Document 50p6bayd4QzR7ek7pO4xe6wYJ

AR226-1753 PFOS: A REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-108 3M LAB REQUEST NO. U2723 F I F M Guideline 714 OECD Guideline 206 AUTHORS: Sean P. Gallagher Raymond L. Van Hoven Joann B. Beavers Mark Jaber STUDY INITIATION DATE: January 22,2001 STUDY COMPLETION DATE: August 20,2003 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St.Pau1, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 USA 1410-822-8600 Page 1 of 196 ~~ ~~~~ wildlife International, Ltd. Project Number 454- 108 -2- GOOD LABORATORYPRACTICE COMPLIANCESTATEMENT 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 analyhcal 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 analyhcal phase performed by Exygen Research under study number 023-066 was performed in compliance with EPA TSCA GLP (40 CFR Part 792). STUDY DIRECTOR: Sean P. Gallagher Senior Biologist, Avian Toxicology SPONSORS REPRESENTATIVE: DATE 3/dY/OY DATE 'wildlife 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 Matrix Fortification January 22,200 1 January 30,2001 January 22,2001 January 31,2001 January 23,2001 January 31,2001 Feed Consumption February 6,2001 February 7,200 1 February 8,2001 Body Weights, Environmental Conditions February 6,2001 February 7,200 1 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 9,200 1 Raw Data, Draf? 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 Jam# H. Coleman Quality Assurance Representative r-ab-03 DATE wildlife International, Ltd. -4- REPORT APPROVAL Project Number 454-108 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: R~$$nd L. Van Hoven, Ph.D. Scientist, Analytical Chemistry MANAGEMENT: DATE l Igal 03 DATE Joann B. Beavers Director, Avian Toxicology Willard B. Nixon, Ph.6. Director of Chemistry wildlife International. Ltd. Project Number 454-108 -5 . TABLE OF CONTENTS Title Page.......................................................................................................................................... 1 Good Laboratory Practice Compliance Statement ........................................................................... 2 QualityAssurance 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 AnalyticalMethod ................................................................................................................... 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 Collectionand Storage..................................................................................................... 18 Candling and Incubation.......................................................................................................... 18 Hatching and Brooding............................................................................................................ 19 OffspringBlood and Tissue Collection................................................................................... 20 Egg Shell Thickness Measurements and Egg Components Collection ................................... 20 StatisticalAnalyses.................................................................................................................. 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 wildlife International, Ltd. Project Number 454-108 -6- TABLE OF CONTENTS PAGE 2 Egg Shell Thickness..................................................................................................... ...........27 OffspringBody Weights.......................................................................................................... 27 OffspringLiver Weights..................................................................................,.......................27 Offspring Observations............................................................................................................ 28 OffspringLiver and Sera.........................................................................................................28 Conclusion.....................................................................................................................................28 References...................................................................................................................................... 29 TABLESAND 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 Studywith PFOS, Adult Birds Found Deamuthanized during the Test....................................................................................... ....................... 31 Table 3. Mean Adult Body Weight (g) from a Northern Bobwhite Reproduction Studywith PFOS ...................................................................................32 Figure 1. Mean Adult Male Body Weight (g) from a Nortbern Bobwhite Reproduction Study with PFOS.................................................. .................33 Figure 2. Mean Adult Female Body Weight (g) from a Northem 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 (ghirdday) from a Northern Bobwhite Reproduction Study with PFOS................................................................... 36 Table 5. Summary of Gross PathologicalObservations from a Northern Bobwhite Reproduction Study with PFOS, Adult Birds Euthanized at Test Termination......................................................................................................37 Table 6. Mean Liver Weights (g) from a Northem Bobwhite Reproduction Study with PFOS .......................................................................................................... 38 Table 7. Summary of ReproductivePerformance from a Northern Bobwhite Reproduction Study with PFOS................................................................... 39 wildlife International, t t d . Project Number 454-108 -7- TABLE OF CONTENTS PAGE 3 Figure 4. Mean Reproductive Performance from a Northern Bobwhite Reproduction Studywith PFOS................................................................... 40 Table 8. Summary of Reproductive Performance, Normalized as Percentages(%), from a Northern Bobwhite Reproduction Study with PFOS............41 Figure 5. Mean ReproductivePerformance, 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 Studywith 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. Certificateof Analysis.................................................................................... 45 Appendix II. Diet and Supplement Formulations................................................................48 Appendix III. Diet Preparation............................................................................................. S O Appendix IV. The Analysis of PFOS in Avian Diet .............................................................51 Appendix V. Diagram of Test Layout.................................................................................. 67 Appendix VI. ReproductiveParameters and Replicate Identification..................................68 Appendix VII. Adult Body Weight (g) from a Northem Bobwhite Reproduction Study with PFOS ..................................................................... 73 Appendix VIII. Feed Consumption (ghirdday) from a Northern Bobwhite Reproduction Studywith 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 Repo...................................................................................... 89 wildlife International, Ltd. Project Number 454-108 -8- TABLE OF CONTENTS PAGE 4 Appendix XIIA. 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 2UV. Reproductive Performance by Week and Pen from a Northem 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 XVIII. Appendix xu(. Appendix XX. Offspring Liver Weights (g) from a Northern Bobwhite Reproduction Study with PFOS ..................................................................... 193 Changes to Study Protocol ............................................................................. 194 Personnel Involved in Study...........................................................................196 wildlijie International, Ltd. -9- SUMMARY 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 virginiunus) 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 33 170-5399 U.S.A. NOMINAL TEST CONCENTRATIONS: 0,10,50, and 150ppm 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. wildlife International, Ltd. Project Number 454-108 - 10 - INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International, Ltd. avian toxicology facility in Easton, Maryland 2 1601. The biological portion of the test was conducted from January 23, 2001 until July 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 virginiunus)of dietary exposureto 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 of a pilot reproduction study (Wildlife International, Ltd. Project Number 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 1 1 1 1 1 1 1 1 wildlife 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 150ppm 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 150/20ppm a.i. test concentration will be referred to as the 150 ppm a.i. test concentration. Adults in the 50 ppm a i . test concentrations were euthanized during Week 7 of the test due to treatment-related mortality and overt signs of toxicity. Therefore, the test was completed with the control group and the 10 ppm a.i. test concentration only. All adult birds were observed daily throughout the test for signs of toxicity or abnormal behavior. Adult body weights were measured at test initiation, on Weeks 2, 4, 6, 8, and at adult termination, and feed consumption was measured weekly throughout the test. At the beginning of Week 8, the photoperiod was increased to induce egg production. Following the start of egg production, eggs were set weekly for incubation. Weekly, eggs were selected by indiscriminate draw for egg shell thickness measurement and all remaining eggs were candled prior to incubation to detect egg shell cracks or abnormal eggs. Eggs were also candled twice during incubation to detect infertile eggs or embryo mortality. On Day 21 of incubation, the eggs were placed in a hatcher and allowed to hatch. Once hatching was completed, hatchlings were removed from the hatcher and the group body weight of the hatchlings by pen was determined. At 14 days of age, the average body weight by parental pen of all surviving offspring was determined. Upon completion of the test, statistical analyses were performed to determine statistically significant differences between groups. MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "PFOS: A Reproduction Study with the Northern Bobwhite". The protocol was based on procedures outlined in the Environmental Protection Agency's Registration Guidelines Pesticide Assessment Guidelines, FIFM wild1ije Xnternational, Ltd. Project Number 454-108 - 12 - Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, Subsection 71-4; OECD Guideline 206; and the ASTM "Standard Practice for Conducting Reproductive Studies with Avian Species" (1,2,3). Test Substance The test substance, PFOS, was received from 3M Corporation on October 29, 1998 and was assigned Wildlife International, Ltd. identification number 4675 upon receipt. The test substance was a white powder and was identified as: FC-95; Lot 217. The test material had a reported purity of 86.9% and expiration date of August 31, 2006 (Appendix I). The test substance was held under ambient conditions in locked storage at the Wildlife International, Ltd. facilities in Easton, Maryland. Concentrations of the test substance in the diet were adjusted to 100% active ingredient. Therefore, dietary concentrationsare 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 initiation (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. wild1ijie Intemational, 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. specificationsby Agway Inc. (Appendix 11, Table 1). The basal ration contained at least 27% protein and 2.5% fat, and no more than 5% fiber. The basal diet contained approximately 1.1% calcium, derived from feedstuffs and the 0.9% limestone used in the formulation of the basal diet by Agway. While this level of calcium is sufficient for growth and maintenance rations, additional calcium is required in the ration of breeding birds for egg shell formation. Therefore, an additional 5% (w/w) of limestone (approximately 38.5% Ca) was added to the basal diet for the adults. This raised the calcium level in the diet for the breeding birds to approximately 3%, slightly above the minimum recommended for quail (2.3%) and mallard (2.75%) (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 (Appendix11,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 Wildlife International, Ltd. Standard Operating Procedures. Diet Preparation Test diets were prepared by mixing PFOS into a premix that was used for weekly preparation of the final diet. Control diet and each 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 111. wildlife International, Ltd. - 14 - Project Number 454-108 Diet Sampling Homogeneity of the test substance in the diet was evaluated by collecting six samples from each of the treated diets and one sample from the control diet on Day 0 of Week 1. Samples were collected from the top, middle and bottom of the left and right sections of the mixing vessel. Control and treatment group diet samples were also collected from the feed troughs on Day 7 of Week 1 to assess stability of the test substance under actual test conditions. Additionally, a sample was collected from the control and treatment group diets, when available, during Weeks 2,3,4,8, 12, 16 and 20 of the test to measureherify test concentrations. The diet samples were stored frozen or transferred immediately to the Wildlife International, Ltd. analytical chemistry facility for analysis. Analytical Method The method used for the analysis of PFOS in avian diet was based upon methodology developed at Wildlife International, Ltd. and entitled "Analytical Method Verification for the Determination of PFOS in Avian Diet" (Wildlife International, Ltd. Project No. 454C-110). Three modifications from the cited methodology were incorporated into the present study. 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 5050 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 HPLCMSMS 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 CIS analytical column (50 mm x 2 mm I.D., 3-pm particle size) wildlye International, Ltd. Project Number 454-108 - 15 - fitted with a Keystone Javelin C18 Guard Cartridge (20 mm x 2 mm I.D.). The instrument parameters are summarized in Appendix IVYTable 1. Calibrationstandards were prepared in 5050 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 fiom 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 sahples. No attempt was made to quantify PFOS on the basis of individual isomeric components. Linear regression equations were generated using the peak area responses versus the respective concentrations of the calibration standards. An example of a calibration curve is presented in Appendix IV, Figure 2. The concentration of PFOS in avian feed samples was determined by substituting the peak areas into the applicable linear regression equation. Typical ion chromatograms of low and high calibration standards are shown in Appendix IV, Figures 3 and 4, respectively. Examples of equations used in calculations are presented in Appendix IV, Table 2. The instrument limit of detection (LOD) for these analyses was 10.0 pg on-column, calculated fiom the product of the injection volume (10.0 pL) and the lowest standard concentration 1.OO pg a.i./L = I .OO pg a.i./pL. The method limit of quantitation (LOQ) for these analyses was 2.00 ppm a i , calculated as the product of the lowest calibration standard analyzed (0.001 mg a.i./L) and the overall dilution factor of the matrix blank sample (2000L K g ) . Examples of calculations are presented in Appendix IV, Table 2. Along with the sample analyses, seven matrix blanks were analyzed to determine possible interferences. No interferences were observed at or above the LOQ during the sample analyses (Appendix IV, Table 3). A typical ion chromatogram of a matrix blank is presented in Appendix IV, Figure 5. Avian diet samples were fortified at the following levels and analyzed concurrently with the samples to determine the mean procedural recovery. The fortification levels were 5.00, 50.0, and 200 ppm a.i. for sampling intervals Week 1, Day 0; Week 1, Day 7; Week 2, Day 0; and Week 3, Day 0. The fortification levels were 5.00, 25.0, and 100 ppm a.i. for sampling interval Week 4, Day 0. The fortification levels were 5.00 and 25.0 ppm a.i. for sampling intervals Week 8, Day 0; Week 12, Day 0; Week 16, Day 0; and Week 20, Day 0. The method yielded mean procedural recoveries of 101%, 101%, wildlife 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 manufactured 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 21.4"C f 1.3"C (SD) with an average relative humidity of 42% f 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 i l d 1ife International, Ltd. Project Number 454- 108 -17- The photoperiod in the adult northern bobwhite room was maintained by a time clock. The photoperiod during acclimation and the first seven weeks of the test was eight hours of light per day. The photoperiod was increased to 17 hours of light per day at the beginning of Week 8 to induce egg laying and was maintained at that length until the adult birds were euthanized. Throughout the test, the birds received a mean of approximately 199 lux (- 18 Et. candles) of illuminationprovided by fluorescent lights that closely approximated noonday sunlight. Observations The test birds were acclimated to the facilities and study pens for 8 weeks prior to initiation of the test. During acclimation, all birds were observed daily. Birds exhibiting abnormal behavior or debilitating physical injuries were not used for the test. During the study, all adult birds were observed daily for signs of toxicity or abnormal behavior. Additionally, all offspring were observed daily from hatching until 14 days of age. A record was maintained of all mortalities and clinical observations. Adult Body Weight and Feed Consumption Adult body weights were measured at test initiation, on Weeks 2,4, 6, 8, and at adult termination. Body weights were not measured during egg laying because of the possible adverse effects handling may have on egg production. Feed consumption for each pen was measured weekly throughout the test. Feed consumption was determined by weighing the freshly filled feeder on Day 0, recording the amount of any additional diet added during the week, and weighing the feeder and remaining feed at the end of the feeding period (Day 7). An attempt was made to minimize feed wastage by the birds by using externally mounted feeders designed with a "feed-saver" lip. The amount of feed wasted by the birds was not quantified, since the wasted feed was normally scattered and mixed with water and excreta. Therefore, feed consumption is presented as an estimate of total feed consumption. Adult Blood Collection An additional four pairs of adult birds in each test level were maintained for collection of blood samples during the test. These pairs received the same diet and were handled in the same manner as other pairs in their test group. However, because of the potential stress of the blood collection procedures, data from the four additional pairs were not used to evaluate treatment related effects. Blood samples were wildlve Xntemational, Ltd. Project Number 454-108 -18- collected from birds in these designated pairs, when possible, at five week intervals throughout the test. At the time of adult termination, blood samples were collected from all surviving birds, when possible, prior to euthanasia. All blood samples were separated into serum and hemacytedplatelets, stored frozen and shipped to the sponsor or sponsor's designate for possible analysis. Adult Necropsy and Tissue Collection When the remaining birds from the 150 ppm a.i. treatment group were euthanized after Week 4, no necropsies were performed. All other adult birds that died or were euthanized during the course of the study were subjected to a gross necropsy. At the conclusion of the exposure period, all surviving adult birds were euthanized with carbon dioxide gas, necropsied, and disposed of by incineration. At the time of necropsy, tissues were collected for histopathological examination and analyses. When available, samples of gall bladder, liver, proventriculus, kidneys, brain, gonads, bursa of Fabricius and adipose tissue were fixed in 10% buffered formalin. Histology samples for the control group and 10 ppm ai. 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 13.5"C f0.2"C (SD) with a mean relative humidity of approximately 69% f6% (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 wildlife 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 37.5"C f 0.0"C (SD) with an average wet bulb temperature of 30.5"C f 0.3"C (SD) (relative humidity of approximately 60%). The incubator was equipped with a pulsator fan and blades that produced a mild 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 2 1 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.inthe hatching compartment was 37.2"C f0.0"C (SD), and the average wet bulb temperature was raised to 33.3"Cf0.1"C (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 wildlife 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 26.0'C f 1.9OC (SD) with an average relative humidity of 59% f 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. wildlife Xntemational, 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 E m s Laid - The number of eggs determined by candling to be cracked divided by the number of eggs laid, per pen. 5 . Viable Embrvos of Ems 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 Embrvos of Viable Embryos - The number of live embryos at the Day 21 candling was divided by the number of viable embryos, per pen. 7. HatchlinFs of 3-Week Embryos - The number of hatchlings removed from the hatcher was divided by the number of live 3-week embryos, per pen. 8. 14-Day Old Survivors of Hatchlings - The number of 14-day old survivors was divided by the number of hatchlings by pen. 9. Hatchlings of Eggs Set - The number of hatchlings was divided by the number of eggs set by pen. wildlife International, Ltd. Project Number 454- 108 - 22 - 10. 14-Dav Old Survivors of Eggs Set - The number of 14-day old survivors was divided by the number of eggs set by pen. 11. Hatchlings of Maximum Set - The number of hatchlings per female divided by the largest number of eggs set from any one female. This transformation was used to convert the number of hatchlings to a percentile value equal to or less than 100. 12. 14-Day Old Survivors of Maximum Set - The number of 14-day old survivors per pen divided by the largest number of eggs set. 13. Egg Shell Thickness - The average egg shell thickness of indiscriminately selected eggs per pen was measured. 14. Offstwing's Bodv Weight - The mean body weights of surviving hatchlings and 14day 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, 150ppm ai. Due to overt signs of toxicity noted at the 150 ppm a.i. test concentration, the dietary concentration for the 150 ppm a.i. treatment level was reduced to 20 ppm a.i. of PFOS at the beginning of Week 3 of the test. The 20 ppm a.i. level was terminated after Week 4 of the test due to the limited recovery of 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 f 0.313 ppm a.i., 50.1 f.0.981 ppm a i . and 147 k 4.31 ppm a i , respectively. The coefficients of variation were 3.21%, 1.96% and w i l d l v e International, Ltd. Project Number 454-108 - 23 - 2.93%, respectively. These values represented 98%, loo%, and 98% of nominal concentrations (Appendix IVYTable 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 f 0.553 ppm ai., 20.8 f. 2.14 ppm a i , 50.9 k 2.04 ppm a.i, and 161 15.6 ppm a i , respectively. The coefficients of variation were 5.44%, 10.3%, 4.01% and 9.66%, respectively. These values represented 102, 104, 102, and 108% of nominal concentrations (Appendix IV, Table 5). Analysis of diet samples collected from feeders after being held at ambient temperature for 7 days averaged 108%, 100% and 103% of the Day 0 values for the 10,50 and 150 ppm a.i. test concentrations, respectively (Appendix IV, Table 6). A typical ion chromatogram of a test sample is shown in Appendix IV, Figure 7. Mean weekly measured concentrations for each test group are presented in Table 1. Mortalities There were no treatment-related mortalities in the 10 ppm a i . treatment group. However, there were three incidental mortalities in both the control group and 10 ppm a.i. treatment group during the course of the study. Control group mortalities occurred during Weeks 14, 19 and 21 of the test. Mortalities in the 10 ppm a.i. test group occurred during Weeks 12,20 and 21 of the test. All mortalities in the control group and 10 ppm a.i. treatment group were noted with head lesions and/or neck injuries that suggest mortality was caused by incidental injury. Due to the nature of the lesions observed at necropsy, none of the mortalities in the 10 pprn ai. 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 ai. 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 150ppm 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. wildlife 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 10ppm 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 ai. 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 i l d 1ife International, Ltd. Project Number 454-108 - 25 - Adult Feed Consumption There were no apparent treatment-related effects upon feed consumption at the 10 ppm a.i. test concentration. No statistically significant differences between the control group and 10 ppm a.i. treatment group were observed at any of the feed consumption intervals. However, treatment-related reductions in feed consumption were observed at the 50 ppm a.i. test concentration during Weeks 1-6 and at the 150 ppm a.i. test concentration during Week 1 and 2 of the test. All of the reductions were statistically significant at p 0.01. The dietary concentration for the 150 ppm a.i. treatment group was reduced to 20 ppm a.i. at the beginning of Week 3 of the test. Following the reduction of the dietary concentration to 20 ppm ai., feed consumption for the group continued to be significantly lower than the control group during Week 3 of the test. Feed consumption at the 20 ppm a i . test concentration was comparable to the control group during Week 4 of the test. Mean feed consumption measurements are shown in Table 4 and Figure 3. Feed consumption measurements by pen are presented in Appendix VIII. Adult Feed Consumption 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. His topathology 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 WildlgeInternational, 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 andor offspring bobwhite from control and 10ppm 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. Su&mary 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-Electrosprayhlass Spectrometry". Blood and liver samples collected at the time of adult termination for the control group and 10 ppm a.i. test concentration were shipped to 3M Environmental Laboratory for analysis. Summary results of sera and liver analyses conducted by 3M Environmental Laboratory are presented in 3M report Tables 1, 2 and 10 through 15 located in Appendix XIIB. The full report for analysis of sera and liver samples collected at the time of adult termination is located in 3M Environmental Laboratory report E01-1245. "Analytical Phase Report for PFOS: A Reproduction Study with the Northern Bobwhite". Reproductive Results When compared to the control group, there were no apparent treatment-related effects upon egg production at the 10 ppm a.i. test concentration. However, while not statistically significant, there were slight reductions in the number of viable embryos as a percentage of eggs set, hatchlings as a percentage w i l d l v e International, Ltd. Project Number 454-108 - 27 - of live 3-week embryos and in 14-day old survivors as a percentage of hatchlings. The slight reductions in fertility, hatchability and offspring survival also resulted in reductions in the numbers of hatchlings and 14-day old survivors as percentages of both the number of eggs set and the maximum number of eggs set. While slight, the reduction in the number of 14-day old survivors as a percentage of eggs set was statistically significant at p < 0.05. Summaries of the reproductive data are presented in Tables 7 and 8, and in Figures 4 and 5. Reproductive parameters by pen are presented in Appendix XI11 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. WiZdZije International, Ltd. -28- Project Number 454-108 Offspring Observations There were incidental observations of neck curl, weakened condition and foodleg 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 of juvenile 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 10ppm 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. wildlijie International, Ltd. - 29 - REFERENCES Project Number 454- 108 1 U.S. Environmental Protection Agency. 1982. Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlye and Aquatic Organisms, subsection 71 4 , Environmental Protection Agency, Office of Pesticide Programs. Washington, D.C. 2 Organization for Economic Cooperation and Development. 1984. Avian Reproduction Test. OECD Guideline for Testing of Chemicals. Guideline 206. Paris. 3 American Society for Testing and Materials. 1986. Standard Practice for Conducting Reproductive Studies with Avian Species. ASTM Standard E1062-86. Annual Book of ASTM Standards. Vol. 11.04. Philadelphia, PA. 15pp. 4 Merck & Co., Inc. 1991. The Merck VeterinaryManual. Merck 8z Co. Rahway, NJ. 1832pp. 5 National Research Council. 1996. Guide for the Care and Use of Laboratory Animals. Washington, DC. National Academy Press. 125 pp. 6 Dunnett, C.W. 1955. A Multiple Comparison's Procedure for Comparing Several Treatments with a Control. Jour. Amer. Statis. Assoc. 50: 1096-1121. 7. Dunnett, C.W. 1964. New Tables for Multiple Comparisons with a Control. Biometrics 20: 482491. 8. Wildlife International, Ltd.. 1994. Avian Reproduction Data System (ARDS) Version 2. Nominal Concentration (ppm a.i.) Table 1 Mean Measured Concentrations(ppm a.i.)of PFOS in Avian Diet from a Northern Bobwhite Reproduction Study Week 1 DayO Day7' Week2 DayO Week3 DayO Interval Week4 DayO Week8 DayO Week12 DayO Week16 DayO Week20 DayO 0 Measured <2.002 <2.0O <2.00 <2.0O <2.w <2.00 C2.00 <2.w <2.00 Mean ' 10 Measured % Nominal 98 108 103 101 97 109 10 1 101 99 Mean - - - 50 Measured %Nominal 100' 100 97 104 104 - Mean ' - - - 150 Measured 'Yo Nominal 98 103 108 96 112 - 'Feed samples were collectedfrom feed troughs at the end of Week 1 to assess stabilityof the test substance under actual test conditions. '<2.00 indicates the value WBS less thanthe limit ofquantitation. 3 The mean of homogeneity samples collected at Week 1, Day 0. 4Thepercent of the mean values for Week 1, Day 0. 5 The 150 ppm a i . treateddiet was reduced to 20 ppm a.i. during this interval. - No values available, test group terminated. I I w 0 I I I z 7 9. 0 sr U R f c 0 00 -31 - A Project Number 454-108 Table 2 Summary of Gross Pathological Observations from a Northern Bobwhite Reproduction Study with PFOS Adult Birds Found D e a d / E u b z e d during the Test ' ' Males 50 150 ppm a.i. ppm a.i. ' Females Control 10 (0 ppm a.i.) ppm a.i. 50 ppm a.i. ' 150 ppm a.i. Number of birds 16 3 Feathers wet around beak 0 0 Feather loss 5 0 Extensive headneck lesions 0 0 Vent - pasty 0 0 Thin 1 0 Emaciated 1 1 Loss of muscle mass 2 1 Prominent keel 1 1 Intracranial bleeding Neck - mid-length fracture Neck - hematoma - Heart - no coronaryfat Spleen small andor pale - Abdominal cavity autolysis - Abdominalcavity small amount of loose blood Crop - empty Gizzard contents - bile-stained Cecal contents - pasty Kidneys - pale - Ovary developing - Ovary regressing 0 1 0 0 0 0 1 0 3 1 1 1 0 0 1 0 0 0 0 1 0 0 - - - Not remarkable 7 1 3 3 16 2 1 0 0 0 0 0 0 0 1 2 0 0 0 0 1 0 0 0 0 0 1 1 2 1 1 1 2 1 1 1 2 1 0 0 0 0 1 0 0 0 1 1 1 0 0 1 1 0 1 2 1 1 1 1 2 1 0 1 0 0 1 1 0 0 0 1 0 0 1 0 0 0 0 1 0 0 0 1 0 0 1 0 0 0 0 0 13 0 Remaining birds in the 50 ppm a.i. test concentrationwere euthanizedduring Week 7 of the test. Gross necropsy observations for birds maintained for collection of blood samples are not reported. `Remaining birds in the 150ppm a.i. test concentrationwere euthanized at the beginning of Week 5. Grossnecropsies were not performed at the termination of the 150 ppm a.i. test concentration. Table 3 M e a n A d u l t Body Weight ( 9 ) from a Northern Bobwhite Reproduction S t u d y w i t h PFOS' Experimental Group (ppm a.i.1 Sex 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 Control 10 50 150 Male Female Male Female Male Female Male Female 2 08 6 210 4 208 2 206 4 205 -8 210 -7 206 -34 212 -41 2 14 -1 214 -1 211 -1 209 -1 197** -13 202 -7 172** 13 172** 3 213 1 213 1 210 1 208 1 185** -8 195* -11 187** 172** - 215 -1 214 4 211 -2 209 -1 179** - 185** - - - - 214 1 218 32 2 09 -1 209 40 - - - - - - - - 21s 7 247 39 208 -1 249 42 I w w - - I - - - - - - The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. * Significantly different from the control at p < 0.05 * * Significantly different from the control at p < 0.01 'Only surviving birds were included in the calculations for each body weight interval. Figure 1 Mean Adult Male Body Ueiqht (g) from a Northern Bobuhite Reproduction Study with PFOS 300 Week 2 Change Week 4 Change Ueek 6 Change Week 8 Change Week T Total Wk 0-2 Wk 2-4 Uk 4-6 Wk 6-8 Wk 8-T Change * Data are not available due to adult mortality U E E K S 00 R 10 Concentrations, ppm a.i. 50 I W W I fs7. B U f P VI c 0 00 Figure 2 Mean hdult Female Body Weight (q) from a Reproduction Study with PFOS Northern Bobwhite I W P I Meek 2 Chanqe Week 4 Chanqe Week 6 Chanqe Week 8 Change U e d c T Tota1 w)c 0-2 Uk 2-4 Uk 4-6 Ut: 6-8 k 8-T Chanqe * Data are not available dw to adult mortality U E E K S 00 Y 10 Concentrations, ppm a.i. Ea50 Table 4 Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Reproduction Study w i t h PFOS Experimental WEEKS Group (ppm a.i.) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Control 12 14 13 13 13 12 12 15 14 14 15 17 18 20 19 20 19 19 19 20 19 10 12 13 13 13 13 12 12 14 13 13 14 16 18 20 20 19 20 20 20 21 19 50 XI** 11** IO** IO** 9** 9** - - - - - - - - - - - - - - - I W VI I 150 7** 6** e** 13 - - - - - - - - - - - - - - - - - - Data are not available due t o adult mortality Significantly different from the control at p e 0.01 0` 1 2 3 t 5 6 7 8 9 10 11 12 13 it 15 16 17 18 19 20 21 Weeks -0 -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 Termination' -Males Control 10 (0 ppm a.i.) ppm a.i. Females ~ . ~ ~ ~ . . ~ _ _ Control lo (0 ppm a.i.) ppm a.i. Number of birds 16 16 Feather loss 3 5 Head lesions 0 0 Foot/leg lesions 0 0 Backhent lesions 0 0 - Liver - pale and mottled 0 0 Abdominal cavity - slight egg yolk peritonitis Intestinal tract area of hyperemia 7 8 - Ceca round worms found - Testes one or both small (-1.0-1.5 cm) 0 3 1 6 Not remarkable 7 3 13 13 6 9 0 1 1 0 2 0 0 1 0 1 0 1 3 - 0 - 6 3 'Gross necropsy observations for birds maintainedfor collectionof blood samplesduringthe test are not reported. Table 6 Mean Liver Weights' (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 0 ppm a.i. 4.427 f 0.496 8.662 f 1.113 0.842 f 0.154 0.850 f 0.087 10 ppm a.i. 4.481 f 0.545 10.884' f 1.596 0.853 f 0.172 0.882 f 0.184 50 ppm a.i. 150 ppm a.i. 'Mean f standard deviation. - Data not available due to mortality. Statistically different &om the control group at p < 0.01. Table 7 Summary of Reproductive Perfonaance from a Northern Bobwhite Reproduction Study w i t h Experimental Group (ppm a.i .) Reproductive Paramter Control 10 50 150 Number of Replicates 13 13 - Total Eggs Laid 605 592 - Eggs Cracked Eggs Set 23 3 - 521 526 - Viable Embryos 4 94 470 - Live 3-Week Embryos 490 466 - Hatchlings 473 414 - 14-Day Old Survivors 457 386 - Eggs Laid/Hen 47 46 - Laid/Hen/Da# 0.50 0.49 - 14-Day Old Survivors/Hen 35 30 - 'Based on 93 days of eggs production. - Data are not available due to adult mortality - - - I w \o - I - - - - - - - h50 ..-..: z w a \ a W m E 3 z z 6 W E Figure 4 Mean Reproductive Performance from a Northern Bobwhite Reproduction Study u it h PFOS Set Embryos available due to adult mortality * rn50 ppm a.i. Live 3-wk Embryos m Hatchlings * lsopPm ai. E lS-Oay Old Survivor s I P 0 I 2 9. f P ch Y 0 00 Table 8 Summary of Reproductive Performance, Normalized as Percentages (%) from a Northern Bobwhite Reproduction Study w i t h PFOS' Experimental Group (ppm a. i . ) Reproductive Parameter Control 10 50 150 Number of Replicates 2. Total Eggs Laid Sggs Laid/Maximum Laid (%) Eggs Cracked/qgs Laid ( % ) Viable ~mbryos/EggsSet (%) Live 3-week Embryos/Viable Embryos (%) Hatchlings/Live 3-Week Embryos (%) 14-Day.Old Survivors/Hatchlings ($1 Hatchlings/Eggs Set (%) 14-Day Old Survivors/Eggs Set (%) Hatchlings/Maximum Set (%) 14-Day Old Survivors/Mdmum Set (%) 13 605 74 3 94 99 97 97 90 87 64 62 13 592 72 0 89 99 89 93 78 72 * 56 52 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A - No eggs available due to adult mortality. Significantly different from the control at p < 0.05 'Values represent pen means for experimentalgroup. Values for each pen are presentedin Appendices VI1 and VIII. 2Represents the total number of eggs laid in each group. z W C!l 6 I- W I 0 a! P h, W a I z a W E: Reproductive parameter Concentrations, ea50 ppm ai. If3g130 Table 9 Mean Egg8hell Thickness Measurements (mm) from a Northern Bobwhite Reproduction Study with PFOS Experimmtal Group (ppm a-i.) NO. Egg8 Measured Shell Thickness Mean (* SD) Control 10 50 150 42 0.229 f 0.021 45 0.227 f 0.010 - - - - - - - Data are not available due to adult mortality Differences between the control and each treatment groups were not significant (p > 0.05). ..... . . . __ T a b l e 10 Mean Body Weight (9)of Hatchlings and 14-Day Old S u r v i v o r s from a Northern Bobwhite Reproduction Study with PFOS Experimental Group (PPM A.I.) Control 10 150 &t chlings Number M e a n ( f ED) 472 412 6 fO 6 f1 - 14-Day Old Survivors Number Mean ( f SD) 457 29 f 2 306 30 f 3 - - Data are not available due to adult mortality The number of hatchhgs weighed may differ from the total number of hatchlings since those hatchlings found dead were not weighed. Differences between the control and each treatment group were not significant (p > 0.05). WildlifeInternational, Ltd. -45- Appendix I Certificate of Analysis Project Number 454-108 1. -ixciwl2. Magnwim 3. sodium 4. Potdm* 5. Nkkcl 6. Iron 1. 0.005wtJwt% 2. 0.001 wt./wt.% 3. 1.439wtJwt.% 4. 6.849wtlw~Yo 5. Q).oo1wuwL% 6. O.OOSwVwt.% 7. <0.001wUwtK 1.91 a t l w t . % 8.41 wt/wt.% 1. Qlloride 2. pluoride 3. Bromide 4. N i i 5. Niitc 6. phorphrte I . sulhtc' orsrnic '(IC) 1. TPA 2. PFPA 3. HFBA 4. NFF'A Elemmtrl M y s i n ' : 1. cubon 2. HyamBcn 3. Nitrogea 4. sulfur 5. Fluorine COA023-018A 1. "heomtical Vduc = 17.8% 2. lhtorrticrlValue =Wh 3. ~rrtiulvllue=o% 4. ti^ Vduc = 5.95% 5. Theoretical Value = 60% 1. -=O.OISwrEwtK 2. 0.59wt.hvt.% 3. ~.04oWLhvL% 4. a.o09wtJ?&% 5. Q).oo6Wt/wt.% 6. 4).007wUut% 7. 8.76wtlwt3c 1. 4.1 w t h % 2. 4 . 1 w t k % 3. O.lOWthvt% 4. 0.28 wthQt.96 1. 12.48 wtJwt.% 2. a m w.% 3. 1.74wtJwt.% 4. 8.84wtJwt.% 5. 54.1 WJWI.% Page I of 3 Wildli-feInternational, Ltd. - 46- Appendix I Page 2 Certificate of Analysis Project Number 454- 108 LWERlM CERTIFICWZ OF ANALYSIS Rwbbn 3 CentreAn.lytiml LabontorlesCOA RefereacerV: 023418A Data ofLast Analysis 08/31/00 ExpirationDatc: 08/31/06 StarageConditions: Frozen Cl0.c Re-aSsasSmmt Date: OK31/06 - 'huity = 1000/. (wrm ofm& impuritica, 1.45% +LC/MSimpurities. 8.41%+horgrmiC Fluaride,0.599/0+NMRimpmitie~l1,.905%-~ acid h@tic~0..38Yo+POM, 0.33%) - Total impurity &omP testp Purity = l W o 13.07%- =i 13.07% 86.9% COAO23618A P8gC 2 Of 3 WildlifeInternational, Ltd. - 47 - - Appendix I Page 3 Certificate of Analysis Project Number 454-108 L4TlWMCERTIFIcQT6 OFANALYSIS Ravldon 3 CentreAnalyticalhbomtorier COA ReferenceU: 023418A CS I .33 C6 4.72 c7 1.14 TOW 8.41 Nots: The 04and C6 valueswen calculatedusing fhcC4 and C6standard calibration curva, rmpcctively. The CS value w u calculated usingthe avaageresult h m the C4 and c6 standardCMes. Likewise,the c7 value was caldptad using the avaage msult from the C6 and C8 standard carves. RcviewedBy: 'Laboratory Manager, centre Analytical Laborptories COAO231)lBA Wildli-feInternational, Ltd. - 48- Project Number 454- 108 Appendix I1 Diet and Supplement Formulations Table 1 Wildlife International, Ltd. Game Bird Ration' INGREDIENTS Fine Corn Meal Soy Bean Meal, 48% Protein Wheat Midds Protein Base Agway Special, 60%Protein Alfalfa Meal, 20% Protein Dried Whey Ground Limestone Eastman CalPhos Methionine Premix -+ Liquid Vitamin and Mineral Premix (see below) GL Ferm (Fermatco)* Salt Iodized Total PERCENT (%) 44.83 30.65 6.50 6.00 4.00 3.00 2.50 0.90 0.60 0.35 0.32 0.25 0.10 100.00 Vitamin and Mineral Premix, when added at 0.32% of the ration, supplied the following amounts per ton: Amount Supplied Per Ton: Vitamin D3 2,000,000 I.C.U. Vitamin A 7,000,000 I.U. Riboflavin Niacin 6 gr40 grams Pantothenic Acid 10 grams Vitamin B 8 mgs Folic Acid 600 mgs Biotin 64 mgs Pyridoxine 1.2 grams Thiamine Vitamin E 1.2 grams 20,000 I.U. Vitamin K (MenadioneDimethylpyrimidinolBisulfite) 5.8 grams Manganese 102 grams Zinc 47 grams Copper Iodine 6.8 grams 1.5 grams Iron 51 grams 'Selenium 182 mgs The guaranteed analysis is a minimum of 27% protein, a minimum of 2.5% crude fat and a maximum of' 5% crude fiber. Fermentation By-products (Source of Unidentified Growth Factors). Wildl-ifeInternational, Ltd. - 49 - Project Number 454-108 Appendix I1 Diet and Supplement Formulations Table 2 Vitamins and Electrolytes Concentrate GUARANTEED ANALYSIS Vitamin A Vitamin D Vitamin E Riboflavin d-Pantothenic Acid Niacin Vitamin B- 12 MSBC Folic Acid Thiamine HC 1 Pyridoxine Hydrochloride Ascorbic Acid Water Soluble Powder Per 4 oz. 2,5000,000 1,000,000 1,000 750 1,250 2,500 2.5 1,000 65 250 250 3,750 Per lb. 10,000,000Iu 4,000,000ICU 4,000 IU 3,000Mg 5,000 Mg 10,000Mg 10.0Mg 4,000Mg 260 Mg 1,000Mg 1,000Mg 15,000Mg INGREDIENTS: Vitamin A Supplement, D-Activated Animal Sterol (source of Vitamin D3),Alpha Tocopheryl Acetate (source of Vitamin E). Riboflavin Supplement, d-Calcium Pantothenate,Niacin Supplement, Vitamin B- 12 Supplement, Menadione Sodium Bisulfite (source of Vitamin K), Folic Acid, Thiamine 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 of approximately 2 grams of Durvet vitamins and electrolytes to approximately 1 gallon of water. WildlifeInternational, Ltd. - 50 - Project Number 454-108 Appendix I11 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: 10 ppm a.i.: 50 ppm ai.: 150ppm ai.: 20 ppm a.i. No premix required. 2.5633 g PFOS + 8097.4 g ration 12.8165g PFOS + 8087.2 g ration 38.4494 g PFOS + 8061.6 g ration 5.1266 g PFOS + 8094.9 g ration Basal ration, 5000.0 g, was weighed into a tared Hobart mixing bowl. The remaining ration was weighed and set aside. The test substance was weighed into a tared weigh boat. Approximately 100 g of set aside ration was placed in a 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: 0 ppm a.i.: 52.25 kg ration + 2.750 kg limestone 10 ppm ai.: 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 150ppm ai.: 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. WildlifeInternational, Ltd. - 51 - - Appendix IV The Analysis of PFOS in Avian Diet Project Number 454-108 WildlifeInternational, Ltd. - 52 - Project Number 454-108 APPENDIX IV ANALYTICAL METHODS AND RESULTS INSTRUMENT: Table 1 Typical HPLCMSMS Operational Parameters Hewlett-PackardModel 1 100 High PerformanceLiquid Chromatographwith a Perkin-ElmerAPI 3000 Mass Spectrometer equipped with a Perkin-ElmerTurboIonSpray ion source. Operated in multiple ion reactionmonitoring(MRM) mode. ANALYTICAL COLUMN: GUARD COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: PFOS RETENTION TIME: PFOS MONITORED MASS: Keystone Betasil CIScolumn (50 mm x 2 mm I.D., 3-pm particle size) Keystone Javelin CIScolumn (20 mm x 2 mm I.D.) 40C 5.00 minutes 250 pLIminute 70.0% Methanol: 30.0%NANOpure@Water containing0.1%Formic Acid 10.0 pL Approximately4.0 minutes 499.0 amu + 99.1 a m u ______~ WildlifeInternational, Ltd. - 53 - Project Number 454-108 APPENDIX IV Table 2 Calculations The concentration of PFOS found at the instrument was determined using the following equation: eak area - (y-intercept) PFOS (mg a.i./L) at instrument = P slope Determination of Sample Residues (PFOS) The concentration, expressed as pprn 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 ai.) in 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.i./L) x overall dilution factor of matrix blank* extract final volume (L) x dilution factor *overall dilution factor of matrix blank sample = initial weight (Kg) Fortification Recoveries The ppm a.i. measured in each sample is divided by the nominal concentration of each sample (fortified level, ppm a.i.). This ratio times 100 is the percent recovery of the method at that level of fortification. m a.i. measured in sample % Recovery = pp ppm a.i. fortified x 100 wildlife International, Ltd. Project Number 454-108 Number (454-1 0%) MAB- 1 MAS- 1 MAS-2 MAS-3 MAB-2 MAS-4 MAS-5 MAS-6 MAB-3 MAS-7 MAS-8 MAS-9 MAB-4 MAS-10 MAS-1 1 MAS-12 APPENDIX IV Table 3 Matrix Blanks and Fortifications Analyzed Concurrently with the Samples Sample Type Interval Concentration of PFOS (ppm ai.) Fortified' Measured',' Percent Recovery Matrix Blank Week 1, Day 0 0.00 < 2.00 Matrix Fortification Week 1, Day 0 5.00 4.85 97 Matrix Fortification Week 1, Day 0 50.0 52.8 106 Matrix Fortification Week 1, Day 0 200 206 103 Matrix Blank Week 2, Day 0 0.00 < 2.00 Matrix Fortification Week 2, Day 0 5.00 4.55 91 Matrix Fortification Week 2, Day 0 50.0 47.4 95 Matrix Fortification Week 2, Day 0 200 189 95 Matrix Blank Week 3, Day 0 0.00 < 2.00 Matrix Fortification Week 3, Day 0 5.00 5.50 110 Matrix Fortification Week 3, Day 0 50.0 52.0 104 Matrix Fortification Week 3, Day 0 200 200 100 Matrix Blank Week 4, Day 0 0.00 < 2.00 Matrix Fortification Week 4, Day 0 5.00 5.20 104 Matrix Fortification Week 4, Day 0 25.0 26.1 104 Matrix Fortification Week 4, Day 0 100 100 100 - 'Less than values correspond to limit of quantitation (LOQ). 'Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly. Mean Recovery (x 1 102 94 105 103 ~ ~~ wildlife Xnternational, Ltd. Project Number 454-108 APPENDIX IV Table 3 (Continued) Matrix Blanks and Fortifications Analyzed Concurrently with the Samples __ - Number (454- 10%) Sample Interval Concentration of PFOS (ppm a.i.) Fortified' Measured'.' Percent Recovery Mean Recoverv MAE35 Matrix Blank Week 8, Day 0 0.00 < 2.00 MAS- 13 Matrix Fortification Week 8, Day 0 5.00 5.40 108 109 MAS-14 Matrix Fortification Week 8, Day 0 25.0 27.5 110 MAB-6 Matrix Blank Week 12, Day 0 0.00 < 2.00 MAS- 15 Matrix Fortification Week 12, Day 0 5 .OO 5.21 104 101 MAS- 16 Matrix Fortification Week 12, Day 0 25.0 24.4 98 MAS- 173 Matrix Fortification Week 16, Day 0 5.26 5.39 102 100 MAS-1 8 Matrix Fortification Week 16, Day 0 25.0 24.5 98 MAB-7 Matrix Blank Week 20, Day 0 0.00 < 2.00 MAS- 19 Matrix Fortification Week 20, Day 0 5.00 4.86 97 97 MAS-20 Matrix Fortification Week 20, Day 0 25.0 24.0 96 'Less than values correspond to limit of quantitation (LOQ). 'Results 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 wildlife International, Ltd. Nominal Concentration (ppm a.i.) 10 Sample I.D. Number (454- 1 OS-) 50 8 9 10 11 12 13 150 14 15 16 17 18 19 Project Number 454-108 APPENDIX IV Table 4 Homogeneityof PFOS in Avian Diet Location Sampled in Mixing Vessel Measured PFOS Concentration (ppm a.i.) Top Left 9.69 Top Right 9.47 Middle Left 9.87 Middle Right 10.2 Bottom Left 9.35 Bottom Right 9.93 Top Left 49.9 Top Right 48.9 Middle Left 51.7 Middle Right 50.4 Bottom Left 50.3 Bottom Right 49.3 Top Left 151 Top Right 153 Middle Left 143 Middle Right 143 Bottom Left 144 Bottom Right 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. C V = 1.96% x = 147ppm a i . SD = 4.3 1 ppm a.i. C V = 2.93% Mean Percent of Nominal 98 100 98 wildlife International, Ltd. Project Number 454-108 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 Concentration' (ppm a.i.) < 2.00 < 2.00 < 2.00 < 2.00 < 2.00 < 2.00 < 2.00 Mean (X) Standard Deviation (SD) Coefficient of Variation (CV) 10 35 2 36 2 42 3 43 3 49 4 50 4 56 8 57 8 61 12 62 12 66 16 67 16 71 20 72 20 'Less than values correspondto limit of quantitationGOQ). 11.0 9.68 9.53 10.7 9.70 9.60 10.6 -x= 10.2ppm a.i. 11.2 SD = 0.553 ppm ai. 10.4 cv = 5.44% 9.86 9.82 10.4 10.1 9.69 Percent of Nominal 110 97 95 107 97 96 106 112 104 99 98 104 101 97 Mean Percent of Nominal 102 wildlife International, Ltd. Project Number 454-108 Nominal Concentration (ppm a.i.) 20 50 150 Sample I.D. Number (454-10%) 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 -) ~ ~~ Measured PFOS Concentration' (ppm a.i.) Mean (X) StandardDeviation (SD) Coefficientof Variation (CV) 18.6 19.8 -x = 20.8 ppm a.i. 23.6 SD = 2.14 ppm a.i. 21.1 CV = 10.3% 2 48.7 2 48.4 3 51.1 -x = 50.9 ppm a.i. 3 53.1 SD = 2.04 ppm a.i. 4 51.1 CV = 4.01% 4 53.1 2 172 -x = 161pprn a.i. 2 150 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 wildlijii International, Ltd. Project Number 454-108 APPENDIX IV Table 6 Ambient Stability of PFOS in Avian Diet During the Northern Bobwhite Reproduction Study Nominal Concentration (ppm a.i.) Sample Number (454-10%) Week 1, Day 0' Mean Measured (ppm a.i.) PFOS Concentration Mean Percent of Nominal Sample Number (454- 10%) Week 1, Day 7 Measured' (ppm a.i.) Mean Measured (ppm a.i.) 0 1 <2.00 10 2-7 9.75 98 50 8-13 50.1 100 150 14-19 147 98 20 < 2.00 <2.00 21 9.74 10.5 22 11.2 23 50.1 50.3 24 50.5 25 148 151 26 154 Day 0 values are from homogeneitysamples presented in Table 4. 'Less than values correspondto limit of quantitation(LOQ). Mean Percent of Day 0 108 100 103 w i l d l v e International, Ltd. - 60 - Project Number 454-108 APPENDIX IV METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN AVIAN DIET Prepare matrix fortification samples in the desired avian feed stock using the dry mix technique. 1 Weigh 10 g samples of the matrix blank, matrix fortification and test samples into weigh boats and transfer to 8oz. French square glass bottles. Record weights. 1 For each sample, measure 100 mL of methanol with a graduated cylinder and transfer into the French square bottle. Cap bottles and place on shaker table. Allow the samples to shake for a minimum of 30 minutes at approximately250 rpm. Vacuum filter with qualitative filter paper and rinse retained feed 3 times with methanol into filtrate. 1 Transfer the filtrate into a 200-mL. volumetric flask and bring to volume with methanol. I Prepare appropriate dilution(s) to bring final concentration into the calibration range of the HPLC/MS/MS methodology. Use methanol for intermediate dilutions, if required. For all final dilutions, use 50% methanol : 50% NANOpure@water dilution solvent. 1 Ampulate and submit sample for HPLC/MS/MS analysis. Figure 1. Analytical method flow chart for the analysis of PFOS in avian diet. wildlife International, Ltd. - 61 - Project Number454-108 APPENDIX IV PRX 499.0-a99.1 No Internal Standard Weighted (llx) Intercept = 883.10 Slope = 11487727.00 Correlation Coeff. = 0.99974 Area 112000 100000 80000- 60000- 0- I d.0 I I I I 1.50 5-00 I I 7.50 1 1 10.0 Cono. pg a.i./L Figure 2. Typical calibration curve for PFOS. Slope = 11487727; Intercept = 883.10; r = 0.99974. Curve is weighted (Ux). wildlijie International, Ltd. - 62 - Project Number 454-108 APPENDIX lV PFOS-1 STD 0.00100 mg r.lA 4675A-0110 -1 m4.98 In 1 period No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 5.0 Quant Thres. 1.O Min. Wdth 3 Mull. Width 6 l3ase.Width 35 'AT Win. (secs) 2 0 Smooth 1 Expected RT 4.02 Area 12195 Height 1171 Start Time 3.79 End Time 4.37 Integration Width 0.59 Retention Time Integration Type -4.02 A RB Wed, 5 b 7, 2001 1758 intensity: 10000 cps 1009080706050. 4030 20- 240 41 81 121 161 201 241 281 Scan 0.69 1.36 2.03 2.70 3.37 4.04 4,71 Time Figure 3. Typical ion chromatogram of a low PFOS calibration standard, 0.00100 mg a.i./L. (Monitored mass 499.0 + 99.1). wildlifeInternational, Ltd. -63 - Project Number 454-108 APPENDIX JY PFOSJ STD 0.0100 mg 8.iA 4675A-011D -5 m4.98 in 1 period No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99. I NoiseThres. 5.0 Quant Thres. 1.o Min. Width 3 Mult. Width 6 Base.Width 35 RTWin. (secs) 20 Smooth 1 ExpectedRT 4.02 Area 114007 Height 10269 Start Time 3.80 End Time 4.39 Wed Feb 7, 2001 18:21 100. 90, 80 70 60 50 40 30 20 4h intensity: 10000 cps 240 Figure 4. Typical ion chromatogramof a high PFOS calibrationstandard,0.0100mg a.i./L. (Monitoredmass 499.0 +99.1). - 64 - wildlife International, Ltd. Project Number 454-108 APPENDIX IV PFOS-7 MA53 454*108,109- Wed, Feb 7, 2001 18:33 4.98 in 1 period rn No Internal standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 NoiseThres. 5.0 QuantThres. 1.0 Min. Width 3 Mull. Width 6 Base. Width 35 RTWin.(secs) 20 Smooth 1 ExpecledRT 4.02 Area 0 Height 0 Start Time 0.00 ~ n ndm 0.00 Integralion Width 0.00 Retention Time 0.00 Integration Type intensity: lo000 cps O0.l 90- 80- 70 60- 1 50. 4& 3P 2@ 1023 39 0-7 , 41 ' 0.69 105 130 8'1 ' lil ' 1.36 2.03 164 205 181 ' 201 I 2.70 3.37 240 274 I I I 241 281 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. (Monitoredmass 499.0 +99.1). WildlifeInternational, Ltd. - 65 - Project Number 454-108 APPENDIX IV Pm-9 MAS8 454-1 08,109- Wed, Feb 7, 2001 1845 m4.98 in 1 period No Internal Standard Use Area 100.1 1: 4.97 MRM, 298 scans I 90 499.0->99.1 Noise Thres. 5.0 80 QuarNThres. 1.0 Min. Width 3 70 Mutt. Width 6 Base. Width 35 60. RTWin.(secs) 20 50. Smooth 1 Expected RT 4.02 40- Area 60579 30- Height 5641 Start Time 3.80 20- End Time 4.39 ~ Integration Width 0.59 ! Retention Time Integration Type 4.02 A-BB intensity: 10000 cps 240 .03 2.70 3.37 4.04 4.71 Time I Figure 6. Typical ion chromatogram of a matrix fortification sample (454-108-MAS-8, 50.0ppm a.i.) (Monitoredmass 499.0 +99.1). - 66 - wild1ife Intemational, Ltd. Project Number 454-108 APPENDIX IV PFOS-13 4 2 454-108,109- Wed, Feb 7, 2001 19:lO m4.98 in 1 period No Internal Standard Use Area O01 1: 4.97 MRM,298 scans 90 499.0->99.1 NoiseThres. 5.0 80 Quant Thres. 1 .O 70 Min. Width 3 Mult. Width 6 60 Base.Width 35 RT Win. (sea) 20 50 Smooth 1 Expected FIT 4.02 40 Area 55642 30 Height 521 3 Start Time 3.80 20 End Time 4.39 Integration Width 0.59 Retention Time Integration Type -4.02 A BB intensity: 10000 cps 240 41 81 121 161 201 241 281 Scan 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Figure 7. Typical ion chromatogramof an avian diet sample on Day 0 (454-108-42, 10.0ppm a.i.). Dilution factor = 2000X. (Monitored mass 499.0 + 99.1). - 67 - Project Number 454-108 Appendix V Diagram of Test Layout from a Northern Bobwhite Reproduction Study with PFOS Room 45 Pen Numbers 221-240 10 ppm a i . ........................ Pen Numbers 241-260 50 ppm a i . ..II I t t t It IIIt II t II t t II I*! Pen Numbers 201-220 0 ppm ai. :a ......................... Pen Numbers 261-280 150 ppm a i . - b One Meter (approximate) a. Four-foot Chroma Lights b. GQF Model 0330 Battery Breeder Note: Pens 217-220,237-240,257-260and 277-280were used to house birds used for blood sampling. wild1$e Intemational, Ltd. -68- Project Number 454-108 Appendix VI Reproductive Parameters 1. Eggs Laid Definition - The number of eggs produced during the breeding season. Sensitivitv - 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. Sensitivitv - 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. Sensitivitv - 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. Wildlije International, Ltd. - 69 - 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. wildlife 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 14day old survivors by parental pen of origin, taken upon removal from the brooder unit at 14 days of age. Fourteen-day old survivors from each weekly lot of eggs are weighed. Mean weight for each week and mean weight by pen are reported. Sensitivitv - 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. wildlife International, Ltd. - 71 - Project Number 454-108 Appendix VI Replicate Identification Experimental Group Control 0 ppm a.i. Report Replicate Nknber 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Report Pen Number Raw Data Band Number Raw Data Pen Number Male Female 20 1 20 1 329 330 202 202 33 1 332 203 203 333 334 204 204 335 336 205 205 337 338 206 206 339 340 207 207 34 1 342 208 208 343 344 209 209 345 346 210 210 347 348 21 1 21 1 349 350 212 212 35 1 352 213 213 353 354 214 214 355 356 215 215 357 358 216 216 359 360 wildrife International, Ltd. - 72 - Project Number 454-108 Appendix VI Replicate Identification Experimental Group Treatment 10ppm a.i. Report Replicate Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Report Pen Number Raw Data Band Number Raw Data Pen Number Male Female 217 22 1 369 370 218 222 37 1 372 219 223 373 374 220 224 375 376 22 1 225 377 378 222 226 379 380 223 227 381 382 224 228 383 384 225 229 385 386 226 230 387 388 227 23 1 389 390 228 232 391 392 229 233 393 394 230 234 395 396 23 1 235 397 398 232 236 399 400 Appendix VI1 - T a b l e 1 A d u l t B o d y Weight (9)f r o m a N o r t h e r n B o b w h i t e R e p r o d u c t i o n S t u d y w i t h PFOS (Control, M a l e s ) ~ Week 0 change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Pa Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 201 202 203 2 04 205 206 '207 208 209 210 211 2 12 213 214 215 216 210 -2 2 14 1 218 6 206 5 206 5 184 6 182 4 205 8 209 4 207 7 213 7 212 5 195 2 196 13 232 9 244 10 208 0 215 -1 224 0 211 -4 211 -2 190 -3 186 4 213 1 213 -2 2 14 2 220 0 217 -1 197 -1 209 -2 241 -1 254 2 208 0 214 1 224 1 207 1 209 3 187 1 190 3 214 2 211 4 216 -1 22 0 -1 216 -1 196 3 207 2 240 -2 256 5 208 1 215 0 225 -1 208 4 2 12 3 188 -3 193 -3 216 1 215 -1 215 - 18 219 -4 215 1 199 7 209 -3 238 4 261 2 209 5 215 224 --15 11 212 8 215 1 185 14 190 -3 217 3 214 5 197 21 215 3 216 -8 206 8 206 -5 242 -1 263 -9 2 14 200 2 13 220 216 199 187 220 219 218 218 208 2 14 2 01 241 2 54 Mean SD 208 6 214 -1 2 13 1 215 -1 2 14 1 215 16 4 17 2 17 2 11 6 19 9 16 _____ The means for body weights and body weight changes are calculated and rounded separately. Total Change 4 - 14 -5 14 10 15 5 15 10 11 5 -4 19 5 9 10 7 9 Appendix VI1 - T a b l e 2 Adult Body Weight (g) from a Northern Bobwhite Reproduction Study with PFOS (Control, Females) Pen 201 202 203 2 04 205 206 207 208 209 210 211 212 213 2 14 215 216 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 191 3 194 -2 192 1 193 0 193 24 217 - 211 -1 210 2 2 12 2 214 6 220 28 248 220 3 223 -3 220 2 222 7 229 - 205 5 210 0 210 1 211 3 214 21 235 201 6 2 07 0 207 3 210 0 210 19 229 205 7 212 -4 208 0 208 -3 205 24 229 224 4 228 0 228 -3 225 11 236 - - 220 2 222 -2 220 0 220 -4 216 48 264 191 6 197 0 197 -1 196 5 201 26 227 187 4 191 -2 189 2 191 25 216 19 235 - 197 4 201 -1 200 -1 199 3 202 57 259 211 7 218 2 220 1 221 4 225 217 1 218 -2 216 6 222 4 226 24 250 226 7 233 -1 232 1 233 -3 230 51 281 218 3 221 -1 220 5 225 5 230 29 259 234 2 236 -1 235 0 235 -3 232 44 276 210 4 2 14 -1 213 1 2 14 4 218 32 247 14 2 14 2 14 2 14 7 13 13 20 Total Change 26 37 - 30 28 24 - 44 36 48 62 - 33 55 41 42 39 11 The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. Appendix VI1 - T a b l e 3 Adult Body Weight ( 8 ) from a Northern Bobwhite Reproduction Study w i t h PFOS ( 1 0 PPM A. I., Males) Pen 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 200 8 225 1 219 2 190 2 198 3 188 1 209 2 205 9 198 2 217 4 209 2 205 -8 2 14 5 211 3 224 4 2 18 -1 208 2 11 4 208 1 226 1 221 -2 192 0 201 -4 189 0 211 3 2 14 -4 200 -3 221 -1 211 -3 197 7 219 -2 214 2 228 -3 217 0 211 -1 12 3 209 4 227 2 219 0 192 -1 197 2 189 2 214 -1 210 6 197 0 22 0 0 208 2 2 04 -2 217 0 216 0 225 0 217 -1 210 1 12 2 2 13 -4 229 -5 219 -2 191 6 199 0 191 -1 213 -4 216 -4 197 -6 220 -6 210 -3 2 02 -1 217 0 216 -1 225 -2 216 -3 211 -2 12 3 209 0 224 12 217 -6 197 3 199 10 190 4 209 -2 212 -11 191 -3 214 -10 207 1 201 10 217 -1 215 1 223 -7 213 -17 209 -1 10 8 209 236 211 200 209 194 207 201 188 2 04 208 211 216 216 216 196 208 11 Total Change 9 11 -8 10 11 6 -2 -4 -10 -13 -1 6 2 5 -8 -22 -1 10 The means for body weights and body weight changes are calculated and rounded separately. Differences between control and this treatment group were not significant (p>0.05). Appendix VI1 - T a b l e 4 A d u l t Body Weight ( 9 ) from a Northern Bobwhite Reproduction S t u d y w i t h PFOS ( 1 0 PPM A . I . , Females) Pen 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Mean SD Week 0 Change Week 0-2 Week 2 Change Week 2-4 Week 4 Change Week 4-6 Week 6 Change Week 8 Week 6-8 ~~ Change Week 8-Term ~ 203 2 220 5 220 7 178 2 180 4 2 13 1 198 0 197 8 194 2 219 7 235 -5 199 7 214 5 196 6 236 4 189 3 205 -3 225 -1 227 -4 180 -1 184 0 2 14 -2 198 0 205 -3 196 1 226 1 230 4 206 -5 219 -3 2 02 1 240 -2 192 1 202 -1 224 -1 223 1 179 0 184 3 2 12 2 198 5 2 02 3' 197 3 227 1 234 -1 201 -2 216 0 2 03 2 238 -4 193 2 201 -1 223 -1 224 3 179 0 187 1 214 -2 203 -3 205 -5 200 2 228 3 233 1 199 0 216 0 205 -2 234 1 195 -6 200 58 222 24 227 20 179 40 188 23 2 12 58 - 200 23 200 - 202 35 231 234 35 199 45 216 55 203 49 - 235 52 189 Test Term 258 246 247 219 2 11 270 223 - 237 - 269 244 271 252 287 - 206 4 209 -1 208 1 209 -1 209 40 249 18 3 17 2 17 2 16 3 17 14 22 Total Change 55 26 27 41 31 57 25 - 43 - 34 45 57 56 51 - 42 12 -The means for body weights and body weight changes are calculatedand rounded separately. Data are not available due to adult mortality. Differences between control and this treatment group were not significant (p> 0.05). Appendix VI1 - T a b l e 5 A d u l t Body Weight ( 9 ) f r o m a Northern Bobwhite Reproduction S t u d y w i t h PFOS ( 5 0 PPM A. I . , M a l e s ) Pa 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term - - 216 0 216 -19 197 -4 193 - - - - 207 -7 200 -9 191 -9 182 - - - - - - 201 12 189 -31 158 13 145 - - - - - 198 -17 181 -29 152 - - - - 206 -5 201 -7 194 1 195 - - - 209 -16 193 -18 175 -16 159 - - - - - - 203 -2 201 -3 198 -8 190 - - - - 204 -7 197 -9 188 -2 186 - - - - 200 -9 191, -1 190 -27 163 - - - - 223 -4 219 -11 208 -5 203 - - - 182 3 185 -10 175 1 176 - - - - 202 -10 192 -15 177 0 177 - - - - 207 -10 197 -10 187 -1 186 - - - - - 204 -9 195 -13 182 -4 178 - 214 -6 208 -5 203 6 209 - - - - - - - 203 -11 192 -11 101 -41 140 - - - - 205 -8 - 9 5 197** -13 10 0 105** -8 15 12 179** 20 - - - - Total Change - - - - - - - - - -The means for body weights and body weight changes are calculatedand rounded separately. Data are not available due to adult mortality. * * Significantly different from the control at p < 0.01 Appendix VI1 - T a b l e 6 A d u l t Body Weight ( g ) from a Northern B o b w h i t e Reproduction S t u d y w i t h PFOS ( 5 0 PPM A. I . , Females) Pa 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term - 199 -1 198 -1 197 2 199 - - 206 -6 200 -5 195 -4 191 - 227 -5 222 -7 215 -3 2 12 - - 211 -7 204 -3 201 -26 175 - 209 -4 205 -6 199 -24 175 - 196 -18 178 0 178 -3 175 - - - 199 -6 193 -5 188 -2 186 - 202 -6 196 -26 170 10 180 - - 235 1 236 -1 235 -6 229 - - - 206 -8 198 -11 187 - 13 174 - 204 -11 193 0 193 -3 190 - - 223 -13 210 -30 180 -51 129 - - - 177 -2 175 -2 173 2 175 - 225 -17 208 -8 200 -31 169 - - - 217 -6 211 -13 198 - 12 186 - - 218 -7 211 1 212 -4 208 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 210 -7 - - 14 5 2 02 -7 15 9 195* -11 17 15 185** 22 - - - - - ~~ Total Change - - - - - - - - - - - The means for body weights and body weight changes are calculated and rounded separately. - Data are not availabledue to adult mortality. * Significantly different from the control at p < 0.05 * Significantly different from the control at p < 0.01 WPndiX VI1 - T a b l e 7 A d u l t Body Weight ( 9 ) from a Northern Bobwhite Reproduction S t u d y w i t h PFOS ( 2 5 0 PPM A . I . , M a l e 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 Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 191 -22 169 7 176 - - - - - - 213 -44 169 - - - - - - - - 198 -15 183 15 198 - - - - - - 218 -48 170 17 187 - - - - - - 209 -34 175 -4 171 - - - - - - 194 -55 139 - - - - - - - - 199 -32 167 24 191 - - - - - - 242 -35 2 07 18 225 - - - - - - 195 -53 142 38 180 - - - - - - 2 14 -28 186 4 190 - - - - - 212 -21 191 -8 183 - - - - - - 194 -23 171 6 177 - - - - - - 207 -25 182 - - - - - - - 211 -35 176 20 196 - - - - - - 196 -47 14 9 31 180 - - - - - - 197 -20 177 1 178 - - - - - - 206 -34 172** 13 187** - - - - - - 13 13 18 14 14 - - - - - - ~ Total Change - - - - - - - - - - - - -The means for body weights and body weight changes are calculated and rounded separately. Data are not availabledue to adult mortality. * * Significantly different from the control at p < 0.01 I 4 W I 7 9. CD 2 f U R P c 0 00 Appendix VI1 - T a b l e 8 Adult Body Weight (9) from a Northern Bobwhite Reproduction Study with PFOS ( 1 5 0 PPM A . I., Females) Pen 249 250 251 252 253 2 54 255 256 257 258 259 260 261 262 263 264 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 203 -44 159 21 180 250 -47 203 - - 201 -36 165 10 175 234 -30 204 -70 134 2 14 -35 179 13 192 232 -26 206 17 223 206 -22 184 -34 150 236 -43 193 -4 189 178 -47 131 6 13 7 213 -40 173 18 191 181 -41 14 0 23 163 223 -62 161 29 190 219 -62 157 10 167 219 -17 202 15 217 182 -44 130 1 13 9 204 -55 149 - 11 138 212 -41 172** 3 172** - - - - - - 21 13 25 26 29 - - - - - - Total Change - - -The means for body weights and body weight changes are calculated and rounded separately. Data are not available due to adult mortality. * * Significantly different from the control at p < 0.01 I 00 0 I 7 9. B $ d e f c 0 00 Appendix VI11 - T a b l e 1 Feed Consumption (g/bird/day) from a Northern Bobwhite Reproduction Study with PFOS (Control) WEEKS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 201 2 02 203 2 04 205 206 207 2 08 209 210 211 2 12 213 2 14 215 216 11 13 12 12 12 12 12 13 12 13 17 18 19 22 18 21 20 21 21 22 17 11 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 21 20 12 13 13 13 13 12 12 15 15 14 12 21 20 24 24 23 19 12 11 16 16 11 12 13 13 12 12 12 13 13 13 16 17 17 19 20 21 20 21 19 19 19 12 14 13 13 13 13 12 16 14 15 15 16 17 20 21 22 21 20 19 21 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 21.- 21 21 22 20 12 15 13 13 12 12 12 15 14 14 17 18 19 22 21 21 21 21 21 21 18 13 14 14 14 14 14 14 16 16 17 17 17 19 21 20 21 21 21 21 22 22 14 14 13 13 13 13 12 13 14 14 17 17 19 20 20 22 21 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 21 21 22 22 21 22 23 21 Mean SD 12 14 13 13 13 12 12 15 14 14 15 17 18 20 19 20 19 19 19 20 19 111111111122223333332 I 00 c I < 7 9. CD 2 8 5 % f c 0 00 Feed Consumption Appendix VI11 - T a b l e 2 (g/bird/day) from a Northern B o h h i te Reproduction ( 1 0 PPM A. I. ) Study with PFOS WEEKS Pen 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 11 14 14 14 14 13 13 13 12 12 12 14 17 18 18 19 18 20 20 21 19 12 13 12 13 13 12 12 14 12 11 16 16 19 21 21 22 22 21 17 18 21 11 14 13 13 12 13 12 14 13 13 12 14 15 19 21 19 21 20 22 20 15 10 12 11 12 11 11 12 13 12 12 12 14 16 17 18 18 18 19 20 20 17 11 12 11 11 11 11 11 13 13 13 14 15 18 17 17 16 18 19 22 21 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 11 13 13 13 14 15 16 18 18 18 17 16 15 14 16 12 14 14 14 13 13 13 14 14 10 9 14 20 17 16 15 15 15 16 15 16 12 14 13 13 14 12 13 14 12 14 14 18 19 22 23 23 22 23 22 25 22 13 15 14 14 14 14 13 15 14 15 16 17 19 21 23 21 23 24 23 27 24 11 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 21 20 22 21 13 14 13 13 13 13 13 15 15 14 17 19 18 23 21 23 22 23 22 24 21 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 21 20 23 21 22 21 21 21 22 20 11 11 11 12 12 11 11 11 10 11 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 21 19 111111112232222323343 Differences between control and this treatment group were not significant (p>0.05). I 0w 0 I Mean 11** 11** IO** IO** g** g** - -- - ---- -- - - -- SD 111122- - - - - - - - - - - - - e Data are not availabledue to adult mortality. P * * Significantly different from the control at p < 0.01 Y 0 00 Appendix VI11 - Table 4 Feed Consumption (g/bird/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 85- - 251 8 6 10 12 252 7 5 5 8 253 6 5 8 12 2 54 7 4 9 17 I 255 8 7 5 13 00 P 256 6 4 6 13 I 257 6 6 11 14 258 7 7 13 13 259 7 5 7 12 260 7 5 9 13 261 7 5 10 12 262 7 8 11 15 263 8 6 8 13 264 7 6 6 13 Mean 7** 6** E** 13 - - - - - - - - - - - - - - SD 1122- - - - - - - - - - - - - - - - - Data are not available due to adult mortality. * Significantly different from the control at p < 0.01 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 Males 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Feather loss - - - - - - -x-xx- Intestinal tract area of hyperemia - x - - - - x - x x x -- -xx - Testes one or both small (-1.0-1.5 a) - - - - - - -x- - I oe Not remarkable x-xxxx-x- -x- - VI I Females Pens 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Feather loss FootAeg lesions - BacWvent lesions Ceca round worms found Not remarkable - X - X X a a X - - - X - a a - X - - - - a a - - - x- - - - - a - -Xa-X- - a - - X-ax- - a - - - - a - - -x -a-X-ax-XXaXX- a - Bird found dead during the test. Necropsyhdmgs are presented in Table 2. Appendix M - Table 2 Individual Gross Pathological Observations from a Northern Bobwhite Reproduction Study with PFOS Adult Birds Euthanized at Test Termination 10 ppm a.i. Males Pens 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Feather loss x - - - x - - - x - x -- x - - - Intestinal tract area of hyperemia - - x -- x - x - x - x x x - x Ceca - round worms found - - - - - - x - x - x -- - - Tesstemsa-llo(n-e1.o0r-1b.o5thcm) - x x - - - - - - x x x x - - 0mII0 Not remarkable - - x -- x -- - - - - - x - Females 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 ~~ ~~ ~~ ~~ Feather loss - -XXXXaXaX-XXXa Head lesions - - x -- - - a - a - - - -a Liver - pale and mottled - - - x -- a - a - - - -a 7 9. - Abdominal Cavity slight egg yolk peritonitis - - - - - a X a -- - -a Intestinal tract - - area of hyperemia - - - - - a - a x -- - - a Not remarkable xx- - - - a - a - X -- - a R e a - Bird founddead during the test. Necropsy findings are presented in Table 2. - 87 - Project Number 454-108 Appendix X - Table 1 Adult Liver Weights (g) from a Northern Bobwhite Reproduction Study with PFOS Control (0 ppm a.i.) Male Pen Liver Female Liver 20 1 202 203 204 205 206 207 208 209 210 211 212 213 2 14 215 216 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 - Data not reported due to mortality. 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 -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 22 1 222 223 224 225 226 227 228 229 230 23 1 232 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 - Data not reported due to mortality. * statisticallydifferent fiom the control group at p < 0.01. 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 - 89 - wild1ife Intemational, Ltd. Project Number 454-108 Appendix XI Histopathology Report - 90 - EPL" EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-108 WILDLIFE INTERNATIONAL, LTD. STUDY NUMBER 454-108 EPL PROJECT NUMBER 212-027 PFOS: A REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE PATHOLOGY REPORT Submitted by: Experimental Pathology Laboratories, Inc. P.O. Box474 Herndon, VA 20172-0474 (703) 471-7060 Submitted to: Wildlife International, Ltd. Easton, MD 21601 December 6,2001 ~~ ~ Project Number 454-108 EXPERIMENTALPATHOLOGY LABORATORIES,INC. TABLE OF CONTENTS PATHOLOGY SUMMARY ................................................................................. COMPLIANCE STATEMENT ............................................................................ QUALITY ASSURANCE FINAL CERTIFICATION............................................ SUMMARY INCIDENCETABLES ADULTS Males ........................................................................................................... Females....................................................................................................... HISTOPATHOLOGY INCIDENCE TABLES Males ........................................................................................................... Females....................................................................................................... CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Males ........................................................................................................... Females....................................................................................................... SUMMARY INCIDENCE TABLES OFFSPRING Males ........................................................................................................... Females....................................................................................................... HISTOPATHOLOGYINCIDENCE TABLES Males ........................................................................................................... Females....................................................................................................... Pane 1 7 1-1 1-2 11-1 11-3 111-1 111-3 IV-1 IV-2 V-1 V-2 - 92 - Project Number 454-108 PATHOLOGY SUMMARY I _ _ - Project Number 454-108 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. WILDLIFE INTERNATIONAL, LTD. STUDY NUMBER 454-108 EPL PROJECT NUMBER 212-027 PFOS: A REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE PATHOLOGY SUMMARY Light microscopic examination was performed on sections of selected tissues from adult male and female Northern bobwhite quail (Colinusvirgnianus) which were untreated or which received 10 ppm a.i. of the test article (Perfluorooctane Sulfonic Acid, Potassium Salt [PFOS]) in the feed for approximately 21 weeks. Selected tissues from untreated 14 day-old offspring of the adult bobwhite were also examined by light microscopy. The objective of this study is to evaluate the effects upon adult bobwhite (Colinus virginiianus)of dietary exposure to the test substance over at least a 21-week period. The experimental design was as follows: Experimental Group Control (0 ppm) 10 ppm a.i. Adultsa Males I Females I 20 20 20 19 Off spring' Males I Females I 6 4 5 5 - 94 - Project Number 454-108 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Wildlife International, Ltd. Study Number 454-108 MATERIALS AND METHODS At the completion of the study interval, all adult bobwhite were euthanized and necropsies were performed by Wildlife International, Ltd. Selected offspring were also euthanized at 14 days of age. Tissues were collected by Wildlife International, Ltd. from adults and offspring, fixed in 10% neutral buffered formalin, and sent to Experimental Pathology Laboratories, Inc (EPL?, where they were embedded in paraffin and made into hematoxylin and eosin-stained sections on glass microscope slides. The following tissues from adult and offspring were examined by light microscopy as available: liver, gallbladder, proventriculus, kidney, brain, gonad (ovary or testis), bursa of Fabricius, and adipose tissue. All tissues required by protocol are presented in the Histopathology Incidence Tables. Microscopic findings for each tissue examined from each bobwhite are listed in the Histopathology Incidence Tables. Microscopic changes were graded one to five depending on severity. Nongradable findings (including autolysis such that a diagnostic interpretationcould not be rendered) are listed as present (P). All findings for all animals are summarized by sex, age group, and treatment group in the Summary Incidence Tables, together with the total number of animals in each group for which tissues were examined. A tabulation of gross observations noted at necropsy with the corresponding microscopic change, if appropriate, is presented in the Correlation of Gross and Microscopic Findings tables. The entries in these tables were transcribed from the gross necropsy observations provided by Wildlife International. Ltd. -2- Project Number 454-108 - 95 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Wildlife International, Ltd. Study Number 454-108 RESULTS Except for three control (334,342,352)and two 10ppm (384,388)adult females, all adults and offspring survived to the end of their respective study intervals. No effects considered possibly related to test article (PFOS) administration were noted in liver, kidney, 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 females; and testes of male offspring. 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. The bursa of Fabricius typically regresses prior to sexual maturity. 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. There were no apparent test-article related effects noted on the gallbladders from the offspring. Testes of all adult males exhibited normal spermatogenesis, characterized by the presence of numerous maturing or mature spermatozoa in the seminiferous tubules. In one (1) control and seven (7) treated males, the testes were observed macroscopicallyas being smaller in site (< I.5 cm) when compared to other birds. In these birds, spermatogenesis was evident microscopically and there was no definitive difference in the morphology of the testes when compared to their unaffected cohorts. Therefore, no definitive microscopic changes could be associated with the size reduction. However, the reduced size of the testes of these birds was consistent with early postreproductive phase regression, a normal physiological phenomenon. The increased incidence of reduced size of the testes in 10 ppm a.i. males when -3- - 96 - Project Number 454-108 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Wildlife International, Ltd. Study Number 454-108 compared to controls, suggests that treatment may have accelerated the normal post-reproductive phase regression. Testes of all offspring were listed as immature because they lacked all evidence of spermatogenesis, had small seminiferoustubules with absent or narrow lumens, and germinal epitheliumwith decreased cell layers (comparedto adult testes). This morphology is consistent with normal physiological immaturity expected in 14 day-old birds. Because of the smaller size of the seminiferous tubules in the offspring testes, the melanin pigment-bearingcells normally present in the testicular interstitiumwere, in effect, more closely packed in a smaller area rather than being widely separated by larger seminiferous tubules (thus the microscopic diagnosis of "pigment concentration" in the testis). Ovaries of adult females exhibited normal oogenesis, characterized by numerousova in various stages of follicular development. All ovaries of offspring females were characterized by numerous oocytes and very small, nonpedunculatedfollicles completely embedded in a prominent ovarian cortex. This morphology is consistent with the normal physiological immaturity expected in 14 day-old birds. Several additional microscopicfindings (described below) were noted in adult and/or offspring bobwhite quail from control and treated groups. Generally, in both adult and offspring males and females, the incidence of these lesions was variable, with some lesions occurring at a higher incidence in controls when comparedto treated birds, and other lesions having the opposite distribution. In addition, there was no significant difference in the mean severity of these lesions in 10 ppm a.i. treated birds when compared to controls. For these reasons, these findings were considered incidental and unrelatedto test article (PFOS) administration. -4- - 97 - Project Number 454- 108 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Wildlife International, Ltd. Study Number454-108 Mononuclear cell infiltrates, observed in various tissues, consisted of foci of lymphocytes and plasma cells generally accompanied by a few heterophils as a minor component. Mineralization in the kidneys of both control and treated adult males was characterized by the presence of basophilic material in the cortex. In most cases this material appeared to be associated with the glomerulus. Vacuolization in the liver was characterized by the presence of vacuoles within the hepatocellular cytoplasm, which varied from usually small, poorly demarcated vacuoles, giving a "lace-like" appearance to the hepatocellular cytoplasm, to variably sized, sharply demarcated, clear vacuoles in the hepatocellular cytoplasm. While special stains were not employed to identify the material in the vacuoles, the latter morphology was consistent with fat accumulation. Hepatocellular vacuolization was observed in the livers of adults and offspring of both sexes. Although there was a slight increase in the incidence of this change in 10 ppm a.i. treated adult males and females, when compared to controls, there was no significant difference in the mean severity between the groups. Since this finding was also commonly observed in the livers from control birds, it was considered to be incidental and not related to test article administration. The apparent increased incidence in the treated birds was considered to be due to biological and/or distributional variability. CONCLUSIONS AND SUMMARY No lesions considered possibly 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 -5- - 98 - Project Number 454-108 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Wildlife International, Ltd. Study Number 454-108 females, and testes of male offspring. The increase in the number of adult males in the 10 ppm a.i. treated group with reduced testicular size, 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. 4x k FREDERICKA. RUECKER, DVM, MS, Diplomate, ACVP Veterinary Pathologis FAWlcp Date I / -6- Project Number 454-108 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. COMPLIANCE STATEMENT Client Name Wildlife International, Ltd. EPL Project Coordinator Dr. Margarita M. Gruebbel Client Study 454-1 08 EPL Pathologist Dr. Frederick A. Ruecker Species Northern Bobwhite EPL Project Number 212-027 Study Title PFOS: A Reproduction Study with the Northern Bobwhite Test Article Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) The Histopathology portions of the above-referenced study were conducted in compliance with the Good Laboratory Practice regulations of the Environmental Protection Agency as stipulated by 40 CFR 160. b EPL Pr4ect Coordinator -7- ~- - I - 100 - Project Number 454-108 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. QUALITY ASSURANCE FINAL CERTIFICATION Study Title: PFOS: A Reproduction Study with the Northern Bobwhite Client Study: 454-108 EPL ProjectCoordinator: Dr. MargaritaM. Gruebbel EPL Project Number: 212-027 EPL Pathologist: Dr. Frederick A. Ruecker The followingaspects of this study were inspectedbythe Quality Assurance Unit of Experimental Pathology Laboratories, Inc. Dates inspections were performed and findings reportedto the EPL ProjectCoordinator and Management are indicatedbelow. Area Inspected Inspection Dates Reporting EPL Project Sheets Project Setup Histology Setup Data Review Draft Report Final Report 10/11,15/01 10112/01 10/19/01 11/1,2,5/01 11/29/01 1/28/03 10/11,15/01 10112/01 10/19/01 11/1,2,5/01 11/29/01 1/28/03 Datereportedto StudyDirectorNanagement Date of last quarterly facility inspection 1E0103 11/02 - EPL/Quality Assurande Unit Form No. 6-2(712439) -8- - 101 - ~~ Project Number 454-108 SUMMARY INCIDENCE TABLES ADULTS - 102- Project Number 454-108 454-108 Adult Sacrifice Mal e Northern Bobwhite SUMMARY INCIDENCE TABLE I GROUP I GROUP I I I CONTROL 10-ppmai ADIPOSE TISSUE (NO. EXAMINED) (20) (20) Inf 1amnation, Chroni c 3 I I I BRAIN (NO. EXAMINED) Inf1amnati on, Meninges, (20) (20) Granulomatous Mineralizing Granuloma, Meni nues 1 2 BURSA OF FABRICIUS (NO. EXAMINED) GALLBLADDER (NO. EXAMINED) (18) (20) Autolysis Precludes Diagnosis 18 18 I KIDNEY (NO. EXAMINED) I (20) I (20) I 1 Infi 1trate, Mononuclear Cell I 1 1 5 I Inflamnation, Chronic I I I I I I b r i m e n t a l PathologyLaboratonicS, Inc. 1-1 454-108 Adult Sacrifice Female Northern Bobwhite - 103 - Project Number 454-108 SUMMARY INCIDENCE TABLE EPL Fkperimental Pathology Laboratories, Inc. - 104 - Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLES ADULTS - 105 - Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE 454-108 Adult Sacrifice Male Northern Bobwhite GROUP ADIPOSE TISSUE Inf1 a n a t ion. Chroni c GAL1 BLADDFR Auto1 vsi s Precludes Diaanosi s LIVER Oeaeneration. Hepatocel 1 ul ar. Centri lobular Infi 1 trate. Mononuc lear Cell Necrosis. Heoatocel lular Vacuo1 ization. Hepatocellular EPL 11-1 Key :X-Not Remarkable N=No Section I-Incomplete A-Autolysis Experimental Pathology Laboratories, Inc. Irinimal 2-sl ight/mi ld fwoderate 4-moderately severe 5.severe/hlgh - 106- Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE 454-108 Adul t Sacri fi ce Male Northern Bobwhite GROUP ADIPOSE TISSUE Inflamnation. Chronic PROVENTRICULUS . Infi 1 trate. Mononuclear Cell Inf1 amnati on. Serosa EPL 11-2 Experimental Pathology Laboratories, Inc. Key :X-Not Remarkable N-No Section I-Incomplete A-Autolysis 1-minimal E-sllght/ai Id 3-moderate 4-moderately severe 5.severelhloh P-Present h e n i g n M-Malignant m-missing one paired organ wunscheduled sac./death - 107 - Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE 454-108 Adult Sacrifice Female Northern Bobwhite GROUP EPL 11-3 Key :X-Not Remarkable N-No Section I-Incomplete A-Autolysis Experimental Pathology Laboratories, Inc. 1-minimal 2-sl ightlmild 3-moderate 4-moderately severe 5.severe,hi g h - 108 - Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE 454-108 Adult Sacrifice Female Northern Bobwhite ADIPOSE TISSUE I nf 1amnation, Chronic A N I M A 3333 L 7777 Xi N/ X 2 ii GROUP URSA OF FABRICIUS I NI NI N I N GAI LBLADDER A u t o l y s i s Precludes Diaanosis AAAX P P P EPL Key :X-Not Remarkable N-No Section I-Incomplete A-Autolysis - 109 - Project Number 454-108 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS ADULTS :! Numb Client Topography/ Site 345 TESTIS Client Gross Observations Microscopic Observations Right testis approximately 1.5 an No Correlating Lesion I c c 0 I e R r 0 00 454-108 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS 37 1 373 1387 393 40 1 Client Topography / Site TESTES TESTES I TESTIS TESTES TESTIS TESTIS TESTIS Client Gross Observations Testes 1.25 cm ~ Testes 1.25 cm Right t e s t i s 1.0 cm Testes 1.0 cm Right t e s t i s 1.0 cm Right t e s t i s 1.0 cm Right t e s t i s 1.0 cm Microscopic Observations No Correlatina Lesion (TESTIS) IN0 Correlatinq Lesion (TESTIS) No Correlating Lesion No Correlating Lesion (TESTIS) No Correlatinq Lesion No Correl a t ing Lesion No Correlating Lesion I c, c, c, I I 11-2 - - 111-3 I c c h) I R f c 0 00 Adult Sacri f i ce Species: Northern Bobwhite Amma Numhlr Client Topography/ Site 378 LIVER 408 LIVER Sex: Females Group Identification: 10-ppmai - Sacri f iced Client Gross Observations Pale, mottled Microscopic Observations Vacuolization, Hepatocellular Pale. mottled Vacuolization, Hepatocellular ~ 111-4 454- 108 CORRELATION OF GROSS AND MlCROSCOPiC FINDINGS Adul t Sacri f i ce Species: Northern Bobwhite nma GUmbLr Client Topography/ Site Sex: Females Client Gross Observations Group Identification: 10-ppmai - Unscheduled Sac. /Death Microscopic Observations 384 * No coronary fat No Comnent Required OVARY &vel opi ng No Correlating Lesi on P 111-5 CI 0 00 - 115 - Project Number 454-108 SUMMARY INCIDENCE TABLES OFFSPRING 454- 108 Offspring Sacrifice Mal e Northern Bobwhite - 116 - Project Number 454-108 SUMMARY INCIDENCE TABLE EPL Experimental PathologyLaboratories, Inc. 454-108 Offspring Sacrifice Female Northern Bobwhite - 117 - SUMMARY INCIDENCE TABLE EPL EkperimentalPathology Laboratories,Inc. - 118 - Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLES OFFSPRING - 119- Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE 454- loa Offspring Sacri f ice Male Northern Bobwhite ADIPOSE TISSUE r GROUP A N I M A2 15 3 N GROUP KIDNEY X I PROVFNTRICULUS X I n f i l t r a t e . Mononuclear Cell TESTIS N Inmature ent Concentration v- 1 I E P L ;:;;;g;imhi;;sl ~ ~pathology La~boratories, ln~c. ~ ight/mi Idi 3-moderate L-~moderately Sever~e ~ - 120 - Project Number 454-108 HISTOPATHOLOGY INCIDENCE TABLE GROUP 454-108 Offspring Sacrifice Female Northern Bobwhite A N I M A 1 ADIPOSE TISSUE BRAIN URSA OF FABRICIUS oid Depletion GALLBLADDER Auto1vsi s Precludes D iaanosi s Y GROUP OVARY Imnature PROVENTRICULUS EPL Key :X-Not Remarkable N-No Section I-Incomplete A-Autolysis Experimental Pathology Laboratories, Inc. 1-minimal 2-slight/mi Id 3-moderate 4-moderately severe 5.severe/hi gh w i l d l i f e International, Ltd. - 121 - Project Number 454-108 Appendix XIIA The Analysis of PFOS in Red Blood Cells and Sera Collected through Week 15 - . LLL - Wildlijie International, Ltd. Project Number 454-108 Exygen Study No.:023-066 Table XXVI. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 5 Sponsor ID 454-108-361-Wk5 454-108-361-Wk5* 454-108-362-Wk5 454-108-362-Wk5* 454-108-363-Wk5 454-108-363-Wk5* 454-108-364-Wk5 454-108-364-Wk5* 454-108-365-Wk5 454-108-365-Wk5* 454-108-366-Wk5 454-108-366-WW* 454-108-368-Wk5 454-108-368-WW* Centre ID 0107077 0107077 0107078 0107078 0107079 0107079 0107080 0107080 010708 I 0107081 0 107082 0107082 0107083 0107083 Set Number 05 1801A 051801A 051801A 051801A 051801A 051801A 05 1801A 051801A 051801A 051801A 05 1801A 05 1 801 A 05 1801A 05 1801A Extraction ' Date Analysis Date 5/18/01 5/18-19/01 5/18/01 5118-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 918-19/01 5/18/01 5/18-19/01 5/18/01 5/18- 19/01 5/18/01 5/18-19/01 5/18/01 5118-19/01 5/18/01 5/18-19/01 5/18/01 5/18-19/01 5/18/01 5/18- 19/01 5/18/01 5/18- 19/0I 5/18/01 5/18-19/01 AVERAGE STANDARD DEVIATION: Analyte Found (pg/mL) NO NQ NQ NQ NQ NQ NQ NQ NQ NQ 0.00292 0.00264 NQ NQ 0.000440 0.000993 * Duplicate injection NQ = Not Quantifiable (A peak was detected at the correspondinganalyte 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 40 of 98 - 123 - 'wildlifeInternational, 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 Centre Set Extraction Analysis Analyte 454-108-401-Wk5 0107084 051801DR 5118/01 5/21-22/01 113 454-108401-Wk5* 0107084 051801DR 5118/01 5/21-22/01 107 454-108-4O2-Wk5 0107085 051801DRD 5118/01 5/2U01 353 454-108-402-Wk5* 0107085 051801DRD 5/18/01 5/22/01 351 454-108403-Wk5 0107086 051801DR 5/18/01 5/2 1-2U01 30.5 454-108-403-Wk5* 0107086 051801DR 5118101 512 1-22/01 30.4 454-108-404-Wk5 0107087 051801DR 5118101 5L2I-22/01 26.9 454- 108-404-Wk5' 0107087 05 1801DR 5/18/01 5121-22/01 25.8 454- 108-405-Wk5 0107088 051801DRD 5118101 5/22/01 415 454-108-405-Wk5* 0107088 051801DRD 5118101 5/22/01 441 454-108-406-Wk5 0107089 051801DRD 5/18/01 5/22/01 1050 454- 108-406-Wk5* 0107089 05180LDRD 5/18/01 5/22/01 1040 454-108-407-Wk5 0107090 051801DRD 5118101 5/2U01 495 454-108-407-Wk5* 0107090 051801DRD 5118/01 5/22/01 501 454-108408-Wk5 0107091 051801DRD 5/18/01 5/22/01 45 1 454-108408-Wk5* 0107091 051801DRD 5118/01 5/22/01 47 1 AVERAGE: 369 STANDARD DEVIATION 324 * Duplicate injection LOQ = 0.01 p g h L for red blood cells and serum Exygen Research Page 41 of 98 - 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 Centre Set Extraction Analysis Analyte 454-108-441-Wk5 0107092 052201CD 5/2U01 5/24/01 166 454-108-441-Wk5* 0107092 052201CD 5/22/01 5/24/01 163 454-108-442-Wk5 0107093 052201CD 5/22/01 5/24/01 108 454-108-442-WW * 0107093 052201CD 5/22/01 5/24/0 1 103 AVERAGE: 135 STANDARD DEVIATION: 34 Table XXIX. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm a.i. for Week 10 Sponsor __ ID 454-108-361-Wk 10 454-108-361-WklO* 454-108-362-Wk10 454-108-362-WklO* 454-108-363-Wkl0 454-108-363-Wkl0* 454-108-364-Wk10 454-108-364-Wk10* 454-108-365-Wk 10 454-108-365-Wk10* 454-108-366-Wkl0 454-108-366-WklO* 454-108-367-Wk10 454- 108-367-Wk10* 454-108-368-Wk10 454-108-368-WklO* Centre ID 0107 100 0107100 0107101 0107101 0107102 0107102 0107103 0107103 0 107 104 0107104 0107105 0107105 0107106 0107 106 0107107 0107107 * Duplicate injection Set Number 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B 051801B Extraction Analysis Date Date 5/18/01 5/18/01 5/19/01 511 910 1 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/18/01 5/19/01 51 1910 1 5/19/01 511 910 1 5/19/01 5/19/01 5/19/01 5/19/01 5/19/01 511 910 1 5/19/0 1 5/19/01 5/19/01 5/19/01 AVERAGE: STANDARD DEVIATION Analyte Found (pg/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 correspondinganalyteretention 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,0.00005pg/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 w i l d l i j e International, Ltd. - 125 - Project Number 454-108 Exygen Study No.: 023-066 Table XXX.Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 10 Sponsor ID 454-108-401-Wk10 454-108-401-Wk10* 454-108-402-Wk10 45eioa-402-wkio* 454-108-403-Wk10 454-108-403-WklO* 454-108-404-Wk10 454- 108-404-wklO* 454-i o a - 4 0 5 - w ~ o 454-108-405-Wl~lW 454- 108-406-Wk10 454-108-406-WklO* 454-108407-Wk10 454-108-4O7-WklO* 454- 108-408-Wk 10 454-108-408-WklO* Centre ID 0107108 oio7ioa 0107109 0107109 01071LO 0107110 0107111 0107111 01071 12 0107 112 0107113 0107113 0107114 0107114 0107115 0107115 * Duplicate injection Set Number 05 18OlDRD 05 180lDRD 051801DRD 051801DRD 05 1801DR 051801DR 05 1801DR 05 1801DR 051801DR 05 1801DR 05 1801DR 05 I801DR 051801DR 05 180 IDR 05180lDR 051801DR Extraction Analysis Date Date 5118/01 5/18/01 5/18/01 5/22/01 5LWO1 5/22/01 511 810 1 5/22/01 5/18/01 5L21-2UOl 5/18/01 5/21-2U01 5/18/01 5/21-22101 511 810 1 5L21-22/01 5/18/01 5l21-22/01 5/18/01 5/21-22/01 5/18/01 5/2 1-22101 5/18/01 5/2 1-22/01 5/18/01 5/21-22/01 5/18/01 5/21-22/01 5/18/01 5/2 1 -22/0 1 5/18/01 5D1-22/01 AVERAGE STANDARD DEVIATION: Annlyte Found (pg/mL.) 612 67 1 203 190 23.0 20.1 19.0 21.2 18.0 20.0 48.9 50.5 22.8 22.3 30.4 30.2 129 220 LOQ = 0.01 pg/mL for red blood cells and serum Exygen Research Page 43 of 98 - 126 - wildlifeInternational, Ltd. Project Number 454-108 Exygen Study No.: 023-066 Table XXXI. Summary of PFOS in Quail Red Blood Cell Samples at 0 ppm ai. for Week 15 Sponsor ID 454-108-361-Wk15 454-108-361-Wk15* 454-108-362-Wk15 454-108-362-Wk15* 454-108-363-Wk15 454-108-363-Wk15* 454-108-364-Wk15 454-108-364-Wk15* 454-108-365-Wk15 454-108-365-Wk15* 454-108-366-Wk15 454-108-366-Wk15* 454-108-367-Wk15 454-108-367-Wk15* 454-108-368-Wk15 454-108-368-Wk15* 454-108-333-Wk15 454-108-333-Wk15* Centre ID 0107116 0107116 01071 17 01071 17 0107118 0107118 0107119 0107119 0107120 0107120 0107121 0107121 0107122 0107122 0107123 0107123 0107124 0107124 Set Number 052101A 052 101 A 052101A 052101A 052101A 052 I01A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052101A 052 10 1 A 052 101 A 0521OlA 052101A Extraction Date Analysis Date 5/21/01 5/21/01 ~nimi 5/21/01 5/21/01 mimi 5/21/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/21/01 5/21/01 5/22/01 5/22/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22l01 5/21/01 5/21/01 5/21/01 5/22/01 5/22/0 1 5/22/01 AVERAGE STANDARD DEVIATION: Analyte Found (pglmL) 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 calibrationstandards (0.0001pg/mL)) LOQ = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 44 of 98 wildlife International, Ltd. - 127 - Project Number 454-108 Exygen Study No.:023-066 Table XXXII. Summary of PFOS in Quail Red Blood Cell Samples at 10 ppm a.i. for Week 15 Sponsor ID 454-108-401-Wk15 454-108-401-Wk15* 454-108402-Wk15 454-108-402-Wk15* 454-108-403-Wk15 454-108-403-Wk15* 454-108404-Wk15 454-108-404-Wk15* 454-108-405-Wk15 454-108-405-Wk15* 454-308406-Wk15 454-108-406-Wkl5* 454- 108-407-WkI5 454-108-407-Wk15* 454-108408-Wk15 454- 108-408-Wk15* 454-108-383-Wk15 454-108-383-Wk15* Centre ID 0107125 0107125 0107126 0107126 0107127 0107127 0107 128 0107128 0107129 0107129 0107 130 0107130 0107131 0107131 0107132 0107 132 0107133 0107133 Set Number 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052101AD 052lOlAD 05210I AD 052101 AD 052101AD 052201CD 052201CD 052101AD 052101AD Extraction Analysis Date Date 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/2 1/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/01 5/22-23/01 5/21/0 I 922-23/01 5/21/01 Y22-23/01 5/21/01 5122-23/01 5/21/01 5/22-23/01 5/21/01 5/24/01 5/2 1/01 5/24/0 1 5/21/01 5122-23101 5/21/01 5/22-23/01 AVERAGE STANDARD DEVIATION: Analyte Found ( p e ) 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 -11. Summary of PFOS in Quail Red Blood Cell Samples at 50 ppm a.i. for TERM Sponsor Centre Set Extraction Analysis Analyte 454-108-409-TERM 0107134 052201CD 5/22/01 5/24/01 361 454-108-409-TERM* 0107134 052201CD 5/22/01 5/24/01 356 454-108-410-TERM 0107135 052201CD 5/2U01 5/24/01 106 454-108-410-TERM* 0107135 052201CD 5/22/01 5/24/01 114 AVERAGE: 234 STANDARD DEVIATION 144 * Duplicate injection LOQ = 0.01 p g / d for red blood cells and serum Exygen Research Page 45 of 98 - 128 - 'Mlildlije Internationat, Ltd. Project Number 454-108 Exygen Study No.: 023-066 Table xxxn7. Summary of PFOS in Quail SerumSamples at 0 ppm ai. for Week 5 Sponsor Centre Set Extraction Analysis Analyte 454-108-361-Wk5* 454-108-362-Wk5 454- 108-362-Wk5' 454-108-363-Wk5 454-108-363-Wk5* 454-108-364-Wk5 454- 108-364-Wk5* 454-108-366-WW 454-108-366-Wk5" 454-108-368-Wk5 454-108-368-Wk5* 0107154 0107155 0107155 0107156 0107156 0107157 0107157 0107159 0107159 0107160 0107160 * Duplicate injection M2301AR M2301AR 072301072301AR 072301AR 07230 I AR 072301AR 072301AR 072301AR 072301AR 072301AR 7/23/03 7125101 7/23/01 7/25/01 7n3101 7nsioi 7123101 7n5101 7n31oi 7~~101 7/23/01 7n5101 71~3101 m i 0 1 1/23/01 7125101 7~3101 7n5101 7/23/01 1/25/01 in310 1 7/25/01 AVERAGE: STANDARD DEVIATION 0.0295 0.0149 0.0134 0.0198 0.0166 0.00510 . 0.00535 0.0135 0.0120 0.0138 0.0156 0.0156 0.00746 LOQ = 0.01 pg/mL for red blood cells and serum Exygen Research Page46 of 98 - 129 - w i l d l v e International, Ltd. Project Number 454-108 Exygen Study No.: 023-066 Table XXXV.Summary of PFOSin Quail Serum Samples at 10 ppm a.i. for Week 5 Spoasor Centre Set Extraction A ~ l y s i s Analyte 454-108-402-WW 0107162 072301AD 7/23/01 7/26/01 45.8 454-108-402-Wk5* 0107162 072301AD 7/23/01 7/26/01 45.6 454-108-403-Wk.5 0107163 072301AD 7/23/01 7/26/01 115 454-108-403-Wk5* 0107163 072301AD 7/23/01 7/26/01 128 454-108-404-Wk5 0107164 072301AD 7/23/01 7/26/01 104 454-108-404-Wk5* 0107164 072301AD 7/23/01 7/26/01 98.3 454-108-405-Wk5 0107165 072301AD 7/23/01 7/26/01 138 454-108-405-WW* 0107165 072301AD 7/23/01 7/26/01 129 454-108-406-WW 0107166 072301AD 7/23/01 7/26/01 106 454-108-406-WW* 0107166 072301AD 7/23/01 7/26/01 107 454-108407-Wk5 0107167 072301AD 7/23/01 7/26/01 42.4 454-108407-Wk5* 0107I67 072301AD 7/23/01 7/26/01 36.3 454-108-408-Wk5 0107168 072301AD 7/23/01 7/26/01 77.6 454-108-408-WW* 0107168 072301AD 7/23/01 7/26/01 84.4 AVERAGE 89.8 STANDARD DEVIATION: 35.0 Table XXXVI.Summary of PFOS in Quail Serum Samples at 50 ppm a.i. for Week 5 Sponsor Centre Set Extraction Analysis Analyte 454-108-441-Wk5 0107092 072301AD 7/23/01 7/26/01 55 1 454-108-441-Wk5* 0107092 072301AD 7/23/01 7/26/01 562 454-108-442-Wk5 0107093 072301AD 7/23/01 7/26/01 529 454-108-442-Wk5* 0107093 072301AD 7/23/01 7/26/01 520 AVERAGE: 541 STANDARD DEVIATION: 19.4 * Duplicate injection LOQ = 0.01 p.lg/mcfor red blood cells and serum Exygen Research Page 47 of 98 I* - 130 - wildlife 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 Centre Set Extraction Analysis Analyte 454-108-361-Wk10 0101171 454-108-361-Wk10* 0107177 454-108-362-Wk10 0107178 454-108-362-WklO* 0107178 454-108-363-Wk10 0107179 454-108-363-Wk10* 0107179 454-108-364-Wk10 0107180 454-108-364-WklO* 0107180 454-108-365-Wk10 0107181 454-108-365-WkIO* 0107181 454-108-366-Wk10 0107182 454-108-366-WklO* 0101182 454-108-361-Wk10 0107183 454-108-361-WklO* 0101183 454-108-368-Wk10 0107184 454-108-368-WklO* 0107184 07230lB 072301B 072301B 072301B 012301B 072301B 072301B 072301B 072301B 072301B 072301B 012301B 072301B 072301B 012301B 072301B 7/23/01 7/24/01 1/23/01 7/24/01 7/23/01 1/24/01 7/23/01 1/24/01 7/23/01 7/24/01 1/23/01 1/24/01 7/23/01 in4101 7/23/01 1/24/01 7n3m 7/24/01 1/23/01 1/24/01 1/23/01 7/24/01 7/23/01 7/24/01 7/23/01 1/24/01 7/23/01 7/24/01 7/23/01 1/24/01 7/23/01 7/24/01 AVERAGE STANDARD DEVIATION: * Duplicate injection LOQ = 0.01 pg/mL for red blood cells and serum 0.00483 0.00489 0.00295 0.00249 0.00467 0.00521 0.00390 0.00375 0.0027 1 0.00292 0.00439 0.00437 0.00563 0.00542 0.00537 0.00526 0.00430 0.00105 Exygen Research Page 48 of 98 - 151 - wildlife 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 Centre Set Extraction Analysis Analyte J ID ID Dat- e Date Found &mL) 454-108-401-Wk10 0107185 072301DD 7/23/01 7/27-28/01 213 454-108-401-WklO* 0107185 072301DD 7/23/01 7/27-28101 207 454-108-402-WklO 0107186 072301DD 7/23/01 7/27-28/01 53.6 454-108-402-Wk10* 0107186 072301DD 7/23/01 7D7-28/01 53.7 454-108-403-Wk10 0107187 072301DD 7/23/01 7/27-28/01 148 454-108-403-WklO* 0107187 072301DD 7/23/01 7/27-28/01 177 454-108-404-Wk10 0107188 072301D 7/23/01 7/27-28/01 19.4 454-108-404-WklO* 0107188 072301D 7/23/01 7/27-28/01 20.3 454-108-405-Wk10 0107189 072301DD 7/23/01 7/27-28/01 157 454-108-405-Wk10t 0107189 072301DD 7/23/01 7/27-28/01 147 454-108-407-Wkl0 0107190 072301DD 7/23/01 7/27-28101 105 454-108-407-Wk1O* 0IO7 190 072301DD 7/23/01 7/27-28/01 110 454-108-408-Wk 10 0107191 072301DD 7/23/01 7/27-28101 97.0 454-108-408-WklO* 0107191 072301DD 7/23/01 7/27-28/01 93.7 AVERAGE: 114 STANDARD DEVIATION: 63.4 * Duplicate injection LOQ = 0.01 pg/mL, for red blood cells and serum Exygen Research Page 49 of 98 - 152 - wildlifeInternational, Ltd. 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 Centre Set Extraction Analysis Analyte 454-108-361-Wk 15 0107192 072301C 703101 7/24-25/01 NQ 454-108-361-Wk15* 0107192 072301C 7R3101 7/24-25/01 NQ 454-108-3623%15 0107193 omoic 703rni 704-25/01 NQ 454- 108-362-Wkl5* 0107193 072301C 7/23/01 7/24-25/01 NQ 454- 108-363-Wk15 0107194 012301C 7/23/01 7/24-25/01 0.00154 454- 108-363-Wk15* 0107194 072301C 7/23/01 7/24-25/01 0.00160 454-108-364-Wkl5 0107195 072301C 7/23/01 7D4-25/01 NQ 454-108-364-Wk15* 0107195 0 7 2 3 0 1 ~ 7/23/01 7/24-25/01 NQ 454-108-365-Wk15 0107196 072301C 1/23/01 7/24-25/01 NQ 454-108-365-Wk15* 0107196 072301C 7/23/01 7/24-25/01 NQ 454- 108-366-Wk15 0107 197 072301C 7/23/01 7/24-25/01 NQ 454-108-366-Wkl5* 0107197 072301C 7/23/01 7124-25101 NQ 454- 108-367-Wk15 0107198 072301C 7/23/01 7/24-25101 0.00227 454-108-367-Wk15* 0107198 072301C 7/23/01 704-25/01 0.00249 454-108-368-Wk15 0107199 M2301C 7/23/01 7/24-25/01 0.00253 454-108-368-Wk15* 0107199 072301C 7/23/01 7124-25101 0.00255 454-108-333-Wk15 0107200 072301C 7/23/01 7/24-25/01 0.00162 . 454-108-333-Wk15* 0107200 072301C 7/23/01 7/24-25/01 AVERAGE 0.00160 0.000928 STANDARD DEVIATION: 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.OOOI &TIL)) LOQ = 0.01 p g / d for red blood cells and serum For values recorded as NQ, 0.00005pg/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 wild1ife Intemational, Ltd. - 133 - 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 Centre Set Extraction Analysis Analyte 454-108-401-Wk15 0107201 072301DD 7/23/01 7n7-28/01 181 454-108-401-Wk15* 0107201 072301DD 7/23/01 7/27-28/01 180 454-108-402-Wk15 0107202 072301DD 7/23/01 7/27-28/01 8.21 454-108-402-Wk15* 0107202 072301DD 7/23/01 7/27-28/01 8.45 454-108403-Wk15 0107203 072301DD 7/23/01 7/27-28/01 246 454-108-403-Wk15* 0107203 072301DD 7/23/01 7/27-28/01 239 454-108-404-Wk15 0107204 072301DD 7/23/01 7/27-28/01 11.1 454-108-404-Wk15* 0107204 072301DD 7/23/01 7/27-28/01 10.7 454-108405-Wkl5 0107205 072301DD 7/23/01 7/27-28/01 141 454-108405-Wk15* 0107205 072301DD 7/23/01 7/27-28/01 156 454-108-406-Wkl5 0107206 072301DD 7/23/01 7/27-28/01 7.04 454-108-406-Wk15* 0107206 072301DD 7/23/01 7/27-28/01 6.98 454-108-407-mi5 0107207 0 7 2 3 0 1 ~ ~ 7/23/01 7/27-28/01 107 454-108-407-Wk15* 0107207 072301DD 7/23/01 707-28/01 114 454- 108-408-Wk15 0107208 072301DD 7/23/01 7127-28101 10.2 454-108-408-Wk15* 0107208 072301DD 7/23/01 7/27-28/01 8.91 454-108-383-Wk15 0107209 072301DD 7/23/01 7/27-28/01 131 454-108-383-Wk15* 0107209 072301DD 7/23/01 7/27-28/01 143 AVERAGE: 95.0 STANDARD DEVIATION 86.6 Table XLI. Summary of PFOS in Quail Serum Samples at 50 ppm ai. for TERM Sponsor ID 454-108-410-TERM 454-108-410-TERM* Centre ID 0107211 010721 1 * Duplicate injection Set Number 072301DD 072301DD Extraction Analysis Date Date 7/23/01 7/23/01 7/27-28/01 7/27-28/01 AVERAGE STANDARD DEVIATION Analyte Found (pg/mL) 476 514 495 26.9 LOQ = 0.01 pg/mL for red blood cells and serum Exygen Research Page 5 1 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 - 135 - wildlife International, Ltd. Project Number 454-108 Report EO1-1245 ROWI E01-1246 PFOS A Reproduction Study with the Nothern Bobwhite PFOS:A Repmd& Shrdyvrithth. "nBobwb SUMMARY The concentration of the C8Fl7S0i anion of peffluorooctane sulfonate, potassium salt (PFOS. ClF1$O;K') wa8 determined in the liver and sera of adult bobwhite quail and their oilspring in both a control group and a 10 ppm, dosed group from Wildlife feeding study t454-108. The followfng tables summarize the C,F,S% concentrations found in the liver and sera of study samples. These results only relate to the samples tested. Matrix-extracted PFOS standards were used to establish the HPLC-MSlMS instrument calibration curve lor C,F,,W. Concentrations of C,F1,So; are reported after correction for the purity and the potassium contributionto the mass of the PFOS reference substance. Table 1summarizes Wa study sample data. Table 2 summarizesstudy data after removing certain results that have been statistically determined to be outliers utilizingthe Dixon 9-Test. Table 1. SummaryTable, QF17SOiConcentrationin Sera and Uver 3M Environmenlll Lsbaratafy 3M EnvironmentalLabomto?y Page701226 Page 7 - 136 - wildlife International, Ltd. Project Number 454-108 Report E01-124s Repat 01-1246 PFOS A Repduction Study with the Nothem Bobwhite PFO%ARopmductlonShldywlth~NorUmmBobwhk. Table 2. SummaryTable, C#lTSot' ConcentradonIn Senand Liverwith Outlkrr Removed No d i e m fmn this dah ret. Refer toTsMe 1for res&. For IndivMual mphvelwa sea Tablea 10to 13 and Atla&fnenlB. Accurpcy: semple ruMs Maccurateto 1- 4-30%. :ere QC earnplea met lhe mmptence cdterla for the method. - Liver aC sanpkrdd not &the H of QC at each levd mud bo- 75% 12S% crlterlaforthe melhod. All Svcn QC sampladl(3) spked at 10ppbwwe>125%rclcovery. M Omer HvaQC wnplm (8)were* cfferla. marebr.he - toEondQC criteria WOI mol In that 213 d a!4 aC ware wllhln X% 125% PURPOSE Tha purposed this analyticalphase of the study is to quantifyC8Ft7S0in the liver and sera of both control and dosed Northern Bobwhite Ouail and thelr offspring. REFERENCSEUBSTANCES Table 3. ReferenceSubstance6 StorageCondiins I 3M Labomtory identificationNumber Frozen-20% *I. 1O'C SD-OW, Lot Y 217 F r o m-20% 4-10% SDWB 3M EnvironrnentslLatmmtoIy 3M EnvironmentalLaboratory - 131 - wildlifeInternational, Ltd. Project Number 454-108 R c p ~ fElO1-1245 Rwll EOl-lZ4S PFOS A Reproduction SLudy with the Nothem Bobwhite PFOS A RspoductrCnStudy whl th.NorthernBobuvhlto 3M Envimnmlntal Laboratory 3M Environmental Laboratory Pege15ot226 Page 15 wiId1ife Intemationa1, Ltd. - 138 - Project Number 454-108 RepoaE01-1245 R0p~tE01-1245 PFOS:A ReproductionStudy withthe NothernBobwhite P m A FWwAzIbn StudyuUl~IhaNorlhem BabwMtr I E01-1245-284961454-1M).388. IOpprnfemak,adukO2-01-02 I EOI-1246-284981464-lWN8,lm f d , uhIR,U2-01-02 0.4 I oa I I 1 1 14700 I 1 1 11600 I EOI-1241285171454-10, lOppnfemak,aciJt. 0201-02 I 0.2 I 1 1 7840 I ,EO1-1245-28630 454-108-525,1Oppmfemek,0tlsphg,~1-02 0.03 1 0.W 1 12100 There are two results reporiedfor these samples. Bothvalues are included and averaged in the "Average of Dup Sample" column. These values mrejected based on the results of the Dixon's Cl-Test. 90% confidence interval, for the rejection of outliers. Accuracy: Sample results are accurateto 100%+I- W h . Sera QC samples met the acceptancecriteria for the method. 3M Envlmnrnmkd Labomtory 3M Environmental Laboratory Page 18 d 226 Page 16 - 139 - wildlijie International, Ltd. Project Number 454-108 Report 301-1245 R.pat EO1-1245 PFOS A Repraduction Study with the Nothern Bobwhite P F m A kpdudbnSlulywIhU1.Nc#wnBobwhi . EO1-124528475 454.108383,oppmmala.adult, 02-0142 . E01424528476 454.108-365.oppmmale,adult.02-01-02 0.1 1 c m c4M) 0.1 1 400 0.4 1 .dXl <400 0.4 1 el00 3M Envlmental labwalq 3M Environmentalhboratov Page 17 wiId1ije Internationa1, Ltd. - 140- Project Number 454-108 R e p ~ tEt O1-1245 R e m f31-1245 PFOS A RepductionStudy withtheNothanr Bobwhite PFO!&A~~WIUW~~ 0.1 . E01-12452WS 454-10&385,lQWImel~&d100,(n-01-02 o.l . EOI-1245.2R497 454-loe3e7,l~mde,&d100P2-01-02 0.3 0.3 ~~ 0.1 . MI-1245.28499 454108388,loppn male,adlt, dlOO,OZ-0i-Cn o.l 0.06 . EOI-124528501 454-108-3QIl.QWImele,&d100,W01-02 0.06 '2 , M1-1245.28503 454-10&399. l@pnmale.acwt.dlW,02-01M 02 03 02 , EM-124528504 45c108399,lOppmmab,~d100,0241@ 0. I o.l 0.1 . E01-124&28510 454-108401,Iappn male, adult dl00,02-0102 o.l . E01-124528512 454-108403, IOppnmaIe. edv!&d100,02-01.W 0.2 o,2 0.1 . 01-124528614 454-108405,1Oppnmale,acMt,d100,0241M 0.1 . EOI-124528516 454-106407,lmd,adukd100,WQl.W , 0.1 o.l 100 100 100 I100 l1o0o0 100 ' 100 loo 100 100 100 .-- 100 100 loo loo loo 100 loo 100 loo loo lIrn 1174Mo 196OOO 113Bo 141Mx) 147000 leMoo 165oM) 127wo 121Ooo 185ooo 132ooo 144000 162Cac 131ooC --1 159ooo ntxloo -1z7m 138OOO loBMl0 117ooO 112Ooo 1 m 215ooc 13xxx 112ooo 105000 3M EnvlronmontslLabocaay 3M Environmental Laboratory Page 18 d 226 Page 18 - 141 - wildlife International, Ltd. Project Number 454-108 Report EO1-1245 Report E01-1245 PFOS: A ReproductionStudy with the Nothem Bobwhite PFOS A iWmfhUmStudy whl the Notihem Bobwrhit. . EO1-124528928 454-108283, apPn &, oltsphg,WOlU? 0a.o0z2 11 . m, EOl-1245128521 454-108-291,Oppnmale, 02-01-02 0.06 1 0.05 1 <400 e400 400 <400 Cuyl G400 .EO1-124528534 ~ 1 0 8 5 5120ppnmete, wng,02-01-02 0.07 1 13800 o.07 1 11600 0.1 1 l2'O0 . EO1-12628537 45C106568,10ppTlmale.Msping,02-01-02 0.1 1 two0 There am tworesubreportedfor these samples. Bothvaluesam includedand averagedinthe "Average of DupSampldcolumn. Accuracy: Sample rssuHs are accurate to 100% +/- 30%. Sera QC samples met the acceptancecriteriafor the method. 12500 11200 3M EnvkonmenlslLaboratocy 3M Environmental Laboratory Pago 19 Of 226 Page 19 wild1ife Intemational, Ltd. - 142- Project Number 454-108 Repoa EO1-1245 Ropoct 01-1245 PFOS A Rqnduction Study with the NothernBobwhite PFOS A RepmducUon Studywlmthn NomemBnbwW Table 12. GF&S Data Summaw. FemaleQuailLiver M u # and OffaMlna) EO1-134240321 0 ppnai.330 F Liver EOI-1342-3W39 0 ppm ai. 332 F Liver EOl13123Ms7 0 nunai.234 F Liver M1-1342.3M15 0 ppllai.336 FLiver EO1-1342- 0 p ~ani.338F L k F O l - I W 2 ~ 1 1OmmaiL340FLivar 1.0749 1 1.0469 1 1.Wm 1 1.0188 1 1.0543 1 1.0182 1 402 <m2- 6029 402 .602* <502* E01-1342-3C609 Oppna.i.362FLiwr EOI-1342-30821 Oppnai.364FUlw 01-1342.30845 0 ppna.l.366FLiver EW-1342.30663 Oppnal.368FuvSr r EO1-1342+W81 10ppnki.370F L h EO1-134230699 I O p p n a i . 3 7 2 F k 17 IOmmal.374FLker 1.0424 1 1.0384 I 1 .a577 1 I .OB20 1 1.0868 1 1.0748 1 1.0790 1 40.2' 402 402 <m2* 5920' 4430' 5030' 3M E m i m s n t a l Laboratay 3M Environmental Laboratory Page 20 - 143 - wildlife International, Ltd. Project Number 454-108 Repart EO1-1245 Fqlod Mi-1246 PFOS A ReproductionStudy withthe Nothem Bobwhite PFOS:ARIprodudbn Studyvdihthe NotthemBobwNt. e These values were rejectedbased on the resultsof the Dixon's Q-Test, 90% confidence intetval. for the dection of outliers. - Accuracy: Sample rebulls are accurateto 100% +/-30%. * Liver QC samples did not meet the W of QC at each level must be within 75% 125% criteria for the method. All liverQC samples (3) spiked at 10 ppbwere >125% recovery. All other liver - QC samples (6) were within were within 75"A 125%. criteria, therefore the second QC criteria was met in that 2/3 of all QC 3M EnvlmnmrntalLaboratory 3M Environmental Liaboratoty Page 21 01 P6 Page 2 1 - 144 - wildlife International, Ltd. Project Number 454-108 Report EOI-1245 RIporl EOl-1245 PFOS:A Reproduction Study with the Nothm Bobwhite PFo8: A R e p d u 3 l m Study wllh the NotWtemBctMIh EO1-1342GW613 0ppmai.SSMLiver EOI-1342- Oppmal.337MLhw EOI-134230402 0 ppm ai. 339 M Liver 1.ax2 1 1.0269 1 0.0886 1 502' 502' 40.T EOI-1342.30800 '0ppmai. XI MLiver MI-134240818 0 ppmai.983MLiver EOI-1342-30638 Omnnai3EMLhw I MI-1342-30872 IlOppmai.369MUver EOI-1342- 10 ppm ai. 371 M Liver EOI-134240708 10 ppm a.i. 373 M Liver EOI-1342-30728 10ppmai.375 M Uver E01-134240744 lOppmai.377MLiver E01-1-82 lOppmai.379MUver MI-1342.30780 fOppnai.381 M h EOl-1342.30798 lOppmai.383MLiver 01-1342-30829 10ppmai.386MLiver EO1-134240847 10ppmai.387MLiver EOI-1342.30885 10ppm ai.389MLiver MI-1342.30883 lOppmai.391 MLhrar MI-134230901 lOppmai.393MUver E01-1342-3OQ19 10ppmai.395MUver EOl-1342.30937 lOppmai397MUver E01-1242~5510ppmai 399M Uvor E01-1342-30973 10ppmai.401 MLiver M1-1342.30ggl 1 O m m ai 4E3M Limr EO1-1342.31008 IlOppmaL 40SM Liver 01-1342-31027 IlOppm a i407 M Liver 3M Emironmm Laboratory 3M EnvironmentalLaboratory 1 .am 1 1.0818 1 1.0916 1 I 1.0010 1 10 I 1.0858 10 1.0278 10 1.1008 10 1.0236 10 1.0312 10 1.0501 10 1.0110 10 1 .m 10 1.0315 10 1.0310 10 1.0947 10 1.0834 10 1.0489 10 1 .om 10 1.On6 10 1.m1 100 1.w19 1M II 1.- I 100 I 1.0%6 1 loo I <502* 402' &2* m I ~~ 78mY am* 7m- 803oO' 9070(r 8ooo6' ~~ 73500' 834o(r 79300' 703oO' 70400' 8M1M)' 651W 501W 63oocr 147000' lruYnn* 138000' 132000. I Pago 12 of 228 Page 22 wiId1ife Intemational, Ltd. - 145 - Project Number 454-108 Rtport EO1-1245 R.port E01-1246 PFOS: A Repmductim Study with the Nothm Bobwhite PFOS:A RepDducllon S W y withthe Nor(hrrn Bobuhii0 These values were rejected based on the results of the Dixon's Q-Test, 90% confidence Interval, for the rejection of outliers. Attachment B contains data summary tables. Accuracy: Sample results are accurate to 100% 4-30%. - Liver QC samples did not meet the % of QC at each level must be within 75% 125%criteria for the method. All liver QC samples (3)spiked at 10ppbwere r125.k recovery. All other liver QC samples (6) were within 75% -were within 125%. criteria, therefom the second QC criteria was met in that 2/3 of all QC STATEMENT OF CONCLUSION Sera results from the male control group portion of this study showed no detectable levels of C.F&Oj in 19 of 19 quail (400ndmL). The sera results from the female control group portion of this study showed no detectable levels of C,Ft7SO; in 16 of 16 birds (c400ng/mL) (Table 15). Analytical results for twenty sera samples from the 10ppm dosed male group showed an average - of 141000 nghnL GF,fiO; (range 94400 226000 nglmL, Table 14). Analytkal resutto for seventeen sera samples from the 1Oppm dosod female group showed an average of 8870 ng/mL - CoFd30; (range4710 14700ng/mL, Table 15). Sera resultsfrom tho mak offspring control group showed no detectable levels of C8Ft,+O; in 6 of 6 birds (c400 nghL). The sera results from the female oflspring control group portion of this study showed no detectablelevels of CIF17SO;in 4 of 4 birds ( ~ 4 0 0ng/mL. Table 14). Analytical - resultsfor five sera samples from the 1Oppm dosed male offspring group showed an average of 12600ng/mL (range 8220 19200nglmL, Table 14). Analytical results for five sera samples from the lOppm dosed female offspring group showed an average of 12400 ng/mL (range 7640 - 18500ne/mL, Table 14). Liver results from the male control group showed no detectable levels of CIFIiSO; in 19 of I9 birds (402nglg, Table 15)and quantifiable concentrations of C,F1,SOJ' in 1 of 20 (209 nglg, Table 14). The liver results from the female control group showed no detectable levels of - (&Fl7S0J' in 20 of 20 birds (40.2ne/& Table 14). Analytical results for twenty h e r samples from the lOppm dosed male group showed an average of 88500 nglg C8F,7SO; (range 50100 - 147000ng/g, Table 14). Analytical results for twenty liver samples from the 1Oppm dosed female group showed an average of 4900 ng/g CeFi7SOj (range 3290 7550 ngg, Table 14). 3M Envimental Laboratory 3M EnvironmentalLaboratory PagE 23 of 226 Page 23 Wildlve International, Ltd. - 146 - Project Number 454-108 Report E01-1245 Rqmrt EO!-1245 PFOS: A Reproduction Study with the Nothem Bobwhite PFOS' A Reprodudon study HAth me Northern Bobwhite Liver results from the male offspring control group portion of this study showed no detectable levels of CBFt7SO; in 6 of 6 birds (~50.2ng/g, Table 14). The liver results from the female offspring control group portion of this study showed no detectable levels of C,F,,SOi in 4 of 4 birds (~20.n0g/g. Table 14). Analytical results for four liver samples from the lOppm dosed male Offspring group showed an average of 5760 ng/g (range 4940 - 6520 nglg, Table 15). Analytical results for four liver samples from the IOppm dosed female offspring group showed an average of 5490 nglg (range 4590 - 7030 ng/g, Table 15). Table 14. SummaryTable, CeF&Oi Concentratlon in Sera and Liver 18 Grap MeOffspring Control Grow Female Offspring 4 contd Grow 9990nghL ' I I NA 4710-31WnmL c 4 M n!$nL (6 of 6) NA c4M)n$mL (4of 4) 20 4900n6Jg' 3290-7550ndg' I 6 <20.0 Wg' c20.0nq'g (6 d 6)' 4 40.0 nqlg' 43.0 nqlg (4d 4)' ICcmGmu~ 5 Female OffspringlOppn 5 amup 126Wn&1L 8220-19203nqhnL 124OOq$mL 7640-185WnghnL 5 7350 n&' 4940 - 13700 nq'g. 5 7180m 4593-13srIwg* For individualsample values see Tables 10 to 13 and Atlachment 8. . Accuracy: Sample resub are accurate to 100% +I. 3%. Sere QC samples met the acceplence criteriafor me method. ' Uver OC samples did not meet the H ol QC at each level must be within 75% 125%criterla for the rnelhcd. All liver OC samples (3) spiked at 10 ppb were >125% recovery. All other liver QC samples (6) were within CritEriB. thereforethe second ac criteria was met in that 2/3 d all ac were within 75% - 125%. OM EnvironmentalLaboratory 3M Environmental Laboratory Rage 24 of 226 Page 24 - 147 - wildlife International, Ltd. Project Number 454- 108 Report E01-1245 Report E01-1245 PFOS: A ReproductionStudy with the Nothern Bobwhite PFOS:A Reproductionstudy with the Northern Bobwhite Table 15. SummaryTable, CBFllSOi Concentrationin Sera and Liver with OutliersRemoved * No outliers from this data set. Refer to Teble 1 lor results. For individualsample values see Tables 10 to 13end Attachment 8. Accuracy: Sample results are accuraleu)100% 4-309% Sera QC samples met the acceptancecriterla for the method. ** Liver QC samples did not meet the H of QC at each level must be within 75% ~ 125% criteria for the method. All liver QC sampias (3)spiked a1 10 ppb were >125% recovery. All other liver QC samples (6) were wlthin criteria, thereforethe second Q criteria was met In thrrt 213 01 all QC were wilhin 75% - 125%. STATISTICAMLETHODS Statistical methods were limited to calculating means, standard deviations. % coefficient of variation and the Dixons Q-Test. The Dixons Q-Test, used lor the rejection of outliers, has values ordered from lowest to highest. A Q-value is determined from the difference of a result and the next closest result which is divided by the difference of the highest and lowest value in the group of values. A critical Q-value is determinedbased on the number of samples in the group and the desired confidence interval. If the Q-value is greater than the critical 0-value then the value will be rejected. STATEMENT OF DATAQUALITY The measurementof accuracy available at this time is the quality control and matrix spike results. For sera data, the accuracy (the observed amount in the QC and MS samples, divided by the amount spiked, expressed as a percent) was found to range from 74.1% to 126%; while precision, expressed as the standard deviation of the recoveries, was found to range from 2.14"/. to 9.56%. For liver data, the accuracy was found to range from 72.6% to 141%; while precision was found to range from 2.1 1% to 5.51%. 3M EnnronmenteJ Laboratory 3MEnvironmental Laboratory Page 25 01 226 Page 25 Reproductive Performance by Pen Appendix XI11 - T a b l e 1 Eggs Laid / Maximum Laid (%) from a Northern B o b w h i t e Reproduction S t u d y w i t h PFOS Eggs Replicate Laid 0 PPM A.I. Max. Laid Arcsin % Trans. Eggs Laid 10 PPM A.I. Max. Laid Arcsin % Trans. Eggs Laid 50 PPM A.I. Max. Laid Arcsin % Trans. Eggs Laid 150 PPM A.I. ax. Laid Arcsin % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 55 63 87 69.12 27 63 43 40.89 - -- - 38 63 60 ,50.95 23 63 37 37-17 32 63 51 45.45 - -- - 56 63 89 70.53 47 63 75 59.74 50 63 79 62.98 54 63 86 67.79 - -- - 53 63 84 66.52 54 63 86 67.79 62 63 98 82.76 54 63 86 67.79 605 819 47 63 74 60.73 12 0 20 13.29 34 63 54 47.28 46 63 73 58.70 43 63 68 55.71 42 63 61 54.74 47 63 75 59.74 53 63 84 66.52 -21 63 33 35.26 - - 54 63 86 67.79 - - - 44 63 70 56.69 44 63 70 56.69 63 63 100 90.00 41 63 65 53.78 -60 63 95 77.40 - - 592 819 46 63 72 60.02 11 0 17 13.50 - - - - - - - -- - - Data are not available due to adult mortalii. Differences between the control and each treatment group were not significant (p > 0.05). I w P 00 I - - - - - - - -- - Eggs Appendix XI11 - Figure la - Laid from a Northern Bobwhite Reproduction Study u i t h PFOS Control 60 50 z W P 40 \ CL W m 530 z 20 * * * 10 n 7 -I- I " 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 * Eggs Laid Data a r e not Pe available n due Number to adult mortality. Eggs Appendix XI11 - Figure l b Laid from a Northern Bobuhite Reproduction Study with PFOS - 10 PPH h.1. 60 90 z W L1 40 \ U Wrn 530 z 20 * * * 10 n I -r 1 " 217 218 213 220 221 222 223 224 225 226 W 228 223 230 231 232 * Eggs Laid Data a r e not Pe available n due Number to adult mortality. Reproductive Performance by Pen Appendix XI11 - T a b l e 2 Eggs Cracked / Eggs Laid (8) from a Northern Bobwhite Reproduction Study with PFOS Replicate Esse Crack 0 PPM A.I. W?" Arcsin Laid % Trans. 10 PPM A.I. Eggs Eggs Crack Laid Arcsin % Trans. 50 PPM A.I. Eggs Eggs Crack Laid Arcsin % Trans. 150 PPM A.I. Eggs Eggs Crack Laid ArCSin % Trans. 1 0 55 0 0.00 0 34 0 0.00 - 2 0 27 0 0.00 1 46 2 8.48 3 - - - 0 43 0 0.00 4 2 38 5 13.26 0 42 0 0.00 5 0 23 0 0.00 0 47 0 0.00 6 0 32 0 0.00 0 53 0 0.00 - 7 - - 0 21 0 0.00 - - 8 0 56 0 0.00 - 9 2 47 4 11.90 0 54 0 0.00 10 1 50 2 8.13 - - - 11 1 54 2 7.82 0 44 0 0.00 12 - 0 44 0 0.00 13 1 53 2 7.90 0 63 0 0.00 14 0 54 0 0.00 0 41 0 0.00 15 16 62 26 30.53 2 60 3 10.52 16 0 54 0 0.00 Total Mean SD 23 605 2 47 4 12 3 6.12 7 8.91 3 592 0 46 1 11 0 1.46 1 3.59 - - - -- - - -- - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). - - - - - - - - - - 2 9. B Eggs Appendix XI11 - F i g u r e 2a - Cracked / Eggs Laid from a Northern Bobwhite Reproduction Study with PFOS Control 50 z LJ a 40 \ 530 rY W m 2 20 10 + * n - 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Pen Number Eggs Laid HEggs Cracked 9: Data are not available due to adult mortality. Eggs Appendix XI11 - Figure 2b Cracked / Eggs Reproduction Laid from a Study with - Northern Bobuhite PFOS 10 PPIl A.I. -t 50 z W n 40 \ f m CT W m z 20 * * e- 10 n I I " 217 218 219 220 221 222 223 '224 225 226 227 228 229 230 231 232 * Eggs Laid Data ar0 not Pe available n Number EEggs Cracked due to adult mortality. from a - Reproductive Performance by Pen Appendix XI11 Table 3 Viable gaabryos / Eggs Set ( % I Northern Bohhite Reproduction Study with PFOS 0 PPM A.I. Viable Eggs Replicate Embr. Set Arcsin % Trans. 10 PPM A.I. Viable sggs Embr. Set Arcsin % Trans. 50 PPM A.I. Viable Eggs Embr. Set Arcsin % Trans. 150 PPM A.I. Viable Eggs Embr. Set Arcsin % mans. 1 47 50 94 75.82 30 30 100 90.00 - - 2 12 23 52 46.25 39 40 97 80.90 3 - - 39 39 100 90.00 4 30 31 97 79.65 38 38 100 90.00 5 21 21 100 90.00 35 42 83 65.91 6 28 28 100 90.00 45 48 94 75 .'52 - - 7 - 17 19 89 71.07 - - - 8 51 51 100 90.00 - 9 39 40 97 80.90 46 48 96 78.22 10 43 43 100 90.00 - - - 11 47 49 96 78.34 39 39 100 90.00 12 27 37 73 58.68 13 47 47 100 90.00 55 57 96 79.20 14 43 49 88 69.52 17 36 47 43.41 - - 15 38 40 95 77.08 43 53 81 64.25 16 48 49 98 81.79 - Total Mean SD 494 521 38 40 94 79.95 12 11 13 12.19 470 526 36 40 89 75.17 11 10 15 14.26 - - - -- - - -- - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). - - - - - - - - - - I c VI P I Bcd 1 9. Uiable Appendix XI11 - Figure 3a Embryos / Eggs Reproduction Set from a Study with - Northern Bobuhite PFOS Control 50 $0 30 20 10 * * * n I --r T- * Pen Number Eggs Set E Uiable Embryos nata a r e not available due to adult mortality. Uiablo Appendix XI11 - F i g u r e 3b Embryos / Eggs Reproduction Set from a Study uith - Northern Bobuhite PFOS 10 PPH h.1. 50 40 z w a \ CY30 w rn S 3 z 20 10 * * * n 217 218 219 220 221 222 I I 2-23 224 2 Z 226 227 228 229 bo I 231 232 * Pen Number Eggs Set E Uiable Embryos Data a r e not available due t o adult mortality. Reproductive Performance by Pen Appendix XI11 - T a b l e 4 L i v e 3-Week Embryos / V i a b l e Ehbryos (%) from a Northern Bobwhite Reproduction Study with PFOS 0 PPM A.I. Live Viable Replicate 3-Week Embr. % Arcsin Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 46 47 98 81.61 12 12 100 90.00 - -- - 30 30 100 90.00 21 21 io0 90.00 - 28 28 100 90.00 - - - 51 51 loo 90.00 38 39 97 80.79 43 43 100 90.00 47 47 100 90.00 - -- - 46 47 98 81.61 43 43 100 90.00 37 38 97 80.66 48 48 100 90.00 490 494 38 38 99 87.28 12 12 1 4.25 10 PPM A.I. Live Viable 3-Week Embr. % Arcsin Trans. 29 30 97 79.48 39 39 100 90.00 39 39 100 90.00 38 38 100 90.00 34 35 97 80.27 45 45 100 90.00 - 16 17 94 75.96 - - - 46 - 46 - 100 - - 90.00 39 39 100 90.00 27 27 100 90.00 55 55 100 90.00 16 17 94 75.96 43 43 100 90.00 - -- - 466 470 36 36 99 86.28 11 11 2 5.91 50 PPM A.I. Live Viable 3-Week Embr. % Arcsin Trans. - - - - - - - - - - - -- - - - - - - - - -- - - -- - - -- - - -- - - -- - - -- - - -- - - -- - - -- - - -- - - - - - - - -- - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). 150 PPM A.I. Live Viable 3-Week Embr. % Arcsin Trans. - - - - - -- - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - -- - - -- - - - - - - - - - - -- - - - - - - - - - - - - -- - Live Appendix XI11 - Figure 4a 3-Meek Embryos / Uiable Reproduction Study - Embryos from with PFOS a Northern Control Bobwhite 40 z W n \ 01-30 W m E 3 z 20 10 * * n I 7 201 202 203 204 205 206 2W 208 209 210 211 212 213 214 215 216 * Pen Number Uiable Embryos Live 3-Ueek Embryos Data are not available due t o adult mortality. Live Appendix XI11 - Figure 4b 3-Week Embryos / Uiable Riaproduction Study - Embryos from u i t h PFDS a 10 Northern PPM A.I. Bobuhite 60 90 40 30 20 10 * * * n I 1 - I 217 218 219 220 221 222 223 224 225 224 227 228 229 230 231 232 * Pen Number Uiable Embryos E Live 3-Ueek Embryos Data are not available due to adult mortality. - Reproductive Performance by Pen Appendix XI11 T a b l e 5 Hatchlings / L i v e 3-Week Ehbryos (%) from a Northern Bobwhite Reproduction Study with PFOS 0 PPM A.I. Number Live Replicate Hatch. 3-Week 8 Arcsin Trans. 10 PPU A.I. Number Live Hatch. 3-Week % Arcsin Trans. 50 PPM A.I. Number Live Hatch. 3-Week % Arcsin Trans. 150 PPM A.I. Number Live Hatch. 3-Week % Arcsin Trans. 1 44 46 96 77.96 20 29 69 56.15 - - - 2 12 12 100 90.00 36 39 92 73.90 3 - 38 39 97 80.79 4 30 30 100 90.00 32 38 84 66.59 5 20 21 95 77.40 31 34 91 72- 72 6 28 28 100 90.00 39 45 87 68.58 - - - 7 - 13 16 81 64.34 - 8 47 51 92 73.74 - - - - - - 9 35 38 92 73.68 42 46 91 72.85 10 42 43 98 81.23 - 11 12 47 47 100 90.00 - -- - 39 39 100 90.00 27 27 100 90.00 13 45 46 98 81.52 55 55 100 90.00 14 42 43 98 81.23 16 16 100 90.00 - - 15 35 37 95 76.56 26 43 60 51.04 16 46 48 96 78.22 - - Total Mean SD 473 490 36 38 97 81.66 11 12 3 6.29 414 466 32 36 89 74.38 12 11 13 13.21 - - - -- - - -- - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). - - - - - - - - - - I c o\ 0 I e P c 0 00 Appendix XI11 - Figure 5a - Hatchlings / Live 3-Ueek Embryos from a Northern Bobuhite Reproduction Studu u i t h PFOS Control 60 50 $0 z W U \ a30 W m S 3 2: 20 10 * * * 0 I I I 202 203 204 205 206 207 208 209 210 212 214 215 216 * Pen Number Live 3-Ueek Embryos E Hatchlings Data are not available due to adult mortality. Appendix XI11 - Figure 5b - Hatchlings / Live 3-Week Embryos from a Northern Bobuhite Reproduction Study u i t h PFOS 10 PPM h.1. bo 40 z W a \ LT 30 W m S 3 z 20 10 * * n I I - 217 218 219 220 221 222 223 22$ 225 226 227 228 229 230 231 232 * Pe L i v e 3-Uoek Embryos Data are not available n Number CI Hatchlings due to adult mortality. Reproductive Perfomance by Pen Appendix XI11 - T a b l e 6 14-Day Old Survivors / Hatchlings (%) from a Northern Bobwhite Reproduction Study w i t h PFOS 0 PPM A.I. 14-Day Number Replicate Old Hatch. % Arcsin Trans. 10 PPM A.I. 14-Day Number Old Hatch. % Arcsin Trans. 50 PPM A.I. 14-Day Number Old Hatch. % Arcsin Trans. 150 PPM A.I. 14-Day Number Old Hatch. % Arcsin Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 43 44 98 81.33 - - 12 12 100 90.00 - - 30 30 100 90.00 19 20 95 77.08 27 28 96 79.11 - -- - 46 47 98 01.61 33 35 94 76.11 40 42 95 77.40 47 47 100 90.00 - -- - 45 45 100 90.00 40 42 95 77.40 35 35 100 90.00 40 46 87 68.83 451 473 35 36 97 82.22 11 11 4 7.09 20 20 100 90.00 36 36 100 90.00 29 30 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 5s 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 - - - - - - -- - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). - - - -- - - -- - I c a\ W I e e 6or $0 z W n \ 30 W m r 3 z 20 10 * * 0 1 I 201 ,202 203 204 205 206 207 2m 210 * I 212 * Hatchlings Data are not Pe available n Number E l4-Day Old Survivors due to adult mortality. 214 215 14-Day Appendix XI11 - Figure 6b - Old Survivors / Hatchlings from a Northern Bobuhite Reproduction Study u i t h PFOS 10 PPM h.1. 60 40 30 20 10 * 0- 217 218 219 220 221 222 1 223 224 225 226 227 228 * Hatchlings Data are not Pe available n Number E 14-Day Old Survivors due to adult nortality. 230 231 232 Reproductive Performaace by Pen Appendix XI11 - T a b l e 7 Hatchlfngs / Eggs Set (%) from a Northern Bobwhite Reproduction Study w i t h PFOS 0 PPM A.I. Number E g g s Replicate Hatch. Set Arcsin % Trans. 10 PPM A.I. Number Eggs Hatch., Set Arcsin % Trans. 50 PPM A.I. Number Eggs Hatch. Set Arcsin % Trans. 150 PPM A.I. Number Eggs Hatch. S e t Arcsin % Trans. 1 44 50 88 69.73 20 30 67 54.74 - - - 2 12 23 52 46.25 36 40 90 71.57 3 - 38 39 97 80.79 4 30 31 97 79.65 32 38 84 66.59 5 20 21 95 77.40 31 42 74 59.22 6 28 28 100 90.00 39 48 81 64.34 - - - 7 - 13 19 68 55.81 - - - - 8 47 51 92 73.74 - - - - 9 35 40 88 69.30 42 48 88 69.30 10 42 43 98 81.23 11 12 47 49 96 78.34 - -- - 39 39 100 90.00 27 37 73 58.68 13 45 47 96 78.10 55 57 96 79.20 14 42 49 86 67.79 16 36 44 41.81 - - - 15 35 40 88 69.30 26 53 49 44.46 16 46 49 94 75.67 - Total Mean SD 473 521 36 40 90 73.58 11 11 12 10.24 414 526 32 40 78 64.35 12 10 18 14.02 - - - -- - - -- - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). - - - -- - - -- - Reproductive Performance by Pen Appendix XI11 - T a b l e 8 14-Day Old Survivors / Egg8 Set (%) from a Northern Bohhite Reproduction Study with PFOS 0 PPM A.I. 14-Day Eggs Replicate Old Set Arcsin % Trans. 10 PPM A.I. 14-Day Eggs Old Set Arcsin % Trans. 50 PPM A.I. 14-Day Eggs Old Set Arcsin % Trans. 150 PPM A.I . 14-Day Eggs Old Set Arcsin % Trans. 1 43 50 86 68.03 2 12 23 52 46.25 3 4 30 31 97 79.65 5 19 21 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 74.60 11 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 11 11 12 9.08 20 30 67 54.74 36 40 90 71.57 29 39 74 59.58 30 38 79 62.69 30 42 71 57.69 38 48 79 62.84 10 19 53 46.51 37 48 77 61.40 - -- - 37 39 95 76.91 27 37 73 58.68 53 57 93 74.64 16 36 44 41.81 23 53 43 41.21 * 386 526 30 40 72 59.25 11 10 17 11.35 - Data are not available due to adult mortality. Significantly different from the control at p C 0.05 - - - -- - -- - - - - -- - - -- - I c a\ 4 I 7 9. s 0 e d 91 P c 0 00 Reproductive Performance by Pen Appendix XI11 - T a b l e 9 Hatchlfngs / Maximum Set (%) from a Northern Bobwhite Reproduction Study with PFOS ~ ~~ ~ ~ 0 PPM A.I. Number Max. Replicate Hatch. Set Arcsin % Trans. 10 PPM A.I. Number M a x . Hatch. Set Arcsin % Trans. ~~ 50 PPM A.I. Number Max. Hatch. S e t Wcsin B Trans. 150 PPM A.I. Number M a x . Hatch. Set Arcsin % Trans. 1 2 3 4 5 6 7 0 9 10 11 12 13 14 15 16 Total Mean SD 44 57 77 61.47 12 57 21 27.31 - -- - 30 57 53 46.51 20 57 35 36.32 20 57 49 44.50 - - - - 47 57 02 65.24 35 57 61 51.59 42 57 74 59.14 - 47 57 02 65.24 - - - 45 57 79 62.69 42 57 74 59.14 35 57 61 51.59 46 57 01 63.94 473 741 36 57 64 53.44 11 0 20 11.95 20 57 35 36.32 36 57 63 52.63 30 57 67 54.74 32 57 56 40.53 31 57 54 47.52 39 57 68 55.81 13 57 23 20.53 - -- - 42 57 74 59.14 - -- - 39 57 60 55.01 27 57 47 43.49 55 57 96 79.20 16 57 20 31.99 26 57 46 42.40 - -- - 414 741 32 57 56 40.94 12 0 20 13.22 - -- - - -- - -- - - -- - - -- - - - - - - - - -- - - -- - - - - - - -- - - -- - - -- - - -- - - -- - - - - -- - - -- - - -- - - -- - - -- - - -- - - -- - - -- - - -- - - -- - - -- - - -- - - - - - - - - - - - - - -- - - - - - - - -- - - -- - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). Reproductive Performance by Pen Appendix XI11 - T a b l e 10 14-Day Old Survivors /Maximum Set (%) f r o m a Northern Bobwhite Reproduction Study with PFOS 0 PPM A.I. 14-Day Max. Replicate Old Set Arcsin % Trans. 10 PPM A.I. 14-Day Max. O l d Set Arcsin % Trans. 50 PPM A.I. 1 4 - ~ a yMax. O l d Set Arcsin % Trans. 150 PPM A.I. 14-Day Max. O l d 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 56.90 457 741 35 57 62 52.04 11 0 19 11.44 20 57 35 36.32 36 57 63 52.63 29 57 51 45.50 30 57 53 46.51 30 57 53 46.51 38 57 67 54.74 10 57 18 24.76 - -- - 37 57 65 53.68 37 57 65 53.68 27 57 47 43.49 53 57 93 74.64 16 57 28 31.99 23 57 40 39.44 386 741 30 57 52 46.45 11 0 19 12.41 - -- - -- - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). - - - - - - - -- - ? 9. B Y % r P 0 00 from Reproductive PerfoIzPaace by Week and Pen Appendix X I V - T a b l e la Eggs Laid by Week and P e n a Northern Bobwhite Reproduction Study with PFOS Experimental Group (ppm a-i.) 1 LOT A B C D Control E F G H I J PENS ~~ ~ 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 2 0 - 0 0 0 - 1 0 0 0 - 2 0 3 1 5 2 - 1 0 0 - 5 1 2 2 - 5 3 4 2 5 5 - 2 0 1 - 4 5 3 4 - 5 4 6 5 5 5 - 3 0 3 - 6 5 4 5 - 5 5 7 6 6 6 - 5 0 3 - 6 7 6 7 - 6 6 7 6 6 1 - 4 3 4 - 7 7 7 7 - 6 7 7 7 7 0 - 5 3 5 - 7 7 8 8 - 6 7 7 7 6 0 - 6 5 6 - 7 4 6 7 - 6 7 7 6 6 4 - 6 5 5 - 7 6 7 6 - 7 7 7 7 7 4 - 6 7 5 - 6 5 7 a - 5 8 7 7 Totals 9 32 49 59 71 73 77 73 80 82 Totals 55 27 - 38 23 32 - 56 47 50 54 - 53 54 62 54 605 - Data are not available due to adult mortality 'LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. I c 4 0 I 7 9. (D Q Reproductive Perfonnance by Week and Pen MPendix X I V - T a b l e lb Eggs Laid by Week and P e n from a Northern Bobwhite Reproduction S t u d y with PFOS Experimental Group (ppm a.i.) 1 LOT A B C D 10 E F G H I J PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 0 0 0 0 5' 0 0 - 2 1 0 4 0 2 - 0 1 0 0 4 3 1 - 2 - 4 3 5 2 5 - 2 3 1 3 5 4 0 - 5 - 6 3 6 2 6 - 2 5 4 4 6 6 4 - 5 - 5 4 6 5 6 - 4 6 6 5 5 5 4 - 7 - 5 5 7 5 8 - 4 7 5 5 4 7 4 - 6 - 0 5 7 5 6 - 5 8 7 7 4 8 4 - 9 - 2 6 8 6 7 - 6 6 6 5 4 6 3 - 6 - 7 5 6 5 7 - 5 6 7 6 4 6 0 - 6 - 7 6 7 6 6 - 6.4 7 7 6 8 1 - 6 - 7 7 7 5 7 - Totals 14 30 46 62 72 65 81 72 72 78 Totals 3 4 46 43 42 47 53 21 - 54 - 44 44 63 4 1 60 - 592 - Data are not available due to adult mortality LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. 7 9. CD B % P r 0 00 Appendix XIV - Figure 1 Eggs Laid per Hen from a Northern Eobuhite Reproduction Studu u i t h PFOS 7 I # B -0 t i I I I I D F 6 H Ueeks 10 .-.I Concentrations, ppm ai. I c 4 h, I 7 9. Appendix XIV - Figure 2 Hens Laying per Pen from a Northern Bobwhite Reproduction Study with PFOS i.a 0. a cT 0.6 w m E: 3 2 0.4 0.i li 6 i 0 I I I I I t B C E F H J Weeks Concentrations, ppm ai. -0 .mmm 10 I c 4 W I R P c 0 00 Appendix X I V - F i g u r e 3 Eggs per Laying Hen from a Northern Bobwhite Reproduction Study u i t h PFOS 7 6 6 E+ m E 3 z 3 2 1 0 I I I I I I I I b B C D E F 0 H I J Weeks . . Concentrations. DDOI ai. -0 m.1.110 Reproductive Performance by Week and Pen Appendix XIV - T a b l e 2a Bggs Cracked by Week and Pen ram a Northen? Bobwhite Reproduction Study with PFOS Experimental Group (ppm a-i.) PENS 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Totals 1 LOT A 0 B 1 C 3 D 2 Control E 2 F 0 G 6 H 2 I 2 J 5 Totals 0 0 - 2 0 0 - 0 2 1 1 - 1 0 16 0 23 - Data are not available due to adult mortalii LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. Reproductive Performaace by Week and Pen Appendix X I V - T a b l e 2b Eggs Cracked by Week and Pen f r o m a Northern Bobwhite Reproduction S t u d y with PFOS Experimental Group (ppm a.i.1 PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Totals A 0 B 0 C 0 D 1 10 E 0 F 0 G 1 H 0 I 1 J 0 ~~ Totals 0 1 0 0 0 0 0 - 0 - 0 0 0 0 2 - 3 ~~ - Data are not available due to adult mortality 1LOT A - Eggs Set During Week 12; LOT B - Eggs-Set During Week 13; etc. 7 9. sCD efY 0 00 Reproductive Performance by Week and Pen Appendix XIV - T a b l e 3a Eggs Set by Week and Pen from a Northern Bobwhite Reproduction Study with PFOS Experimental Group (ppm a . i . ) 1 LOT A B C D Control E F G H I J PENS 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 1 0 - 0 0 0 - 1 0 0 0 - 1 0 2 1 5 1 - 0 0 0 - 4 0 1 2 - 5 2 3 1 4 5 2 0 1 - 4 2 3 3 - 4 4 4 5 5 4 - 2 0 2 - 5 5 3 5 - 5 4 5 5 5 6 - 5 0 3 - 6 6 5 6 - 5 6 5 6 6 0 - 3 3 3 - 6 7 6 7 - 6 6 6 6 6 0 5 2 5 - 7 6 7 7 - 4 7 1 7 6 0 5 4 5 5 5 6 4 5 6 - 6 6 6 5 6 4 5 - 7 5 7 5 - 6 7 4 7 7 3 3 7 4 5 5 6 a - 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 52 1 - Data are not available due to adult mortality LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. I c 4 4 I R f 0 00 R e p r o d u c t i v e Perfonuance b y Week and Pen Appendix XIV - T a b l e 3b Eggs S e t b y Week and Pen from a Northern Bobwhite Reproduction Study with PFOS Experimental Group (ppm a-i.) PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 1 LOT A B C D 10 E F 0 H I J 0 0 0 0 4 0 0 - 1 - 0 0 3 0 1 - 0 0 0 0 4 2 1 - 2 - 4 2 5 1 5 - 1 3 0 3 4 4 0 - 4 - 5 3 5 2 5 - 2 4 4 3 6 5 4 - 5 - 5 2 6 4 5 - 3 6 5 5 4 5 3 - 6 - 4 5 6 5 7 - 4 6 5 4 4 6 4 - 6 - 0 4 7 4 6 - 4 8 6 7 3 8 3 - 7 - 1 6 6 6 5 - 6 5 6 4 4 5 3 - 6 - 7 4 6 4 7 - 4 5 6 6 3 6 0 - 5 - 6 6 6 6 5 - 6 3 7 6 6 7 1 - 6 - 7 5 7 4 7 - ~ ~~ ~~ ~ - Totals 30 40 39 38 42 48 19 - 48 - 39 37 57 36 53 - - Data are not available due to adult mortality I LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. Totals 9 26 39 55 64 60 70 67 64 72 ~~ 52 6 I w 4 00 I I c 0 00 from R e p r o d u c t i v e P e r f o r m a n c e by Week and P e n Appendix X I V - T a b l e 4a V i a b l e Embryos by Week and P e n a Northern Bobwhite Reproduction Study with PFOS Experimental Group (ppm a.i.) 1 LOT A B C D Control E F G H I J PENS 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Totals 1 0 - 0 0 0 - 1 0 0 0 - 1 0 1 1 5 5 1 - 0 0 0 - 4 0 1 2 - 5 0 3 1 22 4 3 - 2 0 1 - 4 2 3 3 - 4 0 4 5 35 5 0 - 2 0 2 - 5 5 3 5 - 5 4 5 4 45 5 2 - 4 0 3 - 6 6 5 6 - 5 6 5 6 59 5 0 - 3 3 3 - 6 7 6 5 - 6 6 5 6 61 6 0 - 5 2 5 - 7 6 7 7 - 4 7 1 7 64 4 0 - 5 5 5 - 6 4 5 6 - 6 6 6 5 63 5 3 - 6 4 5 - 7 5 7 5 - 6 7 4 7 71 7 3 - 3 7 4 - 5 4 6 8 - 5 7 4 6 69 Totals 47 12 - 30 21 28 - 51 39 43 47 - 47 43 38 48 494 ' - - Data are not available due to adult mortality LOT A - Eggs Set During Week 12; LOT B Eggs Set During Week 13; etc. I c1 7 9. B 2 f P VI c 0 00 Reproductive Perfonuance by Week and Pen Appendix XIV - T a b l e 4b Viable Embrps by Week and Pen from a Northern Bobwhite Reproduction Study with PFOS Experimental Group (ppm a.i.) 1 LOT A B C D 10 E F G H I J PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 0 0 0 0 0 0 0 - 0 - 0 0 2 0 0 - 0 0 0 0 1 2 1 - 2 - 4 0 5 0 4 - 1 2 0 3 4 4 0 - 4 - 5 0 5 2 5 - 2 4 4 3 6 5 4 - 5 - 5 0 6 1 5 - 3 6 5 5 4 5 3 - 6 - 4 2 6 2 7 - 4 6 5 4 4 5 3 - 6 - 0 4 6 1 3 - 4 8 6 7 3 7 3 - 7 - 1 6 6 4 1 - 6 5 6 4 4 5 2 - 6 - 7 4 6 1 7 - 4 5 6 6 3 6 0 - 5 - 6 6 6 4 4 - 6 3 7 6 6 6 1 - 5 - 7 5 7 2 7 - Totals 2 19 35 50 58 51 63 63 61 68 Totals 30 39 39 38 35 45 17 - 46 - 39 27 55 17 43 - 470 - Data are not available due to adult mortality ' - LOT A - Eggs Set During Week 12; LOT B Eggs Set During Week 13; etc. I L 00 0 I z 9. B 6 e c 0 00 - Reproductive Performance by Week and Pen Appendix X I V T a b l e 5a L i v e Three-Week Ebbryos by Week and Pen from a Northern Bobwhite Reproduction Study w i t h PFOS Experimental Group (ppm a-i.) 1 LOT A B C D Control E F G H I J PENS 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 0 0 - 0 0 0 - 1 0 0 0 - 0 0 1 1 5 1 - 0 0 0 - 4 0 1 2 - 5 0 3 1 4 3 - 2 0 1 - 4 2 3 3 - 4 0 4 5 5 0 - 2 0 2 - 5 4 3 5 - 5 4 5 4 5 2 - 4 0 3 - 6 6 5 6 - 5 6 5 6 5 0 - 3 3 3 - 6 7 6 5 - 6 6 5 6 6 0 - 5 2 5 - 7 6 7 7 - 4 7 1 7 4 0 - 5 5 5 - 6 4 5 6 - 6 6 6 5 5 3 - 6 4 5 - 7 5 7 5 - 6 7 4 7 7 3 - 3 7 4 - 5 4 6 8 - 5 7 3 6 Totals 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 - Data are not available due to adult mortality 'LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. I c 00 L I efY 0 00 Reproductive Perfonnance by Week and Pen Appendix X I V - T a b l e 5b Live Three-Week Ehbryos by Week and Pen from a Northern Bobwhite Reproduction Study with PFOS Experimental Group (ppm a.i.1 1 TMT A B C D 10 E F G H I J PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 0 0 0 0 0 0 0 - 0 - 0 0 2 0 0 - 0 0 0 0 1 2 1 - 2 - 4 0 5 0 4 - 1 2 0 3 4 4 0 - 4 - 5 0 5 2 5 - 2 4 4 3 6 5 3 - 5 - 5 0 6 1 5 3 6 5 5 4 5 3 - 6 - 4 2 6 2 7 - 4 6 5 4 4 5 3 - 6 - 0 4 6 1 3 - 3 8 6 7 3 7 3 - 7 - 1 6 6 3 1 6 5 6 4 3 5 2 - 6 - 7 4 6 1 7 - 4 5 6 6 3 6 0 - 5 - 6 6 6 4 4 - 6 3 7 6 6 6 1 - 5 - 7 5 7 2 7 - Totals 2 19 35 49 58 51 61 62 61 68 Totals 29 39 39 38 34 45 16 - 46 - 39 27 55 16 43 - 466 - Data are not available due to adult mortality LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. I F 00 N I 7 9. e P F 0 00 Reproductive Performance by Week and Pen Appendix X I V - T a b l e 6a Hatchlings by Week and Pen from a Northern Bobwhite Reproduction Study w i t h PFOS Experimental Group (ppm a.i.) 1 LOT A B C DL Control E F 0 H I J ~~~ PENS 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 0 0 - 0 0 0 - 1 0 0 0 - 0 0 0 1 5 1 - 0 0 0 - 4 0 1 2 - 5 0 3 1 - - 4 3 2 0 1 - 3 2 3 3 4 0 4 5 - - 4 0 2 0 2 - 5 4 3 5 5 3 5 4 - - - 5 2 4 0 3 5 6 5 6 5 6 5 6 - - 5 0 33 3 - 6765 6 6 5 5 - - 5 0 52 5 - 766 7 4 7 1 7 4 0 - 5 4 5 - 6 2 5 6 - 5 6 5 4 - - 5 3 - 6 4 5 7 4 7 5 6 7 4 7 73 - 374 3 4 6 8 - 5 7 3 6 Totals 2 22 34 42 58 60 62 57 70 66 Totals 44 12 - 30 2 0 2 8 - 47 35 42 47 - 45 42 35 46 473 - Data are not available due to adult mortality LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. c -' 2 9. B from Reproductive Performaace by Week and Pen Appendix X N - T a b l e 6b Hatchlings by Week and Pen a Northern Bobbite Reproduction Study w i t h PFOS Ekperimental Group (ppm a.i. ) PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Totals LOT1 A B C D 10 E F G H I J - 0 0 0 0 0 0 0 0 0 00 010 0 -2 0 2 0 1 3 3 0 - 3 1 4 4 1 .6 3 3 - 5 3 6 5 5 3 5 2 - 5 3 5 5 3 4 5 3 - 5 2 7 6 7 3 6 2 - 7 2 4 5 4 2 5 2 - 5 4 5 6 5 3 6 0 - 5 5 3 7 6 6 6 1 5 ~~ ~~ Totals 20 36 30 32 31 39 13 - 42 ~~ '- Data are not available due to adult mortalii LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. - 0 0 2 0 0 - 4 0 5 0 3 - 5 0 5 2 3 - 5 0 6 1 4 - 4 2 6 2 3 - 0 4 6 1 3 - 1 6 6 3 0 - 7 4 6 1 4 - 6 6 6 4 3 - 7 5 7 2 3 - 39 27 55 16 26 -- 2 15 - 27 - 43 - 51 - 47 - 56 - 51 - 59 - 63 - ~~ 414 I + 00 P I e R r 0 00 Reproductive Performance by Week and Pen - Appendix X I V T a b l e 7a ~ 14-Day Old Survivors by Week and Pen from a Northern B o h h i t e Reproduction Study with PFOS Experimental Group (ppm a.i.) PENS 201 202 203 204 205 206 207 200 209 210 211 212 213 214 215 216 Totals A B C D Control E F G H I J 0 0 - 0 0 0 - 1 0 0 0 - 0 0 0 1 2 4 1 - 0 0 0 - 4 0 1 2 - 5 0 3 1 21 4 3 - 2 0 1 - 3 2 2 3 - 4 0 4 3 31 4 0 - 2 0 2 - 5 4 2 5 - 5 3 5 3 40 5 2 - 4 0 3 - 5 5 5 6 - 5 6 5 5 56 5 0 - 3 2 3 - 6 6 6 5 6 6 5 5 58 5 0 - 5 2 4 - 7 6 6 7 - 4 6 1 6 59 4 0 - 5' 4 5 - 6 2 5 6 - 5 6 5 3 56 5 3 - 6 4 5 - 6 4 7 5 - 6 6 4 7 68 7 3 - 3 7 4 3 4 6 8 - 5 7 3 6 66 Totals 43 12 - 30 19 27 - 46 33 40 47 - 45 40 35 40 457 - Data are not available due to adult mortalii 1LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. from Reproductive PerformaPce by Week and Pen *P@naX XIV - T a b l e 7b 14-Day Old Survivors by Week and P e n a Northern Bobwhite Reproduction Study wit& PFOS Experimental Group (ppm a.i.1 PENS 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 1 LOT A B C D 10 E F 0 H I 3 0 0 0 0 0 0 0 - 0 - 0 0 1 0 0 - 0 0 0 0 1 0 0 - 2 - 2 0 5 0 3 - 0 2 0 0 3 3 0 - 3 - 5 0 5 2 3 - 1 4 1 1 6 3 1 - 5 - 5 0 6 1 2 - 3 6 4 5 3 5 2 - 4 - 4 2 6 2 3 - 3 5 4 3 4 5 3 - 5 - 0 4 6 1 3 - 2 7 5 7 3 6 1 - 6 - 1 6 6 3 0 - 2 4 3 4 1 5 2 3 - 7 4 6 1 4 - 4 5 6 5 3 6 0 - 5 - 6 6 6 4 3 - 5 3 6 5 6 5 1 4 - 7 5 6 2 2 - - Totals 20 36 29 30 30 38 10 - 37 - 37 27 53 16 23 - - Data are not available due to adult mortality ' - LOT A - Eggs Set During Week 12; LOT B Eggs Set During Week 13; etc. Totals 1 13 26 36 49 46 53 46 59 57 386 I c 00 Q\ I R e c 0 00 Pa A 201 2 02 2 03 204 205 206 207 208 209 210 211 2 12 213 214 215 216 Appendix X V - T a b l e 1 Eggshell Thichess (mm) p e r P e n by Week from a N o r t h e r n Bobwhite Reproduction S t u d y w i t h PFOS (Control) Weeks B C D E F G H I J - - - - 0.232 0.231 0.219 - 0.229 0.221 0.223 - 0.249 -- -- - 0.183 0.256 0.258 - 0.221 0.221 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 eggs available. Mean and standard deviation of pen means: ~~ Mean SD [nl 0.224 0.006 4 0.230 0.002 2 0.255 0 - 005 3 0.221 0.000 2 0.193 0.009 3 0.238 0 - 007 3 0.186 0.012 4 0.243 0.016 3 0,224 0.005 4 0.232 0.005 4 0.255 0.007 3 0.250 0.003 4 0.222 0.001 3 Mean 0.229 SD 0.021 42 Pen A 217 - 218 219 - 220 221 - 222 223 - 224 225 - 226 227 - 228 229 - 230 231 - 232 A T > p d x 2CV - T a b l e 2 Eggshell Thickness (arm) per P e n by Week from a Northern Bobwhite Reproduction Study with PFOS (10 PpEd A. I. ) Weeks B C D E F G H I J - 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 .22a - 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: - Nopggs available. Differences between the control and this treatment group were not significant (p > 0.05). Mean SD [nl 0.224 0.007 4 0.217 0.006 3 0.237 0.011 4 0.230 0.010 3 0.223 0.019 4 0.248 0.014 3 0.221 0.002 2 0.226 0.005 4 0.212 0.009 4 0.229 0.007 3 0.226 0.012 4 0.238 0.005 3 0.218 0.004 4 Mean 0.227 SD 0.010 45 Appendix XVI - T a b l e 1 Mean Hatchling Body Weight (8) p e r Pen by Week from a Northern Bobwhite Reproduction Study w i t h PFOS (Control) Weeks [No. of Hatchlings] Total Pen At nl BE nl C1 nl DI nl E1 nl F1 nl G [ nl I1 nl I1 nl J[ n] Mean SD Hatch 2 01 202 203 2 04 205 206 207 200 209 210 211 212 213 2 14 215 216 6 1 51 51- 11 1 01 1 01 I 01 - 6 [ 41 1 01 6C 11 6t 21 - 6 1 51 1 01 6 1 31 6 1 11 71 41 6-1 31 6 1 21 t 01 51 11 6 1 31 6 1 21 6 1 31 6-1 31 71 41 1 01 71 41 61 51 71 41 -I 01 6 1 51 61 41 6 1 51 - 61 61 6 [ 21 61 51 71- 61 71 51 61 61 61 SI 71 41 71 71 7 -6 1 31 6 6[ 31 6 6 1 71 6 71 41 6 - 71 31 6 6 [ 41 6 6 [ 61 6 71 81 6 - 7 1 51 7 6 1 71 6 71 31 7 6 1 61 6 Mean 6 0 44 0 12 0 30 0 20 0 27 0 47 0 35 0 42 0 47 0 45 0 42 0 35 0 46 SD 0 472 -The number of hatchlings weighed may differ from the total number of hatchlings since those hatchlings found dead were not weighed. Data are not available due to adult mortality. SO = Standard deviation of mean body weight, by parental pen, by week. Appendix XVI - T a b l e 2 Mean Hatchling B o d y Weight ( 9 ) per Pen by Week from a Northern Bobwhite Reproduction S t u d y w i t h PFOS ( 1 0 PPM A . I. ) Weeks [No. of Hatchlings] Total Pen A [ nl B[ nl C [ nl D[ nl E[ nl F[ nl G[ nl H[ nl I[ nl J[ n] M e a n SD Hatch 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Mean 6 The number of hatchlings weighed may differ from the total number of hatchlingssince those hatchlingsfound dead were not weighed. - Data are not available due to adult mortality. Differencesbetween the control and this treatment group were not significant (p > 0.05). SD = Standard deviation of mean body weight, by parental pen, by week. SD 1 412 I + CD 0 I 8 e + 0 00 Appendix XVII - T a b l e 1 Mean 14-Day O l d Survivors Body Weight (9)p e r Pen by Week from a Northern Bobhi t e Reproduction S t u d y w i t h PFOS (Control I Weeks [No. of 14-Day Old Survivors] 14-Day Pa A [ nl B[ nl C [ nl D[ nl E1 nl F[ nl G[ nl H[ nl I[ nl J[ n] Mean SD Total 2 01 202 203 2 04 205 206 207 2 08 209 210 211 2 12 213 2 14 2 15 216 [ 01 I 01 - E 01 t 01 I 01 - 26[ 11 [ 01 [ 01 -[ 01 01 r 01 [ 01 241 11 - Data are not available due to adult mortality. SD = Standard deviation of mean body weight, by parental pen, by week. 30 24 31 31 30 34 26 27 30 30 30 31 29 Mean 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 SD 2 457 Appendix XVII - T a b l e 2 Mean 14-Day Old S u r v i v o r s Body Weight (9)per Pen by Week frat a Northern Bobwhite Reproduction Study with PFOS ( 1 0 PPM A . r . 1 Pen 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 A I -nl a t nl C t nl D[ nl Weeks [No. of 14-Day Old Survivors] E[ nl F[ nl G [ nl H[ nl I[ nl - Data are not available due to adult mortality. Differences between the control and this treatment group were not significant (p > 0.05). SD = Standard deviation of mean body weight, by parental pen, by week. J[ n] M e a n 14 -Day SD Total 28 5 20 33 2 36 26 2 29 30 3 30 29 1 30 28 2 38 29 5 10 26 1 37 34 3 37 32 2 27 33 2 53 36 3 16 30 3 23 Mean 30 SD 3 386 - 193 - Project Number 454-108 Appendix XVIII Offspring Liver Weights (8) from a Northern Bobwhite Reproduction Study with PFOS Control (0 ppm a.i.) Bird 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 - wildlij2 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 150ppm 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 ai. 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 10ppm ai. 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 wildlife International, Ltd. - 195- Project Number 454-108 9. The protocol was amended to add Attachment A, the analytical phase plan to be used by 3M Environmental Laboratory for analysis of samples, and to add the 3M Environmental Laboratory Project Identification Number to be used for the study.. 10. The protocol was amended to update Attachment A, the analytical phase plan to be used by 3M Environmental Laboratory for analysis of samples. 11. The protocol was amended to change Attachment A, the 3M analytical phase plan, to redefine the peak area precision requirement and the method for calculating the calibration curve. 12. The protocol was amended to indicate the main 3M Environmental Laboratory Project Identification Number (LIMS) for the analytical phase plan results is E01-1245. Additional 3M tracking numbers associated with the study are U2723, E01-1342 and E01-1365. Additionally, the 3M Principal Analytical Investigator was changed from Harold Johnson to Lisa A. Stevenson. 13. The protocol was amended to change the Sponsor Representative from Rochelle R. Robideau to Susan A. Beach. 14. The protocol was amended to change the Sponsor Representative from Susan A. Beach to John L. Newsted. 15. The temperature in Hatcher C, containing eggs for Lot J, was inadvertently not recorded on the afternoon of July 8,2001. 16. The liver weight for an adult female (Band ##428) in the 50 ppm a i . treatment group was inadvertently not recorded. 17. Liver weights were recorded for adults and offspring selected for tissue collection, and data were compared statistically using Dunnett's t-test. Feather samples were also collected, when available, from birds from which tissue samples were collected. The protocol did not require weighing of livers or collection of these samples. 18. On May 28,2001, the temperature in brooder level B35-4, housing offspring from the 10 ppm a.i. treatment group, was inadvertently not recorded. On June 29,2001, the temperatures in brooder levels B04-3,4 and 5, housing offspring from the 10 ppm a.i. treatment group, were inadvertently not recorded. 19. Dietary samples collected on Day 0 of Week 1 were not stored frozen for the entire time interval between collection and processing for analysis. These samples were removed from the freezer and stored at ambient temperature for one day prior to processing. 20. Week 15 blood samples were collected from one male in the control group and one male in the 10 ppm a.i. treatment group who were not designated for blood collection. The additional blood samples were collected from male birds in pens where the female had died. wildlife 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