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BACK TO MAIN PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (Lepomis macrochirus) FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-134 ENVIRONMENTAL LABORATORY REQUEST NUMBER: U2723 U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1730 and OECD Guideline 305 AUTHORS: Kurt R. Drottar Raymond L. VanHoven, Ph.D. Henry O. Krueger, Ph.D. STUDY INITIATION DATE: October 31, 2000 STUDY COMPLETION DATE: June 21, 2001 Submitted to 3M Corporation Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 129 BACK TO MAIN Wildlife InternationalTLtd. 2- - Project Number 454A-134 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: Perfluorooctanesulfonate, Potassium Salt (PFOS): A Flow-Through Bioconcentration Test with the Bluegill (Lepomis macrochirus) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-134 STUDY COMPLETION: June 21, 2001 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Part 160,17 August 1989. STUDY DIRECTOR: Senior Biologist DATE Wildlife International. Ltd. -3 - BACK TO MAIN Project Nnmher 454A-134 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Part 160, 17 August 1989. 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: DATE CONDUCTED: Test Substance Preparation November 29, 2000 Matrix Fortification December 8, 2000 Water Chemistry February 20, 2001 Analytical Data and Draft Report May 14 - 17 and May 2 0 - 2 4 , 2001 Biological Data and Draft Report June 11 - 13,2001 Final Report June 20, 2001 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: November 29, 2001 November 30, 2000 December 8, 2000 December 11, 2000 February 20, 2001 February 22, 2001 May 24, 2001 June 1,2001 June 13,2001 June 20, 2001 June 14, 2001 June 20, 2001 Wildlife InternationalTLtd -4- REPORT APPROVAL BACK TO MAIN Project Number 4 5 4 A -134 SPONSOR: 3M Corporation TITLE: Perfluorooctanesulfonate, Potassium Salt (PFOS): A Flow-Through Bioconcentration Test with the Bluegill (Lepomis macrochirus) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-134 STUDY DIRECTOR: Kurt R. Drottar Senior Biologist MANAGEMENT: Director of Aquatic Toxicology and Non-Target Plants DATE Wildlife International, Ltd.____________________ -5- BACK TO MAIN Project Number 454A-134 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.............................................................................................................................................................. 8 Introduction......................................................................................................................................................... 9 Objective............................................................................................................................................................. 9 Experimental Design.......................................................................................................................................... 9 Materials and Methods..................................................................................................................................... 10 Results and Discussion..................................................................................................................................... 15 Conclusions....................................................................................................................................................... 17 References......................................................................................................................................................... 18 TABLES Table 1 - Means and Ranges of Water Quality Parameters........................................................................ 19 Table 2 - Concentrations of PFOS in Water Samples During the Uptake Phase..................................... 20 Table 3 - Concentrations of PFOS in Water Samples During the Depuration Phase...............................21 Table 4 - PFOS Concentrations in Edible, Nonedible and Whole Fish Tissues of Bluegill Exposed to 0.086 mg a.i./L............................................................................22 Table 5 - Apparent Steady-State BCF Values for Bluegill Exposed to 0.086 mg a.i./L.......................... 25 Table 6 - BIOFAC Model Estimates for Bluegill Exposed to 0.086 mg a.i./L ........................................ 26 Wildlife International, Ltd.____________________ -6- BACK TO MAIN Project Number 454A-134 TABLE OF CONTENTS - Continued - TABLES (Cont'd.) Table 7 - PFOS Concentrations in Edible, Nonedible and Whole Fish Tissues of Bluegill Exposed to 0.87 mg a.i./L............................................................................ 27 Table 8 - Day 28 BCF Values for Bluegill Exposed to 0.87 mg a.i./L......................................................29 FIGURES Figure 1 Concentrations of PFOS in Edible Fish Tissues of Bluegill Exposed to 0.086 mg a.i./L.......................................................................................................................... 30 Figure 2 Concentrations of PFOS in Nonedible Fish Tissues of Bluegill Exposed to 0.086 mg a.i./L.......................................................................................................................... 31 Figure 3 Concentrations of PFOS in Whole Fish Tissues of Bluegill Exposed to 0.086 mg a.i./L.......................................................................................................................... 32 Figure 4 Concentrations of PFOS in Edible, Nonedible and Whole Fish Tissues of Bluegill Exposed to 0.87 mg a.i./L............................................................................................................................. 33 Wildlife International, Ltd.____________________ -7- BACK TO MAIN Project Number 454A-134 TABLE OF CONTENTS - Continued - APPENDICES Appendix 1 Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test....................... 34 Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well W ater..................................................................... 35 Appendix 3 The Analysis of Perflurooctanesulfonate, Potassium Salt (PFOS) Concentrations in Freshwater and Bluegill Sunfish Tissue in Support of Wildlife International, Ltd. Project No.: 454A-134..................................................... 37 Appendix 4 Temperature and pH of Water in the Test Chambers..................................................... 88 Appendix 5 Dissolved Oxygen of Water in the Test Chambers........................................................... 90 Appendix 6 Hardness, Alkalinity, Conductivity and TOC of Water in the Negative Control.......... 94 Appendix 7 Cumulative Mortality and Treatment-Related Effects..................................................... 95 Appendix 8 Changes to Protocol......................................................................................................... 104 Appendix 9 Protocol, Amendments and Deviations............................................................................. 105 Appendix 10 Personnel Involved in the Study....................................................................................... 129 Wildlife International, Ltd.____________________ -8- BACK TO MAIN Project Number 454A-134 SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: SUMMARY 3M Corporation Ms. Susan A. Beach Wildlife International, Ltd. Easton, Maryland 21601 WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: NOMINAL TEST CONCENTRATIONS: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST (0.086 mg a.i./L): LENGTH OF TEST (0.87 mg a.i./L): 454A-134 Perfluorooctanesulfonate, Potassium Salt (PFOS) Perfluorooctanesulfonate, Potassium Salt (PFOS): A Flow Through Bioconcentration Test with the Bluegill (Lepomis macrochirus) Negative Control, 0.10 and 1.0 mg a.i./L Negative Control, 0.086 and 0.87 mg a.i./L. Exposure to 0.87 mg a.i./L ceased after 35 days due to fish mortality and tissue sampling Experimental Start (OECD) - November 29, 2000 Experimental Start (EPA) - December 5, 2000 Biological Termination - April 2, 2001 Experimental Termination - April 11, 2001 118 Days (62-Day Uptake, 56-Day Depuration) 35 Days of Uptake TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: MEASUREMENTS OF 10 NEGATIVE CONTROL FISH COLLECTED AT TEST TERMINATION: WEIGHT (g): TOTAL LENGTH (mm): Bluegill (Lepomis macrochirus) Osage Catfisheries, Inc. 1170 Nichols Road Osage Beach, Missouri 65065 Juveniles Mean = 2.70; Range = 2.03 to 3.32 Mean = 62; Range = 56 to 66 RESULTS: (0.086 mg a.i./L) APPARENT STEADY-STATE BCF: Edible 484 Nonedible 1124 Whole Fish 856 RESULTS: (0.87 mg a.i./L) Edible Nonedible Whole Fish DAY 28 BCF*: 136 386 278 *Note: The BCFs were underestimated due to fish mortality prior to achieving steady-state. Wildlife International, Ltd.____________________ -9- BACK TO MAIN Project Number 454A-134 INTRODUCTION A bluegill sunfish, Lepomis macrochirus, bioconcentration study was conducted for 3M Corporation at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase of the test was conducted from December 5, 2000 to April 2, 2001. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454A-134 in archives located on the Wildlife International, Ltd. site. OBJECTIVE The objective of this study was to determine the bioconcentration potential of perfluorooctanesulfonate, potassium salt (PFOS) in the bluegill sunfish. EXPERIMENTAL DESIGN The bioconcentration test consisted of a 62-day uptake phase followed by a 56-day depuration phase. During the uptake phase, the test organisms were exposed in one of three groups: 1) A negative (dilution water) control; 2) A nominal concentration of 0.10 mg active ingredient (a.i.)/L; or 3) A nominal concentration of 1.0 mg a.i./L. At the start of the depuration phase, stock flow to the treated groups was stopped and the bluegill were exposed to dilution water without PFOS for the remainder of the test. Each test chamber contained 90 bluegill at test initiation, and one replicate was tested for each treatment and the negative control. Water samples were collected on Day -4 (pre-test), Day -1 (pre-test) on uptake Days 0 (0 and ~4 hours), 1, 3, 7, 14, 21, 28, 35, 42, 49, 56 and 62 and on depuration Days 14, 28, 42 and 56 during the test and analyzed for PFOS using liquid chromatography-mass spectrometry (LC/MS). Tissue samples were also collected at selected water sample collection periods during the test and analyzed for PFOS by LC/MS. The results of these analyses were used to calculate the BCF values, uptake rates and depuration rates in edible tissue, nonedible tissue and whole fish. Wildlife International, Ltd.____________________ - 10 - BACK TO MAIN Project Number 454A-134 MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "Perfluorooctanesulfonate, Potassium Salt (PFOS) (Appendix 9): A Flow-Through Bioconcentration Test with the Bluegill (Lepomis macrochirus) ". The protocol was based on procedures outlined in U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1730 (1); ASTM Standard E1022-84 Standard Practice fo r Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve Molluscs (2); and OECD Guideline for Testing of Chemicals 305, Bioconcentration: Flow-Through Fish Test (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, a white powder, was identified as FC-95, Lot #217. Information provided by the Sponsor indicated a purity of 86.9% and an expiration date of August 31, 2001. The test substance was stored under ambient conditions. Preparation of Test Solutions The nominal test concentrations were 0.10 and 1.0 mg a.i./L. Two stock solutions were prepared at concentrations of 10 and 100 mg a.i./L. The appropriate amount of test substance was weighed out and dissolved in dilution water for each stock. The stock solutions were stirred with an electric top-down mixer to aid in the solubilization of the test substance. After mixing, the stock solutions appeared clear and colorless. Stock solutions were prepared at approximately weekly intervals during the uptake phase of the test. The stock solutions were injected into the diluter mixing chambers (at a rate of 3.5 mL/minute) where they were mixed with dilution water (at a rate of 350 mL/minute) to achieve the desired test concentrations. All test solutions appeared clear and colorless. Test Organism The bluegill, Lepomis macrochirus, was selected as the test species for this study. The bluegill is one of the recommended freshwater fish species for use in bioconcentration tests (1, 2, 3). Bluegill used in the test were obtained from Osage Catfisheries, Inc., Osage Beach, Missouri. The fish were approximately 7 months old at test initiation. Identification of the species was verified by the supplier. Wildlife International, Ltd.____________________ - 11 - BACK TO MAIN Project Number 454A-134 The bluegill were held in Wildlife International, Ltd. well water for 103 days prior to testing. The fish were acclimated to test conditions for approximately 49 hours prior to test initiation. During the holding and acclimation periods the fish showed no signs of disease or stress. During the 14-day holding period preceding the test, water temperatures ranged from 22.3 to 22.7C. The pH of the water ranged from 7.8 to 8.2 and dissolved oxygen ranged from 7.6 to 8.2 mg/L. Instrumentation used for water measurements are described in the Environmental Conditions section of this report. At test initiation, the bluegill were collected from the acclimation tank and indiscriminately distributed 1 to 2 at a time into the test chambers until each chamber contained 90 fish. During holding and during the test, the bluegill were fed flake food supplied by Zeigler Brothers, Inc., Gardners, Pennsylvania. The bluegill were fed at least once daily during holding and once daily during the test. Feeding and sampling schedules were coordinated so that fish were sampled at least four hours after feeding. All fish used in the test were from the same source and year class, and the standard length of the longest fish was no more than twice the length of the shortest. The length and weight of fish in the negative control were considered to be representative of all fish used in the test. The mean total length of 10 negative control fish measured at the end of the test was 62 mm with a range of 56 to 66 mm. The average wet weight (blotted dry) was 2.70 grams with a range of 2.03 to 3.32 grams. Loading was defined as the total wet weight of fish per liter of test water that passed through the test chamber in 24 hours, and was determined to be 0.48 g fish/L/day. The loading rate was based on the average weight of the fish at the end of the test and the initial stocking density (90 fish per tank). Test Apparatus A continuous-flow diluter was used to deliver each concentration of the test substance and a negative control. A peristaltic pump (Cole-Parmer Instrument Company, Chicago, Illinois) was used to deliver the test substance stock solutions into mixing chambers assigned to each PFOS treatment. The stock solutions were mixed with dilution water in the mixing chambers prior to delivery to the test chambers. The flow of dilution water to the test chambers was controlled by rotameters. The delivery of water from the rotameters was checked prior to the test and at approximately weekly intervals thereafter. Approximately 6.3 volume additions of test water were delivered to the test chambers every 24 hours. The general operation of the diluter was checked at least two times a day during the test and once on the last day of the test. Wildlife International, Ltd.____________________ - 12 - BACK TO MAIN Project Number 454A-134 Test chambers were 104-L stainless steel aquaria filled with approximately 80 L of test solution. The depth of the test water in a representative chamber was approximately 19 cm. Test chambers were indiscriminately positioned in a temperature-controlled water bath designed to maintain a constant temperature. The water bath was enclosed in a plexiglass ventilation hood in order to minimize any potential for cross contamination. Test chambers were siphoned daily and periodically cleaned during the test to remove excess feed and fecal matter. Test chambers were identified by the project number and test concentration. Dilution W ater The water used for holding and testing was freshwater obtained from a well approximately 45 meters deep located on the Wildlife International, Ltd. site. The well water is characterized as moderately-hard water. The specific conductance, hardness, alkalinity and pH measurements of the well water during the four-week period immediately preceding the test are presented in Appendix 1. The well water was passed through a sand filter to remove particles greater than approximately 25 g,m, and pumped into a 37,800-L storage tank and aerated with spray nozzles. Prior to use, the water again was filtered (0.45g,m) to remove microorganisms and particles. The results of periodic analyses performed to measure the concentrations of selected contaminants in well water used by Wildlife International, Ltd. are presented in Appendix 2. Environmental Conditions The target temperature range for the test was 22 1C. Temperature was recorded continuously in the negative control with a Fulscope ER/C Recorder (1900 J Series model no. A). Temperature was also measured in all test chambers at the beginning and end of the test and at weekly intervals during the test, with a liquid-in glass thermometer. Dissolved oxygen was measured with a Yellow Springs Instrument Company, Inc. Model 51B dissolved oxygen meter. With the exception of uptake Day 24, measurements were made daily in each test chamber. Dissolved oxygen measurements were not made on uptake Day 24 due to biologist oversight. Measurements of pH were made in each test chamber at the beginning and end of the test and at weekly intervals during the test using a Fisher Accumet Model 915 pH meter. Wildlife International, Ltd.____________________ - 13 - BACK TO MAIN Project Number 454A-134 Hardness, alkalinity, conductivity and total organic carbon (TOC) were measured in the negative control at the beginning and end of the test, and at weekly intervals during the test. Hardness and alkalinity were measured by titration based on procedures in StandardMethodsfo r the Examination o f Water and Wastewater (4). Conductivity was measured using a Yellow Springs Instrument Company, Inc. Model 33 SalinityConductivity Temperature meter. Total organic carbon was measured using a Shimadzu model TOC-5000 total organic carbon analyzer. Ambient room light was used to illuminate the test systems. Fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone 50) were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in light intensity. Light intensity at the surface of the water (over the negative control) was 278 lux at test initiation. Light intensity was measured using a SPER Scientific Model 840006 light meter. Observations All fish were observed once each day to evaluate the number of mortalities and the number of individuals exhibiting signs of abnormal behavior. Procedures for Exposure of Fish to PFOS The test chambers were conditioned by delivering PFOS to the diluter system for approximately 5 days before adding the fish. Water samples were collected three times during the pre-test period to confirm that equilibrium concentrations of test substance in the test chambers were achieved prior to adding the fish. At the end of the pre-test period, the uptake phase of the test was initiated on December 5, 2000 by placing the fish in the test chambers. Bluegill were impartially removed from the holding tank in groups of 1 to 2. The groups of bluegill were distributed among the test chambers until each test chamber contained 90 fish. The duration of the uptake phase was 62 days for the low exposure concentration (0.1 mg a.i./L, nominal). The uptake phase for the high concentration (1.0 mg a.i./L, nominal) was terminated after 35 days due to fish mortality. All subsequent references in this report to sampling periods after Day 35 of uptake refer to the 0.1 mg a.i./L (nominal) exposure concentration. At the end of the uptake phase, stock flow to the treatment groups was stopped and the bluegill were exposed to dilution water without PFOS for a period of 56 days. Wildlife International, Ltd. - 14 - BACK TO MAIN Project Number 454A-134 Collection and Analysis of W ater Samples Water samples were collected on Days 0 (0 hours and 4 hours), 1, 3, 7, 14, 21, 28, 35, 42, 49, 56 and 62 of the uptake phase. Water samples were also collected on Days 14, 28, 42 and 56 of the depuration phase. At each water sampling interval, two water samples were collected from the negative control and three samples were collected from each of the two PFOS treatment groups. One negative control sample and two samples from each of the PFOS treatment groups were analyzed for PFOS. The remaining samples were held in reserve as backup samples. All water samples were collected from mid-depth of each test chamber using a glass pipette. The water samples were analyzed for PFOS by liquid chromatography-mass spectrometry (LC/MS). Procedures for analysis of the water samples are provided in Appendix 3. Water samples were analyzed as soon as possible after collection without storage. Collection and Analysis of Tissue Samples Tissue samples were collected on Days 0 (4 hours), 1, 3, 7, 14, 21, 28, 35, 42, 49, 56 and 62 of the uptake phase. Tissue samples were also collected on Days 14, 28, 42 and 56 of the depuration phase. At each tissue sampling interval, a sufficient number of fish were collected to provide two replicate samples of negative control fish and four replicate samples of each PFOS treatment group. Fish were impartially removed from the test chambers and euthanized by severing the spinal cord above the opercular region. The fish were blotted dry and measured for total length and wet weight within approximately 15 minutes of collection, when possible. Each fish was then rinsed with dilution water, blotted dry again and dissected into edible and nonedible tissue fractions. Dissection was accomplished by making an incision from just posterior to the base of the pectoral fin dorsally through the spinal cord. The head, fins and viscera were removed from the body and were considered to be nonedible tissue. The remaining tissue (including skin) was considered the edible tissue. Tissue samples were transferred to tared scintillation vials and weighed. Procedures for extraction and analysis of the tissue samples are provided in Appendix 3. All tissue samples were extracted immediately or stored at approximately -14C until extraction. Tissue Lipid Content Selected fish were collected to determine lipid content (Appendix 3). The determination of percent lipids provides the potential to express BCF values in terms of lipid content. Fish were sampled on Day 0 of uptake, on Day 62 of uptake and on Day 56 of depuration. All fish collected for lipid content were stored at approximately -14C until analysis. In this study, no attempt was made to express bioconcentration in relation to lipid content. Wildlife International, Ltd. - 15 BACK TO MAIN Project Number 454A-134 Data Analysis Whole fish concentrations were calculated based on the sum of the edible and nonedible parts. The steady-state bioconcentration factor (BCF) values were determined from the tissue concentrations at apparent steady-state divided by the average water concentration. Tissue concentrations were considered to be at apparent steady-state if three or more consecutive sets of tissue concentrations were not significantly different (p > 0.05). Tissue concentrations were evaluated for normality and homogeneity of variance using the Shapiro-Wilk's test and Bartlett's test, respectively. If the data did not meet the assumptions, the data was transformed in an attempt to correct the data. Mean tissue concentrations were then compared using analysis of variance and Dunnett's test . The kinetic bioconcentration factor (BCFK), uptake rate (Ki) and depuration rate (K2) were calculated for edible, nonedible and whole fish exposed to 0.1 mg a.i./L (nominal) using BIOFAC computer software (5). BIOFAC is a nonlinear parameter estimation routine which estimates rate constants from a set of sequential time-concentration data. These rate constants are then used to calculate a BCFK (BCFK = K1/K2). RESULTS AND DISCUSSION W ater Chemistry Means and ranges of temperature, dissolved oxygen and pH of the water in the test chambers are presented in Table 1. The individual measurements are given in Appendicies 4 and 5. Water temperatures in the test chambers were within the temperature range of 22 1C established for the test with one exception. On Day 19 of depuration, the continuous temperature recorder measured 20C. The duration of this temperature deviation was approximately 2 hours. Dissolved oxygen concentrations remained > 6.4 mg/L (74 percent of saturation) throughout the test. Measurements of pH ranged from 7.9 to 8.2. Weekly measurements of hardness, alkalinity, conductivity and total organic carbon remained consistent throughout the test (Table 1 and Appendix 6). Observations of M ortality and Clinical Signs Observations of mortality and clinical signs are presented in Appendix 7. Bluegill in the negative control appeared normal and healthy throughout the test. Two bluegill died in the 0.1 mg a.i./L (nominal) treatment group; all other fish appeared normal and healthy. Bluegill in the 1.0 mg a.i./L (nominal) treatment group started to die on Day 9 of the uptake phase. By Day 35 of the uptake phase, all fish in the 1.0 mg/L treatment group had either died or been sampled for tissue analyses. Any fish found dead in the aquaria were stored frozen. Wildlife International, Ltd. - 16 - BACK TO MAIN Project Number 454A-134 Concentrations of PFOS in W ater Concentrations of PFOS in the negative control were <LOQ (0.0500 mg a.i./L) (Tables 2 and 3). Measured concentrations of PFOS during the uptake phase in the 0.10 mg a.i./L treatment group ranged from 68 to 113% of the nominal test concentration (Appendix 3). When concentrations measured during the uptake phase were averaged, the mean measured concentration was 0.086 mg a.i./L which represented 86% of the nominal test concentration. Measured concentrations of PFOS during the uptake phase in the 1.0 mg a.i./L treatment group ranged from 73 to 101% of the nominal test concentration. When concentrations measured during the uptake phase were averaged, the mean measured concentration was 0.87 mg a.i./L, which represented 87% of the nominal test concentration. Concentrations of PFOS during the depuration phase were all <LOQ. Concentrations of PFOS in Fish Tissues Concentrations of PFOS in the negative control tissue samples contained no quantifiable PFOS concentrations (Appendix 3). The concentrations of PFOS in tissues of fish exposed to 0.086 mg a.i./L are presented in Table 4. PFOS concentrations in edible and nonedible tissues appeared to reach steady-state at Day 49 (Figures 1-3). Tissue concentrations from uptake days 49, 56 and 62 were not significantly different (p >0.05). Although not statistically different, the concentrations of PFOS in tissues appeared to still be increasing. The mean measured tissue concentrations during this period of time were 41.6, 96.7 and 73.6 mg a.i./Kg for edible, nonedible and whole fish, respectively. Steady-state BCF values ranged from 484 in edible tissue to 1124 in nonedible tissue (Table 5). BIOFAC estimates of the time to reach 90% of steady state in edible tissue, nonedible tissue and whole fish were 485, 443 and 506 days, respectively (Table 6). During the depuration phase of the test, PFOS was eliminated slowly with estimates of time to reach 50% clearance of 146, 133 and 152 days for edible tissue, nonedible tissue and whole fish, respectively. The concentrations of PFOS in tissues of fish exposed to 0.87 mg a.i./L are presented in Table 7. PFOS concentrations in edible, nonedible and whole fish tissues did not appear to reach steady-state by Day 28 (Figure 4). By Day 35, all fish in the 0.87 mg a.i./L treatment group had been sampled or were dead. Consequently, the only information on bioconcentration that could be determined from the 0.87 mg a.i./L treatment group was the Day 28 BCF. Day 28 BCF values ranged from 136 in edible tissue to 386 in nonedible tissue (Table 8). Based on the length of time required to reach apparent steady-state in the 0.086 mg/L treatment group, these values underestimate the bioconcentration potential of PFOS. Wildlife International, Ltd.____________________ - 17 - BACK TO MAIN Project Number 454A-134 CONCLUSIONS Perfluorooctanesulfonate, potassium salt (PFOS) rapidly bioconcentrated in the tissues of bluegill sunfish (Lepomis macrochirus). Bluegill exposed to 0.87 mg a.i./L were either dead or had been sampled by Day 35 of the uptake phase. Consequently, no reliable information was gained from this treatment group. Although bioconcentration was rapid, apparent steady-state concentrations were not reached in the 0.086 mg a.i./L treatment group until Day 49. In addition, although Day 49, 56 and 62 tissue residues were not significantly different, PFOS concentrations appeared to be increasing during this period of time. Apparent steady-state BCF values for edible, nonedible and whole fish tissues were 484, 1124 and 859, respectively. During depuration, PFOS was eliminated slowly. The BIOFAC estimates for time to reach 50% clearance for edible, nonedible and whole fish tissues were 146, 133 and 152 days, respectively. Wildlife International, Ltd.__________ - 18 - BACK TO MAIN Project Number 454A-134 REFERENCES 1 U.S. Environmental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines (draft), OPPTS Number 850.1730: Fish BCF. 2 ASTM Standard E1022-84. 1988. Standard Practice fo r Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve Molluscs. American Society for Testing and Materials. 3 OECD Guideline for Testing of Chemicals 305. 1996. Bioconcentration: Flow-Through Fish Test. 4 APHA, AWWA, WPCF. 1985. Standard Methods fo r the Examination o f Water and Wastewater. 16th Edition, American Public Health Association. American Water Works Association. Water Pollution Control Federation, New York. 5 BIOFAC. 1991. September 19, 1991 version. The Dow Chemical Company, Midland, Michigan. Wildlife International, Ltd. - 19 - BACK TO MAIN Project Number 454A-134 Table 1 Means and Ranges of Water Quality Parameters Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Uptake Phase Nominal Concentration (mg a.i./L) Temperature1 (C) DO2 (mg/L) PH Conductivity (pmhos/cm) Negative Control 21.9 21.8 - 22.0 7.8 6.8 - 8.6 8.1 7.9 - 8.2 324 310 - 330 Alkalinity (mg/L as CaC03) 180 174 - 185 Hardness (mg/L as CaC03) TOC (mg C/L) 127 104 - 138 <1 <1 - <1 21.8 7.8 8.1 0.10 21.7 - 22.0 6.8 - 8.6 7.9 - 8.2 21.8 7.5 8.1 1.0 21.7 - 21.9 6.4 - 8.2 7.9 - 8.2 1 Temperature measured continuously in the negative control ranged from 20.0 to 22.0C. 2 At a temperature of 22C, the dissolved oxygen saturation concentration is 8.7 mg/L and 60% saturation is 5.2 mg/L. BACK TO MAIN - 20 Table 2 Concentrations of PFOS in Water Samples During the Uptake Phase Project Number 454A-134 Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Nominal _____________________________________________________ Day of Uptake_____________________________________________________ Concentration 0 4 (mg a.i./L) Hour Hours 1 3 7 14 21 28 35 42 49 56 62 Negative Control <LOQ1 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Mean Measured Concentration (mg a.i./L) <LOQ Percent of Nominal -- 0.10 0.0717 0.0791 0.0702 0.0751 0.0826 0.0680 0.110 0.0822 0.0915 0.0983 0.103 0.0853 0.0887 0.086 0.0692 0.0741 0.0734 0.0717 0.0781 0.0709 0.113 0.0843 0.0914 0.110 0.103 0.0948 0.0914 86 1.0 0.797 0.891 0.867 0.813 0.845 0.900 0.988 0.913 0.838 2 0.802 0.930 0.820 0.734 0.818 0.875 1.01 0.925 0.871 2 -- -- -- 'The Limit of Quantitation (LOQ) was 0.0500 mg a.i./L. 2Samples not collected due to 100% mortality. Note: Values presented for each sampling interval are two replicate samples collected from each test chamber. 0.87 87 Wildlife International, Ltd. - 21 - BACK TO MAIN Project Number 454A-134 Table 3 Concentrations of PFOS in Water Samples During the Depuration Phase Sponsor: Test Substance: Test Organism: Dilution Water: Uptake Phase Mean Measured Concentration (mg a.i./L) 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Day of Depuration (mg a.i./L) 14 28 42 Negative Control <LOQ' <LOQ 0.086 <LOQ <LOQ <LOQ <LOQ 'The Limit of Quantitation (LOQ) was 0.0500 mg a.i./L. <LOQ <LOQ <LOQ 56 <LOQ <LOQ <LOQ - 22 - BACK TO MAIN Project Number 454A-134 Table 4 _____________________ PFOS Concentrations in Edible, Nonedible and Whole Fish Tissues of Bluegill Exposed to 0.086 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Sample ID E4/N4 E5/N5 E6/N6 E7/N7 Uptake Day 0 (4 hours) 0 (4 hours) 0 (4 hours) 0 (4 hours) Edible Tissue Concentration (mg a.i./Kg) 0.167 0.155 0.144 0.182 Edible Tissue Weight (gL 2.0545 1.8960 2.5219 1.3561 Nonedible Tissue Concentration (mg a.i./Kg) 0.415 0.519 0.417 0.497 Nonedible Tissue Weight (L 2.1153 2.2103 2.7449 1.8377 Whole Fish Concentration1 (mg a.i./Kg) 0.293 0.351 0.286 0.363 E17/N17 E18/N18 E19/N19 E20/N20 1 1 1 1 0.734 1.6517 1.68 2.0826 1.26 0.726 1.9269 1.85 2.2925 1.34 0.631 1.2074 1.72 1.8419 1.29 0.806 1.0777 2.07 1.4272 1.53 E30/N30 E31/N31 E32/N32 E33/N33 3 3 3 3 1.73 1.7643 4.59 1.8836 3.21 2.07 1.3794 5.50 1.8505 4.04 2.03 1.2621 5.47 2.0994 4.18 2.11 1.1883 5.97 1.7014 4.38 E43/N43 E44/N44 E45/N45 E46/N46 7 7 7 7 3.73 2.2291 10.2 2.7943 7.33 4.25 1.6419 10.6 1.8984 7.66 4.73 1.2994 11.9 1.6429 8.73 6.25 1.2686 15.2 1.7110 11.4 E56/N56 E57/N57 E58/N58 E59/N59 14 14 14 14 11.4 2.0423 27.3 2.5002 20.2 9.07 2.1548 23.2 2.6582 16.9 13.7 1.0709 35.3 1.4046 26.0 12.6 1.1804 32.6 1.7455 24.6 E69/N69 E70/N70 21 21 11.7 2.1161 12.0 1.2984 E71/N71 E72/N72 21 21 12.9 1.4092 10.6 1.6268 iWhole Fish Concentration = (edible wt. X edible conc.) + (nonedible wt. X nonedible conc.) ___________________________________________(edible wt. + nonedible wt.)________________ 33.3 22.7 24.6 24.4 2.4712 1.9396 1.9910 2.1872 23.3 18.4 19.8 18.5 BACK TO MAIN - 23 - Table 4 (Continued) Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Sample ID E82/N82 E83/N83 E84/N84 E85/N85 Uptake Day 28 28 28 28 Edible Tissue Concentration (mg a.i./Kg) 18.3 13.7 23.9 23.1 Edible Tissue Weight (gL 1.4636 1.4113 1.2081 0.8830 E95/N95 E96/N96 E97/N97 E98/N98 35 35 35 35 22.6 1.7888 27.7 1.5897 23.8 1.1136 20.6 1.2226 E103/N103 E104/N104 E105/N105 E106/N106 42 42 42 42 27.6 1.2037 25.3 1.4942 21.2 1.4367 27.6 1.3033 E111/N111 E112/N112 E113/N113 E114/N114 49 49 49 49 33.3 1.7249 36.2 1.2185 39.0 1.5506 30.6 1.4650 E119/N119 E120/N120 E121/N121 E122/N122 56 56 56 56 48.3 1.6058 38.9 1.0946 44.1 1.0521 38.3 1.8592 E127/N127 E128/N128 E129/N129 E130/N130 62 62 62 62 42.4 1.2209 66.2 1.3902 42.2 1.0832 39.2 1.6697 'Whole Fish Concentration = (edible wt. X edible conc.) + (nonedible wt. X nonedible conc.) (edible wt. + nonedible wt.) Nonedible Tissue Concentration (mg a.i./Kg) 49.4 40.7 65.3 57.9 67.1 73.3 62.0 59.1 64.0 68.1 54.4 79.6 85.0 95.1 93.1 77.7 122 94.2 73.2 106 101 112 105 96.4 Nonedible Tissue Weight (g[ 1.7564 1.8903 1.3062 1.3512 2.0434 2.1281 1.6454 1.3627 1.9474 1.9266 2.0920 1.6100 2.2873 1.5993 2.2184 1.9337 2.1688 1.5831 2.0532 2.1739 1.7596 1.9170 1.5965 2.4378 Project Number 454A-134 Whole Fish Concentration1 (mg a.i./Kg) 35.3 29.2 45.4 44.1 46.3 53.8 46.6 40.9 50.1 49.4 40.9 56.3 62.8 69.6 70.8 57.4 90.6 71.6 63.3 74.8 77.0 92.7 79.6 73.1 BACK TO MAIN - 24 - Project Number 454A-134 Table 4 (Continued) PFOS Concentrations in Edible, Nonedible and Whole Fish Tissues of Bluegill Exposed to 0.086 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Sample ID E135/N135 E136/N136 E137/N137 E138/N138 Depuration Day 14 14 14 14 Edible Tissue Concentration (mg a.i./Kg) 48.5 31.8 31.6 42.0 Edible Tissue Weight (g) 1.3634 1.4503 1.2626 0.9340 E143/N143 E144/N144 E145/N145 E146/N146 28 28 28 28 26.0 1.7691 33.3 1.1358 38.7 0.8845 55.8 0.6909 E150/N150 E151/N151 E152/N152 E153/N153 42 42 42 42 24.1 1.7590 31.2 1.1220 30.0 0.8440 33.0 0.8672 E157/N157 E158/N158 E159/N159 E160/N160 'Whole Fish Concentration 56 21.1 1.5471 56 37.6 0.5892 56 32.9 0.6244 56 31.2 0.7223 (edible wt. X edible conc.) + (nonedible wt. X nonedible conc.) (edible wt. + nonedible wt.) Nonedible Tissue Concentration (mg a.i./Kg) 124 79.4 81.8 113 85.7 95.1 85.7 94.8 71.7 80.6 78.3 82.1 57.7 80.3 85.4 84.4 Nonedible Tissue Weight (g) 1.6928 2.1909 1.8708 1.4560 2.0744 1.6772 1.4801 1.2803 2.3578 1.7635 1.5181 1.2687 1.9735 1.2353 1.0439 0.9975 Whole Fish Concentration1 (mg a.i./Kg) 90.3 60.4 61.6 85.3 58.2 70.1 68.1 81.1 51.4 61.4 61.0 62.2 41.6 66.5 65.8 62.1 Wildlife International, Ltd. - 25 - BACK TO MAIN Table 5 Apparent Steady-State BCF Values for Bluegill Exposed to 0.086 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Tissue Type Edible Mean Measured Test Concentration (mg a.i./L) 0.086 Nonedible 0.086 Whole Fish 0.086 Uptake Days at Apparent Steady-State 49, 56 and 62 49, 56 and 62 49, 56 and 62 Mean Measured Apparent Steady- State Tissue Concentration (mg a.i./Kg) 41.6 96.7 73.6 Apparent Steady-State BCF 484 1124 856 Wildlife International, Ltd. - 26 - BACK TO MAIN Table 6 BIOFAC Model Estimates for Bluegill Exposed to 0.086 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Tissue Type Kinetic Bioconcentration Factor (BCFK) Uptake Rate Constant (k1, L .K g^.D ay1) Edible 1866 8.9 Depuration Rate Constant (k2, Day-1) 0.0047 Nonedible 4312 22 0.0052 Whole Fish 3614 16 0.0045 Estimate Time to Reach 90% of Steady State (Days) 485 443 506 Estimated Time to Reach 50% Clearance (Days) 146 133 152 - 27 - BACK TO MAIN Project Number 454A-134 Table 7 PFOS Concentrations in Edible, Nonedible and Whole Fish Tissues of Bluegill Exposed to 0.87 mg a.i./L Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution Water: Well Water Sample ID E9/N9 E10/N10 E11/N11 E12/N12 Uptake Day 0 (4 hours) 0 (4 hours) 0 (4 hours) 0 (4 hours) Edible Tissue Concentration (mg a.i./Kg) 1.46 1.48 1.19 1.39 Edible Tissue Weight (g) 0.9795 1.3226 1.6284 1.5205 E22/N22 E23/N23 E24/N24 E25/N25 1 1 1 1 4.68 1.7060 6.59 1.3724 5.56 1.1272 5.64 1.0507 E35/N35 E36/N36 E37/N37 E38/N38 3 3 3 3 17.3 0.9889 15.8 1.2666 19.0 0.8348 20.8 1.1215 E48/N48 E49/N49 E50/N50 E51/N51 7 7 7 7 42.0 1.7575 44.0 1.5527 57.7 1.3391 46.8 0.9532 E61/N61 E62/N62 14 14 87.1 1.9813 81.6 1.2152 E63/N63 E64/N64 14 14 90.7 1.1290 73.3 1.0811 iWhole Fish Concentration = (edible wt. X edible conc.) + (nonedible wt. X nonedible conc.) (edible wt. + nonedible wt.) Nonedible Tissue Concentration (mg a.i./Kg) 3.52 4.37 4.22 4.06 11.1 14.2 13.3 12.1 39.3 42.0 43.8 51.8 100 102 102 120 177 207 245 214 Nonedible Tissue Weight (g) 1.5183 1.6397 1.9391 1.9489 1.8323 1.7515 1.7081 1.5273 1.4781 1.6727 1.4005 1.6070 2.3064 2.0505 2.2079 1.3562 2.9193 1.8260 1.5376 1.6199 Whole Fish Concentration1 (mg a.i./Kg) 2.71 3.08 2.84 2.89 8.00 10.9 10.2 9.47 30.5 30.7 34.5 39.1 74.9 77.0 85.3 89.8 141 157 180 158 - 28 - BACK TO MAIN Project Number 454A-134 Table 7 (Continued) PFOS Concentrations in Edible, Nonedible and Whole Fish Tissues of Bluegill Exposed to 0.87 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water: Sample ID E74/N74 E75/N75 E76/N76 E77/N77 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Uptake Day Edible Tissue Concentration (mg a.i./Kg) 21 79.4 21 117 21 104 21 102 E87/N87 E88/N88 E89/N89 E90/N90 28 28 28 28 102 131 107 133 Edible Tissue Weight (g)_ 1.8300 1.4526 1.6824 1.4076 1.6679 1.0923 1.1675 1.1466 Nonedible Tissue Concentration (mg a.i./Kg) 201 278 246 229 289 372 320 361 Nonedible Tissue Weight (sl 2.1803 1.9786 2.2130 1.7558 2.0549 1.4045 1.6595 1.5186 iWhole Fish Concentration = (edible wt. X edible conc.) + (nonedible wt. X nonedible conc.) (edible wt. + nonedible wt.) Note: Sampling ended after Day 28. By Day 35 all the fish in this treatment group had died or were previously sampled for tissue analysis. Whole Fish Concentration1 (mg a.i./Kg) 146 210 185 172 205 267 232 263 Wildlife International, Ltd. - 29 - BACK TO MAIN Table 8 Day 28 BCF Values for Bluegill Exposed to 0.87 mg a.i./L Sponsor: Test Substance: Test Organism: Dilution Water: Tissue Type Edible 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Mean Measured Test Concentration (mg a.i./L) Mean Day 28 Tissue Concentration (mg a.i./Kg) 0.87 118 Nonedible 0.87 336 Whole Fish 0.87 242 Note: BCF values presented here are biased low due to fish mortality. Day 28 BCF 136 386 278 Wildlife International, Ltd. BACK TO MAIN Project Number 454A-134 Figure 1. Concentrations of PFOS in Edible Fish Tissues of Bluegill Exposed to 0.086 mg a.i./L. BACK TO MAIN Wildlife International, Ltd. Project Number 454A-134 Figure 2. - 31 Concentrations of PFOS in Nonedible Fish Tissues of Bluegill Exposed to 0.086 mg a.i./L. BACK TO MAIN Wildlife International, Ltd. Project Number 454A-134 Figure 3. - 32 Concentrations of PFOS in Whole Fish Tissues of Bluegill Exposed to 0.086 mg a.i./L. Wildlife International, Ltd. BACK TO MAIN Project Number 454A-134 -33 - Figure 4. Concentrations of PFOS in Edible, Nonedible and Whole Fish Tissues of Bluegill Exposed to 0.87 mg a.i./L. 400 350 /Sd 300 mrn m 250 I 200 | 150 uI 100 50 a Non-Edible x Whole Fish Edible 0 0 10 20 30 Day Wildlife International, Ltd. - 34 - BACK TO MAIN Appendix 1 Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week Period Immediately Preceding the Test Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Specific Conductance (g,mhos/cm) Mean 313 (N = 4) Range 310 - 315 Hardness (mg/L as CaC03) 130 (N = 4) 128 - 132 Alkalinity (mg/L as CaC03) 178 (N = 4) 176 - 178 pH 8.1 (N = 4) 8.0 - 8.2 BACK TO MAIN Wildlife International, Ltd. Project Number 454A-134 - 35 - Appendix 2 Analyses of 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-P 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 1Analyses performed by TNO Nutrition and Food Institute on samples collected on November 15, 2000. Continued BACK TO MAIN Wildlife International, Ltd. Project Number 454A-134 - 36 - Appendix 2 (Continued) Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Pesticides And Organics (Page 2) Component 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 Measured Concentration <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 Component 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 Measured Concentration <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 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 Metals 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, L td.____________________ - 37 - BACK TO MAIN Project Number 454A-134 Appendix 3 THE ANALYSIS OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) CONCENTRATIONS IN FRESHWATER AND BLUEGILL SUNFISH TISSUE IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-134 Wildlife International, Ltd.___________ - 38REPORT APPROVAL BACK TO MAIN Project Number 454A-134 SPONSOR. 3M Corporation TITLE: Perfluorooctanesulfonate, Potassium Salt (PFOS): A Flow-Through Bioconcentration Test with the Bluegill (Lepomis macrochirus) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-134 PRINCIPAL INVESTIGATOR: WILDLIFE INTERNATIONAL. LTD, MANAGEMENT: Date Director, Analytical Chemistry BACK TO MAIN Wildlife International, Ltd.____________________ - 39 - Project Number 454A-134 Introduction Freshwater samples and tissue samples were collected from a flow-through aquatic test to determine the bioconcentration potential of perfluorooctanesulfonate, potassium salt (PFOS) in the bluegill (Lepomis macrochirus). The study was conducted by Wildlife International, Ltd. and identified as Project Number 454A-134. The analyses of freshwater and tissue samples were performed at Wildlife International, Ltd. by high performance liquid chromatography (HPLC) with mass spectrometric detection. Water samples were diluted and analyzed by HPLC with single quadrupole mass spectrometric detection (LC/MS). Tissue samples were homogenized, extracted, diluted, and analyzed by HPLC with triple quadrupole mass spectrometric detection (LC/MS/MS). Freshwater samples were collected and analyzed from December 1, 2000 to April 2, 2001. Tissue samples were collected and analyzed from December 5, 2000 to April 2, 2001. Additional tissue samples were collected and analyzed gravimetrically for lipid content. Samples for lipid content were collected on December 5, 2000, February 5, 2001 and April 2, 2001 and analyzed between April 4 and April 11, 2001. Test Substance and Internal Standard The test substance, PFOS, was used to prepare calibration standards and matrix fortification samples and was identified as Wildlife International, Ltd. identification number 4675. 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 internal standard, referred to hereafter as 4HPFOS, was stored under ambient conditions. Analytical Method Water and tissue samples were analyzed for PFOS using high performance liquid chromatography (HPLC) with mass spectrometric detection. Water samples were analyzed according to the method entitled "Analytical Method Validation for the Determination of Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) in Freshwater, Saltwater and Algal Media" (Wildlife International, Ltd. Project No. 454C-109). Tissue samples were analyzed according to the method entitled "Analytical Method Validation for the Determination of Perfluorooctane Sulfonic Acid, BACK TO MAIN Wildlife International, Ltd.____________________ - 40 - Project Number 454A-134 Potassium Salt (PFOS) in Fish Tissues" (Wildlife International, Ltd. Project No. 454C-119). For water analyses, one minor modification from the validation was incorporated into the present study. This change was use of 10 ppb in place of 100 ppb 4HPFOS internal standard concentration in all calibration standards and study sample dilutions. The lower internal standard nominal concentration was closer to the range of PFOS calibration used in the present work. The analytical methodology implemented for the determination of lipid content in fish tissue is presented on page 42. Freshwater Samples A method flow chart for the analysis of PFOS in freshwater is presented in Figure 1. Samples were diluted in a 50% methanol (HPLC grade, 99.9+%): 50% NANOpure water solution containing 10.0 gg 4H PFOS/L and 0.05% formic acid (v/v) so that they fell within the calibration range of the LCMS methodology. Aliquots of the dilutions were transferred to autosampler vials and submitted for analysis by direct injection. Concentrations of PFOS in freshwater samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) interfaced with a Perkin-Elmer API 3000LC mass spectrometer operated in selective ion monitoring (SIM) detection mode. The mass spectrometer was equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separations were achieved using a Keystone Betasil Ci8 analytical column (50 mm x 2 mm I.D., 3 gm particle size) fitted with a Keystone Javelin C18 Guard Cartridge (20mm x 2 mm). The instrument parameters are summarized in Table 1. Freshwater quality control (QC) samples (matrix blanks and fortifications) were processed in the same manner as the test samples. Freshwater was fortified with the appropriate PFOS in methanol stock solution using a gas-tight syringe. Matrix blank samples were not fortified with the test substance. Tissue QC Samples Bluegill sunfish (Lepomis macrochirus) tissues were obtained from a breeding stock maintained at Wildlife International, Ltd. Approximately 200 individual fish (ages 3 to 5 months) were removed from a breeding tank and euthanized by severing the spinal cord above the opercular region. Heads, fins and viscera were removed from the body and were considered to be nonedible tissue. The balance of the tissue was considered to be edible tissue. The edible and nonedible tissues BACK TO MAIN Wildlife International, Ltd.____________________ - 41 - Project Number 454A-134 were separately homogenized with a Waring stainless steel blender. An appropriate number of 1-gram aliquots of edible and nonedible homogenate were weighed into separate, 20-mL glass scintillation vials. Each vial was uniquely identified and labeled with a facility log identification number. The tissue QC samples were stored frozen until they were used in the preparation of matrix blank and matrix fortification samples, or storage stability samples. Tissue Samples A method flow chart for the analysis of PFOS in fish tissues is presented in Figure 2. Edible and nonedible fish tissue samples were collected from the test in 20-mL glass scintillation vials and stored frozen, if necessary, until analysis. Upon analysis, tissue samples were batched by sampling interval and tissue type. At that time, three of the appropriate tissue QC samples also were removed from the freezer. Any frozen samples were allowed to thaw. One tissue QC sample was designated as the matrix blank sample. The other tissue QC samples were designated as matrix fortification samples and were fortified with the appropriate PFOS stock solution(s) in methanol using gas-tight syringes. Test and QC tissue homogenates were extracted as follows. Ten milliliters of methanol were added to each vial. The samples were homogenized with a tissue shredder for approximately one minute. The samples were then sonicated with a sonic dismembrator for approximately five minutes. The samples were capped, shaken, and centrifuged at approximately 2000 rpm for approximately 5 minutes. Aliquots of the extract were then volumetrically diluted into the calibration range of the LCMS methodology with 50:50 methanol: NANOpure water dilution solvent. Aliquots of the diluted extracts were transferred to autosampler vials and submitted for analysis. Back-up tissue samples were collected and stored frozen. If necessary, the back-up tissue samples were removed from the freezer, allowed to thaw and processed using the same procedures described above. Tissue Storage Stability Samples Stability samples were prepared at test initiation to establish test substance stability in fish tissues stored frozen during the study. Two tissue QC samples of each fish tissue type (edible and nonedible) were removed from the freezer and allowed to thaw. The edible and nonedible fish tissues BACK TO MAIN Wildlife International, Ltd.____________________ - 42 - Project Number 454A-134 were each fortified at 0.100 and 10.0 mg a.i./Kg using the appropriate PFOS stock solution and a gas-tight syringe. The stability samples were returned to the freezer. Following 119 days of frozen storage, the stability samples were removed from the freezer and analyzed. Fresh fortification and matrix blank samples also were prepared and analyzed at this time. The samples (new and old) were processed using the same procedures described for tissue sample analyses. Concentrations of PFOS in fish tissue were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 HPLC interfaced with a Perkin-Elmer API 3000LC Mass Spectrometer operated in multiple ion reaction monitoring (MRM) detection mode. The mass spectrometer was equipped with a Perkin-Elmer TurbolonSpray ion source. Chromatographic separations were achieved using a Keystone Betasil Ci8 analytical column (50 mm x 2 mm I.D., 3 pm particle size) fitted with a Keystone Javelin C18 Guard Cartridge (20 mm x 2 mm). The instrument parameters are summarized in Table 2. Tissue Samples for Lipid Content A method flow-chart for the analysis of lipid content in fish tissue is presented in Figure 3. Edible and nonedible fish tissue samples to be analyzed for lipid content were collected from the test at initiation, on Day 62 of the uptake phase (Day 0 depuration), and at termination (Day 56 depuration). Only fish from the Negative Control and 0.10 mg a.i./L nominal test level were sampled for lipid content. Samples were collected in 20-mL glass scintillation vials and stored frozen until analysis. Sample weights were recorded at the time of collection. Upon analysis, tissue samples designated for lipid content determination were removed from the freezer and allowed to thaw. For each sample, 10 mL of NANOpure water was added to the fish tissue in the vial and the sample was homogenized for approximately one minute using a tissue shredder. Each homogenate was transferred to a 250-mL separatory funnel that contained 25 mL of chloroform and 50 mL of methanol. Each sample vial was rinsed with an additional 10 mL of NANOpurewater and the rinse was poured into the respective separatory funnel. The separatory funnels were shaken with venting for approximately one minute. Fifty milliliters of chloroform followed by 50 mL of saturated sodium chloride were added to each separatory funnel. The separatory funnels were briefly swirled with venting. The phases were allowed to separate. For each sample, the chloroform layer was drained through a powder funnel packed with glass wool and anhydrous sodium sulfate into a 250-mL round- BACK TO MAIN Wildlife International, Ltd.____________________ - 43 - Project Number 454A-134 bottom flask. An additional 50-mL aliquot of chloroform was added to each separatory funnel and the extraction and draining procedures were repeated. The extracts were rotary evaporated in a water bath maintained at approximately 40C to near dryness. Each extract was transferred to a pre weighed labeled scintillation vial. Each 250-mL round-bottom flask was rinsed with a small volume of chloroform and the rinse was transferred to the respective scintillation vial. The remaining solvent in each vial was evaporated under a gentle stream of nitrogen or clean dry air. The vials were reweighed and the weights were recorded. Lipid content was calculated for each sample as the ratio of lipid to fish tissue weights (mg/Kg). Calibration Standards For water analyses, calibration standards of PFOS prepared in a 50% methanol : 50% NANOpure water solution containing 10.0 gg 4H PFOS/L and 0.05% formic acid (v/v), ranging in concentration from 0.500 to 5.00 gg a.i./L, were analyzed with the each sample set. PFOS was fortified into the standards by appropriate dilutions of a 1.00 mg a.i./L stock solution of PFOS in methanol. Five calibration standards (different concentrations) were analyzed with each sample set. The calibration standard series was injected at the beginning and end of each run, and one standard was injected, at a minimum, after every five samples. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS: internal standard) of the calibration standards. A typical calibration curve from water analyses is presented in Figure 4. The concentration of PFOS in the freshwater samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms of low and high calibration standards used for freshwater analyses are presented in Figures 5 and 6, respectively. For tissue analyses, calibration standards were prepared in 50:50 methanol: NANOpure water by appropriate dilutions of either a 1.00 mg a.i./L or a 10.0 mg a.i./L stock solution of PFOS in methanol. Two separate PFOS calibration standard sets were employed in the study. For Days 0 through 7 of the uptake phase, and for stability sample analyses, PFOS calibration standards ranging in concentration from 0.500 to 5.00 gg a.i./L, were analyzed with each sample set. For all subsequent sampling intervals, a higher (5.00 to 50.0 PFOS gg a.i./L) calibration range was required to minimize dilution of the sample extracts. For each sample set, five calibration standards (different BACK TO MAIN Wildlife International, Ltd.____________________ - 44 - Project Number 454A-134 concentrations) were analyzed. The appropriate calibration standard series was injected at the beginning and end of each run, and one standard was injected, at a minimum, after every five samples. Linear regression equations were generated using the peak area responses versus the respective concentrations of the calibration standards. A typical calibration curve from tissue analyses is presented in Figure 7. Concentration of PFOS in fish tissue samples was determined by substituting the peak area response into the applicable linear regression equation. Representative ion chromatograms of low and high calibration standards used for tissue analyses are presented in Figures 8 and 9, respectively. For both water and tissue analyses for PFOS, 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. Fortification Stocks Freshwater and fish tissue homogenates were fortified with the appropriate stock solution of PFOS prepared in methanol. Each stock solution was assigned a unique identification code that was recorded on a stock preparation log sheet. Limits of Quantitation The method limit of quantitation (LOQ) for PFOS in freshwater samples was 0.0500 mg a.i./L, calculated as the product of the lowest PFOS calibration standard (0.0005 mg a.i./L) and the dilution factor of the matrix blank sample (100) analyzed concurrently with the test samples. The LOQ was calculated on an individual basis for each tissue sample since each entire submitted sample (of differing weight) was extracted without an adjustment to a constant weight. The LOQ (mg a.i./Kg) for a given tissue analysis was calculated as the product of the lowest PFOS calibration standard (0.0005 or 0.005 mg a.i./L) and the overall dilution factor (L/Kg) of the tissue sample. To illustrate for a 1.207 gram sample analyzed with the low-level calibration standard set, extraction with 10 mLs of methanol followed by a 10x volumetric dilution (overall dilution factor = 82.85 L/Kg), gives an LOQ = 0.0414 mg a.i./Kg. BACK TO MAIN Wildlife International, Ltd. - 45 Project Number 454A-134 Freshwater Matrix Blank and Fortification Samples Along with the actual freshwater sample analyses, 20 freshwater matrix blank samples were analyzed at periods throughout the study to determine possible interferences (Table 3). No matrix interferences were observed at or above the limit of quantitation (0.0500 mg a.i./L). A representative ion chromatogram of a freshwater matrix blank sample is presented in Figure 10. Freshwater samples were fortified at two levels (0.0750 and 0.500 mg a.i./L) or three levels (0.0750, 0.500 and 2.00 mg a.i./L) at each sampling interval using appropriate stock solutions of PFOS prepared in methanol. Freshwater matrix fortifications were analyzed concurrently with each sample set and the analytical results were not corrected for mean procedural recovery. Recoveries ranged from 84.9 to 120% of nominal concentration (Table 3). The mean and standard deviation of fortification recoveries at the 0.0750, 0.500 and 2.00 mg a.i./L fortification levels were 94.7% 5.12% (n = 20), 98.7% 6.60% (n = 20), and 96.9% 2.95% (n = 12), respectively. A representative ion chromatogram of a freshwater matrix fortification is presented in Figure 11. Tissue Matrix Blank and Fortification Samples Along with the actual tissue sample analyses, 16 edible and 16 nonedible fish tissue matrix blank samples were analyzed to determine possible interferences (Tables 4 and 5, respectively). No interferences were observed at or above the applicable limits of quantitation during the test. Representative ion chromatograms of edible and nonedible fish tissue matrix blank samples are presented in Figures 12 and 13, respectively. Edible and nonedible fish tissue homogenates were fortified with the appropriate stock solution(s) of PFOS prepared in methanol. Tissue matrix fortifications were prepared and analyzed concurrently with each sample set and the analytical results were not corrected for mean procedural recovery. The levels of fortification were chosen to bracket the expected concentration of test samples. Because measured concentrations increased with time for the duration of the uptake phase, the fortification levels were frequently revised. Low-level fortification levels ranged from 0.100 to 5.00 mg a.i./L and high-level fortification levels ranged from 10.0 to 500 mg a.i./L. Edible fish tissue recoveries ranged from 94.8 to 111% of nominal concentration for the low-level fortifications, and from 92.0 to 107% of nominal concentration for the high-level fortifications (Table 4). The mean and standard deviation of fortification recoveries at the low- and high-level fortifications (n = 16) for the BACK TO MAIN Wildlife International, Ltd. - 46 - Project Number 454A-134 edible fish tissue were 102% 4.61% and 100% 4.40%, respectively. Nonedible fish tissue recoveries ranged from 93.9 to 116% and from 90.1 to 122% of nominal concentration for low- and high-level fortifications, respectively (Table 5). The mean and standard deviation of fortification recoveries at the low- and high-level fortifications (n = 16) for the nonedible fish tissue were 106% 5.55% and 103% 8.49%, respectively. Representative ion chromatograms of edible and nonedible fish tissue matrix fortification samples are presented in Figures 14 and 15, respectively. Example Calculations The analytical result and percent recovery for freshwater sample 454A-134-68, from the 0.10 mg a.i./L nominal PFOS treatment group, was calculated using the following equations: Peak Area Ratio = Analyte Peak Area/Internal Standard Peak Area PFOS Area = 593240 Internal Standard Area = 1295042 Peak area ratio = 0.4581 Internal Standard Concentration: 10.0 pg a.i./L Y-intercept = -0.0187 Slope = 5.2182 Initial volume (Vi) = 0.100 mL Final volume (Vf) = 10.0 mL Dilution factor (Vf/Vj) = 100 Peak area ratio - (Y-intercept) PFOS (pg a.i./L) at Instrument = Slope x Internal Standard Concentration = 0.9137 PFOS (mg a.i./L) in sample = PFOS (mg a.i./L) at Instrument x Dilution Factor = 0.0009137x 100 = 0.09137 PFOS (mg a.i./L) in sample x 1 0 0 Percent of Nominal Concentration = PFOS (mg a.i./L) nominal 0.09137 mg a.i./L x 1 0 0 0.10 mg a.i./L = 91.4 % Wildlife International, Ltd.____________________ - 47 - BACK TO MAIN Project Number 454A-134 The analytical result for edible fish tissue sample 454A-134-E-43, from the 0.10 mg a.i./L nominal PFOS treatment group, was calculated using the following equations: Peak Area = 11192 Y-intercept = 713.81 Slope = 6287.33 Primary Dilution: Initial Weight (Wi) = 2.2291 g Extraction Volume (Ve) = 10.0 mL Secondary Dilution: Initial Volume (Vi) = 0.0500 mL Final Volume (Vf) = 25.0 mL Overall Dilution Factor (Ve/Wi x Vf/Vi) = 2240 mL/g = 2240 L/Kg __ . ,, . (peak area - y-intercept) PFOS at instrument (pg a.i./L) = slope = (11192 - 713.81) = 6287.33 = 1.667 pg a.i./L PFOS in sample (mg a.i./Kg) = PFOS at instrument (pg a.i./L) x overall dilution factor x unit conversion factor 1.667 pg a.i. 2240L 1mg PFOS in sample (mg a.i./Kg) L X Kg x 1000 pg = 3.73 mg a.i./Kg RESULTS Freshwater Sample Analysis Freshwater samples were collected and analyzed for PFOS concentrations on Days -4 and -1 (twice) during the pre-uptake phase of the test and Days 0 (0 and 4 hours), 1, 3, 7, 14, 21, 28, 35, 42, 49, 56, and 62 during the uptake phase of the test. Uptake was suspended on Day 62 of the test and depuration began. During the depuration phase of the test, freshwater samples were collected and analyzed for PFOS concentrations on Days 14, 28, 42, and 56. Measured concentrations of PFOS in the pre-test diluter verification (pre-uptake phase) samples were <LOQ in the negative controls. Measured concentrations in pre-test samples ranged from 64.6 to 95.4% of the nominal concentration in the 0.10 mg a.i./L treatment group and from 71.8 to 111% of the nominal concentration in the BACK TO MAIN Wildlife International, Ltd.____________________ - 48 - Project Number 454A-134 1.0 mg a.i./L treatment group (Table 6). During the uptake phase of the test, measured concentrations of PFOS in the negative control freshwater samples were <LOQ. Freshwater samples collected from the 0.10 and 1.0 mg a.i./L treatment groups ranged from 68.0 to 113% and from 73.4 to 101% of the nominal concentration, respectively (Table 7). During the depuration phase of the test, measured concentrations of PFOS in all freshwater samples were <LOQ. A representative ion chromatogram of a freshwater sample is presented in Figure 16. Tissue Sample Analysis Tissue samples were collected on uptake Days 0 (4 hours), 1, 3, 7, 14, 21, 28, 35, 42, 49, 56, and 62 and depuration Days 14, 28, 42, and 56 of the test. Measured concentrations of PFOS in all negative control edible and nonedible fish tissue samples were <LOQ (Tables 8 and 9). The results of analyses of edible and nonedible fish tissue samples collected from the 0.100 and 1.00 mg a.i./L treatment groups are presented in Tables 8 and 9, respectively. Representative ion chromatograms of edible and nonedible tissue samples from the same study fish are presented in Figures 17 and 18, respectively. Stability Sample Analysis Stability samples were prepared at test initiation (uptake Day 0) and stored frozen. The results of stability sample analyses are presented in Table 10. These data indicate that the test substance was stable during relatively long term (119 days) frozen storage at the 0.100 and 10.0 mg a.i./Kg concentration levels. Definitive tissue sample storage did not exceed 7 days during the conduct of the study. Tissue Sample Analysis for Lipid Content Tissue samples were collected on uptake Days 0 (0 hour) and 62 and depuration Day 56 to determine lipid content in fish tissues on a wet-weight basis. The results of lipid analyses in edible and nonedible fish tissues are presented in Tables 11 and 12, respectively. BACK TO MAIN Wildlife International, Ltd. - 49 - Project Number 454A-134 Table 1 Typical HPLC/MS Operational Parameters for Analysis of Aqueous Samples INSTRUMENT: Hewlett-Packard Model 1100 High Performance Liquid Chromato graph (HPLC) with a Perkin-Elmer API 3000 Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Betasil Ci8 column (50 mm x 2 mm I.D., 3-|im particle size) GUARD COLUMN: Keystone Javelin C18 column (20 mm x 2 mm I.D.) OVEN TEMPERATURE: 40C STOP TIME: 5.00 minutes FLOW RATE: 250 ^L/minute MOBILE PHASE: 70.0% Methanol : 30.0% NANOpure Water containing 0.1% Formic Acid INJECTION VOLUME: 10 ^L PFOS RETENTION TIME: Approximately 3.7 minutes INTERNAL STANDARD RETENTION TIME: Approximately 2.5 minutes PFOS MONITORED MASS: 499 amu INTERNAL STANDARD MONITORED MASS: 427 amu BACK TO MAIN Wildlife International, Ltd. - 50 - Project Number 454A-134 Table 2 Typical HPLC/MS/MS Operational Parameters for Analysis of Tissue Samples INSTRUMENT: Hewlett-Packard Model 1100 High Performance Liquid Chromato graph (HPLC) with a Perkin-Elmer API 3000 Mass Spectrometer equipped with a Perkin-Elmer TurbolonSpray ion source. Operated in multiple ion reaction monitoring (MRM) mode. ANALYTICAL COLUMN: Keystone Betasil Ci8 column (50 mm x 2 mm I.D., 3-pm particle size) GUARD COLUMN: Keystone Javelin C18 column (20 mm x 2 mm I.D.) OVEN TEMPERATURE: 40C STOP TIME: 5.00 minutes FLOW RATE: 250 pL/minute MOBILE PHASE: 70.0% Methanol: 30.0% NANOpure Water containing 0.1% Formic Acid INJECTION VOLUME: 10.0 pL PFOS RETENTION TIME: Approximately 4.1 minutes PFOS MONITORED MASS: 499.0 amu ^ 99.1 amu BACK TO MAIN Wildlife International, Ltd. - 51 Project Number 454A-134 Table 3 Matrix Blanks and Fortifications Analyzed Concurrently with Freshwater Samples Sample Number (454A-134-) PT-MAB-1 PT-MAS-1 PT-MAS-2 PT-MAS-3 Sampling Interval (Day) -4 Pretest -4 -4 -4 Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS) (mg a.i./L) Fortified Measured1 0.00 <LOQ 0.0750 0.0707 0.500 0.493 2.00 1.98 Percent Recovery2 -- 94.3 98.6 99.0 PT-MAB-2 PT-MAS-4 PT-MAS-5 PT-MAS-6 -1 -1 -1 -1 Pretest 0.00 0.0750 0.500 2.00 <LOQ 0.0779 0.464 1.91 -104 92.9 95.3 PT-MAB-3 PT-MAS-7 PT-MAS-8 PT-MAS-9 -1 -1 -1 -1 Pretest 0.00 0.0750 0.500 2.00 <LOQ 0.0724 0.483 1.86 -96.5 96.5 93.2 MAB-1 MAS-1 MAS-2 MAS-3 0 Hour 0 - Uptake 0 0 0 0.00 0.0750 0.500 2.00 <LOQ 0.0712 0.488 2.00 -95.0 97.5 99.9 MAB-2 MAS-4 MAS-5 MAS-6 0 Hour 4 - Uptake 0 0 0 0.00 0.0750 0.500 2.00 <LOQ 0.0728 0.490 1.97 -97.1 98.0 98.6 MAB-3 1 Uptake MAS-7 1 MAS-8 1 MAS-9 1 0.00 0.0750 0.500 2.00 <LOQ 0.0745 0.498 1.95 -99.3 99.7 97.6 MAB-4 MAS-10 MAS-11 MAS-12 3 3 3 3 Uptake 0.00 0.0750 0.500 2.00 <LOQ 0.0666 0.509 1.86 -88.9 102 92.8 MAB-5 MAS-13 MAS-14 MAS-15 7 7 7 7 Uptake 0.00 0.0750 0.500 2.00 <LOQ 0.0753 0.508 1.96 -100 102 98.2 MAB-6 MAS-16 14 14 Uptake 0.00 0.0750 <LOQ 0.0671 -89.5 MAS-17 14 0.500 0.450 90.0 MAS-18 14 2.00 1.85 92.7 1The limit of quantitation (LOQ) was 0.0500 mg a.i./L, calculated as the product of the lowest calibration standard (0.0005 mg a.i./L) and the dilution factor of the matrix blank sample (100). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly.__________ BACK TO MAIN Wildlife International, Ltd. - 52 Project Number 454A-134 Table 3 (continued) Matrix Blanks and Fortifications Analyzed Concurrently with Freshwater Samples Sample Number (454A-134-) MAB-7 MAS-19 MAS-20 MAS-21 Sampling Interval (Day) 21 Uptake 21 21 21 Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS) (mg a.i./L) Fortified Measured1 0.00 0.0750 0.500 2.00 <LOQ 0.0710 0.600 1.99 Percent Recovery2 -94.6 120 99.6 MAB-8 MAS-22 MAS-23 MAS-24 28 28 28 28 Uptake 0.00 0.0750 0.500 2.00 <LOQ 0.0657 0.517 1.91 -87.6 103 95.4 MAB-9 MAS-25 MAS-26 MAS-27 35 35 35 35 Uptake 0.00 0.0750 0.500 2.00 <LOQ 0.0727 0.470 2.01 -96.9 94.0 101 MAB-10 MAS-28 MAS-29 42 42 42 Uptake 0.00 0.0750 0.500 <LOQ 0.0734 0.517 -97.9 104 MAB-11 MAS-30 MAS-31 49 49 49 Uptake 0.00 0.0750 0.500 <LOQ 0.0712 0.463 -95.0 92.7 MAB-12 MAS-32 MAS-33 56 56 56 Uptake 0.00 0.0750 0.500 <LOQ 0.0701 0.477 -93.4 95.3 MAB-13 MAS-34 MAS-35 62 62 62 Uptake 0.00 0.0750 0.500 <LOQ 0.0745 0.481 -99.3 96.1 MAB-14 MAS-36 MAS-37 14 Depuration 14 14 0.00 0.0750 0.500 <LOQ 0.0751 0.494 -100 98.7 MAB-15 MAS-38 28 Depuration 28 0.00 0.0750 <LOQ 0.0644 -85.8 MAS-39 28 0.500 0.525 105 1The limit of quantitation (LOQ) was 0.0500 mg a.i./L, calculated as the product of the lowest calibration standard (0.0005 mg a.i./L) and the dilution factor of the matrix blank sample (100). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly.________ BACK TO MAIN Wildlife International, Ltd. - 53 Project Number 454A-134 Table 3 (continued) Matrix Blanks and Fortifications Analyzed Concurrently with Freshwater Samples Sample Number (454A-134-) MAB-16 MAS-40 MAS-41 Sampling Interval (Day) 42 Depuration 42 42 Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS) (mg a.i./L) Fortified Measured1 0.00 0.0750 <LOQ 0.0637 0.500 0.452 Percent Recovery2 -84.9 90.4 MAB-17 56 Depuration 0.00 <LOQ -- MAS-42 MAS-43 56 56 0.0750 0.500 0.0712 0.484 94.9 96.9 1The limit of quantitation (LOQ) was 0.0500 mg a.i./L, calculated as the product of the lowest calibration standard (0.0005 mg a.i./L) and the dilution factor of the matrix blank sample (100). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly.________ BACK TO MAIN Wildlife International, Ltd. - 54 Project Number 454A-134 Table 4 Matrix Blanks and Fortifications Analyzed Concurrently with Edible Fish Tissue Samples Sample Number (454A-134-) E-MAB-1 E-MAS-1 E-MAS-2 Sampling Interval (Day) 0 Hour 4 - Uptake 0 0 Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS) (mg a.i./Kg) Fortified Measured1 0.00 <0.0500 0.100 0.0970 10.0 9.65 Percent Recovery2 -97.0 96.5 E-MAB-2 E-MAS-3 E-MAS-4 1 1 1 Uptake 0.00 0.100 10.0 <0.0500 0.0993 9.77 -99.3 97.7 E-MAB-3 E-MAS-5 E-MAS-6 3 3 3 Uptake 0.00 0.500 50.0 <0.100 0.494 48.6 -98.8 97.2 E-MAB-4 E-MAS-7 E-MAS-8 7 7 7 Uptake 0.00 1.00 100 <0.200 0.948 95.0 -94.8 95.0 E-MAB-5 E-MAS-9 E-MAS-10 14 14 14 Uptake 0.00 5.00 250 <1.00 5.31 268 -106 107 E-MAB-6 E-MAS-11 E-MAS-12 21 21 21 Uptake 0.00 5.00 250 <1.00 5.21 251 -104 101 E-MAB-7 E-MAS-13 E-MAS-14 28 28 28 Uptake 0.00 5.00 250 <1.00 5.27 249 -105 99.5 E-MAB-8 E-MAS-15 E-MAS-16 35 35 35 Uptake 0.00 5.00 100 <1.00 4.92 104 -98.3 104 E-MAB-10 E-MAS-19 E-MAS-20 42 42 42 Uptake 0.00 5.00 100 <1.00 4.97 105 -99.4 105 E-MAB-11 E-MAS-21 E-MAS-22 49 49 49 Uptake 0.00 5.00 100 <1.00 5.27 106 -106 106 E-MAB-12 E-MAS-23 56 56 Uptake 0.00 5.00 <1.00 5.31 -106 E-MAS-24 56 200 208 104 1 Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor of the matrix blank sample. All sample weights = 1.00 gram. 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly.__________ BACK TO MAIN Wildlife International, Ltd. - 55 Project Number 454A-134 Table 4 (continued) Matrix Blanks and Fortifications Analyzed Concurrently with Edible Fish Tissue Samples Sample Number (454A-134-) E-MAB-13 E-MAS-25 E-MAS-26 Sampling Interval (Day) 62 Uptake 62 62 Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS) (mg a.i./Kg) Fortified Measured1 0.00 <1.00 5.00 5.54 200 192 Percent Recovery2 -111 96.0 E-MAB-14 E-MAS-27 E-MAS-28 14 Depuration 14 14 0.00 5.00 200 <1.00 5.37 205 -107 102 E-MAB-15 E-MAS-29 E-MAS-30 28 Depuration 28 28 0.00 5.00 100 <1.00 5.15 104 -103 104 E-MAB-16 E-MAS-31 E-MAS-32 42 Depuration 42 42 0.00 5.00 100 <1.00 4.82 92.0 -96.5 92.0 E-MAB-17 56 Depuration 0.00 <1.00 -- E-MAS-33 56 5.00 4.99 99.7 E-MAS-34 56 100 101 101 1 Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor of the matrix blank sample. All sample weights = 1.00 gram. 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly.________ BACK TO MAIN Wildlife International, Ltd. - 56 Project Number 454A-134 Table 5 Matrix Blanks and Fortifications Analyzed Concurrently with Nonedible Fish Tissue Samples Sample Number (454A-134-) N-MAB-1 N-MAS-1 N-MAS-2 Sampling Interval (Day) 0 Hour 4 - Uptake 0 0 Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS) (mg a.i./Kg) Fortified Measured1 0.00 <0.0500 0.100 0.109 10.0 9.26 Percent Recovery2 -109 92.6 N-MAB-2 N-MAS-3 N-MAS-4 1 1 1 Uptake 0.00 0.100 10.0 <0.0500 0.106 9.36 -106 93.6 N-MAB-3 N-MAS-5 N-MAS-6 3 3 3 Uptake 0.00 0.500 50.0 <0.100 0.470 45.8 -93.9 91.6 N-MAB-4 N-MAS-7 N-MAS-8 7 7 7 Uptake 0.00 1.00 100 <0.200 0.975 90.1 -97.5 90.1 N-MAB-5 N-MAS-9 N-MAS-10 14 14 14 Uptake 0.00 5.00 250 <1.00 5.25 255 -105 102 N-MAB-6 N-MAS-11 N-MAS-12 21 21 21 Uptake 0.00 5.00 500 <1.00 4.98 505 -99.7 101 N-MAB-7 N-MAS-13 N-MAS-14 28 28 28 Uptake 0.00 5.00 500 <1.00 5.25 516 -105 103 N-MAB-8 N-MAS-15 N-MAS-16 35 35 35 Uptake 0.00 5.00 100 <1.00 5.15 108 -103 108 N-MAB-10 N-MAS-19 N-MAS-20 42 42 42 Uptake 0.00 5.00 100 <1.00 5.26 104 -105 104 N-MAB-11 N-MAS-21 N-MAS-22 49 49 49 Uptake 0.00 5.00 100 <1.00 5.40 108 -108 108 N-MAB-12 N-MAS-23 56 56 Uptake 0.00 5.00 <1.00 5.41 -108 N-MAS-24 56 200 245 122 1 Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor of the matrix blank sample. All sample weights = 1.00 gram. 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly.__________ BACK TO MAIN Wildlife International, Ltd. - 57 Project Number 454A-134 Table 5 (continued) Matrix Blanks and Fortifications Analyzed Concurrently with Nonedible Fish Tissue Samples Sample Number (454A-134-) N-MAB-13 N-MAS-25 N-MAS-26 Sampling Interval (Day) 62 Uptake 62 62 Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS) (mg a.i./Kg) Fortified Measured1 0.00 <1.00 5.00 5.68 200 212 Percent Recovery2 -114 106 N-MAB-14 N-MAS-27 N-MAS-28 14 Depuration 14 14 0.00 5.00 200 <1.00 5.29 207 -106 104 N-MAB-15 N-MAS-29 N-MAS-30 28 Depuration 28 28 0.00 5.00 100 <1.00 5.45 111 -109 111 N-MAB-16 N-MAS-31 N-MAS-32 42 Depuration 42 42 0.00 5.00 100 <1.00 5.19 98.9 -104 98.9 N-MAB-17 56 Depuration 0.00 <1.00 -- N-MAS-33 56 5.00 5.80 116 N-MAS-34 56 100 112 112 1 Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor of the matrix blank sample. All sample weights = 1.00 gram. 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly.________ BACK TO MAIN Wildlife International, Ltd. - 58 Project Number 454A-134 Table 6 Measured Concentrations of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Pre-Test Diluter Verification Samples Nominal Test Concentration (mg a.i./L) 0.0 (Negative Control) Sample Number (454A-134-) PT-1 PT-9 PT-17 Phase Pre-Uptake Sampling Time (Day) -4 -1 -1 Measured Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.i./L) < LOQ < LOQ < LOQ Percent of Nominal2 ---- 0.10 PT-3 PT-4 PT-11 PT-12 PT-19 PT-20 -4 0.0850 85.0 -4 0.0844 84.4 -1 0.0695 69.5 -1 0.0646 64.6 -1 0.0954 95.4 -1 0.0937 93.7 1.0 PT-6 PT-7 -4 0.917 91.7 -4 0.949 94.9 PT-14 -1 0.718 71.8 PT-15 PT-22 -1 0.738 73.8 -1 1.11 111 PT-23 -1 1.01 101 1The limit of quantitation (LOQ) was 0.0500 mg a.i./L, calculated as the product of the lowest calibration standard (0.0005 mg a.i./L) and the dilution factor of the matrix blank sample (100). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. BACK TO MAIN Wildlife International, Ltd. - 59 Project Number 454A-134 Table 7 Measured Concentrations of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Freshwater Samples from a Bluegill Sunfish Bioconcentration Test Nominal Test Concentration (mg a.i./L) 0.0 (Negative Control) Sample Number (454A-134-) 1 9 17 25 33 41 49 57 65 73 78 83 88 Phase Uptake Sampling Time (Day) 0, 0 hours 0, 4 hours 1 3 7 14 21 28 35 42 49 56 62 Measured Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.i./L) < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Percent of Nominal2 -- ----------- -- -- 93 Depuration 98 103 108 14 28 42 56 < LOQ < LOQ < LOQ < LOQ --- --- 0.10 3 Uptake 0, 0 hours 4 0, 0 hours 11 0, 4 hours 12 0, 4 hours 19 1 20 1 27 3 28 3 35 7 36 7 43 14 44 14 51 21 52 21 59 28 60 28 67 35 68 35 75 42 76 42 80 49 81 49 85 56 86 56 90 62 91 62 0.0717 0.0692 0.0791 0.0741 0.0702 0.0734 0.0751 0.0717 0.0826 0.0781 0.0680 0.0709 0.110 0.113 0.0822 0.0843 0.0915 0.0914 0.0983 0.110 0.103 0.103 0.0853 0.0948 0.0887 0.0914 71.7 69.2 79.1 74.1 70.2 73.4 75.1 71.7 82.6 78.1 68.0 70.9 110 113 82.2 84.3 91.5 91.4 98.3 110 103 103 85.3 94.8 88.7 91.4 1The limit of quantitation (LOQ) was 0.0500 mg a.i./L, calculated as the product of the lowest calibration standard (0.0005 mg a.i./L) and the dilution factor of the matrix blank sample (100). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. BACK TO MAIN Wildlife International, Ltd.____________________ - 60 - Project Number 454A-134 Table 7 (continued) Measured Concentrations of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Freshwater Samples from a Bluegill Sunfish Bioconcentration Test Nominal Test Concentration (mg a.i./L) 0.10 Sample Number (454A-134-) 95 96 100 101 105 106 110 111 Phase Depuration Sampling Time (Day) 14 14 28 28 42 42 56 56 Measured Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.i./L) < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ Percent of Nominal2 --------- 1.0 6 Uptake 0, 0 hours 7 0, 0 hours 14 0, 4 hours 15 0, 4 hours 22 1 23 1 30 3 31 3 38 7 39 7 46 14 47 14 54 21 55 21 62 28 63 28 70 353 71 353 0.797 0.802 0.891 0.930 0.867 0.820 0.813 0.734 0.845 0.818 0.900 0.875 0.988 1.01 0.913 0.925 0.838 0.871 79.7 80.2 89.1 93.0 86.7 82.0 81.3 73.4 84.5 81.8 90.0 87.5 98.8 101 91.3 92.5 83.8 87.1 1The limit of quantitation (LOQ) was 0.0500 mg a.i./L, calculated as the product of the lowest calibration standard (0.0005 mg a.i./L) and the dilution factor of the matrix blank sample (100). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 3Sampling was suspended after Day 35 Uptake due to 100% mortality at the 1.0 mg a.i./L nominal test level. BACK TO MAIN Wildlife International, Ltd.____________________ - 61 - Project Number 454A-134 Table 8 Measured Concentrations of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Edible Fish Tissue Samples from a Bluegill Sunfish Bioconcentration Test Nominal Test Concentration (mg a.i./L) 0.0 (Negative Control) Sample Number (454A-134-) E-1 E-2 E-14 E-15 E-27 E-28 E-40 E-41 E-53 E-54 E-66 E-67 E-79 E-80 E-92 E-93 E-100 E-101 E-108 E-109 E-116 E-117 E-124 E-125 Phase Uptake Sampling Time (Day) 0, 4 hours 0, 4 hours 1 1 3 3 7 7 14 14 21 21 28 28 35 35 42 42 49 49 56 56 62 62 Measured Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.i./Kg) <0.0510 <0.0665 <0.0960 <0.0835 <0.114 <0.0910 <0.0965 <0.162 <0.468 <0.560 <0.730 <0.710 <0.740 <0.800 <0.970 <0.575 <0.399 <0.590 <0.575 <0.520 <0.775 <0.820 <0.660 <0.615 E-132 E-133 E-140 E-141 E-147 E-148 E-154 E-155 Depuration 14 14 28 28 42 42 56 56 <0.685 <0.710 <0.585 <0.590 <0.830 <0.825 <0.755 <1.07 0.10 E-4 Uptake 0, 4 hours E-5 0, 4 hours 0.167 0.155 E-6 0, 4 hours 0.144 E-7 0, 4 hours 0.182 E-17 E-18 1 0.734 1 0.726 E-19 1 0.631 E-20 E-30 1 0.806 3 1.73 E-31 3 2.07 E-32 3 2.03 E-33 3 2.11 1Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor of the sample (L/Kg). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly.____________ BACK TO MAIN Wildlife International, Ltd.____________________ - 62 - Project Number 454A-134 Table 8 (continued) Measured Concentrations of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Edible Fish Tissue Samples from a Bluegill Sunfish Bioconcentration Test Nominal Test Concentration (mg a.i./L) 0.10 Sample Number (454A-134-) E-43 E-44 E-45 E-46 E-56 E-57 E-58 E-59 E-69 E-70 E-71 E-72 E-82 E-83 E-84 E-85 E-95 E-96 E-97 E-98 E-103 E-104 E-105 E-106 E-111 E-112 E-113 E-114 E-119 E-120 E-121 E-122 E-127 E-128 E-129 E-130 Phase Uptake Sampling Time (Day) 7 7 7 7 14 14 14 14 21 21 21 21 28 28 28 28 35 35 35 35 42 42 42 42 49 49 49 49 56 56 56 56 62 62 62 62 Measured Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.i./Kg) 3.73 4.25 4.73 6.25 11.4 9.07 13.7 12.6 11.7 12.0 12.9 10.6 18.3 13.7 23.9 23.1 22.6 27.7 23.8 20.6 27.6 25.3 21.2 27.6 33.3 36.2 39.0 30.6 48.3 38.9 44.1 38.3 42.4 66.2 42.2 39.2 E-135 E-136 E-137 Depuration 14 14 14 48.5 31.8 31.6 E-138 E-143 E-144 14 42.0 28 26.0 28 33.3 E-145 E-146 E-150 28 38.7 28 55.8 42 24.1 E-151 E-152 42 31.2 42 30.0 E-153 42 33.0 1Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor of the sample (L/Kg). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly.____________ BACK TO MAIN Wildlife International, Ltd.____________________ - 63 - Project Number 454A-134 Table 8 (continued) Measured Concentrations of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Edible Fish Tissue Samples from a Bluegill Sunfish Bioconcentration Test Nominal Test Concentration (mg a.i./L) 0.10 Sample Number (454A-134-) E-157 E-158 E-159 E-160 Phase Depuration Sampling Time (Day) 56 56 56 56 Measured Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.i./Kg) 21.1 37.6 32.9 31.2 1.0 E-9 Uptake 0, 4 hours 1.46 E-10 0, 4 hours 1.48 E-11 0, 4 hours 1.19 E-12 0, 4 hours 1.39 E-22 1 4.68 E-23 1 6.59 E-24 1 5.56 E-25 1 5.64 E-35 3 17.3 E-36 3 15.8 E-37 3 19.0 E-38 3 20.8 E-48 7 42.0 E-49 7 44.0 E-50 7 57.7 E-51 7 46.8 E-61 14 87.1 E-62 14 81.6 E-63 14 90.7 E-64 14 73.3 E-74 21 79.4 E-75 21 117 E-76 21 104 E-77 21 102 E-87 28 102 E-88 28 131 E-89 28 107 E-90 28 133 1Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor of the sample (L/Kg). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly.____________ BACK TO MAIN Wildlife International, Ltd.____________________ - 64 - Project Number 454A-134 Table 9 Measured Concentrations of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Nonedible Fish Tissue Samples from a Bluegill Sunfish Bioconcentration Test Nominal Test Concentration (mg a.i./L) 0.0 (Negative Control) Sample Number (454A-134-) N-1 N-2 N-14 N-15 N-27 N-28 N-40 N-41 N-53 N-54 N-66 N-67 N-79 N-80 N-92 N-93 N-100 N-101 N-108 N-109 N-116 N-117 N-124 N-125 Phase Uptake Sampling Time (Day) 0, 4 hours 0, 4 hours 1 1 3 3 7 7 14 14 21 21 28 28 35 35 42 42 49 49 56 56 62 62 Measured Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.i./Kg) <0.0458 <0.0575 <0.0820 <0.0645 <0.0820 <0.0795 <0.0805 <0.0965 <0.434 <0.457 <0.560 <0.560 <0.580 <0.620 <0.590 <0.417 <0.363 <0.480 <0.555 <0.375 <0.540 <0.520 <0.505 <0.505 N-132 N-133 N-140 N-141 N-147 N-148 N-154 N-155 Depuration 14 14 28 28 42 42 56 56 <0.470 <0.472 <0.449 <0.505 <0.590 <0.570 <0.630 <0.780 0.10 N-4 Uptake 0, 4 hours N-5 0, 4 hours 0.415 0.519 N-6 0, 4 hours 0.417 N-7 0, 4 hours 0.497 N-17 N-18 1 1.68 1 1.85 N-19 1 1.72 N-20 N-30 1 2.07 3 4.59 N-31 3 5.50 N-32 3 5.47 N-33 3 5.97 1Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor of the sample (L/Kg). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly.____________ BACK TO MAIN Wildlife International, Ltd. - 65 Project Number 454A-134 Table 9 (continued) Measured Concentrations of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Nonedible Fish Tissue Samples from a Bluegill Sunfish Bioconcentration Test Nominal Test Concentration (mg a.i./L) 0.10 Sample Number (454A-134-) N-43 N-44 N-45 N-46 N-56 N-57 N-58 N-59 N-69 N-70 N-71 N-72 N-82 N-83 N-84 N-85 N-95 N-96 N-97 N-98 N-103 N-104 N-105 N-106 N-111 N-112 N-113 N-114 N-119 N-120 N-121 N-122 N-127 N-128 N-129 N-130 Phase Uptake Sampling Time (Day) 7 7 7 7 14 14 14 14 21 21 21 21 28 28 28 28 35 35 35 35 42 42 42 42 49 49 49 49 56 56 56 56 62 62 62 62 Measured Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.i./Kg) 10.2 10.6 11.9 15.2 27.3 23.2 35.3 32.6 33.3 22.7 24.6 24.4 49.4 40.7 65.3 57.9 67.1 73.3 62.0 59.1 64.0 68.1 54.4 79.6 85.0 95.1 93.1 77.7 122 94.2 73.2 106 101 112 105 96.4 N-135 N-136 N-137 N-138 N-143 N-144 N-145 N-146 N-150 N-151 N-152 N-153 Depuration 14 14 14 14 28 28 28 28 42 42 42 42 124 79.4 81.8 113 85.7 95.1 85.7 94.8 71.7 80.6 78.3 82.1 1Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor of the sample (L/Kg). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. BACK TO MAIN Wildlife International, Ltd.____________________ - 66 - Project Number 454A-134 Table 9 (continued) Measured Concentrations of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Nonedible Fish Tissue Samples from a Bluegill Sunfish Bioconcentration Test Nominal Test Concentration (mg a.i./L) 0.10 Sample Number (454A-134-) N-157 N-158 N-159 N-160 Phase Depuration Sampling Time (Day) 56 56 56 56 Measured Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS)1 (mg a.i./Kg) 57.7 80.3 85.4 84.4 1.0 N-9 Uptake 0, 4 hours 3.52 N-10 0, 4 hours 4.37 N-11 0, 4 hours 4.22 N-12 0, 4 hours 4.06 N-22 1 11.1 N-23 1 14.2 N-24 1 13.3 N-25 1 12.1 N-35 3 39.3 N-36 N-37 3 42.0 3 43.8 N-38 3 51.8 N-48 7 100 N-49 7 102 N-50 N-51 7 102 7 120 N-61 14 177 N-62 14 207 N-63 14 245 N-64 N-74 14 214 21 201 N-75 21 278 N-76 21 246 N-77 21 229 N-87 28 289 N-88 28 372 N-89 28 320 N-90 28 361 1Less than values correspond to limit of quantitation (LOQ). For each analysis, the LOQ was calculated as the product of the lowest calibration standard and the overall dilution factor of the sample (L/Kg). 2Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly.____________ BACK TO MAIN Wildlife International, Ltd. - 67 - Project Number 454A-134 Table 10 Measured Concentrations of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Tissue Storage Stability Samples from a Bluegill Sunfish Bioconcentration Test Nominal Concentration (mg a.i./Kg) Negative Control Sample Number (454A-134-) E-MAB-184 N-MAB-184 Tissue Type Edible Nonedible Measured Concentration of Perfluorooctanesulfonate, Potassium Salt (PFOS) (mg a.i./Kg) <LOQ1 <LOQ1 Percent of Nominal2, 5 --- 0.100 E-STMAS-13 E-MAS-354 N-STMAS-13 N-MAS-354 Edible Edible Nonedible Nonedible 0.109 0.111 0.114 0.112 109 111 114 112 E-STMAS-23 Edible 8.43 84.3 10.0 E-MAS-364 Edible 11.3 113 N-STMAS-23 Nonedible 9.30 93.0 N-MAS-364 Nonedible 11.3 113 1 Less than values correspond to limit of quantitation (LOQ). The LOQ was 0.0500 mg a.i./Kg, calculated as the product of the lowest calibration standard (0.0005 mg a.i./L) and the overall dilution factor of the matrix blank sample (100 L/Kg). All sample weights = 1.00 gram. 2 Results were generated using MacQuan, version 1.6 software. Manual calculations may differ slightly. 3 The stability samples were fortified on December 5, 2000 and stored in the freezer. The samples were removed from the freezer on April 3, 2001 (after 119 days of frozen storage) and analyzed. 4 These samples were prepared on April 3, 2001 and the results were compared to the stability sample results. 5 The criterion for storage stability was Percent of Nominal between 80 and 120%.______________________________ BACK TO MAIN Wildlife International, Ltd.____________________ - 68 - Project Number 454A-134 Table 11 Lipid Content in Edible Fish Tissue Nominal Test Concentration (mg a.i./L) 0.0 (Negative Control) Sample Number (454A-134-) EL1 EL2 EL3 EL4 Phase Uptake Sampling Time (Day) 0, 0 hour 0, 0 hour 0, 0 hour 0, 0 hour Lipid Weight (g) 0.0314 0.0453 0.0453 0.0463 Fish Tissue Weight (g) 1.7499 2.1445 1.8577 1.8335 Lipid/Fish Tissue Weight1 (mg/Kg) 17,900 21,100 24,400 25,300 EL6 Uptake 62 0.0231 1.5122 15,300 EL7 62 0.0135 1.0670 12,700 EL8 62 0.0134 0.9726 13,800 EL9 62 0.0162 1.0650 15,200 EL16 EL17 EL18 EL19 Depuration 56 56 56 56 0.0254 0.0163 0.0158 0.0256 1.3549 1.0890 1.1234 1.3777 18,700 15,000 14,100 18,600 0.10 EL11 Uptake 62 0.0162 1.0411 15,600 EL12 62 0.0327 1.3551 24,100 EL13 62 0.0329 1.4893 22,100 EL14 62 0.0188 0.9054 20,800 EL21 EL22 EL23 EL24 Depuration 56 56 56 56 0.0172 0.0190 0.0250 0.0252 1.2362 1.3483 1.3303 1.1040 1Ratio calculated as [lipid weight (g) +- fish tissue weight (g)] x 1000 mg/g x 1000 g/Kg. 13,900 14,100 18,800 22,800 BACK TO MAIN Wildlife International, Ltd.____________________ - 69 - Project Number 454A-134 Table 12 Lipid Content in Nonedible Fish Tissue Nominal Test Concentration (mg a.i./L) 0.0 (Negative Control) Sample Number (454A-134-) NL1 NL2 NL3 NL4 Phase Uptake Sampling Time (Day) 0, 0 hour 0, 0 hour 0, 0 hour 0, 0 hour Lipid Weight (g) 0.1363 0.2383 0.2258 0.1992 Fish Tissue Weight (g) 2.0649 2.6355 1.9568 2.0812 Lipid/Fish Tissue Weight1 (mg/Kg) 66,000 90,400 115,400 95,700 NL6 Uptake 62 0.0506 1.8821 26,900 NL7 62 0.0274 1.5901 17,200 NL8 62 0.0298 1.7596 16,900 NL9 62 0.0518 1.7302 29,900 NL16 NL17 NL18 NL19 Depuration 56 56 56 56 0.0616 0.0318 0.0360 0.0966 1.7263 1.6746 1.5847 1.8165 35,700 19,000 22,700 53,200 0.10 NL11 Uptake 62 0.0503 1.5484 32,500 NL12 62 0.1370 1.7642 77,700 NL13 62 0.1276 2.0113 63,400 NL14 62 0.1221 1.2898 94,700 NL21 NL22 NL23 NL24 Depuration 56 56 56 56 0.0343 0.0404 0.0710 0.0714 1.7085 1.8394 1.7652 1.5682 1Ratio calculated as [lipid weight (g) +- fish tissue weight (g)] x 1000 mg/g x 1000 g/Kg. 20,100 22,000 40,200 45,500 Wildlife International, Ltd.____________________ - 70 - BACK TO MAIN Project Number 454A-134 METHOD OUTLINE FOR THE ANALYSIS OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) IN FRESHWATER Prepare matrix fortification samples in freshwater matrix by spiking the requisite volume of PFOS stock solutions directly into freshwater. Perform fortifications with gas-tight syringes and Class A volumetric flasks. I Prepare appropriate dilutions of study and QC samples to within the calibration range of the PFOS LCMS methodology: Partially fill Class A volumetric flasks with 50% methanol/50% water dilution solvent containing 0.0100 mg/L 4H PFOS internal standard and 0.05% v/v formic acid. Add appropriate volume of sample and bring to volume with dilution solvent. Process matrix blank samples for a given matrix using the same dilution and aliquot volumes as for the lowest fortification level in that matrix. Mix well by several repeat inversions. I Ampulate samples and submit for LCMS analysis. Figure 1. Method flow chart for the analysis of Perfluorooctanesulfonate, Potassium Salt (PFOS) in freshwater. Wildlife International, Ltd.____________________ - 71 - BACK TO MAIN Project Number 454A-134 METHOD OUTLINE FOR THE ANALYSIS OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) IN FISH TISSUES Quality control samples are prepared from aliquots (approximately 1 g) of bulk control fish tissue homogenate. Remove appropriate (edible or non-edible) aliquots from frozen storage and allow to thaw. Prepare fortification samples with the appropriate PFOS stock solution using gas-tight syringe(s). The matrix blank sample will be unfortified fish tissue homogenate. i Add 10.0 mL of methanol to each sample with a glass Class A volumetric pipette. Homogenize each test sample for approximately 1 minute using a tissue shredder. Rinse the homogenizer with the appropriate solvent(s) in between samples. i Sonicate each sample for approximately 5 minutes with a sonic dismembrator i Cap the vials and shake well. Centrifuge the vials at approximately 2000 rpm for approximately 5 minutes. i Prepare appropriate dilutions of study and QC samples to within the calibration range of the PFOS LCMS methodology: Partially fill Class A volumetric flasks with 50% : 50% methanol/NANOpure water dilution solvent. Add appropriate volume of sample and bring to volume with dilution solvent. Process matrix blank samples for a given matrix using the same dilution and aliquot volumes as for the lowest fortification level in that matrix. Mix well by several repeat inversions. i Transfer an aliquot of each sample to an autosampler vial and submit for LC/MS/MS analysis. Figure 2. Method flow chart for the analysis of Perfluorooctanesulfonate, Potassium Salt (PFOS) in fish tissues. Wildlife International, Ltd.____________________ - 72 - BACK TO MAIN Project Number 454A-134 METHOD OUTLINE FOR THE ANALYSIS OF LIPIDS IN FISH TISSUES Remove vials to be analyzed from the freezer. Allow samples to thaw. i For each sample, add 10 mL of NANOpure water to fish tissue in vial and homogenize for approximately 1 minute using a hand-held tissue shredder. Rinse the homogenizer with the appropriate solvent(s) in between samples. i Transfer each homogenate to a 250-mL separatory funnel that contains 25 mL of chloroform and 50 mL of methanol. i Rinse each vial with an additional 10 mL of NANOpurewater and pour rinse into respective separatory funnel. i Shake each separatory funnel for approximately one minute with venting. i Add 50 mL of chloroform followed by 50 mL of saturated sodium chloride to each separatory funnel. i Briefly swirl each separatory funnel with venting. i Allow the phases to separate. For each sample, drain the chloroform layer through a powder funnel packed with glass wool and anhydrous sodium sulfate into a 250-mL round-bottom flask. i Add an additional 50-mL aliquot of chloroform to each separatory funnel and repeat the extraction and draining procedures. i Rotary evaporate the extracts in a water bath maintained at approximately 40C to near dryness. i Transfer each extract to a pre-weighed, labeled scintillation vial. i Rinse each 250-mL round-bottom flask with a small volume of chloroform and transfer rinse to respective scintillation vial. i Evaporate the remaining solvent in each vial under a gentle stream of nitrogen or clean dry air. i ___________________________ Reweigh each vial and record weight. Figure 3. Method flow chart for the analysis of lipids in fish tissues. Wildlife International, Ltd.____________________ - 73 - BACK TO MAIN Project Number 454A-134 Figure 4. A typical calibration curve for Perfluorooctanesulfonate, Potassium Salt (PFOS) in freshwater. Slope = 4.64; Intercept = -0.01; r = 0.99932. Curve is weighted (1/x). (monitored masses = 499 amu (PFOS) and 427 amu (4HPFOS internal standard)). Wildlife International, Ltd._____ - 74 - BACK TO MAIN Project Number 454A-134 Figure 5. A representative ion chromatogram of a low-level (0.500 |ig a.i./L) Perfluorooctanesulfonate, Potassium Salt (PFOS) standard for freshwater analyses. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard bottom)). Wildlife International, L td _____ - 75 - BACK TO MAIN Project Number 454A-134 PFOS_5 STD 5.00 ug a.i./L 4675A-011D -15 4.98 in 1 period PFOS Internal Standard: 4HPFOS Use Area Absolute Retention Time 1: 4.97 Q1 Ml, 298 scans 499.0 Noise Thres. Quant Thres. Min. Width Mult. Width Base. Width RT Win. (secs) Smooth Expected RT 20.0 15.0 3 6 40 30 1 3.80 Area 2505786 Height 227264 Start Time End Time Integration Width Retention Time Integration Type 3.61 4.28 0.67 3.83 A - BB Tue, Dec 5, 2000 11:10 PFOS_5 STD 5.00 ug a.i./L 4675A-011D -15 4.98 in 1 period 4HPFOS use as Internal Standard 1: 4.97 Q1 MI, 298 scans 427.0 Noise Thres. Quant Thres. Min. Width 2.0 1.0 3 Mult. Width 6 Base. Width 30 RT Win. (secs) Smooth 20 1 Expected RT 2.53 Area 1052782 Height 133897 Start Time 2.44 End Time 2.95 Integration Width Retention Time 0.50 2.54 Integration Type A - BB Tue, Dec 5, 2000 11:10 intensity: 250000 cps intensity: 250000 cps Figure 6. A representative ion chromatogram of a high-level (5.00 pg a.i./L) Perfluorooctanesulfonate, Potassium Salt (PFOS) standard for freshwater analyses. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard bottom)). Wildlife International, Ltd. - 76 - BACK TO MAIN Project Number 454A-134 Figure 7. A typical calibration curve for Perfluorooctanesulfonate, Potassium Salt (PFOS) in fish tissue. Slope = 4552.41; Intercept = 69.58; r = 0.99940. Curve is weighted (1/x). (monitored mass = 499 amu ^ 99.1 amu). Wildlife International, Ltd.____________________ - 77 - BACK TO MAIN Project Number 454A-134 Figure 8. A representative ion chromatogram of a low-level (0.500 gg a.i./L) Perfluorooctanesulfonate, Potassium Salt (PFOS) standard for fish tissue analyses. From low-range (0.500 to 5.00 gg a.i./L) calibration set. (monitored mass = 499 amu ^ 99.1 amu). Wildlife International, Ltd.____________________ - 78 - BACK TO MAIN Project Number 454A-134 Figure 9. A representative ion chromatogram of a high-level (50.0 gg a.i./L) Perfluorooctanesulfonate, Potassium Salt (PFOS) standard for fish tissue analyses. From high-range (5.00 to 50.0 gg a.i./L) calibration set. (monitored mass = 499 amu ^ 99.1 amu). Wildlife International, Ltd._____ - 79 BACK TO MAIN Project Number 454A-134 PFOS_7 454A-1 34- MAB-1 Tue, Dec 5, 2000 11:22 4.98 in 1 period PFOS Internal Standard: 4HPFOS Use Area Absolute Retention Time 1: 4.97 Q1 Ml, 298 scans 499.0 Noise Thres. Quant Thres. Min. Width Mult. Width Base. Width 20.0 15.0 3 6 40 RT Win. (secs) 30 Smooth Expected RT 1 3.80 Area 0 Height 0 Start Time 0.00 End Time Integration Width 0.00 0.00 Retention Time 0.00 Integration Type intensity: 250000 cps PFOS_7 454A-1 34- MAB-1 4.98 in 1 period 4HPFOS use as Internal Standard Tue, Dec 5, 2000 11:22 1: 4.97 Q1 Ml, 298 scans 427.0 Noise Thres. 2.0 Quant Thres. 1.0 Min. Width 3 Mult. Width 6 Base. Width 30 RT Win. (secs) Smooth 20 1 Expected RT 2.53 Area 1178466 Height 150726 Start Time 2.44 End Time 2.95 Integration Width 0.50 Retention Time 2.56 Integration Type A - BB intensity: 250000 cps Figure 10. A representative ion chromatogram of a freshwater matrix blank sample (454A - 134 MAB - 1, dilution = 100x). The arrow indicates the retention time of Perfluorooctanesulfonate, Potassium Salt (PFOS). (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard bottom)). Wildlife International, Ltd._____ - 80 - BACK TO MAIN Project Number 454A-134 PFOS_9 454A-1 34- MAS-2 Tue, Dec 5, 2000 11:34 4.98 in 1 period pros Internal Standard: 4HPFOS Use Area Absolute Retention Time 1: 4.97 Q1 Ml, 298 scans 499.0 Noise Thres. 20.0 Quant Thres. 15.0 Min. Width 3 Mult. Width 6 Base. Width 40 RT Win. (secs) 30 Smooth 1 Expected RT 3.80 Area 995049 Height 92517 Start Time 3.61 End Time 4.28 Integration Width 0.67 Retention Time 3.83 Integration Type A - BB PFOS_9 454A-134- MAS-2 4.98 in 1 period 4HPFOS use as Internal Standard Tue, Dec 5, 2000 11:34 1: 4.97 Q1 Ml, 298 scans 427.0 Noise Thres. 2.0 Quant Thres. Min. Width Mult. Width Base. Width RT Win. (secs) Smooth Expected RT 1.0 3 6 30 20 1 2.53 Area 1109335 Height 139303 Start Time End Time 2.43 2.93 Integration Width Retention Time Integration Type 0.50 2.56 A - BB intensity: 250000 cps intensity: 250000 cps Figure 11. A representative ion chromatogram of a freshwater matrix fortification sample (454A-134-MAS-2, 0.500 mg a.i./L nominal concentration, dilution = 250x). (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard bottom)). Wildlife International, Ltd.____________________ - 81 - BACK TO MAIN Project Number 454A-134 Figure 12. A representative ion chromatogram of an edible fish tissue matrix blank sample (454A134-E-MAB-1, overall dilution factor = 100 L/Kg). The arrow indicates the retention time of Perfluorooctanesulfonate, Potassium Salt (PFOS). (monitored mass = 499 amu ^ 99.1 amu). Wildlife International, Ltd.____________________ - 82 - BACK TO MAIN Project Number 454A-134 Figure 13. A representative ion chromatogram of a nonedible fish tissue matrix blank sample (454A134-N-MAB-1, overall dilution factor = 100 L/Kg). The arrow indicates the retention time of Perfluorooctanesulfonate, Potassium Salt (PFOS). (monitored mass = 499 amu ^ 99.1 amu). Wildlife International, Ltd.____________________ - 83 - BACK TO MAIN Project Number 454A-134 Figure 14. A representative ion chromatogram of an edible fish tissue matrix fortification sample (454A-134-E-MAS-6, 50.0 mg a.i./Kg nominal concentration, overall dilution factor = 20000 L/Kg). (monitored mass = 499 amu ^ 99.1 amu). Wildlife International, Ltd.____________________ - 84 - BACK TO MAIN Project Number 454A-134 Figure 15. A representative ion chromatogram of a nonedible fish tissue matrix fortification sample (454A-134-N-MAS-8, 100 mg a.i./Kg nominal concentration, overall dilution factor = 50000 L/Kg). (monitored mass = 499 amu ^ 99.1 amu). Wildlife International, Ltd._____ - 85 - BACK TO MAIN Project Number 454A-134 PFOS_13 454A-1 34- 3 Tue, Dec 5, 2000 11:58 4.98 in 1 period PFOS Internal Standard: 4HPFOS Use Area Absolute Retention Time 1: 4.97 Q1 Ml, 298 scans 499.0 Noise Thres. 20.0 Quant Thres. 15.0 Min. Width 3 Mult. Width 6 Base. Width 40 RT Win. (secs) 30 Smooth 1 Expected RT 3.80 Area 355173 Height 33158 Start Time End Time Integration Width Retention Time Integration Type 3.63 4.30 0.67 3.83 A-BB intensity: 250000 cps PFOS_13 454A-1 34- 3 Tue, Dec 5, 2000 11:58 4.98 in 1 period 4HPFOS use as Internai Standard 1: 4.97 Q1 MI, 298 scans 427.0 Noise Thres. 2.0 Quant Thres. 1.0 Min. Width 3 Mult. Width 6 Base. Width 30 RT Win. (secs) 20 Smooth 1 Expected RT 2.53 Area 1094547 Height 138642 Start Time End Time Integration Width Retention Time Integration Type 2.44 2.95 0.50 2.56 A -BB intensity: 250000 cps Figure 16. A representative ion chromatogram of a freshwater sample (454A-134-3, dilution = 100x) from the 0.10 mg a.i./L treatment group. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard bottom)). Wildlife International, Ltd.____________________ - 86 - BACK TO MAIN Project Number 454A-134 Figure 17. A representative ion chromatogram of an edible fish tissue sample (454A-134-E-30, overall dilution factor = 1420) from the 0.10 mg a.i./L treatment group). (monitored mass = 499 amu ^ 99.1 amu). Wildlife International, Ltd.____________________ - 87 - BACK TO MAIN Project Number 454A-134 Figure 18. A representative ion chromatogram of a nonedible fish tissue sample (454A-134-N-30, overall dilution factor = 2650) from the 0.10 mg a.i./L treatment group). (monitored mass = 499 amu ^ 99.1 amu). - 88 - BACK TO MAIN Project Number 454A-134 Appendix 4 Temperature and pH of Water in the Test Chambers Uptake Phase Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution Water: Well Water Uptake Phase Mean Measured Concentration (mg a.i./L) Day 0 Temp.1 (C) pH Day 7 Temp.1 (C) pH Day 14 Temp.1 (C) pH Day 21 Temp.1 (C) pH Negative Control 22.0 8.2 21.9 8.0 21.8 8.0 21.8 8.1 Day 28 Temp.1 (C) 21.9 pH 7.9 0.086 21.9 8.2 21.8 8.0 21.8 8.0 21.8 8.1 21.9 7.9 0.87 21.9 8.2 21.8 8.0 'Continuous measurements of temperature ranged from 20.0 to 22.0C. 21.8 Uptake Phase Mean Measured Concentration (mg a.i./L) Negative Control Day 35 Temp.1 (C) 21.8 pH 8.1 Day 42 Temp.1 (C) 21.8 pH 8.0 7.9 21.8 Day 49 Temp.1 (C) 21.9 pH 8.0 8.1 21.8 Day 56 Temp.1 (C) 21.9 pH 8.1 8.0 0.086 21.7 8.1 21.8 7.9 0.87 21.7 8.2 2 --2 'Continuous measurements of temperature ranged from 20.0 to 22.0C. Measurements discontinued due to 100% mortality. 21.8 8.0 _ -- 21.9 8.1 -- _ - 89 - BACK TO MAIN PROJECT NO.: 439A-111 Appendix 4 (Continued) Temperature and pH of Water in the Test Chambers ________________________________________________ Depuration Phase______________ Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Uptake Phase Mean Measured Concentration (mg a.i./L) _______ Day 1_______ Temp.1 (C) pH ________Day 7_______ Temp.1 (C) pH ______Day 14_____ Temp.1 (C) pH ______ Day 21______ Temp.1 (C) pH Negative Control 21.9 8.1 21.8 8.2 21.9 8.0 21.9 8.1 _______ Day 27 Temp.1 (C) 21.8 pH 8.1 0.086 21.8 8.1 21.8 8.2 'Continuous measurements of temperature ranged from 20.0 to 22.0C. 21.9 Uptake Phase Mean Measured Concentration (mg a.i./L) Negative Control Day 35 Temp.1 (C) 21.9 pH 8.1 Day 42 Temp.1 (C) 21.9 pH 8.1 8.1 21.8 Day 49 Temp.1 (C) 22.0 pH 8.1 8.1 21.7 Day 56 Temp.1 (C) 21.9 pH 8.1 8.1 0.086 21.8 8.1 21.9 8.1 'Continuous measurements of temperature ranged from 20.0 to 22.0C. 22.0 8.1 21.9 8.1 Wildlife International, Ltd. - 90 - BACK TO MAIN Sponsor: Test Substance: Test Organism: Dilution Water: Day Appendix 5 Dissolved Oxygen (mg/L) of Water in the Test Chambers1 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Uptake Phase Negative Control 0.086 mg a.i./L 0.87 mg a.i./L 0 8.2 8.2 8.2 1 6.8 6.8 6.8 2 7.4 7.2 6.9 3 7.3 7.1 6.5 4 7.3 7.2 7.3 5 7.3 6 7.3 7 7.0 8 7.6 9 7.6 10 7.8 11 7.7 12 7.5 13 7.8 14 7.3 15 7.5 16 7.5 17 7.9 18 8.0 19 7.4 20 7.8 21 7.8 22 7.8 23 7.8 24 NC2 25 7.5 26 7.5 27 7.6 28 7.3 29 8.0 30 7.6 31 7.8 32 7.5 33 7.8 34 7.8 35 7.5 7.3 7.3 7.0 7.6 7.4 7.6 7.6 7.3 7.6 7.1 7.2 7.3 7.8 8.0 7.5 7.8 7.8 7.6 7.8 NC 7.4 7.4 7.6 7.2 7.8 7.4 7.6 7.4 7.9 7.8 7.4 7.1 6.8 7.0 7.4 7.4 7.2 7.5 7.0 7.1 6.4 6.7 7.4 8.0 8.0 7.5 7.7 7.8 7.7 7.9 NC 7.4 7.6 7.8 7.4 8.0 7.9 8.0 7.7 7.8 8.2 8.1 1A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation in freshwater at 22C. 2NC - Data inadvertently not collected Wildlife International, Ltd. - 91 - BACK TO MAIN Appendix 5 (Continued) Dissolved Oxygen (mg/L) of Water in the Test Chambers1 Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution Water: Well Water Uptake Phase Day Negative Control 0.086 mg a.i./L 0.87 mg a.i./L 36 7.8 7.6 2 37 7.7 7.8 -- 38 8.2 7.8 -- 39 7.8 7.6 -- 40 7.8 7.8 -- 41 7.8 7.7 -- 42 7.4 7.2 -- 43 7.7 7.4 -- 44 7.3 7.0 -- 45 7.8 7.4 -- 46 7.8 7.2 -- 47 7.6 7.3 -- 48 7.9 7.6 -- 49 8.0 8.0 -- 50 7.5 7.2 -- 51 7.6 7.5 -- 52 7.5 7.1 -- 53 8.0 7.9 -- 54 7.4 7.1 -- 55 7.8 7.2 -- 56 7.8 7.4 -- 57 7.7 7.4 -- 58 7.4 7.4 -- 59 7.6 7.4 -- 60 7.6 7.6 -- 61 7.6 7.6 -- 62 7.6 7.6 -- 1A dissolved oxygen concentration of 5.2 mg/L represents 60% saturation in freshwater at 22C. 2Data not collected due to 100% mortality. Wildlife International, Ltd. - 92 - BACK TO MAIN Sponsor: Test Substance: Test Organism: Dilution Water: Day Appendix 5 (Continued) Dissolved Oxygen (mg/L) of Water in the Test Chambers1 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Depuration Phase Negative Control 0.086 mg a.i./L 1 7.5 2 7.6 3 8.3 4 8.0 5 8.2 6 8.0 7 8.4 8 8.3 9 7.9 10 8.0 11 8.0 12 7.9 13 8.0 14 7.9 15 7.6 16 8.1 17 8.0 18 7.9 19 7.8 20 8.2 21 8.0 22 8.1 23 8.4 24 8.4 25 8.1 26 7.5 27 7.9 28 7.5 29 8.2 30 8.0 31 8.3 32 8.4 33 8.0 34 8.3 35 8.5 7.6 7.7 8.4 8.0 8.2 8.0 8.4 8.4 8.0 8.0 8.0 8.0 8.0 7.9 8.0 8.2 8.0 8.0 7.8 8.2 8.1 8.2 8.4 8.4 8.2 7.6 7.9 7.6 8.2 8.1 8.3 8.4 8.1 8.4 8.5 XA dissolved oxygen concentration of 5.2 mg/L represents 60% saturation in freshwater at 22C. Wildlife International, Ltd. - 93 - BACK TO MAIN Sponsor: Test Substance: Test Organism: Dilution Water: Day Appendix 5 (Continued) Dissolved Oxygen (mg/L) of Water in the Test Chambers1 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Depuration Phase Negative Control 0.086 mg a.i./L 36 8.4 37 8.2 38 8.0 39 8.0 40 8.1 41 7.7 42 7.8 43 8.2 44 7.8 8.5 8.1 8.0 8.0 8.2 7.7 7.8 8.3 7.8 45 8.2 46 8.1 47 8.6 48 8.4 8.3 8.1 8.6 8.4 49 8.5 50 8.1 51 8.6 52 8.1 53 8.2 54 8.4 55 8.2 56 8.2 8.6 8.1 8.6 8.1 8.2 8.4 8.3 8.3 XA dissolved oxygen concentration of 5.2 mg/L represents 60% saturation in freshwater at 22C. Wildlife International, Ltd. - 94 - BACK TO MAIN Appendix 6 Hardness, Alkalinity, Conductivity and TOC of Water in the Negative Control ______________________________________Uptake Phase___________________________ Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution Water: Well Water Parameter Day 0 7 14 21 28 35 42 49 Hardness 132 112 126 130 136 132 104 134 (mg/L as CaCO3) Alkalinity 183 174 179 183 181 183 183 184 (mg/L as CaCO3) Conductivity (pmhos/cm) 310 325 330 330 325 325 310 320 TOC (mg C/L) <1 <1 <1 <1 <1 <1 <1 <1 56 130 181 330 <1 ____________________________________ Depuration Phase Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomis macrochirus Dilution Water: Well Water Parameter Day 1 7 14 21 28 35 42 49 56 Hardness 134 110 126 124 130 132 132 128 126 (mg/L as CaCO3) Alkalinity 179 178 178 180 183 180 175 179 178 (mg/L as CaCO3) Conductivity (pmhos/cm) 330 330 325 320 325 325 330 320 325 TOC (mg C/L) <1 <1 <1 <1 <1 <1 <1 <1 <1 Wildlife International, Ltd. - 95 - BACK TO MAIN Appendix 7 Cumulative Mortality and Treatment-Related Effects1 ______________________ Negative Control - Uptake Phase________ Sponsor: Test Substance: Test Organism: Dilution Water: Day 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Cumulative Number Observations Dead Number Remaining 0 AN 0 90 1 AN 0 85 2 AN 0 80 3 AN 0 80 4 AN 0 75 5 AN 0 75 6 AN 0 75 7 AN 0 75 8 AN 0 70 9 AN 0 70 10 AN 0 70 11 AN 0 70 12 AN 0 70 13 AN 0 70 14 AN 0 70 15 AN 0 65 16 AN 0 65 17 AN 0 65 18 AN 0 65 19 AN 0 65 20 AN 0 65 21 AN 0 65 22 AN 0 60 23 AN 0 60 24 AN 0 60 25 AN 0 60 26 AN 0 60 27 AN 0 60 28 AN 0 60 29 AN 0 55 30 AN 0 55 31 AN 0 55 32 AN 0 55 33 AN 0 55 34 AN 0 55 35 AN 0 55 'Observed Effects: AN = Appears Normal Number Sampled 5 5 0 5 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 Wildlife International, Ltd. - 96 - BACK TO MAIN Appendix 7 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1 ______________________ Negative Control - Uptake Phase___________ Sponsor: Test Substance: Test Organism: Dilution Water: Day 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Cumulative Number Observations Dead Number Remaining Number Sampled 36 AN 0 50 0 37 AN 0 50 0 38 AN 0 50 0 39 AN 0 50 0 40 AN 0 50 0 41 AN 0 50 0 42 AN 0 50 5 43 AN 0 45 0 44 AN 0 45 0 45 AN 0 45 0 46 AN 0 45 0 47 AN 0 45 0 48 AN 0 45 0 49 AN 0 45 5 50 AN 0 40 0 51 AN 0 40 0 52 AN 0 40 0 53 AN 0 40 0 54 AN 0 40 0 55 AN 0 40 0 56 AN 0 40 5 57 AN 0 35 0 58 AN 0 35 0 59 AN 0 35 0 60 AN 0 35 0 61 AN 0 35 0 62 AN 0 35 10 'Observed Effects: AN = Appears Normal. Wildlife International, Ltd. - 97 - BACK TO MAIN Appendix 7 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1 ____________________ Negative Control - Depuration Phase__________ Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Cumulative Number Number Number Remaining Sampled Day Observations Dead 1 AN 2 AN 3 AN 4 AN 5 AN 6 AN 7 AN 8 AN 9 AN 10 AN 11 AN 12 13 AN 14 AN 15 AN 16 AN 17 AN 18 AN 19 AN 20 AN 21 AN 22 AN 23 AN 24 AN 25 AN 26 AN 27 AN 28 AN 29 AN 30 AN 31 AN 32 AN 33 AN 34 AN 35 AN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 25 0 25 0 25 0 25 0 25 0 25 0 25 0 25 0 25 0 25 0 25 0 25 0 25 0 25 5 20 0 20 0 20 0 20 0 20 0 20 0 20 0 20 0 20 0 20 0 20 0 20 0 20 0 20 4 16 0 16 0 16 0 16 0 16 0 16 0 16 0 'Observed Effects: AN = Appears Normal *Biological observations not recorded on this day. Wildlife International, Ltd. - 98 - BACK TO MAIN Appendix 7 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1 ____________________ Negative Control - Depuration Phase__________ Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Cumulative Number Number Number Remaining Sampled Day Observations Dead 36 AN 37 AN 38 AN 39 AN 40 AN 41 AN 42 AN 43 AN 44 AN 45 AN 46 AN 47 AN 48 AN 49 AN 50 AN 51 AN 52 AN 53 AN 54 AN 55 AN 56 AN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 16 0 16 0 16 0 16 0 16 0 16 0 16 4 12 0 12 0 12 0 12 0 12 0 12 0 12 0 12 0 12 0 12 0 12 0 12 0 12 0 12 12 'Observed Effects: AN = Appears Normal. Wildlife International, Ltd. - 99 - BACK TO MAIN Appendix 7 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1 ______________________ 0.086 mg a.i./L - Uptake Phase____________ Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Cumulative Number Number Number Remaining Sampled Day Observations Dead 0 AN 0 90 5 1 AN 0 85 5 2 AN 0 80 0 3 AN 0 80 5 4 AN 0 75 0 5 AN 0 75 0 6 AN 0 75 0 7 AN 0 75 5 8 AN 0 70 0 9 AN 0 70 0 10 AN 0 70 0 11 AN 0 70 0 12 AN 0 70 0 13 AN 0 70 0 14 AN 0 70 5 15 AN 0 65 0 16 AN 0 65 0 17 AN 0 65 0 18 AN 0 65 0 19 AN 0 65 0 20 AN 0 65 0 21 AN 0 65 5 22 AN 0 60 0 23 AN 0 60 0 24 AN 0 60 0 25 AN 0 60 0 26 AN 0 60 0 27 AN 0 60 0 28 AN 0 60 5 29 AN 0 55 0 30 AN 0 55 0 31 AN 0 55 0 32 AN 0 55 0 33 AN 0 55 0 34 AN 0 55 0 35 AN 0 55 5 'Observed Effects: AN = Appears Normal Wildlife International, Ltd. - 100 - BACK TO MAIN Appendix 7 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1 ______________________ 0.086 mg a.i./L - Uptake Phase____________ Sponsor: Test Substance: Test Organism: Dilution Water: Day 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Cumulative Number Observations Dead Number Remaining Number Sampled 36 AN 37 AN 38 AN 39 AN 40 AN 41 AN 42 AN 43 AN 44 AN 45 AN 46 AN 47 AN 48 AN 49 AN, 1X 50 AN 51 AN 52 AN 53 AN 54 AN 55 AN 56 AN 57 AN 58 AN 59 AN, 1X 60 AN 61 AN 62 AN 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 2 2 2 2 50 0 50 0 50 0 50 0 50 0 50 0 50 5 45 0 45 0 45 0 45 0 45 0 45 0 45 5 39 0 39 0 39 0 39 0 39 0 39 0 39 5 34 0 34 0 34 0 33 0 33 0 33 10 Observed Effects: AN = Appears Normal. X =Dead. Wildlife International, Ltd. - 101 - BACK TO MAIN Appendix 7 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1 _____________________ 0.086 mg a.i./L - Depuration Phase___________ Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Cumulative Number Number Number Remaining Sampled Day Observations Dead 1 AN 2 AN 3 AN 4 AN 5 AN 6 AN 7 AN 8 AN 9 AN 10 AN 11 AN 12 13 AN 14 AN 15 AN 16 AN 17 AN 18 AN 19 AN 20 AN 21 AN 22 AN 23 AN 24 AN 25 AN 26 AN 27 AN 28 AN 29 AN 30 AN 31 AN 32 AN 33 AN 34 AN 35 AN 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 23 0 23 0 23 0 23 0 23 0 23 0 23 0 23 0 23 0 23 0 23 0 23 0 23 0 23 5 18 0 18 0 18 0 18 0 18 0 18 0 18 0 18 0 18 0 18 0 18 0 18 0 18 0 18 4 14 0 14 0 14 0 14 0 14 0 14 0 14 0 'Observed Effects: AN = Appears Normal *Biological observations not recorded on this day. Wildlife International, Ltd. - 102 - BACK TO MAIN Appendix 7 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1 _____________________ 0.086 mg a.i./L - Depuration Phase___________ Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Cumulative Number Number Number Remaining Sampled Day Observations Dead 36 AN 37 AN 38 AN 39 AN 40 AN 41 AN 42 AN 43 AN 44 AN 45 AN 46 AN 47 AN 48 AN 49 AN 50 AN 51 AN 52 AN 53 AN 54 AN 55 AN 56 AN 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 14 0 14 0 14 0 14 0 14 0 14 0 14 4 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 10 10 'Observed Effects: AN = Appears Normal. Wildlife International, Ltd. - 103 - BACK TO MAIN Appendix 7 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1 _______________________ 0.87 mg a.i./L - Uptake Phase Sponsor: Test Substance: Test Organism: Dilution Water: Day 3M Corporation PFOS Bluegill, Lepomis macrochirus Well Water Cumulative Number Observations Dead Number Remaining Number Sampled 0 AN 1 AN 2 AN 3 AN 4 AN 5 AN 6 AN 7 AN 8 AN 9 AN, 1X 10 AN 11 AN 12 AN, 1R 13 AN, 1X 14 AN, 6R, 14X 15 AN, 8X 16 AN, 6X 17 AN 18 AN 19 AN 20 AN 21 AN, 3R, 5X 22 AN, 1R, 4X 23 AN, 1R, 2X 24 AN, 1X 25 AN, 5R, 1X 26 AN, 6X 27 AN, 1R 28 AN, 3X 29 AN, 1C, 1X 30 1C, 1X 31 1C 32 1C 33 1C 34 1C 35 1X 0 0 0 0 0 0 0 0 0 1 1 1 1 2 16 24 30 30 30 30 30 35 39 41 42 43 49 49 52 53 54 54 54 54 54 55 90 5 85 5 80 0 80 5 75 0 75 0 75 0 75 5 70 0 70 0 69 0 69 0 69 0 69 0 68 5 49 0 41 0 35 0 35 0 35 0 35 0 35 5 25 0 21 0 19 0 18 0 17 0 11 0 11 5 30 20 10 10 10 10 10 1Observed Effects: AN = Appears Normal, C = Lethargic, R = Lying on Bottom, X =Dead. Wildlife International, Ltd. - 104 - BACK TO MAIN Project Number 454A-134 Appendix 8 Changes to Protocol This study was conducted in accordance with the approved Protocol with the following changes: 1. Amendment: The proposed experimental start and termination dates were added to the protocol. 2. Amendment: The frequency of light intensity measurements was added to the protocol. 3. Amendment: The frequency of TOC measurements was added to the protocol. 4. Amendment: The methodology for TOC measurements was added to the protocol. 5. Amendment: Storage stability QC samples were added to the protocol. 6. Amendment: The protocol was clarified to indicate when fish would be sampled for lipid analysis on Day 0. 7. Amendment: The internal reference standard was added to the protocol. 8. Deviation: Dissolved oxygen was not measured on Day 24 of the uptake phase. 9. Deviation: Test temperature was out of range for approximately 2 hours. 10. Deviation: The 0.87 mg a.i./L test concentration adversely affected the test organisms. 11. Deviation: Biological observations were not recorded on Day 12 of the depuration phase. 12. Deviation: Four fish were collected from each treatment on Days 28 and 42 of the depuration phase. 13. Deviation: The depuration phase was terminated on Day 56. 14 Deviation: Fish in the 0.87 mg a.i./L treatment group were not collected for lipid analysis at the end of the uptake or depuration phases. W ildlife International, Ltd. - 105 - Appendix 9 Protocol, Amendments and Deviations BACK TO MAIN Project Number 454A-134 Wildlife International, Ltd. - 106- BACK TO MAIN Project Number 454A-134 PROTOCOL PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (Lepomis macrochirus) U S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1730 and OECD Guideline 305 Environmental Laboratory Request Number U2723 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8601 October 18, 2000 Wildlife International, Ltd. - 107- BACK TO MAIN Project Number 454A-134 Wildlife International, Ltd. - 2- PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (Lepomis macrochirus) SPONSOR: 3M Corporation Environmental Laboratory P.O.Box 33331 St. Paul, Minnesota 55133 SPONSOR'S REPRESENTATIVE: Ms. Susan A. Beach TESTING FACILITY: Wildlife International Ltd. 8598 Commerce Drive Easton, Maryland 21601 STUDY DIRECTOR: Kurt R. Drottar LABORATORY MANAGEMENT: Henry O. Krueger, Ph D. Director of Aquatic Toxicology & Non-Target Plants FOR LABORATORY USE ONLY Proposed Dates: Experimental Start Date: Experimental Termination Date: Project No.: ^ /S d-A - / 3 4 - Test Concentrations: Neeative Control. 0.1 and 1.0 me a.i./L Test Substance No.: 4675 Reference Substance No. iif applicable!: 1 J PROTOCOL APPROVAL / > /3 //0 O DATE ( ^dudx- SPONSOR'S REPRESNTATIVE DAT PROTOCOL NO.: 454/101800/BLU-BIO/SUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. - 108 - BACK TO MAIN Project Number 454A-134 Wildlife International, Ltd. -3 - INTRODUCTION Wildlife International, Ltd. will conduct a bioconcentration test with the bluegill sunfish (Lepomis macrochirus) for the Sponsor at the Wildlife International Ltd. aquatic toxicology facility in Easton, Maryland. The study will be performed based on procedures in U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1730 (1); ASTM Standard E l 022-84 Standard Practice fo r Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve Molluscs (2); and OECD Guideline for Testing of Chemicals 305, Bioaccumulation: Flow-Through Fish Test (3). Raw data for all work performed at Wildlife Interna tional, Ltd. and a copy of the final report will be filed by project number in archives located on the Wildlife International, Ltd. site, or at an alternative location to be specified in the final report. OBJECTIVE The objective of this study is to obtain laboratory data characterizing the bioconcentration potential of Perfluorooctanesulfonate (hereafter referred to as PFOS) in the bluegill, Lepomis macrochirus. EXPERIMENTAL DESIGN Bluegill will be exposed to two test concentrations and a negative control. Each group will consist of one test chamber with up to approximately 480 grams of fish biomass in each chamber. The test will be divided into an uptake and a depuration phase. During the uptake phase, fish in the treatment groups will be exposed to sublethal concentrations of PFOS, while fish in the control group will be exposed to dilution water. The duration o f the uptake phase (3 hours to 28 days) and the depuration phase (6 hours to 60 days) may vary according to the time required to reach steady-state. During both phases of the study, test organisms and water samples are collected and analyzed for PFOS content. These values will be used to determine the uptake rate constant (ki), the depuration rate constant (k2), the kinetic bioconcentration factor (BCFK) and the steady-state bioconcentration factor (BCF) for whole body tissues, edible tissue (skin on) and nonedible tissue. MATERIALS AND METHODS Test Substance The test substance will be Perfluorobutanesulfonate, Potassium Salt, hereafter referred to as PFOS. Information on the characterization of test, control or reference substances is required by Good PROTOCOL NO.: 454/101800/BLU-BIO/SUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. - 109- BACK TO MAIN Project Number 454A-134 Wildlife International, Ltd. -4 - Laboratory Practice Standards (GLP). The Sponsor is responsible for providing Wildlife International, Ltd. written verification that the test substance has been characterized according to GLPs prior to initiation o f the study. If written verification of GLP test substance characterization is not provided to Wildlife International, Ltd., it will be noted in the compliance statement of the final report. The Sponsor is responsible for all information related to the test substance, including the retention of a reserve sample of the lot or batch of the test substance used in this study. The Sponsor also agrees to accept any unused test substance and/or test substance containers remaining at the end of the study. Preparation of Test Concentrations The test substance will be administered to the test organisms in water. This route of administration was selected because it represents the most likely route of exposure to aquatic organisms A primary stock solution of PFOS will be prepared by transferring the PFOS to a volumetric flask and diluting to an appropriate volume with dilution water. Test Organism The test organism used in this study will be the bluegill, Lepomis macrochirus. Bluegill are one o f the recommended fish species for use in bioconcentration tests (1,2,3). Bluegills will be obtained as juveniles from a commercial supplier, gradually acclimated to Wildlife International, Ltd. well water, and held for a period of at least 14 days prior to the test. If the fish are held at .water temperatures that differ from the test temperature, they will be brought to test temperature at a rate not exceeding 3C per 72 hours during holding. If mortality exceeds 3% during the 48-hour period immediately preceding the test, the entire batch of fish will be rejected or held for an additional 14day period to ensure that they are healthy. Fish will be handled as little as possible, but when handling is necessary, it will be done carefully, gently, and quickly. To control bias, fish will be removed from holding tanks with nets and impartially distributed to the test chambers No other forms o f bias are expected to affect the results of the study PROTOCOL NO.: 454/101800/BLU-BIO/SUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. - 110- BACK TO MAIN Project Number 454A-134 Wildlife International, Ltd. -5 - The minimum acceptable size of individual test organisms will be determined by the quantity of tissue required for measurement of residues. Loading (the total wet weight of fish per liter of test solution) will not exceed 1.0 gram of fish per liter o f solution that passes through a test chamber in 24 hours. Bluegill will be fed flake food or another commercial feed, once daily. Excess feed will be siphoned from the tanks approximately 30 minutes after feeding. Feeding and sampling schedules will be coordinated so that fish will be sampled at least four hours after feeding. Specifications for acceptable levels o f contaminants in fish diets have not been established. However, there are no known levels of contaminants reasonably expected to be present in the diet that are considered to interfere with the purpose or conduct of the test. Dilution W ater Water used for the holding and testing of bluegill will be obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site. The water will be passed through a sand filter and pumped into a 37,800-L storage tank where the water will be aerated with spray nozzles. Prior to use the water will be filtered to 0.45 im in order to remove fine particles. Water used for holding and testing is characterized as moderately hard. Typical values for hardness, alkalinity, pH and specific conductance are approximately: Hardness, mg/L as C aC 03 Alkalinity, mg/L as CaC03 pH Specific Conductance, pmhos/cm 130 170 8.2 320 Hardness, alkalinity, pH and specific conductance will be measured weekly to monitor the consistency of the well water. Means and ranges of the measured parameters for the four-week period preceding the test will be provided in the final report. Analyses will be performed at least once annually to determine the concentrations of selected organic and inorganic constituents of the well water and results of the most recent GLP analyses will be summarized in the final report. Diluter System and Test Conditions A continuous-flow diluter will be used to provide two concentrations of PFOS and a control. The flow of dilution water to control and treatment test chambers will be regulated by rotameters. A PROTOCOL NO.: 454/101800/BLU-BIO/SUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. - in - BACK TO MAIN Project Number 454A-134 Wildlife International, Ltd. - 6- peristaltic pump will be used to inject the test substance working stock solution into a mixing chamber where dilution to the test concentration occurs. Flow rates will be adjusted so that each test chamber receives at least six volume additions of water every 24 hours. Delivery of the test substance working stock solution will be initiated at least 48 hours prior to the test. Duplicate water samples from each test chamber will be collected at least twice to verify the establishment of equilibrium concentrations of PFOS in the test chambers before introduction of the test organisms (i.e., measured concentrations should remain within 70 to 120% of nominal concentrations). The general operation of the diluter will be checked visually at least two times per day during the test. Stainless steel aquaria filled with approximately 80 L of test water will be used as test chambers. Test chambers will be placed in a water bath to maintain a test temperature of 221C. The test chambers and water bath will be enclosed in a plexiglass hood in order to minimize potential cross-contamination. Ambient light during testing will be provided by fluorescent tubes that emit wavelengths similar to natural sunlight (e.g. Colortone 50 or equivalent). A photoperiod of 16 hours of light and 8 hours of darkness will be controlled with an automatic timer. A 30-minute transition period o f low light intensity will be provided when lights go on or off to avoid sudden changes in light intensity. Test chambers will be identified by the project number and test concentration. PROCEDURES The test will be divided into an uptake and a depuration phase. Organisms in the control group will be exposed to well water in the uptake and depuration phases. Organisms in the treatment groups will be exposed to the test substance in the uptake phase and well water in the depuration phase. Selection of Test Concentrations The test concentrations selected will not stress, irritate, or otherwise adversely affect the organisms. The test concentrations will be selected in conjunction with the Sponsor and will be based on acute or chronic toxicity data. The two exposure concentrations should differ by a factor of 10. PROTOCOL NO : 454/101800/BLU-BIO/SUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. - 112- BACK TO MAIN Project Number 454A-134 Wildlife International, Ltd. -7 - Chemical/Physical Measurements Temperature will be monitored and recorded continuously during the entire test in one control replicate using a Fulscope ER/C Recorder (1900 J Series Model A), or equivalent. Recorder measurements will be verified prior to the test and at approximately weekly intervals during the test using a liquid-in-glass thermometer. Temperature also will be measured in every test chamber at the beginning and end of the test and at weekly intervals during the test using a liquid-in-glass thermometer. Dissolved oxygen will be measured daily in every test chamber during the test using a Yellow Spring Instruments Model 5 IB dissolved oxygen meter, or equivalent. In the event that dissolved oxygen levels fall below 60% saturation, appropriate actions will be taken after consultation with the Sponsor. Measurements of pH will be made in every test chamber at the beginning and end of the test and at weekly intervals during the test using a Fisher Accumet Model 915 pH meter, or equivalent. Hardness, alkalinity, and conductivity will be measured in the control treatment at the beginning and end of the test and at weekly intervals during the test. Hardness and alkalinity measurements will be made by titration using procedures based on methods in Standard Methods fo r the Examination o f Water and Wastewater (4). Conductivity will be measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter, or equivalent. Biological Measurements Observations of behavior and mortality will be made daily. For a test to be considered valid, mortality and abnormal behavior should not be apparent in more than 10% of the fish in the treatment or control groups. Duration of Uptake Phase The statistically optimum duration of the uptake phase (u) is near u = 1 6/k2, but not more than 3.0/k2, which is equivalent to 95 percent of steady state. The uptake phase will continue until the concentration o f PFOS residues in body tissues reaches steady-state. The criterion for attainment of steady-state is that three consecutive PFOS residue concentrations in fish are not statistically different from each other. PROTOCOL NO . 454/101800/BLU-BIO/SUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. - 113 - BACK TO MAIN Project Number 454A-134 Wildlife International, Ltd. 8- - Duration of Depuration Phase Two times u is usually a sufficient time for about 95 percent removal of the body burden (t.A= 0.69/k2), however, the depuration phase will continue until the concentration of PFOS residue in the organisms attains any one of the following three conditions: 1. Residues reach less than 10% of steady-state; or 2. Residues fall below the LOQ; or 3. Sixty (60) days o f depuration have elapsed. Sample Intervals for W ater and Fish Collection Fish and water will be sampled no less than five times during the uptake phase and four times during the depuration phase of the study at the same intervals. In addition, control and treated water will be sampled no less than twice prior to addition of fish to the chambers to confirm the concentrations desired. In the absence of information on the bioaccumulation potential of the test substance and/or reliable physico-chemical data from which bioaccumulation potential may be estimated, the recommended sampling schedule is as follows. Phase Sample Interval W ate r*1 Fish2 Action Pre-Uptake3 Uptake -n, -n2 0 ~ 4 hours 1 day 3 day 7 14 21 28 XX- XXX XX XX XX XX XX XX Start Add fish I Transfer Depuration 1 XX 3 XX 7 XX 10 X X 14" X X 1At least five replicates of water samples will be collected at each interval listed for the analysis of PFOS. Additional samples will be drawn as necessary for the determination of pH, hardness, alkalinity, specific conductance, and total organic carbon (TOC). 2 Sufficient fish will be collected to allow the preparation o f four replicate samples from each treatment group and duplicate samples from the control group for the analysis of PFOS. Additional samples will be drawn as necessary for the analysis of lipid content. 3Pre-Uptake water sampling (-m, -n2) for confirmation o f treatment levels prior to addition of fish. 4Depuration phase sampling will continue until the concentration of PFOS residue in the organisms attains any one of the conditions outlined in the duration o f depuration phase on page 8, PROTOCOL NO.: 454/101800/BLU-BIO/SUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. - 114- BACK TO MAIN Project Number 454A-134 Wildlife International, Ltd. -9 - The number and frequency of sample intervals may be changed based on physico-chemical properties of the test substance from which predictions of bioaccumulation rate can be derived and/or at the direction of the Sponsor. At each sampling interval, two water samples will be collected from the control and three water samples will be collected from the PFOS treatment groups. The PFOS concentration will be determined for a minimum of one replicate of a control and two replicates at each treatment level for each interval in which samples are analyzed (as specified by the Study Director). The remaining replicates of samples drawn for PFOS analysis will be held in reserve and may be analyzed at the direction of the Study Director or Chemistry Principal Investigator. The concentration of PFOS in the water at each sample interval will be presented as the average of the replicate measurements, as applicable. Additional samples will be drawn as necessary for the determination o f pH, hardness, alkalinity, specific conductance, and total organic carbon (TOC). Fish Sampling To assess the PFOS content in fish tissues, sufficient fish will be collected to allow the preparation o f four replicate samples from each treatment group and duplicate samples from the control group. One additional sample from each group will be collected and held in reserve and may be analyzed at the direction o f the Study Director or Chemistry Principal Investigator. The concentration o f PFOS in fish at each sample interval will be presented as the average of the replicate measurements. Fish sampled for determination of PFOS content will be impartially removed from the test chambers, rinsed with dilution water, blotted dry, and sacrificed. The weight (wet weight, blotted dry) and total length of each fish will be determined within approximately 15 minutes o f collection, if possible. Each fish will be separated into edible and nonedible tissue and the wet weight of each tissue fraction (i.e., edible and nonedible) will be recorded. Fish will be stored frozen if not analyzed immediately. PROTOCOL NO.: 454/101800/BLU-BIO/SUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. - 115 - BACK TO MAIN Project Number 454A-134 Wildlife International, Ltd. -10- Additional samples will be drawn as necessary for the analysis of lipid content.. Analytical Method Water and tissue samples will be analyzed for PFOS using liquid chromatography-mass spectrometry. Water samples will be analyzed according to the method entitled "Analytial Method Validation for the Determination o f Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) in Freshwater, Saltwater and Algal Media" (Wildlife International, Ltd Project No. 454C-109). Tissue samples will be analyzed according to the method entitled "Analytical Method Validation for the Determination of Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) in Fish Tissues" (Project No. 454C-119). Any modifications to the above methodologies will be documented in the raw data and described in the final report. Tissue Lipid Content At a minimum, fish will be sampled from the control aquarium at Day 0 (upon transfer of fish to aquaria), at an interval where the PFOS concentration in fish has reached steady state (or following 28 days o f exposure), and at the end of the depuration period. Fish will be sampled to generate four samples from the control and two treated aquaria at each of the intervals noted. The lipid content of the tissue homogenates will be determined by chloroform/methanol extraction. Data Analysis Results o f tissue analyses will be presented on a wet weight basis. The steady-state bioconcentration factor for the test substance (BCF), kinetic bioconcentration factor (BCFK) uptake rate (ki), and depuration rate (k2) will be expressed for edible tissue, nonedible tissue and whole fish (5). RECORDS TO BE MAINTAINED Records to be maintained for data generated at Wildlife International, Ltd. will include but not be limited to: 1. A copy of the signed protocol. 2. Identification and characterization of the test substance, if provided by the Sponsor. 3. Dates of initiation and termination o f the test. 4. Test organism holding and acclimation records. PROTOCOL NO.: 454/101800/BLU-BIO/SUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. - 116- BACK TO MAIN Project Number 454A-134 Wildlife International, Ltd. - 11 - 5. Methods used to prepare stock solutions and dilutions of the test substance. 6 Daily observations. 7. Water chemistry calculations (e.g., hardness and alkalinity). 8. Organism weight and length measurements. 9. The methods used to analyze test substance concentrations and the results of analytical measurements. 10. Statistical calculations. 11. A copy of the final report. FINAL REPORT A report o f the results of the study will be prepared by Wildlife International, Ltd. The report will include, but not be limited to, the following: 1. Name and address of the facility performing the study. 2. Dates on which the study was initiated and completed. It is the responsibility of the Sponsor to provide the final date that data are recorded for chemistry, pathology and/or supporting evaluations that may be generated at other laboratories. 3. A statement of compliance signed by the Study Director addressing any exceptions to Good Laboratory Practice Standards. 4. Objectives and procedures stated in the approved protocol, including any changes in the original protocol. 5. Statistical methods employed for analyzing the data. 6. The test, control and reference substances identified by name, chemical abstracts number or code number, strength, purity, and composition or other appropriate characteristics, if provided by the Sponsor. 7. Stability and, when relevant to the conduct of the study, the solubility of the test, control and reference substances under the conditions of administration, if provided by the Sponsor or contracted to Wildlife International, Ltd. 8. A description of the methods used. 9. A description of the test system used. Where applicable, the final report shall include the number o f animals used, body weight range, source of supply, species, age, and procedure used for identification. 10. A description o f the dosage, dosage regimen, route of administration, and duration. 11. A description of all circumstances that may have affected the quality or integrity of the data. PROTOCOL NO.: 454/101800/BLU-BIO/SUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. - 117- BACK TO MAIN Project Number 454A-134 Wildlife International, Ltd. - 12- 12. The name of the Study Director, the names o f other scientists or professionals, and the names of all supervisory personnel, involved in the study. 13. A description o f the transformations, calculations, or operations performed on the data, a summary and analysis of the data, and a statement of the conclusions drawn from the analysis. 14. The signed and dated reports of each of the individual scientists or other professionals involved in the study, if applicable. 15. The location where all specimens, raw data, and the final report are to be stored. 16. A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made and the dates of any findings reported to the Study Director and Management. 17. If it is necessary to make corrections or additions to a final report after it has been accepted, such changes shall be in the form of amendment by the Study Director. The amendment should clearly identify the part of the final report that is being added to or corrected and the reasons for the correction or addition. Amendments shall be signed and dated by the Study Director. CHANGING OF PROTOCOL Planned changes to the protocol will be in the form of written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part of the protocol and will be attached to the final protocol. Any other changes will be in the form o f written deviations signed by the Study Director and filed with the raw data. All changes to the protocol will be indicated in the final report. GOOD LABORATORY PRACTICES This study will be conducted in accordance with Good Laboratory Practice Standards for EPA (40 CFR Part 160); OECD Principles o f Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF (59 NohSan, Notification No. 3850, Agricultural Production Bureau). Each study conducted by Wildlife International, Ltd. is routinely examined by the Wildlife International, Ltd. Quality Assurance Unit for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. A statement o f compliance with Good Laboratory Practices will be prepared for all portions of the study conducted by Wildlife International, Ltd. The Sponsor will be responsible for compliance with Good Laboratory Practices for procedures performed by other laboratories (e g., residue analyses or pathology). Raw data for all work performed at Wildlife International, Ltd. and a copy of the final report will be filed by project number in archives located on the Wildlife International, Ltd. site, or at an alternative location to be specified in the final report. PROTOCOL NO : 454/101800/BLU-BIO/SUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. - 118- BACK TO MAIN Projcct Number 454A-134 Wildlife International, Ltd. - 13 - REFERENCES 1. U.S. Environmental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines (Public Draft), OPPTS Number 850.1730: Fish BCF. 2. ASTM Standard E1022-84. 1988. Standard Practice fo r Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve Molluscs. American Society for Testing and Materials. 3. OECD Guideline for Testing of Chemicals 305. 1996. Bioconcentration: Flow-Through Fish Test. 4. APHA, AWWA, WPCF. 1985. Standard Methods fo r the Examination o f Water and Wastewater. 16th Edition, American Public Health Association. American Water Works Association. Water Pollution Control Federation, New York. 5. G. E. Blau and G. L. Agin 1978. BIOFAC. The Dow Company. Midland, Ml. PROTOCOL NO.: 454/101800/BLU-B10/SUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. -119- BACK TO MAIN Project Number 454A-134 W i l d l i f e In t e r n a t io n a l l t d . PROJECT NO.: 454A-134 Page 1 of 2 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (Lepomis mctcrochirus) PROTOCOL NO.: 454/101800/BLU-BIO/SUB454 AMENDMENT NO.: 1 SPONSOR: 3M Corporation PROJECT NO.: 454A-134 EFFECTIVE DATE: November 7,2000 ENVIRONMENTAL LABORATORY REQUEST NO.: U2723 AMENDMENT: Page 2 Add: Experimental Start Date: 12/5/00 Experimental Termination Date: 1/16/01 REASON: The above information was not known when the Study Director signed the protocol. AMENDMENT: Diluter System and Test Conditions, Page 6 Add: Light intensity will be measured at test initiation with a SPER Scientific Ltd. light meter or equivalent. REASON: To specify the frequency of light intensity mesurement. AMENDMENT: Chemical/Physical Measurements, Page 7 Change: Hardness, alkalinity, and conductivity will be measured in the control treatment at the beginning and end of the test and at weekly intervals during the test. To: Hardness, alkalinity, conductivity and total organic carbon (TOC) will be measured in the control treatment at the beginning and end of the test and at weekly intervals during the test. REASON: To specify the frequency of TOC measurements. AMENDMENT: Chemical/Physical Measurements, Page 7 Add: TOC will be measured using a Shimadzu Model TOC-5000 TOC analyzer. REASON: To add the methodology for TOC measurements. gxfi Q+tc Wildlife International, Ltd. - 120- BACK TO MAIN Project Number 454A-134 W i l d l i f e In t e r n a t io n a l l t d . PROJECT NO.: 454A-134 Page 2 of 2 AMENDMENT: Analytical Method, Page 10 Add: Tissue stability samples will be prepared at test initiation to establish test substance stability in fish tissues stored frozen during the study. REASON: To add storage stability QC samples. AMENDMENT: Tissue Lipid Content, Page 10 Change: At a minimum, fish will be sampled from the control aquaria at Day 0. . . To: At a minimum, fish will be sampled on Day 0. . . REASON: Fish sampled on Day 0 for lipid analysis will be collected prior to distribution to the test chambers. DATE SPONSOR'S REPRESENTATIVE Wildlife International, Ltd - 121 - BACK TO MAIN Project Number 454A-134 W i l d l i f e In t e r n a t io n a l l t d . PROJECT NO.: 454A-134 Page 1 of 1 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (Lepomis macrochirus) PROTOCOL NO.: 454/101800/BLU-BIO/SUB454 SPONSOR: 3M Corporation EFFECTIVE DATE: November 28,2000 AMENDMENT NO.: 2 PROJECTNO .: 454A-134 ENVIRONMENTAL LABORATORY REQUEST NO.: U2723 AMENDMENT: Page 2 Add: Reference Substance No.: 4526 REASON: To add the internal reference standard. DATE SPONSOR'S REPRESENTATIVE 9\-tu n->i-4>o Wildlife International, Ltd. - 122- BACK TO MAIN Project Number 454A-134 W i l d l i f e In t e r n a t io n a l l t d . PROJECT NO.: 454A-134 Page 1 of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (Lepomis macrochirus) PROTOCOL NO: 454/101800/BLU-BIO/SUB454 DEVIATION NO.: 1 SPONSOR: 3M Corporation PROJECTNO .: 454A-134 DATE OF DEVIATION: December 29.2000__________________________________________________ DEVIATION: The protocol states that dissolved oxygen will be measured daily in every test chamber. Dissolved oxygen was not measured on Day 24 of the uptake phase of the test. REASON: Biologist oversight. All dissolved oxygen measurements during the test were >60% of saturation. Consequently, it is the best judgement of the Study Director that this deviation did not adversely affect the results of the study. STUDY DIRECTOR LABI IRY MANAGEMENT -sry ^ /fe/' DATE Wildlife International, Ltd. - 123 - W i l d l i f e In t e r n a t io n a l l t d . BACK TO MAIN Project Number 454A-134 PROJECT NO.: 454A-134 Page 1 of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (Lepomis macrochirus) PROTOCOL NO: 454/101800/BLU-BIO/SUB454 DEVIATION NO.: 2 SPONSOR: 3M Corporation PROJECT NO.: 454A-134 DATE OF DEVIATION: February 24. 2001____________________________________________________ DEVIATION: The protocol states that the test chambers will be placed in a water bath to maintain a test temperature of 22 1C. On Day 19 of depuration, the continuous temperature recorder reached 20C. The temperature was out of range for approximately 2 hours. REASON: Unknown. Based on the short duration of the temperature deviation, it is the best judgement of the Study Director that this deviation did not adversely affect the results of the study. Wildlife International, Ltd. - 124- BACK TO MAIN Project Number 454A-134 W i l d l i f e Iin t e r n a t io n a l l t d . PROJECT NO.: 454A-134 Page 1 of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (.Lepomis macrochirus) PROTOCOL NO: 454/101800/BLU-BIO/SUB454 DEVIATION NO.: 3 SPONSOR: 3M Corporation PROJECT NO.: 454A-134 DATE OF DEVIATION: December 14. 2000___________________________________________________ DEVIATION: The protocol states that the test concentrations selected will not stress, irritate, or otherwise affect the organisms. The 0.87 mg a.i./L treatment group was actually chronically toxic to bluegill. REASON: Information on the chronic toxicity of PFOS to bluegill was not known when the test concentrations were selected. This deviation adversely affected the results of the study because adequate bioconcentration data was not obtained from the 0.87 mg a.i./L treatment group. Col/HlOl DATE Wildlife International, Ltd. - 125 - W i l d l i f e In t e r n a t io n a l l t d . BACK TO MAIN Project Number 454A-134 PROJECT NO.: 454A-134 Page 1 of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (Lepomis macrochirus) PROTOCOL NO: 454/101800/BLU-BIO/SUB454 DEVIATION NO.: 4 SPONSOR: 3M Corporation PROJECT NO.: 454A-134 DATE OF DEVIATION: February 17, 2001____________________________________________________ DEVIATION: The protocol states that observations of behavior and mortality will be made daily. Biological observations were not recorded on Day 12 of depuration. REASON: Biologist oversight. Biological observations on Day 11 and 13 of depuration were the same. Consequently, it is the bestjudgement of the study director that this deviation did not adversely affect the results of the study. JL LABORATORY MANAGEMENT (q M / o( DATE t? J h Je t DATE Wildlife International, Ltd. - 126- W i l d l i f e In t e r n a t io n a l l t d . BACK TO MAIN Project Number 454A-134 PROJECT NO.: 454A-134 Page 1 of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (Lepomis macrochirus) PROTOCOL NO: 454/101800/BLU-BIO/SUB454 DEVIATION NO.: 5 SPONSOR: 3M Corporation PROJECT NO.: 454A-134 DATES OF DEVIATION: March 5 and 19. 2001_______________________________________________ DEVIATION: The protocol states that sufficient fish will be collected to allow the preparation of four replicate samples from each treatment group and duplicate samples from the control group. One additional sample from each group will be collected and held in reserve and may be analyzed at the discretion of the Study Director or Chemistry Principal Investigator. On Days 28 and 42 of depuration, reserve samples were not collected. REASON: Reserve samples were not collected to conserve the number of fish remaining in the study. It is the best judgement of the study director that this deviation did not adversely affect the results of the study. Wildlife International, Ltd. - 127- BACK TO MAIN Project Number 454A-134 W i l d l i f e In t e r n a t io n a l l t d . PROJECT NO.: 454A-134 Page 1 of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (Lepomis macrochirus) PROTOCOL NO: 454/101800/BLU-BIO/SUB454 DEVIATION NO.: 6 SPONSOR: 3M Corporation PROJECT NO.: 454A-134 DATE OF DEVIATION: April 2,2001________________________________________________________ DEVIATION: The protocol states that the depuration phase will continue until the concentration of PFOS residue in the organisms attains any one of the following three conditions: 1) Residues reach less than 10% of steady-state, or 2) Residues fall below the LOQ, or 3) Sixty (60) days ofdepuration have elapsed. The residues did not meet 1 or 2 above and depuration was terminated on Day 56 of depuration. REASON: The Sponsor requested that sampling occur every two weeks during depuration. It is the best judgement of the study director that this deviation did not adversely affect the results of the study. &/JH /o{ DATE LABORATORY MANAGEMENT Wildlife International, Ltd. - 128- BACK TO MAIN Project Number 454A-134 W i l d l i f e In t e r n a t io n a l l t d . PROJECT NO.: 454A-134 Page 1 of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: PERFLUOROOCTANESULFONATE, POTASSKJM SALT (PFOS): A FLOW-THROUGH BIOCONCENTRATION TEST WITH THE BLUEGILL (Lepomis macrochirus) PROTOCOL NO: 454/101800/BLU-BIO/SUB454 DEVIATION NO.: 7 SPONSOR: 3M Corporation PROJECTNO .: 454A-134 DATES OF DEVIATION: February 5 and April 2. 2001__________________________________________ DEVIATION: The protocol states that fish will be samples for lipid analysis at steady-state and at the end ofthe depuration phase. No fish were sampled from the 0.S7 mg a.i./L treatment group. REASON: There were no fish remaining in the 0.87 mg a.i./L treatment group at the above times. It is the best judgement of the study director that this deviation did not adversely affect the results of the study. STUDY DIRECTOR DATE DATE Wildlife International, Ltd. - 129 - BACK TO MAIN Appendix 10 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. Kurt R. Drottar, Senior Aquatic Biologist 4. Cary A. Sutherland, Laboratory Supervisor 5. Raymond L. Van Hoven, Ph.D., Scientist 6. Susan T. Platania, Biologist