Document 8VYn3DqGz13eB6beb1eEGrxDa

PFOS: A D DIETARY ACUTE MALLARD DUCK STUDY TEST SUBSTANCE Identity:Perfluorooctanesulfonatmea;y alsobe referredto as PFOS or FC-95. (I-Octanesulfonicacid,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-p,otassium salt,C.AS # 2795-39-3) Remarks: The testsubstance isa whitepowder. Sample was taken from 3M lotnumber 217. Sample was storedunder ambient conditions priortotestingP.uritydetermined to be 90.49% by LC/MS, 'H-HMR, 19FNMR and elementalanalyses techniques. METHOD Method: OPPTS 850.2200, OECD 205, and FIFRA SubdivisionE. Section 71-2 Type: Dietaryacute GLP: Yes Year completed: 2000 Species:Anas platyrhynchos Supplier:WhistlingWings, Inc.H,anover,IL Analyticalmonitoring:PFOS measured on Day 0 forhomogeneity in feed and verificatioann,d Day 5 forstability. Test phases: Acclimation- 9 days Exposure - 5 days Post-exposureobservation- 3 or 17 days Statisticamlethods: LC5o valuescalculatedby probit analysisusingthe 6omputer softwareof C.E.Stephan. Body weight data were compared by Dunnett'stestusing TOXSTAT software.No statistical analyseswere appliedtofeed consumption data. Test bird age: 10 days Pretreatment: None Test conditions: Housing and environmental conditions: Indoorsinbatteriesof thermostaticallcyontrollebdroodingpens. Floorspace ofeach pen measured approximately62 X 90 cm. Ceilingheightwas approximately25.5 cm. Externalwalls,ceilingsand floorswere constructedofgalvanizedsteelwireand sheeting. IdentificationE:ach group of birdsidentifiebdy pen number and testconcentration.Individualisdentifiebdy wing bands. Number of replicates:Six forcontrolst,'wo foreach treatment group Number of ducks per replicate:five Number of concentrations:Eightplusa negativecontrol Feed and water: Game birdrationformulatedas below,water from the town ofEaston publicwater supply. Both providedad libitum duringacclimationand testing. Game Bird Ration Ingredients Percent FineCom Meal 44.83 Soy Bean Meal,48% Protein 30.65 Wheat Midds 6.50 ProteinBass 6.00 Agway Special,60% Protein 4.00 AlfalfMaeal,20% Protein 3.00 DriedWhey 2.50 Ground Limestone 0.90 Eastman CalPhTs- 0.60 MothioninePremix + Liquid 0.35 Vitaminand MineralPremix(seebelow) 0.32 GL Form (Formatco@ 0.25 SaltIodized 0.10 'Fermentatiobny-products(sourceofunidentifigerdowthfactors) Vitamin and MineralPremix Vitaminor Mineral VitaminD3 VitaminA F;riboflavin Niacin PantothenicAcid VitaminB12 FolicAcid Biobn Pyridoxine Thiamine VitaminE VitaminK (Menadionedimethylpyrimidibnioslulft) Manganese Zinc Copper Iodine Iron Selenium Amount Per Ton 2,000,000I.C.U. 7,000,000l.U. 6 grams 40 grams 10 grams Bmg* 600 mg 64 mg 1.2grams 1.2grams 20.000 l.U. 6.8grams 102 grams 47 grams 6.8grams 1.5grams 51 grams 182 grams Prophylaxis:None Brooding compartment mean temperature:38 + 20C Ambientroom mean temperatur2e5:.2+ 0.70C Averagerelatihvuemidity5:3+ 18% PhotoperioSdi:xteehnourslighp-etrd-ay Lightinfgl:uorescleingthwthsichclosealpyproximantoeon-day sunlighta;verage approximately207 lux. Test dietpreparation:Testsubstancemixed directliyntotherationby means ofa Hobartmixer.No carriewras used. Dietsampling: Homogeneity ofthetestsubstanceinthedietevaluated by collectinsgixsamples from the9.1ppm and sixfrom thelowestand highestconcentrationS.amples collectefdromthetop,middle,and bottom oftheleftand dghtsectionsofthe mixingvessel.These samples alsoservedas,.thveedficatiosnamples fortheseconcentrations.Two verificatisoanmples from theremainingconcentrationasnd one from the controlwere collecteadt preparatioonn Day 0. Stabilitsyamples were collecteadttheend ofthe exposurepedod (Day 5)fromthe control(one sample)and each treatmentgroup(twosamples each). RESULTS Nominal concentrations:Bk control9,.1,18.3,36.6,73.2,146,293, 586, and 1171 ppm Measured concentrations:<LOQ, 9.8,19.5,40.2,74.5,174, 291, 537, and 1196 ppm Element value:DietaryLC5o = 628 (448- 958)ppm No mortalitcyoncentration 146 ppm NOEC (bodyweightgain) 36.6ppm Allelement valuesbased on nominalconcentrations AnalyticalMethodology: Dietsamples were extractedwithmethanol. Analyses oftestsolutionwsere performedatWildlifIenternationLatld. usinghighperformanceliquicdhromatographywithmass spectrometdc detection(HPLC/MS). When determinintgheconcentratioonfthe test substance inthetestsolutionst,hesame and most prominentpeak response forperfluorooctanesulfonawtaes used. No attemptwas made toquantifyon thebasisofindividuailsomericcomponents. The LOQ (limit ofquantitatiowna)s 1.15ppm inthisstudy.The mean percentrecoveryof matrixfortificatiaonnaslyzedconcurrentldyuringsample analysiswas 94.7.Samples collectefdordeterminatioonfhomogeneity indietranged from 102 - 108% ofnominal.Samples collectefdorverificatiionndiethad measured valuesfrom 92 to 119% ofnomi-n-@aM'ela.sured valuesfor ambientstabilistaymples takenatDay 5 rangedfrom 94 - 130% of nominal. Summary ofanalyticaclhemistrydata: Homogeneity inAvian Diet Nominal Test Concentration, PPM Measured Values atDay 0. ppm Mean Measured Percentof Concentration, Nominal PPM 9.1 9.52,9.70,9.79,8.09,10.9,10.5 9.8 108 18.3 18.5,23.4,18.3,17.3,19.41.9.9 19.6 107 1171 1239,1221.1118,1301,1163,1133 1196 102 VerificatioInnAvian Diet Nominal Test Concentration, PPM Measured DuplicateConcentrationsatDay 0. ppm Mean Measured Percentof Concentration, Nominal PPM Negabve Control 36.6 < LOO 46.7,34.6 - - 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 StabilitiynAvian-Diet Day 0 Mean Measured Concentration, PPM Measured DuplicateConcentrationsatDay 5,ppm NegafiveControl < LOQ Mew Measured Mean Concentration, Percentof PPM Day 0 - 9.8 12.3,11.0 11.7 119 19.5 18.2,19.7 19.0 97 40.2 47.9,56.8 52.4 130 74.5 77.6,77.9 77.8 104 174 167,160 1.64 94.3 291 297,293 295 101 537 552,530 541 101 1196 1160,1122 1136 95 Biologicaolbservations Survivaland clinicoablservationsN:o mortalitoicecsurreidnthe controlgroup,and allbirdswere normal inappearance and behaviorthroughoutthe test.The firsdteaths occurredon day 4 in the 1171 ppm treatment.MortalitoyccurredthroughDay 8 inall dose groups > 293 ppm withsome ofthedeaths being duringthe post-exposureperiod.There were no treatment-relatemdortalities or overtsignsoftoxicitayt concentration<s 146 ppm. Birdsatall concentrations> 293 ppm displayedsign@-oftoxicitiyncluding reduced reactionto stimul(isoundand motion),lossof coordinationr,uffleadppearance, lethargyand lower limb weakness. Birdsatthe 1171 ppm levelalsodisplayedprostrate posture,depressionand convulsionsthroughDay 8. Recovery with normal appearance and behaviorwas noted from Day 9 through testtermination Body weight gain:When compared tothe controlgroup,there were no apparenttreatmentrelatedeffectson body weight among the birdsinconcentrations< 36.6 ppm. During the Day 8-15 and Day 15-22 post-exposureperiods,body weight gainappeared comparable among allgroups.There was a statisticaslilgynificant (p < 0.05)reductioninweightgainatthe9.1 ppm levelforthe Day 0-5 and Day 5-8 periods.However, differencefsrom the control group atthe 9.1 ppm levelappear to be due toa lowermean Day 0 body weight forthe 9.1 ppm levela,nd were notdose responsive. Therefore,these differencewsere not consideredtreatmentrelated. Marked, treatment-relatedc,oncentrationresponsiveeffectson body weight was noted inconcentrations> 73.2 ppm forDays 0-5; Day 5-8 post-exposureweightgaincontinuedto be reduced at concentrations> 293 ppm. Feed Cons *um6-tionW:hen compared tothe controlgroup,there was a marked reductioninfeed consumption inthetreatment groups > 293 ppm throughoutthe study. Gross gecropsy: Allbirdsthatdiedduringthe study,halfof those survivingat Day 8 and the restattestterminationwere subjected toa gross necropsy. Necropsy resultsforbirdsfound dead were similari,ncludingthinconditionl,ossofmuscle mass, alteredspleen color,empty crops,and empty gastrointestintarlactsT.hese necropsy findingswere consideredto be treatmentrelated. Percent Cumulative Mortal! Nominal Concentration, PPM Negative Control Day I 0 Day 2 0 Day 3 0 Day 4 0 Day 5 0 9.1 0 0 0 0 0 18.3 0 0 0 0 0 36.6 o@ 0 0 0 0 73.2 0 0 0 0 0 146 0 0 0 0 0 293 0 0 0 0 0 586 0 0 0 0 10 1171 f 0 0 0 20 60 *No mortalitieosccurredinany of thetreatmentlevelsfrom Day 8 to Day 22 Day 6 Day 7 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 10 20 30 80 90 Day 8* 0 0 0 0 0 20 30 90 Body Weight (grams) Exposure Period Recovery Period Nominal Mom Body Concentration, Weight, PPM Day 0 Mean Body Weight Change Day 0-5 Mean Body Weight Change Day 5-8 Mean Body Weight Change Day 8-15 Mean Body Weight Change Day 15-22 Mean Total Body Weight Change Day 8-22 Mean Body Weight Day 22 NegativeControl 135 1 1.44 101 230 183 413 1 823 9.1 119 108 91 230 198 -427 773 18.3 146 131 100 241 186 427 811 36.6 147 128 100 243 208 451 828 73.2 143 117 82 216 203 418 782 146 143 100 89 232 124 356 688 293 129 32 57 256 234 490 701 586 144 -6 36 221 219 439 1 613 1171 124171 - @7 1531 198 251 449 634 Mean Average Feed Consumption Nominal Granis Grams Grams Grams Concentration, FoodfBird/DayFood/Bird/DayFood/Bird/DayFood/BirdfDay PPM Days 0-5 Days 6-8 Days -15 Days 16-22 NegafiveControl 92 125 171 ISO 9.1 73 117 172 1 198 18.3 91 132 186 204 36.6 94 125 165 179 73.2 77 101 148 173 146 105 159 159 164 293 44 63 109 132 586 36 55 1 114 143 1171 22 25 106 154 Gross PathologicalObservations from Birds thatDied In Study Finding 293 ppm 586 ppm 1171ppm N N=2 N=3 =9 Crop empty 0 2 7 Emaciated 1 11 4 G.I.Tract,primarileympty I I I Gizzardcontentsbilestained 0 2 4 Gizzard,empty 0 0 1 Intestincaolntents,blackand tar4ike 0 0 1 Keel prominent 0 0 21 Kidneys,pale 0 0 1 Loss ofmuscle mass 2 2 6 Spleen,gray 0 0 1 Spleen,smalland pale Spleen,pale 11 1 2 0 2 3 Thin 1 1 4 CONCLUSIONS The dietaryLC50 value forMallardDuck exposed to PFOS was determinedtobe 628 ppm witha 95% confidenceintervaolf448 to 958 ppm. The slopeofthe concentration-responsceurvewas 3.67 and the chi-squarevalue was 2.13. The no mortalityconcentrationwas 146 ppm. Based upon reductionsinbody weight gainat the 73.2 ppm test concentrationt,he no observed effectconcentrationwas 36.6 ppm. Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331,,St. Paul,Minnesota,55133 DATA QUALITY ReliabilityK:limischranking= 1 REFERENCES This studywas conducted at WildlifIenternationaLltd.,Easton, MD atthe requestofthe 3M Company. OTHER Last changed: 5/1/00 PFOS: A DIETARY LC50 STUDY WITH THE MALLARD WILDLEFE INTERNATIONAL LTD. PROJECT NO.: 454-102 3M REQUEST NO. U2723 U.S. EnvironmentalProtectioAngency Series850 - EcologicalEffectsTestGuidelines(draft) OPPTS Number.850.2200 FIFRA SubdivisioEn, Section71-2 OECD Guideline205 AUTHORS: Sean P. Gallagher Courtney S. Casey Joann B. Beavers Raymond L. Van Hoven STUDY INITIATION: April21, 1999 STUDY COMPLETION: April26,2000 SUBMITTED TO 3M Corporation EnvironmentalLaboratory 935 Bush Avenue St.Paul,Minnesota55144 WildlifeInternati*onalL,td. 8598 Commerce Drive Easton,Maryland 21601 (410)822-8600. Page I of 65 WILDLIFE INTERNATIONAL, LTD -2- PROJECT NO.: 454-102 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A DietaryLC50 StudywiththeMallard WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-102 STUDY COMPLETION: .-Apri2l6,2000 This studywas conducted incompliance with Good LaboratoryPracticeStandards as publishedby the U.S. EnvironmentalProtectionAgency, 40 CFR Part 160 and 792, 17 August 1989; OECD Principleosf Good LaboratoryPractice(,OCDE/GD(92) 32, EnvironmentMonograph No. 45, Paris, 1992);and Japan MAFF, 59 NohSan, NotificatioNno. 3850, AgriculturaPlroductionBureau, 10 August 1984 with thefollowingexception: 'Me testsubstancewas not characterizeidn accordancewith fullGLP compliance;however, the characterizatiownas performed accordingto 3M Standard OperatingProceduresand Methods, and all raw data are being maintainedin the 3M archives.'Me testsubstanceisbeing recharactetizeidn accordancewith GLP. The stabilitoyf thetestsubstanceand referencestandardunder conditionsof storageatthetestsite was notdeterminedinaccordancewith Good LaboratoryPracticeStandards. STUDY DIRECTOR: Sean P. Gallagher I SeniorBiologistA,vian Toxicology SPONSOR'S REPRESENTATTVE DATE a,_ ()-- /@"A Ms. Susan A. Beach DATE- LI@726LV WILDLIFE INTERNATIONAL, LTD -3- PROJECT NO.: 454-102 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good LaboratoryPracticeStandardsas published by the U.S. EnvironmentalProtectionAgency, 40 CFR Part 160 & 792, 17 August 1989; OECD Principleosf Good 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 findingswere reportedto theStudy Directorand Laboratory Management were as follows: ACTIVITY DATE CONDUCTED Matrix Fortification, Test Substance Prep., AnalyticalSampling April22, 1999 Feed Consumption and AnalyticalSampling April27, 1999 Necropsy, Blood Collection, LiverWeights and Bile Collection May 14, 1999 AnalyticalData and DraftReport July7, 8,and 9, 1999 BiologicalData and DraftReport Aug. 30, 3 1, Sept. 1, 1999 FinalReport April 18,2000 DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT April22, 1999 April27, 1999 April23, 1999 May 4, 1999 May 14,1999 May 21, 1999 July9, 1999 July 16, 1999 September 1, 1999 April 18,2000 September 3. 1999 April19,2000 axLooLn o@.CO&MOLN Susan L. Coleman SeniorQualityAssurance Representative DATE 4-iq-oo WILDLIFE INTERNATIONAL, LTD -4- REPORT APPROVAL PROJECT NO.: 454-102 SPONSOR: 3M Corporation TITLE: PFOS: A DietaryLC50 Study withthe Maud WU,DLIFE E@TERNATIONAL LTD. PROJECT NO.: 454-102 3M LAB REQUEST NO.: U2723 STUDY DIRECTOR: Sean P. Gallagher I SeniorBiologistA,vian Toxicology CEEMISTRY PRINCIPAL INVESTIGATOR: 0 Date RaWont L.'*'VaHnoven, Ph.D ScientistA,nalyticalChemistry REPORT APPROVED BY: Jo @eavers@@@@@@ Director,Avian Toxicology Date 2- /0 Date WillardB. Nixon,Ph.f). Manager, AnalyticalChemistry Date WILDLIFE INTERNATIONAL, LTD -5- PROJECT NO.: 454-102 TABLE OF CONTENTS TITLE PAGE ................................................................P.a.g.e...I................... GOOD LABORATORY QUALITY ASSURANCE PRACTICE COMPLIANCE STATEMENT .................P.a.g.e..2....... STATEMENT .........................................P.a.g.e..3.................... REPORT APPROVAL .........................................................P.a.g.e..4................... TABLE OF CONTENTS .......................................................P.a.g.e..5.................... Tables and Appendices ........................................................P.a.g.e..6.................... SUMMARY .................................................................P.a.g.e...7................... INTRODUCTION ............................................................P.a.g.e...8................... OBJECTIVE .................................................................P.a.ge...8................... MATERIALS AND METHODS .................................................Pa.g.e...8.................... Test Substance...............................................................Pa.g.e...8................... Treatment Groups ............................................................P.a.g.e..9................... Durationof theTest..........................................................P.a.g.e..9.................... Test Birds..................................................................P.a.g.e...9................... AninW Diet......... Page 10 ............*.*.*.*."..*.".*.'..*...*.*.**'**'*."."."*. DietPreparation.............................................................P.a.g.e..1.0................... DietSampling ...............................................................P.a.g.e..1.0................... AnalyticalMethod ...........................................................P.a.g.e..1.0................... Housing and EnvirorunentalConditions...........................................Pa.g.e...1.3................... Observations...................................... Page 13 AninW Body Weights/Feed Consumption .........................................P.a.g.e..1.4................... Gross Necropsy .............................................................P.a.g.e..1.4................... StatisticaAlnalyses...........................................................P.a.g.e..14................... RESULTS ...................................................................P.a.g.e..1.4.................. Diet Analysis................................................................P.a.g.e..1.4.................. Mortalitieasnd ClinicalObservations............................................P.a.g.e..1.5................... Body Weight and Feed Consumption .............................................P.a.g.e..1.6.................. Gross Necropsy ..............................................................P.ag.e..1.7................... CONCLUSION ...............................................................Pa.g.e..1.8................... REFERENCES ...............................................................P.ag.e..1.9................... WILDLIFE INTERNATIONAL, LTD -6- TABLE OF CONTENTS - continued- TABLES AND APPENDICES PROJECT NO.: 454-102 TABLE 1: TABLE 2: TABLE3: TABLE 4: TABLE5: CumulativeMortalityfroma MallardAcute DietaryToxicity Study withPFOS ...............................................P.a.g.e.2.0.................. Mean Body Weight (g)froma MallardAcuteDietaryToxicity Study withPFOS ...............................................P.a.g.e.2.1.................. Mean Feed Consumption (g/bird/dafyr)om a Mallard Acute DietaryToxicityStudywithPFOS ............................P.a.g.e.2.3.................. Gross PathologicaOlbservationsfrom a MallardAcute DietaryToxicityStudywithPFOS .................................P.a.g.e..2.5................. CumulativeMortality(EstimatedCumulativeDose,mg/kg) from a MallardAcute DietaryToxicityStudywithPFOS .....................P.a.g.e.2.6............ APPENDIX 1: CertificatoefAnalysis.......................................P.a.g.e..2.7................. APPENDIX H: DietFormulation............................................P.a.g.e.2.8.................. APPENDIX III: AnalyticalMethods and Results................................P.a.g.e..29.................. APPENDIX IV: DietPreparatio.n...........................................P.a.g.e..4.3................. APPENDIX V: CumulativeMortalityby Pen from a Mallard Acute DietaryToxicityStudywithPFOS ........................P.a.g.e..4.4.............. APPENDIX VI: IndividuaBlody Weights(g)from a Mallard Acute DietaryToxicityStudyvath PFOS ........................P.a.g.e..4.5.............. APPENDIX VII: Feed Consumption (gtbird/dayb)y Pen from a MallardAcute DietaryToxicityStudywithPFOS ..................Pa.g.e..6.0........ APPENDIX VIII: Changes toProtocol.........................................P.a.g.e..6.4................. APPENDIX IX: PersonnelInvolvedinthe Study................................P.a.g.e..65.................. WILDLIFE INTERNATIONAL, LTD -7- PROJECT NO.: 454-102 SUMMARY SPONSOR: 3M Corporation TEST SUBSTANCE: PFOS WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-102 STUDY: PFOS: A DietaryLC50 StudywiththeMallard RESULTS: The dietaryLC50 valueforMallardsexposedto PFOS was determinedto be 628 ppm a.i.witha 95% confidenceintervaolf 448 ppm a.i.to 958 ppm a.i. The slopeof the concentration-responcsuervewas 3.67 and thechisquarevaluewas 2.13. The no mortalityconcentratiownas 146 ppm a.i. Based upon reductionms body weightgain at the 73.2 ppm a.i.test concentrationt,heno-observed-effeccotncentratiownas 36.6ppm a.i. TEST DATES: Hatch - April13,1999 Acclimation- April14-22,1999 ExperimentalStart-April22, 1999 ExperimentalTermination- NOMINAL TEST CONCENTRATIONS: 0,9.1,18.3,36.6,73.2,146,293,58a6n,d 1171ppma.i. TEST ANIMALS: Mallard(Anasplatyrhynchos) AGE TEST ANIMALS: 10 days of age attestinitiation SOURCE TEST ANIMALS: WhistlingWings,Inc. PO Box 1-A 113 Washington St. Hanover,IL 61041-3512 STUDY COMPLETION: April26,2000 WILDLIFE INTERNATIONAL, LTD -8- PROJECT NO.: 454-102 INTRODUCTION This studywas conductedby WildlifeInternationLatld.for3M Corporationat the Wildlife InternationLatld.toxicologyfacilitiyn Easton,Maryland. The in4ifeportionof the testwas conductedfrom April22, 1999 to May 14, 1999. Raw datageneratedatWildlifeInternationLatld. and a copy of the fmal reportare filedunder ProjectNumber 454-102 inarchiveslocatedon the WildlifeInternationLatld.site. OBJECTIVE Ile objectiveof thisstudy was to evaluatethe toxicityof a testsubstanceto the mallard(Anas platyrhynchos)administeretdhroughthe dietfor fivedays. An LC50 valuewillbe calculatedi,f possible. MATERIALS AND METHODS The methods used in conductingthisstudy are based upon proceduresspecifieidn the U.S. EnvironmentalProtectionAgency Series850 EcologicalEffectsTest GuidelinesOPPTS Number 850.2200 (1) Section71-2 of the EnvironmentalProtectionAgency's RegistratioGnuidelines, PesticidAessessment Guidelines,FIFRA SubdivisionE, Hazard Evaluation:Wildlifeand Aquatic Organisms (2);OECD Guideline205,GuidelineforTestingof Chemicals,Avian DietaryToxicity Test(3);and upon ASTM StandardE857-87, "StandardPracticefor ConductingSubacute Dietary ToxicityTestswithAvian Species"(4). Test Substance The testsubstancewas receivedfrom 3M Corporationon October 29, 1998 and was assigned WildlifeInternationLatld.IdentificatiNounmber 4675 upon receipt.The testsubstancewas white powder identifiaesd: FC-95; Lot No.:217. The reportedpurityof thetestsubstancewas 98.9%, with an expiratiodnateof 2008. Followingtestterminationt,hetestmaterialwas reanalyzed.The results of reanalysiisndicatae testsubstancepurityof 90.49%. Alltestconcentrationhsave been adjustedto reflectthe purityreportedon the new Certificaotfe Analysis(Appendix1). The testsubstancewas storedunder ambientconditions. The internalstandardwas receivedfrom 3M Corporationon July 2, 1998 and was assigned WildlifeInternationLatld.identificatinounmber 4526 upon receipt.'Me internasltandarda, granular WILDLIFE INTERNATIONAL, LTD -9- PROJECT NO.: 454-102 materialw,as identifieads: II-LIK 2H, 2H PerfluorooctanSeulfonicAcid, ChemicalAbstract Number: 27619-97-2. The standardwas storedunder ambientconditions TreatmentGroups The testconsistedof a geometricseriesof eighttestconcentrationasnd a controlgroup. Thirty mallardducklingswere assignedto thecontrolgroup and ten mallardducklingswere assignedto each of the treatmentgroups. Birdswere sortedby weight,then chosenindiscriminateflryom withineach representewdeightclassforplacementintocontroland treatmentgroups. The birdswere housed in broodingpens containingfiveducklingseach. On Day 0,followingexperimentasltartt,he Sponsor requestedtheadditionof a 10 ppm testconcentrationB.irdsfrom thesame lotthatremainedaftertest initiatiwoenre used fortheadditionatlestconcentrationN.ominal dietaryconcentrationussed inthis studywere 0, 9.1,18.3,36.6,73.2,146,293, 586 and 1171 partsper millionactiveingredien(tppm a.i.).Ile dietaryconcentrationwsere establishebdased upon known toxicitdyata and information suppliedby the Sponsor. Each group was fedtheappropnatetestor control&et forfivedays. During theexposureperiod the controlgroup receiveduntreatedfeed. Followingthe five-dayexposureperiodallgroups were givenuntreatedbasaldietforthreedays.On Day 8, halfof the treatmentand controlbirdswere eudianizedand liverweightand tissueb,lood,and bilesaznplescollectefdoranalysis.The remaining birdswere fedbasalrationuntilDay 22. On Day 22,thesebirdswere sacrificeadnd alsosampled for liverweightand tissueb,lood,and bile. DurationoftheTest The primaryphases ofthistestand theirdurationswere: 1. Acclimation- 9 days. 2. Exposure- 5 days. 3. Post-exposuroebservation- 3 or 17 days TestBirds All mallard(Anas platyrhynchos) were 10 days of age and appeared to be in good healthat initiatioofnthe test.The birdswere obtainedfrom WhistlingWings Inc.,Hanover,IL. and were hatchedon April13,1999. Birdsrangedinweightfrom 96 to204 grams attestinitiatioTnh.e birds used inthisstudywere immature and couldnot be differentiatbeydsex. All birdswere from the WILDLIFE I*NTERNATIONAL, LTD -10- PROJECT NO.: 454-102 same hatch, pen-rearedand phenotypicalliyndistinguishabflreom wild birds. All birdswere acclimatedto thecaging and facilitifersom theday afterhatchuntilinitiationf thetest. Animal Diet Throughout accliniatioannd testingalltestbirdswere fed a game bird rationformulatedto WildlifeInternationLatld.'sspecificatio(nAsppendix II). Water from the town of Eagon public water supply,and feed were providedad libitumduringacclimationand testing.The birdsreceived no form of antibiotimcedicationduringacclimationortesting. DietPM 'Me testsubstancewas mixed directlyintothe ration.Mixing was done with a Hobart mixer (Model Number AS200T). All dietarytestconcentrationwsere adjustedto 100% PFOS based upon the reportedpurityof the testsubstance. All dietaryconcentrationasnd the LC50 value are reported as ppm a.i.in the diet.Nominal dietarytestconcentrationussed in thisstudy were 9.1, 18.3,36.6, 73.2,146,293, 586 and 1171 ppm a.i.(Appendix Diet SgMRIinSamples of the testdietswere collectedto verifythe testconcentrationsadministeredand to confirm the stabilitaynd homogeneity of the testsubstancein the diets. Homogeneity of the test substancein the dietwas evaluatedby collectinsgix swnples from the 9.1 ppm a.i.a.i.and six samples from the 1171 ppm a.i.a.i.testdietsat preparationon Day 0. Homogeneity samples were collectefdrom the top,middle and bottom of the leftand rightsectionsof the mixing vessel. The homogeneity samples also served as verificatiosnamples. One verificatiosnample was collected from the controldietand two verificatiosnamples were collectedfrom each remainingtreatment group at preparationon Day 0. At theend of the exposure period(Day 5),one sample was collected from the controland two samples were collectedfrom each treatmentgroup to determinestabilitoyf the testsubstanceinthe diet.The stabilitsyamples were collectedfrom feed remainingin the feeders afterbeing at ambient testpen conditionsfor fivedays. Samples were transferredimmediatelyto WildlifeInternationaLltd.analyticaclhemistry. AnalZicalMethod The method used for the analysisof the avian dietsamples was based upon methodology developedatWildlifeInternationaLltd. and entitle"dMethod Outlineforthe Determinationof PFOS WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-102 in Avian Feed". Avian dietsamples were extractedwith methanol. Methanol was added to a requisitqeuantityof feed containedin a French-squareglassbottle.Bottleswere capped and shaken on a shakertable. Samples were vacuum filtereudsing qualitativfeiltepraper. The retainedfeedwas rinsedthreetimeswith methanol intothe filtrateT.he filtratweas transferretdo a volumetnc flask and broughtto volume with methanol. As appropriates,amples were furtherdilutedwith methanol. Each sample then was dilutedwith a 50% methanol : 50% NANOpure(D water solutioncontaining 0.100mg4HPFOS (internasltandard)/Land 0.05% formic acid (v/v)so they fellwithinthe calibratiornange of the PFOS methodology. A method flowchartis providedin Appendix III, Figure 1. Concentrationsof PFOS in the standardsand samples were determinedby reversed-phasehigh performance liquidchromatography usinga Hewlett-PackardModel 1100 High Performance Liquid Chromatograph (BPLC) with a Perkin-EhnerAPI 10OLC Mass Spectrometerequipped with a PerkinElmer TurbolonSpray ion source. HPLC separationswere achievedusing a Keystone BetasilCls analyticaclolumn (100 nim x 2 mm I.D.,3 pm particlseize).The instnimentparametersare summarized inAppendix HI,Table 1. Calibrationstandardsof PFOS prepared in a 50% methanol : 50% NANOpure(D water solution containing0.100mg4HPFOS (internasltandard)/Land 0.05% formic acid (v/v),rangingin concentrationfrom 0.00229 to 0.0457 mg a.i./Lwere analyzedwith the samples. The same and most prominent peak responsefor PFOS was utilizetdo monitor PFOS in allcalibrationq,ualitycontrol, and study samples. No attemptwas made to quantifyPFOS on the basisof individualisomeric components. Linear regressionequationswere generatedusing peak area response ratios(PFOS : internalstandard)versus the respectiveconcentratiornatios(PFOS : internalstandard)of the calibratiosntandards.A typicalcalibratiocnurve is presentedin Appendix III,Figure 2. The concentrationof PFOS in the samples was determinedby substitutintghe peak area responseratios intothe applicablelinearregressionequation. Representativeion chromatograms of low and high calibratiosntandardsarepresentedinAppendix III,Figures3 and 4,respectively. The method limitof quantitatio(nLOQ) fortheseanalyseswas setat 1.15 ppm a.i.calculatedas the productof the lowestcalibratiosntandardanalyzed(0.00229mg a.i./La)nd the dilutionfactorof thematrixblanksamples (500). WILDLIFE INTERNATIONAL, LTD -12- PROJECT NO.: 454-102 Two matrixblank samples were analyzedto determinepossibleinterferencesN.o interferences were observedator above theLOQ duringsampleanalyses(Appendix IH,Table2). An interference inthe feedappearedat approximatelythesame retentiotnime as thepeak of interesbtut itwas well below the LOQ. A representaticvheromatogram of a matrixblank ispresentedin Appendix III, Figure5. Avian dietwas fortifieadt 4.57,183 and 1830 ppm a.i.and analyzedconcurrentlwyith the samples to determinethemean proceduralrecovery(AppendixIII,Table3). Sample concentrations were not correctedforthemean proceduralrecoveryof 94.7%. A representaticvheromatogram of a matrixfortificatiiosnpresentedinAppendix III,Figure6. An example calculatioinspresentedfor sample number 454-102-2,nominal concentratioonf 18.3ppm a.i.inaviandiet. InitiaWleight: 10.0g FinalVolume: 200 mL DilutionFactor:100 (intermediadtielutiofnactoxr fmaldilutiofnactor) PFOS Peak Area: 113568 InternaSltandardPeak Area: 413160 Peak Area Ratio:0.2749 Calibratiocnurveequation. Slope:2.77397 Intercept0:.01894 Curveisweighted(I/x). PFOS (mg a.i./La)tinstmment (Peakarearati-o (Y-interceptx)I).S.Concentration Slope (0.2749- 0.01894)x 0.100 mg/L 2.77397 0.00923mg a.i./L Note:I.S.= intemalstandard. WILDLIFE INTERNATIONAL, LTD - 13- PROJECT NO.: 454-102 PFOS (ppm a.i.i)nsample PFOS (mg a.i./La)tinstnunentx FinalVolume (L)x DilutionFactor InitiaWleight (Kg) 0.00923 x 0.200 x 100 0.01 18.5ppm a.i. PercentofNominal Concentration PFOS (ppm a.i.i)nsample x 100 PFOS (ppm a.i.n)ominal 18.5 - x 100= 101% 18.3 Housing and EnvironmentalConditions During acclimationand testinga,llbirds were housed indoorsin batteriesof thermostatically controlledbrooding pens manufactured by Safeguard Products,Inc. Each pen had floorspace that measured approximately62 X 90 cm. Ceilingheightwas approximately25.5 cm. Externalwalls, ceilingsand floorswere constructedof galvanizedsteelwire and sheeting.Birds were sortedby weight,then chosen indiscriminatelfyrom withineach representedweight classfor assignment to pens. Each group of birdswas identifiebdy pen niunberand testconcentration.Individuablirds were identifiebdy wing bands. During thetestthe average temperatureinthe brooding compartment of the pens was 38'C =L2*C (SD). Average ambient room temperatureforthisstudywas 25.2*C 0.70C (SD) with an average relativheumidityof 53% 18% (SD). The photoperiod(maintainedby a time clock)was sixteen hours of lightper day duringacclimationand throughoutthe test.The lightsource was fluorescent lightswhich closelyapproximatenoon-day sunlight.The birdswere exposed to an average of approximately207 lux of illumination.Housing and husbandry practiceswere based on guidelines establishebdy theNationalResearch Council(5). Observations During acclimationallbirdswere observeddaily.Birdsexhibitingabnormal behavioror physical injurywere not used. Followingtestinitiatioannd continuinguntilterminationa,llbirdswere WILDLIFE INTERNATIONAL, LTD -14- PROJECT NO.: 454-102 observedat leastonce daily. A recordwas maintainedof allmortalitys,ignsof toxicitya,nd abnormal behavior. Animal B& Weiehts/FeedConsmpt'Lon Individuablody weightswere measured atthe imtiatioonf the test,on Day 5, on Day 8 forall birds,and on Days 15 and Day 22 forallremainingbirds.Average feedconsumptionvaluesduring theexposureperiod(Days 0-5)and thepost-exposuroebservatiopneriod(Days 6-8)were determined by pen for each treatmentgroup and the controlgroup. Additionallyf,eed consumptionwas determinedforDays 8-15 and 15-22fortheremaininghalfof thetreatmentand controlbirds.Feed consumptionwas determinedby measuringthe change inthe weight of the feedpresentedto thebirds over a givenperiodof time. The accuracyof feedconsurnptimvaluesmay have been affectedby the unavoidablewastage of feedby thebirds. Gross NecLopsy Alltestbirdsthatdiedduringthe courseof thetestand allbirdsremainingatthe tern-dnatiownere subjectedto a grossnecropsy.Additionallyl,iverweightand livertissues,erum and blood solidsa,nd bilewere collectefdrom birdseudianizedon Day 9 and 22, and when possiblefrom thosethatdied duringthe courseofthe study. StatisticAanlalyses Mortalitydatawere analyzedusingthecomputerprogram of C.E. Stephan(6).The program was desiped to calculattehe LC50 valueand the 95% confidenceintervably probitanalysism,oving averagemethod or thebinomialprobabilitmyethod (7,8, 9). In thisstudythe LC50 valuewas determinedby the probitmethod. The slopeof the concentration-responcsuerveand resultsof the goodness of fittestare reported. Body weight data were compared by Dunnett'stestusing TOXSTAT software(10,11).No statisticaanlalyseswere appliedtofeedconsumptiondata. RESULTS DietAnalysis Avian dietsampleswere collectefdrom the 9.1,18.3and 1171 ppm a.i.testconcentrationasnd analyzedto evaluatehomogeneityof the testsubstancein the avian diet.The analysisof these samples also servedas verificationf testsubstanceconcentrations.Resultingmean measured WILDLIFE INTERNATIONAL, LTD -is- PROJECT NO.: 454-102 concentrationss,tandarddeviationsand coefficientosf variation(CV) for thesetestconcentrations were 9.8 0.969 ppm a.i.(CV = 9.940/o)1,9.5 2.13 ppm a.i.(CV = 10.90/oa)nd 1196 70.2ppm a.i.(CV 5.871/o)r,espectivel(yAppendix IH, Table 4). Controlavian dietsamples collecteduring the testshowed no interferenceasbove the LOQ. Samples collecteduringthe testto verifythe 36.6, 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 valuesrepresented110, 102, 119, 99.3 and 91.6% of the nominal concentrationsr,espectivel(yAppendix III,Table 5). Analysisof avian diet samples collectedfrom feedersafterbeing held at ambient temperaturefor fivedays averaged 119, 97.4,130, 104, 94.3, 101, 101 and 95.0% of theDay 0 valuesforthe 9.1,18.3,36.6,73.2,146, 293, 586 and 1171 ppm a.i.testsubstanceconcentrationsr,espectivel(yAppendix IR, Table 6). A representativcehroniatogramof a testsample isshown inAppendix Ul,Figure 7. Mortalitieasnd ClinicalObservations No mortalitieosccurredin the controlgroup, and allbirds were normal in appearance and behaviorthroughoutthetest.There were no treatment-relatmeodrtalitieosr overtsignsof toxicityin the 9.1,18.3,36.6,73.2,or 146 ppm a.i.treatmentgroups (Table 1 and Appendix V). However, there was 20% (2 of 10)mortalityinthe293 ppm a.i.treatmentgroup,30% (3 of 10)mortalityinthe586 ppm a.i.treatmentgroup and 90% (9 of 10)inthe 1171 ppm a.i.treatmentgroup. In the 293 ppm a.i.treatmentgroup singlemortalitiewsere noted on the mornings of Day 7 and Day 8. Signsof toxicitwyere firstobservedon the morning of Day 4 and continueduntiltermination forthose birdssacrificeodn Day 8 and untilthe afternoonof Day 8 forthose birdssacrificeodn Day 22. All birdsinthistreatmentleveldisplayedsignsof toxicity.These includedreduced reaction to stimuli(sound and motion),lossof coordinationr,uffledappearance,lethargy,and lower Iiinb weakness. All remainingbirdsappeared to have recoveredand were normal in appearance and behaviorfrom the moming of Day 9 untiltesttermination. At the 586 ppm a.i.treatmentleveltherewere threetreatment-relatemdortalitieso,ne each noted on the morning of Day 5,Day 6, and Day 7. Signsof toxicitywere firsotbservedon themorning of Day 3 and continueduntilterminationforthosebirdssacrificeodn Day 8 and untilthe afternoonof Day 8 for thosebirdssacrificeodn Day 22. All birdsin thistreatmentleveldisplayedsignsof toxicity.These includedreduced reactionto stimuli(sound and motion),ruffledappearance,lower WILDLIFE INTERNATIONAL, LTD -16- PROJECT NO.: 454-102 limb weakness,and lethargy.All remainingbirdsappearedto have recoveredand were normal in appearance and behaviorfrom themorning ofDay 9 untiltesttermination. In the 1171 ppm a.i.treatmentgroup therewere nine treatment-relatmeodrtalities'.Me firstwo mortalitiewsere found on the morning of Day 4, followedby four mortalitieosn Day 5, two mortalitieosn Day 6 and the finalmortalityon Day 7. Overt signsof toxicitywere firstobservedon themorning of Day 2 and the singlesurvivingbirdcontinuedto displaysignsof toxicitythrough the afternoonof Day S. Signs of toxicityobservedamong birdsin the 1171 ppm a.i.treatmentgroup includedreduced reactionto stimuli(sound and motion),lossof coordinationp,rostrateposture, depression,convulsions,ruffledappearance,lower limb weakness, and lethargy.The one surviving bird appeared to have recoveredand was normal in appearanceand behavior from the morning of Day 9 untiltesttermination. Body WeiWit and Feed Cons=tion When compared to the controlgroup,therewere no apparenttreatmentrelatedeffectson body weightamong birdsin the 9.1, 18.3,or 36.6 ppm a.i.treatrnengtroups. There was a statistically significan(tp<0.05)reductioninweight gain atthe 9.1 ppm a.i.levelforthe Day 0-5 periodthatwas reflecteidn significantloywer (p< 0.05)mean Day 5 body weight. When compared to the control group,reductionsin weightgainwere alsostatisticaslilgynifican(tp<0.05)forthe Day 5-8 period, resultinign a significant(lpy<0.05)lower Day 8 mean body weight. However, differencefsrom the controlgroup at the 9.1 ppm a.i.levelappear to be due to a lower mean Day 0 body weight forthe 9.1 ppm a.i.leveland were not dose responsive.Therefore,thesedifferenceswere not considered treatmentrelated. There was a marked, treatment-relatecdo,ncentration-responsievfefecton body weight in the 73.2, 146,293, 586, and 1171 ppm a.i.treatmentgroups duringtheexposureperiod(Days 0-5)(Table 2 and Appendix VI). These reductionswere statisticalsliygnificanat p<0.05 forthe 73.2ppm a.i. leveland at p<0.01 atthe 146,293, 586 and 1171 ppm a.i.levels.During the Day 5-8 post-exposure period,weight gain continuedto be reduced for survivingbirdsin the 293, 586 and 1171 ppm a.i. treatmentgroups. These reductionswere statisticalsliygnificanatt p<0.01 forthe 293 and 586 ppm a.i.levels. Differencesat the 1171 ppm a.i.levelwere marked but could not be statistically compared. Body weight lossesand reductionsinweight gainatthe 293, 586 and 1171 ppm a.i.levels were reflecteidn significantl(yp<0.05 or p<0.01) lowermean body weightson Days 5, 8, 15,and 22 WILDLIFE INTERNATIONAL, LTD -17- PROJECT NO.: 454-102 of the test.At the 146 ppm a.i.levelmean body weights on Days 8 and 22 were alsosignificantly (p<0.05)lowerdw thecontrolgroup. During the Day 8-15 and Day 15-22 post-exposureperiods, body weight gainappeared comparable among allgroups. When compared to the controlgroup, therewas a marked reductionin feed consumption in the 293, 586, and 1171 ppm a.i.treatmentgroups duringtheexposureperiod(Days 0-5). A reductionin feed consumption continuedto be observed through Day 15 in the 293, 586 and 1171 ppm a.i. treatmentgroups. Gross NecLopsy During the courseof the test,allbirdsthat died were subjectedto a gross necropsy. Necropsy resultsfor birds found dead were similar. Common observationsincludedthincondition,lossof muscle mass, alteredspleencolor,empty crops,and empty gastrointestintarlacts.'Findings were consideredto be treatmentrelated.Detailsof thenecropsy findingsarepresentedinTable 4. Half of the survivingbirdswere subjectedto grossnecropsyon Day 8 and the remainingbirdson Day 22, followingtestterminationT.he necropsy resultsforbirdseudunized on Day 8 and Day 22 from the 18.3,146 and 1171 ppm a.i.treatmentgroups were unremarkable. One bird inthe control group and one bird in the 36.6 ppm a.i.treatmentgroup were noted with retainedyolk sacs during necropsyof birdson Day 8. A singlebirdinthe 9.1 ppm a.i.treatmentgroup necropsiedon Day 8 was notedas small,with a lack of muscle mass. In the 73.2 ppm a.i.treatmentgroup one bird necropsiedon Day 8 was small,with a lackof muscle mass, and had a 2 x 0.5 cm abscess containing caceous necroticmaterialon the rightsideof the abdominal wall.Due to the nature of the lesions observed in birds,and the isolatedincidenceof occurrence,the findingslistedabove in euthanized birdsfrom the 9.1,36.6,and 73.2 ppm a.i.treatmentgroups were not consideredto be relatedto treatment.In additiona,llremainingbirdsin the control,9.1,36.6,and 73.2 ppm a.i.treatment groups were non remarkable. In the 293 ppm a.i.treatmentgroup, one birdeuthanizedon Day 8 was notedwith an incidental findingof a retainedyolk sac. A second birdinthe 293 ppm a.i.group,necropsiedon Day 8, was noted as emaciatedwith a lack of muscle mass. This lossofmuscle mass was consistentwith reduced weight gain forthe leveland was consideredtreatmentrelated.Another bird in the 293 ppm a.i. treatmentgroup, euthanizedon Day 22, was noted with 2 cm cystscontainingyellow fluidon the WILDLIFE INTERNATIONAL, LTD 18- PROJECT NO.: 454-102 anteriorportionof the leftlobe of the liver.Due to the natureof the cystsand the isolatedincidence ofoccurrence,thisobservationwas not consideredtreatinenrtelated. Tnree birdsinthe 586 ppm a.i.treatmentlevelwere euthanizedon Day 8.At necropsy,they were noted to be thinand lackingmuscle mass. One bird was noted with a slightlpyale liver.These findingsare consideredto be treatrnenrtelated.Four birds in the 586 ppm a.i.treatmentlevel surviveduntilDay 22. Necropsy resultsforthesefourbirdswere not remarkable. CONCLUSION The dietaryLC50 value for Mallardsexposed to PFOS was determinedto be 628 ppm a.i.with a 95% confidenceintervalof 448 ppm a.i.to 958 ppm a.i..]Me slopeof the concentration-response curve was 3.67 and the chi-squarevaluewas 2.13. The no mortalityconcentrationwas 146 ppm a.i.. Based upon reductionsin body weight gain at the 73.2 ppm a.i.testconcentrationt,he no-observedeffectconcentrationwas 36.6 ppm a.i.. WILDLIFE INTERNATIONAL, LTD -19- PROJECT NO.: 454-102 REFERENCES I U. S. Environmental ProtectionAgency, 1996. Series850- EcologicalEffectsTest Guidlelines (draft)O,PPTS Number 850.2200:Avian DietaryToxicityTest. 2 U.S. Environmental Protection Agency. 1982. PesticideAssessment Guidelines,bYFRA Subdivision E, Hazard Evaluation: Wildlifeand Aquatic Organisms, subsection 71-2, EnvironmentalProtectionAgency, Officeof PesticidePrograms. Washington, D.C. 3 Organization for Economic Cooperation and Development. 1984. Aidan Dietary Toxicity Test OECD Guideline for Testing of Chen-dcals. Guideline 205. Paris. 4 American Society for Testing and Materials. 1987. Standard Practicefor Conducting Subacute DietaryToxicityTestswith Avian Species.ASTM StandardE857-87. Annual Book of ASTM Standards,Vol. 11.04. PhiladelphiaP,A. 5 National Research Council. - 1996. Guide for the Care and Use of Laboratory Animals. Washington, D.C. National Academy Press. 125 pp. 6 Stephan, C.E. 1977. Methods for Calculating an LC50. Pages 65-84 In Aquatic Toxicology and Hazard Evaluations, American Society for Testing and Materials. Pub. No. STP 634. PhiladelphiaP,A. 7 Stephan, C.E. 1978. U.S. EPA, EnvironmentalResearch Laboratory,Duluth,MN. Personal Conununication. 8 Finney, D.J. 197 1. StatisticalMethods in Biological Assay, 2nd cd., Griffin Press, London. 9 Thompson, W.R. 1947. BacteriologicalReviews, Vol 2,No.2: 115-145. 10. Dunnet, C.W. 1955. A MultipleComparison'sProcedurefor Comparing SeveralTreatments witha Control.Jour.Amer. StatisA.ssoc.50:1096-1121. 11. Gulley,D.D. 1990. TOXSTAT Release3.2.The UniversityofWyoming. WILDLIFE INTERNATIONAL, LTD -20- PROJECT NO.: 454-102 TABLE I CumulativeMortalityfrom a MallardAcute DietaryToxicityStudy with PFOS Experimental Group (ppm a.i.) Day 0 No. Dead Per No. Exposed Exposure Period Day 1 Day 2 Day 3 Day 4 Day 5 No. Dead Per No. Exposed Post-Exposure Period Day 6 Day 7 Day 8 Control 0 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 Treatment 9.1 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 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 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 2/10 0/10 0/10 0/10 0/10 0/10 0/10 1/10 6/10 0/10 0/10 0/10 0/10 0/10 0/10 2/10 8/10 0/10 0/10 0/10 0/10 0/10 1/10 3/10 9/10 0/10 0/10 0/10 0/10 0/10 2/10 3/10 9/10 The LC50 valuewas calculatedtobe 628 ppm a.i.with a 95% confidenceintervalof 448 ppm a.i.to958 ppm a.i. No mortalitieosccurredin ofthetreatmentlevelsfrom Day 8 to Day 22. WILDLIFE INTERNATIONAL, LTD -21- PROJECT NO.: 454-102 TABLE2 Page I Mean Body Weight (g)from a MallardAcute DietaryToxicityStudy with PFOS Experimental Group (ppm a.i.) Day 0 Exposure Period D!y 5 if-f@ge ay 0-5 Post-ExposurePeriod Day 8 Clmgel Day 5-8 Total Change (Day 0-8)1 Control 0 Mean SD 135 279 144 24 42 22 380 101 53 16 Treatment 9.1 Mean SD 119 226** 108** 14 36 23 317* 91 66 33 18.3 Mean SD 146 277 131 14 26 18 377 100 32 11 36.6 Mean SD 147 275 128 16 32 22 375 100 37 12 73.2 Mean SD 143 260 117* 30 55 33 343 82 90 42 146 Mean SD 143 242 19 37 ioo** 21 331 89 48 16 293 Mean SD 129 161** 32** 17 28 33 220** 57 55 44 586 Mean SD 144 137** -6** 23 27 26 175** 36 34 41 1171 Mean SD 147 112** -37** 25 28 13 185 2 3 12 - - 'Mean changeiscalculatesdeparatelfyrom themean body weightsusingindividuablody weights (SeeAppendixVI). 2n--I,couldnotbe evaluatedstatisticaulsliyngDunnett'st-test. (-)= No dataavailabldeue tomortalit-y. *Statisticadlilfyferenftrom thecontroglroupatp < 0.05(Dunnett'ts-test). **Statisticadll erentfromthecontroglroHEatp < 0.01 (Dunnett'ts-test). 245 34 198* 55 231 25 228 30 200* 73 188** 32 94** 54 39** 36 152 - WILDLIFE INTERNATIONAL, LTD -22- PROJECT NO.: 454-102 TABLE2 Page 2 Mean Body Weight (g)from a Mallard Acute DietaryToxicityStudy with PFOS Experimental Group (ppma.i.) Day 8 Post-ExposurePeriod Change' Day 15 Day 8-15 Day 22 Change' Total Day 15-22 Change(8-22) Control 0 Mean SD Treatment 9.1 Mean SD 18.3 Mean SD 36.6 Mean SD 73.2 Mean SD 146 Mean SD 293 Mean SD 410 640 230 30 56 47 345* 575 230 33 32 20 384 625 241 27 42 22 377 620 243 33 44 40 364 579 .216 82 97 21 332* 564 232 58 56 22 211** 467** 256 49 51 35 823 183 413 49 24 42 773 198 427 38 15 34 811 186 427 81 41 62 928 208 451 78 34 71 782 203 418 135 39 56 688* 124* 356 86 90 106 701* 234 490 41 35 54 586 Mean SD 174** 394** 221 613** 219 439 46 82 37 91 13 47 1171 Mean SD 185' 373 2 1982 634 2 25 12 4492 - - - - 'Mean changeiscalculatesdeparatelfyromthemean bodyweightsusingindividuablodyweights(SeeAppendixVI). 7n=I,couldnotbe evaluatesdtatisticaulsliyngDunnett'st-test. (-)= No dataavailabldeuetomortality. *Statisticadlilfyferefnrtomthecontroglroupatp < 0.05(Dunnett'ts-test). **Statisticadlilfyferefnrtomthecontroglroupatp < 0.01(Dunnett'ts-test). WILDLIFE INTERNATIONAL, LTD -23- PROJECT NO.: 454-102 TABLE3 Page 1 Mean Feed Consumption (glbird/dayf)rom a MaRard Acute Dietary ToxicityStudy with PFOS Experimental Group (ppm a.i.) Control 0 Mean SD Exposure Period Day 0-5 92 10 Treatment 9.1 73 18.3 91 36.6 94 73.2 77 146 105 293 44 586 36 1171 22 No dataavailabldeue tomortality. Post-ExposurePeriod Day 6-8 125 17 117 132 125 101 159 63 55 25 WILDLIFE INTERNATIONAL, L-rD -24- PROJECT NO.: 454-102 TABLE3 Page 2 Mean Feed Consumption (g/bird/dafyr)om a MallardAcute Dietary ToxicityStudy withPFOS Experimental Group (ppm a.i.) Control 0 Mean SD Post-ExposurePeriod Day 8-15 Day 15-22 171 180 13 20 Treatment 9.1 172 198 18.3 186 204 36.6 165 179 73.2 148 173 146 159 164 293 109 132 586 114 143 1171 106 154 No dataavailabldeue tomoruaity. WILDLIFE INTERNATIONAL, LTD -25- PROJECT NO.: 454-102 TABLE4 Gross PathologicalObservations from a Mallard Acute DietaryToxicityStudywith PFOS Birdsthatdiedduringthecourseofthe study Finding Crop empty Emaciated G.I.Tract,primarilyempty Gizzard contentsbilestained Gizzard,empty Intestinaclontents,black and tar-like Keel prominent Kidneys,pale Loss of muscle mass Spleen, grey Spleen,small and pale Spleen,pale Thin Male, Female, and Undetemiined PPM A.I. 293 586 1171 N=2 N=3 N=9 0 2 7 1 1 4 I I I 0 2 4 0 0 1 0 0 1 0 0 2 0 0 1 2 2 5 0 0 1 1 1 2 0 2 3 1 1 4 WILDLIFE INTERNATIONAL, LTD PR( -26- Experimental Group (ppm a.i.) Control 0 0 0 0 0 0 Treatment 9.1 9.1 Pen Dg 0 1 015 2 0/5 3 0/5 4 015 5 0/5 6 015 1 015 2 0/5 Table 5 CumulativeMortality(EstimatedCumulativeDose, mg/kgl)from a Mallard AcuteDi= Toxic!!yStudywithPFnc No. Dead Per No. Exposed (CumulativeDose,mg/kg) Exposure Period Day I Day 2 Day 3 Day 4 Day 5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 015 015 015 0/5 0/5 015 0/5 015 015 015 0/5 015 0/5 0/5 015 0/5 015 015 015 015 015 0/5 0/5 0/5(4) 0/5(4) 0/5(8) 0/5(8) 0/5(12) 0/5(12) 0/5(16) 0/5(16) 0/5(20) 0/5(20) No. Dead Per No. Post-ExposureP Day 6 Day 7 E 0/5 0/5 0 015 015 0 0/5 0/5 0 015 0/5 0 015 015 0 0/5 0/5 0 0/5 0/5 0 015 015 0 18.3 1 015 0/5(8) 0/5(16) 0/5(24) 0/5(32) 0/5(40) 0/5 0/5 0 18.3 2 0/5 0/5(9) 0/5(18) 0/5(27) 0/5(36) 0/5(45) 0/5 0/5 0 36.6 1 015 0/5(19) 0/5(38) 0/5(57) 0/5(76) 0/5(95) 015 015 0 36.6 2 015 0/5(16) 0/5(32) 0/5(48) 0/5(64) 0/5(80) 015 015 0 73.2 1 015 0/5(31) 0/5(62) 0/5(93) 0/5(124) 0/5(155) 015 015 0 73.2 2 0/5 0/5(30) 0/5(60) 015(90) 0/5(120) 015(150) 015 015 0 146 1 015 0/5(74) 0/5(148) 0/5(222) 0/5(296) 0/5(370) 015 0/5 0 146 2 0/5 0/5(101) 0/5(202) 0/5(303) 0/5(404) 015(505) 015 015 0 293 1 0/5 0/5(64) 0/5(128) 0/5(192) 0/5(256) 0/5(320) 015 015 0 293 2 0/5 0/5(126) 0/5(252) 0/5(378) 0/5(504) 0/5(630) 015 1/5 2 586 1 015 0/5(175) 0/5(350) 0/5(525) 0/5(700) 0/5(875) 015 115 1 586 2 0/5 0/5(153) 0/5(306) 0/5(459) 0/5(612) 1/5(765) 2/5 2/5 2 1171 1 0/5 0/5(240) 0/5(480) 0/5(720) 1/5(960) 4/5(1200) 5/5 515 5 1171 2 015 0/5(207) 0/5(414) 0/5(621) 1/5(828) 2/5(1035) 3/5 4/5 4 The LC50 valuewas calculatetdobe approximately628 ppm a.iw.itha 95% confidenceintervaolf448 to958 ppm a.i.. 1-Estimatedcumulativedoseisbasedupon theaveragebodyweightand feedconsumptionoverthe5-dayexposureperiod,and servesasa rough approximationoftheactualamount oftestsubstanceconsumed. 2- No mortalitieosccurredinM of thetreatmentor controloups afterPal B. WILDLIFE INTERNATIONAL, LTD -27- PROJECT NO.: 454-102 "PENDIX I Cert*ificaotfeAnalysis FC-95tLot 217 Mm-ch 9, 2000 RichadK Pofer Ila smpl, was mdymd wine LC&M, l@F-NI&4wA at--antalnalym tecbniqucl&lmre$UUB 9iftNtrASIbOWftMMP]BtOCOOWnft*)UgWh%Wclg'tF'r'n" 'mi"S'tb= wsso 1"c C"(),']:, gff2gav-, - WitSOi*K' c4Fl$@@IC C.7FISS2,Lji: c4FI-isollc Wj#s2L7E c4op2lgO6T, Clip@3SOL7@cCF 0.04% OJ3 % 1.38% 1.30% 3.71% 1.19% 0.49% 0.13% 0.04% O.OS% ois % AddfflonLUdyw,im= disuftu=aftbeso*lowu ddambud using"E-NMR twbniquwandfoundto caatdathefouowingmalepatem couposition: CYS(CE@).-cBW 912EEd ob" vhmo x Isnubbr 11- CF2(CF3)eMCFIXCFi@r S(@-K van X+y isMai* 5.W x o 0.Y!l0) (CPS)O-(CFLI@SLw- r axmropyibnm% stm x b mawy i) Cara*,-Mcpl)g-o; r Mla bmnd% vA= x ismenw m (CPs)sC-(CFOSeW ic t~ kwol wb= x ism&W!Q CPS-(CYS)A-qCFS)r<CS4SrW K' gnftrmlg=-Mmkl bimmobw.bae x+y Ismgdy 4.andx o 0) 10.3% 1.6% 02% 0.2% WILDLIFE INTERNATIONAL, LTD -28- PROJECT NO.: 454-102 APPENDIX II DIET FORMULATION WILDLIFE INTERNATIONAL LTD. GAME BIRD RATION' INGREDIENTS FineCom Meal Soy Bean Meal, 48% Protein Wheat @&dds ProteinBase Agway Special,60% Protein AlfalfaMeal, 20% Protein DriedWhey Ground Limestone Eastman CalPhos MethioninePremix + Liquid Vitaminand MineralPremix (seebelow) GL Ferm (Fennatco)2 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 PREMIX AMOUNT ADDED PER TON VitaminD3 VitaminA Riboflavin Niacin PantothenicAcid Vitamin B12 FolicAcid Biotin Pyridoxine Thiamine VitaminE Vitan-dnK (Menadione DimethylpyrimidinoBlisulfite) Manganese Zinc Copper Iodine Iron Selenium 2,000,000 I.C.U. 7,000,000l.U. 6 grams 40 grams 10 grams 8 mgs 600 mgs 64 mgs 1.2grams 1.2grams 20,000 l.U. 5.8 grams 102 grams 47 grams 6,8 grams 1.5 grazns 5 1 grains 182 mgs 'Tlieguaranteedanalysisisa minimum of 27% protein,a minimum of 2.5% crude fatand a maximum of 5% crude fiber. 2 FermentationBy-Products(Sourceof UnidentifieGdrowth Factors) . WILDLIFE INTERNATIONAL, LTD -29- PROJECT NO.: 454-102 APPENDIX III ANALYTICAL METHODS AND RESULTS . WILDLIFE INTERNATIO NAL, LTD -30- PROJECT NO.: 454-102 INSTRUMENT: ANALYTICAL COLUMN: OVEN TENTERATURE: STOP TIME: FLOW RATE: MOBBLE PHASE: INJECTION VOLUME: PFOS RETENTION TIME: INTERNAL STANDARD RETENTION TIME: PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: Appendix III Table I TypicaliC/MS OperationalParameters Hewlett-PackardModel 1100 High PerformanceLiquidChromatograph with a Perkin-ElmerAPI IOOLC Mass Spectrometerequippedwith a Perkin-Elmer TurboIonSprayionsource.Operatedin selectivieon monitoringmode (SM. KeystoneBetasilCls column (100nim x 2 mm I.D.,3 @= particlseize) 300C 10.0 minutes 0.220 mL/minute 72.0% Methanol: 28.0% NANOpure(D Water containing0.1% Fonnic Acid 25.0 tiL Approximately7.0 minutes Approximately4.8 minutes 498.6 amu 426.7 amu WILDLIFE INTERNATIONAL, LTD -31- PROJECT NO.: 454-102 Appendix IH Table 2 Matrix Blanks Analyzed ConcurrentlyDuring Sample Analysis Number (454-102-) MAB-1 Sample Type MatrixBlank Measured Concentrationof PFOSI (ppm a.i.) < LOQ MAB-2 MatrixBlank < LOQ 'The lin@dotf quantitatio(nLOQ) was 1.15 ppm a.i.based upon the productof the lowestcalibratiosntandard analyzed(0.00229mg a.i./La)nd thedilutiofnactorofthe matrixblank samples(500). WILDLIFE INTERNATIONAL, LTD -32- PROJECT NO.: 454-102 Appendix III Table 3 Matrix FortificationAsnalyzed ConcurrentlyDuring Sample Analysis Sample Number (454-102-) MAS-LA MAS-4A Concentrationsof PFOS (ppm a.i.) Fortified Measured 4.57 4.54 4.57 4.79 Percent Recovered 99.2 105 MAS-2 183 176 96.1 MAS-5 183 162 88.3 MAS-3 MAS-6 1830 1576 1830 1716 Mean = StandardDeviation= CV= N= Note:Resultsand correctionfsor new testsubstancepuritywere generatedusingMacQuan and manual calculationsV.alues-havebeen rounded forn2rting purposes. 86.1 93.7 94.7 6.99 7.38 6 version1.5software WILDLIFE INTERNATIONAL, LTD PROJ -33 Appendix III Table 4 Homogeneity ofPFOS inAvian Diet Nominal Concentration (ppm a.i.) 9.1 Sample Number (S454-102-) 54 55 56 57 58 59 LocationSampled in Mixing Vessel Top Left Top Right Middle Left Middle Right Bottom Left Bottom Right PFOS Measured Concentration (ppm a.i.) 9.52 9.70 9.79 8.09 10.9 10.5 Mean Measured Concentration(.R) StandardDeviation(SD) CoefficienotfVariation(CV)' R = 9.8ppm a.i. SD 0.969 ppm a.i. CV 9.94% 18.3 2 Top Left 18.5 3 Top Right 23.4 4 Middle Left 18.3 5 Middle Right 17.3 6 Bottom Left 19.4 7 Bottom Right 19.9 R = 19.5ppm a.i. SD = 2.13 ppm a.i. CV= 10.9% 1171 18 Top Left 1239 19 Top Right 1221 20 Middle Left 1118 21 Middle Right 1301 22 Bottom Left 1163 23 Bottom Right 1133 R = 1196 ppm a.i. SD = 70.2ppm a.i. CV = 5.87% Note:Resultsand correctionfsor new testsubstancepuritywere generatedusing MacQuan version1.5softwareand manual calculati rounded forreportingpurposes. 'Coefficienotfvariationwas calculateudsingfttlplrecisionof mean and standarddeviationresults. WILDLIFE INTERNATIONAL, LTD PROJ -34Appendix III Table 5 Verificatioonf PFOS ConcentrationsinAvian Diet Nominal Concentration (ppm a.i.) 0.0 9.1 18.3 Sample Number (S454-102-) 1 - Swnpling interval (Day) 0 - - PFOS Measured Concentration' (ppm a.i.) < LOQ Percent of NomitW - - Mean Measured Concentration (ppm a.i.) - 9.82 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 93.8 291 307 105 586 16 0 17 0 550 93.9 537 523 89.4 1171 - - - - 11962 Note:Resultsand correctionfsor.newtestsubstancepurityweregeneratedusingMacQuan version1.5softwareand manualcalculationsV.alueshave purposes. 'Thelimitofquantitati(oLnOQ) was 1.15ppm a.i.basedupon theproductofthelowestcalibratiosntandardanalyzed(0.00229mg a.i.ALa)nd thedilut blanksamples(500). 2 Resultobtainedfrom Table4. WILDLIFE INTERNATIONAL, LTD PROJ -35Appendix III Table 6 Ambient Stabilitoyf PFOS inAvian Diet During theMallard LC50 Study Nominal Concentration (ppm a.i.) 0 9.1 Sample Number (S454-102-) 1 54-59 Day 0' Mean Measured Concentration (ppm a.i.) - 9.8 Mean Percent of Nominal - 109 Sample Number (S454-102-) 39 60 61 Day 5 Measured Concentratio2n (ppm a.i.) < LOQ Mean Measu Concentratio (ppm a.i.) - 12.3 11.7 11.0 18.3 2-7 19.5 107 40 18.2 19.0 41 19.7 36.6 8,9 40.2 110 42 47.9 52.4 43 56.8 73.2 10,11 74.5 102 44 77.6 77.8 45 77.9 146 12,13 174 119 46 167 164 47 160 293 14,15 291 99.3 48 297 295 49 293 586 16,17 537 91.6 50 552 541 51 530 1171 18-23 1196 102 52 1150 1136 53 1122 'Day 0 resultsobtainedfiromTable 4 and Table 5. 2ne limitof quantitatio(nLOQ) was 1.15 ppm a.i.based upon theproductof the lowestcalibratiosntandardanalyzed(0.00229 mg a.i.ALa)nd the dilutionfactoro (500). 3The Day 5 valueisconsistenwtith the Day 0 mean measured concentratiovnalueforthislevel.No analyticamleasuring biasisindicated. WILDLIFE INTERNATIONAL, LTD -36- PROJECT NO.: 454-102 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN AVLAN DIET Preparematrixfortificatisoanmples in theclesireadvianfeedstockusingthe dry n-titxechnique. Dry Mix Technique For thehigh-levemlatrixfortificatisoanmple,weigh the requisitqeuantityofWildlifeInternationaLlAd.(WIL) rationintoa weigh boat 4 Weigh therequisitqeuantityoftestsubstance(PFOS) intoa beaker. Add K ofthe WIL rationand thetestsubstanceto a largerbeaker. Rinse thebeaker ffigcontainedthe PFOS with small portionsofthe remainingrationand hwisferallportionsto the largerbeaker. Mix the contentsofthe largerbeakerwell and tmi,;fetrhemixturetoa Waring blender. Blend themixture for-5 minutesdopping atI niinuteintervaltsoscrapedown the sidesofthe blender. During thedurd intervatlransferthefortifiefdeedtoa beaker,nux weiland returnthe mixturetothe blendertoconpleu trii)dnignthe specified time. Prepare thenexttwo matrixfortificatiloenvelsby serialdilutions. Follow the same proceduredescribedforthehigh4evelmatrixfortificatieoxnceptweigh the appropriatequantityoffortifiemdatrix(high ormid- level)ratherdm theleg substance. Weigh I0-g samples ofthe matrixblank,matrixfortificatiaonnd testsamples intoweigh boats and transfetro 16-oz.French-squareglassbottles. Record theweights. For each sample, measure 100 mLs ofmdhanol with a graduated cylinderand transfervolume to theFrench-squarebottle. Cap bottlesand place on shakertable.Allow the samples to shake fora minimum of30 minutes at 250 rpm. Vacuum filteerach sample with qualitativfeiltepraper and fim retainedfeed3 times withmethanol intothe filtrate. Transferthefiltratteoa 200-mL volumetricflaskand bringtheflasktovolume with methanol. Prepareappropriatedilution(st)obring finalconcentrationintothe calibratiornange ofthe LCMS methodology. Use methanol forintermediatedilutionsi,frequired. For all finaldilutionusse 50% methanol: 50% NANOpure(ID watersolutioncontaining0.100 mg 4H PFOS (internasltandardyl and 0.05% formicacid(v/v). 4 Ampulate and submit samples forLC/MS analysis. Figure1. Analyticamlethod flowcharftortheanalysiosfPFOS inaviandiet. WILDLIFE INTERNATIONAL, LTD -37- Appendix III PROJECT NO.: 454-102 1.50 - 1.00 Cd 0.50- 0 0 0 0 0.00 0.000 0.100 0.200 0.300 0.400 Concentration(Ratio) 0.500 Figure2. AtypicalcalibratiocnurveforPFOS. Slope= 2.77397;Intercept=0.01894;r= 0.9981. Curve isweighted(I/x). WILDLIFE INTERNATIONAL, LTD -38- Appendix III PROJECT NO.: 454-102 100- go. 80, 7060. 50. 41)3C@ 2010. 24 12 C!t. 5.- 3.1 1.30 Intensity1;995 cps 167 145 ;E4 ol IAA'22 1 19 916 61 q'i 15,1 iii 211 Scan 2.55 3.81 5.06 6.32 7.57 8.83 Time Figure3. A representativieonchromatogram ofa low-leve(l0.00229mg a.i./LP)FOS standard. WILDLIFE INTERNATIONAL, L-rD -39- Appendix III PROJF-CT NO.: 454-102 100, so- 8(3- 7060- so40- 3020- 1 (@ 9 0-. 2.4 48 3'1 1.30 75 61 2.55 90 9'1 3.81 Intensity2:6964 cps 166 144 122 121 5.06 151 6.32 190204 230 lil' '2il' * Scan 7.57 8.83 Ilme Figure4. A representativieonchromatogram of a high-leve(l0.0457mg a.i./LP)FOS standard. WILDLIFE INTERNATIONAL, LTD -40- AppendixIII PROJF,CT NO.: 454-102 100- 90. 24 80- 180 166 intensfty2:30 cps 70- 60- 50. 40- 30- 20---y 10ol@L 5,5 79 05 A.A,,AMA Alm A 31 6'1 O'l 1.30 2.55 3.81 lil 5.06 144 AA lil 6.32 I lil 7.57 216 203 .@ _1,1, AA v VY V V IIL 211 ' 8.83 Scan Ilme Figure S. A representaticvheromatogram ofa matrixblank sample (454-102-MAB-1). The arrow indicatestheretentiotnime of PFOS. WILDLIFE INTERNATIONAL, LTD -41- Appendix III PROJECT NO.: 454-102 100. 90. 80- 70- 60,so40- 30- 20- 10- 1 1 2,40 3'1 1.30 54 71 8ra95 ' 6*1 ' ' 9'1 2.55 3.81 intensity6:238 cps 166 144 122 213 121 lil 181 2il Scan 5.06 6.32 7.57 8.83 nme Figure 6. A representaticvheromatogram of a matrixfortificatisoanmple (454-102-MAS- IA). WILDLIFE INTERNATIONAL, LTD -42- Appendix HI PROJECT NO.: 454-102 100908070, 60so4030r 2& 1 ol Intensity7:248 cps 167 145 24 45 31 1.30 63 61 2.55 94 91 3.81 123 iii 5.06 iii 6.32 a iii 7.57 229. 211 Scan 8.83 Tlme Figure 7. A representaticvheromatogram ofa testsample (454-102-2). WILDLIFE INTERNATIONAL, LTD -43- PROJECT NO.: 454-102 APPENDIX IV DIET PREPARATION Weight and volume of constituentussedtopreparetestdiets: Nominal Concentrations (ppm a.i.) 0 9.1 18.3 36.6 73.2 146 293 586 1171 Test Substance (9) - 0.0910 0.1818 0.3638 0.7282 1.4659 2.9123 5.8239 -11.6483 Basal Ration 9000.0 8999.9 8999.8 8999.6 8999.3 8998.5 8997.1 8994.2 8988.4 Dietswere preparedas follows: 95000.0 g of basalrationwas weighed intoa taredHobart mixing bowl. *Tbe testsubstancewas weighed ina taredweigh boat eapproximately 100 g of basalrationwas takenfrom themixing bowl and placedina Waring blender. *The testsubstancewas added totheblenderand theweigh boatwas rinsedwith additionalration, with therinsealsobeingplaced intheblender. othe blendercontentswere blended forapproximately60 secondsand transferredtothemixing bowl. The blenderwas rinsedwith additionalrationw,ith therinsealsobeingplaced inthemixing bowl. othe bowl was placedon a Hobart mixer and thecontentswere mixed forapproximatelysixminutes. The remainingrationas added tothebowl and thecontentswere mixed forsixmore minutes. one dietwas transferretdo a labelledpaper feedbag. WILDLIFE INTERNATIONAL, LTD PROJECT -44- APPENDIX V Cumulative Mortalityby Pen from a MallardAcute DietaryToxicityStudy with PFOS Experimental Group Pen (ppm a.i.) Control 0 1 0 2 0 3 0 4 0 5 0 6 Treatment 9.1 1 9.1 2 18.3 1 18.3 2 36.6 1 36.6 2 73.2 1 73.2 2 146 1 146 2 293 1 293 2 586 1 586 2 1171 1 1171 2 Day 0 0/5 015 015 0/5 0/5 0/5 015 0/5 0/5 O@/5 0/5 0/5 015 015 0/5 015 0/5 015 0/5 015 015 015 No. Dead Per No. Exposed- Exposure Period Day I Day 2 Day 3 D!y 4 015 0/5 015 015 015 015 015 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 015 0/5 0/5 0/5 0/5 015 015 015 015 015 015 015 015 0/5 0/5 015 0/5 015 0/5 015 015 0/5 0/5 015 015 015 0/5 015 015 015 015 015 015 015 015 015 015 015 015 0/5 015 015 015 015 015 015 015 015 015 015 015 0/5 015 015 015 0/5 015 015 015 0/5 0/5 0/5 015 015 0/5 0/5 015 115 015 015 015 115 Day 5 015 015 015 0/5 015 015 015 0/5 015 015 015 015 015 015 015 015 015 015 015 1/5 4/5 2/5 'Me LC50 valuewas calculatetdobe 628 ppina.iw.itha 95% confidenceintervaolf448 ppm a.i.to958 ppm a.i. No mortalitioecscurredinany ofthetreatmenotrcontrolEUU s afterDay 8. No. Dead Per Post-Expos Day 6 Day 7 0/5 015 0/5 0/5 0/5 0/5 0/5 015 0/5 015 015 0/5 015 015 015 0/5 015 0/5 015 0/5 015 015 015 0/5 015 015 015 0/5 015 015 0/5 015 015 015 015 115 015 115 2/5 2/5 515 515 3/5 4/5 WILDLIFE INTERNATIONAL, LTD -45- APPENDIX VI PROJECT NO.: 454-102 IndividuaBlody Weights(g)from a Mallard Acute DietaryToxicityStudywithPFOS Page I Experimental Group (ppm a.i.) Bird Day 0 Day 5 Change Day 0-5 Dg 8 Change Dg 5-8 Total ChE&@ (0-8) 0 1 132 302 170 404 102 272 2 136 292 156 381 89 245 3 154 319 165 415 96 261 4 158 319 161 429 110 271 5 169 309 140 418 109 249 Mean 150 308 158 409 101 260 SD 15 12 12 18 9 12 0 1 136 282 146 399 117 263 2 142 284 142 394 110 252 3 152 299 147 409 110 257 4 152 312 160 430 118 278 5 177 345 168 484 139 307 Mean 152 304 153 423 119 271 SD -16 26 11 37 12 22 0 1 125 265 140 372 107 247 2 134 282 148 371 89 237 3 149 304 155 440 136 291 4 154 292 138 378 86 224 5 166 328 162 430 102 264 Mean 146 294 149 398 104 253 SD 16 24 10 34 20 26 0 1 124 271 147 376 105 252 2 135 263 128 360 97 225 3 151 300 149 408 108 257 4 163 333 170 432 99 269 5 170 351 181 453 102 283 Mean 149 304 155 406 102 257 SD 19 38 21 38 4 22 WILDLIFE INTERNATIONAL, LTD -46- APPENDIX VI IndividuaBlody Weights (g)from a Mallard Acute DietaryToxicityStudy with PFOS Page 2 ExpenLmental Group (ppm a.i.) Bird __Day 0 Day 5 Change Dg 0-5 Da 8 0 1 107 222 115 298 2 126 267 141 360 3 116 203 87 266 4 107 203 96 284 5 ill 248 137 348 Mean 113 229 115 311 SD 8 28 24 41 PROJECT NO.: 454-102 Change D21 5-8 76 93 63 81 100 83 15 Total c@ele 191 234 150 177 237 198 37 0 1 100 235 135 335 100 235 2 100 232 132 328 96 228 3 102 233 131 317 84 215 4 108 267 159 383 116 275 5 102 210 108 299 89 197 Mean 102 235 133 332 97 230 SD 3 20 18 32 12 29 Group Total Mean 135 279 144 380 101 245 SD 24 42 22 53 16 34 WILDLIFE INTERNATIONAL, LTD -47- APPENDIX VI IndividuaBlody Weights (g)from a Maflard Acute DietaryToxicityStudy with PFOS Page 3 PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Bird Day 0 9.1 1 123 2 118 3 151 4 108 5 119 Mean 124 SD 16 Day 5 244 231 286 209 231 240 29 Change Day 0-5 Da 8 121 349 113 334 135 399 101 313 112 332 116 345 13 33 Change Day 5-8 105 103 113 104 101 105 5 Total Change (0-8) 226 216 248 205 213 222 17 9.1 1 96 145 49 146 1 50 2 123 250 127 345 95 222 3 127 237 110 343 106 216 4 112 216 104 316 100 204 5 111 215 104 294 79 183 Mean 114 213 99 289 76 175 Group Total SD 12 41 29 83 43 71 Mean 119 226** 108** 317* 91 198* SD 14 36 23 66 33 55 *Statisticadlilfyferenftromthecontroglroupatp < 0.05(Dunnett'ts-test). **Statisticadlilfyferenftrom thecontroglroupatp < 0.01(Dunnett's-t-test). WILDLIFE INTERNATIONAL, L-rD -48- PROJECT NO.: 454-102 APPENDIX VI IndividuaBlody Weights (g)from a Mallard Acute DietaryToxicityStudy with PFOS Page 4 Experimental Group (ppm a.i.) Bird Day 0 18.3 1 123 2 141 3 150 4 152 5 160 Mean 145 SD 14 Day 5 261 297 270 286 294 282 16 Change Day 0-5 Day 8 138 355 156 421 120 361 134 390 134 391 136 384 13 27 Change Day 5-8 94 124 91 104 97 102 13 Total Change_ 232 280 211 238 231 238 25 18.3 1 130 240 110 327 87 197 2 137 266 129 359 93 222 3 145 268 123 366 98 221 4 151 258 107 366 108 215 5 171 333 162 433 100 262 Group Total Mean 147 273 126 370 97 223 SD 16 35 22 39 8 24 Mean 146 277 131 377 100 231 SD 14 26 18 32 11 25 WILDLIFE INTERNATIONAL, L-rD -49- PROJECT NO.: 454-102 APPENDIX VI IndividuaBlody Weights (g)from a Mallard Acute Diemq ToxicityStudywith PFOS Page 5 Experimental Group (ppm a.i.) Bird Day 0 36.6 1 131 2 132 3 142 4 160 5 1.79 Mean 149 SD 21 Day 5 261 256 269 270 335 278 32 Change Day 0-5 130 124 127 110 156 129 17 Day 8 372 356 369 353 433 377 33 Change Day 5-8 ill 100 100 83 98 98 10 Total Change 241 224 227 193 254 228 23 36.6 1 131 230 99 316 86 185 2 136 287 151 402 115 266 3 147 263 116 379 116 232 4 151 255 104 344 89 193 5 160 324 164 427 103 267 Group Total Mean 145 272 127 374 102 229 SD 12 36 29 44 14 39 Mean 147 275 128 375 100 228 SD 16 32 22 37 12 30 WILDLIFE INTERNATIONAL, LTD -50- PROJECT NO.: 454-102 APPENDIX VI IndividuOBody Weights (g)from a Mallard Acute DietaryToxicityStudy withPFOS Page 6 Experimental Group (ppm a.i.) Bird Day 0 73.2 1 113 2 113 3 142 4 154 5 179 Mean 140 SD 28 Day 5 203 219 286 290 338 267 56 Change Day 0-5 Day 8 90 281 106 288 144 390 136 379 159 480 127 364 28 82 Change Day 5-8 78 69 104 89 142 96 29 Total Change 168 175 248 225 301 223 55 73.2 1 109 222 113 302 80 193 2 129 254 125 332 78 203 3 139 243 104 340 97 201 4 147 196 49 175 -21 28 5 204 352 148 458 106 254 Mean 146 253 108 321 68 176 SD 36 59 37 101 51 86 Group Total Mean 143 260 117* 343 82 200* SD 30 55 33 90 42 73 *Statisticadlilfyferenftrom thecontroglroupatp < 0.05(Dunnett'ts-test). WILDLIFE INTERNATIONAL, L-rD PROJECT NO.: 454-102 APPENDIX VI IndividuaBlody Weights (g)from a Mallard Acute DietaryToxicityStudy withPFOS Page 7 Experimental Group (ppm a.i.) Bird Day 0 146 1 108 2 138 3 151 4 151 5 1.64 Mean 142 SD 21 Day 5 169 248 271 254 282 245 44 Change Day 0-5 61 110 120 103 118 102 24 Day 8 236 330 375 341 380 332 58 Change Day 5-8 67 82 104 87 98 88 14 Total c 128 192 224 190 216 190 38 146 1 115 204 89 280 76 2 139 219 80 287 68 3 144 229 85 348 119 4 159 261 102 358 97 5 158 285 127 374 89 Group Total Mean 143 240 97 329 90 SD 18 33 19 43 20 Mean 143 242 ioo** 331 89 SD 19 37 21 48 16 *Statisticadlilfyferenftrom thecontrolgroupatp < 0.01 (Dunnett'ts-test). 165 148 204 199 216 186 29 188*W32 WILDLIFE INTERNATIONAL, LTD -52- APPENDIX VI IndividuaBlody Weights (g)from a Mallard Acute DietaryToxicityStudy with PFOS Page 8 PROJECT NO.: 454-102 Experimental Group (Ppm a.i.) Bird Day 0 293 1 115 2 139 3 140 4 155 5 112 Mean 132 SD 18 Day 5 142 119 156 187 134 148 26 Change Day 0-5 27 -20 16 32 22 15 21 Day 8 179 165 217 291 203 211 49 Change Day 5-8 27 -20 16 32 22 15 21 Total c 64 26 77 136 91 79 40 293 1 123 205 82 289 2 120 183 63 273 3 151 139 -12 - 4 106 158 52 223 5 124 185 61 138 Mean 125 174 49 Group Total SD 16 Mean 129 SD 17 26 161** 28 36 32** 33 = No dataavailabldeue tomortality. Statisticadli!fyferenftrom thecontrolgroup atp < 0.0 1 (Durmett'ts-test). 231 68 220** 55 82 63 -12 52 61 49 36 57** 44 166 153 - 117 14 113 69 94*W' 54 WILDLIFE INTERNATIONAL, LTD -53APPENDIX VI IndividuaBlody Weights (g)from a Mallard Acute DietaryToxicityStudy with PFOS Page 9 PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Bird Day 0 586 1 122 2 135 3 152 4 154 5 165 Mean 146 SD 17 Day 5 105 143 160 129 195 146 34 Change Day 0-5 -17 8 8 -25 30 1 22 Day 8 126 209 217 143 174 46 Change Day 5-8 21 66 57 -52 23 54 Total Change 4 74 65 .-22 30 47 586 1 105 114 9 159 2 120 130 3 149 - 10 185 - - 4 153 128 -25 185 5 182 128 -54 - Mean 142 125 -15 SD 30 7 31 Group Mean 144 137** -6** Total SD 23 27 26 (-)= No dataavailabldeue tomortality. **Statisticadlilfyferenftrom thecontroglroupatp < 0.01(Dunnett'ts-tcst). 176 15 175** 34 45 55 - 57 - 52 6 36** 41 54 65 - 32 - 50 17 39*i@36 WILDLIFE INTERNATIONAL, LTD -54- APPENDIX VI IndividuaBlody Weights (g)from a Mallard Acute DietaryToxicityStudy with PFOS Page 10 PROJECT NO.: 454-102 Experimental Group (p2m a.i.) Bird Day 0 1171 1 124 2 140 3 145 4 150 5 166 Mean 145 SD 15 Day 5 88 - 96 118 100 17 Change Day 0-5 -39 - -49 -32 -40 9 Day 8 - Change Day 5-8 - Total C!!@nLe - 1171 1 128 87 -41 - - - 2 110 - - - - - 3 145 132 -13 - - - 4 157 - - - - - 5 200 154 -46 185 31 -15 Mean 148 124 -33 185 31 -15 SD 34 34 18 - - Group Total Mean 147 112** -37** 1852 312 152 SD 25 28 13 - - - No dataavailabldeue tomortality. 'No mortalitieosccurredinany ofthetreatmentorcontrolgroupsafteDray 8. Statisticadlilfyferenftrom thecontroglroupatp < 0.01 (Dunnett'st-test). WILDLIFE INTERNATIONAL, LTD -55- APPENDIX VI PROJECT NO.: 454-102 IndividuaBlody Weights (g)from a Mallard Acute DietaryToxicityStudy vith PFOS Page II ExperimenW Group (p2m a.i.) Bird Day 8 Day 15 Change Day 8-15 Day 22 Change DU 15-22 Total Change (8-22) 0 1 404 721 317 895 174 491 2 381 633 252 780 147 399. 3 415 685 270 861 176 446 4 429 692 263 822 130 393 5 418 679 261 847 168 429 Mean 409 682 273 841 159 432 SD 18 32 26 43 20 40 0 1 399 606 207 810 204 411 2 394 604 210 791 187 397 3 409 638 229 819 181 410 4 430 543 113 730 187 300 5 484 752 268 924 172 440 Mean 423 629 205 815 186 392 SD 37 77 57 70 12 54 0 1 372 583 211 779 196 407 2 371 613 242 823 210 452 3 440 636 196 859 223 419 4 378 594 216 794 200 416 5 430 627 197 816 189 386 Mean 398 611 212 814 204 416 SD 34 22 19 31 13 24 Group Mean 410 640 230 823 183 413 Total SD 30 56 47 49 24 42 WILDLIFE INTERNATIONAL, LTD -56- APPENDIX VI IndividuaBlody Weights (g)firoma Mallard Acute DietaryToxicityStudy with PFOS Page 12 PROJECT NO.: 454-102 Experimental Group (Ppm a.i.) Bird 9.1 1 2 3 4 5 Mean SD Day 8 349 334 399 313 332 345* 33 Day 15 Change Day 8-15 Day 22 602 253 821 549 215 730 616 217 802 562 249 767 546 214 744 575 230 773 32 20 38 Change 219 181 186 205 198 198 15 Total CLu ge@ 472 396 403 454 412 427 34 Exper'unental Group (ppm a.i.) Bird Day 8 Day 15 Change Day 8-15 DM 22 Change Day 15-22 18.3 1 355 611 256 811 200 2 421 693 272 938 245 3 361 578 217 713 135 4 390 626 238 794 168 5 391 618 231 799 181 Mean 384 625 242 811 186 SD 27 42 22 81 41 *Statisticadlilfyferenftromthecontroglroupatp < 0.05(Dmmett'st-test). Total Change 465 517 352 404 408 427 62 WILDLIFE INTERNATIONAL, LTD -57- APPENDIX VI Ind@vidualBody Weights (g)from a MaRard Acute DietaryToxicityStudy with PFOS Page 13 PROJIFCT NO.: 454-102 Experimental Group (ppm a.i.) Bird Day 8 36.6 1 372 2 356 3 369 4 353 5 4.33 Mean 377 SD 33 Day 15 Change Day 8-15 Day 22 Change Day 15-22 677 305 942 265 607 251 817 210 613 244 793 180 559 206 746 187 642 209 840 198 620 243 828 208 44 40 73 34 Total Change 570 461 424 393 407 451 71 Experimental Group (ppm a.i.) Bird Day 8 73.2 1 281 2 298 3 390 4 379 5 480 Mean 364 SD 82 Dg 15 Change Day 8-15 Day 22 Change Day 15-22 482 201 648 166 480 192 646 166 636 246 864 228 599 220 796 197 699 219 955 256 579 216 782 203 97 21 135 39 Total ChanE_ 367 358 474 417 475 418 56 WILDLIFE INTERNATIONAL, LTD -58- PROJECT NO.: 454-102 APPENDIX VI IndividuaBlody Weights (g)from a Mallard Acute DietaryToxicityStudy with PFOS Page 14 Experimental Group (ppm a.i.) Bird Day 8 146 1 236 2 330 3 375 4 341 5 380 Mean 332* SD 58 Day 15 Change Day 8-15 Day 22 475 239 659 569 239 739 631 256 738 570 229 754 576 196 550 564 232 688* 56 22 86 Change 184 170 107 184 -26 124* 90 Total Change 423 409 363 413 170 356 106 Experimental Group (ppm a.i.) Bird Da 8 Day 15 Change Day 8-15 Day 22 Change Day 15-22 293 1 179 447 268 731 284 2 165 415 250 646 231 3 217 526 309 748 222 4 291 516 225 704 188 5 203 430 227 676 246 Mean 211** 467** 256 SD 49 51 35 701* 234 41 35 *Statisticadlilfyferenftromthecontroglroupatp < 0.05(Durmett'ts-test). **Statisticadlilfyferenftrom thecontroglroupatp < 0.01(Dumiett'ts-test). Total Chang 552 481 531 413 473 490 54 WILDLIFE INTERNATIONAL, L-rD -59- APPENDIX VI IndividuaBlody Weights (g)from a Mallard Acute DietaryToxicityStudy withPFOS Page 14 PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Bird Day 8 586 1 126 2 209 3 217 4 5 143 Mean 174 SD 46 Day 15 Change Day 8-15 DR 22 Change Day 15-22 308 182 525 217 469 260 706 237 460 243 675 215 340 197 546 206 394 221 613 219 82 37 91 13 Total Change 399 497 458 403 439 47 Experunental Group (ppm a.i.) Bird Day 8 Day 15 Change Day 8-15 Day 22 Change Day 15-22 1171 1 - - - 2 3 4 5 185 383 198 Mean 1851 3831 1982 SD - - - ,In=1,couldnotbe evaluatedstatisticalulsyingDunnett'st-test. - - 634 6342 - 251 2512 - Total Change 449 4492 - WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-102 60- APPE-NDIX VU Feed Consumption (g/bird/dayb)y Pen from a Mallard Acute DietaryToxicityStudy with PFOS Page I ExperimentalGroup (ppm a.i.) Pen Exposure Perio@ Day 0-5 Post-Exposure Period Day 6-8 Control 1 109 145 2 92 140 3 85 117 4 93 132 5 82 100 6 94 116 Mean 92 125 SD 10 17 WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-102 -61APPENDIX VII Feed Consumption (g/bird/dayb)y Pen from a Maffard Acute DietaryToxicityStudy with PFOS Page 2 ExperimenW Group (ppm a.i.) Pen Exposure Period D2X 0-5 Post-Exposure Period DLy 6-8 9.1 1 73 130 2 72 104 Mean 73 117 18.3 1 90 135 2 91 128 Mean 91 132 36.6 1 103 129 2 85 120 Mean 94 125 73.2 1 79 109 2 74 94 Mean 77 101 146 1 89 102 2 121 217 Mean 105 159 293 1 28 61 2 59 65 Mean 44 63 586 1 40 43 2 32 67 Mean 36 55 1171 1 23 25 2 22 26 Mean 22 25 WILDLIFE INTERNATIONAL, LTD -62- PROJECT NO.: 454-102 APPENDIX VII Feed Consumption (g/bird/dayb)y Pen from a Mallard Acute DietaryToxicityStudy withPFOS Page 3 ExperimentalGroup (ppm a.i.) Pen Control 1 2 3 Mean SD Exposure Period Day 8-15 185 161 166 171 13 Post-Exposure Period Day 15-22 159 182 199 180 20 WILDLIFE INTERNATIONAL, LTD -63- APPENDLX VH PROJECT NO.: 454-102 Feed Consumption (g/bird/dayb)y Pen from a Mallard Acute DietaryToxicityStudy with PFOS Page 4 ExperimentalGroup (ppm a.i.) Pen Exposure Period Day 8-15 Post-Exposure Period Day 15-22 9.1 1 172 198 18.3 1 186 204 36.6 1 165 179 73.2 1 148 173 146 1 159 164 293 1 109 132 586 1 114 143 1171 1 106 154 WILDLIFE INTERNATIONAL, LTD -64- APPENDIX VIII PROJECT NO.: 454-102 CHANGES TO PROTOCOL This study was conducted in accordancewith the approved Protocolwith the following changes: 1 . The protocolwas amended toadd an additionaltestconcentrationof 10 ppm a.i.. 2. The protocolwas amended to indicatethatbilewillbe collectedfrom allstudy birds. The protocolwas clarifietdo indicatethecollectioonf liverfrom birdsthatdied duringthe courseof the study. 3. The protocolwas amended to change thetestconcentrationfsrom 0, 10, 20, 40, 80, 160, 320, 640 and 1280 ppm a.i.t,o 0, 9.1,18.3,36.6,73.2,146, 293, 586 and 1171 ppm a.i.Test concentrationwsere changed to reflecthe testsubstancepuritygiven in the new certificatoef analysis. 4. Individuallnyumbered wmg bands were used to uniquelyidentifyeach duckling. The protocol requiredlegbands to be used to identiftyhe birds. 5. Blood samples were collectedon Day 8 and Day 22 in non-heparinized5 ml borosilicatgelass testtubes. The protocolindicatedthatheparinizedvacutainerswould be used. 6. 'Me finaltemperaturefrom one brooderuniton Day 8 and allbrooder unitson Day 22 were not recorded. 7. 'Me afternoonobservationswere inadvertentlnyot recordedfor birdsin a singlebrooder uniton May 8, 1999. 8. Observationsforcontrolbirdsfrom two brooderlevelswere inadvertentlnyot recordedon Day 6 of the test. Temperatures for these brooder levelswere also not recordedon this day. Observationsforcontrolbirdsfrom one brooderlevelwere inadvertentlnyot recordedon Day 22 of thetest. WILDLIFE INTERNATIONAL, LTD PERSONNEL -65APPENDIX IX INVOLVED IN THE STUDY PROJECT NO.: 454-102 The followingkey WildlifeInternationaLltd.personnelwere involvedintheconduct or management of thisstudy: (1) Mark Jaber,WildlifeToxicologist (2)Joann B. Beavers,Director,Avian Toxicology (3)Sean P. Gallagher,SeniorBiologist (4)Courtney Casey, M.S., SeniorBiologist (5) Willard B. Nixon, Ph.D.,Manager, AnalyticalChemistry (6)TirnKendall,Supervisor,AnalyticalChemistry (7)Raymond L Van Hoven, Ph.D., Scientist (8)EllenMank, Chemist WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-102 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A DIETARY LC50 STUDY WITH THE MALLARD PROTOCOL NO.:454/111098fMLCSDT.WC/OPPTS SPONSOR:3M Corporation EFFECTIVE DATE: April22,1999 AMMMENT NO.: I PROJECT NO.: 454-102 ANMWDh4ENT: On page 2 of the protocoladd an additionatlestconcentrationof 10 ppm a.i REASON: Sponsor request. Study Director Laboratory M@migeme-ntSp@nsor PrincipalContact Date c:ot Date Lq Date WILDLIFE INTERNATIONAL, LTD PROJECT NO.: 454-102 AM[ENDMIENT TO STUDY PROTOCOL STUDY TITLE:PFOS:A DIETARY LC50 STUDY WITH THE MALLARD PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS AMENDMENT NO.: 2 SPONSOR- 3M Corporation PROJECT NO.: 454-102 EFFECTIVE DATE: April27,1999 AMENDMENT: On page 8 under necropsy add: When possibleb,ilewillbe collectefdrcm allbirds.Liverswillalsobe collectefdrom all.@irdtshat dieduringthe courseof the test. REASON:, BileisbeingcollecteadtSponsor request.The protocolisbeingclarifietdoindicatethecollection of liverfrom birdsthatche. St*Director Lab, )ratoryM"anag@m-@t Spnokco)rPMrincipDiCaolntact Date Ck Date q@q WILDLIFE INTERNATIONAL, LTD pap-,Ieof1 DEVIATION TO STUDY PROTOCOL STUDYTITLE: PFOS: A DIETARY LC50 STUDY WITH THE MALLARD PROTOCOL NO.: 454/111098/MLVSDT.WC/OPPTS DEVIATION NO.: I SPONSOR- 3M Corporation PROJECTNO.: 454-102 DATE OF DEFACTO DEVIATION: April22-May 14,1999 DEVIATION: huhviduallynumbered wing bands were used touniquelyidentifeyach duckling.The protocol statedthatbirdswould be identifiebdy coloredlogbands. REASON: Protocolerror.Due totheextendeddurationofthisstudy,ducklingswould have outgrown leg bands. The protocolshouldhave been elmifiedtoindicattehatallducldinp would be identified by wing bands. IMEPACR: None. Wing bands areroutineluysed to identifdyucklingsand allbirdswere uniquelyidentified. At@@ f . )DAL STUDYDMECTOR I -f DAff - DWEWATIONAL LT-D.MANAGEMENT DATE Una WILDLIFE INTERNATIONAL, LTD Page I of I DEVIATION TO STUDY PROTOCOL STUDY =E: PFOS: A Dietmy LC50 Studywiththemallard PROTOCOL NO.: 454/111098fi\4LCSDT.WC/OPPTS SPONSOK- 3M Corporation DATE OF DEVIANON: April30 and May 14,1999 DEVIATION NO.: 2 PROJECT NO.: 454-102 DEVIANON: Blood samplescollecteodn Day 8 and Day 22 offfitcestwere storedinnon-hepadnized 5 ml borosillicagtleasstesttubespriortobeingrefrigmateaThe protocolitidicatetdhatblood samples would be placedin labelledheparanizedvacutainers. REASON: Test animalswere too smallto effecientldyraw blood by syringe.Blood was draw by decapitatinbgirdsand drainingblood intotesttubes.Heparin was not added to blood samples due to a Sponsor requestto seperateserum from the blood at WildlifeIntemationd Ltd. Seperatim of serum requirescoagulatim of blood samples. IMEPACT: None. STUDY DIRECTOR WILDLIFE Il@rER@t'A-TIONAL LTD. MANAGENEENT DATE iiATE U4 WILDLIFE INTERNATIONAL, LTD Page I of I DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A DietaryLC50 Study withtheMallard PROTOCOL NO.: 454/111098/MICSDT.WC/OPPTS SPONSOR- 3M Corporation DATE OF DEVIATION: April30 and May 14,1999 DEVIATION NO.: 3 PROJECT NO.: 454-102 DEVIATION: On April30, 1999,the finaltemperaturefrom a brooderunithousing birdsinthe 640 ppni a.it.reatmentlevelwas not recordedpriortotemiinationofthosebirds.Additionallyo,n May 14 thefinaltmnperatm an allbrooderunitswere not recordedpriorto terminationof those birds. REASON: Biologistoversight The brooderpens where ternperatarewsere not recordedwere einpty atthe time of afternoonobservations.Brooder pen temperaturesaret@picallryecordedduring afternoonobservations.Biologistsrecordingafternoonobservationson theorcassionswhere temperatureswere missed neglectedto recordtemperaturesfor empty pens thathoused birds earlietrhe same day. EKIPACT: None. Temperaturesrecordedforbroodersthe day priorto themissed dataindicatedthat thebrooderswere fimctiomngproperly STUDY DIRECTOR WILDLIFE IkTERNkTIONAL LTD. MANAGEMENT r(II DAiz @-3 DATE WILDLIFF- INTE:RNATIONAL, LTD -Page 1 of I DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A DietaryLCSO Studywith theMallard PROTOCOLNO.: 454/111098/MLCSDT.WC/OPPTS SPONSOR- 3M Corporation DATE OF DEVIATION: May 9,1999 DEVIATION NO.: 4 PROJECTNO.: 454-102 DEVIATION: On May 8,1999, theafternoonobservationsforunitS 17-1housingbirdsfrom the 320 ppm a.i.treatmentlevelwere inadvertentlnyot recorded. REASON: Biologistoversight. IMPACR: None. Observationsfrom themorning of May 8 and May 9, 1999 indicatethataR birds inthislevelwere normal in appearanceand behavior.Sincethetemperatureforthatunitwas recorded atthe same time observationswere performed,itisapparentthatthe birdswere observed,but the observationnot recorded. STUDYDIRECTOR IV WILDLME II-TrERNA:T-10lqALLT-D.MANAGENIENT DATE @-3@cic) L% WILDLIFE INTERNATIONAL, L-rD Page I of I DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A DIETARY LC50 STUDY WITH THE MALLARD PROTOCOL NO.: 454/11109&/MLVSDT.WC/OPPTS DEVIATION NO.: 5 SPONSOP, 3M Corporation PROJECT NO.: 454-102 DATE OF DEFACTO DEVIATION: April28 and May 14,1999 DEVIATION.@ On April28,1999,Day 6 ofthetesto,bservationfsorcontroglroupbirdsinbrooderlevelSs62-2 and S62-3 were inadvertentlnyotrecorded.Temperaturesforthesebrooderlevelswere alsonot recordedon thisdate.On May 14,1999,Day 22 ofthetes@observationfsorcontrolgroupbirds in levelS70-3 were inadvertentnloyt recorded.The protocolrequiredthatbirdswould be observedatleastonce dailyand thatthebroodercompartmenttemperaturewould be recorded once daily. REASON: Biologisotversight. IMEPACT: With the exceptionof the missed observationso,bservationisndicateallcontrolbirdswere normal inbehaviorand appearanceand broodertemperaturewserewithintheacceptablreangefor the durationof thet6k Itisreasonabletoexpectthatbirdswerenormal in,brhaviorand appearanceand thatbroodertemperatureswere vathm the acceptablerange atthe time the observationwsere not recorded.Therefore,thisdeviatiocnausedno adverseimpact on the outcome and interpretatioofnthestudy. STUDY DIRECTOR i DATE E. )925t& WILDLIFE INTERNATIONAL LTD. MANAGENEENT DATE PAN WILDLIFE INTERNATIC)NAL, LTD SOP DEVIATION STUDY. TITLE: PFOS: A DIETARY LC50 STUDY WITH THE MALLARD PROTOCOLNO.: 454/111098/MLCSDT.WC/OPPTS SOP NO.: 415 SPONSOR.- 3M Corporation DATE OF DEFACTO DEVIATION: April23,1998 DEVIATION NO.: I PROJECT NO.: 454-102 DEVIATION: On April23, 1999 the temperatureof me brooderlevelused tohouse the 1290 ppm a.i. treatmentgroup was I'F below thespecifiemdinimum of 95'F. There areno recordsofthe respons'ibletechnicianor bicloglstmakmg a thermostatadjustmentto correctthetemperature. However, m thefollowingday thetemperaturewas withinnormal range. REASON: Misadjustnent of thebrooderunitthermostat. INTACR: None. There was no apparenteffed on testbirdbody weight orhealth. STUDYDIRECTOR WILDLWE RMRNATIONAL LTD. MANAGEMENT DATE WILDLIFE INTERNATIONAL, LTD it SOP DEVIATION go STUDY TITLE: PFOS: A DIETARY LC50 STUDY WITH THE MALLARD IN PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS SOP NO.: 423.IA DEVIATION NO.: 2 SPONSOR: 3M Corporation PROJECT NO.: 454-102 DATE OF DEFACTO DEVIATION: April22,1999 DEVIATION: On April??,1999,duringdietpreparatim Day 0 ofthetes@1.4659g oftestsubstance was weighed out for preparationof the 160 ppm ai.testconcentratiodniet.This amount exceeded the targetamountby 0.099g REASON: Dieticiaonversight. IWACR: None. The differenrien the amount of testsubstame weighed resultedin a less infmal dietaryconomtratiorl 1% change STUDYDMECTOR WILDLIFE &RERNATIONAI, T@T MANAGENffiNT DATE PROTOCOL PFOS: A DIETARY LC50 STUDY WITH THE MALLARD U.S. EnvironmentalProtectioAngency Series850 -EcologicalEffectsTestGuidelines OPPTS Number 850.2200 FEFRA SubdivisionE, Section71-2 OECD Guideline205 3M Lab RequestNo. U2723 WILDLIFE Submittedto 3M Corporation EnvironmentalLaboratory P.O. Box 33331 St Paul,Mim=ota 55133 INTERNATIONAL 8598 Commerce I)zive Easton,Maryland 21601 (410)822-8600 LTD. November 10,1998 PROTOCOL NO.: 454/111098/NMSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -2- PFOS: A DIETARY LC50 STUDY WITH nM MALLARD SPONSO SPONSOR!S REPRESENTATIVE: TESTING FACILITY- . STUDY DMECTOR LABORATORY MANAGEMENT: 3M Corporation EnvironmentalLaboratory P.O. Box 33331 St PAUL Minnesota 55133 Ms. Susan A. Beach WildlifbIntm-nationLatld. 8598 Commerce Drive Easton,Maryland 21601 Sean P.Gdlagher, SeniorAvian Biologist Joann B. Beavers Manager ofAvian To;dcology FOR LABORATORY USE ONLY Proposed Dates: Experimental S= Date: ProjecNto - L41sq - 102TestConcentr-a0t1itoolni-so1:.-1$-A6O%1kI0O't"b-0@,1l77%0v- Test Substance-No.: Experimental TmmiinatimDate: StudyRoom: -7(p tVtjL:IDn)a -tLb -7la PROTOCO-L APPROVAL SNMY D CTOR T, LABOI@KTORY CANA MENT DATE ql DATE/ SP SOR!SREPRESINTATIVE DAI PROTOCOL NO.: 454/111098ftvffCSDT.WCJOPPTS 3M LAB REQUEST NO. U2723 aim WILDLIFE INTERNATIONAL LTD. -3- OBJECRIVE Theobjectoifvtehisstudiystoevaluattheetoxicoiftaytesstubstantcotehemallar(dAnay platyrhynchaod.myu)ustertwhrougthedieftorfivdeays.An LC50valuweilblecalculaitfed, possible. SUMMARY Groupsoftm m&lud duckling5s-,10 daysold,willbefeddietcsontainintghetestsubstancaet selectecdoncentratiofnosrfivedays.A controlgroupthatr=ives untreateddietwillbe maintained concurrently.The testsubstancewillbe mixed mto dw che@usuallyin a geometricseriesof concentrafionTsh.e =posure periodwillbe followebdy aperiodofatleasthreedayson untreatedfee& At theend of thethree-daoybservatiopneriodh,alfof thebirdsineachtreatn=tgroupwillbe cuthnizedand liverand bloodcollectefdorresidueanalysisI.lm remainingbirdswillbe maintained on basaldietforan additiona1l4 days and ffimwillbe euthanizedw,ithEverand bloodcollected. Throughoutthetestt,hebirdswillbe observedfortoxicologicraelsponses.When possiblea,n LC50 valuewillbe calewatc& MATERLALS AND- METHODS The methods,specieussedandrouteofadministratidoenscribeidnthisprotocolarebasedupon procedurespecifieidntheU.S.EnvironmentalProtectioAngency Series850 -EcologicalEffectsTest GuidelinesOPPTS Number 850.2200 (1);Section71-2oftheEnvironmentaPlrotmfim Agenc3@s ftgistratiGmuidelinePse,sticidAessess?nenGtuidelinesF,LFRA SubdiWsionF,Hazard Evaluat*ion: WildlifaendaquaticOrganisms(2),ASTM StandardE857-87,"StandardPracticeFor Conducting SubacuteDietaryToxicityTestsWithAWan Species"(3),andOECD Guidelin2e05,AWan Dietary ToxicitTyest(4).Inordertocontroblias,birdswillbe assignetdopensby indiscriminadtreaw. No otherpotentiaslourcesofbiasareexpectedto affectheresultosfffisctudy. The testsubstancewillbe administereidnthediel Thisrouteofadministratiwoans selected becai, itrepresenttshemost likelryouteofexposureto avianspeciesintheenvironment. Test Substance h&rnmUOn on thecharactermtonof testc@ontroolr refe=ce substancesisrequiredby Good LaboratorPyracticSetandards(GLP). The SponsorisresponsiblfeorprovidinWgildlifeInternational IA wnttenvmficationthatthetestsubstancehasbeen charactenzeadccordingto GLPs priortoitsuse inthestudy.IfwrittevnerificatioofnGLP testsubstancceharacterintiiosnnotprovidedtoWildlife PROTOCOL NO.: 454/11109&/ULCSDT.WC/OPPTS 3M LAB REQIJEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -4- InternatiLotnda.il,twilblenoteidnthecomplianscteatemeonftthefinarleporTth.eattachfeodrm IDENTMCATION OF TESTSUBSTANCE BY SPONSOR (Appendii)xistobeusedtoprovide informationne=ssaryforGLP compliance. The Sponsorisresponsiblfeorallinformatiornelatetdothetestsubstanceand agreestoaccept any unused testsubstanceand/ortestsubstancecontainerrsemainingattheend of thestudy. TreatmgmtQ=S Groupsoftenmallardduckling(sfivpeerp=) willbe assignebdy indisaiminatderawtoeach of thetreat3nenatnd cmtrolgroups.The sexofeachindividuwailllnotbe determined.A testwill usuallyconsisotf ageometriscerieosffivedictuyconcentratioannsd atleasthreecontrolgroups.Test concentratiownisllbe selecteadfterevaluatintgoxicitdyataprovidedby theSponsor. The highesttest concentratinoenednotameod 5000 ppm. Each group-isfedtheappropriatteestorcontroldietforfive days.Thiswillbefollowedby a periodofatleasthreedayson untreatefdee& Controlbirdswillreceivaen amount ofthesolvenotrvehiclienffiediiretequivalentothehighest amountinthetreateddiets.Ifmore thanone studyisconductedsimultaneousliynthesame room, with thesame solventorvehiclea,concurrentcontrolmay be ufilizefdorallstudies. Durationof Test The primaryphasesofthetestandtheirdurationasre: I. Acclimatio-nFrom receipotrhatchofhatchlingusntilthestarotf exposure. 2. Exposure-Fivedays. 3. Post-eVosureobservatio-n3 days/17days. TeslBirds The rmffiarrdepresentasn ecologicallsyignificaanntd widelydistributsepdeciesintheUnited States.Tlw mallardhasdemonstratedsensitivittoyffiecffectpsroducedby known toxicchemicals. Moreover,thisspeciehsasproventobe a good laboratcamyodel and a largeamount of baselined,ataare available. AU mallardwsillbefrom thesame hatchandwillbe 5-10 daysoldattestinitiatioOnn.lythose thatareappa=tly healthywillbeusedinthetest Body weightsmay varybetween 75 and 300 grams dependingon theageofthecohortofbirdsused(e.g.5 versus10 days).At leastfivetestand three. PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. controlgroupswillbe used. Each treatmentOrcontrolgroup willcontaintenbirds(two pens of five Yirdseach)witheachgroup ofbirdsidentifiebdy Pcn number and testconcentration.Individualbirds willbe idmfifiebdy coloredlegbands. Birdswillbe obtainedfrom WhistlingWings, 113 Washington Sbw@ Hanover,IL 61041 or fi-omanotherreputablesupplier.AU experimentalbirdswillbe acclimated to thecagmg and facilitifersom the time of hatch or receiptuntilunt:tationf the test. Animal Di AU testbirdswillbe fed a game bird rationformulatedto WildlifeInternationaLltcL specificati(oTnasble1).Feed andwaterwillbe providedad libitumduringacclimatioannd duringthe test.Ilm watersourcewillbe theTown ofEastonmunicipalwatersupply.Feed isanalyzed periodically inaccordancewithWildlifeInternationaLltcLStandardOperatingProcedures.The birdswillreceive no form ofantibiotimcedicationduringthetest Specificationfsoracceptablelevelsof contaminantsingame birdrationfor avian specieshave not been establishedH.owever, thereareno levelsof contaminantsreasonablyexpected to be present in the dietthatareconsideredto interferweith thepurpose or conductof the study. Diet Pmparati Testdietswillbe preparedatthe startof thetestand a sufficienptortionestimatedfor the5-day exposure periodwillbe presentedto the birds.Ifnecessary@appropriatedietwillbe added as needed. Afterfivedays ofexposure,allbirdswillreceiveuntreatedbasalration. The testsubstancewillbe mixed directliyntotherationorby dissolvingor suspendingthetest substanceinacetoneand/ortablegrade com oilpriortonuxing withthefeed.Ifused,acetonewillbe allowedtoVoWiU= from thedietsduringthemixing procedure.Tablegradecom oilwin normallybe incorporateidntothedietata maximum of20/,(wlw) ofthefinaldiet.Mixing isnormally done with a Hobart mixer(Model Number AS200T). AU testsubstancecalculationwsillbe based on thepurityofthetestsubstanceas receivedor cmTecW to100*/oactiviengredienbtased on theinformationprovidedby theSponsor inAppendix Iof thisprotocol. PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -6- DietS=pling samples of the experimentaldietsYnU be rou=ted for chemicalanalysisto determinethe homogeneity and stabilitoyfthetestsubstanceinthe aviandietand toverify/mew= testconcentrations.Allsamples willbe placedinuniquelyidenfifiepdolypropylenejars. Samples willbe collecteodn Day 0, atdietpreparationt,o determinehomogeneity,measure test cOncentrat'Onsa.nd establisDhay 0 valuesforevaluatingstab'UYover the courseof the exposure periocl On Day 5,.atthe end of the exposurephase,samples willbe collectedfi-omthe feederscontainingthe experimentaldietsto assessstabilitoyf the testsubstanceunder'testconditions.The dietsampling scheme forstudieswithfivetestconcentrationisssummarized below. Tlw sampling scheme may be adjustedbased on theactualnumber of concentrationstested(seePage 2) or the optionof the Sponsor. PROPOSED NUMBERS ExperimentalGroup Control Level I -Low Concentration Level 2 Level 3 Level 4 Level5 -High Concentration OF SANTLES Day 0 Day 5 1 1 61 2 2 2 2 2 2 2 61 2 19 11 TotalNumber of Samples 30 Samples collectedfrom.the leftand rightsidesof thetop,middle and bottom layersof feed inthemixing vesselto determinehomogeneity. The above numbers of samplesrepresentthosecollectedfrom the testand do not mclude quality control(QC) samples such as matrixblanks and fortificatiopnrseparedand analyzedduringthe analyticaclhemistryphase of the study. PROTOCOL NO.: 454/111099/MLCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIC)NAL LTD. -7- Diet Analyses Samples ofthemxpaimental dietsmay be ana47,edfOtOwing collectioonr storedin a freezeruntil Prepared and/orextractedfor analysis.Chemical analysesof dietswillbe perforinedby Wildlife InternationLatlcL The analyticamlethod usedwillbe based upon methodology providedby the sponsor and identifieidnAppendix U. The methodology used toanalyzethetestsamples willbe documented intheraw dataand sununarizedinthefinalreporl Maximum sampleholdingtfinesp,riorto analysis, may be specifiedby theSponsor,and ifspecifiedw,illbe added tothisprotocolby anendmcnt Housine and EnvironmentalConditions Testbirdswillbe housed indu=ostaticauy controllebdroodingpens manufactured by Safeguard Products,Inc.or equivalentE.ach pen measures approximately62 X 9.2X 25.5 cm higIL The external waus, floorsand ceilingosf each pen areconstructeodfvinylcoatedwiregriclUpon initiatioonf the test,eachbroodingpen willcontainfivebirds.Two broodingpens willbe assignedtoeach treatment group (10 birdstotal)w,hile atleastsixpens willbe assignedto thecontrol(atleast30 birdstotal). Each group willbe idenfifiebdy pea nwnben On Day 8 of thetes@birdsinone pen from each treatinent group (5birds)and threepens from thecontrolgroup (15birds)willbe eudianized.Remaining birds willbe maintainedforan additiona1l4 days. During thatperiod,remainingbirdsmay be separatedinto two pens. During thefirsteightdays of the tes@the temperaturein thebrooding compartment of the pen willbe maintainedatapproximately29 *C. Brooders willbe turnedoffduringthelast14 days ofthe test. Acceptablerangesfor broodingcompartment temperatureswillbe specifiedin Wildlife InternationLatld.StandardOperatingProco&=. Broodingcompartn=t temperatureswillbe recorded once each day duringthe&st eightdays of thetest.The ambientroom temperaturewillrange between appmximatcly 15-30*C. Tcmpmture and relativheumiditywillbe recordedtwiredailyduring the test. A 16 hoursUght/8hours dark photoperiodwillbe maintained,and theaveragelightintensitpyrovided to thebirdsduringthetestwin be detmm3inc& Housing and husbandrypracticeswillb e conductedso as to adhereto the guidelinesestablished by NationalResearchCouncil(5). PROTOCOL NO.: 454/111098ftvfLCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -8- Observations AU birdswillbe observedatleastonx dailyformortalityand toxicologicarlesponses. Animal B& Mights/Feed Cons=ption Inchviduablody weightswillbe measured atthe imtiationof thetest,atthe end of the exposure periodm Day 5,and m Day 8. Average estimatedfeedconsumption willbe determinedby pen foreach treatn=t and controlgroup fortheexposure(Days 0-5)and post-exposureobservation(Days 6-8) periods. For thosebirdsmaintainedforan additional14 days,body weight and feed consumption measurements willbe performed weeldy and attesttermination.The accuracyof feedconsumption valuesmay be affectebdy unavoidablewastage of feedby birds.No attemptswillbe made to quantify the amount of wasted feed,as itisnormallyscatteredand mixed withwater and excreta. NecmpEy All testbirdsthatdieduringthecourseof thetestand allbirdseuthanizedwillbe subjectedto a grossnecropsy. For thosebirdseuthanizedon Days 8 and 22 of thetest,Evers and - 2 mL ofblood willbe collecteclLiverswillbe weighed,placedin individualabelledcontainersand storedfrozen. Blood willbe placedinlabellehdepafinizevdacutainerasnd placedunder refiigeratiorAlU samples will be transferretdothe Sponsor forresidueanalysis. Di.@positioonf Test Birds At testtermmtion, testbirdswillbe mwmuzed by using carbon dioxidegas,cervicaldislocation or otherappropriatmeethods. The method used willbe doemnented inthe raw data.All carcasseswin be storedfrozenpriorto shipment to the Sponsor. StatisticCaallculations The concentratimresponsedataobtainedfim thetestwillbe evaluatedand,ifpossible,an LCS 0 value and 95% confidencelimitswillbe calculated.When themortalitydata facilitatsetsatistical a,i@yses,one of threemeffiodswiU be used to calculatean LC50 value. The datawiU be analyzed,in orderofpref=am, by probitanalysist,hemoving averagemethod, orthebinomialprobabilitmyethod (6,7, 8,9). The choiceof method forcalculatintgheLC50 valuewillbe based upon themortality patternobserved.When possiblea, no mortalitylevel'wflble determinedand reported. PROTOCOLNO.: 454/111098/MLCSDT.WCIOPPTS 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -9- RECORDS TO BE MAINTAINED Recordstobe maintainedfordatageneratedatWildlifIenternationLatlctvnU includeb,utnot be limitedto: 1. A copy ofthesignedprotocol. 2. Idenfificatainodn characterizatioofnffitcestsubstancei,fprovidedby theSponsor. 3. Datesofinitiatiaonndterminatioonfthetesl 4. Animalhistory. 5. Husbandryandenvironmentaclonditions. 6. Cmcentrationcalculatioannsdrecordsofdietpreparation. 7. Body weightmeasurements. 8. Feed consumptionmeasurements. 9. Dailyobservations. 10. N=opsy findings. 11. Statisticcaallculations. 12. Analyticaclhemistrymethods,resultasnd chromatogramsi,fapplicable. 13. A copyofthefirarleporl FINAL REPORT A reporotftheresultosfthesh* wilblepreparebdyVtrilcMIntematiomtLLtcLThe reportwill includeb,utnotbe limitetdo,thefollowing1. Name and addressofthefacilitpyerformingthestudy. 2. Datesm whichthestudywas initiateadndcompleted.ItistheresponsibiliotfytheSponsorto providethe finaldatethatdataarerecordedfor chenustryp,athologyand/orsupporting evaluationtshatmay be generatedatotherlaboratories. 3. A statementofcompliancesignedby theSh* Directoarddressinagny oxeptionstoGood LaboratoryPracticSetandards. 4. Objwtivm andproce&m stateidntheapprovedprotocolincludinagny changesintheori&al pmtocol 5. Statisticmaelthodsemployedforanalyzintghedataw,hen applicableT.he calculateLdC50 value,95 percentconfidencelimitss,lopeof theroncentration-respocnusreve,theresultosf the goodness-of-fittest(e.g.V test)a,nd a descriptioonf statisticmaelthods usedl The same stafistewsillbe providedforpositivceontrol(swhenused). PROTOCOLNO.: 4s4/illO98/NILCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -10- 6. . The tes%controland referencseubstancesidentifiebdy name, chemicalabstractsnumber or code munber,strengtkpmty, and camposibon or otherappropriatecharacteristicisf,provided by the Sponsor. 7. Stabilitaynd,when relevanto theconduct.ofthestudy,thesolubilitoyfthe test,controland referencesubstancesunder theconditionsof administrationi,fprovidedby the Sponsor. 8. A descriptimofdw test*,om used.Wh= applicablet,hefinalreportshallincludethenumber of animalsused,body weightrange,sourceof supply,species(includinsgcientifincame), age, and procedureused foridmtificatiom .9. A descriptioonf thedosage,dosage regimen,routeof administrationa,nd duration. 10. A descriptioonfthemethods used,includingbut notlimitedto: a) Detaileddescriptioonf thebasaldic@includinsgource,dilumts (ifused),and supplements (ifused).A nutrienatnalysisofthe dietwillbe includedinthetestreport. b) The number of concentrationussed,nomirad and (where required)ineasureddietary concentrationof testsubstancein each level assaymethod used to determine actual concentrationnsu,mber ofbirdsper concentratioannd forcontrol,and names of toxicants used forpositivecontrol(sifapplicable)R.esultsofrange-findintgest(ifcmducted). c,) Acclimationproceduresand methods of assigningbirdstotestpens. d) Frequency,durationand metbo& of observation.Descriptim of signsof intoxicatioannd ofl= abnornudbehaviori,ncludin6g= ofomt, durations,everit(yincludindgeath),and numbers affww inthedffercnt&etary concentraums and controlseach day ofthetest period. e) Description*ofhousing conditionsi,ncluding'types,izeand materialof pen, pen temperatures,apprommatr,testroom humidity,photoperiodand lieibngintensity. f) Average body weightsforbirdsineach pen at thebeginningof the tes%the end of the exposure periodand end of the tesl g) Estimatedfood consmnption forfficexposure periodand forthepostexposure period. 11. A descriptionof allcircumstancetshatmay have affectedthequalityor integritoyf the dat& Anything unusual aboutthetest,any deviationfrom theseprocedures,and any otherrelevant information. 12. Mz name oftheSt* Dhctor, thenames of otherscientistosrprofessionalsa,nd thenames of allsupervisorypersonnel,involvedin thestudy. 13. A descriptioonf the transformationcsa,lculationso,r operationsperformed on the data,a summary and analysisofthedata,and a statementof ft conclusionsdrawn from fficanalysis. PROTOCOL NO.: 4541111098/NILCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. 14. The signedand dated reportsof each of theindividualscientistosr otherprofessionalsinvolved in the study,ffapplicable. 15. The locationwhere allspecimens,raw data,and thefinalreportaretobe stored. 16. A statementprepamd by the QualityAssurance Urnt listintghedatesthatstudy inspectionsand auditswere made and thedatesof any findingsreportedto the Study Directorand Manag=ent. 17. Ifitisnecessarytomake correctionosradditionsto a finalreportaft ithas been accepted,such changesdmU be m theform ofammjmcnt by theSb* Du=Wr The amendment should clearly identifytheparto.f thefinalreportthatisbeingadded to orcorrectedand thereasonsforthe correctioonr addition.Amendments shallbe signedand datedby theStudy Director. CHANGING OF PROTOCOL Planned changes to the protocolwillbe in theform of writtenamendments signed by the Study Directorand theSponsces Representative.Amendments willbe consideredas partof theprotocoland willbe attachedto theEmalprotocol Any otherchanges willbe in theforxnof writtendeviationssigned by the Study Directorand filedwith theraw data.AU changestotheprotocolwillbe indicatedinthe finalreporl GOOD LABORATORY PRACRICES Thisshidywillbe conductedinaccordancewithGood LaboratoryPracticeStandardsforEPA (40 CFR Part 160 and/orPart792);OECD PrincipleosfGood LaboratoryPractice(OCDE/GD (92) 32, Envirorment Monograph No. 45); and Japan MAFF (59 NohSan, NotificatioNno. 3850, AgriculWralProductionBureau). Each sb* conductedby WildlifeInternationLatlcL isroutinely examined by the Wildlifehit=nationaLltd.QualityAssuranceUnit forcompliancewith Good Laboratory PracticesS,tandardOperatingProceduresand the specifiedprotocol.A statementof compliancewith Good LaboratoryPracticeswillbe preparedforaU portionsof the study conducted by WildlifeInternationaLltd. Tbc Sponsor willbe responsiblfeorcompliancewith Good Laboratory Practicefsorprocedurespmfom3cd by otherlaboratorie(se.g.r,esidueanalysesor pathology).Rawdata forallwork perf=ied atVMdl&e InternationLatld.and a copy ofthefinalreportwillbe filedby project number in archiveslocatedon theWildlffeInternationaLltd.siteor atan alternativleocationto be specifiedin thefinalreport. PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. REFERENCES I U.S.EnvironmentalProtectionAgency. 1996. Scrics850-EmlogicalEfefcts Test Guidelinse (draft),OPPTS Number 850.2200: AWan Dietary Toxicity Test 2 U.S. Envirorumental Protection Agency. 1982. PesticideAssessment Guidelines,filFRA Subdi,4sion E, Hazard Evaluation: Wila7ifeand Aquatic Organisms, subsection 71-2, EnvironmentalProtectionAgency, OfficeofPesticideProgmm. Washington,D.C. 3 American Societyfor Testing and Materials. 1997. StandardPracticeforConducting SubacuteDietaryToncity TestsvnthAvian Species.ASTM StandardE857-87. Annual Book of ASTM Standards,Vol. 11.04.PhiladelphiaP,A. 4 Organization for Economic Cooperation and Development 1984. Avian Dietary Toxicity Test. OECD Guddelinefor Testingof Chemicals. Guideline205. Paris. 5 National Research CounciL 1996. Guide for the Care and Use oflaboratory Animals. Washington, DC. National Academy Press. 125 pp. 6 F-mney,D. J. 197 1. StatisticaMlethods in BiologicalAssay, 2nd ed.,GriffinPress,London. 7 Thompson, W. R. 1947. BacteriologicalResiews, Vol 2, No. 2: 115-145. 9 Stephan, C- E. 1977. Methods for Calculatingan LC50. Pages 65-84 JUAquatic Toxicology and Hazard Evaluations, American Society for Testing and Materials. Pub. No. STP 634. PhiladelphiaP,A- 9 Stephan,C- E. 1978. U.S. EPA, Eaviro=ental ResearchLaboratory,Duluth,MN. Personal Communication. PROTOCOL NO.: 454/111098/ULCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 film Um WILDLIFE U4 raw to INTERNATIONAL, L-m. -13TABLE 1:DIET FORMULATION WILDLIFE DMPNATIONAL LTD. GAME BIRD RATION' INGP@EDMNrrS FineCom Meal Soy Bean Meal,48% Protein Wheat Ndds ProteiBnase Agway SpeciaL60% Protein AffWa MeaL 20% Protein DriedWhey Ground Limestone Eastman CalPhos MethioninePremix+ Liquid Vitudn andMineralPremix(seebelow) GL Ferm (Fermatco@.'. SaltIodized Total PERCENR 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 VITAAIIN AND NIINERAL PRENIIX AMOUNT ADDED PER TON VitaniiD3n VitaminA Ribonavin Niacin PantotheniAccid Viwain B,2 FoUc Acid Biotin Pyridoxine Thiamine VitaminE VitmninK (MenadioneDimethylVflimidiBnioslulfite) Manganese Zinc Copper Iodine iron Selenium 2,000,000LC.U. 7,000,000LU 6 grams 40 grams 10 grams 9 mgs 600 mgs 64 mgs 1.2grams 1.2grams 20,000l.U. 5.8grams 102gmm 47 grams 6.8gmms 1.5grams 51 grams 182 mgs The guaranteeadnalysiissa minimum of27% proteiza4nu-nimumof2.5% crudefatand a of5% crudeMer. 2 FermentatioBny-Pmducts PROTOCOL NO.: 454/111098ftALCSDT.WCIOPPTS 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -14- APPENDIXI IDENMICATION OF TEST SUBSTANCE BY SPONSOR To be Completed by Sponsor L TestSubstanceIdentit(ynm= tobe used inthereport):PFOS (PfffluorooctanSeulfonicAcid potassium Bait ReferenceStandard(ifapplicable)A:nalyticalStandard: N/A InternalStandard-.1.1.2-21LH-Hg PerfluorooctanSeulfgnicAcid TestSubstanceSample Code orBatchNumber- Lot 217 Test SubstancePurity(0/AoctiveIngreZent): 98.9 ExpirationDate: 2008 E[. Test SubstanceCharacterization Have theidentity, e gemtty and compositioonrothercharacteristics which 4ppropriatS6'iym_dek c testsubstanceand referencestandardbeen detmmifi6dOriortoitsuseinthissh* inaccordancewithGLP Standards? Yes No X M. Test Substance StorageConditions Pleaseindicatethe recommended storageconditionsatWildlifeIntcmatiormlLtd. Ambient room I=erature Has thestabilitoyf the testsubstanceunder thesestorageconditions been determinedin accordancewith GLP Standards? Yes No -X- Other perfinentstabilitiynformation: IV. TestConcentrations: Adjusttestconcentratiotno 100% ai. X based upon thepurity(*/og)ivenabove. Do not adjusttestconcentratiotno I00*/o &L Testthematerial V. ToxicityInformation: MamnidiatL Aquatic: Rat LD50 251 mwkg Mouse LD50 N/A InvertebratTeoxicity(ECILC50) FishToxicit(yLC50) D;VbWmaamna: 27 me/L DVImia magna: 50 mg& Rainbow Trout: 11 mgll Fdhcid MWnw: 38 mg& Other ToxicityInformation(includingfindingsofchronicand subchromc tests): Pleasesee MSDS PROTOCOL NO.: 454/111098/1@ECSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -15- APPENDIXII Method Outlineforthe Detmm3inationof PFOS inAvian Feed 1. Preparematrixfordficatiosnamples inthe desiredavianfeed stockusingthe dly mix technique. 2. Weigh 10 g samplesof thematrixblank and matrix fortificatifoenedsamples intoa weigh boat and transferto 16 oz. French squareglassbottles.Record weights. 3. For eachweighed sample,measure 100 mLs ofmethanol with.agraduatedcylinderand transfer intothe French square bot'tle, 4. Seal bottleswith screw caps and placeon shakertable.Allow to shake fora minimum of @O minutes. 5. Vacuum filtewrith qualitativfeiltepraper and rinseretainedfeed 3 fi=s with methanol into filtrate. 6. Tran filtratientoa 200-mL volumetricflaskand bringtovolume withmethanol 7. Transfer 5 mLs of 200-mL solutionintoa 10-mL volumetricflaskpartiaUy-fiUewdith 5001c mcdmol:50% water dilutiosnolventcontaining0.05% v/v formicacidand 100 mg a.i./mL 4HPFOS internasltandarclBring tovolume withdilutiosnolvent 8. Mix and transferapproximately1 raL of theI0-mL solutionintoa syringeand filtetrhzougha 0.2izm filteirntoan BPLC autosaniplevrial. 9. Ampulate and submitswnple forLCMS analysis. PROTOCOL NO.: 454/111098/hff-CSDT.WC/OPPTS 3M LAB REQUEST NO. U2723