Document 0qgv0vJ1dJj1wRxL3gBJ9OkNJ

ADIETARY LC30 STUDYWIPTrHoTs:HE NORTHERN BOBWHITE WILDLIFE INMTELRANABTRIEOQNUAELSLTTDN.OP:ROUJEaCT NOS 454103 Serie 8U5S0.EcElPognPeTloESfenNcu)mePro3cT5t0si2oG3n0u0Algeens (ds) FIFRA Subdivision,Scion 71.2. 0RCD Guststne 205 AUTHORS: SCTocouarmnm..eyGSBa.leClvohieesyr Reymond vin Hoven STUDY INITIATION: Apri 21, 1599 STUDYCOMPLETION: Ap26, 2r000 AMENDEDREPORTDATE:March 23,204 EnvSo3UmMBmCeMonrIploTrLaTatibTEoonDrOsory P5a3, BMuinAncae$5144 Wildlife International, Ltd. Ea1s59oa8n,iCMnoamr2ym2aa.n5sd6D02i16e01 Page 1o69 moe GRO52E ------------------------------------ WILDLIFE INTERNATIONAL,LTD _ 2. ___PROJECTNO: 454103 GOOD LABORATORYPRACTICECOMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Dictary LCS0 Study withtheNorthern Bobwhite WILDLIFE INTERNATIONAL LTD, PROJECT NO: 454-103 MLAB REQUEST NO: U2723 STUDY COMPLETION: April 26, 2000 AMENDED REPORT DATE: March 23, 2004 tPrhien"cTUihSps.lsetsEuondfvyiwGraooosndmceLonantbdaoulrcatPterodortieyncPtcrioaomcnptilAcigea,enn(ccOeyC,wDiEt4/h0GGICoDFoXR9d2L)Paab3ro2t,ra1Et6no0vriyarPnordnamc7te9in2ct,e MS1ot7annoAdguarrgaduspsahtpNu1o9b.8l94;i5,sOPEhaCbeiDdys, A1u99g2u)s,ta1n9d84Jawpiatnh tMheAFfoFl,lo5wi9ngNoexhcaenpt,ioNnotification No. 3850, Agcultural Production Burcau, 10 `GLAP bcoempliimaencteh;eheoswtewvears,ctohneducchatreadcttehreiztaettiosnubwsatsanpceerwfaosrmneodtahcacrosrcdeirnigzteod3iMn aSctcaonrddaarndceOpweirtahtifnlgl PsurboscteadnucreeswaasndrecMheatrhaocdtse,izaenddiaallccroarwdandcaetawiatrhc GbLeiPngstamnadianrtdasinFeodlloinwintghest3udMy caorcmhpilveetsi.on.The test `wa"sThnoetstdaebtileirtmyionfedthin faecsctosrudbasntcaencweitahndGoeofdreLnacboersattaonrdyaPrrdacutnidceerSctoannddairidosn.sofstorageatthe et ste: accAonrsdiaynsceeswiotfh GliLvePrstaannddarsdesraansdamrpleesscoofntdhuecsteeadnalbyyse3sMs.eCboirnpogrrsetpoonrtewderseepanraottelpye.rformed in ) STUDYDIRECTOR: SSeenino.r BGiaollolgaigshter `SPONSOR'SREPRESENTATIVE oare__333loy d MIE oA Bf each Bits silaye foy AMENDED GHO5? _-- WILDLIFE INTERNATIONAL, 1 a Es PROJECT NO: 454-103 QUALITY ASSURANCE STATEMENT by This study was examined for compliance with the US. Environmental Protection Agency, 40 Good CER Laborsory Practice Standards Part 160 and 792, 17 August as published 1989; OECD. Principles of Good Laboratory Practice, (OCDE/GD (92) 32, Evironment Monograph No. 45, ars, 1992); snd Japan MAFF, 59 Nohan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The date ofall auditsan inspections an th dacs that any findings were repored to the Study Director and Laboratory Management wee as follows: Actviry T&estAnSaulybtsitcaanlceSaPmreppl,ing Matix Forieation F&eeAdnaCloyntsiucamlptSiaomnpling AnDarlayftticRse!poDrtaa and BiDoralfotgyReDpaotratand Final Report AmReepnodretd Fina DATECONDUCTED April 22,1999 April 22,1999 April 27,1999 uy 7.8,9,1999 August26,27,30,31, 1999. April 17:18,2000 March 19,2004 STUDYDDAITREECRTEOPRORMTEADNATOG:EMENT Aprl22,1909 Aprl22,1999 Apel 23,1999 Aprl23, 1999 AprI27,1999 Mayd,1999 sly, 1999 sly 16,1999 Augusi31,1999 April18,2000 September 13,1999 April 19,2000 March 19,2004 March 19,2004 SusanSuwCosloemnanA. Coleman Senior Quality Assurance Representative DATE___ 3-43-04 AMENDED GRO5DG eee WILDLIFE INTERNATIONAL, to Pp AMENDEDREFORT APPROVAL SPONSOR: 3 Coporstion TITLE: PROS: A DietaryLCS0Studywiththe Nore Bobi WILDLIFEINTERNALTTD,IPOROJNECATNLOS 454103 3M LABREQUESNOT.: U2723 PTS EI , STUDY DIRECTOR: Ssonnt ton visToicoey `CHEMISTRYPRINCIPALINVESTIGATOR: STlmo7ARsS almieCMnrmseHy REPORTAPPROVED BY: H rtatw e NoOpcehreo!n DTiorD7a/hNo2omo2in8nFyrHl ow ,ater Es3Henk 200d oe 9 were LR -------------------------------------------------- WI ILDN LIFT E ERNATtoIONAL,meomcrnos aseiws 5. TITLE PAGE c TABLE OF CONTENTS Fig| GQUOAOLDITLYAABSOSRUARTAONRCYEPRSATCATTIECMEENCTO.M.P.LIANCE STATEMENT... Page 2 Pas3 RETPOARTOFBACPOPNRLTOEVNTAESL...... Pg oo Pa5 TAI SdAPPEIGRE SUMMARY. cer Pog6 Pie1 OIBNJTECRTOIDVUECw TION w oe o Pe Po MATERIALS AND METHODS.. ee Pig a TDosuSrubasotfatnthceieT..o.sn -- SE --III Inn PeeGEes AnTeistmBainDdsi.k a ---- a C----I ------ II ee eo DHoiAuncastliySngeraMnidetEhnovdionmena CoonndoSs --: pi IIIII seeyo I ree GAOrbnosisemsravlNaetBicoordnosypsWye.i.g.r hiFsee d Copinsampion TI III I CI eeaa ea Sates Anas fi Crea RESULTS... SO -- oPage13 DiM BeotdyAWoeTigaShnradnid cFa eeadCoOl nsurmptsison.o.. n.ST .. E I -- PeOeEs1S Grose Necro. -- III ee CONCLUSION. -- POE 18 REFERENCES... SE J Page 19 00500 _--_--m WILDLIFE INTERNATIONAL, LT 6 ROCIO, TAcOonFBtCiOnuNLeTdENETS TABLES AND APPENDICES TABLE 1: TABLE2: TABLES: TABLE4: SCtumyulWatHivePMFoOrSality roma Nore therneBobwhic Ace Disary Toect Page20 MSteuadnywBiotdhyPWFeOiSg.h.t. 9 froma NorthereBobwthiterAcute e Dieay TosicPhy g21 AMceuatneDFietcdarCyonTsouxmipctioSnt(gybiwriddhayP)FOfS aoNovrtmheern Borbwhite Pages `GArcouepDiGcrotsrsyaTtohxiocliotygSctluOdbysweirtvahtPiFoOnsS.fraNoorthmern Bobwhiteet PORES TABLE 5: NCourmtuhleartnivBeoMbowrhtailcitAyc(uEtsetDiimsattaerdyCTuomuxliactyivSetuDdosyew,imhg/PkgO)S.fr.o.am Pugs 26. APPENDIX: CerifoifAcmaytse. ne Page APPENDIIX: Diet Formulation... ne PAG2E8 APPENDIIX: Anayscal Mcthods and RESuS cc Page 29 APPENDIVX: DietPrepasio...... enn ORE APPENDIVX: `CAumcuelaDtiicvtearMyoTorxiaclbiSytPtundyyritohmaPFNOorSthervBobcwhitr. Pe 44 APPENDIX VI: AIcndsiviDduiatlarByodTyoxWieciitgyhSs (y9)wfirtomhPa FNoOrSther Bobwhite Pagdes APPENDIX VII: NoFretodheCronBsoubmwphiiotneA(cguhtireddDieeyt)arbyyTPeoniftoma SUVPFOS oe POE 58 APPENDIXVII: Cha0nPrgotoscol... ---- APPENDIX: Personnel InvalvedintheStudy nn PGE62 APPENDIXX: Revised StudySummary... Pag APPENDIXiX: ReportAmendment. rn PORE 8 AMENDED COOL0n WILOLIFEINTERNATION1A1L0 , a. pROECTNO: ssa SUMMARY SPONSOR: 3M Corporation TEST SUBSTANCE: PFOS WILDLIFE INTERNATIONAL LTD. PROJECT NO: 454-103 STUDY: PFOS: A Dietary LCS0 Study with the Northen Bobwhite RESULTS: The dietary LCSO value determined to be 220ppm for a.. northem bobwhite exposed to witha 95% confidence interval PFOS of 164 was ppm 410 269ppm 2. The slopoftheconcentrationresponse curve was 7.005andthe chi-square valuewas 0.023. The no mortality concentration Was 732 ppm ai. Biscd upon treatment related morality, signs of toxicity and cffects upon body weight gain at the 146 ppm ai. test concentration, the no-observed-Tec concentratwiaosn 73.2ppm ai TESTDATES: Ho~cAprhil 12,1999 Acclimation - April 12:2, 1999 Experimental Start -April 22, 1999 Experimental Termination May 14, 1999 NCOONMCIENNATLRTAETSITONS: 0,183, 36.6, 73.2, 146, 293, 586 ad 1171 ppm. TEST ANIMALS: Norther Bobwhite (Colinus virginia) AGE TEST ANIMALS: 10daysofageattest inition SOURCE TEST ANIMALS: -W8i5l9d8liCfeomInmteermacteioDnrailveLu. Production Flock Easton, Maryland 21601 STUDY COMPLETION: April 26,2000 AMENDED REPORT DATE: March 23, 2004 m7aSteeeriAalppfoelnlodwiiXxngfstoudtyhecormepvliestdionno.minal est concentanid oensslbased ponte sanalyssofthe test AMENDED GRO _--m---mpmnm---- WILDLIFE INTERNATIONAL, LTD a PROJECT NO.: 454-103 This stad was conducted by WillIiNeTnReOmDaUtCioTnIlONLid, for 3M Corporation a the Wiis Inmational Lit. avin oiclogy faci in son, Maryland. Theinfe porn of he test as conducted rom April 2, 19991 May 14, 1999. Raw daa gene Willie erations Lid and copy of the fnl sponar fd under Pre Number 454-103 in archives load on the Wide nrmtonal Ld se he objectiveof his study wast cunts hOeBJoECnTIcVEofa est substance 10 the Norther Bobwhic (Cons virginians) administered through the dio ive days, An LCS0 valewil be clcltd, itposile. The methods use in condusM ingAhsTsE adARNarI De MbaA EsTedL HpOoS DnS procedars specifi in he US. Envionmentl Protection Agcy Sere 850 Ecological Effects Tet Guidelines OPPTS Number 850.2200 (1), Section 71:2 of the Eavionmentl Protcion Agency's Regisraion Guidelines, Pesiide Assent Guidelines, FIFRA Subdivision E, Hazard Evasion: Wills snd Aquatic Orgasms 2; OECD Guideline 205, Guideline for Tsing of Chemical, Avian Dietary Toney Test; 0 upon ASTM Sanda BSS?-7,"Sandard Pract for Condcing Subacute Diary Toschy Tes with Avian Species" (6). "TestTSuhbestteasntcseu:bstance was recived from 3M Corportion on October 29, 1998 and vas assigned Wide Iemaional Lid nificaion Number 4675 up recep. The fst substance was whi powder deified ss: FC.95 Lot No:217. The pri purity fhe est stance ws 98.96, with an xpiaton date of 2008, Following st emination, ets matrel ws rcnayzsd. The resus ofranayi diatea st substance pryof90.49%. Alles conenrtions ave ben dsedo sreofrleecdtuthnedpeuraimtbyierneptocrotnedditoinontsh.e new Certificateof Analysis (Appendix 1'. The test substance was. The icra sandard was rcsived from 3M Carporsion on ly 2, 1998 and vas assigned Wide Icrnionl Ld, dentifcion number 4526 upon recip. The neal standard, gran eelohrvioangoaeymcooo opdel,c yeastyoFt 5A.eaWnhtci brocmoneessscaroe wi GoLd Pnsadpaeaydte Aine n0u07 WILDLIFE INTELR D NATIO PROWN CTNOA :dskL icy, 9 teil, was identifi as 1H, 1H, 2H, 28 Peslorooctane Sulfonc Act, Chemical Abstct Number: 27619:97-2. Thstandard was soredndes ambitconditions Treat"mTheenteGstrocounpssisted of geometric sere of sven fest consetations and contol group, Thin nonhern bobwihic chicks were asigned 0 the contol group and en northern bobwhite chicks were assigned to cach of the treatment groups. The birds were sorted by weight, then chosen airnoduipssc.rimiTnhaetelbyinfdrsomwewriethhinouesaecdh irnepbrreosoednitendg wpeeingshtcocnltaasisnfionrg pfliavceemcehnictksintcoaccho.ntroNlomainndatlredaitemteanrty concenraions used inthis study wer 0, 18.3, 36.6, 73.2, 14, 293, 86 and 117 pats pe millon `active ingredient (ppm a.i.) of PFOS. The dietary concentrations were established based upon known toxicity data and information supplied by the Sponsor. Each grou was dt appropriate esto contol te fo ive days, Dveing the exposes poried he contol group received untested feed. Following the five-day exposure period al groups were ven untested basal ie for three days. On Day 3, halfof the survivin estnent sd conrl bids were chained and ver issue, blood, nd bile samples were collected or analysis, The remaining birds wee fed basal ration unl Day 22. On Day 22, thes bisds were cutbanized and ls sampled fore weigh, bod, and bie `D"uThre oraiftmthaieryToepsnhtasesofthis ts and thei durations were: 1. Accimation- 10 days. 2. Exposure das. 3. Posbonposuschsrvation- 3or 7days TestABirdnsorthern bobwhit (Colin viginians) were 10 days of age and appeared to be in good health at nition ofthe est. The birds were blind from Widife Intemational Lu. Production Flock, Eason, MD and were hatcheond Apel 12, 1999, Birds ranged in wight rom 18 0 23 grams at test iio. The bids sed in his study wer immatare an coukd no be difrised by sex 60001 _--mm WILDLIFE INTERNATIONAL, LD Co PROWCTNO: sum "10. Albindswere from the sme hatch, prscared and phenotypically inistinguisable from wild birds Al bindswereacclimate to the caging and files rom th day of hatch nil nation ofthe est. AnimTahlrDoiugehtout acclimation and testing al tet birds were fed a game bird ron formulaied to Wilde Inemational Lids specifications (Appendix 1. The chicks were given a vitamin supplement in thei water fom th day thy were hatched uni the niisionof th tes, Water rom he town of Exton public vate suppl, and feed were provided ad linn during acclimation and testing, The bindsreceivedno formofnibotc medication durin acclimation or testing. DPiertepaTrhaettiesotnsubstance was mixed dict it te tin. Mixing was done ith a Hobart mixer (Model Number AS200T), All distary test concentrations were adjusted to 100% PFOS based upon ihe reported purty ofthe test substance. All dictry concentrations and he LS0 value are reported a5 ppm ai. in the dict. Nominal dietary test concenrations used in this study were 183, 36.6, 72, 146,293, 586, 1171 ppm ai. (Appendix IV), DietSSaammpplleisngof the test diets wre collected to verify the test concenrutions administered and (0 confirm the stability and homogeneity of the tet substance in the diets. Homogeneity of the test Substancs in the diet was calused by collecting six samples om the 15.3 ppm ai. a. fst diet and six samples from the 1171 ppm a. est dict a preparation on Day 0. Homogeneity samples were collected from th top, middie and bottom of the Ieft and right sections of th mixing vessel, The Homogeneity samples also served as verification samples. One verification sample was collected from the contol dict and two verification samples were collcted from cach reining treatment roup at preparation on Day 0. At the endofthe exposureperiod (Day 5), one sample was collected from the control and two samples wee callie from each reatment grou to detrmine sail of ihe test substance in the diet under test conditions. The stability samples were callcted from feed remaining in the fesders afer beinga ambien test pen conditions for fv days. Samples were transferred immediateltyo Wilde Intemational Lid. analytical chemistry. 600805 WILDLIFE INTERNATIONAL, LTD oo ne PROJECTNO:ast103 AnalvicalMethod "The method used for the analysis of the avian diet samples was based upon methodology developed at Wildlife Intemational Lid. and eniled "Method Outline fr the Determinationof PFOS in Avian Feed" Avian diet samples were extracted with methanol. Methanol was added to a requisite quantity of feed contained in a French-square glass botle. Bottles were capped and shaken on a shaker table. `Samples were vacuum filteredusing qualitativefilerpaper. The retained feedwasrinsed three times with methanol into the filtrate. The fillrae was transferred to a volumetric flask and brought to volume with methanol. As appropriate, samples were further diluted with methanol. Each sample then was diluted with a 50% methanol ; 50% NANOpure water solution containing 0.100 mg 4H PFOS (intemal standard). and 0.05% formic acid (v) 50 tha they fell within the calibration range ofthe PFOS methodology. Amethod flowchart is provided in Appendix 11, Figure 1 Concentrations of PFOS in the standards and samples were detemined by reversed-phase high performance liquid chromatography using a Hewlet-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer APY 100LC Mass Spectrometer cquipped with a PerkinElmer TurbolonSpray ion source. HPLC separations were achieved using a Keystone Basil Cis analytical column (100 mm x 2 mm LD, 3 pm particle size). The instrument parameters are summarized in Appendix II,Table 1. Calibration standards of PFOS prepared in a 50% methanol: 50% NANOpure water solution containing 0.100 mg 4H PFOS (intemal sandard)L and 0.05% formic acid (vv), ranging in concentration from 0.00229 10.0457 me .i/L were analyzed withthe samples. The same and most prominent peak response for POS was utilized to moritor PFOS inal calibration, quality control, and study samples. No atempt was made (0 quantify PFOS on the basis of individual isomeric components. Linear regression equations were generated usin pesk area response ratios (PFOS intemal standard) versus the respecive concentration ratios (PFOS : intemal standard) of the calibration standards. A. typical calibration curve is presented in Appendix Ill, Figure 2. The concentration of PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms of low and high calibration stanardeparesrendtedsin Appendix i, Figures 3 and 4, respectively. GROsNG _--mm WILDLIFE INTERNATIONAL, to ne [------------ The method limit of quanitaon (LOQ) for thse aslyses was st a 1.15 ppm a. auld 1s he productofthe west calibration standard analyzed (0.00229 mg 8.1) and the iaion factor of the matrix blank samples (500). "Two matrix lark samples were analyzed to determine posse inerfeences. No interferences re observed ator above the LOQ during sample analyses (Appendix I, Table 2). An inierference in the feed sppesre at approximately the same etnvion ime as the peak of terest bat it was well below the LOQ. A representative chromatogram ofa mati blak i preseted in Appendis Il Figure 5. Avian dict was fortified at 457, 183 and 1830 ppm .. and analyzed coneumently with the samples to determine the mean procedural recovery (Appendix I, Table 3). Sample concentrations wre no cotectefo he mean prossdurl recovoefry 94.7%. A representative chromatogram ofa matrix fortification i resented in Appendix Il, Figur.e An example calculation is presented for sample number 454-1032, nominal concentration of 183 ppm ai. in avian diet Intel Weigh 100 Fil Volume, 200m. DilutionFacto: 100 eros Guo actorx ial dui tor) PROS Pek Ars: 113568 Inna Sanda esk Ave: 413160 Pek Ara Rt 02749 Catbraion curv cain Sop: 277397 nse 001354 Carve isweighted 15) PROS (mgairyatinsumens = ( EPeekkVarrear nrratiinoo- t (-ineeSrieppe e) xxe1L5S..CpCooncnectnetnrra) ttiioonn ROO? _--m--mm---- WILDLIFE INTERNATIONAL, LTD ie PROJECT NO.: 454-103 Note 15. = tral standard. 2705 gpmaiy mele 2749-00159%)x0.m1g00. wm - oom mgain - Tw PTTOS 088e 17) tv ene x Fi m Volum (t 1) itio on Fir 0009230200 100 ew - Isp PercentofNominal Concematon = rFoOsSO(PomSa)) innosmaimnpalle = 1WEwsS roe toi Housingand Environmental Conditions During acclimation an testing. al birds were housed indoors in bateies of thermastaially conrolled brooding pers manufactured by Beacon Steel Products Co. (Model No. B7350). Eachpen hEaxdtfelmoaolrwsaplalcs,ecethlaitnmgesaasnudrefdloaorpsprwoexriemactoenlstyr7uc2tXedo9f0gcaml.vanCiezielidnsgeheeigwhitrwaansdasphpereotixnigm.atBeilryd2s3wcemr.e sorted by weight, then chosen indiscriminately from within cach represen weight class for assignment o pens. Each group of birds was identificd by pen number and est concenraton. Individual ind were identbyilefgbianedsd. During the test the average temperature in the brooding compartment of the pens was 38C + 2C (SD). Average ambient oom temperature for this study was 273C 4 12C (SD) with an average relative humidity of 31% + 14% (SD). The photoperiod (maintained by a time clock) was sixteen Hours ofligh pr day during acclimation and thoughout the est. The light source was uorscent Highs which closly approximate noonday sunlight. The birds were exposed to an average of Crom -_--m WILDLIFEINTERNALDTIONAL, prosEcTio: 4se03 ae approximately 139 lu ofilumination. Housing and husbandry practices were based on guidelines established by the National Research Council (5). Observations During aclimation all birdswereobserved daly. Bids exhibiting abnormal behavior or physical injury were not used. Following test initiation and continuing until termination, al birds were typically observed at last twice daily. A record was maintainedofall mortality, signsof toxicity, and sbnormal behavior, `AnimalBodyWeightoFecdConsumption Individual body weighs were measured at th initiation of the test, on Day 5, Day 8, and on Days 15 and Day 22 for all remaining birds. Average feed consumption values during the exposure period (Days 0-5) and the post exposure observation period (Days 6-8) were determined by pen for cach treatment group and the control group. Additonal, feed consumption was determined for Days 8:15 and 15:22 for the remaining treatment and control birds, Fed consumption was determined by measuring he change in the weight of th feed presented o the birds over a given period ofime. The accuracy of fed consumption values may have ben affected by the unavoidable wastageof eedby the birds. GrossNeszopey AT test birds that died during the cours ofthe test and al birds remaining tthe termination were Subjcted to a gross necropsy. Addionally, livers were eighcd and liver tissue, blood, and bile wet collected from bids uthanoinDzady 8 and 22, and when possible fom those tht died during the courseofthe sudy. SutisticalAalvses Mortality data were analyzed using the compute program of CE. Siphan (6). The program was designed 1 calculate the LCSO value and the 95% confidence interval by probit analysis, moving average method or the binomial probability method (789). Tn his study, the LCSO value was determined using the probit method. The slop ofthe concentraton-esponse curve and resuls ofthe goodness of ft est are reported. Body weight data were compared by Dunnett' test using TOXSTAT softwar (10,1). No statistical anlyses ere applied to feed consumpion data. 600509 _--m--m---- eee ---- WILDLIFEINTERNATIONAL, LD 1s PROJECT NO; 454-103 RESULTS Dict Anasis Avian dict samples were collcted from the 18.3 and 1171 ppm ai. est concentrations and analyzed samples to evaluate also served homogeacity otfh test subsance in the avian as verification of test subsance concentrations. diet. The analysis of these Resulting mean measured concentrations, standard deviations and coeflicients of variation (CV) for thes tet concentrations were 19:5 2.13 ppm ai. (CV = 109%) and 1196 + 102 ppm ai. (CV = 5.87%), respectively (Appendix I, Table 4). Control avian diet samples cllected duringthe test showed no interferences above the LOQ. Samples collcted during the est o verify the 36.6, 73.2, 146, 293 and 586 ppm ai. fest $37 substance concentrations had mean measured ppm ai, respectively. These values represented concentrations 110, 102, 119, of 40.2, 99.3 and 74.5, 174, 91.6%of the 291 and nominal concearations, respectively (Appendix I, Table 5). Analysis of avian dict samples collcted from feeders after being held at ambient temperature and 10o2ft%he Day 0 valuesforthe 18.3, 36.6, for five days averaged 101, 122, 73.2, 146,293, 586 and 1171 ppm 104, ai. 101, 109, 114 tet substance concentrations, respectively (Appendix I, Table 6). A representative chromatogram oaftest sample is shown in Appendix Il, Figur7e One incidental mortality occurred i the conrl group during the course of the sudy (Table 1 and Appendix V). On the moming of Day 5, one bird was noted with a broken lg and was subsequently euthanizoend Day 6. Additionally, wo birds in the control group were intrmitenly noted with fot Jesions associted with cage mat aggression. Otherwise,alcontrol birds were normal in pearance and behavior througho the test. No treatment related moraiies or overt signs of toxicity were observed in the 18.3, 36.6, or 73.2 ppma. treatmentgroups. Onebird in the 18.3pom i. rcament group was notasleamde rom Day 6 thtough Day 8, nd bloodofundetermined origin was noted on the undersideofane bird rom ihe 73.2 ppm a. ratment group on Day 22. Otherwise, al bids in the 15.3, 36. and 73.2 pom a. weatment groups were normal in appearance and behavior throughout he test period. GROG WILDLIFE INTERNATIONAL, LTD -16- PROJECT NO: 454-103 "There was 11% (1 of 9) mortality in the 146ppm a. treatment group, 80% (8of 10) mortality in the 293 ppm ai. treatment group and 100% (10 of 10) mortality in the 86 and 117) ppm a. treatment groups. In the 146 ppm a.. treatment group, one bird was euthanized on Day 3 after sustaining abroken leg. This incidental mortality was not used inthe calculationofthe LCS0 value. Additionally, there was one treatmentelated mortality in the 146 ppm a.. treatment group, a bird found dead on the moming of Day 7. Clinical signs oftoxicity were observed n this treatment group on Day 5, when two birds displayed wing droop. All other bids at his est concentration were normal in appeaarndabenhacvieorfo th duration ofthe tes. nthe 293 ppm a.. treatment group thre were ighttreatment elated mortalities, occurring on Days 5, 6.and 7. Signs of toxicity were first observed on the morning of Day 4 and continued to be exhibited through the moming of Day 8 for the single bird cuthanized on Day 8, and through the aftemoon of Day 8 for the single bird surviving until Day 22. Signs of toxicity included a ruffled appearance, reduced reaction to stimuli (sound and motion), lethargy, wing droop, loss of coordination, lowes limb weakness and convulsions. The single remaining bird appeared to have recovered and was. nomnal in appearance and behavior from the atcmoon of Day 9 until test termination. In the 586 ppm .i. treatment group mortalities were fist noted on Day 3 and continued to be bserved through Day 7. at which point al birds had died. Overt signs oftoxicity were first observed on the aftemoonofDay 2 and continued through the moming of Day 7, when the final birds were found dead. Signs of tonicity observed among birds in the 586 ppm a.. treatment group included a ruffed appearance, reduced reaction t stimuli (sound and motion), lethargy, depression, wing droop. Tossofcoordination lower limb weakness, lower limb rigidity, prostate pasture, and convulsions In the 1171 ppm ai. treaiment group 100% mortality had occurred by the moming of Day 4. Signs of toxicity in the 1171ppm a.. reatment group were first observed on the afiemoon of Day 2, with the frst. mortalities noted on the moming of Day 3. Signs of toxicity observed prior to death included a ruffed appearance, reduced reaction to stimuli (sound and motion, lethargy, depression. ossof coordination,wingdroop, and lower limb weakness andrigidity. 6002 eee WILDLIFE INTERNATIONAL, LTD 17- PROJECT NO: 454-103 Body WeightandFeedConsumpion When compared 1 the control group, thers werenoapparent treatment related effects upon body weight among birds in the 18.3, 36.6 or 73.2 ppm a. treatment groups. However, there was a concentration responsive reduction in body weight gain or body weight los in the 146, 293 and 586. ppm a. rcament groups during the exposure period (Days 0-5) (Table 2 and Appendix VI). Differences from the control group were satisically significant atp < 0.05 for the 146 ppm a. level and at 0.01 for the 293 and 586 ppm ai. levels. A statistically significant (p<0.01) reduction in body weight gan continued tobeobserved at the 146 ppm .. test concentration through Dayof the study. At the 293 ppm ai. concentration a statistically significant (p<0.01) mean weight loss continued trough Day of the study and a masked reduction in weight gain was noted through Day. 15 ofthe study. Due o total mortality, body weight effects couldnotbe determinedforthe 1171 ppm ai. level during the exposure period or for the 586 and 1171 ppm .. treatment groups for postexposure period. There wereno apparent treatment related effects ponfecdconsumption at the 18.3, 36.6, 73.2 or 146 ppm a. test concentrations (Table 3 and Appendix VII). However, a reduction in feed consumption was noid at the 293, 586 and 1171 ppm ai. treatment groups during the exposure period (Days 0-5). There were no eaimentrlated effects on feed consumption in any of the surviving treatment groups during the Day 6-8 post-exposure period. In the 293 ppm as. treatment s70up only one bird survived Day 22. The reduction infeed consumption observed atthe 293 ppm ai. test concentration during both the Day 8-15 and 15-22 postexposure periods was the result of having only one bird inthe pen, and was not considered obe treatment elated. GrossNecropsy During the course of the test, ll birds that died were subjected 1 a gross necropsy. Necropsy. resus for birds found dead were similar. Common observations included thin condition, Ioss of muscle mass, altered spleen color, autolysis of tissues and pal organs. Details of the necropsy findings are presented in Table 4. HA of the surviving birds were subjected (0 gross ncropsy on Day and the remaining birds were necropsied on Day 22, following test termination. On Day 8, one bird in the 73.2 ppma. restment group was noted witah slighty pale liver. Due to the isolated natureof his finding it was CnOnE -- WILDLIFEINTERNATIONAL, UD pROJECTNO. dsaios Se not cansidred be related to eatment, The single bird etbanized from the 293 ppm a. treatment r0up was observed at necropsy to have witha lackofmuscle mass and general thinness. Since hese findings correlated with an impact upon body weight noted at this concentration, the findings were considered be rcament related. Necropsy results were emarkable for ll other bids euthanized on Day8. Similarly, Day 22 necropsy findings were unremarkable for ll is. CONCLUSION The dietary LC30 value for northern bobwhit exposed to PFOS was determined 0 be 220 ppm ai. With a 95% confidence interval of 164 ppm i. to 289 ppm ai. The slope of the concentatonresponse curve was 7.005 and th chi-square value was 0.023. The no mortality concentration was 73.2 ppm a. Based upon eament related mortality, signs oftoxicity and effects upon body weight gan at the 146 pp a... test concentration, the no-observed-effect concentration was 73.2 ppm ai. Goon WILDLIFE INTERNATIONAL, LT0 -19- PROJECT NO: 454-103 REFERENCES 1. U(dSra.ftE),nOviPrPoTnSmeNnutmablePrro8t5e0c.t2i2o0n0:AgAevnicayn,D1i9t9a.ry STocxriccsi8ty50Te--s.EcologicalEffectsTest Guidelines 2. SUuSb.diEvnivsiiroonn,menHtaazlarPdrotEevacltuiaotnionA:gencyW.ild19e82.andPesAtqiucaitdiecAsOsregsasnmiesnmts,Gusiudbelsiencetsi,onFI71F-R2A, Environmental Protection Agency, Officeof Pesticide Programs. Washinglon, D.C. 3. OTersgta.niOzEatCiDonGforuEciofnoordmTiecsetCinoglopoefriCahteimnoincaealsn.d DGeuvideelloipnmeen20t5.. P1a9r8i4s.. Avian Dietary Toxicity 4. SAumbearciutceaDnieStoacriyetTyoxifcoirtyTeTsetsitsngwiathndAvMiaatnerSipeaclise.s. A19S87T. M SSttaannddaarrddE8P5r7a-c8ti7c.e AfonrnuCaolndBuocotkionfg ASTM Standards, Vol. 11.04. Philadelphia, PA. 5. WNaatsihoinnaglionR,esDe.aC.rchNatCioounnaclilA.cad1e99m6y. PreGsus.ide13f5oprp.the Care and Use of Laboratory Animals. 6. SCtoempmhuanni,caCt.iEo.n 1976. US. EPA, Environmental Research Laboratory,Duluth, MN. Person! 7. SHtaezpahradn,ECv.aEl.uat1i9o7n7s., MAemtehroidcsafnorSCocaieltey ufolraTnteLsitCniSng0g.aPnadgeMsat6eri5alsI.-nAq8uPautb4i.c NToo.xicSoTlPo.gy6a3n4d Philadelphia, PA. 8. Finney, DJ. 1971. StatisticalMethodsin BiologicalAssay. 20ded.,Griffin ress,London. 5. Thompson, W.R. 1947. Bacieriological Reviews, Vol2,No.2: 115-145. 10. wDiutnhnactC,onCt.roWl. Jo1u9r.55.AmeAr.MuSlattisp.leAsCsoocm.pr5i0s:o1n09'6s.1P1r2o1cedure for Comparing Several Treatments. 11. Gulley, D.D. 1990. TOXSTATRelease3.2. The UniveofrWsyoimitngy. Coot dl WILDLIFE INTERNATIONAL, LTD 20. rROECTNO: stay TABLE `Cumulative Mortality froam Norther Bobwhite Acute Dietary Toxicity Study with POS Gpreromuepnal NoDeExpdoPsuerreNoPerBioadpowd | Gps) Do Dwi Dw Dud DwiD3 NoP.oDsetaEdxpPoesruNroe.EPexrpioosded mb Du Di Contest o 030 0% 030 oso 030 030 10 0 130 Treatment 183 366 2 16 ol ol wo 00 om ol oo oo 0 010 om ol ol 00 ono ono ow ow ol wo oo ne 0s om mo ono ono wo ono oo oo ono ono www ES ol wo oo ono ol zo 40 #10 S10 S56 wo ol oi 0 20 $0 m0 oi 1000 un Ol wo 00 Io 1010 10% 100 tno 1010 T27 oBCimr3o0crvuaalbleeiwssascoccnuodynJaanTeobsfeuh5i0Cnpoognsmobr31 wkrieh.se99t 7ocuposnboersa Dmayer1a0DofayTpE25.om WI pT Grout _-- WILDLIFE INTERNATIONAtLm, "ae PROECT No. 4st-103 TPAaBgLeE|? Mean Body Weigh (2 froma Northern Bobwhite Acute Dieury Toxicity Study with PFOS -- oGmraon,) E --S -- al PosExposue Paid Dayo Days Day0:5 Day Day58 Change' Control Me So owT0now PE52 ii Treatment Iby Tos 02 n3 06 Mkyen 0hawhow n3 os! w3 2 Msoem owv2wo! n 3 ai 126 we bMyem m > a53om w FR e i3e mm Mkyen 0[eeAae wPA ea o5e $6 M5em 0Todere dae :. : .: mo Me ! :. .: R SemealrovucCeOnu dfooees rcyiott rouBetp lywe SoHART <0 Due' ac So rE Since tohot pea 1p 001 (Det Gnosis WILDLIFE INTERNATIONAL, LTD 2. PROTENCOT 454-103 TAPaBgLeE?? Mean Body Weight from a Northen Bobwhite Acte isa Toxiity Study with PFOS T TmeGaroiw) T e em m Gee et Dus Dwis Dudas bum Dnln cameo Cont"rolyebyn vwsh on3 ni 23 is Treatment kN y s E4 66 BMyen 3 e (A w w7 n3 a5 w3 u3 5`0 n2 EMYan 37%0 owa i 2 "3 we 5man wHI oos ow! 2 n2 is\ wm pen EE 5s PM 56 5ven .: :. . :. : . wm 5Man :> . .: . e O taoatoe edoa ly wh Dots on e re ad teats get oecorte gop 1p <0.05 Dues ca) Goon? -- ee -- ------------------------ ---------------------- WILDLIFE INTERNATIONAL, LTD 23. PROJECT NO. 454-103 TAPaBgLeE| S `Mean Feed Consumpion (TgohrbiicridtdyayS)ufdryomwiathNoPrFtOhSem Bobwhite Acute Dictary TEeGrou--p (opmai) Conrat Exposure Period Days 0 M3ean 92 Treatment 183 5 366 5 72 0 we 5 m s ss . Je 4 PostExposure Period Days 12 u 2 5 10 9 1 y Tem -- Goats _--nm--nmm WILDLIFE INTERNATIONAL, LTD 2 PROJECTNO.: 454-103 TAPaBgLeE2 3 Mean Feed Consumption (TboxiicridtyyS)tufdryomwiathNoPrFtOhSem Bobwhite Acute Dictary = Group Goma) -- PostExposure Period DTS ou Te -- o IMe)an 20 51 Treatment 183 366 n2 16 EY sto un 0 n " 15 i 1s un " 5 5 . Tm Cross _-- WILDLIFE INTERNATIONAL, LT 25. PROJECT NO: 454-103 Group GrosTsAPBtLhaElo4gical Observations froma Northem Bobwhite Acute Ditary Toxicity Study with PFOS Birds tht died during the courseofthe study TT TrT e mitae Finding PPMAL Cowl TW V Be I m Ni NN Neo Ne Abdominal cavity. some utalyss Abdominal cavity, autolysis throughout 0 0 0 2 o o 2 4 i | Crop, empry Emacited o o 0 2 o 2 s 2 5 5 Fracured eg Gl wet empty 1 o ' o o | 0 o ) 0 Gicaard content ie ssined Hea,ateior portion motied white color o 0 o 0 2 1 5 o 1 0 Hear, pale Intestinal coments ar ike 0 0 0 2 ' 0 0 0 2 0 Keel, promi: Kidneys, ple 0 0 0 0 1 0 3 2 10 o Liver, ple snd motled Loss ofmuscle mass 0 1 0 o 0 0 o hl 7 9 MSmuaslclulinasrustkuereel, pie 0o o' 03 0o o0 Spleen, black Spleen, dark o o o 1 0 o o o o z Spleen rey Spleen, grey-brown o 0 0 1 0 0 0 0 o | SSppliecene,, psamlael 0 0 1 o 1 0 0 0 o | Spleen, small and pale Thin 0 o 0 0 0 o 3 o " 2 Not Remarkable 0 0 | 0 o 60050 _--_-- Z$ I Fh g2 j%of|25 | LE. | _|F 1# ik2 2 | si | . i E2 | JF583 H3Bf4Ee1lSeesd 23s Coa SE p tl 3| ls 2) gdll sr5e5e5r2e8 Sesser ssssms s885ss 8338355 sss58s ,83,85,8,s 38883 $3333 rmene 53 33 33 22 3% 23% 33) sperssesssanay 5332 33 30 55 53 35 j 5835 2 3838 5% 928% 23 3898 3% 32%8 22 58 35 834 i3 & ez ss 2225 8Ei 55 5% 33 5% 53 53 ssf? 2% 55 g 85 2 5% zgiolf l2 i 5232 53 8% 23% 238 33d i sfsZ3E3agsgs5e2s3g8a3a8E3a9gi i i1dy $553 3% 333333 338; 11 cs 2228 88 85segSaE 88l1 iiiI) $3 3% 35 55 53 238 33|f i% $3 3% 58 35 35 23 sgl2fi}d someon cee HHHELH | 8fl occ g22 38 27 23 8x #22 E IaH: &5 Szii] isTE 3S 3 Gros _--mm WILDLIFEINTERNATIONAL, co -27- FROIECTNO: t54403 APPENDIXI Certificate Of Analysis FC-95, Lot 217 Mares, 2000 Rib oe Tem ERINen --1= ------ 1|-- -- ee r mna 1 ----| fred I Ee E rn rkE Ke E Cros _--_-- WILDLIFE INTERNATIONAL, LTD 28 PROJECTNO: 454-103 APPENDIX TI WILDLIFE INTERDNIAETTIFOONARLMULLTDA.TIGOANME BIRD RATION' INGREDIENTS SFionyeBCeoanmMMeeaall, 48% Protein pWheeant Msideds `AAglfwaalfya MSpeeacli,al2,0% Protetienin GDrrioeudnWdhLeimyestone MEeatshtimoanniCnaelPPrheomsix + Liquid GViLtaFmeirnms(nFderMmiantecroa)l"Premix (seebelow) TSoatlatllodized PERCENT (%) a30865 660500 340000 025500 003650 002352 T0001000 VITAMIN AND MINERAL PREMIX AMOUNT ADDED PER TON VViittaammiinn AD, RNiibaocfilnavin VPainttaomtihnenBiyc Acid BiFooltiicnAcid PTyhriiadmionien.e VViittaammiinn KE (Menadione Dimethylpyrimidinol Bisulfc) ZiMnaneganese: TCoodpinpeer Sreolnenium 72000000.00000011UC.U. 460 ggrraammss. 1m0 ggrsams 66040 mmagss 1122ggrraammss.. 200005180g.rams. 14072 ggrraammss 6185ggrraammss. 18512 mgrgasms "Tohfe g5u%arcarnutdceedfiabnera.lysis is a minimumof 27% protein, a minimum of 2.5% crude fa and a maximum FermentationBy-Products(SourceofUnidentified Growth Factors). Cron _--m-- ee WILDLIFE INTERNATIONAL, LTD -29- PROJECTNO: 454-103 | APPENDIXIT ANALYTICAL METHODS AND RESULTS R051 _--_--nmm- WILDLIFE INTERNATIONAL, LTD 30. ee PROJECT NO.: 454-103 INSTRUMENT: ANALYTICAL COLUMN: `OVEN TEMPERATURE: STOPTIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: PROS RETENTION TIME: IRENTTEENRTNIAOLNSTTIAMNE.DARD PROS MONITORED MASS: IMNOTNEIRTNOARLESDTMAANSSD:ARD APPENDIX I Table 1 Typical LC/MS Operations! Parameters HaePwelrkeitnP-EalcmkenrdAPMIo1de0l0L1C10M0aisghSpPeectfroormmeatnecreeqLuiiqpupieddCwhirtohmaPteorgkrianp-hEwlmiehr T(uSrMb)olonSpray ion source. Operated n selective on monitoring mode Keystone Betasi Cy column (100 mmx 2 mm LD, 3 pm particle size) soc 100minutes 0220 mminute 72.0% Methanol : 28.0% NANOpure Water conning 0.1% Formic Acid 25041 Approximately 7.0 minutes Approimately 45 minutes 498.6 amu 4267 am CROSS _--mmmm WILDLIFE INTERNATIONAL, Lo - PROJECT NO. 454-103 APPENDIX Il Table2 Matix Blanks Analyzed Concumenly During Sample Analysis m ed eeee POS! asin Toe MAB Mari Blank Goma) <1oq MAB2 TahnalyTzoedmo(0f.0q02u2a9nmigo4n1/1()LOaGn)d Marx Blank hweasi1i15npofme1t. ofbahreedmwapra lherproadmupclteosf F(0e0) <0 Towesr cabraion Sandor 60086 _--m e WILDLIFE INTERNATIONAL, LTO PROJECT NO.: 454-103 AeveNDIX Tats E --Ma--tiFortificationsAnyE zed Concue r r Ding Sample Anyi-- Sample Number ome: Pee BE (454-103) VASA Fortified pd oEE Measured oe Recovered Naassas 105 i1e6 %"61 --_-- MwAasSE 11880 1i5n1e6 5561 TTITT TETE TITI Aes Sandard DevNte-l bi:os emsmeneeyes xp or Rademsin icnoimoens. TVo omes ToaeIbaencpyrSeepEorepedesn,g NAS ven TT Ron? --_ 23| L5 os . 5 $ is i z , f E 25 feel Ls 2 3: HeE3lH3 5E15H80 2 2ii5Ts [2 | 1, 2 SE Lo NE) HEPESEEFEELER i 11 3 i || H 2 31.7E2f7e|3zr3s5sd7_eld & i=oegdHnafi.il;t 3g i i & iz i2 8J <! Hio Z| eneoor 22mm] i 1id &zz3 % "re fHiwgHdi tL|| HiEF gilZi 38 i 2 600553 _--mm he Z 3 Lil 5 2 8 2 CE 8 3 Eg Hl i ii g il g zz :uy x|b iEki! 3g reese Ea) [ = 1] 1F LH 583 E ile w33 mFoa eszs 75} | gE]: ; . | [ THHrE eee | 3 HE ommmemmme g Kk 3 ifis sa o|3i5 si i: ii Eo fiti ee fig iigi.d Hl Hie 28 8 3 aii 5 3 ai 2al COOnG ee : L; i5 3 s3 8ozr 58z 3: z|[3 | Wels vee oald gt i | 5s i i z3:i HtiA3e1% 5=2 332 3a2r 5e2x ozzzz seyw B2E.l)F3i | ob5LAR [Er nn ee me meme ne]1: Zoe e 3 : LI 1 i: :||Sle zeros oe 2 C1 |sl|ki: e ere seeLdE J Sr it i 5 iH gl [1 EERE g y & u , i =u Jil2ee3e2e0r1e20c|8lii y : 5:8 25 GRO HO WILDLIFE INTERNATIONAL, LTD -36- PROJECT NO: 454-103 METHOD OUTLINIEN FAOVRIATNHDEIAENTALYSIS OF PFOS Pcp ma ifcion sles heetsdvi sdtackig he ichi: . OryMis This Forege i rf, he qtyoi fWhe ean 4(WI) i ms with etqty fi b08)s ok A500 rin dtbe ap ow rtcd 6 TS i pein ig ond lpr BM bk ie cof er er eld tcmts. Wang So During he hBindlhemittesefo r5mdinsdsBpinA,| ieehrticedhoew1hesindesofbedCoep ng iin oobe an eo bPrepo are the nettma ymaiisfiocnateioneses0eby sel drices. gyof mt O40 T Wegh 10slof he e is bak, isfotiosdRecordrhieci s chbosder 16. rch sshs For, esr 10s ofi ih tdpd ven Be hs bose Conpdpctsi hakercal. Allowth mp? 0 hikeor mise 30 ius4250pe Vaec cht thain ieede ted od3lhs cbr i Tote fc 320-mL. lume skeding sk vlitahmanb. ' Fora Pnrl cpsrepirint50%otstl)mto ei4g4nnN e orcmAdewittNluttO iiocnolbi,nS riosunen0g .of004LHEPMFSOSmh(ocdmollogsya.ndy. Bae omesls Ampito LOS sys Figure 1. Analytical method flowchartfo he analysis ofPROS in avian die. r0u3 -_ WILDLIFE INTERNATIONAL, LTD EN APPENDIX 1 PROJECT NO.: 454-103 150 100 | i oso | | o | | ems 000 | 0000 0100 0200 0300 0400 0500 Figure2 CAutryvpeiciawl leibiragthio(en1c)d.ufor PROS. Slap=e 2.77397; Iner=e0p0t18945 = 0.981. GOODIE WILDLIFE INTERNATIONAL, LTD 38. APPENDI1X PROJECT NO.: 454-103 10 167 ntonsy: 1995cps o s 7 & 5 " 3 2 us le 122 # TTTh T2EL5 T3em Tsao Tsek er asls sTmee Figured. Arepresentative on chromatogram of s low-level (0.002o2g9 1) PROS sandard [GRE _- WILDLIFE INTERNATIONAL, LTD 39. APPENDIIX! PROJECTNO: 454-103 - 0 a 7 s 5 4 3 2 1 9 20 48 7s 00 Tf%er25 e3rm 188 Itonsty:26984 cps 14s 122 190204 230 aSzons i6er% 871s zeiss Tsmane Figured. Arepresentative on chromatogram of high-level (00457 m 2:1) PFOS standard. R055 WILDLIFE INTERNATIONAL, LTD ~40APPENDiIX PROJECT NO.: 454-103 100 sol 2 a 0 60 s0 a0 : 0 Intensity:230ope 120 20 144 * 216 FPA . Ad ATAAAR7WA lB P2a00lhi T Te eSeh rSh iGk iSsm iTee aGrs sTaene Figure. AThreepraesrenotadtiviecchtrotmhaetroegcratmioonfaimmeaotfiPbOlaSnk sample (454-103-MAB-1) GROS3Y -_ WILDLIFE INTERNATIONAL, LTD a APPENDIX PROJECTNO: 454-103 0 166 Intonsity:6238cps. a 8 7 o 5 4 a 144 2 1 ohare 122 301 625% 9a1m 1s2o1s a6si% 17815 z8is | Tsiamne Figure6. Arepresentative chromatogramof mati fortificationsample (454-103-MAS-14) aNsyus WILDLIFE INTERNATIONAL, LTD a. APPENDI1X PROJECTNO.: 454-103 " 187 Intensity: 7248cps o 8 2 6 5 4 145 2 1 ob 4 123 107 220 a11% e2r85 ealm z5r06 A6s32 178617 28161s | TSiemme FigureT. Arepresentative chromatogramof ts sample (454-1032). 60057 WILDLIFE INTERNATIONAL, LTD ae DIEATPPPREENPDAIRXAITVION PROJECT NO.: 454-103 Weight and volumeofconstituuesendts0 prepare test dits: ConNcoemnivnaatilons| STubostrance | RBatoiron "3106863 003168-1388 889099099099.650 f7r2 oLamsso 85999998.53 S286 5250213203 989997.412 71 116483 s9884 Diets were prepared as follows: +5000.0 of basal ation was weighed into a tred Hobart mixing bowl. "The test substance was weighed in tred weigh bos. Appbreonxdiemra.tely 100gofbasal ron was taken from themixingbowl and placedi a Waring The"weistthtshuebistnaencaelwsoasbeaidndgepdlatocetdhenbtlheendbelreandnedrthe weigh boat ws rinsed with additions ration, Thebobweln.deTrhceonbtleenndtesrwwearse rbilnesneddedwiftohraadpdptriooxniamlatreatliyon6,0wsietchohndesrainnsdetarlasnosbfeeirnegd ptloatcheedmiinxtihneg minbgowl +The"Tbhoewrlemwaaisnipnlgacreidoonnaa Haodbdaerdt 1mixtheerbaonwdlhaendcotnhteenctosntweentrsewmeirxeedmifxoedapfporrosxiixmmatoerlymsiinxumtiensutes. +The dict was transferred (0a labelled paperfeedba. 000513 33! kEoefi| ssssms sssssseessanas g2| o2f54)f3.] 233s szzzzzossasss | H2e3le sssses sssesssensesss 3 [B za3 E$: 3 2i | sssses sssssssseansgy 2|F | aElH |H ssssss sssssssessesans 5 [fo | [2A2H 5 . gBZaE|TEFH ssssss sssssssi. ssesss [3Bog3 2g |: B | fr= 2 833333 338853383533885c%o 2[:32 ||| d+ 3f|] ss9ess sssssszsssssss 3: 3 5,8 | g|J JT S5:id | J S| 3 |g 888888 58855858885888 3E:E8R 2 3 3Hog | ~vmene ~nommnmannnn-~ [22535 2 3 3; z 532 z Z| i 723]3. f.5 oram == H[=3H;24 wil EI cccccot2=SsrnSERREEES 2, 23 2 ROSY _--mmmm _ WILDLIFE INTERNATIONAL, LTD ase PROJECT NO.: 454-103 APPENDIX VI Individu"aAlcuBteodDyiaWeriyghTtosxi(eg)tyfSurdayNoowritthhmePrFnOBSobwhite Page! TEgo--ort)-- Bed Ono Dus DCranngse pws bChuatngse cTuomm o FPo I oonao3n i5 so0w 53 0 i5 3 i Son Mon 31 5= 5 o 6w % 0 is 5 2is 0 SS s 1 o 5 5 o xCooowsokaox Fs SE ona wo5s Ii1Hs Sio2m03on Ve 30 > n Ss s3% 0s 5 afi] 7 SC ? : 3 1 3 o dvoownoan xxom o s ih w 3;3 2u5 PSooomk 3R Men 1 > HEEa on 5;$ iiss i SC : 3 s : ` o S: oonoww omoh n o[s I w3 :5` iiis Sio om Men TH 02% ii @ 0i 5 3a0 so _ 3 i i : 3 600610 --_--m WILDLIFE INTERNATIONAL, LTD 46 PROJECTNO: 454-103 APPENDIX VI Individu`aAlcuBteodDyieWtaeriygThosxi(cgi)tfyrSotmudayNwoirtthhePrFnOBSobwhite Page? _--E--T m pGmroup Bid Dayo DiS ChDaanyg0es Dws ChDaansges CThoanugle o 2' 21 72 79 336 97 i1e6 h3 22 2 1un0 a: 2- 2- Me5n 220 33 1T2o aWo i0o 25 so 1 3 2 s 2 4 o 2' 12s 330 n1s F3s9 n9 22s 35 219 u1n 0 a3 58 2is Me5n a 220 2 In e 2 10 a 2 m Tow Msepn 2 2 w 02 w 5 s1 Tw3 Tou! so \ 4 3 s 2 4 000622 --_--_--m---- WILDLIFE INTERNATIONAL, LTD PROJECT NO: 454-103 a APPENDIX VI Individu`aAlcuBteodDyiWecigThosx(i)yfSturadyNwooirthhmePrRnOBSobwhite Pages --TToTo-- ormIc, o------------ Chang-- e ----Co-- ng --T-- ow Gmel) Bid Dwo ys Dn0S ows Duds come 13 [E Tm . is Tw www . soa PE 5 w son ow EE 2 u som x ow ww MEeran W T4 HAW4 T " 3 5 183 ew sm 1 1 dw ox sw 5 In sow ow sw 5 N soon uw ow ow 0 2 som as oun ow 5 Tew Men 3 Me I T w 3 nH2 Rwm5 y T | m m2 eS Toull p 0 i a 3 s 2 s > 53 0052 _-- ee WILDLIFE INTERNATIONAL, LTD Lae PROJECT NO: 454-103 Individual Body WeiAghPtPsE(NgDIfX VrIaNoorthmern Bobwhite "AcuteDear ToxPiatgeysStudywith PFOS -- goeoln) -- Bi Dwo -- Dus ChDaunigse -- bys CDownt-- gs cTeome 366 [IE sas s wv [I u ' n sow wow . 1 ia ow PE 5 w soa ow oma s Msesn --W 20 3 2 W 3 v 1 12 306 Cow mw ow . ww : ow mw ow . N soa n u . ww ons Bw 5 2 som now s u Go Meo Mew T 35 0HTH3E i3 w HEl] o+ wm2 Ton s __ 3 3 2 3 : 2 60051 _------ WILDLIFE INTERNATIONAL, LTD Las. PROJECT NO.; 454-103 _--Tpermenial @Grpomuepi,) n2 APPENDIX VI Individu`aAlcuBtoe dDyieWtaeriyghTtossi(cgi)tfyrSotmudayNwoirtthhPeFmOBSobwhite Page's Bid Duo Dus ChDaanyg0eS 'i2 5 21 % 7 sow 5 im 2 un soa 0 5 Msaon T1 H3B | Days ChDaunSgSe CTohuml 2. u ' " 36 ' 16 ' 18 36 6 1s w 3 T 1 Em2 n2 1i n 5a 7 6 2 ow 3 n 5 ? is soa 0 9n ' 1 ion 2 " 7 i soz 2 0 " 5 Mseom 20 02 103 33 7| i"| TGoruolup sMoem 30| 02 5\ 33 7| T2c [LITE _-- WILDLIFE INTERNATIONAL, LTD 50. PROJECT NO: 454-103 APPENDIX VI Individu`aAlcuBteodDyieWtaeriyghTtosxi(c8i)tyfSturdayNoowritthhmePrFOBSobwhite Pages -_Er--permenT gGprmoup Bid Dio Days CDhuaynOgeS DayS ChaDnaygses CThoanugle 6 ' 1 = 5 3s 7 1 2 1 ES 5 + 5 sw x . - . . io n 3 s 1 so s 2 4 9 Msoe TE W 3 F 3 W 3 s3 5 146 1i u .5 7 2m 7 5 3 s 1 31 2 s . " am . . . so x i ES 4 5 Gow Mseon TH2 Mew 20 E2d 56m 3 2em5 I= Ea E T"oSualnicaly den orn he cont g2roup p< 0.053(Dunnd en's ct3), 5 2 s erSiiiecn aom lhe cyont gre<a1t0) (Dunes C06 --_---8mAe se e_- WILDLIFE INTERNATIONAL, LTD sie PROJECT NO.: 454-103 _Exp-- erimem ntal (oGprmoup, 2 APPENDIX VI Individu`aAlcuBteodDyieWteariyghTtosri(cgi)tfy rStoumadyNwoirtthhPeFrOBSobwhite Page? Bid Divo Days ChDaanygoes. Days ChDaanygses ChTaonugle ' is 2 22 ' 3 21 " 2 - - 3 2 in 2 - : `2 : . . . . s2 2 2 : : . EMYe)n w 2 TH2T T : - : 3 '1 1 0 1s " a 21 . - . 52 2 o - . 4 0 un 3 . . s 2 1 El 2 SMDe.n TB H| W2m a 22 - w : J Toul sD : 2 2 IS4 e C 4 a5 ST mellr y diferent rom he con gro st < 0.05 Dunner' Hc)-- . *Saicaly ifr fom heconve roopap 00) Dummer' es G05 G _-- WILDLIFE INTERNATIONAL, LTD s2- PROJECTNO.: 454-103 APPENDIVXI Individu`aAlcuBteodDyieWteariyghTtosxi(cgtyfStruadNyoowritthhmeFrFOBSobwhite Pages --T Eo--E o y ------------C-- hange ------Con-- g --T-- om gout) Bn wo Duws Dubs pus buts chee st6 ow - . . 2 wow a . . . soa ow a . . . sm . . . sow ow s . i . Mao THES 536 Coe es . . 2. . . som wa . . . ow - - . . - soa .i Men T 0 IH o R : E R A : T : T-- oEuEo!mc estoEyA cone gop 1.00:1 Dspe's izs) : : : Year _--mm--------------_--_-- WILDLIFE INTERNATIONAL, LTD PROJECTNO.: 454-103 5. APPENDIX VI Individu`aAlcuBtoedDyicWteriygThotsigci)tfy rStoumadyNwoirtthhPeFnOBSobwhite Page -_--_-- gGprmoaiw), Bid Do DS CDhyanOgSe Dus DChiasngse cThoualm un ow . .. : ow . . . sw - . - N om - - - . . som - - - . . Mseow TH : i : : : un I Toul EE row - . . . : om - - . . iow - : - . in - - - . som . - - - osow T : e i : 3-- : : -- a : : 60058 _--mm WILDLIFE INTERNATIONAL, LTD sae PROJECT NO.: 454-103 APPENDIX VI Individu`aAlcuBteodDyieWteairgyhtTsoxi(cgi)tfyrSotmudayNwoirtthhPeFrOBSobwhite: Page 10 -- Erperimeril Ggprmoauip) Bid Days Duis CDhaanBgIes Dayz DChuaniges cTaoumte o 2' 5 73 s62 i2s 22 o" 2 21 5 3 50 35 0 @7 E 2 E"5 2261 "5 M)en "359 @i Ens) E6l 73 =5 0 21 33 55s 2is 77 21 3 h3 7 o z3 05s 2 0 Me5an 3% a5 EB2) 505 23 so 3 5 o 2 4 ai 5 o 2| 50 rsds i2s @ i2s 93 3i 5 2n 555 @ 2in Fy n7s 5s 25 7 1a 55 SMeoan "35 55 75 71 Eo)4 "s TGoou!w Mreayn 37` %10 Ei5 IH@ 73 Eo5 RELIN:) _--_--m WILDLIFE INTERNATIONAL, LTD oss PROJECT NO.: 454-103 APPENDIX VI Individu`aAlcuBteodDyieWteariyghTtosxi(cg)tyfrSotmudayNwoirtthhePrFnOBSobwhite Page 11 BE Group, pm ai 183 Big -- Change -- Change Toul Diy$ Duis Dubs Dey22 Duis: cham '3 23 3 35 4i s MseDn TW5 5 1 2% 2 " os 2 8s 2 " 2 56 2% 5 z 9s 2 wo n 2 22 " @x m 3 w 2 m 3 w 3 Experimental pGprmouaip. 366 Bid Day$ DuylS CDhaayn$g1eS Day2? DCahyainsg2e2 CThoaunlge ' 35 6 2 81 2 a 2 3 6 2 9 2% 52 3 3 6 2 9 2% 4 4 38 6 2 2 as 5 a n 2 97 2 st M0e)n T3 3 85632 w7 w 2 W 4 600550 _--m WILDLIFE INTERNATIONAL, LD -s6- PROJECT NO.: 454-103 APPENDIX VI Individu`aAlcuBteodDyieWtaeriyghTtosxi(cgi)tyfSturadyNoowritthhmePrnFOBSobwhite Page 12 -E_e--permenal pGrpomup Bid Ds Owls CDhaaynbgles Dey DCwhainsge3 CThoanugle 2 ' 2 se 2 7% 2 " 2 3 58 2 " 9 " 3 3 2 " EY 4 3 6 2 2 5 36 El % 1 --_--n Msaon T 5 5 w 4 4 2 m 2 4 m : 2 m 2 w a2 eee % pGrmouap, Bid Dove Dayls CDhaaynRgles Diy22 CDheaingse: CTohuel 146 ' 3s El 2 st 2 " 2 2 ss x 7" 2 4s 3 . . : .. . 4 38 2 5 is " s 2 El 2 2 6 MsaDn TE 3 w 3 m | m 2 m 2 m ' TOSiscaw llydife erent fGo ethoonmlgroupsp 005(Dunn's ics) GROG _--mmm WILDLIFE INTERNATIONAL, LTD 51. PROJECTNO.: 454-103 TERGrEoup Goma 2 _--O_r--an APPENDIX VI Individu`aAlcuBteodDyieWtaeriyghTtosic(i)tyfSturadyNoowritthhmePrFnOBSobwhite Page 13 Bid Deyo 1 21 2 - 3 4 - s - M>an d om Days ChDaOngSe Dek CDhaaynsges CThoanugle 5s " ss 4 - - - . : . - . - - - 2 . - - . . s TEE Ww: mw R0GET WILDLIFE INTERNATIONAL, LTD -s- PROJECT NO. 454-103 APPENDIX VII Food ConsumptionD(ibcitradydTaoyxi)cbiytyPSenudfyrowmitahNPoFrOthSer Babwhitc Acute Page | TE Bpewereed C Ponbon Feed Gpmai) Pn Dm0s Tb T wes Con | 5 2 2 7 2 3 5 i 2 2 s 5 o 10 MEan)n T 2 m 2 CO0553 WILDLIFE INTERNATIONAL, LTD PROJENCOT 454-103 so. APPENDIX VII Food ConsumptionD(igcbtirryddTsoyx)icbiytyPSntudfyomwitahNPorFtOhSen Bobwhite Acute J Tre Cro Ta-pas Fered To-- ra [r--_ ro TBReRTTTbT wes 1s Me2:n 55: 0iTw 366 2: 53 w5w Men WT na : 0 2 Me>:n l io w is ws 2: 07 is Men 5 io m 2: `` hsy vem 5 5 sts : . 1 Ve:n 000 s 0000 > 0 00 wn 2: 4i Men 5 : ET 600551 WILDLIFE INTERNATIONAL, LTD 60 PROJECTNO.: 454-103 APPENDIX VII Feed ConsumptionD(igebtiarrdyidTaoxyi)cbiytyPSetnudfyrowmitahNPorFtOhSen Bobwhite Acute Page3 ET EEE (pmai) Pa TT BnE To T wnm Control ' 0 on 2 0 2 3 7 2 MseDn T 2 TS hm: BL (oma) be TD T wE mum 153 ' 0 2 366 ' 1 1s n2 ' 1 1s 16 ' n 1 2 ! 5 GOOLSS _-- DWIILLDDLLIIFFEEIINNTTEERRNNAATTIIOONNAALL,,tLToD "61 PRPROOJJEECCTTNNOO.:4435441-10033 APPENDIX VII CHANGES TO PROTOCOL `1.waTs chlarpirfoiteodcotlo iwnadsicaamtenthdeecdoltleictnidoincotftlhvatrbiflreowmiblirbdesctohaltledciteedd druorminatlhstucdoyubrisrdosf.thTehestpurdoytocol t2u.bBesl.ooTdhseamprpolteoscowleriendoiclaltcetdetdhatonheDpaaryin8iaznedd Dvaacyut2a2nienrsonw-ohuelpdarbienuiszeedd: ml borosilicate glass test PTP3.efMTlehc8et,hp1reo0tt0oec,sotls1uw8ba3ss,a3mn6ce6en,dp7eu3rd2i,ytog14ic6vh,eann2g9ie3n,tthh5ee6nteeatnwdccon1ec1e7rn1tcrpaaptomiofnaas.nfarlToyesmsti0s,co2n0c,e4n0i,at80i,on1s60w,e6r40chaanndge1d26f0o n4.otTrheecotredmepderatures from several booder units on Day 4,7, 8, and ll brooderunits on Day 22 were r.eTahmeeanfttgermoouopnoonbsAeprrvialt2i6o,n1s9w9e9r,e inadvertently no recorded or2 birds inthe 640 ppm i. 600656 _--_--------_ WILDLIFE INTERNATIONAL, LTO 62. APPENDIX IX PERSONNEL INVOLVED IN THE STUDY PROJECT NO: 454-103 The following key Wildlife Intemational Ltd. personnel were involved inthe conducotr `managementofthis study: (1) Mark Jaber, Wildlife Toxicologist (2) Joann B. Beavers, Director, Avian Toxicology (3)ScanP. Gallagher, SeniorBiologist (4) Courtney Casey, M.S., Senior Biologist (5) Willard B. Nixon, Ph.D., Manager, Analytical Chemistry (6) TimothyZ. Kendall, Supervisor, Analytical Chemistry (7) RaymonLd. Van Hoven, Ph.D., Scientist (8) Ellen Mank, Chemist GOST --_--m--m WILDLIFE INTERNATIONAL, LTD 6 APPENDIX X REVISED STUDY SUMMARY PROJECT NO: 454-103 This study was reported using a test substance purity of 90.49%. The test substance characterization was revised on 9/7/00, following study completion, and the purity was determined to `be 86.9%. The Sponsor requested that the results of thisstudybe revised to reflect the most recent purity analyses. The resuls previously presented were simply corrected by the ratio of the two, purities ora factorof0.9603. No attemptwas madeto recalculate analytical standard concentrations. or measured concentrations from original raw data. As such, minor errors du to rounding may be present. Atached i the revised summary page from the final report and the most recent certificate of analysis. AMENDED 600853 --_--_---s8w oww---- WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-103 6h REVISED STUDY SUMMARY BASED ON A PURITY OF 86.9% ronson SPONSOR'S REFPRESENTATIVE: LDOACTAATAINODNAOFCSTOUDOYPF, TRYHAEW FINAL REPORT: SM Corporation Ms. Susan A. Beach WEialsdtloinf,eMIanrtyelrnaantdion2a1l60Li1d. "LWTIDL.DPLIRFOEJEICNTTENRUNMABTEIRO:NAL TEST SUBSTANCE: stop. NCOONMCIENNTARLATTEISONTS: TESTDATES: LENGTH OF TEST: asa103 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A Dietary LCS Study with the Northern Bobwhite 0,17.6,35.1,703, 141,281,562and 1125 ppmai. EBixopleorgiimceanltTaelrSmintat-iaAopnrti-lM2a2y, 1149,991999 Experimental Termina-tMiaoyn 14, 1999 24 TEST ORGANISM SOURCEOFTEST ORGANISMS: AGE OFTEST ORGANISMS: "Northem Bobwhite (Coli virgimianss) WEaisltdolni,feMIanrtyelmaantdion2a1l6L0i1d. Production Flock 10 days at est iniation To 95% CONFIDENCE LIMITS: `NCOOMNOCRETNATLRIATTYI.ON CNOON-COBESNETRRVAETDI-OENFFECT- Tappan 158 and 278 ppm aii. 70.3 ppm ai 703 ppm ai AMENDED 6C0859 reer WILDLIFE INTERNATIONAL, 10 B "65. PROJECTNO. 454-103 ro Senrtare Arabica)LaC borata oriesr ,Inc. INTERIM Conte Ani rCLaEearRobTPioGI)OhAFROeIFsACNeAALYT3S0IE1SA Fim ti Ef --em r --nw ---- =n ------] --]| rar om iE LE e orpimndi ra see | 5Sa 5 ay FEE AMENDED GOOGS0 WILDLIFE INTERNATIONAL, LTD i" PROJECT NO.: 454-103 [IN= CTEp RiIMCERTIFICATE OFALo vaAboLrYaStoSries, Inc. ectCs oon mEes rte sha -- R-- E e TTARh m S r EF Ts-- S b SrEmeRe REEEeEt C TBnn pESeE. L eteenR tc ee--e8--00 -- _-- noe CONG ttee tra --------e -------------- WILDLIFE INTERNATIONAL, LTD _ PROJECT NO.: 454-103 ON Zsa INTEPRrIeM:CvdE21R2TIOIFCAaVAA61LT0)3S3115 2v0)81100 ena ComnaeeAn Lebar Con Rees 0350180 Tam i T ErEe eT rity L E og s e able rCoQLhefril]r cm lly rare COOLET _m-- m m WILDLTIF=ETIENRTNERANTAITIOONNAALL,,oLToD -68- PRPROOJJEECCTTNNOO.:445541-01033 APPENDIX XI Report Amendment 1. AOrmiegnindaeldReRpeoprotrt Reason: 2 AOrmiegnindaeldReRpeoprot:rt Reason: 3 AOrmiegnindaeldReRpeoprotr:t Reason: a AOrmiegnindaeldReRpeoprotr:t Reason: s OArmieginndaeldReRpepoorrtt: Reason: 6 Original Report: Amended Report Reason 2 Original Report: RAemaesnond:ed Report: `TTihleeaPmaegneded report date was added. The total numberofpages T`woasicnhdaicnagteedtfhraotmt6h2e.1r0ep6o9r.t was amended and note change in pagination. "PTahgeea2mended report date was added and the exceptions to the `Tcoomsphloiawntcheesaamteemnednetdwreeproertredvaitsee,d.provide new signaturesand dsattaetsemfeonrtthe amended report and 0 updatethe compliance P`Tahgeea3udit dates for the amendedreportwere added. "sTiogsnhatouwrethaenad damteefonrtehpedoarmteaeundddietddartepeosrta.ndtoprovaindeew `PNaegwessignatures anddateswereaffainxdaettdle change was mTaodper.ovide signatures and datesofspproval for the amended report and to update a manager's tle. Pages AApmpeennddmiexntX,, RweevriesacdddSetdutdoytShuemTmaabrlyeo,faCnodntAepnptesn.dix XI, Report aTnodrreevpiosrettahemeTnadblmeeonf.Contes to include the revised summary Page? ASfuomomtanroytpeagaen.dthe amendedreportdatewereadded to the `TsotuddyirseuctmhmaerryeaadnedrttoosAhpopwentdhiexaXm,enwdheidchrecpoonrttaidnatse.the revised Page A"TofdoiortencottehwearseaaddedretdotAoptpheenTdeisxtXS,ubwshtiacnhcecotnetxa.ins the most recent Certificate ofAnalysis. AMENDED. GeOGao YLWOILLDILIFFEEIINNTTEERRNNAATTIIOONNAALL,,uLTDD "o- APPENDIX XI Report Amendment (continued) PPRROOIJEECCTTNNOO.::4455641-10033 8 ReAsmseonnd:edRepom: 5. ReAsmseonndscdRepor: TPoapgersov6i34d6e7a,dRdietviiosneadl SinufdorymaStuiomnm.ary,were added tothe report. TPaogpersov6i8daenddet6a9i,lsReopfcohratnAgmeesnmdamdeen1,0twhereoraidgidneadl troeptohrte.report AMENDMENT SIGNATURES. JuJ b StudyDirctor ERCIY Due Wisltdeern.ematioWalo, Loid.nM)aragerent D2ac3 Mpc 2004 REVIEWED BY: QuaySAussusraancen &. Colman Dae 3-22-04 AMENDED GOoan