Document jBVaXjw0DzZqXddBaaM3z68n2

- - - PFOS: A PILOT REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE AR226-1750 FINAL REPORT WILDLIFE INTERNATIONAL,LTD. PROJECT NUMBER: 454-104 3M LAB REQUEST NO. U2723 FIFRA Guideline 7 1-4 AUTHORS: Sean P. Gallagher Raymond L. VanHoven Joann B. Beavers STUDY INITIATION DATE: February 28,2000 STUDY COMPLETION DATE: December 18,2003 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St.Pau1, Minnesota 55106 Wildlijie International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 125 wildlife International, Ltd. Project Number 454-104 -2GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR 3M Corporation TITLE: PFOS: A Pilot Reproduction Study with the Northern Bobwhite WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-104 STUDY COMPLETION: December 18,2003 The study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S.Environmental Protection Agency, 40 CFR Part 160, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan Notification No. 3850, Agricultural Production Bureau, 10 August 1984, with the following exceptions: The study was conducted under multiple protocols. The in-life portion was conducted under one protocol (Wildlife International, Ltd. study number 454-104), and the analytical portions were conducted under two separate protocols (Exygen Research study numbers 023-041 and 023-063). Exygen Research study number 023-041 was initiated with a separate Study Director. Results of analyses conducted by Exygen Research for study numbers 023-041 and 023-063 are reported separately. STUDY DIRECTOR: Senior Biologk, Avian Toxicology SPONSOR'S REPRESENTATIVE: wildlife International, Ltd. Project Number 454-104 -3- QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR part 160, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all audits and inspections and the dates any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY Test Substance Preparation Sample Preparation Diet Preparation Analytical Data, Draft Report Biological Data, Draft Report Statistics and Final Report DATE CONDUCTED February 28,2000 March 1,2000 March 7,2000 January 16,17,28 and 29,2003 January 16, 17,20, 28-3 1,2003 November 26, December 1-3,2003 DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT February 28,2000 March 1,2000 March 7,2000 March 1,2000 March 1,2000 March 7,2000 January 29,2003 January 31,2003 January 31,2003 February 19,2003 December 3,2003 December 18,2003 All inspectionswere study based unless otherwisenoted. Susan L. Coleman, B.A. Senior Quality Assurance Representative DATE Wildlife International, Ltd. -4REPORT APPROVAL Project Number 454-104 SPONSOR 3M Corporation TITLE: PFOS: A Pilot Reproduction Study with the Northern Bobwhite WILDLIFE INTERNATIONAL, LTD. PROJECT " M B E R : 454- 104 3M LAB REQUEST NO.: U2723 LP,* STUDYDIRECTOR Sean P. Gallagher Senior Biologist, Avian Toxicology DATE MANAGEMENT: A Joann B. Beayers Director, Avian Toxicology #4&ikdd&& Willard B. Nixon, Ph.%. Director of Chemistry ,%&kl DATE Wildlife International. Ltd. Project Number 454-104 -5- TABLE OF CONTENTS Title Page.......................................................................................................................................... 1 Good Laboratory Practice Compliance Statement ........................................................................... 2 Quality Assurance Statement ........................................................................................................... 3 Report Approval ............................................................................................................................... 4 Table of Contents ............................................................................................................................. 5 Summary .......................................................................................................................................... 8 Introduction ...................................................................................................................................... 9 Objectives......................................................................................................................................... 9 Experimental Design ........................................................................................................................ 9 Materials and Methods ................................................................................................................... 11 Test Substance and Internal Standard...................................................................................... 11 Test Organisms ........................................................................................................................ 11 Identification............................................................................................................................ 12 Avian Feed and Water ............................................................................................................. 12 Diet Preparation ....................................................................................................................... 13 Diet Sampling .......................................................................................................................... 13 Analytical Method ................................................................................................................... 13 Housing and Environmental Conditions.................................................................................. 15 Observations ............................................................................................................................ 15 Adult Body Weight and Feed Consumption............................................................................ 16 Adult Blood Collection............................................................................................................ 16 Adult Necropsy and Tissue Collection .................................................................................... 16 Egg Collection and Storage ..................................................................................................... 17 Candling and Incubation.......................................................................................................... 17 Hatching and Brooding............................................................................................................ 17 Offspring Blood and Tissue Collection ................................................................................... 18 Statistical Analyses.................................................................................................................. 19 Results and Discussion................................................................................................................... 20 Analytical Results.................................................................................................................... 20 Mortalities and Clinical Observations ..................................................................................... 21 Adult Body Weight .................................................................................................................. 21 Feed Consumption ................................................................................................................... 22 Gross Necropsy ........................................................................................................................ 22 Histopathology......................................................................................................................... 23 Tissues Analysis ...................................................................................................................... 23 Reproductive Results ............................................................................................................... 24 Offspring Body Weights .......................................................................................................... 24 Liver Weights .......................................................................................................................... 24 Conclusion...................................................................................................................................... 25 References ...................................................................................................................................... 26 wildlije International, Ltd. Project Number 454-104 -6- TABLE OF CONTENTS PAGE 2 TABLES Table Table 2. Table 3. Table 4. Table 5. Table 6. Table 7. Table 8. Mean Measured Concentrations (ppm a.i.) of PFOS in ,vian D et from a Northern Bobwhite Pilot Reproduction Study............................................... 27 Mean Adult Body Weight (g) fiom a Northern Bobwhite Pilot Reproduction Study with PFOS ................................................................................ 28 Mean Feed Consumption (g/bird/day) fiom a Northern Bobwhite Pilot Reproduction Study with PFOS ...................................................... .29 Summary of Gross Pathological Observations from a Northern Bobwhite Pilot Reproduction Study with PFOS, Adult Birds Euthanized at 6 Weeks and Test Termination.............................................................................. 30 Mean Egg Production (Eggs Laid/Hen and Eggs/Hen/Day) from a Northern Bobwhite Pilot Reproduction Study with PFOS........................................ 31 Summary of Reproductive Performance (Eggs Set from Week 5) from a Northern Bobwhite Pilot Reproduction Study with PFOS ............................ 32 Mean Body Weight (g) of Hatchlings and Surviving Offspring from a Northern Bobwhite Pilot Reproduction Study with PFOS ............................ 33 Mean Liver Weights (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS....................................................................................................... 34 Wildlije International. Ltd. Project Number 454-104 -7- TABLE OF CONTENTS PAGE 3 APPENDICES Appendix I. Appendix I1. Appendix I11. Appendix IV. Appendix V. Appendix VI. Appendix VI1. Appendix VI11. Certificate of Analysis ....................................................................................... 35 Diet and Supplement Formulations ................................................................... 38 Diet Preparation................................................................................................. 40 The Analysis of PFOS in Avian Diet ................................................................ 41 Diagram of Test Layout..................................................................................... 55 Adult Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS......................................................................... 56 Feed Consumption (g/bird/day) from a Northern Bobwhite Pilot Reproduction Study with PFOS................................................ 60 Individual Gross Pathological Observations from a Northern Bobwhite Pilot Reproduction Study with PFOS64 ............................ 64 Appendix IX. Histopathology Rep0rt....................................................................................... 68 Appendix X. Egg Production (eggs laidherdweek) from a Northern Bobwhite Pilot Reproduction Study with PFOS .............................. 109 Appendix XI. Reproductive Performance by Pen from a Northern Bobwhite Pilot Reproduction Study with PFOS .............................. 113 Appendix XI1. Mean Offspring Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS .............................................................. 119 Appendix XI11. Adult Liver Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS....................................................................... 120 Appendix X N . Offspring Liver Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS .............................................................. 122 Appendix XV. Changes to Study Protocol............................................................................... 123 Appendix XVI. Personnel Involved in the Study...................................................................... 125 wildlife International, Ltd. -8SUMMARY Project Number 454-104 STUDY: PFOS: A Pilot Reproduction Study with the Northern Bobwhite SPONSOR: 3M Corporation WILDLIFE INTERNATIONAL,LTD. PROJECT NUMBER: 454-104 TEST DATES: Study Initiation-February 28,2000 Experimental Start - February 29,2000 Adult Termination-April 11 & July 13,2000 Biological Termination-July 21,2000 TEST ANIMALS: Northern bobwhite (Colinusvirginiunus) AGE TEST ANIMALS: Approximately 30 weeks of age at the initiation of the test SOURCE TEST ANIMALS: Trace Pheasantry, Inc. 288 Levengood Road Douglassville, PA 19508 U.S.A. NOMINAL TEST CONCENTRATIONS: 0, 1.8,6.2, and 17.6 ppm a.i. RESULTS: Northern bobwhite were exposed to PFOS at dietary concentrations of 1.8, 6.2, and 17.6 ppm a.i. for 6 weeks. The control group and 17.6 ppm ai. treatment groups were maintained on test diets for an additional 13 weeks. No treatment-related mortalities or overt signs of toxicity were observed at any of the test concentrations. There were no apparent treatment-related effects on body weight, feed consumption, or liver weight at the 1.8 and 6.2 ppm a.i. test concentrations. Additionally, there were no apparent treatment-related effects on female body weight or liver weight at the 17.6 ppm a.i. test concentration. There were no apparent treatment-related effects on any reproductive parameters measured during the study for any of the concentrations tested. When compared to the control group, there was a significant reduction in mean male body weight in the 17.6 ppm a.i. treatment group. There was a significant treatmentrelated reduction in feed consumption for the 17.6 ppm a.i. treatment group when compared to the control group. There was also a significant reduction in mean liver weight for adult males at the 17.6 ppm a.i. treatment level that may have been related to the treatment. Histopathological examination of selected tissues also revealed regression of testicular tissue for two adult males in the 17.6 ppm a.i. test group that may have been related to treatment. Based upon the results of this study, the no-observed-effect concentration for northern bobwhite exposed to PFOS in the diet for 6 weeks was 6.2 ppm a.i. wildlife Intemational, Ltd. Project Number 454-104 -9- INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International, Ltd. avian toxicology facility in Easton, Maryland 2 1601. The biological portion of the test was conducted from February 29, 2000 until July 21,2000. Raw data generated at Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454-104 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 exposure to the potassium salt of Perfluorooctane Sulfonic Acid (hereafterreferred to as PFOS) over a period of approximately 6 weeks or 19 weeks. Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects of adult exposure to PFOS on the number of eggs laid, fertility, embryo viability, hatchability and offspring survival were evaluated. Histopathological examination of selected tissues and analyses of blood and tissue samples were also used to evaluate the effects upon adults exposed to PFOS and to their offspring. EXPERIMENTAL DESIGN Northern bobwhite (20 males and 20 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 LC50 study (Wildlife International, Ltd. Project Number 454-103) and additional toxicity information provided by the Sponsor. The original test concentrations selected were 0, 2, 7 and 20 ppm a.i. Following experimental start, the test material was reanalyzed and the final purity was lower than originally reported. Therefore, the actual nominal test concentrations were 0, 1.8, 6.2 and 17.6 ppm a.i. Group 1 2 3 4 - PFOS Treatment Groups Nominal Concentration Pens per (Control) 0 5 1.8 5 6.2 5 17.6 5 Birds ser Pen 1 1 1 1 1 1 1 1 W i l d 1f e International, Ltd. Project Number 454-104 - 10- Each treatment and control group contained five pairs of birds with one male and one female per pen. Three treatment groups were fed diets containing either 1.8, 6.2, or 17.6 ppm a.i. of PFOS. The control group was fed diet comparable to the treatment groups, but without the addition of the test substance. Adult northern bobwhite were exposed to PFOS at nominal dietary concentrations of 1.8, 6.2 and 17.6 ppm a.i. for a period of 6 weeks. A control group, fed non-treated diet, was maintained concurrently with the treatment groups. Each treatment and the control group consisted of five pairs of birds, housed with one male and one female per pen. At the end of Week 6, adult birds in the 1.8 and 6.2 ppm a.i. test concentrations were euthanized and subjected to gross necropsy. Test birds in the control group and 17.6 ppm a.i. treatment group were maintained on the appropriate diets until the beginning of Week 20 of the test, at which time they were also euthanized and subjected to gross necropsy. Effects on adult health, body weight, and feed consumption were evaluated as well as effects upon egg production, embryo viability, hatchability and offspring survival for all test concentrations. The adult birds were observed daily for mortality, abnormal behavior and signs of toxicity. Adult body weights were measured at test initiation, on Weeks 2, 4, 6, 8, 10 and 11, and at adult termination. Feed consumption for each pen was measured over a seven day period each week throughout the test. Necropsies were performed on all adult birds and on 10 offspring fi-om each test concentration. Liver weights were recorded for all necropsied birds. Liver, bile and blood (when available) and feather samples were collected at the time of necropsy for possible analysis. Additional samples of liver, brain, kidney, gonad, proventriculus, gall bladder, adipose tissue, and bursa of fabricius (when available) were fixed in 10% buffered formalin for histopathological examination. During the test, the number of eggs laid in each pen was recorded to evaluate egg production. Eggs laid in Weeks 1, 3 and 6 of the test were collected and separated into shell, shell membrane, yolk and albumen and stored frozen for possible analysis. Eggs laid during Weeks 5 and 6 (Days 31 to 37) of the test were collected and set for incubation. Embryo viability, hatchability, hatchling health and survivability were examined. wild1ife Xnternational, Ltd. Project Number 454-104 - 11 - MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "PFOS: A Pilot Reproduction Study with the Northern Bobwhite". The protocol was based on procedures outlined in the Environmental Protection Agency's Registration Guidelines: Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, Subsection 71-4 and the ASTM "Standard Practice for Conducting Reproductive Studies with Avian Species" (1,2). Test Substance and Internal Standard The test substance, PFOS, was received 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 an original reported purity of 98.9% and had expired at the time of experimental start. An assay of the test material after experimental start indicated a purity of 90.49%. The final assay of the test material indicated a purity of 86.9% and expiration date of August 31,2006 (Appendix I). The test substance was held under ambient condition in locked storage at the Wildlife International, Ltd. facilities in Easton, Maryland. Concentrations of the test substance in the diet were adjusted to 100% active ingredient based upon the original reported purity of 98.9%. Dietary concentrations are expressed as parts per million active ingredient (ppm a.i.) in the diet based upon the final reported purity of 86.9%. The internal standard, 4H PFOS, was received from 3M Corporation on July 2, 1998 and was assigned Wildlife International, Ltd. identification number 4526 upon receipt. The internal standard was a granular material identified as: lH, lH, 2H, 2H Perfluoroctane Sulfonic Acid; CAS No. 27619-97-2. The internal standard was held under ambient conditions in locked storage at the Wildlife International, Ltd. facilities in Easton, Maryland. Test Organisms Forty-eight (24 males and 24 females) pen-reared northern bobwhite were purchased from Trace Pheasantry, Inc., 288 Levengood Road, Douglassville, PA 19508, U.S.A. At the start of acclimation, the bobwhite appeared healthy and were phenotypically indistinguishable from wild type. The birds were from the same hatch, approaching their first breeding season and had not been used in any previous testing. At the start of acclimation, a random number generating function in a spreadsheet program was used to randomize pen assignment for each bird. Immediately prior to test initiation, all potential study wiId1ije InternationaI, Ltd. Project Number 454-104 - 12- 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 approximately 30 weeks of age at test initiation (first day of exposure to test diet) and ranged in weight from 194 to 254 grams at test initiation. Sex of the birds was determined by a visual examination of the plumage. Identification Adult birds were identified by individual leg bands, each pen was identified with a unique number, and groups of pens were identified by project number and concentration. During the test, the number of eggs laid in each pen was recorded to evaluate egg production. All eggs collected for sampling or incubation were marked with the pen number using a permanent ink marking pen for identification. Hatchlings were identified by leg bands so that they could be traced to their parental pen of origin. Avian Feed and Water All adult birds and their offspring were given feed and water ad libitum during acclimation and testing. The basal diet fed to both adults and offspring was formulated to Wildlife International, Ltd. specifications by Agway Inc. (Appendix 11,Table 1). The basal ration contained at least 27% protein and 2.5% fat, and no more than 5% fiber. The basal diet contained approximately 1.1% calcium, derived from feedstuffs and the 0.9% limestone used in the formulation of the basal diet by Agway. While this level of calcium is sufficient for growth and maintenance rations, additional calcium is required in the ration of breeding birds for egg shell formation. Therefore, an additional 5% (w/w) of limestone (approximately 38.5% Ca) was added to the basal diet for the adults. This raised the calcium level in the diet for the breeding birds to approximately 3%, slightly above the minimum recommended for quail (2.3%) and mallard (2.75%) (3). Offspring received basal diet without test substance and without the addition of 5% supplemental limestone. Water was supplied by the town of Easton public water supply. All offspring received a watersoluble vitamin and electrolyte mix in their water (Appendix 11, 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. wild1ife Intemational, Ltd. Project Number 454- 104 -13- Diet Preparation Test diets were prepared by mixing PFOS into a premix that was used for weekly preparation of the final diet. Control diet and each treated diet were prepared weekly beginning on February 28, 2000 and presented to the birds on Tuesday of each week. Dietary concentrations were adjusted for purity of the test substance and are presented as parts per million active ingredient (ppm ai.). Details of the weekly preparation of test and control diets are shown in Appendix III. Diet Sampling Homogeneity of the test substance in the diet was evaluated by collecting six samples from each of the treated diets and two samples from the control diet on Day 0 of Week 1. Samples were collected from the top, middle and bottom of the left and right sections of the mixing vessel. Control and treatment group diet samples were also collected from the feed troughs on Day 7 of Week 1 to assess stability of the test substance under actual test conditions. Additionally, a sample was collected from the control and treatment group diets during Week 6 of the test to measure/verify test concentrations. The diet samples were stored frozen or transferred immediately to the Wildlife International, Ltd. analytical chemistry facility for analysis. Analytical Method The method used for the analysis of PFOS in avian diet was based upon methodology developed at Wildlife International, Ltd. and entitled "Analytical Method Verification for the Determination of PFOS in Avian Diet" (Wildlife International, Ltd. Project No. 454C-110). Samples were extracted with methanol and diluted in a 50% methanol: 50% NANOpure@water solution containing 0.0100 mg lH, lH, 2H, 2H Perfluooroctane Sulfonic Acid (4H PFOS; internal standard)/L and 0.05% formic acid (v/v) so that they fell within the calibration range of the PFOS methodology. A method flow chart is provided in Appendix IV, Figure 1. Concentrations of PFOS in the standards and extracts of the samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer SCIEX API lOOLC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil C18 analytical column (50 mrn x 2 mm I.D., 3-pm particle size). The instrument parameters are summarized in Appendix IV, Table 1. WildlifeInternational, Ltd. - 14- Project Number 454-104 Calibration standards of PFOS prepared in a 50% methanol :50% NANOpure@water solution containing 0.0100 mg 4H PFOS (internal standard)&, and 0.05% formic acid (v/v), ranging in concentration from 0.000439 to 0.00439 mg a.i./L (Week 1, Day 0) or 0.000351 to 0.00439 mg a.i./L (Week 1, Day 7, Week 6, Day 0 and the sample re-extraction set), were analyzed with the samples. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOS on the basis of individual isomeric components. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS : internal standard) of the calibration standards. An example of a calibration curve is presented in Appendix IV,Figure 2. The concentration of test substance in the samples was determined by substituting the peak area response ratios of the samples into the applicable linear regression equation. Typical ion chromatograms of low and high-level calibration standards are shown in Appendix IV, Figures 3 and 4, respectively. Examples of calculations are presented in Appendix IV, Table 2. The method limit of quantitation (LOQ) for the Week 1, Day 0 analysis was set at 0.879 ppm a i , calculated as the product of the lowest calibration standard analyzed (0.000439 mg a.i./L) and the overall dilution factor of the matrix blank sample (2000 LKg). The method LOQ for the Week 1, Day 7 and Week 6, Day 0 analyses and the sample re-extraction set was set at 1.41 ppm ai., calculated as the product of the lowest standard analyzed (0.000351 mg a.i./L) and the overall dilution factor of the matrix blank samples (4000 LKg). Examples of calculations are presented in Appendix IV, Table 2. Along with the sample analyses, four matrix blanks were analyzed to determine possible interferences. No interferences were observed at or above the LOQ during the sample analyses (Appendix IV, Table 3). A typical ion chromatogram of a matrix blank is presented in Appendix IV, Figure 5. Avian diet samples were fortified at 0.879, 8.79, and 22.0 ppm a.i. (Week 1, Day 0) or 1.76, 8.79 and 22.0 ppm a.i. (Week 1, Day 7; Week 6, Day 0 and the sample re-extraction set) and analyzed concurrently with the samples to determine the mean procedural recovery. The method yielded mean procedural recoveries of 105%, 104%, 107% and 102%. These values correspond to each sample set analyzed or reanalyzed during the definitive study (Appendix IV, Table 3). Sample measured WiId1ife Intemational, Ltd. Project Number 454- 104 -15- concentrations were not corrected for the respective mean procedural recovery of that sample set. A typical ion chromatogram of a matrix fortificationis presented in Appendix IV,Figure 6. Housing and Environmental Conditions Housing and husbandry practices were conducted so as to adhere to the guidelines established by the National Research Council (4). The adult birds were housed indoors in batteries of pens manufactured by Georgia Quail Farm Manufacturing (GQFM Model No. 0330), measuring approximately 25 X 5 1 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, suficient 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 22.3 f 1.1"C (SD) with an average relative humidity of 50 f 14% (SD). The air handling system in the study room was designed to vent up to 15 room air volumes every hour and replace them with fresh air. The photoperiod in the adult northern bobwhite room was maintained by a time clock. The photoperiod during acclimation and the test was 17 hours of light per day to induce egg laying. Throughout the test, the birds received a mean of approximately 188 lux (- 17.5 ft. candles) of illuminationprovided by fluorescent lights that closely approximated noon-day sunlight. Observations The test birds were acclimated to the facilities and study pens for approximately 6 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 Wild1$e Intemational, Ltd. Project Number 454-104 -16- from hatching until approximately 12 weeks 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, 10, 11 and at adult termination. Feed consumption for each pen was measured weekly throughout the test. Feed consumption was determined by weighing the freshly filled feeder on Day 0, recording the amount of any additional diet added during the week, and weighing the feeder and remaining feed at the end of the feeding period (Day 7). 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. All remaking birds were euthanized two days after the beginning of Week 20. Therefore, no feed consumption estimate was calculated for Week 20. Adult Blood Collection At the time of adult termination (end of Week 6 for 1.8 and 6.2 ppm a.i. groups; beginning of Week 20 for control group and 17.6 ppm a.i. group), blood samples were collected from all surviving birds, when possible, prior to euthanasia. All blood samples were separated into serum and hemacytes/platelets, and serum was stored frozen and shipped to Centre Analytical Laboratories, Inc. for possible analysis. Adult Necropsy and Tissue Collection At the conclusion of the exposure period, all surviving adult birds were euthanized by cervical dislocation, necropsied, and stored frozen. At the time of necropsy, tissues were collected for histopathological examination (0 and 17.6 ppm a.i. groups only) and possible analyses. When available, samples of gall bladder, liver, proventriculus, kidneys, brain, gonads, bursa of Fabricius and adipose tissue were fixed in 10% buffered formalin. Histology samples were shipped to EPL in Herndon, VA for histopathology. When available, samples of bile and liver were stored frozen and shipped to Centre Analytical Laboratories, Inc. for possible analysis. Any remaining tissue not fixed for histopathology and feather samples were stored frozen for potential analysis. wildlife International, Ltd. Project Number 454-104 -17- Egg Collection and Storage Eggs laid during a seven day period beginning on the second day of Week 5 of the test were collected daily from test pens and stored in a cold room until incubation. The cold room was maintained at a mean temperature of 13.1'C f0.1'C (SD) with a mean relative humidity of approximately 72% f4% (SD). Groups of eggs were identified by an alphabetic lot code. All eggs laid during the week were considered as one lot. Candling and Incubation At the end of the weekly interval, all eggs were removed from the cold room, counted and candled with a Speed King (Model No. 32) egg-candling lamp to detect egg shell cracks or abnormal eggs. Cracked or abnormal eggs were recorded and discarded. All eggs 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 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 were placed in a Petersime Incubator (Model No. SP20). In the incubator the temperature was maintained at an average 37.5 f0.0'C (SD) with an average wet bulb temperature of 30.6 f 0.1"C (SD) (relative humidity of approximately 60%). The incubator was equipped with a pulsator fan and blades that produced a mild breathing air movement designed to eliminate intracabinet temperature and humidity variation during incubation. In order to prevent adhesion of the embryo to the shell membrane, the incubator was also equipped with an automatic egg rotation device, designed to rotate the eggs from 50" off of vertical in one direction to 50' off of vertical in the opposite direction (total arc of rotation was 100') every two hours through Day 21 of incubation. Eggs were candled on \ Day 11 of incubation to determine embryo viability and on Day 21 to determine embryo survival. Hatching and Brooding On Day 21 of incubation, the eggs were placed in a Petersime Hatcher (Model No. S-6H) and allowed to hatch. Pedigree baskets constmcted of galvanized steel wire mesh were used to keep hatchlings separated by parental pen of origin. Eggs were not rotated in the hatcher. The average temperature in the hatching compartment was 37.2 f O.OC (SD), and the average wet bulb temperature was raised to 33.3 0.0"C (SD) (relative humidity of approximately 77%). WildlveInternational, Ltd. Project Number 454-104 -18- 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 approximately 5 weeks of age. All hatchlings were moved to large flight pens, where they were housed approximately 7 weeks. The hatchlings were fed untreated diet without the addition of 5% supplemental limestone. At approximately 12 weeks of age, the average body weight by parental pen of all surviving offspring was determined, and the birds were euthanized with carbon dioxide and disposed of by incineration. Those offspring selected for blood and tissue sampling were stored frozen following necropsy and later 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 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 approximately 30 days of age. Brooding was discontinued once hatchlings were determined to be of sufficient size to thermoregulate. The average ambient room temperature in the room housing brooders was 28.6"C f 1.0"C (SD) with an average relative humidity of 39% f 8% (SD). All hatchlings were removed from brooding pens and transferred to flight pens at approximately 5 weeks of age. Each flight pen measured approximately 1.2 m X 1.2 m, with a ceiling height of approximately 1 m. External walls, floor and ceiling of each pen were constructed of wire mesh. 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 offspring, blood samples were collected from 10 offspring in each treatment group. All blood samples were separated into serum and hemacytes/platelets, stored frozen, and shipped to the Centre Analytical Laboratories, Inc. for possible analyses. Additionally, tissues from the 10 offspring in each group were collected for histopathological examination and analyses. When available, samples of gall bladder, liver, proventriculus, kidneys, brain, gonads, bursa of Fabricius and adipose tissue were fixed in 10% buffered formalin and shipped to EPL in Herndon, VA for histopathology. When available, samples of bile and liver tissue were stored frozen and shipped to Centre Analytical Laboratories, Inc. for possible analysis. wildlijie International, Ltd. Project Number 454- 104 - 19- Statistical Analyses Upon completion of the test, an Analysis of Variance (ANOVA) was performed to determine statistically significant differences between groups. Dunnett's multiple comparison procedure (5, 6) was used to compare the three treatment means with the control group mean and assess the statistical significance of the observed differences. Sample units were the individual pens within each experimental group, except body and liver weights where the sample unit was the individual bird. Percentage data were examined using Dunnett's method following arcsine square root transformation. Two sets of statistical analyses were conducted with the body weight and feed consumption data. One set of analyses only looked at body weight and feed consumption data from the first 6 weeks of the study and evaluated all three-treatment groups. The second set of analysis only evaluated the control and 17.6 ppm a.i. treatment groups and examined data for the fill duration of the study, 20 weeks. Dunnett's multiple comparison procedure was not considered appropriate to compare the control group to a single treatment group. The student's T-test was used to make statistical comparisons in those instances where only the control group and the 17.6 ppm a.i. treatment group were compared. 1. Adult Body Weight - Individual body weight was measured at test initiation, Weeks 2, 4, 6, 8, 10, 11, and at adult termination. Statistical comparisons were made between the control group and each treatment group at each weighing interval by sex for the first 6 weeks. In addition, statistical comparisons were made between the control group and 17.6 ppm a.i. treatment group for weeks 8, 10, 11, and 20. 2. Adult Feed ConsumDtion - Feed consumption expressed as grams of feed per bird per day was examined by pen weekly during the test. Statistical comparisons were made between the control and each treatment group for weeks 1 through 6. In addition, statistical comparisons were made between the control and 17.6 ppm a.i. treatment group for weeks 7 through 19. 3. E-ms Laid - The number of eggs laid per female per treatment group. For the evaluation of reproductive performance, data taken from week 5 eggs set. 4. Viable Embrvos - The number of live embryos determined at Day 11 by candling. 5. Eggs Cracked of Ems Laid - The number of eggs determined by candling to be cracked divided by the number of eggs laid, per pen. 6. Viable Embrvos of Eggs Set - The number of embryos at the Day 11 candling was divided by the number of eggs set, per pen. wildlife International, Ltd. Project Number 454-104 -20- 7. Live 3- Week Embrvos of Viable Embryos - The number of live embryos at the Day 21 candling was divided by the number of viable embryos, per pen. 8. Hatchling- s of 3-Week Embrvos - The number of hatchlings removed from the hatcher was divided by the number of live 3-week embryos, per pen. 9. 14-Day Old Survivors of Hatchlings - The number of 14-day old survivors was divided by the number of hatchlings per week, by pen. 10. Hatchlings of Eggs Set - The number of hatchlings was divided by the number of eggs set per week, by pen. 11. 14-Day Old Survivors of Eggs Set - The number of 14-day old survivors was divided by the number of eggs set per week, by pen. 12. Offs~rinp'sBody Weight - The group body weights of surviving hatchlings and 14day old survivors were measured by parental pen group. 13. Liver Weieht - Individual liver weights were measured at adult termination and at the termination of offspring of week 5. Statistical comparisons of adult liver weights were made by sex between the control and treatment groups. Juvenile liver weights were compared by test group, without regard to sex. RESULTS AND DISCUSSION Adult northern bobwhite were exposed to PFOS at nominal dietary concentrations of 1.8, 6.2 and 17.6 ppm a.i. for a period of 6 weeks. A control group, fed non-treated diet, was maintained concurrently with the treatment groups. Each treatment and the control group consisted of five pairs of birds, housed with one male and one female per pen. At the end of Week 6, adult birds in the 1.8 and 6.2 ppm a.i. test concentrations were euthanized and subjected to gross necropsy. Test birds in the control group and 17.6 ppm a.i. treatment group were maintained on the appropriate diets until the beginning of Week 20 of the test, at which time they were also euthanized and subjected to gross necropsy. Analytical Results None of the control samples showed any indication of the presence of the test substance or of the presence of co-eluting substance at the characteristic retention time of the test substance (Table 1). Diet samples were collected from the 1.8, 6.2 and 17.6 ppm a.i. test concentrations on Week 1, Day 0, and were analyzed to evaluate the homogeneity of the test substance in the diet and to verify test substance concentrations. Means and standard deviations for the three test concentrations were 1.8 f 0.13 ppm a.i., wild1ife Intemational, Ltd. Project Number 454-104 -21 - 6.0 k 0.65 ppm a.i. and 17.6 f 1.46 ppm a i , respectively. The coefficients of variation were 7.2%, 11% and 8.3%, respectively. These values represented 100, 97 and 100% of nominal concentrations (Appendix IV, Table 4). Samples collected during Week 6, Day 0 of the test to verify test substance concentrations for the 1.8, 6.2 and 17.6 ppm a i . diets had means and standard deviations of 2.0 & 0.092 ppm a.i., 6.0 2 0.67 ppm a.i. and 16.8 f 0.608 ppm ai., respectively. The coefficients of variation were 4.6%, 11% and 3.6%, respectively. These values represented 111,97 and 95% of nominal concentrations (Appendix IV, Table 5). Analysis of diet samples collected from feeders after being held at ambient temperature for 7 days averaged loo%, 103% and 98% of the Day 0 values for the 1.8,6.2 and 17.6 ppm a.i. test concentrations, respectively (Appendix IV, Table 6). A typical ion chromatogram of a test sample is shown in Appendix IV, Figure 7. Mortalities and Clinical Observations No adult mortalities occurred in the control group or in any of the treatment groups during the course of the test. Several birds were noted with head or foot lesions and feather loss as a result of pen wear and/or penmate aggression during the course of the test. An incidental clinical sign, lameness, was associated with the injuries. All other birds were normal in appearance and behavior for the duration of the test. Adult Body Weight When compared to the control group, there were no apparent treatment-related effects upon body weight at the 1.8 and 6.2 ppm a.i. test concentrations and any differences between the control group and those two treatment groups were not statistically significant at any of the body weight intervals. However, there were treatment-related reductions in mean body weight for males at the 17.6 ppm a.i. treatment group. With the exception of the Week 2 body weight interval, mean male body weight at the 17.6 ppm a.i. test concentration was statistically (p<0.05) different from the control group at all other intervals (Weeks 4, 6, 8, 10, 11, and 20). For female body weights, none of the differences observed between the treatment groups and the control group were statistically significant for any of the intervals monitored during the study. Mean body weight measurements are presented in Table 2 and individual body weight measurements are presented in Appendix VI. WildEve International, Ltd. Project Number 454-104 -22 - Feed Consumption Due to excessive wastage by some birds, feed consumption was variable between pens. However, when compared to the control group, there were no treatment-related effects upon feed consumption at the 1.8 or 6.2 ppm a.i. test concentrations. While statistically significant (p<0.05) differences between the control and the 1.8 ppm a.i. test group were observed during Week 5 and 6 of the study, these differenceswere not considered treatment-related due to the lack of a concentration-response relationship at the greater doses. At the 6.2 ppm a i . test concentration, there was a statistically significant reduction in feed consumption during Week 4 and Week 6 when compared to the control group. However, these differences were not dose-responsive and the significance of the apparent reduction at Week 6 may have been a consequence of the fact that there was a relatively large increase in feed consumption in the control group at this time interval as compared to that observed at Week 5 or 7. Thus, at Week 6, all treatment group feed consumption rates were significantly reduced from the control level but were actually higher than observed at Week 5. As a result, the reductions in feed consumption at 6.2 ppm a.i. were not considered to be treatment-related. There was a treatment-related effect upon feed consumption at 17.6 ppm a.i. test concentration when compared to the control group. The reduction in feed consumption at 17.6 ppm a i . test concentration was consistent throughout the study and statistically significant (p<0.05) from the control group at Weeks 2, 3, 4, 6, 8 and 15. Mean feed consumption measurements are shown in Table 3, and feed consumption measurements by pen are presented in Appendix VII. Gross Necropsy At the end of Week 6 (Day 42), all adult birds in the 1.8 and 6.2 ppm a.i. treatment groups were euthanized and subjected to gross necropsy. Adult birds in the control and 17.6 ppm a.i. treatment groups were maintained on the appropriate diets until the beginning of Week 20 and were then euthanized and subjected to gross necropsy. When compared to the control group, there was an increased incidence in the number of males with small testes in the 17.6 ppm a.i. group that was considered possibly treatment-related. All other findings observed were considered to be incidental to treatment. Necropsy findings are reported in Table 4 and Appendix VIII. Study offspring were approximately 12 weeks of age at the time of euthanasia. A subsample of 10juvenile birds for each test group were subjected to gross necropsy. Necropsy revealed one to three birds in each treatment level with some feather loss. Additionally, a single bird in the 6.2 ppm a.i. wild1ife Intemational, Ltd. Project Number 454-104 -23 - treatment group was noted with abrasions on the rump. All findings observed were considered to be incidental to treatment. Histopathology Light microscopic examination was performed on selected tissues by a board certified pathologist at Experimental Pathology Laboratories, Inc. Herndon, VA. Sections of liver, brain, kidney, gonad, proventriculus, gall bladder, adipose tissues, and bursa of Fabricius (when available) were collected from adult test birds and from 10 offspring (approximately 12 weeks old at euthanasia) from each test group for examination. No lesions considered possibly related to treatment were noted in liver, kidney, proventriculus, gall bladder, ovary, brain and bursa of Fabricius of adult male and females, or their offspring at any of the concentrations tested. Additionally, there were no lesions considered to be treatment-related noted in adipose tissue of adult males or females and offspring of both sexes, or in the testes of male offspring. Testes of two adult males at the 17.6 ppm a.i. test concentration exhibited decreased seminiferous tubule diameter most consistent with post-reproductive phase regression, a normal physiological phenomenon. The occurrence of testicular regression for adult males in the 17.6 ppm a.i. treatment group may be incidental to treatment, but a treatment-related effect could not be precluded. The full pathology report is provided in Appendix IX. Tissue Analysis The analysis of the egg, blood and tissue samples collected during the study were conducted by Exygen Research (formerly known as Centre Analytical Laboratories) and are reported separately. Blood and liver analytical results are reported in "Extraction of Potassium Perfluorooctanesulfonate from Quail serum and quail liver for analysis using HPLC-Electrosprayhlass Spectrometry. Centre Study Number 023-41". The egg components analytical results are reported in "Extraction of Potassium Perfluorooctanesulfonate from Egg Membrane, Albumen, and Yolk for analysis using HPLCElectrosprayhlass Spectrometry. Centre Study Number 023-065". Wildlqe International, Ltd. Project Number 454-104 - 24 - Reproductive Results There were no apparent treatment-related effects on egg production at any of the concentrations tested. While egg production was highly variable among hens, egg production at the 1.8,6.2 or 17.6 ppm a.i. test concentrations was comparable to or exceeded the control group. Mean egg production by concentration is presented in Table 5. Individual egg production data are presented in Appendix X. When compared to the control group, there were no apparent treatment-related effects on embryo viability, hatchability, hatchling health and survivability at the 1.8, 6.2 or 17.6 ppm a.i. levels. While there was a significant (p<0.05) reduction in the number of viable embryos at the 1.8 ppm a.i. test concentration, the reduction was due to fewer eggs being set for incubation at this level. Pen P207 of the 1.8 ppm a.i. level did not lay during the week that eggs were collected for incubation. As a result, there were fewer hatchlings and 14-day old survivors in the 1.8 ppm a.i. treatment group. However, when the reproductive endpoints were expressed as percentages, no statistically significant differences were observed at any of the concentrations tested. There was slight reduction in embryo viability at the 6.2 ppm a.i. test concentration when compared to the control. However, this reduction was not dose responsive and was not considered treatment-related. Reproductive data are summarized in Table 6. Reproductive data for individual pens is presented in Appendix XI. Offspring Body Weights There were no apparent treatment-related effects upon the body weights of hatchlings in any of the treatment groups. There were also no apparent treatment-related effects upon the body weight of juvenile birds in the 1.8, 6.2 or 17.6 ppm a.i. treatment groups. Offspring body weight data is presented in Table 7 and Appendix XII. Liver Weights When compared to the control group, there were no apparent treatment-related effects on male adult liver weight at the 1.8 or 6.2 ppm a.i. test concentrations, or on female adult liver weights at any of the concentrations tested. However, there was a statistically significant (p<0.05) reduction in mean male adult liver weight at the 17.6 ppm a.i. test concentration. Adult males in the control group had a mean liver weight of 3.402 grams compared to a mean liver weight of 2.527 grams for males at the 17.6 ppm a.i. test concentration. When compared to the control group, there were no apparent treatment-related effects on juvenile liver weight at any of the concentrations tested. Mean adult and offspring liver 'wildlife International, Ltd. Project Number 454-104 - 25 - weights are presented in Table 8. Individual adult liver weights are presented in Appendix XIII. Individual offspring liver weights are presented in Appendix XIV. CONCLUSION Northern bobwhite were exposed to PFOS at dietary concentrations of 1.8, 6.2, and 17.6 ppm a i . for 6 weeks. The control group and 17.6 ppm a.i. treatment groups were maintained on test diets for an additional 13 weeks. No treatment-related mortalities or overt signs of toxicity were observed at any of the test concentrations. There were no apparent treatment-related effects on body weight, feed consumption, or liver weight at the 1.8 and 6.2 ppm a.i. test concentrations. Additionally, there were no apparent treatment-related effects on female body weight or liver weight at the 17.6 ppm a.i. test concentration. There were no apparent treatment-related effects on any reproductive parameters measured during the study for any of the concentrations tested. When compared to the control group, there was a significant reduction in mean male body weight in the 17.6 ppm a i . treatment group. There was a significant treatment-related reduction in feed consumption for the 17.6 ppm a.i. treatment group when compared to the control group. There was also a significant reduction in mean liver weight for adult males at the 17.6 ppm a.i. treatment level that may have been related to the treatment. Histopathological examination of selected tissues also revealed regression of testicular tissue for two adult males in the 17.6 ppm a.i. test group that may have been related to treatment. Based upon the results of this study, the no-observed-effect concentration for northern bobwhite exposed to PFOS in the diet for 6 weeks was 6.2 ppm a.i. w i l d 1ife International, Ltd. -26REFERENCES Project Number 454-104 1 U.S. Environmental Protection Agency. 1982. Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlije and Aquatic Organisms, subsection 71 4 , Environmental Protection Agency, Office of Pesticide Programs. Washington, D.C. 2 American Society for Testing and Materials. 1986. Standard Practice for Conducting Reproductive Studies with Avian Species. ASTM Standard E1062-86. Annual Book of ASTM Standards. Vol. 11.04. Philadelphia, PA. 15 pp. 3 Merck & Co., Inc. 1991. The Merck VeterinaryManual. Merck & Co. Rahway, NJ. 1832pp. 4 National Research Council. 1996. Guide for the Care and Use of Laboratory Animals. Washington, DC. National Acad'emy Press. 125 pp. 5 Dunnett, C.W. 1955. A Multiple Comparison's Procedure for Comparing Several Treatments with a Control. Jour. Amer. Statis. Assoc. 50: 1096-1121. 6 Dunnett, C.W. 1964. New Tables for Multiple Comparisons with a Control. Biometrics 20: 482491. Table 1 Mean Measured Concentrations(ppm a.i.) of PFOS in Avian Diet from a Northern Bobwhite Pilot Reproduction Study Nominal Concentration' (ppm a.i.) Week 1 Day 0 Day 7 ~ Week 6 Day 0 Control Measured < 0.879 < 1.41 MtXll 1.8 Measured 1.8 1.8 2.0 % Nominal 100 100 111 Mean 6.2 Measured 6.0 6.2 6.0 % Nominal 97 103 97 Mean 17.6 Measured 17.6 17.2 16.8 % Nominal 100 98 95 ' Nominal concentrationsand results of diet analysiswere corrected for the change in test substancepurity from 90.49%to 86.9%. The limit of quantitation (LOQ) was equivalent to 0.879 ppm ai. faWeek 1, Day0 and 1.41ppm a.i. for Week 1, Day 7 and Week 6, Day 0. The mean percent of the Day 0 values. Table 2 Mean Adult Body Weight' (g) fiom a Northern Bobwhite Pilot Reproduction Study with PFOS Experimental Group Sex Week 0 Change Week Change Week 0-2 2 2-4 4 Change 4-6 Week 6 Change Change 0-6 6-8 Week 8 Change 8-10 Week 10 Change Week Change Week 10-11 11 11-20 20 Change 0-20 Control M 2 214 -1 (0 ppm a.i.) SD 5 3 F 2 222 7 SD 14 3 213 -1 6 2 229 -3 11 4 1.8 M 8 206 0 ppm a.i. SD 14 3 F 8 221 -1 SD 17 6 207 4 15 3 22 1 1 17 7 6.2 M 8 210 -2 ppm a.i. SD 13 2 F 8 231 3 SD 17 7 208 -1 13 2 234 3 17 6 17.6 ppm a.i. M 8 208 -10 * SD 11 4 F 8 227 1 SD 16 4 198 -3 9 3 227 4 16 4 212 2 6 1 226 8 14 7 211 1 15 2 222 -10 11 30 207 0 12 2 237 -2 23 8 196 * 0 7 2 232 0 14 6 213 -1 7 5 234 12 12 6 212 6 14 5 211 -10 36 34 207 -3 11 4 235 4 23 7 196 * -13 * 6 5 23 1 5 14 3 212 3 6 2 237 -2 9 13 -- -- __ -- -- -- I I __ I -- __ I -- I 196' -1 * 7 2 23 1 4 15 2 1 Mean ( X ) f standard deviation (SD). The means for body weights and body weight changes are calculated and rounded separately. - Data not available. Birds euthanized at the end of Week 6. Statistically different fiom the control group at p<O.OS. -R*hI i? Table 3 Mean Feed Consumption' (g/bird/day)from a Northern Bobwhite Pilot Reproduction Study with PFOS Experimental Group WWkS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Control Mean 20 22 23 24 24 28 24 32 27 31 31 35 30 35 36 34 30 30 31 0 ppma.i. SD 3 2 2 2 3 2 3 4 3 3 4 5 3 5 2 3 2 3 21 EJ \o I 1.8 Mean 18 20 20 24 19 * 22 * ppm a i . SD 2 3 2 2 4 2 6.2 Mean 18 20 20 21 * 22 23 * ppm a.i. SD 3 3 4 2 3 3 17.6 Mean 17 18 * 20 * 21 * 22 23 * 21 25 * 26 28 29 3 1 26 27 28 * 29 21 24 24 ppm a.i. SD 1 1 2 2 2 2 3 4 6 6 5 5 4 5 6 6 5 9 6 1 Mean f standard deviation. - Data not available. Birds euthanizedat the end of Week 6. * Statisticallydifferent kom the control group at ~0.05. Number of birds Feather loss Foot lesions Right wing twisted Thin condition - Right testis small, - Right testis small, - Both testes small, 1.25 cm 1.5 cm 1.5 cm Regressed wary Not remarkable Table 4 Summary of Gross Pathological Observations fiorn a Northern Bobwhite Pilot Reproduction Study with PFOS Adult Birds Euthanized at 6 Weeks and Test Termination Males - Treatment Group (ppm a.i.) Control 1.8 6.2 17.6 5 0 0 0 0 2 0 0 - 3 Females - Treatment Group (ppm a.i.) Control 1.8 6.2 17.6 ~~ 5 3 0 0 0 0 2 Table 5 Mean Egg Production (Eggs Laid/Hen and Eggs/Hen/Day) From a Northern Bobwhite Pilot Reproduction Study with PFOS Experimental Group Weeks 1 2 3 4 5 6Total E/WD2 7 8 9 10 11 12 13 14 15 16 17 18 19 20Total E/WD2 Control Total 21 25 29 27 27 32 161 0ppma.i. Mean 4 5 6 5 5 6 32 0.77 SD 2 1 1 1 1 1 6 0 . 1 4 30 33 33 28 28 26 32 30 34 32 30 30 31 8 566 6 7 7 6 6 5 6 6 7 6 6 6 6 2 113 0.84 21133311012221120.09 17.6 ppma.i. Total 27 27 30 29 33 35 Mean 5 5 6 6 7 7 SD 2 1 1 1 1 0 181 36 0.86 50.13 32 34 35 34 34 34 33 31 28 30 28 25 21 5 585 6 7 7 7 7 7 7 6 6 6 6 5 4 1 117 0.87 10000011333241200.15 1 Total number of eggs laid and mean number of eggs laid per pen f standard deviation. 2 Eggs per Hen per Day -- Data not available. Birds euthanized at the end of Week 6. Differences between the control group and the treatment groups were not statistically significant (e.05) -32- Project Number 454- 104 Table 6 Summary of Reproductive Performance (EggsSet fiom Week 5 ) fiom a Northern Bobwhite Pilot Reproduction Study with PFOS Reproductive Parameter Control (0 ppm a.i.) Experimental Group 1.8ppm a.i. 6.2 ppm a.i. Number of Replicates Eggs Laid Eggs Cracked Eggs Set Viable Embryos Live 3-Week Embryos Hatchlings Offspring Survivors Eggs Laid/Hen Offspring/Hen 5 5 5 29 18 29 0 0 0 29 18 29 29 17 * 25 28 17 25 28 17 25 27 17 20 6 4 6 5 3 4 Eggs Laid/Hen/Day 0.83 0.64 0.83 Eggs Cracked/EggsLaid (%) 0 0 0 Viable EmbryosEggs Set (%) 100 95 86 Live 3-Week EmbryosNiable (YO) 97 100 100 Hatchlingd3-Week Embryos (%) 100 100 100 Offspring Survivors/HatcNings(%) 97 100 86 HatchlingdEggs Set (YO) 97 95 86 offspring Survivors/Eggs Set (%) 93 95 72 HatchlingdHedDay 0.80 0.49 0.71 offspring SurvivordHedDay 0.77 0.49 0.57 ~~~ * Statistically different fkom the control group at ~ 0 . 0 5 . 17.6 ppm a.i. 5 34 0 34 34 33 31 25 7 5 0.97 0 100 97 94 79 91 73 0.89 0.71 Table 7 Mean Body Weight' (g) of Hatchlings and Surviving Offspring from a Northern Bobwhite Pilot Reproduction Study with PFOS Experimental Group Hatchlings Mean SD Surviving Offspring* Mean SD Control (0 ppm a.i.) 6.1 f 0.5 175 f 10 1.8 ppm a.i. 5.6 f 0.5 175 f 1 6.2 ppm a.i. 5.7 f 0.6 179 f 5 17.6 ppm a.i. 6.0 f 0.5 173 f 8 1Mean f standard deviation. 2 Offspring were approximately 12 weeks of age at final body weight interval. Differences between the control group and the treatment groups were not statistically significant Q~0.05). I u u I 7 9. CD !2 3 3 d 8 f P VI w 0 P Experimental Group Control (0 pprn a.i.) Table 8 Mean Liver Weight' (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS Males Liver Mean SD Females Liver Mean SD offspring2 Liver Mean SD 3.402 f 0.446 7.645 f 1.295 3.746 f 0.339 1.8 ppm a.i. 3.501 f 0.514 6.323 f 1.668 3.756 f 0.453 6.2 ppm a.i. 3.684 f 0.686 5.623 f 0.505 4.985 f 1.367 17.6 ppm a.i. 2.527 f 0.359 * 6.851 k 1.880 3.429 f 0.495 1Mean f standard deviation. 2 Offspring were approximately 12 weeks of age at time of euthanasia and tissue collection. * Statistically different from the control group at p<O.OS. I w P I tp 9. 3fd 8 P v1 P c 0 P - 35 - wild1ife Intemational, Ltd. Project Number 454-104 Appendix I Certificate of Analysis Centre Andlytkd Laboratories, Inc. 3048 ResearchDrive State College, PA 16801 www.centrelab.Com Fm:(814) 231-12530r(814)231-1580 INTERIM CERTIFICATE OF ANALYSIS RCVMOR3 Centre Analytical Laboratorlcs COA Referenced: 023-018A 3M Prodnet: PFOS, Lot 217 Reference #: SD-018 TcrtNrmc I Purity' I Purity: 86.9% spedflutlom I Rault I 86.9% Appearance [dCntifiCatiOn NMR White Crystalline Powder Cmfomls Positive Metals (ICPMS) 1... c_ alc_ ium____. ~~ 1. 0.005 wtJwt.% 2. Magnesium 3. Sodium 4. Potassium2 5. Nick1 6-.. Imn I--- 2. 0.001wtlwt.% 3. 1.439 wtlwt.% 4. 6.849wtlwt.% 5. <0.001Wt./Wt.% 6. 0.005 wtlwt.% 7. Manganese Total % Impurity (NMR) Total % Impurity 7. ~ . 0 0 1 ~ w t . % 1.91 wtlwtP9 8.41 wtJwt.99 (LC/MS) Total % I d t v I I None Detected POM Residual Solvents (TGA) Purity by DSC Inorganic Ani- (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate' Organick i d s 'OC) 1. TFA 2. PFPA 1-. W--F-R-A- 4. NFPA 1 . cprbonv 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine 0.33 wtJwt.% None Detected Not Applicable' 1. co.015 wtlwt.% 2. 0.59wtfwt.% 3. 4.o.o4owtJwt.% 4. 4.009wtJwt.?h 5. <0.006wtJwt.% 6. <0.007wthvt.% 7. 8.76wtJwt.% 1. eo.1 wt.lwt.Yn 2. 4.1wt./wt% I 3. 0.10 wtJwt.Yn 4. 0.28wt./wt.% 1. 'IhcorcticalValw = 17.8% 2. Theoretical Value = 0% 3. 'Iheorptical Value = 0% 4. TheonticalValue=5.95% 5. ThcorcticalValue = 60% 1. 12.48 wt./wt.% 2. 0.244 wt,fwt.% 3. 1.74wt./wt.% 4. 8.84wtJwt.% 5. 54.1 wtJwt.% COA023-018A Page 1of 3 -36- 'wildlife International, Ltd. Project Number 454-104 Appendix I Page 2 Certificate of Analysis cntrAnalyticd Laboratories, Inc. 3048ReseerchDrive State College, PA 16801 mmv.centreleb.com Fax: (814) 231-1253 or (814) 231-1580 L V T E W CERTIFICATE OF ANALYSIS Rntisioa 3 Centre Analytical Laboratories COA Reference It: 023-018A Date of Last Analysis: 08/31/00 Expiration Date: 08/3 1/06 Storage Conditions: From 5-1OOC Re-assessment Date: 08/31/06 'Purity = 100% - (sum of metal impurities, 1.45% +LC/MS impurities, 8.41YdInorganic Fluoride. 0.59%+NMRimpurities, 1.905%torganic acid impurities. 0.38%+POAA. 0.33%) - Total impurityfrom all tests = 13.07% Purity = 1Wh 13.07%- 86.9% 2Potassiumis expectedin this salt hrm and is thmforenot consideredan impurity. 'Purity by DSC is generally not applicable to materials of lowpurity. No endothermwas observed for this sample. 4Sulfurin the sample appears to be convertedto SO, and hence detected using the inorganic anion methodconditions. The anion result agreeswell with the sulfur determhtion in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. %FA HFBA NFPA PFPA Triflu~aceticacid Heptafluorobutyric acid Nonafluoropcntanoic acid Pmtafluompropanoic acid 6Theoretical value calculationsbased on the empiricalformula, C~FI,S@K*(MW=~~~) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COAO23-018A Page 2 of 3 -37- wildlife Xntemational, Ltd. Project Number 454- 104 Appendix I Page 3 Certificate of Analysis cntrAnahdaca 3048Rehearch Drive I LaboFatorks, Inc State College, PA 16801 www.centrelab.com Fax: (814) 231-1253 or (814) 231-1560 INTERlM CERTIFICATE OF ANALYSIS Revlslon 3 Centre Anrlytlcal Laboratories COA Reference #: 023418A L C M purity Profile: Impurity c4 c5 C6 c7 Tot81 wwwt Yo 1.22 1.33 4.72 1.14 8.41 Note: The C4 and C6 values w m calculated usingthe C4 andC6 standard calibration CUTVCS, respectively. The C5 value was calculatedusing the avaage result from the C4 and C6 standard curves. Likewise, the C7 value was calculated Using the average rwult frmnthe C6 and CS standard curves. Prepared By: Scientist, Centre Analytical Laboratories /:A/. I Date Reviewed By: `Laboratory Manager,Centre Analytical Laboratories COA023-018A Page 3 of 3 -38 - wildlife International, Ltd. Project Number 454-104 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 grams 40 gams Pantothenic Acid 10 grams Vitamin B 8 mgs Folic Acid 600 mgs Biotin 64 mgs Pyridoxine 1.2 grams Thiamine 1.2 grams Vitamin E 20,000 I.U. Vitamin K (Menadione DimethylpyriniidinolBisulfite) 5.8 grams Manganese 102 grams Zinc 47 grams Copper Iodine 6.8 grams 1.5 grams Iron 51- '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. 2 FermentationBy-Products (Source of Unidentified Growth Factors). -39- wildlife International, Ltd. Project Number 454-104 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 HCl Pyridoxine Hydrochloride Ascorbic Acid Water Soluble Powder Per 4 oz. 2,5000,000 1,000,000 1,000 750 1,250 2,500 2.5 1,000 65 250 250 3,750 Per lb. 10,000,000 IU 4,000,000 ICU 4,000 IU 3,000 Mg 5,000 Mg 10,000 Mg 10.0 Mg 4,000 Mg 260 Mg 1,000 Mg 1,000 Mg 15,000 Mg INGREDIENTS: Vitamin A Supplement, D-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 electrolytesto approximately 1 gallon of water. -40- Wildlife International, Ltd. Project Number 454- 104 Appendix I11 Diet Preparation Premixes for PFOS were prepared on February 28, 2000; April 7, 2000; May 12, 2000; and June 19,2000. Nominal preparation was as follows: Control: 1.8 ppm a i : 6.2 ppm ai.: 17.6 ppm a i : No premix required. 0.3084 g PFOS + 6099.7 g ration 1.0794 g PFOS + 6098.9 g ration 3.0839 g PFOS + 6096.9 g ration Basal ration was weighed into a tared Hobart mixing bowl. Approximately 100 g of ration was transferred to a WaringB blender. The test substance was weighed into a tared weigh boat and a small mortar was taken to crush any areas of consolidation, The test substance was transferred to the Warhg@ blender and the weigh boat was rinsed three times with ration from the mixing bowl, with the rinse being added to the blender. The blender contents were mixed approximately one minute and transferred to the mixing bowl. The blender was rinsed with uncontaminated ration from the bowl, with the rinse being returned to the bowl. The bowl was placed on a Hobart mixer, and the contents were mixed approximately 15 minutes. The premix was weighed into 1000.0 g aliquots, placed in appropriately labeled freezer bags, reweighed, and stored frozen. As needed, the appropriate premix was incorporated into the final diet as follows: 0 ppm a.i.: 23.75 kg ration + 1.250 kg limestone 1.8 ppm ai.: 1000 g Premix + 22.75 kg ration + 1.250 kg limestone 6.2 ppm ai.: 1000 g Premix + 22.75 kg ration + 1.250 kg limestone 17.6 ppm ai.: 1000 g Premix + 22.75 kg ration + 1.250 kg limestone The diets were mixed for approximately 20 minutes in a Patterson-Kelly Twin Shell Blender. -41 - WildlifeInternational, Ltd. Project Number 454-104 Appendix IV The Analysis of PFOS in Avian Diet -42- wild1ijie Intemational, Ltd. Project Number 454-104 APPENDIX IV ANALYTICAL METHODS AND RESULTS Typical LCMS Operational Parameters TABLE 1 INSTRUMENT: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer SCIEX API lOOLC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil CI8column (50 mm x 2 mm I.D., 3-pm particle size) OVEN TEMPERATURE: 30C STOP TIME: 5.00 minutes FLOW RATE: 0.220 mL/minute MOBILE PHASE: 72.0% Methanol :28.0% NANOpure@Water containing 0.1% Formic Acid INJECTION VOLUME: 5.0 pL PFOS RETENTION TIME: Approximately 3.6 minutes INTERNAL STANDARD RETENTION TIME: Approximately 2.6 minutes PFOS MONITORED MASS: 498.6 amu INTERNAL STANDARD MONITORED MASS: 426.7 amu -43 - Wildlife Intemational, Ltd. Project Number 454-104 APPENDIX IV TABLE 2 CALCULATIONS The concentration of PFOS found at the instrument was determined using the following equation: PFOS (mg a.i./L) at instrument = area - (y-intercept)x internal standard conc. (mg a.i./L) slope Determination of Sample Residues (PFOS) The concentration, expressed as ppm a.i., for each sample was determined using the following equation: PFOS (mg a.i./L) at instru. x extract final volume (L) x dilution factor PFOS (ppm ai.) in sample = initial weight (Kg) Determination of Limit of Ouantitation (LOO) The method LOQ, expressed as ppm a i , was determined using the following equation: LOQ (ppm a.i.) = lowest standard concentration (mg a.i./L) x overall dilution factor of matrix blank* extract final volume (L) x dilution factor *overall dilution factor of matrix blank sample = initial weight (Kg) Fortification Recoveries The ppm a.i. measured in each sample is divided by the nominal concentration of each sample (fortified level, ppm a.i.). This ratio times 100 is the percent recovery of the method at that level of fortification. % Recovery m = pp ap.ip. mmeaa.is.ufroerdtiifniesdample x 100 wildlife International, Ltd. Project Number 454-104 APPENDIX N TABLE 3 Matrix Blanks and Fortifications Analyzed Concurrently with the Samples L + Number (454-1 04-) Sample Tme Interval Concentration of PFOS (ppm ai.) Fortified' Measured" Percent Recovery (a Mean Recovery MAB-1 Matrix Blank Week 1, Day 0 0.00 < 0.879 MAS-1 Matrix Fortification Week 1, Day 0 0.879 0.992 113 MAS-2 Matrix Fortification Week 1, Day 0 8.79 8.89 101 MAS-3 Matrix Fortification Week 1, Day 0 22.0 22.0 100 MAB-2 Matrix Blank Week 1, Day 7 0.00 < 1.41 - MAS4 Matrix Fortification Week 1, Day 7 1.76 1.97 112 MAS-5 Matrix Fortification Week 1, Day 7 8.79 9.11 104 MAS-6 Matrix Fortification Week 1, Day 7 22.0 21.3 97.0 105 e I 104 I MAB-3 Matrix Blank Week 6, Day 0 0.00 < 1.41 MAS-7 Matrix Fortification Week 6, Day 0 1.76 1.98 113 107 MAS-8 Matrix Fortification Week 6, Day 0 8.79 9.40 107 MAS-9 Matrix Fortification Week 6, Day 0 22.0 22.0 100 MAB-4 Matrix Blank Re-extraction Set4 0.00 1.41 - MAS-10 Matrix Fortification Re-extraction Set' 1.76 1.84 105 102 MAS-1 1 Matrix Fortification Re-extraction Set4 8.79 8.86 101 MAS-12 Matrix Fortification Re-extraction Set4 22.0 21.7 98.7 'Concentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate of Analysis dated October 11,2001. Nominal test concentrationsbased upon the new test substancepurity are 1.8,6.2 and 17.6ppm a.i. 'Less than values correspond to limit of quantitation (LOQ). Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since fortified and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes. 4Re-extraction of:sample 454-104,105-13 from Week 1, Day&samples 454-104-23 and -26 from Week 1, Day 7 and sample 454-104,105-50 from Week 6,Day 0. WildlgeInternational, Ltd. Project Number 454-104 APPENDIX IV TABLE 4 Homogeneity of PFOS in Avian Diet Nominal Concentration' (ppm a.i.) Sample I.D. Number (S-454- 1043 Location Sampled in Mixing Vessel Measured PFOS Concentration' (ppm a.i.) Mean (X) Standard Deviation (SD) Coefficient of Variation (CV) Mean Percent of Nominal 1.8 Top Left 1.70 Top Right 1.82 Middle Left 1.92 -x = 1.8 ppm a.i 100 Middle Right 1.73 SD = 0.13 ppm a.i. Bottom Left 1.77 CV = 7.2% Bottom Right 2.05 6.2 9 Top Left 5.33 10 Top Right 5.65 11 Middle Left 6.09 -x = 6.0 ppm a.i. 97 12 Middle Right 5.81 SD = 0.65 ppm a.i. 13 Bottom Left 5.87' CV=11% 14 Bottom Right 7.21 17.6 15 Top Left 16.5 16 Top Right 17.7 17 Middle Left 16.5 W = 17.6 ppm ai. 100 18 Middle Right 20.4 SD = 1.46 ppm a.i. 19 Bottom Left 17.0 CV = 8.3% 20 Bottom Right 17.6 'Concentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate of Analysis dated October 11, 2001. Nominal test concentrations based upon the new test substance purity are 1.8,6.2 and 17.6 ppm a.i. *Meanresult of duplicatere-extraction of original sample. -46- wildlife International, Ltd. Project Number 454-104 Nominal Concentration' (ppm a.i.) 0 APPENDIX IV , TABLE5 Verification of PFOS Concentrations in Avian Diet Sample I.D. Number (S-454-104-) Interval (Day 0 of Week -) Measured PFOS Concentration"* (ppm a.i.) Mean (X) Standard Deviation (SD) Coefficient of Variation (CV) Mean Percent of Nominal 1 1 i0.879 N/A 2 1 < 0.879 43 6 < 1.41 N/A 44 6 < 1.41 1.8 3-8 1 45 6 46 6 47 6 6.2 9-14 1 48 6 49 6 50 6 (see Table 4) 100 1.94 -x =2.0 ppm a.i. 111 1.94 SD = 0.092 ppm a.i. 2.10 CV=4.6% (see Table 4) 97 6.3 1 -x = 6.0 ppm a.i. 97 6.52 5.273 SD = 0.67 ppm a.i. cv = 11% 17.6 15-20 1 51 6 52 6 53 6 (see Table 4) 100 17.5 -x = 16.8ppm a.i. 95 16.5 SD = 0.608 ppm a.i. 16.4 CV= 3.6% I Concentrationswere corrected for change in test substance purity (98.9% to 86.9%) per Certificate of Analysis dated October 11,2001. Nominal test concentrations based upon the new test substance purity are 1.8,6.2 and 17.6 ppm a.i. *Less than values correspond to limit of quantitation (LOQ). Mean result of duplicate re-extraction of original sample. w i l d l g e International, Ltd. Project Number 454-104 - APPENDIX IV TABLE 6 Ambient Stability of PFOS in Avian Diet During the Northern Bobwhite Pilot Reproduction Study Nominal Concentration' (ppm a.i.) Sample Number (S-454-104-) Week 1, Day 0' Mean Measured (ppm a.i.) PFOS Concentration Mean Percent of Nominal Sample Number (S-454- 104-) Week 1, Day 7 Measured293 (ppm a.i.) Mean Measured (ppm a.i.) Mean Percent of Day 0 0 1-2 < 0.879 1.8 3-8 1.8 100 6.2 9-14 6.0 97 17.6 15-20 17.6 100 21 < 1.41 22 < 1.41 23 1.974 1.8 24 1.65 25 1.72 26 5.714 6.2 27 7.03 28 5.82 29 18.1 17.2 30 17.2 31 16.2 I t I 100 103 98 'Day 0 values are from homogeneity samples presented in Table 4 and verification samplespresented in Table 5. 'Concentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate of Analysis dated October 11, 2001. concentrationsbased upon the new test substance purity are 1.8,6.2 and 17.6ppm a.i. Less than values correspond to limit of quantitation (LOQ). 4Mean result of duplicate re-extraction of original sample. Nominal test -48 - wild1ffe Xntemational, Ltd. Project Number 454-104 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 10g samples of the matrix blank, matrix fortificationand test samples into weigh boats and transfer to 8-02. 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. 1 Cap bottles and place on shaker table. Allow the samples to shake for a minimum of 30 minutes at approximately 250 rpm. 1 Vacuum filter with qualitative filter paper and rinse retained feed 3 times with methanol into filtrate. Transfer the filtrate into a 200-mL volumetric flask and bring to volume with methanol. J Prepare appropriate dilution(s) to bring final concentration into the calibration range of the LCMS methodology. Use methanol for intermediate dilutions, if required. For all final dilutions, use 50% methanol: 50% NANOpure@water dilution solvent containing 0.0100 mg 4HPFOSL (internal standard) and 0.05% (v/v) formic acid. 1 Ampulate and submit sample for LCMS analysis. Figure 1. Analytical method flow chart for the analysis of PFOS in avian diet. -49 - WildlifeInternational, Ltd. APPENDIX IV Project Number 454- 104 1 0.00 0.00 I I 0.06 III 0.12 'I 1 0.18 ) I' 0.24 'II 0.30 I I' 0.36 II"I1 0.42 0.48 Concentration (Ratio) 1 Figure 2. Typical calibration curve for PFOS. Slope = 2.48679; Intercept = 0.07396; r = 0.9985. Curve is weighted (l/x). - 50 - Wildlife International, Ltd. Project Number 454-104 APPENDIX IV 100. 90 80 70 60 50 40 30 20 10 0, intensity: 20000 cps 212 4 1 81 121 161 201 241 281 Scan 0.69 1.36 2.04 2.71 3.38 4.06 4.73Time Figure 3. Typical ion chromatogram of a low-level PFOS standard, 0.000351mg a.i./L. -51 - wildlife International, Ltd. Project Number 454-104 1o o l APPENDIX IV intensity: 20000 cps 21 4 37 182 41 81 121 161 201 241 281 Scan 0.69 1.36 2.04 2.71 3.38 4.06 4.73Time Figure 4. Typical ion chromatogram of a high-level PFOS standard, 0.00439mg a.i./L. -52 - WildlifeInternational, Ltd. Project Number 454-104 APPENDIX IV intensity: 40000 cps 42 1 41 81 121 161 201 241 281 Scan 0.69 1.36 2.04 2.71 3.38 4.06 4.73Time Figure 5. Typical ion chromatogram of a matrix blank sample (454-104-MAB-1). The arrow indicates approximate retention time of PFOS. -53 - wildlqe Intemutional, Ltd. Project Number 454-104 APPENDIX IV 100 90 80 70 60 50 40 30 20 11 C 0 intensity: 20000 cps 218 34 41 0.69 183 105128 81 121 1.36 2.04 160/1 l l l l 161 201 2.71 3.38 264 t 241 281 Scan 4.06 4.73Time Figure 6 . Typical ion chromatogram of a matrix fortification sample (454-104-MAS-9,22.p0pm ai.). - 54 - wildlife Xntemational, Ltd. Project Number 454-104 APPENDIX IV ~~ 10 9 8 7 6 5 4 3 2 1 intensity: 40000 cps 4 1 81 121 161 201 241 281 Scan 0.69 1.36 2.04 2.71 3.38 4.06 4.73Time Figure 7. Typical ion chromatogram of an avian diet sample on Day 0 (S-454-104-3, 1.8 ppm a.i.). - 55 - Wildlife International, Ltd. Appendix V Diagram of Test Layout Project Number 454-104 b a Pens 1-20 L .................................. b ................. I I = apprOX. 1m a = GQF Battery Breeder Model No. 0330 b = Four X 4 ft. Chroma 50 light bulbs Appendix VI Table 1 Adult Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS Control (0 ppm a i ) - Males Week Change Week Change Week Change Week Change Change Week Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 6-8 8 8-10 10 10-11 11 11-20 20 0-20 20 1 202 203 204 205 Mean SD 2 12 1 210 -2 217 -6 22 1 1 209 0 214 -1 5 3 213 0 208 -4 211 0 222 -1 209 0 213 -1 6 2 213 3 204 0 211 1 22 1 1 209 3 212 . 2 6 1 216 4 204 -6 212 -5 222 1 212 3 213 -1 7 5 -3 213 3 216 -6 2 10 1 211 -1 1 205 6 211 -1 2 10 5 215 5 -3 209 4 213 -3 210 5 215 -2 -1 22 1 2 223 -2 -1 211 1 212 -3 22 1 6 209 2 227 6 211 2 -1 212 3 215 -3 212 4 216 2 1 2 6 2 5 2 5 2 7 4g I Control (0 ppm a i ) - Females Week Change Week Change Week Change Week Change Change Week Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 6-8 8 8-10 10 10-11 11 11-20 20 0-20 20 1 235 5 240 -1 239 12 251 16 -3 248 7 255 -9 246 17 263 28 202 203 5 204 z 205 E Mean e SD 199 11 222 9 225 3 229 5 222 7 14 3 210 -5 231 -8 228 -2 234 3 229 -3 11 4 205 13 223 13 226 3 237 -1 226 8 14 7 218 19 236 14 229 4 236 7 234 12 12 6 6 224 1 225 0 225 -20 205 6 9T . 0 236 2 238 -11 227 5 232 10 10 239 3 242 -5 237 -8 229 4 0 236 -25 211 3 214 18 232 3 d 3 237 -2 234 -4 230 2 232 10 QP 5 9 13 17 6 12 16 21 10 VI c;. 0 P The means for body weights and body weight changes are calculated and rounded separately. Appendix VI Table 2 Adult Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS 1.8 ppm a.i. - Males Week Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 206 194 -1 207 210 6 208 228 0 209 204 -1 210 196 -2 193 7 2 16 2 228 4 203 6 194 1 200 4 218 2 232 -2 209 2 195 0 204 10 220 10 230 2 211 7 195 -1 Mean 206 0 SD 14 3 207 4 15 3 211 1 212 6 15 2 14 5 1.8 ppm a.i. - Females Week Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 206 207 208 209 210 Mean SD 216 9 247 -6 207 -6 228 2 209 -2 221 -1 17 6 225 1 241 -11 201 9 230 2 207 4 22 1 1 17 7 226 12 230 -11 210 4 232 5 211 -62 222 -10 11 30 238 22 219 -28 214 7 237 9 149 -60 211 -10 36 34 The means for body weights and body weight changes are calculated and rounded separately. Appendix VI Table 3 Adult Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS 6.2 ppm a i . - Males Week Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 211 194 -2 192 1 193 3 196 2 212 205 -3 202 -1 201 0 201 -4 213 222 1 223 0 223 -3 220 -2 2 14 204 -1 203 1 204 -1 203 -1 215 225 -4 221 -5 216 1 217 -8 Mean 210 -2 208 -1 207 0 207 -3 I SD 13 2 13 2 12 2 11 4 ul 03 I 6.2 ppm a i . - Females Week Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 211 2 12 213 2 14 215 Mean SD 254 3 211 10 220 -7 241 -2 230 9 231 3 17 7 257 11 221 -2 213 -2 239 8 239 1 234 3 17 6 268 -2 219 -9 211 7 247 5 240 -10 237 -2 23 8 266 12 210 -1 218 -2 252 11 230 0 235 4 23 7 The means for body weights and body weight changesare calculatedand roundedseparately. Appendix VI Table 4 , Adult Body Weight (g) fiorn a Northern Bobwhite Pilot Reproduction Study with PFOS 17.6 pprn a.i. - Males Week Change Week Change Week Change Week Change Change Week Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 6-8 8 8-10 10 10-11 11 11-20 20 0-20 ~ 2 16 211 -13 217 194 -7 218 208 -12 219 204 -5 220 224 -12 198 -5 187 1 196 1 199 -5 212 -6 193 3 188 0 197 -2 194 0 206 -1 196 -15 188 -6 195 -13 194 -10 205 -19 4 200 -4 196 0 ' 196 3 199 -12 -2 186 1 187 -5 182 3 185 -9 0 195 0 195 -2 193 5 198 -10 0 194 0 194 -2 192 5 197 -7 0 205 -2 203 -1 202 3 205 -19 M&Xl SD 208 -10 11 4 198 -3 9 3 196 0 72 196 -13 6 5 0 196 -1 195 -2 193 4 197 -11 2 72 6 2 7 1 7 52 I 17.6 pprn a i . - Females __ Week Change Week Change Week Change Week Change Change Week Change Week Change Week Change Week Change Pen 0 0-2 2 2-4 4 4-6 6 0-6 6-8 8 8-10 10 10-11 11 11-20 20 0-20 216 2 14 5 219 7 226 -9 217 3 -1 216 1 217 -8 209 -25 184 -30 217 220 1 221 -1 220 8 228 8 218 248 3 251 2 253 -2 25 1 3 219 213 -2 211 9 220 1 221 8 -7 22 1 6 227 -6 221 -33 188 -32 7 1 252 6 258 -4 254 6 260 12 9. 4 225 5 230 -2 228 5 233 20 sCD 220 239 -4 235 4 239 1 240 1 1 24 1 4 245 -16 229 7 236 -3 $ M&Xl 227 1 SD 16 4 227 4 16 4 232 0 14 6 231 5 14 3 d 0 231 4 235 -7 228 -8 220 -7 8 4 15 2 16 5 16 19 33 24 P F + g The means for body weights and body weight changes are calculated and rounded separately. Appendix VII Table 1 Feed Consumption (g/birdday) from a Northern Bobwhite Pilot Reproduction Study with PFOS Control (0 ppm a.i.) Weeks ~~ Pen 1 2 3 4 5 67 8 9 10 11 12 13 14 15 16 17 18 19 201 18 20 20 23 22 25 22 27 25 27 27 28 25 29 34 30 27 24 28 202 23 24 25 28 28 30 28 37 31 34 34 37 33 37 38 34 32 31 31 203 19 21 21 24 21 27 23 32 27 29 28 31 29 36 36 35 30 29 31 204 17 22 26 24 25 27 20 36 28 33 37 40 31 43 39 39 31 33 32 205 22 24 24 23 26 29 26 31 25 31 28 37 30 32 33 34 30 30 32 Mean 20 22 23 24 24 28 24 32 27 31 31 35 30 35 36 34 30 30 31 SD 3 2 2 2 3 2 3 4 3 3 4 5 3 5 2 3 2 3 2 Appendix VI1 Table 2 Feed Consumption (g/bird/day) fi-om a Northern Bobwhite Pilot Reproduction Study with PFOS 1.8 ppm a.i. Weeks Pen 1 2 3 4 5 6 206 18 20 20 22 22 23 207 21 25 23 28 14 24 208 17 20 20 24 22 23 209 17 17 20 22 21 22 210 18 19 19 24 17 19 Meall 18 20 20 24 19 22 SD 2 3 2 2 4 2 1 5 1 R f c i? Appendix VI1 Table 3 Feed Consumption (ghirdday) from a Northern Bobwhite Pilot Reproduction Study with PFOS 6.2ppm a.i. Weeks Pen 21 1 20 19 18 22 21 22 212 14 15 15 17 19 19 I a h, 213 18 23 25 24 24 26 I 214 18 20 21 20 21 23 215 22 22 21 21 25 26 Ma 18 20 20 21 22 23 SD 3 3 4 2 3 3 f P VI c 0 P Appmdixw Table 4 Feed Consumption (ghird/day) from a Northern Bobwhite Pilot Reproduction Study with PFOS 17.6ppm a.i. Weeks Pa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1 216 18 19 20 23 22 24 23 27 27 31 30 34 30 24 23 32 28 22 22 217 16 17 18 22 20 21 20 22 22 23 25 28 25 26 25 27 22 13 17 I 218 18 19 22 20 24 27 25 32 34 36 36 38 31 35 38 35 33 35 33 219 16 17 18 17 19 21 18 21 20 23 23 24 21 22 22 20 22 19 20 220 18 19 21 22 22 22 21 24 25 26 29 31 22 30 31 29 28 30 27 Mean 17 18 20 21 22 23 21 25 26 28 29 31 26 27 28 29 27 24 24 SD 1 1 2 2 2 2 3 4 6 6 5 5 4 5 6 6 5 9 6 -64- Project Number 454-104 Appendix VI11 Table 1 Individual Gross Pathological Observations from a Northern Bobwhite Pilot Reproduction Study with PFOS Birds Euthanized at Test Termination Not remarkable Males Control (0 ppm a.i.) ~~ ~ 201 202 203 204 205 X X X X X Females Feather loss Right wing twisted Not remarkable Pens 201 202 203 204 205 -65 - Project Number 454-104 Appendix VI11 Table 2 Individual Gross Pathological Observations from a Northern Bobwhite Pilot Reproduction Study with PFOS Birds Euthanized at 6 Weeks Not remarkable Males 1.8 ppm a.i. Pens 206 207 208 209 210 X X X X X Feather loss Foot lesions Thin condition Regressed ovary Not remarkable Females Pens 206 207 208 209 210 Project Number 454-104 - 66 - Appendix VI11 Table 3 Individual Gross Pathological Observations from a Northern Bobwhite Pilot Reproduction Study with PFOS Birds Euthanized at 6 Weeks Males - Testes small, 1.5cm - Right testis small, 1.5cm Not remarkable 6.2ppm a.i Pens 211 212 213 214 215 - X X - X X X Feather loss Not remarkable Females Pens ~ 211 212 213 214 215 X X X - - X X Project Number 454-104 - 67 - Appendix VI11 Table 4 Individual Gross Pathological Observations from a Northern Bobwhite Pilot Reproduction Study with PFOS Birds Euthanized at Test Termination Males Right testis small, - 1.25 cm Not remarkable 17.6 ppm a.i. Pens 216 217 218 219 220 X-X- - X X X Feather loss Not remarkable Females Pens 216 217 218 219 220 - X X - X X - X -68 - wildlife International, Ltd. Appendix IX Histopathology Report Project Number 454-104 - 69 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER 454-104 EPL PROJECT NUMBER 212-024 PFOS: A PILOT 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 January 25,2001 EPL" - 70 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454- 104 TABLE OF CONTENTS PATHOLOGY SUMMARY ................................................................................. QUALITY ASSURANCE FINAL CERTIFICATION............................................ Ewe 1 8 ADULT SACRIFICE SUMMARY INCIDENCETABLES Males........................................................................................................... I-1 Females........................................................................................................ 1-3 HISTOPATHOLOGY INCIDENCE TABLES Males........................................................................................................... 11-1 Females....................................................................................................... 11-3 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Males........................................................................................................... 111-1 Females....................................................................................................... 111-2 OFFSPRING SACRIFICE SUMMARY INCIDENCETABLES Males............................................................ Females....................................................................................................... HISTOPATHOLOGY INCIDENCETABLES Males.. ......................................................................................................... Females.. ..................................................................................................... CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Males.................................................. .................................................... Females....................................................................................................... IV- 1 IV-2 v- 1 v-2 VI- 1 Vl-5 -71 - Project Number 454-104 PATHOLOGY SUMMARY EPL" - 72 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 WILDLIFE INTERNATIONAL, LTD. STUDY NUMBER 454-104 EPL PROJECT NUMBER 212-024 PFOS: A PILOT REPRODUCTIONSTUDY WITH THE NORTHERN BOBWHITE PATHOLOGY SUMMARY Light microscopic examination was performed on sections of selected tissues from adult male and female Northern bobwhite (Colinus virginianus) which were untreated or which receivedvarious concentrations of the test article (Perfluorooctanesulfonic acid, potassium salt [PFOS]) in the feed for six to 19 weeks. Selected tissues from untreated approximately 12-week-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 virginianus)of dietary exposure to a test substance over at least a six-week period. The experimental design was as follows: Group * Adults Males I Females Offspring ** Males I Females * Bobwhite in the control and 17.6 ppm a.i. groups were treated for 19 weeks; bobwhite in the 1.8 and 6.2 ppm a.i. groups were treated for six weeks. ** Offspring in all groups did not receive the test article. I -73 - Project Number 454-104 EXPERIMENTAL PATHOLOGY LABORATORIES,INC. Wildlife International,Ltd. Study Number 454-104 MATERIALS AND METHODS At the completion of the various study intervals, all adult bobwhite were euthanized, and necropsies were performed by Wildlife International, Ltd. Selected offspring were euthanized at approximately 12 weeks of age using carbon dioxide gas, and samples were collectedfor histopathological evaluation by Wildlife International, Ltd. Selected tissues from adults and offspring were fixed in 10% neutral bufferedformalin and sent to Experimental Pathology Laboratories,Inc. (EPL@)w, here they were embedded in paraffin and made into hematoxylin and eosin-stainedsections on glass microscope slides. The following tissues from adults and offspring were examined by light microscopy as available: liver, gallbladder, proventriculus, kidney, brain, gonad (ovary or testis), bursa of Fabricius, and adipose tissue. In some cases, nonprotocol-required tissues were sectioned along with adjacent protocol-requiredtissues; these were evaluated, and microscopic findings were recorded at the discretion of the pathologist. All tissues required by 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. Nongradabbfindings are listed as present (P) and tissues with extensive autolysis are listed as (A). All findings for all animals are summarized by sex, age group, and treatment group in the Summary Incidence Tables, together with the total number of animals in each group for which the tissues were examined. A tabulation of gross observations noted at necropsy with the corresponding microscopic change, if appropriate, is presented in the Correlation of Gross and Microscopic Findingstables. The entries in these tables were -2- EPL" - 74 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 Wildlife International,Ltd. Study Number 454-104 transcribed from the Gross Necropsy records provided by Wildlife International, Ltd. RESULTS All adults and offspring survived to the end of their respective study intervals. No effects considered possibly relatedto test article (PFOS) administrationwere noted in liver, kidney, adipose tissue, gallbladder, brain, bursa of Fabricius,proventriculus,and ovary of male and female adult bobwhite or their offspring, or in the testes of offspring males. Testes of all adult males exhibitedspermatogenesis characterizedby the presence of numerous mature spermatozoa in the seminiferous tubules. In two 17.6 ppm a.i. males, seminiferous tubules had marginally decreased diameter (though spermatogenesis was still evident) compared to other control and 17.6 ppm a.i. testes, resulting in overall smaller testes which corresponded to grossly observed 1.25 cm right testes. The smaller testes of these birds were most consistent with early post-reproductive phase regression, a normal physiologicalphenomenon. The occurrence of such testicular regressiononly in the 17.6 ppm a.i. dose group may have been a fortuitous event accentuated by the small group sizes, but the possibility of a test article effect cannot be entirely ruled out. Testes of all offspring were listed as immature because they lacked all evidence of spermatogenesis, had small seminiferous tubules with absent or narrow lumens, and had germinal epithelium with decreased cell layers (compared to adult testes). This morphology was consistent with normal physiologicalimmaturity in young (approximately 12-week-old) birds rather than a pathologicaleffect. The overall small diameter of offspring testes corresponded to measurements of less than 1.5 cm recorded at necropsy. Because of the -3- Project Number 454- 104 -75 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Wildlife International,Ltd. Study Number 454-104 smaller size of the seminiferous tubules in offspring testes, the melanin pigmentbearing cells normally present in the testicular interstitium were in effect more closely packed in a smaller area, rather than being widely separated by larger seminiferous tubules as in adult testes. This melanin pigment concentration corresponded to the grossly observed black offspring testes, but was considered a normal age-relatedfinding and not a pathologicaleffect. All ovaries examined in offspring females were characterizedby numerous oocytes and very small, nonpedunculated follicles completely embedded in a prominent ovarian cortex. This morphology is consistentwith normal physiological immaturity in young (approximately 12-week-old) birds rather than a pathological effect. Several additional microscopicfindings (describedbelow) were noted in adult and/or offspring bobwhite from control and treated groups. Incidences and mean severity were similar when groups from each generational cohort were compared with each other. Some of these lesions occurred only in a single bird. For these reasons, these findings were considered incidental and unrelatedto test article (PFOS) administration. Mononuclear cell infiltrates in various tissues consisted of discrete foci of small lymphocytes and plasma cells; a few heterophilswere sometimes also present but were always a minor component. Chronic and chronic active inflammation of adipose tissue were characterizedby infiltrates consisting exclusively or predominantly of macrophages, or mixed populationsof heterophils and marophages, respectively. Scattered necrotic adipocytes were associated with chronic active inflammation in one 6.2 ppm a.i. offspring male. A small focus of cortical tubules exhibited cytoplasmic vacuolizationin the kidney of one adult 17.6 ppm a.i. female; this change in a single bird was considered -4- EPL" - 76 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454- 104 Wildlife International,Ltd. Study Number454-104 incidental. Scattered clusters of coarsely to finely dark basophilic material in the renal cortex of a few adults and offspring denoted mineralization. One control adult female exhibited foreign material and granulomatous inflammation in several tissues including the serosa of the ovary, liver, and gallbladder, and in the adipose tissue. Granulomatous inflammation characterized by small mononuclear cells, macrophages, and occasional multinucleatedgiant cells was associated with foci of foreign material (deep magenta globules and bright pink amorphous material consistent with extrafollicularegg yolk). Collectively,these findings were consistent with the common clinical condition usually referred to as "egg yolk peritonitis." Liver pigment deposition consisted of dust-like to coarsely irregular, brown to golden-brown pigment granules present in hepatocyte cytoplasm, Kupffer cells, and/or periportal areas; the identity of the pigment was not determined, but may have been related to bile. Small single clusters of hyperplastic bile ducts were noted in one adult control female and one 1.8 ppm a.i. offspring female. A small focus of heterophils (acute inflammation) was noted in one 1.8 ppm a.i offspring male. The liver of an adult 17.6 ppm a.i. female exhibited amyloid deposition associated with secondary hepatocellular hypertrophy and necrosis. Liver amyloid deposits consisted of amorphous, pale bluish-violet material which filled and distended the hepatic sinusoids, and compressed adjacent hepatic cords. Additional liver lesions included hepatocellularfatty change and vacuolization. Fatty change denoted variably sized, sharply demarcated, clear vacuoles (consistent with fat accumulation) in the hepatocellular cytoplasm. Vacuolization denoted multiple, usually small, confluent, poorly demarcated vacuoles Gvhich often resulted in a "lace-like" appearance of the hepatocellular cytoplasm. When present in most hepatocytes, these changes were designated -5- EPL" - 77 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 Wildlife International,Ltd. Study Number 454-104 "diffuse," and when present in a few scattered areas, they were designated as "focal." When hepatocellularfatty change occurred predominantly in the areas adjacent to bile channels and associated blood vessels, it was designated as "periportal.I' Hepatocellularfatty change was noted in the livers of adults and offspring, while hepatocellular vacuolization was seen only in offspring. This generational difference was considered to be due to normal age-related variation rather than being a treatment effect. When control and treated groups of each generational cohort were compared, combined incidences of diffuse, focal, and periportal fatty change in male and female adults and offspring were similar. Hepatocellular fatty change was considered incidentaland unrelatedto treatment. There was a relative increase in incidence of hepatocellularvacuolization in female offspring, but the magnitude was small, and the increase was not clearly dose-related. It is most likely that this finding was coincidental and not related to test article administration. CONCLUSIONSAND SUMMARY No lesions considered possibly relatedto test article (PFOS) administration were noted in liver, kidney, adipose tissue, proventriculus, gallbladder, ovary, and brain of adult male and female bobwhite or their offspring, or in the testes of offspring bobwhite. Testes of two high dose (17.6 ppm a i ) adult male bobwhite exhibited decreased seminiferous tubule diameter most consistent with early post-reproductive phase regression, a normal physiological phenomenon. The occurrence of testicular regression only in the 17.6 ppm a.i. dose group may have been a fortuitous event accentuated by the small group sizes, but the possibility of a test article effect cannot be entirely ruled out. The -6- Project Number 454-104 I - 78 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Wildlife International,Ltd. Study Number 454-104 few other changes in various tissues of adult and/or offspring bobwhite from control and treated groups were considered incidental and unrelated to test article (PFOS)administration. MMG/lcp M. GRUEBBEL, DVM, PhD, Diplomate, ACVP I -7- Project Number 454-104 I - 79 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. QUALITY ASSURANCE FINAL CERTIFICATION Study Title: PFOS: A Pilot Reproduction Study with the Northern Bobwhite Client Study: 454-104 EPL Project Coordinator: Dr. Margarita M. Gruebbel EPL Project Number: 212-024 EPL Pathologist: Dr. Margarita M. Gruebbel The following aspects of this study were inspected by the Quality Assurance Unit of ExperimentalPathology Laboratories, Inc. Dates inspectionswere performed and findings reported to the EPL Project Coordinator and Management are indicated below. Area Inspected Inspection Dates Reporting EPL Project Sheets Project Setup Histology Setup Data Review Draft Report Final Report 9111/00; 10/3/00 9/27/00 10/2/00 11I13/00 12/12,27/00 1129101 9111/00; 10/3/00 9/28/00 10/2/00 11/I3/00 12113,27100 1/29/01 Date of last quarterly facility inspection 9/00 EPL Quality Assurance Unit' Date Form No. 6-2 (7/2/99) -8- - 80 - Project Number 454-104 SUMMARY INCIDENCE TABLES ADULT SACRIFICE 454-104 Adult Sacrifice Male Bobwhite - 81 - SUMMARY INCIDENCE TABLE Project Number 454-104 1-1 EPL Experimental Pathology Laboratories, Inc. 454-104 Adult Sacrifice Male Bobwhite - 82 - SUMMARY INCIDENCE TABLE Project Number 454-104 1-2 I Experimental Pathology Laboratorles, 1C. 454-104 Adult Sacrifice Female Bobwhite - 83 - SUMMARY INCIDENCE TABLE ProjectNumber 454-104 1-3 EPL Experimental Pathology Laboratories, Inc. 454-104 Adult Sacrifice Female Bobwhite - 84 - SUMMARY INCIDENCE TABLE Project Number 454-104 1-4 EPL Experimental Pathology Laboratories, Inc. -85 - Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLES ADULT SACRIFICE 454-104 Adult Sacrifice Male Bobwhite - 86 - Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 17.6 BRAIN BURSA OF FABRICIUS CLOACA GALLBLADDER Infiltrate, Mononuclear Cell Serosa, Foreign Material Serosa, Inflammation, I Granulomatous xxxxx X I I I I I I I 1 l N l N l N l N l N l N IIIIIII I I I I I I I I I 1 I l x l x l I I I I I I I xx 21 *2 1 IIIIIII I E P L I Experimental Pathology Laboratories, Inc. 11-1 Key :X-Not Remarkable N-No Sectlon I - I n c a p l e t e A-Autolysis I-aininol Z ~ s l l g h t h i l d f w d s r a t e 4-loderatsly severe 5-$avere/hlgh P-Present 84anfgn M-Ualignant n-missing one paired organ u-unscheduled sac./death 454-104 Adult Sacrifice Male Bobwhite - 87 - Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE GROUP GROUP 1- 7.6 1 IIIIII I E P L Experimental Pathology Laboratories, Inc. 11-2 Key :X=Not Renarkable N-No Section I-Incomplete A-Autolysis lrinimal 2-slighthi Id 3ioderate lmderately severe 5.severe/hlgh P-Present &Benign H=Halipnant wmissing one paired organ u=unscheduled sac./derth 454-104 Adult Sacrifice Female Bobwhite -88 - Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE GROUP mGROUP ADIPOSE TISSUE Foreinn Material Infiltrate, Mononuclear Cell Inflammation. Chronic Inflammation, Granulomatous BRAIN BURSA OF FABRICIUS CLOACA GALLBLADDER Infiltrate, Mononuclear Cell Serosa, Foreign 'Material Serosa, Inflammation, Granulomatous KIDNEY E P L Experimental Pathology Laboratories, Inc. 11-3 Key :X-Not Rednarkable N-No Section I = I n c a p l s t c A-Autolysis liinimal 2.~1i p h t h i l d 3nodarate 4 w d e r a t e l y seven 5-tevere/hlgh P-Present 84cnign H-Halipnant m-misrinp one paired organ u-unscheduled sac./death 454-104 Adult Sacrifice Female Bobwhite - 89 - Project Number 454- 104 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 17.6 A N I M A I IIIIII IIIIII EPL Experimental Pathology Laboratories, Inc. Key :X-Not Rmarkable N-No Section I-Incanplete A-Autolysis 1-minimal Z-slight/nild 3-noderate 4-moderately severe 5=severe/high Papresent 8-Benign M-Malignant wairring one paired organ u-unscheduled sac.ldeath Project Number 454-104 - 90 - CORRELATION OF GROSS AND MICROSCOPIC FINDINGS ADULT SACRIFICE 454-104 Adult Sacrifice CORRELATION OF GROSSAND MICROSCOPICflNDlNGS Animal Number 481 485 Client Topography / Si TESTIS 1TESTIs Client Gross Observations Right 1.25 cm Microscopic Observations Seminiferous Tubules, Decreased Diameter bSeminiferous iameter Tubules, Decreased 111-1 R I f c B 454-104 Adult Sacrif i ce CORRELATIONOF GROSS AND MICROSCOPICFINDINGS Species: Bobwhite I Animal Number Client Topography I Site 454 EXTERNAL 456 EXTERNAL 458 EXTERNAL 460 EXTERNAL EXTERNAL Sex: Females Group IdentiticatiOn: CONTROL - Sacrificed I 1 Client Gross Observations Microscopic Observations Feather loss neck Feather loss neck Feather l o s s neck Right wing twisted Feather loss neck and head No Comment Required N o Comment Required No Comment Required No Comment Required No Comment Required 111-2 454-104 Adult Sacrifice Species: Bobwhite CORRELATIONOF GROSS AND MICROSCOPIC FINDINGS S a Females Group Identification: 1 7 . 6 - Sacrificed Animal Number 482 484 488 490 Client Topography/ Site EXTERNAL EXTERNAL EXTERNAL EXTERNAL Client Gross Observations Feather l o s s neck and back Feather l o s s neck and back Feather l o s s head and neck Feather l o s s neck and back Microscopic Observations N o Comment Required No Comment Required No Comment Required N o Comment Required ~ I \w9 I Y 9. s 0 5 P e R 111-3 E; P - 94 - Project Number 454-104 SUMMARY INCIDENCE TABLES OFFSPRING SACRIFICE 454-104 Offspring Sacrifice Male Bobwhite -95SUMMARY INCSDENCE TABLE ~ ~~ Project Number 454-104 IV-1 EPL Experimental Pathology Laboratories, Inc. 454-104 Offspring Sacrifice Female Bobwhite - 96 - SUMMARY INCIDENCE TABLE Project Number 454-104 Immature 3 5 5 8 PROVENTRICULUS (NO. EXAMINED) (5) (6) (5) (8) Infiltrate, Mononuclear Cell 5 6 5 8 IV-2 EPL Experimental Pathology Laboratories, Inc. - 97 - ~ Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLES OFFSPRING SACRIFICE 454-104 Offspring Sacrifice Male Bobwhite -98- Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 1.8 GROUP 6.2 GROUP 17.6 IADIPOSE TISSUE Infiltrate, Mononuclear Cell Inflammation, Chronic Inflammation, Chronic Active Mine ra 1iza t ion Necrosis BRAIN BURSA OF FABRICIUS lslelslo I _X_ l _X_ l _X_ l _X_ IIII lxlxlxlx IIII lNlXlXlX I I # I I I I I I I GALLBLADDER Infiltrate, Mononuclear Cell KIDNEY Infiltrate, Mononuclear Cell Tubules, Mineralization XAX X 1 xx xx 1 v- 1 I E P L I Experlmental Pathology Laboratories, Inc. Key :X-Not Remarkable N-No Section I-lncmplete A-Autolysis l.mininal Z-sli h t h i l d 3-moderate lioderately severe i-revere/high P-Present B=Ben!gn H=Rlignant n-mlrsing one paired organ u-unscheduled tac./death 454-104 Offspring Sacrifice Female Bobwhite - 99 - Project Number 454- 104 HISTOPATHOLOGY INCIDENCE TABLE GROUP GROUP GROUP 6.2 GALLBLADDER Infiltrate, Mononuclear Cell KIDNEY Infiltrate, Mononuclear Cell Tubules, Mineralization xx 1 12 X X < 11 1 X xx 1 v-2 EPL Experimental Pathology Laboratories, Inc. Key :X-Not R m r k a b l e N*No Section I-Incmplete A-Autolysis 1-minimal Z-slight/ml I d h o d e r a t e 4-nodsratcly severe S-rcvere/high P*Prescnt fJ=Bcnign H=klignant n-missing one paired organ u-unscheduled sac./dtath 454-104 Offspring Sacrifice Female Bobwhite - 100 - Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE , GROUP 17.6 A N I BRAIN BURSA OF FABRICIUS _x_x_x---x--x---x--x--x--- I xxxxxxxx v- 3 I E P L I Experimental Pathology Laboratories, Inc. Key :X-Not Remarkable I-No Section I-Incapletc A-Autolysis l-minimal Z-rlight/mild 3 d c r a t e 4roderately severe tiseverelhigh P-Present 6-Benign H-Halignant miisrlng one paired organ u-unscheduled saceldeath - - 101 - Project Number 454-104 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS OFFSPRING SACRIFICE 454-104 Offspring Sacrifice Species: Bobwhite CORREUllON OF GROSS AND MICROSCOPIC flNDlNGS Sex Males Group Identification: CONTROL - Sacrificed Animal Number 9503 Client Topography/Site TESTIS 9508 9515 9520 TESTIS TESTIS TESTIS 9526 TESTIS Client Gross Observations Microscopic Observations Diffusely black, 0 . 4 - 0.6 x 0.2 cm Immature; Pigment Concentration (both present) (noted at gross trimming 1 . 5 x .3 cm black one present (noted Immature; Pigment Concentration at gross trirmning) - 6 x .2 cm black both testes (noted Immature; Pigment Concentration at gross trimming) 1 One .5 x . 3 cm, other . 7 cm x . 3 cm, both black (noted at gross trimming ) Immature; Pigment Concentration One present .2 x .2 cm black area, Immature; Pigment Concentration .2 cm x .2 cm white area (noted at gross trimming) VI-1 454-104 Offspring Sacrifice CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Species: Bobwhite I %i %r! Client Topography/ Site PESTIS TESTIS EXTERNAL TESTIS TESTIS Sex: Males Group Identificatiox 1.8 - Sacrificed ~ -~ - ~ Client Gross Observations Microscopic Observations One testis - 8 x .4 cm, other .5 x .4 cm, both black (noted at gross trimming) IOne testis .4 x .2 cm, other .5 x .3 cm, both black (noted at gross trimming) Immature; Pigment Concentration IImmature; Pigment Concentration Feather loss on rump No Comment Required Diffusely black, 0.4 x 0.2 cm, Immature: Pigment Concentration bilateral (noted at gross trinnning) One testis .5 x .2 cm, other .5 x .3 cm, both black (noted at gross trimming) Immature; Pigment Concentration E I VI-2 454-104 Offspring Sacrifice CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Species: Bobwhite Sex: Males Group Identification: 6.2 - Sacrificed ~ -~ ANnuimmabler 9549 Client Topography / Site EXTERNAL TESTIS 9551 TESTIS 9552 EXTERNAL TESTIS 9559 TESTIS 9563 EXTERNAL TESTIS Client Gross Observations Microscopic Observatiom Feather loss on rump One present fragmented, black, .7 cm x . 3 cm (noted at gross trimming) No Comment Required Immature; Pigment Concentration One testis . 3 x .2 cm, other .6 x .2 cm, both black (noted at gross trimming) Immature; Pigment Concentration Feather loss on rump No Comment Required One testis .5 x .2 cm, other .4 x .2 cm, both black (noted at gross trimming) Innnature; Pigment Concentration One testis . 4 x .2 cm, other -6 x .2 cm, both black (noted at gross trimming) Immature; Pigment Concentration Feather l o s s abrasions on rump and No Comment Required head One testis .6 x . 3 cm, other - 4 x - 2 cm, both black (noted at gross trimming) Immature; Pigment Concentration VI-3 I c 0 P I `51 9. cb z! $ 8 8 R f c 0 P 454-104 Offspring Sacrifice CORRELATION OF GROSS AND MICROSCOPIC flNDlNGS Species: Bobwhite I Client TopographyI Site -t9582 EXTERNAL TESTIS TESTIS Sex Males Group Identification: 17.6 - Sacrificed Client Gross Observations Microscopic Observations Feather loss on rump One testis .7 x .4 cm, other .5 x .2 cm, both black (noted at gross t rimmine:) One testis . 7 x . 3 cm, other .5 x - 2 cm, both black (noted at gross trimming ) No Comment Required Immature; Pigment Concentration Tnnnature; Pigment Concentration VI-4 454-104 Offspring Sacrifice Species: Bobwhite CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex Females Group Identification: - Sacrificed Animal Number 9512 Client Topography I Site EXTERXAL Client Gross Observations Feather loss on rump Microscopic Observations No Comment Required * --- L VI-5 454-104 O f f spring Sacrifice Species: Bobwhite CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex: Females Group Identification: 1.8 - Sacrificed Animal Number 9531 Client Topography1Site EXTERNAL Client Gross Observations Feather loss on rump and back Microscopic Observations No Comment Required VI-6 454-104 Offspring Sacrifice Species: Bobwhite CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex Females Group Identification: 17.6 - Sacrificed Animal Number 9583 client Topography I Site EXTERNAL Client Gross Observations Feather loss on rump Microscopic Observations No Comment Required I c-r 0 00 I 3 9. scb z5R8 P c VI-7 0 P Appendix X Table 1 Egg Production (eggs laidhedweek) from a Northern Bobwhite Pilot Reproduction Study with PFOS Control (0 ppm ai.) Weeks Pen 1 2 3 4 5 6 Total E/wD' 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total E m ' 201 3 4 5 4 5 6 27 0.64 5 7 6 7 7 7 7 7 7 7 7 7 7 2 117 0.87 202 3 4 5 5 5 6 28 0.67 6 6 7 7 7 6 7 6 7 7 6 6 7 2 115 0.85 203 6 6 7 6 6 7 38 0.90 7 7 7 7 7 6 7 7 7 7 7 7 7 2 130 0.96 204 3 4 6 6 4 6 29 0.69 4 6 7 7 7 7 7 6 6 4 3 3 3 0 99 0.73 205 6 7 6 6 7 7 39 0.93 8 7 6 0 0 0 4 4 7 7 7 7 7 2 105 0.78 Total 21 25 29 27 27 32 161 30 33 33 28 28 26 32 30 34 32 30 30 31 8 566 Mean 4 5 6 5 5 6 32 0.77 6 7 7 6 6 5 6 6 7 6 6 6 6 2 113 0.84 SD 2 1 1 1 1 1 6 0.14 2 1 1 3 3 3 1 1 0 1 2 2 2 1 12 0.09 1 Eggs laid per hen per day Appendix X Table 2 Egg Production (eggs laidhedweek) fiom a Northern Bobwhite Pilot Reproduction Study with PFOS 1.8 ppm a.i. Week Pen 1 2 3 4 5 6 Total E/H/D' 206 3 6 6 6 6 7 34 0.81 207 6 7 7 7 1 0 28 0.67 I c. 208 5 5 6 5 5 6 32 0.76 0 I 209 4 5 5 5 6 7 32 0.76 210 4 6 4 4 2 0 20 0.48 Total 22 29 28 27 20 20 Mean 4 6 6 5 4 4 SD 1 1 1 1 2 4 ' Eggs laid per hen per day 146 29 0.70 6 0.13 Appendix X Table 3 Egg Production (eggs laidherdweek) from a Northern Bobwhite Pilot Reproduction Study with PFOS Pen 1 2 211 5 7 212 0 2 213 6 5 214 5 5 215 4 5 Total 20 24 Mean 4 5 SD 2 2 ' Eggs laid per hen per day 6.2 ppm a.i. Weeks 3 4 5 6 Total E M ' 6 7 7 7 39 0.93 3 4 5 6 20 0.48 7 6 3 7 34 0.81 5 6 6 7 34 0.81 6 4 6 6 31 0.74 27 27 27 33 158 5 5 5 7 32 0.75 2 1 2 1 7 0.17 Appendix X Table 4 Egg Production (eggs laicherdweek) fiom a Northern Bobwhite Pilot Reproduction Study with PFOS 17.6ppm a.i. Weeks Pen 1 2 3 4 5 6 Total EJWD' 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total E m ' ~~ ~~ 216 5 5 5 5 7 7 34 0.81 5 6 7 6 6 6 6 4 0 0 1 3 0 0 84 0.62 217 5 6 7 6 7 7 38 0.90 7 7 7 7 7 7 6 7 7 7 7 2 0 0 116 0.86 I c 218 6 7 7 6 7 7 40 0.95 7 7 7 7 7 7 7 7 7 7 7 7 7 2 133 0.99 c h) I 219 3 4 4 5 5 7 28 0.67 7 7 7 7 7 7 7 7 7 9 6 6 7 1 120 0.89 220 8 5 7 7 7 7 41 0.98 6 7 7 7 7 7 7 6 7 7 7 7 7 2 132 0.98 Total 27 27 30 29 33 35 181 32 34 35 34 34 34 33 31 28 30 28 25 21 5 585 M e a n 5 5 6 6 7 7 36 0.86 6 7 7 7 7 7 7 6 6 6 6 5 4 1 117 0.87 SD 2 1 1 1 1 0 5 0.13 1 0 0 0 0 0 1 1 3 3 3 2 4 1 20 0.15 ' Eggs laid per hen per day Appendix XI Page 1 Reproductive Performance by Pen froma Northern Bobwhite Pilot Reproduction Study with PFOS Table 1 Reproductive Data (Count) By Pen Parameter Control (0 ppm a.i.) Pens 201 202 203 204 205Total 1.8ppm a i . 206 207 208 209 210 Total 6.2 ppm a.i. 211 212 213 214 215 Total 17.6ppm a i . 216 217 218 219 220 Total Eggs Laid 5665729 Eggscracked 0 0 0 0 0 0 Eggs Set 5665729 Viable Embryos Live 3-Wk Embryos Hatchlings 5665729 5565728 5565728 offspring survivors 5555727 6056118 00000 0 6056118 6046117 6046117 6046117 6046117 7557529 00000 0 7557529 5537525 5537525 5 5 3 7 5 25 5532520 7776734 00000 0 7776734 7776734 7676733 7576631 7363625 I c c-. w I T 9. cb 0, ? P c 0 P Replicate 1 2 3 4 5 Total Ma SD Control (0 ppm a.i.) Eggs Eggs/ Laid Days Hen/Day 5 7 0.71 6 7 0.86 6 7 0.86 5 7 0.71 7 7 1.00 29 6 0.83 1 0.12 Replicate Control (0 ppm a.i.) Eggs Eggs Cracked Laid YO 1 0 5 0 2 0 6 0 3 0 6 0 4 0 5 0 5 0 7 0 Total Mean SD 0 29 0 6 0 0 1 0 Appendix XI Page 2 Reproductive Performance by Pen a Northern Bobwhite Pilot ReproductionStudy with PFOS Table 2 Eggs Laid / Hen / Day 1.8 ppm ai. Eggs Eggs/ Laid Days HenlDay 6.2 ppm ai. Eggs Eggs/ Laid Days Hen/Day 6 7 0.86 0 7 5 7 0.71 6 7 0.86 1 7 0.14 18 4 0.64 3 0.34 7 7 1.00 5 7 0.71 5 7 0.71 7 7 1.00 5 7 0.71 29 6 0.83 1 0.16 Table 3 Eggs Cracked / Eggs Laid (%) 1.8 ppm ai. Eggs Eggs Cracked Laid % 0 6 0 0 0 0 5 0 0 6 0 0 1 0 0 18 0 4 0 0 3 0 6.2 ppm ai. Eggs Eggs Cracked &d YO ~~ 0 7 0 0 5 0 0 5 0 0 7 0 0 5 0 0 29 0 6 0 0 1 0 17.6 ppm ai. Eggs Eggs/ Laid Days Hen/Day 7 7 1.00 7 7 1.OO 7 7 1.OO 6 7 0.86 7 7 1.00 34 7 0.97 0 0.06 17.6 ppm ai. Eggs Eg_g_s Cracked Laid % 0 7 0 0 7 0 0 7 0 0 6 0 0 7 0 ~~ 0 34 0 7 0 0 0 0 I c c P I 7 9. CD 2 Z E: 3 q P ? c 0 P Control (0 ppm ai.) Viable Eggs Replicate Embryos Set ?h 5 5 100 6 6 '100 6 6 100 5 5 100 7 7 100 Total Meall SD 29 29 6 6 100 1 1 0 AppendixxI Page 3 Reproductive Performance by Pen h na Northern Bobwhite Pilot ReproductionStudy with PFOS Table 4 Viable Embryos / Eggs Set (%) 1.8 pprn ai. V i l e Eggs Embqos Set % 6.2 ppm a.i. Viable Eggs Embryos Set % 6 6 100 0 0 4 5 80 6 6 100 1 1 100 17 18 3 4 95 3 3 10 5 7 71 5 5 100 3 5 60 7 7 100 5 5 100 25 29 5 6 86 1 1 19 Control (0 ppm ai.) Live Re~licate 3-Week Viable % 5 5 100 5 6 83 6 6 100 5 5 100 7 7 100 Total Meall SD 28 29 6 6 97 1 1 7 Table 5 Live 3-WeekEmbryos I Viable Embryos (%) 1.8 ppm ai. Live 3-Week Viable % 6.2 ppm ai. Live 3-Week Viable % 6 6 100 0 0 4 4 100 6 6 100 1 1 100 17 17 3 3 100 3 3 0 5 5 100 5 5 100 3 3 100 7 7 100 5 5 100 25 25 5 5 100 1 1 0 17.6 ppm a.i. Viable Eg~g-s Embryos Set % 7 7 100 7 7 100 7 7 100 6 6 100 7 7 100 34 34 7 7 100 0 0 0 17.6 ppm a.i. Live 3-Week Viable YO 7 7 100 6 7 86 7 7 100 6 6 100 7 7 100 33 34 7 7 97 1 0 6 Control (0 ppm a.i.) Live Appendix XI page 4 Reproductive Performance by Pen from a Northern Bobwhite Pilot Reproduction Study with PFOS Table 6 Hatchhgs / Live 3-WeekEmbryos (%) 1.8 ppm ai. Live 6.2 ppm ai. Live Total Mean SD 5 5 100 5 5 100 6 6 100 5 5 100 7 7 100 28 28 6 6 100 1 1. 0 control (0ppm ai.) offspring 6 6 100 0 0 4 4 100 6 6 100 1 1 100 17 17 3 3 100 3 3 0 5 5 100 5 5 100 3 3 100 7 7 100 5 5 100 ~ ~~~ 25 25 5 5 100 1 1 0 Table 7 surviving offspring/ Iwchhgs 1.8 ppm ai. 6.2ppm ai. offspring Total Mean SD 5 5 100 5 5 100 5 6 83 5 5 100 7 7 100 27 28 5 6 97 1 1 7 6 6 100 0 0 4 4 100 6 6 100 1 1 100 17 17 3 3 100 3 3 0 5 5 100 5 5 100 3 3 100 2 7 29 5 5 100 ~~~ 20 25 4 5 86 1 1 32 17.6ppm a.i. Live 7 7 100 5 6 83 7 7 100 6 6 100 6 7 86 31 33 6 7 94 1 1 9 17.6ppm a.i. 7 7 100 3 5 60 6 7 86 3 6 50 6 6 100 25 31 5 6 79 2 1 23 I c c o\ I 3 9. 5 3 3 e P ul c 0 P Replicate 1 2 3 4 5 Total Mean SD Control (0ppm ai.) Eggs Hatch Set YO 5 5 100 5 6 83 6 6 100 5 5 100 7 7 100 28 29 6 6 97 1 1 7 Appendix XI Page5 . Reproductive Performance by Pen froma Northern Bobwhite Pilot Reproduction Study with PFOS Table 8 Hatchlings / Eggs Set (`A) 1.8 pprn ai. Eggs Hatch Set % 6 6 100 0 0 4 5 80 6 6 100 1 1 100 17 18 3 4 95 3 3 10 6.2 ppm ai. Eggs Hatch Set YO 5 7 71 5 5 100 3 5 60 7 7 100 5 5 100 25 29 5 6 86 1 1 19 Control (0ppm a.i.) offspring Eggs Replicate S W Set YO 5 5 100 5 6 83 -5 6 83 5 5 100 7 7 100 Total Mean SD 27 29 5 6 93 1 1 9 . Table 9 Surviving Offspring / Eggs Set (`A) 1.8 ppm ai. mn rig Eggs surv set % 6 0 4 6 1 ~~ 17 3 3 6 100 0 5 80 6 100 1 100 ~~ 18 4 95 3 10 6.2 ppm ai. offspring Eggs S W set YO 5 5 3 2 .5 7 71 5 100 5 60 7 29 5 100 20 29 4 6 72 1 1 30 17.6 ppm a i . Eggs Hatch Set YO 7 7 100 5 7 71 7 7 100 6 6 100 6 7 86 31 34 6 7 91 1 0 13 17.6 ppm a.i. Offs-pring- E_m_ s SW Set Yo 7 7 100 3 7 43 6 7 86 3 6 50 6 7 86 25 34 5 7 73 2 0 25 Replicate 1 2 3 4 5 Total Mean SD Control (0 ppm a.i.1 Hatch/ Hatch Days HedDay -5 5 6 5 7 28 6 1 7 0.71 7 0.71 7 0.86 7 0.71 7 1.00 0.80 0.13 Appendix XI Page 6 ReproductivePerformance by Pen from a Northern Bobwhite Pilot Reproduction Study with PFOS Table 10 Hatchlings I Hen I Day 1.8 ppm ai. Hatch Hatcw Days Hen/Day 6.2 ppm a.i. Hatcw Hatch Days HedDay 6 7 0.86 0 7 0.00 4 7 0.57 6 7 0.86 1 7 0.14 17 3 0.49 3 0.40 5 7 0.71 5 7 0.71 3 7 0.43 7 \ 7 1.00 5 7 0.71 25 5 0.71 1 0.20 Control (0 ppm a.i.) Offspring offspring/ Replicate Surv Days Hen/Day 1 5 7 0.71 2 5 7 0.71 3 5 7 0.71 4 5 i 0.71 5 7 7 1.00 Total 27 Mean 5 0.77 SD 1 0.13 Table 11 Surviving Offspring I Hen I Day 1.8 ppm a.i. offspring OfFSpringl Surv Days Hen/Day 6 7 0.86 0 7 0.00 4 7 0.57 6 7 0.86 1 7 0.14 17 3 0.49 3 0.40 6.2 ppm ai, o&phg -rind Surv Days Hemay 5 7 0.71 5 7 0.71 3 7 0.43 2 7 0.29 5 7 0.71 20 4 0.57 1 0.20 17.6ppm ai. HatcW Hatch Days Hen/Day 7 7 1.M 5 7 0.71 7 7 1.00 6 7 0.86 6 7 0.86 31 6 0.89 1 0.12 ~~ ~ ~ 17.6ppm a i . offspring offspringl Surv Days HedDay ~~ 7 7 1.00 3 7 0.43 6 7 0.86 3 7 0.43 6 7 0.86 25 5 0.71 2 0.27 Appendix XII Mean Offspring Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS Mean Hatchling Body Weight (g) Replicate 1 2 3 4 5 Mean SD control (0 ppm a.i.) 5.6 6.2 6.3 6.6 5.6 6.1 0.5 1.8ppm a.i. 5.3 I 6.0 6.0 5.0 5.6 0.5 6.2ppm a.i. 6.6 5.8 5.7 4.9 5.8 5.7 0.6 17.6 ppm a.i. 6.1 5.6 6.7 6.2 5.3 6.0 0.5 Mean Surviving Offspring' Body Weight (g) Replicate 1 2 3 4 5 Mean SD control (0 pprn a.i.) 185 171 160 178 182 175 10 1.8 ppm a.i. 175 -- 174 176 173 175 1 6.2ppm a.i. 181 183 171 181 181 179 5 ' Offspring were approximately 12 weeks of age at final body weight interval. -- No offspringavailable. 17.6ppm a.i. 164 169 175 175 184 173 8 - 120 - Project Number 454-104 Appendix XI11 Adult Liver Weight (g) from a Northern Bobwhite Pilot ReproductionStudy with PFOS Page 1 Pen 20 1 202 203 204 205 Mean SD Control (0 ppm a.i.) Male Liver 3.260 3.156 3.373 4.170 3.051 3.402 0.446 Female Liver 9.210 7.606 7.908 5.612 7.889 7.645 1.295 Pen 206 207 208 209 210 Mean SD 1.8ppm a.i. Male Liver 3.161 3.750 4.211 3.496 2.887 3.501 0.5 14 Female Liver 7.020 8.556 5.744 6.278 4.016 6.323 1.668 - 121 - Project Number 454-104 Appendix XI11 Adult Liver Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS Page 2 Pen 21 1 212 213 214 215 Mean SD 6.2ppm a i . Male Liver 3.552 4.394 4.075 2.591 3.810 3.684 0.686 Female Liver 6.052 5.041 5.404 6.246 5.372 5.623 0.505 ~~ Pen 216 217 218 219 220 Mean SD ~~ 17.6ppm a i . Male Liver 3.056 2.076 2.505 2.625 2.375 2.527 0.359 Female Liver 8.880 4.147 6.768 6.13 1 8.329 6.851 1.880 Appendix XIV Offspring' Liver Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS Control (0 ppm a.i.) Pen Liver 1.8ppm a.i. Pen Liver 6.2 ppm a.i. Pen Liver 20 1 201 202 202 203 203 204 204 205 205 Mean SD 3.905 3.804 3.977 4.125 3.688 2.973 3.612 4.133 3.563 3.680 3.746 0.339 206 206 206 208 208 209 209 209 209 210 Mean SD 3.819 3.300 3.655 4.418 3.45 8 4.128 4.146 3.596 4.095 2.940 3.756 0.453 21 1 21 1 212 212 213 213 214 214 215 215 Mean SD ' Offspring were approximately 12 weeks of age at the time of euthanasia and tissue collection. 3.826 3.533 6.097 4.645 3.899 3.771 7.795 5.996 4.701 5.582 4.985 1.367 17.6ppm a.i. Pen Liver 216 216 217 217 218 218 219 219 220 220 Mean SD 4.370 3.032 2.998 3.050 3.336 3.271 4.281 3.21 1 3.459 3.286 3.429 0.495 - 123 - wild1ijie Intemational, 1:td. Project Number 454-104 Appendix X V Changes to Study Protocol This study was conducted in accordancewith the study protocol signed on February 28,2000 and the following amendments and deviations: 1. The protocol was amended to indicate eggs would be held refrigerated until separated for sampling and eliminated the separation of the shell membrane from the shell. 2. The protocol was amended to reduce the number of eggs collected for analysis from all eggs, to eggs collected during Weeks 1 , 3 and 6 of the test. Eggs collected during Weeks 2 , 4 and 5 will be disposed of by incineration. 3. The protocol was amended to change the test substance purity from 98.9% to 90.49%. Correspondingly, the test concentrations were changed from 0, 2, 7 and 20 ppm a.i to 0, 1.8, 6.4 and 18.3 ppm a.i. 4. The protocol was amended to indicate that for a seven day period beginning March 31 (early Week 5), eggs would be collected daily for incubation, hatching and rearing of offspring. The amendment also detailed the conditions of egg storage, incubation, housing and brooding of hatchlings. Additionally the amendment indicated hatchlings would be uniquely identified and weighed at hatch and at 14 days of age and listed reproductive parameters to be measured. 5. The necropsy section of the protocol was amended to indicate at test termination, samples would be collected from all remaining study adults and from 10 offspring in each test group for histopathological examination. Any remaining tissue not fixed for histopathology would be stored frozen for potential analysis. 6. The protocol was amended to extend the adult portion of the study for the control group and 18.3 ppm a.i. treatment group at least four weeks. Adult birds in the 1.8 and 6.4 ppm a.i. treatment groups will be euthanized at the end of Week 6. During the extension, the number of eggs laid for each pen would be recorded and eggs would be disposed of. Attempts would also be made to collect blood samples from the control group and 18.3 ppm a.i. treatment group birds at the end of Week 6. Adult test birds in the 1.8 and 6.4 ppm a.i. treatment group will be euthanized at the end of Week 6. Additionally, the amendment required collection of feather samples at the time of gross necropsy. 7. The protocol was amended to indicate the raw data and report would be audited by the Quality Assurance Unit and a Good Laboratory Practice compliant final report ,would be prepared. Additionally, the Sponsor's representative was changed to John Newsted, and his address was added. 8. The protocol was amended to indicate analyses of egg, blood and tissue samples will be reported separately. Results of egg, blood and tissue analyses may be amended to the biological results at a later date. - 124 wildlife International, Ltd. Project Number 454-104 Appendix XV Page 2 Changes to Study Protocol 9. The protocol was amended to add statistical analyses of data to determine statistically significant differences between groups. 10. Study offspring were not euthanized, weighed and disposed of at 14 days of age. Instead, offspring were raised to approximately 12 weeks of age prior to being euthanized, weighed, sampled and stored frozen. A protocol amendment was not prepared in a timely manner for this change. 11. The test substance purity changed from 90.49% to 86.9%. Correspondingly, the test concentrations changed from 0, 1.8, 6.4 and 18.3 ppm a.i. to 0, 1.8, 6.2 and 17.6 ppm a i . A protocol amendment was not produced in a timely manner for this change. 12. Additional adult body weight measurements were taken at Weeks 6,8, 10 and 11 of the test. The amendment to extend the test inadvertently did not specify additional body weight measurements. 13. Eggs laid on July 8, 2000 (Day 4 of Week 19) were inadvertently not counted and collected. Instead, eggs laid on July 8,2000 were counted and collected with eggs laid on the following day. - 125 - wildlife International, Ltd. Project Number 454- 104 Appendix XVI Personnel Involved in the Study The following key Wildlife International, Ltd. personnel were involved in the conduct or management of this study: Avian Toxicolom (1) Mark Jaber, Wildlife Toxicologist (2) Joann B. Beavers, Director, Avian Toxicology (3) Linda R. Mitchell, Manager of Ecotox Operations (4) Diana Temple, Laboratory Supervisor ( 5 ) Sean P. Gallagher, Senior Biologist, Avian Toxicology Analytical Chemistry (1) Willard B. Nixon, Ph.D., Director of Chemistry (2) Raymond L. Van Hoven, Ph.D., Scientist, Analytical Chemistry