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17) OECD 203-OPPTS 850.1075, 96-Hour static acute toxicity test with the (fathead minnow), 454A115 PERFLUORO BUTANE SULFONATE, POTASSIUM SALT (PFBS): A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE FATHEAD MINNOW (Pimephulespromelus) FINAL REPORT SANITIZED WILDLIFE INTERNATIONALLTD. PROJECT NUMBER: 454A- 115 3M LAB REQUEST NO. E00-1429 DEC 0 9 zoo3 U. S Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1075 and OECD Guideline 203 AUTHORS: Kurt R. Drottar Henry 0. Krueger, Ph.D. STUDY INITIATION DATE: April 4,2000 STUDY COMPLETION DATE: March 20,200 1 Submitted to 3M Corporation Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, Minnesota 55144 WildlifeInternational, Ltd. 8598 Commerce Drive Easton, Maryland 2 1601 (4 10) 822-8600 Page 1 of 41 'WILDLIFE INTERNATIONLATLD. PROJECT NO.: 454A-115 -2GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT DEC 0 9 2003 SPONSOR: 3M Corporation SANITIZED TITLE: Perfluoro Butane Sulfonate, Potassium Salt (PFBS): A 96-Hour Static Acute Toxicity Test with the Fathead Minnow (Pimephalespromelas) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-115 STUDY COMPLETION: March 20,2001 This study was conducted in compliancewith Good LaboratoryPractice Standardsaspublishedby the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHM (98) 17); and Japan MAFF,' 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984 with the following exceptions: The test substance was not characterized in compliance with Good Laboratory Practices prior to its use in the study. However, subsequent GLP compliant characterization resulted in a purity similar to the original characterization purity. The stability of the test substance under conditions of storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Kurt R Drottar Senior Biologst SPONSOR APPROVAL: DATE 3 h.3Ji I DATE WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A- 115 -3- QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S.Environmental Protection Agency, 40 CFRParts 160and 792,17 August 1989; OECD Principlesof Good Laboratory Practice (ENVMCKHEM (98) 17); and Japan MAFF, 59NohSan, 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: ACTMTY: Test Substance Reparation I Matrix Fortification Observations Analytical Data and Draf? Report Biological Data and Draft Report Final Report DATE CONDUCTED: December 18,2000 December 18,2000 December21,2OOO January 16 and 17,2001 January 18 and 19,2001 March 16,2001 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: December 18,2000 December 20,2000 December 18,2000 December 20,2000 December 21,2000 January 4,2001 January 17,2001 January 18,2001 January 19,2001 March 16,2001 January 23,2001 March 16,2001 I J&es H. CoIeman Quality Assurance Representative DATE WILDLIFE INTERNATIONALTLD. -4REPORT APPROVAL PROJECT NO.: 454A-115 SPONSOR: 3M Corporation TITLE: Perfluoro Butane Sulfonate, Potassium Salt (PFBS): A 96-Hour Static Acute Toxicity Test with the Fathead Minnow (Pimephalespromelas) WILDLIFE INTERNATIONAL,LTD. PROJECT NUMBER: 454A- 115 STUDY DIRECTOR: Kurt R Drottar Senior Biologist MANAGEMENT: Director, Aquatic Toxicology and Non-Target Plants DATE WILDLIFE INTERNATIONALILD . -5- PROJECT .NO. 454A-115 TABLE OF CONTENTS TitleKover Page................................................................................................................................................... 1 Good Laboratory Practice Compliance Statement............................................................................................... 2 Quality Assurance Statement............................................................................................................................... 3 Report Approval................................................................................................................................................... 4 Table of Contents................................................................................................................................................. 5 sumlnaly............................................................................................................................................................... 7 Introduction .......................................................................................................................................................... 8 Objective ................................................................. ..:.......................................................................................... 8 Experimental Design ............................................................................................................................................ 8 Materials and Methods......................................................................................................................................... 9 Results and Discussion...................................................................................................................................... 12 Conclusions....................................................................................................................................................... 13 References ......................................................................................................................................................... 14 TABLES Table i .Summary ofhalyhcal Chemistry Data ........................................................................................... 15 Table 2 .Temperature. Dissolved Oxygen and pH of Water in the Test Chambers ....................................... 16 Table 3 .CumulativePercent Mortality and Treatment-RelatedEffects......................................................... 17 Table 4 .LC50 Values ...................................................................................................................................... 18 WILDLIFE INTERNATIONLTDA. L -6TABLE OF CONTENTS - Continued - PROJECT NO.: 454A- 115 FIGURE Figure 1. Concentration-Response Curve (96-Hour Data) ............................................................................ 19 APPENDICES Appendix 1 - Specific Conductance, Hardness, Alkalinity, and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test .................................. 20 Appendix 2 - Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water ............................................................................. 2 1 Appendix 3 - The Analysis of PFBS in Freshwater in Support of Wildlife International, Ltd. Project No.: 454A- 115 ........................................................................... 23 Appendix 4 - Changes to Protocol ............................................................................................................... 41 Appendix 5 - Personnel Involved in the Study............................................................................................. 42 WILDLIFE INTERNATIONALTLD. SANITIZED PROJECT NO.: 454A-115 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: -7SUMMARY 3M Corporation DEC 0 9 2003 Wildlife International Ltd. Easton, Marvland 2 1601 WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANISMS: 454A-115 Perfluoro Butane Sulfonate, Potassium Salt (PFBS) Perfluoro Butane Sulfonate, Potassium Salt (PFBS): A 96-Hour Static Acute Toxicity Test with the Fathead Minnow (Pimephales promelas) Negative Control, 220,437, 888, 1655 and 3341 mg a.i./L Experimental Start (OECD) - December 11,2000 Experimental Start (EPA) - December 18; 2000 Biological Termination - December 22,2000 Experimental Termination- December 22,2000 96 Hours Fathead Minnow (Pimephalespromelas) Wildlife International, Ltd. cultures Easton, Maryland 2 1601 AGE OF TEST ORGANISMS : MEASUREMENTS OF 10 NEGATIVE CONTROL FISH: WEIGHT (8): TOTAL LENGTH (D): 96-HOUR ~ C 5 0 : 95% CONFIDENCE LIMITS: NO MORTALRY CONCENTRATION: NO-OBSERVED-EFFECTCONCENTRATION: Juveniles Mean = 0.32; Mean = 35: Range = 0.22 to 0.47 Range = 30 to 40 1938 mg a.i./L 888 and 3341 mg a.i./L 888 mg a.i./L 888 mg a.i./L WILDLIFE INTERNATIONALTLD. -8INTRODUCTION PROJECT NO.: 454A-115 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 December 18,2000 to December 22,2000. Raw data generated by Wildlife InternationalLtd.and a copyof the final report are filed under Project Number 454A-115 in archives located on the Wildlife International, Ltd. site. OBJECTIVE The objective of this study was to evaluate the acute toxicity of Perfluoro Butane Sulfonate, Potassium Salt (PFBS) to the fathead minnow, Pimephalespromelas, during a 96-hour exposure period under static test conditions. EXPERIMENTAL DESIGN Fathead minnows were exposed to a geometric series of five test concentrations and a negative (dilution water) control. Two replicatetest chambers were maintained in each treatment and controlgroup,with 10fathead minnows in each test chamber for a total of 20 fathead minnows per test concentration. Nominal test concentrationswere selectedin consultation with the Sponsor, and were based upon the results of an exploratory range findingtoxicity test. Nominal test concentrations selectedwere 204,408,8 16,1632 and 3263 mg active ingredient (a.i.)/L. Mean measured test concentrationswere determined from samplesof testwater collectedffom each treatment and the control goup at the beginning of the test, at approximately 48 hours, and at test termination. Fatheadminnows were indiscriminately assigned to exposurechambersat test initiation. Observationsof mortality and other clinical signs of toxicity were made at approximately 2,24, 48, 72 and 96 hours after test initiation. Cumulativepercent mortality observed in the treatment groups was used to calculateLC50 values at 24,48,72 and 96 hours. The no mortality concentrationand the no-observed-effect-concentration(NOEC)were determined by visual interpretation of the mortality and clinical observation data. WILDLIFE INTERNATIONALILD. SANITIZED -9- DEC 0 9 2083 MATERIALS AND METHODS PROJECT NO.: 454A-115 The study was conducted based on the procedures outlined in the protocol, "PerfluoroButane Sulfonate, Potassium Salt (PFBS): A 96-Hour Static Acute Toxicity Test with the Fathead Minnow (Pimephales promelas)".The protocol was based on procedures outlined in U.S. EnvironmentalProtectionAgency Series 850 - Ecological Effects Test Guidelines, OPPTS Number 850.1075 (1): OECD Guideline for Testing of Chemicals 203: Fish, Acute Toxicity Test (2); and ASTM Standard E729-88a, Standard Guidefor Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians (3). Test Substance The test substance was received from 3M Corporation on June 28, 2000 and was assigned Wildlife International Ltd. identification number 5292. The test substance was described as a white powder. It was identified as Potassium Perfluoro butane sulfonate, AKA Developmental Product, AKA PFBS, fiom lot 2. Informationprovided by the Sponsor indicated a purity of 97.9%. A subsequentrevision of the certificate of analysis indicated a purity of 97.3% and an Expiratiofleassessment Date of January 17, 2002. The test substance was stored at ambient room temperature. Preparation of Test Concentrations Nominal test concentrations were 204,408, 816, 1632 and 3263 mg a.i./L,. All materials which came into contact with the test substance during preparation of test concentrations were constructed of plastic or stainless steel. A 60-L primary stock solutionwas prepared in dllution water at a concentrationof 3263 mg a.i./L. The primary stock solution was mixed with an electric mixer to aid in the solubilization of the test substance. After mixing, the primary stock solution was proportionally diluted with dilution water to prepare the four additionaltest concentrations. The appropriateamount of primary stock was mixed with dilutionwater inthe test chambers. All test solutions appeared clear and colorless. Test concentrations were corrected for the original purity of the active ingredient in the test substance (97.9%). Test Organism The fathead minnow, Pimephalespromelas, was selected as the test species for this study. The fathead minnow is representative of an important group of aquatic vertebrates and was selected for use in the test based upon past history of use in the laboratory. Fathead minnows used in the test were obtained from cultures maintained at Wildlife International, Ltd. WILDLI FE INTERNATIOLNTDA.L PROJECT NO.: 454A-115 Fathead minnows were held at approximately the same temperature as used during the test. The fish were held for approximately 8 months prior to testing. The fish were acclimated to test conditions for approximately 52 hours prior to test initiation. During the holding and acclimationperiods, the fish showed no signs of disease or stress. During the 14-dayholding period preceding the test, water temperaturesranged from 19.3t022.6OC. ThepHofthewaterrangedfrom8.2 to 8.5 anddissolvedoxygenrangedfrom7.5 to 8.2mg/L. Instrumentation and methods used for water measurements are described in the Environmental Conditions section of this report. At test initiation, the fathead minnows were collected from the acclimation tank and transferred to the test chambers. During the holding period, fathead minnows were fed a commercially-prepareddiet (Zeigler Brothers, Inc., Gardners, PA). The fish were not fed during the acclimationperiod (at least 48 hours prior to the test) or during the test. All fish used in the test were fiom the same source and year class, and the total length of the longest fish was no more than twice the length of the shortest. The average total length of 10 negative control fish measured at the end of the test was 35 mm with a range of 30 to 40 mm. The average wet weight (blotted dry) of 10 negative control fish at the end of the test was 0.32 grams with a range of 0.22 to 0.47 grams. Loading was 0.2 1 g fishL of test water present in the test chambers at any given time. Test Apparatus Test chamberswere 25-L polyethylene aquaria containing 15L of test solution. The depth of water in a representative test chamber was approximately 16.5 cm. Test chambers were impartially positioned in an environmentalchamber set to maintab a temperature of 22k2"C. The test chamberswere labeledwith theproject number, test concentration and replicate. Dilution Water The water used for culturing and testing was freshwater obtained from a well approximately40 meters deep located on the Wildlife International, Ltd. site. The well water is characterized as moderately-hard water. The specific conductance, hardness, alkalinity, and pH measurements of the well water during the four-week period immediately preceding the test are presented in Appendix 1. WILDLIFE INTERNATIONALTLD. PROJECT NO.: 454A-115 -11- The well water was passed through a sand filter to remove particles greater than approximately 25 pm, and pumped into a 37,800-L storage tank and aerated with spray nozzles. Prior to use, the water again was filtered (0.45 p)to remove microorganisms and particles. The results of periodic analyses performed to measure the concentrations of selected contaminants in well water used by Wildlife International, Ltd. are presented in Appendix 2. Environmental Conditions Lighting used to illuminate the cultures and test chambers during holding, acclimation 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 of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity at test initiation was approximately 222 lux at the surface of the water. Light intensity was measured using a SPER Scientific Ltd. light meter. Temperature was measured in each test chamber at the beginning of the test and at approximately 24-hour intervals thereafter using a liquid-in-glass thermometer. Temperaturealsowasmeasuredcontinuouslyin one negativecontrol replicate using a Fulscope EWC Recorder. The target test temperature duringthestudy was 22i2"C. Dissolved oxygen and pH measurements were made on water samples from all replicate test chambers of each treatment and control at test initiation and at approximately 24-hour intervals thereafter. Hardness, alkalinity and specific conductance were measured in the dilution water at test initiation. Measurementsof pH were made using a Fisher Accumet Model 915 pH meter, and dissolvedoxygenwas measured using a Yellow Springs Instrument Model 5 1B dissolved oxygen meter. Specific conductance was measured using a Yellow Springs InstrumentModel 33 Salinity-Conductivity-Temperaturemeter. Hardnessand alkalinitymeasurements were made by titrationbased on procedures in StandardMethodsfor the Examination of Water and Wastewater (4). Observations Observations were made to determinethe number of mortalities. The number of individualsexhibiting clinical signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 2, 24,48,72 and 96 hours after test initiation. WILDLIFE INTERNATIONLTDA. L PROJECT NO.: 454A-115 - 12- Statistical Analyses The 24,48,72 and 96-hour LC50 values and the 95% confidenceintervalswere calculatedwhen possible by probit analysis, the moving average method or binomial probability with non-linear interpolation ( 5 , 6 , 7) using the computer software of C.E. Stephan (8). In this study, the binomial method was used to calculate all LC50 values. The no mortality concentrationand NOEC were determinedby visual interpretationof themortality and clinical observation data. Analvtical Chemistrv Water samples were collected at mid-depth from each replicate test chamber of each treatment and control group at the beginning of the test, at 48 hours and at test terminationto measure concentrationsof thetest substance. The samples were collected in plastic vials and analyzed as soon as possible without storage. Analytical procedures used in the analysis of the samples are provided in Appendix 3. RESULTS AND DISCUSSION Measurement of Test Concentrations Results of analyses to measure concentrations of PFBS in water samples collected during the test are presented in Table 1and in the analyticalchemistry report (Appendix 3). Nominal concentrationsselectedforuse in this study were 204,408,816, 1632 and 3263 mg a.i./L. Samples collected at test initiation had measured values that ranged from 100to 105%of nominal values. Measured values for samples taken at 48 hours ranged from 99 to 109% of nominal. Measured values for samples taken at 96 hours ranged from 99 to 123% of nominal. When measured concentrationsof the samples analyzed at test initiation,approximately48 hours and at test terminationwere averaged, the mean measured concentrationsfor this study were 220,437,888,1655 and 3341 mg a.i./L. Mean measured concentrations were used in the calculation of LC50 values. Observations and Measurements Measurements of temperature, dissolved oxygen and pH are presented in Table 2. Temperatures were within the 221t2OC range established for the test. Dissolved oxygen concentrationsremained 17.5m g 5 (86% of saturation) throughout the test. Measurements of pH ranged from 8.3 to 8.5 during the test. Daily observations of mortality and other clinical signs of toxicity observedduringthe test are shownin Table 3. Fathead minnows in the negativecontrol, 220 and 888 mg a.i./Ltreatment groups appearednormaland WILDLIFE INTERNATIONALTLD. PROJECT NO.: 454A-115 -13- healthy during the test. One fish in the 437 mg a.i./L treatment group was observed to be lying on the bottom after 2 hours of exposure. However, throughout the remainder of the test all fish in the 437 mg a.i./L treatment group appearednormal. After 96-hours of exposure,mortality in the 1655and 3341mg a.i./L treatment groups was 30 and loo%, respectively. LC50 values and 95% confidence limits at 24, 48, 72 and 96 hours were calculated from the mortality data, and are shown in Table 4. A graph of the concentration-response curve is presented in Figure 1. CONCLUSIONS The 96-hour LC50 value for fathead minnows (Pimephalespromelas) exposed to Perfluoro Butane Sulfonate, Potassium Salt (PFBS) was 1938 mg a.i./L with 95% confidence limits of 888 and 3341 mg a.i./L. The 96-hour no-mortality concentration and the NOEC were 888 mg a.i./L. \ WILDLIFE INTERNATIONLTAD.L - 14- REFERENCES PROJECTNO.: 454A-115 1 U.S. Environmental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines (drap),OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater andMarine. 2 Organisation for Economic Cooperation and Development. 1993. OECD Guidelines for Testing of Chemicals. Guideline 203: Fish, Acute Toxicity Test. Adopted by the Council on 12 July 1992. 3 ASTM Standard E729-88a. 1994. Standard Guidefor ConductingAcute ToxicityTests withFishes, Macroinvertebrates, and Amphibians. American Society for Testing and Materials. 4 APHA, AWWA, WPCF. 1985. StandardMethodsfor the Examination of Water and Wastewater. 16th Edition. American Public Health Association. American Water Works Association. Water Pollution Control Federation, New York. 5 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. - 6 Finney, D.J. 1971. Statistical Methods in Biological Assay. Second edition. Griffin Press, London. 7 Thompson, W.R 1947. Bacteriological Reviews. Vol. 11, No. 2. Pp. 115-145 8 Stephan, C.E. 1977. "Methods for Calculating an LC50," Aquatic Toxicology and Hazard Evaluations, American Society for Testing and Materials. Publication Number STP 634, pp 65-84. WILDLIFE INTERNATIOLNID.AL -15- PROJECT NO.:454A-115 Sponsor: Test Substance: Test Organism: Dilution Water: Nominal Test Concentration (mg a.i./L) Negative Control Table 1 Summary of Analytical Chemistry Data 3M Corporation PFBS Fathead Minnow, Pimephales promelas Well Water Replicate A B A B A B Sampling Time (Hours) 0 0 48 48 96 96 Measured Concentration (mg a.i./L) <LOQ' <LOQ <LOQ <LOQ <LOQ <LOQ 204 A 0 205 B 0 209 A 48 21 1 B 48 213 A 96 250 B 96 232 408 A 0 426 B 0 407 A 48 43 2 B 48 415 A 96 498 B 96 446 816 A 0 824 B 0 85 1 A 48 888 B 48 876 A 96 962 B 96 926 1632 A 0 692 B 0 674 A 48 665 B 48 617 A 96 67 3 B 96 61 1 3263 A 0 B 0 A 48 B 48 A 96 B 96 'The limit of quantitation (LOQ) was 50.0 mg a.i./L. 'Samples not collected due to 100% mortality. 3354 3417 3327 32q4 -- --2 Mean Measured Concentration (mg a.i./L) <LOQ 220 437 888 1655 3341 Percent of Nominal -- 108 107 109 101 102 - 16- Table 2 Temperature, Dissolved Oxygen and pH of Water in the Test Chambers PROJECT NO.: 454A-115 Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration (mg a.i./L) Nee"ative Control 3M Corporation PFBS Fathead Minnow, Pimephales promelas Well Water 0 Hour' T- e__m__n2 D_ O_3 Replicate (OC) (mg/L) pH A 21.5 8.4 8.4 B 22.1 8.4 8.3 24 Hours Tern0 DO (ocj (mg/L> PH 21.1 8.1 8.4 22.6 8.0 8.4 48 Hours TemD DO ("C) (mg/L) pH 20.7 8.3 8.3 22.5 8.1 8.4 72 Hours Temp Do ( o c j (m&) PH 20.8 7.8 8.3 22.5 7.7 8.3 96 Hours Temp DO (Ocj (mg/L) pH 21.1 8.0 8.4 22.7 7.8 8.4 220 A 22.0 8.3 8.4 22.3 8.0 8.4 22.1 8.2 8.4 22.2 7.8 8.4 22.4 8.0 8.5 B 22.0 8.3 8.4 22.4 7.9 8.4 22.3 8.2 8.4 22.3 7.8 8.4 22.6 7.7 8.5 437 A 22.2 8.3 8.4 22.4 7.7 8.4 22.1 7.8 8.4 22.2 7.5 8.4 22.8 7.5 8.4 B 22.1 8.3 8.4 22.2 7.8 8.4 22.3 7.9 8.4 22.1 7.6 8.4 22.4 7.5 8.5 888 A 22.0 8.3 8.4 21.1 8.1 8.5 20.7 8.0 8.4 20.8 7.9 8.4 21.6 7.8 8.5 B 21.8 8.4 8.4 21.8 7.8 8.4 21.6 7.9 8.4 21.8 7.6 8.4 22.0 7.6 8.5 1655 A 22.0 8.4 8.4 22.1 7.8 8.4 B 22.1 8.4 8.4 22.5 7.6 8.4 21.8 8.1 8.4 22.4 7.6 8.4 22.1 8.0 8.5 22.4 7.5 8.4 22.3 7.9 8.5 22.5 7.5 8.5 3341 A 22.1 8.4 8.4 22.6 7.9 8.4 22.4 - 4 - - - I - -- B 22.0 8.5 8.4 22.5 8.0 8.4 22.4 - 4 - - -- - -- ' The 0-hour dilution water measurements for hardness, alkalinity and specificconductance were 128 mg/L as CaCO3, 182 m& as CaC03and 340 pmhos/cm, respectively. Temperature measured continuously during the test ranged from approximately 21.O to 22.0"C. A dissolved oxygen concentration of 5.2 mg/L represents60% saturation at 22C in freshwater. - 17- Table 3 Cumulative Percent Mortality and Treatment-Related Effects SpOnSOf: Test Substance: Test Organism: Dilution Water: Mean Measured 3M Corporation PFBS Fathead Minnow, Pimephales promelas Well Water 2 Hours Test Concentration No. (mg a.i./L) Replicate Exposed No. Dead' ~ffm' Negative Control A B 10 0 10 AN 10 0 10 AN 24 Hours No. Dead Effects 0 10 AN 0 10 AN 48 Hours No. Dead Effects 0 10 AN 0 10 AN 72 Hours. No. Dead Effects 0 10 AN 0 10 AN Cumulative Percent Mortality 0 220 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 43 7 A 10 0 9AN,IR 0 10 AN 0 10 AN 0 10 AN 0 B IO 0 10 AN 0 10 AN 0 10 AN 0 10 AN 888 A 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 B 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 1655 A 10 0 10 AN 3 7AN 5 5AN 5 5AN 30 B 10 0 10 AN 0 10 AN 0 10 AN 1 9AN - - 3341 A 10 0 10 AN 10 10 - 10 100 - - B 10 0 10 AN 10 10 - 10 ' Cumulative number of dead fish. ' Observed Effects: AN = Appears Normal, R = Lying on Bottom PROJECTNO.: 481A-115 96 Hours No. Dead Effects 0 10 AN 0 10 AN Cumulative Percent Matar%' 0 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 5 5AN 30 1 9" 10 10 - - 100 WILDLIFE INTERNATIONALTLD. - 18- PROJECT NO.: 454A-115 Table 4 LC50 Values Sponsor: Test Substance: Test Organism: Dilution Water: Time 24 Hours 3M Corporation PFBS Fathead Minnow, Pimephales promelas Well Water Lower 95% Confidence LC50 Limits (mg a.i./L) (mg a.i./L) 21 18 1655 48 Hours 1999 1655 72 Hours 1938 888 96 Hours 1938 888 Upper 95% Confidence Limits (mg a.i./L) 3341 3341 334 1 3341 Statistical Method Binomial Binomial Binomial Binomial WILDLI FE INTERNATIO~NAmL. - 19- Figure 1. Concentration-ResponseCurve (96-Hour Data) PROJECT NO.: 454A-115 8 7 n .c t-n 6 a 0, aW 5 .E- e 3z 4 3 2 1 100 1000 Mean Measured Concentration (mg a.i./L) 10000 WILDLIFE INTERNATIOLNTDA. L PROJECT NO.: 454A-115 Appendix 1 Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Fathead Minnow, Pimephales promelas Well Water Specific Conductance . (pnhos/cm) Mean 313 (N=4) Range 310 - 315 Hardness (m& as CaC03) Alkalinity ( m aas CaC03) 131 (N=4) 178 (N = 4) 128 - 132 176 - 178 PH 8.0 (N = 4) 8.0 - 8.1 W I L D L I F E INTERNATIONAL Ln,. - 21 - PROJECT NO.: 454A-115 Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water' Component Aclonifen Alachlor Ametryn Atrazine Azinphosethyl . . Azinphos-methyl Azoxystrobin Bifenthrin Bioallethrin Bitertanol Bromacil Bromophos Bromophosethyl Bromopropylate Bupirimate Carbaryl Carbofuran Carboxin C hlorfenvinphos Chloridazon C hlorpropham C hlorpyrifos Chlorpyrifos-rnethyl Chlorothalonil Coumaphos Cyanazine Cyfluthrin Cypermethrin Cyproconazole Deltamethrin Demeton Demeton-0 Desethylatrazine Desisopropylatrazine Desrnetryn Diazinon Dichlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid Measured Concentration Component Pesticides and Organics 4.03 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4.04pg/L 4 . 0 8 pg/L 4 . 2 5 pg/L 4 . 0 5 pg/L 4.05 pg/L 4 . 0 5 pg/L 4 . 0 5 pg/L 4 . 0 2 pg/L 4 . 0 2 pg/L Dichlorvos Dicofol Diethyltoluamide Difenoconazole Dimethoate Dimethomorph Disulfoton DMST Dodernorph Endosulfan-a Endosulfan-j3 Endosulfan-sulfte Epoxiconazole 4 . 0 2 pg/L <0.05 pg/L 4.05 pgL 4.03pg/L 4 . 0 2 pglL 4.02 ppn <0.05 pglL <0.02 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4.04 Pg/L 4.02pg/L 4 . 0 5 pg/L 4.05 pgn 4 . 2 5 pg/L d.05 pg/L 4 . 0 2 pg/L 4 . 0 2 pg/L 4 . 0 2 pg/L 4.01 p g n 4.02 pg/L 4 . 0 1 pg/L 4.01 pg/L 4 . 0 1 pg/L 4.03 pg/L <0.02 p g 5 <0.01 pg/L 4.03 pg/L EPm Esfenvalerate Ethion Ethofumesate Ethoprophos Etridiazole Etrimfos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb Fenpiclonil Fen propath rin Fenpropirnorph Fenthion Fenvalerate flumi fop-butyl Fluoroglycofen-ethyl Fluroxypyr-meptyl Flutolanil Fonophos Furalaxyl Heptenophos Imazalil Iprodion Kresoxim-methyl Lenacil Lindane Measured Concentration <0.01 pg/L <0.25 p g L <0.02 p g n <0.03 pglL 4 . 0 2 pg/L 4 . 0 5 pg/L 4.02 pg/L 4 . 0 5 pg/L <0.01 pg/L 4.01 pg/L <0.01 pg/L 4 . 0 2 pg/L ~ 0 . 0 p5g/L <0.02 pg/L <0.02 pg/L 4.05 pgiL 4 . 0 2 pg/L 4 . 0 1 pg/L 4 . 0 2 pg/L 4 . 0 5 pg/L <0.05 pg/L 4.01pgn 4.03pg/L ~0.03p g L 4 . 0 5pg/L 4 . 2 5 pg/L 4.01 pglL 4.01 pgiL 4.02 pgL 4 . 0 2 pg/L <0.02 pg/L 4 . 0 5 pg/L 4 . 0 2 pglL 4 . 0 1 pg/L 4.02 pgn 4 . 0 2 pgfL <0.01 p g 5 <0.05 pg/L 4 . 0 2 pg/L <0.05 pg/L <0.02 p g n 'Analyses performed by TNO Nutrition and F d Instituteon samples collectedon October 14 and 15,1999. Continued WILDLIFE INTERNATIOLNTDA. L - 22 - PROJECT NO.:454A-115 Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water' Page 2 PesticidesAnd Organics(Page 2) Component Malathion Metalaxyl Metamitron Metazachlor Methidathion Paclobutazole Parathion Parathion-methyl Penconazole Pendimethalin Permethrin-cis Permethrin-trans Phosalone Phosmet Phosphamidoncis Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazine Propham Propiconazole Propoxur Proppmide Prosulfocarb Pyrazophos Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt Measured Concentration 4 . 0 2 pg/L 4 . 0 5 pg/L 4 . 0 5 pg/L 4 . 0 2 pg/L 4.02 pgL 4 . 0 5 pg/L <0.01 pg/L 4 . 0 1 pg/L 4 . 0 5 pg/L 4 . 0 3 p@ 4 . 0 1 pg/L 4 . 0 1 pg/L 4 . 0 5 pg/L 4 . 0 2 pg/L 4 . 0 5 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4 . 0 2 p@ <0.01 pg/L 4 . 0 1 pg/L 4.01 pg5, 4 . 0 1 pg/L 4.02 ptpn 4 . 0 5 pB/L 4 . 0 3 pg/L 4.02 pg5 4 . 0 2 pg/L 4 . 0 3 pg/L Component Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl Pyrifenox-1 Pyrifenox-2 F'ynmethanil Quizalofopethyl Simazine Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbuthylazine Tetrachlorvinphos Tetrahydroftalimide Tetramethrin Thiabendazole Thiometon Tolclofos-methyl Tolylfluanid Triadimefon Triadirnenol Triallate Triazophos Trifluralin Vamidothion Vinclozolin Metals 11.0 mg/L 18.0 mg/L 29 mg/L 4 . 0 1 5 mg/L 1.1 mg/L 4 . 0 2 mg/L 4 . 1 PE4.L 4 . 2 Pi& <0.5 pg/L 4 . 2 Pg/L Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium Measured Concentration 4 . 0 1 pg/L 4 . 0 1 pg/L <0.02 pg/L 4 . 0 1 pg/L 4 . 0 5 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4 . 0 2 pg/L 4 . 0 1 pg/L <0.02 pg/L <0.05 p@ <0.05 pg/L <0.01 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4 . 0 5 pg/L 4 . 0 1 pg/L 4.05 pg5, <0.04 pg/L d . 0 1 pg/L 4 . 0 4 pg/L 4 . 0 5 pg/L 4.05p a 4 . 0 2 pg/L 4 . 0 2 pg/L 4.02 pg5 4.01 pg/L 4 . 0 1 pg/L 4 . 1 pg/L 4.7 4 . 2 5 pg/L 4.3 4 . 2 Pg/L CO.1 pg/L 4 . 5 Pg/L ~0.025pglL ~ 0 . p5g/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15, 1999. WILDLIFE INTERNATIONLTAD.L - 23 - Appendix 3 PROJECTNO.: 454A-115 THE ANALYSIS OF PFBS IN FRESHWATER IN SUPPORT OF WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-115 WILDLIFE INTERNATIONALTLD. - 24 - PROJECT NO.: 454A-115 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PERFLUORO BUTANE SULFONATE, POTASSIUM SALT (PFBS): A 96-HOUR STATIC ACUTE TOXICITY TEST with the FATHEAD MINNOW (Pimephalespromelas) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-115 3M ENVIRONMENTAL LAB PROJECT NUMBER E00-1429 PRINCIPAL INVESTIGATOR: Ra&d . Van Hoven, Ph.D. scientist MANAGEMENT: Willard B. Nixon, Ph.D. Director, Analytical Chemistry 0 3 -36- 01 DATE DATE WILDLIFE INTERNATIONALTLD. -25 - PROJECT NO.: 454A-115 SANITIZED Introduction DEC 0 9 2003 Freshwater samples were collected from a static acute aquatic toxicity study designed to determine the effects of PFBS (Perfluoro Butane Sulfonate, Potassium Salt) to the fathead minnow (Pimephales promelus). T h ~ study was conducted by Wildlife International, Ltd. and identified as Project No.: 454A- 115. The analyses of these water samples were performed at Wildlife International, Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). Samples were received for analysis on December 18, 20 and 22, 2000 and were analyzed on each sample receipt day. Analvtical Standard The analytical standard was received from 3M Environmental Technology and Safety Services on March 27, 2000, assigned Wildlife International, Ltd. Identification number 5216, and stored under ambient conditions. The analytical standard, a white powder, was identified as: Potassium Perfluorobutane Sulfonate (L-7038, Lot 2), expiration date: March 2010. The analytical standard was further identified with the 3M Environmental Laboratory test control and reference number TCR # The test substance had a reported purity of 97.90%. A subsequent revision of the certificate of analysis indicated a purity of 97.3% and an ExpiratiodReassessment Date of January 17, 2002. The analytlcal standard was the same material and lot number as the test substance (Wildlife International, Ltd. Identification number The analytical standard was used to prepare calibration and matrix fortification samples. Analvtical Method Water samples were analyzed accordmg to the method entitled "Analytical Method Validation for the Determination of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Freshwater" (Wildlife International, Ltd. Project No. 454C-115). Samples were diluted in a 50% methanol : 50% NANOpure" water solution so that they fell within the calibration range of the PFBS methodology. Aliquots of the dilutions were transferred to autosampler vials and submitted for analysis by direct injection. Concentrations of PFBS in freshwater samples were determined by reverse-phase hgh performance liquid chromatography using a Hewlett-Packard Model 1100 figh Performance Liquid Chromatograph (HPLC) interfaced with a PerkmElmer API lOOLC mass spectrometer (single quadrupole) operated in selective ion monitoring (SIM) detection mode. The mass spectrometer was equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separations were acheved using a Keystone PRISM FU column (30 mm x 1.5 mm, 3-pm WILDLIFE INTERNATIONALTLD. - 26 - PROJECT NO.: 454A-115 particle size) fitted with a Keystone Javelin CISGuard Cartridge (20 mm x 2 mm). The instrument parameters are summarized in Table 1 and a method flowchart is provided in Figure 1. Primary and Secondarv Stock Solutions, All primary and secondary stock preparations were adjusted for the purity of the analytical standard (97.90%). A 10.0 mg a.i./mL primary stock solution of PFBS in methanol was prepared by weighing 1.024 g of the analytical standard and bringing to a final volume of 100 mL with methanol. Secondary stock solutions (1000, 100,'10.0, 1.00, and 0.100 mg a.i./L) of PFBS in methanol were prepared by serial volumetric dilution from the primary stock. Calibration Standards and Calibration Curves Calibration standards were prepared in 50:50 methanol: NANOpure" water by appropriate dilutions of the 10.0 mg a.i./L stock solution of PFBS in methanol. The calibration standards of PFBS, ranging in concentration fiom 0.0100 to 0.0500 mg a.i./L, were analyzed with each sample set. Five calibration standards (different concentrations) were analyzed with the samples. The calibration standard series was injected at the beginning and end of each run,and one standard was injected, at a minimum, after every five samples. Linear regression equations were generated using the peak area responses versus the respective concentrations of the calibration standards. A typical calibration curve is presented in Figure 2. The concentration of PFBS in the samples was determined by substituting the peak area responses into the applicable linear regression equation. Representative ion chromatograms of low and high calibration standards are presented in Figures 3 and 4, respectively. Limit of Ouantitation The method limit of quantitation (LOQ) for these analyses was set at 50.0 mg a.i./L calculated as the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (5000). Matrix Blank and Fortification Samples Three matrix blank samples were analyzed to determine possible interference. No interferences were observed at or above the LOQ during samples analyses (Table 2). A representative ion chromatogram of a matrix blank is presented in Figure 5. WILDLIFE INTERNATIONLTAD.L PROJECT NO.: 454A-115 Freshwater was directly fortified (ie. without use of carrier solvent) with PFBS at 100, 1500 and 12000 mg a.i.4, and analyzed concurrently with the samples to determine the mean procedural recovery (Table2). Sample concentrations were not corrected for the mean procedural recovery of 103%. A representative ion chromatogram of a matrix fortification is presented in Figure 6. Example Calculations Sample number 454A-,115-4, nominal concentration of 204 mg a.i./L (208 mg/L) in freshwater. First Initial Volume: 0.200 mL Calibration curve equation: First Final Volume: 10.0 mL Slope: 41637464 Second Initial Volume: 0.250 mL Intercept: 95950.78906 Second Final Volume: 25.0 mL Curve regression weighted l/x Dilution Factor: 5000 PFBS Peak Area: 1835872 - eak area (y-intercept) PFBS (mg a.i./L) measured at instrument = JJ slope PFBS (mg a.i./L) in sample = PFBS measured at instrument (mg a.i./L) x dilution factor - 1835872 - 95950.78906 x 5000 41637464 = 209 PFBS Percent of Nomind Concentrationg:=(',,,, m nomioPal a.i./L in sam le a.i.i) x 100 --209 x 100 = 102% - 204 Calculated with HPLC/MS instrument software: MacQuan, version 1.6. WILDLIFE INTERNATIONALTLD. - 28 - PROJECT NO.: 454A-115 Sample Analvsis RESULTS Freshwater samples were collected from an acute toxicity study with the fathead minnow (Pimephales promelas) at test initiation, December 18, 2000 (Day 0), on December 20, 2000 (Day 2), and at test termination, December 22, 2000 (Day 4). The measured concentrations of PFBS in the samples collected at initiation of exposure of the test organisms (Day 0) ranged from 99.7 to 105% of the nominal concentrations. Samples collected at Day 2 had a measured concentration range of 99.1 to 109% of nominal values. Samples collected at test termination (Day 4) had a measured concentration range of 98.7% to 123% of nominal values (Table 3). A representative ion chromatogram of a test sample is shown in Figure 7. WILDLIFE INTERNATIONAL LTD. - 29 - PROJECT NO.: 454A-115 Table 1 Typical I-KPLC/MS Operational Parameters INSTRUMENT: ~~ Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API lOOLC Mass Specpometer operated in Selective Ion Monitoring (SIM) Mode ION SOURCE: Perkin-Elmer TurboIonSpray ANALYTICAL COLUMN: Keystone PRISM RP (30 mm x 1.5 mm, 3-pn particle size) GUARD COLUMN: Keystone Javelin CIScartridge (20 mm x 2 mm) OVEN TEMPERATURE: 40C STOP TIME: 3.00 min FLOW RATE: 200 pL/min MOBILE PHASE: 25% NANOpure' Water with 0.1% Ammonium Formate: 75% Methanol INJECTION VOLUME: 5.0 pL PFBS PEAK RETENTION TIME: PFBS MONITORED MASS: Approximately 2.3 minutes 299.0 m u WILDLIFE INTERNATIONALTLD. -30- PROJECT NO.: 454A-115 Table 2 Matrix Blanks and Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-115-) MAB- 1 MAB-2 MAB-3 Sample Type Matrix Blank Matrix Blank Matrix Blank Concentrations of PFBS (mg a..i./L) Fortified 0.00 0.00 0.00 Measured' <LOQ' <LOQ <LOQ Recovered' -- MAS-1 Matrix Fortification 100 108 108 MAS-4 Matrix Fortification 100 91.3 91.3 MAS-7 Matrix Fortification 100 101 101 MAS-2 Matrix Fortification 1500 1480 98.7 MAS-5 Matrix Fortification 1500 1650 110 MAS-8 Matrix Fortification 1500 1480 98.4 MAS-3 Matrix Fortification 12000 12800 106 MAS-6 Matrix Fortification 12000 13200 110 MAS-9 Matrix Fortification 12000 1 1900 99.1 Mean= 103 Standard Deviation = 6.38 CV = 6.22% N=9 1 Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 'The limit of quantitation (LOQ) was 50.0 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (5000). W I L D L I F E INTERNATIONAL LTD. -31 - PROJECT NO.: 454A-115 Table 3 Measured Concentrations of PFBS in Freshwater Samples from a Fathead Minnow Static Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 0.0 Sample Number (454A-1 IS-) 1 2 .13 14 25 26 Sampling Time (Day) 0 0 2 2 4 4 PFBS Measured Concentration' (mg a.i./L) < LOQ' < LOQ < LOQ < LOQ c LOQ c LOQ Percent of Nominal' -- 204 3 0 4 0 15 2 16 2 27 4 28 4 205 101 209 102 211 103 213 105 25 0 123 232 114 408 5 0 6 0 17 2 18 2 29 4 30 4 426 105 407 99.7 432 106 415 102 498 122 446 109 816 7 0 8 0 19 2 20 2 31 4 824 101 85 1 104 888 109 876 107 962 118 32 4 926 114 Measured and Percent of Nominal values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. The limit of quantitation (LOQ) was 50.0 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor ofthe matrix blank samples (5000). WILDLIFE INTERNATIONLTD A. L -32- PROJECT NO.: 454A-115 ~~ Table 3 (Continued) Measured Concentrations of PFBS in Freshwater Samples from a Fathead Minnow Static Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 1632 Sample Number (454A-115-) 9 10 21 22 33 34 Sampling Time (Day) 0 0 2 2 4 4 PFBS Measured Concentration' (mg a.i./L) 1692 1674 1665 1617 1673 1611 Percent of Nominal 104 103 102 99.1 103 98.7 32633 11 0 3354 103 12 0 3417 105 23 2 3327 102 24 2 -- 4 -- 4 3264 -- 1-0- 0 -- -- 1 Measured and Percent of Nominal values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. The limit of quantitation (LOQ) was 50.0 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (5000). This treatment group was not sampled on Day 4 due to 100% mortality on Day 1. Fish were not in the test chambers during Day 2 sampling. WILDLIFE INTERNATIONLTAD.L -33 - PROJECT NO.: 45444-1 15 METHOD OUTLINE FOR THE ANALYSIS OF PFBS IN FRESHWATER Prepare each matrix fortification sample by weighing the requisite amount of PFBS test substance on an analytical balance and transferring directly into a Class A volumetric flask partially filled with freshwater. Rinse weighing paper and the sides of the flask with repeat freshwater rinses. Swirl the flask to dissolve the test substance and then bring to final volume with freshwater. Sonicate, as appropriate, and mix with several repeat inversions. The matrix blank is unfortified freshwater. Prepare appropriate dilutions of study and QC samples to within the calibration range of the PFBS methodology: Partially fill Class A volumetric flasks with 50% methanol : 50% NANOpure" water dilution solvent. Add the appropriate volume of sample and bring to volume with dilution solvent. Perform secondary dilutions as necessary. Process matrix blank samples using the same dilution and aliquot volume as for the lowest fortification level. Mix well by several repeat inversions. .1 Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis of PFBS in freshwater. WILDLIFE INTERNATIOLNTDA. L - 34 - Area PROJECTNO.: 454A-115 O+ 0.0 1 1 1 1 1 1 , , , , 1 , , , , , , , ~ 1 ~, 10 20 30 40 50 Concentration pga.i.iL Figure 2. Atypical calibration curve for PFBS. Slope = 41637464; Intercept = 95950.78906; r = 0.9969. WILDLIFE INTERNATIONALTLD. -35- PROJECT NO.: 454A-115 intensity: 500000 cps 137 1 31 61 91 121 151 scan 0.52 1.02 1.52 2.03 2.53 Time Figure 3. A representative ion chromatogram of a low-level (0.0100 mg a.i./L) PFBS standard. WILDLIFE INTERNATIONALTLD. -36- PROJECT NO.:454A-115 intensity: 500000 cps 100 90 80 70 60 50 136 40 30 20 10 0 31 61 91 121 151 scan 0.52 1.02 1.52 2.03 2.53 Time Figure 4. A representative ion chromatogram of a high-level (0.0500mg a.i./L) PFBS standard. WILDLIFE INTERNATIONALTLD. -37- PROJECT NO.: 454A-115 100- 90- 80- 70- 60- 50- 40- 30- 20- 10- 0 ! 131 2I 5 1 41 5 1 1 701 31 61 0.52 1.02 1861 1 91 1.52 intensity: 500000 cps 11 141 1 131 8 1 1 51 6 l 121 151 scan 2.03 2.53 Time Figure 5 . A representative ion chromatogramof a matrix blank sample (454A-115-h4AI3-1). The arrow indicates the retention time of PFBS. WILDLIFE INTERNATIONLTAD.L -38- PROJECT NO.: 454A-115 1009080- 70- 60- 5040- 137 21 42 66 I I I I I I I 31 61 91 121 151 0.52 1.02 1.52 2.03 2.53 Time Figure 6 . A representative ion chromatogramof a matrix fortification sample (454A-115-MAS-2, nominal PFBS concentration of 1500mg a.i./L, dilution factor = 50000~). WILDLIFE INTERNATIONALTLD. - 39 - PROJECT NO.:454A-115 intensity: 500000 cps 37 3 2 1 31 61 91 121 151 Scan 0.52 1.02 1.53 2.03 2.53 Time Figure 7. A representativeion chromatogram of a test sample (454A-115-7n,ominal PFBS concentration of 816 mg a.i./L, dilution factor = 25000~). WILDLIFE INTERNATIONLTAD.L - 40 - PROJECT NO.:454A-115 Appendix 4 Changes to Protocol This study was conducted in accordance with the approved Protocol with the'following changes: 1. The protocol was amended to add the proposed experimental start and termination dates and test concentrations. 2. Unnecessarytemperature measurementswere made in the 3341mg a.i./L treatment group on Day 2 of the test. 3. Feed provided to the fish was not analyzed for contaminants WILDLIFE INTERNATIOLNTDA. L -41 - PROJECT NO.: 454A-115 Appendix 5 Personnel Involved in the Study The followingkey Wildlife International Ltd. personnelwere involved in the conductormanagement of this study: 1. Henry 0.Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Director, Analytical Chemistry 3. Cary A. Sutherland, Laboratory Supervisor 4. Raymond L. Van Hoven, Ph.D., Scientist 5 . Kurt R Drottar, Senior Biologist 6. Susan T. Platania, Biologist