Document QgadoyKL3yYyzmRZ93oMjzqBE

A DIETARY LCS0 STUDY WIpTrOHS:THE NORTHERN BOBWHITE Ar226 _0953 ALiT SN WILDLIFE INT3EMRLNAABTIROENQAULESLTTD.NO:PROUJ2E7C23T NO.: 454-103 Series 8U3S0.- EEncvoliorgoincmaelntEfaflecPtrsotTeecsttioGnuiAdgeelnicneys (draft) `OPPTS Number 850.2200 FIFRA Subdivision E, Section 71-2 OECD Guideline 205 . 3 2 23 T4 3S2E zE358% = AUTHORS: CSoeuarntPn.eyGaSl.laCgahseery JRoaaynmnoBn.dBLe.avVearns Hoven I STUDY COMPLETION: April 26,2000 SUBMITTED TO . Envir3onMmeCnotraplorLaatbioornatory 535 Bush Avenue St. Paul, Minnesota 55144 | & Epa-oTS 0008317900 =s 55 3o Wildlife International, Ltd. 23 & Ea8s5t9o8n,CMoamrmyelracned D2r1i6v0e1 10) 822-8600 Page 1 62 001373 WILDLIFE INTERNATIONAL, LTD 2. PROJECT NO. 454-103 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: ADietary LCS0 Studywith the Northern Bobwhite WILDLIFE INTERNATIONAL LTD. PROJECT NO: 454-103 3M LAB REQUEST NO: 2723 STUDY COMPLETION: April 26, 2000 the"TUh.iSs.stEundvyiwroansmecnotnadlucPtreodtiencctoiomnplAigaennccey,wi4t0h GCoFoRd LPaabrtora1t6o0ryanPdrac7t9i2c,e S1t7anAduagrudsstas1p9u8b9l;isOheEdCbDy P1r9i9n2c)i,plaesnodfJaGpoaond MLAabFoFr,ato5r9y NPorahcStaicne,, N(oOtCifDiEc/atGiDo(n9N2o). 3328,50E,nvAigrroincmueltnutraMlonPorgorduacpthioNno.Bu4r5e,auP,ari1s0, August 1984 withth following exception: charTahceterteisztatsiuobnswtaanscepewrafsormnoetd achcacroarcdtienrgizteod3iMn aSctcaonrddaarndceOpweirtahtifnugllPGroLcPeducroemsplainadncMee;thhoodwse,vearn,dtahlel raw data are being maintained in the 3M archives. The test substance is being recharactrized in accordance with GLP was"nTohetsdtabeilittyeofrithnmeactiecsotnrsdueabnsdcteanwciethanGdooredfeLraebnocreasttoarnydaPrradctuincdeerStcaonnddairtdiosns of storage at the est site STUDYDIRECTOR: Md,' Pll SSeenainorP. BiGoallolgaigshter SPONSOR'S REPRESENTATIVE MI s" Susan A. Beach Leuk, pate Mao pate_74//22 001374 W--_I--L_--DLIFE INTERNATIONrALe, LTD 3. PRPOROCJTENCTON:O4.5454l-01033 QUALITY ASSURANCE STATEMENT `Thisstudywasexaminedforcompliancewith GoodLaboratoryPractice Standardsaspublished by the U.S. Environmental Protection Agency, 40 CER Part 160 and 792, 17 August 1989; OECD PrinciplesofGood Laboratory Practice, (OCDE/GD (92) 32, Environment Monograph No. 45, Paris, 1992); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. Thedatesofallauditsandinspectionsandthedatesthatany findingswerereportedto the Study Director and Laboratory Management were as follows: ACTIVITY DATECONDUCTED DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT Test Substance Prep. &Analytical Sampling April22,1999 April2, 1999 April23, 1999 Matrix Fortification~~Ap2r2,i19l99 April22,1999 April23, 1999 "Feed Consumption &AualyticalSampling April27, 1999 Apri: l 27,1999 May4, 1999 AnDarlayftticRaelpoDratta and ily7,8,9, 1999 July 9, 1999 July 16,1999 Biology Data and DraftReport . August 26,27,30,31,1999 August31,1999 September 13, 1999 Final Report April 17-18, 2000 April 18,2000 April 19, 2000 Supen . Susan L. Coleman Coleman Senior Quality Assurance Representative pate 4-19-00 001375 WILDLIFE INTERNATIONAL, LTD -4- REPORT APPROVAL PROJECTNO.: 454-103 SPONSOR: 3M Corporation TITLE: PFOS: A Dietary LCS0 Studywiththe Northern Bobwhite WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103 3MLAB REQUEST NO.: U2723 STUDYDIRECTOR: Ad 8-120 | Sean P_ Gallagher `Senior Biologist, Avian Toxicology Wve Date CHEMISTRYPRINCIPAL INVESTIGATOR: 4 AL LUfo RSacientist, AnalVyatnicHalovCehne,miPhs.tD ry A200 Date REPORT APPROVED BY: \ >is, Tews Baws __f2e/2e TDioruecltoBr., BAevaivaenrsToxicology Date WMialnlaagredr,B. AnNailxyotni,caPlhC7hemistry Date" 001376 WILDLIFE INTERNATIONAL, LTD 5 PROJECT NO.: 454-103 `TOAFCBONTLENETS TIEPAGE ssmeirmemsammmmsammmmmngrsssn Dg 1 GOODLABORATORY PRACTICE COMPLIANCE STATEMENT............c.... Pag 2 QUALITYASSURANCE STATEMENT... PGE 3 REPORTAPPROVAL... PAGE 4 TABLE OF CONTENTS... GES TaAb008AlPeEsS cers PAGE 6 SUMMARY orsrssssmsssmsmmmmantsssmassssmmmonmmminnn0nR DOIROBUCTIN.cummmsmmmmmsssmassmmommmsnnomnnkogh ORECTIVE ansmmmmmssmmsasmmmmnmssmmommmpsnnkigh MATEANR DMEITHAODL S..S. POG 8 TSB THCER mrmrsmsmrmhmmmmsmmm--m--m--mm--m--s--mm--m--s--gslliiggYh 4 0 eS SE FSURERINRSD,ccr rmmmmrmrsmrm mmr--e----m-------- ------_gPb age 10 DDIIRRABEIAIEPGRRorIoErninrnsremnmnmmnmmmmmmmmmm--mm------------------2 De1 `DHouOsiWngEanRd EavrirsoumentavlCeOngGOiRS.s..a..e..i...r.rsrrmrrmvrrmvrmnrmrrmer--rnr--rr--nrramPTBogSs 13 1 TAOniWmalNBoAdDy HWeightms/mFemcdmCOmBsSUeTPmIOp.a..s..i.mvvmrvermrvmrvmr--rr--rr--rv--e--vr--nrrerenenrePagGeE 1144 BARA AYR ccimsmmmmssmmmmmmonm------ gg 1 BESTS mmorpg 15 FO ----------E--------_-------- B'odyMWeoigrahtnadtnCdliaFnoiecdialCOOtRBSSiUIEPeIHOVsNA..O.R..S...r.e.rr.e.nr.evrrrerrrvrorrrrnsmnrsrsvrnsnervePPA2G8SS 1157 CHRIS crmmmmesmmnmmmmmmmmmmmm----------2egt 11 CONCISE mmm isons S38 18 BEFIREHCES sonnel BG 19 001377 WILDLIFE INTERNATIONAL, LTD "6 PROJECT NO.: 454-103 `TABLE OF CONTENTS - continue-d `TABLES AND APPENDICES TABLE: TABLE2: TABLE3: TABLE4: SCuOmuYlaWtiitvhePMRorOtSal.it.y.from a NortherBobwhite AcutneDientarny Toxicity 388 20 MSOeUanWBiotdhyBWFeOiSg.htn (g)from a Non rthernBobwhe iteAcuteDietr ary Toxicity P28 21 MeanFeed Consumption (g/bird/day) from aNorther Bobwhite AcuteDictaryToxicityStudy With POS. PAGO 23 GroupGross Pathological Observationsfrom a NorthernBobwhite `Acute Dictary Toxicity Study With FOS... riven Page25 TABLES: Cumulative Mortality(EstimatedCumulativeDose,mg/kg)from a Northern BobwhiteAcuteDietary Toxicity Studywith PFOS.................... Page 26 APPENDIIXI: Analytical Methods and RESUHS..........veesevseresnserensPAE29 APPENDIX IV: Diet BIEPAIRON....ccrrecrvrnsnnsnsn PAGS 43 APPENDIVX: CumulativeMortalitybyPenfrom a NorthernBobwhite Acute Dietary Toxicity Study With PFOS ...........ocesvererrr Page44 APPENDIVXI: IndividualBody Weights(g) from a NorthernBobwhite Acute Dietary Toxicity Study With PFOS ......v...vuesrvesvvrrr APPENDIX VII: Feed Consumption (g/bird/day)by Penfroma. Northern Bob Acuw teDh ietai ryTt oxice ity PagE45 APPENDIXVIII: Cha1n0Pg roteocosl ........cusurrsssssmsssssmssmssssssssssssssssssssssssssnses APPENDIIXX: Personnel Involved inthe SEY ....o.ovvvercrrrone S-- PAGE 61 001378 WILDLIFE INTERNATIONAL, LTD -7- PROJECT NO.: 454-103 SUMMARY SPONSOR: 3M Corporation `TEST SUBSTANCE: PFOS `WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103 STUDY: PFOS: A Dietary LCS0 Study withthe Northern Bobwhite RESULTS: The dietary LCSO value for northem bobwhite exposed to PFOS was determined to be 220 ppm a.i. with a 95% confidence interval of 164 ppm ai. 10 289 ppm ai. The slope of the concentration-response curve was 7.005 and the chi-square value was 0.023. The no mortality concentration was 732 ppm ai. Based upon treatment related mortality, signs of toxicity and effects upon body weight gain at the 146 ppm ai. test `concentration, the no-observed-ffect concentration was 73.2 ppm a. TEST DATES: Hat- Acprhil 12, 1999 . Acclimation ~ April 12-22, 1999 Experimental Start -April 22, 1999 Experimental Termination ~ May 14, 1999 NOMINAL TEST CONCENTRATIONS: 0, 18.3, 36.6, 73.2, 146, 293, 586and 1171 ppma.i TEST ANIMALS: Northern Bobwhite (Coins virginianis) AGE TEST ANIMALS: ~ 10daysofage atest initiation SOURCETESTANIMALS:~ W8i5ld9l8ifCeoImnmteerrncaetioDnrailveLtd. Production Flock Easton, Maryland 21601 STUDY COMPLETION: April 26, 2000 001379 WILDLIFE INTERNATIONAL, LD -8. PROJECTNO.: 454-103 INTRODUCTION ThisstudywasconductedbyWildlifeInternationalLtd.for 3MCorporationattheWildlife International Ltd.aviantoxifc aciloitylinoEag stoyn, Maryland. Thein-lifeportionof the testwas condufrcomtAperidl 22,1999toMay 14, 1999.RawdatagenerataWitldelidfe International Lid. and acopyof thefinalreportarefiledunderProject Number 454-103inarchiveslocatedonthe `WildlifeInternationalLtd. site. OBJECTIVE "Theobjectiveofthisstudywastoevaluatethe toxicityof atestsubstancetothe Norther Bobwhite (Colinus virginianus) administeredthrough the diet for five days. An LCSO value will be calculated, if possible. MATEARNDIMEATHLODSS `Themethodsusedin conductingthisstudyarebaseduponproceduresspecifiedinthe U.S. EnvironmentalProtectionAgency Series 850 ~EcologicalEffectsTest Guidelines OPPTS Number 8502200 (1), Section 71-2 ofthe Enviromental Protection Agency's Registration Guidelines, Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard valuation: Wildife and Aquatic Organisms (2); OECD Guideline 205, Guideline for Testingof Chemicals, Avian Dietary Toxicity Test(3); and uponASTM Standard E857-87,"StandardPracticefor Conducting SubacuteDietary "ToxicityTestswith Avian Species" (4). TestSubstance Thetestsubstancewasreceivedfrom 3MCorporationonOctober 29, 1998andwasassigned `Wildiife International Ltd. Identification Number 4675 upon receipt. The test substwaasnwhcitee powderidentified as: FC- Lot9No5 .:2; 17. Thereportedpurityofthetestsubstancewas98.9%,with anexpirdaatetofi2o00n8. Followingtesttermination,thetestmaterialwasreanalyzed.Theresults of reanalysisindicate atestsubstancepurity of 90.49%. Alltestconcenhatvrebeaentadijuostnedtso reflectthepurity reportedon the newCertificate of Analysis (AppendixI). Thetest substawnacse storedunderambieatconditions. `Theinternalstandardwasreceivedfrom 3M CorporationonJuly2, 1998and wasassigned `WildlifeInternationalLtd. identnuimbferi45c26auptonriecoein pt.Theintemal standagarrandul,ar 001380 WILDLIFE INTERNATIONAL, LTD -9- PROJECT NO.: 454-103 matweasridientaifileda,s: 1H, 1H], 2H,2HPerfluorooctane SulAfcido,Cnhemiiccal Abstract Number: 27619-97-2. Thestandardwasstoredunderambientconditions. `TreatmentGroups. `Thetestconsistedof ageometricseries ofseventestconceatrationsand acontrolgroup. Thirty northern bobwhite chickswereassignedtothecontrol groupandtennorthernbobwhitechickswere assignedto cachofthe treatment groups. Thebirds weresortedby weight,thenchosen indiscriminatelyfrom withineachrepreseatedweightclassfor placement into controlandtreatment groups. Thebirdswerehousedinbrooding penscontainingfivechickseach. Nominaldietary conceunsetdirnthaissttuidyowenres0, 18.3, 36.6, 73.2,146,293, 586and 1171partspermillion activeingr(pepmd.ii)oefPnFOtS.Thedietaryconcentrationswereestablishedbased uponknown toxicitydataandinformationsuppliedbythe Sponsor Each group was fedtheappropriatetestorcontroldiet forfivedays.Duringthe exposureperiod thecontrol groupreceiveduntreatedfeed. Followingthefive-dayexposureperiodallgroupswere givenuntreatedbasaldietfor three days. OnDay8, halfofthesurvivingtreatmentandcontrolbirds wereeuthanizedandlivertissue,blood,andbilesampleswerecollectedforanalysis. Theremaining birdswerefedbasalrationuntilDay22. On Day 22,thesebirdswerecuthanizedandalsosampled for liverweight,blood,andbile. `DuroaftthieToesnt "Theprimary phasesofthistestandtheirdurationswere: 1. Acclim-a1t0idaoysn. 2. Expos-5udaryse. 3. Post-exposureobservation- 3or17days TestBirds All northernbobwhite (Colinusvirginianus)were 10daysofageandappearedtobeingood healthatinitioafttheitoestn. Thebirdswereobtainedfrom WildifeInternationalLid.Production Flock, Easton, MDandwerehatchedon April 12, 1999.Birdsrangedinweightfrom 18to23grams. attestinitiation. Thebirds usedinthisstudy wereimmatureand couldnotbe differentiatedbysex. 001381 WILDLIFE INTERNATIONAL, LTD -10- PROJNO.E: 45C4-1T03 Albirdswerefrom thesamehatch,pen-rearedandphenotypicallyindistinguishablefromveldbirds. AXbirdswereacclimatedtoth cagingandfacilitesfromthedayofhatchuntiliniiationofthetet, imal Di Throughoutacclimationandtestingalltestbirdswerefod agamebirdration formulatedto `WildlifeIntemationalLtd's specifications (Appendix I. Thechicksweregiven avitamin supplementintheirwaterfrom thedaytheywerehatcheduntilthe intao ofthetest. Waterfrom thetown ofEastonpublicwatersupply,andfeed wereprovidedadlibitumduringacclimationand testing. Thebirdsreceivednoformofantibioticmedicationduringacclimationortesting. `is Pegaret `Thetestsubstance wasmixeddirectlyintotheration. Mixingwasdonewith aHobartmixer (Model Number AS200T).Alldietarytestconcentrationswereadjustedto 100% PFOSbasedupon the reportedpurityofth testsubstance. AlldietaryconcentrationsandtheLCS0valuearereported asppma. in thediet. Nominaldietarytestconcentrationsusedinthisstudywere18.3,36.6, 73.2, 146,293, 586, 1171ppm a.i. (AppendixIV). : DistSampling. Samplesofthetestdietswerecollectedtoverify thetestconcentrationsadministeredandto confirm thestabilityandhomogeneityofthetestsubstanceinthediets. Homogeonfethiettesyt substanceinthediet wasevaluatedbycollecting six samplesfromthe18.3ppma.i.a. estdictand sixsamplesfromthe 1171ppm a.i. testdietatpreparationonDay0. Homogeneitysamples were collectedfromth top,middleandbottomoftheleftandrightsectionsofthemixingvessel. The homogeneity samplesalsoservedasverificationsamples. Oneverifsaimplcewaastciolloectned from thecontroldietand twoverificationsampleswirecollectedfromeachremainingtreatment groupatpreparoantDiayo0n. Attheendoftheexposureperiod(Day5),onesamplewascollected fromthecontrolandtwosampleswere collectedfromeach treatmentgroupto determine stability of thetestsubstanceinthedietundertestconditions. Thestability sampleswerecollectedfromfeed remaininginthefeedersaflerbeing atambicattestpenconditionsforfivedays. Samples were transferred immeditoaWitldlliyfe International Ltd. analytical chemistry. 001382 WILDLIFE INTERNATIONAL, LTD -u- PROJECT NO.: 454-103 Analytical Method Themethodusedfortheanalysisoftheaviandiet sampleswasbaseduponmethodology devealtoWipldeifdeInternationalLtd.andeatited"MethodOutlinefortheDetermination of PFOS in AvianFeed". `Avian dictsampleswereextractedwithmethanol. Methanolwasaddedto arequisite quantityof feedcontainedinaFrench-sglqasusbaotrtlee. Bottles were cappedandshakenon ashaker table. `Sampleswerevacuumfilteredusingqualitativefilterpaper.Theretainedfocdwas rinsedthreetimes with metihntoathnefialtralte. The filtratewastransferredto avolumetricflaskandbroughtto vowlithumetmhaneol. Asappropriate,sampleswerefurtherdilutedwithmethanol. Eachsample thenwasdilutedvith a50%methanol: 50% NANOpurewate solution containing0.100mg 4H PFOS(intemalstandard) and0.05% formicacid (+)sothattheyfellwithin thecalibrationrange ofthe PFOSmethodology. Amethodflowchartisprovidedin AppendixI,Figure 1 Concentrationsof PFOSinthestandardsandsampleswere determinedbyreversed-phasehigh performanceliquidchromatographyusing aHewlett-PacMkoadredl 1100HighPerformaLinqcuied Chromatograph(HPLC)with aPerkin-ElmerAPY 100LCMassSpectrometerequippedwith aPerkinElmer TurbolonioSnpsoruarcye.HPLCseparationswereachievedusing a KeystoneBetasil Cis analyticalcolumn (100mm x 2mm LD, 3pmparticlesize). Theinstrument parametersare summin AapperndiixIz,Taebled1. Calibrationstandardsof PFOSpreparedin 250%methanol : 50% NANOpurewatersolution containing 0.100 mg 4H PFOS (internal standard)L. and 0.05% formic acid (v/v), ranging in concentration from0.00229to 0.0457mga.i/Lwereanalyzedwiththesamples. Thesaanmdmoest promineatpeakresponsefor PFOSwasutilizedtomonitor PFOSinallcalibration, quality costrol, andstudysamples. Noattempt wasmadetoquantify FOSonthebasisofindividualisomeric components. Linearregression equationsweregeneratedusingpeakarearesponseratios (PFOS intemalstandard)versustherespectiveconcentration ratios (PFOS:intemalstandard)ofthe calibrationstandards. A.typicalcalibration curveispresentedin AppendixII, Figure2. The concentrationof PFOS inthesampleswasdeterminedbysubstitutingthepeakarearesponseratios into the applicable linear regression equation. Representative ion chromatogramsoflow and high calibrationstandardsarepresentedinAppendixII, Figures 3and 4,respectively. 001383 WILDLIFE INTERNATIONAL, LTD -12- PROJECT NO.: 454-103 `Themethodlimitof quantitation (LOQ)for theseanalyseswassetat 1.15 ppm a.i. calculatedas `theproduct of thelowestcalibration standard analyzed (0.00229 mga.i/L) and the dilution factorof thematrixblank samp(l50e0s). `Twomatrixblanksampleswereanalyzedtodeterminepossibleinterferences. Nointerferences wereobservedato abovetheLOQduringsampleanalyses(AppendixIT, Table2),Aninterference inthefeedappearedatapproximatelythesameretention time asthepeakofinterestbutitwaswell belowthe LOQ. A representativechromatogramof amatrixblankispresentedin Appendix III, Figur5e. Aviandietwasfortiafti4.e57d, 183and1830 ppma.. andanalyzed concurrentlywiththe samplestodeterminethemeanprocedural recovery (AppIeIIn,Tadblie3x). Sample concentrations `werenotcorrectedforthemean proceduralrecoveryof 94.7%. Arepresentativechromatogramof a `matrixfortificationispresentedinAppendixIII, Figure6. - An example calculation is presented for sample number 454-103-2, nominal concentration of 18.3ppm a.i.inaviandiet. InitialWeight: 100g FinalVolume: 200 mL Dilution Facor: 100 (intdileutiornfacmtorexidalidilutaiontfacteor) PFOSPeakArea: 113568 InternalStandardPeak Area: 413160 PeakAreaRatio: 02749 Calibcurrvaeetquiatoionn. Slope: 277397 Intercept: 0.01894 Curvesweighted (1). PROS gat Sm (Peakarea ratio - o(vVeintercep) xL1S5.Concentration Sipe 001384 WILDLIFE INTERNATIONAL, LTD BER PROJECT NO.: 454-103 Note: LS. =internal standard. PFOS(ppma.)insample (0.2749 -001854) 0.100mpl. mw = 000923 main PFOS (mg 8.1/1)atinse truR mxFeinate l Volume(L) xDilution Factor 000923 x 0200 x 100 ot =185ppma Pe inal C om PFOS(ppma.i.) insample PFOS(ppm 2.1.)nominal x 100 = 2 11835 0m101% Duringacclimationand testing,allbirdswerehousedindoorsinbatteriesofthermostatically controlledbroodingpensmanufacturedbyBeacon Steel Products Co.(ModelNo. B735Q). Eachpen hadfloorspacethatmeasuredapproxi7m2aXt9e0clmy. Ceilingheightwasapproxima2t3eclmy. Externalwalls,ceilingsandfloorswereconstructedofgalvanizedsteel wireandsheeting.Birdswere sorted by weight,thenchosenindiscriminately from withineachrepresentedweightclassfor assigntmoepennst. Eachgroupofbirdswasidentifiedbypen numberand testconcentration. Individualbirdswereidentbyilefgibaendsd. Duringthetesttheaveragetemperatureinthebroodingcompartmentofthepeaswas 38+2CC (SD). Average ambient room temperature for this study was 27.3C # 12C (SD) with an average relativehumidityof 31% % 14% (SD). Thephotoperiod (maibnyattiamei cloncke )wads sixteen hoursof lightperday duringacclimation andthroughoutthetest. Thelightsourcewasfluorescent lightswhichcloselyappronxooni-dmaysauntligeht. Thebirdswereexposedtoanaveragoof 001385 WILDLIFE INTERNATIONAL, LTD S14 PROJECT NO. 454-103 approximately 139 lux of illumination. Housing and husbandry practices were based on guidelines establishedby theNational Research Council (5). Observations During acclimation allbirdswere observeddaily. Birdsexhibitingabnormalbehaviororphysical injurywere aotused. Followingtestinitiationandcontinuinguntiltermination,allbirdswere typicallyobservedatleasttwice daily. Arecordwasmaintainedofallmorality,signsoftoxicity, andabnormalbehavior. y Riad i Individualbodyweightsweremeasuredattheinitiationofthe test, onDay 5,Day8,andon Days15andDay22forallremainingbirds. Averagefoedconsumptionvaluesduringtheexposure period (Days 0-5)andthe post-exposureobservation period (Days68) weredeterminedbypenfor eachtreatmentgroupandthecontrolgroup. Additionally, feed consuwamsdpettermiineodfnor Days 8-15 and 15:22 for the remaining treatment and control birds. Feed consumption was determinedbymeasuringthechangeintheweightofthefeedpresentedtothebirdsover agiven peroiftoimde. Theaccuracyoffeed consumptionvaluesmayhavebeen affectedbytheunavoidable waosffetedbaythgebireds. GrossNecropsy Alltestbirdsthatdiedduringthecourseofthetestandallbirdsremainingatthetermination were subtjoaegrocsstneceropdsy. Additionally,liverswereweighedandlivertissue,blood,andbile `were collectedfrom birdscuthanizedonDay 8and22,andwhenpossiblefromthosethatdiedduring thecouoftrhesstudey. `Statistical Analyses Mortalitydatawereanalyzedusingthecomputerprogramof C.E.Stephan (6). Theprogramwas designedtocalculatethe LCS valueandthe 95%confidenceinterval byprobitanalysis,moving average method or the binomial probability method (7,8,9). In this study, the LCSO value was detuesinrgthmepirobnitmeethdod. Theslopeofthe concentraticuorvnea-nedrsespulotsnofsthee goodnessof fttestarereported. Bodyweightdatawerecompared byDumnettstestusing `TOXSTATsoftware(10,11).Nostatisticalanalyseswereappliedtofeedconsumption data 001386 WILDLIFE INTERNATIONAL, LTD S15 PROJECTNO. 454-103 RESULTS "Diet Analysis Avian dictsampleswerecollectedfromthe 18.3and 117]ppmai. testconcentratiaonnds analyzedtoevaluatehomogeneityofthetestsubstanceintheaviandiet. Theanalyofsthiesse samplesalsoservedas verification oftestsubstanceconcentrations. Resumelantmeiasnurged `concentrations,standarddeviationsandcoefficientsofvariation (CV)forthesetestconcentrations were 19.5 + 2.13 ppm ai. (CV = 10.9%) and 1196 + 70.2 ppm ai. (CV = 5.87%), respectively (AppIeI,nTadblie 4x).Controlaviandiet samples collectedduringthetestshowed nointerferences abovethe LOQ. Samplescollectedduringthetesttoverifythe 36.6,73.2, 146, 293and 586 ppma.. test substance concentrations had mean measured. concentrations of 402, 74.5, 174, 291 and 537 ppma.respectively. These values represented 110, 102, 119, 99.3and91of.the6 nomi%nal concentrations, respectively (Appendix IIT, Table S). Analoyfsaviiasn diet samples collected from feeders after being held at ambient temperature for five days averaged 101, 122, 104, 101, 109, 114 `and 102%oftheDay 0valuesforthe18.3,36.6, 73.2, 146, 293, 586and 1171ppmai.testsubstance `concreespenctitvelry(AappetndiixIoII,Tnabsle6,). Arepeseatativechromatogramof atestsample isshown inAppeIIn,Fdiguirex7. Oneincidentalmortalityoccurredinthecontrolgroupduringthecourse ofthestudy(Table 1 and AppendixV). OnthemorningofDay 5, onebird wasnotedwith abrokenlegandwassubsequently euthanoniDzaey6d. Additionally,two birdsinthecontrolgroupwereintermittentlynotedwithfoot lesionsassociatedwithcagemateaggression. Otherwise,allcontrolbirdswerenormalinappearance andbehaviorthroughoutthetest. No treatment related mortalities or overt signsoftoxicity were observed in the 18.3, 36.6, or 73.2 ppm a.i.treatmentgroups.Onebirdinthe 18.3ppma. treatmentgroupwasnotedaslamefrom Day6throughDay 8, andbloodofundeterminedoriginwasnotedontheundersideofonebirdfrom the 73.2ppm a.i. treagrotupmoneDany2t2.Otherwise,al birdsinthe 18.3, 36.6and 73.2 ppma.i. treatmentgroupswere normalinappearanceandbehaviorthroughoutthetestperiod. 001387 WILDLIFE INTERNATIONAL, LTD -16- PROJECTNO.: 454-103 Thwaes1r1%e(1of9)mortintahel146ipptmay.i.treatmentgroup, 80% (8of10)mortaliitny the 293 ppm a.i.treatmentgroup and 100% (10 of 10) moralityinthe $86 and 1171 ppm ai. treatmentgroups. Inthe 146ppm a.i. treatmentgroup,onebird waseuthanizedonDay 3after sustaaibrnokiennlegg. Thisincidentalmortalitywasnotusedinthecalculationofthe LCSO0value. Additionally,therewasonetreatmeat-relatedmortalityinthe 145ppma.i. treatmentgroup, abird founddeadonthemorningof Day 7. Clinicalsigns oftoxicitywereobservedinthistreatmentgroup on Day5, whentwobirdsdisplayedwingdroop.~Allotherbirdsatthistestconcentratwieorne `normalinappearanceandbehavior forthedurationofthetest. Inthe293ppm a..treatmentgroup there were eighttreatment related mortalities, occurringon Da5,6yansd7. SigasoftoxicitywerefirstobservedonthemorningofDay 4andcontinuedtobe: exhibitedthroughthemorningofDay forthesinglebirdeuthanizedonDay8,andthroughthe afteroonofDay8forthesinglebird survivinguntilDay22. Signsoftoxicityinclaurdufeflded appearance, reduced reactionto stimuli (sound andmotion), lethargy,wing droop, lossof `coordination, lowerlimbweaknessandconvulsions. Thesingleremainingbirdappearedtohave recoveredandwasnormalinappearanceandbehaviorfromtheafiemoon ofDay 9 ntltest temination. Inthe586ppm a.i.treatmentgroupmortalitieswerefirstnotedonDay 3andcontinuedtobe observedthroughDay 7,atwhichpointallbirdshaddied. Overt signs oftoxicity werefirst dbserved ontheaftemoon ofDay 2andcontinuedthroughthemomingofDay7, whenthefinalbirdswere founddead. Signsoftoxicity observedamongbirdsin the 536ppma.i.treatmentgroupincluded a ruffledappearance,reducedreactiontostimuli(sound andmotion),lethargy,depression,wing droop, lossofcoordination,lowerlimbweakness,lowerlimb rigidity,prostrateposture,andconvulsions. Inthe1171 ppm ai.treatmgeronutp 100%mortality hadoccurredbythe momingofDay4. Signsoftoxicityinthe 1171ppma..treatmentgroupwere firstobservedontheafternoonof Day2, withthefirstmortalities notedonthe morningofDay3. Sigasoftoxicityobservedpriortodeath includedaruffed appearance, reduced reaction to stimuli (sound and motion), lethargy, depression, lossofcoordination,wingdroop,andlowerlimb weaknessandrigidity. 001388 ue BodyWeightandFeedConsumption `Whencomparedtothecontrolgroup,threwereno apparettreatment elatedeffctsuponbody `weightamong birdsinthe 18.3, 36.6or 73.2 ppm a. treatment groups. Howethvereewars,a `concentration responsivereduction in bodyweightgainor body weightloss inthe 146, 293and 586 PPM a.treatmentgroupsduringthe exposure period (Days 0-5) (Table 2and Appendix VI). Differencesfromthecontrolgroupwerestatistically significantat p <0.05forthe 146ppm a.. level andatp<0.01forthe 293and586 ppma.i. levels. A statistically significant (p<0.01) reductiionn body weightgaincontinuedtobe observedatthe146 ppm a..testconcentrationthrough Day 8ofthe study. Atthe293ppmai.concentration astatistically significant (p<0.01)mean weight loss `continuedthroughDay 8ofthestudyand amarkedreductioninweight gainwasnotedthrough Day 15ofthestudy. Due tototalmortality,bodyweighteffectscouldnotbedeterminedforthe 1171 ppm ai. levelduring the exposureperiodorforthe 586and 1171ppma.i. treatmentgroupsforpost- exposureperiod. "Therewerenoapparenttreatmentrelatedeffectsuponfeedconsumptionatthe18.3, 36.6, 73.2 or 146ppmai.testconcentrations(Table 3 andAppendixVI). Howae reduv ctie on ir nfe, ed consumptionwasnoted atthe293, 586and 1171ppm a.. treatment groupsduringtheexposure period (Days 0-5). There were no treatment-related effectson feedconsumptioninany of the surviving treatment groupsduring the Day6-8post-cxposureperiod. Inthe 293 ppm ai. treatment `grouponlyonebird survived toDay22. Thereductioninfeedconsumption observedat the 293 ppm ai. test concentrationduring boththe Day 8-15and 15-22post-exposure periodswastheresult of having onlyonebirdinthepen,andwas notconsideredtobetreatmentrelated. GrossNecropsy Duringthecourseofthetest,allbirdsthatdiedweresubjectedto agrossnecropsy. Necropsy resultsforbirdsfounddeadweresimilar. Common observations includedthin condition,loss of fimnudsicnlgesamarssp,raletesredeisnnpTlateelneecd4o,lor, autolysisoftis ands pau leoe rgas ns. Detailsofthe necropsy HalfofthesurvivingbirdsweresubjectedtogrossnecropsyonDay 8andtheremainingbirds were necropsiedonDay 22,following testtermination. OnDay 8, onebirdinthe 73.2 ppm a. 001389 treatmentgroupwasnotedwith aslightly paleliver. Duetotheisolatednatureof thisfinding,it was WILDLIFE INTERNATIONAL, LTD -18- PROJECTNO.: 454-103 `notcons tobi ered latee dtor tree atmed nt. Thesinglebirdeuthanizedfromthe 293ppma.i. treatment groupwasobservedatnecropsytohavewithalackof musclemassand general thinness.Sincethese findings correlatedwithan impactuponbodyweightnotedatthisconcentration,thefindingswere contsobi etrdeatemenrt re elatded. Necropsyresults wereunremarkablefor all otherbirdseuthanized onDay8.Similarly, Day22necropsyfindingswereunremarkableforallbirds. CONCLUSION ThedietaryLCS0valuefornorthernbobwhiteexposedto PFOSwasdeterminedto be 220 ppma.i. with a 95% confidenceintervalof 164ppm a.i. to 289ppm ai. Theslopeoftheconcentration- responsecurvewas 7.005andthechi-vsalq uewuasa0.r02e3.Thenomortalityconcentration was 73.2 ppm ai. Based upontreareltatemdmoertanlitty, signsoftoxicityand effectsuponbodyweight gainatthe 146ppma.i. a.itestconcentration,theno-observed-effectconcentrationwas 73.2ppma... 001390 i WILDLIFE INTERNATIONAL, LTD PRPOROJJEECCTTNNOO.::445544-110033 19. REFERENCES 1. US. Environmental Protection Agency, 1996. Series 850 - Ecological Effects Test Guidelines (draft), OPPTS Number 850.2200: Avian DietaryToxicityTest. 2. US. Environmental Protection Agency. `1982. Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, subsection 71-2, Environmental Protection Agency, OfficeofPesticide Programs. Washington, D.C. 3. Organization for Economic Cooperation and Development. 1984. Avian Dietary Toxicity Test. OECD GuidelineforTesoftChi emincalgs. Guideline 205. Paris. 4. AmericanSocietyfor Testing andMaterials. 1987. StandardPrafcortCoinducctieng SubacuteDietaryToxicity Testswith Avian Species. ASTM Standard E857-87. AnnualBook of ASTMStandards, Vol. 11.04, PhiladelphiPaA,. 5. WNaatsihoinanlgtRoens,eaD.rC.cNhatCioounncaillA.cad1e9m96y. PreGsus.ide12f5oprp.the Care and UseofLaboratory Animals. 6. SShant, ephan,CCF.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, MN. Personal 7. Stephan, C.E. 1977. Methods for Calculating an LCSO. Pages 65-84In AquaticToxicologyand PHhaizlaadredlphEivaa,luPaAt.ions, American Society for Testing and Materials. Pub. No. STP 634. 8. Finney, D.J. 1971. StatisticalMethods inBiological Assay,20ded., Griffin Press, London. 9. Thompson, W.R. 1947. BacteriologicalReviews, V2o ,Nol .2: 115-145. 10. wDuintnahetC,onCtrWo.l Jo1u9r.55A.meAr. MSutlattiip.lAessCoocm.paSr0i:s1o0n9'6s-1P1r2o1c.edure for Comparing Several Treatments 11. Gulley, D.D. 1990. TOXSTAT Release 3.2. TheUniveorfWsyoimitngy. 001391 WILDLIFE INTERNATIONAL, LTD 20- PROJECT NO.: 454-103 TABL1E `Cumulative Mortality fr aNoo rthm ern Bobwhite Acute Dietary Toxicity Study with PFOS. ErGpreomuepnil To. DEexapdosPeurreNPoeriEoxdposed NoP.oDsetaEdxpPoesurreoPeErxipoodsed (emai) Dy0 Dl Dayz Dy3 Days D3 Dye Du Dui Control 0 030 030 030 030 030 030 10 130 130 Treatment 183 366 n2 O10 O10 010 010 010 00 wo 010 010 00 00 oo 010 010 O10 010 00 oo 010 00 010 ow ol 010 010 on O10 146 ol ono om VIP 09 09 om " EE o0 ol ol 010 00 20 410 80 EO ste 010 010 010 V0 20 S10 0 100 1000 1m 010 on O00 30 1010 1020 10 1010 1000 T--o{_eN--oCm3o0rtvaliotiewsoecccuroedeinday0of5te50cpasel+1Weilam95%rcoopnedfenn Do r etloDoaf1y6242p.resa1. 0289pp a1. 2Bird euthanized on day 3 aftersustaining 8 broken leg. 001392 WILDLIFE INTERNATIONAL, LD 21. PROJECTNO.: 454-103 TABLE2 Page 1 Mean Body Weight (5) from a Northern Bobwhite Acute Dietary Toxicity Study with FOS BeGermouepnl @pmai) Exposure Period Dayo Days CDhayneges Po-- st Period DayS Days ChTaontgale! Cont0rol Mem nw 10 88 18 so 1 4+ 3 52 4 Treatment 183 sMoem a1 a4 u3 o5 92 23 66 Msoem 02 a3 12 393 81 12 B2 MsDen 21 wo2 91 33 71 126 us sMoem 02 a3 3 23 o2n 14% 9 Msem 01 a2e a2e 14 a4n a5n $86 sMDem 01 d2ew a2we -- -- -- un __sSMDDe_n 2- . - . . a 1 - - - - z Wi=eNnochdaangea1vasiclalballeadiucedtsoempoarraatleiltyy.rosth mea bodyweightswin dividual odyweights (5 AppendinVD. ""SSttaattiisstticiaclallyldyifdfifefrreenntrfoontmhtehce tcarrlolgrgoruopuapat pp<< 00..0031(D(uDnunmeett's'sLeLsi)st. 001393 WILDLIFE INTERNNAATTIIOONNAALL,,tLmD "2. PPRROOJJEECCTTNNOO:: 445544--110033 TABLE2 Page2 Mean Body Weight (g) from a Norther Bobwhite Acute Dietary Toxicity Study with PFOS "P BeeGe rmoaupnwBpembeed (Gpmai) TG TDays a Dayls g Daysls DayC z a Daysg ? Cont0rol Mem sD 36 1509 35 8B 23 Treatment 183 sMDen "6 68 23 87 23 66 Mem sD 3 3 6s 5 2%2 87 22 m2 sMDem 3s3 4 22 Com4 22 6 sMDean E3 O)3 2%1 E2 22 2 M5en 2 3s- 1: 55- 2 ChaTnogwes) 48 a3 540+ "2 as1 EY 86 sMDean :- -: :- =- N- :. mm sMDen ;- :- :. :- :- -- "T=hneilc,ohcocuhdlaadnnagoeak1vbsaelileeavbaaeltudatuetdsosetmpaoatrriaasttleiilctyaylTl.yowmithhDeTunonent'osdywesetg.hts ogiv] bodyweights (SosAppend V0 Statisticallydiffrent rom thecontrolgroupat p< 0.03XDunet'sties 001394 WILDLIFE INTERNATIONAL, LD 2. PROJECT NO.: 454-103 TABLE 3 Page 1 Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Acute Dietary `Toxicity Study with PFOS ---- (@Gprmoaupi) `Exposure Period PostExposure Period -- 7-- wes Control o MBeE an 92 12 Treatment 183 366 n2 146 EE ss um 9 n 3 2 10 1 9 : 10 5 9 6 1 4 - = Vodtswa do me eray meee 001395 WILDLIFE INTERNNAATTIIOONNAALL,, TLToD 24 PPRROOJJEECCTTNNOO.:: 445544-110033 TPAaBgLeE2 3 Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Acute Dietary `Toxicity Study with PFOS. TE @Gprmoauip) PostExposure Peri.od ome banm Control 0 Me0an 52 11 Treatment 13 366 n2 146 9 56 mm 10 2 u 1s B 15 unc 14 s 9 - - - - DNR 001396 WILDLIFE INTERNATIONAL, LTD 25. PROJECTNO.: 454-103 TABLE 4 Group GrossPathologicalObservations from a NorthernBobwhiteAcute DietaryToxicity StudywithPFOS `Birdsthatdiedduringthecourseofthe study --_ Finding Vile,Female, 3 Undetermined PPMAL Coml B WE Im Nel N= N=8 N=l0 N= Abdominal cavity,someautolysis Abdominalcavity, autolysisthroughout 0 2 0 2 ` 1 1 Crop,empty Emacited o 2 5 2 o o 2 5 5 Fracturedlg. GLtactempty 1 1 0 0 0 o co 1 1 0 Gizzardcontentsbilestained 0 Hear,anterior portionmattedwhitecolor 0 o 2 0 1 5 1 o Heart,pale Intestinalcontents ar dike 0 0 oo o 2 2 1 Keel,prominent Kidacys,pale 0 o 0 0 1 0 3 10 2 o Liver, paleandmatted Los ofmusclemass 0 0 1 0 0 4 0 7 9 Muscular-skeletl, pale Smallinstature 0 o 1 0 3 0 o 0 `Spleen, black 0 0 0 1 0 `Spleen,dark 0 0 0 J 2 `Spleen, grey o 0 0 1 S`pSlpeleeenn,,pgarleey-brown Spleen,small 0 0 01 0o 11 o o 0 1 Spleen,smallandpale Thin 0 0 0 0 0 3 o 4 2 NotRemarkable o 1 0 0 001397 WILDLIFE INTERNATIONAL, tro s- PROJECTNO: 4ski03 Cumiive rly(cimr eicCmToixteSsBos,omwighPyPoSm NoseBic EGnr aoi,) reBui DDuoi T oEPbxampoion(PCeadmbiesyDos,meeD)ui hai BYue[epoarrbepirenBbtaadt = 0 o3wmooow w ws wws eos sasas wws wws ooss 0 o o3 s w mmoooms ooww uussow awws oomwas oowss oaasswsooas resimeWnot 6 mom a am Wm os ow om 1 ws wo wag osey asm wson os ws os Wa Ws s oan 0s(y wsae son os as os ;% 21 wess wasaayp soeaen swioemn soesen oossees aass ooss ooss m m1 2 wwss wwaoyn woseen sGes0n9 oussawm oossGaieey aass ooss oosn WWo on2 mwss oossuune aessoemn swesmmm iossaew essaae o1ss uWs isus mw 1 os awseap seaawm oossoon oossaes sweosw Aus o4wss euss m11 ooss sosamy wwomw owsemn woseon swseawwy euss ssss us mIo m: wuss wwaolw oosseowmm swoowm wsss 93s He r e P5E ww B 63 eBadcnouirde s ksoBene dyeh fdcmtoveheSconcepio sve gh @OE appranimaton oftheacta] moruonetov ftestsubst e conpsumeedc .hryt. --WI-- LDLIFE TINTTAERTNAOTIMONAALL,tLTeD 2. PPRROOEJECCTTNNOO.::d45d4-l103 APPENDIX Certificate Of Analysis FC95, Lot217 Mar9,c20h00 Rita Poe ayLENE mmes Tore -- ----op 1 -- swo ee-- s --x E enoJ r y RN p|a | S---- F_FoEseo esxox agin iw | ------ -- C -- GRcs-- SA | -- Bo|-- w-- | CTCukeS1 ORTM oodm --7 S-- ---- LS-- Ce aSA atte low abot wing PENMRtochaiquesendfoundto sN te brs ey no e ET 001399 WILDLIFE INTERNATIONAL, LTD 28- PROJECTNO.: 454-103 APPENDIX II `WILDLIFE INTERDNIAETTIFOONRAMLULLTDA.TIGOANME BIRD RATION' INGREDIENTS FSionyeBCeoanmMMeeaall, 48% Protein `PrWohteeaitnMBiadsdes ``AAlgfvailafya MSpeeacli,al2,06%0P%roPtreoitnein D`GrrioeudnWdhLeiymestone EMeatshtimoanninCealPPrheomsix + Liquid VGiLtaFmeirnman(FdeMrimnaetcroa)l'Premix (sce below) TSoalttalodized PERCENT (%) 430865 665000 430000 205500 00.3650 002352 1000.1000 _ VIATNDA MINEMRALI PREN MIX . AMOUNTADDEDPERTON VViittaammiinn DAy RiNbiaocfilnavin `VPiatnatmotihnenBiyc; Acid BiFooltiicnAcid P`yTrhiidamoisniene VViittaammiinn EK (Menadione Dimethylpyrimidinol Bisulfite) MZainngcanese CIaopdipnee r SeIlraennium 27,,000000,,000000L1UC.U. 406 ggrraammss 180 gmrgasms 60604 mmggss 1122 ggrraammss 200005.180g.rams 14072 ggrraammss 6185 ggrraammss 15812 gmrgasms. "Tohefgu5a%racnrtuedeedfainbearl.ysisis aminimumof27%protein, &minimumof2.5%crudefatand amaximum "Fermentation By-Products (Sourceof Uridentified Growth Factors). 001400 WILDLIFE INTERNATIONAL, LTD 29. PROJECT NO.: 454-103 APPENDIX II ANALYTICAL METHODS AND RESULTS . 001401 WILDLIFE INTERNATIONAL, LTD -30- PROJECTNO.: 454-103 . "TTROVENT ANALYTICALCOLUMN: OVENTEMPERATURE: STOPTIVE: FLOW RATE: MOBILE PHASE: * DVECTION VOLUME: 'PFOS RETENTION TIME: RIENTTEENRTNIAOLNSTTIAMNED:ARD PFOSMONITORED MASS: IMNOTNEIRTONOARRLESDMTMAAANSSDS:ARD APPENDIX II Table 1 "Typical LC/MS Operational Parameters Flwien-Packard Model 1100 Fgh Peformance Liquid Cromaiograpwihlho `aPTeurrkbion-lEolnmiAoSePnrIpsor1u0rac0eyL.COMpaesrsatSepdeictnrsoemleetcetrievequiiopnpmeodwniithtaoPremiroknidngs-Elmer (SDV). KeystoneBetasilCyscolumn (100mm x 2mm LD,, 3ymparticlesize) 30C 100 minutes 0220 mUminute 72.0%Met:2h8.0a% NnANOopurle Wate cont0a.1%iFonrmiicnAcgid 25040 | Approximately 7.0 minutes Approximately 4.8 minutes 498.6 amu e26e7 aemu 001402 W_I--LDLIFE INTERNATIOe NAL,s LD S31. PROJECTNO.: 454-103 APPENDIX I Table2 `Matrix Blanks Analyzed Concurrently During Sample Analysis me (45N4u-m1b0e3r) Type MAB-L `Matrix Blank Co Ven d Pn ROS' or (ppmai) <10Q MAB-2 Matrix Blank <L0Q TahnealTyazneid o(f0qu.an0timt0agt2i2o/n21(9)LOaGn)dwthaesdil1u.t1i5ponpfmacao1r.obfatsheedmuaptornix(b6lapnrkodsuacmtpolfesthe(50o0)w.es!calibrationsaadard 001403 R WILDLIFE INTERNATIONAL, LTDp ey eePR ROJECN T NO.:i45i4-103 +&z 3. APPENDIX II Table3 Matrix Fortifications Analyzed Concurratly During Sample Analysis I Sample Number Gpmai) Percent (5103) Forded Feared Recovered TMMs AS4A aa5 4a7s9m w10r5 MMAAsS2S 1188 111662 986.31 MMAasS3S 11883300 11751766 8963.71 - TTStandarm d Deviation = w 699 cNv= 7358 "oeRanedemlasnauadl coarlrceuloaitoinosnsorVahlouwes1h5a1vesbuebesnanrcoeunPduetdtfyowrreerpeogretniengrpiuerdpoSsaensg MacQuamversion 13Sars 001404 WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-103 a sermon mies --o_S--mn-- Staepe LomiHoopmMioyoofPdRrSOoSisniAvan SimpCoey ws :3: ETTootis nwiis ;:$ SNeornnyg mii3 wn niu faoEiut) fi1ir 333 sSeemiigae biioast CeEE et ry io ihng epspsoG f ee odg sroYo SiwehpEsimamns aSoitRa rn 13le hens ls Ve= t on Va re 88528 WILDLIFE ILNTEiRNATIONAL, Tm Coun mei) Nssoetmie) Te ns = 56 .5 na 0i ui LE T E----------PROa JECTNO: 454-103 ae Appr Tabs Verfatonof FOS ConcentrationsinAvion Dic mTomwo'm MasumdRCaeyot Nhose3m ae - - - 0o i" m05s 0o EmHs m1s : i mw Cm Goomme ai)n ns "0 ns m eNnotri te -- wo no ww o8 mw ino s = " wm Rens rin - - ok beywe rd ogMonei 3 - se wor eV r e ei moy BRTe eeshkEi fedfoenionl1e040.)va 15g id pn prthwsleosd ytd0.072 1.1 eifc ofbemi ga33 WILDLIFE INTERNATIONAL, 10 FROJECTNO: 454103 ES Aveo aes AmcSublyo ROSinAvianDi Dig be orderBibiLCSSy C oa, l m SiE ti L e2mim rre] mewme ~~ e=ae Sim T T = = 0 w ns 33 pa ww w wo 2 aGI mmmcesa mcas o&o Eo = > wnid we - ws - n wn ns ne ma o un n w a mn ve ws El - wn ne 23 Eni a w w w " wa we - 3 iTMe = r Hevdre 0et r tetsdO0318 nt 8teik 282 533 L WILr DLIFE HINiTeERrNAToIOn NALy, vLT8D eo -36- PPRROOIJEECCTTNNOO.: 445544-110038 AspNDOCI METHOD OUTLIINNE FAOVIRATNHDEIAENTALYSIS OF PFOS Preparemaisfrictionsamples he deedvi fedstockwing deymi chi. `+ Fochihie mat DryMix Teta nina,iheegyofWHA el14 (VIL) tons weighbos lheeeqty imn(P70) to AfWILrnd mb rebe Rebf od ECSwithlpnofeinrt tr prot Sk. - xnctrof grb veldets es Wt de DingbetidBleevdaolnmniarchreforilmliefesdsp4pbka,|mmixiweelltaerler0 hepiedorvefso sidBesodf ocnomeprle ising ities | Fol thss rochedesePfroepsihoeetioseiirFercautnocnh eelWeyeeols iptoipon.gc of tied steigh Weigh10gmieofth i ak,mai rifcoieon.sdRttse nhmopwedighswihbos1dnt 16.French ls For1c 00iofi eho e vi r sed odedrvhost Fhars le a Violchrli lavehe ep endfed iv on iO Tete i 4200.voliakding fk vob itcan. + Forlla Preparl esprstese3s0o d% bona)oaioebm3e0c%nbNfuoAnNcoOoreeerewadteittseihoGencstltroaueoni,0dg.10o0fmtge4LHCMPFSOSaGbisotdes.tandard). od 003%form cd(OM. Aptssnsob simplefoLOIS yi. Figur1e. Analyticalmethodflowchartfortheanalysisof PFOSinaviandct. 001408 WILDLIFE INTERNATIONAL, LTD 37. APPENDITXL PROJECTNO.: 454-103 150 5 100 ; 050 000 0000 0100 0200 030 0400 0500 Concentration (Ratio) Figure2. C Atypiuciaslwer cigahtv ledi(1e bxc)ru.rvaftoriFoOnS,Slope = 2.77397; ntercpt = 0.01894;r=0.9981 001409 WILDLIFE INTERNATIONAL, LTD 3 APPENDIIXI PROJECTNO.: 454-103 10 o 8 7 s . s 187 Intonity: 1685 cps . 4 a 2 1s ! 24 122 I Sh Sm See GTmAIT7aTRIEasTANm)e Figured. A representative ion chromatogram ofa low-level 0.00229 mg a../L) PFOS standard. 001410 W--I_--LDLIFE INTERNATIONAL,tLp D 39 APPENDIIX PPRROOJJEECCTTNNOO.:: 44s54t-l10033 o s 8 7 ol 5 4 8 21 1 obo 28 4s 5 31130 T2e85 e3n81 186 Intonalty: 26954cps 144 122 190204 230 a5z06 1651% 178517 261613 TSicamne Figured. Arepreonschreomnatotgraamotf ahiighv-leevel (0.0457mga.iL) PFOS standard. 001411 --WI-- LDLI" FE INTT ERNATIW ONAL,A uUDD TONA PPRROOL JJEECCTTNNOO., ::445544-110033 -40- APPENDITXU 100 oof 24 80 70 60 50 . 180 Inten2s20tcyp:s 186 30 144 # oNil A aTg V R e ASJILO S1%er285 earm a6m06 16812 216 |eg20w|8 1n 478s zaits TSmane Figure. A"Trheeparersreonwtaitnidvieccathersotmhaetroegtrenatmioonf iammaetorifxPbFlOaSn.ksample (454-103-MAB-1), 001412 W_ ILDL-- IFE-- INTERNATIONAL, LD a1. APPENDIIXI PPRROOEJECCTTNNOO.:: 445544--110033 101 168 Intensity: 6238cps 9! 8 70 8 sl . 4 3 2 144 1 24 122 a130 a2155 83181 152106 T6183172 "718517 261813 TSiemane Figure6. Arepresentative chromatogramof a matrix fortification sample (454-103-MAS-14). 001413 TWI= LDLIFE TITNTNEARTNAITOINONAALL,,tLTDp "a. APPENDIIXT! PRPROOJJEECCTTNNOO..:4545441-10033 10 167 Intensity: 7248cps . 8 7 .8 . 40 3 2 145 1 o 24 a 4 123 197 229 317 Tet et 121 151 181 211 | Sen 130 255 381 5.08 6.32 7867 8.83 Time Figure 7. Arepressniativechromatogram of atest sample (454-103-2), 001414 WIL2D-LI7FE NITNETRENRNAATTIIOONNAALL,,uLToD ae DIETAPPPREEPNADRIATVXION PPRROOEJCECTTNNOO:. 445544-110033 `Weightandvolumeofconstituentsusedtoprepare testdiets: ConNcoemnitrzaatlions| SubTsetsatnce | RBaatsiaoln ppm02.) g- 3005 00 316863 00.3861388 889999968 71462 0174268529 8999.3 8998.5 258963 5289213293 8899997412 1171 116483 39884 Dietswere prepasafrolleowds: #5000.0 gofbasalrationwasweighedinto atared Hobartmixingbow. Thetestsubstancewasweighedina taredweigh boat. Appbrloexnidemra.tely 100 of basalratonwastakenfromthemixingbowandplacedin a Waring. ThWeitteshttshubrsitnasneacelvsaobsedinegpeltaoctehdenbltehnedbleernadnedr.theweigh bostwasrinsedwithadditonalration, +TbheobwlelndTehrceobnlteenndteswrwearserbilnesneddewdiftohraadpdpirtoixoinmaaltrealtiyo6n0,wsietchotnhdsrainnsdetarsanosbfeerirnegdtpoltahceemdiixnitnhge mixibnogwl. Th"eTbhoerwelmwaaisnipnlgarcaeidoonnaasHaodbdaerdttmoitxheerbaonwdltahnecdotnhteecnotnstwenetrsemweirxeemdifxoeradpfporroxsiixmamtoerleymisnixumteism.es. +Thedietwastransferredto labelled paperfeedbag. 001415 SWLIOLUDILIFFEE IINNTTEERRNNAATTIIOONNAALL,,w1o0 a MPOROETECCTTNNO:O:45e4103 ToCrropr `_CpenumsMraybyPenFofr.oDmEexNdpooPrnedAriProrsErixDnpboipsteedActsDiaryTxisySadyNwoi.hbDoPessEcdOEPSxegorserPErpon Topnaal ) D0 BT Dwi buy Di ys t owe ar hav ooo o 3 Tos eoses owss es 0wss os ooess os oass os oass as 4 os os as os 0s os woss os ooss os ooss os os us os oo Treatment 6soosos eoss aass 0oss 0oss oass v[ esIws 1w8s3 266 2vowsos Uw amss os ooasss o0ss os oaosss aass os wos ws0s ws os aowss os 2mn63a6 lis aVoeoosess ooss es ooss os ooasss ooss os ooosss Ios as as as as os aws as0s os os aass os ws uss n21a 3 aToos ess eaesss ooosss oowsse aos ss siuss swwooowwosss uaisss 5586 m 2v1o0sses ooss ms ooss as ouss ws aass ws aass oa wwooowss owwm sss ws un : sos os as ss ss sss ss T1 ieDToiCmvoalrceamisvscsicdlcdeioessybootfuh0eydpoo1wrieseSnStrougsstsme Day. STE ST TAT BB 2 e eeeoorornrroo @ W_I--LDLIFE INTERNATIONe AL, s LD -45- PROJPEROCJETCNTNOO.:: 445544-110033 APPENDIVXI Individu`aAlcuBteodDiyeWtaeriyghTtosxi(cgi)tfyrSotmudayNwoirtthhePrFnOBSobwhite: Page| T "Experimental eer @Gprmouapi) Bid Dayo Days DChiaynogse Days ChDaaynsgse CThoatnagle 0 . -- 1 19 2% 2 20 2 s 27 7 3 3 7 18" 3 4 21 2 2 2 i 10 a "0 10 3 2118 5 Mean T 2 32 9 11601 350% 3 1& 1 213 sD 22 66 44 99 3 3 17 -- 0 1 2 19 2 26 25 7 5 33 35 109 1156 43 22 2n 19 3a7 103 271 5 Mem T 2 32 1 &91 389 2 58 5 177 SS$D 11 33 22 3 3 11 0 22 1 2 19 20 zn 2 4 6 30 2 43 220 320 39 327 52 35 13 0 sMeDm -- 211 2s9 38 4 s 7 3 1 9 73 1152 5 18 5 2 B4 0 1 19 2 19 2 27 9 3 37 35 9 3 1168 43 2119 02 11 3a6 106 211 52 Mean "20 3 1 30 10 PE} 10 39 9 2 9 -- SD e 1 3 e 1 e s a 2 2 001417 W_I--LDLIFE INTERNATIONAeLe, LD -46- PRPOROIJEECCTTNNOO::4455441-10033 Themen Group. (ppmai) 0 --_-- 0 GToootaadpl APPENDIX VI Individu`aAcluBtoeDdiyeWteariyghTtosx(icgi)tfyroSmtudayNowritthhePrFnBOoSbwhite Page2 Bid Dsyo Days Change De0S Days CDhaaynsgse CThoatnagle 1 2 129 n3 97 3365 79 1166 34 EF2 3320 110 "- 2. 2- 5 Man T 2 2 34 1100 "#0 0 3 10 b 0 2 sS$D 11 33 22 055 22 44 1 18 22 30 12 35 1" 39 9 i! 225 3 19 4 21 29 2 10 1 37 a 8 9 218 5 Mean r 2 T320232 a 10 42 l 190s 2a SD 2 Mem 2 0 2 0 3 1 3 3B SSDD 1 1 4 4 3 3 0s5 3 2 44 001418 W_I--LDLIFE INTERNATIe ONAL,r LD 41. PRPROOJJEECCTTNNOO.:: 445544-110;3 APPENDIX VI Individ`uAacluBteodDiyeWtaeriyghTtosxi(cgi)tfyrSotmudayNowritthhePrFnOBSobwhite Page3 -- Evperimentl ------ pGmroauip) Bid Dsy0 DsyS ChaDanygoes Days CDhaaynsges CThoaungle 183 1 1 2% 7# 8 15 2 1 3 1 39 6 2 32 2 63 8 1 42 E 2 " 2 2 5 2 6 1 "% 10 2 sMaDn 22 34 m4 @6 9 2 1m5 183 TGoruolup 1 1 2% 2 1 2% 32 2 42 3 5 2 35 sMaDm TB2 33 MseDan 211 314 9 38 10 1 9 37 8 98 9 8 2 " 10 =n 1" 9 2 2 @3 T3 05 5 1 B 2 92 03 001319 --W-- I-- LDL-- -- IFE IT NTERNA ATIONT AL, uI p OMAL, PRPROOJJEEt CCTTNNOO.::4D 45564-110033 ae APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS. Paged -_--_-- G@prmoauip) Bid Duo Dys ChDayn0gSe Duys ChDaunsgse CThoatnagle 366 1 1s 2 s 3s 9 n 2 1 EY 1 3 5 3 1 2 10 3 8 1 +m 30 9 38 3 52 5 5 " 9 2 -- M)am TH1 3 B 3 22 053 1 y 1 m22 366 dTGoeouwl 1 19 2 10 = 5 1 21 2 33 5 1s 32 2 10 3 1 4 2 3s 5 " 9 2 5 36 1 " s 2 sMapm Tm I2 3 2 0 @4 8 0 m 2 Me5)n 222 5 330 21 2 3 3 s 1 h 2 2 001420 W-I_--LDLIFE INTERNe ATIOe NAL,' LTO a. PPRROOEJECCTTNNOO.:: 44544-11303 APPENDIX VI Individual BodyWeights(g) from aNorthernBobwhite Acute Dietary Toxicity Study with PFOS Page 5. _Exp--erimemntal = GpGrmoauip) Bi Dio Dys ChDayn0gSe Dus ChDaanysgse CThoanugte 2 1 1s 2 s2 5 1 21 2 73 7 1 32 2 53 7 16 42 n 3 7 1 52 0 5 36 5 15 v 0Msam Ts v W 9m 3o 7 v 2 m2 1 1s z 93 7 16 2 2 31 n8 7 13 32 3 5 7 16 42 1 3 7 18 5 2 2 10 a 9 1 v Gop osMMeann 2 2200 032 30 3 105 3378 77 a niwe Total s1 2 1 3 1 2 001421 W_I--LDLIFE INTERNATIONAL, eLrTD -50- FPRPROOJJEECCTTNNOO.::445544-110033 APPENDIX VI IndividAuaclutBeodDyieWteairyghTtosxi(cgi)tfyrSotmudayNwoirtthhePrFnOBSobwhite: Page6 --~-------- Group (ppmai) Bid Diy0 ee Days CDhiaynogse Days CDhaanygse CThaonlge 146 --_-- 1 1 2% 9 35 7 16 2 1 2 9 2 4 13 3 2 EF 4 - - - 4 2 33 un 38 5 16 5 5 2 5 2 4 9 Mem -- 21 SD 2 2 33 3 8 3 33 1 351 000T33w 146 1 1 2% 6 31 7 2 1 zn 8 35 8 3 1 2 8 3 6 +2 - - - - 52 2 1 2 4 Man --2 SD. 2 2% 2 6 m6 3 3 2 TGortoaulp SMeDan 220 27 3 7 3 El 3 2 *S"ta(t=isNtiocadlaltyadaiffvereantidruolemftaohmeobcrotanlltirteoyl.groupat p< 0.05(Dunnett's test). 2*Siatisticalydifferent from thecontrolgroupat p < 0.01 (Dunnett's Lies), 1B 16 1" s Ca 5 Be 4 001422 WWIILLDDLLIIFFEEININTTEERRNNAATTIIOONNAALL,,tLDO sie PRPROOWJEECCTTNNOO::445544-410033 --_E--rperimenal @Gpromuapi.) 29 APPENDIX VI IndivBoidydWueiaghlts (g) from aNorthern Bobwhite Acute Dietary Toxicity Study with PFOS Page 7 Bid Dwo Dus ChaDnng0eS Dys DChuasngse CThoatnagle 11 2 22 1 3 21 n 2 - - . 3 2 18 2 - . . `2 - - - e . s2 2 2 - . . L s 2 2 E - : : 2 11 1 15 + 4 21 . - . . 3 0 - - - 42 n 3 - . - 5 2 16 El - - . sMepm T 1 2 H 22 B - & B - F - GTroouulp Mesan 201 82 22 84 24 E5 g StEatTisicalydilaton he contol groupstp< 0.05(Duetset), 2*Suisicallydiffe omthe onlgroupat p<001 (Dunnett's Ls 001423 WILDLIFE INTERNATIONAL, LTD 52. PROJECT NO.: 454-103 TEGroup @pmai) EY APPENDIX VI Individual Body Weights (g)from a Northen Bobwhite Acute Dietary Toxicity Study with PFOS Pages Bird Dayo Days CDhaayn0gse Days CDhaanygse CThaontge 1 18 - - - - - 2 2 1B a - - . 3 2 1s 3 - . - s2 - - - - - s2 n - - . . Me0w T3 H2 3 2 - - z ss 1 1 16 3 - - - 2 . - - . . 3 2 16 4 - - . 41 - - - - - s2 - - - - Mam B 4 TCooulp sMeDn 201 I 2 2 >- 2 : 3 - '(-2)S=tnNisoidcaatlalyavGaiillabeletdufreotmotmhoertcaloitiyn.groupat p< 0.01 (Dunnett's Lest 001424 W_I--LDLIFE INTERNATIONAL, LD 53 PRPROOEJCECTTNNOO.:: 445544-110033 APPENDIX VI Individu`aAcluBtoedDyieWteariyghTtosx(igc)itfyroSmtudayNowritthhePrnFBOoSbwhite: Page -------------- Group (ppm .i) Bid _Dsyo Days Change Dey0-S Dsys CDhaaysnge CThoatnagle nn 1 18 - - - - - 22 - - - - * 32 - - - . . 42 - - - - - 52 - - - - - -e _ ssM%eDm -- 21 -- e :- -- - --- un 1 8 - - - - - 22 - - - - - 32 - - . - - +2 - - - - - s2 - - - - - Mam TH. Gow MSeDm 201 - - - - = Total SD. 1 - - - - - = No data availabedue to morta. eee -------------------------- 001425 W_I--_L--DLIFE INTERNATIONAL, LD 54. |PPRROOJFEECCTTNNOO.:: 445544-110:3 APPENDIX VI IndividuAaclutBeodDiyeWteairgyhTtosx(igci)tfyroSmtudayNowritthhePrFBOoSbwhite: Page 10 Grroup (ppm ai) 0 -- Bid 1 2 3 4 5 Mean SD 0 1 2 3 4 5s Men --_-- Ss$D 0 Group TaTotlal 1 2 3 4 5 Mean SD. Mean SSDD Day8 Changg e Dyls Daydls Duy22 2 4 34 36 "2 6 40 6 50 78 39 6 99 o1u 4 15 61 2 7 2 2 5 34 2 104 3 8 5 5s 1166 Change Days: 1 21 2 21 2 2 3 3 Toul Change 34 E] 50 4" 54 as 33 35 55 20 35 El 18 I) 7 1 21 39 39 37 4 6 7 27 85 31 103 21 29 4 60 39 59 "3 6 20 30 21 a 2@ 2 #5 33 88 66 112 4 4 99 30 54 2 2 4s 16 7 25 6 18 49 34 32 3 54 2 id 55 18 5 2 2 4s 38 4 6 28 95 2 3 55 2 78 3 55 5 37 3 39 5 11 3 2) 4 2 8 as 66 1100 0005s 0 11 3033 0000 083 001426 WILDLIFE INTERNATIONAL, LTD _s5- PROJECT NO.: 454-103 APPENDIX VI IndividualBody Weights (g) from a Northern Bobwhite: Acute Dietary Toxicity Study with PFOS Page 11 em ------ tn G@rmoauip) Bi DyS Dyls CDhyasnlgse Duyn ChDaanylgse2 CThoaumlp 183 1 1 EF) 1 n 25 " 2 3 6 2 82 " 3 3s " 2 5 2 51 ` 4 n zn 9s 2 " 5 i n 2 22 46 sMaDw R c s 3 G m7 B3 #3 _--m-- `Experimental Group (ppmai) Bird Day8 366 . 1 35 2 39 3 37 4 38 5a Mean 38 SD 3 Day15 Change Day8-15 Day22 Change Day15:22 60 25 81 21 65 2% or 2 65 28 61 zn n2 91 26 52 97 2 65 26 89 24 5 2 7 2 "Total Change 46 52 54 4s 54 50 4 001427 WILDLIFE INTERNATIONAL, LD os. PROJECT NO.: 454-103 APPENDIX VI IndivBoiddyWueiaghlts(g)from aNorthemBobwhite: Acute Dietary Toxicity Study with PFOS Page 12 Tee mGraouip) Bid Day Dayls CDhaaynSgles Duyn ChDaanygles2 TCootaamlge n2 1 2 El 2 7% 2 " 2 5 2 n1 " 3 36 @ 2 7 n Ls 3 6 2 8 2 i 5 36 El 1 a . Msam m H3m + 2 m ` W2m @2 TE @`pGmraoiup) Bird DwS Dyls CDhyasndgse Dy? ChDaanygles CThoatnagle us 1 35 58 Pl n 46 2 = ss " zn os `8 2a1 5 2 5 2 "2 4 sMDen TI3 m 3 W 1 w2 ma2 BL1 BE Sttisiclly ifrent omth conrolgrovp at <0.-- 05 Dumnet's ies) 001428 --W_I--LDLIFE INTERNATIe ONAL, y LD 57. PRPROOJJEECCTTNNOO.::44554-1303 APPENDIX VI . IndividualBody Weights (g)from a NorthernBobwhite Acute Dietary Toxicity Study with PFOS Page 13 Themen GGprmoauip Bi Dwy0 Dys CDhaungse Dys CDhaunsgse TCohtaangls 2 Ode _--_-- 12 2 3 . 4 - 5 M0en TW1 35 1 55 . . - . y . . . . . . - B : B . B 2 EY . . . . . m 2 m* 001429 W_I--LDLIFE INTERNATIOA NAL, aLeD -s8- PPRROOJJEECCTTNNOO.::445544-110033 APPENDIX VII Feed Consumption (g/bird/day) by Pen from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page | EE -_-- Gpmai) Pa T Dmos TD T mes Control 1 9 n 2 7 2 3 8 7 4 2 2 5 9 5 6 10 5 _ 2 MsaDm T 2 y 2 pm2 001430 WILDLIFE INTERNATIONAL, LD 59 PROJECT NO: 454-103 APPENDIX VII Feed Consumption (g/bird/day) by Pen from aNorthern Bobwhite Acute: Dietary Toxicity Study vith POS. Page? Experimental Group Exposure Period Fost-Exposure Feriod pmai) Pen Days Day 638 183 21 98 110 Mean 9 366 21 87 15" Mean 3 7 n2 21 1100 11 Mean 5 16 21 107 n8 Mean 5 0 203 21 45 180 Mean 5 5 6 21 55 216 Mean s 1 um 1 4 - Me:an 4 T = "amma 001431 SWIe LDLIFE INTERNATOINONAALL,, iLTeD -60- PPRROOJJEECCTTNNOO::445544-110033 APPENDIX VII Feed ConsumptionD(i/ebtiarrdy/Tdoaxyi)cbiytyPSetnufdryowmitahNPoFrOthSer Bobwhite Acute Page3 TT EpcmenlGrp _--_-- Gpmai) perl PonEmom eed Pa TD T HE Dgnm Control 1 10 1 2 10 2 3 7 2 2 --_-- sMaDw TT 2 pim Emme _-- Gpmai) 183 366 na 146 2 Bead FewEpeTed Pa Dye DmnE 1 10 2 1 1 15 1 1 1s 1 u 1 1 8 5 001432 A WILDLIFE INTERNAOTIOANA, L, iLTD "61- FPRROOEJECCTTNNOO..:443541-1m03 APPENDIXVIII CHATN O PG ROTE OCS OL 1, "Theprotocolwasamendedtoindicatethatbilewillbecollected fromallstudy birds. `Theprotocol `wasclarified toindicatethecollectionofliverfrombirdsthatdied duringthecourseofthestudy 2. Bloodsampleswerecollectedon Day 8and Day 22innon-heparinized Smiborosilicateglasstest `tubes. The protocolindicated that heparinized vacutainerswould be used. 3. The protocolwasamended tochangethetestconceatrationsfrom0,20,40, 80, 160,640and 1280 rpefplmecatit,htet0e0s,tsu18b.s3t,a3n6c.e6p,u7r3i.t2y,gi1v4e6,n2i9n3t,h5e8n6eawcnedrt1i1f7ic1atpepomfaa.nia.ly"sTiess.t concentrationswerechangedto 4. Thetemperatures from several brooder units on Day 4, 7, 8,andall brooder unitson Day22were. not recorded 5. The aftemoonobservationswereinadvertentlynotrecordedfor 2birdsinthe640 ppm a. tregarot upomnAeprn il2t 6, 1999. 001433 WILDLIFE INTERNATIONAL, LTD -- APPENDIX IX PERSONNEL INVOLVED IN THE STUDY PROJECTNO.: 454-103 ThefollowingkeyWildlifeInternational Ltd.personnelwereinvolvedintheconductor `managementofthis study: (1) Mark Jaber, Wildlife Toxicologist (2) Joann B. Beavers, Director, Avian Toxicology (3)SeanP. Gallagher, Senior Biologist (4)Courtney Casey, M.S.,SeniorBiologist (5) WillarBd.Nixon,Ph.D., Manager,AnalyticalChemistry (6) TimothZy. Kendall, Supervisor, Analytical Chemistry (7) RaymonLd. VanHoven,Ph.D., Scientist (8) Ellen Mank, Chemist 001434 q WILDLIFE INTERNO ATIONe AL, LTD roe PE ROJECR TNO.: 454-2 102 1i AMENDMENT TO STUDY PROTOCOL 1 STUDY TITLE: PFOS: A DIETARY LCS0 STUDY WITHTHENORTHERN BOBWHITE PROTOCOL NO.: 454/030999/QLC/SUBAS4 AMENDMENT NO: 1 n `SPONSOR: 3M Corporation PROJECTNO: 454-103 i EFFECTIVE DATE: April 27, 1999 i AMENDMENT: Onpage 8under Necropsy add: i io`duWrhienngptohsesicboluer,sbeiloefwthielltbeestc.ollectedfromallbirds.Livers will lsobecollectedfromal irsthat i REASON: i ofBivielr ersobmeibnigrcdosltlheacttdeidea.tSponsorrequest. Theprotocol isbeing clarified toindicatethecollection. rr . ' Bn Lda. PSSpo LL Study Director 5 Laboratory As -- : < L. Principal Contact H: -~ I Date alt Dae #2 ' 001435 WILDLIFE INTERNATIONAL, LTD I" DEVIATION TO STUDY PROTOCOL : STUDY TITLE: PROS: ADietaryLCSO StudyviththeNorthernBobwhite I PROTOCOLNO.: 454/030999/QLC/SUB4S4 . DEVIATION NO: 1 i SPONSOR: 3M Corporation PROJECTNO.: 454-103 i+ DATE OF DEVIATION: April 30 and May 14, 1999 | -_ . AMENDMENT: 1. hepariniBzleodod5smalmbpolroessicloilclaetcetgeldaossnDeastytu8baesnpdrDioaryto2b2eoifngtrheeftieisgtewreatreeds.Tthoreepdriotnolcaoblelilneddicnaotne-d i that blood samples would be placed in labelledheparanized vacutiners. i REASON: By ddeocatpiotTaetasinStgpbaoninisdmoasrlrasenwqdeurderesattitonioonssgmbeapllaorotadotienestfefirtcueisemtnfttulrybodemrs.atwHhbeelpboalorodiobndywaastysrWniionlgtdeal.difdB eedInttl oebwmlaao otsoiddosrno aaamlwpLnd libdey.s. `Seperationofserumrequirescoagulation ofbloodsamples,whichheparinprevents. a IMPACT: i requestB.lThoeordesfaomrpelteshwsdeerveidartiaownncianuasnedenffoiacdiveenrtmsaeninmpearcatnodnstehreusmtvuidaysocuoltlceocmteedopreirneSrppornestao-r tion. y 2,0 E be AR we STUDY DIRECTOR DATE iby |B IDLER ONAL [7D WANAGEMENT ou " PE -- 001436 i 3 WILDLIFE INTERNATIONAL, LTD PROJECTNO.: 454-103 Poel r DEVIATION TO STUDY PROTOCOL : STUDYTITLE: PFOS: ADietaryLCS0Studywithte NorthernBobwhits 1 PROTOCOLNO:: 454030999/QLC/SUB4S4 ~~ DEVIATION NO: 2 ] SPONSOR: 3M Carporation PROJECTNO: 454-103 i DATE OF DEVIATION: April 26- May 14,1999 R: -_ i DEVIATION: B| broodeOrnlesveelvsehroaulsoicncgassitoundsytbeimrpdesratureswereinadvertentlynotrecordedfortwoto eight 1 Ai B B-y a. iN Bn a REASON: `were oBtiorleocogridsetod,vetrhseibgrhota.dWeirptehatshwehxecreeptteimopneroafMtuareys1w,e1r9e9p9o,twrheecnortdherdeweebrreoodpyteamptetrhaetturiemse oofbsaefrtveartniooonsno.bsBieorlvoagtiisotnssr.eBcorrodoidngerapfteenrtneomopneorbasteurrveastiaornostoypnitchalloycrceacsioorndsewdhdeurreintgemapfeireamtouornes t`hweorsemaimsesedadyn.eglectedtorecord temperaturesforemptybrooder levelsthathousedbirdsearlier IMPACT: Datafothesebroodeslevelsonthedaysprior toandlorfollowin themissedtemperatures birnodoicdaetresdvtehratetpeemrpfeorramtiurnegspwreorpeewriltyohinn tthheedacacyespotafbtlheeramnigse.seIdtiosbrserevaatiosnsto.oTnheexrpaeecftobtrhea,lttthehies deviationcausednoadverseimpactontheoutcomeofthe stud. ul : ALa Pow, n- STUDY DIRECTOR olaln DATE oN WILDLIFE Ys TTD. MANAGEMENT DATE : 001437 a a WILDLIFE INTe ERNATIONs AL, Lo PPeage lloef | | : DEVIATION TO STUDY PROTOCOL - STUDY TITLE: PFOS: ADietary LCS50 Study with the Norther Bobwhite | PROTOCOL NO.: 454/030999/QLC/SUB454 DEVIATION NO.: 3 B SPONSOR: 3M Corporation PROJECTNO.: 454-103 " -_-- | DATE OF DEVIATION: April 26, 1999 DEVIATION: " , birdsfOronmtAphrei6l4206,pp19m9a9.,i.Daty 4roftheelesvtaeuldiyn,tatdhveemartfetneetrlnyondoinodbntsoetrivnatciloundsefroercuonridtedBo3b7s-e1rvhaotusiionngs. B of two birds. B REASON: . Biologist oversight. . 2 IMPACT: Bn: None. Observations from themorning of Day 4were made forallbirdsin Pen B37-1, nNIt idtsehtreaeiaolsbisonengravwbahlteiitcoonh.awsesruemdeistphleayywiengrseingonsrmoafltoixniacpitpye.aBraenccaeuasnedobbeshearvvaitoiro,nbsuotfthtewboiobliorgdisswtefarielnedottonroetceodrdit Bnyr Nat,e Lelie. aly STUDY DIRECTOR DATE i `WILDLIFE TM Se Rr LTD. MANAGEMENT DATE 001438 WILDLIFE INTERNATIONAL, LTD BR a SOP DEVIATION PROJECTNO: 454-103 STUDY TITLE: PROS: A DIETARY LCS0 STUDY WITH THE NORTHERN BOBWHITE: a PROTOCOLNO:: 454/030999/QLC/SUBA4S4 a SOPNO:415 DEVIATIONNO:: 1 `SPONSOR: 3M Corporation PROJECTNO: 454-103 A DATE OF DEFACTO DEVIATION: May 10-13, 1999 | --_-- ": DEVIATION: B5 onmayBr1o0o-1d3e,r1u9n9i9t.B2T1h-i1subsreooddteorhuonuistecraonngterdoblegrtowuepebnir1dasnwdas3bbeelloowwtthheespmeicnifiimeudmmiannimthuemrofar9e5noF .- rteecmopredrsatoufretsh.erBersopoodnesriubnlieteBc2h6n-i1cuiesndorfbhiooluosgeisbtirmdaskfirnogm tahteh3e2rm0opsptamta.d.jtursetamtemnetnttgorcoourprwecstth1e a!) bwealsowwtihtheinsptehceifspieecdmifiineidmruanmgoe.f 95FonMay 10,1999. Onthefolowingday th broodertemperature n REASON: : B Misadustmentofthe brooder uit thermostat. n IMPACT: N `Nose. Therewasnoappareateffectontestbirdbodywighto health. Mole ddA Bn STUDY DIRECTOR dln DATE WILDLIFE Eh, TTD. MANAGEMENT BES w 5 001459 i e WILDLIFE INTERNATIONALe , r LFTOD PPRROOJJEECCTTNNOO.: 445544-110033 1: SOP DEVIATION a STUDY TITLE: PFOS: ADIETARY LCS0 STUDY WITH THE NORTHERN BOBWHITE PROTOCONOL: 454/030999/QLC/SUBAS4 " SOPNO: 423.14 DEVIATION NO: 2 . `SPONSOR: 3M Corporation PROJECT NO: 454-103 | DATE OF DEFACTODEVIATION: April22, 1998 - DEVIATION: i OnApril22,1999,duringdictpreparationonDay 0ofthetest, 1.4659 goftestsubstance a. ewxacseweediegdthhedtoaurgtetfaomropurnetbpya0.r0a9o9t3fi.toThnhee1S6O0Pipnpdmicaa.t.esttehsattctohnecteensttrsautbisotnadniceetw.iTlhlbieswaemioguhnetd to within 0.0005 gofthetargetamount. REASON: : 0 Dietician oversight. ) IMPACT: 0 `None. Thediffrenceintheamountoftetsubstanceweighedrelied n lessthan1%change in finaldietaryconcentration. w: i/ b 2d. <x STUDY DIRECTOR. DATE ala . SE aes |B WILDLIFE (ONAL LTD. MANAGEMENT DATE " 001440 i. PFOS: A DIETARY LCS50 STUDY WITH THE NORTHERN BOBWHITE 1 US. Environmental Protection Agency i Series 850-OPEPcoTlSogNiucamlbEefrfe8c5t0s.T2e2s0t0Guidelines [i FIFRASubdivision E,Section 71-2 OECD Guideline 205 B. 3M Lab Request No. U2723 RB Submittedto RB . 3M Corporation Environmental Laboratory 935 Bush Avenue 5 St. Paul, Minnesota 55144. WILDLIFE INTERNATIONAL LTD. 3 8598 Commerce Drive 5 `Easton, Maryland 21601 (410) 822-8600 u[8 March, 1999 001441 A WILDLIFE INTERNATIONAI L LTD ect ~semreeeriereeeeesemseee ees w > a PFOS: A DIETARY LCS0 STUDY WITH THENORTHERN BOBWHITE "n i SPONSOR: | E3nMviCroornpmoernattailonLaboratory SPt..0.PaBulo,xM3i3n3n3e1sota 55133 SPONSOR'SREPRESENTATIVE: Ms. Susan A. Beach H TESTINGFACILITY: " . `8W5i9ld8liCfoe mImnteerrncaetiDornailveLtd. `Easton, Maryland 21601 . STUDYDIRECTOR: Sean P. Gallagher, Senior Avian Biologist nm LABORATORYMANAGEMENT: `JMoaannaagBe.roBfeaAvveirasn Toxicology n FOR LABORATORY USE ONLY a Start Date: alan Termimionpa: _ 5443 a ProjetNo: _HH -103 StudyRoom: 25 " Test Concentrations:0, 30 49, $0160, v0, L%o 1330 yma; wu Test Substance No: 475 Refercace Substance No. iapplicable): _ 4531 un PROTOCOLAPPROVAL 0 Lieed Sb, STUDY DIRECTOR Aha DATE 4 RAT] . x SPONSOR'S REPRESENTATIVE | PROTOCOL NO. 454/030999/QLC/SUB4S4 Sr / 3 IE! Ja 9 Dad 001442 3MLAB REQUEST NO. Uz ] a WILDLIFE INTERNATIONAL LTD " OBJECTIVE B "Theobjectiveofthisstudyistoevaluateth toxicityofa tes substancetothenorthernbobiwhite: (Colinusvirginiarus)administeredthroughthediet orfivedays.AnLCS0valuewillbecalculateidf, un possible. a SUMMARY Groupsoftenbobwitechicks, 10-14daysold,willbefeddietscontainingthe test substanceat B selectedconcentrfoarftivieodanyss. Acontrolgroupthatreceivesuntreateddietwillbemaintained concurrently. Thetestsubstance villbemixedintothediet, usuallyin ageometricseriesof " concentrations.Theexposureperiodwillbefollowedby aperiodofatleastthreedaysonuntreated ' food Attheendofthethree-day observationperiod, halfofth birdsincachtreatmentgroupvillbe euthanizedand liverandbloodcollectedforresidueanalysis.Therembiardsiwilnbeimainntaigned H onbasaldietforanadditional 14daysandthenwillbeeuthanized,vithliverandbloodcollected. "Throughoutth test,thebirdswillbe observedfortoxicological responses.WhenpossiabnLlCeS, Bn valuewillbecalculated. a MATERIALS ANDMETHODS "Themethods,speciesusedandrouteofadministrationdescribe inthisprotocolarebased upon un `proceduresspecifiedintheU.S. EnvironmentalProtectionAgency Series850 -EcologicalEffects Test Guidelines OPPTS Number 850.2200 (1); Section 71-2ofthe Environmental Protection Agency's " Registration Guidelines, Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation - Wildlifeand Aquatic Organisms (2), ASTM Standard E8S7-87,"Standard Practice For Conducting - Subacute Dietary Toxicity Tests With Avian Species" (3), and OECD Guideline 205, Avian Dietary |8 ToxicityTest(4). Taord tocontrolbias,birdswil beassignedtopeasbyindiscriminatedraw. No nN otherpotentialsourcesofbiasare expectedtoaffecttheresultsofthestudy. `Thetestsubstancewillbeadminisintteherdeictd. Thisroutsofadministwrasasteliecotend nN `becauseitrepresentsthemostlikelyrouteofexposuretoavianspeciesinthecavironment. J > |8 BN Informationon thecharacterizationof test, controlorreferencesubstancesisrequiredbyGood LabPoracrticeaStatndaordsr(GLyP).`TheSponsorisresponsibleforproviding WildlifeIntemational Ltd.writtenverification thatthetestsubstancehasbeencharacterizedaccordingtoGLPsprior 1its PROTOCOL NO. 454/030999/QLC/SUBAS4 001443 314pREQUESNOT.12723 n "a B n "a on 0 : | a un un LL}. oH un W_I--LDLIFE INTERNATIONAL LTD an useinthestudy. Ifwrittenverificationof GLPtestsubstance,characterization isnot providedfo `WildifsInternationalLid,itwillbenotedinthecompliancestatementofthefinalreport. The __ attached form IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR (Appendix I) isto be usedtoprovideinformationnecessaryfor GLPcompliance. `TheSponsorisresponsibleforal informationrelatedtothetestsubstanceandagreestoaccept `anyunusedtestsubstance and/ortest substancecontainers remainingattheendofthestudy. TreatmentGroups `Groups oftenbobwhitechicks (fiveperpen)willbeassignedbyindiscriminatedrawtocachof thetreaandtconmtroelgrnoutps.Thesex ofeachindividualwillnotbedetermined. Atestwill usually consistof ageometricseriesoffivedietaryconcentrationsandatleas threscontrolgroups. "Testconcentrationswillbeselectedafterevaluatingtoxicitydataprovidedbythe Sponsor.Thehighest testconcenenedtnotrexcaeetd50i00oppnm.Eachgroup ife theappropriatetestorcontroldietfor fivedays. Thiswillbefollowedbyaperiodofatleastthre daysonuntreated ocd. Controlbirdswill eceiveanamountofthesolventorvehiclein theirdietequivalenttothe highestamountinthetreated dies. Ifmorethanonestudyisconductedsimultaneouslyin thesame room,withthesamesolveatorvehicle, aconcurrentcontrolmaybeutilizedforallstuics. a "Theprimaryphasesofthe testand therdurationsare: 1. Acclimation Fromreceiptorhatchofhatchlingsuntilth statofexposure. 2. Expo-sFivuedrayes. 3. Postexposure observation- 3days/L7days. Test Birds w `Thebobwhiterepresentsanecologically significantand widelydistributedspeciesin the United 3 States. Thebobwitehasdemonstratedsensitivitytotheeffctsproducedbyknowntoxicchemicals. ol Moreover,tisspecieshasproventobe agoodlaboratorymodelandalargeamountofbasclinedata areavailable, ou 001444 i PROTOCOL NO. 454/030999/QLC/SUBA4S4 3MLABREQUENSOT. U2723 iH mw WILDLIFE INTERNATIONAL LD w 5 Albobhitewillbe fromthesamehatchandvillbe 10-14daysoldattestinitiation.Only... x thosethatareapparentlyhealthyvillbeusedinthetest. Bodyweightsmayvarybetween 10and 50 gramsdependingontheageofthecobortofbirdsused (e.g.10ver1s4duayss). Atleivaetesstatnd mn threecontrolgroupswillbeused.Eachtreatmentorcontolgroupvillcontaintenbirds (twopeasof fivebirdscach)witheachgroupof birds identifiedbypen numberandtestconcentration. Individual mn birds wil be identifiedby colored leg bands. Birds will be obtained from Top Flight Quail Farm, Belvidere, NJ07823,fromanotherreputablesupplieror obtainedfromtheWildlifeInternatioLntadl. wn `productionflock. All experimentalbirdsvillbeacclimatedtothe cagingandfacilites from the timeof : hatchorreceiptuntilinitiation ofthetest. iW _-- . Alltestbirdsvillbefed agamebirdrationformulatedto WildifeInternationalLtd. on specifications(Table 1). Fromthetime ofhatchor receiptofthe hicksuntiltheinitiationofthetest, thebirdswill receive awater solublevitaminmixviatheirwater. Feedandwaterwillbeprovidaed mn libitumduringacclianmdduaritngitheotenst,Thewatersourcewillbethe TownofEastonmunicipal watersupply. FeedisanalyzedperiodicallyinaccordancewithWildlifeInternationalLtd.Standard on `Operating Procedures. Thebirdswillreceiveno formofantibioticmedicationduringthetest. m Specificationsforacceptablelevelsofcontaminantsingamebirdrationforavian specieshave = notbeen established.Thereare nolevelsofcontaminantsreasonablyexpectedtobepresent nthediet ow thatareconsideredto interferewiththepurposeorconductofthe study. DiPerteparation ou "Testdietswilbepreparedatthestartofthetestand asufficientportion estimatedforthe 5-day wm `exposureperiodwillbepreseatedtothebirds. Afterfivedaysofexposure,allbirdswillreceive untreatedbasalration. mw `Thetestsubstance willbemixeddirectlyintotherationorbydissolvingor suspendingthetest substanceinacetoneand/ortablegradecommoi priortomixingwiththefeed.Ifused,acetonewillbe ol allowedtovolatilize from thedietsduringthe mixingprocedure. Tablegradecomoil will normally be incorporatedintothedietat amaximumof2% (w/w)ofthefinaldiet.Mixingisnormdoanelwilthya m Hobartmixer(Model Number AS200T). . 002.145 ow PROTOCOL NO. 454/030999/QLC/SUB4S4 3MLABREQUESTNO. U2723 iN WILDLIFE INTERNATIONAL LD wn x m om mn on : mn mn nN wom wo . mw x " = |3 a oIw mE ` Alltestsubstancecalculations willbebased onthepurityofthe testsubstance.asreceivedor correctadto 100% activeingredientbasedontheinformation providedbytheSponsorinAppendix I of thisprotocol. f-- Samplesof theexperimentaldietswillbe collectedforchemicalanalysistodetermine the homogeneityandstabilityofthetestsubstanceinthe avian dietand toverifylmeasuretest `concentrations. Allsampleswillbeplacedin uniquelyideatifiedpolypropylene jars. Sampleswil becollectedonDay0,atdictpreparation,todeterminehomogeneity,measuretest concentrations, andestablishDay 0valuesforevaluatingstabilityoverthecourseoftheexposure period.OnDay5,attheendoftheexposurephase,sampleswillbecollected from thefeeders `containingtheexperimentaldiets10assessstabilityofthetestsubstanceundertestconditions. Ifa separatestudyisconductedwith adifferentspecies (.5.,mallard),a thesametestconcentrations, dietsthatarepreparedmaybedividedbetweenthetwostudies. Therefore,samplescollectedfor homogeneityandverificationwillbecommontobothstudies. However,samplescollefcrotmethde -- feedersinthe studyroom (c.g. Day 5samples)willbeunique to that respectivestudy. Thediet sampling scheme forstudies with fivetestconcentrationsissummarizedbelow. Thesamplingschems `maybeadjustedbasedontheactual purmberofconcentrationstested(seePage2)ortheoptionofthe Sponsor. Experimental Group Dayo Days -- : : Control 1 1 Love 1 -LowConcentration 2 Lewis 2 2 Lewd 2 2 Level 5-HighConcentration 2 1 u "TotalNuomfSabmplees=r30 bSoattmopmlelascyoelrlseocftfeedefdrionmtthheemilexfitanngdvreisgsehlttsioddeestoefrtmhienethopo,momgiedndelietayn.d : 001446 PROTOCOLNO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 N oWOLFE INVERNATIONALGD un " i Theabovenumbersof samplesrepreseatthosecollectedfromthetest anddonotinclude quality contol (QC)samplessuchasmatrixblanksandfortifications preparedandanalyz duringthe m analyticalchemistryphaseofthestudy. TM Di Samplesoftheexperimentaldietsmaybeanalyzedfollowingcollectionorstoredin afreezer untilpreparedandlorextractedforanalysis. Chemicalanalysesofditswil beperformedbyWildlife |} InternationalLtd. Theanalyticalmethod usedvillbebased upon methodologyprovidedbythe `SponsorandidentifiedinAppendiITx. Themethodologyusedtoanalyze thetestsampleswillbe: Bn documentedintherawdataand summarizedinthefinalreport. Masx ampli eholdm ingtiu mes,pm rior toanalysis, maybespecified bytheSponsor,andifspecified,willbeaddedtothisprotocolby BH amendment E Cant 0 "Testbirds willbehousedinthermstatically confrolled broodingpeas manufacturedbythe 0 Beacon SteelCompany(Model B735Q)orequivalent.Eachpenmeasuresapproxim7a2tXe90lXy 23cm high. Theextemalwallsandceilingsof cachpenareconstructedofwiremeshandgalvanized sheeting. Floorsarcofwiremesh. Uponinationofthe test, cachbrooding penwillcontain five H. birds, Twobrooding penswillbeassignedtoeachtreatmentgroup(10birdstotal),whileatleastsix \ peasvil beassignedtothe control(at least 30birdstotal). Eachgroupwillbeidentibfyipeedn o `umber. Duringthetet,th temperatureinthebroodingcompartmentofthepenwillbemaintainedat approximately 38C. Acceptablerangesforbroodingcompartmenttemperatures willbespecifiedin BH `WildlifeInternationalLtd.StandardOperatingProcedures. Broodingcompartmenttemperatureswill berecordedoncecachdayduringthetest.Theambient oom temperaturewillangebetween approxi- 0 `mately 15-30C.Roomtemperatureandrelativehumiditywillberecordedtwicedailyduringthetest. g A16 hoursligh/8hoursdarkphotoperiodwillbemaintained,and theaveragelight intensityprovided ou tothebirdsduringthetstwillbedetermined. . Housingandhusbandrypractioeswillbeconductedsos toadheretotheguidelinesestablished H `by NationalResearchCouncil(5). H 001447 0 PROTOCOL NO. 454/030999/QLC/SUB4S4 3M LAB REQUEST NO. U2723 B WILDLIFE INTERNATIONAL LTD " 5. B Observations a AIbidsvillbeobservedatleastoncedailyformortalityandtoxicologicalresponses. | Necropsy " Alltestbirdsthatdieduringthecourseofthetestandalbirdscuthanized wilbesubjectedtoa. grossnecropsy. ForthosebirdseuthanizedonDays 8and22ofth tet, livesand ~ 2mL.ofblood i: willbecollected. Liverswillbeweighed,placedinindividuallabelledcontainersandstoredfrozen, : Bloodwillbeplacedin labelledheparinized vacutainersand placedunderrefrigeration. All samples. 0 willbetransferredtothe Sponsorforresidueanalysis. : `DispoosfTiesttiBiordns = Attesttermination,testbirdswillbeeuthanized by usingcarbondioxidegas, cervical disolrotohercapparoprtiatiemeothonds. Themethodusedwillbedocumentedintherawdata. All uN carcasseswillbestoredfrozenpriortoshipmenttothe Sponsor. : Statte Celtals a `Theconcentrationresponse dataobtainedfrom thetestwill be evaluated and,ifpossible, an Ho LCS0 value and 95% confidence limits will be calculated. When the mortality data facilitates ` statisticalanalyses,oneofthreemethodswillbeusedtocalculateanLCSOvalue. Thedatawillbe i analyzed,inorderofpreference,byprobitanalysis,themovingaveragemethod,orthebinomialproba- un bility met(6,h7,o8,d9).ThechoiceofmethodforcalculatingtheLCS0valuewillbebaseduponthe ou `mortalitypattern observed.Whenpossible, a no mortalitylevelwillbedeterminedandreported. REC TOO BE MR AIND TAIS NED uo Records tobemaintainedfordatageneratedatWildlife InternationalLid.willinclud,butnot - be limitetod: |] 1. Acopyofthesignedprotocol. . = 2. Wentificationandcharacterizationofthetestsubstance,ifprovidedbytheSponsor. oF 3. Datesofinitiation andtermination ofthetest. 4. Animalhistory. ou 5. Husanb denviaronmnentadlconrditiy ons. 6. Concentrationcalculationsandrecordsofdietpreparation. 001448 o PROTOCOLNO. 454/030999/QLC/SUBAS4 3MLABREQUENSOT. U2723 [1 B WILDLIFE INTERNATIONAL LTD mn | . BH : an - 7. Bodyweightmeasurements 8. Feedconsumptionmeasurements. 9. Daily observations. 10. Statisticalcalculations. 11. Analyticalchemistry methods,resultsandchromatograms,if applicable. 12. Acopyofthefinalreport. i [FINALREPORT n Areportoftheresultsofthestudy willbepreparedby WildlifeInternational, Ltd.The report ou will include, butnotbelimited to, the following: 1. Nameandaddressofthefacilityperformingth study. |. 2. pDartoevsiodntwhhoifchatlhesattuedytwiatsdianittaiaatredarncdcaompleftoerdc.heImtiisstthe epaslpoognyasnodiflotbrheiSpulopnispoitrnytgo . evaluationsthatmaybegeneratedatotherlaboratories. 0 3. AstatementofcompliancesignedbytheStudyDirectoraddressinganyexceptionsto Good | LaboratoryPracticeStandards; : Hn. 4. Objectivesandproceduresstatedintheapprovedprotocol,includinganychangesintheoriginal protocol. 0 5. Statisticalmethodsemployedforanalyzingthedata,whenapplicable. ThecalculatedLCS0 = value,95percentconfidence limits,slopeoftheconcentration-responsecurve,theresultsofthe ol `goodness-of-fittest (e.g. X"test),and adescriptionofstatisticalmethodsused.Thesame "- statisticswillbeprovidedforpositivecontrols (whenused). . 6. Thetest,control andreferencesubstances identified byname,chemical abstractsmumberor on `odenumber, strength,purity,andcompositionorotherappropriatecharacteristic,ifprovided `by the Sponsor. oN 7. Stabilityand,whenrelevanttotheconductofthestudy, thesolubilityofthetest,control and . referencesubstancesundertheconditionsofadministration,if providedbytheSponsor. mw 8. Adescofrthietepststysitemousned.Whereapplicable,thefinalreport shallincludethe : `numberof animals used, bodyweight range,source of supply,species (includingscientific om name),age,andprocedureusedforidentification. - 9. Adescriptionofthedosage,dosageregimen,routsof administration,and duration. om 10. Adescriptionofthemethodsused,includingbutnotlimitedto: 001449 4 PROTOCOL NO. 454/030999/QLC/SUBAS4 3MLAB REQUEST NO. U2723 " W~I~LDLIFE INTERNATIONAL LTD a -10- n a) sDueptpalielemdentdess(crfiupsteido)n. oAfnutthreienbtaasnalalydsieits,ofitnhcldudiicntgvilsolubrecien,clduidleudenitnsth(eitfesutsreedp)o,rt,and i b) cTohnecennturmabteiornoofftceosntcesnutbrsattainocnseiunsecda,cnholmeivneall,aasnsday(mwehterheodrueqsueirdetd)odmeetaesrumriendeadciettuaarly on cuonscdenftorraptoisointsi,vencuomnbterroolsf(bfiarpdpslpiecrabcloen)c.enRtersautlitosnoanfrd faorncougtreol,-taenfsdtni(aifmnceodnsdouifcttnoexdgi)c.ants oN ) Acclimation proceduresand methodsofassigningbirdstotest pens. @) Fortheeqrueanbcnyo,rdmaul brehaaavnitodrm,ietionhcolndusdoinfgobtsiemrevoaftioonns.etD,esdcurriapttioino,nofsevseirgintsyof(iinntcolxuidicnagtidoenatahn)d, i taensdtnpeurmiboed.rsaffectedin thedifferent dietaryconccatrations aadcontrolseach day ofthe u;N tDempeerasturceso,fraphpoiruosxipinmgattceotinedsitotrioononm,hiunmcildiutdyi,ngphtoytpoep,esriiozdeaandndlimghatitngainlteoafsipye.n,pen wom `Averxagebppoedryioowdeaingsdhetsnufdoofbrtihredeseistn. eachpenatthebeginningofthe test,theendofthe [| 8) Estimatedfoodconsumptionfortheexposureperiodandforthepostexposureperiod. ,. 1. Adescriptionofallcircumstancesthat mayhaveafectedth qualityorintegrityofthedata. mn Anythingunusualaboutthetest,anydeviation fromtheseprocedures,andanyotherrelevant on. 12. ThenameoftheStudyDirector, thenamesofotherscientistsorprofessionals,andthenamesof al suppeersornnelv,inivolvsediontherstuydy. w 13. A description ofthe transformations, calculations, or operations performed onthe data, a summaryandanalysisofthedata,ad a statementoftheconclusionsdrawnfromtheanalysis. i 14. Thesignedanddatedreportsof achoftheindividualscientistsorotherprofessionalsinvolved mn inthestiuf adppliycab,le. 2 15. Thelocationwhereal specimens,rawdata,an thefinalreportaretobestored. w 16. AstatementpreparedbytheQualityAssuranceUnitlistingthedatestha studyinspectionsand auditsweremadeandthedatesofanyfindingsreported otheStdyDirectorandManagement. = 17. Ifitisnecessarytomakecorrectionsoradditionsto a finalreportaftrithasbeenaccepted, suchchangesshallbeintheformof amendmentbythe StudyDirector.Theamendmentshould ow clearlyidentifythepartofthe finalreportthatisbeing addedtoorcorrectedandthereasonsfor thecorreocratddiitoionn. Ameadrmentsshallbe goedanddatedbytheStudyDirector. m 001450 [1 PROTOCOL NO. 454/030999/QLC/SUB4S4 3MLABREQUESTNO. 02723 Bn WILDLIFE INTERNATIONAL LTD uN 1- x CHANGING OFPROTOCOL `Plannedchangestotheprotocol willbe ntheformofwrittenamendmentssignedbytheStudy 0 DirectorandtheSponsor'sRepresentative. Amendmentsvillbeconsideredaspastoftheprotocoland. | wil beattachedtothefinalprotocol. Anyotherchangeswillbein theformofwritendeviations 0 signedbytheStudyDirectorandfiledwiththerawdata. Allchangestotheprotocolwillbeindicated. inthefinalreport. w GOOD LABORATORY PRACTICES wm `ThisstudywillbeconductedinaccordancewithGoodLaboratoryPractice Standardsfor EPA (40 CFR Part 160 and/or Part 792); OECD PrinciplesofGood Laboratory Practice (OCDE/GD (92) : 32, Environment Monograph No. 45); and Japan MAFF (59 NohSan, Notification No. 3850, n Agricultural ProductionBureau). Eachstudy conductedby Wildife InternationalLed. isroutinely examinedbythe WildifeInternatioLntadl. Quality AssuranceUnitforcompliancewithGood in Laboratory Practices,StandardOperatingProcedures andthespecifiedprotocol. Astatemeonft `compliance with Good LaboratoryPractices willbepreparedforallportionsofthestudyconductedby mn `WildlifeIntemationalLtd.The Sponsorwillberesponsibleforcompliancewith GoodLaboratory Practicesforprocedures performedby otherlaboratories (e.g,residueanalysesorpathology). Raw on dataforallworkperformed at WildlifeInternational Ltd.and a copy of the final reportwillbe filedby ie `project numberinarchiveslocatedonthe WildlifeIntemationalLtd.siteoratanaltemativelocationto wu bespecified inthe finalreport. mn un = PROTOCOLNO. 454/030999/QLC/SUBAS4 001451 3MLABREQUESNOT. U2723 on WILDLIFE INTERNATIONAL LTD on -n- REFERENCE n 1 U`GSui.delEinnvesir(odnrmae)n,taOlPPPTroSteNcutmiobnerAg8e5n0.c2y2.00:19A9v6i.an DiSeertiaersy T8o5x0i-citEycoTleostg.ical Effects Test 2 US. Environmental Protection Agency. 1982. Pesticide Assessment Guidelines, FIFRA mm SEunbvdiirvoinsmieonntalE,PrHoatezcatriodn AEgvaelnucayt,ioOnf:fice WoiflPdelsitfieciadnedPrAoqguraatmisc. WOarsghainnigstmosn,, sDu.bCs.ection 71-2, 5i 3 ASmubearciuctaenDiSeotcaireytyToxfiocritTyesTtesitnsg waintdh AMvaitaenriSaplesc.ies.19A87S.TMStSatnadnadradrdPEra8cSt7i-c8e7.forAnCnounadlucBtoionkg ofASTM Standards, Vol. 11.04. Philadelphia, PA. om 4 OTersgta.niOzEatCiDoGnuifdorelEicnoenfoomiTc CeoopsoerftaCthieiomnincaanlgsd.DeGvueildoelpimneent20.5. 19P8a4ri.s.Avian Dietary Toxicity wu 5 `NWaatsihoinnaglioRne,sDeCa.rchNaCtoiuonncaillA.ca1d9e96m.y PrGeussi.de1f2o5rptph.e Care and UseofLaboratory Animals, un 6 Finney, D. J. 1971. Stristcal Methods inBiologicalAssay,20d ed,GriffinPress,London. 7 ThompsWo.nR,. 1947. BacteriologicalReviews; Vol 2, No. 2 115-145. m 8 `SatnedphHaanz,arCd. EEv.al1u9a7t7i.onsM,etAhmoedrsifcoarn CSaolcciutlyaftoiranTngesLtCiSn0g.anPdagMeatser6i5a-l8s4.InPuAbg.uaNtoi.c TSoTxPico6l3o4g.y , Philadelphia, PA. nu.: 9 StSeepphhaann,,C.EC.1 . 1978. U.S.EPA,EnvironmentalResearchLaboratory,Duluth, MN. Personal un mE PROTOCOL NO. 454/030999/QLC/SUBASS 001452 3M LAB REQUEST NO, U2723 [+] WILDLIFE INTERNATIONAL LTD H -13- TABLE 1: DIET FORMULATION Bn `WILDLIFE INTERNATIONLATLD,.GAME BIRD RATION! EE -- INGREDIENTS n -- WShoeyaBteMaindMdeasl,48% Proein n Tr 0: Dr``iAAlcgfawdlafyaWMhSepeeaycli,al2,06%0%ProPtreoitnein Fe EGarsotumnadnLCiamlePsthoonse Methionine Premix + Liquid 0 i lL os 0. TeSGaLltFTeodrimze(dFermatco) PERCENaTn(%) 3605605 i]24300%00 005600 035 ox000%1250 0H "VITAMINANDMINERAL PREMIX AMOUNTADDED PERTON 0 VViitaammiinnDA, ; NRiibsocfilnavin un a : ' FVoiltiacmAicniBdy iw PByiroitdionxine ThViitaammiinneE. om Foimation . MZainngcanese x . ICoodpipneer ["]. TSerloennium fem 72,000000,,0000001LCU..U. 460 ggrraammss 60 mmggss 6412mggsrams 20,00102LUg.rams. 14072 ggrraammss 6185 ggrraammss 15812gmrgams - T5h%ecgruuadreanfitbeeer,danalysisis aminimumof 27%protein, amisimumof2.5% crudefatand amaximumof wm * FermentationBy-Products(SourceofUsidentified GrowthFactors). ou . 001453 |: PROTOCOL NO. 454/030999/QLC/SUBA4S4 3M LAB REQUEST NO. U2723 0 WILDLIFE INTERNATIONAL LTD " ue | APPENDIX BR IDENTIFICATION OFTEST SUBSTANCEBYSPONSOR "TobeComplbyeStpoensdor Bl. te Subsancerty cameobosdinte report: EOS.(PrlorotansSufiAc Poesia Sa Reference Standard (if applicable): Analytical Standard: NA 0 InternalStandard: P1.1e2.2r6 HfHlHuorooSuclfotnicaAcind e `Test SubstanceSampleCodeorBatch Number:__ Lot 217 wn Test SubstancePurity (% Active Ingredient): _98.9 ExpirationDate: _2008 n Sites egetpryod openrot domi IL TestSubstanceCharacterization Hayetheic other is `determined prior toitsuse inthisstudyinaccordancewithGLPStandards? -- Yes _xNo Ho HL TestSubsStotragaeCnondictioens. Pleaseindicat therecommendedstorageconditionsatWid nematioalLd n e-- t -------- - [titi Hasthe: oft under these a condition.s on Otherperstabii ninfeormnatiton: Ya x No 0 IV. TestConcentrations: x Adbjusattes st choe encpeuntrdrtayti(u o%n )tgopi1v0eo 0na%bao.nv.e. mn F' DaoimtTaedsitutsetmtaetsrtacolmAmSmIgSt.ionto 100% _V. ToxiiyInformation: ou Mammalian: RatLDSO 250mghg MowssLDSO_NA wu Dag magna:27mul __ ReiobowTrout: Lime Aquatic: Invertebrate Toxicity (BC/LCS0) `FishTondicity(LCS0) P0 laseseMs0 DS 00000 wn `Other Toxicity Information (includingfindings ofchronicandsubchronictests): - 001454 ot PROTOCOLNO. 454/030999/QLC/SUB454 3M LABREQUEST NO. U2723 mn ~WI-LDLIFE INTERNATIONAL LTD ": "15. APPENDITXI mn MethodOutline fortheDeizrminationof BFOSinAvianFeed on 1. Preparematrixfortification samplesinthedesiredavianfoodstockusingthedrymix technique. 0 2. 1W0ei1g6ho1z.0Frgesnacmhpslqeusaorfetglhaesmsabtortitlxebsl.aRnekcaonrddmvaietirgihxifso.rtificationfeed samplesinto aweighboatand transfer Bl 2 FForreaccahchsqwuaerieshboetdles.ample,measur 100mlsofmethanol with 8graduatedcylinderand trans ino he wm 4. Sealbottleswithscrewcapsandplaceonshakertable. Allowtoshakefor aminimumof 30minutes. | 5. Vacuumfilterwith qualitativefilterpaperandrinseretainedfoed 3 timeswithmethanolinto filtrate. n 6. Transferfiltrateinto a 200-mLvolumetricflaskandbringtovolumewithmethanol. mn 7. wTartanesrfedrilu5timoLnssoolfve2n0t0c-omnLtaisnoilnugti0on.0in5t%o va/v10f-ormmLicvoalcuimdetarnidc f1l0a0skppgartai.alilym-Lfil4leHdPwFitOhS5i0n%temmaelthsatnanodla:r5d0.% ; `Bringtovolume withdilution solvent. | 3 iMnitxoaanndHtPrLanCsafeurtapopsroaxmipmavltieeallry. 1mLofthe 10-mLsolutionitoasyringeandfierthrough a0.2umfilter mn Ampulateandsubmit samplefor LCMS analysis. wi " ui PROTOCOLNO. 454/030999/QLC/SUB4S4 : 001455 3MLABREQUENSOT. U2723