Document 4a4XBYZLVvpEOV2p3n6G8RaoR

AR226-1753 PFOS: A REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-108 3M LAB REQUEST NO. U2723 FIFRA Guideline 714 OECD Guideline 206 AUTHORS: Sean P. Gallagher Raymond L. Van Hoven Joann B. Beavers Mark Jaber STUDY INITIATION DATE: January 22,2001 STUDY COMPLETION DATE: August 20, 2003 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St.Paul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 USA M l 0-822-8600 Page 1 of 196 W ildlife International, Ltd. Project Number 454-108 2- GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Reproduction Study with the Northern Bobwhite WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-108 STUDY COMPLETION: August 20, 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 454-108), 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 analytical phase performed by Exygen Research trader study number 023-066 was performed in compliance with EPA TSCA GLP (40 CFR Part 792). STUDY DIRECTOR: Senior Biologist, Avian Toxicology SPONSOR'S REPRESENTATIVE: DATE W ildlife International, Ltd. Project Number 454-108 -3 - - QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR part 160, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all audits and inspections and the dates any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY DATE CONDUCTED DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT Test Substance Preparation January 2 2,2001 January 22, 2001 January 23, 2001 Matrix Fortification January 30, 2001 January 3 1 ,2001 January 31, 2001 Feed Consumption February 6, 2001 February 7,2001 February 8, 2001 Body Weights, Environmental Conditions Candling February 6, 2001 June 12, 2001 February 7,2001 June 12, 2001 February 9, 2001 June 13, 2001 Tissue Collection June 19,2001 June 20, 2001 October 1, 2001 14-Day Old Body Weights June 25, 2001 June 25, 2001 July 5, 2001 Egg Shell Thickness Measurements Hatchling Body W eights June 28, 2001 July 2, 2001 June 28, 2001 July 2, 2001 July 2, 2001 July 5,2001 Data Entry Check August 10-13, 2001 August 13, 2001 August 14, 2001 Analytical Data, Draft Report October 29-November 2 and November 5, 2001 November 5, 2001 November 9, 2001 Raw Data, Draft Report November 27-30 and December 3-5, 2001 December 5, 2001 February 5, 2002 Final Report August 20, 2003 August 20, 2003 August 20, 2003 Jame^ H. Coleman Quality Assurance Representative DATE W ildlife International, Ltd. Project Number 454-108 -4 - REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Reproduction Study with the Northern Bobwhite WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-108 3M LAB REQUEST NO. U2723 STUDY DIRECTOR: Sean P. Gallagher Senior Biologist, Avian Toxicology CHEMISTRY PRINCIPAL INVESTIGATOR: DATE MANAGEMENT: DATE Joann B. Beavers Director, Avian Toxicology Willard B. Nixon, Ph.' Director of Chemistry DATE fp/n DATE W ildlife International, Ltd. Project Number 454-108 -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...................... 11 Test Substance..........................................................................................................................12 Test Organisms..................................................................... 12 Identification............................................................................................................................ 12 Avian Feed and Water............................................................................................................. 13 Diet Preparation....................................................................................................................... 13 Diet Sampling.......................................................................................................................... 14 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.................................................................................... 18 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..................................................................................................................21 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 W ildlife International, Ltd. Project Number 454-108 -6 - TABLE OF CONTENTS PAGE 2 Egg Shell Thickness................................................................................................................ 27 Offspring Body Weights.......................................................................................................... 27 Offspring Liver Weights.................................................. 27 Offspring Observations............................................................................................................ 28 Offspring Liver and Sera......................................................................................................... 28 Conclusion...................................................................................................................................... 28 References...................................................................................................................................... 29 TABLES AND FIGURES Table 1. Mean Measured Concentrations (ppm a.i.) of PFOS in Avian Diet from a Northern Bobwhite Reproduction Study.......................................................... 30 Table 2. Summary of Gross Pathological Observations from a Northern Bobwhite Reproduction Study with PFOS, Adult Birds Found Dead/Euthanized during the Test.............................................................................................................. 31 Table 3. Mean Adult Body Weight (g) from a Northern Bobwhite Reproduction Study with PFOS................................................................................... 32 Figure 1. Mean Adult Male Body Weight (g) from a Northern Bobwhite Reproduction Study with PFOS................................................................... 33 Figure 2. M ean A dult Fem ale B od y W eight (g) from a Northern Bobwhite Reproduction Study with PFOS................................................................... 34 Table 4. Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Reproduction Study with PFOS................................................................... 35 Figure 3. Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Reproduction Study with PFOS................................................................... 36 Table 5. Summary of Gross Pathological Observations from a Northern Bobwhite Reproduction Study with PFOS, Adult Birds Euthanized at Test Termination...................................................................................................... 37 Table 6. Mean Liver Weights (g) from a Northern Bobwhite Reproduction Study with PFOS.......................................................................................................... 38 Table 7. Summary of Reproductive Performance from a Northern Bobwhite Reproduction Study with PFOS................................................................... 39 W ildlife International, Ltd. Project Number 454-108 -7 - TABLE OF CONTENTS PAGE 3 Figure 4. Mean Reproductive Performance from a Northern Bobwhite Reproduction Study with PFOS.................................................................. 40 Table 8. Summary of Reproductive Performance, Normalized as Percentages (%), from a Northern Bobwhite Reproduction Study with PFOS...........41 Figure 5. Mean Reproductive Performance, Normalized as Percentages (%), from a Northern Bobwhite Reproduction Study with PFOS....................................... 42 Table 9. Mean Egg Shell Thickness Measurements (mm) from a Northern Bobwhite Reproduction Study with PFOS................................................................................... 43 Table 10. Mean Body Weight (g) of Hatchlings and 14-Day Old Survivors from a Northern Bobwhite Reproduction Study with PFOS....................................... 44 APPENDICES Appendix I. Certificate of Analysis.....................................................................................45 Appendix II. Diet and Supplement Formulations.................................................................48 Appendix HI. Diet Preparation...............................................................................................50 Appendix IV. The Analysis of PFOS in Avian Diet..............................................................51 A p p en d ix V . D ia g ra m o f T e st L a y o u t............................................................................................................ 6 7 Appendix VI. Reproductive Parameters and Replicate Identification...................................68 Appendix VII. Adult Body Weight (g) from a Northern Bobwhite Reproduction Study with PFOS.....................................................................73 Appendix VIE. Feed Consumption (g/bird/day) from a Northern Bobwhite Reproduction Study with PFOS.....................................................81 Appendix IX. Individual Gross Pathological Observations from a Northern Bobwhite Reproduction Study with PFOS.................................... 85 Appendix X. Adult Liver Weights (g) from a Northern Bobwhite Reproduction Study with PFOS.....................................................................87 Appendix XI. Histopathology Report.....................................................................................89 Wildlife International, Ltd. Project Number 454-108 8- - TABLE OF CONTENTS PAGE 4 Appendix XILA. The Analysis of PFOS in Adult Red Blood Cells and Sera........................... 121 Appendix XIIB. The Analysis of PFOS in Adult and Juvenile Liver and Sera........................ 134 Appendix Xffl. Reproductive Performance by Pen from a Northern Bobwhite Reproduction Study with PFOS.................................................... 148 Appendix XIV. Reproductive Performance by Week and Pen from a Northern Bobwhite Reproduction Study with PFOS.....................................170 Appendix XV. Egg Shell Thickness Measurements (mm) per Pen by Week from a Northern Bobwhite Reproduction Study with PFOS......................... 187 Appendix XVI. Mean Hatchling Body Weight (g) per Pen by Week from a Northern Bobwhite Reproduction Study with PFOS......................... 189 Appendix XVII. Mean 14-Day Old Survivor Body Weight (g) per Pen by Week from a Northern Bobwhite Reproduction Study with PFOS............... 191 Appendix XVffl. Offspring Liver Weights (g) from a Northern Bobwhite Reproduction Study with PFOS.................................................................... 193 Appendix XIX. Changes to Study Protocol..............................................................................194 Appendix XX. Personnel Involved in Study............................................................................196 W ildlife International, Ltd. -9SUMMARY Project Number 454-108 STUDY: PFOS: A Reproduction Study with the Northern Bobwhite SPONSOR: 3M Corporation WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER 454-108 TEST DATES: Study Initiation - January 22, 2001 Experimental Start - January 23, 2001 Photostimulation - March 13,2001 First Eggs Set - April 12, 2001 Analytical Termination - April 15, 2002 Adult Termination - June 19-20, 2001 Biological Termination - July 23, 2001 Experimental Termination - April 15, 2002 TEST ANIMALS: Northern bobwhite (Colinus virgirtianus) AGE TEST ANIMALS: 24 weeks of age at the initiation of the test SOURCE TEST ANIMALS: Morris Quail Farm, Inc. 18370 S.W. 232 Street Goulds, Florida 33170-5399 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 body weight and feed consumption at the 50 and 150 ppm a.i. test concentrations. The 50 and 150 ppm a.i. test concentrations were terminated prior to the reproductive phase of the test. There were no apparent treatment-related effects upon body weight or feed consumption at the 10 ppm a.i. test concentration. There were overt signs of toxicity observed in adults at the 10 ppm a.i. test concentration. Additionally, there were reductions in testes size and treatment-related effects upon overall reproductive performance at the 10 ppm a.i. test concentration. Based on the effects observed at the 10 ppm a.i test concentration, the no-observed-effect concentration for northern bobwhite exposed to PFOS in the diet was determined to be less than 10 ppm a.i., the lowest concentration tested during this study. W ildlife International, Ltd. Project Number 454-108 - 10- INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife Inter national, Ltd. avian toxicology facility in Easton, Maryland 21601. The biological portion of the test was conducted from January 23, 2001 until July 23, 2001. Raw data generated at Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454-108 in archives located on the Wildlife International, Ltd. site. Biological specimens are stored at 3M Corporation, St. Paul, Minnesota 55133. OBJECTIVES The objective of this study was to evaluate the effects upon the adult northern bobwhite (Colinus virginianus) 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 to their offspring. EXPERIMENTAL DESIGN Northern bobwhite (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 o f a pilot reproduction study (W ildlife International, Ltd. Project N um ber 4 5 4 -1 0 4 ) 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 Der Pen Males Females 11 11 11 11 W ildlife International, Ltd. Project Number 454-108 -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 a.i. 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 were noted at the 150 ppm a.i. test concentration, the dietary concentration for the 150 ppm a.i. treatment level was reduced to 20 ppm a.i. of PFOS at the beginning of Week 3 of the test. The 20 ppm a.i. level was terminated after Week 4 of the test due to the limited recovery of birds at this test concentration. For reporting purposes, the 150/20 ppm a.i. test concentration will be referred to as the 150 ppm a.i. test concentration. Adults in the 50 ppm a.i. test concentrations were euthanized during 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 ppm 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 8, 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 21 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. MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "PFOS: A Reproduction Study with the Northern Bobwhite". The protocol was based on procedures outlined in the Environmental Protection Agency's Registration Guidelines Pesticide Assessment Guidelines, FIFRA W ildlife International, Ltd. Project Number 454-108 - 12- 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. Concentra tions 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 One hundred and seventy-seven (90 males and 87 females) pen-reared northern bobwhite were purchased from Morris Quail Farm, Inc., 18370 S.W. 232 Street, Goulds, Florida 33170-5399, U.S.A. At the start of acclimation, the bobwhite 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 initia tion (first day of exposure to test diet) and ranged in weight from 177 to 250 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. Except eggs laid by birds maintained for blood collection, all eggs laid during the study were marked with the pen number using a permanent ink marking pen for identification. Hatchlings were identified by leg bands so that they could be traced to their parental pen of origin. W ildlife International, Ltd. - 13 - Project Number 454-108 Avian Feed and Water All adult birds and their offspring were given feed and water ad libitum dining acclimation and testing. The basal diet fed to both adults and offspring was formulated to Wildlife International, Ltd. specifications by Agway Inc. (Appendix II, Table 1). The basal ration contained at least 27% protein and 2.5% fat, and no more than 5% fiber. The basal diet contained approximately 1.1% calcium, derived from feedstuffs and the 0.9% limestone used in the formulation of the basal diet by Agway. While this level of calcium is sufficient for growth and maintenance rations, additional calcium is required in the ration of breeding birds for egg shell formation. Therefore, an additional 5% (w/w) of limestone (approximately 38.5% Ca) was added to the basal diet for the adults. This raised the calcium level in the diet for the breeding birds to approximately 3%, slightly above the minimum recommended for quail (2.3%) and mallard (2.75%) (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. All offspring received a watersoluble vitamin and electrolyte mix in their water (Appendix II, Table 2). Neither the adults nor offspring received any form of medication in the feed during the test. Feed and water were analyzed periodically in accordance w ith W ild life 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 treated diet 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 III. W ildlife International, Ltd. - 14- Project Number 454-108 Diet Sampling Homogeneity of the test substance in the diet was evaluated by collecting six samples from each of the treated diets and one sample from the control diet on Day 0 of Week 1. Samples were collected from the top, middle and bottom of the left and right sections of the mixing vessel. Control and treatment group diet samples were also collected from the feed troughs on Day 7 of Week 1 to assess stability of the test substance under actual test conditions. Additionally, a sample was collected from the control and treatment group diets, when available, during Weeks 2, 3,4, 8,12, 16 and 20 of the test to measure/verify test concentrations. The diet samples were stored frozen or transferred immediately to the Wildlife International, Ltd. analytical chemistry facility for analysis. Analytical Method The method used for the analysis of PFOS in avian diet was based upon methodology developed at Wildlife International, Ltd. and entitled "Analytical Method Verification for the Determination of PFOS in Avian Diet" (Wildlife International, Ltd. Project No. 454C-110). Three modifications from the cited methodology were incorporated into the present study. The following changes were made. (1) High performance liquid chromatography with triple quadrupole mass detection (HPLC/MS/MS) was used rather than single quadrupole mass detection to reduce potential matrix interferences. (2) The internal standardization procedure was eliminated because an appropriate internal standard could not be identified. (3) A shorter analytical chromatography column (50 mm vs. 100 mm) was used to expedite analysis time. Samples were extracted with methanol, vacuum filtered, diluted in methanol, and then diluted in a 50:50 methanol: NANOpure 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 Qg analytical column (50 mm x 2 mm I.D., 3-pm particle size) W ildlife International, Ltd. Project Number 454-108 -15 - fitted with a Keystone Javelin C l8 Guard 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 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 |xL) and the lowest standard concentration 1.00 pg a.i./L = 1.00 pg a.i./pL. The method limit of quantitation (LOQ) for these analyses was 2.00 ppm a.i., 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 sam ple (2000 L /K g). Exam ples o f calculations are presented in A ppendix 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 ppm 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%, W ildlife International, Ltd. Project Number 454-108 -16 102%, 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. Study Phases The primary phases of the study and their approximate durations were: 1. Acclimation - 8 weeks. 2. Pre-photostimulation - 7 weeks. 3. Pre-egg laying (with photostimulation) - 3 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 manufac tured by Georgia Quail Farm Manufacturing (GQFM Model No. 0330), measuring approximately 25 X 51 cm. The pens had sloping floors that resulted in ceiling height ranging from 20 to 26 cm. The pens were constructed of galvanized wire mesh and galvanized sheeting. A diagram of the test layout is presented in Appendix V. Each pen was equipped with feed and water troughs. 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 troughs were changed and water added as necessary to provide potable water (generally every 2-3 days). Only birds associated with this study were maintained in the study room in order to avoid excessive disturbances. The average temperature in the adult northern bobwhite study room during the course of the test was 21.4C 1.3C (SD) with an average relative humidity of 42% 18% (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. W ildlife International, Ltd. Project Number 454-108 - 17- The photoperiod in the adult northern bobwhite room was maintained by a time clock. The photoperiod during acclimation and the first seven weeks of the test was eight hours of light per day. The photoperiod was increased to 17 hours of light per day at the beginning of Week 8 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 199 lux (~ 18 ft. candles) of illumination provided by fluorescent lights that closely approximated noon-day sunlight. Observations The test birds were acclimated to the facilities and study pens for 8 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). An attempt was made to minimize feed wastage by the birds by using externally mounted feeders designed with a "feed-saver" lip. 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 An additional four pairs of adult birds in each test level were 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 W ildlife International, Ltd. Project Number 454-108 1 8- - 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 the 150 ppm a.i. treatment group were euthanized after Week 4, no necropsies were performed. All other adult birds that died or were euthanized during the course of the study were subjected to a gross necropsy. At the conclusion of the exposure period, all surviving adult birds were euthanized with carbon dioxide gas, necropsied, and disposed of by incineration. At the time of necropsy, tissues were collected for histopathological examination and analyses. When available, samples of gall bladder, liver, proventriculus, kidneys, brain, gonads, bursa of Fabricius and adipose tissue were fixed in 10% buffered formalin. Histology samples for the control group and 10 ppm a.i. treatment group were 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 test pens, cleaned and stored in a cold room until incubation or disposal (for those pens used for blood collection). The cold room was maintained at a mean temperature of 13.5C 0.2C (SD) with a mean relative humidity of approximately 69% 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 selected 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 to be incubated were fumigated with formaldehyde gas in an airtight cabinet with a circulating fan for approximately two hours, to reduce the possibility of pathogen contamination prior to incubation. Formaldehyde gas was generated by W ildlife International, Ltd. Project Number 454-108 - 19- combining 26 g of potassium permanganate and 25 ml of 37% commercial grade formalin in a porcelain bowl at the base of the airtight cabinet. 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 37.5C 0.0C (SD) with an average wet bulb temperature of 30.5C 0.3C (SD) (relative humidity of approximately 60%). The incubator was equipped with a pulsator fan and blades that produced a mild breathing air movement designed to eliminate intracabinet temperature and humidity variation during incubation. In order to prevent adhesion of the embryo to the shell membrane, the incubator was also equipped with an automatic egg rotation device, designed to rotate the eggs from 50 off of vertical in one direction to 50 off of vertical in the opposite direction (total arc of rotation was 100) every two hours through Day 21 of incubation. Eggs were candled on Day 11 or 12 of incubation to determine embryo viability and on Day 21 to determine embryo survival. Hatching and Brooding On Day 21 of incubation, the eggs were placed in a Petersime Hatcher (Model No. S-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 37.2C 0.0C (SD), and the average wet bulb temperature was raised to 33.3C 0.1C (SD) (relative humidity of approximately 77%). All hatchlings, unhatched eggs, and egg shells were removed from the hatcher on Day 25 or 26 of incubation. The group body weight of the surviving hatchlings by pen was determined. Hatchlings were leg 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 chicks was determined. The chicks were euthanized with carbon dioxide and disposed of by incineration. Hatchlings were housed in batteries of brooding pens manufactured by Beacon Steel Company (Model B735Q). Each pen measured approximately 72 X 90 X 23 cm high. The external walls and W ildlife International, Ltd. Project Number 454-108 - 20- ceilings of each pen were constructed of galvanized wire mesh and galvanized sheeting. Floors were of galvanized wire mesh. Thermostats in the brooding compartment of each pen were set to maintain a temperature of approximately 38 C from the time of hatching until the birds were 14 days of age. The average ambient room temperature was 26.0C 1.9C (SD) with an average relative humidity of 59% 13% (SD). The photoperiod for the hatchlings was maintained by a time clock at 16 hours of light per day. Offspring Blood and Tissue Collection Prior to euthanasia of the last group of 14-day old offspring (Lot J), blood samples were collected from 10 offspring in both the control group and the 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 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 analyses. Fecal samples were also collected from droppings pans below the floor of brooders housing Lot J offspring and stored frozen for possible analyses. A single composite sample was collected each for the control group and 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 odd numbered pens during odd numbered weeks (1,3,5, etc.) and from each of 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 and shipped to the Sponsor for possible analysis. Eggs collected during weeks 3-9 were opened at the waist, and their contents were removed. The egg contents were separated into albumen and yolk, stored frozen and shipped to the Sponsor for possible analysis. The shells were thoroughly rinsed with water and then 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 mm. W ildlife International, Ltd. Project Number 454-108 -21 - 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 reproductive parameters). Statistical analysis of the data was performed using Avian Reproduction Data System (ARDS) Software; a validated software package developed by Wildlife International, Ltd. (8). 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 11-12 candling was divided by the number of eggs set, per pen. 6. Live 3-Week Embryos of Viable Embryos - The number of live embryos at the Day 21 candling was divided by the number of viable embryos, per pen. 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-Dav Old Survivors of Hatchlings - The number of 14-day old survivors was divided by the number of hatchlings by pen. 9. Hatchlings of Eggs Set - The number of hatchlings was divided by the number of eggs set by pen. W ildlife International, Ltd. Project Number 454-108 -22 - 10. 14-Dav Old Survivors of Eggs Set - The number of 14-day old survivors was divided by the number of eggs set by pen. 11. Hatchlings 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 hatch lings to a percentile value equal to or less than 100. 12. 14-Dav 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 mean 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 ppm a.i. group. RESULTS AND DISCUSSION Mature northern bobwhite received PFOS at nominal dietary concentrations of 10, 50, 150 ppm a.i. Due to overt signs of toxicity noted at the 150 ppm a.i. test concentration, the dietary concentration for the 150 ppm a.i. treatment level was reduced to 20 ppm a.i. of PFOS at the beginning of Week 3 of the test. T h e 20 ppm a.i. le v e l w a s term inated after Week 4 o f the test due to the lim ited reco v ery o f birds at this level. Adults in the 50 ppm a.i. test concentrations w ere euthanized dining Week 7 of the test due to treatment-related mortality and overt signs of toxicity at this level. Therefore, the test was completed with the control group and 10 ppm 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 ppm a.i. test concentrations on Week 1, Day 0, and were analyzed to evaluate the homogeneity of the test substance in the diet and to verify test substance concentrations. Means and standard deviations for the three test concentrations were 9.75 0.313 ppm a.i., 50.1 0.981 ppm a.i. and 147 4.31 ppm a.i., respectively. The coefficients of variation were 3.21%, 1.96% and W ildlife International, Ltd. Project Number 454-108 -23 - 2.93%, respectively. These values represented 98%, 100%, and 98% of nominal concentrations (Appendix IV, Table 4). Samples collected during 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 0.553 ppm a.i., 20.8 2.14 ppm a.i., 50.9 2.04 ppm a.i, and 161 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 108%, 100% and 103% of the Day 0 values for the 10, 50 and 150 ppm a.i. test concentrations, respectively (Appendix IV, Table 6). A typical ion chromatogram of a test sample is shown in Appendix IV, Figure 7. Mean weekly measured concentrations for each test group are presented in Table 1. Mortalities There were no treatment-related mortalities in the 10 ppm a.i. treatment group. However, there were three incidental mortalities in both the control group and 10 ppm a.i. treatment group during the course of the study. Control group mortalities occurred during Weeks 14, 19 and 21 of the test. Mortalities in the 10 ppm a.i. test group occurred during Weeks 12, 20 and 21 of the test. All mortalities in the control group and 10 ppm a.i. treatment group were noted with head lesions and/or neck injuries that suggest m ortality w as caused b y incidental injury. D u e to the nature o f the lesion s observed at necropsy, none of the mortalities in the 10 ppm a.i. treatment group were considered to be related to treatment. Five mortalities occurred in the 50 ppm a.i. treatment group and three mortalities occurred in the 150 ppm a.i. treatment groups during the test. Two additional birds in the 150 ppm a.i. group were euthanized due to their debilitated conditions. All mortalities in the 50 and 150 ppm a.i. test concentrations exhibited overt signs of toxicity prior to death and were considered treatment-related. All remaining birds at the 150 ppm a.i. test concentration were euthanized at the beginning of Week 5 of the test. Necropsies were not performed on the birds euthanized from the 150 ppm a.i. treatment group. All remaining birds at the 50 ppm a.i. test concentration were euthanized and necropsied during Week 7 of the test. Necropsy findings are summarized in Table 2. W ildlife International, Ltd. Project Number 454-108 -24- Clinical Observations Clinical signs of toxicity were observed at all concentrations tested. At the 10 ppm a.i. test concentration, signs of toxicity were first observed during Week 5 of the test and continued intermittently until adult termination. Signs of toxicity at the 10 ppm a.i. test concentration included reduced reaction to external stimuli (sound and movement), ruffled appearance and lethargy. Signs of toxicity at the 50 ppm a.i. test concentration were first noted during Week 4 of the test, when several birds were noted as ruffled in appearance and lethargic. During body weight measurements at the end of Week 4, several birds in the 50 ppm a.i. exhibited muscle spasms and a loss of coordination following handling. At the 150 ppm a.i. test concentration, signs of toxicity were first noted during Week 2 of the test when one individual was noted as ruffled in appearance and lethargic. All birds in the 150 ppm a.i treatment group exhibited signs of toxicity induced by the stress of handling during body weight measurement at the end of Week 2. In several cases, birds convulsed and became rigid after being captured for body weight measurements. Signs of toxicity in the 50 and 150 ppm a.i. treatment groups continued until euthanasia and included reduced reaction to external stimuli (sound and movement), wing droop, loss of coordination, loss of righting reflex, lower limb rigidity, convulsions, shallow and rapid respiration, ruffled appearance, lower limb weakness, lethargy, gaping, prostrate posture and spasms. Adult Body Weight There were no apparent treatment-related effects upon adult body weight at the 10 ppm 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, concentration-responsive, treatment-related reductions in mean body weight for both males and females at the 50 and 150 ppm a.i. test concentrations during the Week 2 body weight interval. Reductions in mean body weight continued for both males and females in the 50 ppm a.i. treatment group through the Week 6 body weight interval. Following reduction of dietary concentrations to 20 ppm a.i. during Week 3 of the test, males in the 150 ppm a.i. test concentrations made slight weight gains at the Week 4 interval, while females in the 150 ppm a.i. treatment group maintained mean body weight. With the exception of mean female body weight at the 50 ppm a.i. test concentration during Week 2, all differences from the control group were statistically significant at p < 0.05 or 0.01. Mean body weight measurements are presented in Table 3, and Figures 1 and 2. Individual body weight measurements are presented in Appendix VII. W ildlife International, Ltd. Project Number 454-108 -25 - Adult Feed Consumption There were no apparent treatment-related effects upon feed consumption at the 10 ppm a.i. test concentration. No statistically significant differences between the control group and 10 ppm a.i. treatment group were observed at any of the feed consumption intervals. However, treatment-related reductions in feed consumption were observed at the 50 ppm a.i. test concentration during Weeks 1-6 and at the 150 ppm a.i. test concentration during Week 1 and 2 of the test. All of the reductions were statistically significant at p < 0.01. The dietary concentration for the 150 ppm a.i. treatment group was reduced to 20 ppm a.i. at the beginning of Week 3 of the test. Following the reduction of the dietary concentration to 20 ppm a.i., feed consumption for the group continued to be significantly lower than the control group during Week 3 of the test. Feed consumption at the 20 ppm a.i. test concentration was comparable to the control group during Week 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 10 ppm a.i. test concentration were subjected to gross necropsy following adult termination. When compared to the control group, there was an increase in the number of males with small testes in the 10 ppm a.i. treatment group that was considered treatment related. One male in the control group was noted with small testes, compared to seven males in the 10 ppm a.i. treatment 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 apparent treatment-related effects upon the mean weights of male livers at the 10 ppm a.i. test concentration. Differences between male liver weights for the 10 ppm a.i. treatment group and the control group were slight and not statistically significant. There was a statistically significant (p < 0.01) increase in mean female liver weight at the 10 ppm a.i. test concentration, when compared to the control group. 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 liver, kidney, brain, and proventriculus of adult and offspring males and females, bursa of Fabricius in offspring males and females, adipose tissue of adult males and females, ovaries of adult and offspring female, and testes W ildlife International, Ltd. Project Number 454-108 -26- of male offspring. The increase in the number of adult males in the 10 ppm a.i. treated group with reduced testicular size, not accompanied by any morphological change in spermatogenesis, 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 or adipose tissue in male and female offspring could not be determined since none (bursa of Fabricius) or only one (adipose tissue) sample was submitted for evaluation. In addition, any potential test-article effects on the gallbladder from treated adult males and females 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 bobwhite 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 mallard study run concurrently with the same test concentrations. Summary results of blood analyses performed for this study are presented in Exygen report Tables XXVI through XLI 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 ppm 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 E01-1245. "Analytical Phase Report for PFOS: A Reproduction Study with the Northern Bobwhite". Reproductive Results When compared to the control group, there were no apparent treatment-related effects upon egg production at the 10 ppm a.i. test concentration. However, while not statistically significant, there were slight reductions in the number of viable embryos as a percentage of eggs set, hatchlings as a percentage W ildlife International, Ltd. Project Number 454-108 -27- of live 3-week embryos and in 14-day old survivors as a percentage of hatchlings. The slight reductions in fertility, hatchability and offspring survival also resulted in reductions in the numbers of hatchlings and 14-day old survivors as percentages of both the number of eggs set and the maximum number of eggs set. While slight, the reduction in the number of 14-day old survivors as a percentage of eggs set was statistically significant at p < 0.05. 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 XIII and XIV. 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 ppm 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 ppm 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 ppm 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. W ildlife International, Ltd. -28- Project Number 454-108 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. 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 ppm 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, 10 through 15 located in Appendix XIIB. The full report for analysis of sera and liver samples collected at the time ofjuvenile termination is located in 3M Environmental Laboratory report E01-1245. "Analytical Phase Report for PFOS: A Reproduction Study with the Northern Bobwhite". CONCLUSION There were no apparent treatment-related effects upon adult body weight or feed consumption at the 10 ppm a.i. test concentration. However, minor overt signs of toxicity were observed in adults at the 10 ppm a.i. concentration. Additionally, there were reductions in testes size, and slight, but treatmentrelated, effects upon fertility, hatchability and offspring survival at the 10 ppm a.i. test concentration. There were treatment-related mortalities, overt signs of toxicity and treatment-related effects upon body weight and feed consumption at the 50 and 150 ppm a.i. test concentrations. The 50 and 150 ppm a.i. test concentrations were terminated prior to the reproductive phase of the test. Based on the effects observed at the 10 ppm a.i test concentration, the no-observed-effect concentration for northern bobwhite exposed to PFOS in the diet was determined to be less than 10 ppm a.i., the lowest concentration tested during this study. 'Wildlife International, Ltd. -29REFERENCES Project Number 454-108 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 fo r Conducting Reproductive Studies with Avian Species. ASTM Standard E l062-86. Annual Book of ASTM Standards. Vol. 11.04. Philadelphia, PA. 15 pp. 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 o f 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: 482 491.8 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.) o f PFOS in Avian Diet from a Northern Bobwhite Reproduction Study W eekl Day 0 Day 7 1 Week 2 Day 0 Week 3 Day 0 Interval Week 4 Day 0 Week 8 Day 0 W eek 12 W eek 16 W eek 20 Day 0 Day 0 Day 0 Project Number 454-108 0o0 0 Measured <2.00 2 <2.00 < 2 .0 0 <2.00 <2.00 <2.00 <2.00 <2.00 < 2 .0 0 Mean 10 Measured % Nominal 98 3 103 101 97 109 101 101 99 Mean 50 Measured % Nominal oo 100 4 97 104 104 Mean 150 Measured % Nominal 98 3 103 4 108 96 5 112 5 "" " 1Feed samples were collected from feed troughs at the end of Week 1 to assess stability o f the test substance under actual test conditions. 2<2.00 indicates the value was less than the limit o f quantitation. 3The mean of homogeneity samples collected at Week 1, Day 0. 4The percent of the mean values for Week 1, Day 0. 5The 150 ppm a.i. treated diet was reduced to 20 ppm a.i. during this interval. - No values available, test group terminated. -31 - Project Number 454-108 Table 2 Summary of Gross Pathological Observations from a Northern Bobwhite Reproduction Study with PFOS Adult Birds Found Dead/Euthanized during the Test Males 50 1 150 2 ppm a.i. ppm a.i. Females Control 10 50 1 (0 ppm a.i.) ppm a.i. ppm a.i. 150 2 ppm a.i. Number ofbirds Feathers wet around beak Feather loss Extensive head/neck lesions Vent - pasty Thin Emaciated Loss of muscle mass Prominent keel Intra-cranial bleeding Neck - mid-length fracture Neck - hematoma Heart - no coronary fat Spleen - small and/or pale Abdominal cavity - autolysis Abdominal cavity - small amount of loose blood Crop - empty Gizzard contents - bile-stained Cecal contents - pasty Kidneys - pale Ovary - developing Ovary - regressing Not remarkable 16 0 5 0 0 1 1 2 1 0 0 0 1 3 1 0 1 0 0 0 7 3 0 0 0 0 0 1 1 1 1 0 0 0 1 1 0 0 0 1 0 1 3 3 16 2 10 0 0 0000 12 00 0 0 10 0000 112 1 112 1 112 1 0000 10 0 0 1 1 10 0 110 12 11 112 1 0 100 1 10 0 0 100 10 00 0 100 0 100 10 00 0 0 13 0 1Remaining birds in the 50 ppm a.i. test concentration were euthanized during Week 7 of the test. Gross necropsy observations for birds maintained for collection of blood samples are not reported. 2Remaining birds in the 150 ppm a.i. test concentration were euthanized at the beginning of Week 5. Gross necropsies were not performed at the termination o f the 150 ppm a.i. test concentration. Table 3 Mean Adult Body W eigh t (g ) from a N o rth e rn B o b w h ite R e p r o d u c tio n S tu d y w ith PFOS1 Experimental Group (ppm a.i.) Sex Control 10 50 150 Male Female Male Female Male Female Male Female Week 0 Change Week 0-2 Week 2 Change Week 2-4 Week 4 Change Week 4-6 Week 6 Change Week 6-8 Week 8 Change Week 8-Term Test Total Term Change 208 6 210 4 208 2 206 4 205 -8 210 -7 206 -34 212 -41 214 -1 214 -1 211 -1 209 -1 197** -13 202 -7 172** 172** 13 3 213 1 213 1 210 1 208 1 185** -8 195* -11 187** 172** - " 215 -1 214 4 211 -2 209 -1 179** 185** - -- 214 218 209 209 - - 1 32 -1 40 - - - 215 7 247 39 208 -1 249 42 - '-- -- -- Project Number 454-108 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 .0 5 * * Significantly different from the control at p < 0.01 1 Only surviving birds were included in the calculations for each body weight interval. F ig u re 1 Mean A dult Male Body U eight (g ) from a N o rth e rn B obuhite R e p ro d u c tio n S tu d y u it h PFOS MALE BODV HEIGHT (g ) t N ac 1 &c to M- OO3n c** 2= m co 7T 3 - 7 O) O-KIl Oto 2*T= tO3o* oo to iiiiiiiiiiillllllllllllllllillliiiiiiiiilllllllll * 00 EJCTIO. 'StOT3o to % n3Al- Oh*+ ito 3 OSEZ3 US* H I 80I-1717 ioqum^ pafojd - - FEMALE BODV UEIBHT (g ) Figure 2 Mean Adult Female Body Height Cg) from a Northern Bobuhite Reproduction Study uith PFOS 300 200 100 121 Week 0 tW Meek 2 Change Week 4 Change Week 6 14k 0 -2 14k 2 -4 14k 4 -6 Change Data a re not a v a ila b le due to adu lt m o rta lity 14 E E K S Change Week 8 14k 6 -8 Change Ueek T Total 14k 8 -T Change Concentrations, ppm a .i. o S i o [2250 it Rn u> Project Number 454-108 Table 4 Mean F eed C onsum ption ( g / b i r d / d a y ) from a N o rth e rn B o b w h ite R e p r o d u c tio n S tu d y w i th 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 10 50 150 12 14 13 13 13 12 12 15 14 14 15 17 18 20 19 20 19 19 19 20 19 12 13 13 13 13 12 12 14 13 13 14 16 18 20 20 19 20 20 20 21 19 11** 11** 10** 10** 9** 9** _ 7** 6** 8** 13 u> - Data are not available due to adult mortality Significantly different from the control at p < 0.01 Project Number 454-108 Figure 3 Mean Feed Consumption (g/bird/day) from a Northern Bobuhite Reproduction Study uith PFOS Project Number 454-108 _____ 0 ----10 Concentrations, ppm a .i. ........ so .........150 -37- Project Number 454-108 Table 5 Summary of Gross Pathological Observations from a Northern Bobwhite Reproduction Study with PFOS Adult Birds Euthanized at Test Termination1 M ales Control (0 ppm a.i.) 10 ppm a.i. F em ales Control (0 ppm a.i.) 10 ppm a. Number o f birds 16 16 Feather loss 35 Head lesions 00 Foot/leg lesions 00 Back/vent lesions 00 Liver - pale and mottled Abdominal cavity slight egg yolk peritonitis Intestinal tract area o f hyperemia 0 . 7 0 8 Ceca - round worms found Testes - one or both small (~1.0-1.5 cm) 03 16 N ot remarkable 73 13 13 69 01 10 20 01 01 01 30 -- 63 1Gross necropsy observations for birds maintained for collection of blood samples dining the test are not reported. Table 6 Mean Liver Weights1(g) from a Northern Bobwhite Reproduction Study with PFOS Experimental Group Adult M ales Mean SD Adult Females Mean SD Offspring M ales Mean SD Offspring Females Mean SD Control O ppm a.i. 4.427 0.496 8.662 1.113 0.842 0.154 0.850 0.087 10 ppm a.i. 50 ppm a.i. 150 ppm a.i. 4.481 0.545 10.884* 1.596 . __ ._ 0.853 0.172 0.882 0.184 m_ __ ^ e a n standard deviation. - Data not available due to mortality. * Statistically different from the control group at p < 0.01. 00 Project Number 454-108 Table 7 Summary o f R e p r o d u c tiv e P erform an ce from a N o rth e rn B o b w h ite Reproduction S tu d y w ith PROS Reproductive Parameter 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 Control 13 605 23 521 494 490 473 457 47 0.50 35 Experimental Group (ppm a.i.) 10 50 13 592 3 526 470 466 414 386 - 46 0.49 30 - 150 - '* Project Number 454- 1Based on 9 3 days of eggs production. - Data are not available due to adult mortality Figure 4 Mean Reproductive Performance from a Northern Bobuhite Reproduction Study uith PFOS 50 40 LaJ. \ et en 30 ZD Z <c LJ 20 10 0 Eggs Laid * Eggs Eggs Viable L ive 3-ufe Cracked Set Embryos Embryos Data a re not a v a ila b le due to adult m o rta lity H a tch lin g s 14-0ay Old Survivors o Project Number 454-108 Table 8 Summary of R e p r o d u c tiv e Performance, N o rm a lize d a s P e r c e n ta g e s (%) from a N o rth ern B o b w h ite R e p ro d u c tio n S tu d y w ith PFOS1 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 (%) 13 60S 74 3 94 99 97 97 90 87 64 62 Experimental Group (ppm a.i.) 10 50 13 592 72 0 89 99 89 93 78 72* 56 52 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A - No eggs available due to adult mortality. * Significantly different from the control at p < 0 .0 5 / 1Values represent pen means for experimental group. Values for each pen are presented in Appendices VII and VIII. 2 Represents th e total number of eggs laid in each group. 150 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Project Number 454-108 / Figure 5 Mean R eprod u ctive P erfo rm ance, N orm alized as P ercen tag es ( X ) , from a N o r th e r n B o b u h ite R e p r o d u c tio n S tu d y u ith PFOS 00534853535353532348534848534848 U CD U CauJ Cl <c U Eggs L a id / Maximum Laid Eggs C racked / Eggs Laid V iable Embryos/ Live 3 -Week/ Eggs Set V ia b le Embryos H a tc h lin g s / L ive 3-Ueek Reproductive parameter 14-Day O ld/ H a tch lin g s E3o Concentrations, ppm a .i. 10 2350 S511S0 4K^) Project Number 454-108 Table 9 Mean Eggshell Thickness Measurements (mm) from a Northern Bobwhite Reproduction Study with PFOS Experimental Group (ppm a.i.) Control 10 50 150 No. Eggs Measured 42 45 - Shell Thickness Mean ( SD) 0.229 0.021 0.227 0.010 --- - Data are not available due to adult mortality Differences between the control and each treatment groups were not significant (p > 0 .0 5 ). . Project Number 454-108 Table 10 Mean B ody W eight ( g ) o f H a tc h lin g s and 1 4 -D ay O ld S u r v iv o r s from a N o rth ern B obw h ite R e p ro d u c tio n S tu d y w ith PFOS Experimented Group (PPM A.I.) Hatchlings ------------Number Mean ( SD) 14-Day Old Survivors Number Mean ( + SD) Control 50 150 472 6 0 412 6 1 . - ' 457 29 2 386 30 3 - - D ata are not available due to adult mortality The number o f hatchlings weighed may differ from the total number of hatchlings since those hatchlings found dead were not weighed. Differences between the control and each treatment group were not significant (p > 0 .0 5 ). -u ' Project Number 454-108 Wildlife International, Ltd, -45 - Project Number 454-108 Appendix I Certificate o f A nalysis Centre Analytical Laboratories, Inc. 3 048 Research brtve P ho ne:(814)231-8032 State C ollege, R 4 16801 w w w .centreiab.eom Fax: (814) 231-12S3 or (8 14 ) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFO S,Lot217 __________ to t Name Purity1 Refereace #: SD-018 Purity; 86.9%____________________ SpedOeattens Result 86.9% Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium2 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) Related Compounds POAA Residual Solvents (TGA) Purify by DSC Inorganic Amans (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfite4 Organic Acids ' (IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis': 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine White Crystalline Powder 1. Theoretical Value = 17.8% 2. Theoretical Value " 0% 3. Theoretical Value - 0% 4. Theoretical Value = 5.95% 5. Theoretical Value --60% Conforma Positive 1. 0.005 wt/wt% 2. 0.001 wt/wt.% 3. 1.439 wt/wt% 4. 6.849 wt/wt.% 5. <0.001 wt/wt% 6. 0.005 wt/wt.% 7. <0.001 wt/wt% 1.91 wt/wt.% 8.41 wt/wt.% None Detected 0.33 wt/wt% None Detected Not Applicable* 1. <0.015 wt/wt% 2. 0.59 wt/wt.% 3. <0.040 wt/wt% 4. <0.009 wt/wt% 5. <0.006 wt/wt% 6. <0.007 wt/wt% 7. 8.76wt/wt% 1. <0.1 wt/wt% 2. <0.1 wt/wt% 3. 0.10 wt/wt.% 4. 0.28 wt/wt% 1. 12.48 wt/wt% 2. 0.244 wt/wt.% 3. 1.74 wt/wt.% 4. 8.84 wt/wt.% 5. 54.1 wt7wt.% COA023-018A Page 1o f 3 Wildlife International, Ltd -46- Project Number 454-108 Appendix I Page 2 Certifcate o f A nalysis Centre A nalytical Laboratories, Inc 3 048 Research D rive State Collega, PA 16801 w w w .centrelab.oom Phone: (814) 231-8032 Fax: (8 14 ) 231-1253 or (8 14 ) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/06 Storage Conditions: Frozen <-10*C Re-assessment Date: 08/31/06 Purity =>100% - (sum o f metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.905%+organic acid impurities, 0.38%+POAA, 0.33%) Total impurity from all tests = 13.07% Purity -100% - 13.07% - 86.9% 2Potassium is expected in this salt form and is therefore not considered an impurity. ^Purity by DSC is generally not applicable to materials of low purity. No enddtherm was observed for tins sample. `Sulfur in the sample appears to be converted to SO* and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to Ibis interpretation. Based on the results, the SO4 is notconsidered an impurity.5 5TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafloorobutyric acid Nonafluoropentanoic acid Pentafluoropropanoic acid `Theoretical value calculations based on the empirical formula, C tF|7SOj'K+(MW-538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-0I8A Page 2 o f3 Wildlife International, Ltd, -47- Project Number 454-108 A ppendix I Page 3 Certificate o f A nalysis Centre Analytical Laboratories, Inc 3 04a Research Drive Phone: (814) 231-8032 Stalls C ollega, FW 16801 w w w .oentrelab.com Fax: (614) 231-1253 or (8 14 ) 231-1580 INTERIM CERTIFICATE OF ANALYSIS R evisio n i Centre Analytical Laboratori COA Reference #: 023-018A LC/MS Purity Profile: Im purity C4 C5 C6 C7 Total w t/w t % 1.22 1.33 4.72 1.14 8.41 ' Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average result from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average result from the C6 and C8 standard curves. Prepared By: Chartes Sitnofrs Scientist, Centre Analytical laboratories So/fiJo/ D ate ^oLmfLUtReviewed By: ^lohn Flaherty / Date Laboratory Manager, Centre Analytical Laboratories COA023-018A Page 3 o f3 Wildlife International, Ltd -48- Project Number 454-108 Appendix II Diet and Supplement Formulations Table 1 Wildlife International, Ltd. Game Bird Ration1 INGREDIENTS Fine Com Meal Soy Bean Meal, 48% Protein Wheat Midds Protein Base Agway Special, 60% Protein Alfalfa Meal, 20% Protein Dried Whey Ground Limestone Eastman CalPhos Methionine Premix + Liquid Vitamin and Mineral Premix (see below) GL Ferm (Fermatco)2 Salt Iodized Total PERCENT (%) 44.83 30.65 6.50 6.00 4.00 3.00 2.50 0.90 0.60 0.35 0.32 0.25 0.10 100.00 Vitamin and Mineral Premix, when added at 0.32% o f the ration, supplied the following amounts per ton: Amount Supplied Per Ton: Vitamin D3 Vitamin A Riboflavin Niacin Pantothenic Acid Vitamin B ]2 Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (Menadione Dimethylpyrimidinol Bisulfite) Manganese Zinc Copper Iodine Iron Selenium 2.000. 0 0 0 1.C.U. 7.000. 0 0 0 1.U. 6 grams 40 grams 10 grams 8 mgs 600 mgs 64 mgs 1.2 grams 1.2 grams 2 0 ,0 0 0 1.U. 5.8 grams 102 grams 47 grams 6.8 grams 1.5 grams 51 grams 182 mgs 1 The guaranteed analysis is a minimum o f 27% protein, a minimum o f 2.5% crude fat and a maximum o f 5% crude fiber. 2 Fermentation By-Products (Source o f Unidentified Growth Factors). Wildlife International, Ltd, -49- Project Number 454-108 Appendix II 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 oz. 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-A ctivated Animal Sterol (source o f Vitamin D 3), Alpha Tocopheryl Acetate (source o f Vitamin E). Riboflavin Supplement, d-Calcium Pantothenate, Niacin Supplement, Vitamin B-12 Supplement, Menadione Sodium Bisulfite (source o f Vitamin K), Folic Acid, Thiamine HC1, Pyridoxine Hydrochloride, Ascorbic Acid, Sodium Chloride, Calcium Chloride, Magnesium Sulfate, Ferric Ammonium Citrate, Potassium Chloride and Dextrose. MIXING PROCEDURE: The vitamin and electrolyte m ix was prepared as a ration o f approximately 2 grams o f Durvet vitamins and electrolytes to approximately 1 gallon o f water. Wildlife International, Ltd, -50- Project Number 454-108 Appendix III 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: No premix required. 10 ppm a.i.: 2.5633 g PFOS + 8097.4 g ration 50 ppm a.i.: 12.8165 g PFOS + 8087.2 g ration 150 ppm a.i.: 38.4494 g PFOS + 8061.6 g ration 20 ppm a.i. 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 Waring blender. The test substance was transferred to the Waring 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, rew eighed, and stored frozen. As needed, the appropriate premix was incorporated into the final diet as follows: 0 ppm a.i.: 52.25 kg ration + 2.750 kg limestone 10 ppm a.i.: 2000 g Premix + 50.25 kg ration + 2.750 kg limestone 50 ppm 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. Wildlife International, Ltd. -51 - Appendix IV The Analysis of PFOS in Avian Diet Project Number 454-108 Wildlife International, Ltd -52- Project Number 454-108 INSTRUMENT: APPENDIX IV ANALYTICAL METHODS AND RESULTS Table 1 Typical HPLC/MS/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3 0 0 0 Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in multiple ion reaction monitoring (MRM) mode. ANALYTICAL COLUMN: GUARD COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: PFOS RETENTION TIME: PFOS MONITORED MASS: Keystone Betasil C18 column (50 mm x 2 mm I.D., 3-pm particle size) Keystone Javelin C ]8 column (20 mm x 2 mm I.D.) 40C 5.00 minutes 250 pL/minute 70.0% Methanol: 30.0% NANOpure Water containing 0.1% Formic Acid 10.0 pL Approximately 4.0 minutes 499.0 amu -->99.1 amu Wildlife International, Ltd, -53- Project Number 454-108 APPENDIX IV Table 2 Calculations The concentration o f PFOS found at the instrument was determined using the follow ing equation: peak area - (y-intercept) PFOS (m g a.i./L) at instrument = slope Determination of Sample Residues (PFOS) The concentration, expressed as ppm a.i., for each sample w as determined using the follow ing equation: PFOS (mg a.i./L) at instru. x extract final volum e (L) x dilution factor PFOS (ppm a.i.) in sample = initial weight (Kg) Determination of Limit of Quantitation (LOQ) The method LOQ, expressed as ppm a.i., was determined using the follow ing equation: LOQ (ppm a.i.) = low est standard concentration (mg a.i./L) x overall dilution factor o f matrix blank* extract final volume (L) x dilution factor overall dilution factor o f matrix blank sample = initial weight (Kg) Fortification Recoveries The ppm a.i. measured in each sample is divided by the nominal concentration o f each sample (fortified level, ppm a.i.). This ratio times 100 is the percent recovery o f the method at that level o f fortification. ppm a.i. measured in sample % Recovery = ppm a.i. fortified x 100 W ildlife International, Ltd. Project Number 454-108 Number (454-108-) M A B -1 M A S-1 M A S-2 M A S-3 M A B -2 M A S-4 M A S-5 M A S-6 M A B -3 M A S-7 M A S-8 M A S-9 M A B -4 M AS-10 M A S-11 M A S-12 APPENDIX IV Table 3 Matrix Blanks and Fortifications Analyzed Concurrently with the Samples Sample Type Interval Concentration o f PFOS (ppm a.i.) Fortified1 M ea su red 1,2 Percent Recovery 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 W eek 1, D ay 0 W eek 1, D ay 0 W eek 1, D ay 0 W eek 1, D ay 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 0.00 5.00 50.0 200 0.00 5.00 50.0 200 0.00 5.00 50.0 200 0.00 5.00 25.0 100 < 2 .0 0 4.85 52.8 206 < 2 .0 0 4.55 47.4 189 < 2 .0 0 5.50 52.0 200 < 2 .0 0 5.20 26.1 100 97 106 103 _ 91 95 95 _ 110 104 100 _ 104 104 100 1Less than values correspond to limit o f quantitation (LOQ). 2Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly. Mean Recovery (X) 102 94 105 103 W ildlife International, Ltd. Project Number 454-108 APPENDIX IV Table 3 (Continued) Matrix Blanks and Fortifications Analyzed Concurrently with the Samples Number (454-108-) M A B -5 M A S -13 M AS-14 M A B -6 M A S -15 M A S -16 M AS-173 M A S-18 M A B -7 M A S -19 M A S-20 Sample Type Interval Matrix Blank Matrix Fortification Matrix Fortification Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Matrix Fortification Matrix Blank Matrix Fortification Matrix Fortification Week 8, Day 0 Week 8, Day 0 Week 8, Day 0 W eek 12, Day 0 W eek 12, D ay 0 W eek 12, DayO W eek 16, DayO W eek 16, DayO Week 20, Day 0 Week 20, Day 0 Week 20, Day 0 Concentration o f PFOS (ppm a.i.) Fortified12 M e a su r e d 1,2 0.00 5.00 25.0 0.00 5.00 25.0 5.26 25.0 0.00 5.00 25.0 < 2 .0 0 5.40 27.5 < 2 .0 0 5.21 24.4 5.39 24.5 < 2 .0 0 4.86 24.0 Percent Recovery 108 110 104 98 102 98 _ 97 96 Mean Recovery (x) 109 101 100 97 1Less than values correspond to limit o f quantitation (LOQ). 2Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly. 3W eek 16 samples were held and batched with W eek 20 samples. Matrix fortifications were prepared to verify stability. required. A storage matrix blank was not -55 - W ildlife International, Ltd. Nominal Concentration (ppm a.i.) 10 50 150 Sample I.D. Number (454-108-) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Project Number 454-108 APPENDIX IV Table 4 Homogeneity of PFOS in Avian Diet Location Sampled in M ixing Vessel Measured PFOS Concentration (ppm a.i.) 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 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 (x) Standard D eviation (SD ) Coefficient o f 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% - x = 147 ppm a.i. SD = 4.31 ppm a.i. CV = 2.93% Mean Percent of Nominal 98 100 98 -56- W ildlife International, Ltd. Project Number 454-108 -57- Nominal Concentration (ppm a.i.) Sample I.D. Number (454-108-) 34 41 48 55 60 65 70 APPENDIX IV Table 5 Verification of PFOS Concentrations in Avian Diet Interval (Day Oof W eek-) 2 3 4 8 12 16 20 Measured PFOS Concentration1 (ppm a.i.) < 2 .0 0 < 2 .0 0 < 2 .0 0 < 2 .0 0 <2.00 <2.00 <2.00 M ean(x) Standard Deviation (SD) Coefficient o f Variation (CV) - 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 1Less than values correspond to limit o f quantitation (LOQ). 11.0 9.68 9.53 10.7 9.70 9.60 10.6 x = 10.2 ppm a.i. 11.2 SD = 0.553 ppm a.i. 10.4 CV = 5.44% 9.86 9.82 10.4 10.1 9.69 Percent of Nominal - 110 97 95 107 97 96 106 112 104 99 98 104 101 97 Mean Percent of Nominal W ildlife International, Ltd, Project Number 454-108 Nominal Concentration (ppm a.i.) 20 50 150 Sample I.D. Number (454-108-) 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 o f W eek-) 3 3 4 4 2 2 3 3 4 4 2 2 Measured PFOS Concentration1 (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 o f 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 = 161 ppm a.i. S D = 15.6 ppm 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 W ildlife International, Ltd. Project Number 454-108 APPENDIX IV Table 6 Ambient Stability of PFOS in Avian Diet During the Northern Bobwhite Reproduction Study Nominal Concentration (ppm a.i.) Sample Number (454-108-) W eek 1, DayO Mean Measured (ppm a.i.) PFOS Concentration Mean Percent of Nominal Sample Number (454-108-) 0 1 <2.00 - 10 2-7 9.75 98 50 8-13 50.1 100 150 14-19 147 98 20 21 22 23 24 25 26 1D ay 0 values are from hom ogeneity samples presented in Table 4. 2Less than values correspond to limit o f quantitation (LOQ). W eek 1, D ay 7 Measured12 (ppm a.i.) Mean Measured (ppm a.i.) < 2 .0 0 9.74 11.2 50.1 50.5 148 154 < 2 .0 0 10.5 50.3 151 Mean Percent of DayO 108 100 103 -59- 'Wildlife International, Ltd. -60- Project Number 454-108 APPENDIX IV METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN AVIAN DIET Prepare matrix fortification samples in the desired avian feed stock using the dry mix technique. 4 W eigh 10 g samples o f the matrix blank, matrix fortification and test samples into weigh boats and transfer to 8oz. French square glass bottles. Record weights. 4 For each sample, measure 100 mL o f methanol with a graduated cylinder and transfer into the French square bottle. 4 Cap bottles and place on shaker table. A llow the sam ples to shake for a minimum o f 30 minutes at approximately 250 rpm. 4 Vacuum filter with qualitative filter paper and rinse retained feed 3 times with methanol into filtrate. 4 Transfer the filtrate into a 200-m L volumetric flask and bring to volum e with methanol. 4 Prepare appropriate dilution(s) to bring final concentration into the calibration range o f the HPLC/M S/M S m ethodology. U se methanol for intermediate dilutions, i f required. For all final dilutions, use 50% methanol : 50% NANOpure water dilution solvent. 4 Ampulate and submit sample for HPLC/MS/MS analysis. Figure 1. Analytical method flow chart for the analysis of PFOS in avian diet. -61 - W ildlife International, Ltd, Project Number 454-108 APPENDIX IV Figure 2. Typical calibration curve for PFOS. Slope = 11487727; Intercept = 883.10; r = 0.99974. Curve is weighted (1/x). W ildlife International, Ltd. -62- Project Number 454-108 APPENDIX IV PFOSJ STD 0.00100 mg a.IVL 4675A-011D -1 pros4.98 In 1 period No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. Quant Thres. Min. Width Mult. Width Base. Width 'RTWin. (secs) Smooth Expected RT 5.0 1.0 3 6 35 20 1 4.02 Area 12195 Height 1171 Start Time EndTime Integration Width Retention Time Integration Type 3.79 4.37 0.59 4.02 Ai-BB Wed, :eb 7, 2001 17:58 100i 90 80 70 605040 30 20 10 0-- I29 I49 616 I l1111131815I9 41 81 121 161 0.69 1.36 2.03 2.70 intensity: 10000 cps 240 208 201 241 281 Scan 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). W ildlife International, Ltd, -63- Project Number 454-108 APPENDIX IV PFOSJ STD 0.0100 mg JA 4675A-011D -5 4.98 in 1 period PFOS No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 5.0 Quant Thres. 1.0 Min. Width 3 Mult. Width 6 Base. Width RT Win. (secs) Smooth 35 20 1 ExpectedRT 4.02 Area 114007 Height 10269 Start Time EndTime Integration Width Retention Time Integration Type 3.80 4.39 0.59 4.02 Ai-BB Wed Feb 7, 2001 18:21 100 90 80 70 60 50 40 30 20 10 0,,`I--12I-- I--53I--79r--i1--09-1-- 41 81 121 0.69 1.36 2.03 intensity: 10000 cps 240 Figure 4. Typical ion chromatogram of a high PFOS calibration standard, 0.0100 mg a.i./L. (Monitored mass 499.0 --99.1). W ildlife International, Ltd, -64- Project Number 454-108 APPENDIX IV PFOS 7 MAB-3 454-108,109- Wed, Feb 7, 2001 18:33 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 NoiseHires. 5.0 Quant Thres. 1.0 Min. Width 3 Mult. Width 6 Base. Width 35 RTWin. (secs) 20 Smooth 1 ExpectedRT 4.02 Area 0 Height 0 Start Time 0.00 EndTime 0.00 Integration Width 0.00 Retention Time 0.00 Integration Type 10090 80 70 60 50 40 30 20 10 o- intensity: 10000 cps --i--1i--05 I1--301--1r--64 205 240 274 81 121 161 201 241 281 Scan 1.36 2.03 2.70 3.37 4.04 4.71 Time Figure 5. Typical ion chromatogram of a matrix blank sample (454-108-MAB-3). The arrow indicates the approximate retention tim e o f PFOS. (Monitored mass 499.0 -->99.1). W ildlife International, Ltd, -65- Project Number 454-108 APPENDIX IV PFOSJ MAS-8 454-108,109- Wed, Feb 7, 2001 1845 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. Quant Thres. Min. Width 5.0 1.0 3 Mult. Width 6 Base. Width 35 RT Win. (secs) 20 Smooth 1 Expected RT 4.02 Area 60579 Height 5641 Start Time 3.80 End Time 4.39 Integration Width 0.59 Retention Time 4.02 Integration Type fit-BB 100| 96 86 76 66 intensity: 10000 cps 240 Figure 6. Typical ion chromatogram of a matrix fortification sample (454-108-MAS-8, 50.0 ppm a.i.) (Monitored mass 499.0 -->99.1). -66- W ildlife International, Ltd. Project Number 454-108 APPENDIX IV PF0SJ3 42 454-108,109- Wed, Feb 1 2001 19:10 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 NoiseThres. 5.0 Quant Thres. 1.0 Min. Width 3 Mult. Width 6 Base. Width 35 RTWin. (secs) 20 Smooth 1 ExpectedRT 4.02 Area 55642 Height 5213 Start Time End Time Integration Width Retention Time Integration Type 3.80 4.39 0.59 4.02 A -BB intensity: 10000 cps 100i 90- 80- f lr 60- 50- 4(1 3d 20- 10 0+ 21 48 78 -I I 129-1--1-5r7176 41 81 121 161 201 241 281 Scan 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 (454-108-42, 10.0 ppm a.i.). Dilution factor = 2000X. (Monitored mass 499.0 -->99.1). -67- Project Number 454-108 Appendix V Diagram of Test Layout from a Northern Bobwhite Reproduction Study with PFOS Room 45 Note: Pens 217-220,237-240,257-260 and 277-280 were used to house birds used for blood sampling. -68- W ildlife International, Ltd. Project Number 454-108 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 10-12 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. -69- W ildlife International, Ltd. Project Number 454-108 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 25 or 26 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, chicks will stick to their shells. - Temperature: If too hot and humid, chicks 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 D efin ition - 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 D efin ition - 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. W ildlife International, Ltd. -70- Project Number 454-108 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. -71 - W ildlife International, Ltd, Project Number 454-108 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 Raw Data Band Number Report Pen Raw Data Pen Number Number Male Female 201 201 329 330 202 202 331 332 203 203 333 334 204 204 335 336 205 205 337 338 206 206 339 340 207 207 341 342 208 208 343 344 209 209 345 346 210 210 347 348 211 211 349 350 212 212 351 352 213 213 353 354 214 214 355 356 215 215 357 358 216 216 359 360 -72- W ildlife International, Ltd. Project Number 454-108 Appendix VI Replicate Identification Experimental Group Treatment lOppma.i. Report Replicate Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 _ Raw Data Band Number Report Pen Raw Data Pen Number Number Male Female 217 221 369 370 218 222 371 372 219 223 373 374 220 224 375 376 221 225 377 378 222 226 379 380 223 227 381 382 224 228 383 384 225 229 385 386 226 230 387 388 227 231 389 390 228 232 391 392 229 233 393 394 230 234 395 396 231 235 397 398 232 236 399 400 Appendix VII - Table 1 A d u lt B ody W eigh t (g ) from a N o rth e rn B o b w h ite R e p r o d u c tio n S tu d y w i th PFOS (Control, Males) Pen 201 202 203 204 205 206 y207 208 209 210 211 212 213 214 215 216 Mean SD 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 210 214 218 206 206 184 182 205 209 207 213 212 195 196 232 244 208 16 -2 1 6 5 5 6 4 8 4 7 7 5 2 13 9 10 6 4 208 215 224 211 211 190 186 213 213 214 220 217 197 209 241 254 214 17 0 -1 0 -4 -2 -3 4 1 -2 2 0 -1 -1 -2 -1 2 -1 2 208 214 224 207 209 187 190 214 211 216 220 216 196 207 240 256 213 17 0 1 1 1 3 1 3 2 4 -1 -1 -1 3 2 -2 5 1 2 208 215 225 208 212 188 193 216 215 215 219 215 199 209 238 261 215 17 1 0 -1 4 3 -3 -3 1 -1 -18 -4 1 7 -3 4 2 -1 6 209 215 224 212 215 185 190 217 214 197 215 216 206 206 242 263 214 19 5 -15 -11 8 1 14 -3 3 5 21 3 -8 8 -5 -1 -9 1 9 214 200 213 220 216 199 187 220 219 218 218 208 214 201 241 254 215 16 Total Change 4 -14 -5 14 10 15 5 15 10 11 5 -4 19 5 9 10 7 9 The means for body weights and body weight changes are calculated and rounded separately. Project Number 454-108 Appendix VII - Table 2 A d u lt B ody W eigh t ( g ) from a N o rth e rn B o b w h lte R e p r o d u c tio n S tu d y w i th PFOS (Control, Females) Pen 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Mean SD 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 191 3 194 -2 211 -1 210 2 220 3 223 -3 205 5 210 0 2 0 1 6 .207 0 205 7 2 1 2 -4 224 4 228 0 220 2 222 -2 191 6 197 0 187 4 191 - 2 197 4 201 -1 2 1 1 7 218 2 217 1 218 - 2 226 7 233 - 1 218 3 221 -1 234 2 236 - 1 2 10 4 214 - 1 14 2 14 2 192 212 220 210 207 208 228 220 197 189 200 220 216 232 220 235 213 14 1 2 2 1 3 0 -3 0 -1 2 -1 1 6 1 5 0 1 2 193 214 222 211 210 208 225 220 196 191 199 221 222 233 225 235 214 14 0 6 7 3 0 -3 11 -4 5 25 3 4 4 -3 5 -3 4 7 193 220 229 214 210 205 236 216 201 216 202 225 226 230 230 232 218 13 24 28 - 21 19 24 48 26 19 57 24 51 29 44 32 13 217 248 235 229 229 264 227 235 259 250 281 259 276 247 20 Total Change 26 37 30 28 24 44 36 48 62 33 55 41 42 39 11 The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. Project Number 454-108 Appendix VII - Table 3 Adult B ody W eigh t (g ) from a N o rth e rn B o b w h ite R e p r o d u c tio n S tu d y w ith 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 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 200 225 219 190 198 188 209 205 198 217 209 205 214 211 224 218 208 11 8 1 2 2 3 1 2 9 2 4 2 -8 5 3 4 -1 2 4 208 226 221 192 201 189 211 214 200 221 211 197 219 214 228 217 211 12 1 1 -2 0 -4 0 3 -4 -3 -1 -3 7 -2 2 -3 0 -1 3 209 227 219 192 197 189 214 210 197 220 208 204 217 216 225 217 210 12 4 2 0 -1 2 2 -1 6 0 0 2 -2 0 0 0 -1 1 2 213 229 219 191 199 191 213 216 197 220 210 202 217 216 225 216 211 12 -4 -5 -2 6 0 -1 -4 -4 -6 -6 -3 -1 0 -1 -2 -3 -2 3 209 224 217 197 199 190 209 212 191 214 207 201 217 215 223 213 209 10 0 12 -6 3 10 4 -2 -1 1 -3 -10 1 10 -1 1 -7 -17 -1 8 209 236 211 200 209 194 207 201 188 204 208 211 216 216 216 196 208 11 Total Change 9 11 -8 10 11 6 -2 -4 -10 -13 -1 6 2 5 -8 -22 -1 10 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 .0 5 ). Project Number 454-108 Appendix VII - Table 4 A d u lt B ody W eig h t (g ) from a N o rth e rn B o h w h ite R e p r o d u c tio n S tu d y w i th PROS (10 PPM A.I., Females) Pen 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Week 0-2 Week 2-4 Week 4-6 Week 6 -8 Week 8 -Term Test Term 203 220 220 178 180 213 198 197 194 219 235 199 214 196 236 189 206 18 2 5 7 2 4 1 0 8 2 7 -5 7 5 6 4 3 4 3 205 225 227 180 184 214 198 205 196 226 230 206 219 202 240 192 209 17 -3 -1 -4 -1 0 -2 0 -3 1 1 4 -5 -3 1 -2 1 -1 2 202 224 223 179 184 212 198 202 197 227 234 201 216 203 238 193 208 17 -1 -1 1 0 3 2 5 3 3 1 -1 -2 0 2 -4 2 1 2 201 223 224 179 187 214 203 205 200 228 233 199 216 205 234 195 209 16 -1 -1 3 0 1 -2 -3 -5 2 3 1 0 0 -2 1 -6 -1 3 200 222 227 179 188 212 200 200 202 231 234 199 216 203 235 189 209 17 58 24 20 40 23 58 23 - 35 - 35 45 55 49 52 - 40 14 258 246 247 219 211 270 223 - 237 - 269 244 271 252 287 - 249 22 Total Change 55 26 27 41 31 57 25 - 43 - 34 45 57 56 51 - 42 12 The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. Differences between control and this treatm ent group were not significant (p > 0 .0 5 ). Project Number 454-108 Appendix VII - Table 5 A d u lt B ody W eigh t (g ) from a N o rth e rn B o b w h ite R e p r o d u c tio n S tu d y w i th 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 Week 0-2 Week 2-4 Week 4-6 Week 6 -8 Week 8 -Term Test Term 216 207 201 198 206 209 203 204 200 223 182 202 207 204 214 203 205 9 0 -7 -12 -17 -5 -16 -2 -7 -9 -4 3 -10 -10 -9 -6 -11 -8 5 216 200 189 181 201 193 201 197 191 219 185 192 197 195 208 192 -19 1 -9 -31 -29 -7 -18 -3 -9 -1 -1 1 -10 -15 -10 -13 -5 -11 197** 10 -13 8 197 -4 191 -9 158 -13 152 194 1 175 -16 198 -8 188 -2 190 -27 208 -5 175 1 177 0 187 - 1 182 -4 203 6 181 -41 185** 15 -8 12 193 182 145 195 159 190 186 163 203 176 177 186 178 209 140 179** 20 _ - - __ ---------------- -_ -- - _ - 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 Project Number 454-108 Appendix VII - Table 6 A d u lt B ody W eigh t ( g ) from a N o rth e rn B o b w h ite R e p ro d u c tio n S tu d y w i th PFOS (50 PPM A.I., Females) 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 Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Test Term 199 206 227 211 209 196 199 202 235 206 204 223 177 225 217 218 -1 -6 -5 -7 -4 -18 -6 -6 1 -8 -11 -13 -2 -17 -6 -7 198 200 222 204 205 178 193 196 236 198 193 210 175 208 211 211 -1 -5 -7 -3 -6 0 -5 -26 -1 -1 1 0 -30 -2 -8 -13 1 197 2 195 -4 215 -3 2 0 1 -26 199 -24 178 -3 188 -2 170 10 235 -6 187 -13 193 -3 180 -51 173 2 200 -31 198 - 1 2 2 12 -4 199 191 212 175 175 175 186 180 229 174 190 129 175 169 186 208 - __ ---------------- _ - 2 10 -7 14 5 202 15 -7 9 195* 17 -11 15 185** . 22 - - --- - - 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 .05 * Significantly different from the control at p < 0.01 -ooj Project Number 454-108 Appendix VII - Table 7 A d u lt B ody W eigh t ( g ) from a N o rth e rn B o b w h ite R e p r o d u c tio n S tu d y w i th PFOS (150 PPM A.I., Males) Pen 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 Mean SD 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 191 213 198 218 209 194 199 242 195 214 212 194 207 211 196 197 -2 2 -44 -15 -48 -34 -55 -32 -35 -53 -28 -2 1 -23 -25 -35 -47 -20 169 7 169 183 15 170 17 175 -4 139 167 24 207 18 142 38 186 4 191 -8 171 6 182 176 20 149 31 177 1 176 - 198 187 171 191 225 180 190 183 177 196 180 178 _ - _ ---------------- _ ---------------- _ - 206 -34 13 13 172** 18 13 14 187** 14 - - -_ -- _- -- - 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 Project Number 454-108 Appendix VII Table 8 Adult B ody W eigh t ( g ) from a Northern B o b w h ite R e p ro d u c tio n S tu d y w i th PFOS (150 PPM A.I., Females) Pen 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 Mean SD 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 203 250 201 234 214 232 206 236 178 213 181 223 219 219 182 204 212 21 -44 -47 -36 -30 -35 -26 -22 -43 -47 -40 -41 -62 -62 -17 -44 -55 -41 13 159 203 165 204 179 206 184 193 131 173 140 161 157 202 138 149 21 - 10 -70 13 17 -34 -4 6 18 23 29 10 15 1 -11 172** 25 3 26 180 - 175 134 192 223 150 189 137 191 163 190 167 217 139 138 172** 29 _ - - - - __ --------------- -- -- -- _- --------------- - --- - - 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 Project Number 454-108 Appendix VIII - Table 1 Feed Consumption ( g / b i r d / d a y ) from a N o rth e rn B o b w h ite R e p r o d u c tio n S tu d y w ith PFOS (C on trol) WEEKS Pen 1 2 3 4 5 6 7 8 9 10 1 1 1 2 13 14 15 16 17 18 19 20 2 1 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 1 1 13 1 2 1 2 1 2 1 2 12 13 12 13 17 18 19 22 18 2 1 20 2 1 2 1 22 17 1 1 12 12 13 13 12 12 14 12 12 14 16 16 18 18 12 14 16 18 16 15 13 16 14 13 13 13 12 16 16 15 17 18 20 20 13 13 12 14 14 14 13 12 13 12 13 13 12 12 14 13 12 13 15 16 18 17 17 19 20 18 20 17 12 14 12 12 12 12 12 14 13 13 14 14 14 16 16 17 18 20 20 22 19 13 14 14 13 13 13 13 14 14 14 13 14 16 18 18 18 18 20 19 2 1 20 12 13 13 13 13 12 12 15 15 14 12 2 1 20 24 24 23 19 12 1 1 16 16 1 1 12 13 13 12 12 12 13 13 13 16 17 17 19 20 2 1 20 2 1 19 19 19 12 14 13 13 13 13 12 16 14 15 15 16 17 20 2 1 22 2 1 20 19 2 1 20 12 13 13 13 12 12 12 15 13 13 14 16 18 20 20 20 20 20 20 20 19 12 13 13 13 12 12 1 1 14 13 14 15 16 19 2 1 20 20 2 1 - 2 1 2 1 22 20 12 15 13 13 12 12 12 15 14 14 17 18 19 22 2 1 2 1 2 1 2 1 2 1 2 1 18 13 14 14 14 14 14 14 16 16 17 17 17 19 2 1 . 20 2 1 2 1 2 1 2 1 22 22 14 14 13 13 13 13 12 13 14 14 17 17 19 20 20 22 2 1 22 20 19 19 13 14 13 13 13 13 12 16 14 15 15 18 22 23 23 22 23 23 22 23 20 14 16 14 15 14 14 13 16 14 16 17 17 2 1 2 1 2 1 22 22 2 1 22 23 2 1 Project Number 454-108 Mean SD 12 14 13 13 13 12 12 15 14 14 15 17 18 20 19 20 19 19 19 20 19 1 1 1 1 1 1 1 1 1 1 2 2 2 2 33333 32 Appendix VIII - Table 2 F eed C on sum ption ( g / h i r d / d a y ) from a N o r th e r n B o b w h ite R e p r o d u c tio n S tu d y w ith PFOS (10 PPM A . I . ) Pen. 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 WEEKS 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 2 1 1 1 14 14 14 14 13 13 13 12 12 12 14 17 18 18 19 18 20 20 2 1 19 1 2 13 1 2 13 13 12 12 14 12 1 1 16 16 19 2 1 2 1 22 22 2 1 17 18 2 1 1 1 14 13 13 12 13 12 14 13 13 12 14 15 19 2 1 19 2 1 20 22 20 15 10 1 2 1 1 1 2 1 1 1 1 12 13 12 12 12 14 16 17 18 18 18 19 20 20 17 1 1 12 1 1 1 1 1 1 1 1 1 1 13 13 13 14 15 18 17 17 16 18 19 22 2 1 18 12 14 14 13 14 12 13 14 14 14 15 15 18 19 19 20 20 22 23 25 22 12 13 12 13 12 12 1 1 13 13 13 14 15 16 18 18 18 17 16 15 14 16 12 14 14 14 13 13 13 14 14 10 9 14 20 17 16 15 15 15 16 15 16 1 2 14 13 13 14 12 13 14 12 14 14 18 19 22 23 23 22 23 22 25 22 13 15 14 14 14 14 13 15 14 15 16 17 19 2 1 23 2 1 23 24 23 27 24 1 1 14 14 15 13 12 14 17 16 16 18 20 22 23 20 15 23 24 24 26 25 12 13 14 15 13 12 13 14 14 14 13 19 20 20 19 20 20 2 1 20 22 2 1 13 14 13 13 13 13 13 15 15 14 17 19 18 23 2 1 23 22 23 22 24 2 1 13 14 13 13 13 12 12 12 12 12 14 15 17 19 19 18 19 19 20 20 18 13 14 14 14 14 13 12 15 16 16 18 2 1 20 23 2 1 22 2 1 2 1 2 1 22 20 1 1 1 1 1 1 12 12 1 1 1 1 1 1 10 1 1 12 13 15 17 18 17 18 18 17 13 13 Project Number 454-108 Mean SD 12 13 13 13 13 12 12 14 13 13 14 16 18 20 20 19 20 20 20 2 1 19 1 1 1 1 1 1 1 1 2 2 32 2 22 32 334 3 Differences between control and this treatment group were not significant (p > 0 .0 5 ). Appendix VIII - Table 3 F eed C on sum ption ( g / b i r d / d a y ) from a N o rth e rn B o b w h ite R e p r o d u c tio n S tu d y w ith PFOS (50 PPM A . I . ) Pen WEEKS 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 2 1 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 12 9 10 10 12 1 1 10 12 998 9 1 1 10 9 9 10 10 1 1 10 998 5 1 1 12 10 12 10 9 1 1 8 9 9 10 8 13 12 1 1 12 1 1 10 1 1 10 9 9 10 10 14 12 13 12 1 1 8 11 11 998 7 1 1 13 1 2 1 2 1 2 1 1 1 1 9 10 8 8 8 10 10 10 10 1 1 10 1 1 10 10 10 7 8 13 1 1 1 1 1 1 8 12 1 1 10 10 1 1 88 - - Project Number 454-108 Mean SD 1 1 ** 1 1 ** 1 0 ** 1 0 ** 9 ** 9** . 1 1 1 1 2 2- - Data are not available due to adult mortality. * * Significantly different from the control at p < 0.01 Appendix VIII - Table 4 F eed C on sum ption ( g / b i r d / d a y ) from a N o rth e rn B o b w h ite R e p r o d u c tio n S tu d y w ith PFOS (150 PPM A. I . ) WEEKS Pen 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 249 8 8 9 15 250 8 5 251 8 6 10 12 252 7 5 5 8 253 6 5 8 12 254 7 4 9 17 255 8 7 5 13 256 6 4 6 13 257 6 6 11 14 258 7 7 13 13 259 7 5 7 12 260 7 5 9 13 261 7 5 10 12 262 7 8 11 15 263 8 6 8 13 264 7 6 6 13 Mean SD 7** 6** 8** 13 112 2 - Data are not available due to adult mortality. * Significantly different from the control at p < 0.01 oo Project Number 454-108 Males Feather loss Intestinal tract - area of hyperemia Testes - one or both small (~1.0-1.5 cm) Not remarkable Appendix IX - Table 1 Individual Gross Pathological Observations from a Northern Bobwhite 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 X- XX - - X - - - - X - XXX - - - XX - - - - - - - -X- - - - - - X - XXXX - X - - - - X - - - Females Pens 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Feather loss Foot/leg lesions Back/vent lesions Ceca - round worms found Not remarkable XXa X - X a _X . _ a _ _X . - Xa - - - a - - - - a - - - - - a - -Xa -X- - a - - X- aX- - a - - - - a - - -X - - a - X - a X - XX a XX - - a - Bird found dead during the test. Necropsy findings are presented in Table 2. 00 Project Number 454-108 M ales Feather loss Intestinal tract - area o f hyperemia Ceca - round worms found Testes - one or both sm all (--1.0-1.5 cm) N ot remarkable Appendix IX - Table 2 Individual Gross Pathological Observations from a Northern Bobwhite 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 X- - .X- - -X . X - X- - - . . X _ - X -X - X _XXX . X - - - - - - -X -X -X - - - - . XX _ . _XXXX _ _ _ - - -X - -X - - - - - - -X - F em ales Pens 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Feather loss Head lesions Liver - pale and mottled Abdominal cavity - slight egg yolk peritonitis Intestinal tract - area of hyperemia N ot remarkable - . _ XXXX a X a X . XXX a - -X- - - - a - a - - - - - a - - - -X- -a - a - - - - - a - - - - - - - aXa - - - - - a - - - - - - - a - a X - - - - a XX - - - - - a - a - X - - - a a - Bird found dead during the test. Necropsy findings are presented in Table 2. oaos Project Number 454-108 -87- Project Number 454-108 Appendix X - Table 1 Adult Liver Weights (g) from a Northern Bobwhite Reproduction Study with PFOS Control (0 ppm a.i.) Male Pen Liver Female Liver 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Mean SD 5.026 3.391 4.399 3.975 4.545 5.259 3.984 3.775 4.846 4.259 4.608 4.358 4.831 4.085 4.818 4.667 4.427 0.496 6.806 10.843 - 7.132 8.891 7.999 - 8.925 7.670 8.245 9.807 - 9.693 8.835 9.100 8.654 8.662 1.113 - Data not reported due to mortality. -88- Project Number 454-108 Appendix X - Table 2 Adult Liver Weights (g) from a Northern Bobwhite Reproduction Study with PFOS 10 ppm a.i. Male Pen Liver Female Liver 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Mean SD 4.278 4.838 4.119 4.118 4.994 4.532 3.932 4.436 3.931 4.019 4.819 5.042 4.233 4.085 5.989 4.329 4.481 0.545 9.152 10.659 10.238 9.360 13.377 13.525 8.112 - 11.013 - 12.334 11.429 11.352 9.792 11.155 - 10.884 * 1.596 - Data not reported due to mortality. * Statistically different from the control group at p < 0.01. -89- W ildlife International, Ltd. Project Number 454-108 Appendix XI Histopathology Report EPL -90- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-108 W ILDLIFE INTERNATIONAL, LTD. STUDY NUMBER 454-108 EPL PROJECT NUMBER 212-027 PFOS: A REPRODUCTION STUDY W ITH THE NO RTHERN BOBW HITE PATHOLOGY REPORT Submitted by: Experim ental Pathology Laboratories, Inc. P.O. Box 474 Herndon, VA 20172-0474 (703)471-7060 Submitted to: Wildlife International, Ltd. Easton, MD 21601 December 6,2001 Project Number 454-108 EPL ________________________ EXPERIMENTAL PATHOLOGY LABORATORIES, INC. TABLE OF CONTENTS Page PATHOLOGY SUM M ARY........................................................................................ 1 COMPLIANCE STATEM ENT................................................................................... 7 QUALITY ASSURANCE FINAL CERTIFICATION............................................... SUMMARY INCIDENCE TABLES ADULTS Males..................................................................................................................... 1-1 Females................................................................................................................. I-2 HISTOPATHOLOGY INCIDENCE TABLES M ales..................................................................................................................... H-1 Females................................................................................................................ H-3 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Males..................................................................................................................... HI-1 Females................................................................................................................ W-3 SUMMARY INCIDENCE TABLES OFFSPRING Males..................................................................................................................... IV -1 Females............................................................................................ IV-2 HISTOPATHOLOGY INCIDENCE TABLES Males..................................................................................................................... V-1 Females................................................................................................................ V-2 -92- Project Number 454-108 PATHOLOGY SUMMARY EPL -93EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-108 W ILDLIFE INTERNATIONAL, LTD. STUDY NUMBER 454-108 EPL PROJECT NUMBER 212-027 PFOS: A REPRO DUCTIO N STU D Y W ITH TH E NO RTHERN BOBW HITE PATHOLOGY SUMMARY Light microscopic examination was performed on sections of selected tissues from adult m ale and fem ale Northern bobwhite quail (Colinus virginianus) which were untreated or which received 10 ppm a.i. of the test article (Perfluorooctane Sulfonic Acid, Potassium Salt [P FO S ]) in the feed for approximately 21 weeks. Selected tissues from untreated 14 day-old offspring of the adult bobwhite w ere also exam ined by light microscopy. T h e objective of this study is to evaluate the effects upon adult bobwhite (Colinus virginianus) of dietary exposure to the test substance over at least a 21 -w eek period. The experimental design was as follows: Experimental Group Adults Males Females Offspring0 Males Females Control (0 ppm) 20 20 6 4 10 ppm a.i. 20 19 5 5 "Adults were euthanized after receiving the test substance in the diet for approximately 21 weeks. bOffspring did not receive the test substance in the diet. All offspring were euthanized on Day 14 post-hatching. EPL -94- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-108 Wildlife International, Ltd. Study Number 454-108 MATERIALS AND METHODS At the completion of the study interval, all adult bobwhite w ere euthanized and necropsies w ere performed by Wildlife International, Ltd. Selected offspring were also euthanized at 14 days of age. Tissues were collected by Wildlife International, Ltd. from adults and offspring, fixed in 1 0% neutral buffered formalin, and sent to Experimental Pathology Laboratories, Inc (EPL), where they w ere em bedded in paraffin and m ade into hematoxylin and eosin-stalned sections on glass microscope slides. The following tissues from adult and offspring w ere exam ined 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 exam ined from each bobwhite are listed in the Histopathology Incidence Tables. Microscopic changes w ere graded one to five depending on severity. Nongradable findings (including autolysis such that a diagnostic interpretation could not be rendered) are listed as present (P ). All findings for all anim als are sum m arized by sex, a g e group, and treatm ent group in the S um m ary Incidence Tables, together with the total num ber of anim als in each group for which tissues w ere exam ined. A tabulation of gross observations noted at necropsy with the corresponding microscopic change, if appropriate, is presented in the Correlation of G ross and Microscopic Findings tables. T h e entries in these tables w ere transcribed from the gross necropsy observations provided by Wildlife International. Ltd. EPL -95 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-108 Wildlife International, Ltd. Study Number 454-108 RESULTS Except for three control (334, 342, 352) and two 10 ppm (384, 388) adult fem ales, all adults and offspring survived to the end of their respective study intervals. No effects considered possibly related to test article (P FO S ) administration w ere noted in liver, kidney, brain, and proventriculus of adult and offspring m ales and fem ales, bursa of Fabricius in offspring m ales and fem ales, adipose tissue of adult males and fem ales, ovaries of adult and offspring fem ales; and testes of m ale offspring. Any potential test-article effects on the bursa of Fabricius in adult m ales and fem ales or adipose tissue in m ale and fem ale offspring could not be determined since none (bursa of Fabricius) or only one (adipose tissue) sample was submitted for evaluation. The bursa of Fabricius typically regresses prior to sexual maturity. In addition, any potential test-article effects on the gallbladder from treated adult m ales and fem ales could not be definitively determined due to the extensive autolysis that precluded any accurate diagnosis. There were no apparent test-article related effects noted on the gallbladders from the offspring. Testes of all adult m ales exhibited normal sperm atogenesis, characterized by the presence of numerous maturing or m ature sperm atozoa in the sem iniferous tubules. In one (1) control and seven (7) treated m ales, the testes w ere observed macroscopically as being sm aller in size (< 1.5 cm ) when com pared to other birds. In these birds, sperm atogenesis w as evident microscopically and there w as no definitive difference in the morphology of the testes when compared to their unaffected cohorts. Therefore, no definitive microscopic changes could be associated with the size reduction. However, the reduced size of the testes of these birds was consistent with early post reproductive phase regression, a normal physiological phenomenon. The increased incidence of reduced size of the testes in 10 ppm a.i. m ales when 3- - EPL -96EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-108 Wildlife International, Ltd. Study Number 454-108 compared to controls, suggests that treatm ent may have accelerated the normal post-reproductive phase regression. Testes of all offspring w ere listed as im m ature because they lacked all evidence of spermatogenesis, had small seminiferous tubules with absent or narrow lumens, and germinal epithelium with decreased cell layers (compared to adult testes). This m orphology is consistent with normal physiological immaturity expected in 14 day-old birds. Because of the sm aller size of the seminiferous tubules in the offspring testes, the m elanin pigm ent-bearing cells normally present in the testicular interstitium w ere, in effect, m ore closely packed in a smaller area rather than being widely separated by larger seminiferous tubules (thus the microscopic diagnosis of "pigment concentration" in the testis). Ovaries of adult fem ales exhibited normal oogenesis, characterized by num erous ova in various stages of follicular developm ent. All ovaries of offspring fem ales w ere characterized by numerous oocytes and very small, nonpedunculated follicles com pletely em bedded in a prominent ovarian cortex. This m orphology is consistent with the normal physiological immaturity expected in 14 day-old birds. S everal additional microscopic findings (described below) w ere noted in adult and/or offspring bobwhite quail from control and treated groups. Generally, in both adult and offspring m ales and fem ales, the incidence of these lesions w as variable, with som e lesions occurring at a higher incidence in controls when com pared to treated birds, and other lesions having the opposite distribution. In addition, there w as no significant difference in the m ean severity of these lesions in 10 ppm a.i. treated birds w hen com pared to controls. For these reasons, these findings w ere considered incidental and unrelated to test article (P F O S ) administration. 4- - EPL -97- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-108 Wildlife international, Ltd. Study Number 454-108 M ononuclear cell infiltrates, observed in various tissues, consisted of foci of lymphocytes and plasma cells generally accompanied by a few heterophils as a m inor com ponent. Mineralization in the kidneys of both control and treated adult m ales w as characterized by the presence of basophilic m aterial in the cortex. In most cases this material appeared to be associated with the glomerulus. Vacuolization in the liver w as characterized by the presence of vacuoles within the hepatocellular cytoplasm, which varied from usually small, poorly dem arcated vacuoles, giving a "lace-like" appearance to the hepatocellular cytoplasm, to variably sized, sharply dem arcated, clear vacuoles in the hepatocellular cytoplasm. W hile special stains w ere not employed to identify the m aterial in the vacuoles, the latter morphology w as consistent with fat accumulation. Hepatocellular vacuolization w as observed in the livers of adults and offspring of both sexes. Although there w as a slight increase in the incidence of this change in 10 ppm a.i. treated adult m ales and fem ales, w hen com pared to controls, there w as no significant difference in the m ean severity betw een the groups. Since this finding w as also commonly observed in the livers from control birds, it w as considered to be incidental and not related to test article administration. T h e apparent increased incidence in the treated birds w as considered to be due to biological and/or distributional variability. CONCLUSIONS AND SUMMARY No lesions considered possibly related to test article (P FO S ) administration w ere noted in liver, kidney, brain, and proventriculus of adult and offspring m ales and fem ales, bursa of Fabricius in offspring m ales and fem ales, adipose tissue of adult males and fem ales, ovaries of adult and offspring -5-  EPL -98- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-108 Wildlife International, Ltd. Study Number 454-108 fem ales, and testes of m ale offspring. T h e increase in the num ber of adult m ales in the 10 ppm a.i. treated group with reduced testicular size, not accom panied by any morphological change in sperm atogenesis, suggests that P F O S m ay have accelerated early post-reproductive phase regression, a normal physiological phenom enon. Any potential test article effects on the bursa of Fabricius in adult m ales and fem ales or adipose tissue in m ale and fem ale offspring could not be determ ined since none (bursa of Fabricius) or only one (adipose tissue) sample w as subm itted for evaluation. In addition, any potential test-article effects on the gallbladder from treated adult m ales and females could not be definitively determined due to the extensive autolysis that precluded any accurate diagnosis. T h e fe w other changes in various tissues of adult and/or offspring bobwhite from control and 10 ppm a.i. treated groups w ere considered incidental and unrelated to test article (P FO S ) administration. FAR/lcp FR E D E R IC K A. R U EC K ER , DVM, MS, Diplomate, A C VP V eterinary Patholc Date -6- EPL -99EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-108 COM PLIANCE STATEM ENT Client Name Wildlife International, Ltd. EPL Project Coordinator Dr. Margarita M. Gruebbel Client Study EPL 454-108____________________ Pathologist Dr. Frederick A. Ruecker EPL Project Species Northern Bobwhite___________ Number 212-027 Study Title PFOS: A Reproduction Study with the Northern Bobwhite Test Article Perfluorooctane Sulfonic Acid, Potassium Salt (P FO S ) T h e Histopathology portions of the above-referenced study w ere conducted in compliance with the Good Laboratory Practice regulations of the Environmental Protection Agency as stipulated by 4 0 C FR 160. EPL Project Coordinator ^ 1 , ; x 0 03 -7 - EPL - 100- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-108 QUALITY ASSURANCE FINAL CERTIFICATION Study Title: PFOS: A Reproduction Study with the Northern Bobwhite Client Study: 454-108 EPL Project Coordinator: Dr. Margarita M. Gruebbel EPL Project Number: 212-027 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 10/11,15/01 10/12/01 10/19/01 11/1,2,5/01 11/29/01 1/28/03 10/11,15/01 10/12/01 10/19/01 11/1,2,5/01 11/29/01 1/28/03 Date reported to Study Director/Management. Date of last quarterly facility inspection_____ 1/28/03 11/02 (nJA EPL/Quality Assurance Unit Form No. 6-2 (7/2/99) -8- - 101 - Project Number 454-108 SUM M ARY INCIDENCE TABLES ADULTS -102- Project Number 454-108 454-108 Adult Sacrifice Male Northern Bobwhite SUMMARY INCIDENCE TABLE ADIPOSE TISSUE (NO. EXAMINED) Inflammation, Chronic GROUP CONTROL (20) 3 GROUP 10-ppmai (20) BRAIN (NO. EXAMINED) Inflammation, Meninges, Granulomatous Mineralizing Granuloma, Meninges (20) (20) 12 BURSA OF FABRIC IUS (NO. EXAMINED) GALLBLADDER (NO. EXAMINED) (18) (20) Autolysis Precludes Diagnosis 18 18 KIDNEY (NO. EXAMINED) Infiltrate, Mononuclear Cell Inflammation, Chronic Mineralization (20) (20) 15 24 LIVER (NO. EXAMINED) Degeneration, Hepatocellular, Centrilobular Infiltrate, Mononuclear Cell Necrosis, Hepatocellular Vacuolization, Hepatocellular (20) 13 16 (20) 7 20 PROVENTRICULUS (NO. EXAMINED) Infiltrate, Mononuclear Cell Inflam m ation, Serosa (20) (20) 2 TESTIS (NO. EXAMINED) Infiltrate, Mononuclear Cell Reduced Spermatogenesis (20) (20) 11 1 EPL Experimental Pathology Laboratories, Inc. 1-1 -103- Project Number 454-108 454-108 Adult Sacrifice Female Northern Bobwhite SUMMARY INCIDENCE TABLE ADIPOSE TISSUE (NO. EXAMINED) Inflammation, Chronic GROUP CONTROL (19) 3 GROUP 10-ppmai (16) 3 BRAIN (NO. EXAMINED) Inflammation, Meninges, Granulomatous Mineralizing Granuloma, Meninges (20) (19) 1 BURSA OF FABRIC IUS (NO. EXAMINED) GALLBLADDER (NO. EXAMINED) (19) (19) Autolysis Precludes Diagnosis 18 17 KIDNEY (NO. EXAMINED) Infiltrate, Mononuclear Cell Inflammation, Chronic Mineralization (20) (19) 14 1 3 LIVER (NO. EXAMINED) (20) (18) Degeneration, Hepatocellular, Centrilobular 1 Infiltrate, Mononuclear Cell 4 8 Necrosis, Hepatocellular 12 Vacuolization, Hepatocellular 7 12 OVARY (NO. EXAMINED) PROVENTRICULUS (NO. EXAMINED) Infiltrate, Mononuclear Cell Inflammation, Serosa (20) (20) 2 1 (19) (19) 3 EPL Experimental Pathology Laboratories, Inc. 1-2 - 104- Project Number 454-108 HISTO PATHOLOGY INCIDENCE TABLES ADULTS -105- Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE GROUP E PL Experimental Pathology Laboratories, Inc. Key :X-Not Remarkable NNo Section I-Inccmplete A-Autolys1s l-m1n1mal 2-sl1ght/m1Id 3*moderate 4-moderately severe 5-severe/h1gh P-Present B-Ben1gn M-Hal1gnant m-m1ss1ng one paired organ u-unscheduled sac./death -106 - Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE GROUP 10-ppmai 454-108 Adult Sacrifice Male Northern Bobwhite ADIPOSE TISSUE Inflammation. Chronic A N i M A 33333333333333334444 L 6 7777788888 999990 000 9 1 ,3 5 7 9 1 3 3 7 9 1 3 5 7 9 _1_ 3 J l 7 XXXXXXXXXXXXXXXXXXXX RRAIN Inflammation. Meninges. Granulomatous Mineralizina Granuloma. Meninaes RURSA OF FABRICIUS GAI1Rl ADDFR Autolvsis Precludes Diaanosis KIDNEY Infiltrate. Mononuclear Cell Inflammation. Chronic Minerali zation XXX XX X X X X JL X JL JL X X X JL JL pP N N N N N N N N N N N N N _ !L N N N J L J L J L A A A A A A A A A A A A A A A X JL jL X PPPPPP PPPPPPPPP PP P XX XXX XXX X 1 1 11 1 XXX 1 1 u -L 3 FIVER Deaeneration. Hepatocellular. Centrilobular Infiltrate. Mononuclear Cell Necrosis. Hepatocellular Vacuolization. Hepatocellular 1 1 1 JL JL 1 JL 1 ??? 1 1? 1 12 2 1112 J 2 2 _L PROVENTRICULUS Infiltrate. Mononuclear Cell Inflammation. Serosa XXXXXXXX XXX 1 XXXXXXX -- TESTIS Infiltrate. Mononuclear Cell Reduced Soermatoaenesis Xm Xm Xm Xm Xm Xm xm Xm Xm xm Xm Xm Xm m Xm Xm Xm Xm Xm m 3 E PL 11-2 Experimental Pathology Laboratories, Inc. Key :X-Not Remarkable N-No Section I-Incomplete A-Autolys1s l-m1n1mal 2-sl1ght/m1ld 3-moderate 4-moderately severe 5-severe/h1gh P-Present B-Ben1gn M-Mal1gnant m-m1ss1ng one paired organ u-unscheduled sac./death - 107 - Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTRO L 454-108 Adult Sacrifice Female Northern Bobwhite ADIPOSE TISSUE Inflammation. Chronic A N l M A 3 3 3i 3 3 3 3t 3 3 3 3 3i 3 3 3 3 3 3 3 3 L 33333444445555566666 0 ? 4 8 8 0 ? 4 8 8 o ? 4 8 8 0 ? 4 A . _8_ XXXXXNXXXXXXXX XXX 1 21 BRAIN Inflammation. Meninaes. Granulomatous Mineralizina Granuloma. Meninaes X X X X X X X X X X X X X JL JL JL X X JL X BURSA OF FABRICIUS N N N N N N N N N N N N JL JL JL JL N N JL JL GAI 1Rl ADDFR Autolvsis Precludes Diaanosis A X A A A A N A A A A A X JL X A A A JL X P PPPP PPPPPP PPPP PP P KIDNEY Infiltrate. Mononuclear Cell Inflammation. Chronic Mineralization XXXX X 1 X X X X X X X JL X X X 1 XX LIVER Deqeneration. HeDatocellular. Centrilobular Infiltrate. Mononuclear Cell Necrosis. HeDatocellular Vacuolization. HeDatocellular OVARY XX X XX XX XX 1 ? 1 3? 1 _L X 1 1 11 XXXXXXXXXXXXX XXXXXX X PROVENTRICIJI US Infiltrate. Mononuclear Cell Inflammation. Serosa X XX XXXXXXXXX XXXX X 1 _L 2 E PL 11-3 Experimental Pathology Laboratories, Inc. Key iX-Not Remarkable N-No Section I-Incomplete A-Autolys1s l-m1n1mal 2-sl1ght/m1ld 3-moderate 4-moderately severe 5-severe/h1gh P-Present B-Ben1gn M-Mal1gnant m-m1ss1ng one paired organ u-unscheduled sac./death -108 - Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE GROUP 454-108 Adult Sacrifice Female Northern Bobwhite ADIPOSE TISSUE Inflammation. Chronic A N l M A 3 3 3 3 3 3 3 3i 3 3i 3 3 3 3 3 4 4 4 4 L 77 77788888 9999 900 00 Q ? 4 (i fi 0 ? 4 fi fi 0 ? 4 fi fi ? 4 fi 8 XNXN XNXXXXXXXXXX ?1 1 BRAIN Inflammation. Meninaes. Granulomatous Mineralizina Granuloma. Meninaes X X X X X X X X X X X X X X X X X JL 1 RIIRSA OF FARR I d IJS N N N N N N N N N N N N N N N N N JL JL GAI 1Rl ADDFR Autolvsis Precludes Diaanosis A A A X A A A A A A A A A X A A J L JL J L PPP PP P PPPPPP PPPPP KIDNEY Infiltrate. Mononuclear Cell Inflammation. Chronic Mineralization XX X X XXXX X XX XXX 11 1 1 ? LIVER Deaeneration. Heoatocellular. Cent.ri lobular Infiltrate. Mononuclear Cell Necrosis. Heoatocellular Vacuolization. Heoatocellular XN X X XX 1 1?1 111 1 11 1 4 11 11111 12 3 OVARY XXXXXXXX XXXXX XXXXXX PROVFNTR TCIII IJS Infiltrate. Mononuclear Cell Inflammation. Serosa XXXXXX XXXX X X JL X X X 11 1 E PL 11-4 Experimental Pathology Laboratories, Inc. Key :XNot Remarkable N-No Section I-Incomplete A-Autolysis l-m1n1mal 2-sl1ght/m1Id 3-moderate 4-moderately severe 5*severe/h1gh P-Present B-Benlgn M-Halignant m-m1ss1ng one paired organ u*unscheduled sac./death -109- Project Number 454-108 CORRELATION OF GROSS AND M ICRO SCO PIC FINDINGS ADULTS 454-108 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult S a c rific e Species: N orthern Bobwhite__________________________________________ Sex: Males Group Identification: CONTROL - S a c rific e d A nim al Num ber Client Topography / Site Client Gross Observations M icroscopic Observations 345 TESTIS Right testis approximately 1.5 cm No Correlating Lesion Project Number 454-108 III-l 454-108 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS A dult S a c rific e S p e c ie s : N orth ern B o b w h i t e _______ ______________________________ Sex: Males______G r o u p Id e n tific a tio n : 10-ppmai A n im al Num ber Client Topography / Site Client Gross Observations M icroscopic Observations 371 TESTES Testes 1.25 cm No Correlating Lesion (TESTIS) - Sacrificed 373 TESTES Testes 1.25 cm No Correlating Lesion (TESTIS) 387 TESTIS Right testis 1.0 cm No Correlating Lesion 389 TESTES Testes 1.0 cm No Correlating Lesion (TESTIS) 391 TESTIS Right testis 1.0 cm No Correlating Lesion 393 TESTIS Right testis 1.0 cm No Correlating Lesion 401 TESTIS Right testis 1.0 cm No Correlating Lesion Project Number 454-108 111-2 454-108 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS M u lt Sacrifice Species: N orthern Bobwhite__________________________________________ Sex: Females Group Identification: CONTROL - Unscheduled S a c./D eath A nim al" Number Client Topography / Site Client Gross Observations M icroscopic Observations 342 OVARY Regressing No Correlating Lesion Project Number 454-108 111-3 454-108 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult S a c rific e S p e c ie s : N orthern Bobwhite_______ ________________________________ Sex: Females G r o u p Id e n tific a tio n : 10-ppmai A nim al Number Client Topography / Site Client Gross Observations M icroscopic Observations 378 LIVER Pale, mottled Vacuolization, Hepatocellular - Sacrificed 408 LIVER Pale, mottled Vacuolization, Hepatocellular Project Number 454-108 111-4 454-108 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult S a c rific e Species: N orthern B o b w h i t e _______ ___________________________ Sex: Females Group Identification: 10-ppmai A n im al Num ber Client Topography / Site 384 Client Gross Observations No coronary fat M icroscopic Observations No Comment Required - Unscheduled Sac./D eath OVARY Developing No Correlating Lesion Project Number 454-108 111-5 -115- Project Number 454-108 SUM M ARY INCIDENCE TABLES O FFSPRING - 116- Project Number 454-108 454-108 Offspring Sacrifice Male Northern Bobwhite SUMMARY INCIDENCE TABLE ADIPOSE TISSUE (NO. EXAMINED) BRAIN (NO. EXAMINED) GROUP CONTROL (1) GROUP 10-ppmai (6) (5) BURSA OF FABRIC IUS (NO. EXAMINED) Lymphoid Depletion (5> - (5) GALLBLADDER (NO. EXAMINED) Autolysis Precludes Diagnosis (2) (3) 1 KIDNEY (NO. EXAMINED) (6) (5) LIVER (NO. EXAMINED) Infiltrate, Mononuclear Cell Vacuolization, Hepatocellular (6) 2 3 (5) 1 PROVENTRICULUS (NO. EXAMINED) (6) (5) Infiltrate, Mononuclear Cell 1 TESTIS (NO. EXAMINED) Immature Pigment Concentration (3) (5) 35 15 E PL Experimental Pathology Laboratories, Inc. IV-1 - 117- Project Number 454-108 454-108 Offspring Sacrifice Female Northern Bobwhite SUMMARY INCIDENCE TABLE ADIPOSE TISSUE (NO. EXAMINED) GROUP GROUP CONTROL 10-ppmai BRAIN (NO. EXAMINED) (4) (5) BURSA OF FABRIC IUS (NO. EXAMINED) Lymphoid Depletion (4) (5) 1 GALLBLADDER (NO. EXAMINED) Autolysis Precludes Diagnosis (4) (5) KIDNEY (NO. EXAMINED) (4) (5) LIVER (NO. EXAMINED) Infiltrate, Mononuclear Cell Vacuolization, Hepatocellular (4) (5) 1 OVARY (NO. EXAMINED) Immature (2) (5) 24 PROVENTRICULUS (NO. EXAMINED) (4) (5) Infiltrate, Mononuclear Cell EPL Experimental Pathology Laboratories, Inc. IV-2 - 118- Project Number 454-108 HISTO PATHO LO G Y INCIDENCE TABLES O FFSPR IN G - 119- Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE GROUP GROUP 454-108 Offspring Sacrifice Male Northern Bobwhite ADIPOSE TISSUE A N l M A 222222 L 556789 3 8803 1 NNNX N N BRAIN XX XX X X BURSA OF FABRICIUS LvmDhoid Depletion XXXNX X GALLBLADDER Autolvsis Precludes Diagnosis NXXNNN KIDNEY XXXXXX LIVER Infiltrate. Mononuclear Cell Vacuolization. Hepatocellular XX ?1 11 1 PROVENTRICULUS Infiltrate. Mononuclear Cell XX X XX 1 TESTIS Immature Pigment Concentration NN Nw PPp p 55555 23456 ?3 5? 8 NNNNN XXXXX XXXXX NNXXA P XXXXX XXXX 1 XXX X X ni PPPPP PPPPP 1 EPL V-l Experimental Pathology Laboratories, Inc. Key :X-Not Remarkable N-No Section I-Incomplete A-Autolys1$ l-m1nimal 2*sl1ght/m1ld 3-moderate 4-moderately severe 5-severe/high P-Present B-Ben1gn H*Hal1gnant mm1ss1ng one paired organ u-unscheduled sac./death -120 - Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE GROUP GROUP 454-108 - Offspring Sacrifice Female Northern Bobwhite ADIPOSE TISSUE A N 1 M A 2222 L 4577 8Q3 7 NNNN BRAIN XXXX BURSA OF FABRICIUS Lvmphoid Depletion X XX ? GALLBLADDER Autolvsis Precludes Diaanosis XXXX KIDNEY XXXX LIVER Infiltrate. Mononuclear Cell Vacuolization. HeDatocellular XXXX OVARY Immature NN pp PROVENTRICULUS Infiltrate. Mononuclear.Cel 1 XXXX 55555 12 3 5 6 7 58 7 1 N N NNN XXXXX XXXXX XXXXX XXXXX XXXX 1 X p PPP X XXX X ! E PL V -2 ________________________________________________________ Experimental Pathology Laboratories, Inc. Key :X-Not Remarkable N-No Section I*Incomplete A-Autolys1s / hiQ;;s11i',t/"1,d 3`" derate 4'moderately severe P-Present B-Ben1gn M=MaHgnant m*missing one paired organ u-unscheduled sac./death W ildlife International, Ltd. -121 - Project Number 454-108 Appendix XIIA The Analysis of PFOS in Red Blood Cells and Sera Collected through Week 15 W ildlife International, L td - 122- Project Number 454-108 Exygen Study No.: 023-066 Table XXVI. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 5 Sponsor ID 454-108-361-Wk5 454-108-361-Wk5* 454-108-362-Wk5 454-108-362-Wk5* 454- 108-363-Wk5 454-108-363-Wk5* 454-108-364-Wk5 454-108-364-Wk5* 454-108-365-Wk5 454-108-365-Wk5* 454-108-366-Wk5 454-108-366-Wk5* 454-108-368-Wk5 454- 108-368-Wk5* Centre ID 0107077 0107077 0107078 0107078 0107079 0107079 0107080 0107080 0107081 0107081 0107082 0107082 0107083 0107083 Set Number 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A 051801A Extraction Analysis ' Date Date 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ue/mL) NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ 0.00292 0.00264 NQ NQ 0.000440 0.000993 * 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 |ig/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, 0.0001 jrg/mL) was used to calculate the average and standard deviation. Exygen Research Page 40 of 98 - 123 - W ildlife International, Ltd. Project Number 454-108 Exygen Study No.: 023-066 Table XXVII. Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 5 Sponsor ID 454-108-401-Wk5 454-108-401-Wk5* 454-108-402-Wk5 454-108-402-Wk5* 454-108-403-Wk5 454-108-403-Wk5* 454-108-404-Wk5 454-108-404-Wk5* 454-108-405-Wk5 454-108-405-Wk5* 454-108-406-Wk5 454-108-406-Wk5* 454-108-407-Wk5 454-108-407-Wk5* 454-108-408-Wk5 454-108-408-Wk5* Centre ID 0107084 0107084 0107085 0107085 0107086 0107086 0107087 0107087 0107088 0107088 0107089 0107089 0107090 0107090 0107091 0107091 Set Number 051801DR 051801DR 051801DRD 051801DRD 051801DR 051801DR 0518Q1DR 051801DR 051801DRD 051801DRD 051801DRD 051801DRD 051801DRD 051801DRD 051801DRD 051801DRD Extraction Analysis Date Date 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/22/01 5/18/01 5/22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/18/01 5/21-22/01 5/21-22/01 5/18/01 5/22/01 5/18/01 5/18/01 5/22/01 5/22/01 5/18/01 5/22/01 5/18/01 5/22/01 5/18/01 5/22/01 5/18/01 5/22/01 5/18/01 5/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) 113 107 353 351 30.5 30.4 26.9 25.8 415 447 1050 1040 495 501 451 471 369 324 * Duplicate injection LOQ = 0.01 |ig/m L for red blood cells and serum Exygen Research Page 41 o f 98 - 124- W ildlife International, Ltd. Project Number 454-108 Exygen Study No.: 023-066 Table XXVIII. Summary of PFOS in Quail Red Blood Cell Samples at 50 ppm a.i. for Week 5 Sponsor ID 454-108-441-Wk5 454-108-441-Wk5* 454-108-442-Wk5 454-108-442-Wk5* Centre ID 0107092 0107092 0107093 0107093 Set Number 052201CD 052201 CD 052201CD 052201CD Extraction Analysis Date Date 5/22/01 5/22/01 5/22/01 5/22/01 5/24/01 5/24/01 5/24/01 5/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ue/mL) 166 163 108 103 135 34 Table XXIX. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 10 Sponsor ID 454-108-361 -Wk 10 454-108-361-WklO* 454-108-362-Wkl0 454- 108-362-Wkl0* 454-108-363-WklO 454-108-363-Wkl0* 454-108-364-WklO 454-108-364-WklO* 454-108-365-Wkl0 454-108-365-Wkl0* 454-108-366-Wkl0 454- 108-366-Wkl0* 454-108-367-Wkl0 454-108-367-Wk10* 454-108-368-Wkl0 454-108-368-WklO* Centre ID 0107100 0107100 0107101 0107101 0107102 0107102 0107103 0107103 0107104 0107104 0107105 0107105 0107106 0107106 0107107 0107107 * Duplicate injection Set Number 051801B 051801B 051801B 051801B 05180IB 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 05180IB Extraction Analysis Date Date 5/18/01 5/19/01 5/18/01 5/19/01 5/18/01 5/19/01 5/18/01 5/19/01 5/18/01 5/19/01 5/18/01 5/18/01 5/19/01 5/19/01 5/18/01 5/19/01 5/18/01 5/19/01 5/18/01 5/19/01 5/18/01 5/18/01 5/19/01 5/19/01 5/18/01 5/19/01 5/18/01 5/19/01 5/18/01 5/18/01 5/19/01 5/19/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ue/mL) NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NO 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)) 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/mL) was used to calculate the average and standard deviation. Exygen Research Page 42 of 98 -125 - W ildlife International, Ltd, Project Number 454-108 Exygen Study No.: 023-066 Table XXX.Sununary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 10 Sponsor ID 454-108-401-Wkl0 454-108-401-Wkl0* 454-108-402-WklO 454-108-402-WklO* 454-108-403-Wkl0 454-108-403-Wkl0* 454-108-404-WklO 454-108-404-Wkl0* 454-108-405-Wkl0 454-108-405-Wkl0* 454-108-406-WklO 454-108-406-WklO* 454-108-407-WklO 454-108-407-Wkl0* 454-108-408-Wk10 454-108-408-Wkl0* Centre ID 0107108 0107108 0107109 0107109 0107110 0107110 0107111 0107111 0107112 0107112 0107113 0107113 0107114 0107114 0107115 0107115 * Duplicate injection Set Number 051801DED 051801DRD 051801DRD 051801DRD 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR 051801DR Extraction Analysis Date Date 5/18/01 5/22/01 5/18/01 5/22/01 5/18/01 5/22/01 5/18/01 5/22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/18/01 5/18/01 5/18/01 5/21-22/01 5/21-22/01 5/21-22/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/18/01 5/21-22/01 5/21-22/01 5/18/01 5/18/01 5/21-22/01 5/21-22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (iie/mL) 672 671 203 190 23.0 20.7 19.0 21.2 18.0 20.0 48.9 50.5 22.8 22.3 30.4 30.2 129 220 LOQ = 0.01 (ig/mL for red blood cells and serum Exygen Research Page 43 of 98 W ildlife International, Ltd - 126- Project Number 454-108 Exygen Study No.: 023-066 Table XXXI. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 15 Sponsor ID 454-108-361-Wkl5 454-108-361-Wkl5* 454-108-362-Wkl5 454-l08-362-Wkl5* 454-108-363-Wkl5 454-108-363-Wkl5* 454- 108-364-Wkl5 454-108-364-Wkl 5* 454-108-365-Wkl5 454-108-365-Wkl5* 454-108-366-Wkl5 454-108-366-Wkl5* 454-108-367-Wk 15 454-108-367-Wkl5* 454-108-368-Wkl5 454-108-368-Wkl5* 454-108-333-Wkl5 454-108-333-Wkl5* Centre ID 0107116 0107116 0107117 0107117 0107118 0107118 0107119 0107119 0107120 0107120 0107121 0107121 0107122 0107122 0107123 0107123 0107124 0107124 Set Number 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A Extraction Analysis Date Date 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (iig/mL) NQ NQ NQ NQ 0.00226 0.00328 0.00127 NQ 0.00705 0.00391 NQ NQ 0.00795 0.00816 NQ NQ 0.00130 NQ 0.00198 0.00304 * Duplicate injection ND = No peak detected 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 |ig/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, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 44 of 98 W ildlife International, Ltd Project Number 454-108 Exygen Study No.: 023-066 Table XXXII. Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 15 Sponsor ID 454-108-401-Wkl5 454-108-401-Wkl5* 454-108-402-Wkl5 454-108-402-Wkl5* 454-108-403-Wkl5 454-108-403-Wkl5* 454-108-404-Wkl5 454-108-404-Wkl5* 454-108-405-Wkl5 454-108-405-Wkl5* 454-108-406-Wkl5 454-108-406-Wkl 5 * 454-108-407-Wk15 454-108-407-Wkl5 454-108-408-Wkl5 454-108-408-Wkl 5* 454-108-383-Wkl5 454-108-383-Wkl5* Centre ID 0107125 0107125 0107126 0107126 0107127 0107127 0107128 0107128 0107129 0107129 0107130 0107130 0107131 0107131 0107132 0107132 0107133 0107133 Set Number 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052201CD 052201CD 052101AD 052101AD Extraction Analysis Date Date 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/21/01 5/21/01 5/22-23/01 5/22-23/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/24/01 5/21/01 5/21/01 5/21/01 5/24/01 5/22-23/01 5/22-23/01 AVERAGE: STANDARD DEVIATION: Analyte Found (int/mL) 72.2 69.1 2.36 1.65 21.2 18.8 1.30 1.22 15.8 17.9 3.61 3.23 13.3 9.60 1.24 1.55 56.7 54.1 20.3 24.8 Table XXXIII. Summary of PFOS in Quail Red Blood Cell Samples at 50 ppm a.i. for TERM Sponsor ID 454-108-409-TERM 454-108-409-TERM* 454-108-410-TERM 454-108-410-TERM* Centre ID 0107134 0107134 0107135 0107135 Set Number 052201CD 052201CD 052201CD 052201CD Extraction Analysis Date Date 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ua/mL) 361 356 106 114 234 144 * Duplicate injection LOQ = 0.01 |ig/m L for red blood cells and serum Exygen Research Page 45 of 98 W ildlife International, Ltd, - 128 - Project Number 454-108 Exygen Study No.: 023-066 Table XXXIV. Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 5 Sponsor ID 454-108-361-Wk5 454-108-361-Wk5* 454-108-362-Wk5 454-108-362-Wk5* 454-108-363-Wk5 454-108-363-Wk5* 454-108-364-Wk5 454-108-364-Wk5* 454-108-366-Wk5 454-108-366-Wk5* 454-108-368-Wk5 454-108-368-Wk5* Centre ID 0107154 0107154 0107155 0107155 0107156 0107156 0107157 0107157 0107159 0107159 0107160 0107160 * Duplicate injection Set Number 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR 072301 AR 072301AR 072301AR 072301AR 072301AR Extraction Analysis Date Date 7/23/01 7/25/01 7/23/01 7/25/01 7/23/01 7/25/01 7/23/01 7/25/01 7/23/01 7/25/01 7/23/01 7/25/01 7/23/01 7/25/01 7/23/01 7/25/01 7/23/01 7/25/01 7/23/01 7/25/01 7/23/01 7/25/01 7/23/01 7/25/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) 0.0282 0.0295 0.0149 0.0134 0.0198 0.0166 0.00510 0.00535 0.0135 0.0120 0.0138 0.0156 0.0X56 0.00746 LOQ = 0.01 (tg/mL for red blood cells and serum Exygen Research Page 46 of 98 'W ildlife International, Ltd, - 129- Project Number 454-108 Exygen Study No.: 023-066 Table XXXV. Summary of PFOS in Quail Serum Samples at 10 ppm a.i. for Week 5 Sponsor ID 454-108-402-Wk5 454-l08-402-Wk5* 454-108-403-Wk5 454-108-403-Wk5* 454-108-404-Wk5 454-108-404-Wk5* 454-108-405-V/k5 454-108-405-Wk5* 454-108-406-Wk5 454- 108-406-Wk5* 454-108-407-Wk5 454-108-407-Wk5* 454-108-408-Wk5 454-108-408-Wk5* Centre ID 0107162 0107162 0107163 0107163 0107164 0107164 0107165 0107165 0107166 0107166 0107167 0107167 0107168 0107168 Set Number 072301 AD 072301AD 072301AD 072301 AD 072301AD 072301 AD 072301AD 072301AD 072301AD 072301AD 072301AD 072301AD 072301AD 072301AD Extraction Analysis Date Date 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 7/23/01 7/26/01 AVERAGE: STANDARD DEVIATION: 0r*0"i Analyte Found (ue/mL) 45.8 45.6 115 128 104 98.3 129 106 107 42.4 36.3 77.6 84.4 89.8 35.0 Table XXXVI. Summary of PFOS in Quail Serum Samples at 50 ppm a.i. for Week 5 Sponsor 1D 454-108-441-Wk5 454-108-441-Wk5* 454-108-442-Wk5 454-108-442-Wk5 * Centre ID 0107092 0107092 0107093 0107093 * Duplicate injection Set Number 072301AD 072301AD 072301 AD 072301 AD Extraction Analysis Date Date 7/23/01 7/23/01 7/26/01 7/26/01 7/23/01 7/23/01 7/26/01 7/26/01 AVERAGE: STANDARD DEVIATION: Analyte Found (iig/mL) 551 562 529 520 541 19.4 LOQ = 0.01 |ig/m L for red blood cells and serum Exygen Research Page 47 of 98 W ildlife International, Ltd - 130 - Project Number 454-108 Exygen Study No.: 023-066 Table XXXVII. Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 10 Sponsor ID 454-108-361-Wkl0 454-108-361-Wkl 0* 454-108-362-WklO 454-108-362-Wkl 0* 454- 108-363-Wkl0 454-108-363-WklO* 454-108-364-WklO 454-108-364-Wkl0* 454-108-365-WklO 454-108-365-WklO* 454-108-366-Wk10 454-108-366-WklO* 454-108-367-Wkl0 454-108-367-WklO* 454-108-368-Wkl0 454- 108-368-WklO* Centre ID 0107177 0107177 0107178 0107178 0107179 0107179 0107180 0107180 0107181 0107181 0107182 0107182 0107183 0107183 0107184 0107184 * Duplicate injection Set Number 072301B 072301B 072301B 07230IB 072301B 072301B 072301B 072301B 072301B 072301B 072301B 072301B 072301B 072301B 072301B 072301B Extraction Analysis Date Date 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 AVERAGE: STANDARD DEVIATION: Analyte 0.00483 0.00489 0.00295 0.00249 0.00467 0.00521 0.00390 0.00375 0.00271 0.00292 0.00439 0.00437 0.00563 0.00542 0.00537 0.00526 0.00430 0.00105 LOQ = 0.01 (ig/mL for red blood cells and serum Exygen Research Page 48 of 98 - 131 - W ildlife International, Ltd. Project Number 454-108 Exygen Study No.: 023-066 Table XXXVIII. Summary of PFOS in Quail Serum Samples at 10 ppm a.i. for Week 10 Sponsor ID 454- 108-401-Wkl0 454-108-401-Wkl0* 454-108-402-WklO 454-108-402-WklO* 454-108-403-Wkl0 454-108-403-WklO* 454-108-404-Wk10 454-108-404-WklO* 454-108-405-WklO 454-108-405-Wkl0* 454-108-407-WklO 454-108-407-Wk10* 454-108-408-WklO 454-108-408-WklO* Centre ID 0107185 0107185 0107186 0107186 0107187 0107187 0107188 0107188 0107189 0107189 0107190 0107190 0107191 0107191 Set Number 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301D 072301D 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD Extraction Analysis Date Date 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/23/01 7/27-28/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/23/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/27-28/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 AVERAGE: STANDARD DEVIATION: Analyte Found (iie/mL) 213 207 53.6 53.7 148 177 19.4 20.3 157 147 105 110 97.0 93.7 114 63.4 * Duplicate injection LOQ = 0.01 |ig/m L for red blood cells and serum Exygen Research Page 49 of 98 W ildlife International, Ltd -132- Project Number 454-108 Exygen Study No.: 023-066 Table XXXIX. Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 15 Sponsor ID 454-108-361-Wkl5 454-108-361-Wkl5* 454-108-362-Wk15 454-108-362-Wk15* 454- 108-363-Wkl5 454-108-363-Wkl5* 454-108-364-Wkl5 454-108-364-Wkl5* 454-108-365-Wkl5 454-108-365-Wk 15* 454-108-366-Wkl5 454-108-366-Wkl5* 454- 108-367-Wkl5 454-108-367-WU5* 454-108-368-Wkl5 454-108-368-Wkl5* 454-108-333-Wkl5 454-108-333-Wkl5* Centre ID 0107192 0107192 0107193 0107193 0107194 0107194 0107195 0107195 0107196 0107196 0107197 0107197 0107198 0107198 0107199 0107199 0107200 0107200 Set Number 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C 072301C Extraction Analysis Date Date 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/23/01 7/24-25/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/23/01 7/23/01 7/24-25/01 7/24-25/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/23/01 7/23/01 7/24-25/01 7/24-25/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ng/mL) NQ NQ NQ NQ 0.00154 0.00160 NQ NQ NQ NQ NQ NQ 0.00227 0.00249 0.00253 0.00255 0.00162 0.00160 0.000928 0.00105 * 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)) LOQ = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value o f the lowest calibration standard, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 50 of 98 - 133 - W ildlife International, Ltd. Project Number 454-108 Exygen Study No.: 023-066 Table XL. Summary of PFOS in Quail Serum Samples at 10 ppm a.i. for Week 15 Sponsor ID 454-108-401-Wkl5 454-108-401-Wkl5* 454-108-402-Wkl5 454-108-402-Wkl5* 454-108-403-Wkl5 454-108-403-Wkl5* 454-108-404-Wkl5 454-108-404-Wk15* 454-108-405-Wkl5 454-108-405-Wkl5* 454-108-406-Wkl5 454-108-406-Wkl5* 454-108-407-Wkl5 454-108-407-Wkl5* 454-108-408-Wk15 454- 108-408-Wkl5* 454-108-383-Wkl5 454-108-383-Wkl5* Centre ID 0107201 0107201 0107202 0107202 0107203 0107203 0107204 0107204 0107205 0107205 0107206 0107206 0107207 0107207 0107208 0107208 0107209 0107209 Set Number 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301DD 072301 DD 072301DD 072301 DD 072301DD Extraction Analysis Date Date 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 7/27-28/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ue/mL) 181 180 8.21 8.45 246 239 11.1 10.7 141 156 7.04 6.98 107 114 10.2 8.91 131 143 95.0 86.6 Table XLI. Summary of PFOS in Quail Serum Samples at 50 ppm a.i. for TERM Sponsor ID 454-108-410-TERM 454-108-410-TERM* Centre ID 0107211 0107211 * Duplicate injection Set Number 072301DD 072301DD Extraction Analysis Date Date 7/23/01 7/23/01 7/27-28/01 7/27-28/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ue/mL) 476 514 495 26.9 LOQ = 0.01 ng/m L for red blood cells and serum Exygen Research Page 51 o f 98 W ildlife International, Ltd. -134- Project Number 454-108 Appendix XIIB The Analysis of PFOS in Adult and Juvenile Liver and Sera Samples Collected at Termination W ildlife International, Ltd, -135 - Project Number 454-108 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study w ith the Nothcm Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite S ummary T h e concentration oi the C t F ,7S 0 3' anion of perfluorooctane sulfonate, potassium salt (P F O S , C sFtrSO j'K ' ) w as determined in the liver and sera of adult bobwhite quail and their offspring in both a control group and a 10 ppm, dosed group from Wildlife feeding study #454-108. The following tables sum m arize the C eF ,7S 0 3' concentrations found in the liver and sera of study sam ples. Th es e results only relate to the sam ples tested. M atrix-extracted P F O S standards were used to establish the H PLC -M S/M S instrument calibration curve for CFi?SO j. Concentrations of C 3F 17S 0 3' are reported after correction for the purity and the potassium contribution to the m ass of the PFO S 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 th e Dixon Q-Test. Table 1. Summary Table, CSF17SO3' Concentration In Sera and Liver S -v i''..ir\c C.'Vti Male Control Group Nupmi n OF S i f :.A S a m i'U .s 19 A,I I-;:.;. C;FvSO ` CoNC. SCFU R .'in c je o f n e o u it j I----------------------------------- NU' f L i .'f R S am plfs NA <400 ngftnL (19 of 19) 20 Av' K.'-U: CuFiySO j" C o n c. L ive r SOingTg' R.mciC of He^ulls <50.2 ng/g (19 of 20)" 209 ng/g (1 of 20)* Female Control Group 17 <400ngfrnL(ieof17} NA ! 2110ngfrnL(1of17) 20 <502 no's* <502 ng (20of 20)* Male 10ppm Group 20 141000 ngftiA. 94400 - 226000 ng/mL 20 88500 ng/g* 50100-147000 ng/g* 18Female 10ppm Group 9990ngAnl 4710-31900 ngfrnL 20 4900 ngAg* 3290-7550 ng/g* Male Offspring Control Group Female Offspring Control Group ! ! 6 4 Male Offspring 10ppm Group 5 Female Offspring 10ppm Group 5 NA <400ng*nL(6of6) NA <400ngfrnL(4cf4) 12600 ng/mL 8220-19200 ng/mL 12400ngftnL 7640-18500 ngfrnL 6 <20.0 ng^g* ! <20.0 ng/g (6 of 6)* 4 <20.0 ng/g* <20.0 ng/g (4 of 4)* 5 7350 ng/g* 4940-13700 ng/g* 5 7180 ng/g* 4590-13900 ng/g* For Individual sample values see Tables 10 to 13 and Attachment B. Accuracy: Sample results are accurate to 100% +/- 30%. Sera QC samples met the acceptance criteria for the method. * liver QC samples did not meet tie %of QC at each level must be within 75%-125% criteria for the method. Al liver QC samples (3) spiked at 10 ppb were >125% recovery. All other liver QC samples (6) were within criteria, therefore the second QC criteria was mat In that 2/3 of all QC were within 75% 125%. 3M Environmental laboratory 3M EnvironmentalLaboratory Page 7 of 226 Page 7 W ildlife International, Ltd. -136- Project Number 454-108 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 2. Summary Table, CgFiySO*' Concentration In Sera and Liver w ith Outliers Removed S a m p lin g E v ln t N [.till H nr Si ha S amples A iA-,. C,. r ; , 3 0 J Cone, Slra R ange of R e s iilU N i .' Ih l H o rL klr S amples A riiA C .L C .F irS O -, CONC. LiVLH FV .ngc o f R e s u lts MalaControl Group 19' 19 <50.2 ng/g** <50.2 ng/g (19 of 19)** Female Control Group 16 <400ngfrnL <400ngfrnL(16of 16) 20" Mala 10ppm Group 20* 2 tr * * Female IQppm Group 17 8700 ngM . 4710-14700ng/mL 20* * MaleOffspring Control Group Female Offspring ControlGroup 6* 4* * 6* 4* MatoOffspring 10ppm Croup Female Offspring lOppm Group 5* 5* ; *i I * * 4 5760 ng/g" 4940 - 6520 ng^** 4 5490 ngW* 4590-7030 n ^ g - * No outliers from this data sat. Refer toTable 1 tor results. For individual sample values see Tables 10 to 13 and Attachment B. Accuracy: Sample results are accurate to 100% +/- 30%. Sera QC samples met the acceptance criteria for the method. *' Liver QC samples dkl ro t m eet the 14 ol QC at each level must be within 75% -123% criteria for the method. All Svet OC samples (3) spiked at 10 ppb were >125% recovery. All other UverQC samples (6) were within criteria, therefore the second QC criteria was met In that 2/3 ol all QC were within 75% -125%. Purpose T h e purpose of this analytical phase of the study is to quantify C sF,7S 0 3' in the liver and sera of both controland dosed Northern BobwhiteQuailand theiroffspring. R eference S ubstances Table 3. Reference Substances R eference S ubstance PFOS Source Expiration Dale Storage Conditions 3M Laboratory Identification Number Physical Description Purity 3M Internal 08/31/2006 Frozen-20C+/-10"C SD-018, Lo1#217 I While Crystalline Powder 86.9% PFDA Oakwood Products, Inc. 12/01/2010 Frozen-20"C +/-10C SD-036 White Solid 98% PFDA n nonadecafluorodecanolc acid, or Periluorodacanolc add. CioFnOiH and Is used solely to monitor for gross Instrument failure and not as an Internal standard lor quantitation. 3M Environmental Laboratory 3M EnvironmentalLaboratory Paga 8 c l 226 Page 8 - 137- W ildlife International, Ltd. Project Number 454-108 Report E01-1245 Report EG1-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study w lti the Northern Bobwhite Table 10. CsFitSOj'Data Summary, Fmale Quail Sera (Adult and Offspring) vM - .. - ' 1 : ; . 1' ' I: u " '' C ,F wS O , . LU:iLU Lu.MF-'l F ( U l/L ' , o r S era) . E01 -1245-28444 454-108-330, Oppm Iemale, adult, 01-31 -02 . E01-1245-28446 454-108-332, Oppm Iem ale, adult, 01-31-02 . E01-1245-28449 454-108-336, Oppm Iem ale, adult, 01-31-02 . E01-1245-28451 454-105338, Oppm fem ale, adult, 01-31-02 . E01-1245-28453 454-108-340, Oppm female, adult, 01-31 -02 . E01-1245-28466 464-105344, Oppm female, adult, 01-31-02 , E01-1245-28458 454-108-346, Oppm fem ale, ad u t, 02-01-02 . E01-124528460 454-108-348, Oppm fem ale, ad u l, 02-01-02 . E01-124528462 454-108-350, Oppm female, ad u t, 02-01-02 . EOI-124528464 454-105352, Oppm fem ale, ad u t, 02-01-02 , EOI-124528466 454-108-354, Oppm fem ale, ad u t, 0201-02 , EOI-124528468 454-105356, Oppm fem ale, ad u t, 02-01-02 , E01-124528470 454-105358, Oppm fem ale, ad u t, 02-01-02 . E01-124528472 454-105360, Oppm female, ad u t, 02-01-02 . E01-124528474 454-108362, Oppm fem ale, ad u t, 02-01-02 . E01-124528477 454-108366, Oppm female, ad u t, 02-01-02 . E01-1245284781454-108368, Oppm fem ale, adut, 0231-02 E01-124528481 4 5 4 -108370,10ppm fem ale, adult, 0 2 3 1 3 2 E01-1245-284831454-108372,10ppm fem ale, acMt, 0 2 3 1 3 2 E01-1245-2848Si 4 5 4 -108374,10ppm fem ale, adut, 0 2 3 1 3 2 EO1-1245-20487' 454-1 0 5 3 7 6 ,10ppm fem ale, adut, 0 2 3 1 3 2 E01-1245-284681454-108378,10ppm fem ale, adut, 0 2 3 1 3 2 E01-1245-28491 454-1 0 5 3 8 0 ,10ppm fem ale,adut, 0 2 3 1 3 2 0.4 0.4 0.05 0.05 0.3 03 0.3 03 02 02 02 02 0.1 0.1 0.3 0.3 0.4 OA 0.4 0.4 1 0.3 0.3 0 .3 0.3 0.3 0.3 0.2 0.2 0.2 02 0.3 0.3 0.3 0.4 0.2 0.3 0.1 0.1 oo 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 <400 1 2050 1 2180 1 <400 1 <400 1 <400 1 <400 1I <400 1 <400 1 > 9170 1 9140 1 8400 1 9280 1 4980 1 5790 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 i 2110 <400 <400 <400 3M Environmental Laboratory 3M EnvironmentalLaboratory Page 15 01226 Page 15 -138- W ildlife International, Ltd. Project Number 454-108 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study Ml the Northern Bobwhite .r t . ' ' 11 ' c,H , -j 1 ^j 1 vVJP. FS E01-1245-28493 454-108-382, lOppm fem ale, adult, 02-01-02 E01-1245-2B496 454-108-386,10ppm fem ale, adult, 02-01-02 E01-124828498 454-108-388,10ppm female, adult, 02-01-02 E01-1245-28500 454-108390,10ppm female, adult, 02-01-02 E01-1245-28502 454-108392,10ppm female, adult, 02-01-02 E0M 24S-28504 454-108394,10ppm fem ale, adult, 02-01-02 E01-1245-28506 454-108396,10ppm fem ale, adult, 02-01-02 E01-1245-28506 454-108398,10ppm female, adult, 02-01-02 E01-1245-28511 454-108402,10ppm fem ale, adult, 02-01-02 E01-1245-28513 454-108404,10ppm fem ale, adult, 02-01-02 E01-1245-28515 454-108406,10ppm fem ale, adult, 02-01-02 E01-1245-28517 454-108408,10ppm fem ale, adult, 02-01-02 . E01-1245-28518 454-108246, Oppm fem ale, offspring, 02-01-02 . E01-1245-28521 454-108259, Oppm female, offspring, 02-01-02 . E01-1245*28524 454-108273, Oppm female, offspring, 02-01-02 . E01-1245-28525 454-108277, Oppm fem ale, offspring. 02-01-02 . E01-1245-28528 454-108-517,10ppm fem ale, offspring, 02-01-02 , E01-1245-2B530 454-108525,10ppm fem ale, offspring, 02-01-02 . E01-1245-28532 454-108538,10ppm female, offspring, 02-01-02 . E01-1245-28535 454-108557,10ppm fem ale, offspring. 02-01-02 t E01-1245-28536 454-108561,10ppm female, offspring, 0201-02 0.3 0.4 03 0.2 0.3 0.3 0.09 0.1 0.4 0.1 02 0.2 0.1 0.1 0.1 0.1 0.08 0.06 02 02 0.02 0.02 0.03 0.03 0.08 0.08 0.06 0.06 0.05 0.05 10 31900* 1 14700 1 11600 1 6310 1 9400 11400 1 4710 1 I 10500 1 6720 ; 1 9060 i 1 8980 : 1 7840 1 <400 1! 1 <400 <400 1 <400 1 <400 1 <400 1 <400 1 <400 <400 <400 <400 <400 1 18500 17600 16600 1 12800 12500 1 12100 1 14600 13400 1 12200 8330 7900 1 7460 i 1 11500 10800 1 10100 * There are two results reported for these samples. Both values are included and averaged in the `Average of Dup Sam ple" column. These values were rejected based on the results of the Dixon's Q-Test, 90% confidence interval, for the rejection of outliers. Accuracy: Sam ple results are accurate to 100% +/- 30% . Sera Q C sam ples m et the acceptance criteria for the method. 3M Environmental Laboratory 3M Environmental Laboratory Page 16 of 226 Page 16 W ildlife International, Ltd. -139- Project Number 454-108 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhitc PFOS: K Reproduction Study with the Northern Bobwhite Table 11. CgF^SCy Data S w im ary, Male Quail Sera (Adult and Offspring) ! Jl 'L l: . E01-124828443 454-108-329, Oppm n o ie, aduli, 01-31-02 . EOI-1245-28445 454-108-331, Oppm male, adult, 01-31-02 , EOI-1245-28447 454-106-333, Oppm male, adii!, 01-31-02 . E01-1245-28448 454-108-335, Oppm m ale, adii), 01-31-02 . E01-1245-28450 454-108-337, Oppm m ale, aduli, 01-31-02 . EOI-1245-28452 454-108-339, Oppm m ale, aduli, 01-31-02 , E01-1245-284S4 454-108-341, Oppm male, aduli, 01-31-02 . EOI-1245-28455 454-108-343, Oppm m ala, aduli, 01-31-02 . E01-1245-28457 454-108*345, oppm m ale, aduli, 01-31-02 . EOI-1245-28459 454-108-347, Oppm m ale, aduli, 02-01-02 . EOI-1245-28461 454-108-349, Oppm male, aduli, 02-01-02 . E01-1245-28465 454-108-353, Oppm male, aduli, 02-01-02 , E01-1245-28467 454-108-355, Oppm m ala, aduK, 02-01-02 . E01-1245-28469 454-108-357, Oppm male, aduli. 02-01-02 . E01-1245-2B471 454-108359. Oppm m ale, adult, 02-01-02 . E01-1245-28473 454-108361, Oppm m ale, adult, 02-01-02 . E01-1245-28475 454-108363, Oppm m ale, adult, 02-01-02 , E01-1245-28476 454-108-365, Oppm m ala, adult, 02-01-02 . E01-1245-28478 454-108-367, Oppm m ale, aduli, 02-01-02 (.Li 0.2 0.2 0.4 0.4 0.4 0.4 0.2 0.2 0.1 0.1 0.2 02 02 0.2 0.1 0.1 0.2 0J2 0.2 0.2 0.02 0.02 0.3 0.3 0.2 05 0.07 0.07 0.2 0.2 0.2 0.2 0.1 0.1 0.4 0.4 0.1 0.1 C,f:,-LO, -, S...7P1 ;.t : : il of S era) 1 -'i O l. " . 1 1 1 1 1 1 1 1 1 1 i 1 1 1 i 1 1 1 1 i 1 1 1 i 1! 1 i i 1 1 1 1 1 1 1 1 1 1 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 3M Environmental Laboratory 3M Environmental Laboratory Page 17 of 228 Page 17 W ildlife International, Ltd. - 140- Project Number 454-108 Report E01-1245 Report 01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study Mi the Northern BotmMte \L 1 E01-1245-28480 454-108-369,10ppio male, adult, d100,02-01-02 , E01-1245-28482 454-108-371,10ppio male, adult, d100,02-01-02 . E01-1245-28484 454-108-373,10ppio m ale, adult, d100,02-01-02 . E01-1245-28486 454-108-375,10ppm m ale, adult, d100,02-01-02 . E01-1245-28488 454-108-377,10ppm male, aduR, d100,02-01-02 . E01-1245-28490 454-108-379,10ppm male, adult, d100,02-01-02 . E01-1245-28492 454-108-381,10ppm male, adult. d100,02-01-02 . E01-1245-28494 454-108-383, lOppm m ale, adult, d100,02-01-02 ,, E01-1245-28495 454-108-385,10ppm m ala, adult, d100,02-01-02 , E01-1245-28497 454-1084387, lOppm m ale, adult, d100,02-01-02 . E01-1245-28499 454-108-389, lOppm m ale, adult, dlOO,02-01-02 . E01-1245-28501 454-108-391, lOppm m ale, adult, dlOO,02-01-02 . E01-1245-28503 454-108-393,10ppm m ale, adult, dlOO,02-01-02 . E01-1245-28506 464-108-306, lOppm m ale, Adult, d l00,02-01-02 , E01-1245-28507 454-105397,10ppm male, adult, d100,02-01-02 , E01-1245-28509 454-108-399,10ppm m ale, adult, d100,02-01-02 . E01-1245-28510 454-108-401,10ppm male, adult, d100,02-01-02 . EOI-1245-28512 454-105403, lOppm male, adult, d100,02-01-02 . E01-1245-2B514 454-108-405,10ppm male, adult, d100,02-01-02 . E01-1245-28516 454-105407, lOppm m ale, adult, d100,02-01-02 OO 02 02 02 02 0.1 0.1 03 03 0.07 0.07 0.1 0.1 0.1 0.1 03 03 0.1 0.1 03 0.3 0.1 0.1 0.06 0.06 0.2 0.2 0.3 0.3 0.1 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.1 0.1 . cc rr,r.ss oo . O r S :'--I, 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 107000 117000 181000 179000 144000 156000 138000 138000 94400 96100 150000 166000 141000 149000 147000 156000 119000 123000 174000 196000 129000 136000 141000 147000 160000 165000 127000 136000 99600 107000 138000 159000 203000 226000 127000 138000 108000 117000 99200 112000 ~ ~V.1f L l :'j 112000 180000 150000 138000 95300 158000 145000 151000 121000 185000 132000 144000 162000 131000 ; 103000 148000 215000 132000 112000 105000 3M Environmental Laboratory 3M Environmental Laboratory Page 18 of 226 Page 18 - 141 - W ildlife International, Ltd. Project Number 454-108 Report E01-1245 Report E01-1245 PFOS: Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite 1 ' . E01-1245-28519 454-108-253, Oppm m ale, offspring, 02-01-02 i ! , E01-1245-28520 454-108-256, Oppm m ale, offspring, 02-01-02 . E01-1245-28522 454-108-266, Oppm m ate, offspring, 02-01-02 i, . E01-1245-28523 454-108-270, Oppm m ala, offspring, 02-01-02 . E01-1245-28526 454-108-283, Oppm male, offspring, 02-01-02 . E01-1245-28527 454-108-291, Oppm m ale, offspring, 02-01-02 f 0.02 0.02 0.07 0.07 02 02 0.02 0.02 0.05 0.05 PP 88 '" h ' ` 1 1 1 1 1 1 1 1 1 1 1 1 C , F , . S O | -, -L o r S l R\) 1).. -V.i- <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 <400 1 <400 <400 <400 <400 <400 E01-1245-28529 454-108-522,10ppm male, offspring, 02-01-02 0.07 10 19300 . E01-1245-28531 454-108-533,10ppm m ale, offspring, 02-01-02 , E01 -1245-28533 454-108-545, lOppm m ale, offspring, 02-01-02 0.07 0.07 0.04 i 0.04 1 1 1 1 9310 8220 15500 13600 . E01-1245-2B534 454-108-552, lOppm m ale, offspring, 02-01-02 0.07 1 13600 0.07 1 11500 , EOI-1245-28537 454-108-568,10ppm m ale, offspring, 02-01-02 0.1 0.1 1 12100 1 10400 There are two results repotted for these samples. Both values are Included and averaged in the "Average of Dup Sample* column. 19300 8760 14600 12500 11200 Accuracy; Sam ple results are accurate to 100% +/- 30% . Sera Q C samples met the acceptance criteria for the method. 3M Environmental Laboratory 3M EnvironmentalLaboratory Page 19 01226 Page 19 -142- W ildlife International, Ltd, Project Number 454-108 Report E01-1245 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern BobwhKe Table 12. CeFwSOi'Data Summary, Female Quail Liver (AduH and Offspring) N tr-:- ; E01-1342-30321 0 ppm a i. 330 F Liver E01-1342-30339 0 ppm a l .332 F Liver E01-1342-30357 0 ppm a i. 334 F Liver E01-1342-30375 0 ppm a i. 336 F Liver E01-1342-30393 0 ppm a i. 338 F Liver E01-1342-30411 0 ppm a i. 340 F Liver E01-1342-30429 0 ppm a l. 342 F Uver E01-1342-30447 0 ppm a.i. 344 F Liver E01-1342-30465 0 ppm a.1.346 F Liver E01-1342-30483 0 ppm a.1.348 F Liver E01-1342-30501 0 ppm a.!. 350 F Liver E01-1342-30519 0 ppm a.1.352 F Liver E01-1342-30537 0 ppm a.!. 354 F Liver E01 -1342-30555 0 ppm a i. 356 F Liver E01-1342-30573 0 ppm a i. 358 F Liver E01-1342-30591 0 ppm a l. 360 F Liver E01-1342-30609 0 ppm a.i. 362 F Liver E01-1342-30627 0 ppm a i. 364 F Liver E01-1342-30645 0 ppm a.1.366 F Liver E01-1342-30663 0 ppm a i. 368 F Liver E01-1342-30681 E01-1342-30699 E01-1342-30717 E01-1342-30735 E01-1342-30753 E01-1342-30771 E01-1342-30789 EOI-1342-30520 EO1-1342-3O038 E01-1342-30856 E01-1342-30874 E01-1342-30892 E01-1342-30910 E01-1342-30928 E01-1342-30946 E01-1342-30964 E01-1342-30982 E01-1342-31000 E01-1342-31018 E01-1342-31036 10 ppm a l. 370 F Liver 10 ppm a i. 372 F Liver 10 ppm a i. 374 F Liver 10 ppm a i. 376 F Liver 10 ppm a.i. 378 F Liver 10 ppm a.i. 380 F Liver 10 ppm a i. 382 F Liver 10 ppm a i. 384 F Liver 10 ppm a i. 386 F Uver 10 ppm a i. 388 F Liver 10 ppm a i. 390 F Liver 10 ppm a i. 392 F Liver 10 ppm a i. 394 F Liver 10 ppm a i. 396 F Liver 10 ppm a l 398 F Uver 10 ppm a i. 400 F Uver 10 ppm a i. 402 F Liver 10 ppm a i. 404 F Uver 10 ppm a i. 406 F Liver 10 ppm a i. 408 F Liver -r ' / " ii - ; I '1 - L'.. (fi : I L. 1.0749 1 1.0469 1 1.0232 1 1.0188 1.0543 1.0182 1.0904 1.0087 1.0998 1.1038 1.0237 1.1509 1.0578 1.0312 1.1031 1.0586 1.0424 1.0364 1.0577 1.0920 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1.0868 1.0748 1.0790 1.1036 1.0021 ; 1.0825 1.0122 0.6864 1.1215 1.0878 1.0078 1.0060 1.0238 1.0370 1.0240 1.0181 1.0876 1.0173 1.0047 1.0044 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 :v c F .sOj \ S-il II i i (i.rvr, o= l i',FR) <502* <50.2* <502* <502* <502* <502* <502* <502* <502* <502* <50.2* <502* <502* <502* <50.2* <50.2* <502* <502* <502* <502* 5920* 4430* 5430* 3980* 3750* 3990* 3290* 4190* 7550* 3490* 4870* 5940* 5030* 4910* 4400* 5040* 5900* 5420* 5870* 4560* 3M Environmental Laboratory 3M Environmental Laboratory Page 20 of 220 Page 20 - 143- W ildlife International, Ltd. Project Number 454-108 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite c - - c r ho E 01-136531184 E01-1365-31204 E01-1365-31219 E01-1365-31224 E 0 1 -1 3 6 5 3 1 2 4 0 E01-1365-31250 E01-1365-31260 E01-136531275 E01-1365-31280 0 ppm a.i. 246 F Liver, Offspring 0 ppm a.i. 259 F Liver, Offspring 0 ppm e.i. 273 F Liver, Offspring 0 ppm a J. 277 F Liver, Offspring 10 ppm a j. 517 F Liver, Offspring 10 ppm a j. 525 F Uver, Offspring 10 ppm a j. 538 F Liver, Offspring 10 ppm a j. 557 F Liver, Offspring 1 0 p p m aj.5 6 1 F Liver, Offspring 0.7302 0.4434 0.5004 0.4326 0.6570 0.5720 0.5211 0.4654 0.9417 ('.G'GCr Ll.CR) 1 <20.0* <20.0* 1 <20.0* 1 <20.0* 1 5690* 1 7030* 10 13900.* 1 4680* 1 4590* e These values were rejected based on the results of the Dixon's Q-Test, 90% confidence interval, for the rejection of outliers. Accuracy; Sample results are accurate to 100% + /- 30% . * Liver Q C sam ples did not m eet the Vi of Q C at each level must be within 7 5 % 1 25 % criteria for the method. AH liver Q C samples (3 ) spiked at 10 ppb w ere > 1 2 5% recovery. All other liver Q C sam ples (6) w ere within criteria, therefore the second Q C criteria w as m et in that 2 /3 of all Q C were within 7 5 % -1 2 5 % . 3M Environmental Laboratory 3M Environmental Laboratory Page 21 of 226 Page 21 -144- W ildlife International, Ltd. Project Number 454-108 Report E01-1245 Report 01*1245 PFOS: A Reproduction Study with the Nothera Bobwhite PFOS: A Reproduction Study with dia Northern Bobwhite Table 13. CgFuSOj' Data Summary, Male Quail Liver (Adult and Offspring) ,V,J o .'.in f I I-- v. - E01-1342-30312 0 ppm a.1.329 M Liver B01-1342-30330 0 ppm a i. 3 3 1 M Liver E01-1342*30348 0 ppm aL 333 M Liver E01-1342-30366 0 ppm a l. 335 M Liver E01-1342-30384 0 ppm a l. 337 M Liver E01-1342-30402 0 ppm a i. 339 M Liver E01-1342-30420 O ppm ai.341 M Liver E01-1342-3O438 0 ppm a i. 343 M Liver E01-1342-30456 0 ppm a i. 345 M Liver E01-1342-30474 0 ppm a i. 347 M Liver E01-1342-30492 0 ppm a l. 349 M Liver E01-1342-30510 0 ppm a l. 351 M Liver E01-1342-30S28 0 ppm a i. 353 M Liver E01-1342-30546 0 ppm a i. 355 M Liver E01-1342-30564 0 ppm a i. 357 M Liver E01-1342-30582 0 ppm a l. 359 M Liver EQ1-1342-30600 0 ppm a i. 3 6 1 M Liver E01-1342-30618 0 ppm a i. 363 M Liver E01-1342-30636 0 ppm a l. 365 M Liver E01-1342-30654 0 ppm a i. 367 M Liver E01-1342-30672 10 ppm a i. 369 M Liver E01-1342-30690 10 ppm a i. 371 M Liver E01-1342-30708 10 ppm a.i. 373 M Liver E01-1342-30726 10 ppm a i. 375 M Liver E01-1342-30744 10 ppm a i. 377 M Liver E01-1342-30762 10 ppm aJ. 379 M Liver E01-1342-30780 1 0 p p m ai.3 8 1 M Liver E01-1342-30798 10 ppm a l. 383 M Liver E01-1342-30629 10 ppm a i. 386 M Liver E01-1342-30647 10 ppm a i. 387 M Liver E01-1342-30865 10 ppm a l. 389 M Liver E01-1342-30883 10 ppm a i. 391 M Liver E01-1342-30901 10 ppm a i. 393 M Liver E01-1342-30919 10 ppm a i. 395 M Liver E01-1342-30937 10 ppm aL 397 M Liver E01-1342-30955 10 ppm aL 399 M Liver E01-1342-30973 10 ppm aL 401 M Liver E01-1342-30991 10 ppm aL 403 M Liver E01-1342-31009 10 ppm aL 405 M Liver E01-1342-31027 10 ppm a l 407 M Liver (O) 1,0050 1.0530 1.0338 1.0062 1.0269 0.0888 1.0235 1.0090 1.1046 1.0586 1.0358 1.0583 1.0183 1.0899 1.0382 1.0941 1.0084 1.0816 1.0916 1.0327 1.0010 1.0856 1.0276 1.1008 1.0236 1.0312 1.0501 1.0110 1.0678 1.0315 1.0310 1.0947 1.0834 1.0489 1.0309 1.0276 1.0961 1.0019 1.0336 1.0966 c; : C F, S O , r. LI l; -..ri 11 1 1 1 1 1 1 1 1 1 1 1 1 i1 1 1 1 1 1 1 ! (IJ G /G GF Li /GF} <50.2* <502* 209* <502* <502* <50.2* <50.2* <505* <502* <502* <502* <505* <50.2* <502* <502* <502* <502* <502* <502* <50.2* . 10 78600* 10 78500* 10 88300* 10 70000* 10 80300* 10 90700* 10 80000* 10 73500* 10 83400* 10 79300* 10 70300* 10 70400* 10 86800* 10 65100* 10 50100* 10 63000* 100 147000* 100 146000* 100 138000* 100 132000* 3M Environmental Laboratory 3M Environmental Laboratory Page 22 of 226 Page 22 W ildlife International, Ltd. -145 - / Project Number 454-108 Report E01-1245 Raport E01-1245 PFOS: A Reproduction Study with die Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite ..C l ' l l . N . [ ! i E01-136S31194 E01-138531199 E01-1365-31209 01*136531214 E 0 1 -1 3 6531229 E 0 1 -1 3 6 5 3 1 2 3 4 E01-1365-31245 E 0 1 -1 3 6 5 3 1 2 5 5 E 0 1 -1 3 6 5 3 1 2 6 5 E01-1365-31270 E01-1365-31285 0 ppm a l. 253 M Liver, Offspring 0 ppm a.i. 256 MLiver, Offspring 0 ppm a i. 266 MLiver, Offspring 0 ppm a i. 270 MLiver, Offspring 0 ppm a i. 283 M Liver, Offspring O pp m aL 291 M Liver, Offspring 10 ppm a i. 522 M Liver, Offspring 10 ppm a l. 533 M Liver, Offspring 10 ppm a i. 545 M Liver, Offspring 10 ppm a i. 552 M Liver, Offspring 10 ppm a i. 668 M Liver, Offspring C `) 0.3806 0.5947 0.2673 0.5900 0.6666 0.6072 0.7325 0.6903 0.4624 0.5353 0.7581 : t j !I ) 1 1 i1 1 1 1 10 1 1 1 1 C. F /SO, :i L vl-*) <20.0* <20.0* <203* <20.0* <20.0* <20.0* 1 3 7 0 0 .* 4940* 5580* 5990* 6520* These values were rejected based on the results of the Dixon's Q-Test, 9 0% confidence interval, for the rejection of outliers. Attachment B contains data summary tables. Accuracy: Sam ple results are accurate to 100% +/- 30% . * Liver Q C sam ples did not m eet the Vi of Q C at each level must be within 7 5 % - 1 2 5 % criteria for the method. All liver Q C sam ples (3) spiked at 10 ppb w ere > 1 2 5 % recovery. All other liver Q C sam ples (6) w ere within criteria, therefore the second Q C criteria was m et in that 2 /3 of all Q C w ere within 7 5 % -1 2 5 % .*ol S t a t e m e n t o f C o n c l u s io n S era results from the m ale control group portion of this study showed no detectable levels of C 3F ,7S 0 3` in 19 of 19 quail (<4 0 0 ng/mL). T h e sera results from the fem ale control group portion of this study showed no detectable levels of C tF ^ S O j in 16 of 16 birds (< 4 0 0 ng/m L) (Table 15). Analytical results for twenty sera samples from the 10ppm dosed m ala group showed an average ol 1 4 1 0 0 0 ng/mL C ,F 17S 0 3' (range 9 44 0 0 - 2 26 0 0 0 ng/mL, Table 14). Analytical results for seventeen sera samples from the 10ppm dosed female group showed an average of 8670 ng/mL C 9F 17S O i (range 4 7 1 0 - 1 4 7 0 0 ng/mL, Table 15). S era results from the m ale offspring control group showed no detectable levels of C aF 17S 03' In 6 of 6 birds (< 4 0 0 ng/m L). Th e sera results from the fem ale offspring control group portion of this study showed no detectable levels of C eF ,7S 0 3'in 4 of 4 birds (<4 0 0 ng/mL, Table 14). Analytical results for five sera sam ples from the 10ppm dosed m ale offspring group showed an average of 12600 ng/m L (range 8 22 0 -1 9 2 0 0 ng/mL, Table 14). Analytical results for five sera sam ples from the 10ppm dosed fem ale offspring group showed an average of 1 24 0 0 ng/m L (range 7 6 4 0 18500 ng/mL, Table 14). Liver results from the m ale control group showed no detectable levels of C ,F ,7S 0 3' in 19 of 19 birds (<50.2 ng/g, T a b le 15) and quantifiable concentrations of C 3F 17S 0 3' in 1 of 2 0 (2 09 ng/g. Table 14). T h e liver results from the fem ale control group showed no detectable levels of C 8F 17S 0 3' In 2 0 of 2 0 birds (<50.2 ng/g, Table 14). Analytical results for twenty liver sam ples from the 10ppm dosed m ale group showed an average of 885 0 0 ng/g C aF l7S O a' (range 5 0 1 0 0 147000 ng/g, Table 14). Analytical results for twenty liver samples from the 10ppm dosed lem ale group showed an average of 4 90 0 ng/g C eF ,7S 0 3' (range 3 29 0 - 7 5 5 0 ng/g. T a b le 14). 3M Environmental Laboratory 3M EnvironmentalLaboratory Paga 23 at 226 Page 23 - 146 - W ildlife International, Ltd. Project Number 454-108 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Liver results trom the male offspring control group portion of this study showed no detectable levels of CbF17S 0 3' in 6 of 6 birds (<50.2 ng/g, Table 14). The liver results from the female offspring control group portion of this study showed no detectable levels of CBF17S 0 3' In 4 of 4 birds (<20.0 ng/g, Table 14). Analytical results for four liver samples from the 10ppm dosed male offspring group showed an average of 5760 ng/g (range 4940 - 6520 ng/g, Table 15). Analytical results for four liver samples from the 10ppm dosed female offspring group showed an average of 5490 ng/g (range 4590 - 7030 ng/g, Table 15). Table 14. Summary Table, C8Fi 7S03' Concentration in Sera and Liver S ampling E ven t Male Control Group N umber of S ema Samples Average CGF17S O 3 Conc. S era Range of Results ------------------------- 1 19 NA . <400 ng/mL (19of19) N umber of L iver S ample s A ver age C eFv S O / Conc. Liver 20 <50.2 ng/g* Range of Results <50.2 ng/g (19 of 20)* 209 ng/g (1 of 20)* Female Control Group I 17 NA <400 ng/mL (16 of 17) 20 <50.2 ng/g* , <50.2 ng/g (20 of 20)* 2110 ng/mL (1 of 17) Male 10ppm Group 20 141000 ng/mL 94400 - 226000 nmL 20 I 88500 ng/g* ; 50100-147000 ng/g* Female tOppm Group Male Offspring Control Group 18 9990 ng/mL I 6: NA 4710 - 31900 n^nL <400 ng/mL (6 of 6) 20 4900 ng/g* 3290-7550 ng/g* 6 <20.0 ng/g* <20.0 ng/g (6 of 6)* Female Offspring : Control Group 4 NA <400 ng/mL (4 of 4) 4 <20.0 ng'g* <20.0 ng/g {4 of 4)* Male Offspring . 10ppm Group , Female Offspring 10ppm Group 5 5 12600 ng/mL 8220-19200 ngfrnL 12400 ng/mL 7640-18500 ngAnL 5 7350 ngrfg* 4940 -13700 ng/g* 5 7180 ng/g* 4590 -13900 ng/g* For individual sample values see Tables 10 to 13 and Attachment B. Accuracy: Sample results are accurate to 100% +/ 30%. Sera QC samples met the acceptance criteria for the method. Liver QC samples did not meet the V4 of QC at each level must be within 75% * 125% criteria for the melhod. Ail liver QC samples (3) spiked at 10 ppb were >125% recovery. All other liver QC samples (6) were within criteria, therefore the second QC criteria was met in that 2/3 of all QC were within 75% -125%. 3M Environmental Laboratory 3M Environmental Laboratory Page 24 of 226 Page 24 W ildlife International, Ltd. - 147- Project Number 454-108 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 15. SummaryTable, C8F,7S03` Concentration in Sera and Liver with Outliers Removed S am plin g E vent N umber up S fh a Samples A verage C0F 17SO3 Cono. S era Mala Control Group Female Control Group 19* 16 <400 rxmL Male 10ppm Group 20* Female 10ppm Group Male Offspring Control Group Female Offspring Control Group Male Offspring 10ppm Group Female Offspring 10ppm Group 17 ; 8700 n^mL 6' 4* 5* : 5* Range of Resulls Nur/BER OF L iveh Samplcs A verage CgFuS O j C o nc. L iver 19 <50.2 ng/g" <400 ngW . (16 of 16) 20* 20* * 4710'14700 ng/mL 20* * 6* 4* 4 5760 ng/g" 4 5490 ng/g** Range of Results <50.2 ng (19 of 19)** * 4940 *6520 ng/g** 4590 * 7030 ng/g** * No outliers from this data set. Refer to Table 1 for results. For individual sample values see Tables 10 to 13 and Attachment B. Accuracy: Sample results are accurate to 100% +/- 30%. Sera QC samples met the acceptance criteria for die method. ** Liver QC samples did not meet the Vof QC at each level must be within 75% - 125% criteria for the method. All liver QC samples (3) spiked at 10 ppb were >125% recovery. Alt other liver QC samples (6) were within criteria, therefore the second QC criteria was met In that 2/3 ol all QC were within 75% -125%. S tatistical M e t h o d s Statistical methods were limited to calculating means, standard deviations. % coefficient of variation and the Dixons Q-Test. The Dixons Q-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 which is divided by the difference of the highest and lowest value in the group of values. A critical Q-value is determined based on the number ot 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 Data Quality The measurement of accuracy available at this time is the quality control and matrix spike results. For sera data, the accuracy (the observed amount in the QC and MS samples, divided by the amount spiked, expressed as a percent) was found to range from 74.1% to 126%; while precision, expressed as the standard deviation of the recoveries, was found to range from 2.14% to 9.56%. For liver data, the accuracy was found to range from 72.6% to 141%; while precision was found to range from 2.11 % to 5.51 %. 3M Environmental Laboratory 3M Environmental Laboratory Page 25 of 226 Page 25 Reproductive Performance by Pen Appendix XIII - Table 1 Egga Laid / Maximum Laid (%) from a Northern Bobwhite Reproduction Study with PFOS Eggs Replicate Laid 0 PPM A. I. Max. Laid Arcsin % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 55 63 87 69.12 27 63 43 40.89 - -- - 38 63 60 ,50.95 23 63 37 37.17 32 63 51 45.45 - -- - 56 63 89 70.53 47 63 75 59.74 50 63 79 62.98 54 63 86 67.79 - -- - S3 63 84 66.52 54 63 86 67.79 62 63 98 82.76 54 63 86 67.79 605 819 47 63 74 60.73 12 0 20 13.29 Eggs Laid 10 PPM A..1 . Max. Laid Arcsin % Trans. 34 63 54 47.28 46 63 73 58.70 43 63 68 55.71 42 63 67 54.74 47 63 75 59.74 53 63 84 66.52 2 1 63 33 35.26 - -- - 54 63 86 67.79 - -- - 44 63 70 56.69 44 63 70 56.69 63 63 100 90.00 41 63 65 53.78 60 63 95 77.40 - -- - 592 819 46 63 72 60.02 1 1 0 17 13.50 Eggs Laid 50 PPM A.I. Max. Laid Arcsin % Trans. . - -- -- -- -- -- -- -- -- "- -- --- -- -- -- _ - -- __ _ - - _ Eggs Laid 150 PPM A.I. Max. Laid Arcsin % Trans. - -- -- -- -- -- _- -- -- -- -- - -- -- - -- - -- -- .. - -- __ - - - - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0 .0 5 ). Project Number 454-108 Appendix XIII - Figure la Eggs Laid from a Northern Bobuhite Reproduction Study uith PFOS - Control 70 v4*o. NUMBER / PEN Project Number 454-108 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Pen N u m b e r Eggs Laid * Data are not available due to adult m ortality. NUMBER / PEN Appendix XIII - Figure lb Eggs Laid from a Northern Bobuhite Reproduction Study uith PFOS - 10 PPM A.I. 70 * 217 218 219 220 221 222 223 224 225 226 227 228 228 230 231 232 ___ P e n N u m b e r B i Eggs Laid sf( Data are not available due to adult m ortality. o Project Number 454-108 Reproductive Performance by Pen Appendix XIII - Table 2 Eggs Cracked / Eggs Laid (%) from a Northern Bobwhite Reproduction Study with PFOS 0 PPM A. I. Eqqs Eggs Replicate Crack Laid Arcsin % Trans. 10 20 3- 42 50 60 7- 80 92 10 1 11 1 12 - 13 1 14 0 15 16 16 0 55 0 0 .0 0 27 0 0 .0 0 -- - 38 5 13.26 23 0 0 .0 0 32 0 0 .0 0 -- - 56 0 0 .0 0 47 4 11.90 50 2 8.13 54 2 7.82 -- - 53 2 7.90 54 0 0 .0 0 62 26 30.53 54 0 0 .0 0 Total Mean SD 23 605 2 47 4 12 3 6.12 7 8.91 10 PPM A. I. Eggs Eggs Crack Laid Arcsin % Trans. 0 34 1 46 0 43 0 42 0 47 0 53 0 21 -0 54 -0 44 0 44 0 63 0 41 2 60 -- 3 592 0 46 1 11 0 0.00 2 8.48 0 0.00 0 0.00 0 0.00 0 0.00 0 0.00 -- 0 0.00 -- 0 0.00 0 0.00 0 0.00 0 0.00 3 10.52 -- 0 1.46 1 3.59 50 PPM A.I. Eggs Eggs Crack Laid Arcsin % Trans. .. - -- -- -- - '- -- -- -- -- -- -- - -- -- -_ - -- -- _ - -- -- _ - - - _ - - 150 PPM A.I. Eqgs Eggs Crack Laid Arcsin % Trans. ._ - -- -- -- -- - -- - -- -- -- -- -- - -- -- -- -- -- __ - -- -- - - - - - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0 .0 5 ). Project Number 454-108 I Appendix XIII - Figure 2a Eggs Cracked / Eggs Laid from a Northern Bobuhite Reproduction Study uith PFOS - Control 70 Data are not available due to adult mortality. Project Number 454-108 Appendix XIII - Figure 2b Eggs Cracked Eggs Laid from a Northern Bobuhite Reproduction Study uith PFOS - 10 PPM A.I. LO/Jl NUMBER / PEN Project Number 454-108 217 218 220 221 222 223 224 225 2 2 6 227 228 Pen N u mb e r B l Eggs Laid EE3 Eggs Cracked Data are not available due to adult mortality. 229 231 232 Reproductive Performance by Pen Appendix XIII - Table 3 Viable Embryos / Eggs Set (%) from a Northern Bobwhite Reproduction Study with PFOS 0 PPM A. I. Viable Eggs Replicate Embr. Set Arcsin % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 47 50 94 75.82 12 23 52 46.25 - -- - 30 31 97 79.65 2 1 2 1 100 90.00 28 28 100 90.00 - -- - 51 51 100 90.00 39 40 97 80.90 43 43 100 90.00 47 49 96 78.34 - -- - 47 47 100 90.00 43 49 88 69.52 38 40 95 77.08 48 49 98 81.79 494 521 38 40 94 79.95 1 2 1 1 13 12.19 10 PPM A. I. Viable Eggs Embr. Set Arcsin % Trans. 30 39 39 38 35 45 17 46 .39 27 55 17 43 - 30 100 90.00 40 97 80.90 39 100 90.00 38 100 90.00 42 83 65.91 48 94 75.52 19 89 71.07 -- - 48 96 78.22 -- - 39 100 90.00 37 73 58.68 57 96 79.20 36 47 43.41 53 81 64.25 -* - 470 526 36 40 89 75.17 1 1 10 15 14.26 50 PPM A.I. Viable Eggs Embr. Set Arcsin % Trans. _ __ - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- _ - __ - -- -- - 150 PPM A.I. Visible Eggs Embr. Set Arcsin % Trans. .. - -_- - - - -- -- -- -- -- -- -- -- -- -- -- -- -- __ - -- -- _ - - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0 .0 5 ). Project Number 454-108 Appendix XIII - Figure 3a Uiable Embryos / Eggs Set from a Northern Bobuhite Reproduction Study uith PFOS - Control 60 Ui NUMBER PEN Project Number 454-108 Date are not available due to adult mortality. 213 214 215 216 NUMBER PEN Appendix XIII - Figure 3b Uiable Embryos / Eggs Set from a Northern Bobuhite Reproduction Study uith PFOS - 10 PPM ft.I. Hii Eggs Set i-- I Uiable Embryos s|c Data are not available due to adult mortality. Lo/\i Project Number 454-108 Reproductive Performance by Pen Appendix XIII - Table 4 Live 3 -Week Embryos / Viable Embryos (%) from a Northern Bobwhite Reproduction Study with PFOS 0 PPM A. I . Live Viable Arcsin Replicate 3-Week Embr. % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 46 47 98 81.61 1 2 12 100 90.00 - -- - 30 30 100 90.00 2 1 2 1 100 90.00 28 28 100 90.00 - -- - 51 51 100 90.00 38 39 97 80.79 43 43 100 90.00 47 47 100 90.00 - -- - 46 47 98 81.61 43 43 100 90.00 37 38 97 80.66 48 48 100 90.00 490 494 38 38 99 87.28 12 12 1 4.25 10 PPM A..1 . Live Viable Arcsin 3-Week Embr. % Trans. 29 30 97 79.48 39 39 100 90.00 39 39 100 90.00 38 38 100 90.00 34 35 97 80.27 45 45 100 90.00 16 17 94 75.96 - -- - 46 46 100 90.00 - -- - 39 39 100 90.00 27 27 100 90.00 5 5 5 5 100 90.00 16 17 94 75.96 43 4 3 100 90.00 " -- - 466 470 36 36 99 86.28 1 1 1 1 2 5.91 50 PPM A. I . Live Viable Arcsin 3-Week Embr. % Trans. __ - -- -- -- -- -- - -- - -- -- -- -- -- - -- -- -- *- __ - -- -- - - - - - 150 PPM A.I. Live Viable Arcsin 3 -Week Embr. % Trans. . - -- -- -- -- -- -- -- -- --- -- - -- -- -- -- __ - -- _- - - - - - '- - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0 .0 5 ). Project Number 454-108 NUMBER / PEN Appendix XIII - Figure 4a Live 3-Ueek Embryos / Uiable Embryos from a Northern Bobuhite Reproduction Study uith PFOS - Control o so 485353530253232323535348484890485323232348 40 30 20 10 0 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 215 Pen Nu mb e r S Uiable Embryos i-- Live 3-Ueek Embryos $ Data are not available due to adult mortality. oo Project Number 454-108 Appendix XIII - Figure 4b Live 3-Ueek Embryos / Uiable Embryos from a Northern Bobuhite Reproduction Study uith PFOS - 10 PPM A.I. 60 VO NUMBER / PEN Project Number 454-108 ^ data are not available due to adult mortality. Reproductive Performance by Pen Appendix XIII - Table 5 Hatchlings / Live 3 -Week Embryos (%) from a Northern Bobwhite Reproduction Study with PFOS 0 PPM A. I. Number Live Arcsin Replicate Hatch. 3-Week % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 44 46 96 77.96 12 12 100 90.00 - -- - 30 30 100 90.00 20 21 95 77.40 28 28 100 90.00 - -- - 47 51 92 73.74 35 38 92 73.68 42 43 98 81.23 47 47 100 90.00 - -- - 45 46 98 81.52 42 43 98 81.23 35 37 95 76.56 46 48 96 78.22 473 490 36 38 97 81.66 1 1 12 3 6.29 10 PPM A. I. Number Live Arcsin Hatch. 3-Week % Trans. 20 29 69 56.15 36 39 92 73.90 38 39 97 80.79 32 38 84 66.59 31 34 91 72.72 39 45 87 68.58 13 16 81 64.34 - -- - 42 46 91 72.85 - -- - 39 39 100 90.00 27 27 100 90.00 5 5 5 5 100 90.00 16 16 100 90.00 26 43 60 51.04 - *- - 414 466 32 36 89 74.38 12 1 1 13 13.21 50 PPM A. X . Number Live Arcsin Hatch. 3-Week % Trains. _. - -- -- -- -- -- - -- - -- -- -- -- - -- -- -- -* -- _ - -- -- . - - - " - - 150 PPM A.I. Number Live Arcsin Hatch. 3-Week % Trans. . .. - -- -- -- -- - -- -- - -- -- -- -- - -- -- -- -- -* __ - -- -- . - - - - - - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0 .0 5 ). Project Number 454-108 Appendix XIII - Figure 5a Hatchlings / Live 3-Ueek Embryos from a Northern Bobuhite Reproduction Study with PFOS - Control 0 4853534848535348532353235353535353 Os NUMBER / PEN Project Number 454-108 -r 201 202 203 204 205 20 20? 208 209 210 211 212 213 214 215 21 ___ Pen Number B H Live 3-Ueek Embryos EE3 Hatchlings 3c Data are not available due to adult mortality. NUMBER / PEN Appendix XIII - Figure 5b Hatchlings / Live 3-Ueek Embryos from a Northern Bobuhite Reproduction Study uith PFOS - 10 PPM A.I. 222 223 224 225 PenNumber H B Live 3-Ueek Embryos E 3 Hatchlings Data are not available due to adult mortality. OKN> Project Number 454-108 Reproductive Performance by Pen Appendix XIII - Table 6 1 4 -Day Old Survivors / Hatchlings (%) from a Northern Bobwhite Reproduction Study with PFOS 0 PPM A. I. 14-Day Number Arcsin Replicate Old Hatch. % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 43 44 98 81.33 12 12 100 90.00 - -- - 30 30 100 90.00 19 20 95 77.08 27 28 96 79.11 - -- - 46 47 98 81.61 33 35 94 76.17 40 42 95 77.40 47 47 100 90.00 - -- - 45 45 100 90.00 40 42 95 77.40 35 35 100 90.00 40 46 87 68.83 457 473 35 36 97 82.22 1 1 1 1 4 7.09 10 PPM A..1 . 14-Day Number Arcsin Old Hatch. % Trans. 20 20 100 90.00 36 36 100 90.00 29 38 76 60.88 30 32 94 75.52 30 31 97 79.65 38 39 97 80.79 10 13 77 61.29 - -- - 37 42 88 69.82 - -- - 37 39 95 76.91 27 27 100 90.00 53 55 96 79.01 16 16 100 90.00 23 26 88 70.14 - -" - 386 414 30 32 93 78.00 1 1 12 8 10.39 50 PPM A.I. 14-Day Number Arcsin Old Hatch. % Trans. _ _ - -- -- -- -- -- -- -- -- -- -- -- -- -- -- *- __ - -- -- _ - ~ 150 PPM A.I. 14-Day Number Arcsin Old Hatch. % Trans. __ - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- _ - -- -" _ - - Project Number 454-108 - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0 .0 5 ). NUMBER / PEN Appendix XIII - Figure 6a 14-Dag Old Survivors Hatchlings from a Northern Bobuhite Reproduction Study uith PFOS - Control 60 50 ' -to 30 20 10 0 201 202 203 20-t 205 206 207 208 209 210 211 212 213 Pen N u mb e r B Hatchlings E 3 14-Dag Old Survivors $ Data are not available due to adult mortality. 21-t 215 216 Project Number 454-108 Ji Ai ON Appendix XIII - Figure 6b 14-Dag Old Survivors / Hatchlings from a Northern Bobuhite Reproduction Study uith PFOS - 10 PPM A.I. Pen N u mb e r BB Hatchlings E53 14-Dag Old Survivors Data are not available due to adult mortality. o\ Project Number 454-108 Reproductive Performance by Pen Appendix XIII - Table 7 Hatchlings / Eggs Set (%) from a Northern Bobwhite Reproduction Study with PFOS 0 PPM A. I. Number Eggs Replicate Hatch. Set Arcsin % Trans. 10 PPM A..1 . 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 44 50 88 69.73 12 23 52 46.25 - -- - 30 31 97 79.65 20 2 1 95 77.40 28 28 100 90.00 - -- - 47 51 92 73.74 3 5 40 88 69.30 42 43 98 81.23 47 49 96 78.34 - -- - 45 47 96 78.10 42 49 86 67.79 3 5 40 88 69.30 46 49 94 75.67 473 521 36 40 90 73.58 1 1 1 1 12 10.24 20 30 67 54.74 36 40 90 71.57 38 39 97 80.79 32 38 84 66.59 31 42 74 59.22 39 48 81 64.34 13 19 68 55.81 - -- - 42 48 88 69.30 - -- - 39 39 100 90.00 27 37 73 58.68 55 57 96 79.20 16 36 44 41.81 26 53 49 44.46 -- 414 526 32 40 78 64.35 1 2 10 18 14.02 . .. - -- - - - - .- - -- - - -- - - -- - - -- - - -- - - -- - - -- - - -- - - -- - - -- - - -- - - -- - - -- - _ - -" - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0 .0 5 ). 150 PPM A.I. Number Eggs Hatch. Set Arcsin % Trans. - -- -- -- -- -- -- -- -- -- -- -- - -- - -- -- -- __ - -- -- - - - " Project Number 454-108 Reproductive Performance by Pen Appendix XIII - Table 8 14-Day Old Survivors / Eggs Set (%) from a Northern Bobwhite Reproduction Study with. PFOS 0 PPM A. I. 14-Day Eggs Replicate Old Set Arcsin % Trans. 1 43 50 86 68.03 2 1 2 23 52 46.25 3 - -- - 4 30 31 97 79.65 5 19 2 1 90 72.02 6 27 28 96 79.11 7 - -- - 8 46 51 90 71.75 9 33 40 82 65.27 10 40 43 93 74.68 1 1 47 49 96 78.34 12 - -- - 13 45 47 96 78.10 14 40 49 82 64.62 15 35 40 88 69.30 16 40 49 82 64.62 Total Mean SD 457 521 35 40 87 70.13 1 1 1 1 1 2 9.08 10 PPM A. I. 14-Day Eggs Old Set Arcsin % Trans. 20 30 67 54.74 36 40 90 71.57 29 39 74 59.58 30 38 79 62.69 30 42 71 57.69 38 48 79 62.84 10 19 53 46.51 - -- - 37 48 77 61.40 - -- - 37 39 95 76.91 27 37 73 58.68 53 57 93 74.64 16 36 44 41.81 23 53 43 41.21 - -- - * 386 526 30 40 72 59.25 1 1 10 17 11.35 50 PPM A.I. 14-Day Eggs Old Set Arcsin % Trans. _ _. - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- . - -- -- - -- - 150 PPM A.I. 14-Day Eggs Old Set Arcsin % Trans. ._ - -- -- -- -- -- -- -- -- - -- -- -- -- -- -- -- . - -- -- - -- - a\ -a Project Number 454-108 - Data are not available due to adult mortality. Significantly different from the control at p < 0.05 Reproductive Performance by Pen Appendix XIII - Table 9 Hatchlings / Maximum Set (%) from a Northern Bobwhite Reproduction Study with PFOS 0 PPM A. I. Number Max. Replicate Hatch. Set Arcsin % Trans. 10 PPM A.I. Number Max. Hatch. Set Arcsin % Trans. 50 PPM A.I. Number Max. Hatch. Set Arcsin % Trans. 150 PPM A.I. Number Max. Hatch. Set Arcsin % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 44 57 77 61.47 1 2 57 2 1 27.31 - -- - 30 57 53 46.51 20 57 35 36.32 28 57 49 44.50 - -- - 47 57 82 65.24 35 57 61 51.59 42 57 74 59.14 47 57 82 65.24 - -- - 45 57 79 62.69 42 57 74 59.14 35 57 61 51.59 46 57 81 63.94 473 741 36 57 64 53.44 1 1 0 20 11.95 20 57 35 36.32 36 57 63 52.63 38 57 67 54.74 32 57 56 48.53 31 57 54 47.52 39 57 68 55.81 13 57 23 28.53 - -- - 42 57 74 59.14 - -- - 39 57 68 55.81 27 57 47 43.49 55 57 96 79.20 16 57 28 31.99 26 57 46 42.48 " -- - 414 741 32 57 56 48.94 12 0 20 13.22 _ - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- __ - -- -- - _ - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- __ - -_ -_ - - - - - - Project Number 454-108 - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0 .0 5 ). Reproductive Performance by Pen Appendix XIII - Table 10 14-Day Old Survivors / Maximum Set (%) from a Northern Bobwhite Reproduction Study with PFOS 0 PPM A. I. 14-Day Max. Replicate Old Set Arcsin % Trans. 10 PPM A..1 . 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 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 43 57 75 60.29 1 2 57 2 1 27.31 - -- - 30 57 53 46.51 19 57 33 35.26 27 57 47 43.49 - -- - 46 57 81 63.94 33 57 58 49.54 40 57 70 56.90 47 57 82 65.24 - -- - 45 57 79 62.69 40 57 70 56.90 35 57 61 51.59 40 57 70 56.90 457 741 35 57 62 52.04 1 1 0 19 11.44 20 57 35 36.32 36 57 63 52.63 29 57 51 45.50 30 57 53 46.51 30 57 53 46.51 38 57 67 54.74 10 57 18 24.76 - -- - 37 57 65 53.68 - -- - 37 57 65 53.68 27 57 47 43.49 53 57 93 74.64 16 57 28 31.99 23 57 40 39.44 - -- - 386 741 30 57 52 46.45 1 1 0 19 12.41 . - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- __ - -- -- . - - - . __ - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- __ - -- - -- - - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0 .0 5 ). Project Number 454-108 Reproductive Performance by Week and Pen Appendix XIV - Table la Eggs Laid by Week and Pen from a Northern Bobwbite Reproduction Study with PFOS Experimental Group (ppm a.i.) 1 LOT A B C D Control E F G H I J PEN S 2 0 1 202 203 204 205 206 207 208 209 210 2 1 1 2 1 2 213 214 215 216 Totals 2 0 - 00 0 1 000 2 031 52 -1 00-51 22 -5342 55 -2 0 1 -4 534 -5 4 6 5 5 5 -30 3 -6 54 5 - 5 5 7 6 6 6 -50 3 - 6 7 6 7 - 6 6 76 6 1 -434 -7777 -6 7 77 7 0 -53 5 -7 7 8 8 -6 7 7 7 6 0 -656 -7467 -6 776 e4 -655 -76 76 -7 777 74 -675 -6 578 -5 8 77 9 32 49 59 71 73 77 73 80 82 Totals 55 27 - 38 23 32 - 56 47 50 54 - Data are not available due to adult mortality ^LOT A - Eggs Set During W eek 12; LOT B - Eggs Set During W eek 13; etc. - 53 54 62 54 605 Project Number 454-108 Reproductive Performance by Week and Pen Appendix XIV - Table lb Eggs Laid by Week and Pen from a Northern Bobwhite Reproduction Study with PFOS Experimental Group (ppm a .i .) i LOT A B C D 10 E F G H I J PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Totals 0 0 0 0 5' 0 0 2 1 0 4 0 2 01 0 04 3 1 -2 -4 35 2 5- 2 3 1 354 0 -5 -6 36 2 6 - 2 54 46 6 4 -5 -54 6 5 6 - 4 6 6 55 54 -7 -5 5 7 5 8 - 4 7 5 54 7 4 - 6 - 0 5 7 5 6 - 5 8 7 74 8 4 - 9 -2 6 8 6 7 - 6 6 6 54 6 3 - 6 - 7 5 6 5 7 - 5 6 7 64 6 0 -6 -7 6 7 6 6 - 6 4 7 76 8 1 -6 -777 57 - 14 30 46 62 72 65 81 72 72 78 Totals 34 46 43 42 47 53 2 1 - 54 - Data are not available due to adult mortality 1 LOT A - Eggs Set During W eek 12; LOT B - Eggs Set During W eek 13; etc. - 44 44 63 41 60 - 592 Project Number 454-108 Appendix XIV - Figure 1 Eggs Laid per Hen from a Northern Bobuhite Reproduction Study uith PFOS Weeks C o ncentration s, ppm a .i. 0 -- 10 Appendix XIV - Figure 2 Hens Laying per Pen from a Northern Bobuhite Reproduction Study uith PFOS NUMBER Project Number 454-108 --o ---to Appendix XIV - Figure 3 Eggs per Laying Hen from a Northern Bobuhite Reproduction Study uith PFOS C o ncentration s, ppm a .i. o - to Reproductive Performance by Week and Pen Appendix XIV - Table 2a Eggs Cracked by Week and Pen from a Northern Bobwhite Reproduction Study with PFOS Experimental Group (ppm a.i.) PENS --------------------------------------------------------------- 2 0 1 202 203 204 205 206 207 208 209 210 2 1 1 2 1 2 213 214 215 216 1 LOT A B C D Control E F G H I J 0 0 - 00 0 - 0 0 0 0 - 0 0 0 0 0 0 -00 0 - 0 0 0 0 - 001 0 0 0 -00 0 - 0 2 0 0 - 001 0 0 0 - 00 0 - 0 0 0 0 - 002 0 0 0 -00 0 - 0 01 0- 001 0 0 0 -00 0 - 0 0 0 0 - 0000 0 0 - 00 0 - 0 0 0 0 - 1 0 5 0 0 0 - 00 0 - 0 0 0 1 - 0 0 1 0 0 0 - 00 0 - 0 0 0 0 - 0 02 0 0 0 -20 0 -0 00 0 - 0030 Totals 0 1 3 2 2 0 6 2 2 5 Totals 0 0 _ 2 0 0 0 2 1 1 . 1 0 16 0 - Data are not available due to adult mortality 1LOT A - Eggs Set During W eek 12; LOT B - Eggs Set During Week 13; etc. 23 Project Number 454-108 Reproductive Performance by Week and Pen Appendix XIV - Table 2b Eggs Cracked by Week and Pen from a Northern Bobwhite Reproduction Study with PROS E xperim ental (ppm a . i . ) 1 LOT A B C D 10 E F G H I J PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 0 0 0 00 0 0 - 0 - 0 0 0 0 0 0 0 0 00 0 0 - 0 - 0 0 0 0 0 0 0 0 00 0 0 - 0 - 0 0 0 0 0 0 0 .. 0 0 0 0 0 - 0 - 0 0 0 0 1 0 0 0 00 0 0 - 0 - 0 0 0 0 0 0 0 0 00 0 0 - 0 - 0 0 0 0 0 0 0 0 00 0 0 - 0 - 0 0 0 0 1 0 0 0 00 0 0 - 0- 0 0 0 0 0 0 1 0 00 0 0 - 0 - 0 0 0 0 0 0 0 0 00 0 0 - 0 - 00 0 0 0 - T otals 0 0 0 1 0 0 1 0 1 0 T otals 0 1 000 0 0 - 0- 000 02 - - D ata are not available due to adult mortality 1L0T A - Eggs S e t During W eek 12; LOT B - Eggs S e t During W eek 1 3; e tc . 3 < ON Project Number 454-108 Reproductive Performance by Week and Pen Appendix XIV - Table 3a Eggs Set by Week and Pen from a Northern Bobwhite Reproduction Study with PFOS Experimental (ppm a.i.) 1 LOT A B C D Control E F G H I J PEN S 2 0 1 202 203 204 205 206 207 208 209 210 2 1 1 2 1 2 213 214 215 216 Totals 1 0 - 00 0 - 1 0 0 0 - 1 0 2 1 5 1 - 00 0 - 4 0 1 2 - 5 2 3 1 4 5 -20 1 -4 2 33 -4 44 5 54 -20 2 -5535 -54 55 5 6 - 50 3 - 6 6 5 6 - 5 6 5 6 e 0 - 33 3 - 6 7 6 7 - 6 6 6 6 6 0 - 52 5 - 7 6 7 7 - 4 7 1 7 6 0 - 55 5 - 6 4 5 6 - 6 6 6 5 5 4 -64 5 - 7 5 7 5 - 6 7 4 7 7 3 - 37 4 - 5 5 6 8 - 5 7 4 6 6 24 41 50 64 65 64 65 72 70 Totals 50 23 - 31 2 1 28 - 51 40 43 49 - Data are not available due to adult mortality 1 LOT A - Eggs Set During W eek 12; LOT B - Eggs Set During W eek 13; etc. - 47 49 40 49 521 ` Project Number 454-108 Reproductive Performance by Week and Pen Appendix XIV - Table 3b Eggs Set by Week and Pen from a Northern Bobwhite Reproduction Study with PROS Experimental Group i.) PENS --------------------------------------------------------------- 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Totals 1 LOT A B C D E F G H I J 0 0 0 04 0 0 1 0 0 3 0 1 0 0 0 04 2 1 - 2 - 4 2 5 1 5 - 1 3 0 34 4 0 -4 - 5 3 5 2 5 - 2 4 4 36 5 4 - 5 - 5 2 6 4 5 - 3 6 5 54 5 3 - 6 - 4 5 6 5 7 - 4 6 544 6 4 - 6 - 0 4 7 4 6 - 4 8 6 73 8 3 - 7 - 1 6 6 6 5 - 6 5 6 44 5 3 - 6 - 7 4 6 4 7 - 4 5 6 63 6 0 -5 -6 6 6 6 5 - 6 3 766 7 1 -6 -75747 - 9 26 39 55 64 60 70 67 64 72 Totals 30 40 39 38 42 48 19 . 48 - Data are not available due to adult mortality * LOT A - Eggs Set During W eek 12; LOT B - Eggs Set During W eek 13; etc. 39 37 57 36 53 526 oo Project Number 454-108 Reproductive Performance by Week and Pen Appendix XXV - Table 4a Viable Embryos by Week and Pen from a Northern Bobwhite Reproduction Study with PROS Experimental Group (ppm a.i.) PENS --------------------------------------------------------------- 2 0 1 202 203 204 205 206 207 208 209 2 10 2 1 1 2 1 2 213 214 215 216 Totals 1 LOT A B C D Control E F G H I J 1 0 - 00 0 - 1 0 0 0 - 1 0 1 1 5 1 - 00 0 - 4 0 1 2 - 5 0 3 1 4 3 - 2 0 1 '- 4 2 3 3 - 4 0 4 5 5 0 -202 -553 5 -54 54 5 2 -403 -6 6 5 6 -5 6 56 5 0 - 33 3 - 6 7 6 5 - 6 6 5 6 6 0 - 52 5 - 7 6 7 7 - 4 7 1 7 4 0 - 55 5 - 6 4 5 6 - 6 6 6 5 5 3 - 64 5 - 7 5 7 5 - 6 74 7 7 3 -37 4 - 54 6 8 - 5 7 4 6 5 22 35 45 59 61 64 63 71 69 Totals 47 12 _ 30 2 1 28 51 39 43 47 - Data are not available due to adult mortality 1LOT A - Eggs Set During W eek 12; LOT B - Eggs Set During W eek 13; etc. . 47 43 38 48 494 vo Project Number 454-108 Reproductive Performance by Week and Pen Appendix XIV - Table 4b Viable Embryos by Week and Pen from a Northern Bobwhite Reproduction Study with PFOS Experimental (ppm a.i.) 1 LOT A B C D 10 E F G H I J PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 0 0 0 00 0 0 - 0 - 0 0 2 0 0 0 0 001 2 1 - 2 -4 0 5 04 1 2 0 34 4 0 - 4 -5 0 5 2 5 2 4 4 36 54 -5 -5 0 6 1 5 3 6 5 54 5 3 -6 -4 2 6 2 7 4 6 544 5 3 -6 - 0 4 6 1 3 4 8 6 73 7 3 -7 - 1 6 6 4 1 6 S 6 44 5 2 - 6 - 7 4 6 1 7 4 56636 0- 5- 6 6 644 6 3766 6 1 - 5- 7572 7 - Totals 2 19 35 50 58 51 63 63 61 68 Totals 30 39 39 38 35 45 17 - 46 - Data are not available due to adult mortality 1 LOT A - Eggs Set During Week 12; LOT B - Eggs Set During W eek 13; etc. - 39 27 55 17 43 - 470 0o0 Project Number 454-108 Reproductive Performance by Week and Pen Appendix XIV - Table 5a Live Three-Week Embryos by Week and Pen from a Northern Bobwhite Reproduction Study with PFOS Experimental Group (ppm a.i.) 1 LOT A 6 C D Control E F G H I J PEN S 2 0 1 202 203 204 205 206 207 208 209 210 2 1 1 2 1 2 213 214 215 216 Totals 0 0 - 00 0 - 1 0 0 0 - 0 0 1 1 5 1 -00 0 -4 01 2 -503 1 4 3 -2 0 1 -42 33 -4 04 5 5 0 -2 0 2 -5435 - 54 54 5 2 -40 3 - 6 6 5 6 - 5 6 5 6 5 0 -33 3 -6 76 5 - 6 6 5 6 6 0 -52 5 -7 6 77 -4 7 1 7 4 0 -55 5 -6 4 56 - 6 6 6 5 53 -64 5 -7575 -6 74 7 7 3 -374 -546 8 - 573 6 3 22 35 44 59 61 64 63 71 68 Totals 46 12 - 30 21 28 - 51 38 43 47 - Data are not available due to adult mortality 1LOT A - Eggs Set During W eek 12; LOT B - Eggs Set During W eek 13; etc. - 46 43 37 48 490 Project Number 454-108 Reproductive Performance by Week and Pen Appendix XIV - Table 5b Live Three-Week Embryos by Week and Pen from a Northern Bobwhite Reproduction Study with PFOS*1 Experimental Group (ppm a.i.) PENS ------------------------------------------------------------- -- 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 1 LOT A B C D 10 E F G H I J 0 0 0 00 0 0 -0 -0 0 2 0 0 0 0 0 01 2 1 -2 -4 0 5 04 1 2 0 34 4 0 -4 - 5 0 5 2 5 2 4 4 36 53 -5 -5 0 6 1 5 3 6 5 54 5 3 -6 -4 2 6 2 7 4 6 544 5 3 -6 -0 4 6 1 3 3 8 6 73 7 3 -7 -1 6 6 31 e 5 6 43 52 -6 -74 6 1 7 4 5 6 63 6 0 -5 -6 6 6 44 6 3 766 6 1 -5 -7 5 72 7 - Totals 2 19 35 49 58 51 61 62 61 68 Totals 29 39 39 38 34 45 16 - 46 - Data are not available due to adult mortality 1 LOT A - Eggs Set During W eek 12; LOT B - Eggs Set During Week 13; etc. - 39 27 55 16 43 - 466 Project Number 454-108 Reproductive Performance by Week and Pen Appendix XIV - Table 6a Hatchlings by Week and Pen from a Northern Bobwhite Reproduction Study with PFOS Experimental Group (ppm a.i.) PEN S 2 0 1 202 203 204 205 206 207 208 209 210 2 1 1 2 1 2 213 214 215 216 Totals 1 LOT A B C DV Control E F G H I J 0 0 -00 0 1 00 0 0001 5 1 -00 0 -4 0 1 2 - 5 0 3 1 4 3 -2 0 1 -32 33 -4 04 5 4 0 -2 02 -5435 -53 54 5 2 -40 3 -56 5 6 -5 6 5 6 5 0 -33 3 -6 76 5 - 6 6 5 5 5 0 - 52 5 - 7 6 6 7 - 4 7 1 7 4 0 -54 5 -6 2 5 6 - 5 6 5 4 5 3 -64 5 -74 7 5 - 6 74 7 7 3 -37 4 -34 6 8 - 5 7 3 6 2 22 34 42 58 60 62 57 70 66 Totals 44 12 - 30 20 28 - 47 35 42 47 - Data are not available due to adult mortality 1 LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. - 45 42 35 46 473 0U0> Project Number 454-108 Reproductive Performance by Week and Pen Appendix XIV - Table 6b Hatchlings by Week and Pen from a Northern Bobwhite Reproduction Study with PFOS Experimental (ppm a.i.) 1 LOT A B C D 10 E F G H I J PENS 217 218 219 220 2 2 1 222 223 224 225 226 227 228 229 230 231 232 Totals 0 0 0 00 0 0 -0 * 0 0 2 0 0 0 0 0 01 0 0 -2 - 4 0 5 0 3 0 2 01 3 3 0 -3- 5 0 52 3 1 4 4 1 6 3 3 - 5 - 5 0 6 1 4 3 6 5 53 5 2 -5 - 4 2 6 2 3 3 5 5 34 5 3 -5 T 0 4 6 1 3 2 7 6 73 6 2 -7 - 1 6 6 3 0 2 4 542 52 -5 - 74 6 1 4 4 5 6 53 6 0 -5 ' 6 6 6 4 3 5 3 7 66 6 1 -5 -7 5 7 2 3 - 2 15 27 43 51 47 56 51 59 63 Totals 20 36 38 32 31 39 13 42 - Data are not available due to adult mortality i LOT A - Eggs Set During W eek 12; LOT B - Eggs Set During W eek 13; etc. 39 27 55 16 26 . 414 0-r0* Project Number 454-108 Reproductive Performance by Week and Pen Appendix XIV - Table 7a 1 4 -Day Old Survivors b y Week and Pen from a Northern Bobwhite Reproduction Study with PFOS Experimental (ppm a.i.) PEN S 2 0 1 202 203 204 205 206 207 208 209 210 2 1 1 2 1 2 213 214 215 216 Totals LOT1 A B C D Control E F G H I J 00. 00 0 . 1 000 0001 4 1 -00 0 -40 1 2 -5 03 1 4 3 -2 0 1 -32 2 3 -4 0 4 3 4 0 -2 0 2 -542 5 - 5 3 5 3 5 2 -40 3 -555 6 -5 6 5 5 5 0 - 32 3 - 6 6 6 5 - 6 6 5 5 5 0 - 52 4 - 76 6 7 -4 6 1 6 4 0 - 5' 4 5 - 6 2 5 6 - 5 6 5 3 5 3 -64 5 -647 5 -6 6 4 7 7 3 -37 4 -34 6 8 - 5 7 3 6 2 21 31 40 56 58 59 56 68 66 Totals 43 12 - 30 19 27 - 46 33 40 47 - Data are not available due to adult mortality 1 LOT A - Eggs Set During W eek 12; LOT B - Eggs Set During W eek 13; etc. - 45 40 35 40 457 Project Number Reproductive Performance by Week and Pen Appendix XIV - Table 7b 14 -Day Old Survivors by Week and Pen from a Northern Bobwhite Reproduction Study with PFOS Experimental Group (ppm a.i.) PENS --------------------------------------------------------------217 218 219 220 2 2 1 222 223 224 225 226 227 228 229 230 231 232 Totals 1 LOT A B C D 10 E F G H I J 0 0 0 0 0 0 0 - 0 - 0 0 1 0 0 -' 0 0 0 0 1 0 0 - 2 - 2 0' 5 0 3 - 02 0 03 3 0 3-5052 3 - 1 4 1 1 6 3 1 -5 - 5 0 6 1 2 - 3 6 4 53 5 2 -4 -4 2 6 2 3 - 3 5 4 34 5 3 -5 - 0 4 6 1 3 - 2 7 5 73 6 1 -6 - 1 6 G 3 0 - 2 4 341 52 -3 -74 6 1 4 - 4 5 6 53 6 0 -5 - 6 6 6 4 3 - 5 3e 5G51 -4 - 7 5 622 - 1 13 26 36 49 46 53 46 59 57 Totals 20 36 29 30 30 38 10 _ 37 - Data are not available due to adult mortality LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. 37 27 53 16 23 386 ooo Project Number 454-108 Pen 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 A - Appendix XV - Table 1 Eggshell Thickness (jam) per Pen b y Week from a Northern Bobwhite Reproduction Study with PFOS (Control) Weeks B CDE FG H I 0.232 0.219 0.221 0.223 - 0.231 0.229 - --- - - 0.249 0.256 0.258 - - 0 .2 2 1 0.221 - 0.183 0.194 0.202 --- - - 0.230 0.239 0.244 0.194 0.196 0.184 0.169 - 0.238 0.230 0.261 0.228 0.220 0.219 0.228 -- - - 0.238 0.230 0.231 0.228 - 0.248 0.256 0.262 0.250 0.249 0.253 0.247 0.222 0.223 0.221 J - - No eggs available. Mean and standard deviation of pen means: Mean SD [n] 0.224 0.230 0.006 0.002 4 2 0.255 0 .2 2 1 0.193 0.005 0.000 0.009 3 2 3 0.238 0.186 0.243 0.224 0.007 0.012 0.016 0.005 3 4 3 4 0.232 0.255 0.250 0.222 0.005 0.007 0.003 0.001 4 3 4 3 Mean 0.229 SD 0 . 0 2 1 42 Project Number 454-108 Pen 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 A _ - Appendix XV - Table 2 Eggshell Thickness (mm) per Pen b y Week from a Northern Bobwhlte Reproduction Study with PFOS (10 PPM A.I. ) Weeks B C D E FG H' I J 0.214 0.226 0.224 0.231 - 0 .2 2 1 0.210 0.220 0.224 0.234 0.242 0.249 - 0 .2 2 1 0.241 0.228 0.241 0.238 0.202 0.212 - 0.263 0.239 0.240 - 0.222 0.219 - - --- 0.222 0.231 0.228 0 .2 2 1 - --- 0.214 0.201 0.222 0.209 - 0.222 0.229 0.237 0.242 0.220 0.215 0.227 - 0.232 0.241 0.241 0.214 0.214 0.222 0 .2 2 1 - -- - - - - Mean and standard deviation of pen means: - N oeg g s available. Differences between the control and this treatment group were not significant (p > 0 .0 5 ). Mean SD [n] 0.224 0.217 0.237 0.230 0.223 0.248 0 .2 2 1 0.007 0.006 0 .0 11 0.010 0.019 0.014 0.002 4 3 4 3 4 3 2 0.226 0.005 4 0.212 0.229 0.226 0.238 0.218 0.009 0.007 0.012 0.005 0.004 4 3 4 3 4 Mean 0.227 SD 0 . 0 1 0 45 Project Number 454-108 Appendix XVI - Table 1 Mean Hatchling Body Weight (g) per Pen by Week from a Northern Bobwhite Reproduction Study with PFOS (Control) Weeks [No. of Hatchlings] Total Pen A [ n] B [ n] C[ n] D[ n] E[ n] F[ n] G[ nj H[ n] I [ n] J[ n] Mean SD Hatch 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 [ 0] [ 0] [ 0] l 0] [ 0] 5[ 1 ] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] 6[ 1] 6 [ 5] 5[ 1] [ 0] [ 0] [ 0] 6 [ 4] [ 0] 6[ 1] 6 [ 2] 6 [ 5] [ 0] 6 [ 3] 6[ 1] 7 [ 4] 6 [ 3] 61 2] [ 0] 5 [ 1] 6 1 3] 6 [ 2] 6 [ 3] 6 1 3] 7 [ 4] [ 0] 7 [ 4] 6 1 5] 7 [ 4] [ 0] - 6 [ 2] [ 0] 6 [ 2] - 6 [ 5] 6 [ 4] 6 [ 3] 6 1 5] - 7 [ 5] 6 1 3] 7 [ 5] 6 [ 4] 7 [ 5] 6 [ 2] 6 [ 4] [ 0] 7 [ 3] 6 1 5] 61 6] 6 1 5] 6 [ 6] 7 [ 5] 6 [ 6] 6 [ 5] 6 [ 6] 7 [ 5] [ 0] - 6 [ 3] 6 [ 33 6 [ 3] 6 [ 6] 6 [ 7] 61 6] 7 [ 5] 7 [ 6] 7 [ 6] 7 [ 5] 6 [ 5] 7 [ 5] t 0] - 6 [ 5] 61 2] 6 [ 4] 5 [ 7] 61 6] 6 t 6] 6 [ 7] 7 [ 4] 7 [ 7] 6[ 1] 6 [ 7] 7 [ 4] [ 0] - 6 [ 5] 6 1 4] 6 [ 5] 6 [ 6] 6 [ 2] 6 1 5] 7 [ 6] 7 [ 5] 6 [ 6] 6 [ 5] 7 [ 4] 6 [ 5] 6 [ 3] 6 [ 6] 6 [ 4] 6 [ 5] 6 [ 7] 6 [ 4] 6 [ 7] 7 [ 5] 7 [ 6] 6 [ 7] 7 [ 4] 6 [ 7] 7 [ 7] 7 0 44 6 [ 3] 6 0 1 2 - 6 [ 3] 6 0 30 6 [ 7] 6 0 20 7 [ 4] 6 0 27 7 t 3] 6 0 47 6 [ 4] 6 0 35 6 [ 6 ] 6 0 42 7 [ 8] 6 0 47 - 7 [ 5] 7 0 45 6 [ 7] 6 0 42 7 [ 3] 7 0 35 6 [ 6 ] 6 0 46 Mean 6 SD 0 472 The number of hatchlings weighed may differ from the total number of hatchlings since those hatchlings found dead were not weighed. - D ata are not available due to adult mortality. SD = Standard deviation of mean body weight, by parental pen, by week. Project Number 454-108 Appendix XVI - Table 2 Mean Hatchling Body Weight (g) per Pen by Week from a Northern Bobwhite Reproduction Study with PFOS (10 PPM A. I. ) Weeks [No. of Hatchlings] Total Pen A[ n] B[ n] C[ n] D[ n] E[ n] F[ n] G[ n] H[ n] I [ n] J[ n] Mean SD Hatch 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 [ 0] [ 0] [ 0] 6[ 1] 6 [ 3] 6 [ 3] St 2 ] 6 t 2] 6 [ 4] St 5] 6 0 20 [ 0] [ 0] 6 [ 2] 7 1 4] 6 [ 6] 6 [ 5] 7 [ 7] St 4] 7 [ 5] St 3] 6 0 36 [ 0] [ 0] [ 0] 5[ 3] 6 [ 5] 6 [ 5] 6 [ 6] 6 [ 5] St 6 ] St 7] 6 0 37 [ 0] [ 0) 5[ 1 ] 6[ 1] 6 [ 5] 6 t 3] 5[ 7] St 4] St 5] 6 [ S] 6 0 32 [ 0] 6[ 1] 6 [ 3] St 6] 6 [ 3] 7 [ 4] 6 t 3] 7 [ 21 St 31 6 [ 61 6 0 31 [ 0] [ 0] 6 [ 3] 6 [ 3] 6 [ 5] 6 [ 5] St 6] St 51 St 61 7 t 61 6 0 39 [ 0] - [ 0] - [ 0] - 5[ 3] - 6 [ 2] - 6 [ 3] - 5[ 2 ] - St 21 - [ 01 - 5 [ 11 - 6 1 13 [ 0] - 5 [ 2] - 5[ 3] - 6 [ 5] - 5 [ 5] - 5[ 5] - 5[ 7] - 5[ 51 - St 51 - 6 t 5] - 5 0 42 [ 0] 7 [ 4] 7 [ 5] 7 [ 5] 6 [ 4] t 0] 61 1 ] 7 [ 71 7 [ 6] 7 [ 7] 7 0 39 [ 0] t 0] [ 0] [ 0] 6 [ 2] 6 [ 4] 6 [ 6] 7 t 4] 7 [ 6] 7 C 5] 6 0 27 6 [ 1] 6 [ 5] 6 [ 5] 6 [ 6] St 6 ] 6 t 6] 61 6] 7 [ 61 7 [ 6] St 7] 6 0 54 [ 0] [ 0] 8 [ 2] 8[ 1] at 2 ) 6[ 1] 8 t 3] 7[ 1] 8 t 4] 8 [ 21 8 0 16 [ 0] - 6 [ 3] - 6 [ 3] - 6 [ 4] - 6 [ 3] - 6 [ 3] - t 0] St 4] 5 [ 3] 5 [ 3] 6 0 26 - -__ Mean 6 SD 1 412 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. Differences between the control and this treatment group were not significant (p > 0 .0 5 ). SD = Standard deviation of mean body weight, by parental pen, by week. Project Number 454-108 Appendix XVII - Table 1 ATeas 14-Day Old Survivors Body Weight (g) per Pen by Week from a Northern Bobwhite Reproduction Study with PFOS (Control) Weeks [No. of 14-Day Old Survivors] 14-Day Pen A[ n] B [ n] C[ n] D[ n] E[ n] F[ n] G[ n] H[ n] l[ nj J[ n] Mean SD Total 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 [ 0] [ 0] [ 0] [ 0] [ 0] 26 [ 1 ] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] 24 [ 1 ] 29 [ 4] 22 [ 1 ] [ 0] [ 0] [ 0] 28 [ 4] [ 0] 22 [ 1 ] 30 [ 2] 28 [ 5] [ 0] 29 [ 3] 25 [ 1] 29 [ 4] 26 [ 3] 29 [ 2 ] [ 0] 28 [ 1 ] 32 [ 3] 24 [ 2 ] 28 [ 2 ] 26 [ 3] 25 [ 4] [ 0] 23 [ 4] 34 [ 3] 32 [ 4] [ 0] - 28 [ 2 ] [ 0] 31 [ 2 ] - 36 [ S3 25 [ 4] 23 [ 2 ] 31 [ 5] 30 [ 5] 29 [ 3] 32 [ 5] 22 [ 3] 27 [ 5] 261 2 ] T 32 [ 4] [ 0] 30 [ 3] 32 [ 5] 22 [ 5] 23 [ 53 29 [ 61 32 [ 53 30 [ 6] 30 [ 5] 30 [ 5] 33 [ 53 [ 0] - 28 [ 3] 28 [ 2 ] 3 1 1 33 32 [ 6] 23 [ 6] 25 [ 6] 30 [ 5] 301 63 27 [ 63 29 [ 5] 27 [ 5] 29 [ 5] [ 0] - 29 [ 5] 30 [ 23 28 t 43 33 [ 7] 27 [ 61 27 [ 61 30 [ 73 28 [ 4] 31 [ 63 27J 13 30 [ 63 29 [ 4] [ 0] - 31 [ 5] 30 [ 4] 32 [ 5] 33 [ 61 28 [ 2 ] 27 [ 5] 29 [ 61 3 1 1 51 28 [ 61 33 [ 53 28 [ 3] 34 [ 53 22 [ 3] 32 [ 61 34 [ 4] 33 [ 53 40 [ 63 32 [ 43 30 [ 71 30 [ 51 31 [ 61 32 [ 61 37 [ 41 32 [ 71 32 [ 7] 25 [ 33 34 [ 3] 31 [ 73 29 [ 4] 38 [ 3] 30 [ 4] 32 [ 6] 31 [ 8] 34 [ 5] 32 [ 7] 37 [ 3] 27 [ 63 30 24 31 31 30 34 26 27 30 30 30 31 29 2 43 2 12 2 30 2 19 2 27 3 46 3 33 3 40 1 47 2 45 2 40 4 35 3 40 Mean 29 SD 2 457 - Data are not available due to adult mortality. SD = Standard deviation of mean body weight, by parental pen, by week. Project Number 454-108 Appendix XVII - Table 2 Mean 14-Day Old Survivors Body Weight (g) per Pen by Week from a Northern Bobwhite Reproduction Study with PFOS (10 PPM A. I. ) Weeks [No. of 14-Day Old Survivors] 14-Day Pen A[ ~n] B [ n] C[ n] D[ n] E[ n] F[ n] G[ n] H[ n] I[ n] J[ n] Mean SD Total 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] 30 [ 1] [ 03 [ 0] - [ 0] [ 0] [ 0] [ 0] 32 [ 1] [ 03 t 03 26 [ 2] 29 [ 23 [ 0] 36 [ 53 [ o) 31 [ 33 - [ 0] 34 [ 2] t 03 [ 0] 27 [ 3] 28 [ 3] [ 03 26 [ 3] 35 [ 53 [ 0] 32 [ 5] 42 [ 2] 33 [ 3] - 13 [ 1] 34 [ 4] 18 [ 1] 31 [ 1] 28 [ 6] 22 [ 33 24 [ 1] - 27 [ 53 - 35 [ 5] [ 0] 29 [ 6] 36 [ 13 22 [ 2] - 23 [ 33 34 [ 6] 28 [ 4] 32 [ 5] 28 [ 3] 30 [ 53 31 [ 23 27 [ 4] 31 [ 4] 35 [ 2) 31 [ 6] 33 [ 2] 33 [ 33 - 29 ( 3] 32 [ 5] 23 [ 4] 33 [ 33 28 [ 4] 26 [ 53 33 [ 3] 24 [ 5] [ 03 30 [ 43 33 [ 63 31 [ 1] 27 [ 3] - 31 [ 23 35 [ 7] 26 [ 53 28 [ 7] 29 [ 3] 27 [ 6] 32 [ 13 25 [ 63 33 [ 1] 31 [ 63 31 [ 6] 34 [ 3] [ 03 - 20 [ 2] 30 [ 4] 25 [ 3] 25 [ 4] 29 [ 1) 26 [ 5] 22 [ 2] 27 [ 3] 33 [ 7] 32 [ 4] 35 [ 6] 36 [ 1] 30 [ 4] - 31 [ 4) 34 [ 5] 26 [ 6] 31 [ 53 32 [ 3] 30 [ 63 [ 03 29 [ 5] '37 [ 6] 32 [ 6] 34 [ 63 38 [ 4] 32 [ 33 - 311 53 32 [ 3] 26 [ 6] 31 [ 5] 30 [ 6] 30 [ 53 25 [ 1) 28 [ 4] 37 [ 7] 34 [ 5) 34 [ 6] 36 [ 2] 30 [ 23 - 28 33 26 30 29 28 29 26 34 32 33 36 30 5 20 2 36 2 29 3 30 1 30 2 38 5 10 1 37 3 37 2 27 2 53 3 16 3 23 Mean 30 SD 3 386 - Data are not available due to adult mortality. Differences between the control and this treatment group were not significant (p > 0 .0 5 ). SD = Standard deviation of mean body weight, by parental pen, by week. Project Number 454-108 -193- Project Number 454-108 Appendix XVIII Offspring Liver Weights (g) from a Northern Bobwhite Reproduction Study with PFOS Control (0 ppm a.i.) Bird 1 2 3 4 5 6 Mean SD Male Liver 0.672 0.884 0.658 1.058 0.922 0.858 0.842 0.154 Female Liver 0.950 0.789 0.767 0.893 0.850 0.087 Group Mean 0.845 SD 0.125 10 ppm a.i. Male Bird Liver Female Liver 1 2 3 4 5 Mean SD 1.022 0.859 0.637 0.727 1.019 0.853 0.172 1.017 0.747 0.682 0.845 1.121 0.882 0.184 Group Mean 0.868 SD 0.169 The above differences from the control group w ere not statistically significant. - 194- W ildlife International, Ltd. Project Number 454-108 Appendix XIX Changes To Study Protocol This study was conducted in accordance with the study protocol signed on January 22, 2001 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 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 ppm a.i. Birds were euthanized without collection of samples. 4. The protocol was amended to euthanize the 50 ppm a.i. 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 possible analysis. Eggs selected from Lots C through I were opened and the albumen and yolk were 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 delete the reference substance number from page two of the protocol. 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. - 195- W ildlife International, Ltd. Project Number 454-108 9. The protocol was amended to add Attachment A, the analytical phase plan to be used by 3M Environmental Laboratory for analysis of samples, and to add the 3M Environmental Laboratory Project Identification Number to be used for the study.. 10. 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. The temperature in Hatcher C, containing eggs for Lot J, was inadvertently not recorded on the afternoon of July 8, 2001. 16. The liver weight for an adult female (Band #428) in the 50 ppm a.i. treatment group was inadvertently not recorded. 17. Liver weights were recorded for adults and offspring selected for tissue collection, and data were compared statistically using Dunnett's t-test. Feather samples were also collected, when available, from birds from which tissue samples were collected. The protocol did not require weighing of livers or collection of these samples. 18. On May 28, 2001, the temperature in brooder level B35-4, housing offspring from the 10 ppm a.i. treatment group, was inadvertently not recorded. On June 29, 2001, the temperatures in brooder levels B04-3, 4 and 5, housing offspring from the 10 ppm a.i. treatment group, were inadvertently not recorded. 19. Dietary samples collected on Day 0 of Week 1 were not stored frozen for the entire time interval between collection and processing for analysis. These samples were removed from the freezer and stored at ambient temperature for one day prior to processing. 20. Week 15 blood samples were collected from one male in the control group and one male in the 10 ppm a.i. treatment group who were not designated for blood collection. The additional blood samples were collected from male birds in pens where the female had died. - 196- W ildlife International, Ltd. Project Number 454-108 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 of Avian Toxicology (3) Linda R. Mitchell, Manager of Ecotox Operations (4) Diana Temple, Laboratory Supervisor (5) Sean P. Gallagher, Senior Biologist Analytical Chemistry (1) Willard B. Nixon, Ph.D., Director of Chemistry (2) Raymond L. Van Hoven, Ph.D., Scientist