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BACK TO MAIN DIETARY ACUTE NORTHERN BOBWHITE STUDY REVISED Tissue analysis is now included. TEST SUBSTANCE_____________________________ Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC-95. (1-Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-, potassium salt, CAS # 2795-39-3) Remarks field: The test substance is a white powder. Sample was taken from 3M lot number 217. Sample was stored under ambient conditions prior to testing. Purity determined to be 90.49% by LC/MS, 1HHMR, 19F-NMR and elemental analyses techniques. METHOD Method: OPPTS 850.2200, OECD 205, and FIFRA Subdivision E. Section 71-2 Type: Dietary acute GLP: Yes Year completed: 1999 (Test), 2000 (Report) Species: Colinus virginianus Supplier: Wildlife International Ltd. Production Flock, Easton, Maryland Analytical monitoring: Test substance concentration in standards and samples were determined by reversed-phase HPLC and mass spectroscopy. PFOS measured on Day 0 for homogeneity in feed and verification, and Day 5 for stability. Test phases: Acclimation - 10 days Exposure - 5 days Post-exposure observation - 3 or 17 days Statistical methods: Lc50values calculated by probit analysis using the computer software of C.E. Stephan. Body weight data were compared by Dunnett's test using TOXSTAT software. No statistical analyses were applied to feed consumption data. Test bird age: 10 days Pretreatment: None Test conditions: Housing and environmental conditions: Indoors in batteries of thermostatically controlled brooding pens. Each pen's floor space measured approximately 72 X 90 cm. Ceiling height was approximately 23 cm. External walls, ceilings and floors were constructed of galvanized steel wire and sheeting. Identification: Each group of birds identified by pen number and test concentration. Individuals identified by leg bands. BACK TO MAIN Number of replicates : Six for controls, two for each treatment group Number of bobwhite per replicate : five Number of concentrations: seven plus a negative control Feed and water: Game bird ration formulated as below, water from the town of Easton public water supply. Both provided ad libitum during acclimation and testing. Game Bird Ration Ingredients Percent Fine Corn Meal 44.83 Soy Bean Meal, 48% Protein 30.65 Wheat Midds 6.50 Protein Base 6.00 Agway Special, 60% Protein 4.00 Alfalfa Meal, 20% Protein 3.00 Dried Whey 2.50 Ground Limestone 0.90 Eastman CalPhos 0.60 Methionine Premix + Liquid 0.35 Vitamin and Mineral Prem ix (see below) 0.32 GL Ferm (Fermatco) 1 0.25 Salt Iodized 0.10 'Fermentation by-products (source of unidentified growth factors) Vitamin and Mineral Premix Vitamin or mineral Vitamin D 3 Vitamin A Riboflavin Niacin Pantothenic Acid Vitamin B12 Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (Menadione dimethylpyrimidinol bisulfite) Manganese Zinc Copper Iodine Iron Selenium Amount Per Ton 2,000,000 I.C.U. 7,000,000 I.U. 6 grams 40 grams 10 grams 8 mgs 600 mgs 64 mgs 1.2 grams 1.2 grams 20,000 I.U. 5.8 grams 102 grams 47 grams 6.8 grams 1.5 grams 51 grams 182 grams BACK TO MAIN Prophylaxis : None Brooding compartment mean temperature: 38 + 2oC Ambient room mean temperature: 27.3 + 1.2oC Average relative humidity: 31 + 14% Photoperiod: Sixteen hours light per day Lighting: fluorescent lights which closely approximate noon-day sunlight; average of approximately 139 lux of illumination Test diet preparation: Test substance mixed directly into the ration by means of a Hobart mixer. No carrier was used. Diet sampling: Homogeneity of the test substance in the diet evaluated by collecting six samples from the 18.3 ppm concentration and six from the 1171 ppm concentration. Samples collected from the top, middle, and bottom of the left and right sections of the mixing vessel. These samples also served as the verification samples for these concentrations. Verification samples of the other treatment groups (two samples from each) and the control (one sample) were collected at preparation on Day 0. Stability samples were collected at the end of the exposure period (Day 5) from the control (one sample) and each treatment group (two samples each). RESULTS___________________________________________________ Nominal concentrations: Bk control, 18.3, 36.6, 73.2, 146, 293, 586, and 1171 ppm Measured concentrations: <LOQ, 19.5, 40.2, 74.5, 174, 291,537, and 1196 ppm Element value: Dietary LC50 = 220 (164 - 289) ppm No mortality concentration = 73.2 ppm NOEC (body weight gain) = 73.2 ppm All element values based on nominal concentrations Analytical Methodology: Diet samples were extracted with methanol. Analyses of test solutions were performed at Wildlife International Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). When determining the concentration of the test substance in the test solutions, the same and most prominent peak response for perfluorooctanesulfonate was used. No attempt was made to quantify on the basis of individual isomeric components. The LOQ (limit of quantitation) was 1.15 ppm in this study. The mean percent recovery of matrix fortifications analyzed concurrently during sample analysis was 94.7. Samples collected for determination of homogeneity in diet ranged from 102-107% of nominal. Samples collected for verification in diet had measured values from 92 to 119% of nominal. Measured values for BACK TO MAIN ambient stability samples taken at Day 5 ranged from 101-122% of nominal. Summary of analytical chemistry data: Homogeneity in Avian Diet Nominal Test Concentration, ppm Measured Values at Day 0, ppm 18.3 1171 18.5, 23.4, 18.3, 17.3, 19.4, 19.9, 1239, 1221, 1118, 1301, 1163, 1133 Mean Measured Concentration , ppm 19.5 Percent of Nominal 107 1196 102 Verification in Avian Diet Nominal Test Concentration, ppm Negative Control Measured Duplicate Concentrations at Day 0, ppm < LOQ Mean Measured Concentration , ppm - Percent of Nominal - 36.6 45.7, 34.6 40.2 110 73.2 77.8, 71.2 74.5 102 146 176, 172 174 119 293 274, 307 291 99 586 550,523 537 92 Ambient Stability in Avian Diet Day 0 Mean Measured Concentration, ppm Negative Control Measured Duplicate Concentrations at Day 5, ppm < LOQ Mean Measured Concentration , ppm - Mean Percent of Day 0 - 19.5 19.2, 19.9 19.6 101 40.2 44.4, 53.8 49.1 122 74.5 76.4, 77.9 77.2 104 174 177, 174 176 101 291 537 1196 318, 315 560, 665 1260, 1187 317 613 1224 109 114 102 BACK TO MAIN Biological observations Mortalities and clinical observations: One incidental mortality occurred in the control group as a result of a broken leg on the morning of Day 5. It was subsequently euthanized on Day 6. Two other birds in the control group were intermittently noted with foot lesions associated with cage mate aggression. Otherwise, all control birds were observed to be normal in appearance and behavior throughout the test. The first treatment-related mortalities occurred on Day 3 in the 586 and 1171 ppm treatment groups. Mortality occurred through Day 8 in all dose groups > 146 ppm with some of the deaths being during the post-exposure period. There were no treatment-related mortalities or overt signs of toxicity at concentrations < 73.2 ppm. There was 11% mortality in the 146 ppm treatment group, and two additional birds displayed clinical signs of toxicity (wing droop). All other birds in this test group displayed normal appearance and behavior for the duration of the test. Recovery with normal appearance and behavior occurred on Day 9 to test termination. There was 80% mortality (occurring on Days 5,6, and 7) for birds in the 293 ppm treatment group. Signs of toxicity observed prior to death included a ruffled appearance, reduced reaction to stimuli (sound and motion), lethargy, wing droop, loss of coordination, lower limb weakness and convulsions. Recovery with normal appearance and behavior occurred on Day 9 to test termination. There was 100% mortality (occurring from Day 3 through Day 7) for birds in the 586 ppm treatment group. Signs of toxicity observed prior to death included a ruffled appearance, reduced reaction to stimuli (sound and motion), lethargy, depression, wing droop, loss of coordination, lower limb weakness, lower limb rigidity, prostrate posture, and convulsions. There was 100% mortality (occurring from Day 2 (noted on Day 3 for Day 2 afternoon) through Day 4) for birds in the 1171 ppm treatment group. Signs of toxicity observed prior to death included a ruffled appearance, reduced reaction to stimuli (sound and motion), lethargy, depression, wing droop, loss of coordination, lower limb weakness, and lower limb rigidity. BACK TO MAIN Body weight gain: When compared to the control group, there were no apparent treatment related effects on body weight among the birds in concentrations < 73.2 ppm. During Days 0-5, statistically significant reductions in body weight gain or body weight loss occurred in the 146, 293, and 586 ppm treatment groups. Body weight effects could not be determined for test organisms in the 1171 ppm group due to total mortality. Feed Consumption: No apparent treatment related effects were noted for feed consumption for birds in concentrations <146 ppm. Reduced feed consumption was noted for birds in treatment groups > 293 ppm from Days 0-5. No treatment-related effects on feed consumption in any of the surviving treatment groups during the Day 6-8 post-exposure period were observed. Gross Necropsy: All birds that died during the study, half of those surviving at Day 8 and the rest at test termination were subjected to a gross necropsy. Necropsy results for birds found dead were similar, including thin condition, loss of muscle mass, altered spleen color, autolysis of tissues and pale organs. These necropsy findings were considered to be treatment related. The single bird euthanized from the 293 ppm treatment was found to have treatment related necropsy findings. Necropsy results for all other birds euthanized on Day 8 and Day 22 were unremarkable. % Cumulative Mortality Nominal Concentration , ppm Day 1 Day 2 Day 3 Day 4 Day 5 Negative 0 0 0 0 0 Control 18.3 0 0 0 0 0 36.6 0 0 0 0 0 73.2 0 0 0 0 0 146 0 0 10** 0 0 293 0 0 0 0 20 586 0 0 10 20 50 1171 0 0 30 100 100 *No mortalities occurred in any of the treatment levels from Day 8 to Day 22 ** Bird euthanized on day 3 after sustaining a broken leg. Day 6 0 0 0 0 0 40 80 100 Day 7 Day 8* 00 00 00 00 10 10 80 80 100 100 100 100 BACK TO MAIN Summary of Analytical Results for Liver Tissue1(ug/g wet) during Day 1 through Day 7 Test Period2 - Individual Values Reported N om inal Day 3 Liver Day 4 Liver Day 5 Liver Day 6 Liver Day 7 Liver Conc., ppm Conc. Conc. Conc. Conc. Conc. 146 111 293 249 202 153, 124 586 NE NE 126 235 111 1171 134 132,123,124 1Concentrations were not corrected for purity of PFOS standard used (lot 171). No attempt was made to correct for moisture content. 2Reported results are only for concentrations where mortality occurred. During this time period, the negative control and concentrations <293 ppm exhibited no mortality. (One bird euthanized after sustaining a broken leg.) There was also no mortality in concentrations > 293 ppm during days 1 and 2. NE = Not Extracted Summary of Analytical Results for Liver Tissue (ug/g wet) and Serum (ug/mL) at Day 8 and Day 221 - Average Value Reported Nom inal Conc., Day 8 Liver Day 8 Serum Day 22 Liver Day 22 Serum ppm Conc. Conc. Conc. Conc. Negative Control <LOQ (~0.070) <LOQ (0.010) 0.215 0.115 18.3 18.5 <LOQ 3.25 5.89 36.6 25.8 3.04 2.92 7.27 73.2 44.0 41.2 10.0 26.2 146 70.3 41.6 25.8 40.5 293 NE Not applicable Not applicable Not applicable 586 NS NS Not applicable Not applicable 1171 NS NS Not applicable Not applicable "'Concentrations were not corrected for purity of PFOS standard used (lot 171). No attempt was made to correct for moisture content in relevant tissue, liver. NS = No Sample analyzed. NE = Not Extracted Body Weight (grams) Exposure Period Recovery Period Nominal Concentration (ppm) Mean Body Weight, Day 0 Mean Body Mean Body Mean Body Mean Body Weight Weight Weight Weight Change Change Change Change Day 0-5 Day 5-8 Day 8-15 Day 15-22 Mean Total Mean Body Body Weight Weight, Change, Day Day 22 8-22 Negative Control 18.3 20 21 +10 +8 +23 +22 +11 +9 +24 +23 45 47 82 87 36.6 20 +11 +8 +26 +24 50 89 73.2 20 +9 +7 +24 +20 44 79 146 20 +7* +6** +24 +21 45 79 BACK TO MAIN 293 20 2** 1** +14 +20 34 55 586 20 _4** ~ -- -- -- -- 1171 20 ~ ~ -- -- -- - Note: Numbers may not add manually due to rounding. Values for 293 ppm treatment group are impacted by the fact that only one bird remained in that treatment group after Day 8. ` 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 (--) = No data available due to mortality. Mean Average Feed Consumption Nominal Concentration, ppm Grams Grams Grams Grams Feed/Bird/Day Feed/Bird/Day Feed/Bird/Day Feed/Bird/Day Days 0-5 Days 6-8 Days 8-15 Days 15-22 ' Negative Control 9 10 9 13 18.3 9 11 10 12 36.6 8 12 14 15 73.2 10 13 13 15 146 9 10 11 14 293 5 9 8 9 586 6 19 _ _ 1171 4 -- -- -(--) = No data available due to mortality. Gross Pathological Observations from Birds that Died in Study Finding Abdominal cavity, some autolysis Abdominal cavity, autolysis throughout Crop, empty Emaciated Fractured leg G.I. Tract empty Gizzard contents bile stained Heart, anterior portion mottled white color Heart, pale Intestinal contents tar-like Keel, prominent Kidneys, pale Liver, pale and mottled Loss of muscle mass Muscular-skeletal, pale Small in stature Spleen, black Spleen, dark Male, Female, and Undetermined ___________ (ppm)____________ Control 146 293 586 1171 N = 1 N = 2 N = 8 N = 10 N = 1( 0 022 4 0 001 1 0 025 2 0 025 8 1 100 0 0 011 0 0 025 1 0 0 10 0 0 002 1 0 002 0 0 0 1 3 10 0 002 0 0 100 0 0 047 9 0 100 0 0 030 0 0 001 0 0 000 2 Spleen, grey Spleen, grey-brown Spleen, pale Spleen, small Spleen, small and pale Thin Not remarkable 0 001 0 0 000 1 0 0 10 1 0 000 1 0 003 0 0 004 2 0 0 10 0 BACK TO MAIN CONCLUSIONS____________________________________________ The dietary LC50 value for Northern Bobwhite exposed to perfluorooctanesulfonate was determined to be 220 ppm with a 95% confidence interval of 164 to 289 ppm. The slope of the concentrationresponse curve was 7.005 and the chi-square value was 0.023. The no mortality concentration was 73.2 ppm. Based upon treatment related mortality, signs of toxicity and effects upon body weight gain at the 146 ppm test concentration, the no observed effect concentration was 73.2 ppm. Author and/or submitter: 3M Corporation, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY____________________________________________ Reliability: Klimisch ranking = 1. REFERENCES This study was conducted at Wildlife International Ltd., Easton, MD at the request of the 3M Company. The tissue analysis was conducted by 3M Environmental Laboratory, St. Paul, MN. OTHER____________________________________________________ Last changed: 5/3/00, 6/28/01