Document dE4oRD4aZBGXJRmpo0ry9nJ6

PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE RAINBOW TROUT (Oncorhynchusmykiss) FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A- 145 ENVIRONMENTAL LABORATORYPROJECT NUMBER: U2723 U. S Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1075 and OECD Guideline 203 AUTHORS: Susan J. Palmer Raymond L. VanHoven, Ph.D. Henry 0.Krueger, PbD. STUDY INITIATION DATE: May 16,2001 STUDY COMPLETION DATE: January 7,2002 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55 106 Wildlij2International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 42 WildlifeInternational, Ltd. -2- Project Number 454A-145 GOOD LABORATORY PRACIWE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: Perfluorooctanesulfonate,Potassium Salt (PFOS): A 96-HourStaticAcute Toxicity Testwith the Rainbow Trout (Oncorhynchusmykiss) WILDLIFE INTERNATIONAL,LTD.PROJECT NUMBER 454A-145 STUDY COMPLETION: January 7,2002 This studywas conductedin compliancewithGoodLaboratoryPractice Standardsaspublishedby the U.S. Environmental ProtectionAgency in 40 CFR Parts 160and 792,17 August 1989;OECDPrinciplesof Good LaboratoryPractice(ENVNCICHEM (98) 17); and JapanMAFF, 59NohSan,NoScationNo. 3850, Agricultural Production Bureau, 10 August 1984. STUDY DIRECTOR: Senior Biologist 1' SPONSOR APPROVAL: , - 1 . - I WildlifeInternational, Ltd. -3- Project Number 454A-145 QUALITY ASSURANCE STATEMENT This studywas examinedfor compliancewith Good Laboratory PracticeStandardsaspublishedby the U.S.Environmental Protection Agency, 40 CFR Parts 160and 792,17 August 1989;OECD Principles of Good LaboratoryPractice (ENV/MC/CHEM(98) 17); and JapanMAFF,59NohSan, NotificationNo.3850, AgriculturalProduction Bureau, 10August 1984. Thedates of all inspectionsand audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Test Substance Preparation Matrix Fortification,Analytical Sampling and Test Initiation Analytical Data and Draft Report BiologicalData and DraftReport Final Report DATE CONDUCTED: June 8,2001 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: June 8,2001 June 13,2001 June 11,2001 July 11 & 12,2001 July 12,2001 January 7,2002 June 11,2001 July 12,2001 July 12,2001 January 7,2002 June 14,2001 July 13,2001 July 13,2001 January 7,2002 Quality Assurance Program Supervisor I I Wildlife International, Ltd. -4- Project Number 454A- 145 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: Perfluorooctanesulfonate,Potassium Salt(PFOS): A 96-HourStaticAcute Toxicity Test withthe Rainbow Trout (Oncorhynchusmykiss) WJLDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-145 STUDY DIRECTOR: DA Senior Biologist WILDLIFE INTERNATIONAL, LTD. MANAGEMENT: I Henry f l k u e g e r , !h.D. Director, Aquatic Toxicology and Non-Target Plants Wildli$e International. Ltd. -5- Project Number 454A- 145 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 Obj.ecti.ve........................................................................................................................................................ 8 Experimental Design ..................................................................................................................................... 8 Materials and Methods .................................................................................................................................. 9 Test Substance ................................................................................................................................. 9 Preparation of Test Concentrations ................................................................................................. 9 Test Organism.................................................................................................................................. 9 Test Apparatus............................................................................................................................... 10 Dilution Water ................................................................................................................................ 10 Environmental Conditions ............................................................................................................. 11 Observations .................................................................................................................................. 12 StatisticalAnalyses........................................................................................................................ 12 Analytical Chemistry...................................................................................................................... 12 Results and Discussion................................................................................................................................ 12 Measurement of Test Concentrations ............................................................................................ 12 Observations and Measurements ................................................................................................... 13 Conclusions ................................................................................................................................................. 13 References.................................................................................................................................................... 14 Wild1ife Intemationa1, Ltd. -6- Project Number 454A-145 TABLE OF CONTENTS - Continued - TABLES AND FIGURES Table 1. Summary of Analytical Chemistry Data .................................................................................... 15 Table 2. Temperature,Dissolved Oxygen and pH of Water in the Test Chambers................................ 16 Table 3. CumulativePercent Mortality and Treatment-RelatedEffects.................................................. 17 Table 4. LC50 Values............................................................................................................................... 18 Figure 1. Concentration-ResponseCurve (96-Hour Data) ........................................................................ 19 APPENDICES Appendix 1. SpecificConductance, Hardness,Alkalinity, and pH of Well Water Measured During the 4-Week Period Lmmediately Preceding the Test .............................. 20 Appendix 2, Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water................................................................................................. .......................... 21 Appendix 3; The Analysis of PFOS in Freshwaterin Support of Wildlife International,Ltd. ProjectNo.: 454A-145....................................................................................................... 23 Appendix 4. Changes to Protocol.............................................................................................................41 Appendix 5. Personnel Involved in the Study.......................................................................................... 42 WildlifeInternational, Ltd. -7- Project Number 454A-145 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATAAND A COPY OF THE FINAL REPORT: SUMMARY 3M Corporation Ms. Rochelle Robideau Wildlife International, Ltd. Easton, Maryland 21601 WILDLIFF, 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: MEASUREMENTS OF 10 NEGATIVE CONTROL FISH: WET WEIGHT: STANDATiD LENGTH: TOTAL LENGTH: 454A- 145 Perfluorooctanesulfonate, Potassium Salt (PFOS) Perfluorooctanesulfonate, Potassium Salt (PFOS): A 96-Hour Static Acute Toxicity Test with the Rainbow Trout (Oncorhynchus mykiss) Negative Control, 3.0,6.3, 13,25 and 50 mg a.i./L Experimental Start (OECD) -June 10,2001 Experimental Start (EPA) -June 11,2001 Biological Termination - June 15,2001 Experimental Termination - June 18,2001 96 Hours Rainbow Trout (Oncorhynchus mykiss) Thomas Fish Company Anderson, California 96007 Juveniles Mean = 0.34 g Mean=3.1 cm Mean = 3.6 cm Range = 0.25 to 0.47 g Range=2.8to3.4cm Range = 3.4 to 4.0 cm 96-HOUR LC50: 95% CONFIDENCE INTERVAL: NO MORTALITY CONCENTRATION: NO-OBSERVED-EFFECTCONCENTRATION: 22 mg a.i./L 18 to 27 mg a.i./L 6.3 mg a.i.L 6.3 mg a.i./L . Wildlife International, Ltd. Project Number 454A-145 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 fiom June 11,2001 to June 15,2001. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454A-145 in archives located on the Wildlife International, Ltd. site. OBJECTIVE The objective of this studywas to determine the acute effectsof Perfluorooctanesulfonate,Potassium Salt (PFOS) to the rainbow trout, Oncorhynchus mykiss, duringa 96-hour exposure period under statictest conditions. EXPERIMENTAL DESIGN Rainbow trout were exposed to a geometric series of fivetest concentrationsand anegative(dilution water) control. Two replicate test chambers were maintained in each treatment and control group, with 10 rainbow trout in each test chamber for a total of 20 rainbow trout per test concentration. Two abiotic replicate test chambers alsowere maintained at the highest concentration. Nominal test concentrationswere selected in consultation with the Sponsor, and were based upon the results of an exploratory range finding toxicity test:Nominal test concentrationsselected were 3.1,6.3,13,25 and 50 mg active ingredient(a.i.)/L,. Mean measured test concentrationswere determined from samples of testwater collected fiomeachtreatment and the control group at the beginning of the test, at approximately 48 hours, and at test termination. Rainbow trout were indiscriminately assigned to exposure chambers at test initiation. Observations of mortality and other clinical signs of toxicity were made at approximately4,24,48,72 and 96 hours after test initiation. Cumulative percent mortality observed in the treatment groups was used to calculate or estimateLC50 values at 24,48,72 and 96 hours. The no mortality concentration andtheno-observed-effectconcentration(NOEC)were determined by visual interpretation of themortality andclinicalobservationdata. WildlifeInternational, Ltd. -9- Project Number 454A-145 MATERIALS AND METHODS The study was conducted based on the procedures outlined in the protocol, "Perfluoro- octanesulfonate,Potassium Salt (PFOS): A 96-Hour Static Acute Toxicity Test with the Rainbow Trout (Oncorhynchus mykiss)". The protocol was based on procedures outlined in the U.S. Environmental ProtectionAgency Series 850-EcologicalEffectsTest Guidelines, OPPTSNumber 850.1075: FishAcute Toxicity Test, Freshwater andAdavine (1); OECD Guideline for Testing of Chemicals, 203: Fish, Acute Toxicity Test (2); and ASTM StandardE729-88aYStandard Guidefor ConductingAcute Toxicity Tests with Fishes,Macroinvertebrates and Amphibians (3). Test Substance The test substance was received from 3M Corporation on October 29, 1998 and was assigned WildlifeInternational,Ltd. identificationnumber4675. The test substancewas describedasawhitepowder. It was identified as FC-95 from lot number 217(T-6295). Informationprovidedby the Sponsorindicateda purity of 86.9% and an expirationdate of August 31,2001. The test substance was stored at ambientroom temperature. Preparation of Test Concentrations Nominal test concentrationswere 3.1,6.3,13,25 and 50 mg a.i./L, based on a test substancepurity of 86.9%. All materials which came into contact with the test substance during preparation of test concentratiohswere constructed of plastic or stainless steel. A 40-L primary stock solution was preparedin dilution water at a concentration of 150 mg a.i./L. The primary stock solution was mixed with an electric mixer and sonicated for approximately 23 hours to aid in the solubilization of the test substance. Two replicates of eachtest solutionwereprepared at concentrationsof 3.1,6.3, 13,25 and 50mg ai./L by adding the appropriate volume of primary stock to dilution water in the test aquaria to achieve a final volume of 15 L. Each solutionwas stirredwith a stainlesssteelwhisk for approximatelyone minute. All test solutions appeared clear and colorless. Test Organism The rainbow trout, Oncorhynchus mykiss, was selected as the test species for this study. The rainbow trout is representativeof an importantgroup of aquaticvertebrates and was selected for use in the test based upon past history of use in the laboratory. Rainbow trout used in the test were obtained from Thomas Fish Company, Anderson, California. WildlifeInternational, Ltd. - 10- Project Number 454A-145 The fish were held for approximately five weeks prior to the test in water from the same source and at approximatelythe same temperature as used during the test. During the 14-dayholdingperiod preceding the test, water temperatures ranged from 11.9 to 12.8"C. The pH of the water ranged from 8.2 to 8.4 and dissolved oxygenranged from 9.2 to 10.3mg/L (285% of saturation). Instrumentation and proceduresused for water measurements are described in the Environmental Conditionssection of this report. Thefishwere acclimated to test conditions for approximately 51 hours prior to test initiation. During the acclimation period, no mortalities occurred and the fish showed no signs of disease or stress. At test initiation, the rainbow trout were collected fiom the acclimationtank and indiscriminatelydistributed two at a time to the test chambers until each contained 10 fish. During the holding period, the fish were fed a commercially-prepareddiet (Zeigler Brothers, Inc., Gardners, PA). The fish were not fed during the acclimationperiod (at least 48 hours prior to the test) or during the test. All fish used in the test were from the same source and year class, and the length of the longest fish was no more than twice the length of the shortest. The average standard length of 10 negative control fish measured at the end of the test was 3.1 cm with a range of 2.8 to 3.4 cm, with an average total length of 3.6 cm with a range of 3.4 to 4.0 cm. The averagewet weight (blotteddry) of 10negative control fish at the end of the test was 0.34 g with a range of 0.25 to 0.47 g. Loading was 0.23 g fish& of test water. Test Apparatus Test chamberswere 25-L polyethylene aquaria containing 15L of test solution. The depthof water in a representative test chamberwas approximately 17.5cm. Test chamberswereindiscriminatelypositioned in an environmentalchamber set to maintainthe desired temperature throughout the test. The 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 charactabed asmoderatelyhard water. The specificconductance, hardness, alkalinity and pH measurementsof thewellwater duringthe four-week period immediately preceding the test are presented in Appendix 1. WildlifeInternational, Ltd. -11- Project Number 454A-145 The well water was passed through a sand filter to remove particles greater than approximately 25 pm, and pumped into a 37,800-L storagetankwhere the water was aerated with spraynozzles. Prior to use, the water again was filtered(0.45 pm) to remove microorganisms and particles. The results of periodic analyses performed to measure the concentrations of selected contaminantsin the well water arepresented in Appendix 2. Environmental Conditions Lighting used to illuminate the cultures and test chambers during holding, acclimation and testing was provided by fluorescenttubes 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 30minute transition period of low light intensity was provided when lights went on and off to avoid sudden changesin lighting. Light intensity at test initiation was approximately 126lux at the surfaceofthewater of one representative test chamber. Temperature was measured in each test chamber at the beginning of the test and at approximately 24-hour intervals thereafter using a liquid-in-glass thermometer. Temperature also was measured continuously in one negative control replicate using a Fulscope EWC Recorder, which was verified prior to test initiation with a liquid-in-glass thermometer. The target test temperature duringthe study was 12f 1OC. Dissolved oxygen and pH measurements were made on water samples collected from all replicate test chambers of each treatment and control at test initiationand at approximately 24-hourintervals thereafter. Hardness, alkalinity and specific conductance were measured in the dilution water at test initiation. Light intensity was measured using a SPER Scientific Ltd. Model 840006C light meter. Measurements of pH were made using a Fisher Accumet Model 9 15 pH meter, and dissolved oxygen was measured using a Yellow SpringsInstrument Model 51B dissolved oxygen meter. SpecZcconductancewas measuredusing a Yellow SpringsInstrument Model 33 Salinity-Conductivity-Temperaturemeter. Hardness and alkalinity measurements were made by titration based on procedures in Standard Methods for the Examination of Water and Wastewater (4). WildlifeInternational, Ltd. - 12- Project Number 454A-145 Observations All organisms were observed periodically to determine the number of mortalities in each control and treatment group. The numbers of individuals exhibiting signs of toxicity or abnormal behavior also were evaluated. Observations were made approximately4,24,48,72 and 96 hours after test initiation. Statistical Analyses The mortality datawere analyzed, when possible, using the computerprogram of C. E. Stephan(5). The program was designed to calculate the LC50 value and the 95% confidence interval by probit analysis, the moving average method, and binomial probability with nonlinear interpolation (6,7,8). In this study, there was less than 50%mortality in anytreatment group prior to the 96-hour observationperiod. Therefore, the 24,48 and 72-hour LC50 values were estimatedto be greater than the highest concentrationtested. The 96-hour LC50 value was calculated using probit analysis. The no mortality concentration and NOEC were determined by visual interpretation of the mortality and observation data. Analytical Chemistry Water samples were collected at mid-depth fiom each biotic test chamber of each treatment and control group at the beginning of the test, at approximately 48 hours and at test termination to measure concentrations of the test substance. Additional samples were collected on Day 1 of the test from the negative control replicates to confirm the results of the Day 0 analysis. Samples also were collected for analysis at approximately48 and 96 hours from the abiotictest chambers prepared at a concentrationof 50 mg a.i./L. All samples were collected in plastic vials and analyzed as soon as possible without storage. Analytical procedures used in the analysis of the samples are provided in Appendix 3. RESULTSAND DISCUSSION Measurement of Test Concentrations Results of analyses to measure concentrations of PFOS in water samplescollectedduringthetest are presented in Table 1 and in the analytical chemistry report (Appendix 3). Nominal concentrations selected for use in this studywere 3.1,6.3,13,25 and 50 mg a.i./L. Samplescollected at test initiation hadmeasured concentrationsthat ranged from 93 to 103%of nominal concentrations. Measured concentrations for biotic samples taken at 48 hours ranged fiom 94 to 103%of nominal,while the abioticsamplesranged fiom 105to 106%of nominal. Measured concentrationsfor biotic samplestaken at 96 hours ranged from 91to 105%of WildlifeInternational, Ltd. - 13 - Project Number 454A-145 nominal, while the abiotic samples were 102% of nominal, When measured concentrations of the biotic samples analyzed at test initiation, approximately48hours and at test terminationwere averaged, the mean measured concentrationsfor this study were 3.0,6.3,13,25 and 50 mg a.i./L, representing97,100,100,100 and 100% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations. Observations and Measurements Measurements of temperature, dissolved oxygen and pH are presented in Table 2. Temperatures were within the 12 * 2C range established for the test. Dissolved oxygen concentrations remained 29.2 mg/L (85% of saturation)throughout the test. Measurementsof pH ranged fi-om8.1 to 8.5 during the test. Measurements of hardness, alkalinity and specific conductance in the dilution water at test initiation were typical of Wildlife International, Ltd. well water (Table 2). Daily observations of mortality and other signs of toxicity observed during the test are presented in Table 3. Rainbow trout in the negative control group appeared normal and healthy throughout the test period. Rainbow trout in the 3.0 and 6.3 mg a.i./L treatment groups also appearednormal and healthy during the test, with no mortalities or overt signs of toxicity observed. After 96-hours of exposure, mortality in the 13, 25 and 50 mg a.i./L treatment groups was 20, 50 and loo%, respectively. LC50 values and 95% confidence intervals,estimated or calculated from the mortality data at 24,48,72and 96 hours, are shown in Table 4. A graph of the concentration-response curve is presented in Figure 1. CONCLUSIONS The 96-hour LC50 value for rainbow trout (Oncorhynchus mykiss) exposed to Perfluorooctanesulfonate, Potassium Salt (PFOS) was 22 mg a.i./L with a 95% codldence interval of 18 to 27 mg a.i./L. The 96-hour no mortality concentration and the NOEC were 6.3 mg a.i./L. WildlifeIntemational, Ltd. - 14- REFERENCES Project Number 454A- 145 U.S. Environmental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines (draft>,OPPTS Number 850.1075: Fish Acute Toxiciv Test,Freshwater and Marine. Organisationfor Economic Cooperation and Development. 1993. OECD Guidelinesfor Testingof Chemicals. Guideline 203: Fish, Acute Toxicity Test. Adopted by the Council on 12 July 1992. ASTM Standard E729-88a. 1994. Standard Guidefor Conducting Acute Toxicity Tests with Fishes,Macroinvertebrates, and Amphibians. American Society for Testing and Materials. APHA, AWWA, WPCF. 1998. StandardMethodsfor the Examination of Waterand Wastewater. 20th Edition. American Public Health Association. American Water Works Association. Water Pollution Control Federation, New York. Stephan, C.E. 1978. U.S.EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. Finney, D.J. 1971. StatisticalMethods in BiologicalAssay. Second edition. Griffin Press, London. Thompson,W.R 1947. Bacteriological Reviews. Vol. 11,No. 2. Pp. 115-145. 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. WildlifeInternational, Ltd. - 15- Proiect Number 454A-145 Table 1 Summary of Analytical Chemistry Data Nominal Test Concentration' (mg a.i./L) 0.0 (Negative Control) 3.1 Sampling Time (HOW) 0 0 48 48 96 96 0 0 48 48 96 96 PFOSMeasured Concentration' (maa.i./L) < LOQ'f <LW <LOQ <LOQ <LOQ <LOQ 3.15 3.02 2.90 3.01 2.83 2.97 Percent of Nominal' 102 97.5 93.6 97.1 91.4 95.9 MeanMea~ed Test Concentration (mg a.i./L) MeanMeasured Percent of Nominal 3.0 97 6.3 0 6.22 98.7 6.3 100 0 6.21 98.6 48 6.16 97.8 48 6.43 102 96 6.15 97.6 96 6.60 105 13 0 13.2 102 13 100 0 12.1 93.2 48 12.7 97.4 48 12.3 94.9 96 13.1 101 96 12.6 96.7 25 0 25.0 99.9 25 100 0 25.7 103 48 24.3 97.2 48 25.7 103 96 25.7 103 96 26.2 105 50 0 49.7 99.4 50 100 0 49.8 99.5 48 51.1 102 48 51.5 103 96 49.6 99.1 96 50.8 102 50 48 53.1 106 52 104 (Abiotic) 48 52.6 105 96 50.9 102 96 51 .O 102 ' The limit of quantitation (LOQ)was 0.200 mg a.i./L based upon the productofthe lowestcalibrationstandardanalyzed(0.000500 mg a.i./L) and the dilution factor of the matrix blank samples (400). Results were generated using MacQuan version 1.6software.Manual calculationsmay differ slightlysincenominal and measured concentrations were corrected for change in test substance punty and rounded for reporting purposes. Results are mean of duplicate re-preparations. Original Negative Controls were contaminated during analyticalsampling. WildlifeInternational, Ltd. - 16- Project Number 454A-145 Table 2 Temperature, Dissolved Oxygen and pH of Water in the Test Chambers Mean Measured 0 Hour' Concentration (mg a.i.L) * ~emp.2 DO' Rep. ("C) (mgn) PH -~ ~ ~ ~~ ~ Negativecontrol A B 12.5 10.4 8.3 12.6 10.7 8.4 24 Hours Temp. DO ("C) ( m a ) PH 12.1 9.6 8.3 12.1 9.6 8.3 48 Hours Temp. DO ("C) (mgn) PH 12.1 9.4 8.2 12.2 9.4 8.2 72 Hours Temp. DO ("C) ( m a ) PH 12.1 9.4 8.2 12.3 9.4 8.2 96 Hours Temp. DO ("C) ( m a ) PH 12.1 9.4 8.1 12.2 9.4 8.1 3.0 A 12.5 10.6 8.4 12.2 9.4 8.3 12.3 9.4 8.2 12.5 9.4 8.2 12.5 9.4 8.1 B 12.4 10.8 8.4 12.0 9.8 8.3 12.0 9.4 8.2 12.2 9.4 8.2 12.2 9.4 8.1 6.3 A 12.1 10.6 8.4 11.8 9.6 8.4 11.7 9.4 8.2 11.8 9.4 8.2 11.8 9.4 8.1 B 12.1 10.8 8.4 11.7 9.8 8.4 11.7 9.3 8.2 11.7 9.4 8.2 11.7 9.4 8.1 13 A 11.7 10.8 8.4 11.4 9.8 8.4 11.3 9.3 8.2 11.4 9.2 8.3 11.3 9.2 8.2 B 11.9 10.8 8.4 11.6 9.8 8.4 11.6 9.2 8.2 11.6 9.3 8.3 11.6 9.3 8.2 25 A 12.3 10.8 8.4 11.7 9.8 8.4 11.6 9.3 8.2 11.7 9.2 8.3 11.7 9.2 8.2 B 12.5 10.8 8.4 11.8 9.8 8.5 11.7 9.4. 8.2 11.7 9.3 8.3 11.7 9.2 8.2 50 A 12.9 10.8. 8.4 11.8 9.8 8.4 11.8 9.4 8.3 11.9 9.4 8.3 11.9 9.4 8.2 B 12.9 10.8 8.4 11.9 9.6 8.4 11.9 9.2 8.3 11.9 9.2 8.3 11.9 9.2 8.2 C 4 13.8 10.0 8.3 12.0 10.0 8.4 12.0 9.8 8.4 12.0 9.8 8.4 12.0 9.8 8.4 D 4 13.7 10.2 8.3 12.0 10.0 8.5 12.0 9.8 8.4 12.0 9.8 8.4 12.0 9.8 8.4 ' The 0-hour dilution water measurementsfor hardness, alkalinity and specific conductance were 132mg/L as CaC03, 184 mgL as CaC03and 270 pmhos/cm, respectively. ' Temperature measured continuouslyduring the test ranged from approximately 12.0to 12.5"C. A dissolved oxygen concentration of 6.5 mg/L represents 60% saturation at 12C in freshwater. Abiotic redicate. WildlifeInternational, Ltd. - 17- Project Number 454A-145 Table 3 Cumulative Percent Mortality and Treatment-Related Effects ~ Mean Measured Concentration (mg a.i.4,) Negative Control 3.0 6.3 13 25 50 4 Hours No. No. Rep. Exposed Dead' Observed Effects' ~ A 10 0 1 0 AN B 10 0 10 AN A 10 0 1 0 AN B 10 0 10 AN A 10 0 10 AN B 10 0 10 AN A 10 0 10 AN B 10 0 10 AN A 10 0 10 AN B 10 0 10 AN A 10 0 10 AN B 10 0 10 AN 24 Hours No. Observed Dead Effects 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 48 Hours No. Observed Dead Effects 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 1 9AN 0 10 AN 72 Hours No. Observed Dead Effects 0 10AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 3 7AN 4 6AN 1 Cumulative number of dead fish. Observed Effects: AN = appear normal. 96 Hours No. Observed Dead Effects 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 1 9AN 3 7AN 6 4AN 4 6AN lo -- lo -- Cumulative Percent 0 0 0 20 50 100 Wild1ife Intemational, Ltd. - 18- Table 4 LC50 Values Project Number 454A-145 Time 24 Hours LCSO (mg a.i./L) 95% Confidence Interval (mg a.i./L) -- 1 Statistical Method NA 48 Hours NA' 72 Hours NA' 96 Hours 22 18-27 Probit Analysis The LCSO value and 95% confidenceintervalcould not be statistically calculated. The LCSO value was estimatedto be greaterthan the highest concentrationtested. WildlifeInternational, Ltd. Figure 1. Concentration-Response Curve (96-Hour Data) Project Number 454A-145 1 10 100 Concentration (mg a.i.R) WildlifeInternational, Ltd. - 20 - Project Number 454A- 145 Appendix 1 Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test Specific Conductance (pmhoslcm) Hardness (mgLas CaC03) Alkalinity (mg/L as CaC03) Mean 311 (N=4) 130 (N= 4) 177 (N= 4) Range 310-315 128 - 132 176 - 178 PH 8.3 (N= 4) 8.2- 8.4 WildlifeInternational, Ltd. -21- Project Number 454A-145 Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water' Component Pesticidesand Organics Measured Concentration Component Measured Concentration Aclonifen Alachlor Atrazine Azinphos-ethyl Azinphos-methyl Azoxystrobin Bifenthrin Bioallethrin Bitertanol Bromacil Bromophos Bromophos-ethyl Bromopropylate Bupirimate Carbaryl Carbofuran Carboxin C hlorfenvinphos C hloridazon Chlorpropham Chlorpyrifos C hlorpyfifos-methyl C hlorothalonil Coumaphos Cyanazine Cyfluthrin Cypermethrin Cyproconazole Deltamethrin Demeton Demeton-0 Desethylatrazine Desisopropylatrazine Desmetryn Diazinon Dichlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid <0.03 pg/L 4 . 0 1 pg/L <0.01 pg/L 4.01 pgL 4.04 Pg/L 4 . 0 8 pglL ~ 0 . 2p5g/L 4.05 pg/L ~0.05pg/L <0.05pg/L <0.05pg/L <0.02 pg/L <0.02 pg/L 4 . 0 2 pg/L 10.05p g L <0.05pg/L <0.03 pglL <0.02 pgrL 4 . 0 2 pg/L <0.05pg/L <0.02 pg/L CO.01 p g L <0.01 pg/L <0.04 p g 5 <0.02 pg/L 4 . 0 5 pg/L <0.05pg/L 4.25 pg/L <0.05p a <0.02 pg/L C0.02 pg/L <0.02 pg/L <0.01 p g L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.03 p g L 4.02 pg& <0.01 pg/L 4.03pg/L Dichlorvos Dicofol Diethyltoluamide Difenoconazole Dimethoate Dimethomorph Disulfoton DMST Dodemorph Endosulfanu Endosulfan-j3 Endosulfan-sulfate Epoxiconazole Eptam Esfenvalerate Ethion Ethofumesate Ethoprophos Etridiazole Etrimfos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb Fenpiclonil Fen propathrin Fenpropimorph Fenthion Fenvalerate Fluazifopbutyl Fluoroglycofen-ethyl Fluroxypyr-meptyl Flutolanil Fonophos Furalaxyl Heptenophos Imazalil Iprodion Kresoxim-methyl Lenacil Lindane <0.01 pg/L 4 . 2 5 pg/L C0.02 pa ~ 0 . 0 p3 g L <0.02 pglL <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L CO.01 p g L <0.02 pg/L <0.05 pg/L <0.02 p g n <0.02 pg/L ~0.05pg/L <0.02 pglL <0.01 pg/L <0.02 pg/L <0.05pg/L <0.05p g L <0.01 pg/L <0.03p g L ~ 0 . 0p3g/L <0.05p g L <0.25 pg/L <0.01 pg/L <0.01 pg/L c0.02 pg/L 4 . 0 2 pg/L C0.02 pg/L ~0.05pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.05 pg/L C0.02 p g n <0.05 pg/L <0.02 pg/L 'Analvsesuerformed bv TNO Nutrition and Food Institute on samples collected on November 15,2000. Wild1ife Intemational, Ltd. - 22 - Project Number 454A-145 Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International,Ltd. Well Water' ~~ ~ Pesticides And Organics (Page 2) Component Measured Concentration Component Measured Concentration Malathion Metalaxyl Metamitron Metazachlor Methidathion Paclobutazole Parathion Parathion-methyl Penconazole Pendimethalin Permethrincis Permethrin-trans Phosalone Phosmet Phosphamidon4 Pirimicarb Pirimiphosethyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazine Propham' Propiconazole Propoxur Propyzamide Prosulfocarb Pyrazophos 4.02 p g 5 4.05p g 5 <0.05p g 5 <0.02 p g 5 4.02 p g 5 4.05 p g 5 <0.01 p g 5 <0.01 p g 5 4.05 pg/L <0.03p g 5 co.01 p g n 4.01 pg5 <0.05p g 5 <0.02p g 5 <0.05p g 5 4.01 pgn 4 . 0 1 pg/L <0.01 p g n <0.02 p g 5 <0.01 p g 5 4 . 0 1 pg/L <0.01 pg/L <0.01 p g 5 -=0.02pB/L <0.05p g 5 <0.03 pgk. <0.02p g 5 4.02 pg5 ~0.03p g 5 Methoxychlor Metolachlor Metribwin Mevinphos Nitrothal-Isopropyl Pynfenox-1 Pynfenox-2 Pynmethanil Quizalofop-ethyl Simazine Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbuthylazine Tetrachlorvinphos Tetrahydroftalimide Tetramethrin Thiabendazole T hiorneton Tolclofos-methyl Tolylfluanid Triadimefon Triadimenol Triallate Triazophos Trifluralin Vamidothion Vinclozolin 4 . 0 1 pg/L 4.01 pg/L 4.02 pg5 4 . 0 1 pg/L 4.05p g 5 4.01 pg5 4.01 p g 5 4.01 pg/L 4.02 pa 4 . 0 1 pg/L 4 . 0 2p g n 4.05 pg5 4.05 p g 5 4 . 0 1 pg/L 4.01 pg5 4.01 pg5 4.05p g 5 4.01 pg5 4.05 pg/L 4.04 Pg5 4.01 pg5 4.04 Pgn 4.05p g 5 4.05pg/L 4.02 p g 5 a 0 2 pg5 4.02 pg/L 4.01 pg5 4 . 0 1 pg/L Metals Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 13.2 mg/L 21 m g 5 35 m g 5 4.02 m g 5 6.2 m g 5 <0.09m g 5 0.72 p g 5 4 . 3P g 5 4 . 6P g 5 ~ 0 . p4 g 5 Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium 'Analyses performed by TNO Nutrition and Food Institute on samples collected on November 15,2000. WildlifeInternational, Ltd. - 23 - Project Number 454A-145 THE ANALYSIS OF PFOS IN FRESHWATER IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECTNO.: 454A-145 WildlifeInternational, Ltd. - 24 - Project Number 454A-145 REPORT APPROVAL SPONSOR 3M Corporation TITLE: PerfluorooctanesuKonate,PotassiumSalt (PFOS):A 96-HourStaticAcute ToxicityTestwith the Rainbow Trout (Oncorhynchusmykiss) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-145 PRINCIPAL INVESTIGATOR: MANAGEMENT: Willard B. Nixon, Ph.D. Director, Analytical Chemistry 01I07 10% DATE DATE WildlifeInternational, Ltd. - 25 - Project Number 454A-145 Introduction Freshwater sampleswere collectedfrom a 96-howtoxicity test designedto determine the effectsof PFOS (PerfluorooctanesulfonateP, otassium Salt) to the rainbow trout (Oncorhynchusmyhss). This study was conducted by Wildlife International, Ltd. and identified as Project No.: 454A-145. The analyses of these water samples were performed at Wildlife International, Ltd. using high performanceliquidchromatography with mass spectrometric detection (HPLCMS). Sampleswerereceived for analysis on June 11,13,and 15, 2001 and were analyzed between June 11 and June 18,2001. Test Substance and Internal Standard The test substanceused for this study was Wildlife International, Ltd. identificationnumber 4675. The test substance, referred to hereafter as PFOS, was used to prepare calibration and matrix fortification samples. The internal standard was received from 3M Corporation on July 2,1998 and was assignedWildlife International,Ltd. identificationnumber 4526 upon receipt. The internal standard, a granular material,was identified as: lH, lH, 2H, 2H Perfluorooctane SulfonicAcid, ChemicalAbstractNumber: 27619-97-2. The standard, referred to hereafter as 4H PFOS, was stored under ambient conditions. Analytical Method The method usedfor the analysisof the freshwatersampleswas developedat WildlrfeInternational, Ltd. and entitled "Analytical Method for the Determination of PFOS in Freshwater, Saltwater, and Algal Medium". This methodology was included as Appendix I1 of Wildlife International, Ltd. protocol number 454/011299/MVAL/SUl3454. It was based upon methodology provided by 3M Corporation. Several modifications from the validated methodology were implemented for the present study. First, the concentration of the internal standard was changed from 100 pg/L to 10.0 pg/L to better match the calibration and test sample instrumental concentrations. Second, a guard cartridge was used in conjunction with a shorter (100 to 50 mm) analytical column and warmer oven temperature (30 to 40C) for long-term protection of the analytrcal column and faster run times. WildlifeInternational, Ltd. - 26 - Project Number 454A-145 Samples were diluted in a 50% methanol: 50% NANOpure@ water solution containing 10.0 pg 4H PFOS L and 0.05% formic acid (v/v) so that they fell within the calibration range of the PFOS methodology. Concentrations of the PFOS in the standards and samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC)with a Perkin-Elmer API lOOLC Mass Spectrometerequipped with aPerkin-Elmer TurboIonSpray ion source. HPLC separations were achievedusing a Keystone Betasil CISanalyticalcolumn (50 mm x 2 mm I.D., 3 - p uparticle size) fittedwith a KeystoneJavelin C18guardcartridge(20x 2 mm). The instrument parameters are summarized in Table 1. A method flowchart is provided in Figure 1. Calibration Curve and Limit of Quantitation Calibration standards of PFOS prepared in a 50% methanol :50% NANOpure@water solution containing 10.0 pg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v), ranging in concentration fiom 0.500 to 5.00 pg a.i./L, were analyzed with the samples. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOS on the basis of individual isomeric components. Linear regression equations were generatedusing peak area response ratios (PFOS :internal standard)versus therespectiveConcentration ratios (PFOS : internal standard) of the calibration standards. A typical calibration curve is presented in Figure 2. The concentrationof PFOS in the sampleswas determinedby substitutingthe peak area response ratios into the applicable linear regression equation. Representative ion chromatograms of low and high calibration standards are presented in Figures 3 and 4, respectively. The method limit of quantitation (LOQ) for these analyses was set at 0.200mg a.i.L calculated as the product of the lowest calibration standard analyzed (0.000500 mg a.i./L) and the dilution factor of the matrix blank samples (400). Matrix Blank and Fortification Samples Threematrixblank sampleswere analyzedto determinepossible intederence. No interferenceswere observed at or above the LOQ during samples analyses (Table 2). A representative ion chromatogram of a matrix blank is presented in Figure 5 . WildlifeInternational, Ltd. - 27 - Project Number 454A-145 Freshwaterwas fortified at 1.00,lO.Oand 100mg a.i./L and analyzedconcurrentlywiththesamples to determinethe mean procedural recovery (Table 3). Sampleconcentrationswerenot correctedforthe mean procedural recovery of 10 1%. A representative ion chromatogram of a matrix fortification is presented in Figure 6. Example Calculations Sample number 454A-145-18, nominal concentration of 6.3 mg a.i.L in freshwater. Peak Area Ratio =Analyte Peak Areahternal Standard Peak Area ConcentrationRatio =Concentration of AnalytdConcentrationof Internal Standard Internal Standard Concentration: 0.0100 mg/L First Initial Volume: 0.500 mL First Final Volume: 10.0 mL Second Initial Volume: 0.100 Second Final Volume: 10.0 Dilution Factor: 2000 PFOS Peak Area: 349928 Internal Standard Peak Area: 3 19027 Peak Area Ratio: 1.0969 Calibration curve equation. Slope: 3.3385 Intercept: 0.0235 Curve is weighted (l/x) Peak area ratio - PFOS (mg a.i.L) at instrument = Slope -- 1.0969- 0.0235 o~oloo 3.3385 Internal Standard Concentration = 0.003215 WildlifeInternational, Ltd. -28- Project Number 454A-145 PFOS (mg a.i./L) in sample = PFOS (mg a.i./L) at instrument x Dilution Factor = 0.003215 x 2000 = 6.43 PFOS m a.i./L in sam le Percent of Nominal Concentration= pFos((m: no-> x 100 = 102% Quantitation softwarefor recoveries: MacQuan, version 1.6. RESULTS Sample Analysis Freshwater samples were collected from the 96-hour toxicity test with the rainbow trout (Oncorhynchus mykiss) at test initiation, June 11, 2001 (Day 0), on June 13, 2001 (Day 2) and at test termination, June 15,2001 (Day 4). The measured concentrations of PFOS in the samples collected at initiation of exposure of the test organisms (Hour 0) ranged from 93.2 to 103% of the nominal concentrations. Samplescollected at Day 2 had a measured concentration range of 93.6 to 103%of nominal values. Samplescollectedat test termination(Day 4) had a measured concentrationrange of 91.4 to 105%of nominal values (Table 4). Samples from the abiotic 50 mg a.i./L treatment group were comparable to samples from the 50 mg a . i L treatment group with the fish present (Table 4). A representative ion chromatogram of a test sample is shown in Figure 7. WildlifeInternational, Ltd. - 29 - Project Number 454A-145 INSTRUMENT: Table 1 Typical HpLC/MS Operational Parameters with a Perkin-Elmer API lOOLC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: GUARD COLUMN: Keystone Betasil C18 column(50 mm x 2 mm I.D., 3 - p ~ nparticle size) Keystone Javelin CIScolumn (20 x 2 mm) OVEN TEMPERATURE: 40C STOP TIME: 5.00 minutes FLOW RATE: 0.220 mL/minute MOBILE PHASE: 70.0% Methanol :30.0% NANOpure@Water containing 0.1% Formic Acid INJECTION VOLUME: 10.0 pL PFOS RETENTION TIME: Approximately 4.4 minutes INTERNAL STANDARD RETENTION TIME: Approximately 3.0 minutes PFOS MONITORED MASS: 499 m u INTERNAL STANDARD MONITORED MASS: 427 m u WildlifeInternational, Ltd. -30- Project Number 454A-145 Table 2 Matrix Blanks Analyzed ConcurrentlyDuring SampleAnalysis Number (454A-145) MAB- 1 Samde Type Matrix Blank Measured Concentration of PFOS' (mg a.i./L) .C LOQ MAB-2 Matrix Blank < LOQ MAB-32 Matrix Blank < LOQ ' The limit of quantitation (LOQ)was 0.200 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.000500 mg a.i./L) and the dilution factor of the matrix blank samples (400). * Result is mean of duplicate re-preparations. Original matrix blank was contaminated during analytical sample preparation. Wild1ife International, Ltd. -31 - Project Number 454A-145 Table 3 Matrix Fortifications Analyzed ConcurrentlyDuring SampleAnalysis Sample Number (454A- 145-) MAS- 1 MAS-4 MAS-7 Concentrationsof PFOS (. m-ga.i./L) Fortified' 1.oo 1.00 1.00 Measured' 1.15 0.972 1.04 Percent Recovered' 115 97.2 104 MAS-2 10.0 9.61 96.1 MAS-5 10.0 9.69 96.9 MAS-8 10.0 10.4 104 MAS-3 100 91.9 91.9 MAS-6 100 102 102 MAS-9 100 101 101 ~~~ Mean = 101 Standard Deviation = 6.64 CV =6.58% N =9 1 Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly. Wildlqe International, Ltd. - 32 - Project Number 454A-145 Table 4 Measured Concentrationsof PFOS in Freshwater Samples from a Rainbow Trout (Oncorhynchusmykss) 96-Hour Toxicity Test Nominal Test Concentration' (mg a.i./L) 0.0 (Negative Control) Sample Number (454A-145-) 1A 2A 13 14 27 28 sampling Time Pay) 0 0 2 2 4 4 PFOS Measured Concentration2 (mg a.i.iL) Percent of Nominal2 3.1 3 4 15 16 29 30 3.15 102 3.02 97.5 2.90 93.6 3.01 97.1 2.83 91.4 2.97 95.9 6.3 5 6 17 18 31 32 6.22 98.7 6.2 1 98.6 6.16 97.8 6.43 102 6.15 97.6 6.60 105 13 7 0 8 0 19 2 20 2 33 4 34 4 13.2 102 12.1 93.2 12.7 97.4 12.3 94.9 13.1 101 12.6 96.7 1 The limit of quantitation (LOQ) was 0.200 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.000500 mg a.i./L) and the dilution factor of the matrix blank samples (400). Resultswere generatedusing MacQuanversion 1.6software. Manual calculationsmay differslightlysince nominal and measured concentrationswere corrected for change in test substance purity and rounded for reporting purposes. 3 Results are mean of duplicate re-preparations. Original Negative Controls were contaminated during analyhcal sampling. WildlifeInternational, Ltd. -33- Project Number 454A-145 Table 4 (Continued) Measured Concentrations of PFOS in Freshwater Samples from a Rainbow Trout (Oncorhynchus mykiss)96-Hour Toxicity Test Nominal Test concentration' (mg a.i./L) 25 Sample Number (454A-145-) 9 10 21 22 35 36 Sampling Time (Day) 0 PFOS Measured Concentration' (mg a.i./L) 25.0 25.7 24.3 25.7 25.7 26.2 Percent of Nominal' 99.9 103 97.2 103 103 105 50 11 12 23 24 37 38 49.7 99.4 49.8 99.5 51.1 102 51.5 103 49.6 99.1 50.8 102 50 25 2 (Abiotic) 26 2 39 4 40 4 53.1 106 52.6 105 50.9 102 51.0 102 1 The limit of quantitation (LOQ) was 0.200 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.000500 mg a.i./L) and the dilution factor of the matrix blank samples (400). Results were generated using MacQuan version 1.6sofhvare. Manual calculations may differ slightly since nominal and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes. WildlifeInternational, Ltd. - 34 - Project Number 454A-145 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FRESHWATER Prepare matrix fortification samples in freshwater matrix by spiking the requisite volume of PFOS stock solutions directly into freshwater. Perform fortificationswith gas-tight syringesand Class A volumetric flasks. Prepare appropriate dilutions of study and QC samples to withinrange of the PFOS HPLC/MS methodology: Partially fill Class A volumetricflasks with 50% methanol :50% NANOpure@water dilution solvent containing 10.0 pg 4H PFOSL and 0.05% (v/v) formic acid. Add appropriate volume of sample and bring the flask to volume with dilution solvent. Process the matrix blank samples using the same dilution and aliquot volume as for the lowest fortificationlevel. Mix well by several repeat inversions. Ampulate samples and submit for HPLCNS analysis. Figure 1. Analytical method flowchart for the analysis of PFOS in freshwater. WildlifeInternational, Ltd. - 35 - Ffos 499.0 Internal Standard: 4HPFOS Weighted ( l l x ) Intercept = 0.0235 Slope = 3.3385 Correlation Coeff. = 0.9994 I Area(Rati0) Project Number 454A-145 Conc. (Ratio) Figure 2. A typical calibration curve for PFOS. 8 WildlifeInternational, Ltd. - 36 - Project Number 454A-145 PFOS-1 STD 0.500 ug a.lA 4675A-011D -21 4.98 in 1 period pA3s Internal Standard: 4HPFOS Use Area Absdute Retention Time 1: 4.98 Q l MI, 297 scans 499.0 NdieThres. 3.0 Ouanl Thres. 1.0 Min. Wklth 3 Mull. Wkllh 6 Base. Widlh 80 RTWin.(secs) 2 0 Smooth 1 ExpectedRT 4.34 Area 55560 Height 5767 Start Time 4.12 End T i 4.87 lntegratbn Width 0.74 Retention Time Integration Type -4.38 A VB Wed, Jun 13. 2001 1446 10090. 807060. 5040- 30- 20. 2 1 1 8' 41 0.69 intensity: 65000 cps 89 81 ' 1.36 260 121 155 193 E 121 ' 161 ' 261 ' 2 2.04 2.71 3.38 4.06 4.73 Time PFOS-1 STD 0.500 ug alJL 4675A-011D -21 4.98 in 1 period 4tpFos use as Internal Standard 1: 4.98 Q1 MI, 297 scans 427.0 Noise Thres. 2.0 Quant Thres. 1 .O Min. Width ' 3 Mult. Width 6 b.Wdth 30 RTWin. (secs) 20 Smooth 1 EWeCbd RT 2.95 kea 299568 Height 40766 Start Time 283 EM T i i 3.33 Integration Width 0.51 Retention Time tntegratlon Type -2.9s A BB Wed, Jun 13, 2001 14148 1009080. 70. 60. 50. 4e 30. 2010 0 intensity: 55000 cps . .-Aci 28 A 88 ,...* 41 81 0.69 1.36 124 .. 121 2.04 15 .+ 161 2.71 223 262 ,i,.., 201 2 4 1 281 Scan 3.38 4.06 4.73 Time Figure 3. A representativeion chromatogramof a low-level (0.500 pg a.i./L)PFOS standard. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard bottom)). WildlifeInternational. Ltd. - 37 - Project Number 454A-145 PFOS-5 STO 5.00 ug a.IR 4675A-011D -25 4.98 In 1 period Ros Internal Standard: 4HPFOS U s e Area Absolute Retention Time 1: 4.98 Q1 Mi. 297 scans 499.0 Noise Thres. 3.0 Quant Thms. 1.O Min. Width 3 Mult. Width 6 Base. Wdth 80 RTWln.(secs) 2 0 Smooth 1 EnpectedRT 4.34 Area . 507560 Height 60620 Start Time 4.12 End Time 4.90 Integration Width 0.77 Retention Time Integration Type -4.39 A VB Wed. Jun 13, 2001 16:13 intensity: 55000 cps 10 9 8 7 6 6 4 3 2 1 41 8 1 121 161 2 0 1 2 4 1 2 8 1 Scan 0.69 1.36 2.04 2.71 3.38 4.06 4.73 Time PFOS-5 STD 5.00 Ug r.lA 4675A-011D -25 4.98 in 1 period 4HpFDs use as Internal Standard 1: 4.98 Q1 MI, 297 scans 427.0 Noise Thres. 2.0 auantms. 1.0 Mln. Width . 3 Muit. Width 6 Baee. Wdth 30 RTWin. (secs) 20 Smooth 1 EXp38d RT 2.96 Area 304762 Helght 40262 Start Time 2.83 EndTIm 3.35 Integration Width 0.52 Retention Time Integration Type 2.96 A - 88 Wed, Jun 13. 2001 15:13 intensity: 55000 cps 8 176 :il, 7 3 2 1 247 40.169 81.138 204 2 7 1 _ 3.3.8 244.016 248.713 sTcimane Figure 4. A representative ion chromatogram of a high-level (5.00 pg a.i./L) PFOS standard. (monitored masses = 499 m u (PFOS - top) and 427 amu (4HPFOS internal standard - bottom)). Wildlge International, Ltd. - 38 - Project Number 454A-145 PFOS-7 454A- 14 5 - MAE-2 Wed. Jun 13, 2001 1525 4.98 in 1 period Ac6 lnmmalStandard: 4WFOS Use Area Absolute Retention Time 1: 4.98 Q1 MI. 297 f i c a ~ 49Q.O ~ d s e ~ h r e s . 3.0 Quant Thras. 1.O Mh. WMth 3 Mult. Width 6 Base. wdth 80 RTWln. (secs) 2 0 Smooth 1 ErpadedRT 4.34 Area 833 Height 9 4 start Time 4.21 End Tim 4.83 integration Width 0.42 Retention Time Integration Type -4.41 A BB intensity: 55000 cps 10 1 1 44 114 170 217 24tL262 0.69 1.36 2.04 2.71 3.38 4.06 4.73Time PFOS-7 454A-145- MAE-2 Wed, Jun 13, 2001 1525 4.90 in 1 period 4HpFos use as internal Standard intensity: 55000 Cps 1: 4.90 Q l MI. 297 scans 427.0 Noise Threr. 2.0 Quant Thres. 1 .O M i . width 3 Muit. Width 6 0ase. width 30 RT Win. (cecs) 2 0 SmOOth 1 ExpmAed RT 2.95 tuea 318679 Height 42248 Start Time 2.83 EndTim 3.33 lntegratlon Width 0.61 Retention Time integration Type -2.98 A BB iOOl 5 4 3 2 1 96112 236 204 0.89 1.38 2.04 2.71 3.38 4.06 4.73 Figure 5. A representative ion chromatogram of a matrix blank sample (454A-145-MAB-2). Dilution factor = 40Ox. The arrow indicates the retention time of PFOS. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard - bottom)). WildlifeInternational, Ltd. - 39 - Project Number 454A-145 ~~ ~~ PFOS-9 454A-145- MAS4 Wed. Jun 13, 2001 15:38 4.98 h 1 period Ac6 Internal Standard: 4HPFO.S U s s Area AbMkRe Retention Time 1: 4.98 Q1 MI, 297 scam 499.0 Noisemres. 3.0 Quant T b s . 1.0 Mn. width 3 Mul. Wldth 6 Base. Wldth 80 RTWln.(secs) 20 SmoOth 1 ExpU6URT 4.34 Area .264966 Hel@t 27164 Start Time 4.14 End Tlms 4.90 IfltegrPtiCfI Width 0.76 Relention Time Integration Type -4.38 A VB 10 3 2 1 41 0.69 intensity: SO00 cps 2P 81 121 161 201 241 281 Scan 1.36 2.04 2.71 3.38 4.06 4.73Tlme PFOS-9 454A-145- MAS5 Wed, Juri 13. 2001 15:38 4.98 In 1 period 4Wms use a8 Internal Standard 1: 4.98 Q1 MI,297 Scan3 427.0 NoisaThres. 2_ . 0 . Quant Thres. 1.O Mh. Wdth 3 Mult Width 6 h.widUI 30 RTWh(8eca) 20 SroOth 1 Expsded RT 2.95 &ea 318348 Height 43093 Start Tbw 281 EM Tim 3.32 Integration WMth 0.61 Retention Time IntWt'ath TyPa -2.86 A BB '1874 6 1 0.60 108 138 1.36 2.04 2. intensity: 55000 cps 4 226 268 201 3.38 ,241 4.06 281 4.73 Snmcaen Figure 6 . A representative ion chromatogram of a matrix fortification sample (454A-145-MAS-5). Nominal Concentration= 10.0mg a.i./L, Dilution factor = 4000x. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard bottom)). WildlifeInternational, Ltd. Project Number 454A-145 PFOS-22 22 454A-145- Wed, Jun 13, 2001 17:Oo 4.98 in 1 period pR)6 lnlemalStandard: 4HPFOS Use Area Absolute Retention Time 1: 4.98 Q1 MI, 297 scans 499.0 NoiseThres. 3.0 Quant Thres. 1.0 Min. WWlh 3 MUM. Width 6 Base.Widm 80 RTWin.(secs) 20 Smooth 1 Expd8dRT 4.34 Area 281742 Height 28749 Start Time 4.14 End Tbne 4.88 Integration Width 0.74 Retention Time Integration Type A4-.V39B 10 20. 10. n+ Y. intensity: So00 cps 261 42- 88 138 194 0.69 1.36 2.04 2.71 3.38 4.06 4 . 7 3 T i m I PFOS-22 22 454A-145- Wed, Jun 13, 2001 17:OO 4.98 In 1 period 4wFos use as Internal Standard 1: 4.08 Q1 MI, 297 scans 427.0 NoiseThres. 2.0 Quent Thres. 1.0 Mh. Wldth 3 Mult. width ' 6 Bass.W& 30 RTWin.(secs) 2 0 smoom 1 UCpectedRT 2.96 Area 318850 Height 42050 Slart Time 2.83 End Time 3.33 Integration Width 0.51 Retention Time 298 Integration Type A - BB intensiv. 55000 cps Figure 7. A representative ion chromatogram of a test sample (454A-145-22). Nominal Concentration= 25 mg a.i./L, Dilution factor= 1OOOOx. (monitored masses = 499 amu (PFOS-top) and 427 atnu (4HPFOS internal standard- bottom)). WildlifeInternational, Ltd. - 41 - Project Number 454A-145 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, test concentrations, and test and reference substance identification. 2. The test chambers used in the studywere polyethylene,but were not Teflon@-lined.The Teflon@-liners were included in the protocol in error, so this change to the protocol had no impact upon the study. WildlifeInternational, Ltd. - 42 - Project Number 454A-145 Appendix 5 Personnel Involved in the Study The followingkey WildlifeInternational, Ltd. personnel were involvedinthe onductorman gementof this study: 1. Henry 0.Krueger, Ph.D., Director, Aquatic Toxicologyand Non-Target Plants 2. Willard B. Nixon, Ph.D., Director, Analytical Chemistry 3. C q A. Sutherland, Laboratory Supervisor 4. Raymond L. Van Hoven, Ph.D., Scientist 5. Susan J. Palmer, Senior Biologist 6 . Molly McCoy, Biologist 7. Frank J. Lezotte,Chemist