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13) OECD 201-OPPTS 850.5400, Algal toxicity- 96-Hour toxicity test with the freshwater alga, 454A-129 PFBS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA (Selenastrum capricornutum) SANITIZED L 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 E3ffects Test Guidelines OPPTS Number 850.5400 and OECD Guideline 20 1 AUTHORS: Kurt R llrottar Henry 0.KruLegerP, h.D. STUDY INITIATION DATE: October 6,2000 STUDY COMPLETION .DATE: March 20,200 1 Submitted to 3M Corporation Environmental laboratory Building .2-3E-09 935 Bush Avenue St. Paul, Minnesota 55 144 Wildlife International, Ltd. 8598 Commerce Drive Easton, M q l a n d 21601 (410) 822-8600 Page 1 of 56 wildlifeInternational, Ltd. - '.>-- Proiect Number 454A- 129 SANITIZED GOOD LABOMTORY PRACTICE COMPLIANCE STATEMENT D E C 0 9 2003 SPONSOR: 3M Corporation TITLE: PFBS: A 96-Hour Toxicity Test with the Freshwater Alga (SeIenustrum cupricomutum) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-129 STUDY COMPLETION: March 20,200 1 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, Notlfication No. 3850, Agricultural Production Bureau, 10 August 1984 with the following exceptions: The test substancewas not characterized in compliancewith 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 of storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Kurt R Drottar Senior Biologist -a W 3j ! B / d f DATE SPONSOR: DATE . wildlifeInternational, h d . -3- Project Number 454A-129 QUALITY ASSUFWWE STATEMENT This study was examined for compliance with GoodLaboratory Practice Standards aspublished by theUS. 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, Agncultural 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 Quality Aksurance Program &pervisor wildlife International, Ltd. Project Number 454A-129 REPORT APPROVAL SPONSOR 3M Corporation TITLE: PFBS: A 96-Hour Toxicity Test with the Freshwater Alga (Selenustrumcupricornutum) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A- 129 STUDY DIRECTOR: Kurt R Drottar Senior Biologist DATE MANAGEMENT: Director, Aquatic Toxicology and Non-Target Plants WildltfeInternational. Ltd. Proiect Number 454A-129 TABLE OF CONTENTS TitleKover Page.............................................................................................................................................. 1 Good Laboratory Practice Compliance Statement.......................................................................................... 2 Quality Assurance Statement.......................................................................................................................... 3 Report Approval.............................................................................................................................................. 4 Table of Contents ............................................................................................................................................ 5 summary.......................................................................................................................................................... 7 Introduction ..................................................................................................................................................... 9 Objective ......................................................................................................................................................... 9 Experimental Design ....................................................................................................................................... 9 Materials and Methods.................................................................................................................................. 10 Results and Discussion.................................................................................................................................. 14 Conclusion..................................................................................................................................................... 16 References ..................................................................................................................................................... 17 TABLES Table 1 . Summary of Analytical Chemistry Data .................................................................................... 18 Table2 . Temperature Measurements....................................................................................................... 19 Table3 . Light Intensity Measurements.................................................................................................... 20 Table4 . 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-HourInterval During the Test............................................................................................................................ 25 wildlifeInternational, Ltd. -6- Project Number 454A-129 Table 8 - TABLE OF CONTENTS -Continued- .. EC10, EC50 and EC90 Values Based on Cell Density Over the 96-Hour Exposure Period ............................................................................................................................ Table 9 - EC10, EC5O and EC90 Values Based on Arca Under the Growth Curve Over the 96-Hour Exposure Pen........................................................................................................ 27 Table 10- EC10, 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....................................................... 3 1 Figure 3 - Cell Density During the Recovexy Phase .................................................................................... 32 Appendix 1 - Appendix 2 - Appendix 3 - Appendix 4 - Appendix 5 - Appendix 6 Appendix 7 - Appendix 8 - APPENDICES Freshwater Algal Medium ............................................................................................... 33 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water ....................................................................... 34 The Analysis of PFBS in Freshwater Algal Medium in Support of Wildlife International, Ltd. Project N*o.:454A- 129....................................................... 36 Cell Density for Each Replicate Per Treatment Over the 96-Hour Exposure Pe..................................................................................................... 52 Area Under the Growth Curve for Each Replicate Per Treatment Over the 96-Hour Exposure Period ................................................................................................. 53 Growth Rate for Each Replicate Per Treatment Over the 96-Hour Exposure Period .....54 Changes to Protocol.......................................................................................................... 55 Personnel Involved in the Study ....................................................................................... 56 wildlife International, Ltd. -7- Proiect Number 454A-129 SANITIZED SUMMARY DEC 0 9 2OQ3 l SPONSOR: SPONSOR'S REPRESENTATIVE: 3M Corporati.on LOCATION OF STUDY, RAW DATA AND A COPY OF THE Wildlife International, Ltd. Easton, MD 21601 WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST: 454A-129 Perfluorobutslnesulfonate,Potassium Salt (PFBS) PFBS: A 96-Hour Toxicity Test with the Freshwater Alga (Selenastrum capricornutum) Negative Control, 285,563,1077,2216,4561 and 9478 mg a.i./L Experimental Start - January 22,200 1 Exposure Texmination Experimental Termination - Fe r2u6yary25oo,2001 96 Hour Exposure, 6 Day Recovery SOURCE OF TEST ORGANISMS: CELL DENSITY 72-HOUR EC50: 95% CONFIDENCE LIMITS: 96-HOUR EC10: 95% CONFIDENCELIMITS: 96-HOm EC50: 95% CONFIDENCELIMITS: 96-HOUR EC90: 95% CONFIDENCELIMITS: 72-HOUR NOAEC: 96-HOUR NOAEC: Wildlife International, Ltd. 1469 mg a.i./L 8 1 and 28 12 mg a.i./L 528 mg a.i./L, 375 and 1895 mg a.i./L 2347 mg a.i./'L 20 18 and 2707 mg a.i.LL 7390 mg a.i./L 7270 and 7500 rng a.i./L ~ 2 8 r5ng a.i.12 1077 rng a.i./Z WildlgfeInternational, Ltd. -8- SUMMARY (Continued) AREA UNDER THE GROWTH CURVE 72-HOUR EC50: 95% CONFIDENCE LIMITS: 96-HOUR EC 10: 95% CONFIDENCE LIMITS: 96-HOUR EC50: 95% CONFIDENCE LIMITS: 96-HOUR EC90: 95% CONFIDENCE LIMITS: 72-HOUR NOAEC: 96-HOUR NOAEC: 1590 mg a.i./L 328 and 2373 :mg a.i./L 385 mg a.i./L 1 91 and 951 rrtg a.i./L 2 146mg a i / L 1748 and 2706 mg a.i./L 7294 mg a.i.L 6888 and 7673 mg a.i./L a 8 5 mg a.i./L, 285 mg a.i./L GROWTH RATE 7 2 - ~ 0 U REC50: 95% CONFIDENCE LIMITS: 96-HOUR EC 10: 95% CONFIDENCE LIMITS: HOUR EC50: 95% CONFIDENCE LIMITS: HOUR EC90: 95% CONFIDENCE LIMITS: 72-HOUR NOAEC: 96-HOUR NOAEC: 5661 mg a.i./L, 5230 and 606'7 mg a.i./L 1674 mg a.i./L, 1482 and 1839 mg a.i./L 5733 mg a.i./L, 5659 and 581'7 mg a i / L 8849 mg a.i./L, 8656 and 908 1 mg a.i./L 4 3 5 mg a . i L 1077 mg a.i./L Project Number 454A- 129 wildltfeInternational, Ltd. 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 of the test was conducted from January 22,200 1to February 1,200 1. 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 of the study was to evaluate the toxicity of perfluorobutanesulfonate,potassium salt (PFBS) to the growth of the freshwater alga, Selenastrum capricornutum,during a 96-hour exposure period. EXPERIMENTAL DESIGN The freshwater alga, Selenastrum capricornutum, was exposed to a geometric series of 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 additionalreplicatewas alsomaintained for analytical sampling on Day 3 of 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 ingrrdent (a.i.)/L. Mean measured test concentxations were determined fiom samples of test medlum collected fiom each treatment and the control group at test initiation, at approximately 72 hours, and at test termination. At test initiation, an inoculumof the algal cells was prepared at a concentrationof approximately 1.0 X IO6 cells/mL. The concentration of algal cells in the ina:ulum was verified and 1.0 mL was added to each test chamber to achieve a nominal concentration of approximately 1.0X lo4cells/mL. Samples were collected fiom 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 contrcil were calculated for each parameter over the 96-hour exposure period. EC50 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 wildlifeInternational, Ltd. Project Number 454A-129 - 10 - SANITIZED DEC 0 9 2003 the 72 and 96 hour intervals. The no-observed-adverse-effect-concentratio(Nn OAEC) 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. MATEFUALSAND METHODS The study was conducted according to the procedures outlined in the protocol, "PFBS: A 96-Hour Toxicity Test with the Freshwater Alga (Selenastrum capricormtum)". The protocolwas basedon proceduresoutlined in the U.S. Environmental Protection Agency Series 850-Ecological Effects Test Guidelines, OPPTS Number 850.5400: Algal Toxiciv Tiers IandN (draft)(1) and [OECDGuidelines for Testing of Chemicals, 201Algal, 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 I Developmental Product, AKA PFBS, from lot 2. Information provided by the Sponsor indicated a purity of 97.9%. A subsequentrevision of the ceriificate of analysis indicated a purity of 97.3% and an Expiratiofieassessment Date of January 17, 2002. The test substancewas 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 solubilizationof 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 n L 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%). wildlifeInternutional, 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 of an important group of freshwater algae, and was selected for use in the test based upon a past history of use and ease of culturing in the labloratory. 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 negativecontrolorganisms were expected to exhibit exponential growth over the 96-hour exposure period. Exponential growth phase, defined as the period of growth where the algal cells are dividing at a constant rate, is indicated by the linear section of the growth curve (Figure 1). Culture Medium The algal cells were cultured and tested in fieshwater algal medium (3). Stock nutrient solutions were prepared by addingreagent-grade chemicalsto WildlifeInternational,Ltd.well waterpurifiedby reverseosmosis. The test medium was prepared by adding the appropriate volumes of stock nutrient solutions to purified well water (Appendix 1). The pH of the medium was adjusted to 7.5 * 0.1using 10%HCI; the mediumwassterilized by filtration (0.22 pm) prior to use. Analyses were performed at least once annually to determine the concentrationsof selectedorganic and inorganicconstituents in the well water. Theresultsof analysesperformed to measure the concentrations of selected contaminantt; 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 rnL of 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 environmentalchamber designedto maintain the desired test temperature throughoutthe test. Thetest chambers were shaken continuously at 100 rpm. w i l d l tfe International, Ltd. - 12 - Project Number 454A-129 Environmental Conditions Test flasks were held in an environmental chamber at a temperature of 24*2OC. The temperature of a container of water adjacent to the test flasks in the envirconmentalchamber was recorded twice daily during the test using a liquid-in-glass thermometer. The algaewere held under continuouscool-whitefluorescentlightingthroughoutthe test. The target light intensitywas 4300 f430 lux. Light intensitywas measured at the four comers and the middle ofthe shakertable daily during the test. Light intensity was measured using an SPER Scientific Model 840006 light meter. The pH of the medium prepared for each treatment and control group was measured at test initiation and termination using a Fisher Accumet Model 9 15 pH mete:r. Samples for pH measurement at test initiation were collected from the individual batches of 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 controlgroup for pH measurement. Algal Growth Measurements Test medium samples were collected from the treatment and control groups for the determinationof algal cell densities. Single samples were collected from each. of the three "biological" replicates per treatment and control group at 24-hour intervals during the 96-hour exposure, and were counted immediately. Cellcountswere conducted using a hemacytometer and microscope. Each sample was diluted using an electrolyte solution (Isoton@),as needed, to maintain counting accuracy. A small amount of each sample was loaded onto a hemacytometer and 10grids were counted. The mean number of cells per grid was estimatedand this value was used to calculatethe cell density of the sample. Using this technique,the minimumquantifiablecell densitywas 1.0 x io3ceiis/ml. All 96 hour cell count sampleswere examined microscopicallyfor atypical cell morphology (e.g.,changes in cell shape, size or color). Growthof cells in the replicate test chambers also was assessedfor aggregationsor flocculations of cells and adherence of the cells to the test chhber. wildlifeInternational, Ltd. - 13 - Proiect 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 = ( ( N I - N ~ ) / ~ ) ( ~ I ) + ( ( N * + N Z - ~ N ~ ) / ~ ) ( ~ ~ - ~ I ) + ( ( N , . I + N1 , - ~ N ~ ) / ~ ) ( ~ - ~ , - I where: A=Area No=Nominal number of cellslml at t, N1 = Mean measured number of celldml, at tl Nz= Mean measured number of cells/ml, at t 2 N, = Mean measured number of cells/ml, at t, tl = time of first measurement after beginning of test (hours) t2= time of second measurement after beginning of test (hours) t, = time of n* measurement after beginrihg of test (hours) Growth rates were calculated for the control and each treatment group using the following formula: Growth Rate = ("0t)n where: No = Mean measured number of cells/ml, at t, N,, = Mean measured number of cells/mL at t, ,t = Time of 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: x - Percent Inhibition = Mean Cell DeMnseiatnyhChelll I)MenesaintyCcOellnDhlensityTr-ent 100 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 EIC 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 of variances using the Shapiro-Wilk's test wildlifeInternational, Ltd. - 14 - Proiect Number 454A-129 and Bartlett'stest, respectively. Ifthe 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'stest. Results of 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 terminationto measure concentrationsof 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 of the samples are presented in Appendix 3. RESULTS A N I ) DISCUSSION Measurement of Test Concentrations Results of analyses to measure concentrationsof`PFBSin 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. Samplescollected at the beginningof the test had measured concentrationsthat rangedfrom 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 24.7"Cand were withinthe range establishedfor the test (24+2OC).Thelight intensityduring the exposurephase of the test ranged fi-om3930 to 4550 lux and was withinthe desiredrangefor the test (approximately3870 to 4730 lux) (Table 3). Measurementsof 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. wildlifehtemutional, Ltd. - 15- Project Number 454A-129 Data Analyses The effect of 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 of variation for control cell density was 4.5%. After 72 hours of exposure, cell density percent inhibition in the 285,563,1077,2216,4561 and 9478 mg a.i./L treatmentgroups was 32,33,44, 62,83 and 99%, respectively. Dunnett's test showed tlhat cell density was significantly reduced in all treatment groups in comparison to the negative control (p 5 0.05). Consequently, the NOAEC for 72 hour cell density was 4 8 5 mg a.i./L, the lowest concentrationtested. After 96 hours of exposure, cell densitypercent dubition in the 285, 563, 1077, 2216, 4561 and 9478 mg a.i./L treatment groups was -10, 8.3, 5.6,46, 76 and loo%, respectively. Dunnett's test showed that cell density was significantly reduced in the 2216,4561 and 9478 mg a.i./L treatment groups (p 5 0.05). Consequently, the NOAEC for 96 hour cell density was 1077mg a.i./L. After 72 hours of exposure, area under the grovdh curve percent inhibition in the 285,563,1077,2216, 456 1and 9478 mg a.i./L, treatment groups was 26,3 1,42,60,80 and 98%, respectively. Dunnett's test showed that area under the growth curvewas significantlyreduced in all treatment groups (p s 0.05). Consequently,the NOAEC for 72 hour area under the growth curve was 4 8 5 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 5.5,18,21,52,78 and 99%, respectively. Dunnett's test showedthat area under the growth curve was significantly reduced in all treatment groups except the 285 mg a.i./L treatment group (p5 0.05). Consequently, the NOAEC for 96 hour area under the growth curve was 285 mg a.i./L. wildlife International, Ltd. - 16 - Proiect Number 454A-129 After 72 hours of exposure, growth rate percent irhibition in the 285,563,1077,2216,4561 and 9478 mg a.i./L treatment groups was 8.2,8.7, 13,21,38 and 9296, respectively. Dunnett'stest showed that growth rate was significantly reduced in all treatment goups (p S 0.05). Consequently, the NOAEC for 72 hour growth rate was ~ 2 8 m5 g a.i./L, the lowest concentration tested. After 96 hours of exposure, growthratepercent inhibitionin 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 WBSsignificantly reduced in the 2216,4561 and 9478 mg a.i./L treatment groups (p s 0.05). Consequently, the .NOAEC for 96 hour growth rate was 1077 mg a.i./L. Visual and Microscopic Observations After 96 hours of exposure, there were no signs of aggregation, flocculationor adherenceofthe algaeto 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 m.edium 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 11and are illustrated graphically in Figure 3. CONCLi USIONS The conclusions of this study were based on the ,mostsensitive endpoint measured (i.e., cell density, area under the growth curve andor growthrate). The 7 2 - h E~C5~0,based on cell density, was 1469mg a.i./L with 95% confidence limits of 8 1 and 28 12 mg a.i./L. The 96-hour EC 10, based on area under the growth curve, was 385 mg a.i./L with 95% confidence limits of 191 and 95 1 rng 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.,Lwith 95% confidencelimitsof 6888 and 7673mg a.i./L. The 72-hour NOAEC value, based on cell density, area under the growth curve and growth rate, was a 8 5 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. - 1'7 - Proiect Number 454A- 129 REFERENCES 1 U.S.Environmental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines (draft>, OPPTS Number 850.5400: Algal Toxiciv, Tiers I and II. 2 Organization of Economic Cooperation and Development. 1984. Algal, Growth Inhibition Test. OECD Guideline for Testing of Chemicals. Guideline 201. Paris. 3 ASTM StandardGuide 1218-903;Standard Guidefor ConductingStatic 96-Hour ToxicityTestswith Microalgae. August 1990. 4 The SAS System for Windows. 1996. Release: 6.12, TS Level 0020. SAS Institute Inc., Cary, North Carolina. 5 West, 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 ABLE 1 Summary of Analytical Chemistry Data Sponsor: 3M Corporation Test Substance: PFBS Test Organism: Freshwater alga, Selenastruni capricornutum Dilution Water: Freshwater medium Nominal Sampling Measured Concentration Time Concentration' (mg a.i./L) Negative Control (Hours) O2 723 (mg a.i./L) -= LOQ .C LOQ 964 < LOQ Mean Measured Concentration (mg a.i./L) -- Percent of Nom__inal 3 13 0 290 285 91 72 28 1 96 283 625 0 567 563 90 72 563 96 558 1250 0 1146 1077 86 72 993 96 1091 2500 0 2188 22 16 89 72 2209 96 2252 5000 0 4522 4561 91 72 455 1 96 4610 10000 0 9859 9478 95 72 9154 96 942 1 10000 (Abiotic) 72 9467 9484 95 96 950 1 1 Limit of Quantitation (LOQ)was 100 mg a . i L 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. 96-hour samples were composites of test solution collected from each of the three replicates per treatment and control group. wildlifeInternational, Ltd. - 19- Proiect Number 454A-129 Table 2 Temperature Measurements Sponsor: Test Substance: Test Organism: Dilution Water: Time (Day) 0 3M Corporation PFBS Freshwater alga, Selenastmm capricornutum Freshwater medium Temperature ("C) Measurement 1 Measurement 2' 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 ' With the exception of Day 10, temperature measurement 2 was taken at least 4 hours after measurement 1. wi1d1(fe Intemutiona1, Ltd. - 20 - Proiect Number 454A- 129 Tablle 3 Light Intensity Measurements Shaker Table AQL #6 Sponsor: Test Substance: Test Organism: Dilution Water: Test Day 3M Corporation PFBS Freshwater alga, Selenastrum capricornutum Freshwater medium Light Intensity Measurements (lux) No. 1 No. 2 No. 3 No. 4 3960 4010 4490 3990 3980 4030 4400 4350 3930 4030 4120 4050 3960 41 10 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 41 10 4060 4410 3890 No. 5 4370 4260 4550 4280 4030 4020 4030 4160 3910 3940 3970 wildlifeIntemutionul. Ltd. -21 - Project Number 454A-129 ' Table 3 (Clontinued) Light Intensity Measurements Shaker Table AQL #3 Sponsor: Test Substance: Test Organism: Dilution Water: Test Day 0 3M Corporation PFBS Freshwater alga, Selenastrum capricornuturn Freshwater medium Light Intensity Measurements (lux) No. 1 No. 2 No. 3 No. 4 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 wiId1(fe I ntemational, Ltd. - 2;! - Proiect Number 454A-129 Table 4 pH Measurements Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Freshwater alga, Selenastrum capricornutum 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 8.7 456 1 7.1 8.5 9478 7.1 7.5 ' 9484 (Abiotic) -- 7.6 0-hour samples were collected ftom the batches of test solution prepared for the treatment and * control groups at test initiation. 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-Ho1-uInterval During the Test' Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Freshwateralga, Selenastnrm capricomutum Freshwater medium Mean Measured 24 Hours Concentration (mg a.i.L) Negative Control Mean Cell Density 3 1,000 Percent Mubition -- 48 Hours Mean Cell Percent Density 179,000 Inhibition -- 72 Hours Mean Cell Percent Density 1 ,o 15,000 Inhibition -- 285 30,333 2.2 162,333 9.3 688,3332 32 563 1O?? 27,000 13 3n c;&7 *",VU, 27 22 138,667 23 1!9,000 34 673,33 32 34 4 7 1 &L72 2 I A ,vu, 7A-Ar 2216 17,333 44 95,333 47 385,0002 62 4561 15,333 51 62,333 65 175,333* 83 ' 9478 12,000 61 16,000 Values calculated using SAS 6.12. Manual calculationsmay differ slightly 91 14,6672 99 2 Indcates a sigillficant difference&omthe negative control at 72 hours using Dunnett's test (p s 0.05). 3 Indcates a sigillficant difference&omthe negative control at 96 hours using Dunnett's test (p s 0.05). Project Number 454A-129 96 Hours MeanCell Percent Density 3,560,000 Inhibition -_ 3,920,000 -10 3,265,000 8.3 3,360,000 CL 2.V 1,930,0003 46 860,0003 76 15,3333 100 Project Number 454A-129 - 24 - Table 6 Mean Areas Under the Growth Curve and Percent Inhibition for Each 24-Hour Interval During the Test' Sponsor: Test Substance: Test Organism: 3M Corporation PFBS Freshwater alga, Selenastrum capricornutum Dilution Water: Freshwater medium Mean Measured 0 - 24 HOW 0 - 48 Hours Test Concentration Mean Percent Mean Percent (mg a i & ) Area Inhibition Area Inhibition Negative Control 252,000 -- 2,532,000 __ 0 - 72 HOU~S Mean Area Percent Inhibition 16,620,000 -- 285 244,000 3.2 2,3 16,000 8.5 12,284,0002 26 5 A.,?- 204,000 19 1,952,000 23 _1 _1 ,45h,00n2 31 1077 128,000 49 1,564,000 38 9,6 12,0002 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 98 'Values calculated using SAS 6.12. Manual calculations may differ slightly. 'Indicates a sigmficant difference fiom the negative control at 72 hours using Dunnett's test (p < 0.05). 3Indicates a sigmficant differencefiom the negative control at 96 hours using Dunnett's test (p < 0.05). 0 - 96 HOU~S Mean Area Percent Inhibition 71,280,000 -- 67,344,000 5.5 58,476,0003 18 56,552,0003 21 34,264,000' 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 Test' Sponsor: Test Substance: Test Organism: 3M Corporation PFBS Freshwater alga, Selenastmm capricomutum Dilution Water: Freshwater medium Mean Measured 0 - 24 Horn 0 - 48 Horn Test Concentration Mean Percent Meall Percent (mg a.i./L) Negative Control Growth Rate 0.0445 Inhibition Growth Rate 0.0601 __ Inhibition 0 - 72 Horn Mean Percent Growth Rate Inhibition 0.0640 -- 285 0.0458 -3.0 0.0580 3.4 0.05872 8.2 563 nn A i ? V.V.tIJ 7.3 0.0547 8.9 0.05w2 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.O39fl2 38 9478 0.0130 71 0.0095 84 0.00522 92 1Values calculated using SAS 6.12. Manual calculations may differslightly. 21ndicatesa si&icant differencefiom the negative control at 72 hours using Dunnett'stest (p < 0.05). 31ndicatesa si&icant dflerencefiom the negative control at 96 hours using Dunnett's test (p c 0.05). 0 - 96 HOWS 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 - 26 - Project Number 45412- 129 Table 8 EC10, EC50 and EC90 Values Based on Cell Density Over the 96-Hour Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: Time 24 Hours 48 Hours 3M Corporation PFBS Freshwater alga, Selenastmm capricornutum Freshwater medium EClO (mg a.i./L) Not Determined 95% Confidence Limits - (mga.i./L) Not Determined - 72 Hours <285 -- 1 96 Hours 528 375 and 1895 'Confidence limits could not be calculatedwiththe data obtained. EC50 (mg a.i./L) 4366 263 1 1469 2347 95% Confidence Limits (mg a.i./L) a . 0 and 12305 1644 and 3927 81 and 2812 2018and2707 EC90 (mg a.i./L) Not Determined Not Determined 6821 7390 95% Confidence Limits (mg a.i./L) - I 5639and8187 7270 m d 7500 - 27 - Project Number 454A-129 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: Time 24 Hours 3M Corporation PFBS Freshwater alga, Selenastntm capricomutum Freshwater medium EClO 95% Confidence Limits (mg a.i./L) Not Determined (mg a.i./L) - 48 Hours Not Determined - 72 Hours <285 - I 96 Hours 3 85 191 and 951 1Confidence 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) <O.O 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 (me a.i./L) - 6231and8204 6888and7673 - 28 - Table 10 EC10, EC 0 and EC90 Values Based on Growth Rate Over t,e 96- lour Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Freshwater alga, Selenastrum capricomutum Freshwater medium Time 24 Hours EClO (mg a.i./L) Not Determined 95%Confidence Limits (mga i & ) - 48 Hours Not Determined - EC50 (mg a.i./L) 2195 5892 72 Hours 734 4 . 0and 2207 5661 96 Hours 1674 1482 and 1839 5733 'Confidence limits could not be calculated with the data obtained. 95% Confidence Limits (mg a.i./L) a . 0 and 8922 4683 and 7385 5230and6067 5659 and 5817 EC90 (mg a.i./L) Not Determined Not Determined 9303 8849 Project Number 454A-199 95% Confidence Limits (mg a.i.L) - 8571and9664 8656 and 9081 wildlifeInternational, Ltd. -29- Proiect Number 454A-129 Tab1.e 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 Cell Densities Kells/mL2) (mg a.i./L) Day 0 Day 3 Negative Control . 18,000 1,785,000 Day 6 6,140,000 9478' 2,000 3,000 980,000 1 The treatment group was diluted to a concentration of the test substance that theoreticallywould not lnhibit growth. 2 Due to the method used to prepare recovery phase test solutions, initial cell densities were not equivalent throughout the treatment groups. wi1dl{fe I nternationaI , Ltd. - 30 - Project Number 454A-129 Figure 1. Negative Control Algal Growth, Expres:sedin Cell Density, During the 96-Hour Exposure. Mean Cell Density (Cellsm) 0 24 48 72 96 Exposue Duaiion (Horn) WildlifeInternational, Ltd. -31 - Proiect Number 454A-129 Figure 2. Concentration-Response Curve, Expressed in Cell Density Mean Cell Density ( C e k m ) lCC0 0 24 48 72 Exposm Dualion(Hours) +?I,#ivechltml -285 nga1.L +563 rrga1.L -&- 1077ng a1.L +2216 ngaLL -0- 4561 ng a1.L +9478 nga1.L 96 wildlifeInternational, Ltd. - 3:2 - Project Number 454A-129 Figure 3. Cell Density Illring the Recovery Phase ]/ 10000000 - 1000000 100000 - I / 10000 - 41 1000 - -Negative Contol --+9478 mg a.1.R 100 1 wildlife Intemutionul. Ltd. - 33 - Proiect Number 454A-129 Appendix 1 Freshwater Algal Medium' Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Freshwater alga, Selenastrum capricornutum Freshwater medium Nominal Compound Concentration 12.16 4.4 1 0.1855 0.4 154 3.27 0.1598 1.428 7.26 0.012 0.300 25.50 14.70 1.044 15.0 mg/L 1The pH was adjusted to 7.5 f. 0.1 using 110% HC1. wildlife International, Ltd. - 34 - Proiect Number 454A-129 Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water' 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 C hloridazon C hlorpropham C hlorpyriphos C hlorpyriphos-methyl Chlorthalonil Coumaphos C yanazin Cyfluthrin Cypermethrin Cyproconazole Deltamethrin Demeton Demeton-o Desethylatrazin Desisopropylatrazin Desmetryn Diazinon Dichlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid 4 . 0 3 pg/L <0.01 pg/L 4 . 0 1 pg/L 4 . 0 1 pglL < o m pg/L 10.08p a ~ 0 . 2 p5g/L <0.05pg/L 10.05 pg/L <0.05 pg/L 10.05 pg/L 4 . 0 2 pg/L c0.02 pg/L 4 . 0 2 pg/L ~0.0p5g/L 4.05 pg/L <0.03 pg/L 4 . 0 2 pg/L C0.02 pg/L (0.05 pg/L 4 . 0 2 pg/L 4 . 0 1 pg/L <0.01 pg/L co.04 p a - 4 . 0 2 pg/L 4.05 pgL 4.05 pg/L 4.25 PdL 4.05 pg/L 4.02 pgL 4 . 0 2 pg/L 4 . 0 2 pg/L 4.01 pg/L 4 . 0 2 pg/L 4.01 pglL 4 . 0 1 pg/L 4 . 0 1 pg/L 4.03 pg/L 4.02 pg/L 4 . 0 1 pglL 4 . 0 3 pg/L Dimethornorf Disulfoton DMST Dodemorf Endosulfan-a Endosulfan-P Endosulfan-sulfaat Epoxiconazole Eptam Esfenvaleraat Ethion Ethofumesaat Ethoprophos Etridiazole Etrimphos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb Fen piclonil Fenpropathrin Fenpropimorf Fenthion Fenvaleraat Fluazifopbutyl Fluoroglycofencthyl Fluroxypyr-meptyl Flutolanil Fonophos Furalaxyl Heptenophos Imazalil Iprodion Kresoxim-methyl Lenacil Lindane Malathion Metalaxyl Metamitron Metazachlor Methidathion 10.05 pg/L 4 . 0 2 pg/L 4.05pg/L 4 . 0 1 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L 10.05pg/L 10.02 pg/L C0.02 pg/L 4.05 pg/L <0.02 pg/L <0.01 pg/L C0.02 pg/L 10.05pg/L 10.05pg/L 10.01 pglL ~ 0 . 0 p3g/L 10.03 pg/L <0.05 pg/L 4 . 2 5 pg/L 4.01 pglL 4.01 pgL aI.02p g L C0.02 pg/L 4 . 0 2 pa 4.05p g L C0.02 pg/L 4.01 pg/L 4 . 0 2 pg/L 4 . 0 2 pg/L 4.01 pgL <0.05pg/L 4 . 0 2 pg/L <0.05 pg/L 10.02p g n <0.02 pg/L 4.05pg/L ~ 0 . 0 5p g L 10.02 pg/L 4 . 0 2 pg/L 'Analyses performed by TNO Nutrition and Food Instituteon samplescollected on October 14 and 15, 1999. WildltfeInternational, Ltd. - 35 - Proiect Number 454A-129 Appendix 2 (Continued) Analyses of Pesticides,.Organics and Metals in WildlifeIntemationd,~ t dW. ell Water' Pesticides And Organics(Page 2) Component Measured Concentration Component Measured Concentration Dichlorvos Dicofol Diethyltoluamide Difenoconazole Dimethoate Paclo butazole Parathion Parathion-methyl Penconazole Pendimethalin Permethrincis Permethrin-trans Phosalon Phosmet Phosphamidon-cis Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazin Propham Propiconazool Propoxur Propyzamide Prosulfocarb Pyrazophos CO.01 pg/L <0.25 pg/L 4.02pgn <0.03 p g L 4 . 0 2 pg/L 4 . 0 5 pg/L 4 . 0 1 pg/L <0.01 pg/L <0.05 pg/L <0.03 pg/L 4 . 0 1 pg/L <0.01 pg/L <0.05 pg/L 4 . 0 2 pg/L <0.05 pg/L <0.01 pg/L CO.01 pg/L 4 . 0 1 pg/L C0.02 pglL <0.01 pg/L 4.01 pgL 4.01 p g L 4 . 0 1 pg/L 4.02 pgiL 4.05 pg/L 4.03 pg/L 4 . 0 2 pg/L 4.02 pgn 4,03pg/L Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl Pynfenox- 1 F'ynfenox-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 4 . 0 1 pg/L <0.01 pg/L <0.02 pg/L 4 . 0 1 pg/L <0.05 pgA, <0.01 pg/L <0.01 pg/L <0.01 pg/L C0.02 pg/L <0.01 pg/L <0.02 pg/L CO.05 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L CO.01 pg/L <0.05 p g n <0.04 ~ f l <0.01 pg/L <0.04 pg/L 4.05pg/L -==0.0p5g n 4.02 pgL 4 . 0 2 pg/L <0.02 pg/L 4 . 0 1 pg/L 4.01 pgL Metals Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt ll.Orng/L 18.0 mg/L 29 mg/L <0.015 mg/L 1.1 mg/L <0.02 mg/L co.1 pg/L c0.2 p g L <os pg/L 4 . 2 pg/L Nickel CopDer Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium 4 . 1 pg/L <0.7 pg/L <0.25 p g L <0.3 pg/L C0.2 pgrL <0.1 p g n <os pg/L <0.025 p g L <OS pg/L 'Analyses performed by TNO Nutritionand Food Institute on :sunpiescollectedon October 14 and 15,1999. wild1[fe Intemational, Ltd. - 36 - Appendix 3 Project Number 454A- 129 THE ANALYSIS OF PFBS IN FRESHWATERALGAL MEDIUM IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A- 103 WILDLIFE INTERNATIONALIDL. - :37 - PROJECT NO.: 454A-129 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFBS: A 96-HOUR TOXICITY TES'T with the FRESHWATER ALGA (Selenastmm capricornutum) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-129 3M ENVIRONMENTAL LAB PROJECT NUMBER E00-1429 PRINCIPAL INVESTIGATOR: A Raymzd f .Van Hoven, Ph.D. Scientist MANAGEMENT: Willard B. Nixon, Ph.D. 1 Director, Analytical Chemistry 03-90-01 DATE .DATE WILDLIFE INTERNATIONALLTD. PROJECT NO.: 454A-129 -38 - SANITIZED 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, Porassium Salt) to the freshwater alga (Selennstntrn cupricornuturn).This study was conducted by Wildlife International, Ltd. and identified as Project Number 454A- 129. The analyses of these water samples were performed at Wildlife International, Ltd. using high performance liquid chromatography with mass spectrometric detection ("LChIS). Samples were received for analysis on January 22, 25 and 26, 200 1 ;md 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 52 16, and stored under ambient conditions. The analytical standard, a white powder, was identified as: Potassium Perfluorobutane Sulfonate expiration date: March 2010. The analfical 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 certdicate of analysis indicated a purity of 97.3% and an ExpiratiodReassessment Date of 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. Analvtical Method Freshwater medium samples were analyzed according to the method entitled "halytical Method Validation for the Determination of 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 of the PFBS methodology. Aliquots of 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 Perlun-Elmer M I lOOLC 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 acheved using a Keystone ~__ WILDLIFE INTERNATIONALTLD. - 39 - PROJECT NO.: 454A-129 PRISM RP column (30 mm x 1.5 mm, 3-j.un particle size) fitted with a Keystone Javelin CISGuard 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 Secondarv Stock Solutions, All primary and secondary stock preparations were adjusted for the purity of the analflcal standard (97.90%). A 10.0 mg a.i./mL primary stock s01uti:on of PFBS in methanol was prepared by weighmg 1.024 g of the analytical standard and bringing to a final volume of 100 mL with methanol. Secondary stock solutions (1000, 100, 10.0, 1.00, and 0.100 mi; a.i./L) of PFBS in methanol were prepared by serial volumetric ddution from the primary stock. Calibration Standards and Calibration Curves Calibration standards were prepared in 50:50methanol: NANOpure" water by appropriate dilutions of the 10.0 mg a.i./L stock solution of PFBS in methanol. The calibration standards of PFBS, ranging in concentration from 0.0100 to 0.0500mga.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 of 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 of the calibration standards. A typical calibration curve is presented in Figure 2. The concentration of PFBS in the samples was determiried by substituting the peak area responses into the applicable linear regression equation. Representative ion chromatograms of low and high calibration standards are presented in Figures 3 and 4, respectively. Limit of Quantitation The method limit of quantitation (LOQ) for these analyses was set at 100 mg a.i./L calculated as the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (10000). Matrix Blank and Fortification Samdes Three matrix blank samples were analyzed to determine possible interference. No interferences were WILDLIFE INTERNATIOLNTDA. L - 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 dlrectly fortified (i.e. wilhout use of 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 of 93.5%. A representative ion chromatogram of a matrix fortification is presented in Figure 6. Examule Calculations Sample number 454A-129-4, nominal concentration of 1250 mg a.i./L in freshwater medium. First Initial Volume: 0.100 mL Calibr.ationcurve 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 l/x Dilution Factor: 40000 PFBS Peak Area: 1385658 - PFBS (mg a.i./L)measured at instnunent = Peak areaslo(pye-intercept) PFBS (mg a.i./L) in sample = PFBS measured at instrument (mga.i./L) x dilution factor - 1385658 - 147443.62500 4oooo 43223072 = 1146 Percent of Nominal Concentration =PPFFBBSsm((iga.i./L in sam le x 100 ---11124560 :< 100 = 91.7% WILDLIFE INTERNATIOLNTDA. L -41 - PROJECT NO.: 454A-129 Calculated with HPLCMS instrument software: MacQuan, version 1.6. Sample Analysis - RESU- LTS Freshwater medium samples were collected from an acute toxicity study with the freshwater alga (Selenustrum 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 of PFBS in the samples collected at initiation of exposure of the test organisms (Day 0) ranged from 87.5 to 98.6% of the nominal concentrations. Samples collected at Day 3 had a measured concentration range of 79.5 to 94.7% of nominal values. Samples collected at test termination (Day 4) had a measured concentration range of 87.3% to 95.0% of 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.,lL treatment group with the freshwater alga present (Table 3). A representative ion chromatogram of a test sample is shown in Figure 7. WILDLIFE INTERNATIOLNTD A.L PROJECT NO.: 454A-129 Table 1 Typical HPLCNS Operational Parameters INSTRUMENT: Hewlett-Packard Model 1100High Performance Liquid Chromatograph with a Perkin-Elmer API lOOLC Mass Spectrometer operated in Selective Ion Monitoring (SIM) Mode ION SOURCE: Perkin-Elmer TiirboIonSpray ANALYTICAL COLUMN: Keystone PRISM RP (30 mm x 1.5 mm, 3-pnparticle size) GUARD COLUMN: Keystone Javelin CIScartridge (20 mm x 2 mm) OVEN TEMPERATURE: 40C STOP TIME: 3.00 min FLOW RATE: MOBILE PHASE: INJECTION VOLUME: 200 pL/min 25% NANOpurr:" Water with 0.1% Ammonium Formate: 75% Methanol 5.0 pL PFBS PEAK RETENTION TIME: Approximately ;!.2 minutes PFBS MONITORED MASS: 299.0 m u WILDLIFE INTERNATIONALTLD. -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 Measured' <LOQ' <LOQ <LOQ Percent Recovered' --- -- MAS-1 Matrix Fortification 150 141 93.8 MAS-4 Matrix Fortification 150 138 92.1 MAS-7 Matrix Fortification 150 136 90.9 MAS-2 Matrix Fortification 2500 2338 93.5 MAS5 Matrix Fortification 2500 2385 95.4 MAS-8 Matrix Fortification 2500 2302 92.1 MAS3 MAS -6 Matrix Fortification Matrix Fortification 12000 12000 11460 11520 95.5 . 96.0 MAS-9 Matrix Fortification 12000 11050 ` 92.1 Mean = 93.5 Standard Deviation = 1.82 CV =1.95% N=9 1 Measured and Percent Recovered values were CalCUliIted using MacQuan, version 1.6 software. Manual calculations may vary slightly. 'The limit of 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 of the matrix blank samples (10000). r WILDLIFE INTERNATIOLNTDA. L -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 3 13 625 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 Concentration' (mg a.i./L) < LOQ~ < LOQ c LOQ 290 28 1 283 567 5 63 558 1146 993 1091 2188 2209 2252 4522 455 1 4610 9859 9154 942 1 9467 9501 Percent of Nominal' -- 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 Measured and Percent of Nominal values were calculated using MacQuan, version 1.6 s o h a r e . Manual calculations may vary slightly. The limit of 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 of the matrix blank samples (10000). WILDLIFE INTERNATIOLNTDA. L - 45 - PROJECT NO.: 454A-129 METHOD OUTLINE FOR THE ANALYSIS OF PFBS IN FRESHWATER MEDIUM Prepare each matrix fortification sample by weighqg the requisite amount of PFBS test substance on an analytical balance and transferring directly into a Class A volumetric flask partially filled with freshwater medium. U s e weighing paper and the sides of 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. Centrifbge samples, as appropriate, at approximately 2500 rpm for approximately 15 minutes. Prepare appropriate dilutions of study and QC samples to within the calibration range of the PFBS methodology: Partially fill Class A volumetric flasks with 50% methanol : 50% NANOpure" water dilution solvent. Add the appropriate volume of 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. Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis of PFBS in freshwater medium. WILDLIFE INTERNATIONALLTD. - 4r5 - Area 240000 210000 180000 150000 120000 900000/ PROJECT NO.: 454A-129 Figure 2. . A typical calibration curve for PFBS. ;Slope= 43223072; Intercept = 147443.62500; r = 0.9986. WILDLIFE INTERNATIONALTLD. -47- PROJECT NO.: 454A-129 intensity: 500000 cps ' O9O0l 4 131 'j,I , 28 93 I ,66, 8,l , 0 31 61 91 121 151 scan 0.52 1.02 1.52 2.03 2.53 Time Figure 3. A representative ion chromatogram of a low-level (0.0 100 mg a.i./L) PFBS standard. WILDLIFE INTERNATIONALTLD. - 48 - PROJECT NO.: 454A-129 intensity: 500000 cps i5d 131 4400 30- !230- -r 31 61 91 121 151 Scan 0,52 1,02 1,52 2,03 2,53 Tim Figure 4. A representative ion chromatogram of a high-level (0.0500 mg a.i./L) PFBS standard. WILDLIFE INTERNATIOLNTDA. L - 4.9 - PROJECT NO.: 454A-129 intensity: 500000 cps 100- 90. 8 0- I .v 70- 60. 50. 40. 30. 2 0- lo. 12 24 41 55 66 84 107120 134 164 scan o J I I I I I I I I I I , , , , l , I I 31 61 91 121 151 0.52 1.02 1.52 2,03 2.53 Time Figure 5 . A representative ion chromatogram of a matrix blank sample (454A-129-MAB-1). The arrow indicates the retention time o:FPFBS. WILDLIFE INTERNATIOLNTAD.L - 50 - PROJECT NO.: 454A-129 0intensity: 500000 cps 3 30.1 133 20 10. 15 33 44 66 84 Figure 6 . A representative ion chromatogram of a matrix fortification sample (454A-129-MAS-2, nominal PFBS concentration of 2500 mg a.i./L, dilution factor = 100000~). WILDLIFE INTERNATIONLTDA. L -51 - PROJECT NO.: 454A-129 intensity: 500000 cps 90- 60- 50 40- 30- 20 la 0-4 I 1 6I I 37I 31 0.52 67 I I I 61 1.02 31 85 102 ;1 I I I I I . 91 121 I.52 2.03 . . 151 2.53 . Scan Time Figure 7. A representative ion chromatogram of a. test sample (454A-129-4, nominal PFBS concentration of 1250 mg a.i./L, dilution factor = 40000~). wildlifehtemutional, Ltd. Proiect 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 capricomutum Freshwater medium Mean Measured Concentration (mg a.i./L) Replicate 24 Hours Cell Densities (Cells/mL)' 48 Hours 72 Hours Negative Control A .B C 2 1,000 25,000 47,000 168,000 184,000 185,000 1,200,000 1,045,000 800,000 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 7 10,000 3,825,000 5 63 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 2 1,000 118,000 530,000 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 5 1,000 171,000 850,000 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). wildlifeInternational, 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 PFJ3S Freshwater alga, Selenastrum capricornutum Freshwater medium Mean Measured Concentration (mg a.i./L) Replicate Cumulative Area Under the Growth Curve 0 - 24 Hours 0 - 48 Hours 0 - 72 Hours 0 - 96 Hours Negative Control A B C 132,000 180,000 444,000 2,160,000 2,448,000 2,98 8,000 18,336,000 16,956,000 14,568,000 77,3 16,000 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 3 12,000 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 5 1,432,000 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 3 1,884,000 C 108,000 1,308,000 7,020,000 34,560,000 456 1 9478 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 A 84,000 204,000 3 12,000 492,000 B 0 0 132,000 264,000 C 60,000 240,000 384,000 __4_32,000 wild1ife International, Ltd. - 54 - Project Number 454A-129 Append5x 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, Selenustrum cupricornutum Freshwater mediuni Mean Measured Concentration (mg a.i./L) Replicate 0 - 24 Hours Growth Rate 0 - 48 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 - 96 Hours 0.0617 0.0608 0.061 1 285 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 0.022 1 0.0492 0.0533 0.0600 C 0.0309 0.05 14 0.055 1 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 0.05 1 1 0.0548 4561 A 0.0076 0.0339 0.0394 0.0463 B 0.022 1 0.0433 0.0407 0.0465 C 0.022 1 0.0359 0.0391 0.0464 9478 A 0.022 1 0.0055 0.0065 0.0067 B 0.000 0.0084 0.0065 0.0042 C 0.0169 0.0144 0.0025 0.0019 'WildltfeInternational, Ltd. - 55 - Project Number 454A-129 Appendix 7 Changes to Protocol Ths study was conducted in accordancewith the approved Protocol with the followingchanges: 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 fkshwater algal medium constituents. 3. Two light intensity measurements during the recovery phase were outside of the acceptable range. Wild1fe International, Ltd, - 56 - Proiect Number 454A- 129 Appenldix 8 Personnel Involved in the Study The following key personnel were involved in the conduct or management of 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. KLUR-~. Drottar, Senior Biologist 5. Cary A. Sutherland,Laboratory Supervisor 6. Debbie Desjardins, Biologist