Document 69d7Qzjg70dQ3bveza7XOMB4

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 wildlife International, Ltd. Project Number 454-105 -2- GOOD LABORATORY PRACTICE COMPLIANCESTATEMENT 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: Sean P. Gallagher Senior Biologk, Avian Toxicology SPONSOR'S REPRESENTATIVE: A/ I' L. Newsted DATE DATl WildlgeInternational, 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 4th 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,-fi NohSan Notification No. 3850, Agricultural Production Bureau, 10 August 1984. STUDY DIRECTOR L p.& SeanP. Gallagher Senior Biologist, Avian Toxicology SPONSOR'S REPRESENTATIVE: 6 Wildlge International, Ltd. Project Number 454-105 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 28,2000 March 1,2000 March 7,2000 April 11,2000 January 16,17,28 & 29,2003 January 27-31, February 3,2003 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 inspectionswere study-based unless otherwise noted. Quality Assurance gepresentative 12- DATE wildlijie International, Ltd. -4- REPORT APPROVAL Project Number 454-105 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 Senior Biologist, Avian Toxicolo& CHEMSTRY PRINCIPAL INVESTIGATOR &f?J&K/L R a d L . Man Hoven, Ph.D. Scientist, Analytical Chemistry MANAGEMENT: \. v- Joann B. Bhvers Director, Avian Toxicology &kfd/&& Willard B. Nixon, Ph.6. Director of Chemistry DATE b/18/,m DATE DATE WildlifeInternational. 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 Substanceand Internal Standard...................................................................................... 11 Test Organisms........................................................................................................................ 11 Identification............................................................................................................................ 12 Avian Feed and Water ............................................................................................................. 12 Diet Preparation....................................................................................................................... 13 Diet Sampling.......................................................................................................................... 13 Analytical Method ................................................................................................................... 13 Housing and EnvironmentalConditions.................................................................................. 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 Candlingand Incubation.,..................................................... :.................................................. 17 Hatching and Brooding............................................................................................................ 17 OffspringBlood and Tissue Collection................................................................................... 18 StatisticalAnalyses................................................................................................................. -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 ReproductiveResults............................................................................................................... 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 (ghirdday) 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 f'rom 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 WildlgeInternational, Ltd. Project Number 454-105 -7- TABLE OF CONTENTS PAGE 3 APPENDICES Appendix I. Certificate of Analysis........................................................................................ 35 Appendix 11. Diet and SupplementFormulations.................................................................... 38 Appendix 111. Diet Preparation...................................... ............................................................ 39 Appendix IV. The Analysis of PFOS in Avian Diet ................................................................. 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 fiom a Mallard Pilot Reproduction Study with PFOS ................................................... 63 Appendix IX. Histopathology Report.................... .................................................................... 67 Appendix X. Egg Production (eggs laidhedweek) 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 wildlije 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 (Anusplutyrhynchos) 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. 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 ai. 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. were no apparent treatment-related effects on any of the reproductive parameters wdsured. 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. WildlifeInternational, 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 2 1601. The biological portion of the test was conducted from February 29,2000 until July 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 (Anasplatyrhynchos) of dietary exposure to the potassium salt of Perfluorooctane Sulfonic Acid (hereafier 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 concentrationswere selected in consultation with the Sponsor, based upon the results of a LCSO 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 Pens per (Control) 0 5 1.8 5 6.2 5 17.6 5 Birds uer Pen 1 1 1 1 1 1 1 1 wild1ife Intemational, 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 survivability were examined. I wildlife International, Ltd. Project Number 454- 105 - 11 - MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "PFOS: A Pilot Reproduction Study with the Mallard". The protocol was based on procedures outlined in the Environmental 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 fiom 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 ai.) in the diet based upon the final reported purity of 86.9%. The internal standard, 4H PFOS, was received fiom 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: lH, lH, 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 wildlqe 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 acclimateto 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 11, 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. wildlife 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" (WildlifeInternational, Ltd. Project No. 454C-110). Samples were extracted with methanol and diluted in a 50% methanol : 50% NANOpure@water solution containing 0.0100 mg lH, IH, 2H, 2H Perfluooroctane Sulfonic Acid (4H PFOS; 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 1100High Performance Liquid Chromatograph Q-IPLC) with a Perkin-Elmer SCIEX API lOOLC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil CIS analytical column (50 mm x 2 mm I.D., 3-pm particle size). The instrument parameters are summarized in Appendix N ,Table 1. w i l d l y e 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)& and 0.05% formic acid (v/v), ranging in concentration from 0.000439 to 0.00439 mg a i & (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 equationswere 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 LKg). The method LOQ for the Week 1, Day 7 and Week 6, Day0 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 & ) 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 IV,Table 3). A typical ion chromatogram of a matrix blank is presented in Appendix IV, Figure 5. Avian diet samples were fortified at 0.879, 8.79, and 22.0 ppm a.i. (Week 1, Day 0) or 1.76, 8.79 and 22.0 ppm a.i. (Week 1, Day 7; Week 6, Day 0 and the sample re-extraction set) and analyzed concurrently with the samples to determine the mean procedural recovery. The method yielded mean procedural recoveries 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 i l d l v e Xnternational, 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 fortificationis 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 manufactured by Safeguard Products, Inc. (Model No. 5353, 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 f 0.7"C (SD) with an average relative humidity of 48% f 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 fluorescent 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. wildlife 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 f 0.1"C (SD) with a mean relative humidity of approximately 75% f 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 f0.0"C (SD) with an average wet bulb temperature of 30.6 f0.1"C (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 membrane, the incubator was also equipped with an automatic egg rotation device, 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 f 0.0"C (SD), and the average wet bulb temperature was raised to 33.8 f0.2"C (SD) (relative humidity of approximately 77%). Wildlife 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 fiozen 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 f 0.6"C (SD) with an average relative humidity of 71 f 11% (SD). All ducklings were removed from brooding pens and transferred to pens without supplemental heat at approximately 3 weeks of age. These pens were the same model used to house the adult birds. The photoperiod for the offspring was maintained by a time clock at 16 hours 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 hemacytedplatelets, 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 17.6ppm ai. treatment group were compared. 1. Adult Bodv 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 17.6ppm a i . treatment group for weeks 7 through 19. 3. Ems 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. Egzs Cracked of Ems Laid - The number of eggs determined by candling to be cracked divided by the number of eggs laid, per pen. 6. Viable Embwos of Eggs Set - The number of embryos at the Day 14 candling was divided by the number of eggs set, per pen. wildlijie International, Ltd. Project Number 454-105 - 20 - 7. Live 3- Week Embrvos 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 14day old survivors was divided by the number of hatchlings per week, by pen. 10. Hatchlinm 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 14day old survivors were measured by parental pen group. 13. Liver Weierht - 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 verifl test substance concentrations. Means and standard deviations for the three test concentrations were 1.8 +_ 0.13 ppm ai., 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 rf: 0.092 ppm a.i., 6.0 & 0.67 ppm a.i. and 16.8 f 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 andor 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 bleeding at test termination. She also exhibited body tremors and rapid blinking and respiration during the bleeding process. Clinical signs appeared to be triggered by the stress 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 1.8pprn 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 wildlife International, Ltd. Project Number 454- 105 - 22 - 17.6 ppm a.i. treatment group was significantly (p < 0.05) lower at the Week 10, 1 1 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) differencesbetween the control group and the 17.6ppm 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 bird.sin 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. WildlgeInternational, 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 ai. 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 E. 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-Electrosprayhiass 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 HPLCElectrosprayh4as.s Spectrometry; Centre Study Number 023-065". wildlife 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 concentrationis 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 concentrationstested. 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 pprn 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 wildlife 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 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. wildlife International, Ltd. - 26 - REFERENCES Project Number 454-105 ~~ 1 U.S. Environmental Protection Agency. 1982. Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlve 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 for Conducting Reproductive Studies with Avian Species. ASTM Standard E1062-86. Annual Book of ASTM Standards. Vol. 11.04. Philadelphia, PA. 15pp. 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 of 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 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 Concentration' Day 0 Week 1 Interval Day 7 Week 6 Control (0 ppm a.i.) Measured < 0.879 c 1.41 < 1.41 I Mean N 4 1.8 Measured 1.8 1.6 2.0 I (ppm a.i.) % Nominal 100 89 Ill Mean ' 6.4 Measured 6.0 5.2 6.0 (ppm a.i.) % Nominal 97 87 97 Mean ' 18.3 Measured 17.6 18.4 16.8 (ppm a.i.) % Nominal 100 105 95 ~~ ' Nominal concentrations and results of diet analysis were corrected for the change in test substance purity from 90.49% to 86.9' The limit of 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. The mean percent of the Day 0 values. Table 2 Mean Adult Body Weight' (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 1 1 11-20 20 0-20 Control M Z 1120 -36 1084 -2 1082 (0ppma.i.) SD 53 65 59 54 66 F R 998 -36 962 89 1051 SD 106 87 70 84 52 1.8 ppm ai M Z 1093 SD 70 F x 1003 SD 92 -10 1083 52 69 88 1092 64 55 -9 1073 21 77 -13. 1079 57 72 42 1124 3 16 71 41 1 1052 55 59 75 116 63 1136 43 24 78 72 35 1114 110 41 91 106 19 1142 59 1202 40 1242 -38 1204 83 32 81 21 79 40 59 95 97 58 -54 998 26 1025 -10 1015 -10 1005 7 31 61 62 18 44 50 59 23 118 I - - -- I- - I - - -- - - - - - - - I -- - -- I -- - - - -- I -- -- -- - - - 6.2 M Z 1091 -33 1058 39 1098 21 1118 27 - - I -- - _- I - - ppm ai. SD 31 20 40 37 42 73 63 74 - - - - - -_ - - F % 988 66 1054 28 1082 -43 1038 51 -- -I- - - I I I SD 62 69 65 66 105 78 95 111 - - - I - I 17.6 M 2 1095 -41 1053 -4 1049 38 1087 -8 -13 1074 4 1078' -5 1073. -31 1042 -53 ppm ai. SD 107 56 78 14 70 25 51 74 62 24 55 65 18 73 92 94 57 F X 999 23 1022 11 1033 -55 978 -21 SD 150 104 138 53 102 59 149 88 17 995 -27 967 -10 958 62 148 37 172 15 173 -60 897 -102 98 139 90 -1 Mean (X)f standard deviation (SD).The meansfor body weights and body weight changes are calculatedand roundedseparately. Data not avalible. Birds euthanizedat the end of Week 6. statisticallymerent fiomthe control g o y , at p c 0.05. I h) 00 I R e Table 3 Mean Feed Consumption' (g/bird/day) from a Mallard Pilot Reproduction Study with PFOS Experimental Group Weeks 1 2 3 4 5 6 7 8 9 10 1 1 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 ppma.i. SD 28 16 24 37 17 40 53 63 28 71 39 28 19 22 42 28 41 25 44 I h, \o I 1.8 Mean 157 150 154 172 160 177 ppm a.i. SD 47 31 43 47 42 50 r: 17.6 Mean 156 136 200 187 196 234 225 241 253 226 249 218 195 267' 172 191 * 238' 183 * 227' ppma.i. SD 65 38 62 42 57 84 51 79 67 70 69 62 69 49 52 42 51 39 29 T 9. CD 1 Mean & standard deviation. - Data not avalible. Birds euthanized at the end of Week 6. R * Statistically different from the controlgroup at p e 0.05. P v, P L_ Number of birds Molting flight feathers Feather loss & abrasions on neck Bumble foot Impacted feed under tongue Abnormally small body size - Testes small, 1.0-2.0cm - Testes small, 2.0-3.0cm - Testes small, 3.5 cm Regressing ovary Regressed ovary Egg yolk peritonitus Egg remnants in the repro. tract Intrabdominal egg Air sacculitus Not 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 Females - Treatment Group (ppm a.i.) Control 1.8 6.2 17.6 5 5 5 5 3 0 0 0 0 0 0 0 3 1 3 4 0 0 0 1 0 0 0 0 0 0 0 3 2 0 0 1 2 0 0 0 - - - - - - - - - - - - - 0 0 0 0 0 4 2 0 5 5 5 5 2 0 0 3 0 0 1 0 3 3 3 1 0 0 0 0 0 0 0 1 - - - - - 0 1 0 0 4 0 0 5 0 1 3 1 1 0 0 0 0 0 1 0 0 0 0 1 1 1 1 0 Table 5 Mean Egg production (Eggs Laid/Hen and Eggs/Hen/Day) froma Mallard Pilot Reproduction Study with PFOS Experimental Group Weeks 1 2 3 4 5 6Total E/H/D* 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total E/H/D' Control Total 17 15 14 17 22 27 112 2 1 2 6 1 3 1 3 7 5 0 0 0 0 0 0 0 0 85 0ppma.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 1 6 0 . 3 9 3 3 3 4 3 2 0 0 0 0 0 0 0 0 1 5 0 . 1 6 17.6 ppma.i. Total 15 22 33 31 24 22 147 Mean 3 4 7 6 5 4 29 0.70 SD 3 1 1 1 2 3 5 0 . 1 1 1 0 1 5 1 5 1 7 8 7 7 4 3 4 0 0 0 0 90 2 3 3 3 2 1 1 1 1 1 0 0 0 0 18 0.19 34343332120000230.24 ~~ ' Total number of eggs laid and mean number of eggs laid per pen f standard deviation. --'Eggs 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). Project Number 454-105 - 32 - 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 1.8 ppm a.i. 6.2 ppm a i . 17.6ppm a.i. Number of Replicates Eggs Laid Eggs Cracked Eggs Set Viable Embryos Live 3-Week Embryos Hatchlings offspring survivors Eggs Laid/Hen offspring/Hen 5 5 5 5 24 18 12 24 0 1 0 1 24 17 12 23 22 17 12 21 22 17 12 21 21 14 12 18 21 13 12 17 5 4 2 5 4 3 2 3 Eggs LaidMenlDay 0.69 0.51 0.34 0.69 Eggs Cmcked/Eggs Laid (%) 0 5 0 4 Viable EmbryodEggs Set (%) 90 100 100 92 Live 3-Week Embryofliable (%) 100 100 100 100 Hatchlingd3-Week Embryos (%) 92 82 100 83 offspring SurvivordHatchlings(%) 100 94 100 97 HatcNingdEggs Set (%) 85 82 100 76 offspring Survivors/Eggs Set (%) 85 77 100 74 Hatchlings/Hen/Day 0.60 0.40 0.34 0.51 offspring SurvivordI-IenDay 0.60 0.37 0.34 0.49 I Differencesbetweenthe control group and the treatment groups were not statisticallysigruficant(p > 0.05) I Table 7 Mean Body Weight' (g) of Hatchlings and Surviving Offspring fkom a Mallard Pilot Reproduction Study with PFOS Experimental Group Hatchlings Mean SD Surviving Offspring' Mean SD Control (0 ppm a.i.) 28.4 f 2.0 1012 f 72 1.8 ppm a.i. 32.8 f 1.4 1065 k 92 6.2 ppm a.i. 33.3 f 0.4 1036 f 71 17.6 ppm a.i. 35.0 f 1.6 1030 f 35 'Mean f standard deviation. 2 Offspring were approximately 12 weeks of age at final body weight interval. Differencesbetween the control groupand the treatmeng groupswere not statisticallysignificant0,> 0.05) Table 8 Mean Liver Weights' (g) from a Mallard Pilot Reproduction Study with PFOS Experimental Group Control (0 ppm a.i.) Males Liver Mean SD 21.336 rf: 4.793 Females Liver Mean SD 21.240 f 5.382 offspring2 Liver Mean SD ~~ ____ ~ 20.890 f 3.148 I 1.8 25.336 f 1.564 39.801 rf: 8.832 27.661 +_ 4.999 * w P ppm a.i. I 6.2 ppm a.i. 29.205 f 4.900 31.938 rf: 4.462 ' 23.220 f 4.215 17.6 ppm a.i. 23.411 f 8.019 20.624 f 2.947 24.717 rf: 5.489 1Mean f standard deviation. * Offspring were approximately 12 weeks of age at time of euthanasia and tissue collection. * Statistically different from the control group at p < 0.05. - 35 - WildlifeInternational, t t d . Project Number 454-105 Appendix I Certificate of Analysis Andutkd laboratories, Inc. College, PA 18801 www.cenilrelab.com Fax:(814)231-1253or(814)231-1580 XiWERIM CERTIFICATEOF ANALYSIS Revision 3 Centre Annlytlcrl Laboratoriu COA Reference # 023418A 3M Prodnet: PFOS,Lot 217 Referencs#: SD-018 I I Pnw:86.9% Tert Name I Spedfk8dOW I Purity' Appearance I White crystalline Powder ReRllt 86.9% ConformS NMR Metals (IcP/MS) 1. calcium 2. Magnesium 3. sodium 4. Potassium' 5. Nickel 6. Iron 7. Maneanese Positive 1. 0.005 wt.hvt.?/. 2. 0.001 wtlwt?? 3. 1.439 wtJwt.% 4. 6.849 wtfwt.% 5. ~0.001wt/wt% 6. 0.005 wtfwt.% 7. <o.O01wtJwt% Related Compounds - I POAA Residual Solvents (TGA) Purityby DSC .- I. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate I 7. sulfate4 OrganicAcids '(.IC.) 1. TFA 2. PFPA 3. HFBA ~ 4. NFPA Elemental Analysis': 1. C a r h 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine COAO23-018A I 0.33 wtJwt.% None Detected Not Applicable' 1. <0.015 wt./wt% 2. 0.59wtlwt.% 3. < O . W wtJwt.% 4. c0.009wtlwt?4 5. C0.006 wtJwt.% 6. <0.007dwt-% 7. 8.76 wt./wt.% I 1. eo.1 wt./wt.% 2. Ql.1 wt./wt.% 3. O.lOwtJwt.% 4. 0.28 wt./wt?h 1. Theoretical Value = 17.8% 2. ThcorcticalValue = 0% 3. TheoreticalValue=0% 4. lhtoretical Value = 5.95% 5. Thcorctical Value = 60% 1. 12.48 wtJwt.% 2. 0.244 wt./wt.% 3. 1.74wtJwt.% 4. 8.84wt./wt.% 5. 54.1 wtJwt.% Page 1 of3 -36- wiId1ife Internationa1, Ltd. Project Number 454-105 Appendix I Page 2 Certificate of Analysis AWE= CERTIFICATE OF ANALYSIS Revision 3 Centre Annlylicrl Labomtoria COA Reference It: 023-018A Date ofLast Analysis: 08/31/00 ExpirationDate: 08/31/06 Storage Conditions: Frozen G10''C Re-assessmentDate: 08/31/06 'purity = 1ooO/o- (sum ofmetal impurities, 1.45% +LCMS impurities,8.41%+Inorganic Fluoride, 0.59%+NM.Rimpurities, 1.905%+0rganic acid impurities,0.38%+POAA, 0.33%) - Total impurity fiom all tests= 13.07% Purity = 100% 13.07% = 86.9% *Potassiumis expected in this salt form and is thereforenot considered an impurity. 'Purity by DSC is generally not applicableto materials of low purity. No endotherm was observed for this sample. "Sulfurin the sample appears to be convertedto SO4 and hence detectedusing the inorganicanion methodconditions. The anion result agrees wall with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4is not consideredan impurity. %A HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonafluoropentanoic acid Pentafluompropanoic acid h r e t i c a l value calculations based on the empirical formula, CeFl,S@-K+(MW=538) This work was conductedunder JPA Good LaboratoryPractice Standards(40 CFR 160). COA02M18A Page 2 of 3 -37- WildlifeInternational, Ltd. Project Number 454-105 Appendix I Page 3 Certificate of Analysis INTERIM CERTIFICATE OF ANALYSIS Revislon 3 Centre Analytical Laboratorlea COA Reference #: 023-018A I Impnrity c4 WtJWt. Yo 1.22 I c5 I 1.33 I C6 4.72 c7 1.14 Total 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculatedusing the averageresult from the C4 and C6 standardcurves. Likewise,the C7 value was calculatedusing the average result from the C6 and C8 standard curves. Prepared By: Scientist,CentreAnalytical Labonitoties n M Reviewed By: J6hn Flahcrhr Date 5aboratory Manager, cmtre Analytical Laboratories COAO23-018A Page 3 of 3 -38 - WildlifeInternational, Lta. Project Number 454-105 Appendix I1 Diet and Supplement Formulations Table 1 Wildlife International,Ltd. Game Bird Ration' INGREDIENTS Fine Corn Meal Soy Bean Meal, 48% Protein Wheat Midds Protein Base Agway Special, 60% Protein Alfalfa Meal, 20% Protein Dried Whey Ground Limestone Eastman CalF'hos 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% of the ration, supplied the following amounts per ton: Amount Supplied Per Ton: Vitamin D3 2,000,000 I.C.U. Vitamin A 7,000,000 I.U. Riboflavin 6 grams Niacin 40 grams Pantothenic Acid 10 grams Vitamin B12 8 mgs Folic Acid 600 mgs Biotin 64 mgs Pyridoxine 1.2 grams Thiamine 1.2 grams Vitamin E 20,000 I.U. Vitamin K (Menadione Dimethylpyrimidinol Bisulfite) 5.8 grams Manganese 102 grams Zinc 47 grams Copper 6.8 grams Iodine 1.5 grams Iron 51 grams ' Selenium 182 mgs The guaranteed analysis is a minimum of 27% protein, a minimum of 2.5% crude fat and a maximum of 5% crude fiber. Fermentation By-products (Source of Unidentified Growth Factors). -39- wildlife International, Ltd. Project Number 454-105 Appendix I11 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: 1.8 ppm a.i.: No premix required. 0.3084 g PFOS + 6099.7 g ration 6.2 ppm a.i.: 17.6 ppm a i : 1.0794 g PFOS + 6098.9 g ration 3.0839 g PFOS + 6096.9 g ration Basal ration was weighed into a tared Hobart mixing bowl. Approximately 100 g of ration was transferred to a Waring@ blender. The test substance was weighed into a tared weigh boat and a small mortar was taken to crush any areas of consolidation. The test substance was transferred to the Waringo 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 + 1.250kg limestone 1.8 ppm a.i.: 1000 g Premix + 22.75 kg ration + 1.250kg limestone 6.2 ppm a.i.: 1000 g Premix + 22.75 kg ration + 1.250kg limestone 17.6ppm ai.: 1000g Premix + 22.75 kg ration + 1.250kg limestone The diets were mixed for approximately 20 minutes in a Patterson-Kelly Twin Shell Blender. -40- Wildlqe International, Ltd. I Project Number 454-105 Appendix N The Analysis of PFOS in Avian Diet -41 Wildlife International, Ltd. Project Number 454-105 Appendix N ANALYTICAL METHODS AND RESULTS Tpical LC/MS OperationalParameters TABLE 1 INSTRUMENT: Hewlett-Packard Model 1100High Performance Liquid Chromatograph with a Perkin-Elmer SCIEX API 1OOLC Mass Spectrometerequipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil CIScolumn (50 mm x 2 mm I.D., 3-pm particle size) OVEN TEMPERATURE: 3OoC STOP TIME: 5.00 minutes FLOW RATE: 0.220 mL/minute MOBILE PHASE: 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 m u INTERNAL STANDARD MONITORED MASS: 426.7 amu -42 - wildlife International, Ltd. Project Number 454-105 Appendix IV TABLE 2 CALCULATIONS The concentrationof PFOS found at the instrumentwas determined using the following equation: PFOS (mg a.i./L) at instrument = eak area - (y-intercept)x internal standard conc. (mg a.i./L) slope Determination of Sample Residues fPFOS) The concentration, expressed as ppm a.i., for each sample was determined using the following equation: PFOS (mg a.i./L) at instru. x extract final volume (L) x dilution factor PFOS (ppm a i ) in sample = initial weight (Kg) Determination of Limit of Ouantitation(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 ai.). This ratio times 100 is the percent recovery of the method at that level of fortification. m ai. measured in sample % Recovery =Ep ppm a.i. fortified loo wildlife International, Ltd. Project Number 454- 105 Appendix IV TABLE 3 Number (454-1 05-) Matrix Blanks am Fortifications Analyzed Concurrently with the Samples Sample Type Interval Concentrationof PFOS (ppm a.i.) Fortified' Measured',' Percent Recovery ~ Mean Recovery (x1 MAB-1 Matrix Blank Week 1, Day 0 0.00 < 0.879 MAS- 1 Matrix Fortification Week 1,Day 0 0.879 0.992 113 105 MAS-2 Matrix Fortification Week 1,Day 0 8.79 8.89 101 MAS-3 Matrix Fortification Week 1,Day 0 22.0 22.0 100 MAB -2 Matrix Blank Week 1, Day 7 0.00 < 1.41 MAS-4 Matrix Fortification Week 1, Day 7 1.76 1.97 112 104 MAS-5 Matrix Fortification Week 1, Day 7 8.79 9.11 104 MAS-6 Matrix Fortification Week 1, Day 7 22.0 21.3 97.0 MAB-3 Matrix Blank Week 6, Day 0 0.00 < 1.41 MAS-7 Matrix Fortification Week 6, Day 0 1.76 1.98 113 107 MAS-8 Matrix Fortification Week6,DayO , 8.79 9.40 107 MAS-9 Matrix Fortification Week 6, Day 0 22.0 22.0 100 MAB -4 Matrix Blank Re-extraction Set4 0.00 < 1.41 MAS- 10 Matrix Fortification Re-extraction Set4 1.76 1.84 105 102 MAS- 11 Matrix Fortification Re-extraction Set4 8.79 8.86 101 MAS-12 Matrix Fortification Re-extraction Set4 22.0 21.7 98.7 ' Concentrationswere corrected for change in test substancepurity (98.9?? to 869??)per Certificateof Analysis dated October 11,2001. Nominal test concentrationsbased upon the new test substance purity are 1.8, 6.2 and 17.6 ppm ai. 'Less than values correspond to limit of quantitation(LOQ). 'Results were generatedusing MacQuan version 1.6 software. Manual calculations may differ slightly since fortified and measuredconcentrationswere correctedfor change in test substancepurity and rounded for reporting purposes. Re-extraction of:sample454-105,105-13 from Week 1, Day 0; samples454-105-23 and -26 from Week 1, Day 7 and sample454-105,105-50 from Week 6, Day 0. wildlife International, Ltd. Project Number 454-105 Appendix IV TABLE 4 Homogeneity of PFOS in Avian Diet Nominal Concentration' (ppm a.i.) Sample I.D. Number (S-454-105) Location Sampled in Mixing Vessel Measured PFOS Concentration' (ppm a.i.) Mean (x) Standard Deviation (SD) Coefficient of Variation (CV) Mean Percent of Nominal 1.8 Top Left 1.70 Top Right 1.82 Middle Left 1.92 -x = 1.8 ppm a.i 100 Middle Right 1.73 SD = 0.13 ppm a i . Bottom Left 1.77 CV= 7.2% 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 Middle Right 5.81 SD = 0.65 ppm a.i. 13 Bottom Left 5.87' c v =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 of Analysis dated October 11, 2001. Nominal test concentrationsbased upon the new test substancepurity are 1.8,6.2 and 17.6pprn a i 2 Mean result of duplicate re-extraction of original sample. Wildlqe International, Ltd. Project Number 454-105 Nominal Concentration' (ppm a.i.) 0 Sample I.D. Number 6-454- 1054 1 2 43 44 Appendix IV TABLE 5 Verification of PFOS Concentrationsin Avian Diet Interval (Day 0 of Week -) Measured PFOS Concentration',' (ppm a.i.) Mean (X) Standard Deviation (SD) Coefficient of variatioh C C V ~ 1 < 0.879 N/A 1 < 0.879 6 < 1.41 NIA 6 < 1.41 Mean Percent of Nominal 1.8 3-8 1 (see Table 4) 100 45 6 1.94 -x =2.0 ppm a.i. 111 46 6 1.94 SD = 0.092 ppm a.i. 47 6 2.10 CV= 4.6% 6.2 9-14 1 (see Table 4) 97 48 6 6.3 1 -x = 6.0 ppm ai. 97 49 6 6.52 50 6 5.273 SD = 0.67 ppm a.i. c v = 11% 17.6 15-20 1 (see Table 4) 100 51 6 17.5 -x = 16.8ppm ai. 95 52 6 16.5 SD = 0.608 ppm a.i. 53 6 16.4 CV= 3.6% Concentrationswere corrected for change in test substancepurity (98.9% to 86.9%) per Certificateof Analysisdated October 11,2001. Nominal test concentrationsbased upon the new test substancepurity are 1.8,6.2 and 17.6ppm ai. 'Less than values correspond to limit of quantitation(LOQ). Mean result ofduplicatere-extraction of original sample. wildlife International, Ltd. Project Number 454-105 Appendix IV TABLE 6 Ambient Stability of PFOS in Avian Diet During the Mallard Pilot Reproduction Study Nominal Concentration' (ppm a.i.) Sample -Number (S-454-105-) d_ Week 1, Day 0' Mean Mean Percent Measured of (ppm a.i.) Nominal I PFOS Concentration Sample Number (S-454- 105) Week 1, Day 7 Mea~ured',~ (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 32 < 1.41 33 < 1.41 34 1.50 1.6 35 1.49 36 1.85 1 P 01 1 89 6.2 9-14 6.0 97 37 5.48 5.2 87 38 5.01 39 5.03 17.6 15-20 17.6 100 40 18.7 18.4 105 41 18.8 42 17.8 Day 0 values are from homogeneity samples presented in Table 4 and verification samples presented in Table 5. 'Concentrations were corrected for change in test substancepurity (98.9% to 86.9%) per Certificate of 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. Less than values correspond to limit of quantitation (LOQ). -47wildlife 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 mix technique. Weigh 10 g samples of the matrix blank, matrix fortification and test samples into weigh boats and transfer to 8oz. French square glass bottles. Record weights. For each sample, measure 100 mL of methanol with a graduated cylinder and transfer into the French square bottle. 1 Cap bottles and place on shaker table. Allow the samples to shake for a minimum of 30 minutes at approximately 250 rpm. Vacuum filter with qualitative filter paper and rinse retained feed 3 times with methanol into filtrate. 1 Transfer the filtrate into a 200-mL volumetric flask and bring to volume with methanol. Prepare appropriate dilution(s) to bring final concentration into the calibration range of the LCMS methodology. Use methanol for intermediate dilutions, if required. For all final dilutions, use 50% methanol: 50% NANOpure@water dilution solvent containing 0.0100mg 4HPFOSL (internal standard) and 0.05% (vh) formic acid. 1 Ampulate and submit sample for LCMS analysis. Figure 1. Analytical method flow chart for the analysis of PFOS in avian diet. -48 - wildlife International, Ltd. Appendix IV Project Number 454-105 1 0.00 0.06 0.12 0.18 0.24 0.30 0.36 0.42 0.48 Concentration (Ratio) Figure 2. Typical calibration curve for PFOS. Slope = 2.48679; Intercept = 0.07396; r = 0.9985. Curve is weighted (Ux). -49 - w i l d l g e International, Ltd. Project Number 454-105 Appendix IV intensity: 20000 cps 100 90 80 70 60 50 40 30 20 21 2 10 0 4 1 8 1 121 161 201 241 281 Scan 0.69 1.36 2.04 2.71 3.38 4.06 4.73Time 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 '001 Appendix IV intensity: 20000 cps 0.69 1.36 2.04 2.71 3.38 4.06 4.73Time Figure 4. Typical ion chromatogram of a high-level PFOS standard, 0.00439mg a.i./L. -51 - wildlife International, Ltd. Project Number 454-105 Appendix IV intensity: 40000 cps 8"1 100 41 81 121 161 201 241 281 Scan 0.69 1.36 2.04 2.71 3.38 4.06 4.73Time Figure 5. Typical ion chromatogram of a matrix blank sample (454-105-MAB-1). The arrow indicates approximateretentiontime of PFOS. -52- WildlifeInternational, Ltd. Appendix IV Project Number 454-105 100 90 86 70 60 50 218 40 30 20 1 41 81 121 161 201 241 281 Scan 0.69 1.36 2.04 2.71 3.38 4.06 4.73Time Figure 6. Typical ion chromatogram of a matrix fortification sample (454-105-MAS-9,22.0 ppm ai.). -53 - wiId1ge International, Ltd. Project Number 454-105 Appendix IV '"3 intensity: 40000 cps 8 '32 I 228 1 4 1 81 121 161 201 241 281 Scan 0.69 1.36 2.04 2.71 3.38 4.06 4.73Time I Figure 7. Typical ion chromatogramof an avian diet sample on Day 0 (S-454-105-3, 1.8ppm a.i.). - 54 - Wildlije Intemational, Ltd. Appendix V Diagram of Test Layout Project Number 454-105 PENS 198#) PENS 16-18 PENS 13-15 PENS 10.12 PENS 1.9 PENS 1-3 1-1 = approx. Im ' = 6"gutter a = SafeguardProductsInc.Model No. 5355 b = Four X 4 ft. Chroma 50 light bulbs Appendix TI Table 1 Adult Body Weight (g) from a Mallard Pilot Reproduction Study with PFOS Control (0 ppm a i ) - Males ~ ~~~ Week Change Week Change Week Change Week Change Change Week Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 6-8 8 8-10 10 10-11 11 11-20 20 0-20 ~ ~~ 201 1080 10 1090 13 1103 29 1132 52 202 1121 -33 1088 13 1101 47 1148 27 203 1064 -57 1007 -41 966 41 1007 -57 204 1198 -133 1065 71 1136 65 1201 3 205 1139 33 1172 -68 1104 26 1130 -9 ~ ~ Mean 1120 -36 1084 -2 1082 42 1124 3 SD 53 65 59 54 66 16 71 41 -26 1106 16 1164 35 1042 61 1262 8 1138 71 1177 44 1221 -136 1085 5 37 1201 18 1219 44 1263 142 80 1122 107 1229 -116 1113 49 72 1334 10 1344 -58 1286 88 36 1174 21 1195 77 1272 133 ~~ ~~ 19 1142 59 1202 40 1242 -38 1204 83 32 81 21 79 40 59 95 97 58 Control (0 ppm a i . ) - Females Week Change Week Change Week Change Week Change Change Week Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 6-8 8 8-10 10 10-11 11 11-20 20, 0-20 201 1074 -172 902 161 1063 -69 994 -80 202 1027 -50 977 28 1005 -9 996 -31 203 1108 -36 1072 -20 1052 96 1148 40 204 928 28 956 174 1130 -10 1120 192 205 851 52 903 101 1004 -1 1003 152 Mean 998 -36 962 SD 106 87 70 89 1051 84 52 1 1052 55 59 75 116 -47 947 -14 982 -100 1048 44 1076 -64 939 -54 998 31 61 94 1041 39 1021 -3 1045 -65 1011 67 1006 26 1025 62 18 -1 1040 -85 936 23 1068 18 1029 -6 1000 -10 1015 44 50 -19 1021 -53 66 1002 -25 -98 970 -138 1 1030 102 2 1002 151 -10 1005 7 59 23 118 The means for body weights and body weight changes are calculated and rounded separately. Appendix VI Table 2 Adult Body Weight (g) fiom a Mallard Pilot Reproduction Study with PFOS 1.8 ppm a.i. - Males ~~~ Week 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 1093 SD 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 70 44 1128 -19 63 1193 123 96 1073 86 36 1050 -45 63 1136 43 24 78 72 1.8 ppm a.i. - Females Week Change Week 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 1003 SD 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 239 -2 962 -7 36 1109 52 88 1168 204 60 1196 64 35 1114 110 41 91 106 The means for body weights and body weight changes are calculated and rounded separately. Appendix VI Table 3 Adult Body Weight (g) from a Mallard Pilot Reproduction Study with PFOS 6.2 ppm a.i. - Males Week Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 211 1096 212 1077 213 1051 214 1097 215 1135 -50 1046 -57 1020 -17 1034 -27 1070 -13 1122 38 1084 58 1142 46 51 1071 -55 1016 -61 22 1056 128 1184 133 92 1162 -24 1138 41 -7 1115 -4 1111 -24 Mean 1091 -33 1058 39 1098 21 1118 27 SD 31 20 40 37 42 73 63 74 ~ ~ 6.2 ppm a.i. - Females Week Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 211 985 168 1153 39 1192 8 1200 215 212 1032 39 1071 84 1155 -163 992 -40 213 1049 -21 1028 -79 949 36 985 -64 214 890 85 975 18 993 -27 966 76 215 983 59 1042 78 1120 -71 1049 66 Mean 988 SD 62 66 1054 69 65 28 1082 -43 1038 51 66 105 78 95 111 The means for body weights and body weight changes are calculated and rounded separately. Appendix `I Table 4 Adult Body Weight (g) from a Mallard Pilot Reproduction Study with PFOS 17.6 ppm a.i. - Males Week Change Week Change Week Change Week Change Change Week Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 6-8 8 8-10 10 10-11 11 11-20 20 0-20 216 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 -8 25 51 74 72 1085 -13 1072 -19 1053 -111 942 -86 -52 1082 80 1162 8 1170 -8 1162 -96 -87 1048 -68 980 -12 968 80 1048 -90 18 1104 -6 1098 -22 1076 24 1100 38 -15 1050 28 1078 18 1096 -139 957 -30 -13 1074 62 24 4 1078 55 65 -5 1073 -31 1042 -53 18 73 92 94 57 17.6ppm ai. - Females ~~~ Week Change Week Change Week Change Week Change Change Week Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 6-8 8 8-10 10 10-11 11 11-20 20 0-20 216 1075 217 895 218 1112 219 788 220 1125 Mean 999 SD 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 4 7 1077 4 8 -10 958 -60 897 -102 15 173 98 139 90 Themeans for body weights and body weight changes are calculatedand 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 Pa 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 Appendix VII Table 2 Feed Consumption (ghirdday) 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 rn 0 I 2 9. 8 FP f d e 8 m P c 8 Appendix VII Table 3 Feed Consumption (g/bird/day) from a Mallard Pilot Reproduction Study with PFOS 6.2 ppm a.i. 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 Appendix VII Table 4 Feed Consumption (g/birdday) from a Mallard Pilot Reproduction Study with PFOS 17.6ppm 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 -63 - Project Number 454-105 Appendix VIII Table 1 Individual Gross Pathological Observations fkom a Mallard Pilot Reproduction Study with PFOS Birds Euthanized at Test Termination Control (0 ppm a.i.) Males Molting flight feathers Bumble foot - Testes small - Testes small - 2.0 - 3.0 cm 3.5 cm Not remarkable Pens 201 202 203 204 205 xx X xxx X X xx Females ~~ Molting flight feathers Bumble foot Regressed ovary Egg remnants in the reproductive tract Not remarkable Pens 201 202 203 204 205 X X X X X X xxx X X -64- Project Number 454-105 Appendix VIII Table 2 Individual Gross Pathological Observations from a Mallard Pilot Reproduction Study with PFOS Birds Euthanized at Test Termination Bumble foot Not remarkable Males 1.8 ppm a.i. Pens 206 207 208 209 210 X xxxx Females Bumble foot Regressing ovary Egg yolk peritonitus Not remarkable Pens 206 207 208 209 210 xx X X - - X - X -65 - Project Number 454-105 Appendix VIU Table 3 Individual Gross Pathological Observations from a Mallard Pilot Reproduction Study with PFOS Birds Euthanized at Test Termination Bumble foot Not remarkable Males 6.2 ppm a.i. Pens ~~ 211 212 213 214 215 X xx X X Females Feather loss and abrasions on neck Bumble foot Egg yolk peritonitus Extensive egg yolk peritonitus Intrabdominal egg Not remarkable Pens 211 212 213 214 215 X X X X X X - X X X - 66 - Project Number 454-105 Appendix VIII Table 4 Individual Gross Pathological Observations fiom a Mallard Pilot Reproduction Study with PFOS Birds Euthanized at Test Termination 17.6 ppm a.i. Males Pens ~ 216 217 218 219 220 Mass of feed impacted under the tongue Bumble foot - Testes small - Testes small - 1.O - 2.0 cm 2.5 cm Not remarkable X xxxx xxx X Females General condition - abnormally small body' 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 1 Denotes a small body frame, but does not indicate a thin condition. - 67 - w i l d l v e International, Ltd. Project Number 454-105 Appendix M Histopathology Report I EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-105 WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER 454-105 EPL PROJECT NUMBER 212-025 PFOS: A PILOT REPRODUCTION STUDY WITH THE MALLARD PATHOLOGY REPORT Submitted by: Experimental Pathology Laboratories, Inc. P.O. Box 474 Herndon, VA 20172-0474 (703) 471-7060 Submitted to: Wildlife International, Ltd. Easton, MD 21601 January 25,2001 EPL" I - 69 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-105 TABLE OF CONTENTS PATHOLOGY SUMMARY................................................................................. QUALITY ASSURANCE FINAL CERTIFICATION............................................ mJ!2 1 8 ADULT SACRIFICE SUMMARY INCIDENCE TABLES Males........................................................................................................... 1-1 Females....................................................................................................... 1-2 HISTOPATHOLOGY INCIDENCE TABLES Males........................................................................................................... 11-1 Females....................................................................................................... 11-2 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Males........................................................................................................... 111-1 Females....................................................................................................... 111-3 OFFSPRING SACRIFICE SUMMARY INCIDENCE TABLES Males........................................................................................................... IV-I Females....................................................................................................... IV-2 HISTOPATHOLOGY INCIDENCE TABLES Males........................................................................................................... V-1 Females....................................................................................................... V-3 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Males........................................................................................................... VI-1 Females....................................................................................................... VI-5 Project Number 454-105 - 70 - PATHOLOGY SUMMARY -71 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-105 WILDLlFE INTERNATIONAL, LTD. STUDY NUMBER 454-105 EPL PROJECT NUMBER 212-025 PFOS: A PILOT REPRODUCTION STUDY WITH THE MALLARD PATHOLOGY SUMMARY Light microscopic examination was performed on sections of selected tissues from adult male and female mallards (Anas platyrhynchos) which were untreated or which receivedvarious concentrations of the test article (Perfluorooctanesulfonicacid, potassium salt [PFOS]) in the feed for six to 19 weeks. Selected tissues from untreated approximately 12-week-old offspring of the adult mallards were also examined by light microscopy. The objective of this study is to evaluate the effects upon adult mallards (Anas platyrhynchos) of dietary exposure to a test substance over at least a six-week period. The experimental design was as follows: * Mallards in the control and 17.6 ppm a.i. groups were treated for 19 weeks; mallards in the 1.8 and 6.2 ppm a.i. groups were treated for six weeks. ** Offspring in all groups did not receive the test article. MATERIALS AND METHODS At the completion of the various study intervals, all adult mallards were euthanized, and necropsies were performed by Wildlife International,Ltd. Project Number 454-105 - 72 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Wildlife International,Ltd. Study Number454-105 Selected offspringwere euthanized at approximately 12 weeks of age using carbon dioxide gas, and samples for histopathologicalevaluationwere collected by Wildlife International, Ltd. Selected tissues from adults and offspring were fixed in 10% neutral bufferedformalin and sent to Experimental Pathology Laboratories,Inc. (EPL@)w, here they were embedded in paraffin and made into hematoxylin and eosin-stained sections on glass microscope slides. The following tissues from adults and offspring were examined by light microscopy as available: liver, gallbladder,proventriculus,kidney, brain, gonad (ovary or testis), bursa of Fabricius, and adipose tissue. In some cases, nonprotocol-required tissues were sectioned along with adjacent protocol-requiredtissues; these were evaluated, and microscopic findings were recorded at the discretion of the pathologist. All tissues required by protocolare presented in the Histopathology Incidence Tables. Microscopic findings for each tissue examined from each mallard are listed in the Histopathology Incidence Tables. Microscopic changes were graded one to five depending on severity. Nongradable findings are listed as present (P) and tissues with extensive autolysis are listed as (A). All findings for all animals are summarized by sex, age group, and treatment group in the Summary Incidence Tables, together with the total number of animals in each group for which the tissues were examined. A tabulation of gross observations noted at necropsy with the corresponding microscopic change, if appropriate, is presented in the Correlation of Gross and Microscopic Findings tables. The entries in these tables were transcribedfrom the Gross Necropsy records provided by Wildlife International, Ltd. -2- -73 - Project Number 454-105 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Wildlife International,Ltd. Study Number454-105 RESULTS All adults and offspring survived to the end of their respective study intervals. No effects considered possibly related to test article (PFOS) administration were noted in liver, kidney, gallbladder, proventriculus, ovary, brain, and/or bursa of Fabricius of male and female adult mallards or their offspring, in the adipose tissue of adult females and offspring of both sexes, or in the testes of offspring males. Testes of adult males exhibited several features most consistent with post-reproductivephase regression, a normal physiological phenomenon, including aspermia, decreased spermatogenesis, and/or decreased seminiferous tubule diameter. Group differences in the extent of these findings were evident. Aspermia, or complete absence of mature spermatozoa, was noted only in the testes of 315 17.6 ppm a.i. adult males, while decreased spermatogenesis, characterized by lower numbers of mature spermatozoa than expected in actively reproductivetestes, was seen in 1/5 17.6 ppm a.i. males but in 415 control adult males. While seminiferous tubules from both control and 17.6 ppm a.i. adult males had decreased diameter, the mean decrease in 17.6 ppm a.i. adult males was more pronounced than in controls. Correspondingly,the size range of 17.6 ppm a.i. testes recorded at necropsy was 1.O-2.5cm, lower than the 2.0-3.5 cm range recordedfor control testes. Overall, these findings are suggestive of more advanced testicular regression in the 17.6 ppm a.i. adult males compared to controls. This may have been a fortuitous event accentuated by the small group sizes, but the possibility of a test article effect cannot be entirely ruled out. All offspring testes examined were listed as immature because they lacked all evidence of spermatogenesis,had very small seminiferous tubules with absent or narrow lumens, and had germinal epithelium with decreased cell layers -3- EPL" - 74 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-105 Wildlife International,Ltd. Study Number454-105 (compared to adult testes). This morphology was consistentwith normal physiologicalimmaturity in young (approximately 12-week-old) birds rather than a pathological effect. Ovaries of many control and 17.6 ppm a.i. adult females exhibited fibrosis and/or decreased follicular diameter. Decreased follicular diameter corresponded to grossly observed "regressed ovary." Affected follicles were smaller and had shorter stalks than follicles in reproductively active ovaries. Fibrosiswas characterizedby a few nodular foci of mature connective tissue scatteredthrough the ovarian cortex. These ovarian findings were most consistentwith early post-reproductivephase regression, a normal physiological phenomenon. Since the incidence and degree of these findings were similar in control and 17.6 ppm a.i. female adult groups, they were considered incidental and unrelatedto treatment. Ovaries of all offspringfemales were characterizedby numerous oocytes and very small, nonpedunculated follicles completely embedded in a prominent ovarian cortex. This morphology is consistent with normal physiological immaturity in young (approximately 12-week-old) birds rather than a pathological effect. Adipose tissue of adults and offspring consisted of sheets of large white adipocytes with single large lipid droplets, among which were often scattered a few adipocytes which contained multiple, smaller, discrete vacuoles. While almost all samples of adipose tissue exhibited a few of these smaller adipocytes, microvesiculation was diagnosed only when the numbers of these cells were increased. Microvesiculation was noted in both control and treated adult and offspring mallards, and was usually a very subtle, minimal finding. A clearly increased incidence was noted only in 17.6 ppm a.i. adult males compared to control adult males. This increase may have been a fortuitous event accentuated -4- Project Number 454-105 I -75 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Wildlife International,Ltd. Study Number454-105 by small group sizes or possible variability in adipose tissue sampling sites, but the possibility of a test article effect cannot be entirely ruled out. Incidences of adipose tissue microvesiculation in male and female offspring did not exhibit clear, dose-related increases, and were considered incidental and unrelated to treatment. Several additional microscopic findings (described below) were noted in adult and/or offspring mallards from control and treated groups, including mononuclear cell infiltrates in various tissues; kidney mineralization; ureteral lamina propria cyst; and involution of the bursa of Fabricius. Incidences and mean severity were similar when groups from each generational cohort were compared with each other. For these reasons, these findings were considered incidental and unrelated to test article (PFOS) administration. Mononuclear cell infiltrates in various tissues consisted of discrete foci of small lymphocytes with occasional monocytes and macrophages. Scattered clusters of coarsely to finely dark basophilic material in the renal cortex denoted kidney mineralization. In one 6.2 ppm a.i. offspring female, a section of ureter fortuitously sectioned along with kidney exhibited a single lamina propria cyst lined by flattened epithelium and filled with amorphous, pale basophilic material. Only one adult mallard (a control male) had a bursa of Fabricius available for microscopic evaluation. The bursa in this individual exhibited the age-related involution expected in adult birds. The bursa of Fabricius was present and unremarkable in all male and female offspring. Additional findings were also noted in the liver of adults and/or offspring. Liver pigment deposition consisted of dust-like, brown to golden-brown pigment granules present in hepatocyte cytoplasm, Kupffer cells, and/or periportal areas; the identity of the pigment was not determined, but may have been related to bile. The liver of one adult control female exhibited amyloid deposition consisting -5- EPL" - 76 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-105 Wildlife International,Ltd. Study Number454-105 of amorphous, pale bluish-violet material which filled and distended the hepatic sinusoids, and compressed adjacent hepatic cords. Minimal focal hepatocellular degeneration/necrosis occurred only in a few control and treated offspring. It consisted of a few small discrete foci in which hepatocytes were pale to hypereosinophilic,had indistinct outlines, and fragmented or condensed nuclei. Hepatocellulardegeneration/necrosis was considered incidentaland unrelatedto treatment. In adults and offspring, liver fatty change denoted variably sized, sharply demarcated, clear vacuoles (consistent with fat accumulation) in the hepatocellularcytoplasm. Liver hepatocellularvacuolization denoted multiple, usually small, confluent, poorly demarcated vacuoles which often resulted in a "lace-like" appearance of the hepatocellularcytoplasm. When present in most hepatocytes,these changes were designated "diffuse," and when present in a few scattered areas, they were designated as "focal." Hepatocellularfatty change occurred sporadically at low incidences and similar severity in control and treated adults and offspring, and was considered incidental. In adults and offspring, hepatocellularvacuolization had similar incidences when control and treated groups of each sex were compared, but marginal, not always dose-related increases in severity were sometimes noted. It seems more likely that these very minor differences were more related to biological variation accentuated by small group sizes rather than being test article effects. CONCLUSIONS AND SUMMARY No lesions considered possibly related to test article (PFOS) administrationwere noted in liver, kidney, proventriculus, gallbladder, ovary, brain, and/or bursa of Fabricius, brain of adult male and female mallards or their -6- - Project Number 454-105 I 77 - I EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Wildlife International,Ltd. Study Number454-105 offspring, in the adipose tissue of adult females and offspring of both sexes, or in the testes of offspring males. Testes of adult males exhibited several features most consistentwith post-reproductivephase regression, a normal physiologicalphenomenon, including aspermia, decreased spermatogenesis, and/or decreased seminiferous tubule diameter. Group differences in the extent of these findings were evident, with aspermia occurring only in the high dose (17.6 ppm a.i.) adult males, and the degree of decrease in seminiferous tubule diameter being generally more pronounced in 17.6 ppm a.i. adult males than in control adult males. Overall, these findings are suggestive of more advanced testicular regression in the 17.6 ppm a.i. adult males compared to controls. Adult males in the 17.6 ppm a.i. group also exhibited clearly increased incidences of adipose tissue microvesiculation. These testicular and adipose tissue findings in 17.6 ppm a.i. adult males may have been fortuitous events accentuated by the small group sizes, but the possibility that they were related to test article administration cannot be entirely ruled out. The few other changes in various tissues of adult and/or offspring " mallards from control and treated groups were considered incidental and unrelated to test article (PFOS) administration. c hjlARGhRlTA M. GRUEBBEL, DVM, PhD., Di.Dlomate. ACVP Veterinary Pathologist MMG/lcp -7- EPL" -78- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454- 105 QUALITY ASSURANCE FINAL CERTIFICATION Study Title: PFOS: A Pilot Reproduction Study with the Mallard Client Study: 454-105 EPL Project Coordinator: Dr. Margarita M. Gruebbel EPL Project Number: 212-025 EPL Pathologist: Dr. Margarita M. Gruebbel The following aspects of this study were inspected by the Quality Assurance Unit of Experimental Pathology Laboratories, Inc. Dates inspections were performed and findings reported to the EPL Project Coordinator and Management are indicated below. Area Inspected Inspection Dates Reporting EPL Project Sheets Project Setup Histology Setup Data Review Draft Report Final Report 9111/00; 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 Date of last quarterly facility inspection EPL Quality Assurance Unff 9/00 //.4/01 Date -8Form No.6-2(712199) Project Number 454-105 - 79 - SUMMARY INCIDENCE TABLES ADULT SACRIFICE I 454-105 Adu1t Sacrif ice Male Mallard - 80 - SUMMARY INCIDENCE TABLE Project Number 454-105 1-1 I ExperimentalPathologyLaboratories, Inc. 454-105 Adult Sacrifice Female Mallard -81 - SUMMARY INCIDENCE TABLE Project Number 454- 105 1-2 EPL Experimental Pathology Laboratories, Inc, Project Number 454-105 - 82 - HISTOPATHOLOGY INCIDENCE TABLES ADULT SACRIFICE 454-105 Adult Sacrifice Male Mallard E P L Experimental Pathology Laboratories,Inc. 11-1 Key :X-Not Renarkable N-No Section I-Incomplete Adutolysir lnlnimal 2-slighthild Jlaoderate Moderately severe tsevere/high P-Present B-Eenlnn H-Malignant m-mirsing one paired organ u-unscheduled sac./dcath 454-105 Adult Sacrifice Female Mallard - Project Number 454-105 84 - HISTOPATHOLOGY INCIDENCE TABLE GROUP GROUP 17.6 Infiltrate, Mononuclear Cell Microvesiculation BRAIN BURSA OF FABRICIUS Involution 1 GALLBLADDER Infiltrate, Mononuclear Cell KIDNEY Infiltrate, Mononuclear Cell Mineralization I ,I 111 I I I1 1 1I 1 I ) 1 I x x x I xlx X I NNNNN N 1 1 1 111 1 Xmmmm m 1111 1 I E P L I ExperimentalPathologyLaboratories, Inc. 11-2 Key I X=Not Rsmarkable N-No Section I - I n c a p l e t s A-Autolysis l m i n i n a l 2.rli h t h i l d 3-moderate 4-noderately severe tisevere/high P-Present B-Ben?pn M-Maliflnant mmnissing one paired organ u-unscheduled sac./death - Project Number 454-105 - 85 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS ADULT SACRIFICE 454-105 Adult Sacrifice Species: Mallard CORRELATIONOF GROSS AND MlCROSCOPlCFINDINGS Sex Males GroupIdentificationr CONTROL - Sacrificed ANnuimmbaelr Client Topography / Site Client Gross Observations Microscopic Observations 1961 1963 1965 1967 1969 EXTERNAL TESTES EXTJ3RNAL EXTERNAL TESTES EXTERNATA TESTES EXTERNAL EXTERNBL TESTES Molting flight feathers No Comment Required -3.0 cm Seminiferous Tubules, Decreased Diameter (TESTIS) Bumble foot left foot No Comment Required Molting flight feathers No Comment Required -3.5 cm Seminiferous Tubules, Decreased Diameter (TESTIS) Bumble foot both feet No Comment Required -3.5 cm Seminiferous Tubules, Decreased I Diameter (TESTIS) 00 Q\ I Bumble foot right foot No Comment Required Molting flight feathers No Comment Required -2.0 cm Seminiferous Tubules, Decreased Diameter (TESTIS) 111-1 454-105 Adult Sacrifice Species Mallard OORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex Males Group Identificatioz 17.6 - Sacrificed Admaal Number 991 993 clientTopography I Site EXTERNAL EXTERNAL TESTES ,995 Ex- - - - GZ TRACT `TESTES L997 EXTERNAL TESTES 1999 TESTES Client Gross Observations Bumble foot both feet Bumble foot both feet '1.5 cm Bumble foot both feet Mass of feed impacted under the tongue -2.0 cm Bumble foot both feet "1.0 cm -2.5 cm Microscopic Observations No Comment Required No Comment Required Seminiferous Tubules, Decreased Diameter (TESTIS) No Comment Required No Comment Required Seminiferous Tubules, Decreased Diameter (TESTIS) No Comment Required Seminiferous Tubules, Decreased Diameter (TESTIS) Seminiferous Tubules, Decreased Diameter (TESTIS) , 111-2 I 00 4 I 3 9. cb 4 5 f P VI c 0 ul 454-105 Adult Sacrifice CORRELATlON OF GROSS AND MICROSCOPIC FINDINGS Animai Number 1962 clitnt Topography i Site EXTERNAL EXTERNAL OVARY 1966 1968 1970 EXTERNAL OVARY - OVARY REPRODUCTIVE EXTERNAL EXTERNAL OVARY Client Gross Observations Bumble foot both feet Molting flight feathers Regressed ovary Bumble foot both feet Regressed ovary R-egressed ovary Egg remnant Bumble foot both feet Molting flight feathers Regressed ovary Microscopic observatioll$ NO Comment Required NO Comment Required FFoilblroiscilses, Decreased Diameter; No Comment Required Follicles, Decreased Diameter Follicles, Decreased Diameter No Comment Required No Comment Required NO Comment Required Follicles, Decreased Diameter 111-3 454-105 Adult Sacrifice CORRELATION OF GROSS AND MICROSCOPIC flNDlNGS Animal Number Client Topography / Site client Gross obsemtions Microscopic Observatiom 1992 EXTERNAL RESPIRATORY Molting flight feather Air sacculitis No Comment Required Po Comment Required 1994 ABDOMINAL CAVITY OVARY EXTERNAL Egg yolk peritonitis (slight) Regressed ovary Molted flight feathers No Comment Required Follicles, Decreased Diameter -- No Comment Required OVARY Ovary regressed Follicles, Decreased Diameter 1996 EXTERNAL Bumble foot both feet No Comment Required 1998 OVARY EXTERNAL Regressed ovary I Follicles, Decreased Diameter 00 \o Yolting flight feathers No Comment Required I GENERAL CONDITION OVARY Abnormally small Regressed ovary No Comment Required Follicles, Decreased Diameter 2000 OVAXY Regressed ovary Follicles, Decreased Diameter r 111-4 - Project Number 454- 105 - 90 SUMMARY INCIDENCE TABLES OFFSPRING SACRIFICE 454-105 Offspring Sacrifice Male Mallard -91 - SUMMARY INCIDENCE TABLE Project Number 454-105 IV-1 EPL Experimental Pathology Laboratories, Inc. 454-105 Offspring Sacrifice Female Mallard I - 92 - SUMMARY INCIDENCE TABLE Project Number 454- 105 1 GROUP I GROUP I GROUP I GROUP I I I BRAIN (NOt ,EXAMINED) (5) (6) (6) (5) Infiltrate, Mononuclear Cell BURSA OF FABRICIUS (NO. EXAMINED) (5) (6) (6) (5) GALLBLADDER (NO. EXAMINED) (5) (6) (6) (5) Infiltrate, Mononuclear Cell 2 5 5 1 OVARY (NO. EXAMINED) Immature Infiltrate, Mononuclear Cell (5) (6) (6) (5) 5 6 6 5 1 1 IV-2 EPL Experimental PathologyLaboratorles, Inc. Project Number 454-105 -93 - HISTOPATHOLOGY INCIDENCE TABLES OFFSPRING SACRIFICE I 454-105 O f f spring Sacrifice Male Mallard Project Number 454- 105 - 94 - HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 1.8 GROUP 6.2 IADIPOSE L ~ TI~ SS_ UE _ . _ Infiltrate, Mononuclear Cell Microvesiculation BRAIN I Infiltrate. Mononuclear Cell BURSA OF FABRICIUS GALLBLADDER Infiltrate, Mononuclear Cell v- 1 EPL Experimental Pathology Laboratories, Inc. Key I X=Not Raarkable N-No SrctIon I=Incmplete A=AutolyrIr lalninal 2-rlt ht/nlld Swoderate taoderatety severe 5=tevcre/hIgh P-Premnt B-Ben?gn NmNal Ignant m-mioIng one paired organ uwscheduled rac./death 454-105 Offspring Sacrifice Male Mallard - 95 - Project Number 454-105 HISTOPATHOLOGY INCIDENCE TABLE GROUP 17.6 v- 2 EPL Experlmental Pathology Laboratories, Inc. Key :X-Not Renarkable N-No Section 1.Incmplete A-Autolysis 1-ninlml 2-sliqhthild 3lnoderate 4woderately severe S-severe/hlgh P-Present 8 - h i p n H.Mallgnant m i s s i n g one paired organ uwscheduled sac./death 454-105 Offspring Sacrifice Female Mallard - 96 - Project Number 454-105 HISTOPATHOLOGYINCIDENCETABLE GROUP GROUP GROUP v-3 EPL Experimental PathologyLaboratories,Inc Key :+Not Remarkable N-No Section I-Inccmplete A-Autolysis llninimal Z-slight/ni ld 3lnoderate 4-noderatsly severe 5-severelhigh P=Presmt M e n i g n H.Hallgnant m-missing one paired organ u-unscheduled rac./death 454-105 Offspring Sacrifice Female Mallard - Project Number 454-105 - 97 HISTOPATHOLOGY INCIDENCE TABLE GROUP 17.6 v- 4 EPL Experimental Pathology Laboratories, Inc. Key :X=Not Rmarkable H-No Section I*Incmplete A-Autolysis lsinimal Z-sli h t h i l d Jlodrrate 4 4 e r a t e l y severe fi-oevere/hlgh P-Present E=Een!gn Malignant nrmlssing one paired organ u*unscheduled sac-/death -98- Project Number 454- 105 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS OFFSPRING SACRIFICE 454-105 Offspring Sacrifice Species: Mallard coRREmnoFl OF GROSS AND MICROSCOPICFINDINGS Sex Males Group IdentiiicatioE CONTROL - Sacrificed Animal Number 2305 2308 2314 2316 Client Topography1Site EXTERNAL EXTERNAL EXTERNAL EXTERNAL Client Gross Observations Bumble foot both feet Bumble foot both feet Bumble foot both feet Bumble foot both feet Microscopic Observations No Comment Required No Comment Required NO Comment Required No Comment Required I t \9 L 454-105 Offspring Sacrifice - CORRELATIONOF GROSS AND MlCROSCOplCFINDINGS Group Identibication: 1.8 - Sacrificed ANnuimmabler 2323 Client Topography I Site EXTERNAL Client Gross Observations Bumble f o o t both feet Microscopic Observatiom No Comment Required L VI-2 454-105 Offspring Sacrifice Species: Mallard CORREIATION OF GROSS AND NllCROSCOPlC FINDINGS Sex Males Group Identificatioz 6.2 - Sacrificed Animal Number Client Topography / Site Client Gross Observations I Microscopic Observations 2339 EXTEBNAL Bumble foot both feet No Comment Required VI-3 454-105 Offspring Sacrifice S p e c k Mallard CORRELATIONOF GROSS AND MICROSCOPICFINDINGS Sex Males Group Identificatiox 1 7 . 6 - Sacrificed Animal Number 2354 Client Topography I Site EXTERNAL Qient Gross observations Bumble foot both feet hfiaoscopic Observations No Comment Required I c 0 h) I 454-105 ' Offspring Sacrifice CORRELATION OF GROSS AND MICROSCOPICFINDINGS Animal Number 2322 2325 2332 2333 Client Topographyf Site EXTERNAL EXTERNAL EXTEBNAL EXTERNAL Client Gross Observations Bumble foot both f e e t Bumble foot both f e e t Bumble foot both f e e t Bumble foot both f e e t Microscopic Observations No Comment Required No Comment Required No Comment Required No Comment Required I e E: I VI-5 .. I.. 454-105 Offspring Sacrifice Species: Mallard CORREIATION OF GROSS AND MICROSCOPIC FINDINGS Sex: Females GroupIdentificatioE 6 . 2 - Sacrificed ANnuimmbael r 2335 2336 234 2 2344 Client Topography Site FXTERNAL I EXTERNAL EXTERNAL EXTERNAL Client Gross Observations Bumble foot both f e e t I Bumble foot both f e e t Bumble foot l e f t f o o t Bumble foot both f e e t Microscopic obsemtiolls o Comment Required I b o Comment Required o Comment Required N o Comment Required I I c 0 P I VI-6 454-105 Offspring Sacrifice CORRELATIONOF GROSS AND MICROSCOPIC FINDINGS Animal Number 2347 - 23 4 8 Client Topography I Site EXTERNAL EXTERNAL ABDOMINAL CAVITY EXTERNAL Client Gross Observations Picked and broken feathers General unkept condition Retained yolk sac Slight bumble f o o t both f e e t Miaoswpic Observations NO Comment Required NO Comment Required No Comment Required No Comment Required I I-- 0 VI I VI-7 Appendix X Table 1 Egg Production (eggs laidlhedweek) from a Mallard Pilot ReproductionStudy with PFOS Control (0 ppm a.i.) ~~ Weeks ~~ Pen 1 2 3 4 5 6 Total E/H/D' 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total E m ' 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 27 112 M e a n 3 3 3 3 4 5 22 SD 3 3 4 3 3 3 16 1 Eggs laid per hen per day 21261313 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 Appendix X Table 2 Egg Production (eggs laidkedweek) from a Mallard Pilot Reproduction Study with PFOS Pen 1 2 206 0 0 207 0 2 208 5 7 209 0 0 210 6 6 Total 11 15 Ma 2 3 SD 3 3 1 Eggs laid per hen per day 1.8 ppm a i Weeks 3 4 5 6 7 7 6 1 4 0 0 0 7 7 7 7 0 0 0 0 7 6 6 8 25 20 19 16 5 4 4 3 3 4 3 4 Total 21 6 40 0 39 106 21 18 Em' 0.50 0.14 0.95 0.00 0.93 0.50 0.44 Appendix X Table 3 Egg Production (eggs laidhedweek) fiom a Mallard Pilot Reproduction Study with PFOS 6.2 ppm a.i. Weeks Pen 1 2 3 4 5 6 Total E/H/D' 211 0 0 0 0 0 0 0 0.00 I 212 1 4 7 6 8 6 32 0.76 Y, 0 213 0 1 0 0 0 0 1 0.02 00 I 214 0 0 0 0 0 0 0 0.00 215 4 7 6 6 6 2 31 0.74 Total 5 12 13 12 14 8 64 Mean 1 2 3 2 3 2 13 0.30 SD 2 3 4 3 4 3 17 0.41 1 Eggs laid per hen per day Appendix X Table 4 Egg Production (eggs laidhadweek)from a Mallard Pilot Reproduction Study with PFOS 17.6ppm a i . W& Pen 1 2 3 4 5 6 Total E?/H/D' 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total E/H/D' 216 5 6 7 6 6 2 32 0.76 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.00 217 0 4 5 7 6 1 23 0.55 2 7 7 7 7 7 7 4 3 4 0 0 0 0 55 0.59 218 7 4 7 6 2 7 33 0.79 1 7 4 3 0 0 0 0 0 0 0 0 0 0 15 0.16 219 0 3 7 5 6 5 26 0.62 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.00 220 3 5 7 7 4 7 33 0.79 7 1 4 7 1 0 0 0 0 0 0 0 0 0 20 0.21 Total 15 22 33 31 24 22 147 1 0 1 5 1 5 1 7 8 7 7 4 3 4 0 0 0 0 90 M e a n 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 ' Eggs laid per henper day APpendixm Page 1 Reproductive Performance by Pen from a Mallard Pilot ReproductionStudy with PFOS Table 1 Reproductive Data (Count) By Pen Parameter Pens Control(0ppm a.i.) 201 202 203 204 205 Total 1.8ppm a.i. 206 207 208 209 210 Total 6.2ppm a.i. 211 212 213 214 215 Total ~ ~~~~ 17.6ppm a.i. 216 217 218 219 220 Total EggsLaid Eggs Cracked Eggs Set viable Embryos Live 3-wk Embryos HatchIillgs survivors 5760624 0 0 0 0 0'0 5760624 3760622 3760622 2760621 2760621 4070718 00001 1 4070617 4070617 4070617 3050614 3050513 0800412 00000 0 0800412 0800412 0800412 0800412 0800412 7 5 3 5 4 24 00010 1 7534423 6 5 3 4 3 21 6534321 6424218 5424217 I c c-.. 0 I 7 9. sCD e f c 8 Replicate 1 2 3 4 5 Total Mean SD Coml(0 ppm a.i.) Eggs Eggs/ Laid Days H d a y 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 1 2 3 4 5 Total Mean SD Control (0 ppm a.i.) Eggs Eggs Cracked Laid YO 0 5 0 0 7 0 0 6 0 0 0 0 6 0 0 24 0 5 0 0 3 0 Appendix XI Page 2 ReproductivePerformanceby Pen from a Mallard Pilot Reproduction Study with PFOS Table 2 Eggs Laid / Hen / Day 1.8ppm 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 6.2ppm ai. Eggs Eggs/ Laid Days HenlDay 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 / E= Laid ('33) 1.8ppm ai. Eggs Egk3 Cracked Laid Yo 0 4 0 0 0 0 7 0 0 0 1 7 14 1 18 0 4 5 0 4 8 6.2ppm ai. Eggs Eggs Cracked Laid % ~ 0 0 0 8 0 0 0 0 0 0 4 0 0 12 0 2 0 0 4 0 17.6ppm a.i. 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 I 24 c c 5 0.69 c 1 0.21 I 17.6ppm ai. Eggs Eggs Cracked Laid ~ ~~~ 0 7 0 5 0 3 1 5 0 4 1 24 0 5 0 1 % 0 0 0 20 0 ~ ~- 4 9 Replicate 1 2 3 4 5 Total Mean SD Control (0 pprn a.i.) Viable Eggs Embryos Set % 3 5 60 7 7 100 6 6 100 0 0 6 6 100 22 24 4 5 90 3 3 20 Replicate 1 2 3 4 5 Total Meall SD Control (0 ppm ai.) Live 3-Week Viable % 3 3 100 7 7 100 6 6 100 0 0 6 6 100 22 22 4 4 100 3 3 0 Appendixx Page 3 Reproductive Performance by Pen from a Mallard Pilot Reproduction Study with PFOS Table 4 Viable Embryos / Eggs Set (%) 1.8ppm ai. Viable Eggs Embryos Set YO 4 4 100 0 0 7 7 100 0 0 6 6 100 6.2 ppm ai. Viable Eggs Embryos Set ~ ~~ 0 0 8 8 0 0 0 0 4 4 ~ YO 100 100 17 17 3 3 100 3 3 0 12 12 2 2 100 4 4 0 Table 5 Live 3-Week Embryos I Viable Embryos (%) 1.8 ppm a.i. Live 3-Week Viable YO 4 4 100 0 0 7 7 100 0 0 6 6 100 17 17 3 3 100 3 3 0 Live 3-Week 6.2 ppm ai. Viable ~ ~~~~~~ 0 0 8 8 0 0 0 0 4 4 YO 100 100 12 12 2 2 100 4 4 0 17.6 ppm ai. Viable Eggs Embrvos Set % 6 7 86 5 5 100 3 3 100 4 4 100 3 4 75 21 23 4 5 92 1 2 11 17.6 ppm a.i. Live 3-Week Viable YO 6 6 100 5 5 100 3 3 100 4 4 100 3 3 100 21 21 4 4 100 1 1 0 Replicate 1 2 3 4 5 Total Ma SD Control (0 ppm ai.) Live Hatch 3-week % 2 3 67 7 7 100 6 0 6 0 100- 6 6 100 ~~~~~ 21 22 4 4 92 3 3 17 Replicate control (0 ppm ai.) offspring SW. Hatch % 1 2 3 4 '5 2 2 100 7 7 100 6 6 100 0 0 6 6 100 Total Mean SD 21 21 4 4 100 3 3 0 Appendix XI Page 4 Reproductive Performanceby Pen from a Mallard Pilat Reproduction Study with PFOS Table 6 Hatchlugs / Live 3-Week Embryos (%) 1.8 ppm a.i. Live Hatch 3-week % 3 4 75 0 0 5 7 71 0 0 6 6 100 14 17 3 3 82 3 3 16 6.2 ppm ai. Live Hatch 3-week % 0 0 8 8 100 0 0 0 0 4 4 100 12 12 2 2 100 4 4 0 Table 7 survivingoffspring /Hatchlings 1.8ppm ai. otrspring SW. % 3 3 100 0 0 5 5 100 0 0 5 6 83 13 14 3 3 94 3 3 10 6.2ppm ai. offspring SW. YO 0 0 8 8 100 0 0 0 0 4 4 100 12 12 2 2 100 4 4 0 ~~~~ 17.6 ppm a.i. Live Jatch 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 ppm a.i. OfE5Pa surv. Hatch YO 5 6 83 4 4 100 2 2 100 4 4 100 2 2 100 ~ ~~ 17 18 3 4 97 1 2 7 Replicate 1 2 3 4 5 Total Mean SD Control (0 ppm ai.) Eggs Hatch set YO 2 5 40 7 7 100 6 6 100 0 0 6 6 100 21 24 4 5 85 3 3 30 ReDlicate control (0 ppm a.i.) om)* Eggs surv set % 1 2 3 4 5 Total Mean SD 2 5 40 7 7 100 6 6 100 0 0 6 6 100 21 24 4 5 85 3 3 30 Appendix XI Page 5 Reproductive Performance by Pen from a Mallard Pilot ReproductionStudy with PFOS Table 8 Hatchlings/ Eggs Set (%) 1.8ppm ai. Eggs Hatch Set % 3 4 75 0 0 5 7 71 0 0 6 6 100 14 17 3 3 82 3 3 16 6.2pprn a.i. Eggs Hatch set YO 0 0 8 8 100 0 0 0 0 4 4 100 12 12 2 2 100 4 4 0 Surviving Table 9 / Eggs Set (%) 1.8ppm ai. offspring Egps SurV Set YO 3 4 75 0 0 5 7 71 0 0 5 6 83 13 17 3 3 77 3 3 6 6.2ppm ai. OffSpMg Eggs SurV set % 0 0 8 8 100 0 0 0 0 4 4 100 12 12 2 2 100 4 4 0 17.6ppm a.i. Eggs Hatch Set YO ~- 6 7 86 4 5 80 2 3 67 4 4 100 2 4 50 18 23 4 5 76 2 2 19 17.6ppm a.i oapring Eggs Sllrv SPt o/, 5 7 71 4 5 80 2 3 67 4 4 100 2 4 50 17 23 3 5 74 1 2 18 I z c I 8 T 9. co E? Z E: 8 f P VI L 0 VI Replicate 1 2 3 4 5 Total Mean SD Control (0ppm ai.) Hat!& Hatch Days H d a y 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 ai.) f=Tr& Replicate Sarv offspring/ Days Hen/Day 1 2 3 4 5 Total Mean SD 2 7 0.29 7 7 1.oo 6 7 0.86 0 7 0.00 6 7 0.86 21 4 0.60 3 0.43 Appendix X I Page 6 Reproductive Performance by Pen &om a Mallard Pilot Reproduction Study with PFOS Table 10 Hatchhgs / Hen / Day Hatch 1.8 pprn ai. Hatch/ Days HeniDay 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 Hatch 6.2 ppm a i Hatch/ Days HenlDay 0 7 0.00 8 I 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 1.8ppm a.i. orrspring @ - Sum Days HenDay 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 ai. offspring 0ffW-w Surv D m 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 Whd Sum Days Heflay 5 7 0.71 4 7 0.57 2 7 0.29 4 7 0.57 2 7 0.29 17 3 0.49 1 0.19 Appendix XII Mean Offspring Body Weight (g) from a Mallard Pilot Reproduction Study with PFOS Mean Hatchling Body Weight (g) Replicate 1 2 3 4 5 Mean SD control(0 ppm a.i.) 26.5 30.6 29.7 -- 26.8 28.4 2.0 1.8 ppm a.i. 34.3 -- 31.6 -- 32.3 32.8 1.4 6.2 ppm a.i. 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 Mean Surviving Offspring' Body Weight (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 ~ ~~~ ' Offspringwere approximately12 weeks of age at final body weight interval. -- No offspring available. 17.6 ppm a i . 985 105 1 1056 1059 998 1030 35 - 117- Appendix XI11 Page 1 Adult Liver Weight (g) from a Mallard Pilot Reproduction Study with PFOS Project Number 454-105 Pen 20 1 202 203 204 205 Mean SD Control (0 ppm ai.) 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 2 10 Mean SD 1.8ppm 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 XI11 Page 2 Adult Liver Weight (g) from a Mallard Pilot Reproduction Study with PFOS Project Number 454-105 Pen 211 2 12 213 2 14 215 Mean SD 6.2ppm 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 17.6ppm 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 Offspring' Liver 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 20 1 2 1.940 206 24.539 212 20 1 2 1.925 206 33.706 212 202 17.608 206 24.375 212 202 19.435 208 26.543 212 203 17.948 208 26.833 212 203 19.424 208 27.320 212 203 24.555 210 28.403 215 205 16.544 210 26.273 215 205 24.035 210 38.193 215 205 25.485 210 20.424 215 l_l-l I_ Mean SI3 20.890 3.148 27.661 4.999 ' Offspringwere approximately 12 weeks of age at the time of euthanasiaand tissue collection. 29.540 25.845 21.894 27.367 24.851 18.934 23.160 25.229 15.702 19.680 23.220 4.215 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 - 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 fiom the shell. 2. The protocol was amended to reduce the number of eggs collected for analysis fiom 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 concentrationswere changed from 0 , 2 , 7 and 20 ppm a.i to 0, 1.8, 6.4 and 18.3ppm 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 14days of age, and listed reproductiveparameters 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 ta John Newsted, and his address was added. 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. wi - 121ldlge 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 ai. 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 - Wildlve 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 Toxicolorrv (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 Chemistrv (1) Willard B. Nixon, Ph.D., Director of Chemistry (2) Raymond L. Van Hoven, Ph.D., Scientist, Analytical Chemistry