Document YD1ox4wwvdxKKGdj1dERkyyDn

/\@-3,3yts f 3 ( p 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 o f 202 O'tiOOS 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 July 5,2001 Candling July 6, 2001 ARDS data entry check August 14 & 15, 2001 Analytical data and draft report October 29, November 2 & 5, 2001 Biological data and draft report December 5, 7, 10-12, 2001 Final report August 20, 2003 DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT January 22, 2001 January 31, 2001 January 23,2001 January 31,2001 February 7, 2001 M ay 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 December 12, 2001 August 20, 2003 November 9, 2001 March 11,2002 August 21, 2003 Robert N. McGee Quality Assurance Representative DATE c'M007 ,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: Sean P. Gallagher ' Senior Biologist, Avian Toxicology CHEMISTRY PRINCIPAL INVESTIGATOR: DATE Ra^crfortd L. V anH oven, Ph.D . Scientist, Chemistry MANAGEMENT: DATE Joann B. Beavers Director, Avian Toxicology DATE Director of Chemistry 001008 W ildlife 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 W ater................................................................................................................. 13 Diet Preparation................................................................................................................ 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 13 Wildlife 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 R eferences............................................................................................................................................ 29 TABLES AND FIGURES 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 PFO S.......................................................................................32 Figure l . Mean Adult Male Body Weight (g) from a Mallard Reproduction Study with PFO S.......................................................................................33 Figure 2. Mean Adult Female Body Weight (g) from a Mallard Reproduction Study with PFOS.......................................................................................34 Table 4. Mean Feed Consumption (g/bird/day) from a Mallard Reproduction Study with PFO S...................................................................................... 35 Figure 3. Mean Feed Consumption (g/bird/day) from a Mallard Reproduction Study with PFOS.......................................................................................36 Table 5. Summary o f 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 PFO S...............................................................................................................38 Table 7. Summary of Reproductive Performance from a Mallard Reproduction Study with PFOS.......................................................................................39 CC1 0 1 0 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 PFO S...................................................................................... 40 Table 8. Summary o f Reproductive Performance, Normalized as Percentages (%), from a Mallard Reproduction Study with PFO S.............................. 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 PFO S...................................................................................... 43 Table 10. Mean Body Weight (g) of Hatchlings 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 o f PFOS in Avian Diet.................................................................. 51 A p p en d ix V . D iag ra m o f T est L a y o u t....................................................................................................... 67 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 PFO S.......................................................... 85 Appendix X. Adult Liver Weights (g) from a Mallard Reproduction Study with PFOS......................................................................... 87 Appendix XI. Histopathology Report......................................................................................... 89 O '? 0 1 1 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 o f 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 PFO S.......................................................176 Appendix XV. Egg Shell Thickness Measurements (mm) per Pen by Week from a Mallard Reproduction Study with PFO S.........................................193 Appendix XVI. Mean Hatchling Body Weight (g) per Pen by Week from a Mallard Reproduction Study with PF O S .........................................195 Appendix XVII. Mean 14-Day Old Survivor Body Weight (g) per Pen by Week from a Mallard Reproduction Study with PFOS.............................. 197 Appendix XV11I. 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 C'iioia W ildlife 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 o f 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. c e io i.a W ildlife 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 o f 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 ildlife International, Ltd. Project N um ber 454-105) 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 Der Pen Males Females 11 11 11 11 C 'liV A 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 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 o f birds at this test concentration. For reporting purposes, the 150/20 ppm a.i. test concentration will be referred to as the 150 ppm a.i. 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 in cu b atio n . W e ek ly , eggs w ere selected by in d iscrim in a te d ra w fo r eg g shell thickness m e a su re m e n t 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. c "1 0 1 Otr 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 Practicefo r Conducting Reproductive Studies with Avian Species" (1,2,3). Test Substance The test substance, PFOS, was received from 3M Corporation on October 29, 1998 and was assigned Wildlife International, Ltd. identification number 4675 upon receipt. The test substance was a white powder and was identified as: FC-95; Lot 217. The test material had a reported purity o f 86.9% and expiration date 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 o f the test substance in the diet were adjusted to 100% active ingredient. Therefore, dietary concentrations are expressed as parts per million active ingredient (ppm a.i.) in the diet. Test Organisms 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 wild type. The birds were from the same hatch, approaching their first breeding season and had not been used in any previous testing. At the start of acclimation, a random number generating function in a spreadsheet program was used to randomize pen assignment for each bird. Immediately prior to test initiation, all potential study 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 Wildlife 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) o f limestone (approximately 38.5% Ca) was added to the basal diet for the adults. This raised the calcium level in the diet for the breeding birds to approximately 3%, slightly above the minimum recommended for quail (2.3%) and mallard (2.75%) (4). Offspring received basal diet without test substance and without the addition of 5% supplemental limestone. Water was supplied by the town of Easton public water supply. Neither the adults nor offspring rec eiv e d an y fo rm o f m ed icatio n in the feed d u rin g the test. F eed an d w ater w ere an aly zed p erio d ically 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 o f 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 o f Week 1. Samples were collected from C0 1 0 1 7 W ildlife International, Ltd. Project Number 454-109 - 14- the top, middle and bottom o f the left and right sections of the mixing vessel. Control and treatment group diet samples were also collected from the feed troughs on Day 7 o f Week I 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 o f the PFOS m eth o d o lo g y . A m eth o d flo w ch art is p ro v id ed in A p p en d ix IV, Figure 1. C o n cen tratio n s o f 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 Cis analytical column (50 mm x 2 mm I.D., 3-pm particle size) fitted with a Keystone Javelin C |8 Guard Cartridge (20 mm x 2 mm I.D.). The instrument parameters are summarized in Appendix IV, Table 1. Calibration standards were prepared in 50:50 methanol: NANOpure water by appropriate dilutions o f a 1.00 mg a.i./L stock solution 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 C^IOIQ 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, Day 0. The fortification levels were 5.00 and 25.0 ppm a.i. for sampling intervals Week 8, Day 0; Week 12, Day 0; Week 16, Day 0; and Week 20, Day 0. The method yielded mean procedural recoveries of 101%, 101%, 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. C9 W ildlife 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 o f 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. C fM 0 2 0 Wildlife 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 o f the Week 4, no necropsies were performed. All other adult birds that died or were euthanized during the C 1(321 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 o f by incineration. At the time o f 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 v v.>a.i 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 o f galvanized steel wire mesh were used to keep hatchlings separated by parental pen o f origin. Eggs were not rotated in the hatcher. The average temperature in the hatching compartment was 37.2C 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 o f age, the average body weight by parental pen o f 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. C01023 W ildlife 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 were separated into albumen and y o lk and the yolk w as stored frozen. All egg sam p les 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 c7 1 0 2 ' 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 o f 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 o f 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 num ber 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-Dav 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-Dav Old Survivors of Eggs Set - The number of 14-day old survivors was divided by the number of eggs set per week, by pen. 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. c 'I 'V >T W ildlife International, Ltd. Proj ect Number 454-109 -22 - 12. 14-Dav Old Survivors o f 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 o f 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). W ildlife 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 o f 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. c 0 1 .0 W ildlife 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 o f 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. C0 1 0 2 8 W ildlife 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 postreproductive 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 nG29 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 th ro u g h 15 lo cated in A p p en d ix XIIB. T h e full rep o rt for an aly sis of sera and liv e r 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. c ^ 'io a o 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 were incidental observations of neck curl, weakened condition and foot/leg lesions and associated lameness, for offspring in both the control group and 10 ppm a.i. treatment group. 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 01 W ildlife International, Ltd. Project Number 454-109 -28- collected at the time o fjuvenile 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 o f 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. C #Ti032 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 fo r Conducting Reproductive Studies with Avian Species. ASTM Standard E1062-86. 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 fo r 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 20:482491. 8. Wildlife International, Ltd.. 1994. Avian Reproduction Data System (ARDS) Version 2. Cl033 o Nominal Concentration (ppm a.i.) Table 1 lean Measured Concentrations (ppm a.i.) of PFOS inAvian Diet from a Mallard Reproduction Study W kl Day 0 Day 7 1 Week 2 Day 0 Week 3 Day 0 Interval Week 4 Day 0 Week 8 DayO Week 12 Week 16 Week 20 Day 0 Day 0 DayO 0 Measured <2.00 : <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 <2.00 Mean 10 Measured % Nominal 98 100 4 103 101 97 109 101 101 99 Mean 50 Measured % Nominal 100 101 4 97 104 104 Mean 150 Measured % Nominal 98 97 4 108 96 5 112 5 - - - - 1Feed samples were coll ed from feed troughs at the end of Week 1 to assess stability of the test substance under actual test conditions, 2<LOQ indicates the val was less than the limit of quantitation, 3Die mean of homogene samples collected at Week 1, Day 0. 4The percent of the meai allies for Week 1, Day 0. 5The 150 ppm a.i. treate liet was reduced to 20 ppm a.i. during this interval, - No values available, te rroup terminated. o Project Number 454-109 -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 (Oppm a.i.) Males 50 ' 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 A d u lt Body We ght (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 Heek 0 Chan Heek 0 Week 2 Change Heek 4 Change Week 6 Change Heek 8 Change Test Total Heek 2-4 Heek 4-6 Heek 6-8 Heek 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 to i The means for body weights and body weight Tanges are calculated and rounded separately. - Data are not available due to adult mortality * * Significantly different from the control at [ : 0.01 1 Only surviving birds were included in the cal iations for each body weight interval. Project Number 454-109 -> C5 ep oduc tic 50 MALE BODY UEI6HT (g) n oS999?5 --- I------- 1--------1--------1......""I--------1--------1--------1--------1--------1 \ liiiniiiniiniiiiiiiimiiiiHiiiiiiHiiHiinHiimiiiiiiniin S. O S 7f if _D g- k> * i n u m i l i + c I.r..............................................i Dn HAi in 3 P Y KLitoB InniiiiiiiiiiiiiiiiiiiiiiiiuiiuiiiiiiiDiiiiiiiHimiimiiii zz m * 2 .rJI ck J_ rn CD .C no >u C3 -- C fiiiiiiiiiiiirr i 5? A I sm 02020101000148480102005323000101010000 I* I 6U 8E O T G O n n o n3 ri do a D * m <D S x: jr ? 59 S * 71 1* - rn 1 V.1 vC xz _k n &y o> <k* s <A * * EO n 2XT FEMALE BODY UEIBHT (g) QQQBQQOoeS? 5 Iiiiiiiiiiiiiiiiii mu iiiiiiiiiiMiiiiiiiiiiiiiiiiiimill i1 1i "OjJ hi 1 5 .......... . L wwwwwmwwmwM Q i I g.51 1_ 1 3 1 in <D 1 cl 2 mmiiiimimmiiiimimiiiiimiimiiimmiiimii ..........ih...... ............... . J1 t___________________________ _ 1. .......................................... IT r i~ i * - 1 1C 1^ ,, |1 -n>1_ C4 1 -- ip 11 n2 1 41 1 11 1 liimH 1 11 iiiiiiiiiiiiiiiiiimiiiiiiiimiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii! vv a r -K - o n (g/ 5e .24 133 .37 145 .16 124 :ality at p < O.C il at p < 0 - o u <3 i040 .r*yi G f (K 2 Table 7 Summary of R e p r o d u c t iv e 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 ^Based on 7 2 days ot eggs production. - Data are not available due to adult mortality Project Number 454-109 n> Tn~j m u L. rr n -B O2 L. 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B, .............. ....... .... ro . 1 rji m n a y Il II r ~ o JD TJ wg hiiniViiiiiiiiiiriiiiiiiiiMiViiititrmiim c -*1 ^ PI 1 iu 1 Il /7n3 1 'U Ii 3 0i"i *T-n CL -* hiiiNirriiintfflititiirti iiiiiit^viiiViiiiiiiriiH SSI ep od :t: n tu | i ih PF nti to '' 5 5 yiniihiiiiiT'iTiiririimin iiiiriiiii ir ^ iih n iiri triii ^ 3 UJ 101 p t Wa imnmi niiiimiiiiiiimunniiiinimmimmi 601-fP i9quin|v[ p a to jj Tabi 9 Mean Eggshell Thickness Measurements (mm) from a Mallard Reproduction Study with PFOS Experimental Group (ppm a.i.) Control 10 50 150 No. 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). ( j <ryi 0 3 - 9 C01050 C H IO S I C riiO S 2 f>1053  '"1 0 5 4 C'lLO55 O * ' 0 5 6 0 'M 0 5 7 0^1058  n '* 0 ^ 9 f o - O f : o I oj :t il b< 4 4- )S . IEMD3 : V ' be5 ' re\ i c il o i )i I F } > 3 n e n :r it o i i . 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