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PFOS: A PILOT REPRODUCTION STUDY WITH THE MALLARD FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-105 3M LAB REQUEST NO. U2723 FTFRA Guideline 71-4 AUTHORS: Sean P. Gallagher Raymond L. VanHoven Joann B. Beavers STUDY INITIATION DATE: February 28,2000 STUDY COMPLETION DATE: December 18,2003 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St.Paul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 o f 122 000354 W ildlife International, Ltd. Project Number 454-105 - 2GOOD LABORATORY PRACTICE COM PLIANCE STATEM ENT SPONSOR: 3M Corporation TITLE: PFOS: A Pilot Reproduction Study with the M allard WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-105 STUDY COMPLETION. December 18,2003 The study was conducted in compliance with Good Laboratory Practice Standards as published by the U S. Environmental Protection Agency, 40 CFR Part 160, 17 August 1989; OECD Principles o f Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan Notification No. 3850, Agricultural Production Bureau, 10 August 1984, with the following exceptions: The study was conducted under multiple protocols. The in-life portion was conducted under one protocol (Wildlife International, Ltd. study number 454-105), and the analytical portions were conducted under two separate protocols (Exygen Research study numbers 023-042 and 023-063). Exygen Research study number 023-042 was initiated with a separate Study Director. Results o f analyses conducted by Exygen Research for study numbers 023-042 and 023-063 are reported separately. Amendments number 2 and 3 and deviation I for the Exygen Research analytical protocol for study number 023-042 do not have the Sponsor's signature to indicate these changes were approved by the Sponsor. SI USY DIRECTOR: Senior Biologist, Avian Toxicology SPONSOR'S REPRESENTATIVE: / John L. Newsted DATE DATE 000357 W ildlife International, Ltd. Project Number 454-105 - 2GOOD LABORATORY PRACTICE COM PLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Pilot Reproduction Study with the M allard WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-105 STUDY COMPLETION: December 18,2003 The study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR Part 160, 17 August 1989; OECD Principles o f Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MFF, 5?> NohSan Notification No. 3850, Agricultural Production Bureau, 10 August 1984. ' STUDY DIRECTOR: A tL- \ * L .____________ Sean P. Gallagher * Senior Biologist, Avian Toxicology SPONSOR'S REPRESENTATIVE: DATE DATE 00035$ W ildlife International, Ltd. Project Number 454-105 -3 QUALITY ASSURANCE STATEMENT This study w as examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR part 160, 17 August 1989; OECD Principles o f Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all audits and inspections and the dates any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY DATE CONDUCTED DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT Test Substance Preparation February 28,2000 February 28,2000 March 1,2000 Sample Preparation Diet Preparation March 1,2000 March 7,2000 March 1,2000 March 7,2000 March 1,2000 March 7,2000 Blood Collection April 11,2000 April 11,2000 April 12, 2000 Analytical Data & Draft Report January 16,17,28 & 29,2003 January 29,2003 January 31,2003 Biological Data & Draft Report January 27-31, February 3,2003 February 3,2003 March 12,2003 Data and Final Report December 1-3,2003 December 3,2003 December 18,2003 All inspections were study-based unless otherwise noted. Kimabbeerrllyy A. Hoxtery Quality Assurance:RReeipresentative DATE 00035^ W ildlife International, Ltd, Project Number 454-105 -4- REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Pilot Reproduction Study with the Mallard WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-105 3M LAB REQUEST NO.: U2723 STUDY DIRECTOR: Sean P. Gallagher j Senior Biologist, Avian Toxicology CBE M i m Y PRINCIPAL INVESTIQATOR: DATE Scientist, Analytical Chemistry M A NAGEM EN T: Joann B. BWavers Director, Avian Toxicology W illardB . Nixon, Ph.D. Director o f Chemisy DATE 2- //r /i DATE G003> Wildlife International, Ltd. Project Number 454-105 -5TABLE OF CONTENTS Title Page............................................................................................................................................. 1 Good Laboratory Practice Compliance Statement........................................................................... 2 Quality Assurance Statem ent............................................................................................................ 3 Report Approval.................................................................................................................................4 Table of Contents...............................................................................................................................5 Summary............................................................................................................................................ 8 Introduction........................................................................................................................................ 9 Objectives...........................................................................................................................................9 Experimental Design......................................................................................................................... 9 Materials and M ethods...................................................................................................................... 11 Test Substance and Internal Standard........................................................................................ 11 Test Organisms........................................................................................................................... 11 Identification............................................................................................................................... 12 Avian Feed and W ater................................................................................................................ 12 Diet Preparation.......................................................................................................................... 13 Diet Sampling............................................................................................................................. 13 Analytical M ethod...................................................................................................................... 13 Housing and Environmental Conditions.......... .........................................................................15 Observations............................................................................................................................... 15 Adult Body W eight and Feed Consumption..............................................................................16 Adult Blood Collection...............................................................................................................16 Adult Necropsy and Tissue Collection......................................................................................16 Egg Collection and Storage....................................................................................................... 16 Candling and Incubation.............................................................................................................17 Hatching and Brooding.............................................................................................................. 17 Offspring Blood and Tissue Collection.................................................................................... 18 Statistical Analyses.................................. ..................................................................................19 Results and Discussion......................................................................................................................20 Analytical Results.......................................................................................................................20 Mortalities and Clinical Observations.......................................................................................21 Adult Body Weight and Feed Consumption......... ....................................................................21 Gross Necropsy.......................................................................................................................... 22 Histopathology........................................................................................................................... 23 Tissues A nalysis........................................................................................................................ 23 Reproductive Results................................................................................................................. 24 Offspring Body W eights............................................................................................................24 Liver W eights............................................................................................................................ 24 Conclusion.........................................................................................................................................25 References.........................................................................................................................................26 C0036J Wildlife International, Ltd, Project Number 454-105 6- - TABLE OF CONTENTS PAGE 2 TABLES Table 1. Mean Measured Concentrations (ppm a.i.) o f PFOS in Avian Diet from a M allard Pilot Reproduction Study............... ..................................................... 27 Table 2. Mean Adult Body W eight (g) from a Mallard Pilot Reproduction Study with PFOS.............................................................................28 Table 3. Mean Feed Consumption (g/bird/day) from a Mallard Pilot Reproduction Study with PFOS............................................................................. 29 Table 4. Summary o f Gross Pathological Observations from a Mallard Pilot Reproduction Study with PFOS, Adult Birds Euthanized at 6 W eeks and Test Termination...................................................................................30 Table 5. Mean Egg Production (Eggs Laid/Hen and Eggs/Hen/Day) from a M allard Pilot Reproduction Study with PFOS.............................................................. 31 Table 6. Summary o f Reproductive Performance (Eggs Set from Week 5) from a M allard Pilot Reproduction Study with PFOS...................................................32 Table 7. Mean Body W eight (g) of Hatchlings and Surviving Offspring from a M allard Pilot Reproduction Study with PFOS................................................... 33 Table 8. Mean Liver Weights (g) from a M allard Pilot Reproduction Study with PFO S............................................................................................................ 34 I 000362- Wildlife International, Ltd Project Number 454-105 -7- TABLE OF CONTENTS PAGE 3 APPENDICES Appendix I. Appendix n. Appendix in. Certificate o f A nalysis.................................................................... ...................... 35 Diet and Supplement Form ulations............................................... ...................... 38 Diet Preparation.............................................................................. ...................... 39 Appendix IV. The Analysis of PFOS in Avian D iet............................................ ...................... 40 Appendix V. Diagram o f Test Layout.................................................................. Appendix VI. Adult Body W eight (g) from a Mallard Pilot Reproduction Study with PFOS..................................................... ...................... 55 Appendix VH. Appendix vin. Feed Consumption (g/bird/day) from a Mallard Pilot Reproduction Study with PFOS............................................ ...................... 59 Individual Gross Pathological Observations from a M allard Pilot Reproduction Study with PFO S............................. ...................... 63 Appendix DC. Histopathology Report.................................................................... ...................... 67 Appendix X. Appendix XI. Appendix xn. Egg Production (eggs laid/hen/week) from a M allard Pilot Reproduction Study with PFO S............................. ..................... 106 Reproductive Performance by Pen from a M allard Pilot Reproduction Study with PFO S............................. ..................... 110 M ean Offspring Body Weight (g) from a M allard Pilot Reproduction Study with PFOS............................................ ..................... 116 Appendix XIII. Adult Liver W eight (g) from a Mallard Pilot Reproduction Study with PFOS..................................................... ..................... 117 Appendix XIV. Offspring Liver Weight (g) from a Mallard Pilot Reproduction Study with PFOS............................................ ..................... 119 Appendix XV. Changes to Study Protocol............................................................. ..................... 120 Appendix XVI. Personnel Involved in Study.......................................................... ..................... 122 000363 Wildlife International, Ltd. Project Number 454-105 - 8SUMMARY STUDY: PFOS: A Pilot Reproduction Study with the Mallard SPONSOR: 3M Corporation WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-105 TEST DATES: Study Initiation - February 28,2000 Experimental Start - February 29,2000 Adult Termination - April 11 & July 14,2000 Biological Termination - July 25,2000 TEST ANIMALS: Mallard (Anasplatyrhynchos) AGE TEST ANIMALS: Approximately 27 weeks o f age at the initiation of the test SOURCE TEST ANIMALS: W histling Wings, Inc. 113 Washington Street Hanover, IL 61041-0509 U.S.A. NOMINAL TEST CONCENTRATIONS: 0 ,1 .8,6.2, and 17.6 ppm a.i. RESULTS: M allards were exposed to PFOS at dietary concentrations o f 0, 1.8, 6.2 and 17.6 ppm a.i. for 6 weeks. The control group and 17.6 ppm a.i. test group were maintained on test diets for an additional 13 weeks. No treatment-related mortalities were observed at any o f the concentrations tested. No overt signs o f toxicity were noted at the 1.8 or 6.2 ppm a.i. test concentrations. At the 17.6 ppm a.i. test concentration, a single hen was noted with signs o f toxicity that may have been related to treatment. There were no apparent treatment-related effects on body weight amongst males and females in the 1.8 and 6.2 ppm a.i. test concentrations. There were mean body weight losses among males and females in the 17.6 ppm a.i. test group that may have been related to treatment. There were no apparent treatment-related effects on feed consumption or egg production in the 1.8, 6.2 or 17.6 ppm a.i. treatment groups. Thi-e were no apparent treatment-related effects on any of the reproductive parameters measured. Histopathological examination o f selected tissue revealed a higher incidence o f testicular regression and adipose tissue microvesiculation for adult males in the 17.6 ppm a.i. group. While these observations may be incidental to treatment, a treatment-related effect could not be precluded. Based upon the results o f this study, the no-observed-effect concentration for mallards exposed to PFOS in the diet for six weeks was 6.2 ppm a.i. 0 0 0 3 V Wildlife International, Ltd. Project Number 454-105 -9 - INTRODUCTION This study was conducted by W ildlife International, Ltd. for 3M Corporation at the W ildlife International, Ltd. avian toxicology facility in Easton, Maryland 21601. The biological portion o f the test was conducted from February 29,2000 until July 25,2000. Raw data generated at W ildlife International, Ltd. and a copy o f the final report are filed under Project Number 454-105 in archives located on the W ildlife International, Ltd. site. Biological specimens are stored at 3M Corporation, St. Paul, Minnesota 55133. OBJECTIVES The objective o f this study was to evaluate the effects upon the adult Mallard (Anas platyrhynchos) of dietary exposure to the potassium salt o f Perfluorooctane Sulfonic Acid (hereafter referred to as PFOS) over a period o f approximately 6 weeks or 19 weeks. Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects o f adult exposure to PFOS on the number o f eggs laid, fertility, embryo viability, hatchability and offspring survival were evaluated. Histopathological examination o f selected tissues and analyses o f blood and tissue samples were also used to evaluate the effects upon adults exposed to PFOS and to their offspring. EXPERIMENTAL DESIGN M allard (20 males and 20 females) were randomly distributed into one control group and three treatment groups. The test concentrations were selected in consultation with the Sponsor, based upon the results of a LC50 study (Wildlife International, Ltd. Project Number 454-102) and additional toxicity information provided by the Sponsor. The original test concentrations selected were 0, 2, 7 and 20 ppm a.i. Following experimental start, the test material was reanalyzed and the final purity was lower than originally reported. Therefore, the actual nominal test concentrations were 0 ,1 .8 ,6 .2 and 17.6 ppm a.i. Group 1 2 3 4 PFOS Treatment Groups Nominal Concentration (ppm a.i.) (Control) 0 1.8 6.2 17.6 Pens per Group 5 5 5 5 Birds ner Pen Males Females 11 11 11 11 0003*5 Wildlife International, Ltd. Project Number 454-105 -10- Each treatment and control group contained five pairs o f birds with one male and one female per pen. Three treatment groups were fed diets containing either 1.8, 6.2, or 17.6 ppm a.i. o f PFOS. The control group was fed diet comparable to the treatment groups, but without the addition o f the test substance. Adult mallards were exposed to PFOS at nominal dietary concentrations o f 1.8, 6.2 and 17.6 ppm a.i. for a period of 6 weeks. A control group, fed non-treated diet, was maintained concurrently with the treatment groups. Each treatment and the control group consisted o f five pairs of birds, housed with one male and one female per pen. At the end o f Week 6, adult birds in the 1.8 and 6.2 ppm a.i. test concentrations were euthanized and subjected to gross necropsy. Test birds in the control group and 17.6 ppm a.i. treatment group were maintained on the appropriate diets until the beginning o f W eek 20 o f the test, at which dme they were also euthanized and subjected to gross necropsy. Effects on adult health, body weight, and feed consumption were evaluated as well as effects upon egg production, embryo viability, hatchability and offspring survival for all test concentrations. The adult birds were observed daily for mortality, abnormal behavior and signs o f toxicity. Adult body weights were measured at test initiation, on Weeks 2, 4, 6, 8, 10 and 11, and at adult termination. Feed consumption for each pen was measured over a seven day period each week throughout the test. Necropsies were performed on all adult birds and on 10 offspring from each test concentration. Liver weights were recorded for all necropsied birds. Liver, bile and blood (when available) and feather samples were collected at the time o f necropsy for possible analysis. Additional samples o f liver, brain, kidney, gonad, proventriculus, gall bladder, adipose tissue, and bursa o f fabricius (when available) were fixed in 10% buffered formalin for histopathological examination. During the test, the number o f eggs laid in each pen was recorded to evaluate egg production. Eggs laid in Weeks 1, 3 and 6 o f the test were collected and separated into shell, shell membrane, yolk and albumen and stored frozen for possible analysis. Eggs laid during Weeks 5 and 6 (Days 31 to 37) of the test were collected and set for incubation. Embryo viability, hatchability, hatchling health and surviv ability were examined. 000360 Wildlife International, Ltd. Project Number 454-105 -11 - MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "PFOS: A Pilot Reproduction Study with the M allard". The protocol was based on procedures outlined in the Environ mental Protection Agency's Registration Guidelines Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, Subsection 71-4 and the ASTM "Standard Practice for Conducting Reproductive Studies with Avian Species" (1,2). Test Substance and Internal Standard The test substance, PFOS, was received from 3M Corporation on October 29, 1998 and was assigned W ildlife International, Ltd. identification number 4675 upon receipt. The test substance was a white powder and was identified as: FC-95; Lot 217. The test material had an original reported purity o f 98.9% and had expired at the time o f experimental start. An assay o f the test material after experimental start indicated a purity o f 90.49%. The final assay o f the test material indicated a purity o f 86.9% and expiration date o f August 31,2006 (Appendix I). The test substance was held under ambient condition in locked storage at the W ildlife International, Ltd. facilities in Easton, Maryland. Concentrations o f the test substance in the diet were adjusted to 100% active ingredient based upon the original reported purity o f 98.9%. Dietary concentrations are expressed as parts per million active ingredient (ppm a.i.) in the diet based upon the final reported purity o f 86.9%. The internal standard, 4H PFOS, was received from 3M Corporation on July 2, 1998 and was assigned W ildlife International, Ltd. identification number 4526 upon receipt. The internal standard was a granular material identified as: 1H, 1H, 2H, 2H Perfluoroctane Sulfonic Acid; CAS No. 27619-97-2. The internal standard was held under ambient conditions in locked storage at the W ildlife International, Ltd. facilities in Easton, Maryland. Test Organisms Fifty-one (24 males and 27 females) pen-reared mallards were purchased from W histling W ings, Inc., 113 W ashington Street, Hanover, IL 61041-0509, U.S.A. At the start of acclimation, the mallards appeared healthy and were phenotypically indistinguishable from wild type. The birds were from the same hatch, approaching their first breeding season and had not been used in any previous testing. A t the start o f acclimation, a random number generating function in a spreadsheet program was used to randomize pen assignment for each bird. Immediately prior to test initiation, all potential study birds 00036? W ildlife International, Ltd. Project Number 454-105 -12- were examined for physical injuries and general health. Birds that did not appear healthy, either due to injury or inability to acclimate to laboratory conditions, or were outside the weight range for the test, were excluded from the study. All birds were approximately 27 weeks o f age at test initiation (first day o f exposure to test diet) and ranged in weight from 788 to 1258 grams at test initiation. Sex o f the birds was determined by a visual examination o f the plumage. Identification Adult birds were identified by individual leg bands, each pen was identified with a unique number, and groups o f pens were identified by project number and concentration. During the test, the number o f eggs laid in each pen was recorded to evaluate egg production. All eggs collected for sampling or incubation were marked with the pen number using a permanent ink marking pen for identification. Hatchlings were initially identified by wing bands so that they could be traced to their parental pen o f origin. Wing bands were removed and the offspring were banded with leg bands at approximately four weeks o f age. Avian Feed and Water All adult birds and their offspring were given feed and water ad libitum during acclimation and testing. The basal diet fed to both adults and offspring was formulated to W ildlife International, Ltd. specifications by Agway Inc. (Appendix II, Table 1). The basal ration contained at least 27% protein and 2.5% fat, and no more than 5% fiber. The basal diet contained approximately 1.1% calcium, derived from feedstuff's and the 0.9% limestone used in the formulation o f the basal diet by Agway. While this level o f calcium is sufficient for growth and maintenance rations, additional calcium is required in the ration o f breeding birds for egg shell formation. Therefore, an additional 5% (w/w) o f limestone (approximately 38.5% Ca) was added to the basal diet for the adults. This raised the calcium level in the diet for the breeding birds to approximately 3%, slightly above the minimum recommended for quail (2.3%) and mallard (2.75%) (3). Offspring received basal diet without test substance and without the addition o f 5% supplemental lim estone. W ater was supplied by the town o f Easton public water supply. Neither the adults nor offspring received any form o f medication in the feed during the test. Feed and water were analyzed periodically in accordance with W ildlife International, Ltd. Standard Operating Procedures. C003g W ildlife International, Ltd. Project Number 454-105 - 13 - Diet Preparation Test diets were prepared by mixing PFOS into a premix that was used for weekly preparation o f the final diet. Control diet and each treated diet were prepared weekly beginning on February 28, 2000 and presented to the birds on Tuesday o f each week. Dietary concentrations were adjusted for purity o f the test substance and are presented as parts per m illion active ingredient (ppm a.i.). Details o f the weekly preparation o f test and control diets are shown in Appendix III. Diet Sampling Homogeneity o f the test substance in the diet was evaluated by collecting six samples from each o f the treated diets and one sample from the control diet on Day 0 o f Week 1. Samples were collected from the top, middle and bottom o f the left and right sections o f the mixing vessel. Control and treatment group diet samples were also collected from the feed troughs on Day 7 o f Week 1 to assess stability o f the test substance under actual test conditions. Additionally, a sample was collected from the control and treatment group diets during Week 6 o f the test to measure/verify test concentrations. The diet samples were stored frozen or transferred immediately to the W ildlife International, Ltd. analytical chemistry facility for analysis. Analytical Method The method used for the analysis o f PFOS in avian diet was based upon methodology developed at W ildlife International, Ltd. and entitled "Analytical M ethod Verification for the Determination o f PFOS in Avian Diet" (W ildlife International, Ltd. Project No. 454C-110). Samples were extracted with methanol and diluted in a 50% m ethanol: 50% NANOpure water solution containing 0.0100 mg 1H, 1H, 2H, 2H Perfluooroctane Sulfonic Acid (4HPFOS; internal standard)/L and 0.05% formic acid (v/v) so that they fell within the calibration range o f the PFOS methodology. A method flow chart is provided in Appendix IV, Figure 1. Concentrations o f PFOS in the standards and extracts o f the samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer SCIEX API 100LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil C |8 analytical column (50 mm x 2 mm I.D., 3-pm particle size). The instrument parameters are summarized in Appendix IV, Table 1. 00036Q W ildlife International, Ltd. Project Number 454-105 -14- Calibration standards o f PFOS prepared in a 50% m ethanol: 50% NANOpure water solution containing 0.0100 mg 4H PFOS (internal standard)/!, and 0.05% formic acid (v/v), ranging in concentration from 0.000439 to 0.00439 mg a.i./L (Week 1, Day 0) or 0.000351 to 0.00439 mg a.i./L (Week 1, Day 7, W eek 6, Day 0 and the sample re-extraction set), were analyzed with the samples. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOS on the basis o f individual isomeric components. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS : internal standard) o f the calibration standards. An example o f a calibration curve is presented in Appendix IV, Figure 2. The concentration o f test substance in the samples was determined by substituting the peak area response ratios o f the samples into the applicable linear regression equation. Typical ion chromatograms o f low and high-level calibration standards are shown in Appendix IV, Figures 3 and 4, respectively. Examples o f calculations are presented in Appendix IV, Table 2. The method lim it o f quantitation (LOQ) for the W eek 1, Day 0 analysis was set at 0.879 ppm a.i., calculated as the product o f the lowest calibration standard analyzed (0.000439 mg a.i./L) and the overall dilution factor o f the matrix blank sample (2000 L/Kg). The method LOQ for the Week 1, Day 7 and Week 6, DayO analyses and the sample re-extraction set was set at 1.41 ppm a.i., calculated as the product o f the lowest standard analyzed (0.000351 mg a.iTL) and the overall dilution factor o f the matrix blank samples (4000 L/Kg). Examples o f calculations are presented in Appendix IV, Table 2. Along with the sample analyses, four matrix blanks were analyzed to determine possible interferences. No interferences were observed at or above the LOQ during the sample analyses (Appendix IV, Table 3). A typical ion chromatogram o f a matrix blank is presented in Appendix IV, Figure 5. Avian diet samples were fortified at 0.879, 8.79, and 22.0 ppm a.i. (Week 1, Day 0) or 1.76, 8.79 and 22.0 ppm a.i. (Week 1, Day 7; Week 6, Day 0 and the sample re-extraction set) and analyzed concurrently with the samples to determine the mean procedural recovery. The method yielded mean procedural recoveries o f 105%, 104%, 107% and 102%. These values correspond to each sample set analyzed or reanalyzed during the definitive study (Appendix IV, Table 3). Sample measured 00037 W ildlife International, Ltd. Project Number 454-105 - 15- concentrations were not corrected for the respective mean procedural recovery of that sample set. A typical ion chromatogram o f a matrix fortification is presented in Appendix IV, Figure 6. Housing and Environmental Conditions Housing and husbandry practices were conducted so as to adhere to the guidelines established by the National Research Council (4). The adult birds were housed indoors in batteries o f pens manufac tured by Safeguard Products, Inc. (Model No. 5355), measuring approximately 75 X 90 X 45 cm high. The pens were constructed o f vinyl-coated wire mesh. A diagram o f the test layout is presented in Appendix V. Each pen was equipped with a feed trough. Weekly, sufficient feed for the feeding period was placed in the trough for each pen and presented to the birds. During the feeding period additional feed was weighed and added to the troughs as needed. Water was supplied by nipple-type waterers. Only birds associated with this study were maintained in the study room in order to avoid excessive disturbances. The average temperature in the adult mallard study room dining the course o f the test was 25.9 0.7C (SD) with an average relative humidity o f 48% 17% (SD). The air handling system in the study room was designed to vent up to 15 room air volumes every hour and replace them with fresh air. The photoperiod in the adult mallard room was maintained by a time clock. The photoperiod during acclimation and the test was 17 hours of light per day to induce egg laying. Throughout the test, the birds received a mean o f approximately 202 lux (~ 18.8 ft. candles) o f illumination provided by fluo rescent lights that closely approximated noon-day sunlight. Observations The test birds were acclimated to the facilities and study pens for approximately 4 weeks prior to initiation o f the test. During acclimation, all birds were observed daily. Birds exhibiting abnormal behavior or debilitating physical injuries were not used for the test. During the study, all adult birds were observed daily for signs o f toxicity or abnormal behavior. Additionally, all offspring were observed daily from hatching until termination. A record was maintained o f all mortalities and clinical observations. 00037* W ildlife International, Ltd, Project Number 454-105 -16- A dult Body W eight and Feed Consum ption Adult body weights were measured at test initiation, on W eeks 2, 4, 6, 8, 10, 11 and at adult termination. Feed consumption for each pen was measured weekly throughout the test. Feed consumption was determined by weighing the freshly filled feeder on Day 0, recording the amount o f any additional diet added during the week, and weighing the feeder and remaining feed at the end o f the feeding period (Day 7). The amount o f feed wasted by the birds was not quantified, since the wasted feed was normally scattered and mixed with water and excreta. Therefore, feed consumption is presented as an estimate o f total feed consumption. All remaining birds were euthanized three days after the beginning o f Week 20. Therefore, no feed consumption estimate was calculated for Week 20. A dult Blood Collection At the end o f W eek 6, blood samples were collected from all surviving birds, when possible. Following collection o f blood samples, birds in the 1.8 and 6.2 ppm a.i. treatment groups were euthanized. Additional blood samples were also collected, when possible, from birds in the control group and the 17.6 ppm a.i. treatm ent group prior to euthanasia at the beginning of Week 20. All blood samples were separated into serum and hemacytes/platelets, stored frozen and shipped to Centre Analytical Laboratories, Inc. for possible analysis. A dult Necropsy and Tissue Collection A t the conclusion of the exposure period, all surviving adult birds were euthanized by cervical dislocation, necropsied, and stored frozen. At the time o f necropsy, tissues were collected for histopathological examination (0 and 17.6 ppm a.i. groups only) and possible analyses. W hen available, samples o f gall bladder, liver, proventriculus, kidneys, brain, gonads, bursa o f Fabricius and adipose tissue were fixed in 10% buffered formalin. Histology samples were shipped to EPL in Herndon, VA for histopathology. When available, samples of bile and liver were stored frozen and shipped to Centre Analytical Laboratories, Inc. for possible analysis. Any remaining tissue not fixed for histopathology and feather samples were stored frozen for potential analysis. Egg Collection and Storage Eggs laid dining a seven day period beginning on the second day o f Week 5 o f the test were collected daily from test pens and stored in a cold room until incubation. Eggs to be incubated were washed to reduce the possibility o f pathogen contamination before storing them in the cold room. Eggs G0037JL W ildlife International, Ltd. Project Number 454-105 -17- collected for the first four days were washed by hand prior to storage. The remaining eggs were washed in a commercial egg washer (Kuhl Egg Washer) with a chlorine based detergent (Kuhl Super CD). W ater in the washer was wanned to approximately 45C. The eggs were placed in the wash water and soaked for approximately 15 seconds. The washer's circulation motor was then turned on for approximately three minutes. The eggs were removed from the washer, allowed to cool to approximately room temperature and rinsed with fresh water. The cold room was maintained at a mean temperature o f 13.1 0.1 C (SD) with a mean relative humidity of approximately 75% 4% (SD). Groups o f eggs were identified by an alphabetic lot code. All eggs laid during the week were considered as one lot. Candling and Incubation At the end o f the weekly interval, all eggs were removed from the cold room, counted and candled with a Speed King (M odel No. 32) egg-candling lamp to detect egg shell cracks or abnormal eggs. Cracked or abnormal eggs were recorded and discarded. All eggs not discarded were placed in a Petersime Incubator (Model No. SP20). In the incubator the temperature was maintained at an average 37.5 0.0C (SD) with an average wet bulb temperature o f 30.6 0.1C (SD) (relative humidity o f approximately 60%). The incubator was equipped with a pulsator fan and blades that produced a mild breathing air movement designed to eliminate intracabinet temperature and humidity variation during incubation. In order to prevent adhesion o f the embryo to the shell m em brane, the incubator w as also equipped w ith an autom atic egg rotation device, designed to rotate the eggs from 50 off o f vertical in one direction to 50 off o f vertical in the opposite direction (total arc o f rotation was 100) every two hours through Day 24 o f incubation. Eggs were candled on Day 14 o f incubation to determine embryo viability and on Day 21 to determine embryo survival. Hatching and Brooding On Day 24 o f incubation, the eggs were placed in a Petersime Hatcher (Model No. SP-6H) and allowed to hatch. Pedigree baskets constructed o f galvanized steel wire mesh were used to keep hatchlings separated by parental pen o f origin. Eggs were not rotated in the hatcher. The average temperature in the hatching compartment was 37.2 0.0C (SD), and the average wet bulb temperature was raised to 33.8 0.2C (SD) (relative humidity o f approximately 77%). C0037J W ildlife International, Ltd. Project Number 454-105 -18- All hatchlings, unhatched eggs, and egg shells were removed from the hatcher on Day 27 or 28 o f incubation. The group body weight o f the surviving hatchlings by pen was determined. Ducklings were wing banded for identification by pen o f origin and then routinely housed according to the appropriate parental concentration grouping in brooding pens until approximately 3 weeks o f age. All ducklings were moved to pens without supplemental heat, where they were housed approximately 9 weeks. The ducklings were fed untreated diet without the addition of 5% supplemental limestone. A t approximately 12 weeks o f age, the average body weight by parental pen o f all surviving offspring was determined, and the birds were euthanized with carbon dioxide and disposed o f by incineration. Those offspring selected for blood and tissue sampling were stored frozen following necropsy and later disposed o f by incineration. Hatchlings were housed in batteries o f brooding pens manufactured by Safeguard Products, Inc. Each pen measured approximately 62 X 92 X 25.5 cm high. The walls, floors and ceilings o f each pen were constructed o f vinyl-coated wire mesh. Thermostats in the brooding compartment o f each pen were set to maintain a temperature o f approximately 38C from the time o f hatching until the birds were five to seven days o f age, when the temperature was adjusted to maintain a temperature o f approximately 29C. Brooding was discontinued once hatchlings were determined to be o f sufficient size to thermoregulate. The average ambient room temperature in the room housing offspring was 23.5 0.6C (SD) with an average relative humidity o f 71 11% (SD). All ducklings were removed from brooding pens and transferred to pens without supplemental heat at approximately 3 weeks o f age. These pens were the same model used to house the adult birds. The photoperiod for the offspring was maintained by a time clock at 16 hours o f light per day. Offspring Blood and Tissue Collection Prior to euthanasia o f the offspring, blood samples were collected from 10 offspring in each treatment group. All blood samples were separated into serum and hemacytes/platelets, stored frozen, and shipped to the Centre Analytical Laboratories, Inc. for possible analyses. Additionally, tissues from the 10 offspring in each group were collected for histopatholpgical examination and analyses. When available, samples o f gall bladder, liver, proventriculus, kidneys, brain, gonads, bursa o f Fabricius and adipose tissue were fixed in 10% buffered formalin and shipped to EPL in Herndon, VA for histopathology. When available, samples o f bile and liver tissue were stored frozen and shipped to Centre Analytical Laboratories, Inc. for possible analysis. . 00037$ W ildlife International, Ltd. Project Number 454-105 -19- Statistical Analyses Upon completion o f the test, an Analysis o f Variance (ANOVA) was performed to determine statistically significant differences between groups. Dunnett's multiple comparison procedure (5, 6) was used to compare the three treatment means with the control group mean and assess the statistical significance o f the observed differences. Sample units were the individual pens within each experimental group, except body and liver weights where the sample unit was the individual bird. Percentage data were examined using Dunnett's method following arcsine square root transformation. Two sets of statistical analyses were conducted with the body weight and feed consumption data. One set o f analyses only looked at body weight and feed consumption data from the first 6 weeks o f the study and evaluated all three treatment groups. The second set of analysis only evaluated the control and 17.6 ppm a.i. treatment groups and examined data for the full duration o f the study, 20 weeks. Dunnett's multiple comparison procedure was not considered appropriate to compare the control group to a single treatment group. The student's T-test was used to make statistical comparisons in those instances where only the control group and die 17.6 ppm a.i. treatment group were compared. 1. Adult Body W eight - Individual body weight was measured at test initiation, W eeks 2, 4, 6, 8, 10, 11, and at adult termination. Statistical comparisons were made between the control group and each treatm ent group at each weighing interval by sex for the first 6 weeks. In addition, statistical comparisons were made between the control group and 17.6 ppm a.i. treatment group for weeks 8, 10,11, and 20. 2. Adult Feed Consumption - Feed consumption expressed as grams o f feed per bird per day was examined by pen weekly during the test. Statistical comparisons were made between the control and each treatment group for weeks 1 through 6. In addition, statistical comparisons were made between the control and 17.6 ppm a.i. treatment group for weeks 7 through 19. 3- Eggs Laid - The number o f eggs laid per female per treatment group. For the evaluation o f reproductive performance, data was taken from week 5 eggs set. 4. Viable Embryos - The number of live embryos determined at Day 14 by candling. 5. Eggs Cracked o f Eggs Laid - The number o f eggs determined by candling to be cracked divided by the number o f eggs laid, per pen. 6. Viable Embryos o f Eggs Set - The number o f embryos at the Day 14 candling was divided by the number o f eggs set, per pen. C00375T Wildlife International, Ltd. Project Number 454-105 -20- 7. Live 3- Week Embryos o f Viable Embryos - The number o f live embryos at the Day 21 candling was divided by the number o f viable embryos, per pen. 8. Hatchlings o f 3-Week Embryos - The number o f hatchlings removed from the hatcher was divided by the number of live 3-week embryos, per pen. 9. 14-Dav Old Survivors o f Hatchlings - The number o f 14-day old survivors was divided by the number o f hatchlings per week, by pen. 10. Hatchlings o f Eggs Set - The number of hatchlings was divided by the number o f eggs set per week, by pen. 11. 14-Dav Old Survivors of Eggs Set - The number o f 14-Day old survivors was divided by the number o f eggs set per week, by pen. 12. Offspring's Body W eight - The group body weights o f surviving hatchlings and 14-day old survivors were measured by parental pen group. 13. Liver Weight - Individual liver weights were measured at adult termination and at the termination o f offspring o f week 5. Statistical comparisons o f adult liver weights were made by sex between the control and treatment groups. Juvenile liver weights were compared by test group, without regard to sex. RESULTS AND DISCUSSION Adult mallards were exposed to PFOS at nominal dietary concentrations o f 1.8, 6.2 and 17.6 ppm a.i. for a period o f 6 weeks. A control group, fed non-treated diet, was maintained concurrently with the treatm ent groups. Each treatment and the control group consisted of five pairs of birds, housed with one male and one female per pen. At the end o f Week 6, adult birds in the 1.8 and 6.2 ppm a.i. test concentrations were euthanized and subjected to gross necropsy. Test birds in the control group and 17.6 ppm a.i. treatment group were maintained on the appropriate diets until the beginning o f W eek 20 o f the test, at which time they were also euthanized and subjected to gross necropsy. Analytical Results None o f the control samples showed any indication of the presence of the test substance or of the presence o f co-eluting substance at the characteristic retention time o f the test substance (Table 1). Diet samples were collected from the 1.8, 6.2 and 17.6 ppm a.i. test concentrations on W eek 1, Day 0, and were analyzed to evaluate the homogeneity o f the test substance in the diet and to verify test substance concentrations. M eans and standard deviations for the three test concentrations were 1.8 0.13 ppm a.i., 00037'$' W ildlife International, Ltd. Project Number 454-105 -21 - 6.0 0.65 ppm a.i. and 17.6 1.46 ppm a.i., respectively. The coefficients o f variation were 7.2%, 11% and 8.3%, respectively. These values represented 100, 97 and 100% o f nominal concentrations (Appendix IV, Table 4). Samples collected during Week 6, Day 0 o f the test to verify test substance concentrations for the 1.8, 6.2 and 17.6 ppm a.i. diets had means and standard deviations o f 2.0 0.092 ppm a.i., 6.0 0.67 ppm a.i. and 16.8 0.608 ppm a.i., respectively. The coefficients o f variation were 4.6% , 11% and 3.6%, respectively. These values represented 111,97 and 95% of nominal concentrations (Appendix IV, Table 5). Analysis o f diet samples collected from feeders after being held at ambient temperature for 7 days averaged 89%, 87% and 105% o f the Day 0 values for the 1.8, 6.2 and 17.6 ppm a.i. test concentrations, respectively (Appendix IV, Table 6). A typical ion chromatogram o f a test sample is shown in Appendix IV, Figure 7. M ortalities and Clinical Observations No adult mortalities occurred in the control group or in any o f the treatment groups during the course o f the test. Several birds were noted with bumble foot (foot lesions) and feather loss as a result o f pen wear and/or penmate aggression during the course o f the test. A single bird in the 6.2 ppm a.i treatm ent group was noted with a head lesion. An incidental clinical sign, lameness, was associated with the bumble foot. In the 17.6 ppm a.i. treatment group, the hen from pen 218 arched her neck and became rigid when captured for bleeding at test termination. She also exhibited body tremors and rapid blinking and respiration during the bleeding process. Clinical signs appeared to be triggered by the stress o f handling. This hen was observed to be normal in appearance and behavior prior to test termination. All other birds in all test groups were normal in appearance and behavior for the duration o f the test. Adult Body Weight and Feed Consumption When compared to the control group there were no apparent treatment-related effects on adult body weight at the 1.8 or 6.2 ppm a.i. test concentrations and any differences between the control group and 6.2 ppm a.i. test concentration were not statistically significant. There was a statistically significant (p < 0.05) reduction in female body weight at the 1.8 ppm a.i. test concentration from Week 2 to Week 4. However, this reduction was not consistent nor dose-responsive and was not considered treatment-related. There were mean body weight losses among males and females in the 17.6 ppm a.i. treatment group that may have been related to treatment. When compared to the control group, mean male body weight for the W ildlife Internationalt Ltd. Project Number 454-105 -22- 17.6 ppm a.i. treatm ent group was significantly (p < 0.05) lower at the W eek 10, 11 and 20 body weight intervals. There was also a significant loss o f overall mean body weight for males at the 17.6 ppm a.i. test concentration. W hile there was an overall loss o f mean body weight for females in the 17.6 ppm a.i., the difference from the control was not statistically significant (p > 0.05). Mean body weight measurements are presented in Table 2, and individual body weight measurements are presented in Appendix VI. Due to excessive wastage by some birds, feed consumption was variable between pens. However, when compared to the control group, there appeared to be no treatment-related effects on feed consumption at the 1.8, 6.2 or 17.6 ppm a.i. test concentrations. While there were statistically significant (p < 0.05) differences between the control group and the 17.6 ppm a.i. treatment group for W eeks 14,16, 17, 18 and 19, these differences were due to apparent increases in feed consumption for die 17.6 ppm a.i. group. Feed consumption measurements at the 17.6 ppm a.i. test concentration were consistently higher than the control group with a corresponding decrease in body weight, suggesting the increase was the result o f feed wastage. M ean feed consumption measurements are shown in Table 3, and feed consumption measurements by pen are presented in Appendix VII. G ross Necropsy A t the end o f Week 6 (Day 42), all adult birds in the 1.8 and 6.2 ppm a.i. treatm ent groups were euthanized and subjected to gross necropsy. Adult birds in the control and 17.6 ppm a.i. treatm ent groups were maintained on the appropriate diets until die beginning o f Week 20 and were then euthanized and subjected to gross necropsy. All findings observed were considered to be incidental to treatment. Necropsy findings are reported in Table 4 and Appendix VIII. Study offspring were approximately 12 weeks o f age at the time o f euthanasia. A subsample o f 10 juvenile birds for each test group were subjected to gross necropsy. Necropsy revealed several birds in each treatm ent level with bumble foot (foot lesions). Additionally, a female offspring in the 17.6 ppm a.i. treatm ent group was noted with picked and broken feathers and an unkempt appearance. Internally the bird was noted with a retained yolk sac, a remnant o f the natal yolk sac. One male offspring in the 17.6 ppm a.i. treatment group was noted with the posterior portion o f the left kidney absent. All findings observed were considered to be incidental to treatment. GOO370 W ildlife International, Ltd. Project Number 454-105 -23 - H istopathology Light microscopic examination was performed on selected tissues by a board certified pathologist at Experimental Pathology Laboratories, Inc. Herndon, VA. Sections o f liver, brain, kidney, gonad, proventriculus, gall bladder, adipose tissues, and bursa o f fabricius (when available) were collected from adult test birds and from 10 offspring (approximately 12 weeks old at euthanasia) from each test group for examination. No lesions considered possibly related to treatment were noted in liver, kidney, proventriculus, gall bladder, ovary, brain and bursa o f fabricius o f adult male and females, or their offspring at any o f the concentrations tested. Additionally, there were no lesions considered to be treatm ent-related noted in adipose tissue o f adult females and offspring o f both sexes, or in the testes o f male offspring. When compared to the control group, testes o f adult males at the 17.6 ppm a.i. test concentration exhibited a higher incidence o f features consistent with post-reproductive phase regression. Adult males in the 17.6 ppm a.i. group also exhibited an increased incidence o f adipose tissue microvesiculation. The increased incidence o f testicular regression and adipose tissue microvesiculation for adult males in the 17.6 ppm a.i. treatment group may be incidental to treatment, but a treatment-related effect could not be discounted. The full pathology report is provided in Appendix IX. Tissue Analysis The analysis o f the egg, blood and tissue samples collected during the study were conducted by Exygen Research (formerly known as Centre Analytical Laboratories) and are reported separately. Blood and liver analytical results are reported in "Extraction o f Potassium Perfluorooctanesulfonate from Quail serum and quail liver for analysis using HPLC-Electrospray/Mass Spectrometry; Centre Study Number 023-41". The egg components analytical results are reported in "Extraction o f Potassium Perfluorooctanesulfonate from Egg Membrane, Albumen, and Yolk for analysis using HPLCElectrospray/M ass Spectrometry; Centre Study Number 023-065". 0003#f W ildlife International, Ltd. Project Number 454-105 -24- Reproductive Results W hile egg production was highly variable among hens, there were no apparent treatment-related effects upon egg production at any o f the concentrations tested. Any differences between the control group and the treatm ent groups were not statistically significant. When compared to the control group, there was a reduction in egg production at the 6.2 ppm a.i. treatment level. However, this reduction was not dose responsive and was not considered related to treatment. Egg production at the 1.8 and 17.6 ppm a.i. test concentrations was comparable to or exceeded the control group. Mean egg production by concentration is presented in Table 5. Individual egg production data are presented in Appendix X. W hen compared to the control group, there were no apparent treatment-related effects on embryo viability, hatchability, hatchling health and survivability at the 1.8, 6.2 or 17.6 ppm a.i. levels. Any differences between the control group and the treatment groups were not statistically significant for any param eter measured. While there were fewer 14-day old survivors per hen produced at the 1.8 and 6.2 ppm a.i. test concentrations, these reductions were due to fewer eggs being set for incubation at these levels. Reproductive data are summarized in Table 6. Reproductive data for individual pens is presented in Appendix XI. Offspring Body W eights There were no apparent treatment-related effects upon the body weights o f hatchlings in any o f the treatment groups and any differences from the control group were not statistically significant. There were also no apparent treatment-related effects upon the body weight o f juvenile birds in the 1.8, 6.2 or 17.6 ppm a.i. treatment groups. Offspring body weight data is presented in Table 7 and Appendix XII. Liver W eights When compared to the control group, there were no apparent treatment-related effects on adult liver weight at any o f the concentrations tested. There was a statistically significant (p < 0.05) increase in mean female liver weight at the 1.8 and 6.2 ppm a.i. test concentrations. While mean adult liver weights were higher among males and females at the 1.8 and 6.2 ppm a.i. tests concentrations when compared to the control group, these differences were due to the timing o f adult termination for these treatment groups. The 1.8 and 6.2 ppm a.i. test birds were euthanized at the end of Week 6 o f the test, during a period o f high egg production. The control group and 17.6 ppm a.i. treatment group test birds were euthanized at the beginning o f Week 20 o f the test by which time egg production had declined dramatically. One 000380 W ildlife International, Ltd. Project Number 454-105 -25 - would expect birds in full reproductive condition, particularly females, to have higher levels o f fat reserves in the liver and correspondingly higher liver weights. When compared to the control group, there were no apparent treatment-related effects on juvenile liver weight at any o f the concentrations tested. While there was a significant increase in mean offspring liver weight for the 1.8 ppm a.i. treatm ent group, the increase was not dose-responsive and appeared to be related to slightly higher mean body weights for this group. Mean adult and offspring liver weights are presented in Table 8. Individual adult liver weights are presented in Appendix XIII. Individual offspring liver weights are presented in Appendix XIV. CONCLUSION M allards were exposed to PFOS at dietary concentrations o f 0, 1.8, 6.2 and 17.6 ppm a.i. for 6 weeks. The control group and 17.6 ppm a.i. test group were maintained on test diets for an additional 13 weeks. No treatment-related mortalities were observed at any o f the concentrations tested. No overt signs o f toxicity were noted at the 1.8 or 6.2 ppm a.i. test concentrations. At the 17.6 ppm a.i. test concentration, a single hen was noted with signs o f toxicity that may have been related to treatment. There were no apparent treatment-related effects on body weight among males and females in the 1.8 and 6.2 ppm a.i. test concentrations. There were mean body weight losses among males and females in the 17.6 ppm a.i. test group that may have been related to treatment. There were no apparent treatmentrelated effects on feed consumption or egg production in the 1.8, 6.2 or 17.6 ppm a.i. treatm ent groups. There were no apparent treatment-related effects on any o f the reproductive parameters measured. Histopathological examination o f selected tissue revealed a higher incidence o f testicular regression and adipose tissue microvesiculation for adult males in the 17.6 ppm a.i. group. W hile these observations may be incidental to treatment, a treatment-related effect could not be precluded. Based upon the results o f this study, the no-observed-effect concentration for mallards exposed to PFOS in the diet for six weeks was 6.2 ppm a.i. 00038# W ildlife International, Ltd. -26REFERENCES Project Number 454-105 1 U.S. Environm ental Protection Agency. 1982. Pesticide Assessment Guidelines. FJFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, subsection 71-4, Environmental Protection Agency, Office o f Pesticide Programs. W ashington, D.C. 2 A m erican Society fo r Testing and M aterials. 1986. Standard Practice fo r Conducting Reproductive Studies with Avian Species. ASTM Standard E l062-86. Annual Book o f ASTM Standards. Vol. 11.04. Philadelphia, PA. 15 pp. 3 M erck & Co., Inc. 1991. The M erck Veterinary Manual. M erck & Co. Rahway, NJ. 1832 pp. 4 N ational R esearch Council. 1996. Guide fo r the Care and Use o f Laboratory Animals. W ashington, DC. National Academy Press. 125 pp. 5 D unnett, C.W . 1955. A M ultiple Comparison's Procedure for Comparing Several Treatments with a Control. Jour. Amer. Statis. Assoc. 50: 1096-1121. 6 D unnett, C.W . 1964. New Tables for M ultiple Comparisons with a Control. Biometrics 20:482491. 00038& Table 1 Mean M easured Concentrations (ppm a.i.) o f PFOS in Avian Diet from a M allard Pilot Reproduction Study Nominal Concentration1 ___________________ Interval Week 1____________ Day 0 Day 7 Week 6 DayO Control (0 ppm a.L) 1.8 (ppm ai.) 6.4 (ppm ai.) 18.3 (ppm ai.) Measured < 0.8792 Mean Measured % Nominal Mean Measured % Nominal Mean Measured % Nominal 1.8 100 6.0 97 17.6 100 <1.41 1.6 89 3 5.2 87 3 18.4 105 3 <1.41 2.0 111 6.0 97 16.8 95 1Nomina] concentrations and results ofdiet analysis were corrected for die change in test substance purity from 90.49% to 86.9` 2 The lim it o f quantitation (LOQ) was equivalent to 0.879 ppm a i. for Week 1, DayO and 1.41 ppm a.i. for W eek 1, Day 7 and W eek6, DayO. 5The mean percent o f die Day 0 values. Project Number 454-105 fscooo T able 2 M ean A dult B ody W eight1 (g) from a M allard P ilo t R eproduction S tudy w ith PFO S SC 0G 0 Tt XI Experimental Group Sex Week Change: Week Change! Week Change Week Change Change Week Change: Week Char 0 0-2 2 2-4 4 4-6 6 0-6 6*8 8 8-10 10 io-: Week Change Week Change 11 11-20 20 0-20 Control M x 1120 -36 1084 -2 1082 42 1124 3 (O ppm aj.) SD 53 65 59 54 66 16 71 41 998 -36 962 89 1051 1 1052 55 SD 106 87 70 84 52 59 75 116 1.8 p p m a.i M X 1093 -10 1083 -9 1073 SD 70 52 69 21 77 F X 1003 SD 92 88 1092 -13* 1079 64 55 57 72 63 1136 24 78 43 72 35 1114 110 41 91 106 6.2 M X 1091 -33 1058 39 1098 21 1118 27 p p m a.i SD 31 20 40 37 42 73 63 74 F X 988 66 1054 28 1082 -43 1038 51 SD 62 65 66 105 78 95 111 17.6 ppm a.1 M * 1095 -41 1053 SD 107 56 78 F X 999 23 1022 SD 150 104 138 -4 1049 38 1087 -8 14 70 25 51 74 11 1033 -55 53 102 59 978 -21 149 88 19 1142 32 81 -54 998 31 61 _ -- 59 1202 21 79 26 1025 62 18 4C 1242 4C 59 -1C 1015 44 50 -38 1204 95 97 -10 1005 59 23 - -- - -- -, _ 83 58 7 118 _ - -- - - - - - - - -- _ - -- --- -- --_ --- - -13 1074 62 24 4 55 17 995 -27 62 148 37 1078' 65 11 967 -U 172 If 1073' -31 73 92 958 -60 173 98 1042 * -53* 94 57 897 -102 139 90 Project Number 454-105 Mean (5?) standard deviation (SD). The means for body weights and body weight changes are calculated and round separately. - Data not avalible. Birds euthanized atthe rad of Week 6. *Statistically different from die control group at p < 0.05. Table 3 ed Consumption1 (g/bird/day) from a Mallard Pilot Reproduction Study wife PFOS Weeks 6 7 8 9 10 11 12 13 14 15 16 17 18 19 212 200 236 220 211 236 175 139 196 139 123 168 130 170 40 53 63 28 71 39 28 19 22 42 28 41 25 44 177 50 178 56 234 225 241 253 226 249 218 195 267* 172 191 * 238 * 183 * 227 * 84 51 79 67 70 69 62 69 49 52 42 51 39 29 od of Week 6. at p < 0.05. Project Number 454-105 S&cooo Il II 00038$ G0038? 000388 0003% <f 0003fa 0003?/ 0003 -37- W ildlife International, Ltd, Project Number 454-105 Appendix I Page 3 Certificate of Analysis Centre Analytical Laboratories, Inc 3&>iaRomandi Drive Siete Collage, R4 1680l ww.centfetato.coni Phone: (814)231-8032 F* (814)231-1253or (814) 231-1580 INTERIM CERTIFICATE OFANALYSIS Jtevbiom3 Centre Analytical Laboratories COA R eference #: 023-018A LC/MS Purity Profile: Im parity C4 C5 C6 Cl T o ta l W t/Wt % 1.22 05 1.14 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average result from the C4 and C6 standard curvet. Likewise, the C7 value was calculated using the average result from the C6 and C8 standard curves. Prepared By: Charles Simona Scientist, Centre Analytical Laboratories /o/n/vl Date Reviewed By. o k W fh k i *>/*/<>( / 1w b1 Flaherty / Date '"Laboratory Manager, Centre Analytical Laboratories COA023-D1KA Page3of3 C003$3 -38- W ildlife International, Ltd Project Number 454-105 Appendix II Diet and Supplement Formulations Table 1 Wildlife International, Ltd. Game Bird Ration1 INGREDIENTS Fine Com Meal Soy Bean Meal, 48% Protein Wheat Midds Protein Base Agway Special, 60% Protein Alfalfk Meal, 20% Protein Dried Whey Ground Limestone Eastman CalPhos Methionine Premix + Liquid Vitamin and Mineral Premix (see below) GL Ferm (Fermatco)2 Salt Iodized 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, when added at 0.32% o f the ration, supplied the following amounts per ton: Amount Supplied Per Ton: Vitamin D3 Vitamin A R ib o flav in Niacin Pantothenic Acid Vitamin B 12 Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (Menadione Dimethylpyrimidinol Bisulfite) M anganese Zinc Copper Iodine Iron Selenium 2.000. 0001.C.U. 7.000.0 0 0 1.U. 6 grams 40 grams 10 grams 8 mgs 600 mgs 64 mgs 1.2 grams 1.2 grams 20,0001.U. 5.8 grams 102 grams 47 grams 6.8 grams 1.5 grams 51 grams 182 mgs 1 The guaranteed analysis is a minimum o f 27% protein, a minimum of 2.5% crude fat and a maximum o f 5% crude fiber. 2 Fermentation By-Products (Source o f Unidentified Growth Factors). G0039f -39- W ildlife International, Ltd. Project Number 454-105 Appendix III Diet Preparation Premixes for PFOS were prepared on February 28, 2000; April 7, 2000; May 12, 2000; and June 19,2000. Nominal preparation was as follows: Control: No premix required. 1.8 ppm a.i.: 0.3084 g PFOS + 6099.7 g ration 6.2 ppm a.i.: 1.0794 g PFOS + 6098.9 g ration 17.6 ppm a.i.: 3.0839 g PFOS + 6096.9 g ration Basal ration was weighed into a tared Hobart mixing bowl. Approximately 100 g of ration was transferred to a Waring blender. The test substance was weighed into a tared weigh boat and a small mortar was taken to crush any areas of consolidation. The test substance was transferred to the Waring blender and the weigh boat was rinsed three times with ration from the mixing bowl, with the rinse being added to the blender. The blender contents were mixed approximately one minute and transferred to the mixing bowl. The blender was rinsed three times with uncontaminated ration from the bowl, with the rinse being returned to the bowl. The bowl was placed on a Hobart mixer, and the contents were mixed approximately 15 minutes. The premix was weighed into 1000.0 g aliquots, placed in appropriately labeled plastic bags, reweighed, and stored frozen. As needed, the appropriate premix was incorporated into the final diet as follows: 0 ppm a.i.: 23.75 kg ration + 1.250 kg limestone 1.8 ppm a.i.: 1000 g Premix + 22.75 kg ration + 1.250 kg limestone 6.2 ppm a.i.: 1000 g Premix + 22.75 kg ration + 1.250 kg limestone 17.6 ppm a.i.: 1000 g Premix + 22.75 kg ration + 1.250 kg limestone The diets were mixed for approximately 20 minutes in a Patterson-Kelly Twin Shell Blender. 60039$ -40- W ildlife International, Ltd. Project Number 454-105 Appendix IV The Analysis o f PFOS in Avian Diet C 0 0 3 9 -41 - W ildlife International, Ltd. Project Number 454-105 Appendix IV ANALYTICAL METHODS AND RESULTS Typical LC/MS Operational Parameters TABLE 1 IN STR U M EN T: Hewlett-Packard M odel 1100 High Performance Liquid Chromatograph with a Perkin-Elmer SCIEX API 100LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil C|* column (50 mm x 2 mm I.D., 3-pm particle size) OVEN TEMPERATURE: 30C STOP TIME: 5.00 minutes FLOW RATE: 0.220 mL/minute MOBILE PHASE: 72.0% Methanol : 28.0% NANOpure W ater containing 0.1% Formic Acid INJECTION VOLUME: PFOS RETENTION TIME: INTERNAL STANDARD RETENTION TIME: 5.0 pL Approximately 3.6 minutes Approximately 2.6 minutes PFOS MONITORED MASS: 498.6 amu INTERNAL STANDARD MONITORED MASS: 426.7 amu 000397 W ildlife International, Ltd. - 42- Project Number 454-105 Appendix IV TABLE 2 CALCULATIONS The concentration of PFOS found at the instrument was determined using the following equation: PFOS (mg a.i./L) at instrument = Pea^c- ^ ea interceE) x standard cone, (mg a.i./L) Determination o f Sample Residues (PFOS1 The concentration, expressed as ppm a.i., for each sample was determined using the following equation: PFOS (mg a.i./L) at instru. x extract final volume (L) x dilution factor PFOS (ppm a.i.) in sample = initial weight (Kg) Determination o f Limit of Quantitation (LOO) The method LOQ, expressed as ppm a.i., was determined using the following equation: LOQ (ppm a.i.) = lowest standard concentration (mg a.i./L) x overall dilution factor o f matrix blank* overall dilution factor of matrix blank sample = extract final volume (L) x dilution initial weight (Kg) factor Fortification Recoveries The ppm a.i. measured in each sample is divided by the nominal concentration o f each sample (fortified level, ppm a.i.). This ratio times 100 is the percent recovery o f the method at that level of fortification. _. _ ppm a.i. measured in sample % Recovery = ppmTTfortified x 100 I 00039$ V i f t I t i t z \ o \a a l. BEST COPY AVAILABLE Pr set un a-> 4-: 5 O u> 41 A] <ei ix f T kB E in 3 *-] s-: B tt i l in ; anti Ft tif ati as m m c nc .Tt Vl1 t l Si ip s an le T) ! nterv 1 3o e r ltd' ol F( (i n : ) Ft ifii 1 4e un u F ce Rt 3V. lea le< 'e r x) A] i M rix lai W k l .l ay oc < .8: a: i l itri Fo fit ioi w k l,J ay ' 37' 1 >9: 13 05 a: 2 i itri Fo fk 10! w k 1,) ay 79 89 01 a: 3 } itri Fo fit ioi w k 1,1 ay IX 2.0 00 A] 2 M fix laj w k 1,1 ay OC K .4 A4 } itri Fo At ioi w k l ,l ay 76 97 12 04 a: 5 } to Fo fit ioi w k 1,1 ay 79 11 04 a: 6 i .tri Fo fit ioi w k 1,1 ay 2.0 1.3 7.C A] 3 M rix lai w k 6 ,l ay 00 < .4 . a: 7 i :tri Fo fk ioi w k 6 ,l ay 76 98 13 07 a: 8 r .tri Fo fit ioi w k 6 ,1 ay 79 40 07 ai 9 f tri Fo fit ioi w k 6 ,1 ay IX 2.0 00 A1 4 M rix lai e-< tracti n ! t4 00 < .4 _ I AS 0 r tr i Fo fit ioi e-( tracti n! t4 76 84 05 02 ] AS 1 i .tri Fo fic ioi en tracti n i t4 79 86 01 ] AS 2 i tr i Fo f i t ioi tracti n ! t4 2.0 1.7 1.7 ICC` c 1C iti< . Wf CO cte or mg D ti sul wri (98.9i to .9 x r nti te A i jftii ita >ct( t i 20( N im est nc< rati p .1; S3 dl a_i 5 tt va tsc *S| Kit im; .fq itit on 3Q 3P lilt' ere sae ed ag ac< m ' sio .6 i w . f nual c cul am ay Ter gh sis foi ied d r \sxa cc snt ion /e r orr ed n ide or khi 5P< os< 4 b se t fro if:; jp t 5 + '5.1 - i : o ir ,E 0; spies 54- 5-2 ind 6fi V :k! y** an uq 45 105 15-: fro w< 6, iy< ose pos e n t test ista : lan; n ti ubi tee ity l 1 / i i U !i / e . 1 1 r, 1 u 1 1 1 ? e J , j - 6 1 BEST COPY AVAILABLE P ijt t l in >e 41 -] '5 ^ 3; x u L 1'< a e it a it a j )i l i. ) i nps . 1 4 ii it a i 3 4 A -1 ); ^ >] u i ; .! V r. aj B o n >! e n it y '] '1 ( S i i L i u ] li :l L > a it i 5; n p 51 i 1 A x n ; U a el A :i a r< i *] ( S C 3: c 1 r ti >1 1 ( ) ] fi a i. A 'A 1 3 > S a d it 1 k v * .c 1 5 ') 3 e :i i i : f V u a U a 7\ le i l rc it o I >n m .1 < R> : ' ' o j fi .C 1 3| 1 i J .2 tl d 11: A i . 2 3 = 1 .f p Ml .. 1( 1 1 fi l l It I ij h , 3 i C = ( 11 p ai. il tl n 1 -ft .1 < \ = -. 2 X i I 0 ti n 1 i| h >5 i "0 J i3 l ) I j lii ;# 5 1 1 tl d 11: -ft i. s X = f 0 p 11 u .i 9 1 ! 1 fi ii 1 I i| h .1 ! E = ( f 5 > j a a i . U li t i n 1 -ft ; .17 <n 1 4 n 1 c ti 0 1 ij h .1 1 > "( 3 1- f 1 > 1 3 ii i . 1 1 vl ,c 1 1 : -ft 1 1 1 f: li li I ij h n J' tl >1 1 A ft 2 ) 1 c ti d 1 ij h <f .1 . 7 <f c = l' .( 1 p a 1. . 1( !(.4 ! I = H j j j n a i . r, c ( \ - f 2 y, if 1 J i x i n i i v i o x fi l i : :I a: g ! i t 3 ub t B 'A ] U i1 / 2 $ Y t 6 9 4 : ii e it a' o t u a i p t t e m t s s il si u 31 j a ity 1* e 1. i, 6 2 u d 1 . J p a i. . 2 d a a * a It > c j] li : * n -( x n :t o i if o ij ii il s n pit k ' 3 r .f a u i f A s 1; si i i ti 1 C c al r l, (X . 4c iii l 1 st 00040 -45- GC0402 W ildlife International, Ltd. Nominal Concentration1 (ppm a.i.) 0 Sample I.D. Number (S-454-105-) 1 2 43 44 Appendix IV TABLES Verification o f PFOS Concentrations in Avian Diet Interval (Day OofWeek-) Measured PFOS Concentration1'2 (ppm a.i.) Mean (X) Standard Deviation (SD) Coefficient of Variation (CV) 1 <0.879 1 < 0.879 6 <1.41 6 <1.41 N/A N/A 1.8 3-8 1 - 45 6 1.94 46 6 1.94 47 6 2.10 6.2 9-14 1 - 48 6 6.31 49 6 6.52 50 6 S.273 (see Table 4) X =2.0 ppm a.i. SD = 0.092 ppm a.i. CV = 4.6% (see Table 4) x = 6.0 ppm a.i. SD = 0.67 ppm a.i. CV=11% 17.6 15-20 51 52 53 1 6 6 6 - 17.5 16.5 16.4 (see Table 4) X = 16.8 ppm a.i. SD = 0.608 ppm a.i. CV = 3.6% 1Concentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate of Analysis dated October 11, 2001. Nomini the new test substance purity are 1.8,6.2 and 17.6 ppm a-i. 2Less than values correspond to limit of quantitation (LOQ). 3Mean result ofduplicate re-extraction oforiginal sample. 454-105 ercent f inal 0 1 r ; 0 ) ased upon 40. CC040 00040# 00040$ 00040 G00409 -53- W ildlife International, Ltd. Project Number 454-105 Appendix IV intensity: 40000 cps Figure 7. Typical ion chromatogram of an avian diet sample on Day 0 (S-454-105-3,1.8 ppm a.i.). 00040^ -54- Wildlife International, Ltd. Appendix V Diagram o f Test Layout Project Number 454-105 PENS19*20 * PENSIMS PENSI3-U ft PENSHMZ ft PENSI ft PENS4-6 ft PENS1-3 ft j = approx, lm ; 6" gutter a = Safeguard Products Inc. Model No. 5355 b = Four X 4 ft. Chroma 50 light bulbs 0004to Appendix VI Table 1 Adult Body Weight (g) from a Mallard Pilot Reproduction Study with PFOS Control (0 ppm a.i.) - Males W eek Change W eek Change Week Change W eek Change Change Week Change Week Change Week Change W eek Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 6-8 8 8-10 10 10-11 11 11-20 20 0-20 201 1080 10 1090 202 1121 -33 1088 203 1064 -57 1007 204 1198 -133 1065 205 1139 33 1172 Mean 1120 SD 53 -36 1084 65 59 13 1103 13 1101 -41 966 71 1136 -68 1104 -2 1082 54 66 29 1132 47 1148 41 1007 65 1201 26 1130 42 1124 16 71 52 27 -57 3 -9 3 41 -26 1106 16 1164 35 1042 61 1262 8 1138 19 1142 32 81 71 1177 37 1201 80 1122 72 1334 36 1174 59 1202 21 79 44 1221 -136 1085 18 1219 44 1263 107 1229 -116 1113 10 1344 -58 1286 21 1195 77 1272 40 1242 40 59 -38 1204 95 97 5 142 49 88 133 83 58 Project Number 454-105 Control (0 ppm a.i.) - Females Week Change Week Change Week Change W eek Change Change Week Change W eek Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 6-8 8 8-10 10 10-11 11 11-20 20, 0-20 201 1074 -172 902 202 1027 -50 977 203 1108 -36 1072 204 928 28 956 205 851 52 903 Mean 998 -36 962 SD 106 87 70 161 1063 28 1005 -20 1052 174 1130 101 1004 89 1051 84 52 -69 994 -9 996 96 1148 -10 1120 -1 1003 1 1052 59 75 -80 -31 40 192 152 55 116 -47 947 -14 982 -100 1048 -44 1076 -64 939 -54 998 31 61 94 1041 39 1021 -3 1045 -65 1011 67 1006 26 1025 62 18 -1 1040 -85 936 23 1068 18 1029 -6 1000 -10 1015 44 50 -19 1021 66 1002 -98 970 1 1030 2 1002 -53 -25 -138 102 151 -10 1005 59 23 7 118 The means for body weights and body weight changes are calculated and rounded separately. BEST COPY AVAILABLE Project Number 454-105 nap I II j OL/4 s Ph a '5 > *3. VH 'S -s d ` g cS s SjJ II H H i1 y nu y hR ? y ha y h fl y y les Ve ^ II h 3 II R y a n 0<N 0 0 JJ y il CN li hy Il 9 '6 H Il ^ H Il II II X H ; 1 o 1Os m VO i n fO CN 1 tuTS mn ivt 1 w> r * \ =tN 1w w r --4 _w - - 1 .-- 1 1_ . . 1o "H Os o " 4 O 1 *r ' r 1 1 't S 1 pH i i \1-NI w^ 1 h CN n i-t qo w (A v R y y R R R y R y R y y u y \o r> oo on o Q M M ^1 M rvi 4) vni yy y ff A 1 y <-> --' y R M y yy y g. y y fl TT R a y%* -U II IIyfT4>. * & a y II a y J CN y yo o w y y i r i y yO y J 6 II ^ II ^H X --. H 1 OS r - CN 1 O vo 1* ' " N r* . nn =*--4 W> *-- 1 Ml 1 ,1 1 1_ . _ _ 1^4 OS O--4 O 1"4 o 1 1- T 1 1 O < 5 o 1 1 o r* n fi +* W0> W W a vA i>n * **- a so r> w w u ^ CJ a_ <sj rsj ro rsi ro 4) Vi - I II y <y Ri y yy yi y R 5 y R 'S II RBU u R R S, Il u 8 R R B R y &1 ysR yy II II s H .O N to N an II nH C II 000413L A ppendix V I T able 3 Adult Body Weight (g) from a Mallard Pilot Reproduction Study with PFOS 6.2 ppm a.i. -M ales W eek Change W eek Change W eek Change W eek Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 211 1096 212 1077 213 1051 214 1097 215 1135 Mean 1091 SD 31 -50 1046 -57 1020 -17 1034 -27 1070 -13 1122 -33 1058 20 40 38 1084 51 1071 22 1056 92 1162 -7 1115 39 1098 37 42 58 1142 -55 1016 128 1184 -24 1138 -4 1111 21 1118 73 63 46 -61 133 41 -24 27 74 Project Number 454-105 000413 6.2 ppm a.i. - Fem ales Week Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 211 985 212 1032 213 1049 214 890 215 983 Mean SD 988 62 168 1153 39 1071 -21 1028 85 975 59 1042 66 1054 69 65 39 1192 8 1200 84 1155 -163 992 -79 949 36 985 18 993 -27 966 78 1120 -71 1049 28 1082 66 105 -43 1038 78 95 215 -40 -64 76 66 51 111 The means for body weights and body weight changes are calculated and rounded separately. P rn ie c t N u m b e r 4 5 4 -1 0 5 08 jg o S P>4 a* VM '8 'S IS d r -* ' S >> 2 d tie hai 45 -1 6 h Sf>o II i M H h II M JS uIN II II II n 3* CN II h n p"H R h M II II p> II II S' r--l II II II II 15 fl nI U o II h u II h 8 OO II h II 1 H oo II cd II U II & II u . II II JS ri w II II * II H ? II 1 > Vft II 1a 1 II S II II II II r"s1i II H II II > CN II II U II II I U II 1 0/ > II II II 11 a, 11 .. < V on n 1 m r i i ! i 1 ST va s. 0 1 s : CK 1 r"*00 Tf; Os 1 11 N - - - - - 1 - - - - i--* 1 F--4 O s 00 <N r i 00 1 vr> 00 1 c* CN o 00 00 1 00 I - p - 1 c i ooo 0V0 0IM0 1 II 1 0n0 CN OO oo 3 I 1? s 1 c? *1o1 0l 0- wv N p--4 1 rH'i V\ON 1 IA .. PC 1(V4 (VI i1 1m Tf I VO I N *4 C* 1 ^4 00 o 1o l> 00 I 1 VO r o s o\ 1 5 1 *-- i i 1 H 1 1 - 1 Os o*4 VI p" 1 i c~ 1 ooi r-i mi 1-^ CN 1 T v> 1 O cn - Ov 1 S 1 1 c n CN CN CN CN V3 H II II II II II II II II II II II II II II II II II II II II II II II 1 II II II H II II II II II II II o ?P H H II II II H J5 IN H 1 II H S CN II 1 n r-* II fl J* II P !> H II S P"* II II - II II 15 R H II R p4 II H u II II I OO II 1 1l II f i II 0.10 II H II u II B. II S) . II sq H H H NI R II pe fl II S R 1 1 R JS R II ,,> fl II M r1si fl II - H II CN H II 4> II II J II II II II 4/ > fl II II II a, II ,, fSI r%> i m 1 j - *? i I 1 -7 1 tS v C 1 O s f .t i Pi VO 11 1 1 OO 1 - - - 1 -- - P-4 ^4 p^ 1 - O s * C" 1 P I 1 00 H i CN H 1 CN P--1 - 1 ON* so CIi CN 1 rf*s*) r~ Ci 1 rv>- Vi oo iSO OCVi 1 I Os 0^0r 'Tf < Os 1C1i 1 1 H-- so 1 iP*s. \ r s I 1 iW T1 oo V. VO Ci m 1-H p4 1 OO Oi V, 1 \o f4 I CN 1 I O s 11 1 i C5 C: o 1 S3 o Nt T * c i 1 1 - i 81 O s * *4 V O Os 00 1 1 a Ci ooi OS 1 r-* CN 1 CN i-- 1 W1 V 'l w> w 1 1 00 p--4 C ^ Os H 1 1CN CN CN CN C N V3 Il II Il II Il 1 Il H Il II Il II Il II Il II Il II nu uu ui H II ui p u uu H .1 ug Il S 'il Il -g II fl 5 H Il Il II 1 II III 1 3>l 1 ? fl Il I Il - f i Il 'S H U H Il ^ Il H i II Il ^ Il II g II R e 11 M II 00041^ tf Il h l I I-^**- oOos S3 ? 000415 00041# 00041^ 00041$ G004/<7 0004 O C004/ 00042*1 00042J  00042$ 00042 JT 00042^ C042JT 0042g 0004^| 00043 C0043 000430. C0043 C0043f C0040ff 00043# 454-105 A d u lt S a c rific e Fem ale M a lla rd -81 - SUMMARY INCIDENCE TABLE ADIPOSE TISSU E (N O , EXAM INED) I n f i l t r a t e , M ononuclear C e ll M ic ro ve s ic u la tio n BRAIN (N O . EXAM INED) BURSA O F FABRICIU S (NO. EXAM INED) In vo lu tio n GALLBLADDER (N O . EXAM INED) I n f i l t r a t e , M ononuclear C e ll K ID N EY (N O . EXAM INED) I n f i l t r a t e , M ononuclear C e ll M in e ra liza tio n LIV E R (N O . EXAM INED) Am yloid D e p o s itio n H e p ato cyte (8 ), F a tty Change, D iffu s e H e p a to c yte (s), F a tty Change, Focal H e p ato cyte (s), V a c u o liza tio n , D iffu s e I n f i l t r a t e , M ononuclear C e ll Pigm ent D e p o s itio n OVARY (N O . EXAM INED) Fib ro sis F o ll ic l e s , Decreased Diam eter PROVBNTRICULUS (N O . EXAM INED) I n f i l t r a t e , M ononuclear C e ll GROUP CONTROL (5) 1 2 (5) GROUP 1 7 .6 (5) 2 2 (5) (5) (5) 54 (5) (5) 44 1 (5) (5) 1 1 1 34 55 1 (5) (5) 32 45 (5) (5) 54 Project Number 454-105 1-2 EPL Experimental Pathology Laboratories, Inc. G0437 -82- Project Number 454-105 HISTOPATHOLOGY INCIDENCE TABLES ADULT SACRIFICE 000438 -83- Project Number 454-105 HISTOPATHOLOQY INCIDENCE TABLE GROUP CONTROL GROUP 1 7 .6 454-105 A d u lt S a c rific e Male M a lla rd J i M A i ADIPOSE TISSU E I n f i l t r a t e . M ononuclear C e ll M ic ro ve sic u la tio n B R A IN 111i 1 9 99 9 9 66666 135 79 X XXX 1 XXXXX 1i 111 9999 9 9 9 99 9 13579 1121X XXXXX BURSA OP FABRICIUS In vo lu tio n GALLBLADDER I n f i l t r a t e , M ononuclear C e ll K ID N E Y I n f i l t r a t e , M ononuclear C e ll M in e ra lisa tio n LIV ER Am yloid D e p o sitio n H e p ato cyte (s), F a tty Change, D iffu se H e p ato cyte (s), F a tty Change, Focal H e p ato cyte(e), V a c u o lisa tio n , D iffu s e I n f i l t r a t e , M ononuclear C e ll Pigm ent D e p o s itio n P R O V EN T R IC U LU S I n f i l t r a t e , M ononuclear C e ll T ES T IS Asperm ia Sem iniferous Tu b u le s, Decreased Diam eter Sperm atogenesis, Decreased NNN N 4 X 1211 Xnj Xm Xm Xm Xm NN NNN XX 11 1 m Xm X m Xn ii 1 1 22 11112 21 1 2 2 32 X1 1i i 2 2 2 11111 m m m Xu m 22 i 42 3 4. 111i X mm mmm pPP i 4 552 2 EPL II-l Experimental Pathology Laboratories, Inc. Kty !X-Not RHrkil>lf It-lhi Saetto M ncw plt A-Autoly*1i Intintiti {'tlght/llld 3-nodarat *^o<taratoty vara vart/hlgh P'Praiart Sanigli M-Hallgiumt m in in g ont palrad organ n-imackadalad iac./daath 00043* -84- Project Number 454-105 HISTOPATHOLOQY INCIDENCE TABLE GROUP CONTROL GROUP 1 7 .6 454-105 Ad u lt S ac rifice Fem ale M a lla rd i M A l ADIPOSE TISSUE I n f i l t r a t e , M ononuclear C e ll M ic ro ve sicu la tio n B R A IN 11 111 99999 66667 24680 XX 1 11 XXXXX 11112 99990 99990 2 468 0 XX 11 11 XXXX X BURSA OF FABRICIUS In vo lu tio n GALLBLADDER I n f i l t r a t e , M ononuclear C e ll K ID N E Y I n f i l t r a t e , M ononuclear C e ll M in e ra lisatio n LIV ER Am yloid D ep osition H e p a to c yte (s ), F a tty Change, D iffu s e H e p a to c yte (s ), F a tty Change, Focal H e patocyte(s), V ac u o lizatio n , D iffu s e I n f i l t r a t e , M ononuclear C e ll PiRm ent D e p o s itio n OVARY Fib ro sis F o l l i c l e s , Decreased Diam eter P R O V EN T R IC U LU S I n f i l t r a t e , M ononuclear C e ll NNNNN 11 111 X SI m m m 1i i i 2 1 i 23 i 21 i 21 X 3 31 3 333 1 4322 N NN N N X 1 11 1 mm m i i2 1 1 3332 11112 1 11 32 333 X 41 11 EPL II-2 Experimental Pathology Laboratories, Inc. Ky iX-Not RaurklM H-Ho Snctlon Mncanplntn A-AutolyH l t l n l u l 2"il1oM/irn<l 3 nltrjte 4-xfMte1y aevtrn 5-ivm/h1gh P-PniMt Mwlgn H-fteHaiunt m tstlng on* pitrwf organ iMawMHiM aac./Matli 00044* -85- Project Number 454-105 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS ADULT SACRIFICE 000444 454-105 Adult Sacrifice Species: M a l l a r d CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sh e M ales G roup Identification: C O N TR O L - S a c r i f i c e d An im a i Number 1961 Client Topography / Site EXTERNAL rESTES 1963 RTTKEMAT. CTTgBHAT, TESTES 1965 EXTERNAL TESTES 1967 1969 EXTERNAL EXTERNAL TESTES Client Gross Observations Jo ltin g flig h t feathers '3 . 0 cm Bumble f o o t l e f t f o o t b o ltin g flig h t feathers ~ 3 .5 cm ' Bumble fo o t b o th fe e t " 3 .5 cm Bumble fo o t r ig h t fo o t M olting flig h t feathers ~ 2 .0 cm Microscopic Observations To Comment R e q u ir e d Sem iniferous T u b u le s, Decreased Diam eter (T ES T IS ) *o Comment R e q u ire d go Comment R e q u ire d Sem iniferous Tu b u le s, Decreased Diam eter (TES TIS ) No Comment R e q u ire d Sem iniferous Tu b u le s, Decreased Diam eter (T ES T IS ) No Comment R e q u ire d No Comment R e q u ire d Sem iniferous Tu b u le s, Decreased Diam eter (T ES T IS ) I 00 ON Project Number 454-105 III-l 454-105 Adult Sacrifice Species; M a l l a r d CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sec M ales Group Identification; 1 7 . * - S a c r i f i c e d An im a i Number Client Topography/ Sito CEenl Gross Observations Micro: opic Observations 1991 1993 EXTERNAL EXTERNAL TESTES i 1995 EXTERNAL G l TRACT "TESTES 1997 EXTERNAL TESTES 1999 TESTES Bumble f o o t b o th f e e t Bumble fo o t b o th fe e t - 1 . 5 cm Bumble fo o t b o th fe e t Mass o f fe e d im pacted u nder th e tongue " 2 .0 cm Bumble fo o t b o th fe e t ~ 1 .0 cm " 2 .5 cm to Comment Requ. re d 1o Ceminent Requ re d Sem iniferous Tu u le s , Decreased Diam eter (T E S T I ) io Conment Requ re d No Comment Requ re d Sem iniferous Tu iu le s , Decreased D ia m e te r ( T E S T I :) No Comment Requ .re d Sem iniferous Tu >ules, Decreased Diam eter (T E S T I i) S em iniferou s Tu ru le s . D ecreased Diam eter (T E S T I i) 1 oo % \ Project Number 454-105 C0044J III-2 -88- Project Number 454-105 o b e s t c o p y a va ila b le 00044$ 60044$ 0.0 0 4 4 # 4 5 4 -10 5 O ffs p rin g S a c rific e M ale M a lla rd -91 SUMMARY INCIDENCE TABLE Project Number 454-105 ADIPOSE TIS SU E (N O . EXAM INED) I n f i l t r a t e , M ononuclear C e ll M icro ve sicu la tio n BRAIN (N O . EXAM INED) I n f i l t r a t e , M ononuclear C e ll BURSA O F FA B R IC IU S (N O . EXAM INED) GALLBLADDER (N O . EXAM INED) I n f i l t r a t e , M ononuclear C e ll KIDNEY (N O . EXAM INED) I n f i l t r a t e , M ononuclear C e ll LIV ER (N O . EXAM INED) H epatocyteB, D e g e n eratio n /N e crosis, Focal H epatocyteB, F a tty Change, D iffu s e Bepatocytes, F a tty Change, Focal Bepatocytes, V a c u o liza tio n , D iffu s e I n f i l t r a t e , M ononuclear C e ll Pigm ent D e p o s itio n PROVENTRICULUS (N O . EXAM INED) I n f i l t r a t e , M ononuclear C e ll TES TIS (N O . EXAM INED) Iam ature I n f i l t r a t e , M ononuclear C e ll GROUP CONTROL (5) 1 (5) GROUP 1 .8 (4) (4) 1 (5) (4) (5) (4) 33 (5) (4) 44 (5) (4) 1 1 44 54 (5) (4) 34 (4) (4) 34 22 GROUP 6 .2 (4) (4) (4) (4 ) 2 (4 ) 2 (4) 1 1 3 3 1 (4) 4 (4 ) 4 GROUP 1 7 .6 (5 ) 2 (5) (5 ) (5) 2 (5 ) 4 (5 ) 1 1 4 4 (5) 5 (5) 5 2 ' _ ................. IV -1 EPL Experimental Pathology Laboratories, Inc. . 00044 454-105 O ffsp rin g S ac rifice Fem ale M a lla rd -92SUMMARY INCIDENCE TABLE Project Number 454-105 ADIPOSE TIS SU E (NO. EXAM INED) I n f i l t r a t e , M ononuclear C e ll M icro ve sicu la tio n BR AIN (N O . .EXAM IN ED ) I n f i l t r a t e , M ononuclear C e ll BURSA O F FABRICIU S (NO. EXAM INED) GALLBLADDER (N O . EXAM INED) I n f i l t r a t e , M ononuclear C e ll KIDNEY (N O . EXAM INED) I n f i l t r a t e , M ononuclear C e ll LIV ER (N O . EXAM INED) H e p a to c y te s , D egeneration/Necroeis, Focal Hepatocytee, F a tty Change, D iffu s e H epatocytee, F a tty Change, Focal H patocytes, V a c u o liza tio n , D iffu s e I n f i l t r a t e , M ononuclear C e ll Pigm ent D e p o sitio n 3VARY (N O . EXAM INED) Im m ature I n f i l t r a t e , M ononuclear C e ll PROVKNTRICULUS (N O . EXAM INED) I n f i l t r a t e , M ononuclear C e ll JR ETER (N O . EXAM INED) Lam ina P r o p r ia , C y s t(s ) GROUP CONTROL (5) (5) (5) (5) 2 (5) 3 (5) 1 1 3 4 (5) 5 (5) 4 GROUP 1 .8 (6) 1 2 (6) (6) (6) 5 (6) 6 (6) 5 6 (6) 6 1 (6) 5 GROUP 6 .2 (6 ) 2 (6 ) (6 ) (6) 5 (6) 6 (6) 4 6 1 (6 ) 6 1 (6) 5 (1) 1 GROUP 1 7 .6 (5) 2 (5 ) (5 ) (5) 1 (5 ) 3 (3 ) 5 5 (5 ) 5 (5 ) 5 IV-2 EPL Experimental Pathology Laboratories, Inc. 00044.8 -93- Project Number 454-105 HISTOPATHOLOGY INCIDENCE TABLES OFFSPRING SACRIFICE 0004419 -94- Project Number 454-105 HISTOPATHOLOGY INCIDENCE TABLE 4 5 4 -10 5 O ffsp rin g S a c rific e Male M a lla rd J 1 M A l ADIPOSE TISSUE I n f i l t r a t e , M ononuclear C e ll M icro ve8 icu la tion GROUP CONTROL 22222 33333 0001 1 5 7 8 4.A 1 XXXX 1 GROUP 1 .8 2222 3333 2233 381 4 XXXX GROUP 6 .2 -- 2222 3333 3344 891 3 X XX X B R A IN I n f i l t r a t e , M ononuclear C e ll XXXXX X XX 1 XXXX BURSA OF FABRICIUS XXX X X XXXX XXXX GALLBLADDER I n f i l t r a t e , M ononuclear C e ll AX l1 1 A 1 11 AA 11 K ID N EY I n f i l t r a t e , M ononuclear C e ll n m Xoi m m L1 i2 mmmm 1i 11 m m Xtt Xir i2 LIV ER H e p a to c y te s , DeReneration/N ecrosls, Pocal H epatocytes, F a tty Change, D iffu s e Hepatocytes, F a tty Change, Focal Hepatocytes, V a c u o liza tio n , D iffu s e I n f i l t r a t e , M ononuclear C e ll Pigm ent D e p o s itio n 1 2 2i 2 111i 1 1 2i 22 1i 11 i 2 32z 111 1 P R O V EN T R IC U LU S I n f i l t r a t e , M ononuclear C e ll XX 11i 1i 12 11 1 1 TES TIS Im m ature I n f i l t r a t e , M ononuclear C e ll mmm N PP 11 P PpPP i2 PPpP EPL V -l Experimental Pathology Laboratories, Inc. Ky iJHIot RnrUbl< K-Ko Section Mncplt* A-Autolyil* 2-il1gtit/atld 3"WdMt* 4-aodtrattty itvw 5-M**r/h1jh amP-Frumt Mwtga H-H*l1giunt m illin g pg1rd organ u-uniclwdulad iae,/ditt> 000450 -95- Project Number 454-105 HISTOPATHOLOGY INCIDENCE TABLE GROUP 17.6 54-105 O ffsp rin g S a c rifice Male M a lla rd J i M A L ADIPOSE TISSUE I n f i l t r a t e , M ononuclear C e ll M lc rove s Ic u la t io n 22222 33333 55566 457 03 X XX 11 B R A IN I n f i l t r a t e , M ononuclear C e ll XXXXX BURSA OF FABRICIUS XXXXX GALLBLADDER I n f i l t r a t e , M ononuclear C e ll AAA 11 K ID N E Y I n f i l t r a t e , M ononuclear C e ll m m m m Xm iiii LIV ER H p a to c y te s , D e g e n eratio n /N e crosis, Focal H epatocytes, F a tty Change, D iffu se H epatocytes, F a tty Change, Focal H epatocytes, V a c u o liza tio n , D iffu s e I n f i l t r a t e , M ononuclear C e ll Pigm ent D e p o sitio n i 2 222 3 11 1X P R .O V E N T R IC U U JS I n f i l t r a t e , M ononuclear C e ll XXXXX T ES T IS Im m ature I n f i l t r a t e , M ononuclear C e ll P P PP P 11 rz EPL Erperboental Pathology Laboratories, Inc. -L -- K*y i X-Not RMnrkabl* IWto Sictlon l-In co g l'ti A-AutolyiU l<elnM 2>i11gK/a1W J-ao4*nti 4-Mdmly vrg 5*tmert/Mgk M m r t B-Bmlgn tHtatlgmnt n1ilng om pa1r*g organ >ac./Matli 00045$ -96- Project Number 454-105 HISTOPATHOLOQY INCIDENCETABLE GROUP CONTROL GROUP 1 .8 5 4 -10 3 O ffsp rin g S a c rific e Fem ale M a lla rd J i M A l ADIPOSE TISSUE I n f i l t r a t e . M ononuclear C e ll M icro veB icu la tio n 22222 33333 0001 2 1 2370 XXXXX 222222 333333 222333 2 5602 3 XX X 1 21 B R A IN I n f i l t r a t e , M ononuclear C e ll XXXXX XXXXXX BURSA OP FABRICIUS XX XXX XXX XX X GALLBLADDER I n f i l t r a t e , M ononuclear C e ll AX A 11 A 11 1 11 K ID N E Y I n f i l t r a t e , M ononuclear C e ll in m xm m m 1 ii m m m (b m n i i i l1 L LIV ER H e p a to c y te s , D e ge n eratio n/N e crosis, Focal Hepatocytes, F a tty Change, D iffu s e H epatocytes, F a tty Change, Focal H epatocytes, V a c u o liza tio n , D iffu s e I n f i l t r a t e , M ononuclear C e ll Pigm ent D e p o s itio n 2 1l 11l i i i 32 33 3 1111 11 OVARY Im m ature I n f i l t r a t e , M ononuclear C e ll PPppp PPPPPP 1 P R O V EN T R IC U LU S I n f i l t r a t e , M ononuclear C e ll X 11l i X 1 1111 URETER Lam ina P r o p r ia , C y s t(a ) GROUP 6 .2 222 2 2 2 3 3 3 3 3 3' 3334 44 5672 46 XX XX 11 XXX X X X XXXX XX X 111 al mmm m mm 1i i 1 21 22 3 1 11i 111 i' PPpPPP i X 11 1 31 p EPL V -3 Experimental Pathology Laboratories, h e . Key ! X*Hot AMirktbH N-Ho St1on I-lncMptiti A^Autolyili lrartiriMl 2-itldit/aUd 3^nd<rat 4raoderatly r a 5-iviri/h1oh P-Prm nt B-Dm T a-iliilng cm pi1rad orgui g-antclmlulad uc./dMtk C0045 4 5 4 -10 5 O ffs p rin g S a c rific e Fem ale M a lla rd -97- Project Number 454-105 HISTOPATHOLOQY INCIDENCE TABLE GROUP 17.6 ADIPOSE TISSUE I n f i l t r a t e , M ononuclear C e ll M icro ve slcu la tlo n B R A IN I n f i l t r a t e , M ononuclear C e ll BURSA O F FABRICIUS GALLBLADDER I n f i l t r a t e , M ononuclear C e ll K ID N EY I n f i l t r a t e , M ononuclear C e ll LIV ER H e p a td c y te a , D e gen eration/N ecrosls, Focal H epatocyteB , F a tty Change, D iffu s e H patocytes, F a tty Change, Focal Hepatocytes, V a c u o liza tio n , D iffu s e I n f i l t r a t e , Mononufclear C e ll Pigm ent D e p o s itio n OVARY Im m ature I n f i l t r a t e , M ononuclear C e ll P R O V EN T R IC U LU S I n f i l t r a t e , M ononuclear C a ll 2222 3 33 3 4455 7 838 X d Xdi PP EPL V -4 Experimental Pathology Laboratories, Inc. Ky : X-Kot Raurfctblt IHIo SKtton I"Incoelt A-Autolyi1 H rislstl 2-i11gkt/stl<r 4**xl"atly vara 5-aavara/hljli P-Praiaat Maitfot IHUUgaut m in in g on* palnd y u a-anaclwdalai lac./daatt C0045f -98- Project Number 454-105 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS OFFSPRING SACRIFICE 00045$ 454-105 Offspring Sacrifice Species: M a lla r d CORRELATION OF G RO SS AND MICROSCOPIC FINDINGS Sec M ales G roup Identification: CONTROL - S a c r i f i c e d A nim al N um ber 2305 2308 2314 2316 Client Topography / Site EXTERNAL EXTERNAL EXTERNAL EXTERNAL Client Gross Observations taxable f o o t b o th f e e t tum ble f o o t b o th f e e t hum ble f o o t b o th f e e t Bum ble f o o t b o th f e e t M icroscopic O bservations So Comment R e q u ire d So Comment R e q u ire d No Comment R e q u ire d So Comment R e q u ire d Project Number 454-105 V I-1 *5*000 454-105 Offspring Sacrifice Specks: M a lla rd CORRELATION OF G RO SS ANO MICROSCOPIC FINDINGS Sec M ales G roup Identification: 1 .8 - S a c r i f i c e d A nim al N um ber 2323 O ien t Topography / Site EXTERNAL Client G ross Observations Jum ble fo o t, b o th f e e t M icroscopic O bservations io C o n sen t R e q u ire d s Project Number 454-105 V I-2 JS^OOO %g S a c r i f i c e liall& rd CORRELATION OF G RO SS M ID MICROSCOPIC FINDINGS Ses: H a le s Group Identification: 6 .2 - S a c r i f i c e d Clieni Topography / Site EXTERNAL EXTERNAL Client G ross Observations Jum ble f o o t b o th f e e t Bumble f o o t b o th f e e t M icroscopic Observations To Comment R e q u ire d Ho Comment R e q u ire d Project Number 454-105 SvOOO V I-3 60045$ 60045$ COO460 C0046X Appendix X Table 1 Egg Production (eggs laid/hen/week) from a Mallard Pilot Reproduction Study with PFOS Control (0 ppm a.i.) Weeks Pen 1 2 3 4 5 6 Total E/H/D1 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total E/H/D1 201 6 4 0 1 5 6 202 7 7 7 7 7 7 203 4 4 7 6 5 7 204 0 0 0 0 0 0 205 0 0 0 3 5 7 22 0.52 3 6 1 0 0 0 0 0 0 0 0 0 0 0 42 1.00 6 8 6 5 1 0 0 0 0 0 0 0 0 0 33 0.79 6 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0.00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 15 0.36 6 8 6 8 6 5 0 0 0 0 0 0 0 0 10 0.11 26 0.28 10 0.11 0 0.00 39 0.41 Total 17 15 14 17 22 27 M ean 3 "... 3 3 3 4 5 SD 3 3 4 3 3 3 112 22 16 21 26 13 13 7 5 0 0 0 0 0 0 0 0 0.53 4 5 3 3 1 1 0 0 0 0 0 0 0 0 0.39 3 3 3 4 3 2 0 0 0 0 0 0 0 0 85 17 0.18 15 0.16 1Eggs laid p er hen per day -- oo \ 1 Project Number 454-105 00046JL Appendix X Table 2 Egg Production (eggs laid/hen/week) from a Mallard Pilot Reproduction Study with PFOS Pen 1 2 206 0 207 0 208 5 209 0 210 6 0 2 7 0 6 Total M ean SD 11 2 3 15 3 3 1Eggs laid per hen per day 1.8 ppm a.i. W eeks 3 4 5 6 Total E /H /D 1 7 7 6 1 21 0.50 40 0 0 6 0.14 7 7 7 7 40 0.95 00 00 0 0.00 7 6 6 8 39 0.93 25 20 19 16 106 5 4 4 3 21 0.50 3 4 3 4 18 0.44 Project Number 454-105 00046J Jfo vO O O Appendix X Table 3 Egg Production (eggs laid/hen/week) from a Mallard Pilot Reproduction Study with PFOS Pen 1 2 211 0 212 1 213 0 214 0 215 4 0 4 1 0 7 Total 5 12 M ean 1 2 SD 2 3 1Eggs laid per hen per y 6.2 ppm a.i. W eeks 3 4 5 6 Total E /H /D 1 00 00 0 0.00 7 6 8 6 32 0.76 00 00 1 0.02 00 00 0 0.00 6 6 6 2 31 0.74 13 12 14 8 64 3 2 3 2 13 0.30 4 3 4 3 17 0.41 0o0 Project Number 454-105 I 2 3 4 5 6 Total 56 76 62 0 4 576 1 ; 747627 ' 0 37565 1 3 5 774 7 32 23 33 26 33 il 15 22 33 31 24 22 m3 4 76 54 i 3 11123 147 29 5 'S laid per hen per day 9 0 0 0 00046$ 0046JP 000468 0 0 0 4 tff Project Number 454-105 00047 Replicate 1 2 3 4 5 Total M ean SD Control (0 ppm a i.) Eggs Hatch Set % 2 5 40 7 . 7 100 6 6 100 00 - 6 6 100 21 24 4 5 85 3 3 30 Control (0 ppm aL ) Offspring Eggs Replicate Surv Set % 1 2 3 4 5 Total Mean SD 2 5 40 7 7 100 6 6 100 00 6 6 100 21 24 4 5 85 3 3 30 Appendix XI Page 5 Reproductive Performance by Pen from a Mallard Pilot Reproduction Study with PFOS Table 8 Hatchlings / Eggs Set (%) 1.8 ppm a i. Eggs Hatch Set 34 00 57 00 66 14 17 33 33 % 75 - 71 - 100 82 16 6.2 ppm a i. Eggs Hatch Set 00 88 00 00 44 12 12 22 44 % 100 - 100 100 0 Table 9 Surviving Offspring / Eggs Set (%) 1.8 ppm a i. Offspring Eggs Snrv Set 34 00 57 00 56 13 17 33 33 % 75 - 71 - 83 77 6 6.2 rom aiOffspring Eggs Surv Set 00 88 00 00 44 12 12 22 44 % m 100 - 100 100 0 17.6 ppm a.i. Eggs H atch Set % 6 7 86 4 5 80 2 3 67 4 4 100 2 4 50 18 23 4 5 76 2 2 19 17.6 ppm a i Offspring Eggs Surv Set 57 45 23 44 24 17 23 35 12 % 71 80 67 100 50 74 18 Project Number 454-105 00047* Replicate 1 2 3 4 5 Total M ean SD Control (Oppm a.i.) Hatch/ Hatch Days Hen/Day 2 7 029 7 7 1.00 6 7 0.86 0 7 0.00 6 7 0.86 21 4 0.60 3 0.43 Control (Oppm a-t) Offspring Offspring/ Replicate Stnv Days Hen/Day 1 2 3 4 5 Total Mean SD 2 7 0.29 7 7 1.00 6 7 0.86 0 7 0.00 6 7 0.86 21 4 0.60 3 0.43 Appendix XI Page 6 Reproductive Performance by Pen from a Mallard Pilot Reproduction Study with PFOS Table 10 Hatchlings / Hen / Day 1.8 ppm a i. Hatch/ Hatch Days Hen/Day 3 7 0.43 0 7 0.00 5 7 0.71 0 7 0.00 6 7 0.86 14 3 0.40 3 0.40 6.2 ppm a i. Hatch/ Hatch Days Hen/Day 0 7 0.00 8 7 1.14 0 7 0.00 0 7 0.00 4 7 0.57 12 2 0.34 4 0.51 Table 11 Surviving Offspring / Hen / Day 1.8 ppm a i. Offspring Offspring/ Surv Days Hen/Day 3 7 0.43 0 7 0.00 5 7 0.71 0 7 0.00 5 7 0.71 13 3 0.37 3 0.36 6.2 ppm a i Offspring Offspring/ Stnv Days Hen/Day 0 7 0.00 8 7 1.14 0 7 0.00 0 7 0.00 4 7 0.57 12 2 0.34 4 0.51 17.6 ppm a i H atch/ Hjrfrh Days Hen/Day 6 7 0.86 4 7 0.57 2 7 0.29 4 7 0.57 2 7 0.29 18 4 0.51 2 0.24 17.6 ppm a.i. Offspring Offspring/ Stnv Days Hen/Day 5 7 0.71 4 7 0.57 2 7 0.29 4 7 0.57 2 7 0.29 17 3 0.49 1 0.19 Appendix XII Mean Offspring Body Weight (g) from a Mallard Pilot Reproduction Study with PFOS Mean Hatchling Body Weight (g) Replicate 1 2 3 4 5 Mean SD Control (0 ppm a i.) 26.5 30.6 29.7 -- 26.8 28.4 2.0 1.8ppm a.L 34.3 -- 31.6 -- 32.3 32.8 1.4 6.2 ppm a i. ,_ 33.6 -- 33.0 33.3 0.4 17.6 ppm a i. 34.8 33.3 37.5 34.8 34.5 35.0 1.6 Project Number 454-105 M ean Surviving O ffspring1B ody W eight (g) Replicate 1 2 3 4 5 Mean SD Control (0 ppm a i.) 1024 1050 977 -- 999 1012 31 1.8 ppm a i. 995 -- 1170 -- 1031 1065 92 6.2 ppm a i. 1086 -- -- 986 1036 71 ' Offspring were approximately 12 weeks of age at final body weight interval. --No offspring available. 17.6 ppm a i. 985 1051 1056 1059 998 1030 35 Tpi.bOOO -117- Appendix XIII Page 1 Adult Liver Weight (g) from a Mallard Pilot Reproduction Study with PFOS Project Number 454-105 Pen 201 202 203 204 205 Mean SD Control (0 ppm a.i.) Male Liver 18.774 28.913 19.874 22.680 16.441 21.336 4.793 Female Liver 19.198 30.570 19.405 16.724 20.303 21.240 5.382 Pen 206 207 208 209 210 Mean SD 1.8 ppma.i. Male Liver 27.814 24.713 25.891 23.983 24.280 25.336 1.564 Female Liver 46.860 31.905 35.551 33.155 51.532 39.801 8.832 00047$ -118- Appendix XIII Page 2 Adult Liver Weight (g) from a Mallard Pilot Reproduction Study with PFOS Project Number 454-105 6.2 ppm a.i. Male Pen Liver S8SSSSSSS5SS5SSSSSB3SSSSE99ES5SB98EBI 211 27.724 212 25.304 213 32.723 214 35.818 215 24.458 Mean SD 29.205 4.900 Fonale Liver 36.008 30.427 37.337 27.942 27.977 31.938 4.462 Pen 216 217 218 219 220 Mean SD 17.6 ppm a.i. Male Liver 18.845 34.189 29.108 20.358 14.556 23.411 8.019 Female Liver 21.358 21.871 24.518 17.355 18.018 20.624 2.947 C0047# C0047S 000470 G0047 00047