Document O3OKOKw7xw0Gm3jkYkD83jnov

DIETARY ACUTE NORTHERN BOBWHITE STUDY TEST SUBSTANCE Identity:Perfluorooctanesulfonatmea;y alsobe referredto as PFOS or FC-95. (1-Octanesulfoniacid,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-p,otassiumsalt,CAS # 2795-39-3) Remarks field: The testsubstance isa whitepowder. Sample was takenfrom 3M lotnumber 217. Sample was storedunderambient , conditionspriorto testingP.uritydeterminedtobe 90.49% by LC/MS 'HHMR, 'GF-NMR and elementalanalysestechniques. METHOD Method: OPPTS 850.2200,OECD 205, and FIFRA SubdivisionE. Section 71-2 Type: Dietaryacute GLP: Yes Year completed:1999 (Test)2,000 (Report) Species:Colinusvirginianus Supplier: WildlifIenternationLatld.ProductionFlock,Easton,Maryland Analyticalmonitoring:Testsubstanceconcentratioinnstandardsand samples were determinedby reversed-phaseHPLC and mass spectroscopy.PFOS measured on Day 0 forhomogeneityinfeed and verificatiaond, Day 5 forstability. Test phases: Acclimation- 10 days Exposure -5 days Post-exposureobservation- 3 or17 days Statisticamlethods: LC5o valuescalculatebdy probit analysisusingthecomputer softwareofC.E. Stephan. Body weightdata were compared by Dunneft'stestusingTOXSTAT software.No statistical analyseswere appliedtofeed consumption data. Test birdage: 10 days Pretreatment:None Test conditions: Housing and environmental conditions: Indoorsinbatterieosf thermostaticalcloyntrollebdroodingpens. Each pen'sfloorspace measured approximately72 X 90 cm. Ceilingheightwas approximately23 cm. Externalwalls,ceilingasnd floorswere constructedofgalvanizedsteelwireand.sheeting. IdentificatioEna:ch group of birdsidentifiebdy pen number and testconcentrationI.ndividualisdentifibeyd legbands. Number of replicatesS:ixforcontrolst,wo foreach treatment group Number of bobwhite per replicatef:ive Number of concentrations:seven plusa negativecontrol Feed and water:Game birdrationformulatedas below,waterfrom thetown ofEaston publicwatersupply.Both providedad libitum duringacclimatioannd testing. Game BirdRation Ingredients Percent Fine Corn Meal 44.83 Soy Bean Meal, 48% Protein 30.65 Wheat Midds 6.50 ProteinBase 6.00 Agway Special,60% Protein 4.00 AlfalfaMeal,20% Protein 3.00 DriedWhey 2.50 Ground Limestone 0.90 Eastman CalPhos 0.60 Methionine Premix + Liquid 0.35 Vitaminand MineralPremix (see below) 0.32 G L F e rm (Fe rF aFt-c-or-)- 0.25 SaltIodized 0.10 'Fermentationby-products(sourceof unidentifiegdrowth factors) Vitaminand MineralPremix Vitaminor mineral Amount Per Ton Vitamin D3 Vitamin A Riboflavin Niacin PantothenicAcid Vitamin B12 FolicAcid Biotin 2,000,000 I.C.U. 7,000,000 l.u. 6 grams 40 grams 10 grams 8 mgs 600 mgs 64 mgs Pyridoxine Thiamine Vitamin E 1.2 grams 1.2 grams 20,000 l.U. Vitamin K (Menadione dimethylpyrimidinoblisulfite) 5.8 grams Manganese 102 grams Zinc Copper 47 grarris 6.8 granis Iodine Iron 1.5grams 51 grams iselenium 182 grams ProphylaxisN:one Broodingcompartmentmean temperature3:8 + 20C Ambient room mean temperature2:7.3+ 1.20C Averagerelativheumidity3:1 + 14% PhotoperiodS:ixteenhourslig@pte-rday Lightingf:luorescelnitghtwshichcloselaypproximatneoon-day sunlighta;verage ofapproximately139 luxof illumination Test dietpreparation:Test substance mixed directliyntothe rationby means ofa Hobart mixer. No carriewras used. Diet sampling: Homogeneity of the testsubstance inthedietevaluated by collectinsgixsamples from the 18.3 ppm concentrationand sixfrom the 1171 ppm concentration.Samples collectedfrom the top,middle,and bottom ofthe leftand rightsectionsofthe mixing vessel.These samples also served as the verificatisoanmples forthese concentrations. Verificatiosnamples ofthe othertreatmentgroups (two samples from each) and the control(one sample) were collecteadt preparationon Day 0. Stabilitsyamples were collectedat the end ofthe exposure period(Day 5) from the control(onesample) and each treatmentgroup (twosamples each). RESULTS Nominal concentrations: Bk control,18.3,36.6,73.2,146, 293, 586, and 1171 ppm Measured concentrations: <LOQ, 19.5,40.2,74.5,174, 291, 537, and 1196 ppm Element value: DietaryLC5()= 220 (164 - 289) ppm No mortalitcyoncentration 73.2 ppm NOEC (body weightgain) 73.2 ppm Allelement values based on nominal concentrations AnalyticalMethodology: Dietsamples were extractedwithmethanol. Analyses oftestsolutionswere performed atWildlifIenternationaLltd. using high performance liquidchromatography withmass spectrometric detection(HPLC/MS). When determiningthe concentratioonfthe test substance inthe testsolutionst,he same and most prominent peak response forperfluorooctanesulfonawtaes used. No attemptwas made to quantifyon the basisofindividuailsomericcomponents. The LOQ (limit of quantitationw)as 1.15 ppm inthisstudy.The mean percentrecoveryof matrixfortificatiaonaslyzed concurrentlyduringsample analysiswas 94.7.Samples collectedfordeterminationof homogeneity indietranged from 102-107% of nominal. Samples collectedforverificatiiondiethad measured valuesfrom 92 to 119% of nominal. Measured valuesfor ambiensttabilsiatmyplestakenatDay5 rangedfrom101-122%of nominal. Summary ofanalyticcahlemistrdyata: HomogeneityInAvianDiet NominaTlest Concentration, PPM Measured Values atDay 0,ppm 18.3 18.5,23.4,18.3,17.3,19.4,19.9, 1171 1239,1221,1118.1301,1163,1133 Mean Percentof Measured Nominal Concentration -PPM 19.5 107 1196 102 Verificationin Avian Diet Nominal Test Mean Percentof Con. centration, Measured DuplicateConcentrationast Day 0, Measured Nominal PPM PPM Concentration NegativeControl < LOQ @pm 36.6 45.7,34.6 40.2 110 73.2 77.8,71.2 74.5 102 146 176,172 174 119 293 274,307 291 99 586 550,523 537 92 Ambient StabilityIn Avian Diet Day 0 Mean Measured Mean Measured DuplicateConcentrationsat Day 5, Measured Mean Percentof Concentration, PPM Concentration Day 0 PPM NegativeControl < LOQ PPM - - 19.5 19.2,19.9 19.6 101 40.2 44.4,53.8 49.1 122 74.5 76.4,77.9 77.2 104 174 177,174 176 101 291 318,315 317 109 537 560,665 613 114 1196 1260,1187 1224 102 Biologicoablservations Mortalitiaensd clincialobservationsO:ne incidentmaolrtality occurredinthe contr'olgroup as a resultofa broken legon the morning of Day 5. Itwas subsequentlyeuthanizedon Day 6. Two otherbirdsinthe controlgroup were intermittentnloyted withfoot lesionsassociatedwithcage mate aggression.Otherwise,all controlbirdswere observed to be normal inappearance and behaviorthroughoutthe test. The firsttreatment-relatemdortalitieosccurredon Day 3 inthe 586 and 1171 ppm treatmentgroups. Mortalitoyccurredthrough Day 8 inalldose groups > 146 ppm withsome ofthe deaths being during the post-exposureperiod.There were no treatment-related mortalitieosr overtsignsof toxicitaytconcentrations< 73.2 ppm. There was 11% mortalitiynthe 146 ppm treatmentgroup, and two additionablirdsdisplayedclinicaslignsoftoxicit(ywingdroop).All otherbirdsinthistestgroup displayednormal appearance and behaviorforthe durationofthe test.Recovery withnormal appearance and behavioroccurredon Day 9 totesttermination. There was 80% mortalit(yoccurringon Days 5,6,and 7) forbirdsin the293 ppm treatmentgroup. Signs oftoxicitoybserved priorto death includeda ruffleadppearance, reduced reactiontostimuli (sound and motion),lethargyw,ing droop,lossofcoordination, lowerlimbweakness and convulsions.Recovery with normal appearance and behavioroccurredon Day 9 totesttermination. There was 100% mortalit(yoccurringfrom Day 3 through Day 7) for birdsinthe 586 ppm treatmentgroup.Signs oftoxicitoybserved priortodeath includeda ruffledappearance, reduced reactionto stimuli(soundand motion),lethargyd,epression,wing droop,loss of coordinationl,ower limbweakness, lower limbrigiditpyr,ostrate posture,and convulsions. There was 100% mortalit(yoccurrinfgrom Day 2 (notedon Day 3 forDay 2 afternoon)through Day 4)forbirdsinthe 1171 ppm treatmentgroup.Signs oftoxicitoybserved pdor to death includeda ruffleadppearance, reduced reactionto *stimul(isound and motion), lethargyd,epression,wing droop,lossofcoordinationl,ower limb weakness, and lower limbrigidity. Body weightgain:When comparedtothecontroglroup,there were no apparent treatmentrelatedeffectson body weight among the birdsinconcentrations< 73.2 ppm. Dudng Days 0-5, statisticaslilgynificanrteductionsinbody weight gain or body weight lossoccurredinthe 146, 293, and 586 ppm treatment groups. Body weight effectscouldnot be determinedfortest organisms inthe 1171 ppm group due tototalmortality. Feed Consumption: No apparent treatmentrelatedeffectswere noted forfeed consumption forbirdsinconcentrations<146 ppm. Reduced feed consumption was noted forbirdsintreaf-mengtroups > 293 ppm from Days 0-5.No treatment-relateedffectson feed consumption inany of the survivingtreatmentgroups duringthe Day 6-8 post-exposurepedod were observed. Gross Necropsy: Allbirdsthatdied duringthe study,halfof those survivingat Day 8 and the restattestterminationwere subjectedto a grossnecropsy. Necropsy resultsforbirdsfound dead were similari,ncludingthinconditionl,ossofmuscle mass, alteredspleen color,autolysisoftissuesand pale organs.These necropsy findings were consideredto be treatmentrelatedT.he singlebirdeuthanized from the293 ppm treatmentwas found to have treatmentrelated' necropsy findings.Necropsy resultsforallotherbirdseuthanized on Day 8 and Day 22 were unremarkable. % Cumulative Mortality Nominal Concentration I PPM Day 1 Negative 0 Control Day 2 0 Day 3 0 Day 4 0 Day 5 0 18.3 0 0 0 0 0 36.6 0 0 0 0 0 73.2 0 0 0 0 0 146 0 0 10** 0 0 293 0 0 0 0 20 586 0 0 10 20 50 1171 0 0 30 100 100 *No mortalitieosccurredinany of the treatmentlevelsfrom Day 8 to Day 22 Birdeuthanized on day 3 aftersustaininga broken leg. Day 6 0 0 0 0 0 40 80 100 Day 7 Day 8* 0 0 0 0 0 0 0 0 10 10 80 80 100 100 100 100 Body Wei ht(grams) ExposurePeriod Recovery Period Nominal Concentratio (ppm) Mean Body Weight, Day 0 Mean Body Weight Change Day 0-5 Mean Body Weight Change Day 5-8 Mean Body Mean Body Mean Total Mean Body Weight Weight Body Weight Weight, Change Change Change, Day Day 22 Day 8-15 Day 15-22 8-22 Negative 20 +10 +8 +23 +22 45 82 Control 18.3 21 +11 +9 +24 +23 47 87 36.6 20 +11 +8 +26 +24 50 89 73.2 20 +9 +7 +24 +20 44 79 146 20 +7* +6** +24 +21 45 79 293 20 -2" -1** +14 +20 34 55 586 20 -4** - - -- 1171 20 Note: Numbers may not add manually due to rounding. Values for293 ppm by the factthatonlyone birdremained inthattreatmentgroup afterDay 8. *Statisticaldliyfferenftrom the controlgroup at p<0.05 (Dunneft'st-test). **Statisticadlilfyferenftrom the controlgroup atp=<0.01 Punnett'st-test No data availabledue tomortality. treatmentgroup are impacted Mean Average Feed Consumption Nominal Grams Grams Grams Grams Concentration,Feed/Bird/Day Feed/Bird/Day Feed/Bird/Day Feed/Bird/Day PPM Days 0-5 Days 6- Days 8-15 Days 15-22 Negative Control 9 10 9 13 18.3 9 11 10 12 36.6 8 12 14 15 73.2 10 13 13 15 146 9 10 11 14 293 5 9 8 586 6 19 - 1171 1 4 1 - No data availabledue tomortality. GrossPathologicOablservationfsromBirdsthatDiedinStudy Finding Abdominal cavity,some autolysis Abdominal cavity,autolysisthroughout Crop, empty Emaciated Fractured leg G. 1.Tract empty Gizzard contentsbilestained Heart,anteriorportionmottledwhite color Heart,pale . Intestinaclontentstar4ike Keel, prominent Kidneys,pale Liver,paleand mottled Loss of muscle mass Muscular-skeletalp,ale Small instature Spide,n,black Spleen, dark Spleen,grey Spleen, grey-brown Spleen, pale Spleen, small Spleen, smalland pale Thin Not remarkable Male,Female,and Undetermined (ppm) Control 146 293 586 1171 N=l N=2 N=8 N=10 N=10 0 0 2 2 4 0 0 0 1 1 0 0 2 5 2 0 0 2 5 8 1 1 0 0 0 0 0 1 1 0 0 0 2 5 1 0 0 1 0 0 0 0 0 2 1 0 0 0 2 0 0 0 1 3 10 0 0 0 2 0 0 1 0 0 0 0 0 4 7 9 0 1 0 0 0 0 0 3 0 0 0 0 0 1 0 0 0 0 0 2 0 0 0 1 0 0 0 0 0 1 0 0 1 0 1 0 0 0 0 1 0 0 0 3 0 0 0 0 4 2 0 0 1 0 0 CONCLUSIONS The dietaryLC50 valueforNorthernBobwhite exposed to perfluorooctanesulfonawtaes determinedto'be 220 ppm witha 95% confidenceintervaolf164 to289 ppm. The slopeoftheconcentrationresponse curvewas 7.005 and the chi-squarevaluewas 0.023.The no mortalitcyoncentratiownas 73.2 ppm. Based upon treatmentrelated mortalitys,ignsoftoxicitaynd effectsupon body weightgainatthe 146 ppm testconcentrationt,he no observed effectconcentratiownas 73.2 PPM- Author and/or submifter: 3M Corporation,Environmental Laboratory, P.O. Box 33331, St.Paul,Minnesota,55133 DATA QUALITY ReliabilityK:limischranking= 1. REFERENCES Thisstudywas conductedat WildlifIenternationLatld.,Easton,MD atthe requestofthe3M Company. OTHER Last changed: 5/3/00 I PFOS: A Dieta@ PFOS: A DIETARY LC50 STUDY WITH THE NORTHERN BOBWIUTE WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103 3M LAB REQUEST NO.: U2723 U.S. EnvironmentalProtectionAgency Series850 - EcologicalEffectsTest Guidelines(draft) OPPTS Number 850.2200 FIFRA SubdivisionE, Section71-2 OECD Guideline205 AUTHORS: Sean P. Gallagher Courtney S. Casey Joann B. Beavers Raymond L. Van Hoven STUDY INITIATION: April21, 1999 STUDY COMPLETION: SUBMITTED April26,2000 TO 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St.Paul,Minnesota 55144 Wildl@feIntemational,Ltd. 8598 Commerce Drive Easton,Maryland 21601 (410)822-8600 Page I of 62 WILDLIFE INTERNATIONAL, LTD -2- PROJECT NO.: 454-103 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A DietaryLC50 Study withtheNorthernBobwhite WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103 3M LAB REQUEST NO.: U2723 STUDY COMPLETION: April26,2000 @ This study was conducted incompliance with Good Laboratory PracticeStandards as publishedby the U.S. EnvironmentalProtectionAgency, 40 CFR Part 160 and 792, 17 August 1989; OECD Principlesof Good LaboratoryPractice(,OCDE/GD(92) 32, EnvironmentMonograph No. 45, Paris, 1992);and Japan MAFF, 59 NohSan, NotificatioNno. 3850, AgriculturaPlroductionBureau, 10 August 1984 withthefollowingexception: The testsubstancewas not characterize'dinaccordance with fiihGLP compliance;however, the characterizatiownas performed accordingto 3M Standard OperatingProceduresand Methods, and all raw data are being maintained'inthe 3M archives. The testsubstanceisbeing recharacterizeidn accordance with GLP. The stabilitoyf thetestsubstanceand referencestandardunder conditionsof storageatthetestsite was not determinedinaccordancewith Good Laboratory PracticeStandards. STUDY DIRECTOR: L@, ?, Sean P. Gallagher SeniorBiologist SPONSOR'S REPRESENTATIVE Li MS. Susan A. Beach 6CXA DATE DATE WILDLIFE INTERNATIONAL, LTD -3- PROJECT NO.: 454-103 QUALRIY ASSURANCE STATEMENT Tlus studywas examinedforcompliancewith Good LaboratoryPracticeStandardsas published by theU.S. EnvironmentaPlrotectioAngency,40 CFR Part160 and 792, 17 August 1989;OECD PrincipleosfGood LaboratoryPractice(,OCDE/GD (92)32,EnvironmentMonograph No. 45,Paris, 1992);and Japan MAFF, 59 NohSan, NotificatioNno. 3850,AgriculturaPlroductionBureau,10 August 1984. The datesof allauditsand inspectionasnd thedatesthatany findingwsere reportedto theStudyDirectorand LaboratoryManagement wereasfollows: ACTI= DATE CONDUCTED DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT TestSubstancePrep. & AnalyticaSlampling April22,'1999 kpril22, 1999 April23, 1999 MatrixFortification April22, 1999 April22, 1999 April23, 1999 Feed Consumption & AnalyticaSlampling April27,1999 April27, 1999 May 4, 1999 AnalyticaDlata and DraftReport July7,8,9, 1999 July9, 1999 July16,1999 BiologyData and Dmft Report August 26,27,30,3 1,1999 August31, 1999 September 13,1999 FinalReport April17-18,2000 April18,2000 April19,2000 c& Susan L.Coleman SeniorQualityAssuranceRepresentative DATE 4-ici-Ob WILDLIFE INTERNATIONAL, L-rD -4- REPORT APPROVAL PROJECT NO.: 454-103 SPONSOR: 3M Corporation TITLE: PFOS: A DietaryLC50 Studywith theNorthernBobwhite WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103 3M LAB REQUEST NO.: U2723 STUDY DIRECTOR: Sean P. Gallagher SeniorBiologistA,vian Toxicology CHEMISTRY PRINCIPAL INVESTIGATOR: k- i I, @- d, Raynydnd'L.Van Hoven, Ph.D ScientistA,nalyticalChemistry REPORT APPROVED BY: B. Beavers Director,Avian Toxicology Date - 4 Date 4 12-4 Date WillardB. Nixon,Ph.w Date Mamger, AnalyticalChemistry WILDLIFE INTERNATIONAL, LTD -5- PROJECTNO.: 454-103 TABLE OF CONTENTS =E PAGE ............................................................P.a.g.e..I................. GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT ................P.a.g.e.2......... QUALITY ASSURANCE STATEMENT ......................................P.a.g.e..3................. REPORT APPROVAL .....................................................P.a.g.e.4.................. TABLE OF CONTENTS ...................................................P.a.g.e..5................. Tablesand Appendices....................................................P.a.g.e.6.................. I SUNINMY ........................................................................P.a.g.e..7.................. INTRODUCTION ........................................................P.a.g.e..8................. OBJECTRVE ............................................................P.a.g.e..8.................. MATERIALS AND M=ODS .............................................P.a.g.e..8................. TestSubstance..........................................................P.a.g.e..8.................. TreatrnenGtroups........................................................P.a.g.e..9................. DurationoftheTest......................................................P.a.g.e..9.................. TestBirds..............................................................P.a.ge...9................. Animal ....**.*.*'*.*.*....................................P.a.g.e..1.0................. DietPreparatio.n............................. ..........P.a.g.e..1.0.. DietSampling...................................:...............................................P..a..g..e...1..0................... AnalyticaMlethod ..........I......................................................P.a.g.e.1.1................. Housingand EnvironmentalConditions.......................................P.a.g.e..1.3................. Observation.s...........................................................P.a.g.e.1.4................. AnftnalBody Weights/FeedConsumption......................................P.a.g.e.1.4................. Gross Necropsy .........................................................P.a.g.e..1.4................. StatisticAanlalyses.....................................................;...P.kg.e..1.4................. RESULTS ..............................................................P.a.g.e..1.5................. DietAnalysis...........................................................P.a.g.e..1.5................. Mortafifieasnd ClinicaOlbservation.s........................................P.a.g.e.1.5................. Body Weight and Feed Consumption.........................................P.a.g.e..1.7................. Gross Necropsy.........................................................P.a.g.e..1.7................. I CONCLUSION .................................................................................P..a..g..e...1..8.................... REFERENCES ..........................................................P.a.g.e..1.9................. WILDLIFE INTERNATIONAL, LTD -6- TABLE OF CONTENTS continued- TABLES AND APPENDICES PROJECT NO.: 454-103 TABLE 1: TABLE 2: TABLE 3: TABLE 4: TABLE5: CumulativeMortalityfrom a Northern Bobwhite Acute DietaryToxicity Study with PFOS .................................................P.a.g.e.2.0................... Mean Body Weight (g)from a NorthernBobwhite Acute DietaryToxicity Study with PFOS .................................................P.ag.e..2.1................... Mean Feed Consumption (gtbird/dayf)rom a Northern Bobwhite Acute DietaryToxicityStudy with PFOS .............................P.a.g.e..2.3................ Group Gross PathologicalObservationsfrom a Northern Bobwhite Acute DietaryToxicityStudy with PFOS .............................P.a.g.e..2.5................ CumulativeMortality(EstimatedCtunulativDeose, mg/kg) from a Northern Bobwhite Acute DietaryToxicityStudy with PFOS .............Pa.g.e..2.6... APPENDIX 1: CertificatoefAnalysis.........................................P.a.g.e..2.7.................. APPENDEK.U: DietFormulation..............................................Pa.g.e..2.8................... APPENDIX HI: AnalyticalMethods and Results..................................P.a.ge..2.9................... APPENDIX IV: DietPreparation..............................................P.a.g.e..4.3.................. APPENDIX V: Cumulative Mortalityby Pen from a NorthernBobwhite Acute DietaryToxicityStudy with PFOS .........................P.a.g.e..4.4............. APPENDIX VI: IndividuaBlody Weights (g):6roma NorthernBobwhite Acute DietaryToxicityStudy with PFOS .........................P.a.g.e..4.5............. APPENDIX VII: Feed Consumption (gtbird/dayb)y Pen from a NorthernBobwhite Acute DietaryToxicity Study with PFOS .............................................P.a.g.e..58................... APPENDIX VRI: Changes to Protocol...........................................P.a.g.e.6.1................... APPENDIX IX: PersonnelInvolvedinthe Study ..................................P.a.g.e.6.2................... 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 DietaryLC50 Studywith theNorthern Bobwhite RESULTS: The dietaryLC50 value for northernbobwhite exposed to PFOS was determinedto be 220 ppm a.i.with a 95% confidenceintervaolf 164 ppm a.i.to 289 ppm a.i. The slopeof the concentration-responcsuerve was 7.005 and the chi-squarevalue was 0.023. The no mortalityconcentration was 73.2 ppm a.i. Based upon treatmentrelatedmortalitys,ignsof toxicityand effectsupon body weight gain at the 146 ppm a.i.test concentrationt,heno-observed-effecctoncentrationwas 73.2 ppm a.i. TEST DATES: Hatch - April12, 1999 Acclimation-April 12-22,1999 ExperimentalStart-April22, 1999 ExperimentalTermination- May 14, 1999 NOMINAL TEST CONCENTRATIONS: 0, 18.3,36.6,73.2,146,293, 586 and 1171 ppm a.l. TEST ANIMALS: NorthernBobwhite (Colinusvirginianus) AGE TEST ANIMALS: 10 days of age attestinitiation SOURCE TEST ANIMALS: WildlifeInternationLatld.ProductionFlock 8598 Commerce Drive Easton,Maryland 21601 STUDY COMPLETION: April26,2000 WILDLIFE INTERNATIONAL, LTD -8- PROJECT NO.: 454-103 RQMODUCTION This study was conductedby WildlifeInternationaLltd. for 3M Corporationat the Wildlife InternationaLltd. aviantoxicologyfacilitiyn Easton,Maryland. The in-lifpeortionof the testwas conductedfrom April 22,'1999 to May 14, 1999. Raw datageneratedat WildlffeInternationaLltd. and a copy of the finalreportare filedunder ProjectNumber 454-103 in archiveslocatedon the WildlifeIatemationalLtd.site. OBJECTIVE Tle objectiveof thisstudywas to evaluatethe toxicitoyf a testsubstanceto the Northern Bobwhite (ColinusWrginzanus) administeredthrough the dietforfivedays.An LC50 valuewillbe calculated, ifpossible. MATERLKLS AND METHODS The methods used in conductingthisstudy are based upon procedures specifiedin the U.S. EnvironmentalProtectionAgency Series850 - EcologicalEffectsTest GuidelinesOPPTS Number 850.2200 (1), Section71-2 of the EnvironmentalProtectionAgency's Regist@on Guidelines, PesticideAssessment Guidelines,FNRA SubdivisionE, Hazard Evaluation:Wildlifeand Aquatic Organisms (2);OECD Guideline205, Guidelinefor Testingof Chemicals,Avian DietaryToxicity Test (3);and upon ASTM StandardE857-87, "StandardPracticefor Conducting Subacute Dietary ToxicityTestswithAvian Species"(4). Test Substance The testsubstancewas receivedfrom 3M Corporationon October 29, 1998 and was assigned WildlifeInternationaLltd.IdentificatiNounmber 4675 upon receipt.The testsubstancewas white powder identifieads: FC-95; Lot No.:217. The reportedpurityof thetestsubstancewas 98.9*/ow,ith an expirationdate of 2008. Followingtestterminationt,hetestmaterialwas reanalyzed.The results of reanalysisindicatea testsubstancepurityof 90.49%. All testconcentrationhsave been adjustedto reflecthe purityreportedon the new Certificatoef Analysis(Appendix 1). 'Me testsubstancewas storedunder ambient conditions. Tle internalstandardwas receivedfrom 3M Corporationon July 2, 1998 and was assigned WildlifeInternationaLltd. identificatinounmber 4526 upon receipt.'Me internasltandard,a granular WILDLIFE INTERNATIONAL, LTD -9- PROJECT NO.: 454-103 material,was identifieads: 1K 1K 2FL 2H PerfluorooctanSeulfonicAcid, Chemical Abstract Number 27619-97-2. The standardwas storedunder ambientconditions. Treatrn=t Groups 'Me testconsistedof a geometricseriesof seven testconcentrationasnd a controlgroup. Thirty northernbobwhite cluckswere assignedto the controlgroup and t= northernbobwhite chickswere assignedto each of the treatmentgroups. The birds were sortedby weight then chosen mdiscrhmately from withineach representedweight classforplacement intocontroland treatrnent groups. The birdswere housed in broodingpens containingfivechickseach. Nominal dietary concentrationussed in thisstudywere 0, 18.3,36.6,73.2,146, 293, 586 and 1171 partsper million activeingredient(ppm a.i.o)f PFOS. The dietaryconcentrationwsere establishebdased upon known toxicitydataand informationsuppliedby the Sponsor. Each group was fed the appropriatetestor controldietforfivedays. During the exposure period the controlgroup receiveduntreatedfeed. Following the :five-daeyxposure period allgroups were given untreatedbasal dietfor threedays. On Day 8, halfof the survivingtreatrnenatnd controlbirds were eudianizedand livertissue,blood, and bilesamples were collectedforanalysis.The rmnaining birdswere fed basalmtion untilDay 22. On Day 22, thesebirdswere eudmnized and alsosampled for liverweight@blood,and bile. Duration oftheTest The primary phases ofthistestand theirdurationswere: 1. Acclimation- 10 days. 2. Exposure -5 days. 3. Post-exposureobservation- 3 or 17 days Test Birds AR northernbobwhite (Colinus virginianus)were 10 days of age and appeared to be in good healthat initiatiofn the test.The birdswere obtainedfrom WildlifeInternationaLltd.Production Flock,Easton,MD and were hatchedon April12, 1999. Birdsranged inweight from 18 to23 grams at testinitiatioTnh.e birdsused in thisstudywere immature and could not be differentiatbeyd sex. WILDLIFE INTERNATIONAL, LTD -10- PROJECT NO.: 454-103 AU birdswere from the same hatch,pen-rearedand phenotypicalliyndistinguishabflreom wildbirds. Allbirdswere acclimatetdothecagingand facilitifersomtheday ofhatchuntilinitiatioofnthetest. Animal Diet Throughoutacch=tion and testinaglltestbirdswere fed a game birdrationformulatetdo WildlifeInternationLatld.'sspecificatio(nAsppendix]I). The chickswere givena vit=ffi supplementintheirwaterfrom theday theywere hatcheduntitlheinitiatioofnthetest.Water from thetown of Eastonpublicwatersupply,and feedwere providedad libitumduringacclimatioannd testingT.he birdsreceivedno form ofantibiotmiecdicationduringacclimatioonrtesting. Diet PLe,Uaration Ile testsubstancweas nuxed directliyntotheration.Mixingwas donewitha Hobartmixer (ModelNumber AS2001). Alldietartyestconcentratiownesre adjustetdo 100% PFOS basedupon thereportedpurityofthetestsubstance.AU dietaryconcentratioannsd theLC50 valuearereported as ppm a.i.inthediet.Nominal dietartyestconcentratiounssedinthisstudywere 18.3,36.6,73.2, 146,293,586,1171 ppm a.i.(Appendix DietSmRIin Samples of the testdietswere collectetdo verifythetestconcentrationasdministereadnd to confirmthestabilitaynd homogeneityof the testsubstanceinthe chets.Homogeneityof thetest substanceinthedietwas evaluatedby collectinsgixsamplesfrom the 18.3ppm a.i.a.i.testdietand sixsamplesfrom the 1171 ppm a.it.estdietat preparatioonn Day 0. Homogeneitysampleswere collectefdrom thetop,middleand bottomof theleftand rightsectionosf themixingvessel.The homogeneitysamplesalso servedas verificatisoanmples. One verificatisoanmple was collected from the controldietand two verificatisoanmpleswere collectefdrom each remainingtreatment groupatpreparatioonn Day 0. At theendof theexposureperiod(Day 5),one samplewas collected from thecontroland two sampleswere collectefdrom eachtreatmengtroup to determinestabiliotfy thetestsubstanceinthedietundertestconditions.Ile stabilistaympleswere collectefdrom feed remainingin thefeedersafterbeingat ambienttestpen conditionfsor fivedays. Samples were transferreidmmediatelytoWildlifeInternationLatld.analyticaclhemistry. WILDLIFE INTERNATIONAL, L-rD -11- PROJECT NO.: 454-103 AngL)IicMal The method used for the analysisof the aviandietsampleswas based upon methodology developedatWildlifeInternationLatld.and entitle"dMethod OutlinefortheDeterminatioonfPFOS inAvian Feed". Avian dietsampleswere extractewdithmethanol.Med=ol was addedtoa requisitqeuantitoyf feedcontainedina French-squargelassbottle.Bottleswere capped and shakenon a shakertable. Sampleswere vacuum filtereudsingqualitativfeiltepraper.The retainefdeedwas rinsedthreetimes withmethanolintothefiltrateT.he filtrawtaes transferrteod a,voiumetncflaskand broughtto volume withmethanol.As appropriates,ampleswere finthedrilutewdith methanol.Each sample thenwas dilutewditha 50% methanol: 50% NANOpure(D watersolutiocnontainin0g.100 mg 4H PFOS (izftmasltandard)/aLnd 0.05% formicacid(v/v)so thattheyfelwlithinthecalibratiroannge ofthePFOS methodology.A method flowchartisprovidedinAppendixEll,Figure1. Concentrationosf PFOS inthe standardsand sampleswere determinedby reversed-phasheigh performanceliquidchromatographyusinga Hewlett-PackaiMdodel 1100 legh PerformanceLiquid Chramatograph(BPLC) witha Perldn-EhnerAPI 10OLC Mass Spectrometeerquippedwitha PerldnEbner TurbolonSpmy ion source.BPLC separationwsere achievedusinga KeystoneBetasilCis analyticaclolumn(100 mm x 2 nim I.D.,3 pm particlseize).The fimtnnnenptarametersare summarizedinAppendix Ell,Table 1. Calibratiosntandardsof PFOS preparedina 50% methanol:50% NANOpure(D watersolution containin0g.100mg 4H PFOS (intenisatlandard)[aLnd 0.05% fonnicacid(v/v)r,angingin concentratiofnrom 0.00229to 0.0457mg a.i./wLere analyzedwiththesamples.The same and most prominentpeak responseforPFOS was utilizetdo monitor-PFOS inallcalibratioqnu,alitycontrol, and studysamples. No attemptwas made to quantifyPFOS on the basisof individuailsomeric components. Linearregressioenquationswere generatedusingpeak arearesponseratios(PFOS : internasltandardv)ersusthe respectivceoncentratiornatios(PFOS : internasltandard)of the calibratisotnandards.A typicalcalibraticounrveispresenteidn AppendixIII,Figure2. The concentrationf PFOS inthe sampleswas determinedby substitutitnhge peak arearesponsemtios intothe applicablleinearregressioenquation.Representativieonchromatogramsof low and high calibratisotnandardsarepresentedinAppendix HI,Figures3 and 4,respectively. WILDLIFE INTERNATIONAL, LTD -12- PROJECT NO.: 454-103 The method limitofquantitati(oLnOQ) fbrtheseanalyseswas setat 1.15ppm a.i.calculateads the product of the lowestcalibratiosntandardanalyzed(0.00229mg a.i./La)nd the Cution factorof thematrix blank.samples (500). Two matrix blank samples were analyzedto determinepossibleinterferencesN.o interferences were observedator above the LOQ duringsample analyses(Appendix III,Table 2). An interference in the feed appeared at approximatelythe same retentiotnime as the peak of interesbtut itwas well below the LOQ. Figure5. A representativcehromatogram of a matrixblank ispresentedin Appendix Ell, Avian dietwas fortifieadt 4.57, 183 and 1830 ppm a.i.and analyzedconcurrentlywith the samples to determinethe mean proceduralrecovery(Appendix HI, Table 3). Sample concentrations were not correctedfor the mean proceduralrecoveryof 94.7%. A representativcehromatogram of a .matrixfordficatioinspresentedinAppendix HI,Figure 6. An example calculationis presentedfor sample number 454-103-2,nominal concentrationof 18.3ppm a.i.inavian didt. rnitiaWleight: 10.0g FinalVolume: 200 mL DilutionFactor:100 (intermediatdeilutiofnactorx finaldilutiofnactor) PFOSPeakArea: 113568 InternalStandardPeak Area: 413160 Peak Area Ratio:0.2749 Calibratiocnurveequation. Slope:2.77397 Intercept:0.01894 Curve isweighted(1/x). PFOS (mg &i./L)atinstrument (Peakarearatio- (Y-interceptx))I.S.Concentration Slope WILDLIFE INTERNATIONAL, LTD .13- PROJECT NO.: 454-103 (0.2749- 0.01894)x 0.100mg/L 2.77397 0.00923mg ai./L Note:I.S. internasltandard. PFOS (ppm ai.)insample PFOS (mg a.i./La)tinstrumenxtFinalVolume (L)x DilutioFnactor InitiWaelight(Kg) 0.00923x 0.200x 100 0.01 18.5ppm ai PercentofNominal Concentration PFOS (ppm ai.)insample x 100 PFOS (ppm ai.)nominal 18.5 -x 100= 101% 18.3 Housingand EnvironmentalConditions Duringacch=fion and testinga,llbirdswere housedindoorsin batterieosf thermostatically controllebdroodingpensmanufacturedby Beacon SteelProductsCo. (ModelNo. B735Q). Eachpen had floorspacethatmeasuredapproximatel7y2 X 90 cm. Ceftg heightwas approximatel2y3 cyn. Externalwaus, ceilingasnd floorswere constructeodfgalvanizedsteewlireand sheeting.Birdswere sortedby weight then chosenindiscriminateflryom withineach representewdeightclassfbr assignmentto pens. Each group of birdswas identifibeyd pen number and testconcentration. Individuablirdswere identifibeyd legbands. Dunng thetesttheaveragetemperaturem thebroodmg compartn=t ofthepens was 38'C +-2'C (SD). Averageambientroom tmnperaturfeorthisstudywas 27.30C* 1.20C(SD) withan average relativheuxniditoyf 31% :1:14% (SD). The photo@eriod(maintainebdy a time clock)was sbdee@ hoursof lightperday duringacclimatioannd throughouthetest.The lightsourcewas fluorescent lightwshich closelyapproximatenoon-daysunlight.The birdswere exposedto an averageof WILDLIFE INTERNATIONAL, LrD -14- PROJF.CT NO.: 454-103 approximately139 luxof illuminationH.ousing and husbandrypracticewsere basedon guidelines establishebdy theNationalResearchCouncil(5). Observations During acclimatioanllbirdswere observeddaily.Birdsexhibitinagbnormalbehaviororphysical injurywere not used. Followingtestinitiatiaonnd continuinugntilterminatioanl,lbirdswere typicalloybservedat leastwicedaily.A recordwas maintainedof allmortalitys,ignsof toxicity, and abnormalbehavior. Animal B& Weiphts/FeeCdonsumDtion Individuablody weightswere measuredat theinitiatioofnthetesto,n Day 5, Day 8, and on Days 15 and Day 22 forallremainingbirds.Averagefeedconsumptionvaluesduringtheexposure period(Days 0-5)and thepost-exposuroebservatiopneriod(Days 6-8)were determinedby pen for each treatmengtroup and the controlgroup. Additionallfye,edconsumptionwas determinedfor Days 8-15 and 15-22 for the remainingtreatmentand controlbirds. Feed consumptionwas determinedby measuringthechange intheweightof the'feedpresentedto thebirdsovera given periodoftime. 'Me accuracyoffeedconsumptionvaluesmay havebeen affectebdy theunavoidable wasuge offeedby thebirds. Gross NecMpay AR testbirdsthatdiedduringthecourseofthetestand allbirdsremainingattheterminatiownere subjectetdo a'grossnecropsy.Additionalllyi,verwsere weighedand livertissueb,lood,and bfle were collectefdrom birdseuflianizeodn Day 8 and 22,and when possiblefrom thosethatdiedduring thecourseofthestudy. StatisticAanlalyses Mortalitydatawere analyzedusingthecomputerprogram ofC.E. Stephan(6).The program was designedto calculattehe LC50 valueand the95% confidencientervably probitanalysism,oving averagemethod or the binomialprobabilimteythod (7,8,9).In thisstudy,the LC50 valuewas determinedusingtheprobitmethod. The slopeofthe concentration-respocnusreveand resultosfthe goodnessof fittestare reported.Body weightdatawere compared by Dunnett'stestusing TOXSTAT software(10,11).No statistiacnaallyseswereappliedtofeedconsumptiondata. WILDLIFE INTERNATIONAL, LTD -15- PROJECTNO.: 454-103 RESULTS Diet&gLUis Avian dietsampleswere collectefdrom the 18.3and 1171 ppm a.i.testconcentratioannsd analyzedto evaluatehomogeneityof thetestsubstancein theaviandiet.The analysiosf these samplesalso servedas verificatiofn testsubstanceconcentrationsR.esultingmean measured concentrationsst,andarddev=ons and coeffimentosf variatio(nCV) forthesetestconcentrations were 19.5 2.13ppm a.i.(CV = 10.9*/oa)nd 1196 70.2ppm a.i.(CV 5.87vo),respectively (AppendixIH,Table4). Controlaviandietsamplescollectedduringthetestshowed no interferences above theLOQ. Samplescollectedduringthetestto verifythe36.6,73.2,146,293 and 586 ppm a.i. testsubstanceconcentrationhsad mean measured concentrationosf 40.2, 74.5, 174, 291 and 537 ppm a.i.r,espectivelyT.hese valuesrepresente1d10, 102,119,99.3and 91.6% of thenominal concentrationrse,spective(lAyppendixHI,Table5). Analysisof aviandietsamplescollectefdrom feedersafterbeingheldatambienttemperatureforfivedays averaged101,122, 104, 101,109, 114 and 102% oftheDay 0 valuesforthe18.3,36.6,73.2,146,293,586 and 1171 ppm a.it.estsubstance concentratiorness,pective(lAyppendixIILTable6). A repi@s@tive chromatogramofa testsample isshown.inAppendix EII,Figure7. Mortalitieasnd ClinicaOlbservations One incidentamlortalitoyccurredinthecontrolgroupduringthecourseof thestudy(TableI and AppendixV). On themorningof Day 5,one birdwas notedwitha brokenlegand was subsequently euflmizedon Day 6. Additionalltyw,o birdsinthecontroglroupwere intermittenntoltyedwithfoot lesionsassociatewdithcagemate aggression.Otherwise,allcontrolbirdswere normalinappearance and behaviorthroughoutthetest. No treatmenrtelatedmortalitioers overtsignsof toxicitwyere observedin the 18.3,36.6,or 73.2ppm a.it.reatmengtroups.One birdinthe 18.3ppm a.i.treatmengtroup was notedas lame from Day 6 throughDay 8,and bloodofundetemiinedoriginwas notedon theundersideof one birdf.rom the73.2ppm a.i.treatmentgroup on Day 22. Otherwise,allbirdsinthe 18.3,36.6and 73.2ppm a.i. treatmentgroupswere normal inappearanceand behaviorthroughoutthetestperiod. WILDLIFE INTERNATIONAL, LTD -16- PROJECRNO.: 454-103 'Iherweas 11% (1of 9)mortalitiynthe146 ppm ai.treatmengtroup,80% (8of 10)mortalitiyn the293 ppm a.i.treatmengtroup and 100% (10 of 10) mortalitiynthe586 and 1171 ppm a.i. treatmengtroups. In the 146 ppm a.i.treatmengtroup,one birdwas eudmnizedon Day 3 after sustaininagbrokenleg.Thisincidentamlortalitwyas notusedinthecalculationftheLC50 value. Additionalltyh,erewas one treatment-relamtoerdtalitiynthe 146 ppm a.i.treatmentgroup,a bird founddead on themorningofDay 7. Clinicaslignsoftoxicitwyere observedinthistreatmengtroup on Day 5, when two birdsdisplayewding droop. AU otherbirdsat thistestconcentratiownere normal inappearanceand behaviorforthedurationof thetest. In the293 ppm a.i.treatmentgroup therewere eighttreatrnent-relmaotretdalitieosc,curringon Days 5,6 and 7. Signsof toxicitwyere firsotbservedon themorningof Day 4 and continuedto be exhibitetdhroughthemorningof Day 8 forthe singlebirdeuflmnizeodn Day 8, and throughthe afternooonf Day 8 fbrthesinglbeirdsurvivinugntilDay 22. Signsoftoxicitiyncludeda mffled appearancer,educedreactiotno stimuli(sound and motion),lethargyw,ing droop,lossof coordinatiolno,werlimb weakness and convulsions.The singleremainingbirdappearedto have recoveredand was normal in appearanceand behaviorfrom the afternooonf Day 9 untiltest termination. In the586 ppm a.i.treatmentgroup mortalitiweesre firsntotedon Day 3 and continuedto be observedthroughDay 7,atwhichpointallbirdshad died.Overtsignsoftoxicitwyere firsotbserved on fficafternooonf Day 2 and continuetdhroughthemorningof Day 7, when thefinalbirdswere founddead. Signsof toxicitoybservedamong birdsinthe586 ppm a.i.treatmengtroup includeda ruffleadppearance,reducedreactiotno stimul(isoundand motion),lethargyd,epressionw,ing droop, lossofcoordinatiolno,werlimbweakness,lowerlimbrigiditpyr,ostratpeosturea,nd convulsions. In the 1171 ppm a.i.treatmengtroup 100% mortalithyad occurredby themorningof Day 4. Sips of toxicitiynthe1171 ppm a.i.treatmengtroup were firsotbservedon theafternoonof Day 2, withthefirsmtortalitineostedon themorningof Day 3. Signsof toxicitoybservedpriorto death includeda ruffleadppearance,reducedreactiotno stimuli(soundand motion),lethargyd,epression, lossofcoordinatiowni,ng droop,and lowerlimbweaknessand rigidity. WILDLIFE INTERNATIONAL, LTD -17- -PROJECT NO.:454-103 Bgd-yWeisthtand Feed Co u When compared tothecontroglroup,therewere no apparenttreatmentrelateedffectuspon body weightamong birdsinthe 18.3,36.6 or 73.2 ppm a.i.treatmengtroups. However,therewa.Sa concentratiornesponsivreeductioinnbody weightgainor body weightlossinthe146,293 and 586 ppm a.i.treatmengtroupsduringtheexposureperiod(Days 0-5)(Table2 and AppendixVI). Dfferencesfrom thecontrolgroupwere statisticaslilgynificaanttp < 0.05forthe146 ppm a.i.level and atp<0.01 forthe293 and 586 ppm a.i.levels.A statisticaslilgynifica(npt<0.01)reductioinn body weightgaincontinuedtobe observedatthe 146 ppm a.it.estconcentratiotnhroughDay 8 ofthe study. At the293 ppm a.i.concentratiaonstatisticaslilgynifica(npt<0.01)mean weightloss continuedthroughDay 8 ofthestudyand a marked reductioinnweightgainwas notedthroughDay 15 ofthestudy.Due tototalmorlilityb,ody weighteffectcsouldnotbe determinedforthe1171 ppm a.i.levelduringthe exposureperiodor forthe586 and 1171ppm a.i.treatmentgroupsforpostexposureperiod. There were no apparenttreatmentrelatedeffectuspon feedconsumptionatthe 18.3,36.6,73.2or 146 ppm a.i.testconcentratio(nTsable3 and Appenc@xVU). However,a reductioinn feed consumptionwas notedat the293, 586 and 1171 ppm a.i.treatmengtroups duringtheexposure period(Days 0-5). There were no treatment-relaetfefdectosn feedconsumptionin any of the survivintgreatmengtroupsduringtheDay 6-8post-exposurpeeriod.In the293 ppm a*.tir.eatn=t group onlyone birdsurvivedtoDay 22. The reductioinnfeedconswnptionobservedatthe293 ppm a.i.testconcentratiodnuringboth theDay 8-15 and 15-22post-Wosure periodswas theresultof havingonlyone birdinthepen,and was notconsideretdo be treatmentrelated. GrossNec=s During thecourseof thetesta,llbirdsthatdiedwere subjectetdo a grossnecropsy.Necropsy resultfsorbirdsfound dead were similar.Common observationisncludedthinconditionl,ossof muscle mass, alteredspleencolor,autolysiosf tissuesand paleorgans. Detailsof the necropsy findingsarepresentedinTable 4. Half of thesurvivingbirdswere subjectedto grossnecropsyon Day 8 and theremainingbirdswere necropsiedon Day 22,followintgestterminationO.n Day 8, one birdinthe73.2ppm a.i. treatmentgroupwas notedwitha slightlpyaleliver.Due totheisolatendatureofthisfindingi,twas WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-103 notconsideretdobe relatetdotreatment.The singlebirdeudmnizedfrom the293 ppm a.i.treatment group was observedatnecropsytohave witha lackofmusclemass and generalthinness.Sincethese findingscorrelatewdith an impactupon body weightnotedattlusconcentratiotnh,efindingswere consideretdobe treatmentrelated.Necropsy resultwsereunremarkableforallotherbirdseuffianized on Day 8. SimilarlyD,ay 22 necropsyfindingwsere unremarkableforallbirds. CONCLUSION The dietarLyC50 valuefornorthernbobwhiteexposedtoPFOS was determinedtobe 220 ppm a.i. witha 95% confidencientervaolf 164 ppm a.i.to 289 ppm a.i.Ile slopeof the concentrationresponsecurvewas 7.005and thechi-squarvealuewas 0.023. The no mortalitcyoncentratiownas 73.2ppm a.i.Based upon treatmentrelatemdortg4, signsoftoxicitaynd effectuspon body weight gainatthe146ppm a.ia..itestconcentratiotnh,eno-observed-effeccotncentratiownas 73.2ppm a.i.. WILDLIFE INTERNATIONAL, LTD -19- PROJECT NO.: 454-103 REFERENCES 1. U.S. EnvironmentalProtectioAngency, 1996. Series850 - EcologicaElffectTsestGuidelines (draftO)P,PTS Number 850.2200:AvianDietaryToxicityTest. 2. U.S. Environmental Protection Agency. -1982. PesticideAssessment Guidelines,fiYFRA Subdivision E, Hazard Evaluation: Wildl@feand Aquatic Organisnu, subsection 71-2, EnvironmentaPlrotectioAngency,Officeof PesticidPerograms.Washington,D.C. 3. Organization for Economic Cooperation and Development. 1984. AWan Dietary Toxicity Test OECD GuidelineforTestingofChemicals. Guideline205. Paris. 4. American Societyfor Testingand Materials. 1987. StandardPracticefor Conducting SubacuteDietaryToxicityTestswithAvmn Species.ASTM StandardE857-87. Annual Book of ASTM StandardsV,ol.11.04.PhiladelphiPaA,. 5. NationalResearch Council. 1996. Guide for the Care and Use of LaboratoryAnimals. Washington,D.C. NationalAcademy Press.125pp. 6. Stephan,C.E. 1978. U.S.EPA, EnvironmentaRlesearch]LaboratorDyu,luth,NW. Personal Communication 7. Stephan, C.E. 1977. Methods forCalculatingan LC50. Pages 65-84 InAquatic Tojdcologyand Hazard Evaluations, American Society for Testing and Materials. Pub. No. STP 634. PhiladelphiPaA,. 8. Finney, D.J. 1971. StatisticaMlethods in BiologicalAssay, 2nd ed.,GriffinPress,London. 9. Thompson, W.R. 1947. BacteriologicalReWews, Vol 2,No.2: 115-145. 10.Dunnet,C.W. 1955. A MultipleComparison'sProcedureforComparingSeveralTreatments witha ControlJ.our.Amer. StatisA.ssoc.50:1096-1121. 11.Gulley,D.D. 1990.TOXSTAT Release3.2.The UniversitoyfWyoming. WILDLIFE INTERNATIONAL, L-rD -20- PROJECT NO.: 454-103 TABLE I CumulativeMoAal4 from a NorthernBobwhite Acute DietaryToxicityStudy with PFOS Experimental Group No. Dead Per No. Exposed Exposure Period (ppm ai.) Day 0 Day I Day 2 Day 3 Day 4 Day 5 No. Dead Per No. Exposed Post-ExposurePeriod Day 6 Day 7 Day 8 Control 0 0/30 0/30 0/30 0/30 0/30 0/30 1/30 1/30 1/30 Treatment 18.3 36.6 73.2 146 293 586 1171 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0110 0/10 0/10 0/10 0/10 0/10 0/10 0110 0/10 0/10 1/102 0/10 1/10 3/10 0/10 0/10 0/10 0/9 0/10 2/10 10/10 0/10 0/10 0/10 0/9 2/10 5110 10/10 0/10 0/10 0110 0/9 4/10 8/10 10/10 0/10 0/10 0/10 1/9 8/10 10/10 10/10 0110 0/10 0/10 1/9 8/10 10/10 10/10 The LC50 valuewas calculatetdobe220 ppm ai.witha 95% confidenceintmvalof 164 ppm iLi.to289 ppm &i.. 1- No morwitiesoccurredinany ofthecontroolrtreatinengtroupsfrom Day 8 toDay 22. 2- Birdeudmiizedon 3 aftersustaininagbrokenleg. WILDLIFE INTERNATIONAL, LTD -21- PROJECT NO.: 454-103 TABLE2 Page I Mean Body Weight (g)froma NorthernBobwhiteAcuteDietaryToxicityStudywithPFOS Experimental Gmup (ppmai.) ExposurePeriod Cbangel Dg 0 Dg 5 Dg 0-5 Post-ExposurPeeriod Change'--- Total DM8 Dg 5-8 Changel Control 0 Mean 20 30 10 38 8 18 SD 1 4 3 5 2 -4 Treatment 18.3 Mean 21 31 11 40 9 20 SD 1 4 3 5 2 3 36.6 Mean 20 31 11 39 8 19 SD 2 3 2 3 1 2 73.2 Mean 20 30 9 37 7 16 SD 1 2 1 3 1 2 146 Mean 20 27* 7* 33* 6** 13** SD 2 3 3 3 2 4 293 Mean 20 18** -2** 18** -2** -1** SD 1 2 2 4 4 5 586 Mean 20 16** -4** - - - SD 1 2 2 1171 Mean 20 - - SD I Mean changeiscalculatseedparateflryomthemean bodyweightsusingindividubaoldyweights(SeeAppendixVI). (-)= No dataavailabdlueetomortality. *Statisticdailflfyerefnrtomthecontroglroupatp < 0.05(Dumett'st-test). **StatistiEd!i!fIfyerefnrtmnthecontroglroupatp < 0.01(Dumett'st-test). WILDLIFE INTERNATIONAL, LTD -22- PROJECT NO.: 454-103 TABLE2 Page 2 Mean Body Weight (g)from a NorthernBobwhiteAcuteDietmy ToxicityStudywithPFOS Experimental Group (1@pma.i.) Post-ExposurPeeriod Change' DM8 Day 15 Dg 9-15 Da 22 Change' Total Dg 15-22 Changel(8-22) Control 0 Mean SD 37 59 23 6 10 5 82 22 45 13 3 8 Tmatment 18.3 Mean SD 40 68 24 6 8 3 87 23 47 7 3 3 36.6- Mean SD 73.2 Mean SD 38 65 26 3 5 2 35 60 24 3 4 2 89 24 50 7 2 4 79 20 44 4 2 2 146 Mean SD 293 Mean 2 SD 34* 58 24 3 3 1 21 35 14 79 21 45 2 2 1 55 20 34 586 Mean - SD - 1171 Mean - SD - 'Mean changeiscalculatesdeparatelfyromthemean bodywei usingindividubaoldyweights(SeeAppendixVI). 2n=1,couldnotbe evaluatesdtatisticwailtlhyDunnett'ts-test (-)= No dataavailabldeuetomortality. *StatisticdailflfyerefnrtomthecontroEluU atp < 0.05)(Dunnettt'-stest). WILDLIF'E INTERNATIONAL, L-rD -23- PROJECT NO.: 454-103 TABLE3 Page I Mean Feed Consumption(glbird/dafyr)om a NorthernBobwhiteAcuteDietary ToxicityStudywithPFOS Experimental Group (ppma.i.) Control 0 Mean SD Treatment 18.3 36.6 73.2 146 293 586 1171 ExposurePeriod Day 0-5 9 2 9 8 10 9 5 6 4 Post-Expos=Period DE 6-8 10 2 11 12 13 10 9 19 - No dataavailabldeue tomortality. WILDLIFE INTERNATIONAL, LTD -24- PROJECT NO.: 454-103 TABLE3 Page 2 Mean Feed Consumption (glbird/dayf)rom a NorthernBobwhite Acute Dietary ToxicityStudy with PFOS Experimental Gmup (ppm ai.) Control 0 Mean SD Post-ExposurePeriod Day 8-15 Day 15-22 9 13 2 1 Treatment 18.3 36.6 73.2 146 293 586 1171 10 12 14 15 13 15 11 14 9 9 - - No dataavailabldeue tomoftality. WILDLIFE INTERNATIONAL, LTD -25- PROJFCT NO.: 454-103 TABLE4 GroupGronsPathologicOablservations from a Northern Bobwhite Acute DietaryToxicityStudy with PFOS Birdsthatdiedduringthecourseof thestudy Finding Control N=l Male, Female,and Undetermined PPM A.I. 146 293 586 1171 N =2 N =8 N =10 N =10 Abdominal cavity,some autolysis 0 Abdominal cavitya,utolysitshroughout 0 Crop, empty 0 Emaciated 0 Fracturedleg I G.I.traa empty 0 Gi=d contentbsilestained 0 Heart,anterioprortionmottledwhitecolor 0 Heart,pale 0 Intestinaclontentstar-like 0 KeeL prominent 0 Kidneys,pale 0 Liver,paleand mottled 0 Loss ofmuscle mass 0 Musculu-skeletalp,ale 0 Small in stature 0 Spleen,black 0 Spleen,dark 0 Spleen,grey 0 Spleen,grey-brown 0 Spleen,pale 0 Spleen,small 0 Spleen,smalland pale 0 Thin 0 Not Remarkable 0 0 2 0 0 0 2 0 2 1 0 0 1 0 2 0 1 0 0 0 0 0 1 0 0 1 0 0 4 1 0 0 3 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 2 4 1 1 5 2 5 8 0 0 1 0 5 1 0 0 2 1 2 0 3 10 2 0 0 0 7 9 0 0 0 0 1 0 0 2 1 0 0 1 0 1 0 1 3 0 4 2 0 0 WILDLIFE INTERNATIONAL, LTD PRO -26- Experimental Group (jMma.i.) Control 0 0 0 0 0 0 Treatment 18.3 18.3 Table5 CumulativeMortalit(yEstimateCdumulativeDose,mg/kgl)froma NorthernBobwhite AcuteDIS.M_MToxici!Sy@ldlwithPFOS No. Dead PerNo. Exposed(CumulativeDose,mg/kg) No. Dead P ExposurePeriod Post-Exlx Pen -Dff0 D!lI Dg 2 DM 3 DM 4 DM 5 D!z 6 Dm 1 0/5 015 0/5 0/5 015 015 015 015 2 015 015 015 015 015 0/5 015 015 3 0/5 015 0/5 015 0/5 015 015 015 4 015 015 015 015 015 015 015 015 5 015 015 015 015 015 0/5 1/5 115 6 0/5 015 015 015 015 015 0/5 0/5 1 015 2 015 0/5(7) 0/5(6) 0/5(14) 0/5(21) 0/5(28) 0/5(35) 015 0/5 0/5(12) 0/5(18) 0/5(24) 0/5(30) 0/5 0/5 36.6 36.6 1 015 2 015 0/5(13) 015(11) 0/5(26) 0/5(39) 0/5(52) 0/5(65) 015 015 0/5(22) 0/5(33) 0/5(44) 015(55) 015 015 73.2 73.2 1 015 0/5(33) 015(66) 0/5(99) 0/5(132) 0/5(165) 015 015 2 0/5 0/5(32) 0/5(64) 0/5(96) 0/5(128) 0/5(160) 015 0/5 146 1 015 0/5(46) 0/5(92) 0/5(138) 0/5(184) 0/5(230) 015 115 146 2 015 0/5(49) 0/5(98) 1/5(147) 1/5(196) 1/5(245) 115 115 293 1 015 0/5(66) 0/5(132) 0/5(198) 0/5(264) 1/5(330) 115 4/5 293 2 015 015(101) 0/5(202) 0/5(303) .0/5(404) 115(505) 3/5 4/5 586 1 015 0/5(213) 0/5(429) 1/5(639) 2/5(852) 2/5(1065) 4/5 5/5 586 2 015 0/5(178) 0/5(356) 0/5(534) 0/5(712) 3/5(890) 4/5 515 1171 1 0/5 0/5(256) 0/5(512) 3/5(768) 515 515 5/5 515 1171 2 0/5 0/5(256) 015L512) 0/5(7681 515 515 5/5 515 TheLc5ovaluweascalculateobdeapproximat2e2l0yppm a.iw.itha95% confidenicnetervoafl164to289ppm a.i.. Birdwas eudmiizedduetoabrokenleg. Estimtedcumulativdeoseisbasedupontheaveragebodyweightandfeedconswnptionoverthe5-&y exposme perioda,nd serveass arough approximatoonftheactualamount oftestsubstancceonsumed. No mortalitioecscurreidn!U ofthetreatmenotrcontroolu2s afteDray 8. WILDLIFE INTERNATIONAL, LTD -27- PROJE:--@ NO.: 454-103 APPEI*MIX I Certificaotfe Analysis FC-95, I.,o2t17 mu& 9,2000 RiduwdhLPqfa It& smple-asadrod utfusLCJUS.IHMdR. 'PJLMdt.=d elemcub&iIWYM tccbW4ucLI& vttbonUnts&ow ftampb tAWAWM ft *UDWW9 WettaPatcA comP*sMon: c2ysso3-r CAF4011C CAFIS(h%r C,Fiiso,t,r Qpnsos*r 0.04% OM *A 1.38% 1.30% 3.71% -O:W% WHSOSW -0.49% clop2igosw --- 0.13% r'lifnsosw* 0.04% gi-m-'r sp"16sojr 0.05% I - 0.3s% AdCdonaMy,dw;go dbftftttloucfftUM&W=dctmlmdus'ng*FNMRtwlldqm andfomdto CF3(CFdrM pdww uam=mcdclqwL=b. V/=a 14-yIsMabiTZSf.andx 0 -0L2e-q- (CFL)2cl4cFx%srw xr kinc%vbmxbaftd C-V2-R-OKCFa)-SW x- (Cpo,o(CF,),SO,7Kr (t4xdAkgglh wbm X b MdntY CFS-(CF3@,qcLzs)r(CFs2wh(int=alg=zmdwlbmud@6wbmx+Y Iswdnly 4,andx 110) 70.5% 10.3% 1.6% 0.2% 0.2% WILDLIFE INTERNATIONAL, LTD -28- PROJECT NO.: 454-103 APPENDDC H DIET FORMULATION WILDLIFE INTERNATIONAL LTD. GAME BIRD RATION' INGREDIENTS Fine Com Meal Soy Bean Meal, 48% Protein Wheat Nfidds ProteinBase Agway Special,60% Protein Alfal& Meal, 20% Protein Dried Whey Ground Limestone Eastman CalPhos MediioninePremix + Liquid Vitamin and MineralPremix (seebelow) GL Ferm (Fermatco@ SaltIodized Total PERCENT 44.83 30.65 6.50 6.00 4.00 3.00 2.50 0.90 0.60 0.35 0.32 0.25 0.10 100.00 VITAMIN AND MINERAL PRFIVHX AMOUNT ADDED PER TON Vitamin D3 Vitamin A Riboflavin Niacin PantothenicAcid Vitamin B12 FolicAcid Biotin Pyridoxine Thiamine Vitamin E Vit=k K (MenadioneDirnethylpyrimidinBoilsulfite) Manganese Zinc Copper Iodine Iron Selenium 2,000,000I.C.U. 7,000,000 1-U. 6 grams 40 grains 10 grams 8 mgs 600 mgs 64 mgs 1.2 grams 1.2 grams 20,000 l.U. 5.8 grams 102 grams 47 grams 6.8 gram 1.5gram 51 grum 182 mgs 'Tbe guaranteedanalysisisa minimum of 27% proteina, minimum of 2.5% crude fatand a maximum of 5% crudefiber. 2FermentationBy-Products(Sourceof UnidentifiedGrowth Factors). I - WILDLIFE INTERNATIONAL, LTD -29- PROJECT NO.: 454-103 APPENDIX M ANALYRICAL METHODS AND RESULTS WILDLIF'E INTERNATIONAL, LTD -30- PROJECT NO.: 454-103 INSTRUMENT: ANALYRICAL COLUMN: OVEN ISWERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECMON VOLUME: PFOS RETENTION TIME: INTERNAL STANDARD RETENTION TM@ffi: PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: APPENDIX HI Table I TypicalLC/MS OperationalParameters Hewlett-PackardModel 1100 High PerformanceLiquidChromatograph with a Peridn-ElmerAPI 10OLC Mass Spectrometerequippedwitha Perldn-Elmer TurboIonSprayion source.Operatedinselectivieon monitoringmode (SM. KeystoneBeLuff Cls column (100nun x 2 mm I.D.,3 pm particlseize) 300C 10.0 minutes 0.220 mIJminute 72.0% Methanol 28.0% NANOpureOD Water containing0.1% Formic Acid 25.0pL Approximately7.0minutes Approximately4.8 minutes 498.6amu 426.7 WILDLIFE INTERNATIONAL, LTD -31- PROJECT NO.: 454-103 APPENDDC M Table2 MatrixBLuks AnalyzedConcurrentlDyuringSampleAnalysis Number (454-103-) MAB-1 S!aple Type MatrixBlank MeasuredConcentratioonf PFOS, (ppma.i.) < LOQ MAB-2 MatrixBlank <LOQ 'The limitofquantitati(oLnOQ) was 1.15 ppm ai.basedupon theproductofthelowestcallb-mtiiodn@ard Egyzed (0.0022M9g a.i./La)ndthedilutiofnactoorfthematrixblankEMIes (500). WILDLIFE INTERNATIONAL, LTD -32- PROJE%"T NO.: 454-103 APPENDDC Ell Table 3 Matrix FortificatioAnnsalyzed ConcurrentlyDuring Sample Analysis Sample Number (454-103-) MAS-LA MAS-4A ConcentrationsofPFOS (ppm ai.) Fortified Measured 4.57 4.54 4.57 4.79 Percent Recovered 99.2 105 M,kS-2 183 176 96.1 MAS-5 183 162 88.3 MAS-3 M,kS-6 1830 1576 1830 1716 Mean = StandardDeviation= CV= N= Note:Resultsand correctionfsornew testsubstancepunty were generatedusingMacQuan and manual calculationsV.alueshave been rounded forreportingpurposes. 86.1 93.7 94.7 6.99 7.38 6 version1.5 software WILDLIFE INTERNATIONAL, LTD PROT 33- APPENDDC III Table4 Nominal Concentration (ppma.i.) 18.3 Sample Number (S-454-103-) 2 3 4 5 6 7 HomogeneityofPFOS inAvian Diet LocationSampled in Mxing Vessel PFOS MeasuredConcentration (ppma.i.) Top Left 18.5 Top Right 23.4 MiddleLeft 18.3 MiddleRight 17.3 BottomLeft 19.4 BottomRight 19.9 Mean MeasuredConcentratio(n5t) StandarDdeviatio(nSD) CoefficieonftVariatio(nCV)l 5t= 19.5ppm a.i. SD = 2.13ppm a.i. CV = 10.9% 1171 18 Top Left 1239 19 Top Right 1221 20 Middleleft ilig 21 MiddleRight 1301 22 BottomLeft 1163 23 BottomRight 1133 R = 1196 ppm a.i SD 70.2ppm. a.i CV 5.87% Note:Resultsand coffewons"fonrew testsubstancpeuritywere generateudsingMacQuan version1.5softwareand manual calculati roundedforreportinpgurposes. 'Coefficieonftvariatiownas calculateudsingfidlprecisioonfmean and standardeviatiornesults. WILDLIFE INTERNATIONAL, L-rD PROJ 34APPENDIX III Table5 VerificatioofnPFOS Concentratioinns"an Diet Nominal Concentration (ppma.i.) 0.0 Sample Number (S454-103-) 1 sampling Interval (Day) 0 PFOS MeasuredConcentration' (ppma.i.) < LOQ Percent Of Nominal Mean Measured Concentration (ppma-i.) - 19.3 - - - - 19.51 36.6 8 0 45.7 125 40.2 9 0 34.6 94.5 73.2 10 0 11 0 77.8 106 74.5 71.2 97.3 146 12 0 13 0 176 120 174 172 117 293 14 0 15 0 274 93.9 291 307 105 586 16 0 17 0 550 93.9 537 523 89.4 1171 - - - - 1196' Note:Resultsandcorrectiofnosrnew testsubstancpeuritwyeregenerateudsingMacQuan version1.5softwareandmanualcalculationVsa.lueshave purposes. "Me lindotfquantitati(oLnOQ)was 1.15ppm a.ib.asedupontheproducotfthelowestcalibratisotnandaradnalyze(d0.0022m9g a.i./aLn)dthedil blanksamples(500). 'ResulotbtainefdromTable4. WILDLIFE INTERNATIONAL, L-rD PROJE 35APPENDIX III Table6 AmbientStabiliotfyPFOS inAvianDietDuringtheNorthernBobwhiteLC50 Study Nominal Concentration (ppma.i.) 0 Sample Number (S-454-103-) 1 Dayof Mean Measured Concentration (ppma.i.) - Mean Percent Of Nominal - Sample Number (S454-103-) 24 Day 5 Measured Concentratior? (ppmai.) < LOQ Me= Measured Concentration (ppmai.) - 18.3 2-7 19.5 107 25 19.2 19.6 26 19.9 36.6 8.9 40.2 110 27 44A 49.1 28 53.9 73.2 10.11 74.5 102 29 76.4 77.2 30 77.9 146 12.13 174 119 31 177 176 32 174 293 14,15. 291 99.3 33 319 317 34 315 586 16.17 537 91.6 35 560 613 36 665 1171 18-23 1196 102 37 1260 1224 39 1187 'Day0 resulotbstainefdromTable4 andTable5. 2Thelimiotfquanfitati(oLnOQ) was 1.15 ppm a.ib.asedupontheproductofthelowesctalibratisotnandaradnalyze(d0.00229mg a.iJLa)ndthedilutiofnactoorf (500). WILDLIFE INTERNATIONAL, LTD -36- PROJECR NO.: 454-103 METHOD APPENDIX M OUTLINE FOR THE ANALYSIS OF PFOS IN AVLAN DIET preparematrixfortificatisoanmplesinthecloakedavian&ed dock usingto drynux ted=qum Dq Mix Technique For thehigh-levemlatrixfortificatsimovnic, weightherequisitqemdty ofWildlifeInternationLatl& (WIL) radon intoa weigh boal I& Weigh therequisitqeuantityoftestsubstanc(effOS) intoa beaker. Add 3/,ofthewiL mtionand thetestsubstancetoa kW beaker. RinsethebeakerthatcontainedthePFOS withsmallportionsofthercmahiingrationandtransfearllportionstothelargerbc&cr. Mix thecontentosfthekW beakerwellandUmneer thenlixttntoa Waring blender. Blendthemixturefor-5 minutesAOPPing atI nlinutoki-als tos=pe down thesidesoftheblender. Duringthethirdintervatlransfedrw f"uficdfeedtoa beak-.-ix welland returnthemixturetothebluxlertocompletenixinginthe specifietdimc preparethenexttwo matrixfmtificado]nawlsby saW dilutiFollowthesame proceduredescribedforthehigh-levemlatrixfortificatieoxnceptweigh theappropriatqeuantityoffortifiemdatrix(highor mid-levelr)atherthan thetestsubdance, Weigh IO-Ssamplesofthematrixblank,matrixfmifficafiandtestsampla intoweigh boatsand t=dcr to 16-oL French-square91= bottlesR.ecordtheweightl For each sample,measure 100 mLs of methanolwith a graduatedcylinderandtransfevrolume totheFmich-squarebottic Cap bottleasnd placeon shakertable.Allow thesamplesto sh&c fora minimum of 30 minutesat250 rpm Vacuum filteerachsamplewithqualitatifvieltepraperand ritm retainefdeed3 tmm withmed=ol intothefiftratp- TnuLsfertheflitrat"oa 200-mL volumetricflaskand bringtheflaskto volume withmcd=ol Prepareappropriatdeilution(tso)bringfinalconcentratioinntothecalibratiroanngeoftheLCMS methodology. Use methanolforintermediatdeilutionsi,frequire& For allfinaldilutionusse50% medmol: 50% NANOpurcO watersolutiocnontainin0g.100mg 4H PFOS CuA=ualstandard)/L and 0.05% fonnicacid(v/v). Ampulate and submit=Wles forLCMS analysis. Figure 1. Analyticamlethod flowchartfortheanalysisofPFOS in aviandiet. WILDLIFE INTERNATIONAL, LTD -37- APPENDIX M PROJECT NO.: 454-103 1.50 1.00 0.50 - 0 0 0 0 0.00 0.000 .100 0.200 0.300 0.400 .500 Concentration(Patio) Figure 2. AtypicalcalibratiocnurveforPFOS. Slope= 2.77397;Intercept=0.01894;r= 0.9981. Curve isweighted(I/x). WILDLIFE INTERNATIONAL, LTD -38- APPENDDC M PROJECT NO.: 454-103 lntensit1y9'95 cP$ 100- 167 go- 70, 60. so, 40- 30146 20, 10, 24 CLI-1- ;E4 91 10 122 3'1 6'1-' * S'l 2:iPl&9ft-fsc-an 1.30 2.55 3.81 5.06 6.32 7.57 6.83 Time Figure3. A representativoenchromatogramofa low-leve(l0.00229mg a.i./LP)FOS standard. WILDLIFE INTERNATIONAL, LTD -39- APPENDDC M PROJECT NO.: 454-103 Intensit2y6:954 cps 100. 166 go- so- 70- 60- 60- 40- 30- 144 20- 1009 &4 48 75 90' 122 . .. 31 61 * ' 91 121 1.30 2.55 3.61 5.06 a . a 190204 230 151 'lil''2il'* SM 6.32 7.57 8.83 Time Figure4. A representativoenchromatogramofa high-leve(l0.0457mg ai./L)PFOS standard. WILDLIFE INTERNATIONAL, LTD -40- APPENDDC III PROJECT NO.: 454-103 intensit2y3:0 cps iso go. 24 166 80- 70- 60- so, 403020,- IA11 1 i(@ 0 14 55 79 105 A,N. AIV A A AA AA IOA1I';=- v -14Ina-al I.(I 3.1 . .6.1 . .9.1 . .lil- -151 1.30 2.55 3.81 5.06 6.32 216 203.@.pl @loelAV AVY " @l L jai* '2il SCan 7Z7 8.83 Ilme FigureS.- A representaticvheromatogramofa matrixblanksample(454-103-NIAB-1). The arrowindicatetsheretentiotnimeofPFOS. WILDLIFE INTERNATIONAL, L-rD -41APPENDDC M PROJECT NO.: 454-103 100. Intensity6:238 cps 166 go- 80, 70- 60, so40- 30144 20. 10, 11 2,4- 54 71 869.5 122 oll 213 . . 31. . . 61. I 91* ' -'12I1 ' '151 - lal 211 Scan 1.30 2.55 3.81 5.06 6.32 7.57 8.83 Time Figure6. A representaticvheromatogram ofa matrixfortificatisoanmple (454-103-MAS-IA). WILDLIFE INTERNATIONAL, LTD -42- APPENDDC III PROJECT NO.: 454-103 intensity7:248 cps 100- 167 90- 80- 70- 60, so- 40- 30145 20- I(). 2p4 4 63 94 123 197 229 31 6'1 9.1 211 Scan 1.30 2.55 3.81 5.06 6.32 7.67 8.83 Time Figure7. A representaticvheromatogramofa testsample(454-103-2). WILDLIFE INTERNATIONAL, LTD -43- APPENDDC IV DEET PREPARATION PROJECT NO.: 454-103 Weight and volume ofconstituenutsed topreparetestdiets: Nominal Concentrations (ppm a.i.) 0 18.3 36.6 73.2 146 293 586 1171 Test Substance (9) 0.1-818 0.3638 0.7282 1.4659 2.9123. 5.8239 11.6483 Basal Ration (9) 9000.0 8999.8 8999.6 8999.3 8998.5 8997.1 8994.2 8988.4 Dietswere preparedas follows: o5000.Og ofbasalrationwas weighedintoa taredHobartmixingbowl. otbe testsubstancewas weighedina Ured weigh boat. *Approximately100g ofbasalrationwas takenfromthemixingbowl and placedina Waring blender. *The testsubstancewas addedtotheblenderand theweighboatwas rinsedwithadditionamltion, withtherinsealsobeingplacedintheblender. olhe blendercontentwsereblendedforapproximately60 secondsand transferretdothemixing bowl. The blenderwas rinsedwithadditionarlationw,iththerinsealsobeingplacedinthe mixing bowl. *The bowl was placedon a Hobartmixerand thecontentwsere mixed forapproximatelysixminutes. The remainingmtionasaddedtothebowl and thecontentwseremixed forsixmore minutes. *The dietwas transferretdoa labellepdaperfeedbag. WILDLIFE INTERNATIONAL, LTD 44- PROJECT Experimental Group (p2m a.i.) Control 0 0 0 0 0 0 Treatment 18.3 18.3 36.6 36.6 73.2 73.2 146 146 293 293 586 586 1171 1171 Cumulative Mortalt Pen Dg 0 APPENDIX V by Pen from a Northern BobwlfiteAcute DLe!:@gToxi2il@SZtudy withPFOS No. Dead PerNo. Exposed Fxposure Period D!Z I D@y2 DM 3 Day 4 DM 5 No. Dead Per Post-Expos' -D@y 6 Day 1 015 015 015 015 015 0/5 2 015 015 015 015 015 015 3 015 015 015 015 015 015 4 015 015 015 015 015 015. 5 015 0/5 015 0/5 0/5 015 6 0/5 0/5 015 0/5 015 0/5 015 015 0/5 015 0/5 0/5 015 0/5 115* 115 015 0/5 1 015 015 015 015 015 0/5 2 015 015 015 015 015 015 1 0/5 015 015 0/5 015 0/5 2 015 015 015 015 015 015 1 015 015 015 015 0/5 015 2 015 015 015 015 015 015 .1 015 015 015 015 015 015 2. 015 015 0/5 115* 115 115 1 0/5 0/5 0/5 015 015 115 2 015 0/5 015 015 015 115 1 0/5 0/5 0/5 1/5 2/5 2/5 2 015 015 015 015 015 3/5 1 015 015 015 3/5 5/5 515 2 015 015 015 015 5/5 515 0/5 0/5 015 015 0/5 0/5 015 0/5 015 0/5 015 015 0/5 1/5 1/5 1/5 115 4/5 315 4/5 4/5 5/5 4/5 5/5 515 515 515 515 The LC50 value was calculatedto be 220 ppm a.i.with a 95%, confidence intervalof 164 ppm a.i.to 289 ppm a.i. * - Bird was eudianizeddue toa broken leg. I- No mortalitieosccurredinM of thecontrolortreatmentzou2s afterD!y 8. WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-103 -45- APPENDIX VI IndividuaBlody Weights(g)from a NorthernBobwhite Acute DietaryToxicityStudywithPFOS Page I Experimental Group @22m a.i.) Bird Day 0 Da 5 Change Dg 0-5 D!y 8 Change Dg 5-8 0 1 19 24 5 27 3 2 20 27 7 34 7 3 21 32 11 42 10' 4 22 32 10 40 8 5 23 39 16 so 11 Mean 21 31 10 39 8 SD 2 6 4 9 3 0 1 1'9 26 7 35 9 2 20 25 5 35 10 3 20 29 9 37 8 4 22 33 11 43 10 5 22 .31 9 39 8 Mean 21 29 87 38 9 SD 1 3 2 3 1 0 1 19 23 4 30 7 2 20 26 6 29 3 3 20 29 9 32 3 4 22 30 8 37 7 5 22 35 13 40 5 Mean 21 29 8 34 5 SD 1 5 3 5 2 0 1 19 28 9 37 9 2 19 27 8 35 8 3 19 30 11 36 6 4 21 32 11. 42 10 5 22 33 11 43 10 Mean 20 30 10 39 9 SD 1 3 1 4 Total Change 8 14 21 18 27 19 7 16 15 17 21 17 17 2 11 9 12 15 18 13 4 18 16 17 21 21 19 2 WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-103 -46- Experi=nW Group (22Ma.i.) 0 APPENDIX VI individuaBlody Weights(g)from a Northem Bobwlute Acute DietaryToxicityStudywithPFOS Page 2 Bird 1 2 3 4 5 Mean SD Dg 0 19 20 21 20 22 20 1 DE 5 28 27 32 30 34 30 3 Change Dg 0-5 7 11 10 -12 10 2 Dg 8 35 36 44 44 40 5 Change D2X 5-8 7 9 12 1010 2 0 Group Total 1 18 30 12 39 9 2 21 35 14 46 11 3 19 29 10 37 8 4 21 32 11 41 9 5 22 32 10 42 10 Mean 20 32 if 41 9 SD 2 2 2 3 1 Mean 20 30 10 38 8 SD 1 4 3 5 2 Total Chan e 16 16 23 2219 4 21 25 18 20 20 21 3 18 4 WILDLIFE INTERNATIONAL, LTD PRO.TECT NO.: 454-103 -47- APPENDDC VI IndividuaBlody Weights(g)from a NorthernBobwhite Acute DietaryToxicityStudywithPFOS Page 3 Experimental Group @22m a.i.) Bird Day 0 18.3 1 19 2 19 3 21 4 22 5 22 Mean 21 SD 2 DM 5 26 33 27 34 36 31 4 Change DM 0-5 Dg 8 7 34 14 39 6 35 12 46 14 46 11 40 4 6 Change Dg 5-8 8 6 8 12 10 9 2 Total Change 15 20 14 24 24 19 5 18.3 1 19 28 9 38 10 19 2 19 28 9 37 9 is 3 20 29 9 38 9 18 4 22 34 12 44 10 22 5 22 35 13 44 9 22 Mean 20 31 10 40 9 20 SD 2 3 2 3 1 2 Group Mean 21 31 11 40 9 20 Total SD 1 4 3 5 2 3 WILDLIFE INTERNATIONAL, LTD -48- PROJECT NO.: 454-103 APPENDIX VI IndividualBody Weights (g)from a NorthernBobwhite Acute DieL-uyToxicityStudy with PFOS Page 4 Experimental Group (pEm a.i.) Bird D!Y 0 36.6 1 18 2 19 3 19 4 21 5 21 Mean 20 SD 1 Dg 5 26 30 29 30 34 30 3 Change Dg 0-5 DLy 8 8 35 11 39 10 37 9 38 13 43 10 38 2 3 Change Dg 5-8 9 9 8 8 9 9 1 Total Ch!age 17 20 is 17 22 19 2 36.6 1 19 29 10 37 8 is '2 19 29 10 37 8 is 3 21 32 11 40 8 19 4 22 35 13 44 9 22 5 23 36 13 44 8 21 Mean 21 32 11 40 8 20 SD 2 3 2 4 0 2 Group Mean 20 31 11 39 8 19 Total SD 2 3 2 3 1 2 WILDLIFE INTERNATIONAL, LTD -49- PROJECT NO.: 454-103 APPENDIX VI IndividuaBlody Weights(g)froma NorthernBobwhite Acute DietaryToxicityStudywithPFOS Page 5 Experimental Gmup (ppmai.) Bird Day 0 73.2 1 18 2 19 3 20 4 21 5 21 Mean 20 SD 1 Day 5 26 26 29 32 30 29 3 Change Day 0-5 8 7 9 11 9 9 1 Change D!z 8 __gay 5-8 32 6 33 7 36 7 39 7 36 6 35 7 3 1 Total Change 14 14 16 18 15 15 2 .73.2 1 18 27 9 34 7 16 2 20 31 11 38 7 18 3 21 30 9 37 7 16 4 21 32 11 39 7 .18 5 22 32 10 41 9 19 Mean 20 30 10 38 7 17 SD 2 2 1 3 1 1 Gmup Mean 20 30 9 37 7 16 Total SD 1 2 1 3 1 2 WILDLIFE INTERNATIONAL, LTD -50- PROJECT NO.: 454-103 APPENDIX VI IndividuaBlody Weights (g)from a NorthernBobwhite Acute DietaryTo@dcityStudy withPFOS Page 6 Experimental Group (P m a.i.) Bird Day 0 146 1 19 2 .19 3 20 4 22 5 23 Mean 21 SD 2 Day 5 28 28 24 33 28 28 3 Change Day 0-5 Day 8 9 35 9 32 4 - 11 38 5 32 8 34 3- 3 Change Day 5-8 7 4 - 5 4 5 1 Total Change 16 13 16 9 14 3 146 1 18 24 6 31 7 2 19 27 8 35 8 3 19 27 8 33 6 4 21 - - - - 5 23 24 1 28 4 Mean 20 26 6 32 6 SD 2 2 3 3 2 Group Total Mean 20 27* 7* 33* 6** SD 2 3 3 3 2 (-)= No dataavailabldeuetomortahty. *Statisticadlilfyfemtfromthecontroglroupatp < 0.05(Dunnett'ts-test). **Statig@@ differenftrom thecontroglroupatp < 0.01LDunnetVst-test). 13 16 14 5 12 5 13** 4 WILDLIFE INTERNATIONAL, LTD -51- PROJECT NO.: 454-103 APPENDEK VI IndividuaBlody Weights(9)froma NorthernBobwhite Acute DietaryToxicityStudywithPFOS Page 7 ExperimenW Group @n-P-Ma.i.) 293 Bird Day 0 1 18 2 19 3 20 4 2-2 5 22 Mean 20 SD 2 Day 5 20 17 18 20 Change DLy 0-5 Day 8 2 21 -2 - -2 - -2 - -1 21 2 - Change D!y 5-8 1 - 1 - Total c 3 3 293 1 19 19 0 15 -4 -4 2 19 - - - - 3 20 20 0 - 4 20 17 -3 - 5 21 16 .5 - - Group Total Mean 20 18 -2 15 -4 -4 SD 1 2 2 - - Mean 20 18** -2** 18** -2** -1** SD 1 2 4 4 5 (-)= No dataavailabldeuetomortality. *Statisficdailflfyerefnit-otmhecontroglmup atp< 0.05(Dtumett'ts-test). **StatistiEd!i!f!fyerefnrtomthecontroolu2 atp < 0.01(Dunn tt'ts-test). WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-103 -52- APPENDIX VI IndividuaBlody Weights(g)from a NorthernBobwhite Acute DietaryToxicityStadywithPFOS Page 8 Experin=tal Group m a.i.) Bird D!10 596 1 18 2 20 3 21 4 21 5 22 Ukan 20 SD 2 Dg 5 13 18 17 16 3 Change Day 0-5 -7 -3 -5 -5 2 D!y 8 - Change Dg 5-8 Total 586 1 19 16 -3 2 20 - - 3 20 16 -4 4 19 - 5 21 - - Group Total Mean 20 SD 1 Mean 20 SD 1 16 -4 0 1 16** -4*i--- - 2 2 (-)= No dataavailabldeuetomortality. **Statisticdailf!fyerefnrtomthecontroglroup!t 0.01(Dunn tt'ts-test@. WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-103 -53- APPENDIX VI individuaBlody Weights(g)from a NorthernBobwhite Acute DietaryToxicityStudywithPFOS Page 9 Experimental Group PLPm. a.i.) Bird Day 0 1171 1 is 2 20 3 20 4 21 5 21 Mean 20 SD 1 Day 5 Change Day 0-5 Day Change Day 5-8 Total Change 1171 1 18 2 20 3 20 4 22 - - - - 5 21 - - - - Group Total Mean 20 - - - - SD 1 - - - - Mean 20 - - - - SD 1 - - - - No data availableduetomortality. WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-103 -54- APPENDIX VI IndividuaBlody Weights(g)from a NorthernBobwhite Acute DietaryToxicityStudywithPFOS Page 10 Experin=tal Group (22Ma.i.) Bird Dq 8 Change DE 15 Day 8-15 Dg 22 Change Day 15-22 0 1 -27 42 15 61 19 2 34 56 22 77 21 3 42 69 27 92 23 4 40 63 23 84 21 5 50 78 28 104 26 Mean 39 62 23 84 22 SD 9 14 5 16 3 0 1 35 55 20 74 19 2 35 53 18 74 21 3 37 64 27 85 21 4 43 74 31 103 29 5 39 59 20 so 21 Mean 38 61 23 83 22 SD 3 8 6 12 4 0 Group Total 1 .30 54 24 79 25 2 29 .45 16 63 18 3 32 54 22 77 23 4 37 55 18 75 20 5 40 68 28 95 27 Mean 34 55 22 78 23 SD 5 8 5 11 4 Mean 37 59 23 82 22 SD 6 10 5 13 3 Total 34 43 50 44 54 45 8- 39 39 48 60 41 45 9- 49 34 45 38 55 44 8 45 8- WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-103 -55- APPENDIX VI IndividuaBlody Weights(g)froma NorthernBobwhite Acute DietaryToxicityStudywithPFOS Page 11 Experimental Group @EPM ai.) Bird Dg 8 18.3 1 34 2 39 3 35 4 46 5 46 Mean 40 SD 6 Change Change Dg 15 Dg 8-15 Dg 22 DU 15-22 53 19 78 25 65 26 85 20 60 25 86 26 73 27 95 22 71 25 92 21 64 24 87 23 8 3 7 3 Total Change 44 46 51 49 46 47 3 Experimental Group (2P-M a.i.) Bird Day 8 36.6 1 35 2 39 3 37 4 38 5 43 Mean 38 SD 3 Change Change D!y 15 DM 8-15 D@y 22 Day 15-22 60 25 81 21 65 26 91 26 65 28 91 26 61 23 83 22 72 29 97 25 65 26 89 24 5 2 7 2 Total C 46 52 54 45 54 50 4 WILDLIFE INTERNATIONAL$ LTD -56- PROJFCT NO.: 454-103 APPENDIX VI IndividuaBlody Weights(g)froma NorthernBobwhite Acute DietaryToxicityStudywithPFOS Page 12 Experimental Group (ppm a.i.) Bird Day 8 73.2 1 32 2 33 3 36 4 39 5 36 Mean 35 SD 3 Day 15 Change Day 8-15 Day 22 Change Day 15-22 54 22 76 22 58 25 77 19 62 26 79 17 65 26 87 22 59 23 78 19 60 24 79 20 4 2 4 2 Total Change. 44 44 43 48 42 44 2 Experimental Group (am a.i.) Bird DU8 Day 15 Change Dg 8-15 DU 22 Change Day 15-22 146 1 35 58 23 81 23 2 32 55 23 77 22 3 4 38 63 25 81 18 5 32 57 25 78 21 Mean 34* 58 24 79 21 SD 3 3 1 2 2 No dataavailabldeue tomortality. *StatistiLdaikffe=tfromthecontroEluU atp < 0.05(DunnetVst-test) Total Change 46 45 43 46 45 1 WILDLIFE INTERNATIONAL, LTD -57- PROJECT NO.: 454-103 APPENDIX VI IndividuaBlody Weights(g)from a NorthernBobwhite AcuteDietaryToxicityStudywithPFOS Page 13 EVerimental Group (ppma.i.) Bird Day 0 293 1 21 2 3 4 5 Mean 21 SD - No dataavailabldeue tomortality. Day 5 35 Change Day 0-5 14 Day 8 55 35 14 55 - - - Change Day 5-8 20 20 - Total Change 34 34 - WILDLIFE INTERNATIC)NAL, LTD PROJECT NO.: 454-103 -58- APPENDIX VII ]FeedConsumption(glbird/dabyy) Pen from a NorthernBobwhiteAcute DietaryToxicityStudywithPFOS Page I ExperimentalGroup (ppm&i.) Pen ExposurePeriod Day 0-5 Post-ExposurPeeriod Day 6-8 Control 1 9 12 2 7 12 3 8 7 4 12 12 5 9 9 6 10 9 Mean 9 10 SD 2 2 WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-103 -59- APPENDIX VII Feed Consumption(g/bird/dabyy) Pen from a NorthernBobwhite Acute DietaryToxicityStudywithPFOS Pge 2 ExperimentalGroup ExposurePeriod Post-ExposurPeeriod (ppma.i.) Pen Day 0-5 Day 6-8 18.3 1 9 10 2 8 11 Mean 9 11 36.6 1 8 14 2 7 9 Mean 8 12 73.2 1 10 12 2 10 14 Mean 10 13 146 1 7 8 2 10 12 Mean 9 10 293 1 4 8 2 6 10 Mean 5 9 586 1 6 16 2 5 22 Mean 6 19 1171 1 4 - 2 4 Mean 4 No dataavailabldeue toadultmortality. WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-103 -60- APPENDIX Vla Feed Consumption(gibird/dabyy) Pen froma NorthernBobwhiteAcute DietaryToxicityStadywithPFOS Page 3 Expedmntal Group (ppma.i.) PM ExposurePenod Day 8-15 Post-ExposurPeeriod Day 15-22 Control 1 10 14 2 10 12 3 7 12 Mean 9 13 SD 2 EVedmntal Group (ppmai.) Pen 18.3 1 36.6 1 73.2 1 146 1 293 1 ExposurePeriod Day 8-15 10 14 13 11 8 Post-ExposurPeeriod Day 15-22 12 15 15 14 9 WILDLIFE INTERNATIONAL, LTD -61- APPENDIX VM CHANGES TO PROTOCOL PROJECT NO.: 454-103 1."Me protocolwas amendedtoindicattehatbilewillbe collectefdrom allstudybirds.The protocol was clanfietdomdcate thecouectonoflivefrrom birdsthatdieddunng thecourseofthestudy 2.Bloodsampleswerecollecteodn Day 8 and Day 22 innon-heparinize5dml borosilicagtleasstest tubes.The protocolindicatetdhathepannizedvacutainerwsould be used. 3.The protocolwas amended tochangethetestconcentra6onsfrom 0, 20,40,80, 160,640 and 1280 ppm ai.,to0,18.3,36.6,73.2,146,293,586 and 1171 ppm a.i.Testconcentratiownesre changedto reflectthetestsubstancepuritygiveninthenew certificaotfeanalysis. 4.The temperaturefsrom severalbrooderunitson Day 4,7,8,and allbrooderunitson Day 22 were notrecorded 5.'Me aftemoonobservationwsere inadvertentnloyt recordedfor2 birdsinthe640 ppm a.i. treatmengtroup on April26, 1999. WILDLIFE INTERNATIONAL, LTD -62- APPENDICK IX PERSONNEL INVOLVED IN THE STUDY PROJECT NO.: 454-103 The fouowmg key WildlifeInternationLatld.personnelwere involvedintheconductor managen=t ofthisstudy: (1) Mark JaberW,ildlifTeomcologist (2)JoannB. Beavers,DirectorA,vian Toxicology (3)Sean P.CraflagheSre,niorBiologist (4)CourtneyCasey,M.S.,SeniorBiologist (5) WillardB.Nixon,Ph.D.,Manager,AnalyticaClhemistry (6)TimothyZ.Kendall,SupervisorA,nalyticaClhemistry (7)Raymond L.Van Hoven, Ph.D.,Scientist (8)EllenMank, Chmnist WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-102 AMEENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A DIETARY LC50 STUDY WITH THE NORTHERN BOBWHITE PROTOCOL NO.: 454/030999/QLC/SUB454 AMENDMENT NO.: I SPONSOR-- 3M Corporation PROJECT NO.: 454-103 EFFECTIVE DATE: April27,1999 ANIENDNffiNT: On page 8 under Necropsy add: Wlum possibleb,ilewillbe collectefdrom allbirds.Liverswillalsobe collectefdrom allbirdsthat dieduringthecourseof thetest. REASON: Bileisbeing collecteadtSponsor request.The protocolisbeingclarifietdo indicatethe collection of liverfrom birdsthatdie. Study Director LaboratoryM\tAagemmt Splnsor PrincipalContact Date Date bate' WILDLIFE INTERNATIONAL, LTD PaizeI of I DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A DietaryLC50 Studywith theNorthernBobwhite PROTOCOL NO.: 454/030999/QLC/SUB454 DEVIATION NO.: 1 SPONSOR: 3M Corporation PROJECT NO.: 454-103 DATE OF DEVIATION: April.30and May 14,1999 AMENDMENT: Blood samples collecteodn Day 8 and Day 22 of thetestwere storedinlabellednonheparinized5 ml borosillicagtleasstesttubespriorto beingrefrigeratedT.he protocolirdeatrd thatblood samples would be placedin labelledheparanizedvacutainers. REASON: Testanimalswere toosmalltoefficientldyraw bloodby syringe.Blood was drawn by decapitatingbirdsand drainingblood intotesttubes.Heparin was not added toblood samples due to a Sponsor requestto separateserum from the blood at WildlifeInternationaLltd. Seperationofsum requirescoagulationofblood samples,which heparinprevents. IMI?ACR: Blood samples were drawn in an efficienmtamer and senan was collectedper Sponsor request Thereforethisdeviatim causedno adversennpact on the study outcome or mterprotation. STUDY DIRECTOR WILDLIFEENTERNATIONAL LTD. MANAGEHENT DATE DATE WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-103 Pa 1ofI DEVIATION TO STUDY PROTOCOL STUDY TIME: PFOS: A DietaryLC50 StudywiththeNorthem Bobwhite PROTOCOLNO.: 454/030999/QLC/SUB454 DEVIATION NO.: 2 SPONSOR- 3M Corporation PROJECT NO.: 454-103 DATE OF DEVIATION: April26- May 14,1999 DEVIATION: On severaloccasionstemperaturews= inadvertentnloyt recordedfor'twoto eight brooderlevelshousingstudybirds. REASON: BiologisotversightWith theexceptionofMay 1,1999,when threebroodert=peratures were notrecordedt,hebrooderpens whmr, temperaturewsere notrecordedwere empty attheb= of aftanoonobservationsB.rooderpen temperaturesaretypicallryecordedduringafternoon observationsB.iologistrsecordingafternoonobservationosn theoccasionswhere temperatures were missed neglectedtorecordtemperaturesforempty brooderlevelsthathoused birdsearlier the same day. IMTACT: Data forthesebrooderlevelson thedays priorto and/orfollowingthemissed temperatures mdicatedthattemperaturewsere withm thea=ptable range.Itisreasonableto expectthatthe brooderswere performingproperlyon the days of the missed observations.Tbereforet,his deviationcausedno adverseimpact on theoutcome ofthestudy. STUDY DIRECTOR WILDLEFE iNTFIbNATidNAL LTD. MANAGEMENT r DATE 0,\Ck DATE WILDLIFE INTERNATIC)NAL, L-rD PaLe I of I DEVIATION TO STUDY PROTOCOL STUDYTITLE: PFOS: A DietaryLC50 Study withtheNorthernBobwhite PROTOCOL NO.: 454/030999/QLC/SUB454 SPONSOR: 3M Corporation DEVIATION NO.: 3 PROJECT NO.: 454-103 DATE OF DEVIATION: April26,1999 DEVIATION: On April26, 1999,Day 4 ofthestudy,theaftemom observationfsorunitB37-1 housing birdsfrom the640 ppm ai treatmentlevelinadvertentldyidnotincluderecordedobservations oftwo birds. REASON: Biologistoversight. U*IPACR None. Observationsfrom themorning of Day 4 were made forallbirdsinPen B37-1, detailinwghich were displayingsignsof toxicityB.ecause observationsoftwo birdswere notnotedit isreasonableto assune theywere normal inappearanceand behavior,but thebiologisftailedtorecord theobservatiorl r STUDY DIRECTOR DATE WILDLIFE Dn7NMAMiiAL LTD. MANAGENffiNT DATE WILDLIFE INTERNATIONAL, LTD PROJECT No.: 454-103 SOP DEVIATION STUDY TITLE: PFOS: A DIETARY LC50 STUDY WITH THE NORTHERN BOBWH[TE-- - PROTOCOL NO'.: 454/030999/QLC/SUB454 SOP NO.: 415 SPONSOR- 3M Corporation DATE OF DEFACTO DEVIATION: May 10-13,.1999 DEVIATION NO.: I PROJECT NO.: 454-103 DEVIATION: Brooder unitB2 1-1 used tohouse controlgroup birdswas below thespecifiedminimum of 95F on may 10-13,1999. This brooderunitranged between 1*and 3* below theminimum and thereareno recordsof the responsibletechnicianor biologistmaidng a thermostatadjustmentto correctthe temperatures.BrooderunitB26-1 used to house birdsfrom the320 ppm a.it.reatmentgroup was 10 below the spedfiedminimum of 95OF on May 10, 1999. On the followingday thebroodertemperature was withinthe specifierdange. REASON: Misadjustm=t of thebrooderunitthermostat. IMEPACR: None. There was no apparenteffecton testbirdbody weight or health. A.P-@ STUDY DIRECTOR DATE WILDLIFE DiTMNATIO)qAL LTD. MANAGEMENT DATE WILDLIF-E INTERNATIONAL, LTD PROJECT NO.: 454-103 ---------- SOP DEVIATION STUDY TITLE: PFOS: A DIETARY LC50 STUDY WITH THE NORTHERN BOBWH[TE PROTOCOL NO.: 454/030999/QLC/SUB454 SOPNO.: 423.IA DEVIATION NO.: 2 SPONSOR-- 3M Corporation PROJECT NO.: 454-103 DATE OF DEFACTO DEVIATION: April22,1999 DEVIANON: On April22,1999,duringdietpreparationon Day 0 ofthetes41.4659g oftestsubstanor, was weighed out forpreparatioonf the 160 ppm a.i.testconcentratiodniet.This amount exceededthetargetamount by 0.099g.The SOP indicatetshatthetestsubstancewillbe weighed towithin0.0005 g ofthetargetamount. REASON: Dieticiaonversight.. IMEPAC17: None. The diff=nce intheamount oftestsubstanceweighedresulteidna lessthan1% change infinaldietarcyoncentration. STUDY DIRECTOR WELDLIFE NVERNABONAL LTD. MANAGENEENT DATE DATE PROTOCOL PFOS: A DIE7ARY LC50 STUDY WITH TBE NORTBERN BOBWHrrE U.S. EnvironmentalP,rotectionAgency Series850 - EcologicalEffer-tTsest Guidelines OPPTS Number 850.2200 FIFRA SubdivisionE, Section71-2 OECD Guideline205 3M Lab RequestNo. U2723 Submittedto 3M Corporation EnvironmentalLaboratory, 935 Bush Avenue St.Paid,Niinnesota55144 WILDLIFE INTERNATIONAL 8598 Commerce Drive Easton,Maryland 21601 (410)822-8600 LTD. March 9, 1999 WILDLIFE INTERNATIONAL LTD -2PFOS: A DEETARY LC50 STUDY WrrH TBE NORTBERN BOBWHNE EM --.Mom SPONSOP, SPONSOR!S REPRESENTATIVE: TESTING FACELRIY: 3M Corporation Environmental Laboratory P.O. Box 33331 St.PaA Minnesota 55133 Ms. Susan A. Beach WildlifeInternationaLltd. 8598 Conunerce Drive Easton,Maryland 21601 STUDY DIRECTOR-- Sean P. Gallagher,SeniorAvian Biologist LABORATORY MANAGE Joann B. Beavers Manager of Avian Toxicology FOR LABORATORY USE ONLY Proposed Dates: Experimental StartDate: ProjectNo.: L45f-410-S Test Concentratic)ns-,.100 .,Lk',D%,o , 16, Experimental Temdmtim Date: Study Roorw. fy\a. C TestSubstanceNo.: 4LIS memce substanceNo. (ifapplicable): 146@G PROTOCOL APPROVAL --ST@U@DCY T D OR DATE RATO SP6NSOR!S REPRFSENtk-TfVE PROTOCOL NO. 454/030999/QLCISUB454 DAICE 3M LAB REQUEST NO. UA. ffra Em WILDLIFE INTERNATIONAL LTD -3- OBJECTIVE The objectiovfethistudyistoevaluatheetoxiciotfya tesstubstantcoethenortherbnobwhite (Colinuvsirginianaudsm)inistetrherdougthhedieftorfivdeays.An LC50 valuewilble calculateidf, possible.- SUAU"RY Groups of tenbobwhite chicks,10-14 days old,willbe feddietscontainingthe.testsubstanceat selectedconcentrationfsorfivedays. A controlgroup thatremves untreateddietwillbe maintained concurrently.The testsubstancewillbe mixed intothe diet usuallyin a geometricseriesof concentrationsI.le exposureperiodwillbe fb.Uowedby a penod of atleastffim days on untreated feed. At theend oftheth=-day observationperiod,halfof the birdsineach treatrnengtroup willbe cuthanizedand liverand blood collectefdor.residuaenalysis.The rmnafiiinbgirdswillbe maintained on basaldietfbr an additional14 days and then willbe euthanizedw,ith liverand blood r-ouected. Throughout thetest,the birdswillbe observedfortoxicologicarlesponses.When possible,an LC50 valuewillbe calculated. MATERL4LS AND MIETHODS 'Me methods, speciesused and routeof administratiodnescribedinthisprotocolare based upon proceduresspecifiedintheU.S. EnvironmentalProtectionAgency Series950 - EcologicalEffectsTest GuidelinesOPPTS Number 850.2200 (1); Section71-2 of the EnvirormentalProtectionAgency's RegistratioGnuidelinesP,esticideAssessment Guidelines,filFR4 Sub&Wsion E, Hazard Evaluation: Wildlifeandaquatic Organisnu (2),AS'IM StandardE857-87, "StandardPracticeFor Conducting SubacuteDietary ToxicityTestsWith Avian Species"(3),and OECD Guideline205, AWan Dietary TojdcityTest(4).In orderto controlbias,birdswillbe assignedto pens by indiscriminatderaw. No otherpotentialsourc4rsof bias are expectedto affectthe resultsof the study. The testsubstancewillbe admh@sured inthe diet.This routeof administratiownas selected because itrepresentsthe most likelyrouteof exposure to avian speciesin the environment. Test Substance Informationon the characterizatiofn test,controlor refc=ce substancesisrequiredby Good LaboratoryPracticeStandards(GLP). The Sponsor isresponsiblfebrprovidingWildlifeInternational Ltd. writtenverificatiotnhatthetestsubstancehas been chamcter@ accordmg to GLPs pnor to rts PROTOCOL NO. 454/030999/QLCISUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD -4- use in the study. If writtenverificationf GLP testsubstancecharacterizatiiosnnot-prQvided.t-o WildlifeInternationaLltd.,itwillbe noted in the compliancestatementof the finalreport. 'Me attachedform IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR (Appmdix I)isto be used to provideinformationnecessaryforGLP compliance. The Sponsor isresponsibleforallinformationrelatedtothetestsubstanceand agreesto accept any unused testsubstanceand/ortestsubstancecontainersrmnaini% atthe end of the study. Treatment gloups Groups oftenbobwhite chicks(fiveper pen) willbe assignedby indiscriminatderaw to each of thetreatmentand controlgroups. Ile sex of each individuawlillnot be determined. A testwill uTessutallyconsistof a geometricseriesof :fivdeietaryconcent-Aons and at leastthreecontrolgroups. concentrationwsillbe selectedafterevaluatingtoxicitydataprovidedby the Sponsor. The highest testconcentrationeed notexceed 5000 ppm. Each group isfedtheappropriatetestor controldietfor fivedays. This willbe followedby a periodofatleastthreedays on untrated feed. Controlbirdswillreceivean amount of the solventor vehiclein theirdietequivalentto the highestamount 'inthe treateddiets.Ifmore gm one studyis conductedsimultaneouslyin the same room, with thesame solventor vehiclea, concurrentcontrolmay be ufilizofdorallstudies. Duration of Test The primary phases ofthetestand theirdurationsare: 1. Acclimation-From receiptor hatchof hatchhw untilthestartof exposure. 2. Exposure - Five days. 3. Post-Wosure observation- 3 days/I7 days. Test B The bobwhite representsan ecologicallsyignificanatnd widelydistributesdpeciesinflw United States.Ile bobwhitehas dmnonstated sensitivityo theeffectsproduced by known toxicchemicals. Moreover, tlusspecieshas proven to be a good laboratorymodel and a largeamount of baselinedata are available. PROTOCOL NO. 454/030999/QLCISUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD -5- Allb.obwhitweilble.fromthesamehatchandwillbe 10-14daysoldattesitnitiatiOonnl.y. thosethatareapparentlhyealthywillbe usedinthetest.Body weightsmay varybetween 10 and 50 grams dependingm theage ofthecohortofbirdsused (e.g1.0 versus14 days).At leastfivetestand ffim controglroupswillbe used.Each treatmenotr controglroupwillcontaintenbirds(twopens of fivebirdseach)vatheachgroup ofbirdsidentifibeyd pennumber and testconcentrationI.ndividual birdswillbe identifibeyd coloredlegbands. Birdswillbe obtainedfrom Top FlightQuailFarm, BelvidereN,J 07823,from anotherreputablseupplieorr obtainedfromffiWcildlifeInternationLatld. productiofnlock.AU experin=talbirdswillbe acclimatetdothecagn andhxftes fromthetimeof hatchor receiputntilinitiatioofnthetest. AnfinalDi AR tea birdswillbe fed a game birdrationfbrmulatetdo WildlifeInternationLatld. specificatio(nTsable1).From thetimeofhatchor receipotfthechicksuntitlheinitiatioofnthetes@ thebirdswillreceivea watersolublveitaminmix viatheirwater.Feedand waterwillbe providedad libitumduringacclimatioanndduringthetest.The watersourcewillbetheTown of Eastonmunicipal watersupply.Feed isanalyzedperiodicalilnyaccordancewithW"zldldIenternationLatld.Standard OperatingProcedures.Ile birdswillreceivneo formofantibiotmiecdicatiodnuringthetest. Specificatiofnosracceptablleevelosf contaminantsingame birdrationforavianspecieshave notbeen establishedI.bm-eareno levelsof contaminantsreasonablyexpectedto be presentinthediet thatareconsideredtointerferweiththepurposeor conductof thestudy. DietP=arati Testdietswillbe preparedatthestartofthetestand a sufficiepnotrtionestknatedforthe5-day exposureperiodwillbe presentetdo thebirds.Afterfivedaysof exposure,allbirdswillreceive unbraw basalration. The testsubstancevnU be mixeddirectliyntothemtionorby dissolvinogr ovendmg thetest substanceinacetoneand/ortablegradecom oilpriortomixingwiththefeed.Ifused,acetonewillbe allowedtovolifilifzreomthedietsduringthemixingprocedure.Tablegradecom oilwillnormallybe incorporateidntothechetata maximum of2% (w/w)ofthefinadliet.Mixingisnormallydone witha Hobartmixrr(ModelNumber AS200'1). PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD -6- AR testsubstancecalculatiownisllbe based on thepurityofthetestsubstancea-s-receiveodr correctedto 100% activeingredienbtased on theinformationprovidedby the Sponsor inAppendix I of thisprotocol. DietSMRIin Samples of the experimentaldietswillbe collectedfor chemicalanalysisto determinethe homogeneity and stabilitoyf the test substancein the avian chet and to venfy/measure test concentrationsA.ll samples willbe placedinuniquelyidentifiepdolypropylenejars. Samples willbe collectedon Day 0, atdietpreparation,to determinehomogeneity, measure test concentrationsa,nd establishDay 0 valuesfor evaluatingstabilitoyver the course of the exposure period. On Day 5, at the end of the exposurephase,samples willbe collectedfrom the feeders containingthe experimentaldietsto assessstabilitoyf the testsubstanceunder testconditions.Ifa separatestudy isconductedwith a differenstpecies(e.g.m,allard),at the same testconcentrations, dietsthatare prepared may be dividedbetween the two studies.Therefore,samples collectedfor homogeneity and verificatiownillbe common to both studies.However, samples collectedfrom the feedersin the study room (e.g.Day 5 samples)willbe uniqueto thatrespectivestudy. The diet sampling scheme forstudieswith fivetestconcentrationisssununmized below. The sampling scheme may be adjustedbased on theactualnumber of concentrationtsested(seePage.2) or the optionof-the Sponsor. PROPOSED ExpeximentalGroup NUMBERS OF SANHIIIES Day 0 Day 5 Control 1 1 Level 1 -Low Concentration 61 2 Level2 2 2 Level3 2 2 Level4 2 2 Level 5 - High Concentration 61 2 19 11 TotalNumber ofSamples 30 'Samples collectefdrom theleftand rightsidesof thetop,middle and bottom Lvm offeedin themixing vesseltodeterminehomogeneity. PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD -7- The above numbers ofsamplesrepresenthosecollectefdrom thetestand do notincludequality control(QC) samplessuch as matrixblanksand fbrtificatipornesparedand analyzedduringthe analyticaclhemistryphase ofthestudy. DietAnal3Lses Samplesof theexperimentadlietsmay be analyzedfollowingcollectionr storedina freezer untiplreparedand/orextractefdoranalysis.Chemicalanalysesofchetswillbe performedby Wildlife InternationLatld. Ile analyticamlethod used willbe basedupon methodologyprovidedby the Sponsorand identifiiendAppendixR. The methodologyused to analyzethetestsampleswillbe documented intheraw dataand summarizedinthefinalreport.Maximum sampleholdingtimes,prior to analysism,ay be specifiebdy theSponsor,and ifspecifiewdi,llbe added to thisprotocolby amendment. Housing and EnvironmentalConditions Test birdswillbe housed inthermostaticalcloyntrollebdroodingpens manufacturedby the Beacon SteelCompany (ModelB735Q) or equivalentE.ach pen measuresapproximatel7y2 X 90 X 23 cm high.The extemal'wausand ceilingosfeachpen areconstructeodfwiremesh and galvanized sheetingF.loorsareof wiremesh. Upon mrtiatioonfthetrA eachbroodingpen willcontainfive birds.Two broodingpenswillbe assignetdoeacht==ent group(10birdstotal)w,hileatleasstix penswillbe assignedtothecontro(latleast30 birdstotal).Each groupwillbe identiiefdby pen number. Duringthetestt,hetemperaturienthebroodingcompartn=t of'thpeen willbe maintainedat approximatel3y8'C. Acceptablerangesforbroodingcompartmenttemperaturewsillbe specifieidn WildlifeInternationLatld.StandardOpemtng Procedures.Broodmg compartmenttemperaturewsill be recordedonceeachday duringthetest.The ambientroom ternperammwillrangebetweenappmximately15-300C. Room temperatureand relativheumiditywillbe recordedtwicedailyduringthetest. A 16hours fight/h8oursdarkphotoperiowdillbe maintaineda,nd theaveragelightintensitpyrovided tothebirdsduringthetestwillbe ddennined. Housing and husbandrypracticewsillbe conductedso as to adhereto theguidelineesstablished by NationalResearchCouncil(5). PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD Observations All birdswillbe observedatleastonce dailyformortalityand toxicologicarlesponses. Nee= Alltestbirdsthattheduringthecourseofthetestand allbirdscuffianizewdillbe subjectedto a grossnecropsy. For thosebirdseuflmnizedon Days 8 and 22 of the tes@ liversand - 2 mL of blood willbe collected.Liverswillbe weighed,placedin individualabelledcontainersand stcr)ed frozen. Blood willbe placed in labelledhepafinizedva@uwners and placed under refrigerationA.ll samples willbe b-amferredtothe Sponsor forresidueanalysis. DigRositionofTest Birds At testterminationt,estbirdswill be cuthanizedby using carbon dioxidegas, cervical dislocatioonr otherappropriatemethods. The method used willbe documented in the raw data. AU carcasseswillbe storedfrozenpriorto shipmenttotheSponsor. StatisticaClalculations The concentrationresponsedata obtainedfrom the testwillbe evaluatedand, ifpossible,an LC50 value and 95% confidencelimitswillbe calculated.When the mortalitydata facilitates statisticaanlalyses,one of 6= methods willbe used to calculataen LC50 value. The datawillbe analyzed,in orderofpreference,by probftanalysist,hemoving averagemethod, or thebinomialprobabilitymethod (6,7, 8,9). The choiceofmethod forcalculatintghe LC50 valuewillbe based upon the mortalitypatternobserved.When possiblea, no mortalitylevelwillbe determinedand reported. RECORDS TO BE MAINTAINED Records to be mamtmned fordata generatedatWil&fe bitamtional Ltd.willinclude,but not be limitedto: 1. A copy ofthe signedpmtocol.. 2. Identificatiaond characterizatioonfthetestsubstance,ifprovidedby theSponsor. 3. Dates ofinitiatiaonnd tm@on 4. Animal history. ofthetest. 5. Husbandry and environmentalconditions. 6. Concentrationcalculationasnd recordsof dietpreparation. PROTOCOL NO. 454/030999/QLC/SUB454 3M IAB REQUEST NO. U2723 WILDLIFE INTERNATIC)NAL LTD owl P.7,: -9- 7. Body weight measurements. 8. Feed consumption measurements. 9. Daily observations. 10. Statisticcaallculations. 11. Analyticalchemistrymethods,resultsand chromatograms, ifapplicable. 12. A copy ofthefinalreport. FINAL REPORT A reportofthe resultsof the studywillbe prepared by WildlifeInternationaLlt,d. 'ne report willinclude,butnotbe limitedto,thefollowing: I. Name and addressofthefdcilitpyerfoiuiingthestudy. 2. Dates on which thestudywas initiateadnd completed. itisthe responsibiliotfythe Sponsor to providethe ffiw date thatdata are recordedfor 6wastry, pathologyand/or supporting evaluationsthatmay be generatedat otherlaboratories. 3. A statementof compliancesignedby the Study Directoraddressingany exceptionsto Good LaboratoryPracticeStandards. 4. Objectivesand proceduresstatedinthe approved protocol includingany'changesintheoriginal protocol. 5- Statisticamlethods employed foranalyzingthe data,when applicable.The calculatedLC50 value,95 percentconfidencelimitss,lopeof the concent-dfion-responsceurve,the resultsof the goodness-of-fittest(e.g.X2 test),and a descriptionof statisticamlethods used. The same statistiwcisU be providedforpositivecontrols(when used). 60 The test,controland referencesubstancesidentifiebdy name, chemical abstractsnumber or code number, strengthp,urity,and compositionor otherappropriatecharacteristicisf,provided by the Sponsor, 7. Stabilitaynd, when relevanto the conduct of the study,the solubilitoyf the test,controland referencesubstancesunder theconditionsof admwstrafion, ifprovidedby theSponsor. 8. A descriptioonf the testsystem used. where applicablet,he finalreportshallincludethe number of animal used, body weight range,sourceof supply,species(includingscientific name), age,and procedureused foridentification. 9. A descriptioonfthe dosage,dosage regimen,routeof administrationa,nd duration. 10. A descriptioonfthemethods used,includingbut notlimitedto: PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD 10- a) Detaileddescriptionof the basal dic@,includingsource,diluents(if used), and . supplements(ifused).A nutrientanalysisofthedietwillbe includedinthetestreport. b). Tllenumber of concentrationussed, nominal and (where reqwred) measured d@dary cOnce4tratioonf testsubstancein each leve4 assay method used to daennine actual concentrationsn,umber ofbirdsPer concentrationand forcontrol,and names of toxicants used forpositivecontrols(ifapplicable)R.esultsof range-findintgest(ifconducted). c) Acclimationproceduresand methods of assigningbirdsto testpens. d) Frequency@durationand methods of observation.Descriptionof signsof intoxicatioannd otherabnormal behavior,mcluchng time of onse@ duration,seventy (includingdeath), and numbers affectedinthedfferentdietaryconcentrationasnd controlseach day of the testperiod. e) Descriptionof housing conditions,includingtype, size and materialof pen, pen tmnpemftm, approxinatetestmom humidity,pbotoperiodand lightinigntensity. f) Average body weightsforbirdsin each pen at the beginningof the test,the end of the exposure periodand end of the test. g) Estimatedfood consumptionfortheexposure periodand forthepostexposurepcrlod. 11. A descriptioonf allcircumstancesthatmay have affectedthe qual4 or integritoyf the data. Anything unusual about the test,any deviaton fi-orfnlme procedures,and any otherrelevant information. 12. The name ofthe Study Director,the names of otherscientistosrprofessionalsa,nd the names of allsupervisorypersonnel,involvedinthe study. 13. A descriptioonf the t-dmfommfions,calculationso,r operationsperformed on the data, a summary and analysisofthedata,and a statementof theconclusionsdrawn from the analysis. 14. The sigaedand datedreportsof each of the individualscientistosr otherprofessionalisnvolved inthe study,ifapplicable. 15. ne locationwhere allspecimens,raw data,and the finalreportareto be stored. 16. A statementpreparedby the QualityAssurance Unit listintghe datesthatstudyinspectionsand auditswere made and thedatesof any findingsreportedto theStudy Directorand Management. 17. Ifitisnecessaryto make correctionosr additionsto a finalreportafterithas been accepted, such changes shallbe intheform of amendment by theStudy Director.Ilieamendment should clearlyidentiftyhepartofthefinalreportfliaitsbeingadded toor com=ed and the reasonsfor the,rontzbon or addrtim. Amendments shallbe signedand datedby the Study Director. PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 L4 WILDLIFE INTERNATIONAL LTD . 14. CHANGING OF PROTOCOL Planned changes totheprotocolwillbe intheform of writtenamendments signedby the Study Directorand the Sponsoes Representative.Amendments willbe consideredas partofthe protocoland willbe attachedto thefinalprotocol.Any otherchangeswillbe in theform of writtendmations signedby the Study Directorand filedwith the raw data. Allchangesto theprotocolwillbe indicated inthe finalreport. GOOD LABORATORY PRACTICES This studywillbe conducted.in accordancewith Good LaboratoryPracticeStandardsfor EPA (40 CFR Part 160 and/orPart792);OECD Principleosf Cmod LaboratoryPractice.(OCDE/GD (92) 32, EnvirorunentMonograph No. 45); and Japan MAFF (59 NohSan, NotificatioNno. 3850, AgriculturaPlroductionBureau). Each study conductedby WildlifeInternationLatld. is routinely mmninw by the WildlifeInternationLatld. QualityAssurance Unit fbr compliancewith Good Laboratory PracticesS,tandard OperatingProceduresand the specifiedproU=I. A statementof compliance with Good LaboratoryPracticeswillbe preparedforallportionsofthestudy conducted by WildlifeInternationaLltd. The Sponsor willbe responsiblefor compliancewith Good Laboratory Practicesfor proceduresperformed by otherlaboratorie(se.g.r,esidueanalysesor pathology).Raw dataforallwork Performed atWildlifeInternationaLltd.and a copy of thefinalreportwillbe filedby projectnumber inarcluveslocatedon theWildlifeInternationaLltd.siteor atan alternativleocationto be specifiedinthefinalreport. PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD -12'REFERENCES 14 I U.S. Environmental ProtectionAgency. 1996. Series850- F=Iogical EffectsTest Guidelines (draft), OPPTS Number 8.50.2200: AWan Dietary Toxicity Test. 2 U.S. Environmental Protection Agency. 1982. Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, subsection 71-2, EnvironmentalProtectionAgency, Officeof PesticidPerograms. Washington, D.C. 3 American Societyfor Testing and Materials. 1987. StandardPracticefor Conducting SubacuteDietaryToxicityTestswith Avian Species.ASTM StandardE857-87. Annual Book of ASTM Standards,Vol. 11.04. PhiladelphiaP,A. 4 Organization for Economic Cooperation and Development 1984. Awan Dietary Toxicity Test. OECD GuidelineforTestingof Chemicals. Guideline205. Paris. 5 National Research Council. 1996. Guide for the Care and Use of Laboratory Animals. Washington, DC. NationalAcademy Press. 125 pp. 6 FinneyD.J. 1971. StatisticaMlethods in BiologicalAssay, 2nd ed.,GriffinPress,London. 7 Thompson,W.P.. 1947. BacteriologicalReWews,7 Vol 2,No. 2: 115-145. 8 Stephan, C E. 1977. Methods for Calculating an LC50. Pages 65-84 In Aquatic Toxicology and Hazard Evaluatiom, American Society for Testing and Materials. Pub. No. STP 634. PhiladelphiaP,A. 9 Stephan, C. E. 1978. U.S. EPA, EnvironmentalResearchLaboratory,Duluth,MN. Personal Communication. PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD -13- TABLE 1: DMT FORMULANON WILDLIFE PQTERNATIONAL, *LTD.GAME BIRD RAnoN' INGREDIENTS Fine Com Meal Soy Bean MeaL 48% Protein Wheat Mdds ProteinBase Agway Sp=aL 600/oProtein AlfalfaMeaL 20% Protein Dried-Whey Ground Limestone Eastman CalPhos MethioninePremix + Liquid Vitaminand MineralPremix (seebelow) GL FerM (FM=tCO)2 SaltIodized Total PERCENT (0/.) 44.83 30.65 6.50 6.00 4.00 -3.00 2.50 0.90 0.60 0.35 0.32 0.25 -0.10 100.00 VITANEN AND NGNERAL PRENFIX AMOLTNT ADDED PER TON ViLu3inD3 Vitamin A Riboflavin Niacin PantothenicAcid ViLvninB)2 FolicAcid Biotin Pyridoxine Thiamine VitaminE ViLwninK (MenadioneDimethyl"rimidinoBlimffite) Manganese E= Copper Iodine iron Selenium 2,000,000I.C.U. 7,000,000l.U 6 grams 40 grams 10 gram 8 mgs 600 mgs 64 mgs 1.2grams 1.2grams 20,000LU 5.8grams 102 grams 47 grams 6.8grams 1.5gmms 51 grams 182 mgs The guaranteedanalysisisa minimum of 27% pmtein,a minimum of 2.5% crude fatand a maximum of 5% crudefibu. 2 FerMCM=on By_ftdl= (SourceofUMdentLfiedGMWAL Factors). PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST.NO. U2723 WIL.DL.IFE INTERNATIONAL- LTD -14- APPENDIXI IDEq-MCAnON OF TEST SUBSTANCE BY SPONSOR To be CmVlded by Spmsor 1. TestSubstanceIdentity'(natronbee usedinthereport)P:FOS (PerfluoroortaSnuelfonicAcid PotassiumSalt ReferenceStandard(ifapplicableA)n:alyticaSltandard- N/A IntemalStandarcL-1.1.2.2Rl-LI-LPHerfluorooctanSeulfonicAcid TestSubstanceSample Code orBatchNumber Lot 217 TestSubstancePurity(0/AoctiveIngedient): 98.9 ExpiratioDnate: 2008 IL TestSubstar= Have thek gity andcmvos*d*LMor otherdwad=Lqtics YA@ch.appr=odpv.mdryw te@subs== andrcfin= smodardbem dewuni6idpnortiDtussem ft sft*inaccordanwciethGLP Sumdar&? Yes x No M TestSubs=m Swmge Cmditions Pkm mdlcatrt.herecmm=ded storagcrm,ddionsatVadbk hhm&ong lAi Ambicat Has thestabfliotfythetd substa= underth= wrage conditiom bem detmminedinaccmdar= withGLP Sumdards? 003erpmfinensttab&yin@@- -Yes x No IV. TestConcent-atim: Ad-justtestOMOMbafiOn to IOOP/-ai x baiedupm thepmty (0/og)rm above. Do notad4t = cmcmfta6m to1000/o .a.LTestft nia=W AS IS. V. TcDdcithy*nration. MmmoliarL RAII.DSO 251ffmft MmmLD50 N/A Aquafic: kvmtebratTcoxicitMyC4,C50) DgpMA ma=a: 27ma DagMa magna: 50 mg! FishTwdcity0,C50) RainbpwTrout:II mpJL FatheadNlmow 38 nwJL Od= Tmcrtylnffinnat(immckxhpfgMm ofchmme andsubcbm= t:su): PleaseseeMSDS PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 KR WILDLIFE INTERNATIC)NAL LTD -15- -APPENDIX H MR Method OutlinefortheDeiemiinatioonfPFOS inAvian Feed 91 1 Preparematrixfortificatsiaomnplesinthedesiredavianfeedstock usingthedrymix technique. 2. Weigh 10 g samplesofthematrixblankand matrixfortif:ic-faeteidosnamplesintoa weigh boatand transfer 99 to 16 oz.FrenchsquareglassbottlesR.ecordweights. 3.. For each weighed sample,measure 100 mLs of methanolwitha graduatedcylinderand transfemrto the French squarebattle. 4. Sealbottlewsithscrew capsand placeon shakertable.AUow.to shakefora minimum of 30 minutes. 5. Vacuum filtewrithqualitatifvieltepraperand nnseretainefdeed3 timeswithmethanolintofiltrate. 6. Transferfilt-aitnetoa 200-mL volumetrifclaskand bringtovolumewithmethanol. 7. Transfer5 mLs of 200-mL solutioinntoa 10-mL volmnetrifclaskpartially-fiwhietdh 501/omeO=ol:50% waterdilutiosnolvenctontainin0g.05% v/vformicacidand 100 gg a.iJmL 4BPFOS internasltandard. Bringtovolume withdilutiosnolvent. S. Mix and t=fer approximatelIymL ofthe10-mL solutioinntoa syringeand filtetrhrougha 0.2gm filter intoan EPLC autosamplervial. 9. Ampulate and submitsampleforLCMS analysis. PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723