Document nN58K964Qj0kQrL696dbKJyX6

- . Ph226-183( Fro: AREPRODUCTIONSTUDY WITHTHE NORTHERN BOBWHITE FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROTECTNUMBER: 454-108 MLABREQUEST NO. 12723 FIERA Guideline 714 OECD Guideline 206 AUTHORS: SSReoasoyam?m.oBG.naBlesagavheeaneHoven Naik abe STUDY INITIATION DATE: Jea22,r20y01 STUDY COMPLETION DATE: August 20,2003 Submited io Envir3o3nMmeBCnaothpaloArLviaoebnsosry `St.Paul, Minnesota 55106 Wildlife International, Ltd. Eason5,T98MiirCyooismaamnzedr42c166D00i01veUSA Fuge 101156 i CORY? _ WiWidlidiliffeeIInntteerrnnaattiioonnaal',,LLtedd.. =~ e Project Num mber454-108 a GOODLABPO RACTR ICE CAOMPLTIANCOESTR ATEMY ENT SPONSOR: 3MCorporation TITLE: PFOS: AReprSto udyd withu thec Nort theri nBoo bwhn ite: `WILDLIFEINTERNATIONAL, LTD. PROJECT NUMBER: 454-108 STUDY COMPLETION:August 20,2003 CboyothteLaUTbSho,ersatEtuodryymPwraasivcsoned(PiurcEttNeeodcViIicncMnAoCgmtpIomlcCsynH,(ce4E98Mw)CittF17h)RGPoaaaonrddtL1laJ6ba0op,raan1tM7oAAruFyPgFru,asc5tt9i1c9Ne8So3ka;inOadEnaCrNDdoPtsirfaiicpanutibcolniisNphooeef.d $550,Agri ProdcionBuea,10 Augas 1984, with he Following ceptions: 20d 3MCCoomrpploertseisoingnaeedatndidcakeerdepionrthsefroerbpolrot.Sunmdmairsyutaanlalsysreoscionnclduudcetde2d5byppEexnydgieensRaensdaruchl Teports illbesubmited sarily. "Thestudywasconductedundermultipleprotocols.Theini and 3Manalyticalportionswere cRoSnedyuccathemdbauemnrldye02ri3oc-nl0p6e6paornosdtos0c2ow3le0(r7Wei0lc)dolnifceiInatderunnadteiornatwl,Latdddi.tsiotnuadlynseupmabrerp4 ro5ce4s(-Baen1 ydgte0hneR8eEsxe)yagr,ecnh conductAsliysacscoorfdeangcecwointhenGtosocdonLadbcotroadtuornydePrraEcxtiycgeeSntRaensdeaarrtc,hasniddryesnulutmsboefr0h2e3s-e0a7n0awleyreseacret reported separately. Theaslytclphas performedbyBpResearchundestd mumber023-066wasperformed in compliancewithEPATSCA GLP (40 CFRPart792). STUDYDIRECTOR: SaAn Glaligbir h Senior BoAvgian Teoscc,logy de BATE SPONSOR'S REPRESENTATIVE: Tog News4. BA3TEh oy "0738 ---------------------------------- Wildlife International, Ltd. ProjctNumber 454-108 5 QUALITY ASSURANCE STATEMENT by th UTSi.ssEvdiwasseuxamPirnoeedctfioorncoAmgpeloisen,ce4w0iCtFhRGopoadtLa1b60o,ra1t7orAyuPgruasctti1c9e85S;taOndEaCrdDsaPsrpinucbilpilseheodf $Ga5ot5o0sd,anALyagbonnrdaetiontrgyaswPePrarrcoetdiruceecpio(orEntNeBdV1u/0rMheCe,/SC1tH0udEAyMuDgiu(r9se8ts)to11r978)a4;n.danTLdhabeJoadrpasatonersMyofAMaFaFln,gae5um9etNnoathnSwdaenri,enNsaoptfeioclfltiociwasaon:tndiNotohn.e Actviry DATECONDUCTED STUDYDDAITREERCETPOORRTMEDANTOA:GEMENT PTreenpSeunbteitoannce MaricFoistion FeedConnnpton sey 2,2001 Jeary 30,200 Fray6,201 rym Jonay31, 2000 Ronan Jeary 2,200 Jea31r, 20y00 Fearso0l CoBBnotdyionWeoingashts, Cunting TineCollcion B1e4ty0W0e0i8ghs gg Sh= el Ticaes Wscehlning Body Dubna Check DAravaRyepaonDas, ReRaownDua, Dust. FitReport February 6, 2001 Joe 12,200 une 19,200 me25, 2001 Jun 28, 2000 res August 1013,3000 NOovecmbe2tr95N,oo3v0e0bm0beer 2 r12d DeNcoevemmbbeerr 22.753,2000m1d up 20, 2003 Fabry, 2001 ne 12,2000 me20, 2000 Je35,2001 ne2.2001 hy, 200 Apso Noveber5,2001 December5, 2000 Ags February 9,200 ne 13,200 Otter, 2001 Jy ,2000 roe ays, 000 Au 120 November, 2001 Febru5,a2r00y2 Avo. QB ualityi.AE sCoulrenmeasnRe-presentative BATE9003 000789 -_ ee Wildlife International, Ltd. i" REPORTAPPROVAL Pj amber54108 SPONSOR: 3Coporton IME PROS: ARepStyrwillbecNotreBibwn WILDLIFEINTERNATIONAL,LTD, PROTECTNUMBER: 454108 MLABREQUESTNO. 12723 STUDY DIRECTOR: / ehbnav' lanTdoseo ELWel CHEMISTRY PRINCIPALINVESTIGATOR: 1, 'L. VanHoven,Ph.D. `Scicatist, Analytical Chemistry eluks DATE MANAGEMENT: e LN ner At74n/ny78 whV3 Cna790 Wildlife International, Ltd. 5. TAOFBCONLTENETS ProjectNumber 454-108 OYE cere ssesasmsavsrrmsmsmsssessmssmserssemmessemsrmanmmmosa J DIC SATs 14 AdultBody Weight and FedCOBSUIPHOD......ccvnmsmmsrsmsmmsmssmrsssssssssssssnss | T OffspringBloodandTissueCOEEHON nner sss 20 `EggShellThickness Measau ndEr ggCe ompm onee ntsn Collt ects ion ............ p-- Diet ABRIYHCR)RESUS... stenosessss 8 no7oL -_ Wildlife International, Ltd. `TOAPFACGBOEN2TLENETS Project Number 54-108 Oring OBSERVERS .crrercrevrresonssenesoeremorsosoon2n8 `TAABNDFLIGEURSES Table 1. Moeamn MeNoarstuhreerdnBCoonbcwenhtirtaetiRoensp(rppOmGa.ui.e)SioKfoYPnFOr Sin Avir anDietr 30 Tuble 2.SRuepmrmoadurcytoifoGnrSotusdPyawtihtohlPogFiOcSal,AObdsuerlvaBtiirodnssfFrooumndaDeNaodr/BruathBaonbzwehdite Table 3. MeanAdultBodyWeight 8) rom a Nother Bobwhite Fig 1.MeanAdslMleBodyWeight@)fom aNorthern Figur 2. MBoaonbAdsulRt FeomapleBIodoSy WGYeicgWhEttDgPiRrOooSm naNorr ther over "Table 4. MBeobawnhFieteedRConesumpptrionSo(g/yubirWdi/idaoyP)RfnOroSm vr a Northern 35 ! Figure 3. BMoebawnhFieteedRCoenspumprtioondS(gO/ubYircWdH/idRayPo)RfOrnoSm avNorethernn eer 36 Table S. SBuombwmhairtyeRoefprroodsuscPtaitohnolSotguidcyawlOibtsherPvFaOtSio,nAsdfurlotBmiarNiorBtuhtebranized i Table 6. MeanLiver Weighs(2from aNorther BobwhiteReproduction Table 7. SBuombwmhairtyeoRfReepprorSudPodefycodWriimlausoPeRecfnOoSt.maiNovrheera 3) | nz Wildlife International, Ltd. TAOPFBACGOELNTEE NTS PrsjctNumber 454-108 Figure 4. MeanRepro Perfd ormu ancecfrt omaiNov rthe ern Table 8. Summary ofReproductivePerformance, Normalizedas Percentages(%), from a NorthernBobwhiteReproductionStudywith PFOS...........41 Figur .MeanReproductivePerformance,Normalized ssPercentages(%), fr aNoo rthm ern Bobwhite ReproduSctutdyiwiotnh PFOS.........cvwuvesrsninn 42 Table 9. Moan Eg Shel ThicknessMeasurements (mu)fom aNortherBobwhite Table 10. MeanBody Weightg) of Hatchling sd 4-DayOldSurvivors fr aNoo rthm ern Bobwhite ReproSdtuudcyWittihPoROnS ........ccvoevmssnees 88. APPENDICES Appendix IL. DietadSupplementFOMDUIAONS ccc Appendix TV. TheAnaof lPFOySinsAVis Diet.ccvccrrcrreS1 Appendix VI. ReprParaometedrsanudRecplicta GiEaBv6aten. Appendix VIL AdultBody Weight()fom a Nth Bobwhite Appendix VIL FBeoebdwhCiotnesRumepiponr(boinSdddtaIyy)cWirhomPosRNOnoSrthern Appentix TX. NIonrditvhieduralBoGbrowshsiteRoleogpicralOobdSscuyrcvWatitthifirooonmnsa POS... Appendix X. AdultLiverWeights(g)from a Northern Bobwhite cv 68 85 "9793 e ee------------------------ Wildlife International, Ltd. ProjectNumber 454-108 a TABLE OPFAGCEO4NTENTS AppendinXIIA. TheAnalysis ofPEOSin AdultRedBloodCll80058. .cvcerrcrc 21 `Appendix XIB. TheAnalysis of FOSinAdultsadJoveileLiver83d Set... 134. Appendix XII ReBporowdiucetiRveepPreodrufcotrimoanncSebyyPweintfhrPomRa NOorthSern... 148 Appendix XIV. ReproductivePerformancebyWeeksndPenfrom a NortherBobwhiteReprodStuudcyWtitihPoFOnS w...cvvvvesnsns 170 Appendix XV. Appendix XV. AppendixXVIL AppendixXVIIL EoggmSheNlolrTthhieckrnBeosbswMheiatseurReempernotdsi(cmomn)SpaeryPewinbthyFWOeeSk... Meosma HaNotrcthhienrgBBoobdwyhWietiegRhet p2)rporPdeuaScbtuytdWiyewoeiknthPROS... MeWeaenkf1r4o-mDayNOolrdtShuerrvBiovobrwhBiotdoyReWperiogdhutc(ti)opnerSPteuadybwyith OfR fsprinT gLivSerlO WeyigWhthsS (Pg)FfOrSom r a Nortr hemBor bwhite 187 189 PFO... 191 193 Appendix. XX. Personnel InvoilnSvtuedyd. rnl96 chev Wildlife International, Ltd. Project Number454-108 -5SUMMARY STUDY: PFOS: AReprStoudydwithuthecNorttheinBoobwhnite SPONSOR: 3M Corporation WILDLIFE INTERNATIONAL, LTD, PROJECTNUMBER454108 TEST DATES:StEuxpdeyrinmentaal Sat-rJt Juierayro22y,22n30,021001 FPEiholg ostmSg ueltaoAs np-.eat1c2h,21030,12001 AAnadluyTtealrmTeirmninaatt--iiJounosn-eAp19r:i20,1250,021002 ExBpioelroigimceanltTamTiernmaitniaotnio-n lyAp2e3,l21050,12002 TESTAMMALS: Northerbobwit (Cols igi) AGETESTANIMALS: 26weeksofsge at he titoof theest. SOTUESTRANCIMAELS: Mo18r5i70sQSuWa.i2F32aSmeIntc. GUoSulAds, Fisica 33170:5399 "NOMINALTEST CONCENTRATIONS: 0,10, 50, and 150 ppm .. RESULTS: fTheecret wperoenrbcoadmyewnetgrchateadndmofrteaelditcieos,nsouvemrptisoingns1tofheto5xi0cit0y dand15r0epstpmmentr1.eltatted hcoencrenteratpiorns.opdThaush5ec0oatefndtihev1t5e0espt.pmThea.re ewxetrce onoanppcareentnwnretaemrteenartmtiatiiedoperfinfoer0scts uWpeorne obvoedryt swieiggnhtoftoorxifeceidtcyonabssuemrpvteidoninaadutlhtes1a0t tpohm1a04.pptemstac1,onceetntrcaotnicoenn.traTthieorne. foAvdeedricattlilonraoplblrsyeo,ruvtcehidrveeaiwpeeterheforem1da0uncctpieopnamts tianh1e1s0tteeptspsmcizo.en.oaentidmstirocenos,nmceetnnha.ieolnna.toe-dBceafbsfeeicdtes0lyulepetodhne | CJeosncehnatrnat1i0opnfmor.o.trheelowBeosbtwhciteoenxpcoseettnoesPtteRdOdrSuarinottthiisditeotywni.s determin(e0 be n27as5 _--_-- Wildlife International, Ltd. ProjctNumber454-108 on INTRODUCTION : Thistudywasconducted by WifeItrnatonsl,Ld or 3MCorpottreaWitldienonr ational, Lid. avin toxicology faci in Easton, Maryland 21601. The biological portion ofthetet was condromuJacuarty23e, 2d001 wJa ly 23,i2001. Ravedtsgenerated tWildenermsionslLi,d and copyofthefinareport re filedunderPrject Number 454-108inarchiveslcatdonthe Wile Internationa,Ld se. Biological specimens cestore st3CM rporaon, St.Pal,Miaoesota 55133 oBsECTIVES Th objectiveofthis studywastoevaluatetheeffets uponthe altnorthernbabwhite (Colima Virginians)ofdicary exposure ote potassium tof PerlvoroocaneSulfonic Acid (heres ered 10 83 PROS)over aperiodof approxim2a1tweeelksy Eftonalt healt, bodyweight,andfeed consuwmerpetvaiunotend. I addition,the effectsofadultexposeto PEOSonthe umberof gga 1d, fry, cmbryo viabily, hachabily, offspring survival, and egg shel thickness wer evaluated. Histopatologiel examinationofselectedteu sudsoalysesof lodand suesampleswere ls used 0 vasethe fects uponailsexposed 0 FOSand10hioffspring EXPERIMENTAL DESIGN Northerbobwhit (80malsand80females)wererandomlydistributedinfoon contolgroup nd thr seamengroup.Thetest concenrtionswe selected icosultonwittheSpons,based uponth resultsof 8 plot reproductionsy(Wife nematioal, Lic. Projet Number 454104) and additonal oct information provided by he Sponsor. PFOS Treatment Groups Grow NominaGlpCommacmton PGenrsowp BHexmiaPteolnosg-y _ MBalietsspFeermFaelnes 21 10 (Control) 0 1166 4s 1va1 3 s 0 150 16 s ra 1s s FE . i "0796 ------------------------------------------------------ Wildlife International, Ltd. Project Number454-108 "ie Bach treatmentandcontrol groupcontained16pirofbirdswithove oseadonefemaleper pen.Fouraddidonalpairofbinds weeaddedtoeach test level (fora totalof 20paisperleveland maintainedforcollectionofbloodsamplesduringthetest.Thretregaroutps wmereefendditcs containing iter 10, 50,or 150ppai.ofPFOS. Thecontrolgroup wasfdditcomparabletothe retmengroups,botwithouttheaddin ofthe estsubstance. Because avertsignsof arwiercenottedyat the 150pm .. etcocentratteidoitnr,y concentration forte 150pm ..rstmentevel ws duced 020pm ..ofPOSat hebeginning of We3ofethekes.The20 ppm i. levelwastermitealr Week 4ofthe estdueto heltd recoveryofbids ati test conocatation. Forreporting urpass, he 15020pm a.fstconcentration Willberefered 1a th 150ppm a.testconcerto. Alsinte 50pn ai. etcones weecutbanizedduringWeck 7ofthetestdutotreatmentslatedmortalityadovertsignsof icy. Therefore,thetestwascompletedwiththecontrol groupadthe10pp. etconcentrationonly. A adultbidswere observeddalythroughotthetes orsignsoftori orsboormalbehavior: Adult body weights wee messured at test nition, on Weeks 2,4, , 5, an a aul cminaion, and feed consumption wasmessedweekly throughoutthetest. AtthebegiofnWeeik n8,tghe photowpaseinrcrieasoedd10 inedg purodcuctieon. Followingth artof ggprodugctsiweorens,ek weekly for incubation. Weekly, ges were selected by indiscriminate draw fr egg shel thickoess messoremeotandal remaiingeggs werscandiedpro10incubation to detect ggshellcracksox maobrnaolrimtayl. eOggns.DayEgg2s1 owferecuablastoiocna,ntdlhed etgwgiscweedrueripnlgaciendciubnatioancthoedsetnecdt ailnfleortwieled eogghsatocrh.emObnrcyeo aching was completed, htclings were removed fom the bather nd te group body weighofthe Hacbhypelnwaisdnetergminsed. At 14daysofage,th averagebodyweightbyparentalpen ofal surviving oftpriog was deteepined. Upon completo of th ts, satistical analyse were perormed 0 determin satstcaly igaificantdiffrence between goups. MATEARNDIMAETLHOSDS Thestudywasconducted according to theproceduresoundinthe protocol, "POS: A ReproductionStywiththeNorthern Bowie". The protocolwasbasedonproceduresculined i the Environmental Protection Agency's Registtion Guidelines Pesticide Assssment Guidelines, FIFRA nore? c-- e ------------------------------------------------ Wildlife International, Ltd. ProjectNumber454-108 n- Subdivision E,HazardEvluaton: Wildlifeand Ags Organisms, Subsection 714; OECD Guideline 206;andthe ASTM "Standard Practice forConducting ReproductiveStudies with Avian Species" (1,2,3). TestSubstance "Thetestsubstance,PROS,wasreceivedfrom 3M Corporation on October 29, 19n9dwa8s assigned Wildife Inertial, Lid. identification numbes 4675 upon ecept. The fest substance was a whitepowderandwas identifiaesd: FC-95;Lot217. The tstmaterialhad reportedpurityof 86.9% and expiration dae of August 31, 2006 (Appendix ). The tes substance was held under ambient conditions i locked lrageat theWildife ntrtional, Li, faciniExltoni,Mtaryelansd, Concentra tions ofthe test substanceinthedietwereadjusted to 100%activeingredient,Therefore, dictary concentrations ar expressedaspartspesmillionactiveingredient(ppm 1.)intheie. TestOrganisms Onehundredandseventy-seven (90 malesand 7 females)pen-earednorhembobwhitwere purcfrohmMaorsiseQuadil Farm, In. 18370 SW. 232 Stet, Goulds, Florida 33170-5399, US.A. At `wthielsdttayrpteo.fTahccelbiimradtsiowne,rtehferboombwthhiesteamweebraetacphp,aarpepnrtloyach hine g teaa indpfl ihresntt ortyeph ciicnay lglsyeiansdoisntainngduihsahdabnloetfbreoemn usedin anyprevioustesting. At thesar ofacclimation, arandomnumbergeneratingfunctionia 8 spreadsheet programwasusdto randomizepenassgament forcsch bird. Immedpirintolfeyst inion,allpotentialstdybirdswere examined frphysicalinjies sadgeoealhealth.Birdsthttid notappearbea, ithe due oinaryorinabilitytosclimat o laboratory conditions, owere outside he weightrageforth test,wereexcludedfomthe study.Albirdswere24weeksofage atet iiation (frstdayofexposure otetdit) andrangedinweightfrom 177 1 250gramsat etiiaton. Sex ofthebirdswasdeterminedby avisal examination ofth plumage Identification Adu binds were identbyiinfdiiviedudal legbands, schpenwasidentifiedwith auni number, and.grooufppensswereidentifiedbyprojectnumberandconcentration. Excepteggs laidbybids maintained fobloodcollection, all eggs id drin the study weremarked withtephen number using 3 permancat ikmarkingpenforidentification. Hatchling were identified bylegband sothattheycould betac to herparentalpenof orig C0798 - JWYilVdAliyfee IInntteerrnnaattiioonnaall,,LLetdd.. se PPerkojeecNt eNummbbeers4t54h-i10o8s AvFeeid anadWanter testing.AThaedblatsbailnddsienfdedthtio ebooftfhsapdruilntgswaeregofifvsepnrfinog dwaansd wfoartmeulratdeditooWidfureontcecrlmiamtaitoiaoln,sLnid. specbiy AfgviayIcn.a(AptpenidioxI,nTabsle 25% 3dnomore ham 56fie, 1). Th bse aoncontainedat est 27% proteinand Thebsedit contined pprosimately 11%calcium,derivedfromfeedstuffsand he 09% gmreowstohnasduemdinnttahseafcoermraotinosn,aodfdtihtobnaasl adliieabyAi greq.uirWehiilethiesrateivoenlooffbrceeudimngibssidfsfcocfgr shheelblafsoarlmdaiticotn.fTohretrehfoardeu,ltasnaddTihtiisonails5e%dt(hwe/wc)aolf cliummestIovneei(apptrhoexdiimeattelfyr38t.h5e%bCread)iwnags.abdideddttoo approimate3l%y,slighty shove theminum recommended forquail (2.3%) ndmala (75%) 4). Offspringrecivedbas dictwithouttetsubstanceandwithout he addin of 5% supplemental ones. Wate was supplied by th twofEaton public te supply. Al offspring rceveda water solublevitaminand electrolytemixintheirwate(AppiaI, Tble2). Neither te adults or offpring recivedayform ofmedication nhfed dingthets, Feedaad terwer accordancewith WildnteoaiaL,i.StandardOperaProcscires. alyzedperiodical in Dict Preparation Testdietsvere prepa bymining PFOS oto apri.thatwasusdforweeklypreparation of hc iondit.Control dietnd ach rete diewererereweeklybegin on Jue 2, 2001ad presentedtothebinson Tuesdayof cachweek.Ditaryconcewneetdrjsatdftoripitoyonftshe {et substan sdarepresente sparspemilionaciveingredient(pma... Deofahelweeksly repationoftestndctrl isaeshownin Appendix I [gies] _-- WWoihldil.ifc e Inc ternd atio. nal,ScLitd. I Proc jctNumo ber45i 4-108 sa "a Diet Sampling Homogeneyofthe estsubtas ithdietwas exaltedb collectingsixsamples rom cachof thetreateddietsandonesamplefromthecontroldieton Day 0ofWeek1. Sawm erep coll lecte edfrs om thetop,middleandbottomoftheleftandrightsectionsofthemixing vessel. Conantdtr reao tmelnt tgersotupsdubisettasnacmeuplnedsewrercehalasotcesotllceocntdeidtfiornosm.thAedfdieteidotnraloluyg,hsasoanmDpalyew7aosfcWoelleekct1etdofarsosemstsshtcaobntiololifattnhyde reamengroupdics,when vailbl,ding Weeks 2,3, 4, 8, 12, 16 nd20 ofthtett0messureveify testconcentrations. Thedietsamples werestoredfrozenortransferred immediatelytotheWildlife International,Ltd.analyticalchem facii litysfot ranarlysyis. Analy"tTihcalmeMtehtohdodse frthe caysof FOS i avianditwasbaseduponmebodoloydevelopedat Wide atermsional, Lu. an entitled "AnalyticalMethodVerfisio orthe Determinationof PFOS in Avian Die" (Wide Intemational, Ltd. Projct No. 4S4C-10. Tee modifications from the cited methodologywereincorporated nt th presentstudy. Thefollchaongewsweirenmadge. (1) wHaisghuspeedrrfaotrhmearntcheanlsiqiunigdlcehqruoamdartuopgorleapmhayswidthettrciptleonqutarderduupcoelpeomeanstsidlemtaecttriioxni(ntHePrfLeCr/enMcSes/.MS) @)The intemal standardizationprocedurewas limitedbecause a apropeinte cold not be dented eralstandard ) Ashorteranalyticalchromatcoolgumrn(a50pmhmyv8100mm)wasusdfo expodicanalysis . Sampleswereextractedwithmethanol, vacuumfire,iedinmetbanol,and thendivedin P50O:5S0 mmeetthhaonodlo:logNyA.NOApumreethowdatferlodilcuhtairotn isoplrvoveisadotedthiant AtphpeyenfdelilxwiItVh,inFitghuerecal1.ibrCatoinoncernanagteiooftnhoef POS in the sandards aod extractsofthe samples were determined byreverephase HPLOMSMS wing Howe PackardModel 1100 High Performance Liqid Chromsograph netics wih Perkin. Elmer API 3000 Mass Spectrometer operated in mulipl reaction maniiring (MRM) detection mode. he mass spectrometer was equipped with Perkin-Elmer TusboloaSpayfo sure. HPLC separations were achieved using aKeystone Bets Cy nalytical column (50 mam x 2 ms LD., 34m particle siz) 2800 --_--_--,,,----------,----,---- L Wildlife Inteo rnational, Lt4dn . a ProjectNummbere4, 54-.108 -is- itedwith KeystoneJavelinC18GuardCartridge (20mm x2mmLD).Theinstrumentparametersare. summarizeidn AppeIVn,dabilex1. Calibration standardswerpreparedin 0-50methanol; NANwOaebpyapuprorprieditions of8 1.00mg aLstocksolutionofPFOSinmethanol. Thecalbtardardas,rtangoingnin pcromoinenat pcefreormesnp0o.n0ts0e1f0ro01r aP0R.O0tS10w0imasguo8l.in1zL,dw1e0rmeaotno aPFwOliStihyneaazclhlsceaamlpdilbersoet,. Tquhaelistycoa narnodl,mmoasntd Lsitnuedayrsrueigpriess.siNoon aequtattiownsaswemraedgeetnoqeuaanctdifsyiPnFgOtShoe npethae kbaarsesa.ofreisndpiovnisdeusalveisrosmuesrtichceormepsopneecnttisv.e conceonfthte craliabrattioin sotanndarsds, An IV,Figur 2.TheconcentrationofPFOSin avi ampleof 8calbrationcurve ipresatedinAppendix feed sampleswasdeterminedbysubstitiogthepeak acrleiabsranitoonstthaendaaprpdlsiacarblsehloiwnenarinrAepgrpeesnsdioinxIeVqu,aFtiiogn.ureTy3apidcal4,iroenspecchtriovmealtyo.grEamxs oaf mloofwpeaqlnudaetihoisngsh usdincalculations ars presented inAppend IV,Table 2. "Theinstramentlitofdetction(LOD) ortheseanalyseswas 10.0pgon-<olumn, calculatedfrom heproduc ofthe injectionvolume (10.0 uL)andthe loweststandardconcentration1.0 tg :/L= 1.00 988.4L.Themehod ini ofquantitation (LOQ) fo theseanalyseswis 2.0 ppmai clcultedssthe mpraorduxcbloafntkheslaomwpelset2c0a00lLIKi).bstEraxnadamaprtdlaeilsyoozfcenadu(t0i00o1nmsgre1p/re1s)enatneddtihneaAvpepraeladiilIaVt,oTnabflae c2otfothre Along with the sample analyses, seven matrix blanks were analyzed to determine possible | i i(ntAerpernpceesI.Vn,TNdaboilinetx3e)r.feAretnycpeiscwaleroen ocbhsreormvaetdoagrtaomroaf bomvaettrhiexbLlOakQdiusrpirnegsetnthedsianmpAplpeeanndailxyIsVe,s | ! Figures. | Avianditsamples were forifed atthe following levelsandanalyzedcocuretlywiththe samples10 determinethe meanprocedural recovery, The fortificationlevels were 5.0, 500,and ! 200ppmai. forsaminteprvalslWeick1n,Dagy0 We1,eDayk7 Weck2, Day 0; and We3,eDaky. The fortification levels were 500, 250, and 100 ppm .. for sampling interval Week 4,Day 0, The fWoertcikfic1a6t,ioDnalye0ve;ls5wdeWreee5.k0200a,nDda2y50..0 Tphpmem.e.tfboodysaimepkldidngmeinatnerpvraolsceWdeureakl 8e,coDvaeyri0c;sWoefe0k11%2,, D1a0y1%0,; cnosoL WWuidAldty.eInnttoemmaattiioo,nnaall,, L1b1s].~~ vritebnr5e108 iA1i,s1,e0A.p0Tnl5uie,3n,pShecpmepoersms4o,25s0,5i2w0oe10e ,en2085Ar ep ct n bfols Study Phases Ea igeTwe i-- estt--u --- vs. `Theprimaryphasesof thestudyandtheirapproximatedurationswere: 5. joPost-adult termination (final incubation, hatching, and 14-day offspring`rearingperiod) - 5 tHeiNTonromdgREcatemGeossnpC3o.ioon honce etiesteheeles ld srSteuhreeednmnbeTy GeheoorsgsiabQutaSilcoFgaarrmrtMavnmutfacoturiingad(GQeFMlnMooddeldNgo.tt0r330)o, meeoaseurimnsg ape proximataerly 25 X e-- e ------------------------------ Wildlife International, Ltd. Project Number 454-108 a ThephotoperiodintheadultsorthernBobwhiteroomwas maintainedby atime clock. The photoperiodduringacclimation nd he fistsvn week of he festwas igh bursof ghtperday. The a`pnhdotwopaesrmioad iwanseincraetastehdat tloen1g7thhouurssotfhleiaghlt ptebrdiadysawtetrheeebuetghiannniiznegd.ofTWehekr8 too tiunedugectehe,gtogelabuyiitngs recivedameanof approximately 199 ux (18 .candle)of mination provide by fuorescat ight hat closelysppronionm-daaystuneligdht Observations "Thetestbidswereslatedtotefui and studypsfor 8weeks priofo iio ofthe est. During acclimation, al birds were observed dily. Birds exhibiting abnormal behavior or debilitatingphysicainjureswerenotuedforthe est. Duinthe study,all acu bindswereobserved aly for ignof ticity orbaormal behavior, Additilolonffasplrinlgwyer,eobserveddaly rom itchingui 16daysof age. Arecordwasmitoofl mortalsandcll observations. Adult BodyWeight and Feed Consumption Aultbodyweightsweremeasuredat et tintin, on Weeks2,4,, 5, odatsulteminatio. Bodyweightswere otmeasureddurin cg layingbecauseof hepossibleavers fectshandingmay haveo eggproduction Feedconsumption foreachpenwasmesure weeklythroughout he test.Feedconsumptiwoans determinedbyweighingthefshlyfilledoderonDay 0, recordingtheamountofanyaddon!diet `a7d.deAddnuraitnmgpthtewwaesekm,aadnedwtemiignhiimnigztehfefeededewraasntdagreebmyaitnhienbgifrededbaytuthseienngdexotfetrhneafleleymdionugnptereidofede(dDearys deswitiha"gfsntaeve dlp. Theamount offdwastedby tebidswasnotquantified,sncte wastefeed wasnormalyscene and mixed ithvateand excreta, Thefredecofnsuomptrion,s presentedasanestimateof totalfedconsumption. AdBlooud Colllecttion Anadditionalfourpairsof adultbirdsin cachts eve weremaintainedforcollectionofblood samplesduringth test.Thesepaiseeivd he samedietandwerebandiedi hesame aneasabe pairinther estgroup. However,because ofthepotenti tes oftheblood collectionprocedures,daa from thefouraddionspais wre not used toEvaluetesmen else fet.Blodsampleswere 0503 Wildlife International, Ltd. PrsjctNumber 454-108. .cllctdfrom birdsinthesedesignatedpairs,whenpossibleatfiveweek tervals roughontthefst. A te imeofadult tcmiaation, blood samples were collected fom alsurvivingbids, when posse, priotocubasaia. Al bloodsampleswer separated ntoserumand bemacytesltelets,soredfrozen and shippedtothesponsoro sponsor'sdesignateforpossible nays AditNecropry and Tissue Collcton Whenthremabiirndsfironmthge 150ppm a.treatmentgroupwereeuthanizedafer Week , 0 aecwoepepesrfoermesd. All othe dalbidstat dieofwere euthanizeddinthe corseofthe studywere subctd 0 8gros necropsy.ALtheconclusionof heexposurperiod,al survivingalt bidswerechancedwithcarbondosidega,neopsed,anddispsedofbyincineration.At thetime of necropsy, issues were collected for histopathologial examination and analyses. When availble, samples of gal ladder, live, proventriculus, Kidoeys, bai, gonads, burs of Fabricio and adipose tisuewere fixedin 10%bufferedformalin. Histologysamplesforthcontrolgrovpand 10ppm i btirle,atmievnet gprroouvpenwterieclsuh,ipkpieddnetyos,EbPrLioi,nrHeeprmoddounct,ivVeAofrograhnisstaonpatahdoilpoogsye. tWshaenwearveaisltaoblree,d sfaomzpelnessnodf sipped 1th Spofnospososibrle analysis. Fecal samplewers ls collected, when available,fom the lowerdigetrascttanidstvore ed frfoorzposesibnle analysis. Egg CollectionandStorage Eggswerecollecteddaly hom testps,cleaned ndstoredin 3coldroomul incubatiooxn disposal (orthos pens usdforbloodcllction). Thecold roomwasmatin at meantemperature of 135C. 02C(SD)with amean evehumidityofspproximalely69% 6% (SD) Grooufpegsgs were denibey d anspbabei ltcod. Alleggs aidin weskly tervalwereconsidessreond ot. Candling and Incubation Atthe nd oftheweekly interval,al ggswereremovedfrom tecold oom,countedandeggs selectd by indiscriminate draw foreggshell thickness measurement. The remaining eggs were candied with a Speed King (Model No. 32) ggcaning lamp t0 detect eg shell cack or abnormal eggs. Cracks or sbuormal eggs were recorded and discarded. All eggstob incubated were fumigated with pfoossrimbailltdyeohfgyadpseatihnogaenn.airctoignhttacmaibnianieotnwiptrhioarci1r0culianctuibnagtifoann.foraFporpmraolxdeihmyadetetglwaoys hwouarss,tgoernecrdtuecde tbhye ron504 ---- er ---- ------------ -------------- ------ Wildlife International, Ltd. ProjectNumber454-108 combining26ofpotassiumpermanganateand 25mlof37%commercial gradeformal in porcelain Bow athbase ofthe itightcabinet. Aleggs notdiscardedo usdfor ggshellthicknessmeasurementswereplacedin Peers Incubotor(ModelNo.SP20).In heincubatortetemperaturewasainaioed atan average 375C & a0p.p0roCxi(mSaDt)elywi6t0h%)a.nTahveeriagnecbwaettobwualsbetqeumippepraetduwriethof 3p0u.l5stCo+in0.3odC l(SaDd)s(hraeltaptirvoedhuucmeidditmyilodf ratingsirmovement designed eliminateinrcabinettemperatureandhumidityvaiaiodaring incubation. Inonder reve adbesionofthembry 1 teshelmembrane, theincubatewasalso equipped ithen sutomaticeg rotation device,designed torotatethe ggsfrom50 ofofvertialin ne direction to 0 offofvencal ith appositedirston(olarof iainwas100%) verytwo ours through Day21ofincubation. Eggswere candiedanDay 11or 12ofincubationtodetermineembryo vail andonDay21 todetermineembryo survival, `HatancdhBrioodningg aloOtnwoDaehyat2dch1.oPfeidnicgubraeteiobna,sktehtesecgognsstwreurcetepdloacfegdailnvaanPiezteedrstiemleHwaitrcehmeer(sMhowdeerleNwoe.Sd-t6oH)Kaenedp hatchlings separated by parent! pen of origin. Eggs were not rotated in the htcher. The average {temperature i hehatchingcompartmentwas372C 00C (SD), ndthe veragewetbulbtemperature was ised103.3C 0 (SD) lative bidityofapproximately 7%). Abatching,unbachedeggs,aaeg shellswersremoved rom hehatcheronDay25or 26of incubation.Thegroupbodyweigh ofthe survivinghtclogs bypenwasdetermined. Htchiingswere leg banded for identification by peoforigi snd then routinely housed cconin to he sppropriste parental concentration roving inbrodingpes ntl 14daysofage.Theatcwlerifendungiessod it witthheaodudotn of$%spplrcnal limestone, At 14daoyfagse, heavebordyaweiggheby parentalpenofall survivinchicks wsdetenined.The hicks wereeubanizedwithcarbondioxideand disopfbyoincsineeratdion Hatchwelriehnougsesd in batteriesofbropo ensd mani ufan ctug red by Beacon Steel Company (Model B735Q).Eachpenmeasuredappro7x2iX9m0a X 2t3ceml higyh.Theexternalwallsand coosos Wildlife International, Ltd. PrsoctNumber 454-108 nlingsofcachpenweeconsretedof galvanized wiremeshandgalvanizedseeing. Floorsweeof avanizedwiemesh.Thermostatsinthe broodingcompariment ofschpenweeset toaise temperature of sproximately 38Cfomthtim ofhteinguni thebirdswere 14 day ofage. The averageambitromtemptwas260C. 19C(SD)with a averagerelative humidity of 9% & 13%(SD). Thephotoperiodforthbacbingswasmaintained by tim clockat16 ours of ightper a. OffBlosod spndTrissuieColnlectgion Proto cthanasioftels groupof 14ay oldafpria (Lot,blodsampleswerecollected from 10offpringinbotthecontrol groupandthe 10ppm a.treatmentgroup. Allbloodsampleswere separateditoserumsndhemacytepltlts, soredfrozen,an shipped thesponsor fopossible analyses. Additonal suesfromthe10 ffirin in cachgroupwerecolciedforhistpatbologial examination sad posible analyses. When availble, samples of gal bladder, lve, provensicuus, Kidneys,bri,gonads,brs of Fabriciusanadiposetsawerefedin 10%bufferedformal and shipped to EPL in Hemdon, VA for histopathology. When available, samples of bile, live, provenricuus, Kidneys,bri,reproductive organsanadiposetissu weresorefrozeaodshipped0 thespoorpnosisbleaonalyrses.Fecalsampleswerealsocollected romdroppingspansbelowthe floor ofbroodrs housingLot ofpringandtrefzforposible analyses. A inglecompositesample was collctedachfo th control groupan te 10ppm a. eament group. EtsShelThicknessMeasurementsandEggComponents Collection Weeklythroughoutthecg layingperiod,one gwascollected,whenavaiable,fomechofthe oddnumberedpensduring oddmumberedweeks (155,ec) an fromeach oftheeven numberedpens duringthe even numbered weeks (246, cc). The eggs from weeks 1, 2 and 10 were separated ino albumen,yo,shel andmembraneand llcomponentswere soredfrozenaadsipped ote Sponsorfor possible analysis. Eggs collected during weeks 3:9 were openeda the ais, andtercontents were removed. The eggcontentswereseparated nto albumenandyok, storedfrozenandshipped fothe Spoforpnossisbleaonalrysis.The hellwerethroughlyrinsed ithwatesndthenallowed10 idy fort eastoneweekat oomtemperatepirtobengmessedfor ices.The averagetikes of he ried hell plusthemembranewas deteninedbymeasuring ivepoits soundthewaistofte gg i usamiicronmetger. Measurement were made 0 he nearest 0.002 ma. C723506 --_------ Wildlife International, Ltd. ProjectNumber454-108 a StatisticalAnalyses Uponcompletionofthetest,ansnlysisofvariance (ANOVA)wasperformedto determine sasicllysignificantdiffeesesbetweengroups.Dunnettmultiplecomparisonprocedure (6)was used tocomparethethee treatmentmeans with th controlgroupmeanand ascss hesaistcal significance oftheobserveddifrnces. Sample nits werhe indivpiends uwaili ach experimental roup,exceptada bodyweghtswher thesampleuitwastheindividual bird. Percednattawgeree examinedusingDunnett's methodfollowingarcsinesquare roottransformation(see AppendixVIfor reSpyrsotdeumct(iAvReDpSar)aSmoefttewrsa)r. St&avtailsitidcaatleadnasloyfstivsaorfetphaeckdaagteawdaesveplerofpoerdmbeydWusiilnlgAiveiaaenrRaetprioodnuc,tiLoinD.a(t8)a. Eachof he followingparameterswasanalyzedsaitcaly 1. AdultBodyWeight - Individualbodyweight wasmeasuredattestinitiation, Weeks2,4, 6, 8and atadulttermination. Statisticalcomparisonsweremadebetweenthecontrolgroupandeach 2 AtrdeualttmFeenotgdrCoounpsautmepatcihowne-igFhiendgicnotnesruvmalpbtiyosneexx.pressed as gramsoffeedprbirdperdaywas examinedbypenweeklydiving he test.Sica comparisonsweremadebetweenthe contol andeach restmentgroup. 3. EogfgesgLgaiadiodfbMyaanxyiomnuemLfaeimdal-eThTehinsutmrbaenrsfoofrmeagtgisolnawiadspeursfeedmatlceodnivveidredhbey tmhbeleasrgoefstgnusmbneird 018pervcalueeosnthantoreiqual01e00 4. aanCrackedofEgeLad-The umberof gsdetermine by caningfo be cracked dividedby the number of eggs laid, per pen. i 5. ViableEmbryosofEggs Set -TheaumberofvisbleembryosattheDay 11-12candlingwas divided by the number ofeggsset,per pen. 6. Lwiavsed3i-vWiedeedkbEymtbhreynousmboferVoifabvlaebEimbermyborysos-T,hpeer npuem.berofliveembryosattheDay21 candling i 7. Hatchling of3-WeekEmbryos -Thenumber ofhatchlingsremovedfrom the hatcherwasdivided | `bythe numberoflive3-week embryos,perpen. 8. 1S n4-Du auyOor lfmhdav tcbhli ingesv boypfro eHna.tr chlis ngs - The number of 14-day old survivors was divided by the 5. Hotohiing of ge Sct Th umberofbtclingswsdivided byte umberof gssetbype. 4 GnO507 e rer re---------------------- Wildlife International, Ltd. ne PrsjctNumber454-108 10. 1S u4-Du amyOor lfdebgvsi etbv yrpoeo faE.gr gsSs et - The number of 14-day old survivors was divided by the 11. oHaftcehglingestoffoMmaaxniymounmSeefta-eT.heTnhuimsbterraonfsfhoartmcahtliionngwsapesrfuesmadlteocdiovnivdeerdtbtyhethneulmabrgeersotfnbuamtbcehr tingsto pretvalu qu ools an100, 12. 14he-lDaarygOesltduSmurbveirvoorfseogfgsMasext.imumSet -Thenumberof 14-day oldsurvivorsperpendividedby 13. was csr. EggShellThickness - Theaverage eggshellthicknessof indiscriminatelyselectedeggsperpen 14. vivir were messedbypavers en vp. Offspring's BodyWeight - Themeanbodyweightsofsurvivinghatchlingsand14-dayold 15. LeiverlWeLicgohtto~ifInfdiidnvigd.uaSltalitviesrtweigchotmspawreirseonmsewaseurreedaadteabduyltsteexrbmeitnawteieonntahnedcaotnttreromlginraotuipoannodf 10ppm. group. RESANUD DILSCUTSSISON Maturenortherbobwiiteeeived PFOS t nominalitary coneneatonsof10,5, 150pma. Due 0 overtsignsofoxcty note atthe 150pma. testconcethendiettarrycoancetntriatioonfnr,the 150pm .. ratment level was redfou20cpped.. of PROS atthebegianiogof We3eoftkhe test. The20pm.Ivewa emoalr Wee ofthe etd 0 he itdrecoveryof id at i level. dl the50s pma.tskconcenrionswerechanced during Week 7ofthe tetdusto restmeotrelted mortalityaad vertsigns oftoxiciatty tis evel, Theeltohreestwascompleted with the contol group 320 10 ppm .. tt concen only. Th cool group was ed dik comparabletothe resentgroups,butwiles tescion ofte festsubstance. Diet Analytica Rests Noneofthe contol semplesshowedanyindicationoftheprseace ofte estsubstanceorof he presenceofco-luting sbsances f the characeritc etn tie of he est subtace. Dict samples i werecollect omthe 10,031d150pp i. stconcertosonWeck1, Dey0, ndweresulyzed | 0 cvalat the Bomogeaity of te tst substance in th dietand to verity test substances coneatos. Mesos sodstandarddevintions orthetr stconcemtions were 8.15% 0313pmad 50.1 0951 | Pom i. 83d 147 431 ppm 8, respectively. The coefficientsofvariation were 321%, 196% and . "0308 -_-- TWiYldEliEfeIInntteerrnnaattiioonnaall,,TLtdd.. PreojeicteNuembbeer4s54s-1i08s ae 299%, repectively. These values represented 98%, 100%, nd S8% of nominal concentrations (AppIeV,nTadblie 4x). Sample collected in hetest ovr stsusan concernsfo te 10,20,50,asd 150 Pom .. dithdmeasand standarddeviionsof 102 0553pm a.208 2.14pm a,509.2 2.00poma, nd 161 15.ppm .., respectively. Thecoef ofvariwaerte i4o6,n 10.3%, 401% nd 9.66%, respectively. These values represented. 102, 104, 102, and 108% of moms concentration (AppendixIV,Table . Analysisofdietsmplescollectedfomfeedersaftebeing eld atambicattemperaturefr 7days averaged 108%, 100% 8nd 103%of heDay 0values orte 10,50ad 150ppm i. tentconcreespecttivelay(ApipenodixnIV,Tsabl,e6). Atypicalinchromatogorafm festsample ishownin AppendixIV, Figur 7. Meanweeklymeasuredconceaftoricaochnesst roupae preseinntbleed1. Mortaites Therewee a0 eam relatedmorales i the 10pm a.weskmentgroup. However, here were throincidentalmori inboththecontolgroupand 10pm .. rstrmowpedurinng tthe coofuthsstedy. Controlgroupmorales csrd during Weeks 14, 19and 21 ofthtest, Morintahe10ippmsa. ts groupoccduirgWeeedks 12,20and 21 ofthetest.Allmortals ihnattescgonntolmgorroalitaynws10cpavmedab.ytientcimdeennttaglriouypw.erDunostedtwietshhaeardloesfitonhsalensdioorn naebcmkernserdact accopey, one ofthemoralesinth 10ppm as. eae groupwerconsideredfo berelated to -- Fivemolesoars ith 50 ppmai. reamed roupadthreemoralesoecuredi te 150ppm i. team groups rinthe test.Twoadditonalbirdsin he150ppm i. groupwere cuhized du to their debilited condiions. All morals in he 50 and 150 ppm a. tet concentration ibid overt signs fxs pri dent sndwereconsidered eament lated. All retemsta.iNneicnogpbisridessawtetrheeu1o50ppeprmfoar.m.edesotnctohnecebnitdrasticounbwaesirzeeedufthoamnitzeeda15t0hpepbegain.nirneg softWmegeerknutop.ftAhlel |! omsbiidsantthie50pnpmga. test conoawreurteutihaonizned nd necrduorinpg sWeickc7odf thetes.Nesopsy dings aresummariniTacbeled2. 19509 _-- Wildlife International, Ltd. Project Number454-108 a ClinicalObservations Clinicalsignsoftoxicty wereobserved at alconcentrationstested. ACthe 10ppm ai. test conceiration,signsoftrictwerefisobservedduringWeek 5 ofthetest andconfinedntrmitcty `ntl adult termination. Sigsoftoicatthye 10 pm a. etconcentrationincl reducedreactionfo extemal simul (saondmuovenmendt), ruffled sppcarsnacned lethargy Signsoftoxicityatthe50ppm ..tetconcentrationwerefirstnotedduringWeek 4 ofthetest, Whenseveralbindswerenoted a ruffledinappesancesnd ethargi. Duringbodyweightmeasurements atthecadof Week4,severalbird inthe 50ppm .. exhibitedmusclespas and a os ofcoordination following badiing. Atthe 150ppm ..testconcentration, ign ofeicitywer istnotedduringWeek 2ofthe test whenoveindividualwasnotedas ruffle inappearance and lethargic.Albiridnhse 150 pmpmea.iatresatmuoefnttrhgeroeeuapdmoexfheiWbientcedkt2si.gnIsonf tsoexviecriatlycaisneds,ubcibnedydstchoensvturlessseodaf nhdba ecanmdeurd riignigl dbaoi dfyrwn beeiigg nhgt capture fo body weight measurements. Signs oftoxicity nthe 50 and 150 ppm ai.treatmentgroups continued ual uthandaincnhedsrediuced esttoiexotemnal stil (saondmuovenmendt, wing droop, lossofcoordination, lossofrighting reflex, lower limb rigidity, convulsions, shallow and rapid respiration, fled ppesrane, lolimwbweeaknerss, ergy, api,prostote posureadspasms. AdultBodyWeight Therewere nosparenrstneatrelate ffcts uponadultbodyweighta the 10ppm a. test concentration, and nydiffrence betweenthecontrol 7oupandthe 10ppm ..tet concentration were not stdstclly sigificant at any of the body weight intervals. However, there were marked, b`ceon5c0enatnrdat1io5n0-preospmonas.ivet,e sttrecaotnmceenntt-rraetliaotnesd rrientdeuiWnec meekt a2nbbiooddoyywwn eeiiggshhttfotrebrovtalh. males and femaleast Reductiiamoenens bodyweight continuedfor bot males andfemalesin he 50 ppm a. resentgrupthroughthe Week 6 body weight interval. Following reduction ofdietary concentrations to20pm1. dring Week 3 ofthe fest, males in the 150 pom a. tetconcentrations made slight weightgains a the Week interval, while female in the 150 ppm .. restmentgroup maintained meanbodyweight, Withtheexcepion ofmean femalebodyweight tthe 50ppm a. tetconsenraton ringWek2,aldifferencesfromth control groupwer tatisicllysigaificnt a p<0.05or 0.01. Meanbodyweightmeasuraer mreesneetdsin Table 3,andFigures and. IndividalbodyweightmeasurementsaepresentedinAppendix Vl "0510 _--_-- La Wa ildle ife Inn ternationala , Ltd. nTPd nrojevct Neummbern45i4-, 1s08 se Adult FeedConsumption Therewere no apparent treatmentrested ffctsuponfedconsumptionatthe 10ppma. tet conceniion. Nostatisialysignificantdiffcncesbetween th contol groupand 10pomai rcamentgroupwee observedatanyofthfeedconsumption tervals However, reament related eduction iafeedconsumpionwereabserved the 50pm ..testconccottionduring Weeks 1.6 sad athe 150ppm. testconcdeurinngWteekr1aandt2 ioftoh tnest. Al ofte reductiwoenres statisticallysignificant at p< 001 The dietaryconcentrationfo he 150ppm a. restmentgroupwas reduced10.20pm a. tthebeganingof Weck 3ofthts.Followingtereductionofthdicary concentrationto20ppm i.feed consumpionforth groupconfined obesiaificntly lowerthanthe controlgroupduringWeek 3ofthe test.Feed consumpion ath20ppm ..tsk conectionwas compaabietothecontrolgroupding Week 4 of the test.Mean eedconsumpionmessurcments re Shownin Table4and Fire3. Foodconsumption messurenntbypeaar resentedinAppendixVILL Adult NecanrdLoiveprWesighyts Alsurvivingadultsinthecontrolgrovpand10ppmai. tetconcentrationweresubjected 0gross accopsyfollduolwterimiaantiogn, Whencomparedto thecontrol rsp, theewasannese i the umber ofmaleswithsmalltestei the 10pp a. treatmentgrouptht wasconsidtereratemednt related. Onemaleinthcontrolgroupwasnedwithsll ees,compared 0seven alesinte 10 pom a. restmentgroup. Alterfinwdereiconnsigdersed unreflo aeatmeentd. Necropsy dings aeteportedinTable SandAppend IX. Whe comparetothecontrol rou,therswernospare treatmentelated ffsuponthe meanweights ofmale iver atthe 10pm ..tst concentration. Difbeetweeenmanleicvereweigshts for the 10 ppm .. restment group and the control group were sight ad not ttistcally significant. "Therewas aisticaly igaifican(p< 0.01)increas inmes fale iverwelghtat he 10 pm test concentration, when compartehde control group. Mian adult iver weightrse prescatd in Table 6, hileindividualadult iver weighsar prescatdinAppendia X. Histopathology rai,anNdo plresoivoensnctoinsuildseorfeaddruelltat1enddtooftperstioagrmtiacllee(sPaFnOdS)elademsin,isbtrrastioonfFwaebrreicnioutsedi oinfplieviern,gkimdanelys, and females, adipose tseofadult maanldfeemalses, ovariesofadult an ofpring female, and ests a31g --_-- Wildlife International, Ltd. PrsjoctNumber 454-108 5 ofmae offspring. Theincrease atheoumbe ofadult malsinthe 10pm i. ted groupwith reduce tsiclrsize,notsccompaidbyanymophalogiclchangeinspermatogenesis,sugasithat PROS may have scceented carly postreproductive phase regression, normal physiological phenomenon. Anypotentitaesltarticleeffectsontheburs of Fabriciusinadultmalesand elesor `adiposetissueinmaleandfemale offspringcouldnotbe determinedsincenone(bursaofFabricius)or efofnelcytsonoen t(ahdigpaolsbeltaidsdsueer)frsoammplreeswtaedsaslutbmimtatleeds faonr efveamlaulateisonc.oulIdnnaoddibteiodne,fianniytivpeoltyednteitaelrtmeisnt-eadrtdiculee 10th extensive utolyisthtprecludeanysurediagnosis.Thefewfechangesinvaistues ofadultanoroffspringbobwhitfromcontrolsod 10ppmai. residgroupswereconsideredincidetal andunrelatedto testarticle (PFOS)administration. Thefullhitopureptorbtisoprlovoidgediyn AppendiXx AdultLiver andSera Adult blood sample (bot ed lod cel and sr) collected through Wek 1 ofthe et were anabylBxyygenzReseeardch.Theressforanalysisofsamplescollected fortisstudy arereporcd {ogethrwith elsforthemallardstudy ru conuenlywitthe saetestconcentrations, Sma estsofbloodanalysesperformedfortis tidyrepresentedin Ege report Tables XXVIthrough XLlocatedin AppendXiIxA'Thefllreportforsaayss of boasamplesbyEsygenResearch fs TRoecdatBeldouonddCelr lExayngdenSSdyfe NouAmnbau elyrs0i2s3-U0s6i6n.gHP"ELxCt:raBctilonoefPcotraMsosaissusmSppPeecrrtfrlaoumreyotro/yctaneulfonat from Bloodand versamplescollected ttheinofacl rminationfortheconvo groupand10por ai. test concenation wereshippe to 3M EnviromentalLaboratory foranalysis. Summaryresus of seraan iveranalysescondacte by 3MEnvironmentalLaboratoryaepresented in 3Mreport Tables1, 2 nd 10 rough 15locinAappetndeXIdI.Thefllreprforanalysisof srandlvesamples collected at the imeofadult termination located in 3M Environmental Laboratory report EOI-1245. "AnalyticalPhaseReport for PFOS:AReprodSuacdytwiitohtnh Northen Bobwhite. Reproductive Results product`iWohneant ctohmep1a0repdpmtoit.heecsotntcroolngtroauip,nt.herHeowweerveern,o wahpiplaerennott satay treatment-relsaitgenidfeicfafnetc,tstuhpeornweegrge. Slightreductions i the maneofible cbeyoss percentage of eggs se, batchlings a peretage ! Co0B1R ---------- ee eee YWe ildlife International, Ltd. ProjectNumber454-108 - ofive3-weekcmbryosandin14-day odsurvivors asapercentageofhatchling.Theslight reductions infertility, batchabiity andoffspring survival loreal ireductionsinthe umbers ofhtclings nd 14-day oldsurvivorsaspercentagesofbot theumberofoggstandthemaximnumberof 3 ct. Whileslight,thereductioninth umber of 14<ayoldsurvivors 1s percentageofeggssetwas statisticallysignificantat p < 0.05. Summaries of thereproductivedata ar resendin Tables 7and5, andin Figu4anrde5. Reproductiveparsmeters bypenarepresentedin AppendixXIIandXIV. Egg ShellThickness "Therewere10apparent estmentrelated effectsupon ggshel thickness atthe 10ppm a.test concenraton. Whencomparedt tecontol group, ther were nosatiscalysignificant differencesin eashel thicknessin he 10pm a. rcemetgroup.Eg shellthicknessdataaepreseaiedinTable andAppendixXV. OffsBpodry Wieinghtgs Therewer10appar ratmentreltedeffects pon offpringbodyweight te 10ppmai.test h`ceonbceondtryawtieoin.ghWthoefnhtccoimipnagrseodrto1t4h-escyon0trdoslugrrvoiuvpo,rtshfeoremwtehreen1o0pstamtistai.callryesisgeninftigcarnotudipf.feOrefnpcersiign bodyweigh dat represented Table 10,and AppendicsXVIandXVI. OpringLiverWelghts ai.te`tTchonecreenwterarteionno.tArenatymednitf-frreelnatceedbeeftfweecetnstuhpon1t0phepmmeaa.nwtereiagthmtesnotgfrofofuspparnindglhievceorsnattolthgero1u0ppwpams not saisticaly significant. Mean ofspring lvr weights ar presented in Table 6, whie individual ofsrigliverweightsaepresented nAppendixXVIL 0081.3 _--_-- LWiLlUdlife IDnOteOrnAaGtiAonTa,l,SLtld.y eeeProS joctNumber 454-1E 08 a OffspTrhinegrOebsweerrveatoicoindsetalobservation ofneckcs, weakenedconditionandfootie lesionsand asocitedlameness,fo offprininboththecontrol groupand10pma. restmentgroup. All ther offspringwere nolin pearancesndbehavior oughont he et. OffsLipverr aindnSegrn Bloodandfiversamples calcdat the tim of offpringtermination forthecontrolgroup ad 10 pomai.testconcctratonwere hippedto 3MEnviromental Laborstory foranalysis.Summaryrests ofsera an ivranalysesconductedby 3MEnvironmentalLaboratoryaepresented i 3MreportTables 1,210trough 15locatedin AppendixXITB,Thefull eprtfranalysisofser an liversamples clalthcetimtofeuvde termination ilocated in 3MEnvironmentalLborsoyrepotEOLIZ45. "AnalyticalPhas Reportfo PFOS: ARegroducton StudywiththeNorther Bobwhite". concLusion `Therewerenoappareat treatment.relatedeffectsuponadultbodyweightorfeed consumptionat he10pom a. testconcentration. However,minoravertsigns of octwereobserved iadultsat he 10 pm a3. concentration. Addiionly thre wer redactions in tse ie, an sight, but westent elie,effects uponfrit,htchailtyand ffpingsurvivalate 10ppmai. fstcontin. Therewere eatmeotrelat morales,overtsigns of oicty 0d este elated effects vpon bodyweightandfed consumption atthe 50and 150pp .. testconcentrations.The 50and 150ppm a. testconcentrationswereterminedpriortothereprodistivephase ofth est. Baonstheeffdects observed athe 10ppm testconcent, hi o-obscrvecffctconcentfornorebowie expose {0 PROS inthe ictwasdetermined tbe Issthan 10pm ., helowest concentration tested ing tissy. 60534 C2315 Be11. - . i Tr 2 v 3 | fle + i Eiles eool HH li Hl Els 83) 5 o oe os s oe [iiEl : Flat Dad al al) [RE ii = ) 8 ' 0816 _-- s nmem ee 0 ProjoctNumber 454-108 `SummaryofGrossPTaathboll2eogical Observations from`AdauNlotrBtihdersnFBooubnwdhDiteeRaedpr/odBucutitondhSutrauidnngytiwhizetTehePsdFtOS Males Females -- TmT ai mma7i CG Orpmm mai))p0 pamali pmppsTmali ipmT maadi Numberofbirds 16 3 3 3 16 2 Featherswetaroundbesk Feather oss oo s0 1ooo oo Extnsivebead/necklesions. 0 0 12 Veat-pasty Thin 0 1o 1o 0o00 Emaciaied 11 1121 Los ofmusclemass Prominentkeel 21 11 1121 1 121 Intracranial bleoding 1 Neck -mid-lengthfracture o o oo 1ooo Neck -bematoma Heart 0coronary at 00 1o 111 1 1o ASbpdleoemnin-samlacalvaintydl-or pale 3 1 1211 autolysis Abdomcaviiyn-amalll 11 1121 amount of looseblood Crop empty oo 10 o10o 1 10 Gizbzialresdctoanitneentds oo 1 [3 0 Coca contents -pasty 1 1 0 o o Kidoeys-pale 0 o10 Ovary -developing -- o 1 oo Ovary-regressing -- 1 o Not remarkable 71 o 01 0 "Rbesneiing bifrodrbnidhsm5i0pneen.foectolcleocntieomnrotfobnowdeesseputnbiazeeodoGipoorWeeck 7of hs(x.Gromsropey _R`e--wmearienoiontpgebrifrodrsmiedtht1h5e0teprmnina.ti.oneotfctnece1i0ipoenw.e.rteetuctohnacneinzterdaatitohne.beginofWeck. Groseopies Cn0817 i} a ve... 3 Bf as os: oo oo | LI ~ Pl8 iss as Fa : i #3 BE 3 Em goer] i} Pees it . aig ! 1 % 88% &% - bei FL Hi > %% B% EZ E3 HERE] Hide JH : of if ad adad| fEiEE _---- mMMMm M m-m ProioctNomber 454-108 I | | EN ca1 1 =0 "a Se 231 "eel 551 EA a| 22 Hi [It 1. _---- e Io a 1 sa pe----al I 2 Pose Io. i efeSTISSSSSY $ 58 1 vo. gv PoAaow 1 E-- x 0 TT + mop 1 sx mss EL Emm 672349 EEE -- :3 i EE = : : C2820 ---------------- ------------------------------------ " Prject Number45-108 6hnoo5g21 ] GnO82T to '. 60523 C1824 . cross GnO8S26 HlI lefseessessenss| 31} Hy i A Jofroreseanste l il I E GU l CH H IeH Edi H En cnos2?