Document LgV6y60Q5pMj42XmgVrYoNv5X

BACK TO MAIN PFOS: A 7-DAY TOXICITY TEST WITH DUCKWEED (Lemnagibba G3) FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-111 3M LAB REQUEST NO. U2723 U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.4400 AUTHORS : Debbie Desjardins Cary A. Sutherland Ray VanHoven, Ph.D. Henry 0.Krueger, Ph.D STUDY INITIATION DATE: January 28,2000 STUDY COMPLETION DATE: March 26,2001 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55 106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 2 1601 (410) 822-8600 Page 1 of 47 BACK TO MAIN WILDLIFE INTERNATIONAL, LTD. -2- PROJECT NO.: 454A-111 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A 7-Day Toxicity Test with Duckweed (Lemnagibba G3:) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-111 STUDY COMPLETION: March 26,200 1 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); arid Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984 with the following exceptions: The test substance was not characterized in accordance with f i l l GLP compliance; however, the characterization was performed according to 3M Standard Operating Procedures and Methods, and all raw data are being maintained in the 3M archives. The test substance has been recharacterized in accordance with GLP (September 7, 2001). The stability of the test substance under conditions of storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: -- CaKA. Sutherland Laboratory Supervisor - ,/,Lib) DATE SPONSOR: BACK TO MAIN WILDLIFE INTERNATIONALT, D. -3- PROJECT NO.: 454A-111 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of Good Laboratory Practice, (ENV/MC/CHEM (98)17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Test Substance Preparation Matrix Fortifications Counts and Observations Analytical Data and Draft Report Biological Data and Draft Report Analytical Report Second Draft Biological Report Second Draft Final Draft DATE CONDUCTED: March 2,2000 March 3,2000 March 10,2000 May 15 - 16,2000 May 16,2000 March 12,2001 March 14-15,2001 March 23,2001 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: March 2,2000 March 2,2000 March 6, 2000 March 14,2000 March 10,2000 March 16,2000 May 16,2000 May 16,2000 May 16,2000 May 19,2000 March 12,2001 March 19,2001 March 15,2001 March 23,2001 March 23,200 1 March 26,200 1 Kimbdrly A. Ho#ter Quality Assurance Representative 3-db-UI DATE BACK TO MAIN WILDLIFE INTERNATIONAL, LTD. -4- PROJECT NO.: 454A-111 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 7-Day Toxicity Test with Duckweed (Lemna gibba G3) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-1I1 STUDY DIRECTOR: Cary A. Sutherland Laboratory Supervisor MANAGEMENT: Director, Aquatic Toxicology and Non-Target Plants WILDLIFE INTERNATIONLATLD. -5- BACK TO MAIN . PROJECT NO. 454A-111 TABLE OF CONTENTS TitleKover Page ................................................................................................................................. 1 Good Laboratory Practice Compliance Statement ................................................................................ 2 Quality Assurance Statement ............................................................................................................... 3 Report Approval ... ........ ............................... 4 Table of Contents ...................... ............ .............. ...... ... 5 Summary ............................................................................................................................................ 7 Introduction ........................................................................................................................................ 8 ............. .............. .... 8 Experimental Design .............................................................. ......................................................... 8 Materials and Methods ........................................................................................... Results and Discussion...................................................................................................................... 13 Conclusions ...................................................................................................................................... 14 References ........................................................................................................................................ 15 TABLES Table 1 . Summary ofAnalytical Chemistry Data .......................................................................... 16 Table 2 . Temperature Measurements ............................................................................................. 18 Table 3 . pH Measurements ............................................................................................................ 19 Table 4 . Day 7 Plant and Frond numbers. Mean Plant and Frond Numbers. and Percent Inhibition Values ........................................................................................... 20 Table 5 . Mean Percentage of Fronds Observed Dead. Chlorotic or Necrotic Per Treatment .................................................................................................................. 21 Table 6 . IC 10. IC50 and IC90 Values Based on Frond Number Over the 7-Day Exposure Period............................................................................................................... 22 BACK TO MAIN WILDLI FE I NTERNATIONAL, LTD. -6- PROJECT NO.: 454A-11I TABLE OF CONTENTS -Continued- APPENDICES Appendix I - 20X AAP Medium ................................................................................................. 23 Appendix I1 - Analyses of Pesticides, Organics, Metals and Other Inorganics in Wildlife International, Ltd. Well Water................................................................ 24 Appendix 111 - The Analysis of PFOS in 20X AAP Medium in Support of Wildlife International, Ltd. Project No.: 454A-111 ................................................. 25 Appendix IV - Observations by Replicate of Frond and Plant Production, the Numbers of Dead, Chlorotic and Necrotic Fronds, and Other Sublethal Effects....................... 43 Appendix V - Changes to Protocol ................................................................................................ 46 Appendix VI - Personnel Involved in the Study............................................................................... 47 BACK TO MAIN WILDLI FE INTERNATI ONAL, LTD. -7SUMMARY SPONSOR: SPONSOR'S REPRESENTATIVE: l LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: 3M Corporation Rochelle R. Robideau Wildlife International, Ltd. Easton, MD 2 1601 PROJECT NO.: 454A-111 I/ WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: NOMINAL TEST CONCENTRATIONS: MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF EXPOSURE: 454A- 111 PFOS (Perfluorooctanesulfonate, Potassium Salt) IUPAC Name: 1-Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6, 7,7,8,8,8-heptadecafluoro-potassiunsialt; CAS #2795-39-3 PFOS: A 7-Day Toxicity Test with Duckweed (Lernnagibba G3) Negative Control, 11.0, 22.0, 43.9, 87.9, 76 and 351 mg a ./L Negative Control, 7.74, 15.1, 31.9, 62.5, 47 and 230 mg a. ,/L Experimental Start (OECD) - March 2, 2000 Experimental Start (EPA) - March 3, 2000 Exposure Termination - March 10, 2000 Experimental Termination - March 10, 2000 7 Days TEST ORGANISM: SOURCE OF TEST ORGANISMS: Duckweed (Lemna gibba G3) Wildlife International Ltd. Easton, Maryland 2 1601 7-DAY IC50 95% CONFIDENCE LIMITS: NO-OBSERVED-ADVERSEEFFECT-CONCENTRATION: 108 mg a.i./L 45.7 - 144 mg a.i./L 15.1 mg a.i./L BACK TO MAIN WILDLIFE INTERNATIONAL, LTD. -8INTRODUCTION PROJECT NO.: 454A-111 This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase of the test was conducted from March 3, 2000 to March 10,2000. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454A-111 in archives located on the Wildlife International, Ltd. site. OBJECTIVE The objective of the study was to evaluate the toxicity of PFOS (Perfluorooctanesulfonate, Potassium Salt) to duckweed, Lemna gibba G3, during a 7-day exposure period under static test conditions. EXPERIMENTAL DESIGN Fronds of duckweed, Lemna gibba G3, were exposed to a geometric series of six test concentrations and a negative (culture medium) control under static conditions for seven days. Three replicate test chambers were maintained for each treatment and control group. Two additional replicates were also maintained for analytical sampling on Days 3 and 5 of the test. In addition, three "abiotic" replicates (test solution without duckweed plants) were prepared for the highest test concentration. Nominal test concentrations were selected in consultation with the Sponsor and were based upon the results of a range finding test. The nominal test concentrations were 11.0,22.0, 43.9, 87.9, 176 and 351 mg active ingredient (a.i.)/L. Mean measured test concentrations were determined from samples of test medium collected from each treatment and the control group on Days 0, 3, 5 and 7 of the test. Plants that appeared healthy and uniform in size were used for testing. Five plants were added to each replicate test chamber, talung care to ensure that the number of fronds in each test chamber were equal. At the beginning of the test, the total number of fronds in each replicate was 15. Effects upon the duckweed were assessed through direct counts of duckweed frond numbers on Days 3, 5 and 7. Observations of chlorosis, necrosis, break-up of duckweed colonies, root destruction, death and any other BACK TO MAIN WILDLI FE INTERNATIONALL, TD. -9- PROJECT NO.: 454A-111 abnormalities in plant or frond appearance were also performed on those days. Mean frond numbers for each treatment group were used to calculate the lC10, IC50 and IC90 values (Le., the theoretical toxicant concentration that would produce a 10, 50 or 90% reduction in frond number relative to the control) and percent mhibition values relative to the negative control for Days 3, 5 and 7 of the exposure period. The no-observed-adverse-effect-concentration (NOAEC), defined as the highest test concentration that produced no adverse effects upon plant and frond production or appearance, was determined through the evaluation of the statistical results, the concentration-response pattern and other observed effects. MATERIALS AND METHODS The study was conducted based on the procedures outlined in the protocol, "PFOS: A 7-Day Toxicity Test with Duckweed (Lemna gibba G3)". The protocol was based on procedures outlined in the U S . Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines, OPPTS Number 850.4400: Aquatic Plant Toxicity Test Using Lemna spp., Tiers I and II (1). 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 whte powder. It was identified as FC-95 from lot number 217 (T-6295). Information provided by the Sponsor indicated a purity of 98.9% and an expiration date of 2002. The test substance was reanalyzed by the Sponsor and the Certificate of Analysis dated September 7, 2000 indicated a purity of 86.9% and expiration date of August 3 1,200 1. The test substance was stored at ambient room temperature. Preparation of Test Concentrations Nominal test concentrations were 11.0, 22.0, 43.9, 87.9, 176 and 351 mg a.i.L, based on a test substance purity of 86.9%. All materials which came into contact with the test substance during preparation of test concentrations were constructed of plastic or were Teflon@-covered. A primary stock solution was prepared in 20X AAP medium at a concentration of 351 mg a.i./L. The primary stock solution was stirred with a magnetic stir plate for approximately 24 hours to aid in the solubilization of the test substance. After mixing, the primary stock solution was proportionally diluted with 20X AAP medium BACK TO MAIN WILDLIFE INTERNATIONALL,TD. - 10- PROJECT NO.: 454A-11 I to prepare the five additional test concentrations. All final test solutions appeared clear and colorless. Test Organism The duckweed, Lemna gibba G3, was selected as the test species for this study. The species is representative of an important group of aquatic plants, and was selected for use in the test based upon a past history of use, and ease of culturing in the laboratory. The original duckweed cultures were obtained from The United States Department of Agriculture and have been maintained in culture medium at Wildlife International, Ltd., Easton, Maryland. Duckweed plants used in the test were obtained from Wildlife International, Ltd. cultures that had been actively growing in 20X AAP medium for at least two weeks prior to test initiation. Culture Medium Duckweed was cultured and tested in 20X AAP medium (2). Stock nutrient solutions were prepared by adding reagent-grade chemicals to Wildlife International, Ltd. well water purified by reverse osmosis. The test medium was prepared by adding appropriate volumes of the stock nutrient solutions to purified well water (Appendix I). The pH of the medium was adjusted to 7.5 f0.1 using 10% HCl and the medium was sterilized by filtration (0.22 p)prior to use. Analyses are performed at least once annually to determine the concentrations of selected organic and inorganic constituents in the well water used by Wildlife International Ltd. to prepare the medium. The results of the most recent GLP analyses performed to measure the concentrations of selected contaminants in the well water are presented in Appendix 11. Test Apparatus Test chambers were sterile 250-mL plastic beakers covered with disposable petri dish lids, and each contained 100 mL of test or control medium. Beakers were labeled with the project number, test concentration and replicate, and were indiscriminately positioned daily in an environmental chamber designed to maintain the desired test temperature throughout the test. BACK TO MAIN Wi LDLI FE INTERNATIONALL,TD. - 11 - PROJECT NO.: 454A-111 Environmental Conditions Test beakers were held in an environmental chamber at a temperature of 25 f 2C. The temperature of a container of water adjacent to the test beakers in the environmental chamber was recorded twice daily during the test using a liquid-in-glass thermometer. The duckweed was held under continuous warm-white fluorescent lighting throughout the test. The target light intensity was 5000 f 750 lux. Light intensity was measured at five locations surrounding the test chambers on Day 0 of the test using a SPER Scientific Model 840006C light meter. The pH of the medium in each treatment and control group was measured at test initiation and termination using a Fisher Accumet Model 915 pH meter. Samples for pH measurement at test initiation were collected from the individual batches of test solution prepared for each treatment and control group. At test termination, samples of test medium from each of the three replicate test chambers were pooled by treatment group for pH measurement. Growth Measurements and Observations Growth, defined as an increase in the total numbers of fronds in each replicate test chamber, was determined through direct counts on Day 3, Day 5 and at the end of the test. In addition, the total number of duckweed plants in each replicate test chamber was determined on Day 7 of the test. Percent inhibition values were calculated relative to the control based upon the numbers of fronds produced by the end of the 7 d a y exposure period. Observations of effects such as chlorosis, necrosis, dead fronds, root destruction and break-up of duckweed colonies were performed on Days 3, 5 and 7 of the test. Chlorotic fronds were defined as fronds possessing areas of bleached color progressing from green to yellow. Fronds noted as necrotic possessed localized regions of dead or decaying tissue, usually surrounded by healthy tissue. Fronds with no apparent living tissue, usually .all brown or white in color, were considered to be dead. Dead or shortened roots in a treatment group replicate upon comparison to the control replicates was reported as root destruction. The presence of a noticeably greater number of free-floating individual fronds in a treatment group upon comparison to the control replicates was reported as colony break-up. Any other abnormalities in frond or plant appearance were also documented. BACK TO MAIN WILDLIFE INTERNATIONALL,TD. - 12- PROJECT NO.: 454A-111 Statistical Analyses Mean plant and frond numbers, percent inhibition values and the percentages of necrotic, chlorotic and dead fronds were calculated using ``Microsoft Excel Version 5.O" (3), while statistical analyses were conducted using "TOXSTAT Version 3.5" (4). Percent inhibition values were calculated for each treatment group as the percent reduction in mean frond number relative to mean frond number in the control replicates. The following formula was used: Percent Inhibition = Mean Frond NumberconW-olMean Frond NumberTreawent X 100 Mean Frond Numbercont,ol The Day 3, 5 and 7 IClO, IC50 and IC90 values and 95% confidence limits were determined, when there was sufficient reduction in frond numbers relative to the control, using linear interpolation (4) with treatment response (frond number) and exposure concentration data. The percentages of dead, chlorotic and necrotic fronds also were calculated relative to the total number of fronds in each test chamber. The frond number data on Days 3, 5 and 7 were evaluated for normality and homogeneity of variances (p = 0.05) using the Shapiro-Wilks' and Levene's tests, respectively (4). Since the data were normally distributed and the variances were homogeneous, statistically significant differences between the control and treatment groups were identified using analysis of variance (ANOVA) and Dunnett's test (4). Results of the statistical analyses, as well as an evaluation of the concentration-response pattern and other observations of effects (e.g., percentages of chlorotic, necrotic and dead fronds), were used in the determination of the no-observed-adverse-effect-concentration(NOAEC). Analvtical Chemistry Samples of test medium were collected from the control and each treatment group at test initiation, on Days 3 and 5, and at test termination to measure concentrations of the test substance. Samples of test medium collected at test initiation were taken from the individual batches of test solution prepared for each treatment and the control group. Samples collected on Days 3 and 5 were taken from "analytical" test chambers maintained for each treatment and control group. Samples collected at test termination consisted of the pooled test solution from the three "biological" replicates of each treatment and control group. The 351 mg a.i./L abiotic replicates were sampled on Days 3, 5 and 7 to determine the stability of the test BACK TO MAIN WILDLIFE INTERNATIONALL,TD. - 13 - PROJECT NO.: 454A-111 substance under the conditions of administration. The samples were placed in plastic centrifuge tubes and were analyzed immediately without storage. Analytical procedures used in the analysis of the samples are presented in Appendix 111. RESULTS AND DISCUSSION Measurement of Test Concentrations Results of analyses to measure concentrations of PFOS in the test solutions are presented in Table 1 and Appendix 111. Nominal concentrations used in this study were 11.0, 22.0, 43.9, 87.9, 176 and 351 mg a.i./L. The measured concentrations of PFOS in the samples collected at initiation of exposure of the test organisms (Day 0) ranged from 64.2 to 82.6% of the nominal concentrations. Samples collected on Day 3 had a measured concentration range of 67.3 to 83.3% of nominal. Samples collected on Day 5 had a measured concentration range of 65.4 to 85.4% of nominal. Samples collected at test termination (Day 7) had a measured concentration range of 63.9 to 83.8% of nominal. Samples from the abiotic 35 1 mg a.i./L replicates were comparable to samples from the 351 mg a.i./L biotic replicates. When the measured concentrations obtained at test initiation, on Days 3 and 5, and at test termination were averaged, the mean measured test concentrations were 7.74, 15.1, 31.9, 62.5, 147 and 230 mg a.i./L, representing 70.4, 68.6, 72.7, 71.1, 83.5 and 65.5% of nominal concentrations, respectively. Observations and Measurements Measurements of temperature and pH are presented in Tables 2 and 3, respectively. The temperatures ranged from 24.2 to 25.2"C and were within the range established for the test (25 f 2C). Measurements of pH ranged from 7.9 to 8.4 on Day 0 and from 8.6 to 9.0 on Day 7. The light intensity ranged from 4340 to 5070 lux and was within the desired range for the test (4250 to 5750 lux) (Table 2). The toxicity of PFOS to duckweed was determined by evaluating the production of plants and fronds and their general health over a 7-day exposure period. Day 7 plant and frond numbers, mean plant and frond numbers, and frond number inhibition values are shown in Table 4. Percentages of dead, chlorotic and necrotic fronds on Days 3, 5 and 7 are presented in Table 5 . IC10, IC50 and IC90 values and 95% confidence limits calculated for Days 3, 5 and 7 based on frond numbers are presented in Table 6. Observations of plant and frond health by replicate are presented in Appendix IV. BACK TO MAIN WILDLIFE INTERNATIONLATLD, . - 14- PROJECT NO.: 454A-111 Observations of plant and frond health by replicate are presented in Appendix IV. With the exception of one necrotic frond observed on Day 3 and Day 5 of the test, duckweed plants in the negative control replicates appeared healthy and exhibited normal growth throughout the test. Duckweed plants in the 7.74 and 15.1 mg a.i./L treatment groups also appeared healthy through Day 7, with no significant (p > 0.05) inhibition of growth observed in comparison to the negative control. Incidental observations of necrosis and chlorosis in the 15.1 mg a.i./L treatment group were few in number and were not considered treatment-related. Frond numbers on Day 7 in the 3 1.9,62.5, 147 and 230 mg a.i./L treatment groups were inhibited by 24, 32, 65 and 8I%, respectively, in comparison to the negative control. Dunnett's test indicated that frond numbers were significantly reduced (p < 0.05) in those treatment groups 231.9 mg a.i./L when comparedto the negative control. The 7-day IC50 value was calculated to be 108 mg a i./L, with 95% confidence limits of 45.7 and 144 mg a.i./L. Duckweed exposed to PFOS at concentrations of 147 and 230 mg a.i./L exhibited a dose-responsive increase in the incidence of dead, chlorotic or necrotic fronds during the test. By Day 7 of the test, all treatment groups 2 3 1.9 mg a.i./L showed evidence of sublethal effects, including root destruction and/or a cupping of the plant downward on the water surface. CONCLUSIONS The 7-day IC50 for Lemna gibba G3 exposed to PFOS (Perfluorooctanesulfonate, Potassium Salt) was 108 mg a.i./L, with 95% confidence limits of 45.7 and 144 mg a.i./L. The 7-day NOAEC, based on the inhibition of frond production and evidence of sublethal effects, was 15.1 mg a.i./L. BACK TO MAIN WILDLIFE INTERNATIONALL,TD. - 15 - PROJECT NO.: 454A-111 REFERENCES U.S. Environmental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines (drap),OPPTS Number 850.4400: Aquatic Plant Toxicity Test Using Lemna spp., Tiers I and II. ASTM Standard Guide 1415-913. 1991. Standard Guide for Conducting Static Toxicity Tests with Lemna gibba G3. Microsoft Corporation. Microsoft Excel Version 5 . 0 ~ .Copynght 1985 - 1994. West, Inc. and D.D. Gulley. TOXSTAT Version 3.5. Copyright 1996. Western Ecosystems Technology, Inc., Cheyenne, Wyoming. BACK TO MAIN WILDLIFE INTERNATIONALT, D. - 16- PROJECT NO.: 454A-111 Table 1 Summary of Analytical Chemistry Data ~~~ Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Duckweed, Lemna gibba G3 20X AAP Medium Nominal Concentration (mg a.i./L) Sampling Time (Day) Measured Concentration (mg a.i./L) Negative Control 0' 32 52 73 < LOQ~ < LOQ LOQ < LOQ Mean Measured Concentration (mg a.i./L) < LOQ Mean Measured Percent of Nominal __ 11.0 0 7.57 7.74 70.4 3 8.35 5 7.47 7 7.55 22.0 0 15.2 15.1 68.6 3 15.4 5 14.6 7 15.2 43.9 0 32.2 31.9 72.7 3 31.9 5 31.8 7 31.7 ' Day 0 samples were collected from individual batches of test solution prepared for the treatment and control groups at test initiation. 2 Day 3 and Day 5 samples were collected from the additional analytical replicates (biotic). 3 Day 7 samples were composites of test solution collected from each of the three replicates per treatment and control group. 4 Limit of Ouantitation (LOO) was 4.39 mg a.i./L. BACK TO MAIN WILDLI FE INTER NATI ONAL, LTD. - 17- PROJECT NO.: 454A-111 Table 1 - Continued - Summary of Analytical Chemistry Data Sponsor: Test Substance: Test Organism: Dilution Water: Nominal Concentration (mg a.i./L) 87.9 3M Corporation PFOS Duckweed, Lemna gibba G3 20X AAP Medium Sampling Time (Day) Measured Concentration (mg a.i./L) 0' 63.5 32 63.1 52 61.5 73 61.8 Mean Measured Concentration (mg a.i./L) 62.5 Percent of Nominal 71.1 176 0 145 3 146 5 150 7 147 147 83.5 35 1 0 226 3 237 5 232 7 224 230 65.5 35 1 (Abiotic) 3s 225 235 75 232 23 1 65.8 1 Day 0 samples were collected from individual batches of test solution prepared for the treatment and control groups at test initiation. 2 Day 3 and Day 5 samples were collected from the additional analytical replicates (biotic). Day 7 samples were composites of test solution collected from each of the three replicates per treatment and control group. '4 Limit of Quantitation (LOQ) was 4.39 mg a.i./L Day 3, Day 5 and Day 7 samples collected from the additional abiotic replicates. BACK TO MAIN WILDLIFE INTERNATIONLATLD, . -18- PROJECT NO.: 454A-111 Table 2 Temperature Measurements Sponsor: Test Substance: Test Organism: Dilution Water: Time (Day) 3M Corporation PFOS Duckweed, Lemna gibba G3 20X AAP Medium Measurement 1 Temperature ("C) Measurement 2 ' O2 25.1 24.5 1 24.9 24.9 2 24.6 24.4 3 24.6 24.2 4 24.5 24.5 5 24.8 24.6 6 25.2 25.0 7 25.2 25.0 Temperature Measurement 2 was taken at least 4 hours after Measurement 1 with the exception of test termination. 2 Light intensity measurements at test initiation were 4340,4520, 5070, 4560 and 4500 lux. BACK TO MAIN WILDLIFE INTERNATIONALL,TD. - 19- PROJECT NO.: 454A-111 Table 3 pH Measurements Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Duckweed, Lemna gibba G3 20X AAP Medium Mean Measured Test Concentration (mg a.i./L) Day 0 ' pH Measurements Day 7 * Negative Control 7.9 8.9 7.74 8.0 9.0 15.1 8.1 9.0 31.9 8.1 9.0 62.5 8.1 8.9 147 8.3 8.6 230 8.4 8.7 ' Day 0 samples were collected from the individual batches of test solution prepared for the treatment and control groups at test initiation. Day 7 samples were collected from the pooled test solution from the three replicates per treatment and control group. BACK TO MAIN WILDLIFE INTERNATIONALL,TD. - 20 - PROJECT NO.: 454A-111 Table 4 Day 7 Plant and Frond Numbers, Mean Plant and Frond Numbers, and Percent Inhibition Values Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Duckweed,Lemna gibba G3 20X AAP Medium Mean Measured Test Concentration (mg a.i./L) Day 7 Plant Replicate Number Negative Control A 24 B 18 C 16 Mean Plant Number 19 Day 7 Frond Number 182 208 202 Mean Frond Number 197 Frond Number Percent Inhibition` __ 7.74 A 18 18 181 177 10 B 19 171 C 16 180 15.1 A 18 20 210 219 -1 1 B 17 203 C 24 245 31.9 A 14 14 144 151* 24 B 17 151 C 12 157 62.5 A 15 11 176 134* 32 B 8 117 C 9 110 147 A 14 15 52 69* 65 B 17 70 C 12 86 230 A 17 17 37 37* 81 B 16 34 C 17 40 ' Percent inhibition was calculated relative to the negative control replicates. * Statistically significant difference @ < 0.05) from the negative control using ANOVA and Dunnett's test. BACK TO MAIN WILDLIFE INTERNATIONALLT, D. -21 - PROJECT NO.: 454A-111 Table 5 Mean Percentage' of Fronds Observed Dead, Chlorotic or Necrotic Per Treatment Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Duckweed, Lemna gibba G3 20X AAP Medium Mean Measured Day 3 Percentage Test Concentration (mg a.i./L) (N)' Dead Chlorotic Necrotic _ - 1 _ 1 1 Negative Control 35 0.0 0.0 1.0 Day 5 Percentage (N)* Dead Chlorotic Necrotic 74 0.0 0.0 0.51 Day 7 Percentage (N)* Dead Chlorotic Necrotic 197 0.0 0.0 0.0 7.74 33 0.0 0.0 0.0 70 0.0 0.0 0.0 177 0.0 0.0 0.0 15.1 40 0.0 0.0 0.0 87 0.0 0.0 0.40 219 0.0 1.1 0.0 31.9 34 0.0 0.0 0.9 67 0.0 0.0 0.45 151 0.0 0.0 0.23 62.5 35 0.0 0.0 0.0 70 0.0 0.0 0.58 134 0.0 0.9 0.61 147 29 0.0 0.0 8.8 42 0.0 4.3 7.6 69 1.0 11 4.5 230 25 0.0 2.5 12 33 0.0 4.0 26 37 3.8 9.4 19 ' ___ ~_____ Values represent the average percentage of dead, chlorotic or necrotic fronds for the three replicates per treatment. Calculations were performed using Excel 5.0. Manual calculations may vary slightly due to rounding. * N = Mean number of fronds per treatment on each observationday. Mean number of fronds per treatment at test initiation = 15. BACK TO MAIN WILDLIFE INTERNATIONALT, D. PROJECT NO.: 454A-111 - 22 - Table 6 IC10, IC50 and IC90 Values Based on Frond Number Over the 7-Day Exposure Period Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Duckweed, Lemna gibba G3 20X AAP Mehum Time Day 3 IC10 (mg a.i./L) 101 95% Confidence Limits (mg a.i./L) < 0.0 - 212 Day 5 30.7 13.3 - 142 IC50 (mg a.i./L) > 230 182 Day 7 22.1 13.3 - 26.0 108 95% confidence limits could not be calculated from the data obtained. 95% Confidence Limits (mg a.i./L) -- 1 89.1 - 240 45.7 - 144 IC90 (mg a.i./L) > 230 > 230 > 230 95% Confidence Limits (mg a.i./L) -- 1 -- 1 -_1 BACK TO MAIN WILD LIFE INTER NATIONAL, LTD. - 23 - PROJECT NO.: 454A-111 APPENDIX I 20X AAP Medium' Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Duckweed, Lemna gibba G3 20X AAP Medium Component Nominal Concentration 243.2 mgiL 88.0 m g L 3.712 m g L 8.32 mgL 65.6 p g L 3.196 m g L 28.56 pg/L 145.2 pg/L 0.240 pg/L 6.00 m g L 510.0 m g L 294.0 m g k 20.88 mg/L 300.0 m a ' The pH of the medium was adjusted to 7.5 5 0.1using 10%HC1. BACK TO MAIN WILDLIFE INTERNATIONLATLD, . - 24 - PROJECT NO.: 454A-111 APPENDIX I1 Analyses of Pesticides, Organics, Metals and Other Inorganics in Wildlife International, Ltd. Well Water' ANALYSIS Miscellaneous Measurements Total Dissolved Solids AmmoniaNitro en Total Organic &rbonz Total Cyanide MEASURED CONCENTRATION 286 < 0.050 < 1.o < 10.0 O r anochlorines and P'CBs Aljrin Alpha BHC Beta BHC Delta BHC Gamma BHC (Lindane) Chlordane DDD, PP,' DDE, PP DDT pp' Dieldnn Endosulfan, A Endosulfan, B Endosulfan Sulfate Endrin Endrin Aldehvde Heptachlor ' Methoxychlor Heptachlor Epoxide Toxaphene PCB-1016 PCB-1221 PCB- 1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 < 0.005 < 0.005 < 0.005 < 0.005 < 0.006 < 0.025 < 0.006 < 0.005 < 0.008 < 0.005 < 0.005 < 0.005 < 0.018 < 0.010 < 0.005 < 0.005 < 0.007 < 0.005 < 0.500 < 0.260 < 0.260 < 0.260 < 0.720 < 0.720 < 0.720 < 0.720 'Metals ang Other Inorganics Aluminym Arsenic ~~g1j~~ Ca iuy Calcium chro~~p? Cobalt copper3 Iron Lead' Magnesiunf Manganese Iron-' ~elenjum~ Silver SOdip3 Zinc ~~~ < 100 < 25.0 < 0.50 < 1 .o 35.0 < < 21..0o < 20.0 < 100 < 10.0 < 113..o5 < 0.20 < 2.0 < 2.0 6.62 < < 251..o0 21.3 < 20.0 Analyses performed by QST Environmental, Gainesville, Florida for samples collected on November 3 through November 7, 1997. Analyses performed by Wildlife International, Ltd. for the sample collected on November 5, 1997. Analyses performed by Wildlife International, Ltd. for samples collected on November 5 through 7, 1997. BACK TO MAIN WILDLIFE INTERNATIONALT, D. - 25 - PROJECT NO.: 454A-111 APPENDIX I11 THE AT ALYSIS OF PFOS IN 20X P CEDIUA IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-111 BACK TO MAIN WILDLIFE INTERNATIONALT, D. - 26 - REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 7-Day Toxicity Test with Duckweed (Lernnagibba G3) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-111 PROJECT NO.: 454A-11I PRINCIPAL INVESTIGATOR: MANAGEMENT: Willard B. Nixon, Ph.D. / Director, Analytical Chemistry DAp i / BACK TO MAIN WILD LIFE I NTER NATIONAL, LTD. - 27 - PROJECT NO.: 454A-111 Introduction 20X AAP medium samples were collected from a seven-day toxicity test designed to determine the effects of PFOS (Perfluorooctanesulfonate, Potassium Salt) to duckweed (Lernna gibba G3). This study was conducted by Wildlife International, Ltd. and identified as Project No.: 454A-111. The analyses of these water samples were performed at Wildlife International, Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLCMS). Samples were received for analysis between March 3 and 10, 2000 and were analyzed on each sample receipt day. Test Substanceand Internal Standard The test substance used for this study was Wildlife International, Ltd. identification number 4675. The test substance was used to prepare calibration and matrix fortification samples. The internal standard was received from 3M Corporation on July 2, 1998 and was assigned Wildlife International, Ltd. identification number 4526 upon receipt. The internal standard, a granular material, was identified as: lH, lH, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2. The standard was stored under ambient conditions. Analytical Method The method used for the analysis of the 20X AAP medium 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 I1 of Wildlife International, Ltd. protocol number 4541011299/MVAL/SuB454. It was based upon methodology providedby 3M Corporation. Samples were centrifuged, as necessary, and diluted in a 50% methanol : 50% NANOpureO water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v) so that they fell within the calibration range of the PFOS methodology. Concentrations of the PFOS in the standards and samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API lOOLC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. HPLC separations were achieved using a Keystone Betasil CI8analytical column (50 mm x 2 mm I.D., 3-pm particle size). The instrument parameters are summarized in Table 1. A method flowchart is provided in Figure 1. WILDLIFE INTERNATIONALL,TD. - 28 - BACK TO MAIN PROJECT NO.: 454A-111 Calibration Curve and Limit of Ouantitation Calibration standards of PFOS prepared in a 50% methanol : 50% NANOpure@water solution containing 0.100 mg 4H PFOS (internal standard)/L and 0.05% formic acid (vh), ranging in concentration from 0.0439 to 0.879 mg 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 of 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 4.39 mg a.i./L calculated as the product of the lowest calibration standard analyzed (0.0439 mg a.i./L) and the dilution factor of the matrix blank samples (100). Matrix Blank and Fortification SamDles Four 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 . 20X AAP medium was fortified at 8.79, 87.9 and 439 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 104%. A representative ion chromatogram of a matrix fortification is presented in Figure 6. Example Calculations Sample number 454A- 111-5, nominal concentration of 87.9 mg a.i./L in 20X AAP medium Peak Area Ratio = Analyte Peak Areanternal Standard Peak Area BACK TO MAIN WILDLIFE INTERNATIONALL,TD. - 29 - PROJECT NO.: 454A-111 ConcentrationRatio = Concentrationof Analyte/Concentrationof Internal Standard Internal Standard Concentration: 0.100 mg/L Initial Volume: 0,100 mL Final Volume: 25.0 mL Dilution Factor: 250 PFOS Peak Area: 8433545 Internal Standard Peak Area: 2550872 Peak Area Ratio: 3.30614 Calibration curve equation. Slope: 1.22511 Intercept: 0.19508 Curve is weighted (Ux) Peak area PFOS (mg a.i./L) at instrument = Slope(Y-intercept) x Internal Standard Concentration - 3.30614 - 0.19508 x 0.100 1.22511 = 0.2539 PFOS (mg a.i./L) in sample = PFOS (mg a.i./L) at instrument :< Dilution Factor = 0.2539 x 250 = 63.5 PFOS m a.i./L insample Percent of Nominal Concentration= pFos((m\ no,ninal x 100 Calculated recovery: 72.3% Quantitation softwarefor recoveries: MacQuan, version 1.6. Note: manual calculation of recovery may differ. BACK TO MAIN WILDLIFE INTERNATIONLATLD, . - 30 - PROJECT NO.:454A-111 RESULTS Sample Analysis 20X AAP medium samples were collected from the seven-day toxicity test with duckweed (Lemna gibba G3) at test initiation, March 3, 2000 (Day 0), on March 6, 2000 (Day 3), on March 8, 2000 (Day 5) and at test termination, March 10, 2000 (Day 7). The measured concentrations of PFOS in the samples collected at initiation of exposure of the test organisms (Hour 0 ) ranged from 64.2 to 82.6% of the nominal concentrations. Samples collected at Day 3 had a measured concentration range of 64.2 to 83.3% of nominal values. Samples collected at Day 5 had a measured concentration range of 65.4 to 85.4% of nominal values. Samples collected at test termination (Day 7) had a measured concentration range of 63.9 to 83.8% of nominal values (Table 4). Samples from the abiotic 351 mg a.i./L treatment group were comparable to samples from the 351 mg a.i./L treatment group with duckweed present (Table 4). A representative ion chromatogram of a test sample is shown in Figure 7. BACK TO MAIN WILDLIFE INTERNATIONALL, TD. -31 - PROJECT NO.:454A-111 INSTRUMENT: Table 1 Typical HPLCMS Operational Parameters Hewlett-Packard Model 1100 High PerformanceLiquid Chromatograph with a Perkin-Elmer API l00LC Mass Spectrometerequipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: PFOS RETENTION TIME: INTERNAL STANDARD RETENTION TIME: PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: KeystoneBetasil CI8column (50 mm x 2 mm I.D., 3-pm particle size) 30C 5.00 minutes 0.220 mL/minute 72.0% Methanol : 28.0% NANOpureO Water containing 0.1% Formic Acid 5.0 pL Approximately 3.9 minutes Approximately 2.7 minutes 498.6 amu 426.7 amu BACK TO MAIN WILDLIFE INTERNATIONALL,TD. - 32 - PROJECTNO.: 454A- 111 Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-111-) MAB-1 Sample Type Matrix Blank Measured Concentration of PFOS' (mg a.i./L) < LOQ MAB-2 Matrix Blank .c LOQ MAB-3 Matrix Blank < LOQ MAB-4 Matrix Blank < LOQ 1 The limit of quantitation (LOQ) was 4.39 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.0439 mg a.i./L) and the dilution factor of the matrix blank samples (100). BACK TO MAIN WILDLIFE INTERNATIONALLT, D. - 33 - PROJECT NO.: 454A-111 Table 3 Matrix Fortifications Analyzed ConcurrentlyDuring Sample Analysis Sample Number (454A-111-) MAS-1 MAS-4 MAS-7 MAS-10 Concentrationsof PFOS Fortified' (mg a.i./L) Measured' 8.79 9.23 8.79 9.64 8.79 10.1 8.79 9.08 Percent Recovered2 105 110 115 103 MAS-2 87.9 92.7 105 MAS-5 87.9 89.9 102 MAS-8 87.9 91.5 104 MAS-1 1 87.9 90.0 102 MAS-3 439 446 101 MAS4 439 440 100 MAS-9 439 448 102 MAS-12 439 422 96.0 Mean = 104 Standard Deviation = 4.86 CV =4.69% N = 12 1 Concentrations were corrected for change in test substance purity (98.9% to 86.9%) per Certificate of Analysis dated September 7,2000. Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since fortified and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes. BACK TO MAIN WILDLIFE INTERNATIONALL, TD. - 34 - PROJECT NO.: 454A-111 Table 4 Measured Concentrations of PFOS in 20X AAP Medium Samples from a Duckweed Seven-Day Toxicity Test Nominal Test Concentration' (mg a.i./L) 0.0 (Negative Control) 11.0 22.0 43.9 87.9 Sample Number (454A-111-) 1 8 16 24 2 9 17 25 3 10 18 26 4 11 19 27 5 12 20 28 Sampling Time Pay) 0 3 5 7 PFOS Measured Concentration"' (mg a.i./L) < LOQ < LOQ < LOQ < LOQ 7.57 8.35 7.47 7.55 15.2 15.4 14.6 15.2 32.2 31.9 31.8 31.7 63.5 63.1 61.5 61.8 Percent of Nominal3 ----- 66.3 73.1 65.4 66.1 69.2 70.1 66.2 69.0 73.3 72.6 72.4 72.2 72.3 71.8 70.0 70.3 176 6 0 1145 82.6 13 3 :146 83.3 - 21 5 150 85.4 29 7 I47 83.8 P 1 Concentrationswere corrected for change in test substancepurity (98.9% to 86.9%) per Certificate of Analysis dated September 7, 2000. 2 The limit of quantitation (LOQ) was 4.39 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.0439 mg a.i./L) and the dilution factor of the matrix blank samples (100). 3 Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since nominal and measured concentrations were corrected for change in test substance purity and rounded for , reportiwurposes. BACK TO MAIN WILDLIFE INTERNATIONALL,TD. - 35 - PROJECT NO.: 454A-111 Table 4 (continued) Measured Concentrations of PFOS in 20X AAP Medium Samples from a Duckweed Seven-Day Toxicity Test Nominal Test Concentration' (mg a.i./L) 351 Sample Number (454A-111-) 7 14 22 30 Sampling Time P a y1 0 3 5 7 PFOS Measured Concentration's2 (mg a.i./L) 226 237 232 224 Percent of Nominal3 64.2 67.3 66.0 63.9 351 15 3 225 64.2 (Abiotic) 23 5 235 66.9 31 7 232 66.1 f Concentrationswere corrected for change in tesi t substancepurity (98.9%to 86.9%) per Certificateof Analysis dated September 7,2000. 2 The limit of quantitation (LOQ) was 4.39 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.0439 mg a.i./L) and the dilution factor of the matrix blank samples (100). Results were generated using MacQuan version 1.6 software. Manual calculations may differ slightly since - - - nominal and measured concentrations were corrected for change in test substance purity and rounded for reporting purposes. _ _ ___^ P BACK TO MAIN -36- PROJECT NO.:454A-111 METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN 20X AAP MEDIUM Prepare matrix fortification samples by spiking the requisite volume of PFOS stock solutions directly into 20X AAP medium using gas-tight syringes and Class A volumetric flasks. J Centrifuge all samples, as necessary, for approximately five minutes at approximately 1500 rpm. .1 Dilute matrix fortification and test samples into the range of the calibration standards by partially filling Class A volumetric flasks with 50% methanol : 50% NANOpureO water solution containing 0.100mg 4 H PFOS (internal standard)/L and 0.05% formic acid (v/v). Add the appropriate volume of sample and bring the flask to volume with the dilution solvent. Process the matrix blank sample using the same dilution and aliquot volume as for the lowest fortification level. Mix well by several repeat inversions. Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis of PFOS in 20X AAP medium. WILDLIFE INTERNATIONALT, D. - 37 - BACK TO MAIN PROJECT NO.: 454A-111 20 n .r( c, W Ld 0.00 2.00 4.00 6.00 Concentration (Ratio) 8.00 10.00 Figure 2. A typical calibration curve for PFOS. Slope = 1.22511;Intercept = 0.19508; r = 0.9982. Curve is weighted (l/x). WILDLIFE INTERNATIONALT, D. -38- 10 BACK TO MAIN PROJECT NO.: 454A-111 intensity: 2000000 cps 7 6 3 2 1 0.69 1.36 2.04 2.71 3.38 4.06 4.73Time Figure 3. A representativeion chromatogramof a low-level (0.0439 mg a.i./L)PFOS standard. WILDLIFE INTERNATIONALT, D. -39- BACK TO MAIN PROJECT NO.: 454A-11I intensity: 2000000 cps n228 n 193 - - 1 4 3 5 6 5 0-69 -. 1.36 121 2.04 1 6 1 - 2 0 1 - 2 4 1 - 2 8 1 scan 2.71 3.38 4.06 4.73Time Figure 4. A representative ion chromatogram of a hgh-level (0.879 mg a.i./L) PFOS standard. WILDLIFE INTERNATIONALL, TD. -40- BACK TO MAIN PROJECT NO. 454A-111 intensity: 2000000 c p s 1 O! -1 63 168 194 230 a I 1 1 I I I I I I I I I I 41 81 1 2 1 161 201 241 281 Scan 0.69 1.36 2.04 2.77 3.38 4.06 4.73Time Figure 5 . A representativeion chromatogramof a matrix blank sample (454A-111-MAB-1). The arrow indicates the retention time of PFOS. WILDLIFE INTERNATIONAL, LTD. -41 - BACK TO MAIN PROJECT NO.: 454A-111 100 90. 80 70 60 5 4 3 2 1 intensity: 2000000 cps 41 0.69 81 1.36 121 2.04 161 2.71 201 3.38 241 4.06 281 Scan 4.73Time Figure 6. A representative ion chromatogram of a matrix fortification sample (454A-111-MAS-2). WILDLIFE INTERNATIONALL,TD. BACK TO MAIN PROJECT NO.:454A-111 100 90 80 70 60 50 40. 30 20 1 intensity: 2000000 cps 229 41 0.69 81 1.36 121 2.04 161 2.71 201 3.38 241 4.06 2 8 1 Scan 4.73Time Figure 7. A representative ion chromatogramof a test sample (454A-111-5). BACK TO MAIN WILDLIFE INTERNATIONAL,TD. - 43 - PROJECT NO.: 454A-111 APPENDIX IV Observations by Replicate of Frond and Plant Production, the Numbers of Dead, Chlorotic and Necrotic Fronds, and Other Sublethal Effects Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Duckweed,Lemna gibba G3 20X AAP Medium Mean Measured Test Concentration (mg a.i.L) Replicate Initial No. Fronds and Plants' Number Fronds Number Dead Day 3 Number Chlorotic Negative Control A 151 5 36 0 0 B 151 5 33 0 0 C 1515 36 0 0 7.74 A 1515 33 0 0 B 1515 32 0 0 C 1515 33 0 0 15.1 A 1515 39 0 0 B 15f5 36 0 0 C 1515 44 0 0 31.9 A 15f5 36 0 0 B 1515 32 0 0 C 15 f 5 35 0 0 62.5 A 15/5 42 0 0 B 1515 34 0 0 C 15f5 30 0 0 147 A 1515 24 0 0 B 1515 30 0 0 C 151 5 33 0 0 230 A 15 f 5 27 0 2 B 1515 24 0 0 C 1515 25 0 0 ' At test initiation, each replicate contained 5 plants with a total of 15 fronds. 2 Observations: AN = appear normal; P = pale roots in comparisonto the control. Number Necrotic 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 4 2 1 3 3 3 Observations2 AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN P AN AN WILDLIFE INTERNATIONALT, D. BACK TO MAIN PROJECT NO.: 454A-111 APPENDIX IV - Continued - Observations by Replicate of Frond and Plant Production, the Numbers of Dead, Chlorotic and Necrotic Fronds, and Other Sublethal Effects Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Duckweed,Lemna gibba G3 20X AAP Medium Mean Measured Test Concentration (mg a.i./L) Replicate Number Fronds Number Dead Day 5 Number Chlorotic Number Necrotic Observations' Negative Control A B C 82 0 0 0 AN 65 0 0 1 AN 75 0 0 0 AN 7.74 A 77 0 0 0 AN B 65 0 0 0 AN C 67 0 0 0 AN 15.1 A 85 0 0 1 AN B 73 0 0 0 AN C 102 0 0 0 AN 31.9 A 74 0 0 1 AN B 63 0 0 0 AN C 65 0 0 0 AN 62.5 A 96 0 0 0 AN B 56 0 0 0 AN C 57 0 0 1 AN 147 A 36 0 B 41 0 C 50 0 5 R 2 R 2 R 230 A 33 0 4 7 B 33 0 0 9 C 33 0 0 10 1 Observations: AN = appear normal; P = pale roots in comparisonto the control; R = root destruction (small roots). BACK TO MAIN WILDLIFE INTERNATIONAL,TD. - 45 - PROJECT NO.: 454A-111 APPENDIX IV - Continued - Observations by Replicate of Frond and Plant Production, the Numbers of Dead, Chlorotic and Necrotic Fronds, and Other Sublethal Effects Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFOS Duckweed, Lemna gibba G3 20X AAP Medium Mean Measured Test Concentration (mg a.i./L) Replicate Negative Control A B C Number Plants ~~ 24 18 16 Number Fronds ~~ 182 208 202 Day 7 Number Dead Number Chlorotic 0 0 0 0 0 0 Number Necrotic ~ 0 0 0 Observations' ~~~ AN AN AN 7.74 A 18 181 0 0 0 AN B 19 171 0 0 0 AN C 16 180 0 0 0 AN 15.1 A 18 2 10 0 3 0 AN B 17 203 0 4 0 AN C 24 245 0 0 0 AN 3 1.9 A 14 144 0 0 1 PC B 17 151 0 0 0 PC C 12 157 0 0 0 PC 62.5 A 15 176 0 5 0 PC B 8 117 0 0 0 PC C 9 110 0 0 2 PC 147 A 14 52 0 4 7 PC, R B 17 70 2 9 0 PC, R C 12 86 0 11 0 PC, R 230 A 17 37 0 2 8 PC, R B 16 34 3 6 5 PC, R C 17 40 1 2 8 PC, R 1 Observations: AN = appear normal; PC = plant cupped downward on water surface; R = root destruction (small roots). BACK TO MAIN WILDLIFE INTERNATIONAL,LTD. - 46 - PROJECT NO.: 454A-111 APPENDIX V Changes to Protocol This study was conducted in accordance with the approved Protocol with the following changes: 1. The protocol was amended to change the test medium from Hoagland's to 20X AAP medium. 2. The protocol was amended to add the proposed experimental start and termination dates, test concentrations and test substance identification number. 3 . The nominal test concentrations were recalculated based on a test substance purity of 90.49%. 4. The nominal test concentrations were recalculated based on a test substance purity of 86.9%. 5 . The protocol was amended to change the test substance name from Perfluorooctane Sulfonic Acid, Potassium Salt to Perfluorooctanesulfonate, Potassium Salt. BACK TO MAIN WILDLIFE INTERNATIONLATLD, . ~ - 47 - PROJECT NO.: 454A-111 APPENDIX VI Personnel Involved in the Study The following key 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., Manager, Analytical Chemistry 3. Raymond L. VanHoven, Ph.D., Scientist 4. Cary A.Sutherland, Laboratory Supervisor 5 . Debbie Desjardins, Biologist