Document Gzjq619dyXoNLy2OGxqjN3rdY

ARJJ6-H55 PFOS: A REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-108 3M LAB REQUEST NO. U2723 FTFRA Guideline 71-4 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 S tPaul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 USA 1-410-822-8600 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 under 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 22,2001 January 22,2001 January 23, 2001 Matrix Fortification January 30,2001 January 31,2001 January 31, 2001 Feed Consumption February 6, 2001 February 7,2001 February 8,2001 Body Weights, Environmental Conditions February 6,2001 February 7,2001 February 9,2001 Candling June 12, 2001 June 12, 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 June 28, 2001 June 28, 2001 July 2, 2001 Hatchling Body Weights 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 James H. Coleman Quality Assurance Representative DATE if* W ildlife International, Ltd. Project Number 454-108 -4REPORT 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: U J./L ___ W l L. Van Hoven, Ph.D. Scientist, Analytical Chemistry MANAGEMENT: Joann B. Beavers Director, Avian Toxicology m Willard B. Nixon, Ph.D. Director of Chemistry -- DATE t A i/ c DATE DATE lZ . /Mi DATE G O O 'a n Wildlife 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 *'V 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 Adult Female Body 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 CO' Wildlife 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 o f 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 n. Diet and Supplement Formulations................................................................... 48 Appendix HI. Diet Preparation..................................................................................................50 Appendix IV. The Analysis of PFOS in Avian D iet................................................................ 51 A ppendix V . D iag ram o f T est L ay o u t................................................................................................... 67 Appendix VI. Reproductive Parameters and Replicate Identification.....................................68 Appendix VH. Adult Body Weight (g) from a Northern Bobwhite Reproduction Study with PFOS.......................................................................73 Appendix VIII. Feed Consumption (ghird/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 W ildlife 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 XIII. 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 XVUI. 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 virginianus) 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. cm y 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 454-104) 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 C O O ! ;I 7 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 o f 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 C00V9S 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 o f 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 during acclimation and testing. The basal diet fed to both adults and offspring was formulated to Wildlife International, Ltd. specifications by Agway Inc. (Appendix II, Table 1). The basal ration contained at least 27% protein and 2.5% fat, and no more than 5% fiber. The basal diet contained approximately 1.1% calcium, derived from feedstuff's 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 with W ildlife 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. Coi ntrol and treatment group diet samples were also collected from the feed troughs on Day 7 of Week 1 to assess stability o f the test substance under actual test conditions. Additionally, a sample was collected from the control and treatment group diets, 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 Cib 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 18 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 o f detection (LOD) for these analyses was 10.0 pg on-column, calculated from the product of the injection volume (10.0 pL) 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 o f the m atrix 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, DayO. 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%, coe\ 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 pbotoperiod 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 o f the test. During acclimation, all birds were observed daily. Birds exhibiting abnormal behavior or debilitating physical injuries were not used for the test. During the study, all adult birds were observed daily for signs of toxicity or abnormal behavior. Additionally, all offspring were observed daily from hatching until 14 days o f 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 dining 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 - 18- collected from birds in these designated pairs, when possible, at live 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 o f 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 li 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 OCi-i i 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. 0 ^ 0 :3 0 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. Proj ect 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 o f 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. i 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 dietaiy 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 term inated after W eek 4 o f the test due to the lim ited recovery o f birds at this level. 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 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% o f 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 o f 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 mortality was caused by incidental injury. D ue to the nature of the lesions 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 dining 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 VUI. 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 OOO'- Wildlife 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 OOOb.l W ildlife International, Ltd, Project Number 454-108 -27- o f 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 o f 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. cA O ' 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. W ildlife International, Ltd. -29REFERENCES Project Number 454-108 1 U.S. Environmental Protection Agency. 1982. Pesticide Assessment Guidelines, FIFRA Subdivision 1S', 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 o f 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 fo r 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: 482491. 8. Wildlife International, Ltd.. 1994. Avian Reproduction Data System (ARDS) Version 2. G-OO'V* 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 Week 1 Day 0 Day 71 Week 2 DayO Week 3 DayO Interval Week 4 DayO Week 8 DayO Week 12 Week 16 Week 20 Day 0 DayO DayO 0 Measured <2.00 2 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 Mean 10 Measured % Nominal 98 3 108 4 103 101 97 109 101 101 99 Mean 50 Measured % Nominal 100 3 100 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 of the test substance under actual test conditions. 2<2.00 indicates the value was less than the limit of quantitation. 3The mean of homogeneity samples collected at Week 1, Day 0. ` The percent of the mean values for Week 1, Day 0. 5The 1SOppm a.i. treated diet was reduced to 20 ppm a.i. during this interval. - No values available, test group terminated. O U' Project Number 454-108 -31 - Project Number 454-108 Table 2 Summary o f 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 0 0 00 10 0000 1 12 1 112 1 112 1 0000 10 0 0 1 1 10 0 110 12 11 1 12 1 0 10 0 1 100 0 100 100 0 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. Remaining 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 of the 150 ppm a.i. test concentration. (j'OOb.'bb Tabla 3 Mean Adult Body Weight (g) from a Northern Bobwhite Reproduction Study with 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 208 210 6 4 208 206 2 4 205 210 -8 -7 206 -34 212 -41 214 -1 214 -1 211 -l 209 -1 197** -13 202 -7 172** 172** 13 3 213 1 213 1 210 1 208 1 185** -8 195* -11 187** 172** - 215 214 -1 4 211 -2 209 -1 179** 185** - - 214 218 209 209 - - - * 1 32 -1 40 - - - ** 215 247 208 249 - - - - 7 39 -1 42 - - . - UtoJ The m eans for body w e igh ts and body w eight changes are calculated and rounded separately. - Data are not available due to adult m ortality. * Significantly different from the control at p < 0.05 * * Significantly different from the control at p < 0.01 ^Only surviving birds w ere included in the calculations for each body w eight interval. Vi,& Project Number 454-108 -33 - Project Number 454-108 Figure 1 Mean Adult Male Body Ueight (g) from a Northern Bobuhite Reproduction Study uith PFOS CIf 5a iiiiiiiiiiiiiiimiiiiiiiiiimiiiiiiimimiiii C K> i* 8: S (0 i B X H U1 u 3 nnrnfr ? ! lllllllllllllllllllllllllllllllllllllllllllllllll 6a iiiiiiiimiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiin % I o 3 Qi M *f>St 1 <) 1H0I3M AG08 31WU ft !* ft ** 0 5 G ';0 - Project Number 454-108 34 - x3: XO) CD jc=La OL. cO CN I+-. H *> i_ oen 3 ~p D* ^ U . ^ -Ctt 2Ox- s* m2 en OS .5 ?lx. 3U 3 Ec<O " 53484853532353532348532323 (6) JLH0I3I1 AQ08 3iyU3J CU 4 I8 fs *4 % h. 1 * S * 1n-,/'} , Table 4 Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Reproduction Study with PFOS Experimental W E EK S Group (ppm a.i.) 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 2 1 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 1 2 12 14 13 13 14 16 18 20 20 19 20 20 20 2 1 19 1 1 ** 1 1 ** 1 0 ** 1 0 *'k 9** 9*' 7*i 6 ** 8** 13 - D ata are not available due to adult m ortality Significantly different from the control at p < 0.01 '0 i. i .*? Mean figure 3 Feed Consumption (g /b ird /d a y ) from Reproduction Study uith PFOS a Northern Bobuhite --0 -" 10 Concentrat.io..n..s.,.. pgpom a.i. ....... 150 1 OJ ON Project Number 454-108 -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 Number of birds Feather loss Head lesions Foot/leg lesions Back/vent lesions Liver - pale and mottled Abdominal cavity - slight egg yolk peritonitis Intestinal tract - area of hyperemia Ceca - round worms found Testes - one or both small (-1.0-1.5 cm) Not remarkable Males Control 10 (0 ppm a.i.) ppm a.i. 16 16 35 00 00 00 00 -- 78 03 16 73 Females Control 10 (0 ppm a.i.) ppm a.i. 13 13 69 01 10 20 01 01 01 30 .. 63 1Gross necropsy observations for birds maintained for collection of blood samples during the test are not reported. Table 6 Mean Liver Weights1(g) from a Northern Bobwhite Reproduction Study with PFOS Experimental Group Adult Males Mean SD Adult Females Mean SD Offspring Males 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 _ __ 1Mean standard deviation. - Data not available due to mortality. *Statistically different from the control group at p < 0.01. 0.853 0.172 0.882 0.184 m. . - Project Number 454-108 Table 7 Summary of Reproductive Performance from a Northern Bobwhite Reproduction Study with PROS Reproductive Parameter Control Number of Replicates Total Eggs Laid Eggs Cracked Eggs Set Viable Embryos Live 3 -Week Embryos Hatchlings 14-Day Old Survivors Eggs Laid/Hen EggB Laid/Hen/Day1 14-Day Old Survivors/Hen 13 605 23 521 494 490 473 457 47 O.SO 35 1B ased on 93 d ays of e gg s production. - D ata are not available due to adult m ortality Experimental Group (ppm a .i .) 10 50 13 592 3 526 470 466 414 386 46 0.49 30 - - 150 _ - - - Project Number 454- Figure 4 Mean R e p ro d u c tive Perform ance from a N orthern Bobuhite Reproduction Study uith PFOS 50 '' ---- - I O Project Number 454-108 E<w * Lai<J Edg* Eggs U iablv U v * 3-ofe . Crackad Sat Embryos Eabryos * Data a ra not a v a lla b ia due to adult a o r ta litg H atch lin gs 1+-Day Old Su rvivo rs Wf ' Table 8 Summary of Reproductive Performance, N o r m a liz e d a s P e r c e n ta g e s (%) 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 ith PROS1 Reproductive Parameter Control Number of Replicates 2. Total Eggs Laid Eggs Laid/Maxitnum 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 (%) Hatchiings/Maximum Set (%) 1 4 -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 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 N /A - No e g g s available due to adult mortality. * Significantly different from the control at p < 0.05 ^Values represent pen m eans for experim ental group. Values for each pen are presented in Appendices V II and V III. 2 oO Represents the total number of e ggs laid in each group. 1i U5 Project Number 454-108 too " 90 Figure 5 Mean Reproductive Perform ance, Norm alized a s P e rc e n ta ge s (X), from a N o rth e rn Bobuhite R e prod u ction Study uith PFOS 48234853238923022348485353482323 80 70 23482348535353234848484853535323 Li 60 C<CD SO U aCJ La i 40 <E 30 L3"i 20 KL>. 10 0 Eggs Laid / MaxiM ia Laid Egg* Crackedx Egg* Laid V ia b le Eabryosx U v e 3-Ueek/ E ggs Set U iab le Eabryos H atch lin gs/' U v e 3-Week Reproductive paraaeter 14-Oay 01dx H atch lin gs Project Number 454-108 E3o 5 10 Concentrations, ppa a.i. 1771SO G B ISO O o u :; Table 9 Mean E g g a h e ll T h ic k n e s s Measurements (mm) from a N o r th e r n B o b w h lte R e p r o d u c tio n S tu d y w ith 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 - - D ata are not available due to adult m ortality Differences between the control and each treatment groups were not significant (p > 0.05). Project Number 454-108 *v . '**-- (- Tabla 10 Mean B ody W eigh t ( g ) o f H a tc h lin g s and 1 4 -D ay O ld S u r v i v o r s 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 Experimental Group (PPM A.I.) Hatchlings -- ------------------------------ -- -------- Number Mean ( SD) 14-Day Old Survivors ---------------------------- Number Mean ( SD) Control 10 50 150 472 6 0 412 6 1 - _ 457 386 - - 29 2 30 3 - D ata are not available due to adult mortality The number of hatchlings weighed m ay differ from the total number of hatchlings since those hatchlings found dead were not w eighed. Differences between the control and each treatment group were not significant {p > 0.05). Project Number 454-108 Wildlife International, Ltd, -45- Project Number 454-108 Appendix I Certificate of Analysis Centre Analytical Laboratories, Inc 348 Aoaearch belva state cotoga, m i 8801 www.cenkelalxeom Phon (814)231-8082 Fax: (14) 231-1253 or (614)231-1580 INTERIM CERTIFICATE OF ANALYSIS IoM a5 Centre Analytical Laboratorio CO Reference #: 0234)18A 3M Product: PFOS,Lot217 Refertuc* 6: SD-018 Parity: 86.9% 1STO SpedOeatisaa Refait P a rtly ' -----------R 3 5 ----------- Appearance Identification NMR White Cryatalline Powder Conforma 1 Poaitive Itisi 1 pilli j i S 2 2 E lE ifflS I iIi3 a Total % Impurity (LC/MS) Total % Impurity (OOMS) belated Compounds POAA Purity by DSC inoriamo Anione (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate' Organic Acida ' (IC) 1. ITA 2. PFPA 3. HFBA 4. NFPA Elemental Anatyai*4: 1. Carbon 2. Hydrogen 3. Nitrogen 4. SulAir 5. Fluorine I 1. Theoretical Value " 17.834 2. Theoretical Value " 034 3. Theoretical Value-034 4. Theoretical Value =5.95% 5. Theoretical Value = 60% 1. 0.005 wt/wt% 2. 0.001 wt/wt% 3. 1.439 wt/w t% 4. 6.849 wt/w t% 5. <0.001 wt/wt.% 6. 0-005 wt/wt% 7. <0.001 wt/wt.% 1.9 wt/wt.% 8.41 w t/wt% None Detected 0.33 wt/wt.% None detected Not Applicable* I <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. 8.76 wt/wt.% 1. 0 .1 wt/wt% 2. <0.1 wt/wt.% 3. 0.10 wt/wt.% 4. 0.28 wt/wL% 1. 12.48 wt/wt% 2. 0.244 wt/wt.% 3. 1.74 wt/wt.% 4. 8.84 wt/wt.% 5. S4.1 w t/w t% COA023-01IA JPagc I of? Wildlife International, Ltd, -46- Project Number 454-108 Appendix I Page 2 Certificate of Analysis Centre Analytical Laboratories. Inc 30 4 R m in h Drive State College, 1B801 www.centrulab.com Phons: (814)231-8032 F*)C(814)231-1263Of(814)231-1580 INTERIM CERTIFICATE OF ANALYSIS Ktvititm3 C entre A sslytlcal Laboratorie COA Reference #: 01341IA Date o f Last Analysis: 08/31/00 Bxjiintion Date: 08/31/06 Storage Condition*: Frozen <-10*C Re-assessment Date: 08/3 IANS Purity -100% (turn o f metal impuritiea, 1.45% +LC/MS impuritiea, 8.41%+Inorgsnie Fluoride, 0.59%+NMR importtier, 1.905%+organic add impuritiea, 0.38%+POAA, 0.33%) Total impurity from all teste -13.07% Purity -100% -13.07% - 86.9% ^Potassium is expected in this salt form and is therefore not considered an impurity. *Purity by DSC ii generally not applicable to materials o f low purity. No endotherm was observed for this sample. `Sulfur in the sample appears to be converted to SO and hence detected using the inorganic anion method conditions. The anion result agree well with foe aulfor determination in the elemental analysis, lending confidence to this interpretation. Baaed on foe results, foe SO is riotconsidered an impurity. 'TPA HFBA NFPA PFPA Trifluoroscctic arid Heptafleorobiityric arid Nonafluoropentanoic acid Pentafluoroproptnoic arid `Theoretical value calculations based on foe empirical formula, C*F|TSQjK*(MW-538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-IBA Pate 2 of 3 Wildlife International, Ltd, -47- Project Number 454-108 Appendix 1 Page 3 Certificate of Analysis Centre Analytical Laboratories. Inc dd4a Re-- arch Drive State College, R4 1B801 www.centrelab.cpm Phon: (814) 231-8032 Fix: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS JUristoH3 Centre Analytical Laboratories COA Reference #: 023-018A LC7MS Purity Profile: - Impurity ~ wt/wt.% C? 122 C5 o s : e? ------------------ 332------------------;-- C7 1.14 T55 8.41 Note: The C4 end C6 values were calculated using the C4 nd C6 standard calibration curvet, respectively. The C5 value was calculated using tin avenge result from the C4 and C6 standard curvet. Likcwite, the C7 value wa* calculated using the avenge reult from the C6 and C8 standard curve*. Prepared By: -- it ^ jjn Chariee Simom Scientist, Centre Analytical Laboratories Date -Q L R A kReviewed By: Flaherty/ Date Laboratory Manager, Centre Analytical Laboratories COA023-01BA P * |s 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% of the ration, supplied the following amounts per ton: Amount Supplied Per Ton: Vitamin D3 Vitamin A Riboflavin Niacin Pantothenic Acid Vitamin Bi 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 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. Fermentation By-Products (Source o f Unidentified Growth Factors). C C O '::'-' 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 1,0 0 0 ,0 0 0 1,000 750 1,250 2,500 2.5 1,000 65 250 250 3,750 1 Per lb. 10,000,000 IU 4,000,000 ICU 4,000 IU 3,000 Mg 5,000 Mg 10,000 Mg 10.0 Mg 4,000 Mg 260 Mg 1,000 Mg 1,000 Mg 15,000 Mg INGREDIENTS: Vitamin A Supplement, D-Activated Animal Sterol (source o f Vitamin D3), Alpha Tocopheryl Acetate (source of 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 mix was prepared as a ration o f approximately 2 grams o f Durvet vitamins and electrolytes to approximately 1 gallon o f water. G * 0 `3 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 50ppma.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 Flobart 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, reweighed, and stored frozen. As needed, the appropriate premix was incorporated into the final diet as follows: Oppm 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 APPENDIX IV ANALYTICAL METHODS AND RESULTS INSTRUMENT: Table 1 Typical HPLC/MS/MS Operational Parameters Hewlett-Packard M odel 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 M ass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in multiple ion reaction monitoring (M RM ) mode. ANALYTICAL COLUMN: GUARD COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: PFOS RETENTION TIME: PFOS MONITORED MASS: Keystone Betasil C ]8 column (50 mm x 2 mm I.D., 3-p.m particle size) Keystone Javelin C )8 column (20 mm x 2 mm I.D.) 40C 5.00 minutes 250 pLJminute 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 0 0 '.5 - -, 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 following equation: peak area - (y-intercepf) PFOS (mg a.i ) at instrument = slope Determination o f Sample Residues (PFOS) The concentration, expressed as ppm a.i., for each sample was determined using the following equation: PFOS (mg a.i./L) at instru. x extract final volume (L) x dilution factor PFOS (ppm a.i.) m sample = -------- ------------------- initial weight (Kg)------- ---------------------- Determination of Limit of Quantitation (LOQ) The method LOQ, expressed as ppm a.i., was determined using the following equation: LOQ (ppm a.i.) = lowest standard concentration (mg a.i7L) x overall dilution factor of 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 of the method at that level o f fortification. ppm a.i. measured in sample __ % Recovery = " -- _p_p_m___a_.i_. fortified r X 1 0 0 W ildlife International, Ltd Project Number 454-108 -54- APPENDIX IV Table 3 Matrix Blanks and Fortifications Analyzed Concurrently with the Samples Number (454-108-) MAB-1 MAS-1 MAS-2 MAS-3 MAB-2 MAS-4 MAS-5 MAS-6 MAB-3 MAS-7 MAS-8 MAS-9 MAB-4 MAS-10 MAS-11 MAS-12 Sample Type Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Interval Week 1, Day 0 Week 1, Day 0 Week 1, Day 0 Week l,D ayO 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 Concentration o f PFOS (ppm a.i.) Fortified1 Measured1,2 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 < 2.00 4.85 52.8 206 < 2.00 4.55 47.4 189 < 2.00 5.50 52.0 200 < 2.00 5.20 26.1 100 Percent Recovery 97 106 103 91 95 95 110 104 100 _ 104 104 100 Mean Recovery 00 102 94 105 103 1Less than values correspond to limit o f quantitation (LOQ). 2Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly. sJ W ildlife International, Ltd, Project Number 454-108 Number (454-108-) MAB-5 MAS-13 MAS-14 MAB-6 MAS-15 M AS-16 MAS-173 MAS-18 MAB-7 M AS-19 MAS-20 APPENDIX IV Table 3 (Continued) Matrix Blanks and Fortifications Analyzed Concurrently with the Samples Sample Type Interval Concentration o f PFOS (ppm a.i.) Fortified12 Measured1,2 Percent Recovery 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 Week 12, Day 0 Week 12, DayO Week 12, DayO Week 16, DayO Week 16, DayO Week 20, Day 0 Week 20, DayO Week 20, Day 0 0.00 5.00 25.0 0.00 5.00 25.0 5.26 25.0 0.00 5.00 25.0 < 2.00 5.40 27.5 < 2.00 5.21 24.4 5.39 24.5 < 2.00 4.86 24.0 108 110 _ 104 98 102 98 _ 97 96 Mean Recovery 00 109 101 100 97 1Less than values correspond to limit of quantitation (LOQ). 2Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly. 3Week 16 samples were held and batched with Week 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 Mixing 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 Deviation (SD) Coefficient of Variation (CV) x = 9.75 ppm a.i SD = 0.313 ppm a.i. CV = 3.21% x = 50.1 ppm a.i. SD = 0.981 ppm a.i. CV = 1.96% - x = 147 ppm a.i. SD = 4.31 ppm a.i. CV = 2:93% Mean Percent of Nominal 98 100 98 W ildlife Internationalj Ltd. Project Number 454-108 -57- Nominal Concentration (ppm a.i.) 0 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 0 of Week -) 2 3 4 8 12 16 20 Measured PFOS Concentration1 (ppm a.i.) <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 M ean(x) Standard Deviation (SD) Coefficient o f Variation (CV) - Percent of Nominal - Mean Percent of Nominal - 10 35 2 36 2 42 3 43 3 49 4 50 4 56 8 57 8 61 12 62 12 66 16 67 16 71 20 72 20 1Less than values correspond to limit of quantitation (LOQ). 11.0 110 9.68 97 9.53 95 10.7 107 9.70 97 9.60 96 10.6 x= 10.2 ppm a.i. 106 102 11.2 SD = 0.553 ppm a.i. 112 10.4 CV = 5.44% 104 9.86 99 9.82 98 10.4 104 10.1 101 9.69 97 '-V w y' 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 of Week -) 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 of Variation (CV) X = 20.8 ppm a.i. SD = 2.14 ppm a.i. C V = 10.3% x = 50.9 ppm a.i. SD = 2.04 ppm a.i. CV = 4.01% x = 161 ppma.i. SD = 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 -58- 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.) 0 10 50 150 Sample Number (454-108-) 1 2-7 8-13 14-19 Week 1, DayO Mean Measured (ppm a.i.) <2.00 9.75 50.1 147 PFOS Concentration Mean Percent of Nominal 98 100 98 Sample Number (454-108-) 20 21 22 23 24 25 26 Week 1, Day 7 Measured12 (ppm a.i.) Mean Measured (ppm a.i.) < 2.00 9.74 11.2 50.1 50.5 148 154 <2.00 10.5 50.3 151 1Day 0 values are from homogeneity samples presented in Table 4. 2Less than values correspond to limit o f quantitation (LOQ). Mean Percent of DayO 108 100 103 -59- o W U;' Q W ildlife International, Ltd, -60- Project Num ber 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. I 4- Weigh 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. Allow the samples to shake for a minimum o f 30 minutes at approximately 250 rpm. 4 Vacuum filter with qualitative filter paper and rinse retained feed 3 times with methanol into filtrate. 4 Transfer the filtrate into a 200-mL volumetric flask and bring to volume with methanol. 4 Prepare appropriate dilution(s) to bring final concentration into the calibration range of the HPLC/MS/MS methodology. Use methanol for intermediate dilutions, if required. For all final dilutions, use 50% methanol : 50% NANOpure water dilution solvent. 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). -62- W ildlife International, Ltd. Project Number 454-108 APPENDIX IV PFO SJ STD 0.00100 mg a .lA 4675A-011D -1 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 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 3.79 End Time Integration Width 4.37 0.59 Retention Time Integration Type 4.02 A BB Wed, : eb 7, 2001 17:58 100-j 90- 80- 70- 60- 50- 40 30 20 10 29 49 66 111 138159 <H T " I....- I - I------ 1 I I I 41 81 121 161 0.69 1.36 2.03 2.70 Intensity: 10000 cps 281 Scan 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). GCOb W ildlife International, Ltd, -63- Project Number 454-108 APPENDIX IV PF0S_5 STO 0.0100 mg I.IA . 4675A-011D -S 4.96 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 35 RT Win, (secs) 20 Smooth 1 Expected RT 4.02 Area 114007 Height 10269 Start Time End Time Integration Width Retention Time Integration Type 3.80 4.39 0.59 4.02 Ai-B B Wed Feb 7,2001 1B'21 intensity: 10000 cps Figure 4. Typical ion chromatogram of a high PFOS calibration standard, 0.0100 mg a.i./L. (Monitored mass 499.0 -- 99.1). 0-O O fcO -64- W ildlife International, Ltd. Project Number 454-108 APPENDIX IV PFOS 7 DAB-3 454-108,109- Wed, Feb 7, 2001 18:33 4.98 In 1 period pros No Internal Standard Use Area intensity: 10000 cps 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Hires. Quant Hires. Min. Width Mult. Width 5 .0 1.0 3 6 Base. Width RT Win. (secs) Smooth Expected RT 35 20 1 4.02 Area 0 Height 0 Start Hme 0.00 End Time 0.00 Integration Width 0.00 Retention Time 0.00 Integration Type 90- 80 70 60 50 40 1 30 20 10 o4 23 3-9 , I-- --1r0.5.I 1130I 1!64 r 2I 05 I "2"4I "0 I 27- 4I 1...... 41 81 121 161 201 241 281 Scan 0.69 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 time of PFOS. (Monitored mass 499.0 99.1). G' -65- W ildlife International, Ltd. Project Number 454-108 APPENDIX IV PFO SJ MAS-8 454-108,109- Wed, Feb 7, 2001 18 45 4.98 In 1 period PR38 No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres, 5.0 Quant Hires. 1.0 Min. Width 3 Mult. Width 6 Base. Width 35 RT Win. (secs) 20 Smooth 1 Expected RT 4.02 Area 60579 Height 5641 Start Time End Time Integration Width Retention Time Integration Type 3.80 4.39 0.59 4.02 A -B B 100 90 80 70 60 50 40 30 20 10 15 38 O' 41 0.69 intensity: 10000 cps 88 126 A JL209 i 176 ,, 280 81 121 i i 201 241 281 Scan 1.36 2.03 2.70 3.37 4.04 4.71 Time \ 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 P F0 SJ3 42 454-108,109- Wed, Feb 7,2001 19:10 4.98 in 1 period pros No Internal Standard Use Area 1; 4.97 MRM, 298 scans 499.0->99.1 Noise Three. 5 .0 Quant Tines. 1.0 Min. Wkftii 3 Mult. Width 6 Base. Width 35 RT Win. (secs) 20 Smooth 1 Expected RT 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 intensity; 10000 cps 100 90 80 70 60 50 40 30 20 10 210 _ 21 48 78 0 t~ I I i - i ' i 41 81 0.69 1.36 129 157178 / V . t I "I-- T *i " r 121 181 201 241 2.03 2.70 3.37 4.04 281 Scan 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 o f 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. -00 -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. Of -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 Definition - The average weight of hatchlings by parental pen of origin, taken immediately upon removal from the hatcher. Hatchlings from each weekly lot of eggs are weighed. Mean weight for each week and mean weight by pen are reported. Sensitivity - This is an occasionally affected parameter and may reflect some residual or latent toxic effects from chemical residue in the egg. 8. 14-Day Old Survivors Definition - Birds that survive brooding for two weeks following hatch. Sensitivity - This is a seldom affected parameter and is probably more indicative of the conditions under which the birds were reared in battery brooders than the chemical to which the adults were exposed. 0 0 0 IS 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 o f 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. c< tO ' -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 Adult Body Weight (g) from a Northern Bobwhite Reproduction Study with PFOS (C ontrol, M ales) Pen 201 202 203 204 205 206 >201 208 209 210 211 212 213 214 215 216 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 Teat 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 l l l 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 m eans for body w eights and body w eight changes are calculated and rounded separately. Project Number 454-108 Appendix VII - Table 2 Adult Body Weight (g) from a Northern Bobwhlte Reproduction Study with PFOS (C on trol, 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 W e e k 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 0 201 6 .207 0 205 7 212 -4 224 4 228 0 220 2 222 -2 191 6 197 0 187 4 191 -2 197 4 201 - 1 2 11 7 218 2 217 1 218 -2 226 7 233 - 1 218 3 221 - 1 234 2 236 - 1 210 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 m eans for body w eights and body w eight changes are calculated and rounded separately. - Data are not available due to adult mortality. v w Project Number 454-108 Appendix VII - Table 3 Adult Body Weight (g) from a Northern BoJbwhite Reproduction Study with PP03 (1 0 PPM A. I . , M a l e s ) 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 4 227 2 219 0 192 - 1 197 2 189 2 214 - 1 210 6 197 0 220 0 208 2 204 -2 217 0 216 0 225 0 217 - 1 210 1 12 2 213 229 219 191 199 191 213 216 197 220 210 202 217 216 225 216 -4 -5 -2 6 0 -1 -4 -4 -6 -6 -3 -1 0 -1 -2 -3 2 11 -2 12 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 -11 -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 m eans for body w eights and body weight changes are calculated and rounded separately. Differences between control and this treatment group were not significant (p>0.05). Project Number 454-108 Appendix VII - Table 4 Adult Body Weight (g) from a Northern Bobwhite Reproduction Study with PFOS (1 0 PPM A . I . , F e m a le s ) 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 - 1 224 - 1 223 1 179 0 184 3 212 2 198 5 202 3' 197 3 227 1 234 - 1 201 -2 216 0 203 2 238 -4 193 2 208 1 17 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 m eans for body w eights and body weight changes are calculated and rounded separately. - D ata are not available due to adult mortality. Differences between control and this treatment group were not significant (p>0.05). Project Number 454-108 Appendix VII - Table 5 Adult Body Weight (g) from a Northern Bobwhite Reproduction Study with PFOS (5 0 PPM A . I . , M e le e ) 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 S 216 200 189 181 201 193 201 197 191 219 185 192 197 195 208 192 -19 ` -9 -31 -29 -7 -18 -3 -9 -1 -11 -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 1 7 9 *. 20 - - __ ---------------- _ -- _ - _ - Total Change _ - - - - The m eans for body w eights 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 O Project Number 454-108 y ' Appendix VII - Table 6 Adult Body Weight (g) from a Northern Bobwhlte Reproduction Study with PFOS (50 P m A . I . , Females) Pen 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 Mean SD Week o 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 - 1 206 -6 227 -5 2 1 1 -7 209 -4 196 -18 199 -6 202 -6 235 1 206 -8 204 - 1 1 223 -13 177 -2 225 -17 217 -6 218 -7 210 -7 14 5 198 - 1 200 -5 222 -7 204 -3 205 -6 178 0 193 -5 196 -26 236 - 1 198 - 1 1 193 0 210 -30 175 -2 208 -8 2 1 1 -13 211 1 202 -7 15 9 197 195 215 201 199 178 188 170 235 187 193 180 173 200 198 212 195* 17 2 -4 -3 -26 -24 -3 -2 10 -6 -13 -3 -51 2 -31 -12 -4 -11 15 199 191 212 175 175 175 186 180 229 174 190 129 175 169 186 208 185** 22 - - - _ __ ------- -- --------- --- _ - - - - Total Change _ - - - _ _ oo The m eans for body w eights 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 .V '-J? Project Number 454-108 f? ) V'"X' Appendix VII - Table 7 Adult Body Weight (g) from a Northern Bobwhlte Reproduction Study with PROS (1 5 0 PPM A . I . , M a le s ) 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 191 213 198 218 209 194 199 242 195 214 212 194 207 211 196 197 206 13 -22 -44 -15 -48 -34 -55 -32 -35 -53 -28 -21 -23 -25 -35 -47 -20 -34 13 169 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 1 172** 18 13 14 176 - 198 187 171 191 225 180 190 183 177 196 180 178 187** 14 _ - - __ ---------------- -_ -- _ ---------------- _- -- _ - _ - Total Change _ - - - - - - - - - - The m eans for body w eights 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 Body Weight (g) from a Northern BoJbwhite Reproduction Study with PFOS (1 5 0 PPM A . I . , F e m a le s ) 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 -44 -47 -36 -30 -35 -26 -22 -43 -47 -40 -41 -62 -62 -17 -44 -55 159 203 165 204 179 206 184 193 131 173 140 161 157 202 138 149 21 - 10 -70 13 17 -34 -4 6 18 23 29 10 15 1 -11 180 - 175 134 192 223 150 189 137 191 163 190 167 217 139 138 _ - __ --------------- - _ --------- -- ------- - - - 212 -41 2 1 13 172** 25 3 26 172** 29 - - -- -- -- -- - Total Change _ - - - - - _ The m eans for body w eights and body w eight changes are calculated and rounded separately. - D ata are not available due to adult mortality. * * Significantly different from the control at p < 0.01 Project Number 454-108 C0 'A `vj Appendix VIII - Table 1 Feed Consumption (g/bird/day) from a Northern Bohwhlte Reproduction Study with PFOS (Control) HEERS Pen 1 2 3 4. 5 6 7 8 9 10 1 1 12 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 12 12 12 12 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 21 19 19 19 12 14 13 13 13 13 12 16 14 15 15 16 17 20 21 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 11 14 13 14 15 16 19 21 20 20 2 1 2 1 2 1 22 20 12 15 13 13 12 12 12 15 14 14 17 18 19 22 21 21 2 1 21 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 21 2 1 21 22 22 21 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 11 11 1 222233333 32 Appendix VIII - Table 2 Feed Consumption (g/bird/day) from a Northern Bobwhite Reproduction Study with PROS (10 PPM A. I.) Pen 217 218 219 220 221 222 223 224 22S 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 12 13 12 13 13 12 12 14 12 1 1 16 16 19 21 21 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 21 20 22 20 15 10 12 1 1 12 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 IS 16 15 16 12 14 13 13 14 12 13 14 12 14 14 18 19 22 23 23 22 23 22 25 22 13 IS 14 14 14 14 13 15 14 15 16 17 19 21 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 21 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 21 22 21 2 1 2 1 22 20 1 1 1 1 1 1 12 12 1 1 11 1 1 10 1 1 12 13 15 17 18 17 18 18 17 13 13 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 2232222323 3 4 3 Differences between control and this treatment group were not significant (p > 0 .0 5 ). - a. Project Number 454-108 'A ` Appendix VIII - Table 3 Feed Consumption (g/blrd/day) from a Northern Bobwhite Reproduction Study with PFOS (50 PPM A.I. ) Pen 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 WEEKS 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 2 1 12 9 10 10 12 11 10 12 99a 9 1 1 10 9 9 10 10 1 1 10 9 9 8 5 11 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 9987 1 1 13 12 12 12 1 1 1 1 9 10 8 8 8 10 10 10 10 11 10 1 1 10 10 10 7 8 13 1 1 1 1 1 1 8 12 11 10 10 11 88 - --- - --- --- - - -- - - Project Number 454-108 Mean SD 1 1 ** 1 1 ** 1 0 ** 1 0 ** 9** 9** . 1 1 1 1 22 - - Data are not available due to adult mortality. * * Significantly different from the control at p < 0.01 Appendix VIII - Table 4 Feed Consumption (g/blrd/day) from a Northern Bobwhite Reproduction Study with 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 - 00 Project Number 454-108 Mean SD 7** 6** 8** 13 1122 - - O - D ata are not available due to adult mortality. W * Significantly different from the control at p < 0.01 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 - X X a' X X - - a - Bird found dead during the test. Necropsy findings are presented in Table 2. Project Numi 00 ST Males Feather loss Intestinal tract - area of hyperemia Ceca - round worms found Testes - one or both small (~1.0-1.5 cm) Not remarkable Appendix EX - 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 X . XXX _ X X-X- - - - XX * - - -X- -X- XXXX _ . -- - -X- oo ON Project Number 454-108 Females Pens 217 218 219 22 0 221 222 223 224 225 226 227 228 229 230 231 232 Feather loss Head lesions Liver - pale and mottled - . XXXX a X a X . XXX a X- - - -a- a- - - - - a - - X- - a - a - - - - - a Abdominal cavity - slight egg yolk peritonitis . _ aXa _ _a Intestinal tract - area of hyperemia - -. - a . aX . - . - a Not remarkable XX - - - -a - a -X- - - a -V a - Bird found dead during the test Necropsy findings are presented in Table 2. 'A Project Number 454-108 -87 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. `O * -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. cn OS -89- Wildlife International, Ltd. Project Number 454-108 Appendix XI Histopathology Report .o .a .vJ- !*v: EPL -90- EXPERIM ENTAL PATHOLOG Y LABORATORIES, INC. Project Number 454-108 W IL D L IF E IN TE R N A TIO N A L, LTD . S TU D Y NUM BER 454-108 EPL PR O JEC T NUM BER 212-027 PFO S: A R E P R O D U C T IO N S T U D Y W IT H T H E N O R T H E R N B O B W H ITE PATHOLOGY REPORT Subm itted by: Experim ental Pathology Laboratories, Inc. P.O . Box 474 Herndon, VA 20172 -0 474 (703) 471-7060 S ub m itted to: W ildlife International, Ltd. Easton, M D 21601 Decem ber 6,2001 EPL -91 - EXPERIM ENTA L PATHOLOG Y LABORATORIES, INC. Project Number 454-108 TABLE OF CONTENTS P A T H O L O G Y S U M M A R Y .................................................................... C O M P L IA N C E S T A T E M E N T .............................................................. Q U A L IT Y A S S U R A N C E FINAL C E R T IF IC A T IO N ........................ SUM M ARY INCIDENCE TABLES ADULTS M a le s .................................................................................................... Fem ales............................................................................................... HISTO PATHO LOG Y INCIDENCE TABLES M a le s .................................................................................................... Fem ales............................................................................................... CORRELATION OF GROSS AND M ICROSCOPIC FINDINGS M a le s .................................................................................................... F em ales............................................................................................... SUM MARY INCIDENCE TABLES O F F S P R IN G M a le s ................................................................................................... Fem ales............................................................................................... HISTOPATHOLOGY INCIDENCE TABLES M a le s .................................................................................................... F em ales Page 1 7 1-1 I-2 11-1 II- 3 III- 1 III-3 IV -1 IV -2 V-1 V -2 -92- Project Number 454-108 PATHOLOGY SUMMARY U.'- OOOi ' 7.4. - EPL -93- EXPERIM ENTAL PATHOLOG Y LABORATORIES, INC. Project Number 454-108 W IL D L IF E IN TE R N A TIO N A L, LTD. S TU D Y NUM BER 454-108 EPL PR O JEC T N UM BER 212-027 PFO S: A R E P R O D U C T IO N S T U D Y W ITH T H E N O R T H E R N B O B W H ITE PATHOLOGY SUMMARY Light m icroscopic exam ination w as perform ed on sections of selected tis s u e s fro m a d u lt m a le a n d fe m a le N o rth e rn b o b w h ite q u ail (Colinus virginianus) w hich w ere untreated or which received 10 ppm a.i. of th e test article (P e rflu o ro o c ta n e Sulfonic A cid, P o tassiu m S a lt [P F O S ]) in th e fe e d for approxim ately 21 w eeks. S elected tissues from untreated 1 4 day-old offspring of th e adult bobw hite w ere also exam in ed by light m icroscopy. T h e objective of this s tu d y is to e v a lu a te th e effe c ts u pon ad u lt b o b w h ite (Colinus virginianus) of dietary exposure to the test substance over at least a 21 -w eek period. T h e experim ental design w as as follows: Experimental Group Adults* Males F em ales Offsprina0 Males F em ales Control (0 ppm) 20 20 6 4 10 ppm a.i. 20 19 5 5 'Adults were euthanized after receiving t ne test substance in the diet for approximately 21 weeks. bOffspring did not receive the test substance in the diet. All offspring w ere euthanized on D ay 14 post-hatching. e e o ; r * EPL -94- EXPERIM ENTAL PATHOLO GY LABORATORIES, INC. Project Number 454-108 Wildlife International, Ltd. Study Number 454-108 MATERIALS AND METHODS A t the com pletion of the study interval, all adult bobw hite w ere eu thanized an d necropsies w ere perform ed by W ildlife International, Ltd. S elected offspring w ere also euthanized at 14 days of age. Tissues w ere collected by W ildlife In tern atio n al, Ltd. fro m adults a n d offspring, fixed in 1 0 % neutral b u ffered form alin, and sent to Experim ental Pathology Laboratories, Inc (EPL), w here th e y w e re e m b e d d e d in paraffin an d m a d e into hem atoxylin an d eo sin -stain ed sections on glass m icroscope slides. T h e following tissues from adult and offspring w e re exam ined by light m icroscopy a s available: liver, gallbladder, proventriculus, kidney, brain, gonad (ovary or testis), bursa of Fabricius, and adipose tissue. A ll tissues required b y protocol a re p resen ted in th e H istopathology Incidence Tables. M icroscopic findings for each tissue exam ined from each b o b w h ite a re listed in th e H istopathology In cid en ce T a b le s . M icroscopic c h a n g e s w e re grad ed o n e to five depending on severity. N ongradable findings (including autolysis such that a diagnostic interpretation could not b e rendered) a re listed a s p resen t (P ). All findings for all an im als a re su m m arized by sex, a g e group, and tre a tm e n t g ro u p in th e S u m m a ry In cid en ce T a b le s , to g e th e r w ith th e total n u m b e r of an im a ls in e a c h group for w hich tissues w e re exam ined . A tabulation of gross observations noted a t necropsy with the c o rre s p o n d in g m ic ro s c o p ic c h a n g e , if a p p ro p ria te , is p re s e n te d in th e C o rre la tio n of G ro s s a n d M icroscopic Findings ta b le s . T h e en tries in th e s e ta b le s w e re transcribed from th e gross necropsy observations provided by W ildlife International. Ltd. EPL -95 - EXPERIM ENTAL PATHO LO G Y LABORATORIES, INC. Project Number 454-108 Wildlife International, Ltd. Study Number 454-108 results E xcept for th ree control (3 3 4 , 34 2 , 3 5 2 ) and tw o 10 ppm (3 8 4 , 3 8 8 ) adult fem ales, all adults and offspring survived to the end of their respective study intervals. N o effects considered possibly related to test article (P F O S ) i ad m inistration w e re n oted in liver, kidney, brain, and proventricuius of a d u lt an d o ffsp rin g m a le s a n d fe m a le s , b u rs a of F a b ric iu s in offsp rin g m a le s a n d fe m a le s , adipose tissue of adult m ales and fem ales, ovaries of adult an d offspring fem ales; and testes of m ale offspring. A ny potential test-article effects on the b u rs a of F ab riciu s in ad u lt m a le s a n d fe m a le s o r a d ip o s e tis s u e in m a le a n d fe m a le offspring could not be determ ined since none (bursa of Fabricius) o r only one (adipose tissue) sam ple w as subm itted for evaluation. T h e bursa of Fabricius typically reg resses prior to sexu al m aturity. In addition, a n y potential test-article effects on the gallbladder from treated adult m ales and fem a le s could not be definitively determ ined due to th e extensive autolysis that precluded any accurate diagnosis. T h e re w ere no apparent test-article related effects noted on th e gallbladders from th e offspring. T e s te s of all adult m ales exhibited norm al sp erm ato g en esis, c h a ra c te rize d b y th e p re s e n c e of n u m ero u s m aturing o r m a tu re s p e rm a to z o a in th e sem inifero u s tubules. In o n e (1 ) control an d seven (7 ) tre a te d m a le s , th e te s te s w e re o b s e rv e d m acroscopically a s bein g s m a lle r in s iz e (< 1 .5 c m ) w h e n com pared to other birds. In th ese birds, sperm atogenesis w as evident m icroscopically an d th e re w a s no definitive differen ce in th e m orphology o f th e testes w hen com pared to their unaffected cohorts. Th erefo re, no definitive m icroscopic changes could be associated with the size reduction. H o w ever, the reduced size of the testes of these birds w as consistent with early postreproductive ph ase regression, a norm al physiological phenom enon. T h e in c re a s e d in c id e n c e of red u ced s ize of th e te s te s in 1 0 pp m a .i. m a le s w h e n -3 - coo EPL -96- EXPERIM ENTAL PATHOLOG Y LABORATORIES, INC. Project Number 454-108 Wildlife International, Ltd. Study Number 454-108 com p ared to controls, suggests that treatm en t m ay h ave accelerated the norm al post-reproductive phase regression. T e s te s of all offspring w e re listed a s im m atu re b e c a u s e th e y lacked all evidence of sperm atogenesis, had sm all sem iniferous tubules with absent or narrow lum ens, an d germ inal epithelium with d ecreas ed cell layers (com p ared to ad u lt te s te s ). T h is m orphology is consistent with n o rm al physiological im m atu rity e x p e c te d in 1 4 d ay-o ld birds. B e c a u s e of th e sm aller s ize of th e sem iniferous tu b u les in th e offspring testes, th e m elan in p ig m en t-b earin g cells norm ally p re s e n t in th e te sticu lar interstitium w e re , in effect, m o re clo sely p a c k e d in a sm aller area rather than being w idely separated by larger sem iniferous tubules (th u s th e m ic ro s c o p ic d ia g n o s is o f "p ig m e n t c o n c e n tra tio n " in th e te s tis ). O varies of adult fem ales exhibited norm al oogenesis, characterized by n u m e ro u s o v a in various s ta g e s of follicular d e v elo p m en t. All o varies of offspring fem ales w ere characterized by num erous oocytes and very sm all, n o n p e d u n cu lated follicles c o m p letely e m b e d d e d in a p ro m in en t o varian cortex. T h is m orphology is consistent with th e norm al physiological im m aturity exp ected in 14 day-old birds. S e v e ra l ad d itio n a l m ic ro s c o p ic fin d in g s (d e s c rib e d b e lo w ) w e r e n o te d in adult and/or offspring bobw hite quail from control and treated groups. G enerally, in both adult a n d offspring m a le s a n d fe m a le s , th e in cid en ce of th e s e lesions w a s v a ria b le , w ith s o m e lesions occurring a t a h ig h er in cid en ce in controls w h e n co m p ared to treated birds, an d other lesions having the opposite distribution. In ad d itio n , th e re w a s no significant d iffe re n c e in th e m e a n se v e rity o f th e s e lesio n s in 1 0 ppm a .i. tre a te d birds w h e n c o m p a re d to controls. F o r th e s e reaso n s, th ese findings w ere considered incidental and unrelated to test article (P F O S ) adm inistration. 4- - ono EPL -97- EXPERIM ENTAL PATHO LO G Y LABORATORIES, INC. Project Number 454-108 Wildlife International, Ltd. Study Number 454-108 M o n o n u c le a r cell infiltrates, o b s e rv e d in vario u s tissu es, co n sisted o f foci of lym phocytes an d plasm a cells generally accom panied by a fe w heterophils as a m inor com ponent. M ineralization in th e kidneys of both control and tre a te d ad u lt m a le s w a s ch a ra c te rize d b y th e p re s e n c e o f basophilic m a te ria l in th e cortex. In m ost c a ses this m aterial a p p e a re d to b e asso ciate d with th e glom erulus. V a cu o lizatio n in th e liver w a s c h a ra c te rize d b y th e p re s e n c e of v a c u o le s within the hepatocellular cytoplasm , which varied from usually sm all, poorly d e m a rc a te d v a c u o le s , giving a "lace-like" a p p e a ra n c e to th e h e p a to c e llu la r cyto p lasm , to variab ly sized, sharply d e m a rc a te d , c le a r v a c u o le s in th e hepatocellular cytoplasm . W hile special stains w ere not em ployed to identify the m a te ria l in th e vacu o les, th e latter m o rp h o lo g y w a s co n sisten t w ith fa t accum ulation. H e p a to c e llu la r vacuolization w a s o b s e rv e d in th e livers o f ad u lts a n d offspring of both sexes. Although th ere w as a slight in c re a s e in th e incidence of this c h a n g e in 1 0 ppm a.i. tre a te d adult m a le s an d fe m a le s , w h e n c o m p a re d to controls, th e re w a s no significant d ifferen ce in th e m e a n s everity b e tw e e n th e gro u p s. S in c e this finding w a s also c o m m o n ly o b s e rv e d in th e livers from control b ird s , it w a s c o n s id e re d to b e in c id e n ta l a n d n o t re la te d to te s t a rtic le ad m in istratio n . T h e a p p a re n t in creased in cid en ce in th e tre a te d birds w a s considered to b e due to biological an d /o r distributional variability. CONCLUSIONS AND SUMMARY N o lesions considered possibly related to test article (P F O S ) ad m in istratio n w e re noted in liver, kid n ey, brain, a n d p ro ven tricu lu s of a d u lt a n d offspring m a le s an d fem ales, bursa of Fabricius in offspring m a le s a n d fe m a le s , adipose tissue of adult m ales and fem ales, ovaries of adult an d offspring -5- 0 f> _ EPL -98- EXPER IM EN TA L PATHO LO G Y LABORATORIES, INC. Project Number 454-108 Wildlife International, Ltd. Study Number 454-108 fe m a le s , a n d te s te s of m a le offspring. T h e in crease in th e n u m b e r of ad u lt m a le s in th e 1 0 p p m a.i. tre a te d group with re d u ced testicu lar s ize , not a c c o m p a n ie d b y a n y m o rphological c h a n g e in s p e rm a to g e n e s is , suggests th a t P F O S m a y h a v e accelerated early post-reproductive phase regression, a norm al physiological p h e n o m e n o n . A n y potential te s t article effects on th e b u rsa of Fab riciu s in ad u lt m a le s a n d fe m a le s or a d ip o s e tissu e in m a le an d fe m a le offspring could not b e d eterm ined since none (bursa of Fabricius) or only one (adipose tissue) sam p le w a s subm itted for evaluation. In addition, an y potential test-article effects on th e gallbladder from treated adult m ales and fem ales could not b e definitively determ ined d u e to th e extensive autolysis th at precluded an y accurate diagnosis. T h e fe w o th e r c h a n g e s in v ario u s tis s u e s o f ad u lt a n d /o r offspring b o b w h ite fro m control and 10 ppm a.i. treated groups w ere considered incidental and unrelated to test article (P F O S ) adm inistration. FA R /lcp FREDERI V e te rin a ry D a te plm ate, A C V P EPL. -99EXPERIM ENTA L PATHOLOG Y LABORATO RIES, INC. Project Number 454-108 C O M P LIA N C E S T A T E M E N T Client Nam e W ildlife International, Ltd. Client Study 454-108 E P L Project Coordinator Dr. M argarita M. Gruebbel EPL P a th o lo g ist I Dr. Frederick A. Ruecker Species Northern Bobwhite E P L Project Num ber 212-027 Study Title P FO S: A Reproduction Study with the Northern Bobwhite Test Article Perfluorooctane Sulfonic Acid, Potassium S alt (P F O S ) T h e H isto p ath o lo g y portions of th e a b o v e -re fe re n c e d study w e re co n d u cted in com pliance with the G ood Laboratory Practice regulations of the Environm ental Protection A gency as stipulated by 4 0 C F R 160. -7 <H 0 EPL -100- EXPERIM ENTAL PATHO LO G Y LABORATORIES, INC. Project Number 454-108 QUALITY ASSURANCE FINAL C ERTIFICATIO N Study Title: PFO S: A Reproduction Study with the Northern Bobwhfte Client Study: 454-106 EPL Project Coordinator: Dr. M argaritaM . Gruebbel EPL Project N u m b er 212-027 EPL Pathologist: Dr. Frederick A. Ruecker T h e following aspects of this study w ere inspected by the Quality Assurance Unit of Experimental Pathology Laboratories, Inc. Dates inspections w ere performed and findings reported to the EPL Project Coordinator and Managem ent are indicated below. Area Inspected ____________________________ Dates___________________________ ________ Inspection________ _________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 D ate reported to Study D irector/M anagem ent________________ 1/2 8 /0 3 Date of last quarterly facility inspection______________________ 11/02 Form No. 6-2 (7/2/99) -8 - -101 - Project Number 454-108 S U M M A R Y IN C ID E N C E TA B LES 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-ppmal (20) BRAIN (NO. EXAMINED) Inflammation, Meninges, Granulomatous M ineralizing Granuloma, Meninges (20) (20) 12 BURSA OF FABR1C1US (NO. EXAMINED) GALLBLADDER (NO. EXAMINED) (18) (20) Autolysis Precludes Diagnosis 18 18 KIDNEY (NO. EXAMINED) I n f il tr a te , Mononuclear Cell Inflaimiation, Chronic M ineralization (20) (20) 15 24 LIVER (NO. EXAMINED) (20) (20) Degeneration, Hepatocellular, C en trilo b u lar I n f il tr a te , Mononuclear Cell 13 7 Necrosis, Hepatocellular Vacuolization, Hepatocellular 16 20 PROVENTRICULUS (NO. EXAMINED) I n f il tr a te . Mononuclear Cell Inflam nation. Serosa (20) (20) 2 TESTIS (NO. EXAMINED) I n f il tr a te , Mononuclear Cell Reduced Spermatogenesis (20) (20) 11 1 1 EPL Sperimentai Pathology Laboratories, Inc. 1-1 -103- Project Number 454-108 454-108 Adult S acrifice 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 M ineralizing Granuloma, Meninges (20) (19) 1 BURSA OF FABRICIUS (NO. EXAMINED) GALLBLADOER (NO. EXAMINED) (19) (19) Autolysis Precludes Diagnosis 18 17 KIDNEY (NO. EXAMINED) I n f il tr a te , Mononuclear Cell Inflammation, Chronic M ineralization (20) (19) 14 1 3 LIVER (NO. EXAMINED) (20) (18) Degeneration, Hepatocellular, C en trilo b u lar 1 I n f il tr a te , Mononuclear Cell 4 8 Necrosis, Hepatocellular 12 Vacuolization, Hepatocellular 7 12 OVARY (NO. EXAMINED) PROVENTRICULUS (NO. EXAMINED) I n f il tr a te , Mononuclear Cell Inflammation, Serosa (20) (19) (20) (19) 23 1 EPL Experimental Pathology laboratories, Inc. 1-2 - 104- Project Number 454-108 H IS T O P A T H O L O G Y IN C ID E N C E TA B LES ADULTS -105 - Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE GROUP m um . 454-108 Adult S acrifice Male Northern Bobwhite ADIPOSE TISSUE Inflammation. Chronic A N i M A 33333333333333333333 L 2 3 3 3 3 3 4 4 4 4 4 5 5 5 5 5 61 6 6 6 s9 1 3 5 7 <? 1 3 5 7 9 1 3 7 9 1 3 3 7 X X X X X X X X X X X JL X X X X X ?1 X BRAIN Inflamnation. Meninges. Granulomatous M ineralizina Granuloma. Meninges XX X X X X X X X X JL JL X X X X X jX X P BURSA OF FARR1CIUS GALLBLADDER Auto1v sis Precludes Diaanosis N N N N N N N N N N N N N N N N N NX X XA A A A A A A A A N A A J L X X X X X X PPPPPPPPP P P P P PX XXX KIDNEY I n f i l t r a t e . Mononuclear Cell Inflamnation. Chronic M ineralization XXXXXX XX XXX 1 X X X XX X 11 LIVER Degeneration. Hepatocellular. Centrilobular I n f il tr a te . Mononuclear Cell Necrosis. Heoatocellular Vacuolization. Heoatocellular PR0VENTR1CULUS I n f il tr a te . Mononuclear Cell Inflammation. Serosa X 1 1 11 1 11 1 2 1 1 X X ? 1 22 1 1 1?? ? 1 ] 1 XX \ X X X X X X X X X X X X X JL X X X X X X TFSTIS I n f il tr a te . Mononuclear Cell Reduced Spermatogenesis Xm Xm Xm Xm Xm Xm Xm Xm Xm Xm Xm Xm Xm m XflL XflL XflL XflL XflL x. | I E PL ii-i Experimental Pathology Laboratories, Inc. Key : X-Hot Remarkable K-Ko Section I-Ineonplete A-Autolysis l-n1n1al 2*sl1ght/1ld Smoderate 4moderately severe 5-severe/h1gh P-Present B-Benlgn M*Na11gnant n-mlssing one paired organ (^unscheduled sac./death -106- Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE GROUP - Dimu__________________________ 454-108 Adult S acrifice Male Northern Bobwhite ADIPOSE TISSUE Inflammation. Chronic A N I M A 33333333333333334444 l 6 7 7 7 7 7 8 8 8 8 8 9 9 9 9 9 O' 0 0 0 9 13s 7 9 13s 7 9 1357 91 357 X X X X XX XX XX X X XX X X X X X X BRAIN Inflamnation. Meninges. Granulomatous M ineralizing Granuloma. Meninaes BURSA OF FABRICIUS GALLBLADDER Autolvsis Precludes Diaanosis KIDNEY I n f i l t r a t e . Mononuclear Cell Inflamnation. Chronic M ineralization LIVER Deaeneration. Keoatocellular. C en trilo b u lar I n f i l t r a t e . Mononuclear Cell Necrosis. Hepatocellular Vacuolization. Heoatocellular PROVENTRICULUS I n f il tr a te . Mononuclear Cell Inflammation. Serosa XXX XX X X X X X X X JLJ L X X X X Pp t N N N N N N N N N N N N N N N1N N N N X A A A A A A A A A A A A A A A XX AX X PPPPPPPPPPPPp P P PP X XX XXX XXX X 1 1 11 1 X XX 1 JL 1 J _ _____ i1__ 11 11 1] X 1?? ? 112 1 1? ? 1 1 12 1 122 1 X X X X X XX X XXX X X XX XX X 1 _L TESTIS I n f il tr a te . Mononuclear Cell Reduced SDermatoaenesis Xm Xm Xm Xm Xm Xm Xm Xm Xm Xm xm Xm Xm n| Xm Xbl Xm Xbl Xm JIL X 3 | EPL 11-2 Experimental Pathology Laboratories, Inc. Kay : X-Not Remarkable N-No Section I-Incouplete A-Autolys1s 1-mlnlmal Z-s)1ght/1liJ 3-moderate 4-moderately severe 5-severe/high P-Present B-Ben1gn M-Mal1gnant m-mlsslng one paired organ u-unscheduled sac./death -107 - Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL 454-108 Adult S acrifice Female Northern Bobwhite ADIPOSE TISSUE Inflanmation. Chronic A N 1 M A 3 3 3i 3 3 3 3i 3 3 3 3 3i 3 3 3 3 3 3 3 3 l 33333444445555566666 g ? 4 fi a fi ? 4 fi fi g ? 4 fi 8 0 ? 4 fi 8 X X X X X N X X X X X JL X X XXX 1 21 BRAIN Inflanmation. Meninges. Granulomatous M ineralizina Granuloma. Meninges X X X X X X X X X X X XX X X X X X X X BURSA OF FABRICIUS N N N N N N N N N N N N N N N N NJL NJL GALLBLADDER Autolvsis Precludes Oiaanosis A X A A A A N A A A A A A A A A A A A Jl P P P P P P P P P P p JL P P P J L P JL KIDNEY I n f i l t r a t e . Mononuclear Cell Inflanmation. Chronic M ineralization XXXX X 1 X X X X X XXX XX X 1 1X LIVER Degeneration. HeDatocellular. C e n trilo b u la r I n f il tr a te . Mononuclear Cell Necrosis. Henatocellular Vacuolization. HeDatocellular OVARY XX X XX JL X XX 1 ? 1 3j? 3 J ___ 1 X X 1 1X X X X X X : X X X X X X X X X X X X X X JL PROVENTRFCULUS I n f il tr a te . Mononuclear Cell Inflanmation. Serosa X X X X X X X X X X X X X X i X X JL 1X !? I T' j 1 ' |f i 1 E PL n-3 Experimental Pathology Laboratories, Inc. Key : X-Not Renarkabi N-Ho Sectlon M ncaaplete A-Autolysls li1n1al 2-sl 1tiht/rql 1cJ 3-moderate 4noderately severe 5-severe/h1flh P-Present B-Ben1gn M-Mal1gnant m-m1ss1ng one palred organ u*un$cheduied sac./death eoo - 108- Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE GROUP 454-108 Adult S acrifice Female Northern Bobwhite ADIPOSE TISSUE Inflamnation. Chronic BRAIN Inflamnation. Meninges. Granulomatous M ineralizina Granuloma. Meninges 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 7 7 7 7 7 8 8 8 8 8 9 9 9 9 9 0 0' 0 0 0 ? 4 fi fl 0 P 4 fi 8 0 ? 4 fi fi ? 4 fi fi XNX N X NX X X X X XXXX X ?1 ! lX X X X X X X X X X X X X X X X XX X 1 BURSA OF FABRICIUS N N fl N N l N N N N N N N li N N N N N GALLBLADDER Autolvsis Precludes Diagnosis A AAXA AAAAA A AA XAAA A A P PP PP P PP P P P P P P P P P KIDNEY I n f il tr a te . Mononuclear Cell Inflammation. Chronic M ineralization LIVER Degeneration. Hepatocellular. C en trilo b u lar I n f il tr a te . Mononuclear Cell Necrosis. Hepatocellular Vacuolization. Hepatocellular OVARY X X XX 1 XN X X X X X X X X jX X 11 ? j ! X X xX 1 1 1 ?' 1 1 411 111 1 1 1 1 1j 1 1 1? 3 X X X X X X X X X X X! X X X X X X X X PROVENTRICULUS I n f il tr a te . Mononuclear Cell Inflamnation. Serosa X X XX XX X X X X X X X XX X 11 !! i i ! i ; ! E PL 11-4 Key : X-Not Renerkable N-No Section I-Inconplete A-Auto1ys1s Experimental Pathology Laboratories, Inc. 1ht/"nd 3"'od<r`te 4-"oderately 5evere P-Presnt B-Ben1gn M-NaUgnant m-mlsslng one paired organ u-unscheduled sac./death r- -109- Project Number 454-108 C O R R E LA TIO N O F G R O S S A N D M IC R O S C O P IC F IN D IN G S ADULTS 454-108 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult S a c rific e S p ecies: N orthern Bobwhite________________________________________ Sex: Hales G ro u p Id e n tificatio n : CONTROL - S a c rific e d A nim al" N um ber Client Topography / Site Client G ross Observations M icroscopic Observations 345 TESTIS Right te s t i s approximately 1.5 cm No C orrelating Lesion Project Number 454-108 - e* III-l 454-108 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult S a c rific e S p ecies: N orthern Bobwhite________________________________________ Sex: Males G r o u p Id e n tific a tio n : 10-ppmai Animal Number Client Topography / Site Client Gross Observations Microscopic Observations 371 TESTES Testes 1.25 cm No C orrelating Lesion (TESTIS) - Sacrificed 373 TESTES Testes 1.25 cm No C orrelating Lesion (TESTIS) 387 TESTIS Right t e s t i s 1.0 cm No C orrelating Lesion 389 TESTES Testes 1.0 cm No C orrelating Lesion (TESTIS) 391 TESTIS Right t e s t i s 1.0 on No C orrelating Lesion 393 TESTIS Right t e s t i s 1.0 cm No C orrelating Lesion 401 TESTIS Right t e s t i s 1.0 cm No C orrelating Lesion Project Number 454-108 111-2 454-108 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult S acrifice Species: N orthern Bobwhi te _______ ______________________________Sex: Females Group Identification: CONTROL - Unscheduled Sac./Death Animal" Number Client Topography / Site Client Gross Observations Microscopic Observations 342 OVARY Regressing No C orrelating Lesion to Project Number 454-108 I I 1-3 a 454-108 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult Sacrifice Species: Northern Bobwhite________________ __________ _____ Sex: Females G roup Identification: 10-ppmai Animal" Number Client Topography / Site Client Gross Observations Microscopic Observations 378 LIVER Paie, mottled Vacuolization, Hepatocellular - Sacrificed 408 LIVER Pale, mottled Vacuolization, Hepatocellular Project Number 454-108 e IH-4 3 o 454-108 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult S acrifice Species: Northern Bobwhite______________________________________ Sex: Females G ro u p Identification: 10-ppmai A rum ai Number Client Topography / Site 384 Client Gross Observations No coronary fa t Microscopic Observations No Cannent Required - Unscheduled Sac./Death OVARY Developing No C orrelating Lesion Project Number 454-108 _ 111-5 e U" -115- Project Number 454-108 S U M M A R Y IN C ID E N C E TA B LES O F F S P R IN G GOOi:' fe* 454-108 Offspring Sacrifice Hale Northern Bobwhite ^ - 116- Project Number 454-108 SUMMARY INCIDENCE TABLE GROUP GROUP CONTROL lO-DDM E PL Experimental Pathology Laboratories, Inc. IV-1 \Jf if ` - ' -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 FABRICIUS (NO. EXAMINEO) Lymphoid Depletion GALLBLADDER (NO. EXAMINED) Autolysis Precludes Diagnosis (4) 1 (4) (5) 1 (5) KIDNEY (NO. EXAMINED) (4) (5) LIVER (NO. EXAMINED) I n f il tr a te , Mononuclear Cell Vacuolization, Hepatocellular (4) (5) 1 OVARY (NO. EXAMINED) Immature (2) (5) 24 PROVENTRICULUS (NO. EXAMINED) (4) (5) I n f il tr a te , Mononuclear Cell -118- Project Number 454-108 H IS T O P A T H O L O G Y IN C ID E N C E TA B LE S O FFS P R IN G -119- Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE GROUP GROUP EPL Experimental Pathology Laboratories, Inc. Key : X-Not Remarkable N-No Section [-Incomplete A-AutolysH l-m1n1mal 2 * s 1 I d 3-moderate -moderately severe 5-severe/h1gh P*Present B-Ben1gn M-Mel1gnant in-missing one paired organ u-unscheduled sac./death -120- Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE GROUP GROUP E PL Experimental Pathology Laboratories, Inc. Key :X-Hot Remarkable N-No Section I-lncomplete A-Autolysls Im1n1mal 2-sl1ght/m1ld 3-moderate 4-ioderately severe 5-severe/h1gh P-Present B-Benlgn -Malignant m-m1s$1ng one paired organ u-unscheduled sac,/death -121 - W ildlife International, Ltd. Project Number 454-108 Appendix XIIA The Analysis of PFOS in Red Blood Cells and Sera Collected through Week 15 W ildlife International, Ltd -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-WkS 454-l08-361-Wk5* 454-108-362-Wk5 454-108-362-Wk5* 454-l08-363-Wk5 454- 108-363-Wk5* 454-108-364-Wk5 454-108-364-Wk5* 454- 108-365-Wk5 454-108-365-Wk5* 454-108-366-WkS 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 Analyte Date Date Found (w/mL) 5/18/01 5/18-19/01 NQ 5/18/01 5/18-19/01 NQ 5/18/01 V I8-19/01 NQ 3/18/01 5/18-19/01 NQ 5/18/01 5/18-19/01 NQ 5/18/01 5/18-19/01 NQ 5/18/01 5/18-19/01 NQ 5/18/01 5/18-19/01 NQ 5/18/01 5/18-19/01 NQ 5/18/01 5/18-19/01 NQ 5/18/01 5/18-19/01 0.00292 5/18/01 V18-19/01 0.00264 5/18/01 5/18-19/01 NQ 5/18/01 5/18-19/01 ______ m AVERAGE: 0.000440 STANDARD DEVIATION: 0.000993 * Duplicate injection NQ = Noc 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 jig/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 40 of 98 i `}f i -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-l08-402-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-WkS* 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 05I801DRD 05180IDRD 051801DR 051801DR 051801DR 05I801DR 051801DRD 05180IDRD 051801DRD 051801DRD 051801DRD 051801DRD 051801DRD 051801DRD Extraction Analysis Date Date 5/18101 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/22/01 5/18/01 5/18/01 5/22/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/22/01 5/18/01 5/18/01 5/22/01 5/22/01 S/18/01 S/22/01 5/18/01 5/22/01 5/18/01 5/18/01 5/18/01 5/22/0) 5/22/01 5/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (int/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 ng/mL for red blood cells and serum Exygen Research Page 41 of 98 1: f ` (f ' -124 Wildlife 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 052201 CD 052201CD 052201CD 052201CD Extraction Analysis Date Date 5/22/01 5/24/01 5/22/01 5/22/01 5/22/01 5/24/01 5/24/01 5/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (iie/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-Wltl0 454-108-361-Wkl0* 454-108-362-Wk!0 454-108-362-WklO* 454-108-363-WklO 454-108-363-WklO* 454-108-364-WklO 454-108-364-Wk 10* 454-108-365-WklO 454-108-365-Wk10* 454-108-366-Wkl0 454-108-366-WklO* 454-108-367-Wkl0 454-108-367-Wk10* 454- 108-368-WklO 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 0S1801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B Extraction Analysis Date Date 5/18/01 5/18/01 5/19/01 5/19/01 51/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/19/01 5/19/01 5/19/01 5/19/01 5/19/01 5/18/01 5/18/01 5/18/01 5/19/01 5/19/0t S/19/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/19/01 5/19/01 5/19/01 5/19/01 5/19/01 5/19/01 AVERAGE: STANDARD DEVIATION: Analyte Found (uc/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 G0013. .! /* . .afe 125- W ildlife International, Ltd, Project Number 454-108 Exygen Study No.: 023-066 Table XXX.Suminary of PFOS in Quail Red Blood Cell Samples at 10 ppm aJ. for Week 10 Sponsor ID 454-108-401-WklO 454-108-401-WklO* 454-108-402-WklO 454-108-402-WklO* 454- I08-403-Wkl0 454-]08-403-Wkl0* 454-108-404-WklO 454-108-404-WklO* 454-108-405-Wkl0 454-108-405-WklO* 454-108-406-WklO 454-108-406-WklO* 454-108-407-Wkl0 454-108-407-Wkl0* 454-108-408-WklO 454-108-408-WklO* Centre ro 0107108 0107108 0107109 0107109 0107110 0107110 0107111 0107111 0107112 0107112 0107113 0107113 0107114 0107114 0107115 0107115 Set Number 051801DRD 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 5/18/01 5/21-22/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/18/01 5/18/01 5/21-22/01 5/21-22/01 5/21-22/01 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 5/21-22/01 5/21-22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ue/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 * Duplicate injection LOQ = 0.01 fjg/mL for red blood cells and serum Exygen Research Page 43 of 98 e o: 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-Wk15 454-108-362-Wkl5* 454-108-363-Wk!5 454-108-363-Wkl5* 454-108-364-WU5 454-108-364-Wkl5* 454-108-365-Wk15 454-108-365-Wkl5* 454-108-366-Wk15 454-108-366-Wkl5* 454-108-367-Wk 15 454-l08-367-Wkl5* 454- 108-368-Wkl 5 454-108-368-Wkl5" 454-108-333-WU5 454-l08-333-Wkl5* Centre ID 0107116 0107116 0107117 0107117 0107118 0107118 0107119 0107119 0107120 0107120 0107121 0107121 0107122 0107122 0107123 0107123 0107124 0107124 Set Nnmber 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 5/22/01 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 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/21/01 5/22/01 5/22/01 5/21/01 5/22/01 5/21/01 5/21/01 5/21/01 5/21/01 5/22/01 5/22/01 5/22/01 5/22/01 5/21/01 5/21/01 5/22/01 5/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ng/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 pg/mL)) LOQ = 0.01 (ig/mL for red blood cells and serum For values recorded as NQ, 0.00005 (Jg/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, 127- Project Number 454-108 Exygen Sludy No.: 023-066 Table XXXII. Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm aa. for Week 15 Sponsor ID 454-108-401-Wk15 454-108-401-Wkl5* 454-108-402-Wkl5 454-108-402-WklS* 454-108-403-Wkl5 454-108-403-WklS* 454-108-404-Wkl5 454-108-404-WklS* 454-108-405-Wkl5 454-108-405-Wkl5* 454-108-406-Wkl5 454-108-406-Wkl5* 454-108-407-Wk 15 454-108-407-Wkl5* 4S4-108-408-Wkl5 454-108-408-Wk 15* 454-i08-383-Wkl5 454- 108-383-Wk 15* Centra 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 05210IAD 052101AD 052201CD 052201CD 052101AD 052101AD Extraction Analysis Date Date 5/21/01 5/21/01 5/22-23/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/21/01 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 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/22-23/01 5/24/01 5/21/01 5/24/01 5/21/01 5/21/01 5/22-23/01 5/22-23/01 AVERAGE: STANDARD DEVIATION: 1 1 Analyte 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 aj . 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 Date Analysis Date 5/22/01 5/22/01 5/24/01 5/24/01 5/22/01 5/22/01 5/24/01 5/24/01 AVERAGE: STANDARD DEVIATION: Anelyte Found (ua/mL) 361 356 106 114 234 144 * Duplicate injection LOQ = 0.01 Mg/mL for red blood cells and serum Exygen Research Page 45 of 98 coo; W ildlife International, Ltd Project Number 454-108 Exygen Study No.: 023-066 Table XXXTV. Summary of PFOS in Quail Serum Samples at 0 ppm a.i. for Week 5 Sponsor ID 454-108-361-Wk5 454-108-361-WkS* 454-108-362-Wk5 454-108-362-WkS* 454-108-363-Wk5 454-108-363-WkS* 454-108-364-Wk5 454-108-364-Wk5* 454-108-366-Wlc5 454-108-366-Wlc5* 454-108-368-W k 5 454-108-368-Wk5* Centre ID 0107154 0107154 0107155 0107155 0107156 0107156 0107157 0107157 0107159 0107159 0107160 0107160 * Duplicate injection Set Number 072301AR 072301AR 072301AR 072301A R 072301A R 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR 072301AR Extraction Date Analysis 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: 1 Analyte Found (usfaiL) 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 jJg/mL for red blood cells and serum Exygen Research Page 46 of 98 -129- W ildlife International, Ltd. 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 454-108-402-Wk5* 454- 108-403-Wk5 454-108-403-Wk5* 454-108-404-Wk5 454-108-404-Wk5* 454-108-405-Wt 454-108-405-WkS* 454-108-406-Wk5 454-108-406-Wlc5* 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 072301AD 072301AD 072301AD 072301AD 072301AD 072301AD 072301AD 072301AD 072301AD 072301AD 072301AD 072301AD 072301AD 072301AD Extraction Analysis Date Date 7/23/01 7/23/01 7/23/01 7/26/01 7/26/01 7/26/01 7/23/01 7/26/01 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 7/26/01 7/26/01 7/26/01 7/26/01 7/26/01 7/26/01 7/26/01 7/26/01 7/26/01 7/26/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ue/nr>L) 45.8 45.6 115 128 104 98.3 138 129 106 107 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 ID 454-108-441-Wlc5 454-108-441-Wk5* 454-108-442-Wlc5 454-108-442-Wk5* Centre ID 0107092 0107092 0107093 0107093 Set Number 072301AD 072301AD 072301AD 072301 AD Extraction Analysis Date Date 7/23/01 7/23/01 7/23/01 7/23/01 7/26/01 7/26/01 7/26/01 7/26/01 AVERAGE: STANDARD DEVIATION: Analyte Found (uK/mL) 551 562 529 520 541 19.4 * Duplicate injection LOQ = 0.01 ng/mL for red blood cells and serum Exygen Research Page 47 of 98 G X J .. W ildlife International, Ltd, 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-WklO* 454-108-362-WklO 454-108-362-WklO* 454-108-363-Wkl0 454-108-363-Wkl0* 454-108-364-WklO 454-108-364-Wkl0* 454-108-365-WklO 454-108-365-Wkl0* 454-108-366-WklO 454- 108-366-WklO* 454-108-367-Wkl0 454- 108-367-Wkl0* 454-108-368-Wkl0 454-108-368-Wkl0* Centre ID 0107177 0107177 0107178 0107178 0107179 0107179 0107180 0107180 0107181 0107181 0107182 0107182 0107183 0107183 0107184 0107184 Set Number 072301B 072301B 072301B 072301B 072301B 072301B 072301B 072301B 072301B 072301B 072301B 072301B 072301B 072301B 072301B 07230IB Extraction Analysis Date Date 7/23/01 7/23/01 7/24/01 7/24/01 7/23/01 7/24/01 7/23/01 7/24/01 7/23/01 7/23/01 7/23/01 7/24/01 7/24/01 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 7/23/01 7/23/01 7/24/01 7/24/01 7/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (|ig/mL) 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 * Duplicate injection LOQ = 0.01 iig/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-WklO* 454-108-402-Wkl0 454-l08-402-Wkl0* 454-108-403-WklO 434-108-403-Wk10* 454-108-404-Wkl0 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 7/27-28/01 7/23/01 7/27-28/D1 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 7/27-28/01 7/23/01 7/27-28/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 7/23/01 7/27-28/01 7/23/01 7/23/01 7/27-28/01 7/27-28/01 AVERAGE: STANDARD DEVIATION: Analyte Found (wt/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 ng/mL for red blood cells and serum Exygen Research Page 49 of 98 G O O S . ; ' p''0-. Wildlife 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-Wltl5 454-108-361-W115* 454-108-362-Wkl5 454-108-362-W115* 4S4-108-363-Wkl5 454-108-363-WU5* 454-108-364-Wkl5 454-108-364-Wk15* 454-l08-36S-Wkl5 454-108-365-Wk15* 454-108-366-Wkl 5 454-108-366-Wkl5* 454-108-367-WU5 454-108-367-WU5* 454-108-368-Wkl 5 454-108-368-WklS* 454-108-333-Wkl5 454-108-333-Wk!5* 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 7/23/01 7/23/01 7/23/01 7/24-25/01 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 7/24-25/01 7/23/01 7/23/01 7/24-25/01 7/24-25/01 7/23/01 7/24-25/01 7/23/01 7/24-25/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ua/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 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 50 of 98 C* 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 4S4-108-404-Wkl5 454-108-405-Wkl5 454-108-405-Wkl5* 454-108-406-Wkl5 454-108-406-WklS* 454-108-407-Wkl5 454-108-407-Wk 15* 454-108-408-Wkl5 454- 108-408-Wk!5* 454-108-383-Wkl5 454- 108-383-Wk15* 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 072301 DD 07230IDD 072301DD 072301DD 07230IDD 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 7/27-28/01 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 7/23/01 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 AVERAGE: STANDARD DEVIATION: Analyte Found (us/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 7/27-28/01 7/23/01 7/27-28/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ue/mL) 476 514 495 2.9 LOQ = 0.01 ng/mL for red blood ceils and sertim Exygen Research Page 51 of 98 -134- Wildlife International, Ltd. 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 Rspert E01-1246 PFOS: A Reproduction Study w ith (be Nothcm Bobwhite PFOS: A Reproduction Study Mi the Northern PobuHIa Summary The concentration ol the C ,Ft7S O j' anion of perfluorooctane sulfonate, potassium salt (PFO S, C ,F17SO j'IO ) was determined in the Svsr and sera of adult bobwhite quail and their offspring In both a control group and a 10 ppm, dosed group from WiklMe feeding study *454-108. The following tables summarize the C lF,rS O l' concentrations found In the Over and sera of study samples. These results only relate to the samples tested. Matrix-extracted P F O S standards were used to establish the HPLC-MS/MS Instrument calibration curve for CtFirSO s'. Concentrations of CaFtrSOj' are reported after correction for the purity and the potassium contribution to the mass of the PFO S reference substance. Table 1 summarizes a l study sample data. Table 2 summarizes study data after removing certain results that have been statistically determined to be outliers utilizing the Dixon Q-Test, Table 1. Summary Table, C*FitSOj' Concentration in Sera and Liver i. . fi Srj.-.i'i 1 \ C F jC ' CtiNC. Si ;IA Mala Control Group IB NA it 'IV |>` Of r i i . J l / t . <400ngfrnL(19aH9| S.-.i i i ' i r , c r, so Co-.;:. Li/. 20 <S0t2ngipa Ferrala OonM roup 17 <400npmL(18ci17i NA 2110npW .|1oll7) 20 <S02nsn* Mala 10ppm Group 20 141000ngAii. 94400 * 228000 ngftnL 20 BBSOOngfg* Famala Ifopm Group IB 9990ngftnL 4710* 31BOOngM. 20 4900 neb- 1! Il l - i,f II. ..H , <ea2rt{tBof2or 20Sngb(1of20)` <S02myg(20ot20|* S01W -147000 n fto * 3280-7560f W MrieOflqrtig Control Gioif) Famalo OMprins Contact Qroup Male Offspring 10ppm Group Famala Offspring 10ppm Group 6 4 3 5 NA <400rvhrL(6ol8) NA ! 400 nglmL (4of 4) ! 12000 ngfmL 8220* 10200 ngim 12400 rigM. 7940- 1B500ngfrnL 6 SOdngg* 4 <aaon# g r ft 7300 ngdo* 1 5 7180 n^g* <20.0 r q f a (6 ol 6)* <2O-0n^g(4o<4>* 4940 13700 ngijg* 4S90-13900 ng*g* For tndtvtdual Mmpi vskMt s w Tablet 10 to IS and ASachnwnt B. Accuracy: Sample reiuJtaara accurate to 100% */ 30%. Sara QC aampiaa mat Via acceptance criteria for Bie matood * UvarQCaampiaadkJnotmeettwHolQCattachlavalmuatbawithin75% 125%criterialorthamathod. Alltvar QC aampiaa (3) spftad at 10 ppb were >125% raoovary. AHother HvarQC aampiaa (6) warn Rhln criteria. tw aiore tha acond QC oftaria waa mat in that 2/3 of aHQC wara iwttNn 75% 125%. 3M EnMronmenlal laboratory 3M EnvironmentalLaboratory Pegs 7 ol 226 Page 7 136 W ildlife International, Ltd, Project Number 454-108 Report EOI-1245 Report 1-1 2 4 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study Mi Si Northern Boburtilte Tab! 2. Summary TaMa, C*FitSOj' Concentration In Sara and Liver wHh Outlier Removed 0-dV ! n.t "t ,, , ,, w'A!.'i 11 /, (' ' J.C Cu'i'.;. S- lu fi 11;r; . oi H.- ,uli . )\! L ' . l ! c> i : f ~o C ii.u Li.'t si A!w n ]. li' i.ult. Male Consul Qraup Fannia Control Qm MdelOppiiOrtHp 19* 16 20* 16 <602r/p~ <SO^n(A3(16oM9r <400ngftnL 4OOn0ftnL(16of 16) 20* * 2xr Fama*# lOppro Qm? MaJaOMpdnfl Contai Oreup ParnateOflapring | Cental Group mWaOflapring 10ppm Group FemateOftap*n6 IGppmGroup 17 r 45* 5* STOOngtnl 4710* 147Q0ngAnL *! 206* 4* 4 4 STBOnpti** 6460 ngV* 4940 6620 ng/p** 4660-7030 r v V * No ouA tn from this data aat. RafartoTabl* 1 lor raautla. For IndvWual aampla valuta aaa Tabtaa 10 to 13 and Attachmant B. Accuracy: Samplaraadliartacairatato 100%W-30%. Sara QC aamplaa mat tha aooaptanc t erttarta lor tha mathod. Wm** Uvar QC sarnpias <tt not moat tha %of QC at aach lave! mutt bs urfthln75% - 125% ertarta for tha mathod. A l i m OCaafnptas<3)apfcadaMOppbwara>125% racovary. A lothar QCtamplaa<fl)waramhlncritaria.fraratoratha aacond QC criteria waa mat Inthat 2/3 d a) QCwarawtfhln 75% *125%. PUHPOSE The purpose ol this analytical phase ol the study is to quantity C e F u StV in the liver and sera of both control and dosed Northern Bobwhlte Quad and their offspring. Reference S ubstances Tables. Reference Substances Hi rm rii \f. S iH M ^ c r p ro s PI~DA Source 3M Internal Oakwood Producta, Inc. Expiration Date (31/2006 12/01/2010 Storage Conditions Frozen -20*C W- 10*C Frozen 20*C+/-10*C 3M Laboratory IdantHIcatlon Number SD-018, Lot#217 Physical Description I While Crystalline Ponder ; SD-036 WNtaSoUd Purity 6.9% 96% PFDA la nonadacafluorodacanoic add. or Paifluorodacanofc add. CmP kOiH and la uaad soMy to monter lor groaa Inatrumant tater* and not aa an mtamal alandard lor quanMMon. 3M Envtronmantal Laboratory 3M Environmental Laboratory P s g e S riZ Page 8 GOO W ildlife International, Ltd. Project Number 454-108 o V* MO Report E01-1245 RapOlt E01-1245 PFOS: A Reproduction Study w ith theN othem Bobwhhe PF08: A Reproduction Study Ml lw Northern BotMMIt Tabi 10. CdFirSOi' Data Summary, Ruttala Qua! Sara (Adult and Offaprtng) . E01-1245-28444 454-106630, Oppmtornato, adut. 01-31-02 . E01-1245-28448 454-108-332, Oppmternato, adut. 01-31-02 1 t 0.06 1 0.06 1 <400 <400 <400 <400 <400 <400 , 601-1245-28449 454-106-338, Oppmtornato,dut, 01-31-02 , E01-1245-28461 464-106336, Oppmtomaia, adut. 0131-02 03 t <400 <400 03 1 <400 0.3 1 <400 <400 03 1 <400 , E01-1245-284S3 454-106340, Oppmternato, adut, 0131-02 . E01-124528466 464-106-344, Oppmtornato, adut, 01-31-02 , E01-1245-284SS 454-106348, Oppmtornato, adut. 0201-02 . E0M245-2B460 464-106348, Oppmtornato, adut, 0231-02 , E01-1245-28462 454-106360, Oppmtornato, adut, 0231-02 . EOI-1245-28464 464-106362, Oppmtornala, adut, 023132 *o3 o 02 1 <400 <400 02 1 <400 02 1 <400 <400 02 1 <400 0.1 1 <400 <400 0.1 1 <400 1 <400 <400 1 <400 0.4 1 <400 <400 0.4 1 <400 0.4 1 <400 <400 0.4 1 <400 . E0M245-2B468 454-106354. Oppmtornato adut. 0231-02 03 1 <400 <400 03 1 <400 , EOI-1245-28468 454-108366, Oppmtomaia, adut, 023132 , E01-1246-28470 464-106368, Oppmtornala, adut, 023132 , EOI-1246-28472 454-108360. Oppmtornato adut. 023132 . E01-1245-28474 464-108352, Oppmtomaia, adut, 023132 , E01-1246-28477 454-106386, Oppiotornato, adut, 023132 . EOI-1245-28479 454-106-388, Oppmtornato, adut, 023132 0.3 <400 <400 0.3 <400 0 .3 1 400 <40 0 0.3 1 <400 0.3 1 2000 0.3 2190 2110# 02 <400 <400 02 <400 02 <400 <400 02 <400 03 1 <400 <400 0 3 1 <400 E01-1245-28481 454-106370.10ppmtonato,adut. 023132 0 3 1 9170 EOI-1245-28483 464-106372, lOppmtornato,adut, 020132 04 1 9140 E01-1245-28485 454-106374,10ppmtornato, adut, 023132 0.2 i 8400 E01-1245-28487 454-106378, lOppmtornato, adtA 023132 0.3 1 8280 E01-124528489 454-106378, lOppmtornala, adut, 023132 0.1 1 4980 E01-1245-28491 464-106380, lOppmtornato,adut, 023132 0.1 1 5790 3M EnvironmanM Laboratory 3M Environmental Laboratory Paga 15 01228 Page IS 0001 - 138- W ildlife International, Ltd. Project Number 454-108 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothern Bobwhite PROS: A Rapredudlon Study Mi tw Nonham eatmNto E01-124628493 454-103382, lOppmfamals, aduli. 0231-02 EQ1-124628488 454-106-386, lOppmtornale, aduK,0231-02 E01-1245-28408 454-106366. lOppmfamale, aduli, 0231-02 E01-124628600 454-106390. lOppm temala, aduli, 023132 01-1245-28502 454-106392,10ppm(amala, aduK,023132 E01-1245-28604 464-106394, lOppm famals, aduK,023132 Eoi-i245-2aaoe 454-106398. lOppm (amala, ad ii. 023132 EOI-1245-28506 494-106396,10ppmIemale, aduR,023132 E01-1S46-28611 464-106402, lOppm(amala, acu, 023132 E01-1245-28513 454-106404,10ppmfamala. aduK, 023132 E01-124626616 464-106406, lOppm(amala, aduK, 023132 E01-124528517 454-106406,10ppm(amala, aduK, 023132 . E01-1245-2B61B 454-106246. Oppmlamala, dlipm g.023132 , E01-1246-28621 454-106-259, Oppmtornala, oftopring, 023132 . E01-1246-28624 454-106273, Oppmtornato, offaprtng, 023132 . E01-1245-2B525 454-106277, Oppmtornato, oftopring.023132 . E01-1245-28528 454-106617, lOppm(amato, oftopring,023132 . E01-1245-28530 454-106525,10ppmtornato, oftopring,023132 . E01-1245-28532 454-106536,10ppmtornato, oKapring. 020132 . E01-1245-2B535 454-106557. lOppmtornato,ofspring.0231-02 a E01-1245-28530 454-106361, lOppm tornala, ofepring, 023132 0.3 0.4 0.3 0-2 0.3 0.3 0.09 0.1 04 0.1 02 02 0.1 0.1 0.1 0.1 02 02 0.02 0.02 0.03 0.03 0.06 0.06 0.05 0.05 IP P o o ss 8 8 10 31900* 1 14700 1 11600 1 6310 1 9400 1 11400 1 4710 1 10600 1 6720 1 9060 I 1 9990 1 7840 1 <400 <400 <400 1 <400 <400 1 <400 1 <400 <400 1 <400 <400 <400 1 <400 1 1B500 17900 1 16600 12800 12100 12500 1 1 14000 12200 13400 8330 7490 7900 11500 10100 10800 * There are two reeulte reported tor these samples. Both values are Included and averaged In the 'Average of Dup Sam ple' column. These values ware rejected based on the results of the Dixon's Q-Tesl, 90% confidence interval, for the rejection of outliers. Accuracy: Sample results are accurate to 100% +f- 30%. Sera O C samples mat the acceptance criteria for the method. 3M Environmental Laboratory 3M Environmental Laboratory Page 180(228 Page 16 C0 0 9 2 5 W ildlife International, Ltd. 139 Project Number 454-108 Report E01-1245 Rapoit E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: * Reproduction Study tel t a N oitam Botatala Tabla 11. CsFuSOi'Pate Summary, Mala Quail Sara (Adult and Offspring) t .1 , .. . E0M245-28443 464-100-329. Oppilinata. a d * 01-31-02 . E0M2462B446 464-10*331, Oppmmale, aduli, 01-31-02 , E01-124526447 454-106-333, Oppmirate: U , 01-31-02 . E01-1245-29448 454-106-336, Oppmnate a d * 01-31-02 . E01-1245-28450 454-106337. Oppmnato, a d * 01-31-02 . E01-1245-28452 454-106338, Oppmmala, aduli, 01-31-02 . E01-1246-28464 464-106341, Oppmmala, a c t* 01-31-02 . E01-1246-28455 454-106343, Oppmirate: a d *. 01-31-02 , E01-12462S467 464-106345, Oppmmala, a d * 01-31-02 . E0M246-2B469 464-106347. Oppianata, a d * 02-01-02 . E01-12452B461 464-106349, Oppmirate, a d * 02-01-02 . E01-1245-2S4S6 454-106353, Oppmmala, a d * 02-01-02 , EOI-1246-20407 454-106369, Oppmnato, aduli, 02-01-02 . E01-1245-29489 464-106367. (fepmmala, adite 0601-02 . E01-12462B471 464-106369. typm mala. a d *. 024)1-02 . E01-1245-26473 464-106361, Oppitimata, a d * 02-01-02 . E01-1245-28475 454-106363, Oppmmala, ad*, 02-01-02 . E01-1245-2S476 454-106366, Oppmmata, adut, 0201-02 , E01-1245-26478 464-100-307, Oppmmali, ftduft.02-01-02 oo 0.2 1 0.2 1 0.4 1 0.4 1 0.4 1 0.4 1 0.2 1 0.2 1 0.1 1 ai 1 02 1 02 1 0.2 02 01 1 0.1 1 02 1 02 1 02 1 02 1 0.02 1 0.02 1 03 1 03 1 a* 1 0.2 1 0.07 1 0.07 1 0.2 1 0.2 1 1 1 01 1 0.1 1 0.4 1 0.4 1 0.1 1 0.1 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 3MEnvtonmanlal Laboratory 3M Environmental Laboratory Peg* 17 of 226 Page 17 -140 W ildlife International, Ltd. Project Number 454-108 Report EOI-1245 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PP08: A RtproducSonStudywtfi tha Norihun BofcuNI E01-1245-28480 454-106-309.10ppm irato, aduli. d100,02-01-02 . E01-1246-28482 454-108-371, lOpptnmato, aduR, d100,02-01-02 . E01-1245-28484 464-108-373,10ppili irato, aduli, d100,02-01-02 . E0M2464B4M 464-108073 lOppmirato, aduli. 0100,02-01-02 , E01-1246-284 454-108077. lOppmirato. a d * dioo,02-01-02 . E01-1246-28490 454-108079,10ppmirato, a * * d100g(B4>14 . 01-1246-28402 454-108081,10ppm irato, a d * d i00.02-01-02 . E0M245-28494 454-10808310ppmmala, a d * d100,Q2-01-02 . E01-1246-28406 454-106086,10ppmmala, a d * d i00,02-01-02 , E01-1246-28497 454-108087,10ppilimala, a d * d i00,02-01-02 . E01-1246-28499 454-108089, K ftm mala, a d * dlOO,02-01-02 . E01-1245-28501 464-108091,10ppiamela, a d * d i00,02-01-02 . E01-1245-28S03 454-108098, lOppm irato, aduli, di00,02-01-02 . E01-1M5-2S606 464-1 OS-3 0 6 ,10ppm mala, aek*. tfl00,02-01-02 , E01-1246-2B607 464-109-307, lOppm mal, M ; di00,02-01-02 , E01-1245-28509 454-108399,10ppmmala, a d * d100,02-01-02 . E01-1246-28510 454-108401,10ppm irato, aduli. d100,02-01-02 . E01-1246-28512 454-106-403,10ppm mata, sdutt, d100,02-01-02 . EOI-124628614 454-108406,10ppmmala, aduk. d100,02-01-02 . E01-1245-28616 464-108407, lOppmmala, a d * d100,02-01-02 02 02 02 02 0.1 0.1 0.3 03 0.07 0.07 ai 0.1 ai ai 0.3 0.3 0.1 0.1 a3 0.3 0.1 0.1 0.06 0.06 0.2 02 03 0.2 0.3 as ai 0.1 0.1 0.1 03 03 0.1 0.1 0.1 0.1 100 107000 112000 100 117000 100 181000 180000 100 179000 100 144000 150000 100 158000 100 138000 136000 100 139000 100 94400 95300 100 90100 100 150000 1511000 100 180000 100 141000 146000 100 149000 100 147000 161000 100 150000 100 119000 121000 100 123000 100 174000 185000 10O 198000 100 128000 132000 100 138000 100 141000 144000 100 147000 100 160000 162000 100 165000 100 127000 131000 100 130000 100 som 103000 100 107000 100 138000 148000 100 159000 100 203000 215000 100 226000 100 127000 132000 100 136000 100 106000 112000 100 117000 100 99200 105000 100 112000 3M EnvironmartM Laboratory 3M Environmental Laboratory Paga 18 of226 Page 18 G 0 0 !>'*'f 141 W ildlife International, Ltd, Project Number 454-108 Report E01-1245 Report E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A RsproduoBon Study Mi ths Northam Botwtils ' i (. f : o I . E0M24&28619 454-108-253. Cppmmais, oltspring.02-01-02 . E01-1245-28620 454-106-258. Qppmmais, otlsprtng, 02-01-02 . E0M24S-28522 454-108-288, Cppmmais, oflspring. 02-01-02 . E0M24628523 454-106270, oppmmais, Oflsprtlg, 02-01-02 . E01-1245-28526 454-108-283, Oppmmais, oKspring, 02-01-02 . E01-1245-28*27 454-106291, Oppmmais, ofISprtng. 0201-02 0.02 0.02 0.02 0.02 ao7 0.07 012 02 0.02 ao2 0.06 0.05 1 1 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 E01-1245-28529 i 454-108-522,10ppm mala, ollaprng.02-0102 0.07 1 10 19300 19300 . E01-1245-28531 454-106633, lOppm mais, odapring. 020102 , E01-1246-28533 454-106645,10ppm mala, ofrprtng, 020102 0.07 1 9310 0.07 .. 1 .. 8220 aoa ! 1 1 15500 0.04 1 13800 , E01-1245-28534 456106652, ICppm male, oAapring. 020102 007 1 13800 0.07 1 11600 , E01-1248-28537 456106588,10ppmmale, oMprlng, 020102 0.1 0.1 1i 1 12100 10400 * There are two results reported for these samples. Both values ere included and averaged in the "Average at Oup Semple* column. 8780 14800 12500 11200 Accuracy; Sample results are accurate lo 100% +/- 30%. Sera OC samples met the acceptance criteria for the method. 3M Envirannuntal Laboratory M Environmental Laboratory Pegs 18 of 226 Page 19 -142 W ildlife International, Ltd. Project Number 454-108 Report E01-1245 RepWIEOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Repmduetlon Study wM) the Northern BobwNM Table 12. C eFirS O j'D eti Summary, Female Q uell Liver (A dult end O ffspring) E0M342-30321 } ppmaL 330 F Liver E01-1342-30339 0 ppmaL 332 F Uver E01-1342-30367 0 ppma l 334 F Uver E01-1342-30375 0 p o n ! 336 F liver E01-1342-30383 OpernJ. 331F Uver E01-1342-304H 0 ppmai.340 F Liver E01-1342-30429 0 ppma l.342 F Uver B01-1342-30447 0 ppma l 344 F Uwer EO1-1342-aO406 0 ppme i. 340 F ix e r E01-1342-30403 0 ppmal. 340 F Uver E01-1342*30601 0 ppmal. 350 F Lkw E01-1342-30519 0 ppmal. 352 F Uver E01-1342-30537 0 ppmaL 354 F Uver E01-1342-30656 0 ppma i. 356 F Uver E01-1342-30673 OppmaL3S6FUver E01-1342-30691 0 ppmal. 360 F Uver E01-1342-3060B 0ppm al.362FUver E01-1342-30027 0 ppmal. 304 F Lkmr EOI-1342-30045 0 ppmei. 368 F Uver E01-1342-30663 0 ppmal. 368 F Uver E01-1342-30681 E01-1342-30699 E01-1342-30717 E01-1342-30735 EOI-1342-30753 EOI-1342-30771 E01-1342-307B9 E01-1342-30620 E01-1342-30838 10 ppma l 370 F lkmr 10 ppma l. 372 F Uver 10 ppma l 374 F Uver 10 ppm ai.376 FUver 10 ppm a l 370 F Uver 10 ppm a l. 960 F ihm 10 ppmai. 382 F Livor 10 ppma i 384 F Uver 10 ppm al. 386 F Uver E01-1342-308S6 E01-1342-30674 E01-1342-30092 E01-1342-30010 E01-1342-30S28 EOI-1342-30046 E01-1342-30064 E01-1342-30962 E0M342-31000 E01-1342-31018 E01-1342-31030 10 ppmal. 308 F Uver 10 ppmal. 390 F Uver 10ppma l 382 F Uver 10 ppma l 384 F Uver 10 ppm al. 386 F Lkmr 10 ppma l 380 F Uver 10 ppm a l 400 F Uver 10 ppm eJ.408 F Uver 10 DPm a l 404 F Uver 10 ppmal. 406 F Uver 10 ppmai. 400 F Lkmr 3M Environmental Laboratory 3M Environmental Laboratory 1.0749 1.0469 1.0232 1.0186 1.0543 1.0162 1.0804 1.0007 1.0990 1.1036 1.0237 1.1500 1.0570 1.0312 1.1031 1.0500 1.0424 1.0304 1.0677 1.0020 1.0066 1.0748 1.0790 1.1036 1.0021 1.0826 1.0122 0.6664 1.1215 1.0873 1.0078 1.0060 1.0236 1.0370 1.0240 1.0181 1.0676 1.0173 1.0047 1.0044 ........1........... 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11 1r 1 1 1 1 1 1 1 1 1 <50.2* <30.2" <6012* <502* <502* 50.2* <502* <502* <502* <502* <602* <50X <502* <502* <602* <502* <502* <502* <502* <602* 5820* 4430* 5430* 3960* 3730* 3990* 3280* 4180* 7550* 3480* 4870* 5940* 6090* 4910* 4400* 6040* 5900* 5420* 6070* 4500* Pag 20 0(220 Page 20 -143- W ildlife International, Ltd. Project Number 454-108 Report E01-1245 RsptE01-124S PFOS: A Reproduction Study with the Nothero Bobwhite PFOS: A Raproduahm Study itti ths Noriham Botmhlle CF\ SO 601-1365-31164 E01-1365-312O4 E01-136531218 E01-135-31224 E01-136531240 E01-136M1250 E01-1366-31260 E01-1365-31275 E01-1306-312S0 Oppma i. 246 F Livor, Offspring 0 ppmai 258 F Uvsr, Offspring 0 ppma l 273 F Uvsr, Ottering 0 ppmaL 277 F Uvsr, Offspikig to ppmai. 617 F Uvsr, Ofcprkig 10ppmal. 525 F Uvsr, Offspring 10 ppma l 538 F Uvsr, Offspring 10 ppmel. 567 F Uvsr, Offspring 10 ppmaL 561F Uvsr, Offspring ; ,) 0.7302 0.4434 0.5004 0.4326 0.6670 0.5720 0.5211 0.4664 0.9417 I , i..i i O 1 <20.ir 1 <20.tr 1 20.tr 1 m tr 1 seeo* 1 7030' 10 13600.* 1 4660* 1 4660* These values were rejected based on the results of the Dixon's Q-Tset, 90 % confidence Interval, lor the rejection of outliers. Accuracy: Sample resuls are accurate to 100% +/- 30%, * Uver Q C samples did not meet the H of QC at each level must be within 75 % -1 2 5 % criteria forthamethod. A M ve rQ C samples (3) spiked at 10 ppb were >126% recovery. AH other liver Q C samples (6) were within criteria, therefore the second Q C criteria was met in that 2/3 of all Q C were within 7 5 % -1 2 5 % . 3M Envkonrrwntsl Latxnfevy 3M Environmental Laboratory Pag* 21 of 22 Page 21 -144 W ildlife International, Ltd. Project Number 454-108 Report E01-1245 Rapati E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PF06-. A RapraducDon Study Mi 1# Northam BctwhNa Table 13. CgFtrSOj' Data Summary, Mala QuaD Liver (Adult and Oflepring) c F, c;o i 11 . . 1' 1 s i i o E01-1342-30312 Oppma.1.329M Uvar BJ1-1342-30330 0 ppm al. 331 MUvar 601-1342-30348 Qppm aL 333 M Uvar EOI-1342-aOOM 0 ppma i 336 M Uvar E01-1342-30384 0 ppma i 337 MUvar EOI-1342-30402 0 ppma i 330 M Uvar E01-1342-30420 0 ppm ai. 341 MUvar E01-1342-3O438 0 ppmal. 343 M Uvar E01-1342-30466 0 ppma i. 345 M Uvar E01-1342-3O474 0ppm al.347M Um r E01-1342-30492 E01-1342-30610 0 ppm al. 351 M Uvar EQ1-1342-30S28 0 ppm aL 353 M Uvar E01-1342-30S46 0 ppm a t 355 M Uvar 01-1342-30684 0 ppma i 357 M Uvar 601-1342-30682 0 ppm a i 359 M Uvar 601-1342-30600 0 ppma l. 381M Uvar E01-1342-S08ia 0 ppma l. 383M Uvar E01-1342-30638 0 ppmaL 386M Uvar E01-1342-30654 0 ppmaL 367 M Uvar 601-1342-30672 10 ppma l. 389 M Uvar E01-1342-30690 10 ppma l. 371 M Uvar E01-1342-30708 10 ppmai. 373 M Uvar E01-1342-30726 10ppmal.37SMUvar E01-1342-30744 10 ppm ai. 377 M Uvar E01-1342-30762 10 ppm aJ. 379 M Uv*f E01-1342-30780 10 ppma l. 381 MUvar E01-1342-30798 10 ppma l. 383 M Uvar E01-1342-30629 10 ppma l. 386 MUvar E01-1342-30647 10 ppma i 387 MUvar EOI-1342-30685 10 ppm aL 389 M Uvar E01-1342-30683 10 ppm aL 391 MLfcar 601-1342-30901 10ppm aL393 MUvar E01-1342-30919 10 ppma i 395MUvar E01-1342-30937 10 ppmaL 397 M Uvar E01-1342-30B56 10 ppma i 399 MUvar E01-1342-30973 10 ppma i 401 MUvar E01-1342-30991 10 ppm aL 403 M Uvar E01-1342-31009 to ppm aL 406 MUvar E01-1342-31027 10ppm aL 407 MUvar '- 1.0060 1.0530 1 1 1.0338 1 1.0062 1 1.0269 1 0.0888 1 1.0235 1 1.0090 1 1.1046 1 1.0586 i 1 1.0358 1 1.0583 1 1.0183 I 1 1.0890 1 1.0382 1 1.0941 1 14)084 1 1418 1 1.0016 1 1.0327 1 1.0010 1.0856 1.0278 1.1008 10 10 10 10 1 .0 2 1.0912 1.0501 1.0110 1.0678 10 10 10 10 10 1.0315 1.0310 1.0947 1.0834 10 10 10 10 1.0489 1.0306 1.0276 1.0361 1.0019 1.03 1.0966 10 10 10 100 100 100 100 . 1 -J <50-2* <502* 1', 209 * <602* <502* <502* <502* <50JT <5QJ2* <502* <502* <502* <502* <502* <502* <502* <502* <502* <502* <502* 79900* 78500* 58300* 70000* 80300* 60700 80000* 73500* 83400* 79300* 70300* 70400* 861100* 65100* 50100* 63000* 147000* 148000* 138000' 132000* 3M Envlrontnantal Laboratory 3M Environmental Laboratory Pag* 92 01228 Page 22 0 i i O i* > W ildlife International, Ltd. / Project Number 454-108 Report E01-1245 Report EOI-1246 PFOS: A Reproduction Study with flic Nothem Bobwhite WOS: A Reproduction Study wrih9 Northern OoCwhte ;j 1 1 E01-136531194 Ed-136531199 Ed-136531209 Ed-136531214 E01-136531229 E01-136531234 0 ppmal. 263 M Uver, Offspring 0 ppms i. 2S6 M Lknr, Offspring 0 ppma l 206 M liner, OKsprina 0 ppms i 270 M Uver, Ofteprina 0 ppmJ. 283 M Uver, Oflsprlng 0 ppms i. 291 M Uver, Offspring E01-136531245 Ed-136531255 Ed-136531265 E01-136531270 E01-13663128S 10 ppmai. 522 M Uver, Ofhpring 10 ppm aJ. 633 M Uvar, OSepring 10 ppmeX 646 M Uver, Otepring 10 ppmaJ. 562 M Lher, Olhprtnfl 10 ppma i 668 M U rn, Offspring 0.3806 0.5947 0.2673 0.5800 0.6666 0.6072 0.7325 0.6903 0.4624 0.5363 0.7581 r r r.o r >s . t 1 200* 1 <20.0* <2tur 1 <20.0* 1 20.0* 1 <2tur 10 13700* 1 4940* 1 6660* 6990* 1 8520* Thaee values wars rejected based on the results of the Dixon's O-Test, 9 0 % confidence interval, for the rejection of outliers. Attachment B contains data summary tables. Accuracy: Sam ple results are accurals to 100% +/- 30%. * Liver Q C samples did not meet the % of Q C at each level must be within 7 5 % 125% criteria for the method. AD liver Q C samples (3) spiked at 10 ppb were >125% recovery. AH other liver Q C samplee (0) were within criteria, therefore the second Q C criteria was met In that 2/3 ol all Q C were within 7 5 % -1 2 5 % . S ta te m e n t o f C o n c l u s io n Sera results from the male control group portion of this study showed no detectable levels of C*F,tSO j in 19 of 19 quail (<400 ng/mL). The sera results from the female control group portion of this study showed no detectable levels of C ,F17SQ j' in 16 of 16 birds (<400 ng/mL) (Table 15). A nalytical results for tw enty sera sam ples from the 10ppm dosed m ala group show ed an average ol 141000 ng/mL C ,F,7S03' (range 94400 - 226000 ng/mL, Table 14). Analytical results for seventeen sera samples from the 10ppm dosed female group showed an average of 8670 ng/mL C|F,tSOs (range 47 10 -1 4 7 0 0 ng/ml_ Table 15). Sera results from the male offspring control group showed no detectable levels of C|F17S O j' In 6 of 6 birds (<400 ng/mL). The sera results from the female offspring control group portion of this study showed no detectable levels of C|F,jSOs'in 4 of 4 birds (<400 ng/mL, Table 14). Analytical results for five sera samples from tha 10ppm dosed male offspring group showed an average of 12600 ng/mL (range 6220 -19200 ng/mL, Table 14). Analytical results for five sera samples from the lOppm dosed female offspring group showed an average of 12400 ng/mL (range 7640 18500 ng/mL, Table 14). Liver results from the male control group showed no delectable levels of C ,F17SO j' In 19 of 19 birds (<50.2 ng/g, Table 15) and quantifiable concentrations of C*F,7SO j in 1 of 20 (209 ng/g. Table 14). The liver results from the female control group showed no detectable levels of CsFnSO a in 20 of 20 birds (<50.2 ng/g, Table 14). Analytical results for twenty liver samples from the 10ppm dosed male group showed an average of 88600 ng/g CeFirSOs* (range 50100 147000 ng/g, Table 14). Analytical results for twenty liver samples from the lOppm dosed female group showed an average of 4900 ng/g C *FI7S O j' (range 3290 - 7550 ng/g, Table 14). 3M Environment! Laboratory 3M Environmental Laboratory Pag* 23 ol 226 Page 23 V." .* ' . S ; 146- W ildlife International, Ltd, Project Number 454-108 Report EOI-1245 Report EOI-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS A Reproduction Study with t ie Northern Bobwhite Liver results from the male offspring control group portion of this study showed no detectable levels of CjF17S O j' in 6 of 6 birds (<50.2 ng/g. Table 14). The liver results from th e female offspring control group portion of this study showed no detectable levels of C ,F ,7S03' In 4 of 4 birds (<20.0 ng/g, Table 14). Analytical results for four liver sam ples 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 sam ples from the 10ppm dosed female offspring group showed an average of 5490 ng/g (range 4590 - 7030 ng/g, Table 15). Table 14. Sum m ary Table, C aFn SO j Concentration In Sera and U ver S/IMl'Ufr:. fcVENI Male Control Group N'JMIU H OfStHrt SAMPU5 19 Avow.r, C, 1,,S O / Cun. Si hu r NA R.unge of Results <400ngAnL(19af 19) nor Li'-JYii SauI'LES 20 1 Ferrate Control Group I 17 ' NA <400 no*nL (16 o n 7) 2110nglm l(i of 17) j 20 A'/UIAGf C.F- SO,* Cunc. Livi h <S0.2ngl(|" <50.2 , Ranof* of Rrsulls <50.2 n g (19 of 20)* 209 ngtg (1 of20)' <50.2 rg (20 Of20)* mgfa*M a k ic p p m Group 20 141000 ncmL 94400 - 226000 ng/mL 20 88500 ! 50100-147000 ng* Female iOppm Group 18 WSOng/rrt | 4710 - 31900ngftnL 20 4900 i W j 3290-7550 n g V Male Offspring Control Group Female Offspring Control Group Male Offspring tOopm Group Female Offspring IQppm Group 6 4 5 5 NA <400 ng/mL (6 of 6) NA <400 ng/VnL (4 of 4) 12600 ng/mL 8220-19200 ng^nL 12400 ng/foL 7640-18500ng*nL 6 <20.0 n^g* <20.0 npfo <6of 6)* 4 <20.0 ng/g* ! <20.0 nQ (4 of 4)* 1 ngfg'S 7350 npte* 4940 13700 5 7180rqtf j 4590* 13900 rtffl' For Individual sample values see Tables 10 to 13 end Attachment D. Accuracy: Sample results are accurate to 100% */ 30%. Sera OC samples met the acceptance criteria tor t o method. * UverOC samples did no! mart the % of OC at each level must be within ?5%-125%criteria for t o method. All liver OC samples (3) spiked at 10 ppb were >123% recovery. All other liver QC samples (6) were within criteria, therefore the second OC criteria was met In that 2/3 of a l QC ware within 75% * 125%. 3M Environmental laboratory 3M Environmental Laboratory Page 24 ol 226 Page 24 GO W ildlife International, Ltd, - 147 Project Number 454-108 Report E01-1245 Roport E01-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS: A Reproduction Study with the Northern Bobwhite Table 15. Sum m ary Table, C sF i7 S0 3` Concentration in Sera and Liver with O utliers Rem oved Si.iMi ir;(, E vi ni Nur.iui ii l> SbHil Sampu. s A ; e.hacl c ft.-so ; ConC Sena Mala Control Grotp Female Control Group 19* 16 <4Q0ngtnL Mate 10ppm Group Female 1Qppm Qidup Male Offspring Control Group Female Offspring Control Group Male Offspring 10ppm Group Female Offspring 10ppm Group 20* 17 6* 4* 5* 5" 8700ngAnL R .inrji' of Rosolia fJUVftH * Livi, h Sa m p ix ;. Av x a c l c (.Fv, b o r Cnc. Livi r R in g e of Results 19 <50.2 ngffl** <50-2 nfi (19 ol 19)** <400nmL(16of 16) ar 20* 4710-14700 ng/mL 20* 6* 4* 4 5760 ngffc" 4940 * 6520 ngfg** 4 5480 ngfgT 4590 * 7030 ngrr * No outlier from this dele 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 QO samples met the acceptance criteria tor die method. * ' Liver QC samples did not meet the % of QC at each level must be within 75% 125% criteria for the method. Aflfever OC samples (3) spiked at 10 ppb were >125% recovery. AH other liver QC samples (6) were wrtthln criteria, therefore the second QC criteria was met In that 273 ol all QC were within 75% 125%. S t a t ist ic a l M e t h o d s Statistical methods were limited to calculating m eans, standard deviations, % coefficient of variation and the Dixons Q-Test. The Dixons O-Test, used lor the rejection of outliers, has values ordered from lowesl 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 tho number ol sam ples m the group and the desired confidence interval, it the Q-value is greater than the critical Q-value then the value will be rejected. Statement of Data Quality The m easurem ent 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 sam ples, divided by the amount spiked, expressed a s a percent) was found to range from 74.1% to 126%; while precision, expressed a s 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 Laboratoty 3M Environmental Laboratory Pago 25 01226 Page 25 R e p ro d u c tiv e Perform ance b y Pen Appendix XIII - Table 1 E g g s L a id / Maximum L a id (%) fro m 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 Egge Replicate Laid 0 PPM A. I . Max. Laid Arcsin % Trans. 1 2 3 4 5 e 7 8 9 10 11 12 13 14 15 16 Total Mean SD 55 63 87 6 9 .1 2 27 63 43 4 0 .89 - -- - 38 63 60 .5 0 .9 5 23 63 37 3 7 .1 7 32 63 51 4 5 .4 5 - -- - 56 63 89 7 0 .5 3 47 63 75 5 9 .7 4 SO 63 79 6 2 .9 8 54 63 86 6 7 .7 9 - -- - 53 63 84 6 6 .5 2 54 63 86 6 7 .7 9 62 63 98 8 2 .7 6 54 63 86 6 7 .7 9 605 819 47 63 74 6 0 .7 3 12 0 20 1 3 .2 9 Eggs Laid 10 PPM A..1 . Max. Laid Arcsin % Trans. 34 63 54 4 7 .2 8 46 63 73 5 8 .7 0 43 63 68 5 5 .7 1 42 63 67 5 4 .7 4 47 63 75 5 9.74 53 63 84 6 6 .5 2 21 63 33 3 5 .2 6 - -- - 54 63 86 6 7 .7 9 - -- - 44 63 70 5 6 .6 9 44 63 70 5 6 .6 9 63 63 100 9 0 .0 0 41 63 65 5 3 .7 8 60 63 95 7 7 .4 0 - -- 592 819 46 63 72 6 0 .0 2 11 0 17 1 3 .5 0 Eggs Laid 50 PPM A . I . Max. Laid Arcsin % Trans. Eggs Laid 150 PPM A . I . Max. Laid Arcsin % Trans. _ __ - -- -- - -- - -- -- -- -- -- -- -- -- - - - -- -- _- - -- -- _ __ - -- -- -- -- -- -- -- -- -- -- -- -- -- --- -_ - -- -- - - Project Number 454-108 - D ata are not available due to adult m ortality. D ifferences betw een the control and each treatm ent group were not significant (p > 0.05). O ly NUMBER PEN Appendix XIII - Figure la Eggs Laid iron a Northern Bobuhite Reproduction Study uith PFOS - Control 70 60 so 40 30 20 10 * 0 201 202 203 2CH 205 206 207 206 209 210 211 212 213 214 21S 216 I B E ggs Laid Pen N u n b e r if: Oata a re not a v a ila b le due to adult M ortality. . VO Project Number 454-108 o oo CP NUMBER PEN Appendix XIII - Figure lb Eggs Laid from a Northern Bobuhite Reproduction Study uith PFOS - 10 PPM A.I. 70 0 50 ' 20 10 0 217 218 219 220 221 * --I---- I -- 1-----^ ~"T " 224 225 224 227 228 229 230 231 232 Number E ggs Laid Data are not availab le due to adult mortality. o Project Number 454-108 R ep ro d u ctive Perform ance b y Pen Appendix XIII - Table 2 E ggs C ra ck ed / E ggs L a id (%) fro m 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 i t h PFOS 0 PPM A. I. Eggs Eggs Replicate Crack Laid Arcsin t Trans. 10 PPM A..1 . Eggs Eggs Crack Laid Arcsin % Trans. r50 PPM A.] sag aas Crack Laid Arcsin % Trans. 150 PPM A.I. Eggs Eggs Crack Laid Arcsin Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 0 55 0 0.00 0 27 0 0.00 - -- - 2 38 5 13.26 0 23 0 0.00 0 32 0 0.00 - -- - 0 56 0 o .oo 2 47 4 11.90 1 50 2 8.13 1 54 2 7.82 - -- - 1 53 2 7.90 0 54 0 0.00 16 62 26 30.53 0 54 0 0.00 23 605 2 47 4 12 3 6.12 7 8.91 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 __ - -- -- -- -- -- -- -- -- -- -- -- -- -- -* -- -- - -- - -- - - - - _ * - - - _. ---------------" - -- -- - - - - Data are not available due to adult m ortality. D ifferences betw een the control and each treatm ent group were not significant (p > 0.05). c-> w Project Number 454-108 u Appendix XIII - Figure 2a Eggs Cracked Eggs Laid fron a Northern Bobuhite Reproduction Study uith PFOS - Control 70 io 4c Data are not a v a ila b le due to adult n o rta lity . Vi-.? Project Number 454-108 NUMBER / PEN Appendix XIII - Figure 2b Eggs Cracked / Eggs Laid from a Northern Bobuhite Reproduction Study uith PFOS - 10 PPM A.I. 70 60 so 4? Data are not a v a ila b le due to adult n o rtality. o Project Number 454-108 R ep ro d u ctive Perform ance b y Pen Appendix XIII - Table 3 V ia b le E m bryos / Egga Set (%) from a N o rth ern B obtrh ite R ep ro d u c tio n S tu d y w ith PF08 0 PPM A . I . v ia b le Eggs R eplicate actor. Set A rcsin % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 T otal Mean SD 47 50 94 75.82 12 23 52 46.25 - -- - 30 31 97 79.65 21 21 100 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 7 9 .9 5 12 11 13 1 2 .1 9 10 PPM A . I . v ia b le Eggs Embr. Set A rcsin t Trans. 30 30 100 90.00 39 40 97 80.90 39 39 100 90.00 38 38 100 90.00 35 42 83 65.91 45 48 94 75.52 17 19 89 71.07 - -- - 46 48 96 78.22 - -- - 39 39 100 90.00 27 37 73 58.68 55 57 96 79.20 17 36 47 43.41 43 53 81 64.25 - -- - 470 526 36 40 89 7 5.17 11 10 15 1 4 .2 6 50 PPM A . I . V iable Eggs Embr. Set A rcsin % Trans. 150 PPM A . I . V iable Eggs Embr. Set A rcsin % Trans. _ - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- - - __ - --- - -- -- -- -- -- -- -- -- -- -- -- -- -- .. - __ - -- "" -_ - - -- - -" - Project Number 454-108 - D ata are not available due to adult m ortality. D ifferences betw een the control and each treatm ent group w ere not significant (p > 0.05). o Appendix XIII - Figure 3a Uiable Embryos / Eggs Set from a Northern Bobuhite Reproduction Study with PFOS - Control 60 0101482348532348532323535323235353484853 VVii NUMBER / PEN Project Number 454-108 Bata are not available due to adult mortality. 213 214 216 m Uiable Appendix XIII - Figure 3b Embryos / Eggs Set Iro n a Northern Bobuhite Reproduction Study uith PFOS - 10 PPM ft.I. so 50 Kl NUMBER PEN 30 20 10 0 Data are not availab le due to adult mortality. o '**-s^` o IP ON Project Number 454-108 Reproductive Performance b y Pen Appendix XIII - Table 4 Live 3-W eek E m bryos / V i a b l e E m bryos (%) fro m 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 i t h PFOS 0 PPM A. I . L ive V iab le A rcsin R e p lic a te 3-week Embr. t T ran s. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 46 47 98 8 1.61 12 12 100 9 0 .0 0 - -- - 30 30 100 90.00 21 21 100 90.00 28 28 100 90.00 - -- - 51 51 100 9 0.00 38 39 97 8 0 .7 9 43 43 100 9 0 .0 0 47 47 100 90.00 - -- - 46 47 98 8 1.61 43 43 100 9 0 .0 0 37 38 97 8 0.66 48 48 100 9 0 .0 0 490 494 38 38 99 8 7 .2 8 12 12 1 4 .2 5 10 PPM A..1 . L ive V iab le A rcsin 3-Week Embr. % T ra n s . 29 30 97 7 9 .4 8 39 39 100 9 0.00 39 39 100 9 0.00 38 38 100 9 0 .0 0 34 35 97 8 0.27 45 45 100 90.00 16 17 94 7 5 .9 6 - -- - 46 46 100 9 0.00 - -- - 39 39 100 9 0.00 27 27 100 9 0 .0 0 55 55 100 9 0 .0 0 16 17 94 7 5 .9 6 43 43 100 9 0.00 - -- - 466 470 36 36 99 86.28 11 11 2 5 .9 1 50 PPM A . I . L ive V iable A rcsin 3 -Week Embr. % T ra n s . . - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- __ - -- - 150 PPM A . I . L ive V iable A rcsin 3 -Week Embr. % T ra n s . . - -- -- -- -- -- -- -- -- --- -- -- -- -- -- __ - -- _- . - - Project Number 454-108 - D ata are not available due to adutt m ortality. Differences between the control and each treatment group were not significant (p > 0.05). O wA NUMBER PEN Appendix XIII - Figure 4a Live 3-Ueelc Embryos Uiable Embryos from a Northern Bobuhite Reproduction Study uith PFOS - Control ao $ Data are not a va ilab le due to adult mortality. C) V** V? -, Project Number 454-108 NUMBER PEN Appendix XIII - Figure 4b Live 3-Ueek Embryos / Uiable Embryos from a Northern Bobuhite Reproduction Study uith PFOS - 10 PPM ft.I. 60 50 M) 23234853535348234823488953 30 20 10 0 217 218 219 220 221 222 223 224 22B 226 227 228 P e n___N u m b e r Uiable Embryos S I Live 3-Ueek Embryos $ Data are not availab le due to adult mortality. * " I-- 232 'O Project Number 454-108 fV*- V- R e p ro d u c tiv e P erform ance b y Pen Appendix XIII - Table 5 H a tc h lin g s / L iv e 3 -W eek E m bryos (%) from a N orth ern B obvrhlte R ep ro d u ctio n S tu d y w ith PP08 0 PPM A. I. Number Live Replicate Hatch. 3-Week 4 Arcsin 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 11 12 3 6.29 10 PPM A..1. 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 55 55 100 90.00 16 16 100 90.00 26 43 60 51.04 - -- 414 466 32 36 89 74.38 12 11 13 13.21 50 PPM A.I. Number Live Hatch. 3 -Week t Arcsin Trans. _ __ - -- -- -- -- -- -- -- -- -- -- -- -- -- -- ^ __ - -- - -- 150 PPM A.I. Number Live Arcsin Hatch. 3-Week t Trans. _ - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -* _ - _ - -- -- - - - D ata are not available due to adult m ortality. D ifferences betw een the control and each treatm ent group w ere not sign ifican t (p > 0.0 5 ). Project Number 454-108 T Appendix XIII - Figure 5a Hatchlings / Live 3-Ueek Embryos Iro n a Northern Bobuhite Reproduction Study uith PFOS - Control o\ NUMBER / PEN Project Number 454-108 T- 206 207 206 209 210 ___ P e n N u m b e r L ive 3-Ueek Embryos EES Hatchlings 4c Data are not a v a ila b le due to adult m ortality. 216 Appendix XIII - Figure 5b Hatchlings / Live 3-Ueek Embryos from a Northern Bobuhite Reproduction Study uith PFOS - 10 PPM A.I. 232323535323484890532323 O'N Ni NUMBED / PEN Project Number 454-108 222 223 224 228 22S ___ P e n N u m b e r i L ive 3-Ueek Embryos E59 Hatchlings $ Data are not a va ila b le due to adult m ortality. C> o t '"* ( C *i o "'-fr5 f. '-V o R ep ro d u c tiv e P erform ance b y Pen Appendix XIII - Table 6 1 4 -D ay O ld S u r v i v o r s / E a t c h iI n g a (%) fro m 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 i t h PFOS 0 PPM A. I. 14-Day Number Replicate Old Hatch. t Arcsin Trans. 1 43 44 98 81.33 2 12 12 100 90.00 3 - -- - 4 30 30 100 90.00 5 19 20 95 77.08 6 27 28 96 79.11 7 - -- - e 46 47 98 81.61 9 33 35 94 76.17 10 40 42 95 77.40 11 47 47 100 90.00 12 - -- - 13 45 45 100 90.00 14 40 42 95 77.40 IS 35 35 100 90.00 16 40 46 87 68.83 Total Mean SD 457 473 35 36 97 82.22 1 1 1 1 4 7.09 10 PPM A,.1 . 14-Day Number Arcsin Old Hatch. t 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 11 12 8 10.39 50 PPM A.I. 14-Day Number Arcsin O l d Hatch. % Trauis. 150 PPM A. I. 14-Day Number Old Hatch. % Arcsin Trans. _ - -- -- -- -- -- -- -- -- -- -- -- -- -- -- *- _ __ - -- -- -- -- -- -- -- -- -- -- -- -- -- -* -- _ - -. - -- -- -_ - -- -- - - D a ta are not available due to adult m ortality. Differences between the control and each treatm ent group w ere not significant (p > 0.05). Project Number 454-108 NUMBER / PEN M -Day Appendix XIII - Figure 6a Old S u rv iv o rs / H atch lin gs fro a Northern Bobuhite Reproduction Study uith PFOS - Control 0 60 022348535323235348232353232353534823 -to h--* 30 20 10 0 201 202 203 204 205 20 20? 208 209 210 211 212 m Pen Number H a tc h lin gs EEI M - D a y Old S u r v iv o r s * Data are not a va ila b le due to adult mortality. 214 215 216 Project Number 454-108 *-!r *k Appendix XIII - Figure 6b L4-Day Old Survivors / Hatchlings fro a Northern Bobuhite Reproduction Study uith PFOS - 10 PPM A.I. 0 $ Data are not a va ila b le due to adult Mortality. o\ Project Number 454-108 O o 'vy.5 * -i R ep ro d u ctive Perform ance b y Pen Appendix XIII - Table 7 H a tc h lin g s / E ggs S e t (%) fro m 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 t u d y w i t h PFOS 0 PPM A. X. Number Eggs Replicate 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 35 40 88 69.30 42 43 98 81.23 47 49 96 78.34 - -- - 45 47 96 78.10 42 49 86 67.79 35 40 88 69.30 46 49 94 75.67 473 521 36 40 90 73.58 11 1 1 12 10.24 10 PPM A.. 1. Nimber Eggs Hatch. Set Arcsin % Trans. 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 12 10 18 14.02 50 PPM A.]r. Number Eggs Hatch. Set Arcsin % Trans. 150 PPM A.I. Number Eggs Hatch. Set Arcsin % Trans. - -- -- -- -- -- -- -- -- -- -- -- -- -- --- _ - - __ - -- -- -- -- -- -- -- -- -- -- -- -- -- -- *- _ - _- - -- -" -_ _ - - -- -- - - D ata are not available due to adult m ortality. D ifferences between the control and each treatment group w ere not significant (p > 0.05). Project Number 454-108 R ep ro d u c tiv e P erform ance b y Pen Appendix XIII - Table 8 1 4 -D ay O ld S u r v iv o r s / E ggs S e t (%) fro m a N o r th e r n B o b w h lte R e p r o d u c tio n S t u d y w i t h PFOS 0 PPM A. X. 14-Day Eggs Replicate Old Set Arcsin % Trans. 1 43 50 86 68.03 2 12 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 12 9.08 10 PPM A..1 . 14-Day Eggs Old Set Arcsin Trans. 20 36 29 30 30 38 10 37 37 27 53 16 23 - 386 30 11 30 40 39 38 42 48 19 48 39 37 57 36 53 - 526 40 10 67 54.74 90 71.57 74 59.58 79 62.69 71 57.69 79 62.84 53 46.51 -77 61.40 -95 76.91 73 58.68 93 74.64 44 41.81 43 41.21 -- * 72 59.25 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. __ - -- -- -- -- -- -- --- -- -- -- -- -- -- -- _ - *- -* -- - - - D ata are not available due to adult m ortality. Significantly different from the control at p < 0.05 o Project Number 454-108 v*' Reproductive P e rfo rm a n c e b y Pen A ppen dix XIII - Table 9 H a tc h lin g s / Maximum S e t (%) fro m 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 i t h PFOS 0 PPM A. I. Number Max. Replicate Hatch. Set Arcsin % Trana. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SO 44 57 77 61.47 12 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 10 PPM A.,1 . Number Max. Hatch. Set Arcsin % Trans. 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 50 PPM A.I. Number Max. Hatch. Set Arcsin % Trans. 150 PPM A.I. Number Max. Hatch. Set Arcsin Trans. ._ - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- _ - --- - - -- - -* - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- -_ _ - - -- - _ -_ - - - - - - D ata are not available due to adult m ortality. Differences between the control and each treatment group w ere not significant <p > 0.051. Project Number 454-108 O .** R ep ro d u c tiv e Perform ance b y Pen Appendix XIII - Table 10 1 4 -D ay O ld S u r v i v o r s / Maximum S e t (%) fro m 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 i t h PFOS 0 PPM A. I. 14-Day Max. Replicate 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 12 57 21 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 35 11 741 57 0 62 52.04 19 11.44 10 PPM A..1. 14-Day Max. Old Set Arcsin % Trans. 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 11 0 19 12.41 50 PPM A.I. 14-Day Max. Old Set Arcsin % Trans. . - -- -- -- -- -- -- -- -- -- -- -- -- -- -- - _ - -- . -- " 150 PPM A.I. 14-Day Max. Old Set Arcsin Trans. . .. - -- -- -- -- -- -- -- -- -- -- -- -- -- -* *- __ - -- - - - D ata are not available due to adult m ortality. Differences betw een the control and each treatment group w ere not significant (p > 0.05). w-a*. Project Number 454-108 R e p r o d u c tiv e P e rfo rm a n c e b y Week and Pen Appendix XIV - Table la Egga L a id b y Week and Pen fro m 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 i t h PFOS Experimental (ppm a .i .) 1 LOT A B C D Control E F G H I J PENS 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 2 0 . 000 1000 2 031 5 2 -100 -512 2 -5 34 2 55 - 201 4534 _54 65 5 5 -303 -654 5 -5 5 7 6 6 6 -503 - 6767 -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 -74 67 -6 7 7 6 6 4 -655 -7676 -7 77 7 74 -6 75 -6578 -5 87 7 Totals 9 32 49 59 71 73 77 73 80 82 Totals 55 27 - 3B 23 32 - 56 47 50 54 - D ata are not available due to adult m ortality ^LO T A -E ggs Se t During W eek 12; LO T B -Eggs Se t During W eek 13; etc. - 53 54" 62 54 605 1 "0J Project Number R e p r o d u c tiv e P e rfo r m a n c e b y Week a n d Pen Appendix XIV - Table lb E ggs L a id b y Week and Pen fro m a N o r th e r n B o b w h lte R e p r o d u c tio n S t u d y w i t h PFOS Experimental Group (ppm a.i.) 1 LOT A B C D 10 E F G H I J PEN S 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 0 1 0 04 3 1 - 2 - 4 3 5 2 5 2 3 1 354 0 -5 -6 3 62 6 2 5 44664 -5 -54 65 6 4 6 6 5 5 5 4 - 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 7 768 1 -6 -77 7 5 7 - 14 30 46 62 72 65 81 72 72 78 Totals 34 46 43 42 47 53 21 - 54 - D ata are not available due to adult m ortality A B1LO T - E g g s Se t During W eek 12; LO T - Eggs Se t During W eek 13; etc. - 44 44 63 41 60 - 592 Project Number 454-108 Appendix XIV - Figure X Eggs Laid per Hen froit a Northern Bobuhite Reproduction Study with PFOS Project Number 454-108 o .SK -a w c*>fe lin k i Concentration, ppm a.i0 " *1 0 Appendix XIV - Figure 2 Hens Laying per Pen fro a Northern Bobuhite Reproduction Study uith PFOS NUMBER --0 l l t i k f F Concentrations, ppa a.i. 10 o U) Project Number 454-108 Appendix XIV - Figure 3 Eggs per Laying Hen froe a Northern Bobuhite Reproduction Study uith PFOS Concentrations, ppma.i. 0 **-" 10 Project Number 454-108 R e p r o d u c tiv e P erform an ce b y Week and Pen Appendix XIV - Table 2a E ggs C racked b y Week and Pen fro m a N o r th e r n B o b w b lte R e p r o d u c tio n S tu d y w i t h PFOS Experimental Group (ppm a.i.) PENS --------------------------------------------------------------------- 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 1 LOT A B C 0 Control E F G H I J 0 0 -00 0 -0 00 0 - 0000 0 0 - 00 0 - 0 0 0 0 - 0010 0 0 - 00 0 - 0 2 0 0 - 0010 0 0 -00 0 - 0 0 0 0 - 002 0 0 0 -00 0 - 0 01 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 - 1 0 5 0 0 0 -00 0 - 0 0 01 - 0 0 1 0 0 0 - 00 0 - 0 0 0 0 -0 0 2 0 0 0 -20 0 - 0 0 0 0 - 0 0 3 0 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 23 - D ata are not available due to adult m ortality B1LOT A - E g g s S e t During W eek 12; LOT - Eggs S e t During W eek 13; etc. Project Number 454-108 b o l i Li R e p r o d u c tiv e P erform an ce b y Week and Pen Appendix XIV - Table 2b E ggs C racked b y Week and Pen fro m a N o r th e r n B o b w h lte R e p r o d u c tio n S tu d y w i t h PFOS Experimental Group (ppm a.i.) PENS --------------------------------------------------------------------- 217 218 219 220 221 222 223 224 225 225 227 228 229 230 231 232 Totals l LOT A B C D 10 E F G H I J 0 0 0 00 0 0 - 0 - 0 0 0 0 0 0 0 0 00 0 0 - 0 - 00 0 0 0 0 0 0 000 0 - 0 -00 0 0 0 0 0 0 00 0 0 - 0- 0 0 0 0 1 0 0 000 0 0 - 0-00 0 0 0 0 0 0000 0 - 0-00 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 - 0 0 0 1 0 0 1 0 1 0 Totals 0 1 000 0 0 -0 -0 0 0 o' 2 - - D ata are not available due to adult m ortality 1LO T A - E g g s Se t During W eek 12; LO T B - E g g s Se t During W eek 13; etc. 3 Project Number 454-108 O lrO i'V R e p r o d u c tiv e P erform an ce b y Week and Pen Appendix XIV - Table 3a Eggs S e t b y Week and Pen fro m a N o r th e r n B o b w h lte R e p r o d u c tio n S tu d y w i t h PROS Experimental group (ppm a.i.) PENS ------------------------------------------------ -------------------- 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 1 LOT A B C Control D B P G H I J 1 0 - 00 0 - 1 0 0 0 - 1 0 2 1 51 - 00 0 -4 012 -5231 4 5 - 20 1 -4 2 3 3 - 4 4 4 5 5 4 -20 2 - 5 5 3 5 - 5 4 5 5 5 e - 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 Totals 6 24 41 50 64 65 64 65 72 70 Totals 50 23 _ 31 21 28 51 40 43 49 . 47 49 40 49 521 - D ata are n ot available due to adult m ortality 1LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. --jj Project Number 454-108 R e p r o d u c tiv e P erform an ce b y Week and Pen Appendix XIV - Table 3b Eggs S e t b y Week and Pen fro m a N o r th e r n B o b vrh ita R e p r o d u c tio n S tu d y w i t h PFOS O V, . 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 S 4 - 5 - 5 2 6 4 5 3 6 5 54 s 3 - 6 -4 5 6 5 7 - 46 5 44 6 4 - e - 0 4 7 4 6 - 4 8 673 83 -7 -1 6 6 6 5 - 6 5 644 53 -6 -7 4 6 4 7 - 4 5 663 6 0 - 5 -6 6 66 5 - e 3 7 6e7 1 -6 -7 5 7 4 7 - 9 26 39 55 64 60 70 67 64 72 Totals 30 40 39 38 42 48 19 _ 48 39 37 57 36 S3 . 526 - D ata are not available due to adult m ortality ^LOT A - E g g s S e t During W eek 12; LOT B - Eggs S e t During W eek 13; etc. -00j Project Number 454-108 R e p r o d u c tiv e Performance b y Week a n d Pen Appendix XIV - Table 4a V ia b le E m bryos b y Week and Pen fr o m a N o r th e r n B o b w b lte R e p r o d u c tio n S t u d y w i t h PFOS Experimental Group (ppm a.i.) PENS ---------------------------------------------------------------------- 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Totals 1 LOT A 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 -20 1 - 4 2 3 3 - 4 0 4 5 5 0 -202 - 5 53 5 - 5 4 54 5 2 - 4 0 3 - 6 6 5 6 - 5 `6 5 6 5 0 -33 3 - 6 7 6 5 - 6 6 5 6 e 0 - S2 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 7 4 7 7 3 - 37 4 - 5 4 6 8 - S 74 6 5 22 35 45 59 61 64 63 71 69 Totals 47 12 _ 30 21 28 $1 39 43 47 - D ata are not available due to adult m ortality i LO T A - E g g s S e t During W eek 12; LO T B Eggs S e t During W eek 13; etc. 47 43 38 48 494 Project Number 454-108 R e p r o d u c tiv e P erfo rm a n ce b y Week and Pen Appendix XIV - Table 4b V ia b le E m bryos b y Week and Pen fro m a N o r th e r n B o b w h lte R e p r o d u c tio n S t u d y w i t h PROS Experimental Group (ppm a.i.) PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Totale i 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 0 4 - 1 2 0 34 4 0 -4 - 5 0 5 2 5 - 2 4 4365 4 -5 -5 0 6 1 5 - 3 6 5 54 5 3 - 6 -4 2 6 2 7 - 46 544 5 3 -6 -0 4 6 1 3 - 4 8 6 73 7 3 -7 -1 6 6 4 1 - 6 S 6 44 S 2 - 6 -7 4 6 1 7 - 4 5 6 63 6 0 -5 -6 6 6 4 4 - 6 3 766 6 1 -5-75 727 - 2 19 35 50 58 51 63 63 61 68 Totale 30 39 39 38 35 45 17 - 46 - Data are not available due to adult mortality 1L0T A - Eggs S et During W eek 12 ; LO T B - Eggs S e t During W eek 13; etc. - 39 27 55 17~ 43 - 470 oOo Project Number 454-108 R e p r o d u c tiv e P erform an ce b y Week and Pen Appendix XIV - Table 5a L iv e Three-W eek Em bryos b y Week and Pen fr o m a N o r th e r n B o b w h ite Reproduction S t u d y w i t h PFOS Experimental Group (ppm a.i.) PENS ----------- ----------------------------------------------------------- 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Totals 1 LOT A B C D Control E F G H I J 0 0 - 00 0 - 1 000 0011 5 1 - 00 0 -4 012 - 5 0 3 1 4 3 -201 -4233 -4 04 5 50 -202 - 543 5 - 54 5 4 5 2 - 4 0 3 - 6 6 5 6 - 5 -6 5 6 5 0 -33 3 -6 7 6 5 - 6 6 5 6 6 0 -52 5 -7 677 -4 7 1 7 4 0 -55 5 -64 56 - 6 6 6 5 53 -64 5 - 7 57 5 - 6 74 7 73 -374 -5468 - 57 3 6 3 22 35 44 59 61 64 63 71 68 Totals 46 12 _ 30 21 28 _ 51 38 43 47 46 43 37 48 490 - D ata are not available due to adult m ortality 1LOT A - Eggs S et During W eek 12 ; LOT B - Eggs S et During W eek 1 3 ; etc . Project Number 454-108 R e p r o d u c tiv e P erform an ce b y Week and Pen Appendix XIV - Table 5b L iv e T hree-W eek Em bryos b y Week and Pen fro m a N o r th e r n B o b w h lte R e p r o d u c tio n S t u d y w i t h PFOS Experimental Group <ppm a.i.) i UOT 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 00 0 0 0 0 0 2 0 0 . 0 0 0 01 2 1 -2 - 4 0 5 04 1 2 034 4 0 - 4 - 5 0 5 2 5 2 4 4 36 5 3 - 5 - 5 0 6 1 5 3 6554 5 3 - 6 - 4 2 6 2 7 4 6544 5 3 - 6 - 0 4 6 1 3 - 3 8 673 7 3 -7 -1 6 6 3 1 - 6 5 643 52 -6 -7 4 6 17 - 4 5 663 6 0 -5 -6 6 64 4 - 6 3 7 6e S 1 - 5 - 7 5 7 2 7 - 2 19 35 49 58 51 61 62 61 68 Totals 29 39 39 38 34 45 16 - 46 - D ata are not available due to adult m ortality 1LOT A - Eggs Set During W eek 12 ; LOT B - Eggs S et During W eek 13 ; etc. - 39 27 55 16 43 - 466 R e p r o d u c tiv e P e rfo rm a n c e b y Week a n d Pen Appendix XIV - Table 6a E a tc h lin g a b y Week and Pen from a N o rth e rn B o b vrh ite R e p r o d u c tio n S tu d y w i t h PFOS Experimental Group (ppm a.i.) PEN S 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Totals 1 LOT A B C DV Control E P G H I J 00 51 43 40 52 50 50 40 53 73 00 0 100 0 .000 1 00 0 -4 01 2 - 5 0 3 1 20 1 -323 3 -4 04 5 202 -5435 -53 5 4 40 3 -565 6 -5 6 5 6 33 3 -6765 -6 6 5 5 52 5 -7 6 6 7 -4 7 1 7 54 5 -62 5 6 - 5 e 5 4 64 5 - 7 4 7 5 - 6 7 4 7 37 4 -34 6 8 - 5 73 6 2 22 34 42 58 60 62 57 70 66 Totals 44 12 30 20 28 - 47 35 42 47 D ata are not available due to adult m ortality 1LO T A - E g g s Set During W eek 12; LOT B - Eggs S e t During W eek 13; etc. - 45 42 35 46 473 00 Project Number 454-108 SV Ni o R e p r o d u c tiv e Performance b y Week and Pen Appendix XIV - Table 6b H a tc h lin g s b y Week and Pen 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 Experimental Group (ppm a.i.) 1 LOT A B C D 10 E P G H I J 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 0 01 0 0 -2 - 4 0 5 0 3 - 0 2 0 13 3 0 -3 - 5 0 5 2 3 - 1 4 4 16 3 3 -5 ' S 0 6 1 4 - 3 6 5 53 5 2 -5 - 4 2 6 2 3 - 3 5 5 34 S 3 -S 04 613 - 2 7 6 73 6 2 -7 - 1 6 6 3 0 2 4 5 42 5 2 -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 - Totals 2 15 27 43 51 47 56 51 59 63 Totals 20 36 38 32 31 39 13 - 42 - D ata are not available due to adult m ortality LO T A - E g g s Se t During W eek 12; LOT B - Eggs Se t During W eek 13; etc. - 39 27 55 16 26 414 -. Project Number 454-108 R e p r o d u c tiv e P erform ance b y Week and Pen Appendix XIV -- Table 7a 1 4 -Day O ld S u r v iv o r s b y Week and Pen fr o s t a Northern B o b w h ite R e p r o d u c tio n S tu d y w i t h PFOS Experimental Group (ppm a.i.) PENS 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Totals LOT1 A B C D Control E F G H I J 00 41 43 40 52 50 50 40 53 73 Totals 43 12 - 000 -00 0 -20 1 -20 2 -40 3 - 32 3 - S2 4 - s' 4 S - 64 5 -37 4 _ 30 19 27 -100 0 -401 2 -322 3 -542 5 - 555 6 -666 5 -766 7 -625 6 -647 5 -346 8 46 33 40 47 -0 0 0 1 -5 031 -4 043 -5353 - 5 -6 5 5 -6 6 5 5 -4 61 6 -5653 -6 647 -57 3 6 . 45 40 35 40 2 21 31 40 56 58 59 56 68 66 457 D ata are not available due to adult m ortality 1LOT A - Eggs S et During W eek 12 ; LOT B - Eggs S et During W eek 13 ; etc. Project Number 454-108 Reproductive Performance b y Week and Pen Appendix XIV - Table 7b 1 4 -Day O ld S u r v iv o r s b y Week and Pen freon a N o r th e r n B o b w h lte R e p r o d u c tio n S tu d y w i t h PROS Experimental Group (ppm a.i.) 1 LOT A B C D 10 E F G H I J PEN S 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Totals 0 0 0 00 0 0 0 0 0 01 0 0 - 02 0 03 3 0 1 4 1 16 3 1 - 3 6 4 S3 5 2 - 3 5 4 34 5 3 - 2 75736 1- 2 4 341 52 - 45 6 S3 6 0 - 53 E 56 5 1 - 0 2354563- 5- 4- 00100 2 0' 5 0 3 5 0 523 5 0 612 42 623 04 613 1663 0 74 614 6 6643 75 622 __ * - - 1 13 26 36 49 46 53 46 59 57 Totals 20 36 29 30 30 38 10 - 37 MU ! m a i - D ata are not available due to adult m ortality 1 LO T A -E g g s Se t During W eek 12; LOT B-Eggs Se t During W eek 13; etc. - 37 27 S3 16 23 - 386 o *\i C-J 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 E g g s h e ll T h ic k n e s s (zam) p e r Pen b y Week fro m 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 ith PFOS (C ontrol) Weeks B CDBFG H IJ 0.232 0.219 0.221 0.223 - 0.231 0.229 - - -- - - 0.249 0.256 0.258 - - 0.221 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 - - - - No e g g s available. Mean and standard deviation of pen means: Mean SD [n] 0.224 0.230 0.006 0.002 4 2 0.255 0.221 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 0.021 42 00 1 Project Number 454-108 o Appendix XV - Table 2 E g g s h e ll T h ic k n e s s (mm) p e r Pen b y Week fro m 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 t h PFOS (1 0 PPM A . I . ) W e ek s Pen A B CD E F0 H IJ 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 . - 0.214 0.226 0.224 0.231 - 0.221 0.210 0.220 0.224 0.234 0.242 0.249 - 0.221 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 0.221 -- -- 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 0.221 - - _ Mean and standard deviation of pen means: - No .eggs available. Differences betw een the control and this treatm ent group w ere not significant (p > 0.05). Mean SD tn] 0.224 0.217 0.237 0.230 0.223 0.248 0.221 0.007 0.006 0.011 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 0.010 45 Project Number 454-108 Appendix XVI - Table 1 Mean H a tc h lin g B ody W eigh t ( g ) p e r Pen b y Week fro m a N o rth e rn Bobwhi fce R e p r o d u c tio n S tu d y w ith PFOS (C ontrol) Pen At n] B[ n] C[ n] DC n] Weeks [Ho. of Hatchlings] E[ nl F[ n] Gt n] H[ nl It n] J[ nl Mean Total SD Hatch 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 [ 0] [ 01 [ 0] l 0] t 0] S[ 1] l 0] [ 0] C 0] t 0] [ 0] [ 0] 6[ 1] 6[ 5] 5 [ 1] [ 0] [ 0] [ 0] 6[ 4] t 0] 6 [ 1] 6 [ 2J 6 [ 5] t 03 6[ 3] 6[ 1] 7 [ 4) 61 31 6 1 21 t 01 S I 11 - 6 1 3] 6 [ 2] 6 [ 3] 6 1 33 - 7 [ 4] [ 01 7 t 41 6 [ 5] 7 [ 4] C 0] - 6 [ 2] t 0] 6 [ 2] - 6 [ 5] 6 [ 41 6[ 3] 6 [ S] 7 [ 53 6 1 33 7 [ 5] 6 [ 4] 7[ 53 6 [ 21 6 1 4] [ 0] 7 [ 3] 6 [ 51 6[ 6] 6[ 5] 6[ 6] 7 [ 51 6[ 6] 6( 51 6 [ 6] 7 [ 53 [ 0] - 6 [ 3] 6 [ 3] 6 t 3] 6 [ 6] 6 [ 7] 6[ 6] 7 [ 5] 7 [ 61 7 [ 63 7 [ 5] 6 t 5] 7 [ 5] t 0] - 6 [ 53 6 [ 2J 6 [ 4] 5 [ 7] 6 [ 6] 6 [ 63 6 [ 7] 7 [ 4] 7 [ 73 6 [ 13 6 [ 73 7 [ 43 t 0] - 6 [ S3 6 [ 4] 6 [ 5] 6 [ 6] 6 t 2] 6 C 5] 7 t 6] 7 [ 53 6 [ 6] 6 t S3 7 [ 41 6 [ 5] 6 [ 31 6 [ 6] 6 1 41 6[ 5] 6 [ 7] 6 [ 4] 6 [ 7] 7 t 5] 7[ 6] 6 [ 7] 7 1 4] 6 t 71 7 t 7] 7 0 44 6 t 3] 6 0 12 - 6 [ 31 6 0 30 6 [ 73 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 t 8] 6 0 47 - 7 [ 5] 7 0 45 6 [ 71 6 0 42 7 l 3] 7 0 35 6 [ 6] 6 0 46 Mean SD 60 472 The num ber of hatchlings w eighed m ay differ from the total number of hatchlings since those hatchlings found dead were not w eighed. - D ata are not available due to adult m ortality. S D = Standard deviation of m ean body w eight, by parental pen, by week. O w Project Number 454-108 Kf ` Appendix XVI - Table 2 Mean H a tc h lin g B ody W eight (g ) p e r Pen b y Week from a Northern B o b w h ite R e p r o d u c tio n S tu d y w ith PFOS (1 0 PPM A. I . ) Pen 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 At n] [ 0] [ 0] r 0] t 0] t 0] c 0] [ 0] [ 0] [ 0] [ 0] 6t 1] ( 0] [ 0] - B[ n] [ 0] [ 0] [ 0) t 0] 6[ 1] [ 0] ( 0] - 5[ 2] - 7 [ 4] [ 0] 6[ 5] [ 0] 6 [ 3] - C[ n] D[ n] [ 0] 6[ 2] I 0] 5 1 1] 6[ 3] 61 3] [ 0] - 5[ 3] - 7 [ 5] t 0] 6 [ 5] 8[ 2] 6 [ 3] - 6 [ 1] 7 [ 4] 5 [ 3] 6 [ 1] St 6] St 3) 5 [ 3] - 6 [ 5] - 7 t 5] t 0] St S] 8[ 1] St 4] Weeks [No. of Hatchlings] E[ n ] Ft n] Gt nj Ht n] St 3] St S] St 5] 6 t 5] 6 t 31 St 51 6[ 2] - 5 t 5] - S[ 4] St 2] St 6] 8 1 2] 6 1 3] St 3] St S] St S] 6 [ 3] 7 [ 4] 6 [ 5] St 33 _ 5 [ 5] _ t 0] 6 [ 4] St 6] 6 [ 11 6 [ 3] _ St 21 7[ 7] 61 6] St 7] St 3] 6 t 6] St 2] St 7] 6 t 1] St 6] St S] 8 t 3] t 0) 6 t 2] 6 1 4] St S3 6 t 4] 7 f 2] St S3 St 2] St S3 7 [ 7] 7 [ 4] 7 1 s) 7 [ 13 St 43 it n] 6 [ 43 7 [ 53 St 6] St s] St 3] 6 I 6] t 03 St 5] 7 t 6] 7 [ 6] 7 f S3 8 t 43 St 31 Jt n] Mean Total S D Hatch 61 5] 6 0 20 St 33 6 0 36 6 [ 73 6 0 37 St 6] 6 0 32 St 6] 6 0 31 7 [ 6] e 0 39 5 [ 13 6 1 13 6 t 5] 5 0 42 7 [ 7] 7 0 39 7 ( 53 6 0 27 St 7] 6 0 54 8 [ 2] 8 0 16 5 [ 3] e 0 26 Mean 6 SD 1 412 The num ber o f hatchlings weighed m ay differ from the total number of hatchlings since those hatchlings found dead were not w eighed - D ata are not available due to adult m ortality. Differences betw een the control and this treatment group were not significant (p > 0.05). S D = Standard deviation of mean body w eight, by parental pen, by week. Appendix XVII - Table 1 Mean 1 4 -D ay O ld S u r v iv o r s B ody W eight (g ) p e r Pen b y Week fro m 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 ith PROS (C ontrol) Pen A [ n] B [ n] C [ n] D [ n] Weeks [No. of 14-Day Old Survivors] E [ n] F [ n] G [ n] H [ n] I [ n] J [ n] Mean 1 4 -Day SD Total 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 [ 0] [ 0] t 0] [ 0] [ 0] 26 [ 1] [ 0] t 0] [ 0] [ 0] [ 0] [ 0] 24 [ 1] 29 [ 4] 22 t 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 C 1] 32 [ 3] 24 [ 2] 28 [ 2] 26 [ 3] 251 4] [ 0] 23 [ 4] 34 [ 3] 32 [ 4] [ 0] - 28 [ 2] [ 0] 31 [ 2] - 36 [ 5] 25 [ 4] 23 [ 2] 31 [ 5] 301 5] 29 [ 3] 32 [ 5] 22 [ 3] 27 [ 53 261 2] T 32 [ 4] [ 0] 30 [ 31 32 [ 5] 22 [ 5] 23 [ 51 29 [ 6] 3 2 1 5] 30 [ 6] 30 [ 53 30 [ 53 33 t S3 [ 0] - 28 [ 3] 28 [ 2] 3 1 1 33 32 [ 6] 23 [ 6] 25 [ 63 30 [ 5] 3 0 1 63 27 t 6] 29 [ 5] 27 [ S3 29 [ 51 [ 0] - 29 [ 53 30 [ 2] 28 [ 43 33 [ 7] 27 [ 6] 27 [ 6] 30 [ 7] 28 [ 4] 31 [ 63 27J 1] 30 [ 63 29 [ 4] t 0] - 31 [ 5] 30 [ 4] 32 [ 5] 33 [ 6] 28 [ 2] 27 [ 53 29 [ 6] 31 [ 53 28 [ 63 33 [ 5] 28 [ 3] 34 [ 5] 22 [ 3] 32 [ 6] 34 [ 4] 33 [ 5] 40 [ 6] 32 [ 4] 30 t 7] 30 [ 5] 31 [ 6] 32 [ 63 37 [ 4] 32 [ 7] 32 [ 7] 25 [ 3] 34 [ 3] 31 [ 7] 29 [ 4] 38 [ 33 30 [ 4] 32 [ 6] 31 C 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 - D ata are not available due to adult m ortality. S D = Standard deviation o f mean body w eight, by parental pen, by week. Project Number 454-108 C:-, O W Xi Appendix XVII - Table 2 Mean 1 4 -D a y O ld S u r v i v o r s B ody W eig h t ( g ) p e r Pen b y Week 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 (1 0 PPM A . I . ) Peso. 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 A I "n j C 01 [ 0] C 0} t 0] [ 0] [ 0] [ 0] t 0] * I 0] I 0] 30 [ 1] [ 0] [ 0] * B[ n] Cl a] t 0] [ o] [ 0] t 0] 321 1] [ 0] [ 0] * 26 t 2] " 2 9 t 23 t 0] 36 t S] E 0] 3 it 3) - [ 0] 34 [ 2] E 03 I 0] 27 E 3] 28 E 33 E 0] - 2 6 [ 31 35 t 53 E 0] 32 E 5] 421 23 33 [ 33 D[ n] 13 [ 1] 34 [ 4] 18 E 13 31 [ 1] 28 r 63 22 t 3] 24 ( 1) - 2 7 1 5] - 351 5] t 03 29 E 6] 36 [ U 22 t 2] - Weeks [Ho. of 14 -Day Old Survivors] n] F t n ) G[ a ) HE a ) I [ a ] 23 [ 3] 34 [ 6] 28 E 4] 32 t 5] 28 E 3) 30 [ 5] 31 [ 2] - 271 4] - 3 1 E 4] 35 [ 23 31 { 6] 33 f 2] 33 [ 31 - 29 ( 3] 32 t S3 23 E 43 33 [ 33 28 t 43 26 [ 53 33 t 33 - 24 [ 53 - E 0] 301 4] 33 [ 63 31 [ 1] 27 [ 33 - 31E 23 35 [ 7] 2 6 1 S3 28 t 73 29 ( 3] 2 7 1 63 321 13 _ 251 6) _ 33 E 1) 31 [ 6) 3 1 E 6] 34 E 3] [ 03 - 2 0 { 21 30 E 4] 25 E 33 25 E 4] 2 9 1 1] 26 E 5] 22 E 2] 27 E 3] _ 33 f 7] 32 [ 43 35 E 6) 36 E 13 3 0 1 43 3 1 E 4) 34 E 53 2 6 1 63 31 [ 53 32 [ 33 301 63 E 0] _ 29 [ 53 37 E 63 321 63 34 [ 6) 38 4] 32 E 33 J a] Mean 3 1 E 5] 32 t 3] 2 6 63 3 1 [ 53 30 E 63 30 [ 53 25 [ 13 28 4] ___ 37 [ 7) 34 E 53 34 ( 6] 36 t 23 30 [ 23 28 33 26 30 29 28 29 26 34 32 33 36 30 14-Day SD T o ta l 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 D a ta ara not available due to adult m ortality. Differences betw een the control and this treatment group were not significant (p > 0.05). S D = Standard deviatim i of mean body w eight, by parental pen, by week. Mean SD 30 3 386 -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 were not statistically significant. - 194 - Wildlife 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. W ildlife International, Lid. 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 EOl-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. W ildlife International, Ltd, -196- 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