Document B5kLOJX7wkZ8324E0d00E5Q6o

PFOS: AN ACUTE TOXICITY STUDY WITH THE EARTHWORM IN AN ARTIFICIAL SOIL SUBSTRATE FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-111 Environmental Laboratory Project Number U2723 OECD Guideline 207 AUTHORS: Anne B. Sindermann John R. Porch Henry O. Krueger, Ph.D. Raymond L. Van Hoven, Ph D. STUDY INITIATION DATE: October 17, 2001 STUDY COMPLETION DATE: May 10, 2002 SUBMITTED TO: 3M Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 52 Wildlife International, Ltd. Project Number 454-111 2- - GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M TITLE. PFOS: An Acute Toxicity Study with the Earthworm in an Artificial Soil Substrate WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-111 STUDY COMPLETION DATE: May 10, 2002 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice, ENV/MC/CHEM (98) 17, Paris, 1998; and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. STUDY DIRECTOR R. Porch rvisor, Non-Target Plants and Insects 'L' Date Wildlife International, Ltd. Project Number 454-111 -3 - 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, Paris, 1998; and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all inspections and audits, and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Test Substance Preparation DATE CONDUCTED: November 2, 2001 Test Substance Preparation November 19, 2001 Observations - Day 7 Matrix Fortifications Body Weights - Day 14 Analytical Data, Draft Report Raw Data, Draft Report Final Report November 27,2001 November 27, 2001 December 4, 2001 January 11, 2002 January 10 and 11, 2002 May 6, 2002 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: November 2, 2001 November 5, 2001 November 19,2001 November 20, 2001 November 27,2001 November 28, 2001 November 27, 2001 November 28, 2001 December 4, 2001 December 5, 2001 January 14, 2002 January 14, 2002 January 11,2002 January 16, 2002 May 6, 2002 May 10,2002 O J YWSlO |A. les H. Coleman Quality Assurance Representative -I Date W ildlife International, Ltd. Project Number 454-111 SPONSOR: 3M -4 REPORT APPROVAL TITLE: PFOS: An Acute Toxicity Study with the Earthworm in an Artificial Soil Substrate WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-111 STUDY DIRECTOR. JoItiVR. Porch Supjvisor, Non-Target Plants and Insects IQ yiAjVM 0 ^ Date I WILDLIFE INTERNATIONAL. LTD. MANAGEMENT: Henry . Byfueger, Ph.D. Director, Aquatic Toxicology and Non-Target Plants J/ v/X Date W ildlife International, Ltd. Project Number 454-111 -5 - TABLE OF CONTENTS Title Page............................................................................................................................................1 Good Laboratory Practice Compliance Statement............................................................................2 Quality Assurance Statement............................................................................................................3 Report Approval................................................................................................................................ 4 Table of Contents.............................................................................................................................. 5 Summary............................................................................................................................................ 7 Introduction........................................................................................................................................8 Objective ........................................................................................................................................... 8 Experimental Design..........................................................................................................................8 Materials and Methods...................................................................................................................... 9 Test Substance....................................................................................................................... 9 Test Organism.........................................................................................................................9 Artificial Soil and Test Soils Preparation..............................................................................9 Analytical Sampling............................................................................................................. 10 Analytical Testing ............................................................................................................... 11 Test Chambers......................................................................................................................11 Physical Properties of Test Soils..........................................................................................11 Environmental Conditions.................................................................................................... 11 O bservations......................................................................................................................... 12 Body Weights........................................................................................................................12 Reference Toxicity T e s t.......................................................................................................12 Statistical Analyses ............................................................................................................. 12 Results and Discussion................................................................................................................... 13 Environmental Conditions.................................................................................................... 13 Physical Properties of Test Soils..........................................................................................13 Observations......................................................................................................................... 13 Statistical Analyses.............................................................................................................. 14 Reference Toxicity Test........................................................................................................14 Sample Analysis................................................................................................................... 15 Conclusions 15 W ildlife International, Ltd. Project Number 454-111 6- TABLE OF CONTENTS - Continued - References......................................................................................................................................... 16 Table 1. TABLES Moisture, pHw, and Temperature of Test Soils.............................................17 Table 2. Cumulative Mortality and Observations of Earthworms Exposed to PFOS in an Artificial Soil Substrate.............................................................. 18 Table 3. Average Body Weights of Earthworms Exposed to PFOS in an Artificial Soil Substrate..................................................................................................19 Table 4. Day 14 Measured Values for PFOS in Earthworm Tissue ......................... 20 Appendix 1. Appendix 2. Appendix 3. Appendix 4. APPENDICES Personnel Involved in the Study ..................................................................... 21 Changes to the Study Protocol ....................................................................... 22 Artificial Soil and Test Soils Preparation ......................................................23 Analytical Chemistry......................................................................................25 Wildlife International, Ltd. Project Number 454-111 -7SUMMARY SPONSOR 3M Corporation TITLE : PFOS: An Acute Toxicity Study with the Earthworm in an Artificial Soil Substrate WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454-111 TEST DATES. Study Initiation. October 17, 2001 Experimental Start (OECD): November 2, 2001 Experimental Start (EPA): November 20, 2001 Experimental Termination: December 4, 2001 Study Completion: May 10, 2002 TEST ORGANISM : Earthworm (Eiseniafetida) AGE OF TEST ORGANISM : Adult (with clitellum) SOURCE OF TEST ORGANISM : Worm Man's Worm Farm R & K Trading Co. 116 Pergola Ave. Monroe Township, New Jersey 08831 MEAN MEASURED TEST CONCENTRATIONS: Negative Control 77.0 mg a.i./ kg 141 mg a.i./kg 289 mg a.i./ kg 488 mg a.i./ kg 1042 mg a.i./ kg RESULTS: 7-Day LC50: 398 mg a.i./ kg 7-Day NOEC: 289 mg a.i./ kg 7-Day LOEC. 488 mg a.i./ kg 14-Day LC50: 373 mg a i./ kg 14-Day NOEC: 77 mg a.i./ kg 14-Day LOEC: 141 mg a.i./ kg Wildlife International, Ltd. Project Number 454-111 8- - INTRODUCTION Wildlife International, Ltd. conducted this study for 3M at the Wildlife International, Ltd. toxicology facility in Easton, Maryland. The in-life portion of this test was conducted from November 19, 2001 to December 4, 2001. Raw data generated at Wildlife International, Ltd. and a copy o f the final report are filed under Project Number 454-111 in archives located on the Wildlife International, Ltd. site. Key personnel involved in the conduct or management of the study are listed in Appendix 1. OBJECTIVE The objective of this study was to determine the acute effects of Perfluorooctanesulfonate, Potassium Salt (hereafter referred to as PFOS) on earthworms during a 14-day exposure period in an artificial soil substrate. EXPERIMENTAL DESIGN Earthworms (Eisenia fetida) were exposed to a geometric series of five concentrations o f PFOS in soil. A control group was maintained concurrently in soil without the addition of PFOS. This route of administration was selected because it was representative of the natural exposure of earthworms to chemicals. Four replicate test chambers were maintained in each treatment and control group, with 10 worms in each test chamber. Observations of mortality and clinical signs were conducted twice within the first four hours of test initiation, and then on Day 7 and Day 14. The mortality results were analyzed with the moving average method to determine an LC50 value. At Wildlife International, Ltd., reference toxicity tests with a reference toxicant, chloroacetamide, are conducted periodically to assess the sensitivity of the test species and test procedures. These studies are conducted under separate protocols, as independent studies. A summary of the results from the most current reference toxicity test is presented in this report. Wildlife International, Ltd. -9 - Project Number 454-111 MATERIALS AND METHODS The study was conducted based upon the procedures outlined in the protocol, "PFOS: An Acute Toxicity Study with the Earthworm in an Artificial Soil Substrate." The protocol was based upon procedures outlined in the Organization for Economic Cooperation and Development (OECD) Guideline No. 207, Guideline fo r Testing o f Chemicals, Earthworm, Acute Toxicity Tests (1). Changes to the protocol are listed in Appendix 2. Test Substance The test substance used was received from 3M on October 29, 1998 and assigned Wildlife International, Ltd. identification number 4675. The test substance was a white powder identified by the Sponsor as FC-95, lot 217. Information from the Sponsor indicated the purity o f the test substance to be 86.9% with an expiration date of August 31, 2006. The test substance was stored under ambient conditions. Test Organism Adult earthworms (Eisenia ftida) were obtained from Worm Man's Worm Farm, Monroe Township, New Jersey. Approximately 18.5 hours prior to the test, 280 worms were selected and placed in a container of prepared artificial soil substrate adjusted to moisture content of approximately 33% by weight, for the acclimation period. On the day of test initiation, the worms were rinsed briefly with deionized water and indiscriminately distributed by pairs into groups of 10 worms each. Each group of worms was weighed then placed on the soil surface in the appropriate test chamber. The worms were not fed during testing. Artificial Soil and Test Soils Preparation The artificial soil was prepared in bulk by blending 70% sand, 20% kaolin clay and 10% sphagnum peat (Appendix 3). The pH of the bulk soil prior to hydration was adjusted to 6.0 using calcium carbonate. The bulk artificial soil was stored in a sealed container under ambient conditions until used to prepare the test soils. W ildlife International, Ltd. Project Number 454-111 - 10- Test soils were prepared by stirring the test substance into a portion of bulk soil before mixing it with the remainder of the soil and deionized water (Appendix 3). Sufficient deionized water was added to the dry artificial soil to achieve a moisture content of approximately 33% by weight. Test soil components were mixed for a total of 20 minutes to ensure a homogeneous mix. Negative control soil was prepared in the same manner as test soil, but without the addition of test substance. Seven hundred fifty grams of prepared soil were added to each of four test chambers for the control group, with the exception of the 625 mg a.i./ kg concentration in which no more than 650 g would fit into the test chambers. Test concentrations were adjusted for the purity of PFOS, which was reported to be 86.9% by the Sponsor. Therefore, test concentrations and the LC50 values are reported as milligrams o f test substance active ingredient per kilogram of soil on a dry weight basis (mg a.i./kg). Test soils were prepared and held overnight at approximately 20 2C prior to adding worms to allow the soil to become less fluid. The target nominal concentrations were 78.1, 156, 313, 625, and 1250 mg a.i./kg dry soil. Mean measured concentrations at the time of soil preparation were 77.0, 141, 289, 488, and 1042 mg a.i./kg soil. Analytical Sampling During the test, samples of the experimental soils were collected to determine the homogeneity and stability of the test substance in the artificial soil and to verify test concentrations. Samples were collected at soil preparation to determine homogeneity, measure test concentrations, and establish Day 0 values for evaluating stability over the course of the exposure period. On Day 14, samples were collected from one replicate of the control and two replicates of each test concentration to assess stability of the test substance under test conditions. At test termination samples of tissue from the earthworms in each treatment group were analyzed to determine the concentration of PFOS in the worm tissue from each treatment group. All surviving worms from each treatment group were placed on moistened filter paper for approximately 24 hours to purge gut contents. The entire mass of worms collected from each treatment group was composited, euthanized by freezing and stored frozen until analyzed. Wildlife International, Ltd. - 11 - Project Number 454-111 Analytical Testing Chemical analysis o f the soil and worm samples was performed using HPLC/MS/MS methodology (Appendix 4). Test Chambers The test chambers were one-liter Nalgene beakers covered with plastic wrap which was perforated for air exchange. All test chambers were identified with the project number, test concentration and replicate. Physical Properties of Test Soils Soil temperature was measured in one replicate of each treatment and control group at test initiation and termination using a hand-held thermometer. Moisture content and pH measurements were made on soil samples collected from each batch of soil prepared for treatment and control groups at test initiation. An additional soil moisture content measurement was made after test initiation to verify the soil moisture. At test termination, samples for moisture content and pH measurements were collected from one replicate of each control and treatment group. Measurements o f pH were made on suspensions of soil and deionized water using a waterproof hand-held pH meter, and are reported as pHwvalues. Soil moisture content was determined by measuring the initial weight of the soil sample, then weighing the soil sample after drying for at least 24 hours at approximately 105C. The percent moisture was calculated using the following formula: % Moisture = [(wet weight - dry weight) -*wet weight] * 100 Environmental Conditions During the test, the worms were maintained in an environmental chamber set to maintain a temperature of approximately 20 2C. Air temperature was measured at least once daily in the environmental chamber. The photoperiod during the test was 24 hours of continuous light per day provided by overhead fluorescent bulbs. The target light intensity dining the test was approximately 400 to 800 lux, and was verified on Day 7 of the test. Wildlife International, Ltd. Project Number 454-111 - 12- Observations At test initiation, the worms were placed on the surface of the soil in each test chamber and were observed for burrowing behavior. On Day 7 and Day 14 of the test, the contents of each test chamber were removed to determine the number of surviving worms. All surviving worms were observed for behavioral or pathological abnormalities. On Day 7, following observations, test soil was returned to the test chambers and the worms were placed on the soil surface in order to observe burrowing behavior. On Day 14, following observations and body weight determination, surviving worms were placed on moist filter paper in clean test chambers and allowed to purge their gut contents for approximately 24 hours. The worms were then euthanized by freezing and held prior to analytical testing. Body Weights On Day 0, group weights for all replicates were collected prior to being placed in the test chambers. On Day 14, all surviving worms were removed from each Teplicate test chamber and weighed. Prior to weighing, worms were rinsed with deionized water and blotted dry on paper towels. Group body weights were measured from each test chamber, and average individual body weights were calculated. Reference Toxicity Test A reference toxicity test was conducted under a separate protocol to determine the LC50 value for earthworms exposed to the reference toxicant, chloroacetamide, in the soil (2). The test was conducted under conditions similar to those used in this test, and with earthworms from the same source, to monitor the techniques used and sensitivity of the test population. The worms were exposed to chloroacetamide in the soil at nominal concentrations of 7.5, 15, 30 and 60 mg a.i./kg dry soil. Statistical Analyses The LC50 value and 95% confidence interval were calculated using the computer program of C.E. Stephan (3). The program was designed to calculate the LC50 value and the 95% confidence interval by probit analysis, moving average method or the binomial probability method (4,5,6). In this study, the 7-day LC50 value was calculated using the binomial probability method and the 14-Day W ildlife International, Ltd. Project Number 454-111 - 13 - LC50 value was calculated using the moving average method. Mean body weights at test initiation and test termination as well as the mean change in body weight during the test were determined for each treatment and control group. Treatment group means were compared to control means with Dunnett's test (a=0.05) using SAS Version 8 (7) in order to determine if significant differences from the control occurred. RESULTS AND DISCUSSION Environmental Conditions Air temperature in the environmental chamber was within the desired range of approximately 20 2C, remaining at 22-23C throughout the test. The worms were maintained under continuous lighting at an average intensity of 627 83.9 lux, with a range over the surface of the test chambers of 494-775 lux. Physical Properties of Test Soils Measurements o f soil pHw, temperature and moisture content at test initiation and test termination are presented in Table 1. Soil pHwranged from 6.9 to 8.1 at test initiation and from 8.3 to 8.4 at test termination. Soil temperature was 22C, within the desired range of 20 2C at test initiation and at test termination. At test termination the 1042 mg a.i./kg Replicate A soil temperature was 25C. It was suspected that the Replicate A chamber was placed too close to a light ballast and was not indicative of the soil temperature for that treatment group. Replicate B was measured and found to be 22C. Soil moisture content during the test ranged from 29.5 to 33.9% at test initiation and from 31.4 to 34.5% at test termination, indicating that there was little change in soil moisture content dining the test. Observations The data from weekly observations of the worms for mortality and other signs of toxicity are presented in Table 2. There were no mortalities in the negative control group during the 14-day test. All control worms were normal in appearance and behavior throughout the test period. On Day 7 there was high mortality in the 488 and 1042 mg a.i./kg treatment groups. On Day 14 there was some Wildlife International, Ltd. Project Number 454-111 - 14- mortality in the 141 and 289 mg a.i./kg treatment groups and very high mortality in the 488 mg a.i./kg group and complete mortality in the 1042 mg a.i./kg group. Worms in the 488 and 1042 mg a.i./kg treatment groups exhibited an aversion to the soil during observations of burrowing behavior on Day 0. On day 0 after one hour, some worms in the 488 mg a.i./kg group were not burrowed, and in the 1042 mg a.i./kg group many remained on the soil surface. After two hours, all but one worm in the 488 mg a.i./kg group had burrowed but in the 1042 mg a.i./kg group most worms remained out of the soil. On Day 7, all surviving worms burrowed into the soil within 15 minutes. On Day 7, worms found dead in the 1042 mg a.i./kg group were on the side of the test chamber not in the soil. Statistical Analyses A 7-Day LC50 estimation of 398 mg a.i./kg with 95% confidence limits of 289 and 488 mg a.i./kg soil was determined using the binomial probability method, since the data did not fit either the probit method or moving average method. A 14-Day LC50 was determined using the moving average method since the data did not fit the probit method. The moving average method was acceptable except for the indication that the confidence limits may be too close due to the lower mortality in the 289 mg a.i./kg soil than in the 141 mg a.i./kg soil treatment group. Therefore, the 14-day LC50 estimation for earthworms exposed to PFOS in an artificial soil substrate was determined to be 373 mg a.i./kg soil with 95% confidence limits of 316 and 440 mg a.i./kg. After seven days of exposure the no-observed-effect-concentration, NOEC, was 289 mg a.i./kg soil and the lowest-observed-effect-concentration, LOEC, was 488 mg a.i./kg soil. The 14-Day NOEC was 77 mg a.i./kg soil and the LOEC was 141 mg a.i./kg soil. Average individual body weights, and the change in body weight from test initiation to test termination, were calculated from the Day 0 and Day 14 replicate measurements (Table 3). A slight loss in body weight from test initiation to test termination was expected in both the control and treatment groups since the worms were not fed during the test. Mean body weight at test termination Wildlife International, Ltd. Project Number 454-111 - 15- as well as the mean loss in body weight among worms in the treatment groups were statistically significant (p<0.05) at 488 mg a.i./kg dry soil compared to the control group. Table 4 contains the 14 day measured values for PFOS in earthworm tissue. Reference Toxicity Test The 14-day LC50 value for earthworms exposed to the reference substance, chloroacetamide, in an artificial soil substrate was approximately 19.4 mg a.i./kg dry soil with a 95% confidence interval of 15 and 30 mg a.i./kg dry soil (2). These results are consistent with those observed in previous studies, and verify the adequacy and consistency of the methods used in this study (454-111). Sample Analysis Chemical analysis of test soils and earthworm tissue was conducted at Wildlife International, Ltd. and is reported in Appendix 4. CONCLUSIONS The 7-day LC50 estimation for earthworms exposed to PFOS in an artificial soil substrate was determined to be 398 mg a.i./kg. The 7-day NOEC was determined to be 289 mg a.i./kg and the 7-day LOEC was determined to be 488 mg a.i./kg. The 14-day LC50 estimation for earthworms exposed to PFOS in an artificial soil substrate was determined to be 373 mg a.i./kg. The 14-day NOEC was determined to be 77 mg a.i./kg and the LOEC was 141 mg a.i./kg. Wildlife International, Ltd. - 16REFERENCES Project Number 454-111 1 OECD Guideline 207. 1984. Guideline for Testing o f Chemicals, Earthworm, Acute Toxicity Tests. Organization for Economic Cooperation and Development. 2 Wildlife International, Ltd. 2000. Chloroacetamide: An Acute Toxicity Study with the Earthworm in an Artificial Soil Substrate. Project No. 100R-105. 3 Stephan, C.E. 1978. U S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal Communication. 4 Finney, D.J. 1971. Statistical Methods in Biological Assay. Second Edition, Griffin Press, London. 5 Thompson, W.R. 1947. Bacteriological Reviews. Vol. II, 2:115-145. 6 Stephan, C.E. 1977. Methods for Calculating an LC50. Aquatic Toxicology and Hazard Evaluations. American Society for Testing and Materials. Pub. No. STP 634:65-84.7 7 SAS Institute, Inc. 1999. SAS/STAT User's Guide, Version 8. Cary, NC, SAS Institute, Inc. Wildlife International, Ltd. Project Number 454-111 - 17Table 1 Moisture, pHw, and Temperature of Test Soils' Mean Measured Concentration (mg a.i./kg) Negative Control Test Initiation Moisture Content2 (%) 33.9 Test Initiation pHw3 Test Initiation Temperature3 (C) Test Termination Moisture Content (%) Test Termination pHw Test Termination Temperature (C) 6.9 22 32.1 8.3 22 77.0 31.9 7.9 22 31.4 8.4 22 141 29.5 8.1 22 31.5 8.3 22 289 31.4 8.1 22 33.0 8.3 22 488 31.0 8.1 22 32.3 8.3 22 1042 32.5 8.0 22 34.5 'Measurements taken from one replicate test chamber at each concentration. Measurements taken from prepared soil two days after preparation. Measurements taken from prepared soil on Day 0. Measurement of soil temperature in Replicate B was 22C. 8.3 254 W ildlife International, Ltd. Project Number 454-111 -18- Table 2 Cumulative Mortality and Observations of Earthworms Exposed to PFOS in an Artificial Soil Substrate Mean Measured Concentration (mg a.i./kg) Negative Control Day 7 Number Rep. Dead1 Effects2 A 0/10 B 0/10 C 0/10 D 0/10 10 AN 10 AN 10 AN 10 AN Day 14 Number Dead Effects 0/10 10 AN 0/10 10 AN 0/10 10 AN 0/10 10 AN Replicate ~ Percent Mortality 0 0 0 0 Group Percent Mortality 0 77.0 A 0/10 B 0/10 10 AN 10 AN 0/10 10 AN 0/10 10 AN C 0/10 10 AN 0/10 10 AN D 0/10 10 AN 0/10 10 AN 0 0 0 0 141 A 0/10 B 0/10 10 AN 10 AN 2/10 8 AN 0/10 10 AN 20 0 C 0/10 9 AN, 1T+S 1/10 9 AN 10 D 0/10 10 AN 0/10 10 AN 0 0 7.5 289 A 0/10 B 0/10 10 AN 10 AN 0/10 10 AN 0/10 10 AN 0 0 2.5 C 0/10 10 AN 0/10 8 AN, 2 R 0 D 0/10 10 AN 1/10 9 AN 10 488 9/10 1 AN 8/10 2 AN 10/10 8/10 -- 2T+R 100 80 95 C 10/10 -- 10/10 -- 100 D 9/10 IA N 10/10 -- 100 1042 A 10/10 B 10/10 -- -- C 10/10 -- D 10/10 -- 10/10 10/10 10/10 10/10 -- ---- -- 100 100 100 100 100 1 Mortality data are presented as the cumulative number dead per number exposed. 2 Observed Effects: AN = normal in appearance and behavior, T=thin, S=soft, R=reduced reaction to mechanical stimuli W ildlife International, Ltd. Project Number 454-111 - 19- Table 3 Average Body Weights of Earthworms Exposed to PFOS in an Artificial Soil Substrate Mean Measured Concentration (mg a.i./kg) Negative Control Replicate A B C D Mean Std. Dev. Average Earthworm Body Weights (g) Day 0 0.29 0.23 0.23 0.26 0.25 0.029 Day 14 0.21 0.19 0.18 0.20 0.20 0.013 Total Change -0.08 -0.04 -0.05 -0.06 -0.06 0.017 77.0 A 0.25 0.20 -0.05 B 0.25 0.19 -0.06 C 0.26 0.20 -0.06 D 0.25 0.18 -0.07 Mean Std. Dev. 0.25 0.005 0.19 0.010 -0.06 .0.008 141 A 0.26 0.21 -0.05 B 0.27 0.17 -0.10 C 0.23 0.14 -0.09 D 0.26 0.20 -0.06 Mean Std. Dev. 0.26 0.017 0.18 0.031 -0.07 .0.024 289 A 0.27 0.17 -0.10 B 0.25 0.17 -0.08 C 0.23 0.16 -0.07 D 0.25 0.18 -0.07 Mean Std. Dev. 0.25 0.016 0.17 0.007 -0.08 0.014 488 A 0.24 _ _ B 0.25 0.08 -0.17 C 0.23 -- -- D 0.24 -- -- Mean Std. Dev. 0.24 0.008 0.08--1 -0.17 --2 1042 A 0.25 B 0.25 -- C 0.24 -- D 0.25 -- Mean Std. Dev. 0.25 0.005 -- -- -- -- -- -- -- 1Body weight was statistically different from (p<0.05) the control groups using Dunnett's test. 2Change in body weight was statistically significant (p<0.05) at test concentration when compared to the control groups using Dunnett's test. W ildlife International, Ltd. Project Number 454-111 -20- Table4 Day 14 Measured Values for PFOS in Earthworm Tissue Number (454-111-) Sample Nominal Exposure PFOS Concentration (mg a.i./kg) Measured1 Mean Measured (mg a.i./kg) W -l 0.0 < LOQ2 - W-2A W-2B 78.1 78.1 189 195 201 W-3A W-3B 156 156 209 203 196 W-4A W-4B W-5 313 313 625 240 263 1105 252 .. 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. JThe method limit o f quantitation (LOQ) in earthworm tissue was 50.0 mg a.i./kg on a wet-weight basis and was calculated as the product ofthe lowest calibration standard (0.000500 mg a.i./L) and the weight/volume dilution factor of the matrix blank sample (100,000 L/kg). W ildlife International, Ltd. Project Number 454-111 -21 - Appendix 1 Personnel Involved in the Study The following key Wildlife International, Ltd. personnel were involved in the conduct or management of this study:1234 1. Henry 0 . Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. John R. Porch, Supervisor, Non-Target Plants and Insects 3. Anne B. Sindermann, Senior Biologist 4. Raymond L. Van Hoven, Ph.D., Scientist, Analytical Chemistry W ildlife International, Ltd. Project Number 454-111 -22Appendix 2 Changes to the Study Protocol This study was conducted according to the approved study protocol with the following changes:1234567 1. Test soils were prepared, and analytical samples collected, one day prior to test initiation. 2. An additional soil moisture measurement was made. 3. Soil moisture for the 156 mg a.i./kg level was 29.5%, slightly below approximately 33%. 4. Test soil for the 625 mg a.i./kg level weighed 650 g rather than 750 g. 5. Earthworms used for the study weighed less than between 300 and 600 mg. 6. Earthworms were conditioned for approximately 18.5 hours rather than 24. 7. The soil temperature in Replicate A of the 1250 mg a.i./kg treatment group was 25C at test termination, exceeding the 202C range specified in the protocol. Wildlife International, Ltd. Project Number 454-111 -23 - Appendix 3 Artificial Soil and Test Soils Preparation Artificial Soil Preparation The artificial soil was prepared in bulk by mixing the following constituents in a soil mixer for approximately 20 minutes: quartz sand 31.500 kg kaolin clay 9.000kg sphagnum peat 4.500 kg calcium carbonate 1.125 kg Test Soil Preparation A total of approximately 4 kg (wet weight) of test soil with a calculated soil moisture content of approximately 33% was prepared for the negative control and each of the treatment groups. Test substance concentrations were based on the dry weight portion of the soil. The calculated dry weight of 4 kg of prepared soil, with a moisture content o f 33%, was 2.680 kg. To account for the 4% moisture content typically present in the bulk soil stored in ambient conditions, 1208 mL of water were added to 2680 g. of bulk soil minus the weight of test substance. Amounts of test substance, bulk soil and water were as follows: Concentration (mg a.i./kR test soil) Negative control 78.1 156 313 625 1250 N ominal W eights and Volumes to P re p a re T est Soils Required Amount of Actual Amount of Test Substance (g) Test Substance (g) ---- 0.241 0.2410 Bulk Soil (g) 2680.0 2679.8 0.482 0.4819 2679.5 0.965 0.9650 2679.0 1.927 1.9270 2678.1 3.854 3.8541 2676.2 . Water (mL) 1208 1208 1208 1208 1208 1208 The appropriate amount of bulk soil was weighed into a tared Hobart mixer bowl. Approximately a handful of dry soil was removed to a Nalgene beaker and the measured amount of test substance added and mixed in with a metal spatula, after using dry soil to clean test substance off Wildlife International, Ltd. Project Number 454-111 -24- of the weigh boat. The soil and test substance mixture was added to the Hobart bowl. 1208 mL of deionized water was added and the soil mixed for 5 minutes. After the 5-minute mix, the sides of the bowl were scraped and the soil was mixed for an additional 15 minutes. Prepared test soil, 750 g for each replicate, except for the 625 mg a.i./kg concentration, which received 650 g, was placed into test chambers labeled with concentration and replicate. Negative control soil was prepared in the same manner, without the addition of test substance, and 750 g was placed into each negative control test chamber. W ildlife International, Ltd. Project Number 454-111 -25Appendix 4 THE ANALYSIS OF PFOS IN ARTIFICIAL SOIL AND EARTHWORM TISSUE IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454-111 W ildlife International, Ltd. Project Number 454-111 SPONSOR: 3M -26REPORT APPROVAL TITLE: PFOS: AN ACUTE TOXICITY STUDY WITH THE EARTHWORM IN AN ARTIFICIAL SOIL SUBSTRATE WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454-111 3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723 PRINCIPAL INVESTIGATOR: MANAGEMENT: Willard B. Nixon, Ph. Director, Analytical Chemistry 5 - 10- 0 ? DATE W ildlife International, Ltd. Project Number 454-111 -27- Introduction This study was conducted by Wildlife International, Ltd. for 3M at the Wildlife International, Ltd. analytical chemistry facility in Easton, Maryland. Samples were collected for the determination of Perfluorooctanesulfonate, Potassium Salt (PFOS) in artificial soil and earthworm tissue and were analyzed using high performance liquid chromatography with triple quadrupole mass spectrometric detection (HPLC/MS/MS). Samples were prepared and analyzed between November 27 and 28, 2001 and December 13, 2001. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454-111 in archives located on the Wildlife International, Ltd. site. Analytical Methodology Artificial Soil Submitted artificial soil samples were analyzed for PFOS according to the validated procedures documented in WIL study number 454C-120. The entire submitted soil sample was blended for approximately two minutes prior to sub-sampling the requisite 10-g aliquot for the analytical determination. The artificial soil sub-samples were extracted with methanol, agitated for a minimum of 30 minutes on a gyratory shaker table at approximately 250 rpm and then vacuum filtered with qualitative filter paper. The filtrate was then transferred to a 200-mL volumetric flask and brought to volume with methanol. Approximately 20 milliliters of each sample was transferred to a separate vial and centrifuged for approximately 5 minutes at 2000 rpm. Dilutions into the calibration range of the HPLC/MS/MS methodology were performed with a solution of 50% methanol (HPLC grade, 99.9+%) and 50% NANOpure water. Samples were then analyzed by direct injection. Concentrations of PFOS were determined by reverse-phase high performance liquid chromatography using a HewlettPackard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 3000LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separations were achieved using a Keystone Betasil C]8column (50 mm x 2.0 mm, 3 pm particle size) fitted with a Keystone Javelin C)8 guard column (20 mm x 2.0 mm). A method flowchart is provided in Figure 1 and the instrument parameters are summarized in Table 1. W ildlife International, Ltd. Project Number 454-111 Earthworm Tissue -28 - Submitted earthworm tissue samples were analyzed for PFOS according to methodology developed at Wildlife International, Ltd. Approximately one gram o f each earthworm tissue sample was weighed directly into tared scintillation vials to which 10.0 milliliters o f methanol extraction solvent were added. The samples were concurrently extracted and homogenized using a tissue shredder (Janke and Kunkel) followed by sonic dismembration. The sample extracts were centrifuged and diluted into the calibration range of the HPLC/MS/MS methodology. Dilutions were performed with a solution of 50% methanol (HPLC grade, 99.9+%) and 50% NANOpure water. Samples were then analyzed by direct injection HPLC/MS/MS as described previously. A method flowchart is provided in Figure 2. Primary and Secondary Stock Solutions All primary and secondary stock preparations were adjusted for the purity of the test substance (86.9%). A 10.0 mg a.i./mL primary stock solution of PFOS in methanol was prepared by weighing 1.1508 g of the test substance and bringing to a final volume of 100 milliliters with methanol. Secondary stock solutions (1000, 100, 10.0, 1.00, and 0.100 mg a.i./L) of PFOS in methanol were prepared by serial volumetric dilution from the primary stock. Calibration Standards and Calibration Curves Calibration standards were prepared in 50:50 methanol: NANOpure water by appropriate dilutions of the 1.00 mg a.i./L stock solutions of PFOS in methanol. Calibration standards of PFOS, ranging in concentration from 1.00 to 10.0 pg a.i./L, were analyzed with each artificial soil sample set and calibration standards of PFOS, ranging in concentration from 0.500 to 5.00 pg a.i./L, were analyzed with the earthworm tissue sample set. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration and study samples. No attempt was made to quantify PFOS on the basis of individual isomeric components. 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 responses versus the respective concentrations of the calibration standards. A typical calibration curve for PFOS in artificial soil and earthworm tissue is presented in Figure 3 and Figure 4, respectively. Representative ion Wildlife International, Ltd. Project Number 454-111 -29- chromatograms of low and high calibration standards for the artificial soil are presented in Figures 5 and 6, respectively and representative ion chromatograms of low and high calibration standards for PFOS in earthworm tissue are presented in Figure 7 and 8, respectively. The concentrations o f PFOS in the samples were determined by substituting peak area responses into the applicable linear regression equation. Concentrations of PFOS in the samples were calculated on a dry weight (soil samples) or wet weight (earthworm tissue) basis using the at-instrument PFOS concentrations and the appropriate weight/volume dilution and/or moisture correction factors. Matrix Blanks and Matrix Fortifications Artificial Soil Selected 20.0-g aliquots of artificial soil were fortified with the appropriate stock solutions of PFOS prepared in methanol using a gas-tight syringe. The fortified soils were then homogenized using a Waring blender for approximately 2 minutes. A 10-g aliquot of each homogenized fortified soil sample was weighed into a tared weigh boat and transferred to an 8-oz. French-square bottle for extraction. The matrix blanks were unfortified artificial soil. Earthworm Tissue A 1.00-g aliquot of negative control earthworm tissue was fortified with the 1000 mg a.i./L secondary stock solution of PFOS prepared in methanol using a gas-tight syringe. A matrix blanks was prepared with unfortified negative control earthworm tissue. Limits of Quantitation Artificial Soil The method limit of quantitation (LOQ) in artificial soil was 29.3 mg a.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.00100 mg a.i./L), the weight/volume dilution factor of the matrix blank samples (20,000) divided by the percent solids for the test system soil (68.3%). W ildlife International, Ltd. Project Number 454-111 -30- Earthworm Tissue The method limit of quantitation (LOQ) in earthworm tissue was 50.0 mg a.i./kg on a wet-weight basis and was calculated as the product of the lowest calibration standard (0.000500 mg a.i./L) and the weight/volume dilution factor of the matrix blank sample (100,000 L/kg). Example Calculations Example Calculations Sample number 454-111-5, nominal concentration of 78.1 mg a.i./kg in artificial soil. First Initial Weight: 10.0 g First Final Volume: 200 mL Second Initial Volume: 0.0500 mL Second Final Volume: 50.0 mL Dilution Factor: 20000 Mean Soil Moisture Content: 31.7% Percent Solids: 68.3% LCMS Dilution Factor: 20000/0.683 = 29283 PFOS Peak Area: 23914 Calibration curve equation: Slope: 8981195 Intercept: 1011.0532 peak area - (y-intercept) PFOS (mg a.i./kg) measured at instrument = slope PFOS (mg a.i./kg) in sample = PFOS measured at instrument (mg a.i./kg) x LCMS dilution factor 23914- 1011.0532 8981195 x 29283 = 74.67 PFOS (mg a.i./Kg) in sample Percent of Nominal Concentration = PFOS (mg a.i./Kg) nominal x = 74.67 x 100 = 95.6% 78.1 Calculated with HPLC/MS/MS instrument software: MacQuan, version 1.6. W ildlife International, Ltd. Project Number 454-111 -31 - RESULTS Matrix blank samples for each matrix type were analyzed to determine possible interferences. No interferences were observed at or above the corresponding LOQ (Tables 2 and 3). Representative ion chromatograms of artificial soil and earthworm tissue matrix blank samples are presented in Figures 9 and 10, respectively. Matrix fortification samples for artificial soil had recoveries ranging from 75.6% to 103.2% of the nominal concentrations with a mean recovery of 87.3% of nominal (SD=11.2; CV=12.8). A representative ion chromatogram of an artificial soil matrix fortification is presented in Figure 11. The earthworm tissue matrix fortification (Figure 12) had a recovery o f 86.6% of the nominal concentration. Artificial soil samples collected at Day 0 from the 78.1 mg a.i./kg, 156 mg a.i./kg, 313 mg a.i./kg, 625 mg a.i./kg and 1250 mg a.i./kg treatment levels had mean measured PFOS concentrations o f 77.0 mg a.i./kg (SD=13.6; CV=17.7%, n=6), 141 mg a.i./kg (n=2), 289 mg a.i./kg (n=2), 488 mg a.i./kg (n=2), and 1042 mg a.i./kg (SD=201; CV=19.3%, n=6), respectively (Table 4). These values corresponded to mean percent recoveries of 98.6%, 90.4%, 92.3%, 78.1% and 83.4% of the nominal concentrations with an overall mean percent recovery of 89.6% (SD=15.3; CV=17.1%, n=18). A representative ion chromatogram of an artificial soil test sample is presented in Figure 13. Artificial soil samples collected at Day 14 from the 78.1 mg a.i./kg, 156 mg a.i./kg, 313 mg a.i./kg, 625 mg a.i./kg and 1250 mg a.i./kg treatment levels had mean measured PFOS concentrations o f 80.6 mg a.i./kg (n=2), 143 mg a.i./kg (n=2), 305 mg a.i./kg (n=2), 634 mg a.i./kg (n=2) and 1094 mg a.i./kg (n=2), respectively (Table 5). These values corresponded to mean percent recoveries of 103%, 91.7%, 97.4%, 101% and 87.5% of the nominal concentrations with an overall mean percent recovery of 96.3% (SD=10.7; CV=11.2%, n=10). Earthworm tissue analyzed on Day 14 had mean measured concentrations of 195, 203 and 252 mg a.i./kg for nominal exposure levels of 78.1, 156, and 313 mg a.i./kg, respectively (Table 6). The measured value (1105 mg a.i./kg) for the 625 mg a.i./kg exposure level was suspect due to the limited sample size (3 worms, approximately 0.2 g total weight) available from this treatment group. A representative ion chromatogram of an earthworm tissue test sample is presented in Figure 14. W ildlife International, Ltd. Project Number 454-111 -32REFERENCES 1. American Society for Testing and Materials. 1991. Standard Specification for Reagent Water. D 1193-91, ASTM Section II Water and Environmental Technology, Vol. 11.01:45-47. Wildlife International, Ltd. Project Number 454-111 INSTRUMENT: 33- - Table 1 Typical HPLC/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in multiple ion reaction monitoring (MRM) mode. ION SOURCE: Perkin-Elmer TurboIonSpray ANALYTICAL COLUMN: Keystone Betasil Ci8(50 mm x 2.0 mm, 3-pm particle size) GUARD COLUMN: Keystone Javelin Cis cartridge (20 mm x 2 mm) OVEN TEMPERATURE: 40C STOP TIME: 5.00 min FLOWRATE: MOBILE PHASE: 250 pL/min 30% NANOpure Water with 0.1% Formic Acid: 70% Methanol INJECTION VOLUME: 5.0 pL PFOS PEAK RETENTION TIME: Approximately 4 minutes PFOS MONITORED MASS: 499.0 amu 99.1 amu W ildlife International, Ltd. Project Number 454-111 - 34Table 2 Artificial Soil Matrix Blanks and Fortifications Analyzed Concurrently During Sample Analysis Number (454-111-) Sample Type PFOS Concentration (mg a.i./kg) Fortified Measured1 Percent Recovered1 Mean Measured (mg a.i./kg) Mean Percent Recovery M A B -1 Soil M atrix Blank 0.0 < LOQ2 M A B -2 Soil M atrix Blank < LOQ2 -- -- -- M AS- 1 M A S- 4 Soil Matrix Fortification Soil M atrix Fortification 36.6 28.5 29.3 77.7 80.1 28.9 79.0 M AS- 2 M A S- 5 M A S- 3 M A S- 6 Soil M atrix Fortification Soil M atrix Fortification Soil M atrix Fortification Soil M atrix Fortification 586 1464 565 533 1503 1106 96.4 90.9 103 75.6 549 1305 93.7 89.1 Overall Mean = 87.3 Standard Deviation = 11.2 CV = 12.8 N= 6 1Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2The method limit of quantitation (LOQ) in artificial soil was 29.3 mg a.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.00100 mg a.i./L), the weight/volume dilution factor of the matrix blank samples (20,000) divided by the percent solids for the test system soil (68.3%). Wildlife International, Ltd. Project Number 454-111 -35 - Table 3 Earthworm Tissue Matrix Blank and Fortification Analyzed Concurrently During Sample Analysis Number (454-111-) Sample Type PFOS Concentration (mg a.i./kg) Fortified Measured1 Percent Recovered1 WMAB - 1 Worm Tissue M atrix 0.0 < LOQ2 Blank - W M AS-1 Worm Tissue M atrix Fortification 100 86.6 86.6 'Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2The method limit of quantitation (LOQ) in earthworm tissue was 50.0 mg a.i./kg on a wetweight basis and was calculated as the product o f the lowest calibration standard (0.000500 mg a.i./L) and the weight/volume dilution factor of the matrix blank sample (100,000 L/kg). W ildlife International, Ltd. Project Number 454-111 -36- Table 4 Day 0 Recoveries for PFOS in Artificial Soil Sample Number ;454-ll 1-) Nominal Concentration PFOS Concentration (mg a.i./kg) Measured1 Percent Recovered1 Mean Measured (mg a.i./kg) Mean Percent Recovery 1 0.0 < LOQ2 - - -- 2 78.1 3 78.1 4 78.1 5 78.1 6 78.1 7 78.1 74.6 72.7 104 74.7 69.4 66.5 95.5 x =77.0 98.6 93.1 SD= 13.6 133 CV= 17.7% 95.6 88.9 85.1 8 156 9 156 144 92.1 141 90.4 138 88.4 10 313 11 313 312 99.6 289 92.3 266 85.0 12 ' 13 625 625 526 84.1 488 78.1 449 71.9 14 1250 15 1250 16 1250 17 1250 18 1250 19 1250 1152 906 894 930 1405 966 92.1 72.4 71.5 74.4 112 77.3 x = 1042 SD= 201 CV = 19.3% 83.4 Overall Mean = 89.6 Standard Deviation = 15.3 CV = 17.1% N = 18 1Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2The method limit of quantitation (LOQ) in artificial soil was 29.3 mg a.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.00100 mg a.i./L), the weight/volume dilution factor of the matrix blank samples (20,000) divided by the percent solids for the test system soil (68.3%). W ildlife International, Ltd. Project Number 454-111 -37- Table 5 Day 14 Recoveries for PFOS in Artificial Soil Number (454-111-) Sample Nominal Concentration PFOS Concentration (mg a.i./kg) Measured1 Percent Recovered1 Mean Measured (mg a.i./kg) Mean Percent Recovery 20 0.0 21 78.1 22 78.1 23 156 24 156 25 313 26 313 < LOQ2 74.9 86.2 143 142 302 308 - 95.9 110.4 91.4 91.1 96.5 98.3 -- -80.6 103 143 91.7 305 97.4 27 625 28 625 532 85.1 634 101 735 118 29 1250 30 1250 1059 1129 84.7 1094 87.5 90.4 Overall Mean = 96.3 Standard Deviation = 10.7 CV = 11.2% N = 10 1Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2The method limit of quantitation (LOQ) in artificial soil was 29.3 mg a.i./kg on a dry-weight basis and was calculated as the product of the lowest calibration standard (0.00100 mg a.i./L), the weight/volume dilution factor of the matrix blank samples (20,000) divided by the percent solids for the test system soil (68.3%). Wildlife InternationalLtd. Project Number 454-111 - 38- Table 6 Day 14 Measured Values for PFOS in Earthworm Tissue Number (454-111-) Sample Nominal Exposure PFOS Concentration (mg a.i./kg) Measured1 Mean Measured (mg a.i./kg) W -l 0.0 < LOQ2 - W-2A W-2B 78.1 78.1 189 195 201 W-3A W-3B 156 156 209 203 196 W-4A W-4B 313 313 240 252 263 W-5 625 1105 - 1Measured values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2The method limit of quantitation (LOQ) in earthworm tissue was 50.0 mg a.i./kg on a wet-weight basis and was calculated as the product of the lowest calibration standard (0.000500 mg a.i./L) and the weight/volume dilution factor of the matrix blank sample (100,000 L/kg). W ildlife International, Ltd. -39- Project Number 454-111 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN ARTIFICIAL SOIL Weigh approximately 10.0 grams of each sample into a tared weigh boat. Transfer to 8 oz. French square bottles. 4 For each sample, measure 100 mL of methanol with a graduated cylinder and transfer into the French square bottle. i Cap bottles and place on shaker table. Allow the samples to shake for a minimum of 30 minutes at approximately 250 rpm. i Vacuum filter with qualitative filter paper and rinse retained soil 3 times with methanol into filtrate. I Transfer the filtrate into a 200-mL volumetric flask and bring to volume with methanol. Mix well with several repeat inversions. 4 Transfer approximately 20 mL of each sample into a separate glass centrifuge tube or scintillation vial and cap. Centrifuge samples for approximately 5 minutes at 2000 rpm. i Prepare appropriate dilution(s) to bring final concentration into the calibration range of the LCMS methodology. For all dilutions, use 50% methanol: 50% NANOpurewater dilution solvent, gas-tight syringes, and Class A volumetric glassware. Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis of P F O S in artificial soil. W ildlife International, Ltd, -40- Project Number 454-111 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN EARTHWORM TISSUE Weigh approximately 1.00 grams of each sample into a fared scintillation. 4 Add 10.0 mL of methanol to each sample with a glass Class A volumetric pipette. 4 Homogenize each test sample for approximately 1 minute using a hand-held tissue shredder. Rinse the homogenizer with the appropriate solvent(s) in between samples. 4 Sonicate each sample for approximately 5 minutes with a sonic dismembrator. 4 Cap the vials and shake well. Centrifuge the vials at 2000 ipm for approximately 5 minutes. 4 Prepare appropriate dilutions of study and QC samples to within the calibration range of the PFOS LC/MS/MS methodology with 50% NANOpure:50% methanol. 4 Amputate samples and submit for LCMS analysis. Figure 2. Analytical method flowchart for the analysis of PFOS in earthworm tissue. Wildlife International, Ltd. -41 - Area Project Number 454-111 Figure 3. Atypical calibration curve for PFOS (artificial soil). Slope =8981195; Intercept =1011.0532; r =0.994. W ildlife International, Ltd. -42- Area Project Number 454-111 Figure 4. A typical calibration curve for PFOS (earthworm tissue). Slope =23387; Intercept =2337.9114; r =0.999. Wildlife InternationalLtd. -43 - PFOSJ STD 0.00100 mg a.IJL 4675A-01 ID-16 4.S8 in 1 period PFOS No Internal Standard Use Area 1: 4.97 MRM. 298 scans 499.0-99.1 Noise Thres. Quanl Thres. Min. Width Mutt. Width Base. Width RT Win. (secs) Smooth Expected RT 30.0 1.0 3 6 40 20 1 4.04 Area 10695 Height 1 1 4a Start Time End Tene 3.67 4.34 Integration Width Retention Time Integration Type 0.67 4.00 A-BB Thu, Nov 29, 2001 05:52 Project Number 454-111 intensity: 10000 cps Figure 5. A representative ion chromatogram of a low-level (0.00100 mg a.i./L) PFOS standard (artificial soil). W ildlife International, Ltd. -44- PFOS S STD 0.0100 mg a.iJL 4675A-01 ID-20 4.98 in 1 period pros No Internai Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 3 0 .0 Quant Thres. 1.0 Msi. Width 3 Mult. Width 6 Base. Width 40 RT Win. (secs) 20 Smooth 1 Expected RT 4.04 Area 99916 Height 10171 Start Time End Time Integration Width Retention Time Integration Type 3.70 4.37 0.67 3.99 A - VB Thu, Nov 29, 2001 06:16 Project Number 454-111 intensity: 10000 cps Figure 6. A representative ion chromatogram of a high-level (0.0100 mg a.i./L) PFOS standard (artificial soil). W ildlife International, Ltd. -45 - PFOS_1 STD 0.500 ug a.i./L 4675A-0110-6 4.98 in 1 period PROS No Internal Standard Use Area 1: 4.97 MRM. 298 scans 499.0->99.1 Noise Thres. 30.0 Quant Thres. 1.0 Min. Width 3 Mult. Width 6 Base. Width 40 HT Win. (secs) 20 Smooth 1 Expected RT 4.05 Area 13949 Height 1470 Stari Time 3.77 End Time 4.41 Integration Width 0.64 Retention Time 4.05 Integration Type A - BB Fri, Dec 14, 2001 12:43 Project Number 454-111 intensity: 15000 cps Figure 7. A representative ion chromatogram of a low-level (0.000500 mg a.i./L) PFOS standard (earthworm tissue). W ildlife International, Ltd. -46- PFOS.5 STD 5.00 ug a.iJL 4675A-01 ID-10 4.98 in 1 period PFOS No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 30.0 Quant "thres. 1.0 Min. Width 3 Mult. Width 6 Base. Width 40 RT Win. (secs) 20 Smooth 1 Expected RT 4.05 Area 118405 Height 12241 Start Time 3.70 End Time 4.29 Integration Width 0.59 Retention Time 3.94 Integration Type A-VB Fri, Dec 14, 2001 13.07 Project Number 454-111 intensity: 15000 cps Figure 8. A representative ion chromatogram of a high-level (0.00500 mg a.i./L) PFOS standard (earthworm tissue). W ildlife International, Ltd. -47- PFOS 7 454-111- MAB-1 Thu. Nov 29, 2001 06:28 4.98 in 1 period pros No Internai Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 30.0 Quant Thres. 1.0 Min. Width 3 Mult. Width 6 Base. Width 40 RT Win. (secs) 20 Smooth Expected RT 1 4.04 Area 0 Height o Stall Time End Time Integration Width Retention Time Integration Type 0.00 0.00 0.00 0.00 Project Number 454-111 intensity: 10000 cps Figure 9. A representative ion chromatogram of an artificial soil matrix blank sample (454-111-MAB-l). The arrow indicates the approximate retention time of PFOS. W ildlife International, Ltd. -48- PF0S_7 454-111- WMAB-1 Fri, Dec 14, 2001 13:20 4.96 In 1 period PFOS No Internal Standard Use Area 1: 4.97 MRM. 298 scans 499.0->99.1 Noise Thies. 30.0 Quant Thres. 1.0 Min. Width 3 Mult Width 6 Base. Width 40 RT Win. (secs) 20 Smooth 1 Expected RT 4.05 Area 0 Height 0 Start Time 0.00 End Time 0.00 Integration Width 0.00 Retention Time 0.00 Integration Type Project Number 454-111 Intensity: 15000 cps Figure 10. A representative ion chromatogram of an earthworm tissue matrix blank sample (454-111WMAB-1). The arrow indicates the retention time of PFOS. W ildlife International, Ltd. Project Number 454-111 -49- PF0S_9 454-111- MAS-2 Thu, Nov 29. 2001 06:40 4.96 In 1 period FTOS No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. Quant Thres. Min. Width Mult. Width Base. Width RT Win. (secs) 30.0 0.5 3 6 40 20 Smooth 1 Expected RT 4.04 Area 35 671 Height 3641 Start Time End Time 3.69 4.36 Integration Width 0.67 Retention Time Integration Type 4.00 A* - VB IOOi 908070605040 3020to0+ 2I4 4i1 t 72 I r113 14I1 1-6r*5' 41 B1 121 161 0.69 1.36 2.03 2.70 201 3.37 intensity: 10000 cps 239 241 281 Scan 4.04 4.71 Time Figure 11. A representative ion chromatogram of an artificial soil matrix fortification sample (454-111MAS-2, nominal PFOS concentration of 586 mg a.i./kg, dilution factor = 146413x). W ildlife International, Ltd. Project Number 454-111 -50- PFOS_8 454-1 11- WMAS-1 Fri, Oec 14, 2001 13:26 4.9B in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 30.0 Quant Thres. Min. Width 1.0 3 Mult. Width 6 6ase. Width 40 RT Win. (secs) 20 Smooth Expected RT 1 4.05 Area 22590 Height 2367 Start Time 3.70 End Time Integration Width 4.34 0.64 Retention Time Integration Type 3.94 A-VB 10O| 90- 80- 70- 60 50- 40 30- 20 10- o4 1 9 61 79 41 81 0.69 1.36 116 141 176 121 161 2.03 2.70 intensity: 15000 cps 235 .276 281 Scan 4.71 Time Figure 12. A representative ion chromatogram of an earthworm tissue matrix fortification sample (454' 111-WMAS-l, nominal PFOS concentration of 100 mg a.i./kg, dilution factor = lOOOOOx). W ildlife International, Ltd. -51 - PF0S_13 2 Thu, Nov 28. 2001 07:04 454-111- 4.98 In 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 489.0->99.1 Noise Thres. Quant Thres. Min. Width Mult. Width Base. Width RT Wtn. (secs) Smooth 30.0 1.0 3 6 40 20 1 Expected RT 4.04 Area 23BB3 Height 2478 Start Time 3.69 End Time Integration Width 4.36 0.67 Retention Time 3.99 Integration Type A -BB Project Number 454-111 Intensity: 10000 cps Figure 13. A representative ion chromatogram of an artificial soil test sample (454-111-2, nominal PFOS concentration of 78.1 mg a.i./kg, dilution factor = 29283x). W ildlife International, Ltd. Project Number 454-111 - 52- PFOS.10 454-111- W-2A Fri, Dec 14, 2001 13:38 4.98 in 1 period pros No Internal Standard Use Area t: 4.97 MRM. 298 scans 499.0->99.1 Noise Thres. 30.0 Quant Three. 1.0 Min. Width 3 Mull. Width 6 Base. Width 40 RT Win. (secs) 20 Smooth 1 Expected RT 4.05 Area 46647 Height 4953 start Time End Time 3.70 4.37 Integration Width Retention Time Integration Type 0.87 3.94 A-BB 100i 9080 70 6 50 4B 302010o-l intensity: 15000 cps 235 60 91 117 41 81 121 0.69 1.36 2.03 164 161 2.70 207 201 3.37 . 277 281 Scan 4.71 Time Figure 14. A representative ion chromatogram of an earthworm tissue test sample (454-111-W-2A, dilution factor = lOOOOOx).