Document VJ45XwGjg4ZKgxNb3L4MrG4o8

15) OECD 2 1 1-OPPTS 850.1350, Chronic toxicity test (daphnia magna), 454A -130 PFBS: A SEMI-STATIC LIFE-CYCLE TOXICITY TEST WITH THE CLADOCERAN (Daphnia magna) ;T - f/nss C 2 / > o f/rs iss. FINAL REPORT SANITIZED WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-130 3M Environmental Lab Project No. E00-1429 U.S. Environmental Protection Agency Series 850 - Ecological Effects OPPTS Number 850.1300 OECD Guideline 211 DEC 0 9 2003 AUTHORS: Kurt R. Drottar Raymond L. Van Hoven, Ph.D. Henry 0 . Krueger, Ph.D. STUDY INITIATION DATE: October 6,2000 STUDY COMPLETION DATE: April 11, 2001 Submitted to 3M Corporation Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 80 W ildlife International, Ltd. 2- - DEC 0 9 2003 Project Number 454A-130 SANITIZED GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFBS: A Semi-Static Life-Cycle Toxicity Test with the Cladoceran (Daphnia magna) WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-130 STUDY COMPLETION: April 11, 2001 This study was .conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFP. 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, with the following exceptions: The test substance was not characterized in compliance with Good Laboratory Practices prior to its use in the study. However, subsequent GLP compliant characterization resulted in a purity similar to the original characterization purity. The stability of the test substance under conditions o f storage at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Kurt R Drottar Senior Biologist SPONSOR'S REPRESENTATIVE: DATE Wildlife International, Ltd. -3 - Project Number 454A-130 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Part 160 and 792,17 August 1989; OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17; and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates o f all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Test Substance Preparation Analytical Sample Preparation Exposure Initiation, Offspring Observations, Length Measurements and Offspring Counts Analytical Sampling Biological Data and Draft Report Analytical Data and Draft Report Final Report DATE CONDUCTED: February 7,2001 February 9,2001 February 21,2001 February 28,2001 February 28,2001 March 21 - 2 3 and 26,2001 March 26 and 29,2001 April 10 and 11, 2001 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: February 7,2001 February 9,2001 February 9, 2001 February 14, 2001 February 21, 2001 February 22, 2001 February 28, 2001 February 28, 2001 March 26,2001 March 29,2001 April 11,2001 March 2,2001 March 2, 2001 March 27,2001 March 29,2001 April, 11,2001 Kimberay A. HoxJ*r Quality Assurance Representative DATE Wildlife International, Ltd. -4 - Project Number 454A-13Q REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFBS: A Semi-Static Life-Cycle Toxicity Test with the Cladoceran (Daphnia magna) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-130 STUDY DIRECTOR: Kurt R. Drottar Senior Biologist MANAGEMENT : Henry*0. Krueger, Ph.D. Director, Aquatic Toxicology and Non-Target Plants H / / 1/ 0 I DATE DATE W ildlife International, Ltd._______________________________________Project Number 454A-13Q -5 - TABLE OF CONTENTS Title/Cover P ag e...................................................... 1 Good Laboratory Practice Compliance Statem ent............................................................................................ 2 Quality Assurance Statement................................... 3 Report A pproval....................................................................................................................................................4 Table o f Contents...................................................................................................................................................5 Summary................................................................................................................................................................ 7 Introduction............................................................................................................................................................ 8 Objective................................................................................................................................................................ 8 Experimental Design............................................................................................................................................. 8 Materials and Methods.......................................................................................................................................... 9 Results and Discussion.......................................................................... 13 Conclusions.......................................................................................................................................................... 15 References.......................................... 16 TABLES Table 1 - Summary of Analytical Chemistry D a ta ......................................................................................... 17 Table 2 - Temperature of Water in the Test Chambers.................................................................................. 18 Table 3 - Dissolved Oxygen Content o f Water in the Test Chambers......................................................... 20 T able 4 - pH of Water in the Test Chambers...................................................................................................22 W ildlife International, Ltd. 6- - Project Number 454A-130 TABLE OF CONTENTS - Continued - TABLES (Continued) Table 5 - Specific Conductance, Hardness and Alkalinity in the Negative Control and Highest Treatment Group.........................................................................................................24 Table 6 - Summary of Cumulative Percent Mortality and Treatment-Related Effects for First-Generation D aphnids........................................................................................................ 25 Table 7 - Summary o f Daphnia magna Reproduction During the Life-Cycle Toxicity Test.................. 26 Table 8 - Summary o f Length and Dry Weight o f Surviving First-Generation Daphnids....................... 27 Table 9 - Cumulative Percent Mortality/Immobility and Treatment-Related Effects for Second-Generation Daphnids.......................................................................................................... 28 Appendix 1 Appendix 2 Appendix 3 Appendix 4 Appendix 5 Appendix 6 Appendix 7 Appendix 8 APPENDICES - Specific Conductance, Hardness, Alkalinity and pH of Well Water Measured During the 4-Week: Period Immediately Preceding the T est..................... 29 - Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well W ater..................................................................... 30 - The Analysis of PFBS in Freshwater in Support of Wildlife International, Ltd. Project No.: 454A-130..................................................... 32 - Cumulative Percent Mortality and Treatment-Related Effects First-Generation Daphnids............................................. 52 - Neonate Production............................................................................................................ 73 - Length and Dry Weight o f Surviving First-Generation Daphnids.................................77 - Changes to the Protocol..................................................................................................... 79 - Personnel Involved in the Study........................................................................................80 W ildlife International. Ltd. SANITIZED DEC 0 9 2003 Project Number 454A-13Q SPONSOR: SPONSOR'S REPRESENTATIVE: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: SUMMARY 3M Corporation Wildlife International, Ltd. Easton, Maryland 21601 WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: STUDY: NOMINAL TEST CONCENTRATIONS MEAN MEASURED TEST CONCENTRATIONS: TEST DATES: LENGTH OF TEST: TEST ORGANISM: SOURCE OF TEST ORGANISMS: AGE OF TEST ORGANISMS: 454A-130 Perfluorobutanesulfonate, Potassium Salt (PFBS) PFBS: A Semi-Static Life-Cycle Toxicity Test with the Cladoceran (Daphnia magna) Negative Control, 63, 125, 250, 500, 1000 and 2000 mg a.i./L Negative Control, 60, 121, 247, 502, 995 and 1876 mg a.i./L Experimental Start - February 7, 2001 Biological Termination - February 28, 2001 Experimental Termination - March 2, 2001 21 Days Neonate Cladocerans {Daphnia magna) Wildlife International, Ltd. cultures Easton, Maryland 21601 Juveniles < 24 hours old NOEC: LOEC: MATC: 502m ga.i./L 995 mg a.i./L _______________________ 707 mg a.i./L Wildlife International, Ltd. 8- - Project Number 454A-130 INTRODUCTION This study was conducted by Wildlife International Ltd. for 3M Corporation at the Wildlife International, Ltd. aquatic toxicology facility in Easton, Maryland. The in-life phase of the test was conducted from February 7,2001 to February 28,2001. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454A-130 in archives located on the Wildlife International Ltd. site. OBJECTIVE The objective of this study was to determine the effects of Perfluorobutanesulfonate, Potassium Salt (PFBS) on the survival, growth and reproduction of the cladoceran (Daphnia magna) during a 21-day exposure period under semi-static test conditions. EXPERIMENTAL DESIGN Daphnids were exposed to a geometric series of six test concentrations and a negative (dilution water) control. Ten replicate test chambers were maintained in each treatment and control group with one daphnid in each test chamber. Nominal test concentrations were selected in consultation with the Sponsor, and were based upon the results of an acute toxicity test. Nominal test concentrations were 63,125,250,500, 1000 and 2000 mg active ingredient (a.i.)/L. Mean measured concentrations were determined from new and corresponding old solutions during each week o f the test. Daphnids were transferred to new solutions every Monday, Wednesday and Friday during the test. First-generation daphnids were observed daily for survival, the onset ofreproduction, and clinical signs o f toxicity. With the onset of reproduction, the number o f second-generation neonates were counted (number o f live neonates, number of dead or immobile neonates and number o f aborted eggs) every Monday, Wednesday and Friday during the test. If available, twenty second-generation neonates from each treatment and the control were exposed under static test conditions for 48 hours. At test termination, the length and dry weight of each first-generation daphnid was determined. The no-observed-effect-concentration (NOEC) and lowest-observed-effect-concentration (LOEC) were determined by examination of the mortality, growth and reproduction data. The maximum acceptable toxicant concentration (MATC) was calculated as the geometric mean of the NOEC and LOEC. Wildlife International. Ltd. SANITIZED ' y - DEC 0 9 2003 Project Number 454A-130 MATERIALS AND METHODS The study was conducted based on the procedures outlined in the protocol, "PFBS: A Semi-Static Life Cycle Toxicity Test with the Cladoceran (Daphnia magna)". The protocol was based on procedures outlined in the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1300 (1); OECD Guideline 211: Daphnia magna Reproduction Test (2); Standard Evaluation Procedure, Daphnia magna Life-Cycle (21-Day Renewal) Chronic Toxicity Test (3); and ASTM Standard E 1193-96, Standard Guide fo r Conducting Renewal Life-Cycle Toxicity Tests with Daphnia magna (4)... Test Substance The test substance was received from 3M Corporation on June 28, 2000 and was assigned Wildlife International, Ltd. identification number 5292. The test substance, a white powder, was identified as Potassium Perfluoro Butane Sulfonate, AKA Developmental Product, AKA PFBS, lot 2, CAS Number 29420-49-3. Information provided by the Sponsor indicated a purity of 97.90%. A subsequent revision of the certificate of analysis indicated a purity of 97.3% and an Expiration/Reassessment Date of January 17, 2002. The test substance was stored at ambient room temperature. Preparation of Test Concentrations N om in al test concentrations were 63, 125, 2 5 0 , 5 0 0 , 1000 and 2 0 0 0 mg a.i./L. A 4-L primary stock solution was prepared by adding the test substance to UV sterilized dilution water at a concentration o f2000 mg a.i./L. The test solution was stirred with an. electric stainless steel top-down mixer until all the test substance was dissolved. The primary stock solution was proportionally diluted with UV sterilized dilution water to prepare 2-L o f the other five test concentrations. All dilutions were inverted to mix. After mixing, 200 mL of test solution was added to the 10 replicate test chambers for each treatment group. New solutions were prepared for each renewal day. After mixing, all test solutions appeared clear and colorless. All test concentrations were adjusted for the original reported purity of the active ingredient in the test substance (97.9%). Test Organism The cladoceran, Daphnia magna, was selected as the test species for this study. Daphnids are representative of an important group of aquatic invertebrates and were selected for use in the test based upon Wildlife International, Ltd. - 10- Project Number 454A-13Q past history of use and ease of culturing in the laboratory. Daphnid neonates used in the test were less than 24-hours old and were obtained from cultures maintained by Wildlife International, Ltd., Easton, Maryland. Adult daphnids were cultured at approximately the same temperature as used during the test in Wildlife International, Ltd. well water. Daphnids in the cultures were held for at least 15 days prior to collection of the juveniles for testing. The adults showed no signs of disease or stress during the holding period. During the 14-day holding period preceding the test, water temperatures ranged from 19.8 to 2 0 .1C. ThepH ofthe water ranged from 8.1 to 8.3 and dissolved oxygen ranged from 8.3 to 8.6 mg/L. Instrumentation and methods used for water measurements are described in the Environmental Conditions section of this report. Neonate daphnids (<24 hours old) were obtained for testing from four individual adult daphnids. At test initiation, the juvenile daphnids were collected from the cultures and placed in glass beakers one to two at a time until each beaker contained 10 daphnids. The beakers were then impartially assigned to treatment groups. One daphnid was then transferred to each test chamber using a wide-bore pipet underneath the air/water interface. Daphnids in the cultures and those used in the test were fed a mixture of yeast, Cerophyll, and trout chow (YCT, 1800 mg TSS/L), as well as a suspension of the freshwater green alga, Selenastrum capricornutum (approximately 3.5 x 107cells/mL). During the test, each test chamber was fed 0.5 mL of YCT and 1.0 mL of Selenastrum once daily. Test Apparatus Test chambers were 250-mL plastic (Nalgene) beakers containing 200 mL oftest solution. The depth o f test solution in a representative test chamber was approximately 5 cm. Test chambers were indiscriminately positioned by treatment group in an environmental chamber set to maintain a temperature of 20 1C. The test chambers were labelled with the project number, test concentration and replicate. Dilution Water The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site. The well water is characterized as moderately-hard water. The specific conductance, hardness, alkalinity, and pH of the well water during the four-week period immediately preceding the test are presented in Appendix 1. Wildlife International, Ltd. - 11 - Project Number 454A-130 The well water was passed through a sand filter to remove particles greater than approximately 25 pm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water again was filtered to 0.45 pm and passed through a UV sterilizer to remove microorganisms and fine particles. The results o f periodic analyses performed to measure the concentrations of selected contaminants in well water used by Wildlife International, Ltd. are presented in Appendix 2. Environmental Conditions Lighting used to illuminate the cultures and test chambers during culturing and testing was provided by fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone 50). A photoperiod of 16 hours o f light and 8 hours o f darkness was controlled with an automatic timer. A 30-minute transition period o f low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity was measured at test initiation and at weekly intervals thereafter with a SPER Scientific Ltd. light meter. Light intensity ranged from 357 to 512 lux at the surface of the water (Negative Control, Replicate A). Temperature was measured in alternating replicates of each treatment and the control group at test initiation, in old and new test solutions on renewal days, and at test termination using a liquid-in-glass thermometer. Temperature was also measured continuously in one negative control replicate using a F ulscope E R /C Recorder. Recorder m easurem ents were verified w ith a liquid-in-glass thermometer prior to test initiation and at least weekly thereafter. The target test temperature was 20 1C. Dissolved oxygen was measured daily in alternating replicates during the first seven days of the test, in old and new solutions on renewal days, and at test termination. Measurements of pH were made in alternating replicates at test initiation, in old and new solutions on renewal days and at test termination. Hardness, alkalinity and specific conductance were measured in water samples collected from alternating replicates ofthe negative control and the highest test concentration at test initiation and at approximately weekly intervals thereafter (new solutions). Measurements of pH were made using a Fisher Accumet Model 915 pH meter, and dissolved oxygen was measured using a Yellow Springs Instrument Model 5 IB dissolved oxygen meter. Specific conductance was measured using a Yellow Springs Instrument Model 33 Salinity-Conductivity-Temperature meter. W ildlife International, Ltd. - 12- Project Number 454A-130 Hardness and alkalinity measurements were made by titration based on procedures in StandardMethodsfor the Examination o f Water and Wastewater (5). Biological Observations and Measurements The first generation daphnids were observed daily during the test for survival, the onset of reproduction, and clinical signs of toxicity. The criteria for death included white opaque coloration, lack of movement of appendages, absence of heartbeat and lack of response to gentle prodding. Immobilization was defined as lack of movement except for minor activity of the appendages. The presence o f eggs in the brood pouch, aborted eggs, males or ephippia were also recorded. With the onset of reproduction, the number of second-generation neonates were counted (number of live neonates, number o f dead or immobile neonates and number of aborted eggs) and recorded every Monday, Wednesday and Friday during the test:. At each observation period, the live first-generation daphnids were retained and the second-generation daphnids were discarded. On Day 14, the secondgeneration neonates were collected and used for the second-generation acute exposure. At test termination, the length (measured from the apex of the head to the base of the spine) and dry weight were determined for each surviving first-generation daphnid. The individual daphnids were removed from the test chambers and placed into tared aluminum drying pans. The length of each daphnid was determined using a caliper. The pans were then placed in a drying oven at approximately 60C for approximately 4 6 hours. The pans were removed from the drying oven and placed in a desiccator to cool. When cool, the pans were weighed using a Mettler AE-240 analytical balance. Statistical Analyses Statistical analyses were performed on survival of first-generation daphnids, the number of live young and the length and dry weight of the surviving first-generation daphnids. Survival data were analyzed using Fisher's exact test. Treatment groups with significant survival effects were excluded from the analyses of reproduction and growth. Reproduction and growth (length and dry weight) data were evaluated for normality and homogeneity of variance using the chi-square test and Bartlett's test, respectively. If the data met the assumptions of normality and homogeneity, the Bonferroni t-test was used to identify treatment groups that were statistically reduced in comparison to the negative control (p < 0.05). If the data failed the assumptions of normality or homogeneity, transformations were applied to the data in an attempt to correct W ildlife International, Ltd. - 13 - Project Number 454A-130 the condition. If transformations failed, Wilcoxon's rank sum test with the Bonferroni adjustment was used to evaluate the data (p <. 0.05). All statistical tests were performed on a personal computer using TOXSTAT Version 3.5 (6) statistical software. Analytical Chemistry Water samples were collected from new and old solutions each week during the test. Duplicate samples of new solutions were collected from the freshly prepared solutions. Duplicate samples of the old solutions were collected from a composite of the remaining replicate test chambers. Samples were collected in plastic scintillation vials and were analyzed as soon as possible without storage. Analytical procedures used in the analysis of the samples are provided in Appendix 3. RESULTS AND DISCUSSION Verification of Test Concentrations Results of analyses to measure concentrations of PFBS in water samples collected during the test are presented in Table 1 and the analytical chemistry report (Appendix 3). Nominal concentrations selected for use in the study were 63, 125, 250, 500, 1000 and 2000 mg a.i./L. Measured values of new (freshly prepared) samples ranged from 85 to 103% o f nominal values, whereas measured values from the old solutions ranged from 86 to 106% of nominal values. Consequently, PFBS was stable throughout the renewal periods. W hen m easured concentrations o f sam ples collected during each w eek o f the test were averaged, the mean measured concentrations were 60, 121, 247, 502, 995 and 1876 mg a.i./L, which represented 95, 97, 99, 100, 100 and 94% of nominal, respectively. The results of the study were based on mean measured concentrations. . Physical and Chemical Measurements of Water Measurements of temperature, dissolved oxygen and pH are presented in Tables 2, 3 and 4, respectively. Manual water temperature measurements were within the range o f 201C established for the test. However, there were two instances when tine continuous temperature monitor reached 18C. This occurred during renewal o f test solutions and was probably the result of the temperature probe being exposed to air. Dissolved oxygen concentrations remained 7.9 mg/L (87% of saturation) throughout the test. Measurements of pH ranged from 7.9 to 8.7. W ildlife International, Ltd. - 14- Project Number 454A-13Q Measurements o f specific conductance, hardness and alkalinity in the negative control and the highest treatment group are presented in Table 5. No apparent differences were observed for hardness or alkalinity throughout the exposure period. However, conductivity was elevated in the 1876 mg a.i./L treatment group in comparison to the negative control. Survival and Biological Observations A summary of the results of the observations of mortality and sublethal signs of toxicity for first generation daphnids are shown in Table 6. Daily observations are provided in Appendix 4. After 21 days of exposure, survival in the negative control group was 90%. Survival in the 60, 121, 247, 502, 995 and 1876 mg a.i./L treatment groups was 100, 100, 100, 100, 70 and 20%, respectively. Fisher's exact test showed that survival was significantly different in the 1876 mg a.i./L treatment group in comparison to the negative control (p <. 0.05). Reproduction A summary o f the mean number of live young produced by the first-generation daphnids is presented in Table 7. A complete listing o f the number of neonates produced is provided in Appendix 5. Daphnids in the control and PFBS treatment groups 995 mg a.i./L started producing neonates on Day 8. There were no observations o f aborted eggs in any treatment group. Reproduction averaged 86 young per adult in the negative control. Reproduction in the 60, 121, 247, 502 and 995 m g a.i./L treatment groups averaged 173, 149, 167, 89 and 43 young per adult, respectively. The Bonferroni t-test showed that reproduction was significantly reduced in the 995 mg a.i./L treatment group in comparison to the negative control {p < 0.05). The 1876 mg a.i./L treatment group was not included in the statistical analysis ofreproduction or growth data due to a statistically significant effect on survival. Growth A summary of the mean lengths and mean dry weights of the surviving first-generation daphnids are presented in Table 8. Individual measurements are provided in Appendix 6. The mean length and dry weight of first-generation daphnids in the negative control was 4.18 mm and 0.80 mg, respectively. Wilcoxon's rank sum test showed that mean length was significantly reduced in the 995 mg a.i./L treatment group in comparison to the negative control (p < 0.05). Wildlife International, Ltd. - 15 - Project Number 454A-13Q Second-Generation Acute Exposure The results of the second-generation acute exposure are presented in Table 9. After 48-hours of exposure, survival in the negative control was 100%. Survival in the 60, 121, 247, 502 and 995 mg a.i./L treatment groups was 100,100,100,100 and 100%, respectively. A second-generation acute exposure was not performed for the 1876 mg a.i./L treatment group due to a lack of neonates. CONCLUSIONS There were no adverse effects on survival, reproduction or growth o f Daphnia magna exposed to PFBS at concentrations <502 mg a.i./L. Daphnia magna exposed to 1876 mg a.i./L had significantly reduced survival. Daphnia magna exposed to 995 mg a.i./L had significantly reduced reproduction and length. Consequently, the LOEC for this study, based on reproduction and length was 995 mg a.i./L. The NOEC was 502 mg a.i./L and the MATC was calculated to be 707 mg a.i./L. The results of the secondgeneration acute exposure indicated a NOEC of 995 mg a.i./L. Wildlife International, Ltd. - 16- Project Number 454A-13Q REFERENCES 1 U.S. Environm ental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines (draff), OPPTS Number 850.1300: Daphnia Chronic Toxicity Test. 2 OECD Guideline for Testing of Chemicals. 1997. Guideline 211: Daphnia magna Reproduction Test. 3 U.S. E nvironm ental Protection Agency. 1986. Standard Evaluation Procedure, Daphnia magna Life-Cycle (21-Day Renewal) Chronic Toxicity Test. Hazard Evaluation Division. Office of Pesticide Programs, EPA 540/9-86-141. 4 A m erican Society for Testing and M aterials. 1997. Standard Guide fo r Conducting Renewal Life-Cycle Toxicity Tests with Daphnia magna, ASTM E 1193-96. Philadelphia, PA. 5 APHA, AW W A, W PCF. 1985. StandardMethodsfo r the Examination o f Water and Wastewater. 16th Edition, American Public Health Association. American water Works Association. Water Pollution Control Federation, New York. . 6 W est, Inc. and D.D. Gulley. 1996. TOXSTAT, Version 3.5. WesternEcoSystemsTechnology,Inc. Cheyenne, Wyoming. - 17- Project Number 454A-130 Table 1 Summary of Analytical Chemistry Data Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Nominal Concentration (mg a.i./L) Day 0 (New) Day 2 (New) Day 5 (Old) Negative Control <LOQ` <LOQ <LOQ <LOQ <LOQ <LOQ 63 57.1 63.4 63.7 59.4 62.2 64.5 125 117 121 127 118 125 132 250 239 251 265 237 249 262 500 490 498 544 484 498 537 1000 973 1004 1055 980 1007 1067 2000 1893 1955 1879 1908 `The limit o f quantitation (LOQ) was 20.0 mg a.i./L. 2014 2078 Day 9 (New) <LOQ <LOQ 58.3 59.3 115 116 241 230 474 482 956 946 1774 1835 Day 12 (Old) <LOQ <LOQ 58.2 58.7 116 117 235 238 507 516 999 1002 1894 1962 Day 16 (New) <LOQ <LOQ 54.5 53.2 114 119 257 248 497 477 973 960 1805 1836 Day 19 (Old) < LOQ < LOQ 63.9 60.0 121 122 250 248 503 507 1014 1022 1841 1885 Day 21 (Old) <LOQ <LOQ 63.8 64.6 127 127 251 250 525 500 964 991 1719 1734 Mean Measured Concentration (mg a.i./L) <LOQ 60 121 247 502 995 1876 Percent of Nominal 95 97 99 100 100 94 - 18- Table 2 Temperature of Water in the Test Chambers1 Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, D aphnia magna Well Water Mean Measured (mg a.i./L) Negative Control Day 0 New 20.4 Day 2 Old N ew 20.9 21.0 Day 5 Old N ew 21.0 19.4 Temperature (C ) Day 7 Old N ew 20.8 20.4 60 19.9 20.9 20.5 21.0 19.0 20.7 20.0 121 19.8 20.8 20.8 20.8 19.0 20.8 19.7 247 19.9 21.0 20.9 21.0 19.0 20.9 19.8 502 19.9 20.9 20.9 21.3 19.0 20.9 19.8 995 20.0 21.0 21.0 20.9 19.0 21.0 19.9 1876 20.2 21.0 21.0 21.4 19.2 1Temperature measured continuously during the test ranged from 18.0C to 2 1 .0C. 21.0 20.1 Project Number 454A-130 Day 9 Old N ew 20.6 19.9 20.4 19.7 20.6 19.9 20.8 19.4 20.5 20.8 20.4 20.8 20.6 20.8 Day 12 Old N ew 19.5 19.8 19.3 19.6 19.5 19.6 19.4 20.0 19.3 20.0 20.8 20.1 19.6 20.1 - 19- Project Number 454A-130 Table 2 (Continued) Temperature of Water in the Test Chambers1 Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, D aphnia m agna Well Water M ean M easured Tpqt P.nncpntrfltinn (mg a.i./L) Negative Control Day 14 Old 20.9 New 20.9 Temperature (C) Day 16 Old New 20.5 19.8 Day 19 Old N ew 20.7 20.3 60 20.3 20.5 20.4 19.4 21.0 19.7 121 20.8 20.2 19.9 19.3 21.0 19.5 247 20.9 20.0 19.6 19.3 20.9 19.5 502 20.8 20.1 20.7 19.7 20.5 19.6 995 21.3 20.1 19.8 19.7 21.0 19.7 1876 21.1 20.3 20.6 `Temperature measured continuously during the test ranged from 18.0C to 21.0C. 19.6 21.0 19.9 Day 21 Old 21.0 20.8 19.9 21.0 20.8 21.0 21.0 -20- Project Number 454A-130 Table 3 Dissolved Oxygen Content of Water in the Test Chambers1 Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Negative Control Day 0 New 7.9 Day 1 Old 8.2 Day 2 Old New 8.6 8.4 Dissolved Oxygen Concentration (mg/L) Day 3 Day 4 Day 5 Old Old Old New 8.6 8.4 8.4 8.6 60 8.2 8.2 8.6 8.5 8.6 8.4 8.4 8.6 121 8 0 8 2 8.6 8.5 8.6 g A 8.4 8.6 247 8.0 8.2 8.6 8.5 8.6 8.4 8.4 8.6 502 7.9 8.2 8.6 8.6 8.6 8.4 8.4 8.6 995 8.1 8.2 8.6 8.4 8.6 8.4 8.4 8.6 1876 8.2 8.2 8.6 8.5 8.6 8.4 `A dissolved oxygen concentration o f 5.4 mg/L represents 60% saturation at 20C in freshwater 8.4 8.6 Day 6 Old 8.5 8.5 8.5 8.5 8.5 8.5 8.6 Day 7 Old New 8.2 8.1 8.2 8.3 8.2 8.3 8.2 8.2 8.2 8.2 8.2 8.5 8.2 8.8 -21 - Project Number 454A-130 Table 3 (Continued) Dissolved Oxygen Content o f Water in the Test Chambers1 Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured (mg a.i./L) Negative Control Day 9 Old N ew 8.3 8.4 . D issolved Oxygen Concentration (mg/L) Day 12 Day 14 Day 16 Old N ew Old New Old N ew 8.7 8.0 8.2 8.6 8.3 8.4 60 8.3 8.5 8.6 8.7 8.3 8.4 8.2 8.6 121 8.2 8.6 8.6 8.6 8.2 8.4 8.2 8.7 247 8.2 8.6 8.6 8.6 8.2 8.4 8.4 8.6 502 8.3 8.6 8.6 8.6 8.3 8.4 8.2 8.7 995 8.4 8.8 8.6 8.8 8.2 8.4 8.4 8.9 1876 8.3 9.0 8.8 9.0 8.4 9.0 'A dissolved oxygen concentration o f 5.4 mg/L represents 60% saturation at 20C in freshwater. 8.3 9.0 Day 19 Old N ew 8.4 8.1 8.2 8.1 8.4 8.1 8.2 8.2 8.4 8.4 8.5 8.6 8.5 9.0 Day 21 Old 8.3 8.2 83 8.0 8.2 8.1 8.4 -22- Project Number 454A-130 Table 4 pH of Water in the Test Chambers Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, D aphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Negative Control 60 121 247 502 995 1876 Day 0 New 7.9 8.0 8.0 8.0 8.1 8.1 8.2 Day 2 Old N ew 8.5 8.0 8.5 8.2 8.6 8.2 8.6 8.2 8.6 8.3 8.6 8.3 8.7 8.4 Day 5 PH Day 7 Old N ew Old N ew 8.5 ' 8.1 8.4 8.2 8.6 8.1 8.4 8.2 8.6 8.1 8.4 8.2 8.6 8.1 8.4 8.3 8.6 8.1 8.5 8.3 8.7 8.2 8.5 8.4 8.7 8.2 8.6 8.5 Day 9 Old N ew 8.4 8.0 8.5 8.1 8.5 8.1 8.5 8.1 8.5 8.1 8.5 8.1 8.5 8.2 Day 12 Old N ew 8.5 7.9 8.5 8.0 8.5 8.0 8.5 8.0 8.6 8.1 8.6 8.1 8.7 8.3 -23 - Table 4 (Continued) pH of Water in the Test Chambers Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daph nia magna Well Water M ean Measured Test Concentration (mg a.i./L) Negative Control 60 121 247 502 995 1876 Day 14 Old N ew 8.3 8.2 8.3 8.1 8.4 8.1 8.4 8.2 8.5 8.2 8.5 8.2 8.6 8.2 pH Day 16 Old N ew 8.3 8.0 8.3 8.1 8.4 8.1 8.4 8.1 8.5 8.2 8.5 8.3 8.6 8.4 Project Number 454A-130 Day 19 Old N ew 8.3 8.0 8.3 8.0 8.3 8.0 8.4 8.0 8.4 8.1 8.5 8.2 8.6 8.4 Day 21 Old 8.5 8.5 8.6 8.5 8.6 8.6 8.6 Wildlife International, Ltd. -24- Project Number 454A-13Q Table 5 Specific Conductance, Hardness and Alkalinity in the Negative Control and Highest Treatment Group Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Negative Control Parameter Specific Conductance (p-mhos/cm) Day 0 325 Day 7 335 Time Day 14 340 Day 21 330 Hardness 128 128 132 (mg/L as CaC03) 128 Alkalinity 178 180 182 (mg/L as CaC03) 184 1876 mg a.i./L Parameter Specific Conductance (pmhos/cm) Hardness (mg/L as CaC03) Alkalinity (mg/L as CaC03) Day 0 800 Time . Day 7 800 Day 14 800 140 140 128 178 180 176 Day 21 850 132 184 -25- Project Number 454A-130 Table 6 Summary of Cumulative Percent Mortality and Treatment-Related Effects1for First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, D aphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Cumulative Observations to Day 7 % Mortality % Immobile Effects Negative Control 0 0 10 A N Cumulative Observations to Day 14 %% Mortality Immobile Effects 0 0 10 A N 60 0 0 10 A N 0 0 10 A N 121 0 0 10 A N 0 0 10 A N 247 0 0 10 A N 0 0 10 A N 502 0 0 10 A N 0 0 10 A N 995 0 0 6AN, 4D 30 0 5AN, ID, ID ,G 1876 20 0 8 G 80 0 2G 'Observed Effects: ,AN = Appears Normal, D = Discoloration, G = Appears Smaller *Indicates a significant difference from the negative control on Day 21 using Fisher's exact test (p < 0.05). Cumulative Observations to Day 21 % Mortality % Immobile Effects 10 0 8AN, ID 0 0 10 AN 0 0 10 A N 0 0 10 A N 0 0 10 A N 30 0 6AN, ID 80* 0 2 D ,G Project Number 454A-130 -26- Table 7 Summary of Daphnia magna Reproduction During the Life-Cycle Toxicity Test Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Number of Surviving Daphnids Negative Control 9 Mean Live Young/ Surviving Adult Daphnid (SD) 86 (27) First Day of Reproduction 8 Total Number of Dead/Immobile Neonates 0 60 10 173(34) 8 0 1Z1 10 m y (h/j n uft 247 10 167(30) 8 0 502 10 89 (26) 8 0 995 7 43*(28) 8 0 1876' 2 14 (14) 11 'This treatment was excluded from the statistical analyses of reproduction due to a statistically significant survival effect. ^Indicates a statistically significant difference from the control using the Bonferroni t-test (p <. 0.05). 3 Total Number of Aborted Eggs 0 0 0 0 0 0 0 Wildlife International, Ltd. -27- Project Number 454A-130 Table 8 Summary of Length and Dry Weight of Surviving First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Number of Surviving Daphnids Negative Control 9 Total Length (mm) M ean(SD ) 4.18 (0.182) Dry Weight (mg) M ean(SD ) 0.80 (0.29) 60 10 4.89 (0.111) 1.22 (0.31) 121 10 4.78 (0.207) 1.21 (0.34) 247 10 4.77 (0.206) 0.96 (0.16) 502 10 4.26 (0.480) 0.88 (0.16) 995 7 3.69* (0.352) 0.51 (0.16) 1876' 2 . 2.68 (0.106) 0.10 (0.0) 'T his treatm ent w as exclu ded from the statistical analyses o f grow th due to a statistically sign ifican t survival effect. *Indicates a significant difference from the negative control using Wilcoxon's rank sum test (p z 0.05). -28- Project Number 454A-130 Table 9 Cumulative Percent Mortality/Immobility and Treatment-Related Effects1 for Second-Generation Daphnids Sponsor Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Concentration (mg a.i./L) Daphnia/ Number Replicate Replicate Dead Negative Control A B 10 0 10 0 2 Hours Number Immobile 0 0 60 A 10 0 0 B 10 0 0 121 A i B 10 0 0 247 . A B io 0 10 0 0 0 502 A 10 0 0 B 10 0 0 995 A 10 B 10 Observed Effects: AN = Appears Normal 0 0 0 0 Effects 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN Number Dead 0 0 24 Hours Number Immobile 0 0 00 00 00 00 00 00 00 00 00 00 . Effects 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN Percent Immobile and Dead 0 0 0 0 0 0 Number Dead 0 0 48 Hours Number Immobile 0 0 00 00 00 00 00 00 00 00 00 00 Effects 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN 10 AN Percent Immobile and Dead 0 0 0 0 0 0 Wildlife International, Ltd. -29- Project Number 454A-130 Appendix 1 Specific Conductance, Hardness, Alkalinity and pH o f Well Water Measured During the 4-Week Period Immediately Preceding the Test Sponsor: 3M Corporation Test Substance: PFBS Test Organism: Cladoceran, Daphnia magna Dilution Water: Well Water Specific Conductance (yumhos/cm) Mean 309 (N = 4) Range 305 - 3 1 0 Hardness (mg/L as CaC03) 131 (N = 4) 124-136 Alkalinity (mg/L as CaC03) 178 (N = 4) 176-182 00 1 o 00 pH 8.0 (N = 4) Wildlife International, Ltd -30- Project Number 454A-130 Appendix 2 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Component Measured Concentration Component Measured Concentration Pesticides and Organics Aclonifen Alachlor Ametryn Atrazin Azinphos-ethyl Azinphos-methyl Azoxystrobin Bifenthrin Bioallethrin Bitertanol Bromacil Bromophos Bromophos-ethyl Broompropylaat Bupirimaat Carbaryl Carbofuran Carboxin Chlorfenvinphos Chloridazon Chlorpropham Chlorpyriphos Chlorpyriphos-methyl C h lo rth alo n il Coumaphos Cyanazin Cyfluthrin Cypermethrin Cyproconazole Deltamethrin Demeton Demeton-o Desethylatrazin Desisopropylatrazin Desmetryn Diazinon Diehlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid <0.03 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.04 pg/L <0.08 pg/L <0.25 pg/L <0.05 pg/L <0.05 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.03 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L < 0 .0 4 (J.g/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.25 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.03 pg/L <0.02 pg/L <0.01 pg/L <0.03 pg/L Dimethomorf Disulfoton DMST Dodemorf Endosulfan-a Endosulfan-P Endosulfan-sulfaat Epoxiconazole Eptam Esfenvaleraat Ethion Ethofumesaat Ethoprophos Etridiazole Etrimphos Fenarimol Fenchlorphos Fenitrothion Fenoxycarb Fenpiclonil Fenpropathrin Fenpropimorf Fenthion Fenvaleraat Fluazifop-butyl Fluoroglycofen-ethyl Fluroxypyr-meptyl Flutolanil Fonophos Furalaxyl Heptenophos Imazalil Iprodion Kresoxim-methyl Lenacil Lindane Malathion Metalaxyl Metamitron Metazachlor Methidathion <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0.03 pg/L <0.03 pg/L <0.05 pg/L <0.25 pg/L <0.01 pg/L <0.01 pg/L <0.02 /jg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15,1999. Wildlife International, Ltd. - 31 - Project Number 454A-13Q Appendix 2 (Continued) Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water1 Pesticides and Organics (Page 2) Component Measured Concentration Component Measured Concentration Dichlorvos Dicofol Diethyltoluamide Difenoconazole Dimethoate Paclobutazole Parathion Parathion-methyl Penconazole Pendimethalin Permethrin-cis Permethrin-trans Phosalon Phosmet Phosphamidon-cis Pirimicarb Pirimiphos-ethyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazin Propham Propiconazool Propoxur Propyzamide Prosulfocarb Pyrazophos <0.01 pg/L <0.25 pg/L <0.02 pg/L <0.03 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.03 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.02 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 jig/L <0.05 pg/L <0.03 pg/L <0.02 pg/L <0.02 pg/L <0.03 pg/L Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl Pyrifenox-1 Pyrifenox-2 Pyrimethanil Quizalofop-ethyl Simazin Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbutylazin Tetrachlorvinphos Tetrahydroftaalimide Tetramethrin Thiabendazole Thiometon Tolclophos-methyl Tolylfluanid Triadimefon Triadimenol Triallaat Triazophos Trifluralin Vamidothion Vinchlozolin <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0.01 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L <0.05 pg/L <0.04 pg/L <0.01 pg/L <0.04 pg/L <0.05 pg/L <0.05 ffg/L <0.02 pg/L <0.02 pg/L <0.02 pg/L <0.01 pg/L <0.01 pg/L Metals Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 11.0 mg/L 18.0 mg/L 29 mg/L <0.015 mg/L 1.1 mg/L <0.02 mg/L <0.1 pg/L <0.2 pg/L <0.5 pg/L <0.2 pg/L Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium <1.1 pg/L <0.7 pg/L <0.25 pg/L <0.3 pg/L <0.2 pg/L <0.1 pg/L <0.5 pg/L <0.025 pg/L <0.5 pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15, 1999. Wildlife International, Ltd. -32 - Project Number 454A-130 Appendix 3 THE ANALYSIS OF PFBS IN FRESHWATER IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-130 Wildlife International. Ltd. -33 - Project Number 454A-130 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFBS: A SEMI-STATIC LIFE-CYCLE TOXICITY TEST WITH THE CLADOCERAN {Daphnia magna) WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454A-130 3M ENVIRONMENTAL LAB PROJECT NUMBER: E00-1429 PRINCIPAL INVESTIGATOR: MANAGEMENT: Director, Analytical Chemistry >l/ll/o 1 DATE Wildlife International, Ltd. -34- Project Number 454A-130 Introduction Freshwater samples were collected from a semi-static life-cycle aquatic toxicity study designed to determine the effects of PFBS (Perfluorobutane Sulfonate, Potassium Salt) to the cladoceran (Daphnia magna). This study was conducted by Wildlife International, Ltd. and identified as Project Number 454A -130. The analyses o f these water samples were performed at Wildlife International, Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). Samples were received for analysis between February 7, 2001 and February 28, 2001 and were prepared for analysis on each sample receipt day. Analytical Standard The analytical standard was received from 3M Environmental Technology and Safety Services on March 27, 2000, assigned Wildlife International, Ltd. Identification number 5216, and stored under ambient conditions. The analytical standard, a white powder, was identified as: Potassium Perfluorobutane Sulfonate 5, Lot 2), expiration date: March 2010. The analytical standard was further identified with the 3M Environmental Laboratory test control and reference number TCR . The test substance had a reported purity of 97.90%. A subsequent revision of the certificate of analysis indicated a purity o f 97.3% and an Expiration/Reassessment Date o f January 17, 2002. The analytical standard was the same material and lot number as the test substance (Wildlife International, Ltd. Identification number 5292). The analytical standard was used to prepare calibration standards and matrix fortification samples. Analytical Method W ater samples were analyzed according to the method entitled "Analytical Method Validation for the Determination of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Freshwater" (Wildlife International, Ltd. Project No. 454C-115). Samples were diluted in a 50% methanol : 50% NANOpure water solution so that they fell within the calibration range of the PFBS methodology. Aliquots of the dilutions were transferred to autosampler vials and submitted for analysis by direct injection. Concentrations of PFBS in freshwater samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) interfaced with a Perkin- Wildlife International, Ltd. 35 - Project Number 454A-130 Elmer API I00LC mass spectrometer (single quadrupole) operated in selective ion monitoring (SIM) detection mode. The mass spectrometer was equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separations were achieved using a Keystone PRISM RP column (30 mm x 1.5 mm, 3 qm particle size) fitted with a Keystone Javelin Cjg Guard Cartridge (20 mm x 2 mm). The instrument parameters are summarized in Table 1 and a method flowchart is provided in Figure 1. Primary and Secondary Stock Solutions All primary and secondary stock preparations were adjusted for the purity of the analytical standard (97.90%). A 10.0 mg a.i./mL primary stock solution o f PFBS in methanol was prepared by weighing 1.0243 g of the analytical standard and bringing to final volume with methanol. Secondary stock solutions (1000, 100, 10.0, 1.00, and 0.100 mg a.i./L) o f PFBS 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 10.0 mg a.i./L stock solution o f PFBS in methanol. The calibration standards o f PFBS, ranging in concentration from 0.0100 to 0.0500 mg a.i./L, were analyzed with each sample set. Five calibration standards (different concentrations) were analyzed with the samples. The calibration standard series was injected at the beginning and end o f each run, and one standard was injected, at a minimum, after every five samples. Linear regression equations were generated using the peak area responses versus the respective concentrations o f the calibration standards. A typical calibration curve is presented in Figure 2. The concentration of PFBS in the samples was determined by substituting the peak area responses into the applicable linear regression equation. Representative ion chromatograms of low and high calibration standards are presented in Figures 3 and 4, respectively. Limit of Quantitation The method limit o f quantitation (LOQ) for these analyses was set at 20.0 mg a.i./L calculated as the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (2000). Wildlife International, Ltd. -36- Project Number 454A-13Q M atrix Blank and Fortification Samples Eight 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. Freshwater was either fortified with the appropriate PFBS stock solution in methanol (30.0 mg a.i./L), or directly fortified (/. e. without use o f carrier solvent) with PFBS (400 and 2500 mg a.i./L). Direct fortifications were required for the higher fortification levels due to the limitation in preparing a sufficiently concentrated methanol stock of PFBS. The fortified samples were analyzed concurrently with the test samples to determine the mean procedural recovery (Table 3). Sample concentrations were not corrected for the mean overall procedural recovery of 96.3%. A representative ion chromatogram o f a matrix fortification is presented in Figure 6. Example Calculations Sample number 454A-130-9, nominal concentration o f 500 mg a.i./L in freshwater. First Initial Volume: 0.100 mL Calibration curve equation: First Final Volume: 10.0 mL Slope: 10184392 Second Initial Volume: 0.100 mL Intercept: 21932.54 Second Final Volume: 25.0 mL Curve regression weighted 1/x Dilution Factor: 25000 PFBS Peak Area: 221526 peak area - (y-intercept) PFBS (mg a.i./L) measured at instrument = slope PFBS (mg a.i./L) in sample = PFBS measured at instrument (mg a.i./L) x dilution factor 221526 -21932.54 10184392 x 25000 = 489.9 Wildlife International, Ltd. - 37- Project Number 454A-130 PFBS (mg a.i./L) in sample Percent o f Nominal Concentration = PFBS (mg a.i./L) nominal x 100 489,9 ~ 500 * 100 = 98.0% Calculated with HPLC/M S instrument software: MacQuan, version 1.6. RESULTS Sample Analysis Freshwater samples were collected from a semi-static life-cycle toxicity study with the cladoceran ([Daphnia magna) at test initiation, February 7, 2001 (Day 0), on February 9, 2001 (Day 2 - new solutions), on February 12, 2001 (Day 5 - old solutions), on February 16, 2001 (Day 9 - new solutions), on February 19, 2001 (Day 12 - old solutions), on February 23, 2001 (Day 16 - new solutions), on February 26, 2001 (Day 19 - old solutions) and at test termination on February 28, 2001 (Day 21 - old solutions). The measured concentrations of PFBS in the samples collected at initiation o f exposure of the test organisms (Hour 0) ranged from 90.7 to 98.0% o f the nominal concentrations (Table 4). Samples collected at Day 2, Day 5, Day 9, Day 12, Day 16 and Day 19 had measured concentration ranges o f 94.0 to 101%, 101 to 109%, 88.7 to 96.5%, 92.4 to 103%, 84.5 to 103% and 92.0 to 102% o f nominal values, respectively. Samples collected at test termination (Day 21) had a measured concentration range o f 85.9 to 105% o f nominal values. A representative ion chromatogram o f a test sample is shown in Figure 7. Wildlife International, Ltd. -38 - Project Number 454A-13Q Table 1 Typical HPLC/MS Operational Parameters INSTRUMENT: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 100LC Mass Spectrometer operated in Selective Ion Monitoring (SIM) Mode ION SOURCE: Perkin-Elmer TurboIonSpray ANALYTICAL COLUMN: Keystone PRISM RP (30 mm x 1.5 mm, 3-pm particle size) GUARD COLUMN: Keystone Javelin Cjg cartridge (20 mm x 2 mm) OVEN TEMPERATURE: 40C STOP TIME: 3.00 min FLOW RATE: MOBILE PHASE: INJECTION VOLUME: 200 pL/min 25% NANOpure Water with 0.1% Ammonium Formate: 75% Methanol 5.0 pL PFBS PEAK RETENTION TIME: Approximately 2.1 minutes PFBS MONITORED MASS: 299.0 amu Wildlife International, Ltd. -39- Project Number 454A-130 Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454A-130-) MAB-1 Sample Preparation Date (Day)1 0 Measured Concentration of PFBS (mg a.i./L )2 < LOQ MAB-2 2 < LOQ MAB-3 5 < LOQ MAB-4 9 < LOQ MAB-5 12 < LOQ MAB-6 16 < LOQ MAB-7 19 < LOQ MAB-8 21 < LOQ 1 Preparation Date refers to the Day during the definitive test that these quality control samples were prepared concurrent with sampling of the test samples. 2 The limit o f quantitation (LOQ) was 20.0 mg a.i../L based upon the product o f the lowest calibration standard analyzed (0.0100 mg/L) and the dilution factor of the matrix blank samples (2000),_______ Wildlife International, Ltd. -40- Project Number 454A-130 T able 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number (454A-130-) MAS-1 MAS-4 MAS-7 MAS-10 M AS-13 M AS-16 M AS-19 MAS-22 Preparation Date (Day)1 0 2 5 9 12 16 19 21 Concentrations of PFBS (mg a..i./L) Fortified Measured2 30.0 31.6 30.0 31.0 30.0 29.8 30.0 30.6 30.0 30.0 30.0 29.5 30.0 31.2 30.0 31.1 Percent of Nominal 105 104 99.3 102 100 98.4 104 104 MAS-2 0 400 MAS-5 2 400 MAS-8 5 400 M AS-11 9 400 M A S-14 12 400 MAS-17 16 400 MAS-20 19 400 MAS-23 21 400 390 376 348 361 350 361 372 414 97.5 94.0 87.1 90.3 87.6 90.2 93.0 103 MAS-3 MAS-6 MAS-9 M AS-12 M AS-15 M AS-18 MAS-21 MAS-24 0 2500 2 2500 5 2500 9 2500 12 2500 16 2500 19 2500 21 2500 _______________________ ___ ______________ 2355 2346 2275 2346 2212 2409 3 2356 2481 94.2 93.8 91.0 93.8 88.5 96.4 94.2 99.2 Mean = 96.3 Standard Deviation = 5.63 C V = 5.84% N = 24 1 Preparation Date refers to the Day during the definitive test that these quality control samples were prepared concurrent with sampling of the test samples. 2 Measured and Percent o f Nominal values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 3 Mean of duplicate redilutions (2399 and 2419 mg a.i./L) of original fortification. A dilution error occurred during original sample preparation. Wildlife International, Ltd. -41 - Project Number 454A-130 T able 4 Measured Concentrations of PFBS in Freshwater Samples from a Cladoceran Semi-Static Life-Cycle Toxicity Test Nominal Test Concentration (mg a.i./L) 0.0 Sample Number (454A-130-) 1 2 15 16 29 30 43 44 . 57 58 71 72 85 86 99 100 Sampling Time (Day) 0 0 2 2 5 5 9 9 12 12 16 16 19 19 21 21 PFBS Measured Concentration1 (mg a.i./L) <LOQ2 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Percent of Nominal1 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 63 3 0 40 17 2 18 2 31 5 32 5 45 9 46 9 59 12 60 12 73 16 74 16 87 19 88 19 101 21 102 21 57.1 90.7 59.4 94.3 63.4 101 62.2 98.7 63.7 101 64.5 102 58.3 92.6 59.3 94.1 58.2 92.4 58.7 93.2 54.5 86.5 53.2 84.5 63.9 101 60.0 95.2 63.8 101 64.6 103 1 Measured and Percent o f Nominal values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2 The limit o f quantitation (LOQ) was 20.0 mg a.i./L based upon the product of the lowest calibration standard analyzed (0.0100 mg a.i./L) and the dilution factor of the matrix blank samples (2000). Wildlife International, Ltd. -42- Project Number 454A-130 Table 4 (Continued) Measured Concentrations of PFBS in Freshwater Samples from a Cladoceran Semi-Static Life-Cycle Toxicity Test Nominal Test Sample Sampling PFBS Measured Percent Concentration Number Time Concentration1 of (mg a.i./L)______ (454A-13Q-)________ (Day)_____________ (mg a.i./L)__________ Nominal1 50 60 19 2 20 2 33 5 34 5 47 9 48 9 61 12 62 12 75 16 76 16 89 19 90 19 103 21 104 21 117 93.3 118 94.8 121 96.7 125 99.9 127 102 132 105 115 92.4 116 93.0 116 92.5 117 93.7 114 91.2 119 95.5 121 96.6 122 97.7 127 102 127 101 70 80 21 2 22 2 35 5 36 5 49 9 50 9 63 12 64 12 77 16 78 16 91 19 92 19 239 95.7 237 94.8 251 101 249 99.7 265 106 262 105 241 96.5 230 92.1 235 94.1 238 95.0 257 103 248 99.1 250 100 248 99.3 105 21 106 21 251 100 250 100 1 Measured and Percent of Nominal values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly.___________________________________________________ Wildlife International, Ltd. -43 - Project Number 454A-130 Table 4 (Continued) Measured Concentrations of PFBS in Freshwater Samples from a Cladoceran Semi-Static Life-Cycle Toxicity Test Nominal Test Sample Sampling PFBS Measured Percent Concentration Number Time Concentration1 of (mg a.i./L)______ (454A-130-)________ (Day)______________(mg a.i./L)__________ Nominal1 500 9 10 ' 23 24 37 38 51 52 65 66 79 80 93 94 107 108 0 0 2 2 5 5 9 9 12 12 16 16 19 19 21 21 490 98.0 484 96.7 498 99.5 498 99.7 544 109 537 107 474 94.9 482 96.4 507 101 516 103 497 99.5 477 95.4 503 101 507 101 525 105 500 100 1000 11 12 0 0 973 97.3 980 98.0 25 2 26 2 1004 1007 100 101 39 5 1055 106 40 5 1067 107 53 9 956 95.6 54 67 9 12 946 94.6 999 99.9 68 12 1002 100 81 16 973 97.3 82 16 95 19 96 19 960 1014 1022 96.0 101 102 109 21 964 96.4 110 21 991 99.1 1 Measured and Percent o f Nominal values were calculated using MacQuan, version 1.6 software Manual calculations may vary slightly. Wildlife International Ltd. -44- Project Number 454A-130 Table 4 (Continued) Measured Concentrations o f PFBS in Freshwater Samples from a Cladoceran Semi-Static Life-Cycle Toxicity Test Nominal Test Concentration (mg a.i./L) Sample Number (454A-130-) Sampling Time (Day) PFBS Measured Concentration1 (mg a.i./L) Percent of Nominal1 2000 13 14 ' 27 28 41 42 55 56 69 70 83 84 97 98 0 0 2 2 5 5 9 9 12 12 16 16 19 19 1893 1955 1879 1908 2014 2078 1774 1835 1894 1962 1805 1836 1841 1885 94.7 97.8 94.0 95.4 101 104 88.7 91.8 94.7 98.1 90.2 91.8 92.0 94.2 111 21 1719 85.9 112 21 1734 86.7 1 Measured and Percent o f Nominal values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. Wildlife International, Ltd. -45 - Project Number 454A-13Q M ETHOD OUTLINE FOR THE ANALYSIS OF PFBS IN FRESHW ATER Prepare matrix fortification samples in freshwater as follows: For target PFBS concentrations < 200 mg a.i./L, fortify freshwater with the appropriate stock solution o f PFBS. For target PFBS concentrations > 200 mg a.i./L, prepare by weighing the requisite amount of PFBS test substance on an analytical balance and transferring directly into a Class A volumetric flask partially filled with freshwater. Rinse weighing paper and the sides o f the flask with repeat freshwater rinses. Swirl the flask to dissolve the test substance and then bring to final volume with freshwater. Sonicate as appropriate and mix with several repeat inversions. The matrix blank is unfortified freshwater. I Prepare appropriate dilutions o f study and QC samples to within the calibration range o f the PFBS LCMS methodology: Partially fill Class A volumetric flasks with 50% m ethanol: 50% NANOpure water dilution solvent. Add the appropriate volume of sample and bring to volume with dilution solvent. Perform secondary dilutions as necessary. Process matrix blank samples using the same dilution and aliquot volume as for the lowest fortification level. Mix well by several repeat inversions. I Ampulate samples and submit for LCMS analysis. Figure 1. Analytical method flowchart for the analysis of PFBS in freshwater. Wildlife International, Ltd. -46- Area Project Number 454A-130 Figure 2. A typical calibration curve for PFBS. Slope = 10184392; Intercept = 21932.54; r = 0.99818. Wildlife International, Ltd, -47 - Project Number 454A-130 PFBSJ STD 0.0100 mg a.IVL 5216-002C-6 2.97 in 1 period PFBS No Internal Standard Use Area 1: 2.96 Q1 Ml, 178 scans 299.0 Noise Thres. Quant Thres. Min. Width Mult. Width Base. Width RT Win. (secs) Smooth Expected RT 20.0 20.0 3 6 100 10 1 2.16 Area 124515 Height 14822 Start Time End Time Integration Width 2.03 2.43 0.40 Retention Time Integration Type 2.16 A-BB Thu, Feb 8, 2001 19-12 intensity: 120000 cps Figure 3. A representative ion chromatogram o f a low-level (0.0100 mg a.i./L) PFBS standard. Wildlife International, Ltd. -48 - Project Number 454A-130 PFBS_5 STD 0.0500 mg a.l./L 5216-002C-10 2.97 in 1 period PFBS No Internal Standard Use Area 1: 2.96 Q1 Ml, 178 scans 299.0 Noise Thres. Quant Thres. Min. Width Mult. Width Base. Width RT Win. (secs) Smooth Expected RT 20.0 20.0 3 6 100 10 1 2.16 Area 548302 Height 5721 2 Start Time End Time Integration Width Retention Time Integration Type 2.01 2.68 0.67 2.16 A. - BB Thu, Feb 8, 2001 19:28 intensity: 120000 cps Figure 4, A representative ion chromatogram o f a high-level (0.0500 mg a.i./L) PFBS standard. Wildlife International, Ltd, -49- Project Number 454A-130 PFBS_7 454A-130- MAB-1 Thu, Feb 8, 2001 19:37 2.97 in 1 period PFBS No Internal Standard Use Area 1: 2.96 Q1 Ml, 178 scans 299.0 Noise Thres. Quant Thres. Min. Width Mult. Width Base. Width 20.0 20.0 3 6 100 . RT Win. (secs) Smooth Expected RT 10 1 2.16 Area 0 Height 0 Start Time 0.00 End Time Integration Width Retention Time 0.00 0.00 0.00 Integration Type 100-1 908070605040302010OA intensity: 120000 cps 1 --I 381 1--56 I 1 801 1 1 11-1--5-11291 1 1---1-612 1-- 31 61 91 121 151 Scan 0.52 1.02 1.52 2.03 2.53 Time Figure 5. A representative ion chromatogram o f a matrix blank sample (454A-130-MAB-1). The arrow indicates the retention time o f PFBS. Wildlife International, L td . -50- Project Number 454A-13Q PFBS_9 454A-130- MAS-2 Thu, Feb 8, 2001 19:45 2.97 in 1 period PFBS No Internal Standard Use Area 1: 2.96 Q1 Ml, 178 scans 299.0 Noise Thres. 20.0 Quant Thres. 20.0 Min. Width 3 Mult. Width 6 Base. Width RT Win. (secs) Smooth Expected RT 100 1O 1 2.16 Area 419242 Height 43383 Start Time 2.03 End Time 2.63 Integration Width 0.60 Retention Time 2.18 Integration Type A - BB 100i 908070 60 50 4030- 2010- 04 13 24 37 31 0.52 intensity: 120000 cps 130 59 7078 110 61 1.02 91 1.52 Scan Time Figure 6. A representative ion chromatogram o f a matrix fortification sample (454A-130-MAS-2, nominal PFBS concentration o f 400 mg a.i./L, dilution factor = lOOOOx). Wildlife International, L td , -51 - Project Number 454A-13Q PFBS.21 454A-130- Thu, Feb 8, 2001 20:34 2.97 in 1 period PFBS No Internal Standard Use Area 1: 2.96 Q1 Ml, 178 scans 299.0 Noise "Hires. 20.0 Quant "Hires. 20.0 Min. Width 3 Mult. Width 6 Base. Width 100 RT Win. (secs) 10 Smooth 1 Expected RT 2.16 . Area 221526 Height 24774 Start Time End Time Integration Width Retention Time Integration Type 2.04 2.50 0.45 2.18 A - BB intensity: 120000 cps Figure 7. A representative ion chromatogram of a test sample (454A-130-9, nominal PFBS concentration of 500 mg a.i./L, dilution factor = 25000x). -52- Project Number 454A-130 Appendix 4 Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, D a p h n ia m agna W ell Water M ean Measured Test Concentration (m g a.i./L) R ep licate Initial Number Exposed Number Im m ob ile Negative Control A B C D E F G H I J 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 Day 0 C u m ulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IA N IA N IA N IA N IA N IA N IA N IA N IA N IA N 60 A1 0 0 IA N B1 0 0 IA N C1 0 0 IA N D1 0 0 IA N E1 0 0 IA N F1 0 0 IA N G1 0 0 IA N H1 0 0 IA N I1 0 0 IA N J1 0 0 IA N 121 A1 0 0 IA N B1 0 0 IA N C1 0 0 IA N D1 0 0 IA N E1 0 0 IA N F1 0 0 IA N G1 0 0 IA N H1 0 0 IA N I1 0 0 IA N J1 0 0 IA N 1 Observed Effects: A N = Appeared Norm al, D = Discoloration (Pale), G = Small Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 Day 1 C u m u lative Dead 0 0 0 0 0 0 0 0 0 0 Effects IA N IA N 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 IA N 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 IA N 0 0 1 AN 0 0 IA N 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN Day 2 Num ber Cumulative Im m ob ile Dead 00 00 00 00 00 00 00 00 00 00 Effects 1 AN 1 AN IA N IA N IA N IA N 1 AN IA N 1 AN IA N 0 0 1AN 0 0 IA N 0 0 1AN 0 0 IA N 0 0 IA N 0 0 IA N 0 0 IA N 0 0 IA N 0 0 IA N 0 0 1 AN 0 0 IA N 0 0 1AN 0 0 IA N 0 0 IA N 0 0 IA N 0 0 IA N 0 0 1 AN 0 0 IA N 0 0 IA N 0 0 1AN Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 Day 3 C u m ulative Dead 0 0 0 0 0 0 0 0 0 0 E ffects IA N IA N IA N IA N 1 AN 1 AN IA N IA N 1 AN IA N 0 0 IA N 0 0 1 AN 0 0 1 AN 0 0 IA N 0 0 IA N 0 0 IA N 0 0 IA N 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 IA N 0 0 IA N 0 0 IA N 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1AN 0 0 1AN -53- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water. 3M Corporation PFBS Cladoceran, D aphnia m agna W ell Water Mean Measured Test Concentration (m g a.i./L) Replicate Initial Number Exposed N egative Control A B C D E F G H I J 1 1 1 1 1 1 1 1 1 1 Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 Day 4 C u m ulative Dead 0 0 0 0 0 0 0 0 0 0 60 A 1 0 0 B 10 0 C 10 0 D 10 0 E 10 0 F 10 0 G 10 0 H 10 0 I 10 0 J 10 0 121 A 10 0 B 10 0 C 10 0 D 10 0 E 10 0 F 10 0 G 10 0 H 10 0 I 10 0 J 10 0 1 O bserved E ffects: A N == Appeared N orm al, D = D iscoloration (Pale), G = Sm all E ffects 1 AN IA N IA N IA N IA N IA N IA N 1 AN IA N 1 AN IA N 1 AN 1 AN IA N 1 AN 1 AN IA N IA N IA N 1 AN 1 AN IA N IA N IA N 1 AN 1 AN 1AN IA N 1 AN IA N Number Im m obile 0 0 0 0 0 0 0 0 0 0 Day 5 . C u m ulative Dead 0 0 0 0 0 0 0 0 0 0 E ffects IA N IA N IA N IA N IA N IA N 1 AN 1 AN IA N 1 AN 0 0 IA N 0 0 1 AN 0 0 IA N 0 0 1 AN 0 0 IA N 0 0 IA N 0 0 IA N 0 0 1AN 0 0 IA N 0 0 IA N 0 0 IA N 0 0 IA N 0 0 IA N 0 0 IA N 0 0 IA N 0 0 IA N 0 0 IA N 0 0 1AN 0 0 1AN 0 0 IA N Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 6 C u m u lative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1 AN 1 AN IA N 1 AN IA N 1 AN 1 AN 1 AN 1 AN 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN -54- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: D ilution Water: 3M Corporation PFBS Cladoceran, D a p h n ia m agna W ell Water M ean Measured Test Concentration (m g a.i./L) Replicate Initial Number Exposed Negative Control A B C D E F G H I J 1 1 1 1 1 1 1 1 1 1 Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 Day 7 C u m ulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IA N IA N IA N IA N IA N 1 AN IA N IA N 1 AN 1 AN 60 A 10 0 1 AN B 10 0 IA N C 10 0 IA N D 10 0 IA N E 1 0 0 IA N F 1 0 0 IA N G 10 0 IA N H 10 0 1 AN I 1 0 0 1 AN J 1 0 0 IA N 121 A 1 0 0 B 10 0 C 10 0 D 10 0 E 100 F 10 0 G 10 0 H 100 I 100 J 10 0 1 O bserved Effects: A N = Appeared Norm al, D = Discoloration (Pale), G = Small IA N IA N 1 AN IA N 1 AN IA N IA N 1 AN IA N IA N Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 8 C u m u lative Dead 0 0 0 0 0 0 '0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 E ffects IA N IA N IA N IA N IA N IA N IA N IA N IA N IA N IA N IA N IA N IA N IA N IA N IA N IA N 1 AN IA N IA N IA N IA N 1 AN IA N IA N IA N 1 AN 1 AN IA N Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 9 C u m ulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects 1 AN IA N IA N 1 AN IA N 1 AN IA N 1 AN 1 AN 1 AN 1 AN 1 AN IA N IA N 1 AN IA N IA N IA N 1 AN IA N IA N IA N IA N IA N 1 AN IA N 1 AN 1 AN 1 AN 1 AN - 55 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor: Test Substance: Test Organism: D ilution Water: 3M Corporation PEBS Cladoceran, D a p h n ia m agna W ell Water M ean Measured Test Concentration (m g a.i./L) Replicate Initial N um ber Exposed N egative Control A B C D E F G H I J 1 1 1 1 1 1 1 1 1 1 Number Immobile 0 0 0 0 0 0 0 0 0 0 D ay 10 C u m ulative Dead 0 0 0 0 0 0 0 0 0 0 60 A 1 0 0 B 10 0 C 10 0 D 10 0 E 10 0 F 10 0 G 10 0 H 100 I 10 0 J 100 121 A 1 0 0 B 100 C 10 0 D 10 0 E 10 0 F 10 0 G 10 0 H 10 0 I 10 0 J 100 1 O bserved E ffects: A N = Appeared Norm al, D = Discoloration (Pale), G = Small Effects IA N IA N 1 AN 1 AN IA N 1 AN IA N 1 AN 1AN IA N 1 AN 1 AN IA N 1 AN 1 AN IA N 1 AN IA N IA N IA N IA N 1 AN IA N 1 AN 1 AN IA N IA N IA N 1 AN IA N Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 D a y 11 C u m u lative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects IA N IA N 1 AN 1 AN IA N IA N 1 AN IA N 1 AN 1 AN IA N 1 AN 1 AN IA N 1 AN IA N 1 AN IA N 1 AN 1 AN 1 AN IA N IA N 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 12 C u m u lative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects IA N 1 AN 1 AN IA N IA N 1 AN IA N 1 AN 1 AN 1 AN 1 AN 1 AN IA N 1 AN 1 AN 1 AN IA N IA N IA N IA N IA N 1 AN IA N IA N IA N IA N IA N 1 AN 1AN IA N -56- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed Negative Control A B C D E F G H I J 1 1 1 1 1 1 1 1 1 1 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 13 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IA N IA N IA N 1 AN IA N IA N IA N IA N IA N IA N 60 A 1 0 0 1 AN B 10 0 IAN C 10 0 IAN D 10 0 IAN E 1 0 0 IAN F 10 0 IAN G 10 0 IAN H 10 0 1 AN I 1 0 0 1 AN J 10 0 1 AN 121 A 1 0 0 B 10 0 C 10 0 D 10 0 E 10 0 F 10 0 G 10 0 H 10 0 I 10 0 J 10 0 1 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small IA N IA N IA N IA N IA N 1 AN 1 AN IA N 1 AN 1 AN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 14 . Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects IA N IA N IA N IA N 1 AN 1 AN 1 AN 1 AN 1 AN IA N IA N IA N 1 AN IA N IA N IA N 1 AN IA N IA N IA N IA N 1 AN IA N 1 AN 1 AN 1 AN IA N IA N 1 AN 1 AN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 15 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects IA N 1D IA N 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN IA N IA N IA N IA N 1 AN 1 AN 1 AN IA N 1 AN IA N 1 AN 1 AN IA N 1 AN IA N 1 AN IA N 1 AN IA N IA N IA N -57- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed Negative Control A B C D E F G H I J 1 1 1 1 1 1 1 1 1 1 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 16 Cumulative Dead 0 1 0 0 0 0 0 0 0 0 60 A 1 0 0 B 10 0 C 10 0 D 10 0 E 10 0 F 10 0 G 10 0 H 10 0 I 10 0 J 10 0 121 A 1 0 0 B 10 0 C 10 0 D 10 0 E 10 0 F 10 0 G 10 0 H 10 0 I 10 0 J 10 0 1 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Effects IAN - IAN 1 AN IAN IAN 1 AN IAN 1 AN 1 AN IAN 1 AN IAN IAN 1 AN 1 AN IAN IAN 1 AN IAN 1 AN 1 AN IAN IAN 1 AN IAN IAN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 17 Cumulative Dead 0 1 0 0 0 0 0 0 0 0 00 00 0 .0 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 Effects 1 AN -- IAN IAN 1 AN 1 AN IAN IAN IAN IAN IAN IAN IAN IAN 1 AN 1 AN IAN IAN IAN 1 AN 1 AN 1 AN IAN IAN IAN 1 AN 1 AN 1 AN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 18 Cumulative Dead 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects 1 AN ,, 1 AN 1 AN IAN 1 AN 1 AN 1 AN 1 AN IAN IAN 1 AN IAN IAN IAN 1 AN 1 AN 1 AN 1 AN 1 AN IAN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN -58- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration (mg a.i./L) Negative Control 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Initial Replicate Number Exposed A1 B1 C1 D1 E1 F1 G1 H1 I1 J1 Number Immobile 0 0 0 0 0 0 0 0 0' 0 Day 19 Cumulative Dead 0 1 0 0 0 0 0 0 0 0 60 A1 0 0 B1 0 0 C1 0 0 D1 0 0 E1 0 0 F1 0 0 G1 0 0 H1 0 0 I1 0 0 J1 0 0 121 A1 0 0 B1 0 0 C1 0 0 D1 0 0 E1 0 0 F1 0 0 G1 0 0 H1 0 0 I1 0 0 J1 0 0 1 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Effects IAN - IAN IAN IAN 1 AN 1 AN 1 AN IAN IAN IAN IAN 1 AN IAN IAN IAN IAN 1 AN IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 20 . Cumulative Dead 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects IAN - 1 AN IAN IAN IAN IAN IAN IAN IAN 1 AN 1 AN 1 AN IAN 1 AN IAN IAN 1 AN IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 21 Cumulative Dead 0 1 0 0 0 0 0 0 0 0 Effects 1 AN - 1 AN IAN IAN 1 AN IAN IAN ID 1 AN 0 IAN 0 IAN 0 IAN 0 1 AN 0 1 AN 0 1 AN 0 IAN 0 IAN 0 IAN 0 IAN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 1 AN 0 IAN 0 IAN 0 IAN 0 1 AN 0 1 AN -59- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Initial Test Concentration Number (mg a.i./L) Replicate Exposed Number Immobile 247 A1 0 B1 0 C1 0 D1 0 E1 0 F1 0 G1 0 H1 0 I1 0 J1 0 Day 0 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN 502 A1 0 0 IAN B1 0 0 IAN C1 0 0 IAN D1 0 0 IAN E1 0 0 IAN F1 0 0 IAN G1 0 0 IAN H1 0 0 IAN I1 0 0 IAN J1 0 0 IAN 995 A1 0 0 IAN B1 0 0 IAN C1 0 0 IAN D1 0 0 IAN E1 0 0 IAN F1 0 0 IAN G1 0 0 IAN H1 0 0 IAN I1 0 0 IAN J1 0 0 IAN 1 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 1 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN 1 AN IAN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 0 0 IAN 0 0 1 AN 0 0 IAN 0 0 1 AN 0 0 1 AN 0 0 IAN 0 0 1 AN 0 0 IAN 0 0 IAN 0 0 1 AN 0 0 IAN 0 0 1 AN 0 0 IAN 0 0 1 AN 0 0 IAN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN Day 2 Number Cumulative Immobile Dead 00 00 00 00 00 00 00 00 00 00 Effects IAN 1 AN IAN IAN IAN IAN IAN IAN IAN 1 AN 0 0 IAN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 1 AN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 1AN 0 0 IAN 0 0 1 AN 0 0 IAN 0 0 1 AN 0 0 1 AN 0 0 IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 3 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN 1 AN 1 AN 1 AN 1 AN 1 AN IAN 1 AN 1 AN 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 0 1 AN -60- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed 247 A 1 B1 C1 D1 E1 F1 G1 H1 I1 J1 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 4 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 502 A 1 0 0 B 100 C 100 D 100 E 100 F 100 G 100 H 100 I 100 J 100 995 A 1 0 0 B 100 C 100 D 100 E 100 F 100 G 100 H 100 I 100 J 100 1 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Effects 1 AN IAN IAN IAN IAN IAN 1 AN IAN 1 AN 1 AN 1 AN 1 AN IAN 1 AN 1 AN IAN IAN IAN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN IAN 1 AN 1 AN IAN IAN IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 5 . Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1 AN IAN IAN IAN IAN 1 AN IAN IAN IAN 1 AN 0 0 IAN 0 0 IAN 0 0 1 AN 0 0 1 AN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 IAN 0 0 1 AN 0 0 1 AN 0 0 IAN 0 0 1 AN 0 0 1 AN 0 0 IAN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 6 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Effects 1 AN IAN IAN IAN 1 AN IAN IAN 1 AN 1 AN 1 AN IAN 1 AN 1 AN 1 AN IAN IAN IAN IAN IAN IAN 1D 1D IAN 1 AN 1D 1D 1 AN IAN 1 AN IAN -61 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, D ap h n ia m agna Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed 247 ' A B C D E F G H I J 1 1 1 1 1 1 1 1 1 1 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 7 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects 1 AN 1 AN IA N IAN 1 AN IAN 1 AN IAN 1 AN 1 AN 502 A 1 0 0 IA N B 1 0 0 IAN C 1 0 0 IAN D 1 0 0 1 AN E 1 0 0 IAN F 1 0 0 IAN G 1 0 0 IAN H 1 0 0 IAN I 1 0 0 1 AN J 1 0 0 IAN 995 A 1 0 0 B 100 C 100 D 100 E 100 F 100 G 100 H 100 I 100 J 100 1 Observed Effects: AN - Appeared Normal, D = Discoloration (Pale), G = Small 1D 1D IAN IAN 1D 1D 1 AN IAN 1 AN 1 AN Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 8 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 Effects IAN IAN IAN IAN IAN IAN 1 AN 1 AN IAN 1 AN IAN IAN 1 AN 1 AN IAN IAN IAN IAN 1 AN 1 AN 1D 1D IAN IAN 1D 1D 1 AN IAN IAN - Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 9 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 Effects IAN IAN 1 AN 1 AN IAN 1 AN 1 AN 1 AN IAN 1 AN IAN IAN 1 AN IAN 1 AN IAN 1 AN 1 AN 1 AN IAN __ 1D 1 AN IAN 1D 1D 1 AN IAN 1 AN - -62- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, D ap h n ia m agna Well Water Mean Measured Test Concentration (mg a.i./L) Replicate Initial Number Exposed 247 A 1 B1 C1 D1 E1 F1 G1 H1 I1 J1 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 10 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 502 A 1 0 0 B 100 C 100 D 100 E 100 F 1.0 0 G 100 H 100 I 100 J 100 995 A 1 0 1 B 100 C 100 D 100 E 10 1 F 100 G 100 H 100 I 100 J 10 1 1 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Effects 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN IAN IAN 1 AN IAN IAN IAN 1 AN IAN IAN IAN 1 AN 1 AN IAN _ 1D 1 AN 1 AN - 1 D,G 1 AN 1 AN IAN - Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 11 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 Effects IAN 1 AN IAN IAN 1 AN IAN IAN 1 AN 1 AN 1 AN IAN IAN IAN IAN IAN IAN IAN IAN IAN 1 AN _ 1D 1 AN 1 AN - ID ,G 1 AN IAN 1 AN - Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 12 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 Effects 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN IAN 1 AN 1 AN 1 AN 1 AN 1 AN IAN IAN IAN IAN IAN 1 AN _ 1D 1 AN 1 AN -- 1 D,G IAN 1 AN 1 AN - -63 - Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: Mean Measured Test Concentration (mg a.i./L) 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Replicate Initial Number Exposed 247 A 1 B1 C1 D1 E1 F1 G1 H1 I1 J1 Number Immobile 0 0 0 0 0 0 0 0 0 0 Day 13 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IAN IAN IAN IAN 1 AN IAN 1 AN IAN 1 AN 1 AN 502 A 1 0 0 B 10 0 C 10 0 D 10 0 E 10 0 F 10 0 G 10 0 H 10 0 I 100 J 10 0 995 A 10 1 B 100 C 10 0 D 10 0 E 10 1 F 100 G 10 0 H 100 I 100 J 10 1 1 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small 1 AN 1 AN IAN IAN IAN IAN IAN 1 AN 1 AN IAN __ 1D IAN 1 AN - 1 D,G IAN IAN IAN - Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 14 . Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 Effects IAN IAN 1 AN IAN IAN IAN IAN IAN 1 AN 1 AN IAN IAN IAN IAN IAN IAN IAN IAN IAN IAN _ 1D IAN 1 AN -- 1 D,G 1 AN 1 AN 1 AN - Number Immobile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Day 15 Cumulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 Effects IAN IAN IAN IAN 1 AN 1 AN IAN IAN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN IAN 1 AN __ 1D 1 AN 1 AN - 1 D,G 1 AN 1 AN 1D - - 64- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor: Test Substance: Test Organism: D ilution Water: 3M Corporation PFBS Cladoceran, D aphnia m agna W ell Water Mean Measured Test Concentration (m g a.i./L) Replicate Initial Number Exposed 247 A 1 B1 C1 D1 E1 F1 G1 H1 I1 J1 Number Immobile 0 0 0 0 0 0 0 0 0 0 D ay 16 C u m ulative Dead 0 0 0 0 0 0 0 0 0 0 502 A 1 0 0 B 100 C 100 D 100 E 100 F 100 G 100 H 100 I 100 J 100 995 A 10 1 B 100 C 100 D 100 E 10 1 F 100 G 100 H 100 I 100 J 10 1 1 Observed Effects: A N = Appeared N orm al, D = Discoloration (Pale), G = Small Effects IA N IA N 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN IA N 1 AN IA N 1 AN IA N IA N 1 AN IA N 1 AN 1 AN IA N 1 AN _ IA N IA N 1 AN -- 1D IA N IA N 1D - Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 D ay 17 C u m ulative Dead 0 0 0 0 0 0 0 0 0 0 00 00 00 00 00 00 00 00 00 00 01 00 00 00 01 00 00 00 00 01 Effects IA N IA N IA N IA N 1 AN IA N 1 AN IA N 1 AN 1 AN IA N 1 AN IA N 1 AN IA N 1 AN IA N 1 AN 1 AN IA N _ 1 AN IA N 1 AN - IA N IA N 1 AN 1 AN - Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 D ay 18 C u m ulative Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 Effects IA N IA N IA N IA N 1 AN 1 AN 1 AN 1 AN IA N 1 AN 1 AN IA N 1 AN 1 AN IA N IA N IA N IA N IA N IA N _ 1 AN 1 AN IA N -- IA N 1 AN IA N 1 AN - -65- Project Number 454A-130 Appender 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: D ilution Water: M ean Measured Test Concentration (m g a.i./L) 247 3M Corporation PFBS Cladoceran, D a p h n ia m a g n a W ell Water Replicate Initial Number Exposed A1 B1 C1 D1 E1 F1 Q1 H1 I1 J1 Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 D ay 19 C u m u lative Dead 0 0 0 0 0 0 0 0 0 0 502 A1 0 0 B1 0 0 C1 0 0 D1 0 0 E1 0 0 F1 0 0 G1 0 0 H1 0 0 I1 0 0 J1 0 0 995 A1 B1 C1 D1 E1 F1 G1 H1 I1 J1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 1 1 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Effects 1 AN 1 AN 1 AN 1 AN IA N IA N 1 AN IA N 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN 1 AN IA N IA N 1 AN 1 AN 1 AN - 1D IA N 1 AN IA N - Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 Day 20. C u m ulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IA N 1 AN 1 AN 1 AN IA N IA N IA N 1 AN IA N 1 AN 0 0 IA N 0 0 1 AN 0 0 IA N 0 0 IA N 0 0 1 AN 0 0 1 AN 0 0 IA N 0 0 IA N 0 0 IA N 0 0 1 AN 01 0 0 IA N 0 0 IA N 0 0 1 AN 0 1-- 0 0 1D 0 0 1 AN 0 0 1 AN 0 0 1 AN 0 1- Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 Day 21 C u m ulative Dead 0 0 0 0 0 0 0 0 0 0 Effects IA N 1 AN IA N IA N IA N IA N IA N IA N 1 AN 1AN 0 0 1AN 0 0 1 AN 0 0 IA N 0 0 IA N 0 0 IA N 0 0 1AN 0 0 IA N 0 0 1 AN 0 0 IA N 0 0 IA N 01 0 0 IA N 0 0 IA N 0 0 1 AN 0 1-- 0 0 1D 0 0 1 AN 0 0 IA N 0 0 IA N 0 1- - 66- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor: Test Substance: Test Organism: D ilution Water: 3M Corporation PFBS Cladoceran, D a p h n ia m agna W ell Water M ean Measured Test Concentration (m g a.i./L) Replicate Initial Number Exposed Number Im m ob ile Day 0 C u m ulative Dead Effects 1876 A1 B1 C1 D1 0 0 0 0 0 IA N 0 IA N 0 IA N 0 IA N E1 F1 G1 H1 I1 J1 0 0 0 0 0 0 0 IA N 0 IA N 0 IA N 0 IA N 0 IA N 0 IA N Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Number Im m ob ile 0 0 0 0 0 0 1 .o 0 0 Day 1 C u m u lative Dead 0 0 0 0 0 0 0 0 0 0 E ffects IA N IA N IA N IA N IA N 1 AN - IA N IA N IA N Day 2 Number Cumulative Im m ob ile Dead 00 00 00 00 00 00 01 00 00 00 Effects IA N 1 AN 1 AN 1 AN 1 AN 1 AN -- 1 AN 1 AN 1 AN Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 Day 3 C u m ulative Dead 0 1 0 0 0 0 1 0 0 0 E ffects 1G -- 1G 1G 1G 1G -- 1G 1G 1G - 67- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects1- First-Generation Daphnids Sponsor: Test Substance: Test Organism: D ilution Water: 3M Corporation PFBS Cladoceran, D aphnia m agna W ell Water M ean Measured Test Concentration (m g a.i./L) Replicate Initial Number Exposed Number Immobile Day 4 C u m ulative Dead 1876 A 100 B 10 1 C 100 D 100 E 100 F 100 G 10 1 H 100 I 100 J 100 1 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small Effects 1G - 1G 1G 1G 1G - 1G 1G 1G Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 Day. 5 C u m ulative Dead 0 1 0 0 0 0 1 0 0 0 Effects 1G - 1G 1G 1G 1G - 1G 1G 1G Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 Day 6 C u m ulative Dead 0 1 0 0 0 0 1 0 0 0 Effects 1G -- 1G 1G 1G 1G -- 1G 1G 1G -68- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: D ilution Water: 3M Corporation PFBS Cladoceran, D a p h n ia m agna W ell Water Mean Measured Test Concentration (m g a.i./L) Replicate Initial Number Exposed Number Immobile Day 7 C u m ulative Dead 1876 A 100 B 10 1 C 100 D 100 E 100 F 100 G 10 1 H 100 I 100 J 100 1 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G Small Effects 1G - 1G 1G 1G 1G - 1G 1G 1G Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 D ay .8 C u m u lative Dead 0 1 0 0 0 0 1 0 0 0 Effects 1G - 1G 1G 1G 1G - 1G 1G 1G Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 Day 9 C u m u lative Dead 0 1 0 0 0 0 1 0 0 0 Effects 1G -- 1G 1G 1G 1G -- 1G 1G 1G - 69- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water M ean Measured Test Concentration (m g a.i./L) Replicate Initial Number Exposed Number Immobile Day 10 C u m ulative Dead 1876 A 10 1 B 10 1 C 100 D 100 E 1 10 F 100 G 10 1 H 100 I 100 J 10 0 1 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small E ffects -- - 1G 1G 1G 1G - 1G 1G 1G Number Im m ob ile 0 0 0 0 1 1 0 0 0 0 D ay 11 C u m u lative Dead 1 1 0 1 0 0 1 0 0 0 Effects -- -- 1G -- 1G 1G -- 1G 1G 1G Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 D ay 12 C u m u lative Dead 1 1 0 1 1 1 1 0 0 0 Effects __ ,, 1G __ -- __ 1G 1G 1G -70- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: D ilution Water: 3M Corporation PFBS Cladoceran, D a p h n ia m agna W ell Water Mean Measured Test Concentration (m g a.i./L) R ep licate Initial Number Exposed Number Immobile D ay 13 C u m ulative . Dead 1876 A 10 1 B 10 1 C 100 D 10 1 E 10 1 F 10 1 G 10 1 H 100 I 100 J 10 0 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small E ffects - - 1G -- - - - 1G 1G 1 D ,G Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 D ay 14 C u m ulative Dead 1 1 1 1 1 1 1 0 0 1 Effects _ -- -- -- -- - -- 1G 1G -- Number Im m ob ile 0 0 0 0 0 0. 0 0 0 0 Day 15 C u m ulative Dead 1 1 1 1 1 1 1 0 0 1 E ffects -- -- -- -- -- -- -- 1G 1G - -71- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water M ean Measured Test Concentration (m g a.i./L) Replicate Initial Number Exposed Number Immobile Day 16 C u m ulative Dead 1876 A 10 1 B 10 1 C 10 1 D 10 1 E 10 1 F 10 1 G 10 1 H 100 I 100 J 10 1 1 Observed Effects: AN - Appeared Normal, D = Discoloration (Pale), G Small E ffects -- - -- ~ - 1G 1G ~ Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 D a y 1.7 C u m ulative Dead 1 1 1 1 1 1 1 0 0 1 E ffects -- -- _ -- -- -- - 1G 1G - Number Im m ob ile 0 0 0 0 0 .0 0 0 0 0 Day 18 C u m u lative Dead 1 1 1 1 1 1 1 0 0 1 Effects __ -- _ _ -- _ _ 1G 1G -72- Project Number 454A-130 Appendix 4 (Continued) Cumulative Percent Mortality and Treatment-Related Effects' - First-Generation Daphnids Sponsor: Test Substance: Test Organism: Dilution Water: 3M Corporation PFBS Cladoceran, Daphnia magna Well Water M ean Measured Test Concentration (m g a.i./L) Replicate Initial Number Exposed Number Immobile Day 19 C u m u lative Dead 1876 A 10 1 B 10 1 C 10 1 D 10 1 E 10 1 F 10 1 G 10 1 H 100 I 100 J 10 1 1 Observed Effects: AN = Appeared Normal, D = Discoloration (Pale), G = Small E ffects -- -- ~ 1 D, G 1G - Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 Day 20 C u m u lative Dead 1 1 1 1 1 1 1 0 0 1 E ffects -- -- - 1 D, G 1 D, G - Number Im m ob ile 0 0 0 0 0 0 0 0 0 0 Day 21 C u m u lative Dead 1 1 1 1 1 1 1 0 0 1 Effects _ -- -- -- -- -- 1 D, G 1 D, G - - 73 - Project Number 454A-130 Appendix 5 Neonate Production Sponsor: Test Substance: Test Organism: Dilution Water Mean Measured Test Concentration (m g a.i./L) N egative Control 3M Corporation PFBS Cladoceran, D aphnia m agna Well Water Replicate A B C D E F G H I J 0 0 0 0 0 0 0 0 0 0 0 25 00 00 00 00 00 00 00 00 00 00 7 0 0 0 0 0 0 0 0 0 0 Day 9 12 14 16 19 21 0 31 31 14 19 0 9 27 26 - - 0 0 16 0 6 0 0 0 22 36 24 0 0 0 21 27 32 0 0 7 26 40 17 0 0 24 44 28 22 0 0 17 38 30 0 16 0 33 20 28 0 25 0 12 35 0 18 15 60 A 0 0 0 0 B 0 00 0 C 0 00 0 D 0 00 0 E 0 00 0 F 0 00 0 G 0 00 0 H 0 00 0 I 0 00 0 J 0 00 0 This value excluded from calculation of the mean due to adult death. 5 13 57 61 27 0 4 38 50 62 35 0 0 18 45 52 28 0 0 26 44 52 28 0 3 55 0 47 32 0 35 57 0 61 54 25 18 56 0 52 57 30 2 32 54 44 18 0 0 31 44 49 25 0 6 39 15 33 69 45 Total 95 621 22 82 80 90 118 101 106 80 x ( SD) = 86 ( 27) 163 189 143 150 137 232 213 150 149 207 x ( SD ) = 173 ( 34) -74- Project Number 454A-130 Appendix 5 (Continued) Neonate Production Sponsor: Test Substance: Test Organism: Dilution Water Mean Measured Test Concentration (m g a.i./L) 121 3M Corporation PFBS Cladoceran, D aphnia m agna W ell Water _______________ _______ Replicate A B C D E F G H I J 0 0 0 0 0 0 0 0 0 0 0 25 00 00 00 00 00 00 00 00 00 00 7 0 0 0 0 0 0 0 0 0 0 Day 9 12 14 16 19 21 0 15 27 20 0 9 0 18 46 58 30 0 0 12 31 30 10 0 0 74 0 48 51 0 5 50 0 57 55 27 4 55 0 43 33 9 0 74 0 53 34 33 4 39 57 57 51 0 0 34 46 45 29 0 0 16 41 0 43 17 247 A 0 0 0 0 B 0 00 0 C 0 00 0 D 0 00 0 E 0 00 0 F 0 00 0 G 0 00 0 H 0 00 0 I 0 00 0 J 0 00 0 'This value excluded from calculation of the mean due to adult death. s 11 43 47 4 6 0 6 35 47 32 35 0 0 17 21 40 28 0 2 24 40 45 43 53 10 48 0 41 43 16 21 35 0 27 40 37 22 29 0 45 50 32 8 54 0 40 50 33 0 24 41 50 40 0 7 39 54 0 57 52 Total 71 152 83 173 194 144 194 208 154 117 x ( SD) = 149 ( 47) 155 155 106 207 158 160 178 185 155 209 x ( SD) = 167 ( 30) -75 - Project Number 454A-130 Appendix 5 (Continued) Neonate Production Sponsor: Test Substance: Test Organism: Dilution Water 3M Corporation PFBS Cladoceran, Daphnia magna Well Water Mean __________________________________________Day Measured Test Concentration 0 2 5 7 9 12 14 16 19 21 (m g a.i./L) Replicate 502 A 0 0 0 0 0 12 1 41 42 0 B 0 0 0 0 0 22 40 26 25 0 C 0 0 0 0 0 11 22 27 29 0 D 0 0 0 0 6 25 39 26 41 0 E 0 0 0 0 0 0 0 27 35 0 F 0 0 0 0 0 0 0 10 22 25 G 0 0 0 0 0 4 13 19 25 2 H 0 0 0 0 0 20 35 30 30 0 1 0 0 0 0 0 0 3 6 33 41 J 0 0 0 0 0 10 32 0 8 25 995 A 0 0 0 0 B 0 00 0 C 0 00 0 D 0 00 0 E 0 00 0 F 0 00 0 G 0 00 0 H 0 00 0 1 0 00 0 J 0 00 0 This value excluded from calculation of the mean due to adult death. ,, __ __ __ __ 0 0 0 0 19 16 0 6 15 9 26 0 0 4 21 8 18 0 0 - - ------ 0 0 0 0 12 0 0 5 12 7 4 0 5 13 19 24 34 0 0 3 0 5 13 0 -- "" -- " Total 96 113 89 137 62 57 63 115 83 75 x ( SD) = 89 ( 26) 01 35 56 51 O1 12 28 95 21 01 x( SD) = 43 (28) -76- Project Number 454A-130 Appendix 5 (Continued) Neonate Production Sponsor: Test Substance: Test Organism: Dilution Water 3M Corporation PFBS Cladoceran, D aphnia magna W ell Water Mean Day Measured Test Concentration 0 2 5 7 9 12 14 16 19 21 Total (m g a.i./L)_______ Replicate___________________________________________________________________________________________________________________________________ A 0 00 0 0 - - - - -- B 0 00 0 - - - - -- -- C 0 00 0 0 0 - - -- -- D 0 00 0 0 -- -- -- -- -- E 0 00 0 0 -- -- -- -- -- F 0 00 0 0 -- - -- -- G 0 00 0 - - - - -- -- H 0 0 0 0 0 5 0 4 10 5 I 0 00 0 0 0 0 4 0 0 J 0 00 0 0 0 -- - -- -- 'This value excluded from calculation o f the mean due to adult death. o' o' o' o' o' o' o' 24 4 0' x ( S D )= 14 ( 14) W ildlife International, Ltd. -77- Project Number 454A-130 Appendix 6 Length and Dry Weight o f Surviving First-Generation Daphnids Sponsor: T est Substance: T est O rganism : D ilu tio n W ater: 3 M C orporation PFBS C ladoceran, D a p h n ia m a g n a W ell W ater M ean M easured Test C oncentration (m g a.i./L ) In d iv id u a l L en g th (m m ) M ean L en g th (m m SD ) N egative C ontrol . 4.25 4 .0 5 4 .30 3 .9 5 4 .4 0 4 .30 4 .40 4 .0 0 4 .00 4 .1 8 ( 0 .1 8 2 ) 60 4 .90 4 .8 9 ( 0 .1 1 1 ) 4 .90 4 .90 4 .6 5 4 .90 5 .0 0 5 .0 0 4 .8 0 5 .0 0 4 .8 0 121 4 .7 5 4 .7 8 ( 0 .2 0 7 ) 4 .80 4 .5 0 4 .60 4 .8 5 5 .0 0 4 .6 5 4 .9 0 4 .5 5 5 .1 5 In d iv id u a l D ry W eights (m g) 0 .7 1.0 0 .9 0 .9 1.4 0 .5 0 .7 0 .6 0 .5 1.7 1.3 1.4 1.2 1.4 0 .9 0 .6 1 .4 1.3 1.0 0 .9 1.5 1.9 1.6 0 .9 1.1 0 .9 1.2 1.1 1.0 M ean D ry W eights (m g SD ) 0 .8 0 ( 0 .2 9 ) 1.22 ( 0 .3 1 ) 1.21 ( 0 .3 4 ) Wildlife International, Ltd, -78- Project Number 454A-130 Appendix 6 (Continued) Length and Dry Weight of Surviving First-Generation Daphnids Sponsor: T est Substance: T est O rganism : D ilu tion W ater: 3 M C orporation PFBS C ladoceran, D a p h n ia m a g n a W ell W ater M ean M easured Test C oncentration (m g a.i./L ) In d iv id u a l L en g th (m m ) M ean L en g th (m m SD ) 247 . 4 .7 5 4 .7 7 ( 0 .2 0 6 ) 4 .7 5 4 .60 4 .6 5 5 .0 0 4 .3 5 4 .80 5 .0 0 4 .7 5 5 .0 0 502 4 .3 5 4 .2 6 ( 0 .4 8 0 ) 4 .40 4 .3 0 4 .2 5 4 .4 5 2 .9 5 4 .20 4.65 4 .60 4 .40 995 4 .10 3.69 ( 0 .3 5 2 ) 3 .6 5 3 .6 0 3 .3 0 3 .6 5 4 .20 3 .3 0 1876 2 .7 5 2 .6 0 2.6 8 ( 0 .1 0 6 ) In d iv id u a l D ry W eights (m g) 1.0 1.2 1.1 0 .8 0 .9 0 .8 0 .8 0 .9 1.2 0 .9 1 .0 0 .9 1.0 1.1 1 .0 0 .6 0 .9 0.9 0 .7 0 .7 0 .7 0 .7 0 .4 0 .4 0 .3 0 .6 0 .5 0.1 0.1 M ean D ry W eights (m g SD ) 0 .9 6 ( 0 .1 6 ) 0 .8 8 ( 0 .1 6 ) 0.51 ( 0 .1 6 ) 0 .1 0 ( 0.0) Wildlife International, Ltd. -79- Project Number 454A-130 Appendix 7 Changes to Protocol This study was conducted in accordance with the approved Protocol with the following changes: 1. The protocol was amended to add the experimental start and termination dates, test concentrations and test substance number. 2. The protocol was amended to correct the analytical sampling schedule. 3. During the second-generation exposure, neonates were observed at 2, 24 and 48 hours. 4. Treatment groups were indiscriminately positioned. 5. There were two instances where the continuous temperature recorder reached 18C. Wildlife International, Ltd. -80- Project Number 454A-130 Appendix 8 Personnel Involved in the Study The following key Wildlife International Ltd. personnel were involved in the conduct or management of this study: 1. Henry O. Krueger, Ph.D., Director, Aquatic Toxicology and Non-Target Plants 2. Willard B. Nixon, Ph.D., Director, Analytical Chemistry 3. Raymond L. Van'Hoven, Ph.D., Scientist 4. Cary A. Sutherland, Laboratory Supervisor 5. Kurt R. Drottar, Senior Biologist 6. Susan T. Platania, Biologist