Document K6xbjXMeXEzb9R4M35m0aB14N

PFOS: A REPRODUCTION STUDY WITH THE MALLARD FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454- 109 3M LAB REQUEST NO. U2723 FIFRA Guideline 7 1-4 OECD Guideline 206 AUTHORS: Sean P. Gallagher Raymond L. VanHoven Joann B. Beavers Mark Jaber STUDY INITIATION DATE: January 22,2001 STUDY COMPLETION DATE: August 2 1,2003 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 WildlifeInternational, Ltd. 8598 Commerce Drive Easton, Maryland 2 1601 (410) 822-8600 Page 1 of 202 wildlife International, Ltd. Project Number 454-109 -2GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Reproduction Study with the Mallard WILDLIFE INTERNATIONAL,LTD. PROJECT NUMBER: 454-109 STUDY COMPLETION: August 21,2003 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: Complete signed and dated reports for blood and tissue analyses conducted by Exygen Research and 3M Corporation are not included in the report. Summary tables are included as appendices and full reports will be submitted separately. The study was conducted under multiple protocols. The in-life and 3M analytical portions were conducted under one protocol (Wildlife International, Ltd. study number 454-109), and the Exygen Research analytical portions were conducted under two additional separate protocols (Exygen Research study number 023-066 and 023-070). Analyses of egg contents conducted under Exygen Research study number 023-070 were not conducted in accordance with Good Laboratory Practice Standards, and results of these analyses are reported separately. The analyticalphase performed by Exygen Research under study number 023-066 was performed in compliance with EPA TSCA GLP (40 CFR Part 792). STUDY DIRECTOR: Sea6 P. Gallagher I Senior Biologist, Avian Toxicology SPONSOR'S REPRESENTATIVE: DATE bfL. Newsted J DATE wildlge International, Ltd. Project Number 454-109 -3- QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U S . Environmental Protection Agency, 40 CFR part 160, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all audits and inspections and the dates any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY Test substance preparation Matrix fortification Body weight and environmental conditions Hatchling body weight 14-day old body weight Egg shell thickness measurements Candling ARDS data entry check Analytical data and draft report Biological data and draft report Final report DATE CONDUCTED January 22, 2001 January 30,2001 February 6,2001 May 24,2001 May 31,2001 July 5 , 200 1 July 6, 2001 August 14 & 15,2001 October 29, November 2 & 5 , 200 1 December 5 , 7, 10-12, 2001 August 20, 2003 DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT January 22,200 1 January 3 1,2001 January 23,2001 January 3 1,2001 February 7,2001 May 24,2001 May 31,2001 July 5 , 2001 July 6,2001 August 15,2001 February 9,2001 May 25,2001 May 31,2001 July 6, 2001 July 25, 2001 August 15,2001 November 5,2001 December 12,2001 August 20,2003 November 9,2001 March 11, 2002 August 2 1,2003 Robert N. McGee Quality Assurance Representative wildli$e International, Ltd. -4- REPORT APPROVAL Project Number 454- 109 SPONSOR: 3M Corporation TITLE: PFOS: A Reproduction Study with the Mallard WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-109 3M LAB REQUEST NO. U2723 STUDY DIRECTOR: Sean P. Gallagher I Senior Biologist, Avian Toxicology CHEMISTRY PRINCIPAL INVESTIGATOR: /I Y1d.L - Rt&t&adL. VanHoven, Ph.D. Scientist, Chemistry MANAGEMENT: ? 7h Joann B. Beavlrs Director, Avian Toxicology DATE I f c 33 03 DATE Willard B. Nixon, PI&. Director of Chemistry Wildlife International. Ltd. Project Number 454- 109 -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 .......................................................................................................................................... 9 Introduction .................................................................................................................................... 10 Objectives ....................................................................................................................................... 10 Experimental Design ...................................................................................................................... 10 Materials and Methods ................................................................................................................... 12 Test Substance ......................................................................................................................... 12 Test Organisms ........................................................................................................................ 12 Identification ............................................................................................................................ 12 Avian Feed and Water ............................................................................................................. 13 Diet Preparation ....................................................................................................................... 13 Diet Sampling .......................................................................................................................... 13 Analytical Method ................................................................................................................... 14 Study Phases ............................................................................................................................ 16 Housing and Environmental Conditions .................................................................................. 16 Observations ............................................................................................................................ 17 Adult Body Weight and Feed Consumption ............................................................................ 17 Adult Blood Collection ............................................................................................................ 17 Adult Necropsy and Tissue Collection .................................................................................... 17 Egg Collection and Storage ..................................................................................................... 18 Candling and Incubation .......................................................................................................... 18 Hatching and Brooding ............................................................................................................ 19 Offspring Blood and Tissue Collection ................................................................................... 20 Egg Shell Thickness Measurements and Egg Components Collection ................................... 20 Statistical Analyses .................................................................................................................. 20 Results and Discussion ................................................................................................................... 22 Diet Analytical Results ............................................................................................................ 22 Mortalities ................................................................................................................................ 23 Clinical Observations............................................................................................................... 24 Adult Body Weight .................................................................................................................. 24 Adult Feed Consumption ......................................................................................................... 25 Adult Necropsy and Liver Weights ......................................................................................... 25 Histopathology......................................................................................................................... 25 Adult Liver and Sera................................................................................................................ 26 Reproductive Results ............................................................................................................... 26 wildlije International, Ltd. Project Number 454-109 -6- TABLE OF CONTENTS PAGE 2 Egg Shell Thickness ................................................................................................................ 27 Offspring Body Weights ................,.....,............,,..............................,........,.,......,.........,.,...,....27 Offspring Liver Weights ..........................................................................................................27 Offspring Observations............................................................................................................27 Offspring Liver and Sera ......................................................................................................... 27 Conclusion......................................................................................................................................28 References ......................................................................................................................................29 TABLES AND FIGURES Table 1. Mean Measured Concentrations (ppm a i ) of PFOS in Avian Diet from a Mallard Reproduction Study............................................................................. 30 Table 2. Summary of Gross Pathological Observations from a Mallard Reproduction Study with PFOS, Adult Birds Found Deamuthanized during the Test .............................................................................................................. 3 I Table 3 . Mean Adult Body Weight (g) from a Mallard Reproduction Study with PFOS ................................................................................... 32 Figure 1. Mean Adult Male Body Weight (8) from a Mallard Reproduction Study with PFOS ................................................................................... 33 Figure 2. Mean Adult Female Body Weight (g) from a Mallard Reproduction Study with PFOS ................................................................................... 34 Table 4. Mean Feed Consumption (g/bird/day) from a Mallard Reproduction Study with PFOS ................................................................................... 35 Figure 3 . Mean Feed Consumption (g/bird/day) from a Mallard Reproduction Study with PFOS ................................................................................... 36 Table 5. Summary of Gross Pathological Observations from a Mallard Reproduction Study with PFOS, Adult Birds Euthanized at Test Termination ........................................ .............................................................. 37 Table 6. Mean Liver Weights (g) from a Mallard Reproduction Study with PFOS .......................................................................................................... 38 Table 7. Summary of Reproductive Performance from a Mallard Reproduction Study with PFOS ................................................................................... 39 w i l d l q e International, Ltd. Project Number 454-109 -7- TABLE OF CONTENTS PAGE 3 Figure 4. Mean Reproductive Performance from a Mallard Reproduction Study with PFOS ................................................................................... 40 Table 8. Summary of Reproductive Performance, Normalized as Percentages ("A),from a Mallard Reproduction Study with PFOS .............................. 41 Figure 5. Mean Reproductive Performance, Normalized as Percentages (%), from a Mallard Reproduction Study with PFOS .......................................................... 42 Table 9. Mean Egg Shell Thickness Measurements (mm) from a Mallard Reproduction Study with PFOS ................................................................................... 43 Table 10. Mean Body Weight (g) of Hatchlings and 14-Day Old Survivors from a Mallard Reproduction Study with PFOS...........................................................44 APPENDICES Appendix I. Certificate of Analysis .................................................................................... 45 Appendix 11. Diet and Supplement Formulations.................................................................48 Appendix 111. Diet Preparation ..............................................................................................50 Appendix IV. The Analysis of PFOS in Avian Diet .............................................................. 51 Appendix V. Diagram of Test Layout .................................................................................. 67 Appendix VI. Reproductive Parameters and Replicate Identification ...................................68 Appendix VII. Adult Body Weight (8) from a Mallard Reproduction Study with PFOS ...................................................................... 73 Appendix VIII. Feed Consumption (g/bird/day) from a Mallard Reproduction Study with PFOS ...................................................................... 81 Appendix IX. Individual Gross Pathological Observations from a Mallard Reproduction Study with PFOS ........................................................ 85 Appendix X. Appendix XI. Adult Liver Weights (g) from a Mallard Reproduction Study with PFOS ...................................................................... 87 Histopathology Report ......,............................................................................. 89 wiIdlye IntemationaI, Ltd. Project Number 454- 109 -8- TABLE OF CONTENTS PAGE 4 Appendix The Analysis of PFOS in Adult Red Blood Cells an1 Sera........................ 128 Appendix XIIB. The Analysis of PFOS in Adult and Juvenile Liver and Sera..................... 141 Appendix XIII. Reproductive Performance by Pen from a Mallard Reproduction Study with PFOS.................................................................. 154 Appendix XIV. Reproductive Performance by Week and Pen from a Mallard Reproduction Study with PFOS .................................................... 176 Appendix XV. Egg Shell Thickness Measurements (mm) per Pen by Week from a Mallard Reproduction Study with PFOS ........................................ 193 Appendix XVI. Mean Hatchling Body Weight (g) per Pen by Week from a Mallard Reproduction Study with PFOS ........................................ 195 Appendix XVII. Mean 14-Day Old Survivor Body Weight (g) per Pen by Week from a Mallard Reproduction Study with PFOS .............................. 197 Appendix XVIII. Offspring Liver Weights (g) from a Mallard Reproduction Study with PFOS.................................................................. 199 Appendix XIX. Changes to Study Protocol.......................................................................... 200 Appendix XX. Personnel Involved in Study ....................................................................... 202 wildlife International, Ltd. -9SUMMARY Project Number 454-109 STUDY: PFOS: A Reproduction Study with the Mallard SPONSOR: 3M Corporation WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454- 109 TEST DATES: Study Initiation - January 22,200 1 Experimental Start - January 23,2001 Photostimulation - April 4, 2001 First Eggs Set - April 13,2001 Adult Termination - June 20-2 1, 200 1 Analytical Termination - April 11, 2002 13iological Termination - July 26,2001 Experimental Termination - April 11,2002 TEST ANIMALS: Mallard (Anasplatyrhynchos) AGE TEST ANIMALS: 24 weeks of age at the initiation of the test SOURCE TEST ANIMALS: Whistling Wings, Inc. 113 Washington Street Hanover, IL 6 1041-0509 U.S.A. NOMINAL TEST CONCENTRATIONS: 0, 10,50, and 150 ppm a.i. RESULTS : `There were treatment-related mortalities, overt signs of toxicity and treatment-related effects upon adult body weight at the 50 and 150 pprn a i . test concentrations. There were also treatment-related effects upon feed consumption at the 150 ppm a.i. test concentration. The 50 and 150 pprn a.i. test concentrations were terminated prior to the reproductive phase of the test. There were no treatment-related mortalities, overt signs of toxicity, or treatment-related effects upon body weight, feed consumption or reproductive performance at the 10 pprn a i . test concentration. However, there was an increased incidence of small testes for males in the 10 ppm a i test concentration that may have been related to treatment. Based on the effects observed at the 10 ppm a.i test concentration, the no-observed-effect concentration for mallards exposed to PFOS in the diet was determined to be less than 10 ppm a i , the lowest concentration tested during this study. wiId1ife InternationaI, Ltd. Project Number 454-109 -10- 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 January 23, 2001 until July 26, 2001. Raw data generated at Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454-109 in archives located on the Wildlife International, Ltd. site. Biological specimens are stored at 3M Corporation, St. Paul, MN 55133. OBJECTIVES The objective of this study was to evaluate the effects upon the adult mallard (Anusplutyrhynchos) of dietary exposure to the potassium salt of Perfluorooctane Sulfonic Acid (hereafter referred to as PFOS) over a period of approximately 21 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, offspring survival, and egg shell thickness 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 then offspring. EXPERIMENTAL DESIGN Mallard (80 males and 80 females) were randomly distributed into one control group and three treatment groups. The test concentrations were selected in consultation with the Sponsor, based upon the results of a pilot reproduction study (Wildlife International, Ltd. Project Number 454-105) and additional toxicity information provided by the Sponsor. Group 1 2 3 4 PFOS Treatment Groups Nominal Concentration (ppm a.i.) (Control) 0 10 50 150 Pens per Group 16 16 16 16 Extra Pens Hematology 4 4 4 4 Birds per Pen Males Females 1 1 1 1 1 1 1 1 w i l d 1$e International, Ltd. Project Number 454- 109 - 11 - Each treatment and control group contained 16 pairs of birds with one male and one female per pen. Four additional pairs of birds were added to each test level (for a total of 20 pairs per level) and maintained for collection of blood samples during the test. Three treatment groups were fed diets containing either 10, 50, or 150 ppm xi. of PFOS. The control group was fed diet comparable to the treatment groups, but without the addition of the test substance. Because overt signs of toxicity and treatment-related mortalities were noted at the 150 pprn a.i. test concentration, the dietary concentration for the 150 ppm a.i. treatment level was reduced to 20 pprn a i . of PFOS at the beginning of Week 3 of the test. The 20 ppm a.i. level was terminated at the beginning of Week 5 of the test due to the limited recovery of birds at this test concentration. For reporting purposes, the 150/20 pprn a.i. test concentration will be referred to as the 150 ppm a.i. concentration. Adults in the 50 ppm a.i. test concentrations were euthanized at the end of Week 7 of the test due to treatment-related mortality and overt signs of toxicity. Therefore, the test was completed with the control group and the 10 pprn a.i. test concentration only. All adult birds were observed daily throughout the test for signs of toxicity or abnormal behavior. Adult body weights were measured at test initiation, on Weeks 2,4, 6, 8, and at adult termination and feed consumption was measured weekly throughout the test. At the beginning of Week 11, the photoperiod was increased to induce egg production. Following the start of egg production, eggs were set weekly for incubation. Weekly, eggs were selected by indiscriminate draw for egg shell thickness measurement and all remaining eggs were candled prior to incubation to detect egg shell cracks or abnormal eggs. Eggs were also candled twice during incubation to detect infertile eggs or embryo mortality. On Day 24 of incubation, the eggs were placed in a hatcher and allowed to hatch. Once hatching was completed, hatchlings were removed from the hatcher and the group body weight of the hatchlings by pen was determined. At 14 days of age, the average body weight by parental pen of all surviving offspring was determined. Upon completion of the test, statistical analyses were performed to determine statistically significant differences between groups. w iId1ife Internationa1, Ltd. Project Number 454-109 - .I L^- MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "PFOS: A 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; OECD Guideline 206; and the ASTM "Standard Practice for Conducting Reproductive Studies with Avian Species'' (1,2,3). Test Substance The test substance, PFOS, was received from 3M Corporation on October 29, 1998 and was assigned Wildlife International, Ltd. identification number 4675 upon receipt. The test substance was a white powder and was identified as: FC-95; Lot 217. The test material had a reported purity of 86.9% and expiration date of August 31, 2006 (Appendix I). The test substance was held under ambient conditions 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. Therefore, dietary concentrations are expressed as parts per million active ingredient (ppm a i ) in the diet. Test Organisms Approximately 84 pairs of pen-reared mallard were purchased from Whistling Wings, Inc., 113 Washington Street, Hanover, IL 61041-0509, U.S.A. At the start of acclimation, the mallard were apparently healthy and 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 were examined for physical injuries and general health. Birds that did not appear healthy, either due to injury or inability to acclimate to laboratory conditions, or were outside the weight range for the test, were excluded from the study. All birds were 24 weeks of age at test initiation (first day of exposure to test diet) and ranged in weight from 781 to 1444 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. All eggs laid during the study WildlgeInternational, Ltd. Project Number 454-109 - 13 - were marked with the pen number using a permanent ink marking pen for identification. Hatchlings were identified by wing bands so that they could be traced to their parental pen of origin. 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). 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%) (4). 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. 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 of the three treated diets were prepared weekly beginning on January 23, 2001 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 111. 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 wildlijie International, Ltd. Project Number 454-109 -14- 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, when available, during Weeks 2, 3 , 4 , 8, 12, 16 and 20 of the test to measureherify test concentrations. The diet samples were stored frozen or transferred immediately to the Wildlife International, Ltd. analytical chemistry facility for analysis. Analytical Method The method used for the analysis of PFOS in avian diet was based upon methodology developed at Wildlife International, Ltd. and entitled "Analytical Method Verification for the Determination of PFOS in Avian Diet" (Wildlife International, Ltd. Project No. 454C-110). Three modifications from the cited methodology were incorporated into the present study. These changes were (1) the use of high performance liquid chromatography with triple quadrupole mass detection (HPLC/MS/MS) rather than single quadrupole mass detection to reduce potential matrix interferences, (2) the elimination of the internal standardization procedure because an appropriate internal standard could not be identified, and (3) the use of a shorter (50 mm vs. 100 mm) analytical chromatography column to expedite analysis time. Samples were extracted with methanol, vacuum filtered, diluted in methanol, and then diluted in a 50:50 methanol: NANOpureO water dilution solvent 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 HPLC/MS/MS using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph interfaced with a PerkinElmer API 3000 Mass Spectrometer operated in multiple reaction monitoring (MRM) detection mode. The mass spectrometer was equipped with a Perkin-Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil CI8analytical column (50 mm x 2 mm I.D., 3-pm particle size) fitted with a Keystone Javelin Ci8Guard Cartridge (20 mm x 2 mm I.D.). The instrument parameters are summarized in Appendix IV, Table 1. Calibration standards were prepared in 50:50 methanol: NANOpure' water by appropriate dilutions of a 1.00 mg a.i./L stock solution of PFOS in methanol. The calibration standards, ranging in concentration from 0.00100 to 0.0100 mg a.i./L, were analyzed with each sample set. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration, quality control, and WiId1i$e International, Ltd. Project Number 454-109 - 15 - study samples. No attempt was made to quantify PFOS on the basis of individual isomeric components. Linear regression equations were generated using the peak area responses versus the respective concentrations of the calibration standards. An example of a calibration curve is presented in Appendix IV, Figure 2. The concentration of PFOS in avian feed samples was determined by substituting the peak areas into the applicable linear regression equation. Typical ion chromatograms of low and high calibration standards are shown in Appendix IV, Figures 3 and 4, respectively. Examples of equations used in calculations are presented in Appendix IV, Table 2. The instrument limit of detection (LOD) for these analyses was 10.0 pg on-column, calculated from the product of the injection volume (10.0 yL) and the lowest standard concentration 1.OO yg a.i./L = 1.OO pg a.i./yL. The method limit of quantitation (LOQ) for these analyses was 2.00 ppm ai., calculated as the product of the lowest calibration standard analyzed (0.001 mg a.i./L) and the overall dilution factor of the matrix blank sample (2000 LKg). Examples of calculations are presented in Appendix IV, Table 2. Along with the sample analyses, seven 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 the following levels and analyzed concurrently with the samples to determine the mean procedural recovery. The fortification levels were 5.00, 50.0, and 200 ppm a.i. for sampling intervals Week 1, Day 0; Week 1, Day 7; Week 2, Day 0; and Week 3 , Day 0. The fortification levels were 5.00, 25.0, and 100 pprn a.i. for sampling interval Week 4, Day 0. The fortification levels were 5.00 and 25.0 ppm a.i. for sampling intervals Week 8, Day 0; Week 12, Day 0; Week 16, Day 0; and Week 20, Day 0. The method yielded mean procedural recoveries of 101%, 101%, 102%, 100% and 99%, respectively, for the 5.00, 25.0, 50.0, 100, and 200 ppm a.i. fortification levels (Appendix IV, Table 3 ) . Sample measured concentrations were not corrected for the mean procedural recoveries. A typical ion chromatogram of a matrix fortification is presented in Appendix IV, Figure 6. Wildlife International, Ltd. Project Number 454-109 - 16 - Study Phases The primary phases of the study and their approximate durations were: 1. Acclimation - 3 weeks. 2. Pre-photostimulation - 10 weeks. 3. Pre-egg laying (with photostimulation) - 0 weeks. 4. Egg laying - Approximately 10 weeks. 5. Post-adult termination (final incubation, hatching, and 14-day offspring rearing period) - 5 weeks. Housing and Environmental Conditions Housing and husbandry practices were conducted so as to adhere to the guidelines established by the National Research Council (5). The adult birds were housed indoors in batteries of pens manufactured by Safeguard Products, Inc. (Model No. 5355), measuring approximately 75 X 90 X 45 cm high. The pens were constructed of vinyl-coated wire mesh. A diagram of the test layout is presented in Appendix V. Each pen was equipped with a feed trough. Weekly, sufficient feed for the feeding period was placed in the trough for each pen and presented to the birds. During the feeding period additional feed was weighed and added to the troughs as needed. Water was supplied by nipple-type waterers. Only birds associated with this study were maintained in the study room in order to avoid excessive disturbances. The average temperature in the adult mallard study room during the course of the test was 21.7"C f 1.1"C (SD) with an average relative humidity of 40% f 19% (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 timc clock. The photoperiod during acclimation and the first 10 weeks of the test was eight hours or less of light per day. The photoperiod was increased to 17 hours of light per day at the beginning of Week 11 to induce egg laying and was maintained at that length until the adult birds were euthanized. Throughout the test, the birds received a mean of approximately 188 lux (- 17 ft. candles) of illumination provided by fluorescent lights that closely approximated noon-day sunlight. WildlifeInternational, Ltd. Project Number 454-109 - 17 - Observations The test birds were acclimated to the facilities and study pens for approximately 3 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 14 days of age. A record was maintained of all mortalities and clinical observations. Adult Body Weight and Feed Consumption Adult body weights were measured at test initiation, on Weeks 2, 4,6, 8, and at adult termination. Body weights were not measured during egg laying because of the possible adverse effects handling may have on egg production. 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. Adult Blood Collection Four additional pairs of adult birds were added to each test level to be maintained for collection of blood samples during the test. These pairs received the same diet and were handled in the same manner as other pairs in their test group. However, because of the potential stress of the blood collection procedures, data from the four additional pairs were not used to evaluate treatment-related effects. Blood samples were collected from birds in these designated pairs, when possible, at five week intervals throughout the test. At the time of adult termination, blood samples were collected from all surviving birds, when possible, prior to euthanasia. All blood samples were separated into serum and hemacytes/platelets, stored frozen and shipped to the sponsor or sponsor's designate for possible analysis. Adult Necropsy and Tissue Collection When the remaining birds from 150/20 pm a.i. treatment group were euthanized at the end of the Week 4, no necropsies were performed. All other adult birds that died or were euthanized during the wildlife International, Ltd. Project Number 454-109 -18- course of the study were subjected to a gross necropsy. At the conclusion of the exposure period, all surviving adult birds were euthanized by cervical dislocation, necropsied, and disposed of by incineration. At the time of necropsy, tissues were collected for histopathological examination 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 and shipped to EPL in Herndon, VA for histopathology. When available, samples of bile, liver, proventriculus, kidneys, brain, reproductive organs and adipose tissue were stored frozen and shipped to the Sponsor for possible analysis. Fecal samples were also collected, when available, from the lower digestive tract and stored frozen for possible analysis. Egg Collection and Storage Eggs were collected daily from all pens, when available. Eggs to be incubated were washed to reduce the possibility of pathogen contamination before storing them in the cold room. 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 eggs were then stored in a cold room until incubation. The cold room was maintained at a mean temperature of 13.5"C f 0.2"C (SD) with a mean relative humidity of approximately 69% f 6% (SD). Groups of eggs were identified by an alphabetic lot code. All eggs laid in a weekly interval 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 eggs selectcd by indiscriminate draw for egg shell thickness measurement. The remaining eggs were 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 or used for egg shell thickness measurements were placed in a Petersime Incubator (Model No. SP20). In the incubator the temperature was maintained at an average 37.5"C * 0.0"C (SD) with an average wet bulb temperature of 30.5"C f 0.3"C (SD) (relative humidity of approximately 60%). The incubator was equipped with a pulsator fan and blades that produced a mild w i l d 1ife Internationa1, Ltd. Project Number 454-109 - 19- 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 fiom 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 13 or 14 of incubation to determine embryo viability and on Day 2 1 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"C f 0.0"C (SD), and the average wet bulb temperature was raised to 33.8"C * 0.2"C (SD) (relative humidity of approximately 80%). 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. Hatchlings were wing banded for identification by pen of origin and then routinely housed according to the appropriate parental concentration grouping in brooding pens until 14 days of age. The hatchlings were fed untreated diet without the addition of 5% supplemental limestone. At 14 days of age, the average body weight by parental pen of all surviving ducklings was determined. The ducklings were euthanized with carbon dioxide and 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 maintain a temperature of approximately 29C. The average ambient room temperature was 24.2"C i 1.2"C (SD) with an average relative humidity of 77% f 15% (SD). The photoperiod for the hatchlings was maintained by a time clock at 16 hours of light per day. wildlije InternationaI, Ltd. Projec,tNumber 454-109 - 20 - Offspring Blood and Tissue Collection Prior to euthanasia of the last group of offspring (Lot J), blood samples were collected from 10 offspring in both the control group and 10 ppm a i . treatment group. All blood samples were separated into serum and hemacytes/platelets, stored frozen and shipped to the sponsor for possible analyses. Additionally, tissues from the 10 offspring in each group were collected for histopathological examination. 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, liver, proventriculus, kidneys, brain, reproductive organs and adipose tissue were stored frozen and shipped to the sponsor for possible analyses. Fecal samples were also collected from pans below the floor of brooders housing Lot J offspring and stored frozen for possible analysis. A single composite sample was collected for the control group and a single composite sample was collected for the 10 ppm a.i. treatment group. Egg Shell Thickness Measurements and Egg Components Collection Weekly throughout the egg laying period one egg was collected, when available, from each of the test pens. Eggs were collected from odd numbered pens during odd numbered weeks (1,3,5,etc.) and from the even numbered pens during the even numbered weeks (2,4,6, etc.). The eggs from weeks 1, 2 and 10 were separated into albumen, yolk, shell and membrane and all components were stored frozen. Eggs collected during weeks 3-9 were opened at the waist and their contents removed. The egg contents were separated into albumen and yolk and the yolk was stored frozen. All egg samples were transferred to the Sponsor for possible analysis. The shells from Weeks 3 through 9 were allowed to air dry for at least one week at room temperature prior to being measured for thickness. The average thickness of the dried shell plus the membrane was determined by measuring five points around the waist of the egg using a micrometer. Measurements were made to the nearest 0.002 111111. 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 (6,7) 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 adult body weights where the sample unit was the individual bird. Percentage data were examined using Dunnett's method following arcsine square root transformation (see Appendix VI for wildlife International, Ltd. Project Number 454-109 -21 - reproductive parameters). With the exception of liver weight, statistical analysis of the data was performed using Avian Reproduction Data System (ARDS) Software; a validated software package developed by Wildlife International, Ltd. (8). Liver weights were compared by Student's t-test using Microsoft Excel. Each of the following parameters was analyzed statistically: 1. Adult Body Weight - Individual body weight was measured at test initiation, Weeks 2, 4, 6, 8 and at adult termination. Statistical comparisons were made between the control group and each treatment group at each weighing interval by sex. 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. 3. Eggs Laid of Maximum Laid - The number of eggs laid per female divided by the largest number of eggs laid by any one female. This transformation was used to convert the number of eggs laid to a percentile value less than or equal to 100. 4. Eggs Cracked of Eggs Laid - The number of eggs determined by candling to be cracked divided by the number of eggs laid, per pen. 5 . Viable Embryos of Eggs Set - The number of viable embryos at the Day 14 candling was divided by the number of eggs set, per pen. 6. 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. 7. 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. 8. 14-Day Old Survivors of Hatchlings - The number of 14-day old survivors was divided by the number of hatchlings per week, by pen. 9. Hatchlings of Eggs Set - Thc number of hatchlings was divided by the number of eggs set per week, by pen. 10. 14-Day Old Survivors of Eggs Set - The number of 14-day old survivors was divided by the number of eggs set per week, by pen. 11. Hatchling- s of Maximum Set - The number of hatchlings per female divided by the largest number of eggs set from any one female. This transformation was used to convert the number of hatchlings to a percentile value equal to or less than 100. WiId1ye Intemationa1, I,td. Project Number 454-109 - 22 - 12. 14-Day Old Survivors of Maximum Set - The number of 14-day old survivors per pen divided by the largest number of eggs set. 13. Egg Shell Thickness - The average egg shell thickness of indiscriminately selected eggs per pen was measured. 14. Offspring's Body Weight - The group body weights of surviving hatchlings and 14-day old survivors were measured by parental pen group. 15. Liver Weight - Individual liver weights were measured at adult termination and at termination of selected Lot J offspring. Statistical comparisons were made by sex between the control group and 10 pprn a.i. group. RESULTS AND DISCUSSION Mature mallards received PFOS at nominal dietary concentrations of 10, 50, and 150 pprn a.i. Due to extensive overt signs of toxicity and treatment-related mortalities in the 50 and 150 ppm a.i. treatment groups, the experimental design was modified. Adults in the 50 ppm a.i. test concentrations were euthanized at the end of Week 7 of the test. The dietary concentration for the 150 pprn a.i. treatment level was reduced to 20 pprn a.i. of PFOS at the beginning of Week 3 of the test. The 20 pprn a.i. level was terminated at the beginning of Week 5 of the test due to the limited recovery of birds at this level. Therefore, the test was completed with the control group and 10 pprn a.i. test concentrations only. The control group was fed diet comparable to the treatment groups, but without the addition of the test substance. Diet Analytical Results None of the control samples showed any indication of the presence of the test substance or of the presence of co-eluting substances at the characteristic retention time of the test substance. Diet samples were collected from the 10, 50 and 150 pprn a.i. test concentrations on Week 1, Day 0, and were analyzed to evaluate the homogeneity of the test substance in the diet and to verify test substance concentrations. Means and standard deviations for the three test concentrations were 9.75 k 0.313 ppm a.i., 50.1 f 0.981 ppm a.i. and 147 f 4.31 pprn a.i., respectively. The coefficients of variation were 3.21%, 1.96% and 2.93%, respectively. These values represented 98%, 1OO%, and 98% of nominal concentrations (Appendix IV, Table 4). wildlife International, Ltd. Project Number 454-109 - 23 - Samples collected throughout the test to verify test substance concentrations for the 10, 20, 50, and 150 ppm a i diets had means and standard deviations of 10.2 f 0.553 pprn a i , 20.8 f 2.14 ppm a i , 50.9 * 2.04 ppm a i , and 161 f 15.6 ppm a i , respectively. The coefficients of variation were 5.44%, 10.3%, 4.01% and 9.66%, respectively. These values represented 102, 104, 102, and 108% of nominal concentrations (Appendix IV, Table 5). Analysis of diet samples collected from feeders after being held at ambient temperature for 7 days averaged loo%, 101% and 97% of the Day 0 values for the 10, 50 and 150 ppm xi. test concentrations, respectively (Appendix IV, Table 6). A typical ion chromatogram of a test sample is shown in Appendix IV, Figure 7. Mean weekly measured concentrations for each test group are presented in Table 1. Mortalities While there was one incidental mortality in the control group, there were no mortalities in the 10 pprn a.i. treatment group. Three incidental mortalities and ten treatment-related mortalities occurred in the 50 ppm a.i. treatment group and four treatment-related mortalities occurred in the 150 ppm a.i. treatment group. The single incidental mortality in the control group was a male (Pen 2 10) that died during Week 15 of the test. Gross necropsy of the drake revealed foot lesions on both feet. Internally, hemorrhages were noted in the myocardium, the abdominal air sacs were cloudy and the spleen was enlarged and mottled. Additionally, the liver was firm,mottled and pale, the kidneys were slightly pale and a small amount of clear fluid was noted in the abdominal cavity. Necropsy of the bird's penmate at termination of the adult portion of the study revealed a regressed ovary, but was otherwise unremarkable. Three incidental and ten treatment-related mortalities occurred in the 50 pprn a i treatment group and four treatment-related moralities occurred in the 150 pprn a.i. treatment group. Necropsy findings for the treatment related mortalities in the 50 and 150 ppm a.i. test concentrations were similar. Common findings included thin or emaciated condition with a loss of muscle mass and prominent keel, small spleen and pale kidneys. All remaining birds at the 150 ppm a.i. test concentration were euthanized at the beginning of Week 5. Due to the shortened nature of the exposure period, necropsies were not performed. All remaining birds at the 50 ppm a i test concentration were euthanized at the beginning of Week 8 of the test, and necropsies were performed following euthanasia. Necropsy findings are summarized in Table 2. wildlife International, Ltd. Project Number 454-109 - 24 - Clinical Observations No overt signs of toxicity were observed at the 10 pprn a.i. test concentration. With the exception of foot lesions and occasional lameness, all birds in the control group and 10 ppm xi. treatment group were normal in appearance and behavior throughout the test. In the 150 pprn a i . and 50 ppm a.i. treatment groups, signs of toxicity were first noted at the end of Week 2. One male in the 50 pprn a.i. treatment group and several birds in the 150 pm a i . treatment group exhibited signs of toxicity induced by the stress of handling during the Week 2 body weight measurements. In several cases, birds convulsed during body weight measurements. Signs of toxicity in both the 50 and 150 ppm a.i. treatment groups continued to be observed until the birds were euthanized. Clinical signs of toxicity observed included depression, reduced reaction to external stimuli (sound and movement), wing droop, loss of coordination, ruffled appearance, thin appearance, lower limb weakness, lethargy, lacrimation and convulsions. Adult Body Weight There were no apparent treatment-related effects upon adult body weight at the 10 pprn a.i. test concentration, and any differences between the control group and the 10 ppm a.i. test concentration were not statistically significant at any of the body weight intervals. However, there were marked treatmentrelated reductions in mean body weight at both the 50 and 150 pprn a.i. test concentrations. With the exception of the mean male body weight at the Week 2 interval, all differences between the control group and birds in the 50 ppm a.i. treatment group were statistically significant at p < 0.01. At the 150 ppm a i test concentration, mean male and female body weights were statistically different from the control group at p < 0.01 at both the Week 2 and Week 4 body weight intervals. Between the Week 2 and Week 4 body weight intervals, a weight gain was noted for birds at the 150 pprn a.i. test concentration. However, the weight gain observed was a reflection of the reduction of the concentration of PFOS in the diet to 20 ppm a.i. during Week 3 of the test. Mean body weight measurements are presented in Table 3 , and Figures 1 and 2. Individual body weight measurements are presented in Appendix VII. Wildlife International, Ltd. Project Number 454-109 - 25 - Adult Feed Consumption There were no apparent treatment-related effects upon feed consumption at the 10 or 50 pprn a.i. test concentrations. No statistically significant differences between the control group and 10 or 50 ppm xi. treatment groups were observed at any of the feed consumption intervals. However, treatment-related reductions in feed consumption were observed at the 150 ppm a.i. test concentration during Weeks 1 and 2. The reductions were statistically significant at p < 0.05 during Week 1 and at p < 0.01 during Week 2. The dietary concentration for the 150 ppm xi. treatment group was reduced to 20 ppm a i . during Week 3 of the test. There was a statistically significant (p < 0.01) increase in feed consumption at the 150 (20) ppm a i . test concentration during Weeks 3 and 4 of the test. Mean feed consumption measurements are shown in Table 4 and Figure 3. Feed consumption measurements by pen are presented in Appendix VIII. Adult Necropsy and Liver Weights All surviving adults in the control group and the 10 ppm a.i. treatment group were subjected to gross necropsy following adult termination. When compared to the control group, there was an increased incidence of males with small testes (approximate length < 3.5 cm) in the 10 ppm a.i. treatment group for which a treatment-related effect could not be precluded. Two males in the control group were noted with small testes, compared to seven males in the 10 pprn a.i. group. All other findings were considered unrelated to treatment. Necropsy findings are reported in Table 5 and Appendix IX. When compared to the control group, there were no treatment-related effects upon the mean weights of adult livers at the 10 ppm a.i. test concentration. Any difference between the 10 pprn a i . treatment group and the control group was not statistically significant. Mean adult liver weights are presented in Table 6, while individual adult liver weights are presented in Appendix X. Histopathology No lesions considered related to test article (PFOS) administration were noted in the liver, kidney, adipose tissue, brain, and proventriculus of adult and offspring males and females, bursa of Fabricius in offspring males and females, ovaries of adult and offspring females and testes of male offspring. The increase in the number of adult males in the 10 ppm a i treated group with reduced testicular size (with or without a corresponding microscopic observation), suggests that PFOS may have accelerated early postreproductive phase regression, a normal physiological phenomenon. Any potential test-article effects on the bursa of Fabricius in adult males and females could not be determined since samples were not wildlife International, Ltd. Project Number 454-109 - 26 - submitted for evaluation. In addition, any potential test-article effects on the gallbladder from adults and offspring could not be definitively determined due to the extensive autolysis that precluded any accurate diagnosis. The few other changes in various tissues of adult and/or offspring mallards from control and 10 ppm a.i. treated groups were considered incidental and unrelated to test article (PFOS) administration. The full histopathology report is provided in Appendix XI. Adult Liver and Sera Adult blood samples (both red blood cells and sera) collected through Week 15 of the test were analyzed by Exygen Research. The results for analysis of samples collected for this study are reported together with results for the northern bobwhite study run concurrently with the same test concentrations. Summary results of blood analyses performed for this study are presented in Exygen report Tables X through XXIV located in Appendix XIIA The full report for analysis of blood samples by Exygen Research is located under Exygen Study Number 023-066. "Extraction of Potassium Perfluorooctanesulfonate from Red Blood Cells and Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry". Blood and liver samples collected at the time of adult termination for the control group and 10 pprn a.i. test concentration were shipped to 3M Environmental Laboratory for analysis. Summary results of sera and liver analyses conducted by 3M Environmental Laboratory are presented in 3M report Tables 1, 2 and 10 through 15 located in Appendix XIIB. The full report for analysis of sera and liver samples collected at the time of adult termination is located in 3M Environmental Laboratory report E01-1256. "Analytical Phase Report for PFOS: A Reproduction Study with the Mallard". Reproductive Results There were no treatment-related effects upon reproductive performance at the 10 ppm a.i. test concentration as compared to the controls. When compared to the control group, there were no statistically significant differences in any of the reproductive parameters measured in the 10 pprn a.i. test concentration. Summaries of the reproductive data are presented in Tables 7 and 8, and in Figures 4 and 5. Reproductive parameters by pen are presented in Appendix XI11 and XIV. wildlife International, Ltd. Project Number 454-109 -27 - Egg Shell Thickness There were no apparent treatment related effects upon egg shell thickness at the 10 ppm a.i. test concentration. When compared to the control group, there were no statistically significant differences in egg shell thickness in the 10 ppm a.i. treatment group. Egg shell thickness data are presented in Table 9 and Appendix XV. Offspring Body Weights There were no apparent treatment related effects upon offspring body weight at the 10 pprn a.i. test concentration. When compared to the control group, there were no statistically significant differences in the body weight of hatchlings or 14-day old survivors from the 10 pprn a.i. treatment group. Offspring body weight data are presented in Table 10, and Appendices XVI and XVII. Offspring Liver Weights There were no treatment-related effects upon the mean weights of offspring livers at the 10 pprn a.i. test concentration. Any difference between the 10 ppm a i . treatment group and the control group was not statistically significant. Mean offspring liver weights are presented in Table 6, while individual offspring liver weights are presented in Appendix XVIII. Offspring Observations There were incidental observations of neck curl, weakened condition and foot/leg lesions and associated lameness, for offspring in both the control group and 10 ppm a.i. treatment group. At the 10 ppm a i test concentration, a general observation was made that the offspring from Lots C and D were sluggish, with a slight reduced reaction to external stimuli. None of the offspring in the Lots proceeding or following Lots C and D were noted as sluggish or with a reduced reaction to external stimuli. Due to the isolated nature of the clinical signs of toxicity noted, these observations were not considered treatment related. All other offspring were normal in appearance and behavior throughout the test. Offspring Liver and Sera Blood and liver samples collected at the time of offspring termination for the control group and 10 pprn a i . test concentration were shipped to 3M Environmental Laboratory for analysis. Summary results of sera and liver analyses conducted by 3M Environmental Laboratory are presented in 3M report Tables 1, 2 and 10 through 15 located in Appendix XIIB. The full report for analysis of sera and liver samples wildlijie International, Ltd. Project Number 454-109 - 28 - collected at the time of juvenile termination is located in 3M Environmental Laboratory report EO 1-1256. "Analytical Phase Report for PFOS: A Reproduction Study with the Mallard". CONCLUSION There were no treatment-related mortalities, overt signs of toxicity, or treatment-related effects upon body weight, feed consumption or reproductive performance at the 10 ppm a i . test concentration. However, there was an increased incidence of small testes for males in the 10 ppm a.i. test concentration that may have been related to treatment. There were treatment-related mortalities, overt signs of toxicity and treatment-related effects upon adult body weight at the 50 and 150 ppm a i test concentrations. There were also treatment-related effects feed consumption at the 150 pprn a i test concentration. The 50 and 150 pprn a.i. test concentrations were terminated prior to the reproductive phase of the test. Based on the effect observed at the 10 ppm a.i test concentration, the no-observed-effect concentration for mallards exposed to PFOS in the diet was determined to be less than 10 pprn a i , the lowest concentration tested during this study. WildlifeInternational, Ltd. - 29 REFERENCES Project Number 454- 109 1 U.S. Environmental Protection Agency. 1982. Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, subsection 71-4, Environmental Protection Agency, Office of Pesticide Programs. Washington, D.C. 2 Organization for Economic Cooperation and Development. 1984. Avian Reproduction Test. OECD Guideline for Testing of Chemicals. Guideline 206. Paris. 3 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. 15 pp. 4 Merck & Co., Inc. 1991. The Merck Veterinary Manual. Merck & Co. Rahway, NJ. 1832 pp. 5 National Research Council. 1996. Guide for the Care and Use of Laboratory Animals. Washington, DC. National Academy Press. 125 pp. 6 Dunnett, C.W. 1955. A Multiple Comparison's Procedure for Comparing Several Treatments with a Control. Jour. Amer. Statis. Assoc. 50: 1096-1121. 7 Dunnett, C.W. 1964. New Tables for Multiple Comparisons with a Control. Biometrics 20: 482491. 8. Wildlife International, Ltd.. 1994. Avian Reproduction Data System (ARDS) Version 2. Nominal Concentration (ppm a.i.) Table 1 Mean Measured Concentrations (ppm a i ) of PFOS in Avian Diet from a Mallard Reproduction Study Week 1 DayO Day7l Week 2 DayO Week 3 DayO Interval Week 4 DayO Week 8 DayO Week 12 DayO Week 16 DayO Week 20 DayO 0 Measured <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 Mean 10 Measured % Nominal 98 100 103 101 97 109 101 101 99 Mean 50 Measured %Nominal 100 101 97 104 104 Mean 150 Measured % Nominal 98 97 108 96 112 ~~ ~ ~~ 'Feed samples were collected from feed troughs at the end of Week 1 to assess stabilityof the test substance under actual test conditions. z <LOQ indicates the value was less than the limit of quantitation. The mean of homogeneity samples collected at Week 1, Day 0. The percent of the mean values for Week 1, Day 0. 5 The 150 ppm a i . treated diet was reduced to 20 ppm a.1. during this interval. - No values available, test group terminated. -31 - Project Number 454-109 Table 2 Summary of Gross Pathological Observations from a Mallard Reproduction Study with PFOS Adult Birds Found DeadEuthanized during the Test Control (0 ppm a i . ) 'Males 50 ppm a.i. 150 ppm a.i. ' Females 50 150 ppm a.i. ppm a.i. Number of birds 1 17 2 Abrasion on mandible 0 1 0 Foot/leg lesions 1 3 0 Thin 0 4 0 Emaciated 0 0 1 Loss of muscle mass 0 1 1 Prominent keel Breast muscle - hematoma and loose blood Heart - flaccid 0 0 1 0 1 0 0 0 0 Heart - pale 0 3 0 Heart - petechial to ecchymatic hemorrhages 1 0 0 Left lung - yellow plaques on margins 0 1 0 Lungs - pale 0 1 0 Abdominal air sacs - cloudy 1 0 0 Spleen - small a n d o r pale 0 7 0 Spleen - enlarged and mottled 1 0 0 Liver - firm, pale and mottled 1 0 0 Abdominal cavity - autolysis 0 1 0 Abdominal cavity - small amount of clear fluid Abdominal cavity - blood in left thoracic air sac 1 0 0 0 1 0 Gizzard contents - bile-stained 0 3 0 Small intestines - small white ulcers (polyps) 0 1 0 Intestinal contents - pasty 0 0 0 Intestinal contents - firm 0 1 0 Kidneys - pale 1 4 0 Not remarkable 0 7 1 18 2 0 0 0 0 3 0 2 1 3 1 2 1 0 0 1 0 1 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 2 0 0 0 0 2 1 0 0 0 1 1 0 0 1 10 0 I Remaining birds in the 50 ppm a.i. test concentrationwere euthanized at the end of Week 7 of the test. Gross necropsy observations for birds from which blood samples were collected are not reported. 'Remaining birds in the 150 ppm a.i. test concentrationwere euthanized at the beginning of Week 5. Gross necropsies were not performed at the termination of the 150 ppm a i . test concentration. Table 3 Mean Adult Body Weight (9)from a Mallard Reproduction Study with PFOS' Experimental Group (ppm a-i.) sex Week 0 Change Week 0-2 Week 2 Change' Week 4 Change Week 2-4 Week 4-6 Week 6 Change Week 6-8 Week 8 Change Week 8-Term Test Total Term Change Control 10 50 150 Male Female Male Female Male Female Mal e Female 1084 7 984 4 1111 10 982 22 1104 -57 956 -83 1107 985 -195 -206 1091 23 988 14 1121 7 1004 18 1047 -64 873** -60 912** 88 780** 98 1114 13 1002 19 1127 17 1022 19 983** -66 813** -31 1016** 880** - 1128 -3 1020 12 1144 7 1041 9 916** 781** 112 5 52 1033 122 1151 -24 1049 95 1164 92 1155 171 1127 17 1144 162 I w w I The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. * * Significantly different from the control at p < 0.01 'Only surviving birds were included in the calculations for each body weight interval. Figure 1 Mean Adult Male Body Ueight (9) from a M a l l a r d Reproduction Study u i t h PFOS llool 1200r 1000 h cp 900 v 3- 500 I m W W 400 I -J 300 200 100 0 Week 2 Change Week 4 Change Week 6 Change Week 8 Change Week T Total deet 0 Change Wk 2-4 * Wk 0-2 Data are not available due to adult mortality Uk 4-6 WEEKS Wk 6-8 Wk 8-T Change 170 I 10 Concentrations, ppm 50 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 Figure 2 Mean hdult Female Body Weight ( g ) from a M a l l a r d Reproduction Study w i t h PFOS Week 4 Change Week 6 Change Week 8 Change Week T Tot a1 Wk 4-6 Wk 6-8 Wk 8-T Change * Wk 0-2 Data a r e not available due to adult mortality WEEKS 00 Y 10 Concentrations, ppm 50 I w P I P ul f c 0 \o Table 4 Mean Feed Consumption (g/bird/dayl f r o m a Mallard Reproduction Study with PFOS Experimental WEEKS Group (ppm a - i . ) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Control 102 122 119 134 124 133 133 134 135 132 135 178 185 173 178 178 191 212 194 195 205 10 119 146 134 146 137 145 148 145 139 138 129 158 183 175 175 172 180 188 178 189 193 50 101 114 118 129 116 124 112 - 150 73* 75**166**194** - - I - - - - w ul I - - - - Data are not available due to adult mortality * Significantly different from the control at p < 0.05 * * Significantly different from the control at p < 0.01 Figure 3 Mean Feed Consumption ( g / b i r d / d a y ) from a Reproduction Study with PFOS Mallard I w m I 60 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Weeks ..*...., za - 0 11.1 10 Concentrations, ppm 50 _..__.1_50_ G 5 8 -37- Project Number 454-109 Table 5 Summary of Gross Pathological Observations from a Mallard Reproduction Study with PFOS Adult Birds Euthanized at Test Termination' Males Control 10 (0 ppm a.i.) ppm a.i. Females Control 10 (0 ppm a.i.) ppm a i . Number of birds 15 16 Foot/leg lesions 4 4 Breast blister 0 0 Cranium - swollen and bruised 0 0 Heart - pericardial sac fluid-filled 0 0 Spleen - enlarged and mottled 0 0 Spleen - enlarged and firm 0 0 Liver - firm, pale and mottled 0 0 Abdominal cavity - yolk remnants Abdominal cavity - slight egg yolk peritonitis Abdominal cavity - egg yolk peritonitis Abdominal cavity - slight old egg yolk peritonitis Small intestines - area of hyperemia 0 0 Kidneys - pale 0 0 Testes - small (- 1.5-3.5 cm) 2 6 Reproductive tract - multiple fluid filled cysts Ovary - regressing Ovary - regressed Ovary - cystic follicles Ovary - hemorrhagic follicles Not remarkable 9 8 16 16 8 13 0 2 0 1 0 2 1 0 0 1 1 0 1 0 1 0 2 3 5 0 2 0 1 0 1 0 1 0 4 4 1 1 1 0 2 0 'Gross necropsyobservationsfor birds maintained for collectionof blood samplesduring the test are not reported. Table 6 Mean Liver Weights' (g) from a Mallard Reproduction Study with PFOS Experimental Group Adult Males Mean SD Adult Females Mean SD Offspring Males Mean SD Offspring Females Mean SD Control 0 ppm a.i. 29.422 f 3.864 41.648 f 10.512 14.534 f 4.679 14.951 f 2.326 10 ppm a.1. 30.601 f 4.633 42.207 f 10.268 13.652 f 4.398 14.068 f 2.143 50 ppm a i . 150 ppm a.i. 1Mean f standard deviation. - Data not available due to mortality. The above differences from the control group were not statistically significant(Student'st-test). I w 00 I - P ch f 0 \D Table 7 Summary of Reproductive Performance f r o m a Mallard Reproduction Study w i t h PFOS Reproductive Parameter Control Number of Replicates Total Eggs Laid Eggs Cracked Eggs Set Viable Embryos Live 3-Week Embryos Hatchlings 14-Day Old Survivors Eggs Laid/Hen Eggs Laid/Hen/Day' 14-Day Old Survivors/Hen 15 719 10 644 567 564 460 454 4a 0.67 30 'Based on 72 days of eggs production. - Data are not available due t o adult mortality Experimental Group (ppm a.i.1 10 50 16 877 10 781 664 653 522 507 55 0.76 32 150 I w \o I - - 5 3 5 3 P VI P I 60 50 .. .. .. Figure 4 Mean Reproductive Performance from a M a l l a r d Reproduction Study u i t h PFOS n ~~ .. .. .. Z w Q \ a W m E I 3 Z P 0 Z I 6 W E ES Laid Cont r 01 m Eggs ES Viable Cracked Set Embryos * Data are not available due to adult mortality * & ppm Live 3-wk Embryos Hatchlings * 150ppm E 14-Day Old Survivors Table 8 Summary of Reproductive Performance, Normalized as Percentages (%) from a Mallard Reproduction Study with PFOS' Reproductive Parameter Control Number of Replicates 2 Total Eggs Laid Eggs Laid/Maximum Laid ( % ) Eggs Cracked/Eggs Laid ( % ) Viable Embryos/Eggs Set ( % ) Live 3-Week Embryos/Viable Embryos ( % ) Hatchlings/Live 3-Week Embryos ( % ) 14-Day Old Survivors/Hatchlings ( % ) Hatchlings/Eggs Set ( % ) 14-Day Old Survivors/Eggs Set ( % ) Hatchlings/Maximum Set ( % ) 14-Day Old Survivors/Maximum Set ( % ) 15 719 67 2 88 100 81 99 71 70 4a 47 Experimental Group (ppm a.i .) 10 50 16 a77 76 1 a7 98 80 97 69 67 51 50 NIA - No eggs available due to adult mortality. Differences between the control and each treatment groups were not significant (p > 0.05). I Values represent pen means for experimental group. Values for each pen are presented in Appendices VI1 and VIII. 'Represents the total number of eggs laid in each group. 150 I Figure 5 Mean Reproductive Performance, Normalized as Percentages (Z), from a Mallard Reproduction Study with PFOS 1OC 90 80 70 60 50 40 30 20 10 Eggs Laid/ Maximum Laid Eggs Cracked/ Eggs Laid Uiable Embryos/ Eggs Set Live 3-Week/ Uiable Embryos Hatchlings/ Live 3-Week Reproductive parameter 14-Day Old/ Hatchlings Concentrations, ppm I 10 50 150 I P t3 I 0 W Table 9 Mean Eggshell Thickness Measurements (mm) from a Mallard Reproduction Study w i t h PFOS Experimental Group (ppm a . i . ) No. Eggs Measured Shell Thickness Mean (k SD) Control 10 50 150 49 0.392 f 0.034 54 0.396 f 0.014 I - P w I - - Data are not available due to adult mortality Differences between the control and each treatment groups were not significant (p > 0.05). Table 10 Mean Body Weight (9)of Hatchlings and 14-Day Old S u r v i v o r s f r o m a Mallard Reproduction Study with PFOS Experimental Group (PPM A.I. ) Hatchlings Number Mean ( f SD) ~ ~~ 14-Day Old Survivors Number Mean ( f SD) Control 10 50 150 459 35 f 3 521 37 * 2 0 0 454 287 +- 28 I P P I 507 202 f 23 0 0 The number of hatchlings weighed may differ from the total number of hatchlings since those hatchlings found dead were not weighed. Differences between t h e control and each treatment group were not significant Ip > 0.05). - 45 - wildlife International, Ltd. Y Project Number 454-109 Appendix I Certificate of Analysis a a l laboratories, Inc. e College, PA 16801 www.centrelab.com Fax:(814)231-1253OT (814)231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFOS,Lot 217 Reference #: SD-018 Purity: 86.9% SpedfitaiJons Ruult 86.9% AppCaranCe Identification NMR White Crvstalline Powder conforms Positive I . CalciUm 2. Magnesium 3. Sodium 4. Potassium' 5. Nickel 6 . Iron 7. Manganese Total % Impurity (NMR) Total % Imuurity I . (LC/MS) , Total % Imuuntv E?-- Related Compounds- 1. 0,005 wt/WL% 2. 0,001 wtJwt.% 3. 1.439 wtJwt?h 4. 6.849wtJwt.% 5. <0.001 wt/wt% 6. 0.005 wtJwt.% 7. <0.001 wt./wt.% 1.91 wtlwtoh 8.41 wt./wt?h None Detected 0.33 wtlwt.% None Detected Not ADOlicablc' 1. Chloride 2. Fluoride 3. Bromide 4. Nilrate 5. Nitrite 6. Phosphate 7. sulfste' Organic Acids '(IC) 1. TFA 2. PFPA 3. HFBA 4. NFF'A Elemental Analysis`: 1. CJlrbOn 2. Hydrogcn 3. Nitrogen 4. sulfur 5. Fluorine 1. TheoreticalValue = 17.8% 2. Theoretical Value = 0% 3 . Thooretical Value = 0% 4. Thwretical Value = 5.95% 5. Theoretical Value =60% 1. <O.O15Wt./wt.% 2. 0.59wtJwt?h 3. co.040 *% 4. ~O.o09WtJwt% 5. <00.006wt/wt.% 6. <0.007wt/wt% 7. 8.76wtJwtoh I . <0.1 wtM% 2. <0.1 wtlwt.% 3. O . l O w t l w t . % 4. 0.28 wt./wt.% 1. 12.48 wtfwt.% 2. 0.244 wt.lwt.% 3. 1.74wtJwt?h 4. 8.84 wt.fwt.% 5. 54.1 wtfwt.% COA023-018A Page 1 of 3 - 4.6- - Wild1ife Intemational, Ltd. Project Number 454-109 Appendix I Page 2 state Certificate of Analysis ticCaolllegLe, aPAb16o80r1atorwi.cEenstr,eleIbn.cocm 0 Fax:(814)231-1253or(814)231-1580 INTERliU CERTIFICYTE OF ANALYSIS Reviswn 3 CentreAnalytical LaboratoriesCOA Reference #: 023418A Date of Last Analysis: 08/31/00 ExpirationDate: 0801/06 Storage Conditions: Frozen _CloOC Re-assessment Date: 0813 1/06 - 'Purity = 1Wh (sum of metal impurities, 1.45% + 3 M S impurities. IAI%+Inorganic Fluoride, 0.59%+NhfRimpurities, 1.905%+0rganicacid impurities,0,38%+POAA, 0.33%) Total impurity&om all tests = 13.07% Purity = 100%- 13.07%= 86.9% 2Potassiumis expected in this salt form and is therefore not considered an impurity. 'Purity by DSC is generally not applicable to materials of low purity. No endotherm was observed for this sample. 'Sulfur in the sample appears to be converted to SO4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur dctRmination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO, is not considered an impurity. STPA HFBA NFPA PFPA Trifluoroacctio acid Heptafluorobutyricacid Nonafluoropentanoic acid Pentafluompropanoicacid "'heoretical value calculationsbased on the empiricalformula, QP1$3QKt(MW=538) This work was conducted under EPA Good Laboratocy F'ractice Standards (40 CFR 160). COA023-018A Page 2 of 3 - 47 - WildlifeInternational, Ltd. Project Number 454-109 Appendix I Page 3 Certificate of Analysis LPhone: (814) 231-8032 Fax: (814) 231-1253 cv (814) 231-1580 INTERIM CERTIFICATEOF ANALYSIS Revlsion 3 Centre Analytical Laboratories COA Reference #: 023-018A LCMS PurityProfile: cs 1.33 C6 4.72 1 I I c-7. I Total l A 11.. 7 0.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average result horn the C4 and C6 standard c w e s . Likewise, the C7 value wascalculated using the average result from the C6 and C8 standard curves. - /<A/., ReparedBy: CharlesS' Date Scientist, Centre Analytical Laboratories Reviewed By: - __ '~ab~ratoryManager, centre ~ n a l y t x aL~aboratories COA023-018A Page 3 Of 3 w i l d l i f e International, Ltd. - 48 - Project Number 454-109 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 CalPhos Methionine Premix + Liquid Vitamin and Mineral Premix (see below) GL Ferm (Fermatco)2 Salt Iodized Total PERCENT ("A) 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 Riboflavin Niacin Pantothenic Acid Vitamin B Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (Menadione Dimethylpyrimidinol Bisulfite) Manganese Zinc Copper Iodine Iron Selenium 7,000,000 I.U. 6 grams 40 grams 10 grams 8 mgs 600 mgs 64 mgs 1.2 grams 1.2 grams 20,000 I.U. 5.8 grams 102 grams 47 grams 6.8 grams 1.5 grams 51 grams 182 mgs I The guaranteed analysis is a minimum of 27% protein, a minimum of2.5% crude fat and a maximum of 5% * fiber. Fermentation By-products (Source of Unidentified Growth Factors). crude - 49 - WildlgeInternational, Ltd. Project Number 454-109 Appendix I1 Diet and Supplement Formulations Table 2 Vitamins and Electrolytes Concentrate GUARANTEED ANALYSIS Vitamin A Vitamin D Vitamin E Riboflavin d-Pantothenic Acid Niacin Vitamin B-12 MSBC Folic Acid Thiamine HC1 Pyridoxine Hydrochloride Ascorbic Acid Water Soluble Powder Per 4 02. 2,5000,000 1,000,000 1,000 750 1,250 2,500 2.5 1,000 65 250 250 3,750 Per lb. 10,000,000 IU 4,000,000 ICU 4,000 IU 3,000 Mg 5,000 Mg 10,000 Mg 10.0 Mg 4,000 Mg 260 Mg 1,000 Mg 1,000 Mg 15,000 Mg INGREDIENTS: Vitamin A Supplement, D-Activated Animal Sterol (source of Vitamin D3), Alpha Tocopheryl Acetate (source of Vitamin E). Riboflavin Supplement, d-Calcium Pantothenate, Niacin Supplement, Vitamin B-12 Supplement, Menadione Sodium Bisulfite (source of Vitamin K), Folic Acid, Thiamine HC 1, Pyridoxine Hydrochloride, Ascorbic Acid, Sodium Chloride, Calcium Chloride, Magnesium Sulfate, Ferric Ammonium Citrate, Potassium Chloride and Dextrose. MIXING PROCEDURE: The vitamin and electrolyte mix was prepared as a ration of approximately 2 grams of Durvet vitamins and electrolytes to approximately 1 gallon of water. - 50 - wildlife International, Ltd. Project Number 454-109 Appendix 111 Diet Preparation Premixes for PFOS were prepared on January 22, 2001; February 6 and 12,2001; March 2, 6 and 27, 2001; April 12,2001; May 3 and 23,2001; and June 4,2001. Nominal preparation was as follows: Control: 10ppni ai.: 50 ppni ai.: 150 ppm ai.: 20 ppni a.i. No premix required. 2.5633 g PFOS + 8097.4 g ration 12.8165 g PFOS + 8087.2 g ration 38.4494 g PFOS + 8061.6 g ration 5.1266 g PFOS + 8094.9 g ration Basal ration, 5000.0 g, was weighed into a tared Hobart mixing bowl. The remaining ration was weighed and set aside. The test substance was weighed into a tared weigh boat. Approximately 100 g of set aside ration was placed in a WaringB blender. The test substance was transferred to the WaringB blender and the weigh boat was rinsed three times with set aside ration, 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 set aside ration with the rinse being added to the bowl. The remaining set aside ration was added to the bowl, the bowl was placed on a Hobart mixer, and the contents were mixed approximately 15 minutes. If not used immediately after mixing, 2000.0 g aliquots of the premix were weighed, placed in appropriately labeled plastic bags, reweighed, and stored frozen. As needed, the appropriate premix was incorporated into the final diet as follows: 0 ppni a.i.: 52.25 kg ration + 2.750 kg limestone I O pprn ai.: 2000 g Premix + 50.25 kg ration + 2.750 kg limestone 50 pprn a i . : 2000 g Premix + 50.25 kg ration + 2.750 kg limestone 150 ppm a i . : 2000 g Premix + 50.25 kg ration + 2.750 kg limestone 20 ppm a i . 2000 g Premix + 50.25 kg ration + 2.750 kg limestone The diets were mixed for approximately 20 minutes in a Patterson-Kelly Twin Shell Blender. -51 - wildlife Internationa1, Ltd. Project Number 454-109 Appendix JV The Analysis of PFOS in Avian Diet WildlifeInternational, Ltd. - 52 - Project Number 454-109 APPENDIX IV ANALYTICAL METHODS AND RESULTS INSTRUMENT: Table 1 Typical HPLC/MS/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in multiple ion reaction monitoring (MRM) mode. ANALYTICAL COLUMN: Keystone Betasil c18 column (50 mm x 2 mm I.D., 3-pm particle size) GUARD COLUMN: Keystone Javelin c18 column (20 mm x 2 mm I.D.) OVEN TEMPERATURE: 40C STOP TIME: 5.OO minutes FLOW RATE: 250 pL/minute MOBILE PHASE: 70.0% Methanol: 30.0%NANOpure@Water containing 0.1% Formic Acid INJECTION VOLUME: 10.0 pL PFOS RETENTION TIME: Approximately 4.0 minutes PFOS MONITORED MASS: 499.0 amu -+ 99.1 amu WildlifeInternational, Ltd. - 53- Project Number 454-109 APPENDIX IV Table 2 Calculations The concentration of PFOS found at the instrument was determined using the following equation: eak area - (y-intercept) PFOS (mg a.i./L) at instrument = P slope Determination of Sample Residues (PFOS) 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 Quantitation (LOQ) The method LOQ, expressed as pprn a i , was determined using the following equation: LOQ (ppm a.i.) = lowest standard concentration (mg a.i./L) x overall dilution factor of matrix blank* extract final volume (L) x dilution factor *overall dilution factor of matrix blank sample = initial weight (Kg) Fortification Recoveries The ppm a.i. measured in each sample is divided by the nominal concentration of each sample (fortified level, ppm a.i.). This ratio times 100 is the percent recovery of the method at that level of fortification. m a.i. measured in sample % Recovery = pp pprn a.i. fortified Project Number 454-109 APPENDIX IV Table 3 Matrix Blanks and Fortifications Analyzed Concurrently with the Samples Number (454-log-) MAB-1 MAS-1 MAS-2 MAS-3 MAB-2 MAS-4 MAS-5 MAS-6 MAB-3 MAS-7 MAS-8 MAS-9 MAB-4 MAS-10 MAS-1 1 MAS-12 Sample Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Interval Week 1, Day 0 Week 1, Day 0 Week 1, Day 0 Week 1, Day 0 Week 2, Day 0 Week 2, Day 0 Week 2, Day 0 Week 2, Day 0 Week 3, Day 0 Week 3, Day 0 Week 3, Day 0 Week 3, Day 0 Week 4, Day 0 Week 4, Day 0 Week 4, Day 0 Week 4, Day 0 Concentrationof PFOS (ppm a i ) Fortified' Measured' 32 0.00 < 2.00 5.00 4.85 50.0 52.8 200 206 0.00 < 2.00 5.00 4.55 50.0 47.4 200 189 0.00 < 2.00 5.00 5.50 50.0 52.0 200 200 0.00 < 2.00 5.00 5.20 25.0 26.1 100 100 Percent Recovery 97 106 103 91 95 95 110 104 100 104 104 100 'Less than values correspond to limit of quantitation (LOQ). 2Resultswere generatedusing MacQuan version 1.6 software. Manual calculationsmay differ slightly. Mean Recoverv 102 94 105 103 Project Number 454- 109 APPENDIX IV Table 3 (Continued) Matrix Blanks and Fortifications Analyzed Concurrently with the Samples Number (454-109-) MAB-5 MAS- 13 MAS-14 MAB-6 MAS- 15 MAS-16 MAS-173 MAS-18 MAB-7 MAS- 19 MAS-20 Sample me Matrix Blank Matrix Fortification Matrix Fortification Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Matrix Fortification Matrix Blank Matrix Fortification Matrix Fortification Interval Concentrationof PFOS (ppm a i ) I % Fo~ed' Measured',2 Week 8, Day 0 0.00 Week 8, Day 0 5.00 Week 8, Day 0 25.0 Week 12, Day 0 0.00 Week 12, Day 0 5.00 Week 12, Day 0 25.0 Week 16, Day 0 5.26 Week 16, Day 0 25.0 Week 20, Day 0 0.00 Week 20, Day 0 5.00 Week 20, Day 0 25.0 < 2.00 5.40 27.5 < 2.00 5.21 24.4 5.39 24.5 < 2.00 4.86 24.0 Percent Recovery 108 110 104 98 102 98 97 96 Mean Recovery (E) 109 101 100 97 1 Less than values correspond to limit of quantitation (LOQ). 2Resultswere generatedusing MacQuan version 1.6 software. Manual calculationsmay differ slightly. 3Week 16 samples were held and batched with Week 20 samples. Matrix fortifications were prepared to verlfy stability. A storage matrix blank was not required. Nominal Concentration @pm a.i.) 10 50 150 Sample I.D. Number (454-109-) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Project Number 454-109 APPENDIX IV Table 4 Homogeneity of PFOS in Avian Diet Location Sampled in Mixing Vessel Top Left Top Right Middle Left Middle Right Bottom Left Bottom Right Top Left Top Right Middle Left Middle Right Bottom Left Bottom Right Top Left Top Right Middle Left Middle Right Bottom Left Bottom Right Measured PFOS Concentration (ppm a.i.) 9.69 9.47 9.87 10.2 9.35 9.93 49.9 48.9 51.7 50.4 50.3 49.3 151 153 143 143 144 147 Mean (R) Standard Deviation (SD) Coefficient of Variation (CV) x = 9.75 ppm a.i SD = 0.313 ppm a i CV = 3.21% x = 50.1 ppm a.i. SD = 0.981 ppm a.i. CV = 1.96% R = 147 ppm a.i. SD = 4.31 ppm a.i. CV= 2.93% Mean Percent of Nominal 98 100 98 Project Number 454-109 Nominal Concentration (ppm a i . ) 0 Sample I.D. Number (454-109-) 34 41 48 55 60 65 70 APPENDIX IV Table 5 Verification of PFOS Concentrations in Avian Diet Interval (Day 0 of Week -) 2 3 4 8 12 16 20 Measured PFOS Concentration' (ppm a.i.) < 2.00 < 2.00 < 2.00 < 2.00 < 2.00 < 2.00 < 2.00 Mean (R) Standard Deviation (SD) Coefficient of Variation (CV) 10 35 2 36 2 42 3 43 3 49 4 50 4 56 8 57 8 61 12 62 12 66 16 67 16 71 20 72 20 ' Less than values correspondto limit of quantitation (LOQ). 11.0 9.68 9.53 10.7 9.70 9.60 10.6 x = 10.2ppm a.i. 11.2 SD = 0.553 ppm a.1. 10.4 cv = 5.44% 9.86 9.82 10.4 10.1 9.69 Percent of Nominal 110 91 95 107 97 96 106 112 104 99 98 104 101 97 Mean Percent of Nominal 102 Nominal Concentration (ppm a.i.) 20 50 150 Project Number 454-109 Sample I.D. Number - (454-log-) 46 47 53 54 37 38 44 45 51 52 39 40 APPENDIX IV Table 5 (Continued) Verification of PFOS Concentrations in Avian Diet Interval (Day 0 of Week -) 2 2 Measured PFOS Concentration' (ppm a.i.) 18.6 19.8 23.6 21.1 48.7 48.4 51.1 53.1 51.1 53.1 172 150 Mean (x) Standard Deviation (SD) Coefficient of Variation (CV) x = 20.8 ppm a i . SD = 2.14 ppm a i . CV = 10.3% x = 50.9 ppm a i . SD = 2.04 ppm a i . cv = 4.01% x = 161ppm a i . SD = 15.6ppm a i . CV= 9.66% Percent of Nominal 93 99 118 106 97 97 102 106 102 106 115 100 Mean Percent of Nominal 104 102 108 Project Number 454- 109 APPENDIX IV Table 6 Ambient Stability of PFOS in Avian Diet During the Mallard Reproduction Study Nominal Concentration (ppm a.i.) Sample Number (454-109-) Week 1, Day 0' Mean Measured (ppm ai.) PFOS Concentration Mean Percent of Nominal Sample Number (454-109-) Week 1, Day 7 Measured' (ppm a.i.) Mean Measured (ppm a.i.) 0 1 <2.00 10 2-7 9.7s 98 so 8-13 so. 1 100 150 14-19 147 98 1 Day 0 values are from homogeneity samples presented in Table 4. 2 Less than values correspond to limit of quantitation (LOQ). 27 < 2.00 <2.00 28 9.7s 9.71 29 9.67 30 53.4 50.8 31 48.1 32 141 143 33 144 Mean Percent of Day 0 100 101 97 WildlifeInternational, Ltd. - 6o - Project Number 454-109 APPENDIX IV I 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. *1 Weigh 10 g samples of the matrix blank, matrix fortification and test samples into weigh boats and transfer to 8-oz. 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. 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. .L Prepare appropriate dilution(s)to bring final concentration into the calibration range of the HPLC/MS/MS methodology. Use methanol for intermediate dilutions, if required. For all final dilutions, use 50% methanol : 50% NANOpure@water dilution solvent. .1 Ampulate and submit sample for HPLC/MS/MS analysis. Figure 1. Analytical method flow chart for the analysis of PFOS in avian diet. WildlifeInternational,Ltd. - 61 - Project Number 454- 109 APPENDIX IV PFOS 499.0->99.1 No Internal Standard Weighted (llx) Intercept = 883.10 Slope = 11487727.00 Correlation Coeff. = 0.99974 Area 12000c 100000 80000 60000 40000 20000 Conc. pg a.i./~ Figure 2. Typical calibration curve for PFOS. Slope = 11487727; Intercept = 883.10; r = 0.99974. Curve is weighted (Ux). WildlifeXntemational, Ltd. - 62 - Project Number 454- 109 APPENDIX IV PFOS-1 STD 0.00100 mg a.lJL 4675A-011D -1 4.98 in 1 period FU6 No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 5.0 Quant Thres. 1.O Min. Width 3 Mult. Width 6 Base. Width 35 'RT Win. (secs) 2 0 Smooth I Expected RT 4.02 Area 12195 Height 1 171 Start Time 3.79 End Time 4.37 Integration Width 0.59 Retention Time Inlegration Type -4.02 A BB Wed, Ieb 7,2001 17:58 intensity: 10000 cps 20 9I j F k 240 1o i A 0 29 4 9 6 6 111 138159 "X 1 1 1 I I I I I I I 41 81 121 161 201 241 I 281 Scan 0.69 1.36 2.03 2.70 3.37 4.04 4,71 Time + Figure 3. Typical ion chromatogram of a low PFOS calibration standard, 0.00100 mg a.i./L. (Monitored mass 499.0 99.1). WildlifeInternational, Ltd. - 63 - Project Number 454-109 APPENDIX IV PFOS-5 STD 0.0100 mg a.iJL 4675A-01 D -5 m4.98 in 1 period No internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 5.0 Quanl Thres. 1 .O Min. Width 3 Mull. Width 6 Base. Width 35 RT Win. (secs) 2 0 Smoolh 1 Expected RT 4.02 Area 114007 Height 10269 Start Time 3.80 End Time 4.39 Inlegration Width 0.59 Retention Time 4.02 Integration Type A - BE Wed Feb 7, 2001 1821 100 90 80 70 60 50 40 30 lo0-. 12 53 41 0.69 7c I 81 1 1.36 intensity: 10000 cps 240 Figure 4. Typical ion chromatogram of a high PFOS calibration standard, 0.0100 mg a.i./L. (Monitoredmass 499.0 -+ 99.1). Wild1ife InternationaI , Ltd. - 64 - Project Number 454-109 APPENDIX IV PFOS-7 MAE-3 454-108.109- Wed, Feb 7,2001 18:33 m4.98in 1 period No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0.>99.1 Noise Thres. 5.0 Quant Thres. 1.O Min. Width 3 Mull. Width 6 Base. Width 35 RT Win. (secs) 20 Smooth 1 ExpectedRT 4.02 Area 0 Height 0 Start Time 0.00 End Time 0.00 Integration Width 0.00 Retention Time 0.00 Integration Type intensity: 10000 cps 100- 90- 8 0- 70- 60- 50- 40- 2i 30- 1 23 39 O ' 411 ' 0.69 105 130 8'1 ' l i l 1.36 2.03 1 164 205 240 274 161 ' 261 ' 2 i l ' 2 i l scan 2.70 3.37 4.04 4.71 Time Figure 5 . Typical ion chromatogram of a matrix blank sample (454-109-MAB-3). indicates the approximate retention time of PFOS. (Monitored mass 499.0 +99.1). The arrow WildlifeInternational, Ltd. - 65 - Project Number 454-109 APPENDIX IV PFOS-9 MAS8 454-108,109- Wed, Feb 7,2001 18 45 m4,98 in 1 period No lntemal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 5 0 Quant Thres. 1.O Min. Width 3 Mult. Width 6 Base. Width 35 RT Win. (sets) 20 Smooth 1 ExpectedRT 4.02 Area 60579 Height 5641 Start Time 3.80 End Time Integration Width Retention Time Integration Type 4.39 0.59 -4.02 A BB intensity: 10000 cps IODl I 90. 80 70 60 240 50 40- 30- 20- n 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Figure 6. Typical ion chromatogram of a matrix fortification sample (454-109-MAS-8, 50.0 ppm a i . ) (Monitored mass 499.0 -+99.1). WildlifeInternational, Ltd. - 66- Project Number 454-109 APPENDIX IV PFOS-13 42 454-108,109- Wed, Feb 7, 2001 19:lO m4.98 in 1 period No Internal Standard Use Area 1: 4.97 MRM, 298 Scans 499.0->99.1 Noise Thres. 5.0 Quant Thres. 1.O Min. Width 3 Mutt. Width 6 Base. Width 35 RT Win. (secs) 20 Smooth 1 Expected RT 4.02 Area 55642 30 Height 5213 Start Time 3.80 20 End Time 4.39 Integration Width 0.59 Retention Time Integration Type -4.02 A BB intensity: 10000 cps 240 121 161 201 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Figure 7. Typical ion chromatogram of an avian diet sample on Day 0 (454-109-42, 10.0ppm a i ) . Dilution factor = 2000X. (Monitoredmass 499.0 -+99.1). - 67 - Project Number 454-109 Appendix V Diagram of Test Layout from a Mallard Reproduction Study with PFOS Room 56 ..................................... 270-272 273-275 276-278 150 ppm a i . Pens 261 - 280 .................................................................... 279-280 50 ppm a.i. Pens 241 - 260 259-260 256-258 253-255 244-246 241-243 ...................... .......................................... ............................................................................................................................................................. 230-232 233-235 236-238 239-240 10 ppm a.i. Pens 221 - 240 .................................................................... b ii I__..........._..I~.._...__._______~I________________~I_______._.......~ 0 ppm a.i. Pens 201 - 220 "\I 0 tt 0 ct \ I 1-1 = approx. Im a = Safeguard Products Inc. Model No. 5355 = 6"gutter b = Four X 4 ft. Chroma 50 light bulbs Note: Pens 217-220,237-240,257-260and 277-280were used to house birds used for blood sampling. - 68 - w i l d1ife Internationa1, Ltd. Project Number 454-109 Appendix VI Reproductive Parameters 1. Eggs Laid Definition - The number of eggs produced during the breeding season. Sensitivity - This is a parameter that is frequently affected by chemicals that cause reproductive impairment. It is also highly sensitive to the general conditions under which the test is conducted, and may be adversely affected by improper diet, crowding, excessive disturbances, etc. 2. Eggs Cracked Definition - Eggs determined to have cracked shells when inspected with a candling lamp. Fine cracks cannot be detected without utilizing a candling lamp, and if undetected, will bias the data by adversely affecting embryo development. Sensitivity - This parameter is not frequently affected as cracking is directly related to shell thickness, and few chemicals have caused egg shell thinning. Improper pen design, overcrowding, and pecking by the birds can also increase egg cracking. 3. Eggs Set Definition - All eggs placed under incubation, i.e., total eggs laid minus cracked eggs, mechanically damaged eggs, and those selected for egg shell thickness analysis. The reason for presenting this parameter is to establish a base of reference for the following parameter viable embryos. 4. Viable Embryos Definition - Eggs in which fertilization has occurred and embryonic development has begun. This is determined by candling the eggs 13-14 days after the initiation of incubation. It is difficult to distinguish between infertility and an early embryonic death. Sensitivity - This is a frequently affected parameter with infertility or embryonic mortality caused by an unfavorable environment for fertilization in the oviduct, impotent males, or chemical residue in the egg. wiId1ije Int e m ational, Ltd. - 69 - Project Number 454-109 Appendix VI Reproductive Parameters Page 2 5 . Live Three-Week Embryos Definition - These are embryos that are developing normally after three weeks of incubation. This is determined by candling the eggs. Sensitivity - This parameter is seldom affected. 6. Hatchlings Definition - Embryos that mature, pip the shell, and liberate themselves from their eggs on Day 27 or 28 of incubation. Sensitivity - This is a frequently affected parameter which is also highly sensitive to the conditions of incubation, such as rate and angle of rotation, humidity, and temperature. - Humidity: If too dry, ducklings will stick to their shells. - Temperature: If too hot and humid, ducklings will grow too fast and be too large for their shells; thereby not having the intra-egg mobility necessary to pip their shells. 7. Body Weight - Hatchlings Definition - The average weight of hatchlings by parental pen of origin, taken immediately upon removal from the hatcher. Hatchlings from each weekly lot of eggs are weighed. Mean weight for each week and mean weight by pen are reported. Sensitivity - This is an occasionally affected parameter and may reflect some residual or latent toxic effects from chemical residue in the egg. 8. 14-Day Old Survivors Definition - Birds that survive brooding for two weeks following hatch. - Sensitivity - This is a seldom affected parameter and is probably more indicative of the conditions under which the birds were reared in battery brooders than the chemical to which the adults were exposed. - IU - wildlife International, Ltd. Project Number 454-109 Appendix VI Reproductive Parameters Page 3 9. Body Weight - 14-Day Old Survivors Definition - The average body weight of the 14-day old survivors by parental pen of origin, taken upon removal from the brooder unit at 14 days of age. Fourteen-day old survivors from each weekly lot of eggs are weighed. Mean weight for each week and mean weight by pen are reported. Sensitivity - This parameter is seldom affected, and more closely reflects the rearing practices utilized. 10. Egg Shell Thickness Definition - The thickness of the shell and the membrane of the egg at its equator after having been opened, washed, and dried for at least one week at room temperature. Egg shell thickness measurements for each egg measured, and pen means are reported. Sensitivity - This is a seldom affected parameter; however, measurements must be taken very carefully to ensure accuracy. Possible mechanical errors include membrane curling at the measurement surface, and calcium deposits on the measurement surface. - /I - w i l d l v e International, Ltd. Project Number 454- 109 Appendix VI Replicate Identification Experimental Group Control 0 ppm a.i. Report Replicate Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Report Pen Number Raw Data Pen Number Raw Data Band Number Male Female 20 1 20 1 329 330 202 202 33 1 332 203 203 333 334 204 204 335 336 205 205 337 338 206 206 339 340 207 207 341 342 208 208 343 344 209 209 345 346 210 210 347 348 21 1 21 1 349 350 212 212 35 1 352 213 213 353 354 214 214 355 356 215 215 357 358 216 216 359 360 - 72 - Wildlife International, Ltd. Project Number 454-109 Appendix VI Replicate Identification Experimental Group Treatment 10 ppm a i . Report Replicate Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Report Pen Number Raw Data Pen Number Raw Data Band Number Male Female 217 22 1 369 370 218 222 371 372 219 223 373 374 220 224 375 376 22 1 225 377 378 222 226 379 3 80 223 227 381 3 82 224 228 3 83 3 84 225 229 385 386 226 230 387 388 227 23 1 389 390 228 232 391 392 229 233 393 3 94 230 234 395 396 23 1 235 397 398 232 236 399 400 A p p e n d i x VI1 - T a b l e 1 Adult Body Weight (9)from a Mallard Reproduction Study with PFOS (Control, Males) Pen 201 202 203 204 205 206 207 208 209 210 211 2 12 213 2 14 215 216 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 1098 -11 1087 15 1102 - 12 1090 -47 1043 119 1061 -56 1005 22 1027 34 1061 27 1088 96 994 -1 993 24 1017 43 1060 -15 1045 130 1077 56 1133 19 1152 -18 1134 5 1139 53 1094 -25 1069 75 1144 -19 1125 - 54 1071 144 1061 -15 1046 23 1069 -4 1065 - 13 1052 99 1078 25 1103 21 1124 -18 1106 6 1112 42 1073 12 1085 33 1118 51 1169 22 1191 -78 1126 -38 1088 23 1111 42 1153 -5 1148 29 1264 5 1269 26 1295 18 1313 -5 1308 1050 43 1093 -5 1088 0 1088 -3 1085 - 13 1018 59 1077 1 1078 13 1091 12 1103 0 997 41 1038 36 1074 44 1118 25 1143 -30 1164 -21 1143 21 1164 22 1186 -4 1182 85 1139 0 1139 24 1163 1 1164 5 1169 -68 1057 31 1088 16 1104 13 1117 1 1118 167 1162 1184 1175 1192 1215 1151 1154 1113 1177 1072 1103 1113 1267 1101 1285 1084 7 1091 23 1114 13 1128 -3 1125 52 67 34 64 17 65 24 63 22 68 77 1164 60 Total Change 64 123 181 115 121 90 76 40 51 22 85 116 103 -38 228 92 64 The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due t o adult mortality. Appendix VI1 - T a b l e 2 Adult Body Weight (9)from a Mallard Reproduction Study with PFOS (Control, Females) ~ Pen 201 2 02 203 2 04 205 206 207 208 209 210 211 212 213 214 215 216 Week 0 ~ ~_____ 905 857 860 1024 924 1025 1057 1092 1086 968 930 986 1215 863 983 962 Change Week 2 Week 0-2 Change Week 4 Week 2-4 Change Week 6 Week 4-6 Change Week 8 Week 6-8 Change Week 8-Term ~~ 35 940 25 965 -19 946 -4 942 249 - 11 846 32 878 21 899 33 932 101 21 881 6 887 19 906 6 912 246 26 1050 46 1096 8 1104 -7 1097 205 25 949 -4 945 34 979 39 1018 -46 -3 1022 10 1032 29 1061 24 1085 250 -40 1017 27 1044 -11 1033 14 1047 121 36 1128 1 1129 43 1172 15 1187 227 -3 1083 10 1093 31 1124 -7 1117 93 32 1000 3 1003 32 1035 5 1040 -52 23 953 33 986 9 995 5 1000 2 14 - 14 972 1 973 23 996 20 1016 150 -48 1167 12 1179 26 1205 21 1226 28 -4 859 7 866 18 884 1 885 102 15 998 -11 987 6 993 14 1007 55 -20 942 21 963 31 994 18 1012 12 Test Term 1191 1033 1158 1302 972 1335 1168 1414 1210 988 12 14 1166 12 54 987 1062 1024 Mean SD 984 4 98 26 988 14 1002 19 1020 12 1033 122 90 15 90 17 94 14 94 103 1155 134 Total Change 286 176 298 278 48 310 111 322 124 20 284 180 39 124 79 62 171 109 The means for body weights and body weight changes are calculated and rounded separately. Appendix VI1 - T a b l e 3 Adult Body Weight ( g ) from a Mallard Reproduction Study with PFOS (10 PPM A . I . , Males) Pen 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Mean SD Week o Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 966 1273 1153 1193 1038 1148 1049 1003 1146 1148 1097 1231 1079 1081 1121 1042 22 -2 -57 53 10 31 7 2 44 - 13 -11 73 9 -5 -2 3 988 1271 1096 1246 1048 1179 1056 1005 1190 1135 1086 1304 1088 1076 1119 1045 -10 12 12 -34 - 14 18 24 -20 -9 43 3 12 15 26 5 21 978 1283 1108 1212 1034 1197 1080 985 1181 1178 1089 1316 1103 1102 1124 1066 22 20 -11 -2 14 27 -4 2 25 16 61 32 -11 22 39 23 1000 1303 1097 1210 1048 1224 1076 987 1206 1194 1150 1348 1092 1124 1163 1089 - 72 0 30 38 29 -15 -4 -13 -7 5 55 26 55 -30 29 -21 928 1303 1127 1248 1077 1209 1072 974 1199 1199 1205 1374 1147 1094 1192 1068 43 -84 171 68 24 -59 - 63 -25 40 -35 -116 -230 -34 2 -136 54 971 1219 1298 1316 1101 1150 1009 949 1239 1164 1089 1144 1113 1096 1056 1122 1111 10 1121 7 1127 17 1144 7 1151 -24 82 30 94 20 96 19 101 34 115 95 1127 105 Total Change 5 - 54 14 5 123 63 2 -40 - 54 93 16 -8 -87 34 15 -65 80 17 69 The means for body weights and body weight changes are calculated and rounded separately. Differences between control and this treatment group were not significant (p>0.05). Appendix VI1 - T a b l e 4 Adult Body Weight (9)from a Mallard Reproduction Study with PFOS (10 PPM A . I . , Females) Pen 2 17 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 1071 890 984 1083 1068 1079 1148 852 1000 1025 988 1028 827 970 879 824 982 101 93 18 -36 12 - 13 -20 54 -7 87 29 33 44 21 44 7 - 14 22 37 1164 63 908 -2 948 33 1095 23 1055 5 1059 30 1202 27 845 11 1087 8 1054 -22 1021 7 1072 34 848 42 1014 -3 886 -13 810 37 1004 18 117 22 1227 906 981 1118 1060 1089 1229 856 1095 1032 1028 1106 890 1011 873 847 1022 12 3 -61 19 18 141 -11 -23 46 4 -16 -4 41 -25 59 86 13 15 19 48 1166 925 999 1259 1049 1066 1275 860 1079 1028 1069 1081 949 1097 886 862 1041 126 38 22 -4 34 -23 -24 53 2 -25 5 -11 -3 38 39 23 -23 9 27 1204 947 995 1293 1026 1042 1328 862 1054 1033 1058 1078 987 1136 909 839 1049 130 11 237 167 23 86 136 -29 126 129 26 200 144 154 45 76 -18 95 79 1215 1184 1162 1316 1112 1178 1299 988 1183 1059 1258 1222 1141 1181 985 821 1144 12 8 Total Change 144 294 178 233 44 99 151 136 183 34 270 194 314 211 106 -3 162 92 The means for body weights and body weight changes are calculated and rounded separately. Differences between control and this treatment group were not significant (p>0.05). Appendix VI1 - T a b l e 5 Adult: Body Weight ( 8 ) f r o m a Mallard Reproduction Study with PFOS (50 PPM A. I., Males) Pen 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 1013 1039 1444 1023 997 1196 1367 1084 1018 1180 1142 1009 1088 998 936 1129 1104 139 -39 -46 -62 -38 -50 -88 -84 - 74 -69 -83 -89 -10 -42 -42 -5 -95 -57 28 974 993 1382 985 947 1108 1283 1010 949 1097 1053 999 1046 956 931 1034 1047 124 -48 -21 -134 - 62 -11 -100 -77 - 62 -43 -98 -66 -47 -57 -77 -21 -98 - 64 33 926 972 1248 923 936 1008 1206 948 906 999 987 952 989 879 910 936 -79 2 -130 -182 -28 -29 - 54 -109 -58 -44 - 74 -36 -38 -68 -67 983** -66 102 46 847 974 1118 741 908 979 1152 839 848 955 878 953 841 842 869 916** - 109 - - - - Total Change The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. * * Significantly different from the control at p c 0.01 I 4 4 I ICsL . 0 $: Z G El 5 P Ul f + 0 \D Appendix VI1 - T a b l e 6 Adult Body Weight (g) from a Mallard Reproduction Study with PFOS (50 PPM A. I., F e m a l e s ) Pen 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 93 9 919 978 981 969 951 880 887 1002 928 1034 963 849 904 1109 995 956 64 -106 -75 -81 -90 -145 -73 -60 -75 -105 -19 -98 -106 -76 -108 -56 -54 -83 29 833 844 897 891 824 878 820 812 897 909 936 857 773 796 1053 941 -56 -51 -90 -36 -40 -27 -25 -58 -50 -71 -89 -38 -79 -84 -101 -65 873** -60 69 24 777 793 807 855 784 851 795 754 847 838 847 819 694 712 952 876 1 0 -76 -27 -69 -41 26 -37 -103 12 - 15 -51 -39 -39 -6 -38 813** -31 63 34 778 793 731 828 715 810 821 717 744 850 832 768 655 673 946 838 781** 74 - - - - Total Change The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. * * Significantly different from the control at p < 0.01 I 4 00 I ITs. 0 $: 2 c: B 0- 8 P v1 fc1 0 \o Appendix VI1 - T a b l e 7 A d u l t Body Weight ( 8 ) from a Mallard Reproduction Study w i t h PFOS (150 PPM A . I., Males) Pen 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 2 64 Mean SD Week 0 Change Week 2 Week 0-2 , Change Week 2-4 Week 4 Change Week 4-6 Week 6 Change Week 6-8 Week 8 Change Week 8-Term Test Term 1128 1024 1057 1177 898 1229 1012 1094 1222 1276 1218 1060 1178 1050 1008 1073 -145 -242 -198 -246 -208 -263 -239 -185 -174 -228 -158 -152 -209 -132 -192 -143 983 782 859 931 690 966 773 909 1048 1048 1060 908 969 918 816 930 145 276 72 113 102 118 -27 122 -6 88 55 31 50 90 1128 1058 931 1044 1068 891 1021 1170 1054 996 1024 949 866 1020 1107 -195 912** 88 1016** - - 103 41 106 73 85 - - Total Change The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. * * Significantly different from the control at p < 0.01 Pen 249 250 251 252 253 254 255 256 257 258 ' 259 260 261 262 263 264 A p p e n d i x VII - T a b l e 8 Adult Body Weight (g) from a Mallard Reproduction Study with PFOS (150 PPM A. I., Females) Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Test Term 857 -182 675 - 832 -200 632 198 830 891 -143 748 88 836 903 -218 685 12 7 8 12 892 -159 733 124 857 972 -205 767 44 811 1113 1091 -205 -230 908 135 853 1043 - 1027 -266 761 134 895 912 -178 734 103 837 1056 -257 799 86 885 - 1159 -236 923 106 1029 1061 -208 853 -3 850 - - - - 1116 -248 868 21 889 - - - - 701 -136 645 166 811 - - - - 1104 -212 892 46 93 8 - - - Total Change - - The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. * * Significantly different from the control at p < 0.01 Pen 201 2 02 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Mean SD Feed Consumption Appendix VI11 - T a b l e 1 (g/bird/day) from a Mallard Reproduction (ControlI Study with PFOS WEEKS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 ~ 96 100 122 138 144 132 138 155 138 158 152 174 169 168 164 157 162 181 183 211 204 76 82 102 110 90 95 104 107 95 96 79 172 191 171 112 123 129 146 129 155 145 93 106 115 132 91 111 92 115 121 115 139 184 225 252 236 200 253 324 232 223 292 154 159 133 141 124 144 138 116 129 125 158 179 212 158 165 147 164 168 151 176 170 76 89 81 102 92 111 110 91 129 133 163 168 175 188 240 275 243 243 259 260 318 104 127 130 140 126 148 151 148 139 131 141 195 157 141 139 136 166 166 166 164 167 96 107 105 114 104 111 118 110 102 96 105 150 170 108 131 144 140 157 164 162 180 85 155 121 159 168 187 162 175 164 151 114 226 239 214 181 217 242 263 191 239 234 182 174 187 236 195 231 235 230 252 196 227 180 185 171 192 223 224 327 245 245 227 86 101 91 100 106 107 97 108 92 103 96 143 165 127 143 185 210 215 185 115 143 108 148 103 125 115 124 125 116 125 108 123 178 166 144 156 159 144 145 130 180 169 136 149 152 147 141 152 158 174 149 168 171 193 217 195 205 174 220 265 246 226 267 78 119 106 114 113 116 123 130 119 126 117 156 148 162 153 137 155 152 132 168 150 67 102 L O O 103 97 103 101 107 105 114 102 228 179 168 182 168 160 214 261 212 224 113 131 145 160 170 156 170 151 180 164 166 188 215 210 241 244 242 230 253 173 201 78 109 106 122 107 105 101 120 128 123 114 137 151 187 204 159 205 202 179 209 191 102 122 119 134 124 133 133 134 135 132 135 178 185 173 178 178 191 212 194 195 205 31 27 26 33 31 36 37 36 39 29 37 26 28 35 40 43 43 59 48 39 53 Pen 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Feed Consumption Appendix VI11 - T a b l e 2 (g/bird/day) from a Mallard Reproduction (10 PPM A. I. ) Study with PFOS WEEKS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 121 146 93 134 117 110 129 126 136 132 149 171 157 140 136 133 125 129 122 139 145 94 112 107 137 133 131 134 134 131 136 171 166 223 196 215 235 221 232 228 205 233 117 159 125 152 145 163 172 181 166 157 196 173 220 188 158 164 195 178 198 190 218 206 238 205 172 183 229 264 229 245 190 134 226 224 209 234 206 214 242 259 263 274 84 117 109 122 108 133 133 124 124 136 120 176 218 195 189 186 190 216 122 194 190 107 136 129 149 133 138 135 130 124 123 104 146 193 166 140 127 140 172 158 169 161 169 206 191 232 189 171 166 156 151 150 131 143 173 197 221 192 218 216 215 188 216 124 116 121 166 99 110 126 113 115 113 117 126 134 128 113 135 138 134 123 133 131 137 171 156 129 144 147 129 124 108 111 93 132 169 171 176 145 149 160 138 163 170 103 104 109 133 117 113 113 116 101 122 134 138 168 146 148 136 159 150 143 163 186 90 116 115 143 126 138 133 140 134 132 109 148 168 160 168 173 172 172 155 172 155 153 199 182 180 163 169 178 177 194 163 103 168 177 162 141 134 144 136 135 139 187 94 115 123 139 131 149 155 154 136 134 115 184 212 229 226 198 203 219 200 212 240 109 149 146 126 149 154 150 148 136 148 169 166 200 246 250 273 256 276 237 252 225 98 129 112 118 148 145 146 164 136 149 119 132 155 129 142 153 171 196 247 221 190 93 123 115 109 111 126 102 103 86 118 99 142 143 144 144 165 175 173 175 219 174 Mean SD 119 146 134 146 137 145 148 145 139 138 129 158 183 175 175 172 180 188 178 189 193 33 39 33 30 26 29 37 32 38 21 29 25 30 35 42 41 37 43 48 39 38 Differences between control and this treatment group were not significant (p> 0.05). 1 00 h, 1 79 . CD 2 5 a 5 P VI f c 0 W Feed Consumption Appendix VI11 - T a b l e 3 (g/bird/day) from a Mallard Reproduction (50 PPM A . I . ) Study with PFOS WEEKS Pen 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 233 110 121 96 96 80 75 93 - - - - - - - 234 73 108 96 81 87 125 - - - - - - 235 116 123 159 153 123 112 109 - - - - - - 236 159 180 155 144 122 77 109 - - - - - - - 237 90 98 98 122 129 119 134 - - - - - - - 238 91 93 103 117 118 136 110 - - - - - - - - - 239 79 86 100 101 125 136 104 - - 240 117 122 144 148 166 138 156 - I - - - - - 00 W - - - - - - I 241 116 105 129 134 84 80 07 - - - - - - - - - 242 107 149 149 195 163 136 07 - - - - - - - - - 243 77 90 81 102 95 158 63 - - - - - - - - - 244 92 133 141 132 99 182 254 - - - - 245 114 110 114 130 109 123 95 - - - - 246 72 92 107 148 131 161 81 - - - - - - 247 96 131 109 124 90 113 101 - - 240 104 91 114 139 132 107 104 - - - - - - - - Mean SD 101 114 118 129 116 124 112 22 25 24 27 26 29 45 - - - - - - - - - - - - Data are not available due to adult mortality. Differences between control and this treatment group were not significant (p> 0.05). Feed Consumption A p p e n d i x VI11 - T a b l e 4 (g/bird/day) f r o m a Mallard Reproduction Study with ( 1 5 0 PPM A. I. ) PFOS WEEKS 249 250 251 252 253 2 54 255 256 257 258 259 260 261 262 263 264 64 67 116 198 56 34 163 165 i i a 118 198 185 ao 72 172 149 53 71 134 i o 8 39 45 215 171 53 58 222 285 42 47 62 50 63 190 70 77 171 161 7 0 52 106 133 43 69 133 166 166 142 175 194 a3 94 149 225 56 51 164 165 109 159 300 411 Mean SD 73* 75**166**194** 33 36 56 73 - - Data are not available due to adult mortality. * Significantly different from the control at p < 0.05 * * Significantly different from the control at p < 0.01 - - - - - - - - - - - - - I 00 P I P VI f Y 0 \D Males Foot/leg lesions Testes - small (- 1.5-3.5 cm) Not remarkable Appendix IX - Table 1 Individual Gross Pathological Observations from a Mallard Reproduction Study with PFOS Adult Birds Euthanized at Test Termination Control (0 ppm a.i.) Pens 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 xxx- a - -x- -x- -a- -x- - X - X X X X a X X -- x x Females Pens 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Foot/leg lesions Spleen - enlarged and mottled Liver - firm,pale and mottled Abdominal cavity - yolk remnants Abdominal cavity - slight egg yolk peritonitis Abdominal cavity - egg yolk peritonitis Abdominal cavity - slight old egg yolk peritonitis Small intestines - area of hyperemia Kidneys - pale Reproductive tract - multiple fluid filled cysts Ovary - regressing Ovary - regressed Ovary - cystic follicles Ovary - hemorrhagic follicles Not remarkable - x - x x x - x x -- x -x-x- -x -x- -x- -x- -x- - x - x -- x - -xx x-x-x- -x- -x- -x- - x -- x x - -x- -x -x- -x- a - Bird found dead during the test. Necropsy findings are presented in Table 2. I 00 VI I R f L 0 \D Males FootAeg lesions Testes - small (- 1.5-3.5 cm) Not remarkable Appendix IX - Table 2 Individual Gross Pathological Observations from a Mallard Reproduction Study with PFOS Adult Birds Euthanized at Test Termination 10 ppm a.i. Pens 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 -xx- -x- -x- -xx- - x - x --xx x- -xx-xx-x-xx- Females Pens 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Foot/leg lesions xx-xxxxxx-x-xxxx I Breast blister -x- -x- 00 o\ Cranium - swollen and bruised -x- I Heart - pericardial sac fluid-filled -x- -x- Spleen - enlarged and firm -x- Abdominal cavity - egg yolk peritonitis -xxx Ovary - regressed Ovary - cystic follicles - x - x --xx -x- Not remarkable R P e 0 W - 87 - Project Number 454-109 Appendix X - Table 1 Adult Liver Weights (g) from a Mallard Reproduction Study with PFOS Control (0 ppm a i ) Male Pen Liver 20 1 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Mean SD 31.051 32.528 33.915 25.209 33.438 28.829 28.689 27.090 25.919 27.129 30.090 38.599 27.888 26.030 24.921 29.422 3.864 - Data not reported due to mortality. Female Liver 36.870 36.336 46.728 45.564 57.924 46.002 38.662 59.838 52.441 21.509 45.440 47.038 37.995 25.939 34.229 33.845 41.648 10.512 - 88 - Project Number 454-109 Appendix X - Table 2 Adult Liver Weights (g) from a Mallard Reproduction Study with PFOS 10 ppm a.i. Male Pen Liver Female Liver 22 1 222 223 224 225 226 227 228 229 230 23 1 232 233 234 235 236 Mean SD 24.717 30.663 42.118 27.070 32.793 25.430 25.203 27.580 28.447 33.253 30.606 36.870 29.664 30.874 29.018 35.314 30.601 4.663 61.115 36.454 42.668 54.869 41.660 41.253 47.171 44.828 48.620 28.720 48.247 30.419 42.544 52.126 32.634 21.982 42.207 10.268 ~ The above differences from the control group were not statisticallysignificant (Student'st-test). - 89 - wildlife International, Ltd. Project Number 454-109 Appendix XI Histopathology Report EXPERI MENTAL PATHOLOGY LABORATORIES, I NC. Project Number 454-109 WILDLIFE INTERNATIONAL, LTD. STUDY NUMBER 454-109 EPL PROJECT NUMBER 212-028 PFOS: A REPRODUCTION STUDY WITH THE MALLARD PATHOLOGY REPORT Submitted by: Experimental Pathology Laboratories, Inc. P.O. Box474 Herndon, VA 20172-0474 (703) 471-7060 Submitted to: Wildlife International, Ltd. Easton, MD 21601 December 27,2001 Project Number 454-109 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. TABLE OF CONTENTS PATHOLOGY SUMMARY................................................................................. 1 COMPLIANCE STATEMENT............................................................................ 8 QUALITY ASSURANCE FINAL CERTIFICATION............................................ ADULT MALLARDS SUMMARY INCIDENCETABLES Adult Males.................................................................................................. 1-1 Adult Females ............................................................................................. 1-3 HISTOPATHOLOGY INCIDENCETABLES Adult Males.................................................................................................. 11-1 Adult Females ............................................................................................. 11-5 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult Males.................................................................................................. 111-1 Adult Females ............................................................................................. 111-4 OFFSPRING MALLARDS SUMMARY INCIDENCE TABLES Offspring Males ........................................................................................... IV-1 Offspring Females....................................................................................... IV-2 HISTOPATHOLOGYINClDENCE TABLES Offspring Males ........................................................................................... v-1 Offspring Females....................................................................................... v-2 - 92 - ~~ Project Number 454-109 PATHOLOGY SUMMARY - 93 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454- 109 WILDLIFE INTERNATIONAL, LTD. STUDY NUMBER 454-109 EPL PROJECT NUMBER 212-028 PFOS: A REPRODUCTION STUDY WITH THE MALLARD PATHOLOGY SUMMARY Light microscopic examination was performed on sections of selected tissues from adult male and female mallard ducks (Anasplatyrhynchos)which were untreated or which received 10 ppm a.i. of the test article (Perfluorooctane Sulfonic Acid, Potassium Salt [PFOS]) in the feed for approximately21 weeks. Selected tissues from untreated 14-day post-hatchingoffspring 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 the test substance over at least a 21-week period. The experimental design was as follows: aAdultswere euthanized after receivingthe test substance in the diet for approximately 21 weeks. bOffspringdid not receive the test substance in the diet. All offspringwere euthanizedon Day 14 post-hatching. MATERIALS AND METHODS At the completion of the study interval, all surviving adult mallards were euthanized. Selected offspring were also euthanized at approximately 14 days of age. Necropsieswere performed on all birds by Wildlife International, Ltd. EPL~I - 94 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-109 Wildlife International,Ltd. Study Number 454-109 Tissues were collected by Wildlife International, Ltd. from adults and offspring, fixed in 10% neutral buffered formalin, and sent to Experimental Pathology Laboratories, Inc (EPL@)w, here they were trimmed, embedded in paraffin, and made into hematoxylin and eosin-stained sections on glass microscope slides. The following tissues from adult and offspring mallards were examined by light microscopy as available: liver, gallbladder, proventriculus, kidney, brain, gonad (ovary or testis), bursa of Fabricius, and adipose tissue. All tissues required by protocol are 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. Nongradablefindings (including autolysis such that a diagnostic interpretationcould not be rendered) are listed as present (P). All findings for all animals are summarized by sex, age group, and treatment group in the Summary IncidenceTables, together with the total number of animals in each group for which 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 observations in these tables were transcribed from the Gross Necropsy records provided by Wildlife International, Ltd. RESULTS Except for one control (3235) adult male, 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 the liver, kidney, adipose tissue, brain, and proventriculus of adult and offspring males and females, bursa of Fabricius in offspring males and females, ovaries of adult and -2- - 95 - EPL@ EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-109 Wildlife International,Ltd. Study Number 454-109 offspringfemales and testes of male offspring. Any potential test article effects on the bursa of Fabricius in adult males and females could not be determined since no samples were submitted for evaluation. The bursa of Fabricius typically regresses prior to sexual maturity. In addition, any potential test-article effects on the gallbladder from adults and offspring could not be definitively determined due to the extensive autolysis, which precluded any accurate diagnosis. In two (2) control and seven (7) 10 ppm a.i. adult males, the testes were observed macroscopicallyas being reduced in size (< 3.5 cm) when compared to other birds. Of these birds, one control (3225) and four treated birds (3261, 3275, 3279, 3287), exhibited decreased spermatogenesis, characterized by a reduction in the number or absence of maturing/mature spermatozoa in the seminiferous tubules. The other control (3243) and one treated bird (3295) exhibited degeneration/atrophy of the seminiferous tubules characterized by an absence of mature sperm and a decrease in the number of cell layers of the germinal epithelium. The remaining two of seven 10 ppm a.i. males (3263, 3285) with macroscopically noted reduced testicular size and the balance of the control and 10 ppm a.i. males exhibited normal spermatogenesis, characterized by the presence of numerous spermatids and mature spermatozoa in the seminiferous tubules. Reduced testicular size, with or without microscopic evidence of decreased spermatogenesis, is a feature most consistent with early postreproductive phase regressions, a normal physiological phenomenon. The degeneration/atrophy noted in the control and treated adult birds may simply represent a later stage of this normal change. However, the increased incidence of reduced size (with or without a corresponding microscopic observation) of the testes in 10 ppm a.i. adult males, when compared to controls (7 vs. 2), suggests -3- - 96 - Project Number 454-109 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Wildlife International, Ltd. Study Number 454-109 that treatment may have accelerated the normal post-reproductive phase regression. Testes of all offspring examined were listed as immature because they lacked all evidence of spermatogenesis, had small seminiferous tubules with absent or narrow lumens, and had germinal epithelium with decreased cell layers (compared to adult testes). This morphology is consistent with normal physiological immaturity expected in 14 day-old birds. Ovaries of adult females exhibited normal oogenesis, characterized by numerous ova in various stages of follicular development. In five (5)controls and five (5) 10 ppm a.i. adult females, the ovaries were indicated macroscopicallyas "regressed" or "regressing." Microscopically, those ovaries were no different from their cohorts, except that there may have been a reduction in the number of fully developed ova. If present, this change was not recorded microscopically, since it was considered to be consistent with early post-reproductive phase regression, a normal physiological phenomenon. Since the number of females affected was the same in both the control and 10 ppm a.i. groups, there was no indication that treatment had any effect on this phenomena. All ovaries of offspring females were characterized by numerous oocytes and very small, nonpedunculatedfollicles completely embedded in a prominent ovarian cortex. This morphology is consistent with the normal physiological immaturity expected in 14 day-old birds. Vacuolization in the liver was characterized by the presence of vacuoles within the hepatocellular cytoplasm which varied from, usually small, poorly demarcated vacuoles, giving a "lace-like" appearance to the hepatocellular cytoplasm, to variably sized, sharply demarcated, clear vacuoles in the hepatocellular cytoplasm. While special stains were not employed to identify the -4- - 97 - Project Number 454-109 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Wildlife International,Ltd. Study Number 454-109 material in the vacuoles, the latter morphology was consistent with fat accumulation. Hepatocellular vacuolization was observed in the livers of adults and offspring of both sexes. Although there was a slight increase in the incidence of this change in 10 ppm a.i. treated adult males when compared to controls (20 vs. 12), there was no substantial difference in the mean severity between the groups. In addition, the change was not observed in treated male offspring or at a significant difference in treated adult or offspring females when compared to their respective controls. Since this finding was commonly observed in the livers from control birds, it was consideredto be incidental and not related to test article administration. The apparent increased incidence in the treated adult males was probably due to biological and/or distributional variability. Additional incidental findings also noted in the liver included liver pigment deposition in adult males, consisting of dust-like, brown to golden-brown pigment granules within the hepatocellular cytoplasm. The identity of the pigment was not determined, but it may have been biliary in origin. Also, the liver of two control adult males and one control adult female exhibited amyloid deposition consisting of amorphous pale eosinophilic material, which filled the hepatic sinusoids. In the one control male that died (3235),the liver parenchyma was almost completely replaced by the deposited amyloid. Also noted in the liver, at a low incidence in control and/or treated male and female adults and offspring, were foci of hepatocellular degeneration/ necrosis. These lesions were generally single, small, discrete foci of pale to hypereosinophilic hepatocytes with indistinct, fragmented, or condensed nuclei. Although there was generally a slight predominance of the lesion in treated birds, when compared to controls, it was considered incidental and not related to -5- - 98 - EPL@ EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-109 Wildlife International,Ltd. Study Number 454-109 treatment because of the low incidence, limited distribution, and relatively low severity (generally minimal to slight). Additional microscopic findings (described below) noted in adult and/or offspring mallards from control and treated groups included mononuclear cell infiltrates in various tissues, kidney nephropathy, and in the proventriculus, hyperplasia of the glandular epithelium. Generally, in both adult and offspring males and females, there was either no significant difference in the incidence of these lesions between control and treated groups, or the incidence was variable, with some lesions occurring at a higher incidence in controls when compared to treated birds, and other lesions having the opposite distribution. In addition, there was no significant difference in the mean severity of these lesions in treated birds when compared to controls. For these reasons, these findings were considered incidental and unrelated to test article (PFOS) administration. Mononuclear cell infiltrates, observed in various tissues, consisted of foci of lymphocytes and plasma cells generally accompanied by a few heterophils as a minor component. Nephropathy in the kidneys of both control and treated adult males and females was characterized by the presence of basophilic and/or regenerative tubules in the renal cortex. Hyperplasia of the proventricular glandular epithelium in adult females was generally multifocal in distribution, and characterized by increased numbers and/or basophilic staining of the glandular epithelium. In many cases, there was minimal compression of adjacent normal glandular epithelium. The pathogenesis of this lesion was not determined. However, since the incidence was not significantly different between controls and treated birds, it was considered incidental and not related to treatment. -6- - 99 - Project Number 454-109 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Wildlife International, Ltd. Study Number 454-109 CONCLUSIONS AND SUMMARY No lesions considered possibly related to test article (PFOS) administration were noted in the liver, kidney, adipose tissue, brain, and proventriculus of adult and offspring males and females, bursa of Fabricius in offspring males and females, ovaries of adult and offspring females and testes of male offspring. The increase in the number of adult males in the 10 ppm ami. treated group with reduced testicular size (with or without a corresponding microscopic observation), suggests that PFOS may have accelerated early post- reproductive phase regression, a normal physiological phenomenon. Any potential test-article effects on the bursa of Fabricius in adult males and females could not be determined since no samples were submitted for evaluation. In addition, any potential test-article effects on the gallbladder from adults and offspring could not be definitively determined due to the extensive autolysis that precluded any accurate diagnosis. The few other changes in various tissues of adult and/or offspring mallards from control and 10 ppm a.i. treated groups were considered incidental and unrelated to test article (PFOS) administration. R n FREDERICK A. RUECKER, DVM, MS, Dlplomate, I Veterinary Pathologist 1 FAWlcp -7- - 100 - EPL@ EXPER IMENTAL PATHOLOGYLABORATOR IES, INC. Project Number 454-109 COMPLIANCE STATEMENT Client Name Wildlife International, Ltd. EPL Project Coordinator Dr. Margarita M. Gruebbel Client Study 454-109 EPL Pathologist Dr. Frederick A. Ruecker Species Mallard EPL Project Number 212-028 Study Title PFOS: A Reproduction Study with the Mallard Test Article PerfluorooctaneSulfonic Acid, Potassium Salt (PFOS) The histopathologyportions of the above-referencedstudy were conducted in compliance with the Good Laboratory Practice regulationsof the Environmental Protection Agency as stipulated by 40 CFR 160. % c\(-k$ lLfi2t&. EPL Pbject Coordinator -a- Project Number 454-109 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. QUALITY ASSURANCE FINAL CERTIFICATION Study Title: PFOS: A Reproduction Study with the Mallard Client Study: 454-109 EPL Project Coordinator: Dr. Margarita M. Gruebbel EPL Project Number: 212-028 EPL Pathologist: Dr. Frederick A. Ruecker 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 lO/ll/Ol; 10/15/01 10/12/01 10/19/01 11/6,7/01 11/30/01; 12/4/01 1/28/03 lO/ll/Ol; 10/15/01 10/12/01 10/19/01 11/6,7/01 12/4/01 1/28/03 Date reported to Study DirectorIManagement Date of last quarterly facility inspection I12a/o3 1 1/02 Form No. 6-2 (7/2/99) -9- - 102 - Project Number 454-109 ADULT SUMMARY INCIDENCE TABLES I 454-109 Adult Sacri f i ce Male Mallard ADIPOSE TISSU.E_(NO. EXAMINE-D_) E d enia Foreign Body Granuloma Infiltrate, Mononuclear Cell Inf1 a m a t ion -~ Intravascular - 103 - Project Number 454-109 SUMMARY INCIDENCE TABLE GROUP GROUP CONTROL 10-ppmai (20) (20) __ 1 2 1 (20) (20) 1 ____ Malacia 1 BURSA OF FABRICIUS (NO. EXAMINED) ~_____~__ ---- ~ _ _ ~ GALLBLADDER ~ (NO..~EXAMINE-D-)__-~ Autolysis Precludes Diagnosis .~ ~ ~ _ _ __-__~~~____ - KIDNEY (NO. _EXAMI_NED)~ _ _ ~Aniyl aid Deposi ti on ~ Coccoid Organi sms ___. ~ -. .- (20) - (20) .~ ~ 20 - 20 . (20) (20) _ 1 - _ I -___ - - ~ Mineralization Nephropathy 2 3 LIVER (NO. EXAMINED) I (20) I (20) I 2 'Degenerati on/Necrosi s , Hepatocell ular 2 Hyperplasi-a,_Bile Ducts Infi 1 trate, M i xed Inflanmatorv _ _ _ ~ __ _ 20 20 a 7 Vacuo1 ization, Hepatocellular - ____ ~_ _________ Atrophy, Glandular Epithelium 12 20 (20) _ _ (20) EPL Experimental Pathology Laboratories, Inc. 1-1 -~ ___ - 454- 109 Adult Sacri f i ce Male Mallard PROVENTRICULUS (CONTINUED) ________~~~~~ - 104 - Project Number 454- 109 SUMMARY INCIDENCE TABLE . ..~ )3perimental Pathology Laboratories, Inc. 1-2 - 105 - Project Number 454-109 454-109 Adult Sacri fi ce Feniale Mallard SUMMARY INCIDENCE TABLE L--r--- ADIPOSE TISSUE (NO. EXAMINED) Edenia Foreign Body Granulonia Infiltrate. Mononuclear Cell Inf1 aniniati on ____.__~ __ Coccoid Organisms, Intravascular Infiltrate, Mononuclear Cell, I Perivascular Malacia Vascul iti s/Perivascul i ti s L A OF FABRICIUS (NO. L- EXAMINED) - GALLBLADDER (NO. EXAMINED) _ _ -~ / Anivl oi d D ~ D O tSi o~n Coccoid Organi snis ... Infiltrate- , Mono~ nucle_a_r Cell Inf1 anmat i on, Acute I n f 1 anuna t ~~ i on . Granu 1 oniatous Mineralization LIVER (NO. EXAMINED) I Deaeneration/Necrosis. Infiltrate. Mononuclear Cel_l___ ..~ - P_i_gment, Hepatocytes Mononuclear Ce.11 Infi1 trate ~ . Vacuolization, Hepatocellular ~ -(NO, E XAMillED) h-- Aniyloid Deposi tion b r i m c n t a l Pathology Laboratories,Inc. 1-3 454-109 Adult Sacri fice Female Mallard - 106- Project Number 454-109 SUMMARY INCIDENCE TABLE rimental PathologyLaboratories,hc. 1-4 - 107 - Project Number 454-109 ADULT HISTOPATHOLOGY INCIDENCE TABLES 454- 109 Adult Sacrifice Male Mallard - 108 - Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE GROUP EPL 11-1 Experimental Pathology Laboratories, lnc. Key : X-Not Remarkable N-No Section I=Inconiplete A-Autolysis l=minimal E=slight/miId 3=soderate 4=moderately severe 5=severe/hiah P=Present 6-Benign M-Mal ignant m-missing one paired organ u-unscheduled sac./death 454-109 Adult Sacrifice Male Mallard - 109 - Project Number 454- 109 HISTOPATHOLOGY INCIDENCE TABLE GROUP 'ROVENTRICULL A t r o p h ~Glandular Epithel ium Hyperplasia, G 1 andul a r Epi the1 i urn. Mu1t i focal I n f i l t r a t e , Mononuclear Cell Vasculitis/Perivasculitis lESTIS ~ Coccoid Org.a&sms. I ntr a v a s A a r _ . Degenerati on/Atrophy. Seminiferous Tubules ~~ oi 1atat ion, Seminiferous Tubules I n f i 1 trate,.Mpnonuclear Cell ~ Reduced _Spemadoa_enesi s Vascul i t i s/Perivascul iti s rnc. E P L Key : X-Not Remarkable N-No Section I-Incomplete A=Autolysis ~ ~ ~pathol~ogy ~ ~ ~ i b , ~ ~ . 5I--m.wi.nv~ie.mr~ea.,llhia2h=~sl~ight/m~i I d~ 3-m~ode~rate ~4=ml oderaitely sev~ere ~ , P=Present E-Benign M-Malignant m-mi ssing one paired organ u-unschedul ed s a c . /death 454-109 Adult Sacrifi ce Male Mallard - 110- Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE GROUP ADIPOSE TISSUE Edema ~ ~ _ _ _ _ _ _ foreign BoMranulonia ___ Infiltrate, Mononuclear-Cell Inf1 anmation-- BRAIN Coccoid Organi sms, Intravascular Infiltrate, Mononuclear -kid.Perivascular Mal aci Vasculi tisJPerivasculitis BURSA OF FABRICIIJS ~ GALLBLADDER -~ Auto1 vsi s L r K l udes D i aanosis KIDNEY ~ Amyloid D e u ti on Coccoid O r b s n i s Infiltrate. Mononuclear Cell Inflannnation, Acute _ _ ~ Inflanxnation, Granulomatous Mineralization Nephropathy LIVFR ~~ Amyloid Deposition Degeneration/Necrosis, HeDato c e m i r Hyperplasia. Bile Ducts Infiltrate, Mixed InfJmiatory Cell Infi 1 trate, M o n o n u c l e d L C L Intracytoplasnii c Brown .. ~~~ Pi gnient, Hepatocvtes MononucleaEC_ej_l Infi 1 trate Vacuolization. HeDatocellular E P L I ~~ ~~ ~ 11-3 Key : X=Not Remarkable N=No Section I-Incomplete A-Autolysis ~ ~ ~~ ~ ~ ~~ ~t ~ , i ~bln,c.l ~ ~~ S1-=msie~nvimer~ael/h ig2h-~sl i~ght/m~i I d ~ 3-m~oder~ate ~4.mol deratiely sev~ere ~ , P-Present B-Benign M=Malignant ni-missing one paired organ u=unscheduled sac./death 454-109 Adult Sacri fice Male Mallard - 111 - Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE GROUP PROVENTRICULUS Atrophy, Glandular Eoi the1 iurn Hyperplasia. Glandular Epithelium, Multifocal ~ Infiltrate. Mononuclear Cell Vascu 1 it1 s/Per 1 u u l it1 s TESTIS ~ ___ Coccoid Organi snis, Intravascular Degeneration/Atrophy. - S e n i i n i f e m Tubules Dilatation, Seininiferous - Tubules _- _ _ _ _ _ _ _ ~ Infi 1 tratr,_tlononuclear Cell Reduced Spermatogenesis ~ Vascul itis/Perivascul&s-___ ~___- ~~ ~~ ~~ ~- ___ I . __ __ ~~ ___ ~ ~ _ __ - - -- ~~ __ ~ _ ~_ ~~~~~ __ - 1 1 I I1 tl I VI ~- I I 11-4 -Li P-Present 8=Benign M=Malignant m-missing one paired organ u-unscheduled sac./death 454-109 Adult Sacrifi ce Feniale Mallard - 112- Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE GROUP ! Intravasculx...- _~~.-_____ Infi 1 trate_&MononuclearCell, Perivascular Malacia ~~~~~ Vasculitis/Perivasculitis (BURSA OF FABRICIUS LIVER ~~ Amvl oi d De-t i on Degeneration/Necrosi-s HepatoceLldar Hyperplasia, Bile Ducts Infiltrate, Mixed Inflammatory Cell ~~ Infi 1 trate. Mononuclear Cell Intracytoplasmic Brown __ Piqnient. HepL3tocvtes Mononuclear Cell Infiltrate Vacuolization. Hepatocellular EPL Experimental ~ 11-5 ~Laborato~ries, Key : X-Not Remarkable N-No Section I-Incomplete A-Autolysis h 51--smei~vneirmeailhi a2h-sl,ight/mi Id ~3=moderate ~4=moderately severe P=Present 8-Benign M=Malignant m=missing one paired organ u=unscheduled sac./death 454- 109 Adult Sacri f i ce Female Mallard - 113 - Project Number 454- 109 HISTOPATHOLOGY INCIDENCE TABLE GROUP A N I M A L !~P;EN;;;~ LUs Atro h Glandular Epithelium H er lasia,Glandular Epi the1 ium, Mu1 ti focal Infiltrate. Mononuclear CellVasculitisl~rivasculitis___ EPL ~ Key :X=Not Remarkable N=No Section I=Incomplete A-Autolysis ~pathology ~ ~ ~ b ~ ~ 5l==msie~nviemr~ael/h ig2h-~s i ight/mi~i di 3-mo~derate ~4-~moderatiely seve~re~ ~ , ~ P-Present B=Benign M=Mal ignant m=missing one paired organ u-unscheduled sac./death 454-109 Adult Sacri f i ce Feniale Mal lard - 114- Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE GROUP Infiltrate. Mononuclear Cell Intracvtoplasnii c &oyn Pigment, HepdtocytesMononuclearCell Infiltrate Vacuolization. Hepatocellular 1 1J1 ~--T-------- 2 1 1 L ' 1 2 1 1111 I 1 1 1 1 1 , 1 1 1 ~ 11 1 1 1 1 ' ' I -1 1 2 ' 1 I 1, I 1 L 1 3 1 1 11 2 1 EPL ~ 11-7 Key : X=Not Remarkable N-No Section I=Incomplete ASAutolysis ~pathology ~ ~ ~ b ~ ~ 51--mseivn~eimr~eallhi 2a h=~slight/mi~Iid 3-mo~derate ~4~=nioderaitely seve~r~e ~ , ~ P-Present 6-Benign M=Malignant m=missing one paired organ u-unscheduled s a c . / d e a t h 454-109 Adult Sacri f ic e Female Mal l d r d ~~~ - 115 - Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE GROUP 11-8 P=Present 8;Benign M-Malignant m=missing one paired organ u-unscheduled sac./death - 116- Project Number 454- 109 ADULT CORRELATION OF GROSS AND MICROSCOPIC FINDINGS 454-109 Adult Sacri f ice Species: Mal 1a r d Animal Number Client Topography / Site 3225 TESTES CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex: Males Group Identification: CONTROL - Client Gross Observations Approximately 3.0 cm Microscopic Observations Reduced Spermatogenesis (TESTIS) Sacri f iced 3243 TESTES Approximately 2.0 cm DegenerationlAtrophy, Seminiferous Tubules [TESTIS) -~ I r c3 4 I R 111-1 f Species: nima $urn& 3235 Mal 1 ard Client Topography / Site KIDNEY Sex: Client Gross Observations Slightly pale Males Group Identification: CONTROL - Unscheduled Sac. /Deat h Microscopic Observations No Correlating Lesion I c. c. 00 I 454-109 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult Sacri f ice Species: 11 1 ard Sex: Males Group Identification: 10-ppmai rAnimaI Numbei Client Topography / Site Client Gross Observations Microscopic Observations 3261 [ESTES Approximately 3.0 cm Reduced Spermatogenesis (TESTIS) - Sacri f iced 3263 TESTES Approximately 3.0 cm No Correlating Lesion (TESTIS) 3275 TESTES ~~ 3279 TESTES Approxiniately 3.0 cm Approximately 3.0 cm Reduced Spermatoqenesi s (TESTIS) Reduced Spermatogenesis (TESTIS) 3285 TESTES Approximately 3.5 cm No Correlating Lesion (TESTIS) 3287 TESTES Approximately 3.0 cni Reduced Spermatoqenesi s (TESTIS) 3295 TESTES 1.5 em Degenerati on/Atrophy, Seminiferous Tubules (TESTIS) I I 111-3 454-109 Number 3226 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS 11 l a r d Client Topography / Site .IVER Sex: I Client Gross Observations Firm, pale, mottled Fenla1es I G r o u p Identification: CONTROL - Sacri f iced Microscopic Observations Amy 1oid Depo s it ion (IDNEY Pal e Amyloid Deposi t i on I_V_A R Y NARY Rearessed ovary Multiple fluid-filled cysts No C o r r e l a t ing Lesi on No C o r r e l a t i n g Lesion 3236 NARY Regressed ovary No C o r r e l a t i n g Lesion 3242 NARY Regressed ovary Atrophy NARY Cystic f o l l i c l es No C o r r e l a t i n g Lesion 3244 OVARY Regressed ovary No C o r r e l a t i n g Lesion 3246 OVARY Regressing ovary No C o r r e l a t ing Lesi on I 3 2 48 OVARY Hemorrhagic f o l 1ic l e I n f 1a m a t ion L h) 0 I I 11-4 Adult Sacrifice Species: Mal 1a r d nima $umt,Lr Client Topography / Site Sex: Fenidles Group Identification: 10-ppmai - Sacri f iced 1 Client Gross Observations Microscopic Observations 32 76 OVARY Regressed ovary No C o r r e l a t ing Lesi on 3280 OVARY Regressed ovary No C o r r e l a t i n g Lesion 3284 OVARY Cystic follicles No C o r r e l a t i n g Lesion 3286 OVARY Regressed ovary No C o r r e l a t ing Lesi on 3288 OVARY Regressed ovary No C o r r e l a t ing Lesi on 3290 OVARY Regressed ovary No C o r r e l a t i n g Lesion I c E I e P ch 111-5 - 122 - Project Number 454- 109 OFFSPRING SUMMARY INCIDENCE TABLES 454-109 Offspring Sacri f i ce Male Mallard - 123 - Project Number 454-109 SUMMARY INCIDENCE TABLE . IKIDNEY (NO. EXAMINED) Infiltrate, Mononuclear Cell . ( 3 ) ( 3 ) ~ -______. 454-109 Offspring Sacrifice Female Mallard ADIPOSE TISSUE (NO. EXAMINED) ~~ Infiltrate, Mononuclear Cell ~~ ~ ~ BRAIN (NO. EXAMINED) - 124- Project Number 454-109 SUMMARY INCIDENCE TABLE GROUP CONTROL (7) _ (7) GROUP 10-ppmai (7) 1 _ (7) EXAMINED) (7) (7) 1 (6) (6) 4 4 ~P ~P ~- (7) (7) 3 LIVER (NO. EXAMINED) Degeneration/Necrosi s, Hepatocellular, Acute Infiltrate, Mononuclear Cell Vacuo1 ization, Hepatocellular .___ (7) (7) 7 7 P R O V E N T R I C U L ~ N O .EXAMINED) (7) (7) Infiltrate, Mononuclear Cell 5 6 EPL I b r i m e n t a l PathologyLaboratories, Inc. IV-2 - 125 - Project Number 454-109 OFFSPRING HISTOPATHOLOGY INCIDENCE TABLES 454-109 Offspring Sacrifice Mal e Mal 1 ard -~ - 126 - Project Number 454- 109 HISTOPATHOLOGY INCIDENCE TABLE GROUP GROUP Infiltrate. Mononuclear Cell ~~ ~-_____-- BURSA OF FABRICIUS - GALLBLADDER - ~ _ _ ___ ~ I Autolysis Precludes Diagnosis_- KIDNEY Infi 1 trate. Mononuclear Cell cLIVER ~~ Degenerat ionlNecrosis. Hep a tocellaInfi 1 trate. Mononuclear Cell Vacuo1 ization. Hepatocellular +-- + -I 1 t &-- -- I , I I f Ti+ I - EPL I ~ v-1 ~pathology~ ~ ~ bI ,~, ~ . ~ ;~;;hi~m)~~~;;sl ~ ight/mi~Idi 3-mo~derate ~4=~moderatiely Seve~re~ ~ , ~ 454-109 Offspring Sacrifice Feniale Mallard - 127 - Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE GROUP GROUP c ~~ ADIPOSE TISSUE_ Infi 1 trate, Mononuclear Cell -___-__..~ - ___~__________ . ~ . _____-.. . 1.n.fi 1 trate, Mononuclear C e l l - DeQeneration/Necrosi s. E- Imiature ~ Infiltrate. Mononuclear Cell v-2 ;~n~;;;;ni;~~;;sl E P L Key :X=Not Remarkable N-No Section I-Incomplete A-Autolysis ~ ~PatholoWTLa~boratories, ~ ~ ight/mi Iid 3-moderate 4~-moderately sever~e ~ P=Present 8-Benign M=Malignant m-missing one paired organ u-unscheduled sac./death w i l d l i f e International, Ltd. - 128 - Project Number 454-109 Appendix XIIA The Analysis of PFOS in Adult Red Blood Cells and Sera 4 - 129- WildlifeInternational, Ltd. Project Number 454-109 Exygen Study No.: 023-066 Table X. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 5 Sponsor ID 454-109-3249-Wk5 454-109-3249-Wk5* 454- 109-3251-Wk5 454-109-3251-Wk5* 454- 109-3252-Wk5 454-109-3252-Wk5* 454- 109-3253-Wk5 454-109-3253-Wk5* 454-109-3254-Wk5 454- 109-3254-Wk5 * 454-109-3255-Wk5 454-109-3255-Wk5* 454-109-3256-Wk5 454-109-3256-Wk5* Centre ID 0107230 0107230 0 107231 0107231 0 107232 0107232 0107233 0107233 0107234 0 107234 0107235 0107235 0107236 0 107236 Set Number 052101B 052101B 052 101B 052101B 052101B 052101B 052101B 052101B 052101B 052 101B 052101B 052101B 0521018 052 10I B Extraction Date Analysis Date 5/21/01 5/21/01 5/21101 5/21/01 5/21/01 5/21/01 512 1/01 5/21/01 5/21/01 5/2 1/01 5/21/01 5/21/01 5/21/01 512 I10 1 512210 I 512210 1 5/22/0 1 512210 1 5/22/01 5/22/01 512210 1 5/22/01 5/22/01 5/22/01 5/22/01 512210 1 5/22/01 512210 1 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) NQ NQ 0.00197 0.00167 NQ NQ NQ NQ NQ NQ 0.00143 NQ NQ NQ 0.000401 0.000706 * Duplicate irijectwn N Q = Not Quantifiable (A peak was deteclcd at the corresponding analyte Letention time but was less than the lowest coriccntration of the calibration standards (0.OOOl p g l d . ) ) LOQ = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/nL) was used to calcultite the average and standard deviation. Exygen Research Page 28 of 98 Ad" WildliJe International, Ltd. Project Number 454-109 Exygen Study No.: 023-066 Table XI. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 5 Sponsor ID 454-109-3289-Wk5 454-109-3289-Wk5* 454-109-3290-Wk5 454- 109-3290-WkS* 454- 109-3291-Wk5 454-109-3291-WkS* 454- 109-3292-Wk5 454-109-3292-Wk5* 454-109-3293-Wk5 454-109-3293-Wk5* 454- 109-3294-Wk5 454- 10%3294-Wk5* 454-109-3295-WkS 454- 109-3295-Wk5" 454-109-3296-Wk5 454-109-3296-WkS* Centre ID 0107237 0107237 0107238 0107238 0 107239 0107239 0 107240 0 107240 0107241 0107241 0107242 0 107242 0 107243 0107243 0107244 0107244 Set Number 052101BD 0S2101BD 052101BD 052 101BD 052 10 1 BD 052101BD 052I O 1BD 052101BD 0S2101BD 052101BD 052 I O IBD 05210 IBD 052 10I B D 052101BD 052101BD 052101BD Extraction Analysis Date Date 5/21/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/01 512 1101 5/23/01 5/21/01 5/23/0 1 5 / 2 1/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/0 1 5/21/01 5/23/01 5/2 1/01 5/23/01 9 2 1/01 5/23/0 I S I 1I/O I 5/21/01 5/23/01 5/23/01 5/21/01 5/23/01 5/21/01 5/23/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/nL) 24.4 24.7 11 2 10.0 11.8 12.3 15.1 13.1 30.1 30.8 4.88 3.95 18.9 18.8 26.4 28.5 17.8 8.80 * Duplicate injection LOQ = 0.01 pg/mL for red blood cells and scruni Exygen Research Page 29 of 98 - 131 - WildlgeInternational, Ltd. Project Number 454-109 Exygen Study No.: 023-066 Table XII. Summary of PFOS in Mallard Red Blood Cell Samples at 50 ppm a.i. for Week 5 Sponsor ID 454-109-3329-Wk5 454- 109-3329-Wk5* 454-109-3330-Wk5 454-109-3330-Wk5* Centre ID 0107245 0107245 0107246 0107246 Set Number 052201CD 052201Cl) 052201CD 052201CD Extraction Analysis Date Date 5/22/01 5/22/01 5/22/01 5/24/0 I 5/24/01 5/24/0 1 5/22/01 5/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pglmL) 58.9 62.2 112 123 89 33 Table XIII. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 10 Sponsor ID 454-109-3249-Wk 10 454- 109-3249-Wk10* 454-109-3250-Wk I0 354-10`)-3250-Wk I O * 454- 109-325I-WklO 454- 109.325 1-Wk IO* 454-109-3252-Wk10 454- 109-3252-Wk10* 454- 109-3253-Wk10 454-109-3253-WkIO* 454- 109-3254-Wk10 154-10`)-3254-WklO* 4.54-109-3255-Wkl0 454-109-3255-Wk10* 454-I09-3256-Wk10 454-109-3256-Wk10* Centre ID 0107250 0107250 0107251 010725 I 0107252 01 07252 0107253 0107253 0107254 0107254 0107255 0107255 0107256 0107256 0107257 0107257 Set Number 05220 I A 052201A 052201A 052201A 052201A 05220 1A 05220 1A 052201A 05220 I A 052201A 052201A 05220 1 A 05220I A OS220 1 A 05220 1A 052201.4 Extraction Analysis Date Date 5/22/0 1 5/22/0 I 512210 I 5/22/0 1 5/22/0 I 512210 I 5/22/01 5/22/0 I 5/22/0I 5/22/01 5/22/01 5/22/0 I 5/22/01 5/22/01 5/22/0 1 512210 1 5/23/0 1 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pdmL) NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NU NQ NQ NQ 0.00245 0.000200 0.000600 Duplicate injection NQ= Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)j 1.OQ = 0.01 pg/mL for red blood cclls and semin For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) was used to calciilate the average and standard deviation. Exygen Research Page 30 98 - 132 - 'wildlife International, Ltd. Project Number 454-109 Exygen Study No.: 023-066 Table XIV. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 10 Sponsor ID 454-109-3289-Wkl0 454-109-3289-WkIO* 454- 109-3290-Wk10 454-109-3290-WklO* 454-109-3291-Wk 10 454-109-3291-Wk IO* 454- IO-3292-Wk10 454- 109-3292-WkIO* 454-109-3293-Wkl0 454-109-3293-Wk10" 454-109-3294-Wk10 454- 109-3294-WklO* 454-109-3295-Wk I O 454-109-3295-Wkl0* 454- 109-3206.Wk 10 -45_4-_10_ 9-3296-Wk10* Centre ID 0107258 0107258 0107259 0107259 0107260 0 107260 010726I 0 107261 0107262 0107262 0 107263 0107263 0 107264 0107264 0107265 0107265 * Duplicate injection Set Number 052201AD 052201AD 05220I AD 052201AD 05220 I AD 05220 I AD 052201 AD 052201AD 052201AD 052201 AD 052201AD 052201AD 05220 I AD 052201AD 05220 I AD 052201c Extraction Analysis Date Date 5/22/01 5/23-24/01 5/22/01 5/23-24\01 5/2U01 5/23-24/01 5/22/01 5123-24/01 5/22/01 5/23-24/01 5/22/01 5123-24/01 5/22/01 .5/23-24/01 5/22/01 5123-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/0 1 5/22/0 1 5/23-2410 1 5/22/01 5/23-24/01 5/22/01 Y23-24/01 5/22/01 S/23-24/01 5/22/0 1 5123-24/01 5/22/01 5123-24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL,) 19.3 19.6 20.4 20.2 14.1 14.9 67.3 66.0 33.5 34.2 60.8 58.9 16.1 16.0 11.6 11.7 ~ 30.3 20.7 LOQ = 0.01 pp/mL for red blood cells and serum Exygen Research Page 31 of 98 - 133 - wildlife International, Ltd. Project Number 454-109 Exygen Study NO.: 023-066 Table XV. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 15 Sponsor ID 454- 109-3249-Wk15 454- 109-3249-Wk15* 454-109-3250.Wkl5 454-109-3250-Wk15* 454-109-3251-Wk15 454- 109-3251-Wk15* 454- 109-3252-Wk15 454- 109-3252-Wk15* 454- 109-3253-Wk15 454-109-3253-Wk15* 454- 109-3254-Wk15 454-109-3254-Wk15* 454- 109-3255-Wk15 454- 109-3255-Wk15* 454- 109-3256-Wk15 454-109-3256-Wk15* 454- 109-3236-Wk15 454- 109-3236-Wk15" Centre ID 0107266 0107266 0107267 0107267 0107268 0107268 0 107269 0107269 0107270 0107270 0107271 0107271 0107272 0107272 0107273 0 IO7273 0 107274 0 107274 Set Number 052201B 0522010 05220I 8 052201B 052201B 052201B 05220I B 05220 I B 05220I B 052201B 052201B 052201B 052201B 0522018 052201B O52201R 0522018 052201 B Extraction Analysis Date Date 5/22/01 5/23/01 5/22/01 5/23/01 5/22/0 1 5/23/01 512210 I 5/23/01 5/22/01 5/23/01 5/22/01 5/23/01 512210 1 5/23/01 512210 1 512210 I 5/23/01 5/23/01 5/22/01 5/23/01 5/22/01 5/22/01 5/23/01 5/23/01 5/22/01 5/23/01 5/22/01 5/23/01 5/22/01 5/23/01 5/22/01 5/23/01 .5/22/01 5/23/01 _5_/22/01 5/23/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) NQ NQ NQ NQ 0.00179 0.00188 NQ NQ NQ NU ND ND NQ NQ NQ NQ NQ NQ 0.000243 0.000613 * Duplicate injection ND = No peak detected NQ = Not Quantifiable (A peak was detected at the corresponding analyte rctcntion time but \vas less than the lowest concentration of the calibration standards (0.0001 pg/mL)) LOO = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) and for ND, zero \vas used to calculate the average and standard deviation. Exygen Research Page 32 of 98 - 134 - Wildlife InternationaI, Ltd. Project Number 454-109 Exygen Study No.: 023-066 Table XVI. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 15 Sponsor ID 454- 109.3289- Wk 15 454- 109-3289-Wk15* 454-109-3290-Wk15 454-109-3290-WkI 5* 454-109-3291-Wkl5 454- 109-3291-Wk15* 454-109-3292-Wk15 154- 109-3292-Wk15* 454-109-3293-Wk 15 454-109-3293-Wk15* 454- 109-3294-Wk15 454-109-3294-Wk15* 454- 109-3295-Wk15 454- 109-3295-Wkl5* 154-109-3296-Wkl5 454-109-3296-Wk15* Centre ID 0107275 0107275 0107276 0107276 0107277 0107277 0107278 0107278 0107279 0107279 0 107280 0107280 0107281 0107281 0107282 0 I07282 Set Number 052201BD 052201BD 052201BD 052201BD 052201BD 052201BD 05220 1BD 052201BD 052201BDR 052201BDR 052201BDR 05220 1BDR 052201BDR 052201HIX 052201BD 052201 BD Extraction Analysis Date Date 5/22/0 1 5/22/01 5/22/01 5123-24/01 5123-24/01 5123-24/01 5/22/01 5/23-24/01 5/22/01 5/22/0 I 5123-24/01 5123-24/01 5/22/01 5/23-24101 5/22/0I 323-2410 1 5/22/01 5/24/01 5/22/01 5/22/01 5/22/01 5/24/01 5/24/01 5/24/01 512210 I 5/24/01 5/22/01 5/24/0 1 5/22/01 5/23-24/01 5/22/01 5/23-24/01 ~ AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 53.5 48.2 3.98 3.44 60.9 61.4 9.65 8.79 113 I16 I .54 1.61 8.03 6.56 0.623 0.505 31.1 39.8 Table XVII. Summary of PFOS in Mallard Red Blood Cell Samples at 50 ppm a.i. for TERM Sponsor ID 454- 109-3297-TERM 454-109-7297-TERM* 454- 109 3298-'I'EKM 454-10%32C)8-TERM* Centre Set ID Number 0107283 0107287 0107284 052201CD 052201CD 052201BL~R 0107284 - 052201BDK Extraction Analysis Date Date 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 5/22//1- s124/0 1 AVERAGE: STANDARD DEVIATION: Analyte Found (pglniL) 275 272 406 418 343 no Duplicate injection LOQ = 0.01 pg/mL for red blood cells and serum Exygcn Research Page 33 of 98 - 135 - wildlife International, Ltd. Project Number 454-109 Exygen Study No.: 023-066 Table XVIII. Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 5 Sponsor Centre Set Extraction Analysis Analyte 454-109-3249-Wk5 0107303 454-109-3249-Wk5* 0107303 454-109-3251-Wk5 0107304 454-109-3251-Wk5* 0107304 454-109-3252-Wk5 0107305 454-109-3252-Wk5* 0107305 454-109-3253-Wk5 0107306 454-109-3253-Wk5* 0107306 454- 109-3254-Wk5 0107307 454- 109-3254-Wk5" 0107307 454-109-3255-Wk5 0107308 354-109-3255-Wk5* 0107308 454- I09-3256-Wk5 0107309 454-109-3256-Wk5* - 0107309 * Duplicate injection 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001 A 07200 1 A 072001A 072001A 072001A 07200 1.4 072001A 7/20/01 712 110 1 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 712 1101 7/20/01 7/21/01 7/20/01 712 1101 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 AVERAGE: STANDARD DEVIATION: 0.00414 0.00417 0.00566 0.00599 0.00878 0.00943 0.00516 0.00499 0.00432 0.00444 0.00462 0.00392 0.00544 0.005 16 0.00544 0.00167 LOQ = 0.01 pg/mL for red blood cells and scrum Exygen Research Page 34 of 98 - 136 - wildlife International, Ltd. Project Number 454-109 Exygen Study No.: 023-066 Table XIX. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 5 Sponsor Centre Set Extraction Analysis Analyte 454-109-3289-WkS 0107310 072001AD 7/20/01 7/22/0I 128 4.54-109-3289-Wk5* 0107310 072001hD 7/20/01 7/22/01 120 4.54-109-3290-Wk5 0107311 072001AD 7/20/01 7/22/01 79.6 454-109-3290-Wk5* 0107311 072001AD 7/20/01 7/22/01 94.2 454-109-3291-Wk5 0107312 072001AD 7/20/01 7/22/01 104 454- 109-3291-Wk5* 0107312 072001AD 7/20/01 7/22/01 110 454-109-3292-WkS 0107313 072001AD 7/20/01 7/22/01 83.4 454-109-3292-Wk5* 0107313 072001AD 7/20/01 7/22/01 85.8 454- 109-3293-Wk5 0 107314 072001AD 7/20/01 7/22/01 59.4 454-109-3293-WkS* 0107314 072001AD 7/20/01 7/22/01 76.6 454-109-3294WkS 0107315 072001AD 7/20/01 7/22/01 60.6 454- 109-3294-WkS* 0107315 072001AD 7/2O/O I 7/22/0I 57.5 454-109-3295-WkS 01073I6 072001AD 7/20/0 I 7/22/01 73.3 454-109-329S-WkS* 0107316 072001AD 7/20/01 7/22/01 76.9 AVERAGE: 86.4 STANDARD DEVIATION: 22.2 Table XX. Summary of PFOS in Mallard Serum Samples at 50 ppm a i . for Week 5 Sponsor ID 454-109-3329-Wk5 454-109-3329-Wk5* 454-109-3330-WkS 454-109-3330-Wk5* Centre ID 0107317 0107317 01073 18 0107318 -* Ihplicate injection Set Number 072001AD 072001AD 072001AD 072001AD Extraction Analysis Date Date 7/20/01 7/22/01 7/20/01 7/20/01 7/20/01 7/22/01 7/22/01 7122/01 AVERAGE: STANDARD DEVIATION: Analyte Foirnd (pg/mL,) 376 400 320 290 347 50 LOQ = 0.01 pg/mL for red blood cells and scruni Exygen Research Page 35 of 98 - 137 - wildlife International, Ltd. Project Number 454-109 Exygen Study No.: 023-066 Table XXI.Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 10 Sponsor Centre ID ID 454-109-3249-Wk IO 0107322 454-109-3249-Wk10* 0107322 454-109-3250-WkIO 0107323 454-109-3250-WklO* 0107323 454-109-3251-Wk10 0107324 454-109-325I-WklO* 0107324 454- 109-3252-Wk10 454-109-3252-WklO* 454- 109-3253-Wk10 454-109-3253-Wk IO* 0107325 0107325 0107326 0107326 454-109-3254-Wk10 0I07327 454- 109-3254-Wk10* 0107327 454- 109-3255-WklO 0107328 454-109-3255-WkIO* 0I07328 454-109-3256-Wk10 0107329 - 454-!09-3256-Wk10* - 0107329 Set Number 072001B 072001B 07200 IB 0720010 0720018 072001B 072001B 072001B 072001R 072001B 072001B 072001B 072001B 072001B 072001B -07_ 200_ I B Extraction Analysis Date Date 7/20/01 7/2O/O 1 7/2 1-22/01 7121-22/01 7/20/01 7/21-22/01 7/20/01 7121-22/01 7/2010I 7/21-22/01 7/20/01 7/2 1-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/2 1-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/0I 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 .- 7/21-22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 0.00271 0.00372 0.00726 0.00666 0.00659 0.00530 0.00899 0.00940 0.00517 0.00482 ND ND 0.00337 0.00403 0.00419 0.00384 0.00476 0.00266 * Duplicate injection ND = No peak detccted NQ = Not Quantifiable ( A peak was detected at the corresponding analyte retention time hut was less than the lowest conccntration of the calibration standards (0,0001 pglmL)) LOQ = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (hall the value of the lowest calibration standard, 0.0001 pg/mL) and Tor ND, zero w x used to calculate the average and standard deviatI on. Exygen Research Page 36 of 98 - 138 - wildlye International, Ltd. Project Number 454-109 Exygen Study No.: 023-066 Table XXII. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 10 Sponsor In 454-109-3289-Wkl0 454-109-3289-WklO* 454-109-3290-Wkl0 454-109-3290-Wk10* 454-109-3291-Wk10 454-109-3291-Wk10" 454- 109-3292-Wk10 454- 109-3292-Wk1O* 454- 109-3293-Wk10 354-109-3293-Wk IO* 454-109-3294-Wk 10 154-I09-3294-WklOc 454-109-3295-Wkl0 454-109-3295-Wkl0* 454-109-3296-Wk10 454-109-3296-WK- Centre ID 0107330 0107330 0107331 0107331 0107332 0107332 0107333 0 107333 0107334 0107334 0 107335 0107335 0107336 0107336 0107337 0107337 Set Number 072001BD 072001BD 072001BD 072W1BD 072001BD 072001BD 072001 BD 072001BD 072001BD 07200 1B D 07200 1BD 072001BD 072001B1) 072001BD 072001BD 072001BD Extraction Analysis Date Date 7/20/01 7/20/01 712201 7/22/01 7/20/01 7/22/01 7/20/01 712210 1 7/20/01 7/22/0 1 7/20/01 7/22/0 I 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/0 I 7/22/0 I 7/mO I 7/20/01 7/22/0 I 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/20/01 7/22/01 7/22/0 I AVEKAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 145 147 32.7 32.5 93.5 102 107 111 132 136 85.3 86.4 123 120 114 112 105 33.8 * Duplicate injection LOQ = 0.01 p d m L for red blood cclls and scrum Exygen Research Page 37 of 98 - 139 - WildlifeInternational, Ltd. Project Number 454-109 Exygen Study No.: 023-066 Table XXIII. Summary of PFOS in Mallard Serum Samples at 0 ppm a i . orWeek 15 Sponsor ID 454-109-3236-Wk15 454-109-3236-Wk IS* 454- 109-3249-Wk1S 454- 109-3249-Wkl5* 454- 109-3250-Wk15 454- 109-3250-Wk15* 454- 109-3251-Wk15 454- 109-3251-Wkl5* 4.54- 109-3252-Wk15 454- 109-3252-Wk15 * 454- 109-3253-Wk15 454- 109-3253-Wk15* 454-109-3254-Wk15 454- 109-3254-Wk15' 454-109-3255-Wk 15 454-109-3255-Wk15* 454-109-3256-Wk15 _ 454_ - 1_ 09-_ 32.5_ 6-W_ k1.5* Centre ID 0107338 0107338 0107339 0107339 0107340 0 I07 340 0107341 0107341 0107342 0107342 0107343 0 107343 0107344 0 I07344 0107345 0107345 0 I07346 - 010~ 7346 Set Number 07 1901A 07190I A 071901A 071901A 071901A 071901A 071901A 07 1901A 07 1901A 071901A 071901A 071901A 07 1901A 071901A 071901A 071901A 071901A -071901A Extraction Analysis Date Date 7/ 19/01 7/ 19/01 7/19/01 7/19-20/01 7/19-20101 7119-20/01 7/19/01 7/19/01 7/19/01 7/ 1910I 71 19/01 7119/01 7/19/01 7/ 19101 7/19-20/01 7119-20/01 7119-20/01 7119-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7119-20/01 7/19/01 7/19/01 711 9/0 1 7/19/01 7/19/01 7/19/01 7/19/01 7/19-20/01 7/19-20/0I 7119-20/0 I 7/ 19-20/01 7/ 19-20/01 7/19-20/01 7/19-20/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) ND m 0.00729 0.0102 ND ND 0.00466 0.00441 ND ND 0.00649 0.00723 ND ND 0.0233 0.00699 0.0195 ND 0.000503 0.00616 * Duplicate injcction ND = No peak detected NQ = N o t Quantifiable (A peak was detected at the corresponding analyte retention time but was lcss than the lowest concentration of the calibration standards (0.00Ol pg/mL)) LOQ = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0,0001pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 38 of 98 Wildlije International, Ltd. - 140 - Project Number 454-109 Exygen Study No.: 023-066 Table XXIV. Summary of PFOS in Mallard Serum Samples at 10 ppm ai. for Week 15 Sponsor ID 454-109-3289-Wk15 454-109-3289-Wk15* 454-109-3290.Wk 15 454-109-3290-Wk15* 454-109-329I-Wk15 454- 109-3291-Wk15' 454- 109-3292-Wk15 454- 109-3292-Wk15* 454-109-3293-Wk15 454-109-3293-Wk15* 454-109-3294-Wk15 454- 109-3294-Wk15* 454-109-3295-Wk15 454-109-3295-Wk15* 454- 109-3206-Wk15 454- 109-3296-Wk15' Centre ID 0107347 0107347 0107348 0107348 0107349 0107349 0107350 0107350 0107351 0107351 0107352 0107352 0107353 0107353 0107354 0107354 Set Number 07 190I AD 07 1901AD 07 1901AD 071901AD 071901AD 07 1901AD 07 1901AD 07 1901AD 071901AD 071901AD 07 l901AD 07 1901 AD 071901AD 07 I901AD 07 1901AD Extraction Analysis Date Date 7/19/01 7/20-21/0I 7/19/01 7/19/01 7/19/0 I 7/20-21/01 7/20-21101 7/20-21101 7/19/01 7/20-21101 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/19/01 7/19/01 7/19/01 7/20-21/01 7/20-21/01 7/20-21/01 7/20-2 1/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 152 I62 10.1 8.15 152 I42 12.1 11.6 128 132 6.73 4.52 117 I16 9.52 - 11.3 13.5 61.3 Table XXV. Summary of PFOS in Mallard Serum Samples at 50 ppin ah. for TERM Sponsor ID 454-109-3297-TERM 454-109-3297-TERM' 454-109-3298-TERM 454-109-329Y-TERM* Centre ID 0107355 0107355 0107356 0107356 * Duplicate injection Set Number 072001AD 072001N) 072001AD 072001AD Extraction Date Analysis Date 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/0 I 7/20/01 7/22/01 AVEIUGE: STANDARD DHVIA'I'ION: Analyte Found (pdmL) 685 60 1 664 60 1 638 43 LOQ = 0.01 pg/mL for red blood cells and serum Exygen Research Page 39 of 98 - 141 - Wildlife International, Ltd. Project Number 454-109 Appendix XIIB The Analysis of PFOS in Adult and Juvenile Liver and Sera - 142 - Wild1ife International, Ltd. Project Number 454-109 Report E01-1256 R e p a l E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A ReproductionStudy with the Mallard SUMMARY The concentration of the CBF17SOajnion of pemuorooctane sulfonate, potassium salt (PFOS, CaF,7S0;K') was determined in the liver and sera of adult mallards and their offspring in both a control group and a 10ppm dosed group from Wildlife feeding study #454-109. The following tables summarize the C8FlrSO; concentrations bund in the liver and sera of study samples. Extractedmatrix PFOS standards are used to establish the HPLC-ESIMSIMS instrument calibration curve for C8F17SO;. Concentrations of C8F17SOsa' re reportedafter correction for the purity and the potassiumcontributionto the mass of the PFOS reference substance. Table 1 summarizes all study sample data. Table 2 summarizes study data after removing certain results that have been statistically determined to be outliers utilizing the Dixon Q-Test. Table 1. Summary Table, CaF17S03'Concentration in Sera and Liver For individualsample values see Tables 10to 13 and Attachment8. 1 1 1 c-,:.-"....., I "%....-+"", 3M Environmental Laboratory Page 7 - 143 - wildlife International, Ltd. Project Number 454-109 Report E01-1256 Report EOl-1256 PFOS:A Reproduction Study with the Mallard PFOS: A Repmductlon Study with me Mallard Table 2. Summary Table, C8F17SOiConcentrationin Sera and Liver with Outliers Removed kldeConbdGroup 18' 20' FemakconM 17 -P <100nQhnC(15of17) NA 16&172nghnLrange(Zd17) MdeIOppmGrup 18 87300ngEmL ffl7wlO@J00w$mL 20' I ""% I 3' I * Ll ' /*I I " I I I I J * No outliers removedfrom this data set. Refer to Table 1 for resulk. Fof individualsample values seeTables 10 Io 13 and Attachment 8. PURPOSE The purpose of the analytical phase of this study is to quantify C8F,,S0; in the liver and sera of both control and dosed mallards and in their offspring. REFERENCSEUBSTANCES Table 3. Reference Substances I Source I 3M Internal I 0akwoodProducts.Inc. I Expiration Date 08/3112006 12/01/2010 3M Laboratwy SD-018. Lot #217 SD-036 I PhvsicalDesuiotion I WhiteCWliPowder I WhiteSolid I C-..: I I 3M EnvironmentalLaboratory - 144 - Wildlijie International, Ltd. Project Number 454-109 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS:A Reproduc(ionStudywlthlhe Mallard Absolute Recoveries. The absolute recovery of the QC samples Is determined by comparing the results of the extraded quality control spikes quantitated versus an extracted malrix curve and a solvent curve. The sera spikes quantitate higher using a solvent curve than when using a matrixextracted curve. The low spikes are 11.I% higher, the medium spikes 8.3% higher, and the high spikes 8.3%higher. The 9.5% average higher quantitation of the three levels of spikes using a solvent curve versus an extracted maMx curve demonstrates that it is appropriate to use matrix-extracted standards. The same trend is observed for liver samples. The extracted liver matrix curves are 12.2% higher. 7.8% higher, and 5.6% higher respectively for the low, medium and high spikes of the liver matrix, again demonstrating that it is appropriate to use matrix-extracted standards. Internal Standard: The internal standard (PFDA) was added to all samples and standards. PFDA was not used for quantiiation, but was used to monitor for gross instrument failure. 0 Instrument Response Calibration. HPLC-ESIMSIMS instrument response was calibrated using the concentration of the CsFt,SOJanion of periluorooclane sulfonate. potassium salt (PFOS) that was adjusted for purity and the potassium ion contribution to the mass of PFOS used to prepare stock solutions. Any future calculations requiring concentrations to be expressed as a wrcentane of dietaw in-take must adjust the dosed amounts as well. DATASUMMARY Tables 10 through 13 summarize individual sample data. Representative chromatograms are presented in Attachment C. The table displays CaFI7SO3c' oncentrations for individual injections. All data are corrected for purity of the PFOS reference substance used for the extractedmatrix calibration curve. Table IO. CSF&O; Data Summaw. Female Mallard Sera (Adult and Offspring) E01-1256-28693 1454-1043250.0 P p F ~ l eAd, uk EM-1256-28695 )454-109-3252,8PPM, Femele,Adult 3M EnvironmentalLaboratom 3M Environmental Laboratory ' I 0.5 0.5 I 11 I 400 757 Paae 14 of 216 Page 14 - 145 - WiIdlife International, Ltd. Project Number 454-109 Report E01-1256 Report E01-1256 PFOS:A ReproductionStudy with the Mallard PFOS: A Repmdudim Studywith the Mallard EOl-1256-28717 01-1256-28719 E01-125628721 lE01-125628723 01-1256-28725 EO1-1256-28727 01-1256-28729 01-125628731 E01-1256-28733 E01-1256-28735 01-1256-28737 01-1256-28739 E01-1256-28740 E01-1256-28742 01-125628743 E01-125828744 lE01-125&28745 '454-1043274,lO PPM,Female, Adult 454-1093276.10 PPM. Female, Adult 454-109-3278.10 PPM, Female,Adult ~454-109-3280,lOPPM, Female,Adult 454-1043282.10 PPM, Female, Adult 454-1043284,lO PPM, Female, Adult 454-1043286,lO PPM, Female. Adult 454-1043268,lO W M . Female,Adult 454-1043290.10 PPM. Female,Adult 454-1043292,lO PPM, Female, Adult 1454-109-3294.10 PPM. Female, Adult 1454-1043296.10 PPM. Female, Adult 1454-108-9583,O PPM. Female. Oftspring 1454-109-9539.0 PPM, Female, Oflspring 1454-109-9592.0 PPM. Female. opkpnng ~4541049598.0PPM. Female. ORsMlm )454-109-8603,0PPM, Female, Offspring 'I 0.5 I 0.5 1 0.5 1 02 1 0.5 10 10 I 1 10 1 0.5 1 0.5 10 _. 0.5 10 0.5 .-1 0.5 1 I 0.5 1 I 0.5 1 1 0.15 I I 0.2 1 0.2 1 I 0.1 I 1 I 1 1 I 1 I 1 I I 0.2 I 1 29400 40600 1 9840 32200 10800 6570 28900 42800 16000 10400 , 7660 10800 400 400 <loo <lo0 These values were rejected based on the results of the Dixon's 9-Test. 90% confidence interval, for the rejection of outliers 3M EnvironmentalLabmatory 3M Environmental Laboratory Pane 15 d216 Page 15 - 146 - wild1ije International, Ltd. Project Number 454-109 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS:A Reproduction Shrdy with the Mallard Table 11. CSFi7SOi Data Summary, Male MallardSera (Adult and Offspring) lEO1-125628671 E01-125628673 01-125528675 EO1-125628677 LEOI-1256-28680 01-1256-28682 01-125628684 1454-109-3227,O PPM, Male, Adull 454-1043229.0 PPM, Male, Adult 454-1043231.0 PPM, Male,Adult 454-1043233.0 PPM, Male,Adult 1454-1043237.0 PPM. Male. Adult 1454-109-3239,O PPM. Mele. &It 1454-109-3241,O PPM. Male, Adult 0.5 1 Si00 0.5 1 400 0.5 1 400 I 1 0.3 1) I 1 0.5 1 1 05 1 1 1400 t E01-125628700 E01-1256-28702 EO1-1256-28704 E01-1256-28706 01-125628708 01-125628710 E01-125628712 E01-125628714 E01-1256-28716 E01-125628718 E01-1256-28720 E01-125628722 -E01-125628724 E01-1256287% (EO1-125628728 E01-125628730 E01-1256-28732 E01-125628734 lE01-125828736 E01-12S28738 01-1256-28741 (454-1093257,lOPPM, Male,Adult /454-1043259.10PPM, Male. AchIt 454-109-3261,10 PPM, Male, Adult 454-1093263,10PPM, Male,Adult - . - ~ 454-109-3285 10 PPM~ Male Muit (454-1043267,lO PPM,Male. Advn /454-1043269 10 PPM Male Adult 454-1043271.10PPM. Male.Adull 454-lW3273,lO PPM, Male, Adult 454-lW3275,lO PPM, Male, Adult 454-109-3277,lO PPMMale. WIt 454-1043279,lO PPM. Male.Adull 454-109-3281.10 PPM. Male.Mull 4541043283.10 PPM, Male.Adult 1454-109-3285,lOPPM. Male,Adult 454-1043287,lO PPM, Male, Adult 454-109-3289.10 PPM, Male. Adult 4 % - 1 0 ~ 3 ~ 1i,o PPM, Male, Adult 1454-109-3293.10PPM, Male,Adult 1454-109-3295.10 PPM, Male, Adult 1454-109-9591,O PPM, Male, Offspring 3M Environrnenlal Laboratory 3M Environmental Laboratory 0.5 0.5 I 0.5 I I 02 j 0.5 I 10 I 10 10 400 78800 83300 68700 0.06 100 210000. I I I 02 0.5 I 05 I 10 10 10 I 93800 75700 806oO 0.5 10 11300 0.5 10 106ooO 05 10 ~ 0.5 10 80500 804oO 0.5 10 103oOo 0.5 10 74300 I 1 1 I 0.5 10 91m 0.5 10 108000 0.3 10 78700 0.3 10 97900 I 1 0.3 1 0.5 1 1 0.5 1 0.1 II 10 10 10 11 73500 997M) c100 Page 16 of216 Page 16 wiId1v e International, Ltd. - 147- Project Number 454-109 Report E01-1256 RepoctE01-1256 PFOS:A Reproduction Study with the Mallard PFOS:A ReproductionStudy with the Mallard EO1-1256-28750 1454-1049825.10PPM. Male. msprils E01-1256-28756 1454-1099636.10PPM. Male,OlrSprinS 1EOl-1256-26757 /454-1099642,10PPM. Male, o(fSpring I 0.2 1 1 0.1 1 I 0.2 j 11 11 11 5450 3670 4110 These values were rejected based on the results of the Dixon's Q-Test. 90% confidence interval,for the rejection of outliers. 3M EnvironmentalLaboratow 3M EnvironmentalLuboratov Pam 17 of216 Page 17 - 148 - wild1f e International, Ltd. Project Number 454-109 Report E01-1256 Report E01-1256 PFOS:A ReproductionStudy with the Mallard PFOS: A ReproductionStudy with the MaUard E01-1326-29709 E01-132629727 E01-1326-29745 0 pprn a.l.3234 F Liver, aduli 0 ppm a.i. 3236 F Liver, adult 0 pprn a.i. 3238 F Liver, adult 1.1290 1 c50 1 .o800 1 C50 1.1170 1 <50 E01-132630214 E01-1326-30232 EO?-1328-30250 10 pprn a i. 3290 F Liver, adult 10 pprn a i. 3292 F Llver,adult 10 pprn a.i. 3294 F Liver,adult 3M EnvironmentalLaborator, 3M Environmental Laboratory 1.0352 1.0658 1.0627 1 ' 4600 1 4760 Paw 18 of 216 Page 18 - 149 - Wildlqe International, Ltd. Project Number 454-109 Report E01-1256 Report E01-1256 PFOS:A Reproduction Study with the Mallard PFOS: A Reprcdudion SIudy with the Mallard 3M Enviranmenbl L a h o r a t n ~ 3M EnvironmentalLaboratory Paoe 19 d 216 Page 19 - 150 - WildlifeInternational, Ltd. Project Number 454- 109 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS:A ReproductionStudy with the Mallard Table 13. CSF17SOJ'Data Summarv. Male Mallard Liver (Adult and Offsatinn) I 1 326-29574 10 ppm a i 3219 M Liver, adult I I lE_ O1.-1.32.6.-29592 IO ppm a.i. 3221 M Liver,adult 1.1847 1.1181 <50 11 <50 I I E01-132629628 10 pprn a.i. 3225 M Liver,adult I I I E01-132529646 ;O ppm a.i. 3227 M Liver, adult ~ I I lE01-132629684 10ppm a.i. 3229 M Liver, adult 1.1043 1.0486 1.0336 <50 11 40 <50 3M EnvlronmentalLaboratory 3M Environmental Laboratory Paoe M of 216 Page 20 - 151 - wildlife International, Ltd. Project Number 454-109 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS:A RoproducaOnStudywith the Mallard STATEMENT OF CONCLUSION Sera results from the male control group portion of this study showed no detectable levels of CsF17SOj in 14 of 18 mallards (<IOOng/mL) and quantifiable concentrations of CsF17SO; in 4 of 18 (range 107-1400nglmL. Table 14). The sera results from the female control group portion of this study showed no detectable levels of CBFI7SO; in 15 of 17 birds (<100ng/rnL) and quantifiable concentrations of CaF17SOj in 2 of 17 (range 166-172ng/mL. Table 15). Analytical results for eighteen sera samples from the IOppm dosed male group showed an average of 87300nghL CeFI7S0; (range 68700-108000ng/mL, Table 15). Analytical results for nineteen sera samples from the 10ppm dosed female group showed an average of 16600nglmL CaFllSOj (range 6350-42800ng/mL,Table 15). Sera results from the male offspring control group showed no detectable levels of CBFl7S0jIn 2 of 3 birds (<lOOng/mL) and a quantifiable concentration in 1 of 3 (103 ng/mL. Table 14). The sera results from the female offspring control group portion of this study showed no detectable levels of CaF17SO; in 7 of 7 birds (cIWng/mL, Table 14). Analytical results for three sera sampies from the IOppm dosed male offspring group showed an average of 4410ng/mL (range 3670-5450ng/mL, Table 14). Analytical results for seven sera samples from the 10ppm dosed female offspring group showed an average of 4960nglmL (range 3420-6760ng/mL. Table 14). Liver results from the male control group showed no detectable levels of C8FI7SOj in 20 of 20 birds (<50ng/g, Table 14). The liver results from the female control group also showed no detectable levels of C,F,,SO; in 20 of 20 birds (<50ng/g, Table 14). Analytical results for twenty liver samples from the 10ppm dosed male group showed an average of 60900ng/g CSF1,SOO (range 24200-125000ng/g, Table 14). Analytical results for twenty liver samples from the lOppm dosed female group showed an average of 108OOn9/g CeF17SOi (range 2480-30700ng/g. Table 14). Liver results from the male offspring control group portion of this study showed no detectable levels of CeF17S03'in3 of 3 birds (c50nglg. Table 14). The i i e r results from the female offspring control group portion of this study showed no detectable levels of CeF17SO; in 7 of 7 birds (<50ng/g, Table 14). Analytical results for three liver samples from the lOppm dosed male offspring group showed an average of 3170ng/g (range 2200-4640ng/g, Table 14). Analytical results for seven liver samples from the 1Oppm dosed female offspring group showed an average of 3620ng/g (range 2240-5930ng/g, Table 14). 3M EnvironmentalLaboratcq 3M Environmental Laboratoty Page 21 01 216 Page 21 - 152 - wiId1ife Internationa1, Ltd. Project Number 454-109 Report E0l-1256 Report 01-1256 PFOS: A ReproductionStudy with the Mallard PFOS:A ReproducUonStudy with the Mallard Table 14. SummaryTable, CBF,7SOiConcentrationin Sera and Liver Male 1oppn Group For Individual SarnDles values seeTables 10 to 13 and Attachment B. . _ _ *..r_. _ - - - _ _ . _ I I _La__ 3M Environmental Laboratory .-__"-_^..,." Page 22 wiId1ijie International, Ltd. - 153 - Project Number 454-109 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A ReproductlmStudy with Re Mallard Table 15. Summary Table, CSFl7SOiConcentrationin Sera and Liverwith Outliers Removed I * No outliers removedfrom this data set. Reler to Table 14 for results. For individualsamples values see Tables 10 to 13 and Attachment E. STATISTICAML ETHODS Statistical methods included calculating means, standard deviations, % coefficient of variation, linear regressions. percent recoveries, and the Dixons 0-Test. The Dixons 0-Test. used for the rejection of outliers, has values ordered from lowest to highest. A Q-value is determined from the difference of a result and the next closest result that is divided by the difference of the highest and lowest value in the group of values. A crltlcal Q-value is determined based on the number of samples in the group and the desired confidence interval. If the Q-value is greater than the critical Q-value then the value will be rejected. -~ STATEMENT OF DATAQUALITY The only measurement is the quality control and matrix spike results. Accuracy (the observed amount in the QC and MS samples, divided by the amount spiked, expressed as a percent) was f w n d to range from 79.7% to 172%; while precision, expressed as the standard deviation of the recoveries, was found to range from 0.717% to 15.7%. ....v-.2 _,..._..._ __^___.I_I 3M EnvironmentalLaboratory from Reproductive Performance by Pen Appendix XI11 - T a b l e 1 Eggs Laid / Maximum Laid (%) a Mallard Reproduction Study with PFOS Eggs Replicate Laid 0 PPM A . I. Max. Laid Arcsin % Trans. Eggs Laid 10 PPM A.I. Max. Laid Arcsin % Trans. Eggs Laid 50 PPM A.I. Max. Laid Arcsin % Trans. 1 70 72 97 80.41 61 72 a5 66.99 2 51 72 71 57.31 58 72 ai 63.a3 3 53 72 74 59.09 54 12 75 60.00 4 42 72 58 49.a0 72 72 100 90.00 5 19 72 26 30.91 46 12 64 53.06 6 0 72 0 0.00 57 72 79 62.a4 7 66 72 92 73.22 72 72 100 90.00 a 62 72 a 6 68-12 63 72 aa 69.30 9 42 72 58 49.80 56 72 78 61.87 10 - 59 72 a2 64.a5 11 55 72 76 60.93 57 72 79 62.84 12 56 72 78 61-87 47 72 65 53.90 13 67 72 93 74.72 46 72 64 53.06 14 51 72 71 57.31 56 72 78 61.87 15 54 72 75 60.00 43 72 60 50.61 16 31 72 43 41.01 30 72 42 40.20 Total 719 i o a o 877 1152 - - Mean 48 72 67 54.97 55 72 76 62.83 - - SD 19 0 26 19.87 11 o 15 12.87 - - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). Eggs Laid 150 PPM A.I. Max. Laid Arcsin % Trans. - - - I w ch P I 39. ` U CL 0 P ch f Appendix XI11 - Figure la Eggs Laid from a M a l l a r d Reproduction Study with PFOS - Control 70 60 2 W 50 a \ P 40 w x m 3 Z 30 20 10 * n w r) I - 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 * Eggs Laid Data are not Pe available n due Number to adult mortality. I c3 VI VI I R f 80 70 60 50 a \ =21 40 w m 3 Z 30 20 10 Appendix XI11 - F i g u r e lb - Eggs Laid from a Mallard Reproduction Study u i t h PFOS 10 PPM A.I. n 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Pen Number Eggs Laid from Reproductive Performance by Pen Appendix XI11 - T a b l e 2 Eggs Cracked / Eggs Laid (%) a Mallard Reproduction Study with PFOS 0 PPM A.I. E g g s Eggs Replicate Crack Laid Arcsin % Trans. 10 PPM A.I. Eggs Eggs Crack Laid Arcsin % Trans. 50 PPM A.I. Eggs Eggs Crack Laid Arcsin % Trans. 1 1 70 1 6.86 0 61 0 0.00 2 1 51 2 8.05 0 58 0 0.00 3 0 53 0 0.00 0 54 0 0.00 4 2 42 5 12.60 4 72 6 13.63 5 1 19 5 13.26 0 46 0 0.00 6 0 0 0 57 0 0.00 7 0 66 0 0.00 3 72 4 11.78 8 1 62 2 7.30 0 63 0 0.00 9 0 42 0 0.00 3 56 5 13.38 10 - - 0 59 0 0.00 11 2 55 4 10.99 0 57 0 0.00 12 0 56 0 0.00 0 47 0 0.00 13 1 67 1 7.02 0 46 0 0.00 14 0 51 0 0.00 0 56 0 0.00 15 0 54 0 0.00 0 43 0 0.00 16 1 31 3 10.35 0 30 0 0.00 Total 10 719 10 877 - Mean 1 48 2 5.46 1 55 1 2.43 sn 1 19 2 5.26 1 11 2 5.23 - - Data are not available due t o adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). 150 PPM A.I. Eggs Eggs Crack Laid Arcsin % Trans. - - - - - - Appendix XI11 - F i g u r e 2a - Eggs Cracked / Eggs L a i d from a Mallard Reproduction Study u i t h PFOS Control 70 60 Z w 50 I 21 Y \ 00 U 40 I w rn E 3 Z 30 20 10 n - 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Eggs Laid Pen Number E Eggs Cracked * Data are not available due to adult mortality. 80 70 60 50 a \ CT 40 w m E 3 Z 30 20 10 Appendix XI11 - Figure 2b Eggs Cracked / Eggs Laid from a Mallard Reproduction Study with PFOS - 10 PPM 6.1. 0 217 218 219 220 221 222 223 224 225 Pen Number Eggs Laid EEggs Cracked 229 230 231 232 from Reproductive Performance by Pen Appendix XI11 - Table 3 Viable Embryos / Eggs Set ( 8 ) a Mallard Reproduction Study with PFOS 0 PPM A.I. Viable Eggs Replicate Ehbr. Set Arcsin % Trans. 1 0 PPM A.I. Viable Eggs E m b r . Set Arcsin % Trans. 50 PPM A.I. Viable Eggs Embr. Set Arcsin % Trans. 1 61 64 95 77.50 55 56 98 82.32 2 40 45 89 70.53 49 49 100 90.00 3 44 49 90 71.31 46 50 92 73,57 4 34 35 97 80.27 0 62 0 0.00 5 11 14 79 62.42 38 42 90 72.02 6 0 0 50 52 96 78.69 7 57 59 97 79.39 52 63 83 65.30 8 52 56 93 74.50 57 58 98 82.45 9 37 38 97 80.66 45 46 98 81.52 10 39 54 72 58.19 11 47 49 96 78.34 51 53 96 78.80 12 52 52 1 0 0 90.00 35 41 85 67.51 13 60 61 98 82.64 40 43 93 74.68 14 0 47 0 0.00 46 47 98 8 1 . 6 1 15 47 50 94 75.82 39 40 97 80.90 16 25 25 100 90.00 22 25 88 69.73 - Total 567 644 664 781 - Mean 38 43 88 72.39 42 49 87 71.08 - SD 20 18 26 22.06 14 10 24 20.55 - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). 1 5 0 PPM A.I. Viable Eggs Ehbr. Set Arcsin % Trans. - - - I Y b\ 0 I '1U P. 0 2 5 3 5 f P VI c-' 0 \D Appendix XI11 - Figure 3a Uiable Embryos / Eggs Set from' a Mallard Reproduction Study with PFDS - Control 70 60 50 z w n 40 \ 5 a W m 30 z 20 10 * n I 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 * Eggs Set Data a r e not Pe available n Number CI Uiable Embryos due to adult mortality. Appendix XI11 - F i g u r e 3b - Uiable Embryos Eggs Set from a M a l l a r d Reproduction Study u i t h PFOS 10 PPM 19.1. 70 60 50 40 30 20 10 - n 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Pen Number Eggs Set E Uiable Embryos Reproductive Performance by Pen Appendix XIII- T a b l e 4 L i v e 3-Week Fmbryos / Viable Embryos (%) from a Mallard Reproduction Study w i t h PFOS 0 PPM A.I. Live Viable Replicate 3-Week Embr. % Arcsin Trans. 10 PPM A.I. Live Viable 3-Week Embr. % Arcsin Trans. 50 PPM A.I. Live Viable 3-Week Embr. % Arcsin Trans. 150 PPM A.I. Live Viable 3-Week Embr. % Arcsin Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 61 61 100 90.00 40 40 100 90.00 43 44 98 81.33 34 34 100 90.00 11 11 100 90.00 0 0 57 57 100 90.00 52 52 100 90.00 37 37 100 90.00 - - 47 47 100 90.00 51 52 98 82.03 60 60 100 90.00 0 0 46 47 98 81.61 25 25 100 90.00 564 567 38 38 100 88.08 20 20 1 3.66 55 55 100 9 0 - 00 49 49 100 90.00 46 46 100 90.00 0 0 38 38 100 90- 00 50 50 100 90.00 51 52 98 82.03 57 57 100 90.00 44 45 98 81.43 39 39 100 90.00 51 51 100 90.00 35 35 100 90.00 31 40 78 61.68 46 46 100 90.00 39 39 100 90.00 22 22 100 90.00 653 664 41 42 98 87.01 14 14 6 7.58 - - - - - - - R - Data are not available due t o adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). f c. 0 W Appendix XI11 - Figure 4a - Live 3-Meek Embryos / Uiable Embryos from a Reproduction Study with PFOS Control Mallard 70 60 50 z W n 40 \ 5CY W m 30 Z 20 - * - 10 0 1 201 202 203 20t 205 206 207 208 209 210 211 212 213 214 215 216 Pen Number Uiable Embryos E L i v e 3-Ueek Embryos Data are not available due to adult mortality. 70 - 60 ' - 50 z W c1 40 \ cw f m E 30 3 z 20 10 n 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Pen Number Uiable Embryos E L i v e 3-Week Embryos Reproductive Performance by Pen Appendix XI11 - T a b l e 5 Hatchlings / Live 3-Week Embryos (%) from a Mallard Reproduction Study with PFOS 0 PPM A . I . Number Live Replicate Hatch. 3-Week % Arcsin Trans. 1 0 PPM A.I. Number Live Hatch. 3-Week % Arcsin Trans. 50 PPM A . I . Number Live Hatch. 3-Week % Arcsin Trans. 1 2 3 4 5 6 7 a 9 10 11 12 13 14 15 16 Total Mean SD 55 61 90 71.72 29 40 72 58.37 16 43 37 37.59 33 34 97 80.13 a 11 73 58.52 0 0 33 57 58 49.54 47 52 90 71.94 32 37 86 68.43 - - 38 47 ai 64.05 47 51 92 73.74 59 60 98 82.58 0 0 38 46 a3 65.35 25 25 100 90.00 460 564 31 38 ai 67.07 ia 20 i a 14.04 40 55 73 58.52 44 49 90 71.37 3a 46 a3 65.35 0 0 33 3a a7 68.73 46 50 92 73.57 38 51 75 59.68 39 57 68 55.a i 40 44 91 72.45 15 39 3a 38.33 47 51 92 73.74 34 35 97 80.27 a 31 26 30.53 44 46 96 77.96 35 39 90 71.32 21 22 95 77.69 522 653 33 41 ao 65.02 14 14 21 14.43 - - - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). 150 PPM A . I . Number Live Hatch. 3-Week % Arcsin Trans. - - - I I 2 R f e Appendix XI11 - Figure 5a Hatchlings / Live 3-Ueek Embryos from a Reproduction Study with PFOS - Control Mallard 60 50 z w a 40 \ a u m 30 3 z 20 10 * n 7 U 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Pen Number Live 3-Ueek Embryos E Hatchlings $ Data are not available due t o adult mortality. 70 * 60 '- 50 z LI 0 40 \ Cw I m f 30 z 20 10 n -I " 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Pen N u m b e r L i v e 3-Ueek Embryos E Hatchlings Reproductive Performance by Pen 14-Day Old Survivors / Hatchlings ( % I from a Mallard Reproduction Study with PFOS 0 PPM A.I. 14-Day Number Replicate Old Hatch. % Arcsin Trans. 10 PPM A.I. 14-Day Number O l d Hatch. % Arcsin Trans. 50 PPM A.I. 14-Day Number Old Hatch. % Arcsin Trans. 1 55 55 100 90.00 39 40 97 80.90 2 28 29 97 79.30 42 44 95 77.69 3 16 16 100 90.00 37 38 97 80.66 4 31 33 94 75.75 0 0 5 8 8 100 90.00 32 33 97 79.98 6 0 0 45 46 98 81.52 7 33 33 100 90.00 37 38 97 80.66 8 46 47 98 81.61 37 39 95 76.91 9 31 32 97 79.82 37 40 93 74.11 10 - 15 15 100 90.00 11 37 38 97 80.66 47 47 100 90.00 12 47 47 100 90.00 34 34 100 90.00 13 59 59 100 90.00 8 8 100 90.00 14 0 0 42 44 95 77.69 15 38 38 100 90.00 34 35 97 80.27 16 25 25 100 90.00 21 21 100 90.00 Total 454 460 507 522 - Mean 30 31 99 85.93 32 33 97 02.69 SD 18 18 2 5.51 14 14 2 5.67 - Data are not available due t o adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). 150 PPM A.I. 14-Day Number Old Hatch. % Arcsin Trans. - - - - - - - Appendix XI11 - Figure 6a 14-Day Old S u r v i v o r s / Hatchlings from a Reproduction Study u i t h PFOS - Control 6o 5- Mallard 50 40 z w a \ 5w1 30 m E 3 Z 20 10 - *1 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 * Pen Number Hatchlings E ~~~~ i t - D a u- Old S u r v i v o r s Data a r e not available due t o adult mortality. -60 50 '- 40 30 20 10 n- 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Pen Number Hatchlings E 14-Day Old S u r v i v o r s Reproductive Performance by Pen A p p e n d i x XI11 - T a b l e 7 Hatchlings / Eggs Set (%) from a Mallard Reproduction Study w i t h PFOS 0 PPM A.I. Number Eggs Replicate Hatch. Set Arcsin % Trans. 10 PPM A.I. Number Eggs Hatch. Set Arcsin % Trans. 50 PPM A.I. Number Eggs Hatch. Set Arcsin % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 55 64 86 67.98 29 45 64 53.40 16 49 33 34.85 33 35 94 76.17 8 14 57 49.11 0 0 33 59 56 48 - 4 1 47 56 84 66.37 32 38 84 66.59 38 49 78 61.72 47 52 90 71.94 59 61 97 79.57 0 47 0 0.00 38 50 76 60.67 25 25 100 90.00 460 644 31 43 71 59.05 18 18 28 22.11 40 56 71 57.69 44 49 90 71.37 38 50 76 60.67 0 62 0 0.00 33 42 79 62.42 46 52 88 70.14 38 63 60 50.95 39 58 67 55.09 40 46 87 68.83 15 54 28 31.81 47 53 89 70.34 34 41 83 65.59 8 43 19 25.55 44 47 94 75.37 35 40 88 69.30 21 25 84 66.42 522 781 33 49 69 56.35 14 10 28 20.49 - - - - - - Data are not available due t o adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). 150 PPM A.I. Number Eggs Hatch. Set Arcsin % Trans. - I I f P VI Reproductive Performance by Pen Appendix XIIL - Table 8 14-Day O l d Survivors / Eggs Set (%) f r o m a Mallard Reproduction Study with PFOS 0 PPM A.I. 14-Day E g g s Replicate Old Set Arcsin % Trans. 1 0 PPM A.I. 14-Day Eggs O l d Set Arcsin % Trans. 50 PPM A.I. 14-Day Eggs Old Set Arcsin % Trans. 150 PPM A.I. 14-Day Eggs Old Set Arcsin % Trans. 1 55 64 86 67.98 39 56 70 56.57 2 2a 45 62 52.07 42 49 86 67.79 3 16 49 33 34.85 37 50 74 59.34 4 31 35 a9 70.24 0 62 0 0.00 5 8 14 57 49.11 32 42 76 60.79 6 0 0 45 52 a7 68.48 7 33 59 56 48.41 37 63 59 50.03 8 46 56 a2 65.00 37 sa 64 53.01 9 31 3a a2 64.58 37 46 ao 63.75 10 - 15 54 28 31.81 11 37 49 76 60.34 47 53 89 70.34 12 47 52 90 71.94 34 41 83 65.59 13 59 61 97 79.57 8 43 19 25.55 14 0 47 0 0.00 42 47 89 70.96 15 3a 50 76 60.67 34 40 85 67.21 16 25 25 100 90.00 21 25 a4 66.42 Total Mean SD 454 644 30 43 70 58.20 18 18 27 21.74 507 781 32 49 67 54.85 14 10 27 19.69 - Data are not available due t o adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). I + 4 w I - - - - - - - - from Reproductive Performance by Pen Appendix XI11 - T a b l e 9 Hatchlings / Maximum Set (%) a Mallard Reproduction Study with PFOS 0 P P M A.I. Number Max. Replicate Hatch. Set Arcsin % Trans. 1 0 P P M A.I. Number Max. Hatch. Set Arcsin % Trans. 50 P P M A.I. Number M a x . Hatch. Set Arcsin % Trans. 1 55 64 86 67.98 40 64 63 52.24 2 29 64 45 42.31 44 64 69 56.01 3 16 64 25 30.00 38 64 59 50.40 4 33 64 52 45.90 0 64 0 0.00 5 8 64 13 20.70 33 64 52 45.90 6 0 64 0 0.00 46 64 12 57.97 7 33 64 52 45.90 38 64 59 50.40 8 47 64 73 58.98 39 64 61 51.32 9 32 64 50 45.00 40 64 63 52.24 10 - - 15 64 23 28.96 11 38 64 59 50.40 47 64 73 58.98 12 47 64 73 58.98 34 64 53 46.79 13 59 64 92 73.77 8 64 13 20.70 14 0 64 0 0.00 44 64 69 56.01 15 38 64 59 50.40 35 64 55 47.69 16 25 64 39 38.68 21 64 33 34.95 Total 460 960 522 1024 Mean 31 64 48 41.93 33 64 51 44.41 SD 18 0 28 21.68 14 0 22 15.89 - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). - - 150 P P M A.I. Number M a x . Hatch. Set Arcsin % Trans. - - - - l e 4 P I 2 R f Reproductive Performance by P e n Appendix X I I I - T a b l e 1 0 14-Day Old S u r v i v o r s / Maximum Set (%) f r o m a Mallard Reproduction S t u d y w i t h PFOS 0 PPM A.I. 14-Day Max. Replicate Old S e t Arcsin % Trans. 10 PPM A.I. 14-Day Max. Old Set Arcsin % Trans. 50 PPM A.I. 14-Day Max. Old Set Arcsin % Trans. 150 PPM A.I. 14-Day Max. Old Set Arcsin % Trans. 1 55 64 86 67.98 39 64 61 51.32 2 28 64 44 41.41 42 64 66 54.10 3 16 64 25 30.00 37 64 58 49.49 4 31 64 48 44.10 0 64 0 0.00 5 8 64 13 20.70 32 64 50 45.00 6 0 64 0 0.00 45 64 70 56.98 7 33 64 52 45.90 37 64 58 49.49 8 46 64 72 57.97 37 64 58 49.49 9 31 64 48 44.10 37 64 58 49.49 10 15 64 23 28.96 11 37 64 58 49.49 47 64 73 58.98 12 47 64 73 58.98 34 64 53 46.79 13 59 64 92 73.77 8 64 13 20.70 14 0 64 0 0.00 42 64 66 54.10 15 38 64 59 50.40 34 64 53 46.79 16 25 64 39 38.68 21 64 33 34.95 Total 454 960 507 1024 - Mean 30 64 47 41.57 32 64 50 43.54 SD 18 0 28 21.57 14 0 21 15.43 - Data are not available due t o adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). I L 4 v1 I - - - - - - Reproductive Performance by Week and Pen Appendix XIV - Table la Eggs Laid by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a.i.) 1 LOT A B C D Control E F G H I J PENS 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Totals 8 0 0 0 1 0 3 0 0 0 0 4 0 0 0 16 7 4 3 2 3 0 7 6 0 6 0 7 3 6 2 56 7 8 5 7 0 0 7 7 6 - 7 7 7 7 7 5 87 7 6 7 7 1 0 7 7 5 - 7 7 7 7 7 6 88 7 2 7 6 4 0 7 7 7 7 7 7 7 7 5 87 7 7 7 2 0 0 7 7 5 7 7 7 7 7 1 78 7 6 7 7 4 0 7 7 5 6 7 7 7 6 5 88 7 7 7 2 2 0 7 7 5 5 7 7 7 7 1 78 6 5 3 7 2 0 7 7 5 6 7 7 6 6 2 76 7 6 7 2 2 0 7 7 4 4 7 7 0 1 4 65 Totals 70 51 53 42 19 0 66 62 42 - 55 56 67 51 54 31 719 '- Data are not available due t o adult LOT A - Eggs Set During Week 12; mortality LOT B - Eggs Set During Week 13; etc. I Y 4 cn I I7s. 0 2 2 3 5 8 efc. 0 W Reproductive Performance by Week and Pen Appendix XIV - T a b l e lb Eggs Laid by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a . i . ) 1 LOT A B C D 10 E F G H I J PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 2 3 1 232 9 0 0 9 0 0 9 4 0 6 0 0 0 0 0 0 7 6 6 7 3 1 7 7 3 6 4 1 0 2 0 0 7 7 7 7 7 7 7 7 7 7 7 7 0 7 0 2 3 7 7 7 7 7 7 5 7 7 7 7 5 7 3 5 6 6 7 7 3 7 7 6 7 7 7 6 7 7 7 5 7 7 5 7 5 7 7 7 7 6 7 6 7 7 7 3 3 7 7 7 7 7 7 7 7 6 7 7 7 7 7 5 7 7 6 7 7 7 7 a 7 6 7 4 7 7 7 4 5 5 7 7 5 7 7 6 4 6 5 6 6 6 7 2 7 6 2 7 2 7 7 6 7 2 6 3 7 6 5 4 Totals 37 60 93 98 102 102 105 105 91 84 Totals 61 58 54 72 46 57 72 63 56 59 57 47 46 56 43 30 877 'LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. I c 4 4 I P f13 0 W Appendix XIV - Figure 1 Eggs Laid per Hen from a f l a l l a r d R e p r o d u c t i o n Study with PFOS 7 6 5 E4 m E 3 z 3 2 1 0 I I I I I a I I E C D E F G H I Weeks Concentrations, m m a.i. I F 4 00 I P P VI Appendix XIV - Figure 2 Hens Laying p e r Pen from a Flallard Reproduction Study with 1.0 PFOS 0.8 LT 0.6 Id m E 3 z 0.4 0. i I I I I I I I I I t 6 C D E F G H J Weeks Concentrations, tmrn a.i. Appendix X I V - F i g u r e 3 Eggs p e r Laying Hen from a M a l l a r d Reproduction Study u i t h PFOS 8 L 7 x 6 5 a I W m w E 00 34 0 Z I 3 2 1 0 I I I I I I I I B C 0 E F 6 H I Weeks Concentrations, ppm a.1. Reproductive Performance by Week and Pen Appendix XIV - T a b l e 2a Eggs Cracked by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a . i . ) 1 LOT A B C D Control E F G H I J PENS 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Totals 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 - 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 1 0 1 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Totals 1 1 0 2 1 0 0 1 0 ' - - Data are not available due to adult mortality LOT A - Eggs Set During Week 12; LOT B Eggs Set During Week 13; etc. 2 0 1 0 0 1 10 I w 00 w I RPc. 0 \o Reproductive Performance by Week and Pen Appendix XIV - T a b l e 2b Eggs Cracked by Week and Pen from a Mallard Reproduction Study w i t h PFOS Experimental Group (ppm a . i . ) LOT^ A E C D 10 E F G H I J PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 ~ ~~~ Totals 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 2 0 0 2 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 2 Totals 0 0 0 4 0 0 3 0 3 0 0 0 0 0 0 0 10 'LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. I F 00 h) I s2 . CD $: 3 3 0- 2 R P L 0 \D Reproductive Performance by Week and Pen Appendix XIV - T a b l e 3a Eggs Set by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a.i.) 1 LOT A B C D Control E F G H I J PENS 2 0 1 202 203 204 205 206 207 208 209 210 2 1 1 212 213 214 215 216 7 0 0 0 0 0 2 0 0 - 0 0 3 0 0 0 6 3 3 1 3 0 7 5 0 - 5 0 7 2 6 0 6 8 4 7 0 0 5 7 5 6 7 6 7 6 5 7 5 7 6 1 0 7 6 5 7 6 7 6 7 5 6 1 6 6 3 0 6 7 6 6 7 6 7 6 5 7 6 7 1 0 0 7 6 5 7 6 7 6 7 0 6 6 6 6 3 0 5' 7 4 7 6 7 1 1 0 7 6 5 5 5 2 6 1 0 6 6 4 7 5 7 1 2 0 7 6 4 5 7 6 7 5 5 5 6 7 6 7 0 4 7 5 6 5 2 4 6 7 0 1 3 Totals 12 48 79 82 78 72 78 71 64 60 Totals 64 45 49 35 14 0 59 56 38 - 49 52 61 47 50 25 644 - Data are not available due to adult mortality ' LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. I c1 00 w I P CA f Y Reproductive Performance by Week and Pen Appendix XIV - T a b l e 3b Eggs Set by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a.i.) 1 LOT A B C D 10 E F G H I J PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 2 3 1 232 8 0 0 9 0 0 8 4 0 6 0 0 0 0 0 0 7 5 6 6 3 0 7 6 3 5 4 0 0 1 0 0 6 6 6 7 6 7 6 7 6 7 6 7 0 7 0 2 3 6 7 6 7 6 7 4 6 6 7 6 5 6 3 3 5 6 6 5 2 7 4 6 6 7 6 6 6 6 6 5 7 6 5 6 5 6 7 6 6 5 7 5 7 6 7 2 2 7 6 6 6 7 5 7 5 6 6 7 6 6 6 5 7 5 6 6 7 6 7 7 6 5 7 3 7 6 7 3 4 4 6 7 4 7 6 6 2 6 4 5 5 5 6 2 7 4 2 4 2 6 6 5 6 1 6 2 7 4 5 3 Totals 35 53 86 88 89 93 93 95 79 70 Totals 56 49 50 62 42 52 63 58 46 54 53 41 43 47 40 25 781 I LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. I + 00 P I P v, f Y 0 W Reproductive Performance by Week and Pen Appendix X I V - T a b l e 4a Viable Embryos by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a-i.) PENS 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Totals 1 LOT A 5 0 0 0 0 0 1 0 0 - 0 0 3 0 0 0 9 - B 6 0 3 1 1 0 7 3 0 - 5 0 7 0 6 0 39 C 5 6 2 6 0 0 5 7 4 6 7 6 0 5 5 64 I D 7 5 5 6 1 0 7 5 5 - 7 6 7 0 7 5 73 I Control E 6 1 6 6 2 0 6 I 6 6 7 6 0 5 5 69 F 7 6 7 1 0 0 7 6 5 6 6 7 0 7 0 65 G 6 6 6 6 3 0 5 7 4 - 4 7 6 0 5 5 70 H 7 6 6 1 1 0 7 6 5 5 6 7 0 6 0 63 I 5 5 2 6 1 0 6 6 4 4 7 5 0 5 2 58 J 7 5 7 1 2 0 6 5 4 4 6 6 0 1 3 57 Totals 6 1 40 44 34 11 0 57 52 37 - 41 52 60 0 47 25 567 '- Data are not available due to adult LOT A - Eggs Set During Week 12; mortality LOT B - Eggs Set During Week 13; etc. Reproductive Performance by Week and Pen Appendix XIV - T a l e 4b Viable Embryos b y Week and Pen from a Mallard Reproduction Study w i t h PFOS Experimental Group (ppm a.i.) PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 2 3 1 232 Totals 1 LOT A 8 0 0 0 0 0 8 4 0 0 0 0 0 0 0 0 20 B 7 5 5 0 3 0 7 6 3 0 3 0 0 1 0 0 40 C 6 6 6 0 5 7 6 7 6 3 6 3 0 7 0 2 70 I i-r 00 D 3 6 6 0 7 5 7 4 6 6 7 4 5 6 3 3 78 o\ I 10 E 5 6 6 0 1 7 4 6 6 7 6 6 5 6 6 5 82 F 7 6 5 0 4 6 2 6 6 5 6 5 7 6 7 2 80 G 2 7 4 0 5 7 4 7 5 6 6 7 6 6 5 5 82 H 7 5 6 0 7 6 7 7 5 5 7 3 5 6 7 3 86 I 4 4 6 0 4 6 3 6 2 6 4 5 5 5 6 0 66 J 6 4 2 0 2 6 4 4 6 1 6 2 7 3 5 2 60 Totals 5 5 49 46 0 38 50 52 57 45 39 51 35 40 46 39 22 664 LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. Reproductive Performance by Week and Pen Appendix X I V - T a b l e 5a L i v e Three-Week Embryos by Week and Pen from a Mallard Reproduction Study w i t h PFOS Experimental Group (ppm a-i.) 1 LOT A B C D Control E F G H I J PENS 2 0 1 202 203 204 205 206 207 208 209 210 2 1 1 212 213 214 215 216 5 0 0 0 0 0 1 0 0 0 0 3 0 0 0 6 0 3 1 1 0 7 3 0 5 0 7 0 6 0 5 6 2 6 0 0 5 7 4 - 6 6 6 0 5 5 7 5 5 6 1 0 7 5 5 7 6 7 0 7 5 6 1 6 6 2 0 6 7 6 6 7 6 0 5 5 7 6 7 1 0 0 7 6 5 - 6 6 7 0 6 0 6 6 6 6 3 0 5 7 4 - 4 7 6 0 5 5 7 6 6 1 1 0 7 6 5 - 5 6 7 0 6 0 5 5 2 6 1 0 6 6 4 4 7 5 0 5 2 7 5 6 1 2 0 6 5 4 4 6 6 0 1 3 Totals 9 39 63 73 69 64 70 63 58 56 Totals 61 40 43 34 11 0 57 52 37 - 47 51 60 0 46 25 564 ' R - Data are not available due to adult mortality LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. f I 0 W Reproductive Performance by Week and Pen Appendix XIV - T a b l e 5b L i v e Three-Week Embryos by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a.i.1 PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 2 3 1 232 Totals 1 LOT A 8 0 0 0 0 0 8 4 0 0 0 0 0 0 0 0 20 B 7 5 5 0 3 0 7 6 3 0 3 0 0 1 0 0 40 C 6 6 6 0 5 7 6 7 6 3 6 3 0 7 0 2 70 I w 00 D 3 6 6 0 7 5 7 4 6 6 7 4 1 6 3 3 74 00 I 10 E 5 6 6 0 1 7 4 6 5 7 6 6 4 6 6 5 80 F 7 6 5 0 4 6 2 6 6 5 6 5 7 6 7 2 80 G 2 7 4 0 5 7 4 7 5 6 6 7 3 6 5 5 79 H 7 5 6 0 7 6 6 7 5 5 7 3 5 6 7 3 85 I 4 4 6 0 4 6 3 6 2 6 4 5 5 5 6 0 66 J 6 4 2 0 2 6 4 4 6 1 6 2 6 3 5 2 59 Totals 55 49 46 0 38 50 51 57 44 39 51 35 31 46 39 22 653 LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. Reproductive Performance by Week and Pen Appendix XIV - T a b l e 6a Hatchlings by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a . i . 1 PENS 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Totals 1 LOT A 5 0 0 0 0 0 0 0 0 - 0 0 3 0 0 0 8 B 5 0 0 1 1 0 6 2 0 4 0 7 0 6 0 32 I C 4 3 0 6 0 0 2 6 4 6 4 5 0 5 5 50 I 00 D 7 3 0 6 1 0 3 4 5 5 6 7 0 1 5 53 a I Control E 6 0 2 6 0 0 2 5 5 4 6 6 0 5 5 52 F 6 5 3 1 0 0 3 6 4 5 6 7 0 4 0 50 G 6 6 2 6 3 0 2 7 3 H 7 6 4 1 0 0 3 6 4 4 7 6 0 5 5 62 3 6 7 0 6 0 53 I 4 1 0 5 1 0 6 6 4 3 7 5 0 5 2 49 J 5 5 5 1 2 0 6 5 3 4 5 6 0 1 3 51 Totals 55 29 16 33 8 0 33 47 32 - 38 47 59 0 38 25 460 '- Data are not available due to adult mortality LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. Reproductive Performance by Week and Pen Appendix XIV - T a b l e 6b Hatchlings by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a. i.) 1 LOT A B C D 10 E F G H I J PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 2 3 1 232 Totals 7 0 0 0 0 0 8 1 0 0 0 0 0 0 0 0 16 6 4 3 0 2 0 6 5 2 0 2 0 0 1 0 0 31 5 6 4 0 3 5 3 5 4 1 6 3 0 6 0 1 52 3 6 6 0 6 5 2 3 5 1 7 4 0 6 2 3 59 3 5 5 0 0 7 3 3 5 2 6 5 0 6 5 5 60 3 4 4 0 4 6 2 3 6 1 6 5 2 6 7 2 61 2 7 3 0 5 6 3 4 5 2 6 7 0 5 4 5 64 4 5 5 0 7 6 5 7 5 5 7 3 1 6 6 3 75 3 3 6 0 4 5 3 5 2 3 2 5 2 5 6 0 54 4 4 2 0 2 6 3 3 6 0 5 2 3 3 5 2 50 Totals 40 44 38 0 33 46 38 39 40 15 47 34 8 44 35 21 522 LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. I c3 \o 0 I P ul fc3 0 W Experimental Group (ppm a-i.) R e p r o d u c t i v e Performance by Week and Pen Appendix XIV - T a b l e 7a 14-Day Old S u r v i v o r s by Week and Pen f r o m a Mallard Reproduction S t u d y with PFOS I __ PENS 2 0 1 202 203 204 205 206 207 208 209 210 2 1 1 212 213 214 215 216 Totals 1 LOT A 5 0 0 0 0 0 0 0 0 0 0 3 0 0 0 8 B 5 0 0 1 1 0 6 2 0 - 4 0 7 0 6 0 32 C 4 2 0 6 0 0 2 6 4 - 6 4 5 0 5 5 49 D 7 3 0 6 1 0 3 4 5 5 6 7 0 1 5 53 Control E 6 0 2 6 0 0 2 5 5 4 6 6 0 5 5 52 F 6 5 3 0 0 0 3 5 4 4 6 7 0 4 0 47 G 6 6 2 6 3 0 2 7 2 4 7 6 0 5 5 61 H 7 6 4 1 0 0 3 6 4 3 6 7 0 6 0 53 I 4 1 0 4 1 0 6 6 4 3 7 5 0 5 2 48 J 5 5 5 1 2 0 6 5 3 4 5 6 0 1 3 51 Totals 55 28 16 31 8 0 33 46 31 - 37 47 59 0 38 25 454 - Data are not available due t o adult mortality - P 'LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. ul f 0 W Reproductive Performance by Week and Pen Appendix XIV - T a b l e 7b 14-Day Old Survivors by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a . i . ) ~~ PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 2 3 1 232 Totals LOT' A B C D 10 E F G H I J 7 0 0 0 0 0 8 1 0 0 0 0 0 0 0 0 16 6 3 2 0 1 0 6 4 0 0 2 0 0 0 0 0 24 5 6 4 0 3 4 3 5 4 1 6 3 0 6 0 1 51 3 6 6 0 6 5 2 3 5 1 7 4 0 6 1 3 58 3 5 5 0 0 7 3 3 5 2 6 5 0 6 5 5 60 3 4 4 0 4 6 1 3 5 1 6 5 2 6 7 2 59 2 7 3 0 5 6 3 3 5 2 6 7 0 5 4 5 63 4 4 5 0 7 6 5 7 5 5 7 3 1 5 6 3 73 3 3 6 0 4 5 3 5 2 3 2 5 2 5 6 0 54 3 4 2 0 2 6 3 3 6 0 5 2 3 3 5 2 49 I I W N I Totals 39 42 37 0 32 45 37 37 37 15 47 34 8 42 34 21 507 ' LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. ~ Pen A B 201 2 02 203 2 04 205 206 207 208 209 210 211 2 12 213 214 215 216 - No eggs available. Appendix X V - T a b l e 1 Eggshell Thickness (mm) per Pen by Week from a Mallard Reproduction Study with PFOS (ControlI Weeks C D E F G H I J 0.387 0.397 0.391 0.393 0.398 0.382 0.381 - 0.437 0.428 0.440 0.455 0.353 0.357 0.347 - 0.378 - 0.368 0.374 - - 0.455 0.446 0.417 0.415 0.425 0.435 0.433 0.406 0.406 - 0.401 0.409 - 0.366 0.375 0.352 0.344 0.401 0.386 0.408 0.447 0.426 0.438 0.425 0.385 0.384 0.395 0.349 0.369 0.383 0.384 0.363 0.332 0.289 Mean and standard deviation of pen means: ~ ~~ Mean SD In1 0.392 0.387 0.440 0.352 0.373 0.004 4 0.010 3 0.011 4 0.005 3 0.005 3 0.433 0.020 4 0.431 0.005 3 0.406 0.003 4 0.359 0.014 4 0.399 0.011 3 0.434 0.010 4 0.388 0.006 3 0.371 0.017 4 0.328 0.037 3 Mean 0.392 SD 0.034 49 ~ Pen A 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Appendix X V - T a b l e 2 Eggshell Thickness (mm) per Pen by Week from a Mallard Reproduction Study with PFOS (10 PPM A . I . ) Weeks B C D E F G H I J 0.410 0.416 0.386 0.401 0.415 0.379 - - 0.416 0.425 0.384 0.391 0.425 0.386 0.370 0.363 0.388 0.389 0.431 0.415 0.415 0.381 0.406 0.384 0.417 0.418 0.381 0.390 0.380 0.386 0.384 0.426 0.416 0.406 0.416 0.416 0.419 0.382 0.404 0.371 0.407 0.406 0.378 0.377 0.379 0.370 0.387 0.395 0.400 0.401 0.344 0.396 0.428 0.373 0.401 0.380 Mean and standard deviation of pen means: - No eggs available. Differences between the control and this treatment group were not significant (p > 0.05). ~ Mean ~~ ~ SD ~ [nl 0.404 0.413 0.403 0.416 0.396 0.381 0.407 0.386 0.419 0.395 0.379 0.381 0.406 0.380 0.379 0.395 0.012 4 0.006 3 0.012 4 0.009 3 0.041 4 0.003 3 0.010 4 0.008 3 0.006 4 0.026 3 0.004 4 0.010 3 0.002 3 0.009 3 0.007 3 0.032 3 Mean 0.396 SD 0.014 54 Appendix XVI - T a b l e 1 Mean Hatchling Body Weight ( 9 ) per Pen by Week from a Mallard Reproduction Study with PFOS (ControlI Weeks [No. of Hatchlings] Total Pen A [ nl B[ nl C[ nl D[ nl E[ nl F[ nl G [ nl H[ nl I[ nl J[ n] M e a n SD Hatch 201 2 02 203 2 04 205 206 2 07 208 209 210 211 2 12 213 2 14 215 216 33[ 51 34 35[ 51 33 34[ 51 35 33[ 11 38 31[ 21 29 [ 01 34[ 61 35 40[ 51 40 43[ 31 42 34[ 41 35 331 51 34 35[ 61 36 E 01 38[ 11 36 31[ 31 33 Mean 35 The number of hatchlings weighed may differ from the total number of hatchlings since those hatchlings found dead were not weighed. - Data are not available due to adult mortality. SD = Standard deviation of mean body weight, by parental pen, by week. 3 55 3 29 3 16 6 32 3 8 2 33 3 47 3 32 2 38 3 47 3 59 3 38 2 25 SD 3 459 Appendix XVI - T a b l e 2 Mean Hatchling Body Weight (9)per Pen by Week from a Mallard Reproduction Study w i t h PFOS (10 PPM A. I. I ~~ ~ Weeks [No. of Hatchlings] Total Pen A [ nl B [ nl C [ nl DI nl E1 nl F[ nl GI nl HI nl I1 nl J [ n] Mean SD Hatch 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 40[ 41 39 36[ 41 35 36[ 21 36 [ 01 33[ 21 37 351 61 36 42[ 31 39 36[ 31 34 351 61 35 1 01 33 37[ 51 36 40[ 21 41 391 31 36 361 31 38 36[ 51 38 411 21 41 Mean 37 The number of hatchlings weighed may differ from the total number of hatchlings since those hatchlings found dead were not weighed. Differences between the control and this treatment group were not significant (p > 0.05). SD = Standard deviation of mean body weight, by parental pen, by week. 2 40 2 44 2 38 4 33 2 46 4 38 1 39 3 39 2 15 1 47 2 34 3 8 3 44 2 35 2 21 SD 2 521 Appendix X V I I - Table 1 Mean 14-Day Old S u r v i v o r s Body Weight ( g ) p e r Pen by Week from a M a l l a r d R e p r o d u c t i o n S t u d y with PFOS (Control ) Weeks [No. of 14-Day Old Survivors] 1 14-Day Pen A [ nl B [ nl c [ nl D[ nl E[ nl F[ nl G [ nl H[ nl I[ nl J[ n] M e a n SD Total 201 2 02 203 2 04 205 206 207 208 209 210 211 2 12 213 214 215 216 292[ 51 [ 01 I 03 320[ 11 224[ 11 [ 01 268[ 61 3101 21 [ 01 - 286[ 41 t 01 276[ 71 [ 03 289[ 61 [ 01 306[ 41 232[ 21 01 333[ 61 [ 01 [ 01 286[ 21 309[ 61 335[ 41 2901 61 317[ 41 314[ 51 [ 01 302[ 51 289[ 51 3031 71 243[ 31 t 01 320[ 61 2321 11 [ 01 280[ 31 279[ 41 311[ 51 295[ 51 280[ 61 304[ 71 [ 01 255[ 11 286[ 51 289[ 61 [ 01 3211 21 318[ 61 [ 01 [ 01 295[ 21 313[ 51 359[ 51 334[ 41 309[ 61 285[ 61 [ 01 298[ 51 278[ 51 293[ 61 254 [ 51 295[ 31 [ 01 01 [ 01 253[ 31 298[ 51 329[ 41 - 301[ 41 307[ 61 297[ 71 [ 01 2 7 8 [ 41 [ 01 271[ 61 250[ 61 287[ 21 323[ 61 241[ 31 [ 01 258[ 21 291[ 71 333[ 21 267[ 41 272[ 71 275[ 61 [ 01 271[ 51 253[ 51 273[ 71 252[ 61 309[ 41 295[ 11 1 01 [ 01 271[ 31 324[ 61 3371 41 - 327[ 31 305[ 61 306[ 71 [ 01 3001 61 [ 01 2721 41 185[ 11 [ 01 321[ 41 141 11 01 261 61 289 63 332 41 285 31 264[ 71 289[ 51 [ 01 287[ 51 270[ 21 271[ 51 247[ 51 298[ 51 336[ 11 265[ 21 [ 01 258[ 61 299[ 51 335[ 31 2581 41 278[ 51 277[ 61 [ 01 293[ 11 3151 31 288 246 302 323 231 267 301 334 293 290 293 289 281 Mean 287 14 55 13 28 10 16 8 31 39 8 12 33 13 46 14 31. 23 37 19 47 14 59 12 38 18 25 SD 28 454 - Data are not available due t o adult mortality. SD = Standard deviation of mean body weight, by parental pen, by week. Mean Appendix XVII - T a b l e 2 14-Day O l d Survivors Body Weight (g) per from a Mallard Reproduction Study with (10 PPM A . I . ) Pen by PFOS Week Weeks [No. of 14-Day Old Survivors] ~ 14-Day Pen A [ nl B[ nl C[ nl D[ nl E[ nl F[ nl G[ nl H[ nl I[ nl J[ n] M e a n SD Total 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 271[ 71 [ 01 01 [ 01 [ 01 [ 01 291[ 81 270[ 11 [ 01 01 [ 01 01 [ 01 01 [ 01 [ 01 254[ 61 303[ 31 292[ 21 [ 01 243[ 11 [ 01 282[ 61 291[ 41 [ 01 01 2971 21 01 [ 01 01 [ 01 [ 01 266[ 51 347[ 61 287[ 41 [ 01 286[ 31 318[ 41 296[ 31 287[ 51 300[ 41 226[ 11 3091 61 316[ 31 I 01 266[ 61 01 240[ 11 249[ 31 277[ 61 263[ 61 [ 01 268[ 61 244 51 277 21 261 31 259 51 281 11 280 71 323 41 01 265 61 241 11 237 31 243[ 31 310[ 51 289[ 51 [ 01 01 272 71 300 31 290 31 267 51 265 21 309 61 334 51 01 284 61 296 51 311 51 2571 31 303[ 41 309[ 41 [ 01 281[ 41 281[ 61 347[ 11 257[ 31 2501 51 284[ 11 291[ 61 319[ 51 210[ 21 325[ 61 305[ 71 273[ 21 242[ 21 295[ 71 292[ 31 [ 01 290[ 51 261[ 61 301[ 31 299[ 31 273[ 51 255[ 21 288[ 61 304[ 71 [ 01 317[ 51 303[ 41 307[ 51 243[ 41 328[ 41 241[ 51 [ 01 300[ 71 249[ 61 306[ 51 282[ 71 2841 51 268[ 51 266[ 71 293[ 31 260[ 11 345[ 51 303[ 61 313[ 31 239[ 31 316[ 31 2441 61 [ 01 277[ 41 278[ 51 278[ 31 256[ 51 293[ 21 274[ 31 279[ 21 313[ 51 2061 21 309[ 51 303[ 61 01 241[ 31 281[ 41 237[ 21 1 01 320[ 21 275[ 61 3241 31 309[ 31 268[ 61 01 314[ 51 315[ 21 242[ 31 324[ 31 317[ 51 272[ 21 254 306 271 285 271 296 281 272 266 292 315 227 302 303 289 Mean 282 Differences between the control and this treatment group were not significant (p > 0.05). SD = Standard deviation of mean body weight, by parental pen, by week. 11 39 23 42 25 37 16 32 20 45 15 37 16 37 15 37 14 15 16 47 12 34 22 8 28 42 12 34 30 21 SD 23 507 - 199 - Project Number 454-109 Bird Mean SD Appendix XVIII Offspring Liver Weights (g) from a Mallard Reproduction Study with PFOS Control (0 ppm ai.) Male Liver Female Liver 19.845 11.018 12.740 14.534 4.679 18.179 13.219 11.862 14.513 15.982 17.418 13.485 14.951 2.326 Group Mean SD 14.826 2.917 10 ppm a i Male Bird Liver Female Liver 1 2 3 4 5 6 7 Mean SD 11.418 10.819 18.719 13.652 4.398 13.077 13.409 16.095 11.881 11.558 17.125 15.334 14.068 2.143 Group Mean SD 13.944 2.721 The above differences from the control group were not statistically significant (Student's t-test). 200 - Wilalife International, Lta. Project Number 454-109 Appendix XIX Changes To Study Protocol This study was conducted in accordance with the study protocol signed on January 22,200 1 and the following amendments and deviations: 1. The protocol was amended to add four pairs of birds to each test group to be maintained for collection of blood samples during the test. Additionally, the protocol was amended to add collection of selected tissues for analyses and histopathological examination from all adult birds. When available, fecal samples were also to be collected from the lower digestive tract of test birds. 2. The protocol was amended to reduce the dietary concentration for the 150 ppm a.i. test concentration to 20 ppm a.i. at the beginning of Week 3 of the test. The concentration was reduced based upon treatment-related mortality and signs of toxicity at the 150 ppm a.i. concentration. 3 . The protocol was amended to euthanize the 20 ppm a.i. treatment group as soon as practical after the end of Week 4 of the test. Week 4 body weight and observation data indicated birds had not recovered fully following reduction of the dietary concentration from 150 to 20 pprn a.i. Birds were euthanized without collection of samples. 4. The protocol was amended to euthanize the 50 pprn xi. treatment group as soon as practical after the end of Week 6 of the test. The level was terminated due to treatment-related mortality and signs of toxicity at the 50 ppm a.i. level. Blood, liver, bile and fecal samples, when available, were collected from 10 pairs when the level was terminated. 5. The protocol was amended to add collection of samples from all eggs selected for egg shell thickness measurements. Eggs selected for Lots A, B and J were separated into shell, shell membrane, albumen and yolk and stored frozen for analysis. Eggs selected from Lots C through I will be opened and the yolk was separated and stored frozen for possible analysis. 6. The protocol was amended to add collection of blood, selected tissues and fecal samples, when available, from 10 offspring each from the control group and 10 ppm a.i. treatment group. Blood, tissues and feces were collected for possible analysis. Tissues were also taken for histopathological examination. 7. The protocol was amended to remove the reference substance number (4526) from page 2 of the protocol. The use of an internal standard was deemed unnecessary after refinements to the analytical method were made. 8. The protocol was amended to indicate samples collected for analysis would be shipped to 3M Corporation. Tissues collected for histopathological examination from birds in the 50 and 150 ppm a.i. test concentrations will not be shipped to EPL for examination, but will be disposed of. -201 - wildlife International, Ltd. Project Number 454- 109 9. The protocol was amended to add Attachment A, the analytical phase plan to be used by 3M Environmental Laboratory for analysis of samples. IO. The protocol was amended to update Attachment A, the analytical phase plan to be used by 3M Environmental Laboratory for analysis of samples. 11. The protocol was amended to change Attachment A, the 3M analytical phase plan, to redefine the peak area precision requirement and the method for calculating the calibration curve. 12. The protocol was amended to indicate the main 3M Environmental Laboratory Project Identification Number (LIMS) for the analytical phase plan results is E01-1245. Additional 3M tracking numbers associated with the study are U2723, E01-1342 and E01-1365. Additionally, the 3M Principal Analytical Investigator was changed from Harold Johnson to Lisa A. Stevenson. 13. The protocol was amended to change the Sponsor Representative from Rochelle R. Robideau to Susan A. Beach. 14. The protocol was amended to change the Sponsor Representative from Susan A. Beach to John L. Newsted. 15. Feather samples were collected, when available, from adult birds from which tissue samples were collected. The protocol did not require collection of these samples. Additionally, with the exception of liver samples, tissue samples collected for analysis were not weighed prior to being placed in sample containers. 16. On July 12, 2001, the temperature in brooder level S52-3, housing offspring from Lot J of the control group, was inadvertently not recorded. 17. Dietary samples collected on Day 0 of Week 1 of the test were not stored frozen between January 24 and January 25, 2001. Instead, samples were stored in the analytical chemistry laboratory prior to being processed for analysis on January 25, 2001. The protocol indicated that diet samples would be stored frozen until they were prepared for analysis. 18. Three adult birds in the 50 ppm a.i. test concentration, the hen in pen 243 and the pair in pen 246, were inadvertently euthanized at the beginning of Week 5 of the test during euthanasia of the 150 ppm a.i. test level. - 202 - wildlife International, Ltd. Project Number 454-109 Appendix XX Personnel Involved in Study The following key Wildlife International, Ltd. personnel were involved in the conduct or management of this study: Avian Toxicology (1) Mark Jaber, Wildlife Toxicologist (2) Joann B. Beavers, Director (3) Linda R. Mitchell, Manager (4) Diana Temple, Laboratory Supervisor ( 5 ) Sean P. Gallagher, Senior Biologist (6) Larry T. Frey, Senior Biologist (7) Jennie Grimes, Senior Biologist Analytical Chemistry (1) Willard 13. Nixon, Ph.D., Director (2) Raymond L. VanHoven, Ph.D., Scientist