Document 7RMyV0e9ObLwLnq8EKNgGOG6e

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 CLADOCERAN (Daphnia magna) FINAL REPORT SAN ITlZED WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-118A 3M LAB REQUEST NO. E00-1429 U. S Environmental Protection Agency Series 850 - Ecological Elffects Test Guidelines OPPTS Number 850.1010 and. OECD Guideline 202 DE[ 0 9 2003 AUTHORS : Kurt R Lkottar Henry 0.Krueger, Ph.D. STUDY INITIATION DATE: July 10,2000 STUDY COMPLETION DATE: March 20,200 1 3M Corporation Environmental Laboratory Building 2-3E-09 935 Bush ,4venue St Paul, Minnesota 55 144 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 2 1601 (410) 822-8600 Page 1 of 41 WILDLIFE INTERNATIONAL, LTD. -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 T h s study was conducted in compliance with Gcnd Laboratory Practice Standards as published by the U.S. EnvironmentalProtection Agency in 40 CFR Part 160and 792,17 August 1989;OECD Principlesof Good Laboratory Practice (ENVMCKKEM (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 pUr;ty. 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: A - Kurt R. Drottar Senior Biologist 1 - x/aoial DATE SPONSORAPPROVAL: WILDLI FE INTER NATIONAL, LTD. -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 ProtectionAgency in40 CFRPart 160 and 792,17 August 1989;OECDPMciples ofGood Laboratory Practice (ENV/MC/CHEM (98) 17) and Japan MAFF (59 NohSan, Notification No. 3850, Agricultural Production Bureau). The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Managem.ent were as follows: ACTIVITY: DATE REPORTED TO: ' DATE CONDUCTED: STUDY DIRECTOR: MANAGEMENT: Initial Trial (454A-118) 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 Definitive Trial (454A-118A) 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 February 9,2001 February 9,2001 February 12,2001 Final Report March 16,2001 March 16,2001 March 16,2001 Qual& Assurance Program Supervisor WILDLIFE INTERNATIONALT, D. -4- REPORT APPROVAL PROJECT NO. 454A-11SA SPONSOR: 3M Corporation TITLE: PerfluorobutaneSulfonate, Potassium Salt (PFBS): A 48-Hour StaticAcute Toxicity Test with the Cladoceran (Daphnia magna) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-118A STUDY DIRECTOR: Kurt R Drottar Senior Biologist MANAGEMENT: Director, Aquatic Toxicology and Non-Target Plants DATE WILDLIFE INTERNATIONLATLD. -5- PROJECT .NO. 454A-118A TABLE OF CONTENTS TitleKover Page................................................................................................................................................... 1 Good Laboratory Practice Compliance Statement............................................................................................... 2 Quality Assurance Statement............................................................................................................................... 3 Report Approval................................................................................................................................................... 4 Table of Contents ................................................................................................................................................. 5 summary................................................................................................................................................................ 7 Introduction .......................................................................................................................................................... 8 Objective .............................................................................................................................................................. 8 Experimental Design ............................................................................................................................................ 8 Materials and Methods......................................................................................................................................... 9 Results and Discussion...................................................................................................................................... 12 Conclusions ....................................................................................................................................................... 13 References ......................................................................................................................................................... 14 TABLES Table 1 .Summary ofhalytical Chemistry Data ........................................................................................... 15 Table 2 - Temperature, Dissolved Oxygen, and pH of W.ater in the Test Chambers ...................................... 16 Table 3 - Cumulative Percent Mortalityhnrnobility and `Treatment-RelatedEffects...................................... 17 Table 4 - EClO, EC50 and EC90 Values......................................................................................................... 18 WILDLI FE INTERNATIOLNTAD.L, -6- PROJECT NO.: 454A-118A TABLE OF CONTENTS - Continued - FIGUIZE Figure 1. Concentration-Response Curve (48 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-118A.......................................................... 23 Appendix 4 - Changes to Protocol ................................................................................................................ 40 Appendix 5 - Personnel Involved in the Study............................................................................................. 41 WILDLIFE INTERNATIONAL, LTD. -7- SPONSOR: SPONSOR'S KEPPRESENTATM: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: SUMMARY 3M Corporation Wildlife Inte:mational, Ltd. Easton, Maryland 21601 PROJECT NO.: 454A-118A DEC 0 9 ZOQ~ SANITIZED WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: I TEST SUBSTANCE: STUDY: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: I LENGTH OF TEST: n 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 Tt:nnination - January 25,2001 Experimental.Termination - January 25,2001 48 Hours Cladoceran (Daphnia magna) Wildlife International, Ltd. Cultures Easton, Maryland 2 1601 <24 hours at test initiation 95% CONFIDENCELIMITS: NO MORTALITY/JMMOBILITY 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 WILDLIFE INTERNATIONALL,TD. -8- PROJECT NO. 454A-11SA 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, 2001to January 25,200 1. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454A-11SA 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 10daphnids in each test chamber for a total of 20 daphnids per test concentration. Nominal test concentrationswere selectedin consultation with the Sponsor, and were based upon the results of exploratory rangefmding 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 of 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/immobilityand other clinical signs of toxicity were made approximately4,24 and 48 hours after test initiation. Cumulativepercent mortality and immobility o'bservedin the treatment groups were used to calculate EC 10,EC50 and EC90 values at 24 and 48 hours, when possible. The no mortality/imrnobilityconcentrationand the no-observed-effect-concentration (NOEC) were determined by visual interpretation of the mortality, immobility and clinical observation data. WILDLIFE INTERNATIONLATLD, . -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 Toxic~.tyTest with the Cladoceran (Daphniamagna)". The protocol was based on procedures outlined in U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1010 (I), OECD Guideline for Testing of Chemicals, 202: Daphnia sp. Acute Immobilization Test and Reproduction Test (2)and ASTM Standard E-729-88a, Standard Guidefor ConductingAcute ToxicityTests 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 of the certificate of analysis indicated a purity of 97.3% and an ExpiratiodReassessment Date of January 17, 2002. The test substance was stored at ambient room temperature. PreDaration of Test Concentrations Nominal test concentrationswere 250,500, 1000,2000 and 4000 mg a.i./L. A 1-Lprimary stock solution was prepared in dilution water at a concentrationof 4000 mga.i./L. The primary stock solutionwasinvertedand stirred to aid in the solubilization of the test substance. After mixing, appropriate aliquots of the primary stock solution were proportionally diluted with dilution water tci prepare 500 mL of the other four test concentrations. All dilutions were inverted to mix. After mixing, 225 mL of test solution was added to the two replicate test chambers for each treatment group. All test solutions appeared clear and colorless. Observations of 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 forthis study. Daphnidsarerepresentative of an important group of aquatic invertebrates and were selected for use in the test based upon past hstory of use and ease of culturing in the laboratory. Daphnid neonates used in the test were less than 24-hours old and were WILDLIFE INTERNATIONALL,TD. - 10 - PROJECT NO.: 454A-118A obtainedfiom cultures maintained by Wildlife International,Ltd., Easton, Maryland. Identificationof theoriginal brood stock was verified by the Academy of Natural Sciences, Philadelphia, PA. Adult daphnids were cultured in water from the same:source and at approximatelythe sametemperatureas 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 of disease or stress during the holdmg period. During the 14-day period preceding the test, water temperatures ranged from 19.1 to 20.2"C. The pH of the water ranged fiom 8.2 to 8.5, and dissolved oxygen ranged from 8.0 to 8.6mg/L. Instrumentation used for water measurements are described in the Environmental Condifions section of this report. Neonate daphnids were obtained for testing fiom individualadult daphnids which were observedtohaveno neonates present less than 24 hours prior to test initiation. The progeny fiom 5 adults were used in the test. At test initiation, thejuvenile daphnids were collected from the cultures and indiscriminatelytransferred 1to 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 aidwater interface using a wide-bore pipet. Daphnidsin the cultureswere fed a mixture of yeast, Cerophyllm, and trout chow, as well as a suspension of the freshwatergreen 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@b) eakers containing 225 mL of test solution. The depth of test solution in a representative test chamber was .5.5 cm. Test chamberswere indiscriminatelyplaced in a temperature-controlledwater bath set to maintain a temperature of 20 & 1"C. Test chamberswere labeledwith the project number, test concentration and replicate. Dilution Water The water used for culturing and testing was freshwater obtained from a well approximately40 metersdeep 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 approximately25 tun, and pumped into a 37,800-L storage tank where the water Wiilj aerated with spray nozzles. Prior to use, the water WILDLI FE INTERNATI ONAL, LDI . - .. I! - PROJECT NO.: 454A-118A again was filtered (0.45 jm)to remove microorganisms imd particles. The resultsof periodic analysesperformed 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 (Colortonb 50). A photoperiod of 16 hours of light and 8 hours of darkness was controlledwith an automatictimer, A 30-minutetransitionperiod of low light intensity was provided when lights went 011 and off to avoid sudden changes in lighting. Light intensity at test initiation and termination was approximately 144 and 143lux, respectively,at the surface of the water of 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 EWC Recorder. The target test temperature during the study was 20+1"C. ThepH and dissolvedoxygencontent of 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 of pH were made using a Fisher Acciunet Model 915 pH meter, and dissolved oxygen was measured using a Yellow Springs Instrument Model 5 1El dissolved oxygen meter. Specific conductance was measured using a Yellow Springs InstrumentModel 33 Salinity-Conductivity-Temperaturemeter. Hardnessand alkalinitymeasurementswere made by titrationbased on procedures in StandardMethohfor the Examination of Waterand Wastewater(4). Total organic carbon was rneasured using a ShimadZuModelTOC-5000 analyzer. Observations Observations were made to determinethe numbers of mortalities and immobileorganisms. The numbersof individuals exhibiting clinical signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 4,24 and 48 hours after test initiation. WILDLI FE INTERNATIONLATLD,. - 12- PROJECT NO.: 454A-11SA Statistical Analyses The 24 and 48-hour EC50 values and the 95% confidence intervals were calculatedwhen possibleby 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). The no mortality/immobility concentration and NOEC values were determined by visual inspection of 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 of analyses to measure concentrations of 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 tallected at 24 hours had measured values that ranged fi-om 89 to 99% of nominal, whereas measured values for samples taken at 48 hours ranged fiom 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 Measurementsof temperature, dissolved oxygen and pH are presented in Table 2. Temperaturesremained withinthe 20k 1Crange established for the test. Dissolved oxygen concentrationsremained 28.3 mgL (91%of saturation) throughout the test. Measurements of pH ranged fiom 7.9 to 8.5 during the test. Daily observations of 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 hours of exposure, mortality/immobilityin the 234,470, 886, 1707 and 3767 mg a.i./L treatment groups was 5,0, .5,20 and loo%, respectively. EC50 values and 95% confidence limits at 24 and 48 hours were calculated fiom the mortality/immobility data, and are shown in Table 4. WILDLIFE INTERNATIONLATLD, . PROJECT NO.: 45444- 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 of 5% mortality in the control, the mortality/immobilityobserved in the 234 and 886 mg a.i./L (5 and 5%, respectively) was not considered to be treatment-related. Consequentl:y, the no mortality/immobilityconcentrationand NOEC were 886 mg a.i./L. WILDLIFE INTERNATIONLATLD, . PROJECT NO.: 454A-118A REFERENCES 1 U.S. Environmental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines (draft), OPPTS Number 850.1010. Aquatic invertebrate Acute Toxicity Test, Freshwater Daphnids. 2 Organisation for Economic Cooperation and Development. 1984. Daphnia sp. Acute Immobilization Test and Reproduction Test. ClECD Guideline for Testing of Chemicals. Guideline 202. Paris. 3 ASTM StandardE729-88a. 1994. Standard Cuidefor ConductingAcute ToxicityTestswithFishes, Macroinvertebrates, and Amphibians. American Society for Testing and Materials. 4 APHA, AWWA, WPCF. 1985. StandardMethods for the Ejcamination of 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 Thompson, W.R 1947. Bacteriological Reviews. Vol. 11, 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, Environmentall Research Laboratory, Duluth, Minnesota. Personal communication. WILDLIFE INTERNATIONLATLD, . - 15 - PROJECT NO. 454A-1 18A Table I 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,Daphnia magna Well Water Replicate A B .A B A B Sampling Time (Hours) 0 0 24 24 48 48 250 A 0 B 0 A 24 B 24 A 48 B 48 500 A 0 B 0 A 24 B 24 A 48 B 48 1000 A 0 B 0 A 24 B 24 A 48 B 48 2000 A 0 B 0 A 24 B 24 A 48 B 48 4000 A 0 B 0 A 24 B 24 The limit of quantitation (LOQ) was 100mg a.i./L. Measured Concentration (mg a.i./L) < LOQ' < LOQ < LOQ < LOQ < LOQ < LOQ 240 240 233 235 227 23 1 478 46 1 476 496 45 1 456 858 850 92 1 923 862 902 1661 1738 1770 1842 1588 1640 3592 375 1 3835 3888 Mean Measured Concentration (mg a.i./L) < LOQ 234 470 886 1707 3767 Percent of Nominal 94 94 89 85 94 WILDLIFE INTERNATIONALL,TD. - 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.lL) Negative Control 3M Corporation PFBS Cladoceran, Daphnia magna Well Water 0 Hour' Replicate Temp' (oc) DO3 (m@) A 19.9 8.4 B 19.9 8.6 - PH 7.9 8.0 24 Hours Do (mgn) PH 8.6 8.4 8.6 8.4 48 Hours4 Temp DO ("C) (mglL) PH 20.1 8.4 8.3 20.2 8.4 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 8.6 8.5 19.8 8.4 8.5 B 20.1 8.6 8.2 8.6 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 5 - 19.8 - 5 I The 0-hour dilution water measurementsfor hardness, alkalinity, specificconductanceand TOC were 132mg/LasC a C h 181mg/Las CaC03,290 pmhodcm and -4mg CIL, respectively. Temperature measured continuously during the test ranged from approximately 19.5 to 20.5"C. A dissolved oxygen concentration of 5.4 mg/L represents 60% samation at 20C in freshwater. The test termination dilution water measurements for hardness, alkalinity, specificconductance and TOC were 136 mg/L as CaCO3, 184 mg/L as C a C a , 295 pmhodcm and 1.3 mg CIL, respectively. Measurements discontinued due to 100% mortality. - 17- Table 3 Cumulative Percent Mortalityhntnobility and Treatment-Related Effects' PROJECT NO.: 454A-118A Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Concentration Daphnia/ Number (mg a.i./L) Replicate 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 10 0 ' B 10 10 0 Observed Effects: AN = AppearsNormal 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 0 0 0 0 0 0 0 0 10 10 24 Hours Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 . Effects 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN - I Percent Immobile and &ad 0 0 0 0 0 100 Number Dead 0 1 0 0 0 0 0 0 3 0 10 10 48 Houn Number Immobile 0 0 1 0 0 0 0 1 0 1 0 0 Effects 10 AN 9AN 9AN l0AN 10 AN 10 AN 10 AN 9AN 7AN 9AN - - Percent Immobile andlkad 5 5 0 5 20 100 WILDLIFE INTERNATIONALL,TD. -18- PROJECT NO.: 454A-118A Table 4 EC50 Values Sponsor: Test Substance: Test Organism: Dilution Water: Time 24 Hours 3M Corporation PFBS Cladoceran, Daphnia magna Well Water EC50 (mg a.i./L) Lower (95% Confidence Limits ' 2536 1707 Upper 95% Confidence Limits 3767 Statistical Method Binomial 48 Hours 2183 1707 3767 Binomial Note: EC 10 or EC90 values could not be calculated with the concentration-responseobserved. WILDLIFE INTERNATIONALL,TD. - 19- Figure 1. Concentration-Response Curve (48 Hour Data) PROJECT NO.: 454A-118A 7 6 5 4 3 2 1 100 I 1000 Mean Measured Concentration (mg a.i./L) 10000 WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-118A 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 Cladoceran, Daphnia magna Well Water Mean Specific Conductance (pmhos/cm) 315 (N = 4) Range 305 - 335 Hardness (mgL as CaC03) 131 (N = 4) 116- 140 Alkalinity (mgLas CaC03) 178 (N = 4) 172 - 184 PH 8.1 (N:=4) 8.0 - 8.1 WILDLI FE INTER NATIONAL, LTD. -21 - PROJECTNO.: 454A-118A Appendix 2 Analyses of Pesticides, Organics and Metals in WildlifeInternational,~ t dW. ell Water' Component Measured Concentration Component Measured Concentration Pesticides and Organics Aclonifen Alachlor Ametryn Atrazin Azinphosethyl Azinphos-methyl . Azoxystrobin Bifenthrin Bioallethrin Bitertanol Bromacil Bromophos Bromophos-ethyl Broompropylaat Bupirimaat Carbaryl Carbofuran Carboxin Chlorfenvinphos C hloridazon 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 pg/L <0.01 p g n 4 . 0 1 pg/L 4.01 pgn 4.04 P g n <0.08p g L <0.25 p a 4 . 0 5 pg/L 4 . 0 5 pg/L <0.05 pg/L ~ 0 . 0 5pg/L 4.02 pgL <0.02 p g n C0.02 pg/L 4 . 0 5 pglL 4 . 0 5 pg/L <0.03 pg/L 4 . 0 2 pg/L 4 . 0 2 pg/L 4.05 pgL 4.02 pgn CO.01 p g L 4.01 pgn < o m &L 4.02 pgL 4 . 0 5 pglL 4.05 pglL 4.25 pgL 4.05 pgL 4.02 pgn 4 . 0 2 pglL <0.02 p g n 4 . 0 1 pglL <0.02 p g n <0.01 pg/L 4 . 0 1 pg/L co.01 pglL 4 . 0 3 pg/L <0.02 pg/L <0.01 pg/L 4 . 0 3 pg/L Dimethomorf Disulfoton DMST Dodemorf Endosulfan-a Endosulfan-f3 Endosulfan-sulfaat / Epoxiconazole Eptam Esfenvaleraat Ethion Ethofumesaat Ethoprophos Etridiazole Etrimphos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb Fenpiclonil Fenpropathrin Fenpropimorf Fenthion Fenvaleraat Fluazifopbutyl Fluoroglycofenethyl Fluroxypyr-meptyl Flutolanil Fonophos Furalaxyl Heptenophos Imazalil Iprodion Kresoxim-methyl Lenacil Lindane Malathion Metalaxyl Metamitron Metazachlor Methidathion <0.05 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 <0.02 pg/L <0.05 pg/L 4 . 0 2 pg/L 4 . 0 2 pg/L <0.05 pa 4.02 pgn 4 . 0 1 pg/L <0.02 pg/L 4 . 0 5 pg/L 4.05 pg/L <0.01 p g n a . 0 3 pg/L <0.03 p g L <0.05 pfl 4 . 2 5 pglL 4 . 0 1 pg/L 4 . 0 1 pg/L 4.02p g n 4 . 0 2 pgiL 4 . 0 2 pg/L 4.05 p a 4.02 pgn <0.01 p g n 4.02 pgL <0.02 p g n 4 . 0 1 pg/L 4.05 pgL 4.02 pgn 4 . 0 5 pg/L 4 . 0 2 pg/L <0.02 pg/L <0.05 pg/L ~ 0 . 0 5pg/L <0.02 pg/L <0.02 pg/L 'Analyses performed by TNO Nutrition and Food Institute on samplescollectedon October 14 and 15,1999. WILDLIFE INTERNATIONLATLD, . -22- PROJECT NO.: 454A-118A Appendix 2 (Continued) Analyses of Pesticides, Organics and Metals in Wildlifehtemationd, ~ t dW. en Water' Pesticides and Organics(Page 2) Component Measured Concentration Component Measured Concentration Dichlorvos Dicofol Diethyltoluamide Difenoconazde Dimethoate Paclobutazole Parathion Parathion-methyl Penconazole Pendimethalin Permethrincis Permethrin-trans Phosalon Phosmet Phosphamidon-cis Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazin Propham Propiconazool Propoxur Proppmide Prosulfocarb Pyrazophos <0.01 p g n 4 . 2 5 pg/L 4 . 0 2 pg/L 4.03pg/L 4 . 0 2 pg/L 4 . 0 5 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L <0.05 pg/L 4 . 0 3 pg/L 4 . 0 1 pg/L CO.01 p g n 4 . 0 5 p@ 4 . 0 2 pg/L 4.05 pgL <0.01 pg/L 4.01 p g n 4.01 p g n 4.02 pgn CO.01 pg/L 4 . 0 1 pg/L <0.01 pg/L a . 0 1 pg/L c0.02 pg/L 4.05 p g L 4.03pg/L c0.02 p g n 4 . 0 2 pg/L 4 . 0 3 pg/L Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl Pyrifenox-1 Pynfenox-2 Pynmethanil Quizalofop-ethyl Simazin Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbutylazin .. Tetrachlorvinphos Tetrahydroftaalimide Tetramethrin Thiabendazole Thiometon Tolclophos-methyl To1ylfluanid Triadimefon Triadimenol Triallaat Triazophos Trifluralin Vamidothion Vinchlozolin 4 . 0 1 pg/L <oo.olpg/L c0.02 pg/L 4 . 0 1 pg/L 4 . 0 5 pg/L <0.01 pg/L 4 . 0 1 pa 4 . 0 1 pg/L 4 . 0 2 pg/L <0.01 pg/L <0.02 pglL co.05 pg/L <0.05 pgiL <0.01 pg/L 4.01 pgL 4 . 0 1 pg/L (0.05 p g L 4.01 pgn ~ 0 . 0 5pg/L <0.04 pg/L <0.01 pg/L 4 . 0 4 pg/L 4 . 0 5 pg/L d.05 pg/L 4.02 pgL 4.02 pgL 4.02 pgn 4.01pgn 4 . 0 1 pg/L Metals Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 11.0 m a 18.0 rn@ 29 mg/L 4 . 0 1 5 mg/L 1.1 m a <0.02 mg/L <O.l p g n a . 2Ppn ~ 0 . p5glL <0.2 pgL Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium 4 . 1 pg/L 4 . 7Pgn 4 . 2 5 pg/L <0.3 p g L 4 . 2 Pgn 4 . 1 Pgn ~ 0 . p5g/L <0.025 p g 5 4 . 5Pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15, 1999. WILDLIFE INTERNATIONALTLD. - 23 - PROJECT NO.: 454A-118A Appendix 3 THE ANALYSIS OF PFBS IN FRESHWATER IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-118A ~ ~~ WILDLIFE INTERNATIONLTDA. L -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: Ra*& . Van Hoven, Ph.D. Scientist MANAGEMENT: Willard B. Nixon, Ph.D! Director, Analytical Chemistry 63-20-0 \ DATE DATE WILDLIFE INTERNATIONALTLD. - 2.5 - PROJECT NO.: 454A-118A SANITIZED Introduction DEC 09?@@ 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). l h s study was conducted by Wildlife International, Ltd. and identified Project Number 454A- 118A. The analyses of these water samples were performed at Wildlife International, Ltd. using hgh 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. Analvtical Standard The analytical standard was received from 3hI 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 punty of 97.90%. A subsequent revision of the certificate of analysis indicated a purity of 97.3% and an Expiration/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. Analvtical Method Water samples were analyzed according 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 ill 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 high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (J3PLC) interfaced with a PerhnElmer API lOOLC mass spectrometer (single quadrupole) operated in selective ion monitoring (SIM) WILDLIFE INTERNATIONALTLD. 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,a 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 of the analytical standard (97.90%). A 10.0 mg a.i./mL primary stock solution of PFBS in methanol was prepared by weighmg 1.024 g of the analytical standard and bringing to 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 5 0 5 0 methanol: NANOpure@water by appropriate llutions of the 10.0 mg a.i./L stock solution of PFBS in methanol. The calibration standards of PFBS, ranging in concentration from 0.0100 to 0.0500 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 i r j e c t d 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 100 mg a.i./L calculated as the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (10000). WILDLIFE INTERNATIONALIDL. 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 ( i e . without use of 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 of 97.4%. A representative ion chromatogram of a matrix fortification is presented in Figure 6 . Examde 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: 0,100mL Intercept: 44521.44141 Second Final Volume: 10.0 mL Curve regression weighted l/x Dilution Factor: 10000 PFBS Peak Area: 350078 eak area - (y-intercept) PFBS (mg a.i./L) measured at instrument = P slope PFBS (mg a.i./L) in sample = PFBS measured at instrument (mg a.i./L) x dtlution factor - 350078 - 44521.44141 x 10000 13470852 = 226.8 WILDLIFE INTERNATIONALTLD. -28 - PROJECT NO.: 454A-118A ~ PFBS Percent of Nominal Concentration = =,((& m a.i./L innosmaminLlel x 100 --226.8 x 100 = 90.7% - 250 Calculated with HPLCMS instrument soharc:: MacQuan, version 1.6. Sample Analvsis ~ RESUL- TS 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 l), and at test termination, January 25,2001 (Day 2). The measured concentrations of PFBS in the samples collected at initiation of exposure of the test organisms (Day 0) ranged from 83.1 to 96.1% of the nominal concentrations. Samples collected at Day 1 had a measured concentration range of 88.5 'to 99.2% of nominal values. Samples collected at test termination (Day 2) had a measured concentration range of 79.4 to 92.3% of nominal values (Table 3). A representative ion chromatogram of a test sample is shown in Figure 7. WILDLIFE INTERNATIONALTLD. - 29 - PROJECT NO.: 454A-118A 1 Typical HPLC/MS Operational Parameters INSTRUMENT: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API lOOLC Mass Spectrometer operated in Selective Ion Monitoring (SIM) Mode ION SOURCE: Perkin-Elmer TurboIonSpray ANALaYTICAL COLUMN: Keystone PRISM RP (30 nun x 1.5 mm, 3-pm particle size) GUARD COLUMN: Keystone Javelin C18cartridge (20 mm x 2 mm) OVEN TEMPERATURE: 40C STOP TIME: 3.00 min FLOW RATE: MOBILE PHASE: INJECTION VOLUME: 200 pL/min 25% NANOpure:" Water with 0.1% Ammonium Formate: 75% Methanol 5.0 pL PFBS PEAK RETENTION TIME: Approximately 2.3 minutes PFBS MONITORED MASS: 299.0 m u WILDLIFE INTERNATIONALTLD. -30- PROJECT NO.: 454A- 118A Table 2 Matrix Blanks and Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A- 1 18A-) MAB-1 MAB-2 MAB-3 Sample Type Matrix Blank Matrix Blank Matrix Blank Concentrations of PFBS (mg a..i./L) Fortified 0.00 0.00 0.00 Measured' <LOQ~ <LOQ <LOQ Percent Recovered' -- --- MAS-1 Matrix Fortification 200 193 96.5 MAS4 Matrix Fortification 200 194 96.9 MAS-7 Matrix Fortification 200 187 93.3 MAS-2 Matrix Fortification 1000 1074 107 MAS5 Matrix Fortification 1000 935 93.5 MAS-8 Matrix Fortification 1000 895 89.5 MAS3 Matrix Fortification 5000 5566 111 MAS-6 Matrix Fortification 5000 4866 97.3 MAS-9 Matrix Fortification 5000 - 4571 91.4 Mean = 97.4 Standard Deviation = 7.15 cv = 7.34% N=9 Measured and Percent Recovered 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 of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (10000). WILDLIFE INTERNATIONALTLD. - 5 .. 1 - PROJECT NO.: 454A-118A Table 3 Measured Concentrations of PFBS in Freshwater Samples from a Cladoceran Static Acute Toxicity Test Nominal Test Concentration (mg a.i.L) 0.0 Sample Number (454A-l18A-) 1 2 . 13 14 25 26 Sampling Time (Day) 0 0 1 1 2 2 PFBS Measured Concentration' (mg a.i./L) < LOQ' < LOQ < LOQ < LOQ -= LOQ LOQ Percent of Nominal' -- 250 3 0 4 0 15 1 16 1 27 2 28 2 240 96.1 240 96.0 233 93.2 _. 235 94.0 227 90.7 23 1 92.3 500 5 0 6 0 17 1 18 1 29 2 30 2 478 95.6 46 1 92.2 476 95.1 496 99.2 45 1 90.3 456 91.3 1000 7 0 858 85.8 8 0 850 85.0 19 1 92 1 92.1 20 1 923 92.3 31 2 862 86.2 * Measured and Percent 3o2f Nominal values 2were calculated using 9M02acQuan, version 1.69s0o.f2tware. Manual calculations may vary slightly. 2 The limit of quantitation (LOQ) was 100 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (10000). WILDLIFE INTERNATIONALIDL. - 32 - PROJECT NO.: 454A-118A Table 3 (Continued) Measured Concentrations of PFBS in Freshwater Samples from a Cladoceran Static Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 2000 Sample Number (454A-l18A-) 9 10 21 22 33 34 Sampling Time (Day) 0 0 1 1 2 2 PFBS Measured Concentration' (mg a.i./L) 1661 1738 1770 1842 1588 1640 Percent of Nominal' 83.1 86.9 88.5 92.1 79.4 82.0 40003 11 0 3592 89.8 12 0 375 1 93.8 23 1 3835 95.9 24 1 3888 97.2 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 100 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (10000). This treatment group was not sampled on Day 2 due to 100%mortality on Day 1. WILDLIFEINTERNATIONLTAD.L PROJECT NO.: 4.5412-118A METHOD OUTLINE FOR 'THE ANALYSIS OF PFBS IN FRESHWATER Prepare each matrix fortification sample by weighng 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 weighmg 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 withinthe 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 Itxel. Mix well by several repeat inversions. Figure 1. Ampulate samples and submit for LCMS analysis. ~~ ~~ Analytical method flowchart for the analysis of PFBS in freshwater. WILDLIFE INTERNATIOLNTD A. L - 34 - Area PROJECT NO.: 454A-118A 0~~~~111l1,,,,,,,,.,,,,..,,, 0.0 10 20 30 40 50 Figure 2. A typical calibration curve for PFBS. Slope = 13470852; Intercept = 44521.44141; r = 0.9987 WILDLIFE INTERNATIONLTAD.L - 35 - PROJECT NO.: 454A-118A 6 "420 134 '1 scan 0 ,161 ,35, I 67 I I 8, 2 97 I I , 31 61 91 121 151 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 INTERNATIOLNTD A.L -36- PROJECTNO.: 454A-118A intensity: 120000 cps 134 60- 50. 40. 30. 20lo. 1 6 2 7 47 6978 91 114 04 1 I I I I I I 1 1 I I , Figure 4. A representative ion chromatogramof a I@-level (0.0500 mg a.i./L)PFBS standard. WILDLIFE INTERNATIOLNTDA. L - 37 - PROJECT NO.:454A-118A intensity: 120000 cps d 3 26 I 1 -scan 04 1I22I 2 I 40I I55 I661 812 1 ,106I 134 165 31 0.52 61 1.02 91 121 151 1.52 2.03 2.53 Time Figure 5 . A representative ion chromatogram of a matrix blank sample (454A-118A-MAB-3). The arrow indicates the retention time of PFBS. WILDLIFE INTERNATIONALTLD. -38 - PROJECT NO.: 454A-118A ~~ intensity: 120000 cps 1009080 70 60 50. 40 135 31 61 91 121 151 scan 0.52 1.02 1.52 2.03 253 Time Figure 6 . A representative ion chromatogram of a matrix fortification sample (454A-11SA-MAS-8, nominal PFBS concentration of 1000 mg a.i./L, dilution factor = 50000~). WILDLIFE INTERNATIONLTAD.L - 39 - PROJECT NO.: 454A-1 18A intensity: 120000 cps 1 135 31 61 91 121 151 0.52 LO2 1.52 203 233 Time Figure 7. A representative ion chromatogram of a test sample (454A-118A-29, nominal PFBS concentration of 500 mg a.i./L, dilution factor = 25000~). ' WILDLIFE INTERNATIONAL,LTD. - 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 of the test chambers. 3. The protocol was amended to change the test conctntrations. I, I WILDLIFE INTERNATIONLATLD, . PROJECT NO.: 454A- 118A Appendix 5 Personnel Involved in the Study The followingkey Wildlife International, Ltd. personnel were involved in the conduct or management of this study: 1. Henry 0.Krueger, PbD., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Director, Analytical Cht:mistry 3. Kurt R. Drottar,Senior Biologist 4. Raymond L. Van Hoven, PbD., Scientist 5. Cary A. Sutherland, Laboratory Supervisor 6 . Molly McCoy, Biologist