Document mqr4Xwzwwgz65djvLQLwwGMg4

f l& 2 6 -1 6 f t FFOS: 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 StPaul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 o f 125 W ildlife International, Ltd. Project Number 454-104 - 2GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE. PFOS: A Pilot Reproduction Study with the Northern Bobwhite WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-104 STUDY COMPLETION: December 18,2003 The study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR Part 160, 17 August 1989; OECD Principles o f 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 ami 023-063). Exygen Research study number 023-041 was initiated with a separate Study Director. Results o f analyses conducted by Exygen Research for study numbers 023-041 and 023-063 are reported separately. STUDY DIRECTOR: Senior Biologist, Avian Toxicology SPONSOR'S REPRESENTATIVE: S k l t 'i DATE e r o 03.7 W ildlife International, Ltd. Project Number 454-104 -3 - QUALITY ASSURANCE STATEM ENT 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 o f Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates o f 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. S jju sc u rs o ^ . G o & tm o f\ Susan L. Coleman, B.A. Senior Quality Assurance Representative ia-i8-c0 DATE c rooi.8 W ildlife International, Ltd. -4 REPORT APPROVAL Project Number 454-104 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 S IU D Y -D IE B C TO R : P' Sean P. Gallagher Senior Biologist, Avian Toxicology DATE ScifcoliaC, Analytical Chemistry MA M G jMEN I: Joann B. Beavers Director, Avian Toxicology Director o f Chemistry 4 i& \ . DATE c ro o ? . W ildlife International, Ltd. Project Number 454-104 -5 TABLE OF CONTENTS Title Page...............................................................................................................................................1 Good Laboratory Practice Compliance Statem ent............................................................................. 2 Quality Assurance Statem ent.............................................................................................................. 3 Report Approval................................................... 4 Table o f Contents................................................................................................................................. 5 Sum mary.............................................................................................................................................. 8 Introduction.......................................................................................................................................... 9 Objectives............................................................................................................................................. 9 Experimental Design............................................................................................................................9 M aterials and M ethods....................................................................................................................... 11 Test Substance and Internal Standard......................................................................................... 11 Test Organisms............................................................................................................................ 11 Identification................................................................................................................................ 12 Avian Feed and W ater.................................................................................................................12 D iet Preparation........................................................................................................................... 13 D iet Sampling.............................................................................................................................. 13 Analytical M ethod....................................................................................................................... 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 M ortalities and Clinical Observations............................................................... 21 Adult Body W eight..................................................................................................................... 21 Feed Consumption...................................................................................................................... 22 Gross Necropsy............................................................................................................................22 Histopathology.............................................................................................................................23 Tissues A nalysis..........................................................................................................................23 Reproductive Results.................................................................................................................. 24 Offspring Body W eights............................................................................................................. 24 Liver W eights.............................................................................................................................. 24 Conclusion.......................................................................................................................................... 25 References.......................................................................................................................................... 26 CCOCCO W ildlife International, Ltd. Project Number 454-104 6- - TABLE OF CONTENTS PAGE 2 TABLES Table 1. Mean M easured Concentrations (ppm a.i.) o f PFOS in Avian Diet from a Northern Bobwhite Pilot Reproduction Study................................................ 27 Table 2. M ean Adult Body W eight (g) from a Northern Bobwhite Pilot Reproduction Study with PFO S...................................................................................28 Table 3. M ean Feed Consumption (g/bird/day) from a Northern Bobwhite Pilot Reproduction Study with PFOS.........................................................29 Table 4. Summary o f Gross Pathological Observations from a Northern Bobwhite Pilot Reproduction Study with PFOS, Adult Birds Euthanized at 6 W eeks and Test Termination.................................................................................30 Table 5. M ean Egg Production (Eggs Laid/Hen and Eggs/Hen/Day) from a Northern Bobwhite Pilot Reproduction Study with PFOS.........................................31 Table 6. Summary o f Reproductive Performance (Eggs Set from W eek 5) from a Northern Bobwhite Pilot Reproduction Study with PFOS.............................32 Table 7. M ean Body W eight (g) o f Hatchlings and Surviving Offspring from a Northern Bobwhite Pilot Reproduction Study with PFOS.............................33 Table 8. M ean Liver W eights (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS...........................................................................................................34 CC0021 W ildlife International, Ltd. Project Number 454-104 -7 - TABLE OF CONTENTS PAGE 3 APPENDICES Appendix I. Certificate o f A nalysis........................................................................................... 35 Appendix II. D iet and Supplement Form ulations.......................................................................38 Appendix III. Diet Preparation......................................................................................................40 Appendix IV. The Analysis o f PFOS in Avian D iet................................................................... 41 Appendix V. Diagram o f Test Layout............................... 55 Appendix VI. Adult Body W eight (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 PFO S64.............................64 Appendix IX. Histopathology Report............................................................................................68 Appendix X. Egg Production (eggs laid/hen/week) from a Northern Bobwhite Pilot Reproduction Study with P FO S ...............................109 Appendix XI. Reproductive Performance by Pen from a Northern Bobwhite Pilot Reproduction Study with PFO S...............................113 Appendix XQ. Mean Offspring Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS..................................................................119 Appendix XIH. Adult Liver W eight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS...........................................................................120 Appendix XIV. Offspring Liver W eight (g) from a Northern Bobwhite Pilot Rqjroduction Study with PFO S................................................................. 122 Appendix XV. Changes to Study Protocol....................................................... 123 Appendix XVI. Personnel Involved in the Study...........................................................................125 C00G 22 W ildlife 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 (Colima virginianus) AGE TEST ANIMALS: Approximately 30 weeks o f age at the initiation o f 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 o f 1.8, 6.2, and 17.6 ppm a.i. for 6 weeks. The control group and 17.6 ppm a.i. treatm ent groups were maintained on test diets for an additional 13 weeks. No treatm ent-related m ortalities or overt signs o f toxicity were observed at any o f the test concentrations. There were no apparent treatment-related effects on body weight, feed consumption, or liver weight at the 1.8 and 6.2 ppm a.i. test concentrations. Additionally, there were no apparent treatment-related effects on female body weight or liver weight at the 17.6 ppm a.i. test concentration. There were no apparent treatment-related effects on any reproductive parameters measured during the study for any o f 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 treatm entrelated reduction in feed consumption for the 17.6 ppm a.i. treatm ent 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 o f selected tissues also revealed regression o f 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 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. C 009?3 W ildlife International, Ltd. Project Number 454-104 -9- INTRODUCTION This study was conducted by W ildlife International, Ltd. for 3M Corporation at the W ildlife International, Ltd. avian toxicology facility in Easton, Maryland 21601. The biological portion o f the test was conducted from February 29,2000 until July 21,2000. Raw data generated at W ildlife International, Ltd. and a copy o f the final report are filed under Project Number 454-104 in archives located on the W ildlife International, Ltd. site. Biological specimens are stored at 3M Corporation, St. Paul, M innesota 55133. OBJECTIVES The objective o f this study was to evaluate the effects upon the adult northern bobwhite (olinus virginianus) o f dietary exposure to the potassium salt o f Perfluorooctane Sulfonic Acid (hereafter referred to as PFOS) over a period o f approximately 6 weeks or 19 weeks. Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects o f adult exposure to PFOS on the num ber o f eggs laid, fertility, embryo viability, hatchability and offspring survival were evaluated. Histopathological examination o f selected tissues and analyses o f 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 o f a LC50 study (W ildlife 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 per Pen Males Females 11 11 11 11 GCO024 Wildlife International, Ltd. Project Number 454-104 -10- Each treatment and control group contained five pairs o f 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. o f PFOS. The control group was fed diet comparable to the treatment groups, but without the addition o f the test substance. Adult northern bobwhite were exposed to PFOS at nominal dietary concentrations o f 1.8, 6.2 and 17.6 ppm a.i. for a period o f 6 weeks. A control group, fed non-treated diet, was maintained concurrently with the treatment groups. Each treatment and the control group consisted o f five pairs o f birds, housed with one male and one female per pen. At the end o f W eek 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 o f W eek 20 o f 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 m ortality, abnormal behavior and signs o f 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 weights were recorded for all necropsied birds. Liver, bile and blood (when available) and feather samples were collected at the time o f necropsy for possible analysis. Additional samples o f liver, brain, kidney, gonad, proventriculus, gall bladder, adipose tissue, and bursa o f fabricius (when available) were fixed in 10% buffered formalin for histopathological examination. During the test, die number o f eggs laid in each pen was recorded to evaluate egg production. Eggs laid in Weeks 1, 3 and 6 o f the test were collected and separated into shell, shell membrane, yolk and albumen and stored frozen for possible analysis. Eggs laid during W eeks 5 and 6 (Days 31 to 37) o f the test were collected and set for incubation. Embryo viability, hatchability, hatchling health and survivability were examined. Wildlife 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, H azard 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 W ildlife International, Ltd. identification number 4675 upon receipt. The test substance was a white powder and was identified as: FC-9S; Lot 217. The test material had an original reported purity o f 98.9% and had expired at the time o f experimental start. An assay o f the test material after experimental start indicated a purity o f 90.49%. The final assay o f the test material indicated a purity o f 86.9% and expiration date o f August 31,2006 (Appendix I). The test substance was held under ambient condition in locked storage at the W ildlife International, Ltd. facilities in Easton, Maryland. Concentrations o f the test substance in the diet were adjusted to 100% active ingredient based upon the original reported purity o f 98.9%. Dietary concentrations are expressed as parts per million active ingredient (ppm a.i.) in the diet based upon the final reported purity o f 86.9%. The internal standard, 4H PFOS, was received from 3M Corporation on July 2, 1998 and was assigned W ildlife 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 W ildlife 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. A t the start o f acclimation, the bobwhite appeared healthy and were phenotypically indistinguishable from wild type. The birds were from the same hatch, approaching their first breeding season and had not been used in any previous testing. At the start of acclimation, a random number generating function in a spreadsheet program was used to randomize pen assignment for each bird. Immediately prior to test initiation, all potential study C 90926 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 o f age at test initiation (first day o f exposure to test diet) and ranged in weight from 194 to 254 grams at test initiation. Sex o f the birds was determined by a visual examination o f the plumage. Identification Adult birds were identified by individual leg bands, each pen was identified with a unique number, and groups o f pens were identified by project number and concentration. During the test, the number o f 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 o f 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 W ildlife International, Ltd. specifications by Agway Inc. (Appendix n , Table 1). The basal ration contained at least 27% protein and 2.5% fat, and no more than 5% fiber. The basal diet contained approximately 1.1% calcium, derived from feedstuffs and the 0.9% limestone used in the formulation o f the basal diet by Agway. W hile this level o f calcium is sufficient for growth and maintenance rations, additional calcium is required in the ration o f breeding birds for egg shell formation. Therefore, an additional 5% (w/w) o f 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 o f 5% supplemental lim estone. W ater was supplied by the town o f Easton public water supply. All offspring received a watersoluble vitamin and electrolyte mix in their water (Appendix n , Table 2). Neither the adults nor offspring received any form o f medication in the feed during the test. Feed and water were analyzed periodically in accordance with W ildlife International, Ltd. Standard Operating Procedures. C ~0077 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 o f the final diet. Control diet and each treated diet were prepared weekly beginning on February 28, 2000 and presented to the birds on Tuesday o f each week. Dietary concentrations were adjusted for purity o f the test substance and are presented as parts per million active ingredient (ppm a.i.). Details o f the weekly preparation o f test and control diets are shown in Appendix HI. Diet Sampling Homogeneity o f the test substance in the diet was evaluated by collecting six samples from each o f the treated diets and two samples from the control diet on Day 0 o f Week 1. Samples were collected from the top, middle and bottom o f the left and right sections o f the mixing vessel. Control and treatment group diet samples were also collected from the feed troughs on Day 7 o f 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 W eek 6 o f the test to measure/verify test concentrations. The diet samples were stored frozen or transferred immediately to the W ildlife International, Ltd. analytical chemistry facility for analysis. Analytical Method The method used for the analysis o f PFOS in avian diet was based upon methodology developed at Wildlife International, Ltd. and entitled "Analytical Method Verification for the Determination of PFOS in Avian Diet" (W ildlife 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 IH , 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 o f the PFOS methodology. A method flow chart is provided in Appendix IV, Figure 1. Concentrations o f PFOS in the standards and extracts o f 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 Cig analytical column (50 mm x 2 mm I.D., 3-pm particle size). The instrument parameters are summarized in Appendix IV, Table 1. W ildlife International, Ltd. -14- Project Number 454-104 Calibration standards o f PFOS prepared in a 50% m ethanol: 50% NANOpure w ater 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 (W eek 1, Day 7, W eek 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 m onitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOS on the basis o f individual isomeric components. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS : internal standard) o f the calibration standards. An example o f a calibration curve is presented in Appendix IV, Figure 2. The concentration o f test substance in the samples was determined by substituting the peak area response ratios o f the samples into the applicable linear regression equation. Typical ion chromatograms o f low and high-level calibration standards are shown in Appendix IV, Figures 3 and 4, respectively. Examples o f calculations are presented in Appendix IV, Table 2. The method lim it o f quantitation (LOQ) for the Week 1, Day 0 analysis was set at 0.879 ppm a.i., calculated as the product o f the lowest calibration standard analyzed (0.000439 mg a.i./L) and the overall dilution factor o f the m atrix blank sample (2000 L/Kg). The method LOQ for the W eek 1, Day 7 and W eek 6, DayO analyses and the sample re-extraction set was set at 1.41 ppm a.i., calculated as the product o f the lowest standard analyzed (0.000351 mg a.i./L) and the overall dilution factor o f the matrix blank samples (4000 L/Kg). Examples o f 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 o f 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 o f 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 ^0029 W ildlife International, Ltd, Project Number 454-104 -15- concentrations were not corrected for the respective mean procedural recovery o f that sample set. A typical ion chromatogram o f 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 o f pens manufactured by Georgia Quail Farm M anufacturing (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 o f galvanized wire mesh and galvanized sheeting. A diagram o f the test layout is presented in Appendix V. Each pen was equipped with feed and water troughs. Weekly, sufficient feed for die 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. W ater 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 o f the test was 22.3 1.1C (SD) with an average relative humidity o f 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 o f light per day to induce egg laying. Throughout the test, the birds received a mean o f approximately 188 lux (~ 17.5 ft. candles) o f 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 o f the test. During acclimation, all birds were observed daily. Birds exhibiting abnormal behavior or debilitating physical injuries were not used for the test. During the study, all adult birds were observed daily for signs of toxicity or abnormal behavior. Additionally, all offspring were observed daily (rf)030 W ildlife International, Ltd. Project Number 454-104 -16- from hatching until approximately 12 weeks o f age. A record was maintained o f all m ortalities and clinical observations. Adult Body W eight 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 te s t Feed consumption was determined by weighing the freshly filled feeder on Day 0, recording the amount o f any additional diet added during the week, and weighing the feeder and remaining feed at the end o f 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 o f 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 o f total feed consumption. All rem aining birds were euthanized two days after the beginning o f Week 20. Therefore, no feed consumption estimate was calculated for Week 20. Adult Blood Collection At the time o f adult termination (end o f Week 6 for 1.8 and 6.2 ppm a.i. groups; beginning o f 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 A t the conclusion o f the exposure period, all surviving adult birds were euthanized by cervical dislocation, necropsied, and stored frozen. A t the time of necropsy, tissues were collected for histopathological examination (0 and 17.6 ppm a.i. groups only) and possible analyses. W hen available, samples o f gall bladder, liver, proventriculus, kidneys, brain, gonads, bursa o f Fabricius and adipose tissue were fixed in 10% buffered formalin. Histology samples were shipped to EPL in Herndon, VA for histopathology. W hen 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. CC 003 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 o f Week 5 o f 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 o f 13.1C 0.1C (SD) with a mean relative humidity o f approximately 72% 4% (SD). Groups o f eggs were identified by an alphabetic lot code. All eggs laid during the week were considered as one lot. Candling and Incubation A t the end o f the weekly interval, all eggs were removed from die 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 o f pathogen contamination prior to incubation. Formaldehyde gas was generated by combining 26 g o f potassium permanganate and 25 ml o f 37% commercial grade formalin in a porcelain bowl at the base o f 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 o f 30.6 0.1C (SD) (relative humidity o f approximately 60%). The incubator was equipped with a pulsator fan and blades that produced a mild breathing air movement designed to eliminate intracabinet temperature and humidity variation during incubation. In order to prevent adhesion o f 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 o f vertical in one direction to 50 off o f vertical in the opposite direction (total arc o f rotation was 100) every two hours through Day 21 o f incubation. Eggs were candled on Day 11 o f incubation to determine embryo viability and on Day 21 to determine embryo survival. Hatching and Brooding On Day 21 o f 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 o f 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%). 0^0032 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 o f incubation. The group body weight o f the surviving hatchlings by pen was determined. Hatchlings were leg banded for identification by pen o f origin and then routinely housed according to the appropriate parental concentration grouping in brooding pens until approximately 5 weeks o f age. A ll hatchlings were moved to large flight pens, where they were housed approximately 7 weeks. The hatchlings were fed untreated diet without the addition o f 5% supplemental limestone. At approximately 12 weeks o f age, the average body weight by parental pen o f all surviving offspring was determined, and the birds were euthanized with carbon dioxide and disposed o f by incineration. Those offspring selected for blood and tissue sampling were stored frozen following necropsy and later disposed o f by incineration. Hatchlings were housed in batteries o f 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 o f each pen were constructed o f galvanized wire mesh and galvanized sheeting. Floors were o f galvanized wire mesh. Thermostats in the brooding compartment o f each pen were set to maintain a temperature o f approximately 38 C from the time o f hatching until the birds were approximately 30 days o f age. Brooding was discontinued once hatchlings were determined to be o f 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 o f 39% 8% (SD). All hatchlings were removed from brooding pens and transferred to flight pens at approximately 5 weeks o f age. Each flight pen measured approximately 1.2 m X 1.2 m, with a ceiling height o f approximately 1 m. External walls, floor and ceiling o f each pen were constructed o f wire mesh. The photoperiod for the hatchlings was maintained by a time clock at 16 hours o f light per day. Offspring Blood and Tissue Collection Prior to euthanasia o f 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 o f gall bladder, liver, proventriculus, kidneys, brain, gonads, bursa o f 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. G00033 W ildlife International, Ltd. Project Number 454-104 -19- Statistical Analyses Upon completion o f the test, an Analysis o f Variance (ANOVA) was performed to determine statistically significant differences between groups. Dunnett's multiple comparison procedure (5, 6) was used to compare the three treatm ent means with the control group mean and assess the statistical significance o f 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 o f statistical analyses were conducted with the body weight and feed consumption data. One set o f analyses only looked at body weight and feed consumption data from the first 6 weeks o f the study and evaluated all three-treatment groups. The second set o f analysis only evaluated the control and 17.6 ppm a.i. treatment groups and examined data for the frill duration o f 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. treatm ent group were compared. 1. Adult Body W eight - Individual body weight was measured at test initiation, W eeks 2, 4, 6, 8, 10, 11, and at adult termination. Statistical comparisons were made between the control group and each treatment group at each weighing interval by sex for the first 6 weeks. In addition, statistical comparisons were made between the control group and 17.6 ppm a.i. treatment group for weeks 8, 10, 11, and 20. 2. Adult Feed Consumption - Feed consumption expressed as grams o f 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. Begs Laid - The number o f eggs laid per female per treatment group. For the evaluation o f reproductive performance, data taken from week 5 eggs set. 4. Viable Embryos - The number o f live embryos determined at Day 11 by candling. 5. Eggs Cracked o f Eggs Laid - The number o f eggs determined by candling to be cracked divided by the number o f eggs laid, per pen. 6. Viable Embryos o f Eggs Set - The number o f embryos at the Day 11 candling was divided by the number o f eggs set, per pen. C00034 W ildlife International, Ltd. Project Number 454-104 - 20- 7. Live 3- Week Embryos o f Viable Emhrvns - The number o f live embryos at the Day 21 candling was divided by the number o f viable embiyos, per pen. 8. Hatchlings o f 3-Week Embryos - The number o f hatchlings removed from the hatcher was divided by the number o f live 3-week embryos, per pen. 9. 14-Dav Old Survivors o f Hatchlings - The number o f 14-day old survivors was divided by the number o f hatchlings per week, by pen. 10. Hatchlings of Eggs Set - The number o f hatchlings was divided by the number o f eggs set per week, by pen. 11. 14-Dav Old Survivors o f Eggs Set - The number o f 14-day old survivors was divided by the number o f eggs set per week, by pen. 12. Offspring's Body Weight - The group body weights o f surviving hatchlings and 14-day old survivors were measured by parental pen group. 13. Liver W eight - Individual liver weights were measured at adult termination and at the term ination o f offspring of week 5. Statistical comparisons o f 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 o f 1.8, 6.2 and 17.6 ppm a.i. for a period o f 6 weeks. A control group, fed non-treated diet, was maintained concurrently with the treatment groups. Each treatment and the control group consisted o f five pairs o f birds, housed with one male and one female per pen. A t the end o f 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 die appropriate diets until the beginning o f W eek 20 of the test, at which time they were also euthanized and subjected to gross necropsy. Analytical Results None o f the control samples showed any indication o f the presence o f the test substance or o f the presence o f co-eluting substance at the characteristic retention time o f the test substance (Table 1). Diet samples were collected from the 1.8, 6.2 and 17.6 ppm a.i. test concentrations on W eek 1, Day 0, and were analyzed to evaluate the homogeneity o f 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., ccoon s Wildlife 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 o f variation were 7.2%, 11% and 8.3%, respectively. These values represented 100, 97 and 100% o f nominal concentrations (Appendix IV, Table 4). Samples collected during Week 6, Day 0 o f 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 o f 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 o f variation were 4.6%, 11% and 3.6%, respectively. These values represented 111,97 and 95% o f nominal concentrations (Appendix IV, Table 5). Analysis o f 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 o f a test sample is shown in Appendix IV, Figure 7. M ortalities and Clinical Observations No adult mortalities occurred in the control group or in any o f the treatm ent groups during the course o f the test. Several birds were noted with head or foot lesions and feather loss as a result o f pen wear and/or penmate aggression during the course o f 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 W hen compared to the control group, there ware 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 o f 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. W ith the exception o f 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 o f the differences observed between the treatment groups and the control group were statistically significant for any o f 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. C00036 W ildlife 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. W hile statistically significant (p<0.05) differences between the control and the 1.8 ppm a.i. test group were observed during W eek 5 and 6 of the study, these differences were not considered treatm ent-related due to the lack o f a concentration-response relationship at the greater doses. A t the 6.2 ppm a.i. test concentration, there was a statistically significant reduction in feed consumption during W eek 4 and W eek 6 when compared to the control group. However, these differences were not dose-responsive and the significance o f the apparent reduction at Week 6 may have been a consequence o f 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 W eek 5 or 7. Thus, at Week 6, all treatment group feed consumption rates were significantly reduced from the control level but were actually higher than observed at Week 5. As a result, the reductions in feed consumption at 6.2 ppm a.i. were not considered to be treatment-related. There was a treatment-related effect upon feed consumption at 17.6 ppm a.i. test concentration when compared to the control group. The reduction in feed consumption at 17.6 ppm a.i. test concentration was consistent throughout the study and statistically significant (p<0.05) from the control group at Weeks 2, 3, 4, 6, 8 and 15. Mean feed consumption measurements are shown in Table 3, and feed consumption measurements by pen are presented in Appendix VII. Gross Necropsy At the end o f 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 o f W eek 20 and were then euthanized and subjected to gross necropsy. When compared to the control group, there was an increased incidence in the number o f 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 o f age at the time o f 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. 990*17 W ildlife International, Ltd. Project Number 454-104 -23- treatment group was noted with abrasions on the rump. A ll findings observed were considered to be incidental to treatm ent H istopathology Light microscopic examination was performed on selected tissues by a board certified pathologist at Experimental Pathology Laboratories, Inc. Herndon, VA. Sections o f liver, brain, kidney, gonad, proventriculus, gall bladder, adipose tissues, and bursa o f 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 o f Fabricius o f adult male and females, or their offspring at any o f the concentrations tested. Additionally, there were no lesions considered to be treatment-related noted in adipose tissue o f adult males or females and offspring o f both sexes, or in the testes o f male offspring. Testes o f 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 o f testicular regression for adult males in the 17.6 ppm a.i. treatm ent 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 o f 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 o f 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 o f Potassium Perfluorooctanesulfonate from Egg Membrane, Albumen, and Yolk for analysis using HPLCElectrospray/M ass Spectrometry. Centre Study Number 023-065". 0 0 0 ,1 8 W ildlife International, Ltd. Project Number 454-104 -24- R eproductive Results There were no apparent treatment-related effects on egg production at any o f 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. W hen 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. W hile there was a significant (p<0.05) reduction in the number o f viable embryos at the 1.8 ppm a.i. test concentration, the reduction was due to few er eggs being set for incubation at this level. Pen P207 o f die 1.8 ppm a.i. level did not lay during the week that eggs were collected for incubation. As a result, there were fewer hatchlings and 14-day old survivors in the 1.8 ppm a.i. treatment group. However, when the reproductive endpoints were expressed as percentages, no statistically significant differences were observed at any o f the concentrations tested. There was slight reduction in embryo viability at the 6.2 ppm a.i. test concentration when compared to the control. However, this reduction was not dose responsive and was not considered treatment-related. Reproductive data are summarized in Table 6. Reproductive data for individual pens is presented in Appendix XI. Offspring Body Weights There were no apparent treatment-related effects upon the body weights o f hatchlings in any o f the treatment groups. There were also no apparent treatment-related effects upon the body weight o f 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 o f 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 o f 3.402 grams compared to a mean liver weight o f 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 o f the concentrations tested. Mean adult and offspring liver C 99939 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. C O N C L U S IO N Northern bobwhite were exposed to PFOS at dietary concentrations o f 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 o f toxicity were observed at any o f 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 treatm ent-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 o f the concentrations tested. W hen 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 treatm ent-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. treatm ent level that may have been related to the treatment. Histopathological examination o f selected tissues also revealed regression o f 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 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. 1 C C 9040 W ildlife International, Ltd. -26REFERENCES Project Number 454-104 1 U.S. E nvironm ental Protection Agency. 1982. Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, subsection 71-4, Environmental Protection Agency, Office o f Pesticide Programs. W ashington, D.C. 2 A m erican Society fo r T esting and M aterials. 1986. Standard Practice fo r Conducting Reproductive Studies with Avian Species. ASTM Standard E l062-86. Annual Book o f ASTM Standards. Vol. 11.04. Philadelphia, PA. 15 pp. 3 M erck & Co., Inc. 1991. The Merck Veterinary Manual. M erck & Co. Rahway, NJ. 1832 pp. 4 N ational R esearch Council. 1996. Guide fo r the Care and Use o f Laboratory Animals. W ashington, DC. National Academy Press. 125 pp. 5 D unnett, C.W . 1955. A M ultiple Comparison's Procedure for Comparing Several Treatments with a Control. Jour. Amer. Statis. Assoc. 50:1096-1121. 6 D unnett, C.W . 1964. New Tables for Multiple Comparisons with a Control. Biometrics 20:482491. C00041 Table 1 Mean M easured Concentrations (ppm a.i.) o f PFOS in Avian Diet from a Northern Bobwhite Pilot Reproduction Study Nominal Concentration1 (ppma.i.) Day 0 Week 1 Interval Day 7 Week 6 DayO 3RSSSESES Control Measured < 0.879J <1.41 <1.41 Mean 1.8 Measured 1.8 % Nominal 100 Mean 6.2 Measured 6.0 % Nominal 97 Mean 17.6 Measured 17.6 % Nominal 100 1.8 1003 6.2 103 3 17.2 98 3 2.0 111 6.0 97 16.8 95 Project Number 454-104 1 N om inal co n centrations an d resu lts o f diet an aly sts w ere co rrected fo r d ie change in te st su b stan ce p u rity from 90.49% to 86.9% . 1 T he lim it o f q u an titatio n (LO Q ) w as equivalent to 0 .8 7 9 pp m a .i. fo r W eek I , D ay 0 an d 1.41 ppm a .i. fo r W eek 1, D ay 7 and W eek 6 , Day 0. 3T he m ean percent o f th e D ay 0 values. 1>1 a i j x u t ^ K ^ e g t f g : r< n ; 1 fe n i n .' k b v li e H l j l I e n M ^ c i S a l j i T l i P 7)! I iq e ii e &1 3cq inc V ; :i g ' i t i ( l u e V ed I i- 24 4 \ anl M ?V ; i 1 3 >| n j i ) S> 1c s i 1 2 i: -1 : 12 3 <1 fi 7 2 t< - \ :26 3 U\ 14 Il g V e : 3 a g : <b n >e ' f<el il u ji V e : 2 n g I-1 hI I 8 1) 1) U- 1 S3BS3B333S3333333333333BS3SS33S33S3 42 1 i 2 4 A . 1 4 .4 ( i 2 2 3 2 fi l 2 2: 1 l 4 9n 2< 3 <2 24 4 76 b : 7\ 0 2K \ : n 1 22 6 ,, .. 1pa i . s) i 3 L1 1 13 4 A - - i ?V i l 1 211 :22 ( 21 ( - - s> i 6 i: r i l ( t 4 - - _ .. -- --- S= m ce m P k i 1- ) * s s SS s= a s =5 a 12 4 42 3C 2 12 16 26 7 22 1 __ -- --- _ - - lh ige 2 2 4 10 0 _ -- - IV . 7\ m 2 2 . 1 0 7 ( 2 t" .2 _ _ IP a L . s) i 2 i 12 A 1A - - I ?V : 3 2K i 137 a U 2 A - - s) y 7 0 i 23 i .3 t - - __ -- .. -- -- - M -- _ -- --- -- P ro tect N iim h e r 4 S 4-1 04 1 7.5 b } 2): - 0 * 1 >1 - 1 9 6 C 1 fi < - 2 4 C l! 6 * i * i 5 < 2 Ipn .. 5) l 4 (i 72 * 2 7 > fi 2 I ? IX s: ) II 1 4 2 11 1 3 2 .6 > 14 C 21 e4 4 'i C X. 1 \ 2: 5 7 4 5 6 5 1b U (* " , a ic u 1' e ii ti e ( Ci. Tu n a ai fa t x y Vij 111 n il o y d b c n i f s e e a c b te t o 1: a e :i q EBil 1 - Dit i 3i a a la >1 . B n s . i h i r z d e l t l : z l Ve d 6 i t i t i t i a l j d H r n f r a i b c a t r l p ) ii<D.Ci. 93 > 4 1 7 .1 71 75 28 -8 2 0 7 16 19 3 14 " = = = a = dsaF ( SSSSSS8KB E q e: il u I G X i) ' a d n 1 /i a i PS.4.1 > 2" y * '.3 : 4 2 X 22 .. 1 . 4 S 1 1 !< :t :4 ]?< j p d a i. 5) 22 X i. ! 4 a i 1 C : i * 2 1 1p d a i. ) 4 2 !76 : 4 s ] p n a .. x) 1 Il ' C * : i * 2 i 22 : = S 5 = S = = = = a= = S = S B * 8 3 3 B B a B B 8 B B 8 8 S = 1 d i t s i d n <? il i< a. - ]>ea Kt Vi tt c .li i eit i8 ti e i:> ue td f * Sai s icil / ii 'e e t in n tle Xil o f o j a p< ). sBsaaBBSsaaaassaaBSBaBBaBsaaas&a Ci .1 O O 'al ; 3 C bii 'd ) f m N tb i l b? ite du ioi Sti y lh F( > 5 i o 1 : i : 14 > 31 1 : 3< (5 34 :5 COOCM.S CO9 0 AG C 00047 GOOCHS CCOO c ' O- f~< O -36- W ildlife International, Ltd. Project Number 454-104 Appendix I Page 2 Certificate o f Analysis _____ Analytical Laboratortes, Inc 8644 It--aarehbrtva Stata CoSoq, FA 1BB0 wtmosnbalakoom Phon: (814)231-0032 ftBC (814) 231-1253 Of (814) 231-1580 INTERIM CERTIFICATE OFAN ALYSIS XnitUm 3 Ceatre Analytic*! Laboratories COA Reference (ft 023-818A Date ofLast Analysis: 08/31/00 ExpirationDate: 08/31/06 Storage Conditions: Frozen <-UFC R e-assessm ent D ate: 08/31/06 'Purity -100% - (sumofmetal impurities, 1.45%+LC/MS impurities, 8.41%+lnorganic Fluoride, 0_59%+NMRimpurities, 1.905%+organic acid impurities, 0.38%rfPOAA, 0.33%) Total impurityfrom all tests " 13.07% Purity - 100%- 13.07%- 86.9% 2Potassium is expected in this salt Connand is therefore not considered an impurity. 1Purity by DSCis generally not applicable to materials of lowpurity. Noendofhermwas observed forthis sample. 4Sulfur in the lasnple appearsto be convertedto SOandhence detected usingthe inorganic anion methodconditions. The inioa result agreeswdl with the sulfur determination in foeelemental analysis, lending confidence to fob interpretation. Based on the results, foe SO*is not considered an imparity. STFA HFBA NFPA PFPA Trifluoroscetic acid Heptaflnorobutyric acid Ncnafluoropentsnoic acid Pentafluoropropanoic acid `Theoretical value calculations based on foe empirical formula, C*Fi7S03TC'(MW-538) This work was conducted under EPAGood Laboratory Practice Standards (40 CFR 160). COA023-018A Page 2 o f3 CCO051 -37- W ildlife International, Ltd. Project Number 454-104 Appendix I Page 3 Certificate o f Analysis Centre Analytical Laboratories, Inc 3UResearch DrWe Stati otlege. ft lasftt wamn.ontratob.oom Phons: (814)231-6032 Fax:(#14)231*1283Of(814)231-1580 INTERIM CERTIFICATE OF ANALYSIS KevitUm 3 Centre Analytical Laboratories COA Reference #: 023-018A LC/MS Purity Profile: Im parity C4 C5 C6 C7 5 w t/w t % T S --------------------------' i3 T ............. " 75 1 .4 8.41 Note: The C4 and C6 values werecalculated usingthe C4and C6 standard calibration curves, respectively. TheC5 valuewascalculated using the averageresult from the C4 and C6 standardcurves. Likewise, the C7 valuewas calculated using die avenge result from the C6 and C8 standard corves. Prepared By: Charles SHK50 Scientist, Centre A nalytical Laboratories i/ 'M 5Se Reviewed By. John Flaherty _____ - / D ate Laboratory M anager, Centre A nalytical L aboratories COAQ23-018A Page 3 o f 3 C000S2 -38- W ildlife International, Ltd. Project Number 454-104 Appendix n Diet and Supplement Formulations Table 1 W ildlife 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 Fertn (Fennatco)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 ..1.0J0L.0lf0l Vitamin and Mineral Premix, when added at 0.32% of the ration, supplied the following amounts per ton: Amount Supplied Per Ton: Vitamin Dj Vitamin A Riboflavin Niacin Pantothenic Acid Vitamin B)2 Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (Menadione Dimethylpyriniidinol Bisulfite) Manganese Zinc Copper Iodine Iron Selenium 2.000. 0001.C.U. 7.000. 0001.U. 6 grams 40 grams 10 grams 8 mgs 600 mgs 64 mgs 1.2 grams 1.2 grams 20,0001.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 fet and a maximum o f 5% crude fiber. 2 Fermentation By-Products (Source ofUnidentified Growth Factors). -39- W ildlife International, Ltd. Project Number 454-104 Appendix II Diet and Supplement Formulations Table 2 Vitamins and Electrolytes Concentrate GUARANTEED ANALYSIS Vitamin A Vitamin D Vitamin E Riboflavin d-Pantothenic Acid Niacin Vitamin B-12 MSBC Folic Acid Thiamine HC1 Pyridoxine Hydrochloride Ascorbic Acid Water Soluble Powder Per 4 oz. 2,5000,000 1,000,000 1,000 750 1,250 2,500 2.5 1,000 65 250 250 3,750 Per lb. 10,000,000 IU 4,000,0001CU 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 M INGREDIENTS: Vitamin A Supplement, D-Activated Animal Sterol (source of Vitamin D3), Alpha Tocopheryl Acetate (source of Vitamin E). Riboflavin Supplement, d-Calcium Pantothenate, Niacin Supplement, Vitamin B-12 Supplement, Menadione Sodium Bisulfite (source o f Vitamin K), Folic Acid, Thiamine HC1, Pyridoxine Hydrochloride, Ascorbic Acid, Sodium Chloride, Calcium Chloride, Magnesium Sulfate, Feme Ammonium Citrate, Potassium Chloride and Dextrose. MIXING PROCEDURE: The vitamin and electrolyte mix was prepared as a ration of approximately 2 grams of Durvet vitamins and electrolytes to approximately 1 gallon o f water. C^0 0 S 4 -40- W ildlife International, Ltd. Project Number 454-104 Appendix IQ 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 g ration 6.2 ppm a.i.: 1.0794 g PFOS + 6098.9 g ration 17.6 ppm a.i.: 3.0839 g PFOS + 6096.9 g ration Basal ration was weighed into a tared Hobart mixing bowl. Approximately 100 g of ration was transferred to a Waring blender. The test substance was weighed into a tared weigh boat and a small m ortar 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 mixing bowl. The blender was rinsed with uncontaminated ration from the bowl, with the rinse being returned to the bowl. The bowl was placed on a Hobart mixer, and the contents were mixed approximately 15 minutes. The premix was weighed into 1000.0 g aliquots, placed in appropriately labeled freezer bags, reweighed, and stored frozen. As needed, the appropriate premix was incorporated into the final diet as follows: 0 ppm a.i.: 23.75 kg ration + 1.250 kg limestone 1.8 ppm 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 - Wildlife International, Ltd, Project Number 454-104 A ppendix IV The A nalysis o f PFOS in Avian D iet CO056 -42- W ildlife International, Ltd. Project Number 454-104 APPENDIX IV ANALYTICAL METHODS AND RESULTS Typical LC/M S O perational Param eters 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 TurbolonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil Cu column (50 mm x 2 nun 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 CO0057 -43- W ildlife International, Ltd, Project Number 454-104 APPENDIX IV TABLE 2 CALCULATIONS The concentration o f PFOS found at the instrument was determined using the following equation: PFOS (mg a.i./L) at instrument = pea^ 8168 intercept) ^ jntemaj conc (mg a.i./L) Determination n f Sample R esidues tPFOSft 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 ,.) ,n am p le = -------- ------- ----------- ia iM weight (Kg)------- --------------------- Determination o f Limit o f Quantitation (LOO-) The method LOQ, expressed as ppm a.i., was determined using the following equation: LOQ (ppm a.i.) = lowest standard concentration (mg a.i./L) x overall dilution factor o f matrix blank* ............ ,, ,, ............... . extract final volum e (L> x dilution factor overall dilution factor o f matrix blank sample = --------------m itiafweight (Kg)----------- " Fortification Recoveries The ppm a.i. measured in each sample is divided by the nominal concentration o f each sample (fortified level, ppm a.i.). This ratio times 100 is the percent recovery o f the method at that level o f fortification. ppm a.i. measured in sample % Recovery = ^ Ifo ^ ik d x 100 cnoosQ Wildlife International, Ltd. Project Number 454-104 Number (454-104-) APPENDIX IV TABLE3 M atrix Blanks and Fortifications Analyzed Concurrently with the Samples Sample Type Interval Concentration o f PFOS (ppm a.i.) Fortified* Measured1,2 Percent Recovery Mean Recovery <*) MAB-1 MAS-1 MAS-2 MAS-3 MAB-2 MAS-4 MAS-5 MAS-6 MAB-3 MAS-7 MAS-8 MAS-9 MAB-4 MAS-10 MAS-11 MAS-12 Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Week 1, Day 0 Week 1, DayO Week 1, DayO Week 1, DayO Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Week 1, Day 7 Week 1, Day 7 Week 1, Day 7 Week 1, Day 7 Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Week 6, DayO Week 6, DayO Week 6, DayO Week 6, DayO 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 m 105 101 98.7 105 104 107 102 1Concentrations woe corrected for change in test substance purity (98.9% to 86.9%) per Certificate ofAnalysis dated October 11,2001. Nominal test concentrations based upon the new test substance purity are 1.8,6.2 and 17.6ppm a.i. 2Less than values correspond to limit ofquantitation (LOQ). 3Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since fortified and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes. O 4Re-extraction of: sample 454-104,105-13 from Week l.DayO; samples 454-104-23 and -26 from Week 1, Day 7 and sample 454-104,105-50 from Week 6, DayO. -------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------- o o S(3/1 Wildlife International, Ltd. Project Number 454-104 APPENDIX IV TABLE 4 Homogeneity of PFOS in Avian Diet Nominal Concentration1 (ppm a.i.) Sample I.D. Number (S-454-104-) Location Sanqrled in Mixing Vessel Measured PFOS Concentratimi1 (ppma.L) 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 = 1.8ppm 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 = 6.0ppm a.i. 97 12 Middle Rigid 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 'Concentrations were corrected for change in te 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 trityare 1.8, 6.2 and 17.6 ppm a.i. 2Mean result of duplicate re-extraction of original nple. 090G 3- -46- W ildlife International, Ltd. Project Number 454-104 APPENDIX IV TABLE 5 Verification of PFOS Concentrations in Avian Diet Nominal Concentration12 (ppm a.i.) Sample I.D. Number (S-454-104-) 0, 2 43 44 Interval (Day Oof Week-) 1 1 6 6 Measured PFOS Mean(X) Mean Percent Concentration1,2 Standard Deviation (SD) of (ppma-i.) Coefficient ofVariation (CV) Nominal <0.879 <0.879 < 1.41 <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 S.273 (see Table 4) X =2.0 ppm a.i. SD = 0.092 ppm a.i. CV = 4.6% (see Table 4) X **6.0ppma.i. SD = 0.67 ppm a.i. CV=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 S D 0.608 pp m a.i. 16.4 CV = 3.6% 1Concentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate ofAnalysis 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 ofquantitation (LOQ). 3Mean result ofduplicate re-extraction oforiginal sample. C^.OOGl .i i i i 'S i i i ' i i t ^ w *\ lux IM <o ~P.T MSL1. *W* nt mm t w kri S3: w~ 47 i i 1 i8 1 i 1S o 1 i 1T 1 i GHM9 W i 5 On i S 1 1 i 12 1 i* 1 i1 1 id 1 i1 1 i < 1 i -i 1 T? o l 1 * g r; i i CO rs ia i < i S 1 1 w-I NM T3 fUK*'. VA J M F7 " Ml i MV i ti*,. 1 >--=>--r*ii 9 i i X :i i TaT i i " tT 1i i i A i j** i *;* __ i 4s1 nm jaf NoM VH 33 A 9L, e* a Bl uJ x '-- M w> R rU i ** i i i i i i 1111 H t 3 TT 1 a o i w> i1'r*T? i o- i i i i i i i i i i ii -- 1111R111111111111i111111111J^A"ttFRS4\i*,AVO3ttoOwM) *S.w_VJ*^--*Rtth1>4t>a-,:1I1ii1111111111111ii1111iii1i vr vf --' CN * _ c* rx ij --j .j n t n r-i t _ i-: 5- .-*K i<s W (V W C\ i t i I O. i 1111 i _a i I i ca i . ' T' : i_ WSW *-H r> t i *> i SR 111i1i111111111111111fA3tiMtnHQmcV!SRCV"3d?*i tSvAwO*otuO--O.eOf~SaH*t'i.'H_._ycf-eM5d* i a t* ed i1111 1i t t11 i1 111111i I1 i I i ' i11*. t CN Ut t 4>O s *-> 1.3 J cWa 'AiVj V i i 1111 J? i OM* i i ' i i 7*5a i i rA i *S*T 'S i I i ai < i i i i i i i i i i i i i i i >-1 aa3*ii1111i11 na C ST S 'S St 'S ~ 't l1 .U55, 11 1111 I 1R y 111*rcdTJ t3 Ah 111 ' (PI --.t R cd tT _L 1A w 1111111 *_-> 1I111 r! t ri i t .r .Ri 11111 Jwuyl w r r-i *T T t t -- .-- HM 11 R111 1111 8t K H Se3t FA A Rt*jta " -R' . o F ?B O,, ' iSaS-1I1l d t DO1 > d t:: sr-r**Cuh 1i1 <* t 1 r a t i OS '4 - o v-r: 1111111111111111_rJ*unt5FAnfE9S>t?fH*SkTi .>tOoc25'riOt4:i --*.+taRRocWnr5atrd5-i-aTOanQaK4Kev33'd.. cu i t i vi yi il) fld -R 111 t*ht3*i11 ui S di _ I11 i n*i -.r jr s~" i R e r0062 -48- Wildlife International, Ltd. Project Number 454-104 APPENDIX IV M ETHOD OUTLINE FO R THE ANALYSIS O F PFO S IN AVIAN D IET Prepare matrix fortification samples in the desired avian feed stock using the dry mix technique. 4 Weigh 10 g samples o f 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 o f methanol with a graduated cylinder and transfer into the French square bottle. 4 Cap bottles and place on shaker table. Allow the samples to shake for a minimum o f 30 minutes at approximately 250 rpm. 4 Vacuum filter with qualitative filter paper and rinse retained feed 3 times with methanol into filtrate. 4 Transfer the filtrate into a 200-mL volumetric flask and bring to volume with methanol. 4 Prepare appropriate dilutions) to bring final concentration into the calibration range o f 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 Amputate and submit sample for LCMS analysis. Figure 1. Analytical method flow chart for the analysis o f PFOS in avian diet. GO 0 0 6 3 -49- W ildlife 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 w eighted (1/x). C C 0964 C'OOGS G ' O O & G C0 0 0 fi7 CO0 0 7 0 CC 007 C C 0072 n< s 1 ib 3 iu Bc sii t(l 6 n< Me he B m te Pi t F pn uc on tu 'V h i r0 II II II II II II II II II II II II II II II II II H II II II II II II il II XTnrnkM* A K A 1fA II II II II II II II II II II II II II II II II II II II II n II U li II II h 11 II il II II il 11 II h II II h II II h II II il II II h H II 6. pp a -] al< 8 s SS 8 SS 8 8 S S 8 S S S 8 8 8 8 8 Weit C ttt} \ 3d C mi 1 sei C inj Mi sai e =a N 0 -2 l 4 4 -6 3 -6 sa = -- SS = sa -- = SS -- = as 8 ss 8 SS s 2i: 19^ 2 2 i: 20 > 3 2 i: 22 1 2 U 2C1 1 2l.` 22 4 92 i 02 i 22 0 .03 i :21 5 92 3 .01 0 :22 3 .04 1 :i< 1 96 2 01 4 20 2 03 1 .17 8 M( u 21 ) 2 :I II 2 :0i 1 12 2 :0' 0 12 2 07 3 11 4 = SS = = = = = ss sa ss * = - = = = = = >.2 ipi a.i F na s s s sss as 8 8 8 8 S S 8 8 8 8 8 8 ss 8 'ife k c JUj ed C mi ee C mi eel aaj e Pa 0 ================ -2 2 4 4 -6 6 -6 == =* = =* = 8 s SS SS 8 *= *= 8 ** 2 1 2M 3 \5', 1 61 2 : 2 ; i 2i i 0 2] 2 !li 9 :i( 1 21 2i) 7 ti: 2 il: 7 ili 2 : i i n 2 !3! 8 !4' 5 52 1 21 2: ) 9 13! 1 i4( 0 !3C 0 sss s ss s chocos l i a i 2:1 3 I' 7 7 !3` 3 r6 13' 2 2: 8 *.3i 4 2; 7 5S 3 2 8 S 3 3 S S S B S S s ss = ss =o = = = = 8 = = mn = ss ss H e n a ts foi t iy 8 ffn agl i as bo W 5ht ian se SS 8 8 8 8 8 8 ca ila s8 lai TO 88 de 8 8ep ite s 8 II R ss as as Appendix VI Table 4 Adult Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study wife 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 6 2 7 1 7 5 Project Number 454-104 17.6 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 0-20 216 217 218 219 220 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 1 217 -8 209 -25 184 -30 -7 221 6 227 -6 221 -33 188 -32 1 252 6 258 -4 254 6 260 12 4 225 5 230 -2 228 5 233 20 1 241 4 245 -16 229 7 236 -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. ^ ,0 0 3 Appendix VH 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 Mean 20 22 23 24 24 28 24 32 27 31 31 35 30 35 36 34 30 30 31 SD 3 2 2 2 3 2 3 4 3 3 4 5 3 5 2 3 2 3 2 CoT\ Project Number 454-104 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 Mean 20 22 23 24 24 28 24 32 27 31 31 35 30 35 36 34 30 30 31 SD 3 2 2 2 3 2 3 4 3 3 4 5 3 5 2 3 2 3 2 CY o o Cl Project Number 454-104 BEST COPY AVAILABLt 1 sdC III II d H II " I I II u i \n I-" oo CT\ o S /-\ ......................... - II II CCO076 BEST COPY AVAILABLt Feed< ^4 M n **M tn es <N cs CM ! CnQQ' 7 7 BEST COPY AVAILABLE u N t r<}0'78 CO0079 ro co so CO o OS C80083 CC9934 Project Number 454-104 -70- eplT_____________________ 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 Females................................................................................................ VI-5 CO0035 -71 - Project Number 454-104 PATHOLOGY SUMMARY CCO096 -72- EPL EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 W ILD LIFE IN T E R N A TIO N A L, LTD. STU D Y NUM BER 454-104 EPL PR O JEC T NUM BER 212-024 PFO S: A P IL O T R E P R O D U C TIO N S T U D Y W ITH T H E N O R T H E R N B O B W H ITE PATHOLOGY SUMMARY Light m icroscopic exam ination w as perform ed on sections o f selected tissues from adult m ale and fem ale Northern bobwhite (Colinus virginianus) w hich w ere untreated or which received various concentrations o f the te s t article (Perfluorooctanesulfonic acid, potassium salt [P FO S ]) in the feed fo r six to 19 w eeks. S elected tissues from untreated approxim ately 12-w eek-o ld offspring o f the adult bobwhite w ere also exam ined by light microscopy. T h e objective o f this study is to evaluate the effects upon adult bobwhite (Colinus virginianus) of d ietary exposure to a test substance over at least a six-w eek period. T h e experim ental design w as as follows: Group * Control to dditi a. i.) 1 .8 ppm a. i. P FO S 6 .2 ppm a. i. P FO S 1 7 .6 ppm a. i. P FO S A d u lts M ales Fem ales 55 55 55 55 O ffsprina ** M ales Fem ales 55 46 55 28 * Bobw hite in the control and 1 7.6 ppm a.i. groups w ere treated fo r 19 w eeks; bobw hite in the 1.8 and 6.2 ppm a .i. groups w ere treated for six w eeks. ** O ffspring in all groups did not receive the test article. CC0087 EPL -73- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 Wildlife International. Ltd. Study Num ber 4 5 4 -1 0 4 MATERIAlg-ANP METHODS A t the com pletion of the various study intervals, all adult bobwhite w ere eu th anized, and necropsies w ere perform ed by W ildlife International, Ltd. S elected offspring w ere euthanized at approxim ately 12 w eeks of age using carbon dioxide gas, and sam ples w ere collected for histopathological 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. (E P L 9), 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 w ere exam ined by light microscopy as available: liver, gallbladder, proventriculus, kidney, brain, gonad (ovary or testis), bursa of Fabricius, and adipose tissue. In som e cases, nonprotocol-required tissues w ere sectioned along with adjacent protocol-required tissues; these w ere evalu ated , and m icroscopic findings w ere recorded a t the discretion o f the p a th o lo g is t. A ll tissues required by protocol are presented in the Histopathology Incidence Tables. M icroscopic findings for each tissue exam ined from each bobw hite are listed in the Histopathology Incidence T ab les. M icroscopic changes w ere graded one to five depending on severity. N ongradable 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 treatm en t group in the Sum m ary Incidence T ab les, together with the total num ber o f anim als in each group fo r which the tissues w ere exam ined. A tabulation of gross observations noted at necropsy with the corresponding m icroscopic change, if appropriate, is presented in the Correlation o f G ross and M icroscopic Findings tables. T h e entries in these tables w ere -2 - EPL -74- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 Wildlife International, Ltd. Study N um ber 4 5 4 -1 0 4 transcribed from the Gross Necropsy records provided by W ildlife In ternational, Ltd. BESU1TS A ll adults and offspring survived to the end o f th eir respective study intervals. N o effects considered possibly related to test article (P F O S ) 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 bobw hite or th e ir offspring, or in the testes o f offspring m ales. T estes o f all adult m ales exhibited sperm atogenesis ch aracterized by the presen ce of num erous m ature sperm atozoa in the sem iniferous tubules, in two 1 7 .6 ppm a .i. m ales, sem iniferous tubules had m arginally d ecreased d iam eter (though sperm atogenesis w as still evident) com pared to other control and 1 7 .6 ppm a .i. testes, resulting in overall sm aller testes which corresponded to grossly observed 1.25 cm right testes. T h e sm aller testes o f these birds w ere m ost consistent with early post-reproductive phase regression, a norm al physiological phenom enon. Th e occurrence of such testicular regression only in the 1 7 .6 ppm a .i. dose group m ay have been a fortuitous even t accentuated by th e sm all group sizes, but the possibility o f a test article effect cannot b e entirely a ile d out. T estes o f all offspring w ere listed as im m ature because th ey 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 morphology w as consistent with norm al physiological im m aturity in young (approxim ately 12-w eek-old) birds rather than a pathological effect. T h e overall sm all diam eter of offspring testes corresponded to m easurem ents of less than 1.5 cm recorded at necropsy. B ecause o f the EPL -75- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 Wildlife international. Ltd. Study Num ber 4 5 4 -1 0 4 sm aller size of the sem iniferous tubules in offspring testes, the m elanin pigm ent bearing cells norm ally present in the testicular interstitium w ere in effect m ore closely packed in a sm aller area, rather than being w idely sep arated by larger sem iniferous tubules as in adult testes. This m elanin pigm ent concentration corresponded to the grossly observed black offspring testes, but w as considered a norm al ag e-related finding and not a pathological effect. All ovaries exam ined in offspring fem ales w ere characterized by num erous oocytes and very sm all, nonpedunculated follicles com pletely em bedded in a prom inent ovarian cortex. This m orphology is consistent with norm al physiological im m aturity in young (approxim ately 12-w eek-o ld ) birds rath er than a pathological effect. S everal 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 w ere sim ilar w hen groups from each generational cohort w ere com pared with each other. Som e o f th ese lesions occurred only in a single bird. For th ese reasons, th ese findings w ere considered incidental and unrelated to te s t article (P F O S ) adm inistration. M ononuclear cell infiltrates in various tissues consisted o f discrete foci o f sm all lym phocytes and plasm a cells; a few heterophils w ere som etim es also present but w ere alw ays 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, or m ixed populations of heterophils and m arophages, respectively. S cattered necrotic adipocytes w ere 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 - crooso EPL -76- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 Wildlife International, Ltd. Study N u m b er 4 5 4 -1 0 4 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 inflam m ation in several tissues including the serosa of the ovary, liver, and gallbladder, and in th e 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 (d eep m agenta globules and bright pink am orphous m aterial consistent with extrafollicular egg yolk). Collectively, these findings w ere consistent with the com m on clinical condition usually referred to as "egg yolk peritonitis." Liver pigm ent deposition consisted o f dust-like to coarsely irregular, brown to golden-brow n pigm ent granules present in hepatocyte cytoplasm , K upffer cells, an d /o r periportal areas; the identity of the pigm ent w as not determ ined, but m ay have been related to bile. Sm all single clusters of hyperplastic 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 m ale. Th e liver of an adult 1 7 .6 ppm a.i. fem ale exhibited am yloid deposition associated with secondary hepatocellular hypertrophy and necrosis. Liver amyloid deposits consisted o f am orphous, pale bluish-violet m aterial which 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 . Vacuolization denoted m ultiple, usually sm all, confluent, poorly dem arcated vacuoles frhich often resulted in a "lace-like" appearance of the hepatocellular cytoplasm . W hen present in most hepatocytes, these changes w ere designated -5- EPL -77- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 Wildlife International, Ltd. Study Num ber 4 5 4 -1 0 4 "diffuse," and w hen present in a few scattered areas, they w ere designated as "fo cal." W hen hepatocellular fatty change occurred predom inantly in the areas ad jacent to bile channels and associated blood vessels, it w as designated as "p e rip o rta l." H epatocellular fatty change w as noted in the livers o f adults and offspring, w hile hepatocellular vacuolization w as seen only In offspring. This generational difference w as 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 w ere 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 w as considered incidental and unrelated to treatm ent. Th ere w as a relative increase in incidence o f hepatocellular vacuolization in fem ale offspring, but the m agnitude w as sm all, and the increase was not clearly dose-related. It is m ost likely th at this finding w as coincidental and not related to test article adm inistration. CONCLUSIONS AND SUMMARY No lesions considered possibly related to test article (P F O S ) 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 the testes of offspring bobw hite. Testes of two high dose (1 7 .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 norm al 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. Th e -6 - C 99992 EPL -78- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-104 Wildlife International, Ltd. Study Num ber 4 5 4 -1 0 4 few other changes in various tissues of adult and/or offspring bobwhite from control and treated groups w ere considered incidental and unrelated to test article (P F O S ) adm inistration. M M G /lcp W , bV vH //)>jP IIA R ffiA R ITA M . G R U EB B EL, D VM , PhD, D iplom ate, A C V P V eterin ary Pathologist I I -7 - G0 0 0 9 3 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 __________________________ Dates_______________ ________ Inspection Reporting 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 uarterly facility in s p e c tio n . EPL Q\ iulaa lliihty# AA ss ss MurMann ci tea IUInit 8/flfl Date 'at Form No. 6-2 (7/2/99) -8 -80- Project Number 454-104 SUM M ARY INCIDENCE TABLES ADULT SACRIFICE CC0095 454-104 Adult Sacrifice Male Bobvhite 81SUMMARY INCIDENCE TABLE 4DIP0SE 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, Patty Change, Focal Hepatocytes, Fatty Change, Periportal Hepatocytes, Hypertrophy, Diffuse Hepatocytes, Necrosis, Focal Infiltrate, Mononuclear Cell Pigment Deposition Serosa, Poreign 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 E P L ______________________________ I Experimental Pathology Laboratories, Inc. Project Number 454-104 CO0096 434-104 Adult Sacrifice Male Bobwhite -82SUMMARY INCIDENCE TABLE rE S T IS (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 Experim ental Pathology L aboratories, Inc. 434-104 Adult Sacrifice Female Bobwhite -83SUMMARY 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, Mineralisation Tubules, Vacuolization 1.1VER (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 3VARY (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 33 1 1 (5) (5) 1 1-3 EPL Experim ental Pathology Laboratories, Inc. Project Number 454-104 454-104 Adult Sacrifice Female Bobwhite -84SUMMARY INCIDENCE TABLE 5VARY (CONTINUED) Serosa, Inflammation, Granulomatous PROVINTRICULUS (NO. EXAMINED) Infiltrate, Mononuclear Cell GROUP CONTROL GROUP 17,6 1 (5) (5) 34 Project Number 454-104 * 1-4 EPL Experim ental Pathology L aboratories, Inc. C00099 -85- Project Number 454-104 H1STOPATHOLOGY INCIDENCE TABLES ADULT SACRIFICE /> 0 0 86 Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 17.6 454-104 Adult Sacrifice Male Bobwhite jj i M A l ADIPOSE TISSUE Foreign Material Infiltrate, Mononuclear Cell Inflammation, Chronic Inflammation, Granulomatous 44444 55555 13579 XXXXX 44 44 4 85 8a 8 13579 XXXXX BRAIN X X XX X XX X X X BURSA OF FABRICIUS NN NN N N N NN N CLOACA XX GALLBLADDER Infiltrate, Mononuclear Cell Serosa, Foreign Material Serosa, Inflammation, Granulomatous XX 2I 2 NX 112 KIDNEY Infiltrate, Mononuclear Cell Tubule s, Mine ralisation 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 1 1 11 1 22 X 2 1 2 1 1 1 11 1 EPL II-l Experimental Pathology Laboratories, Inc. Kay : X*Not ft* M rlc b lt -S o S K t f m I-In c < w > l te A - A u to ly ili 1-atiitM l 2 -* 1 Ig k t/iin d 3 -M d tr< u 4 -aed trataly u v r t S -tavara/hlgh P - a r t m t B>Baaia I H h llg M a t w i l t i n g o m p a tra d organ a-OM Ckadalad ta c ./d a a tlt C-09101 -87- Project Number 454-104 HISTOPATHOLOQY INCIDENCE TABLE GROUP CONTROL GROUP 17.6 454-104 Adult Sacrifice Male Bobwhite J i M A l PROVENTRICULUS Infiltrate, Mononuclear Cell 44444 55555 13579 XX 1 11 44444 88888 13579 X 111 1 TBSTIS Infiltrate, Mononuclear Cell Seminiferous Tubules, Decreased Diaaeter Spermatogenesis mmmmm pPppp mmmmm i ii pp?Pp BPL II-2 Experimental Pathology Laboratories, Inc. t o y j X-Not R M rk b l* K-Mo S t c t l o n M n c o a p l o U A -A u to ty sf* l-al* fM l S p e d e n te 4 ^ w d e rtU ly t w i t S*M vr/M gh FPrMnt flHtenlgit JWteIgwit r ls s 1 f tg on p o lrtd organ it*n*cM Tad i* c ./d * th C O 01.02 -88- Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 17,6 454-104 Adult Sacrifice Female Bobwhite J i M A t ADIPOSE TISSUE Foreign Material Infiltrate, Mononuclear Cell Inflammation, Chronic Inflammation, Granulomatous 44444 55556 24680 XXN X P 1 44444 8888 9 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, Mineralisation Tubules, Vacuolization XXX 1 1 XX 1 11 1 LIVER Amyloid Deposition Bile Ducts, Hyperplasia Bepatocytes. 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 EPL 11-3 Experimental Pathology Laboratories, Inc. Key s X-Mot t a M r t a b l H-do S e c tio n r - I n c ( p l i t i A*A utoly1i l**1alae) 2 - i l l ( h t / 1 ld 3 n o d lr ta 4 -aodratly lev e r S 'iev ere/K Ig h ! le i(n (W teH jneet e - e lliln j oee p aired o rfae m ie tn d u lid lee ./d e a th C-^0103 Project Number 454-104 -89 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 17.6 454-104 Adult Sacrifice Female Bobwhite * i M A L 44444 55556 24680 OVARY XXX X Serosa, Foreign Material P Serosa, Inflammation, Granulomatous 1 44444 88889 2 468 0 XXXXX PROVENTRICULUS Infiltrate, Mononuclear Cell XX 1 11 X 1 111 EPL 11-4 Experimental Pathology Laboratories, Inc. Key R f lw r U b le fM lo S e c tio n 1 - In c o a p e te A A ito !y i1 * 2 -illo lH /illd 3-oderate 4-oderaU 1y sever S M m re /M ffc P-ppMfft l-aanl fHlallptiit -a ls tlu g m m p a ire d orge* b*unschduled t e c ./t e e t h CO0104 -90- Project Number 454-104 C O R R E L A T IO N O F G R O S S A N D M IC R O S C O P IC F IN D IN G S A D U LT S A C R IF IC E 454-104 Adult Sacrifice Species; Bobwhite CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Sex: Males Group Identification: 17.6 - Sacrificed A nim al Number 481 ClientTopography/Site TESTIS 485 TESTIS ClientGross Observations Light 1.25 cm Light "1.25 cm MicroscopicObservations Seminiferous Tubules, Decreased Diameter Seminiferous Tubules, Decreased Diameter Project Number 454-104 III-l r O }J4 O C*> BEST COPY AVAILABLE o o 1C FINDINGS Group Identification: CONTROL - Sacrificed % MicroscopicObservations Ho Comment Required Ho Comment Required No Comment Required Ho Comment Required No. Comment Required. Project Number 454-104 BEST COPY AVAILABLfc c ro io GO0 1 1 0 cc o n i -97- Project Number 454-104 HISTO PATHO LO G Y INCIDENCE TABLES O FFSPRING SACRIFICE G 00112 -98- Project Number 454-104 HISTOPATHOLOQY INCIDENCE TABLE GROUP CONTROL GROUP 1.8 GROUP 6.2 454-104 Offspring Sacrifice Male Bobwhite jj l M A l ADIPOSE TISSUE Infiltrate, Mononuclear Cell Inflaoraation, Chronic Inflammation, Chronic Active Mineralization Necrosis 99999 55555 00122 38 50 6 XXXXX 9999 5555 2244 8904 XX X 1 99999 55555 45556 91293 X XX X 2 2 BRAIN XXXXX XXXX XXXXX BURSA OF FABRICIUS NXXXX XXXX NXXXX CLOACA X GALLBLADDER Infiltrate, Mononuclear Cell XAX X 1 XX X X 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 pp PP p 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 EPL V-l Experimental Pathology Laboratories, Inc. Ky i X-Not A M rkb) N-lfo $ ct1on I-J n c < p 1 e t8 A -A u to ly tl* l-a tn lM l 2 * s1 1 g h tW ld 3-aoderftt 4*nodr*t1y m n n 5*sevr/h1gh P - P r tw it B-Btnign m i lt in g OM p*1m d o rgan u*nschdtt1i<l s K ./d o o th C 001.1.3 -99- Project Number 454-104 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 1.8 454-104 Offspring Sacrifice Female Bobwhite J i H A l ADIPOSE TISSUE Infiltrate, Mononuclear Cell Inflammation, Chronic Inflammation, Chronic Active Mineralization Necrosis 9 999 9 55555 01122 62825 XN 12 1 9 9 9 999 555555 333344 013513 XXXXXX BRAIN XXXXX X X X XX X BURSA OF FABRICIUS X X NXX 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 Bepatocytes, Fatty Change, Diffuse Bepatocytes, Fatty Change, Focal Hepatocytes, Patty 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 X X 3 334 11 PPPPp 11111 E PL Experimental Pathology Laboratories, Inc. Kay i X-Hot R<*urlibt -Ho S a c tlo a I* I* c o * p )a ta A -A utolyi1< 1-alatM l J^ u d a ra ta 4-aadarataly lav ara S*tavara/klgh W rm u t Mwlga m a t i n g ona gal rad organ o-antclw H ilaa aac./tfaatli C "012.4 -100- Project Number 454-104 HISTOPATHOLOQY INCIDENCE TABLE GROUP 17.6 454-104 Offspring Sacrifice Female Bobwhite J i H A t ADIPOSE TISSUE Infiltrate, Mononuclear Cell Inflammation, Chronic Inflammation, Chronic Active Mineralization Necrosis 99999999 55555555 6 77788 88 7059 0348 X XXXXX 11 BRAIN XXXXXXXX BURSA OF FABRICIUS XXXXXXXX GALLBLADDER Infiltrate, Mononuclear Cell X XXX A 11 1 KIDNEY Infiltrate, Mononuclear Cell Tubules, Mineralization XX X XX 1 12 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 2 12 11 33 11 OVARY Immature ppPPpPPP PROVENTRICULUS Infiltrate, Mononuclear Cell 1 1 1 1 11 12 EPL V-3 Experimental Pathology Laboratories, Inc. Ky r X--Wot A tM rk a b le S ctlM I-In c a a p U ti A -A utoly,1l 3-aoitarati 4-e>aru1y n w t 5-*tr/liUli r * f n i M t ( M ij* N >N igM at i l t i l n t om plT*d or u-mcl4u1i n c . l t u t h -101- Project Number 454-104 CORRELATION OF GROSS AND M ICROSCOPIC FINDING S O FFSPRING SACRIFICE C0 0 1 1 6 454-104 Offspring Sacrifice Species: Bobwhite c o r r e l a t io n o f g r o s s a n d m ic r o s c o p ic f in d in g s Sex: Males Group Identification: CONTROL - Sacrificed Animal Number 9503 ClientTopography/Site TESTIS 9508 9515 9520 TESTIS TESTIS TESTIS 9526 TESTIS ClientGross Observations MicroscopicObservations Diffusely black, 0.4 - 0.6 x 0.2 cm Immature; Pigment Concentration [both present) (noted at gross trinming) .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 trinming) 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, Issaature; Pigment Concentration .2 cm x .2 cm white area (noted at gross trimming) to Project Number 454-104 O vi-i o jk La 454-104 Offspring Sacrifice Species: Bobwhite CORRELATION O F GROSS AND MICROSCOPIC FINDING S Snc Males Group Identification: 1.8 - Sacrificed A nim al Number 9528 ClientTopography/Site TESTIS 9529 TESTIS 9540 9544 EXTERNAL TESTIS TESTIS ClientGross Observations Micros 3ne testis .8 x .4 cm, other .5 x .4 cm, both black (noted at gross trinsniag) Immature ; Pigmei One testis .4 x .2 cm, other .5 x .3 cm. both black (noted at gross trimming) Immature; Pigmei Feather loss on rump No Comment Requ Diffusely black, 0.4 x 0.2 cm, Immature ; Pigme bilateral (noted at gross trimming) One testis .5 x .2 cm, other .5 x .3 cm, both black (noted at gross trimming) limature ; Pigme o Project Number 454-104 r v VI-2 ') ' ,U CD C'tOlA C ':o i2 0 *.t e ro ic a C "01.73 Appendix X Table 1 Egg Production (eggs laid/hen/week) from a Northern Bobwbite 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 27 0.64 5 7 6 7 7 7 7 7 7 7 7 7 7 2 117 0.87 202 3 4 5 5 5 6 28 0.67 6 6 7 7 7 6 7 6 7 7 6 6 7 2 115 0.85 203 6 6 7 6 6 7 38 0.90 7 7 7 7 7 6 7 7 7 7 7 7 7 2 130 0.96 204 3 4 6 6 4 6 29 0.69 4 6 7 7 7 7 7 6 6 4 3 3 3 0 99 0.73 205 6 7 6 6 7 7 39 0.93 8 7 6 0 0 0 4 4 7 7 7 7 7 2 105 0.78 Total 21 25 29 27 27 32 Mean 4 5 6 5 5 6 SD 2 1 1 1 1 1 161 32 6 1Eggs laid per hemper day 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 1 *--* vO Project Number 454-104 G C 01PA 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 perday 1.8ppma.L 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 1124 146 29 0.70 6 0.13 Project Number 454-104 5 E t5 r s <* P*-- 'S 'S y 7 S c s $ & w^ n 1 88 lei et H UR HH nR II l II i II 1 6 II 0wi0n t-H AA AA s i II 1 H i jn *-- n H a II R R H1 H i II 1 .o II en fN m m m vn en i n II R II II 1 II il l 'O a r- >o i vo i en H II H II RJ II i II II R 5 1 . . II N H 1 II i II II i II II B. II 1 R <N II 1 II i i es II II II II II 1 H II 1n II VD i fl r* m vo i tIM-* *n CS II II II II 1 II i II II II 1CS II r- CS n 1 11 II 1W4 II w> O vo *n i in rs i i Q s- es II . R e II 1 8.1 II * Il II 1 II i II SS II cl II H CS en *r ir> 'a g Q II & Il II II H2 II " Il II II II II II II II II II II II II G 003P 6 *: od u II 1 II W W- V "1 ; II II II lia il 1 H II II I H II <1 II 1 II _ II II II 1 TO II m m ^ N ** 00 en II II II 1 II II II II 1 " II K II II 1 II II 1 s-/ II i- * * ' L II II II II II 1 II II II II 1 II . II II II II m II II II 1 II 1 H 11 II 1 II m iA irt IA nv Ift wrf II II II 1 II "i II 11 II 1 II II ^ Il CD 0-127 o Il I A II Il II CO9 1 ^ 9 CC9130 CC0131 C r:0132 ce0133 C C O '1 3 4 -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 3.260 3.156 3.373 4.170 3.051 3.402 0.446 Female Liver 9.210 7.606 7.908 5.612 7.889 7.645 1.295 Pen 206 207 208 209 210 Mean SD 1.8ppm a.i. Male Liver 3.161 3.750 4.211 3.496 2.887 3.501 0.514 Female Liver 7.020 8.556 5.744 6.278 4.016 6.323 1.668 C99135 -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 Groins Appendix. XIV Offspring1Liver W eight (g) from a Northern Bobwhite Pilot Reproduction Study with PFOS Control (0 ppm a.i.) Pen Liver 1.8ppm a.i. Pen Liver 6.2ppm a.i. Pen Liver 201 201 202 202 203 203 204 204 205 205 M ean SD 3 .9 0 5 3 .8 0 4 3 .9 7 7 4 .1 2 5 3 .6 8 8 2 .9 7 3 3 .6 1 2 4 .1 3 3 3 .5 6 3 3 .6 8 0 3 .7 4 6 0 .3 3 9 206 206 206 208 208 209 209 209 209 210 M ean SD 3 .8 1 9 3 .3 0 0 3 .6 5 5 4 .4 1 8 3 .4 5 8 4 .1 2 8 4 .1 4 6 3 .5 9 6 4 .0 9 5 2 .9 4 0 3 .7 5 6 0 .4 5 3 211 211 212 212 213 213 214 214 215 215 M ean SD 3 .8 2 6 3 .5 3 3 6 .0 9 7 4 .6 4 5 3 .8 9 9 3 .7 7 1 7 .7 9 5 5 .9 9 6 4 .7 0 1 5 .5 8 2 4 .9 8 5 1.367 1Offspring were approximately 12 weeks o f age at 0 tim e o f euthanasia and tissue collection. 17.6 ppm a.i. Pen Liver 216 216 217 217 218 218 219 219 220 220 M ean SD 4 .3 7 0 3 .0 3 2 2 .9 9 8 3 .0 5 0 3 .3 3 6 3.271 4.281 3.211 3 .4 5 9 3 .2 8 6 3 .4 2 9 0 .4 9 5 Project Number 454-104 O [Jk C-i <1 -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 o f the shell membrane from the shell. 2. The protocol was amended to reduce the number o f eggs collected for analysis from all eggs, to eggs collected during Weeks 1, 3 and 6 o f the test. Eggs collected during Weeks 2 ,4 and 5 will be disposed o f by incineration. 3. The protocol was amended to change the test substance purity from 98.9% to 90.49%. Correspondingly, the test concentrations were changed from 0, 2, 7 and 20 ppm a.i to 0, 1.8, 6.4 and 18.3 ppm a.i. 4. The protocol was amended to indicate that for a seven day period beginning M arch 31 (early W eek 5), eggs would be collected daily for incubation, hatching and rearing o f offspring. The amendment also detailed the conditions o f 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 o f age and listed reproductive parameters to be measured. 5. The necropsy section o f 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 o f 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 o f Week 6. During the extension, the number o f 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 o f Week 6. Adult test birds in the 1.8 and 6.4 ppm a.i. treatment group will be euthanized at the end o f Week 6. Additionally, the amendment required collection o f feather samples at the time o f gross necropsy. 7. The protocol was amended to indicate the raw data and report would be audited by the Quality Assurance Unit and a Good Laboratory Practice compliant final report would be prepared. Additionally, the Sponsor's representative was changed to John Newsted, and his address was added. 8. The protocol was amended to indicate analyses o f egg, blood and tissue samples w ill be reported separately. Results of egg, blood and tissue analyses may be amended to the biological results at a later date. COO-138 -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 o f data to determine statistically significant differences between groups. 10. Study offspring were not euthanized, weighed and disposed o f at 14 days o f age. Instead, offspring were raised to approximately 12 weeks o f 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 ,1 0 and 11 o f 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 o f W eek 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 W ildlife International, Ltd. personnel were involved in die conduct or management o f this study: Avian Toxicology (1) M ark Jaber, W ildlife Toxicologist (2) Joann B. Beavers, Director, Avian Toxicology (3) Linda R. M itchell, Manager o f Ecotox Operations (4) Diana Temple, Laboratory Supervisor (5) Sean P. Gallagher, Senior Biologist, Avian Toxicology Analytical Chemistry (1) W illard B. Nixon, Ph.D., Director o f Chemistry (2) Raymond L. Van Hoven, Ph.D., Scientist, Analytical Chemistry G C 03-40