] EF FS FS 3 iiise iii Nusber oo Sampled : . o oo o ooo o5 oo oo oo0 o` o 000385 e eee-------------------- Wildlife International, Ltd. %9- ProjectNumber 454A-134 Appendix 7 (Continued) Cumulative Mortality and Treatment-Related Effects' SToSruotos: GE05oro Negative Control ~ Uptake Phase TDiestiOorngiimnieern_: BWltcsWlaLepomismacrohis CNoaober Naber uber b3%y ObeeArNvions Beat SenIe s Semople 53 awaN H1iss "0oo "a aAwN oo 1i6s o o"5 aaIN ooo ii1 ` i"o aaaNN o0 ii1 . poA awwN o ii o s0l aaNN ii oo 25 aaN 11 oo 5%" aanNN IH1i oo0 TC i ANAp Nema 000386 Wildlife International, Ltd. "100 ProjectNumber 454A-134 Appendix 7 (Continued) Cumulative0.Mo0r8t6almigtyaa1nd.T-reaUtpmteankte-PRhealsaeted Effects' `TSpoosnsSoro: b: P"3FMOCSorporation. DTesit Oyrgatnaises:: BwlueelgiWlli,Ltepomismacrochirus. SNoab Dy ObserAvaNtions Dest 21 aan o 3i5 aawNN 00o :` awN oo 85 aIn 0 1i0 aanN 00 I1o aa 0a i1s aann 0 1v16 aaawN Ceo F1la aawNn 0oo z5 aIaN oo0 FH3 aawn 00 za aANN 0 % aaNN o0 32 aanN o EA33 aIaw o Corie BsAN Appel RNeansbieirng %i 77ss i7ss owso oaas &@a "@@ 85 55 555 : SNuempbetr 53 5o 5 5 oo 3 00 0 05 o o 0:HE 000387 Wildlife International, Ltd. "101- ProjectNumber 454A-134 Appendix 7 (Continued) CumMou rtalil ty aa nd Trt eatmi ent-v Relae ted Effects' --ST_E--pO SourbO tm:6PCFmOoSpgaa n i/l-UptskePhase D[ iu oWeimr : WeelWster------ Dy Observations CNDaemasbetmr NRaebmeinrg SNpaibeers Sne rayN S0 %0 o0 33 aa 0 0 oo 3 aaN oo 00 0 aa aaN Psy 5o i"" aaN pA a ii" pd 000 i oa"@ AaoNyI oo1 i"" o0 5 255 PaY 11 a Coa 3 o0 55" aaaxN 11i 3Fy Ss5s aaaNNN 3i1 3Pu 5 5aa AaaINX 233 3u5 0o a Fr a 2 n 1 000388 --_-- Wildlife International, Ltd. 102 ProjectNumber 454A-134 Comlsive MoArptapleintdyiaxnd7TrCeoanttmiennutedR)elatedEft' TSoupSubse: 7CFOSae 0.086mg aL-DepuratonPhase DTeik OorgViee:nB_ Weell t,rLepomismrad NCaboer Number Naber byT ObsmAvaNicns DeT Rem5ing Samaples 25 Aaxw 22 nBn o :i5 aaIN 22z nnz 0o 537 aaawN :32 nnn 0 1w anN 33 nn IIHH1 a=wxn 222 5nn s0 1i%s a 22 i1s 5 1Is poay :: 1188 EFa1 aaa i:Z 1ii8 pnnA aa 233 1ii8ss 0o %3s aww 32 i1s 0 nnn aaNN 23 1is 4 EFyS aaww 32 11 o 23 aa 32 11 35 aaw 32 11 TB O e 3 e AN AppeaaNr Namal: i Biel churvotteornisc isday. 000389 EEE EE-- Wildlife International, Ltd. 103 Prec Number454A-134 Appe7aCondtiionead) CumulativeMortalityandTreatment-ReElfafetcetsd' STTepekoOarrspeen:: BWPHMhOCSpoLeppoamisma0c.r08c6hinamgai/l--] tion Phase Dione: WoW Co Dy Obevaions frBieses) NSuesmbeern % ry 3 mn NSeuo0bmepr 5a5% aaaNwN i3z 111 o aa aawNn 333 i11 ooo aa" aanNN 3:3 1110 i 0 iia" aaaww 3:: 11100 a . 55a aaN 333 1110 oo 2 N 3 10 o s5s5e3 AaaaN 233: i100o0 I0oo TheBi A App Ng 000390 _-- Wildlife International, Ltd. C104e Project Number 4544-134 CumaltiveMoArpalpietnydainxd7TCroenttmienntu-eRde)lsted Eft' TSeSotans: CPROaS pon 087mg 1.-UptaePhase DToixtOorgWiieers, _ WBhengWltLopomlsmarch TE TT Nas ber Number --DSr01 OwAeaNNns De0os Femaiueg 52 aIN 3 aw 00 7s 55 aaN 7 a 0 777ss 15s AaAwx ii oTMwo ni AaINR 1i & IH ANASNR1X1X isz & in1s RMNeBx on2 aN ap35] x1i aawwn % 333 FHa5 AANSIRR a5Xx AR x H5 a 33 a csu AASNRIX1X aa BiH Fznl AAN.I6RX IIt =i ANAINC3XIX 5a i3 30 Cicx 5" 2 ic " 3ii Fl iicc 5S 1i Tred3Efe A=Agp1rsxNol L= ar5RegLyoenBo,i X1 Dead 2 Number Supl5sed 5o 5 o5 0 $ o o00 $ 0o 000391 Ament Wildlife International, Ltd. 105. Prec Number454A-134 Appendix [---- `Thisstudy was conductedinaccordancewiththeapprovedProtocolwiththefollowingchanges: 1. Amendment: The proposedexperimentalstartandterminationdateswereaddedtotheprotocol. 32. AAmmeennddmmeenntt:; TThheeffroeqquuennccyyoofflTigOhtCimnetesnssietmymnenatssuwreamseantoswdatsoatdhdeepdettoathle.protocol. 45.AAmmenednmetnt:: STthoeramgeetshoadloltoygy QfCor TOpClmeeawseurreemadednetdswoatshadpderdtto.theprotocol. 6.Amendment: Taheapryot.ocolwas clarifiedtoindicatewhenfishwouldbesampledforlipidanalysis 7D. eAmvenadnme:ntD:iThseoilnetedranalyrenferweanceosttamnedaasrduwrasoandDdeydt2o4thoeftphroetopcotl.e phase 9. Deviation: A Testtemperaturewas outof rangeforapproximately 2hours. ---- 11.Deviation:BiologicalobservationswerenotrecordedonDay12of thedepurationphase. 12.Deviation: Fourfish werocollectedfrom cachtreatmentonDays28and42ofthedepurationphase. 13. Deviation:The duration phasevas teint on Dy 56. 14Deviation:Fishinthe 0.97 mg 1. resent grupwere no collectedfor idaly st the cod of the uptake or depuratphiasoens. 000392 -_-- Wildlife International, Ltd. "106- ProcNumber 4544-134 Appencis Proto,AmendamdeDevnistionss 000393 --- Wildlife International, Ltd. 107. ProjectNumber 4S4A-134 PROTOCOL PERAPFLLUOORWOA"OTTCATBRAOSNUEGSLHULBFUIONCOARaCTpEOG,oPNsOCmITAEaSrNLSIoTUcLRMT)ESASATTLWITIO(TPNHFCs: U5,Eni tinAp CT a 0500Grin 05 [E--------" : Sebmsntio E5Ci35oAnpiv aar . Ptr=tey i) Wildlife International, Led. 591 mee ee Occtr 18,200 000394 _------ Wildlife International, Ltd. 108 ProjectNumber 454A-134 Wildlife International, Ltd. i" P ULUO"A HRBOBCBTLOAGNoGUpGoSmPIS OTrLASeSItE IiUOnMSoNATLWATnPTRsOES) soso rr E beTE arso SOSA 1Sonn Sk mmoruTy T-- pieCiom=eit syomcror KeakOmer [--BL KAoRT, egy NonTrtPats . Ti son AsOBATORY USE OLY me _ EER. TetCont NotCncl Lm lbmall, Sn: 01 ot SmaGott ences Arma: Eel ~ ESjofui100 . rr alleen FROTOCOLNO. 4501080WBLUIOSUBASA EmLabi otyRegut s Nos r(2723 000395 _---- Wildlife International, Ltd. 15 ProjectNumber4S4A-134 Wildlife International,Led. Wh ri,1. wiesonucS rion r gl2 emmtVmske eaby iet2 penrvdnp1re15y . Emierr rnctSapdyF0S3e340areePeeat fteGmTGa sOFtTosTte517etO SBteemiBsa,tm Aoe v Gh otd0.06RDnOo wTkopgeof oVO h 0, i2rki,r1 n kp ierB pr i mm es td 80 emTeheitfme merisonemwcmredid1m0 0e 0Shn Tr eeep [ re -- rin 3 pt cn. Bp ciaiteedoom ih tpntiptnyisnp, Dmte hh, m mieSrs trieo c.neite bdai i0ong o 11ho8wf 3l 2 brir5 DmiamTnot fh m yteepiewtelniesnibetmpd ercm 8t.eMtmer nroioefhteOdCP e odtn tr Vin Tee arsworms 1908oeer ie Pie n ort t ee btn5ktr rrrer, ydok FE ------ 000396 _--_---- ee -- Wildlife International, Ltd. -110- ProjectNumber4544-134 Wildlife International, Ltd. LaiwnmPot SnoseGLt.Totp tmpiltpidesgeVgA OPiinr, emteetrlm lestt0batmnmadArt. lt o SveTeaSmem esbern tmie e n i nei s ti tei,nis, cTs egph Jrvnerd [ taTioe e-- beaotieestre0etpbye matm,eTt mmiemupiniete.hsmepof 10 ewvbi popne ebeter 5TOE 3 . Tefopotnetnciia0 esbytnbrmignhtGm3aril , Bh o8g ieimf imdahki4 ityeooi.1 i ri 5. mfrrroetafsecemre p,pyeoid ntrp letd ramet, crpb a--id-- ,ty.i--ontltitnS rhno ieg e 38 oem ROTO. ARLHS bmn syot 000397 -_ Wildlife International, Ltd. -11- Prsoct Number454A-134 Wildlife International,Led. "4 teTphiednmeeptoeoofbi.Lebntipnfih ts ebeedhefinpby ga ly --tnotcred fopri of i Peekda 4 ibn tenfm fe ks kn dr epi 3mmoriosmhefifd.,Feuiyg.e Bisfdcwli concnooseofsicimser kbmrbteif.gF,erS,pmiets ot eokopcptt scmiat8 byepi pe 8 cd Daniireerog sd go gl abd fl cin 4nmkurmdpdonemo 7HAe,rmtioi,kLsoe.Twhevrtlopsdi Sh eirn.frbreivndtek ibeScidd 3e045tmyrbin.Teps oie ifnr.bWri,t nt, tan tcmtr ei, Hp arteete)m hod, lBoys,comadpipecnomnin vib nd wey be ite eioo o hwi.Mot mrogd.opfeS.Amytplrb r rnt iak tyedeemoo tceokm0oe idrol o pee nmtip. of 8 BeSemm1dTfotContowis dletr coofFOS dsck. ThfmfGoettr tmnt ar pdby co. A [ET ------ 000398 ree e--. Wildlife International, Ltd. BUN ProjectNumber 454A-134 WildlifeInternational, Ltd. Es pette wph pEtonbte hi 10cotmmeontstcmrb.t lg ck ti 4 cg Fciry 4hl,lbDealrfodf atbchcohebogcvckstontwilheelms ddnos of 3 icttoveerlm chem.Dtupoef pvaitca.iecozmacshfoftPhOmSenhwbxhlaebterenrt amcitnidocntofsb)t.rTphpsofli,spaamnrocfemcctetnwiolnbodehs ivilel0 l1e2t0Ktwof ----y amboSsu.TeksacmbaosewilSbntpewidpewenenbyh10mLeo efet p1ebee odf 2t41C. Tetcham nd bh bec cumin. hint gtding ig illbnipesriooddbyGeerr isee optltitl . okgg1 hsininofi dtsmvdi beecg,Ciollneith9scmve)i..A hASoonoofo6ndbioowns {etdoffo It ty tty Toca ibpried been bhEpgicsmemb0er sd id olindas cmon. Te be ieimerroncdzoduespsrsonpe. Ori i s beeptmtwheicp fn peed ptee wtls.Os0depen pmhe. a Tit-- t comineedvilmtsr, aofc bendyuit pd inso.rTdoeeite ntimtiyndino.nTshwai becspnrd t cre c ihifpfsn by dBs wrilhe0. nd PROTOCONLO: 45UIGIOUBLUBORUBISHBim LaborReeNeerL273 000399 eerie Wildlife International, Led. REN ProjectNumber454A-134 Wildlife International,Ltd. tic mcmiat asics eglas, icre ie tMbleeEiE Rrt(d003p1Sitiym0ysrplii. For -- meermooocn,eTdemteysiiseG sd ey1 ttlogin roimet5eed8mNoeeeke G1idnt,dpennrd iy 8 m idhs e hi ee ndosiYidl ot ed mtycrm iof git BemtlogrFyle etorHdi 1 i,df t shi ebyt ry sbbe.fHl ehttytetitid eietiPnmwi (,ttoitky tsmeBtrokgtYredsSfosBrtmanVed Caco Toneic T BiccloygieonsMmeasiutremenvtsiiimBdshlyiletlda,Frt0f8midi r-- inemtptt hpembon ele= LL, tt 3002,tnttl t15e pr t yi.Tt perttSpor retecre n at0 urbeerkoosm P08 eo et itSk ROTC NG. LOSS B srm oe oe7 000400 - Wildlife International, Ltd. "14 Project Number454A-134 Wildlife International,Led. a Des"t otopnlayehtot dot ke 0p80e,s m$ynedennhrei eewiin: inofECSrin 3[nrR easmr tioe--.---- : PE Fhsdwie weme-- eci pepe de ikespetinpfftty et lniof.BhJia ndd1ectt eimmistdeuhdi1hsodekof destf vi1lbSeobipilloay mi, et og iel he. [owe [ww Tm] T3i":a i i iwl swey dein PH, ESSBEaREERSVAS TTpeET e teLer peR omfesebe cs avid fer h PRGTOGOLNo. AIGHMALLBOBS Bim brRtNeeUE 000401 _-- Wildlife International, Ltd. -115- PrjetNumber S4A-134 `Wildlife International,Ltd. JrtE aeeliter,ebtentrssbyonseurd dmmmiinth atmienerrs etaAin tgbdrf, rFOsRvtet8pre. nd eita enet tv el P78csdimdn eh td t eofsiep bmeeer eerteoiel etyohat Ta ri m ubini gin n sm t vbsemlie s m nd e oi sinne wrgi -- [Mobtmmii sesd isoynor diof, FeeveSanmpetlinegf. l1e0citblm ,onltpphw cltscgfoe 80 | mempit toekritet bty DnntbCholri a tPtc rrr irT ote[ ttkwsnaO tnlAdi.tT nt tied rk mf i wo ekseee fn ekam,eUtke eeee itimk -- [Ep ---------- 000402 Wildlife International, Ltd. -6- ProjoctNumber4S4A-134 Wildlife International,Led. a dnieeecry ir i. Aweesetodemee sf HOH i rddimen tptny.foteDeoiessteofPyetetreiio nmAd, csiidn iTODek Piie,eYetspiine idicd,"Hestrieod.SV19i, nTm HeAmfoayemetnnisvheh idog$4L7b08b) FeiieroOeridd tsi Toemi Cnott 0ei edep Do nr . tectoe,rlte dPCfScpriop.oit ittiydor eritmfpeanedtdetr mebe i lboo lisonik.Te 9 te DamtaaAeonrarmlytisinospfhtirmfoteese8tberr(p7dbievbtnsiiiife cThEtiE) r - ot 0mnE etpnt-- AL rn, 1 bl ishineasasrist 33 DVaemtaro ctattoinatte.ps, ry Sm {Ter in tsi. TOC0: ARALHOURS EnemRieoers2 000403 ----eeeeeeeeeeeeee Wildlife International, Ltd. -u7- PusectNumber 454A-134 Wildlife International, Led. un. P 53.. Wtr eodti ytc rg o hobn bdsil, lofot J5.R oratmsprtme3 airmsssmn 8 so id 0 --Sei--mei. Ui. epson rtfeo typkbprdbyWHE mi,4 Tht i 1Tt Damvorstei ty ceyptnmye. 1 poy fSc fboeniepveepain ntegeipeemipiemsinps [ Lycnr ts. e RR r 5. OSaulmiiti1etsprstsdig` peenrlps, egsydng 4 coen ,cosgkhmpkn ismetnitbry eesiadhncc, rerd 1. Sraoyswhen it1 cs of ty,ocd + a NtriemmtmeT irei ion ton oiby Spt 5X t ionnot yminir.pViry phien,y if,derpd i i 1J0. Rprietmeie omaton sie seiir, ra J ---------- 000404 Wildlife International, Ltd. -us- ProjectNumber454A-134 Wildlife International, Ltd. e 12. ThpmrmeyfeSteylic n, hed m ofc nd lesions, irsof 15. Amdyosid ofpisotiin,s,d atioof,bxscpssspontcn bis3.,8 1.Tnosrt ddticpe.tsof ceo nin is psi 1156.. TAshtomitnparredc yeQwoAi,r sifongctdtberdy peti 1. itt e metemdcocf o dolgs nep Sn pttyDsihcedMbe asmt., htygity npinof fdmfedttbey giedyoDete.eTdh ciemt inci min,Amit al doiby SyDic Fumehg teprci uilaenopFmRofToeCoiL,sb stby Sd . Dk ir nihtpe rseRtptme..oApmtrigsiillbsced pofteoifnpeiss nitbhyoSstytD.i ad ihn dm.ADcharg fo prcod 60CTIeRnl1y0l,0noPGci00dDLcAOBOeeRAaTirORtiYnoPRdPACoTaIbiCoErGStAPVrIeASc8) oAk rocm MdATbyEW3iNsakem,Moens,oLni.Yor35e3c0,tAcpiisdbPyrci4nDri.E,hsLyi CPulvleryrAevdomn eT0iapkiebsirnkcoecome.postywi.tbGiooddbLyfWaarlrlysvPiciGaoeo,d,LSbLti.dToPOSolPnSse W erp i ricos mmpelesio. eCoiL)a.bRsowPdeton4pwtr:oprpieedr Wbyiclkc miien,1asnksLpkoefftp remcSonbtyopr imdtrfisve ca cn PROTOCOLNO: 4BMBLISIODA Eien ebroRemNemoU2 000405 -_-- eee Wildlife International, Ltd. us. Project Number 454A-134 -nrermcss | CURS BavaiaeP,GrPPi ANgk, 01197%5,:SPaiBs1,50 - EBg cTut Ri ASTISoarntt 8a4.1i.RSvrrecss farCbonigBpesten Ts 3 BCDGr tr TogofCamel Xe. 14, Bret:FoTh RAtTIinAWWA1a,63EPHaCilEie.n,,oi105r.nSFuenPdniennMHNaooltwsoosAkfi.rotnh .ExAroiensoWfiWtasand 5. LBBi GL.Ag.1978.DOPAC.TheDowCompa.Mind. PROTOCOLNO. 401 AORLLSOSURAS Bimmer LaboeryRe Nor UES 000406 -_-- Wildlife International, Led. 10 ProjectNumber454A-134 WLDUIPE hTEmATONuAeL. moncrio psuri AmeTosa raonocon TOYTLLPACSSOUATEOTASUMATr7m0 AATOWURCUH HE ONORC MBArT V iNorEe PROTOCOLNO: 454/101400BLU-BIO/SUBAS4 moAmmcoriMoesNTaNOm:| + ENVIRONMENTALLABORATORYREQUESTNO. 273 : mou net AbB RpE .80 REASON. hast ine h Dite t ept EOE,De tmotToConlon os : Li tiy i ER ie e cit REASON up 5 tyet J -------- Cees ehye hdeetyiitm n aine egid. LonfrrereCrTyaal ssem REASON Tr epg10mrs. J ---- AdTO teend i BtidOG TOC i REASON, Tkei TC n mt Renesas b,GA Pee 110 000407 _------------------ Wildlife International, Ltd. a1 ProjectNumber454A-134 WILDLIFE. INTERNATIONwAoL FRoICTNO: hte13: AMENDMENT:AmitMethodPg10 hi To imstatsg erewilepepe REASON:ToddsrestiltQCsg int bh otiste ably ons AMENTnDeMLidECoNstTus:10 Change A i,hil x ledfm cdss SD. Te Ab io il mpl, nD... REASON ihcpl onDy fc a ibecdpero stunt he haber. AT CAnROlRBATlORY MAMAGEENT ee -- WEnjrfee nEE/a omen Afroct, fs 7 000408 ---- Wildlife International, Ltd. me ProjectNumber 454A-134 Wiouire IreroumonusmL. momen:rsoere JsmToos xreROTOrGOL DYTL A FELOROOUACT TOTAASESAASLTLEe8,eArLOWTROUGH [------ mama: STOR: Con Fr-- EFTRCTIVEDATENore 8.300 ENVIRONMENTALLABORATORYREQUEST: ia pro---- AdRt re: 43 RASC Tbber sid L emoEGS pwiees hr of =---- [ pathos 000409 _ Wildlife International, Ltd. 1. ProjectNumber 454A-134 WILDLIFE INTERNATIONLA1oL. FROCTNO: s5tp34 Pageloll DEVTIOSATUTDYPIROTOOCNOL STUDYTITLE: BPEIROSLCUOOTERNSFTOOWCNIOTTSHA,CTFHOTENTABASLSURIENVGIMELSALAULSsTTpUFoOmLISsm)iAOrFLoOiNW-rT)HROUGH PROTOCOKOL:S4I0IOUBLLBOSUBSH DEVIATIONNO: 1 SPONSOR: 34Caponsicn PROICTNO: 4s4r-134 DADYEVEIOAFTDIOENV:IADTYIeOtpNro:sDyeencsemebser2ht 0s.2G0i00veoyewl1bwt omepme eeyfncyttcies REASONf:CDorinytdainortloi,dge.hAlodbrvypdecena hscosyDsrdntoesoveisndoiftsvniey SR TD RECTOR N co dS elrmoma------ Epeles s3o/9n3t/2) 000410 Wildlife International, Led. -124- Project Number 454A-134 WILDLIFE INTERNATIONLvAoL. PROECTNO, esuhize Page lof] DEVIASTTUIDYOPRONTOTCOOL STUDYTITLE:BERSHLUOOROOCCTAONESNTUELSSTCOWNIATETETHFNOETBATLSUSEURGBISLALAGLeTpTon0tSsI}rsArOFoLiONnWeTHROUGH "PROTOCOLNOx 54101800BLUBIOSUBASH DevIATIONNO: 2 SPONSOR: 3 Capontin PROICTNO: sth DATEOFDEVIATION:Pebrusry2e200 DEVIATION:Tp ShCpTrs eoe2pt2n ee1tt.vaOhteDoiatyo1eh9aoasfhprrw iall,bbp poe df3a n 33vm a.rbm omibra nocsitd REASON: selyaDwsBuoknshevshoitdi4n5oAlpyrEssedeevio , betptof i STUY 2 DIRE2 CTOd R ) A w svasw /o1 soit 000411 EE ------ Wildlife International, Ld. "zs. ProjectNumber4544-134 WILDUIFE INTERNATIONAL Lo. FROTECTNO: stn Pagel of Pr DevATIONToSTUDY PROTOCOL STUDYTITLE:PB ERFLUOROOCR TANESULFOWNO AITE,FHOETABSLSUC TSACLLSLALYaFtOsE S ArTeLiOWn-sT)RROUCH PROTOCNOOL: S410HOUBLUHOSUBASK DEviTONN: 3 SPONSOR: 3MCapri rromCTNO: stain DAOFDETVIATEION: Dass 6000 DEVIATION:Tph reot.otTsaot0h7emgtAc .ioppc vo i lyoni a eerie, et REASON:bifmtmtonisonn thsvi oitc erofePFnC5SMs1g7lcsLcbmeot vf womBypw.btoemsinoopnss: SoovoRRToR rE Adm---- scl 000412 _---- WViuliddlliyfeIInntteerrnnational, LLttdd..~~ -126- PeriojeeactuNmubmeberra4s5a44n-i1z34e WILDLIFE INTERNATIONtoA.L PROTECTNOP: aesunTtozlte DEVIATIONTOSTUDY PROTOCOL STUDYTITLE:BPIEORCFOLNCUEONRTORTOEASCTTIWTIOATNHNTHPEOETSABSULSUILEAGMEISLOLA(LNLTAeGpFTrOSEm:icArFaLcOhiWn-sT)RROUGH PROTOCOL NO: 4SUIOINUBLIOBIOISUBASS DEviATIONNO. SPONSOR: 3MCagonsion PROTECTNO: 54-134 DATEOFDEVIATION: Ecbmay17.2000 DEVIATION:TC hspeo te wsun csstoaa shovatn iDcsyofbaovfiepisoodn. altvillededdl, ios "REASON:CBoigmoeveynigh.bBipgsm!churofvahsnsycavDayc11aind 1r3ofdetewveieyesnaik thaestsofthestudy. . : SriZ bvpmep ToR Eaojo hdr ---- td hos 000413 ee Wildlife International, Ltd. 127. ProjoctNumber454A-136 WILDLIFE INTERNATIONUArL. romero:rasi0ha1l34 Devusmtuon rnaoTTocooL STUDYTITLE: OPERFCLUOROOOCTNANECS1U2L5E7IOWNIATTNRFOTBTLAUSESAGBISLALRCLaTpoIFnOS OAFeLsiONW-TRROUGH PROTOCKOOL asuiOUBLLSIOSUDSH DEvATIONNO: 3 sronsam: Capos FROTECT NO. ssh 134 MOTIVATION Metta DEVIATIONS:TihefioreoachmbiehimrnofiuieodbdckcsdsoabeprBpmy csondctficaOv: riirTeoeeyiDwoo etCetyMe vig, uDheiof REASON:BEotRsoleestlc SmnThEbIofcI ing a. is mS um SOSvoRECIOR aE oli sil--o SCE ES 000414 _-- WuI Wdillhdn lyijfeet Intee rnatr ionan l, LLta tdd.. t ~~ ionPproojjeea cctNNuummbbel errds4aS4n, A--i1z3s4 ie WILDLIFE INTERNATIONUAoL. PROIBCTNO: n45o0r13d4 DEvATSTIUDOYPRNOTTOCOOL STUDYTITLE:PBRILOUOCROONOCCTEAVNEISRUALE5FT7OINWOAITTNETFOBTBASSILASAUL(ToBpFtCsOSmoAITrLoOLWs-eTIBOUGH PROTOCOL NO:45410130UBLUBOSUBASA DEVUATIONNO: SPONSOR: 3 Coporn rROmCTNOS asinine DADTEEVIOAFTDIEONV:ITAhTeIpOeN:tiAnpsil2a.20t00tdeyp o hewlacngofnoswbi ncoD cinA aPEGS a Cmotsy oer n ek Ack 003n OCDnsif on REASONTphSt esrttddss ci eaeoonv Sttvvessydne gGrs in fbsebet : SOBA TRRS RATO] R N iy E-- o S ll a&b/1%l/e,e 000415 ---------------------------------------------- Wildlife International, Ltd. 19- Pres Number4S4A-134 `WILDLIFE INTERNATIOLNoA.L momcro: sini Tugelolt [-- STUDYTITLE C PERLLUORO DOCTANEUHN LSFTGWNAToFD OTBALSOSRUOMSLO ALLToEtOsSe:N rAFoLOsW)-THROUGH FROTOCOLG:cunaIBLL BORSA orvumonNo. 7 SPONSOR: SMCapmmten Fre-- DDAETVEIASTOIFOND:EVTE IAhTeIOrNe:FebruahreyShendiApdll220l0e 0 fdseoptic duct REASONf:Tirrwsetrooemienitger7emmieL resptrang sehrri Eistntt me STRa CTOR YAN Sen ots w &/mloo l 000416 mete Wildlife International, Ltd. ProjectNumber 454A-134 ~130Appendi1x0 Personne Involved ntheStd manage"mTehnetfoolfltowiisnsgkcey:Wii IntemationalLid.pessoas wer volvedintheconducto" r . 21 MHeaakrya.bKer,uDgeir,rPehoDfc,RetDsieocacrrochr,Aquatic ToxicologyandNon-TargetPlants 34. 5. KCWoiurllyatrAdDSBr.oNutoartt,oS,hePnoDeLra,AbroDqriulaeaecyioaSruB,ipnoeAlruodvgsiiyss,ttoiralChemisty 67. RaSyumsoen.dPLl.anVaenniHso,Bvieolno,gPiLstD, Scent AMENDED 0004177 _-- Wildlife International, Ltd. Frojeot Number 4547-134 81 Appendi1x1 `ThCeaUlputlaskteiRoantseOCfoTnshteaKnitnset)i,cTChonecDeenpturraattiioonnFRacattoerCo(nBsCtFaKn)t,5), "TheHalfLife ForClearance,AndTheTime To Reach90%Of Steady State. 1. Thodepurationraeconstant Ck, )wasdeterminedfromdaacollectedduringthedepuration pehqausalesatnhdewnaatsudraelfliongeodfstthehislsoupecoofntchenetrreagtrieosns.iRoensiunletwsohferacle aeqlualcsdauywsleorfaedegpetunrearitaitoeondaunnsidsnyg Excel2000infullprecismoidoen.Manual calcumlayaditffiersolignhtlsy. depuatrconsatantt (ki,)ovalnues Non-EFdisihTbislsuee 2. Thehalflifeforclearanceinissuedurinthedepuration phasewasdeterminedbythefollowing quation fp =(lk)n2 HaflorClvearae nce(dsays) Cees 1 mss 1 m7 3. Theuptake atcconstant(A, wasdeterminedfromthefollowingequation: Ck ACen whereC, sth coneenirationoffishtissuefromthe uptakeldepusationcurvenearth uptake: `midpoint(meanconcentrations romday35wereusedi calculations); whereC, isthemeanmeasuredconcentrationin wer (0.086mga.i/L); ndwhte=3r5dayos. AMENDED 000418 _--m--mmmm Wildlife International, Ltd. "2 Appendi1s1 (continued) `uptakeratecons(t,)avanlutes. Coe 1aw Project Number 4544-134 us] 4. Itisassumedthatthetwocompartment,two-parametermodelapplies.Thismetha attn hetis me: to reach90%steadystate inthe uptake phaseis about equal to the timeittakes to reach90%of `depinu therdea purt atii onpohasne.Therefore, &, ,thedepurationrateconstant,wasusedinthe followingequationtodetermine thetimeto reach90%steadystateforuptake: 09=1- 3 1 =231k, : `Timeto reach90%ofsteady-s(tdaaytse) C 1 "387 [wm 5. BCFKvalues (k,/k, )areprovided below: BCFKvalues "NonEdiFisbhTlisseue CC me 1 a3 m6 AMENDED 000419 _--_-- Wildlife International, Ltd. 133 Project Number454A-134 Appendi1x2 Report Amends 1. Original Report: Pages 1-8 Arend: Tospageswerechanged o ncetheamendedreportdt, evisod sn uresc ,3520a AnpepweSngda mat12iesanddatsdoeto th.sadilion of the pest Reason: `Toreflectthe issuing ofan amendedreport. 2. OriginalReport: Page2an4d. `Amendment: Reason oak er `TheStudyDirectorwaschangedto HearyKruegerandManagementwaschanged Reassigumentof esponsibiles ueto th departure of thoriginalStady 3. Original Report: DataAnalysisSection,Page 1S Amendment: Thisscion wasmodifi tocaythatthe estsfornormalityand homogeney meptatihnewahyssheucommppuottfertihperofoegnsrtassm.s IAOFdACdwais nttoheuissecotiloonnswmaasalmodlkifieyedt,to concentrationfactor(BCFK),the uptakerateconstant(ki), thedepurationrate `cc Foonwsetavnetsh(m ,kaa)sh,thpefahas arlrfe-rslief,evfonrc,learaBncIeG,FanAdCthpertoigrmaemtoreeyapcho90b%eothfestbeeatdywsatyat0e. Reason: Afterconsultationwiththe Sponsoritwasbelievedthatthe BIOFACmodel underestimatestheuptakeanddepurationrateconstants,andoverestimates the: kinetic concentrationfactor, theestimated time to reach90% steady state, andthe Boomer `halffor-clleairafncee. Therefore,thedatahavebeenreanalyzedusingthe `equationsandgraphicalmethods outlinedinthedraftOPPTS 850.1730Guidance 4 OrignalReport Pages 17-nd311.933 Amendment: TSheeenrehsaunltgseadndocronlclusiooenseectciolncs,iasdwmealnlbaertshe.Table 6andFigures 1-3 have. Reason: "Toreporttheresultsofthetest basedonrecalculatednumbers. AMENDED 000420 _--m--m m mm Wildlife International, Ltd. EN Project Number4544-134 : Appendix 12 Continued. ReportAmendment 5. Original Report: ~~ AdditionofAppendix 11. Amendment: Append 1 was aedtodtl ow sation i he rend portvere performed. Reason: To provide adequate explanationofhow thenew calculations wereperformed. f omyoKoylPDo ? e AMENDMENT SIGNATURES: Directorof Aquatic Toxicology andNon-Target Plants i : ofResearch $ REVIEWED BY: Quis ATsHsynasomn. : EaTa o) si4, b 77 AMENDED 000421