Document NEL7jx6nOyYrB1kV3RyVDVw2E

BACK TO MAIN TOXICITY TO AQUATIC PLANTS (Freshwater alga, Anabaena flos-aquae) 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: Sample from 3M production lot number 217. The test substance is a white powder. Purity determined to be 86.9% by LC/MS, 1H-HMR, 19F-NMR and elemental analyses techniques. METHOD Method: OPPTS 850.5400 Test: Acute static GLP: Yes Year completed: 2001 Species: Anabaena flos-aquae Source: Originally from UTEX - The Culture Collection of Algae at the university of Texas at Austin, and maintained in culture medium at Wildlife International Ltd., Easton, MD Analytical monitoring: PFOS measured at 0, 72, 96-hours Element basis: Reported three ways: number of cells/ml, area under the growth curve and growth rate Exposure period: 96-hours Start date: 1/28/00 End date: 6/5/00 Test organisms laboratory culture: Algae cultures had been actively growing in algal culture medium for at least two weeks prior to test initiation. Stock nutrient solutions were prepared by adding reagent-grade chemicals to reverse osmosis-purified well water. Solutions were then diluted in purified well water to prepare final growth media. BACK TO MAIN Test Conditions: Freshwater Algal medium Compound MgCl2.6H2O CaCl2.2H2O Nominal Concentration 12.16 4.40 Units mg/L mg/L H3BO3 MnCl2.4H2O 0.1856 0.416 mg/L mg/L ZnCl2 FeCl3.6H20 CoCl2.6H20 Na2MoO4.2H20 CuCl2.2H2O Na2EDTA.2H20 3.28 0.1598 1.428 7.26 0.012 0.300 g/L mg/L g/L g/L g/L mg/L NaNO3 MgSO4.7H2O 25.5 mg/L 14.7 mg/L K2HPO4 1.044 mg/L NaHCO3 15.0 mg/L Dilution water source: The pH of the medium was adjusted to 7.5 + 0.1 and it was sterilized by filtration (0.22m) prior to use. Test solution preparations: Individual test solutions were prepared in algal medium at each of the six nominal concentrations. The solutions were stirred with magnetic stir plates for approximately 18 hours. The final test solutions appeared clear and colorless. Exposure vessels: Sterile 250 mL glass Erlenmeyer flasks plugged with foam stoppers containing 100 mL of test solution. Agitation: Shaken continuously at 100 rpm Number of replicates: six. Initial algal cell loading: 1.0 X 104 cells/mL Cell counts method: hemacytometer and microscope Number of concentrations: six plus a negative control plus an abiotic control at the highest concentration tested Water chemistry: pH range (0 - 96 hours) 7.4 - 7.6 (control exposure) 7.4 - 7.4 (329 mg/L exposure) Test temperature range (0 - 96 hours) 22.8 - 23.8C BACK TO MAIN Light levels: (0 - 96 hours) 1990 - 2310 lux from cool-white fluorescent lighting Photoperiod: 24-hours light Method of calculating mean measured concentrations: arithmetic mean obtained using results obtained at 0-hours, 72hours and 96-hours RESULTS Nominal concentrations: Negative control, 37.9, 58.6, 88.8, 139, 216, 331 mg/L plus 331 abiotic replicate Measured concentrations: <LOQ, 37.9, 63.9, 93.8, 143, 235, 329 mg/L; abiotic replicate = 349 mg/L Element values (95% confidence interval): 24-hour EC50 (cell density) = 105 mg/L (C.I. not calculable) 24-hour EbC50 (area under curve) = 90 (40 - 150) mg/L 24-hour ErC50 (growth rate) = 94 (33 - 145) mg/L 48-hour EC50 (cell density) = 117 mg/L (C.I. not calculable) 48-hour EbC50 (area under curve) = 103 mg/L (C.I. not calculable) 48-hour ErC50 (growth rate) = 128 mg/L (C.I. not calculable) 72-hour EC10 (cell density) = 43 (34 - 84) mg/L 72-hour EbC10 (area under curve) = <38 mg/L (C.I. not calculable) 72-hour ErC10 (growth rate) = 82 (49 - 116) mg/L 72-hour EC50 (cell density) = 120 (92 - 139) mg/L 72-hour EbC50 (area under curve) = 116 (49 - 142) mg/L 72-hour ErC50 (growth rate) = 174 (146 - 208) mg/L 72-hour EC90 (cell density) = 224 (193 - 275) mg/L 72-hour EbC90 (area under curve) = 204 (134 - 226) mg/L 72-hour ErC90 (growth rate) = 275 (162 - 330) mg/L 96-hour EC10 (cell density) = 82 (29 - 123) mg/L 96-hour EbC10 (area under curve) = 56 (26 - 107) mg/L 96-hour ErC10 (growth rate) = 109 (84 - 125) mg/L 96-hour EC50 (cell density) = 131 (106 - 142) mg/L 96-hour EbC50 (area under curve) = 124 (104 - 138) mg/L 96-hour ErC50 (growth rate) = 176 (169 - 181) mg/L 96-hour EC90 (cell density) = 213 (203 - 219) mg/L 96-hour EbC90 (area under curve) = 209 (197 - 218) mg/L 96-hour ErC90 (growth rate) = 225 (220 - 235) mg/L 72-hour NOAEC (cell density, area under curve): 37.9 mg/L 72-hour NOAEC (growth rate): 93.8 mg/L 96-hour NOAEC (cell density, growth rate): 93.8 mg/L 96-hour NOAEC (area under curve): 63.9 mg/L All element values based on mean measured concentrations BACK TO MAIN Statistical methods: Cell densities, area under the growth curve values, growth rates and percent inhibition values were calculated using "The SAS System for Windows", Release 6.12. The EC10, EC50, and EC90 values and 95% confidence limits were calculated by linear interpolation with treatment response and exposure concentration data using TOXSTAT Version 3.5. Cell densities, areas under the growth curve and growth rates at 72 and 96 hours were evaluated for normality and homogeneity of variances using the Shapiro-Wilk's test and Levene's test, respectively. Where the data were normally distributed with equal variances, the treatment groups were compared to the control using Dunnett's test. In the one instance where data were not normally distributed, the nonparametric Kruskal-Wallis test was used. Results of the statistical analyses were used to determine the NOAEC values. Analytical Methodology: 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 4.80 mg/L in this study. The mean percent recovery of matrix fortifications analyzed concurrently during sample analysis was 103%. Samples collected at test initiation had measured values from 100 to 112% of nominal. The measured values for the samples taken at 72-hours were 99.0 - 110% of nominal. The measured values for the samples taken at 96-hours were 99.0 - 109% of nominal. For the abiotic replicate, the measured value for the sample taken at 72-hours was 103% of nominal and for the sample taken at 96hours, 107% of nominal. Summary of analytical chemistry data: Nominal Test Concentration, mg/L Negative Control 37.9 58.6 88.8 139 216 331 331 (abiotic) Measured Values at 0, 72, and 96hours, Respectively, mg/L All < LOQ Mean Measured Concentration, mg/L <LOQ Percent of Nominal - 37.9, 38.2, 37.6 37.9 100 65.6, 62.7, 63.4 63.9 109 94.3, 97.4, 89.8 93.8 106 142, 146, 142 143 103 230, 238, 236 235 109 331, 328, 329 329 99.4 Not analyzed, 342, 356 349 105 BACK TO MAIN Biological observations after 96-hours: Mean Measured Concentration, mg/L Mean Number of Cells per mL Percent Inhibition via Density Percent Inhibition via Area Under the Curve Percent Inhibition via Growth Rate Negative Control 569,167 - - - 37.9 605,000 -6.3 3.0 -1.3 63.9 585,833 -2.9 13 -0.97 93.8 492,500 13 24* 3.6 143 228,833 60* 66* 22* 235 2,333 100* 99* 100* 329 8,500 99* 100* 96* *Indicates a significant difference from the negative control using the appropriate statistical test (p <0.05) Control response: satisfactory Observations: After 96 hours of exposure, there were no signs of aggregation or adherence of the algae to the flasks in the negative control or any treatment group. In addition, there were no noticeable changes in cell morphology when compared to the negative control. Reversibility of Growth Inhibition: Aliquots of the 235 and 329 mg/L test solutions were diluted with algal medium and cultured for nine days after the exposure phase of the study concluded. Based on the increase in growth observed by Day 9 of the recovery phase, the effect on algal growth was algistatic at a concentration of 235 mg/L. However, no algal cells were detected during the recovery phase in the 329 mg/L treatment, indicating that PFOS was algicidal at that concentration. CONCLUSIONS The potassium perfluorooctanesulfonate 96-hour EC50 and 95% confidence interval for Anabaena flos-aquae was determined using three calculation methods. By cell density, it was 131 (106 - 142) mg/L, by area under the growth curve it was 124 (104 - 138) mg/L and by growth rate 176 (169 - 181) mg/L. The 96-hour NOAEC values were determined to be 63.9 mg/L using the area under the growth curve, and 93.8 mg/L with the cell density and growth rate calculation method. No signs of cell aggregation or adherence were noted in any of the test solutions or the controls. PFOS was determined to be algistatic at a concentration of 235 mg/L and algicidal at 329 mg/L. BACK TO MAIN Submitter: 3M Company, 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, Lab Request number U2723. OTHER Last changed: 6/12/01