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z e m - o z o o - } 73 PFOS: A DIETARY LC50 STUDY W ITH THE NORTHERN BOBWHITE WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103 3M LAB REQUEST NO.: U2723 U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines (draft) OPPTS Number 850.2200 FIFRA Subdivision E, Section 71-2 OECD Guideline 205 t'o Crc~nD> odd vm I -a co --!p i ~"D 33 3T. l2 m oo AUTHORS: Sean P. Gallagher Courtney S. Casey Joann B. Beavers Raymond L. Van Hoven STUDY INITIATION: April 21, 1999 STUDY COMPLETION: April 26, 2000 A E P A -O T S 000811790Q DODflllTIDfl SUBMITTED TO 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, M innesota 55144 o o 3S <~ C~> ' .r o r\> , 1 Wildlife International, Ltd. c o J -v -t s "n rv o? c*.; 8598 Commerce Drive Easton, M aryland 21601 (410) 822-8600 Page 1 o f 62 001373 Wildlife International, ltd 2- - PROJECT NO.: 454-103 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Dietary LC50 Study with the Northern Bobwhite W ILDLIFE INTERNATIONAL LTD. PROJECT NO.. 454-103 3M LAB REQUEST NO.: U2723 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 o f 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: S^aii x . uaiiagubi Senior Biologist SPONSOR'S REPRESENTATIVE DATE Ms. Susan A. Beach 001374 Wildlife International, ltd -3 - PROJECT NO.: 454-103 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR Part 160 and 792, 17 August 1989; OECD Principles o f Good Laboratory Practice, (OCDE/GD (92) 32, Environment M onograph 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 Test Substance Prep. & Analytical Sampling April 22, 1999 April 22, 1999 A pril 23, 1999 M atrix Fortification April 22, 1999 April 22, 1999 A pril 23, 1999 Feed Consumption & Analytical Sampling April 27,1999 April 27,1999 M ay 4, 1999 Analytical D ata and D rift Report July 7, 8 ,9 , 1999 July 9,1999 July 16,1999 Biology D ata and D raft Report August 26, 27, 30, 31, 1999 August 31, 1999 September 13, 1999 Final Report April 17-18,2000 April 18,2000 April 19, 2000 Susan L. Coleman Senior Quality Assurance Representative 001375 Wildlife International, ltd -4 - REPORT APPROVAL PROJECT NO.: 454-103 SPONSOR: 3M Corporation TITLE: PFOS: A Dietary LC50 Study with the Northern Bobwhite WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103 3M LAB REQUEST NO.: U2723 STUDY DIRECTOR: f - a^ Sean P. Gallagher ] Senior Biologist, Avian Toxicology o Date ^ CHEMISTRY PRINCIPAL INVESTIGATOR: Raymon L. Van Hoven, Ph.D Scientist, Analytical Chemistry H -2O 00 Date REPORT APPROVED BY: Joann B. Beavers Director, Avian Toxicology 4// D ate _______________ n r ------- W illard B. Nixon, Ph.Et Manager, Analytical Chemistry Date 001376 Wildlife International, ltd -5 - PROJECT NO.: 454-103 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....................................................................................................................Page6 SUMMARY.........................;........................................................................................................ Page 7 INTRODUCTION......................................................................................................................... Page 8 OBJECTIVE.................................................................................................................................. 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 B irds......................................................................................................................................Page 9 Animal D iet........................................................................................................................................Page10 Diet Preparation................................................................................................................. Page 10 Diet Sampling...................................................... .....................:.................................................Page 10 Analytical M ethod....................................................................................................................... Page 11 Housing and Environmental Conditions..........................................................................................Page13 O b serv atio n s....................................................................................................................... Page 14 Animal Body W eights/Feed Consumption...................................................................................... Page14 Gross N ecropsy................................................................................................................................. Page14 Statistical Analyses...................................................................................................................... Page 14 RESULTS............................................................................................................................................ Page15 Diet A nalysis............................. Page 15 M ortalities and Clinical O bservations.............................................................................................Page15 Body W eight and Feed Consumption.............................................................................................. Page17 Gross N ecropsy................................................................................................................................. Page17 C O N C L U S IO N ................................................................................ Page 18 REFERENCES................................................................................................................................... Page19 001377 Wildlife International, ltd 6- - TABLE OF CONTENTS - continued - TABLES AND APPENDICES PROJECT NO.: 454-103 TABLE 1: Cumulative M ortality from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS................................................................................................. Page 20 TABLE 2: Mean Body W eight (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS.................................................................................................Page 21 TABLE 3: Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS........................................................ Page 23 TABLE 4: Group Gross Pathological Observations from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS........................................................ Page 25 TABLE 5: Cumulative M ortality (Estimated Cumulative Dose, mg/kg) from a Northern Bobwhite Acute D ietary Toxicity Study with PFOS.......................Page 26 APPENDIX I: Certificate o f A nalysis.................................... Page 27 APPENDIX II: Diet Formulation..................................................................................................Page28 APPENDIX IH: Analytical Methods and Results.........................................................................Page29 APPENDIX IV: Diet Preparation...................................................................................................Page43 APPENDIX V: Cumulative M ortality by Pen from a Northern Bobwhite Acute Dietary Toxicity Study with PFO S................................................ Page 44 APPENDIX VI: Individual Body W eights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFO S................................................. Page 45 APPENDIX VII: Feed Consumption (g/bird/day) by Pen from a Northern Bobwhite Acute Dietary Toxicity Study with P F O S ............................................................................................... Page58 APPENDIX VIII: Changes to P rotocol....................................................................................... Page 61 APPENDIX IX: Personnel Involved in the S tudy.........................................................................Page62 001378 Wildlife International, ltd -7 - PROJECT NO.: 454-103 SUMMARY SPONSOR: 3M Corporation TEST SUBSTANCE: PFOS WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-103 STUDY: PFOS: A Dietary LC50 Study with the Northern Bobwhite RESULTS: The dietary LC50 value for northern bobwhite exposed to PFOS was determined to be 220 ppm a.i. with a 95% confidence interval o f 164 ppm a.i. to 289 ppm a.i. The slope o f the concentration-response curve was 7.005 and the chi-square value was 0.023. The no mortality concentration was 73.2 ppm a.i. Based upon treatment related mortality, signs of toxicity and effects upon body weight gain at the 146 ppm a.i. test concentration, the no-observed-effect concentration was 73.2 ppm a.i. TEST DATES: H atch -A p ril 12, 1999 Acclimation - April 12-22, 1999 Experimental Start -A pril 22, 1999 Experimental Termination - May 14, 1999 NOMINAL TEST CONCENTRATIONS: 0, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 ppm a.i. TEST ANIMALS: Northern Bobwhite (Colinus virginianus) AGE TEST ANIMALS: 10 days o f age at test initiation SOURCE TEST ANIMALS: W ildlife International Ltd. Production Flock 8598 Commerce Drive Easton, M aryland 21601 STUDY COMPLETION: April 26, 2000 001379 Wildlife International, ltd - 8- PROJECT NO.: 454-103 INTRODUCTION This study was conducted by Wildlife International Ltd. for 3M Corporation at the Wildlife International Ltd. avian toxicology facility in Easton, Maryland. The in-life portion o f the test was conducted from April 22, 1999 to M ay 14, 1999. Raw data generated at W ildlife International Ltd. and a copy o f the final report are filed under Project Number 454-103 in archives located on the W ildlife International Ltd. site. OBJECTIVE The objective o f this study was to evaluate the toxicity o f a test substance to the Northern Bobwhite (Colinus virginiam s) 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 o f the Environmental Protection Agency's Registration Guidelines, Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: W ildlife and Aquatic Organisms (2); OECD Guideline 205, Guideline for Testing o f 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 W ildlife International Ltd. Identification Number 4675 upon receipt. The test substance was white powder identified as: FC-95; Lot No.:217. The reported purity o f the test substance was 98.9%, with an expiration date o f 2008. Following test termination, the test m aterial was reanalyzed. The results o f reanalysis indicate a test substance purity o f 90.49%. All test concentrations have been adjusted to reflect the purity reported on the new Certificate o f 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 W ildlife International Ltd. identification number 4526 upon receipt. The internal standard, a granular 001380 Wildlife International, ltd -9 - PROJECT NO.: 454-103 material, was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical A bstract Number: 27619-97-2. The standard was stored under ambient conditions. Treatment Groups The test consisted o f a geometric series o f seven test concentrations and a control group. Thirty northern bobwhite chicks were assigned to the control group and ten northern bobwhite chicks were assigned to each o f the treatm ent groups. The birds were sorted by weight, then chosen indiscriminately from within each represented weight class for placement into control and treatm ent groups. The birds were housed in brooding pens containing five chicks each. Nominal dietary concentrations used in this study were 0, 18.3, 36.6, 73.2, 146, 293, 586 and 1171 parts per million active ingredient (ppm a.i.) o f PFOS. The dietary concentrations were established based upon known toxicity data and information supplied by the Sponsor. Each group was fed the appropriate test or control diet for five days. During the exposure period the control group received untreated feed. Following the five-day exposure period all groups were given untreated basal diet for three days. On Day 8, half o f the surviving treatment and control birds were euthanized and liver tissue, blood, and bile samples were collected for analysis. The remaining birds were fed basal ration until Day 22. On Day 22, these birds were euthanized and also sampled for liver weight, blood, and bile. Duration o f the Test The primary phases o f this test and their durations were: 1. Acclimation -1 0 days. 2. Exposure - 5 days. 3. Post-exposure observation - 3 or 17 days Test Birds All northern bobwhite (Colinus virginianus) were 10 days o f age and appeared to be in good health at initiation o f the test. The birds were obtained from W ildlife International Ltd. Production Flock, Easton, MD and were hatched on April 12, 1999. Birds ranged in weight from 18 to 23 grams at test initiation. The birds used in this study were immature and could not be differentiated by sex. 001381 Wildlife International, ltd -10- PROJECT NO.: 454-103 All birds were from the same hatch, pen-reared and phenotypically indistinguishable from wild birds. All birds were acclimated to the caging and facilities from the day o f hatch until initiation o f the test. Animal Diet Throughout acclimation and testing all test birds were fed a game bird ration formulated to W ildlife International Ltd.'s specifications (Appendix II). The chicks were given a vitamin supplement in their water from the day they were hatched until the initiation o f the test. W ater from the town o f Easton public water supply, and feed were provided ad libitum during acclimation and testing. The birds received no form o f antibiotic medication during acclimation or testing. Diet Preparation The test substance was mixed directly into the ration. Mixing was done with a H obart 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 18.3, 36.6, 73.2, 146,293, 586, 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 18.3 ppm a.i. a.i. test diet and six samples from the 1171 ppm a.i. test diet at preparation on Day 0. Homogeneity samples were collected from the top, middle and bottom 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. A t 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 o f the test substance in the diet under test conditions. The stability samples were collected from feed remaining in the feeders after being at ambient test pen conditions for five days. Samples were transferred immediately to W ildlife International Ltd. analytical chemistry. 001382 Wildlife International, ltd -11 - PROJECT NO.: 454-103 Analytical Method The method used for the analysis o f the avian diet samples was based upon methodology developed at W ildlife International Ltd. and entitled "Method Outline for the Determination o f PFOS in Avian Feed". Avian diet samples were extracted with methanol. Methanol was added to a requisite quantity o f feed contained in a French-square glass bottle. Bottles were capped and shaken on a shaker table. Samples were vacuum filtered using qualitative filter paper. The retained feed was rinsed three times with methanol into the filtrate. The filtrate was transferred to a volumetric flask and brought to volume with methanol. As appropriate, samples were further diluted with methanol. Each sample then was diluted with a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v) so that they fell within the calibration range o f the PFOS methodology. A method flowchart is provided in Appendix JH, 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) w ith a Perkin-Elmer API 100LC Mass Spectrometer equipped w ith a PerkinElmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil Cu analytical column (100 mm x 2 mm I.D ., 3 pm particle size). The instrument param eters are summarized in Appendix DI, Table 1. Calibration standards o f PFOS prepared in a 50% methanol : 50% NANOpure w ater 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 attem pt 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 E l, 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 o f low and high calibration standards are presented in Appendix IE , Figures 3 and 4, respectively. 001383 Wildlife International, ltd -12- PROJECT NO.: 454-103 The method limit o f quantitation (LOQ) for these analyses was set at 1.15 ppm a.i. calculated as 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). Two matrix blank samples were analyzed to determine possible interferences. No interferences were observed at or above the LOQ during sample analyses (Appendix IE, 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 m atrix blank is presented in Appendix HI, 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 IE, Table 3). Sample concentrations were not corrected for the mean procedural recovery o f 94.7%. A representative chromatogram o f a matrix fortification is presented in Appendix m , Figure 6. An example calculation is presented for sample number 454-103-2, nominal concentration o f 18.3 ppm a.i. in avian diet. Initial Weight: lO.Og 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 001384 Wildlife International, ltd - 13 - PROJECT NO.: 454-103 (0.2749 - 0.01894) x 0.100 mg/L 2.77397 = 0.00923 mg ai./L Note: I.S. = internal standard. PFOS (ppm a.i.) in sample PFOS (mg a.i./L) at instrument x Final Volume (L) x Dilution Factor Initial Weight (Kg) 0.00923 x 0.200 x 100 -- = 18.5 ppm ai. PFOS (ppm a i.) in sample Percent ofNominal 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 therm ostatically controlled brooding pens manufactured by Beacon Steel Products Co. (Model No. B735Q). Each pen had floor space that measured approximately 72 X 90 cm. Ceiling height was approximately 23 cm. External walls, ceilings and floors were constructed 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 leg 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 27.3C 1.2C (SD) with an average relative humidity o f 31% 14% (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 o f 001385 Wildlife International, ltd 14- PROJECT NO.: 454-103 approximately 139 lux o f illumination. Housing and husbandry practices were based on guidelines established by the National Research Council (5). O bservations During acclimation all birds were observed daily. Birds exhibiting abnormal behavior or physical injury were not used. Following test initiation and continuing until termination, all birds were typically observed at least twice daily. A record was maintained o f all mortality, signs o f toxicity, and abnormal behavior. Animal Body W eiehts/Feed Consumption Individual body weights were measured at the initiation o f the test, on Day 5, Day 8, and on Days 15 and Day 22 for all remaining birds. Average feed consumption values during the exposure period (Days 0-5) and the post-exposure observation period (Days 6-8) were determined by pen for each treatm ent group and the control group. Additionally, feed consumption was determined for Days 8-15 and 15-22 for the remaining treatment and control birds. Feed consumption was determined by measuring the change in the weight 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 term ination were subjected to a gross necropsy. Additionally, livers were weighed and liver tissue, blood, and bile were collected from birds euthanized on Day 8 and 22, and when possible from those that died during the course o f the study. Statistical Analyses M ortality data were analyzed using the computer program o f C.E. Stephan (6). The program was designed to calculate the LC50 value and the 95% confidence interval by probit analysis, moving average method or the binomial probability method (7,8,9). In this study, the LC50 value was determined using the probit method. The slope o f the concentration-response curve and results o f the goodness o f fit test are reported. Body weight data were compared by D unnett's test using TOXSTAT software (10,11). No statistical analyses were applied to feed consumption data. 001386 Wildlife International, ltd - 15- PROJECT NO.: 454-103 RESULTS Diet Analysis Avian diet samples were collected from the 18.3 and 1171 ppm a.i. test concentrations and analyzed to evaluate homogeneity o f the test substance in the avian diet. The analysis o f these samples also served as verification o f test substance concentrations. Resulting mean measured concentrations, standard deviations and coefficients o f variation (CV) for these test concentrations were 19.5 2.13 ppm a.i. (CV = 10.9%) and 1196 70.2 ppm a.i. (CV = 5.87% ), respectively (Appendix m , 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 o f 40.2, 74.5, 174, 291 and 537 ppm a.i., respectively. These values represented 110, 102, 119, 99.3 and 91.6% o f the nominal concentrations, respectively (Appendix HI, Table 5). Analysis o f avian diet samples collected from feeders after being held at ambient temperature for five days averaged 101, 122, 104, 101, 109, 114 and 102% o f the Day 0 values for the 18.3, 36.6,73.2, 146, 293, 586 and 1171 ppm a.i. test substance concentrations, respectively (Appendix HI, Table 6). A representative chromatogram o f a test sample is shown in Appendix HI, Figure 7. M ortalities and Clinical Observations One incidental mortality occurred in the control group during the course o f the study CTable 1 and Appendix V). On the morning o f Day 5, one bird was noted with a broken leg and was subsequently euthanized on Day 6. Additionally, two birds in the control group were intermittently noted with foot lesions associated with cage mate aggression. Otherwise, all control birds were normal in appearance and behavior throughout the test. No treatment related mortalities or overt signs o f toxicity were observed in the 18.3, 36.6, or 73.2 ppm a.i. treatment groups. One bird in the 18.3 ppm a.i. treatment group was noted as lame from Day 6 through Day 8, and blood o f undetermined origin was noted on the underside o f one bird from the 73.2 ppm a.i. treatment group on Day 22. Otherwise, all birds in the 18.3, 36.6 and 73.2 ppm a.i. treatment groups were normal in appearance and behavior throughout the test period. 01387 Wildlife International, ltd -16- PROJECT NO.: 454-103 There was 11% (1 o f 9) mortality in the 146 ppm a.i. treatment group, 80% (8 o f 10) m ortality in the 293 ppm a.i. treatment group and 100% (10 o f 10) mortality in the 586 and 1171 ppm a.i. treatment groups. In the 146 ppm a.i. treatm ent group, one bird was euthanized on Day 3 after sustaining a broken leg. This incidental mortality was not used in the calculation o f the LC50 value. Additionally, there was one treatment-related mortality in the 146 ppm a.i. treatm ent group, a bird found dead on the morning o f Day 7. Clinical signs o f toxicity were observed in this treatm ent group on Day 5, when two birds displayed wing droop. All other birds at this test concentration were normal in appearance and behavior for the duration o f the test. In the 293 ppm a.i. treatm ent group there were eight treatment-related mortalities, occurring on Days 5, 6 and 7. Signs o f toxicity were first observed on the morning o f Day 4 and continued to be exhibited through the morning o f Day 8 for the single bird euthanized on Day 8, and through the afternoon o f Day 8 for the single bird surviving until Day 22. Signs o f toxicity included a ruffled appearance, reduced reaction to stimuli (sound and motion), lethargy, wing droop, loss o f coordination, lower limb weakness and convulsions. The single remaining bird appeared to have recovered and was normal in appearance and behavior from the afternoon o f Day 9 until test term ination. In the 586 ppm a.i. treatm ent group m ortalities were first noted on Day 3 and continued to be observed through Day 7, at which point all birds had died. Overt signs o f toxicity were first observed on the afternoon o f Day 2 and continued through the morning o f Day 7, when the final birds were found dead. Signs o f toxicity observed among birds in the 586 ppm a.i. treatment group included a ruffled appearance, reduced reaction to stimuli (sound and motion), lethargy, depression, wing droop, loss o f coordination, lower limb weakness, lower limb rigidity, prostrate posture, and convulsions. In the 1171 ppm a.i. treatm ent group 100% mortality had occurred by the morning o f Day 4. Signs o f toxicity in the 1171 ppm a.i. treatment group were first observed on the afternoon o f Day 2, with the first mortalities noted on the morning o f Day 3. Signs o f toxicity observed prior to death included a ruffled appearance, reduced reaction to stimuli (sound and motion), lethargy, depression, loss o f coordination, wing droop, and lower limb weakness and rigidity. 001388 Wildlife International, ltd -17- PROJECT NO.: 454-103 Bodv W eight and Feed Consumption When compared to the control group, there were no apparent treatment related effects upon body weight among birds in the 18.3, 36.6 or 73.2 ppm a.i. treatment groups. However, there was a concentration responsive reduction in body weight gain or body weight loss in the 146, 293 and 586 ppm a.i. treatment groups during the exposure period (Days 0-5) (Table 2 and Appendix VI). Differences from the control group were statistically significant at p < 0.05 fo rth e 146 ppm a.i. level and at p<0.01 for the 293 and 586 ppm a.i. levels. A statistically significant (p<0.01) reduction in body weight gain continued to be observed at the 146 ppm a.i. test concentration through Day 8 o f the study. A t the 293 ppm a.i. concentration a statistically significant (p<0.01) mean weight loss continued through Day 8 o f the study and a marked reduction in weight gain was noted through Day 15 o f the study. Due to total mortality, body weight effects could not be determined for the 1171 ppm a.i. level during the exposure period or for the 586 and 1171 ppm a.i. treatm ent groups for postexposure period. There were no apparent treatment related effects upon feed consumption at the 18.3, 36.6, 73.2 or 146 ppm a.i. test concentrations (Table 3 and Appendix VII). However, a reduction in feed consumption was noted at the 293, 586 and 1171 ppm a.i. treatm ent groups during the exposure period (Days 0-5). There were no treatment-related effects on feed consumption in any o f the surviving treatment groups during the Day 6-8 post-exposure period. In the 293 ppm a.i. treatment group only one bird survived to Day 22. The reduction in feed consumption observed at the 293 ppm a.i. test concentration during both the Day 8-15 and 15-22 post-exposure periods was the result o f having only one bird in the pen, and was not considered to be treatm ent related. 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 o f muscle mass, altered spleen color, autolysis o f tissues and pale organs. Details o f the necropsy findings are presented in Table 4. H alf o f the surviving birds were subjected to gross necropsy on Day 8 and the remaining birds were necropsied on Day 22, following test termination. On Day 8, one bird in the 73.2 ppm a.i. treatment group was noted with a slightly pale liver. Due to the isolated nature o f this finding, it was 001389 Wildlife International, ltd -18- PROJECT NO.: 454-103 not considered to be related to treatment. The single bird euthanized from the 293 ppm a.i. treatment group was observed at necropsy to have with a lack o f muscle mass and general thinness. Since these findings correlated with an impact upon body weight noted at this concentration, the findings were considered to be treatment related. Necropsy results were unremarkable for all other birds euthanized on Day 8. Similarly, Day 22 necropsy findings were unremarkable for all birds. CONCLUSION The dietary LC50 value for northern bobwhite exposed to PFOS was determined to be 220 ppm a.i. with a 95% confidence interval o f 164 ppm a.i. to 289 ppm a.i. The slope o f the concentrationresponse curve was 7.005 and the chi-square value was 0.023. The no m ortality concentration was 73.2 ppm a.i. Based upon treatment related mortality, signs o f toxicity and effects upon body weight gain at the 146 ppm a.i. a.i test concentration, the no-observed-effect concentration was 73.2 ppm a.i.. 001390 Wildlife International, ltd -19- PROJECT NO.: 454-103 REFERENCES 1. U.S. Environm ental 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, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, subsection 71-2, Environmental Protection Agency, Office o f Pesticide Programs. W ashington, D.C. 3. O rganization for Economic C ooperation and Development. 1984. Avian Dietary Toxicity Test. OECD Guideline for Testing o f Chemicals. Guideline 205. Paris. 4. A m erican Society for Testing and M aterials. 1987. Standard Practice for Conducting Subacute Dietary Toxicity Tests with Avian Species. ASTM Standard E857-87. Annual Book o f ASTM Standards, Vol. 11.04. Philadelphia, PA. 5. N ational R esearch Council. 1996. Guide fo r the Care and Use o f Laboratory Animals. W ashington, D.C. National Academy Press. 125 pp. 6. Stephan, C .E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, MN. Personal Communication 7. Stephan, C .E. 1977. Methods for Calculating an LC50. Pages 65-84 In Aquatic Toxicology and Hazard Evaluations, American Society for Testing and M aterials. Pub. No. STP 634. Philadelphia, PA. 8. Finney, D .J. 1971. Statistical Methods in Biological Assay, 2nd ed., Griffin Press, London. 9. Thom pson, W .R. 1947. Bacteriological Reviews, Vol 2, No.2: 115-145. 10. D unnet, C.W . 1955. A M ultiple Comparison's Procedure for Comparing Several Treatments with a Control. Jour. Amer. Statis. Assoc. 50:1096-1121. 11. G ulley, D.D. 1990. TOXSTAT Release 3.2. The University o f Wyoming. 001391 Wildlife International, ltd - 20- PROJECT NO.: 454-103 TABLE1 Cumulative M ortality from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) No. Dead Per No. Exposed Exposure Period Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 No. Dead Per No. Exposed Post-Exposure Period Day 6 Day 7 Day 8 1 Control 0 0/30 0/30 0/30 0/30 0/30 0/30 1/30 1/30 1/30 T reatm ent 18.3 36.6 73.2 146 293 586 1171 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 1/102 0/9 0/9 0/10 0/10 0/10 0/10 0/10 2/10 0/10 0/10 0/10 1/10 2/10 5/10 0/10 0/10 0/10 3/10 10/10 10/10 0/10 0/10 0/10 0/9 4/10 8/10 10/10 0/10 0/10 0/10 1/9 8/10 10/10 10/10 0/10 0/10 0/10 1/9 8/10 10/10 10/10 The LC50 value was calculated to be 220 ppm a.i. with a 95% confidence interval o f 164 ppm a.i. to 289 ppm a.i.. 1- No mortalities occurred in any o f the control or treatment groups from Day 8 to Day 22. 001392 Wildlife International, ltd -21 - PROJECT NO.: 454-103 TABLE 2 Page 1 Mean Body W eight (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) Exposure Period Change' Day 0 Day g Day 0-5 Post-Exposure Period Change' Day 8 Day 5-8 Total Change1 Control 0 Mean SD 20 30 14 10 3 38 8 52 18 4 T reatm ent 18.3 Mean SD 21 31 1. 4 11 3 40 9 52 20 3 36.6 Mean SD 20 31 23 11 2 39 8 31 19 2 73.2 Mean SD 20 30 12 9 1 37 7 31 16 2 146 Mean SD 20 27* 23 7* 3 33* 6** 32 13** 4 293 Mean SD 20 18** -2** 12 2 18** -2** 44 -l** 5 586 Mean SD 20 16** -4** 12 2 __ -- _ - 1171 Mean SD 20 1- - _ -- _ - 'M ean change is calculated separately from the mean body weights using individual body weights (See Appendix VI). (-) = No data available due to mortality. Statistically different from the control group at p < 0.05 (Dunnett's t-test). Statistically different from the control group at p < 0.01 (D unnetfs t-test). 001393 Wildlife International, ltd -22- PROJECT NO.: 454-103 TABLE2 Page 2 M ean Body W eight (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) Day 8 Post-Exposure Period Day 15 Change1 Day 8-15 Day 22 Change1 Day 15-22 Total Change1(8-22) Control 0 Mean SD 37 59 23 6 10 5 82 22 13 3 45 8 T reatm ent 18.3 Mean SD 40 68 24 683 87 23 73 47 3 36.6 Mean SD 38 65 26 3 52 89 24 72 50 4 73.2 Mean SD 35 60 24 342 79 20 42 44 2 146 Mean SD 34* 58 33 24 1 79 21 22 45 1 293 2Mean SD 586 Mean SD 21 35 m_ -- 14 - 55 20 -- 1171 Mean SD . - - gS S ^B S & ^^^S S 3B H 9E B E & 555S fi5S 555^S S S 5^^^S E S 9E fl^B B H 9B E S 3S S & 9S S S 9S 33S S S S 53S ^^^H 5S ^9^^fl^^S S 8fl8S 3B ttM B S 5^^B inS S S S !^^E B S S 3S 3B eS S S S 53S S ^E S S S S `Mean change is calculated separately from the mean body weights using individual body weights (See Appendix VI). ^1=1, could not be evaluated statistically with Dunnett's t-test. (-) = No data available due to mortality. Statistically different from the control group at p < 0.05 XDunnett's t-test). 34 -. _ . 001394 Wildlife International, ltd -23- PROJECT NO.: 454-103 TABLE 3 Page 1 Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) Control 0 T reatm ent 18.3 36.6 73.2 146 293 586 1171 Exposure Period Day 0-5___ Mean SD 9 2 9 8 10 9 5 6 4 Post-Exposure Period _____ Day 6-8_____ 10 2 11 12 13 10 9 19 - (-) - No data available due to mortality. 001395 Wildlife International, ltd -24- PROJECT NO.: 454-103 TABLE3 Page 2 Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Experimental Group (ppm a.i.) . C ontrol 0 Mean SD Post-Exposure Period Day 8-15 Day 15-22 9 13 21 T reatm ent 18.3 36.6 73.2 146 293 586 1171 10 12 14 15 13 15 11 14 89 -- (-) = No data available due to mortality. 001396 Wildlife International, ltd -25- PROJECT NO.: 454-103 TABLE4 Group Gross Pathological Observations from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Birds that died during the course of the study Finding Control N=1 Male, Female, and Undetermined PPMA.L 146 293 586 1171 N -2 N =8 N =10 N =10 Abdominal cavity, some autolysis 0 0 2 24 Abdominal cavity, autolysis throughout 0 00 11 Crop, empty 0 0 2 52 Emaciated 0 0 2 58 Fractured leg 1 10 00 G.I. tract empty 0 '0 1 1 0 Gizzard contents bile stained 0 02 51 Heart, anterior portion mottled white color 0 0 1 00 Heart, pale 0 00 21 Intestinal contents tar-like 0 0 0 20 Keel, prominent Kidneys, pale 0 0 1 3 10 0 0 0 20 Liver, pale and mottled 0 10 00 Loss of muscle mass 0 0 4 79 Muscular-skeletal, pale 0 10 00 Small in stature 0 0 3 00 Spleen, black 0 0 0 10 Spleen, dark Spleen, grey 0 0 0 02 0 0 0 10 Spleen, grey-brown 0 00 01 Spleen, pale Spleen, small 0 0 101 0 00 01 Spleen, small and pale Thin Not Remarkable 0 0 0 30 0 0 0 42 0 0 1 00 001397 Wildlife International, ltd - 26- PROJECT NO.: 454-103 Experimental GrouP (ppm a.i.) Control 0 0 0 0 0 0 T reatm ent 18.3 18.3 Table 5 Cumulative Mortality (Estimated Cumulative Dose, mg/kg1) from a Northern Bobwhite ___ Acute Dietary Toxicity Study with PFOS ____________________ No. Dead Per No. Exposed (Cumulative Dose, mg/kg) No. Dead Per No. Exposed _________________________Exposure Period_____________________________________ Post-Exposure Period Pen DayO Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8: 1 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 2 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 3 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 4 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 5 0/5 0/5 0/5 0/5 0/5 0/5 1/5 1/5 1/5 6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1 0/5 2 0/5 0/5(7) 0/5 (6) 0/5 (14) 0/5 (12) 0/5 (21) 0/5 (18) 0/5 (28) 0/5 (24) 0/5 (35) 0/5 (30) 0/5 0/5 0/5 0/5 0/5 0/5 36.6 1 0/5 0/5 (13) 0/5 (26) 0/5 (39) 0/5 (52) 0/5 (65) 0/5 0/5 0/5 36.6 2 0/5 0/5(11) 0/5 (22) 0/5 (33) 0/5 (44) 0/5 (55) 0/5 0/5 0/5 73.2 1 0/5 0/5 (33) 0/5 (66) 0/5 (99) 0/5 (132) 0/5 (165) 0/5 0/5 0/5 73.2 2 0/5 0/5 (32) 0/5 (64) 0/5 (96) 0/5 (128) 0/5 (160) 0/5 0/5 0/5 146 1 0/5 0/5 (46) 0/5 (92) 0/5 (138) 0/5 (184) 0/5 (230) 0/5 1/5 1/5 146 2 0/5 0/5 (49) 0/5 (98) 1/5 (147) 1/5 (196) 1/5 (245) 1/5 1/5 1/5 293 1 0/5 0/5 (66) 0/5 (132) 0/5 (198) 0/5 (264) 1/5 (330) 1/5 4/5 4/5 293 2 0/5 0/5 (101) 0/5 (202) 0/5(303) 0/5(404) 1/5 (505) 3/5 4/5 4/5 586 1 0/5 0/5 (213) 0/5 (429) 1/5 (639) 2/5 (852) 2/5 (1065) 4/5 5/5 5/5 586 2 0/5 0/5 (178) 0/5 (356) 0/5 (534) 0/5 (712) 3/5 (890) 4/5 5/5 5/5 1171 1 0/5 0/5 (256) 0/5 (512) 3/5 (768) 5/5 5/5 5/5 5/5 1171 2 0/5 0/5(256) 0/5 (512) 0/5 (768) 5/5 5/5 5/5 5/5 The LC50 value was calculated to be approximately 220 ppm a.i. with a 95% confidence interval o f 164 to 289 ppm a.i.. *- Bird was euthanized due to a broken leg. Estimated cumulative dose is based upon the average body weight and feed consumption over the 5-day exposure period, and serves as a rough approximaton of the actual amount of test substance consumed. _M jgjnM talitiesoccT O ed^iany^fttm _hegtagU O T ^m ol^roups after Day 8. _________________ 5/5 5/5 001398 Wildlife International, ltd -27- PROJECTNO.: 454-103 APPENDIX I Certificate O f Analysis FC-95, Lot 217 M arch 9 2000 Richard M. Payfer This lample m s awdyzcd using LQMS, `H-NMR. lgF-NMR. and elemental analyses techniques. The results tu these tests showthcample to contala the fcUowlflsweightpercent composition: ciP sS O jir c& M n s? CtFSOs'Kr CkFisSOiTC*' C rFliSO ilC CtFitSOsTC CoF hSOiTC c* m CiiFmSOs"K'' Cfc-Q&fK* S F ^ F u S O tir _ ao4% U S% 130% 3.71% 1.19% THJ33`% ' W% 0.13 % 0.04% 0.05% 035% AdrtitfrnvtTly,tho isomerdistributionofthestaplewasdeterminedusing %->IMRtechniques andfoundto contalathe followingmobpercentoompotlticn: c v M a ^ja o ije (Natural chain. where x is nrainhr 7) CFiICFjJrCFCCFsKCKlV-SQs-Kf (Intenal monomethylfcmicb, w herexfy Is mainly 5. andx*0.y** 01 (CF})jCF-(CFi}*- SOj' IC (Tsotawyl teinoh. wherex is mainly 5) C Jftrti^C S P iJ-S Q s-r i AWta branch. where x Is mainly 61 (CF^C K C TiV SC b-lC Ohbntvlbranch. whew x Is mainly 4) C FH C FsM X C ftJH C Par SO*KT (Internal gem-dinsethyl branch, where r ty is mainly 4. and x * 01 70.5% 17.1% 103% 1.6% 03% 0.2% 001399 Wildlife International, ltd -28- PROJECT NO.: 454-103 A P P E N D IX II DIET FORMULATION WILDLIFE INTERNATIONAL LTD. GAME BIRD RATION1 INGREDIENTS Fine Com Meal Soy Bean Meal, 48% Protein W heat Midds Protein Base Agway Special, 60% Protein A lfalfa 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 N iacin Pantothenic Acid Vitamin Bn Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (Menadione Dimethylpyrimidinol Bisulfite) M anganese Zinc Copper Iodine Iron Selenium 2.000. 0 0 0 1.C.U. 7.000. 0 0 0 1.U. 6 grams 40 grams 10 grams 8 mgs 600 mgs 64 mgs 1.2 grams 1.2 grams 2 0 ,0 0 0 1.U. 5.8 grams 102 grams 47 grams 6.8 grams 1.5 grams 51 grams 182 mgs 'The guaranteed analysis is a minimum o f 27% protein, a minimum o f 2.5% crude fat and a maximum o f 5% crude fiber. ferm entation By-Products (Source o f Unidentified Growth Factors). 001400 Wildlife International, ltd -29- PROJECT NO.: 454-103 APPENDEX III ANALYTICAL METHODS AND RESULTS 001401 Wildlife International, ltd -30- PROJECT NO.: 454-103 INSTRUMENT: APPENDIX HI 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 Cu column (100 mm x 2 mm I.D., 3 pm particle size) OVEN TEMPERATURE: 30C STOP TIME: 10.0 minutes FLOWRATE: 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 001402 Wildlife International, ltd____________ -31 - PROJECT NO.: 454-103 APPENDIX HI Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454-103-) MAB-1 Sample Type Matrix Blank Measured Concentration of PFOS1 (ppm a.i.) <LOQ MAB-2 Matrix Blank <LOQ `The limit of quantitation (LOQ) was 1.15 ppm a.i. based upon the product of the lowest calibration standard zed (0.00229 mg a.i./L) and the dilution factor of the matrix blank samples (500). 001403 Wildlife International, ltd -32- PROJECTNO.: 454-103 APPENDIX m Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454-103-) MAS-1A MAS-4A " Concentrations ofPFOS (ppm a.i.) Fortified Measured 4.57 4.54 4.57 4.79 ll l Percent Recovered 99.2 105 MAS-2 183 176 96.1 MAS-5 183 162 88.3 MAS-3 MAS-6 1830 1576 1830 1716 86.1 93.7 Mean = 94.7 Standard Deviation = CV= N= 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. 001404 Wildlife International, l t d PROJECT NO.: 454-103 -33- APPENDDCin Table 4 Nominal Concentration (ppm a.i.) 18.3 Sample Number (S-454-103-) 2 3 4 5 6 7 Homogeneity of PFOS in Avian Diet Location Sampled in Mixing Vessel PFOS Measured Concentration (ppm a.i.) Top Left Top Right Middle Left Middle Right Bottom Left Bottom Right 18.5 23.4 18.3 17.3 19.4 19.9 Mean Measured Concentration (x) Standard Deviation (SD) Coefficient of Variation (CV)1 x = 19.5 ppm a.i. SD = 2.13 ppm a.i. CV = 10.9% Mean Percent of Nominal 107 1171 18 Top Left 19 Top Right 20 Middle Left 21 Middle Right 22 Bottom Left 23 Bottom Right 1239 1221 1118 1301 1163 1133 x = 1196 ppm a.i SD = 70.2 ppm. a.i CV = 5.87% 102 Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. 'Coefficient ofvariation was calculated using full precision of mean and standard deviation results. 001405 Wildlife International, ltd PROJECT NO.: 454-103 -34- APPENDIX m Table 5 Verification ofPFOS Concentrations in Avian Diet Nominal Concentration (ppm a.1) 0.0 Sample Number (S-454-103-) 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 - 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 0 274 0 ' 307 93.8 291 99.3 105 586 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 lim it 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 of the matrix blank samples (500). R esult obtained from Table 4. 001406 Wildlife International, ltd PROJECT NO.: 454-103 - 35 " APPENDIX ffl Table 6 Ambient Stability ofPFOS in Avian Diet During the Northern Bobwhite LC50 Study Nomina] Concentration (ppm a.i.) 0 Sample Number (S-454-103-) 1 Day O' Mean Measured Concentration (ppm a.i.) - Mean Percent of Nominal - Sample Number (S-454-103-) 24 Day 5 Measured Concentration2 (ppm a.i.) Mean Measured Concentration (ppm a.i.) < LOQ - Mean Percent of Day 0 - 18.3 2-7 19.5 107 25 19.2 19.6 101 26 19.9 36.6 8 .9 40.2 110 27 44.4 49.1 122 28 53.8 732 10.11 74.5 102 29 76.4 77.2 104 30 77.9 146 12.13 174 119 31 177 176 101 32 174 293 14,15 291 99.3 33 318 317 109 34 315 586 16.17 537 91.6 35 560 613 114 36 665 1171 18-23 1196 102 37 1260 1224 102 38 1187 'Day 0 results obtained from Table 4 and Table S. T h e limit of quantitation (LOQ) was 1.15 ppm a.i. based upon the product of the lowest calibration standard analyzed (0.00229 mg a.i7L) and the dilution factor of the matrix blank samples (500). Wildlife International, l t d -36- PROJECT NO.: 454-103 APPENDIX III METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN AVIAN DIET Prepare matrix fortification samples in the desired avian feed stock using the dry mix technique. 4 Dry Mix Technique For the high-level matrix fortification sample, weigh the requisite quantity o f Wildlife International Ltd. (WIL) ration into a weigh boat 4 Weigh the requisite quantity o f test substance (PFOS) into a beaker. 4 Add Vi o f the WIL ration and the test substance to a larger beaker. 4 Rinse the beaker that contained the PFOS with small portions o fthe remaining ration and transfer all portions to the larger beaker. 4 Mix the contents o fthe 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, m ix 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 o f 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. For each sample, measure 100 mLs o fmethanol 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 230 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 o f the LCMS methodology. Use methanol for intermediate dilutions, if required. For all final dilutions use 30% methanol : 30% NANOpure water solution containing 0.100 mg 4H PFOS (internal standardyL and 0.03% formic acid (v/v). Amputate and submit samples for LC/MS analysis. Figure 1. Analytical method flowchart for the analysis ofPFOS in avian diet. 001408 Wildlife International, l t d -37- APPENDDCni PROJECT NO.: 454-103 Concentration (Ratio) Figure 2. A typical calibration curve for PFOS. Slope = 2.77397; Intercept = 0.01894; r = 0.9981. Curve is weighted (1/x). 001409 Wildlife International, l t d -38APPENDIX in PROJECT NO.: 454-103 intensity: 1995 cps Figure 3. A representative ion chromatogram o f a low -level (0.00229 m g a.i./L ) PFOS standard. 001410 Wildlife International, l t d -39APPENDDim PROJECT NO.: 454-103 1 Figure 4. A representative ion chromatogram o f a high-level (0.0457 m g a.i./L ) PFOS standard. 001411 Wildlife International, ltd -40APPENDIX m PROJECT NO.: 454-103 1.30 2.55 3.81 5.06 6.32 7JS7 8.83 Time Figure 5. A representative chromatogram of a matrix blank sample (454-103-MAB-l). The arrow indicates the retention time of PFOS. 001412 Wildlife International, l t d 41 APPENDIX III PROJECT NO.: 454-103 100- go- 80- 70- 60- 50- 40- 30- 20- IQ - 1U1 2. r4u v . 31 1.30 4 - 7- r1 ..869r=$=r 61 91 2.55 3.81 122 121 5.06 166 151 6.32 Intensity: 6238 cps I -2-T1*3 211 8.83 Scan Tim e Figure 6. A representative chromatogram o f a matrix fortification sam ple (454-103-M A S-1A ). 001413 Wildlife International, l t d -42APPENDEX HI PROJECT NO.: 454-103 Figure 7. A representative chromatogram o f a test sam ple (454-103-2). 001414 Wildlife International, ltd -43- A P P E N D IX IV DIET PREPARATION PROJECT NO.: 454-103 W eight and volume o f constituents used to prepare test diets: Nominal Concentrations (ppm a.i.) 0 18.3 36.6 73.2 146 293 586 1171 Test Substance (g) 0.1818 0.3638 0.7282 1.4659 2.9123. 5.8239 11.6483 Basal Ration (g) 9000.0 8999.8 8999.6 8999.3 8998.5 8997.1 8994.2 8988.4 | Diets were prepared as follows: 5000.0 g o f basal ration was weighed into a tared H obart mixing bowl. The test substance was weighed in a tared weigh boat. Approximately 100 g o f basal ration w as taken from the mixing bowl and placed in a W aring 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 H obart 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. 001415 Wildlife International, l t d -44- PROJECT NO.: 454-103 Experimental Group (ppm a.i.) C ontrol 0 0 0 0 0 0 Treatm ent 18.3 18.3 36.6 36.6 73.2 73.2 146 146 293 293 586 586 1171 1171 APPENDIX V Cumulative M ortality by Pen from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS ______ No. Dead Per No. Exposed No. Dead Per No. Exposed P e u _______________________ Exposure Period______________________ Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 ______Post-Exposure Period Day 6 Day 7 Day 8 1 0/5 0/5 0/5 0/5 0/5 0/5 2 0/5 0/5 0/5 0/5 0/5 0/5 3 0/5 0/5 0/5 0/5 0/5 0/5 4 0/5 0/5 0/5 0/5 0/5 0/5 5 0/5 0/5 0/5 0/5 0/5 0/5 6 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1/5* 1/5 0/5 0/5 0/5 0/5 0/5 0/5 1/5 0/5 1 0/5 0/5 0/5 0/5 0/5 0/5 2 0/5 0/5 0/5 0/5 0/5 0/5 1 0/5 0/5 0/5 0/5 0/5 0/5 2 0/5 0/5 0/5 0/5 0/5 0/5 1 0/5 0/5 0/5 0/5 0/5 0/5 2 0/5 0/5 0/5 0/5 0/5 0/5 1 0/5 0/5 0/5 ' 0/5 0/5 0/5 2 0/5 0/5 0/5 1/5* 1/5 1/5 1 0/5 0/5 0/5 0/5 0/5 1/5 2 0/5 0/5 0/5 0/5 0/5 1/5 1 0/5 0/5 0/5 1/5 2/5 2/5 2 0/5 0/5 0/5 0/5 0/5 3/5 1 0/5 0/5 0/5 3/5 5/5 5/5 2 0/5 0/5 0/5 0/5 5/5 5/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 1/5 1/5 1/5 1/5 4/5 3/5 4/5 4/5 5/5 4/5 5/5 5/5 5/5 5/5 5/5 0/5 0/5 0/5 0/5 0/5 0/5 1/5 1/5 4/5 4/5 5/5 5/5 5/5 5/5 ~ S S S B B a B B B B ^ B S a S B S S S B S a ? S S S S S 8 ^ S B B S S S S B S ^ ^ S S a a a a B B ^ S S S B S S B S a B S S a B B e S S S S B a H S a B S B S B 8 S I^ H S a S B S B S S S B The LC50 value was calculated to be 220 ppm a.i. with a 95% confidence interval o f 164 ppm a.i. to 289 ppm a.i. * - Bird was euthanized due to a broken leg. oo 1 - No mortalities occurred in any o f the control or treatment groups after Day 8. p ________________ p a Wildlife International, l t d PROJECT NO.: 454-103 -45 - Experimental Group (ppm a.i.) 0 APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 1 1 2 3 4 5 Mean SD DayO 19 20 21 22 23 21 2 Day 5 24 27 32 32 39 31 6 Change Day 0-5 5 7 11 10 16 10 4 Day8 27 34 42 40 50 39 9 Change Day 5-8 3 7 10 8 11 8 3 1 19 26 7 35 9 2 20 25 5 35 10 3 20 29 9 37 8 4 22 33 11 43 10 5 22 31 9 39 8 Mean 21 29 8 38 9 SD 1 3 2 3 1 1 19 23 4 30 2 20 26 6 29 3 20 29 9 32 4 22 30 8 37 5 22 35 13 40 Mean 21 29 8 34 SD 1 5 3 5 7 3 3 7 5 5 2 1 2 3 4 5 Mean SD 19 19 19 21 22 20 1 28 27 30 32 33 30 3 9 37 8 35 11 36 11 42 11 43 10 39 14 9 8 6 10 10 9 2 Total Change 8 14 21 18 27 18 7 16 15 17 21 17 17 2 11 9 12 15 18 13 4 18 16 17 21 21 19 2 001417 Wildlife International, l t d PROJECT NO.: 454-103 -46- Experimental Group (ppma-i.) 0 APPENDEX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 2 Bird 1 2 3 4 5 Mean SD DayO 19 20 21 20 22 20 1 Day 5 28 27 32 30 34 30 3 Change Day 0-5 9 7 11 10 12 10 2 35 36 44 - 44 40 5 300 Change 7 9 12 10 10 2 0 Group Total 1 2 3 4 5 Mean SD Mean SD 18 21 19 21 22 20 2 20 1 30 35 29 32 32 32 2 30 4 12 39 14 46 10 37 11 41 10 42 11' 41 23 10 38 35 9 11 8 9 10 9 1 8 2 Total Change 16 16 23 22 19 4 21 25 18 20 20 21 3 18 4 001418 Wildlife International, l t d PROJECT NO.: 454-103 -47- APPENDIX VI Individual Body W eights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 3 Experimental Group 18.3 1 19 2 19 3 21 4 22 5 22 Mean SD 21 2 Day 5 Change =Da^0-5 Day 8 Change Day 5-8 26 7 34 8 33 14 39 6 27 6 35 8 34 12 46 12 36 14 46 10 31 11 40 446 9 2 Total Change 15 20 14 24 24 19 5 18.3 1 19 28 9 38 10 2 19 28 9 37 9 3 20 29 9 38 9 4 22 34 12 44 10 5 22 35 13 44 9 Group Total Mean 20 31 10 40 SD 2 3 2 3 Mean 21 31 11 40 SD 1 4 3 5 9 1 9 2 19 18 18 22 22 20 2 20 3 00141S Wildlife International, ltd -48- PROJECT NO.: 454-103 APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 4 Experimental Group Bird DayO 36.6 1 18 2 19 3 19 4 21 5 21 Mean SD 20 1 Day 5 Change Day 0-5 Day 8 26 8 35 30 11 39 29 10 37 30 9 38 34 13 43 30 10 38 323 Change Day 5-8 9 9 8 8 9 9 1 Total Change 17 20 18 17 22 19 2 36.6 1 19 29 10 37 2 19 29 10 37 3 21 32 11 40 4 22 35 13 44 5 23 36 13 44 Group Total Mean 21 32 11 40 SD 2 3 2 4 Mean 20 31 11 39 SD 2 3 2 3 8 8 8 9 8 8 0 8 1 18 18 19 22 21 20 2 19 2 001420 Wildlife International, ltd -49- PROJECT NO.: 454-103 APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 5 Experimental Group (ppm a.i.) Bird Day 0 73.2 1 18 2 19 3 20 4 21 5 21 Mean SD 20 1 Day 5 Change Day 0-5 Day 8 Change Day 5-8 26 8 32 26 7 33 29 9 36 32 11 39 30 9 36 29 9 35 3 13 6 7 7 7 6 7 1 Total Change 14 14 16 18 15 15 2 73.2 Group Total 1 2 3 4 5 Mean SD Mean SD 18 20 21 21 22 20 2 20 1 27 31 30 32 32 30 2 30 2 9 34 11 38 9 37 11 39 10 41 10 38 13 9 37 13 7 7 7 7 9 7 1 7 1 16 18 16 18 19 17 1 16 2 001421 Wildlife International, l t d -50- PROJECT NO.: 454-103 APPENDIX VI Individual Body W eights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 6 Experimental Group (ppm a.i.) Bird Day 0 146 1 19 2 19 3 20 4 22 5 23 Mean SD 21 2 Day 5 Change Day 0-5 Day 8 Change Day 5-8 28 9 35 28 9 32 24 4 33 11 38 28 5 32 28 8 34 333 7 4 5 4 5 1 Total Change 16 13 - 16 9 14 3 146 1 18 24 6 2 19 27 8 3 19 27 8 4 21 - - 5 23 24 1 Mean SD 20 2 26 2 6 3 Group Mean 20 27* 7* Total SD 2 3 3 (-) = No data available due to mortality. Statistically different from the control group at p < 0.05 (Dunnett's t-test). Statistically different from the control group at p < Q.Q1 (D um ett's t-test). 31 35 33 - 28 32 3 33* 3 7 8 6 - 4 6 2 6** 2 13 16 14 - 5 12 5 13** 4 001422 Wildlife International, l t d -51- PROJECT NO.: 454-103 APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 7 Experimental Group pm a.i.) Bird Day 0 293 1 18 2 19 3 20 4 22 5 22 Mean SD 20 2 Day 5 Change Day 0-5 20 2 17 -2 18 -2 -20 -2 -1 2 Day 8 21 - 21 - Change Day 5-8 1 - - 1 - Total Change 3 - - 3 - 293 1 19 19 0 2 19 - - 3 20 20 0 4 20 17 -3 5 21 16 -5 Group Total Mean SD Mean SD 20 1 20 1 18 -2 22 18** -2** 22 (-) = No data available due to mortality. Statistically different from the control group at p < 0.05 (Dunnett's t-test). Statistically different from the control group at p < 0.01 (Dunnett's t-test). 15 - - - -' 15 - 18** 4 -4 - - - -4 - -2** 4 -4 - - - -4 - 5 001423 Wildlife International, l t d PROJECT NO.: 454-103 -52- APPENDEX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietaxy Toxicity Study with PFOS Page 8 Experimental Group (ppm a.i.) 586 1 2 3 4 5 Mean SD 18 20 21 21 22 20 2 Change Day 0-5 Day 8 Change Day 5-8 ---13 -7 - - 18 -3 - - -- - 17 -5 - 16 -5 - 32- - T otal Change - - 586 1 19 16 -3 2 20 - - 3 20 16 -4 4 19 - 5 21 - - G roup M ean SD M ean 20 1 20 16 0 16** -4 1 _4*o T o ta l SD 1 2 2 (-) = No data available due to mortality. Statistically different from the control group at p < 0.01 (Dunnett' s t-test). - - - - - - - - - - - - - - - - - - - -. 001424 Wildlife International, ltd PROJECT NO.: 454-103 -53- APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 9 Experimental Group (ppm a.i.) Bird Day 0 1171 1 18 2 20 3 20 4 21 5 21 Mean SD 20 1 Day 5 m '- Change Day 0-5 - - - Day 8. - - Change Day 5-8 . - Total Change . 1171 1 18 2 20 3 20 4 22 5 21 Mean 20 SD 1 Group Mean 20 Total SD 1 (-) = No data available due to mortality. - - - - - - -- -- --- -- - ---- - - - - - - - - - - - 001425 Wildlife International, ltd PROJECT NO.: 454-103 -54- Experimental Group <PPm a i > 0 APPENDEX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 10 1 2 3 4 5 Mean SD 27 34 42 40 50 39 9 Change Day 8-15 Day 22 Change Day 15-22 42 15 61 19 56 22 77 21 69 27 92 23 63 23 84 21 78 28 104 26 62 23 84 22 14 5 16 3 0 1 35 55 20 74 19 2 35 53 18 74 21 3 37 64 27 85 21 4 43 74 31 103 29 5 39 59 20 80 21 Mean 38 61 23 83 22 SD 3 8 6 12 4 0 Group Total 1 30 54 24 79 25 2 29 45 16 63 18 3 32 54 22 77 23 4 37 55 18 75 20 5 40 68 28 95 27 Mean 34 55 22 78 23 SD 5 8 5 11 4 Mean 37 59 23 82 22 SD 6 10 5 13 3 Total Change 34 43 50 44 54 45 8 39 39 48 60 41 45 9 49 34 45 38 55 44 8 45 8 001426 Wildlife International, ltd PROJECT NO.: 454-103 -55- APPENDIX VI Individual Body W eights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 11 Experimental Group (ppm a.i.) Bird 18.3 1 2 3 4 5 Mean SD 34 39 35 46 46 40 6 Change '8-15 Day 22 Change 53 19 78 25 65 26 85 20 60 25 86 26 73 27 95 22 71 25 92 21 64 24 87 23 8 3 73 Total _Cbange 44 46 51 49 46 47 3 Experimental Group Change Change (ppm a.i.) Bird Day 8 Day 15 Day 8-15 Day 22 Day 15-22 36.6 1 35 60 25 81 21 2 39 65 26 91 26 3 37 65 28 91 26 4 38 61 23 83 22 5 43 72 29 97 25 Mean 38 65 26 89 24 SD 3 5 2 7 2 Total Change 46 52 54 45 54 50 4 001427 Wildlife International, ltd -56- PROJECT NO.: 454-103 APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 12 Experimental Group Change Change (ppm a.i.) Bird Day 8 Day 15 Day 8-15 Day 22 Day 15-22 73.2 1 32 54 22 76 22 2 33 58 25 77 19 3 36 62 26 79 17 4 39 65 26 87 22 5 36 59 23 78 19 Mean 35 60 24 79 20 SD 3 4 2 4 2 Total Change. 44 44 43 48 42 44 2 Experimental Group Change Change Day 22 Day 15-22 146 1 35 58 23 2 32 55 23 3 --- 4 38 63 25 5 32 57 25 Mean SD 34* 3 58 3 24 1 (-) = No data available due to mortality. Statistically different from the control group at p < 0.05 (Dunnett's t-test). 81 77 - 81 78 79 2 23 22 - 18 21 21 2 Total Change 46 45 - 43 46 45 1 001428 Wildlife International, ltd -57- PROJECT NO.: 454-103 APPENDIX VI Individual Body Weights (g) from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 13 Experimental Group (ppm a.i.) Bird DayO 293 1 21 2- 3- 4 5- Mean SD 21 - (-) = No data available due to mortality. Day 5 Change Day 0-5 Day 8 Change Day 5-8 35 14 55 20 --- - - --- -- -- --- - 35 14 55 20 --- - Total Change 34 - - 34 - 001429 Wildlife International, ltd PROJECT NO.: 454-103 -58- APPENDIX VII Feed Consumption (g/bird/day) by Pen from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 1 Experimental Group (ppm a.i.) Pen Exposure Period Day 0-5 Post-Exposure Period Day 6-8 Control 1 2 3 4 5 6 Mean SD 9 7 8 12 9 10 92 12 12 7 12 9 9 10 2 001430 Wildlife International, ltd PROJECT NO.: 454-103 -59- APPENDIX VH Feed Consumption (g/bird/day) by Pen from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 2 Experimental Group Exposure Period Post-Exposure Period (ppm a.i.) Pen Day 0-5 Day 6-8 18.3 1 2 Mean 9 8 9 10 11 11 36.6 1 2 Mean 8 7 8 14 9 12 73.2 1 10 2 10 Mean 10 12 14 13 146 1 7 2 10 Mean 9 8 12 10 293 1 4 26 Mean 5 8 10 9 586 1 2 Mean 1171 1 2 Mean - No data available due to adult mortality. 6 5 6 4 4 4 16 22 19 - 001431 Wildlife International, ltd PROJECT NO.: 454-103 -60- APPENDDC VII Feed Consumption (g/bird/day) by Pen from a Northern Bobwhite Acute Dietary Toxicity Study with PFOS Page 3 Experimental Group (ppm a.i.) Pen Exposure Period Day 8-15 Post-Exposure Period Day 15-22 Control 1 2 3 Mean SD 10 10 7 9 2 14 12 12 13 1 Experimental Group (ppm a.i.) Pen 18.3 1 36.6 1 73.2 1 146 1 293 1 Exposure Period Day 8-15 10 14 13 11 8 Post-Exposure Period Day 15-22 12 15 15 14 9 001432 Wildlife International, ltd PROJECT NO.: 454-103 -61 - APPENDIX VIII CHANGES TO PROTOCOL 1. The protocol was amended to indicate that bile will be collected from all study birds. The protocol was clarified to indicate the collection o f liver from birds that died during the course o f the study 2. Blood samples were collected on Day 8 and Day 22 in non-heparinized 5 ml borosilicate glass test tubes. The protocol indicated that heparinized vacutainers would be used. 3. The protocol was amended to change the test concentrations from 0, 2 0 ,4 0 , 80, 160, 640 and 1280 ppm a.i., to 0,1 8 .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 o f analysis. 4. The temperatures from several brooder units on Day 4, 7, 8, and all brooder units on D ay 22 were not recorded 5. The afternoon observations were inadvertently not recorded for 2 birds in the 640 ppm a.i. treatment group on April 26,1999. 001433 Wildlife International, ltd -62A PP E N D IX IX PERSONNEL INVOLVED IN THE STUDY PROJECT NO.: 454-103 The following key W ildlife International Ltd. personnel were involved in the conduct or management o f this study: (1) M ark 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) W illard B. Nixon, Ph.D., Manager, Analytical Chemistry (6) Timothy Z. Kendall, Supervisor, Analytical Chemistry (7) Raymond L. Van Hoven, Ph.D., Scientist (8) Ellen Mank, Chemist 001434 Wildlife International, l t d PROJECT NO.: 454-102 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A DIETARY LC50 STUDY WITH THE NORTHERN BOBWHITE PROTOCOL NO.: 454/030999/QLC/SUB454 AMENDMENT NO.: 1 SPONSOR: 3M Corporation PROJECT NO.: 454-103 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. Study Director Date 00143S Wildlife International, l t d t' DEVIATION TO STUDY PROTOCOL Page 1 o f 1 STUDY TITLE: PFOS: A Dietary LC50 Study with the Northern Bobwhite PROTOCOL NO.: 454/030999/QLC/SUB454 DEVIATION NO.: 1 SPONSOR: 3M Corporation PROJECTNO.: 454-103 DATE OF DEVIATION: April 30 and May 14,1999 AMENDMENT: Blood samples collected on Day 8 and Day 22 o f the test were stored in labelled nonheparinized 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 efficiently draw blood by syringe. Blood was drawn by decapitating birds and draining blood into test tubes. Heparin was not added to blood samples due to a Sponsor request to separate serum from the blood at Wildlife International Ltd. Seperation o f serum requires coagulation o f blood samples, which heparin prevents. JT? IM PACT: Blood samples were drawn in an efficient manner and serum was collected per Sponsor request Therefore this deviation caused no adverse impact on the study outcome or interpreta tion. 4'n STUDY DIRECTOR /" - f r --------- -----------------------------WILDLIFE TERNATIONAL LTD. MANAGEMENT DATE DATE 001436 Wildlife International, l t d PROJECT NO.: 454-103 Page 1 of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A Dietaiy LC50 Study with the Northern Bobwhite PROTOCOL NO.: 454/030999/QLC/SUB454 DEVIATION NO.: 2 SPONSOR: 3M Corporation PROJECT NO.: 454-103 DATE OF DEVIATION: April 26- May 14,1999 DEVIATION: On several occasions temperatures were inadvertently not recorded for two to eight brooder levels housing study birds. REASON: Biologist oversight With the exception o f May 1,1999, when three brooder temperatures were not recorded, the brooder pens 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 occasions where temperatures were missed neglected to record temperatures for empty brooder levels that housed birds earlier the same day. IMPACT: Data for these brooder levels on the days prior to and/or following the missed temperatures indicated that temperatures were within the acceptable range. It is reasonable to expect that the brooders were performing properly on the days o f the missed observations. Therefore, this deviation caused no adverse impact on the outcome o f the study. STUDY DIRECTOR 001437 I Wildlife International, l t d Page I of 1 4 n I i : 4J I i 1 i K FS DEVIATION TO STUDY PROTOCOL STUDY TITLE: PFOS: A Dietary LC50 Study with the Northern Bobwhite PROTOCOL NO.: 454/030999/QLC/SUB4S4 DEVIATION NO.: 3 SPONSOR: 3M Corporation PROJECTNO.: 454-103 DATE OF DEVIATION: April 26,1999 DEVIATION: On April 26,1999, Day 4 o f the study, the afternoon observations for unit B37-1 housing birds from the 640 ppm a.i. treatment level inadvertently did not include recorded observations o f two birds. REASON: Biologist oversight. IMPACT: None. Observations from the morning o f Day 4 were made for all birds in Pen B37-1, detailing which were displaying signs o f toxicity. Because observations o f two birds were not noted it is reasonable to assume they were normal in appearance and behavior, but the biologist failed to record the observation. AiL l -xJLL______ Mu STUDY DIRECTOR "1 DATE WILDLIFE INT A' In a l l t d . m a n a g e m e n t date 001438 Wildlife International, ltd PROJECT NO.: 454-103 SOP DEVIATION STUDY TITLE: PFOS: A DIETARY LC50 STUDY WITH THE NORTHERN BOBWHITE- - PROTOCOL NO.: 454/030999/QLC/SUB454 SOP NO.: 415 DEVIATION NO.: 1 SPONSOR: 3M Corporation PROJECT NO.: 454rl03 DATE OF DEFACTO DEVIATION: May 10-13,1999 DEVIATION: Brooder unit B21-1 used to house control group birds was below the specified minimum of 95F on may 10-13,1999. This brooder unit ranged between 1 and 3 below the minimum and there are no records o f the responsible technician or biologist making a thermostat adjustment to correct the temperatures. Brooder unit B26-1 used to house birds from the 320 ppm a.i. treatment group was 1 below the specified minimum of 95F on May 10,1999. On the following day the brooder temperature was within the specified range. REASON: Misadjustment o f the brooder unit thermostat. IMPACT: None. There was no apparent effect on test bird body weight or health. AjmLr- STUDY DIRECTOR _____ 1 Ut DATE WILDLIFE D 4 ^ to r f o ^ A L LTD. MANAGEM ENT DATE 0 0 l4 K Wildlife International, LTD PROJECT NO.: 454-103 SOP DEVIATION STUDY TITLE: PFOS: A DIETARY LC50 STUDY WITH THE NORTHERN BOBWHITE a PROTOCOL NO.: 454/030999/QLC/SUB454 SOP NO.: 423.1A a SPONSOR: 3M Corporation DEVIATION NO.: 2 PROJECT NO.: 454-103 DATE OF DEFACTO DEVIATION: April 22,1998 DEVIATION: O nA pril22,1999, during diet preparation on Day Oofthe 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. The SOP indicates that the test substance will be weighed to within 0.0005 g o f the target amount. REASON: Dietician oversight.. IMPACT: None. The difference in the amount o f test substance weighed resulted in a less than 1% change in final dietary concentration. STUDY DITRTEPCPTTOfYRP I ______ DATE j W ILDLffeT n t S n ^ IO N A L LTD. MANAGEMENT ^W\^ DATE 001440 PROTOCOL PFOS: A DIETARY LC50 STUDY W ITH THE NORTHERN BOBWHITE 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 935 Bush Avenue S t Paul, Minnesota 55144 Wildlife International ltd. 8598 Commerce Drive Easton, M aryland 21601 (410) 822-8600 M arch 9,1999 001441 Wildlife International ltd - 2PFOS: A DIETARY LC50 STUDY W ITH THE NORTHERN BOBWHITE SPONSOR: 3M Corporation Environmental Laboratory P.O .B ox 33331 St. Paul, M innesota 55133 SPONSOR'S REPRESENTATIVE: M s. Susan A. Beach TESTING FACILITY: W ildlife International Ltd. 8598 Commerce Drive Easton, M aryland 21601 STUDY DIRECTOR: Sean P. Gallagher, Senior Avian Biologist LABORATORY MANAGEMENT: Joann B. Beavers M anager o f Avian Toxicology ___________________________ FO R LABORATORY USE ONLY________________ Proposed Dates: E xperim ental Start Date: ,.L \ , Experimental AL Termination Date: Project No.: ^ /S ^ / ~ ________ _ Study Room: " 7 6 _______ Test Concentrations:^ i >*6 ^ j , l!)V0 , l*H> ^ 0 ^ a - f_______ Test Substance No.: 6 Reference Substance No. (if applicable): ^ PROTOCOL APPROVAL DATE _y/v/ff DATE/ / PROTOCOL NO. 454/030999/QLC/SUB454 001442 3M LAB REQUEST NO. U2 W i l d l i f e In t e r n a t i o n a l ltd - 3- OBJECTIVE The objective o f this study is to evaluate the toxicity o f a test substance to the northern bobwhite (Colinus virginianus) administered through the diet for five days. An LC50 value w ill be nalciilat^ if possible. SUMMARY Groups o ften bobwhite chicks, 10-14 days old, will be fed diets containing the test substance at selected concentrations for five days. A control group that receives untreated diet w ill be maintained concurrently. The test substance will 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 on untreated feed. A t the end o f the three-day observation period, half o f the birds in each treatm ent group will be euthanized and liver and blood collected for residue analysis. The remaining birds will be maintained on basal diet for an additional 14 days and then will be euthanized, with liver and blood collected. Throughout the test, the birds will be observed for toxicological responses. When possible, an LC50 . value will be calculated. M ATERIALS AND M ETHODS HU 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, Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms (2), ASTM Standard E857-87, "Standard Practice For Conducting Subacute Dietary Toxicity Tests With Avian Species" (3), and OECD Guideline 205, Avian Dietary Toxicity Test (4). In order to control bias, birds will be assigned to pens by indiscriminate draw. No other potential sources o f bias are expected to affect the results o f the study. The test substance will be administered in the diet. This route o f adm inistration was selected because it represents the most likely route o f exposure to avian species in the environment. Test Substance 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 PROTOCOL NO. 454/030999/QLC/SUB454 001443 3M LAB REQUEST NO. U2723 Wildlife International ltd - 4- use in the study. If written verification o f GLP test substance, characterization is not_provided Jo W ildlife International Ltd., it will be noted in the compliance statement o f the final report. The attached form ID EN TIFICA TIO N O F TE ST 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 fire 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 bobwhite chicks (five per pen) will be assigned by indiscriminate draw to each o f the treatm ent 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 will be followed by a period o f at least three days on untreated feed. Control birds will receive an amount o f the solvent or vehicle in their diet equivalent to the highest amount in the treated diets. I f 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 o b serv atio n -3 days/17 days. Test Birds The bobwhite represents an ecologically significant and widely distributed species in the United States. The bobwhite has demonstrated sensitivity to the effects produced by known toxic chemicals. M oreover, this species has proven to be a good laboratory model and a large amount o f baseline data are available. 001444 PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 Wildlife International l t d -5 - A llbobw hite will be.from the same hatch and will be 10-14 days old at test initiation. O nly... those that are apparently healthy will be used in the test. Body weights may vary between 10 and 50 grams depending on the age o f the cohort o f birds used (e.g. 10 versus 14 days). At least five test and three 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 concentration. Individual birds will be identified by colored leg bands. Birds will be obtained from Top Flight Quail Farm, Belvidere, NJ 07823, from another reputable supplier or obtained from the W ildlife International Ltd. production flock. All experimental birds will be acclimated to the caging and facilities from the time o f hatch or receipt until initiation o f the test. Animal Diet All test birds will be fed a game bird ration formulated to W ildlife International Ltd. specifications (Table 1). From the time o f hatch or receipt o f the chicks until the initiation o f the test, the birds will receive a water soluble vitamin mix via their water. Feed and water will be provided ad libitum during acclimation and during the test. The water source will be the Town o f Easton municipal w ater supply. Feed is analyzed periodically in accordance with Wildlife International Ltd. Standard Operating Procedures. The birds will 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. There are no levels o f contaminants reasonably expected to be present in the diet that are considered to interfere wife the purpose or conduct of foe study. Diet Preparation Test diets will be prepared at foe start o f foe test and a sufficient portion estimated for foe 5-day exposure period will be presented to foe birds. 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 foe test substance in acetone and/or table grade com oil prior to mixing with the feed. I f used, acetone will be allowed to volatilize from foe diets during foe mixing procedure. Table grade com oil will normally be incorporated into foe diet at a maximum o f 2% (w/w) o f foe final d iet Mixing is normally done with a Hobart mixer (Model Number AS200T). 001445 PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 Wildlife International l t d All test substance calculations w ill 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. Diet Sampling Samples o f the experimental diets will 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 concentrations. 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. Gn 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. I f a separate study is conducted with a different species (e.g., mallard), at the same test concentrations, diets that are prepared may be divided between the two studies. Therefore, samples collected for homogeneity and verification will be common to both studies. However, samples collected from the feeders in the study room (e.g. Day 5 samples) will be unique to that respective study. 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 6' 2 2 2 6' 19 1 2 2 2 2 2 11 Total Number of Samples = 30 'Samples collected from the left and right sides ofthe top, middle and bottom layers of feed in the mixing vessel to determine homogeneity. 001446 PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 Wildlife International ltd The above numbers o f samples represent those collected from the test and do not include quality control (QC) samples such as matrix blanks and fortifications prepared and analyzed during the analytical chemistry phase o f the study. 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 the Beacon Steel Company (Model B735Q) or equivalent. Each pen measures approximately 72 X 90 X 23 cm high. The external walls and ceilings o f each pen are constructed o f wire mesh and galvanized sheeting. Floors are o f wire mesh. Upon initiation o f the test, each brooding pen will 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. During the test, the temperature in the brooding compartment o f the pen will be maintained at approximately 38C. Acceptable ranges for brooding compartment temperatures will be specified in W ildlife International Ltd. Standard Operating Procedures. Brooding compartment temperatures will be recorded once each day during the test. The ambient room temperature will range between approxi mately 15-30C. Room temperature and relative 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 N ational Research Council (5). 001447 PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 Wildlife International l t d 8- - Observations All birds will be observed at least once daily for m ortality and toxicological responses. a* N ecropsy 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 will 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 A t 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. All 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 will be used to calculate an LC50 value. The data will be analyzed, in order o f preference, by probit analysis, the moving average method, or the binomial proba bility method (6, 7, 8, 9). The choice o f method for calculating the LC50 value will be based upon the m ortality pattern observed. When possible, a no mortality level will be determined and reported. RECORDS TO BE M AINTAINED Records to be maintained for data generated at W ildlife International Ltd. will 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 die test. 4. Animal history. 5. Husbandry and environmental conditions. 001448 6. Concentration calculations and records o f diet preparation. PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 q W i l d l i f e In t e r n a t i o n a l ltd -9 - 7. Body weight measurements. q 8. Feed consumption measurements. 9. Daily observations. q 10. Statistical calculations. 11. Analytical chemistry methods, results and chromatograms, if applicable. 12. A copy o f the final report. q FINAL REPORT q A report o f the results o f the study will be prepared by W ildlife International, Ltd. The report will include, but not be limited to, the following: q 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 q provide the final date that data are recorded for chemistry, pathology and/or supporting evaluations that may be generated at other laboratories. q 3. A statement o f compliance signed by the Study Director addressing any exceptions to Good Laboratory Practice Standards. q 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. H ie calculated LCSO value, 95 percent confidence limits, 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 will be provided for positive controls (when used). 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 o f the test system used. W here 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: 001449 PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 Wildlife International l t d - 10- a) Detailed description o f the basal diet, including source, diluents (if used), and supplements (if used). A nutrient analysis o fthe 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, photoperiod 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. i 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. 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. I f it 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 addition. Amendments shall be signed and dated by the Study Director. 001450 PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 W i l d l i f e In t e r n a t i o n a l ltd - ii- CHANGING OF PROTOCOL Planned changes to the protocol will be in the form o f written amendments signed by the Study D irector and the Sponsor's Representative. Amendments will be considered as part o f the protocol and will be attached to the final protocol. Any other changes will be in the form o f w ritten deviations signed by the Study Director and filed with the raw data. All changes to the protocol will be indicated in the final report. GOOD LABORATORY PRACTICES This study will be conducted in accordance with Good Laboratory Practice Standards for EPA (40 CFR P art 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 No. 3850, Agricultural Production Bureau). Each study conducted by Wildlife International Ltd. is routinely examined by the Wildlife 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 will be prepared for all portions o f the study conducted by W ildlife International Ltd. The Sponsor will 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 will 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. 454/030999/QLC/SUB454 001451 3M LAB REQUEST NO. U2723 W i l d l i f e In t e r n a t i o n a l ltd - 12- REFERENCES 1 U.S. Environm ental 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, FIFRA 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 fo r Testing and M aterials. 1987. Standard Practice for Conducting Subacute Dietary Toxicity Tests with Avian Species. ASTM Standard E857-87. Annual Book o f ASTM Standards, Vol. 11.04. Philadelphia, PA. 4 O rganization for Econom ic Cooperation and Development. 1984. Avian Dietary Toxicity Test. OECD Guideline for Testing o f Chemicals. Guideline 205. Paris. 5 N ational R esearch Council. 1996. Guide fo r the Care and Use o f Laboratory Animals. Washington, DC. National Academy Press. 125 pp. 6 Finney, D. J . 1971. Statistical Methods in Biological Assay, 2nd ed., Griffin Press, London. 7 Thom pson, W . R . 1947. Bacteriological Reviews,'Vol 2, No. 2: 115-145. 8 Stephan, C. E . 1977. Methods for Calculating an LC50. Pages 65-84 In Aquatic Toxicology and Hazard Evaluations, American Society for Testing and M aterials. Pub. No. STP 634. Philadelphia, PA. 9 Stephan, C. E . 1978. U.S. EPA, Environmental Research Laboratory, Duluth, MN. Personal Communication. PROTOCOL NO. 454/030999/QLC/SUB454 001452 3M LAB REQUEST NO. U2723 Wildlife International ltd - 13- TA BLE1: DIET FORMULATION W ILDLIFE 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 m Dried Whey Ground Limestone Eastman CalPhos m Methionine Premix + Liquid Vitamin and Mineral Premix (see below) GL Ferra (Fermatco)2 Salt Iodized m 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 A dd 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 minimum of 27% protein, a minimum of 2.5% crude fat and a maximum of 5% crude fiber. 2 Fermentation By-Products (Source of Unidentified Growth Factors). 001453 PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 W i l d l i f e In t e r n a t i o n a l ltd H m m m - 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): PFOS (Perfluorooctane Sulfonic Acid P otassiu m Salt Reference Standard (if applicable): Analytical Standard: N/A______ Internal Standard: 1, L2.2H.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________________ n. Test Substance Characterization Have the identity, strength, purity and composition or other characteristics which appropriately definethe test substance and reference standard been determined priorto its use in this study in accordancewith GLP Standards? m. Test Substance Storage Conditions _____ Yes x No Please indicate the recommended storage conditions atWildlife International Ltd. _____ Ambient_______________________________________________________________________ Has tte stability ofthe test substanceunderthese storage conditions. been determined in accordance with GLP Standards? _____ Yes x No Other pertinent stability information: IV. Test Concentrations: Adjust test concentrationto 100% a i x based upon the purity (%) given above. Do not adjusttest concentrationto 100% ______ai. Testme material AS IS. V. Tcoddty Information: Mammalian: RatLD50 251 me/kg Mouse LD50 N/A Aquatic: Invertebrate Toxicity (ECZLC50) Fish Toxicity (LC50) Daphnia mama: 27 me/L_____________Rainbow Trout: 11 mg/L Daphniamaena: 50 mg/L____________ Fathead Minnow: 38 mg/L OtherToxicity Information (including findings o fchronic and subchronic tests): Please see MSDS______________________________________________________________; 001454 PROTOCOL NO. 454/030999/QLC/SUB454 3M LAB REQUEST NO. U2723 m * m W i l d l i f e In t e r n a t i o n a l ltd - 15A PP E N D IX H Method Outline for the Determination o f PFOS in Avian Feed m 1. Prepare m atrix fortification samples in the desired avian feed stock using the dry mix technique. 2. Weigh 10 g samples o f the matrix blank and matrix fortification feed samples into a weigh boat and transfer p i to 16 oz. French square glass bottles. Record weights. m 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 minimum o f 30 minutes. m 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% w ater dilution solvent containing 0.05% v/v formic acid and 100 pg a.iVmL 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 pm filter into an HPLC autosampler vial. 9. Ampulate and submit sample for LCMS analysis. PROTOCOL NO. 454/030999/QLC/SUB454 001455 3M LAB REQUEST NO. U2723