Document v1MD5exEyoo1YLa7Lxz72a1g6

PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS): A 96-HOUR STATIC-RENEWAL ACUTE TOXICITY TEST WITH THE SHEEPSHEAD MINNOW (Cyprinodon variegatus) FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-146A ENVIRONMENTAL LABORATORY PROJECT NUMBER: U2723 U. S Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1075 AUTHORS: Susan J. Palmer Raymond L. VanHoven, Ph.D. Henry O. Krueger, Ph.D. STUDY INITIATION DATE: May 16, 2001 STUDY COMPLETION DATE: January 7, 2002 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 46 Wildlife International, Ltd. 2- - Project Number 454A-146A GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: Perfluorooctanesulfonate, Potassium Salt (PFOS): A 96-Hour Static-Renewal Acute Toxicity Test with the Sheepshead Minnow (Cyprinodon variegatus) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-146A STUDY COMPLETION: January 7, 2002 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles o f Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. STUDY DIRECTOR: Senior Biologist SPONSOR APPROVAL: Date Wildlife International, Ltd, -3 - Project Number 454A-146A QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR Parts 160 and 792,17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates o f 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: DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: Initial T rial 454A-146: Test Substance Preparation July 6,2001 Matrix Fortifications July 10, 2001 Observations and Analytical Sampling July 12, 2001 July 6,2001 July 11,2001 July 12, 2001 July 11,2001 July 13,2001 July 16,2001 Definitive Test 454A-146A: Test Substance Preparation August 13, 2001 Matrix Fortifications August 13, 2001 Observations and Analytical Sampling August 17, 2001 Analytical Data and Draft Report October 4 - 5 , 2001 Biological Data and Draft Report October 3 - 5 , 2001 Final Report January 7,2002 August 13, 2001 August 13, 2001 August 17, 2001 October 5,2001 October 5,2001 January 7,2002 August 15,2001 August 15, 2001 August 23, 2001 October 8,2001 October 9,2001 January 7,2002 /a /, QnVfy/s/) Kimberly A. Hotter Quality Assurance Representative h n-o# Date Wildlife International, Ltd, -4 - Project Number 454A-146A REPORT APPROVAL SPONSOR: 3M Corporation TITLE: Perfluorooctanesulfonate, Potassium Salt (PFOS): A 96-Hour Static-Renewal Acute Toxicity Test with the Sheepshead Minnow (Cyprinodon variegatus) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-146A STUDY DIRECTOR: Susan J. Palmer f Senior Biologist WI1.DT.IFF. INTERNATIONA!. T.TD MANAGEMENT: Henry*0. Krueger" Ph.D. Director, Aquatic Toxicology and Non-Target Plants L Date 1 Date 2JX>2 Wildlife International, Ltd, -5- Project Number 454A-146A TABLE OF CONTENTS Title/Cover P age............................................................................................................................................ 1 Good Laboratory Practice Compliance Statement....................................................................................... 2 Quality Assurance Statement............................................ 3 Report Approval............................................................................................................................................. 4 Table of Contents........................................................................................................................................... 5 Summary.........................................................................................................................................................7 Introduction.....................................................................................................................................................8 Objective.......................... 8 Experimental Design......................................................................................................................................8 Materials and Methods...................................................................................................................................9 Test Substance...................................................................................................................................9 Preparation of Test Concentrations.................................................................................................9 Test Organism...................................................................................................................................9 Test Apparatus................................................................................................................................10 Dilution Water.................................................................................................................................10 Environmental Conditions..............................................................................................................11 Observations...................................................................................... 12 Statistical Analyses......................................................................................................................... 12 Analytical Chemistry...................................................................................................................... 12 Results and Discussion.................................................................................................................................12 Measurement of Test Concentrations............................................................................................12 Observations and Measurements................................................................................................... 13 Conclusions......................... 13 References 14 Wildlife International, Ltd, 6- - TABLE OF CONTENTS - Continued - Project Number 454A-146A TABLES AND FIGURES T able 1. Summary of Analytical Chemistry D ata................................................................................... 15 Table 2. Temperature, Dissolved Oxygen and pH of Water in the Test Chambers............................. 17 Table 3. Cumulative Percent Mortality and Treatment-Related Effects................................................19 Table 4. LC50 Values................................................................................................................................20 APPENDICES Appendix 1. Salinity and pH of Saltwater Measured During the 4-Week Period Immediately Preceding the T est..........................................................................................21 Appendix 2. Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Saltwater................................................................................................................................ 22 Appendix 3. The Analysis of PFOS in Filtered Saltwater in Support of Wildlife International, Ltd. Project No.: 454A-146A.......................................................24 Appendix 4. Changes to Protocol............................................................................................................ 45 Appendix 5. Personnel Involved inthe Study...........................................................................................46 Wildlife International, Ltd. -7- Project Number 454A-146A sponsor: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: SUMMARY 3M Corporation Ms. Rochelle Robideau Wildlife International, Ltd. Easton, Maryland 21601 WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST: 454A-146A Perfluorooctanesulfonate, Potassium Salt (PFOS) Perfluorooctanesulfonate, Potassium Salt (PFOS): A 96-Hour Static-Renewal Acute Toxicity Test with the Sheepshead Minnow (Cyprinodon variegatus) Negative Control, Solvent Control and 15 mg a.i./L Experimental Start (OECD) - July 6,2001 Experimental Start (EPA) - August 13, 2001 Biological Termination - August 17, 2001 Experimental Termination - August 17, 2001 96 Hours TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: MEASUREMENTS OF 10 NEGATIVE CONTROL FISH: WET WEIGHT: TOTAL LENGTH: Sheepshead Minnow (Cyprinodon variegatus) Aquatic BioSystems, Inc. Fort Collins, Colorado 80524 Juveniles Mean = 0.44 g Mean = 3.0 cm Range = 0.21 to 0.66 g Range = 2.4 to 3.5 cm 96-HOUR LC50: NO MORTALITY CONCENTRATION: NO-OBSERVED-EFFECTCONCENTRATION: >15 mg a.i./L 15 mg a.i./L <15 mg a.i./L Wildlife International, Ltd, 8- - Project Number 454A-146A INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. An initial trial of the study was conducted under static conditions from July 10, 2001 to July 14, 2001. However, the analytical results were <65% of the nominal concentration during the test. Therefore, the test was repeated under staticrenewal test conditions. The in-life phase of the definitive test was conducted from August 13,2001 to August 17,2001. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454A-146A in archives located on the Wildlife International, Ltd. site. OBJECTIVE The objective of this study was to determine the acute effects of Perfluorooctanesulfonate, Potassium Salt (PFOS) to the sheepshead minnow, Cyprinodon variegatus, during a 96-hour exposure period under static-renewal test conditions. EXPERIMENTAL DESIGN Sheepshead minnows were exposed to a single limit concentration, a negative (dilution water) control and a solvent control (0.5 mL methanol/L) for 96 hours, with renewal of test solutions approximately every 24 hours. Three replicate test chambers were maintained in each treatment and control group, with 10 sheepshead minnows in each test chamber for a total of 30 fish per test concentration. Two abiotic replicate test chambers also were maintained at the limit concentration. The nominal test concentration was selected in consultation with the Sponsor, and was based upon the results of exploratory range finding toxicity tests, and the solubility of the test substance in saltwater. The nominal test concentration selected was 20 mg active ingredient (a.i.)/L. The mean measured test concentration was determined from samples of test water collected from each treatment and the control group at the beginning of the test, from new and old test solution at approximately 24,48 and 72 hours of the test, and from old test solution at test termination. Sheepshead minnows were indiscriminately assigned to exposure chambers at test initiation, and were transferred daily to freshly prepared test solutions. Observations of mortality and other clinical signs of toxicity were made at approximately 2.5, 24, 48, 72 and 96 hours after test initiation. Cumulative percent mortality observed in the treatment group was used to estimate LC50 values at 2.5,24,48,72 and 96 hours. The no mortality concentration and the no-observed-effect-concentration (NOEC) were determined by visual interpretation of the mortality and clinical observation data. Wildlife International, Ltd. -9- Project Number 454A-146A MATERIALS AND METHODS The study was conducted based on the procedures outlined in the protocol, "PFOS: A 96-Hour Static Acute Toxicity Test with the Sheepshead Minnow (Cyprinodon variegatus)". The protocol was based on procedures outlined in the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines (draft), OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater and Marine (1); U.S. Environmental Protection Agency, Standard Evaluation Procedure, Acute Toxicity Test fo r Estuarine and Marine Organisms (2); and ASTM Standard E729-88a, Standard Guidefo r Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates and Amphibians (3). Test Substance The test substance was received from 3M Corporation on October 29, 1998 and was assigned Wildlife International, Ltd. identification number 4675. The test substance was described as a white powder. It was identified as FC-95 from lot number 217 (T-6295). Information provided by the Sponsor indicated a purity of 86.9% and an expiration date of August 31,2006. The test substance was stored at ambient room temperature. Preparation of Test Concentrations The nominal test concentration was 20 mg a.i./L, based on a test substance purity of 86.9%. On each of Days 0, 1, 2 and 3 of the test, a 250-mL primary stock solution was prepared in methanol at a concentration of 40 mg a.i./mL. The primary stock solution was sonicated for approximately 20 minutes and inverted to mix. Five replicates of the test solution (three biotic and two abiotic replicates) were prepared at a concentration of 20 mg a.i./L by adding the appropriate volume of primary stock to dilution water in the test aquaria to achieve a final volume of 15 L. Each solution was stirred with a stainless steel whisk for approximately one minute. All test solutions appeared clear and colorless. Three solvent control replicates were prepared by adding the appropriate volume of methanol to dilution water in the aquaria. The solvent concentration in the treatment and solvent control groups was 0.5 mL/L. Test Organism The sheepshead minnow, Cyprinodon variegatus, was selected as the test species for this study. The sheepshead minnow is representative of an important group of aquatic vertebrates and was selected for use in the test based upon past history of use in the laboratory. Sheepshead minnows used in the test were obtained from Aquatic BioSystems, Inc., Fort Collins, Colorado. Identification of the species was verified by the supplier. Wildlife International, Ltd. -10- Project Number 454A-146A The fish were held for approximately seven weeks prior to the test in water from the same source and at approximately the same temperature as used during the test. During the 14-day holding period preceding the test, water temperatures ranged from 22.5 to 22.8C. The pH of the water ranged from 7.9 to 8.1, salinity was 20%o (parts per thousand) and dissolved oxygen ranged from 7.0 to 7.2 mg/L (89% of saturation). Instrumentation and procedures used for water measurements are described in the Environmental Conditions section of this report. The fish were acclimated to test conditions for approximately 51 hours prior to test initiation. During the acclimation period, no mortalities occurred and the fish showed no signs of disease or stress. At test initiation, the fish were collected from the acclimation tank and indiscriminately distributed two at a time to the test chambers until each contained 10 fish. During the holding period, the fish were fed a commercially-prepared diet (Zeigler Brothers, Inc., Gardners, P A ), as well as Artemia nauplii (Summit Artemia, Ogden, Utah). The fish were not fed during the acclimation period (at least two days prior to the test) or during the test. All fish used in the test were from the same source and year class, and the length of the longest fish was no more than twice the length of the shortest. The average total length of 10 negative control fish measured at the end of the test was 3.0 cm with a range of 2.4 to 3.5 cm. The average wet weight (blotted dry) of 10 negative control fish at the end of the test was 0.44 g with a range of 0.21 to 0.66 g. Loading was 0.30 g fish/L of test water. Test Apparatus Test chambers were 25-L polyethylene aquaria containing 15 L of test solution. The depth of water in a representative test chamber was approximately 17.1 cm. Test chambers were positioned in an environmental chamber set to maintain the desired temperature throughout the test. The test chambers were labeled with the project number, test concentration and replicate. Dilution Water The water used for culturing and testing was natural seawater collected at Indian River Inlet, Delaware that was filtered and diluted to a salinity of approximately 20%o with well water. Salinity and pH measurements taken during the four-week period immediately preceding the test are presented in Appendix 1. Wildlife International, Ltd. -11 - Project Number 454A-146A The freshly-collected seawater was passed through a sand filter to remove particles greater than approximately 25 pm, and pumped into a 37,800-L storage tank. The filtered saltwater then was diluted with freshwater from a well on the Wildlife International, Ltd. site and aerated with spray nozzles. The 20%o water was filtered to 0.45 pm to remove microorganisms and fine particles prior to its use in the test. The results of periodic analyses performed to measure the concentrations of selected contaminants in the saltwater used by Wildlife International, Ltd. are presented in Appendix 2. Environmental Conditions Lighting used to illuminate the cultures and test chambers during holding, acclimation and testing was provided by fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone 50). A photoperiod of 16 hours of light and 8 hours of darkness was controlled with an automatic timer. A 30minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity at test initiation was approximately 139 lux at the surface of the water of one representative test chamber. Temperature was measured in each test chamber at the beginning o f the test, prior to and after test solution renewal, and at test termination using a liquid-in-glass thermometer. Temperature also was measured continuously in one negative control replicate using a Fulscope ER/C Recorder, which was verified prior to test initiation with a liquid-in-glass thermometer. The target test temperature during the study w as 22 2C. D issolved oxygen and pH m easurem ents w ere m ade on w ater samples collected from all replicate test chambers of each treatment and control at test initiation, prior to and after test solution renewal, and at test termination. Salinity was measured in the dilution water prior to test initiation and at test termination. Light intensity was measured using a SPER Scientific Ltd. Model 840006C light meter. Measurements of pH were made using a Fisher Accumet Model 915 pH meter, and dissolved oxygen was measured using a Yellow Springs Instrument Model 51B dissolved oxygen meter. Salinity was measured using a Bio-Marine, Inc. Aquafauna refractometer. Wildlife International, Ltd. 12- - Project Number 454A-146A Observations All organisms were observed periodically to determine the number of mortalities in each control and treatment group. The numbers of individuals exhibiting signs o f toxicity or abnormal behavior also were evaluated. Observations were made approximately 2.5,24,48,72 and 96 hours after test initiation. Statistical Analyses The use of a single test concentration precluded the statistical calculation of LC50 values. Therefore, since there were no mortalities during the study, the 2 .5,24,48,72 and 96-hour LC50 values were estimated to be greater than the single concentration tested. The no mortality concentration and NOEC were determined by visual interpretation of the mortality and observation data. Analytical Chemistry Water samples were collected at mid-depth from each biotic test chamber of each treatment and control group at the beginning of the test, prior to and after test solution renewal at approximately 24,48 and 72 hours of the test, and at test termination to measure concentrations of the test substance. Samples also were collected for analysis from the abiotic test chambers prepared at 20 mg a.i./L prior to and after test solution renewal at approximately 24, 48 and 72 hours, and at test termination. All samples were collected in plastic vials and analyzed as soon as possible without storage. Analytical procedures used in the analysis of the samples are provided in Appendix 3. RESULTS AND DISCUSSION Measurement of Test Concentrations Results of analyses to measure concentrations of PFOS in water samples collected during the test are presented in Table 1 and in the analytical chemistry report (Appendix 3). The single nominal concentration selected for use in this study was 20 mg a.i./L. Samples collected from new test solutions at test initiation and at approximately 24, 48 and 72 hours had measured concentrations that ranged from 74.9 to 92.9% of nominal concentrations. Samples collected from old test solutions at approximately 24, 48 and 72 hours and at test termination had measured concentrations that ranged from 55.6 to 89.9% of nominal concentrations. The abiotic 20 mg a.i./L samples had measured concentrations that ranged from 79.4 to 88.5% of nominal in the new test solutions, and from 45.0 to 54.4% of nominal in the old test solutions. The measured concentrations of PFOS in the abiotic samples from the old solutions were Wildlife International, Ltd. -13 - Project Number 454A-146A slightly lower than the concentrations of the 20 mg a.i./L treatment group samples with the fish present. The most plausible reason for this was increased deposition of test substance at the limit of solubility in the absence of the natural mixing action provided by the movement of the fish. When measured concentrations of the 20 mg a.i./L biotic samples analyzed at each sampling interval were averaged, the mean measured concentration for this study was 15 mg a.i./L, representing 75% of the nominal concentration. The results of the study were based on the mean measured concentration. Observations and Measurements Measurements of temperature, dissolved oxygen and pH are presented in Table 2. Temperatures were within the 22 2C range established for the test. Dissolved oxygen concentrations were 7.0 mg/L (89% of saturation) in the new test solutions and ranged from 1.6 to 6.7 mg/L (20 to 86% of saturation) in the old test solutions. Although dissolved oxygen concentrations dropped below 60% of saturation several times during the test, the biological results of the study indicate that this did not have an adverse effect on the results of the study. Measurements o f pH ranged from 7.9 to 8.3 during the test. Salinity measurements of the dilution water at test initiation and termination were 20%o (Table 2). Daily observations of mortality and other signs oftoxicity observed during the test are presented in Table 3. Sheepshead minnows in the negative control and solvent control groups appeared normal and healthy throughout the test period. No mortalities occurred in the 15 mg a.i./L treatment group during the test. H ow ever, upon transfer to new test solution at approxim ately 48 and 72 hours, som e o f the fish were observed swimming erratically and turning a dark color. The fish appeared normal within approximately two hours, although one fish in the 15 mg a.i./L treatment group appeared discolored at test termination. LC50 values estimated from the mortality data at 2.5,24,48, 72 and 96 hours are shown in Table 4. CONCLUSIONS The 96-hour LC50 value for the sheepshead minnow, Cyprinodon variegatus, exposed to Perfluorooctanesulfonate, Potassium Salt (PFOS) was >15 mg a.i./L, the single concentration tested. The 96-hour no mortality concentration was 15 mg a.i./L. Based on the transitory effects observed in the 15 mg a.i./L treatment group after transfer to fresh test solutions, the NOEC was <15 mg a.i./L. Wildlife International, Ltd. - 14- Project Number 454A-146A REFERENCES 1 U.S. Environmental Protection Agency. 1996. Series 850-EcologicalEffects Test Guidelines (draft), OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater and Marine. 2 U.S. Environmental Protection Agency. 1985. Standard Evaluation Procedure, Acute Toxicity Testfo r Estuarine and Maine Organisms (Estuarine Fish 96-Hour Acute Toxicity Test). Hazard Evaluation Division. Office of Pesticide Programs. EPA-540/9-85-009. Washington, DC. 3 ASTM Standard E729-88a. 1994. Standard Guide fo r Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. American Society for Testing and Materials. Wildlife International, Ltd, 15- - Project Number 454A-146A Table 1 Summary of Analytical Chemistry Data Nominal Test Concentration (mg a.i./L) Negative Control Replicate A B C A B C A B C A B C A B C A B C A B C A B C Sampling Time (Hours) 0 0 0 24 (Old) 24 (Old) 24 (Old) 24 (New) 24 (New) 24 (New) 48 (Old) 48 (Old) 48 (Old) 48 (New) 48 (New) 48 (New) 72 (Old) 72 (Old) 72 (Old) 72 (New) 72 (New) 72 (New) 96 96 96 Measured Concentration (mg a.i./L)` <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Percent of Nominal2 -- -------------- Mean Measured Concentration (mg a.i./L) <LOQ Mean Measured Percent of Nominal - Solvent Control A 0 <LOQ B 0 <LOQ C 0 <LOQ A 24 (Old) <LOQ B 24 (Old) <LOQ C 24 (Old) <LOQ A 24 (New) <LOQ B 24 (New) <LOQ C 24 (New) <LOQ A 48 (Old) <LOQ B 48 (Old) <LOQ C 48 (Old) <LOQ A 48 (New) <LOQ B 48 (New) <LOQ C 48 (New) <LOQ A 72 (Old) <LOQ B 72 (Old) <LOQ C 72 (Old) <LOQ A 72 (New) <LOQ B 72 (New) <LOQ C 72 (New) <LOQ A 96 <LOQ B 96 <LOQ C 96 <LOQ -- ---- ------------ -- <LOQ -- 1 The limit of quantitation (LOQ) was 5.00 mg a.i./L. 2 Results generated using MacQuan Version 1.6 software in full precision mode. Manual calculations may differ slightly. Wildlife International, Ltd 16- - Project Number 454A-146A Table 1 (Continued) Summary o f Analytical Chemistry Data Nominal Test Concentration (mg a.i./L) 20 Replicate A B C A B C A B C A B C A B C A B C A B C A B C Sampling Time (Hours) 0 0 0 24 (Old) 24 (Old) 24 (Old) 24 (New) 24 (New) 24 (New) 48 (Old) 48 (Old) 48 (Old) 48 (New) 48 (New) 48 (New) 72 (Old) 72 (Old) 72 (Old) 72 (New) 72 (New) 72 (New) 96 96 96 Measured Concentration (mg a.i./L)' 16.4 15.7 16.0 15.2 13.3 15.2 16.2 15.7 15.0 13.4 11.3 12.7 17.4 18.2 18.6 15.2 18.0 16.2 17.0 16.6 17.4 13.7 11.1 13.4 Percent of N om inal 1 82.0 78.6 80.0 76.1 66.6 76.2 81.2 78.7 74.9 66.9 56.4 63.4 86.9 90.9 92.9 75.8 89.9 80.9 85.2 82.9 87.1 68.4 55.6 67.1 Mean Measured Concentration (mg a.i./L) 15 Mean Measured Percent of Nominal 75 20 (Abiotic) D 24 (Old) 10.9 E 24 (Old) 9.22 D 24 (New) 16.1 E 24 (New) 16.6 D 48 (Old) 9.62 E 48 (Old) 9.69 D 48 (New) 16.6 E 48 (New) 17.5 D 72 (Old) 9.01 E 72 (Old) 9.25 D 72 (New) 17.7 E 72 (New) 15.9 D 96 9.84 E 96 9.22 54.4 46.1 80.3 83.0 48.1 48.5 83.2 87.3 45.0 46.3 88.5 79.4 49.2 46.1 13 65 1 The limit o f quantitation (LOQ) was 5.00 m g a.i./L. 2 Results generated using MacQuan version 1.6 software in full precision mode. Manual calculations may differ slightly. Wildlife International, Ltd, - 17- Project Number 454A-146A Table 2 Temperature, Dissolved Oxygen and pH o f Water in the Test Chambers Mean Measured Concentration (mg a.i./L) ^Replicate Negative Control A B C OHour1________ Temp12 DO3 (C) (mg/L) pH 22.2 7.0 8.2 22.3 7.1 8.2 22.4 7.1 8.3 _____ 24 Hours (Old)_____ Temp DO (C) (mg/L) pH 22.4 6.2 8.1 22.4 6.0 8.1 22.6 5.9 8.2 24 Hours (New) Temp DO (C) (mg/L) pH 22.6 7.1 8.2 22.6 7.2 8.2 22.5 7.2 8.2 Solvent Control A B C 22.5 . 7.1 22.5 7.1 22.6 7.1 8.3 8.3 8.3 22.8 5.8 8.1 22.8 5.9 8.2 22.9 5.9 8.2 22.6 7.3 8.3 22.6 7.3 8.3 22.7 7.3 8.3 15 A 22.7 7.1 8.3 23.0 5.6 8.1 22.6 7.3 8.3 B 22.8 7.1 8.3 23.1 5.6 8.1 22.7 7.3 8.3 C 22.8 7.1 8.3 23.1 5.4 8.1 22.7 7.3 8.3 D 4 22.7 7.1 8.3 23.1 6.7 8.3 22.8 7.3 8.3 E4 22.8 7.1 8.3 23.1 5.2 8.2 22.7 7.3 8.3 1 The 0 and 96-hour dilution water measurements for salinity were 20%o. 2 Temperature measured continuously during the test remained between 21.5 and 22.5C. 3 A dissolved oxygen concentration of 4.7 mg/L represents 60% saturation at 22C in saltwater with a salinity of 20%o. 4 Abiotic replicates. _____ 48 Hours (Old) Temp (C) DO (mg/L) pH 22.0 5.3 8.0 22.1 4.6 7.9 22.1 3.4 8.0 22.3 4.4 8.0 22.4 3.7 8.0 22.4 3.6 8.0 22.5 4.6 8.0 22.5 3.0 8.0 22.6 5.0 8.1 22.7 5.0 8.1 22.7 4.8 8.1 Wildlife International, Ltd. - 18- Project Number 454A-146A Table 2 (Continued) Temperature, Dissolved Oxygen and pH of Water in the Test Chambers Mean Measured Concentration (mg a.i./L) Replicate 48 Hours (New) Temp12 DO3 (C) (mg/L) pH Negative Control A B C 22.1 7.2 8.2 22.1 7.3 8.2 22.2 7.3 8.2 72 Hours (Old) Temp DO (C) (mg/L) pH 22.2 5.5 8.0 22.3 3.9 7.9 22.3 3.7 8.0 72 Hours (New) Temp DO (C) (mg/L) pH 22.1 7.4 8.2 22.0 7.4 8.2 22.1 7.4 8.2 Solvent Control A B C 22.3 . 7.2 22.4 7.2 22.4 7.2 8.2 8.2 8.2 22.3 4.0 8.0 22.4 3.0 7.9 22.4 4.0 8.0 22.2 7.4 8.2 22.2 7.5 8.2 22.3 7.6 8.2 15 A 22.4 7.2 8.2 22.5 3.6 7.9 22.2 7.6 8.2 B 22.5 7.2 8.2 22.5 3.6 8.0 22.2 7.5 8.2 C 22.5 7.2 8.2 22.6 3.3 7.9 22.3 7.4 8.2 D 4 22.6 7.4 8.3 22.6 5.4 8.1 22.3 7.5 8.2 E4 22.5 7.4 8.3 22.6 5.5 8.1 22.3 7.5 8.2 1 The 0 and 96-hour dilution water measurements for salinity were 20%o. 2 Temperature measured continuously during the test remained between 21.5 and 22.5C. 3 A dissolved oxygen concentration of 4.7 mg/L represents 60% saturation at 22C in saltwater with a salinity of 20%o. 4 Abiotic replicates. 96 Hours (Old) 1 Temp DO (C) (mg/L) pH 22.1 3.8 7.9 21.9 3.5 7.9 22.1 2.8 7.9 22.1 2.6 7.9 22.2 2.7 7.9 22.3 1.7 7.9 22.4 1.9 7.9 22.3 1.6 7.9 22.4 3.3 7.9 22.5 3.4 8.0 22.6 3.2 8.0 Wildlife International, Ltd, -19- Project Number 454A-146A Table 3 Cumulative Percent Mortality and Treatment-Related Effects123 Mean Measured Concentration (mg a.i./L) 2.5 Hours Rep. No. Exposed ' No. Dead 2 Observed Effects3 24 Hours No. Observed Dead Effects 48 Hours ' No. Observed Dead Effects 72 Hours ' No. Observed Dead Effects 96 Hours No. Observed Dead Effects Negative Control A 10 0 10 AN B 10 0 10 AN C 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN Solvent Control A 10 0 10 AN B 10 0 10 AN C 10 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN 0 10 AN - 0 10 AN 0 10 AN 0 10 AN 15 A 10 0 10 AN 0 10 AN 0 2E,D; 8AN 0 3EJD; 8AN 0 10 AN B 10 0 W A N 0 10 AN 0 3E,D; IR; 6AN 0 4E,D; 6AN 0 10 AN C 10 0 10 AN 0 10 AN 0 1E,D; 9AN 0 2E,D; 9AN 0 ID; 9AN 1 All fish were transferred to fresh test solution at approximately 24,48 and 72 hours. 2 Cumulative number of dead fish. 3 Observed Effects: AN = appear normal; E = erratic swimming; D = discoloration (dark); R = lying on bottom. Cumulative Percent Mortality 0 0 0 W lld lIn te r n a tio n a l, Ltd,____________________ Project Number 4S4A-146A -20- Table 4 LC50 Values Time LC50 (mg a.i./L) 95% Confidence Interval (mg a.i./L) Statistical Method 2.5 Hours >15 NA2 24 Hours >15 NA2 48 Hours >15 --1 NA2 72 Hours >15 __1 NA2 96 Hours >15 _1 NA2 1 95% confidence limits could not be calculated from the data. 2 NA = not applicable; LC50 value was estimated by visual interpretation of the mortality data. Wildlife International, Ltd, -21 - Project Number 454A-146A Appendix 1 Salinity and pH of Saltwater Measured During the 4-Week Period Immediately Preceding the Test Parameter Salinity (%o) pH Mean 20 (n = 4) 8.2 (n = 4) Range 20-20 8.1-8.3 Wildlife International, Ltd, -22- Project Number 454A-146A Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Saltwater1 Component Pesticides And Organics 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-0 Desethylatrazine Desisopropylatrazine Desmetryn Diazinon Dichlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid <0.03 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.04 pg/L <0.08 pg/L <0.25 pg/L <0.05 pg/L <0.05 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.03 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.04 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.25 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.03 pg/L <0.02 pg/L <0.01 pg/L <0.03 pg/L . Dichlorvos Dicofol Diethyltoluamide Difenoconazole Dimethoate Dimethomorph Disulfoton DMST Dodemorph Endosulfan-a Endosulfan- Endosulfan-sulfate Epoxiconazole Eptam Esfenvalerate Ethion Ethofumesate Ethoprophos Etridiazole Etrimfos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb Fenpiclonil Fenpropathrm Fenpropimorph Fentbion 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 p/L <0.02 pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on November 15,2000. Wildlife International, Ltd, -23- Project Number 454A-146A Appendix 2 (Continued) Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Saltwater1 Component Pesticides And Organics (Page 2) Measured Concentration Component Measured Concentration Malathion Metalaxyl Metamitron Metazachlor Methidathion Paclobutazole Parathion Parathion-methyl Penconazole Pendimethalin Permethrin-cis Permethrin-trans Phosalone Phosmet Phosphamidon-cis Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazine Propham Propiconazole Propoxur Propyzamide Prosulfocarb Pyrazophos <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.03 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.05 [ig/L <0.03 pg/L <0.02 pg/L <0.02 pg/L <0.03 pg/L Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl Pyrifenox-1 Pyrifenox-2 Pyrimethanil Quizalofop-ethyl Simazine Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbuthylazine Tetrachlorvinphos Tetrahydroftalimide Tetramethrin Thiabendazole Thiometon Tolclofos-methyl Tolylfluanid Triadimefon Triadimenol Triallate Triazophos Trifluralin Vamidothion Vinclozolin <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.05 pg/L <0.04 pg/L <0.01 pg/L <0.04 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L Metals Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 730 mg/L 5,800 mg/L 270 mg/L <0.03 mg/L 200 mg/L <0.18 mg/L <1.0 pg/L <3.0 pg/L <6.0 pg/L <4.0 pg/L Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium <14 pg/L <10.0 pg/L <20.0 pg/L <7.0 pg/L <3.0 pg/L <3.0 pg/L 1.2 pg/L <0.025 pg/L <1 pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on November 15, 2000. Wildlife International, Ltd, -24- Appendix 3 Project Number 454A-146A THE ANALYSIS OF PFOS IN FILTERED SALTWATER IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-146A Wildlife International, Ltd. -25- Project Number 454A-146A REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Static-Renewal Acute Toxicity Test with the Sheepshead Minnow (Cyprinodon variegatus) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-146A PRINCIPAL INVESTIGATOR: MANAGEMENT: Director, Analytical Chemistry Wildlife International, Ltd. -26- Project Number 454A-146A Introduction Filtered saltwater samples were collected from a 96-hour static-renewal acute toxicity test designed to determine the effects of PFOS (Perfluorooctanesulfonate, Potassium Salt) to the sheepshead minnow (Cyprinodon variegatus). This study was conducted by Wildlife International, Ltd. and identified as Project No.: 454A-146A. The analyses of these water samples were performed at Wildlife International, Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). Samples were received for analysis on August 13, 14, 15, 16 and 17, 2001 and were analyzed on each sample receipt day. Test Substance and Internal Standard The test substance used for this study was Wildlife International, Ltd. identification number 4675. The test substance, referred to hereafter as PFOS, was used to prepare calibration standards and matrix fortification samples. The internal standard was received from 3M Corporation on July 2,1998 and was assigned Wildlife International, Ltd. identification number 4526 upon receipt. The internal standard, a granular material, was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619 97-2. The standard, referred to hereafter as 4H PFOS, was stored under ambient conditions. Analytical Method The method used for the analysis of the filtered saltwater samples was developed at Wildlife International, Ltd. and entitled "Analytical Method for the Determination of PFOS in Freshwater, Saltwater, and Algal Medium". This methodology was included as Appendix II of Wildlife International, Ltd. protocol number 454/011299/MVAL/SUB454. It was based upon methodology provided by 3M Corporation. Several modifications from the validated methodology were implemented for the present study. First, the concentration of the internal standard was changed from 100 pg/L to 10.0 pg/L to better match the calibration and test sample instrumental concentrations. Second, a guard cartridge was used in conjunction with a shorter (50 mm vs. 100 mm) analytical column for long-term protection of the analytical column and faster run times. Wildlife International, Ltd. -27- Project Number 454A-146A Samples were diluted in a 50% methanol : 50% NANOpure water solution containing 10.0 pg 4H PFOS /L and 0.05% formic acid (v/v) so that they fell within the calibration range of the PFOS methodology. Concentrations of the PFOS in the standards and samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a PerkinElmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil Cig analytical column (50 mm x 2 mm I.D., 3-pm particle size) fitted with a Keystone Javelin Cig guard cartridge (20 x 2 mm). The instrument parameters are summarized in Table 1. A method flowchart is provided in Figure 1. Calibration Curve and Limit of Quantitation Calibration standards o f PFOS prepared in a 50% methanol: 50% NANOpure water solution containing 10.0 pg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v), ranging in concentration from 0.500 to 5.00 pg a.i./L, were analyzed with the samples. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOS on the basis of individual isomeric components. Linear regression equations were generated using peak area response ratios (PFOS : internal standard) versus the respective concentration ratios (PFOS: internal standard) of the calibration standards. A typical calibration curve is presented in Figure 2. The concentration o f PFOS in the samples was determined by substituting the peak area response ratios into the applicable linear regression equation. Representative ion chromatograms of low and high calibration standards are presented in Figures 3 and 4, respectively. The method limit of quantitation (LOQ) for these analyses was set at 5.00 mg a.i./L calculated as the product of the lowest calibration standard analyzed (0.000500 mg a.i./L) and the dilution factor of the matrix blank samples (10,000). Wildlife International, Ltd. -28- Project Number 454A-146A Matrix Blank and Fortification Samples Five matrix blank samples were analyzed to determine possible interference. No interferences were observed at or above the LOQ during samples analyses (Table 2). A representative ion chromatogram of a matrix blank is presented in Figure 5. Filtered saltwater was fortified at 18 and 22 mg a.i./L and analyzed concurrently with the samples to determine the mean procedural recovery (Table 3). Sample concentrations were not corrected for the mean procedural recovery of 99.2%. A representative ion chromatogram of a matrix fortification is presented in Figure 6. Example Calculations Sample number 454A-146A-9, nominal concentration of 20 mg a.i./L in filtered saltwater. Peak Area Ratio = Analyte Peak Area/Intemal Standard Peak Area Concentration Ratio = Concentration of Analyte/Concentration of Internal Standard Internal Standard Concentration: 0.0100 mg/L First Initial Volume: 0.100 mL First Final Volume: 10.0 mL Second Initial Volume: 0.100 mL Second Final Volume: 10.0 mL Dilution Factor: 10,000 PFOS Peak Area: 252985 Internal Standard Peak Area: 469623 Peak Area Ratio: 0.5387 Calibration curve equation. Slope: 3.2154 Intercept: 0.0244 Curve is weighted (1/x) Peak area ratio - (Y-intercept) PFOS (mg a.i./L) at instrument = Slope x Internal Standard Concentration Wildlife International, Ltd. -29- Project Number 454A-146A 0.5387 - 0.0244 3.2154 X 0.0100 = 0.001599 PFOS (mg a.i./L) in sample = PFOS (mg a.i./L) at instrument x Dilution Factor = 0.001599x10,000 = 16.0 PFOS (mg a.i./L) in sample Percent of Nominal Concentration = PFOS (mg a.i./L) nominal x 100 16.0 20 x 100 = 80.0% Quantitation software for recoveries: MacQuan, version 1.6. RESULTS Sample Analysis Filtered saltwater samples were collected from a 96-hour static-renewal acute toxicity test with the sheepshead minnow (Cyprinodon variegatus). New solutions were collected at test initiation, August 13, 2001 (Hour 0). New and old solutions were collected on August 14 (Hour 24), August 15 (Hour 48), and August 16 (Hour 72), 2001. Old solutions were collected at test termination, August 17,2001 (Hour 96). The measured concentrations of PFOS in the samples collected from the 20 mg a.i./L treatment group at initiation of exposure o f the test organisms (Hour 0) had measured concentrations ranging from 15.7 to 16.4 mg a.i./L, corresponding to 78.6 to 82.0% of the nominal concentrations (Table 4). New samples collected at Hours 24, 48, and 72 had measured concentrations ranging from 15.0 to 16.2 mg a.i./L, 17.4 to 18.6 mg a.i./L, and 16.6 to 17.4 mg a.i./L, respectively. These values corresponded to percent of nominal ranging from 74.9 to 81.2%, 86.9 to 92.9%, and 82.9 to 87.1%, respectively. Old samples collected at Hours 24, 48, and 72 had measured concentrations ranging from 13.3 to 15.2 mg a.i./L, 11.3 to 13.4 mg a.i./L, and 15.2 to 18.0 mg a.i./L, respectively. These values corresponded to percent of nominal ranging from 66.6 to 76.2%, 56.4 to 66.9%, and 75.8 to 89.9%, respectively. The Wildlife International, Ltd. -30- Project Number 454A-146A measured concentrations of PFOS in the samples collected at test termination (Hour 96) had measured concentrations ranging from 11.1 to 13.7 mga.i./L, corresponding to 55.6 to 68.4% of the nominal concentrations. The measured concentrations of PFOS from the abiotic 20 mg a.i./L treatment group were slightly lower (mean measured concentration = 1 3 mg a.i./L) than those from the 20 mg a.i./L treatment group (mean measured concentration =15 mg a.i./L) with the fish present (Table 4). The most plausible reason for this was increased deposition of test substance at the limit of solubility in the absence of the natural mixing action provided by the movement of the fish. A representative ion chromatogram of a test sample is shown in Figure 7. Wildlife International, Ltd. -31 - Project Number 454A-146A INSTRUMENT: Table 1 Typical HPLC/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer (HPLC/MS) equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: GUARD COLUMN: OVEN TEMPERATURE: Keystone Betasil Ci8 column (50 mm x 2 mm I.D., 3-pm particle size) Keystone Javelin Cis column (20 x 2 mm) 30C STOP TIME: 5.00 minutes FLOW RATE: 0.220 mL/minute MOBILE PHASE: 72.0% Methanol : 28.0% NANOpure water containing 0.1% Formic Acid INJECTION VOLUME: 25.0 pL PFOS RETENTION TIME: Approximately 4.4 minutes INTERNAL STANDARD RETENTION TIME: Approximately 3.0 minutes PFOS MONITORED MASS: 499 amu INTERNAL STANDARD MONITORED MASS: 427 amu Wildlife International, Ltd, -32- Project Number 454A-146A Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-146A-) MAB-1 MAB-2 MAB-3 MAB-4 Sample Type Matrix Blank Matrix Blank Matrix Blank Matrix Blank Measured Concentration of PFOS1 (mg a.i./L) < LOQ < LOQ < LOQ < LOQ A r o o MAB-5 Matrix Blank The limit of quantitation (LOQ) was 5.00 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.000500 mg a.i./L) and the dilution factor of the matrix blank samples (10,000). Wildlife International, Ltd -33- Project Number 454A-146A Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-146A-) MAS-1 MAS-3 MAS-5 MAS-7 MAS-9 Concentrations o f PFOS (mg a.i./L) Fortified1 Measured1 18.0 16.2 18.0 17.9 18.0 16.6 18.0 18.4 18.0 18.1 Percent Recovered1 89.9 99.4 92.4 102 101 MAS-2 22.0 20.2 92.0 MAS-4 22.0 22.4 102 MAS-6 22.0 22.0 99.9 MAS-8 22.0 23.8 108 MAS-10 22.0 23.0 105 Mean = 99.2 Standard Deviation = 5.92 CV - 5.97% N =10 R esultsw er^enerate^isin^M ac^Jw i^versio^^^tw M ej^^am w al^alculation^n^diff^sli^tl^ Wildlife International, Ltd, -34- Project Number 454A-146A Table 4 Measured Concentrations of PFOS in Filtered Saltwater Samples from a Sheepshead Minnow (Cyprinodon variegatus) 96-Hour Toxicity Test Nominal Test Concentration (mg a.i./L) Replicate Sampling Time (Hours) Measured Concentration (mg a.i./L)' Percent of Nominal2 Mean Measured Concentration (mg a.i./L) Mean Measured Percent of Nominal 0.0 A 0 <LOQ B 0 <LOQ (Negative Control) C A 0 24 (Old) <LOQ <LOQ B 24 (Old) <LOQ C 24 (Old) <LOQ A 24 (New) <LOQ B 24 (New) <LOQ C 24 (New) <LOQ A 48 (Old) <LOQ B 48 (Old) <LOQ C 48 (Old) <LOQ A 48 (New) <LOQ B 48 (New) <LOQ C 48 (New) <LOQ A 72 (Old) <LOQ B 72 (Old) <LOQ C 72 (Old) <LOQ A 72 (New) <LOQ B 72 (New) <LOQ C 72 (New) <LOQ A 96 <LOQ B 96 <LOQ C 96 <LOQ -- -- -- -------- - -- <LOQ -- 1 The limit o f quantitation (LOQ) was 5.00 mg a.iVL. 2 Results were generated using MacQuan version 1.6 software in full precision mode. Manual calculations may differ slightly._____________________________________________________________________________________________ Wildlife International, Ltd, -35 - Project Number 454A-146A Table 4 (Continued) Measured Concentrations of PFOS in Filtered Saltwater Samples from a Sheepshead Minnow (Cyprinodon variegatus) 96-Hour Toxicity Test Nominal Test Concentration (mg a.i./L) 0.0 (Solvent Control) Replicate A B C A B C A B C A B C A B C A B C A B C A B C Sampling Time (Hours) 0 0 0 24 (Old) 24 (Old) 24 (Old) 24 (New) 24 (New) 24 (New) 48 (Old) 48 (Old) 48 (Old) 48 (New) 48 (New) 48 (New) 72 (Old) 72 (Old) 72 (Old) 72 (New) 72 (New) 72 (New) 96 96 96 Measured 1Concentration (mg a.i./L)1 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Percent of Nominal2 -- - -- - ---- - -- - -- - ----~ - Mean Measured Concentration (mg a.i./L) <LOQ Mean Measured Percent of Nominal - 1 The limit o f quantitation (LOQ) was 5.00 mg a.i./L. 2 Results were generated using MacQuan version 1.6 software in full precision mode. Manual calculations may differ slightly. Wildlife International, Ltd -36- Project Number 454A-146A Table 4 (Continued) Measured Concentrations of PFOS in Filtered Saltwater Samples from a Sheepshead Minnow (Cyprinodon variegatus) 96-Hour Toxicity Test Nominal Test Sampling Concentration Time (mg a.i./L)________Replicate_____ (Hours) Measured Concentration (mg a.i,/L)` Percent Mean Measured Mean Measured of Concentration Percent of Nominal12______(mg a.i./L)_________ Nominal 20 A 0 16.4 82.0 B 0 15.7 78.6 C 0 16.0 80.0 A 24 (Old) 15.2 76.1 B 24 (Old) 13.3 66.6 C 24 (Old) 15.2 76.2 A 24 (New) 16.2 81.2 B 24 (New) 15.7 78.7 C 24 (New) 15.0 74.9 A 48 (Old) 13.4 66.9 B 48 (Old) 11.3 56.4 C 48 (Old) 12.7 63.4 A 48 (New) 17.4 86.9 B 48 (New) 18.2 90.9 C 48 (New) 18.6 92.9 A 72 (Old) 15.2 75.8 B 72 (Old) 18.0 89.9 C 72 (Old) 16.2 80.9 A 72 (New) 17.0 85.2 B 72 (New) 16.6 82.9 C 72 (New) 17.4 87.1 A 96 13.7 68.4 B 96 l l . l 55.6 C 96 13.4 67.1 1 The limit o f quantitation (LOQ) was 5.00 mg a.i./L. 2 Results were generated using MacQuan version 1.6 software in full precision mode. Manual calculations may differ slightly._____________________________________________________________________________________________ Wildlife International, Ltd, -37- Project Number 454A-146A Table 4 (Continued) Measured Concentrations of PFOS in Filtered Saltwater Samples from a Sheepshead Minnow (Cyprinodon variegatus) 96-Hour Toxicity Test Nominal Test Concentration (mg a.i./L) Replicate Sampling Time (Hours) Measured Concentration (mg a.i./L)' Percent of Nominal2 Mean Measured Concentration (mg a.i./L) Mean Measured Percent of Nominal 20 (Abiotic) D 24 (Old) 10.9 E 24 (Old) 9.22 D 24 (New) 16.1 E 24 (New) 16.6 D 48 (Old) 9.62 E 48 (Old) 9.69 D 48 (New) 16.6 E 48 (New) 17.5 D 72 (Old) 9.01 E 72 (Old) 9.25 D 72 (New) 17.7 E 72 (New) 15.9 D 96 9.84 E 96 9.22 54.4 46.1 80.3 83.0 48.1 48.5 83.2 87.3 45.0 46.3 88.5 79.4 49.2 46.1 13 65 1 The limit o f quantitation (LOQ) was 5.00 mg a.i./L. 2 Results were generated using MacQuan version 1.6 software in full precision mode. Manual calculations may differ Wildlife International, Ltd, - 38- Project Number 454A-146A METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FILTERED SALTWATER Prepare matrix fortification samples in filtered saltwater matrix by spiking the requisite volume of PFOS stock solutions directly into filtered saltwater. Perform fortifications with gas-tight syringes and class A volumetric flasks. The matrix blank is unfortified filtered saltwater. 1 Prepare appropriate dilutions of study and QC samples to within range of the PFOS HPLC/MS methodology: Partially fill Class A volumetric flasks with 50% methanol : 50% NANOpure water dilution solvent containing 10.0 pg 4H PFOS/L and 0.05% (v/v) formic acid. Add appropriate volume of sample and bring the flask to volume with dilution solvent. Process the matrix blank samples using the same dilution and aliquot volume as for the lowest fortification level. Mix well by several repeat inversions. i A m putate sam ples and subm it for HPLC/M S analysis. Figure 1. Analytical method flowchart for the analysis of PFOS in filtered saltwater. Wildlife International, Ltd, -39- PFOS 4 9 9 .0 Internal Standard: 4HPFOS Weighted (1/x) Intercept = 0.0244 Slope = 3.2154 Correlation Coeff. = 0.9996 Area(Ratlo) Project Number 454A-146A Figure 2. A typical calibration curve for PFS. Wildlife International, Ltd, -40- Project Number 454A-146A PF0S_1 STD 0.S00 ug a.171. 4675A-011D -21 4.98 in 1 period pros Internal Standard: 4HPFOS Use Area Absolute Retention Time 1: 4.98 Q1 Ml, 297 scans 499.0 Noise Thres. Quant Thres. Min. Width Mult. Width 10.0 5.0 3 6 Base. Width 80 RT Win. (secs) Smooth 20 1 Expected RT 4.24 Area 74579 Height 7951 Start Time 4.02 End Time Integration Width 4.60 0.57 Retention Time 4.24 Integration Type A -VB Mon, Aug 13, 2001 14:23 intensity: 80000 cps PFOS_1 STD 0.500 ug a.!7L 4675A-011D -21 4.98 in 1 period 4HPFOS use as Internal Standard 1: 4.98 Q1 Ml, 297 scans 427.0 Noise Thres. 2.0 Quant Thres. 1.0 Min. Width 3 Mult. Width Base. Width 6 30 RT Win. (secs) Smooth Expected RT 20 1 3.00 Area 414322 Height 63986 Start Time End Time Integration Width Retention Time Integration Type 2.78 3.28 0.51 2.93 A -BB Mon, Aug 13, 2001 14:23 100 90- 8070 60- 50- 40- 30- 20- 10- o-l --2T-4 47 -A - r 103 153J 41 81 121 161 0.69 1.36 2.04 2.71 intensity: 80000 cps 218 247 201 241 3.38 4.06 281 Scan 4.73 Time Figure 3. A representative ion chromatogram of a low-level (0.500 pg a.i./L) PFOS standard. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard bottom)). Wildlife International, Ltd, -41 - Project Number 454A-146A PF0S_5 STD 5.00 ug a.IYL 4675A-011D -25 5.00 in 1 period PFOS Internal Standard: 4HPFOS Use Area Absolute Retention Time 1: 4.98 Q1 Ml, 297 scans 499.0 Noise Thres. 10.0 Quant Thres. 5.0 Min. Width 3 Mult. Width 6 Base. Width 80 RT Win. (secs) 2 0 Smooth 1 Expected RT 4.24 Area 680879 Height 68328 Start Time End Time Integration Width Retention Time Integration Type 4.03 4.75 0.72 4.26 A - VB Mon, Aug 13, 2001 14:48 intensity: 80000 cps PFOS_5 STD 5.00 ug a.IVL 4675A-01 ID -25 5.00 in 1 period 4HPFOS use as Internai Standard 1: 4.98 Q1 Mi, 297 scans 427.0 Noise Thres. Quant Thres. Min. Width 2.0 1.0 3 Mult. Width Base. Width 6 30 RT Win. (secs) Smooth Expected RT 20 1 3.00 Area 420532 Height 56584 Start Time End Time Integration Width Retention Time Integration Type 2.78 3.28 0.51 2.93 A -BB Mon, Aug 13, 2001 14:48 100-1 9080 70 60 5040302010- 11 o-l 14^7- 41 0.69 101120 81 121 1.37 2.04 intenslty: 80000 cps 174 218 242 264 241 281 Scan 4.06 4.73 Time Figure 4. A representative ion chromatogram of a high-level (5.00 pg a.i./L) PFOS standard. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard bottom)). Wildlife International, Ltd, -42- Project Number 454A-146A PFOS_7 MAB-1 454A-146A- Mon, Aug 13, 2001 15:01 4.98 in 1 period PROS Internal Standard: 4HPFOS Use Area Absolute Retention Time 1: 4.98 Q1 Ml, 297 scans 499.0 Noise Thres. 10.0 Quant Thres. 5.0 Min. Width 3 Mult. Width - 6 Base. Width 80 RT Win. (secs) 20 Smooth 1 Expected RT 4.24 Area 4434 Height 495 Start Time 4.11 End Time Integration Width Retention Time Integration Type 4.43 0.32 4.26 A-BB 100908070 6050403020100-1 /V 41 0.69 intensity: 80000 cps 1 87 81 1.36 131 121 2.04 171 161 2.71 221 ^ 8 3 201 241 281 Scan 3.38 4.06 4.73 Time PFOS_7 MAB-1 454A-146A- 4.98 In 1 period 4HPF0S use as Internal Standard Mon, Aug 13, 2001 15:01 1: 4.98 Q1 Ml, 297 scans 427.0 Noise Thres. 2.0 Quant Thtes. Min. Width 1.0 3 Mult Width Base. Width RT W in. (secs) Smooth Expected RT 6 30 20 1 3.00 Area 474292 Height 63384 Start Time End Time 2.79 3.30 integration Width 0.51 Retention Time Integration Type 2.93 A - BB intensity: 80000 cps Figure 5. A representative ion chromatogram of a matrix blank sample (454A-146A-MAB-1). Dilution factor = 10,000x. The arrow indicates the retention time of PFOS. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard bottom)). Wildlife International, Ltd, -43- Project Number 454A-146A PF0S.9 MAS-2 454A-146A- Mon, Aug 13, 2001 15:13 4.98 in 1 period pros Internai Standard: 4HPFOS Use Area Absolute Retention Time 1: 4.98 Q1 Ml, 297 scans 499.0 Noise Thres. 10.0 Quant Thres. Min. Width 5.0 3 Mult. Width 6 Base. Width 80 RT Win. (secs) 20 Smooth 1 Expected RT 4.24 Area 319919 Height 32758 Start Time End Time Integration Width Retention Time Integration Type 4.04 4.75 0.71 4.26 A,-V B intensity: BOOOO cps PFOS_9 MAS-2 454A-146A- Mon, Aug 13, 2001 15:13 4.98 in 1 period 4HPFOS use as Internal Standard 1: 4.98 Q1 Ml, 297 scans 427.0 Noise Thres. 2.0 Quant Thres. 1.0 Min. Width 3 Mult. Width 6 Base. Width 30 RT Win. (secs) Smooth Expected RT 20 1 3.00 Area 473996 Height 61883 Start Time 2.79 End Time Integration Width Retention Time Integration Type 3.35 0.56 2.95 A -B B lOOi 9080706060403020100+ 0.69 1.36 2.04 intensity: 80000 cps 175 2.71 3.38 4.06 4.73 Time Figure 6. A representative ion chromatogram of a matrix fortification sample (454A-146A-MAS-2). Nominal Concentration = 22 mg a.i./L, Dilution factor = 10,000x. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard bottom)). Wildlife International, Ltd, -44- Project Number 454A-146A P F O S J I7 454A-146A- 7 Mon, Aug 13, 2001 16:04 4.98 in 1 period PFOS Internal Standard: 4HPFOS Use Area Absolute Retention Time 1: 4.98 Q1 Ml, 297 scans 4 9 9 .0 Noise Thres. 10.0 Quant Thres. 6.0 Min. Width 3 Mult. Width 6 Base. Width 80 RT Win. (secs) 20 Smooth 1 Expected RT 4.24 Area 259764 Height 26978 Start Time End Time integration Width Retention Time Integration Type 4.02 4.68 0.66 4.26 A-VB intensity: 80000 cps PFOS_17 454A-146A- 7 Mon, Aug 13, 2001 16:04 4.98 in 1 period 4HPFOS use as Internal Standard 1: 4.98 Q1 Ml, 297 scans 4 2 7 .0 Noise Thres. Quant Thres. 2.0 1.0 Min. Width Mult. Width Base. Width RT Win. (secs) Smooth Expected RT 3 6 30 20 1 3.00 Area 4 7 1 08 4 Height 62678 Start Time End Time Integration Width Retention Time Integration Type 2.79 3.30 0.51 2.95 A-BB intensity: 80000 cps Figure 7. A representative ion chromatogram o f a test sample (454A-146A-7). Nominal Concentration = 20 mg a.i./L, Dilution factor = 10,000x. (monitored masses = 499 amu (PFOS - top) and 427 amu (4HPFOS internal standard bottom)). Wildlife International, Ltd, -45- Project Number 454A-146A Appendix 4 Changes to Protocol This study was conducted in accordance with the approved Protocol with the following changes: 1. The protocol was amended to add the proposed experimental start and termination dates, test concentrations, and test and reference substance identification. 2. The protocol was amended to include the procedures and sampling scheme for conducting a limit test. 3. The protocol was amended to delete the analysis of feed since historical analyses of Wildlife International, Ltd. aquatic feed have shown that no contaminants are present at levels known to be capable of interfering with the study. 4. The protocol was amended to clarify the analytical methodology to be used during sample analyses. 5. The protocol was amended to change the proposed test dates and to include the procedures and sampling scheme to be used during a static-renewal test since the test was repeated under staticrenewal test conditions to maintain test concentrations during the exposure period. 6. The protocol was amended to include the use of methanol in the preparation of the test solutions, and to include a solvent control group. 7. Salinity of the dilution water was measured at test termination, as well as at test initiation. Since the additional measurement confirmed that the salinity remained constant during the test, this change to the protocol had no impact upon the study Wildlife International, Ltd. - 46- Project Number 454A-146A Appendix 5 Personnel Involved in the Study The following key Wildlife International, Ltd. personnel were involved in the conduct or management of this study: 1. Henry 0 . Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Director, Analytical Chemistry 3. Cary A. Sutherland, Laboratory Supervisor 4. Raymond L. Van Hoven, Ph.D., Scientist 5. Susan J. Palmer, Senior Biologist 6. Michelle Stence, Biologist 7. Frank J. Lezotte, Chemist