Document aJbpMM1dDaDJK4GR5nX96872b

A R U & -0 0* 5 PFOS: A 96-HOUR TOXICITY TEST W ITH THE FRESHWATER ALGA (Selenastrum capricomutum) FINAL REPORT WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER. 454A-103A 3M LAB REQUEST NO. U2723 U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.5400 and OECD Guideline 201 AUTHORS: K urt R. D rottar Henry O. Krueger, Ph.D. STUDY INITIATION DATE: December 4, 1998 STUDY COMPLETION DATE: June 4, 1999 AMENDED REPORT DATE: April 26, 2000 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, M innesota 55144 Wildlife International Ltd. 8598 Commerce Drive Easton, M aryland 21601 (410) 822-8600 Page 1 o f 58 000607 AMENDED W il d l if e In ter n a tio n a l ltd. 2- - PROJECT NO.: 454A-103A GOOD LABORATORY PR A C TIC E COM PLIANCE STATEM ENT SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Freshwater Alga (Selenastrum capricomutum) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-103A STUDY COMPLETION: June 4,1999 REPORT AMENDED: April 26, 2000 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 o f Good Laboratory Practice, OCDE/GD (92) 32, Environment Monograph No. 45, Paris 1992 and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984 with the following exceptions: The test substance was not characterized in accordance with full GLP compliance; however, the characterization was performed according to 3M Standard Operating Procedures and Methods, and all raw - data are being maintained in the 3M archives. The test substance is being recharacterized in accordance with GLP. The stability o f the test substance under conditions o f storage at the test site was not determined in accordance with Good Laboratory Practice Standards. ' STUDY DIRECTOR: Senior Biologist SPONSOR: (1 . iU p d /J ^ DATE y )# 7 / t o DATE AMENDED 000608 W il d l if e In te r n a tio n a l ltd. -3 - PROJECT NO.: 454A-103A QUALITY ASSURANCE STATEM ENT 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 o f Good Laboratory Practice, OCDE/GD (92) 32, Environment Monograph No. 45, Paris 1992; and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates o f all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Initial Trial 454A-103 Test Substance Preparation Instrument Set-up Draft Report and Data Definitive Trial 454A-103A Test Substance Preparation Draft Report and Data Analytical Data and Draft Report Final Report Amended Report DATE CONDUCTED: February 18,1999 February 22 and 23, 1999 March 29,1999 April 9,1999 May 10 and 11,1999 May 10 and 11,1999 June 4,1999 April 19 and 20,2000 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: February 18,1999 February 23, 1999 March 29, 1999 February 19,1999 February 24,1999 March 30, 1999 A p ril'9 ,1999 May 11, 1999 April 12,1999 May 12,1999 May 11,1999 June 4,1999 April 20,2000 May 11, 1999 June 4,1999 April 24,2000 ----- --------------------Timothy A Springer, Ph.D. Manager, Regulatory and Technical Support DATE AMENDED 000609 W il d l if e Inter n a tio n a l ltd. -4 - PROJECT NO.: 454A-103A REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Freshwater Alga (Selenastrum capricornutum) W ILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-103A STUDY DIRECTOR: V / o2/<3<2> DATE MANAGEMENT: and Non-Target Plants AMENDED 000610 W il d l if e In ter n a tio n a l ltd. -5- PROJECT NO.: 454A-103A TABLE OF CONTENTS Title/Cover P age................................................................................................................................................1 Good Laboratory Practice Compliance Statem ent.........................................................................................2 Quality Assurance Statement........................................................................................................................... 3 Report A pproval................................................................................... 4 Table o f Contents.................>v........................................................................................................................ 5 S um m ary............................................................................................................................................................7 Introduction....................................................................................................................................................... 9 O bjective............................................................................................................................................................9 Experimental Design.........................................................................................................................................9 M aterials and M ethods.............................................................................................................................. 10 Results and Discussion.......................................................................................................... 14 C o n clu sio n s............................................................................................................................. 17 R eferen ces...................................................................................................................... 18 TABLES Table 1 - Summary o f Analytical Chemistry D a ta ....................................................................................19 Table 2 - Temperature M easurements....................................................................................................... 20 Table 3 - Light Intensity Measurements..................................................................................................... 21 Table 4 - pH M easurements........................................................................................................................ 22 Table 5 - Mean Cell Densities and Percent Inhibition for Each 24-Hour Interval During the T est.............................................................................................. 23 Table 6 - Mean Areas Under the Growth Curve and Percent Inhibition for Each 24-Hour Interval During the T est.............................................................................................. 24 Table 7 - Mean Growth Rates and Percent Inhibition for Each 24-Hour Interval During the T est............................................................................................................................. 25 AMENDED 000611 W il d l if e In ter n a tio n a l ltd. -6 - PROJECT NO.: 454A-103A TABLE OF CONTENTS -C o n tin u ed - Table 8 - EC10, EC50 and EC90 Values Based on Cell Density Over the 96-Hour Exposure Period............................................................................................................................ 26 Table 9 - EC10, 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 for the Recovery P hase....................................................................................... 29 F IG U R E S 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 - Recovery Phase Response Curve, Expressed in Cell D ensity..................................................32 A PP E N D IC E S Appendix I - Freshwater Algal M edium ................................................... 33 Appendix II - Analyses o f Pesticides, Organics, M etals and Other Inorganics in Wildlife International Ltd. Well W ater......................................................................... 34 Appendix in - The Analysis o f PFOS in Freshwater Algal Medium in Support o f Wildlife International Ltd. Project No.: 454A-103A.....................................................35 Appendix IV - Cell Density for Each Replicate Per Treatment Over the 96-Hour Exposure Period...................................................................................................52 Appendix V - Area Under the Growth Curve for Each Replicate Per Treatment Over the 96-Hour Exposure Period...................................................................................................53 Appendix VI - Growth Rate for Each Replicate Per Treatment Over the 96-Hour Exposure P eriod. 54 Appendix VH - Changes to Protocol........................................... ..............................................................55 Appendix VUI - Personnel Involved in the Study........................ ............................................................. 56 Appendix IX - Report Amendment............................................................................................................57 AMENDED 000612 W il d l if e In t e r n a t io n a l ltd. -7 - PROJECT NO.: 454A-103A SPONSOR: SPONSOR'SREPRESENTATIVE: LOCATIONOFSTUDY, RAW DATAANDACOPYOFTHE 1 FINALREPORT: 1 WILDLIFEINTERNATIONALv. LTD. PROJECTNUMBER: TESTSUBSTANCE: STUDY: MEANMEASUREDTEST CONCENTRATIONS: TESTDATES: LENGTHOFEXPOSURE: TESTORGANISM: SOURCEOFTEST ORGANISMS: CELLDENSITY 72-HOUREC50: 95%CONFIDENCELIMITS: 96-HOUREC 10: 95% CONFIDENCELIMITS: 96-HOUREC50: 95%CONFIDENCELIMITS: 96-HOUREC90: 95% CONFIDENCELIMITS: 72-HOURNOAEC: 96-HOURNOAEC: SUMMARY 3M Corporation Ms. Susan A. Beach W ildlife International Ltd. Easton, MD 21601 454A-103A PFOS (Perfluoroocatane Sulfonic Acid Potassium Salt) PFOS: A 96-Hour Toxicity Test with the Freshwater Alga (Selenastrum capricomutum) Negative Control, 5.5, 11, 2 1 ,4 4 , 86 and 179 mg a.i./L Experimental Start - April 12, 1999 Exposure Termination - April 16, 1999 Experimental Termination -April 21, 1999 96 Hours Freshwater Alga (Selenastrum capricomutum) W ildlife International Ltd. Easton, M aryland 21601 70 mg a.i./L 44 and 78 mg a.i./L 49 mg a.i./L 43 and 50 mg a.i./L 71 m ga.i./L 66 and 73 mg a.i./L 137 mg a.i./L 105 and 153 mg a.L/L 44 mg a.i./L 44 mg a.i./L I AMENDED 000613 W il d l if e In ter n a tio n a l ltd. PROJECT NO.: 454A-103A 1 00 1 AREAUNDERTHEGROWTH CURVE 72-HOUREC50: 95% CONFIDENCELIMITS: 96-HOUREC10: 95% CONFIDENCELIMTTS: 96-HOUREC50: 95% CONFIDENCELIMITS: 96-HOUREC90: 95% CONFIDENCELIMITS: 72-HOURNOAEC: 96-HOURNOAEC: GROWTHRATE 72-HOUREC50: 95% CONFIDENCELIMITS: 96-HOUREC10: 95% CONFIDENCELIMITS: 96-HOUREC50: 95% CONFIDENCELIMITS: 96-HOUREC90: 95% CONFIDENCELIMITS: 72-HOURNOAEC: 96-HOURNOAEC: SUM M ARY (Continued) 74 mg a.i./L 55 and 82 mg a.iVL 49 mg a.iVL 40 and 50 mg a.i./L 71 mg a.i./L 67 and 74 mg a.i./L 145 mg a.i./L 125 and 155 mg a.i./L 44 mg a.i./L 44 mg a.i./L 120 mg a.i./L 103 and 132 mg a.i./L 59 mg a.i./L 54 and 63 mg a.i./L 126 mg a.i./L 115 and 138 mg a.i./L >179 mg a.i./L Not Calculable 44 mg a.i./L 44 mg a.i./L \ AMENDED 000614 W il d l if e Internatio nal ltd. -9 - PROJECT NO.: 454A-103A IN T R O D U C T IO N This study was conducted by W ildlife International Ltd. for 3M Corporation at the Wildlife International Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase o f th test was conducted from April 12, 1999 to April 21, 1999. Raw data generated by Wildlife International Ltd and a copy o f the final report are filed under Project Number 454A-103A in archives located on the Wildlife International Ltd. site. O BJECTIVE The objective o f the study was to evaluate the toxicity o f PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) to the growth o f the freshwater alga, Selenastrum capricornutum, during a 96-hour exposure period. EXPERIM ENTAL DESIGN The freshwater alga, Selenastrum capricomutum, 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, one "abiotic" replicate (test solution without algae) was prepared for the highest test concentration. Nominal test concentrations were selected in consultation with the Sponsor and were based upon the results o f range finding and definitive tests. The nominal test concentrations selected were 5.7, 11, 23, 46, 91 and 183 mg active ingredient (a.i.)/L. Mean measured test concentrations were determined from samples o f test medium collected from each treatm ent and the control group at test initiation, at approximately 72 hours, and at test termination. A t test initiation, an inoculum o f the algal cells was prepared at a concentration o f approximately 1.0 X 106 cells/mL. The concentration o f algal cells in the inoculum was verified and 1.0 mL was added to each test chamber to achieve a nominal concentration o f approximately 1.0 X 104 cells/mL. Samples were AMENDED 000615 W il d l if e In ter n a tio n a l ltd. -10- PROJECT NO.: 454A-103A 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 nnrfw the growth curve and growth rates. Percent inhibition values relative to the control were calculated for each param eter over the 96-hour exposure period. EC50 values based upon cell densities, areas under the growth curve and growth rates were calculated for each 24 hour interval. EC 10 and EC90 values were calculated, if possible, for the 72 and 96 hour intervals. The no-observed-adverse-effect-concentration (NOAEC) was determined based upon statistical evaluation o f the 72-hour and 96-hour results. At the end o f the 96-hour exposure, algistatic effects were differentiated from algicidal effects. M ATERIALS AND M ETHODS The study was conducted according to the procedures outlined in the protocol, "PFOS: A 96-Hour Toxicity Test with the Freshwater Alga (Selenastrum capricomutum)". 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 I find 11(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 October 29, 1998 and was assigned W ildlife International Ltd. identification number 4675. The test substance was described as a white powder. It was identified as FC-95 from lot number 217 (T-6295). Information provided by the Sponsor indicated a purity o f 98.9% and an expiration date o f 2008. The test substance was reanalyzed by the Sponsor and the Certificate o f Analysis dated March 9, 2000 indicated a purity o f 90.49%. The test substance was stored at ambient room temperature. Preparation o f Test Concentrations Nominal test concentrations were 5.7, 11, 23, 46, 91 and 183 mg a.i./L, based on a test substance purity o f 90.49%. All materials which came into contact with the test substance during preparation o f test concentrations were constructed o f plastic or stainless steel. A primary stock solution was prepared in algal AMENDED 000616 W il d l if e Inter n a tio n a l ltd. - 11- PROJECT NO.: 454A-103A medium at a concentration o f 183 mg a.i./L. The primary stock solution was stirred with a magnetic stir plate for approximately 24 hours to aid in the solubilization o f the test substance. After mixing the primary stock solution was proportionally diluted with algal medium to prepare the five additional test concentrations. All final test solutions appeared clear and colorless. Test Organism The freshwater alga, Selenastrum capricomutum, was selected as the test species for this study. The species is representative o f an important group o f 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 UTEX - The Culture Collection o f Algae at the University o f Texas at Austin and have been maintained in culture medium at W ildlife International Ltd., Easton, Maryland. Algal cells used in this test were obtained from W ildlife 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 tq exhibit exponential growth over the 96-hour exposure period. Exponential growth, 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). OECD guidelines recommend that control cell growth should have increased by a factor o f at least 16 within 3 days. 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 W ildlife 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 I). The pH o f the medium was adjusted to 7.5 0.1 using 10% HC1 and 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 13. AMENDED 000617 W il d l if e In ter n a tio n a l ltd. - 12- PROJECT NO.: 454A-103A 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 a mechanical shatTM- table in an environmental chamber designed to maintain the desired test temperature throughout the test. The test chambers were shaken continuously at 100 rpm. Environmental Conditions Test flasks were held in an environmental chamber at a temperature o f 242C. The temperature o f a container o f 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 f the shaker table daily. 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 o f test solution were collected from pooled replicates o f the treatment and control groups for pH measurement. Aleal Growth Measurements 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 000618 W il d l if e In ter n a tio n a l ltd. -13- PROJECT NO.: 454A-103A per grid was estimated and this value was used to calculate the cell density o f the sample. Using this technique, the minimum quantifiable cell density w as 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 o f the cells to the test chamber. 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+N2-2No)/2)(t2-t1)+((Nn.1+Nn-2N0)/2)(t11-tn.1) where: A = Area N0 = Nominal number o f cells/mL at to Ni = Mean measured number o f cells/mL at ti N2= Mean measured number o f cells/mL at t2 Nn= Mean measured number o f cells/mL at L ti = time o f first measurement after beginning o f test (hours) t2 = time o f second measurement after beginning o f test (hours) t,, = time o f n* measurement after beginning o f test (hours) Growth rates were calculated for the control and each treatm ent group using the following formula: Growth Rate = where: N0= Mean measured number o f cells/mL at to N,, = Mean measured number o f cells/mL at L tn = Time o f n* measurement after beginning o f test (hours) Percent inhibition values were calculated for each treatm ent 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: 000619 W il d l if e In t e r n a t io n a l ltd. - 14- PROJECT NO.: 454A-103A Percent Inhibition = Mean Cell D ensitvr^^i - Mean Cell Density^.-- , X 100 Mean Cell DensityControi 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 and Bartlett's test, respectively. 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 o f 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 o f the test substance. Samples o f test medium collected at test initiation were taken from the individual batches o f 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 o f the remaining replicates for each treatment and the control group. The samples were placed in plastic (Nalgene) bottles and were analyzed immediately without storage. Analytical procedures used in the analysis o f the samples are presented in Appendix EH. RESULTS AND DISCUSSION Measurement o f Test Concentrations Results o f analyses to measure concentrations o f PFOS in the test solutions are presented in Table 1 and Appendix HI. Nominal concentrations selected for use in this study were 5.7, 11, 23, 46, 91 and 183 mg a.i./L. Samples collected at the beginning o f the test had measured concentrations that ranged from 82 to 98% o f nominal. Samples collected at 72 hours and test termination had recoveries that ranged from AMENDED 000620 W il d l if e In te r n a tio n a l ltd. - 15- PROJECT NO.: 454A-103A 82 to 105% and 90 to 103% o f nominal, respectively. When the values obtained for test initiation, at 72 hours and at test termination were averaged, the mean measured test concentrations were 5.5, 11, 21, 44, 86 and 179 mg a.i./L. Observations and Measurements Measurements o f temperature and light intensity are presented in Tables 2 and 3, respectively. The temperatures ranged from 23.6.to 25.8C and were within the range established for the test (242C). The light intensity ranged from 3870 to 4540 lux and was within the desired range for the test (approximately 3870 to 4730 lux) (Table 3). Measurements o f pH were 7.4 to 7.5 on Day 0 and ranged from 7.5 to 8.4 at 96 hours (Table 4). The pH o f the "abiotic" replicate at test termination was 7.4. The effect o f PFOS upon Selenastrum capricomutum 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 IV, V and VI, respectively. EC 10, EC50 and EC90 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. 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%. After 72 horns o f exposure, cell density percent inhibition in the 5.5, 11, 21, 44, 86 and 179 mg a.i./L treatment groups was -5.2, -1.9, -5.5, 11, 72 and 97%, respectively. Dunnett's test showed that cell density was significantly reduced in the 86 and 179 mg a.i./L treatment groups in comparison to the negative control (psO.05). Consequently, the NOAEC for 72 hour cell density was 44 mg a.i./L. After 96 hours of exposure, cell density percent inhibition in the 5.5, 11, 21, 44, 86 and 179 mg a.i./L treatment groups was -11, -5.1, -18, -12, 77 and 99%, respectively. Dunnett's test showed that cell density was significantly reduced in the 86 and 179 mg a.i./L treatment groups (ps0.05). Consequently, the NOAEC for 96 hour cell density was 44 mg a.i./L. AMENDED oooezi W il d l if e In tern atio na l ltd. - 16- PROJECT NO.: 454A-103A After 72 hours o f exposure, area under the growth curve percent inhibition in the 5.5, 11, 21, 44, 86 and 179 mg a.i./L treatment groups was -2.9, 1.3, -3.8, 5.8, 68 and 94%, respectively. Dunnett's test showed that area under the growth curve was significantly reduced in the 86 and 179 mg a.i./L treatment groups (p^0.05). Consequently, the NOAEC for 72 hour area under the growth curve was 44 mg a.i./L. After 96 hours o f exposure, area under the growth curve percent inhibition in the 5.5, 11, 21, 44, 86 and 179 mg a.i./L treatment groups was -8.5, -3.3, -13, -5.3, 75 and 98%, respectively. Dunnett's test showed that area under the growth curve was significantly reduced in the 86 and 179 mg a.i./L treatment groups (p<;0.05). Consequently, the NOAEC for 96 hour area under the growth curve was 44 mg a.i./L. After 72 hours o f exposure, growth rate percent inhibition in the 5.5, 11, 21, 44, 86 and 179 mg a.i./L treatment groups was -1.1, -0.68, -0.72, 3.4, 31 and 82%, respectively. Dunnett's test showed that growth rate was significantly reduced in the 86 and 179 mg a.i./L treatm ent groups (psO.05). Consequently, the NOAEC for 72 hour growth rate was 44 mg a.i./L. After 96 hours o f exposure, growth rate percent inhibition in the 5.5, 11, 21, 44, 86 and 179 mg a.i./L treatment groups was -1.9, -0.84, -3.0 _ -2.0, 27 and 79%, respectively. Dunnett's test showed that growth rate was significantly reduced in the 86 and 179 mg a.i./L treatment groups (p^O.05). Consequently, the NOAEC for 96 hour growth rate was 44 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 f the algae to the test flasks in the negative control or any PFOS treatment group. In addition, there were no noticeable changes in cell color or morphology when compared to the negative control, although a few cells appeared enlarged in the 86 and 179 mg a.i./L treatment group. Reversibility o f Growth Inhibition The 179 mg a.i./L treatment group was maximally inhibited at the end o f the 96-hour exposure period. Aliquots o f the test solution were diluted with algal medium and cultured for five 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. AMENDED 000622 W il d l if e In t e r n a tio n a l ltd. -17- PROJECT NO.: 454A-103A C O N C L U S IO N S The conclusions o f 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 70 mg a.i./L with 95% confidence limits o f 44 and 78 mg a.i./L. The 96-hour EC10, based on cell density, was 49 mg a.i./L with 95% confidence limits o f 43 and 50 mg a.i./L. The 96-hour EC50, based on cell density, was 71 mg a.i./L with 95% confidence limits o f 66 and 73 mg a.i./L. The 96-hour EC90, based on cell density, was 137 mg a.i./L with 95% confidence limits o f 105 and 153 mg a.i./L The 72-hour and 96-hour NOAEC values, based on cell density, area under the growth curve and growth rate, were 44 mg a.i./L. Based on the presence o f visible algal growth at recovery termination, PFOS was considered to be algistatic, rather than algicidal, at the concentrations tested. AMENDED 000623 W il d l if e In t e r n a t io n a l l-td. -18- PROJECT NO.: 454A-103A REFERENCES 1 U.S. Environm ental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines {draft), OPPTS Number 850.5400: Algal Toxicity, Tiers I and 11. 2 O rganization of Economic C ooperation and Developm ent. 1984. Algal, Growth Inhibition Test. OECD Guideline for Testing o f Chemicals. Guideline 201. Paris. 3 ASTM S tandard Guide 1218-90E, Standard Guide fo r 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 W est, Inc. and D.D. Gulley. TOXSTAT Version 3.5. Copyright 1996. W estern EcoSystems Technology, Inc., Cheyenne, Wyoming. 000624 W il d l if e In ter n a tio n a l ltd. -19- PROJECT NO.: 454A-103A Table 1 Summary o f Analytical Chemistry Data Sponsor. Test Substance: Test Organism: Dilution Water: Nominal Concentration (mg a.i./L) Negative Control 3M Corporation PFOS Freshwater Alga, Selenastrum capricomutum Freshwater Algal Medium Sampling Measured Time Concentration1 lours) (mg a.i./L) 7 ? < LOQ 723 < LOQ 964 <LOQ Mean Measured Concentration (mg a.i./L) Percent of Nominal 5.7 0 4.73 72 6.04 96 5.84 5.5 96 11 0 10.7 72 11.2 96 12.1 11 100 23 0 19.8 72 23.1 96 20.7 21 91 46 0 42.7 44 96 72 41.9 * 96 46.3 91 0 83.3 72 86.0 96 88.3 86 95 183 0 179 72 186 96 172 179 98 183 I T 150 96s 188 169 92 `Limit of Quantitation (LOQ) was 0.115 mg a.i./L. 20-hour samples were collected from individual batches of test solution prepared for the treatment and control groups at test initiation. 372-hour samples were collected from the additional (D) replicate (biotic). 496-hour samples were composites of test solution collected from each of the three replicates per treatment and control group. jT T j^ J h O T r^ ^ le M lle rte d ^ m ^ ^ t io ^ J E ^ re p lic a te (abiotic) AMENDED 000625 W il d l if e In ter n a tio n a l ltd. 20 PROJECT NO.: 454A-103A Table 2 Temperature Measurements Sponsor: Test Substance: Test Organism: Dilution W ater: Time (Day) 3M Corporation PFOS Freshwater Alga, Selenastrum capricomutum = 2 ^ S ^ S L ^i .^ !L = = = !=========== Temperature (C) V Measurement 1 Measurement 2 0 23.6 24.2 1 24.8 24.2 2 24.8 3 25.1 4 25.8 24.0 25.3 24.9 5 25.7 24.4 6 25.6 24.9 7 24.0 8 23.9 9 23.9 24.0 23.8 23.9 000626 W il d l if e Inter n a tio n a l ltd. - 21- PROJECT NO.: 454A-103A Table 3 Light Intensity Measurements SBBSSSSSSn Sponsor: Test Substance: Test Organism: Dilution Water: Test *~*= Day 0 1 2 3 4 5 6 7 8 9 3M Corporation PFOS Freshwater Alga, Selenastrum capricomutum Freshwater Algal Medium Light Intensity Measurements (lux) No. 1 3940 3910 4000 4120 4200 3910 4180 4300 4010 4150 No. 2 3880 4270 4290 4400 3990 4120 4200 4180 4230 4090 No. 3 4300 4440 4310 4350 3890 4180 4120 3950 4180 4240 SBBSS5SS^^SBS59& No. 4 3900 4610 4540 4160 4070 4200 4200 4430 4230 4350 No.5 3870 4010 4170 4240 4140 4340 4370 4080 4450 4270 000627 W il d l if e In ter n a tio n a l ltd. -22- PROJECT NO.: 454A-103A Table 4 pH Measurements Sponsor: 3M Corporation Test Substance: Test Organism: PFOS Freshwater Alga, Selenastrum capricomutum Dilution W ater: Freshwater Algal Medium Mean Measured pH Measurements Concentration (mg a.i./L) 0 Hours1 Negative Control 7.5 96 Hours2 8.1 5.5 7.4 8.2 11 7.5 8.2 21 7.5 8.4 44 7.4 8.3 86 7.4 7.8 179 7.4 : 7.5 169 (Abiotic) 7.4 1 0-hour samples were collected from the batches o f test solution prepared for the treatm ent and control groups at test initiation. 2 96-hour samples were collected from the pooled replicates per treatment and control group. AMENDED OQOGZS -23- PROJECT NO.: 454A-103A Table 5 Mean Cell Densities and Percent Inhibition for Each 24-Hour Interval During the Test1 Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Freshwater Alga, Selenastrum capricomutum Dilution Water: Freshwater Algal Medium Mean Measured 24 Hours Concentration (mg a.i./L) Mean Cell Density Percent Inhibition Mean Cell Density Negative Control 32,667 - 129,000 48 Hours Percent Inhibition -- _____ 72 Hours Mean Cell " Percent Density Inhibition 603,333 - 5.5 29,000 11 129,333 -0.26 635,000 -5.2 11 29,333 10 121,000 6.2 615,000 -1.9 21 28,000 14 133,667 -3.6 636,667 -5.5 44 29,667 9.2 140,667 -9.0 535,000 11 86 25,667 21 56,000 57 171,0002 72 179 14,333 56 27,667 79 20,6672 1 Values calculated using SAS 6.12. Manual calculations jnay differ slightly 2 Indicates a significant difference from the negative control at 72 hours using Dunnett's test ip <0.05). 3 Indicates a significant difference from the negative control at 96 hours using Dunnett's test (p 0.05). 97 Mean Cell Density 2,740,000 96 Hours Percent Inhibition - 3,040,000 -11 2,880,000 -5.1 3,240,000 -18 3,080,000 -12 626,6673 77 33,6673 99 0C0629 AMENDED - 24- PR0JECTN0.: 454A-103A Table 6 Mean Areas Under the Growth Curve and Percent Inhibition for Each 24-Hour Interval During the Test1 Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Freshwater Alga, Selenastrum capricomutum Dilution Water: Freshwater Algal Medium________________ Mean Measured Test Concentration (mg a.i./L) 0 - 24 Hours Mean Percent Area Inhibition 0 - 48 Hours Mean Percent Area Inhibition Negative Control 272,000 -- 1,972,000 - 0 - 72 Hours Mean 'Percent Area Inhibition 10,520,000 -- 5.5 228,000 16 1,888,000 4.3 10,820,000 -2.9 11 232,000 15 1,796,000 8.9 10,388,000 1.3 21 216,000 21 1,916,000 2.8 10,920,000 -3.8 44 236,000 13 2,040,000 -3.4 9,908,000 5.8 86 188,000 31 928,000 53 3,412,0002 68 179 52,000 81 316,000 84 656,0002 94 1 Values calculated using SAS 6.12. Manual calculations may differ slightly 2 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 i 0.05). Mean Area 0 - 96 Hours Percent Inhibition 50,400,000 - 54,680,000 -8.5 52,088,000 -3.3 57,200,000 -13 53,048,000 -5.3 12,7444,0003 75 1,068,0003 98 000630 AMENDED - 25Table 7 PROJECT NO.: 454A-103A Mean Growth Rates and Percent Inhibition for Each 24-Hour Interval During the Test1 Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Freshwater Alga, Selenastrum capricomutum Dilution Water: Freshwater Algal Medium Mean Measured Test Concentration (mg a.i./L) 9 - 24 Hours Mean Percent Growth Rate Inhibition 0 - 48 Hours Mean Percent Growth Rate Inhibition Negative Control 0.0493 -- 0.0533 - 0 - 72 Hours Mean Percent Growth Rate In h ib itio n 0.0568 -- 5.5 0.0438 11 0.0533 0.0 0.0574 -1.1 11 0.0448 9.1 0.0518 2.7 0.0572 -0.68 21 0.0428 13 0.0540 -1.4 0.0572 -0.72 44 0.0452 8.3 0.0551 -3.3 0.0549 3.4 86 0.0373 24 0.0357 33 0.03922 31 O ooK> 179 0.0138 72 0.0208 61 1 Values calculated using SAS 6.12. Manual calculations may differ slightly 2 Indicates a significant difference from the negative control at 72 hours using Dunnett's test (p 0.05). Indicates a significant difference from the negative control at 96 hours using Dunnett's test (p <. 0.05). 82 0 - 96 Hours Mean Percent Growth Rate Inhibition 0.0585 - 0.0595 -1.9 0.0590 -0.84 0.0602 -3.0 0.0597 -2.0 0.04293 27 0.01243 79 000631 AMENDED - 26 Table 8 PROJECT NO.: 454A-103A EC 10, EC50 and EC90 Values Based on Cell Density Over the 96-Hour Exposure Period Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Freshwater Alga, Selenastrum capricornutum Dilution Water: Freshwater Algal Medium 95% Confidence Time EC 10 (mg a.i./L) Limits (mg a.i./L) 24 Hours Not Determined -- 48 Hours Not Determined -- 72 Hours 37 <0.0 and 64 96 Hours 49 43 and 50 EC50 (mg a.i./L) 163 81 70 71 95% Confidence Limits (mg a.i./L) 74 and 191 72 and 90 44 and 78 66 and 73 EC90 (mg a.i./L) Not Determined Not Determined 153 137 95% Confidence Limits (mg a.i./L) - - 130 and 165 105 and 153 000632 AMENDED - 27 - PROJECT NO.: 454A-103A 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: 3M Corporation PFOS Freshwater Alga, Selenastrum capricomutum 24 Hours Not Determined 95% Confidence Limits (mg a.i./L) - 48 Hours Not Determined - 72 Hours 46 <0.0 and 56 96 Hours 49 40 and 50 EC50 (mg a.i./L) 122 84 74 71 95% Confidence Limits (mg a.i./L) 19 and 176 67 and 146 55 and 82 67 and 74 EC90 (m tyuV ^ Not Determined Not Determined 165 145 95% Confidence Limits (mg fli/L ) -- - 145 and 176 125 and 155 000633 AMENDED -28Table 10 PROJECT NO.: 454A-103A EC 10, EC50 and EC90 Values Based on Growth Rate Over the 96-Hour Exposure Period Sponsor: 3M Corporation Test Substance: Test Organism: PFOS Freshwater Alga, Selenastrum capricormtum Dilution Water: Freshwater Algal Medium 95% Confidence EC10 Limits Time (mg a.i./L) (mg a.i./L) 24 Hours Not Determined - 48 Hours Not Determined -- 72 Hours 53 23 and 64 96 Hours 59 54 and 63 Confidence limits could not be calculated with the data obtained. EC50 (mg a.i./L) 136 142 120 126 .. i 95% Confidence Limits , (mg a.i,/L) 30 and 204 107 and 185 103 and 132 115 and 138 EC90 (mg a.i./L) Not Determined Not Determined >179 >179 95% Confidence Limits (mg a.i./L) - - --1 --1 / 000634 AMENDED W il d l if e In t e r n a t io n a l ltd. -29- PROJECT NO.: 454A-103A T ab lell Cell Densities for the Recovery Phase Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Freshwater Alga, Selenastrum capricomutum Freshwater Algal Medium Mean Measured Test Concentration (mg a.i./L) Day 0 Negative Control Rep. A 10,000 1,220,000 Day 5 6,420,000 1791 . 1,000 3 5 ,0 0 0 _____________ 1,180,000 1 The treatment group was diluted to a concentration o f 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. 000635 AMENDED W il d l if e In t e r n a t io n a l Ltd. -30- PROJECT NO.: 454A-103A Figure 1. Negative Control Algal Growth, Expressed in Cell Density, During the 96-Hour Exposure. Mean Cell Density (Cells/mL) Negative Control 000636 AMENDED W il d l if e In t e r n a tio n a l ltd. -31- Figure 2. Concentration-Response Curve, Expressed in Cell Density PROJECT NO.: 454A-103A Mean Cell Density (Cells/mL) --a-- Negative Control -- --5.5 mg/L --O - l l m g / L --A--21 mg/L --0 --4 4 mg/L -- -- 86 mg/L -- 179 mg/L 000637 AMENDED Wild life International ltd. -32- Figure 3 - Recovery Phase Response Curve, Expressed in Cell Density PROJECT NO.: 454A-103A Cell Density (Cells/mL) Negative Control 179 mg/L 000638 AMENDED W il d l if e Inter n a tio n a l ltd. -33- PROJECT NO.: 454A-103A A P P E N D IX I Freshwater Algal Medium1 Sponsor: Test Substance: Test Organism: 3M Corporation PFOS Freshwater Alga, Selenastrum capricomutum um Nominal Concentration M gCl2*6H 20 12.16 mg/L C aC l22H 20 4.40 mg/L H3BO3 0.1856 mg/L M nC l2*4H 20 0.416 mg/L ZnCl2 : 3.28 Mg/L FeC l36H 20 0.1598 mg/L CoC126H 20 1.428 Mg/L N a2M o042H20 7.26 Mg/L CuC122H 20 0.012 Mg/L N a2ED TA 2H 20 0.300 mg/L N aN 03 25.50 mg/L M gS04*7H20 14.70 mg/L K2HP0 4 1.044 mg/L N aH C 03 15.0 i The pH was adjusted to 7.5 0.1 using 10% HC1. mg/L 000639 W il d l if e In ter n a tio n a l lto. 34 PROJECT NO.: 454A-103A APPENDIX II Analyses o f Pesticides, Organics, M etals and Other Inorganics in W ildlife International Ltd. Well W ater1 ANALYSIS Miscellaneous Measurements Total Dissolved Solids Ammonia Nitrogen Total Organic Carbon2 Total Cyanide MEASURED CONCENTRATION 286 < 0.050 < 1.0 < 10.0 mg/L mg/L mg/L Mg/L Organochlorines and PCBs A larm Alpha BHC Beta BHC Delta BHC Gamma BHC (Lindane) Chlordane DDD, pp' DDE, pp' DDT, pp' Dielcinn Endosulfan, A Endosulfan, B Endosulfan Sulfate Endrin Endrin Aldehyde Heptachlor Methoxychlor Heptachlor Epoxide Toxaphene PCB-1016 PCBH221 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 < 0.005 < 0.005 < 0.005 < 0.005 < 0.006 < 0.025 < 0.006 < 0.005 < 0.008 < 0.005 < 0.005 < 0.005 < 0.018 < 0.010 < 0.005 < 0.005 < 0.007 < 0.005 < 0.500 < 0.260 < 0.260 < 0.260 < 0.720 < 0.720 < 0.720 < 0.720 M g/L &L vmL M g/L M g/L Mg/L Mg/L M g/L Mg/L Mg^ M g M g/L S/L Mg/L M g/L M g/L Mg, M g/L M g/L M g/L Mg/L Mg/L M&L Mg/L M g/L Mg/L M etals and O ther Inorganics Aluminum3 Arsenic3 Beryllium: Cadmium3 Calcium3 Chromium3 Cobalt3, Lead3 Magnesium: Manganese3 Mercury Molybdenum N ick er Iron3 Selenium3 Silver , Sodium3 Zinc3 < < < < < < < A, < < < < < < < < < 100 25.0 0.50 1.0 35.0 2.0 1.0 20.0 100 10.0 13.5 1.0 0.20 2.0 2.0 6.62 25.0 1.0 21.3 20.0 M g/L m& M g/L M g/L mgA M g/L M g/L Mg M g/L M g/L mg/L Mg^ M g/L M&L M g/L mg/L M g/L M g/L mg/L Mg/L Analyse:s performed b y (S T Environmental, Gainesville, Florida lor samples collected on November 3 through November 7,1997. 2 Analyses performed by Wildlife International Ltd. for the sample collected on November 5, 1997. Analyses performed by Wildlife International Ltd. for samples collected on November 5 through 7,1997. 000640 W il d l if e In t e r n a t io n a l Ltd . -35- APPENDIX ffl PROJECT NO.: 454A-103A THE ANALYSIS OF PFOS IN FRESHWATER ALGAL MEDIUM IN SUPPORT OF W ILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-103A 000641 W il d l if e In ter n a tio n a l ltd. -36- PROJECT NO.: 454A-103A REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Freshwater Alga (Selenastrum capricomutum) W ILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-103A PRINCIPAL INVESTIGATOR: W illard B. Nixon, Ph.D. Manager, Analytical Chemistry DATE 000642 AMENDED Wild life International ltd. -37- PROJECT NO.: 454A-103A Introduction Freshwater algal medium samples were collected from an acute toxicity study designed to determine the effects o f PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) to the freshwater alga (Selenastrum capricomutum). This study was conducted by W ildlife International Ltd. and identified as Project No.: 454A-103A. The analyses o f these water samples were performed at W ildlife International Ltd. using high performance liquid chromatography with mass spectrom etric detection (HPLC/MS). Samples were received for analysis on April 12,15 and 16,1999 and were analyzed on each sample receipt day. Test Substance and Internal Standard The test substance used for the analytical portion o f this study was W ildlife International Ltd. identification number 4675. The test substance was used to prepare calibration standards and m atrix fortification samples. The internal standard was received from 3M Corporation on July 2, 1998 and was assigned W ildlife International Ltd. identification number 4526 upon receipt. The internal standard, a granular material, was identified as: IH, IH, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical A bstract Number: 27619-97-2. The standard was stored under ambient conditions. Analytical Method The method used for the analysis o f the freshwater algal medium samples was developed at W ildlife International Ltd. and entitled "Analytical Method V alidation for the- Determination o f PFOS in Freshwater, Saltwater, and Algal Media". This methodology was included as Appendix II o f W ildlife In tern a tio n a l Ltd. protocol number 454/011299/MVAL/SUB454. It was based upon methodology provided by 3M Corporation. Samples were diluted in a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% form ic acid (v/v) so that they fell within the calibration range o f the PFOS methodology. Concentrations o f PFOS in the standards and samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid 000643 W il d l if e Intern atio na l ltd. -38- PROJECT NO.: 454A-103A Chromatograph (HPLC) with a Perldn-Elmer API 100LC M ass Spectrometer equipped with a PerkinElmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil Cig analytical column (100 mm x 2 mm I.D., 3 pm particle size). The instrument parameters are summarized in Table 1. A method flowchart is provided in Figure 1. Calibration Curve and Limit o f Quantitation Calibration standards o f PFOS prepared in a 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v), ranging in concentration from 0.00229 to 0.0457 mg a.i:./L were analyzed with the samples. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS : internal standard) o f the calibration standards. A typical calibration curve is presented in Figure 2. The concentration o f PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms o f low and high.calibration standards are presented in Figures 3 and 4, respectively. The method limit o f quantitation (LOQ) for these analyses was set a t 0.115 mg a.i./L calculated as the product o f the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor o f the matrix blank samples (50.0). M atrix Blank and Fortification Samples Three matrix blank samples were analyzed to determine possible interference. No interferences were observed at or above the LOQ during samples analyses (Table 2). A representative chromatogram o f a matrix blank is presented in Figure 5. Freshwater algal medium was fortified at 0.915, 45.7 and 220 mg a.L/L and analyzed concurrently with the samples to determine the mean procedural recovery (Table 3). Sample concentrations were not corrected for the mean procedural recovery o f 99.1%. A representative chromatogram o f a matrix fortification is presented in Figure 6. 000644 AMENDED W il d l if e In te r n a tio n a l ltd. -39- PROJECT NO.: 454A-103A Example Calculations Sample number 454A-103A-2, nominal concentration o f 5.7 mg a.i./L in freshwater algal medium. Initial Volume: 0.100 mL Final Volume: 25.0 mL Dilution Factor: 250 PFOS Peak Area: 164427 Internal Standard Peak Area: 240533 Peak Area Ratio: 0.6836 ; Calibration curve equation. Slope: 3.43926 Intercept: 0.03327 Curve is weighted (1/x). PFOS (pg a.i./L) at instrument (Peak area ratio - (Y-intercept)) x I.S. Concentration Slope (0.6836 - 0.03327) x 100 pg a.i./L _____ - = 18.9 pg a.i./L Note: I.S. = internal standard. PFOS (mg a.i./L) in sample PFOS (pg a.i./L) at instrument x Dilution Factor 1000 pg/mg 18.9 x 250 000645 AMENDED W il d l if e Internatio nal ltd. -40- PROJECT NO.: 454A-103A = 4.73 mg a.i./L Percent o f Nominal Concentration PFOS (mg a.i./L) in sample = ------------------------------------x 100 PFOS (mg a.i./L) nominal Calculated recovery: 82.0% Note: manual calculation may differ. RESULTS Sample Analysis Freshwater algal medium samples were collected from the acute toxicity study with the freshwater alga (Selenastrum capricomutum) at test initiation, April 12, 1999 (Hour 0), on April 15, 1999 (Hour 72), and at test termination, April 16, 1999 (Hour 96). The measured concentrations o f PFOS in the samples collected at initiation o f exposure of the test organisms (Hour 0) ranged from 82.0 to 98.1% o f the nominal concentrations. Samples collected at Hour 72 had a measured concentration range o f 91.7 to 105% of nominal values. The Hour-72 abiotic sample was 81.9% o f the nominal concentration. Samples collected at test term ination (Hour 96) had a measured concentration range o f 90.3 to 102% o f nominal values. The Hour-96 abiotic sample was 103% o f the nominal concentration (Table 4). A representative chromatogram o f a test sample is shown in Figure 7. 000646 AMENDED W il d l if e Internatio nal ltd. -41 - PROJECT NO.: 454A-103A IN STR U M EN T: Table 1 Typical LC/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC M ass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil C column (100 mm x 2 mm I.D ., 3 pm particle size) OVEN TEMPERATURE: : 30C STOP TIME: 10.0 minutes FLOW RATE: 0.220 mL/minute MOBILE PHASE: 72.0% Methanol : 28.0% NANOpure W ater containing 0.1% Formic Acid INJECTION VOLUME: 25.0 pL PFOS RETENTION TIME: Approximately 6.6 minutes INTERNAL STANDARD RETENTION TIME: Approximately 4.5 minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 426.7 amu 000647 Wil d l if e Intern atio na l ltd. -42- PROJECT NO.: 454A-103A Table 2 M atrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-103A-) MAB-1 Sample Type M atrix Blank Measured Concentration o f PFOS* (mg a.i./L) <LOQ MAB-2 M atrix Blank <LOQ <y ohJ V MAB-3 M atrix Blank 1 The limit o f quantitation (LOQ) was 0.115 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor o f the matrix blank samples (50.0). #0648 AMENDED W il d l if e Intern atio na l ltd. -43- PROJECT NO.: 454A-103A Table 3 M atrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-103A-) MAS-1 MAS-4 MAS-7 Concentrations o f PFOS (mg a.i./L) Fortified M easured 0.915 0.915 0.915 0.884 1.10 1.02 Percent Recovered 96.6 120 112 MAS-2 MAS-5 MAS-8 45.7 , 45.7 45.7 41.3 48.4 . 41.4 90.2 106 90.6 MAS-3A MAS-3B 220 220 210 201 95.2 91.7 MAS-3C MAS-6 MAS-9 220 220 220 209 221 202 95.0 101 91.9 Mean = 99.1 Standard Deviation =9.75 CV =9.84 N = 11 . Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes._____________ 000649 AMENDED W il d l if e In ter n a tio n a l ltd. -44- PROJECT NO.: 454A-103A Table 4 Measured Concentrations of PFOS in Freshwater Algal Medium Samples from a Freshwater Alga Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 0.0 Sample Number (454A-103A-) 1 8 15 Sampling Time (Hours) 0 72 96 PFOS Measured Concentration1 (mg a.i./L) < LOQ < LOQ < LOQ Percent of Nominal --- 5.7 2 0 9 72 16 96 4.73 82.0 6.04 105 5.84 101 11 3 0 10 72 17 96 10.7 89.8 11.2 93.6 12.1 102 23 4 0 11 72 18 96 19.8 86.2 23.1 101 20.7 90.3 46 5 0 12 72 19 96 42.7 93.4 41.9 91.7 46.3 101 91 6 0 13 72 20 96 83.3 91.0 86?0 94.0 88.3 96.5 183 7 0 14 72 21 96 179 98.1 186 102 172 94.0 183 (Abiotic) 14A 22 72 96 150 81.9 188 103 Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 software and manual calculations. Values have been rounded for reporting purposes. 'The limit o f quantitation (LOQ) was 0.115 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.00229 mg a.i./L) and the dilution factor of the matrix blank samples (50.0).______ 000650 AMENDED W il d l if e In t e r n a t io n a l Ltd . -45- PROJECT NO.: 454A-103A M ETHOD O UTLINE FO R TH E ANALYSIS O F PFOS IN FRESH W ATER ALGAL M EDIUM Prepare m atrix fortification samples by spiking the requisite volume o f PFOS stock solutions directly into freshwater algal medium using gas-tight syringes and Class A volumetric flasks. i Dilute m atrix fortification and test samples into the range o f the calibration standards by partially filling Class A volumetric flasks with 50% methanol : 50% NANOpure water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v). Add the appropriate volume of sample and bring the flask to volume with the dilution solvent. Process the matrix blank sample using the same dilution and aliquot volume as for the lowest fortification level. M ix well by several repeat inversions. I Amputate samples and submit for LC/MS analysis. Figure 1. Analytical method flowchart for the analysis o f PFOS in freshwater algal medium. 000651 W il d l if e Internatio nal ltd. -46- PROJECT NO.: 454A-103A Figure 2. A typical calibration curve for PFOS. Slope = 3.43926; Intercept = 0.03327; r = 0.9986. Curve is weighted (1/x). 000652 AMENDED W il d l if e In te r n a tio n a l ltd. -47- PROJECT NO.: 454A-103A intensity: 2849 cps 157 Figure 3. A representative ion chromatogram of a low-level (0.00229 mg a.i./L) PFOS standard. 000653 AMENDED W il d l if e Inter n a tio n a l ltd. -48- PROJECT NO.: 454A-103A Figure 4. A representative ion chromatogram of a high-level (0.0457 mg a.i./L) PFOS standard. 000654 AMENDED Wil d l if e International lto. -49- PROJECT NO.: 454A-103A intensity: 234 cps Figure 5. A representative chromatogram o f a m atrix blank sample (454A-103A-MAB-1). The arrow indicates the retention time o f PFOS. 000655 W il d l if e Internatio nal ltd. -50- PROJECT NO.: 454A-103A 100908070605040302010- o- 24"a"r-.3..2r_ i i 7 i 7i... 3IA' - 31 61 91 1.30 2.55 3.81 115 intensity: 9104 cps 157 1n 906 211 8.83 Scan Time Figure 6. A representative chromatogram o f a matrix fortification sample (454A-103A-MAS-1). 000656 W il d l if e Intern atio na l ltd. -51- PROJECT NO.: 454A-103A F igure 7. A representative chromatogram o f a test sample (454A-103A-2). 000657 Wil d l if e International ltd. -52- PROJECT NO.: 454A-103A A PPEN D K IV Cell Density for Each Replicate Per Treatment Over the 96-Hour Exposure Period Sponsor Test Substance: Test Organism: Dilution W ater 3M Corporation PFOS Freshwater Alga, Selenastrum capricomutum Freshwater Algal Medium Mean Measured Concentration (mg a.i./L) Replicate 24 Hours Negative Control A 34,000 B 33,000 C 31,000 Cell Densities i(Cells/mL)1 48 Hours 132,000 130,000 125,000 72 Hours 710,000 605,000 495,000 5.5 A 29,000 136,000 780,000 ;B 23,000 118,000 . 500,000 *C 35,000 ` 134,000 625,000 11 A 30,000 105,000 560,000 B 27,000 124,000 660,000 C 31,000 134,000 625,000 21 ~ A 30,000 141,000 860,000 B 26,000 132,000 450,000 C 28,000 128,000 600,000 44 A 30,000 136,000 515,000 B 32,000 151,000 700,000 C 27,000 135,000 390,000 86 A 37,000 65,000 215,000 B 18,000 57,000 136,000 C 22,000 46,000 162,000 96 Hours 2,720,000 2,860,000 2,640,000 3,000,000 3,180,000 2,940,000 2,680,000 2,740,000 3,220,000 3,340,000 3,080,000 3,300,000 2,800,000 3,020,000 3,420,000 650,000 760,000 470,000 179 A 15,000 35,000 21,000 45,000 B 18,000 22,000 A 18,000 30,000 C 10,000 26,000 23,000 26,000 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 ofapproximately 10,000 cells/mL at test initiation (0 hours). 000658 AMENDED Wild life International ltd. -53- PROJECT NO.: 454A-103A APPENDIX V Area Under the Growth Curve for Each Replicate Per Treatment Over the 96-Hour Exposure Period Sponsor 3M Corporation Test Substance: PFOS Test Organism: Freshwater Alga, Selenastrum capricomutum DuOTLW^tCT;^^_ Fre^iwater Algal Medium Mean Measured Concentration ====^ Cumulative Area Under the Growth Curve (mg a.i./L) Replicate 0 - 2 4 Hours 0 - 4 8 Hours 0 - 7 2 Hours Negative Control A 288,000 2,040,000 11,904,000 B 276,000 1,992,000 10,572,000 C 252,000 1,884,000 9,084,000 0 - 9 6 Hours 52,824,000 51,912,000 46,464,000 5.5 A 228,000 1,968,000 12,720,000 57,840,000 B 156,000 1,608,000 8,784,000 52,704,000 C 300,000 2,088,000 10,956,000 53,496,000 11 A 240,000 1,620,000 9,360,000 48,000,000 B 204,000 1,776,000 10,944,000 51,504,000 C 252,000 1,992,000 10,860,000 56,760,000 21 A 240,000 2,052,000 13,824,000 63,984,000 B 192,000 1,848,000 8,592,000 50,712,000 C 216,000 1,848,000 10,344,000 56,904,000 44 A 240,000 1,992,000 9,564,000 49,104,000 B 264,000 2,220,000 12,192,000 56,592,000 C 204,000 1,908,000 7,968,000 53,448,000 86 A 324,000 1,308,000 4,428,000 14,568,000 B 96,000 756,000 2,832,000 13,344,000 C 144,000 720,000 2,976,000 10,320,000 179 A 60,000 420,000 852,000 1,404,000 B 96,000 336,000 576,000 912,000 C 0 192,000 540,000 888,000 000659 AMENDED W il d l if e In ter n a tio n a l ltd. -54- PROJECT NO.: 454A-103A APPENDIX VI Growth Rate for Each Replicate Per Treatment Over the 96-Hour Exposure Period Sponsor Test Substance: Test Organism: 3M Corporation PFOS Freshwater Alga, Selenastrumcapricomutum Dilution W ater Freshwater Algal Medium Mean Measured Concentration Growth Rate (mg a.i./L) Replicate _ J ) i 24Houre__ 0 - 4 8 Hours 0 -7 2 Hours Negative Control A 0.0510 0.0538 0.0592 B 0.0497 0.0534 0.0570 C 0.0471 0.0526 0.0542 5.5 -A 0.0444 0.0544 0.0605 B 0.0347 0.0514 . 0.0543 X 0.0522 0.0541 0.0574 11 A 0.0458 0.0490 0.0559 B 0.0414 0.0525 0.0582 C 0.0471 0.0541 0.0574 21 A 0.0458 0.0551 0.0619 B 0.0398 0.0538 0.0529 C 0.0429 0.0531 0.0569 44 A 0.0458 0.0544 0.0547 B 0.0485 0.0566 0.0590 C 0.0414 0.0542 0.0509 86 A 0.0545 0.0390 0.0426 B 0.0245 0.0363 0.0363 C 0.0329 0.0318 0.0387 179 A 0.0169 0.0261 0.0103 B 0.0245 0.0164 /x 0.0082 C 0.0000 0.0199 0.0116 0 - 9 6 Hours 0.0584 0.0589 0.0581 0.0594 0.0600 0.0592 0.0582 0.0585 0.0602 0.0605 0.0597 0.0604 0.0587 0.0595 0.0608 0.0435 0.0451 0.0401 0.0157 0.0114 0.0010 000660 AMENDED W il d l if e In te r n a tio n a l ltd. -55- PROJECT NO.: 454A-103A APPENDIX VH 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, test concentrations and test substance number. 2. The protocol was amended to specify the analytical method. 3. The protocol was amended to repeat the test with a higher test concentration. 000661 W il d l if e In te r n a tio n a l ltd. -56- APPENDIX VIE Personnel Involved in the Study PROJECT NO.: 454A-103A The following key personnel were involved in the conduct or management o f this study: 1. Henry O. Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. W illard B. Nixon, Ph.D., Manager, Analytical Chemistry 3. Raymond L. VanHoven, Ph.D., Scientist 4. K urt R. D rottar, Senior Biologist 5. M ark A. Mank, Laboratory Supervisor 000662 W il d l if e In ter n a tio n a l ltd. -57- PROJECT NO.: 454A-103A APPENDIX DC Report Amendment 1. Original Report: Pages 1-4, 6 and 36 Amendment: The pages were changed to include the amended report date, revised page numbers, and new signatures and dates due to the addition o f the report amendment as Appendix DC. Reason: To reflect the issuing o f an amended report. 2. Original Report: Page 2 Amendment: The compliance statement was revised. Reason: To clarify how the test substance was characterized. 3. Original Report: Page 10 Amendment: Information provided by the Sponsor reflecting the reanalysis o f the test substance, includine the reanalvsis date and the nuritv. was added to the Test Substance section. Reason: To reflect the current test substance information provided by the Sponsor. 4. Original Report: Entire report Amendment: All test substance concentrations were changed to reflect the purity o f the test substance as determined by the Sponsor in a reanalysis o f the test substance (FC95, Lot 217). Test concentrations originally were based on the reported purity o f 98.9%. The certificate o f analysis dated M arch -9, 2000 indicated a purity o f 90.49%. Therefore, all test substance concentrations, including nominal concentrations, measured concentrations, EC values, and NOAEC values were recalculated and reported as mg a.i./L based on the 90.49% purity. Reason: To report the results o f the test based on the test substance purity o f 90.49% at the request o f the Sponsor. 000663 AMENDED W i l d l i f e In t e r n a t i o n a l ltd. PROJECT NO.: 454A-103 Page 1 of 1 AMENDMENT TO STUDY PROTOCOL STU DY T IT L E : PFOS: A 96-HOUR TOXICITY TEST W ITH TH E FRESH W ATER ALGA (Selenastrum capricornutum) P R O T O C O L N O .: 4 5 4 /1 10998/SEL-96H 1/SU B 454 A M E N D M E N T N O .: 1 SPO N SO R : 3M C orporation P R O JE C T N O .: 4 5 4 A -103 E F F E C T IV E D A TE: January 4, 1999 AM ENDM ENT: Page 2 Add: Experim ental S tart D ate: 2/19/99 Experim ental T erm ination D ate: 2/23/99 T est C oncentrations: N egative C ontrol, 6.3, 13, 25, 50 and 100 mg a.i./L T est Substance N o.: 4675 R E A SO N : T he above inform ation w as no t know n w hen the protocol w as signed by th e Study D irector. A M E N D M E N T : A PPEN D IX II, P age 16 Add: L iquid C hrom atography M ass Spectrom etry (LCM S) M ethod for the D eterm ination o f Perfluorooctane Sulfonic A cid, Potassium Salt (PFO S) in Freshw ater, Filtered S altw ater and A lgal M edium . R E A S O N : T o add th e analytical m ethod to be used in th e study. STUDY D IRECTO R DATE a454\103\amendl W il d l if e Internatio nal ltd. PROJECT NO.: 454A-103 Page 1 of 1 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA (Selenastrum capricornutum) PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 AMENDMENT NO.: 2 SPONSOR: 3M Corporation PRO JECT NO.: 454A-103 EFFECTIV E DATE: April 8, 1999_____________________________________________ _ AMENDMENT: Page 2 Change:. Experimental Start Date: 2/19/99 Experimental Termination Date: 2/23/99 Test Concentrations: Negative Control, 6.3, 13,25,50 and 100 mg a.i./L To: Experimental Start Date: 4/12/99 Experimental Termination Date: 4/16/99 Test Concentrations: Negative Control, 6 .3 ,1 3 ,2 5 ,5 0 ,1 0 0 and 200 mg a.i./L REASON: The test will be repeated because cell counts were not made with a hemacytometer and a microscope. An additional test concentration was added in an attempt to obtain EC90 values. AMENDMENT: EXPERIM ENTAL DESIGN. Page 3 Change: Target concentrations will not exceed 100 mg/L or the solubility limit of the test substance in water. To: Target concentrations will not exceed 1000 mg/L or the solubility limit o f the test substance in water. REASON: The new nominal concentrations exceed 100 mg a.i./L. STUDY DIRECTOR f/# IT 7 DATE a4S4\l 03\amend2 000665 & i W ly V ft PROTOCOL 8 PFOS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA (Selenastrum capricornutum) U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.5400 OECD Guideline 201 3M Lab Request No. U2723 Submitted to 3M Corporation Environmental Laboratory P.O. Box 33331 St. Paul, Minnesota 55133 Wildlife International ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 November 9,1998 000666 PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In ter n a tio n a l ltd . -2- PFOS: A 96-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA (Selenastrum capricornutum) SPONSOR: 3M Corporation Environmental Laboratoiy P.O. Box 33331 St. Paul, Minnesota 55133 SPONSOR'S REPRESENTATIVE: Ms. Susan A. Beach TESTING FACILITY: Wildlife International Ltd. 8598 Commerce Drive Easton, Maryland 21601 STUDY DIRECTOR: Kurt Drottar Senior Aquatic Biologist LABORATORY MANAGEIMMI Henry 0 . Krueger, Ph.D. Director o f Aquatic Toxicology & Non-Target Plants FOR LABORATORY USE ONLY Proposed Dates: Experimental Experimental Start Date: ____________________________ Termination Date: ______ Project No.: A - l 0 ^ > __________________________________ Test C oncentrations:______________________________________________ Test Substance No.: __________ Reference Substance No. (if applicable): PROTOCOL APPROVAL STUDY DIRECTOR / w m DATE 000667 PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 3M LAB REQUEST NO. U2723 Wil d l if e Internatio nal ltd. -3 - IN T R O D U C T IO N W ildlife International Ltd. will conduct a 96-hour toxicity test with the freshwater alga, Selenastrum capricomutum, for the Sponsor at the Wildlife International Ltd. aquatic toxicology facility in Easton, Maryland. The study will be performed based on procedures in the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.5400 (1) and OECD Guideline for Testing o f Chemicals, 201: Alga, Growth Inhibition Test (2). Raw data for all work performed at Wildlife International Ltd. and a copy o f the final report will be filed by project n u m b e r in archives located on the Wildlife International Ltd. site or at an alternative location to be specified in the final report. PURPOSE The purpose o fthis study is to determine the toxicity o f a test substance to the freshwater green alga, Selenastrum capricomutum, during a 96-hour exposure period. EXPERIM ENTAL DESIGN The green alga, Selenastrum capricomutum, will be exposed to a geometric series of at least five - test concentrations and a negative (culture medium) control for 96.hours. Target concentrations will not exceed 100 mg/L or the solubility limit of the test substance in water. Generally, the nominal concentration o f each test substance used in the definitive test will be at least 50% o f the next higher treatment, unless information concerning the concentration-effect curve indicates that a different dilution factor would be more appropriate. Test solutions will be inoculated with 10,000 cells/mL. Three "biological" replicates per experimental group will be prepared for evaluating cell densities. One or more additional "analytical" replicates will be included in the test, as needed, to provide test solution for concentration verification on Day 3 o f the exposure. An abiotic replicate at the highest concentration will be included in the test and w ill be sampled on Day 3 and Day 4. In order to control bias, the position of the flasks will be determined by in d iscrim inate draw daily during the exposure period. No other potential sources o f bias are expected to affect the results o f the study. The response of the algae will be measured in terms o f cell density, biomass expressed as area under the growth curve, and growth rate. An EC50 (i.e., the theoretical test concentration that produces a 50% reduction in the measured parameter) value for cell density will be calculated for each 24 hour interval. EC10, EC50 and EC90 values for cell density, biomass (Et,C) and growth rate (ErC) will be 000665 PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 3M LAB REQUEST NO. U2723 W i l d l i f e In t e r n a t i o n a l ltd. -4 - calculated, if possible, at the 72 and 96 hour intervals. The no observed adverse effect concentration (NOAEC), which is the highest test concentration that induces no adverse inhibitory effect on growth, will be determined relative to each parameter at 72 and 96 hours based upon evaluation o f the statistical results and the dose-response pattern. At the end o f the 96-hour exposure, algistatic effects will be differentiated from algicidal effects in those treatments which are maximally inhibited. M ATERIALS AND M ETHODS Test Substance Information on the characterization o f test, control or reference substances is required by Good Laboratory Practice Standards (GLP). The Sponsor is responsible for providing Wildlife International Ltd. written verification that the test substance has been characterized according to GLPs prior to its use in the study. If written verification o f GLP test substance characterization is not provided to Wildlife International Ltd., it will be noted in the compliance statement o f the final report. The attached form IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR (Appendix I) is to be used to provide information necessary for GLP compliance. The Sponsor is responsible for all information related to the test substance and agrees to accept any unused test substance and/or test substance containers remaining at the end of the study. Preparation o f Test Solutions The test substance will be administered to Selenastrum in an aqueous medium. This route of administration was selected because it represents the most likely route of exposure to algae, which exists in aquatic environments. Test solutions willbe prepared by diluting one or more stock solutions with algal culture medium to achieve the target concentrations. Stocks will be prepared in algal culture medium. All weights, volumes and dilutions used to prepare the test solutions will be documented in the raw data. Test Organism The freshwater green alga, Selenastrum capricornutum, will be used in this test. This species is representative of an important group o f algae and was selected for use in the test based upon past use and ease of handling in the laboratory. Stock cultures, obtained from the Culture Collection o f Algae at the University of Texas at Austin or another supplier, will be maintained in culture medium at W ildlife International Ltd. for a minimum of two weeks prior to use in a toxicity test. Algae used in toxicity tests 000669 PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e Intern atio na l ltd. -5 - will be in exponential growth phase, which is defined as the period o f growth when algal cells are dividing at a constant rate. Just prior to beginning the test, an inoculum o f the stock culture will be prepared so that each milliliter o f inoculum contains enough cells to provide an initial cell density o f approximately 10,000 cells/mL in each replicate. Culture Medium Culture medium prepared according to Wildlife International Ltd. Standard Operating Procedures will be used as dilution water. The concentrations of the components in the medium are presented in Table 1 (3). Stock nutrient solutions will be prepared by adding reagent-grade chemicals to Wildlife International Ltd. well water purified by reverse-osmosis. Appropriate volumes of the stock nutrient solutions will then be diluted with purified well water to prepare the medium. The medium will be filter sterilized (0.2 on or autoclaved) prior to use. The final pH o f the medium should be 7.5 0.1. Analy ses w ill be performed at least once annually to determine the concentrations o f selected organic and inorganic constituents o f the well water and results ofthe analyses will be summarized in the final report. Specifications for acceptable levels o f contaminants have not been established for culture medium However, there are no levels of contaminants reasonably expected to be present in the medium that are considered to interfere with the purpose or conduct o f the study. Test Apparatus T est chambers will be sterile, 250-mL polycarbonate Erlenmeyer flasks plugged with foam stoppers. Each test chamber will hold 100 mL o f test solutioa Test chambers will be indiscriminately positioned on a mechanical shaker table in an environmental chamber and will be shaken continuously at approximately 100 rpm. Test chambers will be labeled with the project number, test concentration, and replicate. Environmental Conditions Test flasks will be held at 24 2C under continuous fluorescent lighting (e.g., cool white light tubes) at an intensity o f approximately 4300 10% lux. Light intensity will be measured at five locations on the shaker table daily during the test. Temperature will be measured twice daily in the environmental chamber using a liquid-in-glass thermometer. 0C0G70 PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 3M LAB REQUEST NO. U2723 Wil d l if e In ter n a tio n a l ltd . 6- - The pH of each treatment and the control will be measured at test initiation and termination using a Fisher Accumet Model 915 pH meter or equivalent. Samples will be collected from the batch o f test solution prepared at test initiation for each treatment and control group for the measurement of pH. At test termination, pH will be measured in pooled test solution samples collected from the three replicates o f each treatment and control group. Biological Measurements Cell densities will be monitored during the test by conducting cell counts using a hemacytometer and a microscope. One sample will be collected from each replicate o f the treatment and control groups at approximately 24 hour intervals during the exposure period. The samples will be evaluated immediately. Each sample will be diluted using an electrolyte solution (Isoton), as needed, to maintain counting accuracy. The cell density of the sample will be calculated based on the mean number o f cells per grid. Samples o f test solutions will be collected from each replicate per treatment and control group at the end o f the test. Each replicate sample will be examined microscopically for atypical cell morphology (e.g., changes in shape, size or color). Growth of cells in the flasks also will be assessed for aggregations or flocculations o f cells and adherence of the cells to the test chamber. A t the end o f the test, algistatic effects, which result in the inhibition o f cell growth, will be differentiated from algicidal effects, which result in the death o f cells. Aliquots (0.5 mL) o f test solutions will be taken from each replicate test chamber where growth is maximally inhibited. Maximally inhibited treatments are those in which it cannot be visually determined if live algal cells are present (e.g. absence o f color to the test solution). These aliquots will be combined and diluted with algal medium to a concentration of the test substance that theoretically should not affect growth. A negative control will be prepared by diluting a 0.5-mL aliquot from one negative control replicate to 100 mL with algal medium. Growth o f these subcultures will be monitored for up to 9 days to determine whether inhibition o f growth observed during the test is reversible. Samples will be collected for cell counts at recovery phase initiation, termination and at approximately 3-day intervals between initiation and termination The recovery phase may be terminated once algal growth is sufficient to indicate that the algal cells have fully recovered from effects due to the test substance. This will be determined through visual examination o f the recovery test solutions. PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 000671 3M LAB REQUEST NO. U2723 g Wild life International ltd . -7 - I Sampling for Analytical McaSUTCmCIlia I Samples o f the exposure solutions will be collected on Days 0, 3 and 4 and analyzed fo r test substance concentrations. Day 0 samples w ill be taken to r n each batch o f test solution prior to their I distribution among the replicate test chambers. Day 3 samples w ill be collected from the extra replicate test cham ber included in the test to provide sample for that analysis and from the abiotic replicates. M If more than one replicate is included for the Day 3 sample analysts, then the solutions from each replicate will be composited to provide the sample for that treatm ent. Day 4 samples will be composited solutions from the three replicates remaining a t the end o f the test. Samples collected on Days 3 and 4 may be centrifuged o r filtered to remove algal cells prior to analysis Samples wifi be analyzed immediately or placed in an appropriate storage container (e.g,, polypropylene or polyethylene bottle) and stored until analyzed. The sample scheme is summarized a below: PROPOSED NUMBERS OF VERIFICATION SAMPLES Experimental Group Day 0 Day 3 Day 4 Control 11 1 a Solvent Control (if needed) Level 1-Low Concentration a Level 2 Level 3 Level 4 a Level 5-High Concentration Totals 11 1 11 1 11 1 11 1 11 1 11 1 77 7 aa The above numbers ofsamples represent those collected from the test and do not include quality control (QC) samples such as matrix blanks and fortifications prepared and analyzed during the a analytical chemistry phase o f the study. aa 000872 PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In te r n a tio n a l ltd . 8- - Analvtical Chemistry Chemical analysis ofthe samples will be performed by Wildlife International Ltd. The analytical method used will be based upon methodology provided by the Sponsor and identified in Appendix II. Modifications made to the analytical method will be documented in the raw data and described in the final report. Statistical Analyses Biomass expressed as area under the growth curve will be calculated for the treatment and control groups using the following formula: A = ((N1-No)/2)(t1)+((N1+N2-2N0)/2)(t2-t1)+ -+ ((N n.1+Nn-2No)/2)(tn-tI1.1) Where: A = Area under the growth curve N0 = Measured number o f cells/mL at t0 N ; =Measured number o f cells/mL at t, N2 =Measured number o f cells/mL at t, Nn=Measured number o f cells/mL at ^ tj = Time of first measurement after beginning o f test (hours) tj = Time o f second measurement after beginning o f test (hours) L = Time of nth measurement after beginning o f test (hours) Growth rates (jj) will be calculated for the treatment and control groups using the following formula: lnN n.ln N 0 (U. = ---------------- t,,-to tu. = Average specific growth rate N0 = Measured number o f cells/mL at t0 Nn= Measured number o f cells/mL at ^ to = Time o f beginning o f test (hours) t,, = Time o f nth measurement after beginning o f test (hours) PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 000673 3M LAB REQUEST NO. U2723 [ Wildlife aa In t e r n a t io n a l ltd. -9 - Percent inhibition will be calculated for each treatment group based upon mean cell density, biomass and growth rate using the following formula: i Mean Control - Mean Treatment Percent Inhibition = Response______Response X 100 Mean Control Response EC values will be determined, when possible, using linear interpolation or other suitable techniques with treatment response (i.e. cell densities, biomass and growth rate) and exposure concentration data for each 24-hour interval o f the test. A no observed adverse effect concentration (NOAEC) will be selected based on an evaluation o f the dose-response pattern and the results of the statistical analysis using individual replicate values o f the cell density, biomass and growth rate. Statistically significant differences between the control and the treatment groups will be identified using analysis o f variance (ANOVA) and a test to compare treatment mean responses to the control response (e.g., Dunnett's test or Bonferroni's t-test). Data will be assessed for normality and homogeneity of variances prior to performing the ANOVA. Transforma tions will be used to correct any condition of non-normality or unequal error variances, if possible. If i a solvent control is used in addition to the negative control, their responses will be compared using Student's t-test. Data from the two control groups will be pooled for treatment level analyses if no statistical differences exist. Otherwise, all comparisons will be made to the solvent control group. Statistical comparisons for the ANOVA and the comparison o f mean responses will be carried out using TOXSTAT V3.5 software (4) or equivalent. RECORDS TO BE M AINTAINED Records to be maintained for data generated at W ildlife International Ltd. will include, but not be limited to: 1. Copy o f signed protocol. 2. Identification and characterization o f the test substance, if provided by Sponsor. 3. Dates o f initiation and termination o f the test. 4. Source of algae. 0006*74 5. Culture conditions. PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In ter n a tio n a l ltd . -10- 6 . Growth measurements. 7. Calculation and preparation o f test concentrations. 8. Observations. 9. The methods used to analyze test substance concentrations and the results o f analytical measurements. 10. Statistical calculations. 11. Test conditions and physical/chemical measurements. 12. Copy o f final report. FINAL REPORT A final report of the results ofthe study will be prepared by Wildlife International Ltd. The report will include, but not be limited to the following, when applicable: 1. N ame and address o f the facility performing the study. 2. Dates upon which the study was initiated and completed, and the definitive experimental start and termination dates. 3. A statem ent o f compliance signed by the Study Director addressing any exceptions to Good Laboratory Practice Standards. 4. O bjectives and procedures, as stated in the approved protocol, including all. changes to the protocol. 5. The test substance identification including name, chemical abstract number or code number, strength, purity, composition, and other information provided by the Sponsor. 6 . Stability and solubility o f the test substance under the conditions of administration, if provided by the Sponsor. 7. A description o f the methods used to conduct the test. 8. A description of the test organisms, including the source, scientific name, age or life stage, light intensity and photoperiod. 9. A description o f the preparation o f the test solutions. 10. The methods used to allocate organisms to test chambers and begin the test, the number o f organisms and chambers per treatment, and the duration of the test. 11. A description of circumstances that may have affected the quality or integrity o f the data. 0 0 0 6 7 5 PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 3M LAB REQUEST NO. U2723 Wil d l if e In t e r n a t io n a l ltd. -11 - 12. The name of the Study Director and the names of other scientists, professionals, and supervisory personnel involved in the study. 13. A description of the transformations, calculations, and operations performed on the data, a summary and analysis o f the biological data and analytical chemistry data, and a statement o f the conclusions drawn from the analyses. A graph plotting the concentration response curve. 14. Statistical methods used to evaluate the data. 15. The signed and dated reports o f each o f the individual scientists or other professionals involved in the study. 16. The location where raw data and final report are to be stored. 17. A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made and the dates of any findings reported to the Study Director and Management. 18. If it is necessary to make corrections or additions to a final report after it has been accepted, such changes will be made in the form o f an amendment issued by the Study Director. The amendment will clearly identify the part o f the final report that is being amended and the reasons for the amendment, and will be signed by the Study Director. CHANGING OF PROTOCOL Planned changes to the protocol will be in the form o f written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part o f the protocol and will be attached to the final protocol. Any other changes will be in the form o f written deviations signed by the Study Director and filed with the raw data. All changes to the protocol will be indicated in the final report GOOD LABORATORY PRACTICES This study will be conducted in accordance with Good Laboratory Practice Standards for EPA (40 CFR Part 160 and/or Part 792); OECD Principles o f Good Laboratory Practice (OCDE/GD (92) 32, Environment Monograph No. 45); and Japan MAFF (59 NohSan, Notification No. 3850, Agricultural Production Bureau). Each study conducted by Wildlife International Ltd. is routinely exam ined by the Wildlife International Ltd. Quality Assurance Unit for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. A statement of PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 3M LAB REQUEST NO. U2723 000676 W il d l if e In t e r n a t io n a l ltd. -12- compliance with Good Laboratory Practices will be prepared for all portions o f the study conducted by W ildlife International Ltd. The Sponsor will be responsible for compliance with Good Laboratory Practices for procedures performed by other laboratories (e.g., residue analyses or pathology). Raw data for all work performed at Wildlife International Ltd. and a copy o f the final report will be filed by project num ber in archives located on the Wildlife International Ltd. site or at an alternative location to be specified in the final report. PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 000677 3M LAB REQUEST NO. U2723 I | W i l d l i f e In t e r n a t i o n a l ltd . I -13 - | REFERENCES 1 U.S. Environmental Protection Agency. 1996. Series 850- Ecological Effects Test Guidelines (draft), OPPTS Number 850.5400: Algal Toxicity, Tiers I and II. I 2 OECD Guideline for Testing of Chemicals. 1984. 201: Alga, Growth Inhibition Test. I 3 ASTM S tandard Guide 1218-90E. 1990. Standard Guidefo r Conducting Static 96-Hour Toxicity Tests with Microalgae. 4 W est, Inc. and D. D. Gulley. 1996. TOXSTATRelease 3.5. Western EcoSystems Technology, I Inc. Cheyenne, Wyoming. I I I I I 000678 PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 3M LAB REQUEST NO. U2723 W il d l if e International ltd. -14- TABLE1 FRESHWATER ALGAL MEDIUM CONSTITUENTS Component Nominal C oncentration MgCl2*6H20 CaCl22H20 H3BO3 MnCl2*4H20 ZnCl2 FeCl3*6H20 CoC126H20 N ajM oO ^H jO CuC12*2H20 Na2EDTA-2H20 NaNOj M gS04*7H20 k 2h p o 4 NaHCO, 12.16 mg/L 4.40 mg/L 0.1856 mg/L 0.416 mg/L 3.28 /g/L 0.1598 mg/L 1.428 jug/L 7.26 Mg/L 0.012 Mg/L 0.300 mg/L 25.50 mg/L 14.70 mg/L 1.044 mg/L 1*5.00 mg/L The pH o f the medium will be adjusted, as necessary, to 7.5 0.1 using 0.1 N NaOH or 10% HC1. PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 000679 3M LAB REQUEST NO. U2723 Wil d l if e Internatio nal ltd . - 15- APPENDIXI IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR To be Completed by Sponsor I. Test Substance Identity (name to be used in the report): PFOS CPerfluorooctane Sulfonic Acid Potassium Salt Reference Standard (if applicable): Analytical Standard: N/A____________________________________ Internal Standard: 1.1.2.2H.H.H.H Perfluorooctane Sulfonic Acid Test Substance Sample Code or Batch Number: Lot 217_______________________________________ Test Substance Purity (% Active Ingredient): 98,9______ Expiration Date: 2008_______________ II. Test Substance Characterization Have the identity, strength, purity and composition or other characteristics which appropriately define the test substance and reference standard been determined prior to its use in this study in accordance with GLP Standards? Y es____ No X III. Test Substance Storage Conditions Please indicate the recommended storage conditions at Wildlife International Ltd. Ambient room temperature______________________________________________________________ Has the stability o f the test substance under these storage conditions been determined in accordance with GLP Standards? Y es_____No X Other pertinent stability information: ------------------------------------------------------------------------------------- IV. Test Concentrations: Adjust test concentration to 100% a.i. X based upon the purity (%) given above. Do not adjust test concentration to 100% _____a.i. Test the material AS IS. V. Toxicity Information: Mammalian: Rat LD50 251 mg/kg Mouse LD50 N/A Aquatic: Invertebrate Toxicity (EC/LC50) Fish Toxicity (LC50) Daphniamaana: 27 me/L__________ . RamhQW.JjQUt; U p ig/L Daphnia maana: 50m e/L__________ , Fathead Minnow; 38m g/L Other Toxicity Information (including findings o f chronic and subchronic tests): _ Please see MSDS___________________________________________________________ PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 000680 3M LAB REQUEST NO. U2723 W il d l if e In te r n a t io n a l ltd. - 16- APPENDIXII Analytical Method to be Provided by Sponsor PROTOCOL NO.: 454/110998/SEL-96H1/SUB454 000681 3M LAB REQUEST NO. U2723