Document Z4w4v6DjLJKMbEYXOvnn18r50

. - pRNG-OI03 PFOS: ADIETARY LC50 STUDY WITH THEMALLARD `WILDLIFE INTERNATIONAL LTD. PROJECT NO. 454-102 3M REQUEST NO. U2723 U.S. Environmental Protection Agency `Series 850 -- Ecological Effects Test Guidelines(draft) 'OPPTS Number 850.2200 ; FIFRA Subdivision E, Section 71-2 (OECD Guideline 205 AUTHORS: Sean P. Gallagher Courtney S. Casey JRoaaynmnoBn. dBLe.avVearns Hoven STUDY INITIATION: April 21, 1999 STUDY COMPLETION: April 26, 2000 SUBMITTTEOD 3M Corporation Envi5r3on5meBnutsahlALvaebnouraetory St. Paul, Minnesota 55144 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1065 01275 We ILDLIFE INTERNATIONALe , LTD e PROJECT NO.: e4m54s1-0102 2. `GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Dietary LC50 Study with the Mallard `WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-102 STUDY COMPLETION: April 26, 2000 `This study wasconducted incompliancewith the U.S. Environmental Protection Agency, 40 GoodLaboratory Practice CFR Part 160 and 792, Standards as publishedby 17 August 1989; OECD Principles ofGood 1992); and Japan Laboratory MAFF, 59 Practice, NohSan, (OCDE/GD(92) Notification No. 32, Environment Monograph No. 45, Paris, 3850, Agricultural Production Bureau, 10 August 1984 with the following exception: The test substance was not characterized in accordance with full GLP compliance; however, the rcahwardaactteariazrateiboeniwnagsmapienrtfaoirnmeeddianctcohredin3gMtaorc3hMivSetsa.ndTahredOtpeesrtastuibnsgtaPnrcoceiedsubreeisnagnrdecMhetahroadcst,earnidziealdnl `accordance with GLP. `The stability of the testsubstanacndereference standard underconditions of storage at the test site: `was not determined in accordance with Good Laboratory Practice Standards. STUDYDIRECTOR: Mi`ne P= Blt Sean P_ Gallagher Senior Biologist, Avian Toxicology SPONSOR'SREPRESENTATIVE pate__ [M10 L'Mo SSusnanAe . Beach0. Lond oate_f Ja /60 01276 WILDLIFE INTERNATIONAL, LTD -3- PROJECTNO.: 454-102 QUALITY ASSURANCE STATEMENT "This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CER Part 160 & 792, 17 August 1989; OECD PrinciplesofGood Laboratory Practice, (OCDE/GD(92) 32, Environment Monograph No. 45, Paris, 1992); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. Thedatesofallauditsandinspectionsandthedatesthatanyfindingswere reportedto the StudyDirectorand Laboratory Management wea frolloews: ACTIVITY DATE CONDUCTED ~~ Matrix Fortification, TAneasltyStuicbasltaSnacmeplPrienp.g, April 22, 1999 FeAenadlyCtoincsaulmSpatmipolniangnd April 27, 1999 NeLcirvoerpsWye,igBhltosodanCodlBleiclteion, Collection May 14,1999 ADnralayftitcRaelpDoartta and July 7,8, and 9, 1999 BDirolaofgitcRaelpDoartta and Aug 30,31, Sept 1,199 Final Report April 18, 2000 STUDYDDAITREECRTEOPRORMTEADNATGOEMENT April 2, 1999 April 27, 1999 April 23, 1999 May 4, 1999 May 14, 1999 July 9, 1999 September 11999 April 18, 2000 May 21,1999 July 16, 1999 Septembe3r, 1999 April 19,2000 Swan A. Coleman SSeunsiaonrLQ.uaCloilteymAasnsurance Representative DaTE___4-19-00 0177 WILDLIFE INTERNATIONAL, LTD 4 REPORT APPROVAL PROJECT NO.: 454-102 SPONSOR: 3M Corporation TITLE: PFOS: A Dietary LC50 Study with the Mallard `WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454-102 3M LAB REQUEST NO.: U2723 STUDYDIRECTOR: Ju/an - dled, . Sean P. Gallagher Senior Biologist, Avian Toxicology hoo Date: CHEMISTRYPRINCIPAL INVESTIGATOR: . "CS/an Hoven, PED Scientist, Analytical Chemistry 4-26-00 Bae REPORTAPPROVEDBY: TAOSbmn x vers Dae Director, Avian Toxicology Lil BY `WillarBd. Nixon, Ph. Date Manager, Analytical Chemistry dike Wil : 01273 WILDLIFE INTERNATIONAL, LTD "$e PROJECTNO. dsi-102 `TOA F CB ONL TENE TS TITLEPAGE........... ee Page|. GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT .....................Page 2 QUALITYASSURANCE STATEMENT ............ REPORT APPROVAL... TABLE OF CONTENTS cc `TaandbAplpenedicses re Page3 Pog4 Page 5 en P2BE6 SUMMARY. . mee Page 1 INTRODUCTION. SR -- LR OBTBCTIVE ovum cosnsiviimvsstsborsias moinsambos agosirais .....Pag8e MATAE NDMR ETI HODA S....L ...ocoS mermssuasmssrsssasmmsssssssmmsirsmss Page 8 TESUSUBSIACE Page 8 DurationoftheTest. OE Sad ATAIDIS ee I page10 Ee Page10 AnaDIlCyEtiScAaDlHNMGe.t.h.od ttre PPaagge1100 Observations............. AnimalBody Welghts/FeedCORSUmPHON. Statistical Aalyoes z I page 3 rrr Page14 pages RESULTS -- _-- Page 14 Moratnd aClinlicaliObtseervsations... CT pages Body Weightand Feed COSUMPON..........cc.c.vevvrecevivvnenesssnsnesesreson Page 16 Gross Necropsy ........ . - rveeirseseeseeee. PAGE IT CONCLUSION. ..counmmmmnussrussissrenmssmmmemsmmmmessmsssmmmsmissommmmsimmmmmmssmmi REFERENCES EE 2g 18 Page19 01279 WILDLIFE INTERNATIONAL, LTD -6- PROOIJEECCTNTON.O.: ds-45-1102 `TABLE OF CONTENTS - continued - `TABLES AND APPENDICES TABLE 1: TABLE2: TABLE3: TABLE 4: TABLES: SCRuEmuYlaVtRivAeEMEorOtRality cfrvomvaeMmamllmamrdmAmcumtmemDimeatamrynTroxrivcietysmmnn 2303% SMealn FBoRdRyTWTeOiRght (s) fcrsoammMramllmanrmdAmcmumtemmDimetmamryeTroxsimcimtymsmnd igs Mean Feed Consumption g/bird/day) froam Mallard `AcuteDictaryToxicity SAY ith PROS... PaBE 23 Gross Pathological Observations DietaryToxicity Studywith from a PFOS. Mallard As cute mog28 MCaulmlualradtiAvceutMeorDtiaeltiatryy(TEosxtiicmiattyedStCuudmyulwaittihvPeFDOoSs.e, mg/kg)from a Page 26 APPENDIXTL CertiOff AicBaBtIeSSIS....rsssnn Pago 21 APPENDIIXE: Dit FOMMUHON.....ecreren P2028. APPENDIX I: Analytical Methods and RESUS... P20 29 APPENDIX IV: Diet PROPAIBON. cco: BOGS 43 APPENDIVX: `CAucmuutleDaiteitvaerMyoTrotxailcitiytybyStPuednyvfirtohamPMRallOardS... PIE 44 APPENDIVXI: `IAncduitveidDuiaeltaBroydTyoxWieciigthysSt(u5d)yfwitrhaPMoaFlOlSmard Page 45 APPENDIX VII: MFaeleldaCrodnAscuumtpetiDoient(agr/ybTirodx/idcaiyt)ybSytuPdeynfwriothm PaFOS ............... Page 60 APPENDIXVIII: Cha1n0PgOWeOCsOL cov SE APPENDIX IX: Personnel Involved in the Study ..... - Page 65 01280 WILDLIFE INTERNATIONAL, LTD 1 PROJECT NO.: 454-102 SUMMARY SPONSOR: 3M Corporation "TEST SUBSTANCE: PFOS `WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-102 STUDY: PFOS: A Dietary LC50 StudywiththeMallard RESULTS: "The dietary LCS50 value for Mallards exposed to PFOS was determintedo be628 ppm a.i. with a 95%confiindterevalnofc44e8ppma.i. to 958 ppm ai. The slope ofthe concentration-response curve was 3.67 andthechi square value was 2.13. The no mortality concentration was 146 ppm a.i Baseduponreductions in bodyweightgainatthe 73.2 ppm ai. test `contc hene o-obn sert vedr -effa ectt conci ento ratin onw, as 36.6 ppma.i. TEST DATES: NOMINAL TEST CONCENTRATIONS: TEST ANIMALS: Hatc-h April 13, 1999 Acclimation - April 14-22, 1999 Experimental Start April 22, 1999 Experimental Termination 0,9.1, 18.3, 36.6, 73.2, 146, 293, 586, and 1171 ppma.i. Mallard (Anas platyrhynchos) AGETESTANIMALS: 10daysofageattestinitiation SOURCE TEST ANIMALS: Whistling Wings, Inc. PO Box I-A H1a1n3oWvaesr,hiInLgt6o1n04S1t.-3512 STUDY COMPLETION: April 26, 2000 041281 WILDLIFE INTERNATIONAL, LTD -8- FROJECTNO. dst-102 INTRODUCTION This study was conducted by Wildlife International Ltd. for 3M Corporation at the Wildlife Intemational Ltd. toxicology facility in Easton, Maryland. The in-life portionof the test was conducted from April 22, 1999 to May 14, 1999. Raw data generated at Wildlife International Ltd. and acopy of thefinalreportarefiledunder Project Number454-102inarchiveslocatedonthe `Wildlife International Ltd. site. OBJECTIVE Theobjecotfitvhiesstudy wastoevaluatethetoxicity of atestsubstanceto the mallard (Anas possible. latyrhynchos) administered through the diet for five days. An LCS0 value will be calculated, if MATEARNDIMAETLHODSS Themethodsusedinconductingthisstudyarebaseduponproceduresspecifiedinthe U.S. Environmental Protection Agency Series 850 Ecological Effects Test Guidelines OPPTS Number 850.2200 (1) Section 71-2 of the Environmental Protection Agency's Registration Guidelines, Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms (2); OECD Guideline 205, Guideline for Testing of Chemicals, Avian Dietary Toxicity Test (3); and upon ASTM Standard E857-87, "Standard Practice for Conducting Subacute Dietary Tonicity Tests with Avian Species" 4). TestSubstance The test substance was received from 3M Corporation on October 29, 1998 and was assigned `Wildlife International Ltd. Identification Number 4675 upon receipt. The test substance was white powder identified as: FC-95;Lot No.:217. The reported purityofthetestsubstance was 98.9%, with an expiration date of 2008. Followingtest termination, the test material was reanalyzed. The results of reanalysis indicate atest substance purityof 90.49%. All test concentrations have been adjusted to reflec the purity reported on the new Certificate of Analysis (Appendix I), The tes substance was stored under ambient conditions. The intemal standard was received from 3M Corporation on July 2, 1998 and was assigned `Wildlife International Ltd. identification number 4526 upon receipt. The intemal standard, a granular 01282 WILDLIFE INTERNATIONAL, LTD 5. PROJECT NO: dsi-102 material, was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2. The standard was stored under ambient conditions TreatmentGroups "Thetestconsistedof ageometricseriesofeighttestconcentrationsand acontrolgroup. Thirty `mallard ducklings were assigned tothecontrol groupandten mallard ducklings were assigned to each ofthe treatment groups. Birds were sorted by weight, then chosen indiscriminately from within each represented weight class for placement into control and treatment groups. The birds were housed in brooding pens containing five ducklings each. On Day 0, following experimental start, the Sponsor requtheeadsdittioneofda 10 ppmtestconcentration. Birdsfrom thesamelotthatremainedaftertest initiationwereusedfortheadditionaltestconcentration. Nominaldietaryconcentruasetdiiontnhiss study were 0, 9.1, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 parts per million active ingredient (ppm ai). The dietary concentrations wereestablished based uponknown toxicitydata andinformation supbpy tlheSipoensodr. Eachgroupwasfedtheappropriatetestor controldietforfivedays. Duthr eexpi osurn eperg iod thecontrolgroupreceiveduntreatedfeed. Followingthefive-day exposureperiodallgroups were givenuntreated basaldietforthree days.OnDay 8, halfof the treatment andcontrolbirdswere `euthanized and liver weight and tissue, blood, and bile samples collected for analysis. The remaining `birds were fed basal ration until Day 22. On Day 22, these birds were sacrificed and also sampled for liver weiangdtihssute,blood,andbile. DuraotftiheToesnt `Theprimary phasesof thistestand their durations were: 1. Acclima-t9diaoysn. 2. Exposure - 5 days. 3. Postexposureobservation - 3 or 17 days TestBirds Al mallard (dns playrhynchos ) were 10 days ofage and appeared to be in good health at initiationofth test. The birds were obtained from Whiting Wings Inc., Hanover, IL. and were `hatched on April 13, 1999. Birds ranged in weight from 96 to 204 grams at test initiation. The birds usedinthisstudywereimmatureandcouldnotbedifferentiated bysex. All bi werr efrd omts he 01283 WILDLIFE INTERNATIONAL, LTD -10- FROJECTNO. dsi-l02 same hatch, pen-reared and phenotypically. indistinguishable from wild birds. All birds were acclimatedtothecagingandfacilitiesfromthedayafterhatchuntilinitiation ofth test. AnimalDiet Throughoutacclimation andtestingalltestbirdswerefed agamebirdrationformulatedto Wildife Intemational Ltd's specifications (Appendix II). Water from the town of Easton public water supply, and feed were provided ad libitum during acclimation and testing. The birds received 0foofar ntibimotic medduirincgacaclimtatioinorotestning. DietPreparation Thetestsubstancewasmixeddirectlyintotheration.Mixing wasdonewith aHobartmixer (Model Number AS200T). All dietary test concentrations were adjusted to 100% PFOS based upon thereportedpurityofthetestsubstance. AllditaryconceanndthteLrCSa0vatlueiaroerenporsted asppm ai. inthediet. Nominaldietarytestconcentrationsusedinthsstudywere 9.1, 18.3, 36.6, 73.2, 146,293, 586 and 1171 ppm a.i. (AppendixIV). Dict Sampling Samplesofthetest diets were collectedtoverifythetestconcentrationsadministered and to confirm thestability andhomogeneityofthetestsubstanceinthedicts. Homogeonfethietteyst substanceinthedictwas evaluatedbycollecting sixsamplesfromthe 9.1 ppm a. ai.andsix samples from the 1171 ppm a.. ai. test dicts at preparatioonn Day 0. Homogeneity samples were. collectedfromthetop,middleandbottomofthelefandrightsectionsofthemixingvessel. The homogeneity samples also served as verification samples. One verification sample was collected fromthecontroldiet andtwoverificationsampleswerecollectedfromeachremainingtreatment. group at preparation on Day 0. At the end of the exposure period (Day 5), one sample was collected from the control and two samples were collected from each treatment group to determine stability of thetestsubstinathnecdieet. Thestabilitysampleswerecollectedfromfeed remaininginthefeeders after being at ambient test pen conditions for five days. Samples were transferred immediately to `Wildife Intemational Ltd. analytical chemistry. AnalyticalMethod Themethodusedforthe analysisoftheaviandietsampleswasbased uponmethodology developed at Wildlife International Ltd. and entitled "Method Outline for the Determinationof PFOS CG1281 WILDLIFE INTERNATIONAL, LTD Se PROJECTNO. ds-toz in Avian Feed". Aviandiet samples wereextracted withmethanol, Methanolwasaddefo da requisite quantity of feed contained in a French-square glass bottle. Bottles were capped and shaken ona shaker table. Samples were vacuum filtered using qualitative filte paper. The retained feed was rinsed three times with methanol into the filtrate. The filtrate was transferred to a volumetric flask and brought to volume with methanol. As appropriate, samples were further diluted with methanol Each sample then was diluted with a 50% methanol: 50% NANOpure water solution containing 0.100mg 4H PFOS (internal standard)L and 0.05% formic acid (v/v) so they fell within the calibration range of the PFOS methodology. A method flowchart is provided in Appendix II, Figure 1. Concentrations of PFOS in the standards and samples were determined by reversed-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a PerkinElmer TurbolonSpray ion source. HPLC separations were achieved using a Keystone Betasil Cs analyticalcolumn (100mm x 2mm LD, 3pmparticlesize). Theinstrument parametersare `summarizedinAppendix II, Table 1 Calibration standards of PFOS prepared in a 50% methanol : 50% NANOpure water solution containing 0.100mg 4H PFOS (internal standard). and 0.05% formic acid (v/v), ranging in concentrationfrom 0.00229to0.0457mg a.i/Lwereanalyzedwiththesamples. The saanmdmoest prominent peak response for PFOS was utilized to monitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOSonthe basisofindividual isomeric components. Linear regression equations were generated using peak area response ratios (PFOS intemal standard) versus the respective concentration ratios (PFOS: internal standard) of the calibration standards. A. typical calibration curve is presented in Appendix Il, Figure 2. The concentrationof PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms of low and high calibration standards are presented in Appendix I, Figures 3 and 4, respectively. "Themethodlimitof quantitation(LOQ) fortheseanalyseswassetat 1.15ppma. calculataesd the productofthe lowest calibration standard analyzed (0.00229 mg .i./L) and the dilution factor of thematrix blank samples (S00). 1285 1285 WILDLIFE INTERNATIONAL, LTD "ne PROJECT NO.: 454-102 Two matrix blank samples were analyzed to determine possible interferences. No interferences were observed at or above the LOQ during sample analyses (Appendix III, Table 2). An interference inthefeed appearedatapproximatelythesameretention time asthepeakofinterestbutit waswell below the LOQ. A representative chromatogramof a matrix blank is presented in Appendix III, Figure 5. Avian diet was fortified at 4.57, 183 and 1830 ppm a.i. and analyzed concurrently with the samples to determine the mean procedural recovery (Appendix III, Table 3). Sample concentrations `werenotcorrectedforthemean proceduralrecoveryof94.7%. Arepresentativechromatogramof a `matrix fortification is presented in Appendix III, Figure 6. An example calculation is presented for sample number 454-102-2, nominal concentration of 18.3 ppm a.i.inaviandiet. Intl Weight: 100 Final Volume: 200 mL DilutionFactor: 100 (intermediatedilutionfactor PFOSPeakArea: 113568 Internal StandardPeak Area: 413160 Peak Area Ratio: 0.2749 finaldion factor) Calibration curve equation Slope: 2.77397 Intercept: 001894 Curves weighted (1). PFOS (mg a/L)atinstrument =(( (oPaeakY kaarreee aarraatitoi io=-(tVinte eceSpir to)pe oxx1Lc SS,. CCp oonncceey nnttrraa) ttiioonn _ 02749-00189 x 0.100 mg. 271397 = 000923 mgain Note: 1S. = internalstandard. 001286 WILDLIFE INTERNATIONAL, LTD 1. PROJECTCTONO.:. 454-102 eros Gpmabinsampli e .= PFOS(mgai/L)atiniument x Final Volume 0 x Dilution Factor RD __000923x0200x100 oor = 18sppmai PerocfNeomninital Concentation = P PR FOS (pA e pm a.) a inE sample 100 = "Tm oso `HousingandEnvironmentalConditions During acclimation and testing, all birds were housed indoors in batteries of thermostatically `controled brooding pens manufactured by Safeguard Products, Inc. Each pen had floor space that `measured approximately 62 X 90 cm. Ceiling height was approximately 25.5 cm. Extemal walls, ceilingsandfloorswereconstructedofgalvanizedsteel wireandsheeting. Birdsweresortedby weight, then chosen indiscriminately from within each represented weight class for assignment to pens. Eachgroupofbirds wasidentifiedbypen numberandtestconcentration. Individualbirds were identifiedbywing bands. Duringthetesttheaveragetemperatureinthebroodingcompaorftthempeensnwats 38C 2C (SD). Average ambient room temperature for this study was 25.2C 07C (SD) with an average relative humidityof 53% 18% (SD). The photoperiod (maintained by a time clock) was sixteen hoursof lightperdayduringacclimationandthroughoutthetest. Thelightsourcewasfluorescent lights which closely approximate noon-day sunlight. The birds were exposed to an average of approximately 207 lux of illumination. Housing and husbandry practices were based on guidelines established by the National Research Council 5). Observations During acclimation all birds were observed daily. Birds exhibiting abnormal behavior or physical injury were not used. Following test initiation and continuing until termination, all birds were 01287 WWIILLDDLLIIFFEEIINNTTEERRNNAATTIIOONNAALL,,LLDTD, 1. PROJECTNO.: 454-102 -- observed at least once daily. A record was maintained of all mortality, signs of toxicity, and abnormal behavior AnimalBody Weights/Feed Consumption Individual bod weights were measured at the initiationof the test, on Day S, on Day 8 for all birds, and on Days 15 and Day 22forallremainingbirds. Averagefeedconsumvaplutesiduorinng the exposure period (Days 0-5) and the post-exposure observation period (Days 6-8) vere determined by pen for each treatment group and the control group. Additionally, feed consumption was determined for Days 8-15 and 15-22forthe remhaalfoifthentreiatmnentagnd control birds. Feed consumption wasdeterminedby measuringthe changeintheweightofthefeedpresentedtothebirds over agivenperoiftoimde.Theaccuroaffceeydconsumptionvaluesmayhavebecnaffectedbythe `unavoidablewastageoffed bythebirds. GrossNecropsy Al testbirdsthatdidduringthecourseofthetet andal birdsremainingatthe termination were Subjected to agross necropsy. Additionally, liver weight and liver tissue, serum and blood solids, and bile were collected from birds euthanized on Day 8 and 22, and when possible from those that died durthieconurgseofthestudy. `StatisticalAnalyses Mortality data were analyzed using the computer programofC.E. Stephan (6). Theprogram was designed to calculate the LCS value and the 95% confidence interval by probit analysis, moving average method or the binomial probability method (7, 8, 9). In this study the LCS0 value was detebyrthmepriobintmeethdod. Theslopeoftheconcentraticuornve-anrderessupltosonfsthee goodness of fit test are reported. Body weight data were compared by Dumet's test using TOXSTAT software (10,11). No statistical analyses were applied to fed consumption data. RESULTS Die"tAAvniaalnydsiistsampleswerecollefrcomttheed 9.1,18.3and 1171 ppma.i. testconcentratiaonds analyzed to evaluate homogeneityofthe test substance in the avian diet. The analysisofthese samples also served as verification of test substance concentrations. Resulting mean measured. 012838 WILDLIFE INTERNATIONAL, LTD 1s PROJECTNO. 454-102 concentrations, standard deviations and coefficients of variation (CV) for these test concentrations were9.8 + 0.969 ppm ai. (CV= 9.94%), 19.+52.13 ppmai. (CV = 10.9%)and 1196 70.2 ppm ai. (CV = 5.87%), respectively (Appendix IIL, Table 4). Control avian diet samples collected during the test showed no interferences above the LOQ. Samples collected during the test to verify the 36.6, 73.2, 146, 293 and 586 ppm aii. test substance concentrations had mean measured concentrations of 40.2,74.5, 174, 291 and 537ppmaii. respectively. These values represented 110, 102, 119, 99.3 and 91.6%ofthe nominal concentrations, respectively (Appendix II, Table 5). Analysisofavian diet samples collected from feeders after being held at ambient temperature for five days averaged 119, 97.4, 130, 104, 94.3, 101, 101 and 95.0%ofthe Day 0 values for the 9.1, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 ppm ai. test substance concentrations, respectively (Appendix III, Table 6). A representativechromatogramof atestsampleisshowninAppendixI,Figure7. MoratndCalinlicaliObsterveatisons Nomortalitiesoccurredinthecontrol group,andallbirdswerenormalinappearanceand behavior throughout the test. There were no treatment-related mortalities or overt signsoftoxicity in the 9.1, 18.3, 36.6, 73.2, or 146ppm a.i.treatmentgroups (Table 1and Appendix V). However, there was 20% (2 of 10) mortality in the 293 ppm a.. treatment group, 30% (3 of 10) mortality in the 586 `ppm ai. treatmentgroupand 90% (9 of 10)inthe 1171ppma.. treatmentgroup. Inthe 293 ppam.i.treatment groupsinglemortalitieswerenotedonthemomingsof Day 7and Da8y. Signsoftoxicitywerefirst observedonthemorningofDay 4and contuntiilnterumineatidon forthosebirdssacrificedon Day 8anduntiltheaftermoon ofDay 8forthosebirdssacrificedon Day 22. All birds in this treatment level displayed sigasoftoxicity. These included reduced reaction to stimuli (sound and motion), loss of coordination, ruffled appearance, lethargy, and lower limb weakness. ~Allremainingbirdsappearedtohaverecovered and werenormalin appearanceand behavior fromthemorningofDay 9untiltesttermination. At the 586 ppm a.. treatment level there were threetreatmentrelated mortalities, one cach noted on the morning of Day 5, Day 6, and Day 7. Signsoftoxicity were first observed on the morning of Day 3 and continued unil termination for those birds sacrificed on Day 8 and until the afternoon of Day 8 for those birds sacrificed on Day 22. All birds in this treatment level displayed signs of toxicity. These included reduced reaction to stimuli (sound and motion), ruffled appearance, lower 001289 WILDLIFE INTERNATIONAL, LTD 16 PROJECTNO: 45-102 limb weakness, and lethargy. All remaining birds appeared to have recovered and were normal in `appearanceandbehavior fromthe morningofDa 9uny tiltesttermination. In the 1171 ppm a.i. treatment group there were nine treatment-related mortalities. The first two. `mortalities were found on the moming of Day 4, followed by four mortalities on Day 5, two `mortalities on Day 6 and the final mortality on Day 7. Overt signsoftoxicity were first observed on the morning of Day 2 and the single surviving bird continued to display signs of toxicity through the aftemoonofDay 8. Signsoftoxicity observed among birds in the 1171 ppm a.i. treatment group included reduced reaction to stimuli (sound and motion), loss of coordination, prostrate posture, depression, convulsions, ruffled appearance, lower limb weakness, and lethargy. The one surviving bird appearedtohaverecoveredandwasnommalinappearanceandbehaviorfromthemorningof Day 9 until test termination. Bod Weightand FeedConsumption Whencomparedtothecontrol group,therewerenoapparenttreatmentrelatedeffectsonbody weight among birds in the 9.1, 183, or 36.6 ppm ai. treatment groups. There was a satstcally significant(p<0.05)reductioninweightgainatthe9.1 ppma.i.level forthe Day 0-5periodthatwas reflectedinsignificantlylower(p<0.05) meanDay 5body weight.Whencomp toa thr ecoe ntrdol `group, reductions in weight gain were also statistically significant (p<0.05) for the Day 5-8 period, resulting in a significantly (p<0.05) lower Day 8 mean body weight. However, differences from the controlgroupatthe9.1ppma.i. level appeartobeducto a lowermean Day 0bodyweightforthe 9.1 ppm ai. levelandwerenotdoseresponsive. Therefore,thesedifferenceswerenotconsidered treatment related. There was a marked, tratmentrelated, concentrationtesponsive effct on body weigh in the 732,146, 293, 596, and 1171 ppm .i. treatmentgroups dutrhiexpnosugre period (Days 0-5) (Table 2levaenldanAdppaetnpd<i0x.0V1I).at tThhees14e6,re2d9u3c,ti5o8ns6 waenrde1s1t7a1tisptipcmall.yi.silgenveilfsi.caDnutraitnpg<t0h.e05Dafoyr 5t-h8e p73o.2stpepxpmosua.ri.e preeraitomde,nwtegirgohutpsg.ainThceosnetirnueedductioonbse wreerdeucseidatfsotricsaulryvisviignngifibciarndts aitn pt<h0e.20913,fo5r8t6hoa2n9d31a1n7d1 Sp8p6mpap.im. acio.mpalreveedls.. BoDdifyfewreeingchets loatsetsheand11r7e1ducptpiomnsaiin. weleivgehlt wgeairne amtatrheke2d93,bu5t86coaunldd 1n1o7t1 bpepmstaat.i.stliecvaelllsy were reflected in significantly (p<0.05 or p<0.01) lower mean body weights on Days 5, , 15, and 22 001290 WILDLIFE INTERNATIONAL, LTD 17. FROJECTNO: dsi-102 ofthetest. Atthe 146 ppma.i. levelmeanbodyweigonhDatyssand 22werealso significantly (9<0.05) lower than the control group. During the Day 8-15 and Day 15-22 post-exposure periods, bodyweightgainappearedcomparableamongal groups. `Whencomparedtothecontrol group,therewas amarkedreductioninfeed consumptioninthe 293, 586, and 1171 ppm a. treatment groups during the exposure period (Days 0-5). A reduction in feed consumption continued to be observed through Day 15 in the 293, 586-and 1171 ppm ai treatmentgroups. GrossNecropsy Duringthecourseofthetest,allbirdsthatdiedweresubjectedto agrossnecropsy. Necropsy results for birds found dead were similar. Common observations included thin condition, loss of `muscle mass, altered spleen color, empty crops, and empty gastrointestinal tracts. * Findings were contsobietdreaetmerntreelatded.Detailsofthenecropsyfindingsareprescatedin Table 4 Halfofthe surviving birds were subjectedtogross necropsy on Day andthe remaining birds on Day22,follteottwermiinantiogn. ThenecropsyresultsforbirdseuthanizedonDay and Day 22. from the 18.3, 146 and 1171 ppm a.i. treatment groups were unremarkable. One bird in the control groupandonebirdinthe 36.6ppma.. treatmentgroupwerenotedwithretainedyolksacsduring necrofobiprdssoyn Da8y. Asibnirdginlthee 9.1 ppma.i. treatmentgroupnecroposnDiaeyd was notedassmall, with alackofmusclemass. Inthe 73.2ppm a.i.treatmentgrouponebird necropsiedonDay wassmall,withalackofmusclemass,andhad a 2x 0.5cmabscesscontaining caceousnecroticmaterialontherightsideoftheabdominalwall. Duetothenatureofthelesions observed in birds, and the isolated incidence of occurrence, the findings listed above in euthanized birdsfrom the9.1, 36.6,and73.2 ppm ai. treatmentgroups werenotconsideredtoberelatedto treatment. Inaddition,all remainingbirdsinthecontrol,9.1, 36.6, and 73.2ppma.i. treatment `groupswerenonremarkable. In the 293 ppm a.i. treatment group, one bird euthanized on Day was noted with an incidental findingof aretainedyolksac. Asecondbirdinthe 293ppma.i. group,necropsiedonDay 8,was noted as emaciated with a lack of muscle mass. This lossofmuscle mass was consistent with reduced weightgainforthelevel andwasconsideredtreatmentrelated. Another bird inthe 293ppma. treatment group, euthanized on Day 22, was noted with 2 cm cysts containing yellow fluid on the 01291 WILDLIFE INTERNATIONAL, LTD 18 PROJECT NO; 454-102 anteriorportion ofthe left lobeofthe liver. Duetothenatureofthecysts andthe isolated incidence: ofoccurrence, this observation was not considered treatment related. Three birdsinthe 586 ppm a.i. treatment level were euthanized on Day 8. At necropsy, they were notedtobethinandlackingmusclemass.Onebirdwasnoted with aslightlypaleliver. These findingsareconsideredtobetreatmentrelated. Fourbirdsinthe 586 ppm a.i. treatment level survived until Day 22. Necropsy resultsforthesefour birdswerenotremarkable. CONCLUSION "ThedietaryLCS0valueforMallardsexposedto PFOSwasdeterminedtobe 628 ppma.i. witah 95% confidence interval of 448 ppm a. to 958 ppm a.i.. The slopeofthe concentration-response. curvewas3.67andthechi-squarevaluewas 2.13. Thenomortalityconcentration was 146 ppma. Baseduponreductionsinbodyweightgainatthe73.2ppma.i. testconcenttrheanot-oibsoernve,deffectconcentratwiaosn 36.6ppma. 001292 WILDLIFE INTERNATIONAL, LTD 19- PROJECT NO.: 454-102 REFERENCES 1 U.S. Environmental Protection Agency, 1996. Series 850- Ecological Effects Test Guidlelines (draft), OPPTS Number 850.2200: Avian Dietary Toxicity Test. 2 SUuSb.divEinsvioinronEm,enHtoazlarPdrotEevcatliuoantioAng:ency.Wild1ij9f8e2. andPesAtqiucaitdeicAsOsregsasnmiesnmts,Guisdueblsiencetsi,onFI7F1-R2A, Environmental Protection Agency, Offic of Pesticide Programs. Washington, D.C. 3 Organization for Economic Cooperation and Development. 1984. Avian Dietary Toxicity Test. OECD Guideline for Testingof Chemicals. Guideline 205. Paris. 4 American Society for Testing and Materials. 1987. Standard Practice for Conducting `Subacute Dietary Toxicity Tests with Avian Species. ASTM Standard E857-87. Annual Book of ASTM Standards, Vol. 11.04. Philadelphia, PA. 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 LCS0. Pages 65-84 In Aquatic Toxicology and Hazard Evaluations, American Society for Testing and Materials. Pub. No. STP 634. Philadelphia, PA. 7 SCotmempuhnainc,aCtiEo.n. 1978. US. EPA, Environmental Research Laboratory, Duluth, MN. Personal 8 Finney, D.J. 1971. Statistical Methods in Biological Assay,2nded., Griffin Press, London. 9 Thompson, W.R. 1947. BacteriologicalReviews, V2o ,Nol .2: 115-145. 10. Dunnet, C.W. 1955. A Multiple Comparisons Procedure for Comparing Several Treatments `wai Conttrohl. Jour. Amer. Statis. Assoc. 50:1096-1121. 11. Gulley, D.D. 1990. TOXSTAT Release 3.2. The University of Wyoming. 01293 WILDLIFE INTERNATIONAL, LTD -20- PROJECTNO.: 454-102 TABLE| `Cumulative Mortality froam Mallard Acute Dietary Toxicity Study with PFOS ExGperroiumepntal No DEexapdoPsuerreNPoe.rEiodxposed NoP.oDset-aEdxPpeorsuNroe.PeErxipoodsed @pmai) "Day0 Dayl Dayz Day3 Dayd Days Day Day? Days' Control 0 030 030 030 030 030 030 030 030 030 Treatment 91 183 366 n2 146 293 586 un 010 010 010 010 010 010 oo 00 010 010 010 00 010 010 000 010 010 010 oo 010 010 010 010 010 oo 010 010 010 010 010 oo 010 010 10 00 00 010 010 000 010 010 110 010 010 000 010 210 610 oo ono ono ol 00 ono oo ono ono 010 010 010 0 ono ono 00 mo 20 20 30 30 #10 90 ono "TphomLaCi50value wascalculatedtobe 628ppm a. with a 93%confidenceinterval of 448 ppm a1 to 958 PNGmortalsaccu nanyofthe treatment wel rom Day 810 Day 22. 0429% WILDLIFE INTERNATIONAL, LTD 21 PROJECTNO.: 454-102 TABLE2 Page | Mean Body Weight (g) from a Mallard Acute Dietary Toxicity Study with PFOS "pGermoeurp ar (ppmai) Exposure Period Dayo Days `DChaanygse Post:Exposure Period Day$ `CDhaaysnge! -- ChangeToDtaaly 0-8)' Cont0rol Mean 135 79 144 380 101 25 sD 2% 2 n Ed 16 3 Treatment 91 MseDan 1i"n 23660 1208 6367 391 15958 183 MseDan 14164 m2% 11331 a21 10i0 221 366 sMDean 14176 22s 12n8 33775 11020 2380 B12 sMDean 13403 25650 n3e 394%3 822 27000 #6 Mean sD 13 19 22 3 1000 21 51 816 1288% 293 Mean 129 1610+ 320 200 57 g4en sD 1" 2 3 5" st 58 Mean 144 ad ov mse 36 390 sD 2 27 2% 3% a 36 nm _--_-- MSeDan 1257 m2ee IaE]me 185- a- 15- eanchange 5 aloudseperately Hom he eanbodyweights using dividualbodyweights (nSee,c cAopuplednndtixbVeIe).valuatedstatisustinigcDaunlneltys test *StatNstoicdaaltlaydaivaeirleabnlte druoemttohmocrotnaltirtoyl.groupat p< 0.05 (Dunnel's Hes) = Statidifsfrtentifrcomathelcolnryol graotpu<0p01 (Dunnett's est) 01295 WILDLIFE INTERNATIONAL, LTD 2. PROJECT NO.: 454-102 TAPaBgLeE22 MeanBodyWeight (8)from a MallardAcuteDietary Toxicity Study with PFOS EpGroup @pmai) PostExposure Period Days Dayls ChDaunyg8eis Dayz CDhaaynlgs?2 Cont0ral Mean s 3a00 6056 23a0 m 19 m2 Treatment 91 Mseoan su3se s2s 2030 3m8 os15 183 sMpen E2N o2s 221 s8u1 1a56 366 sMeDan 3337 0" 2"0 m7s u3s 72 MEYen 364 557 2216 1L 35 E39 6 Men m5e s5e6 m2 C 8 T% 299 Msem e0)e gset r 325s6 woare 323s4 $86 sMDen a6e s8e2 e 3 oi0e 119 un M0en 185f wr 8/ af/ os} "Tnh=ie,nccoulhd na1osbnceavlgaculaaetteeddsesptaatriastteilcyalo ly UsihngeTDh uenaneto'dsyLWeesitghtswigindvdial ody Weights (eAeppendisVD) "Su=tiNstocdaallay iavlaieleabnlerdoumettohmcaonrroal group at p< 0.05 (Dunne's est). +*Staisicaly diflcen rom he control group at p<0.01 (Duets est) Toul ChangelG2) a2 o3n e&d as7t a5s6 315066 05 3a ws?: 01296 WILDLIFE INTERNATIONAL, LTD a. PROJECTNO.: 454-102 `TABLE 3 Page 1 Mean Feed Consumption (g/bird/day)from a Mallard Acute Dietary "Toxicity Study with PFOS Eapeimenal Pan --_------ 0 Treatment 01 183 366 na we 2 st un REE Expose Period -- mes vkyan oo P a 5 n 105 " % n Pos posurePeriod owes 17s w n 1s 01 159 a 5s 2 0497 WILDLIFE INTERNATIONAL, LTD "24 PROJECT NO: 454-102 TABLE 3 Page2 Mean Feed Consumption (g/bird/day) from aMallardAcute Dietary Toxicity Study with PFOS `Experimental Grove (Gpmai) Post-Exposure Period "owsDwi@ Caw Mean 7 180 sD 13 20 Treatment 91 183 366 B2 2 198 1356 204 165 1 148 73 146 159 164 293 109 132 586 m4 I) un 106 154 ONomawamk mom, 01298 WILDLIFE INTERNATIONAL, LTD 25 PROJEJCETCNTO.:NO 454-102 TABLE4 Gross Pathological Observations from a Mallard Acute Dietary Toxicity Study with PFOS Birds thatdieddurthieconurgseoftestudy TTT Finding Crop empty Emaciated G1. Tract, primarily empty Gizzard contents bile stained. Gizzard, empty Intestinal contents, black and tar-lke Keel prominent Kidoeys, pale Lossofmuscle mass Spleen, grey Spleen, small and pale: Spleen, pale Thin Maks, Female, and Undetermined PPMAL 95 586 71 N=2 N=3 N=9 0 2 7 1 1 4 1 1 1 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 01299 ER WILDLIFE INTERNATIONAL, LTD PROJECTNO: 454-102 ies To CumaTlaStivAeMortAalituEy(ExsititmyaCteTdCooumayrlaStivyeDosSe,Emg/Skg) rom aMallard. TT Bt pGErooudnp ve Br o e BoEwe eebr ed wm ow ow om hea om ow TePuby pPearitod wow ow CCCooolalEnmowooms w wom osuwm 8oww omwomn mmnooononmao rmCCaoooslEmoowwoowwooosw w ow non mwoamaa BSorwu wwee Wwlw woslm BwEo wwmm ews owes oosw w Bo ew s uslm nwoho uwamn uoslm nesea wns owsunos X we o1 wes omsEm wwohn wGeen ewsee wmsmoy wms oews uose n Baw m mUoEn SwGe wBoerm sWoen woewne wwoowm oowo . wwooo 1 wwos uwoin doseumn NosEam Nwolw WosEo 8os 8os oonu mBoo onew eise wwoemn oWsomm eSsie wWoEw oWn onwomon s No os w wwign wwoew oNsees Swslow wWeEn oNs onwuson QSrm e m1i ow wosaon;e eGiwaen e ssoonm ewiommewgowm swms 8om 4wm ER io oeme on ot oype woeet yct.monov ps ein dvs1 WILDLIFE INTERNATIONAL, LTD PROIECTNO.: 454-102 APPENDIIX Certificate Of Analysis FC-95, Lot 217 March 9, 2000 `RicMh,Paayrfedr Thissaoplewasanalyuzseindg LC/MS, hid mhpov potion H-NMR, "F-NMR,snd elementalanslysestechniques.Theresults eer mx -- geo eax ------ ----cms 7o sxSR wowS-- | --re-- [--oamsox| osm C--Ecesox 1 oss | didnt ores ronselvo dtm 4PERtackssnd und0 MA EER a T we] Feel1= [omiesoenatmao | 01301 WILDLIFE INTERNATIONAL, LTD 28 PROJECTNO.: 454-102 APPENDIX IT WILDLIFE INTERDNIAETTIFOONRALMULTLDA.TGIOANME BIRD RATION INGREDIENTS FSionyeBCeoamn MMeeaall, 48% Protein `PWrohteeaitnMBiadsdes ``AAlgfwailafya MSpeeacli,al2,06%0P%roPtreoitnein GDrrioeudnWdhLeiymestone MEeatshtimoanninCealPPrheomsix + Liquid `GLVFiertman(adFeMmrimnaietrcnaol)remix (seebelow) TSoalttallodized PERCENT (%) 43046853 665000 340000 029500 006305 003225 1000.1000 VITAMIN AND MINERAL PREMIX AMOUNT ADDED PER TON VViittaammiinn DAy RNiibaocfilnavin PVainttaomtihnenBiyc; Acid BiFooltiic nAcid PTyhriiadmoixniene VViittaammiinn KE (Menadione Dimethylpyrimidinol Bisulfits MZainngcanese ToCdoipnpeer SIerloennium 27,,000000,,000000 LLCU.U, 406 ggrraammss 108 gmrgasms 66040 mmggss 11.2 2 ggrraammss 20,005081Ug.rams 14072 ggrraammss 6185 ggrraammss 15812 mgrgasms. "Th5eg%uacrruadnetefeidbaernalysisis aminimumof 27% protein, aminimumof2.5% crudefatand amaximuomf "Fermentation By-Products (SourceofUnidentified Growth Factors) 01302 APPENDIX II ANALYTICAL METHODS AND RESULTS 04393 WILDLIFE INTERNATIONAL, LTD -30- PROJECT NO.: 454-102 INSTRUMENT: ANALYTICALCOLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: 'PFOS RETENTION TIME: IRNETTEERNNTAILONSTTIAMNED:ARD PFOS MONITORED MASS: IMNOTNEIRTNOARLESDTMAANSDS:ARD Appendix II Table 1 `Typical LC/MS Operational Parameters T"aHPueewrrlekbtitno--PElalcomkeanrridSAoMPnp]osodr1ue0ral0ceyL1.C1O0Mp0aeHsrisagtShepdePceitrnrfsoolmreemtcaeinrvcceeqLuiiioqpnumpiedodCnwhiirtothmo2aPtremoirgoknrdianegp.Ehlwmietrh (SIM). KeystoneBetasil Cyscolumn (100mm x 2 mm 1D., 3 umparticlesize) 0c 100 minutes 0220 mUminute: 72.0%Methanol : 28.0% NANOpureWaterconaining0.1%FormicAcid 250 ul `Approximately 7.0 minutes Approximately 48 minutes 4986.amu 4267 amu 01304 WILDLIFE INTERNATIONAL, LTD "31. PROJECT NO.: 454-102 Appendix II Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454-102) MAB-1 Sew Type `Matrix Blank, WesCo PFOS' (ppm ai) <LoQ MAB-2 Matrix Blank <10Q aTnhaelyTsiemdi0o.f0qu0a2n2iimagti2on.4(L)OaQn)dwthaes1d.i13onppfmacat.o1r.obfatsheedumpaornithbelapnkrsoamdpolufesct(ht3e0l0o)west calibration standard. 01305 WILDLIFE INTERNATIONAL, LTD on. PROJECT NO: 454-102 Appendix Il Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample NumE ber E (54-102) Fortied TWAMAT S4A a45e7 (ppmai)Td ewsued a47s9 Percent Recovered w10r5 MMAASS'2S 118833 117662 9863.1 MMAASSSS 11883300 1175176 896.17 -_-- TT Standard Deviation v=as w69a9 cNv== 7358 "oGteo:arRnedsurmlatsnounadlccaltculaitioonsf.onrVanlesuews(hsatveSbUebeSnAnrGoeunpudreidtfyorWreerpeorGteinnegratedpurpsosiensgMacQanversion 15 Somware 01396 WILDLIFE INTERNATIONAL, LT EN Ap"pTenadeix PROJECTNO: 45-102 Homogendyof PFOS in Avian Dict CNoommmniion Geman oT SNuombcer Askin) =i" 55a TocwionaSampled MisngVesd TNiTodotnegn NBoBotdtoomm RRLiigeghhtt Mess PCroosnnon "opmai) 9oom5m 0T1o00y5 MeanSMaemsasrrDdCeovnoenns(iGo1)n (MeanPaeat. CoinorVansion CV) Nominal 3022=9085p5mapimai. 08 vessel w 23i NiTTtooelpLahenn B1isess $a0=210231g5ppmmaais :5 BNBoiorlmoeRiLigeghhht w139i5 Soll 0 1is NTiTootppmegLehetn im1iss $a= m158Tppwmaai i 325 BNBooioomdmRRLiiegghnht niognl vse "NCoo:oRfneimdntesonaodracpoornrcitvinoagnpscufopesno.dtsinbg laesipoanyowfemreaGnr1antdesdansdnrgdMecviQnrvesrs 15softwar14 rncco. Voshave een 222 33 WILDLIFE INTERNATIONAL, LD BN En ApTgaeblnetsiTxL PROIECTNO: 454102 Verification ofPROSConcentrinaAtviiaoDniest T Conmie rtion mSNummbpeer Goma) shi) Tw " - ws - 6 5 Semde ) MesnedMCOSoon me) a= 7 Foem Nomis - ns CMeombiswenl | mai) - or 15 wr MemoPf me Neat 08 or no EY iwo uBnn o0 mnsa 17035 us E oo i1ns 1jd m nw = iw o mon 2o1s 93 P 1i6 00 -mo w2es = a6 k wm - - - - nog wo keRentco contin or ow okbane tyweepte ing McQ veri 1.3 fesd alcles. Valesbvbo oidfopig NRhaeemkHsc ealtsqh (if3o0a0Tnt b(e1400) vas11 75m.bdpon he rodeof be nest cabinsdsed (00229 1.1) he iio ocr fei Q22 QS @ WILDLIFE INTERNATIONAL, LTD AppTesneditx Tacs PROJECTNO: 454-102 AbicntSaitof PFOSin AvinDictDuring theMallardLCS0Sty -- eo CTonne M wRmiEeEr o emaem a MaNFonihem tSamithe vGMemtm WCraeemymi Weior)r e I EE 06 . Pe wo as a 20 = ws = " Ps wn " ae " = - i 0 = su - : Door dio eT T t To tn tt rt i atsd 50 41) eSie oot i boy0m rdsconsfeir Noeste o os l dl 23R 33 tn et tk WILDLIFE INTERNATIONAL, LTD ] "36 PROJECTNO: 454-102 METHOD OUTLIINNE FAVOIRATNHDEIEATNALYSIS OF PFOS Prepsmaefinospeined vi fd cking dyi bis . Dry Tei Feo itn i1s ry ns VL on i Weigh heise iyof bres PFO) ts er. AeVi rin c e Fi POSie mlrfb einrion nr d se oise erg ek, et pi te dti ke ne to fdet ners i sd Fulow haerceoredscbdBpoemastvmeae sEh ihy iFFdi yoed ixOh Ve rnhe ` Hh 10 tr tik fin tttwd 6 rh oe . Fr xh al, esr100mLofeaeihgrid inde de basfreFechaque le . Cadppcostilel. Allow smpletosikfci 10mts4250 ` Acmexhslew uti Bl pes ods3iesihsal hehe. ` ToseSli 30.vole sk ndbinges vob ithmel ` Pps peeito igocontin i ioninsof LEMSmethods: Frat ou ices 307 it 50NASNO octt bdney og 160m 41PES Gitar nb . Api nd esene LENS is Figur1e. Analyticalmethodflowchartfo theanalysisofPOSinaviandiet 01310 WILDLIFE INTERNATIONAL, LTD 3. Appendix II PROJECT NO.: 454-102 1.50 3 _ 10 050 000 000 0100 0200 0300 0400 0500 Concentration (Ratio) Figure. ACtuyrpvieciaslwceailgihbrtaetdio(n1c)u.rve for PROS. Slope = 277397 Inercept = 001894; = 0.9981 01311 WILDLIFE INTERNATIONAL, LTD = Appendix III BROECTNO: ds-'102 0 107 nenaiy: 1995 cp o 7 o 5 - a A 145 " 24 122 # 31to 6S1h 1Sh 1W21h 1C81R 1T81H 2W11e STeiaen Figur3e. A represntativ on chromatogram ofa low-level (000229 mg aL) PFOS standard 01312 WILDLIFE INTERNATIONAL, LTD "ae AppendiIxl PROIECTNO dsi-'102 To 18 Intonsiy:26954 cps 0 . , o 5 3 144 2 ' o 24 48 7590 12 190204__ 230 S 5 e3k TSerm i5E0s 861s i7Ba zaiss TSamn Figured. A repreisoncchrtomaatoigrvam of high-level (0.0457 mg 1/1) PFOS standard. 041343 WILDLIFE INTERNATIONAL, LTM s0- AppendiIIxI PRONJO.E: 45C4-1T02 100 0 24 80 70 60 50 prs . ntan2s3t0ycp:s. Ho 188 144 20 11 218 A A AANAoA 200 Asa oa STG hTE E25A s YeSrUm WoiSpsos 82OP i7Cegzsim STamne Figure. A"ThreeparrersoenwtaitnidviecacthesrtohmeatroegternatmioofntaimmaetorfixPRbOlSan.k sample (454-102-MAB-1) 01313 WILDLIFE INTERNATIONAL, LTD a Appendix II PROJECTNO: 454-102 168 Intensity:6238 cps 9 8 7 o 5 4 a 144 2 1 - 24 122 3110 82155 9a1m 16201s 168312 17815"7 2611s | STiamne Figures, A represenative chromatogram ofa matrix fortification sample (454-102-MA1SA)-. 04315 WILDLIFE INTERNATIONAL, LTD ae AppendIiIx PROJECTNO: 454-102 Jo 187 Intonsty:7248 ops 9 8 7 6 5 tt 3 145 2 1 . 24 4 123 197 22: $1130 e25191881 1521008 165312 178617 26118 `TSicmaen Figure?. Arepresentative chromatogramof atest sample (454-102:2). 64316 WILDLIFE INTERNATIONAL, LTD a3 PROJECT NO: 454-102 APPENDIX IV DIET PREPARATION `Weight and volumeofconstituents used to prepare test diets: Nominal Concentrations Test Substance 001 009>10 183 0.1818 376.26 00.36838 146 1.4659 259836 2598122339 7 11.6483 Basal Ration 5809090909 89998 88999999.36 8998.5 88999974.12 8988.4 Diets were prepared as follows: #5000.0 gofbasalrationwasweighedinto ataredHobartmixingbowl. Thetest substancewasweighedin ataredweigh boat Approximately 100gof basalrationwastakenfromthemixingbowland placedin a Waring. `blender. +Thetest substancewasaddedtotheblenderandtheweighboatwasrinsedwithadditionalration, `withtherinsealso being plaicnethde blender. +Theblender contentswereble forn apprd oxie mated ly 60 secondsandtransferredtothemixing bowl. "Theblenderwasrinsedwithadditionalration,withtherinsealsobeingplacedinthemixing bowl. The bowlwas placedon aHobartmixerandthecontentswere mixedfor approximatelysixminutes. "Theremaining rationasaddedtothebow]andthecontentsweremixedforsixmoreminutes. Thedict wastransferredto alabelledpaperfeedbag. 01317 WILDLIFE INTERNATIONAL, LTD PROJECTNO: 454102 Cumlaive MorleybyPenfom AMaPlPlEaNrdDIAcXutVeDiary Toxicity Sedywih PFOS. ThCrmy cpm _pen Bo M EpoR n perE iod Dai Dwr bys Dwi buy r bu Em up ed Dye Dai Daw Co oro oes os es os as os os as os oo 2seses wwss ooss eess wwss ooss [oFsE as os oo o soeess wos ssos eos sosos ooss 6 os ws os es os os ooss oass oowss os os as Tareatment oes ws es os as os os os ws a11 aPooss ewss oess eos sosws ooss ooss ooss oawss saw6s 366 Pooaoss oass ooss aoss oass ooss 1 es os os as os os ooss owss aass os os as nnaa 16 Powss esos oes sasos aoss oooss Loos esos os os as oos esws wass os os as i E6Y 2 eoess ooss oass aoss ooss ooss ooss ooss aass : m556 2vasesosos ooss oess ooss aoss ooss uusssw suen 2 oeess woss owss ooss oouss ouss wsoowwss oosws nm 2 es os os oss a wows ws Tm T vs m sr ylevamT coc relpeoeseeBaeys Ipe Se 2R&B R@ WILDLIFE INTERNATIONAL, LTD PROJECT NO: 454-102 -a5APPENDIX VI In`dAicvuitdeuaDlieBtoardyyTWoexiigcihttys S(tg)udfyrowmitah MPaFlOlaSrd Page | ExGperroimuenptal @pmai) Bid Dayo Days ChDaanygoes Dmys CDhaaysnge ChanTogteal0.9 o 21 113326 E0a 1m5o6 a 381 w8 m245 33 113548 331199 616s1 as 9160 2m61 5 169 300 Ho ais 100 25 MseDan 15105 5028 Bn w1e8 105 2%260 0 21 110326 m284 114262 93 w1r0 226%3 33 115522 2099 119670 w "uo s 2F5l7 Me5an 1im2 E34d5 11658 w am s1 237017 ) 16 2 u 31 2 2 0 21 113245 26E5A 11408 m En w5 3wr1 33 94mos 1B5s5 3wmo n8e6 2291 Me5an 1"1666 23258 Tio w3o9810140 226545 sD 16 2 10 34 2 26 o 21 11234s pem) 119278 36s0m 09s7 22s3 43 115613 3030 1w0o a=s 91098 22597 Mea5n T17U0 E35S1 118515 W56m 02 2257 ) 1 38 21 38 4 2 04319 WILDLIFE INTERNATIONAL, LTD 46- PROJECTNO.: 454-102 APPENDIX VI In`dAicvuitdeuaDlieBtoadryyTWoexiigchittys S(tg)udfyowmitah MPaFlOlaSrd Page2 TEeGmromuep a Change -- Change Toul @pmai) Bid Dayo Days Damy0s Days Dayss Change 0 21 112067 m267 n18s1 23690 93 213910 43 111067 220033 986 22864 5&1 1m50 5 1m 28 137 348 100 27 Mean TI sD 8 29 28 T2 31a 515 i3ss 0 21 110000 2235 113352 2385 1%00 22385 43 110082 22637 1B5391 8317 18146 221155 5 102 210 108 29 8 197 sMepan 1023 3250 31s 33% 517 23209 [T5otal sMeDan 1235 72 T2a 35% To1l6 235 01320 WILDLIFE INTERNATIONAL, LTD a7. APPENDIX VI In`dAicvuitdeuaDlieBtoadryy TWoexiigchittys S(@t)ufdryowmitah MPaFllOaSrd Page3 PROJECT NO.: 454-102 ee eee e pGraouip Bid Diy0 _ DiyS ChDainyg0Se Dwy8 ChDaanygse8 _ ChaTmoguel09 91 1 23 24 21 349 105 26 2 ns 21 3 334 103 216 3 151 26 135 299 m 28 4 108 209 101 3 104 28 s 19 21 mn m 101 wm M5e)an ~ 12146 2420 1163 a3s 1s5 2272 51 1 9% 14s a 146 1 0 2 13 250 EY 315 9s m 3 2 wr 110 0 106 26 4 nm 26 104 316 100 200 5 m 215 104 204 9 15 Msepn T1IE aalm 29 a5w %# 17B0 GTooaw! M5e)an 1115 3n6ee 21 3 0r3a 351 T55o "SSttaaitiasiteicaallylySifefcnenfroomm theccoonnt]olpgrroopupaatpp<<00..0051((DDuunnnees1tse)t) 04321 WILDLIFE INTERNATIONAL, LTD La8- PROJECT NO.: 454-102 APPENDIX VI InAdciuvitdeuaDlicBtoardyyTWoexiigcihttys S5t)udfyrowmitah MPaFlOlaSrd Page 4 ar @Gprmouapi) Bid Dayo Days ChDaanygoe Days ChDaanygse CThoaunlge 183 1 123 261 138 ass 9 m 2 Io 297 136 en 126 20 3 150 m0 120 361 9 a 4 152 286 1 390 104 28 5 160 294 134 391 9 2 sMeDn TT1 216 m56 azm 1132 3285 183 TGrooaup 1 130 210 10 21 ws 197 2 157 266 9 389 9 m 3 145 28 123 366 08 m 4 151 258 107 366 108 2s 5 m ES 102 EE 100 %2 MsaDn W 16 35 @ 2 0399% W 5 2 Me5)an 14164 727 1B8l E3d 01 B2l 01322 WILDLIFE INTERNATIONAL, LTD 49. PROJECT NO: 454-102 APPENDIX VI Ind`iAvciutdeuaDilcBtoadryy TWoexiigchittsy(Sgt)ufdryowmitahMPaFllOaSrd Pages EGprouap (pm) 366 Bid Dayo Days CDhaayn0gse Days ChDaanygse ChTaonugle 1 131 21 130 mn nm En 2 E 256 124 356 100 m 3 102 20 2 369 100 Ed 4 160 20 110 353 8 193 5 m9 EE 156 EE 98 250 MsaDn TW WO2 22 in m 53 %10 I2m3 366 GTooulw 1 131 2350 9 316 8 185 2 136 1 151 0 ns 26 3 17 2 16 Ed 16 m 151 255 108 4 8 193 5 160 24 164 on 103 21 MseDn 1 W1S 3T6m 2 W2m0 4 m 1 39 sMepan 14176 7725 82 3735 1020 m38 041323 WILDLIFE INTERNATIONAL, LTD -50- APPENDIX VI Individual Body Weights 8) froam Mallard `Acute Dietary ToxPiacgiety Study with PFOS PROJECT NO: 454-102 TEpeGnrmouepna @pmai) n2 Bid Dmy0 DmyS ChaDmnyg0es Duy8 CDhaansgse CThoaunlge 1 3 203 0 281 E 168 2 Ji 29 106 28 2) ns 3 102 286 1 3% 104 us 4 154 2% 136 379 8 25 5 19 338 159 #0 102 301 sMeDn T3 O2% 56 2 m 8 %2 m5m5 n2 1 109 m m3 02 0 195 2 129 254 125 mn 0 3 19 Er 104 30 97 201 4 197 196 ) ns 21 28 5 204 2 148 ss 106 254 M)en 31466 25530 31078 21011 a51 1866 GTortoaulp MseDan 31603 7505 33 00 72 2n0 States Gren om he con] gro ap <0.05 (DUSRel'T Ls). 013241 WIWILLDDLLIIFFEEININTTEERRNNAATTIIOONNAALL,,vLTOO 51 PROJECT NO: 454-102 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 7 -_Experimental Group Change Change Toul Gpmal) Bid Dio Dns Dn0S Diys Dyss Cham 16 1 108 169 a 26 a 1s 2 138 8 10 3% 0 192 3 151 m 10 5 104 me 4 151 254 103 30 150 s 164 wm usm o% 216 Me5a)n TW21 I"S 2 a52 1 3W 146 1 1s 200 20 2 139 1 0 wr 3 4 EY 8s 348 4 15 261 102 358 s 158 25 mw i Me5a)n 11883 3230 91 2i9 TGoupl Msem 1 a52 T2oe m8I SSSttsiclyGeren fom he onl gop tp <001 Du--e/s}view). 76 165 @ 118 9 204 9 199 8 216 0 12896 16 G2E -- 01325 WILDLIFE INTERNATIONAL, LD 52. APPENDIX VI Individual Body Weights (3) from a Mallard `Acute Dietary Toicity Study with PFOS Pages PROJECT NO: 454-102 Te: mGaroiup) Bid Duo Days ChDamnyg0eS Dy8 ChaDunygses CTuoaunlge El 1 ns 102 zn 9 zn 6 2 139 19 20 165 20 2 3 140 156 3 w 16 n 4 155 15 2m 136 5 m2 14 2 203 2 9 Msen TW R1s 2% a B aal B 21 m " EE 1 13 25 0 2 166 2 120 15 a m 153 3 151 19 2 -2 - 4 106 158 2 02 w 5 4 185 a 138 6 u M)ew TB ES1 2T6E 36 6 36 im GToouwl MEen)n 1n9 T2ee a5e a5oe 5 u E4l S-SRutstTclly diferent om th control group at p < 0.01 (Dunnett's is 01326 WILDLIFE INTERNATIONAL, LTD se PROJECT NO: 454-102 APPENDIX VI IndividualBody Weights (g) from aMallard Acute Dietary Toxicity Study with PFOS Page 9 --Ex-- perimental Group ee Change Change Toul (pmai Bid Dsy0 Days Dny0S Days Duyss Change 6 1 m 105 EY 126 2 4 2 135 13 8 209 3 "" 3 12 160 8 wm 5 6s ` 154 19 25 - - 5 165 195 30 16 52 2 Msen TWI W3E 21m 46 B 5 a ss 1 105 1s 9 159 ks st 2 120 130 10 185 ss 6 3 199 . - - . . 4 153 12s 25 185 5 2 5 1 1s 5 - . . Me5)n TW30 B 7 3B 1 1s 2 5 %n T Tou! sD 3 7 T 2 3 a 3 SSuR lGiE ruescE fomahee lcoym] proup st <0.01(user'sies) 04327 WILDLIFE INTERNATIONAL, LTD san PROJECT NO: 454-102 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS. Page 10 pre un @pmai) Bid Dsy0 Days ChDannyg0Se Days CDhaanygse CThoaunlge un ' 124 ss a9 - - - 2 10 - . . . 3 us % " - - . s 150 us x - - s 165 - - - . . Msen 1W5S 0i] @9 -: 5 umn 1 18 0 " - 2 110 . - - - . 3 1s 13 Et} . - - 4 157 - - - - . 5 20 154 "6 185 a as Men TW E m ws ao Gw rToou!p sMseoan o2s145m 7 ow2 a1 . El- 52 P "loSmaE iolrGE iltesloinmaiythoefceohnetoselsgtrecounpttaepc<acne0.l01ugr(oDurpuseatfels iDadcy5. 04323 A WILDLIFE INTERNATIONAL, LTD PROJECT NO: 454-102 eerie sememmttmm-- ss APPENDIX VI In`dAicvuitdeuDalieBtoardyyTWoexiigcihttys S(tg)udfyrwoiatmh MPaFlOlaSrd Page 11 ~Expcrmenal pGmroauip -- Bid Days Chang- e Change Dyyls Duels Dey22 Dayls22 ChaTnougler 0 21 38014 673231 21%7 879850 114s7 "39919 3 a2s 6685s2 2260 8261 117360 a3943 5 Mean 9 aig 6619 m 261 84m 7 168 a2m9 5) 18 32 2 5 2 " 0 21 339094 6004 22007 8m110 210847 a39n7 hn3 4009 58 239 78109 118817 a3000 Me5an Taa8 7e5s2 2w6s8 9s2s4 1m m smwo s 37 7 57 7 1 st o 21 m371 68133 01 279 211906 507 33 w37o8 653964 211566 854 22030 a4116 5 20 Mean 398 on Gn 219m7 381186 128089 6386 Group MseDan 34140 ED2 710 E3x1 1835 a2n Total sD ES 56 a rr 2 2 01379 WILDLIFE INTERNATIONAL, LTD -s6- APPENDIX VI In`dAicvuitdeuDalieBtoardyyTWoexiigchittys S(tg)udfyrowmitah MPaFlOlaSrd Page 12 PROJECT NO.: 454-102 E Groupmma Change Toul @pmai) Bid Days Dayls Dmygls Day Change Changs 91 ! 9 0 253 1 2 n 2 34 si9 25 0 181 39% 3 399 616 m 0 186 0 4 an 62 29 67 205 st s nm si6 2m 4 198 an MseDn T7 I3 S3B2 m m200 3 m 15 a3 T Group T Change Change Toul Gpmai) Bid Days Dayls Diysls Dey Dayis2 Cham 183 1 355 611 256 si 0 165 2 a 693 m os 2s 517 3 361 El wm mons 352 4 390 66 28 6s "0 5 391 618 m1 9% 18 as Mse0n TI27 632 22 88 Wae a@ SisGeiertefomahe cl ontrollgoupyap< 005(Dumais es). 01330 WWIILLDDLLIIFFEEININTTEERRNNAATTIIOONNALA,, 1LTD + 514 PROJECT NO: 454-102 ------------------------ APPENDIX VI InAdciuvtideuaDlieBtoardyyTWoexiigchittys S(gt)udfyrowmitahMPaFlOlaSrd Page 13 --_--Experimental Group Change Change Total ppm 2.1) Bid Dyy8 Dayls Daysis Dayo Days Change 66 1 mn an 305 wm 2s 510 2 356 01 251 sr 210 61 3 369 613 04 9 180 ea ` 353 EY 206 Ct 393 5 rE) 2 209 s0 198 07 Ma3)n TIT3 eaw" 2"8 @38 324% @7l _--Experimental Group Change Change Tol @pmai) Bid _ Day8 Dayls Dmy%ls Dayo Duylsn Change n2 1 1 wn 201 8 166 367 2 28 0 192 66 166 358 3 3% 66 26 ss 8 a 4 5 99 20 6 9 a7 5 0 69 29 955 256 as MseDm I8e S9m 2a16 1m 35 a;3 a5E6 04331 WILDLIFE INTERNATIONAL, LTD ss PROJECT NO: 454-102 Group Gpmai) 146 APPENDIX VI Individual Body Weights (8) fom aMallard Acute Dietary ToxPiacgiety14Study with PFOS hhhALLPL--N Pr- PLD Bid DiS _ Dayls Changs Dysls Day Change CThaonlge 1 26 as 29 659 184 a 2 330 69 29 79 10 09 3 75 1 256 78 107 363 4 301 510 29 754 184 an s 380 76 196 550 26 10 sMeDan --33528* Se56d 2322 Gs3 1 % 31%06 TeGrmouap Change Change Toul pmai) Bid Day8 Dayls Daysds Day Days Change 20 1 9 "wr 28 mm ss 2 165 as 250 os 21 ast 3 wn 26 309 Hs om 1 + 21 S16 2s 001s an 5 203 40 27 CO an SMeDn T rr) E asei E m365 Wae 3BsE 05 S"Statlistiycally deiecnenfofmomBtehccoonilggoroupp Rapp<<O00.01D(uDienntets'sLtees)t 01332 A WILDLIFE INTERNATIONAL, LTD so. APPENDIX VI Individual Body Weights (g)from a Mallard Acute Dietary Toxicity Study with PFOS. Page 14 PROJECT NO: 454-102 GrToupT pm st --_-- Bid Dus Dayls CDhaayngles Day2 CDhaaynigse2 CThoatnagle 1 126 308 152 2s wm 399 2 0 4s x0 ws 2m 07 3 www as us a8 s . . . -. s 143 310 197 sie 26 0 MsaDn 6 TiE w 2 y m5I a 695 B I 1 w w o BE Group Change Change Toul Gpmai) Bid DiS Dis Deyss Dy: Duis: came un 1 - - -. 2 . - 2 .. 3 - . . -. . 4 . . . 5 185 58 198 os wo sD T - ; : T - : T-a -- TT could not be evaluated satstcaly-- wing D-- ams-- est----------em 01333 WILDLIFE INTERNATIONAL, LTD -60APPENDIX VII PROJECT NO: 454-102 Page | Feed Consumption (/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS. --_ "Experimental Group -_ (ppmai) Pen Exposure Period Day0-5 Post-Exposure Period Day 6.8 Control 1 109 145 2 92 140 3 85 n7 4 93 132 5 82 100 6 94 116 Me5a)n * 1902 11275 0132% WILDLIFE INTERNATIONAL, LTD -61- PROJECT NO: 454-102 APPENDIX VII Feed Consumption (g/bird/day) by Pen from a Mallard Acute: Dietary Toxicity Study with PFOS Page 2 Experimental Group __Exposure Period Post-Exposurc Period (ppm a.i) Pen Day 0-5 Day 6:8 91 1 7B 130 2 7 104 Mean 7 117 183 1 9% 135 2 91 128 Mean or 132 366 1 103 129 2 85 120 Mean oF 125 732 1 I] 109 2 7 9% Mean 77 101 146 1 8 102 2 121 217 Mean 105 159 293 SE 2 59 65 Mean 44 63 586 1 40 43 2 32 61 Mean 36 55 um 1 2 2 2 2 26 Mean 2 25 01335 WcILiDLoIFuEsINbTEaRNAlTIOeNAoL,BeLdTD "e2- PROJECT NO: 454-102 APPENDIX VII Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Page 3 `Experimental Group `Exposure Period Post-Exposure Period -- (ppm_ ai) -- Pen _ Day 8--- 15 -- Day -- 1522 Control 1 185 159 2 161 182 3 166 199 --_--s MeSaDnB112 371 01280 04336 WILDLIFE INTERNATIONAL, LTD 6- PROJECT NO: 454-102 APPENDIX VII Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary ToxiciPtaygSet4udy with PFOS Experimental Group. (opmai) Pen Exposure Period PostExposure Period Duis Days 91 1 nm 198 183 1 136 204 366 1 165 1 732 1 148 nm 146 1 159 164 293 1 109 132 586 1 na 143 un 1 106 154 01337 WILDLIFE INTERNATIONAL, LTD -64APPENDIX VIII PROJECTNO.: 454-102 CHATNOPGROTEOCSOL `This study was conducted in accordance with the approved Protocol with the following changes: 1. Theprotocolwasamendedtoaddanadditionaltestconcentrationof 10pprm a. 2. pTrhoetopcrooltwocaoslclawraisfieadmteondienddictaoteintdhiecactoelltehcattionbiolef lwiivlelr bferocmolbliercdtsedthaftrodmiedalldusrtiundgtyhbeicrdosu.rseThoef thestudy. 3. T6h4e0 parnodtoc1o2l80wapspmameani,detd0t0o,c9h.a1n,ge18t.h3e, t3e6s.t6c,on7c3e.2n,tra1t4i6o,ns29f3r,om5806, 1a0n,d201,17410,p8p0m, a1i6.0', 3Te2s0t, congeynsitsa.tion werechaged fo reflectth etsubstance ark giveninthe ew crifcteof 4. rIenqduiivrideudallelgybannumdbsetroebdewuisnegdbtaoniddesnwtiefrytehuesbeidrdtsouniquelyidentifycach duckling. Theprotocol 5. teBsltootdubsesa.mTplheesprwoetroecoclolilnedcitceadtoendtDhaatyhepaarnidniDzeadyva2c2utianinneorns-whoepualrdibniezuesde5d.ml borosilicate glass 6. Trheceofridenda.ltemperaturefromonebrooderunit onDay 8andallbrooderunitsonDay 22werenot 7. MThae ay8,te1m99o9o.n observations were inadvertently not recorded for birds in a single brooder unit on 8. oOfbstehrevatteisotn.s Tfeormpcoenrtaotlurbeirsdfsofrrotmhetsweo bbrrooooddeerrlleevveellsswweerreeinaaldsveortneontt.lyrencootrrdeecdoordnedthoinsDdaayy6 oOfbtsheertveastti.ons for control birds from one brooder level were inadvertently not recorded on Day 22 04338 WILDLIFE INTERNATIONAL, LTD -65- APPENDIX IX PERSONNEL INVOLVED IN THE STUDY PROJECT NO: 454-102 The following key Wildlife Intemational Lid. personnel were involved in the conduct or `managementofthis study: (1) Mark Jaber, Wildife Toxicologist (2) Joann B. Beavers, Director, Avian Toxicology (3) SeaPn. Gallagher, Senior Biologist (4) Courtney Casey, M.S. Senior Biologist (5) WillarBd.Nixon, Ph.D, Manager, Analytical Chemistry (6) Tim Kendal, Supervisor, Analytical Chemistry (7)Raymond L. Van Hoven,Ph.D. Scientist (8) Ellen Mank, Chemist 64329 L i WIE LDLIFE INe TERNR ATIe ONAL, , LTe@Dovtven PREOJETCTNO O.: 454-102 1 AMENDMETNOT STUDY PROTOCOL RA STUDY TITLE: PROS: ADIETARYLCS0 STUDYWITH THE MALLARD : PROTOCOL NO.:454/111098/MLCSDT.WC/OPPTS 1 'SPONSOR:3M Corporation AMENDMENT NO.: 1 PROJECTNO: 454-102 i _--,--, [EFFECTIVE DATE: April 22, 1999 i AMENDMENT: Onpage 2oftheprotoacddoaln additesttcoinceontrnatiaonlof 10 ppmi. REASON: i Sponsor request, 1! Alp- Sad Study Director i |] Laboratory M? ae a - `Principal Contact wl i Date 1)Js)3l alla Date lo/2]29 Date 01340 i WILDLIFE INTERNATIONAL, LTD PROJECTNO.: 454-102 i AMENDMENT TO STUDY PROTOCOL 1 STUDY TITLE: PFAO DIS ETA: RY LC50 STUDYWITHTHEMALLARD "PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS AMENDMENT NO.: 2 1 SPONSOR: 3M Corporation PROJECTNO.: 454-102 i EFFECTIVE DATE: April 27,1999 1 AMENDMENT: die during the coursof thetest. On page under necropsy add: i `Whenpossible,bilewillbecollectedfromallbirds.Livers willalsobecollected fromallbirdsthat 1 REASON: `Bile isbeing collected atSponsor request.The protocolisbeingclarifiedtoindicatethe collection 1 `of liver from birdsthatdie. 1 [Li/se f. Ah `StudyDirector 1 tory 1 . Sy `Principal Contact 1 3 Date allay Duis 10/22/49 Date' ! 01341 a W--I_L--DLIFE INTERNAe TIONe AL, LD PaPaege loLoff1l DEVIATION TO STUDY PROTOCOL STUDY TITLE: ~~ PFOS: A DIETARY LCS0 STUDY WITH THE MALLARD i PROTOCOLNO.: 454/111098/MLVSDT.WC/OPPTS DEVIATION NO: 1 i SPONSOR: 3MCorporation PROJECTNO.: 454-102 i } DATE OF DEFACTO DEVIATION: April 22-May 14, 1999 1 DEVIATION: A Isntdaitveiddtuhaaltlbyirmdusmwboeurleddwbienigdebnatnifdisewdbeyrceoulsoerdetdoluengibqaunedlsyi.dentify achduckling.Theprotocol a || REASON: Protocolerror. `Ducto the extendedduration ofthisstudy, ducklingswould haveoutgrown leg. `bands. The protocol by wing bands. should have been clarified toindicatethat all ducklings would be identified 1 IMPACT: n Li 1. all, ily `None.Wing bandsareroutinelyusedtoidentifyducklingsandall birdswereuniquelyidentified. . 1 STUDY DIRECTOR DATE a alb\ WILDLIFE INVERNATIONAL LTD. MANAGEMENT DATE u 041342 q WILDLIFE INTERNATIONAL, LTD Page lof 1 DEVIATION TO STUDY PROTOCOL a STUDYTITLE: PFOS: ADietary LESO Study witheMtallhard PROTOCOLNO: 454/111038/MLCSDT.WCIOPPTS DEVIATION NO: 2 || SPONSOR: 3M Corporation PROJECTNO; 454102 5 DATE OF DEVIATION: April 30 snd May 14, 1999 a DEVIATION: 5 mlborBolsoioldiscaatmepglleasscstoelsltecttuebedospnrDiaoryto8baenidnDgeryef2ri2goerfattheedt.eTshtewperoetsoocorlediindnicnaotne-dhtehpaatribnliozoedd a `sampleswouldbeplainclabeellded heparanizedvacutainers. REASON: A decapit"aTteisntgabniirmdaslasnwdedrreai0n0insgmballolotdoneftfoetceitetnulbyedsr.aHwepbalroiodnbwyassyrniontgaed.deBdtolbwloaoosddsoarmapwldbeys a duSeepetroati8onSopfonsseorrurmerqeuqeusitrefsocsoaegpuelraattieosneorfumblforoodmsatmhpelebsl.oodat WildlifeInternational Ltd. a IMPACT: un None. 0 Lay ' 21d 5 STUDY DIRECTOR 311 DATE a WILDLIFE FERRO TTD. MANAGEMENT DATE 04343 a WILDLIFE INTERNATIONAL, LTD 7 DEVIATION TO STUDY PROTOCOL a STUDY TITLE: PFOS: ADietary LCS0 StudywiththeMallard PROTOCOL NO: 454/111098/MLCSDT.WC/OPPTS DEVIATION NO: 3 a SPONSOR: 3M Corporation PROJECTNO 454-102 a DATE OF DEVIATION: April 30andMay 14, 1999 a DEVIATION: ppma.OntreAaptrmieln3tl0e,v1e9l9w9a,stnhoeftinraelcotredemdppreirorattfourtoremmemianabtiorn ofoutnhiootsheoubdisridnseg.bAidrrddistiionntahlley,64o0n Hn `bMiardys,14thefina temperaturesonallbrooderunitswerenotrecorded priortotermination ofthose: REASON: a atthe tBiimoeloogfiasfttoevrenrosoingohbts.eTrhvaetbiroonosd.eBrpreondsewrhepreentetmpereatumrespawerereetynrpoitcraaelcloytrredecudovrdererdsdeurpistnyg a atfetmeprenroaotnuorbessewrevrateimoinsss.eBidnoleoggliescttserdtecoorredcionrgdatfetmepmeoroatnuorbessefrovarteimopntsyopnenthsethoactcahsosuisonesdbwihredrse caries thesameday. IMPACT: un . un the brooders w`eNroenef,uTnecmtipoenriantguprreospreercloy.rdedforbroodersthedayprio 1 themisseddataindicatedthat Jo 0allial, un STUDY DIRECTOR 43) DATE 1 ee WILDLIFE \ATIONAL LTD. MANAGEMENT able DATE 0134% a W_I--LDLIFE INTERNATIONAL, eLeTD PPaagegelLoof1fl DEVIATION TO STUDY PROTOCOL " STUDY TITLE: POS: A Dietary LCSO Study with the Mallard PROTOCOLNO.: 454/111098/MLCSDT.WC/OPPTS a SPONSOR: 3M Corporation a DATE OF DEVIATION: May 8, 1999 -_-- DEVIATION NO: 4 PROJECTNO: 454-102 DEVIATION: n n ppm ai.OtnrMeaatyme8n,t1l9o9v9e,wtehreeafintaedmvoeortnenotblsyenrovattrieoncsofrdoedu.nt S17-1 housingbirdsfromthe 320 H REASON: Biologist oversight. IMPACT: a inthislevelwerNeonneo.rmOablsienrvaaptpieoanrsafnrcoematnhdebemhoamviionrg.oSfinMcaeyth8etandeMamy 9,p1f9oe9r9tihratnuandittwitahsacutraaelrcotbridereedds nN aotbtsherevastaimontniotmreeocbosredrevda.tionswereperformed, itisapparentthathebirdsweroobserved,butthe a ( R 3 STUDY DIRECTOR DATE 3% B WILLE TTD. MANAGEMENT DATE EUACT] a 01345 B WILDLIFE INTERNATIONAL, LTD Page lof] DEVIATITOON STUDY PROTOCOL STUDY TITLE: PFOS: A DIETARY LCS0 STUDYWITHTHEMALLARD B PROTOCOLNO.: 454/111098/MLVSDT.WC/OPPTS DEVIATIONNO: B SPONSOR: 3MCorporation PROJECTNO:454-102 DATE OF DEFACTO DEVIATION: April 28andMay 14, 1999 DEVIATION: B OanndA$p6r2i-l32w8,er1e9i99n,Daady v6 eofrtnhteotteersetn,cotorbdlseeydrv.aTtionsefo mcontprfoolrgterhoeusprebbirroadosdeinrtlbervooeudlesrwrelerveeaellssSo6s2n-o2t. BH i`n rleevelco5o7n0tr-h3idswdeeartdee.iOnnaMdayv1e4,rn1ot99te9r,enDcoatrylde2yd2.oTfthheetpersott,oocboslerrveaqtuiionrsefdtohracotnbtirrodlsgwroouuplbdibrdes observed atleastoncedaily andthathe broodercompartmenttemperaturewouldberecorded i oncedaly. H REASON: B Biologist oversight. 2 IMPACT: ``Wniotrhmtahle beexhcaevpitoironaonfdtehpepemairsasneced aonbdsberrvoaotdieorntse,mopbesreartvuatrieosnwseirnedwiictahtienatlhleaccocnetprtoalbblierrdasnwgeeroer H thedurationofthe te. Itis reasonabletoexpectthatbirdswerenormalinbehaviorand appearanceand thatbroodertemperatureswerewithin theacceptablerangeatth timethe obserwveraentotireocorndesd. Therefore, thisdeviationcausednoadverseimpactonthe R outcome ndinterpreofttahtesituodny. u / : " STJUDuY nDIeRECTI OR NA aDATl E e RB WL`tDLIaFEIyNTWEReN(AAtTTIOIN:AOL NAL LTD.MAiNAGEMENT DATESfolos 01346 RN -- WILDLIFE INTERNATIONAL, LTD -- nN SOP DEVIATION n STUDY TITLE: PROS: A DIETARY LCS0 STUDY WITH THE MALLARD PROTOCOLNO:: 454/111098/MLCSDT.WC/OPPTS . n "SOPNO.:415 DEVIATIONNO.: 1 n SPONSOR: 3M Corporation PROJECTNO:: 454-102 DATE OF DEFACTO DEVIATION: April 23, 1958 nn DEVIATION: treatmOenntgArporuipls2a3,s1919F9bteheltoewmtphereastpuecriefoiefd omneibnriomoudm olfev9e5lFu.seTdhteorheouasreetnhoer1e28c0 oppormfdtahs.ei n Hreoswpeonvseirb,leotnetchhenicfiolalnoowribnigdoleoygtishtemtaekmipnegraattuhreerwmaosstwaitthainnodrmajlrtauongcoesr.recttthmeteempernatutre, REASON: n Misadjustmentofthe brounoittdhermeostrat. IMPACT: n None, Therewasnoapparenteffectontestbirdbodyweightorhealth. , a Je, 9. All STUDY DIRECTOR 9 oft DATE ou `Wa ILDLIFE INFERNATIONAL LTD. MANAGEMENT Da ATE las a 01347  WILDLIFE INTERNATIONAL, LD Ny SOP DEVIATION wm STUDY TITLE: PFOS: ADIETARYLCS0 STUDY WITH THE MALLARD PROTOCOL NO.: 454/111098/MLCSDT.WCIOPPTS nN SOPNO:: 423.14 nm `SPONSOR: 3M Corporation DEVIATIONNO:: 2 PROJECTNO:: 454-102 DATE OF DEFACTO DEVIATION: April 2, 1998 n DEVIATION: was weOignhAepdriolu22,fo1r99p9r,deuprairndaiotefitporhenepar1a6t0iopnponm Day a. 0teosfttchoentceestn,tr1at4i6o5n9dgioetf.tTehstissubasmtoaunn:t "a exceededthetargetamountby 0.099 ae n Dietician oversight. n IMPACT: un iNnonfien.aTlhdieetdairfyfceornecnecneitrnatthieona.mountoftestsubstanceweighed resultedin alessthan1%change un Ao bral) STUDY DIRECTOR J n WILDLIFE (ATIONAL LTD, MANAGEMENT n sls DATE __DA_TEalslag 04348 [| rroTOCOL a PFOS: A DIETARY LC50 STUDY WITH THE MALLARD H SeriUeSsS8.5E0nviorloongmiecnatallEPfrfoetcetcstTieosntAGgueindceylines | 'OPPTS Number 850.2200 FIFRA Subdivision E, Section 71-2 " OECDGuideline205 1 rE: H H n n = || Submitted to WILDLIFE 3M Corporation Environmental Laboratory P.O. Box 33331 `St. Paul, Minnesota 55133 INTERNATIONAL LTD. Eu859n#81C0No)ma8m2er2r.cd3e60D10r6i8v1e Novembe 10,1998 04349 PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 3MLAB REQUEST NO. U2723 D N e WIE LDLIFE ITNTR ERNAAe TIOA NAL LTD. e : . n > mn PFOS: A DIETARYLC50 STUDYWITH THEMALLARD || SPONSOR: n 3MCorporation EnPv.Oi,roBnomxen3t3a33lL1aboratory St. Paul,Minnesota 55133 SPONSOR'S REPRESENTATIVE: Ms.SusanA. Beach mn TESTINGFACILITY: WildlifeIntemationalLid 8598 CommerceDrive un Easton,Maryland 21601 STUDY DIRECTOR: ScanP. GalSlenaiorgAvihanBeiolrogi,st Hn LABORATORYMANAGEMENT: MJoananB.onBfeAavaveiresngToxiecolrogy n --------------_FORIABORATORYUSEONLY ce [zim daly gm Lm | Bn | ProposedDates: . 1l n i .__4sq-/ FreeNog, TO StudyBova : = 3 m| a | sTest Csoncesntri:c: 6S 130,640md (Ge peopeionme Seehh, hls | mn PROTOCOLAPPROVAL n A( . STUDYDI `OR Yaad DATE n LABORATORY wt 7 a en en EER % 20 q L 04350 a SPONSOR'S REPRESENTATIVE A5 fd Qed The Qi re He ME Fa loge al PROTOCOLNO.: 454/111098/MLCSDT.WC/OPPTS 3MLABREQUENSOT. U2723 " e W--I_--LDLIFE INTERNATIONAL LTD. r n " nu "Th eobjectiveoft h i sst udyistoev OBJECTIVE aluat th toxicity of test substancetothemallard(Anas nm platyrhynchos) possible. adminisece through th diet for five days. An LCSO value will be calelated, i un i selecteGdrcooupnsocfetennmtalrfloaarrftdidviuecodkalnyisns.gs,A5c-o1n0tdraoylsgorlodu,pwitlhlabreecfedivdeistusnctornetaatiendidnigetthwoitlelbtesmuabisntatnacienaetd an concureatly. Thetestsubstancewillbemixedintothediet,usuallyin ageometricseries of n concentrations.Theexposurepeiod willbefollowedby periodof a eastthredaysoruntreatedfood. Attheendofthethree-dayobservationperiod,halfofthebird ineachtreatmentgroupwillbe on `eountbhaasnaizldeidaetnfdolriavenraadndidtbiloonoadlco1l4ldeactyesdfaonrdrtehseindwuiealnlableyseiust.hTahniezerd,weithmlibviearrdasnwidibllnboeodimacoilnnlteacigtneedd. n "Throughoutthetet, thebirdswillbeobserved fortoxicological responses.WheapossiabnlLCe5,0 valuewill becalculated. . MATEARNDIMEATHLODSS a "Themethods,speciesusedandrouteofadministrationdescribedinthi protocolarebased upon proceduresspecifiedi theU.S. EnvironmentalProtectionAgencySeries 850 -EcologicalEffectsTest oH Guidelines OPPTS Number 850.2200 (1);Section71-2ofthe Environmental Protection Agency's a RegistrationGuidelines,PesticideAssessment Guidelines,FIFRASubdEi,HvazairdsEvailuaotionn: WildanldAigufateic Organisms (2), ASTMStandardE8S7-81, "StandardPractice For Conducting Subacute Dietary Toicity Tests With Avian Species" (3), and OECD Guideline 205, AvianDietary a ToxicityTest (4).Inordertocontrolbias,birdswillbeassignedtopens byindiscriminatedraw. No otherpotentialsourcesofbiasarecxpectedtoaffectth realsofthestay. u Thetes substancevillbeadminisitnteherdieetd. Thisrouteofadministwarsasetliecotend becauseitrepresentsthemostlikelyrouteofexposuretoavianspeciesinthe eaviromment. u Test Substance H formationonthecharacerizationoftest,controlo referencesubstancesisrequiredbyGood Laboracric Satantdarods(rGLyP).TheSponsorisresponsiblefo providing Wilde International H Lad.writinverificationthatthetostsubstancehasbeencharacterizedaccordingto GLP prirtoitsuse inthestudy.Ifwrittenverificationof GLPtestsubstancecharacterizationisnot providedto Wildlife H PROTOCOLNO: 454/111098/MLCSDT.WCIOPPTS 3MLABREQUESTNO,U2723 61351 -- a WI-- LDLIFE --I-NTrERNNAATTIOONMAALLLTwD. 0000000 0 * Intemational Lid.it willbenotedinthecompliancestatementofthe inal report.Theattachedform a IDENTIFICATION OFTEST SUBSTANCE BY SPONSOR (Appendix I is to be used to provide B informationnecessary forGLPcompliance. `TheSponsorsresponsibleforal information elatedtothtetsubstanceandagreestoaccept |] | `anyunusedtestsubstanceand/ortestsubstancecontainersremainingattheendofthestudy. TreatmentGroups H Groupsoftea mallardducklings (fiveperpen)willbeassignedbyindiscriminatedrawtocach ofthetreaantdcomatreolgnroutps.Thesex:ofeachindividualvillnotbedetermined. Atestwill H `usuallyconsisofa geometricserieoffive ditaryconcentrationsandat lastthreecontrolgroups. Test concentwirllabetseilecotendafte evaluatingtoxicitydataprovidedbytheSponsor,Thehighest test H `concneeednnott erxceaedtS00i0popmn. Eachgroupisfdtheappropriatetestorcontroldietforfive: days. Thisvillbefollowedby aperiodofatleastthreedaysonuntreatedfeed. H Controlbirdswill ceiveanamount ofthesolventorvehicleintheirdietequivalenttothehighest aminotheurestneddtiet. 1fmorethananestudy isconductedsimltancouslyinthesameroom,with || thesamesolveatorvehicle,a concurrentcontrolmaybeutilized orallsudics. H `DuntoifToesnt "Theprimary phasesofthetestandtheirdurationsarc: 0 1. Acclimation - Fromreceipto hatchofhatchlingsuntilthestart ofexposure. 2. Exposure-Fivedays. un 3. Postcxposureobservation - 3days/1 days. Test Birds Hu `Themallard representsanecologicalysignificantandwidelydistributedspeciesinthe United States.Themallardhasdemonstratedsensitivitytoth effectsproducedbyknowntoxicchemicals. BH Moreover,thisspecieshasproventobe agoodlaboratorymodeland # large amountofbaselinedataare. available. Bn Allmallard willbefromthesemehatchandwillbe5-10daysoldattetinitiation. Olythose: thatrcapparentlyhealthywillbeusedi thetest. Bodyweightsmayvarybetwoca75and 300grams Bn dependingontheageofthecohortofbirdsused (c.g. ver1s0duayss). Atleastfivetestandthree B PROTOCOL NO: 454/111098/MLCSDT.WC/OPPTS 3MLABR=EQUESTNO. U2,Th1as52  -- WILDL_ IFE -- INTER-- NATI-- ONAL_ LTD. --_--_-------- | > nm controlgroupswillbe used.Eachtreatmentorcontrolgroupwillcontaintenbirds(twopensoffive birdscach) ithcachgroupofbirdsidentifiedbypen numberandtestconcentration.Individualbirds willbeidentifi bycolored Ie bands.Birds willbeobtainedfromWhistlingWings, 113 Washington un Stree,Hasoves,IL.61041orfrom antherreputablesupplier. Allexperimentalbirdsvilbeacclimated : 10thecagingandfacilitesfrom th timeofhatchorreceiptuntilinitiation ofthetest. mn `Animal Dict AItest birdswillbefod a gamebird ration formulatedto WildifeInternationalLid. a specifications(Table1). Feedandwatervil beprovidedad Hbiturduringacclimationandduringthe test.ThewatersourcevillbetheTownofEastonmunicipalwatersupply. Feedisanalyzedperiodically mn inaccordance ith WidifeInternationalLid.StandardOperatingProcedures.Thebivirllrdecisve 10form ofantibioticmedicationduringthetest. a Specificationsforacceptablelevels ofcontaminantsingamebirdrationforavianspecieshave ao notbeen established.However,theeare no levels ofcontaminantsreasonablyexpectedtobepreseat inthediethat areconsideredtointerferewiththepurposeorconductofthestudy. a `DictPreparation Tes dieswillbepreparedatthestartofthetestand asuficietportionestimatedforthe 5-day A `exposureperiod willbepresentedtothebirds. Ifnecessary,appropriatedictwillbeaddedasneeded. After fivedaysofexposure, ll birdswill receiveuntreatedbasalration. a Thetest substancewillbemixeddirectly ntothe ationorbydissolvingorsuspendingthetest substanceinacetoneand/ortablegradecormoilprior tomixingwiththefeed. If used, acetwoilnlbes H allowedtovolatilize romthedietsduringthemixingprocedure.Tablegradecomoilwillnormallybe incorporated ntoth dict at &miuof2% (w/w)ofthefinaldiet.Mixingisnormdoanelwilthya H Hobart mixer(ModelNumber AS200T). a Alltestsubstance calculations will be based on the purityofthe testsubstance as received or `correctedto 100% active ingredientbasedontheinformationprovidedbytheSponsorin Appendix Iof 0 hisprotocol. | 04353 a PROTOCOLNO.: 454/111098/MLCSDT.WC/OPPTS 3MLABREQUEST NO. U2723 | WILDLIFE INTERNATIONAL LTD. mn -6- fr n `Samplesoftheexperimentaldietswillbecollectedforchemical analysistodeterminethe homogeneityandstabilityofthetestsubstanceintheaviandietandfo verifymessuretestconentra- H tions. Allsampleswillbeplacedinuniquelyidentified polypropyleas jars. H Sampleswillbecollected on Day 0, atdiet preparation,to determine homogeneity, measuretest conceztrations,andestablishDay 0vahesfoxevaluatingstabilityovrthecourseoftheexposureperiod. OnDay 5,attheendofthe exposurephase,sampleswillbecollectedfromthe feederscontainingthe i `oxperimentaldictstoassessstabilityofthetestsubstanceundertestconditions. Thedictsampling schemeforstudieswithfivetestconcentrationsissummarizedbelow.The samplingschememaybe: H adjustedbasedontheactual numberofconcentrationstested(seePage2)o theoption oftheSponsor. d PROPOSEDNUMBERSOFSAMPLES ExperimentalGroup Dayo Days oH Control 1 1 Level 1 -LowConcentration @ 2 H Level 2 2 2 Level 3 2 2 0 Level 4 2 2 Level 5 -HighConcentration 2 d 19 1 `TotalNuomfSabmplees=r30 H b"oStatmopmlleasyceorlsleocftefdeefdriomntthheemliexfitanngdvreisgshetlsitoddeestoefrtmhineetohpo,mmoigdednleeitaynd H "The above mmbersof samplesrepresentthosecollected from the test and do not include quality control (QC) samples suchasmatrixblanksandfortificationspreparedandanalyzedduringthe H analytical chemistryphaseofthestudy. "| 01351 oi PROTOCOL NO: 454/111098/MLCSDT.WC/OPPTS 3MLABREQUEST NO, U2723 mw = i WILDLIFE INTERNATIONAL LTD. a " n y `Samplesoftheexperimentaldietsmaybeanalyzedfollowingcollectionorstoredin afreezer until prepared andlor extractedforanalysis. Chemicalanalysesofdiets villbe performedby Wildlife A InternationalLtd.Theanalyticalmethodusedwillbebaseduponmethodologyprovidedby theSponsor andidenitniApfpeindiexIdI.Themethodology usedtoanalyzethetestsampleswillbe documented A inthe raw dataandsumma intrhei finz al reepodrt. Maximum sample holding times, priorto analysis, nu `maybespecifiedbythe Sponsor,andifspecified,willbe addedtothisprotocolbyamendment. ad 3 Con Hn `Testbirdswillbehousedinthermostaticallycontrolledbroodingpensmanufacturedby Safeguard ProIdnc.uorcequtivaslen,t. Eachpen measures appro6x2 Xi92mXa25t.5cemlhigyh.Theexternal H `walls,floorsandceilingsofeachpenareconstructedofvinylcoatedwiregrid. Uponinitiaotfitohne test,eachbroodingpenwillcontainfivebirds. Two broodingpens willbe assignedtoeachtreatment H group (10 birdstotal),whileat least six penswillbeassignedtothecontrol (atleast 30birdstotal). Each group willbeidentified by pen number. OnDay 8ofthetest,birdsin one pen fromcachtreatment nH group( birds)andthreepensfromthe conrolgroup (1Sbirds)villbeeuthanized.Remainingbirds | two pens. `willbemain fort anada diti ionan l14edad ys.Duringthatperiod,remainingbirdsmaybeseparated into Ha `Duringthefirsteightdaysofthetest,thetemperatureinthe broodingcompartmentofthe pen `willbemain atat ppra oxii matn elye 29dC.Brooderswillbeturnedoffduringthe last14daysofthe. H test. Acceptablerangesfor broodingcompartmenttemperatures willbespecifiedin Wildlife IntemationalLtd.StandardOperating Procedures.Broodingcompartmenttemperatures willberecorded a `onceeachdayduringthefist eightdaysof thetest. Theambientroomtempewirllarantgebuetrweeen `approximately 15-30C. Temperature andrelativehumiditywillberecordedtwice dailyduringthetest. nn A16hours light/3hoursdarkphotoperiodwillbemaintained,and theaveragelightintensityprovided tothebirdsduringthetestwillbedetermined. H `Housingandhusbandrypracticeswillbe conductedsoastoadheretothe guidelinesestablished H `by NationalResearchCouncil(5). 01355 Hn PROTOCOLNO.: 454/111098/MLCSDT.WC/OPPTS 3MLABREQUESNOT. U2723 B WILDLIFE INTERNATIONAL LTD. ______ B * Obscrtions . Allbirdswillbeobserved atfeast oncedalyformortalityandtoxicologicalresponses. Individualbodyweightswillbemeasuredatth initiationofte est,attheendoftheexposure: RK periodonDay5,and onDay8. Averageestimatedfeedconsumptionwillbedeterminedbypenforeach treatmentandcontrol group forthe exposure (Days 0-5) and post-exposure observation (Days 6-8) N periods. Forthosebirdsmaintainedforanadditional 14 days,bodyweiagndhfeted consumption `measurementsvilbeperformedweeklyandattesttermination.The accoffueedrconasumcptiyon valuesmaybeaffectedbyunavoidable wastageoffeedbybirds.Noattwillebemmadeptoqtuantsify R theamountof wastedfeed,asit is normally scatteredandmixedwithwater andexcreta. R jo Alltestbidstha dieduringthecourseofthetestandal birdscuthanizedwillbesubjectedto R agossnecropsy.ForthosebirdscuthanizedonDays 8and22ofthe test, liversand ~2mLof blood will becollected. Liversvillbeweighed,placedinindividuallabelledcontainersandstoredfrozen. a Bloodwillbeplacedinlabeled heparinized vacutanersandplacedunderrefrigeration.Allsamples will betransferredtotheSponsorforresidue analysis. RN a R Disposition of Test Birds Attesttermination,testbirdswillbecuthanizedbyusingcarbondioxidegas,ervica dislocation n oxotberappropriatemethods. Themethoduse willbedocumentedintherawdata. Allcarcasswielsl bestoredfrozenpriortoshipmentfo the Sponsor. nH `Theconcentrationrespdatoaonbtasineedfom theestwillbeevaluatedand,if possible,anLCSO valueand95%confidence limitsvillbecalculated.Whenthemortalitydatafacilitates statistical asnlyses,aneofthreemethodswillbe sedtocalculateanLCS0 value, Thedata willbeanalyzeid,n H onderofpreferbeynpcroebi,tanalysis, themovingaveragemethod,o thebinomialprobabilitymethod (6,7,8,9). ThechoiceofmethodforcalculatingtheLCSOvaluevillbebaseduponthemortality H `pattem observed.Wheapossinobmolrtaeli,tylevelvillbedeterminedandreported. = 01356 H PROTOCOLNO.: 454/111098/MLCSDT.WCIOPPTS 3MLABREQUEST NO.U2723 H WILDLIFE INTERNATIONAL LTD. 0 9 RECTO O BEMRAIND TAIS NED H RecordstobemaintainedfordatageneratedatWildlifeInternationalLtd.will include,but not belimitetod: H 1. Acopyofthesigaedprotocol. 2. Identiafndichacraactteriizaotinonofthetestsubstance,if probvytiheSdpoensodr. HB 3. Datesofinitiation andterminoaftthietoesnt. 4. Animalhistory. H 5. Husbandry andenvironmentalconditions. 6. Concentrationcalculationsendrecofdoitprrepadratison. 7. Bodyweightmeasurements. H 8. Feedconsumptionmeasurements. 9. Dailyobservations. " 10. Necropsyfindings. 11. Statisticalcalculations. H 12. Analytical chemistry methods, results and chromatograms,if applicable. 13. Acofto hefinp alrepyort. | EINALREPORT nu Arepextoftheresultsofthestudyvillbe preparedbyWideIntemational,Lid.Thereportwill include,butnotbelimitedto thefollowing: 1. Nameandaddressofthefacility pecformingthestudy. H 2. Datesonwhichthestudywas niiatedandcompleted. Iti theresponsibilityofthe Sponsorto provide the finaldatethatdataarerecordedfor chemistry,pathology and/or supporting oH evaluationsthatmaybegeneratedatother aboratories. 3. Astatementofcompliance signedbytheStudyDirectoraddressinganyexceptionstoGood NH LaboratoryPracticeStandards. 4. Objetivesandproceduressatedintheapprovedprotocol,incdinganychangesinthe original H protocol. . 5. Statisticalmethodsemployedforanalyzingthedata,when applicable. Thecalculated LCS0 value,95 peroeatconfidencelimit,slopeofthe conoentraton-esponsecurve,theresultsofthe un oodaess-of-fttest (e.g. X*test),and adescriptionofstatisticalmethodsused.Thesamo statisticswillbeprovidedforpositivecontrols (whenused). nH 01357 " PROTOCOLNO: 454/111098/MLCSDT.WC/OPPTS 3MLABREQUEST NO. U2723 m\ WILDLIFE INTERNATIONAL LTD. B -10- 6. Thetest,controlandreferencesubstances identifiedbyname,chemical abstracts numberorcode H number, strength, purty,andcomposition orotherappropriatecharacteristic, if provibdytehde Sponsor, HB 7. Stabilityand,whe relevant totheconductofthestuds,thesolubilityofthetest,controland referencesubsuntdearthnecocndietiosn ofadminiifsprotvirdedabytthieSoponnso,r. HB 8. Adescorfthie epststysitemousend.Whereapplicable,the finalreportshalincludethenumber ofaniusemd,boadylweisghtrange, sourceofsupply, species (including scientific name), age, = andproucsedefordidenutifircatieon. 9. Adescriptionofthedosage,dosageregimen,routeof administration,andduration. 10. Adescriptionofthemethodsused,includingbutnotlimitedto: H #) De(tfauisleedd).desAcrniupttriioennotfatnhaelbyassiaslofdtiehte,idniceltuwdiinlglsboeuirnccel,uddieudianttsh(eifeusstedr)e,paonrdt.supplements H b) cTohnceennturmabteironooffctonectensturbasttiaonncseuisnecda,cnohmlienvaell,aanssda(ywmheetrheoredquusierdetd)omdeeatseurrmiendedaiccttuaarly H ucosnecdefntorraptoisoints,ivneucmobnetrxoolfsb(iirfdaspppelirccaobnlcee)n.trRaetsiuolntasnodffroraconntogl,ean-tdenfsat(imiefnscoodnfduitcotxneicdga)n.ts ) Acclimation proceduresandmethodsofassigningbidsto testpens. H &) oFtrhoeqrueanbcayc,rdumraabteihoanviaonrd,mientclhuoddisngofiombeseorfvantsieotn,.dDurateion,ssevceorfitryga(isioncfpuidnitntogxdieciaatthio)o,naannndd " `upmoboerrsaffectedinthe diffrent dietaryconcentrations andcontrolscachdayof the test H )" teDmepscerriapttuiroenso,afphporuosxiimnagtceotnedsittiroonosm,hiunmcilduidtiyn,gpthyopteo,pesriizoedaannddlmiagthetriinaglinotfeapseiny., pen H Aveeraxgebppoerdiyoowdeainsgdhetnsudfoorfrbtihredeseisnt.cachpenatthebeginningofthe test,theendofthe # Estimatedfoodconsumptionfortheexposureperiod andforthepostexposureperiod. H 11. Adescriptionofallcircumstancesthatmayhaveaffectedthequalityorintegrityofthedata. `Anythingunusualaboutthetest,anydeviationfromthese procedures,andanyotherrelevant H information. 12. ThenameoftheStyDirecto,thenamesofotherscientistsorprofessionals,andthenamesof ] all supervisorypersonne,involvedinthestudy. 13. A description ofthetransformations,calculations,oroperationsperformedonthedats, a B summaryandanalysisofthedata, nd astatementoftheconclusionsdrawn romtheanalysis. 04358 H PROTOCOLNO.: 454/11109/MLCSDT.WC/OPPTS 3MLABREQUESTNO.U2723 8 Lo H WILDLIFE INTERNATIONAL LTD. = -1- BH 14. iTnhtesoisgnuedenidfpdatledcrep.ortsofcachoftheindividualscientistso otherprofessionalsinvolved 15. Thelocation where llspecimens,raw data,andthe inalreportar tobestored. BH 16. Astatementpreparedbythe QualityAssuranceUnitlistingthedate tha studyinspectionsand aditsweremadeandthedatesofanyfindingsreport totheStudyDirectorand Management. H 17. fitsnecessaryto makecorrectionsoradditionstoa final eportaferithasbeen accepted,such changes shallbentheform ofamendmentbyth StudyDirector Theamendshomuledcnleartly H identifythe prtofthefinalreportthei being addedtoorcorectedandthereasonsforthe correocratddiitoionn.Amendmentsshallbesignedand datedbytheStudyDirector. n CHANGINGOFPROTOCOL `Plannedchanges totheprotocolvillbeinthe formofwrittensmendmeatssignedby theStudy Hu Directoran the Sponsor'sRepresentative.Amendmentswillbeconsideredaspartoftheprotocoland `willbeattached the finalprotocol.Any heschangeswillbe i th form ofwritendeviationssigned nu bythe StudyDirectorandfled withtheraw data. Allchangestotheprotocolwillbeindicatedinthe final report. nu GOODLABORATORYPRACTICES oH "Thisstudywillbeconductedin accordancewithGoodLaboratoryPractice Standardsfor EPA (40 CFR Past 160 andlorPart792); OECDPrinciplesofGoodLaboraPtraoctricye (OCDE/GD (52) 32, Environment Monograph No. 45); end Japan MAFF (59 NohSan, Notification No. 3850, ou AgriculturalProduction Bureav),Each studyconductedbyWildiifeInternationalLi.isroutinely examinedbythe WildifeIntemationalLtd. QualityAssuranceUnitforcompliancewithGood ou LaboratoryPractices,StandardOperatingProceduresandthespecifiedprotocol. Astatementof `compliancewithGoodLaboratoryPracticeswillbepreparedforal portionsofthestudyconductedby oN `WildlifeInternationalLtd.TheSponsorwillberesponsibleforcompliancewithGoodLaboratory `Practicesfor procedures performedbyotherlaboratories (c.g,residue analyses or pathology). Raw data nH foal work pecformedaWildeIntemationalLd.an a opy ofthe finalreportwillbe filedbyproject `numberinarchives locatedontheWiklifeIntemationalLid.siteor stensltemativelocationtobe uN specifiedinth finalreport. nH 01359 oH PROTOCOLNO.: 454/11109/MLCSDT.WC/OPPTS 3MLABREQUEST NO. U2723 m D WILDE LIFE A INTERNATIONAL LTD. m eee -12- mn H 1 U(Sdr.afEhn,vOirPoPnTmeSnNtaulmPbreorte8c5t0i.o2n2A0g0e:nAcvyi.an19D9i6e,tSaerryieTso8xi5c0i-tyETecstlogEffiectcsTaelst Guidelines Bn 2 SUuSb.diEvnisviiornonEm,enHtaazlarPrdotEevcatliuoantiAogne:ncyWi.ldl1i9f8e2.anPdesAtqiucaidteicAsOsregsasnmiesnmts,Guisdueblsiencetsi,onFI71F-R2A, Environmental Protection Agency, OfficeofPesticide Programs. Washington, D.C. 0 3 `ASmubearciucteaDnietSaorcyieTtoyxifcoirtyTTeessttisnwgiathndAvMiaatnerSipaelcisc.s. 1A98S7.TMSSttaannddaarrddPEr8aSc7ti-c8e7.forACnoandlucBtoionkg HB of ASTMStandards, Vol. 11.04. PhiladelphiPaA,. 4 Organization for Economic Cooperation and Development. 1984. Avian Dietary Toxicity Hn Test. OECDGuidelinefor TestingofChemicals. Guideline 205. Paris. H 5 NWaasthiionngatlonR,esDeCa.rcNhatCioounnacliAl.cad1e9m%y.PrGeussi.de1f2o5rptph.e Care and UseofLaboratoryAnimals. 6 Finney, D.J. 1971.StatisticalMethods inBiologicalAssay, 2nd. ed., Griff Press, London. n 7 Thompson, W.R. 1947.BacteriologicalReviews, Vol 2, No2. 115-145. 8 aStnedphHaanz,aCr.dE.Eva1l9u7a7t.ioMnest,hAomdesrfiocraCnalScoucliaettiyngToarnTLeCstSiOn.g aPnadgeMsat6e5r-ia8l4sI.nAPguub.atNioc.ToSxTiPcol63o4g.y Philadelphia, PA. H 9 Stephan,C.E. 1978. Communication. U.S.EPA, EaviroReusemarcehLnabotraatorly,Duluth, MN. Personal H I] PROTOCOL NO: 454/111098/MLCSDT.WC/OPPTS 01350 3MLABREQUESTNO, U2723 m WILDLIFE INTERNATIONAL, Lo. i- R WILDLIFE INTTAEBRLNEAIT: IDOINEATLFLOTRD.MUGLAAMTEIOBNIRD RATION" BR INGREDIENTS m Imi Rn SoFiyneBCeaonmMMeeaa,l48%Protein WPhreotaetinMBiadsdes `Agway Spe6c0i% Parolte,in GDrrioeudndWhLeimyestone H MEeatshtimoanninCealPPrheomsix + Liquid Vitamin and Mineral Premix (sezbelow) n SGaLltFleordimze(dFermatco) Total PERCENT (4) i3w0u6s5s 660500 400 025500 003650 032 002150 100.00 a VITAMIN AND MINERAL PREMIX un ii, H RViibtoafmlianviAn NiPaanctoitnhenic Acid Vitamin By H ys PByirodtionrine H `VTihtiaammiinneE | MTVaiintegaamniensKe(MenadioneDimethylpyrimidinol Bisulfite) ICoodpipneer H Tron Selenium AMOUNT ADDED PER TON -- 7,000,0060gLrUa.ms 40 grams 180 mgrgasms "ss641.2 mggsrams 20,0010.2LUg.rams 140f52.8ggarraammmss 68 grams 511.g5rgarmasms. 182 mgs H ! * ToFhfeer$m%cgeurnaturaadtnietoenfBeidbyearnP.arloydsuicsitss aminimumof 27%protein, aminimumof2.5% crudefatand a maximum i 0 PROTOCOLNO:: 454/111058/MLCSDT.WC/OPPTS 04361 3MLABREQUENSOT, U2723 0 WILDLIFE INTERNATIONAL LTD. Bn -14- APPENDIXI mR IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR To be Completed by Sponsor . a L TestSubstance entity (nametobeusedinthereport): PEQS(PerfluorooctaneSulfonieAcidPotassiumSalt nu Reference Standard (if applicable): Analytical Standard: NA Internal Standard: _P1.1.e2 2rHHlHuH oroocSultfonaicAncied a `Test Substance SampleCodeor BatchNumber:__ Lot217 ---------------- `Test Substance Purity (% Active Ingredient): _98 _9 ExpirationDate: _2008 m I. Test Substance Characterization Have etpheridoenmtit Se Bane d itionorotherechatracaterifsotics determinedprior0its useinthisstudyinaccordancewith GLPStandards? Yes No_X_ mn IL Test Substance Storage Conditions m Pleaseindicatetherecommendedstorageconditionsat WildlifeInternational Ltd. --Ambieatroomtemperaty00ee om BE Has the stabily oaf the test: nee under these conditions Yes__No_X Other pertinent stability information: un IV. TestConcentrations: oH --X_Abdajsuesdtutpeostncotnhceepnutrriattyi(o%n)togi1v0e0n%sab.oiv.e. Dontadusttest concentratioton 100% ai. Testthe material AS IS. un V. Toxicity Information: `Mammalian: RatLDSO_251mghkg MouseLDSO_N/A un Aquatic: InvertebrateToxicity (EC/LCS0) FishToxicity (LCS0) D2 uplni7 amam gs gle RaTirouc t Umbglloe w DS upimiO amam gs gle FatheadMinnow:38mefl, n OtherToxicityInformation (includingfindingsofchronicendsubchronictests): un mn 01362 un PROTOCOLNO.: 454/111098/MLCSDT.WC/OPPTS 3MLABREQUENSOT. U2723 HB WILDLIFE INTERNATIONAL LTD. = -15- APPENDITXI n MethodOutlinefortheDeterminationof PFOSin Avian Feed N 1. Preparematrixfotficaton samplesinthedesiredavianfeedstockusingthedrymix technidue. HN 2. anWeditgrha1n0sgfsotarom1pl6eoszo.fFtrheenmcahtsrixqblaugnlkaaasnsbdortmtalteersi.xRfoerctoifridcawteiiognfhtese.d samplesinto aweighboat | 3. FiorctnahcehFtwreeinogchhedsqsuaamrpelbeot,tmleea.sure 100ms ofmethwaithnaogralduatedcylianndtdranesfrer Hn 4. Sealbottles `minutes. withscrewcapsandplaceonshakertable. Allowtoshakefor aminimouf3m0 un 5. Vacuum frat. filterwithqualitativefilerpaper andrinseretained fe3teimdeswithmethanolinto un 6. Transfer filtrate into a 200-mL volumetric flask and bring to volume with methanol. 7. mTreatnhsafneorl:S5m0%Lswoatfer20d0il-umtiLosnolsuotliveonntinctoontaai1ni0n-gm0L.v0o5l%umevt/vrifcofrlmaisck apcaritdiaalnlyd-f1il0l0edjuwgitah.i5/0m%L nu 4HPFOSinternalstandard.Bringtovolumewithdilution solvent. 8. M0.i2xaamndftirlaenrsifnetroaapnprHoPxLimCataeulyto1msaLmopfvltiehalre.10-mLsolutioninto asyringe andfilterthrough a 9. AmpulateandsubmitsampleforLCMS analysis. n 01353 n PROTOCOLNO.: 454/111098/MLCSDT.WC/OPPTS 3MLABREQUEST NO. U2723