Document bOvxxn82R8dwv8am7KjnZL2Vk

PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE RAINBOW TROUT (<Oncorhynchus mykiss) FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-145 ENVIRONMENTAL LABORATORY PROJECT 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 O. Krueger, Ph.D. STUDY INITIATION DATE: May 16,2001 STUDY COMPLETION DATE: January 7,2002 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 42 ,Wildlife International Ltd. 2- - Project Number 454A-145 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: Perfluorooctanesulfonate, Potassium Salt (PFOS): A 96-Hour Static Acute Toxicity Test with the Rainbow Trout (Oncorhynchus mykiss) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-145 STUDY COMPLETION: January 7,2002 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, NotificationNo. 3850, Agricultural Production Bureau, 10 August 1984. STUDY DIRECTOR: LU&44. Susan J. Palmi Senior Biologist SPONSOR APPROVAL: Wildlife International, Ltd. -3 - Project Number 454A-145 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 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. The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Test Substance Preparation M atrix Fortification, Analytical Sampling and Test Initiation Analytical D ata and Draft Report DATE CONDUCTED: June 8,2001 June 11,2001 July 11 & 12,2001 DATE REPORTED TO. STUDY DIRECTOR: MANAGEMENT: June 8,2001 June 13, 2001 June 11,2001 July 12,2001 June 14,2001 July 13, 2001 Biological D ata and D raft Report July 12,2001 July 12,2001 July 13,2001 Final Report January 7, 2002 January 7,2002 January 7, 2002 Date Wildlife International, Ltd. -4 - Project Number 454A -145 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: Perfluorooctanesulfonate, Potassium Salt (PFOS): A 96-Hour Static Acute Toxicity Test with the Rainbow Trout (Oncorhynchus mykiss) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-145 STUDY DIRECTOR: Susan J. Palmer, Senior Biologist WILDLIFE INTERNATIONAL. LTD, MANAGEMENT: Henry 0? Krueger,t E T ' Director, Aquatic Toxicology and Non-Target Plants D ate/ U. 2tX)Z. 0 1 Ja. Date ol Wildlife International, Ltd, -5 - Project Number 454A-145 TABLE OF CONTENTS Title/Cover Page..................................................................................................................................................... 1 Good Laboratory Practice Compliance Statement......................................................................................... 2 Quality Assurance Statement................................................................................................................................3 Report Approval.....................................................................................................................................................4 Table of Contents...................................................................................................................................................5 Summary................................................................................................................................................................. 7 N Introduction........................................................................................................................................................ 8 Objective........................................................................................................................... Experimental Design............................................................................................................................................. 8 Materials and M ethods.......................................................................................................................................... 9 Test Substance......................................................................................................................................... 9 Preparation o f Test Concentrations....................................................................................................... 9 Test Organism.......................................................................................................................................... 9 Test Apparatus....................................................................................................................................... 10 Dilution W ater.........................................................................................................................................10 Environmental Conditions........................................................................................... O b s e r v a tio n s ...........................................................................................................................................12 Statistical Analyses................................................................................................................................ 12 Analytical Chemistry.............................................................................. Results and Discussion............................................................................................................................ Measurement of Test Concentrations.................................................................................................. 12 Observations and M easurements..........................................................................................................13 12 Conclusions.......................................................................................................................................................... 13 References. 14 11 Wildlife International, Ltd. 6- - TABLE OF CONTENTS - Continued - Project Number 454A-145 TABLES AND FIGURES Table 1. Summary o f Analytical Chemistry D ata........................................................................................ 15 T able 2. Temperature, Dissolved Oxygen and pH o f Water in the Test Cham bers.................................16 Table 3. Cumulative Percent Mortality and Treatment-Related Effects.................................................... 17 T able 4. LC50 Values......................................................................................................................................18 Figure 1. Concentration-Response Curve (96-Hour D ata)...................... ....................................................19 APPENDICES Appendix 1. Specific Conductance, Hardness, Alkalinity, and pH of Well Water Measured During the 4-Week Period Immediately Preceding the T est...............................20 Appendix 2. Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well W ater...................................................................................................................................21 A p p en d ix 3. T he A nalysis o f PFO S in Freshw ater in Support o f W ildlife International, Ltd. Project No.: 454A -145.... ........................................................................................................ 23 Appendix 4. Changes to Protocol...................................................................................................................41 Appendix 5. Personnel Involved in the Study...............................................................................................42 Wildlife International, 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 WILDLIFEINTERNATIONAL, LTD. PROJECT NUMBER: TEST su b st a n c e: STUDY: MEAN MEASURED TEST CONCENTRATIONS: TEST d a tes: LENGTH OF TEST: 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 TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: MEASUREMENTS OF 10 NEGATIVE CONTROLFISH: WET WEIGHT: STANDARD LENGTH: TOTAL LENGTH: 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 = 2.8 to 3.4 cm 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. 8- - 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 from 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 study was to determine the acute effects o f Perfluorooctanesulfonate, Potassium Salt (PFOS) to the rainbow trout, Oncorhynchus mykiss, during a 96-hour exposure period under static test conditions. EXPERIMENTAL DESIGN Rainbow trout were exposed to a geometric series o f five test concentrations and a negative (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 also were maintained at the highest concentration. Nominal test concentrations were selected in consultation with the Sponsor, and were based upon the results of an exploratory range finding toxicity test. Nominal test concentrations selected were 3.1 ,6 .3 ,1 3 ,2 5 and 50 mg active ingredient (a.i.)/L. Mean measured test concentrations were determined from samples of test water collected from each treatment and the control group at the beginning of the test, at approximately 48 hours, and at test termination. Rainbow trout were indiscriminately assigned to exposure chambers at test initiation. Observations of mortality and other clinical signs o f toxicity were made at approximately 4 ,2 4 ,4 8 ,7 2 and 96 hours after test initiation. Cumulative percent mortality observed in the treatment groups was used to calculate or estimate LC50 values at 24,48,72 and 96 hours. The no mortality concentration and the no-observed-eflectconcentration (NOEC) were determined by visual interpretation of the mortality and clinical observation data. Wildlife International, Ltd. -9 - Project Number 454A-145 MATERIALS AND METHODS The study was conducted based on the procedures outlined in the protocol, "Perfluorooctanesulfonate, 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 Protection Agency Series 850 - Ecological Effects Test Guidelines, OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater and Marine (1); OECD Guideline for Testing o f Chemicals, 203: Fish, Acute Toxicity Test (2); and ASTM Standard E729-88a, Standard Guide fo r Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians (3). Test Substance The test substance was received from 3M Corporation on October 29, 1998 and was assigned Wildlife International, Ltd. identification number 4675. The test substance was described as a white powder. It was identified as FC-95 from lot number 217 (T-6295). Information provided by the Sponsor indicated a purity of 86.9% and an expiration date of August 31,2001. The test substance was stored at ambient room temperature. Preparation of Test Concentrations Nominal test concentrations were 3.1,6.3,13,25 and 50 mg a.i./L, based on a test substance purity of 86.9%. All materials which came into contact with the test substance during preparation o f test concentrations w ere constructed o f plastic or stainless steel. A 40-L prim ary stock solution w as prepared in 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 o f the test substance. Two replicates o f each test solution were prepared at concentrations o f 3 .1 ,6 .3 ,1 3 ,2 5 and 50 mg a.i./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 solution was stirred with a stainless steel whisk for approximately one 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 representative o f an important group o f aquatic vertebrates 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. Wildlife International, 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 approximately the same temperature as used during the test. During the 14-day holding period preceding the test, water temperatures ranged from 11.9 to 12.8C. The pH o f the water ranged from 8.2 to 8.4 and dissolved oxygen ranged from 9.2 to 10.3 mg/L (>85% o f saturation). Instrumentation and procedures used for water measurements are described in the Environmental Conditions section o f this report. The fish were 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 from the acclimation tank and indiscriminately distributed two at a time to the test chambers until each contained 10 fish. During the holding period, the fish were fed a commercially-prepared diet (Zeigler Brothers, Inc., Gardners, PA). The fish were not fed during the acclimation period (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 o f the test was 3.1 cm with a range of 2.8 to 3.4 cm, with an average total length o f 3.6 cm with a range o f 3.4 to 4.0 cm. The average wet weight (blotted dry) of 10 negative control fish at the end o f the test was 0.34 g with a range o f 0.25 to 0.47 g. Loading was 0.23 g fish/L of test water. Test Apparatus Test chambers were 25-L polyethylene aquaria containing 15 L o f test solution. The depth ofwater in a representative test chamber was approximately 17.5 cm. Test chambers were indiscriminately positioned in an environmental chamber set to maintain the 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 characterized as moderatelyhard water. The specific conductance, hardness, alkalinity and pH measurements o fthe well water during the four-week period immediately preceding the test are presented in Appendix 1. Wildlife International, 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 storage tank where the water was aerated with spray nozzles. 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 contaminants in the well water are presented in Appendix 2. Environmental Conditions Lighting used to illuminate the cultures and test chambers during holding, acclimation and testing was provided by fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone 50). A photoperiod o f 16 hours o f light and 8 hours o f 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 changes in lighting. Light intensity at test initiation was approximately 126 lux at the surface ofthe water of one representative test chamber. Temperature was measured in each test chamber at the beginning o f 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 ER/C Recorder, which was verified prior to test initiation with a liquid-in-glass thermometer. The target test temperature during the study was 12 1C. D isso lv ed o x y g en an d pH measurements were 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. 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 915 pH meter, and dissolved oxygen was measured using a Yellow Springs Instrument Model 5 IB dissolved oxygen meter. Specific conductance was measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter. Hardness and alkalinity measurements were made by titration based on procedures in Standard Methods fo r the Examination o f Water and Wastewater (4). Wildlife International, 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 o f toxicity or abnormal behavior also were evaluated. Observations were made approximately 4, 2 4 ,4 8 ,7 2 and 96 hours after test initiation. Statistical Analyses The mortality data were analyzed, when possible, using the computer program o f 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 any treatment group prior to the 96-hour observation period. Therefore, the 24,48 and 72-hour LC50 values were estimated to be greater than the highest concentration tested. 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 from each biotic test chamber o f each treatment and control group at the beginning of the test, at approximately 48 hours and at test termination to measure concentrations o f the test substance. Additional samples were collected on Day 1 o f the test from the n e g a tiv e co n tro l rep licates to c o n firm th e re su lts of th e Day 0 analysis. S am p le s also were collected for analysis at approximately 48 and 96 hours from the abiotic test chambers prepared at a concentration o f 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. RESULTS AND DISCUSSION Measurement of Test Concentrations Results of analyses to measure concentrations of PFOS in water samples collected during the test are presented in Table 1 and in the analytical chemistry report (Appendix 3). Nominal concentrations selected for use in this study were 3.1,6.3,13,25 and 50 mg a.i./L. Samples collected at test initiation had measured concentrations that ranged from 93 to 103% of nominal concentrations. Measured concentrations for biotic samples taken at 48 hours ranged from 94 to 103% of nominal, while the abiotic samples ranged from 105 to 106% o f nominal. Measured concentrations for biotic samples taken at 96 hours ranged from 91 to 105% of Wildlife International, Ltd. -13- Project Number 454A-145 nominal, while the abiotic samples were 102% of nominal. When measured concentrations o f the biotic samples analyzed at test initiation, approximately 48 hours and at test termination were averaged, the mean measured concentrations for this study were 3 .0,6.3,13,25 and 50 mg a.i./L, representing 97,100,100,100 and 100% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations. Observations and Measurements Measurements o f 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 >9.2 mg/L (85% of saturation) throughout the test. Measurements o f pH ranged from 8.1 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 o f 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 appeared normal and healthy during the test, with no mortalities or overt signs o f toxicity observed. After 96-hours of exposure, mortality in the 13, 25 and 50 mg a.i./L treatment groups was 20, 50 and 100%, respectively. LC50 values and 95% confidence intervals, estimated or calculated from the mortality data at 2 4 ,4 8 ,7 2 and 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% confidence interval o f 18 to 27 mg a.i./L. The 96-hour no mortality concentration and the NOEC were 6.3 mg a.i./L. Wildlife International, Ltd. - 14- Project Number 454A-145 REFERENCES 1 U.S. Environm ental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines {draft), OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater and Marine. 2 O rganisation for Economic C ooperation and Development. 1993. OECD Guidelines for Testing of Chemicals. Guideline 203: Fish, Acute Toxicity Test. Adopted by the Council on 12 July 1992. 3 ASTM S tan d ard E729-88a. 1994. Standard Guide fo r Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. American Society for Testing and Materials. 4 APHA, AW W A, W PCF. 1998. StandardM ethodsfor the Examination o f Water and Wastewater. 20th Edition. American Public Health Association. American Water Works Association. Water Pollution Control Federation, New York. 5 Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. 6 Finney, D.J. 1971. StatisticalMethods in BiologicalAssay. Second edition. Griffin Press, London. 7 Thom pson, W .R. 1947. Bacteriological Reviews. Vol. II, No. 2. Pp. 115-145. 8 Stephan, C.E. 1977. "Methods for Calculating an LC50", Aquatic Toxicology and Hazard Evaluations, American Society for Testing and Materials. Publication Number STP 634, pp 65-84. Wildlife International, Ltd. - 15- Project Number 454A-145 Table 1 Summary o f Analytical Chemistry Data Nominal Test Concentration1 (mg a.i./L) 0.0 (Negative Control) Sampling Time (Hours) 0 0 48 48 96 96 PFOS Measured Concentration2 (mg a.i./L) < L O Q 1'3 < LOQ1 < LOQ < LOQ < LOQ <LOQ Percent of Nominal2 -- -- - - Mean Measured Test Concentration (mg a.i./L) <LOQ Mean Measured Percent of Nominal _ 3.1 0 3.15 102 3.0 97 0 3.02 97.5 48 2.90 93.6 48 3.01 97.1 96 2.83 91.4 96 2.97 95.9 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 (Abiotic) 48 48 96 96 53.1 106 52.6 105 50.9 102 51.0 102 52 104 1 The limit o f quantitation (LOQ) was 0.200 mg a.i./L based upon the product o f the lowest calibration standard analyzed (0.000500 mg a.i./L) and the dilution factor o f the matrix blank samples (400). 2 Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since nominal and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes. 3 Results are mean o f duplicate re-preparations. Original Negative Controls were contaminated during analytical sampling. Wildlife International, Ltd. -16- Project Number 454A-145 Table 2 Temperature, Dissolved Oxygen and pH of Water in the Test Chambers Mean Measured Concentration (m g a.i./L) Rep. O H our1 Tem p.12 D O 3 (C) (mg/L) PH Negative Control A B 12.5 10.4 8.3 12.6 10.7 8.4 3.0 A 12.5 10.6 8.4 B 12.4 10.8 8.4 6.3 A 12.1 10.6 8.4 B 12.1 10.8 8.4 13 A 11.7 10.8 8.4 B 11.9 10.8 8.4 25 A 12.3 10.8 8.4 B 12.5 10.8 8.4 50 A 12.9 10.8- 8.4 B 12.9 10.8 8.4 C 4 13.8 10.0 8.3 D 4 13.7 10.2 8.3 24 Hours Temp. DO (C) (mg/L) pH 12.1 9.6 8.3 12.1 9.6 8.3 12.2 9.4 8.3 12.0 9.8 8.3 11.8 9.6 8.4 11.7 9.8 8.4 11.4 9.8 8.4 11.6 9.8 8.4 11.7 9.8 8.4 11.8 9.8 8.5 11.8 9.8 8.4 11.9 9.6 8.4 12.0 10.0 8.4 12.0 10.0 8.5 48 Hours Temp. DO (C) (mg/L) pH 12.1 9.4 8.2 12.2 9.4 8.2 12.3 9.4 8.2 12.0 9.4 8.2 11.7 9.4 8.2 11.7 9.3 8.2 11.3 9.3 8.2 11.6 9.2 8.2 11.6 9.3 8.2 11.7 9 .4 . 8.2 11.8 9.4 8.3 11.9 9.2 8.3 12.0 9.8 8.4 12.0 9.8 8.4 72 Hours Temp. DO (C) (mg/L) pH 12.1 9.4 8.2 12.3 9.4 8.2 12.5 9.4 8.2 12.2 9.4 8.2 11.8 9.4 8.2 11.7 9.4 8.2 11.4 9.2 8.3 11.6 9.3 8.3 11.7 9.2 8.3 11.7 9.3 8.3 11.9 9.4 8.3 11.9 9.2 8.3 12.0 9.8 8.4 12.0 9.8 8.4 96 Hours Temp. DO (C) (mg/L) pH 12.1 9.4 8.1 12.2 9.4 8.1 12.5 9.4 8.1 12.2 9.4 8.1 11.8 9.4 8.1 11.7 9.4 8.1 11.3 9.2 8.2 11.6 9.3 8.2 11.7 9.2 8.2 11.7 9.2 8.2 11.9 9.4 8.2 11.9 9.2 8.2 12.0 9.8 8.4 12.0 9.8 8.4 1 T he 0-hour dilution w ater m easurem ents for hardness, alkalinity and specific conductance w ere 132 mg/L as CaC03, 184 mg/L as C aC 03 and 270 |im hos/cm , respectively. 2 Tem perature m easured continuously during the test ranged from approxim ately 12.0 to 12.5C. 3 A dissolved oxygen concentration o f 6.5 mg/L represents 60% saturation at 12C in freshwater. 4 Abiotic replicate. Wildlife International, Ltd, - 17- Project Number 454A-145 Table 3 Cumulative Percent Mortality and Treatment-Related Effects Mean Measured Concentration (mg a.i./L) Rep. No. Exposed 4 Hours No. D ead1 Observed E ffects12 Negative Control A 10 0 10 AN B 10 0 10 A N 3.0 A 10 0 10 AN B 10 0 10 A N 6.3 A 10 0 10 AN B 10 0 10 A N 13 A 10 0 10 A N B 10 0 10 A N 25 A 10 0 10 A N B 10 0 10 A N 50 A 10 0 10 A N B 10 0 10 A N 1 Cumulative num ber o f dead fish. 2 Observed Effects: A N = appear normal. 24 Hours No. Observed Dead Effects 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 A N 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 A N 0 10 AN 48 Hours No. Observed Dead Effects 0 10 AN 0 10 A N 0 10 AN 0 10 A N 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 A N 0 10 AN 1 9 AN 0 10 A N 72 Hours No. Observed Dead Effects 0 10 AN 0 10 A N 0 10 AN 0 10 A N 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 A N 3 7 AN 4 6 AN 96 Hours No. Observed Dead Effects 0 10 AN 0 10 A N 0 10 AN 0 10 AN 0 10 AN 0 10 A N 1 9 AN 3 7 AN 6 4 AN 4 6 AN 10 -- 10 -- Cumulative Percent M ortality 0 0 0' 20 50 100 Wildlife International, Ltd. -18- Table4 LC50 Values Project Number 454A-145 Time 24 Hours LC50 (mg a.i./L) >50 95% Confidence Interval (mg a.i./L) J Statistical M ethod NA1 48 Hours 72 Hours >50 >50 __1 NA1 NA1 96 Hours 22 18-27 Probit Analysis 1 The LC50 value and 95% confidence interval could not be statistically calculated, The LC50 value w as estimated to be greater than the highest concentration tested. Wildlife International, Ltd. -19- Figure 1. Concentration-Response Curve (96-Hour Data) Project Number 454A-145 1 100 Wildlife International, 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 (pmhos/cm) Hardness (mg/L as 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 Wildlife International, Ltd, - 21- Project Number 454A-145 Appendix 2 Analyses o f Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Pesticides and Organics Component Measured Concentration Component Measured Concentration Aclonifen Alachlor Ametryn Atrazine Azinphos-ethyl Azinphos-methyl Azoxystrobin Bifenthrin Bioallethrin Bitertanol Bromacil Bromophos Bromophos-ethyl Bromopropylate Bupirimate Carbaryl Carbofuran Carboxin Chlorfenvinphos Chloridazon Chlorpropham Chlorpyrifos Chlorpyrifos-methyl Chlorothalonil Coumaphos Cyanazine Cyfluthrin Cypermethrin Cyproconazole Deltamethrin Demeton Demeton-O Desethylatrazine Desisopropylatrazine Desmetryn Diazinon Dichlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid <0.03 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.04 pg/L <0.08 pg/L <0.25 pg/L <0.05 pg/L <0.05 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.03 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.04 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.25 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.03 pg/L <0.02 pg/L <0.01 pg/L <0.03 pg/L Dichlorvos Dicofol Diethyltoluamide Difenoconazole Dimethoate Dimethomorph Disulfoton DMST Dodemorph Endosulfan-a Endosulfan- Endosulfan-sulfate Epoxiconazole Eptam Esfenvalerate Ethion Ethofumesate Ethoprophos Etridiazole Etrimfos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb Fenpiclonil Fenpropathrin Fenpropimorph Fenthion Fenvalerate Fluazifop-butyl Fluoroglycofen-ethyl Fluroxypyr-meptyl Flutolanil Fonophos Furalaxyl Heptenophos Imazalil Iprodion Kresoxim-methyl Lenacil Lindane <0.01 pg/L <0.25 pg/L <0.02 pg/L <0.03 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0.03 pg/L <0.03 pg/L <0.05 pg/L <0.25 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/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 <0.02 pg/L <0.05 pg/L <0.02 pg/L `Analyses performed by TNO Nutrition and Food Institute on samples collected on November 15,2000. Wildlife International, Ltd, -22- Project Number 454A-145 Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Component Pesticides And Organics (Page 2) Measured Concentration Component Measured Concentration Malathion Metalaxyl Metamitron Metazachlor Methidathion Paclobutazole Parathion Parathion-methyl Penconazole Pendimethalin Permethrin-cis Permethrin-trans Phosalone Phosmet Phosphamidon-cis Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazine Propham Propiconazole Propoxur Propyzamide Prosulfocarb Pyrazophos <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.03 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.03 pg/L <0.02 pg/L <0.02 pg/L <0.03 pg/L Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl Pyrifenox-1 Pyrifenox-2 Pyrimethanil Quizalofop-ethyl Simazine Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbuthylazine Tetrachlorvinphos Tetrahydroftalimide Tetramethrin Thiabendazole Thiometon Tolclofos-methyl Tolylfluanid Triadimefon Triadimenol Triallate Triazophos Trifluralin Vamidothion Vinclozolin <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.05 pg/L <0.04 pg/L <0.01 pg/L <0.04 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L Metals Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 13.2 mg/L 21 mg/L 35 mg/L <0.02 mg/L 6.2 mg/L <0.09 mg/L 0.72 pg/L <0.3 pg/L <0.6 pg/L <0.4 pg/L Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium <1.4 pg/L <1.0 pg/L <2.3 pg/L <0.7 pg/L <0.3 pg/L <0.3 pg/L <0.25 pg/L <0.025 pg/L <1 pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on November 15,2000. Wildlife International, Ltd. -23- Appendix 3 Project Number 454A-145 THE ANALYSIS OF PFOS IN FRESHWATER IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-145 Wildlife International, Ltd. -24- Project Number 454A-145 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: Perfluorooctanesulfonate, Potassium Salt (PFOS): A 96-Hour Static Acute Toxicity Test with the Rainbow Trout (Oncorhynchus mykiss) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-145 PRINCIPAL INVESTIGATOR: MANAGEMENT: Willard B. Nixon, Ph.D. Director, Analytical Chemistry yCl Ion -- DATE DATE Wildlife International, Ltd. -25- Project Number 454A-145 Introduction Freshwater samples were collected from a 96-hour toxicity test designed to determine the effects of PFOS (Perfluorooctanesulfonate, Potassium Salt) to the rainbow trout (Oncorhynchus mykiss). This study was conducted by Wildlife International, Ltd. and identified as Project No.: 454A-145. The analyses ofthese water samples were performed at Wildlife International, Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). Samples were received for analysis on June 11,13, and 15, 2001 and were analyzed between June 11 and June 18,2001. Test Substance and Internal Standard The test substance used for this study was Wildlife International, Ltd. identification number 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 assigned Wildlife International, Ltd. identification number 4526 upon receipt. The internal standard, a granular material, was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2. The standard, referred to hereafter as 4H PFOS, was stored under ambient conditions. Analytical Method The method used for the analysis o f the freshwater samples was developed at Wildlife International, Ltd. and entitled "Analytical Method for the Determination o f PFOS in Freshwater, Saltwater, and Algal Medium". This methodology was included as Appendix II of Wildlife International, Ltd. protocol number 454/011299/MVAL/SUB454. It was based upon methodology provided by 3M Corporation. Several modifications from the validated methodology were implemented for the present study. First, the concentration o f 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 o f the analytical column and faster run times. Wildlife International, Ltd. -26- Project Number 454A-145 Samples were diluted in a 50% methanol: 50% NANOpure water solution containing 10.0 pg 4H P FO SIL 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 100LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil Cig analytical column (50 mm x 2 mm I.D., 3-pm particle size) fitted with a Keystone Javelin Cig guard cartridge (20 x 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 o f PFOS prepared in a 50% m ethanol: 50% NANOpure water solution containing 10.0 pg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v), ranging in concentration from 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 generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratio s (PFOS : internal standard) of th e calib ratio n standards. A typical calib ratio n curve is p resen ted in Figure 2. The concentration o f PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms o f low and high calibration standards are presented in Figures 3 and 4, respectively. The method limit o f quantitation (LOQ) for these analyses was set at 0.200 mg a.i./L calculated as the product o f 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 Three matrix blank samples were analyzed to determine possible interference. No interferences were observed at or above the LOQ during samples analyses (Table 2). A representative ion chromatogram of a matrix blank is presented in Figure 5. Wildlife International, Ltd. -27- Project Number 454A-145 Freshwater was fortified at 1.00,10.0 and 100 mg a.i./L and analyzed concurrently with the samples to determine the mean procedural recovery (Table 3). Sample concentrations were not corrected for the mean procedural recovery of 101%. A representative ion chromatogram o f a matrix fortification is presented in Figure 6. Example Calculations Sample number 454A-145-18, nominal concentration o f 6.3 mg a.i./L in freshwater. Peak Area Ratio = Analyte Peak Area/Intemal Standard Peak Area Concentration Ratio = Concentration o f Analyte/Concentration o f 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: 319027 Peak Area Ratio: 1.0969 Calibration curve equation. Slope: 3.3385 Intercept: 0.0235 Curve is weighted (1/x) Peak area ratio - (Y-intercept) PFOS (mg a.i./L) at instrument = Slope x Internal Standard Concentration = 0.003215 Wildlife International, 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 (mg a.i./L) in sample Percent of Nommai Concentration = ?pQS (fflg a i /L) nominaI x 100 6.43 6.3 x 100 = 102% Quantitation software for 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 o f PFOS in the samples collected at initiation o f exposure of the test organisms (Hour 0) ranged from 93.2 to 103% of the nominal concentrations. Samples collected at Day 2 had a measured concentration range o f 93.6 to 103% of nominal values. Samples collected at test termination (Day 4) had a measured concentration range 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. Wildlife International, Ltd. -29- Project Number 454A-145 INSTRUMENT: Table 1 Typical HPLC/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perldn-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: GUARD COLUMN: Keystone Betasil C u column (50 mm x 2 mm I.D., 3-pm particle size) Keystone Javelin Cis column (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 |xL PFOS RETENTION TIME: Approximately 4.4 minutes INTERNAL STANDARD RETENTION TIME: Approximately 3.0 minutes PFOS MONITORED MASS: 499 amu INTERNAL STANDARD MONITORED MASS: 427 amu Wildlife International, Ltd. -30- Project Number 454A-145 Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-145-) MAB-1 Sample Type Matrix Blank Measured Concentration of -- PPD1 (mg a.i./L) <LOQ MAB-2 Matrix Blank <LOQ MAB-32 Matrix Blank <LOQ 1 The limit o f 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). 2 Result is mean o f duplicate re-preparations. Original matrix blank was contaminated during analytical sample preparation. Wildlife International, Ltd. -31- Project Number 454A-145 Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-145-) MAS-1 MAS-4 MAS-7 Concentrations of PFOS (mg a.i./L) Fortified1 Measured1 1.00 1.15 1.00 0.972 1.00 1.04 Percent Recovered1 115 97.2 104 MAS-2 10.0 9.61 MAS-5 10.0 9.69 MAS-8 10.0 10.4 96.1 96.9 104 MAS-3 MAS-6 MAS-9 100 100 100 91.9 102 101 91.9 102 101 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. Wildlife International, Ltd. -32- Project Number 454A-145 Table 4 Measured Concentrations of PFOS in Freshwater Samples from a Rainbow Trout (Oncorhynchus mykiss) 96-Hour Toxicity Test Nominal Test Concentration1 (mg a.i./L) 0.0 (Negative Control) Sample Number (454A-145-) 1A 2A 13 14 27 28 Sampling Time (Day) 0 0 2 2 4 4 PFOS Measured Concentration12 (mg a.i./L) < LOQ1,3 <LOQ3 <LOQ <LOQ <LOQ <LOQ Percent of Nominal2 -- - -- 3.1 3 0 40 15 2 16 2 29 4 30 4 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 0 60 17 2 18 2 31 4 32 4 6.22 98.7 6.21 98.6 6.16 97.8 6.43 102 6.15 97.6 6.60 105 13 7 0 80 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 o f the matrix blank samples (400). 2 Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since nominal and measured concentrations were 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 analytical sampling. Wildlife International, Ltd. 33- - Project Number 454A-145 Table 4 (Continued) Measured Concentrations o f PFOS in Freshwater Samples from a Rainbow Trout (Oncorhynchus mykiss)96-Hour Toxicity Test Nominal Test Concentration1 (mg a.i./L) 25 Sample Number (454A-145-) 9 10 21 22 35 36 Sampling Time (Day) 0 0 2 2 4 4 PFOS Measured Concentration12 (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 0 12 0 23 2 24 2 37 4 38 4 49.7 99.4 49.8 99.5 51.1 102 51.5 103 49.6 99.1 50.8 102 50 (Abiotic) 25 26 39 40 2 2 4 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). 2 Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since nominal and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes. Wildlife International, 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 o f PFOS stock solutions directly into freshwater. Perform fortifications with gas-tight syringes and Class A volumetric flasks. Prepare appropriate dilutions of study and QC samples to within range of the PFOS HPLC/MS methodology: Partially fill Class A volumetric flasks with 50% methanol : 50% NANOpure water dilution solvent containing 10.0 pg 4H PFOS/L and 0.05% (v/v) formic acid. Add appropriate volume o f 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 fortification level. Mix well by several repeat inversions. Ampulate samples and submit for HPLC/MS analysis. Figure 1. Analytical method flowchart for the analysis o f PFOS in freshwater. Wildlife International, Ltd. 35- - PF06 499.0 Internal Standard: 4HPFOS Weighted (1/x) Intercept = 0.0235 Slope 3.3385 Correlation Coeff. > 0.9994 Area(Ratio) Project Number 454A-145 Figure 2. A typical calibration curve for PFOS. Wildlife International, Ltd. -36- Project Number 454A-145 PF0S_1 STD 0.500 ug a.IVL 4675A-011D -21 4.98 in 1 period PROS Internal Standard: 4HPFOS Use Area Absolute Retention Time 1: 4.98 Q1 Ml. 297 scans 499.0 Noise Thres. 3.0 Quant Thres. 1.0 Min. Width Mult. Width Base. Width RT Win. (secs) Smooth Expected RT 3 6 80 20 1 4.34 Area 55560 Height 5767 Start Time End Time Integration Width Retention Time Integration Type 4.12 4.87 0.74 4.38 A-VB Wed. Jun 13. 2001 14:48 intensity: 55000 cps PFOSjl STD 0.500 ug a.lVL 4675A-011D -21 4.98 in 1 period 4HPF0S use as Internal Standard 1: 4.98 Q1 Ml, 297 scans 427.0 Noise Thres. 2.0 Quant Thres. 1.0 M in. W idth ' 3 Mutt. Width 6 Base. Width 30 RT Win. (secs) Smooth Expected RT 20 1 2.95 Area 299568 Height 40766 Start Time 2.83 End Time 3.33 Integration Width Retention Time 0.51 2.96 Integration Type A-B8 Wed. Jun 13. 2001 14:48 intensity: 55000 cps Figure 3. A representative ion chromatogram o f a low-level (0.500 pg a.i./L) PFOS standard. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard bottom)). Wildlife International, Ltd, -37- Project Number 454A-145 PFOS_5 STD 5.00 ug a.IJL 4675A-011D -25 4.98 in 1 period PROS Internal Standard: 4HPFOS Use Area Absolute Retention Time 1: 4.98 Q1 Ml, 297 scans 499.0 Noise Thres. 3.0 Quant Thres. 1.0 Min. Width 3 Mult. Width 6 Base. Width 80 RT Win. (secs) 20 Smooth 1 Expected RT 4.34 Area 507550 Height 50920 Start Time End Time Integration Width Retention Time Integration Type 4.12 4.90 0.77 4.39 A -VB Wad. Jun 13, 2001 15:13 100908070 60504030 2010o- PFOS_5 STD 5.00 ug a.IJL 4675A-011D -25 - 4.98 in 1 period 4HPFOS use as Internal Standard 1: 4.98 Q1 Ml, 297 scans 427.0 Noise Thres. 2.0 Quant Thres. Min. Width . Mult. Width 1.0 3 6 Base. Width 30 RT Win. (secs) 20 Smooth 1 Expected RT 2.95 Area 304762 Height 40262 Start Time 2.83 End Time 3.35 Integration Width 0.52 Retention Time 2.96 Integration Type A-BB Wed, Jun 13, 2001 15:13 Intensity: 55000 cps 261 Intensity: 55000 cps Figure 4. A representative ion chromatogram of a high-level (5.00 jig a.i./L) PFOS standard. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard bottom)). Wildlife International, Ltd. -38- Project Number 454A-145 PF0S_7 MAB-2 454A-145- Wed. Jun 13, 2001 15:25 4.98 In 1 period FFOS Internal Standard: 4HPFOS Use Area Absolute Retention Time 1: 4.98 Q1 Ml, 297 scans 499.0 Noise Thres. Quant Thres. Min. Width Mult. Width 3.0 1.0 3 6 Base. Width 80 RT Win. (secs) 20 Smooth 1 Expected RT 4.34 Area 833 Height 94 Start Time End Time Integration Width Retention Time Integration Type 4.21 4.83 0.42 4.41 A - BB 100908070605040302(7 10o-l PFOS_7 MAB-2 454A-145- 4.98 in 1 period 4HPF0S use as Internal Standard Wed, Jun 13, 2001 15:25 1: 4.98 Q1 Ml, 297 scans 427.0 Noise Thres. 2.0 Quant Thres. Min. Width M ult. W idth Base. Width RT Win. (secs) 1.0 3 6 30 20 Smooth Expected RT 1 2.95 Area 318679 Height 42248 Start Time 2.83 End Time 3.33 Integration Width Retention Time Integration Type 0.51 2.98 A-BB intensity: 55000 cps 1 114 "81 ' 121 1.38 2.04 178 217 240.262,, 161 201 241 281 Scan 2.71 3.38 4.06 4.73 Time Intensity: 55000 cps Figure 5. A representative ion chromatogram of a matrix blank sample (454A-145-MAB-2). Dilution factor = 400x. The arrow indicates the retention time of PFOS. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard - bottom)). Wildlife International, Ltd, -39- Project Number 454A-145 PF0S_9 MAS-5 464A-145- Wed, Jun 13, 2001 15:38 4.98 in 1 period PROS Internal Standard: 4HPFOS Use Area Absolute Retention Time 1: 4.98 Q1 Ml, 297 scans 499.0 Noise Titres. 3.0 Quant Titres. 1.0 Min. Width Mult. Width 3 6 Base. Width 80 RT Win. (secs) 20 Smooth 1 ExpectedRT 4.34 Area 264966 Height 27164 Start Time 4.14 End Time Integration Width Retention Time 4.90 0.76 4.38 Integration Type A - VB PFOS_9 MAS-5 454A-145- 4.98 In 1 period 4HPFOS use as Internal Standard Wed, Jun 13. 2001 15:38 1: 4.98 Q1 Ml, 297 scans 427.0 Noise Thres. 2.0 Quant Thres. 1.0 Min. Width 3 Mult Width 6 Base. Width 30 RT Win. (secs) Smooth Expected RT 20 1 2.95 Area 318348 Height 43093 Start Time EndTime 2.81 3.32 Integration Width 0.51 Retention Time 2.96 Integration Type A - BB intensity: 55000 cps intensity: 55000 cps Figure 6. A representative ion chromatogram o f a matrix fortification sample (454A-145-MAS-5). Nominal Concentration = 10.0 mg a.i./L, Dilution factor = 4000x. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard - bottom)). Wildlife International, Ltd. -40- Project Number 454A-145 PFOS_22 454A-145- 22 Wed, Jun 13, 2001 17:00 4.98 in 1 period FFOS InternalStandard: 4HPFOS Use Area Absolute Retention Time 1: 4.98 Q1 Ml. 297 scans 499.0 Noise Thres. 3.0 Quant Thres. Min. Width Mult. Width 1.0 3 6 Base. Width 80 RT Win. (secs) 20 Smooth 1 Expected RT 4.34 Area 281742 Height 28749 Start Time 4.14 End Time 4.88 Integration Width 0.74 Retention Time 4.39 Integration Type A -VB PFOS_22 454A-145- 22 Wed, Jun 13, 2001 17:00 4.98 in 1 period 4HPFOS use as Internal Standard 1: 4.98 Q1 Ml, 297 scans 427.0 Noise Thres. 2.0 Quant Thres. Min. Width Mult. Width ' 1.0 3 6 Base. Width RT Win. (secs) 30 20 Smooth Expected RT 1 2.95 Area 319B58 Height 42050 Start Time 2.83 End Time 3.33 Integration Width 0.51 Retention Time 2.98 Integration Type A-BB intensity: 55000 cps intensity. 55000 cps Figure 7. A representative ion chromatogram of a test sample (454A-145-22). Nominal Concentration = 25 mg a.i./L, Dilution factor = lOOOOx. (monitored masses = 499 amu (PFOS --top) and 427 amu (4HPFOS internal standard -- bottom)). Wildlife International, 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 study were 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. Wildlife International, Ltd. - 42- Project Number 454A-145 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 O. Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Director, Analytical Chemistry 3. Cary A. Sutherland, Laboratory Supervisor 4. Raymond L. Van Hoven, Ph.D., Scientist 5. Susan J. Palmer, Senior Biologist 6. Molly McCoy, Biologist 7. Frank J. Lezotte, Chemist