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I ros: NORTHERN 'AREPRODUCTION BSOTBUWDHYIWTIETH THE FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-108 3M LAB REQUEST NO. U2723 FRA Guideline 14 (OECD Guideline 206 AUTHORS: SoRaannm.o.nCaaBliegVoavesrHoven Nk er STUDY INITIATION DATE: aay22,200 STUDY COMPLETION DATE: August20, 2003 subido ESnevip53m3am5MmBMCeounirahptomArLavastebioonrn5es5t1o0r6y Wildlife International, Ltd. 3598 Commerc Drive `Easton, T2860 Maryland 21601 USA Page 106196 COO _-- Wildlife International, Ltd. Project Number 454-108 2 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TLE PROS: AReprStoudyvdithuthecNortberiaBoobwiinte WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-108 ! STUDY COMPLETION: August 20,2003 by the U"STh.esEtnuvdiyrwoanmsecnoanlduPrcotteedcitinocnoAmgpelnicayn,ce4w0iCtFhGRoPoadtLa1b6o0r,at1o7rAyuPgruascttic19e8S9t;anOdEarCdDsaPsrpiunbcliipshloeedfs G58o5o0d, ALgarbiocrualttourraylPPrraocdtuicctei(onEBNuVr/eaMuC,/1C0HAEuMgus(t981)98147),;wiatnhdthJeapfaonllMowAiFnFge,xc5ep9tiNoonhsS:an Notification No. anTedp3orCCtoiomlsrppbloeresattueibsominigtneereddasnnetpdiadanaetlleya.diredporttsheforerpbolrotodaSnumdmtaisrsyuetabnlalsysreesicnocnlduudcetded5bayppEexnydgiecensRe0sdearflclh conductTedheusntduedryownaescporontdouecdtled(uWnidefremulItntiepmlaetpiroontaolc,olLsi.d.Thsteuidny-infuemabnedr 34M54a-n1a0l8y)t,icaalnpdortthieonEsxwyegreen. Resstcudayrncuhmabnealryt0ica2l p3ort0-idon00s23w6.e0r7e60c)onductedundertwoadditionalseparateprotocols (ExygenResearch AsslysesofeggcontentsconductedunderExygenResearchsty mumber 023-070werenot report scparacly. conductedinaccordancewithGoodLaboratoryPracticeStandards,andresultsoftheseanalysesare incompliTahnecaenawliylthicEaPlpAhTasSeCpAerGfoLrPme(4d0bCyFERxyPgaetnR7e52s)earchunderstudy number023-066wasperformed STUDYDIRECTOR , SSeenainorP.BiGoallolgaigsth,erAvian Toxicology DATE SPONSOR'S REPRESENTATIVE: Top Nowstod4. BA3TE/5 [04 COD _- Wildlife International, Ltd. Project Number 454-108 "a QUALITY ASSURANCE STATEMENT Gbyootdhe LUahSbi.orsaEtsnovdriyyroPnrwmaacestnitecaxela(mPiErnoNeeVdctMfioCornIcCAogmHepEnlMciyan,(c94e80)wiC1t7Fh;RGaopnaodrdtJLa1ap6ba,onra1Mt7AoAFruyFg,Pursa$tc9ti1Nc9oe8h9S;StaaOnn,EdaCNrdDosiPisrsaitnpciuibopllniessNhoeo.df 3850, Agricul Production Butea, 10 August 1984. The dtes ofal audits an inspections nd the dates any findings were reported to the Study Director and Laboratory Management were as follows: AcTvITy DATECONDUCTED STUDYDDAITREERCETPOORRTMEADNTOA:GEMENT PrTeepsanSiubisotnance MatixFoicuion Feed Consumption CoBBnroivdiiyooWnnemisegnhasl, Cunting Tie Collection January2,200 Janay30,2000 Febuary 6,200 Febraay6, 2001 Jone 12,2000 ane 19,2000 oy2,200 Jy 3L,2000 Fema 7,000 Febrary7, 2000 Jae 12,2000 June 20,2000 Jamary 23,2000 Jea31r,20y00 Febru8,a2r00y0 February, 2001 me 13,2001 October, 2000 1B4oDdeyyWe0idghs ENgegsmSmheelmeThtieckaess Joe 25,200 Jone 28, 2000 Je 25,2000 ne 28, 2000 Jy, 2001 sup2.2001 WHaeticghlhisng Body Jay2,200 DusBouyChesk~~ August 10413, 2001 ADvraaiyRiecapoDat, ONcotvoebnebre2r58-N,o3v0e1mber 2d RReapwonDus, Dra Fins Reon NoDveecmebemrb2e7r:350,s20d0 ug 20,200 Jay2,200 Mops 13,2000 November52001 Decenbers, 2001 Aupn202000 ly,2000 Aug 14,2000 November, 200 February 5, 2002 Augen 20,2008 c Qualityio .AsCsoulreat mnacenReGpreseenatie ve n DATE Cho -_ Wildlife International, Ltd. we REPORT APPROVAL Project Number 454-108 SPONSOR: 3M Corporation TILE: PFOS: AReprStoudywdithuthecNorttheir Boobwhnite WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-108 3M LAB REQUEST NO. U2723 STUDY DIRECTOR: / SanP.rGaellaghe,r PAIR Senior Biolog, Avian Toricology ols3 BATE CHEMISTRYPRINCIPAL INVESTIGATOR: [IL1f Scleaist, ALn.alVyatnicHalovCehne,miPsht.rDy. slals DATE MANAGEMENT: JDoiraencntoBr.,aAvSian Toxicology WillarHd BiNolxonl, Pfo.p DirecorofChemisty DATE fh Bal nen Wildlife International, Ltd. 5 `TOAFCBONTLENETS Project Number 454-108 a QUAliLYASSUFANCESUEMICNE o.oo sss settee sess d AAdGuU BBoI0d0y8WCeOilgehCtHsOnDd. Food CrOerncSunpsioen.s.soenvserenroemonenr 1117 AdultNec0rdTiosspCOGseioyn. servers 18 CanadndlInciubnatigon. Te OffBlosodapndTrissuieCOnUGCg HON orverrerererreserenrncnrmemsererrenerr 20 `EggShell Thickness Measurements and Egg Componcats COICtion.........rnsn20. Diet Analytical RESUMS .....vvvovrvomsssisssnss cones oso assesses 2d AP IFedCOMSUTI rcV s 2s noe - W Wildilifd e Initerj natieonal l, ILn tdd.,rg __r_nT aProt ject Nui mbe ero 454-1n 08 a, "6 TAOPFB ACGOEN2LTENETS a A-- OffSPrng ODSCTVALONS.....cvcssssssssssssssssmmsnsnns IR-- TAB ANDL FIE GURS ES Table 1. Mreoamn MNeoarstuhreerdCBoonbcewnhtirtaetiRoEnsP(TppOmGU.iE.S)HoOYf P D r F inAvO ianr DS iet r 30 Table 2. SRepruoducomtifoGnrmSomssdaPyawthirotlhogPyiFcOaSl,OAdbulst BeirrdsvfFroaoumntdDiNeoaortdnhEeurstnhaBnoibzwheidte: Table 3.Mean Adult Body Weiggh) rom Northern Bobwhite Figur 1. MBoetaonwhAitdeuReMparoldeuBcotdioynWS eigWhO itt)hfPrFoY mOSaNorrheerra Figur 2. Mean Adsl Feral Body Weigh (5) fom a Norihern BobwhiteReprodStuudcy Wtitih on PFOS........covmrrvmrres 33 sesaminSA Table 4.M`eBoabnwhFieteedRCeoprnosdSt(uguubdicyrmwdtidtaihpyP)oFftOrnSom.iaNoortrhnernr 3S Figure 3. Mean FeedConsumptiongird) rom a Northern `BobwhiteReprodStuudcyWtitih on PFOS.........ccwwwssmsmsmmmmnssnss 36 Table 5. SumofGmrossaPathrologyical ObservfraomtaiNoortnhersn Bobwhite Reproduction Study with PFOS, Adult Birds Euthanized Table 6. Mean Liver Weights (9) fiaNoorthmer Bobwhite Reproduction Table 7. SBobubimeoRfmeRperpoardoudcrutcitoyinveSRPeWirthfPoRfrrOoSmm.#aNonrrtcheer rn r 39 enon Wildlife International, Ltd. TABLE OPFAGCEO3NTENTS Project Number 454-108 Figure 4. MBebawnhRieptrRoduectipverPerofSodrlmauyncWechftPFiraOSNooornthmeervnr i) Table 8. Summaryof ReproductivePerformNoarmanliczeed ,3 Percentages(%),from aNorthernBobwhiteReproductionStudywith PFOS.........41 Figor 5. MreoamnaRNeoprrotdhuecrtBiovebwPheriftoerRmeapnrcoed,uNcotrimoanliSzueddysWiPtehrPcEeSn.ta(g1e4s), Table 5. MReeapnroEGgugsiSohnel1l Thi0ckwniedhsPsFM)Oeasuremerntse(ic)foomraNortherenmBobewhcitse rc Table 10.MeanBody Weight()ofHatchlinagnds 14O-dDSuravivyors fr aNoo rthm ern Bobwhite Reproduction Study With PFOS ...c..c..vcumnssssssssmasisnn84 APPENDICES Appendix L Certificate OfARBIYSIS........covwvssmorisssssmosssssmsssssmsssssssssssssssnsssidS. Appendix IL Dictand Supplement FOMMUIBHONS .....c.ovvvsvrssssssssssssmsssssnsn 8. Appendin TL Diet PIAIION corr SD Appendix TV. The Analysis of PFOSinAVADieter 51 Appendix V. DiaofTgestrLogoaut m rr1 Appendix VI. Reproductive ParamanedRtepelircaste Identification ..........wim.r68 Appendix VIL ARdueltpBordyoWedSitgeuhdtycw(ght)lf iFFor OaSnNoo rthm cemrBeobwehirte TS Appendix VII. FBeoebdwhCiotnesRumpetipon (rg/boiSrtdd/uddauyyW)icftrhotPmRaiONoSro.thnern 81 Appendix IX. NIortnhedrniBGorvoowshiiPtdaetRheuoplroagoidcluacliOobnseSrvaatyiWointshfProRmOaS... 85 Append X. Adu LiverWeigh (5) roma Northern Bobwhite Reproduction StudyWith PFOS .cc.ocumssesssmssssssssssmisss -- Appendix XL Histopathology Rocce 89 Goo DE _ Wildlife International, Ltd. Project Number 454-108 5 `TABLE OF CONTENTS PAGE 4 Appendix XIA. Appendin XIIB. Appendix XIIL Appendix XIV. The Analysis ofPFOS in Adult Red Blood Cell and Serd cv 21 TheAnalysisofPFOS in Adit sad Juri Liverand Se... 134 ReproductivePerformancebyPenfrom a Norther Bobwhite Reproduction Study with PFOS.. sonsrsmsmrseses esses JB RNeoprrtohdeurcntBiovbewPheirtfeorRmeapnrcoedbuyeiWoeneSkuadnydwPietnhfPrEoOmSa vv 170 Appendix XV. Appendix XVL `Appendix XVIL. Appendix XVIIL rEgogmSaheNlolrTthhiecmknBeosbswMheiatseurReepmreondtusct(imomn)SpteurdyPewnibthyPWeFeOkS......187 Meso Hatchling Body Weight ( per Pen by Week fr aNoo rthmer Bobwhite Reproduction Study with PFOS......c........189 WMeeakn1f4r-oDmaayONolrdthSeurrnviBvoobrwBhoitdeyRWeepirgohdtu(cgt)iopnerSPuednybWyi.th PROS... 191 Offspring Liver Weights (g) from a Norther Bobwhite Appendix XIX. CHANGE 10 Sy PIOOROL rrr 198 Appendix XX. Personnel ION it SUY..ccoeccorermsmmomsmsnson 196 Gon: - Wildlife International, Ltd. -5SUMMARY Project Number 454-108 STUDY: PFOS: AReproduction Study with the Northern Bobwhite SPONSOR: 3M Corporation WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER 454-108 TEST DATES: SExtpuedryiImneinitsailSona-rJanuJamar22y,2230,021001 FFihrosttoEsgigmsulSaio-nAt -p.riMlar1c2h,210,012001 Analyte Termination pl 15,2002 BEAdxiupoleltroiTgmeireemnitTnaeaTtrimeoinrn-maJitunnieaoJ1utn-9lA.y-i2po02r,3ni,220100501,12002 TEST ANIMALS: Norther bobwhite (Cols virginia) AGE TEST ANIMALS: 24 weeksofages the ntationofthe test SOURCE TEST ANIMALS: M1o8rr3i7s0Q5us.il2F3a2rSmr,eotc. Goulds, Florida 33170-5399 USA. NOMINAL TEST CONCENTRATIONS: 0,10, 50, and 150 ppm a. RESULTS: Tehcerse wueproentrbeoadtmyenwtesilgahttedanmdorfeteadlsco,nsouvemrpttisoingnsatofthteox5s0tyanadnd15r0capmpemntar1e.latteesdt {Cioencreenptrroadtuocrtsi,veTphhea5s0oafndthe15e0s.ppmThae1.reewsterceonTcoentarpaptairoennst nweesretmteenrmtirnelaatteedd preifofrecttso uwperoen obvoedryt wseigingshtofortofxiecditycoonbsseurmvpeidoninaadtuhles 1a0t pthpem1a0.pptemst.c.ontceesntrctoinocnen.traTthieorne. Novaedrailolnlrelpyr,duecirievewpeerreforremdaunctcieonastihn (1c0epspsmiz4e..antdestecsomnecennttrractliaont.edBeafsfeecdtsonuptohne CCohneccenstraatpisoenrvfeord noarthtehren bo10b.whpiptemex5p1oseedxtocPoFncOeSntrianttiohn,diehte.wasnodoebteiremrivneeddftofebte Tks thn 10 ppma. the lowest concentration estedduring is study: neon Wildlife International, Ltd. Project Number454-108 -10- INTRODUCTION - `This study was conducted by Wildlife International, Ltd. for 3M Corporatiaotn the Wildlife Inter- ncaotnidouncalt,edLtfd.oamviJaannutaorxyic2o3l,og2y00fa1ciulintiy iJnuElays2t3o,n,20M0a1rylRaanvde2d1i6t0a1e.neTrhsetbeidolsotgWicailfpoertiIonntoefmatthieontaels,t wLda.s aInnteadmatcioopnyaolf,tLhied fsiena.l rBeipoolrotgaircealfislpeedcuinmdeenrsParoejescotrNedumsbt e3rM4C5o4r-p1o0r8atiinona,rcSht.ivPeasul,ocMaitnendeosnottahe$5W1i3l3dl.ife omECTIVES The abjective of tis study was to value the effects upon the adult northern bobwhite (Canis Virginians)ofGeary exposure oth ptassiom siltofPerfluooctan Sulfnic Acid (herafe reid 1085 PEOS) over a period of approximately 21 weeks Effects on adult healt, body weigh, snd fd consumption were evaluated. In addition, the ffs of adult exposure to PFOS on the numberofeg63 Jad, frilty, embryo viability, hichaily, offpring survival, and egg shell thickness were valued. Histopathologial examination ofselected tissues and analysesofblood and tssu samples were also used 10 evaluate he efets upon adults exposed 0 POS and to thei offspring. EXPERIMENTAL DESIGN Norther bobwhit (80 males and 80 females) were randomly distributed into one contol group and thre treatment groups. The et concentrations were sleted in consultation withthe Sponsor, based upon the result ofa pit reproduction study (Wie Intemational, Lid. Project Numbes 454-109) and additional trict information provided by the Sponsor PROS Treatment Groups Group Nomimal Comeentraion -- Pew per Giowp bw Pers Hematology _ MaBliesspaFrebmealnes 2 10 (Control) 0 3 0 s 150 116 44 i1 11 1 4 1, 16 4 :: nod Wildlife International, Ltd. Project Number454-108 ue Each treatment and contol group contained 16 paisofbirds with oe male and one female pe pen. Four additonal pars of binds were added to cach test level (fr a total of 20 pais per level) and `maintained for collection of blood samples during the test. Three treatment groups were fed diets rcocnattamiennitnggreoiutphesr, b1u0t, w5i0,thoorut1t5h0e apdpdmitiao.in.ofofthPeFOeSs.tsuTbhseanccoen.trol group was fed diet comparable to the Because overt signs of toxicity were noted at the 150 ppm .i. test concentration, the dietary concentration forthe 150 ppm .. testment evel wasreduced0.20ppm ai. ofPEOS at the beginning of Week 3 ofthe tet. The 20 ppm. level was terminated aftr Week 4ofthe test duetothe limited recoveryofbird a tis test concentration. For reportingpupose,th 150220ppm a.testconcentration will be refe1ra ethd150ppma. testconcentration, Adultsinthe 50ppm ai. testconcentrations were euthanized during Weck 7ofth tes du fotreatment elated mortality and overt signs ofoxic. Therefore,tthestwascompleted with the control group nd the 10ppm i.tetconcentration ly. All adult birds were observed daily throughout the test for signsof toxicity or abnormal behavior. Adult body weighs were measured at test nition, on Weeks 2,4, 6, , anda adult termination, and fend consumption was measured weekly thoughout the tet, At the beginning of Week 5, the photoperiod was increased 1 induce gg production. Following he satofegg producion, eggs were st weekly for incubation. Weekly, cggs were selected by indiscriminate draw for cgg shell thickness. measurement and all remaining eggs were candied prior to incubation 1 detect egg shell racks or abnormal eggs. Eggs wer also candied twice during incubation to detect infrle eggso cmbryo morality. On Day 21 of incubation, the eggs were placed in a htcher and allowed to hatch. Once hatching was completed, hatchlings wer removed fiom the hatcher and th goup body weigh of the hatchlings by pen was determined. At 14 days of age, the average body weight by parental penofall surviving offspring was determined. Upon completion of th test, statistical analyses were performed to determine statistically significant differences between groups. MATERIALS AND METHODS The study was conducted according to the. procedures oulined in the protocol, "PFOS: A Reproduction Study with the Northern Bobwhite" The protocol was based on procedures ound inthe Environmental Protection Agency's Regisration Guidelines Peicide Assssment Guideline, FIFRA GRO'05 ------,,,,,-------------------------------- Wildlife International, Ltd. Project Number454-108 "2 Subdivision E, Hazard Evaluation: Wille and Aquatic Organisms, Subsction 71; OECD Guideline 206; and the ASTM "Standard Practice for Conducting Reproductive Stes with Avian Species" 12,3) Test Substance "The test substance, PROS, was received fiom 3M Corporation on October 29, 1998 and was assigned Wilde International Lid. identification number 4675 upon receipt. The fest substance was a whit powder and was identified as: FC-95; Lot 217. The tet material hada reported puiy of 86.9% and expiration de of August 31, 2006 (Appendix 1). The tsk substance was held under ambicot conditions in locked storage at the Wildlife Intemational, Ltd. facilities in Easton, Maryland. Concentrations of the test substance in the diet were adjusted to 100% active ingredient. Therefore, dietary `concentrations are expressed as parts per million active ingredient (ppm a...) in the diet. Test Organisms One hundred and seventy-seven (90 males and 87 female) penreared northem bobuwhite were purchased from Moris Qusit Frm, Inc, 18370 S.W. 232 Ser, Goulds, Florida 33170-5399, USA. AC the tartofselimatin, the bobwhite were apparently bealy and phenotypcaly inisinguishable from wild type. The bids were from th same atch, approaching ther fs breeding season an had not been used in any previous esting. At the star of acclimation, a random number generating function in 8 spreadsheet program was used to randomize pen assignment or cach bird, Immediately prior to test initiation, all potential study birds were examined for physical nies and general besth. Birds that did aot appear aly, thr due to injury or inability 10 acclimate to laboratory condiions, or were outside the weight rangfoe the test,were excludedfrom the study. All bids were 24 weeks of age at est nia tio (fst day of exposure ots it) an ranged in weight fom 177 to250gram a fest nition. Sex ofthe birds was determibnyed visualexaminationofthe plumage. Ldentitcation Adult binds were identified by individual og bands, cach pon was identified with unique umber, and groups of pen were identified by project mumber and concentration. Except eggs lid by birds maintained fo blood collection, al eggs ad during the study were marked with the pen number sinag `permanent ink marking pen for identification. Hatchlings were identified by leg bands so tha they could betraced this parental pn of rin cn oan Wildlife International, Ltd. i. Project Number 454-108 Avian Feed and Water AT adult birds and this offspring were given feed and water ad Ibi uring aclimation and {esting The basa diet fed o both adults and offspring was formulated to Wildlife International, Ld. specificatiobnys Agway Inc. (Appendix II, Table 1). The basal ration containaetd least 27% protein and 2.5%fat,andnomore than 5% fiber. The basal diet contained approximately 1.1% calcium, derived from feedstuffs and the 0.9% Timestone used nth formulation ofthebasal dit by Agway. While this evelofcalcium is suficent for row and maintenance rations, addional calcium is required in th ration of breeding birds for gg shell formation. Therefore, an additional 5% (w/w)oflimestone (approximately 38.5% Ca) was added to tahpeprboasiamlatdileyt 3f%o,r ithegahdultasb.oveTthihs mriairsiedamthereccaolmcmiuemndleedveflo inquatihle (d2i.e3t%)foarntdhemalblraereddi(n2g75bi%r)ds4)t.o Offprng received basal diet without est substance and without th addition of $% supplemental mest. Water was supplied by the town of Easton public water supply. All offspring reccived a water- soluble vitanadelmectirolynte mix in thei water (Appendix I, Table 2). Neither the adults nor offpring receivedanyformofmedication nthe fed uring he tes. Feedsnd wate were analyzed periodically in accordance with Wide Inermatons, Lu. Standard Operating Procedures. Diet Preparation Test dit were prepared by mixing PFOS ino a premix hat was usdfo weekly preparation of he ial diet, Control det aenach treated ie were prepared weekly beginningonJanuary 23, 2001 and presented to the birds on Tuesday of each week. Dietary concentrations were adjusted for purity of the {est substance an are presented ss parts per million active ingredient (ppm 3.4). Details ofthe weskly preparationoftestand controldietsare shown in Appendix II Cnn Wildlife International, Ltd. re Project Number454-108 Diet Sampling `Homogeneityofthe test substance in the diet was evaluated by collecting six samples from each of the treated diets andonesamplefrom the control diet on Da0y of Week 1. Samples were collected from the top, middie and bottom of the lef and right sections of the mixing vessel. Control and treatment `group diet samples were also collected from the feed troughs on Day 7of Week 1 to assess stabilityofthe {est substance under actual test conditions. Additionally, a sample was collected from the control and reatment group dics, when available, uring Weeks 2, 3,4, 12, 16.and 20of he fst 0 measurciveriy test concentrations. The dict samples were stored frozen or transfered immediately to the Wide International, Ltd. analytical chemistry facility for analysis. Analytical Method Wild`eTheInmteetmhaotdiounsaeldLftdo.r tahnedaneanlytseisdo"fAnPaFlyOtSicianlaMvieatnhodidetVewraisfibcaastieodnufoponthmeetDheotedromliongaytidoenveoflPopFeOd Sat in Avian Diet" (Wildlife Intemational, Ltd. Project No. 454C-110). Three modifications from the cited `methodology were incorporated into the present study. The following changes were made. (1) High performance liquid chromatography with wipe quadrupole mass detection (HPLCIMS/MS) was usedrather than single quadrupole mass detection to reduce potential matrix interferences. 2) The internal standardization prossdore was eliminated becouse sn appropriate intemal andar could nt be denied, ) A shorter analytical chromatography coum (50 mam 8.100 mm) was usd 1 cxpeditc analysis ime. Samples were extracted with methanol, vacuum fiered, diluted in methanol, and then dihted in 8 50:50 methanol: NANOpurc water dilution solvent so that they fell within the calibration rangeofthe PROS methodology. A method flow chart is provided in Appendix IV, Figure 1. Concentrations of PROS in the standards and extracts of the samples were determined by reverse-phase HPLCMSMS using a Hewlett-Packard Model 1100 High Performance Liquid Chromtograph nerfed with a PeinEimer API 3000 Mass Spectrometer operated in mulipl rection monitoring (MRM) dtetion made, "The mass spectromewtaesr equipped with PerkinElmer TurbolonSpray on sours, HPLC separations `were achieved using a Keystone Betasil Cy analytical column (50 mm x 2 mm 1.D., 3-um particle size) enon --_-- Wildlife International, Ltd. Project Number 454-108 "1s fittedwith aKeystoneJavelin C18 Guard Cartridge (20mm x 2 mm LD.).Theinstrumentparametersare summarized in Appendix IV, Table 1. Calibration standards were prepared in 50:50 methanol: NANOpure water by appropriate dilutions of a 100 mg ai/L stock solution of PFOS in methanol. The calibration standards, ranging in concentrationfrom0.00to10.001000 mga../L,wereanalyzed with each sample st. The sameand most `prominent peak response for PFOS was utilized to monitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOS on the basisof individual isomeric components. Linear regression equations were generated using the peak. arca responses versus the respective concentrationsofthe calibration standards. An exampleof a calibration curve is presented in Appendix IV, Figure 2. The concentration ofPFOS in avian feed samples was determined by substituting the peak areas into the applicable linear regression equation. Typical ion chromatograms of low and high calibration standards are shown in Appendix IV, Figures 3 and 4, respectively. Examples of equations used in calculations are presented in Appendix IV, Table 2. "The instrument limitofdetection (LOD) fo these analyses was 10.0 pg on-column, calculated rom he productofthe injection volume (10.0 uL) andthe lowest sandard concentration 1.pg0aL0. = 100 Pg8.i/L. The method limitofquantitation (LOQ) for these analwyass2e.0s0 ppm a.., calculatedasthe productofthe lowest calibration standard analyzed (0.001 mg ../L) and the overal dilution factorofthe moti blank sample (2000 L/Kg). Examplesof calculationsaepresented in Appendix IV,Table 2. Along with the sample analyses, seven matrix blanks were analyzed (0 determine possible interferences. No interferences were observed at or sbove the LOQ during the sample analyses (Appendix IV, Table 3). A typical fon chromatogram ofa matrix blank is presented in Appendix IV, Figure 5. Avian diet samples were fortified at the following levels and analyzed concurrently with the samples 10 determine the mean procedural recovery. The fortification levels were 500, 50.0, aod 200 ppm a.. for sampling intervals Week 1, Day 0; Week 1, Day 7; Weck 2, Day 0; and Week 3, Day 0. "The forificaion levels were 5.00, 25.0, and 100 ppm a.. for sampling interval Week 4, Day0. The fortification levels were 5.00 and 25.0 ppm a.i. for sampling intervals Weck 8, Day 0; Week 12, Day 0; Week 16, Day 0; and Week 20, Day 0. The method yielded mean procedural recoveries of 101%, 101%, Grove: Wildlife International, Ltd. Project Number 454-108 "16- (1A0p2p%e,nd1i0x0I%V,anTdab9l9e%,3).resSpaemctpilveelym,esfosrurthde c5o.0n0c,en2r5.a0t,on50s.0w,er1e00n,oancdom2e0c0tepdpmfora.it. hefomrteiafincaptrioonceldeuvrealls recoveries. A typical ion chromatogroafma mati friction is rested in Appendix IV, Figure 6 Study Phases Theprimarypassofthe studyandther approximatedurations were: 1.2 APcrcolpihmoattoisoinm-u8lawieoenks7. weeks 3. Procgg laying (vith phoostimulatio-n3) week, 4 Baglaying- proximal 10 weeks 5. wee. Post-adult termination (final incubation, hatching, and 14-day offspring rearing period) - Housing and Environment Conditions Housing and husbandry practice were conducted 0a 0 adhere to te guidelines established by he Nationa Research Council (5). The adult birds were housed indoors in baci of pens manufac. t5u1rcedm.byTGheeorpgeinas QhuuadilslFoapirnmg Mfalnourfsactthuariengs(dGQiFMclMiondgelheNiog.ht03r3a0n)g,inmgerasoumri2n0g1a0p.p2r6oxcimm.ateTlhye2p5eaXs were constructed of galvanized wire mesh nd gabvanized shesting. A digram of the test layout is presented in Appendix . Each pen was equipped ith feed and water troughs, Weekly, sufficient feed for te fediog period was placed inthe trough for cach pen and rested to the birds. During the feeding period additional fed was weighed and added 10 the troughs ss needed. Wate troughs wer changed snd water addedasnecessarytoprovide ptabewater (generally very 23 days). Only birds associated with tis study were maintained in the study room in order avoid excesiv disturbances, The average temperature in the adult northern bawhite study room during the courseof the air banding test was system in 21.4C + 1.3C the stad room (SD) was with an average designed 10 vent ruelpatti0ve1Shurmoidoimtys of 42% # ic volumes 18% ever (SD). y bo The and replace them with fe air Groen Wildlife International, Ltd. Project Number454-108 u- "The photoperiod in the adult northern bobwhite room was maintained by a time clock. The photoperiod during acclimation and the first seven weeksofthe test was eight hours oflight per day. The. photoperiod was increased to 17 hours of light per day at the beginning of Week 8 to induce egg laying andwasmaintained atthatlengthuntiltheadultbirdswere euthanized. Throtuhegtesh,tohebuirtds receivead meanofproximately 199 lu (~ 18 . candles)ofluminaton provided by fluorescent lights hat closely approximated noon-day sunlight Observations "The test birdswere acclimated to the facilities andstudypensfor 8weeks priorto initiaotfitohne test. During acclimation, all birds were observed daily. Birds exhibiting abnormal behavior or debilitating physical injuries were not used for the test. During the study, all adult birds were observed daily for signs of toxicity or abnormal behavior, Additionally, al offspring were observed daly from hatching until 14 days ofage. A record was maintained ofall mortalities and clinical observations. Adult Body Weight and Feed Consumption Adult body weights were measured at test nition, on Weeks 2, 4, 6, 3, and at adult termination. Body weights were not measured during egg laying becauseofthe posible adverse effects handling may have on eggproduction. Feod consumption fo cach pen was measured weekly throughout the tet. Feed consumption was determined by weighing the freshly filled feeder on Day 0, recording the amount of any additional diet `7)a.ddeAdndusrtinegmptthewwaesekm,aadned twoeimgihniinmgizteheffeeeeddewrasatnadgerebmyaitnhiengbifredesd baty tuhseiengndoexfttehrnealfleyedmionugnpteerdiofdee(dDearys designed with a "feed-saver" lip. The amount of feed wasted by the birds was not quantified, since the `wasted feed was normally scattered and mixed with water and excreta. Therefore, feed consumption is presentedas an stimatoftotal fed consumption. Adult Blood Callection An additional four pairs of adult birds in each tet level were maintained for collection of blood Samples during the test. These pir received the same diet and were handed in the same manner as other pais in thir test group. However, becauseofthe potential stress ofthe blood coletion procedurdesst, from the four additional pais were not used to evaluate trestment related effect. Blood samples were cnet Wildlife International, Ltd. Project Number 454-108 ES cAolltehcetetdimferoomf baidrudlst sinxmtihneasteidoens,igbnlaotoeddspaamirpsl,eswhweenrepocsosilbllte,edatffriovme awleeskurivnitveirnvgalbsitrdhsr,ouwghheountptohsesitbelset,. prio to cuthanasia. All blood samples wer separted nto serum andhemacytesplatelets, sored frozen and shipped othe sponsor or sponsor's deigase fo posible alysis. Adult Necropsy and Tissue Collection ! When theremabiirndsfironm hge150ppm .. estmentgroupwer cuthanizedafterWeek4,00 necropses were performed. All other adult birds that ded or were euthanized during the couseofthe `study were subjected to a gross necropsy. At the conclusionofthe exposure period, all surviving adult birds were euthanized with carbon dioxide gas, necropsied, and disposedofby incineration. Atthe time of necropsy, tissues were collected for histopathological examination and analyses. When available, samples of gall bladder, liver, proventriculus, kidneys, brain, gonads, bursa of Fabricius and adipose ttirsesautemewnetregrfoiuxpedweinre1s0h%ipbpuefdfetroeEdPfLorminalHine.rndHoins,toVloAgyfosramhpislteospaftohroltohgey,contWrholengraovuapilabnled, 1s0amppplmesa.oif. `bile, liver proventriculus, kidneys, brain, reproductive organs and adipose tissue were stored frozen and shipped to the Sponsor for possible analysis. Fecal samples were also collected, whe available, from the lower digestive tract and stored frozen for possible analysis. Egg Collection and Storage Tags wer collected daly rom test ps, seaned and stored it 8 cok oom unl incubation or isposa (for those pens usedfo blood cllection). The col room was maintained st a mean temperature `of 13.5C + 0.2C (SD) with a mean relative humidityof approximately 69% & 6% (SD). Groupsof eggs were identified by an alphabetic lot code. All eggs laid in a weekly interval were considered as one lot. Candiing and Incubation A the cn of the weekly interval, al eggs were removed fom the cold room, counted and eggs selected by indiscriminate draw for gg shell thickness measurcnent. Th remaining eggs were candied with a Speed King (Model No. 32) gg<andiing lamp to detect egg shel cracks or abnormal eggs. Cracked or abnommal eggs wer recorded an discarded. All eggs to be incubated were fumigated with formaldehyde gas in an aright cabinet witha circulating fan for approximately two hours, to reduc the possibly of paibogen contamination prior (0 incubation. Formaldehyde gas was generated by Cao Wildlife International, Ltd. Project Number 454-108 oe combining 26gofpotassium permanganate nd 25 ml of 36 commercial grade formalin in a porcelain bowlat hebaseofte aight cabinet. Al eggs not discarded or used for egg hel thickness measurements were placed a a Petersime Incubator (Model No. SP20). In the incubator the temperature was maintained at an average 37.5C & 0.0C (SD) with an average wet bulb temperature of 30.5C + 03C (SD) (relative humidity of approximately 60%). The incubator was equipped with a pulsator fan and blades that produced a mild breathing air movement designed to eliminate intracabinet temperature and humidity variation during incubation. In order {0 prevent adhesion f the embryo f the shell membrane, he incubator was so quipped with an aomatc eg tation device, designed orotatethe ggsfrom SUofofvertialin one direction to S0 off of vertical in the opposite direction (oa arof rotation was 100) every two hours vitahbrioluigthysDnady o2n1 Doafyin2c1ubtaotidoent.ermEigngesewmebrreyocasnurdvlievdal,on Day 11 or 12of incubation to determine embryo Hatching and Brooding On Day 21 of incubation, the cggs were placed in a Petrsime Hatcher (Model No. S-6H) and allowed to hatch, Pedigree baskets constructed of galvanized tel wire mesh were wed fo keep hatchlings separated by parental pen of origi. Eggs were not rotated in the hatcher. The average {emperatur nthe hatching compartment was 3724C 00C (SD), andthe average wf bulb fperatre was raised 1333 01C (SD) (lative humidity ofapproximately 77%). incubatAiloln.haTtchhelignrgso,upunbhoadtychweediegghgos,ftahned seugrgvisvhienllgshwaetcrhelirnegmsobvyedpferaowmatshedehtaetrcmhienredo.n DHiatych2l5inogrs2w6eroef leg banded for identification by pen of origin and then routinely housed according to the approrite parental concentration grouping in brooding pn ul 14 days of sg. The hachlings wee ed untested dit without the additionof 5% suplemenal imestone. A 14 daysofage, the average body weigh by `parental penofal surviving chicks was determined. The chicks were euthanized with carbon dioxide and disposed ofby incineration Hatchlings were housed in batteries of brooding peas manufactured by Beacon Steel Company (Model B735Q). Each pen measured approximately 72 X 90 X 23 cm high. The extemal walls and GONG Wildlife International, Ltd. ProjectNumber 454-108 nceilingsofeach pen were constructed of galvanized wire mesh and galvanized sheeting. Floors were of galvanized wire mesh. Thermostats in the brooding compartmentofeach pen were set to maintain temperature of approximately 38 C from the timeofhatching until the birds were 14 days of age. The `average ambient room temperature was 26.0C % 1.9C (SD) with an average relative bumidityof 59% + 13% (SD). The photoperiod for the hatchlings was maintained by a time clock at 16 hours of light per day. Offspring Blood and Tissue Calletion `Prior to cuthanasiaofthe last group of 14-day old offspring (Lot J), blood samples were collected from 10 offspring in both the control group and the 10ppm .i. treatment group. All bloodsampleswere separated into serum and hemacytes/platelets, stored frozen, and shipped to the sponsor for possible analyses. Additonal, issues fom the10 offspring in each group were collected for histopathlogical examination and possible analyses. When availabe, samples of gal bladder, lives, proveatiulus, Kidneys, brn, gonads, bursa of Fabricius and adipose tissu wee fixed in 10% buffered formalin and shipped to EPL in Herndon, VA for histopathology. When available, samples of bile, iver, proventriculus, kidneys, brain, reproductive organs and adipose tissue were storedfrozenand shipped to the sponsor for possible analyses. Fecal samples were also collected from droppings pans below the floor ofbrooders housing LotJ offspring and stored frozen for possible analyses. A single composite sample `was collected each for the control group and the 10 ppm a.i. treatment group. Ege Shell Thickness Measurements and Egg Components Collection Weekly throughout the egg laying period, ane gg was collected, when availabe, fom each ofthe 04d numbered pens during odd numbered weeks (1,3,5, etc.) and from eachofthe even numbered pens during the even numbered weeks (2,4,6, etc.). The eggs from weeks 1,2 and 10 were separated into albumen, yolk, shell and membrane and all components were stored frozen and shipped to the Sponsor for possible analysis. Eggs collected uring weeks 3:9 wer apeneda he vais, and thei contents were removed, The egg contents were separated ino albumen and yok, sored frozen and hipped o the Sponsor for possible analysis. The shells were thoroughly rinsed ith water and then allowed ai dy fort las on week at room temperature rif being measured for thickness, The average thickness of ne ried shell plus the membrane was determined by casing fv points around the wait ofte eg8 using a micrometer. Measurements wer made oth nearest 0.002 mm Cron? Wildlife International, Ltd. Projct Number 454-108 a Statistical Analyses ststcaUlploynsicgonmifpilceatnitodnifoffertehnecetsesbt,eaenenanaglryosuipss.ofDvuanrnieatntc'es m(uAlNipOlVeAc)omwpaasrispoenrfporromceeddutroed(e6t7e)rmwianse used to compar the (hee rcament means with the control group mean and assess the statistical significance ofthe observed difrcnes. Sample units wer the individual pens within each experimental run, except adult body weights where th sample ua was the individual bird. Persetage daa were examined using Dunnett's method following arcsine square root transformation (see Appendix V1 for reproductive parameters). Statistical analysisofthe data was performed using Avian Reproduction Data ESyxsctheomf h(AeRfDoSll)owSionfgtpwaarrae;meatevrasliwdaasteadnasolfytzweadrae ptatckaag:e developed by Wildlife International, Lid. (8). 1. AaduladtuBltodteyrWmieniagtihotn.- InSdiavtidtuaal]bocdoympwaeriigshotnswawsemreeasmuardeed batettweseteinnittihaeticono,nrWoelekgsro2u,p4,an6,d 8caanchd treatment group at cach weighing interval by sex. 2. ACduoltnFseeudmption - Feed consumption expressed as grams of feed per bird per day was aenxdameixncehdwbaytmpeenntwegreokulpy. during the test. Statistical comparisons were made between the control 3. EMasaLaixdofimuLmaid- The numberofogg sid per femle divided by the largest umber of cage laid by any one female. This transformation was use to convert th numberofggs aid toapercentile valu esthoarcqnual 0 100, 4. EC ghgesr sua meocfoefk gEggsge isdL,adipde-pTenh.e numberofeggs determined by candling to be cracked divided by 5. VEiablme brofEyggsoSet - The number of vible embryos at th Day 11-12 candiing was divided by the numberofeggs set, per pen. 6. Live3-Week EmbryosofViableEmbryos - The number of live embryos at the Day 21 candling `was divided by the numberofviable embryos, per pen. 7. Hatcofh3-lWeeikEnmbgrysos - The numberofhatchlings removed from the hatcher was divided . Lb&y-tDhaeynOulmdbeSruorfvilviovres3-owfeHeaktecmhblrnygoss,- pTehrepennu.mber of 14-day old suivors was divided by he `numberofhatchlings by pen. 9. HatcholfEigngsgSest -The numberofhatchwlasidinvigdesdbythe numofbegegssretbypen. CRoais Wildlife International, Ltd. Project Number 454-108 "a. 10. 1S4DuayOrIdvivoofFirpsSse- The number of 14-day od survivors was divided by the `numberofeggs set by pen. 11. oHfeaggts c setohrfoMlmaxiainmnyuomgnSeesftem-aTlh.e nTuhmsbetrraonfhsfaotrcmhaltiinognswpaesr female used to dividedbythe largest number conver the umber of atch lings toapercentilevalueequaltoo lessthan 100. ) 12. 14:DayOdSurvivorsofMaximumSet -The umber of 14-dayoldsuvivosperpendividedby 13. E`gthee SlahreglesltTnhuicmkbneerosfs egTghseseatv.erage egg shell tiekness of indiscriminately selected eggs pee pen was measured. 14. OffirBoidynWegigsht - The mesn body weighs of suviving haichlings and 14-day old survivors were measured by parental pen group. 15. LsievleecrtWedeiLgohtJ ~ofIfnspdriivnigd.ualStlaitviesricwaeligchotmspawreirseonmseawseurreemdaadteadbuyltstxerbmeintawteieonn tanhdcoatnttreorlmignraotuiponanodf 10 ppm ai. grovp RESULTS AND DISCUSSION Mature northern obit recived PROS at nominal dietary conectratons of 10, 50, 150 ppm a. Due to overt signsoftoxicity noted at the 150 pm test concentration, the diary concentration forthe 150 ppm ai. rtm evel was reduced 120 pp ai. of PFOS a he beginingof Weck 3 ofthe es. The 20 ppm .. level was terminated after Weck 4 of the est due 0th limited recovery of bids at tis evel. Adults in the SO ppm ai. tet concentrations were euthanized ding Week 7 ofthe test due fo ratment elated morality and overt signs of toscty at this level, Therefore, the est ws completed with the contol group and 10 ppm .. test concentrations only. The control group was fed dit `comparable to the treatment groups, but without the additionofthe test substance. Diet ANnaolnyetiocfalthReescuolntst.ol samples showed any inictionof te presence ofthe test substanc or ofthe presenceofco-luing subsiances st the characteristic retention imeofthe test substance. Dit samples wer cllcted from he 10,50 and 150 pm a. et concentrations on Week 1, Day 0, and were analyzed to cvaluste the homogeneity of te tet substancei he die ad very test substan concentrations. Means and standard deviations for the thee (est concenrstions were 75 0313 ppm ai 50.1 0.981 ppm ad. and 147.4 431 ppm a, respectively. The coefficients of variation were 3.21%, 1.96% and [ee] Wildlife International, Ltd. Project Number 454-108 "ne 293%, respectively. These values represented 98%, 100%, and 98% of nominal concentrtions (Appendix IV, Table 4). Samples collected durin th estto very test substance concentrationsfo the 10, 20, 50, od 150 ppm ai. diets had means and standard devitons of 102 2 0.553 ppm a, 208 + 2.14 ppm a, 09 2.00 ppm a, and 161 15.6 ppm ., respectively. The coefcents of variston were 544%, 10.3%, 401% and 9.66%, respectively. These values rpresnted 102, 104, 102, and 108% of nominal concentrations (Appendix IV, Table 5). Analysisofdiet samples collected from feeders after being held at ambient temperature f7odarys averaged 108%, 100% and 103%ofthe Day0 values for the 10,50 and 150 ppm .. test concentrations, respectively (Appendix IV, Table 6. A typical ion chromatogram of test sample is shown in Appendix IV, Figure 7. Mean weekly measured concentrations fo cach test ou aepresented n Table 1. Mortaites There were no reamentrelated mori in th 10 ppm ai. rcatment group. However, here were thre incidental moralite in both the control group and 10 ppm 8. treatment group ding the courseofthe study. Conrol group morales occured during Weeks 14, 19 asd 21ofthe es Mortalities in the 10 ppm a.. testgroupoccurred during Weeks 12, 20 and 21 of the test. All mortalities in the control group and 10 ppm a.i. treatment group were noted with head lesions and/or neck injuries that suggest mortality was caused by incidental injury. Due to the natureof the lesions observed at necropsy, nonofthe morale in the 10 ppm a. testment group were considered to be related to eatment Five mortals occured in the 50 ppm .. treatment group and three mortals occured n the 150 ppm .. treatment groups durin the test. Two additonal birds in the 150 ppm ai. group were euthanized due to their debilitated conditions. All mortalities in the 50 and 150 ppm ai. test `concentrations exhibited overt signs oftoxicity prior to death and were considered treatment-related. All t`erste.maiNneicnrgobpiridessawtetrhee n1o5t0ppeprfmoar.im.edteostntcohncbeinrtdrsateiuotnhwaenirzeedeuftrhoamntihzeed 1at50thpepbmeg.i.nniensgtomfenWteegrkou5po.ftAhlel remaining birds at the 50 ppm a.i. test concentration were euthanized and necropsied during Week 7 of the test. Necropsy findings are summarized in Table 2. Gn0ai ee ------------------------------------------ Wildlife International, Ltd. Project Number 454-108 ue Clinica) Observations Clinical signs of toxicity were observed at all concentrations ested, AC the 10 ppm a. test concenttion, signs of toxicity wer first observed durin We ofthe et and continued ntermitctly unl adult termination. Signs ofexicity st he 10 pm . ts concentration inched reduced reactiton externalsimul sound ndmovement), fledsppearaneand leary. } hen sSeivgernaslobifrtdosxwiceitey antottheed 5a0s rpopffmlead.ii. taepstpecaornacnecntersantidonletwhearrgeicf.irDstinnotgedbodudryinwgeWigehetkme4aosfutrheemetnestts, ath cadofWeek , several birds inthe 50 ppm .. exhibited muscle spasas aad a 10s ofcoordination following handing. At the 150 ppm .. tst concentration, signs of axcity wer ist ned during Week 2 fth test when on individual was noted ss ruffed in sppesance and lethargic. Al bids i he 150 PP a. reament group exited signs of toxiiy induced by the sessofhandling during body weight messurement a the end of Week 2. In several cass, birds convulsed and became rigid ater being capturedfo body weight measurements. Signs of toxicity inthe 0 and 150 ppm 4. eament groups continued nil uthanasia and included reduced reaction to extemal stimu (sound and movement) wing droop, loss of coondintion, los of righting ref, lower lm rigidity, convulsions, shallow and rapid respirason, ruffled pearance, lower lm weskoss, lethargy, gaping, prostate posture and spasms. Adult Body Weight There were no apparent neament elated effects upon adult body weight at the 10 ppm ai. tet concentration, and any iffrnces between the contol group and the 10 ppm .. fest concentration were not ssicaly significant at any of the body weight tervals. However, there were marked, concentrationresponsive, treatmentrelated reductions in mean body weight for both males and females at ihe 50 and 150 ppm .. test concnratons durin the Week 2 body weight neva. Reductionsinmean body weight continuedfo both mals nd females in the 0 ppm i. restnat group through the Week 6 body weight interval. Following reduction of dietary concentration 0 20 pr .. during Week 3ofthe fest, mas in th 150 ppm a. test concentrations made slight weight pins at the Week 4 interval, while females inthe 150 ppm 8. tesment group maintainedmeanbody weight, Wilh the exceptionofmean female body weight at he 50 ppm .. tst concentration dring Week 2, all diffirences from the coral eroup were sastically significant at p< 0.05or 0.01. Mean body weight measurementsarepresented in Tabl3e, and Fires 1 and 2. Individualbodyweight measurements ae presented in Appendix VIL. Chon L Wildlife International, Ltd. Project Number 454-108 xs. Adult Fesd Consumption Thre were no apparent wreaimentrelated effects upon fd consumptiona the 10 ppm i. test concenation. No statistically significant difreces between the control group and 10 ppm ai. rterdeuacttmieonntsgirofuepedwecroensoubmspetrivoendwaetreanoybsoefrvtehde afetehdec5o0npspummptaiio.nteisnttcerovnaclesn.traHtoiwoenvdeurr,intgrWeaetemkenst-1r-e6laatnedd athe 150 pom a. test conccation durin Week 1 3nd 2ofthe tes. Allofthe reductions were statistically significant reduced 1020 ppm ai. atp a < he 0.01. begi The dic ningof tary concentration Weck3 ofhe tes ,for Ftahlelo1w5i0n gpptme ae.di.ucttreiaotnm eonftt hgreoudpi awrays concentration20 pom ., fed consumptionfo the group cotioued tobe sigaificantly lower than the control group ring Weck 3 ofthe test. Feed consumption at the 20 ppm ai. fest concentration was comparable 0 the contol group during Week 4of he test. Mean fed consumption measurements are shown in Table 4 and Figur3e. Feed consumption measurements bypenar reseinnAptpeeoddis VII, Adult Necropsy and Liver Weights Al surviving adults in the control group and 10ppm i. tet concentrationweresubjected o ross necropsy following adult termination. When comparetdthe contol grou, here was an increase in the number of males with small tse in the 10 ppm .. rstment group that was considered restment related. One ma in the control group was noted with small estes, compare fo seven ales in the 10 ppm 8. westment group. Al ther findings were considered unrelated to estment, Necropsy findings re reported in Tale 5 and Appendix IX. When compare tothe control group, there were no apparent estment cated effects upon the mean weghts of mle iver at th 10 pom a. test concentration. Differences between mal ive weights for the 10 ppm 8. estment group ad the control group were light and ot statistically significant There was sista significant (p< 0.01 increase in mean femal vr weights the 10ppm a. est concentration, when compared to the contol group. Mean adult Iver weights are prscrd in Table 6, While dividual dot ver weights ar resid nAppendix X, Histopathology No lesions considered related to test article (PFOS) administration were noted in liver, kidney, rain, and proventriculus of adult and ffping males and females, burs of Fabricius in offpring males and females, adipose tissueof adult males and females, ovaries of adult and offspring female, and testes Grays -- ee -- Wildlife International, Ltd. Project Number454-108 "aon of male offspring. Th increas in the umber of adult male in the 10 ppm ai. reted group with reduced esicuar size, ot accompanied by any morphological change in spermatogenesis, suggests that PFOS may have acccesed carly. posteproducive phase rogresion, 8 normal physiological phenomenon. Any potenisl test aril eft on the bursa of Fabricius in adult males and femles or adipose sue in male ad femal ofpring could ot be determined sine none (usa of Fabricius) or only one (adipose tisue) sample was submited fo evahation. In addin, any potential testaricle effects on te gallbladder from tested alt mals an females coud not be definitively determined due 10th extensive autolysisthat recuded ay acre diagnosis. The fwotherchanges in various fisues of adult andlor offspring bobwhite fom control and 10 ppm a.. ated groups were considered incidental and unelted to test ance (PFOS) administration. The full histopathology rport is provided in Appendix X1. Adult iver and Sera Adu blood samples (hth rd blood cells an see) collected through Week 15 ofthe test were analyzed by Exygen Reseach. The ressfo analysisofsamples collected for this study are reported {ogether with els for the mallard study run concurrently with the same et concentrations. Sunmary results of blod analyses performed for this study sr presented in Exygen report Tables XXVI through XL located in Appendix XIIA The fll report for analysis of blood samples by Exygen Research i located under Exygen Study Number 023-066, "Extraction of Ptasium Pefluorooeanesulfonste from Red Blood Cells and Serum for Analysis UsingHPLC:lecsospray Mass Spectrometry" Bloodand liver samples collected at th timofaut termination or the control group and 10pr a. test concentration were shipped to 3M Environmental Laboratory for analysis. Summary results of sea and lve analyses conducted by 3M Environmental Laborstory are preseoted in 3Mreport Tables 1, 2 and 10 through 1 located in Appendix XIIB. The fll report fo anlyss ofsera and iver samples collected a the time of adut termination i lossted in 3M Environmental Laboratory report EOL-1245. "Analytical Phase Report for PROS: A Reproduction Sady with th Norther Bobwhite" Reproductive Results `When compared to the control group, there were no apparent treatment-related effects upon egg pslriogdhutcrteidonocattonthsein10thpepnmuma.bi.ertoesft cvoinacbelnetrcabtiyono.saHowepveerrc,enwihaigleeonfoetgsgtastisslti,cahlalytcshilgininfgicaasnta, ptehrerceenwteargee GROLAT ------ee_------------------------------------------ Wildlife International, Ltd. Project Number 454-108 ae ofive week embryos and in 14-day old survivors as a percentage ofhatchlings. Th sigh reductions in fertility, hatchability and offspring survival also resulted in reductions in the numbers ofhatchlings and 14-day old survivors as percentagesofboththenumberofeggs set and the maximum numberofeggs se. While slight, the reduction in the number of 14-day old survivors as a percentage of eggs set was sutisically significant a p< 005. Summarisofthereproductivedats are presented in Tables 7 and 8, and in Figures 4 and 5. Reproductive parameters by pen are presented in Appendix XIII and XIV. Egg Sh"eTlhleTrehiwceknreessn.o apparent treatment related effcts upon egg shell thicknessa the 10 ppm ai. tet ccoangcesnhterlaltitohni.cknWeshseinntchoemp1a0rpepdmtoa.thetcroenattrmoelntgrgoruopu,p.theEregweshreellntohisctaktniesstsicdalaltyasairgnipfriecsanetntdeidffienreTnacebslien and Appendix XV. Offpring Body Weights There were no apparent treatment related effects upon offspring body weight at the 10 ppm ai. test concentration. When compared 1 the control group, there were no statistically significant differences in the body weight of hatchlings or 14-day od survivors fom the 10 pp a. treatment group. Offspring body weight data ae presented in Table 10, and Appendices XVI and XVII Offpring Liver Weights There were notreatment.related effects upon the mean weights of offspring livers at the 10 ppm. 4. test conceniraion. Any diffrence betweenth 10ppm .. treatment group and the conrol group was not statistically significant. Mean offspring liver weights are presented in Table 6, while individual offspring liver weights are presented in Appendix XVIII. nos i eee Wildlife International, Ltd. pe. Project Number 454-108 Oftpring Observations There were ocidenal observations of neck curl, weakened condition and footleg lesions and associated lameness, offspring werenorma for in offspring in both the control group and 10 ppm appearanceandbehavior throughout thetest. a.i. ) treatment group. All other Offring Liver and Sera Blood and liver samples collected at the imeof offspring terminationfo the control group and 10 ppm ai. test concentration wee hipped to 3M Environmental Laboratory for analysis. Summary results ofsera and iver analyses conducted by 3M Environmental Laboratory are presented in 3M report Tables 1,2, 10 through 15 located in Appendix XIIB. The full report for analysis of ser and liver samples collecteda the timeofjuvenile temination i located in 3M EnvironmentalLaboratoryreportEOI-1245. "Analytical Phase Report for PEOS: A Reproduction Study with the Norther Bobwhite" CONCLUSION There were no apparent rcatment.related effects upon adult body weigho feed consumption at he 10 ppm i. test concentration. However, minor overt signsof ticity were observed in adults at te 10 ppm ai. concentration. Addiionaly, there were reductions in tests size, and light, but retmentelated, effects upon erty, hatchability ndofprin survival at the 10 pm .. tet concentration. `There weretreatment related mortalities, overt signsoftoxicity and treatment-related effects upon bodyweightandfeed consumption at the 50 and 150 ppm ai. test concentrations. The 50and 150ppm 4. test concentrations wre terminated prior to th reproductive phase of the tel. Based onth effects observed at he 10 pm a est concentration, he no-obscrved-<ffct concentration or porter bobwhie exposed to PROS in the diet was detenmined to be es than 10 ppm ., the lowest concentation teed during this sty. anal -------------------------------------------- Wildlife International, Ltd. 2. REFERENCES Project Number454-108 1 UESunSbv.diirvoEinnsmvieionnrtoanElm,PernoHttazelcatriPodrnoAtEgevecantlicuoyan,tiOoAfngf:eincceoyfW. iPeds1t9ei8c2i.daenPdPreosgAtrqiaucmaistd.iecWAsaOssreghsansmgmieionontsn,DGuCsi.udblsiencesso,n.FI7F1R4A, 2 OOrEgaCnDiGzuatiidolnnfeorfoErcToenstoimnigcofCCohoepmeircaatliso.n sGnuiddeDleivneel2o0p6m.enPtr.is1984. Avian Reproduction Test. 5 ARmeperroidcuacntivSeocSlteteysfowritTheAsvtiinagn SapnecdieMs.ateAriSlsT. St1a9nd8a.rd ES1t0a6n2d-a8r6d. PrAancntuiacel fBoorokCoofndAicStTinMg Standards. Vol. 11.04. PhiladelphPiaA,. 15 pp. 4 Merck& Co, Inc. 1991. The Merck Veterinary Mamial, Merck& Co. Ralway, NI. 1832 pp. 5 aWtasohniangltoRn,esDaCr.chNatCioounanlclA.csd1e9m9y6.PreGsu.id1e3f3o.r the Care and UseofLaboratory Animals 6 DwuintnheattC,ontCrWol.. Jo1u9r5.5.AmeAr.MSualtisp,leAsCsoom.pa30r:is1o0n96s-1P1r3o1c.edure for Comparing Several Treatments 7. Dwulnne, C:W. 1964. New Talesfor Maliple Comparisons wit Control. Biomelrics 20: 4825 WOfe International Ld. 1994. Avin ReprDtosSdystuem(cARtDS)iVerosionn2. Cross no. Project Number454-108 gle cl lalee +ol|] EEE 5 | + =x ox flog SLI 3 2 is eg zg 5 oz Ht HE | HHl es =o [Rfhpe Fall all | 3 38 GR0R3T7 --_--_--m--mmm n ProjectNumber 454-108 `SummaryofGrossPTaatbhloel2ogical Observations from`AdaulNtorBtihredrsnFBooubnwdhDietaedR/eBpurtohdauncitzieodndSutruidnygwtihethTePsFtOS wT Males wm" Females Cor1e 0 l 50 wT --------------J-- OE S-- ES, Numberofbirds 16 3 3 3 16 2 Feathers wetaroundbeak 00 1 0 o0 Feather loss: 50 0o00 Extensiveheadnecklesions o 0 12oo eat-pasty 00 00 1 o Thin '0 0000 Emaciaed '1 1 1 2 1 Lossofmusclemass 21 1 1 2 1 [------ 11 1121 Inscranial bleeding 01 00o0 Neck -midHength fracture o0 ' 0 o0 Neck -hematoma 00 111o Heart -00coronaryfat 'o o11o AbSpdloeemnin-aslmcaalvlitayndl-orpale 31 '211 autolysis '1 ''2' Aabmodunotcomafvioitoynk-bsalmoaolldl 00 o 1 0o GCirzozpa-redmcpotnytents - '0 1 1 0 0 bie sained 00 o1 oo Ceca contents-pasty o1 1ooo Kidneys -pale 00 1o0 Ovary -developing, .. 0'oo Ovary -regressing -. 1000 Not remasiable 71 o 01 o aRbesmraivntiingonbfirotbnidshma5i0nptanine2.dfotrcsoclotnoncoefbnowdrseemcpuebsaasreentdrnepgortWeedc.k 7ofbe st.Grosnecropsy Remaining irds ith150ppm ..testconcentrationwereebanizd thbegining of Week 5. rossnecrpsies wereooperformeda heteminalion of he 150ppm a. tsconcentration Gra CE ere 3Eagles as oo FE5 IFIP IEE EERE LE Flies oar BE : lel Bi. osg | - fRaRs E3Rs Rbi. A3iEd I 8331 i 3 Bl ae ve oaann| 3 pc ii 4 |g Ifssoss g e PBR: e% t g | Ble [HE py i 28%3 R8888 R$838 3R%A 1g3i : l i | of sf. afaiao. d| Fiiiifly Uggilhl . .. [[iHiHi ereeeeeeeeeeeeeeeeeeeeeeeeeee-------------- 3. ProjectNumber454-108 : 3 2 ~: i2$3% 38 5ES . #2 3 8 i 13 Ai i2 8 mr mh eee i ot i = Ee pid p eee a iro-FI ofH 8 |: i ii EE | Fa f 63 SS 1 son, 8worn soos gTwa iPog ;0 h ni _--_-- 4 ProjectNumber454-108 i pm i2 - BE i me I i iti i=ooi2 J TEsp1 i. i: s im rE n | 2z : i22 LP op E F i Fit i A 1 0 a ERE e i3 2 pm ee m | i Pe 8worn 4cos 8Twins .i 0 , Croan BN i af a 3 + sE lp EE EElH gar ERNE E CH RE. E Libis Elo ee bo omen i EFlle]] e 2a 2t s | 4I Three 3 13 = & = I B Se ree e A -36- Project Number454-108 :3 L2}B 3 2 5 . q N2E fg wis X sag 233s \ =|f 23 & - i i; CE & . 3d ndFu, MN aoe sue cnenin _--_--_mm-- 3 ProjectNumber 454-108 Summary ofGrossTPaabthloelsogicalObservations from aNortherBobwhiteReproduction Sucywith PFOS Adult Birds Euthanized at Test Termination" FeNautmhbeerroosfsits Vadlesions FBoaockivgentlleseisoinosns LiAvvdeSormpiinlaecl caanvYoidtmye-etretdniis Isanionpgeta CToemsoesraoene5rbo1ortoe3mynd Notrmatable CoNas TO Opmai) pmsl 66 3s 0o 0o o o0 .. ,s o1 5: ,3 CTFeO e Opmai) ppmai 3 .s ! 1 2 o\ o\ o1 31 o? .3 -- !Grossnecropsyobservationsforbirdsmaintainedforcollectionof bloodsamplesduringthe cont Pope = fog & J fr i7 23 . . JH iHllz i splle.d didi bai ei oeb oer| "3 3 : 1 EE it I FREEREEREE | Hd Sl, pall iIH P| HEHHHIHHEL _--_---- --w- ProjectNumber454-108 i: ii ns 8 } 8 : 2 liis is = eB 2252 I 6sa;f of EESTI es 5| E NNNEE ON i I | BE F SS rSi) es Waosu8ama WnRos ed CROs" 7 slesssssssssss iy Sefssssesesesse| 11 |: 38 il renal iil <5 Blafagreosssaniss : i ii t gre LR 282225588538 B1y | er. offi oyyf sazitd| foErl2i3 88 i ils Pp ialdgian chg iifpaiig Colin DISSESEER ER ERRIE tin Wiinniiitiegifitd, 60% -- rr -------------------- ite ProjectNumber454-108 3s 4 I3 $ EEE nm | 2 52|E J mma: i . 8 z gE i LE 53 J He. 2 Rn 8 i. 88 332 I}: i 23283| of p Ie E r TAsPEs3 i 5i pi Hi 33 (OE i fEmEmE 8 Cosy ES dE HERURE ga iil g3 siiqlb ll. ! iH | 3 ii Hy i HH IRRRNEE ii il Cada. SE -- " ProjectNumber454-108 gH CC } ie ig EE Ee | ii } Sg Ig ii is 28 i 5 af |] bi Siei 1H oer ii]t fc iy i17g iiiHf E[Ali...2 ih iH ill Ghost ---------------------------------- Wil1dli1fT3 e Internatio. nal, Ltd. a-s%e Project Number 454-108 Appendi1x CertioffiAcnaalytsies ble Herma rm 0B CoInNtrTeAEnsRtIy MCLbEorRsaokTtessICnO3AFROeIfFerCAenNcAAe:LTYCS2IE0S18 SMRPerteedceiccFn9:f08So,DL41t8317 I.-- ----------] 55 Ms= aakegn = 55 Nam Fre a I5i Sdoaesvmimin |= &5 SSSlomorresyt ane fe LoTsrR 1 ---- aa en | iT3i ceeae i3{sSSR osesmmm mnneau i iam Sa 5 Sm i3 Comma 5ISSaun mmiim oI3 tNrermi 5 Porn IL3 eTmemvmomiivmennoaosn | |il B emiE tll p54 Reertmve BR rir common ey Chraga Wild4life4 Internatioinal, Ltd. -46- Project Number454-108 ApPpaegn2deixI Certificate ofAnalysis Centre Analytical Laboratories. Inc. aa 128 A CIaNtTeEARsaIiMtCnLERaiTbnICO3uFAROeIaFerAmCNcnAALYT0S2-IE4S184 Dust Assi: p10 Epica Du: 043106 StangeCoins:Pr 10 [------ PPaety 10P%A.NO oef ptilp,io,14t5%LlCSipr,io,SATOAcri ToPluaypeoRbo 37e50n-8615350,75 Peano peed ito tf ror skconcur. "c Putty byDSe Cs rgoinly ot ptomcusi offwrapity. No thermwas dSiuunfpn istieosnsmphtedspcioob.ocTohmai tidng5oc 04ng0ea beswcelidln iikctheedit minln. Ded ri, 50, etcm i IBNAPAR DfNoipoaetttriesonsitd FA Poteet haili cleanaenhcps Brin,CFOS"OIW=38) hn rsconcedeBPAGodabnor Pci Sd (40CPR160). conan Peters Chul Wildlife International, Ltd. _-*'- ProjectNumber 454-108 ApPpaegned3i|x CertifofiAncaalytsies SePirezeeri Ans2ltcalCLaeboratories.inc. INTERIMCERRToIn3FOIFACNAALYTSIES CatsAnayaLaboCOrNReasrasb: 230188 LOMSPky rosie py mw t e o m m Ce wm ------ SNtovtS:s,ThremeOpi4r0n4.OeTeheLoCes3vwvoeinsecvacci oliiogosihngi CfsOnigsnmcc feconlgoin n4. Eo 0God 3snacn Prot i. = abl SET Antti RevieetBh y:Larnoryp Vioge,Ci oesApi Libri ah suman esas Chon L Wildlife International, Ltd. ~*~ Project Number 454-108 Diet and SuAppppleemnednitxFormations `Wildife IoemationaTla,blLei.1 Game Bird Raton' INGREDIENTS SoFiyneBCeoamnMMeeaslt, 45%Procin WPrhoetaeitnMBiadsed:s A`AIpvFayMSesp, 2e06%0Pc%roPtrieoitne,in DGrrioeudndWhLeimyestone MEeatsstimoanninCealrPheomsix+ Liquid V GL Fierman(adFeMrimnantecroa'Premisebelow) TSoaullodized PERCENT 0) u30s85 55000 43000 205300 003850 00x2 Too010 `sVpiptlaimsidnathdeMFionlleorwainPgrameomu,niwshpeenlaodnd:ed st 032%oftheration, AmountSuppliedPer Ton: VViiaamniinnDA, 27000000000001100.0. NRiibcofalavin 406garmamss PVainaimoibnenBircyAcid 1S0mg5sm FBoiloiicnAcid 66040 mmeess TPhyriiadmoixniene 1122ggrraammss VViitaammiinnKE (Menadione Dimehylpyrimidinl Bisufe) 200005180p.ans MZianneganese: 14072ggarmasms Codpipneer 61S 5ggurmamss Seolennium 15812gmrgasms "Tehneegubaers,otoedanlyss i minofi27m%purosme, a minimumof2.5% crude ft and amaximofu%m Fermentation By-Products (SuseofUnidentified row Factors) cra _-- Wildlife International, Ltd. -%- Project Number 454-108 Diet and SuAppppleemnednitx FIIormulations Vitamins and ElTeacbtrloel2ytes Concentrate `GUARANTEED ANALYSIS VViitaanmiinaAD RiVbiofalnaivniEn NPiasnctionthenic Acid VMiStBamCin 8-12 FTohliiscmAicniedHCI AsPcyorridboixciAneciHyddrochloride Water Soluble Powder Pardon 12.5000000.000000 1070500 21520500 100205 2o5s0 2725500 - Perlb. 1400000000000001 ICU. 430000001M0g 150000000 MMgg 400100M0gMg 1206000MMgg 151000000MMgg VoiIfaNVmGiiRtEanmDiAInESENu).pTpSRl:iebmoefnlta,viDn-ASucpipvlaetmeedntA,niCmaallcSiteurmolPsaotuortcheenofteV, iNisainDyS)u,pApllepmheanTt,ocioptherrylBAc1e2tate (source SHCyuhdparpoelce,hmlePonrtai,desM,esAnsacdCoirhobinieociSAdocdiadin,udmSDoBedixsituurlmofisCtehel(osroiudrec,eoCaflVciituammCihnloKr)i,dFe,oMiagAcaieds,iTohmiaSmuifnte,HCFe,riPcyrAimdmoxoinnie um M"TIheXvIiNtaGmiPnRaOndCeElDecUtrRoEly:te mix was prepared as a ration ofspproimately 2 gramsofDurvet vitamins and clecuolytes o spproximatly 1 galloofwate, Cone ---------------------------------------- Wildlife International, Ltd. _-%- Project Number 454-108 Appendix III Diet Preparation Premixes for PFOS were prepared on January 22, 2001; February 6and 12, 2001; March 2, 6 `faonldlo2w7s,: 2001; April 12, 2001; May 3 and 23, 2001; and June 4, 2001. Nominal preparation was as Control: No premix required. 10ppmai: 25633 gPFOS +809g7ra.tio4n Soppmai: 12gP8 FOS+18086 72gra5 tion 150ppmai: 38.4494 PFOS + 8061.6 gration 20ppm i 5.1266 8 PFOS+809g4ra.ti9on wasweiBgashaeldarantdiosne,ta5s0i00d.e0. gT,hweaesstwesiugbhsetadnicnetwoastawreeidghHeodbanrttomitxairnegdbwoewilg.h Tbohset.reAmpapirnoixnigmraatteiloyn 100 gofsetaside rtion was placed in a Waring blender. The test substance was transfered to the aWdadreidngtotbhleelnednedrearn.dTthheewbeliegnhdebrosctonwtaesnrtisnwseerdetrmeiexetdiampsprwoixtihmasttelaysiodneeramtiionnu,tweiatnhdtthrarninssfeerbeedintgo othtehmeixbiownlg.bowTlh.e rTehmeaibnlienngdesretwaassidreinrsaetdiotnhwreaestaidmedsedwittohtshetbaosiwd,e trhaetibonowwitwhatsheplraicnesed boeniangHaodbdaerdt `mixer, and the contents were mixed 2r0e0w0ei.g0hegd,aalnidqusottosreodffrtohzeen.premix approximately 15 minutes. were weighed, placed in Iafpnportopursieadteilmymeldaibaetleeldy palfatsetrimcixbianggs,, As needed, the appropriate premix was incorporated intoth final diet as follows: Oppmai 52kg2ati5on+ 2.750kg limestone. 10ppmai: 2000 Premix +50.25kgration+ 2.750ke limestone Soppmai: 2000 g remix +5025keration+ 2750ke limestone 150ppmei: 2000 g Premix + 50.25 kg ation + 2.750 kg limestone. 20ppmai 2000 gPre+m5i0.2x5 kg ation + 2.750kg limestone "The diets were mixed for approximately 20 minutes ina Paterson-Kelly Twin Shell Blender. 6005.77 --_--mm-- MAoo Wildlife International, Ltd. _-5'AppendixIV `The Analysis ofPFOS in Avian Diet Project Number 454-108 Chen _-- Wildlife International, Ltd. ~*~ Project Number454-108 APPENDIX IV ANALYTICAL METHODS AND RESULTS Table1 -- INSTRUMENT. `Typical HHPeLwlCe/tMPSa/cMkSarOdpreM-- roadterilona1lr10P0arH--aimgehtePresrforma`nce Liquid Chromatograph witha Perkin-Elmer API 3000 Mass Spectrometer `quipped witah PerkinElmerTurbolonSpray ion source. Operated in mpl ion reaction monitoring (MRM) mode. ANALYTICAL COLUMN: GUARD COLUMN: OVEN TEMPERATURE: STOPTIME: FLOW RATE: MOBILE PHASE: INIECTION VOLUME: PFOS RETENTION TIME: PFOSMONITOREDMASS: Keystone Betas Cy coma (50 mm x 2 mm LD, im particle ie) Keysone Javelin Cycolumn 20mm x2mm LD.) we 5.00 mines 250 uLiminute 700% Methanol: 30.0% NANOpure Water containing 0.1% Formic Acid 1004 Approximately 4.0 mines 459.0amu = 99.1 amu GOs --_--_-- Wildlife International, Ltd. _-%- Project Number 454-108 APPENDIX IV Table2 Calculations "The concenroaftPRoOnS foundat he instwrasudemteermninted using the following eqution: PFOS (ng 01) tnsramens =BSKAES (rnin) Determinationof ample Residues (PFOS) Theconcentration expressedssppma. foreach sample was determinedusingthe followingequation: PROS(ppma.) in sampl=e FOS(mg4.4/1) atintr,SSextraficnt volume(1m) adiluttion efactor DeterminationofLimitofQuantitation (LOQ) ThemethodLOG, exp2prpme.1,swassdeteermidned sithe fnollogwing equation: L0G (ppm = lowest sandard conceiraton (mg 1x) overal dilaton faocfmtatoi banks overall dition foto ofmtx blank sample -E24E08 clue(7 ion fcr Fortncaton Recoveries "The ppm a. measincuocrseampd i divbyithednomeindal concenratinofeach sample (fried levelppm a... Thi to times 100ithe percem recovery ofthemetha otdavel offotficaton . ppm messiseampdle voRecorery= ppm. foried 10 nos _--_----,-- :i ig 2 g g 3 BP IHI 588 sss 238 338 | il. sg oioif| 3|| 8asesn saasesn zsasass manmsese| i:f C | E | |g] E295e% a299a% aweve EvitEe b B| || Hamm nn a| 3 E ess ss sss | 38 33 Fl| | 2a2d3: agi2Ef ga2i00 4gi0g0s0 (2 HEE CHEE GHEE GEE H i iN2 B2a2]) 25332347 223383%3 3283833% 33323g% |(g%f8 COO a Ea ---- 2 E : i 2 zg 8 5 j 3 i : Bi `82 3x 85 ms| 3< | 1 3 | 13 04s 3 | i g2 EI!: : aHl EREvos ses ne eae| 1 IH 3 3 1z) i i |g 32328 833% a3%2 a3Ea| GE {=I $3 HH | i of 2005 30505 455 1d5 2ifa: E HEERRERIE H15 E3T83 2s= 1E] E2E 22 2t3H3 Ts 44 33% | 23 |aixy 5 ie Lng 3 : ! i i2 g 2 z f 88 ,. 4 ik ik ik | i La Lg | 3 pic 28] Fl) sLaauBud g333353F5 pi59n8%d = " i i A i Ii - ' 5 gl B =| =| a i. 3j Ll i * Ei : 253552822sxisy g8ify |Zi| ivl2 ELE gn EN CFE H3 E i & ai Nneede] -~ z :3 53 h,w 3 2k i33, i3i aanmvesesneegg)l oo) 5 < iI: 3 [GTIERS she :| i 2 gz i ] I+] seg ssEsEs og LEE ef i i i kg ik il xg %g CEEiEg | g2 |LE i 1:i| 2337 s3zaza cass | iHlA 3 ; ~ 3 S| aX| iIEi3l| :" :3 3 =| oes 59 3 i 3 5| . |3 i 3 . 5515 3: = 3 [IME ERIE ET 3 F |} |e Eag| one noe EZ = 3 = 3 2HI Bal ee 1 5. 58 Sakae so: IT El aTe TTE CE Zz Es i - 3 232 a= s g : i 5 i =~ EES m s |5: }5 H2 ye 2 a| 2 Ches 5 Wilsdlif. e Internati.onal, Ltd. -60- Proj"ect Number 454-108 APPENDIX IV METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN AVIAN DIET Preparemaifontfationsample i thedesired avianfed stock ingthedeymis echnigue. 1 Weigh 10 samplesofthe matrioxnlFnrke,ehmastaireorgfacsatbioonensa.ndReectorsdamwpeliegshntto weigh bouts nd want 5- 1 Fo ach sample, messire 100 mLofmean wthoegrads ines and str note French spare 4 (Cp bottles nd place an seker table.AAplplooithne atmp3l5e0smto.shake for a minum of30 minutes st. 1 acum fir with quale le pape and sine esned fd 3 times with ethan to file 1 Tranter the Fite no 20-nL. volumetric fas nd bring to volume with methanol. . mePneapdoelsotprLioktmeadintieonosr)ebrringenlgcoenc,enafieoqnuvneod.teFaorllrsilon ndeioof hne HSP0L%CmMetShManSol 50% NANOpure water dilution solvent. i Ample snd submit sample or HPLCMSMS analysis Figure 1. Analytical method low chat forth anlyssofPROS in avian dit. 0057 Wiloldlif. e Internati.onal, Ltd. 1+ Project Number454-108 TT es EI. ot= 883.10 APPENDIX IV Mos . Tov re a oon avon ~ o 2.50 c3o.0m0 bo att 7.30 10.0 Figur2e. Typical calibration curve for PFOS. Slope = 11487727; Intercept = $83.10; = 0.99974. Curve is weighted (1/x). cnn Wildlif_ e Internati.onal, Ltd. -62- Proje} ctNumber454-108 APPENDIX IV HOLL Som eget A6TSADIID 1 - rr [i-- Use Area. {+:594008951 7298s4ams NoQaisweTuhhves. 35100 ABWwWfiloh ) 52s0 SEopnon d1az ae ores [reli Exon a Height 171 `Start Time. an vlanTe A-B8 Wet ce, R18 10 ny--_-- 0000 5 L.L PE `1 .x 20 uiTTeAtx ofnnsd Zo mGa p AA ahim `Figure 3. T(yMpoinciatlorieodnmcahsrsom4at3ogr9-amo.5f9.0a1).low PFOS calibration standard,0.00100 mg a.i./L. Coon Wil0]dlifn e Internati.onal, Ltd. -63- Project Number454-108 ToRStS00m~g wil 5nn ona pri Sms pro hiNi +: 4sa5d 7own 20ware 50 mt 10 MnwBA.hlWiW taah 3 3250 Son SExaP ptecTnedAwr T"oaks40P2 e Exit i agrakonTips A-:88. APPENDIIXV Vos Fo 7, 01 1821 10a0)) w oslo 5 o2f0 w inF TonI th mHTwW T vii 0 240 a w Bd, Hem Figure 4. T(yMpoinciatlorieodn cmharsosm4a9t9o.g0r--am 9o9.fa1).high PFOS calibration standard0,.0100mg a.i./L. Gn A----------------------------------------reeeeeeeeteens Wil'dlif. e Internati.onal, Ltd. -6- `Proj.ect Number 454-108 APPENDIX IV ooTn wABa woAsnmistspousr Wed Foor, or 180 ay 1000 1 Tame 2 bi ina] rom 2 ve NG[NoseieotTwvleans 51.00 8 ln 3 ATW (secs) 20 EphHSeacoteodimnRoT 402 om EExTen o0m38 Hts pn x100 o :kL ! x | 09 wos ve aos m0 ane te te Se Th SOUR Figure 5. hTyepiacpaplroixoinmcatherormeattaotgironamtoifmeaofmaPtFrOiSx.blank sample (454-108-MAB-3). The arrow indicates (Monitored mass 499.0 = 99.1). Ghden Wilodleife Internati.onal, Ltd. "65 Project Number 454-108 APPENDIIXV eWEtSe. wise wearoer0, tTwoovnnuia Sturied ry 10 3 i+:4n5t7e1n cre wo Ju GNaenoe 1500 oVna 3 n sa oeWidth 2305 = Id Smooth = 1 wpShooewTcstt"aSer 402 wm " prs Ene is wl i" Hio ntial.! B83TR88 1s mg 20 pha oe ie Ph Bh Bh AM Tim Figur .S0Ty0pipcaplmoan.c)hromatogramof matrix fortification sample (454-108:MASS, (Monitored mass 499.0 -- 99.1). Croan: ---- WiWilldndplliiffeeIInntteerrnnaattiioonnaall,,LLttdd.. -66- PProoj.eoctlNuemberr454W-1H08 APPENDIIVX. ToOiTs. a WeaBrn , [siwens-- Tn , fT4oi7w1nn Z5e0 . . Geaawinee 10 o , . BMSoienmni) 230s do EpocedRsT"szais 402 "s ExStartTime. i28s0 2. VeiinFakiardTietee 4-46%9 33 FGe ITfee ni: 1000 uo no TTyhmTstTsR shE UE Figure 7. DTiylpuitciaolnifoanctcohrr=om2a0t0o0gXr.amofanavian diet smplc on Day 0 (654-108-42, 100pm a.. (Monaisst49o9.0r9e9.d1). CHOBE -_-- -67- Project Number 454-108 Appendix V DiagramofTest Layout from a Northern Bobwhite Reproduction Study with PFOS Room 45 Pen N2u1m.b2e4r0s 10 ppm. prem i Pen N2u1m2b6er0s 50 ppmad. NumPbaers |G ma| Sppatels} 1.2... ; Pen I N2u6m1b.2e8r0s pp1m5a0i. b primi Door (aOpnperoxMiemtaetre) a. Fourfoot Chroma Lights b. GQF Model 0330 Battery Breeder NotesPens217-220,237-240, 257-260 and277-280were usedtobouse birds used for blood sampling. CHOEaL WildlifTs e Internati.onal, Ltd. -68- Proj.ect Number454-108 Appendix VI Reproducive Parameters 1. EggsLaid Definit-iTohne numobfeeggrs producedduring thebreedseiasnong. `rSeeparsoidiuicttiyve- iTmphaiisrmiesnt.a pIariamaeltseorhitghhalty siesnsfirteiqvueetnotltyheagfefneectreadl cboyndcihoenmsicuanldserthwahticchautshee dteissttuirsbacnocneds,ucette.d, and may be adversely affected by improper diet, crowding, excessive 2. Eggs Cracked Definition - Eggs determined to bave cracked shells when inspected with a candiog lap. tFhiendeactraabcyksacdavnernsoetlbyeafdfeetcetcitnegdewmibtrhyooutdeuvtielliozpimnegnat.candling lamp, andifundetected, will bias h`Siecaksnieisvsie,y a-nTdhisfepwarcamheetmeircailssnhoavfeequceanutsleyd afofgegcteshdaell ctrhaicnkniinngg.s dIimrpecrtolpyerrelpateend odessihgenl,l overerowding and pecking by the birds can slo increase gg cracking. 3 Esa DmTeoerfcihrnaeintsiiccoaunlil-nygAdltalhmisaeggpegasdraepmglgaisc,redainudtnodtechorysaeibnlscilucbcatateibodna,sfeorio.e.f.grgetfosteharelelnleceiggefoskn1etashides fmaoinlanlluoyswsiincsgrapTcahkreeadmreeetgsegrosn,Viable embryos. 4 Viable Embryos DTdihififnsiicuiilstodtneot-deirEsmgtiignnsegduiinsbhywhbcieactnhwdelfeiennrginltezharetiigolenyshasn1d0o-ac1nc2uedrxaelydys eaamnfbdterrcymotbnhrieycnoidntieicaot.dieonveolfopImnceunbtathiosn. begItunis cSaenussietdivibtyy a-nThuifsaviosraabflreeqeunevnliyonamfefencttefdo pafrearmleitzeratwointhinitfheerlovtiyduocrt,emimbproytoennitc mmaolretsa,liotyr Chemical residue inthe es [Gh ER Wildli1fs. e Internati:onal, Ltd. "85 Proj. ct Number454-108 pr---- Reproductive Parameters Page2 5. LiveThree Week Embryos hDiesisntdoet-nerTmihneeed bayeceambnryionsthgetheaggase. developing normalyaftertree weeks of incubation `Sensitivit-y This parameter is seldom affected. 6 Hatchling D3e5fionri2ti6oonf-iEnmcubbratyioosn.that mature, pip the shell, and liberate themselves from their eggs on Day Seasiivity - This is a frequently affected parameter which is also highly sensitive to the `conditions of incubation, such as rate and angle ofrotation, humidity, and temperature. ~ Humidity: 17100 ry, chicks will ick 0 thi sels Tempheerlatlusrteh:ere1bFy10n0othhoatvainndg thhuemiind,r-cchgigcmkobwiilliltygrnoecwestsoaoryfaospti andtbheeitsohoella,rge for their 7. Body Weight - Hatchlngs Dupfoinnrieomnov-alThfeomavetrheaghetewheeir.ghHoaftchhalcihnlginfgrsobmyeapcahrewneaelklpyenotooffeorgiggisna,rteawkeeingihmemde.diMseealny ight for cach week and mean weight by penar reported `{Soeinseitcifviettys- fThoims icshaemioccaclasrioensalilynaftfhecteegdg.parameter and may rflct some residual or ato 5. 14-Day Od Survivors Definiion Birds ha survive brooding for two weeks folowing hatch SSeonnsdiitlivointsy u-ndTehriswhiiscah tsheeldbiormdsafwfeecrterdeapraerdamientberatearnyd birsoopdreorbsabtlhyanmtohreechiendmiiccaatliv1e0 owfhitchhe the adults were exposed. [OES Wild_l--ife Internati.onal, Ltd. "70- Project Number 454-108 Appoint Reproductive Parameters Page 5. Body Weight- 4-Day Old Survivrs Definition - The average body weight of the 14-day old survivors by parental pen of origin, tCraeakpheonrweuedp.eoknlyreTmootvoal fgrsowmethweebirgohoedde.r Muneisthatwe1i4gdhatysorofaagceh. weFoeukrtaenednm-deaaysowldeisgurhvibvyorpsenfrroem Sieedn.si - Tis paramees s seldom ated, and mare closely reflects the resin practices 10. Egg Shel Thickness Demeefeaisninotpeiemonenend-tT,shweafosrtheSida,cchne6ds08roMfitedhSeoTsreheadllanasnddttpoheneenwmmeeeamnebsrkaatnreoerooefpmorhtteeedcm.pgeartst.s eFqiuatosrhfeleichkanviensgs SSmeoaeeshuinrleyimetno tTeshnuisrsufaeicseaa, csacnluddccoya.mlacffuPeocdstieebdplpeoarsmaemcoehntaetnrhi,ecahmloewaeosvurerres,meminentcalssuuudrreefcmmeee.nmtbsruanset cbuerltiankgenatvetrhye GREY Wildlife International, Led. Projct Number 454-108 ExpGerioumpenl Control opmai Appendix VI Replicate Identification. NRaepbpeotre __ReNpuomrbtePren 1 wm 2 wm 3 0 4 0 s 0s 6 206 7 w s 208 5 19 10 20 un m n ps 5 2 " 14 is us 16 26 RaNuDmubnerpe m wm um 04 05 06 w 08 9 m0 m m m 214 as 216 MaDlieaBand NFeummableer wm wm om mm ns m6 wm mw ww wo ow us ws ws wo 30 mm i as ims as was we 0noas Wildlife International, Ltd. Projct Number454-108 Appendix VI [Tp -- Euprinenal Reghe Repo? RawDuapep--aDlaBedNumber Group Number Number Number Male Female Treatment \ aw 2 wm ppm 2 a m mom 3 ne m mm ` m PY Ea s 2 ns wm wm s m 26 wow 78 P2n3 2P7I 3w8o1 3w8m2 190 225 22209 3w8o5 3a8m6 n m Bl wo 2 m m wm om 5 Ps m wm In 10 4 ws 9% is nl zs wm 16 m 26 wa Crest ii jesagegsanranos ce : E|ARRRAISRARARIEIR 80 3 i ns 5 nageesannaneenie| PL] 1 3 $3 | |FERRAEERRARNARAERR i EE meee| RA 1 eli] i 1 FARRRGRIAAARARIE| | i I eretnq nono "i & i SARFERZAAARRARER FF $ 53 Fo2l reomrecoernonans ell 3i 3 235838885 ANE%RE 80 25 Borman i 3 |ssazsensezaaazs fa i CROEEE : : 53 ARRARARERAE|FF }i) 5 a 3 EERE ELLER ELLE I 3 5 gnmnne nen onan fan i GnoBaL i. PrtNumber 454-108 HH saesgevTggvecneg|ve : $8) asasnannannansng a" 3 i z i ETEFREFLIEREELEEE ERS i"osi 3i i|: fVoryi |i j3 i8 z5 |[7i i ii &i i3 5: P5ol i A|aNsAsRaAsAsTaAsAsRseAsRsAsNaNsR||aRs| i if REa iz |ssasszasasaanzaz(sn| ii} mugoyomregrrgnge|an i |erssanananananns oe il enmmaanengann"foe I [seesrenassenzans os | H 31 33 CROC he ProjectNumber454-108 if AERI IA AeG GEE A | 90 i Bifess5aodaEesass (3d i ; 5 { i 5 RIRSRAR'B ASAIN |S i $f |i |eesnmarazsaniuny as i Piel ii chrg $8 5% ! Ji|seeemmennernenn fn] | g ii |i |ssszaazsanaszass|an |} I fr egr ere ne gn i g 7 i 3 po s i R |eAsNaRsRsAaAsRsAaRnRzAsRzAaRssA(Szs| BE Hi i i i|ssassanaasrseanneans is} fe| J3f COE Tm. Project Number 454-108 3 fief [E 11 [il [mmml | ! Fileresrannraszaana|an is i ii TRRTETINYCEgTIG |? IH i jit 2 if is RoE: se Project Number454-108 I FL 31 g 3 ii8 ! 8 3 Z : [7 |esennernnnannnen ie i "TTgFTUSIRTEORST 58 i BEREERESRREERRRE 8% i degoyeeguzeguge |e |remernnannannan|en| 3 i; tegsrgeemeaeneT |v. i sgragssgnsIansns| ss i I ] I gpd jiet il $38: ai [LH ER fol it LEAN { 98 ! 15 leans snnnnsnar|ie |g Jif -esrrazereasn it Hg Is FIFITLIAIFINAYIR |} HH 3 3} GRO Eafe 4 SiEEh nt Di 1 3 RIEASERESR4EES (ER i yo i dragangressasncg ' je : h GE45RE3RARIEREAS [ER Hi ;2 ifg) r sere ennsneeel r lin i: oi i |zgszassssasaaasg|s aill Bi iERREAsAaEgRaEzReEsYeses (ds ii [a -81- ProjectNumber 454-108 g HEEEEEREEEEREEERRE ER] go line 3 sla2zgagsdRRAARRAR [an 3 HEEEEEEREEEEERREE NER g HEEEREE RRE EEE EEEENERY hE HEEEEEEEEEERREEEENER i HEEEEEFEEEERESEERNER] HLL: EREEEEEE EEE E E ENEE - gifs). nAsazaIRAIASSAAN [8 i' "lalassaazaazazzsang [20 . alansanssazaazsszz|z- ! "lazzazzazssaszass|s~ % ~leaaannsnanagzana|an { "laszssasssssssans|s- | [freee 8g alsszsnzzazaazsazs|a~ g "lassassasssssxxzs|3~ i HB EEEEEEEEEEEEEEER ER ! P| %ER38ER2R3ARARAR 4 Groat romain 2 4 a LEEREERERE REEEEEE] a- i BEEEEEEEEEREEEELEERE RS i 2 IEEEEEEEREEREER an Hal lofi, gil ER |a|a|axnnanzoazaanzaa a2$ vis "lelasnnazaszaszzasns aff Hh olssasazrznasazazs|a~|f ! HEREEEEE EEEEE EER RNE RS 3 elnanasssngsssngazs|a~| g HEEEEEEEEEEEEEERE] al < - i :Df IEEEEEREEERERRER] EEEEEEEEEEEEEEER] ae 3 lt i 5 J EARERERIRERAERAN |], ; COATT -. Pl yl i Se ProjectNumber 454-108 i: Noo $ laeogmceggerges ns. Ee 3 S:| srecarzeaca-aosga]s 3 "laces ozennazssas a HE] i UEEEEEREEEEERE-E-EE in ii 3 "Jasadaanazanasanalan i I HEE EEEEERRR ERRR RAT i Caos . `Project Number454-108. i 9 Apron rrr rn nnn nn pe gol LL jit No Sl 3 : 2 MEREREEEEEEEEEEEEN ERS ES : if Cid g ale igmenavaaraggae bales 3 RE Conn Jil gla A385E3|BR]| | fee hija zl ii: al. 2EOE & gl ox x LC 0x feo 2]eena-| CERN | Rei 3 dabld|] ati l1nl HUH EH Cron gl ox. glovoen on El {OO Blo xEETE gl ox fx ol fi] | I hiHEiLEOlEo aLl E l Balp loof ae1ds [1 dIahl CORTE --_--mnm--m----e-- 57 Project Number 454-108 AAdpulptenLdiivxerXWe-iTgahbtlse1) from a Northern Bobwhite Reproduction Study with PFOS Control (0 ppm a.i) -------- Nae ------Fema-- le -- Fa Liver Liver on w s33o01 jarsoso 2004 4375 um- 220056 ssasss s2a9mmt 2207 ssomm s aos 00 20 priesis 7s6a7is0 mm apeos sar. wm 2 icsons ssaesns 2211s6 p1r66tn s10 ses : Mean os4427 18.662 Dot repre oe oat, cnet -_ Project Number 434-108 -88- Appendix X -Table 2 Adult Liver Weights (g) from a Northern Bobwhite Reproduction Study with PFOS 10 ppm ai. _ e ---- ee Male Female sree rei est aeert m am 9.152 218 a3 10659 29 any 10238 20 aus 9360 m as 133m m as 13.525 Ed 393 2 4436 812. 2s 3931 26 409 1013. wm 4819 1231 8 soe 149 29 423 3m 20 4085 9792 31 5989 m 4x9 ss- Mean aan 108s" FY 0545 15% _-- ~Datanotreporieddoetomortality _-- * Stsicalydifferentfrom he contogop tp <0.01 Cran spi cpa on eee: nour --_-- EPL] "00. EXPERIMENTAL PATHOLOGY LABORATORIES, ING, Project Number 454-108 WILD`LSITFUEDYINNTUEMRBNEATRI4O5N4A-L1,08LTD. EPL PROJECT NUMBER 212.027 PFOS: ARENPORROTDHUECRTNIBOONBSWTHUIDTYE WITHTHE PATHOLOGY REPORT Submitted by: Experimental PPa.tOh.olBoogxy4L7ab4oratories, nc. Hernd(o7n0,3)V4A712-07107820-0474 Submited to: WiEfaestoInn,terMnDati2o1na6l0,1Ltd. December 6, 2001 CROE7? EPL EBL ES mmm Prec Number454-108 TAOFB CONL TENE TS Page PATHOLOGYSUMMARY... 1 COMPLIANCE STATEMENT... 1 (QUALITY ASSUFINRALCEARTINFICACTIONE... outs SUMMARYINCIDENCE TABLES HISTOPATHOLOGY INCIDENCE TABLES CORORF GREOSSLANDA MICRTOSCOIPICFOINDN INGS SUMMARY INCIDENCE TABLES OFFSPRING HISTOPATHOLOGY INCIDENCE TABLES 00g "02. Proect Number 454-108 PATHOLOGY SUMMARY 600579 er ee EI "0 Project Number 454-108 EXPERMENTAL PATHOLOGY LABORATORIES, ING. WILDSLTIUFDEYINNTUEMRBNEATRI4O5N4A-L1,08LTD. EPL PROJECT NUMBER 212-027 PFOS: A REPRODUCTION STUDY WITH `THE NORTHERN BOBWHITE PATHOLOGY SUMMARY Light microscopic examination was performoendsectionsof selected `twihsiscuhewsefrreoum andutltrmealoaertwanhedicfdhemraelceeiNvoerdth1e0rnppbombLwih.itofe quail (Colinus tthest arlclo virginianus) (Perfluorooctane Sulfonic Acid, Potassium Salt [PFOS]) in the feed for `tahpepardouxlitmabtoeblwyh2i1teweweekrsa. alSseoleexctaemditniesdsubeysflrigohtmmuinctrroesactoepdy.14Tdhaey-oobljdeocftfisvperiofngtoifs `studyis itary toevaluate the exposure tothe teefsftecstusbusptoanncaeduolvterboabtwlheiatset (C2o1l-iwnuosekviprgeirniioadn.usT)hoof experimental design was as follows: [oExcoerimomntaloGrosup|| WalesS| Fe omaies T | Wao les r |e Fomal] es [comoiopem) | 20 | 20 | oe | 4 | *321O5wtosowspo.iirdnunhgtarroIcEaevdeSthr resotcsauNbgsthaanTcnoestthseUBdHiItE.RAGSl1o7ff0sprGintgw1e1raepporuehsainmiazreedly on Day 14posthatening. Cosy i-- EPL' Project Number 454-108 A MATEARNDIMEATHLODSS At the completion of the study interval, all adult bobwhite were euthanized and necropsies were performed by Wildiife International, Ltd. Selected offspring `were also euthanized at 14daysof age. Tissues were collected by Wildlife International, Ltd. from adults and offspring, fixed in 10% neutral buffered formalin, and sent to Experimental Pathology Laboratories, Inc (EPL), where they were embedded in paraffin and made into hematoxylin and eosin-stained `sections on glass microscope slides. The following tissues from adult and rovreiA o.to Hare, ri goS (oreo ei es fRFAi,Yan offspring were examined by light microscopy as available: liver, gallbladder, Incidence Tables. Microscopic findings for each tissue examined from each `bobwhite are listed in the Histopathology Incidence Tables. Microscopic changes ----------T------------_t `were graded one to five depending on severity. Nongradable findings (including present (P). All findings for all animals are summarizedbysex, age group, and cBomH RopAe----A----iyroA aTe rIoao r ARD STAR treatment group in the Summary Incidence Tables,together with the total number of Gross and Microscopic Findings tables. The entries in these tables were transcribed from the gross necropsy observations provided by Wildiife International. Ltd. EPL' I a - mon sr50 ---- een nen RESULTS Except for three control (334, 342, 352) andtwo 10 ppm (384, 388) adult administration were noted in liver, kidney, brain, and proventriculusofadult and offspring males and females, bursa of Fabricius in eros offspring malesand females, females; and testes of male offspring. Any potential test-article effects on the bursa of Fabricius in adult males and females or adipose tissue in male and ee ree female offspring could not be determined since none (bursa of Fabricius) or only Fabricius typically regressespriorto sexual maturty. In addition, any potential test-article effects on the gallbladder from treated adult males and females could notbedefinitively determined due to the extensive autolysis that precluded any `accurate diagnosis. There were no apparent test-arlicle related effects noted on the gallbladders from the offspring. ----Testes of all adult males exhibited normal ten spermatogenesis, the seminiferous tubules. In one (1) control and seven(7)treated males, the testes were observed ATA macroscopically as being smaller in size (< 1.5 cm) when `microscopically and there was no definitive difference in the morphology of the testes when comparedtotheir unaffected cohorts. Therefore, no definitive microscopic changes could be associated with the size reduction. However, the reduced size of the testes of these birds was consistent with early post- reproductive phase regression, anormal physiological phenomenon. The increased incidence of reducedsizeof the testes in 10 ppm a.i. males when Cron ro Number 45-108 EPL' PRATT hterater5,6 me 410 r`coodmhpaermeaddtouccoinetroplhsa,sseugfgoegsstssothna.t treatment may have accelerated the normal Testes of all offspring were listed as immature because they lacked all `evidence of spermatogenesis, had small seminiferous tubules with absent or ea. narrow lumens, and germinal a -- epithelium with decreased pT cell layers (comparteod expected in 14 day-old birds. Becausoef the smaller size of the seminiferous tubules in the offspring testes, the melanin pigment-bearing cells normally present in the testicular interstitium were, in effect, more closely packed in a `smaller area rather than being widely separated by larger seminiferous tubules Sdvars of afteHA xEd SormTcagAes,A chrsctnied by (thus the microscopic diagnosis of "pigment concentration" in the testis). Tote morals nonpedunculated nts tv nml phsloglimatty secid follicles completely embedded in a prominent ovarian cortex. in 14 day-old birds. Aaoto`aSehveraaltadfditpiornral mTmiacrtooscaopnicafmineditng,s o(dnersccrakibniednb--eloofwv) weereeSnsottedrwina Ba rate `comptoatr reaetedd birds, and other lesions havingtheopposite distribution. In `addition, there was no significant difference in the mean severity of these lesions in 10 ppm a. treated birds when compared to controls. Forthese reasons, these findings were considered incidental and unrelated 0 test article (PFOS) `administration. a. AoE -- EPL' A---- ftNeri 8 [EXPERIMENTAL PATHOLOGY LABORATORIES, INC. ttt Stror518 `Mononuclear cell infiltrates, observed in various tissues, consisted of foci iBone crameponerA ne,rMtiA ranAnthenTsoA fEboth aAdO Sand vst of lymphocytes and plasma cells generally accompanied by a few heterophils as Vecotzaton i the erws characaize bythe presencs of vcues `within the hepatocellular cytoplasm, which varied from usually small, poorly `demarcated vacuoles, giving a "lace-like" appearance to the hepatocellular `cytoplasm, to variably sized, sharply demarcated, clear vacuoles in the `hepatocellular cytoplasm. conn, a ---- While special stains were not employed to identify the Hepatocellular vacuolization was observed in the livers of adults and offsprionfg both sexes. Although there was a slight increaseinthe incidence of this change in 10 ppm a... treated adult males and females, when compared to controls, there was no significant difference in the mean severity between the groups. Since this findivrata rt amr ng was also commonly observed in theliversfrom control `administration. The apparent increased incidence in the treated birds was `considered to be due to biological and/or distributional variability. CONCLUANSDI SUOMMNARSY No lesions considered possibly related to test article (PFOS) `administration were noted in liver, kidney, brain, and proventriculus of adult and offspring males and females, bursa of Fabricius in offspring males and females, `adipose tissue of adult males and s females, ovaries of adult and offspring noes o- EPL' EXPERIMENTAL PATHOLOGY LABORATORIES, ING. Project Number 454-108 Wiki tomaons, Li. StudyNumber454-108 females, andtestesof male offspring. The Increase In the numberofadit males in the 10 ppma. reated group with reduced testicular size, not accompanied by any morphological change in spermatogenesis, suggests that PFOS may have acoslerated early post.raproductive phase regression, a nomal physiological phenomenon. Any potent tst ance effectsonthe bursa of Fabricius in adult males and females or adipose tisus in male and female offspring couldnotbe determined sinoe none (bursa of Fabricius) or only one (aciposo tissuo) sample was submited fo evaluation. In addition, any polentltestarile effects on the gallbladder from treed adult males and females could not be definitively determined due to the extensive autolysis that prociuded any accurals diagnosis. Tho few other changes in various tissuss of adult and/or ofspring bobwite from control and 10 ppm aL. treated groups ware considered incidental and unrolated 10 test arc (PFOS) acministration. VFeRtEerDiEnRarIyGPKetAhoRloUgEGKER, OVW, MS, Dipole, ACVP Tate FARIGp ([>x/o> "6- CRO ---------------------------------------------------- A EPL] SERENA PROLOG (ABORT `COMPLIANCE STATEMENT 2% senses 22% cess Client Study 454-108 EPL Pathologist ' _Dr. Frederick A. Ruecker sown Sos -- TEP 002 aur An eRete teat - Anicle _PerfluorooctaneSulfonicAcid, Potassium Salt (PFOS) The Hitopthotgy prions hs above rrr scvers conited `compliance with the Good Laboratory Practice regulationsofthe Environmental ProtectionAgencyas stipulated by 40 CFR 160. (ov EPL Prject Coordinator Jor 12,2003 Cnonh -------------------------------------- a rNane 40108 EPL' ER aT ON `QUALITY ASSURANCE FINAL -- CERTIFICATION co -- oe r----r--ED REPLPapo, CSLDr, AEgatSao.nGroth 1e0slloonngsgr ppccsof e tyceytsgPTeudeybybaCualyyeecnccoet FA SA ue SE poms roto 5 aramn son onan itnSk ees ou ren anor zs frrater wvieson Sor -- DOut ct ttartty styppnptt maes p-- m2 pd i dad = 1/28/03 romesam _-- Project Number 454-108 -101- SUMMARY INCIDENCE TABLES ADULTS i Onde 45Ka4al-ne1e08aSrerpiaficBeoeite "102 ProjectNumber 454-108 `SUMMARY INCIDENCE TABLE Er fTeTmme rm I-- -- --1 ---- C0 no fTemtiEon DbenFg e ] e |T | Fva errtsng Smo, T |T ] I Fe -- JS-- S ---- foEF Crpe A -- r -- 1--1------ [o(teoatnyts wproecoiowsogeress||w[e 8|[|| P Joeyoowen [Intro Roomere Gan[1[| G5o [ T| T | TTTFor -- SC-- A---- -- ---- -- [ToHe veepemrateioan.mees sa|| G1 [] Gt m1 TT e S eTo tiateE r -- I [hecrE asts,teptocetior [e || n S | s | [(io nFeeseRowwseeenen [|--GA n1|a2|[ [| | [inflation serosa 1 fess on aE -- 1 ew T | T [[ihneTerdssemrmtoemgemress 1[ 1|r|[ | | 1rrrrr ---- rr rrr t r t rr t r C------------r -- r rrr r1 r1r ] OT --Tre----r---- Cri _--m------,--,--,--,--TM "103+ `Project Number454-108 K4T5ae4ra-e1l0es8uNroirtfhiecren Sombite `SUMMARY INCIDENCE TABLE CO coonm| | EE B[s] | [CC inflamtion, Grr ane {3 r 3 r [| 1 inmtomamtion,mmenS es, ----1| T|-- E -- -- r ---- --r o enimes m --1I -- T e IT ---- RSE OF IETS G0 I fPE eotmeepo ewe) |I mm [wmE T 1 | [hCoTtoeryCessoprecrutesbagrors|10[vw 0 [ IBR || [io nirttratec, dGomrmeesGen |S 1 --0&| r [| Ive e exe |@ [aS w [| [[oeCgoeerratioonnwaetscstor, [iarttrae, dovsmeterGen | |T +[L8 _ [| || [hecnroosttss,tifoeng,mopceattsoler o[|1 1|[ e ||| | | | f-- o e --e ---- am --S-- ------ -- Fr rr [[iPnRCOfvAtENrTTaRItCUeL,UfSo(mWoom.eCtuewair-- NecDo) || (2a 20)| |-- (319)||---- E-- E1 [--T -- ---- 1 --ES------1 IIr S-- T -- -- r E I r E S---- So -- r rr r r 1 rr rrr r T | r Cr r rrrr rT EPL meron ga 104- Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLES ADULTS Cran -105- ProjectNumber 454-108 HISTOPATHOLOGY INCIDENCE TABLE wow XsatiunSacre Wo riers tobaite A Halalalas) 3: (ois amfteemation treme-- s J 12TTaTa TLlOC DTa e CT pEo TO lTaTxdd i 0A fo PO RRO] at aman Senin I NT ror RS I0 0 A [Hie neernatiizing Granta r LLTCCr P CrP r rrr ------ (0 psams--------LCape haa ssaaaa ["p Astalyatspraciudes Diagnosis ||prerpree lrelrplreir p|Ler ieler lelerlerlr[elt eledl Pft aa meefount ea d TT LTCTTP oaUdLa TTadEa] dT adTaTda]] [Cnflamation.Grome --~--LTTT EE a TTT TTT A5 OO| m { E E H ee er e r ei re r rr [afiltrate. nenactear a1 fra uaa} no hn oh td rrr I [Fiscalzation.tematic_| 2202zppaae ep Ti lEa e e--es-------- a pr Poatsitrate. Bosenaimae ce JLTTTTTCTTTTTTT Pastimion Seco HHH rr TTT rd [SIS linlin|in inia in iainln a etreemma Pr LTT a[0ban ajabinYaJo bin CELT reeererreeeeerr-- [ee rrr rr A rrrrr HH rrr rr eee HEE rrr 000 na Err Experimental FabaeyLabortoin oe,Cr rCAo tee wesarcs arCembasins I pittCo scutes fons Cnet --- -106- Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE car HHaasaltueetio0S8rutehefrinceBabtee . | alsalsilsnia)lals H3lE3H3E)3H]E3 pm Ph ots engi p Tro rrrnO i rrr fi m wm | PPn P PPE TXx] [oisetatimsinag brn amateoms, eats Tr TTrr r T rr rr T Tr eT oASAe GF FRsIES {RATrAHrUARCATTAn T AaT ) [efrarsatesFracsivdesDiaamais T[a2TA2lpATeATlAeTAlAelleA[TpA[IpAI[AeIAIpAlpA[TpA]K[AoLlAe]i[A2]] fem Torr B i Ix JR] aaaV a VE a |ipeTraFtiEsatTtin TTT rr A nda i |TSeS i ert--ion--s--es--am--e--, |A TTTT TT TAT | [[inPtaiSltsetracittee.ommronaneartesesGn o|H T L1C 01CH aC d TE 7 avctioons sepatosers ars[[a1x2K 2ZIa I T2K 22 i z oz oz d 7 E rom o ra om Tm iis 3a 333 ARfEp .| [[RserdputcsesS rmtogse nestsr [TTI [r |rr rE A A e ------ rrre ---- I -- 0 12 EPL peer Paiaiog LaborairTeon, to 2Faarmatee ensdtets sLaT peaAednes LhTeRac t Poms e TvOaricosate fons CAs. Bett -107- ProjectNumber 454-108. HISTOPATHOLOGY INCIDENCE TABLE i (A01PG Suon. on) nn xT TTT F OO O IFY A EO OO A pr af laomation, Hening boon EO OO I [randomatans [PPizealzinsania. rrC r TT HEE EE HH Meninges rrrrrrrrT euRsaor eageicis {I aon waY co-- onaCe oo O o aa Dw fe ios------ A0 IxiAlAlAlAINIAIATAIATATAATATATAALTAA] [autolysis Preciuges Oiagnosis |#1 LepieleIelplelplelpeelleeellep] fom -- xi-- x a x OY D I -- oo -- d ld [Cintiviate, nomi cent LLTa CTT TTT [CinflaChmroanitc ion,|EEE TTT EER [CP aimeraltzatton TT Gr TCO OY hhh ve [oepeneration. epmeeniutar, | x |[TT CTTCTT TTT [festcitowiar TCT TTT [Tafiltrate.Mononuclear CelnH TTI TTT EH [Thecrosis, eaatocellulae | |[1 pte |"Vacuolization. Mepstoceltatar| 20 [alat2l | rrrrnd I TLCT+or Thi] me pool i ATO OO I [prenatal aaa x [Cinfiamation,Serosa || | lt HHH rrr rr r r r r r r r T rrrd rrr rrr rr e r H I| on S EAEERERnAm N: FE Bove worms grant -108- Project Number454-108 HISTOPATHOLOGY INCIDENCE TABLE aon sHTostutieSaNcorritfhiecrne sotte 4 . mELEalalals]als 3allss)as m Fores p Hne E NT RH N e A H ETamma oe --ha e a EEaR im!| a [[ieseeatisamsamie -- Jas cE aa Ial ala aa AA ALAA AAA aA ALATA] (tf aintspeciutse Simamats|$12h #LTE[Sh EI+nEELPTF[2][pTa e[p eT]| P F: rHo oo ran --t Y a0 t t AYo A tTH]| f semaines r L h e r a p[ir etitbeatesmsr nesescea r zat r ] Era Tr mE A TEE| fh a h PAPA h RA A A) rr sant Sr [Rae reeleaeH | 0 e -- rr H r rr rrre r r A rS r S r nt EPL rem eb ere orLc mErTt Ske siT n LinsR ok neni 109 Project Number 454-108 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS ADULTS Chi re---------------------------------------------------- "o- Proje Number 456-108 ji g | g 38s 43 gd |: i24l1: [IEE [35 E[gHt 3gE19k LH] il& sil zRB [TIT] CODE ----E-- E-- EE -- ------A ---- ---------------- A------ me ProjectNumber 454-108 qf fee a | $ 2z5 l825s 88 ER HE EEE E E E 2g3 f2]22 2 2 E 8 | ls|R slsl sE leEs]E i)gye Ea & |8 88 godess gs 8 edd5 8 g sHl LlLil LLL =z TTT][1] Crp ---------- ETE ProjetNumber 56-108 g : 1 3 g2f 3 43%4, | | i ; 83 | gioi lo z 4 t: |h : H3E as32l55d[1]1]TTT ona eereeeer ---------------------------- na Project Number 454-108 3 31 HEE ElEl 2! . 3 Sls S$5ikHas E GRO500 ee ProjectNumber 454-108 32 1 2 i4i3 g4 HE dLgEE. 2go8:12s 2 zl lole : 1 h2 1l3 ; 2 4 HE 2 2 {8 ssf3 33464 [TTTLITTTTTTIIITT| 600501 e-- e ------------------------ BiH Project Number 454-108 SUMMARY INCIDENCE TABLES OFFSPRING CROs eee t-------------------- Projct Number454-108 -116- 4Ho5t4e-i1e0oi8rn.tgheSranriSthiciete SUMMARY INCIDENCE TABLE 1 [ JGoew[e Tm T[1 fpw E oo re Tseeo oe n | r n T ] o ]| LSC oome EsG | [o o ||| ] |] [L Pombo 11 Cr o |] [tC yesreceovr ate| 1 [7 1 1] ] [owsommee ToCorp[e | [iC niitate,fomometesr aorry{a B 2 r s [I[fPirirosvtee,sfGonomaE etearecel| |1 |T----] fC istsomen mm1 1] a I-- -- -- ---- -------- -- -- r rT r I-- ] r ---- -- -------- r rT r + i-- -- 1 -- -- r ----rrT r rT t rr t T] ] T r Tr r ] t T t TT Eel Form PegDoT m, T 1] GROG: `Project Number454-108 -n7- 454-108 `SUMMARY INCIDENCE TABLE m ras. EZ TT fF oesTe teenr y | r [ rr | ee ee 1 1 esaorpsnics00. |_|] 11 Comme [omorgoepietion | [1 | |[| 1 1| rr rrr (ere po.ewer @_| | [Psa reteset |] rT [er ioney Go.pwmeny--_|_@I | r |rrr emowe |w | [1| [iniierste,Fonte een 1 [ocottaation,Repstocettaler || 1 [| || fr o open | r @|| r 1 Comore [2 TT [r pRovEwicsto.omr e |@ |r | | ] | | ee 1 -- 1111 rrr rrr rrr r 1 r rr r Tr r 11 rrr rrr trrrr rr prr r r Er E r --A A r I -- -- rrr rd rrr rrr rr -- 1 r rT rr rr rrrrr `Project Number 454-108 -18- HISTOPATHOLOGY INCIDENCE TABLES OFFSPRING ee nse Per Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE cu Gun woHomESwri. tee i || Hafaellafaalsal]a) |aslas eser (ROIPOSETSS IWAN MNT FEA EB A A A A TTT] a A a hahJ ah a OO O LO r emermame Pe ett [aaa xa CTT rrr rrrrrr A lames I [wala awxxa TTTTTT f [e s l srPreeeciugeess pAagsJT ||T[ T TeT TT TTT TTTTH fan hhh J aaaTT TTT OO A mm [arta TIL Te TT ALTTTCL TTT] [oeS eostocS eiar 1 SOT Nr OE CT OT OY PO memes -- xo {Finite manateeesl aa xe rrr HH rrr rTTY EE Mee TTh Pa Tetpe ielm telpir plele T r TTr 11 a rtrarion SS 1TTT TelTelelpiolpl TTTC1} I 0J OO OL r r r rr r r r rr T ed rrr rer -------------- 5WS 0 0OS rrr rrrrrr rr rrr errr ---- r IO Y rr O er SSI 0 rrr A ere C r -- i rr r re rrrrT rrr O r O r TO Nr S OO rrr rd T re T a oH t dmE a toe saS t eacpR ite Ieletalts EE Ba, mar es - Project Number 454-108 -120- HISTOPATHOLOGY INCIDENCE TABLE ce Gur aSThiaapserr steritiocete 4 | H SlelLelOalel 1slsfslsial | | HE Re | (ES Ts me ATATAC TaneyTTT | hhh hae err (aumsaor eames i mebcidpegietion Jaa] daa CC CTE TrCrTTCCCTCTTTTT IA PO fms] Xrrrr [AutolvsfsPreciudesDiagnosis| +| || [A ee ee tr | || I rrr rrr my ume hx xs red rrr rrrrrrrrr em hp a rrr rrr] TTTT TTR ["iacuotization. Mepatocoritar 1+L TTTTL I TCT TTC b ais e P retere r e r eo bT o omps CCo E neCrCTHHH [infiltrate Wommactear cen | TT[TCTTTT TOTPTET ee FF EH Tr YO OA rrr rrr re | 0 OO rrr rr rrr rrrrrr rrr rd SS rr A rrrr r r r r r rrrr rr rr eH A I ------A IT | r f r rr re rrr rr C---- rrr v2 ET Te Taree, or aA en, ss CA c tse rem--" RE oweee------------------------------------------------------------------ WWiillddlliiffeeIInntteerrnnaattii.oonnaall,, LLttdd.. se ~~ PPoroj]eectwNumbbeer 454-H108 Appendix XIIA The Ansyss of PFOS in Red Blood Cell ndSea ColltehrocugthWeeedk 15 ee YaWilndligfe Innwteermnaattioonnala,LLtdd.. -2- eee Project Number454-108 oo Ear uyNo:02.006. `Table XXVI.pSpummmaa.ryfoorfWPFeOeSkinQuail Red BloodCellSamplesat 0 er om Su im hee D aTewsReeESooSoonn NBwoemrweben D DIwwnwmme TDamuammee feohhpeeen SiChamemees GoGoeoosn mowauaan mwwwmm mwmswumem ne Ne eSComiwsss oOoooooomno maDuwaaan uwsmme wemwwaeee NNee i hww ws oGowoewon miGaomana wwwmem ramoawem oaNomeme SShweea oSwesw DGiuxn lwee waawmeE Sonreeok SADA DIVIATION. 44103 [-- QetaeQuaen ow(eeckonwenatduieoncodf cshmcoonmpreansl(e20c00lWoonLe 100.001 or ed odsl siden Fores eecooptyNGw,a0.e0e00d3psl.elheevelesofdewdersScvlaotn, EpRass Psion Chpsng Wildlife International, Ltd. - ProjectNumber 454-108 Exp Sud No:028080 `Table XXVIL. S10upmpmmarayio.ffoPrFWOSeienkQuail RedBloodCellSamplesat S>r Sr "llioa ownss Samelaeonasunwses S"Ccilooraooiswmnss SSClliheaaisnmseer SCClllliornwse CD in eeSe mDaum Abmwi ooomo eGnUnD wSweer Tmaaee oooloooso comuussooRbwR>> asuwweee sssmaaame Ooowos om Gououbk hwle oueoe wwe ssmaalmmeeel Oooooomno Goouemboebieo> weneellr masmemelen oommowee sGmeoowwp wwweee msSmmoeo) oon cern anes me J-- inion remctepen 100 in ms xspob 0i lpoorto po *Onpenscion L0Q=001 posed bodcel andser BuypnReseach Pedtorn CaoarG -_ WWiillddlliiffeeIInntteerrnnaattiioonnaall,,LLttdd.. ES PProojeect Number 454-108 BostgyNae:02n08 "TableXXVIT.SSOupmpmmaray.offoPrRWOeSisnkQuailRedBloodCellSamples at prry GBoen STaR w S oovsen ShalD Sor mSee ac mbmu ue nmewe reutseete moomgncc vommmm u m wm m em m e J-- oe Table XXIX.pSpummmaadr.fyoorfPWReOeS1k0In QuallRed Blood Cel Samplesat 0 aETAT Con meSe e"omne OE Ie homee remap w rmae ne gooooomw ooomuaonn mawemm wmwemmm NNveee e eCneenn oooowom ooommweennn weammm w mmwe we e S me a e ooomooemn a ooawn mawewe wmmmwe aNNee e GCeeen wen Seoooss ooomnwoenns wmemmm maamem a e w Cae ew Sooownoey Sowihomnn mwmuewm am mmw mha -- smooe vieeho 0 =NotQunane o(weesckonceans tieodf a cheacobrmspoSnatianrmr(e3000nDiErLe 100001 un. or Hood ndsen Feorerl oeoroesdekNCw,a0e0d000i5.ehfoteeweeglfndheaersti cation Eup Reeach 28 coon Wildlife International, Ltd. use Project Number 454-108 Bo Sty Nya: 02e.066 "TableXXX.SummaryofPFOSinQuailRed BloodCellSamplesat 10 `ppm a.for Wee1k0 poo>e CBaem te50mo BaDeim Aomuv rensmiuge SaChiRioT. ooomloea ooBiulndd swneeell m sSoweeamna SLSeaemmmaaen oooomolalp mmmeawmoaD wmwewm smsaowww mmow aCCoinalesiweo omoonnn ooomoahnn smmuwee mmmamaemm mw1o SSCilmaewiowo ocoouomin> omomweenas mesemw ammmaamewo aumse SCCoomlmoaaorrrownirso oOooomhmss oomwooskh wwwmew mmmaaawww 2omme Sasi oon: cies swveomonimmvaaTmcOr 5 Dre cnn L0Q=001 py. or ed bod cl rd eum Eryn Fue satss Cronin use WiWillddlliyfee IInntteerrnnaattiioonnaal',, 2Ltd.. Project Number454-108 BstyNn o 02065 TableXXL.SummaryofPOS inQualRed BloodCelSamples a0 ppm a. for Wee1k5. rw=re T Cuomowe eSw Be paem e Abuv sAsmep hSCohwawem mooopmn Ce own oommaamsn omen m waem emmmmw am mw W om SS Sheewmm mowonbm ommuaammaaa amamee aammmmanoowmn SCmiuonmw aoomoommmo ooommmeaennn mamemm mmmmww oawnw SSe Cheam wwwe ooowwonnm om ooommmeeann amamme aa am mmmmmwmm owNmmM ow S S Gesy bwwh nowonom unmoamsmaa amaemm aammme owoao StaommonStTIEon doiwe "ein ND =Nogest sdtedve snk iwhtndnoc oh clamspdrssa0501ei aLUe 100-001 yin or bodcl mdse Foni r vnies ocornepd LNO,t0o0N0D.00. rlwhhe o ae flreesisginedsnnr Bog eh Pa sastos CROs WWiillddlliiffeeIInntteerrnnaattiioonnaall,,LLttdd.. ~ure~ TPrsjoct Noumbmer 4e54-1r08 -- "TableXXXIISummaryofPFOSinQuail RedBloodCellSamples at. 10ppm ai. for Week 15 FE SSieorn TowBETn SnCieewmw mooommma SSCohtoooewwws oooommmm Se ehwem,m GaGommip SSS Chhleaaamem ooGwoome nom S Sews a i . aooms a mmaes mm"Bema TmhDe0ae mreeTam Goomneamnss amamem mmmmammmm bi1es omommaowawp sammme mmmaeammm 1i%a commamomane aaamem mmmmaammm boaem ooGmmehnas ammmmm mmmaoamwm om oe goomemonwcop msmaeem smawaemm Sa aie mmm w1ir sas swoooa ttoay2 Table XXXL.SSumm0aardyp.offoPprFTOESmRiMnQualRed BloodCellSamples at TnieT CB um eSr BT"aheare wehrre saemmei i Seiartne oGwomos oommmmcc mmmeee m e me em ui otnse 4 -- 100001ym.fo ed bondcessd rm Bopha -- CHOG ee------------------------------------------ Wildlife Internati.onal, Ltd. -128- Project Number 454-108 FasNo 23o056 TableXXXIV.TSourmWmeaerky'osf FOS nQuallSerum Samples at 0pmai. rar Can Se Bees A adie D SR i hT aw vmoonns tn omeenscm bvm ewm bTaec remoiomgen Se shoawm ws. ouommuess owooomwme TTTooemw TmTaewm otooomwma Cons oom wom The THe oom iseeuews Cems toonwnes poommaaer Tvmaeemle Tvsememi aeooonmmmss [r-- SoARo IEnom or L0Q 001 untor edbloodcols nd em Eo heus [ Wildlife International, Ltd. u9- ProjectNumber454-108 Besoyeos n TableXXXV`.aSiuefmomrarWyeoefkPFOSInQuail SerumSamplesat 10ppm e=r CuBem tStae SR ChraiT sm E ocooinas Towmoeamn SPereoornvrseeeriooiooilmilmomceeowwn> SCmhoos rwess oooomote mmmoemows Sone im oooooynn oTmmeewww> m Cany s s oola mowm uThnemk A`rbak poAsemyepen vooammm e eawm e QTvaCwm am Tem im oTmmwe e emm em w mm mem m m Tm em m m svnmoE wews owe Table XXXVI SwiTuormWemeokfaPROrSyin Qual Serum Samplesa 50 ppm > GP un rfr Dwlw go ler SRa hTaIs tTooTn TToeae Tvmeome Tmeamw a= ye snoe oioenr a ---- L0Q=001 ua tos ed lod cls nde Exp sans ead a0 5 _-- WWialddiliiffee IInntteerrnnaattiioonnaall,, LLtedd., -10=~ FPoroot%Nuemberr454-108 0000 [ET Table XXXVIL.aSdu.mtmoarrWyoefeP1kF0OSinQuail SerumSamplesat 0 ppm Ta= om GoB n s mG0 bmbaeern An`uheirek stpsoiioegs ) SSSrieowwowinwn ooomommmm oovoowaany oommeeeTwwomaoduonnme SSeenomoowuiwe ooommmmm ooommoeann mmTmme Teuammw eoamwmm SSerweenie onmowmw oommmoaeann mvTmeomaaeme mooomwm SSSheeocvwse Doomome ooommmeeannn ToTeomem mmaeem oaoommmee SS Cheomaw aommoebn mmovonenn mw7mmeeTswe aosamwmes ER imac ston Oenttn L0Q=001 pin fo ed dels dsm BpReh Faster Cron --er-- e -------------------------------- me Wildlife International, Ltd. Project Number454-108 Bote soyNo:03066 TableXXXVIIL,aSluomfomrarWyeofe1Pk0FOS inQuailSerumSamplesat 10ppm SrSRyrT SCaBorm Otm5tee a Sam Sono a omens -- C Ceom -- mn oaoo.o omen omen SSp hhower inn i oommw noommeonhAs m a a o woomwn ooomnaoyn BTi"Dedeee Ta`wmnen rdergee Tooemomommrmm Obe om vmmommmaamm bo am mam MoThmwe mmmauwmm ie im Tom ww JE-- "Dteien 100 =001py forred boddeesm pnRss Pesan C05 ee -- WiWlidldlliiffee IInntteerrnnaattiioonnaal',, 2Lt4dd., 12_____ --Pro--ject Number454-108 -- BoasyNo: 0.06 for Week 15 `TableXXXIX. Summaryof PFOSinQuail SerumSamplesat 0 ppm a.i. RpS>rT CBaen tSete DaDuce Apuu veumscugen ome Tak NG S C Sim mos moammeeu ooommmueccc mmmmem mmmssmee NMeW SCClhaesem oGDomemese ooommmeemcce mmmewwl memaemes oomm ne SSCeihmmes aeomosee oopmmeamccc mommmme Tsmasmeme MWbWa S eCeoe mss hooomwoes omodbmeeccc mmmemm Tmmaaanem oNwen oSCaiimews ooooowpsn oombmmeecc mvmewee oamsmeee socoommw - SCOnS Gbiomwes womeac mmme eumsacme osoemsae [EE Ores in NCQ No Quail ( peakwsdeses heceoresponmingsaaccuntwioeite L0Q001 wn. tos ed ondclnd erm Feomr lus0c0or0dedmLN)Oo,od0f0o0N8Dyrmo.alSf Ehe 0ilacoufthe weestncgabndisnanded vn Boge Resnch ------ Caunig . 1m. Wildlife International, Ltd. ProjectNumber 454-108 Be StyNeos2n 086 for Week 15 `TableXL. SummaryofPFinOQuS ail Serum Samples at 10 ppm ai. So>me Co>m Gneianeise ooooormm aSCorilivoaamnn; oooooomn GGhlioanvtsssinooooomn SSheiwnviaisn oooooommm SCievimerssin ooooovmm cSGoenvmoemorneiisn ooovowipns teSwbe Bubuen mwoemoienn> moem ommmoooonvmsyo mTmoeee mmToaoobnyomoemeonl omoomoooonvhyh TTTooeem wooooooomhomevmneel Tooomwoevnss> immmeee ER wDpue seAsuibgeen mmT aae hAn mmmaaamen b3s6 mmmaaamme 07 mmTaaemee iTeo mmmaaamme lhows mmmmaammm ibinin Wemon vin Table XLLfoSurmTmEaRryMofPROS in Quail Serum Samplesat0 ppm a. Sryr CBoDm iE vaT oTe GoTw Dutton J wSam Dubunn Abmuork suAsmiaen oopesn m ime eTa nTa e hofene svoaotnon 65 ---- Eun Reser [ CHROG _------------------------------TM-- SC Te -- Appendix XIIB rosie Wildlife International, Ltd. 13s. Projct Number454-108 [r--sent PROS: AReStpy itetNahBetbo EO Sowa EEcmNempeestS csute810 dbir sopfsrieoSasRe BrsbtheS,tkocL1ynSaheirag7g8 Pe tE e Lry o eriaFeTT st,Ma irae, daFSCSSrSSact T Toi tte ys tTe tin en hc Sie Tal1. GurmayTabi,4,505ConomrtSairsoadnLivn EEEEEE Se EE =E Ee (ETT =] RE [mm[men +[oer[ome | L EE EE e [I ----me--m--es ve res? GROG Wildlife International, Ltd. -136- Project Number 454-108 Repu E245 Sn PROS: A Repro Sty wi Noh Bibi 7h Ahm typr it Table 2SummaryTae,Co 80 Conoeraton i ersandLivewihOutreReroved [momma] w | = 1 =To |www [ammo] [[m= eme|=[To-mT[o- maT n] w= r| 71--+T1|] [ [s=s[v(mT[om~en[[=-]-TT--T1|] == 1 TT 1 [Sr msr [wr[ ] J J| [oemw|[omorr||] EEE ERE Funross Fooliea srt A EePn bnsto th G0 nb rds Ferenonce SussTances -- Tue.-- Rte-- Subsm tances--e --n r [|SE C [ oom o o[wn omse reet [| oamasr mmre || a Te =e --c-- oRTTR SIE re yr eo -- 0050 ae Wildlife International, Ltd. Project Number454-108 Repo oL1205 win PROS:AReedocinSty withhoNhBow mon Ancornn Tube 10.C40;Dat Summary,FameGulors(dkan Ofpring) promomdoo|mmofm|m|a[m&o[n=e] formsms amma |G3|}| |e| [ermendecnm|o8m1eVmTman|r]s |omel eonm|o 8TmLo]m[n| pormencioms|aGom[m1a[n5 so[o|a er fomenato mseomemnemee |3] 3 | % ] foaer ra |8m ] s | | frmenelea|sm8m]m1m]me[nwea] [emma mmm--ana |8] [ermendorm|mmSmT en]as2nToowa] Er Sli we] eP ames Tnan nw TE = TE [ [erm eE rrmcamet ese ||S &o [fE n a] EEE mas Te [[EFoeoveoastintileerrison . ppmrnsaant eaorcs || 003| 4v 1Toeom| |]| [mmmmmtnmes 101 [owe |] [eer nesasarion 0037s, ppmmma sam2oree | 03 |1| ww | 108.380,10ppem emaac k2e01,2 1 ew 1 | etme Emin Labor rsa Pets COOS. Wildlife International, Ltd. 1s. Project Number454-108 J-- `ReportE01-1245 ok PROS: Aey t er in AReproductionStudywiththeNothernBobwhite [I Eovrisne ons a totvnoom oopmmenst anceine|r 03 | i voT |wv we e| | [Eovioeanesluceonsen 1oppmiwnansancaorca | es |v| vew|) [remiammmme--tne |oo |1 se | [BFt mades tomtmmne sa asoere TeTee] | [Ceon1266201 lise 00a ppmwma san wore | oa | 1| em | |Eot1adsoms1slus 108400, 1oppm mainwd G20 | 01 | | ew | | [isonftonemsator |Eo12462mm8/4 108-400, voppm nanstm202 | [e0s2 | |v || emw e 7] orvomomrammm aman [3TTT8Tw| fore msen | 8 oon| |ormemsdesmom rsmon| 03 |e | |evans mmmema|o21m%a| r 21s] [rm] rer meremperasmo 28TT Tome | frome]somesom pesco | 35 [1[fe [en| [enmomed]m aomom n tmm2s 3 |m VToiaTe]n] ER ar mseeed n ten dite 5 rs To re eSnGesgSot o tospree eerr a3 vinnie BrionLabora ---- nets Cross Wildlife International, Ltd. "19- Project Number 454-108 ---- sire F705 ARetinSywi heNr ebm ro strmen Tube 1.G80 DataSumario QlSr AstandOpin) tg |efromuemmmafloucmmm1|eom3Smm]T mm[ mami5 nno[nwa|a| ET |Loss ommseea |ermemelowmenormstnne |nS| |] To | ||oorrenealmamsemedmacsomnam|oSmTT mAmT T neST ze|| |:memelmmoprmaanne ST] a |oE vomE efoumomo|m3 ma8 are] on 1 | cro EEoer-12esanes| r re e |] =T+]EL |evensmana |on | forme | 3 ||Leerroeancmnesoenmlfoaesmemmeo|||m8 Gm8o1mT m1 m3am]rm]sT a8m|oTo=msa]|| wam------ TT vn GL0B8 --_--m-- Wildlife International, Led, PrtNber 54108 _-- -- e-- r-- I [evel [vse mmmmmmseerrenea]] 4|| 2|| 22TTme]] [ [ [r r ro o o moccsaesm m m mceees]]]32e e e||= 25r r rC] 2o o o [Teom] E r e emmme mm reE e 3|Ee Tm | E| E[ E esmv secmeee ]T 41n 3E | s E [oevemrffroeermmmmmmmenmeea]sS3]rES2e|||aeT]|]en] [[oo[ rememmior romoomrrmme mmmsmaairrrmmemeesam a]]|S&4|||e 2|||=m efe]]m] [Foomrnmiassmermmsa]lSH|E2| f[een]] bn TT TT rn CLOGS _-- sw? mm Wildlife International, Ltd. ~~" Project Number454-108 [-- J-- PROS ro A Repro cn Sty ith be oy en Norats [rm mmm EE |e elI NF I memarmroa| te foes[oom mee|0|| =] foams ooomena [[ 2 |] femme form mmna |2 || [fo oFrre reemee re T[T 0o8e[ m r |]| fe mmm mesam | 28|e |5s| frome ome mr = | 05|| || EE A SKeeva0r:aSraroeeoatsntocecnr on M mimtieerrrryan oor fo Pr] - WilTdid lifJOe TIRntEeOrPnHaRtiU.oOnNaAlY, LLtHd.e -1u2- resePmrojectNmuemebmeree45e4m-e10e8teters Repo 011245 rma PFS:ARepr ih iNotnBotbi #108A ey rn bl 12.C05DataSummaryFama QualLive Ackan Ofpring) FFFooobsaremaasmnfoampmmsaassmtoarrroiieem 1 | tow tom 1| [wr werr]|| EFaeross ammaetserrie | Toiw [|| aarr || Foraamae:f mmasere oe---- |s ttoowm as [[aoe rr || e[oeinsamssalfmfoemiatsrmea errm] oonme| a-- [ v|a-- r der || C HEorre aas -- omrmaeretreire-- s |t -- tono-- w | e-- aerr|| [oerroesssraeomsfimrramateeerriiea--[-- toomoa|--[| --w--earor | [ora opm mrie | mo [we] [FEEfiorerpraiassmaessosmTF oie ooargmaaT sfwoumm-- tmeai emme [--1--|m_se er ||| [E C ormemsR srfomTF mairsT mtie--|C [ tow m a -- 1m [[BBirsassestfoonmmaalrseortriees ttaiem esme]| [orssEsJeo-- gmarsartam--rion-- | -- F[[a mop reen asmfTroomFmarrasxarroien---- m --tmomoa[ --[ --i = --| --e -- {enJoga rim 1 ow [01] avin IEmme Laborstory ren ne CHO. U ------------------------------------------ WiWilldadlliiff. eeIInntteerrnnaattii:oonnaall,,LLatdd., ZZ -143- Pe rojeict Number4o 54-108 ---- Ju-- PROS: AReprithbi eNotnBt le Ho hn ts rnb feor-rmeariss lopmmai |s oreer|o1m| onaporey| [Eo 1361204 JogpometEFimOmprg| owen | 1| aos | [Eoviossiare lopmur|zm asoor s |u1 mo |mp aorg| {eovissssizaeJoppmatzr mpg|_osas| ano | [Eovisssonaio [roppmatsrr npg| am || ww | [Eovisssst3so roppm aicri,Otmg| agro[1| mw | [Eorismsariso[iommersriv Otprg| 0mm | 0 | sor | E+E a Nonrscmtcn eirTarr ove pasmscmgmoraetsn4se0e[a ae0m.beoen hs1 r0tn 0nTn00a.y1r] 5o9ano%bcsneeec [Est-some31ers 1ogpmal657 i.Oleg| ames | 1| wer iEmtomesnutrLmabarsssirmyony ronan fo Ga 4 Wildlife International, Ltd. Project Number454-108 f-- setae PROS: RichtonSty ithbsNhsBelte 10 rt pn Tai 18.750; utSummary seOu Livectsndfri) [eorsasmsjopmatsmuim [Eras opmumiuom [erasme lommumoue | tooo | i | ser | | ew| | ar | | om|v| ee | EE Jopomal rum | EE ---- [Eorromananlopomat sotuiim | vom |v[wer | [(Eorisesoe fogma swim | vow | [eer [Eovrsaaoss lopmarsuim | ow| Teer [[risasom opm souvm | os| TT aor Feoe m T [Eovisasooopmatsiuvm [ovis opmarsouom [Eotisasss ompmatsssuime [eorisasoons joppmat vim [Eoris@sem lopmalmouiw [or12asomon oppmarsmruim | ow |1| eer | ow || aer | ome | 1| ser | | owe |1| ser |ve|v eer | | vom |1| ser | E g n= [(Eovismsommlopmusmouow [[eovisieaoms liopmarsriuiv {Erion opmamu [[ovisnsoms opusum oe ee e o Te [(Eovisesnulopmatmum ooo 0| tome | ve | | vom | 0| | vow|e| | ome|e| er | mew | wr | wer] wo | Eames= [[Eorsasorse[1oppmarmmivee [Eorrsmsoem ficppmatmeni [Eorromcone fioppmatamuiiee | tomo | wo | meer | | tam| 0 | wer | | tow | wo| mer | [maromarovine |r| [oes omea0 [Eoriseammsf1ommal sum [eoniaasoestToppa rue [or 15 s00ssTvoppmatmomiee [[Eorisasonms [1oppmatdriver [corso Jiopmat iim [Eragon opm oouvm ome [vo ees | | vow | vo | ear | | vom |vo| wer | | vee| wo | eo | | sew | wo| amor | | tow |iw | wes | | roo |e| ese | bonnaet amriisn -------- [ GEG. . . "use Wildlife International, Ltd. ProjectNumber 454-108 f---- soni PROS: AReprbeionSly wi teNotherBobi ors -------- fEorimsanios loppmelumOpry|ods| | aoe | [Eorisessne loppmeleum.ONgrg |amr | 1| aoe | [Eorvimsarae Joppmutmouvmomprg | oan|1 | as | [Eorimssorere fopomes ML.OMprg | om |1| ase | [Eor-130631220 logonatdSww.OMprg|cms |1| ave | {Eon13059123Josomat31Ww,Org10mm| 1 an0 [EovisessiaeTioppmuizumonprg | orm| vo | wmer | [Eorismssss Jioppmaisumong| 0mm|1 | ww | [Evian [ioppmalbmOpry| came | 1| wer | [Eor-msaizm io matsm vm OBpay| ams|| ewer S et P + CeeenCep ae oSaeurta re et srsertonecte tm tmssvr aotenasdi4er C .GCosn 3i%e.nDsObeToTlki8T0%S,.cSr20cLnhsCkic [Eovismsaries TromatooMime ote| ome || eo | StoorGoweLuemon e pat15 BAS te ree lpr ge ie n TyESD st sSn ct CT OA oE rnTOR iCntrseshb[toangerSr1e0t0ip2p2s0h000vnwyr1mae1rTeaccot.1o48n),.nA AseDeGoNLECH80e r0e%5s0f28o e a e aEeate a hSetaeeaoSolAooneoCrTocus? CoLEntLrroEiooonnhsT B3 aeonnteyrpSrvs 1 ce S irSolLSRE548051310 Newaeno iaslio3nosnCo 3M0ohee osSrt ly teSn 1 SMEvoanerl Lebrary J CROs - Wildlife , International, Ltd. RR Project Number454-108 f---- J-- 705: ARprSycvileisoBonbi sers---------- re eCtro mntsayLcdErnppsne te tyT ehadsom0dtshacEnot e rR Toh e smaTeks1a.sAro Tale 14.Summ Table,Gu 0ConcanaSatrsosnndLiivner ee |v | |Soa Sm| wen wm [ee] Ww | mo | women ee| |v| re| cme |_| ww |_mor |B | emer| seer T= sets SMEmil ants mies Past CHOSE SE Wildlife International, Ltd. w- ---- Project Number454-108 ReponoL1245 storia PROS:AReStyp wilec Noth Bo oke sam------------ Table 15 Summary Tbl,C505Concenraton inSa snd ivewthOtfiarsRemoved [m[ ormo| me] ow[|o~ m [~ omonT] w=|[wr |corosmar|1 e rm | 5 -l Tor | er | me T~ 1 [ow [ow] Ebner. S A BR IS RE S erBE e TSeinaTheGapseseGees cSCepIa7as sioon tirsa.0o3s55 CoTredcobs ei onsves Se Coata0 wb S o Tew dm me e br e n Se enlbt egtaoC ma a t inge l am SE ens Toemwiisain SM EonartoLnabroriatsoreyres san Peas CONG34 FE -- fl] 2d 55 ih 8i El ! [5 | presnen erergen| 3) %|jg| sessaesseaass ges i Bi seeseaararizses' geo i nlasemen| ah 4 .| ssresacsers zens] 2% i Ta EERETERERETEEELRTAY HH ; Cal i| 25 seecsseacases) ges i: 1 Bl Bie | 82 CROLL a ------------------------------ oe rjcNorer 454108 i Do He i 2g is i 5s = id BS ils 2 88 yo: 3k t 33 3 * a 8 I* ee aR+va aRm A ee& C005 Proc Number 54-108 -150- fq 8 . 8 5 8 2 No Hl " it nl [5 8: 3 23 fs Zak 8g 583 ig 38 83 the Fes aaF+owsen oR 2 oo CAROLE? li Parana ar eas Ca 8 Cra a ree Ca 7) gi dle 8 BELHE |)lmeoamo2 i ii I 2e2YSAN A IICIS gen i i iH i enoocesioieacen: [men i oleae) uh "|5 BR RRA CESRI GIR [FEN i ! Annemerengzgnzng mH ------------------------------------------------------------ 2 ProjectNumber 454-108 [= 33 El 33 x" d38 HS 38 . 2 Fe 888 ag + umn Losi 8). bea o3s og LI dz: 8 83 # I= 2 8 00% ---------------------------------------------- Lise [m---- 8 f : 8 A i vz 2 LI al, p88 iBlH LTEE NEHH 3 85s ig Fog 2538 ios 38 LEH 2 3 gw Fes vos 8 =e GOOG -154- Project Number 454-108 i:4 sa] l] HERI ti fl - 5 i lps| i2 ihi zif Ju & |Z .] fiBt i ERR HH gsggszs grasa | meozes e ans enae ELE 21 : ges i eran an) ah saas erA ANs ANs e | gen R$ su-sgg-gnga-gasz| za 3 Bes) 87 ARR #$TF 598% gen il FER REEE ERE E EE EE EE ii iBl veneer engEiEEgEnEzLnEs] ie 2il GEOG "15s. Project Number 454-108 . <E sig Eo PF EHnd 18s Bd ;I 3 8 Tm Fr I I nmmmmmm--; Se Id | 5 & Tr i3 Re os Or) 5 2 Fim vy ummm PE III I I 8 waa / 8wasn | 2 CRnG 156- Poet Number 54-108 { qm JE 32 == 2 SE = 5% ig EH mm 1 <gC is Ey 3 5 Tm : 33 EE 5 N mm == Er CROs 157 ProjectNumber454-108 SElHll ji31| le PALS [F-|| cesesgrgiagaeg| av |g iH Ei sassass sass |gsa| | g -83 EH]3 saagye3 ang nus s | gun { : . 2 g fs] daiamran) un i H Hy RE 888 853g gg a i |3| verescoses sess] gan | 2s i THOSE AROS EYRE gan i ! anmvascessannzas| $18 il CHO%5 _-- -158- Project Number 454-108 . i c 3 8 ig 35. V83 ig1e3d2 gs E23 3is & $ Er rr = i Er = #8 Ey = 22 mm III 6 =o3 EE) = BsAgos mm =5 I + 4+ prin C8 Ei3Nz3 "my 8 & 8 2 9 Na + wun GO -159- ee -- ProjectNumber 454-108 , I] 32 mm 8 co I i: I y 2 2 fs i CO *1 o3l nEEeHd moo i5 5OSs 3% CT 2 832 Cm E3x CT + 32:8 : Er = 5 2 : I m-- avWaa + swiosnso: oo CRONE Te 13 HEH oo ooan . n | El. Co EL pn | Ba 51 iiss 3 58 i& 1s}h 2 i N; aagsnsiestsfe aiin il i HEE ers i34t) H 32 EI 2k x5 aes aE nesI | oa:i | -| | : || re i | ityi p n eesrsessi |g i: ! ) aerengzanzg | gy A "161+ -- Project Number454-108 or TT = . OI = 3 I= H52H I=Ys= Bay 5is8t8 IE OUI | 6 aEfE: i m--------r Ed Res 43s I 5 3,3 mm i 33 I 8 8 ia + wun pnI8 E2 mm" = - -162- Project Number 454-108 3. 3 CE 5 IE]8 ijiis ia Egaeg] 338 8 Ni 2 : 8 g i mm ey Tm 8 Ql I 2z --------w0 PaEE 5. J <fs := III, 3 Mm ; 2 mr III = 2 CT = 3 [mm a0 + 8wan R 2 CO0GAY -- "lig Cee JI Sgro sibs HI iki i ii LI FE i 340 Fl =|} 5h gge3 8593588F8| 8 i gszaaaacerarass'|3an i EELEEEERLELEEELEN ELL | ng ssa ensg essa| 83 i nd sniaRasAassoeeny| gan i cn gan $A IRs| GRA i i Mememvcongananny fie il 60050 -- "164 Project Number 454-108 i1 i i $5 alg je VE 25 dus gz 3 F $3 mr Tm i3 IE] LN I =f E [I 52 I + 33 Cr LH Rs a I 5 2 IIIT. 5 OE =8 iE OI 8 Na + wun R 2 hd I -------- Project Nbr454-108 ~165- 2 i. 5: ia 82s jez E8T3E33s 33 53d v3 8 f mmm III I CO + EI 2 asst Cl + 2 3 qosArg I3 III 5 - CI CC = %2 I = 3+ CI = 8 ? [roRpp-- R e e CHOBE ProjectNumber 454-108 ME Eo : mi gid EH FI IRE i131 i Pil g Cd:: Blo le EL mer 2 ELi even] on i i[E)N ecreeesreesrzersgesy2 sc| ) es 3 23889S2 3 ARLED E J o arananngoraannass [gan i: i GsiRUfEfiRTAAsIiBEs IEIFeEIaY il EYEETEPRRTTE OaH H!i i |k E.ERsrEreRrEeEEsT eens gee Hi 'f wmvmersagannzas ifs EH GOOBHY on Projct Number454-108 3H Sw pediment 8is gif snazeia yan ogy tii | Pragass fdxdd 47 HH i RS wn Eg4] - " wonRRaanc | gaa] fun aa] ii i pre sn i: i ee wsss sess| gsa i a sn R35 $RES EINE| pan i H$il waneavroaggaansazax| ifie ii 60557 -168- Project Number454-108 [ofBpO gE pela | : 2e8s5%d ||iE] -] soevssn nrssnne | 8 pHi *li GEGHHB E GHG ELE G | ERC : & ik i gaanaass asyz-| gan i 5 LEE Hl EE rarens ners ares) 2a i ljg| os mss snes sens| gn. i i TR RRR SRE REAR| gen i J nememersoginnIny ifs il Lh iH | en] i1f4 s 5|485) 3sisevis iiagosz a gassz 3indze d-|| e3a3) o:g i .| seunge a ess aRae| wa : i i Fla SEGE5 GGEGEEEE G | E5 i i dz) ssssszacnosnesa gaa) 24 dj) 39'gngrazax-eRan) 23 it | B5UBREBRGE GHEE| 6 i 5 FERRETEREFELSREEEANT 11 i : MumeseroognnIng ie il CRNGEG Hi EBEEEEERER ERE] pli i FE eee HEBah Ia! 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J I 1 i a EERE EEE TH8a do Tree RL III REieEd [EEELN I $f3e0d F |]g . |] FORE I cece] i ii g| cccceececs "lz 3 cee "lis Z af cece |i Aol 1; <ooamaomnn i i A = is GROOM _ Project Number 454-108 plc 2o35133% f|= F coor BReg fl ooo 8 il | ORM IF ELiEl rsik favre i i HE is se ProjectNumber 454-108 i csansesRo IR |g pf 3iF RFgl FREEBN FL cil commen: EE 1h ] "vvmee rene is } HEE Ah 2] Hca canons i if Ii x is aout --_----_ ee `ProjectNumber 454-108 i wgseacsess 3 oF ch ; g | fs] ceemeeeen]s Val Fl Fou 20 LLL BEEP Laas Lie (bl i: PEE | IEceEeEeeEnEaEnensERNE I i BLE ee EVEL coeeneennn sd : Ui Blood Fo|lemcomnonnn i iif C005 i. `Project Number 454-108 i ~assadces |g Vad |p| fog ff) reeddyl [fFfl 1 do SEELH$9i|s Coil 0D cna] EERE oe] d ln . Gamma :|4i lr Mi : Hi is J ---------------- i namssczors 3 p : i: gos [Ef Cron FETS I HC I Lid HE ELEN AIIR ER |b] ee ills ly 3 -|<avamno an ili J iit g| creeveeeen|s VALLI | Ptairg |f coe shee i ELF at flfalter is 3 i! 0059 ------ i "gze88EHRE (E ph as ; gfe] cere] ye3E fEE| corr (Pa & gf crm |e]3d JE a 1 LE I i is CO0S?0 ee oy `Project Number454-108. i FEEEEEEEENE pF Body Lf 3 i 8 sre E EM EEN : Pop LE 1] al g3 ad } . af ceemanaee|a] d ont oceans] 2i ce meen ean lid seems 3 false ju ; i 1d 3 Cnoue J i RET EEN pF cll Bag fe] eee EE NE . bes ad |[FBl ee] iz E } EVEL. TI LIRRREERERI IE i i is I. I Tati 5 : of? crete : Cl | fegs is ie Tpist d e3 g B aa l | Bi : Seema 1 {4 : co a Hl : i oC F REE fiis: Fev :"we oom Hon i i NH Coors - pepo 5410 % 43 ~1 82.0%] 82 R 8 883% i I] Lig . Cr$3e53t5 e ots FU i FBpRlE|gse]F fc 1 0 8 C8 T iBpisffl rrarrone il Cease i fl grazasnnaaanaaas 1F } GROG - Jibhg = Ce 8a 8 eia i. i| l Hi s eo s : iif A] gBgE8E8 + 1%8 | } | EEE EEEEEER REE EE i - es. `ProjectNumber 454-108 3] Ix 28% RUS ~ g ; I EE.IEDEEER EES i Il Ez asi e i & 2| 55 wes sss mses rh i FT UTEETEEERREEN 14 Hales Ee3l3ieH]E eE enA enerE eee 1 i3 i i: IEEE Rei =p | 3a 3 53n 3 si ssE sss gf) srs-wrwEEE ERY i | |i i 1s ii :i I 11] 3 Foe i Cu09s SE -- 0. `ProjectNumber 454-108 3%] ssssaan anzass | 8 fa ; Iloniieiio iE go | [fmm 3 Reo |.|s|zzsessmzseeenz i: f E TESERET CW FFEFEFS $j8o3r ([ifFls|E zrerze enmzaszs i Wie soe, i flel zmmzgszszezann Bl fp| mmm i13! ss|f zsEaessrEsm=oze esRsnTenEze 4 af seme sme in T| "eTEEOERT ETEEE No i 3 [3 i |lgy i(Eg1 Ih (iFhE |H 33] fil GON" - i en sas $ass 38% Tal oo saw amma qaen|ge i 2% #28 3888 R'3aR i i; 5) 25 | sl 5d FE 1g fife) FE FEECEEEHE FEET 3 nm as sz sas sees see EEAEEEREEEL REEL 83 [5|3| Fae nEe Ens ERE [375 wea amnanne { 305| If|oopmm | Hi ferme | 20 Les ner mer eer i gs S| FE ETEOERRECRTRE ii de ser vere vere [1 of serasazrazaanany [4 CHOYPS J -- i. re i 8522282 & RRBER t g| woman 0 mann ge FH EEAEEEE reas ; homme gd |3|F| EEE EEE 30 [ilo) peeren aves, %5 F858. 1 [55] mrrunevsanpaenneins |g i PEEL|E7| sLavensaenai5zsEeemaassn : I PH| flames (4 ol blezmran |B HOi 2 s3ss=sauzsasn ih i Fp CCUUETTURRTETES | sesszes = seams iHihi Gong --_--m--------e_--_-- 193 Project Number 454-108 Appendix XVIII Offspring Liver Weights (g) from a Norther Bobwhite Reproduction Study with PFOS --- Bird Control (0 ppm a.i.) Male Female Liver Liver 1 [X32 2 [0 3 0658 4 108 5 0922 6 0858 --_-- Mean 0842 sD 0154 0.950 0789 0767 0893 ------ 0350 0087 Gow Mean 0845 SD 0125 10ppm ai Male Female Bird Liver Liver 1 1022 2 0859 3 0637 + om s 1019 --_-- Mean 0853 sD om 1017 07 0682 0845 1121 ---------- 0882 0.184 Cw Mean 0868 SD 0.169 Theabovediferencesfom thecontolgroupwereno statistically significant. [RR ----Er------------------------------------------ C104. Wildlife International, Ltd. Project Number 454-108 Appendix XIX Changes To Study Protocol the folTlhoiwsisntguadmyenwdamsecnotnsduacntdeddeivniaatcicoonrsd:ance with thestudyprotocol sigonnJaneua;dry22, 2001 and 1. Tcholelpecrtoitooncoolf bwlaosoadmseanmdpeledstodaurdidngfotuherptaesit.soAfdbdiirtidosn1a0llyc,acthheteptrogtroocoulp w0abseammaeinndteadinteodfaodrd `coWlhleenctiaovniolfabsleel,ecfteecdaltissasmupeslefsorwaenraelyaselssaond bheisctoolplaetchtoeldogfircaolm etxhaemilnoawteirondifgresotmivalel tardauclot fbtiedss.t birds, 2. The protocol was amended to reduce the ditary concentration for the 150 ppm ai. test rcoendcuecnetdrabtaisoend tuopo2n0 spigpnmsoaf.it.oxaitcitthye abtetghienn1i5n0gppomf Waei.eckon3coefnttrhateiotnest. The concentration was 3. TthheepernodtoocfoWleweaks4amoefnthdeedtestto. cuWlehacnkiz4etbhoedy20wpeipgmhtaa.n.dtroebastemrevnattgiornoudpaata isnodoicnaatsepdrbaicrtdischalaadfntoetr wreecroeveeruetdhafnuilzleydfwoiltlhoowuitngcorleldeuccttioinonoofsfatmhpleds.itay concentration rom 15010 20 ppm a. Birds 4. TthheeepnrdotoofcoWleweaks 6amoefntdheedtetso.utThhaenilzeevetlhewa5s0 tpeprmmian.atetdredautemetnottgrreoautpmeanstseloaotaensd pmroarcatilciatlyafatnedr wsiegrnesocofllteocxtiecditfyraotmt1h0e p5a0irsppwmhean.thleevleelv.el Bwlaosodt,erlmvien,edb.ile and fecal samples, when available, 5. tThhieckpnreostsocmoelaswuarsemeanmtesn.dedEgtgosadsedleccotleldecftoironLootfssAa,mpBlesanfdroJmwaelrleegsgespasnetleecdteidntfoorshcgeg sshheellll t`mhermoburgahnIe,wearlebuompeenneadndanydoltkheanadlbstuomreend farnodzeynolfkorwpeorsesisbelpeaarnaatleydsisa.ndEsgtgosresdelfercotzeedn ffroormpoLsositblCse analysis 6. The protocol was amended to add collection ofblood, selected tissues and fecal samples, when tahviiassistluoaepbsalteah,onfldroogmcfael1c0eesoxfafmwsiepnrraeitnigocnoe.lalcehctferdomftohre cpoonstsriobllgeroaunpalaynsids. 10 pTpimssau.ei.strewaetrmeentaglrsooupt.akeBnloofdo,r 7. Tprhoetopcrooltocolwasamendedto deletethreference substance number from page two ofthe 8. The protocol was amended to indicate samples collected for analysis wouldbe shipped 0 3M pCpormpoar.ai.titoens.t c oTnicsesnutersactoilolnescwteidlfnoorthbiestosphaitphpoeldotgoicEaPl Lexfaomrineaxtaimionnaftrioomn,bibrudtswiilnltbhee 50 and 150 disposed of. ARONA -- er-------------------------------- Wildlirfe e Internati,onal, Ltd. -105- Project Number 454-108 9. TEhneviprroontmoecnotlalwaLsaboa ratom ry ftoe or saadn ldyAsitd staocfhe smaemnptd lAes,, atnhdean10aldytdichaelpShMaseEnpvliarnotnmoebnetaulsLeidbboryto3rMy Project Identification Number to be used for the study. 10. ETnhveirporontmoecnotlalwaLsabaomresnodreydfotroaunpadlaytsesAotfsscahmmpeleast.A,th analytical pase plan 0 be used by 3M 11. The protocol was amended to change Attachment A,the 3M analytical phase plant,oredefinethe peak area precision requirement and the method for calculating the calibration curve. 12. Tdheentfpcraottoicoonl Nwuamsberam(eLnIdMeSd)foto tihnediacnaatleytitchael pmaaisne p3lMn fEenuvilrsonismeEnOtIa-1l24L5a.boArdadoirtyonParlojSecMt tthreac3kiMngPrniunmcibpearlsAnaaslsyotciicaatledIwviestthigthaetosrtwudays acrheanUg2e7d23f,roEm01Ha-r1o3l4d2JaonhdnsEo0n1-f1o3L6i5s.s AA.dSditteivoennaslolny., 13. TShuenproAt.ocBoelacwhas amended to change th Sponsor Represcatative from Rochelle R. Robideau to 14. TNhoewipreodtocol was amended to change the Sponsor RepresentativefromSusanA. Bea tocJohhn L. 15. The temperature in Hatcher C, containing eggs fo Lot J, was inadvertently not recorded on the `afternoon ofJuly 8, 2001. 16. nTahdevelriiveeartlwyeingohttrefcoorrdaend.adult female (Band #428) in the 50 ppm a.i. treatment group was 17. cLiovmeprarweedi:ghstisawtetrcellreycoursdiendgfoDruamdnueltts'sandisotff.spriFnegastehleerctsedamfpolretsisswueerceollaelcstoionc,olaentdedda,awwheerne waveaiiglhaiblnego, ffiloivmerbsirodrscofliloemctwiohniocfhthiessseuesasmapmlpelses wre colscted. The protoce id not equine 18. rOcnaMmaenyt26gr,o2u0p0,1,watshentaemdpveeraetnulreyinnobtrroeocdoerrdelde.velOBn3h5u,eh2o9u,s2in0g0o1ffhsepri(ncgmpferroamttrhees1i0npbprmooad.e.r nloevterlescBorOdeAd,. 4 nd 5, housing offspringfom he 10ppm .. eaten group, were inadvertently 19. bDeiettwaereynscaomlplleecsticoonlalencdtepdroocnesDsaiyng0foofr Wanealeyksis1.weTrehenoetssaomrpeldesfrwoezrene froermtohveeedntfirreomtihmee tfeerevsaelr and toed at ambien emperare for on day r(oProcs esKng 20. PWPeMek..1 tbrleoaotdmesnatmpglreosupwewrheocolwleercetendofrdoemsoignneamteadlefoirntbhleoocdontcorlollecgtrioonu.p anTdhonaedmdiatlioinn tbhleoo1d0 samples were collected from male birds in pens where the female had died. Pr --_---- Wil+dlif. e Internati:onal, Ltd. -196- Proje" ct Number454-108 Appendix XX Personnel Involved in Study managerement of is dy: The following key Wildlife International, Ltd. personnel were involved in the conduct or (1) Mark Jab, Wii Toxcologist @) Joann B. Beavers, DirectorofAvisn Toxicology (3) Linda R Michel, Managerof Ecoox Operations (4) Diana Temple, Laboratory Supervisor (5) Sean P. Gallagher, Senior Biologist Aualica Chemistry (1 Wild B. Nixon, Ph.D, Director of Chemistry @ RaymonLd. Van Hoven, PhD, Sciatis Cranes