Document 6RkMD8yBmkqomM7nnZXrzQdpg

PFOS: A REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-108 3M LAB REQUEST NO. U2723 FIFRA Guideline 71-4 OECD Guideline 206 AUTHORS: Sean P. Gallagher Raymond L. Van Hoven Joann B. Beavers Mark Jaber STUDY INITIATION DATE: January 22,2001 STUDY COMPLETION DATE: August 20,2003 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue StPaul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 USA 1-410-822-8600 Page 1 of 196 -00787 Wildlife International, Ltd. Project Number 454-108 - 2GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Reproduction Study with the Northern Bobwhite WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-108 STUDY COMPLETION: August 20,2003 The study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR Part 160, 17 August 1989; OECD Principles 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: Complete signed and dated reports for blood and tissue analyses conducted by Exygen Research and 3M Corporation are not included in the report. Summary tables are included as appendices and full reports will be submitted separately. The study was conducted under multiple protocols. The in-life and 3M analytical portions were conducted under one protocol (Wildlife International, Ltd. study number 454-108), and the Exygen Research analytical portions were conducted under two additional separate protocols (Exygen Research study number 023-066 and 023-070). Analyses of egg contents conducted under Exygen Research study number 023-070 were not conducted in accordance with Good Laboratory Practice Standards, and results of these analyses are reported separately. The analytical phase performed by Exygen Research under stuffy number 023-066 was performed in compliance with EPA TSCA GLP (40 CFR Part 792). STUDY DIRECTOR: Senior Biologist, Avian Toxicology SPONSOR'S REPRESENTATIVE: DATE 00798 W ildlife International, Ltd, Project Number 454-108 -3 - QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR part 160, 17 August 1989; OECD Principles 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 DATE CONDUCTED DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT Test Substance Preparation Matrix Fortification Feed Consumption January 22,2001 January 30,2001 February 6,2001 January 22,2001 January 31,2001 February 7,2001 January 23,2001 January 31,2001 February 8,2001 Body Weights, Environmental Conditions Candling Tissue Collection February 6,2001 June 12,2001 June 19,2001 February 7,2001 June 12,2001 June 20,2001 February 9,2001 June 13,2001 October 1,2001 14-Day Old Body Weights Egg Shell Thickness Measurements Hatchling Body W eights Data Entry Check Analytical Data, Draft Report Raw Data, Draft Report Final Report June 25,2001 June 28,2001 July 2,2001 August 10-13,2001 October 29-November 2 and November 5,2001 November 27-30 and December 3-5,2001 August 20, 2003 June 25,2001 June 28, 2001 July 2,2001 August 13,2001 November 5,2001 December 5,2001 August 20,2003 July 5,2001 July 2,2001 July 5,2001 August 14,2001 November 9,2001 February 5,2002 August 20,2003 J a m ^ H. Colem an Quality Assurance Representative DATE 0-00789 W ildlife International, Ltd, Project Number 454-108 -4REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Reproduction Study with the Northern Bobwhite WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-108 3M LAB REQUEST NO. U2723 STUDY DIRECTOR: Sean P. Gallagher Senior Biologist, Avian Toxicology CHEMISTRY PRINCIPAL INVESTIGATOR: ^ l L. Van Hoven, Ph.D. Scientist, Analytical Chemistry MANAGEMENT: Joann]B. Beavers Director, Avian Toxicology Willard B. Nixon, Ph.D. Director o f Chemistry DATE flak3 DATE ^ j*^-l I DATE Mi DATE C00790 W ildlife International, Ltd. Project Number 454-108 -5 TABLE OF CONTENTS Title Page............................................................................................................................................... 1 Good Laboratory Practice Compliance Statement............................................................................. 2 Quality Assurance Statement.............................................................................................................. 3 Report Approval....................................................................................... 4 Table of Contents................................................................................................................................. 5 Summary.............................................................................................................................................. 9 Introduction......................................................................................................................................... 10 Objectives............................................................................................................................................10 Experimental Design............................................................................... 10 Materials and Methods....................................................................................................................... 11 Test Substance............................................................................................................................. 12 Test Organisms.... ....................................................................................................................... 12 Identification................................................................................................................................ 12 Avian Feed and W ater.................................................................................................................13 Diet Preparation................................................................................................................*.........13 Diet Sampling.............................................................................................................................. 14 Analytical M ethod....................................................................................................................... 14 Study Phases................................................................................................................................ 16 Housing and Environmental Conditions..................................................................................... 16 Observations................................................. 17 Adult Body Weight and Feed Consumption...............................................................................17 Adult Blood Collection................................................................................................................17 Adult Necropsy and Tissue Collection....................................................... 18 Egg Collection and Storage........................................................................................................ 18 Candling and Incubation..............................................................................................................18 Hatching and Brooding................................................................................................................19 Offspring Blood and Tissue Collection..................................................................................... 20 Egg Shell Thickness Measurements and Egg Components Collection.................................... 20 Statistical Analyses......................................................................................................................21 Results and Discussion.......................................................................................................................22 Diet Analytical Results............................................................................................................... 22 Mortalities........... ........................................................................................................................23 Clinical Observations.................................................................................................................. 24 Adult Body Weight..................................................................................................................... 24 Adult Feed Consumption............................................................................................................ 25 Adult Necropsy and Liver Weights............................................................................................25 Histopathology.............................................................................................................................25 Adult Liver and Sera....................................................................................................................26 Reproductive Results...................................................................................................................26 C00791 W ildlife International, Ltd. Project Number 454-108 -6 - TABLE OF CONTENTS PAGE 2 Egg Shell Thickness................................................................................................................... 27 Offspring Body Weights............................................................................................................. 27 Offspring Liver Weights................................................... 27 Offspring Observations............................................................................................................... 28 Offspring Liver and Sera............................................................................................................ 28 Conclusion......................................................................................................................................... 28 References......................................................................................................................................... 29 TABLES AND FIGURES Table 1. Mean Measured Concentrations (ppm a.i.) o f PFOS in Avian Diet from a Northern Bobwhite Reproduction Study...........................................................30 Table 2. Summary of Gross Pathological Observations from a Northern Bobwhite Reproduction Study with PFOS, Adult Birds Found Dead/Euthanized during the Test..................................................................................................................31 Table 3. Mean Adult Body Weight (g) from a Northern Bobwhite Reproduction Study with PFOS......................................................................................32 Figure 1. Mean Adult Male Body Weight (g) from a Northern Bobwhite Reproduction Study with PFOS..................................................................... 33 Figure 2. M ean A dult Fem ale B ody W eight (g) from a N orthern Bobwhite Reproduction Study with PFOS..................................................................... 34 Table 4. Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Reproduction Study with PFOS..................................................................... 35 Figure 3. Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Reproduction Study with PFOS..................................................................... 36 Table 5. Summary of Gross Pathological Observations from a Northern Bobwhite Reproduction Study with PFOS, Adult Birds Euthanized at Test Termination......................................................................................................... 37 Table 6. Mean Liver Weights (g) from a Northern Bobwhite Reproduction Study with PFOS............................................................................................................. 38 Table 7. Summary of Reproductive Performance from a Northern Bobwhite Reproduction Study with PFOS..................................................................... 39 C-00792 W ildlife International, Ltd, Project Number 454-108 -7 - TABLE OF CONTENTS PAGE 3 Figure 4. Mean Reproductive Performance from a Northern Bobwhite Reproduction Study with PFOS......................................................................40 Table 8. Summary o f Reproductive Performance, Normalized as Percentages (%), from a Northern Bobwhite Reproduction Study with PFOS............41 Figure 5. Mean Reproductive Performance, Normalized as Percentages (%), from a Northern Bobwhite Reproduction Study with PFOS......................................... 42 Table 9. Mean Egg Shell Thickness Measurements (mm) from a Northern Bobwhite Reproduction Study with PFOS....................................... 43 Table 10. Mean Body Weight (g) o f Hatchlings and 14-Day Old Survivors from aNorthem Bobwhite Reproduction Study with PFO S.........................................44 APPENDICES Appendix I. Certificate o f Analysis.........................................................................................45 Appendix n . Diet and Supplement Formulations....................................................................48 Appendix HL Diet Preparation................................................................................................... 50 Appendix IV. The Analysis o f PFOS in Avian D iet.................................................................51 A p p e n d ix V . D ia g ra m o f T e s t L a y o u t............................................................ 67 Appendix VI. Reproductive Parameters and Replicate Identification.................................... 68 Appendix VQ. Adult Body Weight (g) from a Northern Bobwhite Reproduction Study with PFO S........................................................................ 73 Appendix VTH. Feed Consumption (g/bird/day) from a Northern Bobwhite Reproduction Study with PFOS....................................................... 81 Appendix IX. Individual Gross Pathological Observations from a Northern Bobwhite Reproduction Study with PFOS...................................... 85 Appendix X. Adult Liver Weights (g) from a Northern Bobwhite Reproduction Study with PFO S........................................................................ 87 Appendix XI. Histopathology Report.........................................................................................89 cn0733 W ildlife International, Ltd. Project Number 454-108 8- - TABLE OF CONTENTS PAGE 4 Appendix X IIA The Analysis o f PFOS in Adult Red Blood Cells and Sera............................121 Appendix XIIB. The Analysis o f PFOS in Adult and Juvenile Liver and Sera........................ 134 Appendix XIII. Reproductive Performance by Pen from a Northern Bobwhite Reproduction Study with PFOS...................................................... 148 Appendix XIV. Reproductive Performance by Week and Pen from a Northern Bobwhite Reproduction Study with PFO S...................................... 170 Appendix XV. Egg Shell Thickness Measurements (mm) per Pen by Week from a Northern Bobwhite Reproduction Study with PFOS..........................187 Appendix XVI. Mean Hatchling Body Weight (g) per Pen by Week from a Northern Bobwhite Reproduction Study with PFOS..........................189 Appendix XVII. Mean 14-Day Old Survivor Body Weight (g) per Pen by Week from a Northern Bobwhite Reproduction Study with PFOS................ 191 Appendix XVEL Offspring Liver Weights (g) from a Northern Bobwhite Reproduction Study with PFO S....................................................................... 193 Appendix X3X. Changes to Study Protocol................................................................................. 194 Appendix XX. Personnel Involved in Study............................................................................... 196 rv W ildlife International, Ltd. -9SUMMARY Project Number 454-108 STUDY: PFOS: A Reproduction Study with the Northern Bobwhite SPONSOR: 3M Corporation WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER 454-108 TEST DATES: Study Initiation - January 22,2001 Experimental Start - January 23,2001 Photostimulation - March 13,2001 First Eggs Set - April 12,2001 Analytical Termination - April 15,2002 Adult Termination - June 19-20,2001 Biological Termination - July 23,2001 Experimental Termination - April 15, 2002 TEST ANIMALS: Northern bobwhite (Colinus virginianus) AGE TEST ANIMALS: 24 weeks o f age at the initiation of the test SOURCE TEST ANIMALS: Morris Quail Farm, Inc. 18370 S.W. 232 Street Goulds, Florida 33170-5399 U.S.A. NOMINAL TEST CONCENTRATIONS: 0,10, 50, and 150 ppm a.i. RESULTS: There were treatment-related mortalities, overt signs o f toxicity and treatment-related effects upon body weight and feed consumption at the 50 and 150 ppm a.i. test concentrations. The 50 and 150 ppm a.i. test concentrations were terminated prior to the reproductive phase of the test. There were no apparent treatment-related effects upon body weight or feed consumption at the 10 ppm a.i. test concentration. There were overt signs of toxicity observed in adults at the 10 ppm a.i. test concentration. Additionally, there were reductions in testes size and treatment-related effects upon overall reproductive performance at the 10 ppm a.i. test concentration. Based on the effects observed at the 10 ppm a.i test concentration, the no-observed-effect concentration for northern bobwhite exposed to PFOS in the diet was determined to be less than 10 ppm a.i., the lowest concentration tested during this study. 000795 W ildlife International, Ltd. Project Number 454-108 -10- INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife Inter national, Ltd. avian toxicology facility in Easton, Maryland 21601. The biological portion o f the test was conducted from January 23, 2001 until July 23, 2001. Raw data generated at Wildlife International, Ltd. and a copy o f the final report are filed under Project Number 454-108 in archives located on the Wildlife International, Ltd. site. Biological specimens are stored at 3M Corporation, St. Paul, Minnesota 55133. OBJECTIVES The objective o f this study was to evaluate the effects upon the adult northern bobwhite (Colinus virginianus) of dietary exposure to the potassium salt of Perfluorooctane Sulfonic Acid (hereafter referred to as PFOS) over a period o f approximately 21 weeks. Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects o f adult exposure to PFOS on the number o f eggs laid, fertility, embryo viability, hatchability, offspring survival, and egg shell thickness 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 (80 males and 80 females) were randomly distributed into one control group and three treatment groups. The test concentrations were selected in consultation with the Sponsor, based upon the results o f a pilot reproduction study (Wildlife International, Ltd. Project Number 454-104) and additional toxicity information provided by the Sponsor. Group 1 2 3 4 PFOS Treatment Groups Nominal Concentration (ppm a.i.) (Control) 0 10 50 150 Pens per Group 16 16 16 16 Extra Pens Hematology 4 4 4 4 Birds per Pen Males Females 11 11 11 11 CC0736 W ildlife International, Ltd. Project Number 454-108 -11 - Each treatment and control group contained 16 pairs o f birds with one male and one female per pen. Four additional pairs of birds were added to each test level (for a total of 20 pairs per level) and maintained for collection o f blood samples during the test. Three treatment groups were fed diets containing either 10, 50, or 150 ppm a.i. of PFOS. The control group was fed diet comparable to the treatment groups, but without the addition o f the test substance. Because overt signs of toxicity were noted at the 150 ppm a.i. test concentration, the dietary concentration for the 150 ppm a.i. treatment level was reduced to 20 ppm a.i. o f PFOS at the beginning of Week 3 of the test. The 20 ppm a.i. level was terminated after Week 4 of the test due to the limited recovery o f birds at this test concentration. For reporting purposes, the 150/20 ppm a.i. test concentration will be referred to as the 150 ppm a.i. test concentration. Adults in the 50 ppm a.i. test concentrations were euthanized during Week 7 of the test due to treatment-related mortality and overt signs of toxicity. Therefore, the test was completed with the control group and the 10 ppm a.i. test concentration only. All adult birds were observed daily throughout the test for signs o f toxicity or abnormal behavior. Adult body weights were measured at test initiation, on Weeks 2, 4, 6, 8, and at adult termination, and feed consumption was measured weekly throughout the test. At the beginning of Week 8, the photoperiod was increased to induce egg production. Following the start of egg production, eggs were set weekly for incubation. Weekly, eggs were selected by indiscriminate draw for egg shell thickness measurement and all remaining eggs were candled prior to incubation to detect egg shell cracks or abnormal eggs. Eggs were also candled twice during incubation to detect infertile eggs or embryo mortality. On Day 21 of incubation, the eggs were placed in a hatcher and allowed to hatch. Once hatching was completed, hatchlings were removed from the hatcher and the group body weight of the hatchlings by pen was determined. At 14 days o f age, the average body weight by parental pen o f all surviving offspring was determined. Upon completion of the test, statistical analyses were performed to determine statistically significant differences between groups. MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "PFOS: A Reproduction Study with the Northern Bobwhite". The protocol was based on procedures outlined in the Environmental Protection Agency's Registration Guidelines Pesticide Assessment Guidelines, FIFRA 0^0797 Wildlife International, Ltd Project Number 454-108 -12- Subdrvision E, H azard Evaluation: Wildlife and Aquatic Organisms, Subsection 71-4; OECD Guideline 206; and the ASTM "Standard Practice for Conducting Reproductive Studies with Avian Species" (1,2,3). Test Substance The test substance, PFOS, was received from 3M Corporation on October 29, 1998 and was assigned Wildlife International, Ltd. identification number 4675 upon receipt. The test substance was a white powder and was identified as: FC-95; Lot 217. The test material had a reported purity o f 86.9% and expiration date o f August 31, 2006 (Appendix I). The test substance was held under ambient conditions in locked storage at the Wildlife International, Ltd. facilities in Easton, Maryland. Concentra tions o f the test substance in the diet were adjusted to 100% active ingredient. Therefore, dietary concentrations are expressed as parts per million active ingredient (ppm a.i.) in the diet. Test Organisms One hundred and seventy-seven (90 males and 87 females) pen-reared northern bobwhite were purchased from Morris Quail Farm, Inc., 18370 S.W. 232 Street, Goulds, Florida 33170-5399, U.S.A. At the start of acclimation, the bobwhite were apparently healthy and phenotypically indistinguishable from wild type. The birds were from the same hatch, approaching their first breeding season and had not been used in any previous testing. At the start o f acclimation, a random number generating function in a spreadsheet program was used to randomize pen assignment for each bird. Immediately prior to test initiation, all potential study birds were examined for physical injuries and general health. Birds that did not appear healthy, either due to injury or inability to acclimate to laboratory conditions, or were outside the weight range for the test, were excluded from the study. All birds were 24 weeks of age at test initia tion (first day o f exposure to test diet) and ranged in weight from 177 to 250 grams at test initiation. Sex o f the birds was determined by a visual examination of the plumage. Identification Adult birds were identified by individual leg bands, each pen was identified with a unique number, and groups o f pens were identified by project number and concentration. Except eggs laid by birds maintained for blood collection, all eggs laid during the study were marked with die 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. 0^0798 W ildlife International, Ltd. -13 - Project Number 454-108 Avian Feed and Water All adult birds and their offspring were given feed and water ad libitum during acclimation and testing. The basal diet fed to both adults and offspring was formulated to Wildlife International, Ltd. specifications by Agway Inc. (Appendix 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. While 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%) (4). Offspring received basal diet without test substance and without the addition of 5% supplemental limestone. Water was supplied by the town o f Easton public water supply. All offspring received a watersoluble vitamin and electrolyte mix in their water (Appendix II, Table 2). Neither the adults nor offspring received any form o f medication in the feed during the test. Feed and water were analyzed periodically in accordance w ith W ildlife International, Ltd. Standard O perating Procedures. Diet Preparation Test diets were prepared by mixing PFOS into a premix that was used for weekly preparation of the final diet. Control diet and each treated diet were prepared weekly beginning on January 23,2001 and presented to the birds on Tuesday of each week. Dietary concentrations were adjusted for purity of the test substance and are presented as parts per million active ingredient (ppm a.i.). Details of the weekly preparation of test and control diets are shown in Appendix III. C0(??39 W ildlife International, Ltd. -14- Project Number 454-108 Diet Sampling Homogeneity of the test substance in the diet was evaluated by collecting six samples from each of the treated diets and one sample from the control diet on Day 0 of Week 1. Samples were collected from the top, middle and bottom o f the left and right sections of the mixing vessel. Control and treatment group diet samples were also collected from the feed troughs on Day 7 o f Week 1 to assess stability o f the test substance under actual test conditions. Additionally, a sample was collected from the control and treatment group diets, when available, during Weeks 2 , 3 , 4 , 8,12,16 and 20 of the test to measure/verify test concentrations. The diet samples were stored frozen or transferred immediately to the Wildlife International, Ltd. analytical chemistry facility for analysis. Analytical Method The method used for the analysis of PFOS in avian diet was based upon methodology developed at Wildlife International, Ltd. and entitled "Analytical Method Verification for the Determination o f PFOS in Avian Diet" (Wildlife International, Ltd. Project No. 454C-110). Three modifications from the cited methodology were incorporated into the present study. The following changes were made. (1) High performance liquid chromatography with triple quadrupole mass detection (HPLC/MS/MS) was used rather than single quadrupole mass detection to reduce potential matrix interferences. (2) The internal standardization procedure was eliminated because an appropriate internal standard could not be identified. (3) A shorter analytical chromatography column (50 mm vs. 100 mm) was used to expedite analysis time. Samples were extracted with methanol, vacuum filtered, diluted in methanol, and then diluted in a 50:50 methanol: NANOpure water dilution solvent so that they fell within the calibration range of the PFOS methodology. A method flow chart is provided in Appendix IV, Figure 1. Concentrations of PFOS in the standards and extracts o f the samples were determined by reverse-phase HPLC/MS/MS using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph interfaced with a PerkinElmer API 3000 Mass Spectrometer operated in multiple reaction monitoring (MRM) detection mode. The mass spectrometer was equipped with a Perkin-Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil Cjg analytical column (50 mm x 2 mm I.D., 3-jxm particle size) cc m o o W ildlife International, Ltd. Project Number 454-108 -15 - fitted with a Keystone Javelin C 18 Guard Cartridge (20 mm x 2 mm I.D.). The instrument parameters are summarized in Appendix IV, Table 1. Calibration standards were prepared in 50:50 methanol: NANOpure water by appropriate dilutions o f a 1.00 mg a.i./L stock solution o f PFOS in methanol. The calibration standards, ranging in concentration from 0.00100 to 0.0100 mg a.i./L, were analyzed with each sample set. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOS on the basis o f individual isomeric components. Linear regression equations were generated using the peak area responses versus the respective concentrations of the calibration standards. An example o f a calibration curve is presented in Appendix IV, Figure 2. The concentration o f PFOS in avian feed samples was determined by substituting the peak areas into the applicable linear regression equation. Typical ion chromatograms of low and high calibration standards are shown in Appendix IV, Figures 3 and 4, respectively. Examples o f equations used in calculations are presented in Appendix IV, Table 2. The instrument limit of detection (LOD) for these analyses was 10.0 pg on-column, calculated from the product of the injection volume (10.0 pL) and the lowest standard concentration 1.00 pg a.i./L = 1.00 pg a.i./pL. The method limit o f quantitation (LOQ) for these analyses was 2.00 ppm a.i., calculated as the product of the lowest calibration standard analyzed (0.001 mg a.i./L) and the overall dilution factor of the m atrix blank sample (2000 L/K g). Exam ples o f calculations are presented in A ppendix IV, Table 2. Along with the sample analyses, seven matrix blanks were analyzed to determine possible interferences. No interferences were observed at or above the LOQ during the sample analyses (Appendix IV, Table 3). A typical ion chromatogram of a matrix blank is presented in Appendix IV, Figure 5. Avian diet samples were fortified at the following levels and analyzed concurrently with the samples to determine the mean procedural recovery. The fortification levels were 5.00, 50.0, and 200 ppm a.i. for sampling intervals Week 1, Day 0; Week 1, Day 7; Week 2, Day 0; and Week 3, Day 0. The fortification levels were 5.00, 25.0, and 100 ppm a.i. for sampling interval Week 4, Day 0. The fortification levels were 5.00 and 25.0 ppm a.i. for sampling intervals Week 8, Day 0; Week 12, Day 0; Week 16, Day 0; and Week 20, Day 0. The method yielded mean procedural recoveries of 101%, 101%, C 'O S O iftmimalhi Project Number 454-108 -16- 102%, 100% and 99%, respectively, for the 5.00, 25.0, 50.0, 100, and 200 ppm a.i. fortification levels (Appendix IV, Table 3). Sample measured concentrations were not corrected for the mean procedural recoveries. A typical ion chromatogram o f a matrix fortification is presented in Appendix IV, Figure 6. Study Phases The primary phases of the study and their approximate durations were: 1. Acclimation - 8 weeks. 2. Pre-photostimulation - 7 weeks. 3. Pre-egg laying (with photostimulation) - 3 weeks. 4. Egg laying - Approximately 10 weeks. 5. Post-adult termination (final incubation, hatching, and 14-day offspring rearing period) - 5 weeks. Housing and Environmental Conditions Housing and husbandry practices were conducted so as to adhere to the guidelines established by the National Research Council (5). The adult birds were housed indoors in batteries o f pens manufac tured by Georgia Quail Farm Manufacturing (GQFM Model No. 0330), measuring approximately 25 X 51 cm. The pens had sloping floors that resulted in ceiling height ranging from 20 to 26 cm. The pens were constructed of galvanized wire mesh and galvanized sheeting. A diagram o f the test layout is presented in Appendix V. W ildlife International, Ltd. Project Number 454-108 -17- The photoperiod in the adult northern bobwhite room was maintained by a time clock. The photoperiod during acclimation and the first seven weeks o f the test was eight hours o f light per day. The photoperiod was increased to 17 hours o f light per day at the beginning of Week 8 to induce egg laying and was maintained at that length until the adult birds were euthanized. Throughout the test, the birds received a mean o f approximately 199 lux (~ 18 ft. candles) o f illumination provided by fluorescent lights that closely approximated noon-day sunlight. O bservations The test birds were acclimated to the facilities and study pens for 8 weeks prior to initiation of the test. During acclimation, all birds were observed daily. Birds exhibiting abnormal behavior or debilitating physical injuries were not used for the test. During the study, all adult birds were observed daily for signs o f toxicity or abnormal behavior. Additionally, all offspring were observed daily from hatching until 14 days o f age. A record was maintained o f all mortalities and clinical observations. Adult Body W eight and Feed Consumption Adult body weights were measured at test initiation, on Weeks 2,4, 6, 8, and at adult termination. Body weights were not measured during egg laying because of the possible adverse effects handling may have on egg production. Feed consumption for each pen was measured weekly throughout the test. Feed consumption was determined by weighing the freshly filled feeder on Day 0, recording the amount of any additional diet added during the week, and weighing the feeder and remaining feed at the end 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 of total feed consumption. Adult Blood Collection An additional four pairs o f adult birds in each test level were maintained for collection o f blood samples during the test. These pairs received the same diet and were handled in the same manner as other pairs in their test group. However, because of the potential stress of the blood collection procedures, data from the four additional pairs were not used to evaluate treatment related effects. Blood samples were G00303 W ildlife International, Ltd. Project Number 454-108 -18- collected from birds in these designated pairs, when possible, at five week intervals throughout the test. At the time o f adult termination, blood samples were collected from all surviving birds, when possible, prior to euthanasia. All blood samples were separated into serum and hemacytes/platelets, stored frozen and shipped to the sponsor or sponsor's designate for possible analysis. Adult Necropsy and Tissue Collection When the remaining birds from the 150 ppm a.i. treatment group were euthanized after Week 4, no necropsies were performed. All other adult birds that died or were euthanized during the course o f the study were subjected to a gross necropsy. At the conclusion o f the exposure period, all surviving adult birds were euthanized with carbon dioxide gas, necropsied, and disposed o f by incineration. At the time of necropsy, tissues were collected for histopathological examination and analyses. When available, samples o f gall bladder, liver, proventriculus, kidneys, brain, gonads, bursa of Fabricius and adipose tissue were fixed in 10% buffered formalin. Histology samples for the control group and 10 ppm a.i. treatment group were shipped to EPL in Herndon, VA for histopathology. When available, samples of bile, liver proventriculus, kidneys, brain, reproductive organs and adipose tissue were stored frozen and shipped to the Sponsor for possible analysis. Fecal samples were also collected, when available, from the lower digestive tract and stored frozen for possible analysis. Egg Collection and Storage Eggs were collected daily from test pens, cleaned and stored in a cold room until incubation or disposal (for those pens used for blood collection). The cold room was maintained at a mean temperature o f 13.5C 0.2C (SD) with a mean relative humidity of approximately 69% 6% (SD). Groups o f eggs were identified by an alphabetic lot code. All eggs laid in a weekly interval were considered as one lot. Candling and Incubation At the end of the weekly interval, all eggs were removed from the cold room, counted and eggs selected by indiscriminate draw for egg shell thickness measurement. The remaining eggs were candled with a Speed King (Model No. 32) egg-candling lamp to detect egg shell cracks or abnormal eggs. Cracked or abnormal eggs were recorded and discarded. All eggs to be incubated were fumigated with formaldehyde gas in an airtight cabinet with a circulating fan for approximately two hours, to reduce the possibility o f pathogen contamination prior to incubation. Formaldehyde gas was generated by r00804 W ildlife International, Ltd, Project Number 454-108 -19- combining 26 g o f potassium permanganate and 25 ml o f 37% commercial grade formalin in a porcelain bowl at the base of the airtight cabinet. All eggs not discarded or used for egg shell thickness measurements were placed in a Petersime Incubator (Model No. SP20). In the incubator the temperature was maintained at an average 37.5C 0.0C (SD) with an average wet bulb temperature of 30.5C 0.3C (SD) (relative humidity of approximately 60%). The incubator was equipped with a pulsator fen and blades that produced a mild breathing air movement designed to eliminate intracabinet temperature and humidity variation during incubation. In order to prevent adhesion of the embryo to the shell membrane, the incubator was also equipped with an automatic egg rotation device, designed to rotate the eggs from 50 off of vertical in one direction to 50 off o f vertical in the opposite direction (total arc of rotation was 100) every two hours through Day 21 of incubation. Eggs were candled on Day 11 or 12 of incubation to determine embryo viability and on Day 21 to determine embryo survival. Hatching and Brooding On Day 21 o f incubation, the eggs were placed in a Petersime Hatcher (Model No. S-6H) and allowed to hatch. Pedigree baskets constructed o f galvanized steel wire mesh were used to keep hatchlings separated by parental pen of origin. Eggs were not rotated in the hatcher. The average temperature in the hatching compartment was 37.2C 0.0C (SD), and the average wet bulb temperature was raised to 33.3C 0.1C (SD) (relative humidity o f approximately 77%). All hatchlings, unhatched eggs, and egg shells were removed from the hatcher on Day 25 or 26 o f incubation. The group body weight of the surviving hatchlings by pen was determined. Hatchlings were leg banded for identification by pen of origin and then routinely housed according to the appropriate parental concentration grouping in brooding pens until 14 days of age. The hatchlings were fed untreated diet without the addition of 5% supplemental limestone. At 14 days of age, the average body weight by parental pen of all surviving chicks was determined. The chicks were euthanized with carbon dioxide and disposed o f by incineration. Hatchlings were housed in batteries of brooding pens manufactured by Beacon Steel Company (Model B735Q). Each pen measured approximately 72 X 90 X 23 cm high. The external walls and r i'A Q A l- W ildlife International, Ltd. Project Number 454-108 -20- ceilings of each pen were constructed o f galvanized wire mesh and galvanized sheeting. Floors were of galvanized wire mesh. Thermostats in the brooding compartment o f each pen were set to m aintain a temperature of approximately 38 C from the time o f hatching until the birds were 14 days o f age. The average ambient room temperature was 26.0C 1.9C (SD) with an average relative humidity o f 59% 13% (SD). 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 of the last group of 14-day old offspring (Lot J), blood samples were collected from 10 offspring in both the control group and the 10 ppm a.i. treatment group. All blood samples were separated into serum and hemacytes/platelets, stored frozen, and shipped to the sponsor for possible analyses. Additionally, tissues from the 10 offspring in each group were collected for histopathological examination and possible analyses. When available, samples 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, liver, proventriculus, kidneys, brain, reproductive organs and adipose tissue were stored frozen and shipped to the sponsor for possible analyses. Fecal samples were also collected from droppings pans below the floor o f brooders housing Lot J offspring and stored frozen for possible analyses. A single composite sample was collected each for the control group and the 10 ppm a.i. treatment group. Egg Shell Thickness Measurements and Egg Components Collection Weekly throughout the egg laying period, one egg was collected, when available, from each of the odd numbered pens during odd numbered weeks (1,3,5, etc.) and from each of the even numbered pens during die even numbered weeks (2,4,6, etc.). The eggs from weeks 1, 2 and 10 were separated into albumen, yolk, shell and membrane and all components were stored frozen and shipped to the Sponsor for possible analysis. Eggs collected during weeks 3-9 were opened at the waist, and their contents were removed. The egg contents were separated into albumen and yolk, stored frozen and shipped to the Sponsor for possible analysis. The shells were thoroughly rinsed with water and then allowed to air dry for at least one week at room temperature prior to being measured for thickness. The average thickness of the dried shell plus the membrane was determined by measuring five points around the waist of die egg using a micrometer. Measurements were made to the nearest 0.002 mm. W ildlife International, Ltd. Project Number 454-108 -21 - Statistical Analyses Upon completion of the test, an analysis o f variance (ANOVA) was performed to determine statistically significant differences between groups. Dunnett's multiple comparison procedure (6,7) was used to compare the three treatment means with the control group mean and assess the statistical significance of the observed differences. Sample units were the individual pens within each experimental group, except adult body weights where the sample unit was the individual bird. Percentage data were examined using Dunnetfs method following arcsine square root transformation (see Appendix VI for reproductive parameters). Statistical analysis o f the data was performed using Avian Reproduction Data System (ARDS) Software; a validated software package developed by Wildlife International, Ltd. (8). Each of the following parameters was analyzed statistically: 1. Adult Body Weight - Individual body weight was measured at test initiation, Weeks 2 , 4 , 6 , 8 and at adult termination. Statistical comparisons were made between the control group and each treatment group at each weighing interval by sex. 2. Adult Feed Consumption - Feed consumption expressed as grams of feed per bird per day was examined by pen weekly during the test. Statistical comparisons were made between the control and each treatment group. 3. Eggs Laid o f Maximum Laid - The number o f eggs laid per female divided by the largest number o f eggs laid by any one female. This transformation was used to convert the number of eggs laid to a p e rc e n tile v alu e less th a n o r eq u al to 100. 4. 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. 5. Viable Embryos o f Eggs Set - The number of viable embryos at the Day 11-12 candling was divided by the number o f eggs set, per pen. 6. Live 3-Week Embryos of Viable Embryos - The number o f live embryos at the Day 21 candling was divided by the number of viable embryos, per pen. 7. Hatchlings of 3-Week Embryos - The number o f hatchlings removed from the hatcher was divided by the number of live 3-week embryos, per pen. 8. 14-Dav Old Survivors of Hatchlings - The number of 14-day old survivors was divided by the number o f hatchlings by pen. 9. Hatchlings o f Eggs Set - The number o f hatchlings was divided by the number of eggs set by pen. C0G807 W ildlife International, Ltd. Project Number 454-108 -22- 10. 14-Day Old Survivors of Eggs Set - The number o f 14-day old survivors was divided by the number of eggs set by pen. 11. Hatchlings n f Maximum Set - The number of hatchlings per female divided by the largest number of eggs set from any one female. This transformation was used to convert the number o f hatch lings to a percentile value equal to or less than 100. 12. 14-Dav Old Survivors o f Maximum Set - The number of 14-day old survivors per pen divided by the largest number o f eggs set. 13. Egg Shell Thickness - The average egg shell thickness o f indiscriminately selected eggs per pen was measured. 14. Offspring's Body Weight - The mean body weights of surviving hatchlings and 14-day old survivors were measured by parental pen group. 15. Liver Weight - Individual liver weights were measured at adult termination and at termination o f selected Lot J offspring. Statistical comparisons were made by sex between the control group and 10 ppm a.i. group. RESULTS AND DISCUSSION Mature northern bobwhite received PFOS at nominal dietary concentrations o f 10,50, 150 ppm a.i. Due to overt signs o f toxicity noted at the 150 ppm a.i. test concentration, the dietary concentration for the 150 ppm a.i. treatment level was reduced to 20 ppm a.i. of PFOS at the beginning o f Week 3 o f the test. The 20 ppm a.i. level was terminated after Week 4 of the test due to the limited recovery o f birds at this level. Adults in the 50 ppm a.i. test concentrations were euthanized during Week 7 o f the test due to treatment-related mortality and overt signs o f toxicity at this level. Therefore, the test was completed with the control group and 10 ppm a.i. test concentrations only. The control group was fed diet comparable to the treatment groups, but without the addition of the test substance. Diet Analytical Results None o f the control samples showed any indication of the presence of the test substance or of the presence o f co-eluting substances at the characteristic retention time of the test substance. Diet samples were collected from the 10, 50 and 150 ppm a.i. test concentrations on Week 1, Day 0, and were analyzed to evaluate the homogeneity of the test substance in the diet and to verify test substance concentrations. Means and standard deviations for the three test concentrations were 9.75 0.313 ppm a.i., 50.1 0.981 ppm a.i. and 147 4.31 ppm a.i., respectively. The coefficients of variation were 3.21%, 1.96% and 000808 W ildlife International, Ltd, Project Number 454-108 -23 - 2.93%, respectively. These values represented 98%, 100%, and 98% o f nominal concentrations (Appendix IV, Table 4). Samples collected during the test to verify test substance concentrations for the 10,20, 50, and 150 ppm a.i. diets had means and standard deviations o f 10.2 0.553 ppm a.i., 20.8 2.14 ppm a.i., 50.9 2.04 ppm a.i, and 161 15.6 ppm a.i., respectively. The coefficients of variation were 5.44%, 10.3%, 4.01% and 9.66%, respectively. These values represented 102, 104, 102, and 108% o f nominal concentrations (Appendix IV, Table 5). Analysis of diet samples collected from feeders after being held at ambient temperature for 7 days averaged 108%, 100% and 103% o f the Day 0 values for the 10, 50 and 150 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. Mean weekly measured concentrations for each test group are presented in Table 1. M ortalities There were no treatment-related mortalities in the 10 ppm a.i. treatment group. However, there were three incidental mortalities in both the control group and 10 ppm a.i. treatment group during the course o f the study. Control group mortalities occurred during Weeks 14, 19 and 21 o f the test. Mortalities in the 10 ppm a.i. test group occurred during Weeks 12,20 and 21 o f the test. All mortalities in the control group and 10 ppm a.i. treatment group were noted with head lesions and/or neck injuries that suggest m ortality w as caused b y incidental injury. D ue to the nature o f the lesions observed at necropsy, none of the mortalities in the 10 ppm a.i. treatment group were considered to be related to treatment. Five mortalities occurred in the 50 ppm a.i. treatment group and three mortalities occurred in the 150 ppm a.i. treatment groups during the test. Two additional birds in the 150 ppm a.i. group were euthanized due to their debilitated conditions. All mortalities in the 50 and 150 ppm a.i. test concentrations exhibited overt signs of toxicity prior to death and were considered treatment-related. All remaining birds at the 150 ppm a.i. test concentration were euthanized at die beginning of Week 5 of the test. Necropsies were not performed on the birds euthanized from the 150 ppm a.i. treatment group. All remaining birds at the 50 ppm a.i. test concentration were euthanized and necropsied during Week 7 o f the test. Necropsy findings are summarized in Table 2. C00909 W ildlife International, Ltd. Project Number 454-108 -24- Clinical Observations Clinical signs o f toxicity were observed at all concentrations tested. At the 10 ppm a.i. test concentration, signs of toxicity were first observed during Week 5 of the test and continued intermittently until adult termination. Signs o f toxicity at the 10 ppm a.i. test concentration included reduced reaction to external stimuli (sound and movement), ruffled appearance and lethargy. Signs o f toxicity at the 50 ppm a.i. test concentration were first noted during Week 4 of the test, when several birds were noted as ruffled in appearance and lethargic. During body weight measurements at the end of Week 4, several birds in the 50 ppm a.i. exhibited muscle spasms and a loss of coordination following handling. At the 150 ppm a.i. test concentration, signs of toxicity were first noted during Week 2 of the test when one individual was noted as ruffled in appearance and lethargic. All birds in the 150 ppm a.i treatment group exhibited signs o f toxicity induced by the stress of handling during body weight measurement at the end o f Week 2. In several cases, birds convulsed and became rigid after being captured for body weight measurements. Signs of toxicity in the 50 and 150 ppm a.i. treatment groups continued until euthanasia and included reduced reaction to external stimuli (sound and movement), wing droop, loss o f coordination, loss o f righting reflex, lower limb rigidity, convulsions, shallow and rapid respiration, ruffled appearance, lower limb weakness, lethargy, gaping, prostrate posture and spasms. Adult Body Weight There were no apparent treatment-related effects upon adult body weight at the 10 ppm a.i. test concentration, and any differences between the control group and the 10 ppm a.i. test concentration were not statistically significant at any of the body weight intervals. However, there were marked, concentration-responsive, treatment-related reductions in mean body weight for both males and females at the 50 and 150 ppm a.i. test concentrations during the Week 2 body weight interval. Reductions in mean body weight continued for both males and females in the 50 ppm a.i. treatment group through the Week 6 body weight interval. Following reduction of dietary concentrations to 20 ppm a.i. during Week 3 o f the test, males in the 150 ppm a.i. test concentrations made slight weight gains at the Week 4 interval, while females in the 150 ppm a.i. treatment group maintained mean body weight. With the exception o f mean female body weight at the 50 ppm a.i. test concentration during Week 2, all differences from the control group were statistically significant at p < 0.05 or 0.01. Mean body weight measurements are presented in Table 3, and Figures 1 and 2. Individual body weight measurements are presented in Appendix VII. c o s io W ildlife International, Ltd, Project Number 454-108 -25- Adult Feed Consumption There were no apparent treatment-related effects upon feed consumption at the 10 ppm a.i. test concentration. No statistically significant differences between the control group and 10 ppm a.i. treatment group were observed at any of the feed consumption intervals. However, treatment-related reductions in feed consumption were observed at the 50 ppm a.i. test concentration during Weeks 1-6 and at the 150 ppm a.i. test concentration during Week 1 and 2 of the test. All o f die reductions were statistically significant at p < 0.01. The dietary concentration for the 150 ppm a.i. treatment group was reduced to 20 ppm a.i. at the beginning o f Week 3 of the test. Following the reduction o f the dietary concentration to 20 ppm a.i., feed consumption for the group continued to be significantly lower than the control group during Week 3 o f the test Feed consumption at the 20 ppm a.i. test concentration was comparable to the control group during Week 4 of the test. Mean feed consumption measurements are shown in Table 4 and Figure 3. Feed consumption measurements by pen are presented in Appendix VIII. Adult Necropsy and Liver Weights All surviving adults in the control group and 10 ppm a.i. test concentration were subjected to gross necropsy following adult termination. When compared to the control group, there was an increase in the number o f males with small testes in the 10 ppm a.i. treatment group that was considered treatment related. One male in the control group was noted with small testes, compared to seven males in the 10 ppm a.i. treatment group. All other findings were considered unrelated to treatment. Necropsy findings are reported in Table 5 and Appendix DC. When compared to the control group, there were no apparent treatment-related effects upon the mean weights of male livers at the 10 ppm a.i. test concentration. Differences between male liver weights for the 10 ppm a.i. treatment group and the control group were slight and not statistically significant. There was a statistically significant (p < 0.01) increase in mean female liver weight at the 10 ppm a.i. test concentration, when compared to the control group. Mean adult liver weights are presented in Table 6, while individual adult liver weights are presented in Appendix X. Histopathology No lesions considered related to test article (PFOS) administration were noted in liver, kidney, brain, and proventriculus of adult and offspring males and females, bursa of Fabricius in offspring males and females, adipose tissue o f adult males and females, ovaries of adult and offspring female, and testes Wildlife International, Ltd, Project Number 454-108 -26- o f male offspring. The increase in the number o f adult males in the 10 ppm a.i. treated group with reduced testicular size, not accompanied by any morphological change in spermatogenesis, suggests that PFOS may have accelerated early post-reproductive phase regression, a normal physiological phenomenon. Any potential test article effects on the bursa o f Fabricius in adult males and females or adipose tissue in male and female offspring could not be determined since none (bursa o f Fabricius) or only one (adipose tissue) sample was submitted for evaluation. In addition, any potential test-article effects on the gallbladder from treated adult males and females could not be definitively determined due to the extensive autolysis that precluded any accurate diagnosis. The few other changes in various tissues o f adult and/or offspring bobwhite from control and 10 ppm a.i. treated groups were considered incidental and unrelated to test article (PFOS) administration. The full histopathology report is provided in Appendix XI. Adult Liver and Sera Adult blood samples (both red blood cells and sera) collected through Week 15 o f the test were analyzed by Exygen Research. The results for analysis of samples collected for this study are reported together with results for the mallard study run concurrently with the same test concentrations. Summary results o f blood analyses performed for this study are presented in Exygen report Tables XXVI through XLI located in Appendix XIIA The full report for analysis o f blood samples by Exygen Research is located under Exygen Study Number 023-066. "Extraction of Potassium Perfluorooctanesulfonate from Red Blood Cells and Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry". Blood and liver samples collected at the time o f adult termination for the control group and 10 ppm a.i. test concentration were shipped to 3M Environmental Laboratory for analysis. Summary results o f sera and liver analyses conducted by 3M Environmental Laboratory are presented in 3M report Tables 1, 2 and 10 through 15 located in Appendix XIIB. The full report for analysis of sera and liver samples collected at the time of adult termination is located in 3M Environmental Laboratory report E01-1245. "Analytical Phase Report for PFOS: A Reproduction Study with the Northern Bobwhite". Reproductive Results When compared to the control group, there were no apparent treatment-related effects upon egg production at the 10 ppm a.i. test concentration. However, while not statistically significant, there were slight reductions in the number of viable embryos as a percentage o f eggs set, hatchlings as a percentage 0-00812 W ildlife International, Ltd. Project Number 454-108 -27- o f live 3-week embryos and in 14-day old survivors as a percentage o f hatchlings. The slight reductions in fertility, hatchability and offspring survival also resulted in reductions in the numbers of hatchlings and 14-day old survivors as percentages of both the number of eggs set and the maximum number o f eggs set. While slight, the reduction in the number of 14-day old survivors as a percentage of eggs set was statistically significant at p < 0.05. Summaries of the reproductive data are presented in Tables 7 and 8, and in Figures 4 and 5. Reproductive parameters by pen are presented in Appendix XIII and XIV. Egg Shell Thickness There were no apparent treatment related effects upon egg shell thickness at the 10 ppm a.i. test concentration. When compared to the control group, there were no statistically significant differences in egg shell thickness in the 10 ppm a.i. treatment group. Egg shell thickness data are presented in Table 9 and Appendix XV. Offspring Body Weights There were no apparent treatment related effects upon offspring body weight at the 10 ppm a.i. test concentration. When compared to the control group, there were no statistically significant differences in the body weight o f hatchlings or 14-day old survivors from the 10 ppm a.i. treatment group. Offspring body weight data are presented in Table 10, and Appendices XVI and XVII. Offspring Liver Weights There were no treatment-related effects upon the mean weights o f offspring livers at the 10 ppm a.i. test concentration. Any difference between the 10 ppm a.i. treatment group and the control group was not statistically significant. Mean offspring liver weights are presented in Table 6, while individual offspring liver weights are presented in Appendix XVIII. 000813 W ildlife International, Ltd. - 28- Project Number 454-108 Offspring Observations There were incidental observations of neck curl, weakened condition and foot/leg lesions and associated lameness, for offspring in both the control group and 10 ppm a.i. treatment group. All other offspring were normal in appearance and behavior throughout the test. Offspring Liver and Sera Blood and liver samples collected at the time of offspring termination for the control group and 10 ppm a.i. test concentration were shipped to 3M Environmental Laboratory for analysis. Summary results o f sera and liver analyses conducted by 3M Environmental Laboratory are presented in 3M report Tables 1, 2, 10 through 15 located in Appendix XIIB. The full report for analysis of sera and liver samples collected at the time o fjuvenile termination is located in 3M Environmental Laboratory report E01-1245. "Analytical Phase Report for PFOS: A Reproduction Study with the Northern Bobwhite". CONCLUSION There were no apparent treatment-related effects upon adult body weight or feed consumption at the 10 ppm a.i. test concentration. However, minor overt signs of toxicity were observed in adults at the 10 ppm a.i. concentration. Additionally, there were reductions in testes size, and slight, but treatmentrelated, effects upon fertility, hatchability and offspring survival at the 10 ppm a.i. test concentration. There were treatment-related mortalities, overt signs of toxicity and treatment-related effects upon body weight and feed consumption at the 50 and 150 ppm a.i. test concentrations. The 50 and 150 ppm a.i. test concentrations were terminated prior to the reproductive phase of the test. Based on the effects observed at the 10 ppm a.i test concentration, the no-observed-effect concentration for northern bobwhite exposed to PFOS in the diet was determined to be less than 10 ppm a.i., the lowest concentration tested during this study. 000814 000815 Nominal Concentration (ppma.i.) Table 1 Mean Measured Concentrations (ppm a.i.) of PFOS in Avian Diet from a Northern Bobwhite Reproduction Study __________________________________ Interval______________________ ________ Week 1 Week 2 Week 3 Week 4 Week 8 Week 12 W< c 16 Week 20 DayO Day?1 DayO DayO DayO DayO DayO E '0 DayO 0 Measured <2.001 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <3 0 <2.00 Mean 10 Measured % Nominal 983 108 4 103 101 97 109 101 1 99 Mean 50 Measured % Nominal 1003 100 4 97 104 104 Mean 150 Measured % Nominal 98 3 103 4 108 96' 112' 1Feed samples were collected from feed troughs at the end of Week 1 to assess stability of the test substance und 2<2.00 indicates the value was less than the limit of quantitation. 3The mean of homogeneity samples collected at Week 1, Day 0. *The percent of the mean values fix Week 1, Day 0. 5The 150 ppm a.i. treated diet was reduced to 20 ppm a.i. during this interval. - No values available, test group terminated. actual test conditions. 1 01 Project Number 454-108 05 Uk -31- Project Number 454-108 Table 2 Summary of Gross Pathological Observations from a Northern Bobwhite Reproduction Study with PFOS Adult Birds Found Dead/Euthanized during the Test HSUS3 Males_______ ___________ Females 5 0 1 150 2 ppm a.i. ppm a.i. Control 10 (0 ppm a.i.) ppm a.i. 50 1 ppm a.i. 1502 ppm a.i. Number of birds Feathers wet around beak Feather loss Extensive head/neck lesions Vent-pasty Thin Emaciated Loss of muscle mass Prominent keel Intra-cranial bleeding Neck - mid-length fracture Neck - hematoma Heart - no coronary fat Spleen - small and/or pale Abdominal cavity - autolysis Abdominal cavity - small amount of loose blood Crop - empty Gizzard contents - bile-stained Cecal contents - pasty Kidneys-pale Ovary - developing Ovary - regressing Not remarkable 16 0 5 0 0 1 1 2 1 0 0 0 1 3 1 0 1 0 0 0 7 3 0 0 0 0 0 1 1 1 1 0 0 0 1 1 0 0 0 1 0 1 3 3 16 2 10 0 0 00 0 0 12 00 0 0 10 0000 112 1 112 1 112 1 0000 10 0 0 11 10 0 1 10 12 11 112 1 0 100 1 100 0 100 10 00 0 100 0 10 0 10 00 0 0 13 0 1Remaining birds in the 50 ppm a.i. test concentration were euthanized during Week 7 of the te st Gross necropsy observations for birds maintained for collection of blood samples are not reported. Remaining birds in the 150 ppm a.i. test concentration were euthanized at the beginning of Week 5. Gross necropsies were not performed at the termination of the 150 ppm a.i. test concentration. 7 Table 3 Mean Adult Body Weight (g) from a N orthern Bobwhite R eproduction S tu d y w ith PFOS1 Experimental Group {ppm a.i.) Sex Control 10 50 ISO Hale Franale Male Female Male Female Male Franale Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Total Week 0-2 Week 2-4 week 4-6 Week 6-8 Week 8-Term Term Change 208 6 210 4 208 2 206 4 205 -8 210 -7 206 -34 212 -41 214 -1 214 -1 211 -1 209 -1 197** -13 202 -7 172** 13 172** 3 213 1 213 1 210 1 208 1 185** -8 195* -11 187** 172** - 215 -1 214 4 211 -2 209 -1 179** 185** - - *"- 214 1 218 32 209 -1 209 40 -- -- -- 215 7 247 39 208 -1 249 42 , -- - -* The m eans for body w eights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. * Significantly different from the control at p < 0.0 5 * * Significantly different from the control at p < 0.01 ^Only surviving birds w ere included in the calculations for each body weight interval. Project Number 454-108 o i-l S Proiect Num ber 454-108 j: "o c -C z I* ' - Mu 3n. <O1H,0. =>"2 CO CO tu ; "5 r Jimiiimiimmmimiiiiiiiiiiiimiiiiiiil I &A S rninnimi iinimiiiiiniiiiiiiiini i iiiiiiiiml 11111II 11II 111!111111111111!1111111Iti !11111!i111 ft * (0 u <r i 4 sg c 13 f ...............................................HI...........ninnimi * ft N -u o M ft * o II C0S19 34- 1 t 1 .. *C m r a L. JS tw . n w M Q ~ x: 1 1 1 1 1 1 t i i i i 1 1 1 1 I 1 1 1 i 1 i i Sf -m 1 n XI 1 IJU -- i - Ul i O ru i; V -------1 1 f-- w b- * ! 1 iimmi T----i ------, i i r -..................................................................................... -----------------------------------------------------1 1 \ n i ________________________________________ =_j --...... p* 11 11 1 L-- tu 1 _1 mu XA ?~ W -* m * t. * <n * W m* **> 9 st o. yX 9 U3 .... _ --iv w N tN W W ^ W N ^ v w v - v i *" "1 ........... J tu u f 3 . -- V J. 9 O3 H O ~ n Mi 4 V CL 0 .b LN C kJ H 1 ; K C O -ftiA iu iu iA u iiiiiiiia iiiiiiiiiiia iiiiiitiiiiiiiii w tw t'm w m w m 'w w iiiiiiiniimiiiiiinnniiiiiinviimiimiiii i -- .......................... 1 i C 00S2O Project Number 454-108 C-<70321 o o o s?, (o0823 cnosP A CC0S25 C00S26 Table 8 Summary o f Reproductive Performance, N orm alized as Perce fcagres /%/ from a Northern Bobwhlte R eproduction S tu dy w ith 70S*12 Reproductive Parameter Control Number of Replicates 2. Total Eggs Laid Eggs Laid/Maximum Laid (%) Eggs Cracked/Eggs Laid (%) Viable Embryos/Eggs Set (%) Live 3-Week Embryos/Viable Embryos (%} Hatchlings/Live 3-Week Embryos (%) 14-Day.Old Survivors/Hatchlings (I) Hatch]ings/Bggs Set (%) 14-Day Old Survivors/Eggs Set (*) Hatchlings/Maximum Set (%) 14-Day Old Survivors/Maximum Set (%) 13 605 74 3 94 99 97 97 90 87 64 62 Experimental Group (ppm a . i . ) 10 SO 13 592 72 0 89 99 89 93 78 72* 56 52 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 150 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A - No eggs available due to adult mortality. %Significantly different from the control at p < 0.0 5 1V alues represent pen m eans for experimental group. Values for each pen are presented in Appendices VII id VII 2 Represents the total number of eggs laid in each group. F) --J O si Project Number 454-108