Document ba61x2Kevq1Q6emYjqKDRJBQg

AR226-1742 PFOS: A PILOT REPRODUCTION STUDY WITH THE MALLARD FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-105 3M LAB REQUEST NO. U2723 FIFRA 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 of 122 W ildlife International, Ltd. Project Number 454-105 2- GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Pilot Reproduction Study with the Mallard 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 of 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 of analyses conducted by Exygen Research for study numbers 023-042 and 023-063 are reported separately. Amendments number 2 and 3 and deviation 1 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. STUDY DIRECTOR: Senior Biologist, Avian Toxicology SPONSOR'S REPRESENTATIVE: DATE 3/^-V/W DATE W ildlife International, Ltd. Project Number 454-105 2- GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Pilot Reproduction Study with the Mallard 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 of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MFF, ^ NohSan Notification No. 3850, Agricultural Production Bureau, 10 August 1984. STUDY DIRECTOR: Atiw ttU U L ._________ SeanP. Gallagher Senior Biologist, Avian Toxicology SPONSOR'S REPRESENTATIVE: '^1^3 DATE DATE Wildlife International, Ltd. Project Number 454-105 -3 - QUALITY ASSURANCE STATEMENT This study was 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 of 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 Sample Preparation Diet Preparation Blood Collection Analytical Data & Draft Report Biological Data & Draft Report Data and Final Report February 28, 2000 March 1,2000 March 7,2000 April 11,2000 February 2 8,2000 March 1,2000 March 7,2000 April 11,2000 January 1 6 ,1 7 ,2 8 & 29, 2003 January 27-31, February 3 ,2 0 0 3 January 29, 2003 February 3,2003 December 1-3,2003 December 3, 2003 March 1,2000 March 1, 2000 March 7, 2000 April 12, 2000 January 31, 2003 March 12, 2003 December 18,2003 All inspections were study-based unless otherwise noted. QrJJffrb/) Kimberly A. Hoxter Quality Assurance Representative 2- fZ-Q3 DATE W ildlife International, Ltd. Project Number 454-105 -4REPORT 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: I .\ j U r c - Sean P. Gallagher Senior Biologist, Avian Toxicolog 4 ^ DATE CHEMISTRY PRINCIPAL INVESTIGATOR: Scientist, A nalytical Chem istry M * DATE MANAGEMENT: Joaxm B. Beavers Director, Avian Toxicology fe//g /s3 DATE Director of Chemistry Wildlife International, Ltd. Project Number 454-105 -5 - TABLE OF CONTENTS Title Page...........................................................................................................................................1 Good Laboratory Practice Compliance Statement........................................................................... 2 Quality Assurance Statement........................................................................................................... 3 Report Approval............................................................................................................................... 4 Table of Contents............................................................................................................................. 5 Summary.......................................................................................................................................... 8 Introduction...................................................................................................................................... 9 Objectives......................................................................................................................................... 9 Experimental Design........................................................................................................................ 9 Materials and Methods....................................................................................................................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 Method....................................................................................................................13 Housing and Environmental Conditions.................................................................................. 15 Observations............................................................................................................................. 15 Adult Body Weight 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 Analysis...................................................................................................................... 23 Reproductive Results............................................................................................................... 24 Offspring Body Weights.......................................................................................................... 24 Liver Weights.......................................................................................................................... 24 Conclusion...................................................................................................................................... 25 References...................................................................................................................................... 26 Wildlife International, Ltd. Project Number 454-105 6- - TABLE OF CONTENTS PAGE 2 TABLES Table 1. Mean Measured Concentrations (ppm a.i.) of PFOS in Avian Diet from a Mallard Pilot Reproduction Study................................................................... 27 Table 2. Mean Adult Body Weight (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 of Gross Pathological Observations from a Mallard Pilot Reproduction Study with PFOS, Adult Birds Euthanized at 6 Weeks and Test Termination................................................................................ 30 Table 5. Mean Egg Production (Eggs Laid/Hen and Eggs/Hen/Day) from a Mallard Pilot Reproduction Study with PFOS............................................................31 Table 6. Summary of Reproductive Performance (Eggs Set from Week 5) from a Mallard Pilot Reproduction Study with PFOS.................................................32 Table 7. Mean Body Weight (g) of Hatchlings and Surviving Offspring from a Mallard Pilot Reproduction Study with PFOS................................................. 33 Table 8. Mean Liver Weights (g) from a Mallard Pilot Reproduction Study with PFOS.........................................................................................................34 / W ildlife International, Ltd. Project Number 454-105 -7- TABLE OF CONTENTS PAGE 3 APPENDICES Appendix I. Certificate of Analysis...........................................................................................35 Appendix II. Diet and Supplement Formulations...................................................................... 38 Appendix III. Diet Preparation.................................................................................................... 39 Appendix IV. The Analysis of PFOS in Avian D iet................................................................... 40 Appendix V. Diagram of Test Layout........................................................................................ 54 Appendix VI. Adult Body Weight (g) from a Mallard Pilot Reproduction Study with PFOS.......................................................................... 55 Appendix VII. Feed Consumption (g/bird/day) from a Mallard Pilot Reproduction Study with PFOS................................................................ 59 Appendix VIII. Individual Gross Pathological Observations from a Mallard Pilot Reproduction Study with PFOS.................................................. 63 Appendix IX. Histopathology Report.......................................................................................... 67 Appendix X. Egg Production (eggs laid/hen/week) from a Mallard Pilot Reproduction Study with PFOS................................................. 106 Appendix XI. Reproductive Performance by Pen from a Mallard Pilot Reproduction Study with PFOS................................................. 110 Appendix XII. Mean Offspring Body Weight (g) from a Mallard Pilot Reproduction Study with PFOS................................................................ 116 Appendix XIII. Adult Liver Weight (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 W ildlife International, Ltd. Project Number 454-105 8- SUMMARY 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 of age at the initiation of the test SOURCE TEST ANIMALS: Whistling 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: Mallards were exposed to PFOS at dietary concentrations of 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 of the concentrations tested. No overt signs of 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 of 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. There were no apparent treatment-related effects on any of the reproductive parameters measured. Histopathological examination of selected tissue revealed a higher incidence of 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 of this study, the no-observed-effect concentration for mallards exposed to PFOS in the diet for six weeks was 6.2 ppm a.i. W ildlife International, Ltd. Project Number 454-105 -9- INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International, Ltd. avian toxicology facility in Easton, Maryland 21601. The biological portion of the test was conducted from February 29, 2000 until M y 25, 2000. Raw data generated at Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454-105 in archives located on the Wildlife International, Ltd. site. Biological specimens are stored at 3M Corporation, St. Paul, Minnesota 55133. OBJECTIVES The objective of this study was to evaluate the effects upon the adult Mallard {Anas platyrhynchos) of dietary exposure to the potassium salt of Perfluorooctane Sulfonic Acid (hereafter referred to as PFOS) over a period of approximately 6 weeks or 19 weeks. Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects of adult exposure to PFOS on the number of eggs laid, fertility, embryo viability, hatchability and offspring survival were evaluated. Histopathological examination of selected tissues and analyses of blood and tissue samples were also used to evaluate the effects upon adults exposed to PFOS and to their offspring. EXPERIMENTAL DESIGN Mallard (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 per Pen Males Females 11 11 11 11 W ildlife International, Ltd. Project Number 454-105 -10- Each treatment and control group contained five pairs of 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. of PFOS. The control group was fed diet comparable to the treatment groups, but without the addition of the test substance. Adult mallards were exposed to PFOS at nominal dietary concentrations of 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 of five pairs of birds, housed with one male and one female per pen. At the end of 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 of Week 20 of the test, at which time 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 of 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 of necropsy for possible analysis. Additional samples of liver, brain, kidney, gonad, proventriculus, gall bladder, adipose tissue, and bursa of fabricius (when available) were fixed in 10% buffered formalin for histopathological examination. During the test, the number of eggs laid in each pen was recorded to evaluate egg production. Eggs laid in Weeks 1, 3 and 6 of 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. W ildlife 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 Mallard". 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 Wildlife 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 of 98.9% and had expired at the time of experimental start. An assay of the test material after experimental start indicated a purity of 90.49%. The final assay of the test material indicated a purity of 86.9% and expiration date of August 31, 2006 (Appendix I). The test substance was held under ambient condition in locked storage at the Wildlife International, Ltd. facilities in Easton, Maryland. Concentrations of the test substance in the diet were adjusted to 100% active ingredient based upon the original reported purity of 98.9%. Dietary concentrations are expressed as parts per million active ingredient (ppm a.i.) in the diet based upon the final reported purity of 86.9%. The internal standard, 4H PFOS, was received from 3M Corporation on July 2, 1998 and was assigned Wildlife 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 Wildlife International, Ltd. facilities in Easton, Maryland. Test Organisms Fifty-one (24 males and 27 females) pen-reared mallards were purchased from Whistling Wings, Inc., 113 Washington 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. At the start of 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 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 of age at test initiation (first day of exposure to test diet) and ranged in weight from 788 to 1258 grams at test initiation. Sex of the birds was determined by a visual examination of the plumage. Identification Adult birds were identified by individual leg bands, each pen was identified with a unique number, and groups of pens were identified by project number and concentration. During the test, the number of 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 of origin. Wing bands were removed and the offspring were banded with leg bands at approximately four weeks of 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 Wildlife 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 feedstuffs and the 0.9% limestone used in the formulation of the basal diet by Agway. While this level of calcium is sufficient for growth and maintenance rations, additional calcium is required in the ration of breeding birds for egg shell formation. Therefore, an additional 5% (w/w) of 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 of 5% supplemental limestone. Water was supplied by the town of Easton public water supply. Neither the adults nor offspring received any form of medication in the feed during the test. Feed and water were analyzed periodically in accordance with Wildlife International, Ltd. Standard Operating Procedures. 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 of the final diet. Control diet and each treated diet were prepared weekly beginning on February 28, 2000 and presented to the birds on Tuesday of each week. Dietary concentrations were adjusted for purity of the test substance and are presented as parts per million active ingredient (ppm a.i.). Details of the weekly preparation of test and control diets are shown in Appendix III. Diet Sampling Homogeneity of the test substance in the diet was evaluated by collecting six samples from each of the treated diets and one sample from the control diet on Day 0 of Week 1. Samples were collected from the top, middle and bottom of the left and right sections of the mixing vessel. Control and treatment group diet samples were also collected from the feed troughs on Day 7 of Week 1 to assess stability of the test substance under actual test conditions. Additionally, a sample was collected from the control and treatment group diets during Week 6 of the test to measure/verify test concentrations. The diet samples were stored frozen or transferred immediately to the Wildlife International, Ltd. analytical chemistry facility for analysis. Analytical Method The method used for the analysis of PFOS in avian diet was based upon methodology developed at Wildlife International, Ltd. and entitled "Analytical Method Verification for the Determination of PFOS in Avian Diet" (Wildlife International, Ltd. Project No. 454C-110). Samples were extracted with methanol and diluted in a 50% methanol : 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 of the PFOS methodology. A method flow chart is provided in Appendix IV, Figure 1. Concentrations of PFOS in the standards and extracts of 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 Ci8 analytical column (50 mm x 2 mm I.D., 3-pm particle size). The instrument parameters are summarized in Appendix IV, Table 1. W ildlife International, Ltd. Project Number 454-105 - 14- Calibration standards of PFOS prepared in a 50% methanol: 50% NANOpure water solution containing 0.0100 mg 4H PFOS (internal standard)/L 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, Week 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 of individual isomeric components. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS : internal standard) of the calibration standards. An example of a calibration curve is presented in Appendix IV, Figure 2. The concentration of test substance in the samples was determined by substituting the peak area response ratios of the samples into the applicable linear regression equation. Typical ion chromatograms of low and high-level calibration standards are shown in Appendix IV, Figures 3 and 4, respectively. Examples of calculations are presented in Appendix IV, Table 2. The method limit of quantitation (LOQ) for the Week 1, Day 0 analysis was set at 0.879 ppm a.i., calculated as the product of the lowest calibration standard analyzed (0.000439 mg a.i./L) and the overall dilution factor of 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 of the lowest standard analyzed (0.000351 mg a.i./L) and the overall dilution factor of the matrix blank samples (4000 L/Kg). Examples of 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 TV, Table 3). A typical ion chromatogram of a matrix blank is presented in Appendix TV, 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 of 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 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 of 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 of pens manufac tured by Safeguard Products, Inc. (Model No. 5355), measuring approximately 75 X 90 X 45 cm high. The pens were constructed of vinyl-coated wire mesh. A diagram of 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 during the course of the test was 25.9 0.7C (SD) with an average relative humidity of 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 of approximately 202 lux (~ 18.8 ft. candles) of 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 of 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 of toxicity or abnormal behavior. Additionally, all offspring were observed daily from hatching until termination. A record was maintained of all mortalities and clinical observations. W ildlife International, Ltd. Project Number 454-105 -16- Adult Body Weight and Feed Consumption Adult body weights were measured at test initiation, on Weeks 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 of any additional diet added during the week, and weighing the feeder and remaining feed at the end of the feeding period (Day 7). The amount of 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 of total feed consumption. All remaining birds were euthanized three days after the beginning of Week 20. Therefore, no feed consumption estimate was calculated for Week 20. Adult Blood Collection At the end of Week 6, blood samples were collected from all surviving birds, when possible. Following collection of 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. treatment 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. Adult Necropsy and Tissue Collection At the conclusion of the exposure period, all surviving adult birds were euthanized by cervical dislocation, necropsied, and stored frozen. At the time of necropsy, tissues were collected for histopathological examination (0 and 17.6 ppm a.i. groups only) and possible analyses. When available, samples of gall bladder, liver, proventriculus, kidneys, brain, gonads, bursa of 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 during a seven day period beginning on the second day of Week 5 of 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 of pathogen contamination before storing them in the cold room. Eggs Wildlife 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). Water in the washer was warmed 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 of 13.1 0.1C (SD) with a mean relative humidity of approximately 75% 4% (SD). Groups of 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 of the weekly interval, all eggs were removed from the cold room, counted and candled with a Speed King (Model 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 of 30.6 0.1C (SD) (relative humidity of 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 of the embryo to the shell m em brane, the incubator w as also equipped w ith an autom atic egg rotation d evice, designed to rotate the eggs from 50 off of vertical in one direction to 50 off of vertical in the opposite direction (total arc of rotation was 100) every two hours through Day 24 of incubation. Eggs were candled on Day 14 of incubation to determine embryo viability and on Day 21 to determine embryo survival. Hatching and Brooding On Day 24 of incubation, the eggs were placed in a Petersime Hatcher (Model No. SP-6H) and allowed to hatch. Pedigree baskets constructed of galvanized steel wire mesh were used to keep hatchlings separated by parental pen of 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 of approximately 77%). 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 of incubation. The group body weight of the surviving hatchlings by pen was determined. Ducklings were wing banded for identification by pen of origin and then routinely housed according to the appropriate parental concentration grouping in brooding pens until approximately 3 weeks of 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. At approximately 12 weeks of age, the average body weight by parental pen of all surviving offspring was determined, and the birds were euthanized with carbon dioxide and disposed of by incineration. Those offspring selected for blood and tissue sampling were stored frozen following necropsy and later disposed of by incineration. Hatchlings were housed in batteries of brooding pens manufactured by Safeguard Products, Inc. Each pen measured approximately 62 X 92 X 25.5 cm high. The walls, floors and ceilings of each pen were constructed of vinyl-coated wire mesh. Thermostats in the brooding compartment of each pen were set to maintain a temperature of approximately 38C from the time of hatching until the birds were five to seven days of age, when the temperature was adjusted to maintain a temperature of approximately 29C. Brooding was discontinued once hatchlings were determined to be of 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 of 71 11% (SD). All ducklings were removed from brooding pens and transferred to pens w ithout supplem ental heat at approxim ately 3 w eeks o f age. T hese pens w ere the same model used to house the adult birds. The photoperiod for the offspring was maintained by a time clock at 16 hours of light per day. Offspring Blood and Tissue Collection Prior to euthanasia of 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 histopathological examination and analyses. When available, samples of gall bladder, liver, proventriculus, kidneys, brain, gonads, bursa of Fabricius and adipose tissue were fixed in 10% buffered formalin and shipped to EPL in Herndon, VA for histopathology. When available, samples of bile and liver tissue were stored frozen and shipped to Centre Analytical Laboratories, Inc. for possible analysis. Wildlife International, Ltd. Project Number 454-105 -19- Statistical Analyses Upon completion of the test, an Analysis of 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 of 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 of analyses only looked at body weight and feed consumption data from the first 6 weeks of 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 of 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 the 17.6 ppm a.i. treatment group were compared. 1. Adult Body Weight - Individual body weight was measured at test initiation, Weeks 2, 4, 6, 8, 10, 11, and at adult termination. Statistical comparisons were made between the control group and each treatment 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 of 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 of eggs laid per female per treatment group. For the evaluation of 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 of Eggs Laid - The number of eggs determined by candling to be cracked divided by the number of eggs laid, per pen. 6. Viable Embryos of Eggs Set - The number of embryos at the Day 14 candling was divided by the number of eggs set, per pen. W ildlife International, Ltd. Project Number 454-105 -20 7. Live 3- Week Embryos of Viable Embryos - The number of live embryos at the Day 21 candling was divided by the number of viable embryos, per pen. 8. Hatchlings of 3-Week Embryos - The number of hatchlings removed from the hatcher was divided by the number of live 3-week embryos, per pen. 9. 14-Dav Old Survivors of Hatchlings - The number of 14-day old survivors was divided by the number of hatchlings per week, by pen. 10. Hatchlings of Eggs Set - The number of hatchlings was divided by the number of eggs set per week, by pen. 11. 14-Dav Old Survivors of Eggs Set - The number of 14-Day old survivors was divided by the number of eggs set per week, by pen. 12. Offspring's Body Weight - The group body weights of 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 of offspring of week 5. Statistical comparisons of 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 of 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 of five pairs of birds, housed with one male and one female per pen. At the end of 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 of Week 20 of the test, at which time they were also euthanized and subjected to gross necropsy. Analytical Results None of the control samples showed any indication of the presence of the test substance or of the presence of co-eluting substance at the characteristic retention time of the test substance (Table 1). Diet samples were collected from the 1.8, 6.2 and 17.6 ppm a.i. test concentrations on Week 1, Day 0, and were analyzed to evaluate the homogeneity of the test substance in the diet and to verify test substance concentrations. Means and standard deviations for the three test concentrations were 1.8 0.13 ppm a.i., Wildlife 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 of variation were 7.2%, 11% and 8.3%, respectively. These values represented 100, 97 and 100% of nominal concentrations (Appendix IV, Table 4). Samples collected during Week 6, Day 0 of 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 of 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 of variation were 4.6%, 11% and 3.6%, respectively. These values represented 111, 97 and 95% of nominal concentrations (Appendix IV, Table 5). Analysis of diet samples collected from feeders after being held at ambient temperature for 7 days averaged 89%, 87% and 105% of 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 of a test sample is shown in Appendix IV, Figure 7. Mortalities and Clinical Observations No adult mortalities occurred in the control group or in any of the treatment groups during the course of the test. Several birds were noted with bumble foot (foot lesions) and feather loss as a result of pen wear and/or penmate aggression during the course of the test. A single bird in the 6.2 ppm a.i treatment 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 b leed in g at test term ination. She also exhibited b ody tremors and rapid blinking and respiration during the bleeding process. Clinical signs appeared to be triggered by the stress of 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 of 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 International, Ltd. Project Number 454-105 -22 - 17.6 ppm a.i. treatment group was significantly (p < 0.05) lower at the Week 10, 11 and 20 body weight intervals. There was also a significant loss of overall mean body weight for males at the 17.6 ppm a.i. test concentration. While there was an overall loss of 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 Weeks 14, 16, 17, 18 and 19, these differences were due to apparent increases in feed consumption for the 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 of feed wastage. Mean feed consumption measurements are shown in Table 3, and feed consumption measurements by pen are presented in Appendix VII. Gross Necropsy At the end of Week 6 (Day 42), all adult birds in the 1.8 and 6.2 ppm a.i. treatment groups were euthanized and subjected to gross necropsy. Adult birds in the control and 17.6 ppm a.i. treatment groups were maintained on the appropriate diets until the beginning of 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 of age at the time of euthanasia. A subsample of 10juvenile birds for each test group were subjected to gross necropsy. Necropsy revealed several birds in each treatment level with bumble foot (foot lesions). Additionally, a female offspring in the 17.6 ppm a.i. treatment group was noted with picked and broken feathers and an unkempt appearance. Internally the bird was noted with a retained yolk sac, a remnant of the natal yolk sac. One male offspring in the 17.6 ppm a.i. treatment group was noted with the posterior portion of the left kidney absent. All findings observed were considered to be incidental to treatment. W ildlife International, Ltd. Project Number 454-105 -23 - Histopathology Light microscopic examination was performed on selected tissues by a board certified pathologist at Experimental Pathology Laboratories, Inc. Herndon, VA. Sections of liver, brain, kidney, gonad, proventriculus, gall bladder, adipose tissues, and bursa of 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 of fabricius of adult male and females, or their offspring at any of the concentrations tested. Additionally, there were no lesions considered to be treatment-related noted in adipose tissue of adult females and offspring of both sexes, or in the testes of male offspring. When compared to the control group, testes of adult males at the 17.6 ppm a.i. test concentration exhibited a higher incidence of features consistent with post-reproductive phase regression. Adult males in the 17.6 ppm a.i. group also exhibited an increased incidence of adipose tissue microvesiculation. The increased incidence of 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 of 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 of 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 of Potassium Perfluorooctanesulfonate from Egg Membrane, Albumen, and Yolk for analysis using HPLCElectrospray/Mass Spectrometry; Centre Study Number 023-065". W ildlife International, Ltd, Project Number 454-105 -24 - Reproductive Results While egg production was highly variable among hens, there were no apparent treatment-related effects upon egg production at any of the concentrations tested. Any differences between the control group and the treatment 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. When 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 parameter 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 Weights There were no apparent treatment-related effects upon the body weights of hatchlings in any of 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 of 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 Weights When compared to the control group, there were no apparent treatment-related effects on adult liver weight at any of 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 of 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 of the test, during a period of high egg production. The control group and 17.6 ppm a.i. treatment group test birds were euthanized at the beginning of Week 20 of the test by which time egg production had declined dramatically. One W ildlife International, Ltd. Project Number 454-105 -25 - would expect birds in full reproductive condition, particularly females, to have higher levels of 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 of the concentrations tested. While there was a significant increase in mean offspring liver weight for the 1.8 ppm a.i. treatment 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 Mallards were exposed to PFOS at dietary concentrations of 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 of the concentrations tested. No overt signs of 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 of 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. treatment groups. There w ere no apparent treatment-related effects on any o f the reproductive parameters m easured. Histopathological examination of selected tissue revealed a higher incidence of 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 of this study, the no-observed-effect concentration for mallards exposed to PFOS in the diet for six weeks was 6.2 ppm a.i. W ildlife International, Ltd. -26REFERENCES Project Number 454-105 1 U.S. Environmental Protection Agency. 1982. Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, subsection 71-4, Environmental Protection Agency, Office of Pesticide Programs. Washington, D.C. 2 American Society for Testing and Materials. 1986. Standard Practice fo r Conducting Reproductive Studies with Avian Species. ASTM Standard E l062-86. Annual Book of ASTM Standards. Vol. 11.04. Philadelphia, PA. 15 pp. 3 Merck & Co., Inc. 1991. The Merck Veterinary Manual. Merck & Co. Rahway, NJ. 1832 pp. 4 National Research Council. 1996. Guide for the Care and Use o f Laboratory Animals. Washington, DC. National Academy Press. 125 pp. 5 Dunnett, C.W. 1955. A Multiple Comparison's Procedure for Comparing Several Treatments with a Control. Jour. Amer. Statis. Assoc. 50: 1096-1121.6 6 Dunnett, C.W. 1964. New Tables for Multiple Comparisons with a Control. Biometrics 20: 482 491. Table 1 Mean Measured Concentrations (ppm a.i.) of PFOS in Avian Diet from a Mallard Pilot Reproduction Study Nominal Concentration1 DayO Week 1 Interval Day 7 Week 6 DayO Control (0 ppm a.i.) 1.8 (ppm a.i.) 6.4 (ppm a.i.) 18.3 (ppm a.i.) Measured <0.879 2 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 .4 1 2.0 111 6.0 97 16.8 95 1Nominal concentrations and results of diet analysis were corrected for the change in test substance purity from 90.49% to 86.9` 2 The limit o f quantitation (LOQ) was equivalent to 0.879 ppm a.i. for Week 1, Day 0 and 1.41 ppm a.i. for Week 1, Day 7 and Week 6, Day 0. 3The mean percent o f the Day 0 values. Project Number 454-105 Table 2 Mean Adult Body W eight1 (g) from a Mallard Pilot Reproduction Study with PFOS Experimental Group Sex Week Change Week Change Week Change Week Change Change Week Change Week Change Week Change Week Change 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 Control M X 1120 -36 1084 -2 1082 42 1124 3 (O ppm a.i.) SD 53 65 59 54 66 16 71 41 F X 998 -36 962 89 1051 1 1052 55 SD 106 87 70 84 52 59 75 116 1.8 ppm ai. M X 1093 -10 1083 -9 1073 SD 70 52 69 21 77 F X 1003 SD 92 88 1092 -1 3 * 1079 64 55 57 72 63 1136 24 78 43 72 35 1114 110 41 91 106 6.2 ppm ai. M X 1091 -33 1058 SD 31 20 40 F X 988 SD 62 66 1054 69 65 39 1098 37 42 21 1118 73 63 27 74 28 1082 -43 1038 51 66 105 78 95 111 19 1142 32 81 -54 998 31 61 59 1202 21 79 26 1025 62 18 40 1242 40 59 -10 1015 44 50 -- _ __ . - -- - - - -- -- -- -- -- --. - - - - -- - _ _ , _ ._ - -- _ -- - __ - -- -- - -38 1204 95 97 -10 1005 59 23 _ -- _ - -- __ -- __ -- 83 58 7 118 _ - _ - _ -- _ - 17.6 ppm ai. M X 1095 -41 1053 SD 107 56 78 F X 999 23 1022 SD 150 104 138 -4 1049 14 70 38 1087 25 51 -8 74 11 1033 -55 53 102 59 978 -21 149 88 -13 1074 62 24 4 1078* -5 1073 ' -31 1042 * -53 * 55 65 18 73 92 94 57 17 995 -27 967 -10 958 -60 897 -102 62 148 37 172 15 173 98 139 90 Project Number 454-105 1Mean (X) standard deviation (SD). The means for body weights and body weight changes are calculated and rounded separately. --Data not avalible. Birds euthanized at the end ofWeek 6. *Statistically different from die control group at p < 0.05. Table 3 Mean Feed Consumption1 (g/bird/day) from a Mallard Pilot Reproduction Study with PFOS Experimental Group __________________________________________________________W eeks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Control Mean 146 124 150 178 186 212 200 236 220 211 236 175 139 196 139 123 168 130 170 0 ppm a.i. SD 28 16 24 37 17 40 53 63 28 71 39 28 19 22 42 28 41 25 44 1.8 Mean 157 150 154 172 160 177 _ ,, _ __ __ __ __ _ __ __ ppm a.i. SD 47 31 43 47 42 50 "" 6.2 Mean 145 137 168 191 195 178 ppm a.i. SD 34 39 29 4 0 78 56 __ ,, ... ,, ,,, ---- -- -------- 17.6 M ean 156 136 200 187 196 234 225 241 253 226 249 218 195 2 6 7 * 172 191 * 238 * 183 * 227 * ppm a.i. SD 65 38 62 42 57 84 51 79 67 70 69 62 69 4 9 52 42 51 39 29 Mean standard deviation. -- Data not avalible. Birds euthanized at the end o f Week 6. * Statistically different from the control group at p < 0.05. Project Number 454-105 Number o f birds M olting flight feathers Feather loss & abrasions on neck Bumble foot Impacted feed under tongue Abnorm ally sm all body size Testes sm all, ~ 1.0-2.0 cm Testes small, ~ 2.0-3.0 cm Testes small, - 3.5 cm Regressing ovaiy Regressed ovary E gg yolk peritonitus E gg remnants in the repro. tract Intrabdominal egg Air sacculitus N ot remarkable Table 4 Summary of Gross Pathological Observations from a Mallard Pilot Reproduction Study with PFOS Adult Birds Euthanized at 6 Weeks and Test Termination Males - Treatment Group (ppm a.i.) Control 1.8 6.2 17.6 Fem ales - Treatment Group (ppm a.i.) Control 1.8 6.2 1 76 55 5 5 30 0 0 00 0 0 313 4 00 0 1 00 0 0 00 0 3 20 0 1 20 0 0 -- - - -- - - -- - - -- - - -- - - 00 0 0 04 2 0 5555 2003 00 10 3331 0 0 00 000 1 ---- ---- ---- 0 10 0 4005 0 13 1 10 00 00 10 000 1 1 1 10 Project Number 454-105 Table 5 Mean Egg Production 1(Eggs Laid/Hen and Eggs/Hen/Day) from a Mallard Pilot Reproduction Study with PFOS Experimental Group _________________________________________________ W eeks 1 2 3 4 5 6 Total E/H/D2 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total E/H /D 1 Control Total 17 15 14 17 22 27 112 21 26 13 13 7 5 0 0 0 0 0 0 0 0 85 0 ppm a.i. Mean 3 3 3 3 4 5 22 0.53 4 5 3 3 1 1 0 0 0 0 0 0 0 0 17 0.18 SD 3 3 4 3 3 3 16 0.39 3 3 3 4 3 2 0 0 0 0 0 0 0 0 15 0.16 1.8 ppm a.i. Total 11 15 25 20 19 16 106 Mean 2 3 5 4 4 3 21 SD 3 3 3 4 3 4 18 0.50 0.44 - - -- -- -- -- -- -- -- - -- - - -- -- -- -- -- -- -- -- - - -- 6.2 Total 5 12 13 12 14 8 64 ppm a.i. Mean 1 2 3 2 3 2 13 0.30 SD 2 3 4 3 4 3 17 0.41 _ __ - - - -- -- -- -- -- -- - -- - -- -- -- -- --- -- 17.6 Total 15 22 33 31 24 22 147 10 15 15 17 8 7 7 4 3 4 0 0 0 0 90 ppm a.i. Mean 3 4 7 6 5 4 29 0.70 2 3 3 3 2 1 1 1 1 1 0 0 0 0 18 0.19 SD 3 1 1 1 2 3 5 0.11 3 4 3 4 3 3 3 2 1 2 0 0 0 0 23 0.24 1Total number o f eggs laid and mean number of eggs laid per pen standard deviation. 2Eggs per hen per day. -- Data not available. Birds euthanized at the end of Week 6. Differences between the control group and the treatment group were not statistically significant (p > 0.05). OJ Project Number 454-105 -32- Project Number 454-105 Table 6 Summary of Reproductive Performance (Eggs Set from Week 5) from a Mallard Pilot Reproduction Study with PFOS Reproductive Parameter Control (0 ppm a.i.) Experimental Group 1.8ppm a.i. 6.2 ppm a.i. Number o f Replicates Eggs Laid Eggs Cracked Eggs Set Viable Embryos Live 3-Week Embryos Hatchlings Offspring Survivors Eggs Laid/Hen Offspring/Hen 555 24 18 12 0 10 24 17 12 22 17 12 22 17 12 21 14 12 21 13 12 542 .4 3 2 Eggs Laid/Hen/Day E ggs Cracked/Eggs Laid (%) V iable Embryos/Eggs Set (%) L ive 3-W eek Embryos/Viable (%) H atchlings/3-W eek Embryos (%) Offspring Survivors/Hatchlings (%) H atchlings/E ggs Set (%) Offspring Survivors/Eggs Set (%) Hatchlings/Hen/Day Offspring Survivors/Hen/Day 0.69 O 90 100 92 100 85 85 0.60 0.60 0.51 5 100 100 82 94 82 77 0.40 0.37 0.34 0 100 100 100 100 100 100 0.34 0.34 Differences between the control group and the treatment groups were not statistically significant (p > 0.05) 17.6 ppm a.i. 5 24 1 23 21 21 18 17 5 3 0.69 4 92 100 83 97 76 74 0.51 0.49 Table 7 Mean Body Weight1(g) of Hatchlings and Surviving Offspring from a Mallard Pilot Reproduction Study with PFOS Experimental Group Hatchlings Mean SD Surviving Offspring2 Mean SD Control (0 ppm a.i.) 28.4 2.0 1012 72 1.8 ppm a.i. 32.8 1.4 1065 92 6.2 ppm a.i. 33.3 0.4 1036 71 Project Number 454-105 17.6 ppm a.i. 35.0 1.6 1030 35 'Mean standard deviation. 2Offspring were approximately 12 weeks o f age at final body weight interval. Differences between the control group and the treatmeng groups were not statistically significant (p > 0.05) Experimental Group Control (0 ppm a.i.) 1.8 ppm a.i. 6.2 ppm a.i. 17.6 ppm a.i. Table 8 Mean Liver Weights1(g) from a Mallard Pilot Reproduction Study with PFOS Males Liver Mean SD Females Liver Mean SD O ffs p r in g *2 Liver Mean SD 21.336 4.793 21.240 5.382 20.890 3.148 25.336 1.564 39.801 8.832 * 27.661 4.999 * 29.205 4.900 31.938 4.462 * 23.220 4.215 23.411 8.019 20.624 2.947 24.717 5.489 'Mean standard deviation. 2 Offspring were approximately 12 weeks o f age at time o f euthanasia and tissue collection. * Statistically different from the control group at p < 0.05. UbJ. Project Number 454-105 -35- W ildlife International, Ltd, Project Number 454-105 A ppendix I Certificate o f A nalysis Centre Analytical Laboratories. Inc. 3048 Research Drive State College, PA16801 www.centrelab.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratoryei COA Reference #: 023-018A 3M Product: PFO S,Lot217 Reference#: SD-018 _____________________Parity: 86.9%____________________ ----------T ot Name---------- Specifications Purity1 Result 86.9% Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium1 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) Related Compounds POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate4 Organic Acids ' (IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis*: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine White Crystalline Powder 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% Conforms Positive 1. 0.005 wt/wt.% 2. 0.001 wt/wt.% 3. 1.439 wt/wt.% 4. 6.849 wt/wt.% 5. <0.001 wt/wt.% 6. 0.005 wt/wt.% 7. <0.001 wt/wt.% 1.91 wt./wt.% 8.41 wt./wt.% None Detected 0.33 wt/wt.% None Detected Not Applicable1 1. <0.015 wt./wt% 2. 0.59 wtywt.% 3. <0.040 wtywt.% 4. <0.009 wtywt.% 5. <0.006 wtywt.% 6. <0.007 wt/wt% 7. 8.76 wt/wt.% 1. <0.1 wt/wt.% 2. <0.1 wt/wt.% 3. 0.10 wt/wt.% 4. 0.28 wt/wt.% 1. 12.48 wtywt.% 2. 0.244 wt/wt.% 3. 1.74 wtywt.% 4. 8.84 wt/wt.% 5. 54.1 wtywt.% COA023-018A Page 1 o f 3 -36- W ildlife International, Ltd Project Number 454-105 A ppendix I Page 2 Certificate o f A nalysis C eitre Analytical Laboratories, Inc 3048 Research ttrlve State College, PA 16801 www.centrelab.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OFANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A Date ofLast Analysis: 08/31/00 Expiration Date: 08/31/06 Storage Conditions: Frozen <-l 0"C Re-assessment Date: 08/31/06 'Purity = 100% - (sum ofmetal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.905%+organic acid impurities, 0.38%+POAA, 0.33%) Total impurity from all tests = 13.07% Purity = 100% - 13.07% = 86.9% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials of low purity. No endotherm was observed for this sample. 4Sulfur in the sample appears to be converted to SO4 and hence detected using the inorganic anion m ethod conditions. T h e anion re su lt agrees w ell w ith the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO* is not considered an impurity.56 5TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonafluoropentanoic acid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, CgFnSQjK* (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-018A Page 2 o f 3 -37- W ildlife International, Ltd, Project Number 454-105 A ppendix I Page 3 Certifcate o f A nalysis Centre Analytical Laboratories, Inc 3048 Research Drive State College, PA 16801 www.oentieteb.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A LC/MS Purity Profile: Impurity C4 C5 C6 C7 Total wfc/wt. % 1.22 1.33 4.72 1.14 8.41 Note: The 04 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 curves. Likewise, the C7 value was calculated using the average result front the C6 and C8 standard curves. Prepared By: Charles Sii Scientist, Centre Analytical Laboratories to /ft /o t Date Reviewed By::ciLrnfhJeCt John Flaherty / Date 'Laboratory Manager, Centre Analytical Laboratories COA023-018A Page 3 of 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 Alfalfa 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 follow ing amounts per ton: Amount Supplied Per Ton: Vitamin D3 Vitamin A Riboflavin Niacin Pantothenic Acid Vitamin B 12 Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (Menadione Dimethylpyrimidinol Bisulfite) Manganese Zinc Copper Iodine Iron Selenium 2.000. 0 0 0 1.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 2 0 ,0 0 0 1.U. 5.8 grams 102 grams 47 grams 6.8 grams 1.5 grams 51 grams 182 mgs The guaranteed analysis is a minimum o f 27% protein, a minimum o f 2.5% crude fat and a maximum o f 5% crude fiber. 2 Fermentation By-Products (Source o f Unidentified Growth Factors). 39- - Wildlife 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 gration 6.2 ppm a.i.: 1.0794 g PFOS + 6098.9 gration 17.6 ppm a.i.: 3.0839 g PFOS + 6096.9 gration 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. W ildlife International, Ltd. 40- - Project Number 454-105 Appendix IV The Analysis of PFOS in Avian Diet W ildlife International, Ltd. -41 - Project Number 454-105 Appendix IV ANALYTICAL METHODS AND RESULTS Typical LC/MS Operational Parameters TABLE 1 INSTRUMENT: 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]8 column (50 mm x 2 mm I.D., 3-pm particle size) OVEN TEMPERATURE: 30C STOP TIME: 5.00 minutes FLOW RATE: MOBILE PHASE: 0.220 mL/minute 72.0% Methanol : 28.0% NANOpure Water containing 0.1% Formic Acid INJECTION VOLUME: PFOS RETENTION TIME: 5.0 pL Approximately 3.6 minutes INTERNAL STANDARD RETENTION TIME: Approximately 2.6 minutes PFOS MONITORED MASS: 498.6 amu INTERNAL STANDARD MONITORED MASS: 426.7 amu -42- W ildlife International, Ltd. Project Number 454-105 Appendix IV TABLE 2 CALCULATIONS The concentration o f PFOS found at the instrument w a s d eterm in ed u sin g th e fo llo w in g equation: peak area ratio - (y-intercept) PFOS (mg a.i./L) at instrument = slope x internal standard cone, (mg a.i./L) Determination of Sample Residues (PFOS1 The concentration, expressed as ppm a.i., for each sample was determined using the following equation: ,, , . x . , PFOS (mg a.i./L) at instru. x extract final volume (L) x dilution factor PFOS (ppm a.,.) m sample --------- L- s ------- 1----------W tial weight (Kg)------ -------------------- Determination of 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 of matrix blank* extract final volume (L) x dilution factor *overall dilution factor of matrix blank sample = initial weight (Kg) Fortification Recoveries The ppm a.i. measured in each sample is divided by the nominal concentration of each sample (fortified level, ppm a.i.). This ratio times 100 is the percent recovery of the method at that level of fortification. % Recovery ppm = a.i. measured in sample ppm a.i. fortified x 100 W ildlife International, Ltd. Project Number 454-105 Appendix IV TABLE 3 Matrix Blanks and Fortifications Analyzed Concurrently with the Samples Number (454-105-) Sample Type Interval Concentration o f PFOS (ppm a.i.) Fortified1 M ea su red 1,2 Percent Recovery Mean Recovery (X) M A B -1 M A S-1 M A S-2 M A S-3 M A B -2 M AS-4 M A S-5 M AS-6 M A B -3 M AS-7 M A S-8 M AS-9 M A B -4 M A S-10 M A S -11 M A S-12 Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification W eek 1, D ay 0 W eek 1, D ay 0 W eek 1, D ay 0 W eek 1, D ay 0 Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification W eek 1, D ay 7 W eek 1, D ay 7 W eek 1, D ay 7 W eek 1, D ay 7 Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification W eek 6, D ay 0 W eek 6, D ay 0 W eek 6, D ay 0 W eek 6, D ay 0 Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Re-extraction Set4 Re-extraction Set4 Re-extraction Set4 Re-extraction Set4 0.00 0.879 8.79 22.0 0.00 1.76 8.79 22.0 0.00 1.76 8.79 22.0 0.00 1.76 8.79 22.0 < 0.879 0.992 8.89 22.0 < 1.41 1.97 9.11 21.3 < 1.41 1.98 9.40 22.0 < 1.41 1.84 8.86 21.7 113 101 100 112 104 97.0 . 113 107 100 105 101 98.7 105 104 107 102 1Concentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate of Analysis dated October 11,2001. Nominal test concentrations based upon 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). 3Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since fortified and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes. 4Re-extraction of: sample 454-105,105-13 from Week 1, Day 0; samples 454-105-23 and -26 from Week 1, Day 7 and sample 454-105,105-50 from Week 6, Day 0. 'Wildlife International, Ltd. Project Number 454-105 Appendix IV TABLE 4 Homogeneity of PFOS in Avian Diet N om inal Concentration1 (ppm a.i.) Sample I.D. Number (S-454-105-) Location Sampled in M ixing V essel Measured PFOS Concentration1 (ppm a.i.) Mean (x) Standard D eviation (SD) Coefficient o f Variation (CV) Mean Percent of Nominal 1.8 3 Top Left 1.70 4 Top Right 1.82 5 Middle Left 1.92 x = 1.8 ppm a.i 100 6 Middle Right 1.73 SD = 0.13 ppm a.i. 7 Bottom Left 1.77 CV = 7.2% 8 Bottom Right 2.05 6.2 9 Top Left 5.33 10 Top Right 5.65 11 Middle Left 6.09 x = 6.0 ppm a.i. 97 12 M iddle Right 5.81 SD = 0.65 ppm a.i. 13 Bottom Left 5.872 CV=11% 14 Bottom Right 7.21 17.6 15 Top Left 16.5 16 Top Right 17.7 17 Middle Left 16.5 x = 17.6 ppm a.i. 100 18 Middle Right 20.4 SD = 1.46 ppm a.i. 19 Bottom Left 17.0 CV = 8.3% 20 Bottom Right 17.6 'Concentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate o f Analysis dated October 11, 2001. Nominal test concentrations based upon the new test substance purity are 1.8, 6.2 and 17.6 ppm a.i. 2M ean result o f duplicate re-extraction o f original sample. W ildlife International, Ltd. Project Number 454-105 Nominal Concentration1 (ppm a.i.) 0 Sample I.D. Number (S-454-105-) 1 2 43 44 Appendix IV TABLE 5 Verification of PFOS Concentrations in Avian Diet Interval (Day 0 of Week -) 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 Mean Percent of Nominal _ 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 5.273 (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% 100 111 97 97 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% 100 95 1Concentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate of Analysis dated October 11, 2001. Nominal test concentrations based upon 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 of duplicate re-extraction of original sample. W ildlife International, Ltd. Project Number 454-105 -46- Appendix IV TABLE 6 Ambient Stability of PFOS in Avian Diet During the Mallard Pilot Reproduction Study Nominal Concentration23 (ppm a.i.) Sample Number (S-454-105-) W eek 1, D ay 01 Mean Measured (ppm a.i.) PFOS Concentration Mean Percent of Nominal Sample Number (S-454-105-) W eek 1, D ay 7 M ea su red 2,3 (ppm a.i.) Mean Measured (ppm a.i.) Mean Percent of Day 0 0 1-2 < 0.879 1.8 3-8 1.8 100 6.2 9-14 6.0 97 17.6 15-20 17.6 100 32 < 1.41 33 < 1 .4 1 34 1.50 1.6 35 1.49 36 1.85 37 5.48 5.2 38 5.01 39 5.03 40 18.7 18.4 41 18.8 42 17.8 89 87 105 1D ay 0 values are from hom ogeneity samples presented in Table 4 and verification samples presented in Table 5. 2Concentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate o f Analysis dated October 11, 2001. Nominal test substance concentrations based upon the new test substance purity are 1.8, 6.2 and 17.6 ppm a.i. 3Less than values correspond to limit o f quantitation (LOQ). -47- W ildlife International, Ltd, Project Number 454-105 Appendix IV METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN AVIAN DIET Prepare matrix fortification samples in the desired avian feed stock using the dry m ix technique. 4 W eigh 10 g samples o f the matrix blank, matrix fortification and test samples into w eigh boats and transfer to 8oz. French square glass bottles. Record weights. I For each sample, measure 100 mL o f methanol with a graduated cylinder and transfer into the French square bottle. 4 Cap bottles and place on shaker table. A llow the samples to shake for a minimum o f 30 minutes at approximately 250 rpm. 4 Vacuum filter with qualitative filter paper and rinse retained feed 3 times with methanol into filtrate. 4 Transfer the filtrate into a 200-mL volumetric flask and bring to volume with methanol. 4 Prepare appropriate dilution(s) to bring final concentration into the calibration range o f the LCMS m ethodology. U se methanol for intermediate dilutions, i f required. For all final dilutions, use 50% methanol: 50% NANOpure water dilution solvent containing 0.0100 mg 4HPFOS/L (internal standard) and 0.05% (v/v) formic acid. 4 Ampulate and submit sample for LCMS analysis. Figure 1. Analytical method flow chart for the analysis o f PFOS in avian diet. 48- - W ildlife International, Ltd. Appendix IV Project Number 454-105 Figure 2. Typical calibration curve for PFOS. Slope = 2.48679; Intercept = 0.07396; r = 0.9985. Curve is weighted (1/x). -49- W ildlife International, Ltd. Project Number 454-105 Appendix IV intensity: 20000 cps Figure 3. Typical ion chromatogram of a low-level PFOS standard, 0.000351 mg a.i./L. -50- Wildlife International, Ltd. Project Number 454-105 Appendix IV intensity: 20000 cps Figure 4. Typical ion chromatogram of a high-level PFOS standard, 0.00439 mg a.i./L. -51 - Wildlife International, Ltd. Project Number 454-105 Appendix IV intensity: 40000 cps Figure 5. Typical ion chromatogram of a matrix blank sample (454-105-MAB-l). The arrow indicates approximate retention time of PFOS. 52- - W ildlife International, Ltd. Project Number 454-105 Appendix IV intensity: 20000 cps Figure 6. Typical ion chromatogram of a matrix fortification sample (454-105-MAS-9, 22.0 ppm a.i.). -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.). 54- - W ildlife International, Ltd. A ppendix V Diagram o f Test Layout Project Number 454-105 = approx, lm = 6" gutter a = Safeguard Products Inc. Model No. 5355 b = Four X 4 ft. Chroma 50 light bulbs 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 W eek Change W eek Change Change 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 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 SD 1120 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 W eek Change W eek Change W eek Change W eek Change Change 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 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 -14 -100 -44 -64 947 982 1048 1076 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. Appendix VI Table 2 Adult Body Weight (g) from a Mallard Pilot Reproduction Study with PFOS 1.8ppma.i. - Males W eek Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 206 1165 207 1147 208 1070 209 987 210 1095 Mean SD 1093 70 -12 1153 -27 1120 54 1124 19 1006 -85 1010 -10 1083 52 69 8 1161 -36 1084 6 1130 -29 977 4 1014 -9 1073 21 77 74 1235 44 1128 63 1193 96 1073 36 1050 63 1136 24 78 70 -19 123 86 -45 43 72 1.8 ppm a.i. - Fem ales W eek Change W eek Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 206 895 207 969 208 1057 209 964 210 1132 Mean SD 1003 92 178 1073 76 1045 11 1068 122 1086 55 1187 88 1092 64 55 69 1142 -81 964 5 1073 -6 1080 -51 1136 -13 1079 57 72 -8 1134 -2 962 36 1109 88 1168 60 1196 35 1114 41 91 239 -7 52 204 64 110 106 The means for body weights and body weight changes are calculated and rounded separately. Os Project Number 454-105 Appendix VI Table 3 Adult Body Weight (g) from a Mallard Pilot Reproduction Study with PFOS 6.2 ppm a.i. - Males 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 SD 1091 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 6.2 ppm a.i. - Fem ales W eek Change Week Change Week Change W eek 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. Appendix VI Table 4 Adult Body Weight (g) from a Mallard Pilot Reproduction Study with PFOS 17.6 ppm a.i. - Males W eek Change W eek Change Week Change W eek Change Change 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 6-8 8 8-10 10 10-11 11 11-20 20 0-20 216 1028 -82 946 217 1258 -111 1147 218 1138 -33 1105 219 1062 -10 1052 220 987 29 1016 Mean SD 1095 107 -41 1053 56 78 19 965 -13 1134 -4 1101 -6 1046 -18 998 -4 1049 14 70 48 1013 -15 0 1134 -124 34 1135 -3 40 1086 24 67 1065 78 38 1087 25 51 -8 74 72 1085 -52 1082 -87 1048 18 1104 -15 1050 -13 1074 62 24 -13 1072 80 1162 -68 980 -6 1098 28 1078 4 1078 55 65 -19 1053 -111 942 8 1170 -8 1162 -12 968 80 1048 -22 1076 24 1100 18 1096 -139 957 -5 1073 18 73 -31 1042 92 94 -86 -96 -90 38 -30 -53 57 Project Number 454-105 17.6 ppm a.i. - Fem ales W eek Change W eek Change W eek Change W eek Change Change 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 6-8 8 8-10 10 10-11 11 11-20 20 0-20 216 1075 217 895 218 1112 219 788 220 1125 Mean SD 999 150 -84 991 97 992 -95 1017 78 866 120 1245 23 1022 104 138 43 1034 -126 908 -167 -6 986 -76 910 15 60 1077 -1 1076 -36 32 898 -85 813 25 -74 1171 12 1183 58 11 1033 -55 978 -21 53 102 59 149 88 49 957 95 1005 32 1108 -48 765 -44 1139 17 995 62 148 -29 928 16 1021 -53 1055 -73 692 2 1141 -27 967 37 172 -31 897 54 951 -124 -5 1016 -123 893 -2 9 1064 -191 873 -239 -5 687 6 693 -95 -17 1124 -47 1077 -48 -10 958 -60 897 -102 15 173 98 139 90 The means for body weights and body weight changes are calculated and rounded separately. Appendix VII Table 1 Feed Consumption (g/bird/day) from a Mallard Pilot Reproduction Study with PFOS Control (0 ppm a.i.) Weeks Pen 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 201 183 132 178 216 190 262 258 282 229 233 263 223 112 232 125 110 145 116 155 202 123 137 157 164 188 185 163 188 224 190 168 149 129 190 86 111 154 133 151 203 117 135 128 130 157 169 130 149 172 121 240 168 154 182 126 91 121 114 141 204 140 98 164 216 191 244 238 278 237 196 249 166 159 200 158 143 198 116 248 205 167 117 122 165 203 198 209 282 238 317 261 169 139 174 198 159 220 172 155 Mean 146 124 150 178 186 212 200 236 220 211 236 175 139 196 139 123 168 130 170 SD 28 16 24 37 17 40 53 63 28 71 39 28 19 22 42 28 41 25 44 Project Number 454-105 Appendix VII Table 2 Feed Consumption (g/bird/day) from a Mallard Pilot Reproduction Study with PFOS 1.8 ppm a.i. Weeks Pen 1 2 3 4 5 6 206 130 130 174 185 156 151 207 110 109 105 94 90 106 208 189 158 149 167 173 187 209 135 167 127 215 183 228 210 223 188 216 200 199 215 Mean 157 150 154 172 160 177 SD 47 31 43 47 42 50 I OOn Project Number 454-105 Appendix VII Table 3 Feed Consumption (g/bird/day) from a Mallard Pilot Reproduction Study with PFOS 6.2 ppm a.i. Weeks Pen 1 2 3 4 5 6 211 137 133 168 176 208 196 212 122 92 187 177 182 127 213 194 186 197 247 312 260 214 110 109 122 143 96 125 215 164 166 168 213 179 181 Mean 145 137 168 191 195 178 SD 34 39 29 40 78 56 Project Number 454-105 a\ Appendix VII Table 4 Feed Consumption (g/bird/day) from a Mallard Pilot Reproduction Study with PFOS 17.6 ppm a.i. Weeks Pen 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 216 121 138 269 209 266 219 266 303 284 221 193 173 188 211 112 146 265 175 216 217 102 96 132 133 135 127 156 150 195 160 248 206 178 234 207 191 163 131 223 218 161 135 218 223 244 267 238 299 306 290 350 324 296 338 222 170 294 228 274 219 130 114 139 150 154 200 190 159 168 155 179 177 104 288 118 259 213 165 195 220 265 198 242 220 180 355 277 293 314 306 275 211 209 266 200 188 254 216 228 Mean 156 136 200 187 196 234 225 241 253 226 249 218 195 267 172 191 238 183 227 SD 65 38 62 42 57 84 51 79 67 70 69 62 69 49 52 42 51 39 29 Project Number 454-105 -63- Project Number 454-105 Appendix V m Table 1 Individual Gross Pathological Observations from a Mallard Pilot Reproduction Study with PFOS Birds Euthanized at Test Termination Control (0 ppm a.i.) Males Pens 201 202 203 204 205 Molting flight feathers Bumble foot Testes small - ~ 2.0 - 3.0 cm Testes small - ~ 3.5 cm N ot remarkable XX - -X - XXX - X- - -X - XX - - ----- Females Molting flight feathers Bumble foot Regressed ovary Egg remnants in the reproductive tract N ot remarkable Pens 201 202 203 204 205 X - _ _X X -X -X X - XXX - - -X -X - - - -64- Project Number 454-105 Appendix VIH Table 2 Individual Gross Pathological Observations from a Mallard Pilot Reproduction Study with PFOS Birds Euthanized at Test Termination M ales Bumble foot N ot remarkable 1.8 ppma.i. Pens 206 207 208 209 210 _ _ _ _X XXXX - Females Bumble foot Regressing ovary Egg yolk peritonitus N ot remarkable Pens 206 207 208 209 210 XX _ _X -X - - - - - -X - - -X- - -65- Project Number 454-105 Appendix VIE Table 3 Individual Gross Pathological Observations from a Mallard Pilot Reproduction Study with PFOS Birds Euthanized at Test Termination Males Bumble foot N ot remarkable 6.2 ppm a.i. Pens 211 212 213 214 215 _X X- _X X- X - Females Feather loss and abrasions on neck Bumble foot Egg yolk peritonitus Extensive egg yolk peritonitus Intrabdominal egg N ot remarkable Pens 211 212 213 214 215 .. X __ X- X- X X - -X - - -X - - X- - -- -X - - - -66- Project Number 454-105 Appendix VIE Table 4 Individual Gross Pathological Observations from a Mallard Pilot Reproduction Study with PFOS Birds Euthanized at Test Termination 17.6 ppm a.i. Males Mass o f feed impacted under the tongue Bumble foot Testes small - ~ 1.0 - 2.0 cm Testes small - ~ 2.5 cm N ot remarkable Pens 216 217 218 219 220 _ _X - _ XXXX - - XXX - X- - - - ----- Females General condition - abnormally small body1 Molting flight feathers Bumble foot Air sacculitis Slight egg yolk peritonitus Regressed ovary Not remarkable Pens 216 217 218 219 220 X- - - - XX - X - - -X - - X- - - - X- - - - XXXXX ----- l Denotes a small body frame, but does not indicate a thin condition. -67- W ildlife International, Ltd. Project Number 454-105 Appendix IX Histopathology Report EPL -68- E X P E R IM E N TA L P A TH O LO G Y LAB O R ATO RIES, INC. Project Number 454-105 W ILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER 454-105 EPL PROJECT NUMBER 212-025 PFOS: A PILOT REPRODUCTIO N STUDY W ITH THE M ALLARD PATHOLOGY REPORT Subm itted by: Experim ental Pathology Laboratories, Inc. P.O. Box 474 Herndon, VA 20172-0474 (703) 471-7060 Subm itted to: W ildlife International, Ltd. Easton, MD 21601 January 25, 2001 Project Number 454-105 -69- E P L ___________________________ E X P E R IM E N T A L P A TH O LO G Y LA B O R A TO R IES, INC. TABLE OF CONTENTS P A T H O L O G Y S U M M A R Y ................................................................................................... Page 1 Q U A L IT Y A S S U R A N C E FIN A L C E R T IF IC A T IO N ..................................................... 8 ADULT SACRIFICE SUM M AR Y IN C ID EN CE TABLES M a le s .................................................................................................................................... 1-1 F e m a le s ............................................................................................................................... I-2 H ISTO PATHO LO G Y IN C ID EN CE TABLES M a le s .................................................................................................................................... 11-1 F e m a le s ............................................................................................................................... II-2 CO R RELATIO N O F G R O SS AND M IC R O SC O PIC FINDING S M a le s .................................................................................................................................... 111-1 F e m a le s ............................................................................................................................... III-3 OFFSPRING SACRIFICE S U M M A R Y IN C ID EN CE TABLES M a le s .................................................................................................................................... IV - 1 F e m a le s ............................................................................................................................... IV -2 H ISTO PATHO LO G Y IN C ID EN CE TABLES M a le s .................................................................................................................................... V -1 F e m a le s .............................................................................................................................. V -3 C O RRELATIO N OF G R O SS AND M ICR O SC O PIC FINDING S M a le s ................................................................................................................................... V I-1 F em a le s .............................................................................................................................. V I-5 -70- Project Number 454-105 PATHOLOGY SUMMARY EPL -71 - E X P E R IM E N T A L P A TH O LO G Y LAB O R A TO R IES, INC. Project Number 454-105 W ILDLIFE INTERNATIONAL, LTD. STUDY NUMBER 454-105 EPL PROJECT NUMBER 212-025 PFOS: A PILOT REPRODUCTION STUDY W ITH THE M ALLARD PATHOLOGY SUMMARY Light m icroscopic exam ination was perform ed on sections o f selected tissu e s fro m adult m ale and fem a le m allards (Anas platyrhynchos) w h ich w e re untreated or which received various concentrations of the test article (P e rflu o ro o cta n e su lfo n ic acid, potassium salt [P F O S ]) in the fe e d fo r six to 19 w eeks. Selected tissues from untreated approxim ately 12-week-old offspring of the adult m allards w ere also exam ined by light m icroscopy. The objective o f this stu d y is to evaluate the effects upon adu lt m allards (Anas platyrhynchos) o f dietary exposure to a test substance over at least a six-w eek period. The experim ental design was as follows: Group * C o ntrol (0 ppm a. i.) 1.8 ppm a. i. PFO S 6 .2 ppm a. i. PFO S 17.6 ppm a. i. P FO S A d u lts M a le s F e m a le s 55 55 55 55 O ffspring ** M ales Fem ales 55 46 46 55 * M allards in th e control and 17.6 ppm a.i. group s w e re tre a te d fo r 19 w eeks; m a lla rd s in th e 1.8 and 6.2 ppm a.i. g roup s w e re tre a te d fo r six w e eks. ** O ffsp rin g in all group s did not receive the te st article. MATERIALS AND METHODS A t the com pletion o f the various study intervals, all adult m allards w ere euthanized, and necropsies w ere perform ed by W ildlife International, Ltd. EPL -72- E X P E R IM E N TA L P A TH O LO G Y LA B O R A TO R IES, INC. Project Number 454-105 Wildlife International, Ltd. Study Number 454-105 Selected offspring were euthanized at approxim ately 12 weeks of age using carbon dioxide gas, and sam ples for histopathological evaluation w ere collected by W ildlife International, Ltd. Selected tissues from adults and offspring w ere fixed in 10% neutral buffered form alin and sent to Experim ental Pathology Laboratories, Inc. (EPL), w h e re th e y w e re em bed ded in paraffin and m ade into hem atoxylin and eosin-stained sections on glass m icroscope slides. The follow ing tissues from adults and offspring w ere exam ined by light m icroscopy as available: liver, gallbladder, proventriculus, kidney, brain, gonad (ovary o r testis), bursa o f Fabricius, and adipose tissue. In som e cases, nonprotocol-required tissues were sectioned along with adjacent protocol-required tissues; these w ere evaluated, and m icroscopic findings w ere recorded at the discretion of the pathologist. All tissues required by protocol are presented in the H istopathology Incidence Tables. M icroscopic findings fo r each tissue exam ined from each m allard are listed in the H istopathology Incidence Tables. M icro sco p ic changes w ere graded one to five depending on severity. N ongradable findings are listed as present (P) and tissues with extensive autolysis are listed as (A). All findings fo r all anim als are sum m arized by sex, age group, and trea tm ent group in the S um m ary Incidence Tables, together w ith the total num ber o f anim als in each group for which the tissues w ere exam ined. A tabulation of gross observations noted at necropsy with the corre spond ing m icroscopic change, if appropriate, is presen ted in the C orrelation o f G ross and M icroscopic Findings tables. T he entries in these table s w ere transcribed from the G ross Necropsy records provided by W ildlife International, Ltd. -2- EPL -73 - E X P E R IM E N TA L P A TH O LO G Y LA B O R ATO RIES, INC. Project Number 454-105 Wildlife International, Ltd. Study Number 454-105 RESULTS All adults and offspring survived to the end of their respective study intervals. No effects considered possibly related to test article (PFOS) adm inistration w ere noted in liver, kidney, gallbladder, proventriculus, ovary, brain, and/or bursa o f Fabricius of male and fem ale adult m allards or their offspring, in the adipose tissue o f adult fem ales and offspring o f both sexes, o r in the testes o f offspring males. Testes of adult m ales exhibited several features m ost consistent with post-reproductive phase regression, a norm al physiological phenom enon, including asperm ia, decreased sperm atogenesis, and/or decreased sem iniferous tu bule diam eter. G rou p differences in the extent o f these fin d in g s w ere evident. Asperm ia, or com plete absence of mature sperm atozoa, was noted only in the testes o f 3/5 17.6 ppm a.i. adult males, w hile decreased sperm atogenesis, characterized by low er num bers o f m ature sperm atozoa than expected in actively re p ro d u ctive testes, w a s seen in 1/5 17.6 ppm a.i. m ales b u t in 4 /5 control adult m ales. W hile sem iniferous tubules from both control and 17.6 ppm a.i. adult m ales had decreased diam eter, the m ean decrease in 17.6 ppm a.i. adu lt m ales w as m ore pronounced than in controls. C orrespondingly, the size range of 17.6 ppm a.i. testes recorded at necropsy was 1.0-2.5 cm, lo w er than the 2.0-3.5 cm range recorded fo r control testes. O verall, these findings are suggestive of m ore advanced testicular regression in the 17.6 ppm a.i. adult m ales com pared to controls. This m ay have been a fortuitous event accentuated by the small group sizes, but the possibility of a te st article effect cannot be entirely ruled out. All offspring testes exam ined were listed as im m ature because they lacked all evidence of sperm atogenesis, had very small sem iniferous tubules with absent or narrow lumens, and had germ inal epithelium with decreased cell layers -3- EPL 74- E X P E R IM E N TA L P A T H O L O G Y LAB O R A TO R IES, INC. Project Number 454-105 Wildlife International, Ltd. Study Number 454-105 (com pared to adult testes). This m orphology was consistent with normal physiological im m aturity in young (approxim ately 12-w eek-old) birds rather than a pathological effect. O varies o f m any control and 17.6 ppm a.i. adult fem ales exhibited fibrosis and/or decreased follicular diam eter. Decreased follicular diam eter corresponded to grossly observed "regressed ovary." Affected follicles were sm a lle r and had shorter stalks th a n follicles in reproductive ly active ovaries. Fibrosis was characterized by a few nodular foci of m ature connective tissue scattered through the ovarian cortex. These ovarian findings were most consistent with early post-reproductive phase regression, a norm al physiological phenom enon. Since the incidence and degree o f these findings w ere sim ilar in control and 17.6 ppm a.i. fem ale adult groups, they w e re considered incidental and unrelated to treatm ent. O varies of all offspring fem ales w ere characterized by num erous oocytes and very small, nonpedunculated follicles com pletely em bedded in a prom inent ovarian cortex. This m o rp hology is consistent w ith norm al physiological im m atu rity in young (a pproxim ately 12-w eek-old) birds rath er than a pathological effect. A dipose tissue o f adults and offspring consisted o f sheets o f large white adipocytes with single large lipid droplets, am ong which w ere often scattered a few adipocytes which contained m ultiple, sm aller, discrete vacuoles. W hile alm ost all sam ples o f adipose tissue exhibited a few o f these sm aller adipocytes, m icrovesiculation was diagnosed only w hen the num bers of these cells were increased. M icrovesiculation w as noted in both control and treated adult and offspring m allards, and w as usually a very subtle, m inim al finding. A clearly increased incidence w as noted only in 17.6 ppm a.i. a d u lt m ales com pared to control adult males. This increase m ay have been a fortuitous event accentuated -4- EPL -75- E XP E R IM E N TA L P A TH O LO G Y LAB O R ATO RIES, INC. Project Number 454-105 Wildlife International, Ltd. Study Number 454-105 by sm a ll g ro u p sizes o r possible variab ility in adipose tissue sam pling sites, but the possibility of a test article effect cannot be entirely ruled out. Incidences o f adipose tissue m icrovesiculatio n in m ale and fe m a le offspring did not exhibit clear, dose-related increases, and w ere considered incidental and unrelated to treatm ent. S e ve ra l add itio n a l m ic ro sco p ic findings (describe d below ) w e re n oted in adult and/or offspring m allards from control and treated groups, including m onon uclea r cell infiltrates in various tissues; kidney m ineralization; ureteral lam ina propria cyst; and involution of the bursa of Fabricius. Incidences and m ean severity w ere sim ilar when groups from each generational cohort were com pared with each other. For these reasons, these findings were considered incidental and unrelated to test article (PFOS) adm inistration. M ononuclear cell infiltrates in various tissues consisted o f discrete fo ci of sm all lym phocytes with occasional m onocytes and m acrophages. Scattered clusters o f coarsely to finely dark basophilic m aterial in the renal cortex denoted kidney m ineralization. In one 6.2 ppm a.i. offspring fem ale, a section o f ureter fortuitously sectioned along with kidney exhibited a single lam ina propria cyst lined by flattened epithelium and filled with am orphous, pale basophilic m aterial. O nly one adult m allard (a control m ale) had a bursa o f Fabricius available for m icroscopic evaluation. The bursa in this individual exhibited the age-related involution expected in adult birds. T he bursa of Fabricius w as present and u n rem arkable in all m ale and fem a le offspring. A d d itio n a l fin d in g s w ere also noted in the liver o f adults a n d /o r offspring. Liver pigm ent deposition consisted of dust-like, brown to golden-brow n pigm ent granules present in hepatocyte cytoplasm , K upffer cells, a nd /or periportal areas; the identity of the pigm ent was not determ ined, but m ay have been related to bile. The liver of one adult control fem ale exhibited am yloid deposition consisting -5- EPL -76- E XPER IM EN TA L P A TH O LO G Y LAB O R ATO RIES, INC. Project Number 454-105 Wildlife International, Ltd. Study Number 454-105 of am orphous, pale bluish-violet m aterial which filled and distended the hepatic sinusoids, and com pressed adjacent hepatic cords. M inimal focal hepatocellular d e g e n e ra tio n /n e c ro s is occu rre d only in a fe w control and tre a te d offspring. It co n siste d o f a fe w sm all discre te foci in w hich hepatocytes w e re pale to hyperosinophilie, had indistinct outlines, and fragm ented or condensed nuclei. H epatocellular degeneration/necrosis w as considered incidental and unrelated to treatm ent. In adults and offspring, liver fatty change denoted variably sized, sharply dem arcated, cle a r vacuoles (consisten t w ith fa t accum ulation) in the hepatocellular cytoplasm . Liver hepatocellular vacuolization denoted m ultiple, usually sm all, confluent, poorly d em arcated vacuoles w hich often resulted in a "lace-like" appearance o f the hepatocellular cytoplasm . W hen present in m ost hepatocytes, these changes w e re designated "diffuse," and w hen present in a few scattered areas, they w ere designated as "focal." Hepatocellular fatty change occurred sporadically at low incidences and sim ila r severity in control and tre a te d adults and offspring, and w as co n sidered incidental. In adults and offspring, hepatocellular vacuolization had sim ilar incidences when control and treated groups of each sex were com pared, but m arginal, not alw ays dose-related increases in severity w ere som etim es noted. It seem s m ore likely that these very m inor differences w ere m ore related to biological variation accentuated by sm all group sizes rather than being test article effects. CONCLUSIONS AND SUMMARY No lesions considered possibly related to test article (PFOS) adm inistration w ere noted in liver, kidney, proventriculus, gallbladder, ovary, brain, and/or bursa o f Fabricius, brain o f adult m ale and fem ale m allards or their -6- EPL -77- E X P E R IM E N TA L P A TH O LO G Y LAB O R ATO RIES, INC. Project Number 454-105 Wildlife International, Ltd. Study Number 464-105 offspring, in the adipose tissue o f a du lt fem a les and offspring o f both sexes, o r in the testes o f offspring males. Testes of adult m ales exhibited several features m ost consistent with post-reproductive phase regression, a norm al physiological phenom enon, including asperm ia, decreased sperm atogenesis, and/or decreased sem iniferous tu bule diam eter. G rou p differences in the extent o f th e se fin d in g s w e re evident, w ith aspe rm ia occurring only in the high dose (17.6 ppm a.i.) a d u lt m ales, and the deg re e o f d ecrea se in sem inife ro us tubule dia m e te r being g e n e ra lly m ore pron o u n ce d in 17.6 ppm a.i. adult m ales than in control adult m ales. O verall, these findings are suggestive o f m ore advanced testicula r regression in the 17.6 ppm a.i. adu lt m ales com pared to controls. A d u lt m ales in th e 17.6 ppm a.i. group also exhibited clearly increased incidences of adipose tissue m icrovesiculation. These testicula r and adipose tissue findings in 17.6 ppm a.i. adult m ales m ay have been fortuitous events accentuated by the sm all group sizes, but the possibility that they w ere related to test article adm inistration cannot be entirely ruled out. T he fe w o th e r c han ges in various tissues o f adult a n d /o r offsp ring " m allards from control and treated groups were considered incidental and unrelated to test article (PFO S) adm inistration. M M G /lcp M ARG AR ITA M. G RUEBBEL, DVM, PhD, Diplom ate, A C VP Veterinary Pathologist 1/3-$/>1 -7- EPL -78- E X P E R IM E N T A L P A T H O L O G Y LA B O R A TO R IES, INC. Project Number 454-105 Q U A LITY A SS U R A N C E FINAL C ER TIFIC A TIO N Study Title: PFO S: A Pilot Reproduction Study with the Mallard Client Study: 4 5 4 -1 0 5 EPL Project Coordinator: Dr. Margarita M. Gruebbel EPL Project Number: 212-025 EPL Pathologist: Dr. Margarita M. Gruebbel T h e following aspects o f this study w ere inspected by the Quality A ssurance Unit of Experimental Pathology Laboratories, Inc. Dates inspections were performed and findings reported to the EPL Project Coordinator and M an ag em en t are indicated below. Area Inspected _____________________________ D ates_________________ __________Inspection_______ ______ Reporting EPL Project Sheets Project Setup Histology Setup Data Review Draft Report Final Report 9/11/00; 10/3/00 9/28/00; 10/3/00 10/3/00 11/13,15/00 12/13,14,27/00 1/29/01 9/11/00; 10/3/00 9/28/00; 10/3/00 10/3/00 11/13,15/00 12/14,27/00 1/29/01 D ate of last quarterly facility inspection y IC b tA EPL Quality A ssuranicce U nl am tf a l o t Date Form No. 6-2 (7/2/99) -8- -79- Project Number 454-105 SUMM ARY INCIDENCE TABLES AD U LT SACRIFICE 454-105 A dult S a c rific e Male M a lla rd -80SUMMARY INCIDENCE TABLE ADIPOSE TISSUE (NO. EXAMINED) I n f i l t r a t e , M ononuclear C ell Mic ro v e s i c u l a t i o n BRAIN (NO. EXAMINED) BURSA OF FABRICIUS (NO. EXAMINED) In v o lu tio n 3ALLBLADDER (NO. EXAMINED) I n f i l t r a t e , M ononuclear C e ll KIDNEY (NO. EXAMINED) I n f i l t r a t e , M ononuclear C e ll M in eralization LIVER (NO. EXAMINED) Am yloid D e p o sitio n H e p a to c y te (s), F a tty Change, D iffuse H e p ato cy te(8 ), F a tty Change, Focal H ep ato cy te(s), V acuolization, D iffuse I n f i l t r a t e , M ononuclear C e ll Pigm ent D ep o sitio n PROVENTRICULUS (NO. EXAMINED) I n f i l t r a t e , M ononuclear C e ll TESTIS (NO. EXAMINED) A sperm ia S em iniferous Tubules, D ecreased D iam eter Sperm atogenesis, D ecreased GROUP CONTROL (5) 1 (5) GROUP 17.6 (5) 5 (5) (1) 1 (5) 4 (5) (5) 1 1 2 5 4 (5) 5 (5) 4 3 (5) 3 (5) 2 (5) 4 5 2 (5) 5 (5) 3 5 .1 Project Number 454-105 1-1 EPL Experimental Pathology Laboratories, Inc. 454-105 A dult S a c rific e Fem ale M allard -81 SUMMARY INCIDENCE TABLE ADIPOSE TISSUE (NO. EXAMINED) I n f i l t r a t e , M ononuclear C ell M icrovesiculation BRAIN (NO. EXAMINED) BURSA OF FABRICIUS (NO. EXAMINED) In v o lu tio n 3ALLBLADDER (NO. EXAMINED) I n f i l t r a t e , M ononuclear C ell KIDNEY (NO. EXAMINED) I n f i l t r a t e , M ononuclear C ell M ineralization LIVER (NO. EXAMINED) Amyloid D e p o sitio n H e p a to c y te (s), F a tty Change, D iffuse H e p a to c y te (s), F a tty Change, Focal H epatocyte(s ), V acuolization, D iffuse I n f i l t r a t e , M ononuclear C ell Pigm ent D ep o sitio n OVARY (NO. EXAMINED) F ib ro sis F o llic le s , D ecreased D iam eter PROVENTRICULUS (NO. EXAMINED) I n f i l t r a t e , M ononuclear C ell GROUP CONTROL (5) 1 2 (5) GROUP 17.6 (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. -82- Project Number 454-105 HISTO PATH O LO G Y INCIDENCE TABLES ADULT SACRIFICE -83- Project Number 454-105 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 17.6 454-105 A dult S a c rific e Male M a lla rd jj i M A l ADIPOSE TISSUE I n f i l t r a t e , M ononuclear C e ll M icrovesiculation 1 1 11 1 99999 6 6 66 6 13579 X XXX 1 11111 9 999 9 99999 13579 11211 BRAIN XXXXX XXXXX BURSA OF FABRICIUS In v o lu tio n NNN N 4 NNNNN GALLBLADDER I n f i l t r a t e , M ononuclear C e ll X 1211 XX 11 1 KIDNEV I n f i l t r a t e , M ononuclear C e ll M ineralization Xir Xir Xm Xm Xir m Xm X m Xm ii LIVER Amyloid D ep o sitio n H ep a to c y te (s), F atty Change, D iffuse H ep ato cy te(s), F a tty Change, Focal H epatocyt e ( s ), V a c u o liz a tio n , D iffuse I n f i l t r a t e , M ononuclear C e ll Pigm ent D ep o sitio n PROVENTRICULUS I n f i l t r a t e , M ononuclear C e ll 1 1 22 11112 211 2 11111 2 32 2 111i 2 i2 111i 1 TESTIS Asperm ia Sem iniferous Tubules, D ecreased D iam eter Sperm atogenesis, D ecreased m m m Xm m 22 1 42 3 4. mmmmm p PP 14552 2 1 E PL Experimental Pathology Laboratories, Inc. II-l 1 Key : X -N o t Remarkable H-No S ection I-In c om p lete A -A u to ly s 1s M in im a l 2 -sllg h t/m lId 3-moderate M o d e r a te ly severe 5-severe/h1gh P-Present B-Ben1gn H-Mallgnant m-1ss1ng one paired organ u-unscheduled sac./death -84- Project Number 454-105 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 17.6 454-105 A dult S a c rific e Female M allard * N i M A l ADIPOSE TISSUE I n f i l t r a t e , M ononuclear C ell M icrovesiculation 11111 99 9 99 66 66 7 2 46 8 0 XX 1 11 11112 99990 99990 2 468 0 XX 11 11 BRAIN XXXXX X XX X X BURSA OF FABRICIUS In v o lu tio n NNNNN NNNNN GALLBLADDER I n f i l t r a t e , M ononuclear C ell KIDNEY I n f i l t r a t e , M ononuclear C ell M ineralization 11111 Xmmmm i i 1i X 1 111 mmm 1 i 21 1 LIVER Amyloid D e p o sitio n H e p a to c y te (s), F atty Change, D iffuse H e p a to c y te (s), F a tty Change, Focal H epatocyte(s), V acuolization, D iffuse I n f i l t r a t e , M ononuclear C ell Pigm ent D ep o sitio n OVARY F ibrosis F o llic le s , D ecreased D iam eter 2 1 i 23 1 21i 21 X 3 31 3 333 3332 11112 1 11 32 333 PROVENTRICULUS I n f i l t r a t e , M ononuclear C ell 14322 X 41 11 EPL II-2 Experimental Pathology Laboratories, Inc. _ _ J ____ _ Key iX - N o t Reeiarkeble N-Ho S ection I -I n c m p le te A -A u to 1y s 1s 1-m ln lM l 2-sl1ght/m 11d 3-moderate 4-ioderately severe 5-severe/h1gh P-Present B-Benlgn H-H alignant ie-m1ss1ng one paired organ u-unscheduled sac ,/death -85- Project Number 454-105 CO RRELATION O F GROSS AN D M ICRO SCO PIC FINDINGS ADULT SACRIFICE 454-105 Adult Sacrifice Species: M a l l a r d CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex: M ales Group Identification: CONTROL - S a c r i f i c e d Animal Number 1961 Client Topography / Site EXTERNAL TESTES 1963 EXTERNAL EXTERNAL TESTES 1965 EXTERNAL TESTES 1967 1969 EXTERNAL EXTERNAL TESTES Client Gross Observations M olting f l i g h t fe a th e rs " 3 .0 cm Bumble f o o t l e f t f o o t M olting f lig h t fe a th e rs ~ 3 .5 cm Bumble f o o t b o th f e e t " 3 .5 cm Bumble f o o t r i g h t f o o t M olting f lig h t fe a th e rs ~ Z . O cm Microscopic Observations do Comment R e q u ire d Sem iniferous T ubules, D ecreased D iam eter (TESTIS) No Comment R e q u ire d No Comment R e q u ir e d Sem iniferous T ubules, D ecreased D iam eter (TESTIS) No Comment R e q u ire d Sem iniferous T ubules, D ecreased D iam eter (TESTIS) No Comment R e q u ire d No Comment R e q u ire d Sem iniferous T ubules, D ecreased D iam eter (TESTIS) Project Number 454-105 III-l 454-105 Adult Sacrifice Species: M a l l a r d CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex: M ales Group Identification: 1 7 .6 - S a c r i f i c e d Animal Number 1991 1993 Client Topography / Site EXTERNAL EXTERNAL TESTES 1995 EXTERNAL 6 1 TRACT TESTES 1997 EXTERNAL TESTES 1999 TESTES Client Gross Observations Bumble f o o t b o th f e e t Bumble f o o t b o th f e e t " 1 .5 cm Bumble f o o t b o th f e e t Mass o f fe e d im p acted u n d e r th e tongue " 2 .0 cm Bumble f o o t b o th f e e t " 1 .0 cm " 2 .5 cm Microscopic Observations Jo Comment R e q u ir e d Jo Comment R e q u ire d Sem iniferous T ubules, D ecreased D iam eter (TESTIS) Jo Comment R e q u ire d (Jo Comment R e q u ir e d Sem iniferous T ubules, D ecreased D iam eter (TESTIS) No Comment R e q u ire d Sem iniferous T ubules, D ecreased D iam eter (TESTIS) Sem iniferous T ubules, D ecreased D iam eter (TESTIS) S Project Number 454-105 III-2 454-105 A dult S a c rific e Species: M a l l a r d CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex: F em ales Group Identification: CONTROL - S a c r i f i c e d Animal Number 1962 Client Topography / Site EXTERNAL EXTERNAL OVARY 1966 1968 1970 EXTERNAL OVARY OVARY REPRODUCTIVE EXTERNAL EXTERNAL OVARY Client Gross Observations IJumble f o o t b o th f e e t lo ltin g flig h t feathers Regressed ovary Bumble f o o t b o th f e e t Regressed ovary R egressed ovary Egg rem nant Bumble f o o t b o th f e e t M olting f lig h t fe a th e rs R egressed ovary Microscopic Observations i (o Comment R e q u ir e d lo Comment R e q u ire d !f o l l i c l e s , D e c r e a s e d D ia m e te r ; F ib ro sis do Comment R e q u ir e d F o llic le s , D ecreased D iam eter F o llic le s , D ecreased D iam eter No Comment R e q u ir e d No Comment R e q u ir e d No Comment R e q u ir e d F o llic le s , D ecreased D iam eter Project Number 454-105 III-3 454-105 Adult Sacrifice Species: M a l l a r d CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex: F em ales Group Identification: 1 7 .6 - S a c r i f i c e d Animal Number 1992 1994 1996 1998 2000 Client Topography / Site EXTERNAL RESPIRATORY ABDOMINAL CAVITY OVARY EXTERNAL OVARY EXTERNAL OVARY EXTERNAL GENERAL CONDITION OVARY OVARY Client Gross Observations fe ltin g flig h t feath er A ir s a c c u litis Egg y o lk p e r i t o n i t i s ( s l i g h t ) R egressed ovary M olted f l i g h t fe a th e rs Ovary re g re sse d Bumble f o o t b o th f e e t R egressed ovary M olting f lig h t fe a th e rs A bnorm ally sm all R egressed ovary R egressed ovary Microscopic Observations to Comment R e q u ir e d [to Comment R e q u ir e d No Comment R e q u ir e d F o llic le s , D ecreased D iam eter So Comment R e q u ir e d F o llic le s , D ecreased D iam eter No Comment R e q u ir e d F o llic le s , D ecreased D iam eter No Comment R e q u ir e d No Comment R e q u ir e d F o llic le s , D ecreased D iam eter F o llic le s , D ecreased D iam eter Project Number 454-105 III-4 -90- Project Number 454-105 SU M M AR Y INCIDENCE TABLES O FFSPRING SACRIFICE 454-105 O ffspring S a crifice Male M allard -91 SUMMARY INCIDENCE TABLE Project Number 454-105 ADIPOSE TISSUE (NO. EXAMINED) I n f i l t r a t e , M ononuclear C ell M icrovesiculation BRAIN (NO. EXAMINED) I I n f i l t r a t e , M ononuclear C ell 1 BURSA OF FABRICIUS (NO. EXAMINED) GALLBLADDER (NO. EXAMINED) I n f i l t r a t e , M ononuclear C ell 1 KIDNEY (NO. EXAMINED) I I n f i l t r a t e , M ononuclear C ell 1 LIVER (NO. EXAMINED) I H epatocytes, I D egeneration/N ecro8is, Focal 1 H epatocytes, F a tty Change, I D iffuse H epatocytes, F a tty Change, Focal H epatocytes, V acuolization, I D iffuse I I n f i l t r a t e , M ononuclear C ell I Pigm ent D ep o sitio n 1 jPROVENTRICULUS (NO. EXAMINED) 1 I n f i l t r a t e , M ononuclear C ell 1 fTESTIS (NO. EXAMINED) 1 Immature I n f i l t r a t e , M ononuclear C ell GROUP CONTROL (5) 1 (5) GROUP 1.8 (4) (4) 1 GROUP 6.2 (4) (4) GROUP 17.6 (5). 2 (5) (5) (5) 3 (5) 4 (5) 1 4 5 (5) 3 (4) 3 2 (4) (4) 3 (4) 4 (4) 1 4 4 (4) 4 (4) 4 2 (4) (4) 2 (4) 2 (4) 1 1 3 3 1 (4) 4 (4) 4 (5) (5) 2 (5) 4 (5) 1 1 4 4 (5) 5 (5) 5 2 1 1 IV-1 E PL Experimental Pathology Laboratories, Inc. 454-105 O ffspring S acrifice Female M allard -92SUMMARY INCIDENCE TABLE Project Number 454-105 ADIPOSE TISSUE (NO. EXAMINED) I n f i l t r a t e , M ononuclear C ell M icrovesiculation BRAIN (NO. EXAMINED) I n f i l t r a t e , M ononuclear C ell BURSA OF FABRICIUS (NO. EXAMINED) GALLBLADDER (NO. EXAMINED) I n f i l t r a t e , M ononuclear C ell KIDNEY (NO. EXAMINED) I n f i l t r a t e , M ononuclear C e ll LIVER (NO. EXAMINED) H epatocytes, D egeneration/N ecrosis, Focal H epatocytes, F atty Change, D iffuse H epatocytes, F atty Change, Focal H epatocytes, V acuolization, D iffuse I n f i l t r a t e , M ononuclear C ell Pigm ent D ep o sitio n OVARY (NO. EXAMINED) Im m ature I n f i l t r a t e , M ononuclear C e ll PROVENTRICULUS (NO. EXAMINED) I n f i l t r a t e , M ononuclear C ell URETER (NO. EXAMINED) Lamina P ro p ria , C y st(s) GROUP CONTROL (5) (5) GROUP 1.8 (6) 1 2 (6) (5) (6) (5) (6) 25 (5) (6) 36 (5) (6) 1 1 35 46 (5) (6) 56 1 (5) (6) 45 GROUP 6 .2 (6) 2 (6) (6) (6) 5 (6) 6 (6) 4 6 1 (6) 6 1 (6) 5 (1) 1 GROUP 17.6 (5) 2 (5) (5) (5) 1 (5) 3 (5) 5 5 (5) 5 (5) 5 IV-2 EPL Experimental Pathology Laboratories, Inc. -93 - Project Number 454-105 HISTO PATHO LO G Y INCIDENCE TABLES O FFSPRING SACRIFICE -94- Project Number 454-105 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 1.8 GROUP 6.2 454-105 O ffspring S acrifice Male M a llard n* i H A l ADIPOSE TISSUE I n f i l t r a t e , M ononuclear C ell M icrovesiculation 22222 33333 00011 5 78 46 XXXX 1 2222 3333 2233 3814 XXXX 2222 3333 3344 8913 XXXX BRAIN I n f i l t r a t e , M ononuclear C ell XXXXX X XX 1 XXXX BURSA OF FABRICIUS XXX X X XXXX XXX X GALLBLADDER I n f i l t r a t e , M ononuclear C ell AX 11 1 A 1 11 AA 11 KIDNEY I n f i l t r a t e , M ononuclear C ell n m Xm m m L1 i2 mmmm 1111 m m Xir Sir 12 LIVER H epatocytes, D egeneration/N ecrosis, Focal H epatocytes, F atty Change, D iffuse H epatocytes, F atty Change, Focal H epatocytes, V acuolization, D iffuse I n f i l t r a t e , M ononuclear C e ll Pigm ent D ep o sitio n 1 2 2i 2 111i 1 1 2122 1111 1 2 322 111 1 PROVENTRICULUS I n f i l t r a t e , M ononuclear C ell XX 11i 1112 1111 TESTIS Im m ature I n f i l t r a t e , M ononuclear C e ll mmm N PPP 11 PPPP 12 PPPP 1 | _____________ :________________ . EPL V -l Experimental Pathology Laboratories, Inc. Key tX -B o t RsM rkable N-No Section I-Incom plete A -A u to ly s ls lu In liM l 2-sl1ght/in1ld 3noderate 4noderatTy severe 5-severe/hlgh P-Present B-Beelgn H-Malignant Hissing one paired organ unscheduled sac./d eath 454-105 O ffspring S ac rific e Male M allard -95- Project Number 454-105 HISTOPATHOLOGY INCIDENCE TABLE GROUP 17.6 ADIPOSE TISSUE I n f i l t r a t e , M ononuclear C ell Mic ro v e 8i c u l a t io n BRAIN I n f i l t r a t e , M ononuclear C ell BURSA OF FABRICIUS GALLBLADDER I n f i l t r a t e , M ononuclear C ell KIDNEY I n f i l t r a t e , M ononuclear C ell LIVER H epatocytes, D egeneration/N ecrosis, Focal H epatocytes, F a tty Change, D iffuse H epatocytes, F a tty Change, Focal H epatocytes, V acuolization, D iffuse I n f i l t r a t e , M ononuclear C e ll Pigm ent D ep o sitio n PROVENTRICULUS I n f i l t r a t e , M ononuclear C ell TESTIS Im m ature I n f i l t r a t e , M ononuclear C ell 22 33 55 57 XX AA 11 m m m m Xn PP 11 EPL Experimental Pathology Laboratories, Inc. Key ! X -N o t Remarkable N-No S ection I-In c om p lete A -A u to ly s 1s l-1n1m al 2 -s U g h t/ n lld 3-moderate 4-noderately severe 5-severe/M gh P-Present B-Ben1gn M-Ma11gnant -m is s in g one paired organ u-unscheduled sac ./d eath -96- Project Number 454-105 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 1.8 GROUP 6 .2 E PL Experimental Pathology Laboratories, Inc. V-3 Key : X -N o t Remarkable N-No S ection I-Inc om plete A -A u to ly s is l u l n l m l 2-sl1gh t/m 1ld 3-moderate 4-moderately severe 5-severe/h1gh P-Present B-Ben1gn M-Mal1gnant m-mlsslng one paired organ u-unscheduled sac./d eath 454-105 O ffspring S a c rific e Female M allard -97- Project Number 454-105 HISTOPATHOLOGY INCIDENCE TABLE GROUP 17.6 ADIPOSE TISSUE I n f i l t r a t e , M ononuclear C ell M icrovesiculation BRAIN I n f i l t r a t e , M ononuclear C ell BURSA OF FABRICIUS GALLBLADDER I n f i l t r a t e , M ononuclear C ell KIDNEY I n f i l t r a t e , M ononuclear C ell LIVER H epatocytes, D egeneration/N ecrosi8, Focal H epatocytes, F a tty Change, D iffuse H epatocytes, F a tty Change, Focal H epatocytes, V acuolization, D iffuse I n f i l t r a t e , M ononulear C ell Pigm ent D ep o sitio n OVARY Im mature I n f i l t r a t e , M ononuclear C e ll PROVENTRICULUS I n f i l t r a t e , M ononuclear C ell XX 31 m m Xir m Xir 11 EPL V-4 Experimental Pathology Laboratories, Inc. Key ; X-H ot Renarkeble N-No Section I-In c o n p le te A -A u to ly s1s lm ln lM l 2 -s H o h t/ 1 li Snoderete 4no dera te ly severe 5*severe/M gh P-Present B-Benign M-MaUgnant m-m1ss1ng one paired organ u-unscheduled s ac ./d e ath -98- Project Number 454-105 CO RR ELATION O F GROSS AN D M ICRO SCO PIC FINDINGS OFFSPRING SACRIFICE 4 5 4 -1 0 5 Offspring Sacrifice Species: M a l l a r d CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex: M ales Group Identification: CONTROL - S a c r i f i c e d Animal Number 2305 2308 2314 2316 Client Topography / Site EXTERNAL EXTERNAL EXTERNAL EXTERNAL Client Gross Observations Itumble f o o t b o th f e e t Jumble f o o t b o th f e e t Jumble f o o t b o th f e e t Bumble f o o t b o th f e e t Microscopic Observations 1lo Comment R e q u ir e d Jo Comment R e q u ire d Jo Comment R e q u ire d Jo Comment R e q u ire d Project Number 454-105 .. VI-1 4 5 4 -1 0 5 Offspring Sacrifice Species: M a l l a r d CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex: M ales Group Identification: 1 .8 - S a c r i f i c e d Animiti Number 2323 Client Topography / Site EXTERNAL Client Gross Observations Jumble f o o t b o th f e e t Microscopic Observations Io Comment R e q u ire d Project Number 454-105 ' VI-2 454-105 O ffspring S acn fx ce Species: M a l l a r d CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex: M ales Group Identification: 6 .2 - S a c r i f i c e d Animal Number 2338 2339 O ient Topography / Site EXTERNAL EXTERNAL Oient Gross Observations IJumble f o o t b o th f e e t Jumble f o o t b o th f e e t Microscopic Observations Jo Comment R e q u ir e d Jo Comment R e q u ire d Project Number 454-105 VI-3 4 5 4 -1 0 5 Offspring Sacrifice Species: M a l l a r d CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex: M ales Group Identification: 1 7 .6 - S a c r i f i c e d Animal Number 2354 Client Topography / Site EXTERNAL Client Gross Observations Ijum ble f o o t b o th f e e t Microscopic Observations o Comment R e q u ire d Project Number 454-105 VI-4 454-105 Offspring Sacrifice Species: M a l l a r d CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex: F em ales Group Identification: 1 .8 - S a c r i f i c e d Animal Number 2322 2325 2332 2333 Client Topography / Site ISXTERNAT. iXTERNAL EXTERNAL EXTERNAL Client Gross Observations Bumble f o o t b o th f e e t Bumble f o o t b o th f e e t Bumble f o o t b o th f e e t Bumble f o o t b o th f e e t Microscopic Observations ilo Comment R e q u ir e d 1Jo Comment R e q u ir e d Jo Comment R e q u ire d Jo Comment R e q u ire d Project Number 454-105 1 VI-5 4 5 4 -1 0 5 Offspring Sacrifice Species: M a l l a r d CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex: F em ales Group Identification: 6 .2 - S a c r i f i c e d Animai Number 2335 2336 2342 2344 Client Topography / Site EXTERNAL EXTERNAL EXTERNAL EXTERNAL Client Gross Observations ISumble f o o t b o th f e e t IJumble f o o t b o th f e e t Jumble f o o t l e f t f o o t Sumble fo o t b o th f e e t Microscopic Observations tlo Comment R e q u ire d to Comment R e q u ire d to Comment R e q u ire d . to Comment R e q u ire d Project Number 454-105 VI-6 454-105 Offspring Sacrifice Species: M a lla r d CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex: F em ales Group Identification: 1 7 .6 - S a c r i f i c e d Animal Number 2347 2348 Client Topography / Site EXTERNAL EXTERNAL ABDOMINAL CAVITY EXTERNAL Client Gross Observations Picked and broken fe a th e rs General unkept co n d itio n R etained yolk sac S lig h t bumble fo o t b o th f e e t Microscopic Observations Jo Comment R e q u ir e d Jo Comment R e q u ire d Jo Comment R e q u ire d No Comment R e q u ir e d H- o Project Number 454-105 VI-7 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 /D 1 201 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 per hen per day H -* o ON Project Number 454-105 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 Mean SD 11 2 3 15 3 3 1Eggs laid per hen per day 1.8 ppm a.i. Weeks 34 5 6 Total E /H /D 1 776 1 21 0.50 40 0 0 6 0.14 7 7 7 7 40 0.95 00 0 0 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 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 Mean 1 2 SD 2 3 1Eggs laid per hen per day 6.2 ppm a.i. Weeks 3 4 5 6 Total E /H /D 1 00 0 0 0 0.00 7 6 8 6 32 0.76 00 0 0 1 0.02 00 0 0 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 Project Number 454-105 Appendix X Table 4 Egg Production (eggs laid/hen/week) from a Mallard Pilot Reproduction Study with PFOS 17.6 ppm ai. 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 216 5 6 7 6 6 2 217 0 4 5 7 6 1 218 7 4 7 6 2 7 219 0 3 7 5 6 5 220 3 5 7 7 4 7 32 0.76 0 0 0 0 0 0 0 0 0 0 0 0 0 0 23 0.55 2 7 7 7 7 7 7 4 3 4 0 0 0 0 33 0.79 1 7 4 3 0 0 0 0 0 0 0 0 0 0 26 0.62 0 0 0 0 0 0 0 0 0 0 0 0 0 0 33 0.79 7 1 4 7 1 0 0 0 0 0 0 0 0 0 0 0.00 55 0.59 15 0.16 0 0.00 20 0.21 Total Mean SD 15 22 33 31 24 22 34 76 54 3 11123 147 29 5 10 15 15 17 8 7 7 4 3 4 0 0 0 0 0.70 2 3 3 3 2 1 1 1 1 1 0 0 0 0 0.11 3 4 3 4 3 3 3 2 1 2 0 0 0 0 90 18 0.19 23 0.24 1Eggs laid per hen per day Project Number 454-105 Appendix XI Page 1 Reproductive Performance by Pen from a Mallard Pilot Reproduction Study with PFOS Table 1 Reproductive Data (Count) By Pen Parameter ______ Control (0 ppm a.i.)_____ Pais 201 202 203 204 205 Total ________ 1.8ppma.i.________ 206 207 208 209 210 Total ________ 6.2 ppm a.i.________ 211 212 213 214 215 Total ________17,6 ppm a.i.________ 216 217 218 219 220 Total Eggs Laid Eggs Cracked Eggs Set Viable Embryos Live 3-Wk Embryos Hatchlings Offspring Survivors 5 7 6 0 6 24 00000 0 5 7 6 0 6 24 3 7 6 0 6 22 3 7 6 0 6 22 2 7 6 0 6 21 2 7 6 0 6 21 4 0 7 0 7 18 00001 1 4 0 7 0 6 17 4 0 7 0 6 17 4 0 7 0 6 17 3 0 5 0 6 14 3 0 5 0 5 13 0 8 0 0 4 12 00000 0 0 8 0 0 4 12 0 8 0 0 4 12 0 8 0 0 4 12 0 8 0 0 4 12 0 8 0 0 4 12 7 5 3 5 4 24 0 0 0 10 1 7 5 3 4 4 23 6 5 3 4 3 21 6 5 3 4 3 21 6 4 2 4 2 18 5 4 2 4 2 17 Project Number 454-105 Il Replicate 1 2 3 4 5 Total Mean SD Control (0 ppm a.i.) Eggs Eggs/ Laid Days Hen/Day 5 7 0.71 7 7 1.00 6 7 0.86 0 7 0.00 6 7 0.86 24 5 0.69 3 0.40 Replicate Control (0 ppm a.i.)______ Eggs Eggs Cracked Laid % 1 2 3 4 5 Total Mean SD 05 07 06 00 06 0 24 05 03 0 0 0 0 0 0 Appendix XI Page 2 Reproductive Performance by Pen from a Mallard Pilot Reproduction Study with PFOS Table 2 Eggs Laid / Hen / Day 1.8 ppm ai. Eggs Eggs/ Laid Days Hen/Day 6.2 ppm ai. Eggs Eggs/ Laid Days Hen/Day 4 7 0.57 0 7 0.00 7 7 1.00 0 7 0.00 7 7 1,00 18 4 0.51 4 0.50 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 3 Eggs Cracked / Eggs Laid (%) _________1.8 ppm ai._________ Eggs Eggs Cracked Laid % 040 00 - 07 0 00 - 1 7 14 1 18 04 04 5 8 _________ 6,2 ppm a.i._________ Eggs Eggs Cracked Laid % 00 08 00 00 04 0 12 02 04 0 - 0 0 0 17.6 ppm ai. Eggs Eggs/ Laid Days Hen/Day 7 7 1.00 5 7 0.71 3 7 0.43 5 7_ 0.71 4 7 0.57 24 5 0.69 1 0.21 17.6 ppm ai. Eggs Eggs Cracked Laid 07 05 03 15 04 1 24 05 01 % 0 0 0 20 0 4 9 Project Number 454-105 Control (0 ppm a.i.) Viable Eggs Replicate Embryos Set % 1 2 3 4 5 Total Mean SD 3 5 60 7 7 100 6 6 100 00 6 6 100 22 24 4 5 90 3 3 20 Replicate Control (0 ppm a.i.) Live 3-Week Viable % 1 2 3 4 5 Total Mean SD 3 3 100 7 7 100 6 6 100 00 - 6 6 100 22 22 4 4 100 330 Appendix XI Page 3 Reproductive Performance by Pen from a Mallard Pilot Reproduction Study with PFOS Table 4 Viable Embryos / Eggs Set (%) 1.8 ppma.i. Viable Eggs Embryos Set 44 00 77 00 66 17 17 33 33 % 100 - 100 - 100 100 0 6.2 ppm a.i. Viable Eggs Embryos Set 00 88 00 00 44 12 12 22 44 % 100 - 100 100 0 Table 5 Live 3-Week Embryos / Viable Embryos (%) 1.8ppma.i. Live 3-Week Viable 44 00 77 00 66 17 17 33 33 % 100 - 100 - 100 100 0 6.2 ppm ai. Live 3-Week Viable 00 88 00 00 44 12 12 22 44 % 100 - 100 100 0 17.6 ppm ai. Viable Eggs Embryos Set 67 5.5 33 44 34 21 23 45 12 % 86 100 100 100 75 92 11 17.6 ppm a.i. Live 3-Week Viable % 6 6 100 5 5 100 3 3 100 4 4 100 3 3 100 21 21 4 4 100 1 10 Project Number 454-105 Replicate 1 2 3 4 5 Total Mean SD Control (0 ppm ai.) Live Hatch 3-Week % 2 3 67 7 7 100 6 6 100 00 6 6 100 21 22 4 4 92 3 3 17 Control (0 ppm ai.) Offspring Replicate Surv. Hatch % 1 2 3 4 '5 Total Mean SD 2 2 100 7 7 100 6 6 100 00 - 6 6 100 21 21 4 4 100 330 Appendix XI Page 4 Reproductive Performance by Pen from a Mallard Pilot Reproduction Study with PROS Table 6 Hatchlings / Live 3-Week Embryos (%) 1.8 ppma.i. Live Hatch 3-Week 34 00 57 00 66 14 17 33 33 % 75 - 71 - 100 82 16 6.2 ppm ai. Live Hatch 3-Week 00 88 00 00 44 12 12 22 44 % _ 100 - 100 100 0 Table 7 Surviving Offspring / Hatchlings 1.8 ppm a.i. Offspring Surv. Hatch 33 00 55 00 56 13 14 33 33 % 100 - 100 - 83 94 10 6.2 ppm ai. Offspring Surv. Hatch 00 88 00 00 44 12 12 22 44 % 100 - 100 100 0 17.6 ppm a.i. Live Hatch 3-Week % 6 6 100 4 5 80 2 3 67 4 4 100 2 3 . 67 18 21 4 4 83 2 1 17 17.6 ppma.i. Offspring Surv. Hatch % 5 6 83 4 4 100 2 2 100 4 4 100 2 2 100 17 18 3 4 97 12 7 Project Number 454- Replicate 1 2 3 4 5 Total Mean SD Control (0 ppm ai.) 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 a.i.) 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 ai. 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 (%) 1.8ppma.i. Offspring Eggs Surv Set 34 00 57 00 56 13 17 33 33 % 75 - 71 - 83 77 6 6.2 ppm ai. Offspring Eggs Surv Set 00 88 00 00 44 12 12 22 44 % _ 100 - 100 100 0 17.6 ppm a.i. Eggs Hatch 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 Replicate 1 2 3 4 5 Total Mean SD Control (0 ppm a.i.) Hatch/ Hatch Days Hen/Day 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 Control (0 ppm a.i.) Offspring Offspring/ Replicate Stuv 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 1.8ppma.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/ Surv 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 ai. Hatch/ Hatch 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/ Surv 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 Project Number 454-105 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 1.8 ppm a.i. 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 M ean Surviving O ffspring1 B od y 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 1Offspring were approximately 12 weeks o f age at final body weight interval. -- No offspring available. 17.6 ppm a.i. 985 1051 1056 1059 998 1030 35 o\ Project Number 454-105 -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 ppm a.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 -118- Appendix XIII Page 2 Adult Liver Weight (g) from a Mallard Pilot Reproduction Study with PFOS Project Number 454-105 Pen 211 212 213 214 215 Mean SD 6.2 ppm a.i. Male Liver 27.724 25.304 32.723 35.818 24.458 29.205 4.900 Female 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 Appendix XIV Offspring1Liver Weight (g) from a Mallard Pilot Reproduction Study with PFOS Control (0 ppm a.i.) Pen Liver 1.8 ppm a.i. Pen Liver 6.2 ppm a.i. Pen Liver 201 201 202 202 203 203 203 205 205 205 Mean SD 21.940 21.925 17.608 19.435 17.948 19.424 24.555 16.544 24.035 25.485 20.890 3.148 206 24.539 206 33.706 206 24.375 208 26.543 208 26.833 208 27.320 210 28.403 210 26.273 210 38.193 210 20.424 27.661 4.999 212 29.540 212 25.845 212 21.894 212 27.367 212 24.851 212 18.934 215 23.160 215 25.229 215 15.702 215 19.680 23.220 4.215 1Offspring were approximately 12 weeks o f age at the time o f euthanasia and tissue collection. 17.6 ppm a.i. Pen Liver 216 28.132 216 19.205 217 30.096 217 28.920 218 34.326 218 23.248 219 23.808 219 22.787 220 19.251 220 17.399 24.717 5.489 Project Number 454-105 -120- Wildlife International, Ltd. Project Number 454-105 Appendix XV Changes To Study Protocol This study was conducted in accordance with the study protocol signed on February 28,2000 and the following amendments and deviations: 1. The protocol was amended to indicate eggs would be held refrigerated until separated for sampling and eliminated the separation of the shell membrane from the shell. 2. The protocol was amended to reduce the number of eggs collected for analysis from all eggs, to eggs collected during weeks 1, 3 and 6 of the test. Eggs collected during Weeks 2, 4 and 5 will be disposed of by incineration. 3. The protocol was amended to change the test substance purity from 98.9% to 90.49%. Correspondingly, the test concentrations were changed from 0, 2, 7 and 20 ppm a.i to 0, 1.8, 6.4 and 18.3 ppm a.i. 4. The protocol was amended to indicate for a seven day period beginning March 31 (early Week 5), eggs would be collected daily for incubation, hatching and rearing of offspring. The amendment also detailed the conditions of egg storage, incubation, housing and brooding of hatchlings. Additionally the amendment indicated hatchlings would be uniquely identified and weighed at hatch and at 14 days of age, and listed reproductive parameters to be measured. 5. The necropsy section of the protocol was amended to indicate at termination samples would be collected from all remaining study adults and from 10 offspring in each test group for histopathological examination. Any remaining tissue not fixed for histopathology will be stored frozen for potential analysis. 6. The protocol was amended to extend the adult portion of the study for the control group and 18.3 ppm a.i. treatment group at least four weeks. During the extension, the number of eggs laid for each pen would be recorded and eggs would be disposed of. Attempts would also be made to collect blood samples from the control group and 18.3 ppm a.i. treatment group birds at the end of Week 6. Adult test birds in the 1.8 and 6.4 ppm a.i. treatment groups will be euthanized at the end of Week 6. Additionally, the amendment required collection of feather samples at the time of gross necropsy. 7. The protocol was amended to indicate the raw data and report would be audited by the Quality Assurance Unit and a Good Laboratory Practice compliant final report would be prepared. Additionally, the Sponsor's representative was changed to John Newsted, and his address was added.8 8. The protocol was amended to indicate analyses of egg, blood and tissue samples will be reported separately. Results of egg, blood and tissue analyses may be amended to the biological results at a later date. -121 - Wildlife International, Ltd. Project Number 454-105 9. The protocol was amended to add statistical analyses of data to determine statistically significant differences between groups. 10. Study offspring were not euthanized, weighed and disposed of at 14 days of age. Instead, offspring were raised to approximately 12 weeks of age prior to being euthanized, weighed, sampled and stored frozen. A protocol amendment was not prepared in a timely manner for this change. 11. The test substance purity changed from 90.49% to 86.9%. Correspondingly, the test concentrations changed from 0, 1.8, 6.4 and 18.3 ppm a.i. to 0, 1.8, 6.2 and 17.6 ppm a.i. A protocol amendment was not produced in a timely manner for this change. 12. Additional adult body weight measurements were taken at Weeks 6, 8, 10 and 11 of the test. The amendment to extend the test inadvertently did not specify additional body weight measurements. 13. Eggs laid on July 8, 2000 (Day 4 of Week 19) were inadvertently not counted and collected. Instead, eggs laid on July 8, 2000 were counted and collected with eggs laid on the following day. 14. Wing bands were removed from study offspring at approximately five weeks of age and offspring were reidentified with leg bands. The protocol indicated offspring would be identified with wing bands. - 122- W ildlife International, Ltd. Project Number 454-105 Appendix XVI Personnel Involved in Study The following key personnel were involved in the conduct or management of this study: Avian Toxicology (1) Mark Jaber, Wildlife Toxicologist (2) Joann B. Beavers, Director, Avian Toxicology (3) Linda R. Mitchell, Manager of Ecotox Operations (4) Diana Temple, Laboratory Supervisor (5) Sean P. Gallagher, Senior Biologist, Avian Toxicology Chemistry (1) Willard B. Nixon, Ph.D., Director of Chemistry (2) Raymond L. Van Hoven, Ph.D., Scientist, Analytical Chemistry