Document gbY3vO3wKMok0399mKg7B1YVV

PFOS: A DIETARY LC50 STUDY WITH THE MALLARD WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-102 3M REQUEST NO. U2723 U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines (draft) OPPTS Number 850.2200 FIFRA Subdivision E, Section 71-2 OECD Guideline 205 AUTHORS: Sean P. Gallagher Courtney S. Casey Joann B. Beavers Raymond L. Van Hoven STUDY INITIATION: April 21, 1999 STUDY COMPLETION: April 26, 2000 AMENDED REPORT DATE: March 23, 2004 SUBMITTED TO 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 72 AMENDED W ild lif e In ter n atio n al, ltd 2- - PROJECT NO.: 454-102 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Dietary LC50 Study with the Mallard WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-102 STUDY COMPLETION: April 26, 2000 AMENDED REPORT DATE: March 23, 2004 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR Part 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice, (OCDE/GD(92) 32, Environment Monograph No. 45, Paris, 1992); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984 with the following exception: At the time the test was conducted the test substance was not characterized in accordance with full GLP compliance; however, the characterization was performed according to 3M Standard Operating Procedures and Methods, and all raw data are being maintained in the 3M archives. The test substance was recharacterized in accordance with GLP standards following study completion. The stability of the test substance and reference standard under conditions of storage at the test site was not determined in accordance with Good Laboratory Practice Standards. Analyses of liver and sera samples conducted by 3M Corporation were not performed in accordance with GLP standards and results of these analyses are being reported separately. STUDY DIRECTOR: -A P _________________ DATE Sean P. Gallagher ' Senior Biologist, Avian Toxicology ________ SPONSOR'S REPRESENTATIVE DATE 3 J o lty /& j AMENDED W ild lif e In ter n atio n al, ltd -3 - PROJECT NO.: 454-102 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 & 792, 17 August 1989; OECD Principles of Good Laboratory Practice, (OCDE/GD(92) 32, Environment Monograph No. 45, Paris, 1992); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all audits and inspections and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY DATE CONDUCTED DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT Matrix Fortification, Test Substance Prep., Analytical Sampling April 22, 1999 April 22, 1999 April 23, 1999 Feed Consumption and Analytical Sampling April 27, 1999 April 27, 1999 May 4, 1999 Necropsy, Blood Collection, Liver Weights and Bile Collection May 14, 1999 May 14, 1999 May 21, 1999 Analytical Data and Draft Report July 7, 8, and 9, 1999 July 9, 1999 July 16, 1999 Biological Data and Draft Report Aug. 30, 31, Sept. 1, 1999 September 1, 1999 September 3, 1999 Final Report April 18, 2000 April 18, 2000 April 19, 2000 Amended Final Report March 19, 2004 March 19, 2004 March 19, 2004 S u h f t n r ^ . Q d U j m a n __________ date Susan L. Coleman Senior Quality Assurance Representative 3-(2<3-04 AMENDED W ild lif e In ter n atio n al, ltd -4- PROJECT NO.: 454-102 AMENDED REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Dietary LC50 Study with the Mallard WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-102 3M LAB REQUEST NO.: U2723 STUDY DIRECTOR: Senior Biologist, Avian Toxicology CHEMISTRY PRINCIPAL INVESTIGATOR: Raymona L. Van Hoven, Ph.D Scientist, Analytical Chemistry REPORT APPROVED BY: YyjzEtaJ Linda R. Mitchell Manager of Ecotox Operations 3 03-4 Date Z3 Date Director of Chemistry AMENDED W ild lif e In ter n atio n al, ltd -5 - PROJECT NO.: 454-102 TABLE OF CONTENTS TITLE PAGE........................................................................................................................ Page 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT...................................Page2 QUALITY ASSURANCE STATEMENT........................................................................... Page 3 REPORT APPROVAL............................................................................................................. Page4 TABLE OF CONTENTS...................................................................................................... Page 5 Tables and Appendices........................................................................................................... Page6 SUMMARY.............................................................................................................................. Page7 INTRODUCTION.................................................................................................................... Page8 OBJECTIVE............................................................................................................................. Page8 MATERIALS AND METHODS.............................................................................................. Page8 Test Substance........................................................................................................................ Page8 Treatment Groups................................................................................................................... Page9 Duration of the Test................................................................................................................ Page9 Test Birds................................................................................................................................ Page9 Animal D iet........................................................................................................................ Page 10 Diet Preparation.................................................................................................................. Page 10 Diet Sampling.......................................................................................................................... Page10 Analytical Method................................................................................................................... Page10 Housing and Environmental Conditions............................................................................. Page 13 Observations............................................................................................................................ Page13 Animal Body Weights/Feed Consumption.............................................................................. Page14 Gross Necropsy........................................................................................................................ Page14 Statistical Analyses.................................................................................................................. Page14 RESULTS.................................................................................................................................. Page14 Diet Analysis........................................................................................................................... Page14 Mortalities and Clinical Observations................................................................................ Page 15 Body Weight and Feed Consumption...................................................................................... Page16 Gross Necropsy........................................................................................................................ Page17 CONCLUSION.......................................................................................................................... Page18 REFERENCES.......................................................................................................................... Page19 W ild lif e In ter n atio n al, ltd 6- - PROJECT NO.: 454-102 TABLE OF CONTENTS - continued - TABLES AND APPENDICES TABLE 1: TABLE 2: TABLE 3: TABLE 4: TABLE 5: Cumulative Mortality from a Mallard Acute Dietary Toxicity Study with PFOS.........................................................................................Page 20 Mean Body Weight (g) from a Mallard Acute Dietary Toxicity Study with PFOS......................................................................................... Page 21 Mean Feed Consumption (g/bird/day) from a Mallard Acute Dietary Toxicity Study with PFOS................................................... Page 23 Gross Pathological Observations from a Mallard Acute Dietary Toxicity Study with PFOS............................................................. Page 25 Cumulative Mortality (Estimated Cumulative Dose, mg/kg) from a Mallard Acute Dietary Toxicity Study with PFOS..................................... Page 26 APPENDIX I: Certificate of Analysis................................................................................. Page27 APPENDIX II: Diet Formulation.......................................................................................... Page28 APPENDIX III: Analytical Methods and Results..................................................................Page29 APPENDIX IV: Diet Preparation...........................................................................................Page43 APPENDIX V: Cumulative Mortality by Pen from a Mallard Acute Dietary Toxicity Study with PFOS............................................ Page 44 APPENDIX VI: Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS............................................ Page 45 APPENDIX VII: Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS.................................... Page60 APPENDIX VIII: Changes to Protocol.....................................................................................Page64 APPENDIX IX: Personnel Involved in the Study..................................................................Page65 APPENDIX X: Revised Study Summary..............................................................................Page66 APPENDIX XI: Report Amendment.................................................................................Page 71 AMENDED W ild lif e In ter n atio n al, ltd -7- PROJECT NO.: 454-102 SUMMARY SPONSOR: 3M Corporation TEST SUBSTANCE: PFOS WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-102 STUDY: PFOS: A Dietary LC50 Study with the Mallard RESULTS1: The dietary LC50 value for Mallards exposed to PFOS was determined to be 628 ppm a.i. with a 95% confidence interval of 448 ppm a.i. to 958 ppm a.i. The slope of the concentration-response curve was 3.67 and the chisquare value was 2.13. The no mortality concentration was 146 ppm a.i. Based upon reductions in body weight gain at the 73.2 ppm a.i. test concentration, the no-observed-effect concentration was 36.6 ppm a.i. TEST DATES: Hatch - April 13, 1999 Acclimation - April 14-22, 1999 Experimental Start-April 22, 1999 Experimental Termination - May 14, 1999 NOMINAL TEST CONCENTRATIONS: 0, 9.1, 18.3, 36.6, 73.2, 146, 293, 586, and 1171 ppm a.i. TEST ANIMALS: Mallard (Anasplatyrhynchos) AGE TEST ANIMALS: 10 days of age at test initiation SOURCE TEST ANIMALS: Whistling Wings, Inc. PO Box 1-A 113 Washington St. Hanover, IL 61041-3512 STUDY COMPLETION: April 26, 2000 AMENDED REPORT DATE: March 23, 2004 1See Appendix X for the revised nominal test concentrations and results based upon the reanalysis o f the test material following study completion. AMENDED W ild lif e In ter n atio n al, ltd 8- - PROJECT NO.: 454-102 INTRODUCTION This study was conducted by Wildlife International Ltd. for 3M Corporation at the Wildlife International Ltd. toxicology facility in Easton, Maryland. The in-life portion of the test was conducted from April 22, 1999 to May 14, 1999. Raw data generated at Wildlife International Ltd. and a copy of the final report are filed under Project Number 454-102 in archives located on the Wildlife International Ltd. site. OBJECTIVE The objective of this study was to evaluate the toxicity of a test substance to the mallard (Anas platyrhynchos) administered through the diet for five days. An LC50 value will be calculated, if possible. MATERIALS AND METHODS The methods used in conducting this study are based upon procedures specified in the U.S. Environmental Protection Agency Series 850 Ecological Effects Test Guidelines OPPTS Number 850.2200 (1) Section 71-2 of the Environmental Protection Agency's Registration Guidelines, Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms (2); OECD Guideline 205, Guideline for Testing of Chemicals, Avian Dietary Toxicity Test (3); and upon ASTM Standard E857-87, "Standard Practice for Conducting Subacute Dietary Toxicity Tests with Avian Species" (4). Test Substance The test substance was received from 3M Corporation on October 29, 1998 and was assigned Wildlife International Ltd. Identification Number 4675 upon receipt. The test substance was white powder identified as: FC-95; Lot No.:217. The reported purity of the test substance was 98.9%, with an expiration date of 2008. Following test termination, the test material was reanalyzed. The results of reanalysis indicate a test substance purity of 90.49%. All test concentrations have been adjusted to reflect the purity reported on the new Certificate of Analysis (Appendix I)1. The test substance was stored under ambient conditions. The internal standard was received from 3M Corporation on July 2, 1998 and was assigned Wildlife International Ltd. identification number 4526 upon receipt. The internal standard, a granular 1Following study completion, the test material was recharacterized in accordance with GLP standards and the results o f reanalysis indicate a purity o f 86.9%. While test concentrations have not been adjusted to reflect the newest purity, the revised study summary in Appendix X summarizes the effect o f the change in purity. AMENDED W ild lif e In ter n atio n al, ltd -9 - PROJECT NO.: 454-102 material, was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2. The standard was stored under ambient conditions Treatment Groups The test consisted of a geometric series of eight test concentrations and a control group. Thirty mallard ducklings were assigned to the control group and ten mallard ducklings were assigned to each of the treatment groups. Birds were sorted by weight, then chosen indiscriminately from within each represented weight class for placement into control and treatment groups. The birds were housed in brooding pens containing five ducklings each. On Day 0, following experimental start, the Sponsor requested the addition of a 10 ppm test concentration. Birds from the same lot that remained after test initiation were used for the additional test concentration. Nominal dietary concentrations used in this study were 0, 9.1, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 parts per million active ingredient (ppm a.i.). The dietary concentrations were established based upon known toxicity data and information supplied by the Sponsor. Each group was fed the appropriate test or control diet for five days. During the exposure period the control group received untreated feed. Following the five-day exposure period all groups were given untreated basal diet for three days. On Day 8, half of the treatment and control birds were euthanized and liver weight and tissue, blood, and bile samples collected for analysis. The remaining birds were fed basal ration until Day 22. On Day 22, these birds were sacrificed and also sampled for liver weight and tissue, blood, and bile. Duration of the Test The primary phases of this test and their durations were: 1. Acclimation - 9 days. 2. Exposure - 5 days. 3. Post-exposure observation - 3 or 17 days Test Birds All mallard (Anas platyrhynchos ) were 10 days of age and appeared to be in good health at initiation of the test. The birds were obtained from Whistling Wings Inc., Hanover, IL. and were hatched on April 13, 1999. Birds ranged in weight from 96 to 204 grams at test initiation. The birds used in this study were immature and could not be differentiated by sex. All birds were from the W il d l if e In te r n a tio n a l, ltd 10- - PROJECT NO.: 454-102 same hatch, pen-reared and phenotypically indistinguishable from wild birds. All birds were acclimated to the caging and facilities from the day after hatch until initiation of the test. Animal Diet Throughout acclimation and testing all test birds were fed a game bird ration formulated to Wildlife International Ltd.'s specifications (Appendix II). Water from the town of Easton public water supply, and feed were provided ad libitum during acclimation and testing. The birds received no form of antibiotic medication during acclimation or testing. Diet Preparation The test substance was mixed directly into the ration. Mixing was done with a Hobart mixer (Model Number AS200T). All dietary test concentrations were adjusted to 100% PFOS based upon the reported purity of the test substance. All dietary concentrations and the LC50 value are reported as ppm a.i. in the diet. Nominal dietary test concentrations used in this study were 9.1, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 ppm a.i. (Appendix IV). Diet Sampling Samples of the test diets were collected to verify the test concentrations administered and to confirm the stability and homogeneity of the test substance in the diets. Homogeneity of the test substance in the diet was evaluated by collecting six samples from the 9.1 ppm a.i. a.i. and six samples from the 1171 ppm a.i. a.i. test diets at preparation on Day 0. Homogeneity samples were collected from the top, middle and bottom of the left and right sections of the mixing vessel. The homogeneity samples also served as verification samples. One verification sample was collected from the control diet and two verification samples were collected from each remaining treatment group at preparation on Day 0. At the end of the exposure period (Day 5), one sample was collected from the control and two samples were collected from each treatment group to determine stability of the test substance in the diet. The stability samples were collected from feed remaining in the feeders after being at ambient test pen conditions for five days. Samples were transferred immediately to Wildlife International Ltd. analytical chemistry. Analytical Method The method used for the analysis of the avian diet samples was based upon methodology developed at Wildlife International Ltd. and entitled "Method Outline for the Determination of PFOS W ild lif e In ter n atio n al, ltd -11 - PROJECT NO.: 454-102 in Avian Feed". Avian diet samples were extracted with methanol. Methanol was added to a requisite quantity of feed contained in a French-square glass bottle. Bottles were capped and shaken on a shaker table. Samples were vacuum filtered using qualitative filter paper. The retained feed was rinsed three times with methanol into the filtrate. The filtrate was transferred to a volumetric flask and brought to volume with methanol. As appropriate, samples were further diluted with methanol. Each sample then was diluted with a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v) so they fell within the calibration range of the PFOS methodology. A method flowchart is provided in Appendix III, Figure 1. Concentrations of PFOS in the standards and samples were determined by reversed-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a PerkinElmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil C)8 analytical column (100 mm x 2 mm I.D., 3 pin particle size). The instrument parameters are summarized in Appendix III, Table 1. Calibration standards of PFOS prepared in a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v), ranging in concentration from 0.00229 to 0.0457 mg a.i./L were analyzed with the samples. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOS on the basis of individual isomeric components. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS : internal standard) of the calibration standards. A typical calibration curve is presented in Appendix III, Figure 2. The concentration of PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms of low and high calibration standards are presented in Appendix III, Figures 3 and 4, respectively. The method limit of quantitation (LOQ) for these analyses was set at 1.15 ppm a.i. calculated as the product of the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor of the matrix blank samples (500). W ild lif e In ter n atio n al, ltd 12- - PROJECT NO.: 454-102 Two matrix blank samples were analyzed to determine possible interferences. No interferences were observed at or above the LOQ during sample analyses (Appendix III, Table 2). An interference in the feed appeared at approximately the same retention time as the peak of interest but it was well below the LOQ. A representative chromatogram of a matrix blank is presented in Appendix III, Figure 5. Avian diet was fortified at 4.57, 183 and 1830 ppm a.i. and analyzed concurrently with the samples to determine the mean procedural recovery (Appendix III, Table 3). Sample concentrations were not corrected for the mean procedural recovery of 94.7%. A representative chromatogram of a matrix fortification is presented in Appendix III, Figure 6. An example calculation is presented for sample number 454-102-2, nominal concentration of 18.3 ppm a.i. in avian diet. Initial Weight: 10.0 g Final Volume: 200 mL Dilution Factor: 100 (intermediate dilution factor x final dilution factor) PFOS Peak Area: 113568 Internal Standard Peak Area: 413160 Peak Area Ratio: 0.2749 Calibration curve equation. Slope: 2.77397 Intercept: 0.01894 Curve is weighted (1/x). PFOS (mg a.i./L) at instrument (Peak area ratio - (Y-intercept)) x I.S. Concentration Slope (0.2749 - 0.01894) x 0.100 mg/L 2.77397 = 0.00923 mg a.i./L Note: I.S. = internal standard. W ild lif e In ter n atio n al, ltd - 13 - PROJECT NO.: 454-102 PFOS (ppm a.i.) in sample PFOS (mg a.i./L) at instrument x Final Volume (L) x Dilution Factor Initial Weight (Kg) 0.00923 x 0.200 x 100 0.01 = 18.5 ppm a.i. Percent o f Nominal Concentration PFOS (ppm a.i.) in sample = ----------- ----------------------------- x 100 PFOS (ppm a.i.) nominal 18.5 ----- x 100= 101% 18.3 Housing and Environmental Conditions During acclimation and testing, all birds were housed indoors in batteries of thermostatically controlled brooding pens manufactured by Safeguard Products, Inc. Each pen had floor space that measured approximately 62 X 90 cm. Ceiling height was approximately 25.5 cm. External walls, ceilings and floors were constructed of galvanized steel wire and sheeting. Birds were sorted by weight, then chosen indiscriminately from within each represented weight class for assignment to pens. Each group of birds was identified by pen number and test concentration. Individual birds were identified by wing bands. During the test the average temperature in the brooding compartment of the pens was 38C 2C (SD). Average ambient room temperature for this study was 25.2C 0.7C (SD) with an average relative humidity of 53% 18% (SD). The photoperiod (maintained by a time clock) was sixteen hours of light per day during acclimation and throughout the test. The light source was fluorescent lights which closely approximate noon-day sunlight. The birds were exposed to an average of approximately 207 lux of illumination. Housing and husbandry practices were based on guidelines established by the National Research Council (5). Observations During acclimation all birds were observed daily. Birds exhibiting abnormal behavior or physical injury were not used. Following test initiation and continuing until termination, all birds were W ild lif e In ter n atio n al, ltd - 14- PROJECT NO.: 454-102 observed at least once daily. A record was maintained of all mortality, signs of toxicity, and abnormal behavior. Animal Body Weights/Feed Consumption Individual body weights were measured at the initiation of the test, on Day 5, on Day 8 for all birds, and on Days 15 and Day 22 for all remaining birds. Average feed consumption values during the exposure period (Days 0-5) and the post-exposure observation period (Days 6-8) were determined by pen for each treatment group and the control group. Additionally, feed consumption was determined for Days 8-15 and 15-22 for the remaining half of the treatment and control birds. Feed consumption was determined by measuring the change in the weight of the feed presented to the birds over a given period of time. The accuracy of feed consumption values may have been affected by the unavoidable wastage of feed by the birds. Gross Necropsy All test birds that died during the course of the test and all birds remaining at the termination were subjected to a gross necropsy. Additionally, liver weight and liver tissue, serum and blood solids, and bile were collected from birds euthanized on Day 8 and 22, and when possible from those that died during the course of the study. Statistical Analyses Mortality data were analyzed using the computer program of C.E. Stephan (6). The program was designed to calculate the LC50 value and the 95% confidence interval by probit analysis, moving average method or the binomial probability method (7, 8, 9). In this study the LC50 value was determined by the probit method. The slope of the concentration-response curve and results of the goodness of fit test are reported. Body weight data were compared by Dunnett's test using TOXSTAT software (10,11). No statistical analyses were applied to feed consumption data. RESULTS Diet Analysis Avian diet samples were collected from the 9.1, 18.3 and 1171 ppm a.i. test concentrations and analyzed to evaluate homogeneity of the test substance in the avian diet. The analysis of these samples also served as verification of test substance concentrations. Resulting mean measured W ild lif e In ter n atio n al, ltd - 15 - PROJECT NO.: 454-102 concentrations, standard deviations and coefficients of variation (CV) for these test concentrations were 9.8 0.969 ppm a.i. (CV = 9.94%), 19.5 2.13 ppm a.i. (CV = 10.9%) and 1196 70.2 ppm a.i. (CV = 5.87%), respectively (Appendix III, Table 4). Control avian diet samples collected during the test showed no interferences above the LOQ. Samples collected during the test to verify the 36.6, 73.2, 146, 293 and 586 ppm a.i. test substance concentrations had mean measured concentrations of 40.2, 74.5, 174, 291 and 537 ppm a.i., respectively. These values represented 110, 102, 119, 99.3 and 91.6% of the nominal concentrations, respectively (Appendix III, Table 5). Analysis of avian diet samples collected from feeders after being held at ambient temperature for five days averaged 119, 97.4, 130, 104, 94.3, 101, 101 and 95.0% of the Day 0 values for the 9.1, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 ppm a.i. test substance concentrations, respectively (Appendix III, Table). A representative chromatogram of a test sample is shown in Appendix III, Figure 7. Mortalities and Clinical Observations No mortalities occurred in the control group, and all birds were normal in appearance and behavior throughout the test. There were no treatment-related mortalities or overt signs of toxicity in the 9.1, 18.3, 36.6, 73.2, or 146 ppm a.i. treatment groups (Table 1 and Appendix V). However, there was 20% (2 of 10) mortality in the 293 ppm a.i. treatment group, 30% (3 of 10) mortality in the 586 ppm a.i. treatment group and 90% (9 of 10) in the 1171 ppm a.i. treatment group. In the 293 ppm a.i. treatment group single mortalities were noted on the mornings o f D ay 7 and Day 8. Signs of toxicity were first observed on the morning of Day 4 and continued until termination for those birds sacrificed on Day 8 and until the afternoon of Day 8 for those birds sacrificed on Day 22. All birds in this treatment level displayed signs of toxicity. These included reduced reaction to stimuli (sound and motion), loss of coordination, ruffled appearance, lethargy, and lower limb weakness. All remaining birds appeared to have recovered and were normal in appearance and behavior from the morning of Day 9 until test termination. At the 586 ppm a.i. treatment level there were three treatment-related mortalities, one each noted on the morning of Day 5, Day 6, and Day 7. Signs of toxicity were first observed on the morning of Day 3 and continued until termination for those birds sacrificed on Day 8 and until the afternoon of Day 8 for those birds sacrificed on Day 22. All birds in this treatment level displayed signs of toxicity. These included reduced reaction to stimuli (sound and motion), ruffled appearance, lower W ild lif e In ter n atio n al, ltd - 16 - PROJECT NO.: 454-102 limb weakness, and lethargy. All remaining birds appeared to have recovered and were normal in appearance and behavior from the morning of Day 9 until test termination. In the 1171 ppm a.i. treatment group there were nine treatment-related mortalities. The first two mortalities were found on the morning of Day 4, followed by four mortalities on Day 5, two mortalities on Day 6 and the final mortality on Day 7. Overt signs of toxicity were first observed on the morning of Day 2 and the single surviving bird continued to display signs of toxicity through the afternoon of Day 8. Signs of toxicity observed among birds in the 1171 ppm a.i. treatment group included reduced reaction to stimuli (sound and motion), loss of coordination, prostrate posture, depression, convulsions, ruffled appearance, lower limb weakness, and lethargy. The one surviving bird appeared to have recovered and was normal in appearance and behavior from the morning of Day 9 until test termination. Body Weight and Feed Consumption When compared to the control group, there were no apparent treatment related effects on body weight among birds in the 9.1, 18.3, or 36.6 ppm a.i. treatment groups. There was a statistically significant (p<0.05) reduction in weight gain at the 9.1 ppm a.i. level for the Day 0-5 period that was reflected in significantly lower (p< 0.05) mean Day 5 body weight. When compared to the control group, reductions in weight gain were also statistically significant (p<0.05) for the Day 5-8 period, resulting in a significantly (p<0.05) lower Day 8 mean body weight. However, differences from the control group at the 9.1 ppm a.i. level appear to be due to a lower mean Day 0 body weight for the 9.1 ppm a.i. level and were not dose responsive. Therefore, these differences were not considered treatment related. There was a marked, treatment-related, concentration-responsive effect on body weight in the 73.2, 146, 293, 586, and 1171 ppm a.i. treatment groups during the exposure period (Days 0-5) (Table 2 and Appendix VI). These reductions were statistically significant at p<0.05 for the 73.2 ppm a.i. level and at p<0.01 at the 146, 293, 586 and 1171 ppm a.i. levels. During the Day 5-8 post-exposure period, weight gain continued to be reduced for surviving birds in the 293, 586 and 1171 ppm a.i. treatment groups. These reductions were statistically significant at p<0.01 for the 293 and 586 ppm a.i. levels. Differences at the 1171 ppm a.i. level were marked but could not be statistically compared. Body weight losses and reductions in weight gain at the 293, 586 and 1171 ppm a.i. levels were reflected in significantly (p<0.05 or p<0.01) lower mean body weights on Days 5, 8, 15, and 22 W ild lif e In ter n atio n al, ltd - 17 - PROJECT NO.: 454-102 of the test. At the 146 ppm a.i. level mean body weights on Days 8 and 22 were also significantly (p<0.05) lower than the control group. During the Day 8-15 and Day 15-22 post-exposure periods, body weight gain appeared comparable among all groups. When compared to the control group, there was a marked reduction in feed consumption in the 293, 586, and 1171 ppm a.i. treatment groups during the exposure period (Days 0-5). A reduction in feed consumption continued to be observed through Day 15 in the 293, 586 and 1171 ppm a.i. treatment groups. Gross Necropsy During the course of the test, all birds that died were subjected to a gross necropsy. Necropsy results for birds found dead were similar. Common observations included thin condition, loss of muscle mass, altered spleen color, empty crops, and empty gastrointestinal tracts. Findings were considered to be treatment related. Details of the necropsy findings are presented in Table 4. Half of the surviving birds were subjected to gross necropsy on Day 8 and the remaining birds on Day 22, following test termination. The necropsy results for birds euthanized on Day 8 and Day 22 from the 18.3, 146 and 1171 ppm a.i. treatment groups were unremarkable. One bird in the control group and one bird in the 36.6 ppm a.i. treatment group were noted with retained yolk sacs during necropsy of birds on Day 8. A single bird in the 9.1 ppm a.i. treatment group necropsied on Day 8 was noted as small, with a lack of muscle mass. In the 73.2 ppm a.i. treatment group one bird necropsied on Day 8 was small, with a lack of muscle mass, and had a 2 x 0.5 cm abscess containing caceous necrotic material on the right side of the abdominal wall. Due to the nature of the lesions observed in birds, and the isolated incidence of occurrence, the findings listed above in euthanized birds from the 9.1, 36.6, and 73.2 ppm a.i. treatment groups were not considered to be related to treatment. In addition, all remaining birds in the control, 9.1, 36.6, and 73.2 ppm a.i. treatment groups were non remarkable. In the 293 ppm a.i. treatment group, one bird euthanized on Day 8 was noted with an incidental finding of a retained yolk sac. A second bird in the 293 ppm a.i. group, necropsied on Day 8, was noted as emaciated with a lack of muscle mass. This loss of muscle mass was consistent with reduced weight gain for the level and was considered treatment related. Another bird in the 293 ppm a.i. treatment group, euthanized on Day 22, was noted with 2 cm cysts containing yellow fluid on the W ild lif e In ter n atio n al, ltd - 18 - PROJECT NO.: 454-102 anterior portion of the left lobe of the liver. Due to the nature of the cysts and the isolated incidence of occurrence, this observation was not considered treatment related. Three birds in the 586 ppm a.i. treatment level were euthanized on Day 8. At necropsy, they were noted to be thin and lacking muscle mass. One bird was noted with a slightly pale liver. These findings are considered to be treatment related. Four birds in the 586 ppm a.i. treatment level survived until Day 22. Necropsy results for these four birds were not remarkable. CONCLUSION The dietary LC50 value for Mallards exposed to PFOS was determined to be 628 ppm a.i. with a 95% confidence interval of 448 ppm a.i. to 958 ppm a.i.. The slope of the concentration-response curve was 3.67 and the chi-square value was 2.13. The no mortality concentration was 146 ppm a.i.. Based upon reductions in body weight gain at the 73.2 ppm a.i. test concentration, the no-observedeffect concentration was 36.6 ppm a.i.. W ild lif e In ter n atio n al, ltd - 19- PROJECT NO.: 454-102 REFERENCES 1 U. S. Environmental Protection Agency, 1996. Series 850- Ecological Effects Test Guidlelines {draft), OPPTS Number 850.2200: Avian Dietary Toxicity Test. 2 U.S. Environmental Protection Agency. 1982. Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, subsection 71-2, Environmental Protection Agency, Office of Pesticide Programs. Washington, D.C. 3 Organization for Economic Cooperation and Development. 1984. Avian Dietary Toxicity Test. OECD Guideline for Testing of Chemicals. Guideline 205. Paris. 4 American Society for Testing and Materials. 1987. Standard Practice for Conducting Subacute Dietary Toxicity Tests with Avian Species. ASTM Standard E857-87. Annual Book of ASTM Standards, Vol. 11.04. Philadelphia, PA. 5 National Research Council. 1996. Guide for the Care and Use o f Laboratory Animals. Washington, D.C. National Academy Press. 125 pp. 6 Stephan, C.E. 1977. Methods for Calculating an LC50. Pages 65-84 In Aquatic Toxicology and Hazard Evaluations, American Society for Testing and Materials. Pub. No. STP 634. Philadelphia, PA. 7 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, MN. Personal Communication. 8 Finney, D.J. 1971. Statistical Methods in Biological Assay, 2nd ed., Griffin Press, London. 9 Thompson, W.R. 1947. Bacteriological Reviews, Vol 2, No.2: 115-145. 10. Dunnet, C.W. 1955. A Multiple Comparison's Procedure for Comparing Several Treatments with a Control. Jour. Amer. Statis. Assoc. 50:1096-1121.1 11. Gulley, D.D. 1990. TOXSTAT Release 3.2. The University of Wyoming. W ild lif e In ter n atio n al, ltd -20- PROJECT NO.: 454-102 TABLE 1 Cumulative Mortality from a Mallard Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) No. Dead Per No. Exposed Exposure Period Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 No. Dead Per No. Exposed Post-Exposure Period1 Day 6 Day 7 Day 8 Control 0 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 Treatment 9.1 18.3 36.6 73.2 146 293 586 1171 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 1/10 0/10 0/10 0/10 0/10 2/10 6/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 1/10 2/10 2/10 3/10 3/10 8/10 9/10 9/10 The LC50 value was calculated to be 628 ppm a.i. with a 95% confidence interval of 448 ppm a.i. to 958 ppm a.i. 1- No mortalities occurred in any of the treatment levels from Day 8 to Day 22.____________________ W ild lif e In ter n atio n al, ltd -21 - PROJECT NO.: 454-102 TABLE 2 Page 1 Mean Body Weight (g) from a Mallard Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) Exposure Period Change1 Day 0 Day 5 Day 0-5 Post-Expo;sure Period Change1 Day 8 Day 5-8 Control 0 Mean SD 135 279 24 42 144 22 380 101 53 16 Treatment 9.1 Mean SD 119 226** 108** 14 36 23 317* 66 91 33 18.3 Mean SD 146 277 14 26 131 18 377 100 32 11 36.6 Mean SD 147 275 16 32 128 22 375 100 37 12 73.2 Mean SD 143 260 30 55 117* 33 343 82 90 42 146 Mean SD 293 Mean SD 143 242 19 37 100** 21 129 161** 32** 17 28 33 331 48 220** 55 89 16 57 44 586 Mean SD 144 137** -6** 23 27 26 175** 34 36 41 1171 Mean SD 147 112** -37** 25 28 13 1852 - 'Mean change is calculated separately from the mean body weights using individual body weights (See Appendix VI). 2n=l, could not be evaluated statistically using Dunnett's t-test. (-) = No data available due to mortality. ^Statistically different from the control group at p < 0.05 (Dunnett's t-test). **Statistically different from the control group at p < 0.01 (Dunnett's t-test)._________________ 312 - Total Change (Day 0-8)' 245 34 198* 55 231 25 228 30 200* 73 188** 32 9^** 54 39** 36 152 - W ild lif e In ter n atio n al, ltd -22 - PROJECT NO.: 454-102 TABLE 2 Page 2 Mean Body Weight (g) from a Mallard Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) Day 8 Post:-Exposure Period Change1 Day 15 Day 8-15 Day 22 Change1 Day 15-22 Total Change'(8-22) C ontrol 0 Mean SD 410 640 230 30 56 47 823 183 49 24 Treatm ent 9.1 18.3 Mean SD Mean SD 345* 33 384 27 575 32 625 42 230 20 241 22 773 198 38 15 811 186 81 41 36.6 Mean SD 377 620 243 33 44 40 828 208 78 34 73.2 Mean SD 364 579 216 82 97 21 782 203 135 39 146 Mean SD 332* 58 564 56 232 22 688* 86 124* 90 293 Mean SD 211** 49 467** 51 256 35 701* 41 234 35 586 Mean SD 174** 46 394** 82 221 37 613** 91 219 13 1171 Mean 1852 3732 1982 6342 2512 SD - -- -- 'Mean change is calculated separately from the mean body weights using individual body weights (See Appendix VI). 2n=l, could not be evaluated statistically using Dunnett's t-test. (-) = No data available due to mortality. ^Statistically different from the control group at p < 0.05 (Dunnett's t-test). **Statistically different from the control group at p < 0.01 (Dunnett's t-test). 413 42 427 34 427 62 451 71 418 56 356 106 490 54 439 47 4492 - Wild life International, ltd -23 - PROJECT NO.: 454-102 TABLE 3 Page 1 Mean Feed Consumption (g/bird/day) from a Mallard Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) C ontrol 0 Exposure Period Day 0-5 Mean SD 92 10 Treatm ent 9.1 18.3 36.6 73.2 146 293 586 1171 (-) = No data available due to mortality. 73 91 94 77 105 44 36 22 Post-Exposure Period Day 6-8 125 17 117 132 125 101 159 63 55 25 W ild life International, ltd - 24- PROJECT NO.: 454-102 TABLE 3 Page 2 Mean Feed Consumption (g/bird/day) from a Mallard Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) C ontrol 0 Mean SD Post-Exposure Period Day 8-15 Day 15-22 171 180 13 20 Treatm ent 9.1 18.3 36.6 73.2 146 293 586 1171 (-) = No data available due to mortality. 172 186 165 148 159 109 114 106 198 204 179 173 164 132 143 154 W ild lif e In ter n atio n al, ltd -25 - PROJECT NO.: 454-102 TABLE 4 Gross Pathological Observations from a Mallard Acute Dietary Toxicity Study with PFOS Birds that died during the course of the study Finding Crop empty Emaciated G.I. Tract, primarily empty Gizzard contents bile stained Gizzard, empty Intestinal contents, black and tar-like Keel prominent Kidneys, pale Loss of muscle mass Spleen, grey Spleen, small and pale Spleen, pale Thin Male, Female, and Undetermined _________ PPM A.I.__________ 293 586 1171 N=2 N=3 N=9 027 1 14 111 024 00 1 00 1 002 00 1 225 00 1 1 12 023 l 14 W ild life International, ltd -26- PROJECT NO.: 454-102 Experimental Group (ppm a.i.) C ontrol 0 0 0 0 0 0 Treatm ent 9.1 9.1 Table 5 Cumulative Mortality (Estimated Cumulative Dose, mg/kg1) from a Mallard _______________ Acute Dietary Toxicity Study with PFOS________________ No. Dead Per No. Exposed (Cumulative Dose, mg/kg) No. Dead Per No. Exposed ____________________________ Exposure Period______________________________________ Post-Exposure Period Pen Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 82 1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 3 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1 0/5 2 0/5 0/5 (4) 0/5 (4) 0/5 (8) 0/5 (8) 0/5(12) 0/5 (12) 0/5 (16) 0/5 (16) 0/5 (20) 0/5 (20) 0/5 0/5 0/5 0/5 0/5 0/5 18.3 1 0/5 0/5 (8) 0/5(16) 0/5 (24) 0/5 (32) 0/5 (40) 0/5 0/5 0/5 18.3 2 0/5 0/5 (9) 0/5(18) 0/5 (27) 0/5 (36) 0/5 (45) 0/5 0/5 0/5 36.6 1 0/5 0/5(19) 0/5 (38) 0/5 (57) 0/5 (76) 0/5 (95) 0/5 0/5 0/5 36.6 2 0/5 0/5(16) 0/5 (32) 0/5 (48) 0/5 (64) 0/5 (80) 0/5 0/5 0/5 73.2 1 0/5 0/5 (31) 0/5 (62) 0/5 (93) 0/5 (124) 0/5(155) 0/5 0/5 0/5 73.2 2 0/5 0/5 (30) 0/5 (60) 0/5 (90) 0/5 (120) 0/5(150) 0/5 0/5 0/5 146 1 0/5 0/5 (74) 0/5 (148) 0/5 (222) 0/5 (296) 0/5 (370) 0/5 0/5 0/5 146 2 0/5 0/5(101) 0/5 (202) 0/5 (303) 0/5 (404) 0/5 (505) 0/5 0/5 0/5 293 1 0/5 0/5 (64) 0/5 (128) 0/5 (192) 0/5 (256) 0/5 (320) 0/5 0/5 0/5 293 2 0/5 0/5(126) 0/5 (252) 0/5 (378) 0/5 (504) 0/5 (630) 0/5 1/5 2/5 586 1 0/5 0/5 (175) 0/5 (350) 0/5 (525) 0/5 (700) 0/5 (875) 0/5 1/5 1/5 586 2 0/5 0/5(153) 0/5 (306) 0/5 (459) 0/5 (612) 1/5 (765) 2/5 2/5 2/5 1171 1171 1 0/5 2 0/5 0/5 (240) 0/5 (207) 0/5 (480) 0/5 (414) 0/5 (720) 0/5 (621) 1/5 (960) 1/5 (828) 4/5 (1200) 2/5 (1035) 5/5 3/5 5/5 4/5 The LC50 value was calculated to be approximately 628 ppm a.i. with a 95% confidence interval o f448 to 958 ppm a.i.. '- Estimated cumulative dose is based upon the average body weight and feed consumption over the 5-day exposure period, and serves as a rough approximation of the actual amount of test substance consumed. 2- No mortalities occurred in any of the treatment or control groups after Day 8.____________________________________________________ 5/5 4/5 W ild lif e Inter natio n al, ltd -27- PROJECT NO.: 454-102 APPENDIX I Certificate O f Analysis FC-95, Lot 217 March 9, 2000 Richard M. Payfer th is was analyzed using LC/MS, lH-NMR, ISF-NMR, and elemental analyses techniques. The results 91 these tests show the.sample to contain the following weight percent composition: C iF iS O j-K * c jS sS T ' CtFSOa"K+ CiFiiSO jIC* C iFisSO tTC O F h SO j-K4 C tFnSO jT C C ^F itS O jlT C ioFiiSO alC C ' C Fj -C O zT ? SF^.FiSC hT C 0 .0 4 % 0.83 % 1.38 % 1 .3 0 % 3.71 % 1 .1 9 % "9039% 0749% 0.13 % 0.04 % 0 .0 5 % 0.35 % Additionally, the isomer distribution o fthe sample was determined using l9F-NMR techniques and found to contain the following mole percent con^osltion: CFj(CF*)-SOj' KT (Normal chain, wJk o x is mainly 7) CFiiCFiVCFiCFjKCFiV SOj'KT (Internal monomethyl branch, where x+v is mainly 5. and xwO. yw 01 (C F^C P-iC FzV SO /ir(Isopropyl branch, where x is mainly 5) CF2*h-CF(CFj)- SO,' IT (Aloha branch. where x Is mainly 6) (CF,),C-(CF2)* .S 0 3-K+ (t-butvi branch, where x is mainly 41 CF,-(CFj>-C(CFi)a-(CFj)y SO,' 1C (Internal gem-dimethyl branch, where x+y is mainly 4. and x * 01 70.5% 17.1% 10.3% 1 .6% 0.2% 0.2% W il d l if e In t e r n a t io n a l , ltd -28 - PROJECT NO.: 454-102 APPENDIX II DIET FORMULATION WILDLIFE INTERNATIONAL LTD. GAME BIRD RATION1 INGREDIENTS Fine Com Meal Soy Bean Meal, 48% Protein Wheat Midds Protein Base Agway Special, 60% Protein Alfalfa Meal, 20% Protein Dried Whey Ground Limestone Eastman CalPhos Methionine Premix + Liquid Vitamin and Mineral Premix (see below) GL Ferm (Fermatco)2 Salt Iodized Total PERCENT (%) 44.83 30.65 6.50 6.00 4.00 3.00 2.50 0.90 0.60 0.35 0.32 0.25 0.10 100.00 VITAMIN AND MINERAL PREMIX AMOUNT ADDED PER TON Vitamin D3 Vitamin A Riboflavin Niacin Pantothenic Acid Vitamin Bn Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (Menadione Dimethylpyrimidinol Bisulfite) Manganese Zinc Copper Iodine Iron Selenium 2.000. 000 I.C.U. 7.000. 000 I.U. 6 grams 40 grams 10 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 182 mgs `The guaranteed analysis is a minimum of 27% protein, a minimum of 2.5% crude fat and a maximum of 5% crude fiber. fermentation By-Products (Source of Unidentified Growth Factors) W ild lif e In ter n atio n al, ltd -29- PROJECT NO.: 454-102 APPENDIX III ANALYTICAL METHODS AND RESULTS W ild lif e In ter n atio n al, ltd -30- PROJECT NO.: 454-102 INSTRUMENT: Appendix III Table 1 Typical LC/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil Qg column (100 mm x 2 mm I.D., 3 pm particle size) OVEN TEMPERATURE: 30C STOP TIME: 10.0 minutes FLOW RATE: 0.220 mL/minute MOBILE PHASE: 72.0% Methanol : 28.0% NANOpure Water containing 0.1% Formic Acid INJECTION VOLUME: 25.0 pL PFOS RETENTION TIME: Approximately 7.0 minutes INTERNAL STANDARD RETENTION TIME: Approximately 4.8 minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 426.7 amu W ild lif e In ter n atio n al, ltd -31 - PROJECT NO.: 454-102 Appendix III Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454-102-) MAB-1 Sample Type Matrix Blank Measured Concentration of PFOS1 (ppm a.i.) < LOQ MAB-2 Matrix Blank < LOQ 1The limit o f quantitation (LOQ) was 1.15 ppm a.i. based upon the product o f the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor o f the matrix blank samples (500)._______________________ W ild lif e In ter n atio n al, ltd -32- PROJECT NO.: 454-102 Appendix III Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454-102-) MAS-1 A MAS-4A Concentrations o f PFOS (ppm a.i.) Fortified Measured 4.57 4.54 4.57 4.79 Percent Recovered 99.2 105 MAS-2 183 176 96.1 MAS-5 183 162 88.3 MAS-3 MAS-6 1830 1830 1576 1716 86.1 93.7 Mean = Standard Deviation = CV = N= 94.7 6.99 7.38 6 Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. W ild life Internatio nal, ltd -33Appendix III Table 4 Homogeneity of PFOS in Avian Diet PROJECT NO.: 454-102 Nominal Concentration (ppm a.i.) 9.1 Sample Number (S-454-102-) 54 55 56 57 58 59 Location Sampled in Mixing Vessel Top Left Top Right Middle Left Middle Right Bottom Left Bottom Right PFOS Measured Concentration (ppm a.i.) 9.52 9.70 9.79 8.09 10.9 10.5 Mean Measured Concentration ( X ) Standard Deviation (SD) Coefficient o f Variation (CV) 1 X = 9.8 ppm a.i. SD = 0.969 ppm a.i. CV = 9.94% Mean Percent of Nominal 108 18.3 2 Top Left 18.5 3 Top Right 23.4 4 Middle Left 18.3 5 Middle Right 17.3 6 Bottom Left 19.4 7 Bottom Right 19.9 X = 19.5 ppm a.i. SD = 2.13 ppm a.i. CV = 10.9% 107 1171 18 Top Left 19 Top Right 2 0 Middle Left 2 1 Middle Right 2 2 Bottom Left 23 Bottom Right 1239 1221 1118 1301 1163 1133 X = 1196 ppm a.i. SD = 70.2 ppm a.i. CV = 5.87% 102 Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. 1 Coefficient o f variation was calculated using full precision o f mean and standard deviation results. W ild life Internatio nal, ltd -34Appendix III Table 5 Verification of PFOS Concentrations in Avian Diet PROJECT NO.: 454-102 Nominal Concentration (ppm a.i.) 0.0 Sample Number (S-454-102-) 1 Sampling Interval (Day) 0 PFOS Measured Concentration' (ppm a.i.) < LOQ Percent of Nominal - Mean Measured Concentration (ppm a.i.) - Mean Percent of Nominal - 9.1 - - - - 9.8*2 1082 18.3 - - - - 19.52 1072 36.6 8 0 45.7 125 40.2 110 90 34.6 94.5 73.2 10 11 0 0 77.8 106 74.5 102 71.2 97.3 146 12 13 0 0 176 120 174 119 172 117 293 14 0 15 0 274 93.8 291 99.3 307 105 586 16 0 17 0 550 93.9 537 91.6 523 89.4 1171 __ __ __ 11962 1022 Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. 'The limit of quantitation (LOQ) was 1.15 ppm a.i. based upon the product of the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor of the matrix blank samples (500). 2Result obtained from Table 4. W ild life Internatio nal, ltd -35Appendix III Table 6 PROJECT NO.: 454-102 Ambient Stability of PFOS in Avian Diet During the Mallard LC50 Study Nominal , Concentration Sample (ppm a.i.) Number (S-454-102-) 01 Day 01 Mean Measured Concentration (ppm a.i.) - Mean Percent of Nominal - Sample Number (S-454-102-) 39 Day 5 Measured Concentration2 (ppm a.i.) Mean Measured Concentration (ppm a.i.) < LOQ - Mean Percent of Day 0 - 9.1 54-59 9.8 108 60 12.3 11.7 119 61 11.0 18.3 2-7 19.5 107 40 18.2 19.0 97.4 41 19.7 36.6 8, 9 40.2 110 42 47.9 52.4 1303 43 56.8 73.2 10, 11 74.5 102 44 77.6 77.8 104 45 77.9 146 12, 13 174 119 46 167 164 94.3 47 160 293 14, 15 291 99.3 48 297 295 101 49 293 586 16, 17 537 91.6 50 552 541 101 51 530 1171 18-23 1196 102 52 1150 1136 95.0 53 1122 'Day 0 results obtained from Table 4 and Table 5. The limit of quantitation (LOQ) was 1.15 ppm a.i. based upon the product of the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor of the matrix blank samples (500). The Day 5 value is consistent with the Day 0 mean measured concentration value for this level. No analytical measuring bias is indicated. W ild lif e In ter n atio n al, ltd -36- PROJECT NO.: 454-102 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 Dry Mix Technique For the high-level matrix fortification sample, weigh the requisite quantity of Wildlife International Ltd. (WIL) ration into a weigh boat. 4 Weigh the requisite quantity of test substance (PFOS) into a beaker. 4 Add Vi of the WIL ration and the test substance to a larger beaker. 4 Rinse the beaker that contained the PFOS with small portions of the remaining ration and transfer all portions to the larger beaker. 4 Mix the contents of the larger beaker well and transfer the mixture to a Waring blender. 4 Blend the mixture for -5 minutes stopping at 1 minute intervals to scrape down the sides of the blender. During the third interval transfer the fortified feed to a beaker, mix well and return the mixture to the blender to complete mixing in the specified time. 4 Prepare the next two matrix fortification levels by serial dilutions. Follow the same procedure described for the high-level matrix fortification except weigh the appropriate quantity of fortified matrix (high or mid level) rather than the test substance. 4 Weigh 10-g samples of the matrix blank, matrix fortification and test samples into weigh boats and transfer to 16-oz. French-square glass bottles. Record the weights. 4 For each sample, measure 100 mLs of methanol with a graduated cylinder and transfer volume to the French-squarc bottle. 4 Cap bottles and place on shaker table. Allow the samples to shake for a minimum of 30 minutes at 250 rpm. 4 Vacuum filter each sample with qualitative filter paper and rinse retained feed 3 times with methanol into the filtrate. 4 Transfer the filtrate to a 200-mL volumetric flask and bring the flask to volume with methanol. 4 Prepare appropriate dilution(s) to bring final concentration into the calibration range of the LCMS methodology. Use methanol for intermediate dilutions, if required. For all final dilutions use 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v). 4 Ampulate and submit samples for LC/MS analysis. Figure 1. Analytical method flowchart for the analysis of PFOS in avian diet. W ild lif e In ter n atio n al, ltd -37Appendix III PROJECT NO.: 454-102 Figure 2. A typical calibration curve for PFOS. Slope = 2.77397; Intercept = 0.01894; r = 0.9981. Curve is weighted (1/x). W ild lif e In ter n atio n al, ltd - 38- Appendix III PROJECT NO.: 454-102 100 90 80 70 60 50 40 30 20 10- 24 0-T 311.30 intensity: 1995 cps Figure 3. A representative ion chromatogram of a low-level (0.00229 mg a.i./L) PFOS standard. W ild lif e In ter n atio n al, ltd -39- Appendix III PROJECT NO.: 454-102 Figure 4. A representative ion chromatogram of a high-level (0.0457 mg a.i./L) PFOS standard. W ild lif e In ter n atio n al, ltd - 40Appendix III PROJECT NO.: 454-102 Figure 5. A representative chromatogram o f a matrix blank sample (454-102-M AB-l). The arrow indicates the retention time o f PFOS. W ild lif e In ter n atio n al, ltd -41 - Appendix III PROJECT NO.: 454-102 Figure 6. A representative chromatogram of a matrix fortification sample (454-102-MAS-1A). W ild lif e In ter n atio n al, ltd - 42Appendix III PROJECT NO.: 454-102 intensity: 7248 cps 100 90- Figure 7. A representative chromatogram of a test sample (454-102-2). W ild lif e In ter n atio n al, ltd -43 - PROJECT NO.: 454-102 APPENDIX IV DIET PREPARATION Weight and volume of constituents used to prepare test diets: Nominal Concentrations (ppm a.i.) 0 9.1 18.3 36.6 73.2 146 293 586 1171 Test Substance (g) 0.0910 0.1818 0.3638 0.7282 1.4659 2.9123 5.8239 11.6483 Basal Ration (g) 9000.0 8999.9 8999.8 8999.6 8999.3 8998.5 8997.1 8994.2 8988.4 Diets were prepared as follows: 5000.0 g of basal ration was weighed into a tared Hobart mixing bowl. The test substance was weighed in a tared weigh boat Approximately 100 g of basal ration was taken from the mixing bowl and placed in a Waring blender. The test substance was added to the blender and the weigh boat was rinsed with additional ration, with the rinse also being placed in the blender. The blender contents were blended for approximately 60 seconds and transferred to the mixing bowl. The blender was rinsed with additional ration, with the rinse also being placed in the mixing bowl. The bowl was placed on a Hobart mixer and the contents were mixed for approximately six minutes. The remaining ration as added to the bowl and the contents were mixed for six more minutes. The diet was transferred to a labelled paper feed bag. W ild life Internatio nal, ltd PROJECT NO.: 454-102 -44- APPENDIX V Cumulative Mortality by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) Control 0 0 0 0 0 0 Treatment 9.1 9.1 18.3 18.3 36.6 36.6 73.2 73.2 146 146 293 293 586 586 1171 1171 No. Dead Per No. Exposed Pen_____________________________Exposure Period__________________________ Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 1 0/5 0/5 0/5 0/5 0/5 0/5 2 0/5 0/5 0/5 0/5 0/5 0/5 3 0/5 0/5 0/5 0/5 0/5 0/5 4 0/5 0/5 0/5 0/5 0/5 0/5 5 0/5 0/5 0/5 0/5 0/5 0/5 6 0/5 0/5 0/5 0/5 0/5 0/5 1 0/5 0/5 0/5 0/5 0/5 0/5 2 0/5 0/5 0/5 0/5 0/5 0/5 1 0/5 0/5 0/5 0/5 0/5 0/5 2 0/5 0/5 0/5 0/5 0/5 0/5 1 0/5 0/5 0/5 0/5 0/5 0/5 2 0/5 0/5 0/5 0/5 0/5 0/5 1 0/5 0/5 0/5 0/5 0/5 0/5 2 0/5 0/5 0/5 0/5 0/5 0/5 1 0/5 0/5 0/5 0/5 0/5 0/5 2 0/5 0/5 0/5 0/5 0/5 0/5 1 0/5 0/5 0/5 0/5 0/5 0/5 2 0/5 0/5 0/5 0/5 0/5 0/5 1 0/5 0/5 0/5 0/5 0/5 0/5 2 0/5 0/5 0/5 0/5 0/5 1/5 1 0/5 0/5 0/5 0/5 1/5 4/5 2 0/5 0/5 0/5 0/5 1/5 2/5 The LC50 value was calculated to be 628 ppm a.i. with a 95% confidence interval o f448 ppm a.i. to 958 ppm a.i. * - No mortalities occurred in any of the treatment or control groups after Day 8.__________________________ No. Dead Per No. Exposed _______ Post-Exposure Period Day 6 Day 7 Day 8 * 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1/5 0/5 1/5 2/5 2/5 5/5 5/5 3/5 4/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2/5 1/5 2/5 5/5 4/5 W il d l if e In te r n a t io n a l, ltd -45 - APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 1 PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Bird Day 0 Day 5 Change Day 0-5 Day 8 Change Day 5-8 Total Change (0-8) 0 1 132 302 170 404 102 2 136 292 156 381 89 3 154 319 165 415 96 4 158 319 161 429 110 5 169 309 140 418 109 Mean 150 308 158 409 101 SD 15 12 12 18 9 272 245 261 271 249 260 12 0 1 136 282 146 399 117 2 142 284 142 394 110 3 152 299 147 409 110 4 152 312 160 430 118 5 177 345 168 484 139 Mean 152 304 153 423 119 SD 16 26 11 37 1 2 263 252 257 278 307 271 22 0 1 125 265 140 372 107 2 134 282 148 371 89 3 149 304 155 440 136 4 154 292 138 378 8 6 5 166 328 162 430 102 Mean 146 294 149 398 104 SD 16 24 10 34 2 0 247 237 291 224 264 253 26 0 1 124 271 147 376 105 2 135 263 128 360 97 3 151 300 149 408 108 4 163 333 170 432 99 5 170 351 181 453 1 0 2 Mean 149 304 155 406 102 SD 19 38 2 1 38 4 252 225 257 269 283 257 22 W ild lif e In ter n atio n al, ltd -46- APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 2 Experimental Group (ppm a.i.) Bird Day 0 Day 5 Change Day 0-5 Day 8 0 1 107 2 2 2 115 298 2 126 267 141 360 3 116 203 87 266 4 107 203 96 284 5 1 1 1 248 137 348 Mean 113 229 115 311 SD 8 28 24 41 PROJECT NO.: 454-102 Change Day 5-8 76 93 63 81 100 83 15 Total Change 191 234 150 177 237 198 37 0 Group Total 1 1 0 0 235 135 335 1 0 0 2 1 0 0 232 132 328 96 3 1 0 2 233 131 317 84 4 108 267 159 383 116 5 1 0 2 2 1 0 108 299 89 Mean 102 235 133 332 97 SD 3 2 0 18 32 1 2 Mean 135 279 144 380 101 SD 24 42 2 2 53 16 235 228 215 275 197 230 29 245 34 W ild lif e In ter n atio n al, ltd -47 - APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 3 PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Bird Day 0 9.1 1 123 2 118 3 151 4 108 5 119 Mean SD 124 16 Day 5 Change Day 0-5 Day 8 244 121 349 231 113 334 286 135 399 209 1 0 1 313 231 1 1 2 332 240 116 345 29 13 33 Change Day 5-8 105 103 113 104 101 105 5 Total Change (0-8) 226 216 248 205 213 222 17 9.1 1 96 145 49 2 123 250 127 3 127 237 1 1 0 4 1 1 2 216 104 5 1 1 1 215 104 Mean 114 213 99 SD 1 2 41 29 Group Mean 119 226** 108** Total SD 14 36 23 *Statistically different from the control group at p < 0.05 (Dunnett's t-test). **Statistically different from the control group at p < 0.01 (Dunnett's t-test). 146 345 343 316 294 289 83 317* 66 1 95 106 100 79 76 43 91 33 50 222 216 204 183 175 71 198* 55 W ild lif e In ter n atio n al, ltd -48- PROJECT NO.: 454-102 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 4 Experimental Group (ppm a.i.) Bird Day 0 18.3 1 123 2 141 3 150 4 152 5 160 Mean SD 145 14 Day 5 Change Day 0-5 Day 8 261 138 355 297 156 421 270 1 2 0 361 286 134 390 294 134 391 282 136 384 16 13 27 Change Day 5-8 94 124 91 104 97 102 13 Total Change 232 280 211 238 231 238 25 18.3 Group Total 1 130 240 110 327 87 2 137 266 129 359 93 3 145 268 123 366 98 4 151 258 107 366 108 5 171 333 162 433 100 Mean 147 273 126 370 97 SD 16 35 2 2 39 8 Mean 146 277 131 377 100 SD 14 26 18 32 1 1 197 222 221 215 262 223 24 231 25 W ild lif e In ter n atio n al, ltd -49- PROJECT NO.: 454-102 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 5 Experimental Group (ppm a.i.) Bird Day 0 36.6 1 131 2 132 3 142 4 160 5 179 Mean SD 149 21 Day 5 Change Day 0-5 Day 8 261 130 372 256 124 356 269 127 369 270 1 1 0 353 335 156 433 278 129 377 32 17 33 Change Day 5-8 111 100 100 83 98 98 10 Total Change 241 224 227 193 254 228 23 36.6 Group Total 1 131 230 99 316 86 2 136 287 151 402 115 3 147 263 116 379 116 4 151 255 104 344 89 5 160 324 164 427 103 Mean 145 272 127 374 102 SD 1 2 36 29 44 14 Mean 147 275 128 375 100 SD 16 32 2 2 37 1 2 185 266 232 193 267 229 39 228 30 W ild lif e In ter n atio n al, ltd -50- PROJECT NO.: 454-102 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 6 Experimental Group (ppm a.i.) Bird Day 0 73.2 1 113 2 113 3 142 4 154 5 179 Mean SD 140 28 Day 5 Change Day 0-5 Day 8 203 90 281 219 106 288 286 144 390 290 136 379 338 159 480 267 127 364 56 28 82 Change Day 5-8 78 69 104 89 142 96 29 Total Change 168 175 248 225 301 223 55 73.2 1 109 2 2 2 113 2 129 254 125 3 139 243 104 4 147 196 49 5 204 352 148 Mean 146 253 108 SD 36 59 37 Group Mean 143 260 117* Total SD 30 55 33 *Statistically different from the control group at p < 0.05 (Dunnett's t-test). 302 332 340 175 458 321 101 343 90 80 78 97 -21 106 68 51 82 42 193 203 201 28 254 176 86 200* 73 W ild lif e In ter n atio n al, ltd -51 - PROJECT NO.: 454-102 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 7 Experimental Group (ppm a.i.) Bird Day 0 146 1 108 2 138 3 151 4 151 5 164 Mean SD 142 21 Day 5 Change Day 0-5 Day 8 169 61 236 248 1 1 0 330 271 1 2 0 375 254 103 341 282 118 380 245 1 0 2 332 44 24 58 Change Day 5-8 67 82 104 87 98 88 14 Total Change 128 192 224 190 216 190 38 146 1 115 204 89 2 139 219 80 3 144 229 85 4 159 261 1 0 2 5 158 285 127 Group Total Mean SD Mean SD 143 18 143 19 240 33 242 37 97 19 1 0 0 ** 21 ^Statistically different from the control group at p < 0.01 (Dunnett's t-test). 280 287 348 358 374 329 43 331 48 76 68 119 97 89 90 20 89 16 165 148 204 199 216 186 29 188** 32 W ild lif e In ter n atio n al, ltd -52- APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 8 PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Bird Day 0 293 1 115 2 139 3 140 4 155 5 112 Mean SD 132 18 Day 5 Change Day 0-5 Day 8 142 27 179 119 - 2 0 165 156 16 217 187 32 291 134 2 2 203 148 15 2 1 1 26 2 1 49 Change Day 5-8 27 -2 0 16 32 22 15 21 Total Change 64 26 77 136 91 79 40 293 1 123 205 82 2 1 2 0 183 63 3 151 139 - 1 2 4 106 158 5 124 185 52 61 Mean 125 174 49 SD 16 26 36 Group Mean 129 161** 32** Total SD 17 28 33 (-) = No data available due to mortality. ^Statistically different from the control group at p < 0.01 (Dunnett's t-test). 289 273 - 223 138 231 68 2 2 0 ** 55 82 63 -1 2 52 61 49 36 57** 44 166 153 - 117 14 113 69 9 4 ** 54 W ild lif e In ter n atio n al, ltd -53 - APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 9 PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Bird Day 0 586 1 122 2 135 3 152 4 154 5 165 Mean SD 146 17 Day 5 Change Day 0-5 Day 8 105 -17 126 143 8 209 160 8 217 129 -25 - 195 30 143 146 1 174 34 2 2 46 Change Day 5-8 21 66 57 - -52 23 54 Total Change 4 74 65 - -2 2 30 47 586 1 105 114 9 2 1 2 0 130 10 3 149 - - 4 153 128 -25 5 182 128 -54 Mean 142 125 -15 SD 30 7 31 Group Mean 144 137** -6 ** Total SD 23 27 26 (-) = No data available due to mortality. ^Statistically different from the control group at p < 0.01 (Dunnett's t-test). 159 185 - 185 - 176 15 175** 34 45 55 - 57 - 52 6 36** 41 54 65 - 32 - 50 17 3 9 ** 36 W ild lif e In ter n atio n al, ltd -54APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 10 PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Bird Day 0 1171 1 2 3 4 5 Mean SD 124 140 145 150 166 145 15 Day 5 Change Day 0-5 Day 8 8 8 -39 -- 96 -49 118 -32 -1 0 0 -40 17 9 . - - Change Day 5-8 . - - Total Change - - 1171 1 128 87 -41 2 110 -- 3 145 132 -13 4 157 - - 5 200 154 -46 Group Mean SD Mean 148 34 147 124 34 1 1 2 ** -33 18 _37** Total SD 25 28 13 (-) = No data available due to mortality. 2No mortalities occurred in any of the treatment or control groups after Day 8. ^Statistically different from the control group at p < 0.01 (Dunnett's t-test). - - - 185 185 - 1852 - - - - 31 31 - 312 - -15 -15 152 - W ild lif e In ter n atio n al, ltd PROJECT NO.: 454-102 -55 - APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 11 Experimental Group (ppm a.i.) Bird Day 8 Day 15 Change Day 8-15 Day 22 Change Day 15-22 Total Change (8-22) 0 1 404 721 317 895 174 2 381 633 252 780 147 3 415 685 270 861 176 4 429 692 263 822 130 5 418 679 261 847 168 Mean 409 682 273 841 159 SD 18 32 26 43 2 0 491 399 446 393 429 432 40 0 1 399 606 207 810 204 2 394 604 2 1 0 791 187 3 409 638 229 819 181 4 430 543 113 730 187 5 484 752 268 924 172 Mean 423 629 205 815 186 SD 37 77 57 70 1 2 411 397 410 300 440 392 54 0 Group Total 1 372 583 2 1 1 779 196 2 371 613 242 823 2 1 0 3 440 636 196 859 223 4 378 594 216 794 2 0 0 5 430 627 197 816 189 Mean 398 611 212 814 204 SD 34 22 19 31 13 Mean 410 640 230 823 183 SD 30 56 47 49 24 407 452 419 416 386 416 24 413 42 W ild lif e In ter n atio n al, ltd -56- APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 12 PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Bird Day 8 Day 15 Change Day 8-15 Day 22 Change 9.1 1 349 602 253 821 219 2 334 549 215 730 181 3 399 616 217 802 186 4 313 562 249 767 205 5 332 546 214 744 198 Mean 345* 575 230 773 198 SD 33 32 2 0 38 15 Total Change 472 396 403 454 412 427 34 Experimental Group (ppm a.i.) Bird Day 8 Day 15 Change Day 8-15 Day 22 Change Day 15-22 18.3 1 355 611 256 2 421 693 272 3 361 578 217 4 390 626 238 5 391 618 231 Mean 384 625 242 SD 27 42 2 2 *Statistically different from the control group at p < 0.05 (Dunnett's t-test). 811 938 713 794 799 811 81 200 245 135 168 181 186 41 Total Change 465 517 352 404 408 427 62 Wild life International, ltd -57- PROJECT NO.: 454-102 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 13 Experimental Group (ppm a.i.) Bird Day 8 36.6 1 372 2 356 3 369 4 353 5 433 Mean SD 377 33 Day 15 Change Day 8-15 Day 22 Change Day 15-22 677 305 942 265 607 251 817 2 1 0 613 244 793 180 559 206 746 187 642 209 840 198 620 243 828 208 44 40 73 34 Total Change 570 461 424 393 407 451 71 Experimental Group (ppm a.i.) Change Change Bird Day 8 Day 15 Day 8-15 Day 22 Day 15-22 73.2 1 281 482 2 0 1 648 166 2 288 480 192 646 166 3 390 636 246 864 228 4 379 599 2 2 0 796 197 5 480 699 219 955 256 Mean 364 579 216 782 203 SD 82 97 2 1 135 39 Total Change 367 358 474 417 475 418 56 W ild lif e In ter n atio n al, ltd -58- PROJECT NO.: 454-102 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 14 Experimental Group (ppm a.i.) Bird Day 8 Day 15 Change Day 8-15 Day 22 Change 146 1 236 475 239 659 184 2 330 569 239 739 170 3 375 631 256 738 107 4 341 570 229 754 184 5 380 576 196 550 -26 Mean 332* 564 232 688* 124* SD 58 56 2 2 8 6 90 Total Change 423 409 363 413 170 356 106 Experimental Group (ppm a.i.) Bird Day 8 Day 15 Change Day 8-15 Day 22 Change Day 15-22 293 1 179 447 268 2 165 415 250 3 217 526 309 4 291 516 225 5 203 430 227 Mean 2 1 1 ** 467** 256 SD 49 51 35 ""Statistically different from the control group at p < 0.05 (Dunnett's t-test). """"Statistically different from the control group at p < 0.01 (Dunnett's t-test). 731 646 748 704 676 701* 41 284 231 222 188 246 234 35 Total Change 552 481 531 413 473 490 54 W ild lif e In ter n atio n al, ltd -59- APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 14 PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Bird Day 8 586 1 126 2 209 3 217 4- 5 143 Mean SD 174 46 Day 15 Change Day 8-15 Day 22 Change Day 15-22 308 182 525 217 469 260 706 237 460 243 675 215 - - -- 340 197 546 206 394 221 613 219 82 37 91 13 Total Change 399 497 458 403 439 47 Experimental Group (ppm a.i.) Bird Day 8 Day 15 Change Day 8-15 Day 22 Change Day 15-22 1171 1 2 "" 3 4 5 185 383 198 Mean SD 18? - 38? - 19? - n = l, could not be evaluated statistically using Dunnett's t-test. " 634 63? - 251 25? _ Total Change 449 44? _ W ild lif e In ter n atio n al, ltd PROJECT NO.: 454-102 -60APPENDIX VII Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Page 1 Experimental Group (ppm a.i.) Pen Exposure Period Day 0-5 Post-Exposure Period Day 6-8 Control 1 2 3 4 5 6 Mean SD 109 92 85 93 82 94 92 10 145 140 117 132 100 116 125 17 W ild lif e In ter n atio n al, ltd -61 APPENDIX VII PROJECT NO.: 454-102 Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Page 2 Experimental Group (ppm a.i.) Pen Exposure Period Day 0-5 Post-Exposure Period Day 6-8 9.1 1 73 2 72 Mean 73 130 104 117 18.3 1 90 2 91 Mean 91 135 128 132 36.6 1 103 2 85 Mean 94 129 120 125 73.2 1 79 2 74 Mean 77 109 94 101 146 1 89 2 121 Mean 105 102 217 159 293 1 28 2 59 Mean 44 61 65 63 586 1 40 2 32 Mean 36 43 67 55 1171 1 23 2 22 Mean 22 25 26 25 W ild lif e In ter n atio n al, ltd PROJECT NO.: 454-102 -62 APPENDIX VII Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Page 3 Experimental Group (ppm a.i.) Pen Control 1 2 3 Mean SD Exposure Period Day 8-15 185 161 166 171 13 Post-Exposure Period Day 15-22 159 182 199 180 20 W ild lif e In ter n atio n al, ltd -63 APPENDIX VII PROJECT NO.: 454-102 Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Page 4 Experimental Group (ppm a.i.) Pen Exposure Period Day 8-15 Post-Exposure Period Day 15-22 9.1 1 172 198 18.3 1 186 204 36.6 1 165 179 73.2 1 148 173 146 1 159 164 293 1 109 132 586 1 114 143 1171 1 106 154 W ild lif e In ter n atio n al, ltd -64APPENDIX VIII PROJECT NO.: 454-102 CHANGES TO PROTOCOL This study was conducted in accordance with the approved Protocol with the following changes: 1. The protocol was amended to add an additional test concentration of 10 ppm a.i.. 2. The protocol was amended to indicate that bile will be collected from all study birds. The protocol was clarified to indicate the collection of liver from birds that died during the course of the study. 3. The protocol was amended to change the test concentrations from 0, 10, 20, 40, 80, 160, 320, 640 and 1280 ppm a.i., to 0, 9.1, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 ppm a.i. Test concentrations were changed to reflect the test substance purity given in the new certificate of analysis. 4. Individually numbered wing bands were used to uniquely identify each duckling. The protocol required leg bands to be used to identify the birds. 5. Blood samples were collected on Day 8 and Day 22 in non-heparinized 5 ml borosilicate glass test tubes. The protocol indicated that heparinized vacutainers would be used. 6. The final temperature from one brooder unit on Day 8 and all brooder units on Day 22 were not recorded. 7. The afternoon observations were inadvertently not recorded for birds in a single brooder unit on May 8, 1999. 8. Observations for control birds from two brooder levels were inadvertently not recorded on Day 6 of the test. Temperatures for these brooder levels were also not recorded on this day. Observations for control birds from one brooder level were inadvertently not recorded on Day 22 of the test. W ild lif e In ter n atio n al, ltd -65 APPENDIX IX PERSONNEL INVOLVED IN THE STUDY PROJECT NO.: 454-102 The following key Wildlife International Ltd. personnel were involved in the conduct or management of this study:1 (1) Mark Jaber, Wildlife Toxicologist (2) Joann B. Beavers, Director, Avian Toxicology (3) Sean P. Gallagher, Senior Biologist (4) Courtney Casey, M.S., Senior Biologist (5) Willard B. Nixon, Ph.D., Manager, Analytical Chemistry (6) Tim Kendall, Supervisor, Analytical Chemistry (7) Raymond L. Van Hoven, Ph.D., Scientist (8) Ellen Mank, Chemist W ild lif e In te r n a tio n a l, ltd -66APPENDIX X REVISED STUDY SUMMARY PROJECT NO.: 454-102 This study was reported using a test substance purity of 90.49%. The test substance characterization was revised on 9/7/00, following study completion, and the purity was determined to be 86.9%. The Sponsor requested that the results of this study be revised to reflect the most recent purity analyses. The results previously presented were simply corrected by the ratio of the two purities or a factor of 0.9603. No attempt was made to recalculate analytical standard concentrations or measured concentrations from original raw data. As such, minor errors due to rounding may be present. Attached is the revised summary page from the final report and the most recent certificate of analysis. AMENDED W ild lif e In te r n a tio n a l, ltd PROJECT NO.: 454-102 -67- REVISED STUDY SUMMARY BASED ON A PURITY OF 86.9% SPONSOR: SPONSOR'S REPPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: 3M Corporation Ms. Susan A. Beach Wildlife International Ltd. Easton, Maryland 21601 WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: NOMINAL TEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISM S: 454-102 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A Dietary LC50 Study with the Mallard 0,8.8, 17.6,35.1,70.3, 141, 281, 562 and 1125 ppm a.i. Experimental Start - April 22, 1999 Biological Termination - May 14, 1999 Experimental Termination - May 14, 1999 22 days Mallard (Anus platyrhynchos) Whistling Wings, Inc. Hanover, IL 61041-3512 10 days at test initiation LC50: 9 5 % CONFIDENCE LIMITS: NO MORTALITY CONCENTRATION: N O -O B SE R V E D -E F F E C T CONCENTRATION: 6 0 3 p p m a.i. 4 3 0 an d 9 1 9 p p m a.i. 141 p p m a.i. 3 5 .1 p p m a.i. AMENDED W ild lif e In ter natio n al, ltd -68- PROJECT NO.: 454-102 Centre Analytical Laboratories, Inc. 3048 Research Drive Stats College, PA 18801 www.centrelab.oom Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OFANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-OI8A 3M Product: PFOS,Lot217 Reference#! SD-018 ____________________Parity; 86.%___________________ Test N a m e S p e c ifica tio n s ------------ RHUfi------------ Parity1 86.9% Appearance [dentification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium1 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LOWS) Total % Impurity (GC/MS) Related Compounds POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (1C) 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 wt/wt% 3. 1.439 wt/wt.% 4. 6.849 wt/wt.% 5. <0.001 wt/wt.% 6. 0.005 wt/wt.% 7. 0 .001 wt./wt% 1.91 wt/wt.% 8.41 wUwt.% None Detected 0.33 wt./wt.% None Detected Not Applicable1 1. <0.015 wt/wt.% 2. 0.59 wt/wt.% 3. <0.040 wt/wt% 4. 0 .0 0 9 wt/wt% 5. 0 .0 0 6 wt/wt.% 6. <0.007 wt/wt% 7. 8.76 wt/wt.% 1. <0.1 wtVwt.% 2. <0.1 wt7wt% 3. 0.10wt./wt% 4. 0.28 wt/wt% 1. 12.48 wtVwt.% 2. 0.244 wt/wt.% 3. 1.74 wt/wt.% 4. 8.84 wt/wt.% 5. 54.1 wt7wt% COA023-018A Page 1 of 3 AMENDED W ild lif e In ter n atio n al, ltd -69- PROJECT Centre Analytical Laboratories, Inc 3048 Research' D"ri've Pitone: (814) 231-8032 Stats CollegeF,aPxA: (8161B40)1231-125w3 worw(.Bc1e4n)tr2e3la1b-1.c5o8m0 INTERIM CERTIFICATE OFANALYSIS 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 <-l 0C Re-assessment Date: 08/31/06 'Purity = 100% - (sum o f metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.905%+organic acid impurities, 0.38%+POAA, 0.33%) Total impurity from all tests = 13.07% Purity = 100% -13.07% = 86.9% 2Potassium is expected in this salt form and is therefore not considered an impurity. 'Purity by DSC is generally not applicable to materials o f low purity. No endatherm 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. TFA HFBA NFPA PFPA Trifluoroacetio acid Heptafluorobutyrie acid Nonafluoropentanoic acid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, CsFnS03'K+(MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160), COA023-018A Page 2 o f3 W ild lif e In ter n atio n al, ltd -70- PROJECT NO.: 454-102 Centre Analytical Laboratories, Inc 3048 Research Drive Stale Collega, PA 16801 www.centrelab.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A LC/MS Purity Profile: Impurity C4 C5 C6 C7 Total wt/wt.% 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 B y : Scientist, Centre Analytical Laboratories /Om /x>/ Date LmfUUciqReviewed By: John Flaherty 7 D a te Laboratory Manager, Centre Analytical Laboratories COA023-OI8A Page 3 of 3 AMENDED W il d l if e In t e r n a t io n a l, ltd -71 APPENDIX XI PROJECT NO.: 454-102 Report Amendment 1. Original Report: Amended Report: Reason: 2. Original Report: Amended Report: Reason: 3. Original Report: Amended Report: Reason: 4. Original Report: Amended Report: Reason: 5. Original Report: Amended Report: Reason: 6. Original Report: Amended Report: Reason: 7. Original Report: Amended Report: Reason: Title Page The amended report date was added. The total number of pages was changed from 65 to 72. To indicate that the report was amended and note change in pagination. Page 2 The amended report date was added and the exceptions to the compliance statement were revised. To show the amended report date, provide new signatures and dates for the amended report and to update the compliance statement. Page 3 The audit dates for the amended report were added. To show the amended report audit dates and to provide a new signature and date for the amended report. Page 4 New signatures and dates were affixed and a title change was made. To provide signatures and dates of approval for the amended report and to update a manager's title. Page 6 Appendix X, Revised Study Summary, and Appendix XI, Report Amendment, were added to the Table of Contents. To revise the Table of Contents to include the revised summary and report amendment. Page 7 A footnote and the amended report date were added to the Summary page. To direct the reader to Appendix X, which contains the revised study summary and to show the amended report date. Page 8 A footnote was added to the Test Substance text. To direct the reader to Appendix X, which contains the most recent Certificate of Analysis. AMENDED W ild lif e In ter n atio n al, ltd -72APPENDIX XI Report Amendment (continued) PROJECT NO.: 454-102 8. Amended Report: Reason: 9. Amended Report: Reason: Pages 66-70, Revised Study Summary, were added to the report. To provide additional information. Pages 71 and 72, Report Amendment, were added to the report. To provide details of changes made to the original report. AMENDMENT SIGNATURES: Study Director AoW 3W W \ Date' Wildlife International, Ltd. Management .*>&*/ Date REVIEWED BY: Q julcxo q (. d c A - m o n Quality Assurance 3 -3 5 -0 4 Date AMENDED