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PFOS: A REPRODUCTION STUDY WITH THE MALLARD FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-109 3M LAB REQUEST NO. U2723 FIFRA Guideline 71-4 OECD Guideline 206 AUTHORS: Sean P. Gallagher Raymond L. VanHoven Joann B. Beavers Mark Jaber STUDY INITIATION DATE: January 22, 2001 STUDY COMPLETION DATE: August 21, 2003 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 202 Wildlife International, Ltd. Project Number 454-109 -2 - GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Reproduction Study with the Mallard WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-109 STUDY COMPLETION: August 21, 2003 The study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR Part 160, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan Notification No. 3850, Agricultural Production Bureau, 10 August 1984, with the following exceptions: Complete signed and dated reports for blood and tissue analyses conducted by Exygen Research and 3M Corporation are not included in the report. Summary tables are included as appendices and full reports will be submitted separately. The study was conducted under multiple protocols. The in-life and 3M analytical portions were conducted under one protocol (Wildlife International, Ltd. study number 454-109), 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 study number 023-066 was performed in compliance with EPA TSCA GLP (40 CFR Part 792). STUDY DIRECTOR: Senior Biologist, Avian Toxicology SPONSOR'S REPRESENTATIVE: W ildlife International, Ltd Project Number 454-109 -3 - QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR part 160, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all audits and inspections and the dates any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY DATE CONDUCTED Test substance preparation January 22, 2001 Matrix fortification January 30, 2001 Body weight and environmental conditions February 6, 2001 Hatchling body weight May 24, 2001 14-day old body weight May 31, 2001 Egg shell thickness measurements Candling July 5, 2001 July 6, 2001 ARDS data entry check August 14 & 15, 2001 Analytical data and draft report October 29, Novem ber 2 & 5, 2001 Biological data and draft report Final report December 5, 7, 10-12, 2001 August 20, 2003 DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT January 22, 2001 January 31, 2001 January 23, 2001 January 31, 2001 February 7, 2001 May 24, 2001 May 31, 2001 July 5, 2001 July 6, 2001 August 15, 2001 February 9, 2001 May 25, 2001 May 31, 2001 July 6, 2001 July 25, 2001 August 15, 2001 November 5, 2001 Decem ber 12, 2001 August 20, 2003 November 9, 2001 March 11, 2002 August 21, 2003 Robert N. McGee Quality Assurance Representative DAT 2 ', Z a ? 3 W ildlife International, Ltd. Project Number 454-109 -4 - REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Reproduction Study with the Mallard WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-109 3M LAB REQUEST NO. U2723 STUDY DIRECTOR: Senior Biologist, Avian Toxicology CHEMISTRY PRINCIPAL INVESTIGATOR: Scientist, Chemistry MANAGEMENT: DATE W /Q3 DATE Director, Avian Toxicology DATE Director of Chemistry Wildlife International, Ltd. Project Number 454-109 -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................................................................................................................... 12 Test Substance......................................................................................................................... 12 Test Organisms........................................................................................................................ 12 Identification............................................................................................................................ 12 Avian Feed and Water............................................................................................................. 13 Diet Preparation....................................................................................................................... 13 Diet Sampling.......................................................................................................................... 13 Analytical Method................................................................................................................... 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.................................................................................... 17 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..................................................................................................................20 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 W ildlife International, Ltd. Project Number 454-109 6- - TABLE OF CONTENTS PAGE 2 Egg Shell Thickness................................................................................................................27 Offspring Body Weights..........................................................................................................27 Offspring Liver Weights.......................................................................................................... 27 Offspring Observations............................................................................................................ 27 Offspring Liver and Sera......................................................................................................... 27 Conclusion...................................................................................................................................... 28 References...................................................................................................................................... 29 TABLES AND LIGURES Table 1. Mean Measured Concentrations (ppm a.i.) of PFOS in Avian Diet from a Mallard Reproduction Study............................................................................. 30 Table 2. Summary of Gross Pathological Observations from a Mallard Reproduction Study with PFOS, Adult Birds Found Dead/Euthanized during the Test.............................................................................................................. 31 Table 3. Mean Adult Body Weight (g) from a Mallard Reproduction Study with PFOS................................................................................... 32 Figure 1. Mean Adult Male Body Weight (g) from a Mallard Reproduction Study with PFOS...................................................................................33 Figure 2. Mean Adult Female Body Weight (g) from a Mallard R ep rod u ction S tu dy w ith P F O S .............................................................................................................3 4 Table 4. Mean Feed Consumption (g/bird/day) from a Mallard Reproduction Study with PFOS...................................................................................35 Figure 3. Mean Feed Consumption (g/bird/day) from a Mallard Reproduction Study with PFOS...................................................................................36 Table 5. Summary of Gross Pathological Observations from a Mallard Reproduction Study with PFOS, Adult Birds Euthanized at Test Termination...................................................................................................... 37 Table 6. Mean Liver Weights (g) from a Mallard Reproduction Study with PFOS..........................................................................................................38 Table 7. Summary of Reproductive Performance from a Mallard Reproduction Study with PFOS................................................................................... 39 W ildlife International, Ltd. Project Number 454-109 -7 - TABLE OF CONTENTS PAGE 3 Figure 4. Mean Reproductive Performance from a Mallard Reproduction Study with PFOS...................................................................................40 Table 8. Summary of Reproductive Performance, Normalized as Percentages (%), from a Mallard Reproduction Study with PFOS............................. 41 Figure 5. Mean Reproductive Performance, Normalized as Percentages (%), from a Mallard Reproduction Study with PFOS......................................................... 42 Table 9. Mean Egg Shell Thickness Measurements (mm) from a Mallard Reproduction Study with PFOS...................................................................................43 Table 10. Mean Body Weight (g) of Flatchlings and 14-Day Old Survivors from a Mallard Reproduction Study with PFOS.......................................................... 44 APPENDICES Appendix I. Certificate of Analysis..................................................................................... 45 Appendix II. Diet and Supplement Formulations.................................................................. 48 Appendix III. Diet Preparation............................................................................................... 50 Appendix IV. The Analysis of PFOS in Avian Diet............................................................... 51 A p p en d ix V . D iagram o f T est L a y o u t............................................................................................................. 6 7 Appendix VI. Reproductive Parameters and Replicate Identification.................................... 68 Appendix VII. Adult Body Weight (g) from a Mallard Reproduction Study with PFOS...................................................................... 73 Appendix VIII. Feed Consumption (g/bird/day) from a Mallard Reproduction Study with PFOS...................................................................... 81 Appendix IX. Individual Gross Pathological Observations from a Mallard Reproduction Study with PFOS........................................................ 85 Appendix X. Adult Liver Weights (g) from a Mallard Reproduction Study with PFOS...................................................................... 87 Appendix XI. Histopathology Report..................................................................................... 89 Wildlife International, Ltd. Project Number 454-109 8- - TABLE OF CONTENTS PAGE 4 Appendix XIIA. The Analysis of PFOS in Adult Red Blood Cells and Sera........................128 Appendix XIIB. The Analysis of PFOS in Adult and Juvenile Liver and Sera.....................141 Appendix XIII. Reproductive Performance by Pen from a Mallard Reproduction Study with PFOS...................................................................154 Appendix XIV. Reproductive Performance by Week and Pen from a Mallard Reproduction Study with PFOS.....................................................176 Appendix XV. Egg Shell Thickness Measurements (mm) per Pen by Week from a Mallard Reproduction Study with PFOS....................................... 193 Appendix XVI. Mean Hatchling Body Weight (g) per Pen by Week from a Mallard Reproduction Study with PFOS....................................... 195 Appendix XVII. Mean 14-Day Old Survivor Body Weight (g) per Pen by Week from a Mallard Reproduction Study with PFOS............................. 197 Appendix XVIII. Offspring Liver Weights (g) from a Mallard Reproduction Study with PFOS...................................................................199 Appendix XIX. Changes to Study Protocol..........................................................................200 Appendix XX. Personnel Involved in Study.......................................................................202 Wildlife International, Ltd. -9 SUMMARY Project Number 454-109 STUDY: PFOS: A Reproduction Study with the Mallard SPONSOR: 3M Corporation WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-109 TEST DATES: Study Initiation - January 22, 2001 Experimental Start - January 23, 2001 Photostimulation - April 4, 2001 First Eggs Set - April 13, 2001 Adult Termination - June 20-21, 2001 Analytical Termination - April 11, 2002 Biological Termination - July 26, 2001 Experimental Termination - April 11, 2002 TEST ANIMALS: Mallard (Anasplatyrhynchos) AGE TEST ANIMALS: 24 weeks of age at the initiation of the test SOURCE TEST ANIMALS: Whistling Wings, Inc. 113 Washington Street Hanover, IL 61041-0509 U.S.A. NOMINAL TEST CONCENTRATIONS: 0, 10, 50, and 150 ppm a.i. RESULTS: There were treatment-related mortalities, overt signs of toxicity and treatment-related effects upon adult body weight at the 50 and 150 ppm a.i. test concentrations. There were also treatment-related effects upon feed consumption at the 150 ppm a.i. test concentration. The 50 and 150 ppm a.i. test concentrations were terminated prior to the reproductive phase of the test. There were no treatment-related mortalities, overt signs of toxicity, or treatment-related effects upon body weight, feed consumption or reproductive performance at the 10 ppm a.i. test concentration. However, there was an increased incidence of small testes for males in the 10 ppm a.i. test concentration that may have been related to treatment. Based on the effects observed at the 10 ppm a.i test concentration, the no-observed-effect concentration for mallards exposed to PFOS in the diet was determined to be less than 10 ppm a.i., the lowest concentration tested during this study. Wildlife International, Ltd. Project Number 454-109 - 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 of the test was conducted from January 23, 2001 until July 26, 2001. Raw data generated at Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454-109 in archives located on the Wildlife International, Ltd. site. Biological specimens are stored at 3M Corporation, St. Paul, MN 55133. OBJECTIVES The objective of this study was to evaluate the effects upon the adult mallard (Anas platyrhynchos) of dietary exposure to the potassium salt of Perfluorooctane Sulfonic Acid (hereafter referred to as PFOS) over a period of approximately 21 weeks. Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects of adult exposure to PFOS on the number of eggs laid, fertility, embryo viability, hatchability, offspring survival, and egg shell thickness were evaluated. Histopathological examination of selected tissues and analyses of blood and tissue samples were also used to evaluate the effects upon adults exposed to PFOS and then offspring. EXPERIMENTAL DESIGN Mallard (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 (W ild life International, Ltd. Project N um ber 4 5 4 -1 0 5 ) 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 W ildlife International, Ltd. Project Number 454-109 - 11 - Each treatment and control group contained 16 pairs of birds with one male and one female per pen. Four additional pairs of birds were added to each test level (for a total of 20 pairs per level) and maintained for collection of blood samples during the test. Three treatment groups were fed diets containing either 10, 50, or 150 ppm a.i. of PFOS. The control group was fed diet comparable to the treatment groups, but without the addition of the test substance. Because overt signs of toxicity and treatment-related mortalities 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. of PFOS at the begirming of Week 3 of the test. The 20 ppm a.i. level was terminated at the beginning of Week 5 of the test due to the limited recovery of 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. concentration. Adults in the 50 ppm a.i. test concentrations were euthanized at the end of Week 7 of the test due to treatment-related mortality and overt signs of toxicity. Therefore, the test was completed with the control group and the 10 ppm a.i. test concentration only. All adult birds were observed daily throughout the test for signs of toxicity or abnormal behavior. Adult body weights were measured at test initiation, on Weeks 2, 4, 6, 8, and at adult termination and feed consumption was measured weekly throughout the test. At the beginning of Week 11, the photoperiod was increased to induce egg production. Following the start of egg production, eggs were set weekly for incubation. W eekly, eggs w ere selected by indiscriminate draw for egg shell thickness m easurem ent 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 24 of incubation, the eggs were placed in a hatcher and allowed to hatch. Once hatching was completed, hatchlings were removed from the hatcher and the group body weight of the hatchlings by pen was determined. At 14 days of age, the average body weight by parental pen of all surviving offspring was determined. Upon completion of the test, statistical analyses were performed to determine statistically significant differences between groups. W ildlife International, Ltd. Project Number 454-109 - 12 - MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "PFOS: A Reproduction Study with the Mallard". The protocol was based on procedures outlined in the Environ mental Protection Agency's Registration Guidelines Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, Subsection 71-4; OECD Guideline 206; and the ASTM "Standard Practicefor Conducting Reproductive Studies with Avian Species" (1,2,3). Test Substance The test substance, PFOS, was received from 3M Corporation on October 29, 1998 and was assigned Wildlife International, Ltd. identification number 4675 upon receipt. The test substance was a white powder and was identified as: FC-95; Lot 217. The test material had a reported purity of 86.9% and expiration date of August 31, 2006 (Appendix I). The test substance was held under ambient conditions in locked storage at the Wildlife International, Ltd. facilities in Easton, Maryland. Concentra tions of 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 Approximately 84 pairs of pen-reared mallard were purchased from Whistling Wings, Inc., 113 Washington Street, Hanover, IL 61041-0509, U.S.A. At the start of acclimation, the mallard were apparently healthy and phenotypically indistinguishable from w ild type. The birds w ere from the sam e hatch, approaching their first breeding season and had not been used in any previous testing. At the start of acclimation, a random number generating function in a spreadsheet program was used to randomize pen assignment for each bird. Immediately prior to test initiation, all potential study birds were examined for physical injuries and general health. Birds that did not appear healthy, either due to injury or inability to acclimate to laboratory conditions, or were outside the weight range for the test, were excluded from the study. All birds were 24 weeks of age at test initiation (first day of exposure to test diet) and ranged in weight from 781 to 1444 grams at test initiation. Sex of the birds was determined by a visual examination of the plumage. Identification Adult birds were identified by individual leg bands, each pen was identified with a unique number, and groups of pens were identified by project number and concentration. All eggs laid during the study W ildlife International, Ltd. Project Number 454-109 - 13 - were marked with the pen number using a permanent ink marking pen for identification. Hatchlings were identified by wing bands so that they could be traced to their parental pen of origin. Avian Feed and Water All adult birds and their offspring were given feed and water ad libitum during acclimation and testing. The basal diet fed to both adults and offspring was formulated to Wildlife International, Ltd. specifications by Agway Inc. (Appendix II). The basal ration contained at least 27% protein and 2.5% fat, and no more than 5% fiber. The basal diet contained approximately 1.1% calcium, derived from feedstuffs and the 0.9% limestone used in the formulation of the basal diet by Agway. While this level of calcium is sufficient for growth and maintenance rations, additional calcium is required in the ration of breeding birds for egg shell formation. Therefore, an additional 5% (w/w) of limestone (approximately 38.5% Ca) was added to the basal diet for the adults. This raised the calcium level in the diet for the breeding birds to approximately 3%, slightly above the minimum recommended for quail (2.3%) and mallard (2.75%) (4). Offspring received basal diet without test substance and without the addition of 5% supplemental limestone. Water was supplied by the town of Easton public water supply. Neither the adults nor offspring received any form o f m edication in the feed during the test. Feed and water w ere analyzed periodically in accordance with Wildlife International, Ltd. Standard Operating Procedures. Diet Preparation Test diets were prepared by mixing PFOS into a premix that was used for weekly preparation of the final diet. Control diet and each of the three treated diets 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. 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 Wildlife International, Ltd. Project Number 454-109 - 14 - the top, middle and bottom of the left and right sections of the mixing vessel. Control and treatment group diet samples were also collected from the feed troughs on Day 7 of Week 1 to assess stability of the test substance under actual test conditions. Additionally, a sample was collected from the control and treatment group diets, when available, during Weeks 2, 3, 4, 8, 12, 16 and 20 of the test to measure/verify test concentrations. The diet samples were stored frozen or transferred immediately to the Wildlife International, Ltd. analytical chemistry facility for analysis. Analytical Method The method used for the analysis of PFOS in avian diet was based upon methodology developed at Wildlife International, Ltd. and entitled "Analytical Method Verification for the Determination of PFOS in Avian Diet" (Wildlife International, Ltd. Project No. 454C-110). Three modifications from the cited methodology were incorporated into the present study. These changes were (1) the use of high performance liquid chromatography with triple quadrupole mass detection (HPLC/MS/MS) rather than single quadrupole mass detection to reduce potential matrix interferences, (2) the elimination of the internal standardization procedure because an appropriate internal standard could not be identified, and (3) the use of a shorter (50 mm vs. 100 mm) analytical chromatography column 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 P F O S m eth o d o lo g y . A m eth o d flo w chart is p rovid ed in A p p en d ix IV, F igu re 1. C on cen tration s of PFOS in the standards and extracts of 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 Qg analytical column (50 mm x 2 mm I.D., 3-|im particle size) fitted with a Keystone Javelin Cig 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 of a 1.00 mg a.i./L stock solution of 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 W ildlife International, Ltd. Project Number 454-109 - 15 - study samples. No attempt was made to quantify PFOS on the basis of individual isomeric components. Linear regression equations were generated using the peak area responses versus the respective concentrations of the calibration standards. An example of a calibration curve is presented in Appendix IV, Figure 2. The concentration of PFOS in avian feed samples was determined by substituting the peak areas into the applicable linear regression equation. Typical ion chromatograms of low and high calibration standards are shown in Appendix IV, Figures 3 and 4, respectively. Examples of equations used in calculations are presented in Appendix IV, Table 2. The instrument limit of detection (LOD) for these analyses was 10.0 pg on-column, calculated 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 of quantitation (LOQ) for these analyses was 2.00 ppm a.i., calculated as the product of the lowest calibration standard analyzed (0.001 mg a.i./L) and the overall dilution factor of the matrix blank sample (2000 L/Kg). Examples of calculations are presented in Appendix IV, Table 2. Along with the sample analyses, seven matrix blanks were analyzed to determine possible interferences. No interferences were observed at or above the LOQ during the sample analyses (Appendix IV, Table 3). A typical ion chromatogram of a matrix blank is presented in Appendix IV, Figure 5. Avian diet samples were fortified at the following levels and analyzed concurrently with the samples to determine the mean procedural recovery. The fortification levels were 5.00, 50.0, and 200 ppm a.i. for sampling intervals Week 1, Day 0; Week 1, Day 7; Week 2, Day 0; and Week 3, Day 0. The fortification levels were 5.00, 25.0, and 100 ppm a.i. for sampling interval Week 4, DayO. 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%, 102%, 100% and 99%, respectively, for the 5.00, 25.0, 50.0, 100, and 200 ppm a.i. fortification levels (Appendix IV, Table 3). Sample measured concentrations were not corrected for the mean procedural recoveries. A typical ion chromatogram of a matrix fortification is presented in Appendix IV, Figure 6. Wildlife International, Ltd. Project Number 454-109 - 16 - Study Phases The primary phases of the study and their approximate durations were: 1. Acclimation - 3 weeks. 2. Pre-photostimulation - 10 weeks. 3. Pre-egg laying (with photostimulation) - 0 weeks. 4. Egg laying - Approximately 10 weeks. 5. Post-adult termination (final incubation, hatching, and 14-day offspring rearing period) - 5 weeks. Housing and Environmental Conditions Housing and husbandry practices were conducted so as to adhere to the guidelines established by the National Research Council (5). The adult birds were housed indoors in batteries of pens manufac tured by Safeguard Products, Inc. (Model No. 5355), measuring approximately 75 X 90 X 45 cm high. The pens were constructed of vinyl-coated wire mesh. A diagram of the test layout is presented in Appendix V. Each pen was equipped with a feed trough. Weekly, sufficient feed for the feeding period was placed in the trough for each pen and presented to the birds. During the feeding period additional feed was weighed and added to the troughs as needed. Water was supplied by nipple-type waterers. Only birds associated with this study were maintained in the study room in order to avoid excessive disturbances. The average temperature in the adult mallard study room during the course of the test was 21.7C 1.1C (SD) with an average relative humidity of 40% 19% (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 mallard room was maintained by a time clock. The photoperiod during acclimation and the first 10 weeks of the test was eight hours or less of light per day. The photoperiod was increased to 17 hours of light per day at the beginning of Week 11 to induce egg laying and was maintained at that length until the adult birds were euthanized. Throughout the test, the birds received a mean of approximately 188 lux (~ 17 ft. candles) of illumination provided by fluorescent lights that closely approximated noon-day sunlight. W ildlife International, Ltd. Project Number 454-109 - 17 - Observations The test birds were acclimated to the facilities and study pens for approximately 3 weeks prior to initiation of the test. During acclimation, all birds were observed daily. Birds exhibiting abnormal behavior or debilitating physical injuries were not used for the test. During the study, all adult birds were observed daily for signs of toxicity or abnormal behavior. Additionally, all offspring were observed daily from hatching until 14 days of age. A record was maintained of all mortalities and clinical observations. Adult Body Weight and Feed Consumption Adult body weights were measured at test initiation, on Weeks 2, 4, 6, 8, and at adult termination. Body weights were not measured during egg laying because of the possible adverse effects handling may have on egg production. Feed consumption for each pen was measured weekly throughout the test. Feed consumption was determined by weighing the freshly filled feeder on Day 0, recording the amount of any additional diet added during the week, and weighing the feeder and remaining feed at the end of the feeding period (Day 7). The amount of feed wasted by the birds was not quantified, since the wasted feed was normally scattered and mixed with water and excreta. Therefore, feed consumption is presented as an estimate of total feed consumption. Adult Blood Collection Four additional pairs of adult birds were added to each test level to be maintained for collection of blood samples during the test. These pairs received the same diet and were handled in the same manner as other pairs in their test group. However, because of the potential stress of the blood collection procedures, data from the four additional pairs were not used to evaluate treatment-related effects. Blood samples were collected from birds in these designated pairs, when possible, at five week intervals throughout the test. At the time of adult termination, blood samples were collected from all surviving birds, when possible, prior to euthanasia. All blood samples were separated into serum and 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 150/20 pm a.i. treatment group were euthanized at the end of the Week 4, no necropsies were performed. All other adult birds that died or were euthanized during the W ildlife International, Ltd. Project Number 454-109 - 18 - course of the study were subjected to a gross necropsy. At the conclusion of the exposure period, all surviving adult birds were euthanized by cervical dislocation, necropsied, and disposed of by incineration. At the time of necropsy, tissues were collected for histopathological examination and possible analyses. When available, samples of gall bladder, liver, proventriculus, kidneys, brain, gonads, bursa of Fabricius and adipose tissue were fixed in 10% buffered formalin and shipped to EPL in Herndon, VA for histopathology. When available, samples of bile, liver, proventriculus, kidneys, brain, reproductive organs and adipose tissue were stored frozen and shipped to the Sponsor for possible 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 all pens, when available. Eggs to be incubated were washed to reduce the possibility of pathogen contamination before storing them in the cold room. Eggs were washed in a commercial egg washer (Kuhl Egg Washer) with a chlorine based detergent (Kuhl Super CD). Water in the washer was warmed to approximately 45C. The eggs were placed in the wash water and soaked for approximately 15 seconds. The washer's circulation motor was then turned on for approximately three minutes. The eggs were removed from the washer, allowed to cool to approximately room temperature and rinsed with fresh water. The eggs were then stored in a cold room until incubation. The cold room was maintained at a mean temperature of 13.5C 0.2C (SD) with a mean relative humidity of approximately 69% 6% (SD). Groups of eggs were identified by an alphabetic lot code. All eggs laid in a weekly interval were considered as one lot. Candling and Incubation At the end of the weekly interval, all eggs were removed from the cold room, counted and eggs selected by indiscriminate draw for egg shell thickness measurement. The remaining eggs were candled with a Speed King (Model No. 32) egg-candling lamp to detect egg shell cracks or abnormal eggs. Cracked or abnormal eggs were recorded and discarded. All eggs 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 fan and blades that produced a mild W ildlife International, Ltd. Project Number 454-109 - 19 - breathing air movement designed to eliminate intracabinet temperature and humidity variation during incubation. In order to prevent adhesion of the embryo to the shell membrane, the incubator was also equipped with an automatic egg rotation device, designed to rotate the eggs from 50 off of vertical in one direction to 50 off of vertical in the opposite direction (total arc of rotation was 100) every two hours through Day 24 of incubation. Eggs were candled on Day 13 or 14 of incubation to determine embryo viability and on Day 21 to determine embryo survival. Hatching and Brooding On Day 24 of incubation, the eggs were placed in a Petersime Hatcher (Model No. SP-6H) and allowed to hatch. Pedigree baskets constructed of galvanized steel wire mesh were used to keep hatchlings separated by parental pen of origin. Eggs were not rotated in the hatcher. The average temperature in the hatching compartment was 37.2C 0.0C (SD), and the average wet bulb temperature was raised to 33.8C 0.2C (SD) (relative humidity of approximately 80%). All hatchlings, unhatched eggs, and egg shells were removed from the hatcher on Day 27 or 28 of incubation. The group body weight of the surviving hatchlings by pen was determined. Hatchlings were wing 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 ducklings was determined. The ducklings were euthanized with carbon dioxide and disposed of by incineration. Hatchlings were housed in batteries of brooding pens manufactured by Safeguard Products, Inc. Each pen measured approximately 62 X 92 X 25.5 cm high. The walls, floors and ceilings of each pen were constructed of vinyl-coated wire mesh. Thermostats in the brooding compartment of each pen were set to maintain a temperature of approximately 38C from the time of hatching until the birds were five to seven days of age, when the temperature was adjusted maintain a temperature of approximately 29C. The average ambient room temperature was 24.2C 1.2C (SD) with an average relative humidity of 77% 15% (SD). The photoperiod for the hatchlings was maintained by a time clock at 16 hours of light per day. Wildlife International, Ltd. Project Number 454-109 -20 - Offspring Blood and Tissue Collection Prior to euthanasia of the last group of offspring (Lot J), blood samples were collected from 10 offspring in both the control group and 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. When available, samples of gall bladder, liver, proventriculus, kidneys, brain, gonads, bursa of Fabricius and adipose tissue were fixed in 10% buffered formalin and shipped to EPL in Herndon, VA for histopathology. When available, samples of bile, liver, proventriculus, kidneys, brain, reproductive organs and adipose tissue were stored frozen and shipped to the sponsor for possible analyses. Fecal samples were also collected from pans below the floor of brooders housing Lot J offspring and stored frozen for possible analysis. A single composite sample was collected for the control group and a single composite sample was collected for 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 test pens. Eggs were collected from odd numbered pens during odd numbered weeks (1,3,5, etc.) and from the even numbered pens during the even numbered weeks (2,4,6, etc.). The eggs from weeks 1, 2 and 10 were separated into albumen, yolk, shell and membrane and all components were stored frozen. Eggs collected during weeks 3-9 were opened at the waist and their contents removed. The egg contents w ere separated into album en and yolk and the yolk w as stored frozen. A ll egg sam ples w ere transferred to the Sponsor for possible analysis. The shells from Weeks 3 through 9 were allowed to air dry for at least one week at room temperature prior to being measured for thickness. The average thickness of the dried shell plus the membrane was determined by measuring five points around the waist of the egg using a micrometer. Measurements were made to the nearest 0.002 mm. Statistical Analyses Upon completion of the test, an analysis of variance (ANOVA) was performed to determine statistically significant differences between groups. Dunnett's multiple comparison procedure (6,7) was used to compare the three treatment means with the control group mean and assess the statistical significance of the observed differences. Sample units were the individual pens within each experimental group, except adult body weights where the sample unit was the individual bird. Percentage data were examined using Dunnett's method following arcsine square root transformation (see Appendix VI for W ildlife International, Ltd. Project Number 454-109 -21 - reproductive parameters). With the exception of liver weight, statistical analysis of the data was performed using Avian Reproduction Data System (ARDS) Software; a validated software package developed by Wildlife International, Ltd. (8). Liver weights were compared by Student's t-test using Microsoft Excel. Each of the following parameters was analyzed statistically: 1. Adult Body Weight - Individual body weight was measured at test initiation, Weeks 2, 4, 6, 8 and at adult termination. Statistical comparisons were made between the control group and each treatment group at each weighing interval by sex. 2. Adult Feed Consumption - Feed consumption expressed as grams of feed per bird per day was examined by pen weekly during the test. Statistical comparisons were made between the control and each treatment group. 3. Eggs Laid of Maximum Laid - The number of eggs laid per female divided by the largest number of eggs laid by any one female. This transformation was used to convert the number of eggs laid to a percentile value less than or equal to 100. 4. Eggs Cracked of Eggs Laid - The number of eggs determined by candling to be cracked divided by the number of eggs laid, per pen. 5. Viable Embryos of Eggs Set - The number of viable embryos at the Day 14 candling was divided by the number of eggs set, per pen. 6. Live 3-Week Embryos of Viable Embryos - The number of live embryos at the Day 21 candling w as divided by the number o f viable em bryos, per pen. 7. Hatchlings of 3-Week Embryos - The number of hatchlings removed from the hatcher was divided by the number of live 3-week embryos, per pen. 8. 14-Day Old Survivors of Hatchlings - The number of 14-day old survivors was divided by the number of hatchlings per week, by pen. 9. Hatchlings of Eggs Set - The number of hatchlings was divided by the number of eggs set per week, by pen. 10. 14-Day Old Survivors of Eggs Set - The number of 14-day old survivors was divided by the number of eggs set per week, by pen. 11. Hatchlings of Maximum Set - The number of hatchlings per female divided by the largest number of eggs set from any one female. This transformation was used to convert the number of hatch lings to a percentile value equal to or less than 100. Wildlife International, Ltd. Project Number 454-109 -22 - 12. 14-Dav Old Survivors of Maximum Set - The number of 14-day old survivors per pen divided by the largest number of eggs set. 13. Egg Shell Thickness - The average egg shell thickness of indiscriminately selected eggs per pen was measured. 14. Offspring's Body Weight - The group 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 of selected Lot J offspring. Statistical comparisons were made by sex between the control group and 10 ppm a.i. group. RESULTS AND DISCUSSION Mature mallards received PFOS at nominal dietary concentrations of 10, 50, and 150 ppm a.i. Due to extensive overt signs of toxicity and treatment-related mortalities in the 50 and 150 ppm a.i. treatment groups, the experimental design was modified. Adults in the 50 ppm a.i. test concentrations were euthanized at the end of Week 7 of the test. The dietary concentration for the 150 ppm a.i. treatment level was reduced to 20 ppm a.i. of PFOS at the beginning of Week 3 of the test. The 20 ppm a.i. level was terminated at the beginning of Week 5 of the test due to the limited recovery of birds at this level. Therefore, the test was completed with the control group and 10 ppm a.i. test concentrations only. The control group was fed diet comparable to the treatment groups, but without the addition of the test substance. Diet Analytical Results None of the control samples showed any indication of the presence of the test substance or of the presence of co-eluting substances at the characteristic retention time of the test substance. Diet samples were collected from the 10, 50 and 150 ppm a.i. test concentrations on Week 1, Day 0, and were analyzed to evaluate the homogeneity of the test substance in the diet and to verify test substance concentrations. Means and standard deviations for the three test concentrations were 9.75 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 2.93%, respectively. These values represented 98%, 100%, and 98% of nominal concentrations (Appendix IV, Table 4). Wildlife International, Ltd. Project Number 454-109 -23 - Samples collected throughout the test to verify test substance concentrations for the 10, 20, 50, and 150 ppm a.i. diets had means and standard deviations of 10.2 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% of nominal concentrations (Appendix IV, Table 5). Analysis of diet samples collected from feeders after being held at ambient temperature for 7 days averaged 100%, 101% and 97% of the Day 0 values for the 10, 50 and 150 ppm a.i. test concentrations, respectively (Appendix IV, Table 6). A typical ion chromatogram of a test sample is shown in Appendix IV, Figure 7. Mean weekly measured concentrations for each test group are presented in Table 1. Mortalities While there was one incidental mortality in the control group, there were no mortalities in the 10 ppm a.i. treatment group. Three incidental mortalities and ten treatment-related mortalities occurred in the 50 ppm a.i. treatment group and four treatment-related mortalities occurred in the 150 ppm a.i. treatment group. The single incidental mortality in the control group was a male (Pen 210) that died during Week 15 of the test. Gross necropsy of the drake revealed foot lesions on both feet. Internally, hemorrhages were noted in the myocardium, the abdominal air sacs were cloudy and the spleen was enlarged and mottled. Additionally, the liver was firm, mottled and pale, the kidneys were slightly pale and a small amount of clear fluid was noted in the abdominal cavity. Necropsy of the bird's penmate at termination of the adult portion of the study revealed a regressed ovary, but was otherwise unremarkable. Three incidental and ten treatment-related mortalities occurred in the 50 ppm a.i. treatment group and four treatment-related moralities occurred in the 150 ppm a.i. treatment group. Necropsy findings for the treatment related mortalities in the 50 and 150 ppm a.i. test concentrations were similar. Common findings included thin or emaciated condition with a loss of muscle mass and prominent keel, small spleen and pale kidneys. All remaining birds at the 150 ppm a.i. test concentration were euthanized at the beginning of Week 5. Due to the shortened nature of the exposure period, necropsies were not performed. All remaining birds at the 50 ppm a.i. test concentration were euthanized at the beginning of Week 8 of the test, and necropsies were performed following euthanasia. Necropsy findings are summarized in Table 2. Wildlife International, Ltd. Project Number 454-109 -24 - Clinical Observations No overt signs of toxicity were observed at the 10 ppm a.i. test concentration. With the exception of foot lesions and occasional lameness, all birds in the control group and 10 ppm a.i. treatment group were normal in appearance and behavior throughout the test. In the 150 ppm a.i. and 50 ppm a.i. treatment groups, signs of toxicity were first noted at the end of Week 2. One male in the 50 ppm a.i. treatment group and several birds in the 150 pm a.i. treatment group exhibited signs of toxicity induced by the stress of handling during the Week 2 body weight measurements. In several cases, birds convulsed during body weight measurements. Signs of toxicity in both the 50 and 150 ppm a.i. treatment groups continued to be observed until the birds were euthanized. Clinical signs of toxicity observed included depression, reduced reaction to external stimuli (sound and movement), wing droop, loss of coordination, ruffled appearance, thin appearance, lower limb weakness, lethargy, lacrimation and convulsions. 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 treatmentrelated reductions in mean body weight at both the 50 and 150 ppm a.i. test concentrations. With the exception of the mean male body weight at the Week 2 interval, all differences between the control group and birds in the 50 ppm a.i. treatment group were statistically significant at p < 0.01. At the 150 ppm a.i. test concentration, mean male and female body weights were statistically different from the control group at p < 0.01 at both the Week 2 and Week 4 body weight intervals. Between the Week 2 and Week 4 body weight intervals, a weight gain was noted for birds at the 150 ppm a.i. test concentration. However, the weight gain observed was a reflection of the reduction of the concentration of PFOS in the diet to 20 ppm a.i. during Week 3 of the test. Mean body weight measurements are presented in Table 3, and Figures 1 and 2. Individual body weight measurements are presented in Appendix VII. Wildlife International, Ltd. Project Number 454-109 -25 - Adult Feed Consumption There were no apparent treatment-related effects upon feed consumption at the 10 or 50 ppm a.i. test concentrations. No statistically significant differences between the control group and 10 or 50 ppm a.i. treatment groups were observed at any of the feed consumption intervals. However, treatment-related reductions in feed consumption were observed at the 150 ppm a.i. test concentration during Weeks 1 and 2. The reductions were statistically significant at p < 0.05 during Week 1 and at p < 0.01 during Week 2. The dietary concentration for the 150 ppm a.i. treatment group was reduced to 20 ppm a.i. during Week 3 of the test. There was a statistically significant (p <0.01) increase in feed consumption at the 150 (20) ppm a.i. test concentration during Weeks 3 and 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 the 10 ppm a.i. treatment group were subjected to gross necropsy following adult termination. When compared to the control group, there was an increased incidence of males with small testes (approximate length <3.5 cm) in the 10 ppm a.i. treatment group for which a treatment-related effect could not be precluded. Two males in the control group were noted with small testes, compared to seven males in the 10 ppm a.i. group. All other findings were considered unrelated to treatment. Necropsy findings are reported in Table 5 and Appendix IX. When compared to the control group, there were no treatment-related effects upon the mean weights of adult 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 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 the liver, kidney, adipose tissue, brain, and proventriculus of adult and offspring males and females, bursa of Fabricius in offspring males and females, ovaries of adult and offspring females and testes of male offspring. The increase in the number of adult males in the 10 ppm a.i. treated group with reduced testicular size (with or without a corresponding microscopic observation), suggests that PFOS may have accelerated early post reproductive phase regression, a normal physiological phenomenon. Any potential test-article effects on the bursa of Fabricius in adult males and females could not be determined since samples were not W ildlife International, Ltd. Project Number 454-109 -26 - submitted for evaluation. In addition, any potential test-article effects on the gallbladder from adults and offspring could not be definitively determined due to the extensive autolysis that precluded any accurate diagnosis. The few other changes in various tissues of adult and/or offspring mallards 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 of the test were analyzed by Exygen Research. The results for analysis of samples collected for this study are reported together with results for the northern bobwhite study run concurrently with the same test concentrations. Summary results of blood analyses performed for this study are presented in Exygen report Tables X through XXIV located in Appendix XIIA The full report for analysis of blood samples by Exygen Research is located under Exygen Study Number 023-066. "Extraction of Potassium Perfluorooctanesulfonate from Red Blood Cells and Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry". Blood and liver samples collected at the time of adult termination for the control group and 10 ppm a.i. test concentration were shipped to 3M Environmental Laboratory for analysis. Summary results of sera and liver analyses conducted by 3M Environmental Laboratory are presented in 3M report Tables 1, 2 and 10 through 15 lo ca ted in A p p en d ix XIIB. T he fu ll report for a n a ly sis o f sera and liv er sam p les collected at the time of adult termination is located in 3M Environmental Laboratory report E01-1256. "Analytical Phase Report for PFOS: A Reproduction Study with the Mallard". Reproductive Results There were no treatment-related effects upon reproductive performance at the 10 ppm a.i. test concentration as compared to the controls. When compared to the control group, there were no statistically significant differences in any of the reproductive parameters measured in the 10 ppm a.i. test concentration. 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. Wildlife International, Ltd. Project Number 454-109 -27 - Egg Shell Thickness There were no apparent treatment related effects upon egg shell thickness at the 10 ppm a.i. test concentration. When compared to the control group, there were no statistically significant differences in egg shell thickness in the 10 ppm a.i. treatment group. Egg shell thickness data are presented in Table 9 and Appendix XV. Offspring Body Weights There were no apparent treatment related effects upon offspring body weight at the 10 ppm a.i. test concentration. When compared to the control group, there were no statistically significant differences in the body weight of hatchlings or 14-day old survivors from the 10 ppm a.i. treatment group. Offspring body weight data are presented in Table 10, and Appendices XVI and XVII. Offspring Liver Weights There were no treatment-related effects upon the mean weights of offspring livers at the 10 ppm a.i. test concentration. Any difference between the 10 ppm a.i. treatment group and the control group was not statistically significant. Mean offspring liver weights are presented in Table 6, while individual offspring liver weights are presented in Appendix XVIII. Offspring Observations There w ere incidental observations o f neck curl, w eakened condition and foot/leg lesions and associated lameness, for offspring in both the control group and 10 ppm a.i. treatment group. At the 10 ppm a.i. test concentration, a general observation was made that the offspring from Lots C and D were sluggish, with a slight reduced reaction to external stimuli. None of the offspring in the Lots proceeding or following Lots C and D were noted as sluggish or with a reduced reaction to external stimuli. Due to the isolated nature of the clinical signs of toxicity noted, these observations were not considered treatment related. All other offspring were normal in appearance and behavior throughout the test. Offspring Liver and Sera Blood and liver samples collected at the time of offspring termination for the control group and 10 ppm a.i. test concentration were shipped to 3M Environmental Laboratory for analysis. Summary results of sera and liver analyses conducted by 3M Environmental Laboratory are presented in 3M report Tables 1, 2 and 10 through 15 located in Appendix XIIB. The full report for analysis of sera and liver samples W ildlife International, Ltd. Project Number 454-109 -28 - collected at the time of juvenile termination is located in 3M Environmental Laboratory report E01-1256. "Analytical Phase Report for PFOS: A Reproduction Study with the Mallard". CONCLUSION There were no treatment-related mortalities, overt signs of toxicity, or treatment-related effects upon body weight, feed consumption or reproductive performance at the 10 ppm a.i. test concentration. However, there was an increased incidence of small testes for males in the 10 ppm a.i. test concentration that may have been related to treatment. There were treatment-related mortalities, overt signs of toxicity and treatment-related effects upon adult body weight at the 50 and 150 ppm a.i. test concentrations. There were also treatment-related effects feed consumption at the 150 ppm a.i. test concentration. The 50 and 150 ppm a.i. test concentrations were terminated prior to the reproductive phase of the test. Based on the effect observed at the 10 ppm a.i test concentration, the no-observed-effect concentration for mallards exposed to PFOS in the diet was determined to be less than 10 ppm a.i., the lowest concentration tested during this study. Wildlife International, Ltd. -29 - REFERENCES Project Number 454-109 1 U.S. Environmental Protection Agency. 1982. Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, subsection 71-4, Environmental Protection Agency, Office of Pesticide Programs. Washington, D.C. 2 Organization for Economic Cooperation and Development. 1984. Avian Reproduction Test. OECD Guideline for Testing of Chemicals. Guideline 206. Paris. 3 American Society for Testing and Materials. 1986. Standard Practice for Conducting Reproductive Studies with Avian Species. ASTM Standard E l062-86. Annual Book of ASTM Standards. Vol. 11.04. Philadelphia, PA. 15 pp. 4 Merck & Co., Inc. 1991. The Merck Veterinary Manual. Merck & Co. Rahway, NJ. 1832 pp. 5 National Research Council. 1996. Guide for the Care and Use o f Laboratory Animals. Washington, DC. National Academy Press. 125 pp. 6 Dunnett, C.W. 1955. A Multiple Comparison's Procedure for Comparing Several Treatments with a Control. Jour. Amer. Statis. Assoc. 50: 1096-1121. 7 Dunnett, C.W. 1964. New Tables for Multiple Comparisons with a Control. Biometrics 20:482 491. Wildlife International, Ltd.. 1994. Avian Reproduction Data System (ARDS) Version 2. Project Number 454-109 r'i oo Nominal Concentration (ppm a.i.) Table 1 Mean Measured Concentrations (ppm a.i.) of PFOS in Avian Diet from a Mallard Reproduction Study Week 1 Day 0 Day 71 Week 2 Day 0 Week 3 Day 0 Interval Week 4 Day 0 Week 8 Day 0 Week 12 Week 16 Week 20 Day 0 Day 0 Day 0 0 Measured <2.00 12 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 Mean 10 Measured % Nominal 98 3 100 4 103 101 97 109 101 101 99 Mean 50 Measured % Nominal 101 4 97 104 104 Mean 150 Measured % Nominal 98 3 97 4 108 96 5 112 5 1Feed samples were collected from feed troughs at the end o f Week 1 to assess stability of the test substance under actual test conditions. 2<LOQ indicates the value was less than the limit of quantitation. 3The mean of homogeneity samples collected at Week 1, Day 0. 4The percent of the mean values for 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. coo -31 - Project Number 454-109 Table 2 Summary of Gross Pathological Observations from a Mallard Reproduction Study with PFOS Adult Birds Found Dead/Euthanized during the Test Control (0 ppm a.i.) Males 50 1 ppm a.i. 150 2 ppm a.i. Females 50 1 ppm a.i. 150 2 ppm a.i. Number of birds Abrasion on mandible Foot/leg lesions Thin Emaciated Loss of muscle mass Prominent keel Breast muscle - hematoma and loose blood Heart - flaccid Heart - pale Heart - petechial to ecchymatic hemorrhages Left lung - yellow plaques on margins Lungs - pale Abdominal air sacs - cloudy Spleen - small and/or pale Spleen - enlarged and mottled Liver - firm, pale and mottled Abdominal cavity - autolysis Abdominal cavity - small amount of clear fluid Abdominal cavity - blood in left thoracic air sac Gizzard contents - bile-stained Small intestines - small white ulcers (polyps) Intestinal contents - pasty Intestinal contents - firm Kidneys - pale Not remarkable 1 0 1 0 0 0 0 0 0 0 1 0 0 1 0 1 1 0 1 0 0 0 0 0 1 0 17 1 3 4 0 1 0 1 0 3 0 1 1 0 7 0 0 1 0 1 3 1 0 1 4 7 2 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 18 2 00 00 30 21 31 21 00 10 10 00 00 00 00 50 00 00 02 00 00 21 00 01 10 01 10 0 1Remaining birds in the 50 ppm a.i. test concentration were euthanized at the end of Week 7 of the test. Gross necropsy observations for birds from which blood samples were collected 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. Table 3 Mean Adult Body Weight (g) from a Mallard Reproduction Study with PFOS1 Experimental Group (ppm a .i .) Sex Control 10 50 150 Male Female Male Female Male Female Male Female 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 1084 984 7 4 1111 982 10 22 1104 956 -57 -83 1107 985 -195 -206 1091 988 23 14 1121 1004 7 18 1047 -64 873** -60 912** 780** 88 98 1114 1002 13 19 1127 1022 17 19 983** -66 813** -31 1016** 880** - _ 1128 1020 -3 12 1144 1041 7 9 916** 781** - - -- __ 1125 1033 52 122 1151 1049 -24 95 -- -- -- __ 1164 1155 1127 1144 - - _ _ 92 171 17 162 - - - _ UtoJ Project Number 454-109 The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. * * Significantly different from the control at p < 0.01 1 Only surviving birds were included in the calculations for each body weight interval. Figure 1 Mean Adult Male Bodg Ueight (g) from a M a lla rd R e p r o d u c t io n Study u i t h PFOS ( B) 1H9I3M AOa 31UN -33 - Project Number 454-109 CIf S CL . I0 *DU n Project Number 454-109 -34 CI a Cl 0 CO Ul Figure <6) lH 90n AQ09 31UN3J Table 4 Mean Feed Consumption (g/bird/day) from a Mallard Reproduction Study with PFOS Experimental WEEKS Group ---------------------------------------------------------- (ppm a. i. ) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Control 10 50 150 102 122 119 134 124 133 133 134 135 132 135 178 185 173 178 178 191 212 194 195 205 119 146 134 146 137 145 148 145 139 138 129 158 183 175 175 172 180 188 178 189 193 101 114 118 129 116 124 112 73* 75**166**194** - Data are not available due to adult mortality * Significantly different from the control at p < 0.05 * * Significantly different from the control at p < 0.01 Project Number 454-109 Mean Figure 3 Feed Consumption (g /b ir d /d a y ) from R e p r o d u c t io n S tu dy u i t h PFOS a M a lla rd UCTJs Project Number 454-109 -37- Project Number 454-109 Table 5 Summary of Gross Pathological Observations from a Mallard Reproduction Study with PFOS Adult Birds Euthanized at Test Termination1 Males Control 10 (0 ppm a.i.) ppm a.i. Number ofbirds Foot/leg lesions Breast blister Cranium - swollen and bruised Heart - pericardial sac fluid-filled Spleen - enlarged and mottled Spleen - enlarged and firm Liver - firm, pale and mottled Abdominal cavity - yolk remnants Abdominal cavity - slight egg yolk peritonitis Abdominal cavity - egg yolk peritonitis Abdominal cavity - slight old egg yolk peritonitis Small intestines - area of hyperemia Kidneys - pale Testes - small (~ 1.5-3.5 cm) Reproductive tract - multiple fluid filled cysts Ovary - regressing Ovary - regressed Ovary - cystic follicles Ovary - hemorrhagic follicles Not remarkable 15 4 0 0 0 0 0 0 _ _ _ _ 0 0 2 - 9 16 4 0 0 0 0 0 0 _ _ _ _ 0 0 6 - 8 Females Control 10 (0 ppm a.i.) ppm a.i. 16 16 8 13 02 01 02 10 01 10 10 10 23 50 20 10 -- 10 10 44 11 10 20 1Gross necropsy observations for birds maintained for collection of blood samples during the test are not reported. Table 6 Mean Liver Weights1(g) from a Mallard Reproduction Study with PFOS Experimental Group Adult Males Mean SD Adult Females Mean SD Offspring Males Offspring Females Mean SD Mean SD Control 0 ppm a.i. 29.422 3.864 41.648 + 10.512 14.534 4.679 14.951 2.326 10 ppm a.i. 30.601 4.633 42.207 10.268 13.652 + 4.398 14.068 + 2.143 50 ppm a.i. -- -- -- -- 150 ppm a.i. -- -- -- -- 'Mean standard deviation. - Data not available due to mortality. The above differences from the control group were not statistically significant (Student's t-test). Uoo) Project Number 454-109 Table 7 Summary of Reproductive Performance from a Mallard Reproduction Study with PFOS Reproductive Parameter Number of Replicates Total Eggs Laid Eggs Cracked Eggs Set Viable Embryos Live 3-Week Embryos Hatchlings 14-Day Old Survivors Eggs Laid/Hen Eggs Laid/Hen/Day1 14-Day Old Survivors/Hen Control 15 719 10 644 567 564 460 454 48 0.67 30 Experimental Group (ppm a.i.) 10 50 16 877 10 781 664 653 522 507 55 0.76 32 _ - 150 - - - Project Number 454-109 1Based on 72 days of eggs production. - Data are not available due to adult mortality l Figure 4 Mean R e p ro d u c tiv e P erfo rm ance from R e p r o d u c t io n S tu d y u i t h PFOS 60 Eggs Laid Eggs Eggs Uiable Cracked Set Embryos T" Data a re not a v a ila b le due to adult m o rta lity Live 3-uk Embryos a Mallard H Hat chlings * 150ppm 14-Day Old Survivors -P* o Project Number 454-109 Table 8 Summary o f R e p r o d u c t i v e Performance, N o r m a l i z e d as P e r c e n t a g e s (%) f rom a M a l l a r d R e p r o d u c t i o n S t u d y wit h P F O S 1*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 (%) Hatchlings/Eggs Set (%) 14-Day Old Survivors/Eggs Set (%) Hatchlings/Maximum Set (%) 14-Day Old Survivors/Maximum Set (%) 15 719 67 2 88 100 81 99 71 70 48 47 Experimental Group (ppm a.i.) 10 50 16 N/A 877 N/A 76 N/A 1 N/A 87 N/A 98 N/A 80 N/A 97 N/A 69 N/A 67 N/A 51 N/A 50 N/A N/A - No eggs available due to adult mortality. Differences between the control and each treatment groups were not significant (p > 0.05). 1Values represent pen means for experimental group. Values for each pen are presented in Appendices VII and VIII. 2 Represents the total number of eggs laid in each group. 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 ' Project Number 454-109 Figure 5 Mean R eprod u ctive P erfo rm a n c e , N orm alized as P e rc e n ta g e s C/.), from a M a l l a r d R e p r o d u c t i o n S tu d y u i t h PFOS 4N^J Project Number 454-109 EUo S io Concentrations, ppm Z3 so tm iso T ab le 9 M e a n Eggshell Thickness Measurements (mm) from a Mallard Reproduction Study with PFOS Experimental Group (ppm a.i.) Control 10 50 150 N o . Eggs Measured 49 54 - Shell Thickness Mean ( SD) 0.392 0.034 0.396 0.014 --- - Data are not available due to adult mortality Differences between the control and each treatment groups were not significant (p > 0.05). Project Number 454-109 Table 10 M e a n B o d y Weight (g) of H a t c h l i n g s and 1 4 -Day Old S u r v i v o r s from a Mallard Reproduction Study with PFOS Experimental (PPM A.I.) Hatchlings Number Mean i! SD) 14-Day Old Survivors Number Mean ( SD) Control 10 50 150 459 35 3 521 37 2 0 0 454 287 28 507 282 23 0 0 Project Number 454-109 The number of hatchlings weighed may differ from the total number of hatchlings since those hatchlings found dead were not weighed. Differences between the control and each treatment group were not significant (p > 0.05). -45 - W ildlife International, Ltd Project Number 454-109 Appendix I Certificate of Analysis Centre Analytical Laboratories, Inc. 3048 Research brive Phone: (814) 231-8032 State College, PA 16801 www.centrelab.com Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference if: 023-018A 3M Product: PFOS,Lot217 Reference#: SD-018 ____________________ Purity: 86.9%____________________ Test Name Specifications Purity* Result 86.9% Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium2 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) Related Compounds POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate' Organic Acids*(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis': 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine White Crystalline Powder 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% Conforms Positive 1. 0.005 wt/wt% 2. 0.001 wtywt.% 3. 1.439 wt/wt.% 4. 6.849 wt/wt.% 5. <0.001 wt/wt.% 6. 0.005 wtywt.% 7. <0.001 wt./wt% 1.91 wt/wt.% 8.41 wt/wt.% None Detected 0.33 wt/wt.% None Detected Not Applicable* 1. <0.015 wt/wt% 2. 0.59 wtywt.% 3. <0.040 wt/wt% 4. <0.009 wtywt% 5. <0.006 wt/wt.% 6. <0.007 wt/wt% 7. 8.76 wtywt.% 1. <0.1 wt./wL% 2. <0.1 wt/wt.% 3. 0.10 wt/wt.% 4. 0.28 wt/wt.% 1. 12.48 wtywt.% 2. 0.244 wt/wt.% 3. 1.74 wtywt.% 4. 8.84 wt/wt.% 5. 54.1 wtywt.% COA023-018A Page 1 o f 3 -46- W ildlife International, Ltd Project Number 454-109 Appendix I Page 2 Certificate of Analysis C & itre Analytical Laboratories, Inc 3 040 Research Drive State College, PA 16801 www.centrelab.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231 -1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/06 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/06 'Purity = 100% - (sum o f metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.905%+organic acid impurities, 0.38%+POAA, 0.33%) Total impurity from all tests = 13.07% Purity = 100% - 13.07% = 86.9% ^Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials of low purity. No endotherm was observed for this sample. 4Sulfiir in the sample appears to be converted to SO, and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. STFA HFBA NFPA PFPA Trifhioroacetic acid Heptafluorobutyric acid Nonafluoropentanoic acid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, CgFi7SOj'K'>(MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-0I8A Page 2 o f3 W ildlife International, Ltd. -47 - Project Number 454-109 Appendix I Page 3 Certificate of Analysis S S jS S S ^ Centre Analytical Laboratories, Inc 3046 Research Drive Stale College, PA 16801 www.centrelab.com (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A LC/MS Purity Profile: Im purity C4 C5 C6 C7 Total w t/w t % 1.22 1.33 4.72 1.14 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average result from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average result from the C6 and C8 standard curves. Prepared By: Charles Simofrs Scientist, Centre Analytical Laboratories So/vi fo t Date Reviewed By: 6hn Flaherty / Date ' Laboratory Manager, Centre Analytical Laboratories COA023-0I8A Page 3 of 3 W ildlife International, Ltd. -48 - Project Number 454-109 Appendix II Diet and Supplement Formulations Table 1 Wildlife International, Ltd. Game Bird Ration1 INGREDIENTS Fine Com Meal Soy Bean Meal, 48% Protein Wheat Midds Protein Base Agway Special, 60% Protein Alfalfa Meal, 20% Protein Dried Whey Ground Limestone Eastman CalPhos Methionine Premix + Liquid Vitamin and Mineral Premix (see below) GL Ferm (Fermatco)2 Salt Iodized Total PERCENT (%) 44.83 30.65 6.50 6.00 4.00 3.00 2.50 0-90 0-60 0-35 0.32 0.25 -- QAQ 100.00 Vitamin and Mineral Premix, when added at 0.32% o f the ration, supplied the following amounts per ton: Amount Supplied Per Ton: Vitamin D3 Vitamin A Riboflavin Niacin 2 ,0 0 0 ,0 0 0 1.C.U. 7,000,000 I.U. 6 grams 40 grams Pantothenic Acid Vitamin B n Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (Menadione Dimethylpyrimidinol Bisulfite) Manganese Zinc Copper Iodine Iron 16 grams 8 mgs 600 mgs 64 mgs 1-2 grams 1-2 grams 20,000 I.U. 5.8 grams 102 grams 47 grams 6.8 grams 1.5 grams 51 grams Selenium 1^2 mgs 1 The guaranteed analysis is a minimum o f 27% protein, a minimum o f 2.5% crude fat and a maximum o f 5% crude fiber. 2 Fermentation By-Products (Source o f Unidentified Growth Factors). W ildlife International, Ltd. -49 - Project Number 454-109 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 4,000,000 ICU 4,000 IU 3,000 Mg 5,000 Mg 10,000 Mg 10.0 Mg 4,000 Mg 260 Mg 1,000 Mg 1,000 Mg 15,000 Mg INGREDIENTS: Vitamin A Supplement, D-Activated Animal Sterol (source o f Vitamin D3), Alpha Tocopheryl Acetate (source o f Vitamin E). Riboflavin Supplement, d-Calcium Pantothenate, Niacin Supplement, Vitamin B-12 Supplement, Menadione Sodium Bisulfite (source o f Vitamin K), Folic Acid, Thiamine HC1, Pyridoxine Hydrochloride, Ascorbic Acid, Sodium Chloride, Calcium Chloride, M agnesium Sulfate, Ferric Am monium Citrate, Potassium Chloride and Dextrose. MIXING PROCEDURE: The vitamin and electrolyte mix was prepared as a ration o f approximately 2 grams o f Durvet vitamins and electrolytes to approximately 1 gallon o f water. -50 - W ildlife International, Ltd. Project Number 454-109 Appendix III Diet Preparation Premixes for PFOS were prepared on January 22, 2001; February 6 and 12, 2001; March 2, 6 and 27, 2001; April 12, 2001; May 3 and 23, 2001; and June 4, 2001. Nominal preparation was as follows: Control: No premix required. 10 ppm a.i.: 2.5633 g PFOS + 8097.4 g ration 50 ppm a.i.: 12.8165 g PFOS + 8087.2 g ration 150 ppm a.i.: 38.4494 g PFOS + 8061.6 g ration 20 ppm a.i. 5.1266 g PFOS + 8094.9 g ration Basal ration, 5000.0 g, was weighed into a tared Hobart mixing bowl. The remaining ration was weighed and set aside. The test substance was weighed into a tared weigh boat. Approximately 100 g of set aside ration was placed in a Waring blender. The test substance was transferred to the Waring blender and the weigh boat was rinsed three times with set aside ration, with the rinse being added to the blender. The blender contents were mixed approximately one minute and transferred to the mixing bowl. The blender was rinsed three times with set aside ration with the rinse being added to the bowl. The remaining set aside ration was added to the bowl, the bowl was placed on a Hobart mixer, and the contents were mixed approximately 15 minutes. If not used immediately after mixing, 2000.0 g aliquots of the premix were weighed, placed in appropriately labeled plastic bags, reweighed, and stored frozen. As needed, the appropriate premix was incorporated into the final diet as follows: 0 ppm a.i.: 52.25 kg ration + 2.750 kg limestone 10 ppm a.i.: 2000 g Premix + 50.25 kg ration + 2.750 kg limestone 50 ppm a.i.: 2000 g Premix + 50.25 kg ration + 2.750 kg limestone 150 ppm a.i.: 2000 g Premix + 50.25 kg ration + 2.750 kg limestone 20 ppm a.i. 2000 g Premix + 50.25 kg ration + 2.750 kg limestone The diets were mixed for approximately 20 minutes in a Patterson-Kelly Twin Shell Blender. -51 - W ildlife International, Ltd. Project Number 454-109 Appendix IV The Analysis of PFOS in Avian Diet ,Wildlife International Ltd. - 5 2 - Project Number 454-109 APPENDIX IV ANALYTICAL METHODS AND RESULTS INSTRUMENT: Table 1 Typical HPLC/MS/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in multiple ion reaction monitoring (MRM) mode. ANALYTICAL COLUMN: GUARD COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE INJECTION VOLUME: PFOS RETENTION TIME. Keystone Betasil Ci8column (50 mm x 2 mm ID ., 3-pm particle size) Keystone Javelin Ci8column (20 mm x 2 mm I.D.) 40C 5.00 minutes 250 pL/minute 70.0% Methanol: 30.0%NANOpure Water containing 0.1% Formic Acid 10.0 pL Approximately 4.0 minutes PFOS MONITORED MASS: 499.0 amu - 99.1 amu Wildlife International, Ltd. -53 - Project Number 454-109 APPENDIX IV Table 2 Calculations The concentration of PFOS found at the instrument was determined using the following equation: peak area - (y-intercept) PFOS (mg a.i./L) at instrument slope Determination of Sample Residues (PFOS) The concentration, expressed as ppm a.i., for each sample was determined using the following equation: PFOS (mg a.i./L) at instru. x extract final volume (L) x dilution factor PFOS (ppm a.i.) in sample = ^ 1 initial weight (Kg) Determination of Limit of Quantitation (LOQ) The method LOQ, expressed as ppm a.i., was determined using the following equation: LOQ (ppm a.i.) = lowest standard concentration (mg a.i./L) x overall dilution factor of matrix blank* extract final volume (L) x dilution factor *overall dilution factor of matrix blank sample = initial weight (Kg) Fortification Recoveries The ppm a.i. measured in each sample is divided by the nominal concentration of each sample (fortified level, ppm a.i.). This ratio times 100 is the percent recovery of the method at that level of fortification. ppm a.i. measured in sample , ,,,, % Recovery- = " -----p--p-m---a-.-i. ffortified,-- x 100 Project Number 454-109 APPENDIX IV Table 3 Matrix Blanks and Fortifications Analyzed Concurrently with the Samples Number (454-109-) 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 Sample Type Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Interval Week 1, Day 0 Week 1, Day 0 Week 1, Day 0 Week 1, Day 0 Week 2, Day 0 Week 2, Day 0 Week 2, Day 0 Week 2, Day 0 Week 3, Day 0 Week 3, DayO Week 3, Day 0 Week 3, Day 0 Week 4, Day 0 Week 4, Day 0 Week 4, Day 0 Week 4, Day 0 Concentration of PFOS (ppm a.i.) Fortified1 Measured1,2 0.00 <2.00 5.00 4.85 50.0 52.8 200 206 0.00 <2.00 5.00 4.55 50.0 47.4 200 189 0.00 <2.00 5.00 5.50 50.0 52.0 200 200 0.00 <2.00 5.00 5.20 25.0 26.1 100 100 Percent Recovery 97 106 103 _ 91 95 95 110 104 100 _ 104 104 100 1Less than values correspond to limit of quantitation (LOQ). 2Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly. Mean Recovery (*) 102 94 105 103 -54- Project Number 454-109 APPENDIX IV Table 3 (Continued) Matrix Blanks and Fortifications Analyzed Concurrently with the Samples Number (454-109-) MAB-5 MAS-13 MAS-14 MAB-6 MAS-15 MAS-16 MAS-173 MAS-18 MAB-7 MAS-19 MAS-20 Sample Type Interval Matrix Blank Matrix Fortification Matrix Fortification Matrix Blank Matrix Fortification Matrix Fortification Matrix Fortification Matrix Fortification Matrix Blank Matrix Fortification Matrix Fortification Week 8, Day 0 Week 8, Day 0 Week 8, Day 0 Week 12, Day 0 Week 12, Day 0 Week 12, Day 0 Week 16, Day 0 Week 16, Day 0 Week 20, Day 0 Week 20, Day 0 Week 20, Day 0 Concentration of PFOS (ppm a.i.) Fortified1 Measured1,2 0.00 <2.00 5.00 5.40 25.0 27.5 0.00 <2.00 5.00 5.21 25.0 24.4 5.26 5.39 25.0 24.5 0.00 <2.00 5.00 4.86 25.0 24.0 Percent Recovery 108 110 _ 104 98 102 98 _ 97 96 Mean Recovery (x) 109 101 100 97 1Less than values correspond to limit of quantitation (LOQ). 2Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly. 3Week 16 samples were held and batched with Week 20 samples. Matrix fortifications were prepared to verify stability. A storage matrix blank was not required. Nominal Concentration (ppm a.i.) 10 50 150 Project Number 454-109 Sample I.D. Number (454-109-) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 APPENDIX IV Table 4 Homogeneity of PFOS in Avian Diet Location Sampled in Mixing Vessel Measured PFOS Concentration (ppm a.i.) Top Left Top Right Middle Left Middle Right Bottom Left Bottom Right Top Left Top Right Middle Left Middle Right Bottom Left Bottom Right Top Left Top Right Middle Left Middle Right Bottom Left Bottom Right 9.69 9.47 9.87 10.2 9.35 9.93 49.9 48.9 51.7 50.4 50.3 49.3 151 153 143 143 144 147 Mean (x) Standard Deviation (SD) Coefficient of Variation (CV) x = 9.75 ppm a.i SD = 0.313 ppm a.i. CV = 3.21% x = 50.1 ppm a.i. SD = 0.981 ppm a.i. CV= 1.96% x = 147 ppm a.i. SD = 4.31 ppm a.i. CV= 2.93% Mean Percent of Nominal 98 100 98 -56- Project Number 454-109 Nominal Concentration (ppma.i.) Sample I.D. Number (454-109-) 34 41 48 55 60 65 70 APPENDIX IV Table 5 Verification of PFOS Concentrations in Avian Diet Interval (Day 0 of W eek-) 2 3 4 8 12 16 20 Measured PFOS Concentration1 (ppma.i.) <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 M ean(x) Standard Deviation (SD) Coefficient of Variation (CV) - Percent of Nominal - Mean Percent of Nominal 35 2 36 2 42 3 43 3 49 4 50 4 56 8 57 8 61 12 62 12 66 16 67 16 71 20 72 20 1Less than values correspond to limit of quantitation (LOQ). 11.0 110 9.68 97 9.53 95 10.7 107 9.70 97 9.60 96 10.6 x = 10.2 ppm a.i. 106 11.2 SD = 0.553 ppm a.i. 112 10.4 CV = 5.44% 104 9.86 99 9.82 98 10.4 104 10.1 101 9.69 97 -57- Project Number 454-109 Nominal Concentration (ppm a.i.) 20 50 150 Sample I.D. Number (454-109-) 46 47 53 54 37 38 44 45 51 52 39 40 APPENDIX IV Table 5 (Continued) Verification of PFOS Concentrations in Avian Diet Interval (Day 0 of Week -) 3 3 4 4 2 2 3 3 4 4 2 2 Measured PFOS Concentration1 (ppm a.i.) 18.6 19.8 23.6 21.1 48.7 48.4 51.1 53.1 51.1 53.1 172 150 Mean (x) Standard Deviation (SD) Coefficient of Variation (CV) X = 20.8 ppm a.i. SD = 2.14 ppm a.i. C V= 10.3% X = 50.9 ppm a.i. SD = 2.04 ppm a.i. CV = 4.01% x = 161 ppm a.i. SD = 15.6 ppm a.i. CV = 9.66% Percent of Nominal 93 99 118 106 97 97 102 106 102 106 115 100 Mean Percent of Nominal 104 102 108 -58- Project Number 454-109 APPENDIX IV Table 6 Ambient Stability of PFOS in Avian Diet During the Mallard Reproduction Study Nominal Concentration (ppma.i.) Sample Number (454-109-) Week 1, Day 01 Mean Measured (ppm a.i.) PFOS Concentration Mean Percent of Nominal Sample Number (454-109-) Week 1, Day 7 Measured12 (ppm a.i.) Mean Measured (ppm a.i.) 0 1 <2.00 - 10 2-7 9.75 98 50 8-13 50.1 100 150 14-19 147 98 27 <2.00 <2.00 28 9.75 9.71 29 9.67 30 53.4 50.8 31 48.1 32 141 143 33 144 1Day 0 values are from homogeneity samples presented in Table 4. 2Less than values correspond to limit of quantitation (LOQ). Mean Percent of Day 0 100 101 97 -59- ,Wildlife International Ltd. - 6 0 - Project Number 454-109 APPENDIX IV METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN AVIAN DIET Prepare matrix fortification samples in the desired avian feed stock using the dry mix technique. 4 Weigh 10 g samples of the matrix blank, matrix fortification and test samples into weigh boats and transfer to 8-oz. French square glass bottles. Record weights. For each sample, measure 100 mL of methanol with a graduated cylinder and transfer into the French square bottle. i Cap bottles and place on shaker table. Allow the samples to shake for a minimum of 30 minutes at approximately 250 rpm. I Vacuum filter with qualitative filter paper and rinse retained feed 3 times with methanol into filtrate. I Transfer the filtrate into a 200-mL volumetric flask and bring to volume with methanol. I Prepare appropriate dilution(s) to bring final concentration into the calibration range of the HPLC/MS/MS methodology. Use methanol for intermediate dilutions, if required. For all final dilutions, use 50% methanol : 50%NANOpure water dilution solvent. I Ampulate and submit sample for HPLC/MS/MS analysis. Figure 1. Analytical method flow chart for the analysis of PFOS in avian diet. Wildlife International, Ltd. -61 - APPENDIX IV Project Number 454-109 Figure 2. Typical calibration curve for PFOS. Slope = 11487727; Intercept = 883.10; r = 0.99974. Curve is weighted (1/x). ,Wildlife International Ltd - 6 2 - Project Number 454-109 APPENDIX IV PFOSJ STD 0.00100 mg a.i./L 4675A-01 ID -1 4.98 in 1 period pros No Inlemal Standard Use Area 1: 4.97 298 scans 499.0->99.1 Noise Thres. 5.0 Quant Thres. 1.0 Min. Width 3 Mult. Width 6 Base. Width 'RT Win. (secs) Smooth 35 20 1 Expected RT 4.02 Area 12195 Height 1171 Start Time End Time 3.79 4.37 Integration Width Retention Time Integration Type 0.59 4.02 A-BB Wed, :eb 7, 2001 17:58 intensity: 10000 cps Figure 3. Typical ion chromatogram of a low PFOS calibration standard, 0.00100 mg a.i./L. (Monitored mass 499.0 99.1). Wildlife International, Ltd, -63 - Project Number 454-109 APPENDIX IV PF0S.5 STD 0.0100 mg a.iyL 4675A-011D -5 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 5.0 Quant Thres. 1.0 Min. Width 3 Mult. Width 6 Base. Width 35 RT Win. (secs) 20 Smooth 1 Expected RT 4.02 Area 114007 Height 10269 Start Time End Time Inlegration Width Retention Time Integration Type 3.80 4.39 0.59 4.02 A,-BB Wed Feb 7, 2001 18 21 intensity: 10000 cps Figure 4. Typical ion chromatogram of a high PFOS calibration standard, 0.0100 mg a.i./L. (Monitored mass 499.0 --> 99.1). ,Wildlife International Ltd, - 6 4 - Project Number 454-109 APPENDIX IV PFOS 7 MAB-3 454-108,109- Wed, Feb 7, 2001 18:33 4.98 in 1 period PFOS No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 5.0 Quant Thres. 1.0 Min. Width 3 Mult. Width 6 Base. Width 35 RT Win. (secs) 20 Smooth 1 Expected RT 4.02 Area 0 Height 0 Start Time End Time 0.00 0.00 Integration Width Retention Time 0.00 0.00 Integration Type 100-1 908070605040302010On intensity: 10000 cps 1 105 130 164 205 240 274 --I--- 1----- 1---- 1---- 1---- 1---- 1--- 1-- r~ I----- 1---81 121 161 201 241 281 Scan 1.36 2.03 2.70 3.37 4.04 4,71 Time Figure 5. Typical ion chromatogram of a matrix blank sample (454-109-MAB-3). The arrow indicates the approximate retention time of PFOS. (Monitored mass 499.0 -> 99.1). Wildlife International, Ltd, -65 - Project Number 454-109 APPENDIX IV PF0S_9 MAS-8 454-108,109- Wed, Feb 7, 2001 18 45 4,98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Hires. 5.0 Quant Hires. 1.0 Min. Width 3 Mult. Width 6 Base. Width 35 RT Win. (secs) 20 Smooth 1 Expected RT 4.02 Area 60579 Height 5641 Start Time End Time Integration Width Retention Hme Integration Type 3.80 4.39 0.59 4.02 A -BB 100-| 90 80 70 60- intensity: 10000 cps 240 Figure 6. Typical ion chromatogram o f a m atrix fortification sample (454-109-M AS-8, 50.0 ppm a.i.) (Monitored mass 499.0 - 99.1). ,Wildlife International Ltd, - 6 6 - Project Number 454-109 APPENDIX IV PF0S.13 42 454-108,109- Wed, Feb 7, 2001 19:10 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. Quant Thres. 5.0 1.0 Min. Width Mult. Width Base. Width 3 6 35 RT Win. (secs) 20 Smooth 1 Expected RT 4.02 Area 55642 Height 5213 Start Time End Time Integration Width Retention Time Integration Type 3.80 4.39 0.59 4.02 A,-BB intensity: 10000 cps Figure 7. Typical ion chromatogram o f an avian diet sample on Day 0 (454-109-42, 10.0 ppm a.i.). Dilution factor = 2000X. (Monitored mass 499.0 - 99.1). -67 - Project Number 454-109 Appendix V Diagram of Test Layout from a Mallard Reproduction Study with PFOS Room 56 Pens 261-263 a Pens 264-266 Pens 267-269 Pens 270-272 Pens 273-275 Pens 276-278 150 ppm a.i. Pens 261 - 280 Pens 279-280 Pens 259-260 a 50 ppm a.i. Pens 241 - 260 Pens 256-258 Pens 253-255 Pens 250-252 Pens 247-249 Pens 244-246 Pens 241-243 Pens 221-223 a Pens 224-226 Pens 227-229 Pens 230-232 Pens 233-235 Pens 236-238 10 ppm a.i. Pens 221 - 240 Pens 239-240 Pens 219-220 a 0 ppm a.i. Pens 201 - 220 Pens 216-218 Pens 213-215 Pens 210-212 Pens 207-209 Pens 204-206 Pens 201-203 --------- 1 = approx, lm = 6" gutter a = Safeguard Products Inc. Model No. 5355 b = Four X 4 ft. Chroma 50 light bulbs Note: Pens 217-220,237-240,257-260 and 277-280 were used to house birds used for blood sampling. -68 - W ildlife International, Ltd. Project Number 454-109 Appendix VI Reproductive Parameters 1. Eggs Laid Definition - The number of eggs produced during the breeding season. Sensitivity - This is a parameter that is frequently affected by chemicals that cause reproductive impairment. It is also highly sensitive to the general conditions under which the test is conducted, and may be adversely affected by improper diet, crowding, excessive disturbances, etc. 2. Eggs Cracked Definition - Eggs determined to have cracked shells when inspected with a candling lamp. Fine cracks cannot be detected without utilizing a candling lamp, and if undetected, will bias the data by adversely affecting embryo development. Sensitivity - This parameter is not frequently affected as cracking is directly related to shell thickness, and few chemicals have caused egg shell thinning. Improper pen design, overcrowding, and pecking by the birds can also increase egg cracking. 3. Eggs Set Definition - All eggs placed under incubation, i.e., total eggs laid minus cracked eggs, mechanically damaged eggs, and those selected for egg shell thickness analysis. The reason for presenting this parameter is to establish a base of reference for the following parameter viable embryos. 4. Viable Embryos Definition - Eggs in which fertilization has occurred and embryonic development has begun. This is determined by candling the eggs 13-14 days after the initiation of incubation. It is difficult to distinguish between infertility and an early embryonic death. Sensitivity - This is a frequently affected parameter with infertility or embryonic mortality caused by an unfavorable environment for fertilization in the oviduct, impotent males, or chemical residue in the egg. -69- W ildlife International, Ltd. Project Number 454-109 Appendix VI Reproductive Parameters Page 2 5. Live Three-Week Embryos Definition - These are embryos that are developing normally after three weeks of incubation. This is determined by candling the eggs. Sensitivity - This parameter is seldom affected. 6. Hatchlings Definition - Embryos that mature, pip the shell, and liberate themselves from their eggs on Day 27 or 28 of incubation. Sensitivity - This is a frequently affected parameter which is also highly sensitive to the conditions of incubation, such as rate and angle of rotation, humidity, and temperature. - Humidity: If too dry, ducklings will stick to their shells. - Temperature: If too hot and humid, ducklings will grow too fast and be too large for their shells; thereby not having the intra-egg mobility necessary to pip their shells. 7. Body Weight - Hatchlings Definition - The average weight of hatchlings by parental pen of origin, taken immediately upon removal from the hatcher. Hatchlings from each weekly lot of eggs are weighed. Mean weight for each week and mean weight by pen are reported. Sensitivity - This is an occasionally affected parameter and may reflect some residual or latent toxic effects from chemical residue in the egg. 8. 14-Day Old Survivors Definition - Birds that survive brooding for two weeks following hatch. Sensitivity - This is a seldom affected parameter and is probably more indicative of the conditions under which the birds were reared in battery brooders than the chemical to which the adults were exposed. -70- W ildlife International, Ltd. Project Number 454-109 Appendix VI Reproductive Parameters Page 3 9. Body Weight - 14-Day Old Survivors Definition - The average body weight of the 14-day old survivors by parental pen of origin, taken upon removal from the brooder unit at 14 days of age. Fourteen-day old survivors from each weekly lot of eggs are weighed. Mean weight for each week and mean weight by pen are reported. Sensitivity - This parameter is seldom affected, and more closely reflects the rearing practices utilized. 10. Egg Shell Thickness Definition - The thickness of the shell and the membrane of the egg at its equator after having been opened, washed, and dried for at least one week at room temperature. Egg shell thickness measurements for each egg measured, and pen means are reported. Sensitivity - This is a seldom affected parameter; however, measurements must be taken very carefully to ensure accuracy. Possible mechanical errors include membrane curling at the measurement surface, and calcium deposits on the measurement surface. W ildlife International, Ltd. -71 - Project Number 454-109 Appendix VI Replicate Identification ^ ,F Experimental Replicate Group _____ Number Control 0 ppm a.i. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 _ Raw Data Band Number Report Pen Raw Data P e n -------------------------------- Number______ Number______ Male______ Female 201 201 329 330 202 202 331 332 203 203 333 334 204 204 335 336 205 205 337 338 206 206 339 340 207 207 341 342 208 208 343 344 209 209 345 346 210 210 347 348 211 211 349 350 212 212 351 352 213 213 353 354 214 214 355 356 215 215 357 358 216 216 359 360 -72- Wildlife International, Ltd Project Number 454-109 Appendix VI Replicate Identification Experimental Group Treatment 10 ppm a.i. Report Replicate ,, _ Raw Data Band Number Report Pen Raw Data Pen Number______ Number Number Male Female 1 217 221 369 370 2 218 222 371 372 3 219 223 373 374 4 220 224 375 376 5 221 225 377 378 6 222 226 379 380 7 223 227 381 382 8 224 228 383 384 9 225 229 385 386 10 226 230 387 388 11 227 231 389 390 12 228 232 391 392 13 229 233 393 394 14 230 234 395 396 15 231 235 397 398 16 232 236 399 400 Appendix VII - Table 1 Adult Body Weight (g) from a Mallard Reproduction Study with PFOS (C o n t r o l , Males) Pen 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 1098 -11 1087 15 1102 -12 1090 -47 1043 119 1061 -56 1005 22 1027 34 1061 27 1088 96 994 -1 993 24 1017 43 1060 -15 1045 130 1077 56 1133 19 1152 -18 1134 5 1139 53 1094 -25 1069 75 1144 -19 1125 -54 1071 144 1061 -15 1046 23 1069 -4 1065 -13 1052 99 1078 25 1103 21 1124 -18 1106 6 1112 42 1073 12 1085 33 1118 51 1169 22 1191 -78 1126 -38 1088 23 1111 42 1153 -5 1148 29 1264 5 1269 26 1295 18 1313 -5 1308 - 1050 43 1093 -5 1088 0 1088 -3 1085 -13 1018 59 1077 1 1078 13 1091 12 1103 0 997 41 1038 36 1074 44 1118 25 1143 -30 1164 -21 1143 21 1164 22 1186 -4 1182 85 1139 0 1139 24 1163 1 1164 5 1169 -68 1057 31 1088 16 1104 13 1117 1 1118 167 1084 7 1091 23 1114 13 1128 -3 1125 52 67 34 64 17 65 24 63 22 68 77 1162 1184 1175 1192 1215 1151 1154 1113 1177 - 1072 1103 1113 1267 1101 1285 1164 60 Total Change 64 123 181 115 121 90 76 40 51 - 22 85 116 103 -38 228 92 64 The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. Project Number 454-109 Appendix VII - Table 2 Adult Body Weight (g) from a Mallard Reproduction Study with PFOS (C o n t r o l , F e m a l e s ) Pen 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 905 35 940 25 965 -19 946 -4 942 249 857 -11 846 32 878 21 899 33 932 101 860 21 881 6 887 19 906 6 912 246 1024 26 1050 46 1096 8 1104 -7 1097 205 924 25 949 -4 945 34 979 39 1018 -46 1025 -3 1022 10 1032 29 1061 24 1085 250 1057 -40 1017 27 1044 -11 1033 14 1047 121 1092 36 1128 1 1129 43 1172 15 1187 227 1086 -3 1083 10 1093 31 1124 -7 1117 93 968 32 1000 3 1003 32 1035 5 1040 -52 930 23 953 33 986 9 995 5 1000 214 986 -14 972 1 973 23 996 20 1016 150 1215 -48 1167 12 1179 26 1205 21 1226 28 863 -4 859 7 866 18 884 1 885 102 983 15 998 -11 987 6 993 14 1007 55 962 -20 942 21 963 31 994 18 1012 12 984 4 98 26 988 14 1002 19 1020 12 1033 122 90 15 90 17 94 14 94 103 1191 1033 1158 1302 972 1335 1168 1414 1210 988 1214 1166 1254 987 1062 1024 1155 134 Total Change 286 176 298 278 48 310 111 322 124 20 284 180 39 124 79 62 171 109 The means for body weights and body weight changes are calculated and rounded separately. Project Number 454-109 Appendix VII - Table 3 Adult Body Weight (g) from a Mallard Reproduction Study with PFOS (10 P P M A. I. , Males) Pen 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 966 1273 1153 1193 1038 1148 1049 1003 1146 1148 1097 1231 1079 1081 1121 1042 1111 82 22 -2 -57 53 10 31 7 2 44 -13 -11 73 9 -5 -2 3 10 30 988 1271 1096 1246 1048 1179 1056 1005 1190 1135 1086 1304 1088 1076 1119 1045 1121 94 -10 12 12 -34 -14 18 24 -20 -9 43 3 12 15 26 5 21 7 20 978 1283 1108 1212 1034 1197 1080 985 1181 1178 1089 1316 1103 1102 1124 1066 1127 96 22 20 -11 -2 14 27 -4 2 25 16 61 32 -11 22 39 23 17 19 1000 1303 1097 1210 1048 1224 1076 987 1206 1194 1150 1348 1092 1124 1163 1089 1144 101 -72 0 30 38 29 -15 -4 -13 -7 5 55 26 55 -30 29 -21 7 34 928 1303 1127 1248 1077 1209 1072 974 1199 1199 1205 1374 1147 1094 1192 1068 1151 115 43 -84 171 68 24 -59 -63 -25 40 -35 -116 -230 -34 2 -136 54 -24 95 971 1219 1298 1316 1101 1150 1009 949 1239 1164 1089 1144 1113 1096 1056 1122 1127 105 Total Change 5 -54 145 123 63 2 -40 -54 93 16 -8 -87 34 15 -65 80 17 69 The means for body weights and body weight changes are calculated and rounded separately. Differences between control and this treatment group were not significant (p > 0.05 ). Project Number 454-109 Appendix VII - Table 4 Adult Body Weight (g) from a Mallard Reproduction Study with PFOS (10 PP M A.I., Females) Pen 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 1071 890 984 1083 1068 1079 1148 852 1000 1025 988 1028 827 970 879 824 982 101 93 18 -36 12 -13 -20 54 -7 87 29 33 44 21 44 7 -14 22 37 1164 908 948 1095 1055 1059 1202 845 1087 1054 1021 1072 848 1014 886 810 1004 117 63 -2 33 23 5 30 27 11 8 -22 7 34 42 -3 -13 37 18 22 1227 906 981 1118 1060 1089 1229 856 1095 1032 1028 1106 890 1011 873 847 1022 123 -61 19 18 141 -11 -23 46 4 -16 -4 41 -25 59 86 13 15 19 48 1166 925 999 1259 1049 1066 1275 860 1079 1028 1069 1081 949 1097 886 862 1041 126 38 22 -4 34 -23 -24 53 2 -25 5 -11 -3 38 39 23 -23 9 27 1204 947 995 1293 1026 1042 1328 862 1054 1033 1058 1078 987 1136 909 839 1049 138 11 237 167 23 86 136 -29 126 129 26 200 144 154 45 76 -18 95 79 1215 1184 1162 1316 1112 1178 1299 988 1183 1059 1258 1222 1141 1181 985 821 1144 128 Total Change 144 294 178 233 44 99 151 136 183 34 270 194 314 211 106 -3 162 92 The means for body weights and body weight changes are calculated and rounded separately. Differences between control and this treatment group were not significant (p> 0 .0 5 ). Project Number 454-109 Appendix VII - Table 5 Adult Body Weight (g) from a Mallard Reproduction Study with PFOS (50 P P M A. I. , Males) Pen 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 1013 1039 1444 1023 997 1196 1367 1084 1018 1180 1142 1009 1088 998 936 1129 -39 -46 -62 -38 -50 -88 -84 -74 -69 -83 -89 -10 -42 -42 -5 -95 974 993 1382 985 947 1108 1283 1010 949 1097 1053 999 1046 956 931 1034 -48 -21 -134 -62 -11 -100 -77 -62 -43 -98 -66 -47 -57 -77 -21 -98 926 972 1248 923 936 1008 1206 948 906 999 987 952 989 879 910 936 -79 2 -130 -182 -28 -29 -54 -109 -58 -44 -74 -36 -38 -68 -67 847 974 1118 741 908 979 1152 839 848 955 878 953 841 842 869 _ - - - - - - ,_ _ ----- -- -- -- --------- _ - - - - - - 1104 139 -57 28 1047 124 -64 33 983** -66 102 46 916** 109 - --- - Total Change _ - - - - The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. * * Significantly different from the control at p < 0.01 Project Number 454-109 Appendix VII - Table 6 Adult Body Weight (g) from a Mallard Reproduction Study with PFOS (50 PPM A . I . , Females) Pen 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 939 -106 833 -56 777 1 778 _ 919 -75 844 -51 793 0 793 - 978 -81 897 -90 807 -76 731 - 981 -90 891 -36 855 -27 828 - 969 -145 824 -40 784 -69 715 - 951 -73 878 -27 851 -41 810 - 880 -60 820 -25 795 26 821 - 887 -75 812 -58 754 -37 717 - 1002 -105 897 -50 847 -103 744 - 928 -19 909 -71 838 12 850 - 1034 -98 936 -89 847 -15 832 - 963 -106 857 -38 819 -51 768 - 849 -76 773 -79 694 -39 655 - 904 -108 796 -84 712 -39 673 - 1109 -56 1053 -101 952 -6 946 - 995 -54 941 -65 876 -38 838 - -_ ---------------- . - - _ - - - 956 -83 64 29 873** -60 69 24 813** -31 63 34 781** 74 - --- _ Total Change . - - - - - - The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. * * Significantly different from the control at p < 0.01 Project Number 454-109 Appendix VII - Table 7 Adult Body Weight (g) from a Mallard Reproduction Study with PFOS (150 PPM A.I., Males) Pen 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 1128 1024 1057 1177 898 1229 1012 1094 1222 1276 1218 1060 1178 1050 1008 1073 -145 -242 -198 -246 -208 -263 -239 -185 -174 -228 -158 -152 -209 -132 -192 -143 1107 103 -195 41 983 782 859 931 690 966 773 909 1048 1048 1060 908 969 918 816 930 145 276 72 113 102 118 -27 122 -6 88 55 31 50 90 912** 106 88 73 1128 1058 931 1044 1068 891 - 1021 1170 1054 996 1024 949 866 1020 1016** 85 _ - - - __ ---------------- -- -- __ --------------- --- _ - - - _ - Total Change __ - _ - The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. * * Significantly different from the control at p < 0.01 Project Number 454-109 Appendix VII - Table 8 Adult Body Weight (g) from a Mallard Reproduction Study with PFOS (150 PPM A.I., Females) Pen 249 250 251 252 253 254 255 256 257 258 '259 260 261 262 263 264 Mean SD Week 0 Change Week 2 Change Week 4 Change Week 6 Change Week 8 Change Test Week 0-2 Week 2-4 Week 4-6 Week 6-8 Week 8-Term Term 857 832 891 903 892 972 1113 1091 1027 912 1056 1159 1061 1116 781 1104 -182 -200 -143 -218 -159 -205 -205 -238 -266 -178 -257 -236 -208 -248 -136 -212 985 -206 119 39 675 _ 632 198 748 88 685 127 733 124 767 44 908 135 853 761 134 734 103 799 86 923 106 853 -3 868 21 645 166 892 46 780** 95 98 56 _ 830 836 812 857 811 1043 895 837 885 1029 850 889 811 938 880** 76 _ - - __ ---------------- -- -" __ ---------------- -- - - - Total Change _ - - The means for body weights and body weight changes are calculated and rounded separately. - Data are not available due to adult mortality. * * Significantly different from the control at p < 0.01 Project Number 454-109 Pen 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Mean SD Appendix VIII - Table 1 Feed Consumption (g/bird/day) from a Mallard Reproduction Study with PFOS (Control) WEEKS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 96 100 122 138 144 132 138 155 138 158 152 174 169 168 164 157 162 181 183 211 204 76 82 102 110 90 95 104 107 95 96 79 172 191 171 112 123 129 146 129 155 145 93 106 115 132 91 111 92 115 121 115 139 184 225 252 236 200 253 324 232 223 292 154 159 133 141 124 144 138 116 129 125 158 179 212 158 165 147 164 168 151 176 170 76 89 81 102 92 111 110 91 129 133 163 168 175 188 240 275 243 243 259 260 318 104 127 130 140 126 148 151 148 139 131 141 195 157 141 139 136 166 166 166 164 167 96 107 105 114 104 111 118 110 102 96 105 150 170 108 131 144 140 157 164 162 180 85 155 121 159 168 187 162 175 164 151 114 226 239 214 181 217 242 263 191 239 234 182 174 187 236 195 231 235 230 252 196 227 180 185 171 192 223 224 327 245 245 227 86 101 91 100 106 107 97 108 92 103 96 143 165 127 143 185 210 215 185 115 143 108 148 103 125 115 124 125 116 125 108 123 178 166 144 156 159 144 145 130 180 169 136 149 152 147 141 152 158 174 149 168 171 193 217 195 205 174 220 265 246 226 267 78 119 106 114 113 116 123 130 119 126 117 156 148 162 153 137 155 152 132 168 150 67 102 100 103 97 103 101 107 105 114 102 228 179 168 182 168 160 214 261 212 224 113 131 145 160 170 156 170 151 180 164 166 188 215 210 241 244 242 230 253 173 201 78 109 106 122 107 105 101 120 128 123 114 137 151 187 204 159 205 202 179 209 191 102 122 119 134 124 133 133 134 135 132 135 178 185 173 178 178 191 212 194 195 205 31 27 26 33 31 36 37 36 39 29 37 26 28 35 40 43 43 59 48 39 53 Project Number 454-109 Appendix VIII - Table 2 Feed Consumption (g/bird/day) from a Mallard Reproduction Study with PFOS (10 PPM A. I. ) Pen 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 WEEKS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 121 146 - 93 134 117 110 129 126 136 132 149 171 157 140 136 133 125 129 122 139 145 94 112 107 137 133 131 134 134 131 136 171 166 223 196 215 235 221 232 228 205 233 117 159 125 152 145 163 172 181 166 157 196 173 220 188 158 164 195 178 198 190 218 206 238 205 172 183 229 264 229 245 190 134 226 224 209 234 206 214 242 259 263 274 84 117 109 122 108 133 133 124 124 136 120 176 218 195 189 186 190 216 122 194 190 107 136 129 149 133 138 135 130 124 123 104 146 193 166 140 127 140 172 158 169 161 169 206 191 232 189 171 166 156 151 150 131 143 173 197 221 192 218 216 215 188 216 124 116 121 166 99 110 126 113 115 113 117 126 134 128 113 135 138 134 123 133 131 137 171 156 129 144 147 129 124 108 111 93 132 169 171 176 145 149 160 138 163 170 103 104 109 133 117 113 113 116 101 122 134 138 168 146 148 136 159 150 143 163 186 90 116 115 143 126 138 133 140 134 132 109 148 168 160 168 173 172 172 155 172 155 153 199 182 180 163 169 178 177 194 163 103 168 177 162 141 134 144 136 135 139 187 94 115 123 139 131 149 155 154 136 134 115 184 212 229 226 198 203 219 200 212 240 109 149 146 126 149 154 150 148 136 148 169 166 200 246 250 273 256 276 237 252 225 98 129 112 118 148 145 146 164 136 149 119 132 155 129 142 153 171 196 247 221 190 93 123 115 109 111 126 102 103 86 118 99 142 143 144 144 165 175 173 175 219 174 Project Number 454-109 Mean SD 119 146 134 146 137 145 148 145 139 138 129 158 183 175 175 172 180 188 178 189 193 33 39 33 30 26 29 37 32 38 21 29 25 30 35 42 41 37 43 48 39 38 Differences between control and this treatment group were not significant (p > 0.05 ). Appendix VIII - Table 3 Feed Consumption (g/bird/day) from a Mallard Reproduction Study with PFOS (50 PPM A. I . ) Pen 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 WEEKS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 110 121 96 96 80 75 93 - - - - - - - - - - - - - - 73 108 96 81 87 125 - - - - - - - - - - - - - - - 116 123 159 153 123 112 109 - - - - - - - - - - - - - - 159 180 155 144 122 77 109 - - - - - - - - - - - - - - 90 98 98 122 129 119 134 - - - - - - - - - - - - - - 91 93 103 117 118 136 110 - - - - - - - - - - - - - - 79 86 100 101 125 136 104 - - - - - - - - - - - - - - 117 122 144 148 166 138 156 - - - - - - - - - - - - - - 116 105 129 134 84 88 87 - - - - - - - - - - - - - - 107 149 149 195 163 136 87 - - - - - - - - - - - - - - 77 90 81 102 95 158 63 - - - - - - - - - - - - - - 92 133 141 132 99 182 254 - - - - - - - - - - - - - - 114 110 114 130 109 123 95 - - - - - - - - - - - - - - 72 92 107 148 131 161 81 - - - - - - - - - - - - - - 96 131 109 124 90 113 101 - - - - - - - - - - - - - - 104 91 114 139 132 107 104 - - - - - - - - * - -- Project Number 454-109 Mean SD 101 114 118 129 116 124 112 - - - 22 25 24 27 26 29 45 - - - ------ - -- - -- ---- - ----- - Data are not available due to adult mortality. Differences between control and this treatment group were not significant (p > 0.05 ). Appendix VIII - Table 4 Feed Consumption (g/bird/day) from a Mallard Reproduction Study with PFOS (150 PPM A.I.) Pen 1 2 3 4 WEEKS 9 10 11 12 13 14 15 16 17 18 19 20 21 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 64 67 116 198 56 34 163 165 118 118 198 185 80 72 172 149 53 71 134 108 39 45 215 171 53 58 222 285 42 47 62 50 63 190 70 77 171 161 70 52 106 133 43 69 133 166 166 142 175 194 83 94 149 225 56 51 164 165 109 159 300 411 Mean SD 73* 75** 166** 194** 33 36 56 73 - Data are not available due to adult mortality. * Significantly different from the control at p < 0.05 * * Significantly different from the control at p < 0.01 CO Project Number 454-109 Males Foot/leg lesions Testes - small (--1.5-3.5 cm) Not remarkable Appendix IX - Table 1 Individual Gross Pathological Observations from a Mallard Reproduction Study with PFOS Adult Birds Euthanized at Test Termination Control (0 ppm a.i.) Pens 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 X XX - - - - - - a - - X - - - - - - -X- - - - a - - -X- - - - - X - XXXX a XX - XX Females Pens 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Foot/leg lesions Spleen - enlarged and mottled Liver - firm, pale and mottled Abdominal cavity - yolk remnants Abdominal cavity - slight egg yolk peritonitis Abdominal cavity - egg yolk peritonitis Abdominal cavity - slight old egg yolk peritonitis Small intestines - area of hyperemia Kidneys - pale Reproductive tract - multiple fluid filled cysts Ovary - regressing Ovary - regressed Ovary - cystic follicles Ovary - hemorrhagic follicles Not remarkable - X - XXX - XX - - X - - - X - -- -X- - - - - - - - - - - -- -X- - - - - - - - - - - . _ _ _X _ _ _ _ _ _ _ _ _ X _ __ - - X - - - - - -X- - - _ _X - X - - X - - - - - - XX X -X - -- -X- - - - - - - - - - - - -- - -X- - - - - - - - - X- - - - - X - - - - X - - XX - - - - - - - - - - - - -X- - - X - - - - - -X- " -X- - " - - a - Bird found dead during the test. Necropsy findings are presented in Table 2. 0C0fl Project Number 454-109 Males Foot/leg lesions Testes - small (~ 1.5-3.5 cm) Not remarkable Appendix IX - Table 2 Individual Gross Pathological Observations from a Mallard Reproduction Study with PFOS Adult Birds Euthanized at Test Termination 10 ppm a.i. Pens 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 - XX - - - X - - - - X - - - - -XX - - - - - X - X - - XX X - - - XX - XX - X - XX - - Females Foot/leg lesions Breast blister Cranium - swollen and bruised Heart - pericardial sac fluid-filled Spleen - enlarged and firm Abdominal cavity - egg yolk peritonitis Ovary - regressed Ovary - cystic follicles Not remarkable Pens 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 XX - XXXXXX - X - XXXX -X- - - - - - - - - - -X- - -X - - - - - - - - - - - - - - - - - - - -X- - -X- - - - --X- - - - - - - - - - - - - - XXX X - X - - XX - - - - - - - - - - - - -X- - Project Number 454-109 -87- Project Number 454-109 Appendix X - Table 1 Adult Liver Weights (g) from a Mallard Reproduction Study with PFOS Control (0 ppm a.i.) Male Pen Liver 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Mean SD 31.051 32.528 33.915 25.209 33.438 28.829 28.689 27.090 25.919 27.129 30.090 38.599 27.888 26.030 24.921 29.422 3.864 - Data not reported due to mortality. Female Liver 36.870 36.336 46.728 45.564 57.924 46.002 38.662 59.838 52.441 21.509 45.440 47.038 37.995 25.939 34.229 33.845 41.648 10.512 -88 - Project Number 454-109 Appendix X - Table 2 Adult Liver Weights (g) from a Mallard Reproduction Study with PFOS 10 ppm a.i. Male Pen Liver Female Liver 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 Mean SD 24.717 30.663 42.118 27.070 32.793 25.430 25.203 27.580 28.447 33.253 30.606 36.870 29.664 30.874 29.018 35.314 30.601 4.663 61.115 36.454 42.668 54.869 41.660 41.253 47.171 44.828 48.620 28.720 48.247 30.419 42.544 52.126 32.634 21.982 42.207 10.268 The above differences from the control group were not statistically significant (Student's t-test). -89- Wildlife International, Ltd. Project Number 454-109 Appendix XI Histopathology Report EPL -90- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-109 WILDLIFE INTERNATIONAL, LTD. STUDY NUMBER 454-109 EPL PROJECT NUMBER 212-028 PFOS: A REPRODUCTION STUDY WITH THE MALLARD PATHOLOGY REPORT Submitted by: Experimental Pathology Laboratories, Inc. P.O. Box 474 Herndon, VA 20172-0474 (703) 471-7060 Submitted to: Wildlife International, Ltd. Easton, MD 21601 December 27, 2001 Project Number 454-109 E P L __________________________ EXPERIMENTAL PATHOLOGY LABORATORIES, INC. TABLE OF CONTENTS Page PATHOLOGY SUMMARY..................................................................................... 1 COMPLIANCE STATEMENT................................................................................ 8 QUALITY ASSURANCE FINAL CERTIFICATION.............................................. ADULT MALLARDS SUMMARY INCIDENCE TABLES Adult Males....................................................................................................... Adult Females................................................................................................. HISTOPATHOLOGY INCIDENCE TABLES Adult Males....................................................................................................... Adult Females.................................................................................................. CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult Males....................................................................................................... Adult Females.................................................................................................. 1-1 I-3 11-1 II-5 111-1 III-4 OFFSPRING MALLARDS SUMMARY INCIDENCE TABLES Offspring Males.............................................................................................. Offspring Females........................................................................................... HISTOPATHOLOGY INCIDENCE TABLES Offspring Males............................................................................................... Offspring Females........................................................................................... IV-1 IV-2 V-1 V-2 -92- Project Number 454-109 PATHOLOGY SUMMARY EPL -93 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-109 WILDLIFE INTERNATIONAL, LTD. STUDY NUMBER 454-109 EPL PROJECT NUMBER 212-028 PFOS: A REPRODUCTION STUDY WITH THE MALLARD PATHOLOGY SUMMARY Light microscopic examination was performed on sections of selected tissues from adult male and female mallard ducks (A n a s platyrhynchos) which were untreated or which received 10 ppm a.i. of the test article (Perfluorooctane Sulfonic Acid, Potassium Salt [PFOS]) in the feed for approximately 21 weeks. Selected tissues from untreated 14-day post-hatching offspring of the adult mallards were also examined by light microscopy. The objective of this study is to evaluate the effects upon adult mallards (A n a s platyrhynchos) of dietary exposure to the test substance over at least a 21-week period. The experimental design was as follows: Adults3 Offspring0 | | Experimental Group 1 Control (0 ppm) 1 10 ppm a.i. Males 20 20 Females 20 20 Males 3 3 7 _IFemales 7 aAdults were euthanized after receiving the test substance in the diet for approximately 21 weeks. bOffspring did not receive the test substance in the diet. All offspring were euthanized on Day 14 post-hatching. MATERIALS AND METHODS At the completion of the study interval, all surviving adult mallards were euthanized. Selected offspring were also euthanized at approximately 14 days of age. Necropsies were performed on all birds by Wildlife International, Ltd. EPL -94EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-109 Wildlife International, Ltd. Study Number 454-109 Tissues were collected by Wildlife International, Ltd. from adults and offspring, fixed in 10% neutral buffered formalin, and sent to Experimental Pathology Laboratories, Inc (EPL), where they were trimmed, embedded in paraffin, and made into hematoxylin and eosin-stained sections on glass microscope slides. The following tissues from adult and offspring mallards were examined by light microscopy as available: liver, gallbladder, proventriculus, kidney, brain, gonad (ovary or testis), bursa of Fabricius, and adipose tissue. All tissues required by protocol are presented in the Histopathology Incidence Tables. Microscopic findings for each tissue examined from each mallard are listed in the Histopathology Incidence Tables. Microscopic changes were graded one to five depending on severity. Nongradable findings (including autolysis such that a diagnostic interpretation could not be rendered) are listed as present (P). All findings for all animals are summarized by sex, age group, and treatment group in the Summary Incidence Tables, together with the total number of animals in each group for which tissues were examined. A tabulation of gross observations noted at necropsy with the corresponding microscopic change, if appropriate, is presented in the Correlation of Gross and Microscopic Findings tables. The observations in these tables were transcribed from the Gross Necropsy records provided by Wildlife International, Ltd. RESULTS Except for one control (3235) adult male, all adults and offspring survived to the end of their respective study intervals. No effects considered possibly related to test article (PFOS) administration were noted in the liver, kidney, adipose tissue, brain, and proventriculus of adult and offspring males and females, bursa of Fabricius in offspring males and females, ovaries of adult and -2- EPL -95 - EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-109 Wildlife International, Ltd. Study Number 454-109 offspring females and testes of male offspring. Any potential test article effects on the bursa of Fabrlcius in adult males and females could not be determined since no samples were submitted for evaluation. The bursa of Fabricius typically regresses prior to sexual maturity. In addition, any potential test-article effects on the gallbladder from adults and offspring could not be definitively determined due to the extensive autolysis, which precluded any accurate diagnosis. In two (2) control and seven (7) 10 ppm a.i. adult males, the testes were observed macroscopically as being reduced in size (< 3.5 cm) when compared to other birds. Of these birds, one control (3225) and four treated birds (3261, 3275, 3279, 3287), exhibited decreased spermatogenesis, characterized by a reduction in the number or absence of maturing/mature spermatozoa in the seminiferous tubules. The other control (3243) and one treated bird (3295) exhibited degeneration/atrophy of the seminiferous tubules characterized by an absence of mature sperm and a decrease in the number of cell layers of the germinal epithelium. The remaining two of seven 10 ppm a.i. males (3263, 3285) with macroscopically noted reduced testicular size and the balance of the control and 10 ppm a.i. males exhibited normal spermatogenesis, characterized by the presence of numerous spermatids and mature spermatozoa in the seminiferous tubules. Reduced testicular size, with or without microscopic evidence of decreased spermatogenesis, is a feature most consistent with early post reproductive phase regressions, a normal physiological phenomenon. The degeneration/atrophy noted in the control and treated adult birds may simply represent a later stage of this normal change. However, the increased incidence of reduced size (with or without a corresponding microscopic observation) of the testes in 10 ppm a.i. adult males, when compared to controls (7 vs. 2), suggests -3- EPL -96- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-109 Wildlife International, Ltd. Study Number 454-109 that treatment may have accelerated the normal post-reproductive phase regression. Testes of all offspring examined were listed as immature because they lacked all evidence of spermatogenesis, had small seminiferous tubules with absent or narrow lumens, and had germinal epithelium with decreased cell layers (compared to adult testes). This morphology is consistent with normal physiological immaturity expected in 14 day-old birds. Ovaries of adult females exhibited normal oogenesis, characterized by numerous ova in various stages of follicular development. In five (5) controls and five (5) 10 ppm a.i. adult females, the ovaries were indicated macroscopically as "regressed" or "regressing." Microscopically, those ovaries were no different from their cohorts, except that there may have been a reduction in the number of fully developed ova. If present, this change was not recorded microscopically, since it was considered to be consistent with early post-reproductive phase regression, a normal physiological phenomenon. Since the number of females affected was the same in both the control and 10 ppm a.i. groups, there was no indication that treatment had any effect on this phenomena. All ovaries of offspring females were characterized by numerous oocytes and very small, nonpedunculated follicles completely embedded in a prominent ovarian cortex. This morphology is consistent with the normal physiological immaturity expected in 14 day-old birds. Vacuolization in the liver was characterized by the presence of vacuoles within the hepatocellular cytoplasm which varied from, usually small, poorly demarcated vacuoles, giving a "lace-like" appearance to the hepatocellular cytoplasm, to variably sized, sharply demarcated, clear vacuoles in the hepatocellular cytoplasm. While special stains were not employed to identify the -4- EPL -97- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-109 Wildlife International, Ltd. Study Number 454-109 material in the vacuoles, the latter morphology was consistent with fat accumulation. Hepatocellular vacuolization was observed In the livers of adults and offspring of both sexes. Although there was a slight increase in the incidence of this change in 10 ppm a.i. treated adult males when compared to controls (20 vs. 12), there was no substantial difference in the mean severity between the groups. In addition, the change was not observed in treated male offspring or at a significant difference in treated adult or offspring females when compared to their respective controls. Since this finding was commonly observed in the livers from control birds, it was considered to be incidental and not related to test article administration. The apparent increased incidence in the treated adult males was probably due to biological and/or distributional variability. Additional incidental findings also noted in the liver included liver pigment deposition in adult males, consisting of dust-like, brown to golden-brown pigment granules within the hepatocellular cytoplasm. The identity of the pigment was not determined, but it may have been biliary in origin. Also, the liver of two control adult males and one control adult female exhibited amyloid deposition consisting of amorphous pale eosinophilic material, which filled the hepatic sinusoids. In the one control male that died (3235), the liver parenchyma was almost completely replaced by the deposited amyloid. Also noted in the liver, at a low incidence in control and/or treated male and female adults and offspring, were foci of hepatocellular degeneration/ necrosis. These lesions were generally single, small, discrete foci of pale to hyperosinophilie hpatocytes with indistinct, fragmented, or condensed nuclei. Although there was generally a slight predominance of the lesion in treated birds, when compared to controls, it was considered incidental and not related to -5- EPL -98- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-109 Wildlife International, Ltd. Study Number 454-109 treatment because of the low incidence, limited distribution, and relatively low severity (generally minimal to slight). Additional microscopic findings (described below) noted in adult and/or offspring mallards from control and treated groups included mononuclear cell infiltrates in various tissues, kidney nephropathy, and in the proventriculus, hyperplasia of the glandular epithelium. Generally, in both adult and offspring males and females, there was either no significant difference in the incidence of these lesions between control and treated groups, or the incidence was variable, with some lesions occurring at a higher incidence in controls when compared to treated birds, and other lesions having the opposite distribution. In addition, there was no significant difference in the mean severity of these lesions in treated birds when compared to controls. For these reasons, these findings were considered incidental and unrelated to test article (PFOS) administration. Mononuclear cell infiltrates, observed in various tissues, consisted of foci of lymphocytes and plasma cells generally accompanied by a few heterophils as a minor component. Nephropathy in the kidneys of both control and treated adult males and females was characterized by the presence of basophilic and/or regenerative tubules in the renal cortex. Hyperplasia of the proventricular glandular epithelium in adult females was generally multifocal in distribution, and characterized by increased numbers and/or basophilic staining of the glandular epithelium. In many cases, there was minimal compression of adjacent normal glandular epithelium. The pathogenesis of this lesion was not determined. However, since the incidence was not significantly different between controls and treated birds, it was considered incidental and not related to treatment. -6- EPL -99- EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-109 Wildlife International, Ltd. Study Number 454-109 CONCLUSIONS AND SUMMARY No lesions considered possibly related to test article (PFOS) administration were noted in the liver, kidney, adipose tissue, brain, and proventriculus of adult and offspring males and females, bursa of Fabricius in offspring males and females, ovaries of adult and offspring females and testes of male offspring. The increase in the number of adult males in the 10 ppm a.i. treated group with reduced testicular size (with or without a corresponding microscopic observation), suggests that PFOS may have accelerated early post reproductive phase regression, a normal physiological phenomenon. Any potential test-article effects on the bursa of Fabricius in adult males and females could not be determined since no samples were submitted for evaluation. In addition, any potential test-article effects on the gallbladder from adults and offspring could not be definitively determined due to the extensive autolysis that precluded any accurate diagnosis. The few other changes in various tissues of adult and/or offspring mallards from control and 10 ppm a.i. treated groups were considered incidental and unrelated to test article (PFOS) administration. FAR/lcp FREDERiu k a . H U tU K tH , u v m , Mt>, uiplomate, ACVP Veterinary Path 1 ' ' Date -7- EPL - 100 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-109 COMPLIANCE STATEMENT Client Name Wildlife International, Ltd. EPL Project Coordinator Dr. Margarita M. Gruebbel Client Study EPL 4 5 4 -1 0 9 _______________________ Pathologist Dr. Frederick A. R u ecker EPL Project Species M allard_______________________ Number 212-028 Study Title PFOS: A Reproduction Study with the Mallard Test Article Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) The histopathology portions of the above-referenced study were conducted in compliance with the Good Laboratory Practice regulations of the Environmental Protection Agency as stipulated by 40 CFR 160. jjl*. -T- -8- EPL - 101 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Project Number 454-109 QUALITY ASSURANCE FINAL CERTIFICATION Study Title: PFOS: A Reproduction Study with the Mallard Client Study: 454-109 EPL Project Coordinator: Dr. Margarita M. Gruebbel EPL Project Number: 212-028 EPL Pathologist: Dr. Frederick A. Ruecker The following aspects of this study were inspected by the Quality Assurance Unit of Experimental Pathology Laboratories, Inc. Dates inspections were performed and findings reported to the EPL Project Coordinator and Management are indicated below. Area Inspected Inspection Dates Reporting EPL Project Sheets Project Setup Histology Setup Data Review Draft Report Final Report 10/11/01; 10/15/01 10/12/01 10/19/01 11/6,7/01 11/30/01; 12/4/01 1/28/03 10/11/01; 10/15/01 10/12/01 10/19/01 11/6,7/01 12/4/01 1/28/03 Date reported to Study Director/Management______________ 1/28/03 FormNo. 6-2(7/2/99) -9- - 102 - Project Number 454-109 ADULT SUMMARY INCIDENCE TABLES - 103 - Project Number 454-109 454-109 Adult Sacrifice Male Mallard SUMMARY INCIDENCE TABLE ADIPOSE TISSUE (NO. EXAMINED) Edema Foreign Body Granuloma I n f i l t r a t e , Mononuclear Cell Inflammation GROUP CONTROL (20) 1 GROUP 10-ppmai (20) 2 1 BRAIN (NO. EXAMINED) Coccoid Organisms, Intravascular I n f i l t r a t e , Mononuclear Cell , Perivascular Malacia Vasculiti s/Peri vasculi t i s (20) 1 1 1 1 (20) BURSA OF FABRICIUS (NO. EXAMINED) GALLBLADDER (NO. EXAMINED) Autolysis Precludes Diagnosis (20) 20 (20) 20 KIDNEY (NO. EXAMINED) Amyloid Deposition Coccoid Organisms I n f i l t r a t e , Mononuclear Cell Inflammation, Acute Inflammation, Granulomatous Mi ne r a l i z a t i on Nephropathy (20) 1 6 1 1 2 (20) 1 3 LIVER (NO. EXAMINED) Amyloid Deposition Dgnrt i on/Necros i s , He pat oc e l l ul ar Hyperplasia, Bi le Ducts I n f i l t r a t e , Mixed Inflammatory Cell I n f i l t r a t e , Mononuclear Cell Intracytoplasmic Brown Pigment, Hepatocytes Mononuclear Cell I n f i l t r a t e Vacuolization, Hepatocellular (20) 2 20 8 12 (20) 2 20 7 20 PROVENTRICULUS (NO. EXAMINED) Atrophy, Glandular Epithelium (20) (20) EPL Experimental Pathology Laboratories, Inc. 1-1 454-109 Adult Sacrifice Mal e Mai l a r d PROVENTRICULUS (CONTINUED) Hyperplasia, Glandular Epithelium, Multifocal I n f i l t r a t e , Mononuclear Cell Vasculi ti s/Peri vascul i t i s TESTIS (NO. EXAMINED) Coccoid Organisms, Intravascular Degeneration/Atrophy, Seminiferous Tubules Di l atat i on, Seminiferous Tubules I n f i l t r a t e , Mononuclear Cell Reduced Spermatogenesis Vasculi ti s/Peri vascul i t i s - 104- Project Number 454-109 SUMMARY INCIDENCE TABLE GROUP CONTROL GROUP 10-ppmai 2 1 (20) 1 1 3 1 1 5 (20) 1 1 5 4 EPL Experimental Pathology Laboratories, Inc. 1-2 - 105 - Project Number 454-109 454-109 Adult Sacrifice F ema l e Mai l a r d SUMMARY INCIDENCE TABLE ADIPOSE TISSUE (NO. EXAMINED) Edema Foreign Body Granuloma I n f i l t r a t e , Mononuclear Cell Inflammation GROUP CONTROL (20) GROUP 10-ppmai (20) 1 1 41 BRAIN (NO. EXAMINED) Coccoid Organisms, Intravascular I n f i l t r a t e , Mononuclear Cell , Perivascular Malacia Vasculitis/Perivasculitis (20) (20) BURSA OF FABRICIUS (NO. EXAMINED) GALLBLADDER (NO. EXAMINED) Autolysis Precludes Diagnosis (20) 20 (19) 19 KIDNEY (NO. EXAMINED) Amyloid Deposition Coccoid Organisms I n f i l t r a t e , Mononuclear Cell Inflammation, Acute Inflammation, Granulomatous Mi ne r a l i z a t i o n Nephropathy L I V E R (NO. E X A M I N E D ) Amyloid Deposition Degenerati on/Necrosi s, Hepatocellular Hyperplasia, Bi le Ducts I n f i l t r a t e , Mixed Inflammatory Cell I n f i l t r a t e , Mononuclear Cell Intracytoplasmic Brown Pigment, Hepatocytes Mononuclear Cell I n f i l t r a t e Vacuolization, Hepatocellular (20) 1 6 3 (20) 1 1 1 17 1 17 (20) 6 1 5 (20) 3 1 15 16 OVARY (NO. EXAMINED) Amyloid Deposition Atrophy (20) 1 1 E PL Exprimental Pathology laboratories, Inc. (20) 1-3 - 106- Project Number 454-109 454-109 Adult Sacrifice F ema l e Mai l a r d SUMMARY INCIDENCE TABLE OVARY (CONTINUED) Inflammation GROUP CONTROL GROUP 10-ppmai 11 PROVENTRICULUS (NO. EXAMINED) Atrophy, Glandular Epithelium Hyperplasia, Glandular Epithelium, Multifocal I n f i l t r a t e , Mononuclear Cell Vasculiti s/Peri vasculi ti s (20) 1 3 5 (20) 1 4 6 EPL Experimental Pathology Laboratories, Inc. 1-4 - 107 - Project Number 454-109 ADULT HISTOPATHOLOGY INCIDENCE TABLES - 108 - Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE GROUP 454-109 Adult Sacri f i ce Male Mai lard A Ni l M 3 3 3 3 3 3 3 3 3 3l 3 3 3 3 3 3 3 3 3 3 A 22222222222222222222 L 1 1222223333344444555 ADTPOSF TISSI1F 7 9 1 3 5 7 9 3 5 7 9 1 3 5 7 9 1 3 t-5- XXXXXXXXX X XX XX XX XXX Fdema 2 Foreian Bodv Granuloma I n f i l t r a t e . Mononuclear Cell Inflammati on 1 BRAIN Coccoid Oroanisms. Tnt.ravascul ar I n f i l t r a t e . Mononuclear Cell . Perivascular Malacia Vascul i t.i s/Peri vascul i ti s BURSA OF FARRICIIIS GAI 1Rl ADDFR Aut.olvsis Precludes Diaanosis KIDNFY Amvloid Deposition Coccoid Organisms I n f i l t r a t e . Mononuclear Cel 1 Inflammation. Acute Inflammation. Granulomatous Minerali zation Nephropat.hv 1 Ti/FR Amvloid Deposition Deoenerati on/Necrosis. He pa t o c e l l ul a r Hvperplasia. Rile Ducts I n f i l t r a t e . Mixed Inflammatory Cell I n f i l t r a t e . Mononuclear Cell Intracvtoplasnric Rrown Pi ament. Hepatocvtes Mononuclear Cell I n f i l t r a t e Vacuoli zati on. Henatocel1ular X x: x X X X X X X X X X X X X X X X i :i !.i il 'P 1 !1 i i 2.j ! !4 ' !! | N1 N N N: N N! N Ni N N N : N N! N N N N I N N N | A A A Ai A: A A A! A A A A A A iA A A A A A P : P I P P^P P P ---P- 1[ P P P P P P : P P P i P P P ; m Xm m m Xm m Xm Xm m m Xm X Xm Xm 1 X m Xm Xm m Xm 1 ? | 1 1 1 ! p 3 ? ! i1 jj 3 i ,j ! i ; 5 1 ! ! 1 1 1 1 1 ? 1 113 I 1 11 1 1 1 1 1 1 1 11 ii ; 1i ''! 1i 2 ! 2 : 2 ___ 1_______ 1__2____ L __ L L _1_ i _2_ 2 :1 EPL II-l Experimental Pathology Laboratories, Inc. Key :X=Not Remarkable N=No Section I-Incomplete A-Autolysis l=mininial 2=slight/miId 3-moderate 4-moderately severe 5-severe/high P-Present B-Benign M-Malignant m-missing one paired organ u-unscheduled sac./death - 109 - Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL 454-109 Adult Sacri f i ce Male Mai lard A N 1 M A L PROVENTRICULUS AtroDhv. Glandular EDithelium HvDerDlasia. Glandular Fpit.helium. Multifocal I n f i l t r a t e . Mononuclear Cell Vascul i t . i s/Peri vascul i t i s 3 3 3 3 3 3 3 3 3 3i 3 3 3 3 3 3 3 3 3 3 2 2 2 222 2 222 2 22 22 2 2 22 2 1 1222223333344444555 791 3 57913579 1 3 579 1 3 5 X XXXXXX XXXXXXXXXX 11 ? TESTIS Coccoid Oraanisms. Intravascular Oeoenerati on/Atrophv. Seminiferous Tubules Dilatation, Seminiferous Tubules I n f i l t r a t e . Mononuclear Cell Reduced Spermatoaenesis Vasculitis/Perivasculitis Xm Xm Xm Xm m m Xm Xm Xm m Xm Xm Xm m Xm Xm m Xm Xm Xm , i ;: p1 4 !: i! i 3 !l i ii 1 i :! j ! !!| : i ; i . 1 ____ 1__ ! EPL 11-2 Experimental Pathology Laboratories, Inc. Key :X=Not Remarkable N>No Section I-Incomplete A'Autolysis l=minimal 2=sl1ght/mi1d 3"moderate 4=moderately severe 5=severe/high p.present B-Benign M-Malignant m=missing one paired organ u=unscbeduled sac./death - 110- Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE i--T--- GROUP 10-DPmai 454-109 Adult Sacri fi ce Male Mai lard A N 1 ADIPOSE TISSUE Edema M 3 33 3 33 3 33 3 33 3 3 33 3 33 3 A 22222222222222222222 L 556666677 7 7788888999 / 9 1 3 5 / 9 1 3 5 7 9 1 3 5 7 9 _Li_3_ 5 X X X! X X X X X X X X X X X X X ! X X i Foreian Bodv Granuloma I n f i l t r a t e . Mononuclear Cell 11 1 Inflammation BRAIN Coccoid Oraanisms. Intravascular 1I n f i l t r a t e . Mononuclear Cel . Peri vascular Malacia Vasculi ti s/Peri vasculi ti s BURSA OF FABRICIUS GA1 1Rl ADDFR Autolvsis Precludes Diaonosis X X X X X 1i X X X X X X X X X X X X X X X ! ;! ! ! ; ; : ili , N 1N! N : N N ; N N N 1N N N N N N N N ! N N N 1N ! AAA AA AAAAAAA A AAA AA AA P P P p P P P P P P P P P _E_ P P P P P P KIDNEY Amvloid Deposition Coccoid Ornanisms I n f i l t r a t e . Mononuclear Cell Inflammation. Acute Inflammation. Granulomatous Minerali zation Nephropathv m mXm Xm Xm m Xm Xm Xm Xm m Xm Xm Xm Xm Xm Xm Xm Xm Xm __1 i ?i i LIVER Amvloid Deposition .Deaenerati on/Necrosi s Heoatocellular Hvperplasia. Bile Ducts I n f i l t r a t e . Mixed Inflammatory Cell I n f i l t r a t e . Mononuclear Cel 1 Intracvtoolasmic Brown Pi ament, Heoatocvtes Mononuclear Cell I n f i l t r a t e Vacuolization. Hepatocellular 1 -- i 1 1 1 21r i i i 1 1 1 i :i i 1 1 ? 1 1 1 1 !1 2:2. !2 ; 1 ! 1i1 : 1 ! :' :2 3 2 1 1 ! 1 2 1 122 122 i 1 22 1 1 E PL 11-3 Experimental Pathology Laboratories, Inc. Key :XNot Remarkable N=No Section 'Incomplete A=Autolysi$ 'minimal 2=s1ight/mi1d 3=moderate 4=moderately severe 5=severe/high Represent B=Benign M'Malignant m=missing one paired organ u=unscheduled sac./death - Ill - Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE GROUP 10-DPmai 454-109 Adult Sacri fi ce i Male Mai lard A N l M 3; 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 A 22222222222222222222 L 55666667 7 77 788888999 PROVENTRICULUS 7 9 1 3 ,5 7 9 1 3 5 7 9 1 3 5 7 9 1 3 5 XXXX XX XX XXXXX XX Atrophv. Glandular Epithelium HvDerDlasia. Glandular Epithelium. Multifocal ; I n f i l t r a t e . Mononuclear Gel 1 ? 11 1 1 Vascul i t.i s/Peri vascul i t i s TESTIS Coccoid Oraanisms. Intravascular Deoenerat. i on /Atronhv. Seminiferous Tubules Dilatation. Seminiferous Tubules I n f i l t r a t e . Mononuclear Cell Reduced Soermatooenesis Vasculi ti s/Perivasculi ti s i Xm Xm m Xm Xm Xm m Xm ' m m Xm : m Ixm Xm m m Xm m Xm m !: 3 ! -- i i 1i 1 ?3 ,i ;| 5 i 3 i ! [j i 'i -- i-- ' !i i' ;: ' 1 E PL 11-4 Experimental Pathology Laboratories, Inc. Key :X=Not Remarkable N=No Section I-Incomplete A"Autolysis l=minimal 2=slight/mild 3moderate 4-moderately severe 5=severe/high PPresent B-Benign MMalignant mmissing one paired organ u=unscheduled sac./death - 112 - Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL 454-109 Adult Sacri fi ce Female Mai lard A N I ADIPOSE TISSUE M3333333333333333333 3 A 22222222222222222222 L 12222233333444445555 B 0 ? 4 6 8 0 ? 4 fi 8 0 ? 4 fi fi 0 ? 4 fi XXX XXXX X X X XX XXX Edema Foreian Bodv Granuloma P I n f i l t r a t e . Mononuclear Cell Inflammation 1 ? ?? BRAIN Coccoid Oraanisms. Intravascular I n f i l t r a t e . Mononuclear Cel 1. Perivascular Malacia Vasculitis/Perivasculitis XX XXX XXXXX XX X XX X X XX X .; ! j ; :! i1 '1 . : BURSA OF FABRICIUS N N N N N!N N N N N N N N N N N N 1N N N GA1 1Bl ADDFR Aut.olvsis Precludes Diannosis KIDNEY Amvloid DeDOsition Coccoid Oraanisms I n f i l t r a t e . Mononuclear Cel 1 Inflammation. Acute Inflam m ation. Granulomatous Minerali zati on NeDhrooathv AAAAAA AA AAAAAAA AAAAA PPPPPPPPPP PPPPPPPPPP ! X Xm m m Xm 3 m m m Xm Xm X m m X m Xm Xm Xm i ii ii i 1 1i LIVER Amvloid Deposition De a ener at i on/ Nec r os i s . Heoatocellular HvDerDlasia. Bile Ducts I n f i l t r a t e . Mixed Inflammatorv Cell I n f i l t r a t e . Mononuclear Cel 1 Intracvtoolasmic Brown Pi ament. Heoatocvtes Mononuclear Cell I n f i l t r a t e Vacuolization. Heoatocel1ular 3 i i ? |i i 1 1 j 1 : 1 ; 1 ! 1 i i i i JL 1 i : i 1 3! ! 1, !^ ji 2 1 2! 1! 2: 1 1i 4 3 22 1 ! i 21i 1 1 !1 2!2 EPL 11-5 Experimental Pathology Laboratories, Inc. Key :X=Not Remarkable NNo Section Incomplete A=Autolysi$ minimal 2=slight/mi1d 3=moderate 4=moderately severe 5severe/high P=Present B=Benign M=Malignant m=missing one paired organ u*unscheduled sac./death - 113 - Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE 454-109 Adult Sacri fi ce Female Mai lard OVARY Amvloid Deposition Atrohv Inflammation GROUP CONTROL i-- A N 1 M 33333 3333333 33333 333 A 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 I2 2 L 1 2222233333444445 55 5 (3 0 ? 4 ft 8 0 ? 4 (i 8 0 ? 4 fi 8 0 ? 4 f) XXXX X X XX XX X XX XXXX ? 41 3 PROVENTRICULUS AtroDhv. Glandular EDithelium HvDerDlasia. Glandular EDithelium. Multifocal I n f i l t r a t e . Mononuclear Cell Vasculi ti s/ Peri v ascul i t i s XX XXXXX XX X ? | XX XX 2 1 t1 1 11 ! ?1 2 ii ; ; ; Ii .'j ii : I ' 1' ! 1i : _____ i_______ ;1 1 j EPL 11-6 Experimental Pathology Laboratories, Inc. Key :X=Not Remarkable N=No Section 'Incomplete A=Autolysis l*niinimal 2=$1ight/miId 3*moderate 4=moderately severe 5*severe/h1gh P*Present B=Benign M'Malignant msmissing one paired organ u*unscheduled sac./death -114- Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE GROUP 10-ppmai 454-109 Adult Sacri fi ce Female Mallard A N 1 ADIPOSE TISSUE Edema M 333 33 33 3 33 3 3 3 3 33 3 3 3 3 A 22222222222222222222 L 5666667 7 7 77888889999 8 0 ? 4 fi a 0 ? 4 fi 8 0 ? 4 fi 8 0 ? 4 fi X XXXXXX XXXXXXXXXXX Foreian Bodv Granuloma I n f i l t r a t e . Mononuclear Cel 1 ---- p 1 Inti animati on 1 BRAIN Coccoid Oraanisms. Intravascular I n f i l t r a t e . Mononuclear Cel 1. Peri vascular Malacia Vasculitis/Perivasculitis BURSA OF FABRICIUS GAI 1 Bl ADDFR Autolvsis Precludes Diaanosis XXXXXXXXXXXXXXXXXXX X .:j. i ;! Ni N N ! N ' N N *: A A 1 N: A A A PP P p! p N A P N N1 N N N N N N N N! N Ni N 1 __ 1__ A A A Ai A A A AiA!A A A A P P P P P _Ej P P 1 P P P P P KIDNEY Amyloid Deposition Coccoid Oraanisms I n f i l t r a t e . Mononuclear Cell Inflammation. Acute Inflammation. Granulomatous Minerali zati on Neohronathv Xm Xm m Xm Xm Xm Xm Xm m X m m Xm Xm m m Xm m m ! ! 1!1 i ii ,i l 1 ? 21 i 1 IVFR Amvloid Deposition Deaenerati on/Necrosi s . He oa t oc e l 1 ul a r Hvoerolasia. Bi le Ducts I n f i l t r a t e . Mixed Inflammatory Cell I n f i l t r a t e . Mononuclear Cell Intracvtoplasmic Brown Pi ament. Hepatocvtes Mononuclear Cell I n f i l t r a t e Vacuolization. Hepatocellular 21 2 1 2 1 j 1! 1 . 111 i : , ! 1l 1 1 ii 1 1 1;l i i 1 i 12 1 1 : 2 1 ! 1 i 1 i ; 3 i i i 1 1? i EPL 11-7 Experimental Pathology Laboratories, Inc. Key :X=Not Remarkable N*No Section Incomplete A=Autolysis l=minima1 2slight/mi1d 3=moderate 4=moderately severe 5=severe/high P*Pre$ent B=Benign M=Halignant m=missing one paired organ u=unscheduled sac./death - 115 - Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE GROUP EPL Experimental Pathology Laboratories, Inc. Key :X-Not Remarkable N=No Section -Incomplete A=Autolysis -minimal 2-slight/mild 3-moderate 4-moderately severe 5=severe/high P=Present B=Benign M-Malignant m=missing one paired organ u-unscheduled sac./death - 116- Project Number 454-109 ADULT CORRELATION OF GROSS AND MICROSCOPIC FINDINGS 454-109 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult Sac ri f i c e Species: Mallard___________________________________________ Sex: Males G roup Identification: CONTROL - Sacrificed A nim al Number Client Topography / Site Client Gross Observations M icroscopic Observations 3225 TESTES Approximately 3.0 cm Reduced Spermatogenesis (TESTIS) 3243 TESTES Approximately 2.0 cm Degenerati on/Atrophy, Semi niferous Tubules (TESTIS) -- Project Number 454-109 III-l 454-109 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult Sacrifice Species: Mallard__________________________________________________ Sex: Males G roup Identification: CONTROL - Unscheduled Sac./Death A nim al Number Client Topography / Site Client Gross Observations M icroscopic Observations 3235 KIDNEY Slightly pale No Correlating Lesion Project Number 454-109 l i 1-2 454-109 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult Sacrifice Species: Mallard____________________________________________ Sex: Males _. G roup Identification: 10-ppmai Animal Number Client Topography / Site Client Gross Observations M icroscopic Observations 3261 TESTES Approximately 3.0 cm Reduced Spermatogenesis (TESTIS) - Sacrificed 3263 TESTES Approximately 3.0 cm No Correlating Lesion (TESTIS) 3275 TESTES Approximately 3.0 cm Reduced Spermatogenesis (TESTIS) 3279 TESTES Approximately 3.0 cm Reduced Spermatogenesis (TESTIS) 3285 TESTES Approximately 3.5 cm No Correlating Lesion (TESTIS) 3287 TESTES Approximately 3.0 cm Reduced Spermatogenesis (TESTIS) 3295 TESTES 1.5 cm Degeneration/Atrophy, Seminiferous Tubules (TESTIS) Project Number 454-109 11 1-3 454-109 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult Sacri f ice Species: Mallard___________________________________________ Sex: Females ,_ G roup Identification: CONTROL - Sacrificed Animal Number Client Topography / Site Client Gross Observations M icroscopic Observations 3226 LIVER Firm, pale, mottled Amyloid Deposition KIDNEY Pale Amyloid Deposition OVARY Regressed ovary No Correlati ng Lesion 3228 OVARY Multiple f l u i d - f i l l e d cysts No Correlati ng Lesion 3236 OVARY Regressed ovary No Correlati ng Lesion 3242 OVARY Regressed ovary Atrophy OVARY Cystic foll ic le s No Correlati ng Lesion 3244 OVARY Regressed ovary No Correlati ng Lesion 3246 OVARY Regressing ovary No Correlat i ng Lesion 3248 OVARY Hemorrhagic f o l l i c l e Inflammation Project Number 454-109 1 1 1-4 454-109 CORRELATION OF GROSS AND MICROSCOPIC FINDINGS Adult S a c r i f i c e Species: Mallard___________________________________________ Sex: Females __ G roup Identification: 10-ppmai A nim al Number Client Topography/ Site Client Gross Observations M icroscopic Observations 3276 OVARY Regressed ovary No Correlati ng Lesion - Sacrificed 3280 OVARY Regressed ovary No Correlati ng Lesion 3284 OVARY Cystic f o l 1icles No Correlati ng Lesion 3286 OVARY Regressed ovary No Correlati ng Lesion 3288 OVARY Regressed ovary No Correlati ng Lesion 3290 OVARY Regressed ovary No Correlat i ng Lesion Project Number 454-109 111-5 - 122 - Project Number 454-109 OFFSPRING SUMMARY INCIDENCE TABLES - 123 - Project Number 454-109 454-109 Offspring Sacrifice Male Mai lard SUMMARY INCIDENCE TABLE ADIPOSE TISSUE (NO. EXAMINED) I n f i l t r a t e , Mononuclear Cell GROUP CONTROL (3) GROUP 10-ppmai (3) BRAIN (NO. EXAMINED) (3) (3) BURSA OF FABRICIUS (NO. EXAMINED) (3) (3) GALLBLADDER (NO. EXAMINED) Autolysis Precludes Diagnosis (3) 3 (3) 2 KIDNEY (NO. EXAMINED) I n f i l t r a t e , Mononuclear Cell (3) (3) LIVER (NO. EXAMINED) Degeneration/Necrosi s, Hepatocellular, Acute I n f i l t r a t e , Mononuclear Cell Vacuolization, Hepatocellular (3) 2 3 (3) 1 1 PROVENTRICULUS (NO. EXAMINED) (3) (3) I n f i l t r a t e , Mononuclear Cell 1 3 TESTIS (NO. EXAMINED) Immature (2) (3) 23 EPL Experimental Pathology Laboratories, Inc. 1 IV-1 - 124 - Project Number 454-109 454-109 Offspring Sacrifice Female Mai lard SUMMARY INCIDENCE TABLE ADIPOSE TISSUE (NO. EXAMINED) I n f i l t r a t e , Mononuclear Cell GROUP CONTROL (7) GROUP 10-ppmai (7) 1 BRAIN (NO. EXAMINED) (7) (7) BURSA OF FABRICIUS (NO. EXAMINED) GALLBLADDER (NO. EXAMINED) Autolysis Precludes Diagnosis (7) (6) 4 (7) (6) 4 KIDNEY (NO. EXAMINED) I n f i l t r a t e , Mononuclear Cel 1 (7) 3 (7) LIVER (NO. EXAMINED) Degeneration/Necrosi s, Hepatocellular, Acute I n f i l t r a t e , Mononuclear Cell Vacuolization, Hepatocellular (7) 3 5 3 (7) 2 3 2 OVARY (NO. EXAMINED) Immature (7) (7) 77 PROVENTRICULUS (NO. EXAMINED) (7) (7) I n f i l t r a t e , Mononuclear Cell 5 6 EPL Experimental Pathology Laboratories, Inc. IV-2 - 125 - Project Number 454-109 OFFSPRING HISTOPATHOLOGY INCIDENCE TABLES - 126 - Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE GROUP GROUP 454-109 Offspring Sacrifice Male Mallard A N 1 M A L ADIPOSF TISSIJF I n f i l t r a t e . Mononuclear Cel 1 99 55 79 a1 XX 9 6 0 6 X BRAIN Xi X X BURSA OF FABRICIUS Xi X X 999 666 2 34 5 fi ? XXX XXX i X X X i 1 GA1 1BI.AODFR Autolvsis Precludes Diaanosis KIDNFY I n f i l t r a t e . Mononuclear Cell AAA P: P P Xm Xm Xm x ! A: A pI p !: Xm Xm Xm 1 LIVER Oenenerati on/Necrosi s . Hepatocellular. Acute I n f i l t r a t e . Mononuclear Cell Vacuol i 7at:i on. Hepatocellular PROVFNTRICUI US I n f i l t r a t e . Mononuclear Cell TFSTIS Immature | 11 111 i XX !l N PP X 1 1 s ---- j---- 211 ; pPp : ; j ; ! ! . ' 1j .j.... :__ !i :. ----- !------------ ;------ 1------!------ 1 1'. ' ! i --- ! ------- !------ ------: 1 i I1 ! i :' I j! | I ; :!j ,1 i i . ! , I' ;i1 EPL V-l Experimental Pathology Laboratories, Inc. Key :X=Not Remarkable N=No Section I-Incomplete A'Autolysis l=minimal 2=slight/miId 3moderate 4-moderately severe 5-severe/high P=Present B=Ben1gn M=Malignant m=missing one paired organ u-unscheduled sac./death - 127 - Project Number 454-109 HISTOPATHOLOGY INCIDENCE TABLE GROUP CONTROL GROUP 454-109 Offspring Sacrifice Female Mallard A N 1 M A L ADIPOSE TISSUE I n f i l t r a t e . Mononuclear Cell 9999999 5555566 788 990 1 3 La. ? 8 3 0 X X1 X X X X X i 1 !: BRAIN X X x;x!x:x X 99 66 23 93 X :i i X' X 99 66 34 45 X X Xi X 99 66 45 84 XX X: X 9 6 5 9 X X ! ; BURSA OF FABRICIUS GALLBLADDER Autolvsis Precludes Diaanosis KIDNEY I n f i l t r a t e . Mononuclear Cell x ! x X x i x 1x x : ! X X A! A A A' N P p Pj P ! Xm Xm Xm Xm m m ii m i 1 x ! x X! X1X X^ X ; N X a!A A X A PPP P Xm Xm Xm Xm Xm Xm Xm i LIVER Deaenerat i on/Necrosi s . Hepatocellular. Acute I n f i l t r a t e . Mononuclear Cell Vacuoli7ation. Hepatocellular 1 .3 ? 1 11 i i 11 i X 11 11 1 11 -- OVARY Immature PPPP p p p PP P P P PP PROVENTRICULUS I n f i l t r a t e . Mononuclear Cell XX 11 1? i X 1 ? 11 1 1 \ i '1 ! ; 1' j i . i! i1 i i I; EPL V-2 ..... .. Experimental Pathology Laboratories, Inc. Key :X*Not Remarkable N=No Section -Incomplete A*Auto1ysis 5: " / h i g h Sll9ht/m11d " rate " rately severe P*Present B'Benign M-Malignant m*missing one paired organ u*unscheduled sac./death ,W ildlife International Ltd. - 128 - Project Number 454-109 Appendix XIIA The Analysis of PFOS in Adult Red Blood Cells and Sera - 129- Wildlife International, Ltd. Project Number 454-109 Exygen Study No.: 023-066 Table X. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 5 Sponsor ID 454-109-3249-Wk5 454-109-3249-Wk5* 454-109-3251-Wk5 454-109-3251-Wk5* 454-109-3252-Wk5 454-109-3252-Wk5 * 454-109-3253-Wk5 454-109-3253-Wk5* 454- 109-3254-Wk5 454-109-3254-Wk5* 454-109-3255-Wk5 454-109-3255-Wk5 * 454-109-3256-Wk5 454-109-3256-Wk5* Centre ID 0107230 0107230 0107231 0107231 0107232 0107232 0107233 0107233 0107234 0107234 0107235 0107235 0107236 0107236 Set Number 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B 052101B Extraction Analysis Date Date 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 5/21/01 5/21/01 5/21/01 5/22/01 5/22/01 5/22/01 5/21/01 5/22/01 5/21/01 5/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) NQ NQ 0.00197 0.00167 NQ NQ NQ NQ NQ NQ 0.00143 NQ NQ NQ 0.000401 0.000706 * Duplicate injection NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) LOQ = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) was used to calculate the average and standard deviation. Exygen Research Page 28 of 98 - 130 - W ildlife International, Ltd, Project Number 454-109 Exygen Study No.: 023-066 Table XI. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 5 Sponsor ID 454-109-3289-Wk5 454-109-3289-Wk5 * 454-109-3290-Wk5 454-109-3290-Wk5* 454-109-3291-Wk5 454-109-3291 -Wk5* 454-109-3292-Wk5 454-109-3292-Wk5* 454-109-3293-Wk5 454-109-3293-Wk5* 454-109-3294-Wk5 454-109-3294-Wk5* 454-109-3295-Wk5 454-109-3295-Wk5* 454-109-3296-Wk5 454-109-3296-Wk5* Centre ID 0107237 0107237 0107238 0107238 0107239 0107239 0107240 0107240 0107241 0107241 0107242 0107242 0107243 0107243 0107244 0107244 Set Number 052101BD 052101BD 052I01BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD 052101BD Extraction Analysis Date Date 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/21/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ig/mL) 24.4 24.7 11.2 10.0 11.8 12.3 15.1 13.1 30.1 30.8 4.88 3.95 18.9 18.8 26.4 28.5 17.8 8.80 * Duplicate injection LOQ = 0.01 (ig/mL for red blood cells and serum Exygen Research Page 29 of 98 - 131 - W ildlife International, Ltd. Project Number 454-109 Exygen Study No.: 023-066 Table XII. Summary of PFOS in Mallard Red Blood Cell Samples at 50 ppm a.i. for Week 5 Sponsor ID 454- 109-3329-Wk5 454-109-3329-Wk5* 454-109-3330-Wk5 454-109-3330-Wk5* Centre ID 0107245 0107245 0107246 0107246 Set Number 052201CD 052201CD 052201CD 052201CD Extraction Analysis Date Date 5/22/01 5/22/01 5/22/01 5/22/01 5/24/01 5/24/01 5/24/01 5/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 58.9 62.2 112 123 89 33 Table XIII. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 10 Sponsor ID 454-109-3249-Wk 10 454-109-3249-Wkl0* 454-109-3250-WklO 454-109-3250-WklO* 454-109-3251 -WklO 454-109-3251-Wkl0* 454-109-3252-Wkl0 454-109-3252-WklO* 454-109-3253-Wkl0 454-109-3253-Wkl0* 454-109-3254-WklO 4 5 4 -1 09-3254-W k 10* 454-109-3255-Wk 10 454-109-3255-WklO* 454-109-3256-WklO 454-109-3256-WklO* Centre ID 0107250 0107250 0107251 0107251 0107252 0107252 0107253 0107253 0107254 0107254 0107255 0107255 0107256 0107256 0107257 0107257 * Duplicate injection Set Number 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A 052201A Extraction Analysis Date Date 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ NQ 0.00245 0.000200 0.000600 NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) I .OQ = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) was used to calculate the average and standard deviation. Exygen Research Page 30 of 98 - 132 - W ildlife International, Ltd Project Number 454-109 Exygen Study No.: 023-066 Table XIV. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 10 Sponsor ID 454-109-3289-Wkl0 454-109-3289-WkI0* 454-109-3290-Wk 10 454-109-3290-WklO* 454-109-3291-Wk 10 454-109-3291 -Wk10* 454-109-3292-Wkl0 454-109-3292-Wkl0* 454-109-3293-Wkl0 454-109-3293-WklO* 454-109-3294-WklO 454-109-3294-WklO* 454-109-3295-WklO 454-109-3295-Wk 10* 454-109-3296-Wk 10 454-109-3296-WklO* Centre ID 0107258 0107258 0107259 0107259 0107260 0107260 0107261 0107261 0107262 0107262 0107263 0107263 0107264 0107264 0107265 0107265 * Duplicate injection Set Number 052201AD 052201AD 052201 AD 052201AD 052201AD 052201AD 052201AD 052201 AD 052201 AD 052201 AD 052201AD 052201AD 052201AD 052201 AD 052201 AD 052201 AD Extraction Analysis Date Date 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23-24/01 5/23-24/01 5/23-24/01 5/23-24/01 5/22/01 5/22/01 5/23-24/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ng/mL) 19.3 19.6 20.4 20.2 14.1 14.9 67.3 66.0 33.5 34.2 60.8 58.9 16.1 16.0 11.6 11.7 30.3 20.7 LOQ = 0.01 |ig/m L for red blood cells and serum Exygen Research Page 31 of 98 - 133 - W ildlife International, Ltd, Project Number 454-109 Exygen Study No.; 023-066 Table XV. Summary of PFOS in Mallard Red Blood Cell Samples at 0 ppm a.i. for Week 15 Sponsor ID 454-I09-3249-Wkl5 454-109-3249-Wk 15* 454-109-3250-Wkl 5 454-109-3250-Wkl5* 454-109-3251 -Wkl 5 454-109-3251-Wkl5* 454- 109-3252-Wk 15 454-109-3252-Wkl5* 454-109-3253-Wkl5 454-109-3253-Wkl5* 454-109-3254-WkI5 454- 109-3254-Wkl5* 454-109-3255-Wkl5 454-109-3255-Wkl5* 454-109-3256-Wkl5 454-109-3256-Wkl5* 454-109-3236-Wk 15 454-109-3236-WkI5* Centre ID 0107266 0107266 0107267 0107267 0107268 0107268 0107269 0107269 0107270 0107270 0107271 0107271 0107272 0107272 0107273 0107273 0107274 0107274 Set Number 052201B 05220IB 05220IB 052201B 05220IB 05220IB 052201B 052201B 05220IB 05220IB 052201B 05220IB 05220IB 05220 IB 052201B 05220 IB 052201B 052201B Extraction Date Analysis Date 5/22/01 5/23/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 5/23/01 AVERAGE: STANDARD DEVIATION; Analyte Found (jig/mL) NQ NQ NQ NQ 0.00179 0.00188 NQ NQ NQ NQ ND ND NQ NQ NQ NQ NQ NQ 0.000243 0.000613 * Duplicate injection ND = No peak detected NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration o f the calibration standards (0.0001 pg/mL)) LOQ = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value o f the lowest calibration standard, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 32 of 98 - 134- Wildlife International, Ltd Project Number 454-109 Exygen Study No.: 023-066 Table XVI. Summary of PFOS in Mallard Red Blood Cell Samples at 10 ppm a.i. for Week 15 Sponsor ID 454-109-3289-Wkl 5 454- 109-3289-Wkl5* 454-109-3290-Wkl5 454-109-3290-Wkl 5* 454-109-3291-Wkl5 454-109-3291-Wkl5* 454-109-3292-Wkl5 454-109-3292-Wkl5* 454-109-3293-Wk 15 454-109-3293-Wk 15* 454-109-3294-Wk 15 454-109-3294-Wkl5* 454-109-3295-Wkl5 454-109-3295-Wkl5* 454-109-3296-Wk 15 454-109-3296-Wkl 5* Centre ID 0107275 0107275 0107276 0107276 0107277 0107277 0107278 0107278 0107279 0107279 0107280 0107280 0107281 0107281 0107282 0107282 Set Number 052201BD 052201BD 052201BD 052201BD 052201BD 052201BD 052201BD 052201BD 052201BDR 052201BDR 052201BDR 052201BDR 052201BDR 052201BDR 052201BD 052201BD Extraction Analysis Date Date 5/22/01 5/22/01 5/23-24/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/22/01 5/23-24/01 5/23-24/01 5/22/01 5/23-24/01 5/22/01 5/24/01 5/22/01 5/22/01 5/22/01 5/22/01 5/22/01 5/24/01 5/24/01 5/24/01 5/24/01 5/24/01 5/22/01 5/23-24/01 5/22/01 5/23-24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/rnl.) 53.5 48.2 3.98 3.44 60.9 61.4 9.65 8.79 113 116 1.54 1.61 8.03 6.56 0.623 0.505 31.1 39.8 Table XVII. Summary of PFOS in Mallard Red Blood Cell Samples at 50 ppm a.i. for TERM Sponsor ID 454-109-3297-TERM 454-109-3297-TERM* 454-109-3298-TERM 454-109-3298-TERM* Centre ID 0107283 0107283 0107284 0107284 HDuplicate injection Set Number 052201 CD 052201CD 052201BDR 052201BDR Extraction Analysis Date Date 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 5/22/01 5/24/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 275 272 406 418 343 80 LOQ = 0.01 |4g/mL for red blood cells and serum Exygen Research Page 33 of 98 - 135 - W ildlife International, Ltd Project Number 454-109 Exygen Study No.: 023-066 Table XVIII. Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 5 Sponsor ID 454-109-3249-Wk5 454-109-3249-Wk5 * 454-109-3251-Wk5 454-109-3251 -Wk5* 454-109-3252-Wk5 454-109-3252-Wk5* 454-109-3253-Wk5 454-109-3253-Wk5* 454-109-3254-Wk5 454-109-3254-Wk5* 454-109-3255-Wk5 454- !09-3255-Wk5* 454-109-3256-Wk5 454-109-3256-Wk5* Centre ID 0107303 0107303 0107304 0107304 0107305 0107305 0107306 0107306 0107307 0107307 0107308 0107308 0107309 0107309 * Duplicate injection Set Number 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A 072001A Extraction Date Analysis Date 7/20/01 7/20/01 7/20/01 7/20/01 7/21/01 7/21/01 7/21/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 7/20/01 7/21/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ug/mL) 0.00414 0.00417 0.00566 0.00599 0.00878 0.00943 0.00516 0.00499 0.00432 0.00444 0.00462 0.00392 0.00544 0.00516 0.00544 0.00167 LOQ = 0.01 |ig/mL for red blood cells and scrum Exygen Research Page 34 of 98 - 136 - .W ildlife International, Ltd Project Number 454-109 Exygen Study No.: 023-066 Table XIX. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 5 Sponsor ID 454-109-3289-Wk5 454-109-3289-Wk5* 454-109-3290-Wk5 454-109-3290-Wk5* 454-109-3291-Wk5 454-109-3291 -Wk5* 454-109-3292-Wk5 454-109-3292-Wk5 * 454- 109-3293-Wk5 454-109-3293-Wk5* 454-109-3294-Wk5 454-109-3294-Wk5 * 454-109-3295-Wk5 454-109-3295-Wk5* Centre ID 0107310 0107310 0107311 0107311 0107312 0107312 0107313 0107313 0107314 0107314 0107315 0107315 0107316 0107316 Set Number 072001 AD 072001 AD 072001 AD 072001 AD 072001AD 072001AD 072001 AD 072001 AD 072001 AD 072001 AD 072001AD 072001 AD 072001 AD 072001 AD Extraction Analysis Date Date 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 7/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (MR/mL) 128 120 79.6 94.2 104 110 83.4 85.8 59.4 76.6 60.6 57.5 73.3 76.9 86.4 22.2 Table XX. Summary of PFOS in Mallard Serum Samples at 50 ppm a.i. for Week 5 Sponsor ID 454- 109-3329-Wk5 454-109-3329-Wk5* 454-109-3330-Wk5 454-109-3330-Wk5* Centre ID 0107317 0107317 0107318 0107318 Set Number 072001AD 072001 AD 072001 AD 072001 AD Extraction Analysis Date Date 7/20/01 7/22/01 7/20/01 7/20/01 7/20/01 7/22/01 7/22/01 7/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 376 400 320 290 347 50 * Duplicate injection LOQ = 0.01 |ug/mL for red blood cells and scrum Exygen Research Page 35 of 98 - 137- ,W ildlife International, Ltd Project Number 454-109 Exygen Study No.: 023-066 Table XXLSummary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 10 Sponsor ID 454-109-3249-Wk 10 454-109-3249-WklO* 454-109-3250-Wk 10 454-109-3250-Wk 10* 454-109-3251-Wkl0 454-109-3251-Wkl0* 454-109-3252-Wk 10 454-109-3252-WklO* 454-109-3253-Wkl0 454- 109-3253-Wk 10* 454-109-3254-WklO 454-109-3254-Wkl0* 454-109-3255-Wkl0 454-109-3255-Wkl0* 454-109-325 6-Wkl0 454-109-3256-WktO* Centre ID 0107322 0107322 0107323 0107323 0107324 0107324 0107325 0107325 0107326 0107326 0107327 0107327 0107328 0107328 0107329 0107329 * Duplicate injection Set Number 072001B 072001B 07200 IB 07200IB 07200IB 07200IB 07200 IB 07200IB 07200IB 07200IB 07200IB 07200IB 07200IB 072001B 07200IB 07200IB Extraction Analysis Date Date 7/20/01 7/20/01 7/20/01 7/21-22/01 7/21-22/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/20/01 7/21-22/01 7/21-22/01 7/21-22/01 7/21-22/01 7/21-22/01 7/21-22/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 7/20/01 7/21-22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (pg/mL) 0.00271 0.00372 0.00726 0.00666 0.00659 0.00530 0.00899 0.00940 0.00517 0.00482 ND ND 0.00337 0.00403 0.00419 0.00384 0.00476 0.00266 ND = No peak detected NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) LOQ = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 36 of 98 - 138 - ,W ildlife International, Ltd Project Number 454-109 Exygen Study No.: 023-066 Table XXII. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 10 Sponsor ID 454-109-3289-WklO 454-109-3289-WklO* 454-109-3290-Wk 10 454-109-3290-WklO* 454-109-3291-WklO 454-109-3291 -WklO* 454-109-3292-WklO 454- 109-3292-Wk 10* 454-109-3293-Wkl0 454-109-3293-WklO* 454-109-3294-Wkl0 454-109-3294-WklO* 454-109-3295-Wkl0 454-109-3295-Wkl0* 454-109-3296-Wkl0 454-109-3296-WklO* Centre ID 0107330 0107330 0107331 0107331 0107332 0107332 0107333 0107333 0107334 0107334 0107335 0107335 0107336 0107336 0107337 0107337 Set Number 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001 BD 072001BD 072001BD 072001BD 072001BD 072001BD 072001 BD Extraction Analysis Date Date 7/20/01 7/20/01 7/20/01 7/20/01 7/22/01 7/22/01 7/22/01 7/22/01 7/20/01 7/20/01 7/22/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/20/01 7/22/01 7/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found ((ig/mL) 145 147 32.7 32.5 93.5 102 107 111 132 136 85.3 86.4 123 120 114 112 105 33.8 * Duplicate injection LOQ = 0.01 ng/mL for red blood cells and scrum Exygen Research Page 37 of 98 - 139- .W ildlife International, Ltd Project Number 454-109 Exygen Study No.: 023-066 Table XXIII. Summary of PFOS in Mallard Serum Samples at 0 ppm a.i. for Week 15 Sponsor ID 454-109-3236-Wkl5 454-109-3236-Wkl5* 454-109-3249-Wk 15 454-109-3249-Wkl5* 454-109-3250-Wkl5 454-109-3250-WM5* 454-109-3251-Wk 15 454-109-3251 -Wkl 5* 454-109-3252-Wk 15 454-109-3252-Wkl5* 454-109-3253-Wkl5 454-109-3253-Wkl5* 454-109-3254-Wkl5 454- !09-3254-Wk 15* 454-109-3255-Wk 15 454-109-3255-Wkl 5* 454-109-3256-Wk !5 454-109-3256-Wki5* Centre ID 0107338 0107338 0107339 0107339 0107340 0107340 0107341 0107341 0107342 0107342 0107343 0107343 0107344 0107344 0107345 0107345 0107346 0107346 * Duplicate injection Set Number 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A 071901A Extraction Analysis Date Date 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 7/19-20/01 AVERAGE: STANDARD DEVIATION: Analyte Found (jifi/mL) ND ND 0.00729 0.0102 ND ND 0.00466 0.00441 ND ND 0.00649 0.00723 ND ND 0.0233 0.00699 0.0195 ND 0.000503 0.00616 ND = No peak detected NQ = Not Quantifiable (A peak was detected at the corresponding analyte retention time but was less than the lowest concentration of the calibration standards (0.0001 pg/mL)) LOQ = 0.01 pg/mL for red blood cells and serum For values recorded as NQ, 0.00005 pg/mL (half the value of the lowest calibration standard, 0.0001 pg/mL) and for ND, zero was used to calculate the average and standard deviation. Exygen Research Page 38 of 98 - 140 - .W ildlife International, Ltd Project Number 454-109 Exygen Study No.: 023-066 Table XXIV. Summary of PFOS in Mallard Serum Samples at 10 ppm a.i. for Week 15 Sponsor ID 454-109-3289-Wkl5 454-109-3289-Wkl5* 454-109-3290-Wkl5 454-109-3290-Wkl5* 454-109-3291-Wki5 454-109-3291 -Wk15* 454-109-3292-Wkl5 454-109-3292-Wk 15* 454-109-3293-Wkl5 454-109-3293-Wkl5* 454-109-3294-Wk 15 454-109-3294-Wkl5* 454-109-3295-Wk 15 454-109-3295-Wkl5* 454-109-3296-Wkl5 454-109-3296-Wkl5* Centre ID 0107347 0107347 0107348 0107348 0107349 0107349 0107350 0107350 0107351 0107351 0107352 0107352 0107353 0107353 0107354 0107354 Set Number 071901 AD 071901AD 071901AD 071901AD 071901 AD 071901 AD 071901AD 071901 AD 071901AD 071901 AD 071901AD 071901 AD 071901 AD 071901 AD 071901AD 071901AD Extraction Analysis Date Date 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/20-21/01 7/20-21/01 7/20-21/01 7/20-21/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/20-21/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/19/01 7/20-21/01 7/20-21/01 7/20-21/01 7/20-21/01 7/20-21/01 7/20-21/01 7/20-21/01 7/20-21/01 AVERAGE: STANDARD DEVIATION: Analyte Found (jig/mL) 152 162 10.1 8.75 152 142 12.1 11.6 128 132 6.73 4.52 117 116 9.52 11.3 73.5 67.3 Table XXV. Summary of PFOS in Mallard Serum Samples at 50 ppm a.i. for TERM Sponsor ID 454-109-3297-TERM 454-109-3297-TERM* 454-109-3298-TERM 454-109-3298-TERM* Centre ID 0107355 0107355 0107356 0107356 * Duplicate injection Set Number 072001 AD 072001 AD 072001 AD 072001 AD Extraction Date Analysis Date 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 7/20/01 7/22/01 AVERAGE: STANDARD DEVIATION: Analyte Found (ig/mL) 685 601 664 601 638 43 LOQ = 0.01 |Jg/mL for red blood cells and serum Exygen Research Page 39 of 98 - 141 - W ildlife International, Ltd. Project Number 454-109 Appendix XIIB The Analysis of PFOS in Adult and Juvenile Liver and Sera - 142- W ildlife International, Ltd, Project Number 454-109 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS; A Reproduction Study with the Mallard S ummary T h e concentration of the C 8F 17S 0 3' anion of perfluorooctane sulfonate, potassium salt (P FO S , C8F 17S 0 3'K ') was determined in the liver and sera of adult mallards and their offspring in both a control group and a 10ppm dosed group from Wildlife feeding study # 4 5 4 -1 0 9 . The following tables sum m arize the C 8F t7S 0 3` concentrations found in the liver and sera of study samples, Extracted-m atrix P F O S standards are used to establish the H P L C -E S /M S /M S instrument calibration curve for C 8F 17S 0 3\ Concentrations of CsF^SOa' are reported after correction for the purity and the potassium contribution to the m ass of the P FO S reference substance. Table 1 sum m arizes all study sam ple data. Table 2 summarizes study data after removing certain results that have been statistically determined to be outliers utilizing the Dixon Q -Test. Table 1. Summary Table, CaFirSO]' Concentration In Sera and Liver Sampling Event Male Control Group Number of S era Samples A verage C jF u S O ;' Conc. Sera Rango of Results Number of L iver Samples 18 ! NA <100 ng/mL (14 of 18) 107-1400 ng/mL range (4 of 18) 20 A verage CFrSO i" Conc. L iver <50 ng/g Range of Results NA Female Control Group Male lOppm Group Female 10ppm Group 20 j, NA <100 ng/mL (15 of 20) 166-2680 ngfrnL range (5 of 20) i 20 89700 ng/mL 11300-210000 ng/mL 20 20300 ng/mL 6350-91400 ng/mL 20 20 20 <50ngrg NA 60900 ng/g 24200-125000 ng/g 10800 ng/g 2480-30700 ng/g Male Offspring <100 ng/mL (2 of 3) Control Group 3 NA 103 ng/mL (1 of 3) 3 <50 ng/g NA Female Offspring Control Group 7 NA <100 ng/mL (7 of 7) 7 <50 ng/g NA Male Offspring 10ppm Group 3 4410 ng/mL 3670 - 5450 ng/mL 3 3170 ng/g 22004640 ng/g Female Offspring 10ppm Group 7 J 4960 ng/mL 3420-6760 ng*nL For individual sample values see Tables 10 to 13 and Attachment B. 7 ' 3620 ng/g 2240-5930 ng/g I 3M Environmental Laboratory D iiu 7 D4C Page 7 - 143 - .W ildlife International, Ltd Project Number 454-109 Report E01-1256 Report 01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 2. Summary Table, CBF17S03~Concentration in Sera and Liver with Outliers Removed Sampling frvtNi NUMBER orStKA Samples Avt-KAGk C hF,,-S03' Conc. Sera Range of Results N umber of Liver Samples A verage CaFtrSOj" C onc. Liver Range of Results Male Control Group Female Control Group 18* 17 20* NA <100ngAnL(15oM7) 166-172 ngAnL range (2 Of 17) 20* Male iOppm Group 18 87300 ng/mL 68700-108000 ng/mL 20' Female IOppm Group 19 16600 ng/mL 6350-42800 ng/mL 20* Male Offspring Control Group Female Offspring Control Group 3* 7* * *; * 3* 7* Male Offspring IOppm Group 3* * * 3* ! Female Offspring iOppm Group 7* i _________ I * T i, * No outliers removed from this data set. Refer to Table 1 for results. For individual sample values see Tables 10 to 13 and Attachment B. Purpose T h e purpose of the analytical phase of this study is to quantify C sF ^ S C V in the liver and sera of both control and dosed mallards and in their offspring. Reference Substances Table 3. Reference Substances Reference Substance PFOS PFDA Source Expiration Date Storage Conditions 3M Laboratory Identification Number Physical Description Purity 3M Internal 08/31/2006 Frozen -20"C +/-10C SD-018,Lot#217 Whte Crystalline Powder 8 6 .9 % Oakwood Products, Inc. 12/0 1 /2 0 1 0 Frozen -20C +/- 10`C SD-036 White Solid 98% PFDA is nonadecafluorodecanoic acid, or Perfluorodecanoic acid, C10F19O2H and is used solely to monitor for gross instrument failure and not as an internal standard for quantitation. 3MEnvironmental Laboratory Page 8 - 144- ,W ildlife International, Ltd Project Number 454-109 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Absolute Recoveries. T h e absolute recovery of the Q C samples is determined by comparing the results of the extracted quality control spikes quantitated versus an extracted matrix curve and a solvent curve. The sera spikes quantitate higher using a solvent curve than when using a matrix-extracted curve. Th e low spikes are 11.1% higher, the medium spikes 8 .3 % higher, and the high spikes 8.3% higher. T h e 9.5 % average higher quantitation of the three levels of spikes using a solvent curve versus an extracted matrix curve demonstrates that it is appropriate to use matrix-extracted standards. Th e sam e trend Is observed for liver samples. The extracted liver matrix curves are 12.2% higher. 7.8 % higher, and 5.6% higher respectively for the low, medium and high spikes of the liver matrix, again demonstrating that it is appropriate to use matrix-extracted standards. Internal Standard: The internal standard (P FD A ) w as added to all samples and standards. P F D A was not used for quantitation, but was used to monitor for gross instrument failure. Instrument Response Calibration. H P LC -E S /M S /M S instrument response w as calibrated using the concentration of the CaFt7S 0 3 anion of perfluorooctane sulfonate, potassium salt (P FO S ) that was adjusted for purity and the potassium ion contribution to the mass of P FO S used to prepare stock solutions. Any future calculations requiring concentrations to be expressed as a percentage of dietary in-take must adjust the dosed amounts as well. D ata S ummary Tables 10 through 13 summarize individual sample data. Representative chromatograms are presented in Attachment C. The table displays C sF i.S O j' concentrations for individual injections. All data a re corrected for purity of the PFO S reference substance used for the extracted-m atrix calibration curve. Table 10. CgF^SOj' Data Summary, Female Mallard Sera (Adult and Offspring) 3 M S lJI'i F N i IM -HR SAMPLE: DESCRIPTION E01-1256-28663 EOI-1256-28665 E01-1256-28667 E01-1256-28669 E01-1256-28670 E01-1256-28672 E01-1256-28674 E01-1256-28676 E01-1256-28678 E01-1256-28679 E01-1256-28681 E01-1256-28683 E01-1256-28685 E01-1256-2B687 E01-1256-2B689 E01-1256-28691 E01-1256-28693 E01-1256-28695 454-109-3218,0 PPM, Female, Adult 454-109-3220,0 PPM, Female, Adult 454-109-3222,0 PPM, Female, Adult 454-109-3224,0 PPM, Female, Adult 454-109-3226,0 PPM, Female, Adult 454-109-3228.0 PPM. Female. Adult 454-109-3230,0 PPM, Female, Adult 454-109-3232,0 PPM, Female, Adult 454-109-3234,0 PPM, Female, Adult 454-109-3236,0 PPM, Female, Adult 454-109-3238,0 PPM, Female, Adult 454-109-3240,0 PPM, Female, Adult 454-109-3242,0 PPM, Female, Adult 454-109-3244,0 PPM, Female, Adult 454-109-3246,0 PPM, Female, Adult 454-109-3248,0 PPM, Female. Adult 454-109-3250,0 PPM, Female, Adult 454-109-3252,0 PPM, Female, Adult At- 'OUNT OF ADDITIONAL SETA USED DILUTION <VL) 0.5 0.5 i 0.5 0.5 i 0.5 1 1 1 1 1 Cono, of CyFuSOa IN S a m p l e (ng /m L of Sera) <100 <100 <100 <100 <100 0.5 1 2 6 8 0 * 0.5 1 0.5 1 0.5 1 1 0.5 ! 0.5 1 1 0.5 1 0.5 1 , 0.5 1 0.5 1 <100 172 <100 <100 166 <100 <100 <100 <100 0.5 1 398 0.5 1 <100 0.5 I 1 757* 3M Environmental Laboratory 3M Environm ental Laboratory Paca 14 o f 216 Page 14 - 145- W ildlife International, Ltd Project Number 454-109 Report E01-1256 Report E01 -1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M S ampll N'.Jr.M-H S A U R I F D E S C R IP TIO N E01-1256-28697 E01-1256-28699 E01-1256-28701 E01-1256-28703 E01-1256-28705 E01-1256-2S707 E01-1256-28709 E01-1256-28711 E01-1256-28713 E01-1256-28715 E01-1256-28717 E01-1256-28719 E01-1256-28721 E01-1256-28723 E01-1256-28725 E01-1256-28727 E01-125628729 E01-1256-28731 E01-1256-28733 E0t-t256-28735 E01-125628737 E01-1256-28739 E01-1256-28740 E01-125628742 01-4256-28743 EO1-1256-20744 E01-125628745 E01-125628747 E01-125628748 E01-1256-28751 E01-1256-28752 E01-125628753 E01-1256-28754 E01-1256-28755 E01-125628758 Ed-125628759 454-109-3254,0 PPM, Female, Adult 454-1063256,0 PPM, Female, Adult 454-109-3258,10 PPM, Female, Adult 454-109-3260,10 PPM, Ferrate, Adult 454-109-3262,10 PPM, Female, Adult 454-109-3264,10 PPM, Female, Adult 454-109-3266.10 PPM, Female, Adult 454-1063268,10 PPM, Female, Adult 454-109-3270,10 PPM, Female, Adult 454-1063272,10 PPM, Female, Adult 454-1063274,10 PPM, Female, Adult 454-1063276,10 PPM, Female, Adult 454-1063278,10 PPM, Female, Adult 454-1063280,10 PPM, Female, Adult 454-1063282,10 PPM, Female, Adult 454-1063284,10 PPM, Female, Adult 454-1063286,10 PPM, Female, Adult 454-1063268,10 PPM, Female, Adult 454-1063290,10 PPM, Female, Adult 454-1063292,10 PPM, Female, Adult 454-109-3294,10 PPM, Female, Adult 454-1063296,10 PPM, Female, Adult 454-109-9583,0 PPM, Female, Offspring 454-1069589,0 PPM, Female, Offspring 454-109-9592,0 PPM, Female, Offspring 454-109-9598,0 PPM, Female, Offspring 454-109-9603,0 PPM, Female, Offspring 454-109-9610,0 PPM, Female, Offspring 454-1069573,0 PPM, Female, Offspring 454-1069629,10 PPM, Female, Offspring 454-109-9633, 10 PPM, Female, Offspring 454-109-9634,10 PPM, Female, Offspring 454-109-9645,10 PPM, Female, Offspring 454-1069648,10 PPM, Female, Offspring 454-109-9654,10 PPM, Female, Offspring 454-109-9659,10 PPM, Female, Offspring AVO' INT OF SFR/v =0 (vL) AtMUTiONAL OIUT.ON C o t e . OF C,,Fl7SO;'l\ S a m p le (n g /v L of Sera) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 j 0.5 0.5 I 0.5 0.5 0.5 0.2 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.15 0.2 0.2 j 0.1 0.2 0.2 0.05 0.1 ! 0.1 0.5 0.1 0.2 0.1 0.1 i i ! 1 1 1 1 1 1 1 1 10 1 10 10 j 1 10 T 1 10 10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1' 1 1 1 <100 <100 13600 9420 17000 6350 7900 6730 91400* 7710 29400 40600 9840 32200 10800 6570 28900 42800 16000 10400 7660 10800 <100 <100 <100 <100 <100 <100 <100 5130 3420 6760 3750 4900 5450 5350 These values w ere rejected based on the results of the Dixon's Q-Test, 9 0% confidence interval, for the rejection of outliers 3M Environmental Laboratory 3M Environmental Laboratory Paae 15 of 216 Page 15 - 146- ,W ildlife International, Ltd Project Number 454-109 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 11. C8F17SO3' Data Summary, Male Mallard Sera (Adult and Offspring) 3M SA'.in? Nii'Vnn; SAMPLE DESCRIPHOri E01-1256-28662 E01-1256-28664 E01-1256-28666 E01-1256-28668 E01-1256-28671 EQ1-1256-28673 E01-1256-28675 E01-1256-28677 E01-1256-26680 E01-1256-28682 E01-1256-28684 E01-1255-28686 E01-1256-28688 E01-1256-28690 E01-1256-28692 E01-1256-28694 E01-1256-28696 E01-1256-28698 E01-1256-28700 E01-1256-28702 E01-1256-28704 E01-1256-28706 E01-1256-28708 E01-1256-28710 E01-1256-28712 E01-1256-28714 E01-1256-28716 E01-1256-28718 E01-1256-2B720 E01-1256-28722 E01-1256-28724 E01-1256-28726 E01-1256-28728 E01-1256-28730 E01-1256-28732 E01-1256-28734 E01-1256-28736 E01-1256-28738 E01-1256-2B741 454-109-3217,0 PPM, Male, Adult 454-109-3219,0 PPM, Male, Adult 454-109-3221,0 PPM, Male, Adult 454-109-3223,0 PPM, Male, Adult 454-109-3227,0 PPM, Male, Adult 454-109-3229,0 PPM, Male, Adult 454-109-3231,0 PPM, Male, Adult 454-1093233,0 PPM, Mala, Adult 454-109-3237,0 PPM, Male, Adult 454-109-3239,0 PPM, Male, Adult 454-109-3241,0 PPM, Male, Adult 454-109-3243,0 PPM, Male, Adult 454-109-3245,0 PPM. Male, Adult 454-109-3247,0 PPM, Male, Adult 454-109-3249,0 PPM, Male, Adult 454-109-3251,0 PPM, Male, Adult 454-109-3253,0 PPM, Male, Adiit 454-109-3255,0 PPM, Male, Adult 454-109-3257,10 PPM, Male, Adult 454-1093259,10 PPM, Male, Adult 454-109-3261,10 PPM, Male, Adult 454-1093263,10 PPM, Male, Adult 454-109-3265,10 PPM, Male, Adult 454-109-3267, 10 PPM, Male, Adult 454-109-3269,10 PPM, Male, Adult 454-109-3271,10 PPM, Male, Adult 454-109-3273,10 PPM, Male, Adult 454-109-3275,10 PPM, Male, Adult 454-109-3277,10 PPM, Male, Adult 454-109-3279,10 PPM, Male, Adult !454-109-3281,10 PPM, Male, Adult 454-109-3283,10 PPM, Male, Adult 454-109-3285,10 PPM, Male, Adult 454-109-3287,10 PPM, Male, Adult 454-1093289,10 PPM, Male, Adult 454-109-3291,10 PPM, Male, Adult 454-1093293,10 PPM, Male, Adult 454-109-3295,10 PPM, Male, Adult 454-109-9591,0 PPM, Male, Offspring AMOUNTO5EPA USED (ML) tO U i k DILUTION C ong o C aF , , S O j` lN S am ple (n o /m L OF SFRA) 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.3 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.5 1 0.1 1 0.5 1 107 <100 <100 <100 <100 <100 <100 <100 <100 <100 1400 <100 <100 162 1070 <100 <100 <100 0.5 10 78800 02 10 83300 0.5 10 68700 0.06 02 0.5 , 100 10 10 210000. 93800 75700 0.5 10 80600 0.5 10 11300. 0.5 10 106000 0.5 10 80500 0.5 10 80400 0.5 10 103000 0.5 10 I 74300 0.5 10 91800 0.5 10 , 1QB0 0 0 0.3 10 78700 0.3 10 97900 0.3 10 73500 0.5 ; 10 96800 0.5 10 99700 0.1 1 <100 3M Environmental laboratory SM Environmental Laboratory Page 16 of 216 Page 16 - 147- ,W ildlife International, Ltd Project Number 454-109 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3M S avple fJUl.'UER S A M P JE D E S C R IP T IO N E01-1256-28746 E01-1256-28749 E01-1256-28750 E01-1256-28756 E01-1256-28757 454-109-9606,0 PPM, Male, Offspring 454-109-9578,0 PPM, Male, Offspring 454-109-9625,10 PPM, Mate. Offspring 454-109-9636,10 PPM, Male. Offspring 454-109-9642,10 PPM, Male, Offspring AMOUNTO " i)C -A USl-D (M L) ADD If. NAL dilution Como of CaFuSOj'lN S a m p le (n g /m L of S era) 0.2 ; 1 103 0.2 1 <100 0.2 1 0.1 1 0.2 1 5450 3670 4110 These values w ere rejected based on the results of the Dixon's Q-Test, 90% confidence interval, for the rejection of outliers. 3M Environmental Laboratory 3M Environmental Laboratory Paqe 17 of 216 Page 17 - 148 - W ildlife International, Ltd. Project Number 454-109 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 12. C8F17S03' Data Summary, Female Mallard Liver (Adult and Offspring) 3M Sam 'u N.MTf < SAM PLE DESC RIPTIO N E01-1326-29482 E01-1326-29583 E01-1326-29601 E01-1326-29619 E01-1326-29637 E01-1326-29655 E01-1326-29673 E01-1326-29691 E01-1326-29709 E01-1326-29727 E01-1326-29745 E01-1326-29763 E01-1326-29781 E01-1326-29799 E01-1326-29817 E01-1326-29835 E01-1326-29853 E01-1326-29871 E01-1326-29889 E01-1326-29907 E01-1326-29925 E01-1326-29943 E01-1326-29961 E01-1326-29980 E01-1326-29998 01-1326-30016 E01-1326-30034 E01-1326-30052 E01-1326-30070 E01-1326-30088 E01-1326-30106 E01-1326-30124 E01-1326-30142 E01-1328-30160 E01-1326-30178 E01-1326-30196 E01-1326-30214 E01-132G-30232 E01-132630250 0 pom a.i. 3218 F Liver, adult 0 ppm a.i. 3220 F Liver, adult 0 ppm a.i. 3222 F Liver, adult 0 ppm a.i. 3224 F Liver, adult 0 ppm a.i. 3226 F Liver, adult 0 ppm a.i. 3228 F Liver, adult 0 ppm a.i. 3230 F Liver, adult 0 ppm a.i. 3232 F Liver, adult 0 ppm a.i. 3234 F Liver, adult 0 ppm a.i. 3236 F Liver, adult 0 ppm a.i. 3238 F Liver, adult 0 ppm a.i. 3240 F Liver, adult 0 ppm a.i. 3242 F Liver, adult 0 ppm a.i. 3244 F Liver, adult 0 ppm a.i. 3246 F Liver, adult 0 ppm a.i. 3248 F Liver, adult 0 ppm a.i. 3250 F Liver, adult 0 ppm a.i. 3252 F Liver, adult 0 ppm a.i. 3254 F Liver, adult 0 ppm a.i. 3258 F Liver, adult 10 ppm a.i. 3258 F Liver, adult 10 ppm a.i. 3260 F Liver, adult 10 ppm a.i. 3262 F Liver, adult 10 ppm a.i. 3264 F Liver, adult 10 pom a.i. 3266 F Liver, adult 10 ppm a.i. 3268 F Liver, adult 10 ppm a.i. 3270 F Liver, adult 10 ppm a.i. 3272 F Liver, adult 10 ppm a.i. 3274 F Liver, adult 10 ppm a.i. 3276 F Liver, adult 10 ppm a.i. 3278 F Liver, adult 10 ppm a.i. 3280 F Liver, adult 10 ppm a.i. 3282 F Liver, adult 10 ppm a.i. 3284 F Liver, adult 10 ppm a.i. 3286 F Liver, adult 10 ppm a.i. 3288 F Liver, adult 10 ppm a.i. 3290 F Liver, adult 10 ppm a.i. 3292 F Liver, adult 10 ppm a.i. 3294 F Liver, adult I - r o j n t : >r i_iv:n I F\DD:r,i'iNAI j W r IG H E L ) ((.',) 1 D U JII'JN CONG. OF ChF i7S O j IN Sa m p le 1.0095 1.0944 1.0435 1.0425 1.0703 1.0526 1.0411 1.1704 1.1290 1.0800 1.1170 1.0086 1.0907 1.0291 1.1294 1.1075 1.1709 1.0761 1.1344 1.0669 i 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 | (ng/g of Liver ) <50 <50 . <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 1.0034 1 7080 1.1331 1 6780 1.1294 10 13600 1.1829 1 6080 1.0487 1.1214 1 1 5380 5730 1.0751 10 13200 1.0658 1 5800 1.1226 10 13800 | 1.0953 10 30700 : 1.1956 1 5960 ' 1.0600 1 2480 i 1.0275 1 4560 i 1.0915 1 6330 1.0657 10 28200 1.0330 10 24100 1.0352 10 19800 1.0658 4600 1.0627 1 4760 3M Environmental Laboratory 3M Environmental Laboratory Pacte 18 of 216 Page 18 - 149 - ,W ildlife International, Ltd Project Number 454-109 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard 3-VI Sal **i e: Nur.'iiE*' SA kPLF DF.SCRPTIOM E01-1326-30266 10 ppm a.i. 3296 F Liver, adult E01-1367-31295 E01-1367-31307 E01-1367-31313 E01-1367-31319 E01-1367-31325 E01-1367-31337 E01-1367-31343 ,0 ppm a.i. 9583 F Liver, otfsprina 0 ppm a.i. 9589 F Liver, offspring 0 ppm a.i. 9592 F Liver, offspring 0 ppm a.i. 9598 F Liver, offspring 0 ppm a.i. 9603 F Liver, offspring 0 ppm a.l. 9610 F Liver, offspring 0 ppm a.i. 9573 F Liver, offspring E01-1367-31362 E01-1367-31368 E01-1367-31374 E01-1367-31380 E01-1367-31386 E01-1367-31404 E01-1367-31410 10 ppm a.i. 9629 F Liver, offspring 10 ppm a.i. 9633 F Liver, offspring 10 ppm a.i. 9634 F Uver, offspring 10 ppm a.i. 9645 F Liver, offspring 10 ppm a.i. 9648 F Liver, offspring 10 ppm a.i. 9654 F Uver, offspring 10 ppm aJ. 9659 F Liver, offspring I AMCIINI OF L Vl K ! AEiCK.D |C) J A D D iT iO .\A _ D iL U ' O N COM C. OF CeF tSCL'iN S a m p le (ng/g of Liver I 1.1238 1 1 6330 1.1308 1.0005 1.0390 1.1204 1.1550 1.0217 1.0122 1 1 1 1 1 1 1 <50 <50 <50 <50 <50 <50 <50 1.1676 1 2950 1.0510 1 2240 1.1638 1 4520 1.1510 1 2560 1.1125 1 3500 1.0770 1 5930 1.0581 1 3580 3M Environmental Laboratory 3MEnvironmental Laboratory Pane 19 of 216 Page 19 - 150 - W ildlife International, Ltd, Project Number 454-109 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 13. C8F17SO3' Data Summary, Male Mallard Liver (Adult and Offspring) 1 3M Sami ae I Number E01-1326-29473 E01-1326-29574 01-1326-29592 E01-1326-29610 E01-1326-29628 E01-1326-29646 E01-1326-29664 E01-1326-29682 E01-1326-29700 E01-1326-29718 E01-1326-29736 E 0 1-1326-29754 E01-1326-29772 E01-1326-29790 E01-1326-29808 E01-1326-29826 E01-1326-29844 E01-1326-29662 E01-1326-29880 E01-1326-29898 S A M P LE D E S C R IP T IO N I 0 ppm a.i. 3217 M Liver, adult 0 ppm a.i. 3219 M Liver, adult 0 ppm a.i. 3221 M Liver, adult 0 ppm a.i. 3223 M Liver, adult 0 ppm a.i. 3225 M Liver, adult 0 ppm a.i. 3227 M Liver, adult 0 ppm a.i. 3229 M Liver, adult 0 ppm a.i. 3231 M Liver, adult 0 ppm a.i. 3233 M Liver, adult 0 ppm a.i. 3235 M Liver, adult 0 ppm a.i. 3237 M Liver, adult 0 ppm a.i. 3239 M Liver, adult 0 ppm a.i. 3241 M Liver, adult 0 ppm a.i. 3243 M Liver, aduft 0 ppm a.i. 3245 M Liver, adult 0 ppm a.i. 3247 M Liver, adult 0 ppm a.i. 3249 M Liver, adult 0 ppm a.i. 3251 M Liver, adult 0 ppm a.i. 3253 M Liver, adult 0 ppm a.i. 3255 M Liver, adult E01-1326-29916 E01-1326-29934 E01-1326-29952 E01-1326-29970 E01-1326-29989 E01-1326-30007 E01-1326-30025 E01-1326-30043 E01-1326-30061 E01-1326-30079 E01-1326-30097 E01-1326-30115 E01-1326-30133 E01-1326-30151 E01-1326-30169 E01-1326-30187 E01-1326-30205 E01-1326-30223 E01-1326-30241 E01-1326-30259 10 ppm a.i. 3257 M Liver, adult 10 ppm a.i. 3259 M Liver, adult 10 ppm a.i. 3261 M Liver, adult 10 ppm a.i. 3263 M Liver, adult 10 ppm a.i. 3265 M Liver, aduK 10 ppm a.i. 3267 M Liver, adult 10 ppm a.i. 3269 M Liver, adult 10 ppm a.i. 3271 M Liver, adult 10 ppm a.i. 3273 M Liver, adult 10 ppm a.i. 3275 M Liver, adult 10 ppm a.i. 3277 M Liver, adult 10 ppm a.i. 3279 M Liver, adult 10 ppm a.i. 3281 M Liver, adult 10 ppm a.i. 3283 M Liver, adult 10 ppm a.i. 3285 M Liver, adult 10 ppm a.i. 3287 M Liver, adult 10 ppm a.i. 3289 M Liver, adult 10 ppm a.i. 3291 M Liver, adult 10 ppm a.i. 3293 M Liver, adult 10 ppm a.i. 3295 M Liver, adult A '.'OUMT c r I A nn in oN A i LIVLK WEIGHFD I DILUTION ! () CoNJC OB C,F|7SO;'lN I S ample I (NG/G O f L VER) 1.1169 1.1847 1.1181 1.0788 1.1043 1.0486 1.0336 1.0165 1.0395 1.0373 1.1679 1.1493 1.0619 1.0263 1.1541 1.0813 1.0263 1.0088 1.0294 1.0926 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 <50 1.0654 1.1316 1.1330 1.1404 1.1480 1.0620 1.1687 1.0313 1.1399 1.1143 1.0062 1.0380 1.0669 1.0955 1.1981 : 1.0500 1.0251 1.0441 1.0979 1.0815 10 10 10 100 10 10 10 10 10 10 10 10 10 10 10 ; 10 j 10 10 I 10 10 . 72100 49200 70100 125000 61700 49900 57900 63000 85000 54900 53900 69000 49900 67100 55500 52600 59300 46500 24200 50400 3M Environmental Laboratory 3M Environmental Laboratory Pace 20 of 216 Page 20 - 151 - ,W ildlife International, Ltd Project Number 454-109 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with die Mallard 3 M S a i ---' p N j m e iC - SAMPLE U E S L H IP I ION -:o\ 'jpo~ E01-1367-31301 E01-1367-31331 E01-1367-31349 E01-1367-31356 E01-1367-31392 E01-1367-31398 0 ppm a.i. 9591 M Liver, offsprinq 0 ppm a.i. 9606 M Liver, offsprinq 0 ppm a.i. 9578 M Liver, offsprinq 10 ppm a.i. 9625 M Liver, offsprina 10 ppm a.i. 9636 M Liver, offspring 10 ppm a.i. 9642 M Liver, offsprinq 1.0411 1.1314 1.0413 1.0166 1.1367 1.1252 C o-.c Oi C F- SO. iti S r.- ING/G OF LVER) 1 <50 1 <50 1 <50 1 4640 1 2200 1 2680 Statem ent o f C onclusion S era results from the m ale control group portion of this study showed no detectable levels of C 8F ,7S 0 3` in 14 of 18 mallards (<100ng/m L) and quantifiable concentrations of C 8F ,7S 0 3' in 4 of 18 (range 107-1400ng/m L, Table 14). T h e sera results from the fem ale control group portion of this study showed no detectable levels of C 8F17S 0 3' In 15 of 17 birds (<100ng/m L) and quantifiable concentrations of C 8F 17S 0 3' in 2 of 17 (range 166-172ng/m L, Table 15). Analytical results for eighteen sera sam ples from the 10ppm dosed male group showed an average of 87300ng/m L C 8F 17S 0 3' (range 6 8 7 0 0 -1 08000ng/m L, Table 15). Analytical results for nineteen sera sam ples from the 10ppm dosed female group showed an average of 16600ng/m L C 8F 17S 0 3 (range 6350-42800ng/m L, Table 15). Sera results from the m ale offspring control group showed no detectable levels of C 8F ,7S 0 3' In 2 of 3 birds (<100ng/m L) and a quantifiable concentration in 1 of 3 (1 03 ng/m L, Table 14). The sera results from the fem ale offspring control group portion of this study showed no detectable levels of C 8F ,7S 0 3' in 7 of 7 birds (<100ng/m L, Table 14). Analytical results for three sera sam ples from the 10ppm dosed m ale offspring group showed an average of 4410ng /m L (range 3670-5450ng/m L, Table 14). Analytical results for seven sera sam ples Irom the 10ppm dosed fem ale offspring group showed an average of 4960ng/m L (range 3420-6760ng/m L, Table 14). Liver results from the m ale control group showed no detectable levels of C 8F 17S 0 3' in 20 of 2 0 birds (<50ng/g, Table 14). Th e liver results from the female control group also showed no detectable levels of C 8F ,7S 0 3 in 2 0 of 20 birds (<50ng/g. Table 14). Analytical results for twenty liver sam ples from the 10ppm dosed m ale group showed an average of 60900ng/g C8F ,7S 0 3` (range 24200-125000ng/g, Table 14). Analytical results for twenty liver samples from the 10ppm dosed fem ale group showed an average of 10800ng/g C8F 17S 0 3 (range 2480-30700ng/g, Table 14). Liver results from the m ale offspring control group portion of this study showed no detectable levels of C 8F |7S 0 3' in 3 of 3 birds (<50ng/g. Table 14). Th e liver results from the fem ale offspring control group portion of this study showed no detectable levels of C8F 17S 0 3' in 7 of 7 birds (<50ng/g, Table 14). Analytical results for three liver sam ples from the 10ppm dosed m ale offspring group showed an average of 3170ng/g (range 2200-4640ng/g, Table 14). Analytical results for seven liver samples from the 10ppm dosed fem ale offspring group showed an average of 3620ng/g (range 2240-5930ng/g, Table 14). 3M Environmental Laboratory 3M Environmental Laboratory Page 21 01216 Page 21 - 152 - ,W ildlife International, Ltd Project Number 454-109 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 14. Summary Table, CaF17S 0 3' Concentration in Sera and Liver SAMPLING E ven t Male Control Group Nu'vIHFROrS era S a VRLLS Average C*Fr,SO r Ccnc. Sera Range of Results Number of Liver S ample s A verage CaFi7S03 Co n c . Liver <100 ng/mL (14 of 18) 18 NA 20 <50 ng/g 107-1400 ng/mL range (4 of 18) Rango of Results NA Female Control Group 20 <100 ng/mL (15 of 20) NA 20 166-2680 ng/mL range (5 of 20) <50ng/g NA Male 10ppm Group ; 20 ! 89700 ng/mL Female 10ppm Group 20 20300 ng/mL 11300-210000 ng/mL 6350-91400 ng/mL 20 60900 ng/g 24200-125000 ng/g 20 10800 ng/g . 2480-30700 ng/g | Male Offspring Control Group Female Offspring Control Group i Male Offspring 10ppm Group Female Offspring 10ppm Group 3 7 3 7 ' NA ! NA 4410 ng/mL 4960 ng/mL <100 ng/mL (2 of 3) 103ngAnL (1 of 3) <100 ng/mL (7 of 7) 3670 - 5450 ng/mL 3420 - 6760 ng/mL , 3 <50 ng/g 7 <50 ng/g I 3 3170 ng/g 7 3620 ng/g NA NA 2200-4640 ng/g 2240-5930 ng/g For Individual samples values see Tables 10 to 13 and Attachment B. 3MEnvironmental Laboratory Page 22 - 153 - W ildlife International, Ltd. Project Number 454-109 Report E01-1256 Report E01-1256 PFOS: A Reproduction Study with the Mallard PFOS: A Reproduction Study with the Mallard Table 15. Summary Table, C8F17S 0 3' Concentration in Sera and Liver with Outliers Removed Sampling Event N um ber of S era S a m p le s Average C[F,,SO ,' Conc. Sera Mate Control Group 18* Rango of Results N um ber of L iv e r S a m p le s 20* A verag e C a F .rS O / CONC. Liver Range of Results Female Control Group 17 <100ngfmL(15of 17) NA 166-172 ng/mL range (2 of 17) Male 10ppm Group 18 87300 ng/mL 68700-108000 ng*nL Female 10ppm Group 19 16600 ng/mL 635042800 ng/mL 20* 20* 20* ..... I Male Offspring ! Control Group ! 3* ! 3* Female Offspring Control Group V 7" Male Offspring 1Qppm Group 3* 3* ; Female Offspring 10ppm Group 7* ! r * No outliers removed from this data set. Refer to Table 14 for results. For individual samples values see Tables 10 to 13 and Attachment B. S tatistical M eth o ds Statistical methods included calculating means, standard deviations, % coefficient of variation, linear regressions, percent recoveries, and the Dixons Q -T e st T h e Dixons Q -T e st, used for the rejection of outliers, has values ordered from lowest to highest. A Q-value is determ ined from the difference of a result and the next closest result th a t is divided by the d iffe re n c e of the h ig h e s t and lo w e s t value in the gro u p o f values. A critical Q -value is determined b a s e d on the number of sam ples in the group and the desired confidence interval. If the Q -value is greater than the critical Q -value then the value will be rejected. S tatem ent o f Data Q uality The only m easurem ent is the quality control and matrix spike results. Accuracy (the observed amount in the Q C and M S samples, divided by the amount spiked, expressed as a percent) was found to range from 79.7% to 172%; while precision, expressed as the standard deviation of the recoveries, was found to range from 0.717% to 15,7% . 3M Environmental Laboratory Page 23 Reproductive Performance by Pen Appendix XIII - T able 1 E g g s Laid / M a x i m u m L a i d (%) from a Mallard Reproduction Study with PFOS Eggs Replicate Laid 0 PPM A. I. Max. Laid Arcsin % Trans. Eggs Laid 10 PPM A..1. Max. Laid Arcsin % Trans. Eggs Laid 50 PPM A.I. Max. Laid Arcsin % Trans. Eggs Laid 150 PPM A.I. Max. Laid Arcsin % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 70 72 97 80.41 51 72 71 57.31 53 72 74 59.09 42 72 58 49.80 19 72 26 30.91 0 72 0 0.00 66 72 92 73.22 62 72 86 68.12 42 72 58 49.80 - -- - 55 72 76 60.93 56 72 78 61.87 67 72 93 74.72 51 72 71 57.31 54 72 75 60.00 31 72 43 41.01 719 1080 48 72 19 0 67 54.97 26 19.87 61 72 85 66.99 58 72 81 63.83 54 72 75 60.00 72 72 100 90.00 46 72 64 53.06 57 72 79 62.84 72 72 100 90.00 63 72 88 69.30 56 72 78 61.87 59 72 82 64.85 57 72 79 62.84 47 72 65 53.90 46 72 64 53.06 56 72 78 61.87 43 72 60 50.61 30 72 42 40.20 877 1152 55 72 11 0 76 62.83 15 12.87 . ---------------- __ - '-- - - - - - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- _ - -- *- - - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). Project Number 454- Appendix XIII - Figure la Eggs Laid from a M a lla rd R e p r o d u c tio n S tu dy u it h PFOS - C o n tr o l 80 NUMBER / PEN Project Number 454-109 201 202 203 204 205 20 207 208 209 210 211 212 213 214 215 216 Pen Number i M Eggs Laid 4c D a ta a r e not a v a i l a b l e due to a d u l t m o r t a l i t y . NUMBER / PEN Appendix XIII - Figure lb Eggs Laid Irom a M allard R e p r o d u c tio n S tudy u i t h PFOS - 10 PPM A.I. 80 S U Eggs Laid P en Number ON Project Number 454-109 Reproductive Performance by Pen Appendix XIII - Table 2 E g g s Cracked / Egg s L a i d (%) from a Mallard Reproduction Study with PFOS 0 PPM A. I. Eqqs Eggs Replicate Crack Laid Arcsin % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 1 70 1 51 0 53 2 42 1 19 00 0 66 1 62 0 42 -2 55 0 56 1 67 0 51 0 54 1 31 10 719 1 48 1 19 1 6.86 2 8.05 0 0.00 5 12.60 5 13.26 0 0.00 2 7.30 0 0.00 -4 10.99 0 0.00 1 7.02 0 0.00 0 0.00 3 10.35 2 5.46 2 5.26 10 PPM A..1. Eggs Eggs Crack Laid Arcsin % Trans. 0 61 0 58 0 54 4 72 0 46 0 57 3 72 0 63 3 56 0 59 0 57 0 47 0 46 0 56 0 43 0 30 10 877 1 55 1 11 0 0.00 0 0.00 0 0.00 6 13.63 0 0.00 0 0.00 4 11.78 0 0.00 5 13.38 0 0.00 0 0.00 0 0.00 0 0.00 0 0.00 0 0.00 0 0.00 1 2.43 2 5.23 50 PPM A.I. Eggs Eggs Crack Laid Arcsin % Trans. _ ._ - -- -- -- -- - -- -- - -- -- -- -- - -- -- -- -- -- __ - -- -" _ - - - - * 150 PPM A.I. Eggs Eggs Crack Laid Arcsin % Trans. . _. - -- -- -- -- - -- -- - -- -- -- -- - -- -- -- -- -- __ - -- -- . - - - - - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). Project Number 454-109 Appendix XIII - Figure 2a Eggs Cracked / Eggs Laid from a M allard R e p r o d u c tio n S tudy u i t h PFOS - C o n tr o l 80 NUMBER / PEN Project Number 454-109 P en Number m i Eggs Laid Eggs Cracked Data are not a v a ila b le due to adult m o rtality. Appendix XIII - Figure 2b Eggs Cracked / Eggs Laid from a M allard R e p r o d u c tio n S tu dy u i t h PFOS - 10 PPM A .I. Lv/i Project Number 454-109 Reproductive Performance by Pen Appendix XIII - Table 3 Viable E m b r y o s / E g g s Set (%) from a Mallard Reproduction Study with PFOS 0 PPM A. I. Viable Eggs Replicate Embr. Set Arcsin % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 61 64 95 77.50 40 45 89 70.53 44 49 90 71.37 34 35 97 80.27 11 14 79 62.42 00 57 59 97 79.39 52 56 93 74.50 37 38 97 80.66 - -- - 47 49 96 78.34 52 52 100 90.00 60 61 98 82.64 0 47 0 0.00 47 50 94 75.82 25 25 100 90.00 567 644 38 43 88 72.39 20 18 26 22.06 10 PPM A..1. Viable Eggs Embr. Set Arcsin % Trans. 55 56 98 82.32 49 49 100 90.00 46 50 92 73.57 0 62 0 0.00 38 42 90 72.02 50 52 96 78.69 52 63 83 65.30 57 58 98 82.45 45 46 98 81.52 39 54 72 58.19 51 53 96 78.80 35 41 85 67.51 40 43 93 74.68 46 47 98 81.61 39 40 97 80.90 22 25 88 69.73 664 781 42 49 87 71.08 14 10 24 20.55 50 PPM A.I. Viable Eggs Embr. Set Arcsin % Trans. . - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- _ - --~ - - 150 PPM A.I. Viable Eggs Embr. Set Arcsin % Trans. - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- __ - -- -- - - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). Project Number 454-109 Appendix XIII - Figure 3a Uiable Embryos / Eggs Set from a M allard R ep ro d u ctio n Study u ith PFDS - C o n tro l 70 60 50 NUMBER / PEN to 30 20 10 0 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 P en Number Eggs Set E 3 U iable Embryos Data are not a v a ila b le due to adult m o rtality Project Number 454-109 NUMBER / PEN Appendix XIII - Figure 3b Uiable Embryos Eggs Set from a M allard R e p r o d u c tio n S tu dy u i t h PEOS - 10 PPM A .I. OKN> Project Number 454-109 Reproductive Performance by Pen Appendix XIII- Table 4 L i v e 3 -Week E m b r y o s / Viable E m b r y o s (%) from a Mallard Reproduction Study with PFOS 0 PPM A. I. Live Viable Arcsin Replicate 3-Week Embr. % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 61 61 100 90.00 40 40 100 90.00 43 44 98 81.33 34 34 100 90.00 11 11 100 90.00 00 57 57 100 90.00 52 52 100 90.00 37 37 100 90.00 - -- - 47 47 100 90.00 51 52 98 82.03 60 60 100 90.00 00 46 47 98 81.61 25 25 100 90.00 564 567 38 38 100 88.08 20 20 1 3.66 10 PPM A..1. Live Viable Arcsin 3-Week Embr. % Trans. 55 55 100 90.00 49 49 100 90.00 46 46 100 90.00 00 38 38 100 90.00 50 50 100 90.00 51 52 98 82.03 57 57 100 90.00 44 45 98 81.43 39 39 100 90.00 51 51 100 90.00 35 35 100 90.00 31 40 78 61.68 46 46 100 90.00 39 39 100 90.00 22 22 100 90.00 653 664 41 42 98 87.01 14 14 6 7.58 50 PPM A.I. Live Viable Arcsin 3-Week Embr. % Trans. _ __ - -- -- -- -- -- -- -- -- - -- - -- -- -- -- -- -* __ - -- *" _ - _ - - 150 PPM A.I. Live Viable Arcsin 3-Week Embr. % Trans. _ .. - -- -- -- -- -- -- -- -- -- -- -- -- -- -- "- __ - -- -- . - - Project Number 454- - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0 .0 5 ). NUMBER / PEN Appendix XIII - Figure 4a Live 3-Ueek Embryos / U iable Embryos from a R e p r o d u c tio n Study u i t h PFOS - C o n tr o l M a lla rd 70 60 so 40 30 20 10 0 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 ___ P en Number i m U iable Embryos ^ 3 L iv e 3-Ueek Embryos 4s Data are not a v a ila b le due to adult m o rtality. O-N Project Number 454-109 NUMBER / PEN Appendix XIII - Figure 4b Live 3-Ueek Embryos / U iable Embryos from a R e p r o d u c tio n S tu dy u i t h PFOS - 10 PPM A .I. M a lla rd 70 60 ' 50 40 30 20 10 0 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 P en Number S i U ia b le Embryos EE3 L iv e 3-U eek Embryos CTl Project Number 454-109 Reproductive Performance by Pen Appendix XIII - Table 5 H a tchlings / L ive 3 -Week Embr y o s (%) from a Mallard Reproduction Study with PFOS 0 PPM A. I. Number Live Arcsin Replicate Hatch. 3-Week % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 55 61 90 71.72 29 40 72 58.37 16 43 37 37.59 33 34 97 80.13 8 11 73 58.52 00 33 57 58 49.54 47 52 90 71.94 32 37 86 68.43 - -- - 38 47 81 64.05 47 51 92 73.74 59 60 98 82.58 00 38 46 83 65.35 25 25 100 90.00 460 564 31 38 81 67.07 18 20 18 14.04 10 PPM A..1. Number Live Arcsin Hatch. 3-Week % Trans. 40 55 73 58.52 44 49 90 71.37 38 46 83 65.35 00 33 38 87 68.73 46 50 92 73.57 38 51 75 59.68 39 57 68 55.81 40 44 91 72.45 15 39 38 38.33 47 51 92 73.74 34 35 97 80.27 8 31 26 30.53 44 46 96 77.96 35 39 90 71.32 21 22 95 77.69 522 653 33 41 80 65.02 14 14 21 14.43 50 PPM A.I. Number Live Arcsin Hatch. 3-Week % Trans. ._ - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- _ - -" -" - - 150 PPM A.I. Number Live Arcsin Hatch. 3-Week % Trans. . - -- --- - -- - -- - -- -- -- -- -- -- -- -- -- -- __ - -- -- - - - _ - - - - - - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). Project Number 454-109 - 167- Project Number 454-109 4: Data are not a v a ila b le due to adult m ortality. Hatchlings TJ LOJ. In)I ro O o L. cL. 4-* Ut uo LM30(OI3) Jo3kD_*CoLaO_. X III H Ixj I ja nCOo0>IO>3f "C333OO* TaaaXHJ), \ W3 u C3 - -a %2 o a 1 >o tH CN 10 N OfN N3d / tOaWIN ^ Live 3-Ueek Embryos Appendix XIII - Figure 5b Hatchlings / Live 3-U eek Embryos from a M allard R e p r o d u c tio n S tu dy w ith PFOS - 10 PPM A .I. NUMBER / PEN Project Number 454-109 Reproductive Performance by Pen Appendix XIII - Table 6 1 4 -Day Old Sur v i v o r s / H a t c h l i n g s (%) from a Mallard Reproduction Study with PFOS 0 PPM A. I. 14-Day Number Arcsin Replicate Old Hatch. % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 55 55 100 90.00 28 29 97 79.30 16 16 100 90.00 31 33 94 75.75 8 8 100 90.00 00 33 33 100 90.00 46 47 98 81.61 31 32 97 79.82 - -- - 37 38 97 80.66 47 47 100 90.00 59 59 100 90.00 00 38 38 100 90.00 25 25 100 90.00 454 460 30 31 99 85.93 18 18 2 5.51 10 PPM A.. 1 . 14-Day Number Arcsin Old Hatch. % Trans. 39 40 97 80.90 42 44 95 77.69 37 38 97 80.66 00 32 33 97 79.98 45 46 98 81.52 37 38 97 80.66 37 39 95 76.91 37 40 93 74.11 15 15 100 90.00 47 47 100 90.00 34 34 100 90.00 8 8 100 90.00 42 44 95 77.69 34 35 97 80.27 21 21 100 90.00 507 522 32 33 97 82.69 14 14 2 5.67 50 PPM A.I. 14-Day Number Arcsin Old Hatch. % Trans. .. - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- _ - -- -- - - 150 PPM A.I. 14-Day Number Arcsin Old Hatch. % Trans. - -- -- -- -- -- - -- - -- -- -- -- -- -- -- -- -- __ - -- -- - - Project Number 454-109 - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0 .0 5 ). NUMBER / PEN Appendix XIII - Figure 6a 1 4-D ag Old S u r v i v o r s / H a t c h lin g s from a R e p r o d u c tio n Studg u i t h PFOS - C o n tr o l M a lla rd 0 50 40 30 20 10 0 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 P en Number Hatchlings EES 1 4 - D a y Old S u r v i v o r s Data a re not a v a ila b le due to adult m ortality. O'- l Project Number 454-109 Appendix XIII - Figure 6b 14-Dag Old S u rv iv o rs / Hatchlings from a M allard R e p r o d u c tio n S tu dy u i t h PFOS - 10 PPM A.I. 60 50 ' 40 NUMBER / PEN 30 20 10 Project Number 454-109 0 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Ems H a t c h lin g s P en Number Ezz3 1 4 - D a y Old S u r v i v o r s Reproductive Performance by Pen Appendix XIII - Table 7 H a t c h l i n g s / E g g s Set (%) from a Mallard Reproduction Study with PFOS 0 PPM A. I. Number Eggs Replicate Hatch. Set Arcsin % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 55 64 86 67.98 29 45 64 53.40 16 49 33 34.85 33 35 94 76.17 8 14 57 49.11 00 33 59 56 48.41 47 56 84 66.37 32 38 84 66.59 - -- - 38 49 78 61.72 47 52 90 71.94 59 61 97 79.57 0 47 0 0.00 38 50 76 60.67 25 25 100 90.00 460 644 31 43 71 59.05 18 18 28 22.11 10 PPM A..1. Number Eggs Hatch. Set Arcsin % Trans. 40 56 71 57.69 44 49 90 71.37 38 50 76 60.67 0 62 0 0.00 33 42 79 62.42 46 52 88 70.14 38 63 60 50.95 39 58 67 55.09 40 46 87 68.83 15 54 28 31.81 47 53 89 70.34 34 41 83 65.59 8 43 19 25.55 44 47 94 75.37 35 40 88 69.30 21 25 84 66.42 522 781 33 49 69 56.35 14 10 28 20.49 50 PPM A.I. Number Eggs Hatch. Set Arcsin % Trans. . .. - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- _ - --- - - 150 PPM A.I. Number Eggs Hatch. Set Arcsin % Trans. . - -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- _ - -- - " Project Number 454-109 - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0.05). Reproductive Performance by Pen Appendix XIII - Table 8 1 4 -Day Old S u r v i v o r s / Eggs Set (%) from a Mallard Reproduction Study with PFOS 0 PPM A.I. 14-Day Eggs Replicate Old Set Arcsin % Trans. 10 PPM A.I. 14-Day Eggs old Set Arcsin % Trans. 50 PPM A.I. 14-Day Eggs Old Set Arcsin % Trans. 150 PPM A.I. 14-Day Eggs Old S e t Arcsin % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 55 64 86 6 7 .9 8 28 45 62 5 2 .0 7 16 49 33 3 4 .8 5 31 35 89 7 0 .2 4 8 14 57 4 9 .1 1 00 33 59 56 4 8 .4 1 46 56 82 6 5 .0 0 31 38 82 6 4 .5 8 - -- - 37 49 76 6 0 .3 4 47 52 90 7 1 .9 4 59 61 97 7 9 .5 7 0 47 0 0 .00 38 50 76 6 0 .6 7 25 25 100 9 0 .0 0 454 30 18 644 43 18 70 5 8 .2 0 27 2 1 .7 4 39 56 70 5 6 .5 7 42 49 86 6 7 . 7 9 37 50 74 5 9 .3 4 0 62 0 0 .0 0 32 42 76 6 0 . 7 9 45 52 87 6 8 .4 8 37 63 59 5 0 .0 3 37 58 64 5 3 . 0 1 37 46 80 6 3 . 7 5 15 54 28 3 1 .8 1 47 53 89 7 0 .3 4 34 4 1 83 6 5 . 5 9 8 43 19 2 5 .5 5 42 47 89 7 0 . 9 6 34 4 0 85 6 7 . 2 1 21 25 84 6 6 .4 2 507 32 14 781 49 10 67 5 4 .8 5 27 1 9 .6 9 .- -- -- -- -- -- -- -- -- -- -- -- -- -- -- - -- _ - -- -- . - - - . .. - -- -- -- -- -- -- -- -- -- -- -- -- -- -- ~- __ - -- -- 7 - - - Project Number 454-109 - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0 .0 5 ). Reproductive Performance by Pen Appendix XIII - Table 9 H a t c h l i n g s / M a x i m u m Set (%) from a Mallard Reproduction Study with PFOS 0 PPM A.I. Number Max. Replicate Hatch. Set Arcsin % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 55 64 86 67.98 29 64 45 42.31 16 64 25 30.00 33 64 52 45.90 8 64 13 20.70 0 64 0 0.00 33 64 52 45.90 47 64 73 58.98 32 64 50 45.00 - -- - 38 64 59 50.40 47 64 73 58.98 59 64 92 73.77 0 64 0 0.00 38 64 59 50.40 25 64 39 38.68 460 960 31 64 48 41.93 18 0 28 21.68 10 PPM A.I. Number Max. Hatch. Set Arcsin % Trans. 40 64 63 52.24 44 64 69 56.01 38 64 59 50.40 0 64 0 0.00 33 64 52 45.90 46 64 72 57.97 38 64 59 50.40 39 64 61 51.32 40 64 63 52.24 15 64 23 28.96 47 64 73 58.98 34 64 53 46.79 8 64 13 20.70 44 64 69 56.01 35 64 55 47.69 21 64 33 34.95 522 1024 33 64 14 0 51 44.41 22 15.89 50 PPM A.I. Number Max. Hatch. Set Arcsin % Trans. _ __ - -- -- -- -- -- -- -- -- -- -- -- -- -- --- -- - -- - -- _ - - - 150 PPM A.I. Number Max. Hatch. Set Arcsin % Trans. _ -, - _ - -_ -- -- _ - - - _ - - - Project Number 454-109 - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0 .05). Reproductive Performance by Pen Appendix XIII - Table 10 1 4 -Day Ol d S u r v i v o r s / M a x i m u m Set (%) from a Mallard Reproduction Study with PFOS 0 PPM A.I. 14-Day Max. Replicate Old Set Arcsin % Trans. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total Mean SD 55 64 86 6 7 .9 8 28 64 44 4 1 .4 1 16 64 25 3 0 .0 0 31 64 48 4 4 .1 0 8 64 13 2 0 .7 0 0 64 0 0 .0 0 33 64 52 4 5 .9 0 46 64 72 5 7 .9 7 31 64 48 4 4 .1 0 - -- - 37 64 58 4 9 .4 9 47 64 73 5 8 .9 8 59 64 92 7 3 .7 7 0 64 0 0.0 0 38 64 59 5 0 .4 0 25 64 39 3 8 .6 8 454 30 18 960 64 0 47 4 1 .5 7 28 2 1 .5 7 10 PPM A. I. 14-Day Max. Old Set Arcsin % Trans. 39 64 61 5 1 .3 2 42 64 66 5 4 . 1 0 37 64 58 4 9 . 4 9 0 64 0 0 .0 0 32 64 50 4 5 . 0 0 45 64 70 5 6 .9 8 37 64 58 4 9 . 4 9 37 64 58 4 9 . 4 9 37 64 58 4 9 . 4 9 15 64 23 2 8 . 9 6 47 64 73 5 8 .9 8 34 64 53 4 6 . 7 9 8 64 13 2 0 .7 0 42 64 66 5 4 . 1 0 34 64 53 4 6 . 7 9 21 64 33 3 4 . 9 5 507 32 14 1024 64 0 50 4 3 .5 4 21 1 5 .4 3 50 PPM A. I. 14-Day Max. Old Set Arcsin % Trans. _ - -- -- -- -- -- -- -- -- - -. - -- -- -- -- -- -- __ - -- -- - - 150 PPM A.I. 14-Day Max. Old Set Arcsin % Trans. - -- -- -- -- -- -- -- -- -- -- -- -- -- -" -* __ - -- -- 1.- - - Data are not available due to adult mortality. Differences between the control and each treatment group were not significant (p > 0 .0 5 ). Project Number 454-109 Reproductive Performance by Week and Pen Appendix XIV - Table la Eggs Laid by Week and Pen from a Mallard Reproduction Study with PFOS E x p e rim e n ta l G ro u p (ppm a . i .) _ PENS ------------------------------------------------------------------------------------------------------------------------------------------------------------ 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 1 LOT A B C D C o n tro l E F G H I J 8 0 0 01 0 3 0 0 - 0 0 4 0 0 0 7 4 3 23 0 7 6 0 - 6 0 7 3 6 2 7 8 5 70 0 7 7 6 - 7 7 7 7 7 5 7 6 7 71 0 7 7 5 - 7 7 7 7 7 6 7 2 7 64 0 7 7 7 - 7 7 7 7 7 5 7 7 7 20 0 7 7 5 - 7 7 7 7 7 1 7 6 7 74 0 7 7 5 - 6 7 7 7 6 5 7 7 7 22 0 7 7 5 - 5 7 7 7 7 1 6 5 3 72 0 7 7 5 - 6 7 7 6 6 2 7 6 7 22 0 7 7 4 - 4 7 7 0 1 4 T o tals 16 56 87 88 87 78 88 78 76 65 T o tals 70 51 53 42 19 0 66 62 42 - Data are not available due to adult mortality LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. 55 56 67 51 54 31 719 -Ojs Project Number 454-109 Reproductive Performance b y Week and Pen Appendix XIV - Table lb E ggs L a id b y Week and Pen fr o m a M a l l a r d R e p r o d u c t i o n S t u d y w i t h PFOS E x p e rim en tal G ro u p (ppm a . i .) 1 LOT A B C D 10 E F G H I J 217 218 219 220 221 222 223 224 PEN S 225 226 227 228 229 230 231 232 T o tals 9 0 0 90 0 94 0 6 0 0 0 0 0 0 7 6 6 73 l 7 7 364 1 0 2 00 7 7 7 77 7 7 7 7 7 7 7 0 7 0 2 3 7 7 77 7 7 5 7 7 7 7 5 7 3 5 6 6 7 73 7 7 6 7 7 7 6 7 7 7 5 7 7 5 75 7 7 7 7 6 7 6 7 7 7 3 3 7 7 77 7 7 7 7 6 7 7 7 7 7 5 7 7 6 77 7 7 8 7 6 7 4 7 7 7 4 5 5 7 75 7 7 6 4 6 5 6 6 6 7 2 7 6 2 72 7 7 6 7 2 6 3 7 6 5 4 37 60 93 98 102 102 105 105 91 84 T o t a l s 61 58 54 72 46 57 72 63 56 59 57 47 46 56 43 30 877 LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. O Project Number 454-109 Appendix XIV - Figure 1 Eggs L a id p e r Hen from a M a l l a r d R e p r o d u c t io n Studg u i t h PFOS Concentrations, ppm a .i. 10 -J oc Project Number 454-109 Appendix XIV - Figure 2 Hens L a y in g p e r Pen from a M a l l a r d R e p r o d u c t io n S tu dy u i t h PFOS NUMBER Project Number 454-109 Appendix XIV - Figure 3 Eggs p e r L a y in g Hen from a M a l l a r d R e p r o d u c tio n S tudy u it h PFOS C oncentrations, ppm a .i. 0 10m m m m Project Number 454-109 Reproductive Performance by Week and Pen Appendix XIV - Table 2a Eggs Cracked by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a .i .) PENS ----------------------------------------------------------- 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Totals 1 LOT A B C D Control E F G H I J 0 0 0 00 0 0 0 0 -0 0 0 0 0 0 1 0 0 00 0 0 0 0 -1 0 0 0 0 1 0 0 0 00 0 0 0 0 - 0 0 0 0 0 0 0 0 000 0 0 0 0 -0 0 0 00 0 0 1 0 00 0 0 0 0 - 0 0 0 0 0 0 0 0 000 0 0 00 -0 0 0 0 0 0 0 0 010 0 0 0 0 -0 0 0 0 0 0 0 0 0 01 0 0 0 0 - 0 0 0 0 0 0 0 0 0 10 0 0 1 0 -1 0 1 00 0 0 0 0 00 0 0 0 0 - 0 0 0 0 0 0 0 3 0 0 1 0 1 1 4 0 Totals 1 1 0 2 1 0 0 1 0 . 2 0 1 0 0 1 10 - Data are not available due to adult mortality ^LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. Project Number 454-109 Reproductive Performance by Week and Pen Appendix XIV - Table 2b Eggs Cracked by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a .i .) 1 LOT A B C D 10 E F G H I J PEN s 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 Totals 0 0 0 00 0 0 000 0 0 0 0 0 0 0 0 0 00 0 0 0 00 0 0 0 0 0 0 0 0 0 00 0 0 0 00 0 0 0 0 0 0 0 0 0 00 0 0 010 0 0 0 0 0 0 0 0 020 02 0000 0 0 00 0 0 0 000 0 0 01 0 0 0 0 0 0 0 0 0 000 0 1 0 10 0 0 0 0 0 0 0 0 000 0 0 0 00 0 0 0 0 0 0 0 0 0 00 0 0 0 00 0 0 0 0 0 0 0 0 020 0 0 0000 0 0 0 0 0 0 0 0 1 4 1 2 0 0 2 Totals 0 0 0 4 0 0 3 0 3 0 0 0 0 0 0 0 10 ^LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. ONCi Project Number 454-109 Reproductive Performance by Week and Pen Appendix XIV - Table 3a Eggs Set by Week and Pen from a Mallard Reproduction Study with PFOS E x p e rim en tal (ppm a . i .) 1 LOT A B C D C o n tro l E F G H I J 201 202 203 204 205 206 207 208 PENS 209 210 211 212 213 214 215 216 7 0 0 00 0 2 0 0- 0 0 3 0 0 0 6 3 3 13 0 7 5 0 - 5 0 7 2 6 0 6 8470 0 5 75 - 6 7 6 76 5 7 5 7 61 0 7 6 5 - 7 6 7 6 7 5 6 1 663 0 6 76 - 6 7 6 7 6 5 7 6710 07 65 - 76 7 67 0 6 6 6 63 0 5' 7 4 - 5 7 6 7 5 5 7 6 7 11 0 7 6 5 - 5 6 7 6 7 0 5 5 2 61 0 6 64 - 4 7 5 6 5 2 7 5 7 12 0 7 64 - 4 6 7 0 1 3 T o tals 12 48 79 82 78 72 78 71 64 60 T o tals 64 45 4 9 3 5 14 0 59 56 38 - Data are not available due to adult mortality 1LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. - 49 52 61 47 50 25 644 Project Number 454-109 Reproductive Performance by Week and Pen Appendix XIV - Table 3b Eggs Set by Week and Pen from a Mallard Reproduction Study with PFOS E x p e rim en tal G ro u p (ppm a . i . ) 1 LOT A B C D 10 E F G H I J 217 218 219 220 221 222 223 224 PEN S 225 226 227 228 229 230 231 232 T o ta ls 8 0 090 0 8 4 06 0 0 0 0 0 0 7 56 63 0 7 6 354 0 0 1 0 0 6 6 676 7 6 7 67 6 7 0 7 0 2 3 6 767 6 7 4 66 7 6 5 6 3 3 5 6 6 52 7 4 6 6 7 6 6 6 6 6 5 7 6 5656 7 6657 5 7 672 2 7 666 7 5 7 566 7 6 6 6 5 7 5 6 67 6 7 7 65 7 3 7 6 7 3 4 4 6 74 7 6 6 2 64 5 5 5 6 2 7 4 242 6 6 5 61 62 7 4 5 3 35 53 86 88 89 93 93 95 79 70 T o tals 56 4 9 50 62 42 52 63 58 46 54 53 4 1 43 4 7 4 0 2 5 781 ^LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. 00 Project Number 454-109 Reproductive Performance by Week and Pen Appendix XIV - Table 4a Viable Embryos by Week and Pen from a Mallard Reproduction Study with PFOS E x p e rim e n ta l G ro u p (ppm a . i .) PENS ------------------------------------------------------------------------------------------------------------------------------------------------------------ 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 1 LOT A B C D C o n tro l E F G H I J 5 0 0 00 0 1 0 0 00 300 0 6 0 3 11 0 7 3 0 - 5 0 7 0 6 0 5 6 2 60 0 5 7 4 - 6 7 6 0 5 5 7 5 5 61 0 7 5 5 - 7 6 7 0 7 5 6 1 6 62 0 6 7 6 - 6 7 6 0 5 5 7 6 7 10 0 7 6 5 - 6 6 7 0 7 0 6 6 6 63 0 5 7 4 - 4 7 6 0 5 5 7 6 6 11 0 7 6 5 - 5 6 7 0 6 0 5 5 2 61 0 6 6 4 - 4 7 5 0 5 2 7 5 7 12 0 6 5 4 - 4 6 6 0 1 3 T o tals 9 39 64 73 69 65 70 63 58 57 T o ta ls 61 40 44 34 11 0 57 52 37 - Data are not available due to adult mortality ^LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. 47 52 60 0 47 25 567 oo Oft Project Number 454-109 Reproductive Performance by Week and Pen Appendix XIV - Table 4b Viable Embryos by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a.i.) 1 LOT A B C D 10 E F G H I J PEN S 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 8 0 000 0 84 0 0 0 0 0 0 0 0 7 5 5 03 0 7 6 3 03 0 0 1 0 0 6 6 6 05 7 6 7 6 3 6 3 0 7 0 2 3 6 6 07 5 7 4 6 6 7 4 5 6 3 3 5 6 6 01 7 4 6 6 7 6 6 5 6 6 5 7 6 5 04 6 2 6 6 5 6 5 7 6 7 2 2 7 4 05 7 4 7 5 6 6 7 6 6 5 5 7 5 6 07 6 7 7 5 5 7 3 5 6 7 3 4 4 6 04 6 3 6 2 6 4 5 5 5 6 0 6 4 2 02 6 4 4 6 1 6 2 7 3 5 2 Totals 20 40 70 78 82 80 82 86 66 60 Totals 55 49 46 0 38 50 52 57 45 39 51 35 40 46 39 22 664 LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. 00 ON Project Number 454-109 Reproductive Performance by Week and Pen Appendix XIV - Table 5a Live Three-Week Embryos by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a.i.) 1 LOT A B C D Control E F G H I J PENS 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 5 0 000 0 100 -0 0 3 0 0 0 6 0311 073 0 -5 0 7 06 0 5 62 60 0 574 -6 6 6 05 5 7 5 5 61 0 7 55 -7 6 7 0 7 5 6 1 6 62 0 6 76 -6 7 6 0 5 5 7 6710 0 765 -6 6 7 0 6 0 6 6 6 63 0 5 74 -4 7 6 0 5 5 7 6611 0 765 -5 6 70 6 0 5 52 61 0 6 64 -4 7 5 0 5 2 7 5 6 12 0 6 54 -4 6 6 0 1 3 Totals 9 39 63 73 69 64 70 63 58 56 Totals 61 40 43 34 11 0 57 52 37 - Data are not available due to adult mortality LOT A - Eggs Set During Week 12; LOT B -Eggs Set During Week 13; etc. - 47 51 60 0 46 25 564 Project Number 454-109 Reproductive Performance by Week and Pen A pp en dix XIV - T a b le 5b Live Three-Week Embryos by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a .i .) PENS ----------------------------------------------------------- 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 1 LOT A B C D 10 E F G H I J 8 0 0 00 0 84 0 0 0 0 0 00 0 7 5 5 03 0 763 03 0 01 0 0 6 6 6 05 767636 3 07 02 3 6 6 07 5 74 6 67 4 1 63 3 5 6 601 74 657 6 64 66 5 7 6 5 04 6 2 6 6 5 6 5 7 6 7 2 2 74 05 74 7 5 66 7 36 5 5 7 5 6 07 6 67 5 5 7 3 5 6 7 3 4 4 6 04 6 3 62 64 5 55 6 0 6 4 2 02 64 4 6 1 6 2 6 3 5 2 Totals 20 40 70 74 80 80 79 85 66 59 Totals 55 49 46 0 38 50 51 57 44 39 51 35 31 46 39 22 653 ^LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. Project Number 454-109 Reproductive Performance by Week and Pen A pp en dix XIV - T a b le 6a Hatchlings by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a .i .) 1 LOT A B C D Control E F G H I J PENS 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 Totals 5 0 0 00 0 0 00 - 0 0 3 00 0 5 0011 0 620 -4 07 06 0 4 3 060 02 64 -6 4 505 5 7 3 0 61 0 34 5 -5 6 7 01 5 6 02 60 02 55 -4 6 605 5 6 53 10 0364 -5 6 7 04 0 6 6 2 63 0 2 73 -4 7 6 05 5 7 64 10 0 364 -3 67 06 0 4 1 0 51 0 6 64 -3 7 5 05 2 5 5 5 12 0 6 53 -4 5 6 01 3 8 32 50 53 52 50 62 53 49 51 Totals 55 29 16 33 8 0 33 47 32 - Data are not available due to adult mortality 1LOT A - Eggs Set During Week 12; LOT B- Eggs Set During Week 13; etc. - 38 47 59 0 38 25 460 00 'O Project Number 454-109 Reproductive Performance by Week and Pen Appendix XIV - Table 6b Hatchlings by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a .i .) PENS ------------------------------------------------------ -- 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 1 LOT A B C D 10 E F G H I J 7 000008100000000 64 3 020 652 02 0 010 0 5 64 035354163 0601 3 6 6065235174 062 3 3 5 50073352 650655 3 4 4 04 6 2 3 6 1 6 5 2 67 2 2 730563452 6 7054 5 4 550765755731663 3 3 6 04 5 3 52 3 2 5 2 5 6 0 4 4 2 02 6 3 3 6 0 5 2 3 3 5 2 Totals 16 31 52 59 60 61 64 75 54 50 Totals 40 44 38 0 33 46 38 39 40 15 47 34 8 44 35 21 522 ^LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. Project Number 454-109 Reproductive Performance by Week and Pen Appendix XIV - Table 7a 14-Day Old Survivors by Week and Pen from a Mallard Reproduction Study with PFOS Experimental Group (ppm a.i.) 1 LOT A B C D Control E F G H I J PENS 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 5 00000000-0 03000 5 0011 0620-4 07060 4 2 060 02 64 - 6 4 5 0 5 5 7 3 061 0345 -5 6 701 5 6 0260 02 55 -4 6 605 5 6 53 00 03 54 -4 6 704 0 6 6263 0272 -4 7 60 5 5 7 6 410 0364 -3 6 706 0 4 1 041 0664 -3 7 5052 5 5512 0653 -4 5 601 3 Totals 8 32 49 53 52 47 61 53 48 51 Totals 55 28 16 31 8 0 33 46 31 - Data are not available due to adult mortality ^LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. - 37 47 59 0 38 25 454 Project Number 454 Reproductive Performance by Week and Pen Appendix XIV - Table 7b 14 -Day Old Survivors by Week and Pen from a Mallard Reproduction Study with PROS Experimental Group (ppm a. i .) 1 LOT A B C D 10 E F G H I J PEN S 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 7 00 000 810 00 0 000 0 6 3 2 01 0 64 0 02 0 0 0 0 0 5 64 034 354 16 3 060 1 3 6 6 06 5 2 35 17 4 0 6 1 3 3 55 007 33526 5 065 5 3 4 4 04 6 1 3 5 1 6 5 2 6 7 2 2 7 3 05 6 335 2 6 7 054 5 4 4 5 07 6 575 5 7 3 1 5 6 3 3 3 6 04 5 3 52 3 2 5 2 5 6 0 3 4 2 02 6 3 3 6 0 5 2 3 3 5 2 Totals 16 24 51 58 60 59 63 73 54 49 Totals 39 42 37 0 32 45 37 37 37 15 47 34 8 42 34 21 507 ^LOT A - Eggs Set During Week 12; LOT B - Eggs Set During Week 13; etc. to Project Number 454-109 Pen A 201 . 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 B - - No eggs available. Appendix XV - Table 1 Eggshell T h i ckness (nan) p e r Pen b y Week from a Mallard Reproduction Study with PFOS (C o n t r o l ) Wee k s CDE FGH I 0.387 0.437 0.455 0.406 0.366 0.447 0.349 0.398 0.353 0.425 0.401 0.385 0.363 0.397 0.428 0.378 0.446 0.406 0.375 0.426 0.369 0.382 0.357 0.435 0.386 0.384 0.332 0.391 0.440 0.368 0.417 0.401 0.352 0.438 0.383 0.381 0.347 0.433 0.408 0.395 0.289 0.393 0.455 0.374 0.415 0.409 0.344 0.425 0.384 J - Mean and standard deviation of pen means : Mean SD [n] 0.392 0.387 0.440 0.352 0.373 0.004 0.010 0.011 0.005 0.005 4 3 4 3 3 0.433 0.431 0.406 0.020 0.005 0.003 4 3 4 0.359 0.399 0.434 0.388 0.371 0.328 0.014 0.011 0.010 0.006 0.017 0.037 4 3 4 3 4 3 Mean SD 0.392 0.034 49 Project Number 454-109 Appendix XV - Table 2 Eggshell Thickness (mm) p e r Pen b y Week from a Mallard Reproduction Study with PFOS (10 P P M A. I. ) Pen A B C Wee k s DE FGH IJ 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 0.410 0.388 0.416 0.400 - 0.416 0.417 0.407 0.416 0.389 0.406 0.401 - 0.425 0.418 0.406 0.386 0.437 0.416 0.344 - 0.384 0.381 0.378 0.401 0.415 0.416 0.396 - 0.391 0.390 0.377 0.415 0.415 0.419 0.428 - 0.425 0.380 0.379 0.379 0.381 0.382 0.373 - 0.386 0.386 0.370 - 0 .406 0 .404 0.407 - 0.370 0.384 0.387 - 0.384 0.371 0.380 0.363 0.426 0.395 - Mean and standard deviation of pen means : - No eggs available. Differences between the control and this treatment group were not significant (p > 0.05). Mean SD [n] 0.404 0.413 0.403 0.416 0.396 0.381 0.407 0.386 0.419 0.395 0.379 0.381 0.406 0.380 0.379 0.395 0.012 0.006 0.012 0.009 0.041 0.003 0.010 0.008 0.006 0.026 0.004 0.010 0.002 0.009 0.007 0.032 4 3 4 3 4 3 4 3 4 3 4 3 3 3 3 3 Mean SD 0.396 0.014 54 Project Number 454-109 Appendix XVI - Table 1 M e a n H a t c h l i n g B o d y Weight (g) p e r Pen b y Week from a Mallard Reproduction Study with PFOS (C o n t r o l ) Weeks [No. of Hatchlings] Total Pen A[ n] B [ n] C [ n] D[ n] E [ n] F[ n] G[ n] H[ n] I [ n] J[ n] Mean SD Hatch 201 35 [ 5] 35 [ 5] 36 [ 4] 37 [ 7] 29 [ 6] 33 [ 6] 37 [ 6] 34 [ 7] 29 [ 4] 33 [ 53 34 3 55 202 [ 0] [ 0] 31 [ 3] 35 [ 3] [ 0] 31 [ 5] 34 [ 6] 3 6 63 23 [ 1] 35 [ 5] 33 3 29 203 [ 0] [ 0] [ 0] [ 0] 30 [ 2] 33 [ 3] 36 [ 2] 39 [ 4] [ 0] 34 [ 5] 35 3 16 204 [ 0] 43 [ 1] 4 2 [ S] 43 [ 6] 34 [ 6] 30 [ 1] 43 [ 6] 35 [ 1] 28 [ 4] 33 [ 13 38 6 32 205 [ 0] 30 [ 1] [ 0] 26 [ 1] [ 0] [ 0] 30 [ 3] [ 0] 22 [ 13 31 [ 2] 29 3 8 2 0 6 [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] 207 [ 0] 35 [ 6] 39 [ 2] 38 [ 3] 34 [ 2] 36 [ 3] 40 [ 2] 37 [ 3] 34 [ 6] 34 [ 63 35 2 33 208 [ 0] 38 [ 2] 39 [ 6] 41 [ 4] 39 [ 5] 34 [ 6] 43 [ 7] 42 [ 6] 41 [ 6] 40 [ 5] 40 3 47 209 [ 0] [ 0] 42 [ 4] 44 [ 5] 37 [ 5] 4 1 [ 4] 4 8 [ 3] 4 5 [ 43 4 1 [ 4] 4 3 [ 3] 42 3 32 210 - - - - - - - f -- 211 [ 0] 36 [ 4] 37 [ 6] 39 [ 5] 34 [ 4] 32 [ 5] 34 [ 4] 37 [ 3] 33 [ 3] 34 [ 4] 35 2 38 212 [ 0] [ 0] 37 [ 4] 37 [ 6] 31 [ 6] 31 [ 6] 37 [ 7] 36 [ 6] 34 [ 7] 33 [ 53 34 3 47 213 35 [ 3] 35 [ 7] 35 [ 5] 40 [ 7] 31 [ 6] 34 [ 7] 41 [ 6] 38 [ 7] 34 [ 53 35 [ 6] 36 3 59 2 1 4 [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 03 215 [ 0] 36 [ 6] 36 [ 5] 37 [ 1] 34 [ 5] 30 [ 4] 39 [ 5] 40 [ 6] 35 [ 5] 38 [ 1] 36 3 38 216 [ 0] [ 0] 32 [ 5] 34 [ 5] 33 [ 5] [ 0] 35 [ 5] [ 0] 30 [ 2] 31 [ 3] 33 2 25 Mean 35 SD 3 459 The number of hatchlings weighed may differ from the total number of hatchlings since those hatchlings found dead were not weighed. - Data are not available due to adult mortality. SD = Standard deviation of mean body weight, by parental pen, by week. Project Number 454-109 A pp en dix XVI - T a b le 2 M e a n H a t c h l i n g B o d y Weight (g) p e r P e n b y Week from a Mallard Reproduction Study with PFOS (10 P P M A. I. ) Weeks [No. of Hatchlings] Total Pen A[ n] B [ n] C [ n] D[ n] E [ n] F [ n] G[ n] H [ n] I [ n] J [ n] Mean SD Hatch Project Number 454-109 CO ro 217 39 [ 7] 6] 37 [ 5] 42 [ 3] 37 [ 3] 36 [ 3] 42 [ 2] 42 [ 4] 39 [ 3] 40 [ 4] 39 2 40 218 [ 0] 35 [ 4] 34 [ 6] 35[ 6] 35 [ 5] 28 [ 4] 37 [ 7] 35 [ 5] 38 t 3] 36 [ 4] 35 2 44 219 [ 0] 34 [ 3] 33 [ 4] 37 [ 6] 35 [ 5] 35 [ 4] 40 [ 3] 36 [ 5] 35 [ 6] 36 [ 2] 36 2 38 220 [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] E 0] E 0] 221 [ 0] 35 [ 2] 36 [ 3] 34 [ 6] [ 0] 34 [ 4] 41 [ 5] 42 [ 7] 36 [ 4] 33 [ 2] 37 4 33 222 [ 0] [ 0] 38 [ 5] 38 [ 5] 37 [ 7] 36 [ 6] 38 E 6] 37 [ 6] 32 [ 5] 35 [ 6] 36 2 46 223 37 [ 8] 36 [ 6] 37 [ 3] 39 [ 2] 36 [ 3] 38 [ 2] 47 [ 3] 40 [ 5] 45 [ 3] 42 [ 3] 39 4 38 224 32 [ 1] 36 [ 5] 34 [ 5] 33 [ 3] 35 [ 3] 33 [ 3] 36 [ 4] 33 [ 7] 33 [ 5] 36 [ 3] 34 1 39 225 [ 0] 34 [ 2] 34 [ 4] 35 [ 5] 36 [ 5] 31 [ 5] 39 [ 5] 36 [ 5] 41 [ 2] 35 [ 6] 35 3 39 226 [ 0] [ 0] 29 [ 1] 33 [ 1] 32 [ 2] 30 [ 1] 35 [ 2] 33 [ 5] 34 [ 3] E 0] 33 2 15 227 [ 0) 36 [ 2] 35 [ 6] 35 [ 7] 35 [ 6] 35 [ 6] 37 [ 6] 36 E 7] 38 [ 2] 37 [ 5] 36 1 47 228 [ 0] [ 0] 38 [ 3] 41 [ 4] 41 [ 5] 45 [ 5] 42 [ 7] 39 [ 3] 39 [ 5] 40 [ 2] 41 2 34 229 [ 0] [ 0] [ 0] [ 0] [ 0] 33 [ 2] [ 0] 35 [ 1] 35 [ 21 39 [ 3] 36 3 8 230 [ 0] 37 [ 1] 32 [ 6] 39 [ 6] 37 [ 6] 41 [ 6] 37 [ 5] 39 E 6] 41 [ 5] 36 [ 3] 38 3 44 231 [ 0] [ 0] [ 0] 34 [ 2] 35 [ 5] 41 [ 7] 35 [ 4] 39 [ 6] 40 [ 6] 36 [ 5] 38 2 35 232 [ 0] [ 0] 47 [ 1] 42 [ 3] 42 [ 5] 43 [ 2] 38 [ 5] 42 [ 3) E 0] 41 [ 2] 41 2 21 Mean 37 SD 2 521 The number of hatchlings weighed may differ from the total number of hatchlings since those hatchlings found dead were not weighed. Differences between the control and this treatment group were not significant (p > 0.05). SD = Standard deviation of mean body weight, by parental pen, by week. A ppen dix XVII - T a b le 1 M e a n 1 4 -Day Old S u r v i v o r s B o d y Weight (g) p e r Pen b y Week from a Mallard Reproduction Study with PFOS (C o n t r o l ) Weeks [No. of 14-Day Old Survivors] 14-Day Pen A [ n] Bt n] C[ n] D[ n] E[ n] F[ n] .G[ n] H [ n] I[ n] J[ n] Mean SD Total 201 308 [ 5] 292 [ 5] 306 [ 4] 303 [ 7] 289 [ 63 293 [ 6] 271 [ 6] 273 [ 7] 272 [ 4] 271 [ 5] 288 14 55 202 [ 0] [ 0] 232 [ 2] 243 [ 3] [ 0] 254 [ 5] 250 [ 6] 252 [ 6] 185 [ 1] 247 [ 5] 246 13 28 203 [ 0] [ 0] [ 0] [ 0] 321 [ 2] 295 [ 3] 287 [ 2] 309 [ 4] [ 0] 298 [ 5] 302 10 16 204 [ 0] 320 [ 1] 333 [ 6] 320 [ 6] 318 [ 6] [ 0] 323 [ 6] 295 [ 1] 321 [ 43 336 [ 1] 323 8 31 205 [ 0] 224 [ 1] [ 0] 232 [ 1] [ 0] [ 0] 241 [ 3] [ 0] 141 [ 1] 265 [ 2] 231 39 8 206 [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] 207 [ 0] 268 [ 6] 286 [ 2] 280 [ 3] 295 [ 2] 253 [ 3] 258 [ 2] 271 [ 3] 261 [ 6] 258 [ 6] 267 12 33 208 [ 0] 310 [ 2] 309 [ 61 279 [ 4] 313 [ 5] 298 [ 5] 291 [ 7] 324 [ 6] 289 [ 6] 299 [ 5] 301 13 46 209 [ 0] [ 0] 335 [ 4] 311 [ 5] 359 [ 5] 329 [ 4] 333 [ 2] 337 [ 4] 332 [ 4] 335 [ 33 334 14 31, 210 - - - - - - - - - - 211 [ 0] 286 [ 4] 290 [ 6] 295 [ 5] 334 [ 4] 301 [ 4] 267 [ 4] 327 [ 3] 285 [ 3] 258 [ 43 293 23 37 212 [ 0] [ 0] 317 [ 4] 280 [ 6] 309 [ 6] 307 [ 6] 272 [ 7] 305 [ 6] 264 [ 7] 278 [ 5] 290 19 47 213 317 [ 3] 276 [ 7] 314 [ 5] 304 [ 7] 285 [ 6] 297 [ 7] 275 [ 6] 306 [ 7] 289 [ 5] 277 [ 61 293 14 59 214 [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 0] [ 03 215 [ 0] 289 [ 6] 302 [ 5] 255 ( 1] 298 [ 5] 278 [ 4] 271 [ 5] 300 [ 6] 287 [ 5] 293 [ 1] 289 12 38 216 [ 0] [ 0] 289 [ 5] 286 [ 5] 278 [ 5] [ 0] 253 [ 5] [ 0] 270 [ 2] 315 [ 3] 281 18 25 Mean 287 SD 28 454 - Data are not available due to adult mortality. SD = Standard deviation of mean body weight, by parental pen, by week. -J Project Number 454-109 Appendix XVII - Table 2 M e a n 1 4 -Day Old S u r v i v o r s B o d y Weight (g) p e r Pen b y Wee k from a Mallard Reproduction Study with PFOS (10 P P M A. I. ) Weeks [No. of 14-Day Old Survivors] 14-Day Pen A [ n] B [ n] C[ n] D[ n] E[ n] F[ n] G[ n] H [ n] I[ n] J[ n] Mean SD Total 217 271 [ 7} 254 [ 6] 266 [ 5] 249 [ 3] 243 [ 3] 257 [ 33 242 [ 2] 243 [ 43 239 [ 3] 241 [ 3] 254 11 39 218 [ 0] 303 [ 3] 347 [ 6] 277 [ 6] 310 [ 5] 303 [ 4] 295 [ 7] 328 [ 4] 316 [ 3] 281 [ 43 306 23 42 219 [ 0] 292 [ 2] 287 [ 4] 263 [ 6] 289 [ 5] 309 [ 4] 292 [ 3] 241 [ 5] 244 [ 63 237 [ 2] 271 25 37 220 [ 0] [ 0] [ 0] [ 0] [ 0] [ 03 [ 0] [ 0] [ 0] [ 0] 221 [ 0] 243 [ 13 286 [ 3] 268 [ 6] [ 0] 281 [ 4] 290 [ 53 300 [ 7] 277 [ 4] 320 [ 2] 285 16 32 222 [ 0] [ 0] 318 [ 4] 244 [ 5] 272 [ 7] 281 [ 6] 261 [ 6] 249 [ 6] 278 [ 5] 275 [ 6] 271 20 45 223 291 [ 8] 282 [ 6] 296 [ 3] 277 [ 2] 300 [ 3] 347 [ 13 301 [ 3] 306 [ 5] 278 [ 3] 324 [ 33 296 15 37 224 270 [ 1] 291 [ 4] 287 [ 5] 267 [ 3] 290 [ 3] 257 [ 3] 299 [ 33 282 [ 7] 256 [ 5] 309 [ 3] 281 16 37 225 [ 0] [ 03 300 [ 4] 259 [ 5] 267 [ 5] 250 [ 53 273 [ 5) 284 [ 5] 293 [ 2] 268 [ 6] 272 15 37 226 [ 0] [ 0] 226 [ 1] 281 [ 1] 265 [ 2] 284 [ 13 255 [ 2] 268 [ 5] 274 [ 3] [ 0] 266 14 15 227 [ 0] 297 [ 2] 309 [ 6] 280 [ 7] 309 [ 6] 291 [ 6] 288 [ 6] 266 [ 7] 279 [ 2] 314 [ 5] 292 16 47 228 [ 0] [ 0] 316 [ 3] 323 [ 4] 334 [ 53 319 [ 5] 304 [ 73 293 [ 3] 313 [ 5] 315 [ 2] 315 12 34 229 [ 0] [ 0] [ 0] [ 0] [ 0] 210 [ 2] [ 0] 260 [ 13 206 [ 2] 242 [ 3] 227 22 8 230 [ 0] [ 0] 266 [ 6] 265 [ 6] 284 [ 63 325 [ 63 317 [ 5] 345 [ 5] 309 [ 53 324 [ 3] 302 28 42 231 [ 0] [ 0] [ 0] 241 [ 1] 296 [ 5] 305 [ 73 303 [ 4] 303 [ 6] 303 [ 63 317 [ 5] 303 12 34 232 [ 0] [ 0] 240 [ 1] 237 [ 3] 311 [ 5] 273 [ 2] 307 [ 5] 313 [ 3] [ 0) 272 [ 2] 289 30 21 Mean 282 SD 23 507 Differences between the control and this treatment group were not significant (p > 0.05). SD = Standard deviation of mean body weight, by parental pen, by week. , GO Project Number 454-109 - 199 - Project Number 454-109 Bird 1 2 3 4 5 6 7 Mean SD Appendix XVIII Offspring Liver Weights (g) from a Mallard Reproduction Study with PFOS Control (0 ppm a.i.) Male Liver 19.845 11.018 12.740 14.534 4.679 Female Liver 18.179 13.219 11.862 14.513 15.982 17.418 13.485 14.951 2.326 Group Mean SD 14.826 2.917 10 ppm a.i. Bird 1 2 3 4 5 6 7 Mean SD Male Liver 11.418 10.819 18.719 13.652 4.398 Female Liver 13.077 13.409 16.095 11.881 11.558 17.125 15.334 14.068 2.143 Group Mean SD 13.944 2.721 The above differences from the control group were not statistically significant (Student's t-test). -200 - W ildlife International, Ltd. Project Number 454-109 Appendix XIX Changes To Study Protocol This study was conducted in accordance with the study protocol signed on January 22, 2001 and the following amendments and deviations: 1. The protocol was amended to add four pairs of birds to each test group to be maintained for collection of blood samples during the test. Additionally, the protocol was amended to add collection of selected tissues for analyses and histopathological examination from all adult birds. When available, fecal samples were also to be collected from the lower digestive tract of test birds. 2. The protocol was amended to reduce the dietary concentration for the 150 ppm a.i. test concentration to 20 ppm a.i. at the beginning of Week 3 of the test. The concentration was reduced based upon treatment-related mortality and signs of toxicity at the 150 ppm a.i. concentration. 3. The protocol was amended to euthanize the 20 ppm a.i. treatment group as soon as practical after the end of Week 4 of the test. Week 4 body weight and observation data indicated birds had not recovered fully following reduction of the dietary concentration from 150 to 20 ppm a.i. Birds were euthanized without collection of samples. 4. The protocol was amended to euthanize the 50 ppm a.i. treatment group as soon as practical after the end of Week 6 of the test. The level was terminated due to treatment-related mortality and signs of toxicity at the 50 ppm a.i. level. Blood, liver, bile and fecal samples, when available, were collected from 10 pairs when the level was terminated. 5. The protocol was amended to add collection of samples from all eggs selected for egg shell thickness measurements. Eggs selected for Lots A, B and J were separated into shell, shell membrane, albumen and yolk and stored frozen for analysis. Eggs selected from Lots C through I will be opened and the yolk was separated and stored frozen for possible analysis. 6. The protocol was amended to add collection of blood, selected tissues and fecal samples, when available, from 10 offspring each from the control group and 10 ppm a.i. treatment group. Blood, tissues and feces were collected for possible analysis. Tissues were also taken for histopathological examination. 7. The protocol was amended to remove the reference substance number (4526) from page 2 of the protocol. The use of an internal standard was deemed unnecessary after refinements to the analytical method were made. 8. The protocol was amended to indicate samples collected for analysis would be shipped to 3M Corporation. Tissues collected for histopathological examination from birds in the 50 and 150 ppm a.i. test concentrations will not be shipped to EPL for examination, but will be disposed of. -201 - W ildlife International, Ltd. Project Number 454-109 9. The protocol was amended to add Attachment A, the analytical phase plan to be used by 3M Environmental Laboratory for analysis of samples. 10. The protocol was amended to update Attachment A, the analytical phase plan to be used by 3M Environmental Laboratory for analysis of samples. 11. The protocol was amended to change Attachment A, the 3M analytical phase plan, to redefine the peak area precision requirement and the method for calculating the calibration curve. 12. The protocol was amended to indicate the main 3M Environmental Laboratory Project Identification Number (LIMS) for the analytical phase plan results is E01-1245. Additional 3M tracking numbers associated with the study are U2723, E01-1342 and E01-1365. Additionally, the 3M Principal Analytical Investigator was changed from Harold Johnson to Lisa A. Stevenson. 13. The protocol was amended to change the Sponsor Representative from Rochelle R. Robideau to Susan A. Beach. 14. The protocol was amended to change the Sponsor Representative from Susan A. Beach to John L. Newsted. 15. Feather samples were collected, when available, from adult birds from which tissue samples were collected. The protocol did not require collection of these samples. Additionally, with the exception of liver samples, tissue samples collected for analysis were not weighed prior to being placed in sample containers. 16. On July 12, 2001, the temperature in brooder level S52-3, housing offspring from Lot J of the control group, was inadvertently not recorded. 17. Dietary samples collected on Day 0 of Week 1 of the test were not stored frozen between January 24 and January 25, 2001. Instead, samples were stored in the analytical chemistry laboratory prior to being processed for analysis on January 25, 2001. The protocol indicated that diet samples would be stored frozen until they were prepared for analysis. 18. Three adult birds in the 50 ppm a.i. test concentration, the hen in pen 243 and the pair in pen 246, were inadvertently euthanized at the beginning of Week 5 of the test during euthanasia of the 150 ppm a.i. test level. -202 - Wildlife International, Ltd. Project Number 454-109 Appendix XX Personnel Involved in Study The following key Wildlife International, Ltd. personnel were involved in the conduct or management of this study: Avian Toxicology (1) Mark Jaber, Wildlife Toxicologist (2) Joann B. Beavers, Director (3) Linda R. Mitchell, Manager (4) Diana Temple, Laboratory Supervisor (5) Sean P. Gallagher, Senior Biologist (6) Larry T. Frey, Senior Biologist (7) Jennie Grimes, Senior Biologist Analytical Chemistry (1) Willard B. Nixon, Ph.D., Director (2) Raymond L. VanHoven, Ph.D., Scientist