Document LJ5qLKpxkNn8Z6zZK886vVxO7

19) OPPTS 850.1035,96 hour Static acute toxicity with (saltwater mysid), 454A-128 PFBS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE SALTWTER MYSID (Mysicfopsisbahia) FINAL REPORT SANiTlZED DEC 0 9 2003 WILDLIFE MTERNATIONAL, LTD. PROJECT NUMBER: 4541-3-128 3M EnSionmental Lab Project No. E00-1429 U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1035 AUTHORS: Kurt R Drottar Raymond L. Van Hoven, Ph.D. Henry 0.Krueger, PbD. STUDY INITIATION DATE: October 6 , 2000 STUDY COMPLETION DATE: March 21,2001 Submined to 3M Corporation Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55144 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 2 1601 (410) 822-8600 Page 1 of 39 WildlifeInternational, L t d . Project Number 454A-128 -2- DEC 0 9 2003 SANITIZED GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFBS: A 96-Hour Static Acute Toxicity Test with the Saltwater Mysid (Mysidopsisbahia) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-128 STUDY COMPLETION: March 2 1,2001 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984, with the following exceptions: The test substance was not characterized in compliancewith Good Laboratory Practices prior to its use in the study. However, subsequent GLP compliant characterization resulted in a purity similar to the original characterization purity. The stability of the test substance under conditions of storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Kurt R Drottar Senior Biologist SPONSOR APPROVAL: DATE WildlcfeInternational, h d . -3- Project Number 454A-128 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S.Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10August 1984. The dates of all inspections and auhts and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Test Substance Preparation Matrix Fortification Observations Biological Data and Draft Report Analytical Data and Draft Report Final Report DATE CONDUCTED: January 29,2001 January 29,2001 February 2,2001 February 15 and 16,2001 February 15 and 16,2001 March 21,2001 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: January 29,2001 February 1,200 1 January 29,2001 January 3 1,2001 February 2,2001 February 8,2001 February 16,2001 February 16,2001 February 16,2001 March 21,2001 February 16,200 1 March 21,2001 `Robert N. McGee Quality Assurance Representative Az-.+ DATE 2 / , zoo / wildlifeInternational, Ltd. -4- REPORT APPROVAL Proiect Number 454A-128 SPONSOR: 3M Corporation TITLE: PFBS: A 96-Hour Static Acute Toxicity Test with the Saltwater Mysid (Mysidopsis bahia) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A- 128 STUDY DIRECTOR: . Kurt R DroW Senior Biologist MANAGEMENT: Director, Aquatic Toxicology and Non-Target Plants WildlcfeInternational. L t d. -5- Project Number 454A-128 TABLE OF CONTENTS TitleXover 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 ...................................................................................................................................... 8 Results and Discussion.................................................................................................................................... 12 Conclusions ..................................................................................................................................................... 12 .. References ....................................................................................................................................................... 13 TABLES Table 1 .Summary of Analytical Chemistry Data......................................................................................... 14 Table 2 - Temperature, Dissolved Oxygen and pH of Water in the Test Chambers..................................... 15 Table 3 - CumulativePercent Mortality and Treatment-Related Effects ...................................................... 16 Table 4 - LC50 Values ................................................................................................................................... 17 . WildlifeInternational, L t d . -6- Proiect Number 454A-128 TABLE OF CONTENTS - Continued - APPENDICES Appendix 1 - Salinity and pH of SaltwaterMeasured During the 4-Week Period Immediately 'Preceding the Test ........................................................................................... 18 Appendix 2 - Analyses of Pesticides, Organics and Metals in Wildlife International,Ltd. Saltwater.............................................................................. 19 Appendix 3 - The Analysis of PFBS in Filtered Saltwaterin Support of Wildlife International, Ltd. Project No.: 454A-128 ......................................................................... 21 Appendix 4 - Changes to Protocol............................................................................................................. 38 Appendix 5 - Personnel Involved in the Study .......................................................................................... 39 wildlife hternationul, Ltd. -7- Project Number 454A-128 SANITIZED SPONSOR' SPONSOR'S REPRESENTATIVE: . LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: 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: 96-HOUR LC50: 95% CONFIDENCELIMITS: NO MORTALITY CONCENTRATION: NO-OBSERVED-EFFECTCONCENTRATION: SUMMARY 3M Corporation DEC 0 9 2003 Wildlife International, Ltd. Easton, Maryland 2 1601 454A- 128 Peffluorobutanesulfonate, Potassium Salt (PFBS) PFBS: A 96-Hour Static Acute Toxicity Test with the Saltwater Mysid (Mysidopsisbahia) Negative Control; 32, 64, 127,269, 554 and 1071mg a.i./L Experimental Start - January 29,200 1 Biological Termination - February 2,200 1 Experimental Termination - February 2,200 1 96 Hours Saltwater Mysid (Mysidopsisbahia) Wildlife International, Ltd. cultures Easton, Maryland 2 1601 Juveniles 372 mg a.i./L 314 and 440 mg a.i./L 127 mg a.i./L 127 mg a.i./L wildlife International, Ltd. -8- Project Number 454A-128 INTRODUCTION This study was conductedby Wildlife International,Ltd. for 3M Corporation at the WildlifeInternational, Ltd. aquatic toxicologyfacility in Easton, Maryland. The in-life phase of the testwas conductedfiomJanuary 29, 200 1to February 2,200 1. Raw data generated by Wildlife International,Ltd. and a copy of the final report are filed under Project Number 454A-128 in archves located on the Wildlife International, Ltd. site. OBJECTIVE The objective of this studywas to evaluate the acute toxicity of Perfluorobutanesulfonate,Potassium Salt (PFBS) to the saltwater mysid,Mysidopsis bahia, during a 96-hour exposure period under statictest conditions. EXPERIMENTAL DESIGN Saltwater mysids were exposed to a geometric series of six test concentrations and a negative (saltwater) control. Two replicate test chambers were maintained in each treatment and control group, with 10 mysids in each test chamber for a total of 20 mysids per test concentration. Nominal test concentrations were selected in consultation with the Sponsor, and were based upon the results of an exploratory rangefinding toxicity test. Nominal test concentrations selected were 3 1,63, 125,250,500 and 1000mg active ingredient (a.i.)/L. Mean measured test concentrations were determined Erom samples of test water collected from each treatment and control group at the beginning of the test, at approximately 48 hours and at test tennination. The mysids were indiscriminately assigned to exposure chambers at test initiation. Observations of mortality and other clinical signs were made at approximately 4,24,48, 72, and 96 hours after test initiation. Cumulative percent mortality observed in the treatment groups was used to calculate LC50 values at 24,48,72 and 96 hours. The no mortality concentration and no-observed-effect-concentration(NOEC)were determinedby visual interpretation of the mortality and clinical observation data. MATERIALSA N D METHODS The study was conducted based on the procedures outlined in the protocol, "PFBS: A 96-HourStatic Acute Toxicity Test with the Saltwater Mysid (Mysidopsisbahia)". The protocol was based on procedures outlined in U.S. Environmental Protection Agency Series 850 Ecological Effects Test Guidelines, OPPTS Number 850.1035 (1); U.S. Environmental ProtectionAgency, StandardEvaluation Procedure,Acute Toxicity WildlifeInternational, Ltd. -9- Proiect Number 454A-128 Testfor Estuarine andMarine Organisms (2); and ASTM Standard E729-88a Standard Guidefor Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians (3). Test Substance The test substance was received from 3M Corporation on March 27, 2000 and was assigned Wildlife International, Ltd. identification number 5216. The test substance was described as a white powder. It was identified as Potassium Perfluorobutane Sulfonate from lot number 2. Information provided by the Sponsor indicated a purity of 97.90%, and an expiration date of March 20 10. 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. Preparation of Test Concentrations Nominal test concentrations were 3 1,63, 125,250,500 and 1000mg a.i./L. Allmaterialswhichcameinto contact with the test substance during preparation of test concentrations were constructed of plastic or stainless steel. A 7-L primary stock solution was prepared in dilution water at a concentration of 1000 mg a.i.L. The primary stock solutionwas mixed with an electricmixer for approximately22 minutes to aid inthe solubilization of the test substance. After mixing, the primary stock solution was proportionally diluted with dilution water to prepare 3-L of the other five concentrations. After mixing, 1500 mL of test solution was added to the two replicate test chambers for each treatment group. All test solutions appeared clear and colorless. Test concentrations were corrected for the original reported purity of the active ingredient in the test substance (97.9%). Test Organism The saltwater mysid,Mysidopsis bahia, was selected as the test speciesfor thisstudy, The saltwatermysid is representative of an important group of aquatic marine invertebrates and was selected for use in the test based upon past history of use and ease of culturing in the laboratory. Mysids used in the test were obtained as juveniles from cultures maintained by Wildlife International, Ltd., Easton, Maryland. The identification of the species was provided by the supplier of the original brood stock. wildlife International, Ltd. - 10- Proiect Number 454A-128 Adult mysids were held at approximately the same temperature as used during the test. During the holding and acclimation periods, the adults showed no signs of disease or stress. Dunng the 14-day holding period precedmg the test, water temperatures ranged from 24.6 to 25.9"C. The pH of the water ranged from 8.0 to 8.2, salinity ranged from 20 to 21%0 (parts per thousand) and dissolved oxygen ranged from 6.6 to 7.0 mgL. Instrumentationused for water measurements is described in the EnvironmentulConditionssectionof this report. At test Initiation, the juvenile mysids were carefully collected from the cultures and indiscriminately distributed one to two at a time into the test chambersuntil each chambercontained 10mysids. Thejuvenilemysidswere fed live brine shrimp (Artemiu sp.) nauplii daily during the test to prevent cannibalism. Test Apparatus Test chambers were 2-L polyethylene buckets filled with 1500 mL of test solution. The depth of the test water in a representative test chamber was approximately 8.8 cm. Test chamberswere impartiallypositionedin a temperature-controlledwater bath set to maintain a temperature of 25*2"C. The water bath was covered with a plexiglass hood to reduce the potential for cross-contamination. The test chambers were labeled with the project number, test concentration and replicate. Dilution Water The water used for holding, acclimation and testing was natural seawater collected at Indian River Inlet, Delaware, and diluted to a salinity of approximately 20%0 with well water. Salinity and pH measurements taken .. / during the four-weekperiod immediately preceding the test are presented in Appendix 1. The freshly-collected seawater was passed through a sand filter to remove particles greater than approximately 25 pn, and pumped into a 37,800-L storage tank. The filtered seawater then was diluted with freshwater from a well on the Wildlife International, Ltd. site and aerated with spray nozzles. Prior to use, the water again was filtered (0.45 p)to remove microorganisms and particles. The results of periodic analyses performed to measure the concentrationsof selected contaminantsin saltwaterused by WildlifeInternational,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 controlledwith an automatic timer. A 30-minutetransition period WildlifeInternational, Ltd. - 11 - Project Number 454A-128 of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity at test initiation was approximately 159 lux at the surface of the water. Light intensity was measured using a SPER Scientific Ltd. light meter. Temperature was measured in each test chamber at the beginning and end of the test using a liquid-in-glass thermometer. Temperature also was measured continuously in one negative control replicate using a Fulscope ElUC Recorder. The target test temperature during the studywas 25k2"C. Dissolvedoxygenmeasurementswere made on water samples collected from all replicate test chambers of each treatment and control at test initiation, and at approximately 24-hour intervals thereafter. Measurements of pH were made in alternating replicates of each treatment and the control group at test initiation and at approximately 24-hour intervals thereafter. Salinity was measured in the dilution water at test initiation. Measurements of pH were made using a Fisher Accumet Model 9 15 pH meter, and dissolved oxygen was measured using a Yellow Springs InstrumentModel 5 1Bdissolved oxygen meter. Salinitywasmeasuredusing a Bio-marine, Inc. Aquafauna refractometer. Observations Observations were made to determinethe number of mortalities. The number of individualsexhibiting clinical signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately 4, 24,48, 72 and 96 hours after test initiation. Statistical Analyses The 24,48,72 and 96-hour LC50values and the 95% confidenceintervalswere calculatedwhenpossible by probit analysis, the moving average method or binomial probability withnon-linearinterpolation(4,5,6)using the computer software of C.E. Stephan (7). In this study, the binomial method was used to calculatethe 24-hour LC50 value and the probit method was used to calculate the 48,72 and 96-hour LC50values. The no mortality concentrationand NOEC were determined by visual interpretation of the mortality and clinical observationdata. Analytical Chemistry Water samples were collected at mid-depth from each replicate test chamber of each treatment and the control group at the beginningof the test, at 48 hours and at test termination to measure concentrationsof the test wildlife International, Ltd. - 12- Proiect Number 454A-128 substance. The samples were collected in plastic scintillation vials and analyzed as soon as possible without storage. Analytical procedures used in the analysis of the samples are provided in Appendix 3. RESULTS AND DISCUSSION Measurement of Test Concentrations Results of analyses to measure concentrations of PFBS in water samples collected during the test are presented in Table 1and in the analyticalchemistry report (Appendix 3). Nominalconcentrationsselectedfor use in t h ~ study were 31, 63, 125,250,500-and 1000mg a.i./L. Samples collected at test initiation had measured values that ranged from 99 to 110%of nominal values. Measured values for samples taken at 48 hours ranged from 99 to 112% of nominal. Measured values for samples taken at 96 hours ranged from 102 to 112% of nominal. When measured concentrations of the samples analyzed at test initiatioq approximately48 hours and at test termination were averaged,the mean measured concentrationsfor this study were 32,64,127,269,554 and 1071 mg a.i./L. Mean measured concentrations were used in the calculation of LC50 values. Observations and Measurements Measurements of temperature, dissolved oxygen and pH are presented in Table 2. Temperatures were withinthe 25i2"C range established for the test. Dissolved oxygen concentrationsremained 26.5 m& (88%of saturation)throughout the test. Measurements of pH ranged from 8.0 to 8.3 during the test. Daily observations ofmortality and other clinical signs of toxicity observedduring the test are shownin Table 3. Mysids in the negative control appeared normal and healthy during the test. Mysids in the 32,64 and 127 mg a.i./L treatment groups also appeared normal and healthy during the test. M e r 96-hours of exposure, mortality in the 269,554 and 1071mg a.i./L treatment groups was 15,90 and loo%, respectively. LC50 values and 95% confidence limits at 24,48,72 and 96 hours were calculatedfrom the mortality data, and are shown in Table 4. CONCLUSIONS The 96-hour LC50 value for saltwater mysids (Mysidopsisbahia) exposed to Perfluorobutanesulfonate, Potassium Salt (PFBS) was 372 mg a.i./L. The 95% confidencelimiG were 3 14 and 440 mg a.i./L, and the slope of the concentration-response curve was 7.4. The 96-hour no mortality concentration and the NOEC were 127 mg a.i./L. wi1d1(fe InternationaI , Ltd. - 13,- Proiect Number 454A-128 REFERENCES 1 U.S. Environmental Protection Agency. 1996. Series 850 -Ecological Effects Test Guideilnes(draj), OPPTS Number 850.1035: MysidAcute Toxicity Test. 2 U.S. Environmental Protection Agency. 1985. Standard Evaluation Procedure, Acute Toxicity Test for Estuarine and Marine Organisms (Shrimp 96-Hour Acute Toxicity Test). Hazard Evaluation Division. Office of Pesticide Programs. EPA 540/9-85-0 10. Washington, D.C. 3 ASTM Standard E729-88a. 1994. Standard Guidefor ConductingAcute Toxicity Tests with Fishes, Macroinvertebrates; and Amphibians. American Society for Testing and Materials. 4 Thompson, W.R 1947. Bacteriological Reviews, Vol. 11,No. 2. Pp. 115-145. 5 Stephan, C.E. 1977. "Methodsfor Calculatingan LC50",Aquatic Toxicology andHazardEvaluations. American Society for Testing and Materials. Publication Number STP 634, pp 65-84. 6 Finney, D.J. 1971. Statistical Methoh in Biological Assay. Second edition. Griffin Press, London. 7 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. .~ wildltfe International, Ltd. - 14- Proiect Number 454A- 128 Table 1 S6mrnary of Analytical Chemistry Data Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Saltwatermvsid. Mvsido'osis bahia Filtered Salkater ' Nommal Test C(omncaean.tir.a/Ltio) n Negative Control Replicate A B A B A B g Time 0 0 48 48 96 96 31 A 0 B 0 A 48 B 48 A 96 B 96 63 A 0 B 0 A 48 B 48 A 96 B 96 125 A 0 B 0 A 48 B 48 A 96 B 96 250 A 0 B 0 A 48 B 48 A 96 B 96 500 A 0 B 0 A 48 B 48 A 96 B 96 1000 A 0 B 0 'The limit of quantitation (LOQ) was 10 mg a.i./L. Measured Concentration (maa.i./L) . <<LLOo% < LOQ < LOQ <-= LLOOQQ 32.8 33.4 31.7 32.4 32.4 31.9 65.9 64.1 63.9 63.5 65.1 64.3 125 124 125 124 132 131 267 264 277 267 270 266 552 54 1 558 558 560 556 1071 1070 Mean Measured Concentration (mg a.i./L) < LOQ 32 64 127 269 554 1071 _ _Percent of Nominal 103 102 102 108 111 107 - 15 - Table 2 Temperature, Dissolved Oxygen and pH of Water in the Test Chambers Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Saltwater mysid, Mysidopsis bahia Filtered Saltwater Mean Measured Test Concentration (mg a.i./L) Replicate Temp* (OC) 0 Hour' Dd ( m ~ ) PH Negative Control A 24.5 7.4 8.0 B 24.6' 7.4 24 Hours DO (m&) PH 6.8 I 6.7 8.2 48 Hours DO ( m a ) PH 6.5 8.1 6.6 I - - 32 A 24.4 7.4 8.2 6.8 - 6.6 8.2 B 24.4 7.6 6.8 8.3 6.6 - 64 A 24.5 7.5 8.2 6.8 6.6 8.2 - - B 24.5 7.5 6.8 8.3 6.5 127 - A 24.4 7.5 8.2 B 24.5 7.6 6.8 I 6.8 8.3 - 6.6 8.2 6.6 269 A B 24.5 7.6 24.5 7.6 -8.2 - 6.8 6.8 8.3 6.6 8.2 6.6 - 554 A B 24.5 7.6 24.4 7.6 -8.3 6.8 - 6.8 8.3 6.5 6.6 8-.2 1071 A B 24.4 7.6 24.5 7.7 8-.3 - 6.8 6.8 8.3 - 4 - - 4 - 'The 0-hour dilution water (negative control) measurement for salinity was 200h. 'Temperature measured continuously during the test ranged fium approximately23.0 to 255C. 'A dissolved oxygen concentration of 4.4 me/L represents 60% saturation at 25C in saltwater i t h a salinityof 200h. 'Measurements discontinued due to 100%mortality. 72 Hours DO - ( m a ) PH 6.6 6.6 8.1 6.6 - 6.6 8.1 6.6 - 6.6 8.2 6.5 - 6.6 8.2 6.6 -- 6.6 8.2 - 6.6 6.6 8.2 - I - - Project Number 454A-128 Temp ("C) 24.7 24.7 24.7 24.7 24.7 24.7 96 Hours DO 6.5 6.6 6.6 6.6 6.6 6.5 24.7 6.5 24.7 6.5 24.5 6.5 24.6 6.5 24.6 6.6 24.6 6.6 - - - - PH 8.1 - 8.2 - 8.2 - 8.2 - 8-.2 8.2 I - - - - 16- Project Number 454A-128 Table 3 CumulativePercent Mortality and Treatment-Related Effects Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Saltwater mysid, Mysidopsis buhiu Filtered Saltwater Mean Measured Test Concentration 4 Hours No. No. YO (mg ai./L) Replicate Exposed Dead' Effects* Dead Negative Control A B 10 0 1oAN 0 10 0 10 AN 32 A 10 0 1oAN 0 B 10 0 10 AN 64 A 10 0 ,1oAN 0 B 10 0 10 AN 127 A 10 0 IOAN 0 B 10 0 10 AN 269 A 10 0 10" 0 B 10 0 10 AN 554 A 10 0 1oAN 5 B 10 1 9AN 1071 A 10 1 8C.IAN 5 B 10 0 1 oc ' Observed Effects: AN = Appears Normal, C = Lethargic, E = Enatic Swimming No. Dead 0 0 0 0 0 0 0 0 0 0 1 4 10 10 24 Hours Effects 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 7AN,IE, 1C 6AN - - YO Dead 0 0 0 0 0 25 100 48 Hours No. YO Dead Effects Dead 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 0 0 10 AN 0 10 AN 5 1 9AN 3 5AN,2E 45 6 4AN 10 10 - - 100 72 Hours No YQ Dead Effects Dead 0 10" 0 0 10" 0 IOAN 0 0 IOAN 0 1oAN 0 0 1oAN 0 l0AN 0 0 1oAN 1 9AN 15 2 8AN 6 3C, 1E 65 7 3c - 10 10 - 100 96 Hours No. Dead Effects 0 IOAN 0 1oAN 0 IOAN 0 IOAN 0 1oAN 0 1oAN 0 IOAN 0 IOAN 1 9AN 2 8" 10 - 8 2c 10 10 - - Cumulative Percent Mom&' 0 0 0 0 I5 90 100 wildlife International, Ltd. - 17- Table 4 LC50 Values Sponsor: Test Substance: Test Organism: Dilution Water: Time 24 Hours 48 Hours 72 Hours 96 Hours 3M Corporation PFBS Saltwater mysid,Mysidopsis bahia Filtered Saltwater Lower 95% Confidence LC5 0 (mg a.i./L) Limits (mg a.i./L) 66 1 554 540 450 44 1 3 62 372 3 14 Upper 95% Confidence Limits (mg a.i./L) 1071 643 533 440 Project Number 454A-128 Statistical Method Binomial Probit Probit Probit wildlife International, L t d . -18- Project Number 454A-128 Appendix 1 Salinity and pH of Saltwater Measured During the 4-Week Period Immediately Preceding the Test Sponsor: Test Substance: Test Organism: Dilution Water: Salinity (%o) 3M Corporation PFBS Saltwater mysid, Mysidopsis bahia Filtered Saltwater ' Mean 20(N=4) Range 20 - 20 PH 8.1 (N = 4) 8.0 - 8.2 wildlife International, Ltd. - 19- Proiect Number 454A-128 Appendix 2 Analyses of Pesticides, Organics and Metals in Wildhfe International, Ltd. Saltwater' Component Measured Concentration Component Measured Concentration PesticidesAnd Organics Aclonifen Alachlor Ametryn Atrazine Azinphos-ethyl Azinphos-methyl Azoxystrobin Bifenthrin Bioallethrin Bitertanol Bromacil Bromophos Bromophos-ethyl Bromopropylate Bupirimate Carbaryl Carbofuran Carboxin Chlorfenvinphos Chloridazon C hlorpropham C hlorpyrifos C hlorpyrifos-methyl Chlorothalonil Coumaphos Cy anazine Cfluthrin Cypermethrin Cyproconazole Deltamethrin Demeton Demeton-O Desethylatrazine Desisopropylatrazine Desmetryn Diazinon Dichlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid 4 . 0 3pg/L 4 . 0 1 pg/L CO.01 pg/L 4 . 0 1 pg/L 4.04 pg/L 4.08pg/L 4 . 2 5 pg/L <0.05 pg/L 4 . 0 5 pg/L 4.05 pg/L ~ 0 . 0 5pg/L 4 . 0 2 pa <0.02 pg/L 4.02 pgL <0.05 pg/L 4.05 pg/L 4 . 0 3 pg/L 4 . 0 2 pg/L C0.02 pg/L ~ 0 . 0 5pg/L 4 . 0 2 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4.04 pg/L 4 . 0 2 pg/L 4.05 pg/L 4.05 pg/L d.25 pg/L 4.05p g k 4.02 pgn c0.02 pg/L 4 . 0 2 pg/L 4 . 0 1 pg/L 4 . 0 2 pg/L 4.01 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4 . 0 3 pg/L <0.02 pg/L 4 . 0 1 pg/L 4.03 pg/L Dichlorvos Dicofol Diethyltoluamide Difenoconazole Dimethoate Dimethomorph Disulfoton DMST Dodemorph Endosulfana Endosulfan-p Endosulfan-sulfte Epoxiconazole Eptam Esfenvalerate Ethion Ethofumesate Ethoprophos Etridiazole Etrimfos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb Fenpiclonil Fenpropathrin Fenpropirnorph Fenthion Fenvalerate Fluazifop-butyl Fluoroglycofen-ethyl Fluroxypyr-meptyl Flutolanil Fonophos Furalaxyl Heptenophos Imazalil Iprodion Kresoxim-meth yl Lenacil Lindane 4 . 0 1 pg/z 4.25 pg/L 4 . 0 2 pg/L 4 . 0 3 pg/z <0.02 pg/L 4.05 pg/L 4 . 0 2 pglL 41.05 pg/L 4 . 0 1 pg/L CO.01 pg/L 4 . 0 1 pg/L 4 . 0 2 pg/L 4.05 pg/L 4.02 pgn <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 p g n 4 . 0 2 pg/L 40.05 pg/L 4 . 0 5 pglL 4.01 pglL 4 . 0 3 pg/L 4.03 pg/L 4 . 0 5 pg/L d.25 pg5, 4.01 pglL 4.01 pg/L 4.02pg/L 4 . 0 2 pg/L 4 . 0 2 pg/L ~ 0 . 0 5p g L 4 . 0 2 pg/L 4.01 pglL 4.02 pgn 4 . 0 2 pg/L 4.01 pgn 4 . 0 5 pg/L 4 . 0 2 pg/L 4.05 pg/L 4 . 0 2 pg/L 'Analyses performed by " 0 Nutrition and Food Institute on samplescollected on October 14 and 15,1999. wildlife International, Ltd. - 20 - Proiect Number 454A-128 Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd.Saltsvat&' Page 2 Pesticides And Organics Component Measured Concentration Component Measured Concentration Malathion Metalaxy l Metarnitron Metazachlor Methidathion Paclobutazole Parathion Parathion-methyl Penconazole Pendimethalin Permethrincis Permethrin-trans Phosalone Phosmet Phosphamidon-cis Pirimicarb Pirirniphos-ethyl Pirirniphos-methyl Prochloraz Procymidon Prornetryn Propachlor Propazine Propham Propiconazole Propxur Propyzamide Prosulfocarb F'yrazophos 4 . 0 2 pg/L 4 . 0 5 pg/L (0.05 pg/L 4 . 0 2 pglL c0.02 pa (0.05 pglL <0.01 pg/L 4 . 0 1 pg/L (0.05 pg/L 4.03pg/L 4.01 pgL 4.01 pgL (0.05 pg/L 4 . 0 2 pg/L 4 . 0 5 pg/L <0.01 pg/L 4.01 pgn 4 . 0 1 pg/L 4.02 pgn (0.01 pa 4 . 0 1 pg/L <0.01 pg/L 4 . 0 1 pg/L 4.02 pgn 4.05 pglL 4.03 pg/L 4.02 pgn 4 . 0 2 pg/L 4.03 pfl Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl Pyrifenox-1 Pynfenox-2 Pyrimethanil Quizalofop-ethyl Simazine Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbuthylazine Tetrachlorvinphos Tetrahydroftalirnide Tetramethrin Thiabendazole Thiorneton Tolclofos-methyl Tolylfluanid Triadimefon Triadimenol Triallate Triazophos Trifluralin Vamidothion Vinclozolin 4 . 0 1 pg/L 4 . 0 1 pg/L 4 . 0 2 pg/L <0.01 pg/L 4 . 0 5 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4 . 0 2 pg/L 4 . 0 1 pg/L 4 . 0 2 pg/L ~ 0 . 0 5pg/L 4 . 0 5 pg/L 4 . 0 1 pg/L <0.01 pg/L (0.01 pg/L 4.05 pg/L 4 . 0 1 pg/L 4.05 pg/L 4.04 4.ni pgn 4.04 Pg/L 4.05 pgn (0.05 pg/L 4 . 0 2 pg/L 4.02 pgn 4 . 0 2 pg/L co.01 p g n 4 . 0 1 pg/L Metals Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 520 m@ 4,400rng/L 181 rng/L 4.015 rng/L 3.6 mg/L (0.02 mgL (1.0 p g n Q.0 Pg/L 11.5 pg/L Q.0 pglL Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium (11 Pgn c7sl Pg/L (5.0 pglL 4 . 0 Pgn Q.0 Pgn 4 . 0 pg/L 0.7 Pgn 4.025 pgk 4)s Pg/L `Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15, 1999. wildlife hternationul,k d . -21 - Appendix 3 Proiect Number 454A-128 THE ANALYSIS OF PFBS JN FILTERED SALTWATER IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-128 WILDLIFE INTERNATIONAL LTD. - 22 - PROJECT NO.: 454A-128 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFBS: A 96-HOUR STATIC ACUTE TOXICITY TEST with the SALTWATER MYSID (Mysidopsisbahia) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-128 3M ENVIRONMENTAL LAB PROJECT NUMBER: E00-1429 PRINCIPAL INVESTIGATOR: .. Scientist MANAGEMENT: ' Willard B. Nixon, Ph.D. Director, Analytical Chemistry 03-21 -01 DATE WILDLIFE INTERNATIONALLTD. - 23 - PROJECT NO.: 454A-128 Introduction Saltwater samples were collected from a static acute aquatic toxicity study designed to determine the effects of PFBS (Perfluoro Butane Sulfonate, Potassium Salt) to the saltwater mysid (Mysidopsisbahia). This study was conducted by Wildlife International, Ltd. and identified as Project Number 454A- 128. The analyses of these water samples were performed at Wildlife International, Ltd. using high performance liquid chromatography with mass spectrometric detection O L C / M S ) . Samples were received for analysis on January 29, 31, and February 2, 2001. The submitted samples were prepared for analysis on each sample receipt day. Analyses were completed on the three sampling intervals on January 30, February 1, and February 2, 200 1, 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 condtions. The analytical standard, a white powder, was identified as: Potassium Perfluorobutane Sulfonate Lot 2), expiration date: March 2010. The analytcal standard was further identified with the 3M Environmental Laboratory test control and reference number TCR The test substance had a reported purity of 97.90%. A subsequent revision of the certificate of analysis indicated a purity of 97.3% and an ExpiratiodReassessment Date of January 17, 2002. The analyt~casltandard was the same material and lot number as the test substance. The analytical standard was used to prepare calibration and matrix fortification samples. Analytical Method Water samples were analyzed according to the method entitled "Analytical Method Validation for the Determination of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Saltwater and Algal Media" (Wildlife International, Ltd. Project No. 454C-117). Samples were diluted in a 50% methanol : 50% NANOpure@water solution so that they fell within the calibration range of the PFBS methodology. Aliquots of the dilutions were transferred to autosampler vials and submitted for analysis by direct injection. Concentrations of PFBS in saltwater samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) interfaced with a Perkin-Elmer API 3000 mass spectrometer operated in selective ion monitoring (SIM) detection mode. The mass spectrometer was equipped with a Perkin-Elmer WILDLIFE INTERNATIONALTLD. - 24 - PROJECT NO.: 454A-128 TurboIonSpray ion source. Chromatographic separations were achieved using a Keystone PRISM RP column (30 mm x 1.5 mm, 3 - p particle size) fitted with a Keystone Javelin CI8Guard Cartridge (20 mm x 2 mm). The instrument parameters are summarized in Table 1 and a method flowchart is provided in Figure 1. Priman and Secondaw 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 a final volume of 100 mL with methanol. Secondary stock solutions (1000, 100, 10.0, 1.00, and 0.100 mg a.i./L) of PFBS in methanol were prepared by serial volumetric ddution from the primary stock. Calibration Standards and Calibration Curves Calibration standards were prepared in 50:50 methanol: NANOpure@water by appropriate dilutions of the 10.0 mg a.i./L stock solution of PFBS in methanol. The calibration standards of PFBS, ranging in concentration 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 injected, at a minimum,after every five samples. Linear regression equations were generated using the peak area responses versus the respective concentrations of the calibration standards. A typical calibration curve is presented in Figure 2. The concentration of PFBS in the samples was determined by substituting the peak area responses into the applicable linear regression equation. Representative ion chromatograms of low and high calibration standardsare presented in Figures 3 and 4, respectively. Limit of Ouantitation The method limit of quantitation (LOQ) for these analyses was set at 10.0 mg a.i./L calculated as the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (1000). ~~~ WILDLIFE INTERNATIONALIDL. - 25 - PROJECT NO.: 454A-128 Matrix Blank and Fortification Samules 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. Saltwater was either fortified with the appropriate PFBS stock solution in methanol (15.0 and 200 mg a.i./L), or directly fortified ( i e . without use of carrier solvent) with PFBS (1250 mg a.i./L). The fortified samples were analyzed concurrently with the test samples to determine the mean procedural recovery (Table 2). Sample concentrations were not corrected for the mean procedural recovery of 109%. A representative ion chromatogram of a matrix fortification is presented in Figure 6. Examule Calculations Sample number 454A-128-9, nominal concentration of 250 mg a.i./L in saltwater. First Initial Volume: 0.100 mL Calibration curve equation: First Final Volume: 10.0 mL Slope: 713895744 Second Initial Volume: 0.100 mL Intercept: 678 1268 Second Final Volume: 10.0 mL Curve regression weighted l/x Dilution Factor: 10000 PFBS Peak Area: 25861574 - e& area (y-intercept) PFBS (mg a.i./L) measured at instnunent = P slope PFBS (mg a.i.4,) in sample = PFBS measured at instrument (mg a.i./L) x dilution factor - 25861574 - 6781268 loooo - 713895744 = 267 WILDLIFE INTERNATIONALIDL. - 26 - PROJECT NO.: 454A-128 PFBS m a.i./L in sam le Percent of Nominal Concentration = pFBs((:g nomin:l x 100 _-267 x 100= 107% - 250 Calculated with HpLC/MS instrument software: MacQuan, version 1.6. Sample Analysis RESULTS Saltwater samples were collected from a static acute toxicity study with the saltwater mysid (M'sidopsis bahia) at test initiation, January 29, 2001 (Day 0), on January 31, 2001 (Day 2), and at test termination, February 2, 2001 (Day 4). The measured concentrations of PFBS in the samples collected at initiation of exposure of the test organisms (Day 0) ranged from 98.9 to 110% of the nominal concentrations. Samples collected at Day 2 had a measured concentration range of 99.4 to 112% of nominal values. Samples collected at test term-ination (Day 4) had a measured concentration range of 102% to 112% of nominal values (Table 3). A representative ion chromatogram of a test sample is shown in Figure 7. WILDLIFE INTERNATIONLATLD. -27- PROJECT NO.: 454A-128 Table 1 Typical HPLCMS Operational Parameters ~ INSTRUMENT: ~~ ~ Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 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 CIScartridge (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 2.1 minutes .. PFBS MONITORED MASS: 299.0 m u W I L D L I F E INTERNATIONAL LTD. -28 - PROJECT NO.: 454A-128 Table 2 Matrix Blanks and Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A- 12%) 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 15.0 16.4 109 MAS-4 Matrix Fortification 15.0 18.9 1263 MAS-7 Matrix Fortification 15.0 18.0 120 MAS-2 Matrix Fortification 200 218 109 MAS-5 Matrix Fortification 200 219 110 MAS-8 Matrix Fortification 200 2 14 107 MAS-3 Matrix Fortification 1250 1348 108 MAS -6 Matrix Fortification 1250 1306 105 MAS-9 Matrix Fortification 1250 1325 106 Mean = 109 Standard Deviation = 4.65 CV = 4.26% N=8 1 Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 0 'The limit of quantitation (LOQ) was 10.0 mg a.i./Lbased upon the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (1000). Recovery outside 80-120% (suspect fortification or contamination). Sample not included in the statistical analysis of the data. WILDLIFE INTERNATIONALTLD. - 29 - PROJECT NO.: 454A-128 Table 3 Measure1 Concentrations of PFBS in Saltwater Samp,ds from a Mysid Static Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 0.0 Sample Number (454A-128-) 1 2 . 15 16 27 28 Sampling Time (Day) 0 0 2 2 4 4 PFBS Measured Concentration' (mg a.i./L) < LOQ' < LOQ < LOQ < LOQ < LOQ < LOQ Percent of Nominal' ---- -- --- 31 3- 4 17 18 29 30 32.8 106 33.4 108 31.7 102 32.4 104 32.4 105 31.9 103 63 5 0 6 0 19 2 20 2 31 4 32 4 65.9 105 64.1 102 63.9 101 63.5 101 65.1 103 64.3 102 125 7 0 125 100 8 0 124 98.9 21 2 125 100 22 2 124 99.4 33 4 132 106 ' 34 4 13 1 105 Measured and Percent of Nominal values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. T h e limit of quantitation (LOQ) was 10.0 mg a . i L based upon the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (1000). W I L D L I F EINTERNATIONALTLD. PROJECT NO.: 454A-128 Table 3 (Continued) Measured Concentrations of PFBS in Saltwater Samples from a Mysid Static Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 250 Sample Number (454A-128-) 9 10 .23 24 35 36 Sampling Time (Day) 0 0 2 2 4 4 PFBS Measured Concentration' (mg a.i./L) 267 264 277 267 270 266 Percent of Nominal' 107 106 111 107 108 I07 500 11 0 12 0 25 2 26 2 37 4 38 4 552 110 54 1 108 558 112 558 112 5 60 112 556 111 1000 13 0 14 0 1071 107 1070 107 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 10.0 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (1000). WILDLI FE INTERNATIONALLTD. -31 - PROJECT NO.: 454A-128 METHOD OUTLINE FOR THE ANALYSIS OF PFBS IN SALTWATER Prepare matrix fortification samples in saltwater as follows: For target PFBS concentrations 2 200 mg a.i./L, fortify saltwater with the appropriate stock solution of PFBS. For target PFBS concentrations > 200 mg a.i./L, prepare by weighing the requisite amount of PFBS test substance on an analytical balance and transferring directly into a Class A volumetric flask partially filled with saltwater. Rinse weighmg paper and the sides of the flask with repeat saltwater rinses. Swirl the flask to dissolve the test substance and then bring to h a 1 volume with saltwater. Sonicate as appropriate and mix with several repeat inversions. The matrix blank is unfortified saltwater. Prepare appropriate dilutions of study and QC samples to within the calibration range of the PFBS methodology: Partially fill Class A volumetric flasks with 50% methanol : 50% NANOpure@water dilution solvent. Add the appropriate volume of sample and bring to volume with dilution solvent. Perform secondary dilutions as necessary. Process matrix blank samples using the same dilution and aliquot volume as for the lowest fortification level. Mix well by several repeat inversions. Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis of PFBS in saltwater. ~~ WILDLIFE INTERNATIONALTLD. Area I PROJECT NO.: 454A-128 0 10 20 30 40 so concentration pga.ifL Figure 2. A typical calibration curve for PFBS. Slope = 713895744; Intercept = 6781268; r = 0.99625 WILDLIFE INTERNATIOLNTDA. L - 33 - PROJECT NO.: 454A-128 intensity: 5000000 cps 1 Ob 90. 80- 70. 6@ 50- 40. snl 125 0.52 1.02 2,03 2.53 Time Figure 3. A representative ion chromatogram of a low-level (0.0100 mg a.i./L) PFBS standard. WILDLIFE INTERNATIONALTLD. -34- PROJECT NO.: 454A-128 , intensity: 5000000 cps 0.52 1.02 1.52 2.03 2.53 Time Figure 4. A representative ion chromatogram of a high-level (0.0500 mg a.i./L) PFBS standard. WILDLIFE INTERNATIONALTLD. - 35 - PROJECT NO.: 454A-128 intensity: 5000000 cps 100- 90- 80- 7c- 60- 50. 40. 30- 20. 10. O J6 l 1l5 l27l , 39, , , , 6, 6, , , 8,8 , , , , 125 31 61 91 121 0.52 1.02 1.52 2.03 161 151 smn 2.53 Time Figure 5. . A representative ion chromatogram of a matrix blank sample (454A-128-MAB-1). The arrow indicates the retention time of PFBS. WILDLIFE INTERNATIONALTLD. -36- PROJECT NO.: 454A-128 intensity: 5000000 cps 100 90 80 70 60 50 124 40 30 20 10 0.52 1,02 I .52 2.03 2.53 Time Figure 6. A representative ion chromatogram of a matrix fortification sample (454A-128-MAS-2, nominal PFBS concentration of 200 mg a.i./L, dilution factor = 10000~). WILDLIFE INTERNATIONLATLD. - 37 - PROJECT NO.: 454A-128 intensity: 5000000 cps 5 4 3 2 1 31 61 91 121 151 scan 0.52 1,02 1.52 2.03 2.53 Time Figure 7. A representative ion chromatogram of a test sample (454A-128-9, nominal PFBS concentration of 250 mg a.i./L,dilution factor = 1OOOOx). wildlifeInternational, Ltd. - 38 - Project Number 454A-128 Appendix 4 Changes to Protocol Ths study was conducted in accordancewith 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. Water samples were not collectedfrom the 1071mg a.i./L treatment group on Day 2 of thetestdueto 100% mortality on Day 1. f I,' i r- wildlifeInternational, k d . - 39 - Project Number 454A-128 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, PbD., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, PbD., Manager, Analytical Chemistry 3. Cary A. Sutherland, Laboratory Supervisor 4. Raymond L. Van Hoven, PbD., Scientist 5 . Kurt R Drottar, Senior Biologist 6. Amy S. Blankinshp, Biologist -. I