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PFOS: A DIETARY LC50 STUDY WITH THE MALLARD WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-102 3M REQUEST NO. U2723 U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines (draft) OPPTS Number 850.2200 FIFRA Subdivision E, Section 71-2 OECD Guideline 205 AUTHORS: Sean P. Gallagher Courtney S. Casey Joann B. Beavers Raymond L. Van Hoven STUDY INITIATION: April 21, 1999 STUDY COMPLETION: April 26, 2000 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 o f 65 001275 W il d l if e In t e r n a t io n a l , 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 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 o f 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: 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 is being recharacterized in accordance with GLP. The stability of the test substance and reference standard under conditions o f storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: f- jjjUjU/v _______ J Sean P. Gallagher Senior Biologist, Avian Toxicology SPONSOR'S REPRESENTATIVE DATE v\i V-G D O DATE C01276 W il d l if e In t e r n a t io n a l , ltd -3 - PROJECT NO.: 454-102 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR Part 160 & 792, 17 August 1989; OECD Principles o f 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 o f all audits and inspections and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY DATE CONDUCTED DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT Matrix Fortification, Test Substance Prep., Analytical Sampling April 22, 1999 April 22, 1999 April 23, 1999 Feed Consumption and Analytical Sampling April 27, 1999 April 27, 1999 May 4, 1999 Necropsy, Blood Collection, Liver Weights and Bile Collection May 14, 1999 May 14, 1999 May 21, 1999 Analytical Data and Draft Report July 7, 8, and 9, 1999 July 9, 1999 July 16, 1999 Biological Data and Draft Report Aug. 30, 31, Sept. 1, 1999 September 1, 1999 September 3, 1999 Final Report April 18, 2000 April 18, 2000 April 19, 2000 O o 3 juu*xo G o& unnon Susan L. Coleman Senior Quality Assurance Representative DATE 4*19' 001277 W il d l if e In t e r n a t io n a l , ltd -4 - 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: Date PROJECT NO.: 454-102 Scientist, Analytical Chemistry REPORT APPROVED BY: <C JoaflrrBrBeavers Director, Avian Toxicology Willard B. Manager, Analytical Chemistry Date Date C01278 W il d l if e In t e r n a t io n a l , ltd -5 - PROJECT NO.: 454-102 TABLE OF CONTENTS TITLE PAGE................................................................................................................................. Page 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEM ENT.................................Page 2 QUALITY ASSURANCE STATEM ENT.............................. Page 3 REPORT APPROVAL......................................................................................................................Page4 TABLE OF CONTENTS............................................................................................................. Page 5 Tables and Appendices............................................................................................................... Page 6 SUMMARY................................................................................................................................... Page 7 INTRODUCTION......................................................................................................................... Page 8 O B J E C T IV E ........................................................................... Page 8 MATERIALS AND M ETHODS.................................................................................................Page 8 Test Substance..............................................................................................................................Page 8 Treatment Groups......................... Page 9 Duration o f the T est.....................................................................................................................Page 9 Test Birds......................................................................................................................................Page 9 Animal D iet........................................................................................................................................Page10 Diet Preparation.................................................................................................................................Page10 Diet Sampling.................................................................................................................................... Page10 Analytical M ethod............................................................................................................................ Page10 Housing and Environmental Conditions......................................................................................... Page13 Observations...................................................................................................................................... Page13 Animal Body Weights/Feed Consumption...................................................................................... Page14 Gross Necropsy................................................................................................................................. Page14 Statistical Analyses........................................................................................................................... Page14 RESULTS............................................................................................................................................ Page14 Diet Analysis..................................................................................................................................... Page14 Mortalities and Clinical Observations.............................................................................................Page15 Body Weight and Feed Consumption.................................. Page 16 Gross Necropsy................................................................................................................................. Page17 CONCLUSION................................................................................................................................... Page18 REFERENCES................................................................................................................................... Page19 001279 W il d l if e In t e r n a t io n a l , ltd 6- - TABLE OF CONTENTS - continued - TABLES AND APPENDICES PROJECT NO.: 454-102 TABLE 1: Cumulative Mortality from a Mallard Acute Dietary Toxicity Study with PFOS.................................................................................................Page 20 TABLE 2: Mean Body Weight (g) from a Mallard Acute Dietary Toxicity Study with PFOS.................................................................................................Page 21 TABLE 3 : Mean Feed Consumption (g/bird/day) from a Mallard Acute Dietary Toxicity Study with PFOS........................................................ Page 23 TABLE 4: Gross Pathological Observations from a Mallard Acute Dietary Toxicity Study with PFOS....................................................................Page 25 TABLE 5: Cumulative Mortality (Estimated Cumulative Dose, mg/kg) from a Mallard Acute Dietary Toxicity Study with PFOS..........................................Page 26 APPENDIX I: Certificate o f Analysis..................... Page 27 APPENDIX II: Diet Formulation................................................................................................. Page28 APPENDIX III: Analytical Methods and Results.........................................................................Page29 APPENDIX IV: Diet Preparation.................................................................................................. Page43 APPENDIX V: Cumulative Mortality by Pen from a Mallard Acute Dietary Toxicity Study with PF O S ...................................... ..........Page 44 APPENDIX VI: Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PF O S ................................................ Page 45 APPENDIX VII: Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with P F O S ........................................ Page60 APPENDIX VIII: Changes to Protocol............................................................................................ Page64 APPENDIX IX: Personnel Involved in the Study.........................................................................Page65 C012S0 W il d l if e In t e r n a t io n a l , 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 o f 448 ppm a.i. to 958 ppm a.i. The slope o f 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: H atch -A p ril 13, 1999 Acclimation - April 14-22, 1999 Experimental Start-A pril 22, 1999 Experimental Termination- NOMINAL TEST CONCENTRATIONS: 0, 9.1, 18.3, 36.6, 73.2, 146, 293, 586, and 1171 ppm a.i. TEST ANIMALS: Mallard (Anas platyrhynchos) AGE TEST ANIMALS: 10 days o f age at test initiation SOURCE TEST ANIMALS: Whistling Wings, Inc. PO Box 1-A 113 Washington St. Hanover, IL 61041-3512 STUDY COMPLETION: April 26, 2000 C012S1 W il d l if e In t e r n a t io n a l , ltd 8- - PROJECT NO.: 454-102 INTRODUCTION This study was conducted by Wildlife International Ltd. for 3M Corporation at the Wildlife International Ltd. toxicology facility in Easton, Maryland. The in-life portion o f 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 o f this study was to evaluate the toxicity o f a test substance to the mallard (Anas platyrhynchos) administered through the diet for five days. An LC50 value will be calculated, if possible. MATERIALS AND METHODS The methods used in conducting this study are based upon procedures specified in the U.S. Environmental Protection Agency Series 850 Ecological Effects Test Guidelines OPPTS Number 850.2200 (1) Section 71-2 of the Environmental Protection Agency's Registration Guidelines, Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms (2); OECD Guideline 205, Guideline for Testing of Chemicals, Avian Dietary Toxicity Test (3); and upon ASTM Standard E857-87, "Standard Practice for Conducting Subacute Dietary Toxicity Tests with Avian Species" (4). Test Substance The test substance was received from 3M Corporation on October 29, 1998 and was assigned Wildlife International Ltd. Identification Number 4675 upon receipt. The test substance was white powder identified as: FC-95; Lot No.:217. The reported purity of the test substance was 98.9%, with an expiration date of 2008. Following test termination, the test material was reanalyzed. The results o f 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 C012S2 W il d l if e In t e r n a t io n a l , ltd -9 - PROJECT NO.: 454-102 material, was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2. The standard was stored under ambient conditions Treatment Groups The test consisted of a geometric series o f eight test concentrations and a control group. Thirty mallard ducklings were assigned to the control group and ten mallard ducklings were assigned to each o f 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 o f 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 o f 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 o f 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 0012S3 W il d l if e In t e r n a t io n a l , ltd - 10- PROJECT NO.: 454-102 same hatch, pen-reared and phenotypically indistinguishable from wild birds. All birds were acclimated to the caging and facilities from the day after hatch until initiation o f the test. Animal Diet Throughout acclimation and testing all test birds were fed a game bird ration formulated to Wildlife International Ltd.'s specifications (Appendix II). Water from the town o f 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 o f the test substance. All dietary concentrations and the LC50 value are reported as ppm a.i. in the diet. Nominal dietary test concentrations used in this study were 9.1, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 ppm a.i. (Appendix IV). Diet Sampling Samples o f the test diets were collected to verify the test concentrations administered and to confirm the stability and homogeneity o f the test substance in the diets. Homogeneity o f the test substance in the diet was evaluated by collecting six samples from the 9.1 ppm a.i. a.i. and six samples from the 1171 ppm a.i. a.i. test diets at preparation on Day 0. Homogeneity samples were collected from the top, middle and bottom o f the left and right sections o f 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 o f 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 o f the avian diet samples was based upon methodology developed at Wildlife International Ltd. and entitled "Method Outline for the Determination o f PFOS C 01234 W il d l if e In t e r n a t io n a l , ltd - 11 - PROJECT NO.: 454-102 in Avian Feed". Avian diet samples were extracted with methanol. Methanol was added to a requisite quantity of feed contained in a French-square glass bottle. Bottles were capped and shaken on a shaker table. Samples were vacuum filtered using qualitative filter paper. The retained feed was rinsed three times with methanol into the filtrate. The filtrate was transferred to a volumetric flask and brought to volume with methanol. As appropriate, samples were further diluted with methanol. Each sample then was diluted with a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v) so they fell within the calibration range o f the PFOS methodology. A method flowchart is provided in Appendix TIT, Figure 1. Concentrations o f PFOS in the standards and samples were determined by reversed-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a PerkinElmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil C]8 analytical column (100 mm x 2 mm I.D., 3 pm particle size). The instrument parameters are summarized in Appendix III, Table 1. Calibration standards o f 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 o f individual isomeric components. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS : internal standard) o f the calibration standards. A typical calibration curve is presented in Appendix III, Figure 2. The concentration o f PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms of low and high calibration standards are presented in Appendix III, Figures 3 and 4, respectively. The method limit o f 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). ^1285 W il d l if e In t e r n a t io n a l , ltd - 12- PROJECT NO.: 454-102 Two matrix blank samples were analyzed to determine possible interferences. No interferences were observed at or above the LOQ during sample analyses (Appendix III, Table 2). An interference in the feed appeared at approximately the same retention time as the peak o f interest but it was well below the LOQ. A representative chromatogram o f 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 o f 94.7%. A representative chromatogram of a matrix fortification is presented in Appendix HI, Figure 6. An example calculation is presented for sample number 454-102-2, nominal concentration of 18.3 ppm a.i. in avian diet. Initial Weight: 10.0 g Final Volume: 200 mL Dilution Factor: 100 (intermediate dilution factor x final dilution factor) PFOS Peak Area: 113568 Internal Standard Peak Area: 413160 Peak Area Ratio: 0.2749 Calibration curve equation. Slope: 2.77397 Intercept: 0.01894 Curve is weighted (1/x). PFOS (mg a.i./L) at instrument (Peak area ratio - (Y-intercept)) x I.S. Concentration Slope (0.2749 - 0.01894) x 0.100 mg/L 2.77397 = 0.00923 mg a.i./L Note: I.S. = internal standard. 001286 W il d l if e In t e r n a t io n a l , ltd - 13 - PROJECT NO.: 454-102 PFOS (ppm a.i.) in sample PFOS (mg a.i./L) at instrument x Final Volume (L) x Dilution Factor Initial Weight (Kg) 0.00923 x 0.200 x 100 I = 18.5 ppm a.i. PFOS (ppm a.i.) in sample Percent of Nominal Concentration = ----------------------------------- 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 o f 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 o f 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 o f 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 o f the pens was 38C 2C (SD). Average ambient room temperature for this study was 25.2C 0.7C (SD) with an average relative humidity o f 53% 1 8 % (SD). The photoperiod (maintained by a time clock) was sixteen hours o f 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 o f 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 0G12S7 W il d l if e In t e r n a t io n a l , ltd - 14- PROJECT NO.: 454-102 observed at least once daily. A record was maintained o f all mortality, signs of toxicity, and abnormal behavior. Animal Body Weiehts/Feed Consumption Individual body weights were measured at the initiation o f the test, on Day 5, on Day 8 for all birds, and on Days 15 and Day 22 for all remaining birds. Average feed consumption values during the exposure period (Days 0-5) and the post-exposure observation period (Days 6-8) were determined by pen for each treatment group and the control group. Additionally, feed consumption was determined for Days 8-15 and 15-22 for the remaining half of the treatment and control birds. Feed consumption was determined by measuring the change in the weight o f the feed presented to the birds over a given period o f time. The accuracy o f feed consumption values may have been affected by the unavoidable wastage o f feed by the birds. Gross Necropsy All test birds that died during the course o f 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 o f 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 o f the concentration-response curve and results o f the goodness o f fit test are reported. Body weight data were compared by Dunnett's test using TOXSTAT software (10,11). No statistical analyses were applied to feed consumption data. RESULTS Diet Analysis Avian diet samples were collected from the 9.1, 18.3 and 1171 ppm a.i. test concentrations and analyzed to evaluate homogeneity o f the test substance in the avian diet. The analysis o f these samples also served as verification of test substance concentrations. Resulting mean measured 001258 W il d l if e In t e r n a t io n a l , ltd -15 - PROJECT NO.: 454-102 concentrations, standard deviations and coefficients o f variation (CV) for these test concentrations were 9.8 0.969 ppm a.i. (CV = 9.94%), 19.5 2.13 ppm a.i. (CV = 10.9%) and 1196 70.2 ppm a.i. (CV = 5.87%), respectively (Appendix III, Table 4). Control avian diet samples collected during the test showed no interferences above the LOQ. Samples collected during the test to verify the 36.6, 73.2, 146, 293 and 586 ppm a.i. test substance concentrations had mean measured concentrations of 40.2, 74.5, 174, 291 and 537 ppm a.i., respectively. These values represented 110, 102, 119, 99.3 and 91.6% of the nominal concentrations, respectively (Appendix III, Table 5). Analysis o f avian diet samples collected from feeders after being held at ambient temperature for five days averaged 119, 97.4, 130, 104, 94.3, 101, 101 and 95.0% of the Day 0 values for the 9.1, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 ppm a.i. test substance concentrations, respectively (Appendix III, Table). A representative chromatogram of a test sample is shown in Appendix III, Figure 7. Mortalities and Clinical Observations No mortalities occurred in the control group, and all birds were normal in appearance and behavior throughout the test. There were no treatment-related mortalities or overt signs o f toxicity in the 9.1, 18,3, 36.6, 73.2, or 146 ppm a.i. treatment groups (Table 1 and Appendix V). However, there was 20% (2 of 10) mortality in the 293 ppm a.i. treatment group, 30% (3 o f 10) mortality in the 586 ppm a.i. treatment group and 90% (9 o f 10) in the 1171 ppm a.i. treatment group. In the 293 ppm a.i. treatment group single mortalities were noted on the mornings o f Day 7 and Day 8. Signs o f 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 o f Day 8 for those birds sacrificed on Day 22. All birds in this treatment level displayed signs o f toxicity. These included reduced reaction to stimuli (sound and motion), loss o f 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 o f Day 5, Day 6, and Day 7. Signs o f 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 001289 W il d l if e In t e r n a t io n a l , ltd - 16- PROJECT NO.: 454-102 limb weakness, and lethargy. All remaining birds appeared to have recovered and were normal in appearance and behavior from the morning o f 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 o f 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 o f coordination, prostrate posture, depression, convulsions, ruffled appearance, lower limb weakness, and lethargy. The one surviving bird appeared to have recovered and was normal in appearance and behavior from the morning of Day 9 until test termination. Body Weight and Feed Consumption When compared to the control group, there were no apparent treatment related effects on body weight among birds in the 9.1, 18.3, or 36.6 ppm a.i. treatment groups. There was a statistically significant (p<0.05) reduction in weight gain at the 9.1 ppm a.i. level for the Day 0-5 period that was reflected in significantly lower (p< 0.05) mean Day 5 body weight. When compared to the control group, reductions in weight gain were also statistically significant (p<0.05) for the Day 5-8 period, resulting in a significantly (p<0.05) lower Day 8 mean body weight. However, differences from the control group at the 9.1 ppm a.i. level appear to be due to a lower mean Day 0 body weight for the 9.1 ppm a.i. level and were not dose responsive. Therefore, these differences were not considered treatment related. There was a marked, treatment-related, concentration-responsive effect on body weight in the 73.2, 146, 293, 586, and 1171 ppm a.i. treatment groups during the exposure period (Days 0-5) (Table 2 and Appendix VI). These reductions were statistically significant at p<0.05 for the 73.2 ppm a.i. level and at p<0.01 at the 146, 293, 586 and 1171 ppm a.i. levels. During the Day 5-8 post-exposure period, weight gain continued to be reduced for surviving birds in the 293, 586 and 1171 ppm a.i. treatment groups. These reductions were statistically significant at p<0.01 for the 293 and 586 ppm a.i. levels. Differences at the 1171 ppm a.i. level were marked but could not be statistically compared. Body weight losses and reductions in weight gain at the 293, 586 and 1171 ppm a.i. levels were reflected in significantly (p<0.05 or p<0.01) lower mean body weights on Days 5, 8, 15, and 22 001290 W il d l if e In t e r n a t io n a l , 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 o f 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. H alf 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 o f 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 o f muscle mass, and had a 2 x 0.5 cm abscess containing caceous necrotic material on the right side o f the abdominal wall. Due to the nature o f the lesions observed in birds, and the isolated incidence of occurrence, the findings listed above in euthanized birds from the 9.1, 36.6, and 73.2 ppm a.i. treatment groups were not considered to be related to treatment. In addition, all remaining birds in the control, 9.1, 36.6, and 73.2 ppm a.i. treatment groups were non remarkable. In the 293 ppm a.i. treatment group, one bird euthanized on Day 8 was noted with an incidental finding of a retained yolk sac. A second bird in the 293 ppm a.i. group, necropsied on Day 8, was noted as emaciated with a lack of muscle mass. This loss of muscle mass was consistent with reduced weight gain for the level and was considered treatment related. Another bird in the 293 ppm a.i. treatment group, euthanized on Day 22, was noted with 2 cm cysts containing yellow fluid on the 001291 W il d l if e In t e r n a t io n a l , ltd - 18 - PROJECT NO.: 454-102 anterior portion o f the left lobe o f the liver. Due to the nature of the cysts and the isolated incidence of occurrence, this observation was not considered treatment related. Three birds in the 586 ppm a.i. treatment level were euthanized on Day 8. At necropsy, they were noted to be thin and lacking muscle mass. One bird was noted with a slightly pale liver. These findings are considered to be treatment related. Four birds in the 586 ppm a.i. treatment level survived until Day 22. Necropsy results for these four birds were not remarkable. CONCLUSION The dietary LC50 value for Mallards exposed to PFOS was determined to be 628 ppm a.i. with a 95% confidence interval of 448 ppm a.i. to 958 ppm a.i.. The slope o f the concentration-response curve was 3.67 and the chi-square value was 2.13. The no mortality concentration was 146 ppm a.i.. Based upon reductions in body weight gain at the 73.2 ppm a.i. test concentration, the no-observedeffect concentration was 36.6 ppm a.i.. 001292 W il d l if e In t e r n a t io n a l , ltd -19- PROJECT NO.: 454-102 REFERENCES 1 U. S. Environmental Protection Agency, 1996. Series 850- Ecological Effects Test Guidlelines (draff), 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 o f Pesticide Programs. Washington, D.C. 3 Organization for Economic Cooperation and Development. 1984. Avian Dietary Toxicity Test. OECD Guideline for Testing o f Chemicals. Guideline 205. Paris. 4 American Society for Testing and Materials. 1987. Standard Practice for Conducting Subacute Dietary Toxicity Tests with Avian Species. ASTM Standard E857-87. Annual Book of ASTM Standards, Vol. 11.04. Philadelphia, PA. 5 National Research Council. 1996. Guide for the Care and Use o f Laboratory Animals. Washington, D.C. National Academy Press. 125 pp. 6 Stephan, C.E. 1977. Methods for Calculating an LC50. Pages 65-84 In Aquatic Toxicology and Hazard Evaluations, American Society for Testing and Materials. Pub. No. STP 634. Philadelphia, PA. 7 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, MN. Personal Communication. 8 Finney, D.J. 1971. Statistical Methods in Biological Assay, 2nd ed., Griffin Press, London. 9 Thompson, W .R. 1947. Bacteriological Reviews, Vol 2, No.2: 115-145. 10. Dunnet, C.W. 1955. A Multiple Comparison's Procedure for Comparing Several Treatments with a Control. Jour. Amer. Statis. Assoc. 50:1096-1121. 11. Gulley, D.D. 1990. TOXSTAT Release 3.2. The University o f Wyoming. C01293 W il d l if e In t e r n a t io n a l , ltd -20- PROJECT NO.: 454-102 TABLE1 Cumulative Mortality from a Mallard Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) No. Dead Per No. Exposed Exposure Period Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 No. Dead Per No. Exposed Post-Exposure Period Day 6 Day 7 Day 8 Control 0 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 0/30 Treatment 9.1 18.3 36.6 73.2 146 293 586 1171 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 1/10 0/10 0/10 0/10 0/10 2/10 6/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 1/10 2/10 2/10 3/10 3/10 8/10 9/10 9/10 The LC50 value was calculated to be 628 ppm a.i. with a 95% confidence interval o f 448 ppm a.i. to 958 ppm a.i. 1- No mortalities occurred in any of the treatment levels from Day 8 to Day 22,_____________________ 003-294 W il d l if e In t e r n a t io n a l , ltd -21 - PROJECT NO.: 454-102 TABLE 2 Page 1 Mean Body Weight (g) from a Mallard Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) _________ Exposure Period______ Change1 Day 0 Day 5 Day 0-5 Post-Exposure Period Change' Day 8 Day 5-8 Total Change (Day 0-8)1 Control 0 Mean SD 135 279 24 42 144 22 380 101 53 16 Treatment 9.1 Mean SD 119 226** 108** 14 36 23 317* 91 66 33 18.3 Mean SD 146 277 14 26 131 18 377 100 32 11 36.6 Mean SD 147 275 16 32 128 22 375 100 37 12 73.2 Mean SD 143 260 30 55 117* 33 343 82 90 42 146 Mean SD 143 242 19 37 100** 21 331 89 48 16 293 Mean SD 129 161** 32** 17 28 33 220** 55 57 44 586 Mean SD 144 137** -6** 23 27 26 175** 34 36 41 <N 00 1171 Mean SD 147 112** -37** 25 28 13 312 -- `Mean change is calculated separately from the mean body weights using individual body weights (See Appendix VI). 3n=l, could not be evaluated statistically using Dunnett's t-test. (-) = No data available due to mortality. Statistically different from the control group at p < 0.05 (Dunnett's t-test). Statistically different from the control group at p < 0.01 (Dunnett's t-test). 245 34 198* 55 231 25 228 30 200* 73 188** 32 94** 54 39** 36 152 - 001295 W il d l if e In t e r n a t io n a l , ltd -22- PROJECT NO.: 454-102 TABLE2 Page 2 Mean Body Weight (g) from a Mallard 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 Change1(8-22) 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* 33 384 27 377 33 364 82 332* 58 211** 49 575 32 625 42 620 44 579 97 564 56 467** 51 230 20 241 22 243 40 216 21 232 22 256 35 823 183 49 24 773 198 38 15 811 186 81 41 828 208 78 34 782 203 135 39 688* 124* 86 90 701* 234 41 35 413 42 427 34 427 62 451 71 418 56 356 106 490 54 586 Mean SD 174** 46 394** 82 221 37 613** 91 219 13 1171 Mean 1852 3732 1982 6342 2512 SD - -- -- `Mean change is calculated separately from the mean body weights using individual body weights (See Appendix VI). 2n=l, could not be evaluated statistically using Dunnett's t-test. (-) = No data available due to mortality. *Statistically different from the control group at p < 0.05 (Dunnett's t-test). **Statistically different from the control group at p < 0.01 (Dunnett's t-test). 439 47 4492 - 001296 W il d l if e In t e r n a t io n a l , ltd -23 - PROJECT NO.: 454-102 TABLE 3 Page 1 Mean Feed Consumption (g/bird/day) from a Mallard Acute Dietary Toxicity Study with PFOS Experimental Group (ppma.i.) Control 0 Exposure Period Day 0-5 Mean SD 92 10 Treatment 9.1 18.3 36.6 73.2 146 293 586 1171 (-) = No data available due to mortality. 73 91 94 77 105 44 36 22 Post-Exposure Period Day 6-8 125 17 117 132 125 101 159 63 55 25 001297 W il d l if e In t e r n a t io n a l , ltd -24- PROJECT NO.: 454-102 TABLE 3 Page 2 Mean Feed Consumption (g/bird/day) from a Mallard Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) 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 (-) = No data available due to mortality. 172 186 165 148 159 109 114 106 198 204 179 173 164 132 143 154 001298 W il d l if e In t e r n a t io n a l , ltd -25- PROJECT NO.: 454-102 TABLE4 Gross Pathological Observations from a Mallard Acute Dietary Toxicity Study with PFOS Birds that died during the course o f the study Finding Crop empty Emaciated G.I. Tract, primarily empty Gizzard contents bile stained Gizzard, empty Intestinal contents, black and tar-like Keel prominent Kidneys, pale Loss of muscle mass Spleen, grey Spleen, small and pale Spleen, pale Thin Male, Female, and Undetermined _____________ PPM A.I.__________ 293 586 1171 N=2 N=3 N=9 027 1 14 111 024 001 001 002 00 1 225 00 1 1 12 023 1 14 C01299 W il d l if e In t e r n a t io n a l , ltd -26- PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Control 0 0 0 0 0 0 Treatment 9.1 9.1 Table 5 Cumulative Mortality (Estimated Cumulative Dose, mg/kg1) from a Mallard Acute Dietary Toxicity Study with PFOS No. Dead Per No. Exposed (Cumulative Dose, mg/kg) _________________________Exposure Period________________________________ Pen DayO 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 0/5 0/5 0/5 6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1 0/5 2 0/5 0/5 (4) 0/5 (4) 0/5 (8) 0/5 (8) 0/5 (12) 0/5 (12) 0/5 (16) 0/5 (16) 0/5 (20) 0/5 (20) 0/5 0/5 0/5 0/5 0/5 0/5 18.3 1 0/5 0/5 (8) 0/5 (16) 0/5 (24) 0/5 (32) 0/5 (40) 0/5 0/5 0/5 18.3 2 0/5 0/5 (9) 0/5 (18) 0/5 (27) 0/5 (36) 0/5 (45) 0/5 0/5 0/5 36.6 1 0/5 0/5 (19) 0/5 (38) 0/5 (57) 0/5 (76) 0/5 (95) 0/5 0/5 0/5 36.6 2 0/5 0/5 (16) 0/5 (32) 0/5 (48) 0/5 (64) 0/5 (80) 0/5 0/5 0/5 73.2 1 0/5 0/5(31) 0/5 (62) 0/5 (93) 0/5 (124) 0/5 (155) 0/5 0/5 0/5 73.2 2 0/5 0/5 (30) 0/5 (60) 0/5 (90) 0/5 (120) 0/5 (150) 0/5 0/5 0/5 146 1 0/5 0/5 (74) 0/5 (148) 0/5 (222) 0/5 (296) 0/5 (370) 0/5 0/5 0/5 146 2 0/5 0/5 (101) 0/5 (202) 0/5 (303) 0/5 (404) 0/5 (505) 0/5 0/5 0/5 293 1 0/5 0/5 (64) 0/5 (128) 0/5 (192) 0/5 (256) 0/5 (320) 0/5 0/5 0/5 293 2 0/5 0/5 (126) 0/5 (252) 0/5 (378) 0/5 (504) 0/5 (630) 0/5 1/5 2/5 586 1 0/5 0/5 (175) 0/5 (350) 0/5 (525) 0/5 (700) 0/5 (875) 0/5 1/5 1/5 586 2 0/5 0/5 (153) 0/5 (306) 0/5 (459) 0/5(612) 1/5 (765) 2/5 2/5 2/5 1171 1 0/5 0/5 (240) 0/5 (480) 0/5 (720) 1/5 (960) 4/5 (1200) 5/5 5/5 1171 2 0/5 0/5 (207) 0/5 (414) 0/5 (621) 1/5 (828) 2/5 (1035) 3/5 4/5 The LC50 value was calculated to be approximately 628 ppm a.i. with a 95% confidence interval of448 to 958 ppma.i.. Estimated cumulative dose is based upon the average body weight and feed consumption over the 5-day exposure period, and serves as a rough approximation of the actual amount of test substance consumed. 2- No mortalities occurred in any of the treatment or control groups after Day 8._________________________________________________ 5/5 4/5 QQ13Q0 W il d l if e In t e r n a t io n a l , ltd -27- PROJECT NO.: 454-102 APPENDIX I Certificate O f Analysis FC-95, Lot 217 March 9,2 0 0 0 Richard M. Payfer th is sample was analyzed using LC/MS, 'H-NMR, :^F-NMR, end elementalanalyses techniques. The results Vithese tests show the.m ple to contain the following -weight percent composition; CiFsSOj-KT c S tS v F C XeSO H ? c j F i.s d i'i r C iF iiS O j-r S SS*? C.F.7SOJTKT CyFiiSO jTC cio P iiS O jir CiiFm SOjT T CFi-CO^Tt? 0 .0 4 % 0 .8 3 % 1 .3 8 % 1.30% 3 .7 1 % _ _ W1.(1499%% ^49% 0 .1 3 % 0 .0 4 % <T5S% 0.35 % Additionally, the iscm ia distribution o fth csaitp lew ss determined using 19F-MMR technlquea and found to contain die M ow ing molo percent corapositkm: ---- ,, (Normal chain, where x la mainly T) 735S CFi(CFiVCT(CFiK C F A r S t* K7 (E nteral monomethylbranch, where x fy is mainly 5. and x # 0 . y * 0 ) (C F ihC P -(C F ^rS Q |-K r (Isonrow i branch, w ho* x b mainly 5) C ^ rC T (C F ,)- S C V l? (Aloha branch. where x is mainly 6) (CFjJjCKCFj)** SOj* KT (t-fautvi branch. -where x Is mainly 41 CFsKCFaVCiCFsM CPA- SO j KT (Internal gem-dimethyl branch, where xH-y is mainly 4. and x e 01 17.1% 103% 1.6% 03% 0.2% C01301 W il d l if e In t e r n a t io n a l , ltd -28- PROJECT NO.: 454-102 APPENDIX II DIET FORMULATION WILDLIFE INTERNATIONAL LTD. GAME BIRD RATION1 INGREDIENTS Fine Com Meal Soy Bean Meal, 48% Protein Wheat Midds Protein Base Agway Special, 60% Protein Alfalfa Meal, 20% Protein Dried Whey Ground Limestone Eastman CalPhos Methionine Premix + Liquid Vitamin and Mineral Premix (see below) GL Ferm (Fermatco)2 Salt Iodized Total PERCENT (%) 44.83 30.65 6.50 6.00 4.00 3.00 2.50 0.90 0.60 0.35 0.32 0.25 0.10 100.00 VITAMIN AND MINERAL PREMIX AMOUNT ADDED PER TON Vitamin D3 Vitamin A Riboflavin Niacin Pantothenic Acid Vitamin B)2 Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (Menadione Dimethylpyrimidinol Bisulfite) Manganese Zinc Copper Iodine Iron Selenium 2.000. 000 I.C.U. 7.000. 000 I.U. 6 grams 40 grams 10 grams 8 mgs 600 mgs 64 mgs 1.2 grams 1.2 grams 20,000 I.U. 5.8 grams 102 grams 47 grams 6.8 grams 1.5 grams 51 grams 182 mgs `The guaranteed analysis is a minimum of 27% protein, a minimum of 2.5% crude fat and a maximum of 5% crude fiber. ferm entation By-Products (Source o f Unidentified Growth Factors) 001302 W il d l if e In t e r n a t io n a l , ltd -29- PROJECT NO.: 454-102 APPENDIX III ANALYTICAL METHODS AND RESULTS 001303 W il d l if e In t e r n a t io n a l , ltd -30- PROJECT NO.: 454-102 INSTRUMENT: Appendix III Table 1 Typical LC/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil Qg column (100 mm x 2 mm I.D., 3 pm particle size) OVEN TEMPERATURE: 30C STOP TIME: 10.0 minutes FLOW RATE: 0.220 mL/minute MOBILE PHASE: 72.0% Methanol : 28.0% NANOpure Water containing 0.1% Formic Acid INJECTION VOLUME: 25.0 pL PFOS RETENTION TIME: Approximately 7.0 minutes INTERNAL STANDARD RETENTION TIME: Approximately 4.8 minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 426.7 amu 001304 W il d l if e In t e r n a t io n a l , ltd -31 - PROJECT NO.: 454-102 Appendix III Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454-102-) MAB-1 Sample Type Matrix Blank Measured Concentration of PFOS1 (ppm a.i.) < LOQ MAB-2 Matrix Blank < LOQ 1The limit 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). 001305 W il d l if e In t e r n a t io n a l , ltd -32- PROJECT NO.: 454-102 Appendix III Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454-102-) MAS-1A MAS-4A 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 MAS-6 1830 1716 86.1 93.7 Mean = Standard Deviation = CV = N= 94.7 6.99 7.38 6 Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. 00130G W il d l if e In t e r n a t io n a l , ltd -33Appendix HI 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) 1 x = 9.8 ppm a.i. SD = 0.969 ppm a.i. CV = 9.94% Mean Percent of Nominal 108 18.3 2 Top Left 18.5 3 Top Right 23.4 4 Middle Left 18.3 5 Middle Right 17.3 6 Bottom Left 19.4 7 Bottom Right 19.9 x = 19.5 ppm a.i. SD = 2.13 ppm a.i. CV= 10.9% 107 1171 18 Top Left 19 Top Right 2 0 Middle Left 2 1 Middle Right 2 2 Bottom Left 23 Bottom Right 1239 1221 1118 1301 1163 1133 x= 1196 ppm a.i. SD = 70.2 ppm a.i. CV= 5.87% 102 Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. 1 Coefficient of variation was calculated using full precision of mean and standard deviation results. 001307 W il d l if e In t e r n a t io n a l , ltd -34Appendix III Table 5 Verification of PFOS Concentrations in Avian Diet PROJECT NO.: 454-102 Nominal Concentration (ppm a.i.) 0.0 Sample Number (S-454-102-) 1 Sampling Interval (Day) 0 PFOS Measured Concentration1 (ppm a.i.) < LOQ Percent of Nominal - Mean Measured Concentration (ppm a.i.) - Mean Percent of Nominal - 9.1 - - - - 9.82 1082 18.3 - - - - 19.52 1072 36.6 8 9 0 0 45.7 125 40.2 110 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 15 0 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 of quantitation (LOQ) was 1.15 ppm a.i. based upon the product of the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor of the matrix blank samples (500). 2Result obtained from Table 4. C0130S W il d l if e In t e r n a t io n a l , ltd -35Appendix III Table 6 Ambient Stability o f PFOS in Avian Diet During the Mallard LC50 Study PROJECT NO.: 454-102 Concentration (ppm a.i.) 0 Sample Number (S-454-102-) 1 Day 0 1 Mean Measured Concentration (ppm a.i.) - Mean Percent of Nominal - Sample Number (S-454-102-) 39 Day 5 Measured Concentration3 (ppm a.i.) Mean Measured Concentration (ppm a.i.) < LOQ - Mean Percent of Day 0 - 9.1 54-59 9.8 108 60 12.3 11.7 119 61 11.0 18.3 2-7 19.5 107 40 18.2 19.0 97.4 41 19.7 36.6 8 ,9 40.2 110 42 47.9 52.4 1303 43 56.8 73.2 10, 11 74.5 102 44 77.6 77.8 104 45 77.9 146 12, 13 174 119 46 167 164 94.3 47 160 293 14, 15 291 99.3 48 297 295 101 49 293 586 16,17 537 91.6 50 552 541 101 51 530 1171 18-23 1196 102 52 1150 1136 95.0 53 1122 'Day 0 results obtained from Table 4 and Table 5. 'The limit of quantitation (LOQ) was 1.15 ppm a.i. based upon the product o f the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor o f the matrix blank samples (500). T h e Day 5 value is consistent with the Day 0 mean measured concentration value for this level. No analytical measuring bias is indicated. 001309 W il d l if e In t e r n a t io n a l , ltd -36 - PROJECT NO.: 454-102 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN AVIAN DIET Prepare matrix fortification samples in the desired avian feed stock using the dry mix technique. 4 Dry Mix Technique For the high-level matrix fortification sample, weigh the requisite quantity o f Wildlife International Ltd. (WIL) ration into a weigh boat i Weigh the requisite quantity oftest substance (PFOS) into a beaker. 4 Add 'Ao f the WIL ration and the test substance to a larger beaker. 4 Rinse the beaker that contained the PFOS with small portions o f the remaining ration and transfer all portions to the larger beaker. 4 Mix the contents o f 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 o fthe 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 o f the matrix blank, matrix fortification and test samples into weigh boats and transfer to 16-oz. French-square glass bottles. Record the weights. 4 For each sample, measure 100 mLs o f 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 o f 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 dilutions) to bring final concentration into the calibration range of the LCMS methodology. Use methanol for intermediate dilutions, if required. For all final dilutions use 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v). 4 Amputate and submit samples for LC/MS analysis. Figure 1. Analytical method flowchart for the analysis of PFOS in avian diet. 001310 W il d l if e In t e r n a t io n a l , ltd -37- Appendix III PROJECT NO.: 454-102 Concentration (Ratio) Figure 2. A typical calibration curve for PFOS. Slope = 2.77397; Intercept = 0.01894; r = 0.9981. Curve is weighted (1/x). 001311 W il d l if e In t e r n a t io n a l , ltd -38- Appendix III PROJECT NO.: 454-102 Figure 3. A representative ion chromatogram of a low-level (0.00229 mg a.i./L) PFOS standard. 001312 W il d l if e In t e r n a t io n a l , ltd -39- Appendix III PROJECT NO.: 454-102 Figure 4. A representative ion chromatogram of a high-level (0.0457 mg a.i./L) PFOS standard. 001313 W il d l if e In t e r n a t io n a l , ltd -40- Appendix III PROJECT NO.: 454-102 Figure 5. A representative chromatogram of a matrix blank sample (454-102-MAB-l). The arrow indicates the retention time of PFOS. 001314 W il d l if e In t e r n a t io n a l , ltd -41 - Appendix III PROJECT NO.: 454-102 Figure 6. A representative chromatogram of a matrix fortification sample (454-102-MAS-1A). C01315 W il d l if e In t e r n a t io n a l , ltd -42- Appendix III PROJECT NO.: 454-102 Figure 7. A representative chromatogram of a test sample (454-102-2). C01316 W il d l if e In t e r n a t io n a l , ltd -43 - PROJECT NO.: 454-102 APPENDIX IV DIET PREPARATION Weight and volume o f constituents used to prepare test diets: Nominal Concentrations (ppm a.i.) 0 9.1 18.3 36.6 73.2 146 293 586 1171 Test Substance (g) 0.0910 0.1818 0.3638 0.7282 1.4659 2.9123 5.8239 11.6483 Basal Ration (g) 9000.0 8999.9 8999.8 8999.6 8999.3 8998.5 8997.1 8994.2 8988.4 Diets were prepared as follows: 5000.0 g of basal ration was weighed into a tared Hobart mixing bowl. The test substance was weighed in a tared weigh boat Approximately 100 g o f 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. 01317 W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-102 -44- APPENDIX V Cumulative Mortality by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Experimental Group Pen No. Dead Per No. Exposed Exposure Period (ppm a.i.) Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Control 0 1 0/5 0/5 0/5 0/5 0/5 0/5 0 2 0/5 0/5 0/5 0/5 0/5 0/5 0 3 0/5 0/5 0/5 0/5 0/5 0/5 0 4 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 6 0/5 0/5 0/5 0/5 0/5 0/5 Treatment 9.1 1 0/5 0/5 0/5 0/5 0/5 0/5 9.1 2 0/5 0/5 0/5 0/5 0/5 0/5 18.3 1 0/5 0/5 0/5 0/5 0/5 0/5 18.3 2 0/5 0/5 0/5 0/5 0/5 0/5 36.6 1 0/5 0/5 0/5 0/5 0/5 0/5 36.6 2 0/5 0/5 0/5 0/5 0/5 0/5 73.2 1 0/5 0/5 0/5 0/5 0/5 0/5 73.2 2 0/5 0/5 0/5 0/5 0/5 0/5 146 1 0/5 0/5 0/5 0/5 0/5 0/5 146 2 0/5 0/5 0/5 0/5 0/5 0/5 293 1 0/5 0/5 0/5 0/5 0/5 0/5 293 2 0/5 0/5 0/5 0/5 0/5 0/5 586 1 0/5 0/5 0/5 0/5 0/5 0/5 586 2 0/5 0/5 0/5 0/5 0/5 1/5 1171 1 0/5 0/5 0/5 0/5 1/5 4/5 1171 2 0/5 0/5 0/5 0/5 1/5 2/5 No. Dead Per No. Exposed Post-Exposure Period Day 6 Day 7 Day 8 * 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1/5 0/5 1/5 2/5 2/5 5/5 5/5 3/5 4/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2/5 1/5 2/5 5/5 4/5 The LC50 value was calculated to be 628 ppm a.i. with a 95% confidence interval o f448 ppm a.i. to 958 ppm a.i. * - No mortalities occurred in any of the treatment or control groups after Day 8.___________________________ 8TET00 W il d l if e In t e r n a t io n a l , ltd -45 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 1 Experimental Group (ppm a.i.) Bird Day 0 Day 5 Change Day 0-5 Day 8 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) 272 245 261 271 249 260 12 0 1 136 282 146 399 117 2 142 284 142 394 1 1 0 3 152 299 147 409 1 1 0 4 152 312 160 430 118 5 177 345 168 484 139 Mean 152 304 153 423 119 SD -16 26 11 37 1 2 263 252 257 278 307 271 22 0 1 125 265 140 372 107 2 134 282 148 371 89 3 149 304 155 440 136 4 154 292 138 378 8 6 5 166 328 162 430 1 0 2 Mean 146 294 149 398 104 SD 16 24 10 34 2 0 247 237 291 224 264 253 26 0 1 124 271 147 376 105 2 135 263 128 360 97 3 151 300 149 408 108 4 163 333 170 432 99 5 170 351 181 453 1 0 2 Mean 149 304 155 406 1 0 2 SD 19 38 2 1 38 4 252 225 257 269 283 257 22 C01319 W il d l if e In t e r n a t io n a l , ltd -46APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 2 Experimental Group (ppm a.i.) Bird Day 0 Day 5 Change Day 0-5 Day 8 0 1 107 2 2 2 115 298 2 126 267 141 360 3 116 203 87 266 4 107 203 96 284 5 1 1 1 248 137 348 Mean 113 229 115 311 SD 8 28 24 41 PROJECT NO.: 454-102 Change Day 5-8 76 93 63 81 100 83 15 Total Change 191 234 150 177 237 198 37 0 Group Total 1 1 0 0 235 135 335 1 0 0 2 1 0 0 232 132 328 96 3 1 0 2 233 131 317 84 4 108 267 159 383 116 5 1 0 2 2 1 0 108 299 89 Mean 102 235 133 332 97 SD 3 2 0 18 32 12 Mean 135 279 144 380 101 SD 24 42 2 2 53 16 235 228 215 275 197 230 29 245 34 001320 W il d l if e In t e r n a t io n a l , ltd -47APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 3 PROJECT NO.: 454-102 Experimental Group (PPma-i.) Bird Day 0 9.1 1 123 2 118 3 151 4 108 5 119 Mean SD 124 16 Day 5 Change Day 0-5 Day 8 244 1 2 1 349 231 113 334 286 135 399 209 1 0 1 313 231 1 1 2 332 240 116 345 29 13 33 Change Day 5-8 105 103 113 104 101 105 5 Total Change (0-8) 226 216 248 205 213 222 17 9.1 1 96 145 49 2 123 250 127 3 127 237 1 1 0 4 1 1 2 216 104 5 1 1 1 215 104 Group Total Mean SD Mean SD 114 12 119 14 213 41 226** 36 99 29 108** 23 Statistically different from the control group at p < 0.05 (Dunnett's t-test). **Statistically different from the control group at p < 0.01 (Dunnett's t-test). 146 345 343 316 294 289 83 317* 66 1 95 106 100 79 76 43 91 33 50 222 216 204 183 175 71 198* 55 001321 W il d l if e In t e r n a t io n a 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 SD 145 14 Day 5 Change Day 0-5 Day 8 261 138 355 297 156 421 270 1 2 0 361 286 134 390 294 134 391 282 136 384 16 13 27 Change Day 5-8 94 124 91 104 97 102 13 Total Change 232 280 211 238 231 238 25 18.3 Group Total 1 130 240 1 1 0 327 87 2 137 266 129 359 93 3 145 268 123 366 98 4 151 258 107 366 108 5 171 333 162 433 1 0 0 Mean 147 273 126 370 97 SD 16 35 2 2 39 8 Mean 146 277 131 377 100 SD 14 26 18 32 11 197 222 221 215 262 223 24 231 25 C01322 W il d l if e In t e r n a t io n a l , ltd -49- PROJECT NO.: 454-102 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 5 Experimental Group (ppm a.i.) Bird Day 0 36.6 1 131 2 132 3 142 4 160 5 179 Mean SD 149 21 Day 5 Change Day 0-5 Day 8 261 130 372 256 124 356 269 127 369 270 1 1 0 353 335 156 433 278 129 377 32 17 33 Change Day 5-8 111 100 100 83 98 98 10 Total Change 241 224 227 193 254 228 23 36.6 Group Total 1 131 230 99 316 86 2 136 287 151 402 115 3 147 263 116 379 116 4 151 255 104 344 89 5 160 324 164 427 103 Mean 145 272 127 374 102 SD 1 2 36 29 44 14 Mean 147 275 128 375 100 SD 16 32 2 2 37 1 2 185 266 232 193 267 229 39 228 30 001323 W il d l if e In t e r n a t io n a l , ltd -50- PROJECT NO.: 454-102 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 6 Experimental Group (ppm a.i.) Bird Day 0 73.2 1 113 2 113 3 142 4 154 5 179 Mean SD 140 28 Day 5 Change Day 0-5 Day 8 203 90 281 219 106 288 286 144 390 290 136 379 338 159 480 267 127 364 56 28 82 Change Day 5-8 78 69 104 89 142 96 29 Total Change 168 175 248 225 301 223 55 73.2 1 109 2 2 2 113 2 129 254 125 3 139 243 104 4 147 196 49 5 204 352 148 Mean 146 253 108 SD 36 59 37 Group Total Mean 143 260 117* SD 30 55 33 Statistically different from the control group at p < 0.05 (Dunnett's t-test). 302 332 340 175 458 321 101 343 90 80 78 97 -2 1 106 68 51 82 42 193 203 201 28 254 176 86 200* 73 C01324 W il d l if e In t e r n a t io n a l , ltd -51 - PROJECT NO.: 454-102 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 7 Experimental Group (ppm a.i.) Bird Day 0 146 1 108 2 138 3 151 4 151 5 164 Mean SD 142 21 Day 5 Change Day 0-5 Day 8 169 61 236 248 1 1 0 330 271 1 2 0 375 254 103 341 282 118 380 245 1 0 2 332 44 24 58 Change Day 5-8 67 82 104 87 98 88 14 Total Change 128 192 224 190 216 190 38 146 1 115 204 89 2 139 219 80 3 144 229 85 4 159 261 1 0 2 5 158 285 127 Group Total Mean 143 240 97 SD 18 33 19 Mean 143 242 1 0 0 ** SD 19 37 2 1 **Statistically different from the control group at p < 0.01 (Dunnett's t-test). 280 287 348 358 374 329 43 331 48 76 68 119 97 89 90 20 89 16 165 148 204 199 216 186 29 188** 32 C01325 W il d l if e In t e r n a t io n a l , ltd -52- APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 8 PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Bird Day 0 293 1 115 2 139 3 140 4 155 5 112 Mean SD 132 18 Day 5 Change Day 0-5 Day 8 142 27 179 119 - 2 0 165 156 16 217 187 32 291 134 2 2 203 148 15 2 1 1 26 2 1 49 Change Day 5-8 27 -2 0 16 32 22 15 21 Total Change 64 26 77 136 91 79 40 293 1 123 205 82 2 1 2 0 183 63 3 151 139 - 1 2 4 106 158 52 5 124 185 61 Mean SD 125 16 174 26 49 36 Group Total Mean SD 129 17 161** 28 32** 33 (-) = No data available due to mortality. **Statistically different from the control group at p < 0.01 (Dunnett's t-test). 289 273 - 223 138 231 68 2 2 0 ** 55 82 63 -1 2 52 61 49 36 57** 44 166 153 - 117 14 113 69 9 4 ** 54 001326 W il d l if e In t e r n a t io n a l , ltd -53 - APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 9 PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Bird Day 0 586 1 1 2 2 2 135 3 152 4 154 5 165 Mean SD 146 17 Day 5 Change Day 0-5 Day 8 105 -17 126 143 8 209 160 8 217 129 -25 - 195 30 143 146 1 174 34 2 2 46 Change Day 5-8 21 66 57 - -52 23 54 Total Change 4 74 65 - -2 2 30 47 586 1 105 114 9 2 1 2 0 130 10 3 149 - - 4 153 128 -25 5 182 128 -54 Group Total Mean SD Mean SD 142 30 144 23 125 7 137** 27 -15 31 -6 ** 26 (-) = No data available due to mortality. Statistically different from the control group at p < 0.01 (Dunnett's t-test). 159 185 - 185 - 176 15 175** 34 45 55 - 57 - 52 6 36** 41 54 65 - 32 - 50 17 3 9 *+ 36 C01327 W il d l if e In t e r n a t io n a l , 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 (ppm a.i.) Bird Day 0 1171 1 124 2 140 3 145 4 150 5 166 Mean SD 145 15 Day 5 Change Day 0-5 Day 8 8 8 -39 -- 96 -49 118 -32 -1 0 0 -40 17 9 - - Change Day 5-8 - - - Total Change _ - 1171 1 128 87 -41 2 110 - - 3 145 132 -13 4 157 - - 5 200 154 -46 Mean 148 124 -33 SD 34 34 18 Group Total Mean SD 147 25 112** 28 -37** 13 (-) = No data available due to mortality. *No mortalities occurred in any of the treatment or control groups after Day 8. Statistically different from the control group at p < 0.01 (Dunnett's t-test). - - - - 185 185 - 852 - " - - - 31 31 - 31' - -15 -15 15' - C0132S W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-102 -55 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 11 Experimental Group (ppm a.i.) 0 Bird 1 2 3 4 5 Mean SD Day 8 404 381 415 429 418 409 18 Day 15 721 633 685 692 679 682 32 Change Day 8-15 317 252 270 263 261 273 26 Day 22 895 780 861 822 847 841 43 Change Day 15-22 174 147 176 130 168 159 20 Total Change (8 -2 2 ) 491 399 446 393 429 432 40 0 1 399 606 207 810 204 2 394 604 2 1 0 791 187 3 409 638 229 819 181 4 430 543 113 730 187 5 484 752 268 924 172 Mean 423 629 205 815 186 SD 37 77 57 70 1 2 411 397 410 300 440 392 54 0 Group Total 1 372 583 2 1 1 779 196 2 371 613 242 823 2 1 0 3 440 636 196 859 223 4 378 594 216 794 2 0 0 5 430 627 197 816 189 Mean 398 611 212 814 204 SD 34 22 19 31 13 Mean 410 640 230 823 183 SD 30 56 47 49 24 407 452 419 416 386 416 24 413 42 0 0 1 3 .T 3 W il d l if e In t e r n a t io n a l , ltd -56APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 12 PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Bird Day 8 Change Day 15 Day 8-15 Day 22 Change 9.1 1 349 602 253 821 219 2 334 549 215 730 181 3 399 616 217 802 186 4 313 562 249 767 205 5 . 332 546 214 744 198 Mean 345* 575 230 773 198 SD 33 32 2 0 38 15 Total Change 472 396 403 454 412 427 34 Experimental Group (ppm a.i.) Bird Day 8 Change Change Day 15 Day 8-15 Day 22 Day 15-22 18.3 1 355 611 256 2 421 693 272 3 361 578 217 4 390 626 238 5 391 618 231 Mean 384 625 242 SD 27 42 2 2 *Statistically different from the control group at p < 0.05 (Dunnett's t-test). 811 938 713 794 799 811 81 200 245 135 168 181 186 41 Total Change 465 517 352 404 408 427 62 001330 W il d l if e In t e r n a t io n a l , ltd -57- PROJECT NO.: 454-102 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 13 Experimental Group (ppm a.i.) Bird Day 8 36.6 1 372 2 356 3 369 4 353 5 433 Mean SD 377 33 Change Change Day 15 Day 8-15 Day 22 Day 15-22 677 305 942 265 607 251 817 2 1 0 613 244 793 180 559 206 746 187 642 209 840 198 620 243 828 208 44 40 73 34 Total Change 570 461 424 393 407 451 71 Experimental Group (ppm a.i.) Bird Day 8 73.2 1 281 2 288 3 390 4 379 5 480 Mean SD 364 82 Change Change Day 15 Day 8-15 Day 22 Day 15-22 482 2 0 1 648 166 480 192 646 166 636 246 864 228 599 2 2 0 796 197 699 219 955 256 579 216 782 203 97 2 1 135 39 Total Change 367 358 474 417 475 418 56 001331 W il d l if e In t e r n a t io n a l , ltd -58- PROJECT NO.: 454-102 APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietaiy Toxicity Study with PFOS Page 14 Experimental Group (ppm a.i.) Bird Day 8 Change Day 15 Day 8-15 Day 22 Change 146 1 236 475 239 659 184 2 330 569 239 739 170 3 375 631 256 738 107 4 341 570 229 754 184 5 380 576 196 550 -26 Mean 332* 564 232 688* 124* SD 58 56 2 2 8 6 90 Total Change 423 409 363 413 170 356 106 Experimental Group (ppm a.i.) Bird Day 8 Change Change Day 15 Day 8-15 Day 22 Day 15-22 293 1 179 447 268 2 165 415 250 3 217 526 309 4 291 516 225 5 203 430 227 Mean 2 1 1 ** 467** 256 SD 49 51 35 Statistically different from the control group at p < 0.05 (Dunnett's t-test). Statistically different from the control group at p < 0.01 (Dunnett's t-test). 731 646 748 704 676 701* 41 284 231 222 188 246 234 35 Total Change 552 481 531 413 473 490 54 C01332 W il d l if e In t e r n a t io n a l , ltd -59APPENDIX VI Individual Body Weights (g) from a Mallard Acute Dietary Toxicity Study with PFOS Page 14 PROJECT NO.: 454-102 Experimental Group (ppm a.i.) Bird Day 8 586 1 126 2 209 3 217 A 5 143 Mean SD 174 46 Change Change Day 15 Day 8-15 Day 22 Day 15-22 308 182 525 217 469 260 706 237 460 243 675 215 340 197 546 206 394 2 2 1 613 219 82 37 91 13 Total Change 399 497 458 403 439 47 Experimental Group (ppm a.i.) Bird Day 8 Change Change Day 15 Day 8-15 Day 22 Day 15-22 1171 1 -- 2*1 J 4 5 185 383 198 Mean 1852 383* I983 SD - - - 2n=l, could not be evaluated statistically using Dunnett's t-test. -- 634 251 634s 25P -- Total Change - 449 449s - C01333 W il d l if e In t e r n a t io n a l , ltd -60APPENDIX VII PROJECT NO.: 454-102 Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Page 1 Experimental Group (ppm a.i.) Pen Exposure Period Day 0-5 Post-Exposure Period Day 6-8 Control 1 2 3 4 5 6 Mean SD 109 92 85 93 82 94 92 10 145 140 117 132 100 116 125 17 001324 W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-102 -61 APPENDIX VII Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Page 2 Experimental Group (ppm a.i.) Pen Exposure Period Day 0-5 Post-Exposure Period Day 6-8 9.1 1 73 2 72 Mean 73 130 104 117 18.3 1 90 2 91 Mean 91 135 128 132 36.6 1 103 2 85 Mean 94 129 120 125 73.2 1 79 2 74 Mean 77 109 94 101 146 1 89 2 121 Mean 105 102 217 159 293 1 28 2 59 Mean 44 61 65 63 586 1 40 2 32 Mean 36 43 67 55 1171 1 23 2 22 Mean 22 25 26 25 G01335 W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-102 -62APPENDIX VII Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Page 3 Experimental Group (ppm a.i.) Pen Control 1 2 3 Mean SD Exposure Period Day 8-15 185 161 166 171 13 Post-Exposure Period Day 15-22 159 182 199 180 20 001336 W il d l if e In t e r n a t io n a l , ltd -63 APPENDIX VII PROJECT NO.: 454-102 Feed Consumption (g/bird/day) by Pen from a Mallard Acute Dietary Toxicity Study with PFOS Page 4 Experimental Group (ppm a.i.) Pen Exposure Period Day 8-15 Post-Exposure Period Day 15-22 9.1 1 172 198 18.3 1 186 204 36.6 1 165 179 73.2 1 148 173 146 1 159 164 293 1 109 132 586 1 114 143 1171 1 106 154 001337 W il d l if e In t e r n a t io n a l , ltd -64APPENDIX VIII PROJECT NO.: 454-102 CHANGES TO PROTOCOL This study was conducted in accordance with the approved Protocol with the following changes: 1. The protocol was amended to add an additional test concentration o f 10 ppm a.i.. 2. The protocol was amended to indicate that bile will be collected from all study birds. The protocol was clarified to indicate the collection of liver from birds that died during the course of the study. 3. The protocol was amended to change the test concentrations from 0, 10, 20, 40, 80, 160, 320, 640 and 1280 ppm a.i., to 0, 9.1, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 ppm a.i. Test concentrations were changed to reflect the test substance purity given in the new certificate of analysis. 4. Individually numbered wing bands were used to uniquely identify each duckling. The protocol required leg bands to be used to identify the birds. 5. Blood samples were collected on Day 8 and Day 22 in non-heparinized 5 ml borosilicate glass test tubes. The protocol indicated that heparinized vacutainers would be used. 6. The final temperature from one brooder unit on Day 8 and all brooder units on Day 22 were not recorded. 7. The afternoon observations were inadvertently not recorded for birds in a single brooder unit on May 8, 1999. 8. Observations for control birds from two brooder levels were inadvertently not recorded on Day 6 o f 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 o f the test. C01338 W il d l if e In t e r n a t io n a l , ltd -65 APPENDIX IX PERSONNEL INVOLVED IN THE STUDY PROJECT NO.: 454-102 The following key W ildlife International Ltd. personnel were involved in the conduct or management o f this study: (1) Mark Jaber, W ildlife Toxicologist (2) Joann B. Beavers, Director, Avian Toxicology (3) Sean P. Gallagher, Senior Biologist (4) Courtney Casey, M .S., Senior Biologist (5) Willard B. Nixon, Ph.D., Manager, Analytical Chemistry (6) Tim Kendall, Supervisor, Analytical Chemistry (7) Raymond L. Van Hoven, Ph.D., Scientist (8) Ellen Mank, Chemist W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-102 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A DIETARY LC50 STUDY WITH THE MALLARD PROTOCOL N O .:454/l 11098/MLCSDT.WC/PPTS AMENDMENT NO.: 1 SPONSOR:3M Corporation PROJECT NO.: 454-102 EFFECTIVE DATE: April 22,1999 AMENDMENT: On page 2 o f the protocol add an additional test concentration o f 10 ppm a i. REASON: Sponsor request L Jt)r- Study Director Laboratory Management Sponsor Principal Contact Date Date l 001340 W il d l if e In t e r n a t io n a l , ltd PROJECT NO.: 454-102 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A DIETARY LC50 STUDY WITH THE MALLARD PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS AMENDMENT NO.: 2 SPONSOR: 3M Corporation PROJECT NO.: 454-102 EFFECTIVE DATE: April 27,1999 AMENDMENT: On page 8 under necropsy add: When possible, bile will be collected from all birds. Livers will also be collected from all birds that die during the course o f the test. REASON: Bile is being collected at Sponsor request. The protocol is being clarified to indicate the collection o f liver from birds that die. Laboratmy^^agOTenT^ Date Date U sId h i Date 001341 W il d l if e In t e r n a t io n a l , ltd Page 1 of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A DIETARY LC50 STUDY WITH THE MALLARD PROTOCOL NO.: 454/111098/MLVSDT.WC/OPPTS DEVIATION NO.: 1 SPONSOR: 3M Corporation PROJECTNO.: 454-102 DATE OF DEFACTO DEVIATION: April 22-May 14,1999 DEVIATION: Individually numbered wing bands were used to uniquely identify each duckling. The protocol stated that birds would be identified by colored leg bands. REASON: Protocol error. Due to the extended duration o f this study, ducklings would have outgrown leg bands. The protocol should have been clarified to indicate that all ducklings would be identified by wing bands. IM PA C T: None. Wing bands are routinely used to identify ducklings and all birds were uniquely identified. WILDLEF _________ AL LTD. MANAGEMENT DATE 001342 W il d l if e In t e r n a t io n a l , ltd Page 1 of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A Dietary LC50 Study with the Mallard PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS SPONSOR: 3M Corporation DATE OF DEVIATION: April 30 and May 14,1999 DEVIATION NO.: 2 PROJECTNO.: 454-102 DEVIATION: Blood samples collected on Day 8 and Day 22 o f the test were stored in non-heparinized 5 ml borosillicate glass test tubes prior to being refrigerated. The protocol indicated that blood samples would be placed in labelled heparanized vacutainers. REASON: Test animals were too small to effeciently draw blood by syringe. Blood was draw by decapitating birds and draining blood into test tubes. Heparin was not added to blood samples due to a Sponsor request to seperate serum from the blood at W ildlife International Ltd. Seperation o f serum requires coagulation o f blood samples. IM PA C T: None. DATE DATE 001343 W il d l if e In t e r n a t io n a l , ltd Page 1 of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A Dietary LC50 Study with the Mallard PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS SPONSOR: 3M Corporation DATE OF DEVIATION: April 30 and May 14,1999 DEVIATION NO.: 3 PROJECTNO.: 454-102 DEVIATION: On April 3 0 ,1 9 9 9 , the final temperature from a brooder unit housing birds in the 640 ppm a.i. treatment level was not recorded prior to termination o f those birds. Additionally, on May 14 the final temperatures on all brooder units were not recorded prior to termination o f those birds. REASON: Biologist oversight. The brooder pons where temperatures were not recorded were empty at the time o f afternoon observations. Brooder pen temperatures are typically recorded during afternoon observations. Biologists recording afternoon observations on the occassions where temperatures were missed neglected to record temperatures for empty pens that housed birds earlier the same day. IM PA C T: None. Temperatures recorded for brooders the day prior to the missed data indicated that the brooders wore functioning properly. DATE 001344 W il d l if e In t e r n a t io n a l , ltd Page 1 of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A Dietary LC50 Study with the Mallard PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS SPONSOR: 3M Corporation DATE OF DEVIATION: May 8,1999 DEVIATION NO.: 4 PROJECTNO.: 454-102 DEVIATION: On May 8 ,1 9 9 9 , the afternoon observations for unit S 17-1 housing birds from the 320 ppm a.i. treatment level were inadvertently not recorded. REASON: Biologist oversight. IM PA C T: None. Observations from the morning o f May 8 and May 9,1999 indicate that all birds in this level were normal in appearance and behavior. Since the temperature for that unit was recorded at the same time observations were performed, it is apparent that the birds were observed, but the observation not recorded. ( AlLtvv, A STUDY DIRECTOR DATE WILDLIFE INTERNATIONAL LTD. MANAGEMENT A DATE 001345 W il d l if e In t e r n a t io n a l , l td Page 1 o f 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A DIETARY LC50 STUDY WITH THE MALLARD PROTOCOL NO.: 454/111098/MLVSDT.WC/OPPTS DEVIATION NO.: 5 SPONSOR: 3M Corporation PROJECTNO.: 454-102 DATE OF DEFACTO DEVIATION: April 28 and May 14,1999 DEVIATION: On April 28,1999, Day 6 o f the test, observations for control group birds in brooderlevels S62-2 and S62-3 w o e inadvertently not recorded. Temperatures for these brooder levels were also not recorded on this date. On May 14,1999, Day 22 o f the test, observations for control group birds in level S70-3 were inadvertently not recorded. The protocol required that birds would be observed at least once daily and that the brooder compartment temperature would be recorded once daily. REASON: Biologist oversight. IM PA C T: With the exception o f the missed observations, observations indicate all control birds were normal in behavior and appearance and brooder temperatures werewithin the acceptable range for the duration o f the test. It is reasonable to expect that birds were normal in behavior and appearance and that brooder temperatures were within the acceptable range at the time the observations were not recorded. Therefore, this deviation caused no adverse impact on the outcome and interpretation o f the study. WILDLIFE INTERNATIONAL LTD. MANAGEMENT DATE DATE C01346 W il d l if e In t e r n a t io n a l , ltd fl SOP DEVIATION 1 STUDY TITLE: PFOS: A DIETARY LC50 STUDY WITH THE MALLARD PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 1 SOP NO.: 415 DEVIATION NO.: 1 SPONSOR: 3M Corporation PROJECT NO.: 454-102 1 DATE OF DEFACTO DEVIATION: April 23,1998 1 1 DEVIATION: On April 23,1999 the temperature o f one brooder level used to house the 1280 ppm a.i. treatment group was 1F below the specified minimum o f 95 F. There are no records o f the responsible technician or biologist making a thermostat adjustment to correct the temperature. However, on the following day the temperature was within normal range. V REASON: f l Misadjustmenl o f the brooder unit thermostat IM PA C T: None. There was no apparent effect on test bird body weight or health. fl fl DATE fl WILDLIFE INTERNATIONAL LTD. MANAGEMENT DATE " fl f l C01347 W il d l if e In t e r n a t io n a l , ltd SOP DEVIATION STUDY TITLE: PFOS: A DIETARY LC50 STUDY WITH THE MALLARD PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS SOP NO.: 423.1A DEVIATION NO.: 2 SPONSOR: 3M Corporation PROJECT NO.: 454-102 DATE OF DEFACTO DEVIATION: April 22,1998 DEVIATION: On April 22,1999, during diet preparation on Day 0 o fthe test, 1.4659 g o f test substance was weighed out for preparation o f the 160 ppm a.i. test concentration diet. This amount exceeded the target amount by 0.099g REASON: Dietician oversight.. IM PA C T: None. The difference in the amount o f test substance weighed resulted in a less than 1% change in final dietary concentration. STUls X X S X X V L X W X u x \ DATE DATE 001348 PROTOCOL PFOS: A DIETARY LC50 STUDY WITH THE MALLARD U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.2200 FIFRA Subdivision E, Section 71-2 OECD Guideline 205 3M Lab Request No. U2723 Submitted to 3M Corporation Environmental Laboratory P.O .B ox 33331 S t Paul, Minnesota 55133 Wildlife International ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 November 10,1998 001349 PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l ltd. -2 - PFOS: A DIETARY LC50 STUDY WITH THE MALLARD SPONSOR: SPONSOR'S REPRESENTATIVE: TESTING FACILITY: STUDY DIRECTOR: LABORATORY MANAGEMENT: 3M Corporation Environmental Laboratory P.O. Box 33331 S t Paul, Minnesota 55133 Ms. Susan A. Beach W ildlife International Ltd. 8598 Commerce Drive Easton, Maryland 21601 Sean P. Gallagher, Senior Avian Biologist Joann B. Beavers Manager o f Avian Toxicology FOR LABORATORY USE ONLY Proposed Dates: Experimental Start Date: __ 4 4Project N o.^ S -/OZ Experimental Termination Date: Study Room: __ Test Concentrations: 3 1 0 , \V tfo S n i / r w ^ Test Substance No. t a 5 \ 4 <YY 7(p * V A VA 7t PROTOCOL APPROVAL PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l l t d . -3 - O B JE C T IV E The objective o f this study is to evaluate the toxicity o f a test substance to the mallard (A nas p la ty rh y n c h o s) administered through the diet for five days. An LC50 value w ill be if possible. SUMMARY Groups of ten mallard ducklings, 5-10 days old, will be fed diets containing the test substance at selected concentrations for five days. A control group that receives untreated diet will be m a in ta in e d concurrently. The test substance w ill be mixed into the diet, usually in a geometric series o f concentrations. The exposure period will be followed by a period o f at least three days oh untreated feed. A t the end o f the three-day observation period, half o f the birds in each treatment group w ill be euthanized and liver and blood collected for residue analysis. The remaining birds w ill be m a in ta in e d oh basal diet for an additional 14 days and then will be euthanized, with liver and blood collected. Throughout the test, the birds w ill be observed for toxicological responses. When possible, an LC50 value w ill be calculated. MATERIALS AND METHODS The methods, species used and route o f administration described in this protocol 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 o f the Environmental Protection Agency's Registration Guidelines,P e stic id e A ssessm en t G u idelin es, FIFRA S u bdivision E, H a za rd E va lu a tio n : W ildlife a n d A q u a tic O r g a n is m (2), ASTM Standard E857-87, "Standard P ra c tic e F o r C o n d u ctin g S u bacu te D ie ta ry T o x icity T ests W ith A via n S p ecies" (3), and OECD Guideline 205, A via n D ie ta r y T o x icity T e st (4). In order to control bias, birds w ill be assigned to pens by indiscriminate draw. N o other potential sources o f bias are expected to affect the results o f the study. The test substance w ill be administered in the diet This route o f administration was selected because it represents the m ost likely route o f exposure to avian species in the environment Jgst.Substang Information on the characterization o f test, control or reference substances is required by Good Laboratory Practice Standards (GLP). The Sponsor is responsible for providing W ildlife International Ltd. written verification that the test substance has been characterized according to GLPs prior to its use in the study. If written verification o f GLP test substance characterization is not provided to W ildlife PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 001351 W il d l if e In t e r n a t io n a l ltd . 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 -4 - Intemational Ltd., it w ill be noted in the compliance statement o f the final report. The attached form IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR (Appendix I) is to be used to provide information necessary for GLP compliance. The Sponsor is responsible for all information related to the test substance and agrees to accept any unused test substance and/or test substance containers remaining at the end o f the study. Treatment Groups Groups o f ten mallard ducklings (five per pen) will be assigned by indiscriminate draw to each o f the treatment and control groups. The sex o f each individual will not be determined. A test will usually consist o f a geometric series o f five dietary concentrations and at least three control groups. Test concentrations will be selected after evaluating toxicity data provided by the Sponsor. The highest test concentration need not exceed 5000 ppm. Each group is fed the appropriate test or control diet for five days. This w ill be followed by a period o f at least three days on untreated feed. Control birds will receive an amount of the solvent or vehicle in their diet equivalent to the highest amount in the treated diets. If more than one study is conducted simultaneously in the same room, with the same solvent or vehicle, a concurrent control may be utilized for all studies. Duration o f Test The primary phases o f the test and their durations are: 1. Acclimation - From receipt or hatch o f hatchlings until the start o f exposure. 2. Exposure - Five days. 3. Post-exposure observation - 3 days/17 days. Test-Birds The mallard represents an ecologically significant and widely distributed species in the United States. The mallard has demonstrated sensitivity to the effects produced by known toxic chemicals. Moreover, this species has proven to be a good laboratory model and a large amount o f baseline data are available. All mallards will be from the same hatch and will be 5-10 days old at test initiation. Only those that are apparently healthy will be used in the test. Body weights may vary between 75 and 300 grams depending on the age o f the cohort o f birds used (e.g. 5 versus 10 days). A t least five test and three PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 3M LAB REQUEST NO. W il d l if e In t e r n a t io n a l ltd . -5 - control groups will be used. Each treatment or control group will contain ten birds (two pens o f five birds each) with each group o f birds identified by pen number and test concentratioa Individual birds will be identified by colored leg bands. Birds w ill be obtained from Whistling W ings, 113 Washington Street, Hanover, IL 61041 or from another reputable supplier. All experimental birds w ill be wmliTwatwH to the caging and facilities from the time o f hatch or receipt until initiation o f the test. Animal Pigt A ll test birds will be fed a game bird ration formulated to W ildlife In te rn a ti o n a l Ltd. specifications (Table 1). Feed and water will be provided a d libitu m during acclimation and during the test The water source will be the Town o f Easton municipal water supply. Feed is analyzed periodically in accordance with W ildlife International Ltd. Standard Operating Procedures. The birds w ill receive no form o f antibiotic medication during the test Specifications for acceptable levels o f contaminants in game bird ration for avian species have not been established. However, there are no levels o f contaminants reasonably expected to be present in the diet that are considered to interfere with the purpose or conduct o f the study. Piet Preparation Test diets will be prepared at the start o f the test and a sufficient portion estimated for the 5-day exposure period will be presented to the birds. If necessary, appropriate diet w ill be added as needed. After five days o f exposure, all birds will receive untreated basal ration. The test substance will be mixed directly into the ration or by dissolving or suspending the test substance in acetone and/or table grade com oil prior to mixing with the feed If used, acetone w ill be allowed to volatilize from the diets during the mixing procedure. Table grade com oil w ill normally be incorporated into the diet at a maximum o f 2% (w/w) o f the final diet M ixing is normally done with a Hobart mixer (Model Number AS200T). A ll test substance calculations will be based on the purity o f the test substance as received or corrected to 100% active ingredient based on the information provided by the Sponsor in Appendix I o f this protocol. 001353 PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 W i l d l if e In t e r n a t io n a l l t d . 6- - Diet Sampling Samples o f the experimental diets w ill be collected for chemical analysis to determine the homogeneity and stability o f the test substance in the avian diet and to verify/measure test concentra tions. All samples will be placed in uniquely identified polypropylene jars. Samples will be collected on Day 0, at diet preparation, to determine homogeneity, measure test concentrations, and establish Day 0 values for evaluating stability over the course o f the exposure period. On Day 5, at the end o f the exposure phase, samples will be collected from the feeders containing the experimental diets to assess stability o f the test substance under test conditions. The diet sampling scheme for studies with five test concentrations is summarized below. The sampling scheme may be adjusted based on the actual number o f concentrations tested (see Page 2) or the option o f the Sponsor. PROPOSED NUMBERS OF SAMPLES Experimental Group Day 0 Day 5 Control Level 1 - Low Concentration Level 2 Level 3 Level 4 Level 5 - High Concentration 1 61 2 2 2 61 1 2 2 2 2 2 19 11 Total Number o f Samples = 30 1Samples collected from toe left and right sides o f toe top, middle and bottom layers o f feed in the mixing vessel to determine homogeneity. The above numbers o f samples represent those collected from toe test and do not include quality control (QC) samples such as matrix blanks and fortifications prepared and analyzed during toe analytical chemistry phase o f toe study. PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 001354 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l ltd . -7 - Diet Analyses Samples o f the experimental diets may be analyzed following collection or stored in a freezer until prepared and/or extracted for analysis. Chemical analyses o f diets will be performed by W ildlife International Ltd. The analytical method used will be based upon methodology provided by the Sponsor and identified in Appendix II. The methodology used to analyze the test samples w ill be documented in the raw data and summarized in the final report. Maximum sample holding times, prior to analysis, may be specified by the Sponsor, and if specified, will be added to this protocol by amendment Housing and Environmental Conditions Test birds will be housed in thermostatically controlled brooding pens manufactured by Safeguard Products, Inc. or equivalent Each pen measures approximately 62 X 92 X 25.5 cm high. The external walls, floors and ceilings o f each pen are constructed o f vinyl coated wire grid. Upon initiation o f the test, each brooding pen w ill contain five birds. Two brooding pens will be assigned to each treatment group (10 birds total), while at least six pens will be assigned to the control (at least 30 birds total). Each group will be identified by pen number. On Day 8 o f the test, birds in one pen from each treatment group (5 birds) and three pens from the control group (15 birds) will be euthanized. Remaining birds will be maintained for an additional 14 days. During that period, remaining birds may be separated into two pens. During the first eight days o f the test, the temperature in the brooding compartment o f the pen will be maintained at approximately 29C . Brooders will be turned o ff during the last 14 days o f the test. Acceptable ranges for brooding compartment temperatures will be specified in W ildlife International Ltd. Standard Operating Procedures. Brooding compartment temperatures w ill be recorded once each day during the first eight days of die test. The ambient room temperature w ill range between approximately 15-30 C. Temperature andrelative humidity will be recorded twice daily during the test. A 16 hours light/8 hours dark photoperiod will be maintained, and the average light intensity provided to the birds during the test will be determined. Housing and husbandry practices will be conducted so as to adhere to the guidelines established by National Research Council (5). r \ r* A. PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 W i l d l i f e In t e r n a t io n a l l t d . 8- - Observations All birds will be observed at least once daily for mortality and toxicological responses. Animal Body Weiehts/Feed Consumption Individual body weights will be measured at the initiation o f the test, at the end o f the exposure period on Day 5, and on Day 8. Average estimated feed consumption will be determined by pen for each treatment and control group for the exposure (Days 0-5) and post-exposure observation (Days 6-8) periods. For those birds maintained for an additional 14 days, body weight and feed consumption measurements will be performed weekly and at test termination. The accuracy o f feed consumption values may be affected by unavoidable wastage o f feed by birds. N o attempts will be made to quantify the amount o f wasted feed, as it is normally scattered and mixed with water and excreta. Nssrspsy All test birds that die during the course o f the test and all birds euthanized will be subjected to a gross necropsy. For those birds euthanized on Days 8 and 22 o f the test, livers and ~ 2 mL o f blood w ill be collected. Livers will be weighed, placed in individual labelled containers and stored frozen. Blood will be placed in labelled heparinized vacutainers and placed under refrigeration All samples will be transferred to the Sponsor for residue analysis. Disposition o f Test Birds At test termination, test birds will be euthanized by using carbon dioxide gas, cervical dislocation or other appropriate methods. The method used will be documented in the raw data. A ll carcasses will be stored frozen prior to shipment to the Sponsor. Statistical Calculations The concentration response data obtained from the test will be evaluated and, if possible, an LC50 value and 95% confidence limits will be calculated. When the mortality data facilitates statistical analyses, one o f three methods w ill be used to calculate an LC50 value. The data w ill be analyzed, in order o fpreference, by probit analysis, the moving average method, or the binomial probability method (6, 7, 8, 9). The choice o f method for calculating the LC50 value w ill be based upon the mortality pattern observed. When possible, a no mortality level w ill be determined and reported. 001356 PROTOCOLNO.: 454/1X1098/MLCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l l t d . -9 - RECORDS TO BE MAINTAINED Records to be maintained for data generated at W ildlife International Ltd. w ill include, but not be limited to: 1. A copy o f the signed protocol. 2. Identification and characterization o f the test substance, if provided by the Sponsor. 3. Dates o f initiation and termination o f the test 4. Animal history. 5. Husbandly and environmental conditions. 6. Concentration calculations and records o f diet preparation. 7. Body weight measurements. 8. Feed consumption measurements. 9. Daily observations. 10. . Necropsy findings. 11. Statistical calculations. 12. Analytical chemistry methods, results and chromatograms, if applicable. 13. A copy o f the final report. |7TNAL REPORT A report o f the results o f the sturfy will be prepared by Wildlife International, Ltd. The report w ill include, but not be limited to, the following: 1. Name and address o f the facility performing the study. 2. Dates on which the study was initiated and completed. It is the responsibility o f the Sponsor to provide the final date that data are recorded for chemistry, pathology and/or supporting evaluations that may be generated at other laboratories. 3. A statement o f compliance signed by the Study Director addressing any exceptions to Good Laboratory Practice Standards. 4. Objectives and procedures stated in the approved protocol, including any changes in the original protocol. 5. Statistical methods employed for analyzing the data, when applicable. The calculated LC50 value, 95 percent confidence lim its, slope o f the concentration-response curve, the results o f the goodness-of-fit test (e.g. X2 test), and a description o f statistical methods used. The same statistics w ill be provided for positive controls (when used). 001357 PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 W i l d l if e In t e r n a t io n a l l t d . - 10- 6. The test, control and reference substances identified by name, chemical abstracts number or code number, strength, purity, and composition or other appropriate characteristics, if provided by the Sponsor. 7. Stability and, when relevant to the conduct o f the study, the solubility o f the test, control and reference substances under the conditions o f administration, if provided by the Sponsor. 8. A description of the test system used. Where applicable, the final report shall include the number o f animals used, body weight range, source o f supply, species (including scientific name), age, and procedure used for identification 9. A description o f the dosage, dosage regimen, route o f administration, and duration 10. A description o f the methods used, including but not limited to: a) Detailed description o fthe basal diet, including source, diluents (if used), and supplements (if used). A nutrient analysis o f the diet will be included in the test report b) The number o f concentrations used, nominal and (where required) measured dietary concentration o f test substance in each level, assay method used to determine actual concentrations, number o f birds per concentration and for control, and names o f toxicants used for positive controls (if applicable). Results o f range-finding test (if conducted). c) Acclimation procedures and methods o f assigning birds to test pens. d) Frequency, duration and methods o f observation. Description o f signs o f intoxication and other abnormal behavior, including time o f onset, duration, severity (including death), and numbers affected in the different dietary concentrations and controls each day o f the test period. e) Description o f housing conditions, including type, size and material o f pen, pen temperatures, approximate test room humidity, photqperiod and lighting intensity. f) Average body weights for birds in each pen at the beginning o f the test, the end o f the exposure period and end o f the test g) Estimated food consumption for the exposure period and for the postexposure period. 11. A description o f all circumstances that may have affected the quality or integrity o f the data. Anything unusual about the test, any deviation from these procedures, and any other relevant information. 12. The name o f the Study Director, the names o f other scientists or professionals, and the names o f all supervisory personnel, involved in the study. 13. A description o f the transformations, calculations, or operations performed on the data, a summary and analysis o f the data, and a statement o f the conclusions drawn from the analysis. C01358 PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l ltd . - 11 - 14. The signed and dated reports o f each o f the individual scientists or other professionals involved in the study, if applicable. 15. The location where all specimens, raw data, and the final report are to be stored. 16. A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made and the dates o f any findings reported to the Study Director and Management. 17. Ifit is necessary to make corrections or additions to a final report after it has been accepted, such changes shall be in the form o f amendment by the Study Director. The amendment should clearly identify the part o f the final report that is being added to or corrected and the reasons for the correction or additioa Amendments shall be signed and dated by the Study Director. CHANGING OF PROTOCOL Planned changes to the protocol w ill be in the form o f written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part o f the protocol and will be attached to the final protocol Ary other changes will be in the form o f written deviations signed by the Study Director and filed with the raw data. A ll changes to the protocol will be indicated in the final report. GOOD LABORATORY PRACTICES This study w ill be conducted in accordance with Good Laboratory Practice Standards for EPA (40 CFR Part 160 and/or Part 792); OECD Principles o f Good Laboratory Practice (OCDE/GD (92) 32, Environment Monograph No. 45); and Japan MAFF (59 NohSan, Notification N o. 3850, Agricultural Production Bureau). Each study conducted by W ildlife International Ltd. is routinely examined by the W ildlife International Ltd. Quality Assurance Unit for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. A statement o f compliance with Good Laboratory Practices w ill be prepared for all portions o f the study conducted by W ildlife International Ltd. The Sponsor w ill be responsible for compliance with Good Laboratory Practices for procedures performed by other laboratories (e.g., residue analyses or pathology). Raw data for all work performed at Wildlife International Ltd. and a copy o f the final report w ill be filed by project number in archives located on the W ildlife International Ltd. site or at an alternative location to be specified in the final report PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 001353 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l l t d . - 12- KEFERENCES 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. Environm ental Protection Agency. 1982. Pesticide Assessment Guidelines, F1FRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, subsection 71-2, Environmental Protection Agency, Office o f Pesticide Programs. Washington, D.C. 3 A m erican Society for Testing and M aterials. 1987. Standard Practice for Conducting Subacute Dietaxy Toxicity Tests with Avian Species. ASTM Standard E857-87. Annual Book o f ASTM Standards, Vol. 11.04. Philadelphia, PA. 4 Organization for Econom ic Cooperation and D evelopm ent 1984. Avian Dietary Toxicity Test. OECD Guideline for Testing o f Chemicals. Guideline 205. Paris. 5 N ational Research Council. 1996. Guide fo r the Care and Use o fLaboratory Animals. Washington, DC. National Academy Press. 125 pp. 6 Finney, D .J . 1971. StatisticalMethods in Biological Assay, 2nd ed., Griffin Press, London. 7 Thompson, W . R. 1947. Bacteriological Reviews, Vol 2, No. 2: 115-145. 8 Stephan, C .E . 1977. Methods for Calculating an LC50. Pages 65-84 la Aquatic Toxicology and Hazard Evaluations, American Society for Testing and Materials. Pub. No. STP 634. Philadelphia, PA. 9 Stephan, C E. 1978. U .S. EPA, Environmental Research Laboratory, Duluth, MN. Personal Communication. PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 001360 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd. - 13- T A B L E l: 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 q. iq 100.00 VITAMIN AND MINERAL PREMIX AMOUNT ADDED PER TON Vitamin D3 Vitamin A Riboflavin Niacin Pantothenic Acid Vitamin BI2 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 20,0001.U. 5.8 grams 102 grams 47 grams 6.8 grams 1.5 grams 51 grams 182 mgs 1 The guaranteed analysis is a m inim um of 27% protein, a m inim um of 2.5% crude fat and a m ax im u m of 5% crude fiber. Fermentation By-Products C013G1 PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l ltd . . 14- APPENDIX i IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR To be Completed by Sponsor I. Test Substance Identity (name to be used in the report): PEQJSiPerfluorooctane Sulfonic Acid Potassium Salt Reference Standard (if applicable): Analytical Standard: N/A___________ Internal Standard: _I.1.2.2H.H.H.H Perfluorooctane Sulfonic Acid Test Substance Sample Code or Batch Number: Lot 217________________________________________ Test Substance Purity (% Active Ingredient): 98.9 Expiration Date: 2008________________ II. Test Substance Characterization Have the identity, strength, purity and composition or other characteristics which appropriately define the test substance and reference standard been determined prior to its use in this study in accordance with GLP Standards? Y es_____ N o X HI. Test Substance Storage Conditions Please indicate the recommended storage conditions at Wildlife International Ltd. ____ Ambient room temperature_________________________________________________________________ Has the stability o f the test substance under these storage conditions been determined in accordance with GLP Standards? Y es_____N o X Other pertinent stability information: __________________________________________________________ IV. Test Concentrations: Adjust test concentration to 100% a.i. X based upon the purity (%) given above. Do not adjust test concentration to 100% _____ a.i. Test fne material AS IS. V. Toxicity Information: Mammalian: Rat LD50 251 mg/kg Mouse LD50 N/A Aquatic: Invertebrate Toxicity (EC/LC50) Fish Toxicity (LC50) Daphnia mapna: 27 me/L___________ Rainbow Trent; 11 m g/L , Daphniamagna: 50m e/L ___________ FatfrgadMufflpw ;..llLm g/L Other Toxicity Information (including findings o f chronic and subchronic tests): Please see MSDS___________________________________________________ __________ 001362 PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l ltd. - 15- APPENDIX n Method Outline for the Determination o f PFOS in Avian Feed 1. Prepare matrix fortification samples in the desired avian feed stock using the dry m ix technique. 2. Weigh 10 g samples o f the matrix blank and matrix fortification feed samples into a weigh boat and transfer to 16 oz. French square glass bottles. Record weights. 3. For each weighed sample, measure 100 mLs o f methanol with a graduated cylinder and transfer into the French square bottle. 4. Seal bottles with screw caps and place on shaker table. Allow to shake for a m in im u m o f 30 minutes. 5. Vacuum filter with qualitative filter paper and rinse retained feed 3 times with methanol into filtrate. 6. Transfer filtrate into a 200-mL volumetric flask and bring to volume with methanol. 7. Transfer 5 mLs o f 200-mL solution into a 10-mL volumetric flask partially-filled with 50% methanol:50% water dilution solvent containing 0.05% v/v formic acid and 100 pig a.i./mL 4HPFOS internal standard. Bring to volume with dilution solvent. 8. M ix and transfer approximately 1 mL o f the 10-mL solution into a syringe and filter through a 0.2 pan filter into an HPLC autosampler vial. 9. Ampulate and submit sample for LCMS analysis. PROTOCOL NO.: 454/111098/MLCSDT.WC/OPPTS 001363 3M LAB REQUEST NO. U2723