Document 5DE2B3d7xM5n82D89xyj4wE58

14) OECD 202-OPPTS 850.1010, 48-hour static acute toxicity test with the Cladoceran (daphnia magna), 454A-118A PERFLUOROBUTANE SULFONATE, POTASSIUM SALT (PFBS): A 48-HOUR STATIC ACUTE TOXICITY TEST WITH THE CLADOCEFAN (Daphnia magna) FINAL REPORT SANITIZED WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-118A 3M LAB REQUEST NO. E00-1429 U. S Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1010 and OECD Guideline 202 o EC 0 9 2003 AUTHORS: Kurt R. Drottar Henry O. Krueger, Ph.D. STUDY INITIATION DATE: July 10, 2000 STUDY COMPLETION DATE: March 20,2001 Submitted to 3M Corporation Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410)822-8600 Page 1 of 41 W il d l if e In t e r n a t io n a l, Lt d . 2- - PROJECT NO.: 454A-118A SANITIZED GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT DEC 0 9 2003 SPONSOR: 3M Corporation TITLE: Perfluorobutane Sulfonate, Potassium Salt (PFBS): A 48-Hour Static Acute Toxicity Test with the Cladoceran (Daphnia magna) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-118A STUDY COMPLETION DATE: March 20,2001 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFRPart 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), with the following exceptions: The test substance was not characterized in compliance with Good Laboratory Practices prior to its use in the study. However, subsequent GLP compliant characterization resulted in a purity similar to the original characterization purity. The stability of the test substance under conditions o f storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Kurt R. Drottar Senior Biologist DATE SPONSOR,APPROVAL: DATE Wil d l if e In ter n a tio n a l, Lt d . -3 - PROJECT NO.: 454A-118A 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 CFRPart 1(30 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). The dates o f all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: DATE REPORTED TO: DATE CONDUCTED: STUDY DIRECTOR: MANAGEMENT: In itial T rial C454A-1181 Test Substance Preparation and Analytical Sampling January 16, 2001 Sample Preparation January 16, 2001 January 16, 2001 January 16, 2001 January 18, 2001 January 17, 2001 D efinitive T rial (454A -118A 1 Test Substance Preparation January 23, 2001 January 23, 2001 February 5, 2001 Analytical Data and Draft Report February 7 and 8, 2001 February 8, 2001 February 9,2001 Biological Data and Draft Report Final Report February 9,2001 March 16, 2001 February 9, 2001 March 16, 2001 February 12,2001 March 16, 2001 Marshall T. Hynson / Quality Assurance Program Supervisor DATE < * Wil d l if e In ter n a tio n a l, Lt d . -4 - REPORT APPROVAL PROJECT NO.: 454A-118A SPONSOR: 3M Corporation TITLE: Perfluorobutane Sulfonate, Potassium Salt (PFBS): A 48-Hour Static Acute Toxicity Test with the Cladoceran {Daphnia magna) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-118A STUDY DIRECTOR: MANAGEMENT: Director, Aquatic Toxicology and Non-Target Plants DATE Wil d l if e In ter n a tio n a l, Lt d . -5- PROJECT NO.: 454A-118A TABLE OF CONTENTS Title/Cover Page............................................................................................................................................................1 Good Laboratory Practice Compliance Statement...................................... 2 Quality Assurance Statement...................................................................................................................................... 3 Report Approval.............................................................................................................. 4 Table of Contents......................................................................................................................................................... 5 Summary........................................................................................................................................................................7 Introduction...................................................................................................................................................................8 O b je c tiv e ....................................................................................................................................................................... 8 Experimental Design....................................................................................................................................................8 Materials and Methods.................................................................................................................................................9 Results and Discussion...............................................................................................................................................12 Conclusions............................................................................................................... 13 R e f e re n c e s ................................................................................................................................................................... 14 TABLES Table 1 - Summary o f Analytical Chemistry D a ta .................................................................................................15 Table 2 - Temperature, Dissolved Oxygen, and pH of Water in the Test Chambers......................................... 16 Table 3 - Cumulative Percent Mortality/Immobility and Treatment-Related Effects......................................... 17 Table 4 - EC 10, EC50 and EC90 Values................................................................................................................18 Wil d l if e In tern a tio n a l, Lt d . 6- - PROJECT NO.: 454A-118A TABLE OF CONTENTS - Continued FIGURE Figure 1. Concentration-Response Curve (48 Hour D ata)................................................................................. 19 APPENDICES Appendix 1 - Specific Conductance, Hardness, Alkalinity, and pH o f 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 W ater.................................................................................... 21 Appendix 3 - The Analysis of PFBS in Freshwater in Support of Wildlife International, Ltd. Project No.: 454A-118A............................................................23 Appendix 4 - Changes to Protocol......................................................................... ...........................................40 Appendix 5 - Personnel Involved in the Study................................................................................................. 41 Wil d l if e In ter n a tio n a l, Lt d . -7 - SPONSOR: SPONSOR'S REPPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: SUMMARY 3M Corporation Wildlife International, Ltd. Easton, Maryland 21601 PROJECT NO.: 454A-118A DEC 0 9 2003 SANITIZED WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: 454A-118A Perfluorobutane Sulfonate, Potassium Salt (PFBS) Perfluorobutane Sulfonate, Potassium Salt (PFBS): A 48-Hour Static Acute Toxicity Test with the Cladoceran (Daphnia magna) Negative Control, 234, 470, 886, 1707 and 3767 mg a.i./L Experimental Start (OECD) - January 15, 2001 Experimental Start (EPA) - January 23,2001 Biological Term ination-January 25, 2001 Experimental Termination - January 25, 2001 48 Hours Cladoceran (Daphnia magna) Wildlife International, Ltd. Cultures Easton, Maryland 21601 <24 hours at test initiation 48-HOUR EC50: 95% CONFIDENCELIMITS: NOMORTALITY/IMMOBILITY CONCENTRATION: NO-OBSERVED-EFFECTCONCENTRATION: 2183 mg a.i./L 1707 and 3767 mg a.i./L 886 mg a.i./L 886 mg a.i./L Wil d l if e In ter n a tio n a l, Lt d . 8- - PROJECT NO.: 454A-118A 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 January 23, 2001 to January 25,2001. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454A-118A in archives located on the Wildlife International, Ltd. site. OBJECTIVE The objective of this study was to evaluate the acute toxicity of Perfluorobutane Sulfonate, Potassium Salt (PFBS) to the cladoceran, Daphnia magna, during a 48-hour exposure period under static test conditions. EXPERIMENTAL DESIGN Daphnids were exposed to a geometric series of five concentrations and a negative (dilution water) control. Two replicate test chambers were maintained in each treatment and control group, with 10 daphnids in each test chamber for a total o f 20 daphnids per test concentration. Nominal test concentrations were selected in consultation with the Sponsor, and were based upon the results of exploratory rangefinding toxicity tests. Nominal test concentrations selected were 250,500,1000,2000 and 4000 mg active ingredient (a.i.)/L. Mean measured test concentrations were determined from samples o f test water collected from each treatment and the control group at the beginning of the test, at approximately 24 hours, and at test termination. Daphnids were indiscriminately assigned to exposure chambers at test initiation. Observations of mortality/immobility and other clinical signs of toxicity were made approximately 4 ,2 4 and 48 hours after test initiation. Cumulative percent mortality and immobility observed in the treatment groups were used to calculate EC 10, EC50 and EC90 values at 24 and 48 hours, when possible. The no mortality/immobility concentration and the no-observed-effect-concentration (NOEC) were determined by visual interpretation of the mortality, immobility and clinical observation data. Wil d l if e In ter n a tio n a l, Lt d . -9 - PROJECT NO.: 454A-118A MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "Perfluorobutane Sulfonate, Potassium Salt (PFBS): A 48-Hour Static Acute Toxicity Test with the Cladoceran (Daphnia magna)". The protocol was based on procedures outlined in U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1010 (1), OECD Guideline for Testing of Chemicals, 202: Daphnia sp. Acute Immobilization Test and Reproduction Test (2) and ASTM Standard E-729-88a, Standard Guide fo r 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, from lot 2. Information provided by the Sponsor indicated a purity of 97.9%. A subsequent revision o f the certificate o f analysis indicated a purity o f 97.3% and an Expiration/Reassessment Date of January 17, 2002. The test substance was stored at ambient room temperature. Preparation of Test Concentrations Nominal test concentrations were 250,500,1000,2000 and 4000 mg a.i./L. A 1-L primary stock solution was prepared in dilution water at a concentration o f 4000 mg a.i./L. The primary stock solution was inverted and stirred to aid in the solubilization o f the test substance. After mixing, appropriate aliquots o f the primary stock solution were proportionally diluted with dilution water to prepare 500 mL of the other four test concentrations. All dilutions were inverted to mix. After mixing, 225 mL o f test solution was added to the two replicate test chambers for each treatment group. All test solutions appeared clear and colorless. Observations o f test solutions at test termination indicated that they appeared the same as at test initiation. Test concentrations were corrected for the original reported purity of the active ingredient in the test substance (97.9%). Test Organism The cladoceran, Daphnia magna, was selected as the test species for this study. Daphnids are representative of an important group o f aquatic invertebrates and were selected for use in the test based upon past history of use and ease of culturing in the laboratory. Daphnid neonates used in the test were less than 24-hours old and were Wil d l if e In ter n a tio n a l, Lt d . - 10- PROJECT NO.: 454A-118A obtained from cultures maintained by Wildlife International, Ltd., Easton, Maryland. Identification ofthe original brood stock was verified by the Academy o f Natural Sciences, Philadelphia, PA. Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. Adult daphnids in the cultures were held for at least 14 days prior to collection of the juveniles for testing. The adults showed no signs o f disease or stress during the holding period. During the 14-day period preceding the test, water temperatures ranged from 19.1 to 20.2C. The pH o f the water ranged from 8.2 to 8.5, and dissolved oxygen ranged from 8.0 to 8.6 mg/L. Instrumentation used for water measurements are described in the Environmental Conditions section of this report. Neonate daphnids were obtained for testing from individual adult daphnids which were observed to have no neonates present less than 24 hours prior to test initiation. The progeny from 5 adults were used in the test. At test initiation, the juvenile daphnids were collected from the cultures and indiscriminately transferred 1 to 2 at a time to 10-mL glass beakers. The daphnids then were transferred from the beakers to the test compartments. All transfers were performed below the air/water interface using a wide-bore pipet. Daphnids in the cultures were fed a mixture of yeast, Cerophyll, and trout chow, as well as a suspension of the freshwater green alga, Selenastrum capricornutum. The adults were fed prior to test initiation, but neonates were not fed during the test. Test Apparatus Test chambers consisted of 300-mL plastic (Nalgene) beakers containing 225 mL of test solution. The depth o f test solution in a representative test chamber was .5.5 cm. Test chambers were indiscriminately placed in a temperature-controlled water bath set to maintain a temperature of 20 1C. Test chambers were labeled with the project number, test concentration and replicate. Dilution Water The water used for culturing and testing was freshwater obtained from a well approximately 40 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 of the well water during the four-week period immediately preceding the test are presented in Appendix 1. 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 where the water was aerated with spray nozzles. Prior to use, the water Wil d l if e In ter n a tio n a l, Lt d . - 11 - PROJECT NO.: 454A-118A again was filtered (0.45 Jim) to remove microorganisms and particles. The results ofperiodic 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 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 of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity at test initiation and termination was approximately 144 and 143 lux, respectively, at the surface of the water o f the negative control (replicate A). Light intensity was measured using a SPER Scientific Ltd. light meter. Temperature was measured in each test chamber at the beginning and end of 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 20+1 C. The pH and dissolved oxygen content o f the water were measured in each test chamber at test initiation, at approximately 24 hours after test initiation and at the end of the test. Hardness, alkalinity, specific conductance and total organic carbon (TOC) were measured in the dilution water at test initiation and at test termination. Measurements o f pH were made using a Fisher Accumet Model 915 pH meter, and dissolved oxygen was measured using a Yellow Springs Instrument Model 5 IB dissolved oxygen meter. Specific conductance was measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter. Hardness and alkalinity measurements were made by titration based on procedures in StandardMethodsfo r the Examination o f Water and Wastewater (4). Total organic carbon was measured using a Shimadzu Model TOC-5000 analyzer. Observations Observations were made to determine the numbers o f mortalities and immobile organisms. The numbers of individuals exhibiting clinical signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 4, 24 and 48 hours after test initiation. Wil d l if e In ter n a tio n a l, Lt d . -12- PROJECT NO.: 454A-118A Statistical Analyses The 24 and 48-hour EC50 values and the 95% confidence intervals were calculated when possible by probit analysis, the moving average method or binomial probability with non-linear interpolation (5, 6, 7) using the computer software o f C.E. Stephan (8). The no mortahty/immobility concentration and NOEC values were determined by visual inspection o f the mortality, immobility and clinical observation data. EC 10 and EC90 values could not be calculated with the concentration-response observed. RESULTS AND DISCUSSION Measurement of Test Concentrations Results o f analyses to measure concentrations o f PFBS in water samples collected during the test are presented in Table 1 and in the analytical chemistry report (Appendix 3). Nominal concentration selected for use in this study were 250,500, 1000, 2000 and 4000 mg a.i./L. Samples collected at test initiation had measured values that ranged from 83to 96% of nominal. Samples collected at 24 hours had measured values that ranged from 89 to 99% of nominal, whereas measured values for samples taken at 48 hours ranged from 79 to 92% of nominal. When measured concentrations of the samples collected at test initiation, 24 hours, and at test termination were averaged, the mean measured concentrations for this study were 234, 470, 886, 1707 and 3767 mg a.i./L. Mean measured concentrations were used in the calculation of EC50 values. Observations and Measurements Measurements of temperature, dissolved oxygen and pH are presented in Table 2. Temperatures remained within the 20 1C range established for the test. Dissolved oxygen concentrations remained >8.3 mg/L (91% of saturation) throughout the test. Measurements o f pH ranged from 7.9 to 8.5 during the test. Daily observations o f mortality, immobility and other clinical signs observed during the test are shown in Table 3. Daphnids in the negative control appeared healthy and normal during the test. However, 5% control mortality was observed at test termination. After 48 horns of exposure, mortality/immobility in the 234, 470, 886,1707 and 3767 mg a.i./L treatment groups was 5 , 0 , 5 , 2 0 and 100%, respectively. EC50 values and 95% confidence limits at 24 and 48 hours were calculated from the mortality/immobility data, and are shown in Table 4. Wil d l if e In ter n a tio n a l, Lt d . - 13 - PROJECT NO.: 454A-118A CONCLUSIONS The 48-hour EC50 value for Daphnia magna exposed to Perfluorobutane Sulfonate, Potassium Salt (PFBS) was 2183 mg a.i./L. The 95% confidence limits were 1707 and 3767 mg a.i./L. Based on the presence o f 5% mortality in the control, the mortality/immobility observed in the 234 and 886 mg a.i./L (5 and 5%, respectively) was not considered to be treatment-related. Consequently, the no mortality/immobility concentration and NOEC were 886 mg a.i./L. Wil d l if e In ter n a tio n a l, Lt d . - 14- PROJECT NO.: 454A-118A REFERENCES 1 U.S. Environm ental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines (draft), OPPTS Number 850.1010. Aquatic invertebrate Acute Toxicity Test, Freshwater Daphnids. 2 O rganisation for Economic Cooperation and Development. 1984. Daphnia sp. Acute Immobilization Test and Reproduction Test. OECD Guideline for Testing o f Chemicals. Guideline 202. Paris. 3 ASTM Stan d ard E729-88a. 1994. Standard Guidefo r Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. American Society for Testing and Materials. 4 APHA, AW W A, W PCF. 1985. Standard Methods fo r the Examination o f Water and Wastewater. 16th Edition, American Public Health Association. American Water Works Association. Water Pollution Control Federation, New York. 5 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. 6 Thom pson, W .R. 1947. Bacteriological Reviews. Vol. II, No. 2. Pp. 115-145. 7 Finney, D.J. 1971. Statistical Methods in Biological Assay. Second Edition. Griffin Press, London. 8 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. Wil d l if e In t e r n a t io n a l, Ltd - 15 - PROJECT NO.: 454A-118A Table 1 Summary of Analytical Chemistry Data Sponsor: Test Substance: Test Organism: Dilution Water: Nominal Test Concentration (mg a.i./L) Negative Control 3M Corporation PFBS Cladoceran, D a p h n ia m a g n a Well Water Replicate Sampling Time (Hours) A0 B0 A 24 B 24 A 48 B 48 250 A 0 B0 A 24 B 24 A 48 B 48 500 A 0 B0 A 24 B 24 A 48 B 48 1000 A0 B0 A 24 B 24 A 48 B 48 2000 A0 B0 A 24 B 24 A 48 B 48 4000 A0 B0 A 24 B 24 ` The limit of quantitation (LOQ) was 100 mg a.i./L. Measured Concentration (mg a.i./L) <LOQ` <LOQ <LOQ <LOQ <LOQ <LOQ 240 240 233 235 227 231 478 461 476 496 451 456 858 850 921 923 862 902 1661 1738 1770 1842 1588 1640 3592 3751 3835 3888 Mean Measured Concentration (mg a.i./L) <LOQ 234 470 886 1707 3767 Percent of Nominal 94 94 89 85 94 Wil d l if e In ter n a tio n a l, Lt d . - 16- PROJECT NO.: 454A-118A Table 2 Temperature, Dissolved Oxygen and pH of Water in the Test Chambers Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Concentration (mg a.i./L) Negative Control 3M Corporation PFBS Cladoceran, Daphnia magna Well Water 0 Hour1 Replicate Temp2 DO3 (C) (mg/L) A 19.9 8.4 B 19.9 8.6 pH 7.9 8.0 24 Hours DO (mg/L) PH 8.6 8.4 8.6 8.4 Temp (C) 20.1 20.2 48 Hours45 DO (mg/L) 8.4 8.4 pH 8.3 8.3 234 A 20.0 8.6 8.1 8.6 8.4 20.0 8.4 8.4 B 19.8 8.6 8.1 8.6 8.4 19.8 8.4 8.5 470 A 19.8 8.6 8.2 8.6 8.4 19.8 8.4 8.5 B 19.8 8.6 8.2 8.6 8.4 19.8 8.3 8.5 886 A 19.9 8.6 8.2 8.6 8.5 19.8 8.4 8.5 B 19.9 8.6 8.2 8.6 8.5 19.8 8.4 8.5 1707 A 19.9 8.6 8.2 B 20.1 8.6 8.2 8.6 8.5 8.6 8.5 19.8 8.4 8.5 19.8 8.4 8.5 3767 A 20.1 8.6 8.3 B 20.0 8.6 8.3 8.6 8.5 8.6 8.4 19.8 19.8 _5 _5 - ' The 0-hour dilution water measurements for hardness, alkalinity, specific conductance and TOC were 132 mg/L as CaCO^ 181 mg/L as CaCOj, 290 pmhos/cm and <1 mg C/L, respectively. 2 Temperature measured continuously during the test ranged from approximately 19.5 to 20.5C. 3 A dissolved oxygen concentration o f 5.4 mg/L represents 60% saturation at 20C in freshwater. 4 The test termination dilution water measurements for hardness, alkalinity, specific conductance and TOC were 136 mg/L as CaCOj, 184 mg/L as CaCOj, 295 |imhos/cm and 1.3 mg C/L, respectively. 5 Measurements discontinued due to 100% mortality.______________________________________________________________________ PROJECT NO.: 454A-118A - 17- Table 3 Cumulative Percent Mortality/Immobility and Treatment-Related Effects1 Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Concentration (mg a.i./L) Replicate Daphnia/ Number Replicate Dead Negative Control A B 10 0 10 0 4 Hours Number Immobile 0 0 234 A 10 0 0 B 10 0 0 470 A 10 0 0 B 10 0 0 886 A 10 0 0 B 10 0 0 1707 A 10 0 0 B 10 0 0 3767 A 10 B 10 1 Observed Effects: AN = Appears Normal 10 10 0 0 Effects 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN -- Number Dead 0 0 24 Hours Number Immobile 0 0 00 00 00 00 00 00 00 00 10 0 10 0 Effects 10 AN 10 AN Percent Immobile and Dead 0 10 AN 10 AN 0 10 AN 10 AN 0 10 AN 10 AN 0 10 AN 10 AN 0 -- - 100 Number Dead 0 1 0 0 0 0 0 0 3 0 10 10 48 Hours Number Immobile 0 0 1 0 0 0 0 1 0 1 0 0 Effects 10 AN 9 AN 9 AN 10 AN 10 AN 10 AN 10 AN 9 AN 7 AN 9 AN _ -- Percent Immobile and Dead 5 5 0 5 20 100 Wil d l if e In ter n a tio n a l, Lt d . - 18- PROJECT NO.: 454A-118A Table4 EC50 Values Sponsor: Test Substance: Test Organism: Dilution Water: Time 24 Hours 3M Corporation PFBS Cladoceran, Daphnia magna Well Water ______________________ Lower 95% Upper 95% EC50 Confidence Confidence (mg a.i./L) Limits Limits 2536 1707 3767 Statistical Method Binomial 48 Hours 2183 1707 3767 Binomial Note: EC 10 or EC90 values could not be calculated with the concentration-response observed. Wil d l if e In ter n a tio n a l, Lt d . - 19- Figure 1. Concentration-Response Curve (48 Hour Data) PROJECT NO.: 454A-118A Wil d l if e In ter n a tio n a l, Lt d . -20- PROJECT NO.: 454A-118A Appendix 1 Specific Conductance, Hardness, Alkalinity and pH o f Well Water Measured During the 4-Week Period Immediately Preceding the Test Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Specific Conductance ((imhos/cm) 315 (N = 4) Range 305-335 Hardness (mg/L as CaC03) 131 (N = 4) 116-140 Alkalinity (mg/L as CaC03) 178 (N = 4) 172-184 pH 8.1 (N==4) 8.0-8.1 Wil d l if e In ter n a tio n a l, Lt d . -21 - PROJECT NO.: 454A-118A Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Component Measured Concentration Component Measured Concentration Pesticides and Organics Aclonifen Alachlor Ametryn Atrazin Azinphos-ethyl Azinphos-methyl Azoxystrobin Bifenthrin Bioallethrin Bitertanol Bromacil Bromophos Bromophos-ethyl Broompropylaat Bupirimaat Carbaryl Carbofuran Carboxin Chlorfenvinphos Chloridazon Chlorpropham Chlorpyriphos Chlorpyriphos-methyl Chlorthalonil Coumaphos Cyanazin Cyfluthrin Cypermethrin Cyproconazole Deltamethrin Demeton Demeton-o Desethylatrazin Desisopropylatrazin Desmetryn Diazinon Dichlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid <0.03 gg/L <0.01 gg/L <0.01 gg/L <0.01 gg/L <0.04 gg/L <0.08 gg/L <0.25 gg/L <0.05 gg/L <0.05 gg/L <0.05 gg/L <0.05 gg/L <0.02 gg/L <0.02 gg/L <0.02 gg/L <0.05 gg/L <0.05 gg/L <0.03 gg/L <0.02 gg/L <0.02 gg/L <0.05 gg/L <0.02 gg/L <0.01 gg/L <0.01 gg/L <0.04 gg/L <0.02 gg/L <0.05 gg/L <0.05 gg/L <0.25 gg/L <0.05 gg/L <0.02 gg/L <0.02 gg/L <0.02 gg/L <0.01 gg/L <0.02 gg/L <0.01 gg/L <0.01 gg/L <0.01 gg/L <0.03 gg/L <0.02 gg/L <0.01 gg/L <0.03 gg/L Dimethomorf Disulfoton DMST Dodemorf Endosulfan-oc Endosulfan- Endosulfan-sulfaat Epoxiconazole Eptam Esfenvaleraat Ethion Ethofumesaat Ethoprophos Etridiazole Etrimphos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb Fenpiclonil Fenpropathrin Fenpropimorf Fenthion Fenvaleraat Fluazifop-butyl Fluoroglycofen-ethyl Fluroxypyr-meptyl Flutolanil Fonophos Furalaxyl Heptenophos Imazalil Iprodion Kresoxim-methyl Lenacil Lindane Malathion Metalaxyl Metamitron Metazachlor Methidathion <0.05 gg/L <0.02 gg/L <0.05 gg/L <0.01 gg/L <0.01 gg/L <0.01 gg/L <0.02 gg/L <0.05 gg/L <0.02 gg/L <0.02 gg/L <0.05 gg/L <0.02 gg/L <0.01 gg/L <0.02 gg/L <0.05 gg/L <0.05 gg/L <0.01 gg/L <0.03 gg/L <0.03 gg/L <0.05 gg/L <0.25 gg/L <0.01 gg/L <0.01 gg/L <0.02 gg/L <0.02 gg/L <0.02 gg/L <0.05 gg/L <0.02 gg/L <0.01 gg/L <0.02 gg/L <0.02 gg/L <0.01 gg/L <0.05 gg/L <0.02 gg/L <0.05 gg/L <0.02 gg/L <0.02 gg/L <0.05 gg/L <0.05 gg/L <0.02 gg/L <0.02 gg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15,1999. Wil d l if e In ter n a tio n a l, Lt d . -22- PROJECT NO.: 454A-118A Appendix 2 (Continued) Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Pesticides and Organics (Page 2) Component Dichlorvos Dicofol Diethyltoluamide Difenoconazole Dimethoate Paclobutazole Parathion Parathion-methyl Penconazole Pendimethalin Permethrin-cis Permethrin-trans Phosalon Phosmet Phosphamidon-cis Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazin Propham Propiconazool Propoxur Propyzamide Prosulfocarb Pyrazophos Measured Concentration <0.01 pg/L <0.25 pg/L <0.02 pg/L <0.03 pg/L <0.02 pg/L ' <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.03 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.03 pg/L <0.02 pg/L <0.02 pg/L <0.03 pg/L Component Measured Concentration Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl Pyrifenox-1 Pyrifenox-2 Pyrimethanil Quizalofop-ethyl Simazin Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbutylazin Tetrachlorvinphos Tetrahydroftaalimide Tetramethrin Thiabendazole Thiometon Tolclophos-methyl Tolylfluanid Triadimefon Triadimenol Triallaat Triazophos Trifluralin Vamidothion Vinchlozolin .. ` <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.05 pg/L <0.04 pg/L <0.01 pg/L <0.04 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L Metals Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 11.0 mg/L 18.0 mg/L 29 mg/L <0.015 mg/L 1.1 mg/L <0.02 mg/L <0.1 pg/L <0.2 pg/L <0.5 pg/L <0.2 pg/L Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium <1.1 pg/L <0.7 pg/L <0.25 pg/L <0.3 pg/L <0.2 pg/L <0.1 pg/L <0.5 pg/L <0.025 pg/L <0.5 pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15,1999. Wil d l if e In tern a tio n a l ltd. -23- PROJECT NO.: 454A-118A Appendix 3 THE ANALYSIS OF PFBS IN FRESHWATER IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-118A W il d l if e In tern a tio n a l ltd. -24 - PROJECT NO.: 454A-118A REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PERFLUORO BUTANE SULFONATE, POTASSIUM SALT (PFBS): A 48-HOUR STATIC ACUTE TOXICITY TEST with the CLADOCERAN {Daphnia magna) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-118A 3M ENVIRONMENTAL LAB PROJECT NUMBER: E00-1429 PRINCIPAL INVESTIGATOR: ~l (lrx > * ----- Ra; kF'L Van Hoven, Ph.D. Scientist MANAGEMENT: 'fa ' Willard B. Nixon, Ph.D Director, Analytical Chemistry 0 3-x> -o\ DATE DATE Wil d l if e In tern atio n al ltd. -25 - PROJECT NO.: 454A-118A SANITIZED Introduction DECO 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 cladoceran (Daphnia magna). This study was conducted by Wildlife International, Ltd. and identified as Project Number 454A- 118A. The analyses of these water samples were performed at Wildlife International, Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). Day 0, Day 1, and Day 2 samples were received for analysis on January 23, 24, and 25, 2001, respectively. The submitted samples were prepared for analysis on each sample receipt day. Analyses were completed on January 23, 25, and 26, respectively. Analytical Standard The analytical standard was received from 3M Environmental Technology and Safety Services on March 27, 2000, assigned Wildlife International, Ltd. Identification number 5216, and stored under ambient conditions. The analytical standard, a white powder, was identified as: Potassium Perfluorobutane Sulfonate ( 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 o f 97.90%. A subsequent revision of the certificate of analysis indicated a purity o f 97.3% and an Expiraition/Reassessment Date of January 17, 2002. The analytical standard was the same material and lot number as the test substance (Wildlife International, Ltd. Identification number The analytical standard was used to prepare calibration and matrix fortification samples. Analytical Method W ater samples were analyzed according to the method entitled "Analytical Method Validation for the Determination o f 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 o f the PFBS methodology. Aliquots o f the dilutions were transferred to autosampler vials and submitted for analysis by direct injection. Concentrations o f PFBS in freshwater samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) interfaced with a PerkinElmer API 100LC mass spectrometer (single quadrupole) operated in selective ion monitoring (SIM) Wil d l if e In tern a tio n a l ltd. -26- PROJECT NO.: 454A-118A detection mode. The mass spectrometer was equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separations were achieved using a Keystone PRISM RP column (30 mm x 1.5 mm, 3 - pm particle size) fitted with a Keystone Javelin C )it Guard Cartridge (20 mm x 2 mm). The instrument parameters are summarized in Table 1 and a method flowchart is provided in Figure 1. Primary and Secondary Stock Solutions. All primary and secondary stock preparations were adjusted for the purity o f the analytical standard (97.90%). A 10.0 mg a.i./mL primary stock solution o f PFBS in methanol was prepared by weighing 1.024 g o f the analytical standard and bringing to final volume o f 100 mL with methanol. Secondary stock solutions (1000, 100, 10.0, 1.00, and 0.100 mg a.i./L) o f PFBS in methanol were prepared by serial volumetric dilution from the primary stock. Calibration Standards and Calibration Curves Calibration standards were prepared in 50:50 methanol: NANOpure water by appropriate dilutions of the 10.0 mg a.i./L stock solution o f PFBS in methanol. The calibration standards o f PFBS, ranging in concentration from 0.0100 to 0.0500 mg a.i./L, were analyzed with each sample set. Five calibration standards (different concentrations) were analyzed with the samples. The calibration standard series was injected at the beginning and end o f each run, and one standard was injected, at a minimum, after every five samples. Linear regression equations were generated using the peak area responses versus the respective concentrations o f the calibration standards. A typical calibration curve is presented in Figure 2. The concentration o f PFBS in the samples was determined by substituting the peak area responses into the applicable linear regression equation. Representative ion chromatograms o f low and high calibration standards are presented in Figures 3 and 4, respectively. Limit of Quantitation The method limit o f quantitation (LOQ) for these analyses was set at 100 mg a.i./L calculated as the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor o f the matrix blank samples (10000). Wil d l if e In tern a tio n a l ltd. -27- PROJECT NO.: 454A-118A 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. Freshwater was directly fortified (/. e. without use o f carrier solvent) with PFBS at 200, 1000 and 5000 mg a.i./L and analyzed concurrently with the samples to determine the mean procedural recovery (Table 2). Sample concentrations were not corrected for the mean procedural recovery o f 97.4%. A representative ion chromatogram o f a matrix fortification is presented in Figure 6. Example Calculations Sample number 454A-118A-27, nominal concentration of 250 mg a.i./L in freshwater. First Initial Volume: 0.100 mL Calibration curve equation: First Final Volume: 10.0 mL Slope: 13470852 Second Initial Volume: 0.100 mL Intercept: 44521.44141 Second Final Volume: 10.0 mL Curve regression weighted 1/x Dilution Factor: 10000 PFBS Peak Area: 350078 peak area - (y-intercept) PFBS (mg a.i./L) measured at instrument = slope PFBS (mg a.i./L) in sample = PFBS measured at instrument (mg a.i./L) x dilution factor = 226.8 Wil d l if e In tern a tio n a l ltd. -28- PROJECT NO.: 454A-118A PFBS (mg a.i./L) in sample Percent o f Nominal Concentration = PFBS (mg a.i./L) nominal x 100 226.8 = W x 100 = 90.7% Calculated with HPLC/MS instrument software: MacQuan, version 1.6. Sample Analysis RESUITS Freshwater samples were collected from an acute toxicity study with the cladoceran {Daphnia magna) at test initiation, January 23, 2001 (Day 0), on January 24, 2001 (Day 1), and at test termination, January 25, 2001 (Day 2). The measured concentrations of PFBS in the samples collected at initiation of exposure o f the test organisms (Day 0) ranged from 83.1 to 96.1% o f the nominal concentrations. Samples collected at Day 1 had a measured concentration range o f 88.5 to 99.2% o f nominal values. Samples collected at test termination (Day 2) had a measured concentration range o f 79.4 to 92.3% o f nominal values (Table 3). A representative ion chromatogram o f a test sample is shown in Figure 7. Wil d l if e In ter n a tio n a l ltd. -29- project no.: 454A-1isa Table 1 Typical HPLC/MS Operational Parameters INSTRUMENT: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer operated in Selective Ion Monitoring (SIM) Mode ION SOURCE: Perkin-Elmer TurboIonSpray ANALYTICAL COLUMN: Keystone PRISM RP (30 mm x 1.5 mm, 3-pm particle size) GUARD COLUMN: Keystone Javelin Cjg cartridge (20 mm x 2 mm) OVEN TEMPERATURE: 40C STOP TIME: 3.00 min FLOW RATE: MOBILE PHASE: INJECTION VOLUME: 200 pL/min 25% NANOpure W ater with 0.1% Ammonium Formate: 75% Methanol 5.0 pL PFBS PEAK RETENTION TIME: Approximately 2.3 minutes PFBS MONITORED MASS: 299.0 amu Wil d l if e In tern atio n al ltd. -30- PROJECT NO.: 454A-118A Table 2 Matrix Blanks and Fortifications Analyzed Concurrently During Sample Analysis Concentrations o f PFBS (mg a..i./L) Sample Number Sample Type (454A-118A-)_______________________ Fortified Measured1 MAB-1 MAB-2 MAB-3 Matrix Blank Matrix Blank Matrix Blank 0.00 0.00 0.00 < L O Q 12 <LOQ <LOQ Percent Recovered1 -- -- -- MAS-1 MAS-4 MAS-7 Matrix Fortification Matrix Fortification Matrix Fortification 200 200 200 193 194 187 96.5 96.9 93.3 MAS-2 MAS-5 MAS-8 Matrix Fortification Matrix Fortification Matrix Fortification 1000 1000 1000 1074 935 895 107 93.5 89.5 MAS-3 MAS-6 MAS-9 Matrix Fortification Matrix Fortification Matrix Fortification 5000 5000 5000 5566 4866 - 4571 111 97.3 91.4 Mean = 97.4 Standard Deviation = 7.15 CV = 7.34% N=9 1Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2The limit o f quantitation (LOQ) was 100 mg .a.i./L based upon the product o f the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor o f the matrix blank samples (10000),______ Wil d l if e In tern a tio n a l ltd. -31 - PROJECT NO.: 454A-118A Table 3 Measured Concentrations o f PFBS in Freshwater Samples from a Cladoceran Static Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 0.0 Sample Number (454A-118A-) 1 2 . 13 14 25 26 Sampling Time (Day) 0 0 1 1 2 2 PFBS Measured Concentration1 (mg a.i./L) < LOQ2 < LOQ < LOQ < LOQ < LOQ < LOQ Percent of Nominal1 -- -- -- -- -~ 250 3 4 15 16 27 28 0 0 1 1 2 2 240 96.1 240 96.0 233 93.2 235 94.0 227 90.7 231 92.3 500 5 6 17 18 29 30 0 0 1 1 2 2 478 95.6 461 92.2 476 95.1 496 99.2 451 90.3 456 91.3 1000 7 0 80 858 85.8 850 85.0 19 1 20 1 31 2 921 92.1 923 92.3 862 86.2 32 2 902 90.2 1 Measured and Percent of Nominal values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2 The limit o f quantitation (LOQ) was 100 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (10000). Wil d l if e In tern atio n al lTM. -32- PROJECT NO.: 454A-118A Table 3 (Continued) Measured Concentrations o f PFBS in Freshwater Samples from a Cladoceran Static Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 2000 Sample Number (454A-118A-) 9 10 21 22 33 34 Sampling Time (Day) 0 0 1 1 2 2 PFBS Measured Concentration1 (mg a.i./L) 1661 1738 1770 1842 1588 1640 Percent of Nominal1 83.1 86.9 88.5 92.1 79.4 82.0 40003 11 12 23 24 0 0 1 1 3592 3751 3835 3888 89.8 93.8 95.9 97.2 1 Measured and Percent o f Nominal values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2 The limit of quantitation (LOQ) was 100 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor o f the matrix blank samples (10000). 3 This treatment group was not sampled on Day 2 due to 100% mortality on Day 1.__________________ Wil d l if e In tern a tio n a l ltd. -33 - PROJECT NO.: 454A-118A M ETHOD OUTLINE FOR THE ANALYSIS OF PFBS IN FRESHW ATER Prepare each matrix fortification sample by weighing the requisite amount o f PFBS test substance on an analytical balance and transferring directly into a Class A volumetric flask partially filled with freshwater. Rinse weighing paper and the sides o f 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 LCMS 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. I Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis o f PFBS in freshwater. Wil d l if e In tern a tio n a l ltd. -34- Area PROJECT NO.: 454A-118A Figure 2. A typical calibration curve for PFBS. Slope = 13470852; Intercept = 44521.44141; r = 0.9987 Wil d l if e In tern atio n al ltd. -35 - PROJECT NO.: 454A-118A intensity: 120000 cps Figure 3. A representative ion chromatogram of a low-level (0.0100 mg a.i./L) PFBS standard. Wil d l if e In tern a tio n a l ltd. - 36- PROJECT NO.: 454A-118A Figure 4. A representative ion chromatogram o f a high-level (0.0500 mg a.i./L) PFBS standard. Wil d l if e In tern a tio n a l ltd. - 37- PROJECT NO.: 454A-118A intenslty: 120000 cps Figure 5. A representative ion chromatogram o f a matrix blank sample (454A-118A-MAB-3). The arrow indicates the retention time o f PFBS. Wil d l if e In tern a tio n a l ltd. - 38- PROJECT NO.: 454A-118A intensity: 120000 cps Figure 6. A representative ion chromatogram o f a matrix fortification sample (454A-118A-MAS-8, nominal PFBS concentration o f 1000 mg a.i./L, dilution factor = 50000x). Wil d l if e In ter n a tio n a l ltd. - 39- PROJECT NO.: 454A-118A intensity: 120000 cps Figure 7. A representative ion chromatogram of a test sample (454A-118A-29, nominal PFBS concentration of 500 mg a.i./L, dilution factor = 25000x). Wil d l if e In ter n a tio n a l, Lt d . - 40- PROJECT NO.: 454A-118A 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. The protocol was amended to correct the size o f the test chambers. 3. The protocol was amended to change the test concentrations. Wil d l if e In ter n a tio n a l, Lt d . -41 - PROJECT NO.: 454A-118A Appendix 5 Personnel Involved in the Study The following key Wildlife International, Ltd. personnel were involved in the conduct or management of this study: 1. Henry 0 . Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Director, Analytical Chemistry 3. Kurt R. Drottar, Senior Biologist 4. Raymond L. Van Hoven, Ph.D., Scientist 5. Caiy A. Sutherland, Laboratory Supervisor 6. Molly McCoy, Biologist