Document 0Lz49r41qpxq2wDKJojr5Lpvm

AR226-1747 PFOS: A DIETARY LC50 STUDY WITH THE NORTHERN BOBWHITE WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103 3M LAB 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 69 AMENDED W il d l if e In t e r n a t io n a l , ltd 2- - PROJECT NO.: 454-103 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Dietary LC50 Study with the Northern Bobwhite WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103 3M LAB REQUEST NO.: U2723 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. A nalyses o f liver and sera sam ples conducted by 3M Corporation w ere not performed in accordance w ith GLP standards and results o f these analyses are being reported separately. STUDY DIRECTOR: Sean P. Gallagher Senior Biologist DATE SPONSOR'S REPRESENTATIVE 9 DATE AMENDED W il d l if e In t e r n a t io n a l , ltd -3 - PROJECT NO.: 454-103 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 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. 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 Test Substance Prep. & Analytical Sampling April 22, 1999 April 22, 1999 April 23, 1999 Matrix Fortification April 22, 1999 April 22, 1999 April 23, 1999 Feed Consumption & Analytical Sampling April 27, 1999 April 27, 1999 May 4, 1999 Analytical Data and Draft Report July 7, 8, 9, 1999 July 9, 1999 July 16, 1999 Biology Data and Draft Report August 26, 27, 30,31, 1999 August 31, 1999 September 13, 1999 Final Report April 17-18, 2000 April 18, 2000 April 19, 2000 Amended Final Report March 19, 2004 March 19, 2004 March 19, 2004 Susan L. Coleman Senior Quality Assurance Representative DATE 3 ~3i- \ AMENDED W il d l if e In t e r n a t io n a l , ltd -4- PROJECT NO.: 454-103 AMENDED REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Dietary LC50 Study with the Northern Bobwhite WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103 3M LAB REQUEST NO.: U2723 STUDY DIRECTOR: Sean P. Gallagher 0 Senior Biologist, Avian Toxicology CHEMISTRY PRINCIPAL INVESTIGATOR: j j j . l L - RaymiinfPL.'Van Hoven, Ph.D Scientist, Analytical Chemistry REPORT APPROVED BY: Linda R. Mitchell Manager of Ecotox Operations Date 3-33 Date 23 Date JQ&H Willard B. Nixon, Ph. Director of Chemistry Date AMENDED W il d l if e In t e r n a t io n a l , ltd -5 - PROJECT NO.: 454-103 TABLE OF CONTENTS TITLE PAGE........................................................................................................................ Page 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT................................... Page2 QUALITY ASSURANCE STATEMENT........................................................................... Page 3 REPORT APPROVAL............................................................................................................. Page4 TABLE OF CONTENTS.......................................................................................................... Page5 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..............................................................................................................................Page10 Diet Preparation...................................................................................................................Page 10 Diet Sampling.......................................................................................................................... Page10 Analytical Method...............................................................................................................Page 11 Housing and Environmental Conditions............................................................................. Page 13 Observations............................................................................................................................ Page14 Animal Body Weights/Feed Consumption.............................................................................. Page14 Gross Necropsy........................................................................................................................ Page14 Statistical Analyses.................................................................................................................. Page14 RESULTS.................................................................................................................................. Page15 Diet Analysis...................................................................................................................... Page 15 Mortalities and Clinical Observations................................................................................ Page 15 Body Weight and Feed Consumption...................................................................................... Page17 Gross Necropsy........................................................................................................................ Page17 CONCLUSION.......................................................................................................................... Page18 REFERENCES.......................................................................................................................... Page19 W il d l if e In t e r n a t io n a l , ltd 6- - PROJECT NO.: 454-103 TABLE OF CONTENTS - continued - TABLES AND APPENDICES TABLE 1: Cumulative Mortality from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS......................................................................................... Page 20 TABLE 2: Mean Body Weight (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS............................................................................................... Page21 TABLE 3: Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS................................................... Page 23 TABLE 4: Group Gross Pathological Observations from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS................................................... Page 25 TABLE 5: Cumulative Mortality (Estimated Cumulative Dose, mg/kg) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS......................... Page26 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 Northern Bobwhite Acute Dietary Toxicity Study with PFOS............................................ Page 44 APPENDIX VI: Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS............................................ Page 45 APPENDIX VII: Feed Consumption (g/bird/day) by Pen from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS........................................................................................ Page58 APPENDIX VIII: Changes to Protocol....................................................................................Page61 APPENDIX IX: Personnel Involved in the Study................................................................Page62 APPENDIX X: Revised Study Summary............................................................................. Page63 APPENDIX XI: Report Amendment..................................................................................... Page68 AMENDED W il d l if e In t e r n a t io n a l , ltd -7- PROJECT NO.: 454-103 SUMMARY SPONSOR: 3M Corporation TEST SUBSTANCE: PFOS WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103 STUDY: PFOS: A Dietary LC50 Study with the Northern Bobwhite RESULTS1: The dietary LC50 value for northern bobwhite exposed to PFOS was determined to be 220 ppm a.i. with a 95% confidence interval of 164 ppm a.i. to 289 ppm a.i. The slope of the concentration-response curve was 7.005 and the chi-square value was 0.023. The no mortality concentration was 73.2 ppm a.i. Based upon treatment related mortality, signs of toxicity and effects upon body weight gain at the 146 ppm a.i. test concentration, the no-observed-effect concentration was 73.2 ppm a.i. TEST DATES: Hatch - April 12, 1999 Acclimation - April 12-22, 1999 Experimental Start-April 22, 1999 Experimental Termination - May 14, 1999 NOMINAL TEST CONCENTRATIONS: 0, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 ppm a.i. TEST ANIMALS: Northern Bobwhite (Colinus virginianus) AGE TEST ANIMALS: 10 days of age at test initiation SOURCE TEST ANIMALS: Wildlife International Ltd. Production Flock 8598 Commerce Drive Easton, Maryland 21601 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 il d l if e In t e r n a t io n a l , ltd 8- - PROJECT NO.: 454-103 INTRODUCTION This study was conducted by Wildlife International Ltd. for 3M Corporation at the Wildlife International Ltd. avian 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 454-103 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 Northern Bobwhite (Colinus virginianus) 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 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 o f the change in purity. AMENDED W il d l if e In t e r n a t io n a l , ltd -9- PROJECT NO.: 454-103 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 seven test concentrations and a control group. Thirty northern bobwhite chicks were assigned to the control group and ten northern bobwhite chicks were assigned to each of the treatment groups. The 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 chicks each. Nominal dietary concentrations used in this study were 0, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 parts per million active ingredient (ppm a.i.) of PFOS. 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 surviving treatment and control birds were euthanized and liver tissue, blood, and bile samples were collected for analysis. The remaining birds were fed basal ration until Day 22. On Day 22, these birds were euthanized and also sampled for liver weight, blood, and bile. Duration of the Test The primary phases of this test and their durations were: 1. Acclimation - 10 days. 2. Exposure - 5 days. 3. Post-exposure observation - 3 or 17 days Test Birds All northern bobwhite (Colinus virginianus) were 10 days of age and appeared to be in good health at initiation of the test. The birds were obtained from Wildlife International Ltd. Production Flock, Easton, MD and were hatched on April 12, 1999. Birds ranged in weight from 18 to 23 grams at test initiation. The birds used in this study were immature and could not be differentiated by sex. W il d l if e In t e r n a t io n a l , ltd - 10 - PROJECT NO.: 454-103 All birds were from the same hatch, pen-reared and phenotypically indistinguishable from wild birds. All birds were acclimated to the caging and facilities from the day of 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). The chicks were given a vitamin supplement in their water from the day they were hatched until the initiation of the test. 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 18.3, 36.6, 73.2, 146, 293, 586, 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 18.3 ppm a.i. a.i. test diet and six samples from the 1171 ppm a.i. test diet 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 under test conditions. 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. W il d l if e In t e r n a t io n a l , ltd - 11 - PROJECT NO.: 454-103 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 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 that 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 Flewlett-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 Ci8 analytical column (100 mm x 2 mm I.D., 3 pm particle size). The instrument parameters are sum m arized in A ppendix 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. W il d l if e In te r n a tio n a l , ltd - 12 - PROJECT NO.: 454-103 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). 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 454-103-2, 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 W il d l if e In t e r n a t io n a l , ltd - 13 - PROJECT NO.: 454-103 (0.2749 - 0.01894) x 0.100 mg/L 2.77397 = 0.00923 mg a.i./L Note: I.S. = internal standard. 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 Beacon Steel Products Co. (Model No. B735Q). Each pen had floor space that measured approximately 72 X 90 cm. Ceiling height was approximately 23 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 leg 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 27.3C 1.2C (SD) with an average relative humidity of 31% 14% (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 W il d l if e In t e r n a t io n a l , ltd - 14- PROJECT NO.: 454-103 approximately 139 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 typically observed at least twice 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, Day 8, 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 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, livers were weighed and liver tissue, blood, 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 using 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. W il d l if e In t e r n a t io n a l , ltd - 15 - PROJECT NO.: 454-103 RESULTS Diet Analysis Avian diet samples were collected from the 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 concentrations, standard deviations and coefficients of variation (CV) for these test concentrations were 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 verily 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 101, 122, 104, 101, 109, 114 and 102% of the Day 0 values for the 18.3, 36.6, 73.2, 146, 293, 586 and 1171 ppm a.i. test substance concentrations, respectively (Appendix III, Table 6). A representative chromatogram of a test sample is shown in Appendix III, Figure 7. Mortalities and Clinical Observations One incidental mortality occurred in the control group during the course of the study (Table 1 and Appendix V). On the morning of Day 5, one bird was noted with a broken leg and was subsequently euthanized on Day 6. Additionally, two birds in the control group were intermittently noted with foot lesions associated with cage mate aggression. Otherwise, all control birds were normal in appearance and behavior throughout the test. No treatment related mortalities or overt signs of toxicity were observed in the 18.3, 36.6, or 73.2 ppm a.i. treatment groups. One bird in the 18.3 ppm a.i. treatment group was noted as lame from Day 6 through Day 8, and blood of undetermined origin was noted on the underside of one bird from the 73.2 ppm a.i. treatment group on Day 22. Otherwise, all birds in the 18.3, 36.6 and 73.2 ppm a.i. treatment groups were normal in appearance and behavior throughout the test period. W il d l if e In t e r n a t io n a l , ltd - 16- PROJECT NO.: 454-103 There was 11% (1 of 9) mortality in the 146 ppm a.i. treatment group, 80% (8 of 10) mortality in the 293 ppm a.i. treatment group and 100% (10 of 10) mortality in the 586 and 1171 ppm a.i. treatment groups. In the 146 ppm a.i. treatment group, one bird was euthanized on Day 3 after sustaining a broken leg. This incidental mortality was not used in the calculation of the LC50 value. Additionally, there was one treatment-related mortality in the 146 ppm a.i. treatment group, a bird found dead on the morning of Day 7. Clinical signs of toxicity were observed in this treatment group on Day 5, when two birds displayed wing droop. All other birds at this test concentration were normal in appearance and behavior for the duration of the test. In the 293 ppm a.i. treatment group there were eight treatment-related mortalities, occurring on Days 5, 6 and 7. Signs of toxicity were first observed on the morning of Day 4 and continued to be exhibited through the morning of Day 8 for the single bird euthanized on Day 8, and through the afternoon of Day 8 for the single bird surviving until Day 22. Signs of toxicity included a ruffled appearance, reduced reaction to stimuli (sound and motion), lethargy, wing droop, loss of coordination, lower limb weakness and convulsions. The single remaining bird appeared to have recovered and was normal in appearance and behavior from the afternoon of Day 9 until test termination. In the 586 ppm a.i. treatment group mortalities were first noted on Day 3 and continued to be observed through D ay 7, at w hich point all birds had died. Overt signs o f toxicity w ere first observed on the afternoon of Day 2 and continued through the morning of Day 7, when the final birds were found dead. Signs of toxicity observed among birds in the 586 ppm a.i. treatment group included a ruffled appearance, reduced reaction to stimuli (sound and motion), lethargy, depression, wing droop, loss of coordination, lower limb weakness, lower limb rigidity, prostrate posture, and convulsions. In the 1171 ppm a.i. treatment group 100% mortality had occurred by the morning of Day 4. Signs of toxicity in the 1171 ppm a.i. treatment group were first observed on the afternoon of Day 2, with the first mortalities noted on the morning of Day 3. Signs of toxicity observed prior to death included a ruffled appearance, reduced reaction to stimuli (sound and motion), lethargy, depression, loss of coordination, wing droop, and lower limb weakness and rigidity. W il d l if e In t e r n a t io n a l , ltd - 17 - PROJECT NO.: 454-103 Body Weight and Feed Consumption When compared to the control group, there were no apparent treatment related effects upon body weight among birds in the 18.3, 36.6 or 73.2 ppm a.i. treatment groups. However, there was a concentration responsive reduction in body weight gain or body weight loss in the 146, 293 and 586 ppm a.i. treatment groups during the exposure period (Days 0-5) (Table 2 and Appendix VI). Differences from the control group were statistically significant at p < 0.05 for the 146 ppm a.i. level and at p<0.01 for the 293 and 586 ppm a.i. levels. A statistically significant (p<0.01) reduction in body weight gain continued to be observed at the 146 ppm a.i. test concentration through Day 8 of the study. At the 293 ppm a.i. concentration a statistically significant (p<0.01) mean weight loss continued through Day 8 of the study and a marked reduction in weight gain was noted through Day 15 of the study. Due to total mortality, body weight effects could not be determined for the 1171 ppm a.i. level during the exposure period or for the 586 and 1171 ppm a.i. treatment groups for post exposure period. There were no apparent treatment related effects upon feed consumption at the 18.3, 36.6, 73.2 or 146 ppm a.i. test concentrations (Table 3 and Appendix VII). However, a reduction in feed consumption was noted at the 293, 586 and 1171 ppm a.i. treatment groups during the exposure period (Days 0-5). There were no treatment-related effects on feed consumption in any of the surviving treatment groups during the Day 6-8 post-exposure period. In the 293 ppm a.i. treatment group only one bird survived to D ay 22. The reduction in feed consum ption observed at the 293 ppm a.i. test concentration during both the Day 8-15 and 15-22 post-exposure periods was the result of having only one bird in the pen, and was not considered to be treatment related. 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, autolysis of tissues and pale organs. 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 were necropsied on Day 22, following test termination. On Day 8, one bird in the 73.2 ppm a.i. treatment group was noted with a slightly pale liver. Due to the isolated nature of this finding, it was W il d l if e In t e r n a t io n a l , ltd - 18- PROJECT NO.: 454-103 not considered to be related to treatment. The single bird euthanized from the 293 ppm a.i. treatment group was observed at necropsy to have with a lack of muscle mass and general thinness. Since these findings correlated with an impact upon body weight noted at this concentration, the findings were considered to be treatment related. Necropsy results were unremarkable for all other birds euthanized on Day 8. Similarly, Day 22 necropsy findings were unremarkable for all birds. CONCLUSION The dietary LC50 value for northern bobwhite exposed to PFOS was determined to be 220 ppm a.i. with a 95% confidence interval of 164 ppm a.i. to 289 ppm a.i. The slope of the concentrationresponse curve was 7.005 and the chi-square value was 0.023. The no mortality concentration was 73.2 ppm a.i. Based upon treatment related mortality, signs of toxicity and effects upon body weight gain at the 146 ppm a.i. a.i test concentration, the no-observed-effect concentration was 73.2 ppm a.i.. W il d l if e In t e r n a t io n a l , ltd - 19- PROJECT NO.: 454-103 REFERENCES 1. U.S. Environmental Protection Agency, 1996. Series 850 - Ecological Effects Test Guidelines (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. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, MN. Personal Communication 7. 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. 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 il d l if e In t e r n a t io n a l , ltd -20 - PROJECT NO.: 454-103 TABLE 1 Cumulative Mortality from a Northern Bobwhite 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 Period Day 6 Day 7 Day 8 1 Control 0 0/30 0/30 0/30 0/30 0/30 0/30 1/30 1/30 1/30 Treatment 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 1/102 0/9 0/9 0/10 0/10 0/10 0/10 0/10 2/10 0/10 0/10 0/10 1/10 2/10 5/10 0/10 0/10 0/10 3/10 10/10 10/10 0/10 0/10 0/10 0/9 4/10 8/10 10/10 0/10 0/10 0/10 1/9 8/10 10/10 10/10 0/10 0/10 0/10 1/9 8/10 10/10 10/10 The LC50 value was calculated to be 220 ppm a.i. with a 95% confidence interval of 164 ppm a.i. to 289 ppm a.i.. 1- No mortalities occurred in any of the control or treatment groups from Day 8 to Day 22. 2- Bird euthanized on day 3 after sustaining a broken leg.________________________________________________ W il d l if e In t e r n a t io n a l , ltd -21 - PROJECT NO.: 454-103 TABLE 2 Page 1 Mean Body Weight (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) Exposure Period Change1 Day 0 Day 5 Day 0-5 Post-Exposure Period Change1 Day 8 Day 5-8 Total Change1 Control 0 Treatment 18.3 36.6 Mean SD Mean SD Mean SD 20 30 14 21 31 14 20 31 23 10 3 11 3 11 2 38 8 52 40 9 52 39 8 31 18 4 20 3 19 2 73.2 Mean SD 20 30 12 9 1 37 7 31 16 2 146 Mean SD 293 Mean SD 586 Mean SD 20 27* 23 7* 3 33* 6** 32 20 18** -2** . 18** _2** 12 2 44 20 16** _4** 12 2 _. -- 13** 4 _ J ** 5 . - 1171 Mean SD 20 _ 1- _ - __ -- . - 'Mean change is calculated separately from the mean body weights using individual body weights (See Appendix VI). (-) = 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). W il d l if e In t e r n a t io n a l , ltd -22- PROJECT NO.: 454-103 TABLE 2 Page 2 Mean Body Weight (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) Post-Exposure Period ________________________________ ___ Change1 Day 8 Day 15 Day 8-15 Day 22 Change1 Day 15-22 Total Change'(8-22) Control 0 Mean SD Treatment 18.3 Mean SD 36.6 Mean SD 37 59 23 6 10 5 40 68 24 6 83 38 65 26 352 82 22 13 3 87 23 73 89 24 72 45 8 47 3 50 4 73.2 Mean SD 35 60 24 342 79 20 42 44 2 146 Mean SD 34* 58 33 24 1 79 21 22 45 1 293 Mean 2 SD 586 Mean SD 21 35 14 ___ --- 55 20 _- -- 34 - - 1171 Mean ___ _- 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 with Dunnett's t-test. (-) = No data available due to mortality. ^Statistically different from the control group at p < 0.05 )(Dunnett's t-test).___________________________________ - - W il d l if e In t e r n a t io n a l , ltd -23 - PROJECT NO.: 454-103 TABLE 3 Page 1 Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) Control 0 Treatment 18.3 36.6 73.2 146 293 586 1171 Exposure Period Day 0-5 Mean SD 9 2 9 8 10 9 5 6 4 Post-Exposure Period Day 6-8 10 2 11 12 13 10 9 19 " (-) = No data available due to mortality. W il d l if e In t e r n a t io n a l , ltd -24- PROJECT NO.: 454-103 TABLE 3 Page 2 Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) Control 0 Mean SD Post-Exposure Period Day 8-15 Day 15-22 9 13 21 Treatment 18.3 36.6 73.2 146 293 586 1171 10 12 14 15 13 15 11 14 89 -"" (-) = No data available due to mortality. W il d l if e In t e r n a t io n a l , ltd -25 - PROJECT NO.: 454-103 TABLE 4 Group Gross Pathological Observations from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Birds that died during the course of the study Finding Control N=1 Male, Female, and Undetermined PPM A.I. 146 293 586 1171 N =2 N =8 N =10 N =10 Abdominal cavity, some autolysis Abdominal cavity, autolysis throughout Crop, empty Emaciated Fractured leg G.I. tract empty Gizzard contents bile stained Heart, anterior portion mottled white color Heart, pale Intestinal contents tar-like Keel, prominent Kidneys, pale Liver, pale and mottled Loss o f muscle mass Muscular-skeletal, pale Small in stature Spleen, black Spleen, dark Spleen, grey Spleen, grey-brown Spleen, pale Spleen, small Spleen, small and pale Thin Not Remarkable 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 24 0 0 11 0 2 52 0 2 58 1 0 00 0 1 10 0 2 51 0 1 00 0 0 21 0 0 20 0 1 3 10 0 0 20 1 0 00 0 4 79 1 0 00 0 3 00 0 0 10 0 0 02 0 0 10 0 0 01 0 101 00 01 0 0 30 0 0 42 0 1 00 W il d l if e In te r n a tio n a l , ltd -26- PROJECT NO.: 454-103 Experimental Group (ppm a.i.) Control 0 0 0 0 0 0 Treatment 18.3 18.3 Table 5 Cumulative Mortality (Estimated Cumulative Dose, mg/kg1) from a Northern Bobwhite ____________________ Acute Dietary Toxicity Study with PFOS_____________________ Pen Day 0 No. Dead Per No. Exposed (Cumulative Dose, mg/kg) Exposure Period Day 1 Day 2 Day 3 Day 4 Day 5 No. Dead Per No. Exposed Post-Exposure Period 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 1/5 1/5 1/5 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 (7) 0/5 (6) 0/5 (14) 0/5 (12) 0/5 (21) 0/5(18) 0/5 (28) 0/5 (24) 0/5 (35) 0/5 (30) 0/5 0/5 0/5 0/5 0/5 0/5 36.6 1 0/5 0/5 (13) 0/5 (26) 0/5 (39) 0/5 (52) 0/5 (65) 0/5 0/5 0/5 36.6 2 0/5 0/5 (11) 0/5 (22) 0/5 (33) 0/5 (44) 0/5 (55) 0/5 0/5 0/5 73.2 1 0/5 0/5 (33) 0/5 (66) 0/5 (99) 0/5(132) 0/5 (165) 0/5 0/5 0/5 73.2 2 0/5 0/5 (32) 0/5 (64) 0/5 (96) 0/5 (128) 0/5 (160) 0/5 0/5 0/5 146 1 0/5 0/5 (46) 0/5 (92) 0/5 (138) 0/5(184) 0/5 (230) 0/5 1/5 1/5 146 2 0/5 0/5 (49) 0/5 (98) 1/5(147) 1/5 (196) 1/5 (245) 1/5 1/5 1/5 293 1 0/5 0/5 (66) 0/5(132) 0/5 (198) 0/5 (264) 1/5 (330) 1/5 4/5 4/5 293 2 0/5 0/5 (101) 0/5 (202) 0/5 (303) 0/5 (404) 1/5 (505) 3/5 4/5 4/5 586 1 0/5 0/5 (213) 0/5 (429) 1/5 (639) 2/5 (852) 2/5 (1065) 4/5 5/5 5/5 586 2 0/5 0/5 (178) 0/5 (356) 0/5 (534) 0/5 (712) 3/5 (890) 4/5 5/5 5/5 1171 1 0/5 0/5 (256) 0/5 (512) 3/5 (768) 5/5 5/5 5/5 5/5 1171 2 0/5 0/5 (256) 0/5 (512) 0/5 (768) 5/5 5/5 5/5 5/5 The LC50 value was calculated to be approximately 220 ppm a.i. with a 95% confidence interval of 164 to 289 ppm a.i.. *- Bird was euthanized due to a broken leg. Estimated cumulative dose is based upon the average body weight and feed consumption over the 5-day exposure period, and serves as a rough approximaton 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 5/5 W il d l if e In t e r n a t io n a l , ltd -27- PROJECT NO.: 454-103 APPENDIX I Certificate O f Analysis FC-95, Lot 217 March 9, 2000 Richard M. Payfer th is m r i a s analyzed using LC/MS, `H-NMR, 19F-NMR, and elem ental analyses techniques. The results 0 i these tests show the.sample to contain the following weight percent co ^ o sitio n : CjFsSO jTC C iF iS O aT C CiFSOiTv+ CjF u SOjTC CsF ijSOjTC C jFisSOjTC C sF itSO j-K' C gF ltS O jlC C ioF nS O ilC C u FsiSOaT T CFi-CtVK T 1 SFsCtFiiSOs'K*' 0 .0 4 % 0.83 % 1.38 % 1 .3 0 % 3.71 % 1 .1 9 % -90749% 15.49 % 0.13 % 0.0 4 % 0.05 % 0.35 % Additionally, the isomer distribution o fthe sample was determined using 19F-NMR techniques and found to contain the following mole percent congusitfcm: CK,(CFi),,-SQj- KT (N orm al chain, w here x is m ainly 7) C F jiC F rl^C F iC F jK C F iV SOj' K7 (Internal m onom ethyl branch, w here x ty is m ainly S. and x w 0. y w 0) (C P & O H C P ^ SOj P flBopropyl bnm bh, whero x is m ainly 5) C J 2x,,-C F(C Fj )- SOs' F " (A lnha branch, w here x Is m ainly 6) (t-butvi branch, w here x is m ainly 45 C F j-tC F jV C iC F iM C F iJr SOj` K7 (Internal gem -dim ethyl branch, w here x+y is m ainly 4. and xwO) 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.: 454-103 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 B12 Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (Menadione Dimethylpyrimidinol Bisulfite) Manganese Zinc Copper Iodine Iron Selenium 2.000. 0001.C.U. 7.000. 0001.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 il d l if e In t e r n a t io n a l , ltd -29- PROJECT NO.: 454-103 APPENDIX III ANALYTICAL METHODS AND RESULTS W il d l if e In t e r n a t io n a l , ltd -30- PROJECT NO.: 454-103 INSTRUMENT: A PPE N D IX 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 C18 column (100 mm x 2 mm I.D., 3 |J.mparticle 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 RETENTIO N TIM E: A pproxim ately 4.8 m inutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 426.7 amu W il d l if e In t e r n a t io n a l , ltd -31 - PROJECT NO.: 454-103 APPENDIX III Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454-103-) MAB-1 Sample Type Matrix Blank Measured Concentration o f PFOS1 (ppm a.i.) < LOQ MAB-2 Matrix Blank < LOQ 'The 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 il d l if e In t e r n a t io n a l , ltd -32- PROJECT NO.: 454-103 APPENDIX III Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454-103-) MAS-1A 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 Mean = Standard Deviation = CV = N= 86.1 93.7 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 il d l if e In te r n a tio n a l , ltd PROJECT NO.: 454-103 -33 - APPENDIX III Table 4 Nominal Concentration (ppm a.i.) 18.3 Sample Number (S-454-103-) 2 3 4 5 6 7 Homogeneity o f PFOS in Avian Diet Location Sampled in Mixing Vessel PFOS Measured Concentration (ppm a.i.) Top Left Top Right Middle Left Middle Right Bottom Left Bottom Right 18.5 23.4 18.3 17.3 19.4 19.9 Mean Measured Concentration (x) Standard Deviation (SD) Coefficient o f Variation (CV)1 x = 19.5 ppm a.i. SD = 2.13 ppm a.i. C V = 10.9% Mean Percent of Nominal 107 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 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. 'Coefficient o f variation was calculated using full precision o f mean and standard deviation results. W il d l if e In te r n a tio n a l , ltd PROJECT NO.: 454-103 -34- APPENDIX III Table 5 Verification o f PFOS Concentrations in Avian Diet Nominal Concentration (ppm a.i.) 0.0 Sample Number (S-454-103-) 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 - 18.3 - - - -- 19.52* 1072 36.6 8 0 45.7 125 40.2 110 9 0 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 17 0 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 o f 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 il d l if e In te r n a tio n a l , ltd PROJECT NO.: 454-103 -35 - APPENDIX III Table 6 Ambient Stability o f PFOS in Avian Diet During the Northern Bobwhite LC50 Study Nominal . Concentration Sample (ppm a.i.) Number (S-454-103-) 01 Day O' Mean Measured Concentration (ppm a.i.) - Mean Percent of Nominal - Sample Number (S-454-103-) 24 Day 5 Measured Concentration2 (ppm a.i.) Mean Measured Concentration (ppm a.i.) < LOQ - Mean Percent of Day 0 - 18.3 2-7 19.5 107 25 19.2 19.6 101 26 19.9 36.6 8,9 40.2 110 27 44.4 49.1 122 28 53.8 73.2 10, 11 74.5 102 29 76.4 77.2 104 30 77.9 146 12, 13 174 119 31 177 176 101 32 174 293 14, 15 291 99.3 33 318 317 109 34 315 586 16, 17 537 91.6 35 560 613 114 36 665 1171 18-23 1196 102 37 1260 1224 102 38 1187 '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). W il d l if e In t e r n a t io n a l , ltd -36 - PROJECT NO.: 454-103 APPENDIX III 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 Zi 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. For each sample, measure 100 mLs of methanol with a graduated cylinder and transfer volume to the French-square 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% NANOpurc water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v). Ampulate and submit samples for LC/MS analysis. Figure 1. Analytical method flowchart for the analysis of PFOS in avian diet. W il d l if e In t e r n a t io n a l , ltd -37- APPENDIX III PROJECT NO.: 454-103 Figure 2. A typical calibration curve for PFOS. Slope = 2.77397; Intercept = 0.01894; r = 0.9981. Curve is weighted (1/x). W il d l if e In te r n a tio n a l , ltd - 38- APPENDIX III PROJECT NO.: 454-103 Figure 3. A representative ion chromatogram of a low-level (0.00229 mg a.i./L) PFOS standard. W il d l if e In t e r n a t io n a l , ltd - 39APPENDIX III PROJECT NO.: 454-103 Figure 4. A representative ion chromatogram of a high-level (0.0457 mg a.i./L) PFOS standard. W il d l if e In t e r n a t io n a l , ltd - 40- APPENDIX III PROJECT NO.: 454-103 Figure 5. A representative chromatogram o f a matrix blank sample (454-103-M AB-l). The arrow indicates the retention time o f PFOS. W il d l if e In t e r n a t io n a l , ltd -41 - APPENDIX III PROJECT NO.: 454-103 Figure 6. A representative chromatogram of a matrix fortification sample (454-103-MAS-1A). W il d l if e In t e r n a t io n a l , ltd -42APPENDIX III PROJECT NO.: 454-103 intensity: 7248 cps 100 167 90- Figure 7. A representative chromatogram of a test sample (454-103-2). W il d l if e In t e r n a t io n a l , ltd -43 - APPENDIX IV DIET PREPARATION PROJECT NO.: 454-103 Weight and volume of constituents used to prepare test diets: Nominal Concentrations (ppm a.i.) 0 18.3 36.6 73.2 146 293 586 1171 Test Substance (g) 0.1818 0.3638 0.7282 1.4659 2.9123 5.8239 11.6483 Basal Ration (g) 9000.0 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 il d l if e In te r n a tio n a l , ltd -44- PROJECT NO.: 454-103 Experimental Group (ppm a.i.) Control 0 0 0 0 0 0 Treatment 18.3 18.3 36.6 36.6 73.2 73.2 146 146 293 293 586 586 1171 1171 APPENDIX V Cumulative Mortality by Pen from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS No. Dead Per No. Exposed No. Dead Per No. Exposed Pen_______________________Exposure Period____________________ _____ Post-Exposure Period Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 1 0/5 2 0/5 3 0/5 4 0/5 5 0/5 6 0/5 1 0/5 2 0/5 1 0/5 2 0/5 1 0/5 2 0/5 1 0/5 2 0/5 1 0/5 2 0/5 1 0/5 2 0/5 1 0/5 2 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 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* 1/5 1/5 0/5 0/5 0/5 0/5 1/5 0/5 0/5 0/5 0/5 1/5 0/5 0/5 1/5 2/5 2/5 0/5 0/5 0/5 0/5 3/5 0/5 0/5 3/5 5/5 5/5 0/5 0/5 0/5 5/5 5/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1/5* 1/5 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 1/5 1/5 1/5 4/5 3/5 4/5 4/5 5/5 4/5 5/5 5/5 5/5 5/5 5/5 0/5 0/5 0/5 0/5 1/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1/5 1/5 4/5 4/5 5/5 5/5 5/5 5/5 The LC50 value was calculated to be 220 ppm a.i. with a 95% confidence interval of 164 ppm a.i. to 289 ppm a.i. * - Bird was euthanized due to a broken leg.1 1 - No mortalities occurred in any of the control or treatment groups after Day 8.__________________________ W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-103 -45 - Experimental Group (ppm a.i.) 0 APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 1 Bird 1 2 3 4 5 Mean SD Day 0 19 20 21 22 23 21 2 Day 5 24 27 32 32 39 31 6 Change Day 0-5 5 7 11 10 16 10 4 Day 8 27 34 42 40 50 39 9 Change Day 5-8 3 7 10 8 11 8 3 0 1 19 26 7 35 9 2 20 25 5 35 10 3 20 29 9 37 8 4 22 33 11 43 10 5 22 31 9 39 8 Mean 21 29 8 38 9 SD 1 3 2 3 1 0 1 19 23 4 30 7 2 20 26 6 29 3 3 20 29 9 32 3 4 22 30 8 37 7 5 22 35 13 40 5 Mean 21 29 8 34 5 SD 1 5 3 5 2 0 1 19 28 9 37 9 2 19 27 8 35 8 3 19 30 11 36 6 4 21 32 11 42 10 5 22 33 11 43 10 Mean 20 30 10 39 9 SD 1 3 14 2 Total Change 8 14 21 18 27 18 7 16 15 17 21 17 17 2 11 9 12 15 18 13 4 18 16 17 21 21 19 2 W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-103 - 46- Experimental Group (ppm a.i.) 0 APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 2 Bird 1 2 3 4 5 Mean SD Day 0 19 20 21 20 22 20 1 Day 5 28 27 32 30 34 30 3 Change Day 0-5 9 7 11 10 12 10 2 Day 8 35 36 44 - 44 40 5 Change Day 5-8 7 9 12 - 10 10 2 0 Group Total 1 18 30 12 39 9 2 21 35 14 46 11 3 19 29 10 37 8 4 21 32 11 41 9 5 22 32 10 42 10 Mean 20 32 11 41 9 SD 2 2 2 3 1 Mean 20 30 10 38 8 SD 1 4 3 5 2 Total Change 16 16 23 - 22 19 4 21 25 18 20 20 21 3 18 4 W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-103 -47- APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 3 Experimental Group (ppm a.i.) Bird Day 0 18.3 1 19 2 19 3 21 4 22 5 22 Mean SD 21 2 Day 5 Change Day 0-5 Day 8 Change Day 5-8 26 7 34 8 33 14 39 6 27 6 35 8 34 12 46 12 36 14 46 10 31 11 40 446 9 2 Total Change 15 20 14 24 24 19 5 18.3 Group Total 1 2 3 4 5 Mean SD Mean SD 19 19 20 22 22 20 2 21 1 28 28 29 34 . 35 31 3 31 4 9 9 9 12 13 10 2 11 3 38 37 38 44 44 40 3 40 5 10 9 9 10 9 9 1 9 2 19 18 18 22 22 20 2 20 3 W il d l if e In t e r n a t io n a l , ltd -48 - PROJECT NO.: 454-103 APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 4 Experimental Group (ppm a.i.) Bird Day 0 36.6 1 18 2 19 3 19 4 21 5 21 Mean SD 20 1 Day 5 Change Day 0-5 26 8 30 11 29 10 30 9 34 13 30 10 32 Day 8 35 39 37 38 43 38 3 Change Day 5-8 . 9 9 8 8 9 9 1 Total Change 17 20 18 17 22 19 2 36.6 1 19 29 10 37 2 19 29 10 37 3 21 32 11 40 4 22 35 13 44 5 23 36 13 44 Group Total Mean 21 32 11 40 SD 2 3 2 4 Mean 20 31 11 39 SD 2 3 2 3 8 8 8 9 8 8 0 8 1 18 18 19 22 21 20 2 19 2 W il d l if e In t e r n a t io n a l , ltd -49- PROJECT NO.: 454-103 APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 5 Experimental Group (ppm a.i.) Bird Day 0 73.2 1 18 2 19 3 20 4 21 5 21 Mean SD 20 1 Day 5 Change Day 0-5 Day 8 Change Day 5-8 26 8 32 26 7 33 29 9 36 32 11 39 30 9 36 29 9 35 3 13 6 7 7 7 6 7 1 Total Change 14 14 16 18 15 15 2 73.2 1 18 27 9 34 2 20 31 11 38 3 21 30 9 37 4 21 32 11 39 5 22 32 10 41 Group Total Mean SD Mean SD 20 2 20 1 30 2 30 2 10 38 13 9 37 13 7 7 7 7 9 7 1 7 1 16 18 16 18 19 17 1 16 2 W il d l if e In t e r n a t io n a l , ltd -50- PROJECT NO.: 454-103 APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 6 Experimental Group (ppm a.i.) Bird Day 0 146 1 19 2 19 3 20 4 22 5 23 Mean SD 21 2 Day 5 Change Day 0-5 28 9 28 9 24 4 33 11 28 5 28 8 33 Day 8 35 32 38 32 34 3 Change Day 5-8 7 4 5 4 5 1 Total Change 16 13 16 9 14 3 146 1 18 24 6 2 19 27 8 3 19 27 8 4 21 - - 5 23 24 1 Mean 20 26 6 SD 2 2 3 Group Mean 20 27* 7* Total SD 2 3 3 (-) = 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). 31 35 33 - 28 32 3 33* 3 7 8 6 - 4 6 2 6** 2 13 16 14 - 5 12 5 13** 4 W il d l if e In t e r n a t io n a l , ltd -51 - PROJECT NO.: 454-103 APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 7 Experimental Group (ppm a.i.) Bird Day 0 293 1 18 2 19 3 20 4 22 5 22 Mean SD 20 2 Day 5 Change Day 0-5 Day 8 Change Day 5-8 20 2 21 17 -2 - 18 -2 - --- 20 -2 - -1 21 2- 1 1 - Total Change 3 3 - 293 1 19 19 0 2 19 3 20 20 0 4 20 17 -3 5 21 16 -5 Mean 20 18 -2 Group SD Mean 1 20 2 18** 2 _2** Total SD 1 2 2 (-) = 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). 15 - - - 15 - 18** 4 -4 - - - - -4 - _2** 4 -4 - - - - -4 - -1** 5 W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-103 -52 - APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 8 Experimental Group (ppm a.i.) Bird Day 0 586 1 18 2 20 3 21 4 21 5 22 Mean SD 20 2 Day 5 Change Day 0-5 Day 8 Change Day 5-8 13 -7 - - 18 -3 - - ---- 17 -5 - - 16 -5 32 - - Total Change - - 586 1 19 16 -3 2 20 - - 3 20 16 -4 4 19 - - 5 21 - - Mean SD 20 1 16 0 -4 1 Group Total Mean SD 20 1 16** 2 2 (-) = No data available due to mortality. ^Statistically different from the control group at p < 0.01 (Dunnett's t-test). - - - - - - - - - - - - - - - - - - - W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-103 -53 - APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 9 Experimental Group (ppm a.i.) Bird Day 0 1171 1 2 3 4 5 Mean SD 18 20 20 21 21 20 1 Day 5 Change Day 0-5 Day 8 __ -------- - ----- Change Day 5-8 _ - - Total Change _ - - 1171 1 18 - - - 2 20 - - - 3 20 - - - 4 22 - - - 5 21 - - - M ean 20 - - - SD 1 - - - Group Mean 20 - - - Total SD 1 - - - (-) = No data available due to mortality. - - - - - - W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-103 -54- Experimental Group (ppm a.i.) 0 APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 10 Bird 1 2 3 4 5 Mean SD Day 8 27 34 42 40 50 39 9 Day 15 42 56 69 63 78 62 14 Change Day 8-15 15 22 27 23 28 23 5 Day 22 61 77 92 84 104 84 16 Change Day 15-22 19 21 23 21 26 22 3 0 1 35 55 20 74 19 2 35 53 18 74 21 3 37 64 27 85 21 4 43 74 31 103 29 5 39 59 20 80 21 Mean 38 61 23 83 22 SD 3 8 6 12 4 0 Group Total 1 30 54 24 79 25 2 29 45 16 63 18 3 32 54 22 77 23 4 37 55 18 75 20 5 40 68 28 95 27 Mean 34 55 22 78 23 SD 5 8 5 11 4 Mean 37 59 23 82 22 SD 6 10 5 13 3 Total Change 34 43 50 44 54 45 8 39 39 48 60 41 45 9 49 34 45 38 55 44 8 45 8 W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-103 -55 - APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 11 Experimental Group Change Change (ppm a.i.) Bird Day 8 Day 15 Day 8-15 Day 22 Day 15-22 18.3 1 34 53 19 78 25 2 39 65 26 85 20 3 35 60 25 86 26 4 46 73 27 95 22 5 46 71 25 92 21 Mean 40 64 24 87 23 SD 6 8 3 7 3 Total Change 44 46 51 49 46 47 3 Experimental Group Change Change (ppm a.i.) Bird Day 8 Day 15 Day 8-15 Day 22 Day 15-22 36.6 1 35 60 25 81 21 2 39 65 26 91 26 3 37 65 28 91 26 4 38 61 23 83 22 5 43 72 29 97 25 Mean 38 65 26 89 24 SD 3 5 2 7 2 Total Change 46 52 54 45 54 50 4 W il d l if e In t e r n a t io n a l , ltd -56- PROJECT NO.: 454-103 APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 12 Experimental Group Change Change (ppm a.i.) Bird Day 8 Day 15 Day 8-15 Day 22 Day 15-22 73.2 1 32 54 22 76 22 2 33 58 25 77 19 3 36 62 26 79 17 4 39 65 26 87 22 5 36 59 23 78 19 Mean 35 60 24 79 20 SD 3 4 2 4 2 Total Change 44 44 43 48 42 44 2 Experimental Group Change Change (ppm a.i.) Bird Day 8 Day 15 Day 8-15 Day 22 Day 15-22 146 1 35 58 23 2 32 55 23 3--- 4 38 63 25 5 32 57 25 Mean SD 34* 3 58 3 24 1 (-) = No data available due to mortality. ^Statistically different from the control group at p < 0.05 (Dunnett's t-test). 81 77 81 78 79 2 23 22 18 21 21 2 Total Change 46 45 43 46 45 1 W il d l if e In t e r n a t io n a l , ltd -57- PROJECT NO.: 454-103 APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 13 Experimental Group (ppm a.i.) Bird Day 0 293 1 21 2 3 4 5 Mean 21 SD - (-) = No data available due to mortality. Day 5 Change Day 0-5 Day 8 Change Day 5-8 35 14 55 20 35 14 55 20 ---- Total Change 34 34 - W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-103 -58 - APPENDIX VII Feed Consumption (g/bird/day) by Pen from a Northern Bobwhite 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 9 7 8 12 9 10 9 2 12 12 7 12 9 9 10 2 W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-103 -59 - APPENDIX VII Feed Consumption (g/bird/day) by Pen from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 2 Experimental Group Exposure Period Post-Exposure Period (ppm a.i.) Pen Day 0-5 Day 6-8 18.3 1 9 28 Mean 9 36.6 1 2 Mean 8 7 8 10 11 11 14 9 12 73.2 1 10 2 10 Mean 10 146 1 7 2 10 Mean 9 293 1 4 26 Mean 5 12 14 13 8 12 10 8 10 9 586 1 6 25 Mean 6 16 22 19 1171 1 2 Mean - No data available due to adult mortality. 4 4 4 - W il d l if e In te r n a tio n a l , ltd PROJECT NO.: 454-103 -60- APPENDIX VII Feed Consumption (g/bird/day) by Pen from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 3 Experimental Group (ppm a.i.) Pen Exposure Period Day 8-15 Post-Exposure Period Day 15-22 Control 1 2 3 Mean SD 10 10 7 9 2 14 12 12 13 1 Experimental Group (ppm a.i.) Pen 18.3 1 36.6 1 73.2 1 146 1 293 1 Exposure Period Day 8-15 10 14 13 11 8 Post-Exposure Period Day 15-22 12 15 15 14 9 W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-103 -61 - APPENDIX VIII CHANGES TO PROTOCOL 1. 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 2. 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. 3. The protocol was amended to change the test concentrations from 0, 20, 40, 80, 160, 640 and 1280 ppm a.i., to 0, 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. The temperatures from several brooder units on Day 4, 7, 8, and all brooder units on Day 22 were not recorded 5. The afternoon observations were inadvertently not recorded for 2 birds in the 640 ppm a.i. treatment group on April 26, 1999. W il d l if e In t e r n a t io n a l , ltd -62APPENDIX IX PERSONNEL INVOLVED IN THE STUDY PROJECT NO.: 454-103 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) Timothy Z. Kendall, Supervisor, Analytical Chemistry (7) Raymond L. Van Hoven, Ph.D., Scientist (8) Ellen Mank, Chemist W il d l if e In t e r n a t io n a l , ltd -63APPENDIX X REVISED STUDY SUMMARY PROJECT NO.: 454-103 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 il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-103 -64- 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: NOM INAL TEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: LC50: 95% CONFIDENCE LIMITS: NO MORTALITY CONCENTRATION: NO-OBSERVED-EFFECTCONCENTRATION: 454-103 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A Dietary LC50 Study with the Northern Bobwhite 0, 17.6, 35.1,70.3, 141, 281, 562 and 1125 ppma.i. Experimental Start - April 22, 1999 Biological Termination - May 14, 1999 Experimental Termination - May 14, 1999 22 days Northern Bobwhite (Colinus virginianiis) Wildlife International Ltd. Production Flock Easton, Maryland 21601 10 days at test initiation 212 ppm a.i. 158 and 278 ppm a.i. 70.3 ppm a.i. 70.3 ppm a.i. AMENDED W il d l if e In t e r n a t io n a l , ltd -65 - PROJECT NO.: 454-103 Centre Analytical Laboratories, Inc. 048 Research Drive Phona: (814) 231-8032 state College, p a 16801 www.centrelab.com Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OFANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-O18A 3M Product; PFO S,Lot217 Reference#: SD-018 _____________________ Parity: 88.9*/._____________________ Test Name Specifications ------------ USSR------------ Purity* 86.9% Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium2 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) Related Compounds POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate J. Nitrite 6. P hosphate 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 wtVwt.% 8.41 wt/wt.% None Detected 0.33 wtVwt.% None Detected Not Applicable1 1. <0.015 wt/wt*/. 2. 0.59 wt/wt.% 3. <0.040 wt/wt*/. 4. <0.009 wt/wt */. 5. <0.006 wt./wt.% 6 . < 0.007 w t/w t% 7. 8.76 wt/wt.% 1. <0.1 wt7wt.% 2. <0.1 wtVwt% 3. 0.10wt./wt% 4. 0.28 wt/wt.% 1. 12.48 wt/wt.% 2. 0.244 wt/wt.% 3. 1.74 wt/wt.% 4. 8.84 wt/wt.% 5. 54.1 wt./wt.% C O A 02 3 -01 8 A Page I of 3 AMENDED W il d l if e In t e r n a t io n a l , ltd - 66 - PROJECT NO.: 454-103 Centre Analytical Laboratories, Inc. 3048 Research' D" ri've State Collage, PA 1BB01 www.centrelab.com Phone: (814)231-8032 Fax: (814) 231-1253 or (814) 231-1580 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 <-10C Re-assessment Date: 08/31/06 'Purity = 100% - (sum o f metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.905%+organic acid impurities, 0.38%+POAA, 0.33%) Total impurity from all tests = 13.07% Purity = 100%-13.07% = 86.9% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials o f low purity. No enddtherm was observed for this sample. 'Sulfur in the sample appears to be converted to SO4 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 SO, is not considered an impurity. 5T F A HFBA N l'i'A PFPA Trifluoroacetic acid Heptafluorobutyrie acid Nonailuoropentanoic acid Pentafluoropropanoic acid - Theoretical value calculations based on the empirical formula, CsF nSOj"K* (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-018A Page 2 of3 AMENDED W il d l if e In t e r n a t io n a l , ltd -67- PROJECT NO.: 454-103 Centre Analytical lab o rato ries, Inc 304a Research Driva State Colleao, PA 16801 www.centrelab.com Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OFANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A LC/MS Purity Profile: Impurity C4 C5 C6 C7 Total w t/w t-% 1.22 1.33 4.72 1.14 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average result from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average result from the C6 and C8 standard curves. Prepared By: Scientist, Centre Analytical Laboratories /C w /o t Date Reviewed By: q L /n fL U d , John Flaherty ~~7 Date Laboratory Manager, Centre Analytical Laboratories C O A 0 2 3 - 0 I8 A Page 3 of 3 AMENDED W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-103 -68APPENDIX XI 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 62 to 69. 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 il d l if e In t e r n a t io n a l , ltd -69APPENDIX XI Report Amendment (continued) PROJECT NO.: 454-103 8. Amended Report: Reason: 9. Amended Report: Reason: Pages 63-67, Revised Study Summary, were added to the report. To provide additional information. Pages 68 and 69, Report Amendment, were added to the report. To provide details of changes made to the original report. AMENDMENT SIGNATURES: Study Director J U bcaI vn y. > i W Wildlife International, Ltd. Management REVIEWED BY: O^LLOUT Quality Assurance Q s c J U x n QUO Date 3 M + t+ kw i Date - 4 Date AMENDED