Document zz3NoDEYb8e36MYwVOX2M6D3B

--- Fos: APILOT REPRODUCTION STUDY WITH THE `NORTHERN BOBWHITE FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-104 3M LAB REQUEST NO. U2723 FIFRA Guideline 714 AUTHORS: SReyamno.ndGLa.lgVhaebrloven Jou B. Beavers STUDY INITIATION DATE: February 25,2000 STUDY COMPLETION DATE: December 15,2003 Submited to Evi5r35o5MusCmonepnoArLsvaieboonnreatory Supa Mianeson 55106 Wildlife International, Ltd. Ea$s5t9o8n,Caommnerdce D2r1i6v0e1 #10) 22-8600 Page 10f 125 # -- 600015 Wildlife International, Ltd. Project Number 454-104 "2 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Carporsion me PFOS: APilotReproduction StudyviththeNorteBobwhite `WILDLIFE INTERNATIONAL, LTD. PROJECTNUMBER: 454-104 STUDY COMPLETION: December 18, 2003 by the "UTSh.esEtnuvdiyowamaesnctoanlduPrcotteedcitinocnoAmgpelnicayn,ce40wiCthFGRoPoadrtLa1b6o0r,at1o7ryAuPgruacsttic1e9S89t;anOdEarCdDsaPsripnucbilpilsesheodf G38o50d, ALgarbiocrualttourryaPPrraocdtuicctei(onEBNrVeMaC,/C10HAEuMgus(t98)198147,)w1itnhdtphesfonlloMwAiFngF,exc$e9ptNioonksS:an Noticaion No. protocol"T(hWeisltddewIanstecmaotnidounaclt,edLiudn.desrtmuduyltniupmlebperrot4o5c4o-l10s4.)T,hane int-hiefeapnoalryttiiocnalwaposrctioonsnwedreuucnocdnedruecotndeed SUtnuddeyrntwumbesrep0a3r-a04p1rwotaosonlsta(tBodyewihRe3sseepaarrastuSduydnyuDmibreresct0o2r3-041 and 023-063), xy Research eporedRseseupltasorfaanalyses conducted by Exygen Research for study numbers 023-041 and 023-063 are STUDY DIRECTOR: Me, t. Dade SSeenino?r BiGoallolgaigsth,erAvian Toxicology SPONSOR'S REPRESENTATIVE BAJTEude TfL Newsiod " BaATEgo GNOG19  Wildlife International, Ltd. Project Number 454-104 a QUALITY ASSURANCE STATEMENT GbyootdheLTUabhSoi.rsaEstoouvrdyioPwnramacesincxaealm(PiErnoNteeVdcMtoiCronIcoCAmHgplEciMysn,(c948w0)iC1thF7RGo4ponaddtaL1ap6b0a,or1Ms7tArFAyoEPg,rua5ss9i1cN9eo8kS9StaaOnnEd,CarNDd,soPtaripiuiobplneissNhoeo.df 3da8t5e0s,mAygricnudlitnurgaslwPrroedurceptoirotnedBuoreahu, S1u0 dAuDgiursetct1o9r8s4a. dLThiebdraatersoyfMaalnlagauedmitesntanwdeirnesapsecftoilolnoswsand the acviry DATECONDUCTED PTeenpSuibncs Feruy 2,100 Sump Pepsin Ma1c,30h00 Dicbrgntin Mach7, 200 DAryihoeeDnn, fdeardy 16,1,281d PDritgkaeDnus, Jaaye16,172, Fsouuieposnot DeNcoevmebemrb2e164r,,1003 STUDYDDAITREECRTEOPRORTMEADNTAo:GEMENT Fnay2000 Mah, 200 Mune Much1, 2000 buch,2000 Maar, 200 Jy25,200 seey 31,2003 my, Ferry 19,2008 Deccnber3 200 December 18,208 Aispecons were say based unless ther oted SalSuConlmaann. BXA.Colman `Senior Quality Assurance Represeatative BATE 12-18-03 006.0 _--_--mmmm WiWlilddlliiffeeIInntteerrnnaattiioonnaall,,LLttdd.. ~~ - REPORTAPPROVAL PPorjojeerctNNuusmbbeerra4s54e-i1o04s SPONSOR: 3MCopan TILE: PROS: A Pilot ReproductionStudywithth Northern Bobs `WILDLIFEINTERNATIONAL, LTD. PROJECTNUMBER: 454-104 3MLABREQUESNOT.: U2723 STUDYDIRECTOR: SSeeapon?BGraingosAvliSaoTolxigcoAl,oegry CHEMISTRY PRINCIPAL INVESTIGATOR: 7 /) Sa Re ay 7A PA = RayIfnVadnHoven, Ph.D. bate RY jig /03 DATE MANAGEMENT: DR eTveriEnTooxS ico,log-- y -- Findd,Noion, l7% DieofsChmoisrey sr iehales BREhh cones -_-- WWiildlliefe Inteerrnnaatnioonnaal,, LtAd,. 5. TAOFBCOLNTEENTS TProjOectmNumbeers454-w104 GoodLaboriry rack Compliance Semen 2 TOOFCOMEERS ersersesee8 Test SubstanceadTrl SWB 1] TYTY HousingandEavironmetalCondiions... ree 13 AAAddduuultBNBoelcdoroyodpWCseoyilgaehncidosTnnd.iFedCOCToecnisonu-m.pioC ......er.ILI IT 16 J CinacnaahndddBirlcouanbdiaitngigno.n.g.. ---- CII CII 17 ROfA fseparnindgDsBilsoos cduasndiTiosnsu.e.Co.llection. rnsEE -- -------- weremessrmsssnsnsessses 18 J ------ ST) ! |i AGS By Weigh. --T21 ; Gross Necropay e-------- FEReproductive Resi... -- A CII LiOvefr fWseBpodiryWegiighnhts.g.s. ... IIIIII 006 --_-----m-sry-enven Wildlife International, Ltd. ProjectNumber 454-104 -. TAOPFBACGOEN?LTENETS TABLES Table 1. MreoamnMNerastubreerdnCBoobnwcheintreaFoinlso(RpEmpaO)GofueSEhOGSoi.nn AvinDit 21 Table 2. MReaenAdpulrtBSooudycWdyewisgihhcP9F)OifSomoanNorthernr Bobwhite e ilo er Table 3. BoMbewahniFteeedPClotnsRumeptipont(boiSdddyeuyw)sitirhoPmoFaOnSNo.r.tv her r 29 Table 4. 1BSou6bmwmWheaiertkyesoPafinGordoRTseeptPraoEtdhuoclTtoigoiOncaSltNOubdsyerwviahtie PonRsOSf,oAmr dNoBritthserur thaniedr30 Table. NMoerainbEergngBPorbowdhuictteioPnil(oEtgRgespLroadudcteinonaSnuddEygwgishHen/Dfrnoym)PROS... 31 Tble 6. fSruommmaarNyoortfeRresprBoodbuwchtiivteePFeriftorRmeaprnocdeo(cEtgone SSetyfrowmihWeck ) PEO.. 32 Table 7. MreoamnsBNoodnybeWreniBgohbw5h)iotfeHPlilcoitiRnegprsoadnudctSiuornvSivuidnygwOfifhspring PROS... 3 Table. SMUeanMLAivRerPWOeiSghs 5) fraNoorthmern Bowie iloP Reproduc3 tion 4 nerd _--m roo---_ YWyinldaltiyfee`Imnetrernnaattiioonnaall,, lLtmd.. 0 2. TAOPFBACGOENLTENETS APPENDICES WW PPreojeecet NNumebtere45s4-e10w4 ADDED 1 Appendix 1. APPEAL Appendix IV. ADDER V. Append VL Appendix VIL. Appia VII ComOef AStIAs. DitandSuplemeat Formulations... Die POISON... 35 S-- TheAnalysis Of PFOSio AVish Diet crc] DiaOFgTertLaaymout. 55 ARdslEBoPdyTWSetOiugdhyUtW)HCroPtmFOaSoN.o.r.ns..ersBobwis Flor r ors$6 FBeoebdwhCiotnesuPmilpottiRoenpr(ogdhuicntdidoanyS)tyromwaihNoPrRtOhSencc... 0 NoIrtnhedn BiGobrvwohsiitPdeoiluooaiRcelplroOdbuscetrivoantSitoundsyrwoimtah PFOSSH..c.....6.4. APPERGE IX. Appendix X. Appendix XI. Appendix XII Appendix XIIL Appendix XIV. Appendix XV. Appendix XVI HiSOPABOIORYREPO. ENgogtPherrBoobwhidetge PuiaoicRdehpetrnohdwueicckt)ioofnrSonumday with PEOS vv 109 RNepororduectBivoewPiefeorPmlaontcRebeyPpenrfoomdStauudcywtiliPoFOnS cc 113 PMieoanROffeirpinrgBoodSdyuWdueyicgwihtth8PiFOorSonmaNortr hern e Bobwhe ite ce 119 ARdulEtLipverIWeSiOugdhGtygW)EHfRFoFOraSn.N.o .orthmer BobwhitePilot rr 120 OFfiflsoprRingeLipverrWoeidSgUhudty(cgw)fitohimFoOsNSno.rt.h.ern Bobwhite 122 Canes Study Protocole 123 PersvalovendinntheeSudl cc t2S N064 --_-- Y Wildilifde Ii ntergnate ionall,lLitdd,.v~ on ~aioSPrn Oojevcta eNubmbb eer 4s54, i-10w4 " SUMMARY STUDY: PROS: APlot ReproducionStuywith he Northern Bobwhite SPONSOR: 3M Copan WILDLIFE INTERNATIONAL, LTD, PROJECT NUMBER: 454-104 TESTDATES: SEAtuxdypTn eerrmiSimuns raeFtenbAiFruptoearrnayro 21ly81,82290,0t2y000103,2000 Bicloict Terminalyt21i2o00n0 TEST ANIMALS: Nore bobwhite (Collis igri) AGETESTANIMALS: Approximately 30weoefkges ah nition ofthe cst SOTUESTRANCIMAELS: T2r6a8cLeePveensgsonotdyo,uned. DUsokuglaPsA 19508 NOMINAL TEST CONCENTRATIONS: 0,1.8, 6.2,and17.6ppm a.i. RESULTS: NProoimttha5en3redfboorobnw6heiwtcedswie.erseTfeohxapeoncsoensdtrotoolmPgFraOoSua1pt3 dawenicdaksr.7y.c6Nonopcoemnmetraoa.mtimonwnsteoisfnmecd1n.8,m6p.o2o,pvasnidnw1r7oe6e opvaerrtensitgnscaofmteoxicciatytweedeiteesronebadocyanWyoeifghh:e1fosd ccoonncsnarmaptioonns.o TTvheerrwweiirgehosyt CPtehoaaenrmcm1eee.n8tnmetiaroncndml.ea6s2dsThoeprdpemtu wa.eio.rneteeh5sn et0aSclsoopncypbeornftderyotatwrieoeyniasgf.mhheoesArdccvdoiantsticeoendwaaeltilegiyeh,octntttshsehtroeenw.1seer6ye rpnpoosadppiacreinet WorehdleyiendWcrEoemBpcatrineodnhteiont1hf7ee6ocdopnCptoonl0t.ugmrepoiuso,mnetnhfortrertwhoaesu.1a7.s6Tigopneipfsmicwa+nh3tt.r5erdSucegtiRomCn OinNrmomeuapenwsmhleten Citohmgpsaoaremrdtta,otlhHeilscoopnuae nhto0loouic1.1l.6eTpxonrae0m.wiaasnsoafssetei3toSeeniegthhtthmrshyeechvoenhsDnicerwipaTose orCofencceednatrtalotiotnsrefsoemrenrfto,hswBt.as0beodbwulipotnee etxhnepohsTeeedu1lf7so6POpFpO1ShantshudesS,diothpeTorn6 owmegeokshbe ais 6e3 -- GROG _--_-- EWS diLlocdal.ifoe Ih nteN rmnatiioncal, oLst. od. . Projec. t Number454-104 "9. Thi study was condocted by INTRODUCTION Wilde Inston], Li. for 3M Corporation at the Wilde WInatsercnoantdiuocnat,edLir.omaviFaenbrtuoaxriyco2l9o,gy20f0c0ilntiylinJuElays2to1n,,2M00a0r,laRnadw21d6i0t1a.geTnehrabeidstlgiWeil doreioInntoefmatthieoneasl,t LWudi. daendInceorpmyaotifotnh,efLiod.alsitr.epoBritoalroegficlaeldsupnedecriPmreenccesstoNruemdbaetr34M54C-o1r0p4oirantiaornc,hiStv.esPlaou,ctMeinonnestohtea ssi, Th abjestiveoftis stdy was to cvaOlBaItECthTeIeVffEecSts pon te adult northern bobwhite (Colin 1v0.irgPiRnOsS)) oofivtrarpyerieoxdpoofssuprero0stiemaptoctalsys6iuwmskalst oofrPe19clwueoerko.ociEafnfeecStslfoonnideuAlcedl(the,eabfoedryrweefiegrhet,d 2of2efggds colinds,mptfieoinlyw,reemvbarlyou.edv.aIbinliayd,ditiboanc,thhaebiflfy saondf. aouffliperxipnogsusruerv0ivPalFOwSeornetheevualmuabteedr, H10isetopatvholtohgeaiceaflfelectxsuapaomniatdnuolaftstseeilxepcootsenedds0uPeRsOSanadnadtonheaeofolbflfopyordinasgn.detsuse samweprelalseoussed Northen bowtie (20 mals EanXdP2E0RfIeMmaElNe)TAwLereDErSaInGdNy isbuted nt ane contol group eSnpdotnhhree rreesaulttmsnotfgroCupSs.O Tsthuedyes(t cWonicedntrIanttieomnastiwoenrael,sLie. lPireeceocnsNtuulmetbaedr 4w5i4t1h0t3he)Sapnodnsaori,nbseid o20xpipimy ain.forFmoaltlioowninpgroevxipdeerdimbeynttahle tSaprotn,sotre. teTshtemaoterrigalawaesstrceaonnacleynzerdtaontnseseleicnteadpwuerirty0ws2,7 nlowdes than orgialy reported. Therefore, the actual nominal test contation were 0,1, 6.2 an 17.6 por PROS Trestment Groups Grow NommaloCmonace)ntration PGarmopwer T Conta) 0 s MDateis"pmFPeami To 2 1s 3 62 s 5 [I roa s --_-- s 6 s s La o conan - Wildlife International, Ltd. Project Number 454-104 "10Each trstment ndconirlgroupcontainedfivepairsof bidswit onemaleand onefemaleper pen. Three rcament groups were fd dit containing either 1.8,6.2, or 17.6ppm ai.of PFOS, The control group was fed die comparable o he treatment groups, but without the addition of the tet substance Adult northern bobwite wer exposed fo PFOS at nominal dietary concentrations of 15, 62 and 176ppmai. for apeoif6oweedks. Acontrol group fednon-vesteddit,wasmaintainedconcureoly withthe came groups. Each estmentanthecontrolgroup consisted offivepirofbids, housed withonemaleadonefemalepe pea. A theendofWeek 6, adultbisinthe 1.8 0d 62ppam.est concentrations were cuthanized and subjected 0 gros necropsy. Tet birdsi the control group and 17.6 pm a. restment group were maintained on the sppropiste diets unl the beginning ofWeek 20 of {be test, at whic ime they were also euthanized and subjected to gros necropsy. Effects on adult alt, body weight, and fecd consumption were evaluated as well as efets upon egg production, embryo viability, btchabilty and ofsprin survivl forall et concentrations. "The adult binds wer absered daly for martlty, abnorbehaviorand signsof toxicity. Adult body weights were measured at test nition, on Weeks 2,4, 6,8, 10 and 1, and t adult termination. Feed consumption for cach pen was measured over a sven day period cach weck throughout the ts. Nectopics were performed on all adult birds and on 10 offspring from cach est concentration. Liver weights were corded fo al necropsied isd. Live, bile and blood (when available) and feather samples were collected at the time of neropey for possible analysis, Addiional samples oflve, brain, dn, gonad, proventriculus, gal bladde, adipose tissu, nd bursaoffabricus (when avilable were fied in 10%buffered formalinfor hisopatholoicl examination. Duintetest, thenumberof eggs idin achpnwasrecorded 0evaluate ggproduction.Figs ad in Weeks 1,3 ad 6ofthe tet were collected and parted oo shel, sel membrane, yolk and albumen and sored frozen for possible analysis Eggs aid during Weeks $ and 6 (Days 3110 37)ofth test were collected and st fo incubation. Embryo viability, bathability, hatchling heshth snd survivability were py CnGy Wildlife International, Ltd. Project Number 454-104 ue MATERIALS AND METHODS "The study was conducted according to the procedures outlined in the protocol, "PFOS: A Pilot REenpvrioodnucmteinotnaStPurdoytewcittihonthAegeNnocryth'esrnReBgoibstwrhaittieo"n. GTuhiedlpirnoetso:coPleswtaiscbiadseeAdsosenspsrmoecnetduGrueisdeoluitnleisn,edFiInFtRhAe Subdivision , Hazard Evaluation: Wide andAquatic Organisms, Subsection 71-4 and the ASTM "Standard Practice for Conducting Reproductive Studies with Avian Species" (1,2). "Test Substance and Internal Standard The test substance, PFOS, was received from 3M Corporation on October 20, 1998 and was asigned Wiki Intemaionl Li. identification mumber 4675 upon receipt. The est substance was a white powder and was entified as; FC-95; Lot 217. The est material had an original reported parity of 98.9% and had expire at the time of experimental star. An asayofthe test material afer experimental srt indicted purty of90.45%. The final assayofthe tet materia indicted a purity of 86.9% and espintion datofAugust 31, 2006 (AppendixT). The tet substancewas hed under ambient condition in Tocked storage atthe Wilde Inerationa, Li. facilis in aston, Maryland. Concentrationsofthe fest Substance inthe dietwereadjustedto 100% active ingredientbased upon heoriginalreported puriyof 98.9%. Dicay concentrations ae expressed as pars per million active ingredient (ppm 4. i the diet based upon the fina epored purity of 86.9%. he intemal standard, 4H PFOS, was recived from 3M Corporation on July 2, 1998 and was signed Wilde Intemational, Lid. identification umber 4526 upon receipt, The intemal standard was granular materi] identified as: 1H, 1, 2H, 2H Perfluoroctane Sulfonic Aci; CAS No. 27619.97:2 "The inernal standard ves hed under ambient conditions in locked storag at the Widife Internation, Li. facili in Eston, Maryland. "Test Organisms Forty-cight (24 males and 24 female) peared northern bobuhit wee purchased from Trace bPhoebawshainttcrya,pIpneca.,re2d88heLaelvtheyngaonoddwReoraed,phDeonuogtlyapsivcialllley,nPdAist1i9n5g0u8s,haUb.lSe.A.froAmtwtihledstyapreto.faTchcleimbaitdisonw,etrhee fromthesame hatch, approachingthirfirst breedingsesson andhadnotbeenused in any previous testing. Atth srt of acclimation, a random number generating function ina spreadsheet rogram was used to randomize pen assignmentfo cach bird. Immodistey ri to tst ntatin, al potential study C063 Wildlife International, Ltd. Project Number 454-104 ne birds were examined for physical injuries and genera halt. Birds that did not sppesr heathy, etherdue to injury or inability to acclimate to aboratory conditions, o were outside the weight range for the est, were excluded fromthe study. Al birds were approximately 30 weeksofag at tet initition (frst day of `exposure0testdiet)andranged in weight from 194to 254 grams at test initiation. Sexofthe birds was determined by a visual examinationofthe plumage. Identification Adult birds were identified by individual leg bands, each pen was identified with a nique umber, and groupsofpens were identified by project umber and concentration. During the test the number of cags Ind in cach pen was recorded to evaluate egg production. Al eggs collected for sampling or incubation were marked with the pen number using a permanent ink marking pen for identification. Hatchlings were identified by leg bands so that they could be traced totheir parental penoforigin. Avian Feed and Water AI adult birds snd thei offspring were given feed and watera libitum during acclimation and testing. The basal diet fd (0 bo adults and offspring was formulated to Willie Intemational, Ld `specifications by Agway Inc. (AppendixII, Table 1). The basalration contained at least 27% protein and 2.5% fat,andnomorethan 5% fiber "The basal dict contained approximately 1.1% calcium, derived from feedstuffs and the 0.9% limestone used inthe formulationofthe basal dit by Agway. While this levelofcalcium is sufficient for roth and maintenance rations, additonal calcium is required in the ration of breding birds for cg shell formation. Therefore, an additional 5% (w/w) of limestone (approximately 38.5% Ca) was added to the basal dict for the adult. This rised the calcium level in the dict for the breeding bids to `approximately 3%, slightly above the minimum recommended for quail (2.3%) and mallard (2.75%) (3). Offspring received basal die without test substance and without the addition of $% supplemental limestone `Water was supplied by th townofEaston public water supply. All offspring received a watersoluble vitamin and electrolyte mix in ther water (Appendix I, Table 2). Neither the aduls no offspring received any form ofmedication inthe fod during he test Fecd and water were analyzed periodically in `accordance with Wildlife Intemational, Ltd. Standard Operating Procedures. hore Wildlife International, Ltd. Project Number454-104 ie Diet Preparation he foa`lTedstied,ietCsonwterroel pdrietpaarnedd ebxychmiexitngePFdiOeSwienrteo aprperpeamriexd twheaetkwlaybseugsiendifnogrowneeFkelbyrupraerpyar2a8t,io2n00o0f and presenied othe birds on Toesdsy ofech week. Dietary concentrations were adjusted for purity of twheeetkelsyt ps repau ratiob anonfds taesrtt enprdea csoenntn toeldac dspiae ratersesptheorswminlilniAopnpeacntdiivesinTgIr.edient(ppm a.i.). Detailsofthe Diet Sampling Homogeneity ofthe test substance inthedietwscvaluted by collectingsixsampfolm eexcsh of he rested iesandtwosamples from he control diet on D0afWyerk 1. Sampleswerecollected rom he top, middie and bottom of the ef and right sections otfh mixing vessel. Control and restmeat groupdietsamples werealso collected from the feed trouongDahys7ofWee1k toassessstabilityofthe test substance under actual test conditions. Addionaly, a sample was cllcted from th control and treatment group diets during Week ofthe ts 0 measure est concentrations. The diet samples fwaecrileitsytfoorredamflryoszsen. or transferred immediately to the Wildlife International, Lid. analytical chemistry Analytical Method "The method usdforthe saslyss ofPFOS in visdiet wasbasedupon methodology developed st. Wild Iotermaionsl, Lid. and ented "Analytical Method Verification for the DeterminationofPFOS in Avian Dit" (Wilde Intemational Lu. Project No. 454C-110). `Samples were extracted with methanol and diluted in a 50% methanol: 50% NANOpure water Solution containing 0.0m1g01H0, 1H, 2H, 2H Perfucorociane Slforic Acid (8 PFOS; inernal sandard)L and 0.05% formic acid (v) so that they fll within the calibration range ofthe PFOS methodology. A method lw charts provided in Appendix IV, Figure 1. Conceratons ofPOS inthe standards and extracts of the samples were determined by reverse-phase high performance liquid chromatographyusing a Hewlett-Packard Model 1100High Performance Liquid Chromatograph (HPLC) with a PerkinElmer SCIEX API 100LC Mass Spectrometer equipped with a Perkin-Elmer TurbolonSprsy ion source. HPLC separations were schived using a Keystone Betasil Cv analytical column (80 mm x 2 mm LD., um parce size). The instrument parameters ae summarized in Appendix IV, Table 1 noose Wildlife International, Ltd. ue PrsjctNumber 454-104 Calibration standards of PFOS prepared in a 50% methanol: 50% NANOpure water solution containing 0.01mg040H PFOS (ermal standaxd)L snd 005% formic acd (vA), ranging in `concentration from 0.000439 to 0.00439 mg a.i/L (Week 1, Day 0) or 0.000351 to 0.00439 mg a.i/L (Week 1,Day7, Week6, Day0 and the sample re-extraction set), wereanalyzedwiththesamples. The. `same and most prompieankreespnontse for PFOS was utiltoimoznietodr PFOSinall calibration, quality control, and study samples, No step was made o quantify PEOS on the baofsindiividsual isomeric `components. Linear regression equations were generatedusing peak arearesponse ratios (PFOS : intemal ssttaannddaarrdd). vAernsuesxatmhpelereosfpaecctailviebrcaotnicoenncturravteioins prraetsioesnte(dPFinOSApp: einndtiexrnaIlV, sFtiangduarrd2). oTfhethceonccaelnitbrraattiioonn of test substance in the samples was determined by substituting the peak area response ratiosofthe `samples into the applicable lincar regression equation. Typical ion chromatogramsof low and high-level calibration sandard are shown in Appendix IV, Figures 3 sad 4, respectively. Examplesofcalculations aeprsinAeppenndixtIV,eTablde2 `The method limitofquantitation (LOQ) for the Week1, Day 0 analysis was set at 0.879ppma.i., nlculted as the product ofthe lowest calibration standard scalyzed (0.0439 mg 1)andtheaverll luton factor ofthe mtx blak sample (2000 LI). The method LOQ for the Week 1, Day7 and Week 6, Da0y analyses and the sample re-cxracion et was set at 141 ppm ..,clelated as the productofthe lowest standard analyzed (0.000351 mg 1) adtheoral dilutionfcorofthemarx blank samples (4000 LIKg). ExamplesofcalcaureplresaenttediinAoppenndsix IV, Table 2. Along with the sample analyse, four matrix blanks were analyzed to determine posible interferences, No itrfrences were observed at or above the LOQ during the sample analyses (Appendix IV, Table 3. A typical ion chromatogram ofa mtx blank i presented in Appendix IV, Figwes, Avian diet samples were fortified at 0.879, 8.79, and 22.0 ppm a.i. (Week 1, Day 0) or 1.76, 8.79 and 22.0 ppm ai. (Week 1, Day 7;Week 6, Day 0andthe sample re-cxtractionsci)and analyzed concur withthe samples to determine the mean procedural recovery. The method yielded mean procedural recoveries of 105%, 104%, 107% and 102%. These values correspond to each sample set analyzed or reanalyzed during the definitive study (Appendix IV, Table 3). Sample measured 0n0GIL Wildlife International, Ltd. Project Number 454-104 "se concentrations were not comet for th respecive mean procedrs recovery of that sample st. A typical on chromatogramof matrix friction is presented in Appendix IV, Figure Housing and Environmental Conditions Housing and husbandry practices were conducted $04 0 dbertothe guidelines established by he Nona! Reserch Council (4). The aduk birds were housed indoors i bares of pens manufactured by Georgia Quail Fam Mamufacuring (GQFM Model No. 0330), measuring approimately 25 X S1 cm.Thepehadalisng floors that esate cling eightragingfom 20 26 cm. The pens were constructedofgalvanizedwire mesh and galvanized shcting. A dingram ofthe {estlayouts presineApnpetndeixdV. Bach pen was cuipped with fod ndwatertroughs. Weekly, suficiea fed for the esting periodwasplacedin thetoughforcachpenandpresented 1thbirds. Duther feediingpeg riod additionalfeedwasweighedand dod10 he ougas nheedsed. Watetroweuregchahngsed and wter addedas ncestosprroviydepotablewater (acrcavelry 23 days). Only birds associsted wilh this study were maintained in the study room in order to avoid excessive isubances. The average tempeatrs i the adult nore bobuite sndy room during the courseofthe est was 22.3 11C (SD) with a average relative humidity of S0 + 14% (SD). The aie. handing sysem in th study room was designed to vent up 10 15 room ir volumes every hour and replace them with fresh sir The photoperiod in the adult north bobwhie oom was maintained by a time clock. The photoperiod uring acclimation and the test was 17 hows of light per day to induce cg lying. Throughout the test, the bids received mean of approximately 188 lux (~ 17.5 f. candles) of lumination provided by orescnt gh at closely spproximated noon-day sunlight Observatons "Th est bids were cclimate fo the fciliis and sd peasfo approximately 6 weeksprio (0 ininion of the test. During acclimation, al birds were observed daily. Birds cxibiing abnormal behavior or deilatng physical uri wer not used forthe test. During he study, al adult bids were observed diy forsgnsofoct o abnormal bebavir, Addins, all offpring wre observed daly CNG Wildlife International, Ltd. Project Number 454-104 "6+ rom hatching unl approximately 12 weeks of age. A record was maintained ofall morales and clinical observations. AdultBody Welght and Feed Consumption termina`tAidounl.t bFeoeddy. wceoingshutmspiwieorne mfeoarsucraecdh aptetnestwainsitimateiaosn,uroend Wweeeckksly2, t4h,ro6u,gh8,ou1t0,th1e1 atensdt. at aFdeueldt consumptionwas detebrywmeiighninegtdh fehl ld feeder on Day 0, recotrheadmoiunnt ofany addidiieoadndead dutrhewieenk,agndweighingth feederandremainingfeed at theend ofthe feeding period (Day 7). Anstcmptwis made to minimize fs wastage by the birds by sing externally mountedfeedersdesigned with a"fec-savelri".Theamountoffeed wastedbythebirdswas not quantified, since the wasted feed was normaly seated nd mixed with wter and excreta. Therefor, feedconsumption spresented asan cuimatoftotalfeed consumpion. All rensbiirdiswoegre othanized two days afte the beginning of Weck 20. Therefore, no feed consumpion cstmte vas calculated for Week 20. Adult Blood Calection A he time of adult temminton (end of Week 6 for 1.8 and 62 ppm a. groups; begining of Wek 20 for control group and 17.6 ppm a. group), blood samples were collected from al surviving binds, when possible, prior to cuhanssia, All blood samples were seputed nto serum and Hemacyesplatelts, and seum was sored fuze and shipped o Cente Ansytial Laboratories, oc. for ---- Adult Necropsy and Tissue Collection A he conclusion of the expose period, all surviving adult binds were eubanized by cerviel diocatin, nectopsied, and sored fozen. At the. time of necuapsy, tissues were collected for isopathologeal examination (04nd 17.6 ppm i. groups nly) and possible analyses. When availabe, samples of gall bladder, iver, proventriculus, kidneys, brain, gonads, bursa of Fabricius and adipose issue wer fxd i 10% buffered formalin. Histology samples were shipped to EPL in Herndon, VA for Histopathology. When available, samplesof bile and lve were sored fosen and shipped 0 Centre Analytica Laboratories, nc or possible analysis. Any reminin issue not fxd fr isopabology and esther samwperletorse froforpzoteentni analysis. noe Wildlife International, Ltd. Projct Number 454-104 1 EgeCollectiaonnd Storage Eggs id uring seven day period begin o the sccond day of Week ofthe test were collected dyfom test ens and stored in cold roo util ncbaton. The cold room was mainsioed 10meantemperatoufre 131C 2 01C (SD)with amea elie um of proximately 72% 4% (SD). Groups of eggs were denied by an alphabetic It code. Al egg ad during the week were considered ss one ot. Candin and Incubation Abe cndofthe weekly iteval, eggs wereremfoomvtheecdodroom,covated and candied witha Spesd King (ode No. 72)cg <andin lap to dete ca sel crackso sboormal gs. Cracked or abnormal eggs wee recorded and discarded. All es to be incubated were fumigated with formaldehyde gas in an aight cabinet withcirculatingfn or approximately two bo, reduce the possibilty of pathogen contamination prior to incubation. Formaldehyde gas was generated by combining 26g ofpotassium permanganate and 25 ml of 37% commercial grade formalin porcelain bowatthe aseofthe srigh caine Al eggs not discarded wer placed ina Petrsim Incubator (Model No. SP20). In the incubator he tempevrasatsuirane tan avers 37.5 00C(SD) withan verge we bulb emperatr of 306: 01C (SD) Gelaive humidityofspproximatly 60%). The icubstor ws quipped witha pustor fn and blades that produced mild breathing ir movement designed to liminate inracabioe empersture and midi virion durin incubation. In onder prevent adhesoifohne embryo othe hell membrane, he incubator was iso equipped with sn automatic cg rotation deve, designed 1 rose th eggs from 50 oof vetial in on diction to O of of verieal i the oppsic direction (ttl ar of rotaon was 100) very wo bouts (ough Day 21 of incubation. Eggs were candied on Day 1oficubsion to determine embryo iit andon Da2y1to determine embryo survival. Hatching and Brooding On Day 21 of incubsion, the cggs were placed in Petrie Hatcher (Model No. 6H) and allowed to hoch, Pedigree basket consicted of galvanized see wire mesh were usd keep batching scpared by paral pen of rin. Eggs were no rotated in the batcher, The average \emprstur inthe thing compartment was 37.2 + 00C (SD), ad the average wet bulb temperature was ised 103.3 00C (SD) Gelative humidityofspproximately 7%) 0061 Wildlife International, Ltd. Project Number 454-104 "Al htcbling, usbatched ggsan, cg shellwereremovedfrom te baicher on Day 25 or 26 of incubation, The group body weightofth surviving hatchlings by pen was determined. Hatchings were leg banded for dentition by pen of origin and then routinely housed according to the appropriate parental concentration grouping in brooding peu nil approximately weeksofage. All haichlngs were moved to large flight pens, wher they wee housed approximately 7 weeks. The hatchlings were fod treatedditwithouttheadditionof 5% supplemental limestone. At approximately 12weeksofae, the average body weight by parental penofall surviving offspring was determined, and the birds were. cutbanized with carbon dioxide snd disposed ofby incineration. Thoseofspring selectedfor blood and tissuesampling wer stored frozen following necropsyand ate dispaosfebdy incineration Hatchings were housed in baterisofbrooding pens manufactured by Bescon Steel Company (Model BT350). Esch pen measured approximately 72 X 90 X 23cm high. The extemal walls and ceilings ofeach penwere constructed ofgalvanized wir mesh and galvanized shetio. Floors were of galvanized wire mesh. Thermostats in the brooding compartment of each pen wee se fo maintain 8 {tempoefrspaprtosuimrateey 35C romth imeofbatching unl thebirdswere approximate3l0y days of age. Brooding was discontinued once hatchlings were determined to be of sufficient size 10 thermoregulte. The average ambien room temperature in he room housing brooders was 28.6C + 10C (SD) with un aversge relative humidity of 39% + 8% (SD). All hatchlings were removed from broodingpensand transfered to flight pens at approximately weeksof age. Each flight pen measured approximately 1.2 m X 1.2 m, with ceiling height of approximatcly 1 m. External wall, floor and ceiling ofeachpenwereconstructed of wire mesh. Th photoperiodfr thehatchlingswas maintained by atimeclock a 16 hoursofigpehr dtay. Offspring Blood and Tissue Collection Prior to cuthansiaof the offipring, blood samples were collected from 10 offspring in cach ncatment group. All blood samples were separ ino serum and hemacytespacls, sored frozen, and shipped othe Centre Analytical Laboratories, Ic. for posible analyses Additonal, issues rom the 10 offspring in each group were calected for hisopathologcal examination and analyses. When availble, samples of gallbladder, lve, proventriculus, Kidneys, brio, gonads, bursa of Fabricius and adipose tissue were fixed in 10% buffered formalin and shipped (0 EPL in Hemdon, VA for Histopathology. When availble, samplesof bileandlive ssowerestoredfrozenand sipped t Centre Analytica Laboratories, oc fo posible analysis Cane: Wildlife International, Ltd. Project Number 454-104 "19- Statistical Analyses Upon completionofthe test, an Analysis of Variance (ANOVA) was performed to determine snsicaly significant diffrence between groups. Duanet's muliple comparison procedure (5,6) was used to compare the thre treatment means with the control group mean and sssss the sisal significance ofthe observed differences. Sample units were th individual pens within ach experimental group, except body an liver weights wher th sample unit ws th individual bird. Percentage data were amined using Dunn's method following aresine square roo trnsformation. Twa sts of tatsticalaalyses were conductedwiththe bodyweigh andfedconsumpion data. Opesetofanalyses onylooked atbody weightandfeedconsumptiondatafomthefirst 6weeks ofthestudyandcvalusted llthree eaiment groups. The second set of analysis only cvahated the control and 17.6 pom a. eaiment groups aad examined dua for the ful duration of the study, 20 weeks. Dummet's multle comparisonprocedurewasnotconsidered spproprsetocomparthecontol group 08singlewrestment group. The suden's Test was used to make satis! compass in those instances where nly the contolgroupsndthe17.6ppm i. tregarouptwemreecomnparted 1. Adult10B,o1d1y, Wnedigahtta~dulItndtievrimdiunaaltiboon.dy wSeaiigshtticwaalscmoempaasruirseodnsat wteesrteinmiatidaetiobn,etWweeeeknsth2e, 4c,o6n,to8l, group and cach treatment group at cach weighing interval by sex for the first 6 weeks. In `eaaddtimtieonn,tgsrtoatuipstifcowaleceokmpsar8iso10n,s 1w1e,r2edm2a0d.e between the control group and 17.6 ppm a.i. 2. ACduoltnFsedumpion Feed consumption expressed ss gramsofeed per bid per day was examined by pen weekly durin the tes. Siaitcl comparisons were made betweca the control and each restment groupfwo eeks 1 through 6. In adiion, statistical comparisons were madebetthwecoentreolannd 17.6pm. tregroaupftorwmeekes?tnhrotugh 19, 3. EggsLaid~ The oumber of eggs lid per female per rstment group. For the valuation of eproductive performance, data takenfrom week eggs se. 4. ViableEmbryos -Thenumbe of liv embryos determineadt Day 1 bycandi. 5. ECggsraocfEggksLaei -dThe number ofeggs determine bycandingtobe cracked divided bythe numberofeggs id,perpen. 6. VEisembrofEyggsSoet~sTh oumberofembryos atheDay 1 caning was divided by the [------ 60006 Wildlife International, Ltd. Project Number454-104 20- 7. Live3-WeekEmbryosofViableEmbryos - The numbofelirve embryosattheDay21 candling was divided by the umberof viable embryos,perpen. 8. Hatchlingsof3-WeskEmbryos --Thenumber ofhatchlings removedfromthe haiche was divided by the umboeflirve 3weck embryos, perpen. 5. 14:DayOdSurvivor ofHatching - The number of 14-day old suvivors was divided by the numberofhatchlings per wesk, by pen. 10. Hatchlings ofEggsSet ~The numberofhatcblingswasdividedbythe nuomfebggsesetprer week, by pen. 11. 14:DayOldSurvivorsofFigesSc--The numberof 14-dayoldsurvivorswasdivided bythe umbofeegrgs set er week, by pen. 12. OfsfursvivporrBs owiedrnyeWgmeei'agshsutre--d bTyh epagrrotusp body weig pe group. hts of surviving hatchlings and 14-day old 13. LiverWeigh - Individualliverweightsweremeasuredat adult terminaatniosntthetermination of offspring of week 5. Statistical comparisons of adult liver weights were made by sex between the control nd treatment groups. Juvenile liver weights wee compared by test `group, without regard to sex. RESULTS AND DISCUSSION Adult northern babwhite were exposed 0 PFOS at nominal dietary concentrationsof 18, 62 and 17.6 ppm .. for a period of 6 weeks. A control group, fed non-treated diet, was maintained concurrently withthe treatment groups. Esch testment nd the control group consisted of fv pars ofbids, bose ith nemaleandonefemaleperpen. Attheend ofWeek6, adultbirdsinthe 1.8and 6.2ppm a. est concentrations were cubanizd and subjected to gross necropsy. Test birds in the control group and 17.6 ppm a.i. treatment group were maintained on the appropriate dicts until the beginningofWeek 20 of he test, at whic ime hey were so cutbanized and subictd 0 10s crops. Analytical Results `None of the control samples showed any indicationofthepresenceofthe test substanceo ofthe presence of co-eluting substance a the charscerstic etnton meofthe test subsance (Table 1). Diet Samples were coleted fiom the 1.8, 6.2 and 17.6 ppm a. test concentrtions on Week 1, Day 0, and were analyzed to evaluat the homogeneity ofth test substani the dict and to verify tet substance concentrations. Means and standard dvintions orthe thee tst concentrations were 1.8 0.13 ppma. noeI? _ Wildlife International, Ltd. Project Number454-104 a 6040.65ppm ai. and 17.6: 1.46pm a, respectively. The coefficientsofvariationwere7.2%, 11% and 83%, respectively. These values represented 100, 97 and 100% of nominal concenations (Appendin 1V, Table 4), Samples callcted during Weck 6, Day 0 ofthe tes 0 very test substance concentrations frthe 1.8, 6.2 and 17.6 ppm i. dicts bad means and standard deviations of 20+ 0092 ppm ai 60 0.67 ppm a. and 163 0.p6pm0s8i, respectively. Thecoefiofcvariiatniontwerse 469%11%and3.6%,respecively. Thesevalesrepresented 111,972nd98%ofnominalconcentrations (Appendix IV, Tble 5), Analysisofdit samples collected from feeders afer being helda ambient {empfoer7draysaavteraeged 100%,103%sd 98%oftheDay 0valuesfrthe1.8, 62and 17.6pom a. test concentrations, respectively (Appendix IV, Tab ). A typical ion chromatogram ofa st sample is shown in Appendix IV, Figure 7 Mortals and Clic Observations Noadultmorales oecurdinthecontrolgroupofin nyofthtcamentgroupsduring the course ofthe test. Several birds wer ned with bead foot lesions and feather los 8 result ofpo wer ado peomate aggression during he courseofthe est. An incidental clinical sig, lameness, was sociated with the injrics All othr bids were normal in appearance ad behaviorfor the drsion of detest Adult Body Weight When compared tote conol grou, ere Wee no sppaset etme related fects pon body weight at the 1.8 nd 62 ppm a. test concentrations and any diffrence between he conrol group and those two ratment groups were not stisicaly significant st any of the body weight intervals. However, here were eatment.related eduction in mean body weight for males at the 17.6 pom a. rtm group. With he exception of the Wel 2 body weight interval, mean mle body weight a he 17.6 pom ai. test concentration was satisialy (p<0.05) diffent from th control group atal ther intervals (Weeks 4, 6, 8, 10, 11, aad 20. For female body weighs, none of the diffrence observed between th treatment groups and he control group were siaisialy significantfo anyof the intervals monitored durin th study. Mean body weight measurements st presented in Table2 and iodividual Body weight messrements se presented in Appendix VL. Anos _--nm---- Wildlife International, Ltd. Project Number454-104 2. Feed Consumption HoweverD,uewheton ecxocmepssairveed1wasttahgeecobnytoslomgeroubpi,rdsh,efeeedwercoensnuomtpetaiomnenwtaselvaatreidabelfefebctestwuepeonn. pefnes.d consumption at the 18 or 62 ppm .. text concentrations, While satistialy significant (p<0.05) iffbertweeenthnecocntolendsthe1.8 ppm a. test rovp werobserved rigWeek nd ofthe sty, these ferences were not considered etme relateddie 0 the ack of concerto: sponse eltoattnhesgrehateridops. Ath 6.2ppmi. test concentrationthers was satay iia reduction in fd consumption during Weck 4 and Weck 6 when compared 0 th contol group. However, thes difrcnces were nodos responsive and the significance ofthe spparent reduction ot Week 6 may have ben a consequenceofte fc that there ws a relatively large icras in feed consumption thcontrol groupat his meintervalascompare fothtobservedst Weeok7. Thus, at Week , all reatment group fed consumption tes were sgaificantly reduced from te control level ut were actully higher thanobsera vWeeedk 5. As eslthereductionsinfedconsumptait6on2 PP a. were not considered to be rcamentcated. Ther was a resimentrelated effec upon fd consumption st 17.6 ppm a. et concentration when compre to te conto group. The eduction in feud consumption at 17.6 ppm :. et concentration was consistent roughout he sody snd satstically sigifcant (<0) fom the contol group at Weeks 2,3, 4, 6, and 15. Mean food consumpion measurements ar shown in Table 3, and feed consumption measurements by pen are prescaied in Appendix VIL Gros Necropsy AecodofWeek 6 (Day42),al aol bid in the 1.8 3nd 62ppm .. resegrnoutps ere euthanized and subjced (0 gross necropsy. Adult birds in the control and 17.6 pom a. restment upsweremainainedoth appropri diets nl he beiaoifWnecgk 20 ad werthe cubnized and subected (0 gross necropsy. When compared to the control group thee was an increased inidence in th number of males with smal tests in the 17.6 ppm a. group that was considered pasibly ream lated. All ther findings observed were considered to be incidental to eatment. Necropsy findings re reported in Tb4sadlAppeendix VIL Study offping were appoximatcly 12 weeks of age at the ime of cubanssia, A subsample of 10oni birds or cach et group were subjced to ross necropsy. Necropsy reveled one to tree binds in ach ramen lve with some feather los. Additonal, a single bid in the 6.2 ppm as. roo a _-- Wildlife International, Ltd. Project Number 454-104 a rcament group was noted with sbrsionson the ump. All findings observed were considered to be incidentaltoreatment. Histopathology Light microscopic examination was pecformedonseedtssusby sboardcertified pathlogit at Experimental Pathology Laboratories, Inc. Herndon, VA. Sections of liver, brain, kidoey, gonad, proventriculus, gall badder, dipos issues, and bursof Fabricius (when availabe) wee collected fom `eaxdaumltintaetsitboinr.dsanNdo frleosmio1n0soffcsopnrsiidnegr(eadppproosxsiimbaltyelrye1l2iwedeektosorledtamteenutthawnearsiea)nfortoemdeaicnhtlievsetr,grKoiudpnefyo,r proventriculus, gall bladder, ovary, brsn and bursa of Fabricius of adult male and females, or their offing at any of the concentrations tested. Additionally, thre were no lesions considered {0 be reatmentrelated ote in adipose issue of adult maleso female and offporfibnog sexes, or i the testes ofmale offpring Teteoftwo adult males at th 17.6 ppm ai. test concentration exited decreased seminiferous whole diameter most consistent with postreproductive phase regression, a normal physiological Phenomenon. The occurrenceoftesticular regression or alt males ith 17.6ppm .. reament group may be incidents] to reste, but rstment elated ffct could nobte precluded. The full pathology reports proviniApdpeendidx IX. Tisue Analysis "The analysisofth egg, blood and tissu samples cllcid during the study were conducted by Exygen Research (formerly known ss Centre Anlytial Laboratories) and are reported sepraely. Blood and liver analytical results ar reported in "Extraction of Potassium Perfuorooctaneslfonate from Quail serum snd qual liver or analysis usingHPLC-ElectrosprayMass Spectrometry. Centre Study Number 02341" The cps components amlytcal ress are repored in "Extcton of Potassium Perfuorooctanesulfonate from Egg Membrane, Albumen, and Yolk for analysis wing HPLC ElecrosprayMassSpecromery. Centre Study Normber 023-065". CROG:0 _-- Wildlife International, Ltd. Project Number454-104 4 Reproductive Results Thre were no apparent resment elated fects on egg production at any ofthe cocoons Tested, While egg productionwas highly varibleamonghens,g productionsthe 1.8, 6.2o 17.6 ppm 8 tet conceniratons was comparable to or exceded he coirol group. Mean cg production by concentration presatedin abl. Individual ggproduction aaa presenteidn Appendix X. When compared tothe control group, ter werenoapparent resented effects on embryo abil, batchabiit, bachling esl and survivability a the 18, 62 or 17.6 ppm a. eels. While there was 2 sigificant (p<0.0) reduction in te number of viable embryos at the 18 ppm a. test concentration, th reduction was du 0 fowe gs beng set fr ocubaion at is evel, Pn P207 ofthe 1.8 ppm ai. evel did ot aydurinte wes ta gs were collected for incubation. As esl there wee fewer htclings snd 14-day od survivors inthe 1.8ppm ai. eam group. However, when te reproductive endpoints were expressed 15 percenage, no saiselly sigaifican diffrnces wee observe at an of the conceniraions ested. There was slight reduction in embryo vibilfsy at te 62 pom ai. test concenrtion when compared o the control. However, his reduction was not dose Rreespproondsuicvteivaenddatwaafsorinontdivciodnusaildeerends treaptrmeesnetn-treeldatiendA.ppeRnedpirxodXuc.tive data are summarized in Table 6. OffsprinTgheBrodwyerWeei5g0htaspart trstmentead fects up th body weightsofbachlings in any of the estment groups. There were also no appareot samen related effects upon the body weight of eabidse inth 18, 6.2 or 17.6ppm ai. restmentgroups. Ofspringbody weightdtispresented in Table7 snd Appendix XL Liver Weights When compared to the contol group, tere were 10 ppareat trcmentrelied effects on male adultliver wigsthhte 1.8 or 6.2ppami. stconcoonefemnaleardultiFiveorweinghsasan,yof theconcentrations tested. However, thre was a statically sigifican (p<0.05) reduction in man mle adult vr weight tte 17.6 pm i. fest concenrion. Adal males i the control group had a mean liver weight of 3.402 grams compared 1 a mean lveweigh of 2.527 grams for mals at the 17.6 ppm a3 test concentration. When compared 10 te contol group, tere were 7 apparent ramen clid ffs on juvenile liver weigh a anyofthe concentmions tested. Mean adul ad offspring liver 0061 _------ Wildlife International, Ltd. Project Number454-104 25. weighs ae presented in Table . Individual adult vr weghs are presented fn Appendix XI Individual offpring liver weiaegprshenised in Appendix XIV. concLusio Norther bobwite were exposeto PFOS at dietary concentaionsof 1, 6.2, snd 17.6 ppami fo 6week. The contol groupand 17.6pom .. ucatmentgroupsweremaintainedonestdictsfo an additions 13 week. Notreatmentrelted moraleso overt ignsoftoxicty were observed at any of he test concentrations, There wee no apparent resented effects on body weght, feed consumption, oliver weightat he 18 and 6.2ppm a. tet cocenitons. Additonalterewere 0 appent ncamentelated cffsis on female body weight or liver weighta the 176 ppm ai. test concenation. There were mo. apparent ream clted effets on any repoducive parameter measured ditnh gdy or ny of he concnttons ested. When compared to the control groupthersws significant reductiioan mean mae bey weight in the 17.6 ppm 8. trestment group. There was a significant eatmentelated reduction in food Consumpion fo the 17.6 ppm. tment group when compared 0 he control group. There was alsao Significant reduction in ean ver weight for adult moles a he 17.6 ppm a. raiment vel tht may Have ben rested to the ratment. Hisiopstological cxaminton of selected tissues lo revealed eegtgedssiotneofatemsti,cularBtaissesdu fuopontwtoheadrulst smaolefs hiinsthseud1s7,.6tphepmmoa-..bsefrsvtegdr-ooufpechtatcornoscyenhaaivonbeocx aonhernbobwhit exposedtoPFO in he dietfo 6 weekswas 62pma. [nr _-- Wildlife International, Ltd. 26. REFERENCES Project Number 454-104 | UESuSnbv.divriEosnuivmoienrooauElm,ePnoHttaaczleairPodrnoAtEgevceatnlicuoaynt,ioOAng:efncfyo.WfiiPdeis1ctf9i8ec2ie.daenPdPreosgAtrgiaucmiasdt.ecWAasOssrehgsiasnnmgieoonnnts,,GDuCiSdu.eblsiencetsi,onFI1F1R2A4 2 ARmeeprriocdauncvSeocSiueteysfowriTteAsvtiinagn SapnedcieM.ateArSiaTlMs St1a9n8d6a.rd ES1t06n2d.6r6d. PArniicea oBorokCoonfdAucStTinMg `Standards. Vol. 11.04. PhiladelphiPaA,. 15pp. 3 Mer&cCoku lnc. 199. The Merck VeterinaryMail. Mer&cCok.Rabay, NI. 1832 pp. 4 NaWtaiosnhalinRgesoeDnaC,r.chNaCtoiuonncsllAcad19e9m6y.PrGtui.de13f5o7r.he Care and UseofLaboratory Animals 5 DwuminleatC,onCrWol.. J1o9u5r,5.AmAer.MuSltaitpsl.eACsomepar30i:so1n0s96P-r1o1c2e1dure for Comparing Several Treatments 6 Duoninett, CW. 1964.NewTabes for Muliple CompawritihsaCoontnrosl. Biometri2c0s 482- C2062 Hs i . ii Pk sal ii Bl Ii "lHaisg 3f 82 85 z 28 33 oB ose oes Pilial vos = 3@ Hii ii gk i tH ze fp lihy = fil:l Ii noo _-- Projst Number 454104 fa] 2m gs co 0 Be] eee Eega eas f=] sms von 0 ge ge $85] meee i 1- gems ro | ge ge |] i Ie Ten rin em ee|} iss is Te ome lr sae H fof 27 8% 3 lds] omen #7] 8 8% I TN Ge I~] 8 &8= IH] Te mm fo 2" 8% 5 cee vn sg ov ee z= 3% gzas smesees meeaeee 5% HF R RR em omc name 5% 8 g= 3% em ce %% 8s x8 "8 me oar g gc x8 xa fe owe |} kegx|f ieas 1A ene i . i 2" = 2 ge ee iif g e ii be -w g= = x8 x8 $2 i $1; | {RFE SETI 62061 2 ae ra ge i d= n Loon is || a g4 i i 3 - se 11 11 Re > a" tr 1 ge ol ae Ee Re ae | - a B+ an ae | FE " R&R ge Ee 3 1" =a 8ooha= fi 8 8 ge ge itl Bl 3st oe oa Gn0G8 {fh | j ji 2 fi i glnececenin Pee fos iiAtNT] GnoG 7 -_-- roe0 HET EEE g| == 0 gaa stl af 0c 0 rE 5% 2] ge- ti 0 ge BH E ae no n n e ii FAS1 ER fEeWf l ga em a o1c o0 oim n am gmgeeeeee Sf loge i in ge- PBF ose 22 os ae H dI iN aE mR e "| Rem Re- me- gens ml ae ge- geo ge"ff 8 Rem zen ge Hs 2s 2e Be Sl mee mes gee geo 1PIEz =ii e3 eded y i fy i8 i ii fot i HE BR] Ii ici sei i [313] TEE G06 ------ "32. `ProjectNumber 454-104 Table 6 `SuomfRmepraodurctiyvePerformance(EggsSetfromWeek 5) from aNorthern BobwhitePilotReproductionStudywith PFOS --eeee --_R--ep_r--oductive Parameter E Ceoentroo l Oppu mai) i18pm pmaem T62phpl mael |GTo 7e6pommw al NumberofReplicates EggsLaid Eggs Cracked Eggs Set `Viable Embryos Live 3-Week Embryos Hatchlings Offspring Survivors: s s 5 s 2 8 2 * 0 0 0 2 1' E u ne 2 El = " 2 n = 1" 2 3 n 7 E) 2 EggsLaidHen Oftspring/Hen 6 " 6 7 s 3 + s Eggs LaidHenDay 083 ok 08 097 `EggsCrackedEggs Laid (%) 0 0 0 Visble Embryos/EgSgest (%) 100 9 % 100 Live3-WoekEmbryos/Viable (%) Ed 100 100 0 `Hatchlings/3-Week Embryos (%) 100 100 100 % Offspring SurvivorsHatchlings (%) ~~ 97 100 % " Hatchlingo/Eggs Set (%) 7 9 6 9 OffspringSurvivorsEggsSet(%) % 9 n HatchiingsHea/Day 080 049 on 089 Offspring Survivors/Hen/Day om 049 057 on --_-- ------ * Sutistc-- llydiffrent-- fromtheco-- ntrolgroupe stp.15. ee 61069 B= ne Project Number 454-104 ) "!. Bp :lle : : ccoii | ii Er qa = i IEEEi2 | 3I i CH 2 wi [A ii ] if il Conese _--rr-- i i Clos 6 os Jblg Il3hel S0 , le HY g = os 2&5 g38 3@S { i 2 8 2| 0i PPplEy ord os 8g I [Il g 3 in i 13 i! it wd TE i CE EE4 Hi i: GROGSL ss. Wildlife International, Ltd. Project Number454-104 Appendix] Cerificate ofAnalysis a fonaenAnsitienlLai boratord esbe CIoNnTeAERvIyM CLaEborRrTonCIOsAFROIFmACsNA:ALY2T5S0IE1S84 oe P MeioesinToskanLia e Mower L 33? YSiommrin :i 3 3 L1 ooarsmsee SSSraeen?ei -- e=try i hm abe | pgm 1 Shon i sJmuno T3E Ema | iai3 tSim emmms) : | jiL= Tr C=muabexr. LPIPI. oomTmhemamomrVmneaelutem i=en1c.7a.0l% |||| b&lf1 2sewasawAsdwmm% -- J choos _--_--nmm--m-- 36 Wildlife International, Ltd. Project Number454-104 ApPpaeandei2x] CerificateofAnalysis fo CentrreaAnmal:ytical La aboratories.EInc. CoIntNreTAERyIaMlCELeRbTerIiFInCAGsTOEROeFeAcNeA:LY2S3I01S85 Dacor Anais: 043100 Binion De. cng SugeCoditon,Bram 10 Ress De: U1 Ea roiCty1E0% Nmfemimp(i,m45%dLISi puto 1TORp R ToEtprityaosm luer= 1e3.07% "romieni pct ikom Brestord my. Pauyba yDSCin sgroly plicitmalopy. No darvas P ii ntr teeblee eSseeoe esansgowmeblg5b 0edbpdceterwrien. o ri eS. os ANThAs NpTaaetreabrmosit TIA reseed cetvi ctor ednecp minISOCOWS) i ork aoda crBAGndLubomirrc Suds(10CTR160) rv. rates GRNGRTE --_--_--mm 3. Wildlife International, Ltd. ProjectNumber454-104 ApTpoeesnd CaritofcAamtyes Centre Analytical Laboratories, inc fe a COre] CoInNeTAERsIyMaCaEbR oorTtnCIsOAFROItFAC eNA:ALYT 5S1IE8SA Losryt E ar S m ------ em am oo tw CT sr Nok: Th 4mrteae etievesr cBahs ae iencCoBedeo sd rtvcomaeh 8rsn Pro or LpLm lle Sewiriatiuoie ver HClShrdFlgee,o]reAvr Lorie& hy como rrr 005 _-- as. Wildlife International, Ltd. Projct Number454-104 Dict and SuAppplpeemneinct F1ormalons Tae 1 Wildlife International, Ltd. Game Bird Ration" oREDIENTS Foe Comet WP`nhSroayeoByceaSnnouMieelal,, 48% PPrrotieinn CDrremWLheeeysse2n0e rin NErVaoianiCnduoinid+Pirnid (ee low) Soults GLFerm (Fermatco)' rence 00 us 503065 i3So%oo ooasns 0% no0oi2e5 Vaiamien odMlienevrigPsrisa,whrenied 032% he tion, AreSuplid PrTn pVVirianeeinadDas 2T0ommo01r0n0s RFVoiaanimneBr Acs ooSoopemm FBPoordene oSoSinnoe m iVViitaerinynneK MeadeDimeiypyrinidocBini omiS1tS3,aanms NCaaonmger 0p2fpino=w Sforonneim r5Iepsmom * TFRnmeeeptaironeBdy lysisi rcs mii of274 pri, iio of25% Soo fUide enid Grow Faces. re iand ia of re Gonos _--_-- 3. Wildlife International, Ltd. Project Number 454-104 Dict and SuApplpempenetlFormltions Vitamins snd ElTcabtloely2esConctate GUARANTEED ANALYSTS VVViiinnaimnnnaED RPNiiaoatnaavbiinc Acd FVMoiSaeBmACciindg12 TFRhiaorcinoneecHiyLdbohiride Water Sule Powder Perdoz 250r0e0"0r0000 215209 L00]02s 37535%0 Batlb, 1"0a0o"o00o11o c0u aS500o0000e05 Ag2t06o00v0i5% 1fL50r00a00vN0gd5 wVVioirRiEinDIA)E.SNuRTpiSpbol:felmaevnin,DS-Apcekievnee,d CAnaimlalteaoPanisetceo,f VNicnsS)o,plAelpih,a TVoicaopihnerBy1A3cSepslcuoeen. of iMCehncidsoanNmcS:oDdoSormoeia.nCeos(o.utCsoolfoVmiCmh)i,hFMoecgAac,nsSoen PCehs oFatdoo FCdoocehoFreo,i TMChneeatcNaGmeiPRaaOnsdCpEcrDslUiRomEit:lymi| aalsoroaesd ion ofsprint grofaDuvmet iain sd 0656 --_-- 0. Wildlife International, Ltd. Project Number454-104 DiAcptpPernedpiarxat1ion 19,200P0.reNioxmeisnafolrpPrRepOrSatwiornewparsepsarfeodlloonwsF:ebruary 28, 2000; Apel 7, 2000; May 12, 2000; and Jue Cont: No premix required. ISpmais 03084 PROS + 60997 graion G2pmai: L0794+6g09P89gRrOaiSn DSppmaii 3089 gPFOS + 60969 gration rantBeadsatlora8tiWoanrwiansgwebilgehndeedr.inTohestatersetdsuHbosbtaarntcemiwxaisngwebiogwhled iAnptporoaxairmeadtewleyig1h00bogoatfarndo&nsmwaalsl mloerntdaerwaansdttheaweki0gcehrbunosahtawnaysrrcisnsoedfchoenesltidiamteisonw,ihTrahtioensftrsoumbsthaencmeiwnasg buonwsl,ewriethotheheriWsarbinegn amdidxeidng10botwe.bleTnhdeer.bleTnhdeerbweansderincsoendtewnittshwuenrceonmliaxmeidnaaipepdrtooxinmaftoeomnleyteabtoew,anwidtha(hne isse fboeitnhge rapeptruomxeidmattoeltyhe15bomwiln.es.TheThbeowplrewmaixs aplsacewdeiagnhed8 iHnotboar1t000m.i0x&,aalnodtt,h laCcoemderisn awpperroeprimaitxeeldy Iabeld recaer bag, reweighed, and stored froze. As needed, the appoprine premix was incorporated ot th final dias lows: Oppmai: 2375kgrtion + 1250 ke limestone Leppmaic 1000gPremix +2275kg tion +1250kelimestone G2ppmais 100gPremix+ 22k.gra7io5n 250kg limesone 176ppmai: 1000 remix + 2275kgration + 12kgli5mest0one The iets were mixed for spproimately 20 minutes in PatersonKelly Twin Shel Blender. ---------- a Wildlife International, Ltd. Prsjct Number454-104 Append V The AnalysisofFOS in Avian Dit CN0GEY --_-- "a. Wildlife International, Ltd. Project Number 454-104 ArrENDIXTY ANALYTICAL METHODS AND RESULTS TABLES `Typical LC/MS OperationalParameters STRUMENT PH+PeeekrikeninlEPeavceskarTrbdSoColIdoEXneSApP1Y1e0y010oH0inLgsChuMPreest,oSrOpanepcseemLodmitdcSChqheurpsemeadotnwoghapah wih onsorngmde GI). ANALYTICALCOLUMN: Keysos Bes Cy clu 50m 2 LD, 3m parice ize) ovenTEMPERATIRE: 20C sropTME: soomnues Flow RATE: 0220 mimioue MOBILE PHASE: 72.0% Methanol: 28.0% NANOpure Watercontaining 0.1% Formic Acid INECTIONVOLUME: ~~ Sout eRrEoTsENTION TIME: Approximacy 36 mints RIENTTEENRTNIAOLNSTThAEN,DARD Approval2 mins NrOoNsITORED MASS: 43m6amm MINOTNEIRTNOARLESDTMAANSDSA:RD 42673ms 0059 _--_-- . Wildlife International, Ltd. Project Number 454-104 ArrENDIX TY Tame: caLcuLaTIoNs "The concentrationofPFOS found athe instrument was detrmined sing the following equation: 'PFOS (mg 8./L) at instrument = Desk are t= i (ynAMEIeCEDO)rept internal standardconc. (mg 8/. 1) DeterofmSaimplneReasitdueis (oBPFnOS) The concentration, expressed 8 pin. or ach sample was determined usin te following equation: PROS (pm ai1 iinnsammppl=ePFS(5 8571) at nir,extrtac nltovnoelume (1) (x ditionge ter DeteorfLmimiitofnQuanitaioin oL0nQ) "The method LOG, expres s ppm .. was determined usin theFollowingequation: LOGQ (ppm .. = lowest standard concentration (ng ..L) x over lation ctorofmales blank ovriio foraokpeS la 0dL a ce -- The ppm .. measured in cach sample s divided by the nominal conectionofsch sample (fortified eel, ppm. This ati times 100i the percent recovery ofthe method at hat eve of friction. Boma messed sere %Racavary: `ppmai. fortified * 100 ROvGe : i! ~ rssoe ||] i i ; : i Hi HEE (23% .288 e8s3p3 X|2} ii: 1 i13] fi % J exes fag aZsegdy fIaan 1 IEsR ` Jf i 85 B| ji]l, 1] Ei eaer eeatzs ened mand iii |} ii | | g* f $i ii Ca) | sE2322 535% 3338 BE ny g1 : ii e E bf 1 R i lil : 5 5 iy i bt | IE 3 HE ii ii fi *333 3 oY il g| Hi H8EF2E3R3E3 H333EE3E33833 31333323 |i: g3 | HHai [hiner -- 32 i 1 bl 8 5 2 ] i : : 8 4 i +1 | il od | | ow wg | ii i1f5 2g3esareRRsy ffa8g8gshkysE ggomgezzeasn |: Hy i | 33] 5auask qpuned oanisy |G |CE AE ii 3 3 8 i menere agzmme senmes 3 fi | I i ii z Bil = 3 2 i =| [iL TUS a "s6. Wildlife International, Ltd. Projct Number 454-104 CoGNnceeoyrn! o rrENDIIY TALES saee ee-- eeSe em-- RTiE s -- eee ecwer)eeeeememeoamPme t cDasNio daulty ey Co"mGMiEn? TP coSae lnde e tonnd , omae :: :\ <aonm a . oi `: <ia a . fn ss : . bien - i8" ::. pi1i5s]0 PioxtVtromEeamst un a sae : men " "o2 .`` 3oan xB-wcooepo " ns 10 : . Getic - 5ai ::. ji1fsd oPCpTaetl --C1o12-- m01m, iN-- sowteetccrceoeocr aoenieeeodcspeon hne t psyhReP a7 y 14G ,631 176ps1s. dGo J Liein noine tacponacent nofoposgA on p(0e0) , 0027 ------------ 2 i i| bs C8 8 2 i plc ee | ii5 2 ils " + i : Ii 3 Lee kaw zag A gE CCE3% Ea,(8 e | HE | f Herre = 3 BERET 3 Z ] ; | = Tr g ifi: 2 ee fiiH54 i os ili Hil all 3 3 jegl = fl fa CNNOGA _--_--mmm a. Wildlife International, Ltd. Project Number 454-104 APPENDIX IV METHOD OUTLINE FORTHE ANALYSIS OF PROS IN AVIAN DIET Prepare mati friction sample inthe desired avian eed sock using the ey mi echniue. 1 Weigh 10g sarmaosfttpehretmloa8t-ie0 lFasrekn,chmsaqtuairfergiacstiboonsa.ndRteecsotrsdamwpeligehstis.tweighboss nd 1 For cach sample, measure 100 mLofmethansoqluawriethbogtr.adusted cylinder and rast into he French + (Cop bole and place on shaker abe.apAprlooximhaeclsya2l5e0s1p0m.shake ora misma of 30 mints at 1 Vocoum iter with qualaive fie apesan rine retained fed 3 ime with metho nto iste. 4 Transfer the file oto a 200-mL volumetric Nak an bin to volume with methanol. 1 Prmeeptahroedaoplporgoyp.riUasteemdiettihoannso)lfoobriinntgefrmsedicoanicetnitroanti,oneiqutiteed.caliFborratlilonfrnansiotfhioens,LvCcM 50% methanol: 50% NANOpurSetanwdaatretradnildut0i.o0n3s5ol(vieent)cfoonrmtiacinaicnsg 0.0100 mg 4HPFOS/L (intemal 1 Ampulte and submit sample for LCM analysis. Figure 1. Analytical method flow chart for the analysisof PFOS in avian diel 66a _-- a9. Wildlife International, Ltd. APPENDIXV ProjectNumber454-104 |= | wl os 52 060 o ; fogs | 02 | 0 meme pemtepemet 000 006 012 018 026 030 036 042 048 | Consenaon (Ra) Figure2. Tisywpeiicgahltceadli1br9a)t.ion cufrorPvROeS. Sl=o24p867e3; Int=0e.07r39c6;re=0p.9t85. Curve CnoGns -s0Wildlife International, Ltd. Project Number454-104 APPENDIIXV intensity: 20000 cps. 10( 9 8 7 8 5 4 3 2 212 10) 28 88 io3131 11s 266 0 41 81 121 161 201 241 281 Scan 069 136 204 271 338 406 473Time Figure 3. Typical ion chromatogram ofa low-level PFOSstandard, 0.000351mg8.1.1. R06 si Wildlife International, Ltd. Project Number 454-104 APPENDIX IV intensity: 20000 cps 100 90 ? 14 80 fi 70 i 60 # f 50 40 iiBdl 30 182 Ji 20 i. 10N0 po2,7 [Nepeti144 a AB.,..28.2500 | 41 81 121 161 201 241 281 Scan 069 136 204 271 338 406 4.73Time Figure. Typical on chromatogram ofa igh level PFOS standard, 0.0m0g483.19. noses Wildlife International, Ltd. 52. Projoct Number454-104 APPENDIIXV intensity: 40000cps 100 9 8 7 5 5 4 3 2 42 ! 78 1931, 180 228253 290 o 40619 81136 122014 126711 280318 244016 248713STemne Figure. TaypppircoaxlimoantechrertoemntaitoongirmaemooffPaFOmaSt.tis blank sample (54-104MAB-D). Theamow indices CRoenT --_--_--mm 5. Wildlife International, Ltd. Project Number 454-104 APPENDIIXV intensity: 20000 cps 10 9 8 7 6 5 218 4 3 2 34 183 1 o 68 105128 160 264 40169 718135 "212014 127611208138 244016 28A147S3aTmno Figure 6. Typical on chromatogram ofa matrix fortification sample (454-104-MAS., 22.0ppm a.) C0670 sae Wildlife International, Ltd. ProjectNumber454-104 APPENDIXV 10 9 8 7 6 5 4 3 2 |4 OTE069 intensity: 40000 cps 7290128156 1s 20 265 T1e36 123014 128711 230318 244016 284173STeamno Figure 7. Typical ion chromatogramof an vin diet sample an Day 0 (S454- 1043, 18 ppm. nou Wildlife International, Ltd. ss. DisgrAapmpoefnTdeistaVLyout Project Number 454-104 . Pens1a0 I . aor fd appro Tm 2=FGoOuPrBXa4t1.y rreondseSMoldeglhNuobt.e053 CROOeTT se. is cogs fav is 2x] sRzaE|8" f=] Ba] oeen|en J3| $=| zsz88 a" 3=| Ji] wren on Ji] $=| szz8z a" $=] Lol je] is FIER IEEE El I LER 5F53 H2L lBg| cTmaTn]Tan z385] #i i bs A fy i Fo] sauna 31. 3 fol vomen|o- 3] 3i-| szssa|s" i3-| fa evene|an fr] io] zazae|s 3o| Bel -ov-e|n fof 3] zasaa|z- 1-| HIEFEERIRE 5) ProjectNumber454-104 gogvn les szErE (as saeee|es =zarz a= cose] arazs as EEL El qweeeegse| nn szze~]ne EERE momen [en ! aearga|g> i -eee|oe 13 z5a8a|8= i czenn en i szazs a= 1 seaze|ge l roo? 5. Project Number 454-104 I: gear len i: ngog ea i it1, EN zilil 3: 1;] FM ESE Ela) fHHsI F5R 3a go] lf] ele EEEERIEEE 3] azans|se | f3l2-]| Azo zree |z 1 sweacneess E.EN H momen) EEEEA EE IBgE eEgrBq IE= EEE ETe E i; f 3 ATERR| 8 oo on $a] ovven|ae |} anna 1| saaaa fae [3 LIRLELEIE & g| sazes|ge | GANG: ss. Project Number454-104 i PH IEEEEEI EEN I FN IFEEEEI EE l gi A 31] Ba i Ba] mony |on enna se woman Eel varus [an is 3225850 LE zones |ne ! 3 <i 3iz 3a2] SSREF|g oe- ava EN EEEER ED i ite:l mceennnuee| (oene 3| i- ZENE [8= i. A588 (8 3 | R833 i2 8] RS83% is ! ROG? ------------ i Is] $2 3=| $5] of i gl Ely. 1| fi iEi geJa|] i "lo 3 Ie 1-| fa] } go] Jaf $-| g| L590. geese sn Iz s2883|5" Ja) een |e $3] s2saz|sn =| conan 33] s2z3z a" $=| rmeen an 2] ssszzs- EB wconneeese]e[snn| i Jaf fE] ge] ezeza|se| lio] mono lon 5: szezg 2" 3-| maven Fa] zEzez zo $~| sosea|sr fof zzgza|a= 3-| BRERE[8 g| Project Number 454-104 gg8R7| 5% 2888%|8% saenn|ne sasaz|as vevos|en zrgaz|as eens |en EEEEE ER meen |e- nenen| [i1 smmag|s: 1 qeqnn] ow i sarsa|a i|i cmon] 3 zauzgn|& i oooen | |} zazza|as : snzzg|ge |; R055 -- . Project Number 454-104 al saann| an 2] amans|s~ =| smr=s| 8~ 2] samaz| an 5 =| angen a~ : " aRRER | 8" <% EH r = ls de sxaaa|an Fil 5 | amssa] sn if Sof snags ax 2 ~| sass a~ 8 | srssa|a~ z wl nsang| an | +] sszas|a~ i ~] ssxza| a "| saznaa~ "| =ae=a| an gl s3z38 {a 600677 a. Project Number 454-104 ] iz 5% sks "iiis 5 i2 i - {R38 8 wl sass| =~ * a83a3 ae |} | sms |g" : | graze] an -| sasse| =n | 2522s gs nNNoe8 L ail238 i MEEEEEENE : UT -| gzzaa| =n g| zzzzz ie GROG? Pe Project Number 454-104 o| wenan| an o| wwann| an o| anans| ee o) suns |e of mama] ae iif | anxan fan = by i fe] sasnn|a- By EI saxan ae 5 "| numas|a- i of arras|an o| wanna an o| nanan 2| z5228 fe CNNGRO --_--_----_-- " Project Number454-104 Appendix VII Table| fiom aNortIhnedrinviBduoabl wGriotssPPkatthoRleogpircoadlucOtbisoenrvSattuidoynwsithPFOS Birds EuthatTaetnTercmineatidon _--_ Con0tprpmoali) Males -- Wm Pwmm wr ws -- Notremarable e x x-- x x x --_--_-- Fens FReiagbhterwilnogsvsised No remark -- Wm Pwwm wh ws Sox x x x X o o.x .. CnouHt _--_-- . Project Number 454-104 AppendixVII Tabl2e fiom aNoIrntdhievrindBuoablGwriotsc Pathological Obsrvations PilotReproductionStudywith PFOS Birds Buthaant6izWoeckds _-- 18ppmai --_--_--m-- Mates -- we 0 Pwms 9 70 _ Notrena-- riatic ----m Xx x -- x x -- x --_---------- _-- _-- Fenales _ ww Pmme a 30 rz FoFeaxthteersloonsss S SoL x. D..x RTehgirnecsoenddiotviaorny S Soo . xx -- Notremarkaic -- Xx o-- x x - --_---- COOGHT _--_--m -66- Project Number454-104 Appendix VIII Tabl3e IndividualGrossPathologicalObservations fraNoorthmem Bobwhite Pilot Reproduction Studywith PFOS BirdsEuthaanti6zWeeekds _--nmmm 62ppm _--_-- Males Testessmall, ~ 1.5cm Right stssmal, ~1.5 em. _--Notremarkable p womrr eEowmae o e as Sx. xX + + + 4 Sox. x x --_--_--nnm-- --_-- Pens _ Female-- s wr am-- as ae as Feather loss: x x. x _-- Notremarkable ee So. x - x --_--_-------- Gn00as -_-- - ProjectNumber 454-104 Ap"apbele4nVId from NoIntdievidBualhGriosPPattholRoegpircoadlucOtbisoenrvSattuidoynwsith PFOS Birt BubaTnotiTezrmineatidon Mae Notre `Righttestissmall, ~1.25cm 6pmai -- Tew am wm -------- xSox- xsox- x- Fens --_W-- m am wm -- [ --No-- t-- remate--------x --x-- .ox -- x x" anos _--m--m a Wildlife International, Ltd. Appendix X Histopathology Report Project Number 454-104 CNNGEL _--_-- EPL' -. FR WLS BLPE Io TEe RAT] Ie ON TD. SAS nT AARP -- ovie Po gpe amp a oea 474 Suma: tee TIaL CnNG3G _-- EPL' ER 0. rr ThoFBCONLTENETS `ProjectNumber454-104 Page SYNCS TEJ------ J } va as ws GROG? --_--m--mmm---- " Project Number 454-104 PATHOLOGY SUMMARY CN0GS8 -_-- n EPL" EXPERMENTAL PATHOLOGY LABORATORIES, WG. ProjectNumber 454.104 WEIPLLDSPLTIRUFODEJYEINNCTUTEMNRBNUEAMRTBI4EO8RN4A.2L11,024.L0T2O.4 PROWSI:THAPTIHLEONTORRETPHREORDNUCSTOIBOWNHISTTEUDY PATHOLOGY SUMMARY tasues LfioghmtamdiuclrtosmcaolpeicanedxafmaimnaalteioNnorwtahserpnebrfoobrwmehdia(nCsoelctiinonvsigofinsealneucst)ed (whPiecrhuowreoroecutnainreesaitefdoiocr wahciidc,hprocteaisvsieudmvsaraiou[sPcFoOnSc]a)niirnatthieofnsooeftfhreioxsttoa1rt8icle twheeeakdsu.ltSoelbewctheidetiwsesrueesaflroomexunatmrienaetdedbaypgprhotximmiactreolsyco1p2y-.weTehk-eoladbofjfsoproicnfgiohvfis e`sttaudryyisetxopeovsaulrueattoe attheesetffseucbtsstuapnocne aodvuelrt abtoebwahsitte s(Ciolcinuks pviarigidn.ianTush)eof experimental design was as folows: Growp' [Walesf|aFsomaT les |o Talesm |Femae les | Fanaa -- -- [G2Cppmi aiPz FOS6T | es 5p | 1m5 5a1 | i5 5 p1r |5 o5] s| "=*BbOofobfbuswptriiiltnegiIinnnttahlhlee1gc.roo8nutpaoslniadnd6n.2 lp1p7r.m6eapc.oemgrt.o.nupgtsroowtueparsewireeraetsrdaftoerdsfiorwe1e9wkseek; CONGAD _-- . EPL' ERENT PATHOLOGYCARaRATORES TG ------ w------LR ---- cMthAanT ze,iEheaAnR cdNoeDImcMaEiApTniHLOfsDSwrSorvapriaours smtayoty wnitas,r6masnt4e. were cSalrotcontdiadfeignes,earned saurtehrncweetrssloast o1p2awealkegiofnotpesitgot fonyaWdiin e10%emnautoanlb,or4.efSoermcatoaenvaesofomExaprlmeranadliPanrgtywre fNLaaobmooarwtaiotnonryieienvs,aenIsndc. ae(moEPLas)c,ilwnhaerseeacttheopyrwsgeormewgeaamsbseedmxdiezademsissnoppoaraigfidhietn .ainndTchmeaodpeyinotso Vravaassiulaaebsolefw:Feralivrseres,ctg,ailolanbenldadladoecrno,gnprwoitvheenateric,cunlIutsn,pnkrisodnfeocyas,sbberrsar,iune,kpegraonatatodom(teovrsaorPynoetrotsedsotiss), poxoahtniteodn, and micrescapeFringwereocordot he davonfe IncidenAce Tsaubse, eMirseoscboyiprdotnocgol aorte pseasatusi sterHieapafotmrlogyh wberutrecasealn dovntehedHopiintgaanpsvItachdtn.icReeoTggarbaycsa.iWfioneseeowlsortes fooerarllseannitma[lPasnrisssuimmsariwzieh ebyxtsre.a26a0gyrusp, arnesItaomoantSroopgen rSuomumaorryidtocnesTuaeios,wgeeehextamvinienr o tt mmerofaril so ocfoGrrsopsoAnadntiadbnuglMamiticionrroofsegrcsosFsicnohibnasgerrgvaa,itiseonp,stnaTotteoed,aet nsepcreospsoeyedwsiitehatbtheeowCeroelon GROLTY Promos 50108 EPL' SHS. ansrbed omhe Gross Nesepyrcrdsprovid byWile HREaSnULeTSmctsrsncSopinAg sIn oTaon fTretreegsRpONsy isrie smt aiainEtes tve rLepS. aEseAtain,e gaBiRba,S r--Ton--s --ot c--t t--an--ex--t--sp--rog chascazot ya es A a --------E foooot OE 17.6 ppm a.i. males, seminiferous tubules had marginally decreased diameter (though spermatogenesis was still evident) comparedto othercontrol and 17.6 ppm a.i. testes, PR resulting in rE overall 0 eaTnan 27tr5s c7TE smaller testes which corresponded to rm e 175 p-- rea. d-- on srsTmvyavTs nn a aoanv--nst--unted rierestmoas rou size, bth pss f stri alotanttly ainToofsrpmosangtanoo.wnet mols iaitatSdonsiahyonatn Carooeenm,statthaagiTnapenlaih d-- isseset yes oT Toa SE `physiological immaturity in young (approximately 12-week-old) ET birdsratherthan a COHGIL EPL 75. Project Number 454-104 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. hot mations, Lis. Sy Noes 45.104 smaller sizeof the seminferous tubules in offspring este, the melanin pgmentbearing cls normaly present nthe testicular intrstum were ineffectmore closely packed na smaller area rather than bing widely separated by larger seminiferous tubules as in adult testes. This melain pigment concentration cortesponded othe grossly bserved black offspring testes, bu was considered a normal age-elsted fing and not apathologicalfect, All ovaries examine in offspring females wero characerzed by numerous oocytes and very small, nonpedunculated follicles completely `neomrmbaelddpehdysiinolaogpircoamliniemnmtalovuarryiainn cyoorutenxg. (Tahpipsromxoirmpatheollyog1y2:iws ecsokns-i0sdt)enitcwsith rather thana pathlcgial fect `Several additional microscopic findings (described below) were noted in `maedualtnasnedvleorritoyffwseprreinsgmbioabwrhwihteenfrgormocuopnstrroolmanedactrheagteendergartoiuopnsa.l cIonhcoirdtenwceerseand comparedwith cach other. Same fthese sions occured nly na single bid. For these reasons, hese findings were considered incidental and unrelated to test artiMcloen(oPnFuOcSe)aradcmeinlinstirtartaitone.s in various tssuss consistedof discrete fod of smallymphocytes and pasma cals; few heterophis were sometimes also presentbutwere ahvays a minor component. Chronic and conic active inflammationofadipose tissue were characterized by infiltrates consisting `exclusively or predominantly of macrophages, or mixed populations of ahestseorcoipahtields wainhd cmharroonpihcaagecse, riesnpfecltaivteliyo.nScaotnteer6e.d2npepcrmot1ic.aodfisppociyntgesmaweer.e.A osmnaelladfuolctus17.6ofpcpormticaal.tufbeumlaelseexthhiibsicthedancgyetoipnlaassmiincglveacbuiorldiwzaatsiocnoninsitdheerkeiddney of 4 6206972 . F------ EPL' Sr ES rcedatoto.otcofafotaactrs aanotfctrtnlgsysohctyatmdirranogotisonh maar famOnae oinnseovaedurltsfsounseccixhnogtohreeeigonsmefenoaaanvdy.rear,namnda gobioddr ann re apo ese. rancor mamma A characterized by small mononuclear cells, macrophages, and occasional cemaogmxemnetina cgllobiue5le3saYaonkdo,briCgsohtttpeicyntkvlatmoerrpeheotusrmacteariawloicokensspisteenStwmithwaihnha brown tovgeadmeevosnnSepmmeonnt gras resuentsineapartteeycoopta,, B Kuster cao,A ator esgoA na res waar pigments ot hyperplastic bile ducts were noted in one adult control female and one wes rete ncn 1.8 poifpming al. The ramact 17.8 pom a 1.8 ppm a.i. offspring female. A small fo ofc heteu rophs ils (acute inflammation) . efhematlse esxhitbietedraamyaelo,itd dWeHpnosttitiSmoTnsaIesIsnocidatsed wiSthesEecpoT ndSaryUhRR epa,tocelS lular V`ceomnprteisosteondnaadajtancwyentdehrerpastoica cnoradsd. oVheYpaoScaohtirSaotyrhSanges an,d oe vacuoles (consistent with fat accumulation) in the hepatocellular cytoplasm. ciVaecteuotlosinza.tiioWhnhdeeonnotnreedssmeuelntittplme,omsuatsuahalelvypasamatillo,p,copnnfeelsueacntR,hpoaororlyegdeP eremeardcaer tnedted . CN00D3 [E------ EPL' ERENT PATROLE3YDORON WG. Va------------------ a"GciulseWaewnhaopntrscenattinatryowccngtcrcracs,ahywcee deoshesr sdoans cen chart an esoctad ootessa, rues dosedon Wepstocse "periportal r ty change as odin ers fats and fig fiwhrilaoe mheepeatrwoecoenllctuloaarrtvaetcuroliitzmoabeteiosn wavsosmaenearntnoenlsayeigtn roeffersprnsingaA. roTehvtirsoggneunteeeraritioaonnmatl cchoantngveermslceamapnieo,mcaomatitnsaanndacesrofiwiaess,anfro.c. aHopratrceary freetlneaenrowsaiconncsoind ionfcheprattaseahnaaeevsacaulzaieonsil.eToarsoonse outt thmeatgaa4viesssmaw,aeacntoccroannvtaes neckecelatyosdtrmirclaeted 1s pr `iCsOoNNonC leLnAUNcoDS enSsIitUdMeOerMsNArpRSYoHsarle paosteteaucke(vPFSr) , gorainnthaetrestoovsaoryf.fanodrbgrots. mTlsenoafwtoehha d(o 7t 8ohpoe amig utmlebowieachtod deademousdamemot dr`cnoonorsrirsatnoenotsnw.imtehTeyahrelyrcpeobsret-nereeapdrtodeouctutivse opehvasroegraregsrscesosnimoan,neya vnorrmals1pv7hty8sri7orl9eogsai1cal te. 5t ro possibly of ate rileact cnn hierly mig ot. The o- C064 PfNambe 54.08 EPL' A ------ ST---- coTonwtotlhrtcearsgotsirnuagi ve4rs0S8orofeas nartcsloramitgebeidtoebosm `article (PFOS) administration. wn 2b vod) Veterihary Pathologist 1jrs)el i. Cross i. EPL' EERO LORATORES Prior 5100 RRNEPRA ATOR StyThe: PES: APltRepco Stywith NanSoe cos: evens J" ELProeuntn 22024 E9L Pat Marra. Grae EEIeTtlowoingepctysefSahtsmysweoeraDppaice atmeicusunetsenvaseeUniso ues sess = ones = arose boson Woocsyonn onarosen atten roorgen Sn ne szm00 nme 1ursen az vom antonono wasoo ame rono an avon ones mos an E ak E seo sermatim CNOGRG _--_--m 80 Project Number 454-104 `SUMMARY INCIDENCE TABLES ADULT SACRIFICE COG? aAdsuil-t100Sacettico Male Bobuhite -81`SUMMARY INCIDENCE TABLE Project Number 454-104 [oROrTsEGiSgE nTEMSaTtEerhioa.lB__a_n_s |[ 0 6| || [[ICnofniffietmemaatteio,nM,onGohmesoenlioew| ecorr[| | [| [ibr nefLemafon,Gramulosstons r |[| r ]| brES mrormmmmme -- F -------- F ---- Posen rrr FLELGRC--Kth= o.E= NED------1p ey [ 11 | ] [IBnAfLlLEiLeAeTaGtERe,hMoo.nBenouwelmeeasnCe| ll[) 5 [ |3------[ T--1 ----------[ ]| [[C[iSeercoossss.,TFoorfeliegmnabtaitoenc,t-- ar [ [ T | T E es eoo ---- -- ------ -- -- [IRnEfDitEera0t.eB,aMownonueTeneCer| t|)[m 7 e] [[rCuobuubsileess,VWalcmueroallziaztaitoinon [[ 3[3 1 [] | Ef Go.gmante |om -- -- ---- B-- E Co rr77 -- ---- ---- -- -- ------ -- -- -- ------ b r re T ET p ---- r Ir ----r -- -- [Eep5 atocyess Pavey-- Change, |------|-- [ -- -- -- 1 ---- I [Ho-- patocytes, Hypertrophy,~-- ~ |[ ---- -- | ---- [[HCeIpnaftioleceyattees MWoencornourctlse,arpcoeoalrl[[5| [[ 5 [ 1| ] [[C"PSlegrmoosnse.Fboerpeoingtntimoancertar [|| 2 -- [3__--|| [Setossr.sInflam-- ation, -- [ ------ | ----| [ePReOVENTRICULTS(Wo.BXwtneny | 91|r &y [ r | T r -- C r ---- ---- ----1-- [= EpL ErpePuriboiogymLabeoratnorites,a1. nouns --e -- ee i" NE commswopsce ae Em | Iafilteate, Monomcloar cot | |Seminiferous Tubules, | [1| | | ----] | --------] |Decressed Dlameter | [3 | | [Sf permatog-- enseds ---- [5 -- [5 ---- rrf -- --r--r -- -- -- ---- ---- -- --] ] Je --t --e --r --e ---- me --e -- re-- ---- ( -- -- ---- -- -- ---- -- m-- f-- -- e---- m[ emesrr --mssm-- memee J mem-- e core-- ere {ee -- m e -- eee} -- eeeee-- eete men] ---------- rftr 1T 1 I-- r m-- eer rl--r ---- [re--e-- rrr rrr Hf e e rc ------ ---- -- ---- -- r-- f efe -- -- -- e] -- -- e-- -- -- -- -- -- -- e -- o ---- ------ ---- --em-- ee eet-- ete[eeeee-- e ef f e e -- r f e ee ---- e m e -- | ee ee-- e{a-- etes -- e eett eset -- e-- e esr e] eeeeeeee]} eo emer I e--{r----]---- P [ --_------ ----e -- T e r ---- er eefeem-- j r r-- -- r ee-- r-- r i-- e} I f ------ -- ---- -- ---- | -- e-- r}rm ---- -- -- ------ --] F-- e ------J-- -- ---- -- ( 1 Pr ----i---- |fI-- r -- ---------- ------------ ---- E -- TE--e -- if o,W-- -- o--r W-- T -- -- -- fee ee e-------- I -- [-- -- -- ee -- e re-- ef-- ee=mee -- te t-- l e eeee -- ey _-- rr ------ r----------|m -- -- -- ---- -- I--e ---- --1 ---- ---- ---- ---- ---- T-- ---- -- eee r l -- r e-- r -------- re -- el ----| 1 ----T---- r-- r]---- -- ---- A CROGHe _--_-- AFiesmtieasioenSSaocbreinfiiccne o SUMMARY INCIDENCE TABLE Projct Number 454-104 FomrFoosrE errsrsoarwo-- . gem J-- o(m6 [o-- 5 e| -- -- -- -- T-- [[[CCCiiinnnfftlieiTmtemrmaaatttieio,onnW,,oGGsehoermnsosenTltooenssatGonesr[3 1 5 1 -- ] p--------------------]} CSe E e-- eiRs R-- S -- -- -- ---- -- b po[ m e orpeeo wee, wner] [InRfLiClRLEArBaOsRs,GWioon_snEsEcyl--oa--s5SIT r 1 | 4 || r ------------]] B [s[eFsseocsC osn,sT.ntfioafmeai-- mtpiaocno,rts~r---- {[ 3-- -- ---- | ---- -------- [Ie TeDfRiTeehaot.eEWEonoSmr seTsgegar{1| r 1 s] [[rCbrealteess,VWaicmueorlaiitssaattiloonn~~3 3 3 -- ] --] -- -------- ----] [[hIvoEoRGfos.Bpepeosstt)on----------[1 -- ] [[HECehEpmaptaotoecyyeieess,,FFaacceeyycShh--aum--ngge--,, || ----r ---- -- r -- --1-- ---- I [[oHs-- epp= aatteocc=yyteeesr-- FFapcoecyerSah-- pahnyg.-- , -- | ------ -- -- --|---- -- ---------- ---- -- -- ---- ---- -- -- ---- -- -- -- ----| F [[[HPCeilpfgsitmHioeecneEytaitsbesep,oFFnootsctorimoourncttso,azfGoacalrl |r3 1e 3 -- | 1 + p ] -- --------]] [[oo-- S srCmosTnsT.iFoiraeTig-- ntacorer 1-- 7 | -- y -- -- ] -- -- -- rr PFT r1 y 1 r 1 T 1 18 ere TerenCa ce 0100 --_--_-- Aadsuilat00Saceteice Pesale sobwhite 4 `SUMMARY INCIDENCE TABLE Project Number 454-104 Lo Jor ROmE [oROTe" |r [T e T] e TTCT rv C---- --e -- | -- -- r ---- e -- -- -- -- s -- -- -- 1 t -- -- [PIReOfViElNcTrRaItGeU,LSFo(nWoon.uBewliosieecnayrl[|5(5| | oy [ [| || o oo ------p --i--e fe eeeee eee ee e el r rer ed] ] t o7t --f ee eree er e fT re e r r re r eee eo e eer free}oer] -- t ---------- -- --t -- r m fm e -- r -- ] r Te r r e --} -- -- -- -- eooo -- -- -- -- r f -- -- er -- -- -- -- r -- --=1] r r ------ r r tp r r ------ re r o re t r ] 7 Pr ere r etfs} T r eee et ee er e r] b bo fet r fe] fr t e r ----t -- r 1 7 -- ------ -- -- -- -- ---- r r r rr r rr r ot r o fer r et -- f 7 r fe ------ e --r r ff r r m r } -- r ---- t I Ere Eerkacntl rthlogy borates, oc. CNNEDL _--mmmm -8s- ProjectNumber 454-104 HISTOPATHOLOGY INCIDENCE TABLES ADULT SACRIFICE Rn10T _-- h Enn - reNenbrs04 HSTOPATo cHoOLnOGYWCm IOENGoE rTLE : orPosETTSSEE froreign Material ---- [xx xxx]xxx xx TTTT TTT] -- |TFT TT] [Infilteate.Mononuclear Coll [Inflammation Cheontc || 1| [|[| [TT 11] 1| 1 TTTTTTTTTT] |Inflesmation, Granulomatous || | | [| I TTTTTT] pam AA NO A x Ix ew xTH Ere [BURSA OF wABRICTUS teil i eroT TTT [ww[wWlw[W[ Jwiw{w[ww TT] Coax AA A xx TT OO A TT OAI LoS rEE1) mJ|==S p pe e 3 ee]| {Infiltrate, WomonuelearCell [Seross, ForeignMatertal [203 T J2([3][3 Ja TT1111] |T I TT TTTTTT TTTTTTTT1] {"Sercss. Inflammation, || 7CTT -- SSTTTTTT TTT111] b L r 3 TT 2 rrr3 rr rE t et a B e PPP eana aaa | fubules, Mineralization [Tubules,Vacuolization t------ [ATE rererrge {Amiel Deposition [BileDucte, Hyperplasia || | 137 | I TT TTT 1 TT1T Tr rT A TT TT TT | I TT TTT T1111 || | | [I 11TT TT 11] |0 Hepa7 tocytes, PaveyChange, || I | | I [TTTT 1TTTTTT1] [Eepatocytes Furey Change, |I I [| TT TT 7TYSY SO TTT] I| |Hopatocytes,Hypertrophy, || | | [I 1 | [TIT 111] fputeee | Hepatocytes, Wecroads, Foear | | | TT TTT TTT | | [TT TTT] [Infiltrate, WononuclearCol [3(3 T[1{3|Jal | Jalal[T1111] [PigmentDeposition |Soross, ForeignMatertal TTTop T ar T1111 | TT [| TTT TTT1 [Seross, Toflamsation. -- 7.)CTT | | [|[TT TT TT] 0SS 0 en TT i5F geESmERses CNOL0g --_--_-- . Project Number454.104 TAaasntie-sssonSoamciritfeice HISTOPATHOLOGY INCIDENCE TABLE coarnoto aFrsaue : : fs1o[5fa0lss [roveRTRICHLGE | Infiltcate, Mononuclear conn 1 xT T Ix TT Ix TTT [71 [33] | [aja] [a [1 TTT] 111] E ip r m ppE eiw-- lreeltel-- el(siFae-- ltat lae la-- T lTT -- Ti t TT] [InfiTtsste, Mononuclearcori |Seminiferous fubsles, | | | TT 1 [af[1 |TIT TTT [TT11] 1 T1111 {E C -- DT ecreasTedTDiameter O | | I | | [al[afX | I TTT 11] 1 e ee tbel oe f fpf ele] t SA ------------------ NW SC OW S NR -- A UWT S W C Y CSE C WC 0| me--e --e ---------- d --T p {p {fflt FEE A A ---------- ee {yf} " T me r ri te e pt pe y i [r-------- -------- W-- -- S OTWt W-- WO T t-- S} EE--]a 5A A -- [-- ---- -- -- -- ---- F ------ f E ff I e rrrrrrrrrrrrrrr e tti Ec 0A A AA OY AA SS AA ---- rr-- rrr rrr-- rTr TTT T T7 -- NO WW -- OOOS OOO [ t re--= -- r p -- rrf r ] rp rre r rr ---- 0 ------ EE na LL Ermabg abitoe. rBiR! STeeEr,aTIr,eSn nn104 _-- Aadsutl-tt00Sacettice Fenale Sobunite " Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE covroTvieoL r1o.v6e : | o5 | c F T e r rTe T -- Ca tF [[iinnffllaammmmaattiioonn GGehaenosaltoeastowsA ||[| [ 11a{ [TT1T TT TT pe [PsaoFFaRRIEToE w5w 0 0 www ww x A EE Www TT fe e x s ee C[FiE nitecasS,So-- nona-- cten-- ecor-- r YE 3 035 | [31 11 [rele [econ roresgntacertal 1 1Tv [TTT TTT T C TTTTP CYT-- -- 2 O fy S IEe S H [aubules, Winecelzation 1 oe [AmytetdSepesttion 1 TTT TTT| I (C FHspateo cyess.--7Fac--eyCC h--ang-- e, -- |{ -- T 3 T A O T| I [[FFEEseppeeccooecSrytcaSers,,FFaattetyyGCh--haann--ggee,, [T [|{T TT | 1{TT [FFoEleepcstcoecorrtaeasl.Bppertroy, abit |5|113151TT 51 TTTTs TT 1T] rrr [[EFerpaaftioteeocyamtcaer,,WooLncorCnosaet-- tse,atFcoeciall [|iA []( TOT1 ax aaTa]a[ l{| 1 T TTT] E [C7 oT ons FT oreignT materia-- | TT] A EPL ms `Experimental Pathology Laboratories, nc. fn tm re im rs ER Bat ---- " C030: _-- hiFeasmvtaileerSuBcoebseshiictee "8. Project Number454-104 HISTOPATHOLOGYINCIDENCE TABLE ocroontuapc cbaoo : [ [Seross,ForelgnWatert--atIx|x|I|x[x / | I ITx T T1T1T x TTT] [serosa Inflammation, TCT TT11 1+ T TTT TTTTTT] 3 feRovERTRICw es SO 1 Ix 1 far x TTT] | Infilerate.Wononwclearcolt (1] [i(31 | [a1(xpala TTT] E A s 0 A OO A A f E ee A s A A TTT] EE </</ mm------ -- ---- r-- ao e er rrrrr rrtt r r r tr rr i f-- FrP--P------------ --------le -- rt T e TC Ty f-- r e -- r ed-- t=f rre r rfrf r-- rrr~r~] rrr rrrrrr Ee ------------ TT 1 EE E F A 0 A [---- teed rT -- ] eee PF -- e ---- r ---- r-- rrp r-- p rp rr-- rr=r ~ -- r-- r --_ EE rrr rrr rrrrrTH SSSS A rrr rrrrr | F -- -- r ------ r rpr tr r ee rrl rrt rr I A f -- ---- r rr rr r-- r rrr r r r -- rr rrr r-- rr ---- WS rrr tn SS S00 rrr WSO SO SO rrrrrr NO UO SS SSSS Er ee Erma Fg abrtoes,n ye ta an et, cm, ss iBR, EL 01.06 --_--_--mmm-- -90- aijest Number454-104 `CORRELATION OF GROSS AND MICROSCOPIC FINDINGS ADULT SACRIFICE n0107 | Jl LNin :i | H3: ! ENAN | n030H FE | il i Nil . ig [ 11: 3d i | LIT $3 ! wnN3iog - Project Number454-104 ; His ] z i HR 1 iE ii 7 1if #1 [Wa [TTI] cnN01i0 --_--_--mm--e---- 94. Project Number454-104 SUMMARY INCIDENCE TABLES OFFSPRING SACRIFICE noLrs --_--mmm--m--,,--,----_----_--_ 95. Project Number454-104 Oafsftu-p1r0i0ng Sacrifice Hale Bobwhite SUMMARY INCIDENCE TABLE Pa z rr ECTE0 ys bPiPr _ F lseeceeo soi isttess nn -- ionG--ri eat t -- p1 eer e -- 1-- 1-- -- 5 p-- 1 fp 1 1-- 1 bS of-- rrN omeer -- ------T--w -- JTw1w -- -- w -- -- ] -- tram efeeep ef] }--} [F efeFiwLrlCeAtrLeAoDaOcReEm,(W40m o.roEme IweNtEeDseYG--or[ --C 3 1 Ty1t -- ---- T-- --1 Tny-- ---- --| IB[[FFlrmoevfbielleGeeesoa.crWBei,nMesoasoamieeealteiworn gorT [--o 31 { w1 e 3 |T--w----5 ---------- ---- ] [[m CiHelgae etomccyttm ess:bFpae cpeeygcr--haaneg--e, --11 --51 wm1ew 1 -- 1 ----------| [FEey T posltooicyltceem,Fatty Ghny ge, r [ p s 1 1 ] [[eEpepeeteooccyptteesr,. VFaacrweoylGaannc-- gtson,, | |1 -- -- 1 ------ -- ----1---------- [[PpIProaefmlseetmersreesion,Mg foomuetnety ue s gae r--|--[ n 5 s p o --13 1 wms m --7 --+----| E srr Co L se H Ce y t H1 e0BE--e e1I ny --| [HR[Fgiomsfeiobnesnnnctoen,ceWnotmroantsieotnon Gar 13 5 sTes Ts15 7 7 1 1 ----11 ---- r -- -- ------t r r t r r t tj --1 Ra Ere Experimenta uteg Laborato,oe. Cana _--_--m FOfefrnftueipereinBgonsiuceeeitice SUMMARY INCIDENCE TABLE Projct Number 454-104. ema EE [ineErLites-- cC Ce,rFono-- sr ietonsc-- or--{-- C -- 0--0---- vr -- 0I--es --|| [PPriiasfeefesersmatsrisnae> icointnence=ntegemtve [+ --------| bEBr-- -- REA oGi-- mEoE-- S) -- -- --o 1---- -- w ----+-- e ---- + -- r ---- ] EI : e orn ms--re oTo --Te -- --o--f-- --om--+t-- e ----1-- ---- [PRLTSTASBEYoS 5 ||oe I ee ee 2ppL E [[ICoasdr biateees,ntssoeMomrsoamtueiToernr ~Garr 3 1 ---- 5g| Fe G TC esvma e ---- ----------e --e -- a ---- --ee-- -- a E-- eatgeetttcpreerea ace>domme, ----------------| F [ompem btiactyetesesC=,oeVemewaett-- arnete on, ---- r --|------] ---- -------- --| b PPOe ET meB rE-- -- Eo S E-- 0 m -- --Q--f -- e --S-- [Ir e ntiecate, Foe nomictom ns ce ell[5 n t 1 e|e --e6 ----3 +----| 1 r rr f -------- rf ere ErmaFabi "0123 --_--_--m-- 97. Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLES OFFSPRING SACRIFICE @no1rg --_-- Sif ProjectNumber 454-104 WTopATHw OLOGY MGa IDEHCE TABLE aw om : FF og rmar H aE m [OTROS ITS [Iafiltrate WononwclesrGell Ix[xX[XXX][xx][x Tx xa |I | | [ITifTTTTTTTT11] finflemation, Gheonic [Inflasmation, ChronteActive |TTT TTT TTTTTTTTTT] || [|[TTT TT[aT] [L RuseY caliC sstion -- TT O TTTT TTTTTT 3 be---------- pa ww ee x rH ee EE trrormare (BURSAoFFaBRICTOE LTT TT TTI [wx[Xx|x|[x[w xia][wixheix er xia] a rr Ir rrr TAH Errtritel [EALCBLADDER SS A A Ix[ax x]eae ex {Inffleeste Wonomacteascet | [ I ATIT TTTTTToT] poms IA Ix xx] x(a fe xx EE |Infiltcate,Wonomacteaccet [Frabules, Mineralization || | | (13 I TT13 TT TTITT 111] Ta T1111 peerepr------msmeremmefrmfer mior eY ro Tf Tf T a r} T [BileBucks, Typerptasts [Hepatocyes, Facey change, {11 |TTT] 1 {TTT TTT TTT111TH f {Hepatocyi tes Faceyhnf ge, | | t I IT [Ie [ITTe T TTTn +11 ree TTT Te jHepstocytes, Vacuellaatlon, (pipe |I I | IT TT TTTTTTTT] 1 TTT [Iefilecate, MonomscTosrGeli Inflmmstion Awe [31 11] x apa] pha fTTTTOTT TTT fferroovvaeReTrRrIEGgREGEE=A | I IIx TTT TT TTT TT TTT aT] [eEs F s e e e ememsee tesr Gilll oebifn re Lo[ eb] oTC IT e rrr a I ---- [isfiteeass, memes carr TT1 IE) Pe Eo] (pigment Concentration -- -- [FIFIv(v]Jvle PIP [PieFllel TPP] - Tr i SE EEA TsS sSe R05; _---- GFreefnoapsrinngisnacerifice 9. PrjectNumber 454108 HISTOPATHOLOGY INCIDENCE TABLE couarrotuee, arioue ainaue : [oreoseFSS 1TXT IW [Infiiccate, Wonomselesrcor | | | IT I xreba x Tx] xx] TTI IT TTTTT 11 {Inflammation chronic [Inflammation ChronicActive [31 || 7|[TTT TTT | | I TT I [TTTTTT] PeEC eTsCToTsTtVEsTTL --O TTT| frg----------------t fram EI Ix x ew {~~} x a EEE PETOrTREE [BURSAOFFABRICIUS irre turret oth tltTT= oloy TS [xx[wlw|x|[x[x|%[xxx] [w[w[x[x]x] 1] for rege r TT rrre rr r rrrrr Fame (CALEBLADDER TTT T111111] IRR x refx xx]exw] [Infiierate, Wononactearcorn eres | TTAT TTasa TTT teh Idd rT tT ET ~-- my | Infileeate xx Wonomactearcori |I | Iw x Eee | [x1 Jalal[il TT[ILTT TT |Tubules. Mineralization | (31a [| I | [TT T1111] Pes -- mmm -- P AWC-- 0 i-- ` 0 H -- TW -- J0 [oileDucts Wpperpiaeta | Hepatocytes, Pacey Change, | [| I3TTT TT] | | | | TT | TTT] foie [FEepseeereen FI e Gee, TTT TT ST Epistarass [HOeW NpstocyTteTs, Vacuorization, | | [| | S ITT TTT1110 1]| fpf Hates eteralt Ear TT BS Ton se nrrH [Infhl wa s mma 1T tTi T on TTTTT] fom rr TTT wh Tw pe eee ep f[ pRovERTRICMGS T 1d T T T TT T HT rT T r T T T ] EH er vr primaFuhog aborts oe. Ya eC EO HE BL " 6003.16 _--_-- -100- ProectNumber454-104 Sfetrapering saceitice Female Bobwhite HISTOPATHOLOGYINCIDENCE TABLE a1n7osr 4 [OTPGSE FSS xT fInfilteate Mononwclear Gell | xxx xx TT TTT TT 1 I"TTT TTT71 {Inflemation, Chronic [Inflesmation, ChronicActive I [A131 [ TTTTT TTT | | | I [| TT TT[TTT] [Pmoralization Plessis 11T+ TTT TT T1T71]1 Felele elwtrtetet--t--t-- TT IE ex BTTRBEgE=etrrre relelririgimimimi LTT] l BUREAOFe FABRICs IUS se Xe IXf[hw e [xI{|wT(T xT(Tx1(1x1| 1 1 [CLUETUT SO [Infilerate, WononucTearcolt {i 3 [a] | 3 3 7S 0 | [ {a | TT TTT pe-- owper -- I are e xT xL x -- T TT TT [Infiltate, MononuclearGol {Tubsles,Mineralization || | [a]Jz [[TT] |[| | alTT TTT11711 TT ---- 0 WC 10 0,O90 WW (217 SO 3I [site sees mggmepteets 1111 |Hepatocytes,PattyChange. || |T 1ITT 1 TTT1111 [T "HepTaYt;ocytes,FattyChange, _ | | I [I [I[I[ T I TTTT 117 fopeeal TCA rr [Rptwe San HEE EERRER S SN 73TTY S rr a fooiffue Te ESS {Tnfilecate,MononuclearColl || [ala ja Tal| [Inflammation Ate -- TTT TTT [TT 11111 ! b Jc ORoRYw.+r------m ------------ e e+ lg+ore LereteF Te LdF LT E T1 S NOSTT E TS A A | jpovewmmrenee |Tnfiltrete. WononuclearColl TTTr rer [3(a3(aa aol] | | 1 TT] | ~-- rrr rrr | Ee ------ SS : a-------- rr rrr r ere Era FlagLobai yatoms,1 ee ns HE RR in " nna? I. ProjctNumber 454-104 : CORRELATION OF GROSS AND MICROSCOPIC FINDINGS OFFSPRING SACRIFICE [LUEEE) FE 3 FLERE 6 fH Virolls pililaa dadlss5 3 3 ig 82) 8 Lag 0 1i 4 [olesJ TTT n0320 A-- .. Project Number 454-104 | | LEE | Ho 8gla]la 5|g 3 E |} dE 2aiin E faa g# i dd | 4 1 [a | JLL GnNi0 EE -- he Project Number454-104 allel 0 Ea Tal Talal : I fg giBlailad|un|)aidsa : REE EEE EE i i 144, y fd d fe ldB28LT ConioL SE - , ES 1 Hefdl i Hd | i] HI glug Ls LTTTTTTO noi a Hl fi fih|ln I i {i yl SE HR1 Ei } i, i dHy E[TTT G03. . ep ---------- ee -------------- a La | Hil gil! : :3 5, 3 dy LITT n0LlG 109 Project Number454-104 | zzzss| 33 3| szses| gas rein] nen BoE ios Ll iii fall i ceeno i i oi i | sees] =: 3 i RRz2aa ga Ati ev evoer | enone] namaenn ix 3 | ssszz Fis |B an01l6 To `Project Number454-104 3 3 FH IRALELE IE Lh 41 } Al eoeen] nen H2 iig 3Efren ee 3 af crane] ge- = 3 Af reer a-| z HIEELIEER FIR 3 Cong an Project Number 454-104 5] szzsz| =: i HEALER Lh = PTO ER HIEEEEERE FL 3 GNOLG ne Project Number454-104 HEHE HIEEEEEA REE al conn ann oli hoof x =2 $114 iA 8 :| 4iii) 2it Bla] errr] ace Jal] != H s| EzRszEesS| 2s2 # HIRAAARA EL Js of cernen|me- IigL eens | gen i g| 2zzz28 2is |} 600129 fii 3 sooo te | Fo? c o 8 or oR oR IN BE SShEalTl EoEo Eo 0 UBIL EEREEEE : foc Bosc rms oss 5 coc onnn fii Sore ee ee Pl4a1s ali5,i08 Bf 00010 ;TM5 gal Talore] ; ~ -- faa oer Plager] z J sser| maf |fem) Tog fpf omens bi i | 583) 13 B |e BH His omer) ff srese| zg) BaI ler ful |e) A [lg]green Re  ceron|gne 2 romero : 1 sgsgs| 22 gy nme] | of pean] = i g]omrenfan- jal if Hi | MERTEN {3d el ; i ceven|gan] # Teg wosen|pun J | sane = s|oeeer (eel J ozone = |B geen Jud | Hm enor ry ProjectNumber 454-104. Tglromeslme- Ty meneame ak szses| 2 J ssa] =n :g i [E] H 8 2sg| 2 i } i 1 eosonfann k g nee if BERTH EY ! i gels bf -eere| [Hg ween gen f= i= Hae) | Hw 6008.52 Taf ps worn yg ER lg ! 3" i 3 | mr : 1 Pad 2 szg] == i or |2 szg| == . iE I TET il i TT ie J TTT ae f TM - Bi gze3z| 33 gl ores ae ; ! saz] 3 iTM ETT RT dala =) 5 | Bf 7 szses| -ae ; f ls HB ' 5538s) 58 | glen } Wy FE 4 [jzees| = fA ~ szace| -83 if is or ! 5gse 3 Be =TT } I : i z on i en 3Hoiof E|eanealns } il i coe I i 2 "w Fol i i ils : flrs] i i ye ie i =m ge 0001.6 _--m n0- Project Number454-104 AdultLPiivleort RWeepirogdhAupcptge)inofdnrSioxtmXuaIyINIwoirtthhePrFBOoSbwhite Page 1 Control (0 ppm 8.) ren J Liver Liver 00 2213060 7sa0160 000 ammo 7s5o0n8 5 2051 209 Mean asuas 71024958 18 ppmai Pa --MaLlivee 106 2176%0 2. 2o.n06 m 2807 Mean a3s5i00 FemLaiele a2050 osums "ois oLenst noir _-- i. Project Number454-104 Append XI AdultLPiivleotrRWeepirgohdtu(cgt)PiofanrgoeSm?taadNyowritthhePnRBOoSbwhite S2pmai Po aTeer FTeime pwoe r35e9 asoonn awns pr2e5s0 aSi2on as S30 sm MeFnS soaste ossas 6pmai re Laoer retinvee 2wm6 2200% 0sss0 2as 225008 opme 22 so jo p0r3s a1ss0t 6n01.15 _------ ee -- -- le| szzzzzzass ge 5i: 8 i3 bi8 i ij = Ye] zzsscezzes ge "ll 43P| s s88s 3gan esey ag | ;i | g| seemmrazaz go |fi i : de) mmzmaszazz go | Ge01:19 _-- ame Wildlife International, Ltd. Project Number 454-104 Appendix XV Changes to Study Protocol hefolTlhoiwsinsgtaudmyweascnondduamcntdeeddeivnniasttcicoonsrs:danc witth studyprotocol signedon Februa2r8y,2000and 1. Tshaempplriontgocaonld wcaslamienmdtheedistepoanirnatdtiiocnafeteeedggsshwelolumledmbbreanheefdrrefoihmgeerhaeeld wilseparatedfor 2. cb"aTethesdipscrpootosoecldoolwfldabuseyriiacnnmgcietWnneerdeeaetkdidsotno.r1e3dauncedth6oefutmhbetersto.fEeggggssccoollllesctteeddfdourrianmglWeyeskssfr2o.m4aalnledggsw,i0 3. T0(hCdeompe1sr8po3otnopdciponlgmlwy.a,staemteesntdceodncetnotracthiaonngsewtehree cehsatngseudbsftraonmce0,pu2r,i7tyanfdr2o0mp98m.9a.% 10o05,105.,46%4 4. TaWhmeeeepnkrdom5te)o,nctgolaawlaswosoaduemltedainbldeedecdohletloeicctnoedndidicdtaaitolenytshfaootrfficonarcsaubssaetoivroegn,en,dhaaiyencphueobragitioaodnnbd,egrheioanursniiinngnggofMnoafdrfcbihrn3ogo1d.i(negTarholefy Whaecihgihnegdsa. aAtdcdhitainodnaaltly14tdhaeyasmoefnadgmeenatndisndtiecdatreedprboadiucchtiivnegspawroaumledtebresfuonbiqeuemleyasiudreendti.fied and 5. The ncropey section ofthe protocol was amended 10indicateat est termination, samples would btheiosetcdoolplfaertcohtzoeeldnogffiorcopmoctxaealnmltiinraaeltmaiaoninan.liynsgiAssntyudryemaadiulntisngantidssfureonmt10fioxfefdspfroirngsisiniopeaatchholtoegsty wgroouupd fboer 6. TPghPreouppr3so.twoiclrolclabwmeescnutatghmarenoniudzpeeaddtsft0aetshxettecfnnodutrhowfeeWaedekueslk.t pA6a,drutlDotunrbiionrfgtdshheienstteuxhdteye1nf.os5iaotnhnedhceo6.n4mtpuomlpbgersoau.opf racnodtm1e6n.t3 gs ad cfoWolrelceeatck6bh.lpoAeodnduwlsotaumtlpesldtebbsiefrrdrseocmiontrthdheeecdo1ann8tdraonelgdggrs6.wo4oupuppalnddb.e1.8d.ni3espappotsmmee3dn.ot1fg.r.roAueptwesilltgbrwmoouuupetblbdianis1astoedtboefhmeatehdneedno1df0 rofosWsecnekcr6o.psyA.dditonal, the amendment required collection of eather samples tthe time of 7. `ATsAhsdeudrpiartonotcnoeacloUl,niwtthaesaSnapdmoen3nsdoGerod'otdroepiLrnaedbseoinrctttaotrtihvyee PrwraaawsctdciachteaansagonemddprloeapnoJtrothinwnoaNulelrwdespbtoeerdatuwdainotdeudhdibsypae.tdhdeprrQeeuspaalrwieatssy. added 5. tsTpheaerrsperlodtyto.c.olRewsausltasmoefngdge,dtbioionddiscnadtiasnsaluysaensaoyfseegsm,abylbooed aamnedntdseudetostahmeplbeislawgiillcableerelprstaetd 600120 _--m 124 Wildlife International, Ltd. Projct Number 454-104 Apepaine2XV Changes o Study Protocol. 5. Tihfelepnrocteoscoblewtawseeanmrenodveed to add aise anlyss ofdatatodetermin statically sgnfant 10. osSftfuidapyrinomagfnfwpdeprsrioengrluewsdeeefrrdeozd0neno.apcpAhrpoarxioitmsoaectoe,llyswme1ei2gahdwemedeeknastnwdoefsdiasgnpeootspperideopoar1afedbne1i4sngdtaoylwslyhoamfiaaasegdhe,erwfeoiosgehe,d Chane. 11.coTPnrhcoeetoncteorslattaimsoeunnbsdstcmahenancntegwepdhurfinrtooytmpcr0h,oad6nugc,eedd6.i4r3o3m0edl91y60.43a9p%pmntoa3f.o8rs60i.90s%,.c1h8a,nCgo6em2esapnodnd1in7glpy,mthae1. teAst 12. aAddmiioenalnadoduelxttmbeondedytnwheeittgehstt imnaedvaertsenutlyriewdenmeoetasnpketecnissfytdWeieoksn,],Bo1y0 awnedgh11tomfetaseurteesmt.enTthse 13. IEngsgtsead1,cdgon Jiudlyo8n,l2y00,0 D20a0y0w4eroef cWoeucnkted192)5dwecraellitneaddvweirhctclaysnoidcoonatteedFaonlldowcianlgldeoyd. CooL ns Be WoiblbdlifsetIanttetrinan ntsional, Ltd. d. Project Number4c54s-1i04 Personne AppveonldviexdXiVntIheStudy manTahegfoeoflmtloiwesinsngtutdkeyy Wildife Intemational, Li. personnel wee involved in the conduct or a (0) Mark Iber, WiiToxicologis @) JosanB. Besvers,DirAviean tTococlo,gy ) Linda R. Michell, MaonfEotaoxOpgerateions ((94)) DSicaanna'.TeGmaplllaeg,hLearb,oSreantioorrBy SupierviosoArvlianoToxigcol,ogy An(a1)lytWiicalllCahreBdm.iNsitxroyn, Ph.D, DirectoofChemisty (2) Raymond L. Van Hoven, Ph.D., Scientist, Analytical Chemistry 6012