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13) O ECD 201-OPPTS 850.5400, Algal toxicity- 96-Hour toxicity test w ith the freshw ater alga, 454A-129 PFBS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA (Selenastrum capricornutum) SANITIZED FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-129 DEC 0 9 2003 3M Environmental Lab Project No. E00-1429 U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.5400 and OECD Guideline 201 AUTHORS: Kurt R Drottar Henry O. Krueger, Ph.D. STUDY INITIATION DATE: October 6,2000 STUDY COMPLETION DATE: March 20, 2001 Submitted to 3M Corporation Environmental laboratory Building 2-3E-09 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife International, Ltd. 8598 Commerce Drive Easton, Mary land 21601 (410)822-8600 Page 1 o f 56 Wildlife International, Ltd. 2- - Project Number 454A -129 SANITIZED GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT DEC 0 9 2003 SPONSOR: 3M Corporation TITLE: PFBS: A 96-Hour Toxicity Test with the Freshwater Alga (Selenastrum capricomutum) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-129 STUDY COMPLETION: March 20, 2001 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984 with the following exceptions: The test substance was not characterized in compliance with Good Laboratory Practices prior to its use in the study. However, subsequent GLP compliant characterization resulted in a purity similar to the original characterization purity. The stability of the test substance under conditions o f storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Kurt R Drottar Senior Biologist SPONSOR: DATE DATE Wildlife International, Ltd. -3 - Project Number 454A-129 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Test Substance Preparation Sample Preparation Biological Data and Draft Report Analytical Data and Draft Report Final Report DATE CONDUCTED: January 22, 2001 January 22, 2001 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: January 22, 2001 January 22, 2001 January 22, 2001 January 24, 2001 February 15 and 16,2001 February 16, 2001 February 19, 2001 February 15 and 16, 2001 March 19, 2001 February 16, 2001 March 19, 2001 February 16, 2001 March 20, 2001 Wildlife InternationalLtd, -4 - Project Number 454A-129 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFBS: A 96-Hour Toxicity Test with the Freshwater Alga (Selenastrum capricornutum) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-129 STUDY DIRECTOR: DATE MANAGEMENT: and Non-Target Plants DATE Wildlife International, Ltd. Project Number 454A-129 TABLE OF CONTENTS Title/Cover Page.......................................................................................................................................................1 Good Laboratory Practice Compliance Statement............................................................................................... 2 Quality Assurance Statement.................................................................................................................................3 Report Approval...................................................................................................................................................... 4 Table o f Contents.................................................................................................................................................... 5 Sum m ary................................................................................................................................................................... 7 Introduction..............................................................................................................................................................9 O b je c tiv e ..................................................................................................................................................................9 Experimental Design...............................................................................................................................................9 Materials and Methods..........................................................................................................................................10 Results and Discussion......................................................................................................................................... 14 Conclusion.............................................................................................................................................................. 16 R e f e re n c e s ............................................ 17 TABLES Table 1 - Summary of Analytical Chemistry D ata........................................................................................ 18 Table 2 - Temperature Measurements............................................................................................................ 19 Table 3 - Light Intensity Measurements........................................................................................................ 20 Table 4 - pH Measurements............................................................................................................................ 22 Table 5 - Mean Cell Densities and Percent Inhibition for Each 24-Hour Interval During the Test.................................................................................................... 23 Table 6 - Mean Areas Under the Growth Curve and Percent Inhibition for Each 24-Hour Interval During the Test.................................................................................................... 24 Table 7 - Mean Growth Rates and Percent Inhibition for Each 24-Hour Interval During the Test...................................................................................................................................25 Wildlife International, Ltd. -6 - Project Number 454A-129 TABLE OF CONTENTS -Continued- Table 8 - EC10, EC50 and EC90 Values Based on Cell Density Over the 96-Hour Exposure Period............................................................................................................................... 26 Table 9 - EC 10, EC50 and EC90 Values Based on Area Under the Growth Curve Over the 96-Hour Exposure Period..........................................................................................................27 Table 10- EC 10, EC50 and EC90 Values Based on Growth Rate Over the 96-Hour Exposure Period.................................................................................................................................28 Table 11- Cell Densities During the Recovery Phase...................................................................................... 29 FIGURES Figure 1 - Negative Control Algal Growth, Expressed in Cell Density, During the 96-Hour Exposure.......................................................................... 30 Figure 2 - Concentration-Response Curve, Expressed in Cell Density..........................................................31 Figure 3 - Cell Density During the Recovery Phase.........................................................................................32 APPENDICES Appendix 1 - Freshwater Algal M ediu m .................................................................................................... 33 Appendix 2 - Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well W ater...........................................................................34 Appendix 3 - The Analysis of PFBS in Freshwater Algal Medium in Support of Wildlife International, Ltd. Project No.: 454A -129..........................................................36 Appendix 4 - Cell Density for Each Replicate Per Treatment Over the 96-Hour Exposure Period...................................................................................................... 52 Appendix 5 - Area Under the Growth Curve for Each Replicate Per Treatment Over the 96-Hour Exposure Period...................................................................................................... 53 Appendix 6 - Growth Rate for Each Replicate Per Treatment Over the 96-Hour Exposure Period.... 54 Appendix 7 - Changes to Protocol................................................................................................................55 Appendix 8 - Personnel Involved in the Study............................................................................................56 Wildlife International, Ltd. -7 - Project Number 454A-129 SANITIZED SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: .. STUDY: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANISMS: CELL DENSITY 72-H O U R EC50: 9 5 % CONFIDENCE LIMITS: 96-H O UR EC 10: 9 5 % CONFIDENCE LIMITS: 96-H O UR E C 50: , 9 5 % CONFIDENCE LIMITS: 96-H O UR E C 90: 9 5 % CONFIDENCE LIMITS: 7 2 -H O U R NOAEC: 9 6 -H O U R NOAEC: SUMMARY 3M Corporation - . Wildlife International, Ltd. Easton, MD 21601 DEC 0 9 2003 454A-129 Perfluorobutanesulfonate, Potassium Salt (PFBS) PFBS: A 96-Hour Toxicity Test with the Freshwater Alga (Selenastrum capricomutum) Negative Control, 285, 563,1077, 2216,4561 and 9478 mg a.i./L Experimental Start - January 22,2001 Exposure Termination - January 26, 2001 Experimental Termination - February 5, 2001 96 Hour Exposure, 6 Day Recovery Freshwater Alga {Selenastrum capricomutum) Wildlife International, Ltd. Easton, Maryland 21601 1 4 6 9 m g a.i./L 81 and 2 8 1 2 m g a.i./L 5 2 8 m g a.i./L, 3 7 5 and 1895 m g a.i./L 2 3 4 7 m g a.i./L 2 0 1 8 and 2 7 0 7 m g a.i./L 7 3 9 0 m g a.i./L 7 2 7 0 and 7 5 0 0 m g a.i./L < 2 8 5 m g a.i./L 1 0 7 7 m g a.i./L Wildlife International, Ltd, SUMMARY (Continued) AREA UNDER THE GROWTH CURVE 72-HOUR E C 50: 9 5 % CONFIDENCE LIMITS: 1 5 9 0 m g a.i./L 3 2 8 and 2 3 7 3 m g a.i./L 96-HOUR EC 10: 9 5 % CONFIDENCE LIMITS: 96-h o u r EC50: 9 5 % CONFIDENCE LIMITS: 96-h o u r EC90: 9 5 % CONFIDENCE LIMITS: 72-H O UR NOAEC: 96-H O UR NOAEC: 3 8 5 m g a.i./L 191 and 9 5 1 m g a.i./L 2 1 4 6 m g a .i./L 1 7 4 8 and 2 7 0 6 m g a.i./L 7 2 9 4 m g a.i./L, 6 8 8 8 and 7 6 7 3 m g a.i./L < 2 8 5 m g a.i./L, 2 8 5 m g a.i./L GROWTH RATE 72-HOUR E C 50: 9 5 % CONFIDENCE LIMITS: 9 6 -h o u r E C 10: 9 5 % CONFIDENCE LIMITS: 96-h o ur EC 50: 9 5 % CONFIDENCE LIMITS: 96-h o u r EC90: 9 5 % CONFIDENCE LIMITS: 72-H O U R N O A E C : 96-H O UR NOAEC : 5 6 6 1 m g a.i./L, 5 2 3 0 and 6 0 6 7 m g a.i./L 1 6 7 4 m g a.i./L, 1 4 8 2 and 1 8 3 9 m g a.i./L 5 7 3 3 m g a.i./L, 5 6 5 9 and 5 8 1 7 m g a.i./L 8 8 4 9 m g a.i./L 8 6 5 6 and 9 0 8 1 m g a.i./L < 2 8 5 m g a.i./L 1 0 7 7 m g a.i./L Project Number 454A-129 Wildlife International, Ltd. -9 - Project Number 454A-129 INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The in-life exposure phase o f the test was conducted from January 22,2001 to February 1,2001. Raw data generated by Wildlife International Ltd and a copy of the final report are filed under Project Number 454A-129 in archives located on the Wildlife International, Ltd. site. OBJECTIVE The objective o f the study was to evaluate the toxicity of perfluorobutanesulfonate, potassium salt (PFBS) to the growth o f the freshwater alga, Selenastrum capricornutum, during a 96-hour exposure period. EXPERIMENTAL DESIGN The freshwater alga, Selenastrum capricornutum, was exposed to a geometric series o f six test concentrations and a negative (culture medium) control under static conditions for 96 hours. Three replicate test chambers were maintained for each treatment and control group. One additional replicate was also maintained for analytical sampling on Day 3 o f the test. In addition, two "abiotic" replicates (test solution without algae) were prepared at the highest test concentration for analytical sampling. Nominal test concentrations were selected in consultation with the Sponsor and were based upon the results of a range finding test. The nominal test concentrations selected were 313, 625, 1250, 2500, 5000 and 10000 mg active ingredient (a.i.)/L. Mean measured test concentrations were determined from samples o f test medium collected from each treatment and the control group at test initiation, at approximately 72 hours, and at test termination. At test initiation, an inoculum of the algal cells was prepared at a concentration of approximately 1.0 X 106 cells/mL. The concentration of algal cells in the inoculum was verified and 1.0 mL was added to each test chamber to achieve a nominal concentration of approximately 1.0 X 104cells/mL. Samples were collected from each replicate test chamber at approximately 24-hour intervals during the test to determine cell densities. Cell densities were measured for each replicate and were used to calculate areas under the growth curve and growth rates. Percent inhibition values relative to the control were calculated for each parameter over the 96-hour exposure period. EC5 0 values for cell density were calculated for each 24 hour interval. EC 10, EC50 and EC90 values were calculated, if possible, based upon cell densities, areas under the growth curve and growth rates for Wildlife International, Ltd. Project Number 454A-129 -lo- SANITIZED DEC 0 9 2003 the 72 and 96 hour intervals. The no-observed-adverse-effect-concentration (NOAEC) was determined, when possible, based upon statistical evaluation of the 72-hour and 96-hour results. At the end of the 96-hour exposure, algistatic effects were differentiated from algicidal effects by performing a recovery phase. MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "PFBS: A 96-Hour Toxicity Test with the Freshwater Alga (Selenastrum capricornutum)". The protocol was based on procedures outlined in the U.S. Environmental Protection Agency Series 850-Ecological Effects Test Guidelines, OPPTS Number 850.5400: Algal Toxicity Tiers la n d II (draft)(l) and OECD Guidelines for Testing o f Chemicals, 201 Algal, Growth Inhibition Test (2). Test Substance The test substance was received from 3M Corporation on June 28, 2000 and was assigned Wildlife International, Ltd. identification number 5292. The test substance was described as a white powder. It was identified as Potassium Perfluoro Butane Sulfonate, AKA . Developmental Product, AKA PFBS, from lot 2. Information provided by the Sponsor indicated a purity of 97.9%. A subsequent revision of the certificate of analysis indicated a purity of 97.3% and an Expiration/Reassessment Date o f January 17, 2002. The test substance was stored at ambient room temperature. Preparation of Test Concentrations Nominal test concentrations were 313,625,1250,2500,5000 and 10000 mg a.i./L. A 2-L primary stock solution was prepared in algal medium at a concentration of 10000 mg a.i./L. The primary stock solution was inverted at least 20 times aid in the solubilization o f the test substance. After mixing, the primary stock solution was proportionally diluted with algal medium to prepare 500 mL of the five other test concentrations. All dilutions were inverted to mix. After mixing, 100 mL of test solution was added to the four replicate test chambers for each treatment group. All test solutions appeared clear and colorless. Test concentrations were corrected for the original reported purity of the active ingredient in the test substance (97.9%). Wildlife International, Ltd. -11 - Project Number 454A-129 Test Organism The freshwater alga, Selenastrum capricornutum, was selected as the test species for this study. The species is representative o f an important group of freshwater algae, and was selected for use in the test based upon a past history o f use and ease o f culturing in the laboratory. Original algal cultures were obtained from the University of Toronto and have been maintained in culture medium at Wildlife International, Ltd., Easton, Maryland. Algal cells used in this test were obtained from Wildlife International, Ltd. cultures that had been actively growing in culture medium for at least two weeks prior to test initiation. The negative control organisms were expected to exhibit exponential growth over the 96-hour exposure period. Exponential growth phase, defined as the period o f growth where the algal cells are dividing at a constant rate, is indicated by the linear section o f the growth curve (Figure 1). Culture Medium The algal cells were cultured and tested in freshwater algal medium (3). Stock nutrient solutions were prepared by adding reagent-grade chemicals to Wildlife International, Ltd. well water purified by reverse osmosis. The test medium was prepared by adding the appropriate volumes o f stock nutrient solutions to purified well water (Appendix 1). The pH o f the medium was adjusted to 7.5 0.1 using 10% HC1; the medium was sterilized by filtration (0.22 pm) prior to use. Analyses were performed at least once annually to determine the concentrations o f selected organic and inorganic constituents in the well water. The results o f analyses performed to measure the concentrations o f selected contaminants in well water used by Wildlife International, Ltd. are presented in Appendix 2. Test Apparatus Test chambers were sterile, 250-mL polycarbonate Erlenmeyer flasks plugged with foam stoppers and contained 100 mL o f test or control algal medium. The test chambers were labeled with the project number, concentration and replicate, and were indiscriminately positioned on two mechanical shaker tables in an environmental chamber designed to maintain the desired test temperature throughout the test. The test chambers were shaken continuously at 100 rpm. Wildlife International, Ltd. -12- Project Number 454A-129 Environmental Conditions Test flasks were held in an environmental chamber at a temperature of 242C. The temperature of a container of water adjacent to the test flasks in the environmental chamber was recorded twice daily during the test using a liquid-in-glass thermometer. The algae were held under continuous cool-white fluorescent lighting throughout the test. The target light intensity was 4300 430 lux. Light intensity was measured at the four comers and the middle o fthe shaker table daily during the test. Light intensity was measured using an SPER Scientific Model 840006 light meter. The pH o f the medium prepared for each treatment and control group was measured at test initiation and termination using a Fisher Accumet Model 915 pH meter. Samples for pH measurement at test initiation were collected from the individual batches o f test solution prepared for each treatment and control group. At test termination, samples of test solution were collected from pooled replicates of the treatment and control group for pH measurement. Algal Growth M easurements Test medium samples were collected from the treatment and control groups for the determination o f algal cell densities. Single samples were collected from each o f the three "biological" replicates per treatment and control group at 24-hour intervals during the 96-hour exposure, and were counted immediately. Cell counts were conducted using a hemacytometer and microscope. Each sample was diluted using an electrolyte solution (Isoton), as needed, to maintain counting accuracy. A small amount o f each sample was loaded onto a hemacytometer and 10 grids were counted. The mean number o f cells per grid was estimated and this value was used to calculate the cell density of the sample. Using thi s technique, the minimum quantifiable cell density was 1.0 X 103 cells/mL. All 96 hour cell count samples were examined microscopically for atypical cell morphology (e.g., changes in cell shape, size or color). Growth o f cells in the replicate test chambers also was assessed for aggregations or flocculations of cells and adherence of the cells to the test chamber. Wildlife International, Ltd. - 13 - Project Number 454A -129 Statistical Analyses 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 (4). Area under the growth curve was calculated for the control and treatment group using the following formula: A = ((N1-N0)/2)(t1)+((N1+N2-2N0)/2)(t2-tI)+((Nn.1+Nn-2N0)/2)(tn-tn.1) where: A = Area N0= Nominal number o f cells/mL at to N] = Mean measured number o f cells/mL at ti N2= Mean measured number of cells/mL at t2 Nn = Mean measured number o f cells/mL at t,, ti = time o f first measurement after beginning of test (hours) t2= time of second measurement after beginning of test (hours) t,, = time o f n* measurement after beginning of test (hours) Growth rates were calculated for the control and each treatment group using the following formula: In (Nq/Nq) Growth Rate = t,, where: N0= Mean measured number o f cells/mL at to Nn = Mean measured number o f cells/mL at tn t,, = Time o f n* measurement after beginning of test (hours) Percent inhibition values were calculated for each treatment group as the percent reduction in cell density, area under the growth curve and growth rate relative to the control replicates. The following formula was used: Percent Inhibition = Mean Cell Densitycomroi - Mean Cell Densitv-iwTM,,. Mean Cell Densitycomroi X10 0 Cell densities, areas under the growth curve and growth rates were analyzed statistically to estimate the EC10, EC50 and EC90 values (i.e., the theoretical test concentrations that would produce a 10, 50 or 90% reduction in each parameter, respectively) and 95% confidence limits at 72 and 96 hours. EC50 values were also calculated for the 24 and 48 hour time intervals. The EC values and 95% confidence limits were calculated by linear interpolation using TOXSTAT Version 3.5 (5). Cell densities, areas under the growth curve and growth rates at 72 and 96 hours were evaluated for normality and homogeneity o f variances using the Shapiro-Wilk's test Wildlife International. Ltd. -14- Project Number 454A-129 and Bartlett's test, respectively. If the data did not meet the assumptions of normality or homogeneity, the data was transformed to correct the condition. The treatment groups were then compared to the control using Dunnett's test. Results o f the statistical analyses were used to determine the NOAEC values. Analytical Chemistry Samples of test medium were collected from the negative control and each treatment group at test initiation, at approximately 72 hours and at test termination to measure concentrations of the test substance. Samples of test medium collected at test initiation were taken from the individual batches of test solution prepared for each treatment and the control group. Samples collected at 72 hours were collected from the additional "analytical" replicates. Samples collected at test termination were a composite of the remaining replicates for each treatment and the control group. The samples were placed in plastic scintillation vials and were analyzed as soon as possible without storage. Analytical procedures used in the analysis o f the samples are presented in Appendix 3. RESULTS AND DISCUSSION Measurement of Test Concentrations Results of analyses to measure concentrations of PFBS in the test solutions are presented in Table 1 and Appendix 3. Nominal concentrations selected for use in this study were 313, 625, 1250, 2500, 5000 and 10000 mg a.i./L. Samples collected at the beginning o f the test had measured concentrations that ranged from 88 to 99% of nominal. Samples collected at 72 hours and test termination had recoveries that ranged from 80 to 92% and 87 to 94% of nominal, respectively. When the values obtained for test initiation, at 72 hours and at test termination were averaged, the mean measured test concentrations were 285, 563, 1077, 2216, 4561 and 9478 mg a.i./L. Measurements Measurements of temperature and light intensity are presented in Tables 2 and 3, respectively. The temperatures ranged from 23.8 to 24.7C and were within the range established for the test (242C). The light intensity dining the exposure phase of the test ranged from 3930 to 4550 lux and was within the desired range for the test (approximately 3870 to 4730 lux) (Table 3). Measurements of pH ranged from 6.9 go 7.1 on Day 0 and ranged from 7.5 to 8.7 at 96 hours (Table 4). The pH of the "abiotic" replicate at test termination was 7.6. Wildlife International, Ltd. - 15- Project Number 454A-129 Data Analyses The effect o f PFBS upon Selenastrum capricornutum was determined by evaluating differences in cell densities, areas under the growth curve and growth rates. Mean values were used to calculate growth inhibition for each 24-hour period. Mean cell densities, areas under the growth curve and growth rates and their corresponding percent inhibition values are presented in Tables 5 ,6 and 7, respectively. Cell density, area under the growth curve and growth rate values for each individual replicate are presented in Appendices 4, 5 and 6, respectively. EC50 values and 95% confidence limits calculated for each 24-hour interval based on cell density, area under the growth curve and growth rate are presented in Tables 8 ,9 and 10, respectively. EC 10 and EC90 values were also calculated for the 72 and 96-hour intervals, where possible. Changes in cell density indicated that exponential growth occurred in the negative control replicates (Figure 1). The coefficient o f variation for control cell density was 4.5%. After 72 hours o f exposure, cell density percent inhibition in the 285,563,1077,2216,4561 and 9478 mg a.i./L treatment groups was 32,33,44, 62, 83 and 99%, respectively. Dunnett's test showed that cell density was significantly reduced in all treatment groups in comparison to the negative control (p <. 0.05). Consequently, the NOAEC for 72 hour cell density was <285 mg a.i./L, the lowest concentration tested. After 96 hours o f exposure, cell density percent inhibition in the 285, 563, 1077, 2216, 4561 and 9478 mg a.i./L treatment groups was -10, 8.3, 5.6, 46, 76 and 100%, respectively. Dunnett's test showed that cell density was significantly reduced in the 2216,4561 and 9478 mg a.i./L treatment groups (p <. 0.05). Consequently, the NOAEC for 96 hour cell density was 1077 mg a.i./L. After 72 hours o f exposure, area under the growth curve percent inhibition in the 285,563,1077,2216, 4561 and 9478 mg a.i./L treatment groups was 2 6 ,3 1 ,4 2 ,6 0 ,8 0 and 98%, respectively. Dunnett's test showed that area under the growth curve was significantly reduced in all treatment groups (p s 0.05). Consequently, the NOAEC for 72 hour area under the growth curve was <285 mg a.i./L, the lowest concentration tested. After 96 hours of exposure, area under the growth curve percent inhibition in the 285, 563, 1077, 2216, 4561 and 9478 mg a.i./L treatment groups was 5 .5 ,1 8 ,2 1 ,5 2 ,7 8 and 99%, respectively. Dunnett's test showed that area under the growth curve was significantly reduced in all treatment groups except the 285 mg a.i./L treatment group (p < 0.05). Consequently, the NOAEC for 96 hour area under the growth curve was 285 mg a.i./L. Wildlife International, Ltd. -16- Project Number 454A-129 After 72 hours o f exposure, growth rate percent inhibition in the 285,563,1077,2216,4561 and 9478 mg a.i./L treatment groups was 8.2, 8.7,13, 21, 38 and 92%, respectively. Dunnett's test showed that growth rate was significantly reduced in all treatment goups ip < 0.05). Consequently, the NOAEC for 72 hour growth rate was <285 mg a.i./L, the lowest concentration tested. After 96 hours of exposure, growth rate percent inhibition in the 285, 563, 1077, 2216, 4561 and 9478 mg a.i./L treatment groups was -1.6, 1.5, 1.0, 10, 24 and 93%, respectively. Dunnett's test showed that growth rate was significantly reduced in the 2216,4561 and 9478 mg a.i./L treatment groups ip s 0.05). Consequently, the NOAEC for 96 hour growth rate was 1077 mg a.i./L. Visual and Microscopic Observations After 96 hours o f exposure, there were no signs o f aggregation, flocculation or adherence o fthe algae to the test flasks in the negative control or any PFBS treatment group. However, algal cells in the 2216, 4561 and 9478 mg a.i./L treatment groups appeared enlarged when compared to the negative control. Reversibility of Growth Inhibition The 9478 mg a.i./L treatment group was maximally inhibited at the end of the 96-hour exposure period. Aliquots of the test solution were diluted with algal medium and cultured for six days. Based on the growth observed in the recovery phase, the effect on algal growth was found to algistatic. Cell densities for the recovery phase are presented in Table 11 and are illustrated graphically in Figure 3. CONCLUSIONS The conclusions of this study were based on the most sensitive endpoint measured (i.e., cell density, area under the growth curve and/or growth rate). The 72-hour EC50, based on cell density, was 1469 mg a.i./L with 95% confidence limits o f 81 and 2812 mg a.i./L. The 96-hour EC 10, based on area under the growth curve, was 3 8 5 mg a.i./L with 95%confidence limits o f 191 and 951 mg a.i./L. The 96-hour EC50, based on area under the growth curve, was 2146 mg a.i./L with 95% confidence limits of 1748 and 2706 mg a.i./L. The 96-hour EC90, based on area under the growth curve was 7294 mg a.i./L with 95% confidence limits o f6888 and 7673 mg a.i./L. The 72-hour NOAEC value, based on cell density, area under the growth curve and growth rate, was <285 mg a.i./L, the lowest concentration tested. The 96-hour NOAEC value, based on area under the growth curve, was 285 mg a.i./L. Based on the algal growth observed during the recovery phase, PFBS was considered to be algistatic, rather than algicidal, at the concentrations tested. Wildlife International, Ltd. - 17- Project Number 454A-129 REFERENCES 1 U.S. Environm ental Protection Agency. 1996. Series 850 -Ecological Effects Test Guidelines {draft), OPPTS Number 850.5400: Algal Toxicity, Tiers I andII. 2 O rganization of Economic C ooperation and Development. 1984. Algal, Growth Inhibition Test. OECD Guideline for Testing o f Chemicals. Guideline 201. Paris. 3 ASTM Stan d ard Guide 1218-90E, Standard Guidefor Conducting Static 96-Hour Toxicity Tests with Microalgae. August 1990. 4 The SAS System for W indows. 1996. Release 6.12, TS Level 0020. SAS Institute Inc., Cary, North Carolina.5 5 W est, Inc. and D.D. Gulley. TOXSTAT Version 3.5. Copyright 1996. Western EcoSystems Technology, Inc., Cheyenne, Wyoming. Wildlife International, Ltd. -18- Project Number 454A-129 Table 1 Summary of Analytical Chemistry Data Sponsor: 3M Corporation Test Substance: PFBS Test Organism: Freshwater alga, Selenastrum capricornutum Dilution Water: Freshwater medium Nominal Concentration Sampling Time Measured Concentration1 Mean Measured Concentration (mg a.i./L) (Hours) (mg a.i./L) (mg a.i./L) Negative Control O2 <LOQ 723 <LOQ 964 <LOQ Percent of Nominal -- 313 0 290 285 91 72 281 96 283 625 1250 0 567 72 563 96 558 563 90 0 1146 1077 86 72 993 96 1091 2500 0 2188 2216 89 72 2209 96 2252 5000 0 4522 4561 91 72 4551 96 4610 10000 0 9859 9478 95 72 9154 96 9421 10000 (Abiotic) 72 96 9467 9501 9484 95 1 Limit o f Quantitation (LOQ) was 100 mg a.i./L. 2 0-hour samples were collected from individual batches of test solution prepared for the treatment and control group at test initiation. 3 72-hour samples were collected from the additional (D) replicate. 4 96-hour samples were composites of test solution collected from each of the three replicates per treatment and control group.____________________________________________________________ Wildlife International, Ltd. - 19- Project Number 454A -129 Table 2 Temperature Measurements Sponsor: Test Substance: Test Organism: Dilution Water: Time (Day) 0 3M Corporation PFBS Freshwater alga, Selenastrum capricornutum Freshwater medium Temperature (C) M easurem ent! Measurement 2 1 24.1 24.1 1 24.1 23.8 2 24.3 24.4 3 24.3 24.3 4 24.5 24.5 5 (Recovery) 24.5 24.5 6 24.5 24.5 7 24.5 24.5 8 24.7 24.1 9 24.3 24.4 10 24.4 24.5 1 With the exception o f Day 10, temperature measurement 2 was taken at least 4 hours after measurement 1. Wildlife International, Ltd, -20- Project Number 454A-129 Table 3 Light Intensity Measurements Shaker Table AQL #6 Sponsor: Test Substance: Test Organism: Dilution Water: Test Day 0 3M Corporation PFBS Freshwater alga, Selenastrum capricomutum Freshwater medium Light Intensity Measurements (lux) No. 1 No. 2 No. 3 No. 4 3960 4010 4490 3990 1 3980 4030 4400 4350 2 3930 4030 4120 4050 3 3960 4110 4380 4100 4 3990 4230 4190 4200 5 (Recovery) 4010 4020 3990 3980 6 3980 4100 4240 4000 7 4970 4990 4130 3980 8 3880 3890 4450 3950 9 3890 3910 3960 4380 10 4110 4060 4410 3890 No. 5 4370 4260 4550 4280 4030 4020 4030 4160 3910 3940 3970 Wildlife International, Ltd, -21 - Project Number 454A-129 ' Table 3 (Continued) Light Intensity Measurements Shaker Table AQL #3 Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Freshwater alga, Selenastrum capricornutum Freshwater medium Test uay No. 1 Light Intensity Measurements (lux) No. 2 No. 3 No. 4 0 4030 3990 4000 4090 1 4000 4610 3890 4040 2 3940 3940 4470 3910 3 3960 4180 4390 4230 4 3980 4060 4410 4180 No. 5 4070 4410 4070 3930 3890 Wildlife International, Ltd. Project Number 454A-129 Table 4 pH Measurements Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Freshwater alga, Selenastrum capricomutum Freshwater medium Mean Measured Concentration (mg a.i./L) pH Measurements 0 Hours' Negative Control 6.9 96 Hours2 8.7 285 6.9 8.7 563 7.0 8.7 1077 7.0 8.7 2216 7.0 8.7 4561 7.1 8.5 9478 7.1 7.5 9484 (Abiotic) --. 7.6 1 0-hour samples were collected from the batches of test solution prepared for the treatment and control groups at test initiation. 2 96-hour samples were collected from the pooled replicates per treatment and control group. - 23- Table 5 Mean Cell Densities and Percent Inhibition for Each 24-Hour Interval During the Test1 Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Freshwater alga, S e le n a stru m c a p r ic o m u tu m Freshwater medium Mean Measured Concentration (mg a.i./L) 24 Hours Mean Cell Percent Density Inhibition Negative Control 31,000 -- 48 Hours Mean Cell Density Percent Inhibition 179,000 - 72 Hours Mean Cell Density Percent Inhibition 1,015,000 -- 285 30,333 2.2 162,333 9.3 688,3332 32 563 1077 27,000 on I 13 33 138,667 ! 19 000 23 1A /T 673,3332 / 1 jV /U / 34 AA TT 2216 17,333 44 95,333 47 385,0002 62 4561 15,333 51 62,333 65 175.3332 83 9478 12,000 61 16,000 91 14,6672 1 Values calculated using SAS 6.12. Manual calculations may differ slightly Indicates a significant difference from the negative control at 72 hours using Dunnett's test (p <. 0.05). 3 Indicates a significant difference from the negative control at 96 hours using Dunnett's test (p <. 0.05). 99 Project Number 454A-129 96 Hours Mean Cell Density Percent Inhibition 3,560,000 - 3,920,000 -10 3,265,000 *i<r\ r\r\r\ 8.3 < 1,930,0003 860,0003 15,333s 46 76 100 Project Number 454A-129 - 24- Table 6 Mean Areas Under the Growth Curve and Percent Inhibition for Each 24-Hour Interval During the Test1 Sponsor: Test Substance: Test Organism: 3M Corporation PFBS Freshwater alga, Selenastrum capricomutum Dilution Water: Freshwater medium Mean Measured 0 - 2 4 Homs 0 - 48 Hours Test Concentration (mg a.i./L) Mean Area Percent Inhibition Mean Area Percent Inhibition Negative Control 252,000 -- 2,532,000 - . 0 - 7 2 Homs Mean Area Percent Inhibition 16,620,000 -- 285 244,000 3.2 2,316,000 8.5 12,284,0002 26 563 9*'nva*)nvovwn 19 1,952,000 23 11j45 6-j00*02 31 1077 128,000 49 1,564,000 38 9,612,0002 42 2216 88,000 65 1,200,000 53 6,724,0002 60 4561 64,000 75 756,000 70 3,368,0002 80 9478 48,000 81 148,000 94 276,0002 'Values calculated using SAS 6.12. Manual calculations may differ slightly. 2Indicates a significant difference from the negative control at 72 hours using Dunnett's test (p <. 0.05). 3Indicates a significant difference from the negative control at 96 hours using Dunnett's test (p 0.05). 98 0 - 9 6 Hours Mean Percent Area Inhibition 71,280,000 -- 67,344,000 5.5 58 476 0003 18 56,552,0003 21 34,264,0003 52 15,552,0003 78 396,0003 99 Project Number 454A-129 -25 - Table 7 Mean Growth Rates and Percent Inhibition for Each 24-Hour Interval During the Test1 Sponsor: 3M Corporation Test Substance: PFBS Test Organism: Freshwater alga, Selenastrum capricomutum Dilution Water: Freshwater medium Mean Measured Test Concentration (mg a.i./L) 0 - 2 4 Hours Mean Percent Growth Rate Inhibition 0 - 4 8 Hours Mean Percent Growth Rate Inhibition Negative Control 0.0445 -- 0.0601 - ___ 0 - 72 Hours Mean Percent Growth Rate Inhibition 0.0640 - 285 0.0458 -3.0 0.0580 3.4 0.05872 8.2 53 va/.Au*-mtu n/. jn 0.0547 Qu.Qy 0.05842 8.7 1077 0.0298 33 0.0515 14 0.05602 13 2216 0.0225 49 0.0469 22 0.05062 21 4561 0.0173 61 0.0377 37 0.03982 38 9478 0.0130 71 0.0095 84 0.00522 1Values calculated using SAS 6.12. Manual calculations may differ slightly. 2Indicates a significant difference from the negative control at 72 hours using Dunnett's test {p <. 0.05). 3Indicates a significant difference from the negative control at 96 hours using Dunnett's test (p z 0.05). 92 0 - 9 6 Homs Mean Percent Growth Rate Inhibition 0.0612 - 0.0622 -1.6 0.0603 1.5 0.0606 1.0 0.05483 10 0.04643 24 0.00433 93 Project Number 454A-129 - 26- Table 8 EC 10, EC50 and EC90 Values Based on Cell Density Over the 96-Hour Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Freshwater alga, Selenastrum capricomutum Freshwater medium Time EC10 (mg a.i./L) 95% Confidence Limits (mg a.i./L) 24 Hours Not Determined - 48 Hours 72 Hours Not Determined <285 1 96 Hours 528 375 and 1895 'Confidence limits could not be calculated with the data obtained. EC50 (mg a.i./L) 4366 2631 1469 2347 95% Confidence Limits (mg a.i./L) <0.0 and 12305 1644 and 3927 81 and 2812 2018 and 2707 EC90 (mg a.i./L) Not Determined Not Determined 6821 7390 95% Confidence Limits (mg a.i./L) 5639 and 8187 7270 and 7500 Project Number 454A-129 - 27- Table 9 EC 10, EC50 and EC90 Values Based on Area Under the Growth Curve Over the 96-Hour Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Freshwater alga, Selenastrum capricomutum Freshwater medium Time EC10 (mg a.i./L) 95% Confidence Limits (mg a.i./L) 24 Hours Not Determined - 48 Hours 72 Hours Not Determined <285 1 96 Hours 385 191 and 951 'Confidence limits could not be calculated with the data obtained. EC50 (mg a.i./L) 1,134 2,009 1,590 2,146 95% Confidence Limits (mg a.i./L) <0.0 and 4774 24 and 3992 328 and 2373 1748 and 2706 EC90 (mg a.i./L) Not Determined Not Determined 7274 7294 95% Confidence Limits (mg a.i./L) - - 6231 and 8204 6888 and 7673 - 28- Table 10 EC 10, EC50 and EC90 Values Based on Growth Rate Over the 96-Hour Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Freshwater alga, Selenastnim capricomutum Freshwater medium Time EC10 (mg a.i./L) 95% Confidence Limits (mg a.i./L) 24 Hours Not Determined - EC50 (mg a.i./L) 2195 48 Hours Not Determined - 5892 72 Hours 734 <0.0 and 2207 5661 96 Hours 1674 1482 and 1839 'Confidence limits could not be calculated with the data obtained. 5733 95% Confidence Limits (mg a.i./L) <0.0 and 8922 4683 and 7385 5230 and 6067 5659 and 5817 EC90 (mg a.i./L) Not Determined Not Determined 9303 8849 Project Number 454A-129 95% Confidence Limits (mg a.i./L) - 8571 and 9664 8656 and 9081 Wildlife International, Ltd, -29- Project Number 454A-129 Table 11 Cell Densities During the Recovery Phase Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Freshwater alga, Selenastrum capricornutum Freshwater medium Mean Measured Test Concentration (mg a.i./L) Day 0 Cell Densities (Cells/mL2) Day 3 Negative Control . 18,000 1,785,000 Day 6 6,140,000 94781 2,000 3,000 980,000 1 The treatment group was diluted to a concentration of the test substance that theoretically would not inhibit growth. 2 Due to the method used to prepare recovery phase test solutions, initial cell densities were not equivalent throughout the treatment groups. Wildlife International, Ltd. -30- Project Number 454A-129 Figure 1. Negative Control Algal Growth, Expressed in Cell Density, During the 96-Hour Exposure. Mean Cell Density (Cells/mL) *--N egd veG C rid Wildlife International, Ltd. -31 - Project Number 454A-129 Figure 2. Concentration-Response Curve, Expressed in Cell Density M ean C ell D en sity (C els/tnL ) Exposive D uation (Hours) --a-- Ffegitive Gbntrol -------285mgaI./L --I-- 563 ipgaL/L --0-- 1077 ipgal./L ---- 2216 ipgaL/L --o-- 4561 mgaL/L -- 9478irgaI./L Wildlife International, Ltd. -32- Project Number 454A-129 Figure 3. Cell Density During the Recovery Phase Wildlife International, Ltd. -33- Project Number 454A-129 Appendix 1 Freshwater Algal Medium1 Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Freshwater alga, Selenastrum capricornutum Freshwater medium Compound Nominal Concentration MgCl2*6H 20 CaCl22H 20 H3B3 MnCl2*4H 20 ZnCl2 FeCl36H 20 CoC12*6H 20 Na2M o 0 4*2H 20 CuCl22H 20 N a 2E D T A * 2 H 20 NaN03 M gS047H 20 k 2h p o 4 NaHC03 12.16 4.41 0.1855 0.4154 3.27 0.1598 1.428 7.26 0.012 0.300 25.50 14.70 1.044 15.0 mg/L mg/L mg/L mg/L M-g/C mg/L pg/L pg/L pg/L mg/L mg/L mg/L mg/L mg/L 'The pH was adjusted to 7.5 0. 1 using 10% HC1. Wildlife International, Ltd. -34- Project Number 454A-129 Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Component Measured Concentration Component Measured Concentration Pesticides and Organics Aclonifen Alachlor Ametryn Atrazin Azinphos-ethyl Azinphos-methyl Azoxystrobin Bifenthrin Bioallethrin Bitertanol Bromacil Bromophos Bromophos-ethyl Broompropylaat Bupirimaat Carbaryl Carbofuran Carboxin Chlorfenvinphos Chloridazon Chlorpropham Chiorpyriphos Chlorpyriphos-methyl Chlorthalonil Coumaphos Cyanazin Cyfluthrin Cypermethrin Cyproconazole Deltamethrin Demeton Demeton-o Desethylatrazin Desisopropyiatrazin Desmetryn Diazinon Dichlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid <0.03 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.04 pg/L <0.08 pg/L <0.25 pg/L <0.05 pg/L <0.05 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.03 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.04 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.25 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.03 pg/L <0.02 pg/L <0.01 pg/L <0.03 pg/L Dimethomorf Disulfoton DMST Dodemorf Endosulfan-a Endosulfan-|3 Endosulfan-sulfaat Epoxiconazole Eptam Esfenvaleraat Ethion Ethofumesaat Ethoprophos Etridiazole Etrimphos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb Fenpiclonil Fenpropathrin Fenpropimorf Fenthion Fenvaleraat Fluazifop-butyl Fluoroglycofen-ethyl Fluroxypyr-meptyl Flutolanil Fonophos Furalaxyl Heptenophos Imazalil Iprodion Kresoxim-methyl Lenacil Lindane Malathion Metalaxyl Metamitron Metazachlor Methidathion <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0.03 pg/L <0.03 pg/L <0.05 pg/L <0.25 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 ur/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15,1999. Wildlife International, Ltd, -35- Project Number 454A-129 Appendix 2 (Continued) Analyses of Pesticides., Organics and Metals in Wildlife International, Ltd. Well Water1 Pesticides And Organics (Page 2) Component Measured Concentration Component Measured Concentration Dichlorvos Dicofol Diethyltoluamide Difenoconazole Dimethoate Paclobutazole Parathion Parathion-methyl Penconazole Pendimethalin Permethrin-cis Permethrin-trans Phosalon Phosmet Phosphamidon-cis Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazin Propham Propiconazool Propoxur Propyzamide Prosulfocarb Pvrazophos ' <0.01 pg/L <0.25 pg/L <0.02 pg/L <0.03 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.03 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.03 pg/L <0.02 pg/L <0.02 pg/L <0.03 pg/L Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl Pyrifenox-1 Pyrifenox-2 Pyrimethanil Quizalofop-ethyl Simazin Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbutylazin Tetrachlorvinphos Tetrahydroftaalimide Tetramethrin Thiabendazole Thiometon Tolclophos-methyl Tolylfluanid Triadimefon Triadimenol Triallaat Triazophos Trifluralin Vamidothion Vinchlozolin <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.05 pg/L <0.04 pg/L <0.01 pg/L <0.04 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L Metals Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 11.0 mg/L 18.0 mg/L 29 mg/L <0.015 mg/L 1.1 mg/L <0.02 mg/L <0.1 pg/L <0.2 pg/L <0.5 pg/L <0.2 pg/L Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium <1.1 pg/L <0.7 pg/L <0.25 pg/L <0.3 pg/L <0.2 pg/L <0.1 pg/L <0.5 pg/L <0.025 pg/L <0.5 pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15,1999. Wildlife International, Ltd. 36- Appendix 3 Project Number 454A-129 THE ANALYSIS OF PFBS IN FRESHWATER ALGAL MEDIUM IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-103 W il d l if e In t e r n a t io n a l l t d . -37 PROJECT NO.: 454A-129 REPORT APPROVAL - SPONSOR: 3M Corporation TITLE: PFBS. A 96-HOUR TOXICITY TEST with the FRESHWATER ALGA (,Selenastrum capricornutum) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-129 3M ENVIRONMENTAL LAB PROJECT NUMBER: EOO-1429 PRINCIPAL INVESTIGATOR: 6 3 - 0 0 - oi DATE MANAGEMENT: Willard B. Nixon, Ph.D. / Director, Analytical Chemistry _____________ J /U /u DATE W il d l if e In t e r n a t io n a l ltd . PROJECT NO.: 454A-129 - 3 8 - SANITIZED T, . Introduction DEC 0 9 2003 Freshwater medium samples were collected from an acute toxicity study designed to determine the effects of PFBS (Perfluoro Butane Sulfonate, Potassium Salt) to the freshwater alga (Selenastrum capricornutum). This study was conducted by Wildlife International, Ltd. and identified as Project Number 454A- 129. The analyses o f these water samples were performed at Wildlife International, Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). Samples were received for analysis on January 22, 25 and 26, 2001 and were analyzed on each sample receipt day. Analytical Standard The analytical standard was received from 3M Environmental Technology and Safety Services on March 27, 2000, assigned Wildlife International, Ltd. Identification number 5216, and stored under ambient conditions. The analytical standard, a white powder, was identified as: Potassium Perfluorobutane Sulfonate expiration date: March 2010. The analytical standard was further identified with the 3M Environmental Laboratory test control and reference number ' The test substance had a reported purity of 97.90%. A subsequent revision of the certificate of analysis indicated a purity o f 97.3% and an Expiration/Reassessment Date o f January 17, 2002. The analytical standard was the same material and lot number as the test substance (Wildlife International, Ltd. Identification number The analytical standard was used to prepare calibration and matrix fortification samples. Analytical Method Freshwater medium samples were analyzed according to the method entitled "Analytical Method Validation for the Determination o f Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Saltwater and Algal Media" (Wildlife International, Ltd. Project No. 454C-117). Samples were diluted in a 50% methanol : 50% NANOpure water solution so that they fell within the calibration range o f the PFBS methodology. Aliquots o f the dilutions were transferred to autosampler vials and submitted for analysis by direct injection. Concentrations of PFBS in freshwater medium samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) interfaced with a Perkin-Elmer API 100LC mass spectrometer (single quadrupole) operated in selective ion monitoring (SIM) detection mode. The mass spectrometer was equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separations were achieved using a Keystone W il d l if e In t e r n a t io n a l l t d . -39- PROJECT NO.: 454A-129 PRISM RP column (30 mm x 1.5 mm, 3-pm particle size) fitted with a Keystone Javelin Ci8 Guard Cartridge (20 mm x 2 mm). The instrument parameters are summarized in Table 1 and a method flowchart is provided in Figure 1. Primary and Secondary Stock Solutions. All primary and secondary stock preparations were adjusted for the purity o f the analytical standard (97.90%). A 10.0 mg a.i./mL primary stock solution o f PFBS in methanol was prepared by weighing 1.024 g o f the analytical standard and bringing to a final volume o f 100 mL with methanol. Secondary stock solutions (1000, 100, 10.0, 1.00, and 0.100 mg a.i./L) of PFBS in methanol were prepared by serial volumetric dilution from the primary stock. Calibration Standards and Calibration Curves Calibration standards were prepared in 50:50 methanol: NANOpure water by appropriate dilutions of the 10.0 mg a.i./L stock solution o f PFBS in methanol. The calibration standards o f PFBS, ranging in concentration from 0.0100 to 0.0500 mg a.i./L, were analyzed with each sample set. Five calibration standards (different concentrations) were analyzed with the samples. The calibration standard series was injected at the beginning and end o f each run, and one standard was injected, at a minimum, after every five samples. Linear regression equations were generated using the peak area responses versus the respective concentrations o f the calibration standards. A typical calibration curve is presented in Figure 2. The concentration o f PFBS in the samples was determined by substituting the peak area responses into the applicable linear regression equation. Representative ion chromatograms o f low and high calibration standards are presented in Figures 3 and 4, respectively. Limit of Quantitation The method limit o f quantitation (LOQ) for these analyses was set at 100 mg a.i./L calculated as the product o f the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor o f the matrix blank samples (10000). Matrix Blank and Fortification Samples Three matrix blank samples were analyzed to determine possible interference. No interferences were W il d l if e In t e r n a t io n a l l t d . -40- PROJECT NO.: 454A-129 observed at or above the LOQ during samples analyses (Table 2). A representative ion chromatogram of a matrix blank is presented in Figure 5. Freshwater medium was directly fortified (i.e. without use o f carrier solvent) with PFBS at 150, 2500 and 12000 mg a.i./L and analyzed concurrently with the samples to determine the mean procedural recovery (Table 2). Sample concentrations were not corrected for the mean procedural recovery o f 93.5%. A representative ion chromatogram o f a matrix fortification is presented in Figure 6. Example Calculations Sample number 454A-129-4, nominal concentration o f 1250 mg a.i./L in freshwater medium. First Initial Volume: 0.100 mL Calibration curve equation: First Final Volume: 100 mL Slope: 43223072 Second Initial Volume; 0.250 mL Intercept: 147443.62500 Second Final Volume: 10.0 mL Curve regression weighted 1/x Dilution Factor: 40000 PFBS Peak Area: 1385658 peak area - (y-intercept) PFBS (mg a.i./L) measured at instrument slope PFBS (mg a.i./L) in sample = PFBS measured at instrument (mg a.i./L) x dilution factor 1385658 - 147443,62500 43223072 x 40000 = 1146 PFBS (mg a.i./L) in sample Percent o f Nominal Concentration = PFBS (mg a.i./L) nominal x 100 _ 1146 1250 X 100 = 91.7% W il d l if e In t e r n a t io n a l l t d . -41 - PROJECT NO.: 454A-129 Calculated with HPLC/MS instrument software: MacQuan, version 1.6. Sample Analysis RESULTS Freshwater medium samples were collected from an acute toxicity study with the freshwater alga (Selenastrum capricornutum) at test initiation, January 22, 2001 (Day 0), on January 25, 2001 (Day 3), and at test termination, January 26, 2001 (Day 4). The measured concentrations o f PFBS in the samples collected at initiation o f exposure o f the test organisms (Day 0) ranged from 87.5 to 98.6% o f the nominal concentrations. Samples collected at Day 3 had a measured concentration range o f 79.5 to 94.7% of nominal values. Samples collected at test termination (Day 4) had a measured concentration range o f 87.3% to 95.0% o f nominal values (Table 3). Samples from the abiotic 10000 mg a.i./L treatment group were comparable to samples from the 10000 mg a.i./L treatment group with the freshwater alga present (Table 3). A representative ion chromatogram o f a test sample is shown in Figure 7. W il d l if e In t e r n a t io n a l l t d . -42- PROJECT NO.: 454A-129 Table 1 Typical HPLC/MS Operational Parameters INSTRUMENT: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer operated in Selective Ion Monitoring (SIM) Mode ION SOURCE: Perkin-Elmer TurboIonSpray ANALYTICAL COLUMN: Keystone PRISM RP (30 mm x 1.5 mm, 3-pm particle size) GUARD COLUMN: Keystone Javelin C u cartridge (20 mm x 2 mm) OVEN TEMPERATURE: 40C STOP TIME: 3.00 min FLOW RATE: MOBILE PHASE: INJECTION VOLUME: 200 pL/min 25% NANOpure W ater with 0.1% Ammonium Formate: 75% Methanol 5.0 pL PFBS PEAK RETENTION TIME: Approximately 2.2 minutes PFBS M O N ITO R ED M A SS. 299.0 amu W il d l if e In t e r n a t io n a l ltd. -43 - PROJECT NO.: 454A-129 Table 2 Matrix Blanks and Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-129-) MAB-1 MAB-2 MAB-3 Sample Type Matrix Blank Matrix Blank Matrix Blank Concentrations of PFBS (mg a.i./L) Fortified 0.00 0.00 0.00 M easu red *1 <LOQ2 <LOQ <LOQ Percent Recovered -- -- - MAS-1 MAS-4 MAS-7 Matrix Fortification Matrix Fortification Matrix Fortification 150 150 150 141 138 136 93.8 92.1 90.9 MAS-2 MAS-5 MAS-8 Matrix Fortification Matrix Fortification Matrix Fortification 2500 2500 2500 2338 2385 2302 93.5 95.4 92.1 MAS-3 MAS-6 MAS-9 Matrix Fortification Matrix Fortification Matrix Fortification 12000 12000 12000 11460 11520 11050 , 95.5 96.0 92.1 _______________________________________________ Mean = 93.5 Standard Deviation =1.82 CV =1.95% N=9 1Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2The limit o f quantitation (LOQ) was 100 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor o f the matrix blank samples (10000).______ W il d l if e In t e r n a t io n a l l t d . -44- PROJECT NO.: 454A-129 Table 3 Measured Concentrations of PFBS in Freshwater Medium Samples from a Freshwater Alga Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 0.0 313 625 1250 2500 5000 10000 10000 (Abiotic) Sample Number (454A-129-) 1 8 16 2 9 17 3 10 18 4 11 19 5 12 20 6 13 21 7 14 22 15 23 Sampling Time (Day) 0 3 4 0 3 4 0 3 4 0 3 4 0 3 4 0 3 4 0 3 4 3 4 PFBS Measured Concentration1 (mg a.i./L) < LOQ2 < LOQ < LOQ 290 281 283 567 563 558 1146 993 1091 2188 2209 2252 4522 4551 4610 9859 9154 9421 9467 9501 Percent of Nominal1 -- -- -- 92.6 89.9 90.3 90.7 90.2 89.3 91.7 79.5 87.3 87.5 88.3 90.1 90.5 91.0 92.2 98.6 91.5 94.2 94.7 95.0 1 Measured and Percent o f Nominal values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2 The limit o f quantitation (LOQ) was 100 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor o f the matrix blank samples (10000), W il d l if e In t e r n a t io n a l l t d . -45 - PROJECT NO.: 454A-129 M ETHOD OUTLINE FOR THE ANALYSIS OF PFBS IN FRESHW ATER MEDIUM Prepare each matrix fortification sample by weighing the requisite amount o f PFBS test substance on an analytical balance and transferring directly into a Class A volumetric flask partially filled with freshwater medium. Rinse weighing paper and the sides o f the flask with repeat freshwater medium rinses. Swirl the flask to dissolve the test substance and then bring to final volume with freshwater medium. Sonicate, as appropriate, and mix with several repeat inversions. The matrix blank is . unfortified freshwater medium. i Centrifuge samples, as appropriate, at approximately 2500 rpm for approximately 15 minutes. 4 Prepare appropriate dilutions o f study and QC samples to within the calibration range o f the PFBS methodology: Partially fill Class A volumetric flasks with 50% methanol : 50% NANOpure water dilution solvent. Add the appropriate volume o f sample and bring to volume with dilution solvent. Perform secondary dilutions as necessary. Process matrix blank samples using the same dilution and aliquot volume as for the lowest fortification level. Mix well by several repeat inversions. i Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis of PFBS in freshwater medium. W il d l if e In t e r n a t io n a l l t d . -46- PROJECT NO.: 454A-129 Figure 2. A typical calibration curve for PFBS. Slope = 43223072; Intercept = 147443.62500; r = 0.9986. W il d l if e In t e r n a t io n a l l t d . -47- PROJECT NO.: 454A-129 intensity: 500000 cps Figure 3. A representative ion chromatogram o f a low-level (0.0100 mg a.i./L) PFBS standard. W il d l if e In t e r n a t io n a l l t d . -48 - PROJECT NO.: 454A-129 100 90 80 70 60 50 40 30 20 10 22 43 63 78 Ot i i I I ' I j "7 31 61 0.52 1.02 ; 91 1.52 intensity: 500000 cps Scan Time Figure 4. A representative ion chromatogram o f a high-level (0.0500 mg a.i./L) PFBS standard. W il d l if e In t e r n a t io n a l l t d . -49- PROJECT NO.: 454A-129 intensity: 500000 cps m.V Figure 5. A representative ion chromatogram o f a matrix blank sample (454A-129-MAB-1). The arrow indicates the retention time o f PFBS. W il d l if e In t e r n a t io n a l l t d . -50- PROJECT NO.: 454A-129 intensity: 500000 cps 100- 9^ 80 70 60 50 40 30 20 10- 15 33 44 66 84 108 " i i-- i-- i-- i-- i-- i-- i-- i-- i-- t 1 31 61 91 121 0.52 1.02 1.52 2.03 Scan Time Figure 6. A representative ion chromatogram o f a matrix fortification sample (454A-129-MAS-2, nominal PFBS concentration o f 2500 mg a.i./L, dilution factor = lOOOOOx). W il d l if e In t e r n a t io n a l l t d . -51 - PROJECT NO.: 454A-129 intensity: 500000 cps Figure 7. A representative ion chromatogram o f a test sample (454A-129-4, nominal PFBS concentration o f 1250 mg a.i./L, dilution factor = 40000x). Wildlife International, Ltd. -52- Project Number 454A-129 Appendix 4 Cell Density for Each Replicate Per Treatment Over the 96-Hour Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS . Freshwater alga, Selenastrum cctpricornutum Freshwater medium Mean Measured Concentration Cell Densities (Cells/mL)1 (mg a.i./L) Replicate 24 Hours 48 Hours 72 Hours Negative Control A 21,000 168,000 1,200,000 B 25,000 184,000 1,045,000 C 47,000 185,000 800,000 96 Hours 3,735,000 3,420,000 3,525,000 285 A 29,000 157,000 625,000 4,005,000 B 26,000 152,000 730,000 3,930,000 C 36,000 178,000 710,000 3,825,000 563 A 25,000 136,000 645,000 3,120,000 B 30,000 126,000 625,000 3,195,000 C 26,000 154,000 750,000 3,480,000 1077 A 24,000 133,000 720,000 3,750,000 B 17,000 106,000 465,000 3,180,000 C 21,000 118,000 530,000 3,150,000 2216 A 19,000 88,000 430,000 2,025,000 B 14,000 97,000 330,000 1,845,000 c 19,000 101,000 395,000 1,920,000 4561 A 12,000 51,000 171,000 850,000 B 17,000 80,000 188,000 870,000 C 17,000 56,000 167,000 860,000 9478 A 17,000 13,000 16,000 19,000 B 4,000 15,000 16,000 15,000 C 15,000 20,000 12,000 12,000 1 The initial cell density of the stock culture was determined and an inoculum volume was administered to each test chamber to yield a cell density of approximately 10,000 cells/mL at test initiation (0 hours). Wildlife International, Ltd. -53- Project Number 454A-129 Appendix 5 Area Under the Growth Curve for Each Replicate Per Treatment Over the 96-Hour Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Freshwater alga, Selenastrum capricornutum Freshwater medium Mean Measured Concentration (mg a.i./L) __________ Cumulative Area Under the Growth Curve Replicate 0 - 2 4 Hours 0 - 4 8 Hours 0 - 7 2 Hours 0 - 9 6 Hours Negative Control A B C 132,000 180,000 444,000 2,160,000 2,448,000 2,988,000 18,336,000 16,956,000 14,568,000 77,316,000 70,296,000 66,228,000 285 A 228,000 2,220,000 11,364,000 66,684,000 B 192,000 2,088,000 12,432,000 68,112,000 C 312,000 2,640,000 13,056,000 67,236,000 563 A 180,000 1,872,000 11,004,000 55,944,000 B 240,000 1,872,000 10,644,000 56,244,000 C 192,000 2,112,000 12,720,000 63,240,000 1077 A 168,000 1,812,000 11,808,000 65,208,000 B 84,000 1,320,000 7,932,000 51,432,000 C 132,000 1,560,000 9,096,000 53,016,000 2216 A 108,000 1,152,000 7,128,000 36,348,000 B 48,000 1,140,000 6,024,000 31,884,000 C 108,000 1,308,000 7,020,000 34,560,000 4561 A 24,000 540,000 2,964,000 14,976,000 B 84,000 1,008,000 3,984,000 16,440,000 C 84,000 720,000 3,156,000 15,240,000 9478 A 84,000 204,000 312,000 492,000 B0 0 132,000 264,000 C 60,000 240,000 384,000 432,000 Wildlife International, Ltd. -54- Project Number 454A-129 Appendix 6 Growth Rate for Each Replicate Per Treatment Over the 96-Hour Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Freshwater alga, Selenastrum capricornutum Freshwater medium Mean Measured Concentration (mg a.i./L) Growth Rate Replicate 0 - 2 4 Hours 0 - 4 8 Hours 0 - 72 Hours Negative Control A B C 0.0309 0.0382 0.0645 0.0588 0.0607 0.0608 0.0665 0.0646 0.0609 0 - 9 6 Hours 0.0617 0.0608 0.0611 285 A 0.0444 0.0574 0.0574 0.0624 B 0.0398 0.0567 0.0596 0.0622 C 0.0534 0.0600 0.0592 0.0619 563 A 0.0382 0.0544 0.0579 0.0598 B 0.0458 0.0528 0.0574 0.0601 C 0.0398 0.0570 0.0600 0.0610 1077 A 0.0365 0.0539 0.0594 0.0617 B 0.0221 0.0492 0.0533 0.0600 C 0.0309 0.0514 0.0551 0.0599 2216 A 0.0267 0.0453 0.0522 0.0553 B 0.0140 0.0473 0.0486 0.0544 C 0.0267 0.0482 0.0511 0.0548 4561 A 0.0076 0.0339 0.0394 0.0463 B 0.0221 0.0433 0.0407 0.0465 C 0.0221 0.0359 0.0391 0.0464 9478 A 0.0221 0.0055 0.0065 0.0067 B 0.000 0.0084 0.0065 0.0042 C 0.0169 0.0144 0.0025 0.0019 Wildlife International, Ltd. -55 - Project Number 454A-129 Appendix 7 Changes to Protocol This study was conducted in accordance with the approved Protocol with the following changes: 1. The protocol was amended to add the proposed experimental start and termination dates and test concentrations. 2. The protocol was amended to provide the correct freshwater algal medium constituents. 3. Two light intensity measurements during the recovery phase were outside of the acceptable range. Wildlife International, Ltd. -56- Project Number 454A-129 Appendix 8 Personnel Involved in the Study The following key personnel were involved in the conduct or management o f this study: 1. Henry 0 . Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Director, Analytical Chemistry 3. Raymond L. VanHoven, Ph.D., Scientist 4. Kurt R. Drottar, Senior Biologist 5. Cary A. Sutherland, Laboratory Supervisor 6. Debbie Desjardins, Biologist