Document NEGzo7nd6ORZ14zrJJGbEVnmV

AR226-1750 PFOS: A PILOT REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-104 3M LAB REQUEST NO. U2723 FIFRA Guideline 71-4 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.Paul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 125 W ildlife International, Ltd. Project Number 454-104 2- GOOD 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 D IRECTO R: Sean P. Gallagher Senior Biologist, Avian Toxicology SPONSOR'S REPRESENTATIVE: DATE DATE W ildlife 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 DATE CONDUCTED February 28, 2000 March 1,2000 March 7, 2000 January 16,17, 28 and 29,2003 Biological Data, Draft Report January 16,17,20, 28-31,2003 Statistics and Final Report 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 inspections were study based unless otherwise noted. QjjL&aJf\ Go&LroafN Susan L. Coleman, B.A. Senior Quality Assurance Representative |& -1 8 "0 3 DATE 'Wildlife International, Ltd. Project Number 454-104 -4REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Pilot Reproduction Study with the Northern Bobwhite WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-104 3M LAB REQUEST NO.: U2723 STUDY DIRECTOR: P ' ___________ ;_____________ Sean P. Gallagher Senior Biologist, Avian Toxicology CHEMISTRY PRINCIPAL INVESTIGATOR: DATE Scientist, Analytical Chem istry MANAGEMENT: Joaxm B. Beavers Director, Avian Toxicology Willard B. Nixon, Ph Director of Chemistry _____K IftW DATE 1 DATE W ildlife International, Ltd. Project Number 454-104 -5TABLE 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 W ildlife International, Ltd. Project Number 454-104 6- - TABLE OF CONTENTS PAGE 2 TABLES Table 1. Mean Measured Concentrations (ppm a.i.)ofPFOS in Avian Diet from a Northern Bobwhite Pilot Reproduction Study..............................................27 Table 2. Mean Adult Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS............................................................................... 28 Table 3. Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Pilot Reproduction Study with PFOS...................................................... 29 Table 4. Summary of Gross Pathological Observations from a Northern Bobwhite Pilot Reproduction Study with PFOS, Adult Birds Euthanized at 6 Weeks and Test Termination.............................................................................30 Table 5. Mean Egg Production (Eggs Laid/Hen and Eggs/Hen/Day) from a Northern Bobwhite Pilot Reproduction Study with PFOS....................................... 31 Table 6. Summary of Reproductive Performance (Eggs Set from Week 5) from a Northern Bobwhite Pilot Reproduction Study with PFOS.......................... 32 Table 7. Mean Body Weight (g) of Hatchlings and Surviving Offspring from a Northern Bobwhite Pilot Reproduction Study with PFOS.......................... 33 Table 8. Mean Liver Weights (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS......................................................................................................34 Wildlife International, Ltd. Project Number 454-104 -7- TABLE OF CONTENTS PAGE 3 APPENDICES Appendix I. Certificate of Analysis........................................................................................ 35 Appendix II. Diet and Supplement Formulations.................................................................... 38 Appendix III. Diet Preparation..................................................................................................40 Appendix IV. The Analysis of PFOS in Avian D iet.................................................................41 Appendix V. Diagram of Test Layout...................................................................................... 55 Appendix VI. Adult Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS.........................................................................56 Appendix VII. Feed Consumption (g/bird/day) from a Northern Bobwhite Pilot Reproduction Study with PFOS............................................... 60 Appendix VIII. Individual Gross Pathological Observations from a Northern Bobwhite Pilot Reproduction Study with PFOS64...........................64 Appendix IX. Histopathology Report........................................................................................ 68 Appendix X. Egg Production (eggs laid/hen/week) from a N o rth e rn B o b w h ite P ilo t R e p ro d u ctio n S tu d y w ith P F O S ....................................109 Appendix XI. Reproductive Performance by Pen from a Northern Bobwhite Pilot Reproduction Study with PFOS............................. 113 Appendix XII. Mean Offspring Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS...............................................................119 Appendix XIII. Adult Liver Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS........................................................................120 Appendix XIV. 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. 8- SUMMARY 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 (Colinus virginianus) 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 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 w ere 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. W ildlife International, 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 21601. 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 virginianus) of dietary exposure to the potassium salt of Perfluorooctane Sulfonic Acid (hereafter referred 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 (ppm a.i.) (Control) 0 1.8 6.2 17.6 Pens per Group 5 5 5 5 Birds Der Pen Males Females 11 11 11 11 W ildlife 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 from each test concentration. Liver w eights w ere recorded for all necropsied birds. Liver, bile and blood (w hen 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. W ildlife International, 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 PFO S, w as 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: 1H, 1H, 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 fimction in a spreadsheet program was used to randomize pen assignment for each bird. Immediately prior to test initiation, all potential study W ildlife International, 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. Dining 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 II, Table 1). The basal ration contained at least 27% protein and 2.5% fat, and no more than 5% fiber. The basal diet contained approximately 1.1% calcium, derived from feedstuff's and the 0.9% limestone used in the formulation of the basal diet by Agway. While this level of calcium is sufficient for growth and maintenance rations, additional calcium is required in the ration of breeding birds for egg shell formation. Therefore, an additional 5% (w/w) of limestone (approximately 38.5% Ca) was added to the basal diet for the adults. This raised the calcium level in the diet for the breeding birds to approximately 3%, slightly above the minimum recommended for quail (2.3%) and mallard (2.75%) (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 II, Table 2). Neither the adults nor offspring received any form of medication in the feed during the test. Feed and water were analyzed periodically in accordance with Wildlife International, Ltd. Standard Operating Procedures. W ildlife International, 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 a.i.). 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 W ild life In tern atio n al, L td. an d e n titled "A n aly tical M e th o d V e rificatio n fo r the D ete rm in a tio n o f 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 1H, 1H, 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 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer SCIEX API 100LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil Ci8 analytical column (50 mm x 2 mm I.D., 3-pm particle size). The instrument parameters are summarized in Appendix IV, Table 1. Wildlife International, 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)/L 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 L/Kg). The method LOQ for the Week 1, Day 7 and Week 6, DayO analyses and the sample re-extraction set was set at 1.41 ppm a.i., calculated as the product o f the low est standard analyzed (0.000351 m g a.i./L) and the overall dilution factor o f the m atrix blank samples (4000 L/Kg). 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 W ildlife International, 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 fortification is 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 51 cm. The pens had sloping floors that resulted in ceiling height ranging from 20 to 26 cm. The pens were constructed of galvanized wire mesh and galvanized sheeting. A diagram of the test layout is presented in Appendix V. Each pen was equipped with feed and water troughs. Weekly, sufficient feed for the feeding period was placed in the trough for each pen and presented to the birds. During the feeding period additional feed was weighed and added to the troughs as needed. Water troughs were changed and water added as necessary to provide potable water (generally every 2-3 days). Only birds associated with this study were maintained in the study room in order to avoid excessive disturbances. The average temperature in the adult northern bobwhite study room during the course of the test was 22.3 1.1C (SD) with an average relative humidity of 50 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 illumination provided 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 Wildlife International, 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 remaining 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. W ildlife 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 13.1C 0.1C (SD) with a mean relative humidity of approximately 72% 4% (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 0.0C (SD) with an average wet bulb temperature of 30.6 0.1C (SD) (relative humidity of approximately 60%). The incubator was equipped with a pulsator fan and blades that produced a m ild breathing air m ovem ent designed to elim inate 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 constructed of galvanized steel wire mesh were used to keep hatchlings separated by parental pen of origin. Eggs were not rotated in the hatcher. The average temperature in the hatching compartment was 37.2 0.0C (SD), and the average wet bulb temperature was raised to 33.3 0.0C (SD) (relative humidity of approximately 77%). W ildlife International, 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 28.6C 1.0C (SD) with an average relative humidity of 39% 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. W ildlife 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 full 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.123456 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. treatm en t group fo r weeks 8, 10, 11, and 20. 2. Adult Feed Consumption - Feed consumption expressed as grams of feed per bird per day was examined by pen weekly during the test. Statistical comparisons were made between the control and each treatment group 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. Eggs 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 Embryos - The number of live embryos determined at Day 11 by candling. 5. Eggs Cracked o f Eggs Laid - The number of eggs determined by candling to be cracked divided by the number of eggs laid, per pen. 6. Viable Emhrvns o f Eggs Set - The number of embryos at the Day 11 candling was divided by the number of eggs set, per pen. W ildlife International, Ltd. Project Number 454-104 -20 7. Live 3- Week Embryos of Viable Embryos - The number of live embryos at the Day 21 candling was divided by the number of viable embryos, per pen. 8. Hatchlings of 3-Week Embryos - The number of hatchlings removed from the hatcher was divided by the number of live 3-week embryos, per pen. 9. 14-Dav 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 Eees Set - The number of hatchlings was divided by the number of eggs set per week, by pen. 11. 14-Dav 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. Offspring's Body Weight - The group body weights of surviving hatchlings and 14-day old survivors were measured by parental pen group. 13. Liver Weight - 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 0.13 ppm a.i., W ildlife International, Ltd. Project Number 454-104 -21 - 6.0 0.65 ppm a.i. and 17.6 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 TV, 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 0.67 ppm a.i. and 16.8 0.608 ppm a.i., 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 100%, 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. Wildlife 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 differences were 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. A sa 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 10 juvenile 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. W ildlife International, 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-Electrospray/Mass 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 HPLCElectrospray/Mass Spectrometry. Centre Study Number 023-065". Wildlife 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 dining 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 w ere no apparent treatm ent-related effects upon the body w eights o f hatchlings in any o f 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 W ildlife 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 dining 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 treatm ent. Based upon the results o f 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 ildlife International, Ltd. -26REFERENCES Project Number 454-104 1 U.S. Environmental Protection Agency. 1982. Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, subsection 71-4, Environmental Protection Agency, Office of Pesticide Programs. Washington, D.C. 2 American Society for Testing and Materials. 1986. Standard Practice for Conducting Reproductive Studies with Avian Species. ASTM Standard E l062-86. Annual Book of ASTM Standards. Vol. 11.04. Philadelphia, PA. 15 pp. 3 Merck & Co., Inc. 1991. The Merck Veterinary Manual. Merck & Co. Rahway, NJ. 1832 pp. 4 National Research Council. 1996. Guide fo r the Care and Use o f Laboratory Animals. Washington, DC. National Academy 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 6 Dunnett, C.W. 1964. New Tables for Multiple Comparisons with a Control. Biometrics 20: 482 491. Table 1 Mean Measured Concentrations (ppm a.i.) o f PFOS in Avian Diet from a Northern Bobwhite Pilot Reproduction Study Nominal Concentration1 (ppm a.i.) Interval Week 1 Day 0 Day 7 Week 6 Day 0 Project Number 454-104 o 00 Control 1.8 6.2 17.6 Measured <0.879 2 Mean Measured % Nominal Mean Measured % Nominal Mean Measured % Nominal 1.8 100 6.0 97 17.6 100 <1.41 1.8 6.2 103 3 17.2 <1.41 2.0 111 6.0 97 16.8 95 1Nominal concentrations and results o f diet analysis were corrected for the change in test substance purity from 90.49% to 86.9%. 2The limit of quantitation (LOQ) was equivalent to 0.879 ppm a.i. for Week 1, Day 0 and 1.41 ppma.i. for Week 1, Day 7 and Week 6, Day 0. 3The mean percent o f the Day 0 values. Table 2 Mean Adult Body Weight1(g) from a Northern Bobwhite Pilot Reproduction Study with PFOS Experimental Group Sex Week Change Week Change Week Change Week Change Change Week Change Week Change Week Change Week 0 0-2 2 2-4 4 4-6 6 0-6 6-8 8 8-10 10 10-11 11 11-20 20 Change 0-20 Control (0 ppm a.i.) MX SD 214 5 F X 222 SD 14 -1 3 7 3 213 -1 62 229 -3 11 4 1.8 ppm a.i. M X 206 SD 14 F X 221 SD 17 0 3 -1 6 207 4 15 3 221 1 17 7 6.2 ppm a.i. M X 210 SD 13 F X 231 SD 17 -2 2 3 7 208 -1 13 2 234 3 17 6 17.6 ppm a.i. M X 208 -10 * 198 -3 SD 11 4 93 F X 227 SD 16 1 4 227 4 16 4 212 2 61 226 8 14 7 211 1 15 2 222 -10 11 30 207 0 12 2 237 -2 23 8 196 * 7 232 14 0 2 0 6 213 -1 75 234 12 12 6 212 6 14 5 211 -10 36 34 207 -3 11 4 235 4 23 7 196 * -13 * 65 231 5 14 3 -1 212 3 2 62 3 237 -2 5 9 13 __ .. -- -- -- ---- -- -- - _ __ -- -- --- ---- -- -- - 215 -3 212 4 216 52 52 7 2 4 234 -4 230 2 232 10 17 6 12 16 21 10 _ __ -- ---- -- -- _ -- -- ---- -- __ __ -- 1 _ - -- ... -- .. -- -- -- _ -- --- .. -- -- ... __ - -__ -- -- 0 196* -1 * 195 * -2 193 * 4 197 * -11 * 2 72 62 71 75 0 231 4 4 15 2 235 -7 228 -8 220 -7 16 5 16 19 33 24 Project Number 454-104 Mean (X ) 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 from the control group at p<0.05. Table 3 Mean Feed Consumption1 (g/bird/day) from a Northern Bobwhite Pilot Reproduction Study with PFOS E x perim ental Group _______________________________________________________________W eek s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Control M ean 20 22 23 24 24 28 24 32 27 31 31 35 30 35 36 34 30 30 31 0 ppm a.i. SD 3 2 2 2 32 3 4 3 3 4 5 3 5 2 3 2 3 2 1.8 M ean 18 20 20 24 19* 22 * ppm a.i. SD 2 32 2 42-- -- ---- ---- -- --" " " " " 6.2 M ean 18 20 20 21 * 22 23 * ppm a.i. SD 3 3 4 2 3 3 -- -- -- " " " " -- " " ---- 00 # 17.6 M ean 17 18 * 20 * 21 * 22 23 * 21 25 * 26 28 29 31 26 27 ppm a.i. SD 1 1 2 2 2 2 3 4 6 6 5 5 4 5 29 27 24 24 66596 1Mean standard deviation. -- Data not available. Birds euthanized at the end ofWeek 6. * Statistically different from the control group at p<0.05. Project Number 454-104 Number o f birds Feather loss Foot lesions Right wing twisted Thin condition Right testis small, ~ 1.25 cm Right testis small, ~ 1.5 cm Both testes small, ~ 1.5 cm Regressed ovary Not remarkable Table 4 Summary of Gross Pathological Observations from a Northern Bobwhite Pilot Reproduction Study with PFOS Adult Birds Euthanized at 6 Weeks and Test Termination Males - Treatm ent Group (ppm a.i.)_________ Control 1.8 6.2 17.6 55 5 5 00 0 0 00 0 0 00 0 0 00 0 0 00 0 2 00 10 00 10 - - - - 55 3 3 ________ Females - Treatm ent Group (ppm a.i.) Control 1.8 6.2 17.6 5 4 0 1 0 0 1 55 13 10 00 10 ---10 32 5 4 0 0 0 0 1 Project Number 454-104 Table 5 Mean Egg Production 1(Eggs Laid/Hen and Eggs/Hen/Day) From a Northern Bobwhite Pilot Reproduction Study with PFOS Experimental Group ______________________________________________________ W eeks 1 2 3 4 5 6 Total E/H/D12 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total E/H/D2 Control 0 ppm a.i. Total 21 Mean 4 SD 2 25 29 27 27 32 56556 11111 161 32 6 0.77 0.14 30 33 33 28 28 26 32 30 34 32 30 30 31 6776656676666 2 1 13 33 1 10 12 2 2 8 566 2 113 1 12 0.84 0.09 1.8 ppm a.i. Total 22 29 28 27 20 20 Mean 4 6 6 5 4 4 SD 1 1 1 1 2 4 146 29 6 0.70 0.13 ---- __ ___ _ -------------- -- - -- - - - - -- 6.2 ppm a.i. Total 20 24 27 27 27 33 158 M ean 4 5 5 5 5 7 32 SD 2 2 2 1 2 1 7 0.75 0.17 -- _____ ---- ----- _ --- _ - - 17.6 ppm a.i. Total 27 Mean 5 SD 2 27 30 29 33 35 56677 1 1 1 10 181 36 5 0.86 0.13 32 34 35 34 34 34 33 31 28 30 28 25 21 67 7 7777 6 6 6 6 54 10 0 0 00 1 13 3 3 2 4 5 585 1 117 1 20 0.87 0.15 Project Number 454-104 1Total number of eggs laid and mean number of eggs laid per pen standard deviation. 2Eggs 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 (p>0.05) -32- Project N um ber 454-104 Table 6 Summary of Reproductive Performance (Eggs Set from Week 5) from 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 555 29 18 29 000 29 18 29 29 17 * 25 28 17 25 28 17 25 27 17 20 646 534 Eggs Laid/Hen/Day Eggs Cracked/Eggs Laid (%) Viable Embryos/Eggs Set (%) Live 3-Week Embryos/Viable (%) Hatchlings/3-Week Embryos (%) Offspring Survivors/Hatchlings (%) Hatchlings/Eggs Set (%) Offspring Survivors/Eggs Set (%) Hatchlings/Hen/Day Offspring Survivors/Hen/Day 0.83 0 100 97 100 97 97 93 0.80 0.77 * Statistically different from the control group at p<0.05. 0.64 0 95 100 100 100 95 95 0.49 0.49 0.83 0 86 100 100 86 86 72 0.71 0.57 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 Weight1(g) o f Hatchlings and Surviving Offspring from a Northern Bobwhite Pilot Reproduction Study with PFOS Experimental Group Hatchlings Mean SD Surviving Offspring2 Mean SD Control (0 ppm a.i.) 6.1 0.5 175 10 1.8 ppm a.i. 5.6 0.5 175 1 6.2 ppm a.i. 5.7 0.6 179 5 Project Number 454-104 17.6 ppm a.i. 6.0 0.5 173 8 *Mean standard deviation. 2Offspring were approximately 12 weeks of age at final body weight interval. Differences between the control group and the treatment groups were not statistically significant (p>0.05). Experimental Group Control (0 ppm a.i.) Table 8 Mean Liver Weight1(g) from a Northern Bobwhite Pilot Reproduction Study with PFOS Males Liver Mean SD Females Liver Mean SD Offspring2 Liver Mean SD 3.402 0.446 7.645 1.295 3.746 0.339 1.8 ppm a.i. 3.501 0.514 6.323 1.668 3.756 0.453 6.2 ppm a.i. 3.684 0.686 5.623 0.505 4.985 1.367 17.6 ppm a.i. 2.527 0.359 * 6.851 1.880 3.429 0.495 Mean standard deviation. 2 Offspring were approximately 12 weeks o f age at time o f euthanasia and tissue collection. * Statistically different from the control group at p<0.05. 4*. Project Number 454-104 -35- W ildlife International, Ltd, Project Number 454-104 Appendix I Certificate of Analysis Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 wvyw.centrelab.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFO S,Lot217 Reference#: SD-018 ___ ________________ Parity: 86.9%____________________ TestName Specifications Purity1 Result 86.9% Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium2 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) Related Compounds - POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IQ 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate4 Organic Acids5(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis': 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine White Crystalline Powder 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% Conforms Positive 1. 0.005 wt/wt.% 2. 0.001 wtywt.% 3. 1.439 wtVwt.% 4. 6.849 wtywt.% 5. <0.001 wt/wt.% 6. 0.005 wt/wt.% 7. <0.001 w t/w t% 1.91 wtVwt.% 8.41 wtywt.% None Detected 0.33 wtywt.% None Detected Not Applicable1 1. <0.015 w t/w t% 2. 0.59 wtywt.% 3. <0.040 wtywt.% 4. <0.009 w t/w t% 5. <0.006 wtywt.% 6. <0.007 wt/wt.% 7. 8.76 wt/wt.% 1. <0.1 wt/wt.% 2. <0.1 wt/wt.% 3. 0.10 wtywt.% 4. 0.28 wt/wt.% 1. 12.48 wt/wt.% 2. 0.244 wt/wt.% 3. 1.74 wt/wt.% 4. 8.84 wtywt.% 5. 54.1 wtywt.% COA023-018A Page t o f 3 -36- W ildlife International, Ltd. Project Number 454-104 Appendix I Page 2 Certificate of Analysis Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 www.centrelab.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS R evision 3 Centre Analytical Laboratories COA Reference #: 023-018A Date of Last Analysis: 08/31/00 Expiration Date: 08/31/06 Storage Conditions: Frozen <-10"C Re-assessment Date: 08/31/06 'Purity = 100% - (sum o f metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.905%+organic acid impurities, 0.38%+POAA, 0.33%) Total impurity from all tests = 13.07% Purity = 100% -13.07% - 86.9% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials o f low purity. No endotherm was observed for this sample. 4Sulfur in the sample appears to be converted to SO and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity.5 5TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonafluoropentanoic acid Pentafluoropropanoic acid `Theoretical value calculations based on the empirical formula, CsF 17S03_K+(MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-018A Page 2 o f 3 3 7- - W ildlife International, Ltd. Project Number 454-104 Appendix I Page 3 Certificate of Analysis Centre Analytical Laboratories, Inc 3048 Research Drive State Coll ge, PA 16801 www.centrelab.com Phone: (814) 231-8032 Fax; (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A LC/MS Purity Profile: Im purity C4 C5 C6 C7 Total w t/w t % 1.22 1.33 4.72 1.14 8.41 Note: The 0 4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average result from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average result from the C6 and C8 standard curves. Prepared By: Charles Simdfis Scientist, Centre Analytical Laboratories /o/et/o t Date .aLfnfULd,Reviewed By: _____ _ . John Flaherty / 1 ^Laboratory Manager, Centre Analytical Laboratories COA023-018A Page 3 of 3 3 8- - W ildlife International, Ltd, Project Number 454-104 Appendix II Diet and Supplement Formulations Table 1 Wildlife International, Ltd. Game Bird Ration1 INGREDIENTS Fine Com Meal Soy Bean Meal, 48% Protein Wheat Midds Protein Base Agway Special, 60% Protein Alfalfa Meal, 20% Protein Dried Whey Ground Limestone Eastman CalPhos Methionine Premix + Liquid Vitamin and Mineral Premix (see below) GL Ferm (Fermatco)2 Salt Iodized Total PERCENT (%) 44.83 30.65 6.50 6.00 4.00 3.00 2.50 0.90 0.60 0.35 0.32 0.25 0.10 100.00 Vitamin and Mineral Premix, when added at 0.32% o f the ration, supplied the following amounts per ton: Amount Supplied Per Ton: Vitamin D3 Vitamin A Riboflavin Niacin Pantothenic Acid Vitamin B 12 Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (Menadione Dimethylpyriniidinol Bisulfite) Manganese Zinc Copper Iodine Iron Selenium 2.000. 0 0 0 1.C.U. 7.000. 0 0 0 1.U. 6 grams 40 grams 10 grams 8 mgs 600 mgs 64 mgs 1.2 grams 1.2 grams 2 0 ,0 0 0 1.U. 5.8 grams 102 grams 47 grams 6.8 grams 1.5 grams 51 grams 182 mgs 1 The guaranteed analysis is a minimum o f 27% protein, a minimum o f 2.5% crude fat and a maximum o f 5% crude fiber. 2 Fermentation By-Products (Source o f Unidentified Growth Factors). -39- W ildlife International, Ltd. Project Number 454-104 A ppendix II D iet and Supplem ent Form ulations Table 2 V itam ins and Electrolytes Concentrate GUARANTEED ANALYSIS Vitamin A Vitamin D Vitamin E Riboflavin d-Pantothenic Acid Niacin Vitamin B-12 MSBC Folic Acid Thiamine HC1 Pyridoxine Hydrochloride Ascorbic Acid Water Soluble Powder Per 4 oz. 2,5000,000 1,000,000 1,000 750 1,250 2,500 2.5 1,000 65 250 250 3,750 Per lb. 10,000,000 IU 4,000,000 ICU 4,000 IU 3,000 Mg 5,000 Mg 10,000 Mg 10.0 Mg 4,000 Mg 260 Mg 1,000 Mg 1,000 Mg 15,000 Mg IN G R E D IE N T S : Vitamin A Supplement, D-Activated Animal Sterol (source o f Vitamin D3), Alpha Tocopheryl Acetate (source o f Vitamin E). Riboflavin Supplement, d-Calcium Pantothenate, Niacin Supplement, Vitamin B-12 Supplement, Menadione Sodium Bisulfite (source o f Vitamin K), Folic Acid, Thiamine HC1, Pyridoxine Hydrochloride, A scorbic A cid, Sodium C hloride, C alcium Chloride, M agnesium Sulfate, Ferric A m m onium Citrate, Potassium Chloride and Dextrose. MIXING PROCEDURE: The vitamin and electrolyte mix was prepared as a ration of approximately 2 grams o f Durvet vitamins and electrolytes to approximately 1 gallon o f water. - 40- W ildlife International, Ltd. Project Number 454-104 Appendix III 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: No premix required. 1.8 ppm a.i.: 0.3084 g PFOS + 6099.7 gration 6.2 ppm a.i.: 1.0794 g PFOS + 6098.9 gration 17.6 ppm a.i.: 3.0839 g PFOS + 6096.9 gration Basal ration was weighed into a tared Hobart mixing bowl. Approximately 100 g of ration was transferred to a Waring 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 Waring 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 m ixing 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 a.i. : 1000 g Premix + 22.75 kg ration + 1.250 kg limestone 6.2 ppm a.i.: 1000 g Premix + 22.75 kg ration + 1.250 kg limestone 17.6 ppm a.i.: 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 - W ildlife International, Ltd. Project Number 454-104 Appendix IV The Analysis of PFOS in Avian Diet -42- W ildlife International, Ltd. Project Number 454-104 APPENDIX IV ANALYTICAL METHODS AND RESULTS Typical LC/MS Operational Parameters TABLE 1 INSTRUMENT: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer SCIEX API 100LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil C ]8 column (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 - W ildlife International, 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 = peak area ratio - (y-intercept) slope x internal standard cone, (mg a.i./L) Determination of Sample Residues (PFOS1 The concentration, expressed as ppm a.i., for each sample was determined using the following equation: ___ . PFOS (mg a.i./L) at instru. x extract final volume (L) x dilution factor PFOS (ppm a.i.) m sample = initial weight (Kg) Determination of Limit of Quantitation CLOOl 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 volum e (L) x dilution factor overall dilution factor of matrix blank sample = initial w eight (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. ppm a.i. measured in sample % Recovery = ppm a.i. fortified x 100 W ildlife International, Ltd, Project Number 454-104 APPENDIX IV TABLE 3 M atrix Blanks and Fortifications Analyzed Concurrently w ith the Samples Number (454-104-) Sample Type Interval Concentration o f PFOS (ppm a.i.) Fortified1 M easured1,2 Percent Recovery Mean Recovery (X) MAB-1 MAS-1 MAS-2 MAS-3 M A B -2 MAS-4 MAS-5 MAS-6 M A B -3 M A S-7 MAS-8 MAS-9 MAB-4 M A S-10 M A S-11 MAS-12 Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification W eek 1, Day 0 W eek 1, Day 0 W eek 1, Day 0 W eek 1, Day 0 Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification W eek 1, Day 7 W eek 1, D ay 7 W eek 1, Day 7 W eek 1, Day 7 Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification W eek 6, Day 0 W eek 6, D ay 0 W eek 6, Day 0 W eek 6, Day 0 Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Re-extraction Set4 Re-extraction Set4 Re-extraction Set4 Re-extraction Set4 0.00 0.879 8.79 22.0 0.00 1.76 8.79 22.0 0.00 1.76 8.79 22.0 0.00 1.76 8.79 22.0 < 0.879 0.992 8.89 22.0 < 1.41 1.97 9.11 21.3 <1.41 1.98 9.40 22.0 <1.41 1.84 8.86 21.7 113 101 100 _ 112 104 97.0 113 107 100 _ 105 101 98.7 105 104 107 102 1Concentrations 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. 2Less than values correspond to limit o f quantitation (LOQ). 3Results w ere generated using M acQ uan version 1.6 software. M anual calculations m ay differ slightly since fortified and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes. 4 Re-extraction of: sample 454-104,105-13 from Week 1, Day 0; sam ples 454-104-23 and -26 from W eek 1, D ay 7 and sample 454-104,105-50 from W eek 6 , Day 0. W ildlife International, Ltd Project Number 454-104 -45- APPENDIX TV TABLE 4 Homogeneity of PFO S in Avian Diet Nominal Concentration1 (ppm a.i.) Sample I.D. Number (S-454-104-) Location Sampled in Mixing Vessel Measured PFOS Concentration1 (ppm a.i.) M ean(X ) Standard Deviation (SD) Coefficient o f Variation (CV) Mean Percent of Nominal 1.8 3 Top Left 1.70 4 Top Right 1.82 5 Middle Left 1.92 X = 1.8 ppm a.i 100 6 Middle Right 1.73 SD = 0.13 ppm a.i. 7 Bottom Left 1.77 CV = 7.2% 8 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 M iddle Right 5.81 SD 0.65 ppm a.i. 13 Bottom Left 5.872 CV=11% 14 Bottom Right 7.21 17.6 15 Top Left 16.5 16 Top Right 17.7 17 Middle Left 16.5 X = 17.6 ppm a.i. 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 'C oncentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate o f 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. 2M ean result o f duplicate re-extraction o f original sample. -46- W ildlife International, Ltd. Project Number 454-104 Nominal C oncentration12 (ppm a.i.) 0 APPENDIX IV , TABLE 5 Verification of PFOS Concentrations in Avian Diet Sample I.D. Number (S-454-104-) Interval (Day 0 of W eek -) Measured PFOS C o n c e n tr a tio n 1,2 (ppm a.i.) Mean (x ) Standard Deviation (SD) Coefficient o f Variation (CV) Mean Percent of Nominal 1 1 < 0.879 2 1 < 0.879 43 6 < 1.41 44 6 < 1.41 N /A N/A _ 1.8 3-8 1 45 6 46 6 47 6 6.2 9-14 1 48 6 49 6 50 6 - 1.94 1.94 2.10 - 6.31 6.52 5.273 (see Table 4) X =2.0 ppm a.i. SD = 0.092 ppm a.i. CV = 4.6% (see Table 4) x = 6.0 ppm a.i. SD = 0.67 ppm a.i. C V = 11% 100 111 97 97 17.6 15-20 1 51 6 52 6 53 6 - (see Table 4) 100 17.5 x = 16.8 ppm a.i. 95 16.5 SD = 0.608 ppm a.i. 16.4 CV = 3.6% 1Concentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate o f 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. 2Less than values correspond to limit o f quantitation (LOQ). 3Mean result o f duplicate re-extraction o f original sample. _ - Y Wildlife International, Ltd. Project Number 454-104 -Lf- APPENDIX IV TABLE 6 A m bient Stability of PFOS in Avian Diet D uring the N orthern Bobwhite Pilot Reproduction Study Nominal C o n c e n tr a tio n 123 (ppm a.i.) Sample Number (S-454-104-) W eek 1, Day 01 Mean Measured (ppm a.i.) PFOS Concentration Mean Percent of Nominal Sample Number (S-454-104-) W eek 1, Day 7 M ea su re d 2,3 (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 16.2 100 103 98 1D ay 0 values are from homogeneity samples presented in Table 4 and verification samples presented in Table 5. 2Concentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate o f Analysis dated October 11, 2001. concentrations based upon the new test substance purity are 1.8, 6.2 and 17.6 ppm a.i. 3Less than values correspond to limit o f quantitation (LOQ). 4M ean result o f duplicate re-extraction o f original sample. Nominal test -48- W ildlife International, 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. I Weigh 10 g samples of the matrix blank, matrix fortification and test samples into weigh boats and transfer to 8-oz. French square glass bottles. Record weights. 4 For each sample, measure 100 mL of methanol with a graduated cylinder and transfer into the French square bottle. 4 Cap bottles and place on shaker table. Allow the samples to shake for a minimum of 30 minutes at approximately 250 rpm. 4 Vacuum filter with qualitative filter paper and rinse retained feed 3 times with methanol into filtrate. 4 Transfer the filtrate into a 200-mL volumetric flask and bring to volume with methanol. 4 Prepare appropriate dilution(s) to bring final concentration into the calibration range of the 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 4HPFOS/L (internal standard) and 0.05% (v/v) formic acid. 4 Ampulate and submit sample for LCMS analysis. Figure 1. Analytical method flow chart for the analysis of PFOS in avian diet. -49- W illife International, Ltd. APPENDIX IV Project Number 454-104 Figure 2. Typical calibration curve for PFOS. Slope = 2.48679; Intercept --0.07396; r - 0.9985. Curve is weighted (1/x). -50- Wildlife International, Ltd Project Number 454-104 APPENDIX IV intensity: 20000 cps Figure 3. Typical ion chromatogram o f a low-level PFOS standard, 0.000351 mg a.i./L. -51 - W ildlife International, Ltd. Project Number 454-104 APPENDIX IV intensity: 20000 cps Figure 4. Typical ion chromatogram of a high-level PFOS standard, 0.00439 mg a.i./L. -52- "Wildlife International, Ltd. Project Number 454-104 APPENDIX IV intensity: 40000 cps Figure 5. Typical ion chromatogram of a matrix blank sample (454-104-MAB-l). The arrow indicates approximate retention time of PFOS. -53- W ildlife International, Ltd. Project Number 454-104 APPENDIX IV intensity: 20000 cps Figure 6. Typical ion chromatogram of a matrix fortification sample (454-104-MAS-9, 22.0 ppm a.i.). -54- W ildlife International, Ltd. Project Number 454-104 APPENDIX IV intensity: 40000 cps Figure 7. Typical ion chromatogram o f an avian diet sample on Day 0 (S-454-104-3, 1.8 ppm a.i.). -55- W ildlife International, Ltd, Appendix V Diagram of Test Layout Project Number 454-104 | = approx, lm 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 0-20 201 202 203 204 205 Mean SD 212 1 210 -2 217 -6 221 1 209 0 214 -1 53 213 0 208 -4 211 0 222 -1 209 0 213 -1 62 213 3 204 0 211 1 221 1 209 3 212 2 61 216 4 204 -6 212 -5 222 1 212 3 213 -1 75 -3 213 3 216 -6 210 1 211 -1 1 205 6 211 -1 210 5 215 5 -3 209 4 213 -3 210 5 215 -2 -1 221 2 223 -2 221 6 227 6 -1 211 1 212 -3 209 2 211 2 -1 212 3 215 -3 212 4 216 2 , 2 62 52 52 7 A Ul ^ On Project Number 454-104 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 0-20 201 202 203 204 205 Mean SD 235 5 199 11 222 9 225 3 229 5 222 7 14 3 240 -1 210 -5 231 -8 228 -2 234 3 229 -3 11 4 239 12 205 13 223 13 226 3 237 -1 226 8 14 7 251 16 218 19 236 14 229 4 236 7 234 12 12 6 -3 248 7 255 -9 246 17 263 28 6 224 1 225 0 225 -20 205 6 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 3 237 -2 234 -4 230 2 232 10 5 9 13 17 6 12 16 21 10 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 0-6 206 207 208 209 210 Mean SD 194 -1 210 6 228 0 204 -1 196 -2 206 0 14 3 193 7 216 2 228 4 203 6 194 1 207 4 15 3 200 4 218 2 232 -2 209 2 195 0 211 1 15 2 204 10 220 10 230 2 211 7 195 -1 212 6 14 5 Project Number 454-104 1.8 ppm a.i. - Fem ales 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 4 221 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 0-6 211 212 213 214 215 Mean SD 194 -2 205 -3 222 1 204 -1 225 -4 210 -2 13 2 192 1 202 -1 223 0 203 1 221 -5 208 -1 13 2 193 3 201 0 223 -3 204 -1 216 1 207 0 12 2 196 2 201 -4 220 -2 203 -1 217 -8 207 -3 11 4 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 0-6 211 212 213 214 215 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 changes are calculated and rounded separately. K0J0i Project Number 454-104 Appendix VI Table 4 Adult Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS 17.6 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 0-20 216 217 218 219 220 Mean SD 211 -13 194 -7 208 -12 204 -5 224 -12 208 -10 11 4 198 -5 187 1 196 1 199 -5 212 -6 198 -3 93 193 3 188 0 197 -2 194 0 206 -1 196 0 72 196 -15 188 -6 195 -13 194 -10 205 -19 196 -13 65 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 0 196 -1 195 -2 193 4 197 -11 2 72 62 7 1 7 5 Project Number 454-104 17.6 ppm a.i. - Fem ales 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 0-20 216 217 218 219 220 Mean SD 214 5 220 1 248 3 213 -2 239 -4 227 1 16 4 219 7 221 -1 251 2 211 9 235 4 227 4 16 4 226 -9 220 8 253 -2 220 1 239 1 232 0 14 6 217 3 228 8 251 3 221 8 240 1 231 5 14 3 -1 216 1 -7 221 6 1 252 6 4 225 5 1 241 4 217 -8 227 -6 258 -4 230 -2 245 -16 209 -25 221 -33 254 6 228 5 229 7 184 -30 188 -32 260 12 233 20 236 -3 0 231 4 235 -7 228 -8 220 -7 4 15 2 16 5 16 19 33 24 The means for body weights and body weight changes are calculated and rounded separately. Appendix VII Table 1 Feed Consumption (g/bird/day) from a Northern Bobwhite Pilot Reproduction Study with PFOS Control (0 ppm a.i.) Weeks Pen 1 2 3 4 5 6 7 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 M ean 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 Project Number 454-104 Appendix VII Table 2 Feed Consumption (g/bird/day) from 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 Mean 18 20 20 24 19 22 SD 2 3 2 2 4 2 Project Number 454-104 Appendix VII Table 3 Feed Consumption (g/bird/day) from a Northern Bobwhite Pilot Reproduction Study with PFOS 6.2 ppm a.i. Weeks Pen 1 2 3 4 5 6 211 20 19 18 22 21 22 212 14 15 15 17 19 19 213 18 23 25 24 24 26 214 18 20 21 20 21 23 215 22 22 21 21 25 26 Mean 18 20 20 21 22 23 SD 3 3 4 2 3 3 Project Number 454-104 Appendix VII Table 4 Feed Consumption (g/bird/day) from a Northern Bobwhite Pilot Reproduction Study with PFOS 17.6 ppm a.i. Weeks Pen 1 2 3 4. 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 216 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 17 218 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 M ean 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 Project Number 454-104 -64- Project Number 454-104 Appendix VIII 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.) Pens 201 202 203 204 205 XXXXX Females Feather loss Right wing twisted Not remarkable Pens 201 202 203 204 205 - XXXX - - - -X X"- - - -65- Project Number 454-104 Appendix VIII 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 ppma.i. Pens 206 207 208 209 210 XXXXX Feather loss Foot lesions Thin condition Regressed ovary Not remarkable Females Pens 206 207 208 209 210 _X __ - - - -X - - - -X - - - -X X - XX - -66- Project Number 454-104 Appendix VIII Table 3 Individual Gross Pathological Observations from a Northern Bobwhite Pilot Reproduction Study with PFOS Birds Euthanized at 6 Weeks Males Testes small, ~ 1.5 cm Right testis sm all, ~ 1.5 cm Not remarkable 6.2 ppm a.i. _________________ Pens_______________ 211 212 213 214 215 - -X -- X - - -- -X - XX Feather loss Not remarkable Females Pens 211 212 213 214 215 XX _X _ - -X -X -67- Project Number 454-104 Appendix VIII 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 - XX Feather loss Not remarkable Females Pens 216 217 218 219 220 XX . XX - -X - " -68- W ildlife International, Ltd. A ppendix IX H istopathology Report Project N um ber 454-104 EPL -69- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 W ILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER 454-104 EPL PROJECT NUMBER 212-024 PFOS: A PILO T REPRODUCTIO N STUDY W ITH TH E NO RTHERN BO BW HITE PATHOLOGY REPORT Subm itted by: Experim ental Pathology Laboratories, Inc. P.O. Box 474 Herndon, V A 20172-0474 (703) 471-7060 Subm itted to: W ildlife International, Ltd. Easton, MD 21601 January 25, 2001 -70- Project Number 454-104 Ce p ______ ______________________________________________________________ EXPERIMENTAL PATHOLOGY LABORATORIES, INC. TABLE OF CONTENTS Page PATHOLOGY SUMMARY.................................................................................... 1 QUALITY ASSURANCE FINAL CERTIFICATION............................................. 8 ADULT SACRIFICE SUMMARY INCIDENCE TABLES Males................................................................................................................ 1-1 Females................................................................................................ I-3 HISTOPATHOLOGY INCIDENCE TABLES Males................................................................................................................ 11-1 Females............................................................................................................ II-3 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Males................................................................................................................ 111-1 Females............................................................................................................ III-2 OFFSPRING SACRIFICE SUMMARY INCIDENCE TABLES Males......................................... IV-1 Females........................................................................................................... IV-2 HISTOPATHOLOGY INCIDENCE TABLES Males................................................................................................................ V-1 Females........................................................................................................... V-2 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Males........................................... .................................................................... VI-1 F e m a le s .................................................................................................................................. V I-5 -71 - Project Number 454-104 PATHOLOGY SUMMARY EPL -72- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 W ILDLIFE INTERNATIONAL, LTD. STUDY NUMBER 454-104 EPL PROJECT NUMBER 212-024 PFOS: A PILOT REPRODUCTION STUDY W ITH THE NORTHERN BOBW HITE PATHOLOGY SUMMARY Light m icroscopic exam ination was perform ed on sections o f selected tissu e s fro m adult m ale and fem a le N orthern bobw hite (Colinus virginianus) w hich w ere untreated or which received various concentrations o f the te st article (P erfluo ro o cta n e su lfo n ic acid, potassium salt [PFO S]) in the feed fo r six to 19 weeks. Selected tissues from untreated approxim ately 12-week-old offspring of the adult bobw hite w ere also exam ined by light m icroscopy. The objective of this s tu d y is to eva lu a te th e effects upon adult bobw hite (Colinus virginianus) o f dietary exposure to a test substance over at least a six-week period. The experim ental design w as as follows: Group * Control (0 ppm a. i.) 1.8 ppm a. i. P F O S 6 .2 ppm a. i. P F O S 17.6 ppm a. i. P F O S A d u lts M a le s F e m ales 55 55 55 55 O ffspring ** M a le s Fem ales 55 46 55 28 * B obw hite in the control and 17.6 ppm a.i. groups were treated fo r 19 w eeks; bobw hite in the 1.8 and 6.2 ppm a.i. groups w ere treated fo r six w eeks. ** O ffspring in all groups did not receive the test article. EPL -73- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 Wildlife International, Ltd. Study Number 454-104 MATERIALS AND METHODS A t the com pletion of the various study intervals, all adult bobwhite were euthanized, and necropsies w ere perform ed by W ildlife International, Ltd. Selected offspring w ere euthanized at approxim ately 12 weeks of age using carbon dioxide gas, and sam ples were collected for histopathologlcal evaluation by W ildlife International, Ltd. Selected tissues from adults and offspring w ere fixed in 10% neutral buffered form alin and sent to Experim ental Pathology Laboratories, Inc. (EPL), w here they w ere em bedded in paraffin and m ade into hem atoxylin and eosin-stained sections on glass m icroscope slides. The follow ing tissues from adults and offspring were exam ined by light m icroscopy as available: liver, gallbladder, proventriculus, kidney, brain, gonad (ovary or testis), bursa o f Fabricius, and adipose tissue. In som e cases, nonprotocol-required tissues w ere sectioned along with adjacent protocol-required tissues; these w ere evaluated, and m icroscopic findings were recorded at the discretion of the p a th o lo g is t. A ll tissue s required by protocol are presented in the H istopathology Incidence Tables. M icroscopic findings for each tissue exam ined from each b o b w h ite a re listed in th e H isto p a th o lo g y Incid ence Tab les. M icroscop ic chan ges w ere graded one to five depending on severity. Nongradable findings are listed as present (P) and tissues with extensive autolysis are listed as (A). All findings fo r all anim als are sum m arized by sex, age group, and trea tm ent group in the S u m m a ry Incid ence T ab les, to g e th e r w ith the to ta l n u m b e r o f an im a ls in each group fo r which the tissues were exam ined. A tabulation of gross observations noted at necropsy with the corresponding m icroscopic change, if appropriate, is presented in the C orrelation o f G ross and M icroscopic Findings tables. 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 G ross Necropsy records provided by W ildlife International, Ltd. RESULTS All adults and offspring survived to the end of their respective study intervals. No effects considered possibly related to test article (PFOS) adm inistration w ere noted in liver, kidney, adipose tissue, gallbladder, brain, bursa o f Fabricius, proventriculus, and ovary of m ale and fem ale adult bobwhite o r th e ir o ffsp rin g , o r in th e te ste s o f offsp ring m ales. T estes o f all adult m ales exhibited sperm atogenesis characterized by the presen ce o f num erous m ature sperm atozoa in the sem iniferous tubules. In tw o 17.6 ppm a.i. m ales, sem iniferous tubules had m arginally decreased diam ete r (though sperm atogenesis w as still evident) com pared to other control and 17.6 ppm a.i. testes, resulting in overall sm aller testes w hich corresponded to grossly observed 1.25 cm right testes. The sm aller testes o f these birds w ere m ost consistent with early post-reproductive phase regression, a norm al physiological phenom enon. T he occurrence o f such te sticu la r regression only in the 17.6 ppm a.i. dose group m ay have been a fortuitous even t accentuated by the sm all group sizes, but the possibility o f a test article effect cannot be entirely ruled out. Testes o f all offspring w ere listed as im m ature because they lacked all evidence of sperm atogenesis, had sm all sem iniferous tubules with absent or narrow lum ens, and had germ inal epithelium with decreased cell layers (com pared to adult testes). This m orphology was consistent with normal p h ysio lo g ica l im m a tu rity in young (a pproxim a tely 12-w eek-old) birds ra th e r th a n a pathological effect. The overall sm all diam eter of offspring testes corresponded to m easurem ents of less than 1.5 cm recorded at necropsy. Because o f the -3- EPL -75- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 Wildlife International, Ltd. Study Number 454-104 sm aller size of the sem iniferous tubules in offspring testes, the m elanin pigm ent bearing cells norm ally present in the te sticular interstitium w ere in effect m ore c lo se ly packe d in a s m a lle r area, ra th e r th a n being w id e ly se p a ra te d by larger sem iniferous tubules as in adult testes. This m elanin pigm ent concentration corresponded to the grossly observed black offspring testes, but was considered a norm al age-related finding and not a pathological effect. All ovaries exam ined in offspring fem ales w ere characterized by num erous oocytes and very small, nonpedunculated follicles com pletely em bedded in a prom inent ovarian cortex. This m orp hology is consistent with norm al physiological im m aturity in young (approxim ately 12-w eek-old) birds rather than a pathological effect. Several additional m icroscopic findings (described below ) w ere noted in adult and/or offspring bobwhite from control and treated groups. Incidences and m ean severity were sim ilar when groups from each generational cohort were com pared with each other. Som e o f these lesions occurred only in a single bird. For these reasons, these findings w ere considered incidental and unrelated to test article (PFOS) adm inistration. M ononuclear cell infiltrates in various tissue s consisted o f discrete foci of sm all lym phocytes and plasm a cells; a few heterophils w ere som etim es also present but w ere always a m inor com ponent. C hronic and chronic active inflam m ation of adipose tissue w ere characterized by infiltrates consisting exclusively or predom inantly of m acrophages, o r m ixed populations of heterophils and m arophages, respectively. Scattered necrotic adipocytes were associated with chronic active inflam m ation in one 6.2 ppm a.i. offspring m ale. A sm all focus o f cortical tubules exhibited cytoplasm ic vacuolization in the kidney o f one adult 17.6 ppm a.i. fem ale; this change in a single bird w as considered -4- EPL 7 6- - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 Wildlife International, Ltd. Study Number 454-104 incidental. S cattered clusters of coarsely to finely dark basophilic m aterial in the renal cortex of a few adults and offspring denoted m ineralization. O ne control adult fem ale exhibited foreign m aterial and granulom atous in fla m m a tio n in se ve ra l tissu e s including the serosa of the ovary, liver, and gallbladder, and in the adipose tissue. G ranulom atous inflam m ation characterized by sm all m ononuclear cells, m acrophages, and occasional m ultinucleated giant cells was associated with foci of foreign m aterial (deep m agenta globules and bright pink am orphous material consistent with extrafollicular egg yolk). Collectively, these findings were consistent with the com m on clinical condition usually referred to as "egg yolk peritonitis." Liver pigm ent deposition consisted of dust-like to coarsely irregular, brown to golden-brow n pigm ent granules present in hepatoeyte cytoplasm , K upffer cells, and/or periportal areas; the identity of the pigm ent w as not determ ined, but m ay have been related to bile. Small single clusters of hyperp lastic bile ducts w ere noted in one adult control fem ale and one 1.8 ppm a.i. offspring fem ale. A sm all focus o f heterophils (acute inflam m ation) w as noted in one 1.8 ppm a.i offspring male. T he liver o f an adult 17.6 ppm a.i. fem ale exhibited am yloid deposition associated with secondary hepatocellular hypertrophy and necrosis. Liver am yloid deposits consisted of am orphous, pale bluish-violet m aterial w hich filled and distended the hepatic sinusoids, and com pressed adjacent hepatic cords. Additional liver lesions included hepatocellular fatty change and vacuolization. Fatty change denoted variably sized, sharply dem arcated, clear vacuoles (consistent w ith fat accum ulation) in the hepatocellular cytoplasm . V acuolization denoted multiple, usually small, confluent, poorly dem arcated vacuoles Which often resulted in a "lace-like" appearance of the hepatocellular cytoplasm . W h e n present in m ost hepatocytes, these changes w e re designated -5- EPL 7 7- - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 Wildlife International, Ltd. Study Number 454-104 "diffu se ," and w hen present in a fe w scattered areas, they w e re designated as "fo c a l." W hen hepatocellular fa tty change occurred predom inantly in the areas a d ja c e n t to bile chan n e ls a n d a ssociated blood vessels, it w a s d esign ate d as " p e rip o rta l." H epatocellular fatty ch ange w as noted in the livers o f adults and offspring, w hile hepatocellular vacuolization w as seen only in offspring. This generational difference was considered to be due to normal age-related variation rather than being a treatm ent effect. W hen control and treated groups o f each generational cohort were com pared, com bined incidences of diffuse, focal, and periportal fatty change in m ale and fem ale adults and offspring w ere sim ilar. H epatocellular fatty change was considered incidental and unrelated to treatm ent. There was a relative increase in incidence o f hepatocellular vacuolization in fem ale offspring, b u t th e m a g n itu d e w a s sm all, and th e incre ase w as not c le a rly dose -related. It is m ost likely that this finding w as coincidental and not related to test article adm inistration. CONCLUSIONS AND SUMMARY No lesions considered possibly related to test article (PFOS) adm inistration w ere noted in liver, kidney, adipose tissue, proventriculus, gallbladder, ovary, and brain o f adult m ale and fem ale bobwhite or their offspring, o r in th e testes o f offspring bobw hite. Testes o f tw o high dose (17.6 ppm a.i.) adult m ale bobwhite exhibited decreased sem iniferous tubule diam eter m ost consistent with early post-reproductive phase regression, a normal physiological phenom enon. The occurrence o f testicular regression only in the 17.6 ppm a.i. dose group m ay have been a fortuitous event accentuated by the sm all group sizes, but the possibility o f a test article effect cannot be entirely ruled out. The -6- -78- EPL EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 Wildlife International, Ltd. Study Number 454-104 few o ther changes in various tissues of adult and/or offspring bobw hite from control and treated groups were considered incidental and unrelated to test article (PFO S) adm inistration. M M G /lcp W , bv M AR G AR ITA M. GRUEBBEL, DVM, PhD, Diplom ate, AC VP Veterinary Pathologist jifl bi -7- EPL -79- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 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 Experimental Pathology Laboratories, Inc. Dates inspections were performed and findings reported to the EPL Project Coordinator and Management are indicated below. Area Inspected Inspection Dates Reportinq EPL Project Sheets Project Setup Histology Setup Data Review Draft Report Final Report 9/11/00; 10/3/00 9/27/00 10/2/00 11/13/00 12/12,27/00 1/29/01 9/11/00; 10/3/00 9/28/00 10/2/00 11/13/00 12/13,27/00 1/29/01 D a te o f la s t Q u a rterly fa cilitv in sD e ctio n )0 , / (U \ jlc Lo u * - EPL Quality Assurance Unit ________ SZQQ_________________ Date ilz q jo t Form No. 6-2 (7/2/99) -8- -80- Project Number 454-104 SUM M ARY INCIDENCE TABLES AD U LT SACRIFICE 454-104 Adult Sacrifice Male Bobwhite -81 SUMMARY INCIDENCE TABLE ADIPOSE TISSUE (NO. EXAMINED) Foreign Material Infiltrate, Mononuclear Cell Inflammation, Chronic Inflammation, Granulomatous BRAIN (NO. EXAMINED) BURSA OF FABRICIUS (NO. EXAMINED) CLOACA (NO. EXAMINED) GALLBLADDER (NO. EXAMINED) Infiltrate, Mononuclear Cell Serosa, Foreign Material Serosa, Inflammation, Granulomatous KIDNEY (NO. EXAMINED) Infiltrate, Mononuclear Cell Tubules, Mineralization Tubules, Vacuolization LIVER (NO. EXAMINED) Amyloid Deposition Bile Ducts, Hyperplasia Hepatocytes, Fatty Change, Diffuse Hepatocytes, Fatty Change, Focal Hepatocytes, Fatty Change, Periportal Hepatocytes, Hypertrophy, Diffuse Hepatocytes, Necrosis, Focal Infiltrate, Mononuclear Cell Pigment Deposition Serosa, Foreign Material Serosa, Inflammation, Granulomatous PROVENTRICULUS (NO. EXAMINED) Infiltrate, Mononuclear Cell GROUP CONTROL (5) GROUP 17.6 (5) (5) (5) (2) (5) (4) 33 (5) (5) 2 21 (5) (5) 22 12 1 53 22 (5) (5) 34 1-1 EPL Experimental Pathology Laboratories, Inc. Project Number 454-104 454-104 Adult Sacrifice Male Bobwhlte -82- SUMMARY INCIDENCE TABLE TESTIS (NO. EXAMINED) Infiltrate, Mononuclear Cell Seminiferous Tubules, Decreased Diameter Spermatogenesis GROUP CONTROL (5) 5 GROUP 17.6 (5) 1 2 5 Project Number 454-104 1-2 EPL Experimental Pathology Laboratories, Inc. 454-104 Adult Sacrifice Female Bobwhite -83- SUMMARY INCIDENCE TABLE ADIPOSE TISSUE (NO. EXAMINED) Foreign Material Infiltrate, Mononuclear Cell Inflammation, Chronic Inflammation, Granulomatous BRAIN (NO. EXAMINED) BURSA OF FABRICIUS (NO. EXAMINED) CLOACA (NO. EXAMINED) GALLBLADDER (NO. EXAMINED) Infiltrate, Mononuclear Cell Serosa, Foreign Material Serosa, Inflammation, Granulomatous KIDNEY (NO. EXAMINED) Infiltrate, Mononuclear Cell Tubules, Mineralization Tubules, Vacuolization LIVER (NO. EXAMINED) Amyloid Deposition Bile Ducts, Hyperplasia HepatoCytes, Fatty Change, Diffuse Hpatocytes, Fatty Change, Focal Hepatocytes, Fatty Change, Periportal Hepatocytes, Hypertrophy, Diffuse Hepatocytes, Necrosis, Focal Infiltrate, Mononuclear Cell Pigment Deposition Serosa, Foreign Material Serosa, Inflammation, Granulomatous OVARY (NO. EXAMINED) Serosa, Foreign Material GROUP CONTROL (4) 1 1 (5) GROUP 17.6. (4) 2 1 (5) (2) (5) (5) 14 1 1 (5) (5) 11 12 1 (5) (5) 1 1 3 41 1 22 '3 3 1 1 (5) (5) 1 1-3 EPL Experimental Pathology Laboratories, Inc. Project Number 454-104 1 454-104 Adult Sacrifice Female Bobwhite -84- SUMMARY INCIDENCE TABLE OVARY (CONTINUED) Serosa, Inflammation, Granulomatous PROVENTRICULUS (NO. EXAMINED) Infiltrate, Mononuclear Cell GROUP CONTROL GROUP 17.6 1 (5) (5) 34 Project Number 454-104 \ 1-4 E PL Experimental Pathology Laboratories, Inc. -85- Project Number 454-104 HISTO PATHOLO GY INCIDENCE TABLES A D U LT SACRIFICE -86- Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 17.6 454-104 Adult Sacrifice Male Bobwhite * N i M A i ADIPOSE TISSUE Foreign Material Infiltrate, Mononuclear Cell Inflammation, Chronic Inflammation, Granulomatous 44444 55555 13579 XXXXX 44444 88888 13579 XXXXX BRAIN XXXXX XXXXX BURSA OF FABRICIUS NNNNN NNNNN CLOACA XX GALLBLADDER Infiltrate, Mononuclear Cell Serosa, Foreign Material Serosa, Inflammation, Granulomatous XX 21 2 NX 112 KIDNEY Infiltrate, Mononuclear Cell Tubules, Mineralization Tubules, Vacuolization XX X 11 XX 11 1 LIVER Amyloid Deposition Bile Ducts, Hyperplasia Hepatocytes, Fatty Change, Diffuse Hepatocytes, Fatty Change, Focal Hepatocytes, Fatty Change, Periportal Hepatocytes, Hypertrophy, Diffuse Hepatocytes, Necrosis, Focal Infiltrate, Mononuclear Cell Pigment Deposition Serosa, Foreign Material Serosa, Inflammation, Granulomatous 22 1 4 11111 22 | X 2 1 2 1 1 1 11 1 E PL Experimental Pathology Laboratories, Inc. II-l Key : X-Not Remarkable N*No Section I-Incoaplete A-Autolysls 1-Blnlmal 2-$11ght/ni1ld Smoderate 4-eoderately severe 5-severe/h1gh Represent BBen1gn KHal1gnant Bisslng one paired organ u*unschedu1ed sac./death 8 7- - Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 17.6 454-104 Adult Sacrifice Male Bobwhite A N i M A i PROVENTRICULUS Infiltrate, Mononuclear Cell 44444 55555 13579 XX 1 11 44444 88888 13579 X 111 1 TESTIS Infiltrate, Mononuclear Cell Seminiferous Tubules, Decreased Diameter Spermatogenesis mmmmm PpppP mmmmm i ii pppPp EPL Experimental Pathology Laboratories, Inc. II-2 Key : X-Not Remarkable N-No Section I-lncoaplete A-Autolysis li#1n1ial 2-s11flht/m1ld 3-soderate 4-moderately severe 5-severe/h1gh PPresent B-Ben1gn M-MaUgnant -missing one paired organ u-unscheduled sac./death -88- Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 17.6 454-104 Adult Sacrifice Female Bobwhite A N l M A L ADIPOSE TISSUE Foreign Material Infiltrate, Mononuclear Cell Inflammation, Chronic Inflammation, Granulomatous 44444 55556 24680 XXN X P 1 44444 88889 24680 NXX 11 1 BRAIN XXXXX XXXXX BURSA OF FABRICIUS NNNNN NNNNN CLOACA XX GALLBLADDER Infiltrate, Mononuclear Cell Serosa, Foreign Material Serosa, Inflammation, Granulomatous XX X 1 P 2 X 1221 KIDNEY Infiltrate, Mononuclear Cell Tubules, Mineralization Tubules, Vacuolization XXX 1 1 XX 1 11 1 LIVER Amyloid Deposition Bile Ducts, Hyperplasia Hepatocytes, Fatty Change, Diffuse Hepatocytes, Fatty Change, Focal Hepatocytes, Fatty Change, Periportal Hepatocytes, Hypertrophy, Diffuse Hepatocytes, Necrosis, Focal Infiltrate, Mononuclear Cell Pigment Deposition Serosa, Foreign Material Serosa, Inflammation, Granulomatous 1 333 2 11 11 1 P 1 4 23 3 3 2 11 3 1 1 I EPL Experimental Pathology Laboratories, Inc. II-3 Key sX-Not Remarkable N-No Section I-Incomptete AAutolys1s I*a1n1ia1 2s1Ight/nMId 3"*oderate 4-noderately severe 5*$evere/h1gh PPre$ent 8-Benlgn H*Na11gnant *i1s$1ng one paired organ "unscheduled sac./death -89- Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 17.6 4 5 4 -1 0 4 Adult Sacrifice Female Bobwhite A N i M A t 44444 55556 OVARY 24680 XXX X Serosa, Foreign Material P Serosa, Inflammation, Granulomatous 1 e- co ** 444 889 260 XXXXX O- CO CO PROVENTRICULUS Infiltrate, Mononuclear Cell XX 1 11 X 1 111 EPL 1 1 -4 Experimental Pathology Laboratories, Inc. Key : X-Not Remarkable N-No Section Wncomplete AAutolys1s l-m1n1mal 2-sl1ght/m1 Id 3-moderate 4-moderately severe 5*$evere/h1gh P-Present B-Benlgn MMal1gnant m>m1ss1ng one paired organ u-unscheduled sac./death -90- Project Number 454-104 CO RR ELATIO N OF G RO SS AN D M IC RO SCO PIC FINDINGS A D U LT SACRIFICE 454-104 Adult Sacrifice Species: Bobwhite CORRELATION O F GRO SS AND M ICROSCOPIC FINDINGS Sex: Males Group Identification: 17.6 - Sacrificed Animal Number 481 ClientTopography/Site ESTIS 485 TESTIS ClientGross Observations light 1.25 cm Right "1.25 cm Microscopic Observations Seminiferous Tubules, Decreased Diameter Seminiferous Tubules, Decreased Diameter - Project Number 454-104 III-l 454-104 Adult Sacrifice Species: Bobwhite CORRELATION O F G RO SS AND M ICROSCOPIC FINDINGS Sex: Females Group Identification: CONTROL - Sacrificed Animal Number 454 456 458 460 ClientTopography/Site EXTERNAL EXTERNAL EXTERNAL EXTERNAL EXTERNAL ClientGross Observations leather loss neck Feather loss neck Feather loss neck Right wing twisted Feather loss neck and head Microscopic Observations So Comment Required So Comment Required So Comment Required No Comment Required No Comment Required. Project Number 454-104 IX I-2 454-104 Adult Sacrifice Species: Bobwhite CORRELATION OF GRO SS AND M ICROSCOPIC FINDINGS Sex: Females Group Identification: 17.6 - Sacrificed Animal Number 482 484 488 490 ClientTopography/Site EXTERNAL EXTERNAL EXTERNAL EXTERNAL ClientGross Observations Feather loss peck and back Feather loss neck and back Feather loss head and neck Feather loss neck and back Microscopic Observations No Comment Required No Comment Required No Comment Required No Comment Required Project Number 454-104 III-3 -94- Project Number 454-104 SUM M ARY INCIDENCE TABLES O FFSPRING SACRIFICE 454-104 Offspring Sacrifice Hale Bobwhite -95SUMMARY INCIDENCE TABLE Project Number 454-104 ADIPOSE TISSUE (NO. EXAMINED) Infiltrate, Mononuclear Cell Inflammation, Chronic Inflammation, Chronic Active Mineralization Necrosis BRAIN (NO. EXAMINED) BURSA OF FABRICIUS (NO. EXAMINED) CLOACA (NO. EXAMINED) 3ALLBLADDER (NO. EXAMINED) Infiltrate, Mononuclear Cell KIDNEY (NO. EXAMINED) Infiltrate, Mononuclear Cell Tubules, Mineralization LIVER (NO. EXAMINED) Bile Ducts, Hyperplasia Hepatocytes, Fatty Change, Diffuse Hepatocytes, Fatty Change, Focal Hepatocytes, Fatty Change, Periportal Hepatocytes, Vacuolization, Diffuse Infiltrate, Mononuclear Cell Inflammation, Acute PROVENTRICULUS (NO. EXAMINED) Infiltrate, Mononuclear Cell TESTIS (NO. EXAMINED) Immature Infiltrate, Mononuclear Cell Pigment Concentration GROUP CONTROL (5) GROUP 1.8 (4) 1 (5) (4) (4) (4) (5) (4) 1 (5) (4) 1 11 (5) (4) 31 1 3 33 1 (5) (4) 44 (5) (4) 54 1 54 GROUP 6.2 (5) 1 1 (5) (4) (1) (5) 1 (5) 1 (5) 3 2 5 (5) 5 (5) 5 2 5 GROUP 17.6 (2) (2) (1) (1) (2) (2) (2) 1 1 (2) 1 (2) 2 2 IV-1 E PL Experimental Pathology Laboratories, Inc. 454-104 Offspring Sacrifice Female Bobwhite -96- SUMMARY INCIDENCE TABLE Project Number 454-104 ADIPOSE TISSUE (NO. EXAMINED) Infiltrate, Mononuclear Cell Inflammation, Chronic Inflammation, Chronic Active Mineralization Necrosis BRAIN (NO. EXAMINED) BURSA OF FABRICIUS (NO. EXAMINED) CLOACA (NO. EXAMINED) GALLBLADDER (NO. EXAMINED) Infiltrate, Mononuclear Cell KIDNEY (NO. EXAMINED) Infiltrate, Mononuclear Cell Tubules, Mineralization LIVER (NO. EXAMINED) Bile Ducts, Hyperplasia Hepatocytes, Fatty Change, Diffuse Hepatocytes, Fatty Change, Focal Hepatocytes, Fatty Change, Periportal Hepatocytes, Vacuolization, Diffuse Infiltrate, Mononuclear Cell Inflammation, Acute DVARY (NO. EXAMINED) Immature PROVENTRICULUS (NO. EXAMINED) Infiltrate, Mononuclear Cell GROUP CONTROL (4) 2 1 GROUP 1.8 (6) (5) (6) (4) (6) (1) (5) (6) 12 (5) (6) 13 2 (5) (6) 1 11 23 11 23 (3) (5) 35 (5) (6) 56 GROUP 6.2 (5) 1 (5) (3) (3) 1 (5) 1 (5) 1 3 2 (5) 5 (5) 5 GROUP 17.6 (8) 2 (8) (8) (8) 3 (8) 3 1 (8) 1 1 1 4 4 (8) 8 (8) 8 IV-2 EPL Experimental Pathology Laboratories, Inc. -97- Project Number 454-104 HISTO PATH O LO G Y INCIDENCE TABLES O FFSPRING SACRIFICE -98- Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 1,8 GROUP 6.2 454-104 Offspring Sacrifice Male Bobwhite A N I M A L ADIPOSE TISSUE Infiltrate, Mononuclear Cell Inflammation, Chronic Inflammation, Chronic Active Mineralization Necrosis 99999 55555 00122 38506 XXX X X 9999 5555 2244 8904 XX X 1 99999 55555 45556 91293 XXXX 2 2 BRAIN XXXXX XXXX XXXXX BURSA OF FABRICIUS NXXXX XXXX NXXXX CLOACA X GALLBLADDER Infiltrate, Mononuclear Cell XAX X 1 XXXX XXXX 2 KIDNEY Infiltrate, Mononuclear Cell Tubules, Mineralization XX XX 1 XX X 1 1 XX XX 1 LIVER Bile Ducts, Hyperplasia Hepatocytes, Fatty Change, Diffuse Hepatocytes, Fatty Change, Focal Hepatocytes, Fatty Change, Periportal Hepatocytes, Vacuolization, Diffuse Infiltrate, Mononuclear Cell Inflammation, Acute X 21 1 2 2 2 32 1 1 1 111 1 223 43 11111 PROVENTRICULUS Infiltrate, Mononuclear Cell X 1112 1112 11211 TESTIS Immature Infiltrate, Mononuclear Cell Pigment Concentration mm PppPp PppPp PPPP 1 PPPP m pPPPP 32 pPPPP GROUP 17.6 99 55 88 12 XX XX XN X XX XX 1 2 X 1 pP pP E PL Experim ental Pathology Laboratories, Inc. Key : X-Hot Remarkable N-No Section I-Incomplete A-Auto1ys1s H iln lM l 2-s11ght/i11d 3-moderate 4-moderately severe 5-severe/h1gh P-Present 0-Benlgn M-Mal1gnant mi*1ss1ng one paired organ u-un$chedu1ed sac./death -99- Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 1.8 454-104 Offspring Sacrifice Female Bobwhite * N i M A l ADIPOSE TISSUE Infiltrate, Mononuclear Cell Inflammation, Chronic Inflammation, Chronic Active Mineralization Necrosis 99999 55555 01122 62825 XN 12 1 999999 555 555 333344 013513 XXXXXX BRAIN XXXXX XXXXXX BURSA OF FABRICIUS X X NX X XXXXXX CLOACA X GALLBLADDER Infiltrate, Mononuclear Cell XXXX 1 XXXX 11 KIDNEY Infiltrate, Mononuclear Cell Tubules, Mineralization XX 1 12 X XX 11 1 LIVER Bile Ducts, Hyperplasia Hepatocytes, Fatty Change, Diffuse Hepatocytes, Fatty Change, Focal Hepatocytes, Fatty Change, Periportal Hepatocytes, Vacuolization, Diffuse Infiltrate, Mononuclear Cell Inflammation, Acute X 1 1 1 1 1 11 2 22 11 111 OVARY Immature NN PPp N PpppP PROVENTRICULUS Infiltrate, Mononuclear Cell 11111 111111 GROUP 6.2 9999 9 5555 5 45 66 6 8512 4 X XXX 1 XXXX X NNXXX X XNN 1 X XXX 1 X 3 334 11 PPPPp 11111 EPL V-2 Experim ental Pathology Laboratories, Inc. Key X-Not Renurkable N*No Section Wncwplete A-Autolys1s l*Kln1ie1 2-sl1ght/1ld 3moderate 4-oderetely severe 5-severe/h1gh P-Present B-Ben1gn M*Hel1gnant is-mlsslng one paired organ u-unscheduled sac./death -100- Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE GROUP 17.6 454-104 Offspring Sacrifice Female Bobwhite A N I M A L ADIPOSE TISSUE Infiltrate, Mononuclear Cell Inflammation, Chronic Inflammation, Chronic Active Mineralization Necrosis 99 99 99 99 5 5 55 55 55 67778888 7059 03 48 X XXXXX 11 BRAIN XXXXXXXX BURSA OF FABRICIUS XXXXXXXX GALLBLADDER Infiltrate, Mononuclear Cell X XXX A 11 1 KIDNEY Infiltrate, Mononuclear Cell Tubules, Mineralization LIVER Bile Ducts, Hyperplasia Hepatocytes, Fatty Change, Diffuse Hepatocytes, Fatty Change, Focal Hepatocytes, Fatty Change, Periportal Hepatocytes, Vacuolization, Diffuse Infiltrate, Mononuclear Cell Inflammation, Acute XX X XX 1 12 1 X 1 1 2 12 11 33 11 OVARY Immature ppPPpPPP PROVENTRICULUS Infiltrate, Mononuclear Cell 11111112 EPL V-3 Experim ental Pathology Laboratories, Inc. Key :X-Not Remarkable N-No Section Mnconplete A-Autolysls lm ln lM l 2-sllght/wlId 3noderate 4MOderate1y severe 5-severe/h1gh Represent BBen1gn M*Mal1gnant nwrtsslng one paired organ u-unscheduled sac./death -101- Project Number 454-104 CO RR ELATIO N OF G RO SS AND M ICRO SCO PIC FINDINGS O FFSPRING SACRIFICE 454-104 Offspring Sacrifice Species: Bobwhite CORRELATION OF GRO SS AND M ICROSCOPIC FINDINGS Sex: Males Group Identification: CONTROL - Sacrificed Animal Number 9503 Client Topography /Site TESTIS 9508 9515 9520 TESTIS TESTIS TESTIS 9526 TESTIS ClientGross Observations Microscopic Observations Iliffusely black, 0.4 - 0.6 x 0.2 cm Immature; Pigment Concentration [both present) (noted at gross trimming) .5 x .3 cm black one present (noted Immature; Pigment Concentration at gross trimming) .6 x .2 cm black both testes (noted Immature; Pigment Concentration at gross trimming) 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) 1o N> Project Number 454-104 VI-1 454-104 Offspring Sacrifice Species: Bobwhite CORRELATION O F GROSS AND M ICROSCOPIC FINDINGS Sex: Males Group Identification: 1.8 - Sacrificed A n im al Number 9528 ClientTopography/Site TESTIS 9529 TESTIS 9540 9544 EXTERNAL TESTIS TESTIS ClientGross Observations Microscopic Observations Dne testis .8 x .4 cm, other .5 x .4 cm, both black (noted at gross trimming) Immature; Pigment Concentration One testis .4 x .2 cm, other .5 x .3 cm, both black (noted at gross trimming) Immature; Pigment Concentration Feather loss on rump No Comment Required Diffusely black, 0.4 x 0.2 cm, Immature; Pigment Concentration bilateral (noted at gross trimming) One testis .5 x .2 cm, other .5 x .3 cm, both black (noted at gross trimming) Immature; Pigment Concentration o Project Number 454-104 VI-2 454-104 Offspring Sacrifice Species: Bobwhite CORRELATION OF GRO SS AND M ICROSCOPIC FINDINGS Sex: Males Group Identification: 6.2 - Sacrificed Animal Number 9549 Client Topography /Site EXTERNAL TESTIS 9551 TESTIS 9552 EXTERNAL TESTIS 9559 TESTIS 9563 EXTERNAL TESTIS ClientGross Observations Microscopic Observations ''eather loss on rump Yo Comment Required Dne present fragmented, black, .7 cm x .3 cm (noted at gross trimming) 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) Immature; Pigment Concentration One testis .4 x .2 cm, other .6 x .2 cm, both black (noted at gross trimming) Immature; Pigment Concentration Feather loss 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 o Project Number 454-104 VI-3 454-104 Offspring Sacrifice Species: Bobwhite CORRELATION OF GROSS AND M ICROSCOPIC FINDINGS Sex: Males Group Identification: 17.6 - Sacrificed Animal Number 9581 ClientTopography /Site EXTERNAL TESTIS . 9582 TESTIS ClientGross Observations Microscopic Observations ?eather loss on rump One testis .7 x .4 cm, other .5 x .2 cm, both black (noted at gross trimming) One testis .7 x .3 cm, other .5 x .2 cm, both black (noted at gross trimming) Vo Comment Required Immature; Pigment Concentration Immature; Pigment Concentration O to Project Number 454-104 VI-4 *54-104 . Offspring Sacrifice Species: Bobwhite CORRELATION OF GRO SS AND M ICROSCOPIC FINDINGS Sex: Females Group Identification: CONTROL - Sacrificed Animal Number 9512 ClientTopography /Site EXTERNAL ClientGross Observations leather loss on rump Microscopic Observations Ho Comment Required , O ON Project Number 454-104 VI-5 454-104 Offspring Sacrifice Species: Bobwhite CORRELATION OF GRO SS AND M ICROSCOPIC FINDINGS Sex: Females Group Identification: 1.8 - Sacrificed Animal Number 9531 ClientTopography /Site EXTERNAL ClientGross Observations leather loss on rump and back MicroscopicObservations io Comment Required o -j Project Number 454-104 VI-6 454-104 Offspring Sacrifice Species: Bobwhite CORRELATION O F GRO SS AND M ICROSCOPIC FINDINGS Sex: Females Group Identification: 17.6 - Sacrificed Anim al Number 9583 ClientTopography /Site EXTERNAL ClientGross Observations Feather loss on rump Microscopic Observations 'lo Comment Required Project Number 454-104 VI-7 Appendix X Table 1 Egg Production (eggs laid/hen/week) from a Northern Bobwhite Pilot Reproduction Study with PFOS Control (0 ppm a.i.) Weeks Pen 1 2 3 4 5 6 Total e /h/d ' 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total E/H/D1 201 3 4 5 4 5 6 202 3 4 5 5 5 6 203 6 6 7 6 6 7 204 3 4 6 6 4 6 205 6 7 6 6 7 7 27 0.64 5 7 6 7 7 7 7 7 7 7 7 7 7 2 117 28 0.67 6 6 7 7 7 6 7 6 7 7 6 6 7 2 115 38 0.90 7 7 7 7 7 6 7 7 7 7 7 7 7 2 130 29 0.69 4 6 7 7 7 7 7 6 6 4 3 3 3 0 99 39 0.93 8 7 6 0 0 0 4 4 7 7 7 7 7 2 105 0.87 0.85 0.96 0.73 0.78 Total 21 25 29 27 27 32 Mean 4 5 6 5 5 6 SD 2 1 1 1 1 1 161 32 6 30 33 33 28 28 26 32 30 34 32 30 30 31 8 566 0.77 6 7 7 6 6 5 6 6 7 6 6 6 6 2 113 0.14 2 1 1 3 3 3 1 1 0 1 2 2 2 1 12 0.84 0.09 1Eggs laidperhen perday Project Number 454-104 Appendix X Table 2 Egg Production (eggs laid/hen/week) from a Northern Bobwhite Pilot Reproduction Study with PFOS Pen 1 2 206 3 207 6 208 5 209 4 210 4 6 7 5 5 6 Total Mean SD 22 4 1 29 6 1 1Eggs laid per hen per day 1.8ppma.i. Weeks 3 4 5 6 Total E/H/D1 6 6 6 7 34 0.81 7 7 1 0 28 0.67 6 5 5 6 32 0.76 5 5 6 7 32 0.76 4 4 2 0 20 0.48 28 27 20 20 6544 1 124 146 29 0.70 6 0.13 Appendix X Table 3 Egg Production (eggs laid/hen/week) from a Northern Bobwhite Pilot Reproduction Study with PFOS Pen 1 2 211 5 212 0 213 6 214 5 215 4 7 2 5 5 5 Total Mean SD 20 4 2 24 5 2 1Eggs laid per hen per day 6.2 ppm a.i. Weeks 3 4 5 6 Total E/H/D1 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 12 1 7 0.17 Project Number 454-104 Appendix X Table 4 Egg Production (eggs laid/hen/week) from a Northern Bobwhite Pilot Reproduction Study with PFOS 17.6 ppm a.i. Weeks Pen 1 2 3 4 5 6 Total E/H/D1 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Total E/H /D 1 216 5 5 5 5 7 7 217 5 6 7 6 7 7 218 6 7 7 6 7 7 219 3 4 4 5 5 7 220 8 5 7 7 7 7 34 0.81 5 6 7 6 6 6 6 4 0 0 1 3 0 0 84 0.62 38 0.90 7 7 7 7 7 7 6 7 7 7 7 2 0 0 116 0.86 40 0.95 7 7 7 7 7 7 7 7 7 7 7 7 7 2 133 0.99 28 0.67 7 7 7 7 7 7 7 7 7 9 6 6 7 1 120 0.89 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 Mean 5 5 6 6 7 7 SD 2 1 1 1 1 0 181 36 5 32 34 35 34 34 34 33 31 28 30 28 25 21 5 585 0.86 6 7 7 7 7 7 7 6 6 6 6 5 4 1 117 0.13 1 0 0 0 0 0 1 1 3 3 3 2 4 1 20 0.87 0.15 1Eggs laidperhen perday 1 Project Number 454-104 Appendix XI Page 1 Reproductive Performance by Pen from a Northern Bobwhite Pilot Reproduction Study with PFOS Table 1 Reproductive Data (Count) By Pen Param eter Control (0 ppm a.i.) Pens 201 202 203 204 205 Total ________ 1 .8 p p m a.i._________ 206 207 208 209 210 Total ________ 6.2 ppm a.i._________ 211 212 213 214 215 Total ________ 17.6 ppm a.i.________ 216 217 218 219 220 Total Eggs Laid 5 6 6 5 7 29 Eggs Cracked 0 0 0 0 0 0 Eggs Set 5 6 6 5 7 29 Viable Embryos Live 3-Wk Embiyos Hatchlings 5 6 6 5 7 29 5 5 6 5 7 28 5 5 6 5 7 28 Offspring Survivors 5 5 5 5 7 27 6 0 5 6 1 18 00000 0 6 0 5 6 1 18 6 0 4 6 1 17 6 0 4 6 1 17 6 0 4 6 1 17 6 0 4 6 1 17 7 5 5 7 5 29 00000 0 7 5 5 7 5 29 5 5 3 7 5 25 5 5 3 7 5 25 5 5 3 7 5 25 5 5 3 2 5 20 7 7 7 6 7 34 00000 0 7 7 7 6 7 34 7 7 7 6 7 34 7 6 7 6 7 33 7 5 7 6 6 31 7 3 6 3 6 25 Project Number 454-104 Appendix XI Page 2 Reproductive Performance by Pen from a Northern Bobwhite Pilot Reproduction Study with PFOS Table 2 Eggs Laid / Hen / Day Replicate Control(0ppm a.i.) Eggs Eggs/ Laid Days Hen/Day 1.8ppm a.i. Eggs Eggs/ Laid Days Hen/Day 6.2ppm a.i. Eggs Eggs/ Laid Days Hen/Day 1 2 3 4 5 Total Mean SD 5 6 6 5 7 29 6 1 7 0.71 7 0.86 7 0.86 7 0.71 7 1.00 0.83 0.12 6 7 0.86 07 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 Control(0ppm a.i.) Eggs Eggs Replicate Cracked Laid % 1 2 3 4 5 Total Mean SD 05 06 06 05 07 0 29 06 01 0 0 0 0 0 0 0 Table 3 Eggs Cracked / Eggs Laid (%) 1.8ppm ai. Eggs Eggs Cracked Laid % 06 00 05 06 01 0 18 04 03 0 0 0 0 0 0 6.2ppm a.i. Eggs Eggs Cracked Laid % 07 05 05 07 05 0 29 06 01 0 0 0 0 0 0 0 17.6ppm ai. Eggs Eggs/ Laid Days Hen/Day 7 7 1.00 7 7 1.00 7 7 1.00 6 7 0.86 7 7 1.00 34 7 0.97 0 0.06 17.6ppm ai. Eggs Eggs Cracked Laid % 07 07 07 06 07 0 34 07 00 0 0 0 0 0 0 0 Project Number 454- o Control(0ppm a.i.) Viable Eggs Replicate Embryos Set % 1 2 3 4 5 Total Mean SD 5 5 100 6 6 100 6 6 100 5 5 100 7 7 100 29 29 6 6 100 1 10 Appendix XI Page 3 Reproductive Performance by Pen from a Northern Bobwhite Pilot Reproduction Study with PFOS Table 4 Viable Embryos / Eggs Set (%) 1.8ppm ai. Viable Eggs Embiyos Set % 6 6 100 00 4 5 80 6 6 100 1 1 100 17 18 3 4 95 3 3 10 6.2ppm ai. Viable Eggs Embryos Set % 17.6ppm a.i. Viable Eggs Embiyos Set % 5 7 71 / 5 5 100 3 5 60 7 7 100 5 5 100 25 29 5 6 86 1 1 19 7 7 100 7 7 100 7 7 100 6 6 100 7 7 100 34 34 7 7 100 000 Control(0ppm ai.) Live Replicate 3-Week Viable % 1 2 3 4 5 Total Mean SD 5 5 100 5 6 83 6 6 100 5 5 100 7 7 100 28 29 6 6 97 1 17 Table 5 Live 3-Week Embryos /Viable Embryos (%) 1.8ppm ai. Live 3-Week Viable % 6 6 100 00 4 4 100 6 6 100 1 1 100 17 17 3 3 100 330 6.2ppm ai. Live 3-Week Viable % 5 5 100 5 5 100 3 3 100 7 7 100 5 5 100 25 25 5 5 100 1 10 17.6ppm ai. Live 3-Week Viable % 7 7 100 6 7 86 7 7 100 6 6 100 7 7 100 33 34 7 7 97 10 6 t/l Project Number 454-104 Replicate Control (0ppm a.i.) Live Hatch 3-Week % 1 2 3 4 5 Total Mean SD 5 5 100 5 5 100 6 6 100 5 5 100 7 7 100 28 28 6 6 100 1 10 Appendix XI Page 4 Reproductive Performance by Pen from a Northern Bobwhite Pilot Reproduction Study with PFOS Table 6 Hatchlings / Live 3-Week Embryos (%) 1.8ppm ai. Live Hatch 3-Week % 6 6 100 00 4 4 100 6 6 100 1 1 100 17 17 3 3 100 330 6.2ppm ai. Live Hatch 3-Week % 5 5 100 5 5 100 3 3 100 7 7 100 5 5 100 25 25 5 5 100 1 10 Control(0ppm ai.) Offspring Replicate Surv. Hatch % 1 2 3 4 5 Total Mean SD 5 5 100 5 5 100 5 6 83 5 5 100 7 7 100 27 28 5 6 97 1 17 Table 7 Surviving Offspring / Hatchlings 1.8ppm ai. Offspring Surv. Hatch % 6.2ppm ai. Offspring Surv. Hatch % 6 6 100 00 4 4 100 6 6 100 1 1 100 17 17 3 3 100 3 30 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 ai. Live Hatch 3-Week % 7 7 100 5 6 83 7 7 100 6 6 100 6 7 86 31 33 6 7 94 119 17.6ppm a.i. Offspring Surv. Hatch % 7 7 100 3 5 60 6 7 86 3 6 50 6 6 100 25 31 5 6 79 2 1 23 ON t Project Number 454-104 Replicate Control(0ppm a.i.) Eggs Hatch Set % 1 2 3 4 5 Total Mean SD 5 5 100 5 6 83 6 6 100 5 5 100 7 7 100 28 29 6 6 97 1 17 Appendix XI Page 5 . Reproductive Performance by Pen from a Northern Bobwhite Pilot Reproduction Study with PFOS Table 8 Hatchlings / Eggs Set (%) ______ 1.8ppm a.i.______ Eggs Hatch Set % 6 6 100 00 4 5 80 6 6 100 1 1 100 17 18 3 4 95 3 3 10 ______ 6.2ppm a.i.______ Eggs Hatch Set % 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 Surv Set % 1 2 3 4 5 Total Mean SD 5 5 100 5 6 83 5 6 83 5 5 100 7 7 100 27 29 5 6 93 1 19 Table 9 Surviving Offspring / Eggs Set (%) 1.8ppm a.i. Offspring Eggs Surv Set % 6 6 100 00 4 5 80 6 6 100 1 1 100 17 18 3 4 95 3 3 10 6.2ppm ai. Offspring Eggs Surv Set % 5 5 3 2 .5 20 4 1 7 71 5 100 5 60 7 29 5 100 29 6 72 1 30 ______ 17.6ppm a.i. Eggs Hatch Set % 7 7 100 5 7 71 7 7 100 6 6 100 6 7 86 31 34 6 7 91 1 0 13 17.6ppm a.i. Offspring Eggs Surv Set % 7 7 100 3 7 43 6 7 86 3 6 50 6 7 86 25 34 5 7 73 2 0 25 1 -4 Project Number 454-104 Appendix XI Page 6 Reproductive Performance by Pen from a Northern Bobwhite Pilot Reproduction Study with PFOS Table 10 Hatchlings / Hen / Day Replicate Control(0ppm a.i.) Hatch/ Hatch Days Hen/Day 1 2 3 4 5 Total Mean SD '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 1.8ppm a.i. Hatch/ Hatch 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 6.2ppm a.i. Hatch/ Hatch Days Hen/Day 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 17.6ppm ai. Hatch/ Hatch Days Hen/Day 7 7 1.00 5 7 0.71 7 7 1.00 6 7 0.86 6 7 0.86 31 6 0.89 1 0.12 Control(0ppm ai) Offspring Offspring/ Replicate Surv Days Hen/Day 1 2 3 4 5 Total Mean SD 5 7 0.71 5 7 0.71 5 7 0.71 5 7 0.71 7 7 1.00 27 5 0.77 1 0.13 Table 11 Surviving Offspring / Hen / Day 1.8ppma.i. Offspring Offspring/ Surv Days Hen/Day 6.2ppm ai Offspring Offspring/ 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 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 a.i. Offspring Offspring/ Surv Days Hen/Day 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 M *-* oo Project Number 454-104 A ppendix X II M ean O ffspring B ody W eight (g) from a Northern Bobw hite Pilot Reproduction Study w ith PFOS M ean H atchling B ody W eight (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 1.8 ppm a.i. 5.3 -- 6.0 6.0 5.0 5.6 0.5 6.2 ppm 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 Project Number 454-104 M ean Surviving O ffspring1B ody W eight (g) Replicate 1 2 3 4 5 Mean SD Control (0 ppm a.i.) 185 171 160 178 182 175 10 1.8 ppm a.i. 175 -- 174 176 173 175 1 6.2 ppm a.i. 181 183 171 181 181 179 5 1Offspring w ere approximately 12 weeks o f age at final body weight interval. --N o offspring available. 17.6 ppm a.i. 164 169 175 175 184 173 8 -120- Project Number 454-104 Appendix XIII Adult Liver Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS Page 1 Pen 201 202 203 204 205 Mean SD Control (0 ppm a.i.) Male Liver Female Liver 3.260 3.156 3.373 4.170 3.051 3.402 0.446 9.210 7.606 7.908 5.612 7.889 7.645 1.295 Pen 206 207 208 209 210 Mean SD 1.8 ppm a.i. Male Liver 3.161 3.750 4.211 3.496 2.887 3.501 0.514 Female Liver 7.020 8.556 5.744 6.278 4.016 6.323 1.668 -121 - Project Number 454-104 Appendix XIII Adult Liver Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS Page 2 Pen 211 212 213 214 215 Mean SD 6.2 ppm 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 17.6 ppm 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 6.131 8.329 6.851 1.880 Appendix XIV Offspring1Liver Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS Control (0 ppm a.i.) Pen Liver 1.8 ppm a.i. Pen Liver 6.2 ppm a.i. Pen Liver 201 201 202 202 203 203 204 204 205 205 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 3.458 4.128 4.146 3.596 4.095 2.940 3.756 0.453 211 211 212 212 213 213 214 214 215 215 Mean SD 3.826 3.533 6.097 4.645 3.899 3.771 7.795 5.996 4.701 5.582 4.985 1.367 1Offspring were approximately 12 weeks of age at the time of euthanasia and tissue collection. 17.6 ppm 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 3.211 3.459 3.286 3.429 0.495 Project Number 454-104 -123- W ildlife International, Ltd. Project Number 454-104 Appendix XV Changes to Study Protocol This study was conducted in accordance with 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 ppma.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 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 - W ildlife 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- W ildlife 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 Toxicology (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