Document w14qDKr95Rj5EpaRYERNMgg3

ARR26-- 1106 ACUTE TOXICITY TO THE EARTHWORM (Eisenia fefide) TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC-95. (1-Octanesulfonic acid, 1,1,2,2,3,3.4,4.5,5,6,6,7.7.8.8,8heptadecafiuoro-, potassium salt, CAS # 2795-39-3) R3eMmalortknsu:mbTehre2t1es7t.sSubasmtpalnecewiassastwohrietde upnodwedrera.mbSiaemnptlceonwdaistitoanksepnriforrotmo etelsetmiengn.taPluraintayldyesteesrtmeicnhendiqtouebse. 86.9% by LC/MS, 'H-HMR, *F-NMR and METHOD oMuettlhinoedd:inTthhee sOtrugdayniwzaatsiconofnodrucEtceodnuosmiincgCaooprpoetroactoilobnaasnedd Doenvperloocpemdeunrtes (TeOsEtCtDy)peG:uiSdteatliicn,eaNrtoi.fic2i0al7.soil substrate GLP: Yes Year Completed: 2002 Species: Eisenia fofida Analytical monitoring: Test substance concentrations in soil measured by LC/MS/MS at Days 0 and 14; in earthworm tissues after test termination. Statistical methods: LCs values calculated by moving average method osrofbtiwnaormeioaflpCr.oEba.bSiltietpyhwaint.h Tnrone-altimneenatr ginrtoeurppomlaetaionnsuwseinrgetchoemcpoamrpeudtetor dcoentterromlimneeaifnssigwniitfhicDauntnndeitftf'esretnecsets(far=om0.t0h5e)cuosnitnroglSoAccSurVreerds.ion 8 to TTreasdtiongrgCao.n,isMmonsroouercTeo:wnOsbhtiapi,neNdJ from Worm Man's Worm Farm, R & K Test organism ageatstudy initiation: Adult (with citellum) Test conditions. Test media: Artificial soilof thefollowing composition: KQauoalritnzcslaanyd 321.0.k5gkg `Sphagnum peat 4.5 kg Calcium carbonate 1.125 kg Aviificial soil prepared in bulk by blending 70% sand, 20% kaolin clay and 10% sphagnum peat. The pHofthe bulk soil prior to hydration was adjusted to 6.0 using calcium carbonate. Moisture content adjusted to ~33% by weight. 25 eS9302 w ZB z om = Re 2? 3 CONTAIN NO CBI 000197 LPihgohttoipnegr:ioFdluwoarses2c4e-nhtoluirghstso,ficnotnetnisintuyomusealisguhtr.ed as 494-775 lux. Stock andtestsolutions preparation: The test substance was fciornstcednrtyrmaitixoendawnitdhtthheenteegsattsiovile. coAnptprroolxwiamasteplryep4arkegdofseepaarcahtetleys.t The soil test substance and deionized water were mixed together fvoerss2e0l.minTuetsetspsorlilosrwteorseepparreaptairoend ianntodthheeladpporvoeprrniiaghtte aetxposure approximately 20C prior to adding worms to allow the soil to Ebxepcoosmuerleesvsesflsueidl.s: One ter Nalgene beakers containing approximately 750 gof prepared soil, exceforpthte 625 mg/kg (measured) concentration, where only 650 g wouldfitin the vessel. The beakers were covered with plastic wrap, which was perforated for air exchange. NNuummbbeerr ooff treesptliocratgeasn:ifsomusr per replicate: ten Numbeorf concentrations: five plus a negative control Test media parameters: Initial and final % moisture (controls): 339,321 Initial and final % moisture (1042 mg/kg): 325,345 Initial and final soil pH in water (controls): 69,83 Initial and final soil pH in water (1042 mglkg): ~~ 8.0, 83 Initial and final Temp., C (controls): 2/22 Initial and Final Temp., C (1042 mg/kg): 22,25" 7 One outlier of 26C Element Basis: LCs - mortality. NOEC - burrowing behavior, body `weight and clinical signsoftoxicity. At test initiation, worms were placed on the surfaceofthe soil in each test chamber and observed for burrowing behavior. On Day 7 and Day 14, the contents of each test chamber were removed to determine the numberof surviving worms and to evaluate sublethal effects. On cDhaaym7b,efrosllaonwdingthoebsweorrvmastiwoenrs,etpesltacsoeidl wonasthreetsouirlnseudrtfoacteheinteosrtder tMoeatghaoidnoofbcsaelrcvuelbautrirnogwimnegabneshaovfiomre.asured concentrations: Avithmetic mean 060198 RESOLTS 00000000 MNoemaisnuarledcocnocnecnetnrtartaitoinosn:s <(LDaOyQ,0)7:8.<1L,O1Q5,6,737.103,, 164215,, 218295,04m88g,/k1g042 mglkg (as dry weight) Element values: 7-day LCe (95% C.L.}: 398 (289 - 488) mgikg 77--ddaayy LNOOEECC:: 428889 mmgg/kkgg 14-day LCs (95% CL): 373 (3-14460) mgkg 14-day NOEC: 77 mgkg 14-day LOEC: 141 mgikg All element values were calculated using the Day 0 measured concentrations in the test soils. Statistical Evaluation: The 7-day LCs; value and 95% confidence interval `were calculated by binomial probability. The 14-day element values were determined using the moving average method. Apnearlfyotrimceadl aMteWtihloddiofeloIgntye:rnaAtniaolnaylseLtsd.ofutseisntgsohiilgshapnedrfeoarrmtahnwcoermlisquwiedre chromatography with triple quadrupole mass spectrometric detection (HPLC/MSIMS). When determining the concentrationofthe test substance in the samples, the same and most prominent peak response for perfluorooctanesuifonate was used. Noattemptwas made to quaniify on the basisof individual isomeric components. On Day 14, following observations and body weight determination, all surviving worms from each treatment group were placed on moistened fhioltuerrsp.apTehreawnodramlslowweerdettohpeunreguetthhaeniirzgeudt cboynftreenetzsinfgoraanpdprsotxoirmeadtferloyze24n. priorto analysis. The LOQs (imitsof quaniitation) in soil and earthworms were 29.3 and 50.0 mg/kg, respectively, in this study. The mean percent recovery of matrix fortifications analyzed concurrently during sample analysis was 87.3% in the soil and 86.6% in the earthworm tissue. Soil `samples collected at test initiation had measured values from 78.1% to 98.6%of nominal. Measured values for samples taken on Day 14 ranged from 87.5 to 103%ofnominal. 000199 EEfar Summary of analytical chemistry dafortsoia l,Day 0: Conta, ok Gywei) cou [rr |wenion|some s|ewes| [se [| woe [wi [ we| [oo| woe | wo [a [os | ooue | wo |me| [oo |womomm|oouom|owes| `Summary of analytical chemistry dafortsola l,Day 14: Comaorisin, "mova wea) |mCo ooncony ton,| "Normnt [re| come | we|w| [oe | woe [ow [or [on| wewe [ws [oa| [es | some [ of [we| [en| wom | ou |ws| `Summary of analytical chemistry dataforearthwoDarym14,: re moi retweigh) Cocantvion Lome | wa [we | [on| mw [ow| Low | ew |ow | [ee| vee | vem | : Lb % re PGTGROST pape foraproimatt2y4 Pours1 ur ut contents. Th entice mas ofwormsolecie tracedvyfez andses zon ulanaes. omeachresent Fupwas cod, 2)Restsmaybn veala med amberofwormswere vaioanalyst 000200 cBoinotlroolg,icaanldotbhseer7v7.a0timognksgafttrerea9t6m-ehnotusrasp:pEeaarrtehdwhoeramlsthiyn athnednneogramtailve tDharyou7ghthoeurtetwheaste9st0w%itahnndo 1m0or0t%alimtoyrtoarliotvyerint tchlieni4c8al8 smg/ikogfggtorxnoiucpistay.ndOtnhe g1r0o4u2pmsge/xkhgibgirtoedup7,.5r,es2p.e5ctainvdel9y.5%OnmorDtaalyit1y4,, the 141, 289, respectively. and 488 mg/kg Worms in the 488 and 1042 mg/kg groups exhibited an aversion to the sail during sobosmeervwaotiromnssoifnbtuhrer4o8w8inmgg/bkehgagvriooruponweDraeyn0o.t bOunrrDoawyed0, aafntderionnteheho1u0r4,2 `mogn/ekwgogrrmouipn tmhaen4y88remmga/ikngedgroonutphheasdoiblusrurrfoawceed.Abfutteirn tthweo 1h0o4ur2sg,raolulpb.ut most worms remainedoutofthesoil. On Day 7, al surviving worms burrowed into the soil within 15 minutes. On Day 7, worms found dead in the 1042 mg/kg group were on the sideof the test chamber, not in the soil. ETT= Cumulative percent mortality: eemos [me o 0 o Lm [Ww Tw os [ww Lowe [wm Tw| Control response: Satisfactory Average Earthworm Body Weights: Concer9taon, [=|omonomon|omonamon | om | [ro |omomomon|omomomon | ow | [uw |omomonom|omomomon | aor | [ow [om|o omom momwo on |mo om n| [aw |omompmon| ow | ow | Lo"Stoatnsical|yafofemreontm(po<n0.o05n) f|rom Twhoe cuonntmroil gmrwouopmus| ing Due. ts lest | 000201 CONCLUSIONS The perfluorooctanesulfonate, potassium salt 14-day LCsfor the Earthworm, Eisenia fefida, was determined to be 373 mg/kg with a 95% confidence intervalof 31-4640 mg/kg. The 14-day no observed effect concentration was 77 mglkg. SStu.bmPaiutlt,erM:inne3sMotCao,rp5o5r1at3i3on, Environmental Laboratory, P.O. Box 33331, DATA QUALITY Reliability: Kiimisch ranking 1. REFERENCES "rTehqiusessttudofy twhaes3cMonCdoumcptaendya.t Wildife International, Ltd. Easton, MD at the OTHER Last changed: 6/17/02 000202 reeeeeeeeeeeeemeeeeee eet. PFOS: AN ACUTE TOXICITY STUDY WSIUTBHSTTRHAETEEARTHWORM IN AN ARTIFICIAL SOIL. FINAL REPORT `WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-111 Environmental Laboratory reject Number U2723 `OECD Guideline 207 AUTHORS: Tom Anne B. RS.inPdoerrcmhann Heary O. Krueger, Ph.D. Raymond L. Van Hoven, Ph.D. STUDY INITIATION DATE: October 17,2001 . STUDY COMPLETION DATE: May 10, 2002 SUBMITTED TO: sm 535Bush Aveo Environmental Laboratory St. Paul, Minnesota 55106 Wildlife International, Ltd. 598 Commerce Drive `Easton, Maryland 21601 (410) 822-8600 Page 062 000203 eee meee Wildlife International, Ltd. Project Number 454-111 2. GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M TITLE: POS: AnAcuteToxicityStudywiththeEarthworminanArtificialSoil Substrate: `WILDLIFE INTERNATIONAL, LTD. PROJECTNUMBER: 454-111 STUDY COMPLETION DATE: May 10,2002 GUoSo.d ELavbihoirroasuttnoarecynyrawilaPcsrscoo,tncdEouncNAVgienIccyoMm1pGl4i0(InC0cE8CwR1iH7tP,hGaEPoaoMr1d,6L01aba9o%wr;daa7t5od3r,yJPa1rp7AaaucMgcAuSsFtFa,d195a8%r,NoOhE5SCapDnoP,birNsiihneccdaitbpyiloehnoefs No. 385,AgraProductionBren, 10 Avg: 1984 STUDY DIRECTOR; TShbrvRisor, porNchoo-PlTatasard gsecets en SPONSOR APPROVAL-- 7 ro eratim ve m) p 0 lore 000204 RE EE -------- Wildlife International, Ltd. a Project Number454-111 QUALITY ASSURANCESTATEMENT Thisstudywasexaminedforcompliancewith GoodLaboratoryPractice Standardsaspublished bPyrtihenUc.Sio.fpEGlnoveoisdronLmaebnotraaltoPrryotPercatcitoince,AgEeNncVy/,M4C0/CCFHREMPar(t9s8)11670, aPnardis7,921,99187;aAnudguJsatpa1n98M9A;FOFE,C5D9 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The datesofall inspectionsandaudits, andthedatesthatanyfindingswerereportedtotheStudyDirectorand LaboratoryManagementwereasfollows: sem. TotSPbrati Tot StsPepin OtisDy? Mati Forts ByWeighsDy14 DATECOUCTED, Novenber2, 200 Never 19,2001 Nember071 November 2,201 Dente.201 DATEREORTEDTO: STUDYDRECTOR MANAGEMENT: Norbert, 01 over,201 [I ---- Novo 21,2001 Noveer28,2001 Neves 7,001 Nov2,0e01 Deobir0d01 Dumber,200 AnalDaytst,DiractReaporlt. KewDinDuiom Father January 11,2002 eyOwl00 Mays2 Janary14,2002 ese (150 January 14, 2002 Je16 y00 p- Qt ualyAssue r.eRCeporelsemntaae tnive n I S-10-R 0% 000205 eres Wildlife International, Led. SPONSOR: 3M 4 REPORT APPROVAL Project Number 454-111 TITLE: PROS: AnAct Tock StudywitthExhworm nan Atifiial Si Subsite WILDLIFE INTERNATIONAL, LTD. PROJECTNUMBER: 454-111 STUDYDIRECTOR: . Jolf\R.jor, PorNcohn-Target PlaandnIntsecsts 0 Mes HY Date oeMo `IWILDNLIFTE ERNATLIID.MOANANGEAMENLT: ey TD. Director,Aquatic ToxiacnodNoln-oTagrgyet Plants fBu ife 000206 _--_ Wildlife International, Led. se TAOFBCONLTENETS PrjectNunbes 454-111 000207 _-- nmnmnmnm--_---,------ Wildlife International, Ltd. "6TAOFBCONLTENETS -Continue-d ProjectNumber 454-111 Table1. Moisture,pH,and TemTpABeLrEofaSTtesut$r0e vv 11 Table2. FCuOmuSliatniavneMAeCritiacliiatly SnodlOSbUsBSeTrRvoeaf.tE.asivthovwonvrnss Exposetdo A Table. AverageBodyWeightsofEartiwormsExposedto PFOSinan Artifical Tabled. Day14Measured Valsfor PFOSinEarthworm Te rrr 20 Appendix 1. Persomel IVOIVEd ithAePSPHENYDIc CES n 21 Appendix3, ArtificialSoil22d TestSO PREDATION rvs23 000208 ---------- Er---- ---------- ---------- -------- ---- Wildlife International, Ltd. a. SUMMARY Projct Number 456-111 SPONSOR: 3M Corporation TITLE. PROS: AnActsToictyStywiththeEarthworminanAsificilSoi Substrate WILDLIFE INTERNATIONAL, LTD. PROTECT NUMBER: 454-111 TEST DATES: ExperimentalSStuadty t(aOtEiCoDn):, NOcoovbee1m72,b,220e000r11 EExppeeiimmneatlaTSearrmsi(naEtPiAo)n:: NDeosevmbeerr24n0,,b202e001r01 Study Completion: May 10, 2002 TEST ORGANISM: AGE OF TEST ORGANISM SOURCE OF TEST ORGANISM: Earthworm (Bisentaferia) Adult (ith cll) WRo&rnKMTarnad'isnWgoCor.m Fara M1o6aProoregoTloawnAsvhei.p, New sey 08831 MEAN MEASURED TEST CONCENTRATIONS: NTe0gamtgivaeiClonttreol 141 mgai/kg 289mg ai/kg 4T8o8dammgeaai/lkgke RESULTS: 77--DDaayyLNCOSE0C::39288m9 mgg/a.kkge DDaayyLLOCESCO,, 438783mmggaa//bb. 14-DayNOEC: 77mgai/kg 14-Day LOEC: 141 mgai/kg 000209 m _--_-- --_ mm m--_--_--_------ Wildlife International, Ltd. ProjectNumber 454-11 i" INTRODUCTION to`WxildiliifyecInitneomEaattlioonn,aolM,aLrgtydl.aycnodn,dTuchtedntihifsstourtdoynfofri3sMaetsttwhaesWcioldnldiufcetIendtfermaotmiNoonavle,mLbtedr. 19,2001toDecem4,b20e0r1.RawdatageneratedatWildlifeInternational,Ltd. anda copyof the finalreportarefiledunderProject Number 454-111inarchiveslocatedontheWildlifeInternational, Ltd.site. KeypersonnelinvolvedintheconductormanagementofthestudyarelistedinAppendix1. `OBJECTIVE Potassi"TuhmeSoabljtec(tirveeorfethfisrsotudtyowsassPtRoOdSet)eornmicnaedthhweoarcmusteuerfifnecgts1o4f<Paeyrflxupoorosoucrtapneisuldfonnaaten, artisfoiil scubsitraatle. EXPERIMENTAL DESIGN Barthworms(Eiseniafetida)wereexposedto ageometricseries offiveconcentrationsofPFOS oinf asodiml.iniAsctoranttiroonlwgarsosueplwecatsedmabienctaauisneedicoancsurrrenetlsyeinnsotilwoiftthhouetntshtueardadleitxipoonsourfePoFfOsSa.thTwhoirsmrsout0e chicas. : Four epctstetchamberswersmaintained i cachteament andcontrolgroup,with 10 twhoerfmissitneoauchhotuesrtschoafmtbetr. nObisteirovna,taionndstohfemnoortnalDiatyya7ndacnlidnDicaalysi1g4n,sTwehreemcoonrdaulcitteydtrweiacleswwitehrin analyzedwith themovingaveragemethodtodeterminean LC50value. At Vide Itemations, Lid reicencetoxic stswih a refoce. oviant, cloronctanide,ar conductedperiodicallyto ass thesentvity of th stspecies ad est oft resusfom themostcroton eyes procedures. Thesestudiesareconductedunderseparateprsoptorceoslesn,taesdindteipesndpeonrtts.tudies. Asummary 000210 _-- Wildlife International, Led. -5- Projct Number 454-111 MATERIALS AND METHODS "Thestudywasconductedbaseduponthproceduresoutedi theprotocol, "PROS:AnActs Toxicity StudywiththeEarthworm inanArtificialSilSubstrate"Theprowtasobascedouplon proceduresoutlinedinthe Organizationfor EconomicCooperation andDevelopment.(OECD) Guideline No. 207, Guideline for Testing of Chemicals, Earthworm, Acuie Tozicly Tests (1). ChangestotheprotocolaelsdinAppendix2. Test Substance: Thetestsubstanceusedwas receivedfrom 3MonOctober 29, 1998andassignedWildife InterLidn.idaenttificiatioon numbaerl46,75.Thetetsubstancewas awhitepowderidentifiedby the SpoasFnC-9s5,loot2r17.InformationfromtheSpousorindicatedthe purityofthetestsubstance 10be 86.9%withanexpirationdateofAugust31,2006. Thetes substancewasstored underambicat conditions. TestOrganism Adult cartiwoms (Eisenia ftida) tere obtainedfromWorm Man's Wom Fam, Monroe TownsNehwiJerpse,y. Approximately 18.5hoursprior tho st,280 womms werosclctedand placedina containerofpreparedartificialsoilsubstrateadjustedtomoisturecontentofapproximately 33%byweight,for th acclimapteriiodn.Onthedayoftestntstion,thewormswererinsedbriely withdeionizedwaterandindiscimioatelydisebuted bypais ntogroupsof 10wormscach. Each 8510pofwormswasweighedthnplacedonthesoilsurfaceinth appropriatetestchamber. The wormswero notfod duringtesting. Artifical Sil and Test Soils Preparation Theartificialsoil waspreparedinbulkbyblending 70%sand, 20%kaolinclayand10% sphpeaat(gApnpenudixm3).ThepH ofthebulksilpriortohydrationwasadjusted t6.0using. calciumcarbonate. Thebulkartificialsilwas tore in asealedcontainerunderambitconditions `untilusdtopreparethe estsoil. 000211. _--nmnm Wildlife International, Ltd. ProjectNumber 454-111 "10- Testsoils were preparedbystirring thetestsubstanceinto aportionofbulksoilbeforemixingit `withtheremainderofthe soilanddeionizedwater (Appendix 3). Sufficientdeiowan teriwazsae ddd ed tcootmphoendernytasrvtiefriscmiailsxoeidlftooraacthoitealveofam2oimsitutreescotnotenstaorfeaaphpormooxgiemnaetoeulsy m33i%.byNewgeaitigvhetc.onTtersotlssaoiill `bwuansdprreedpiatreydgirnatmheosfparmeepmaarnendseorialswteersetasdodiel,dbtutcwaicthhoofutftouhretasdkdcithiaomnboefrtsefsotrstuhbsctoanntcreo.lgrSoeuvpe,n iththe exception of he625mga..kg cononzaton iswhich nomore ha 630.3wouldfit ntothe estchambers. tho Spo"nTessotrc,onTcheentrrfaotrioentsweetrceoancdejnutsrtaetdifoonrsatnhedptuhreitLyoSf0PvFaOlSu,ewhairchrweapsorrteepdoarstmeidtlloibgream8s6o.9f%tbeyst `substanceactiveingredient perkilogram ofsoil on adryweightbasis (mga.i/kg). Testsoilswere. preparedandheldovernightatapproximately 2042Cpriorto addingwormstoallow thesoilto becamels fli. Thetargetnomial concnrawterieo78n.1s, 156, 313, 625,and1250mg akg diysil. Meanmeasuredconcetrations ath imeofsoilpreparationwero 7.0, 141,284898,a,nd 1042mg a.i./kgsoil. Analytical Sampling `Duringthetest, samplesoftheexperimentalsoilswerecollectedtodeterminethehomogencity `andstabilityofthetestsubstanceintheartificialsoilandto verifytestconcentrations. Samples were `cdllectedatsailpreparationtodeterminehomogeneity,measuretestconcentrations,andestablish Day 0valuesforevaluatingstabilityoverthecourseoftheexposureperiod.OnDay 14, samplweerse collectedfromonereplicateofthecontrolandtworeplicatesofeachtestconcentrationto assess `stabilityofthetest substanceundertestconditions. At testtermination samplesoftissue fromthe earthwormsincach treatmentgroup were analyze toermine theconection of PROSinthewormtsafromcachrstmentgroup. All surviving wormsfromcachtrtmentgroupwereplaodcamoistenedierpaperfo approsimately 24hours topurgegutcontents, Theentiremas ofwormscalledfrom cachtreatmentgroupwas composited, euthanized by freezing andstored frozen untilanalyzed. 000212 -------- een. ZWYiildAlyifeeIInntteerrnnaattiioonnaal',, LLttdd.. ue PoPriojeecttNNaummbbeer4a5s4-t1i1n1 Anaiytical Testing methodology(Appendix 4). Chemicalanalysisofthesoilandwormsampleswasperformedusing HPLC/MS/MS TestChambers Thetestchamberswereone-liter Nalgenebeakerscovered withplasticwrap whichwas p coenr cefnfootarnrr adiarraetepxlit icchoaae tnen.gde. Alltest chamberswereidentified with theproject number,test PhysicalPropeorfTtesit Seoisls Soiltemperaturewas measuredinonereplicateofeachtreatmentand controlgroupattest i`nwietrieatmioandanodtseirlmisnaamtpiloensucsoillnegctaehdafnrdo-mheeladcthhberamohmeotfesro.l pMroepiasrteudrfecoonttreeanttmaenntdapnHdmcoenatsruolregmreonutpss atestnation. Anadlifonlsilmoirecontentmeasurementwas madeaft tet tiation to c`ovelrliefcyttehdefsioomilmooiesrteuprel.icAattetoefsetatcerhmcionnattrioonla,snadmtprleesstfmeonrtmgoriosutpu.recMonteenatasndpuHormfepaeHsuwmreeremeemnnatsdtweeosren: suspensionsofsoilanddeionizedwateusing awaterproofhand-heldpHmeter,andarereportedas PhHe,nvwaeliugehsi.nSgotielsmooiilsstuarmepcloenatfetnetrwdraysidnegteorrmsitneldebasyt2m4eahsouursina gtahpepirnoitxiiamlawteeilgyht1o0f5tCh.esTohielpsearmcpelnet, %Mostar = (vtweight-diyweight +wetweight] 100 Environmental Conditions teDumrinpgoftaehpeptrerosxti,amtahtetewloy2rum0sr+we2reeCm.aiAnlteatienmepdeirnataunreenwvaisromnemaesnutraelchaamlbaesrtseonttodmaaliynitanitnhea pernovviirdoendmenbtyaolvcehrahmebaedrf.oTrhcepehotboplesrioTdhduertianrggtethegthesttwtaasst2y4hdoouirsnotfhceotnettinwuaosusalpipgrhotxpiemartdealyy 400and0 asver8 ifiedo0 nDay0 7of h, e est. 000213 ee eee e e ee Wildlife International, Ltd. Projoct Number 454-111 2Observations Attestinitiation,thewormswereplacedonthesurfaceof thesoilineach testchamberandwere: obs fore burrr owiv ngbee havd ior.OnDay 7andDay14of thetest,thecontentsofeach testchamber `wereremovedto determinethenumberofsurvivingworms. Allsurviving wormswereobservedfor `beoh r paa thov logii calo abnor rmaa litil es. OnDay7,followingobservations,testsoilwasreturnedto the testchambersandthe wormswereplacedonthe soilsurfaceinorderto observeburrowing pl`baeohoavdioro.n mOoniDsatyf1i4e,r pfaolpleowiinngcoblssernvtaetisotncshaanmdbberosdaynwdeigahltldoewteedrtmoinpautirogne, tshuerivrigvuintgwcoonrtmesntwseforre testing approxim2a4thoeulrsy. Theworms weretheneuthanizedbyfreezingandheldpriortoanalytical `Body Weights OnDay0,groupweightsfor allreplicateswerecollectedpriortobeingplacedinthetest chambers. OnDay14,all survivingwormswereremovedfromcachreplicatetestchamberand weighed. Priorto weighing,wormswererinsedwithdeionizedwaterandblotteddryonpapertowels. `Groupbodyweightsweremeasuredfromeachtestchamber,andaverage individualbodyweightswere calculated. - Reference Toxicity Test A referencetoxicitytestwasconductedunderaseparate protocoltodeterminethe LCS0value forcanthwormsexposedtothereferencetoxicant,chloroacetamide,inthe soil(2). Thetestwas conductedundercondition sil tothos uso itistet, and ithcathwormsfromthesame source,tomonitorth techniquesusdandscsitvty ofth tetpopulation.Thewowerre mexpossed 0cloroactamide ithsilstnominal conoenatonsof 7.5,15, 30 and60mga. dy sil. StatisticalAnsyses The LCS0valueand 95% confidenceintervalwerecalculated using thecomputerprogramof CE. Stephan (3). TheprogramwasdesignedtocalculatetheLCS0valueandthe95%confidence ioervlbyprobitnly,movin avesgemethodoth isosprobablymethod(4,56).Tntis study,the7-day LCS0valuewascalculatedusingthebinomialprobabilitymethodandthe14-Day 000214 _--_------ Wildlife International, Ltd. n- Project Number454-111 LesCtSt0evramlinuaetwioanscaalwceullaltaedutshienmgetshenmcohvainnggeianvebroadgyemweetihgohdtd.Mureianntbhodtyewsetiwgehtesdaettteersmtiinnietdifaotriocnaancdh. caadm controle group. Treatmentgroupmeanswerecompared 0controlmeans ithDuets occured. test (a=0.05)usingSASVersion 8 (7)in order to determineifsignificantdifferencesfromthecontrol Environmeatsl Conditions "REAS NDU DISL CUT SSIS ON +2C,remainingat22-7C throughoutthetest, Thewormswersmaintainedundercontinuous lightingatanaverageintensityof 627 83.9hu,with arangeoverthesurficeofthetestchambersof 97S ox. Physical Properties of TestSoils teMreasmuarieempenrnetsscoatfdsiioniTlobplHn,1,.tSeimlpepraHt,urraenagenddfrmooimst6u.r9e1c8o.n1teatntteasttntietstiionnitaiantdifonraomnd8t.3etsot 8.4attestteminsion. Solltemperaturewas22C withinthedesired angeof20 %2Cattest inaindatttestitermoinatnion. AL estteinationthe 1042mg .ikgReplicate Asoltemperatare was25C. ItwassuspectedthattheReplicate Achamberwasplaced toocloseto alightballastand `wasnotindicativeofthesoiltemperatureforthattreatmentgroup. RepBlwai smc easa uretdae nd fotou ben 22d C. Soilmoisturecontentduringthetestrangedfrom29.5to33.9% attestinitiation contentduringthe est. `andfrom31.4to34.5%attesttermination,indicatingthattherewaslittlechangeinsoilmoisture. Observasions "Thedatafromweekly observationsofthewormsfo metal andatersigns of toxic are preseinnTatbeled2.Therewerenomortalitiesinthenegativecontrolgroupduringthe 14-daytest. Al controlwormswer nomain appearancoandbehaviorthroughoutthotstpaid.OnDa7thyere washighmortalityinthe488and 1042 mga.i/kgtreatmentgroups. OnDay14therewassome 090215 --_--_-- ee Wildlife International, Ltd. Prec Number 454-11 motaliy inthe 141and289mg i. restmentgroupsandveyigh mrtalty nthe488mg kg rou andcompletemortalityinth 1042mg2. group. Worms ithe 438and 1042mg. tmentgroupseibitedanaversion totes during `ko g gb rous powfe ebrurrorotv wina gmboewt headv,i inordo oinDn taheys010.42Onndga.y 0aftgerrooupnmehaonuyrr,asoimneewdoramseinstohle s48u8rm.g Aftertobuts,al butane oer inthe 488mg. groupbadburrowedbtnth 1042mg roupmstworms remain ut of th si. OnDy 7, ll uring wormsburrowed nothe sil t`estwci1ht5mahinnuimttensib. tOehnsDria.y7, wormsfounddeadinthe 1042mga.i./kg groupwereonthesideofthe A7-DayLCS0 `StatisticalAnalyses imationof 398mg... with 5%confidence hits of 289 od 488mg 2. 0 vasdetermined usinthbinomialprobitymethod, sincethedat idno titerthe probit method omoving vege mth. A 14-DayLCS0wasdeterminedusingthemoving average f`omertthhoedisnidnicceattihoendtahtattdhidencootfnifttihdeeplroibistmmeathyobdo.1T0h0emcloovsiendgusavertahgeemoewtehromdwaarsaaccenpttahble2e8x9cmepgt caa.rit/ikvgosromilstheaxnpionstehdeto1P41FmOSgia.ni.a/nkgastoiiflitiraelastomienltsgruobups.tTwhearsedfeortee,rtmhiene1d4t-odbasyL3CS7530megstii.matsifioolnr ith 95% onde lit f 316an 440 3... Afarsvendaysof exposurethnaobsrvd-fftccaNOcECr,vtasr28a9imgoa.nb,g 'soNiOlEaCnwdasth7o7lmowgesti-obksesroveidl-aenfdfetchte-cLoOncEeCntwraatsion1,41LmOgEaC,.wassl4.88 mg a.i/kg soil. The 14-Day Averageindividualbodyweight,andthe cansinbodyweightfom tst nition 10ts {erminton,weecausedfromtheDay 0andDay 14 replicatemeasurements(Table3), A slight lorsessiimnebnogdroyuwpesisgihntcftrhoemwtoerstmisnwiteiateioonttoftesuttrernmitnhattiosnkw.aMseeaxnpeoctdeydwienigbhottahttthtecornmtirnoaltainodn 000216 _--mm---- eee Wildlife International, Led. Lis Project Number 454-111 aswellasthemeanlossinbody weightamong wormsinthetreatmentgroupswere statistically significant(p<0.05)at488mga.1./kgdrysoilcomparedtothecontrolgroup. Table4continth 14 daymeasuredvas oxPFOSincartwor tsa, ReferenceTosicty Test he 1-dayLCS0valu orcrthwormsexposedtothesefrnce substance,horostanide,in anartificialsoilsubstratewasapproximately19.4mga.i/kgdrysoilwith a95%confidenceintervalof 15and 30mg a.i./kgdry soil(2). Theseresultsare consistent withthose observed in previous studies, `andverifytheadequacy andconsistencyofthemethodsusedinthisstudy (454-111). Sample Analysis Chemica analysis of est soils an catworm ss was codictedatWil nmin, Ltd. andisrepionArppt endeixd 4. concLusIons "The 7-day LCS50estimationfor earthwormsexposedto PFOSinanartificialsoilsubstratewas Ld OBeCwtasetdor beem 39ti 8men gtoabme .io.4/d ik3g8.mnThgee.7-dday NTOhEeCw14a-sdadyeLtCerSmDineesdtitmoabtieon2f8o9 msgaat.ih/okmgasndetxhpeos7e-ddtaoy dPeFtOerSmiinnan0arbtifi7cimalsgoiilsubsntrdattehweaLsOdEeCtweramsine1d4t1 ombge33473.mg a.i/kg. The 14-day NOECwas 000217 ------------------------------------------------ Wildlife International, Ltd. "16 REFERENCES Project Number 454-111 1 OTEesCtsD. OGuidreligne a20f7o.nrE1ci9o8n4.ozmiGacuaCdoetalpieniretfiooornTanensdtiDngeovefloCphmeemnitc.als, Earworm, Acsts Tasiity 2 WEiarfthewoIrnmteirnannaAtironialtLtad.So2i0lS00i.bsCahtoer.oaPccsjteacnitdNeo:.A1r00ARc1s0t5e,Titty Sudy withthe 3 StPeeprhsaonn,aCCEo.mmu1n9i7c8.atiUoSn. EPA,EnvironmentalReseachLaboratory,Duluth,Minnesota. 4 FLionnnedyo,n.D.J. 1971, SatsicalMethodsnBiologicalAssy. Second Bltion, ifn Press, 5 Thompson, W.R. 1947.BacteriologicalReviews. Vol.I, 2:115-145. 6 StEevpalhuaant,ioCnsE.. Am19e7r7i,cMaentShoocdisefyorfoCrTaesltincgalnaadniMLaStne)rgi.alsA.quuba.tTNoo. SrTiP 6c34ao:n65ld-H8o4a.zgaryd 7 SAS InstituItnec,. 1999. SASISTATUser'sGide, Version 8Cary, NC, SASIst,Inc. 000218 _-- Wildlife International, Ltd. ProjectNumber 454-111 1. Table1 Moisture, pH,,,andTemperature ofTest Soils" MounMeaswed Concentration @gaikd) niTteiston Moisture Co?en' Test Test eo Tomiomion Initiation 7, 53590 Moisture Content pH, TCTR0CEW ) Negative Contd 339 I) n 21 m0 319 9 ns ut s a sus En 51 2 30 is 310 81 2 n3 100 2s 10 2 us "Mecasurcracats takenfromonereplicate testchamberai each concentration. "Measurements taken from prepared soil twodaysaferpreparation. "Measurementstaken from prepared soilonDay0. "Measurementofsoil temperinaRetpluicratee B was 22C. TermTiensattion pH, ~~ 5 8 5 8 83 53 TermTineastion Tempcemoture n 2 zn zn 2 2st 000219 _-- Wildlife International, Ltd. se Project Number 454-111 Table 2 `Cumulative Mortality and ObservationsofEarthworms Exposed to PFOS e = r i7nan Artifircial Sroil Srubstporaot)enmee e----t--ees oy tins R Dad R peas T Dead EieR s Mfoseatly Mofrei NSehsee ABEbwooib lldaoaan 5M la ooomwm dwdaaavyy oo0 om doa v mo ABSooowmn0 dwRaaAyx ooomkw l dDaAwao o Book Ba oh Gao WAS Bob ommo salwmaas Eo CAAT bafmeeo mb bowamww ow moow as w ABSoabbow dwdaaarw Boh Ba oooWmnee ab oRmTaaraooo00 as waBFI odmee aama BoM am wwweee amCTe wtowo am = Am tos2 F5BI 1d0a1m0 -= wo we1010 -== ww 1Ii0Ho0 *3 Cfhoarr Ee 0 so Mortalitydataarepresentedasthepan ha, in 31h ered cumulativenumber deadpernumberexposed. ection och 000220 ------------------------------------------------ Wildlife International, Ltd. ProjectNumber 454-111 "19+ Table3 AverageBodyWeiofgEahrthtworsms ExpotosPeROdS inan ArtiSofiliSucbstaralte MJeomo pMjeusosresd (gifs) NCeogantive Repene xb Mens>ocu Der sere Aveage actor BodyWeighttse2()emetic Dayo Devi To Change 0o2z5 on 0a2i1o ois "ooootr 0s 0250430s029 0200420003 00654006017 mo a5 0022ss 001290 "0008s 026 020 206 MansS>Der. 025o5z0s805 019a0is010 0060270008 0 A5e a0032z36 o0oinr 02001ss0 MensS>wDer. 0260530s077 010300 0 0070026004 w a5 c 0T2Ys 023 oorr 016 20180 oo MemiSbuDer. 0250520s016 OIT1i800 005007 M a A5 c 0oo3zt5 a_am MensS>uDe. 02004304008 oo=m o_n ar=a? 10a A5< a0022zs =-z -== Mema5S Der 0250430s005 i= re=ze CBhoadnygweeiingbhotdvyawseisgahitivacslsyadtiis t in(i0i.ca0n5t()5<eD.c05o)notetgrcoovnceevsiinvagtoDnwuheelcsotmep.re th contol groups wing Duets est. 000221 ee Wildlife International, Ltd. "20. ProjetNumber 454-111 Table 4 erDay-- 14 Measr-- edValue-- sfor PRO-- FSrionET Cstr-- oE en mTis-- s ---- Tasoien Sense Nomi nin NNind [OY >) Wl 00 <LoQ* - Vwaana EmA1 1on 1s wwaanr 11556 0ft P wwiasr Ea1H P0 w ws as nos - R T vyie itoqo 100)i toW wAnoeva 00mVg e enaoWnASH fetcihepeiesfof posve ak on(s00n3r00(050)000 211 000222 - ----------e--e-------------- WWiildllifee Ibnetmearniaitoionnaall,, LLetdd., a ProjectNumber 454-111 Append Pesome volved inthe Study The following key Wildlife Intemational, Ltd. personnel were involved in the conductormanagement. ofthis study: RaymonLd. VanHoven,PLD,Sims,Analytical Chemis 1. Henry O. Krueger, Ph.D., Director,AquaticToxicanod Nlono-Tagrgyet Plants 2. JohnR. Porch, Supervisor, Non-TargetPlantsandInsects 3. Anne B. Sindermann, Senior Biologist 000223 - Wildlife International, Ltd. ProjectNumber454-111 2 Appendi2x ChtaothneStgyPerotoscol Thisstudywascondotedaccording th approvedstudyproocolwithth followingchanges: 21.. 3 SATonetadsmdoiistltoonwraelresopirrletmphoaeir1se5tdu6,ramenmgdea1naalkystliuecvarelsleavwmaampsslmeea2sn9dc.oetl5.l%e,cltgedh,olynbedlaoywparipoprrotoxtiemsattetlayt3i3o%n.. 54. ETxesnttshoirfmrsuthsee6d2f5ormthgLstugdy woevigehweediJghsedth6a5n0bgertawteheenr3th0a0ann7d5600g0mg. 2.. TEhanotswoolrtmcmspweerarteacronidintRieopnleidcaftoersApopfrtohxeim1a2s5l0ymg18.5hokugrsrrcaattemsetthgarno2u4p.was25Cattsk termination,exceedingth 2052Crangespecified nth protocol. 000224 - Wildlife International, Ltd. ProjctNumber 454-111 a Appendie3 AstificialSoilandTestSoilsPreparation AsSoli Prepaa ration appr"Tohexaritifm2i0caimatilsteoeillswy:aspreparedin bulkby mixingthefollowingconstituentsin asoilmixerfor qFouiencnaty 395000kk Spacgonmacapfenaatte 415.10205155 "TetSol Preparation approxiAmtaotteallyof3a3%pprwoaxismparteeplayr4ekdgfo(rtwhetewneeiggahtti)voeftceostosolilawnidthsacchalocfutlahtedtsroeialtmmoeinsttgurroeucposn,tenTteosft bstancoconcentrationswerebasedonthedyweightportionofthesi,Thecalcdurylwesigth eof kof preparedsil ith amoisturecentof33%,was2.630kg. Toaccuseforth 4%moisture contenttypicallypresent ithebulksisoemambienconditions,1208mL.ofwateweraddedto 2680 g.ofbulksoil mimstheweightoftestsubstance.Amounts of testsubstance, bulksoiland water reas follows: [Nmap mein [ |Te~ Semm~ es | Ta--s[wes[wm | [[wom m [eewm ewwene mwness [eEew [ [o os w [wwwe wmee wweaer mw) am (mw we en |we[mw Theappropriateamountofbulk sail was weighedinto ataredHobart"mixerbowl. Approximately a handful of dry soil was removed to a Nalgenc beaker and themeasured amountof testsubstance addedandmixedinwith ametalspatula,afterusingdrysoiltocleantestsubstanceoff 000225 ----_--_--_--,-- Wildlife International, Ltd. Project Number 454-111 ae oftheweighboat. The soilandtestsubstancemixturewasaddedtotheHobart bowl. 1208mLof deionizedwaterwasaddedandthesoilmixedfor 5minutes. Afterthe S-minutemix,the sides ofthe ebaocwhlrwepelriecastcer,aepxecdeapntdotrhtehsoo6il2w5amsgmiaxiedfocornacneandtdriattiioonn,awl h1i5 mcirneucteeisv.e Pretespt soa il,r 750eg fdor 650g,wasplacedintotsk chambers labeled withconcctardartepiliocatne.Negativecontolsoilwaspreparedin thesame `manner,withoutthe additionoftestsubstance, and750 gwasplacedintoeachnegativecontroltest chamber. 000226 ee Wildlife International, Ltd. PrsjctNumber 454-111 25 Appendix 4 THE ANALYSIS OF PFOS IN ARTIFICIAL SOIL AND EARTHWORM TISSUE IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROTECTNO: 454-111 000227 - Wildlife International, Ltd. "25e REPORT APPROVAL SPONSOR: 3M ProjectNumber 454-111 "TITLE: APFORS: TAINSFOAICILUSTCEUBITSOTXARIACLTIETY STUDY WITH THE EARTHWORM IN AN WILDLIFE INTERNATIONAL, LTD. PROJECT NO; 454-111 SMENVIROLANBPMROJEECNTNTUMABELR: U2723 PRINCIPAL INVESTIGATOR: JY rl onHoven, Po. MANAGEMENT: ll ; -- Director, AnalyticalChemistry sun BATE Hh 000228 - Wildlife International, Ltd. ProjectNumber454-111 1 Introduction Led. a"nTahliytsisctaludcyhweaissecyonfdaulctieydinbEyaWisltdolni,fMeaIrnytlerannadt.iSonaamlp,Llteds.vfeorrec3oMllaectttedhefoWiltdhelidfeetIenrtemmiantiaotniaoolf,n Perfluorooctanesulforate,Potassium Salt(PROS) inartificial soilandearthwormtissueandwere analyzedusinghigh performanceliuidchromatographyWithtriplequadrupolsmass spectrometric detection(HPLC/MS/MS). Sampleswerepreparedandanalyzedbetween November27and28,2001 andDece1m3b,2e00r1.RawdatageneratedbyWildlifeInternational, Ltd. and a copyofthefinal seportarefiledunderProject Number 454-111inarchiveslocatedontheWildlife International,Ltd. site. `Analytical Methodology Artificial Soil `Submittedartificialsoilsampleswere analyzedforPFOSaccordingtothe validatedprocedures documentedin WIL study number 454C-120.Theentiresubmitted soilsample was blendedfor approximatelytwomimespriortosb-sampling the reise 10-5aliquotforthe analytical ctminaton. Theartificalsol subsamplswere extractedwithmethanol, giatedfor aminim of q30umianlutietsioatnetpiaagpvyerrea.toTrhyeshSalkaetretwaablsctahtenatprparnosxfiemraetdetloy2852000r-pmmLavnoldutmheetrnivcaBcausukmanfidlbtreoruedgwhitttho olwiathmmethaenol. Approximately20miltersofeachsamplewastransferedto separatevial HaPnLdCceIntMrSif/uMgeSdmfeotrhaodoplopgyr weroepx5emriifnoummteedsavaittt2h0e0s0ollruptymio.nDoiflut50i%onsmientthoatnhoelc(HaPlLCigrbadrrsaa,ng9te9oi.f9o4t3hn)e and 50% NANOpuwrateer, Sampleswerethenanalyzed bydirectinjection. Concentratioonfs PFOS weredeterminedbyreverse-phasehighperformanceliquidchromatographyusing aHewlettPackardModel 1100HighPerformanceLiquidChromatograph(HPLC)with aPerkin-ElmerAPI 3000LC Mass Spectrometer equipped with a Perkin-Elmer TusbolonSpray ion source. Chromatograpiseparationswerachievedusing KeystoneBetas icone (50mm x 2.0mm, 3- jmparticle size)fittedwith aKeystoneJavelin Cyyguardcolumn(20 mm x 2.0mm). Amethod flowchartisprovidedinFigure | andtheinstrumentparametersarcsummarizedinTable1. 000229 _ Wildlife International, Ltd. he PrsjctNumber 454-111 [Earthworm Tissue develoSpuebmaittWeidleadrstThwaortmtrisas,uesLaimdp.leAs wepreapnalryzoeodofgorraxmPoFfiOeSacahcmccosrtadiiwnogtrtmotmeiestsuhelosdaomlypolgye wasweigheddiel ito ardscnllaionvals fo which 10.0milofmlethaneol erxtracstion solventversadded.Thesampleswore concurrently estrctdndhomogenizedusin ssoshredder (JankeandKunkel)followedbysonicdismembration. Thesampleextractswerecentrifugaend dilutedintothecalibrationrangeoftheHPLC/MS/MSmothodology. Dilweu re t peri foro medwnits ha a`nsaolluytzieondobfy d50i%emtetnhjacntoilo(nHHPPLLCCIgMraSdIeM,S9a9.s9d4e%s)crainbded5p0re%viNouAsNlOyp.uArcmewtahtoedrl.mcSbaamprlteissweprroevtihdeend nFigue2. PrimaryandSecoSn tocdkSa olur tioyns Alprimaryan sccondary sock preparationswer adjustedfothpuriyofth etsubstance (869%. A 100mg mLprimarystock solutionofPROSinmethanolwaspreparedbyweighing 1.1508 ofthetetsubstanceadbring 0. inalvolumeof 100millers withrthan. pr`opS asee dbyc stsoo ecikssnolvud otlioaa nmse(r t10i0dy0il,at1i0on0,r1o0m.0,t1h.e0p0,ranady0s.t1o0c0km.ga.i/L)of PFOSin methanolwere Caliraion Standardsand Calibratrivoens iuCoaloinbfrtsahtei1on0st0amndgar2d.s1wLesrteopcrkespoalrtecdinsn 5of0:P5O0mSetihnanmoelt:a'oNoAlN.OCpaluirberatwiaotnesrtbayndaaprpdrsoopfriPaFtOeS, arnandgcianlgiibrnactioon nstcodaerdnsfrtoforPm1Oa.S0t,0trionog10i.nn0g.iingcao.in.s/Lt,wrearienanfaolymze0d.w5i0t0he5a.ch0a0rti1fic8ia/ls1o,ilwsearmepleset analyzed with he cawsusamp et.Thsmoandmotprosepes respons forPFOS wasulizdtomritor PROSinalcalfraonandstysamples. Noatvaesmamdetopquanetity PROSonthebassofindividualisomericcomponentsTh calibration standardsrevasjctedst. thebeginning andendofcachrun, andonestandardwasinjected,at a minimum, aftereveryfive. co`nscamepnlters.atLioinnseaorfrteghrceaslsiibornateiqounatsitoannsdwaerdrse.geAnteyrpaitceadlucsailnigbpraetaioknacreuarrvesipxoPnsFeOsveirnsuasrttihfiecriaelsspielctaivde cantordese presctdinFigure 3adFigur 4, respectively. Represatativoen 000230 FE -- Wildlife International, Ltd. a. ProjctNumber 454-111 acnhrdo6m,arteopgercatmisvoeflylaonwdarnedprhsieghtcaatliivberaotinocnshtraonmdaatrodgsrfaomrstohfeaortwifaicnidalhsiogihlcaarleipbrreatsieonntesdtianndFairgdusrfeosr 5 PROSincarthvorm isoarepresentedinFigaro 7and 5 respecivey. TheconcentraotfiPoEnOsS nth sampleswerdtrmindbysubstitutingpeakre responses totheapplicable nerrogrsin cquaon.Concentration of PFOSinthesampleswer cleltedonadyweight solspe)or `wt eight (caiworntise)bassusinthest nsrumentPROSconcentrationsan the pproprise `weight/volume dilution and/ormoisturecorrection factors. MatrixBlanksandMatrix Fortifications Arica Sol Selected 20.0-galiquots of artificialsoilwerefortifiedwith the appropriate stocksolutionsof PFOSpreparedinmethanolusing agas-tightsyringe.Thefortifiedsoilswerethenhomogeniuzsiendg a Waring blenderforapproximately 2 minutes. A10-galiquotofeachhomogenizedfortifiedsoil samplewasweighedinto a ardweighbost andtransfered 0an 1-2. Freachsqarebtfor extraction. Themarblankswer fortifiedarilsi A100 EarthwormTissuequotof egativectrlc: hor soewas tifiedwith he 1000mg iL socondanysock seo ofPOSpreparedin han wing ga-Gg ying. Amexblanks was preparewith fortified egaiv ono thomse. Ariciat Sol Liom f Qui antit tats ion Thomethodlimitof qustiaion(LOG)inarficilsolwas 29.3mg aigon ady-weight b`awesiigshat/nvdolwuamsecialuculatfeadctaosrtohfetpheroadutctxobftahkelsoawmepsltecsa(l2i0b,ra0t0i0o)nsitvaindddarbdyt(h0e.p0e0r1c0e0nmtgsoal.iids/Lf)o,rtthhee `testsys soit l (6e 8.3m %). 000231 ----- Wildlife International, Ltd. ProjctNumber454-111 wn Earthworm Tissue basaTnhdewmaetscholdleimitofsqtuahntiptraotdiuocn(oLfOtQh)eilnoweeasrtthcawloirbmratiisosnusetwaandsar5d0.(00m.0g00a5.0./0kmggon a1we)ta-wneditghhet `ightvolum ditionfor ofthmatiblanksample (100,000LAG). ExampleCalculations ExamSpalmeplCealncuumlbaetrio4ns541-5,noraconcrzationof 78. mg 1K nailsol. FFiiSsstteIninocitlaViooWliVugonmhletu::dme21:000000m55L00mL. CSaiple:f35r8r1vt195iunoionn: Tne: 10110522 MSDeeictonsniSdooFniiFlnaaMcoloiVrs:otlu2ur0meC0e0:o0n5t0.e0:mL317% PLoOrMSnSDilla:tion68F3ac%tor, 200000683 = 29283 'PFOSPeakArea: 23914 PROS (nga.)messedatnumer = ESE PROS (ng kg)inspl = PEOS eased anumer (ng2.) x LCMS dilutionfactor BsoOu tTones ory ner -za3a mss Percentof Nominal Concentration =EPaFOsS(mga2-.i/.K/gK)gi)nnosmuimnpalle * 100 Calculatedwith HPLC/ insM truS ment/sofMtwaS re:MacQuan,version 1.6. 000232 _mm _-- Wildlife International, Ltd. Sie Pusect Number 454-11 RESULTS NoinMtaertfreirexncbelsanwkerseamapblseesvfeoraetaocrhsmbaotvreitx htycpoerweesrpoenadnianlgyzLedOtQo(dTeatbelremsin2eapnodss3i)b.leRinetperrefesrneantcevse. F{iognucrhesromaatndogr1a0,mseosfperciiec.aMsaotirliaxnfdrciacstiiwoonrsmatmapsleesmoartiaxr.ibaarlkssialmhplaedsraeceoveprrieessernatnegdiingn fiom 75.6%to 103.2%ofthenominalconcentrationswith 8meanrcevey of 87.3% ofnominal p(SDr=1s12ci;noCFVit=g1u2er.e81d)1.. TAhroepcraestehnvtaotrivetiisosnu cmhraotmiatofgorrtaimfocaftoann(airtgirfiecia1l2s)oiblamdatrreicxofvoerrtyifoifca8ti6o.n6i%s `ofthenominal concentration. AnicasclsamplescollectedatDay Ofromthe 78.1 mg ag,156mg akg,313mg. oa.fi/7k7g.,06m2g5maig k2g./(kSgDa=n1d3.1;25C0mV=g1.7i1/%kgt1r-e6a)t,me14n1tlmevgelashkadm(e4a-2n)m,e2a8s9umregd aPRgOSc(o0n-c2e)n,t4ra8t8iomngs coamekpo(n3-d2e),taonde1a0n4p2ermcgen2t. ecv(eSrD=e20of1;9C.V6%=,1990..34%%,=9)2,3%re,sp7ec.t1iv%ealyd(Ta8b3l.e44%).ofTthheeneomvialnuaels concentrations with an overall mean percent recovery of 89.6% (SD=15.3; CV=17.1%, n=18). A representative jonchromatogramofan artificialsoiltest sampleispreseatedinFigure13. AnicasclsamplescollectedstDay 14fomthe74.1mg.31k 156mgai3g13m,g oaf.i/8k0g,66m2g5magkag.i/(k1g2a),nd141325m0maga..i/k(g6t2r)e,at3m0e5nmtlgeveals Khad(m=e)a,nm6e3a4smurgedaPiFgOS(vco2n)ceantnrdat1i0o5ns4 mg2k(2), respect(iTvaeblley).Thesevaluescorespooded tomeanpercent esoverisof e1c0o3v%,ey9o1.f7%,9976..3%4(%S,D=110017%;aCnVd=1871..52%o%f1-t1h0e),nominalconcentrationswith anoverallmeanpercent 1m 2.`EartfhowxonrommtiinsaslueeaxnpaolsyuzreedeolnDsaoyf 174.h1a,d1m5e6,aannmdea3s1u3rmedgconcKengt,rarteispoencstoifvel1y9(5T,2a0bl3ean)d.2T5h2e mseaamspulreseidzvealu3ew(o1r1m0s5m,gpapr.ov)imafiorltyh0:6225 tmogt;alwekiggehx)poasvuariellblveefrwoamstsiusspetcrtedatumenttogtrhoeupl.tAd representative ion hromatogram ofanctor teu estsample presntedfnFigur 14. 000233 _-- WWiilddliefe bIintteerrnnattiioonndall,, LLtad. eiPS retN NumberdS-111 "a2 REFERENCES I. American Society for Testing and Materials. 1991. Standard Specificatfiorn Reagent `Water. D1193-91, ASTM Section Il Water and Environmental Technology, Vol. 11.01:4547. 000234 ------------------------------------------------------------------------ Wildlife International, Ltd. ProjectNumber 54-111 a. Tabet i Typical HPLECIrMS OeE peTroLn ParametersEE Sree INSTRUMENT: "SCoHphreerweclt.eumtettm-oOtPgaeprcrkaaeshrqdrwMiitoaphdpenlPwdei1tk1sh0i0nHfPEoilrngmiehenPsretAEiPormIsfe3rmo0r0Tru0mtMtbLaaoinsglqoscuniMeSdpRraMy)ion -- ION SOURCE: ANALYTICALCOLUMN: Perkin-Elmer TurbolonSpray KeystoneBeas Cp (50mm x 2.0mm am pari siz) `GUARD COLUMN: OVENTEMPERATURE: Keystone Javelin Cyscartridge(20mm x 2mm) 40C STOP TIME: 5.00 min FLOW RATE: MOBILE PHASE: 250 pl/min 3700%% Mian NANOpure Waterwith 0.1% FormicAcid: INJECTION VOLUME: PFOS PEAK RETENTION TE: 50pL Approsimatly4 mins PFOS MONITOREDMASS: 49am9u--.990.1 amu. 000235 PY Wildlife International, Ltd. ne Project Number 454-111 Tabl2e Anil SolMatixBlanksandFortificationsAnalyzedConcurraly DingSampleAnalysis LEote Sengi e r7-- 705pCearm-- ierose-- eiPronr t mM--eMr aamn--nd --r PNaemt (454-111) Type Fortified Measured! _ Recovered' (mgnike) Recovery MAB-1 MAB-2 SoiSlaMlaMtiaxeBBlaankk 00 pLi0rG = -z -= VMAASS-41 SoSlloMlaMtaiixkFoFxoifiifcaitiionn 366 B33s0 7m1 m9 M0 VMAASSZ-52 SoSloMlaMtiaxkFFiocfttiioonn S86 E6f5 %4 sk 97 MMAAS-S3 SoSloMlMaaigsiFFootriiiSccttoinn Met l10e3e m6 10s 81 e seSmanedCaearDe eogvinesliosSn=pAgW1F1I2pmaette emeeerenatr TT SNou__s6 a a calcumlayavtaryisloignahtilsiy.o (vcsQnsiftoinelspildwa(m020031s10g02Ggo1g.tothermwyeti(6gb0a3h5ieaddvas cor 000236 _--_-- ee ---- Wildlife International, Ltd. Project Number 454-111 xe Table3 EarthwormTissasMati BlaaandFortificationAnalyzedConcurrently DuringSampleAnsys --_-- Tor (454-111) saaple Type oaesin Fortified Measured" Facet Recovered" WAB-1 WemBTliaekMaix 00 <L0G - WMAS-1 VoFmorTisiasteiMonaix 100 86 66 R Nua catiaywoeynHe EsH e e eeree *Theametabhnodsdtlhimeidtiaogfhqucantltailtmaoetiddoint(iLoOhnQe)irtnoeoadroefthowhoetmormatoisiwsteiacwaaksi50.op0lmeSge12n0.di0/ak0g0o(n0La0t0w)e0t5-00 000237 _--nmmmm Wildlife International, Led. Projct Number 454-111 36 Tabled Day 0Reser foxPFOSinAvil Sil -- sunt S anm [-- Pao (54-111) Concentration Measured' Recovered' MVeend (mgai/ke) Peamt Recovery 1 00 ::2 wwniii ;:5 mwwiii ;. I56 i0 awn iBo aas iius 235000 Hii 1222 00 <Lo@ - - = nnorsn 2r[ 1E A sais G"o7 1fsesd iww wa1e Wo ses a2n0 =woe om ms i52 iwi om om nu0sem0 Zr ae1 = sEeeaem oma Sv5es0 f7ii3e Sundaa weo w1i53n ia y vyTe he eTVMa Yes TE eeiniec hkertorshemoiishmobw1o00rieeiyfneodlta ,2t030.s10o8reig13y , wehightlbiddovans. ck 000238 ---------------------------------------------------------------------------------------------- Wildlife International, Ltd. ProjectNumberdS-11 a. Table Day14Reco forv PFOe Sinr Arti ifice ial Ss oil GNoTr) SamplceNoommi]n ngma e Pot MVeend eMetn Monet Reoend _gasing Reso 2 00 <L0t - - - u2 m11 xn 115%6 %2 nn n= aass nosH fsoos me om 10 aon wo oan oPnA E56Ss ws me == 8u5s1 @ wm % jiuxsd 110125s9 Swort ww ws EE Sundari poviion= 107 Pe Rr eeSE TWNnaseoiRmR TT VT calculationsmay vary slightly. *ToheomieptshoodlS imhietofqcueoni ifita3thieono(nLcOQo)ainelarttbiofyinceisalasponidlwas02.9d0.03s1m0g0a4.i/1kkgo,ntadrwy-wieiggh(htb3ats5i:saandowans. tr 000239 ------------------------------------------------------ee Wildlife International, Led. Pret Number 454-111 38. Tubes Day 14MeasuredValuefox FOSinEarthworm Tissue EE Tr Sunsc svn -- PAT Messed MeVrmeaed (mgaifg) wet 00 <Logt - Wwaasn mmi 0110 1s wwaasa 11556 0% m wwaanr mwn p2b0 2 ws as nos - Tvei, EEVo re eo *Tihemeithoadlimiutofnmqeutadntietahtneiopne(dLOaQh)ineaprlthoewvoetrym ktissluewotasea5Gn0R.dO0mGg0(D&0.Li0/Ak5)g0on0aweLt-wyeight 000240 ------eeeeeee. Wildlife International, Ltd. 3. ProjctNumber 454-111 METHOD OUIN ARTTFIOFRLITCIHAIELASNONAILL.YESIS OFFOS TaFr h ro s `Weighapproximately 10.0gramsofeachsampleinto ataredweighboat. Forcachsupee mers 1t00meL.oefe thanavith granted coir CTaosipplaoceo3n0hterteeblr e.All thom s0pc7sake Voc lt vith itam tept e nd ie sind ntshtholnt,ti 3 200nL.ves vtrfpksh srdbring,shuns iranotspn pesm.tly2C0mi,oofexchssploE t seps tlascen 80ber ndGo tp Prepareappropriatedilution(s)tobringfinal concentration intothecalibration rangeoftheLCMS `methodology. Foralldilutions,use50% methanol: 50%NANOpurcwaterdilutionsolvent,gas-tightsyringes, `Ampulate samiples and submitfor LCMSanalysis. Fire1. Ayalmethodflowchartanys ofPOS isic sll. 000241 ------ ee-------- Wildlife International, Ltd. a. Project Number 454-111 METILOD OUITNLEIANERFTOARWTOHRMETAINSASLUYESIS OF PROS Whpinay 110 gn gh mae tardiin 00100. ft ch li gsOiA licig Homogenizzeachtestsampleforapproximately 1minutousing ahand-heldtissu shredder. inte thekop get ih ie ppRspitte gives ababs mongol Sess susshale ie speriamsiay simp mesdinamo. Capthevialsand shake well.Comite 2 000 ys spay S wien `PrepareappropriatedilutionsofstudyandQCsamplestowithinthecalibrationrange: oH ARTRISLOMSMS studios ieS154 3h if ois) `Ampulatesamplesandsubmitfor LCMS analysis. Figure2. Analyticalmethodflowchartfortheanalysisof PFOSinearthwormtissue. 000242 ----------------e--e------------------------------------------------------------------------------ Wildlife International, Ltd. a ProjectNumber 454-111 P. 1000 . `6000 1 sono 0.0 Is ooo Tw Too Conc. pg 8.4/1 Figur3e. Acteypoic.alcalibrationcureforPROS (urificialsoi).Slaps 598In1rs1pt 91015105;32; 000243 eeeseesese------------------------------------ ZWTilEdlifeIInntteerrnnaattiioonnaall,, TLtAd.. a a 12000 _-- PProoijcNteNummbbeerd4s5e4-a1n1 ooo pe 2000 ook TE whe Czoee vasihne ako ade ahs Figure. AT0p9i99c.tcalbration urefo PROS carro tise) Slope23387; erp=23379116; 000244 -------------------------------------- -------------- Wildlife International, Ltd. gees aso ar ries Lg ees a To woe Project Number 454-111 nay: 1000cn SEFohme,ns l8a . :- E [Fr L g. ny pp = Te A [ONAN Figures. Aanedparresa(taatrivtiocnscoahlrl)o.matogram of a owe] (0.00100mg a1)PFOS 000245 --------------------------ee------ Wildlife International, Ltd. TRSSTO A Faeirae a Hr 3 Wm =inL1|0 Eftoimme la LSE Inisgratonwin Sere. 057 oc] Project Number454-111 ue ern te J-- : x J "- "gusTe etn4e10 dT1T:h To8, Tr1 aGlth SrhEyR Figure. Ampreetatve onchromatogram ofahghlevl 0.0100mg 4/1) PFOSstandard (artificial soil). 000246 ----eeee Wildlife International, Ltd. Projct Number454-111 ss. ERSOSA Can Eh ies a mses FiAw 300 3da.v 58 EEEB-- nacsouaenys dos iggEy igorTwn A-58 Ft 301 2 aa 60 om pe pu :o - : sve ove zoApe aus Se th Hh Wh Wh Wo Wim Figur7e. Arepreios nche romnatotgra amotfai lowv -lee vel (0.mg0 a.i0 /L)0 PFO5 Sst0 and0 ard (earthwormtissue). 000247 --_--_-- Wiilddllifee I`nntieerrnnataidoennadl,, lLetdd., vPrmsecmt Nummbeer 4e54-n11 we ER OSA r4o 2na n Bortmens [-- EBEFReAEE r Fc] E =us SthpEanoToevn a. a2im gion Type AVE. E: T :p . i: git 208 ta th Un Yo 3H UW UE Figur8e. (avr se), Areprescatativeionchromatogramof ahigh-level(0.00500mga.i/L)PFOSstandard 000248 _--_--mmmm-------------- Wildlife International, Ltd. mas we fLoignin ae ummamen ; ProjectNumber 454-111 troon Ea g Eisemesf L83o0w0 Tmyw lw Fn [ [oSiLeEE8=, = l ::3o xp vn wm we mom REET Figures. "hAirpsrrseoatniviecooa chherpompartooigrnastoftaantaironitalesiolPmOaSti. bark sample (4SH-111-MAB-, 000249 --------------------------------------e-- e-- e-- . Wildlife International, Ltd. Projct Number 56-11 man wae gDohmioowen hEgiopra mewe fm EilR, l8e E[ FEn,2gh = mEme 2 #8 rows Eb =: ny: vn l . gic Terr Figure 10. NArAepBes.ntTahtevrioonwcihnrdoimcaattoegstrahmreotfeantniocnamhewoorfnFtOSs.imat Hank sample(454-111- 000250 _--_-------- WVilidldifee IInntteerrnnaattiioonnaall,,LLtedd.. | ePorowjeicNt Neubmeberrs4s5t4-i1n11 -- moehs mss memo fioJ s . hEiRE en oa ":. oma0 mhBoaea,adn l8n EmeR om Er Te TeaganWas 4Z"z bp w ;a BRT 12 11a serves T2NX Ra.m Figme1. MAAsSp,rnaomiinnalfPoROcShcroomnstgogaam ooffan50arifctlskigl, maioxn dlolrc- HGHm)pl: (54-11 000251 -------------------------------------- WIildIliefeIInnttoerrnnaavtiioonnaall,, LLetdd.. 50 PPerkecaNNeummbbere4s54s-1t1m1 ns ws fi-- F BmfJyenaenn|-- FBFaEeEE , oi =e Booelmtg LL osm . :. p : i 10 on F loganTa AVR te th Uh Uh FW UE E TICHMAS-),mami PROScometsE of 00mg 1/6, Aio--n --tr--= 10--000 000252 e --_---- e ee ZWTivlddltiyfee`Inntteerrnnaattiioonnaall,, LLttdd.. si moan 4 mesma LFaeorniiaomen . 0 ow . FFEA . w[Ae 8 BE S2h0 ns l,a :- mHoTev.en 55 8 Nes aw LT PPorojiecNt Nwumbbeerd45s4-s11n1 i: ctom a 12 5 Sd hm Figure13. CAseopeensaicve7f8oe.1cmhmgrom1amtKogtrlaemoiofannoiatrnria-2lo9s2i8fl3e5.tsample (54-112,nominalPROS 000253 _--mm--eeeeeeee WViilddallifeIInntteerrnnaattiioonnaall,, LLetdd.. 52. mmaee wa =prras--us fi o -- Ee 7 E EEB rowan } .. B ;. oWSE EeightngeT49a 63d EE Wh .2x: . Th Uh pPreojieectaNNambbeerrd45s4e-1i1n1 a. T 2A Sh Bh th IhES J ---------------- dihfacutort=1i000o00%n). 000254