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Mll'Oioi PFOS: A FLOW -THROUGH LIFE-CYCLE TOXICITY TEST WITH THE SALTWATER MYSID (Mysidopsis bahia) FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-107 3M LAB REQUEST NO. U2723 U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1350 AUTHORS: Kurt R. Drottar Henry 0 . Krueger, Ph.D. STUDY INITIATION DATE: May 21,1999 STUDY COMPLETION DATE: April 26,2000 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, MN 55144 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 60 001191 W il d l if e In t e r n a t io n a l , Lt d . 2- - PROJECT NO.: 454A-107 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Flow-Through Life-Cycle Toxicity Test with the Saltwater Mysid (Mysidopsis bahia) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-107 STUDY COMPLETION: 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 of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984, with the following exceptions: The test substance was not characterized in 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 of storage at the test site, was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Kurt R. Drottar Senior Biologist SPONSOR APPROVAL: DATE Sponsor 001192 W il d l if e In t e r n a t io n a l , Lt d . -3 - PROJECT NO.: 454A-107 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Protocol Test Substance Preparation Light Meter Reading and Analytical Sampling Salinity Measurements Calibration Standards Preparation Biological Data and Draft Report Analytical Data and Draft Report Final Report DATE CONDUCTED: May 24,1999 June 10,1999 June 16,1999 June 17,1999 July 21,1999 October 1,4 and 5,1999 October 4 -7 ,1 9 9 9 April 20,2000 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: May 24,1999 July 16,1999 June 14,1999 June 16,1999 June 17,1999 June 18,1999 June 18,1999 June 18,1999 July 21,1999 July 21,1999 October 6,1999 October 7,1999- October 7,1999 October 8,1999 April 20,2000 April 24,2000 k'r'J uaL . 'U n tisi. Kimberly A. Hoxjer Quality Assurance Representative DATE 001193 W il d l if e In t e r n a t io n a l , Lt d . -4 - PROJECT NO.: 454A-107 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Flow-Through Life-Cycle Toxicity Test with the Saltwater Mysid (.Mysidopsis bahia) V WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-107 STUDY DIRECTOR: MANAGEMENT: Director, Aquatic Toxicology and Non-Target Plants DATE 001194 W il d l if e In te r n a tio n a l, Lt d . -5 - PROJECT NO.: 454A-107 TABLE OF CONTENTS Title/Cover Page..................................................................................... Good Laboratory Practice Compliance Statement................................ Quality Assurance Statement................................................................. Report Approval..................................................................................... V/ Table of Contents................................................................................... Summary................................................................................................. Introduction............................................................................................ Objective................................................................................................ Experimental Design.............................................................................. Materials and Methods........................................................................... Results and Discussion........................................................................... Conclusions............................................................................................. References...................................... ................... .................................... ..1 ..2 ..3 ..4 ..5 .7 .8 .8 .8 .9 14 15 17 TABLES Table 1 - Summary o f Analytical Chemistry D ata...................................................................................... 18 Table 2 - Salinity o f Water in the Negative Control and Highest Treatment Group Test Chambers..... 19 Table 3 - pH of Water in the Test Chambers...............................................................................................20 Table 4 - Temperature (C) o f Water in the Test Chambers...................................................................... 21 Table 5 - Dissolved Oxygen Content (mg/L) of Water in the Test Chambers......................................... 22 001195 W il d l if e In t e r n a t io n a l, Lt d . 6- - PROJECT NO.: 454A-107 TABLE OF CONTENTS - Continued - Table 6 - Survival of Juvenile Mysids Exposed to PFOS (Day 0 Through Pairing on Day 20)............................................................................................. 23 Table 7 - Survival of Adult Mysids Exposed to PFOS (Day 20 Through Test Termination on Day 35).... ..................................................................... 24 Table 8 - Mean Number of Young Produced Per Reproductive Day..........................................................25 Table 9 - Mean Total Length o f Adult Mysids at Test Termination...........................................................26 Table 10 - Mean Dry Weight o f Adult Mysids at Test Termination............................................................ 27 Table 11 - Survival of Second-Generation Mysids Exposed to PFOS During a 96-Hour Static Exposure............................................................................................... 28 APPENDICES Appendix I - Dilution Water Salinity Measured During the ' 4-Week Period Immediately Preceding the Test................................................................. 29 Appendix II - Analyses of Pesticides, Organics, Metals and Other Inorganics in Wildlife International, Ltd. Saltwater............................................................................. 30 Appendix III- The Analysis o f PFOS in Saltwater in Support o f Wildlife International, Ltd. Project No.: 454A-10 7 .................................. 31 Appendix IV - Mysid Reproduction.............................................................................................................52 Appendix V - Mysid Total Length (m m )....................................................................................................55 Appendix VI - Mysid Dry Weight (m g)...................................................................................................... 57 Appendix VH - Changes to Protocol............................................ ................................................................. 59 Appendix VIII - Personnel Involved in the Study........ ................................................................................. 60 001196 W il d l if e In t e r n a t io n a l , Lt d . -7 - PROJECT NO.: 454A-107 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: 'vj WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: NOMINAL TEST CONCENTRATIONS: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF FIRSTGENERATION EXPOSURE: TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: NOEC: LOEC: MATC: SUMMARY 3M Corporation Susan A. Beach Wildlife International, Ltd. Easton, Maryland 21601 | 454A-107 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A Flow-Through Life-Cycle Toxicity Test with the Saltwater Mysid (.Mysidopsis bahia) Negative Control, 0.086,0.17,0.34,0.69,1.4 and 2.7 mg a.i./L Negative Control, 0.057,0.12,0.25,0.55,1.3 and 2.6 mg a.i./L Experimental Start (OECD) - May 26,1999 Experimental Start (EPA) - June 16,1999 Biological Termination - July 25,1999 Experimental Termination - July 25, 1999 1 35 Days Saltwater Mysid (Mysidopsis bahia) Wildlife International, Ltd. Cultures Easton, Maryland 21601 Juveniles <24 hours old 0.25 mg a.i./L 0.55 mg a.i./L 0.37 mg a.i./L II U . ------- 1 001197 W il d l if e In te r n a tio n a l, Lt d . 8- - PROJECT NO.: 454A-107 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 phase of the test was conducted from June 16, 1999 to July 25,1999. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454A-107 in archives located on the Wildlife International, Ltd. site. v. ' OBJECTIVE The objective of this study was to evaluate the effects of Perfluorooctane Sulfonic Acid Potassium Salt (PFOS) on the survival, growth and reproduction o f the saltwater mysid (Mysidopsis bahia) under flow-through test conditions. EXPERIMENTAL DESIGN Mysidopsis bahia neonates, less than 24 hours old, were exposed to a geometric series of six test concentrations and a negative (saltwater) control for 35 days. Nominal test concentrations were selected in consultation with the Sponsor, and'were based upon known toxicity data. Nominal test concentrations were 0.086, 0.17, 0.34, 0.69, 1.4 and 2.7 mg active ingredient (a.i.)/L. Mean measured test concentrations were determined from samples o f test water collected from each treatment and control group at the beginning of the test, at weekly intervals during the test and at test termination. Delivery of the test substance was initiated approximately 51 hours prior to the introduction of the neonate mysids to the test water in order to achieve equilibrium of the test substance in the test chambers. Four replicate test chambers, each containing one compartment with 15 mysids, were maintained for each treatment and control group. To begin the test, neonate mysids were impartially distributed in groups of one or two among glass beakers until each beaker contained 15 mysids. The mysids were then transferred to the test compartments. A total of 60 mysids were exposed in each treatment and the control group. On Day 20 of the test, female and male adults were paired, and the reproduction of the paired mysids was monitored through Day 35. Observations of mortality, clinical signs of toxicity, and reproduction were made 001198 W il d l if e In t e r n a t io n a l , Lt d . -9 - PROJECT NO.: 454A-107 daily. At test termination, the lengths and dry weights of all surviving first-generation mysids were measured All young produced in the test were removed to a separate test chamber at the same test concentration on a daily basis. The second-generation mysids were exposed for 96 hours under static test conditions. The no-observed-effect-concentration (NOEC) and lowest-observed-effect-concentration (LOEC) were determined by examination of the mortality, growth and reproduction data. The maximum acceptable toxicant concentration (MATC) was calculated as the geometric mean of the NOEC and LOEC. MATERIALS AND METHODS The study was conducted based on the procedures outlined in the protocol, "PFOS: A Flow-Through LifeCycle Toxicity Test with the Saltwater Mysid (Mysidopsis bahia)". The protocol was based on procedures outlined in U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines, OPPTS Number 850.1350 (1); and ASTM Standard E l 191-90 Standard Guide fo r Conducting Life-Cycle Toxicity Tests with Saltwater Mysids (2). Test Substance The test substance was received from 3M Corporation on October 29, 1998 and was assigned Wildlife 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 of 90.49%, and an expiration date of 2008. The test substance was stored at ambient room temperature. Preparation of Test Concentrations One stock solution was prepared for each of the six concentrations tested. A primary stock was prepared by dissolving the test substance in dilution water at a concentration of 0.0895 mg a.i./mL. The primary stock was stirred with an electric stainless steel top-down mixer for approximately 24 hours to aid in the solubilization of the test substance. After mixing, the primary stock appeared clear and colorless with a foam on the surface. Aliquots of the primary stock solution were proportionally diluted with dilution water to prepare five additional stocks at concentrations of 0.0447, 0.0224, 0.0112, 0.00559 and 0.00280 mg a.i./mL. The six stocks were injected into the diluter mixing chambers (at a rate of 4.60 mL/minute) where they were mixed with dilution water 001199 W il d l if e In t e r n a t io n a l, Lt d . -10- PROJECT NO.: 454A-107 (at a rate of 150 mL/minute) to achieve the desired test concentrations. All test solutions appeared clear and colorless. Test Organism The saltwater mysid, Mysidopsis bahia (recently renamed Americamysis bahia), was selected as the test species for this study. The saltwater mysid is representative of an important group of aquatic organisms and was selected for use in the test based upon past history of use and ease of culturing in the laboratory. Mysids used in the test were neonates less than 24 hours old and were obtained from cultures maintained by Wildlife International, Ltd., Easton, Maryland. Adult mysids were held in water from the same source as used during the test. Adult mysids were fed live brine shrimp (Artemia sp.) two or three times daily during holding. Brine shrimp were periodically enriched with a fatty acid supplement (ALGAMAC-2000, Aquafauna Bio-Marine, Inc., Hawthorne, California). During the 14-day holding period preceding the test, water temperatures ranged from 25.0 to 25.4C. The pH o f the water ranged from 8.0 to 8.1, salinity ranged from 20 to 23%o (parts per thousand), and dissolved oxygen ranged from 6.8 to 7.4 mg/L. Instrumentation used for water measurements is described in the Environmental Conditions section of this report. At test initiation, the neonate mysids were carefully collected from the cultures and transferred one and two at a time to glass beakers. Mysids then were transferred from the beakers to the test compartments. All transfers were made using a wide-bore pipette below the surface of the water. The mysids were fed live brine shrimp nauplii three to four times a day during the test to prevent cannibalism (except on the last day of the test). Test Apparatus A continuous-flow diluter was used to deliver each concentration of the test substance and a negative (saltwater) control. A peristaltic pump (Cole-Parmer Instrument Company, Chicago, IL) was used to deliver the six test substance stock solutions into mixing chambers assigned to each treatment group. The stock solutions were diluted with dilution water in the mixing chambers in order to obtain the desired test concentrations. The flow of dilution water to the mixing chambers was controlled by rotameters. Rotameters were calibrated prior to test initiation and at weekly intervals thereafter during the test. The flow of test water from each mixing chamber was split and allowed to flow into replicate test chambers. The proportion of test water that was split into each W il d l if e In t e r n a t io n a l, Lt d . - 11- PROJECT NO.: 454A-107 replicate was checked prior to the test and at weekly intervals thereafter during the test to ensure that flow rates varied by no more than 10% of the mean for the four replicates. The diluter was adjusted so that each test chamber received approximately 11 volume additions oftest water every 24 hours. The delivery pump was calibrated before the test and at approximately weekly intervals during the test. The general operation of the diluter was checked visually at least two times per day during the test and once at the end of the test. Prior to sexual maturity, mysids were held in one compartment placed in each replicate test chamber (15/compartment). The compartments were glass beakers with nylon mesh screen attached to two holes on opposite sides. After mysids attained sexual maturity, reproductive pairs were placed in reproductive compartments (one pair per compartment). The reproductive compartments were glass petri dishes with sides of nylon mesh screen attached with silicone adhesive. The test compartments were placed in 9-L glass aquaria test chambers containing approximately 5 L of test solution. Prior to pairing, the depth of water in a representative test compartment was 6.2 cm. After pairing, the depth of water in a representative test compartment was 5.5 cm.. The test chambers were impartially positioned in a temperature-controlled environmental chamber designed to maintain a temperature of 252C. Test compartments were uniquely identified and the test chambers were labeled with the project number, test concentration and replicate. The test chambers for the second generation exposure were 2-L beakers with 1 L of test solution which was dipped out o f a test chamber from the appropriate treatment group. Dilution Water The water used for culturing and testing was natural seawater collected at Indian River Inlet, Delaware, and was diluted to a salinity of approximately 20%o with well water. Salinity measurements during the four-week period immediately preceding the test are presented in Appendix I. The freshly-collected seawater was passed through a sand filter to remove particles greater than approximately 25 pm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to delivery to the diluter system, the water again was filtered (0.45 pm) to remove microorganisms and particles. The results of periodic analyses performed to measure the concentrations o f selected contaminants in saltwater used by Wildlife International, Ltd. are presented in Appendix II. G01201 W il d l if e In te r n a tio n a l, Lt d . - 12- PROJECT NO.: 454A-107 Environmental Conditions Lighting used to illuminate the cultures and test chambers during culturing and testing was provided by fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone 50). A photoperiod of 16 hours of light and 8 hours of darkness was controlled with an automatic timer. A 30-minute transition period o f low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity ranged from 623 to 815 lux over the surface of the negative control, replicate A test chamber. Light intensity was measured weekly^using a SPER Scientific Model 840006C light meter. Temperature was measured in each test chamber at the beginning and end of the test and at weekly intervals during the test using a liquid-in-glass thermometer. Temperature also was measured continuously in one negative control replicate using a Fulscope ER/C Recorder. The target test temperature during the study was 252C. Dissolved oxygen and pH measurements were measured in alternate replicates of each treatment and control group at the beginning and end of the test and at weekly intervals during the test. Salinity was measured daily in alternate replicates of the negative control and the highest treatment group with surviving mysids. Dissolved oxygen was measured using a Yellow Springs Instrument Model 5 IB dissolved oxygen meter, and measurements ofpH were made using a Fisher Accumet Model 915 pH meter. Salinity was measured using a Bio-Marine, Inc., Aquafauna refractometer. Biological Observations and Measurements Observations of the survival and behavior of each first-generation mysid were made daily throughout the test. The criteria for death included lack of movement, absence of respiratory movements, and lack ofreaction to gentle prodding. At the time of pairing (Day 20), the sex and maturity of each mysid was determined by microscopic examination, and, when possible, 5 male/female pairs were made for each replicate test chamber. Any immature mysids or extra females were discarded at this time. Sexually mature males, which were left over after pairing were maintained in a separate compartment within that replicate. After mysids were paired, the number of second-generation mysids were counted and recorded daily until test termination. Second-generation mysids were also observed for abnormal development and aberrant behavior. After each observation, second-generation mysids were collected and exposed at the same test concentration under static test conditions for 96 hours. If a male in a male/female pair died, it was replaced with a male, if 001202 W il d l if e In t e r n a t io n a l, Lt d . -13- PROJECT NO.: 454A-107 available, from the pool of males maintained in the same replicate. At test termination, the sex of each surviving first-generation mysid was confirmed and the length of each mysid was measured using calipers. Each surviving first-generation was then placed in a drying oven at approximately 60C for approximately 24 hours. The dry weight of each surviving first-generation mysid was then determined using an analytical balance. Statistical Analyses Statistical analyses were-performed on survival of the first and second-generation mysids, the number of young released per reproductive day, and the length and dry weight of each surviving first-generation mysid. Survival was evaluated prior to pairing (Day 0 through Day 20) and after pairing (Day 20 through Day 35). Survival data were analyzed using 2 x 2 contingency tables and the Chi-square test to identify treatment groups that were statistically different from the control group. The analyses of reproduction (number o f live young produced per reproductive day) and growth (dry weights and lengths) data included those treatments which did not exhibit a statistical reduction in survival. Analyses included the evaluation o f homogeneity of variances using Bartlett's test and the assessment of normality using the Shapiro-Wilk's test. When data were deemed normal and homogeneous, an analysis ofvariance test was used to determine whether or not statistically significant differences existed among experimental groups. Those treatments statistically different from the control group were identified using Dunnett's test. The results of the statistical analyses were used to aid in the determination of the NOEC and LOEC. The MATC was calculated as the geometric mean of the NOEC and LOEC. All statistical tests were performed using a personal computer with SPSS/PC Version 2.0 (3) or "TOXSTAT Release 3.5" statistical software (4). Analytical Chemistry Prior to test initiation, two sets o f pretest water samples were collected from two replicate test chambers of both the low and high level treatment groups to determine if nominal concentrations had been achieved. Water samples were also collected from two alternating replicates on Days 0 ,7 ,1 4 ,2 1 ,2 8 and 35. Water samples were collected from mid-depth of the test chamber and placed in plastic (Nalgene) bottles. Samples were analyzed as soon as possible without storage. Analytical procedures used in the analysis o f the samples are provided in Appendix HI. 001203 W il d l if e In te r n a tio n a l, Lt d . -14- PROJECT NO.: 454A-107 RESULTS AND DISCUSSION Measurement of Test Concentrations Results o f analyses to measure concentrations of PFOS in water samples collected during the test are presented in Table 1 and the analytical chemistry report (Appendix HI). Nominal concentrations used in this study were 0.086,0.17,0.34,0.69,1.4 and 2.7 mg a.i./L. When measured concentrations ofsamples collected on Days 0 ,7,1 4 ,2 1 ,2 8 and 35 were averaged, the mean measured concentrations were 0.057,0.12,0.25,0.55,1.3 and 2.6 mg a.i./L, which represented 66, 71,74, 80, 93 and 96% of the nominal concentrations, respectively. Mean measured concentrations were used to express the NOEC, LOEC and MATC. Pretest samples collected to verify diluter performance (Appendix ID) were not used in the calculation of mean measured concentrations. Physical and Chemical Measurements o f Water Measurements of salinity in the negative control and the highest treatment group ranged from 19 to 2 l/oo throughout the test (Table 2). Measurements of pH ranged from 8.2 to 8.4 (Table 3) and temperature was maintained within the 252C range established for the test (Table 4). Dissolved oxygen concentrations remained 2=5.8 mg/L (79% of saturation) throughout the test (Table 5). Survival A summary of survival from test initiation to pairing on Day 20 is presented in Table 6. In general, all surviving mysids appeared normal. After 20 days of exposure, survival in the negative control group was 78%. Survival in the PFOS treatment groups s0.55 mg a.i./L ranged from 75 to 92% and were not statistically different from the negative control. Survival in the 1.3 and 2.6 mg a.i./L treatment groups was 32 and 0%, respectively, and was statistically different from the negative control ip <. 0.05). Observations of survival after pairing (from Day 20 to test termination on Day 35) are presented in Table 7. In general, all surviving mysids appeared normal. Survival in the negative control was 92%. Survival percentages in the PFOS treatment groups *0.55 mg a.i./L ranged from 90 to 97% and were not statistically different from the negative control. Survival in the 1.3 mg a.i./L treatment group was 57% and was statistically different from the negative control ip <. 0.05). C01204 W il d l if e In t e r n a t io n a l, Lt d . -15- PROJECT NO.: 454A-107 Reproduction A summary of the mean number of young produced per reproductive day is presented in Table 8. Young production for individual test compartments is presented in Appendix IV. For each female, the number of reproductive days was defined as the number of days that the female was alive from the day o f first brood release of any female in the test to the end of the test. The day of first brood release in this study was Day 22. The mean number o fyoung produced per reproductive day in the negative control groups was 0.315. Reproduction rates in the 0.057, 0.12, 0.25 and 0.55;<mg a.i./L treatment groups were 0.261, 0.361, 0.252 and 0.0559 young per reproductive day, respectively. Dunnett's test showed that reproduction was significantly reduced in the 0.55 mg a.i./L treatment group when compared to the negative control (p z 0.05). The 1.3 and 2.6 mg a.i./L treatment groups were not included in the statistical analysis of the reproduction data due to a statistically significant difference in survival. Growth Summaries of the lengths and dry weights of the surviving adult mysids are presented in Tables 9 and 10, . respectively. Individual measurements are provided in Appendices V and VI. The mean length and mean dry weight in the negative control group were 6.43 mm and 0.63 mg, respectively. Mysids exposed to PFOS at concentrations 0.25 mg a.i./L showed no statistically significant reductions in length or dry weight (p > 0.05). Mysids exposed to 0.55 mg a.i./L showed statistically significant reductions in both length and dry weight ip <. 0.05). The 1.3 and 2.6 mg a.i./L treatment groups were not included in the statistical analyses of growth due to a statistically significant difference in survival. Second Generation Acute Exposure The results of the second generation exposure are presented in Table 11. After 96-hours, control survival was 96%. Survival in all PFOS treatment groups was ;>95% and was not statistically different from the controls. All surviving mysids in the second generation exposure appeared normal with no overt signs of toxicity. CONCLUSIONS There were no statistically significant effects on survival, reproduction or growth of mysid shrimp (Mysidopsis bahia) exposed to PFOS at concentrations of s 0.25 mg a.i./L for 35 days. Reproduction, length and C01205 W il d l if e In t e r n a tio n a l, Lt d . -16- PROJECT NO.: 454A-107 dry weight were the most sensitive biological endpoints in this study. Mysid shrimp exposed to 1.3 and 2.6 mg a.i./L had significantly reduced survival in comparison to the negative control. Mysids exposed to 0.55 mg a.i./L had significantly reduced reproduction, length and dry weight in comparison to the negative control. Consequently, the LOEC, based on reproduction, length and dry weight, was 0.55 mg a.i./L. The NOEC was 0.25 mg a.i./L and the MATC was calculated to be 0.37 mg a.i./L. Second generation mysids exposed to PFOS during a static 96-hour exposure showed no adverse effects. C0120G W il d l if e In t e r n a t io n a l , Lt d . -17- PROJECT NO.: 454A-107 REFERENCES 1 U.S. Environmental Protection Agency. 1996. Series 850 -Ecological Effects Test Guidelines (draft), OPPTS Number 850.1350: Mysid Chronic Toxicity Test. 2 ASTM Standard E1191-90. 1991. Standard Guide for Conducting Life-Cycle Toxicity Tests with Saltwater Mysids, American Society for Testing and Materials. 3 SPSS Inc. 1988. SPSS/PC+Version 2.0. Chicago, Illinois. 4 West, Inc. and D. D. Gulley. 1996. TOXSTAT Version 3.5. Western EcoSystems Technology, Inc. Cheyenne, Wyoming. 001207 PROJECTNO.: 454A-107 -18- Table 1 Summary of Analytical Chemistry Data Sponsor 3M Corporation Test Substance: PFOS Test Organism: Saltwater Mysid,Mysidopsis bahia Dilution Water: Filtered Saltwater Nominal Test Concentration (mg a.i./L) DayO* Day 73 Negative Control <LOQ3 <LOQ <LOQ <LOQ Measured Concentration (mg a.i./L) Day 141 Day213 <LOQ <LOQ <LOQ <LOQ Day 28* <LOQ <LOQ 0.086 0.0694 0.0578 0.0478 0.0619 0.0606 0.0614 0.0554 0.0509 0.0515 0.0569 0.17 0.125 0.0778 0.124 0.0970 0.122 0.114 0.125 0.127 0.112 0.128 034 0389 0331 0.276 0327 0362 0386 0.197 0353 0.212 0371 Day 352 <LOQ <LOQ 0.0580 0.0514 0.124 0.119 0.278 0.251 Mean (mg a.i./L) -- 0.057 0.12 0.25 Percent of Nominal -- 66 71 74 0.69 0.562 0.581 0.543 0.516 0.529 0.556 0.55 80 0.659 0.450 0.542 0.528 0.544 0.583 1.4 133 1.13 1.35 133 1.39 1.26 1.3 93 1.32 1.20 1.27 1.15 1.39 130 2.7 2.56 2.79 2.58 2.54 2.30 2.69 _4 _4 __ - - 2.6 96 1 Replicates A and C measured. 3 Replicates B and D measured. 3 LOQ - Limit of quantitation was 0.0458 mg a.i./L 6 too m W il d l if e In t e r n a t io n a l, Lt d . -19- PROJECT NO.: 454A-107 Table 2 Salinity of Water in the Negative Control and Highest Treatment Group Test Chambers Sponsor: 3M Corporation Test Substance: Test Organism: PFOS Saltwater Mysid, Mysidopsis bahia Dilution Water: Filtered Saltwater Negative Control 2.6 mg a.i./L/ 1.3 m, Day Replicate Salinity (%o) Salinity (%o) 0 .. A 1 'B 20 21 20 21 2C 3D 20 20 20 19 4A 20 20 5B 20 20 6C 7D 8A 20 20 20 20 20 20 9B 20 20 10 C 11 D 20 20 20 20 12 A 13 B 20 20 20 20 14 C 21 20 151 D 20 20 16 A 17 B 20 20 -20 20 18 C 19 D 20 20 20 20 20 A 20 20 21 B 22 C 23 D 20 20 20 20 20 20 24 A 20 20 25 B 20 20 26 C 27 D 20 20 20 20 28 A 20 20 29 B 20 20 30 C 20 20 31 D 20 20 32 A 20 20 33 B 20 20 34 C 35 D 20 20 20 20 1 On Days 15-35, salinity was measured in the 1.3 mg a.i./L treatment group due to 100% mortality in the 2.6 mg a.i./L treatment group. 001209 W il d l if e In t e r n a t io n a l, Ltd -20- PROJECT NO.: 454A-107 Table 3 pH o f Water in the Test Chambers Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Saltwater Mysid, Mysidopsis bahia Filtered Saltwater Mean Measured Test Concentration (mg a.i./L) V Replicate ___ o Negative Control A 8.4 B C D 0.057 0.12 A 8.4 BC D- A 8.4 BCD- 0.25 A 8.4 BC- D- 0.55 A 8.4 BCD- 1.3 A 8.4 BCD- 2.6 A 8.4 B C- i ...... ......... .. i D `--" -- '- Measurements discontinued due to 100% mortality. 7 8.2 8.2 8.2 8.2 - 8.2 8.3 - 8.3 ^^T es^ 14 21 8.3 8.3 8.3 8.3 8.3 8.3 - 8.3 8.3 -8.3 8.3 8.3 8.3 8.3 28 8.3 8.3 8.3 . 8.3 8.3 8.3 35 8.3 8.3 8.3 8.3 8.3 8.3 001210 W il d l if e In t e r n a t io n a l , Lt d . - 21- PROJECT NO.: 454A-107 Table 4 Temperature (C) of Water in the Test Chambers Sponsor Test Substance: 3M Corporation PFOS Test Organism: Dilution Water Saltwater Mysid, Mysidopsis bahia Filtered Saltwater Mean Measured Test Concentration (mg a.i./L) Replicate 0 Negative Control A1 25.0 B 25.0 C 25.0 D 25.0 0.057 A 25.0 B 25.0 C 25.0 D 25.0 0.12 A 25.0 B 24.9 C 25.0 D 25.0 0.25 A 24.8 B 24.8 C 24.9 D 24.9 0.55 A 24.9 B 24.7 C 24.8 D 24.7 1.3 A 24.6 B 24.7 C 24.9 D 25.1 2.6 A 24.9 B 24.8 C 24.8 D 24.8 ^^^easurementsJisMntinuedduetolOO^mo^li^^ 7 25.2 25.1 25.0 25.1 25.2 25.3 25.2 25.2 25.1 25.1 25.2 25.2 25.1 25.0 25.0 25.0 25.0 25.0 25.0 25.0 24.8 24.9 25.0 25.1 25.1 25.0 25.0 25.1 Test Day 14 21 24.8 24.9 24.8 24.9 24.6 24.9 24.5 24.9 24.8 25.0 25.0 25.0 24.9 25.0 25.0 25.0 24.8 25.1 24.9 25.1 24.9 25.1 24.9 25.0 24.7 25.1 24.9 25.1 24.8 25.1 24.9 25.1 24.9 .25.0 24.8 25.0 24.8 25.0 24.8 25.0 24.4 24.5 24.7 24.5 24.5 24.7 25.1 25.0 25.0 - 2 __2 25.1 JL 25.1 JL 28 25.2 25.2 25.2 25.1 25.1 25.1 25.1 25.2 25.1 25.0 25.0 25.1 25.0 -25.1 '25.0 25.1 25.1 25.0 25.0 25.0 24.8 24.8 24.8 25.0 .. -- -- - 35 25.0 24.9 25.0 25.0 24.9 25.0 25.1 25.0 24.9 24.9 24.9 24.9 25.0 25.0 25.0 24.9 25.0 24.9 24.9 24.9 24.8 24.8 24.9 24.9 ... ~ - 001211 W il d l if e In t e r n a t io n a l, Ltd -22- PROJECT NO.: 454A-107 Table 5 Dissolved Oxygen Content (mg/L) of Water in the Test Chambers1 Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Saltwater Mysid, Mysidopsis bahia Filtered Saltwater Mean Measured Test Concentration (mg a.i./L) Replichte 0 Test Day 7 14 21 28 35 Negative Control A B C D 6.1 -- -- -- 6.0 -- -- 6.4 -- -- -- . 6.3 -- -- 6.2 -- -- -- - - - 6.0 - - 0.057 A 6.1 __ __ 6.0 _ B -- 6.3 -- -- -- 6.2 C -- -- 6.0 -- -- -- D - - - 6.0 - - 0.12 A 6.1 __ _ 6.0 B -- 6.1 -- -- -- 6.3 C -- -- 6.3 -- -- - D - - - 6.0 - - 0.25 A 6.1 _ _ _ 5.8 B -- 6.1 -- -- 6.3 C -- -- 6.2 -- -- -- D - -- 5.8 - -- 0.55 A 6.1 _ . -- L- 6.0 B -- 6.2 -- -- -- 6.2 C -- -- 6.2 ---- D - - - 6.0 - -- 1.3 A 6.1 6.0 B -- 6.2 -- -- -- 6.3 C -- -- 6.2 -- -- -- D - - - 5.9 - " 2.6 A 6.1 _ _ _ -- B -- 6.2 -- -- -- -- C -- _ 6.3 _ __ -- D - -- J1 - .- A dissolved oxygen concentration of 4.4 mg/L represents 60% saturation at 25 C in saltwater with a salinity of 20%o. 2Measurements discontinued due to 100% mortality._____ ______________;______________ _ 001212 W il d l if e In t e r n a t io n a l , Lt d . -23- PROJECT NO.: 454A-107 Table 6 Survival of Juvenile Mysids Exposed to PFOS (Day 0 Through Pairing on Day 20) Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Saltwater Mysid,Mysidopsis bahia Dilution Water: Filtered Saltwater Mean Measured Test Concentration (mg a.i./L) 'V " Replicate Survival on Day 20 (Pairing) No. Alive/No. Exposed Total Negative Control A B C D 14/15 10/15 14/15 9/15 47/60 0.057 A B C D 14/15 13/15 15/15 13/15 55/60 0.12 A 14/15 B 10/15 C 9/15 D 12/15 45/60 0.25 A 13/15 B 10/15 C 14/15 D` 12/15 49/60 . 0.55 A 11/15 B 13/15 C 13/15 D 13/15 50/60 1.3 A 7/15 B 4/15 C 4/15 D 4/15 19/60 2.6 A 0/15 B 0/15 C 0/15 D 0/15 0/60 ^^tadi^tes^ji^^^^d^renM jram A e^ne^ive^contool^using^X J^ntin^nc^taW es^^O ^O S^ Percent Survival 78 92 75 82 - 83 32* 0* 001213 W il d l if e In t e r n a t io n a l, Lt d . -24- PROJECT NO.: 454A-107 Table 7 Survival o f Adult Mysids Exposed to PFOS (Day 20 Through Test Termination on Day 35) Sponsor: Test Substance: Test Organism: 3M Corporation PFOS Saltwater Mysid, Mysidopsis bahia Mean Measured Test Concentration (mg a.i./L) Negative Control Replicate A B C D Survival at Test Termination No. Alive/No. Exposed 11/12 7/8 10/11 8/8 Total 36/39 0.057 A B C D 12/13 12/12 10/11 10/10 44/46 0.12 A B C D 0.25 A B C D 12/12 7/9 7/9 10/10 10/10 8/8 9/10 9/9 36/40 '36/37 0.55 A B C D 7/8 10/10 10/10 8/9 35/37 1.3 A 3/7 8/14 B 4/4 C 0/0 D 1/3 i a significant difference from the negative control using a 2 X 2 contingency table (p <. 0.05). Percent 92 96 90 97 95 57* 001214 W il d l if e In t e r n a t io n a l , Ltd -25- PROJECT NO.: 454A-107 Table 8 Mean Number of Young Produced Per Reproductive Day Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Saltwater Mysid, Mysidopsis bahia Dilution Water: Filtered Saltwater Mean Measured Test Concentration (mg a.i./L) Replicate Number of Reproductive Days Negative Control A B C D 70 53 70 42 Number of Young Produced 18 14 20 19 Mean Number of Young/Reproductive Day 0.257 0.264 0.286 0.452 Overall Mean s 0.315 0.0925 0.057 A 60 17 0.283 0.261 0.0873 B 70 14 0.200 C 70 13 0.186 D 56 21 0.375 0.12 A 70 21 0.300 0.361 0.101 B 46 22 0.478 C 54 22 0.407 D 70 18 0.257 0.25 A 70 19 0.271 0.252 0.0723 B 56 12 0.214 C 61 21 0.344 D 56 10 0.179 0.55 A 54 3 ' 0.0556 0.0559* 0.0376 B 56 6 0.107 C 70 3 0.0429 D 56 1 0.0179 1.31 A 22 0 0.000 0.000 0.000 B 14 0 0.000 C 00 0.000 D 11 0 0.000 This treatment group was not included in the statistical analyses of reproduction due to a statistically significant difference in survival. Indicates a significant difference from the negative control using Dunnett's test (p s 0.05). C01215 W il d l if e In t e r n a t io n a l, Lt d . -26- PROJECT NO.: 454A-107 Table 9 Mean Total Length of Adult Mysids at the End of the 35-Day Test Period Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Saltwater Mysid, Mysidopsis bahia Filtered Saltwater Mean Measured Concentration (mg a.i./L) Replicate Negative Control A B C D Replicate Mean (mm) 6.45 6.34 6.46 6.48 Overall Mean s (mm) 6.43 0.0634 0.057 A B C D 6.38 6.45 6.43 0.0729 6.52 6.36 0.12 A 6.55 B 6.65 6.56 0.105 C 6.62 D 6.42 0.25 A 6.48 B 6.38 6.40 0.0548 C 6.36 D 6.38 0.55 A 6.05 B 6.11 6.14* 0.0794 C 6.16 D 6.24 1.31 A 5.93 B 5.98 5.85 0.178 C-- D 5.65 This treatment group was not included in the statistical analyses of total length due to a statistically significant difference in survival. * Indicates a significant difference from the negative control using Dunnett's test (p <, 0.05). C01216 W il d l if e In t e r n a t io n a l , Lt d . -27- PROJECT NO.: 454A-107 Table 10 Mean Diy Weight of Adult Mysids at the End of the 35-Day Test Period Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Saltwater Mysid, Mysidopsis bahia Filtered Saltwater Mean Measured Concentration (mg a.i./L) v Replicate Negative Control A B C D Replicate Mean (mg) 0.599 0.706 0.596 0.636 Overall Mean s (mg) 0.634 0.0510 0.057 A 0.616 0.599 0.0276 B 0.627 C 0.590 D 0.565 0.12 A 0.647 0.641 0.0241 B 0.664 C 0.644 D 0.607 .. 0.25 A 0.644 0.622 0.0227 B 0.601 C 0.603 D 0.639 0.55 A 0.556 0.562* 0.00624 B 0.558 C 0.563 D 0.570 1.31 A 0.440 0.436 0.0441 B 0.478 C-- D 0.390 1 This treatment group was not included in the statistical analyses of dry weight due to a statistically significant difference in survival. * Indicates a significant difference from the negative control using Dunnett's test (p s O.OS'). B B B 3S ^^SS B ^^^B ^B S S!SC SS 8B 9^^B B SIB 3B 8SB 8SB B nS^B H 99M H B M B nB nB ^B ^B n083B SS ^^^8S SH H B B S B H I^^B S B B 9B 98R B B 8^^SS B S SB B SS E nB S SB S B B SB S SS B S SS & B S E 3S 001217 W il d l if e In t e r n a t io n a l , Lt d . -28- PROJECT NO.: 454A-107 Tablell Survival o f Second-Generation Mysids Exposed to PFOS During 96-Hour Static Exposures Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Saltwater Mysid, Mysidopsis bahia Filtered Saltwater Mean Measured Test Concentration Total No. Alive After 96 Hours o f Exposure (mg a.i./L) No. Exposed (Observations') Negative Control 71 68 (AN) Percent Survival 96 0.057 65 63 (AN) 97 0.12 83 79 (AN) 95 0.25 62 59 (AN) 95 0.55 A N = Appears Normal. 13 13 (AN) 100 C01218 W il d l if e In t e r n a t io n a l, Lt d . -29- PROJECT NO.: 454A-107 Sponsor: Test Substance: Test Organism: Dilution Water: Salinity (%o) APPENDIXI Dilution Water Salinity Measured During the 4-Week Period Immediately Preceding the Test 3M Corporation PFOS Saltwater Mysid, Mysidopsis bahia Filtered Saltwater Mean 20 (N = 4) Range 20-20 001219 W il d l if e In t e r n a tio n a l, Ltd -30- PROJECT NO.: 454A-107 APPENDIXII Analyses of Pesticides, Organics, Metals and Other Inorganics _________ in Wildlife International, Ltd. Saltwater1_________ A N A L Y S IS Miscellaneous Measurements Total Dissolved Solids Ammonia Nitrogen Total Organic Carbon2 Total Cyanide M EASURED CONCENTRATIO N 23,500 < 0.050 < 1.0 < 10.0 mg/L mg/L mg/L Aig/L Organochlorines and PCBs Aldrin Alpha BHC Beta BHC Delta BHC Gamma BHC (Lindane) Chlordane DDD, pp' DDE, pp' DDT, pp' Dieldnn Endosulfan, A Endosulfan, B Endosulfan Sulfate Endrin Endrin Aldehyde Heptachlor Methoxychlor Heptachlor Epoxide Toxaphene PCB-1016 PCB-1221 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 Aig/L /ig/L Aig/L Aig/L Aig/L Aig/L Aig/L Aig/L Aig/L Aig/L Aig/L Aig/L Aig/L. Aig/L Aig/L Aig/L Aig/L Aig/L Aig/L Aig/L Aig/L Aig/L Aig/L Aig/L Aig/L Aig/L Metals and Other Inorganics Aluminum3 Arsenic3 Beryllium3 Cadmium3 Calcium3 Chromium3 Cobalt5 Copper Iron5 Lead3 Magnesium3 Manganese3 Mercury Molybdenum3 Nickel3 Potassium3 Selenium3 Silver Sodium3 Zinc3 < 100 Aig/L < 25.0 Aig/L < 0.50 Aig/L < 1.0 Aig/L 235 mg/L < 2.0 Aig/L < 1.0 Aig/L < 20.0 Aig/L < 100 Aig/L < 10.0 Aig/L 760 mg/L < 4.0 Aig/L < 0.20 Aig/L < 2.0 Aig/L < 20.0 Aig/L 277 mg/L < 25.0 Aig/L < 1.0 Aig/L 6,010 mg/L < 20.0 /ig/L `Analyses performed by QST Environmental, Gainesville, Florida for samples collected on November 3 through November 7, , 1997. 2Analyses performed by Wildlife International, Ltd. for the sample collected on November 5,1997. 3Analyses performed by Wildlife International, Ltd. for samples collected on November 5 through 7,1997. 001220 W il d l if e In t e r n a t io n a l , Lt d . -31- APPENDEX IE PROJECT NO.: 454A-107 THE ANALYSES OF PFOS IN SALTWATER IN SUPPORT OF 'v' WILDLIFE INTERNATIONAL PROJECT NO.: 454A-107 001221 W il d l if e In t e r n a t io n a l ltd. -32- PROJECT NO.: 454A-107 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Flow-Through Life-Cycle Toxicity Test with the Saltwater Mysid (Mysidopsis bahia) V' WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-107 PRINCIPAL INVESTIGATOR: J Scientist MANAGEMENT: Manager, Analytical Chemistry DATE 001222 W il d l if e In t e r n a t io n a l l t d . -33 - PROJECT NO.: 454A-107 Introduction Saltwater samples were collected from a flow-through life-cycle toxicity study designed to determine the effects o f PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) to the saltwater mysid {Mysidopsis bahia). This study was conducted by Wildlife International Ltd. and identified as Project No.: 454A-107. The analyses o f these water samples were performed at Wildlife International Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). Samples were received for analysis between June 15, 1999 and July 21, 1999 and were analyzed between June 15,1999 and July 22, 1999. Test Substance and Internal Standard The test substance used for this study was Wildlife International Ltd. identification number 4675. The test substance was used to prepare calibration and matrix fortification samples. The internal standard was received from 3M Corporation on July 2, 1998 and was assigned Wildlife International Ltd. identification number 4526 upon receipt. The internal standard, a granular material, was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2. The standard was stored under ambient conditions. Analytical Method The method used for the analysis o f the saltwater samples was developed at Wildlife International Ltd. and entitled "Analytical Method for the Determination o f PFOS in Freshwater, Saltwater, and Algal Medium". This methodology was included as Appendix II o f Wildlife International 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% formic acid (v/v) so that they fell within the calibration range o f the PFOS methodology. Concentrations o f the PFOS in the standards and samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin- 001223 W il d l if e In te r n a tio n a l ltd . -3 4 - PROJECT NO.: 454A-107 Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil Cig analytical column (50 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 ofJPFOS 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 0.000915 to 0.00915 mg a.i./L, were analyzed with the samples. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOS on the basis o f individual isomeric components. 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 of 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 at 0.0458 mg a.i./L calculated as the product o f the lowest calibration standard analyzed (0.000915 mg a.i./L) and the dilution factor o f the matrix blank samples (50.0). Matrix Blank and Fortification Samples Six 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 ion chromatogram o f a matrix blank is presented in Figure 5. Saltwater was fortified at 0.0823, 0.366 and 3.66 mg a.i./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 92.8%. A representative ion chromatogram o f a matrix fortification is presented in Figure 6. 001224 W il d l if e In t e r n a tio n a l l t d . -35 - PROJECT NO.: 454A-107 Example Calculations Sample number 454A-107-3, nominal concentration of 0.086 mg a.i./L in saltwater. Peak Area Ratio = Analyte Peak Area/Intemal Standard Peak Area Concentration Ratio = Concentration of Analyte/Concentration of Internal Standard Internal Standard Concentration: 0.100 mg/L Initial Volume: 0.500 mL Final Volume: 25.0 mL Dilution Factor: 50.0 PFOS Peak Area: 12963 Internal Standard Peak Area: 145150 Peak Area Ratio: 0.08931 Calibration curve equation. Slope: 5.76026 Intercept: 0.00940 Curve is weighted (1/x). Peak area ratio - (Y-intercept) PFOS (mg a.i./L) at instrument Slope x Internal Standard Concentration 0,08931 -0.00940 5.76026 x 0.100 = 0.001387 PFOS (mg a.i./L) in sample = PFOS (mg a.i./L) at instrument x Dilution Factor = 0.001387 x 50.0 = 0.06935 001225 W il d l if e In te r n a tio n a l lt d . -36- PROJECT NO.: 454A-107 PFOS (mg a.i./L) in sample Percent o f Nominal Concentration = PF 0S (mg a.i./L) nominal x 100 _ 0.06935 0.086 x 100 = 80.6% RESULTS Sample Analysis Saltwater samples were collected from the flow-through life-cycle toxicity study with the saltwater mysid {Mysidopsis bahia) at pre-test, June 15, 1999, at test initiation, June 16, 1999 (Day 0), and weekly during the test through test termination on July 21, 1999 (Day 35). The measured concentrations of PFOS in the samples collected at pre-test ranged from 57.3 to 99.3% o f the nominal concentrations (Table 4). The measured concentrations o f PFOS in the samples collected at initiation o f exposure o f the test organisms (Hour 0) ranged from 67.1 to 103% o f the nominal concentrations (Table 5). Samples collected at Day 7, Day 14, Day 21 and Day 28 had measured concentration ranges o f 45.8 to 95.6%, 70.5 to 99.6%, 57.1 to 87.9% and 59.9 to 99.3% o f nominal values, respectively. Samples collected at test termination (Day 35) had a measured concentration range of 59.8 to 90;0% of nominal values. A representative ion chromatogram of a test sample is shown in Figure 7. 001226 W il d l if e In t e r n a t io n a l Lt d . -37- PROJECT NO.: 454A-107 INSTRUMENT: Table 1 Typical HPLC/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in V .' selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil Cis column (50 mm x 2 mm I.D., 3 pm particle size) OVEN TEMPERATURE: 30C STOP TIME: 5.00 minutes FLOWRATE: 0.220 mL/minute MOBILE PHASE: 72.0% Methanol : 28.0% NANOpure Water containing 0.1% Formic Acid INJECTION VOLUME: 25.0 pL PFOS RETENTION TIME: Approximately 3.8 to 4.5 minutes INTERNAL STANDARD RETENTION TIME: Approximately 2.6 to 3.0 minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 426.7 amu W il d l if e In t e r n a t io n a l l t d . -38- PROJECT NO.: 454A-107 Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-107-) MAB-1 MAB-2 Sample Type s./ Matrix Blank Matrix Blank Measured Concentration of PFOS1 (mg a.i./L) <LOQ <LOQ MAB-3 Matrix Blank < LOQ MAB-4 Matrix Blank <LOQ MAB-5 Matrix Blank < LOQ MAB-6 Matrix Blank ________________ < L o q _ : _____ 1 The limit o f quantitation (LOQ) was 0.0458 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.000915 mg a.i./L) and the dilution factor of the matrix blank samples (50.0). 001228 W il d l if e In t e r n a tio n a l l t d . -39- PROJECT NO.: 454A-107 Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-107-) MAS-1 MAS-4 MAS-7 MAS-10 MAS-13 MAS-16 MAS-2 MAS-5 MAS-8 MAS-11 MAS-14 MAS-17 MAS-3 MAS-6 MAS-9 MAS-12 MAS-15. M AS-18 Concentrations of PFOS (mg a.i./L) Fortified Measured 0.0823 0.0823 0.0823 0.0823 0.0823 0.0823 0.0748 0.0828 0.0673 0.0662 0.0693 0.0787 Percent Recovered 90.9 101 81.8 80.4 84.2 95.6 0.366 0.366 0.366 0.366 0.366 0.366 0.350 0.359 0.314 0.303 0.360 0.287 95.6 98.1 85.8 82.8 98.4 78.4 3.66 3.29 3.66 3.54 3.66 3.39 3.66 3.88 3.66 3.80 3.66 3.98 89.9 96.7 92.6 106 104 109 Mean = 92.8 Standard Deviation = 9.19 CV =9.90 N = 18 001229 W il d l if e In te r n a tio n a l l t d . -40- PROJECT NO.: 454A-107 Table 4 Measured Concentrations o f PFOS in Pre-test Diluter Verification Samples from a Saltwater Mysid Life-Cycle Toxicity Test Nominal Test Concentration (mga.i./L) 0.086 Sample Number (454A-107-) PT-1 PT-2 PT-5 PT-6 Sampling Time (Hours) -24 -24 -24 -24 PFOS Measured Concentration (mg a.i./L)1,2 0.0581 0.0493 0.0627 0.0606 Percent of Nominal 67.6 57.3 72.9 70.5 2.7 PT-3 -24 PT-4 -24 2.21 81.9 2.34 86.7 PT-7 PT-8 -24 -24 2.56 94.8 2.68 99.3 1 The limit o f quantitation (LOQ) was 0.0458 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.000915 mg a.i./L) and the dilution factor o f the matrix blank samples (50.0). 2 Measured values o f matrix blanks and matrix fortification samples analyzed concurrently with pre-test samples were <LOQ, 83.0, 95.6, 100, <LOQ, 93.4, 85.0 and 106 percent o f nominal concentrations for pre-test 1 and pre-test 2, respectively._________________________________ : ______________ 001230 W il d l if e In t e r n a t io n a l Lt d . -41 - PROJECT NO.: 454A-107 Table 5 Measured Concentrations of PFOS in Saltwater Samples from a Saltwater Mysid Life-Cycle Toxicity Test Nominal Test Concentration (mg a.i./L) 0.0 Sample Number (454A-107-) 'i 2 15 16 29 30 43 44 55 56 67 68 Sampling Time (Day) 0 0 7 7 14 14 21 21 28 28 35 35 PFOS Measured Concentration (mg a.i./L)1 < LOQ <LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ <LOQ < LOQ <LOQ < LOQ Percent of Nominal -- -- -- -- -- -- -- -- -- . -- -- 0.086 3 0 0.0694 80.7 40 0.0578 67.2 17 7 0.0478 55.6 18 ' 7 0.0619 72.0 31 14 0.0606 70.5 32 14 0.0614 71.4 45 21 0.0554 64.4 46 21 0.0509 59.2 57 28 0.0515 59.9 58 28 0.0569 66.2 69 35 0.0580 67.4 70 35 0.0514 59.8 1 The limit of quantitation (LOQ) was 0.0458 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.000915 mg a.i./L) and the dilution factor of the matrix blank samples (50.0). 001231 W il d l if e In t e r n a t io n a l l t d . -42- PROJECT NO.: 454A-107 Table 5 (continued) Measured Concentrations o f PFOS in Saltwater Samples from a Saltwater Mysid Life-Cycle Toxicity Test Nominal Test Concentration (mga.i./L) Sample Number (454A-107-) '5 6 19 20 33 34 47 48 59 60 71 72 Sampling Time (Day) 0 0 7 7 14 14 21 21 28 28 35 35 PFOS Measured Concentration (m ga.i./L )1 0.125 0.114 0.0778 0.125 0.124 0.127 0.0970 0.112 0.122 0.128 0.124 0.119 Percent of Nominal 73.5 67.1 45.8 73.5 72.9 74.7 57.1 65.9 71.8 75.3 72.9 70.0 70 0.289 85.0 80 0.286 ' 84.1 21 7 0.231 67.9 22 ' 7 0.197 57.9 35 14 0.276 81.2 36 14 0.253 74.4 49 21 0.227 66.8 50 21 0.212 62.4 61 28 0.262 77.1 62 28 0.271 79.7 73 35 0.278 81.8 74 35 0.251 73.8 1 The limit of quantitation (LOQ) was 0.0458 mg a.i./L based upon the product of the lowest calibration standard analyzed (0,000915 mg a.i./L) and the dilution factor of the matrix blank samples (50,0). 001232 W il d l if e In t e r n a t io n a l l t d . -43 - PROJECT NO.: 454A-107 Table 5 (continued) Measured Concentrations o f PFOS in Saltwater Samples from a Saltwater Mysid Life-Cycle Toxicity Test Nominal Test Concentration (mg a.i./L) 0.69 : Sample Number (454A-107-) '9 10 23 24 37 38 51 52 63 64 75 76 Sampling Time (Day) 0 0 7 7 14 14 21 21 28 28 35 35 PFOS Measured Concentration (mg a.i./L)1 0.562 0.659 0.581 0.450 0.543 0.542 0.516 0.528 0.529 0.544 0.556 0.583 Percent of Nominal 81.4 95.5 84.2 65.2 78.7 78.6 74.8 76.5 76.7 78.8 80.6 84.5 1.4 11 0 1.23 87.9 12 0 1.32 . ' 94.3 25 7 1.13 80.7 26 - 7 1.20 85.7 39 14 1.35 96.4 40 14 1.27 90.7 53 21 1.23 87.9 54 21 1.15 82.1 65 28 1.39 99.3 66 28 1.39 99.3 77 35 1.26 90.0 78 35 1.20 85.7 1The limit of quantitation (LOQ) was 0.0458 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.000915 mg a.i./L) and the dilution factor of the matrix blank samples (50.0). 001233 W il d l if e In t e r n a t io n a l l t d . -44- PROJECT NO.: 454A-107 Table 5 (continued) Measured Concentrations o f PFOS in Saltwater Samples from a Saltwater Mysid Life-Cycle Toxicity Test Nominal Test Concentration (mg a.i./L) Sample Number (454A-107-) Sampling Time (Day) PFOS Measured Concentration (mg a.i./L)1 Percent of Nominal 2.7 "13 14 27 28 41 42 0 0 7 7 14 14 2.56 94.8 2.79 103 2.58 95.6 2.30 85.2 2.54 94.1 2.69 99.6 1 The limit o f quantitation (LOQ) was 0.0458 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.000915 mg a.i./L) and the dilution factor of the matrix blank samples (50.0), 001234 W il d l if e In t e r n a t io n a l l t d . -45- PROJECT NO.: 454A-107 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN SALTWATER Prepare matrix fortificatioji samples by spiking the requisite volume o f PFOS stock solutions directly into filtered saltwater using gas-tight syringes and Class A volumetric flasks. 4 Dilute matrix 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 o f 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. Mix well by several repeat inversions. 4* Amputate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis o f PFOS in saltwater. 001235 W il d l if e In t e r n a t io n a l l t d . -46- PROJECT NO.: 454A-107 Figure 2. A typical calibration curve for PFOS. Slope = 5.76026; Intercept = 0.00940; r = 0.9974. Curve is weighted (1/x). 001236 W il d l if e In t e r n a t io n a l ltd AVAILABLE 4 PROJECT NO.: 454A-107 BEST COPY AVAILABLE Mk Oum. vandon l.a M M * Thu. Jun 17. ItM QC29 OMmM m H k Cal PPM lilU NH P a h API 100LC HD*UaaCtam 1.0:494A107_1JUN3t: CoihhmM: 4MA-107 PPM MyaMUN CycN - Day 0 MJUNM. Samptai dadgnalffl M OC N M M l paraart a t i m M oonoartraUom. PPMIdM PFO S.101 6TO 0.00100 407SA-004D-1 4 * 7 b l 1 parted 4HPF06 v. 1: 4 *4 Q1 ML 110 cam 4 2 0 .7 N o iM lh M . 2 .0 Mn.WMft 0Quanl Thraa. 0.2 MOL WkMh B aat.V A dth 0 IS RT W bi (aaes) 2 0 S m ooth 1 E ip a cte d flT 2.70 Araa 100200 H aight 1400$ S ta ll Tima 2 *9 End Tim a 0 *2 Intagratten W idth 0 *3 RatanOonTfcna 2 *0 M agnaten T yp* A -B 8 W ad, Jun 1. 1099 10:10 10* 90 90 70 00 SO 40 30 20 10 o- 15 21 0 *0 31 41 1.7 IntanaRy: 14060 cpa 101 101 4 *3 112 * Scan Tim a Hk O m . wM on 1 * PiW i i Urn, Juft 17.1000 00*0 CaftnOon Hk CWPPM HJUNM Pate API 100LC MfcMaaaCtnm 1**S 4A107..19JUN9t; OaawmiM 404A-M7 PPM Myatd UN OycM Pay o 10JUN90. OanpNo dadgnatad aa OC to cafcuMa p m m t of PFOS_191 STD 0 *0 1 0 0 40 7 B A 0 0 4 0 -1 4 * 7 fe ll OrfDd PROS H mdO S fe n tM <t o n e U m Aim AbaaM a RrtanSon 1)m 1: 4 * 4 0 1 M , 1 10earn 4 0 0 .0 Notea Thraa. 1 0 .0 Q uant Thraa. 1 .0 M KO M h 0 MuR. W U h B a a a . M h 2S R T1M n.(ates) 2 1 tm oaO i 1 BsaoM R T 0J7 Aim 07S 4 H aight 0 7 0 S tart Tfeaa 0 *0 E n d lfe a a 4 *3 IntagraOon M M h 0 *3 fiatanOon Tfena 3 *3 M agraSon Typa A -V B W ad. Jun 10. 1090 10:10 Papal*** Figure 3. A representative ion chromatogram of a low-level (0.000915 mg a.i./L) PFOS standard. 001237 W il d l if e In t e r n a t io n a l ltd PROJECT NO.: 454A-107 BEST COPY AVAILABLE f W w S n Cat P F O S U Jt N M Path: API lOOCC HOANaaCNem 1.P:454A107.1MJNBt: Cuuawaiar 464*107 PPOS U|Mfi UN CyaN Oay 0 14JUNM. 6arwplaa daUprated m Q C H ealn ia le pare at nemfewl eoneanMtona. P PO S .10C STD 04100 4675* 0040-5 fp u m aa Marnai Standard 1: 4.04 0 1 Hi. 116 te a m 426.7 NoiMTtwaa. 2.0 Quant TTwaa. 0.2 M n-V M ti 8 lULWMtfi 6 BaN. WM6l 16 RTWIn.(Mcal 20 8mooth 1 ExpacNdRT 2.76 Ana 173028 Haight 14211 Start Tima EndTkna lntagra>on Width Retanion Tkna Magradon Typa 2-S6 S 22 048 2J 0 4 SB Wad, Jun 1. I960 16:41 100 60 0 70 60 60 40 60 20 an 201.68 41 1-72 66 9 6 1 0 ' toi r 4-25 TSTcmana INcQ um .vantoal.0 e 414*107 P P 0 6 Upritf UN OyeN Oag 0 16JUNN. S w e p N a d e rig n a N ia a O C N e a p a n a al ncntMtaBnaankudMa P P O t.1 0 6 STD 04100 4675*00406 Wad. Jun 16, 1690 16:41 Paga Oaf Figure4. A representative ion chromatogram of a high-level (0.00915 mg a.i./L) PFOS standard. 001238 W il d l if e In t e r n a t io n a l ltd -49- PROJECT NO.: 454A-107 BEST COPY AVAILABLE M M k T h . J M 17. 1M V M M n a il-- nw H tt C M P T O t 1M W P f l h : A P I 100L C H O M a e a C tirw n 1.0* S 4A 10r .i e J U N W : C o m m a : 4U A r\tfT P f 0 5 MyaW L N C ycle - D e y 0 l J U N t e . e m p ie e 4ealgnMed m O C to celc u M e p a te n t I 5F O S .107 4 5 4A -107- M AS-1 4J 7 in 1 period v , usVoOsSe T uea ee H em al Standard W ed. A n 16, 1606 16:54 1: 4 * 4 Q i M , 116 acane 4 2 6 .7 Notce Three. 2 .0 Q uant Three. M n. W W ti MuM. W idth 0 .2 S 5 S eat. W idth 16 RTW )n.<aeea) Sm ooth Expected FTT 20 1 2.76 Area 17110 H eight 14656 S tart 71me End Tima Integration W idth Retention Time Integration Type 2 *5 2.1 043 2.76 Ji ' BB 100 0 SO 70 60 50 40 0 20 IO 0* IS 20 201J6 IrteneH y; 146S6 epe 66 f7 41 1.72 6t1M M1 57 106 101 y 4 .2 3 s' een Tim e iflk e C M 5 5 0 6 16J U N M 5e B t A P I 100L C H D tte e e C tv w n 14M 64A 107. 16J U N M : c 4M A M 7 5506 M ya M LB e O y d e . O e y 1C I U N M . B em ple d e e ip a le d a O C to celreloti percent o f nominai concern atto ia. PFOS_107 4S 4A -107- U AB-1 w ed, Jun 16, 1556 1 6 *4 Figure 5. A representative ion chromatogram of a matrix blank sample (454A-107-MAB-1). The arrow indicates the retention time o f PFOS. 001233 W il d l if e In te r n a tio n a l ltd PROJECT NO.: 454A-107 BEST COPY AVAILABLE H k C X W Q I ItJ U N tt PaMi: API 100CC HOtMaaaOOTWi tA4MA1T.1M JMHs 4C4A-147 P P M MyaU ( J a Oyda * Day 0 1CJUNM. Satrptai M p i M aa OC la PFOS.109 4S4A-107* MA$1 Wad. Jun 16. 199 17:00 447 In 1 patted V' 1: 444 Q1 ML 116 acara 426.7 Motet Tim a. Quanl Tim a. 2 .0 0 .2 MbuWMh 3 M tiL WhMi Bata. WMh RTW In.(aacs) 6 IS 20 Smoodi Expacted RT 1 2.76 Atm 18*70 Haight 12918 Start Tima End Tima 241 3.14 Integration Width 043 R atanlonTlm a Intagradon Typa 2.72 A - B6 Intenaty: 12916 cpa 049 1.72 248 348 443 Tima \ MaeQuan, vanloa 1J Pteaa* Ttet J m it . ia a t oaaa CMSadlea Fd* Cal P P 06 WJUNea Pteh: API 100LC HD*taeeChrom l44S4A107.1*AINea: Oanam nte: 464A-1Q7 P P 0 6 Mydd Ute Cycte P ay 0 laJU N at. Ba w p f tlu lpnditl a tQ C lp rateHate pwaani ot nomteal oonoamradona. PagaW te*! PFO 9.109 484A-107- MAS-1 447 te l patted Wad. Jun 16. 199 1740 tetenaftr. 1086 cpa Figure 6. A representative ion chromatogram of a matrix fortification sample (454A-107-MAS-1). 001240 W il d l if e In t e r n a t io n a l ltd PROJECT NO.: 454A-107 BEST COPY AVAILABLE t D ll. Jlei 17. 18M M M II N k Cal PPQ8 MJUNM PMb API IOOCC HPiMa--Cftrowi A 4MAI07. 1AJUNM: B 4MA-10T PP08 Upldl LM Ofdm . O ar 0 1UNM . Sampte* daalgnated a a O C l e a PP08_114 464A -107- 8 4 J 7 to 1 parted PF08 u m m Internal Standard W ad. Jun id . 1*99 17.3d Vr 1: 4 4 4 Q1 M . 118 acana 4 2 8 .7 Notea T haa. Q uant Three. 2 .0 0 .2 Mbk W td ti 3 IA A . W M8I 8 Baaa. W idth 15 RT W in. (aaca) 2 0 S m ooti 1 E xacted RT 2.78 Area 145160 H aight 11825 Stan TWna End Tima 241 3.14 Intagradon W Mlh Ratenden Tima 043 2.72 Integration Type A BO 100 80 80 70 80 50 40 30 20 in 0-h 21 048 85 40 48 41 1.72 81 LS i 81 3.39 In te n sity: 11825 epa 100 108 101 443 M a te * H a l. Am 17. 1M M M C atenas' F*K Cat PP0 8 tfJU N M PMcAPIIOOLC HDMaaaCtaam 1434d4A107..ieJUNM: Caa-- Idr 484A-187 PP08 MyMd Ute O rateDay a idJUMM. tamptes d n lp ia lid aa PC te nalnM pareaot or nwntnal eoncanaaMam. PPO S.114 454A -107- t W08 4 4 7 In i parted Um A n a W ad, J in 18, 1889 17:38 fettanaty: 1 7 *7 cpa Figure 7. A representative ion chromatogram of a test sample (454A-107-3). 001241 W il d l if e In t e r n a t io n a l , Lt d . -52- PROJECT NO.: 454A-107 APPENDIXIV Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration (mp a.i./L) Negative Control Mysid Reproduction 3M Corporation PFOS Saltwater Mysid, Mysidopsis bahia Filtered Saltwater________________ Replicate Test Compartment Number of Reprodutive Days' Al U A9* 14 3 14 4 14 5 14 B1 2 3 4 14 14 14 11 Number of Young Produced 3 9 6 5 2 4 8 2 0 Number of Young/Reproductive Day 0.257 0.264 C1 2 3 4 5 14 6 14 3 14 4 14 5 14 2 0.286 D1 2 3 14 9 14 6 14 4 0.452 0.057 A1 2 3 4 5 14 3 14 6 14 3 14 5 40 0.283 B1 2 3 4 5 14 T 14 3 14 5 14 0 14 4 0.200 C1 2 3 4 5 14 3 14 3 14 3 14 0 14 4 0.186 D1 2 3 14 2 14 10 14 7 0.375 4 14 2 1 The number o f reproductive days is the number o f days that the female was alive from first brood release to the end o f the test. 001242 W il d l if e In t e r n a t io n a l, Lt d . -53- PROJECT NO.: 454A-107 APPENDIXIV (Continued) Mysid Reproduction Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration ___ (mg a.i./L) 0.12 3M Corporation PFOS Saltwater Mysid, Mysidopsis bahia Filtered Saltwater Test Compartment Number of Reproductive Days A"-' 1 2 3 4 5 14 14 14 14 14 Number of Young Produced 6 0 8 6 1 Number of Y oung/Reproductive Day 0.300 B1 2 3 4 14 7 14 8 14 5 42 0.478 C1 2 3 4 14 7 14 9 14 4 12 2 0.407 D1 2 3 4 5 14 9 14 2 14 0 14 5 14 2 0.257 0.25 A 1 2 3 4 5 14 2 14 4 14 2 14 6 14 5 0.271 B1 2 3 4 14 4 14 1 14 7 14 0 0.214 C1 2 3 4 5 14 2 52 14 7 14 6 14 4 0.344 D1 14 1 0.179 2 14 0 3 14 3 4 14 6 5 T The number of reproductive days is the number of days that the female was alive from first brood release to the end o f the test.__________ ____ 001243 W il d l if e In t e r n a t io n a l , Lt d . -54- PROJECT NO.: 454A-107 APPENDIXIV (Continued) SKsaacBSK Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration (mg a.i./L) 0.55 Mysid Reproduction 3M Corporation PFOS Saltwater Mysid, Mysidopsis baha Filtered Saltwater Replicate Test Compartment Number of Reproductive Days1 A1 2 3 4 14 14 12 14 Number of Young Produced 0 3 0 0 Number of Young/Reproductive Day 0.0556 B1 2 3 4 14 0 14 0 14 2 14 4 0.107 C1 2 3 4 5 14 0 14 0 14 0 14 3 14 0 0.0429 D1 2 3 4 14 0 ' 0.0179 14 0 14 0 14 1 1.3 A 1 2 3 30 11 0 80 0.000 B1 Lp 14 0 0.000 D1 11 0 0.000 ^em im bCTofr^^^^tive^^s_is e^^A CTofd^A atAe^m akw ^^ivefr^_^m to^m drfftete^ 001244 -55APPENDIX V Mysid Total Length (nun) PROJECT NO.: 454A-107 Mysid Number 1 2 3 4 5 6 7 8 9 10 11 12 Mean Rep A 6.80 6.85 6.30 6.45 6.30 6.75 6.15 6.65 6.30 6.50 5.85 - 6.45 Negative Control Rep B 6.20 6.65 6.55 6.95 5.90 6.10 6.00 - 6.34 RepC 6.50 6.85 6.20 6.60 6.25 6.40 6.15 6.30 6.70 6.65 - - 6.46 RepD 6.55 7.10 6.35 6.10 6.40 6.45 6.30 6.55 - 6.48 Rep A 6.15 6.30 6.35 6.15 ' 6.55 6.90 6.20 6.65 6.25 6.40 6.15 6.50 6.38 0.57 mg a.i./L RepB 6.35 6.20 6.15 6.70 6.45 6.60 6.30 6.80 6.30 6.40 6.50 6.65 6.45 RepC 6.05 6.80 6.55 6.70 6.45 6.65 6.50 6.70 6.30 6.50 -- - 6.52 RepD 6.30 6.45 6.40 6.90 6.55 6.45 5.90 6.35 5.95 6.35 -- - 6.36 Rep A 6.25 6.J0 6.40 6.55 6.60 7.10 6.35 6.40 6.25 7.00 6.55 6.40 6.55 0.12mga.i./L RepB 6.75 6.60 6.55 6.80 6.45 6.90 6.50 __ -- __ -- - 6.65 RepC 6.90 6.70 6.45 7.15 6.35 6.60 6.20 _ __ -- -- 6.62 RepD 6.30 6.75 6.40 6.40 6.10 6.15 6.30 6.80 6.40 6.55 _ 6.42 I O O H* N in Mysid Number 1 2 3 4 5 6 7 8 9 10 11 12 Mean Rep A 6.55 6.40 6.35 6.45 6.50 6.25 6.55 6.70 6.45 6.60 6.48 0.25 mg a.i./L RepB 6.25 6.40 6.30 6.15 6.45 6.90 6.35 6.20 - RepC 5.95 6.35 6.80 6.10 6.75 6.20 6.40 6.35 6.35 -- 6.38 6.36 RepD 6.20 6.45 6.50 635 6.75 6.55 6.05 6.80 5.75 - 6.38 - 56- PROJECT NO.: 454A-107 APPENDIX V (Continued) Mysid Total Length (nun) Rep A 6.05 5.85 6.10 5.90 5.80 6.35 6.30 - - - 6.05 0.55 mg a.i./L RepB 6.05 635 6.35 6.20 6.00 6.20 6.15 6.60 5.05 6.25 RepC 630 630 5.85 6.00 6.10 6.35 6.25 6.40 635 6.00 6.11 6.16 RepD 6.20 6.45 5.95 6.10 6.25 6.40 6.10 6.45 -- - 634 Rep A 5.85 ./ 6.05 5.90 -- -- -- -- -- -- - 5.93 1.3 mg a.L/L RepB 5.75 6.25 5.85 6.05 -- _ -- -- RepC -- -- -- -- -- _ -- -- -- - 5.98 - RepD 5.65 -- -- -- -- __ -- - 5.65 i. TOO & CD Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Saltwater Mysid, M ysidopsis bahia Dilution Water:________ Filtered Saltwater__________ Mysid Number Rep A Negativez Control Rep Rep BC Rep D i 0.51 0.57 0.49 0.68 2 0.70 0.77 0.78 0.86 3 0.52 0.70 0.60 0.54 4 0.64 0.86 0.57 0.59 5 0.56 0.61 0.51 0.53 6 0.75 0.75 0.72 0.67 7 0.44 0.68 0.50 0.60 8 0.79 0.67 0.62 9 0.58 -- 0.52 -- 10 0.69 -- 0.60 -- 11 0.41 -- -- -- 12 - Mean 0.599 0.706 0.596 0.636 -57- APPENDIX VI Mysid Dry Weight (mg) PROJECTNO.: 454A-107 Rep A 0.53 0.62 0.58 0.64 0.82 0.79 0.51 0.66 0.49 0.57 0.50 0.68 0.616 0.057 mg a.i./L Rep Rep BC 0.65 0.45 0.50 0.78 0.55 0.60 0.69 0.56 0.47 0.63 0.84 0.59 0.52 0.54 0.73 0.71 0.61 0.49 0.72 0.55 0.66 __ 0.58 - 0.627 0.590 Rep D 0.59 0.51 0.46 0.80 0.57 0.52 0.46 0.67 0.49 0.58 - 0.565 < Rep A 0.52 0.71 0.65 0.70 0.72 0.80 0.54 0.58 0.59 0.83 0.49 0.63 0.647 0.12 mg a.L/L Rep Rep BC 0.87 0.78 0.62 0.52 0.55 0.56 0.68 0.88 0.64 0.55 0.71 0.72 0.58 0.50 _ _ ---- 0.664 0.644 Rep D 0.54 0.68 0.52 0.67 0.46 0.55 0.64 0.71 0.74 0.56 _ 0.607 Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Saltwater Mysid, M ysidopsis bahia Dilution W ater________ Filtered Saltwater_______________ Mysid Number Rep A 0.25 ms a.L/L Rep Rep BC Rep D 1 0.72 0.45 0.50 0.61 2 0.60 0.68 0.62 0.67 3 0.54 0.62 0.86 0.59 4 0.55 0.53 0.45 0.53 5 0.63 0.S8 0.71 0.84 6 0.48 0.79 0.55 0.73 7 0.79 0.60 0.51 0.52 8 0.71 0.56 0.67 0.80 9 0.67 -- 0.56 0.46 10 0.75 - - - 11 " Mean 0.644 0.601 0.603 0.639 - 58- APPENDIX VI (Continued) Mysid Diy Weight (mg) Rep A 0.62 0.51 0.54 0.45 0.43 0.71 0.63 -- -- 0.556 0.55 mg a.:i./L Rep Rep BC 0.50 0.57 0.48 0.42 0.67 0.39 0.62 0.61 0.57 0.51 0.61 0.60 0.54 0.81 0.59 0.65 0.36 0.48 0.64 0.59 "" " 0.558 0.563 Rep D 0.54 0.66 0.52 0.68 0.55 0.60 0.49 0.52 -- - "" 0.570 PROJECT NO.: 454A-107 Rep ,A 0.36 0.52 0.44 -- H -- __ -- - 0.440 1.3 mg a.i./L Rep Rep BC 0.40 0.57 _ 0.43 0.51 __ . -- 0.478 __ -- ~ Rep D 0.39 .. __ __ __ -- ~ 0.39 W il d l if e In t e r n a t io n a l , Lt d . -59- PROJECT NO.: 454A-107 APPENDIX VH Changes to Protocol This study was conducted in accordance with the approved Protocol with the following changes: 1. The proposed experimental start and termination dates were amended to the protocol. v/ 2. Analysis of feed for PFOS was deleted by amendment. 3. The proportion o f water split to each replicate was not checked at the end o f the test. 4. Temperature was not measured in the B replicate of the 2.7 mg a.i./L treatment group on Day 14 o f the test. 5. Nominal test concentrations were 0.086,0.17,0.34,0.69,1.4 and 2.7 mg a.i./L. 001249 W il d l if e In t e r n a tio n a l, Lt d . -60- PROJECT NO.: 454A-107 APPENDIXVm Personnel Involved in the Study The following key Wildlife International, Ltd. personnel were involved in the conduct or management of this study: 1. Henry O. Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Manager, Analytical Chemistry 3. Jon A. MacGregor, Scientist 4. Mark A. Mank, Laboratory Supervisor 5. Kurt R. Drottar, Senior Biologist 001250 PROTOCOL PFOS: A FLOW-THROUGH LIFE-CYCLE TOXICITY TEST WITH THE SALTWATER MYSID (Mysidopsis bahia) U.S. Environmental Protection Agency v.Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1350 3M Lab Request No. U2723 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife International ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 May 13,1999 W il d l if e In te r n a tio n a l, ltd 2- - PFOS: A FLOW-THROUGH LIFE-CYCLE TOXICITY TEST WITH THE SALTWATER MYSED {Mysidopsis bahia) SPONSOR: 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 SPONSOR'S REPRESENTATIVE: Ms. Susan A. Beach TESTING FACILITY: V Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 STUDY DIRECTOR: KurtDrottar Senior Aquatic Biologist LABORATORY MANAGEMENT: Henry O. Krueger, Ph.D. Director o f Aquatic Toxicology & Non-Target Plants FOR LABORATORY USE ONLY Proposed Dates: I Experimental Start D a te :___ Experimental Termination Date: Project No.: __ Test Concentrations: Negative Control. 0.094.0.19,0.38,0,75.1.5 and 3.0 mg a.i./L Test Substance No.: 4675______ Reference Substance No. (if applicable): 4526 PROTOCOL APPROVAL STUDY DIRECTOR DATE LABORATORY MANAGEMENT ( l - K L s jk SPONSOR'S REPRESENTATIVE PROTOCOL NO.: 454/051399/MYS-LC/SUB454 DATE 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd -3- INTRODUCTION Wildlife International, L td will conduct a flow-through life-cycle toxicity test with the saltwater mysid, Mysidopsis bahia, 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 850.1350 (1); and AS7MStandard E l 191-90 Standard Guidefor Conducting Life-Cycle Toxicity Tests with SaltwaterM ysids (2). Raw data for all work performed at Wildlife International, L td and a copy of the final report will be filed by project number in archives located on the Wildlife International, L td site, car at an alternative location to be specified in the final report. PURPOSE The purpose o f this study is to determine the effects o f a test substance on the saltwater mysid, Mysidopsis bahia, during chronic exposure. Effects on growth, survival, and reproduction will be evaluated EXPERIMENTAL DESIGN Mysids will be exposed to a series of six test concentrations, a negative (dilution water) control beginning at the juvenile stage (<24 hours old) and continuing for at least 7 days after the median time o f first brood release in the control treatment. The test will not be terminated until at least 28 days and may be continued at the request of the Sponsor. Test concentrations will be chosen in consultation with the Sponsor and will be based upon known toxicity data. Each test concentration will be 50% o f the next higher treatment. Four replicate test chambers will be used for each treatment and control group, and each test chamber will contain one compartment holding 15 mysids. In order to control bias, mysids will be indiscriminately assigned to exposure chambers at test initiation. No other potential sources of bias are expected to affect the results of the study. Mysids will be indiscriminately distributed in groups o f one to three among glass beakers representing each test compartment until each beaker contains 15 mysids. The mysids will then be gently transferred into the test compartments. Thus, a total o f 60 mysids will be exposed in each treatment and control group. After mysids attain sexual maturity (usually 10 to 14 days after the beginning of the test), female and male adult mysids will be paired, and the reproduction o f the paired mysids will be monitored. 001253 PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l, ltd -4 - Observations for mortality and clinical signs of toxicity will be made daily. The number o f live young produced by each pair will be counted, recorded, and removed to a separate test compartment at the same test concentration on a daily basis. The young will be exposed under static conditions for 96 hours. The dry weight and length of first-generation rnysids alive at the end of the test will be determined. Data on survival, growth, and reproduction will be analyzed to determine the effects of the test substance on these parameters. MATERIALS AND METHODS 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 of 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 including the retention of a reserve sample o f the lot or batch of the test substance used in this study. The Sponsor also agrees to accept any unused test substance and/or test substance containers remaining at the end o f the study. Preparation of Test Concentrations The test substance will be administered to the test organism in water. This route o f administration was selected because it represents the most likely route of exposure to aquatic organisms. Test Organism The mysid, Mysidopsis bahia, will be used in this test. This species is representative of an important group of organisms and was selected for use in the test based upon past use and ease of handling in the laboratory. Mysids will be obtained from Wildlife International, Ltd. cultures. All rnysids used in the test will be from the same brood stock. 001254 PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In te r n a tio n a l, ltd -5 - Tests will be initiated with mysids less than 24-hours old (juveniles). Cultures will be maintain^ in a recirculating saltwater system kept at approximately 25C. Saltwater will be filtered through a biofilter and UV sterilized prior to entering the recirculating system. All mysids will be held in the laboratory for at least 14 days before juveniles are collected for testing. Mysids will not be subjected to more than a 3C change in temperature or a 3 !00 change in salinity in any 12-hour period during the 14 days before test initiatioa Mysids will be fed live brine shrimp nauplii (Artemia sp .) ad libitum daily. Brine shrimp will be periodically enriched with ALGAMAC-2000 (fatty acid supplement). Feed (brine shrimp) provided to the mysids will be analyzed for PFOS. Excess food will be removed from the test chambers daily. Mysids will be handled as infrequently as possible but when handling is necessary it will be done gently, carefully, and quickly. A siphon collector system will be used to collect neonate mysids directly from brood tanks. Young mysids will be transferred using large-bore pipettes. Dilution Water Natural seawater collected at Indian River Inlet, Delaware will be the source o f die dilution water used in the culture facility and test system The seawater will be filtered through a sand filter prior to its delivery to a 37,800-L holding tank The salinity o f the seawater will be diluted to approximately 20 7, (parts per thousand) with Wildlife International Ltd. wellwater in the holding tank The 20 7 saltwater will be aerated using spray nozzles and filtered (0.45 pm) prior to its delivery to the test system Salinity will be measured weekly to monitor the consistency o f the saltwater. Means and ranges o f the measured parameters for the four-week period preceding the test will be provided in the final report. Analyses will be performed to determine the concentrations o f selected organic and inorganic constituents o f the seawater and results o f the analyses will be summarized in the final report. Test Apparatus A continuous-flow diluter will be used to provide each concentration o f the test substance and a negative (dilution water) control. A syringe pump, peristaltic pump, or a similar device will be used to deliver the test substance to mixing chambers where the test substance will be mixed with dilution water. The flow of dilution water into each mixing chamber will be controlled using rotameters. The rotameters will be calibrated prior to the test and verified and/or calibrated at least once a week during the test. After mixing, PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 C01255 W il d l if e In t e r n a tio n a l, ltd 6- - test solutions will be split to each replicate chamber. The proportion o f water split to each replicate will be checked prior to the test, at approximately weekly thereafter and at the end o f the test to ensure that these flow rates vary by no more than 10% o f the mean. The diluter will be adjusted so that each test chamber receives at least 5 volume additions o f test solution every 24 hours. Peristaltic pumps will be calibrated prior to test initiation and at approximately weekly intervals thereafter and-syringe pumps will be calibrated prior to test initiation. The delivery o f the test substance to the test chambers will begin at least 48 hours prior to the test in order to establish equilibrium concentrations of the test substance. The general operation o f the diluter will be checked visually at least two times per day during the test. Test chambers will be 9-L glass aquaria filled with approximately 5 L o ftest solution. Each treatment and control group will have four replicate test chambers. Test chambers will be impartially positioned in an environmental chamber designed to maintain a temperature o f 25 2C. Test chambers will be labelled with the project number, test concentration and replicate. Prior to sexual maturity, mysids will be held in one compartment placed in each replicate test chamber (15/compartment). The compartments consist o f glass beakers with nylon mesh screen attached to two holes on opposite sides o f the beaker. All attachments o f the nylon mesh screen and glass will be made with silicone adhesive. After mysids attain sexual maturity (usually 10 to 14 days after die beginning o f the test), up to 5 male and 5 female adult mysids will be paired per replicate test chamber. Individual pairs will be held in reproductive compartments (one pair per compartment) placed in each test chamber. The reproductive compartments will be glass petri dishes with sides o f nylon mesh screen attached with silicone adhesive. Immature and additional females will be discarded. Additional male mysids will be maintained in a separate test compartment o f each replicate test chamber. Additional males will be used to replace dead males in the same replicate during the exposure. The test will not be terminated before 7 days past the median time o f first brood release in the control(s). The test will continue for at least 28 days and may be extended longer at the request o f the Sponsor. 00125G PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd -7 - Environmental Conditions Ambient room light will be used to illuminate the test systems. Fluorescent light tubes (Colortone SO or equivalent) that emit wavelengths similar to natural sunlight will be controlled by an antnmatir timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 15-to 30-minute transition period will be provided when lights go on and off to avoid sudden changes in light intensity. Light intensity will be measured at test initiation and at weekly intervals thereafter with a SPER Scientific Ltd. light meter or equivalent v The target test temperature will be 25 2C. Temperature will be monitored and recorded continuously in a negative control using a Fulscope ER/C Recorder (1900 J Series Model A) or equivalent during the entire test. Recorder measurements will be verified with a liquid-in-glass thermometer prior to test initiation and at least weekly thereafter. Temperature will also be measured in each test chamber at the beginning and end of the test and at weekly intervals during the test using a liquid-in-glass thermometer. Dissolved oxygen and pH will be measured at the beginning and the end o f the test and at weekly intervals during die test in alternating replicate test chambers o f each treatment level. In the event that dissolved oxygen levels fall below 60% saturation, appropriate actions will be taken after consultation with the Sponsor. Salinity will be measured daily in alternating replicates o f the negative control treatment and the highest PFOS treatment group. If 100% mortality occurs in any treatment, then dissolved oxygen, pH and temperature measurements will be made at that time and then discontinued. Dissolved oxygen will be measured using a Yellow Springs Instrument Model 5 IB dissolved oxygen meter or equivalent, water pH will be measured using a Fisher Accumet Model 915 pH meter or equivalent, and salinity will be measured using a refractometer or equivalent instrument. Biological Measurements Observations of mortality and clinical signs of toxicity in first-generation mysids will be made daily. The criteria for death include lack o f movement, absence o f respiratory movements, and lack o f reaction to gentle prodding. Live young produced in each compartment will be counted, recorded, and removed daily. Live young will be exposed to the same concentration o f the test substance under static test conditions for approximately 96 hours. At the end of the test, the dry weight and total body length o f each first-generation 001257 PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd 8- - mysid will be determined. Observations o f abnormal development and aberrant behavior will be made for first-and second-generation mysids throughout the test. Sampling for Analytical Measurements Water samples will be collected from two replicate test chambers o f both the low and high level concentrations prior to exposure. Water samples also will be collected from two alternating replicate test chambers o f each treatment-group and the control at the beginning o f the test, at weekly intervals during the test, and at the end o f the test to determine concentrations o f the test substance. In the event that 100% mortality occurs in any treatment, then sampling of that treatment will terminate following the next sampling interval. Samples will be collected at mid-depth from each test chamber, and analyzed immediately. The sample scheme is summarized below: PROPOSED NUMBERS OF VERIFICATION SAMPLES Experimental Group Pretest* DayO Day 7 Day 14 Day 21 Day 28 End Control Level 1-Low Concentration Level 2 Level 3 Level 4 Level 5 Level 6-High Concentration - 2 2 2 2 22 2b 2 2 2 2 2 2 - 2 2 2 2 22 - 22 2 2 22 - 2 2 2 2 22 - 2 2 2 2 22 2 2 2 2 2 22 Totals 4 14 14 14 14 14 14 *Prc-tcst samples will be collected after conditioning ofthe diluter. More than one pre-test sampling interval may be required, depending upon the Sponsor's needs, and additional sampling and results will be documented in the raw data and included in the final report bOne sample from the A and B replicate test chambers. Total Number ofVerification Samples " 88 The above numbers o f samples 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 analytical chemistry phase of the study. At the discretion o f the Study Director, Water samples also will be collected from at least one appropriate chamber whenever a malfunction is detected in any part o f the test substance delivery system. 001.258 PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd -9 - Analvtical Measurements Chemical analysis of the 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 H Modifications made to the analytical method will be documented in the raw data and described in the final report Data Analyses Data to be analyzed include the survival o f first-generation rnysids, the numb' o f young released per reproductive day, the dry weight and length o f each first-generation mysid alive at the end o f the test. If deemed necessary, clinical signs o f toxicity in the adult rnysids may also be analyzed statistically. Concentration-response curves will be fitted to the adult survival, reproduction and growth data. To evaluate whether or not survival is affected by the treatments, 2 X 2 contingency tables and the chi-square test will be used on survival data (i.e., numbers o f alive and dead rnysids). Analyses o f reproduction (number o f young produced per surviving adult) and growth (weight and length) data will be evaluated for normality and homogeneity o f variances. If the data are deemed normal with homogeneous variances, hypothesis testing using analysis of variance (ANOVA) and multiple means tests (e.g., Dunnett's, Bonferroni, Scheffe) will be used. If the data fail the test for normality or homogeneity, then data transformations will be tried in an attempt to correct the condition. When the data transformations fail to correct for non-normality or heterogeneity o f variances, nonparametric procedures will be used to identify statistically significant differences among the experimental groups. The statistical analyses o f survival, growth and reproduction data will aid but may not be exclusively used in the determination o f the no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC). The maximum acceptable toxicant concentration (MATC) will be calculated as the geometric mean o f the NOEC and LOEC. All statistical evaluations will be done using commercially available computer software programs such as TOXSTAT (3) OR SPSS/PC+ (4). RECORDS TO BE MAINTAINED Records to be maintained for data generated by Wildlife International, Ltd. will include, but not be limitedto: 003.259 PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a tio n a l, ltd - 10 - 1. A copy o f the signed protocol. 2. Identification and characterization o f the test substance, if provided by the Sponsor. 3. Dates o f initiation and termination o f the test. 4. History o f the test organism. 5. Test organism weight and length measurements at the end o f the test. 6. Stock solution calculation and preparation. 7. Daily observations. v 8. Water chemistry results (e.g., salinity and pH). 9. If applicable, the methods used to analyze test substance concentrations and the results o f analytical measurements. 10. Statistical calculations. 11. Test conditions (light intensity, photoperiod, etc.). 12. Calculation and preparation o f test concentrations. 13. Copy o f final report. FINAL REPORT A final report o f the results o f the study will be prepared by Wildlife International, Ltd. The report will include, but not be limited to the following, when appropriate: 1. Name and address of the facility performing the study. 2. Dates upon which the study was initiated and completed. It is the responsibility o f the Sponsor to provide the final date that data are recorded for chemistry pathology and/or supporting evaluations that may be generated at other laboratories. 3. A statement o f compliance signed by the Study Director addressing any exceptions to Good Laboratory Practice Standards. 4. Objectives and procedures as stated in the approved protocol, including any changes in the original protocol. 5. The test substance identification, including name, chemical abstract number or code number, strength, purity, composition, and other characteristics 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 o f the test organisms, scientific name, age, life stage, means and ranges o f weights and lengths, observed diseases, treatments, holding proce dures, and feeding regime. 0 0 1 -2 6 0 PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In te r n a tio n a l, ltd - n- 9. A description o f the preparation o f the test solutions, the methods used to allocate organism s to test chambers and begin the test, the number o f organisms and chambers per treatment, and the duration of the test. 10. A description o f circumstances that may have affected the quality or integrity o f the data. 11. The name of die Study Director and the names o f other scientists, professionals, and supervisory personnel involved in the study. 12. A description o f the transformations, calculations, and operations performed on the data, a summary and analysis o f die biological data and analytical chemistry data, and a statement o f the conclusions drawn from the analyses. 13. Statistical methods used to evaluate the data. 14. The signed and dated reports o f each o f the individual scientists or other professionals involved in the study. 15. The location where raw data and final report are to be stored. 16. A statement preparedby the Quality Assurance Unit listing the dates that study inspections and audits were made and the dates o f any findings reported to the Study Director and Management. 17. 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 alteration. Amendments will be signed and dated 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 w ill 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 (ENV/MC/CHEM (98) 17); and Japan MAFF (59 NohSan, Notification No. 3850, Agricultural Production Bureau). Each study conducted by Wildlife International, Ltd. is routinely examined by the Wildlife International, Ltd. Quality Assurance Unit for compliance with Good Laboratory Practices, Standard Operating Procedures and the 0012G1 PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd -12- specified protocol. A statement o f compliance with Good Laboratory Practices will be prepared for all portions o f the study conducted by Wildlife 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 die final report will be filed by project number in archives located on the Wildlife International, Ltd. site, or at an alternative location to be specified in the final report PROTOCOL NO.: 454/051399/MYS-LC/SUB454 001262 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l, ltd -13- REFERENCES 1 U.S. Environmental Protection Agency. 1996. Series 85 0 -Ecological Effects Test Guidelines (draft), OPPTS Number 850.1350: M ysid Chronic Toxicity Test. 2 ASTM Standard E1191-90. 1991. Standard Guidefo r Conducting Life-Cycle Toxicity Tests with Saltw ater M ysids, American Society for Testing and Materials. 3 W est, Inc. and D. D. Gulley. 1996. TOXSTATRelease 3.5. Western EcoSystems Technology, Inc. Cheyenne, Wyoming. 4 Norusis, MJ . 1988. SPSS/PC+ V2.0 base manual for die IBM PC/XT/AT and PS/2. C012G3 PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd -14- APPENDIXI IDENTIFICATION OF TEST SUBSTANCEBY SPONSOR Tobe Completedby Sponsor I. Test Substance Identity (name to be used in the report): PFOS (Perfluorooctane Sulfonic Acid Potassium Salt Reference Standard (if applicable): Analytical Standard: N/A_______________________________ Internal Standard: 1.1.2.2KH.H.H Perfluorooctane Sulfonic Acid Test Substance Sample Code or Batch Number: Lot 217____________________________________ Test Substance Purity (% Active Ingredient): 98.9______ Expiration Date: 2008____________ E Test Substance Characterization Havetheidentity,strength, purityandcompositionorothercharacteristics which appropriatelydefinethetest substance andreferencestandardbeen determinedpriorto itouse inthis studyin accordancewith GLP Standards? _____ Yes x No EL Test Substance StorageConditions Pleaseindicatetherecommendedstorageconditions atWildlifeInternational,Ltd. Ambient__________ ;____________________________________________________________________________ Has the stabilityofthetest substanceunderthese storageconditions been determinedin accordancewith GLP Standards? Otherpertinent stabilityinformation: _____ Yes x No IV. TestCorxentratians: _____ x Adjusttestcanomlratioato 100%a i basedupontte purity(%1givenabove. ___________ Do ai nToetsatdmjuesmttaetretrciaolnAceSntIrSa.tionto 100% V. ToxicityInformation: Mammalian: RatLD50 251 me/kg MouseLD50 N/A Aquatic: InvertebrateToxicity (EC/LC50) Fish Toxicity (LC50) Daphnia maena: 27 mg/L__________ Rainbow Trout: 11 mg/L Daphnia maena: 50 me/L__________ Fathead Minnow: 38 mg/L OtherToxicityInformation (includingfindings ofchronic andsubdbronictests): Please see MSDS_______________________________________________________________ 001264 PROTOCOL NO.: 454/051399/MYS-LC/SUB454 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l , ltd -15- APPENDIXn Analytical Method Provided by Sponsor Samples will be analyzed based upon procedures provided by the Sponsor in the following analytical methods: 1. Liquid Chromatography Mass Spectrometry (LCMS) Method for the Determination o f Perfluorooctane Sulfonic Acid Potassium Salt (PFOS) In Freshwater, Saltwater and Algal Medium A copy o f the above method will be maintained in the raw data. The actual methodology used to analyze the test samples will be documented in the raw data and summarized in the final report. PROTOCOL NO.: 454/051399/MYS-LC/SUB454 001265 3M LAB REQUEST NO. U2723 W il d l if e In t e r n a t io n a l l td . PROJECT NO.: 454A -107 Page 1 o f 1 AMENDMENT TO STUDY PROTOCOL STU D Y TITLE: PFOS: A FLOW-THROUGH LIFE-CYCLE TOXICITY TEST WITH THE SALTWATER MYSID (Mysidopsis bahia) PR O TO C O L N O .: 454/051399/M Y S-LC /SU B 454 A M EN DM ENT N O .: 1 SPO N SO R : 3M Corporation PR O JEC T N O .: 454A -107 EFFEC TIV E D ATE: June 16, 1999 A M EN DM ENT: Page.? ~" Add: Proposed Dates: Experimental Start Date: 6/16/99 Experimental Termination Date: 7/21/99 R EA SO N : The above information w as not known when the protocol w as signed by the Study D irector. A M EN D M E N T: Test Organism. Page 5 D elete: Feed (brine shrimp) provided to the mysids will be analyzed for PFOS. R EA SO N : The feed has already been screened for PFOS. STUDY DIRECTOR -pinm DATE labcK t o r y l& agement ( -fh d s A SPONSOR'S REPRESENTATIVE a454\l07\amendl ikyh i DATE 0012G6 ftev.4u.ed biy