Document qaXEMRde0ejxxp2jGO7a71eOR

AR226-1735 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 7 1-2 OECD Guideline 205 AUTHORS: Sean P. Gallagher Courtney S. Casey Joann B. Beavers Raymond L. Van Hoven STUDY INITIATION: April 2 1, 1999 STUDY COMPLETION: April 26,2000 AMENDED REPORT DATE: March 23,2004 SUBMITTED TO 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 5 5 144 WildlifeInternational, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 72 AMENDED WILDLIFE INTERNATIONAL, 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: i l L k r P . U k Sean P. Gallagher Senior Biologist, Avian Toxicology DATE SPONSOR'S REPRESENTATIVE I A :I/ A 1 / AMENDED WILDLIFE INTERNATIONALL,TD -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 Matrix Fortification, Test Substance Prep., Analytical Sampling April 22, 1999 Feed Consumption and Analytical Sampling April 27, 1999 Necropsy, Blood Collection, Liver Weights and Bile Collection May 14, 1999 Analytical Data and Draft Report July 7, 8, and 9, 1999 Biological Data and Draft Report Aug. 30, 31, Sept. 1, 1999 Final Report April 18, 2000 Amended Final Report March 19,2004 DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT April 22, 1999 April 27, 1999 April 23, 1999 May 4, 1999 May 14, 1999 May21, 1999 July 9, 1999 July 16, 1999 September 1, 1999 April 18, 2000 September 3, 1999 April 19, 2000 March 19, 2004 March 19, 2004 Susan L. Coleman Senior Quality Assurance Representative DATE 3-aa-04 AMENDED WILD LIFE INTERNATIONAL , 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: Sean P. Gallagher Senior Biologist, Avian Toxicology CHEMISTRY PRINCIPAL INVESTIGATOR: L. Van Hoven, Ph.D Scientist, Analytical Chemistry REPORT APPROVED BY: Date 3 -236-4 Date Linda R. Mitchell Manager of Ecotox Operations Willard B. Nixon, Ph.D./ Director of Chemistry Date Date ' / ' AMENDED WILDLIFE INTERNATIONALLT.D -5- PROJECT .NO. 454-102 TABLE OF CONTENTS TITLE PAGE........................................................................................................................ GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT............................... QUALITY ASSURANCE STATEMENT ........................................................................... REPORT APPROVAL ......................................................................................................... TABLE OF CONTENTS...................................................................................................... Tables and Appendices ....................................................................................................... SUMMARY .......................................................................................................................... INTRODUCTION ................................................................................................................ OBJECTIVE ......................................................................................................................... MATERIALS AND METHODS.......................................................................................... Page 1 Page 2 Page 3 Page 4 Page 5 Page 6 Page 7 Page 8 Page 8 Page 8 Test Substance .................................................................................................................... Treatment Groups ............................................................................................................... Duration of the Test ............................................................................................................ Test Birds ............................................................................................................................ Animal Diet ........................................................................................................................ Diet Preparation .................................................................................................................. Diet Sampling ..................................................................................................................... Analytical Method .............................................................................................................. Housing and Environmental Conditions ............................................................................. Observations ....................................................................................................................... Animal Body WeightdFeed Consumption ......................................................................... Gross Necropsy................................................................................................................... Statistical Analyses ............................................................................................................. Page 8 Page 9 Page 9 Page 9 Page 10 Page 10 Page 10 Page 10 Page 13 Page 13 Page 14 Page 14 Page 14 RESULTS ............................................................................................................................. Page 14 Diet Analysis ...................................................................................................................... Mortalities and Clinical Observations ................................................................................ Body Weight and Feed Consumption................................................................................. Gross Necropsy................................................................................................................... Page 14 Page 15 Page 16 Page 17 CONCLUSION..................................................................................................................... Page 18 REFERENCES ..................................................................................................................... Page 19 WILDLIFE INTERNATIONAL. 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 i r d d a y ) 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 ........................................................................... Page 27 APPENDIX 11: Diet Formulation.................................................................................... Page 28 APPENDIX 111: Analytical Methods and Results ............................................................ Page 29 APPENDIX IV: Diet Preparation ..................................................................................... Page 43 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 ............................... Page 60 APPENDIX VIII: Changes to Protocol ............................................................................... Page 64 APPENDIX IX: Personnel Involved in the Study............................................................ Page 65 APPENDIX X: Revised Study Summary........................................................................ Page 66 APPENDIX XI: Report Amendment................................................................................ Page 7 1 AMENDED WILDLIFE INTERNATIONAL, 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 RESULTS' : 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, 5 8 6 , a n d 1171 ppma.i. TEST ANIMALS: Mallard (Anusplutyrhynchos) AGE TEST ANIMALS: 10 days of age at test initiation SOURCE TEST ANIMALS: Whistling Wings, Inc. PO BOX1-A 113 Washington St. Hanover, IL 61041-3512 STUDY COMPLETION: April 26,2000 AMENDED REPORT DATE: March 23,2004 See Appendix X for the revised nominal test concentrationsand results based upon the reanalysis of the test material following study completion. AMENDED WILDLIFE INTERNATIONALL,TD -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 (Anus plutyrhynchos) 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)'. 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 1 Following study completion, the test material was recharacterized in accordance with GLP standards and the results of reanalysis indicate a purity of 86.9%. While test concentrations have not been adjusted to reflect the newest purity, the revised study summary in Appendix X summarizes the effect of the change in purity. AMENDED WILD LIFE INTER NATIONAL , LTD -9- PROJECT NO.: 454-102 material, was identified as: IH, lH, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 276 19-97-2. 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 WILDLIFE INTERNATIONALL,TD - 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 11). 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 pprn 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 pprn 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 pprn 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 WILDLIFE INTERNATIONAL, LTD - 11 - PROJECT NO.: 454- I02 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 111, 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 lOOLC Mass Spectrometer equipped with a PerkinElmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil CIS analytical column (100 mm x 2 mm I.D., 3 ym particle size). The instrument parameters are summarized in Appendix 111, Table 1. Calibration standards of PFOS prepared in a 50% methanol : 50% NANOpureB 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 111, 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 111, 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). WILDLIFE INTERNATIONAL, 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 111, 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 111, 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 111, 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 111, 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 (lix). 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. WI LD LIFE INTERNATIoNAL , LTD - 1.5 - 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 of 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 25.2"C * 0.7"C (SD) with an average relative humidity of 53% f 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 WILDLIFE INTERNATIONAL, 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 WeighWFeed 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 a11 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,ll). 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 WILDLIFE INTERNATIONAL, LTD - 15 - PROJECT NO.: 454- 102 concentrations, standard deviations and coefficients of variation (CV) for these test concentrations were 9.8 k 0.969 ppm a i . (CV = 9.94%), 19.5 k 2.13 ppm a i . (CV = 10.9%) and 1196 k 70.2 pprn a.i. (CV = 5.87%), respectively (Appendix 111, 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, I19,99.3 and 91.6% of the nominal concentrations, respectively (Appendix 111, 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 pprn a i . test substance concentrations, respectively (Appendix 111, Table 6). A representative chromatogram of a test sample is shown in Appendix 111, 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 pprn 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 pprn a.i. treatment group single mortalities were noted on the mornings of Day 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 WILDLIFE INTERNATIONALL,TD - 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 (pc0.05) 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 pprn 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<:O.Ol 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 pprn a.i. treatment groups. These reductions were statistically significant at p<O.O1 for the 293 and 586 pprn a.i. levels. Differences at the 1171 pprn 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 pprn a i levels were reflected in significantly (p<0.05 or p<O.OI) lower mean body weights on Days 5 , 8, 15, and 22 WILDLIFE INTERNATIONAL, 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 pprn 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 pprn 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 pprn 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 WILD LIFE INTER NATI ONAL , 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 pprn 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 xi. 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 pprn 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 . . ~~ ~ ~~ WILDLIFE INTERNATIONALL,TD -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 7 1-2, 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 of Laboratory Animals. Washington, D.C. National Academy Press. 125 pp. 6 Stephan, C.E. 1977. Methods for Calculating an LC50. Pages 65-84 InAquatic 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, Vol2, No.2: 115-145. 10. Dunnet, C.W. 1955. A Multiple Comparison's Procedure for Comparing Several Treatments with a Control. Jour. Amer. Stutis. Assoc. 50:1096-1121. 11. Gulley, D.D. 1990. TOXSTAT Release 3.2. The University of Wyoming. WILDLIFE INTERNATIONAL, LTD - 20 - PROJECT NO.: 454-102 TABLE 1 Cumulative Mortality from a Mallard Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) Day0 No. Dead Per No. Exposed Exposure Period Day 1 Day2 Day3 Day4 Day5 No. Dead Per No. Exposed Post-Exposure Period Day6 Day7 Day8' Control 0 0130 0130 0130 0130 0130 0130 0/30 0130 0130 Treatment 9.1 18.3 36.6 73.2 146 293 586 1171 0/10 0110 0110 0110 0110 0110 0110 0/10 0110 0110 0110 0110 0/10 0110 0110 0/10 0/10 0110 0/10 0110 0110 0110 0/10 0110 0110 0110 0110 0/10 0110 0110 0110 0/10 0/10 0/10 0110 0110 0110 0110 0/10 0110 0110 1/10 0110 0/10 0110 0110 2/10 6/10 0/10 0110 0110 0110 0110 0110 0110 0110 0/10 0/10 0110 0110 0110 0110 0110 0110 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 pprn a.i. I - No mortalities occurred in any of the treatment levels from Day 8 to Day 22. WILDLIFE INTERNATIONALL,TD -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.) Day 0 Exposure Period Change' Day 5 Day 0-5 Post-Exposure Period Change' Day 8 Day 5-8 Total Change (Day 0-8)' Control 0 Mean SD 135 279 144 24 42 22 380 101 53 16 Treatment 9.1 Mean SD 119 226** 108** 14 36 23 317* 91 66 33 18.3 Mean SD 146 277 131 14 26 18 377 100 32 11 36.6 Mean SD 147 275 128 16 32 22 375 100 37 12 73.2 Mean SD 143 260 117" 30 55 33 343 82 90 42 146 Mean SD 143 242 19 37 loo** 21 33 1 89 48 16 293 Mean SD 586 Mean SD 129 161** 32** 17 28 33 144 137** dj** 23 27 26 220** 57 55 44 175** 36 34 41 1171 Mean SD 147 112** -37** 25 28 13 185' 31' 'Mean change is calculated separately from the mean body weights using individual body weights (See Appendix VI). %=1, 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). 245 34 198* 55 23 1 25 228 30 200* 73 188** 32 94** 54 39** 36 1.5' WILDLIFE INTERNATIONALL,TD - 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 Change' Day 15 Day 8-15 Day 22 Change' Day 15-22 Control 0 Treatment 9.1 18.3 36.6 73.2 146 293 Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD 410 640 230 30 56 47 345* 575 230 33 32 20 3 84 625 24 1 27 42 22 377 620 243 33 44 40 364 579 216 82 97 21 332* 564 232 58 56 22 211** 467** 256 49 51 35 823 183 49 24 773 198 38 15 81 1 186 81 41 828 208 78 34 782 203 135 39 688* 86 124* 90 701* 234 41 35 586 Mean SD 174** 394** 22 1 46 82 37 613** 219 91 13 1171 Me an S-~D 1852 373* 198* 634' 2512 'Mean change is calculated separately froin the inean body weights using individual body weights (See Appendix VI). *n=1, 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). Total Change'(8-22) 413 42 427 34 42 7 62 45 1 71 41 8 56 356 106 490 54 439 47 4492 WILDLIFE INTERNATIONALL,TD - 23 - PROJECT NO.: 454-102 TABLE 3 Page 1 Mean Feed Consumption (ghirdday) from a Mallard Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) Control 0 Exposure Period Day 0-5 Mean 92 SD 10 Treatment 9.1 73 18.3 91 36.6 94 73.2 77 146 105 293 44 586 36 1171 22 (-) = No data available due to mortality. Post-Exposure Period Day 6-8 125 17 117 132 125 101 159 63 55 25 WILDLIFE INTERNATIONAL, LTD - 24 - PROJECT NO.: 454-102 TABLE 3 Page 2 Mean Feed Consumption (ghirdday) from a Mallard Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) Control 0 Mean SD Post-Exposure Period Day 8-15 Day 15-22 171 180 13 20 Treatment 9.1 18.3 36.6 73.2 146 293 586 1171 72 198 86 204 65 179 148 73 159 64 109 32 114 143 106 154 (-) =z No data available due to mortality. WILDLIFE INTERNATIONAL, 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 P 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 0 2 7 1 1 4 1 1 1 0 2 4 0 0 1 0 0 1 0 0 2 0 0 1 2 2 5 0 0 1 1 1 2 0 2 3 1 1 4 WILDLIFE NTERNATIONAL, LTD - 26 - PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Control 0 0 0 0 0 0 Treatment 9.1 9.1 Pen Day0 1 015 2 015 3 015 4 015 5 015 6 015 1 015 2 015 Table 5 Cumulative Mortality (Estimated Cumulative Dose, mglkg') from a Mallard Acute Dietary Toxicity Study with PFOS No. Dead Per No. Exposed (Cumulative Dose, mgkg) Exposure Period Day 1 Day 2 Day 3 Day 4 Day 5 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 ( 4 ) 015 ( 4 ) 015 ( 8 ) 015 ( 8 ) 015 (12 ) 015 (12) 015 (16 ) 015 (16 ) 015 (20) 015 (20) No. Dead Per No. Exposed Post-Exposure Period Day6 Day7 Day8* 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 18.3 1 015 015 ( 8 ) 015 ( 1 6 ) 015 (24) 015 (32) 015 (40) 015 015 015 18.3 2 015 015 ( 9 ) 015 ( 1 8 ) 015 (27) 015 (36) 015 (45) 015 015 015 36.6 1 015 015 (19) 015 ( 3 8 ) 015 (57) 015 (76) 015 (95) 015 015 015 36.6 2 015 015 (16) 015 ( 3 2 ) 015 (48) 015 (64) 015 (80) 015 015 015 73.2 1 015 015 (31) 015 ( 6 2 ) 015 (93) 015 (12 4 ) 015 (155) 015 015 015 73.2 2 015 015 (30) 015 ( 6 0 ) 015 (90) 015 (120) 015 (150) 015 015 015 146 1 015 015 (74) 015 (148) 015 (222) 015 (296) 015 (370) 015 015 015 146 2 015 015 (101) 015 (202) 015 (303) 015 (404) 015 (505) 015 015 015 293 1 015 015 (64) 015 (128) 015 (192) 015 (256) 015 (320) 015 015 015 293 2 015 015 ( 126) 015 (252) 015 (378) 015 (504) 015 (630) 015 115 215 586 1 015 015 (175) 015 (350) 015 (525) 015 (700) 015 (875) 015 115 115 586 2 015 015 ( 153) 015 (306) 015 (459) 015 (612) 115 (765) 215 215 215 1171 1171 1 015 015 (240) 015 (480) 015 (720) 115 (960) 415 (1200) 515 515 515 2 015 015 (207) 015 (414) 015 (621) 115 (828) 215 (1035) 315 415 415 '-The LC50 value was calculatedto be approximately 628 ppm a.i. with a 95% confidence interval of 448 to 958 ppm ai.. Estimated cumulative dose is based upon the average body weight and feed consumption over the 5-day exposure period, and serves as a rough approximationof the actual amount of test substanceconsumed. *- No mortalities occurredin any of the treatment or control groups after Day 8. WILDLIFE INTERNATIONAL, LTD - 27 - PROJECT NO.: 454-102 APPENDIX I Certificate Of Analysis FC-95, Lot 217 I -Isximinty4, and x + 0) I WILD LIFE INTERNATIoNA L, LTD - 28 - PROJECT NO.: 454-102 APPENDIX I1 DIET FORMULATION WILDLIFE INTERNATIONAL LTD. GAME BIRD RATION' INGREDIENTS PERCENT ("A) Fine Corn 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 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 B12 Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (MenadioneDimethylpyrimidinolBisulfite) 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 I The guaranteed aiialysis is a minimum of 27% protein, a minimum of 2.5% crude fat and a maximum of 5% crude fiber. 2Fermentation By-products (Source of Unidentified Growth Factors) WILD LIFE INTER NATIoNAL , LTD - 29 - PROJECT NO.: 454-102 APPENDIX I11 ANALYTICAL METHODS AND RESULTS WILDLIFE INTERNATIONAL, LTD - 30 - PROJECT NO.: 454-102 INSTRUMENT: Appendix I11 Table 1 Typical LCMS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 1OOLC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil CI8column (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% NANOpureB 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 WILDLIFE INTERNATIONAL, LTD -31 - PROJECT NO.: 454-102 Appendix I11 Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454- 102-) MAB-1 Sample Type Matrix Blank Measured Concentration of PFOS' (ppm a.i.) P < LOQ MAB-2 Matrix Blank < LOQ '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). WILDLIFE INTERNATIONALL,TD - 32 - PROJECT NO.: 454-102 Appendix I11 Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454-102-) MAS- 1A MAS4A Concentrations of 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 1830 1576 86.1 MAS-6 1830 1716 93.7 Mean = 94.7 Standard Deviation = 6.99 cv = 7.38 N= 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. WILDLIFE INTERNATIONAL, LTD - 33 Appendix 111 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 of Variation (cv)' - 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. 107 CV = 10.9% 1171 18 Top Left 19 Top Right 20 Middle Left 21 Middle Right 22 Bottom Left 23 Bottom Right 1239 1221 1118 1301 1163 1133 - x = 1196 ppm a.i. SD = 70.2 pprn a.i. 102 C V = 5.87% 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 of variation was calculated using full precision of mean and standard deviation results. WILDLIFE INTERNATIONALLT,D - 34 Appendix I11 Table 5 Verification of PFOS Concentrations in Avian Diet PROJECT NO.: 454-102 Nominal Concentration (ppm a.i.) 0.0 9.1 18.3 Sample Number (S-454- 102-) 1 -- __ Sampling interval (Day) 0 _- __ PFOS Measured Concentration' (ppm ai.) < LOQ __ -- Percent of Nominal __ -- -- Mean Measured Concentration (ppm a.i.) __ 9.82 19.5* Mean Percent of Nominal _- 108' 107' 36.6 8 0 9 0 45.7 125 40.2 1I O 34.6 94.5 73.2 IO 0 11 0 77.8 106 74.5 102 71.2 97.3 146 12 0 13 0 176 120 174 1I9 172 1I7 293 14 0 15 0 274 93.8 29 1 99.3 307 105 586 16 0 17 0 1171 -- __ 550 93.9 537 91.6 523 89.4 -- __ 11962 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. '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). 'Result obtained from Table 4. WILDLIFE INTERNATIONAL, LTD - 35 Appendix I11 Table 6 PROJECT NO.: 454-102 Ambient Stability of PFOS in Avian Diet During the Mallard LC50 Study Nominal Concentration (ppm a i ) 0 Sample Number (S-454-102-) 1 - Day 0' Mean Measured Concentration - (ppm a.i.) __ Mean Percent of Nominal __ Sample Number (S-454-102-) P 39 Day 5 Measured Concentration2 - (ppm a.i.) < LOQ Mean Measured Mean Percent Concentration of - (ppm a.i.) _- 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 I10 42 47.9 52.4 1303 43 56.8 73.2 IO, 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 29 1 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. 2The 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). 3The Dav 5 value is consistent with the Day 0 mean measured concentration value for this level. No analytical measuring bias is indicated. WILD LIFE INTERNAT IoNAL , LTD - 36 - PROJECT NO.: 454-102 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN AVIAN DIET Prepare matrix fortification samples in the desired avian fced stock using thc dry mix technique 1 Dry Mix Tcchniquc For the high-level matrix fortification sample, weigh the requisite quantity of Wildlifc lntcmational Ltd. (WIL) ration into a weigh boat. 1 Weigh the requisite quantity of test substancc (PFOS) into a beaker. 1 Add !4 of the WIL ration and the test substance to a larger beaker. 1 Rinsc the beakcr that containcd the PFOS with small portions of the remaining ration and transfer all portions to the larger beaker. 1 Mix the contents of the larger beaker well and transfcr the mixture to a Waring blender. 1 Blend the mixture for -5 minutes stopping at 1 minutc intcrvals to scrapc down the sides of the blender. During the third interval transfer the fortified feed to a beaker, mix well and rcturn the mixturc to thc blcnder to complete mixing in the spccificd time. 1 Prepare the next two matrix fortification levels by serial dilutions. Follow the same procedure described for the high-level matrix fortification except wcigh thc appropriatc quantity of fortified matrix (high or mid- level) rather than the test substancc. 1 Weigh IO-g samples of the matrix blank, matrix fortification and test samplcs into wcigh boats and transfer to 16-02. Frcnch-square glass bottles. Rccord thc wcights. I For each sample, measure 100 mLs of methanol with a graduatcd cylindcr and transfcr volume to the French-squarc bottle. 1 Cap bottles and place on shaker table. Allow the samples to shakc for a minimum of 30 minutes at 250 rpm 1 Vacuum filter each sample with qualitative filter paper and rinsc rctaincd fccd 3 times with methanol into the filtratc 1 Transfcr the filtrate to a 200-mL volumetric flask and bring the flask to volume with mcthanol. 1 Prcparc appropriatc dilution(s) to bring final concentration into thc calibration range of the LCMS methodology. Use methanol for intermediate dilutions, if required. For all final dilutions usc 50% mcthanol : 50% NANOpureB water solution containing 0.100 mg 4H PFOS (internal stan rd)/L and 0.05% formic acid (viv). J Ampulatc and submit samples for LCiMS analysis. Figure 1. ~~ Analytical method flowchart for the analysis of PFOS in avian diet. WILDLIFE INTERNATIONAL, LTD - 37 - Appendix 111 PROJECT NO.: 454-102 1.50 1 0.000 0.100 0.200 0.300 Concentration (Ratio) ~ ~~~~ ~ 0.400 0.500 ~ Figure 2. A typical calibration curve for PFOS. Slope = 2.77397; Intercept = 0.01894; r = 0.9981. Curve is weighted (l/x). WILDLIFE INTERNATIONAL, LTD - 38 - Appendix 111 PROJECT NO.: 454- 102 intensity: 1995 cps 167 10 1 9 9 8 7 6 5 4 3 2 1 .- < - '.I - 04 1. v-r--: 31 1.30 61 2.55 a -*--rs 1. I 1- 91 121 151 3.81 5.06 6.32 181 7.57 21 1 8.83 scan Time Figure 3. A representative ion chromatogram of a low-level (0.00229 mg a.i./L) PFOS standard. WILDLIFEINTERNATIONAL, LTD - 39 - Appendix Ill PROJECT NO.: 454-102 100 90 80 70 60 50 40 30 20 10 0 intensity: 26954 cps 166 1.30 2.55 3.81 5.06 6.32 7.57 8.83 Time Figure 4. A representative ion chromatogram of a high-level (0.0457 mg a.i./L) PFOS standard. WILDLIFE INTERNATIONAL, LTD - 40 Appendix I11 PROJECT NO.: 454-102 10 9 4 8 7 6 5 4 3 2 1 IL 180 I166 intensity: 230 cps 1.30 2.55 3.81 5.06 6.32 7.57 8.83 Time Figure 5. A representative chromatogram of a matrix blank sample (454-102-MAB-1). The arrow indicates the retention time of PFOS. WILDLIFE INTERNATIONAL, LTD -41 - Appendix I11 PROJECT NO.: 454-102 intensity: 6238 cps 1 "3O 7 7 is4 3 I 144 1 2 1 31 61 91 1.30 2.55 3.8 1-2.1 5.06 151 6.32 181 7.57 21 1 8.83 scan Time Figure 6. A representative chromatogram of a matrix fortification sample (454-102-MAS-l A). WILD LIFE INTER NATIONAL , LTD - 42 - Appendix I11 PROJECT NO.: 454-102 intensity: 7248 cps 1.30 2.55 3.81 5.06 6.32 7.57 8.83 Time Figure 7. A representative chromatogram of a test sample (454-102-2). WILDLIFE INTERNATIONAL, 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 (R) 0.0910 0.1818 0.3638 0.7282 1.4659 2.9123 5.8239 1 1.6483 Basal Ration (a> 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. WILDLIFE INTERNATIONAL,LTD PROJECT NO.: 454-102 - 44 - APPENDIX V Cumulative Mortality by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Experimental No. Dead Per No. Exposed Group Pen Exposure Period (ppm ai.) Day0 Day 1 Day2 Day3 Day4 Day5 Control 0 1 015 015 015 015 015 015 0 3 L 015 015 015 015 015 015 0 3 015 015 015 015 015 015 0 4 015 015 015 015 015 015 0 5 015 015 015 015 015 015 0 6 015 015 015 015 015 015 Treatment 9.1 1 015 015 015 015 015 015 9.1 2 015 015 015 015 015 015 18.3 1 015 015 015 015 015 015 18.3 2 015 015 015 015 015 015 36.6 1 015 015 015 015 015 015 36.6 2 015 015 015 015 015 015 73.2 1 015 015 015 015 015 015 73.2 2 015 015 015 015 015 015 146 1 015 015 015 015 015 015 146 2 015 015 015 015 015 015 293 1 015 015 015 015 015 015 293 2 015 015 015 015 015 015 586 1 015 015 015 015 015 015 586 2 015 015 015 015 015 1I5 1171 1 015 015 015 015 1I5 415 1171 2 015 015 06 015 1 I5 215 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 . * - No mortalities occurred in any of the treatment or control groups after Day 8. No. Dead Per No. Exposed Post-Exposure Period Day6 Day7 Day&!* 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 01.5 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 015 1I5 215 015 1I5 1I5 215 215 215 515 515 515 315 415 415 WILDLIFE INTERNATIONALL,TD - 45 - APPENDIX VI Individual Body Weights (8) from a Mallard Acute Dietary Toxicity Study with PFOS Page 1 Experimental Group (ppm a i ) Bird Day 0 Day 5 Change Day O-5 Day8 0 1 132 302 170 404 2 136 292 156 381 3 154 319 165 415 4 158 319 161 429 5 169 309 140 418 Mean 150 308 158 409 SD 15 12 12 18 PROJECT NO.: 454-102 Change Day 5-8 102 89 96 110 109 101 9 Total Change (0-8) 272 245 261 27 1 249 260 12 0 1 136 282 146 399 117 263 2 142 284 142 394 110 252 3 152 299 147 409 110 257 4 152 312 160 430 118 278 5 177 345 168 484 139 307 Mean 152 304 153 423 119 27 1 SD 16 26 11 37 12 22 0 1 125 265 140 372 107 247 2 134 282 148 37 1 89 237 3 149 304 155 440 136 29 1 4 154 292 138 378 86 224 5 166 328 162 430 102 264 Mean 146 294 149 398 104 2.53 SD 16 24 10 34 20 26 0 1 124 27 1 147 376 105 252 2 135 263 128 360 97 225 3 151 300 149 408 108 257 4 163 333 170 432 99 269 5 170 35 1 181 453 102 283 Mean 149 304 155 406 102 257 WILDLIFE INTERNATIONALL,TD - 46 APPENDIX VI Individual Body Weights (8) from a Mallard Acute Dietary Toxicity Study with PFOS Page 2 Experimental Group (ppm a.i.) 0 Bird 1 2 3 4 5 Mean SD Day 0 107 126 116 107 111 113 8 Day 5 222 267 203 203 248 229 28 Change Day 0-5 115 141 87 96 137 115 24 Day 8 298 360 266 284 348 311 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 1 100 235 135 335 100 235 2 100 232 132 328 96 228 3 102 233 131 317 84 215 4 108 267 159 383 116 275 5 102 210 108 299 89 197 Mean 102 235 133 332 97 230 SD 3 20 18 32 12 29 Group Total Mean 135 279 144 3 80 101 245 SD 24 42 22 53 16 34 ~~~ ~ - 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.) 9.1 ~ ~ ~ ~ ~ ~~~~~ ~ Bird Day 0 Day 5 1 123 244 2 118 23 1 3 151 286 4 108 209 5 119 23 1 Mean 124 240 SD 16 29 Change Day 0-5 121 113 135 101 112 116 13 Day 8 349 334 399 313 332 345 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 49 146 1 2 123 250 127 345 95 3 127 237 110 343 106 4 112 216 104 316 100 5 111 215 104 294 79 Group Total Mean 114 213 99 289 76 SD 12 41 29 83 43 Mean 119 226** 108** 317* 91 SD 14 36 23 66 33 *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). 50 222 216 204 183 175 71 198* 55 WI LDLI FE I NTERNATI oNA L, 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 145 SD 14 Day 5 26 1 297 270 286 294 282 16 Change Day0-5 138 156 120 134 134 136 13 Day8 355 42 1 361 390 39 1 3 84 27 Change Day 5-8 94 124 91 104 97 102 13 Total Change 232 280 21 1 238 23 1 238 25 18.3 1 130 240 110 327 87 197 2 137 266 129 359 93 222 3 145 268 123 366 98 22 1 4 151 258 107 366 108 215 5 171 333 162 43 3 100 262 Group Total Mean 147 273 126 370 97 223 SD 16 35 22 39 8 24 Mean 146 277 131 377 100 23 1 SD 14 26 18 32 11 25 WILDLIFE INTERNATIONALL,TD - 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 149 SD 21 Day 5 26 1 256 269 270 335 278 32 Change Day 0-5 130 124 127 110 156 129 17 Day 8 3 72 356 369 353 433 3 77 33 Change Day 5-8 111 100 100 83 98 98 10 Total Change 24 1 224 227 193 254 228 23 36.6 1 131 230 99 316 86 185 2 136 287 151 402 115 266 3 147 263 116 379 116 232 4 151 255 104 344 89 193 5 160 324 164 427 103 267 Group Total Mean 145 272 127 3 74 102 229 SD 12 36 29 44 14 39 Mean 147 275 128 375 100 228 SD 16 32 22 37 12 30 WILDLIFE INTERNATIONALL,TD - 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 140 SD 28 Day 5 203 219 286 290 338 267 56 Change Day 0-5 90 106 144 136 159 127 28 Day 8 28 1 288 390 379 480 364 82 Change Day 5-8 78 69 104 89 142 96 29 Total Change 168 175 248 225 301 223 55 73.2 1 109 222 113 302 80 2 129 254 125 332 78 3 139 243 104 340 97 4 147 196 49 175 -2 1 5 204 352 148 458 106 Mean 146 253 108 321 68 SD 36 59 37 101 51 Group Mean 143 260 117* 343 82 Totai SD 30 55 33 90 42 *Statistically different froin the control group at p < 0.05 (Dunnett's t-test). 193 203 20 1 28 254 176 86 200* 73 WILDLIFE INTERNATIONAL, LTD -51 - PROJECT NO.: 454-102 APPENDIX VI Individual Body Weights (8) 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 142 SD 21 Day 5 169 248 27 1 254 282 245 44 Change Day 0-5 61 110 120 103 118 102 24 Day 8 236 330 375 34 1 380 332 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 89 280 76 2 139 219 80 287 68 3 144 229 85 348 119 4 159 26 1 102 358 97 5 158 285 127 3 74 89 Mean 143 240 97 329 90 SD 18 33 19 43 20 Group Totai Mean 143 242 loo** 33 1 89 SD 19 37 21 48 16 P **Statistically different froin the control group at p < 0.01 (Dunnett's t-test). 165 148 204 199 216 186 29 - 188** 32 WILDLIFE INTERNATIONAL, 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.) 293 Bird 1 2 3 4 5 Mean Day 0 115 139 140 155 112 132 Day 5 142 119 156 187 134 148 Change Day0-5 27 -20 16 32 22 15 Day8 179 165 217 29 1 203 21 1 Change Day 5-8 27 -20 16 32 22 15 Total Change 64 26 77 136 91 79 293 1 123 205 82 2 120 183 63 3 151 139 -12 4 106 158 52 5 124 185 61 Mean 125 174 49 SD 16 26 36 Group Total Mean 129 SD 17 161** 32** 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 23 1 68 220** 55 82 63 -12 52 61 49 36 57** 44 166 153 117 14 113 69 94** 54 WILDLIFE INTERNATIONALL,TD - 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.) 586 Bird 1 2 3 4 5 Mean SD Day 0 122 135 152 154 165 146 17 Day 5 105 143 160 129 195 146 34 Change Day 0-5 -17 8 8 -25 30 1 22 Day 8 126 209 217 143 174 46 Change Day 5-8 21 66 57 -52 23 54 Total Change 4 74 65 -22 30 47 586 1 105 114 9 2 120 130 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. **Statisticallydifferent 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 39** 36 WILDLIFE INTERNATIONAL, LTD - 54 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 10 PROJECT NO.: 454-102 Experimental Group Change Change Total 1171 1 124 88 -39 2 140 3 145 96 -49 4 150 I18 -32 5 166 Mean 145 IO0 -40 SD 15 17 9 1171 1 128 87 -4 1 2 110 3 145 132 -13 4 157 5 200 154 -46 185 31 -1 5 Mean 148 124 -33 185 31 -1 5 SD 34 34 18 Group Total Mean 147 112** -37** 1852 312 15' 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). WI LD LIFE INTERNATIoNAL , LTD - 55 - APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 11 Experimental Group Change (ppm a.i.) Bird Day 8 Day 15 Day 8-15 Day 22 0 1 404 72 1 317 895 2 381 633 252 780 3 415 685 270 86 1 4 429 692 263 822 5 418 679 26 1 847 Mean 409 682 273 84 1 SD 18 32 26 43 PROJECT NO.: 454-102 Change Day 15-22 174 147 176 130 168 159 20 Total Change (8-22) 49 1 399 446 393 429 432 40 0 1 399 606 207 810 204 41 1 2 394 604 210 79 1 187 397 3 409 638 229 819 181 410 4 430 543 113 730 187 300 5 484 752 268 924 172 440 Mean 423 629 205 815 186 392 SD 37 77 57 70 12 54 0 1 372 583 21 1 779 196 407 2 371 613 242 823 210 452 3 440 636 196 859 223 419 4 378 594 216 794 200 416 5 430 627 197 816 189 386 Mean 398 61 1 212 814 204 416 SD 34 22 19 31 13 24 Group Mean 410 640 230 823 183 41 3 Total SD 30 56 47 49 24 42 WILDLIFE INTERNATIONALLT,D - 56 APPENDIX VI Individual Body Weights (8) from a Mallard Acute Dietary Toxicity Study with PFOS Page 12 PROJECT NO.: 454-102 Experimental Group (ppm a i ) 9.1 Bird Day 8 1 349 2 334 3 399 4 313 5 332 Mean 345* SD 33 Day15 Change Days-15 Day22 602 253 82 1 549 215 730 616 217 802 562 249 767 546 214 744 575 230 773 32 20 38 Change 219 181 186 205 198 198 15 Total Change 472 396 403 454 412 427 34 Experimental Group Change Change (ppm a i ) Bird Day 8 Day 15 Day 8-15 Day 22 Day 15-22 18.3 1 355 61 1 256 81 1 200 2 42 1 693 272 938 245 3 361 578 217 713 135 4 3 90 626 238 794 168 5 39 1 618 23 1 799 181 Mean 384 625 242 81 1 186 SD 27 42 22 81 41 *Statistically different from the control group at p < 0.05 (Dunnett's t-test). Total Change 465 517 352 404 408 427 62 WILDLIFE INTERNATIONALLTD, - 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.) 36.6 Bird Day 8 1 372 2 356 3 369 4 353 5 433 Mean 377 SD 33 Day 15 Change Day 8-15 Day 22 Change Day 15-22 677 305 942 265 607 25 1 817 210 613 244 793 180 559 206 746 187 642 209 840 198 620 243 828 208 44 40 73 34 Total Change 570 46 1 424 393 407 45 1 71 Experimental Group (ppm a.i.) 73.2 Bird Day 8 1 28 1 2 288 3 390 4 379 5 480 Mean 364 SD 82 Day 15 Change Day 8-15 Day 22 Change Day 15-22 482 20 1 648 166 480 192 646 166 636 246 864 228 599 220 796 197 699 219 955 256 579 216 782 203 97 21 135 39 Total Change 367 358 474 41 7 475 41 8 56 WILDLIFE INTERNATIONALL,TD - 58 - PROJECT NO.: 454-102 APPENDIX VI Individual Body Weights (8) from a Mallard Acute Dietary Toxicity Study with PFOS Page 14 Experimental Group (ppm a i ) 146 Bird Day 8 1 2 3 4 5 Mean SD 236 330 375 34 1 380 332* 58 Day 15 Change Day8-15 Day22 475 239 659 569 239 739 63 1 256 738 570 229 754 576 196 550 564 232 688* 56 22 86 Change 184 170 107 184 -26 124* 90 Total Change 423 409 3 63 413 170 356 106 Experimental Group Change Change (ppm a.i.) Bird Day 8 Day 15 Day 8-15 Day 22 Day 15-22 293 1 179 447 268 73 1 284 2 165 415 250 646 23 1 3 217 526 309 748 222 4 29 1 516 225 704 188 5 203 430 227 676 246 Mean 211** 467** 256 701* 234 SD 49 51 35 41 35 *Statistically different from the control group at p < 0.05 (Dunnett's t-test). **Statistically different froin the control group at p < 0.01 (Dunnett's t-test). Total Change 552 48 I 53 1 413 473 490 54 WILDLI FE INTERNATIONALLT,D - 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 ai.) 586 Bird 1 2 3 4 5 Mean SD Day 8 126 209 217 143 174 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 I97 546 206 394 22 1 613 219 82 37 91 13 Total Change 399 497 45 8 403 439 47 Experimental Group Change Change (ppm a i . ) Bird Day8 Day 15 Day 8-15 Day 22 Day 15-22 1171 1 2 3 4 5 185 383 198 634 25 1 Mean 185' 383' 19S2 634' 2512 SD 2n=1, could not be evaluated statistically using Dunnett's t-test. Total Change 449 4492 WILDLIFE INTERNATIONALL,TD - 60 APPENDIX VI1 PROJECT NO.: 454-102 Feed Consumption (ghirdday) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Page 1 Experimental Group (ppin a.i.) Pen Exposure Period Day 0-5 Post-Exposure Period Day 6-8 Control 1 109 145 2 92 140 3 85 117 4 93 132 5 82 100 6 94 116 Mean 92 125 WILDLIFE INTERNATIONAL, LTD -61 - PROJECT NO.: 454-102 APPENDIX VI1 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 73 130 2 72 104 Mean 73 117 18.3 1 90 135 2 91 128 Mean 91 132 36.6 1 103 129 2 85 120 Mean 94 125 73.2 1 79 109 2 74 94 Mean 77 101 146 1 89 102 2 121 217 Mean 105 159 293 1 28 61 2 59 65 Mean 44 63 586 1 40 43 2 32 67 Mean 36 55 1171 1 23 25 2 22 26 Mean 22 25 WILDLIFE INTERNATIONALL,TD - 62 APPENDIX VI1 PROJECT NO.: 454-102 Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Page 3 Experimental Group (ppm ai.) 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 WILDLI FE INTERNATI oNA L, LTD -63 APPENDIX VI1 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 WILDLIFE INTERNATIONAL, LTD - 64 APPENDIX VI11 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 pprn ai.. 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. WILDLIFE INTERNATIONALL,TD - 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) 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 Mark, Chemist W I L D L I F E INTERNATIONAL, LTD - 66 APPENDIX 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 WILDLIFE INTERNATIONAL, 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: NOMINAL TEST CONCENTRATIONS : TEST DATES: 454-1 02 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A Dietary LC50 Study with the Mallard 0,8.8, 17.6,35.1,70.3, 141,281,562and1125ppma.i. Experimental Start - April 22, 1999 Biological Termination - May 14, 1999 Experimental Termination - May 14, 1999 I TEST ORGANISM: SOURCE OF TEST ORGANISMS: Mallard (Anusplatyrhynchos) Whistling Wings, Inc. Hanover, IL 61041-3512 AGE OF TEST ORGANISMS: 10 days at test initiation I 95% CONFIDENCE LIMITS: NO MORTALITY CONCENTRATION: 430 and 919 ppm a.i. 141 ppma.i. AMENDED WILDLIFE INTERNATIONAL, LTD - 68 - PROJECT NO.: 454-102 C a t r e Analytkal Laboratories, Inc. e College, PA 18801 w.oBnlreIat).m Fax'(814) 231-1253 (814) 231-1580 INTERM CERTIFICATEOF ANALYSIS Rcvisbon 3 Centre Analytical Laboratorla C O A Reference#: 023-018A 3M Produet: PFOS,Lot 217 Reference #: SD-018 Test Nlmc I Pllrlty' I Purity: 86.9% spcdflCleO M Ruult 86.9% coafonns Poaitive 1. Calcium 2. Magnesium 3. sodium 4. Potassium' 5. Nickcl 7. Mangancsc Total % Impurity @MR) Total % Im.purit.y (Lc/MV Total % Imouritv I 1. O.O05wt/w&% 2. 0.001 wtM% 3. 1.439wtM.% 4. 6.849 wtJwi.% 5. <0.001wt/Wt% 6. 0.005 wt./wt.% 7. <0.00lwt.Iwt% 1.91 wt/wt.% 8.41 wt/wt.% None Detected POAA RcsidoalSolventsW A ) I puritlby DSC I Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. N m 5. Nitrite 6. Phorphate I ' 7. SUl$tc' 3rganic Acids (IC) I . "FA 2. PFPA 2. Hydmgcn 3. Nibugcn 4. Sulfur 5. Fluorine - TheoreticalValuc 17.8% Thmrctical Value = 0% lkoretical Value = 0% 4. Theoretical Value = 5.95% 5. ThmrrticplValue =W A 1. ~0.015Wt/wt% 2. 0.59 wtlwt.% 3. ~ . 0 4 0 w t / w r % 4. <0.009wtJwt% 5 . <0.006wt/wt.% 6. <O.O07dwt% 7. 8.76wtlwt.% I . <O.I MJ\IR% 2. <0.1 wiM% 3. O . I O w t . / w l % 4. 028Wt/wl% 1. 12.48 wt./wt.% 2. 0.244wt.M.% 3. 1.74wtJwt.% 4. 8.84wt/wt.% 5 . 54.1 wtJwt.% CUA023-018A Plgc I of 3 AMENDED WILDLI FE INTERNATIONAL, LTD - 69 - PROJECT NO.: 454-102 afi!!l\Cartrv Anamical laboratorks, Inc. 3048ResearchDrlve State College,PA 16801 www.centrelab.com Phone: (814) 231-8032 ax: (814)231-1253or(814)231-1580 lNTERLU CERTIFICQTEOFANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023418A DateofLast Analysis: 08/31/00 Expiration Date: OW3 1/06 StorageConditions: F r o m 5 10C Re-assessment Date: 08/3 1/06 'Purity = 1 W o - (sum of metal impurities, 1.45%+LCIMS impurities, 8Al%+Inorganic Fluondc, 0.59%+h'h4R impuritics, 1.905%+0rganic acid impuritiw, 0.38%+POAA, 0.33%) Total impurity &om all tests = 13.07% Purity = 100%- 13.07%=86.9% zPotassium is expected in this salt form and is therefore not considered an impurity. 'Purity by DSC is gencrallynot applicableto materialsof low purity. No endothermwas observed for this sample. 'Sulfur in the sample appears to be convnted to SO4 and hence detectedusing the inorganic anion method conditions. Theanionresult agreeswell with the sulfur determination in the elancntalanalysis, lending confidence to this intapretation. Basal on the results, the SO4 is no1 considered an impurity. %A HFBA NFPA PFPA Trifluomacetio acid Heptaflnombutyric acid Nonafluorwpentanoic acid Pen~uotupmpanoicacid ?heoretical value calculations based on thc empirical formula, GF17S@X'(MW=538) This work was conductedunder EPA Good LabomtoryPractice Standards(40 O R 160). COA023-018A Page 2 or3 AMENDED WILDLIFE INTERNATIONAL, LTD - 70 - PROJECT NO.: 454-102 INTERIM CERTIFICATEOF ANALYSIS Revhion 3 Centre Analytical Laboratories COA Reference #: 023-018A LCMS Purity Profile: Imparity c4 cs C6 Wwt Yo 1.22 1.33 4.72 Note: The C4 and C6 values were calculatedusingthe C4 and C6 standard calibration curves, respectively. The C5 value was calculatedusing the avemge result fiom the C4 and C6 standard curves. Likewise,the C7 value was calculated using the avenge result from the C6 and C8 standard c w e s . Prepared By: Charlcs Si Scientist, Centre AnalyticalLaboratoris hn Flahcrty Date !fhratory Manager, Cenh-e Analytical Laboratories COAOZ3-OISA Page 3 of 3 AMENDED WILDLIFE INTERNATIONAL,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 WILDLIFE INTERNATIONAL, ~m - 72 APPENDIX 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: JXA- 9 . SaLL Study Director Date' Wildlife International, Ltd. Management REVIEWED BY: Quality Assurance 3-aa- 04 Date AMENDED